ISTRIBUTION OF IONIZABLE GROUPS - RWTH Publications

AMPHOLYTE MICROGELS WITH CONTROLLED

DISTRIBUTION OF IONIZABLE GROUPS

VON DER FAKULTÄT FÜR MATHEMATIK, INFORMATIK UND NATURWISSENSCHAFTEN DER

RWTH AACHEN UNIVERSITY ZUR ERLANGUNG DES AKADEMISCHEN GRADES EINER DOKTORIN

DER NATURWISSENSCHAFTEN GENEHMIGTE DISSERTATION

VORGELEGT VON

DIPLOM-CHEMIKERIN

RICARDA SCHRÖDER

AUS DÜSSELDORF-HEERDT, DEUTSCHLAND

BERICHTER:

UNIVERSITÄTSPROFESSOR PROF. DR. ANDRIJ PICH

UNIVERSITÄTSPROFESSOR PROF. DR. WALTER RICHTERING

TAG DER MÜNDLICHEN PRÜFUNG: 17.02.2016

DIESE DISSERTATION IST AUF DEN INTERNETSEITEN DER UNIVERSITÄTSBIBLIOTHEK ONLINE

VERFÜGBAR.

III

Für meine Eltern

IV

Table of Contents

1 Introduction…………………………………………………………………………. 1

2 Thesis Outline……………………………………………………………………….. 3

3 State of the Art……………………………………………………………………… 5

3.1. Microgels 5

3.2. Poly-N-vinylcaprolactam and Poly-N-isopropylacrylamide 8

3.3. PVCL and PNIPAm microgels 11

3.4. Literature 16

4 Experimental Part………………………………………………………………….. 20

4.1. Chemicals 20

4.2. Analytical Instrumentation and Experimental Procedures 20

4.3. Microgel Synthesis 24

PART I: Synthesis and Characterization

5 Zwitterionic Microgels…………………………………………………...………… 25

5.1. Introduction 25

5.2. Synthesis Procedure 28

5.3. Precipitation Polymerization…………………………………...……... 30

5.3.1. Introduction 30

5.3.2. Synthesis Procedure 32

5.3.3. Results and Discussion 33

5.3.4. Summary 43

5.4. Emulsion Polymerization…………………………………...………….. 44




5.4.4. Summary 59

5.5. Literature 60

6 Ampholyte Microgels with Statistical Distribution of Ionizable Groups………. 63



6.3. Results and Discussion 67

6.4. Summary 76

6.5. Literature 77

V

7 Ampholyte Microgels with Core-Shell Structure……………………………….. . 79




7.4. Summary 92

7.5. Literature 93

8 Ampholyte Janus-like Microgels………………………………………………….. 94




8.4. Summary 105

8.5. Literature 105

PART II: Interactions with Proteins

9 Interactions with Proteins………………………………………………………….. 106


9.2. Experimental Part 109


9.4. Summary 122

9.5. Literature 123

PART III: Microgel-Based Materials

10 Biomineralization / Biohybrid systems………………………………………...….. 124

10.1. Growth of CaCO3 in Zwitterionic Microgels………………………..... 124




10.1.4. Summary 142

10.1.5. Literature 143

10.2. Microgels and β-TCP as Bone Substitute Material…………...……… 144




10.2.4. Summary 155

10.2.5. Literature 156

VI

11 Coating of Surfaces: Protein-Repellant Surfaces with Zwitterionic Microgels… 158


11.2. Experimental Part 159


11.4. Summary 166

11.5. Literature 167

12 Summary……………………………………………………………………...…….. 168

13 Nomenclature……………………………………………………………………….. 171

14 Appendix…………………………………………………………………………….. 173

Danksagung 178

Schlusserklärung 179

1

1 Introduction

While manifold examples of the synthesis and characterization of ampholyte microgels can be

found in literature, only little attention has been paid to the spatial distribution of ionizable

groups within the three-dimensional polymer network of microgel particles and their

influence on the particles’ properties. Often, microgels are praised as smart drug delivery

systems. In this regard, the location of ionizable groups in the microgels is an essential

parameter to control and predict effective release of drugs and proteins.

T. Hoare and R. Pelton were the first to introduce the problem of different distributions of

ionizable groups. They synthesized microgels based on poly-N-isopropylacrylamide

(PNIPAm) with different acids as comonomers and showed that different microgel

morphologies were obtained depending on the type of comonomers used due to different

reactivity parameters of the monomers [1]. They summarized that a comonomer that reacts

slower than NIPAm is more localized on or close to the microgel surface.

A later work by the same authors analyzed the influence of the distribution of ionizable

groups in PNIPAm-based microgels on the uptake and release of various drugs [2]. Here, the

microgels were functionalized with different acids such as acrylic acid, methacrylic acid,

vinylacetic acid, and fumaric acid. Again, the different reactivity ratios between the respective

acid and NIPAm resulted in microgels with varying distribution of ionizable groups.

Microgels containing vinylacetic acid led to particles with a high surface-functionalization,

while methacrylic acid containing microgels led to particles with ionizable groups located in

the core of the particle. Uptake and release experiments revealed that a higher amount of

drugs could be loaded in the microgels when the charges were situated in the core. Charges on

the surface of the particles lead to a so-called “skin layer” on the surface of the particles that

prevents further uptake of drugs.

In 2006, T. Hoare and D. McLean attempted to develop a kinetic model in order to predict the

distribution of ionizable groups in microgel particles [3]. They state that predictions can be

made on copolymerization ratios, homo-polymerization constants, and initial monomer

concentrations alone. A comparison between the radial density diagrams based on kinetic

model predictions and TEM images (in which carboxylic groups are stained with U(Ac)3)

show a good correlation with regard to the distribution of carboxylic groups. The model could

also be used to predict the radial distribution of crosslinker density within the particle.

The work by T. Hoare and R. Pelton show that the distribution of ionizable groups influences

the swelling and electrophoretic properties of microgel particles and is crucial for many

applications such as drug delivery or chemical sensing.

2

1.2 Literature

[1] T. Hoare, R. Pelton, Langmuir 2004, 20, 2123–33.


[3] T. Hoare, D. McLean, J. Phys. Chem. B 2006, 110, 20327–20336.

3

2 Thesis Outline

The aim of this work is the understanding of how the distribution of functional groups (i.e.

basic and acid moieties added through copolymerization) within the polymer network of

environmentally responsive microgels influences the microgels’ properties. Applications of

microgels are manifold and range from encapsulation/controlled release of drugs and genes

[1][2] and chemical sensing and/or filtration in membranes [3][4] to coating for non-biofouling

and protein-repellent surfaces [5][6] and magnetic resonance imaging [7][8]. With regard to each

application, the microgel architecture plays a crucial role as discussed in the previous chapter.

This work is therefore divided into three parts:

(1) Synthesis and characterization of ampholyte microgel particles.

Microgels are based on poly(N-vinylcaprolactam) (PVCL) and poly(N-

isopropylacrylamide) (PNIPAm) with various distributions of basic and acid moieties

within the microgel particle. Here, zwitterionic as well as ampholyte microgels are

synthesized and the functional groups are incorporated either statistically, as core-shell

or Janus-like (see Figure 2.1). Depending on the amount of hydrophilic comonomers

used, either surfactant-free free-radical precipitation polymerization in water or free-

radical inverse mini-emulsion in heptane/water are used as polymerization techniques.

The synthesis of statistical microgels of both zwitterionic and ampholyte nature

requires a one-step synthesis, while core-shell particles are prepared by a two-step

synthesis. For the synthesis of Janus-like microgels, the particle formation in the

beginning of the synthesis is closely studied to predict the right moment for mixing

separate microgel dispersion at the right time so they form Janus-like particles.

Figure 2.1 Various distributions of ionizable groups aimed at in this work.

The influence of the distribution of ionizable groups as well as their amount in the

microgel on the microgels’ properties such as particle size distribution, hydrodynamic

radius, temperature- and pH-sensitivity, and softness is looked at. Another focus is on

the special behavior and the degradation potential of non-covalently crosslinked

zwitterionic microgels after the addition of salt.

4

(2) Interactions of ampholyte microgels with charged species.

The second part looks closely at the influence of different microgel architecture on the

uptake and release of charged species. Subsequently, the size of the charged species is

increased from simple salts to proteins. The microgels’ properties such as

hydrodynamic radius and swelling behavior are greatly influenced by the addition of

charged species. In case of the protein, where cytochrome c is used as a model protein,

not only the uptake is studied, but also the release using various triggers such as

temperature, the addition of salt and a change in pH. Here, the focus is on how

different distributions of ionizable groups lead to different uptake and release

mechanisms.

(3) Microgel-based materials.

Hybrid materials of the above discussed microgels are studied in this part of the work.

First, ionizable microgels are used as nano-containers for biomineralization of CaCO3

and for bone substitution with β-TCP. Furthermore, zwitterionic microgels are

deposited on surfaces to study the influence of the betaine amount on the surface’s

hydrophilicity, protein-repellency, and self-healing properties.

2.1 Literature

[1] X. Li, J. Yuan, H. Liu, L. Jiang, S. Sun, S. Cheng, J. Colloid Interface Sci. 2010, 348,

408–15.

[2] G. Liu, X. Li, S. Xiong, L. Li, P. K. Chu, S. Wu, Z. Xu, J. Fluor. Chem. 2012, 135, 75–

82.

[3] D. Menne, F. Pitsch, J. E. Wong, A. Pich, M. Wessling, Angew. Chemie - Int. Ed.

2014, 53, 5706–5710.

[4] L. Zhang, M. W. Spears, L. A. Lyon, Langmuir 2014, 30, 7628–7634.

[5] A. W. Bridges, N. Singh, K. L. Burns, J. E. Babensee, L. Andrew Lyon, A. J. García,

Biomaterials 2008, 29, 4605–15.

[6] C. M. Nolan, C. D. Reyes, J. D. Debord, A. J. García, L. A. Lyon, Biomacromolecules

2005, 6, 2032–9.

[7] L. Zhang, H. Xue, Z. Cao, A. Keefe, J. Wang, S. Jiang, Biomaterials 2011, 32, 4604–8.

[8] X. Zheng, J. Qian, F. Tang, Z. Wang, C. Cao, K. Zhong, ACS Macro Lett. 2015, 4,

431–435.

5

3 State of the Art

3.1 Microgels

Microgels are intermolecular crosslinked, three-dimensional colloidal polymer networks with

a porous structure and a high capability to absorb water, hence the name “gel”. The amount of

water that can be absorbed ranges from 10 – 90 wt-% and depends on the hydrophilicity,

presence of charges, and the crosslinking density within the microgel particle [1]. Their sizes

lie in the range of 50 nm to 5 µm [1][2]. Therefore, the term “microgel” is misleading since the

part “micro” is referring to the size of the particles. Though “nanogel” would be the more

appropriate term, in this work, the word “microgel” is still used in the following. The color of

microgel dispersions is highly dependent on the particles’ sizes as well as the degree of

swelling: Dispersions with small or collapsed particles are milky white, while dispersions

with larger or highly swollen particles are nearly transparent, since their refractive index is

close to that of water [3]. Though the swelling of microgels in water is a result of their high

hydrophilicity, they do not dissolve in water due to their internal crosslinking. This

crosslinking can be of covalent nature, but also – dependent on the kind of polymers used -

originate from hydrogen bonding, electrostatic interactions or entanglement of polymer chains

[4]. Microgels that are crosslinked covalently are also referred to as chemically crosslinked

particles, while the others are physically crosslinked. The latter ones are only stable under

certain conditions: changes in the environment such as salt or temperature will lead to the

dissolution of microgels into loose polymer chains. Covalently crosslinked microgels can be

obtained by either the copolymerization of a crosslinker such as N,N’-

Methylenebis(acrylamide) (BIS) [5][6] or a chemical reaction between functional groups in the

polymer chain [7][8]. The latter is often done in a post-polymerization and leads to microgels

that are only crosslinked on the surface.

Microgel particles obtain their colloidal stability by three means: steric hindrance,

electrostatic repulsion or a combination of both [9]. Steric hindrance is caused by the loose

ends of polymer chains on the particle’s surface or by post-modification, e.g. via grafting with

PEGMA [10][11] or PEO [9][12]. Electrostatic repulsion is based on the repulsion forces between

charges on the particle surface and is mostly gained by the use of a charged initiator during

the synthesis, e.g. 2,2'Azobis (2-methylpropionamidine) dihydrochloride (AMPA) or the use

of charged surfactants such as sodium dodecyl sulfate (SDS). The third method combines the

preceding two methods. The good colloidal stability goes so far that microgels are able to re-

disperse in water after being freeze-dried or centrifuged [1].

6

Microgels can be divided into non-responsive and stimulus-responsive gels. This chapter will

only focus on the latter. Stimulus-sensitive microgels undergo volumetric changes when

responding to small variations in the environment. This fascinating behavior has been

extensively studied experimentally and theoretically over the past decades. Most studies in

literature concerning sensitive microgels focus on temperature-sensitive microgels, i.e. they

swell or shrink in size dependent on the solution temperature. The temperature at which the

chemical and physical characteristics change dramatically is called Volume Phase Transition

Temperature (VPTT). Typical examples for temperature-sensitive polymers are poly-N-

isopropyl acrylamide (PNIPAm) and poly-N-vinylcaprolactam (PVCL)-based systems. Both

have a VPTT of around 32 °C [13][14]. This closeness to body temperature makes these

monomers therefore very attractive for biomedical applications.

By the use of comonomers further sensitivities for example to pH, ionic strength, light,

magnetic or electric field, can be incorporated. Because their behavior can be controlled and

tuned externally, they are often called “smart materials” [15][16]. In contrast to macroscopic

gels, micro- and nanogels respond very fast to external stimuli, usually within few seconds

[17][18] due to their high surface area-to-volume ratio [19]. The reaction of macroscopic gels

usually is within several hours because their reaction is dependent on the diffusion speed into

the network. Many authors have reported on the relationship between size and response time

[20][21]. The responsiveness of microgels to external stimuli makes them attractive for a wide

range of applications. They range from targeted drug delivery [22][23][24], catalysis [25][26], tissue

engineering [27], to chemical separation processes [28] and color-tunable colloidal crystals [29].

Microgels have a bulk phase behavior very much different than that of hard microspheres

such as polystyrene (PS) [30][31]. They are liquid at low volume fractions, and show crystal [32]

and glassy phases at high volume fractions. With regard to their softness, they are located

between molecular polymers and hard colloidal particles. As a result, they have a higher

packing density that is beyond the random close packing fraction of hard spheres due to their

ability to deform [33].

The softness of microgels can be changed by variation of the amount of crosslinker as well as

through its response to environmental changes like temperature, pH, and ionic strength. A

different approach was used by Z. Zhou et al. who prepared microgels containing a PS core

and a PNIPAm shell [33]. They could tune the softness of the particles by variation in

temperature and shell thickness. The softness of particles greatly influences their

deformability. Contrary to hard spheres, deformation and interpenetration of polymer chains

can occur for microgels. The softness and deformability of particles are an important factor

7

for the stabilization of Pickering emulsions. Classically, Pickering emulsions are emulsions

stabilized by solid particles. In the last years, thermosensitive microgels were used as

stabilizers due to their high deformability in order to obtain responsive emulsions [34]. In

contrast to hard spheres, microgels deform strongly at the oil-water interface and loose their

spherical shape. The extent of deformation is greatly influenced by the degree of crosslinking.

This effect was intensively studied by M. Destribats et al. [35]. They showed that a higher

deformability leads to kinetically well stabilized emulsions, while a higher crosslinking or an

increase in temperature (resulting in an increase in crosslinking density due to particle

shrinkage) reduces the stability. The degree of deformability also becomes important with

regard to substrate deposition. Deformable microgels can be deposited on surfaces that are not

accessible for rigid particles such as PS or silica [19].

8

3.2 Poly-N-vinylcaprolactam and Poly-N-isopropylacrylamide

N-Vinylcaprolactam (VCL) is a seven-membered vinylamide with a lactam ring that is

soluble in both polar and non-polar solvents. It is a white, crystalline solid with a melting

point of 35 - 38 °C [36]. It is amphiphilic due to its hydrophilic carboxylic and amide group

and its hydrophobic vinyl group. N-isopropylacrylamide (NIPAm) is an acrylamide with a

melting point of 60 – 63 °C [37]. Both monomers are commercially available. The structures of

both VCL and NIPAm can be seen in Figure 3.1.

Figure 3.1 Chemical structures of (left) N-Vinylcaprolactam (VCL); (right) N-

isopropylacrylamide (NIPAm).

In comparison to NIPAm, VCL is more biocompatible. When NIPAm hydrolyses, it produces

toxic amines of low molecular weight [38][39]. Hydrolysis of VCL can occur under strong

acidic conditions and results in the formation of caprolactam (see Figure 3.2) [40].

Most articles in literature discuss the polymerization of VCL via radical polymerization

[42][43][44]. M. Hurtgen et al. prepared thermo-responsive block copolymers of VCL and

vinylacetate in a cobalt-mediated radical polymerization [45]. The reported polydispersity of

Ð = 1.1 confirms the advantage of this method. M. Beija et al. report the polymerization of

PVCL via Reversible Addition-Fragementation Chain Transfer Polymerization (RAFT) and

Macromolecular Design via the Interchange of Xanthates (MADIX) [46]. They were able to

synthesize polymers with Mn of 150,000 g/mol and dispersities below Ð < 1.2. A different

approach was followed by S. C. Cheng et al. They prepared PVCL polymers via γ-radiation in

high yields. They demonstrated how the molecular weight could be tuned with the radiation

dose as well as the monomer concentration [47].

Synthesis procedures to obtain un-crosslinked PNIPAm polymers are rare in literature since

most authors focus on the polymer’s thermosensitivity for the preparation of PNIPAm gels. T.

H. Ho et al. obtained PNIPAm polymer chains with low Ð of 1.05 via RAFT polymerization

[48]. There are, however, many reports about using PNIPAm homo- or copolymers as brushes

or monolayer for the stabilization of nanoparticles. X. Jiang et al., for instance, prepared

poly(acrylic acid)-g-poly(N-isopropylacrylamide) graft copolymers to coat water-soluble iron

oxide nanoparticles [49]. The polymer was obtained in three-step synthesis and served as a

9

template for the preparation of well-defined Fe3O4 nanoparticles. It was shown that the length

of the graft polymer chain influenced the size of the nanoparticles. B.-Y. Zhang et al. prepared

a copolymer of PNIPAM and poly(ethylene glycol) via ATRP [50]. In a subsequent step, this

block copolymer was added to poly(N,N-dimethylamino-ethylmethacrylate) in a click

reaction. The final terpolymer was both temperature- and pH-sensitive.

Figure 3.2 Hydrolysis of N-vinylcaprolactam [41].

Both poly-N-vinylcaprolactam (PVCL) and poly-N-isopropylacrylamide (PNIPAm) are non-

ionic, water-soluble, and temperature-sensitive polymers with a lower critical solution

temperature (LCST) at around 32 °C in water [13][51][52]. The temperature-sensitivity is based

on the presence of both hydrophobic (alkyl chains or vinyl backbone) and hydrophilic groups

(amide or carbonyl groups). Below the LCST, non-crosslinked PVCL polymer chains form

random coils in water because its hydrophilic carbonyl group forms hydrogen bonds with the

surrounding water molecules (polymer-solvent interactions) giving a favorable contribution to

the free mixing energy (ΔHm < 0). Simultaneously, the hydrophobic alkyl ring forms

polymer-polymer interactions. Since the hydrogen bonds are stronger than the hydrophobic

interactions, PVCL is soluble in water. When the temperature reaches the LCST, PVCL

undergoes dramatic changes in its interactions with the surrounding water molecules. The

10

polymer-solvent bonds are disrupted and the polymer-polymer interactions become stronger.

PVCL turns from a random coil to a globular polymer that is insoluble in water. As formerly

ordered water molecules are released by this process, an increase in entropy results. This

process is similar for non-crosslinked PNIPAm polymer chains. The LCST can be tuned by

the ratio between hydrophobic and hydrophilic groups in the polymer. The higher the number

of hydrophobic segments, the lower the LCST becomes [21][53][54].

Berghman et al. distinguish three different types of LCST: type I, type II, and type III [55][56].

PVCL shows a type I LCST transition behavior, i.e. the position of the critical point can be

shifted towards lower concentrations by an increase in the polymer chain length [38].

Since PVCL has a type I transition behavior, its LCST is dependent on the molecular weight

of the polymer as well as the concentration. An increase in Mw and concentration leads to a

decrease of the LCST of PVCL [56][46]. M. Beija et al. could show how the cloud point of

PVCL microgels can easily be tuned [46]. By increasing the molecular weight from 18.000 to

150.000 g/mol, the LCST was shifted from 33 to 46 °C. The incorporation of comonomers is

another possibility to influence the LCST of polymers [14][57]. For instance, A. Kermagoret et

al. synthesized copolymers of PVCL with N-vinylamide and vinylester and were able to shift

the LCST from 32 °C to 81 °C and 19 °C for molar fractions of 0.57 % of N-vinylacetamide

and 0.45 % of vinylacetate, respectively [52].

PNIPAm exhibits an LCST in the same range as PVCL. In contrast to PVCL, PNIPAm is a

type II polymer, i.e. its critical point is almost independent of the polymer chain length.

Poly(vinyl methyl ether) (PVME) is a typical polymer having a type III transition behavior.

Type III polymers have two critical points: one for low and one for high polymer

concentrations, thus showing type I and type II behavior, i.e. the LCST of PVME is

depending both on the polymer molar mass and the concentration [58][59].

Polymers having an LCST are of great interest as their behavior resembles the denaturation

process of proteins and enzymes. As I. Bischotberger et al. explain, the globule-to-coil

transition of a polymer is similar to the denaturation of proteins. Above the LCST, a polymer

is in a compact, “folded” state but it unfolds to an expanded coil below the LCST [60] .

Polymers based on PVCL are used in a wide range of applications. The great potential of

PVCL for the preparation of thermosensitive microgels is discussed in the next chapter.

Copolymers of VCL and N-vinylpyrrolidone (VP) are sold by BASF under the trademark

LUVITEC© and are used as hot-melt adhesives, glue sticks and hydrogel adhesives [61][62].

Copolymers of VCL and 1-vinylimidazole (Vim) were used to prepare protein-like

11

copolymers with hydrophobic units in the core and polar units in the shell of a globule [63].

VCL copolymers are also used in hairsprays and hair-fixation resins [64].

12

3.3 PVCL and PNIPAm microgels

The first microgels were synthesized via dispersion polymerization by R. Pelton in 1986 [65].

They obtained PNIPAM microgels with sizes of about 1000 nm below the VPTT.

Generally, PNIPAm and PVCL microgels are obtained in a free radical polymerization at

synthesis temperatures above the LCST of the respective monomer, i.e. 60 – 80 °C. The main

requirement is that the polymer is insoluble at this temperature, otherwise a macrogel instead

of a microgel will form. At this temperature, the water-soluble, free-radical initiator

decomposes and starts the growth of a monomer radical to a polymer chain via radical

propagation (see Figure 3.3). Through the use of an ionic initiator such as AMPA or

potassium persulfate (KPS), the charge at the polymer chain end will contribute to the

resulting microgel’s colloidal stability. In contrast to the synthesis of PNIPAm microgels,

however, initiators such as KPS cannot be used to produce PVCL microgels due to the

sensitivity of the VCL ring to oxidation (see Figure 3.2) [41][66]. Beyond a critical chain length

of the collapsed polymers, phase separation occurs due to the polymer’s insolubility in water

and the aggregates form precursor particles that grow in size by further aggregation, by the

addition of monomer molecules or by the addition of growing oligo-radicals. The reaction

stops when the amount of monomers is exhausted. During the cooling down of the solution at

the end of the polymerization to T < LCST, the microgel network swells in water [1] [67] [26]

[68]. After synthesis, the microgel dispersion needs to be purified due to the presence of

unreacted monomers, surfactants and/or linear or slightly branched polymer chains. Common

techniques are centrifugation, filtration, decantation, and dialysis.

Figure 3.3 Microgel formation during free radical precipitation polymerization.

The synthesis with free radical polymerization technique is advantageous since it produces

monodisperse particles and particle properties such as particle size and charge are easily

controlled [68]. Still there are some limitations that make other polymerization techniques more

attractive. First, the incorporation of comonomers is very much dependent on their

hydrophilicity [67]. Very hydrophilic monomers may not precipitate at the critical chain length

to form precursor particles. Differences in polymerization parameters between different

13

comonomers as well as between monomer and crosslinker lead to the formation of

inhomogeneous particles. Usually, microgels have a higher crosslinking density in the core

than in the outer shell of the particles [69]. Furthermore, J. Gao and B. J. Frisken have shown

that monomers are able to self-crosslink [70]. They could prepare PNIPAM microgels without

the use of the crosslinker BIS and obtained particles with a narrow particle size distribution.

The authors explained this result by chain transfer reactions preferably at the hydrogen atom

on the tert-C of the main chain backbone. This leads to problems regarding the possible

application as a drug delivery system for microgels containing cleavable crosslinkers (with

regard to biocompatibility and removal of particles from the body) where a self-crosslinking

of monomers results only in partial degradation. Another drawback is the encapsulation of

temperature-sensitive drugs during the synthesis due to the high temperatures used during

precipitation polymerization (usually 60 °C [71] - 70 °C [72][73][74]). X. Hu et al. proposed a

different mechanism to obtain microgels via precipitation polymerization at low temperatures

[68]. They assumed that because the LCST of PNIPAm is at 32 °C, nucleation of particles

should be possible at temperatures far below 60 °C, but above 32 °C. Classical precipitation

polymerization is carried out at high temperatures since the number of radicals decreases with

decreasing temperature. X. Hu bypassed this using two different approaches: (a) the use of the

redox initiator system ammonium persulfate (APS)/ N,N,N’,N’-tetramethylethylenediamine

(TEMED) which allows initiation below the thermal decomposition of APS. As already

shown by X. D. Feng, the mechanism is based on a contact charge transfer complex and a

cyclic transition state between APS und TEMED [75]; (b) the decomposition of the initiator

2,2’-azobis isobutyronitrile (AIBN) was realized by UV radiation. Both variations allowed the

preparation of spherical PNIPAM microgels with a narrow particle size distribution.

The size of the particles can be controlled by various means. An increase in the synthesis

temperature leads to the formation of smaller particles [76][77]. Smaller particles can also be

obtained by the addition of an ionic surfactant [5]. D. Dupin et al. describe how the surfactant

absorb onto the PNIPAm surface and thus lead to stabilization at an early stage of particle

formation [78]. A higher concentration of surfactants leads to even smaller particles. Nonionic

surfactants are usually used for steric stabilization [79]. A. Lee et al. used poly(vinyl alcohol)

of various molecular weight and showed that PNIPAm microgels could be stabilized over a

wide ionic strength. If, in contrast, the size of particles is to be increased, electrolytes can be

added during the synthesis procedure. H. Shimizu et al. added sodium chloride to the

monomer solution to obtain particles with sizes between 0.55 and 1.60 µm [80]. An increase in

size, however, is accompanied with an increase in polydispersity. The particle size of

14

PNIPAM microgels prepared by T. Still et al. in a semi-batch emulsion polymerization could

be tuned between 0.8 – 4 µm depending on the charge concentration [5]. Since the particle size

increases linearly with time, the reaction can be stopped at specific times in order to obtain the

desired particle size.

Micro- or hydrogels based on polymers possessing a LCST such as NIPAm and VCL show a

volume phase transition temperature (VPTT) themselves. That means that particles are

swollen and hydrophilic below the VPTT, above the VPTT they collapse and expel water

from the polymer network (see Figure 3.4). Similar to their polymers, the VPTT of microgels

can be tuned by varying the ratio between hydrophobic and hydrophilic comonomers.

Figure 3.4 Hydrodynamic radius RH of PVCL microgels as a function of temperature.

This phenomena for non-ionic microgels was first shown for PNIPAm microgels by

Hirokawa et al. [81]. PNIPAm-based microgels exhibit a VPTT of 32-34 °C [14][81][82]. Below

the VPTT, the behavior of crosslinked PNIPAm is similar to that of non-crosslinked PNIPAm

as described above. Crosslinked microgels are surrounded by structured water molecules and

thus swollen. I. Bischotberger et al. have discussed the increased solubility of the hydrophobic

parts of PNIPAm at low temperatures resulting from the formation of a hydration shell around

the hydrophobic groups that involves stronger hydrogen bonds between the water molecules

than in the bulk [60]. With an increase in temperature, however, contact between the

hydrophobic groups becomes thermodynamically more favorable than contact between the

hydrophobic groups and water [83]. Subsequently, the polymer network undergoes a

conformation change so that hydrophobic groups are surrounded by more hydrophilic groups.

In contrast to non-crosslinked polymers, however, crosslinked polymer networks cannot

dissolve in water above the VPTT due to the crosslinks.

When NIPAm is modified with an additional hydrophobic methyl group, it shows a much

higher phase transition temperature. N-isopropylmethacrylamide (NIPMAm) has an LCST of

15

38 - 45 °C which is attributed to the steric hindrance of the methyl group that hinders

favorable association due to restraints of the chain conformational change [84][85].

Microgels based on PVCL retain its monomers’ reversible thermosensitivity and most authors

agree that PVCL microgels have a VPTT at around 32 °C in water [9][12][41], tough their

transition is not as sharp as the transition of PNIPAm microgels [86][87]. High-sensitivity

differential scanning calorimetric studies by L.M. Mikheeva et al., however, show that PVCL

microgels reveal two separate transition temperatures: at 31.5 °C and at 37.6 °C [83]. The

authors assign the higher transition to the gel volume collapse, while the formation of

hydrophobic domains within the microgel occurs at the lower temperature. These

hydrophobic domains are a result of the amphiphilic character of the PVCL polymer chain. It

consists of cyclic amide with a hydrophobic polymer backbone (see Figure 3.1). The

formation of the hydrophobic domains is thermodynamically more favored than the complete

collapse of the overall gel which occurs only at a higher temperature of 37.6 °C.

The VPTT is influenced by changing the polymer-solvent interactions. This can be realized

by a variation in crosslinking density [88], pH, solvent [89], the presence of salt [90] or

surfactants [13], and the amount of ionic [91], hydrophobic [92] or hydrophilic groups [93][94] in

the polymer. For instance, Mikheeva et al. studied the effect of NaCl and sodium dodecyl

sulfate (SDS) on the transition temperature of PVCL microgels. They showed that the

presence of NaCl results in increased hydrophobic interactions, hence both transition

temperatures are shifted to lower temperatures, i.e. room temperature. Amphiphilic SDS on

the other hand promotes the formation of hydrophobic domains, therefore pronouncing the

difference between the two separate transition temperatures while at the same time shifting

them to higher temperatures. At an SDS concentration of 5.2 mM, the transition temperatures

shift to 67.6 °C and 78.8 °C, respectively. A. Pich et al. showed that the amount of ethanol in

an aqueous PVCL microgel solution greatly influences the transition temperature [89].

Depending on the amount of ethanol, the VPTT decreases, increases or vanishes completely.

All these examples, however, are not suitable if it comes to applications under physiological

conditions. Variations in solvent or ionic strength, for instance, are hardly or not at all feasible

within the body. Also the use of ionic surfactants is not without difficulty since they cause the

denaturation of proteins [95]. Therefore, the most practicable approach is the incorporation of

hydrophobic or hydrophilic comonomers. Hydrophilic units change the VPTT to higher

temperatures, while hydrophobic comonomers shift it to lower temperatures. A. Pich et al., for

instance, prepared VCL-co-AAEM-co-Vim microgels and showed that a higher amount of

Vim leads to an increase in the VPTT. At pH = 4, the protonation of the Vim-segments leads

16

to a drastic shift of the VPTT to ~ 50 °C compared to ~ 28 °C for pure PVCL-co-AAEM

microgels [93].

While properties of PNIPAm microgels such as a VPTT close the human body temperature

are important for applications in the biomedical field, the problem of colloidal stability under

physiological conditions must be solved. H.-Y. Tsai report the aggregation of PNIPAm

microgels in buffer solution when the temperature rises over the transition temperature [96].

Steric stabilization or the incorporation of acid or basic moieties improves the stability.

Another drawback of PNIPAm microgels is the toxicity of the monomer NIPAm and its

carcinogenetic properties [97]. PNIPAm microgels therefore are neither biocompatible nor

biodegradable. VCL, on the other hand, is more suitable for bio-applications since it is stable

against hydrolysis [40]. Its biocompatibility [47][51], non-toxicity [98][99], solubility in both water

and organic solvents, inexpensiveness [46], and high absorption ability [100] makes its use

attractive for many medical and biochemical applications. S. Peng and C. Wu showed that

microgels of PVCL and sodium acrylate are able to build complexes with Hg2+, Cu2+, Ca2+,

and Na+ making it suitable for water treatment [101]. Y. Wang et al. prepared nanogels based

on PVCL with ketal linkages that are acid cleavable. They studied the drug release behavior

using the drug doxorubicin (DOX) and demonstrated the fast and efficient release at low pH.

17

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21

4 Experimental Part

4.1. Chemicals

N-vinylcaprolactam (VCL, 98 %, destilled before use), [2-(methacryloyloxy)ethyl]-dimethyl-

(3-sulfopropyl)-ammonium hydroxide (sulfobetaine, SB, 97 %), N,N‘-

methylene(bis)acrylamide (BIS, 99 %), itaconic acid (IA, ≥ 99 %), 1-Vinylimidazole (Vim, ≥

99 %), N-isoproylacrylamide (NIPAm, 97 %), Span®80 (sorbitan monooleate), Tween®80

(polyethylene glycol sorbitan monooleate), 2,2‘-azobis(2-methylpropionamidine)

dihydrochloride (AMPA, 97 %), ammonium carbonate (≥ 30 % NH3 basis), phosphate

buffered saline (PBS, tablets), (3-aminopropyl)trimethoxysilane (APTMS, 97 %), 2-

(methacryloyloxy)ethyl acetoacetate (AAEM, 95 %), 2-(N,N’-dimethylamino)ethyl

methacrylate (DMAEM, 98 %), cytochrome c (from equine heart, ≥ 95 %), and acetone (for

HPLC, ≥ 99.8 %) were purchased from Sigma Aldrich and were used without further

purification. 2-(methacryloyloxy)ethyl 2-(trimethylammonio)ethyl phosphate (phospho-

betaine, PB, ≥ 98 %) was purchased from Santa Cruz Biotechnology. β-propiolactone (97 %)

was obtained from Alfa Aesar. Albumin from borine serum tetramethylrhodamine conjugate

(BSA) was purchased from Life technologiesTM. Toluene (min. 99.5 %) was obtained from

VWR Chemicals. Calcium chloride (95 %, granulated) was purchased from Riedel-de-Haёr.

Potassium chloride (pro analysi) and sodium chloride (for analysis) were obtained from

Merck Millipore. Water used in the experiments was purified using a Millipore water

purification system with a minimum resistivity of 18 MΩ*cm. For light scattering

experiments, sterile water from Roth was used.

4.2 Analytical Instrumentation

Dynamic Light Scattering (DLS) The hydrodynamic radius RH of the microgel particles was

measured using an ALV/CGS-3 goniometer with an ALV/LSE 7004 Tau Digital Correlator

and a JDS Uniphase laser operating at 633 nm. The measurements were taken at 25 °C at an

angle of 90 °C after equilibrating the samples for at least 10 min. Temperature trends were

measured in a temperature range of 15 °C to 50 °C in 3 °C-steps with at least five

measurements per temperature step. Before all measurements, the samples were filtered with

a 1.2 µm PTFE filter. Water was filtered with 5 µm, 1.2 µm, and 0.45 µm filters prior to use.

Another device for dynamic light scattering is the Zetasizer Nano ZS (Malvern Instruments,

UK) equipped with a 532 nm laser. Measurements were performed at a scattering angle of

22

173 °. Measurement conditions (equilibration time, measurement time, number of

measurements, temperature range) are identical to the measurements performed at the ALV.

Static Light Scattering (SLS) The molecular weight of the microgel particles was measured

with static light scattering using a Fica goniometer (SLS Systemtechnik, Germany) with a

laser of 543 nm. The samples were measured in an angle range of 25 ° to 145 ° with 5 °

intervals. For each sample, five dilutions were prepared with concentration from 0.1 mg/mL

to 2 mg/mL. Prior to SLS measurements, the change of the refractive index (dn/dc) was

determined using a refractometer (SLS Systemtechnik, Germany). Both measurements were

performed at 20 °C.

Electrophoretic Mobility The electrophoretic mobility was measured at a Zetasizer NanoZS

(Malvern Instruments, UK). Each sample contained 1 mM NaCl and was measured at 25 °C

after an equilibration of at least 10 min. The size of microgels at different pH were measured

using 0,1 M HCl and NaOH to adjust the pH. Measurements were taken between pH = 3 and

10 in 0.5 steps at 25 °C. Before all measurements, the samples were filtered with a 1.2 µm

PTFE filter.

Nuclear Magnetic Resonance (NMR) To determine the comonomer content in microgels,

1H-NMR spectra were taken with a Bruker DPC at a frequency of 300 MHz. 5 wt-% of

freeze-dried microgels were dissolved in D2O and measured at room temperature.

Fourier-transformed Infrared Spectroscopy (FTIR) FTIR measurements were performed on

freeze-dried microgel samples at a Bruker, Alpha-P apparatus at room temperature. The

samples were mixed with KBr powder and then pressed to form a transparent KBr pellet.

UV-Vis Spectroscopy (UV-Vis) UV-Vis spectra were taken at a UV-visible

spectrophotometer using a CARY 100 Bio (Agilent Technologies, USA). A given

concentration of microgel solution in water was measured at room temperature in the range of

900 – 200 nm using 1 cm path length quartz cuvettes.

For the determination of the UCST and LCST of a solution, the temperature was controlled

using a heating circulator and a cooler. The temperature was increased from 5 °C to 70 °C

with a heating rate of 0.4 K/min. The UCST or LCST was defined as the temperature at the

maximal slope for the absorbance against the temperature.

Field Emission Scanning Electron Microscopy (FE-SEM) FE-SEM images were taken at a

S4800 (Hitachi) equipped with a field emission cathode. The electron high tension was set to

1-2 keV. One drop of the microgel solution was put on aluminum foil and dried overnight to

prepare samples for FE-SEM investigation. To improve the electric conductivity, a gold layer

was sputtered for 100 s onto the samples prior to measurements.

23

Transmission Electron Microscopy (TEM) TEM images were taken using a Zeiss Libra 100

(Zeiss, Germany). For the sample preparation one drop of the microgel solution was deposited

at room temperature on a TEM copper grid (Formvar/ carbon film, 200 mesh, Plano GmbH,

Germany) the day before and dried overnight. The TEM was operated at an acceleration

voltage of 120 kV. The images were recorded using a CCD camera system (Ultra Scan 1000,

Gatan, Germany).

Atomic Force Microscopy (AFM) Atomic Force Microscopy images were taken at an

Asylum Research MFP-3D (Santa Barbara, CA) in AC mode. Cantilevers of silicon nitride

were purchased from NanoWorld (Neuchatel, Switzerland) with a force constant of 42 N/m.

Images (10.0 x 10.0 µm²) were acquired using the tapping mode. Force mapping was done in

contact mode. The root-mean-square (rms) roughness gives the height deviation from a mean

line and was determined to quantitatively describe the roughness of the surface:

𝑹𝒒 = [(𝟏

𝑳) ∫ 𝒁(𝒙𝟐)𝒅𝒙

𝑳

𝟎]𝟏𝟐⁄

(1)

with L = length; Z(x) = profile height function.

Thermogravimetric Analysis (TGA) Thermogravimetric analysis (TGA) measurements were

performed using a Netzsch TG 209C unit (Netzsch Gerätebau GmbH, Germany) operating

under nitrogen atmosphere with a flow rate of 10 mL/min. 20–30 mg of samples were placed

in standard Netzsch alumina 85 μL crucibles and heated at 20 K/min from 20 °C to 850 °C.

Differential Scanning Calorimetry (DSC) Measurements were performed at a Netzsch DSC

204 (Netzsch Gerätebau GmbH, Germany). Samples were solved in 1 – 2 drops of D2O in

aluminum crucibles and weighed. The crucibles were closed with an aluminum lid. As

reference, pure D2O was used. The scanning was done from 5 – 70 °C at a heating rate of

2 K/min under nitrogen flow. An isotherm was recorded for 2 min before the sample was

cooled down again to 5 °C at a heating rate of 2 K/min. The cycle was repeated once.

Isothermal Titration Calorimetry (ITC) The measurements were performed at a TAM III

from TA Instruments company, Germany. A given amount of microgel and protein were

solved in PBS buffer, respectively. A solution of pure PBS buffer was used as a reference. All

samples were degassed for 10 min at room temperature and 635 mmHg to remove CO2 from

the solution. 500 µL of microgel solution were filled in the ampoule of stainless steel. The

stirrer in the reaction ampoule was set to 200 rpm. 640 µL (i.e. 500 µL + half of volume of

titrated volume) of the reference solution were filled in the reference ampoule. The ampoules

are lowered stepwise into the calorimeter chamber. After being lowered completely, the

system was allowed to equilibrate for at least 60 min. 280 µL of aqueous CaCl2 (c = 1

24

mg/mL) solution was filled into a glass syringe. It is important that no gas bubbles are inside

the syringe. Measurements were performed at 25 °C and 37 °C. The temperature could be set

with an accuracy of ± 0.0001 °C. 28 injections of 10 µL each were done, each injection lasted

10 s. Between measurements, the signal was allowed to return to the baseline in 15 min. Each

measurement was repeated with pure buffer solution in the syringe to determine dilution heat.

Flow Field-Flow Fractionation (F-FFF) Measurements were done using an AF 2000 MT

separation system (Postnova Analytics, Germany). 20 mL of aqueous microgel solutions with

a concentration of 1 mg/mL were filled into the channel that had a thickness of 294 µm and a

length of 30 cm. The eluent was filtered deionized water. Particles were collected using a

regenerated cellulose membrane (30,000 MWCO). The eluting particles were detected online

by static light scattering.

Colloidal stability measurements The stability of the microgel particles was studied with a

LUMiFuge 1112-33 (L.U.M. GmbH, Germany). The centrifuge measures sedimentation

velocities of dispersions under centrifugal force. Microgel samples were measured in a 2 mm

LUM, rectangular Polycarbonate cell. Centrifugation was done at acceleration velocity

2000 rpm (corresponds to 800 g) at intervals of 10 s at T = 25 °C and 50 °C. The slope of

sedimentation curves was used to calculate the sedimentation velocity and to gain information

about colloidal stability of the samples.

IGASorp Moisture sorption-desorption isotherms were measured using an IGAsorp moisture

sorption analyzer (Hiden Isochema) at 37 °C. The sample was put in a ultra-sensitive balance

and the relative humidity (RH) was increased from 2 % RH to 94 % RH in steps of 10 %.

Plasma activation and spin-coating The substrates were cleaned prior to use with water and

ethanol and dried with nitrogen to remove impurities. Then they were put in an air plasma

oven (Plasma Activate Flecto 10 USB) and activated at 0.2 mbar for 60 s at room

temperature. The substrates were coated with 30 µL of microgel sample via spin-coating with

a rotational speed of 1000 rpm/s for 60 s at room temperature.

Contact angle measurements Measurements were conducted at a Contact Angle Measuring

System G2-Mk4 from Krüss GmbH, Germany. A water droplet of Millipore water of 5 µL

was dropped on a coated Si-wafer. 10 images of the droplet on the surface were immediately

taken and the contact angle was measured with the software.

Protein adsorption tests To determine protein repellent properties of a modified surface, the

wafers were incubated with 700 µL of a 5 µG/mL solution of BSA, BODIFY FL conjugate

for 20 min. The protein solution was removed and substituted with 700 µL of PBS buffer for

10 min. This step was repeated twice. At last, 700 µL of water were added for 10 min and

25

removed again. The measurements were conducted at a fluorescence microscope (Carl Zeiss

Axioplan 2) with 10-fold magnification. A halogen lamp XBO 75 was used and the exposure

time was set to 20 s. As a reference, uncoated wafers were used.

Circular dichroism (CD) spectroscopy CD spectra were recorded on a JASCO J-1100 CD

spectropolarimeter using a quartz cuvette with a 0.1 cm path-length at room temperature.

Protein solutions of cytochrome c with a concentration of 0.0005 g/mL were used in 10 mM

PBS buffer with pH = 3 and pH = 8, respectively. The reported CD profiles were recorded

from 190 to 300 nm to measure secondary protein structure and are an average of three

successive scans obtained at a 10 nm min−1 scan rate corrected by a baseline. The measured

spectra are converted to mean residue ellipticity (MRE) as follows:

[𝜽] = 𝜽

𝟏𝟎∙𝒏∙𝒄∙𝒍 (2)

with [θ] = mean residue ellipticity; θ = observed ellipticity (in mdeg); n = amino acid residue

(104 for cytochrome c [1]); c = protein concentration (in mol); l = pathlength of cuvette.

The content of α-helix, beta sheet, turn, and random coil conformations are determined via the

Structural Estimation Application software using Yang’s reference.

4.3 Microgel synthesis

The syntheses of the microgel particles and their purification are described in the respective

chapters.

26

5 Zwitterionic Microgels

5.1. Introduction

Zwitterionic microgels contain the same amount of cationic and anionic groups and are thus

electrically neutral. In contrast to ampholyte microgels, both charges are located in the same

monomer unit (see Figure 5.1). This ensures that always an equal amount of charges is present

in the particle. In general, the cation does not have an associated hydrogen atom.

A prominent group of zwitterions are betaines. Betaines consist of a quaternary ammonium or

pyridinium group [1] and a negative pendant group. In most cases, the latter is either a sulfo-,

carboxy- or phosphogroup. Hence, the correspondent betaines are called sulfobetaine (SB),

carboxybetaine (CB), and phosphobetaine (PB) (see Figure 5.1).

The main difference between zwitterionic and ampholyte microgels becomes apparent by

looking at their behavior in aqueous solution. In water, ampholyte microgels are swollen

through the electrostatic repulsion of the charged groups. The addition of salt or the change of

pH leads to a collapse of the ampholyte microgel structure because the charges are screened.

This phenomenon is called the polyelectrolyte effect. Zwitterionic microgels, on the other

hand, behave differently. Their structure expands after the addition of salt or the change of pH

which is called the anti-polyelectrolyte effect [2].

SB is similar to taurine, an amino acid found in the human body [3]. It is an essential part of

the muscles and the nervous system [4]. Contrary to other betaines, SB is electrically neutral

over a wide range of pH due to its high acid dissociation constant. CB is similar to glycine

betaine which is an essential part of the osmotic regulation in living organisms [3]. PB is

similar to phosphorylcholine [3] which is an essential part of biological membranes [1]. Thus

much effort has been done to study the behavior of phospholipid analogues. However, all

phosphobetaines described in the literature are not analogues of the described sulfo- and

carboxybetaines, i.e. while SB and CB have the anionic group at the tail of each monomer

unit, PB has the positively charged ammonia group at the end. Even in publications

comparing all three betaines, e.g. by N.Y. Kostina [5], no remark concerning the different

structures is given.

Betaines are well known for their ultra low-fouling properties [5][6][7]. These properties will be

discussed in more detail in chapter 11. In addition to their possible functionalization and their

anti-polyelectrolyte effect, polymers containing betaines are highly interesting for biomedical

applications such as drug delivery [6] and tissue engineering [8].

27

Figure 5.1 Betaine structures: (a) sulfobetaine; (b) carboxybetaine; (c) phosphobetaine.

General structure of (a) zwitterions; (b) ampholytes.

Y. Chang et al. used SB and PNIPAm to obtain zwitterionic copolymers [9]. They stress the

usefulness of the particles due to their biocompatibility and their temperature-sensitivity. The

copolymer exhibits both UCST and LCST depending on the copolymer composition, solution

concentration, solution polarity, and ionic strength. For instance, a copolymer composition of

29.0 mol-% polySB and 71.0 mol-% PNIPAm exhibits an UCST of 15 °C and an LCST of

41 °C in water at a polymer concentration of 5 wt-%. Furthermore, the authors could show

that the same copolymer exhibits extremely low protein adsorption on surfaces. Y. Su et al.

synthesized poly(acrylonitrile)-co-SB copolymers for the preparation of zwitterionic

membranes [10]. The on-off behavior for transport of proteins such as lysozyme and BSA can

be controlled by the electrolyte concentration. If no electrolytes are present, attractive

interactions between the proteins and the sulfobetaine exists, while those interactions are

screened at higher electrolyte concentrations.

The synthesis of zwitterionic hydrogels has been reported by many authors [11][12][13]. In order

to improve mechanical stability and incorporate further desired properties these hydrogels are

often copolymerized. One drawback is the decreased non-fouling properties when other

monomers are inserted. N.Y. Kostina et al. present brushes containing SB, CB or PB and 2-

hydroxyethyl methacrylate (HEMA) [5]. They showed that only CB-co-HEMA copolymers

exhibited a sufficient fouling resistance and thus used them to obtain hydrogels. Hydrogels

with a CB content of 10 mol-% retained the good mechanical stability characteristic for pure

PHEMA hydrogels while at the same time possessing a four-fold amount of water promoting

protein-repellent properties.

a b c d e

28

M. Das et al. were the first to synthesize zwitterionic microgels based on PNIPAm using

sulfobetaine as the zwitterionic comonomer [2]. They showed that only a limited amount of SB

can be incorporated into the microgel network using free radical precipitation polymerization.

With varying pH, no change in particle size could be observed due to the permanent negative

charge of the sulfo group. Interestingly, the authors could detect no anti-polyelectrolyte

behavior of the particles. Furthermore, the authors used the zwitterionic microgels for in-situ

synthesis of gold and silver nanoparticles.

The great potential of zwitterionic structures to synthesize, trap, encapsulate and/or release

nanoparticles is also shown in other literature [14]. L. Zhang et al. used polyCB nanogels of

~ 110 nm in size to encapsulate monodisperse Fe3O4 nanoparticles that can be used for

enhancing Magnetic Resonance Imaging (MRI) performances [6]. They showed that only 3 %

of the nanoparticles are released in PBS, but much faster (80 %) in the presence of the

reducing agent dithiothreitol representing an intracellular environment. The release of

nanoparticles occurred through the cleavage of the disulfide crosslinker. Furthermore, the

nanogels are able to degrade into small polyCB polymers that can be removed from the body.

29

5.2. Synthesis Procedure

Synthesis of Carboxybetaine Carboxybetaine was synthesized as described in my diploma

thesis following the synthesis procedure by Z. Zhang [15].

50 mL of dried acetone were ice-cooled in a flask. DMAEM (1.54 g, 9.9 mmol) and β-

propiolactone (0.99 g, 13.7 mmol) were slowly added with a syringe pump and the mixture

was allowed to stir for 5 h at 15 °C. The product was obtained as a white precipitate. The

solvent was removed and the product was washed three times with anhydrous ether and

acetone.

1H-NMR (300 MHz): δ = 6.04 (s, 1H, H-2), 5.65 (s, 1H, H-1), 4.53 (t, 2H, H-4), 3.69 (t, 2H,

H-7), 3.55 (t, 2H, H-5), 3.07 (s, 6H, H-6), 2.61 (t, 2H, H-8), 1.82 (s, 3H, H-3) ppm.

Figure 5.2 1H-NMR spectrum of carboxybetaine.

Synthesis of Phosphobetaine Commercially available phosphobetaine has its two ionizable

groups in reverse order compared to the other betaines used in this work, i.e. the quaternary

ammonium group is terminal. Therefore, phosphobetaine with a terminal phosphogroup was

synthesized and friendly provided by Rüdiger Bormann (IOC, RWTH Aachen).

Figure 5.3 Structure of phosphobetaine. (a) As commercially available. (b) As synthesized by

R. Bormann; numbers refer to the NMR spectrum in Figure 5.4.

30

Figure 5.4 1H-NMR spectrum of phosphobetaine. Numbers above the spectrum refer to the

peak position, numbers below the spectrum refer to the integral.

1H-NMR (300 MHz): δ = 6.03 (s, 1H, H-1), 5.64 (s, 1H, H-2), 4.51 (s, 2H, H-4), 3.91 (m, 2H,

H-8), 3.76 (m, 2H, H-7), 3.68 (m, 2H, H-5), 3.12 (s, 6H, H-6), 2.08 (t, 3H, H-3) ppm.

31

5.3. Precipitation Polymerization

5.3.1. Introduction

Precipitation polymerization is the main synthesis procedure for microgel synthesis.

Zwitterionic microgels with sulfo-, carboxy-, and phosphobetaines as comonomers were

already synthesized and characterized in my diploma thesis. In addition to crosslinked

zwitterionic microgels, samples without crosslinker BIS were analyzed regarding their

particle size.

In this work, however, non-covalently crosslinked (hence called non-crosslinked) samples

with varying betaine content were further studied with regard to their degradability upon salt

addition and the particle formation process in comparison to conventionally BIS-crosslinked

microgels.

It is postulated that zwitterionic compounds form internal ionic bridges of two different types

(see Figure 5.5)[16]: (a) The molecular structure is rigid and no internal bridges between the

ionic groups can be formed. (b) Intermolecular bridges between two different zwitterionic

groups are formed through antiparallel arrangement. (c) Intramolecular bridges between the

charged groups of one zwitterionic unit are formed; this type is dependent on the length of the

methylene units between the charged groups.

Figure 5.5 Types of ion pairing within zwitterionic structures: (a) rigid structure; (b)

intermolecular bridges; (c) intramolecular bridges.

M. Szafran described the factors that lead to bridges of either type (b) or (c): (1) flexibility of

the chain; (2) bulkiness and hydration of the charged groups; and (3) electrical properties of

both the charged group and the solvent [17]. S. Chen et al. suggested that the charged groups

arrange antiparallel as to minimize net dipole moments [18].

The formation of ionic bridges of type (b) lead to the suggestion that it is possible to

synthesize microgels without the use of conventional crosslinker such as BIS because

intermolecular bridges are expected to form sufficiently strong ion pairing. These bridges,

however, should easily be dissolved by the addition of saline solution.

In literature, degradation of microgels mostly occurs by the use of a degradable crosslinker

that degrades by the use of UV light [19][20][21], pH [22][23], and enzymes [24][25][26]. V. Bulmus for

a b c

32

instance synthesized microgels based on poly(hydroxyethyl methacrylate) (pHEMA) and used

several diacrylate- and dimethylacrylate-functionalized crosslinkers to obtain acid-cleavable

microgels [27]. They studied the hydrolysis time at two different pH values, neutral and acidic,

and showed that particles are stable at neutral pH, while they degrade in acidic medium in less

than 60 min. The authors explained this by the cleavage of the acetal crosslinks resulting in

free linear crosslinker chains. In this procedure, the precursor polymers are recovered and the

degradation of the microgel does not lead to toxic or harmful products which is an essential

criterion for the use as bio- and cytocompatible materials. A. M. Hawkins et al. specifically

focused on the toxicity of the degradation products of poly (β-amino ester) hydrogels and

analyzed the cellular response of mesenchymal cells [28]. M. Patenaude and T. Hoare show in

their work the possible application of NIPAm hydrogels that can be injected over a syringe to

act as a drug delivery system [29]. They point out two challenges: The microgels have to be

robust enough to withstand the shear stress in a syringe. On the other hand, it must be ensured

that the hydrogel degrades without the generation of toxic products and without accumulating

in the body. They could show that their hydrogels degrade in acid and form the formerly used

precursor copolymers. No considerable toxicity or only slight toxicity at high concentrations

was observed in cell tests.

There are only few examples in literature concerning the degradation of zwitterionic

polymers. L. Zhang et al. synthesized carboxybetaine nanogels using a disulfide crosslinker

that can be cleaved in the presence of dithiothreitol (DTT) [6]. Furthermore, they tested the

stability of their particles in PBS buffer for a period of six month. They showed that the

hydrodynamic radius of about 110 nm did not change during this time.

So far, no examples for the synthesis of zwitterionic microgels without the use of crosslinker

can be found in the literature. In contrast to L. Zhang’s work, these microgels are expected to

decompose in saline solution because the crosslinking ionic groups of the zwitterions are

screened and results in a disintegration of the microgel structure. The use of saline solution

offers a simple, non-toxic, and low-cost method for degradation.

33

5.3.2. Synthesis Procedure

Microgel Synthesis in Reactor VCL (1.877 g, 13.49 mmol), the respective betaine (see

Table 5.1), and AMPA (0.06 g, 0.221 mmol) are added in 150 mL of water in a double-walled

reactor. While stirring at 200 rpm, the mixture is purged with nitrogen at 70 °C. The reaction

is started by adding the initiator AMPA. The reaction is allowed to stir at 70 °C for 5 h.

Microgel solutions are cleaned via dialysis in a composite regenerated cellulose membrane

from Millipore (NMWCO 30,000) for 3 d against water.

Table 5.1 Amounts of betaines used for microgel synthesis.

m (Betaine) / g wt-% * n (SB) / mmol n (CB) / mmol n (PB) / mmol

0.02 1 0.072 0.083 0.071

0.04 2 0.143 0.165 0.142

0.06 3 0.215 0.248 0.213

0.08 4 0.286 0.330 0.285

0.10 5 0.358 0.413 0.356

* of betaines with respect to amount of VCL and BIS.

Microgel Synthesis in Calorimeter For calorimetric studies, the same amounts of monomers

were used. They were performed out in a reaction calorimeter RCle from Mettler Toledo with

a 500 mL 3-wall AP01-0.5-RTCal reactor equipped with a Hastelloy® stirrer, a baffle and a

Turbido™ turbidity probe from Solvias. The measurements were done in isothermal mode, in

which the desired reaction temperature (Tr) is set at a constant value and the jacket

temperature (Tj) changes automatically to maintain Tr at the desired value. Data were

evaluated with the software iControl RC1e™ 5.0. For the determination of the net reaction

time the end of the reaction was defined to be when the heat flow was back to a value close to

zero and constant.

For in-situ DLS studies, the amounts of monomers were down-scaled to 100 mL H2O.

Immediately after the addition of the initiator, the size of the particles was determined in 99

steps with 42 s intervals (30 s measurement + 12 s processing time).

34

5.3.3. Results and Discussion

PVCL-based microgels without crosslinker and varying betaines content (up to 5 wt-%) were

successfully synthesized via precipitation polymerization. No aggregation occurred during the

synthesis or after cooling down of the solution. FTIR measurements confirmed the successful

incorporation of SB into the microgel polymer network (graph see appendix).

In my diploma thesis, it was shown that the size of the particles changes in the same way as

microgels containing the crosslinker BIS: While carboxybetaine microgels increase in size

with increasing CB content, SB microgels do not change in size, and PB microgels decrease

in size. This very same behavior is also observed for non-crosslinked particles (see Figure

5.6).

Figure 5.6 Change of particle size RH with increasing amount of betaine for non-crosslinked

particles.

Both crosslinked and non-crosslinked sulfobetaine microgels were further analyzed with SLS

to obtain information about the radius of gyration (see Figure 5.7). Experiments were

performed below the VPTT (i.e. 20 °C).

Figure 5.7 SLS data for crosslinked and non-crosslinked zwitterionic microgels with 5 wt-%

SB.

35

SLS measurements provide the radius of gyration RG. It is 118 nm and 102 nm for crosslinked

and non-crosslinked samples, respectively. The ratio RG/RH gives information about the shape

parameter. It is 0.775 for an ideal, hard sphere [30][31]. The ratio RG/RH is 0.55 and 0.46 for

crosslinked and non-crosslinked microgels, respectively, and is thus much smaller than the

ratio for a hard sphere. V. Boyko gives the range of 0.3 - 0.6 as a typical range for microgels

[32]. He explained this behavior by the inhomogenous crosslinking density in a microgel

particle: it has a dense inner core with a less dense outer shell of dangling polymer chains.

Figure 5.7 shows that zwitterionic microgels crosslinked with BIS are closer to the value of a

hard sphere due to the fact that they are both chemically and physically crosslinked.

Zwitterionic microgels that were prepared without the use of the crosslinker BIS are only

physically crosslinked over intermolecular ionic bridges are thus less compact and have more

pending segments on the particle surface. M. Antonietti et al. already demonstrated that an

increase in crosslinker results in an increase in the ratio RG/RH [33]. They state that a microgel

with RG/RH < 0.6 is highly swollen both in the core and the shell.

AFM measurements give the particle size in dried state. Both crosslinked and non-crosslinked

samples with 1 and 5 wt-% SB were spin-coated on Si-wafer to see differences in the softness

of the particles (see Figure 5.8). These measurements were done solely for SB microgels since

a variation in betaine content does not lead to a change in the particle size (see Figure 5.6).

The following trends can be drawn from the AFM images: (1) Overview images show that

non-crosslinked microgels lead to a better and more even coating than crosslinked particles.

This might be caused by the fact that non-crosslinked samples are softer and can thus adapt

their structure to the surface in a better way than crosslinked particles. (2) The height profiles

reveal that both for 1 and 5 wt-% SB microgels, crosslinked particles have a bigger height

than non-crosslinked samples: 141 nm and 121 nm, respectively, for 1 wt-% SB, and 150 nm

and 144 nm, respectively, for 5 wt-% SB. Furthermore, non-crosslinked samples spread more

on the surface than crosslinked samples: 682 nm and 467 nm, respectively, for 1 wt-% SB,

and 387 nm and 366 nm, respectively, for 5 wt-% SB. The hydrodynamic radius as obtained

from DLS measurements for all samples is ~ 220 nm. This behavior arises from the fact that

crosslinked particles are better able to retain their shape when in contact with a solid surface

than non-crosslinked particles. The latter spread pancake-like and loose thus in height

compared to their crosslinked analogues (see Figure 5.8d). Furthermore, it is obvious that

non- crosslinked particles with a betaine content of 5 wt-% do not spread as pronounced as

microgels with 1 wt-% SB. The higher betaine content and thus the higher amount of internal

36

Figure 5.8 AFM images of 1 and 5 wt-% SB microgels, crosslinked and non-crosslinked.

From top to bottom: (a) Overview of Si-wafer (225 µm²); (b) Zoom-in with lines for height

profiles (25 µm²); (c) Height profile of marked particles in (b) with average sizes; (d)

Schematic draft of microgel shape on surface, in proportion to each other; (e) Zoom-in

(1 µm²); (f) Phase image of (d).

ionic bridges lead to a better stability and thus higher rigidity of the particles. (3) The phase

images of the zoomed-in images reveal that crosslinked particles have a “flat” surface, while

non-crosslinked particles have a “globular” sub-structure. This argues for a homogenous

distribution of the crosslinker BIS in the particles, while the sulfo groups lead to a

deformation and distortion of the network.

37

As shown in the AFM images above, the use of BIS as a crosslinker leads to different

properties of the microgels regarding their size and morphology. In the following,

calorimetric and kinetic studies were performed to understand differences during the synthesis

of crosslinked and non-crosslinked microgels. First, the synthesis was done in a calorimeter

measuring the polymerization heat and the change of turbidity during the synthesis. Second,

in-situ DLS measurements were performed to study the particle growth. Those experiments

were done for PVCL-SB (5 wt-% SB) microgels with and without BIS. Reference

measurements for several monomers and monomer combinations were done to study the

influence and contribution of each monomer to the particle formation process.

Figure 5.9 Heat profile qr (black), hydrodynamic radius RH (red), and turbidity (blue); (a)

non-crosslinked 5 wt-% SB microgels; (b) crosslinked 5 wt-% SB microgels. Measurements

were performed at 70 °C under nitrogen atmosphere.

Figure 5.9 gives the change of the heat profile qr, the hydrodynamic radius RH, and the

turbidity for the copolymerization of VCL/SB without the crosslinker BIS (a) and VCL/SB

with BIS (b). For the heat profile (black curve), two peaks can be identified, belonging to two

separate steps in the polymerization process: one flat peaks shortly after the start of the

reaction (0 to ~5 min and 5 to ~10 min), and one sharp peak (10 to ~ 15 min). The first peak is

not clearly distinguishable. Integration of the curve of the heat profile curves gives the

polymerization heat: it is 0.844 kJ and 0.735 kJ for crosslinked and non-crosslinked

microgels, respectively. Regarding the particle growth (red curve), three steps can be

observed which occur simultaneously with the two peaks seen for the reaction heat: first, the

particles grow for ~ 6 min (to 88 nm and 141 nm for non-crosslinked and crosslinked

particles, respectively), then the particle size remains stable until ~ 12 min, followed by

another growth in particle size (145 nm and 187 nm for non-crosslinked and crosslinked

particles, respectively). The main difference between non-crosslinked and crosslinked

38

particles can be observed during the last part: crosslinked particles grow more rapidly in size

than non-crosslinked particles. For non-crosslinked particles, the particle size remains stable

after ~ 24 min, for crosslinked particles already after ~ 12 min. The turbidity only shows two

separate phases: first a slow increase from ~ 10 % to 11 % after 10 min, then a faster increase

to 14.5 and 16 % for non-crosslinked and crosslinked particles, respectively.

In order to identify the two heat peaks, the monomers were synthesized separately with the

initiator AMPA (see Figure 5.10a). Here, it can be seen that SB does not react when

individually polymerized with AMPA. It is believed that AMPA is not the right initiator if SB

is polymerized alone. Probably, a non-ionic initiator such as azobisisobutyronitrile (AIBN)

would be better suited for this task. Contrary, VCL reacts immediately after the addition of

the initiator resulting in a narrow and sharp peak. The reaction is complete after ~ 10 min.

Integration of the peak gives a polymerization heat of 0.997 kJ.

Figure 5.10 Heat profile qr as measured with calorimetry (a) of monomers SB and VCL with

AMPA; (b) combinations of VCL with BIS and SB; (c) crosslinked and non-crosslinked 5 wt-

% SB microgels.

Furthermore, VCL was copolymerized with BIS alone (see Figure 5.10b). It can be seen that

the reaction is clearly delayed compared to the homopolymerization of VCL. Furthermore, the

former sharp peak is broadened. Its form resembles the broad second peak in Figure 5.10c.

The polymerization heat is 0.802 kJ. All polymerization heats are summarized in Table 5.2.

39

The following conclusions can be drawn from these observations for the identification of the

three peaks as observed in the copolymerization of VCL-SB with and without BIS: The first

peak is caused by SB. In a second step, VCL is incorporated into the microgel network.

Therefore, it can be assumed that the resulting microgel has a diffuse core-shell structure with

SB mostly located in the core of the particle, while VCL is located in the shell.

Table 5.2 Polymerization heats of copolymerization of VCL with SB, and/or BIS.

Monomers used for polymerization Polymerization heat / kJ

VCL-SB-BIS 0.844

VCL-SB 0.735

SB no reaction

VCL 0.997

VCL -BIS 0.802

As already explained above, the particles are crosslinked internally through ion pairing of the

quaternary ammonium group and the sulfo group (see Figure 5.5). This is indirectly proven by

the fact that DLS and AFM images show the formation of microgel particles that would be

loose polymer chains instead if no internal crosslinking would have taken place. Another

possibility to show the presence of those internal ion pairing is FTIR. Y. Su et al. showed for

their poly(acrylonitrile)-based zwitterionic membranes that FTIR is able to detect differences

between dissociated and undissociated sulfo groups [10]. Depending on the degree of

dissociation, the characteristic peak at 1042 cm-1 belonging to the symmetric stretch vibration

is split into two separate peaks.

Therefore, a crosslinked sample with 5 wt-% SB was analyzed with FTIR in three different

states: In dried state, in water, and in 1 M KCl solution that was stirred overnight. For this

experiment, a crosslinked sample was analyzed to ensure that the addition of KCl does not

lead to the degradation of the microgel particle which would influence not only the peaks of

interest but the whole spectrum.

40

Figure 5.11 FTIR spectra of 5 wt-% SB microgel sample in (black) solid state; (blue) in pure

water; (red) in 1 M KCl solution.

The peak of the sulfo group at 1040 cm-1 is most prominent in solid state (see Figure 5.11,

black curve). The spectra for the aqueous samples (blue curve) were subtracted with the

spectrum of pure water. After subtraction, the peak of the sulfo group is very weak due to the

small amount of sulfo groups in the sample. The positions of the peaks were identified by

determining the first deflection. It is 1038 cm-1 in pure water and 1045 cm-1 in 1 M KCl

solution. In agreement with the results shown by Y. Su et al., the peak at 1038 cm-1 is

associated with the undissociated sulfo group with the charged groups being oriented in an

anti-parallel way. The peak at 1045 cm-1 is induced by the dissociated sulfo groups, i.e. a

sulfo group being surrounded by counterions, in this case potassium. In contrast to Y. Su et

al., no splitting into two different peaks could be observed. This suggests that the dissociation

in 1 M KCl is complete and no transition states exist.

The uncrosslinked samples with the highest amount of betaine (i.e. 5 wt-%) were analyzed

concerning their change in size with increasing KCl content. It is expected that the

intramolecular ion bridges are dissolved by the addition of salt because attractive charge-

dipole and dipole-dipole interactions are screened leading to the degradation of the microgel

particles. Freeze-dried microgel samples were dissolved in saline solution and stirred for 3 d

to ensure complete disintegration of the polymer network.

It can be seen in Figure 5.12 that the particle size of all betaine microgels are unaffected at

low salt concentrations, i.e. up to an KCl concentration of 0.05 M KCl. Beginning at 0.1 M

KCl for all samples, a second, smaller peak at around 20 nm can be observed. This indicates

the disintegration of the microgel network to loose polymer chains.

41

Figure 5.12 Change of particle size distribution with varying salt concentration. (a)

sulfobetaine; (b) carboxybetaine; (c) phosphobetaine.

Images taken with FESEM support the data obtained with DLS. Figure 5.13 shows images of

particles with 5 wt-% SB (a) before the addition of salt and (b) after the addition of salt.

Figure 5.13a shows monodisperse particles with a diameter of 200 nm (the particles are in a

dried state and thus slightly smaller then they appear with DLS), while the particles in Figure

5.13b show aggregates and single particles of different sizes. Probably, the small particles of

~ 20 nm form aggregates during the drying process and are thus not seen in the image.

Figure 5.13 FE-SEM images of sulfobetaine microgels (5 wt-%) without crosslinker BIS. (a)

before the addition of salt; (b) after the addition of salt.

a b

c

198 nm

a b

2.00 µm 3.00 µm

42

This behavior can also be observed optically. Figure 5.14a shows non-crosslinked PVCL

microgels at 70 °C and 25 °C. At 70 °C, the microgels are in a collapsed state thus appearing

milky. At 25 °C, however, the swelling of the polymer network causes a rupture leading to

single polymer chains. Non-crosslinked PVCL-co-SB microgels, in contrast, are stable at both

70 °C and 25 °C supporting the assumption that zwitterionic bridges lead to an internal

crosslinking of the particles (Figure 5.14b). These internal ionic bridges are destroyed at room

temperature through the addition of salt indicated by the mostly colorless solution.

Figure 5.14 (a) Non-crosslinked PVCL microgels at 70 °C (left) and 25 °C (right); (b) non-

crosslinked PVCL-co-SB microgels at 70 °C (left), 25 °C (middle), and 25 °C in the presence

of salt (right).

Figure 5.15 (a) Sedimentation velocities of crosslinked (X) and non-crosslinked (non-X)

samples with 1 and 5 wt-% SB, respectively, with and without the addition of salt; (b)

Sedimentation front for sample 1 wt-% SB, non-crosslinked, with salt; drawn in are the

positions of air, water and dispersion for better clarification; (c) Sedimentation front for

sample 1 wt-% SB, non-crosslinked, without salt.

Since no apparent difference could be observed between SB, CB, and PB, the colloidal

stability of the particles was quantified with LUMiSizer measurements only for samples

containing 1 wt-% and 5 wt-% SB. Images of the samples after centrifugation show that there

43

is almost no difference between the crosslinked and the non-crosslinked samples if no salt is

present. In the presence of salt, the non-crosslinked samples are considerably less colloidally

stable than the respective crosslinked sample.

This behavior is also mirrored in the sedimentation fronts given in Figure 5.15b+c. The

sedimentation front is defined as the distance from the rotor center to the phase boundary

between sediment and supernatant [34]. After transforming these curves into transmission-time

curves (not shown), the sedimentation velocity can be calculated through the slope of the

curves. Figure 5.15b shows the sedimentation front for the non-crosslinked sample 1 wt-% SB

after the addition of salt. The sedimentation front moves from the left to the right side with

time indicating a fast precipitation of the particles. The same sample without the presence of

salt (Figure 5.15c) does not exhibit a moving sedimentation front thus showing a higher

colloidal stability.

The sedimentation velocities are summarized in Figure 5.15a. The following conclusions can

be drawn: (1) Samples with salt are less colloidally stable, i.e. they have a higher

sedimentation velocity, than samples that contain no added salt. For crosslinked samples, this

can be explained by the fact that the particles increase in size after the addition of salt due to

the antipolyelectrolyte effect [35][36][37]. The relation between particle size r and sedimentation

velocity ν is as follows [38]:

𝜈 = 2𝑟² ∆𝑝 ×𝑔

9𝜂 (1)

With Δp = density difference between the particle and the medium; g = centrifugal force; η =

viscosity of the medium.

Presuming that both the centrifugal force (for the sedimentation velocity of 2000 rpm and a

cuvette radius of 2 mm) and the viscosity are constant, it follows that the sedimentation

velocity is dependent on both the particle size and the density difference between the particle

and the medium. Assuming further that the change of density of the particle is negligible, the

sedimentation velocity is increasing with increasing particle size. (2) Non-crosslinked samples

are considerably less stable in the presence of salt than crosslinked samples: for the sample

1 wt-% SB, the sedimentation velocity is approximately 30 times higher than for the

respective crosslinked sample (115.7 µm/s and 4.324 µm/s, respectively). This is due to the

degradation of the microgel network. (3) The sample 1 wt-% SB is less stable in the presence

of salt than the sample 5 wt-% SB (115.7 µm/s and 66.67 µm/s, respectively). This is because

the sample with the higher amount of betaine is better able to preserve the internal ionic

bridges than the sample with a lower amount of betaine.

44

5.3.4. Summary

In this chapter, non-covalently crosslinked (i.e. without the use of the crosslinker BIS)

zwitterionic microgels were studied. Zwitterionic microgels can be synthesized without the

use of a crosslinker since the positively charged ammonium group and the negatively charged

sulfo groups are expected to build internal ionic groups that are sufficiently strong enough to

hold the microgel network together. The analysis focuses on two main aspects: first, the

polymerization process itself and second, the degradability of the particles in saline solution.

The polymerization process was studied with calorimetry and in-situ DLS. For both

crosslinked and non-crosslinked particles, two steps could be identified: first, SB was

polymerized, followed by VCL. This suggests that the resulting microgels have a diffuse core-

shell structure with SB located in the particle interior. Furthermore, it could be seen that the

presence of the crosslinker BIS fastens the polymerization process.

Differences in morphology and rigidity between crosslinked and non-crosslinked particles

could be seen with AFM. Particles without the crosslinker spread in a higher extent on a solid

surface than crosslinked particles. This is expected since crosslinked particles are more rigid

than non-crosslinked particles. Furthermore, differences could be observed between particles

with a different content of SB: particles with more SB are more rigid and do not spread in the

same extent than particles with a lower amount of SB.

Since the microgels are crosslinked via internal ionic bridges, the particles were expected to

degrade after the addition of salt that is able to screen the charges. First, the existence of these

internal bridges was verified with FTIR. It could be shown that the peak of the sulfo group at

1038 cm-1 in water is shifted to 1045 cm-1 in 1 M KCl. The first peak indicates the presence of

the undissocaiated sulfo groups with internal ionic bridges. The shift suggests the dissociation

of the sulfo group that is surrounded by potassium ions.

After the addition of KCl, DLS measurements showed the formation of smaller peaks of about

20 nm at higher KCl concentrations indicating the presence of loose polymer chains. Since no

apparent difference could be observed between SB, CB, and PB, colloidal stability

measurements were done solely for SB microgels. Again, it could be seen that non-

crosslinked microgels are considerably less stable in KCl solution that crosslinked samples,

the sedimentation velocity was up to 30 times higher. Eventually, FE-SEM images supported

the assumption of the degradation of non-crosslinked microgel particles in saline solution.

45

5.4. Inverse Mini-Emulsion Polymerization

5.4.1. Introduction

An important factor in precipitation polymerization is the hydrophobicity of the formed

polymers. It is difficult to incorporate larger amounts of hydrophilic comonomers in

microgels by precipitation polymerization [39]. This behavior was shown in chapter 5.3 where

microgels with up to only 5 wt-% of betaines could be synthesized via precipitation

polymerization. To increase the amount of the zwitterionic comonomer in the microgels, a

different technique, namely inverse mini-emulsion (or nano-emulsion) free radical

polymerization, is used and discussed in this chapter. This polymerization technique allows

the incorporation of betaine amounts of 30 mol-% and higher.

Mini-emulsions are aqueous dispersions of oil droplets that are stabilized by a surfactant [40].

They are formed mostly through shear forces implied by ultrasonication, microfluidizer or a

homogenizer that break the monomer droplets which results in the formation of droplets in a

size range of 20 – 500 nm [41]. In this work, ultrasonication is used to create small droplets.

With every wave, ultra sound produces a compression and rarefaction of molecules. During

the rarefaction, small cavitation bubbles form and grow that implode again in one of the

following compressions. The collapse of the cavitation bubbles forms powerful shock waves

in the surrounding liquid. The resulting liquid jets of high speed are the reason for larger

monomer droplets to disperse in the continuous phase. The longer the ultrasonication time and

the higher the ultrasound power, the smaller the droplet size becomes [42][43].

The system usually consists of a dispersed phase, a continuous phase, one or more surfactants

and a strong hydrophobe. The surfactant protects droplets from diffusional degradation

through steric hindrance. Due to the small droplet size, the resulting large droplet surface area

leads to the main amount of surfactant being absorbed on the droplet surface. Thus, only a

small amount of surfactant is able to form micelles. The surfactant greatly influences the

resulting particle size. The more surfactant is used, the more the particles are covered by the

surfactant and subsequently the smaller the particles become. Generally, a smaller size will

also lead to a broader particle size distribution [44]. Due to their size, dispersions appear

transparent or translucent. Syntheses found in literature use anionic, cationic, non-ionic or a

mixture of surfactants, though the first are the most common used. Sodium dodecyl sulfate

(SDS) is often use for the preparation of mini-emulsions [43][45][46]. Non-ionic surfactants are

rarely used in classical emulsions since they are often soluble in the monomer droplets. This

is different for mini-emulsions: Here, the larger surface area leads to a larger fraction of non-

46

ionic surfactant at the surface in a thermodynamic equilibrium. They are mostly used in

controlled living polymerization with atom transfer radical polymerization (ATRP) where

anionic surfactants cannot be used [47].

A co-stabilizer is mostly a low-molecular weight compound that is insoluble in water and

prevents diffusional degradation [48]. The hydrophobe is the key agent in a mini-emulsion. It is

monomer-soluble, but highly water-insoluble (less than 10-7 ml/mL [42]) and thus does not

diffuse between the different monomer droplets through the aqueous phase [44]. In a classical

emulsion polymerization, the diffusion of monomers from small droplets to larger droplets

(i.e. Ostwald ripening) reduces the total surface energy. In a mini-emulsion, however, the

monomer diffusion will increase the concentration of the hydrophobe and thus result in an

increase in free energy. An osmotic pressure is created inside the monomer droplet that

counterveils the Laplace pressure that is induced by the interface energy (i.e. the pressure

difference between inside and outside the droplet). This ideally stabilizes the monomer

droplets completely against Ostwald ripening. In reality, however, the hydrophobe only

retards the monomer diffusion, though the destabilization of droplets is in the range of months

[49]. It was shown that in contrast to the surfactant, the hydrophobe does not influence the

resulting droplet size [40][50]. Usually, high-molecular weight compounds such as long-chain

alkanes and long-chain alcohols are used as hydrophobes [51]. In some papers, the hydrophobe

is also called co-surfactant. This, however, is a misleading term, since the hydrophobe

stabilizes the monomer droplet not on the surface but in the bulk [48]. Due to the lack of

diffusion between droplets they act as “nanoreactors” [52]. As a result, the size as well as the

composition of the latex particles is identical with the composition of the monomer phase. In

contrast to a classical emulsion polymerization, the polymerized particles have the same size

as the initial monomer droplets as demonstrated by K. Landfester et al. with surface tension

measurements and conductometry (see Figure 5.16) [45].

Figure 5.16 Schematic representation of polymerization processes for mini-emulsion.

Water-in-oil mini-emulsions are called inverse mini-emulsions and are used for the formation

of hydrophilic, water soluble polymers such as polyacrylamide, polyacrylic acid or

polymethacrylic acid. Here, initiation can occur in the hydrophobic phase or within the water

47

droplet. Instead of a strong hydrophobe as required in direct mini-emulsions, a lipophobe such

as water or salt is used. Because oil is used as the continuous phase, non-ionic surfactants

with a low HLB (hydrophilic-lipophilic balance) are favorable [51].

Inverse mini-emulsions were first introduced by K. Landfester et al. in 2000 [40]. They

prepared hydroxyethyl methacrylate polymers in cyclohexane as the continuous phase. They

showed that the minimal concentration for the surfactant was 1.6 wt-% with respect to the

monomer. For the synthesis of polyacrylamide, an ionic salt was added as a lipophobe and

greatly increased the stability of the emulsion droplets.

Inverse mini-emulsions are used to synthesize a spectrum of polymers. Many examples in

literature deal with the synthesis of acrylamides by inverse mini-emulsion. S. Wiechers and

G. Schmidt-Naake, for instance, prepared pH-sensitive copolymers of 2-acrylamido-2-methyl-

1-propanesulfonic acid and 1-vinylimidazole with a particle size of 200 nm and demonstrated

that the conversion is dependent on the type of surfactant used during the reaction [53]. D.

Klinger et al. prepared enzymatically degradable microgels based on acrylamide and dextran

methacrylate in water and cyclohexane [54]. The use of two different initiators allowed the

synthesis at 70 °C and 37 °C to obtain monodisperse, spherical particles as confirmed with

SEM.

There are several examples for the preparation of microgels with various sensitivities via

mini-emulsion. To obtain microgels, they have to be crosslinked before they are transferred to

the continuous phase [55]. H. Dong, for instance, prepared magnetic microgels based on

di(ethylene glycol) methyl ether methacrylate via nano-emulsion [56]. The authors used

activators generated by electron transfer atom transfer radical polymerization (AGET ATRP)

as a polymerization technique to obtain degradable, temperature-sensitive microgels that were

used for the entrapment of iron oxide nanoparticles. D. Klinger and K. Landfester synthesized

photo-cleavable PMMA-based microgels via mini-emulsion by free radical polymerization

[52]. They obtained stable emulsion with particles with a size of 140 to 200 nm. Later, the

same authors prepared microgels based on 2-hydroxyethyl methacrylate and methacrylic acid

that were both sensitive to pH and UV light [39]. In contrast to the previous work, these

particles were formed in an inverse mini-emulsion.

48

5.4.2. Synthesis Procedure

VCL, the appropriate amount of sulfobetaine (Table 5.3), 3 wt-% BIS, and 5 wt-% NaCl as a

lipophobe were dissolved in 3.8 mL H2O. In a second flask, 0.60 g (1.400 mmol) Span 80 and

0.20 g (0.153 mmol) Tween 80 (ratio 3:1) were dissolved in 100 mL heptane and dispersed

with ultrasonic. The aqueous solution was added dropwise to the oil phase while

ultrasonicating for 5 min at level 4, 10 %, under ice cooling to obtain a stable emulsion.

Afterwards, polymerization was carried out within 5 min. The emulsion was heated up to

70 °C under nitrogen atmosphere and vigorous stirring. The initiator AMPA was solved in

0.2 mL H2O and added to the emulsion. The reaction was carried out for 2 h.

After cooling down, heptane was removed from the emulsion via centrifugation at 11,000 rpm

at room temperature. The precipitate was washed and centrifuged alternately with water and

heptane for three times each. Between centrifugation, the microgels were shaken in the

respective solvent for 30 min. Afterwards, the samples were dialyzed against water for 7 d.

Table 5.3 Amounts of betaines used for microgel synthesis.

Ratio

VCL:SB

n(VCL)

/ mmol

m(VCL)

/ g

n(betaine)

/ mmol

m(betaine)

/ g *2

2:1 2.443 0.340 1.222 0.341

1:1 2.443 0.340 2.443 0.682

1:2*1 1.222 0.170 2.443 0.683

*1 For this batch, half of the amount of solvents and monomers was used.

*2 Before synthesis, it was ensured that SB is not soluble in heptane in the concentration

range used for the syntheses.

Further experiments were conducted in the same way as described above except synthesis was

performed in a smaller batch using 50 mL of heptane and 2 mL of water (a) varying the

crosslinker amount (see Table 5.4) and (b) substituting VCL for N-vinylpyrrolidone (VPy)

and N-vinylpiperidone (VPi) (see Table 5.5).

Table 5.4 Variation in crosslinker concentration. Amounts of other monomers are identical to

amounts given in Table 5.3. The ratio VCL:SB was 1:1.

m (BIS) / g n (BIS) / mmol % BIS in microgel

0.002 0.015 0.5

0.005 0.031 1

0.008 0.053 2

0.013 0.083 3

49

Table 5.5 Amounts of N-vinylpyrrolidone (VPy) and N-vinylpiperidone (VPi). The ratio

vinylamid:SB was 1:1 in all cases. 3 wt-% of crosslinker was used.

n(VPi)

/ mmol

m(VPi)

/ g

n(VPy)

/ mmol

m(VPy)

/ g

n(betaine)

/ mmol

m(betaine)

/ g *2

1.173 0.147 - - 1.178 0.329

- - 1.19 0.131 1.183 0.330

Samples were purified in the same way as described above.

50

5.4.3. Results and Discussion

Inverse mini-emulsion was performed using Span 80 and Tween 80 as surfactants. They are

both non-ionic and biodegradable [57]. Tween 80 is more hydrophilic than Span 80 due to its

high number of PEG groups. In combination with Tween 80, Span 80 acts as a co-emulsifier.

Span 80 and Tween 80 have HLB (Hydrophile Lipophile Balance) values of 4.3 and 15.0,

respectively. In a ratio of Tween80/Span80 = 1:3, an overall HLB of ~ 7 is achieved

according to the following equation:

(2)

with X = overall HLB.

According to literature, the mixture of a non-ionic surfactant with a low HLB and a nonionic

one with a high HLB improves the stability of inverse mini-emulsions [58]. The overall HLB

should ideally be between 3 and 8 [59].

Figure 5.17 Schematic structure of surfactant orientation in water droplets.

AMPA was chosen as the initiator because of its solubility in water. It therefore starts the

reaction within the water droplets.

Before the synthesis of microgels via inverse mini-emulsion, various reactions were carried

out to analyze the stability of the emulsion droplets. Therefore, the oil and the water phase

were ultrasonicated and the emulsion measured with DLS for 60 min. As expected, the

stability of the droplet size could be influenced by the amount of surfactant used in the

reaction (graph see appendix).

51

An emulsion with a ratio between Tween80/Span80 = 0.1/0.3 breaks immediately. A fourfold

amount of surfactant, in contrast, is stable over the complete measurement time of 60 min.

Since a high amount of surfactant requires a long time of cleaning via dialysis, a third sample

was analyzed. Here, the amount of surfactant was 0.2/0.6. Measurements showed that the

droplets are only stable up to 20 min. This time, however, is thought to be sufficient to initiate

and formulate monodisperse microgel particles. Before breaking, the droplets have a

hydrodynamic radius of 257 ± 25 nm. Breaking of the mini-emulsion can be attributed to two

factors: (1) Droplets coalescence due to Brownian motion and van der Waals forces; (2)

Droplet degradation through monomer diffusion [48]. Coalescence occurs when the adhesion

energy between two droplets is equal or larger than the turbulent energy that keeps the

droplets separated.

The concentrations of surfactants used in the syntheses are above the cmc of both Span 80 and

Tween 80 (1.8 * 10-5 mol/L and 1.2 * 10-5 mol/L, respectively). A test measurement using

concentrations below the cmc’s of both surfactants showed that the monomer droplets are not

stable and aggregate quickly with a PDI between 0.7 and 1.0 (graph shown in the appendix).

Microgel samples were synthesized via inverse mini-emulsion polymerization. After

synthesis, they were cleaned via repeated centrifugation and dialysis. 1H-NMR measurements

(not shown) showed that removal of the surfactants took approximately 1 week. Re-dispersion

in water after freeze-drying resulted in stable microgel dispersions. Colloidal stabilization was

obtained through the use of the cationic initiator AMPA.

First, the incorporation of betaine in the microgel was measured with FTIR (for spectrum see

appendix). The peak of PVCL at 1672 cm-1 is set in proportion to the peak of SB at 1050 cm-1.

By using a calibration line (see appendix), the actual ratio between VCL and SB can be

determined. The data is in good agreement with the theoretical values (see Table 5.6).

Table 5.6 Amount of incorporated SB as determined with FTIR spectroscopy.

Sample name n(VCLtheo)

/ mol

n(SBtheo)

/ mol

n (VCLFTIR)

/ mol

n (SBFTIR)

/ mol

1:2 1 2 2 3*

1:1 1 1 1 1

2:1 2 1 1.4 1

* The correct ratio is 61/100 ~ 2/3.

The size of the particles was then determined with DLS (Table 5.7). Particles become slightly

smaller with increasing content of SB, while the PDI remains constant at ~ 0.04. According to

52

K. Landfester, the narrow particle size distribution is attributed to the fact that all droplets

nucleate nearly at the same time [44]. The microgel size obtained for the sample VCL:SB =

1:1, i.e. 227 nm, correlates closely with the size of the water droplets before the

polymerization.

Table 5.7 Hydrodynamic radius RH and polydispersity index (PDI) for samples of different

composition.

VCL:SB Size (TEM)

/ nm

R,dried (TEM)

/ nm

RH (DLS) /

nm

PDI

(DLS)

RH (DLS) /

nm (after 3

months)

PDI (DLS)

(after 3

months)

2:1 - - 242 ± 16 0.043 243 ± 7 0.052

1:1 with BIS 459 ± 18 230 ± 9 227 ± 19 0.044 221 ± 8 0.048

1:2 - - 216 ± 8 0.047 211 ± 11 0.041

The particle size was measured a second time after a time period of 3 months. The sizes of all

particles as well as their PDI remained stable.

In order to obtain an optical confirmation of this result, TEM images were taken of the sample

with a ratio VCL:SB = 1:1. Figure 5.18 shows that the particles have a spherical shape and are

monodisperse. In dried state, they have a radius of 230 ± 9 nm. This is very close to the result

obtained via DLS. One might expect the size in dried state is smaller than the hydrodynamic

radius in aqueous solution, but particles presumably spread pancake-like on the surface, thus

appearing bigger in size.

Next, the influence of varying temperature on the particles’ properties was investigated. Pure

PVCL microgels have a broad transition with a VPTT of 30.7 °C (Figure 5.18a). This

temperature is well reported in literature [60][61]. The VPTT is shifted towards higher

temperatures for a ratio of VCL:SB = 2:1 to 37.7 °C. Interestingly, a second VPTT appears at

low temperatures if the content of SB is further increasing. For a ratio of VCL:SB = 1:1,

transition temperatures can be seen at 9.7 °C and 42.9 °C. Both are shifted further to higher

temperatures for a ratio of VCL:SB = 1:2, 18.7 °C and 48.5 °C. Below the first VPTT and

above the second VPTT, particles are in a collapsed state, while they are swollen in the

temperature range in between. This behavior for linear VCL-co-SB copolymers was already

described by B. Yang et al [12]. While the transition temperatures are similar, a major

difference between linear copolymers and microgels is visible: While the microgel particles

described here are stable within the whole temperature range, linear copolymers aggregate

53

above and below the LCST and UCST, respectively. The latter’s hydrodynamic diameter

increases rapidly from ~ 40 nm to above 2 µm.

An increase of both VPTTs can be explained by the weakening of the hydrophobic effects of

VCL induced by the caprolactam ring with an increasing amount of zwitterionic, hydrophilic

SB in the polymer network. This effect is not yet seen for a VCL:SB ratio of VCL:SB = 2:1

indicating that a specific amount of SB is needed for the formation of a doubly

thermosensitive microgel.

Figure 5.18 Hydrodynamic radius RH of PVCL-co-SB microgels as a function of temperature

measured with DLS. (a) Pure PVCL microgel; (b) VCL:SB = 2:1; (c) VCL:SB = 1:1; (d)

VCL:SB = 1:2. The TEM image belongs to the sample VCL:SB = 1:1.

B. Yang et al. describe the arrangement of different polymer chain segment depending on the

temperature: Between the UCST and LCST, both VCL and SB are hydrophilic, so that long

linear chains can be observed [12]. If the temperature falls below the UCST, SB becomes

hydrophobic due to intra- and intermolecular bridges between the zwitterionic groups and

subsequently, aggregates with an SB core and a VCL shell are formed. Analogously, at

temperatures above the LCST, VCL becomes hydrophobic whereby hydrogen bonds between

VCL and water break and subsequently, aggregates with a VCL core and an SB shell are

formed.

This behavior can only partly be transferred to VCL-co-SB microgels. Since the polymer

network is chemically crosslinked a complete “inversion” of an SB/VCL core/shell is not

54

possible. Still, due to the change in particle size, a “shifting” of polymer segments to more

favorable positions in a less pronounced way as for linear polymer chains is expected.

Figure 5.19 Absorbance of VCL-co-SB microgels as a function of temperature. (a) VCL:SB

= 2:1; (b) VCL:SB = 1:1; (c) VCL:SB = 1:2.

The position of a phase transition of the microgels can also be observed in UV-Vis

measurements as a change of absorbance (Figure 5.19). At the transition point where microgel

particles go from a swollen to a collapsed state, the turbidity of the microgel dispersion

increases, thus leading to an increase in absorbance.

Similar to DLS measurements, two transition temperatures can be seen for the samples

VCL:SB = 1:1 and 1:2. These measurements confirm the results obtained with DLS.

Figure 5.20 Hydrodynamic radius RH (right axis, upper curves) and electrophoretic mobility

EM (left axis, lower curves) as a function of pH.

55

The sulfo group of sulfobetaine has a low pKa value of -3 whereby the it remains

deprotonated over the whole pH range [62]. Therefore, the electrophoretic mobility is negative

for all samples within the pH 3 to 10 and hardly pH-dependent (see Figure 5.20). The same

can be said about the particle size. Due to the formation of an internal salt within the microgel

network, the particles do not swell with varying pH.

The colloidal stability of the microgel solutions was examined measuring their sedimentation

velocities below and above the VPTT (i.e. T = 25 °C and 50 °C). The samples were

centrifuged while a laser detects the de-mixing of the samples over time. The higher the

sedimentation speed is, the lower is the colloidal stability of the solution.

Figure 5.21 (a) Sedimentation velocities indicating colloidal stability for mini-emulsion

samples with different monomer ratios. Measurements were conducted at T = 25 °C (black

bars) and 50 °C (red bars) and pH = 7. (b) Sedimentation diagram for VCL:SB = 1:1.

Figure 5.21a shows that all crosslinked samples have approximately the same sedimentation

velocities. Below the VPTT, the sedimentation velocities are ~ 20 µm/s. They increase by ca.

50 % at T = 50 °C. This is caused by a loss of steric repulsion and an increase of Van-der-

Waals attractions. Because swollen microgels have a Hamaker constant close to that of water

at room temperature, attractive Van-der-Waals attractions can be neglected [63]. Attractive

forces increase with increasing temperature, therefore leading to a decrease in colloidal

stability above the VPTT. A complete aggregation is, however, prevented by an increase in

electrostatic repulsion between charges on the surface originating from the initiator.

The polydispersity of non-crosslinked samples can also be seen in the sedimentation curves.

Vertical lines that move to the right side of the diagram as seen in Figure 5.21b are caused by

a slow sedimentation of monodisperse particles.

The swelling behavior of the particles can be tuned by varying the crosslinking content.

Samples discussed above contained 3 wt-% of BIS. The amount was decreased to 2 wt-%,

a

b

56

1 wt-% and 0.5 wt-%. L. A. Shah et al. claim that a small increase in crosslinker content does

not influence the position of the VPTT [64]. Beginning from 10 wt-% of crosslinker content,

the transition temperature is increased due to the solubility of BIS in the polymer network. A

change in the crosslinker content, however, changes the extent of swelling of particles. A

higher crosslinker content leads to a higher particle stiffness and thus a suppressed swelling

ability [65][66]. Figure 5.22 shows that the position of both transition temperatures is hardly

effected by a variation in crosslinker content. While all microgel dispersions have roughly the

same size of ~ 205 nm at 5 °C, their degree of swelling is considerably different in the range

of 14 °C to 35 °C. The higher the crosslinking content, the smaller are the particle size in this

range due to an increase in polymer network stiffness.

Figure 5.22 Thermosensitive behavior of VCL-co-SB microgels (VCL:SB = 1:1) with

varying crosslinker content. (a) Hydrodynamic radius RH (measured with DLS). (b)

Absorbance (measured with UV-Vis). Microgels containing only PVCL are given as a

reference.

AFM images (see Figure 5.23) confirm the variation in microgel stiffness. Height profiles

show that the particles spread more the lower the content of crosslinker is, thus appearing

smaller on the Si-wafer surface. While particles have an average inter-particle distance of ~

90 nm for crosslinker contents of 2 and 3 wt-%, there are no gaps between particles with the

lowest crosslinker content of 0.5 wt-%.

a b

57

Figure 5.23 Height profiles and AFM images of VCL:SB = 1:1 microgels with various

crosslinker content.

Both PVCL and PNIPAm polymers exhibit an LCST of ~ 31 - 37 °C [67]. For some

applications, a shift of this narrow temperature range to different temperatures is necessary. It

is well known in literature that the copolymerization with a hydrophobic comonomer lowers

the transition temperature, while the incorporation of hydrophilic comonomers shifts the

transition to higher temperatures. In the following, a different approach will be followed

where PVCL is substituted for its five- and six-ring homologues (see Figure 5.24).

Figure 5.24 Structures of N-vinylpyrrolidone (VPy), N-vinylpiperidone (VPi), and N-

vinylcaprolactam (VCL).

N-vinylpyrrolidone (VPy) contains a five carbon ring. Its polymer, poly-N-vinylpyrrolidone

(PVPy) is water-soluble, hydrophilic, non-ionic and exhibits good biocompatibility and low

cytotoxicity [68][69]. Its transition temperature in aqueous solution is above 100 °C. Adding salt

to the dispersion decreases the transition temperature considerably [70].

5 µm

5 µm

5 µm

5 µm

58

Compared to PVCL and PVPy, few reports have been published about the

(co-)polymerization of N-vinylpiperidone (VPi) and the properties of its polymer, poly(N-

vinylpiperidone) (PVPi). In 2010, V. N. Kizhnyaev et al. were the first to polymerize VCL

with VPi [71]. Ieong et al. described the synthesis of homo- and copolymers via RAFT

polymerization [72]. The homopolymer exhibits a sharp LCST between 87 – 68 °C depending

on the molecular weight.

Figure 5.25 1H-NMR spectra of monomers N-vinylpyrrolidone, N-vinylpiperidone, N-

vinylcaprolactam, and sulfobetaine. Graphs in inset are close-ups of the respective microgel

spectra (vinylamide:SB = 1:1) that show peaks of the respective vinylamide and sulfobetaine

that were used to calculate the ratio between the two monomers.

1H-NMR spectra confirm the successful copolymerization of the respective vinylamide and

SB. For the calculation of the ratio between the two monomers, the peaks of the vinylamide

and SB at 2.99 and 3.25 ppm, respectively, were used (see Figure 5.25). The calculated ratios

are in accordance with the feed ratios (see Table 5.8).

Table 5.8 Feed and calculated ratios between monomers as obtained from 1H-NMR.

Sample Vinylamidefeed VinylamideNMR SBfeed SBNMR

VCL:SB 1 1 1 0.94

VPi:SB 1 1 1 1.07

VPy:SB 1 1 1 1.18

59

In the following, the influence of the lactam-ring size on the temperature behavior is

examined. Microgels containing homologues of VCL with five- and six-ring are prepared in

the same way via inverse mini-emulsion. A ratio of vinylamide:SB of 1:1 with 3 wt-% BIS is

used.

First, the temperature sensitivity is studied with DLS and UV-Vis. DLS measurements reveal

that the LCST of the vinylamide is shifted to higher temperatures with decreasing ring size.

PVCL and PVPi have a sharp LCST of 42.9 °C and 73.0 °C, respectively, while no LCST can

be observed for PVPy. The UCST of SB remains constant at around 10 °C. It is noticeable

that additionally, the UCST becomes broader with decreasing ring size. UV-Vis

measurements confirm the results obtained via DLS (see Figure 5.26).

Figure 5.26 Thermosensitive behavior of vinylamide-co-SB microgels (vinylamide:SB = 1:1)

with varying ring-size content. (a) Hydrodynamic radius RH (measured with DLS). (b)

Absorbance (measured with UV-Vis).

60

5.4.4. Summary

In this chapter, zwitterionic microgels that were synthesized via inverse mini-emulsion

polymerization were discussed. It was shown that it is possible to incorporate a higher amount

of sulfobetaine (SB) than is possible with precipitation polymerization. Here, even ratios of

VCL:SB = 1:2 were accomplishable. The successful incorporation of high amounts of SB was

verified with FTIR. Samples with a ratio of VCL:SB = 1:1 and 1:2 do not show only an LCST

at 42.9 °C and 48.5 °C, respectively, as is typical for VCL-based microgels, but also an UCST

at 9.7 °C and 18.7 °C, respectively. Both UCST and LCST increase with increasing amount of

SB. This behavior was also confirmed by UV-Vis measurements since absorbance increases

for particles in a collapsed state. As expected for SB microgels, the particle size and their

electrophoretic mobility are not dependent on pH due to the low pKa value of the sulfo group.

Samples synthesized with varying amount of crosslinker were analyzed with DLS and AFM.

DLS measurements confirm that the degree of swelling is dependent on the amount of

crosslinker in the microgel particles. An increase in crosslinker leads to a suppressed swelling

while the positions of the phase transition temperatures remain constant. The decreasing

stiffness with decreasing content of crosslinker was also observed with AFM. Particles with

the lowest amount of crosslinker spread flat on the surface of a Si-wafer, while stiffer

particles were better able to retain their spherical shape.

Furthermore, the influence of the caprolactam-ring size on the temperature-sensitive behavior

was studied. Therefore, the VCL homologues N-vinylpyrrolidone (VPy) and N-

vinylpiperidone (VPi) were used as copolymers for the preparation of vinylamide-co-SB

microgels. 1H-NMR measurements confirmed the successful incorporation of both monomers

in ratios in accordance with the feed ratios. It was shown that the position of the UCST

originating from the SB moieties remains constant for all samples, while the LCST

originating from the vinylamide moieties are strongly dependent on the ring size. VCL- and

VPi-based samples have LCSTs of 42.9 °C and 73.0 °C, respectively, while VPy-based

microgels have no LCST below 92 °C.

61

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64

6 Microgels with Statistically Distribution of Ionizable Groups

6.1. Introduction

Microgels that contain both acidic and basic functional groups are ampholytes [1]. They

exhibit different behavior than nonionic, anionic, cationic or zwitterionic microgels.

Polyelectrolyte microgels having only basic or acidic groups swell with a change to acidic or

basic pH, respectively, due to the ionization of the respective groups resulting in a repulsion

of like charges. Upon the addition of salt, they collapse due to the screening of electrostatic

repulsion forces leading to aggregation [1]. The particle size of ampholyte microgels with

anionic and cationic groups based on strong bases and acids, in contrast, is independent on the

pH [2]. The particles do not swell or de-swell with a change in pH. More common, however,

are ampholyte microgels with anionic and cationic groups based on weak bases and acids:

they swell at both high and low pH. In between, both charges are present creating a

zwitterionic microgel that results in a de-swelling of the particle due to attractive interactions

between positive and negative charges [3]. The presence of an isoelectric point (IEP) is a

characteristic feature of ampholyte microgels. The position of the IEP can be shifted by

varying the composition of the microgel [2]. Ampholyte microgels de-swell in a smaller extent

or even swell if salt is added because intramolecular ionic crosslinks that are present in the

absence of salt or at low salt concentrations are broken. This behavior is called “anti-

polyelectrolyte” effect.

Many reports can be found in literature on the synthesis of ampholyte microgels, they have,

however, mostly a core-shell structure [4][5][6]. If no core-shell structure is prepared, the

distribution of acidic and basic groups is hardly investigated or mentioned [7][8][9]. K.

Christodoulakis et al. prepared 2-(diethylamino)ethyl methacrylate-co-methacrylic acid

microgels (DEA-co-MAA) with DEA and MAA either located in a core-shell structure or

randomly distributed [4]. The latter structure was prepared by the simultaneous addition of

both monomers during the reaction. The ratio MAA:DEA was 56:44. SEM images reveal the

spherical shape of the monodisperse particles that have a size of ~ 350 nm. The particles were

stained with potassium hexachloroplatinate or cadmium nitrate. PtCl62- and Cd2+ selectively

stain the positively charged DEA and the negatively charged MAA groups, respectively. TEM

images show uniformly stained particles, however, a close look-up at a single particle for

better visualization is lacking.

In this chapter, ampholyte microgels with statistically, i.e. homogenously, distributed

ionizable groups are synthesized and characterized. N-Vinylcaprolactam (VCL) was used as a

main monomer to obtain temperature-sensitive microgels. N-isopropylacrylamide (NIPAm)

65

microgels were prepared for comparison. The particles are moreover sensitive to changes in

pH and ionic strength [3][7][4][10] influencing their properties such as particle size and

electrophoretic mobility. To obtain ampholyte microgels, itaconic acid (IA) and 1-

vinylimidazole (Vim) (see Figure 6.1) were used as comonomers. In this work, Vim and IA

were used because of their favorable pKa values. Itaconic acid has a pKa of 3.84 and 5.55

[11][12], 1-vinylimidazole has a pKa of 6.0 [13]. This means that microgel particles containing

both monomers are expected to be positively charged below pH 6.0 and negatively charged

above pH 5.5. The isoelectric point (IEP) is expected to be between pH 5.5 and 6.0,

depending on the amount of IA and Vim incorporated in the microgel particles.

Figure 6.1 Temperature, pH, and salt sensitivity of ampholyte microgels.

C. Erbil et al. have shown for copolymers of NIPAm and IA that copolymers with a statistical

distribution of the monomers in the copolymer are obtained [14]. A. Pich have demonstrated

for NIPAm and Vim that the latter is homogenously distributed in particles [15]. For VCL and

Vim, on the contrast, Vim is thought to be in the loosely crosslinked shell. The authors

explain this by the presence of AAEM. A. Pich and his group have shown that VCL and

AAEM form microgels with core-shell structure with AAEM in the center and VCL in the

shell due to different reactivity ratios [15][16][17]. This “template” forces Vim to accumulate in

the shell and not homogenously throughout the whole particle. Therefore, AAEM was not

used a comonomer because this structure is thought to hinder the formation of microgels

where IA and Vim are homogenously distributed in the particle.

Itaconic acid can easily be used as a comonomer [11] and increases the hydrophilicity of the

microgel due to the presence of two carboxylic groups in the side chain (see Figure 6.2) [18]. 1-

vinylimidazole is a weak base having a nitrogen atom that is protonated at low pH. In

literature, it is often used as comonomer to NIPAm-based microgels due to its metal-binding

properties [19][20].

66

Figure 6.2 Chemical structure of itaconic acid and 1-vinylimidazole.

While the synthesis and characterization of microgels based on NIPAm with either IA [21] or

even more so with Vim [15][22][23][24] have been studied in literature, only S. Schachschal et al.

have – to our knowledge – prepared microgels containing both IA and Vim [5]. They prepared

core-shell microgels based on VCL with IA in the core and Vim in the shell and studied the

effect of pH and temperature change on the swelling behavior. The properties of and

preparation techniques for core-shell microgels is more closely looked at in chapter 7.

Experimental data described in this chapter were published in Macromolecules, 2015 (DOI:

10.1021/acs.macromol.5b01305).

67


Microgels were synthesized via precipitation polymerization in water. Experiments showed

that for microgels with a higher amount of IA, the synthesis performed at pH 10, and for

microgels with a higher amount of Vim or equal amounts of IA and Vim, the synthesis done

at pH 3 lead to colloidal stable particles. Appropriate amounts (see Table 6.1 and 6.2) of

NIPAm/VCL, IA, Vim, and BIS were dissolved in 80 mL water and heated up to 70 °C while

purging with N2. After 1 h, the initiator AMPA was added and the reaction carried out for 5 h

under constant stirring. After synthesis, microgel solutions were cleaned via dialysis using a

composite regenerated cellulose membrane from Millipore (NMWCO 30,000) for 3 d against

water and subsequently lyophilized.

Table 6.1 Amounts of monomers used for PVCL microgel synthesis.

Molar ratio

Vim:IA

VCL / g BIS / g IA / g Vim / g AMPA / g

0:20 0.8 0.040 0.187 0 0.03

5:15 0.8 0.040 0.140 0.034 0.03

10:10 0.8 0.040 0.093 0.068 0.03

15:5 0.8 0.040 0.047 0.101 0.03

20:0 0.8 0.040 0 0.135 0.03

5:5 0.8 0.030 0.042 0.030 0.03

15:15 0.8 0.038 0.160 0.116 0.04

20:20 0.8 0.044 0.249 0.180 0.04

25:25 0.8 0.053 0.374 0.270 0.04

Table 6.2 Amounts of monomers used for PNIPAm microgel synthesis.

Molar

ratio

Vim:IA

NIPAm / g BIS / g IA / g Vim / g AMPA / g

0:20 0.8 0.041 0.230 0 0.03

5:15 0.8 0.042 0.173 0.042 0.03

10:10 0.8 0.041 0.115 0.083 0.03

15:5 0.8 0.041 0.058 0.125 0.03

20:0 0.8 0.041 0 0.166 0.03

68

6.3. Results and Discussion

In this part, ampholyte microgels based on either PVCL or PNIPAm were synthesized. In

both cases, the ratio between IA and Vim was varied (see Table 6.1 and Table 6.2).

Furthermore, for PVCL microgels, particles with equal amount of ionizable groups were

synthesized. Here, the molar amount of IA and Vim was increased from 5 to 25 mol-%.

PVCL and PNIPAm microgels with unequal amount of ionizable groups

The molar ratio between the two ionizable groups is thought to influence the properties of the

microgel particles. Therefore, FTIR measurements were done to ensure that the actual ratio is

in agreement with the theoretical value. The FTIR spectra of the monomers VCL, NIPAm, IA

and Vim are shown in Figure 6.3.

N-Vinylcaprolactam shows peaks at 2934 cm-1 (C-H stretching band), 1654 cm-1 (C=O

stretching), 1620 cm-1 (amide I band), and 1481 cm-1 (C-N stretching). The following bands

derive from the deformation of –CH2: 1433 cm-1, 1391 cm-1, 1320 cm-1, 1256 cm-1, 1183 cm-1,

967 cm-1, and 874 cm-1. These results are in good agreement with literature [25][26][27].

N-isopropylacrylamide has peaks at 2962 cm-1 (C-H stretching band), 1657 cm-1 (amide I),

1623 cm-1 (C=O stretching), 1548 cm-1 (amide II), and 1241 cm-1 (amide III). These results

are in accordance with literature [28].

Itaconic acid shows peaks at 3070 cm-1 (CH2 vibration), 1675 cm-1 (C=O stretching), 1372

cm-1 (C-O-H in-plane bending), 1212 cm-1 (C-O stretching), 1066 cm-1 (O-H out-of-plane

bending). These results can found in literature [29] [30] [31].

1-vinylimidazole shows peaks at 3112 cm-1 (N=CH, C=CH stretching), 3005 cm-1 (CH2

asymmetric stretching), 1649 cm-1 (vinyl off-ring), 1511 cm-1 (C=N, C=C stretching), 1496

cm-1 (CH2 bending and CH2 stretching), 1420 cm-1 (CH2 bending and ring stretching), 1372

cm-1 (CH2 und CH wagging), 1326 cm-1 (CH2 and CH wagging), 1281 cm-1 (ring vibration),

1228 cm-1 (ring vibration), 1106 cm-1 (CH in-plane bending), 1081 cm-1 (CH in-plane

bending), 960 cm-1 (CH out-of-plane bending), 820 cm-1 (CH out-of-plane bending), 735 cm-1

(CH2 rocking), 655 cm-1 (ring torsion), and 597 cm-1 (N=CH and C=CH wagging). These

results are in good agreement with spectra found in literature [32].

Peaks at ca. 3400 cm-1 in all spectra derive from OH from water.

Table 6.3 and 6.4 give the amounts of IA and Vim incorporated into the microgel particle as

obtained from FTIR measurements. The amount of VCL and PNIPAm is set to 80 mol%,

respectively. For PVCL and PNIPAm, the peaks of the carbonyl group at 1654 and 1657 cm-1,

69

Figure 6.3 FTIR spectra of (a) monomers VCL, NIPAm, IA, and Vim; (b) VCL:Vim:IA =

80:10:10 microgel as a representative sample. Measurements were conducted at 25 °C.

respectively, were used as references. In all cases, the amounts of IA and Vim as obtained

from FTIR are close to the theoretical values verifying that the incorporation of IA and Vim

in the desired amounts was successful.

Table 6.3 Amounts of ionizable groups in PVCL microgels as obtained with FTIR.

Ratio

VCL:Vim:IA

VCL %

(1654 cm-1

)

Vim %

theor.

Vim % FTIR

(1228 cm-1

)

IA % theor. IA % FTIR

(1675 cm-1

)

80:0:20 80 0 0 20 16

80:5:15 80 5 4 15 14

80:10:10 80 10 10 10 11

80:15:5 80 15 13 5 6

80:20:0 80 20 18 0 0

Table 6.4 Amounts of ionizable groups in PNIPAm microgels as obtained with FTIR.

Sample NIPAm %

(1657 cm-1

)

Vim %

theor.

Vim % FTIR

(1228 cm-1

)


(1675 cm-1

)

80:0:20 80 0 0 20 16

80:5:15 80 5 5 15 13

80:10:10 80 10 6 10 7

80:15:5 80 15 17 5 6

80:20:0 80 20 21 0 0

a b

70

Microgel particles with different amounts of Vim and IA are expected to behave differently

with varying pH with regard to their electrophoretic mobility and their size. Therefore, those

measurements were conducted between pH 3 and 10.

Figure 6.4 shows the dependence of the electrophoretic mobility on the variation of pH. The

black and red curves belong to the mono-ionic microgels with only IA and Vim, respectively.

Microgels with solely IA as comonomer (black curve) are negatively charged throughout the

whole pH range. No differences can be detected between PVCL and PNIPAm microgels.

Microgels with solely Vim (red curve) as comonomer are positively charged with varied pH.

Microgel particles with both IA and Vim are positively charged at low pH and negatively

charged at high pH, thus exhibiting an isoelectric point (IEP). The position of the IEP is

dependent on the amount of IA and Vim present. The more IA is incorporated the lower the

IEP. Thus, PVCL microgels with 5 mol% IA have an IEP of 6.5, 10 mol% of 6.2, and

15 mol% of 5.0. As for PNIPAm microgels, the respective IEPs are at 5.9, 4.8, and 3.9 and

thus slightly lower.

Figure 6.4 Electrophoretic mobility of PVCL (left side) and PNIPAm (right side) particles

with varying pH. The horizontal line at 0 µmcm/Vs is a visual help to better identify the

isoelectric point (IEP). Error bars are not included in order not to overload the graphic, but are

in average ± 0.04 µmcm/Vs.

The shifts as well as the height of the measured electrophoretic mobilities further support that

varying amount of ionizable groups were incorporated for different samples.

The presence of charges influences the size of the particles. Ampholyte microgels swell in

acidic and basic regions due to electrostatic repulsion. On the other hand, the particles

collapse at or close to the IEP.

71

Figure 6.5 shows the change in size with varying pH. To better foreground the dependence

between electrophoretic mobility and particle size, the electrophoretic mobility is shown for

each sample again.

Figure 6.5 Change of particle size (RH, black curve) and electrophoretic mobility (EM, red

curve) with varying pH. Left side: PVCL microgels. Right side: PNIPAm microgels. The

content of itaconic acid is decreasing from top (20 mol%) to bottom (0 mol%).

For samples containing both acidic and basic groups, a minimum in size (black curve) can be

observed that overlaps with the IEP (red curve). At the IEP, no charges are present in the

microgel leading to a collapse in the internal structure. PVCL microgels with a Vim:IA ratio

of 10:10 have an IEP of 6.2 and a hydrodynamic radius of 94 nm.

At pH 3 and pH 10, particles are swollen due to electrostatic repulsion between charged

groups. Vim is positively charged at low pH, while IA is negatively charged at high pH. For

samples with a Vim:IA ratio of 10:10, the change in size from pH 3 to pH 6.2 and from pH

6.2 to pH 10 should be equal if the acidic and basic moieties are distributed equally. An

unequal change in size would indicate a core-shell structure where the charged monomer that

leads to a more prominent change in size if situated in the shell. Figure 6.5 shows an equal

change in size for both PVCL and PNIPAm microgels indicating a homogenous distribution

of ionizable groups. From their minimum at the IEP, PVCL microgels swell 77 % at pH 3 and

90 % at pH 10. For PNIPAm, the swelling degree is 71 % at pH 3 and 79 % at pH 10.

VCL:Vim:IA =

72

In all cases, the change in size with varying pH is very broad. Particles do not change their

size abruptly but gradually over the whole pH range. This indicates a homogeneous

distribution of ionizable groups. Because of their distribution all over the particle, ionizable

groups do not interact with the environment simultaneously, but gradually.

These results show that it is possible to synthesize particles that are highly pH-sensitive and

that their response to changes in pH can be tuned and controlled by the amount of

incorporated ionizable groups.

Furthermore, it is tested whether the presence of charges has an influence on the temperature

sensitivity of the particles. These trends are measured at pH 3 because the particles are more

stable due to the presence of positive charges. Measurements at the IEP of each sample were

not possible because the particles aggregated when the temperature was increased.

Measurements at pH 10 are expected to mirror the results obtained at pH 3 and were thus not

performed.

As shown in various works by A. Pich [5][33], pure PVCL microgels have a Volume Phase

Transition Temperature (VPTT) of 28-35 °C at neutral pH. S. Schachschal [5] et al. have

shown that the incorporation of Vim lead to a shift of the VPTT at pH 4 to over 35 °C. A

charged microgel is more hydrophilic and thus better able to contain the surrounding water

molecules and shift the collapse to higher temperatures.

Figure 6.6 Change of RH with varying temperature at pH 3. (a) PVCL microgels; (b)

PNIPAm microgels.

Figure 6.6 shows that all samples exhibit broad temperature transitions between 30 and 35 °C

with no distinguishable trend. It was expected that the VPTT would differ dramatically

between samples that are fully charged (80:20:0) and samples that have no charges (80:0:20)

at pH 3. The sample containing only Vim was expected to have a significantly higher VPTT

because the protonated Vim groups show a better solvation in water and thus a higher

temperature is needed to disrupt hydrogen bridges.

a b

73

While the VPTT does not change significantly, the extent of de-swelling differs from pure

PVCL microgels containing no ionizable groups. As shown in previous works by A. Pich,

PVCL microgels are twice as big in the swollen as in the de-swollen state [34] (i.e. the ratio

between the size at 20 °C and 40 °C, R40°C/R20°C, is approximately 2.0). Similar behavior can

be observed for the samples 80:0:20, i.e. with IA as the only comonomer. At pH 3, no charges

are present and the microgel particle is thus neutral. As a consequence, it behaves like pure

PVCL microgels with R50°C/R20°C = 2.12 for PVCL and 2.05 for PNIPAm. The opposite can

be seen for the samples 80:20:0 where Vim as the only comonomer is positively charged. For

PVCL and PNIPAm, R50°C/R20°C is 1.54 and 1.57, respectively. The positive charges prevent a

complete collapse of the network. The same is true for the ampholyte microgels with both

Vim and IA. Here, the ratio R50°C/R20°C is between 1.56 and 1.70.

Figure 6.7 AFM in-situ images of microgels with a ratio of VCL:Vim:IA = 80:5:15. First

row: zoom-in images of particles; second row: force constants of particles shown in the first

row; third row: cross sections (see red line in first row). Force constant images are slightly

shifted due to adjustments between measurement modes. (a) pH 3; (b) pH ~ 6.5; (c) pH 10.

All measurements were conducted in water at 20 °C.

AFM in-situ measurements were conducted in water at pH 3, 6.5, and 10. Images in

Figure 6.7 confirm that the particles are spherical and monodisperse and swollen in water at

pH 3 and pH 10, while they are in collapsed state at pH close to the IEP. This agrees with the

data obtained via DLS. Additionally, the force constants were measured to evaluate

a b c

276 ± 50 nm 156 ± 9 nm 291 ± 36 nm

74

mechanical properties of the particles (see second row in Figure 6.7). Hereby, the darker a

spot in the image, the softer the spot is. Purely white spots are generated by the glass

substrate. All particles have two areas: a lightly colored, soft shell and a darker, stiffer core.

This does not display a “real” core-shell structure, but is caused by different thicknesses of

those two areas due to the spreading of the soft microgel particles: the cantilever “senses” the

thick core and the thinner shell by indentation. Determination of the force constants gives the

Young’s Modulus of the particle at each spot. The data is given in Figure 6.8. Since “core”

and “shell of the polymer particle differ considerably, they are analyzed separately.

Figure 6.8 (a) Young’s Modulus for the sample VCL:Vim:IA = 80:5:15. Error bars were

determined by evaluating a representable number of data points in Figure 6.7. (b) AFM

cantilever approaching the surface. The Young’s Modulus E is defined as E = F/z with F =

force and z = distance.

Figure 6.8a shows the Young’s moduli (YM) for VCL:Vim:IA = 80:5:15 microgels at various

pH. A low YM is attributed to a high stiffness indicating that particles are in a collapsed state.

While the YM for the shell does not change significantly, the YM of the core is strongly

dependent on the pH of the dispersion. At low and high pH, particles have a high YM above

1000 kPa. At pH = IEP, the stiffness increases due to attractive forces between opposite

charges that induces the collapse of the particle network. A decrease in stiffness correlates

with a swelling of the particles at low and high pH and confirm the previous DLS

measurements (see Figure 6.5). It can also be seen that particles have a higher YM at pH 10

than at pH 3. At pH 10, particles are swollen due to the deprotonation of the carboxylic

groups. The asymmetric ratio of Vim:IA = 5:15 leads to a higher swelling at pH 10 and thus a

lower YM.

a b

75

PVCL microgels with equal amount of ionizable groups

It was shown that microgel particles with a changing ratio between Vim and IA express

different behavior regarding their location of the IEP and their swelling behavior with varying

temperature and pH. Particles with an equal ratio between Vim and IA, but an increasing

amount of ionizable groups should show similar behavior regarding the location of the IEP,

but different extends in swelling behavior. In the following, samples with Vim:IA ratios of

5:5, 10:10, 15:15, 20:20, and 25:25 are discussed. Since no significant differences were

detected between PVCL and PNIPAm microgels for a Vim:IA ratio of 10:10 in the chapter

above, these particles were only prepared with VCL.

To ensure that the desired amount of ionizable groups is incorporated in the particles, FTIR

measurements were performed.

Table 6.5 Amounts of ionizable groups in PVCL microgels as obtained with FTIR.

Sample VCL %

(1619 cm-1)

Vim %

theor.

Vim % FTIR

(665 cm-1

) IA % theor.

IA % FTIR

(1454 cm-1

)

90:5:5 90 5 6 5 4

80:10:10 80 10 10 10 11

70:15:15 70 15 14 15 15

60:20:20 60 20 16 20 16

50:25:25 50 25 21 25 22

As seen in Table 6.5, FTIR data confirm that the desired amounts of IA and Vim are in

agreement with the actual amounts.

TEM images support the data obtained with FTIR. The sample based on PVCL with a Vim:IA

ratio of 20:20 is taken as an example to visualize the distribution of ionizable groups. The

sample is stained with uranyl acetate that binds to the negatively charged carboxylic groups.

The heavy metal ion U3+ enhances the contrast by increasing the electron density [35] and

black dots visualize its location and distribution in the microgel particle. This method is used

by many groups such as Jones and Lyon [36] and Xing [37].

Figure 6.9 shows the distribution of ionizable groups for the sample with a Vim:IA ratio of

20:20. The image shows slightly deformed spherical particles due to the removal of water

while drying. The images support the thesis of a random distribution of itaconic acid in the

microgel particle. Particles also appear to be rather polydisperse. According to Lyon [36], this

is an illusion caused by an differing spreading of the particles on the TEM grid. Information

about the polydispersity of a sample is better analyzed with DLS.

76

Figure 6.9 Transmission electron image of PVCL microgel particles with a ratio of

VCL:Vim:IA = 60:20:20. The image on the right side is a zoom-in.

Figure 6.10 Change of (a) electrophoretic mobility EM and (b) hydrodynamic radius RH of

PVCL microgels with equal amount of Vim:IA.

In the previous section, the behavior of PVCL microgels with a Vim:IA ratio of 10:10 was

discussed. Samples with an equal but increasing ratio should show very similar behavior

when the pH of the solution is changed. Figure 6.10 shows the change of the electrophoretic

mobility EM and the hydrodynamic radius RH as a function of pH. The graphs indicate that

the degree of swelling becomes more pronounced with a higher concentration of ionizable

groups. This shows that the electrostatic repulsion becomes stronger when more charges are

incorporated. Furthermore, it can be seen the size of particles in collapsed state decreases with

increasing amount of ionizable groups. Particles with a ratio of Vim:IA = 5:5 have a

hydrodynamic radius of ~ 100 nm at pH 6.2, while particles with a ratio of 25:25 have a

hydrodynamic radius of ~ 55 nm.

a b

77

6.4. Summary

This chapter showed that it is possible to synthesize ampholyte microgels with balanced as

well as unbalanced amounts of acidic and basic moieties with statistical distribution of

ionizable groups. Both PVCL and PNIPAm microgels were prepared with itaconic acid (IA)

and 1-vinylimidazol (Vim) as comonomers. The successful incorporation of both

comonomers in desired amounts was verified with FTIR spectroscopy. Also TEM images of

stained samples indicated the random distribution of ionizable groups. It was further shown

that the size of the particles can be tuned with the pH of the solution. For all ampholyte

microgels, a V-shaped curve could be obtained, this means that particles were swollen at high

and low pH and collapsed at the isoelectric point (IEP). These data correlated very well with

measurements for the determination of the electrophoretic mobility. The position of the IEP

and thus the minimum of the size curve could be shifted to higher or lower pH depending on

the amount of IA and Vim. Samples containing only Vim or IA as a comonomer were only

swollen in one pH regime. Microgels with a balanced ratio between IA and Vim, but different

amounts all showed the typical V-shaped curve for their sizes with varying pH with an IEP at

~ 6.2. It could be seen, however, that particles with the highest amount of comonomers

(VCL:Vim:IA = 50:25:25) were smallest in the collapsed state compared to samples with a

lower amount of ionizable groups (VCL:Vim:IA = 90:5:5). Furthermore, the degree of

swelling at pH 3 and pH 8, respectively, compared to pH = IEP was higher for samples with a

higher amount of ionizable groups. This was explained thereby that a higher amount of

charges leads to more electrostatic repulsion between the charged groups thus extending the

microgel network.

AFM images in buffer were taken of samples with an unbalanced amount of ionizable groups

and verified the production of spherical, monodisperse microgel particles. Force maps of the

same particles revealed that particles are stiffer, that means they have a higher Young’s

modulus, at pH = IEP than at pH 3 and pH 8. These results correlate with the results obtained

with DLS. At pH = IEP, the particles are in a collapsed state and are therefore stiffer than in

the swollen state.

78

6.5. Literature

[1] T. Hoare, R. Pelton, Biomacromolecules 2008, 9, 733–740.

[2] M. Bradley, B. Vincent, G. Burnett, Aust. J. Chem. 2007, 60, 646–650.


[4] K. E. Christodoulakis, M. Vamvakaki, Langmuir 2010, 26, 639–47.



[6] W. Richtering, A. Pich, Soft Matter 2012, 8, 11423.

[7] H. Ni, H. Kawaguchi, T. Endo, Macromolecules 2007, 6370–6376.

[8] M. Kashiwabara, K. Fujimoto, H. Kawaguchi, Colloid Polym Sci 1995, 273, 339–345.

[9] K. Ogawa, A. Nakayama, E. Kokufuta, Langmuir 2003, 19, 3178–3184.

[10] M. Bradley, B. Vincent, G. Burnett, Colloid Polym. Sci. 2008, 287, 345–350.

[11] T. Willke, K.-D. Vorlop, Appl. Microbiol. Biotechnol. 2001, 56, 289–295.

[12] J. Li, T. B. Brill, J. Phys. Chem. A 2001, 105, 10839–10845.

[13] T. Hakamatani, S. Asayama, H. Kawakami, Nucleic Acids Symp. Ser. (Oxf). 2008,

677–8.

[14] C. Erbil, B. Terlan, Ö. Akdemir, A. T. Gökçeören, Eur. Polym. J. 2009, 45, 1728–

1737.

[15] L. Shen, A. Pich, D. Fava, M. Wang, S. Kumar, C. Wu, G. D. Scholes, M. a. Winnik, J.

Mater. Chem. 2008, 18, 763.

[16] G. Agrawal, M. Schürings, X. Zhu, A. Pich, Polymer (Guildf). 2012, 53, 1189–1197.

[17] C. Cheng, X. Zhu, A. Pich, M. Möller, Langmuir 2010, 26, 4709–16.

[18] M. Babic, J. Jovasevic, J. Filipovic, S. Tomic, Hem. Ind. 2012, 66, 823–829.

[19] M. Muratalin, P. F. Luckham, J. Colloid Interface Sci. 2013, 396, 1–8.

[20] P. Chen, L. Cao, G. Wang, J. Wang, Polym. Adv. Technol. 2013, 24, 764–771.

[21] M. K. Krušić, J. Filipović, Polymer (Guildf). 2006, 47, 148–155.

[22] L. Li, C. Cheng, M. P. Schürings, X. Zhu, A. Pich, Polymer (Guildf). 2012, 53, 3117–

3123.

[23] S. Bhattacharya, F. Eckert, V. Boyko, A. Pich, Small 2007, 3, 650–7.


[25] Y. Maeda, T. Nakamura, I. Ikeda, Macromolecules 2002, 35, 217–222.

[26] J. Spěváček, J. Dybal, L. Starovoytova, A. Zhigunov, Z. Sedláková, Soft Matter 2012,

8, 6110.

[27] S. Sun, P. Wu, J. Phys. Chem. B 2011, 115, 11609–18.

[28] B. Özkahraman, I. Acar, S. Emik, Polym. Bull. 2010, 66, 551–570.

[29] N. B. Milosavljević, M. Đ. Ristić, A. a. Perić-Grujić, J. M. Filipović, S. B. Štrbac, Z. L.

Rakočević, M. T. K. Krušić, Colloids Surfaces A Physicochem. Eng. Asp. 2011, 388,

59–69.

79

[30] T. Betancourt, J. Pardo, K. Soo, N. A. Peppas, J. Biomed. Mater. Res. A 2010, 93, 175–

88.

[31] R. M. Silverstein, F. X. Webster, D. Kiemle, Spectrometric Identification of Organic

Compounds, John Wiley & Sons, 2005.

[32] J. E. Hansen, B. I. Rickett, J. H. Payer, H. Ishida, J. Polym. Sci. Part B Polym. Phys.

1996, 34, 611–621.


2003, 44, 7821–7827.

[34] A. Balaceanu, D. E. Demco, M. Martin, A. Pich, Macromol. React. Eng. 2011, 44,

2161–2169.

[35] T. Hoare, R. Pelton, Curr. Opin. Colloid Interface Sci. 2008, 13, 413–428.

[36] C. Jones, L. Lyon, Macromolecules 2000, 33, 8301–8306.

[37] Z. Xing, C. Wang, J. Yan, L. Zhang, L. Li, L. Zha, Colloid Polym. Sci. 2010, 288,

1723–1729.

80

7 Microgels with Core-Shell Structure

7.1. Introduction

Due to the broad application range for microgels, microgel architectures tend to become more

and more complex in order to meet multiple requirements in one multifunctional microgel

polymer network. The next step from microgels with a statistical distribution of ionizable

groups that were discussed in the last chapter, with regard to advanced architecture are

microgels with a core-shell structure with ionizable groups spatially separated. These are the

subject of this chapter.

Core-shell microgels consist of two chemically crosslinked networks [1]. Particles can be

prepared either in a one-step synthesis using monomers of different reactivity ratios or in a

two-step synthesis by adding the monomers for the shell after the reaction of the core is

complete. Both steps are carried out at T > LCST. The latter method ensures a strict spatial

separation of core and shell and a well-defined surface between them [2]. A two-step synthesis

is possible because the particles of the first step act as seeds for the second polymerization

step where oligomers precipitating from the aqueous phase are connecting preferably to the

existing particle cores by chain transfer due to their relatively higher hydrophobicity [3][4]. The

total particle number remains constant during the addition of the shell [3].

Due to the soft nature of microgels, core and shell influence each other greatly [1]. Swelling or

collapsing of one network either causes or hinders the swelling or collapsing of the other. I.

Berndt et al. prepared core-shell microgels with PNIPAm in the core and poly(N-

isopropylmethacrylamide) (PNIPMAm) in the shell [5]. PNIPAm and PNIPMAm exhibit

LCSTs of 34 °C and 44 °C, respectively. The core thus collapses at 34 °C, while the shell

remains hydrophilic until a temperature of 44 °C is reached. In between, core and shell

counteract each other creating a field of stress within the particle. SANS measurements

revealed that the shell pulls the core outwards which is intensified with increasing shell

thickness.

Not only the synthetic protocol is important for the successful preparation of core-shell

particles, another important factor is the compatibility of the two monomers as well as the

interfacial tension between the two polymers [6].

In literature, a wide range of analysis techniques are used to gain information about the

internal structure of core-shell microgels. TEM and cryo-TEM measurements give an optical

conformation of the core-shell nature of particles and their morphology [4]. Staining with

phosphate tungsten acid [7], cadmium nitrate, potassium hexachloroplatinate [8] or uranyl

acetate [9] greatly enhances the contrast between core and shell. If nanoparticles are already

81

embedded in the microgel network, no further staining is necessary [10]. Cryo-TEM images

presented by M. Ballauff and L. Yu of poly(styrene) core and PNIPAm shell microgels

clearly distinguish between core and shell and show that the shell is slightly deformed due to

density fluctuations of the shell [4]. Their data confirm results for the same microgel system

that were obtained earlier in the same group by small angle X-ray scattering (SAXS) [11] and

that were predicted in theory by M. Shibayama [12]. These inhomogeneities are explained by

thermal fluctuations of the polymer chains that become stiff when crosslinked. Furthermore, it

was shown that the shell of those particles that were prepared in a two-step synthesis is not

continuously attached to the core and detaches slightly when the particles swell below the

VPTT [2].

Further methods for the characterization of core-shell microgels are SANS [13], NMR [14],

AFM [15][16] or titration [17][18].

Multi-responsive microgels react to more than one external stimulus, mostly pH and

temperature. T. Hu et al. synthesized core-shell particles with a PNIPAm-co-acrylic acid 2-

hydroxyethyl ester (HEA) core and a PNIPAm shell via free-radical RAFT living

polymerization [19]. They could show that core and shell exhibited different transition

temperatures. DLS measurements revealed that the copolymer core collapsed between 28 –

32 °C, while the collapse of PNIPAM shell is shifted to 32 – 25 °C. A dual temperature-

sensitive core-shell microgel was also prepared by A. Balaceanu et al [20]. They prepared

poly(vinylcaprolactam) (PVCL) core and poly(N-isopropylmethacrylamide) (PNIPMAm)

shell and reverse microgels. PVCL and PNIPMAm have volume transition temperatures at

32 °C and 45 °C, respectively. In PNIPMAm core/PVCL shell microgels, DLS measurements

giving the ratio of the core Rcore to the hydrodynamic radius RH indicate that the PVCL shell

collapses first upon an increase in temperature as can be seen from an increase in the ratio

Rcore/RH. A further increase in temperature leads to a collapse of the PNIPMAm core,

decreasing the Rcore/RH ratio.

So, far only few examples for ampholyte core-shell microgels can be found in literature. K. E.

Christodoulakis and M. Vamvakaki prepared 2-(diethyl-amino)ethyl methacrylate (DEA) or

tert-butyl methacrylate (t-BuMA) core and t-BuMA or DEA shell, respectively, in a two-step

emulsion polymerization [17]. Both core and shell could be protonated and deprotonated

separately. They compared the swelling/ de-swelling results to a microgel of the same

composition where t-BuMA and DEA are randomly distributed and found that in the latter the

basic and acid moieties are protonated/ deprotanated simultaneously. S. Schachschal et al.

synthesized ampholyte microgels with anionic core and a cationic shell based on VCL in a

82

one-step synthesis [14]. The core-shell structure arises from different reactivities and water

solubilities of the comonomers itaconic acid dimethyl ester (IADME) and vinylimidazole

(Vim). The ampholyte character of the particles was obtained after hydrolyzation of IADME

to itaconic acid (IA). Using proton high-resolution transverse magnetization relaxation under

magic angle sample spinning (MAS), the authors could show that IA is mostly located in the

core of the microgels. Both the VPTT and the IEP of the particles can be tuned by variation of

the IA and Vim content.

Based on the last work, core-shell microgels with NIPAm or VCL as main monomers in the

core or shell and IA and Vim as comonomers in the core or shell were prepared. Figure 7.1

shows the structure of the particles and the sample names used in this chapter.

In contrast to the work done by S. Schachschal, the core-shell microgels are prepared in a

two-step synthesis to ensure a strict spatial separation between core and shell. Particles with

basic moieties in the core and acid moieties in the shell as well as their reverse counterparts

were prepared to study the influence of distribution of ionizable groups on swelling

properties. The location of ionizable groups will further be important for uptake and release

studies in later chapters. Moreover, the location of VCL and NIPAM was varied. VCL is less

cytotoxic than NIPAm [21][22], its location in the shell might therefore enhance

biocompatibility of the whole core-shell particle. NIPAm-based microgels, however, have a

lower polydispersity and a sharper VPTT than PVCL-based microgels [22]. The

copolymerization of two temperature-sensitive monomers is a facile method for the

preparation of new smart materials.

Figure 7.1 Structure of core-shell microgels and the sample names used in this chapter

(c = core, s = shell).

83


Microgels were synthesized in a two-step precipitation polymerization in water. For the

synthesis of the core, appropriate amounts (see Table 7.1) of NIPAm/VCL, IA/Vim, and BIS

were dissolved in 150 mL water and heated up to 70 °C while purging with N2. After 1 h, the

initiator AMPA was added and the reaction carried out for 3 h under constant stirring. The

core-microgels were left in the reactor without cooling down or purification. For the addition

of the shell, the appropriate amounts (see Table 7.2) of VCL/NIPAm, Vim/IA, BIS and

AMPA were added to the aqueous solution without addition of further water. The reaction

was carried out for further 3 h under N2. After synthesis, microgel solutions were cleaned via

dialysis using a composite regenerated cellulose membrane from Millipore (NMWCO 30,000)

for 3 d against water and subsequently lyophilized for 3 d.

Table 7.1 Amounts of monomers used for synthesis of the core.

Monomer

in core

VCL/NIPAm / g IA / g Vim / g BIS / g AMPA / g

VCL 1.897 - 0.169 0.062 0.053

1.876 0.175 - 0.065 0.053

NIPAm 1.568 - 0.156 0.062 0.049

1.501 0.179 - 0.063 0.050

Table 7.2 Amounts of monomers used for synthesis of the shell (added to the core

dispersion).

Monomer

in shell

VCL/NIPAm / g IA / g Vim / g BIS / g AMPA / g Sample name

NIPAm 1.532 0.182 - 0.062 0.058 cVCL+/sNIPAm-

1.572 - 0.126 0.062 0.056 cVCL-/sNIPAm+

VCL 1.891 0.181 - 0.061 0.051 cNIPAm+/sVCL-

1.871 - 0.104 0.062 0.050 cNIPAm-/sVCL+

Further, core-shell microgels cVCL-/sNIPAm+ were prepared with an increasing shell

thickness. Core particles as prepared according to Table 7.1 were synthesized. The amounts of

the shell can be seen in Table 7.3. For the samples with a core:shell ratio of 1:2.5 and 1:5, the

monomers for the shell were dissolved in 190 mL and 375 mL of water (pre-heated to 70 °C),

respectively, and instantaneously added to the core dispersion.

84

Table 7.3 Amounts of monomers used for synthesis of the shell with increasing shell

thickness (added to the core dispersion).

Ratio

core:shell

NIPAm / g Vim /

g

BIS / g AMPA / g Sample name

1:1 1.532 0.104 0.062 0.050 cVCL-/sNIPAm+ (1:1)

1:2.5 3.802 0.250 0.149 0.132 cVCL-/sNIPAm+ (1:2.5)

1:5 7.581 0.526 0.312 0.260 cVCL-/sNIPAm+ (1:5)

85


The synthesis of core-shell microgels led to colloidally stable dispersions. Measurements of

the colloidal stability (see appendix) reveal that all particles have the same colloidal stability

regardless of the distribution of monomer and ionizable groups.

To ensure that the desired amounts of monomers are incorporated in the microgels, samples

were analyzed with FTIR (see Figure 7.2). The discussion of the monomer spectra and the

respective peak assignment can be found in chapter 6.

Figure 7.2 FTIR spectra for core-shell PVCL-co-Vim-co-IA microgels.

Table 7.4 Amounts of ionizable groups in core-shell microgels as obtained with FTIR.

Sample name VCL %

(1654 cm-1

)

Vim %

theor.

Vim % FTIR

(1284 cm-1

)


(1153 cm-1

)

cVCL+/sNIPAm- 80 10 10 10 8

cVCL-/sNIPAm+ 80 10 9 10 9

cNIPAm+/sVCL- 80 10 11 10 10

cNIPAm-/sVCL+ 80 10 9 10 11

In all cases, the amounts of IA and Vim as obtained from FTIR are close to the theoretical

values indicating that the incorporation of IA and Vim in the desired amounts was successful.

TEM images confirm the formation of core-shell microgels (see Figure 7.3). The particles

were stained with uranyl acetate, U(Ac)3, which binds to the carboxylic groups. Therefore,

regions with IA appear darker than regions with Vim. Figure 7.3a shows that IA is mostly

located in the core of the particles. A thin shell can be seen surrounding the particles. Figure

7.3c shows particles which are completely stained. IA is located in the shell and thus masks

the inner core.

86

Figure 7.3 TEM images of core-shell particles stained with U(Ac)3. (a) cVCL-/sNIPAm+ (b)

cVCL-/sNIPAm+, closer look; (c) cVCL+/sNIPAm-; (d) cVCL+/sNIPAm-, closer look.

Figure 7.4 (a) Hydrodynamic radius RH and (b) electrophoretic mobility EM of core

microgels at 20 °C with varying pH. The horizontal line at 0 µmcm/Vs is a visual help to

better identify the isoelectric point.

Particles were analyzed regarding their pH-sensitivity. First, the pure core particles were

examined (see Figure 7.4). Particles with basic moieties swell at low pH and collapse at high

pH from ~ 270 nm to ~ 100 nm (i.e. - 63 %). Particles with acid moieties swell at high pH due

to the deprotonation of the carboxylic groups and collapse at low pH from ~ 230 nm to ~

125 nm (i.e. - 46 %). The electrophoretic mobility reflects this behavior. While the EM of the

cores containing Vim decreases from 2.1 µmcm/Vs to 0 µmcm/Vs from pH 3 to pH 10, it

decreases less significantly for cores containing IA: from 0 µmcm/Vs at pH 3 to -

0.5 µmcm/Vs at pH 10. This asymmetric behavior is reflected in the FTIR data. Slightly less

IA is incorporated into the core polymer network than Vim. The swelling of both particle

a c

b d

a b

shell

core

87

types is very broad and constant indicating a homogenous distribution of the acidic and basic

moieties in the particles.

Pure IA and Vim have pKa and pKb values of 3.84/5.55 and 6.0, respectively. In the core

microgels, the swelling already starts at pH ~8 for Vim-core particles and at pH ~ 6 for IA-

core particles. The “premature” swelling between pH 6 and pH 8 for Vim-core microgels is

caused by the greater hydrophilicity of VCL compared to Vim due to the carbonyl group of

VCL. Hydrophilic VCL-co-Vim microgels will more readily swell in water than pure Vim. IA

on the other hand is already more hydrophilic than VCL due to its two carboxylic groups,

therefore, no “premature” swelling can be observed. This behavior is in accordance with

results obtained by M. Bradley et al [23].

Figure 7.4 also shows that there are no differences between PVCL- and PNIPAm-based core

microgels regarding their EM with varying pH. Looking at their RH, PNIPAm microgels are

slightly bigger in size than the respective PVCL microgel in the swollen state, while both

have the same size in collapsed state. Since FTIR data confirm that both samples have the

same amount of basic or acid moieties incorporated, the difference in swelling might be

attributed to different crosslinker densities. A higher crosslinker density in the core for VCL-

based microgels would suppress the swelling behavior.

Figure 7.5 shows the pH-responsive behavior of the respective core-shell particles that were

discussed in Figure 7.4. Figure 7.5b shows that core-shell particles show very different

behavior depending on the location of ionizable groups. For particles with Vim located in the

core with IA in the shell (black and blue curves), the particle size constantly decreases from

pH 3 to pH 10. For this system, the core is positively charged at low pH, while the shell is

negatively charged at high pH. Therefore, a V-shaped curve as observed for microgels with

statistical distribution of ionizable groups (see chapter 6) was expected. The collapse of the

shell at low pH exerts an inward pressure on the swollen core, still a strong increase in

particle size (compared to neutral state at pH ~ 6.2) can be observed indicating that the

collapse of the shell has only a small effect. The fact that particles constantly de-swell with

increasing pH indicates that at high pH, the negatively charged shell is not “strong” enough to

counteract the collapse of the neutral core. While the IA-shell supposably increases in size

due to swelling, the overall particle size decreases because the Vim-core pulls the shell

inwards (see Figure 7.6). Though the particle size decreases from low to high pH, two distinct

areas can be differentiated. A rapid decrease occurs from pH 3 to pH 6 and a slower decrease

from pH 6 to pH 10. This indicates that the swelling/ de-swelling transitions of the core and

the shell are independent from each other.

88

Figure 7.5 (a) Hydrodynamic radius RH and (b) electrophoretic mobility EM at 20 °C with

varying pH of core-shell microgels. The horizontal line at 0 µmcm/Vs is a visual help to

better identify the isoelectric point.

Figure 7.6 Schematic illustration of pH-dependent behavior of core-shell particles. (a) core:

Vim, shell: IA; (b) core: IA, shell: Vim. Arrows indicate direction of swelling (green) and de-

swelling (red). X and y denote the particle size at low (x) and high (y) pH.

A different situation arises if IA is located in the core and Vim in the shell (red and magenta

curves). Additionally, a difference can be seen between NIPAm and VCL. The system

cNIPAm-/sVCL+ shows a slight V-shaped curve indicating that particles swell at high and

low pH. The system cVCL-/sNIPAm+, in contrast, reaches a plateau in size at pH > 6.5. For

both systems, results indicate that the positively charged shell at low pH is able to

“counteract” the collapse of the neutral core by exerting a strong outward pressure on the

latter. This is in contrast to the “weak” IA-shell discussed above. The reason for this is likely

to be the nature of the core. The Vim-cores discussed above de-swells highly at high pH and

subsequently pulls the shell inwards. The IA-core, on the other hand, does not de-swell in the

same extent as the Vim-core when neutralized due to its higher hydrophilicity. This could

already be seen in Figure 7.4. Neutral IA-cores (i.e. at pH 3) have an RH of 125 nm, while

neutral Vim-cores (i.e. at pH 10) have a lower RH of 102 nm.

The swelling degrees q can be seen in Table 7.5. They are calculated as follows:

a b

89

𝑞𝑎𝑐𝑖𝑑𝑖𝑐 = (𝑅𝐻,𝑝𝐻3)³

(𝑅𝐻,𝑝𝐻6)³ (1)

𝑞𝑏𝑎𝑠𝑖𝑐 = (𝑅𝐻,𝑝𝐻10)³

(𝑅𝐻,𝑝𝐻6)³ (2)

The results show that particles with PNIPAm in the shell swell in a higher extent than

particles with PVCL in the shell. This is in accordance with the results obtained for the core-

particles where NIPAm-based particles swelled more than VCL-based microgels (see Figure

7.4). This is attributed to the different reaction parameters between BIS with VCL and

NIPAm, respectively, leading to different incorporation of the crosslinker in the polymer

network and thus a different swelling behavior. The table also emphasizes the difference

between particles having Vim or IA in the shell. Particles with IA in the shell have a ratio

below 0 indicating that the particles become smaller in volume.

Particles with Vim in the core and IA in the shell have an IEP of pH ~ 5.5, while particles

with a reverse distribution of ionizable groups have an IEP of pH 6.5. This discrepancy can be

explained by the fact that electrophoresis measures the amount of charges on the particle

surface. Particles with IA in the shell have therefore an IEP hat is closer to the pKa of IA than

to the pKa of Vim. Accordingly, particles with Vim in the shell have a pKa closer that to of

Vim than to the one of IA. For all samples, regardless of the charge location, the absolute

value for the electrophoretic mobility is below 2 µmcm/Vs. M. Semmler et al. state that the

typical value for hard spheres is ~ 4 µmcm/Vs [24]. This indicates that the particles are swollen

therefore showing no distinct, but a diffuse surface typical for microgels.

Table 7.5 Swelling ratio q of core-shell microgels at pH 3 and 10, respectively, compared to

the minimum in size at pH 6.2 (~ IEP). Swelling ratios were calculated as follows:

q1 = RH(pH3)³/RH(pH6.2)³ and q2 = RH(pH10)³/RH(pH6.2)³. The table also shows the swelling ratios for

selected microgels with statistical distribution of ionizable groups.

Sample qacidic qbasic

cNIPAm-/sVCL+ 3.70 1.57

cNIPAm+/sVCL- 3.99 0.44

cVCL-/sNIPAm+ 4.05 1.06

cVCL+/sNIPAm- 4.68 -0.40

VCL:Vim:IA = 80:10:10 statistical 5.50 6.91

VCL:Vim:IA = 80:0:20, statistical - 1.75

90

Comparing the swelling degrees at low and high pH of core-shell particles with particles with

a statistical distribution of ionizable groups (see Chapter 6 and Table 7.5), it can be observed

that microgels with a core-shell distribution swell less at pH 3 and considerably less at pH 10

than microgels with a statistical distribution of ionizable groups. According to T. Hoare and

R. Pelton, microgels with a homogeneous distribution throughout the particle swell in a less

extent [25]. The polyion concentration is lower due to a greater volume that is available. In

core-shell microgels, in contrast, charges are localized in a smaller volume thus repelling each

other in a higher degree resulting in a more pronounced swelling.

This, however, is not observed for the above discussed system. Assuming that the core has a

higher influence on the swelling/ de-swelling behavior, the swelling degrees of microgels with

a statistical distribution of ionizable groups is best compared to core-shell particles with a

swollen core and a neutral shell. For particles with Vim in the core, q is approximately in the

same range as for particles with statistical distribution of ionizable groups. The smaller value

can be explained by the stiff shell of neutral IA suppressing further swelling. For particles

with IA in the core, however, q is considerably lower. A comparison with the sample

VCL:Vim:IA = 80:0:20 with statistical distribution of ionizable groups (it should be noted,

however, that this sample has a twofold amount of IA incorporated), shows that this value is

comparable with the value for core-shell particles containing IA in the core.

Figure 7.7 Hydrodynamic radius RH as a function of temperature for (a) cVCL+/sNIPAm-

and the respective core, measured at pH 3; (b) cVCL-/sNIPAm+ and the respective core,

measured at pH 10. Pure PVCL microgels are given as a reference.

In the following, the hydrodynamic radius of the core-shell microgels is compared to the

respective core-microgels. Measurements were only conducted for particles with PVCL in the

91

core and PNIPAm in the shell and at a pH where the core is charged, i.e. at pH 3 for cores

containing Vim and at pH 10 for cores containing IA. Measurements at the isoelectric point

Table 7.6 Volume phase transition temperatures for core and core-shell microgels.

Sample Measured at pH VPTT Core / °C VPTT Core-Shell / °C

cVCL+/sNIPAm- 3 31.8 33.8

cVCL-/sNIPAm+ 10 31.8 33.0

cNIPAm+/sVCL- 3 31.8 34.4

cNIPAm-/sVCL+ 10 31.7 33.5

led to aggregation at higher temperatures due to the lack of charges. All samples show a broad

transition temperature compared to pure PVCL microgels (see Figure 7.7). Pure PVCL

microgels have a transition temperature of 31 °C. The incorporation of basic or acid moieties

into the core particles does not change the transition temperature significantly (see Table 7.6).

Also, at all pH, core and core-shell microgels are larger in size than pure PVCL microgels due

to charged moieties repelling each other.

In the following, the thickness of the shell in the system cVCL-/cNIPAm+ was increased to

study its effect on the particle’s properties. As shown in Figure 7.4, the size of this sample

decreases until a pH of 6.5, above this pH, the particle size remains constant. To study

whether the swelling behavior at high pH can be influenced, the same particles with an

increasing shell thickness were prepared. The ratio core:shell was increased from 1:1 (as

discussed above) to 1:2.5 and 1:5.

First, an increasing amount of 1-vinylimidazole was confirmed with FTIR (see appendix). It

can be seen that the peak corresponding to Vim at 1284 cm-1 increases with an increasing

shell thickness.

Figure 7.8 TEM images of core-shell microgels (cVCL-/sNIPAm+) with increasing shell

thickness. (a) Ratio core:shell = 1:1, (b) 1:2.5; (c) 1:5. Images with single particles are shown

to better visualize the increase in shell thickness.

TEM images confirm that the particles increase in size (see Figure 7.8). Dark spots in the

corona come from “entangled” U3+ aggregates in the shell polymer network. The thickness of

the shell increases from 24 nm to 50 nm and 71 nm.

c a b

92

Figure 7.9 RH and EM at 20 °C with varying pH of core-shell microgels with increasing shell

thickness.

Figure 7.9 gives the hydrodynamic radius and the electrophoretic mobility for samples with

increasing shell thickness as a function of pH. RH remains constant for a pH > 6.5 for samples

with a ratio core:shell = 1:1 as already discussed above. For a ratio core:shell = 1:2.5, the

particle size slightly increases for pH > 7.5. For a ratio core:shell = 1:5, the particle size

increases in a more pronounced way for a pH > 7. This shows that the inward force exerted by

the collapse of the neutral core on the negatively charged, swollen shell is diminished by an

increase in shell thickness. Still, the degree of swelling at high pH is considerably lower than

for samples with a statistical distribution of ionizable groups. The electrophoretic mobility is

shifted to higher pH for an increasing ratio core:shell while the absolute value increases at low

pH. This can be explained by the higher number of positively charged groups in the particle

shell.

The size of the particles was also measured varying the solution temperature. As for the core-

shell microgels discussed above, no significant changes in the VPTT can be detected. The

graphs are shown in the appendix.

93

7.4. Summary

This chapter discussed the preparation and analysis of ampholyte core-shell microgels. N-

vinylcaprolactam (VCL) and N-ispropylacrylamide (NIPAm) were used as main monomers in

either the core or the shell. Itaconic acid (IA) and 1-vinylimidazole (Vim) were used as

comonomers. Particles were prepared in a two-step synthesis to ensure a strict spatial

separation between the core and the shell. The core-shell structure was visualized with TEM

after selectively staining of the carboxylic groups with U(Ac)3. The successful incorporation

of desired amounts of IA and Vim were confirmed with FTIR spectroscopy. The pH-

sensitivity of the cores was analyzed with regard to the particles’ size and their electrophoretic

mobility. Particles containing IA swell at high pH due to repulsion of negatively charged

groups. Particles containing Vim, in contrast, swell at low pH due to the repulsion of

positively charged groups. Core-shell particles, on the other hand, do not show a V-shaped

curve for the particle size as expected for ampholyte microgels. It was shown that particles

swell greatly at low pH, while the particle size either decreases continuously with increasing

pH (for particles containing IA in the shell) or is constant in size above a pH ~ 7. Also the

degree of swelling greatly differs from particles containing a statistical distribution of

ionizable groups. It could be observed that a neutral shell hinders the complete swelling of the

core. Temperature-dependent size measurements showed that particles have a broadened

transition while particles are larger in size compared to neutral PVCL microgels due to the

repulsion of like charges. An increase of the shell thickness led to an improved degree of

swelling at high pH. Finally, the thermal decomposition of core-shell particles was studied

with TGA. A two-step decomposition was detected caused by different decomposition

temperatures of PVCL and PNIPAm.

94

7.5. Literature

[1] W. Richtering, A. Pich, Soft Matter 2012, 8, 11423.

[2] J. J. Crassous, M. Ballauff, M. Drechsler, U. Schmidt, Y. Talmon, Langmuir 2006, 22,

2403–2406.

[3] D. Gan, L. A. Lyon, Prog. Colloid Polym. Sci. 2006, 133, 1–8.

[4] M. Ballauff, Y. Lu, Polymer (Guildf). 2007, 48, 1815–1823.

[5] I. Berndt, J. S. Pedersen, P. Lindner, W. Richtering, Langmuir 2006, 22, 459–468.

[6] P. Marion, Macromolecules 1997, 30, 123.

[7] Z. Zhou, J. V. Hollingsworth, S. Hong, G. Wei, Y. Shi, X. Lu, H. Cheng, C. C. Han,

Soft Matter 2014, 10, 6286-6293.


[9] C. Jones, L. Lyon, Macromolecules 2000, 33, 8301–8306.

[10] Y. Lu, S. Proch, M. Schrinner, M. Drechsler, R. Kempe, M. Ballauff, J. Mater. Chem.

2009, 19, 3955.

[11] N. Dingenouts, C. Norhausen, M. Ballauff, Macromolecules 1998, 31, 8912–8917.

[12] M. Shibayama, Macromol. Chem. Phys. 1998, 199, 1–30.

[13] K. Kratz, T. Hellweg, W. Eimer, Polymer (Guildf). 2001, 42, 6631–6639.



[15] G. Li, P. D. Pandya, S. S. Seo, Int. J. Polym. Anal. Charact. 2009, 14, 351–363.

[16] A. Burmistrova, M. Richter, M. Eisele, C. Üzüm, R. von Klitzing, Polymers (Basel).

2011, 3, 1575–1590.

[17] K. E. Christodoulakis, M. Vamvakaki, Langmuir 2010, 26, 639–47.

[18] R. Saito, K. Ishizu, T. Fukutomi, J. Appl. Polym. Sci. 1991, 43, 1103–1109.

[19] T. Hu, Y. You, C. Pan, C. Wu, J. Phys. Chem. B 2002, 106, 6659–6662.

[20] A. Balaceanu, Y. Verkh, D. E. Demco, M. Möller, A. Pich, Macromolecules 2013, 46,

4882–4891.

[21] L. Hou, P. Wu, Soft Matter 2015, 11, 2771–2781.


[23] M. Bradley, B. Vincent, G. Burnett, Langmuir 2007, 23, 9237–9241.

[24] M. Semmler, E. K. Mann, J. Ricka, M. Borkovec, Langmuir 1998, 14, 5127–5132.


95

8 Janus-like Microgels

8.1. Introduction

Janus is the name of the old Roman god of end and beginning, of entrance and exit, of old and

new. Not only is the month “January” named after him (January being the month linking the

old and the new year), but due to his two faces (that he used to look in both directions

simultaneously) his name is used in modern-day science to term things or phenomena that

have two “faces” or two aspects. The term is mostly used in chemistry. Here, Janus particles

are colloids with clearly distinguished hemispheres with different chemical or physical

properties such as form [1], material, chemical functionalization, or polarity [2].

Janus particles exhibit many different shapes and forms (see Figure 8.1).

Figure 8.1 Different forms of Janus particles: a Bicompartmental; b Dumbbell; c Half-

raspberry; d Acorn; e Snowman.

In nature, dissymmetry in a molecule offers specific properties. The best examples are

proteins that have an asymmetric distribution of ionizable groups and thus a large dipole

moment. For Janus particles, the dissymmetry offers an additional level of freedom: the

particles can distinguish between left and right, between up and down [1]. This behavior can be

utilized for arranging particles or for the self-assembly of complex structures. Tuning the

physical or chemical properties of each hemisphere, new materials with dual characteristics

are possible. Simulations predict that these highly complex structures are different from

traditional materials [3]. While in “old” materials, atoms and molecules are the building

blocks, anisotropic particles open the pathway to novel supermolecules with exactly the

desired features.

Different synthesis routes have developed addressing the desired properties of the respective

particles. The most common three approaches are discussed in the following.

Immobilization on a substrate. In this process, the particles are deposited on a surface and are

immobilized. Then, “functionalization” is induced from one side leading to an altered upper

hemisphere of the particles. The release of the particles from the surface results in Janus

particles. M. Bradley and J. Rowe used a similar technique to prepare Janus microgels [4].

Instead of a flat surface, they used negatively charged poly(2-vinylpyridine-co-styrene) latex

particles as a surface to mask one side of the positively charged poly(2-vinylpyridine)

microgels that aggregated onto the much larger latex particles. The exposed side of the

96

microgels was functionalized with PNIPAm. They obtained pH- and temperature-sensitive

microgel particles after the dissolution of the latex particles by a reduction of the pH.

Modification of colloids at liquid-liquid interfaces. The functionalization of the particles takes

place at the liquid-liquid interface. H. Kawaguchi et al. who were the first to prepare Janus-

like microgels. PNIPAm microgels were copolymerized with acrylic acid (AAc) and amino

groups were introduced via a carbodiimide coupling reaction on the side of the particles in

contact with water.[5]. To prevent the microgels from “wobbling” at the interface, Y. Umeda

et al. stabilized their PNIPAm-co-AAc microgels at the interface by cooling down the

emulsion to 4 °C [6].

Figure 8.2 Different swelling behavior of a microgel particle in different solvents.

However, the synthesis of Janus microgels via emulsion polymerization entails certain

problems, the main one being that microgels swell differently in different solvents, thus

leading to an inhomogeneous surface functionalization (see Figure 8.2).

Microfluidics The relatively new method of microfluidics mixes two non-miscible phases

with a photoinitiator to form droplets due to surface tension effects in a co-flowing aqueous

phase at the Y-junction of a microchannel. A UV source ensures the polymerization of the

desired particle morphology. Shape, size, and functionality are determined by microfluidic

set-up, i.e. the geometry and the flowing rate of the media. This approach, however, has two

main drawbacks: firstly, it only allows producing a restricted amount of particles at a time,

secondly, usually only spherical shapes or deformed spheres can be realized [7]. Microgels in

micrometer size using microfluidics were prepared by S. Seiffert et al [8]. They mixed pre-

modified and unmodified precursor PNIPAm polymers and crosslinked them via exposure to

UV light. Functionalization with red and green fluorescence labels enabled the verification of

the Janus-like structure with confocal scanning laser fluorescence microscopy.

Further, less frequent used techniques are self-assembly [9][10], phase separation [11][12], and

controlled surface modification [13][14].

Janus particles find applications in microfluidics, stabilization of emulsions, drug delivery,

electronics, and catalysis [2].

In this chapter, ampholyte Janus particles based on N-isopropylacrylamide (NIPAm) were

synthesized using a novel technique. Based on phase separation of the monomers in the first

moments after initiation, the precursor particles or microgel nuclei are mixed at the moment

97

when two different kinds of precursor particles are not able to completely mix with each other

(see Figure 8.3).

Figure 8.3 Synthesis procedure for Janus particles. In Reaction 1 microgel A reacts with

functional group A’, in reaction 2 microgel B reacts with functional group B’. tmix is the time

at which the mixing of dispersions lead to the formation of Janus particles (b). (a) The

dispersion are mixed before tmix leading to one particle with homogenous functional group

distribution. (c) The dispersions are mixed after tmix leading to separate particles A and B

bearing separate functional groups A’ and B’.

a b c

98


Calorimetric measurements were performed in a reaction calorimeter RCle from Mettler

Toledo with a 500 mL 3-wall AP01-0.5-RTCal reactor equipped with Hastelloy® stirrer, a

baffle, and a TurbidoTM turbidity probe from Solvias. The measurements were done in

isothermal mode, in which the desired reaction temperature (Tr) is set at a constant value and

the jacket temperature (Tj) changes automatically to maintain Tr at the desired value. The

amounts of monomer used can be seen in Table 8.1. 300 mL of water was heated up to 70 °C

under nitrogen atmosphere and constant stirring at 200 rpm. After 30 min, the respective

amounts of monomers were added. Simultaneously, a probe recorded the heat generated. If a

stable base line in the produced heat was reached (minimum waiting time was 30 min), the

initiator AMPA was added. The production of heat and the turbidity were recorded in-situ

during the reaction using the iControl RCLeTM 5.0 software. The reaction was allowed to

continue for 3 h and subsequently cooled down to room temperature. For the determination of

the net reaction time the end of the reaction was defined to be when the heat flow was back to

a value close to zero and constant. The fractional conversion can be obtained integrating the

reaction heat curve.

Size kinetics were measured in a 100 mL reactor equipped with a Nano-Flex probe (Particle

Matrix company, Germany) measuring in-situ dynamic light scattering. The aqueous

monomer solution was heated up to 70 °C and degassed under vigorous stirring to avoid

bubbles on the probe surface. After equilibrating under nitrogen atmosphere at 200 rpm for

30 min, the initiator was added. The stirring was stopped after 5 s to ensure accurate DLS

measurements in 33 s intervals. The reaction was allowed to continue for 99 measurements.

The data was analyzed using the Microtrac Felx (v. 11.0.0.4) software providing the

hydrodynamic radius and the polydispersity index.

For the synthesis of Janus particles, two separate reactions were carried out in two different

reactors (see Table 8.1).

Synthesis 1. NIPAm, Vim, and BIS were added in 100 mL H2O. After heating up to 70 °C, the

solution was stirred at 200 rpm under nitrogen atmosphere for 1 h.

Synthesis 2. NIPAm, IA, and BIS were added in 100 mL H2O. After heating up to 70 °C, the

solution was stirred at 200 rpm under nitrogen atmosphere for 1 h.

99

Table 8.1 Amounts of monomers used for the reaction.

Chemical Synthesis 1 Synthesis 2

Amount / g

NIPAm 1.003 1.004

Vim 0.158 -

IA - 0.293

BIS 0.049 0.049

AMPA 0.038 0.037

In both reactors, the initiator AMPA was added simultaneously. 0.9 mL of solution was

removed from each solution after 1 min, 2 min, 3 min, 4 min, 5 min, 6 min, 8 min, 10 min,

12 min, 14 min, 16 min, and 18 min. The two solutions were immediately combined in one

flask and allowed to stir for 3 h at 70 °C at 200 rpm.

To visualize the success of the reaction, transmission electron microscopy (TEM) was

performed at a Zeiss Libra TM 120 (Carl Zeiss, Oberkochen, Germany). 20 µL of each

sample was stained with 5 µL of uranyl acetate U(Ac)3 and one drop was put on a carbon

coated copper grid. Samples were then dried overnight.

100


Janus-like particles based on PNIPAm with itaconic acid (IA) and 1-vinylimidazole (Vim) as

comonomers are discussed in this chapter. An anisotropic distribution of acidic and basic

moieties is aimed at. In contrast to core-shell particles, where one kind of ionizable group is

located in the core and the other in the center of the microgel, Janus-like particles have their

ionizable groups on opposite “sites”.

The synthesis of Janus-like microgels is based on phase separation phase separation of the

precursor particles. The approach via substrate immobilization and emulsion polymerization

lead to particles with only surface-modified functionalization. The aim in this work, however,

is the synthesis of ampholyte microgels with ionizable groups located throughout the whole

microgel.

First preliminary experiments with either VCL or NIPAm as main monomer and the

subsequent analysis with TEM showed that the synthesis process works better for NIPAm

microgels (TEM images for NIPAm will be discussed later in this chapter). Therefore, the

following experiments were solely performed for NIPAm microgels.

Before the synthesis of the actual Janus-like particles, experiments to understand the

polymerization kinetics and particle formation were performed. Therefore, separate reactions

for NIPAM-IA and NIPAm-Vim as well as pure NIPAm microgels were carried out in a

calorimeter and the change of heat flow and particle size were measured starting with the

addition of the initiator (start of the reaction). Syntheses were performed at different pH

according to the type of comonomer (pH 3 for Vim and pH 8 for IA).

First, the change in reaction heat was measured. It was shown that it was not possible to

copolymerize NIPAm with IA in the calorimeter, contrary to the reactions that were

successfully carried out in the double-walled glass reactors. Instead of a stable microgel

dispersion, a sticky hydrogel-like material was obtained. The reason for this is unknown,

though a reaction between itaconic acid and the metal probes is likely. Both the curves for

pure PNIPAm and PNIPAm-co-Vim microgel look similar, though the reaction time is

slightly longer for PNIPAm-co-Vim microgels. Both reactions are finished after ~ 30 min.

It was, however, possible to measure in-situ time-dependent DLS for all three samples. Figure

8.4 shows that the reaction for pure PNIPAm microgels is the fastest and that particles have

reached a hydrodynamic radius of ~ 140 nm after already 4 min. PNIPAm-co-Vim microgels

need a slightly longer reaction time of ~ 8 min at which point they have obtained a

hydrodynamic radius of ~ 250 nm. At first glance, these results are contrary to the results

101

Figure 8.4 (a) Calorimetric measurements for pure PNIPAm microgels (black) and PNIPAm-

co-Vim microgels (red). The numbers give the reaction heat that was created. (b) In-situ time-

dependent DLS measurements for pure PNIPAm (black), PNIPAm-co-IA (blue), and

PNIPAm-co-Vim (red) microgels.

obtained with calorimetry which showed that both reactions continue until ~ 30 min. This

shows, however, that the particles obtain their final size after a very short time and that further

addition of monomers (generating more heat) does not contribute to an increase in particle

size. Microgels containing PNIPAm and IA need about 40 min to reach a hydrodynamic

radius of ~ 500 nm.

The synthesis of Janus particles was realized by mixing PNIPAm-co-IA and PNIPAm-co-Vim

microgel dispersions after a certain amount of time. The results in Figure 8.4 suggest that the

point of mixing time is a crucial factor that influences the form and properties of the resulting

microgel particles. Both reactions (1. NIPAm + IA; 2. NIPAm + Vim) were done

simultaneously in two different reactors. After the addition of the initiator, samples were

taken from both reactions and mixed immediately at various reaction times. The reactions

were allowed to continue for 4 h in a shaker at 200 rpm and 70 °C. Depending on the time of

mixture, colorless (early mixing time) or milky (late mixing time) dispersions were obtained.

Afterwards, samples were stained with U(Ac)3 and analyzed with transmission electron

microscopy (TEM) to confirm the success of the formation of Janus particles. U(Ac)3 binds to

the carboxylic groups of the itaconic acid and gives a clear distinction between parts with

itaconic acid and parts with 1-vinylimidazole.

The reaction times before mixing were changed:

a. NIPAm-co-IA and NIPAm-co-Vim were initiated simultaneously. 1 mL of

each sample was taken after 1, 2, 3, 4, and 5 min from each reactor and mixed

immediately.

a b

102

b. NIPAm-co-Vim was initiated 2.5 min after the initiation of NIPAm-co-IA.

1 mL of each sample was taken after 0.5, 1, 2, 3, and 4 after the initiation of

NIPAm-co-Vim min from each reactor and mixed immediately.

c. NIPAm-co-Vim was initiated 5 min after the initiation of NIPAm-co-IA. 1 mL

of each sample was taken after 0.5, 1, 2, 3, and 4 min after the initiation of

NIPAm-co-Vim from each reactor and mixed immediately.

Figure 8.5 shows TEM images for a batch of samples that were started simultaneously

through the addition of the initiator AMPA (case a). PNIPAm-co-Vim and PNIPAm-co-IA

solution were mixed after 1, 2, 3, 4, and 5 min. TEM images reveal that different mixing

times lead to the formation of microgels with different architectures. When mixed after 1 min,

particles with a homogenous distribution of both IA and Vim are obtained. Mixing after 2 min

leads to particles with one side clearly darker than the other. This suggests that the stained

carboxylic group of IA is located only on one side of the microgel leading to the formation of

Janus-like particles. Later mixing times lead to the formation of two separate particles that

exist side by side. Large aggregates are visible for PNIPAm-IA microgels (see Figure 8.5f).

Figure 8.5 TEM images of PNIPAm-co-IA-co-Vim microgels obtained following route a.

PNIPAm-co-IA and PNIPAm-co-Vim solutions were mixed after (a) 1 min; (b) 2 min; (c) 2

min – zoom-in; (d) 3 min; (e) 4 min; (f) 5 min.

The synthesis was repeated with different initiation times of the two reactions. As shown in

Figure 8.4b, the reaction NIPAm-co-IA is slower than the reaction NIPAm-co-Vim.

Therefore, the reaction NIPAm-co-Vim was started 2.5 min after the initiation of NIPAm-co-

IA (case b). Samples were mixed after 0.5, 1, 2, 3, and 4 min of the initiation of NIPAm-co-

Vim.

a b

d e f

c

103

Figure 8.6 (a+b) TEM images of PNIPAm-co-IA-co-Vim microgels obtained following route

b. Mixing time was 0.5 min. (c) NIPAm-co-Vim microgels; (d) NIPAm-co-IA microgels as

reference.

TEM images reveal that only the mixture of 0.5 min produced Janus-like particles (see Figure

8.6). TEM images for different mixing times are not shown as they resemble images shown in

Figure 8.5 and are of no further interest.

The synthesis following route c (NIPAm-co-Vim initiated 5 min after initiation of NIPAm-co-

IA) did not result in the formation of Janus-like particles independent of the mixing time.

(It was also tried to vary the amount of volume of NIPAm-co-Vim and NIPAm-co-IA,

respectively. Therefore, separate solutions were mixed after simultaneous initiation, but

mixed in different ratios, i.e. NIPAm-co-IA:NIPAm-co-Vim = 2:1 and 1:2. However, in

neither case TEM images revealed the formation of Janus-like particles.)

Particles obtained via the synthesis route b were further characterized with temperature-

dependent DLS to obtain the particle’s hydrodynamic radius (see Figure 8.7a). It can be seen

that the VPTT is ~ 29 °C. In comparison to ampholyte microgels with statistical or core-shell

distribution of ionizable groups, the VPTT is only slightly lower.

Furthermore, the particles’ pH sensitivity was examined. Figure 8.7b shows that particles are

swollen at high (~ 550 nm) and low (~ 650 nm) pH, while being in a collapsed state at pH 5

(430 nm). This and the fact that the absolute value of the electrophoretic mobility is higher at

pH 3 than at pH 10, indicates that slightly more Vim is incorporated than IA.

a b

c d

104

Figure 8.7 (a) RH with varying temperature at pH 6; (b) Electrophoretic mobility EM and RH

with varying pH at 25 °C. Janus-like particles were obtained via synthesis route b.

In the following, the pH-sensitivity of the Janus-like microgels is compared to the pH-

sensitivity of particles with statistical and core-shell distribution of ionizable groups (see

chapters 6 and 7). Figure 8.8 therefore shows the electrophoretic mobility and the

hydrodynamic radius as a function of pH for microgels with various distributions of ionizable

groups.

Figure 8.8 RH and EM as a function of pH for microgels with various distribution if ionizable

groups: Janus-like (black), core-shell (red), and statistical (blue). All samples have a monomer

ratio of VCL:Vim: IA = 80:10:10. Measurements were conducted at T = 25 °C. The

horizontal line (EM = 0 µmcm/Vs) helps to identify the isoelectric point.

All samples have a similar isoelectric point (IEP) of pH ~ 5 - 6. At lower pH, the microgels

are positively charged, while they are negatively charged at higher pH. A difference can be

seen between Janus-like and statistical on the one side and core-shell particles on the other

side. The latter has a more negative EM than the other two because here itaconic acid is

exclusively located in the shell.

a b

105

Furthermore, the hydrodynamic radius RH is within the same size range, i.e. between 100 and

300 nm. Differences can be seen in the swelling behavior. Samples with a statistical

distribution of ionizable groups show a V-shaped behavior, i.e. they are swollen at low and

high pH, while the particles collapse at the IEP. Since the sample has a symmetric ratio

between Vim and IA, the particles swell in the same extent at low and high pH. Core-shell

microgels, on the other hand, swell only at low pH, while a constant decrease in size can be

observed with increasing pH. The reasons are explained in chapter 7. The pH-dependency of

Janus-like microgels is V-shaped for a pH range of 3 – 6.5, while RH remains constant at pH >

6.5. This comparison shows that the distribution of ionizable groups within the microgel

particles is of great importance and highly influences the pH behavior.

Similarly, the colloidal stability of all three samples was studied and compared.

Measurements were conducted at pH 8 because the greatest differences between the above

discussed samples is expected to be seen at high pH for the following reason: All samples are

negatively charged at high pH. The core-shell sample has itaconic acid groups located

exclusively in the shell, Janus-like particles exclusively on one “side”, while particles with a

statistical distribution have itaconic acid located throughout the polymer network. Colloidal

stability obtained via electrostatic interactions is therefore likely to differ considerably. These

assumptions are confirmed in Figure 8.9. Core-shell microgels have the lowest sedimentation

velocity, i.e. they have the highest colloidal stability, while microgels with a statistical

distribution of ionizable groups have the highest sedimentation velocity. Note, however, that

overall, the sedimentation velocities are relatively low for all samples regardless of their

architecture indicating their good colloidal stability.

Figure 8.9 Sedimentation velocities for microgels with various distribution of ionizable

groups. Samples have a monomer ratio of VCL:Vim: IA = 80:10:10.

106

8.4. Summary

This chapter showed that it is possible to synthesize Janus-like particles by mixing two

separate microgels solutions shortly after initiation of each solution. For this, PNIPAm-co-IA

and PNIPAm-co-Vim microgels were synthesized in different reactors, initiated by the

addition of AMPA and subsequently mixed at a certain mixing time. TEM images confirmed

that Janus-like particles were formed at the right mixing time. The formation was possible if

both reactions were initiated simultaneously or shortly after each other. For simultaneous

initiation, Janus-like particles were obtained after 2 min after the addition of AMPA. In a

second experiment, PNIPAm-co-IA was initiated 2.5 min before PNIPAm-co-IA. Mixing

after 3 min after the initiation of PNIPAm-co-IA also led to the formation of Janus-particles.

Since it was shown that this synthesis procedure leads to a spatial separation of acidic and

basic moieties, further studies are needed so that various mixing times lead to variable ratios

between both functional groups resulting in structures such as bicompartmental, dumbbell or

snowman.

8.5. Literature

[1] A. Perro, S. Reculusa, S. Ravaine, E. Bourgeat-Lami, E. Duguet, J. Mater. Chem.

2005, 15, 3745.

[2] A. Walther, A. H. E. Müller, Soft Matter 2008, 4, 663.

[3] S. C. Glotzer, Science 2004, 306, 419–20.

[4] M. Bradley, J. Rowe, Soft Matter 2009, 5, 3114-3119.

[5] D. Suzuki, S. Tsuji, H. Kawaguchi, J. Am. Chem. Soc. 2007, 129, 8088–9.

[6] Y. Umeda, T. Kobayashi, T. Hirai, D. Suzuki, Colloid Polym. Sci. 2010, 289, 729–737.

[7] D. Dendukuri, D. C. Pregibon, J. Collins, T. A. Hatton, P. S. Doyle, Nat. Mater. 2006,

5, 365–9.

[8] S. Seiffert, M. B. Romanowsky, D. A. Weitz, Langmuir 2010, 26, 14842-14847.

[9] Y. Yin, Y. Lu, Y. Xia, J. Am. Chem. Soc. 2001, 123, 771–772.

[10] Y. Yin, Y. Lu, B. Gates, Y. Xia, J. Am. Chem. Soc. 2001, 123, 8718–8729.

[11] P. Mulvaney, M. Giersig, T. Ung, L. M. Liz-Marzán, Adv. Mater. 1997, 9, 570–575.

[12] H. Gu, R. Zheng, X. Zhang, B. Xu, J. Am. Chem. Soc. 2004, 126, 5664–5.

[13] S. Reculusa, C. Poncet-Legrand, S. Ravaine, C. Mingotaud, E. Duguet, E. Bourgeat-

Lami, Chem. Mater. 2002, 14, 2354–2359.

[14] H. Yu, M. Chen, P. M. Rice, S. X. Wang, R. L. White, S. Sun, Nano Lett. 2005, 5,

379–82.

107

9 Interactions with Proteins

9.1 Introduction

The low solubility of many drugs in water is a major problem modern, intelligent polymer

systems should address when utilized as drug carriers. Microgels can be employed due to the

great potential of precisely designing their properties. Functionalization allows tuning the

interactions between microgel and drug that determines the uptake and release of the latter.

Microgels can be tailored to possess crucial characteristics an intelligent drug carriers must

fulfill: hydrophilicity/ hydrophobicity, sensitivity to environmental changes, biocompatibility,

degradability, and specific size. Using microgels not only offers the possibility of using a

smart polymer network for controlled delivery, they also protect proteins against

environmental hazards such as drastic pH or changes in redox potential by providing a

hydrophilic environment that preserves the protein’s activity [1][2]. Additionally, microgels are

capable to store large amounts of proteins in a relatively small volume [3].

The interactions between microgels and proteins are complex. Proteins can undergo

conformational changes, while microgels can swell or collapse upon the sorption of proteins.

The uptake/ release of proteins can be controlled by the following: (a) diffusion; (b) osmotic

pressure; (c) degradability of the polymer; (d) responsiveness of the polymer to environmental

changes such as pH or enzymes [1]. Covalent binding between microgel and protein is avoided

because covalent attachment may change the drug’s properties such as activity.

Diffusion The driving force behind diffusion of protein molecules into oppositely charged

microgels is the gain in free energy. Initially, after mixing of protein and microgel, the strong

binding causes a strong increase in protein concentration at the interface between microgel

and surrounding solution. After time, with proceeding diffusion of protein into the microgel,

the concentration becomes more homogenous throughout the particle.

Swelling/ Osmotic pressure The uptake of protein by a charged microgel causes a release of

counterions. This lowers the osmotic swelling of the microgel’s osmotic swelling pressure. A

collapse of the charged microgel network can induce an increase in charge density, thus

promoting further protein uptake.

Degradability of microgel The use of a cleavable crosslinker enables the degradation of the

microgel with the help of e.g. enzymes or acids [4]. Ideally, the particle consists of a shell that

is impermeable to the protein. Degradation of the microgel particle leads to the release of the

protein.

108

Responsiveness of microgel pH-sensitive microgels can bind proteins electrostatically. The

increase of ionic strength leads to a screening of the electrostatic attractions between protein

and the oppositely charged groups in the microgel, resulting in a release of the protein from

the polymer network. Temperature-sensitive microgels can either release proteins upon

heating by “squeezing” them out or contrary retain the protein through entrapment or

increased charge density [5].

Usually, a combination of all factors affects the degree of uptake and release of proteins to

and from microgels.

V. A Kabanov et al. prepared poly(acrylic acid) (pAA) and N,N-dimethyl-N-ethylaminoethyl

methacrylate bromide) hydrogels and studied the uptake of different proteins [6]. They

described that the uptake of protein takes place primarily in the microgel shell. This leads to a

collapse of the shell while the particle core is still highly swollen. Eventually, the protein-

microgel complex propagates inward leading to further de-swelling of the microgel interior

until the complete polymer networks collapses. This was confirmed by C. Johansson et al.

who further investigated the diffusion of lysozyme into pAA microgels [3]. Microgels were 50

– 150 µm in size which enables visualization with confocal microscopy. They could see that a

shell of lysozyme initially forms rapidly preventing additional uptake of lysozyme. After ~

180 s, however, lysozyme diffuses deeper into the microgel core while no further de-swelling

of the polymer network can be observed. They state that lysozyme molecules diffusing to the

core do not originate from the lysozyme adsorbed in the shell, but from “new” lysozyme

molecules that diffuse through the already existing shell.

H. Bysell and M. Malmsten looked closer at the interaction between lysine and pAA

microgels as a function of ionic strength, pH, and protein concentration [7]. They state that the

de-swelling of the microgel network as described by V. A. Kabanov is greatly depending on

the mesh size of the polymer network and the protein size. A protein that is larger than the

mesh size is exclusively located in the microgel shell, while smaller proteins can penetrate

into the microgel core. They confirm that the formation of a protein-microgel complex in the

shell lead to the collapse of the shell. This happens faster than the protein diffusion, thereby

preventing further uptake into the microgel core. The mesh size in turn is dependent on the

crosslinking density within a microgel particle. The relation between crosslinking density and

protein uptake was investigated by G. E. Eichenbaum et al [8]. They determined the pore size

of the poly(methacrylic acid-co-acrylic acid) microgel via protein size exclusion. They found

out that the loading of proteins is highly dependent on the crosslinking density.

109

A both pH- and temperature-dependent uptake of proteins to microgels was studied by C. S.

O. Silva et al [9]. They used human immunoglobin G (IgG) and cationic PNIPAm microgels

that were functionalized with amine groups. They found out that the high degree of swelling

of the microgel network at low temperatures reduced the uptake of IgG, while ~ 90 % of the

initial IgG concentration could be adsorbed at temperatures above the VPTT. The authors

explained this by the higher charge density in the microgel at higher temperatures inducing

favorable electrostatic interactions.

The first step in testing a polymer’s ability as drug carrier is performing studies using a model

drug that is well described in literature. Many authors use cytochrome c (cyt-c) as a model

protein to study the interactions between proteins and microgels. Cyt-c is found e.g. in the

human body. It is for instance the main initiator of programmed cell death [10]. It has 104

amino acids and a heme group with the Fe(III) being six-coordinated (see Figure 9.1). It is

surrounded by four alpha helices and is covalently bound by two cysteine side chains [11]. The

properties of cyt-c are given in Table 9.1. It is expected that microgels and proteins interact

over electrostatic interactions between the negatively charged carboxylic groups in the

microgel network and the positively charged groups on the outside surface of cyt-c.

Figure 9.1 (a) Structure of cytochrome c; (b) Electrostatic surface properties of cytochrome c,

color-coded by electrostatic potential, blue: positive, red: negative. Images were generated

using the program Discoverstudio.

Table 9.1 Properties of cytochrome c [11][12][13].

Molecular weight 12.3 kDa

Isoelectric point pH 10.2

Radius of gyration 12.8 nm (native state)

Overall net charge at neutral pH +6.9 (at pH 10.2)

a b

110

9.2 Experimental Part

Specifications regarding DLS, SLS, and UV-Vis measurements can be found in chapter 4

(experimental part).

UV-Vis measurements were conducted in order to analyze the uptake and release kinetics of

cytochrome c. 0.005 g of microgel were dissolved in 3 mL of water at three different pH

values: 3, IEP (i.e. ~ 6), and 8. For each sample, the theoretical amount of carboxylic groups

was calculated and cytochrome c in a ratio of 1:1 was added assuming that each carboxylic

group is able to bind one protein molecule. For instance, for the microgel with statistical

distribution of ionizable groups and a monomer ratio VCL:Vim:IA = 60:20:20, 0.005 g

contain 7.26 * 10-6 mM of carboxylic groups. Therefore, 7.26 * 10-6 mM (0.089 g) of

cytochrome c was added.

For kinetic release studies, the respective amounts of microgel and protein in water at three

different pH values were allowed to stir overnight to reach equilibrium. After several hours of

stirring, UV-Vis measurements at 25 °C from 400 to 900 nm were carried out.

Afterwards, the samples were dialyzed in a composite regenerated cellulose membrane from

Millipore (NMWCO 30,000) in water with the respective pH. UV-Vis measurements were

repeated after specific times and the dialysis resumed (note: the dialysis water was not

changed during the dialysis process). UV-Vis measurements of the dialysis water were also

conducted under the same conditions to analyze the amount of cytochrome c released from

the microgel.

For controlled release studies, microgel samples and cytochrome c were dissolved in water of

pH = 8 with the same amounts explained above. After stirring overnight, UV-Vis

measurements was carried out. For dialysis, several parameters were changed:

(1) The pH of the dialysis water was changed from pH 8 to pH 3.

(2) The temperature was changed from T = 25 °C to T = 37 °C (i.e. above VPTT).

(3) NaCl was added.

Again, the samples were measured with UV-Vis after specific times.

To study the effect of ionic salt on the particle size and molecular weight, DLS and SLS

measurements were performed. Microgel and protein were mixed in buffer with pH 8 of the

according NaCl concentration (0, 30, 50, 75, 100, and 125 mM). The solution was stirred for

2 d to allow the sample to reach equilibrium. The samples were centrifuged to remove

unbound protein from the solution. For SLS measurements, the samples were lyophilized to

determine the exact concentration and re-dispersed in buffer with the according salt

concentration. SLS measurements were performed at 25 °C.

111

9.3 Results and Discussion

In this chapter, the interactions between microgels with statistical distribution of ionizable

groups as well as core-shell structure and the protein cytochrome c (cyt-c) are looked at.

Cyt-c is used for this study because it is positively charged over a broad pH range (see Figure

9.3). The microgels that are used, however, are positively charged at pH 3, neutral at pH ~ 6,

and negatively charged at pH = 8. Thus, interactions between microgels and protein can be

studied at these three different pH values. Furthermore, the red color of cyt-c makes it suitable

for UV-Vis measurements to analyze the binding kinetics between protein and microgels.

First, however, the conformational structure of cytochrome c at pH 3 and pH 8 is measured

via circular dichroism (CD) spectroscopy (see Figure 9.2).

Amount of conformation / %

pH 3 pH 8

α-helix 41.3 48.5

β-sheet 13.6 4.6

turn 20.1 26.3

random 25.1 20.6

Figure 9.2 CD spectra of cytochrome c in PBS buffer with different pH and structural

analysis using Yang’s reference.

Both spectra have two negative minima at around 222 and 208 nm and a positive maximum at

around 194 nm. The double minimum is typical for a helical structure [14][15]. A small

shoulder at around 217 nm can be attributed to pleated β-sheet conformation. The structures

were analyzed using Yang’s reference structural analysis (see Figure 9.2).

Yang’s reference predicts that the globular domain can support a mixture of all conformations

at both pH, though α-helix and random coil conformation are dominant. The amount of β-

sheets is decreased slightly at pH 8 compared to pH 3. The positions of the peaks do not vary

when the pH of the solution is changed indicating that cyt-c does not change its conformation

dramatically.

Figure 9.3 shows that cytochrome c is positively charged over a broad pH range. Its

isoelectric point (IEP) is above pH 8. Figure 9.3 gives also the electrophoretic mobility of the

ampholyte PVCL-co-Vim-co-IA microgel with a statistical distribution of ionizable groups

112

and a monomer ratio of 60:20:20. It is positively charged at low pH, while being negatively

charged at high pH. The IEP is at ~ 6.2.

Figure 9.3 Electrophoretic mobility of cytochrome c (black curve) and microgel with

statistical distribution of ionizable groups as an example (monomer ratio VCL:Vim:IA =

60:20:20, red curve). The measurements were conducted at T = 25 °C.

Next, UV-Vis spectra of pure cyt-c were measured at pH 3, 6, and 8 (spectrum shown in the

appendix). Cyt-c exhibits two prominent peaks at 402 and 416 nm (γ-band). These peaks are,

however, prone to change from one to two peaks depending on the oxidation state of iron: the

peak at 402 nm belongs to Fe(III), while the peak at 416 nm can be assigned to Fe(II) [16].

Therefore, the smaller peak at 530 nm (α-band) was used as reference peak. Pure microgel

samples show no peaks in this range (for spectrum see appendix).

Microgel samples with statistical distribution of ionizable groups and a monomer ratio of

VCL:Vim:IA = 60:20:20 were mixed with cyt-c in buffer of pH 3, 6 or 8 at room temperature.

After incubating for 24 h, the samples were dialyzed against buffer of the respective pH.

Depending on the pH, the protein was either bound by the microgel and could thus not be

washed out the dialysis bag or not bound by the microgel and could thus be washed out the

dialysis bag. The release of cyt-c was studied with UV-Vis spectroscopy after various time

intervals. Samples were taken both inside and outside the dialysis bag.

Figure 9.4 shows the change of the UV-Vis spectra over time. The absorbance of the peak at

530 nm before the dialysis is the same for all samples indicating that the concentration in all

samples is the same. Already after 30 min of dialysis, the absorbance for pH 3 is decreased

clearly, while at the same time, the same peak increases in the dialysis water. This indicates

that cytochrome c is not bound within the microgel particle and is able to penetrate through

the dialysis membrane. After 360 min of dialysis, no cyt-c is left in both the samples at pH 3

and pH 6.2. This is also confirmed optically: only the solution at pH 8 maintains its red color.

113

Figure 9.4 UV-Vis measurements of microgel sample with statistical distribution of ionizable

groups and monomer ratio VCL:Vim:IA = 60:20:20. Black curve: pH 3; red curve: pH 6.2;

blue curve: pH 8. (a) Before the start of dialysis; (b) After 30 min of dialysis; (c) After 180

min of dialysis; (d) After 360 min of dialysis. Inset graphs show UV-Vis measurements of

dialysis water. Inset images give optical confirmation of protein release.

The decrease of the peak at 530 nm is analyzed over time and shown in Figure 9.5 for the

sample with statistical distribution of ionizable groups. The graphs show the kinetics of the

dialysis process. An absorbance of 1 means that all carboxylic groups within the microgel

particle bind cyt-c (i.e. 100 %). The measurements were repeated for microgels with a

different monomer ratio as well as a core-shell structure (graphs see appendix). The amount of

cyt-c remaining in the microgel after 24 h of dialysis is analyzed with regard to microgels

with varying distributions of ionizable groups and various amounts of incorporated basic and

acidic moieties at different pH values (see Table 9.2). No binding (i.e. absorbance = 0) can be

seen at pH 3 and pH 6.2 for microgels with statistical distribution of ionizable groups as well

as for the core-shell particles with itaconic acid in the shell. The situation, however, is

different at pH 8 where all of them bind ~ 50 % of the theoretical amount of cyt-c. This

surprisingly low amount can be explained as follows: The first cyt-c molecules that bind to a

microgel particle will bind to the first carboxylic group it “meets”, i.e. on the particle surface.

c d

a b

114

Figure 9.5 Change of absorbance with dialysis time for ampholyte microgel with statistical

distribution of ionizable groups (VCL:Vim:IA = 60:20:20). Black curve: pH 3; red curve: pH

6.2; blue curve: pH 8.

As more and more protein is bound, they will eventually block the polymer network for

further cyt-c to enter. So, though more protein could be bound theoretically, the microgel

interior is probably relatively protein-free. This behavior was already seen by T. Hoare and R.

Pelton [17]. They analyzed the uptake of a cationic drug to PNIPAM microgels containing

carboxylic groups. The authors observed a local collapse of the polymer network in response

to the binding of the protein to the anionic groups. The decreased charge density reduces

repulsion forces between like charges and leads to a phase transition within the microgel.

Further diffusional uptake of protein into the microgel network is hindered or limited.

Table 9.2 Amount of cyt-c (in %) remaining after end of dialysis for microgel samples with

different distributions of ionizable groups. Measurements were conducted at T = 25 °C.

Statistical Core-Shell (IA:Vim = 10:10)

80:10:10 60:20:20 Core +

Shell -

Core -

Shell + (1:1)

Core -

Shell + (1:5)

pH 3 0 0 0 12 14

pH 6.2 0 0 0 28 40

pH 8 46 53 41 52 71

Core-shell particles with itaconic acid in the core, however, show a different behavior. Even

at pH 3, with the shell being positively charged and the core being neutral, ~ 13 % cyt-c is

bound by the microgel. At pH 6, both core and shell are neutral. 28 % and 40 % of cyt-c are

bound by core-shell particles with a ratio between core and shell of 1:1 and 1:5, respectively.

At pH 8, the sample with the thicker shell (1:5) is able to bind ~ 70 % of cyt-c, though the

neutral shell is not expected to bind cyt-c over electrostatic interactions. These latter results

show that cyt-c is bound by the microgels even in the absence of electrostatic

115

forces supporting binding (pH 6) and even so in the presence of electrostatic forces opposing

binding (pH 3). This leads to the assumption that cyt-c is bound to the microgel particle not

only over electrostatical interactions, but suggests the existence of another type of interaction,

hydrophobic interactions, between cyt-c and microgel. The idea of hydrophobic interactions

was first introduced by W. Kauzmann in 1959 [18]. He described them as the tendency of

nonpolar solutes to adhere to each other in aqueous solutions.

In order to analyze the influence of hydrophobic interactions on the binding of cyt-c, the

release kinetics between neutral core-shell microgels (i.e. containing no acid and basic

moieties) and cyt-c were studied with UV-Vis.

Figure 9.6 reveals that ~ 11 % of cyt-c remains in the microgel at pH 3 and 8 at T = 25 °C.

The measurements indicate clearly that interaction between neutral microgel and positively

charged protein takes place at both pH. Since the microgel has no charges at both pH, these

results suggest that hydrophobic, non-coulombic interactions play an important role in the

binding of cyt-c. Hydrophobic interactions between microgels and proteins were also

observed by other authors [19][20][21]. Hydrophobic interactions between cyt-c and the

ampholyte microgel take place between the hydrophobic amino acid residues of the N- and C-

terminal α-helices of cyt-c and the hydrophobic backbone of the polymer chain in the

microgel [22].

Figure 9.6 Change of absorbance with dialysis time for core-shell microgels without

ionizable groups. Black curve: pH 3; blue curve: pH 8.

The hydrophobicity of a microgel increases when the solution temperature is increased from

25 °C to 37 °C (i.e. above the VPTT) [23]. At 37 °C, microgels are in a collapsed state

(combined with a decrease in size of ~ 61 %) exhibiting more hydrophobic domains. No

specific driving force for the release of cyt-c is expected at this elevated temperature. Figure

9.6 reveals, however, that at higher temperatures significantly more cyt-c remains in the

116

microgel after 48 h. Compared to the amount of protein remaining at 25 °C, about 73 % more

protein is bound at 37 °C. This was already observed by other authors [23]. N. Shamim for

instance prepared thermosensitive Fe3O4 nanoparticles and studied the uptake of lysozyme

below and above the VPTT of the PNIPAm shell [24]. They confirmed that hydrophobic

interactions are the driving forces behind the uptake of the protein at elevated temperatures.

This strongly supports the assumption that the increase of hydrophobicity with rising

temperature increases the hydrophobic interactions and leads to more hydrogen bonds

between polymer network and protein [23]. Hydrophobic interactions that occur at high

temperatures are an entropically driven process, while electrostatic interactions at lower

temperatures at pH 8 are enthalpically driven processes [20].

As it was shown that protein uptake is possible at pH 8 due to the opposite charges of

microgel and cyt-c, the release from the microgel was studied using various triggers. This is

studied for microgels with a statistical distribution of ionizable groups (VCL:Vim:IA =

60:20:20) as well as for core-shell microgels with a thick shell (cVCL-/sNIPAm+, 1:5). All

microgels were loaded with cyt-c at pH 8 in the same way as explained above.

The first trigger studied is temperature. Due to the particles’ temperature sensitivity, they

shrink at temperatures above ~ 32 °C. It is a likely scenario that the particles “squeeze” out

the protein at higher temperatures. Therefore, the loaded microgel particles were heated up to

37 °C and dialyzed. If protein is released from the microgel, it will leave the dialysis tube.

Again, UV-Vis measurements were performed to monitor this process. Figure 9.7a shows that

most protein is staying inside the microgel and cannot be released by a change in temperature.

Even after three days of dialysis, almost 80 % of the initial protein amount remains. This

behavior is attributed to hydrophobic interactions between protein and microgel that are

pronounced at elevated temperatures as discussed above.

A different trigger is the addition of salt. Salt ions can shield the interactions between the

carboxylic groups and the protein. Figure 9.7b shows that almost 70 % of the previously

bound cyt-c is released within the first 4 hours. It indicates further that other than electrostatic

interactions between microgel and protein play a role and prevent the complete release of cyt-

c form the microgel. This non-electrostatic part is attributed to hydrophobic interactions as

already discussed above.

In a next experiment, the pH of the microgel dispersion was changed from pH 8 to pH 3

(Figure 9.7c). Carboxylic groups that are negatively charged in basic conditions, become

117

neutral at low pH. After almost 24 h, no protein remains in the microgel particle indicating

that a change in pH results in a full release of cyt-c.

Figure 9.7 Change of absorbance with dialysis time for ampholyte microgel with statistical

distribution of ionizable groups (VCL:Vim:IA = 60:20:20). (a) Change of temperature from

25 to 37 °C; (b) Addition of 0.1 M NaCl; (c) Change of pH 3 to 8; and (d) core-shell structure

cVCL-/sNIPAm+ (1:5), change of pH = 3 to 8.

These results lead to the following conclusions about the release of cyt-c by ampholyte

microgels with a statistical distribution of ionizable groups:

- Temperature is no effective trigger for the release of cyt-c from the microgel. This

indicates that the protein is mainly bound on the surface. At elevated temperatures,

hydrophobic interactions between microgel and protein increase preventing a complete

release.

- The addition of salt suppresses the interaction between cyt-c and microgel. As a

consequence, cyt-c is released from the polymer network. The release, however, is

only partial (~ 70 %) and further suggests the presence of non-coulombic,

hydrophobic interactions.

- For a complete release of protein, a change in pH from 8 to 3 proved to be most

a

c d

b

118

effective. In this case, hydrophobic interactions are overcome by electrostatic

repulsion.

The release of protein through a change in pH from core-shell particles was also studied. For

this, core-shell microgels with a negative core and a positive, thicker shell were taken (cVCL-

/sNIPAm, 1:5). The results can be seen in Figure 9.7d. A clear difference between statistical

and core-shell particles is discernible. While all protein is released from microgel with a

statistical distribution of ionizable groups, this is only about 50 % for core-shell particles.

Obviously, a different mechanism prevents the complete release of cyt-c.

At pH 8, the core is negatively charged, while the shell is neutral. Protein is bound in the core.

A change in pH to acidic conditions leads to a neutralization of the core, while the shell

becomes positively charged. The results indicate that the protein is trapped inside the particle

because the positively charged shell prevents release from the interior.

In the following, was studied how the uptake of cyt-c as well as the presence of salt influences

the particle size. The particle size was measured with increasing ionic strength (0 – 125 mM)

at pH 8. At pH 8.0, the microgel particles are negatively charged, while cyt-c is positively

charged, thus interaction between both is expected. With an increase in ionic strength,

however, the charges of both are screened and less interaction is anticipated. This behavior is

tested for four different samples, namely core-shell particles cVCL-/sNIPAm+ (1:1) and

cVCL-/sNIPAm+ (1:5) and microgels with a statistical distribution of ionizable groups and a

monomer ratio of VCL:Vim:IA = 80:10:10 and 60:20:20.

Figure 9.8 shows that unloaded microgels with statistical distribution of ionizable groups and

a monomer ratio of VCL:Vim:IA = 80:10:10 decrease in size from 230 nm at 0 mM NaCl to

200 nm at 125 mM NaCl correlating to a decrease in volume of 34 %. At pH ~ 8, the microgel

particles are negatively charged. The charges repel each other resulting in a higher particle

size. With increasing ionic strength these charges become more and more screened leading to

a contraction of the polymer network. For the sample 60:20:20, the decrease in volume is only

14 %. Loaded microgels, in contrast, increase in size with increasing ionic strength (34 vol-%

and 18 vol-% for 80:10:10 and 60:20:20, respectively, from 0 mM to 125 mM NaCl). With no

salts present, the negatively charged carboxylic groups and the positively charged cyt-c

interact. With increasing ionic strength, the electrostatic affinity between charges of the

carboxylic groups and the protein decreases. As a result, the microgel becomes more and

more negative and increases in size. For unloaded and loaded microgels, the change in

volume generated by the addition of salt is less pronounced for microgels with a higher

119

Figure 9.8 Influence of ionic strength on particle radius RH for microgel with statistical

distribution of ionizable groups with monomer ratio VCL:Vim:IA = (a) 80:10:10, (b)

60:20:20. Black curve: unloaded microgel; red curve: loaded microgel. All measurements

were conducted in buffer at pH 8.0. Microgel sketches in (a) illustrate the interaction between

salt and cytochrome c .

amount of ionizable groups. The less pronounced change in size for microgels with a high

amount of ionizable groups (60:20:20) further supports the assumption explained above. The

uptake of cyt-c is primarily taking place in the outer shell of a microgel particle, while the

core is mostly unaffected. A higher amount of “unaffected” charges in the core thus results in

a higher particle size of VCL:Vim:IA = 60:20:20 compared to 80:10:10. The release of cyt-c,

therefore, leads to a less pronounced size change for highly charged microgels.

Core-shell particles show a similar behavior (see Figure 9.9). The particle size decreases for

unloaded microgels with increasing ionic strength due to negative charges being screened at

pH 8. Particles loaded with cyt-c increase in size with increasing ionic strength which is

attributed to negative charges being generated as cyt-c is released from the polymer network.

It is noticeable that the degree of swelling of loaded microgels with increasing ionic strength

is considerably lower than for microgel with a statistical distribution of ionizable groups.

Figure 9.10. shows the curves for loaded microgels of all architectures discussed above.

- -

-

- -

- -

- -

-

- -

-

a b

120

Figure 9.9 Influence of ionic strength on particle radius RH for microgel with core-shell

distribution of ionizable groups with increasing shell thickness. Core:shell ratio: (a) 1:1, (b)

1:5. Black curve: unloaded microgel; red curve: loaded microgel. All measurements were

conducted in buffer at pH 8.0.

Instead of the hydrodynamic radius RH, the ratio between the radius at an ionic strength = 0

mM (R0) and the radius at the respective ionic strength (Rx) is given to better visualize the

particle swelling. Note that before each measurement, all samples were allowed to reach

equilibrium. Core-shell particles have a considerably lower slope than microgel with s

statistical distribution of ionizable groups. This suggests that particles with a statistical

distribution easily release cyt-c upon the addition of salt resulting in a swelling of the polymer

network. Core-shell particles, in contrast, release cyt-c in a less pronounced way suggesting

that the neutral shell hinders the complete release of protein. This behavior is more

pronounced for core-shell microgels with a higher shell thickness.

Figure 9.10 Influence of ionic strength on ratio R0/Rx (R0 = RH at ionic strength = 0 mM; Rx

= RH at respective ionic strength) for microgels loaded with cytochrome c. Graphs are shown

for particles with statistical as well as core-shell charge distribution. All measurements were

conducted in buffer at pH 8.0 and at T = 25°C.

a b

121

The release of cyt-c upon the increase of the ionic strength also influences the molecular

weight MW of the microgels. This was already studied by M. Smith and A. L. Lyon for the

interactions between cyt-c and negatively charged PNIPAm-co-AAc microgels [25]. They

showed that the molecular weight decreases with increasing ionic strength due to the release

of cyt-c.

Therefore, MW of microgels with various distributions of ionizable groups (statistical and

core-shell) and various monomer ratios (80:10:10 and 60:20:20 for statistical distribution) in

the absence of cyt-c (black dots), in the presence of cytochrome c at low ionic strength

(30 mM, red dots) and high ionic strength (125 mM, blue dots) was measured at pH 8 (see

Figure 9.11). Note that samples were centrifuged before each measurement to remove

unbound protein.

In the absence of cyt-c, MW of microgels with statistical distribution of ionizable groups and

monomer ratios of 80:10:10 and 60:20:20 does not differ significantly (~ 2x 108 g/mol, blue

dots). MW increases continuously for core-shell microgels with increasing shell thickness

(blue dots).

A loading of the respective microgel particles with cyt-c leads to an increase in MW (red dots).

The sample 80:10:10 increases ~ 1.6 times in MW indicating that a large amount of cyt-c was

incorporated. Samples with twice the amount of ionizable groups (60:20:20) increase only 1.9

times in MW. This discrepancy between theoretical capacity of protein uptake and measured

capacity was already observed discussed for release experiments and confirms the formation

of a protein layer on the microgel surface hindering further uptake. The amount of protein that

is bound by core-shell particles is strongly dependent on the shell thickness. An increase in

shell thickness leads to an increase in MW, though the addition of a thicker shell does not add

further acidic moieties (i.e. itaconic acid) that are able to bind an increasing amount of cyt-c.

This indicates that hydrophobic interactions between protein and neutral shell play an

important role. The difference between unloaded and loaded microgels allows to estimate the

amount of cyt-c (MW = 12.300 g/mol) bound by microgels. The results are given in Table 9.3.

An increase in ionic strength from 30 mM (red dots) to 125 mM (blue dots) at pH 8 leads to a

release of cyt-c due to screening of attractive forces accompanied with an increase in particle

size. For microgel with a statistical distribution of ionizable groups, only small differences

between unloaded microgels (black dots) and loaded microgels (blue dots) in 125 mM salt

solution can be seen suggesting an almost complete release of cyt-c in high salt solution. The

difference between unloaded and loaded microgels at 125 mM salt concentration is more

pronounced for core-shell microgels and rises significantly with increasing shell thickness.

122

This suggests that the protein is not released completely through the addition of salt

supporting the results shown in Figure 9.7 and Figure 9.9.

Table 9.3 Amount of cytochrome c bound by microgels at pH 8 and T = 25 °C.

Sample Number of bound

protein at 30 mM

VCL:Vim:IA = 80:10:10,

statistical

8.5 x 103

VCL:Vim:IA = 60:20:20,

statistical

3.9 x 104

cVCL-/sNIPAm+, c:s = 1:1 1.1 x 105

cVCL-/sNIPAm+, c:s = 1:2.5 9.5 x 105

cVCL-/sNIPAm+, c:s = 1:5 2.0 x 106

Figure 9.11 Relationship of molecular weight MW and microgel architecture in the absence

(black dots) and presence (red dots) of cytochrome c in 30 mM NaCl and 125 mM NaCl (blue

dots). The dashed line is a visual help to separate microgels with core-shell structure from

microgels with statistical distribution of ionizable groups.

123

9.4 Summary

This chapter investigated the use of ampholyte microgels as container for the uptake and

release of proteins. As a model protein, cytochrome c (cyt-c) was used due to its red color and

its positive charge over a wide pH range. The interactions between ampholyte microgels and

cyt-c were studied at pH 3, 6, and 8. UV-Vis measurements confirmed that no cyt-c was

bound at pH 3 for microgels with statistical distribution of ionizable groups and core-shell

microgels with IA in the shell. A small amount of protein, however, remained in the microgel

at this pH for core-shell microgels with Vim in the shell. Since both shell and protein are

positively charged at this pH, the interaction was attributed to hydrophobic interactions. This

was confirmed by UV-Vis studies performed with neutral core-shell microgels. About 50 %

of cyt-c was bound at pH 8.

After loading the microgels with cyt-c at pH = 8, various triggers for the release of cyt-c from

the microgel network were studied. A raise in temperature did not prove to be an effective

trigger to release cyt-c. About 80 % of the initial concentration remained in the particle. The

addition of salt released about 70 % of the bound protein. The non-complete release indicated

the presence of hydrophobic interactions that are not influenced by the addition of salt.

Finally, the pH of the solution was changed from pH 8 to pH 3. For microgels with a

statistical distribution of ionizable groups, this led to a complete release of protein. Core-shell

microgels were able to “trap” the protein inside the particle and thus hindering its release.

The addition of salt also influences the microgel size. Unloaded particles decrease in size with

increasing ionic strength. Since measurements were performed at pH 8, this reflects that

negative charges are shielded by the salt ions and thus leading to a collapse of the microgel

structure. Loaded particles in contrast increased in size with an increasing ionic strength. This

can be attributed to the release of cytochrome c from the microgel, generating more and more

negative charges that repel each other leading to an expansion of the microgel structure. The

change of molecular weight MW for loaded und unloaded microgel in the presence (125 mM)

and absence (30 mM) of salt supported these results. While MW is identical for unloaded and

loaded microgels with statistical distribution of ionizable groups in 125 mM salt solution

indicating a complete release of protein, MW for loaded core-shell microgels in 125 mM salt

solution is higher than for unloaded core-shell microgels. The difference was shown to

increase with increasing shell thickness suggesting that the shell hinders a complete release of

the protein.

The results discussed in this chapter illustrated the importance of different distribution of

ionizable groups on the uptake and release of proteins from the polymer network.

124

9.5 Literature

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149–173.

[2] S. H. Gehrke, J. P. Fisher, M. Palasis, M. E. Lund, Ann. N. Y. Acad. Sci. 1997, 831,

179–207.

[3] C. Johansson, P. Hansson, M. Malmsten, J. Phys. Chem. B 2009, 113, 6183–6193.

[4] N. Murthy, M. Xu, S. Schuck, J. Kunisawa, N. Shastri, J. M. J. Fréchet, Proc. Natl.

Acad. Sci. U. S. A. 2003, 100, 4995–5000.

[5] H. Bysell, R. Månsson, P. Hansson, M. Malmsten, Adv. Drug Deliv. Rev. 2011, 63,

1172–1185.

[6] V. a Kabanov, V. B. Skobeleva, V. B. Rogacheva, a B. Zezin, J. Phys. Chem. B 2004,

108, 1485–1490.

[7] H. Bysell, M. Malmsten, Langmuir 2006, 22, 5476–5484.

[8] G. M. Eichenbaum, P. F. Kiser, D. Shah, S. a. Simon, D. Needham, Macromolecules

1999, 32, 8996–9006.

[9] C. S. O. Silva, R. P. Baptista, A. M. Santos, J. M. G. Martinho, J. M. S. Cabral, M. Â.

Taipa, Biotechnol. Lett. 2006, 28, 2019–2025.

[10] R. Sardari, R. Rezaee, Int. J. Electrochem. Sci. 2013, 8, 11578–11587.

[11] I.-J. Hsu, Y.-J. Shiu, U.-S. Jeng, T.-H. Chen, Y.-S. Huang, Y.-H. Lai, L.-N. Tsai, L.-Y.

Jang, J.-F. Lee, L.-J. Lin, et al., J. Phys. Chem. A 2007, 111, 9286–9290.

[12] O. Kimhi, H. Bianco-Peled, Langmuir 2007, 23, 6329–35.

[13] J. D. Carbeck, I. J. Colton, J. R. Anderson, J. M. Deutch, G. M. Whitesides, J. Am.

Chem. Soc. 1999, 121, 10671–10679.

[14] T. K. Das, S. Mazumdar, S. Mitra, Eur. J. Biochem. 1998, 254, 662–670.

[15] C. P. Lee, Current Topics in Bioenergetics, Elsevier, 2014.

[16] M. Hulko, I. Hospach, N. Krasteva, G. Nelles, Sensors (Basel). 2011, 11, 5968–80.


[18] W. Kauzmann, Adv. Protein Chem. 1959, 14, 1–63.

[19] X. Chen, S. Chen, J. Wang, Analyst 2010, 135, 1736–41.

[20] K. H. Kim, E. K. Lee, Biotechnol. Bioprocess Eng. 2007, 12, 366–371.

[21] Y. Liu, Y. Liu, R. Guo, J. Colloid Interface Sci. 2010, 351, 180–9.

[22] P. P. Parui, M. S. Deshpande, S. Nagao, H. Kamikubo, H. Komori, Y. Higuchi, M.

Kataoka, S. Hirota, Biochemistry 2013, 52, 8732–8744.

[23] N. Welsch, A. Wittemann, M. Ballauff, J. Phys. Chem. B 2009, 113, 16039–45.

[24] N. Shamim, H. Liang, K. Hidajat, M. S. Uddin, J. Colloid Interface Sci. 2008, 320, 15–

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[25] M. Smith, L. A. Lyon, Macromolecules 2011, 44, 8154-8160.

125

10. 1 Biomineralization of CaCO3 in Zwitterionic Microgels

10.1.1 Introduction

Nature is an excellent teacher when it comes to combining the mechanical strength and

toughness of inorganic material with the softness and formability of polymers to form

complex structures with unique properties and specific functions. The control of organic

material over inorganic material to form minerals is called biomineralization. Examples are

manifold – so far over 60 different minerals have been detected [1] - and vary from magnetic

crystals of magnetotactic bacteria, shells, and corals to tooth enamel and bones in humans.

Biomineralization is very complex and many factors such as the organic matrix or the ion

concentration influence the nucleation process and the mineral orientation, size, density, and

morphology [2].

In general, biomineralization is divided into two categories: Biologically induced

mineralization (BIM) and biologically controlled mineralization (BCM) [1]. Biologically

induced mineralization occurs extracellularly or epicellularly (i.e. on the cell surface) when

the mineralization is little controlled and an organism segregates a metabolic by-product that

interacts with the environment leading to the precipitation of the mineral [3]. The resulting

minerals are poorly crystallized and polydisperse in size, have no specific morphology, but

often impurities. Biologically controlled mineralization arises when the organism exerts a

high degree of control over the mineralization process. This happens for instance if a defined

space such as vesicles or macromolecules exists and leads to minerals that are monodisperse

in size with a well-ordered crystal morphology. BCM can occur intracellularly meaning that

the mineralization process is not as sensitive to environmental conditions such as pH,

temperature, pressure, and redox potential as BIM [3].

Most minerals in nature are based on calcium. This can be explained by the abundance of

calcium in the world’s oceans as well as the low water solubility of calcium salts [1]. Calcium

carbonate CaCO3 is used in nature to strengthen the skeletons of mussel shells, snails, or

corals. It is found in six different modifications: amorphous (i.e. unordered, isotropic) CaCO3

(ACC), the three anhydrous crystalline forms vaterite, aragonite, and calcite, and the two

hydrated crystalline forms calcium carbonate monohydrate and calcium carbonate

hexahydrate (ikaite) [1]. Though aragonite and calcite are the most stable forms, stabilization

of less stable forms can be achieved by the addition of other ions such as Mg2+ or Li+ or by

enclosing it in an organic matrix.

In literature, different types of polymers are used for mimicking the biomineralization

process: Peptides [4][5], dendrimers [6][7], synthetic polymers and monolayers. In the following,

126

two of them are described in more detail: Self-assembled monolayers (SAMs) and hydrogels.

Though none of the approaches are able to mimic the complex processes that enfold in nature,

they can be used to gain insights in the principles of biomineralization.

Hydrogels enable a stable transport via diffusion that is advantageous for nucleation. They,

however, consist of many pores where precursor ions are isolated. Nucleation is thus more

hindered than in solution because nucleation only occurs if a certain limit of supersaturation is

reached [2]. Still, this approach enables a better control of mineral growth than from

supersaturated solutions.

For instance, K. Furuichi et al. prepared a poly(acrylic acid) hydrogel containing phosphate

ions [8]. Through diffusion of calcium ions into the matrix, nanocrystalline hydroxylapatite

were formed. They observed that the soft hydrogel was transformed into a solid disk after

72 h of calcification. After heating up of the hybrid material to 700 °C, a porous

hydroxylapatite structure remained. O. Grassmann and P. Löbmann synthesized hydrogels

containing sulfonic acid to mineralize CaCO3 [9]. They showed that an increase in sulfonic

acid content leads to a change in morphology. While a low content of sulfonic acid leads to

the formation of pseudo-octahedral morphology, a higher content leads to the formation of

cuboctahedron morphology.

P. Liu and J. Song mention the possibility to use zwitterionic hydrogels for the growth of

hydroxyapatite [10]. They emphasize the anti-polyelectrolyte behavior and the ability of

zwitterionic polymers to bind both cationic and anionic precursor ions and predict a lower

activation energy for nucleation and growth. A comparison with other charged and uncharged

comonomers revealed that nucleation of hydroxylapatite is possible in all hydrogels, though

differences could be observed in the mineralization degree: in uncharged hydrogels,

mineralization occurred on the surface, while for anionic and cationic hydrogels, spherical

mineral nodules were formed. Sulfobetaine hydrogels, however, had a three times higher

mineral content than anionic or cationic hydrogels. The authors attribute this to two factors:

first, the anti-polyelectrolyte effect leads to a swelling of the hydrogels a facilitation in

electrolyte uptake, and secondly, the ability of zwitterions to bind and stabilize cationic and

anionic precursor ions. Furthermore, the authors could show the influence of the crosslinker

degree on the crystal size: the higher the amount of crosslinker, the smaller the formed

nodules. They assume that a higher crosslinking degree hinders a free diffusion of precursor

ions which leads to a gradient in the concentration of precursor ion in the hydrogel network

resulting in a lower mineral growth rate.

127

This chapter is inspired by the work done by P. Liu and J. Song. Here, phosphobetaine

microgels were used to study their influence on the mineralization process of CaCO3. CaCO3

was used as a standard model mineral because of its well studied properties and high

abundance in nature [11][12]. Phosphobetaine was used due to its known high affinity for

calcium ions [13][14]. The complexation of Ca2+ leads to a saturation of ions in a confined space

that is essential for the nucleation of CaCO3 crystals. The use of PVCL-based zwitterionic

phosphobetaine microgels offers other important advantages apart from their low cytotoxicity

and anti-polyelectrolyte effect that was not studied by P. Liu and J. Song: their temperature-

and pH-sensitivity as well as their confined volume. PVCL microgels shrink at temperatures

around 32 °C [15][16]. Phosphobetaine is pH-sensitive [17] and a change of the pH of the

surrounding medium leads to a change in particle size. Both effects are likely to influence the

nucleation and mineralization process of CaCO3.

128

10.1.2 Synthesis Procedure

For this chapter, zwitterionic microgels based on PVCL with phosphobetaine as a comonomer

were used. The microgel synthesis via precipitation polymerization is described in chapter 5.

For the synthesis of CaCO3, the pH of the solution is required to be above ~ 6. In acidic

medium, CaCO3 decomposes according to the following equation:

CaCO3 + 2 HCl → CaCl2 + H2O + CO2 (1)

Biomineralization is initiated in two different ways: through direct synthesis and via gas phase

diffusion (see Figure 10.1). In one batch prepared via gas diffusion, the samples were dialyzed

before being put in a desiccator.

Figure 10.1 Overview of the synthesis routes for mineralization of CaCO3. (1) Gas diffusion;

(2) Gas diffusion with prior dialysis; (3) Direct synthesis.

(1) Direct Synthesis

The respective microgel was dissolved in destilled water. CaCl2·2H2O was dissolved in 1 mL

of destilled water and added to the aqueous microgel solution. (NH4)2CO3 was dissolved in

5 mL of destilled water and added dropwise under vigorous stirring to the aqueous

microgel/CaCl2 solution. After 10 min of stirring the solution was allowed to stand for 24 h at

room temperature. The product was dialyzed against water for 3 d. The respective amounts of

educts can be seen in Table 10.1.

129

Table 10.1 Amounts of educts used for direct synthesis of CaCO3.

Sample name Theor. amount of

CaCO3 / wt-%

m(CaCl2·2H2O)/

g

m((NH4)2CO3)/

g

m(microgel)/g

D(2)13* 13.0 0.015 0.009 0.065

D(2)28 28.4 0.040 0.026 0.065

D(2)37 37.6 0.058 0.038 0.065

D(2)50 50.4 0.097 0.064 0.065

D(2)67 67.0 0.195 0.127 0.065

* Explanation of the sample name: “D” = “Direct synthesis”; “(2)” = Microgel contains 2 wt-

% PB; “13” = Theoretical amount of CaCO3 is 13 wt-%.

(2) Gas Phase Diffusion

The respective microgel was dissolved in destilled water. CaCl2·2H2O was dissolved in 1 mL

of destilled water and added to the aqueous microgel solution. The sample amounts

correspond to the ones given in Table 10.1 (the sample names are e.g. E(2)13: “E” =

Exsikkator = Desiccator). The aqueous microgel/CaCl2 solution is put in a sealed desiccator

with solid (NH4)2CO3 and allowed to stand for 24 h at room temperature. The product was

dialyzed against water for 3 d.

The following variations from the above described procedure were performed:

(a) Samples with a higher microgel concentration were prepared. Therefore, freeze-dried

microgel samples were dissolved in water with a ratio of 1:3. The viscous solution was

treated as described above. Here, the time in the desiccator was increased to 48 h. The

sample amounts are given in Table 10.2.

(b) After the addition of CaCl2, the samples were dialyzed to remove all Ca2+ that is not

bound by the anionic groups in the microgel. After dialyzing for 2 d, the samples were

put in a desiccator for 24 h and dialyzed anew. The sample names are e.g. E(2)13v:

“v” = vordialysiert = dialyzed before addition of (NH4)2CO3.

(c) The synthesis of pre-dialyzed samples was performed at 40 °C, i.e. above the VPTT of

the microgel. This was done for the sample E(10)37.

(d) The pH of the solution was varied. The pH of the original samples is ~ 8.5. It was

adjusted to pH 3 and pH 10 with 1 M NaOH and 37 % HCl, respectively. This was

done for the sample E(10)67.

130

Table 10.2 Amounts of educts used for gas diffusion synthesis of CaCO3.

Sample

name

Amount of PB /

wt-%

m(microgel) /

g

Theor. amount

of CaCO3 / wt-

%

m(CaCl2·2H2O)/g

E(2)13H* 2 0.287 13.0 0.064

E(4)13H 4 0.337 13.0 0.076

E(4)7H 4 0.416 7.0 0.050

E(4)5H 4 0.852 5.0 0.073

E(10)5H 10 0.476 5.0 0.041

* Explanation of the sample name: “E” = “Exsikkator = Desiccator”; “(2)” = Microgel

contains 2 wt-% PB; “13” = Theoretical amount of CaCO3 is 13 wt-%; “H” = Highly

concentrated.

131

10.1.3 Results and Discussion

In this chapter, the process of biomineralization of CaCO3 in zwitterionic microgels is

analyzed stepwise. Therefore, the behavior of the particles is analyzed as follows:

(a) Pure PB Microgels

(b) Microgels with CaCl2

(c) Microgels with CaCO3

(a) Pure PB Microgels

The properties of phosphobetaine microgels obtained via precipitation polymerization were

analyzed and discussed in my diploma thesis. It was shown that the particle size was strongly

dependent on the amount of phosphobetaine incorporated. The size of particles with 2 wt-%

of PB at 25 °C was 190 nm and decreased to 130 nm with 10 wt-% PB. AFM images show

the formation of spherical, monodisperse particles (see Figure 10.2). Due to the low amount

of crosslinker (3 wt-%), the particles spread widely on a solid surface.

Figure 10.2 (a) AFM image of microgel particles with 5 wt-% PB; (b) Cross-section of

particles marked in the left image.

The particle size was analyzed dependent on the temperature and the pH (see Figure 10.3).

PVCL-based microgels shrink with increasing temperature. The Volume Phase Transition

Temperature (VPTT) of the zwitterionic particles is slightly shifted to higher temperatures

(30.7 °C to 31.2 °C for 0 wt-% and 10 wt-% PB, respectively) due to the increased

hydrophilicity that retards the shrinkage of the of the zwitterionic particles. Furthermore, the

temperature range of the phase transition becomes broader with increasing PB content. While

pure PVCL-microgels show a sharp transition, microgels with 10 wt-% PB have a very broad

a

b

132

transition range. This further suggests that the shrinkage of the microgels is retarded by the

incorporation of the hydrophilic PB comonomer.

Figure 10.3 (1) Temperature- and (2) pH-sensitivity of microgels with different PB content.

A change in pH, however, does not lead to a change in particle size. It is expected that the

hydroxyl group of the PB is protonated at low pH while the quaternized ammonium group

leads to an overall positive charge of the molecule. Within the microgel network, these

charges are thought to repel each other leading to a swelling in particle size. At high pH, PB is

neutral leading to shrinkage in particle size. The absence of pH-sensitivity in phosphobetaine

microgels, however, is due to the low acid dissociation constant of diethyl phosphoric acid of

1.39 [18] meaning that PB is able to maintain its zwitterionic nature at a wide range of pH.

(b) Microgels with CaCl2

It was shown that microgels with 5 wt-% PB start to swell from 110 nm in water to 165 nm at

a concentration of 1 M CaCl2 (i.e. by 50 %). Complementary, the saturation limit of the

particles was determined gravimetrically. Therefore, a known amount of freeze-dried

microgel (1 and 5 wt-% PB) is solved in water to which a known amount of CaCl2 is added.

The samples are shaken overnight and centrifuged three times to remove unbound CaCl2.

Afterwards, the samples are freeze-dried and weighed.

Figure 10.4 shows that microgels with a PB content of 1 wt-% reach a saturation limit at a low

ratio of CaCl2:microgel, i.e. 2.6. Samples with 5 wt-% PB, however, reach the saturation limit

at a higher ratio of 15.5. This approximately corresponds to the fivefold amount of 1 wt-% PB

microgels.

133

Figure 10.4 Gravimetrically determined saturation limit of PB microgels with CaCl2.

The interactions between Ca2+ and microgels were analyzed in a more detailed way with ITC.

Here, CaCl2 solution of a known concentration is titrated to a microgel solution of known

concentration. The raw data, i.e. the generated heat over time, is given in Figure 10.5.

Figure 10.5 ITC raw data for titration of CaCl2 to microgel solution containing 1, 5 or 10 wt-

% PB. The concentration for CaCl2 was 1 g/L in all measurements. Titration was performed at

T = 25 °C.

The graphs show that microgel samples with increasing amount of PB need increasing amount

of CaCl2 to become saturated. The graph for 10 wt-% PB was fitted to calculate the saturation

limit.

With respect to the given concentration and volumes, the ratio between Ca2+ and microgel

gives the binding constant Kb. At the equilibrium

A + B ↔ AB (2)

Kb is defined as:

𝐾𝑏 = [𝐴𝐵]

[𝐴]∙[𝐵] (3)

134

with [A] = concentration of species A that can be bound; [B] = concentration of free binding

sites; [AB] = concentration of occupied binding sites.

At 25 °C, Kb increases with increasing amount of incorporated content of PB. The linear

relationship can be seen in Figure 10.6. Additionally, the measurements were repeated at

37 °C, i.e. above the VPTT of the microgels. It is noticeable that the affinity is about one

order of magnitude lower than at room temperature. This is attributed to the collapse of the

polymer network preventing the uptake of larger amounts of Ca2+.

Figure 10.6 Binding constants Kb of binding of Ca2+ to zwitterionic microgels with varying

PB content as determined from ITC analysis at different temperatures.

(c) Microgels with CaCO3

- Direct synthesis

The synthesis via direct synthesis leads to the precipitation of the microgels forming a white,

ductile disk (see Figure 10.7).

Figure 10.7 Mineralized microgel.

These plates retain the thermosensitivity that is typical for PVCL microgels. They shrink

when heated up to 50 °C and swell again when cooled to room temperature (see Figure 10.8).

The extent of swelling and shrinking is less pronounced when compared to pure microgels

due to the hybrid material’s macroscopic size.

The crystal structure of CaCO3 inside the particles was determined with XRD. For all

samples, independent on PB or CaCO3 content, calcite was found to be the only crystal

135

Figure 10.8 Thermosensitivity of mineralized microgels. (1) 25 °C; (2) 50 °C; (3) 25 °C.

structure according to standard JCPDS files. The reflections at 2Θ = 29.5 (104), 36.1 (110),

and 39.5 (113) confirm the formation of calcite and are in good agreement with the literature.

Figure 10.9 shows the XRD diffraction pattern for the sample D(4)50 as an example. The

assignments of the peaks can be found in the appendix. This result is not surprising as calcite

is the thermodynamically favored modification which exists at ambient temperature and

pressure [19][20][21]. The sharp peaks suggest that CaCO3 was well-crystallized.

Figure 10.9 XRD diffraction pattern of microgel with 4 wt-% PB and 50 wt-% CaCO3 via

direct synthesis. Black lines indicate the peak positions for calcite as found in literature

(JCPDS: 86-2334).

Another means to determine the crystal structure of CaCO3 is FTIR (see Figure 10.10).

Calcite, vaterite, and aragonite show different peaks in the FTIR spectrum. The peaks found

in Figure 10.10 agree with the data obtained with XRD, namely that only calcite is present in

the sample. The peaks belonging to calcite are highlighted in the spectrum above. The peaks

at 1420 cm-1, 875 cm-1, and 711 cm-1 correspond to the asymmetric stretching, out-of-plane

bending, and in-plane-bending of the carbonate group, respectively [22]. The microgel has a

characteristic peak at 1620 cm-1, induced by the carbonyl group of PVCL.

Apart from the crystal structure, an increase in the peak intensity of calcite can be observed.

Thus, it can be qualitatively, though not quantitatively, stated that the amount of CaCO3 is

increasing with increasing theoretical amount.

1 2 3

136

Furthermore, the form and morphology of the hybrid microgels was examined with

microscopy.

Figure 10.10 (a) FTIR spectrum of microgels with 10 wt-% PB and varying CaCO3 content,

synthesized via direct synthesis. (Red) Pure CaCO3 (calcite); (green) Unmineralized microgel

with 10 wt-% PB; (black) Microgel with 13 wt-% CaCO3; (blue) Microgel with 37 wt-%

CaCO3; (turquois) Microgel with 67 wt-% CaCO3. (b) FESEM image of 10 wt-% PB

microgels with 28 wt-% of CaCO3.

The images in Figure 10.10 show large, trigonal-rhombohedral crystals (ca. 4 µm) that are

covered completely or partly by a microgel film. The sample D(10)28 is characteristic for all

samples obtained with direct synthesis. They clearly indicate that the microgel structure has

no influence on the crystal growth. It can be assumed that the direct synthesis is too fast to

enable a controlled biomineralization process and CaCO3 crystals grow randomly with no size

control. Thus, no further characterization of these hybrid particles was performed.

Therefore, a different route to obtain CaCO3 crystals whose sizes are controlled by the

microgel network is followed. Since a fast reaction between CaCl2 and (NH4)2CO3 leads to

large crystals of about 3 – 5 µm, a slow reaction via gas diffusion might enable the microgel

network to influence the crystal formation process.

- Gas Phase Diffusion

According to N. Gehrke, the slow crystallization process via gas diffusion leads to well-

facetted calcite crystals [23]. In the desiccator, (NH4)2CO3 slowly disintegrates:

(NH4)2CO3 ↔ 2 NH3 + CO2 + H2O (4)

a b

137

The synthesis of CaCO3 in PB microgels via gas phase diffusion in a desiccator resulted in

white disk that retain the microgel’s thermosensitivity identical to the results shown in Figure

10.7 and Figure 10.8.

XRD and FTIR measurements reveal that calcite is the only crystal structure obtained with

gas diffusion. Changes in microgel concentration, temperature or pH lead to the same results.

All XRD diffraction patterns as well as the FTIR spectra can be found in the appendix.

FESEM images show the location, size and form of the formed calcite crystals. Figure 10.11

(1) shows 10 wt-% PB microgels with 67 wt-% CaCO3. The sample was dialyzed before it

was put in the desiccator to react with (NH4)2CO3 in order to remove all Ca2+ that is unbound

by the phosphogroup inside the microgel. The image reveals that small CaCO3 minerals were

formed within the microgel structure. These crystals have sizes between 20 and 60 nm. The

microgel structure remains spherical, but disrupted. (2) shows a sample that was not dialyzed

before being put in the desiccator. Here, the image resembles the images taken after the direct

synthesis. Large, trigonal-rhombohedral crystals were formed that exceed the size of

microgels (size (CaCO3) > 2 µm) so that it can be assumed that the microgel network had no

influence on the mineral growth. In the next step, the influence of PB amount on the crystal

growth was studied. (3) shows hybrid particles with less CaCO3 compared to image (1). In a

“broken” microgel particle, small crystals with a size of ~ 30 nm can be seen (red arrow). It

seems that a lower content of PB does not lead to a deformation of the particle in the same

extent as can be observed for microgels with 67 wt-% PB. Also, less crystals with a smaller

size are formed. EDX measurements confirm that less Ca is present in the sample E(10)37

compared to E(10)67 as indicated by the increased Ca/C ratio (Figure 10.12). (4) shows the

biomineralization in microgels without PB. Here, the crystals and completely covered in a

layer of microgels and thus resemble the crystals obtained in (2). The microgel obviously has

no influence on the mineral growth. This indicates that the presence of zwitterionic PB plays a

crucial role in the controlled growth of CaCO3. Finally, (5) and (6) show the influence of pH

on the biomineralization process. (6) shows the results if the microgel/CaCl2 solution was

acidified after the pre-dialysis (pH 3). It is obvious that no mineral was formed since only

spherical, monodisperse microgel particles can be seen. This is expected since CaCO3

decomposes in acidic medium as follows:

CaCO3 + 2 HCl → CaCl2 + H2O + CO2 (5)

(5) shows that crystals inside the microgel structure were also formed under basic conditions

(pH 10). Compared to (1), (pH 8.5), the crystals are smaller in size (~ 30 nm) and more

monodisperse.

138

Figure 10.11 FESEM images. (1) E(10)67, pre-dialyzed before placing into desiccator, pH

8.5; (2) Same sample as in (1), undialyzed; (3) E(10)13, pre-dialyzed; (4) E(0)67; (5)

E(10)67, pH 10; (6) E(10)67, pH 3.

To sum up, the best results for the formation of CaCO3 were obtained if the following

requisitions were met:

(1) The presence of PB is crucial for a controlled mineralization within the microgel

network. The polar phosphogroup effectively immobilizes Ca2+ leading to a

supersaturation of ions that is essential for mineralization. Furthermore, it can be

assumed that due to the anti-polyelectrolyte effect, diffusion of Ca2+ ions into the inner

microgel network is facilitated.

(2) The sample has to be dialyzed before being put in the desiccator to remove all

unbound Ca2+.

139

(3) The pH of the solution has to be pH > 8.5. At low pH, CaCO3 decomposes to CaCl2.

Figure 10.12 EDX analysis of (a) E(10)13 and (b) E(10)67. Traces of Al, P, and Cl derive

from the sample holder.

Figure 10.13 TEM images of (a) 0 wt-% PB; (b) 5 wt-% PB; (c) 10 wt-% PB. Each sample

contains 67 wt-% CaCO3. The images in the second row (‘) are a zoom-in of the respective

image in the first row.

The influence of the PB content on the crystal size and amount is further studied with TEM

(see Figure 10.13). The images show that the mineralization of CaCO3 in uncharged PVCL

microgels leads to a crystal growth on the surface of the particle. Contrary, zwitterionic

microgels induced a crystal growth within the microgel network. A higher content of PB leads

to an increased amount of crystals while the spherical shape of the microgel is still

maintained. The ability of the zwitterionic microgels to highly swell after the addition of Ca2+

supports the internal growth of CaCO3.

a b

200 nm

500 nm 500 nm 200 nm

200 nm 100 nm

a b c

b‘ a‘

1

c‘

140

A quantitative conclusion about the incorporated amount of CaCO3 can be drawn from

thermogravimetric analysis. Here, the dried sample is heated up to 900 °C. At temperatures

around 450 °C, the microgel is decomposed. CaCO3 is decomposed at around 750 °C

according to the following equation:

CaCO3 → CaO + CO2 (6)

Looking at the remaining mass after the microgel’s decomposition (600 °C, see black line in

Figure 10.14), one can read the amount of CaCO3 in the sample. The amounts obtained from

TGA measurements are in good agreement with theoretical values.

Further TGA measurements were performed for hybrid particles that were synthesized at

different temperatures as well as pH (see Figure 10.15). (1) shows that ~ 14 % less CaCO3 are

formed within the microgel particle at 40 °C compared to 25 °C. At 40 °C, the microgels are

collapsed, thus hindering a free diffusion of Ca2+ within the microgel network. These results

are in agreements with ITC data (see Figure 10.5).

Figure 10.14 Decomposition of 10 wt-% PB microgels with different CaCO3 content. The

black vertical line shows the point where the remaining amount of CaCO3 is determined

(600 °C).

Figure 10.15b demonstrates that the highest amount of CaCO3 crystals is gained at pH 8.5 At

pH 10, ~ 11 % less CaCO3 is formed, while at pH 3 no CaCO3 is formed at all. The remaining

4.9 % seen in the graph agree with the remains of pure PB microgels at 600 °C (see (a)).

141

f Figure 10.15 Decomposition of 10 wt-% PB microgels. (a) With 37 % CaCO3, synthesized in

desiccator at different temperatures. (b) With 67 % CaCO3, synthesized at different pH. The

black vertical line shows the point where the remaining amount of CaCO3 is determined

(600 °C).

Figure 10.7 suggest that the hybrid particles retain their temperature sensitivity even after

mineralization. This was studied in more detail with DLS. Figure 10.16 shows that pure PB

microgels have a VPTT of 30.9 °C. The respective crystallized sample as well shrinks with

increasing temperature. The constant decrease in size beginning at 15 °C makes it difficult to

give an exact VPTT for the hybrid particles. Interestingly, the hybrid particles have roughly

the same size in collapsed state than the unmineralized particles.

Figure 10.16 Temperature-sensitivity of pure 2 wt-% PB microgels (red line) and sample

E(2)13v (black line).

The obvious temperature-sensitivity of the hybrid microgels leads to the assumption that

temperature can be used as a trigger to release CaCO3 crystals “on demand”. Therefore, the

sample E(2)13v was heated up to 50 °C and analyzed. TEM measurements (Figure 10.17)

reveal small particles of a size of 59 ± 8 nm and larger particles of a size of 168 ± 14 nm.

Since the smaller particle did not appear in images taken at room temperature, they are

assumed to be CaCO3 crystals that are “squeezed” out of the microgel particles. The crystals

a b

142

are relatively monodisperse and spherical (a). The microgels as well retain their spherical

structure after the removal of CaCO3 (b).

Figure 10.17 TEM images of E(10)67 after heating up to 50 °C. (b) is a zoom-in of (a).

The sample was fractionated with F-FFF to separate particles with different sizes (Figure

10.18). Furthermore, this method gives the radius of gyration, RG.

Figure 10.18 Fractionation of sample E(10)67 at (a) 25 °C and (b) 50 °C, the arrow marks the

peaks generated by small particles. The left-most peak represents the void peak.

The measurement at 25 °C (Figure 10.18a) reveals no distinct, but a broad and undefined

peak. The presence of CaCO3 in the microgel particle leads to a disruption of the polymer

network and thus high polydispersity as was already seen in TEM (Figure 10.13). Contrary, at

50 °C, two separate peaks can be seen (Figure 10.18b). The first peak has its maximum after

9 min corresponding to an RG of ~ 60 nm. This size is identic with the value obtained from

TEM measurements. The main peak has its maximum at t = 23 min corresponding to an RG of

140 nm. This corresponds to the value obtained from DLS. The ratio between RH and RG

gives information about the hardness of microgels. W. Burchard specifies the value of

microgels with 0.3 to 0.6 [24]. The examined sample has a ratio of 0.74 that is close to the

value of a hard sphere (0.775 [25]). This surprisingly high value can be explained by the

collapsed state of the microgel particle at 50 °C which is intensified by the electrostatic

attraction of positive and negative charges that lead to a further “hardening” of the polymer

network.

a b

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10.1.4 Summary

This chapter elaborated the important parameter necessary for successful biomineralization of

CaCO3 in zwitterionic microgels based on PVCL. The most convenient method was slow gas

diffusion. At pH above 8.5 and room temperarure, crystals with sizes of ~ 60 nm could be

detected inside the microgel particle. This required that the microgel sample in aqueous CaCl2

solution was dialyzed before being put in a desiccator in order to remove all unbound Ca2+.

The crystal structure was found to be calcite and confirmed with XRD and FTIR. TGA

measurements showed that the CaCO3 amount was found to be in accordance with the

theoretical values.

Since the hybrid particles retain their thermosensitivity, temperature was used as a trigger to

“squeeze” CaCO3 crystals out of the microgel particles. Both TEM and F-FFF confirmed that

it is possible to separate microgels and CaCO3 at temepratures above the VPTT. This offers

possible applications of this hybrid material as CaCO3 carrier where the CaCO3 can be

released on demand.

144

10.1.5 Literature

[1] F. C. Meldrum, Int. Mater. Rev. 2003, 48, 187–224.

[2] H. Li, L. a Estroff, J. Am. Chem. Soc. 2007, 129, 5480–3.

[3] R. B. Frankel, Rev. Mineral. Geochemistry 2003, 54, 95–114.

[4] D. B. Deoliveira, R. A. Laursen, J. Am. Chem. Soc. 1997, 119, 10627–10631.

[5] D. L. Masica, S. B. Schrier, E. A. Specht, J. J. Gray, J. Am. Chem. Soc. 2010, 132,

12252–12262.

[6] J. J. J. M. Donners, E. W. Meijer, R. J. M. Nolte, C. Roman, A. P. H. J. Schenning, N.

A. J. M. Sommerdijk, B. R. Heywood, Chem. Commun. 2000, 1937–1938.

[7] D.-K. Keum, K. Naka, Y. Chujo, Bull. Chem. Soc. Jpn. 2003, 76, 1687–1691.

[8] K. Furuichi, Y. Oaki, H. Ichimiya, J. Komotori, H. Imai, Sci. Technol. Adv. Mater.

2006, 7, 219–225.

[9] O. Grassmann, P. Löbmann, Chem. Eur. J. 2003, 9, 1310–1316.

[10] P. Liu, J. Song, Biomaterials 2013, 34, 2442–54.

[11] F. C. Meldrum, H. Cölfen, Chem. Rev. 2008, 108, 4332–432.

[12] A.-W. Xu, M. Antonietti, H. Cölfen, Y.-P. Fang, Adv. Funct. Mater. 2006, 16, 903–

908.

[13] V. Wintgens, F. Dalmas, B. Sébille, C. Amiel, Carbohydr. Polym. 2013, 98, 896–904.

[14] F. Degryse, B. Ajiboye, R. D. Armstrong, M. J. McLaughlin, Soil Sci. Soc. Am. J.

2013, 77, 2050.



[17] L. Nong, C. Xiao, Z. Zhong, J. Surfactants Deterg. 2011, 14, 433–438.

[18] W. D. Kumler, J. J. Eiler, J. Am. Chem. Soc. 1943, 65, 2355–2361.

[19] X. Wang, H. Sun, Y. Xia, C. Chen, H. Xu, H. Shan, J. R. Lu, J. Colloid Interface Sci.

2009, 332, 96–103.

[20] X. Li, Q. Shen, Y. Su, F. Tian, Y. Zhao, D. Wang, Cryst. Growth Des. 2009, 9, 3–9.

[21] C. Cheng, Z. Shao, F. Vollrath, Adv. Funct. Mater. 2008, 18, 2172–2179.

[22] T. Z. Forbes, a. V. Radha, A. Navrotsky, Geochim. Cosmochim. Acta 2011, 75, 7893–

7905.

[23] N. Gehrke, H. Cölfen, N. Pinna, M. Antonietti, N. Nassif, Cryst. Growth Des. 2005, 5,

1317–1319.

[24] W. Burchard, Light Scattering from Polymers, Springer Berlin Heidelberg, Berlin,

Heidelberg, 1983.

[25] W. Burchard, K. Kajiwara, M. Chemie, A. Ludwigs, J. Polym. Sci. Part B Polym.

Phys. 1982, 20, 157–171.

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10.2 Microgels and β-TCP as Bone Substitute Material

10.2.1 Introduction

Materials that are used for bone repair are substances that support, promote or stimulate the

healing process of bone either on their own or in combination with other materials. Ideally, it

will also involve the surrounding soft tissue to stimulate the differentiation of osteogenic stem

cells. For this, the material has to be as similar as possible to natural bone in its properties [1].

For the synthetic fabrication of such material the following parameters must be addressed:

biocompatibility, bioresorbability, surface texture (influencing the response of the cell’s, bone

and surrounding tissue), geometry, the presence of pores with defined size and volume, facile

flow and incorporation of oxygen, nutrients or drugs, and chemical purity [2][3].

Synthetic inorganic material

Calcium phosphates are promising materials for biomedical application and the fabrication of

biomaterials. For instance, in the field of bioceramics, hydroxyapatite (HA, (Ca10(PO4)6(OH)2,

Ca/P ratio 1.67) and tricalcium phosphate are used as possible materials for bone substitution,

fixation of implant, and tooth remineralization due to their similarity to human bone apatite

(Ca/P ratio ~ 1.6, depending on the type and age of the bone) [4][5]. β-tricalcium phosphate (β-

TCP, Ca3(PO4)2, Ca/P ratio 1.5) is a polymorph form of tricalcium phosphate and is

characterized by its high biocompatibility with surrounding living tissue, high solubility, and

bioactivity [3][6][7][8]. It is also osteoconductive. In contrast to HA, it decomposes in the human

body and releases a high concentration of Ca2+ and PO4- ions [2]. Ca2+ is essential blood

clotting [9], stabilization of cell membranes [10], and muscle contraction [11]. Chang et al. could

show that an increase in the local Ca2+ and PO4- concentration supports the mineralization

process [12]. Already in 1977, Cameron et al. used β-TCP as bone implantation material [8].

Depending on the temperature during the sintering process, both α- and β-TCP can be

produced working at 1370 °C and 1200 °C, respectively [13]. α- and β-TCP differ in

mechanical strength and their solubility in water. The latter is important for the decomposition

of the ceramic.

A big difference between biologically and synthetically fabricated β-TCP is that the latter

does not possess interconnecting pores that are essential the success of osteoconductive

treatment since they facilitate tissue infiltration. For those, the ceramic has to be submitted to

special treatments. F. Zhang et al. for instance produced macroporous β-TCP that they used

for bone regeneration [14]. They were able to tune the porosity from 55 to 70 % with pore sizes

between 300 and 500 µm using spark plasma sintering that allows the fast production of solid

146

nanostructured ceramics. In their review that illuminates the connection between porosity,

pore sizes and bone regeneration, V. Karageorgiou and D. Kaplan state that the ideal pore size

is considered to be ~ 300 µm, but concede that the minimum pore size is ~ 100 µm to

regenerate new bone [15].

Pure β-TCP as bone substitute material is not well suited due to its gradual, still fast and

unpredictable resorption [3]. A decomposition that is too fast is both detrimental to bone

formation [16] and may lead to inflammation due to high concentration of ceramic particles [2].

In acid environment, β-TCP degrades as follows:

Ca3(PO4)2 𝐻+

→ Ca2+ + 2 CaHPO4 𝐻+

→ 3 Ca2+ + 2 PO43- (1)

An acid environment is generated by H+ secreting macrophages that are the first cells that

cover a new ceramic implantation site. Calcium as the degradation product is either excreted

or incorporated in the body. K. Lin, however, showed that it is possible to slow down the

degradation process through control of the powder size. The authors used nano-sized β-TCP

instead of micro-sized powder [17]. A second drawback of pure β-TCP is its low mechanical

strength [1] and elasticity and its high brittleness [18], it is therefore often used in composites.

Composites

Due to the manifold combination possibilities between inorganic and organic material, a

classification of composites is difficult. Literature offers countless examples for composite

materials.

Pure inorganic materials such as HA and β-TCP have low mechanical strength and low

toughness, so composite materials, i.e. the blending of advantageous properties of both

inorganic and organic materials, are studied to develop new materials suitable for bone

substitution and regeneration. A number of researchers mix β-TCP with bioresorbable and

biocompatible polymers that act as a binder [19]. Crosslinkers are further added for two

reasons: to increase the persistence in tissue and to improve mechanical properties. C.-H. Yao

et al. for instance used gelatin as a binder and glutaraldehyde as a crosslinker. They found out

that their composite materials need at least four days of soaking to improve biocompatibility

[20]. In a later study, the authors substituted glutaraldehyde with genipin, a crosslinker that is

bound in gardenia and shows very low toxicity [19][21][22]. They obtained a formable filling

material that causes no inflammation and leads to and participates in the formation of new

bone. C. Zou et al. used the natural polymer collagen for the fabrication of porous β-

TCP/collagen composites [23]. They were able to achieve a porosity of 95 % with pore sizes

of 50-100 µm. The authors reported that the bone was fully regenerated after 12 weeks in

147

animal testing. Z.-J. Zhu et al. prepared β-TCP/ polylactide composites that showed promising

results [24]. The authors used this combination to prevent early degradation through the

neutralization of alkaline degradation products of β-TCP and the acidic degradation product

of polylactide. Kikuchi et al. added poly(L-lactide-co-glycolide-co-ε-caprolactone) to β-TCP

to stabilize the pH of 6-7 during the acidic degradation of the polyester through the

dissolution of β-TCP [25]. The pH of pure polymer solutions, in contrast, decreased to pH 3

after the degradation to glycolic and L-lactic acid.

So far, only few examples of composites that combine β-TCP with microgels or microspheres

exist in literature. S.M. Kuo prepared chitosan microspheres of roughly 200 – 400 µm in

diameter and used them to encapsulate β-TCP in the interior [26]. Chitosan is an antibacterial

natural polymer that is able to facilitate wound healing. Many studies have been performed to

examine its release properties, primarily of drugs [27][28][29]. M. Kucharska et al. pursued this

idea and demonstrated the potential of β-TCP/chitosan microspheres as possible bone

scaffolds [30]. They obtained a 3D structure through the acidic dissolution and consequent

agglomeration of the chitosan spheres. The material had pores with sizes of ~ 280 µm for the

highest β-TCP concentration (i.e. 10 wt-%), but the porosity of 20 – 25 % failed to copy the

typical porosity of bone (20 – 90 %). Y. Wang prepared microspheres of poly(lactid acid) and

added β-TCP to increase the biocompatibility [31]. The spheres had a higher pore volume

compared to pure poly(lactic acid) spheres, but did not improve the compressive modulus.

In this chapter, PVCL-based microgels were chosen as a binder for β-TCP because of their

high elasticity and water adsorption capacity. In water, they are thought to bind β-TCP

particles through intermolecular and interparticular hydrogen bondings. The size and the

content of microgels in the mixture are likely to influence the porosity of the composite.

Results discussed in this chapter were already published in Biomed. Tech. 2015, DOI:

10.1515/bmt-2014-0141, pii: /j/bmte.ahead-of-print/bmt-2014-0141/bmt-2014-0141.xml.

148

10.2.2 Synthesis Procedure

Microgel synthesis Microgels were prepared via precipitation polymerization in water.

Appropriate amounts of VCL, Vim, BIS, and AAEM (see Table 10.3) were mixed in 500 mL

of water and heated up to 70 °C while stirring at 200 rpm under nitrogen atmosphere. After

stirring for 60 min, the initiator AMPA (0.16 g, 0.59 mmol) was added. The reaction was

allowed to carry on for 4 h. After the synthesis, the samples were purified with dialysis for 3 d

with a composite regenerated cellulose membrane from Millipore (NMWCO 30,000). Four

different samples with varying amount of Vim were prepared.

Table 10.3 Amounts of monomers used for the microgel synthesis.

Amount of monomer / g

Sample VCL Vim BIS AAEM

0 % Vim 6.000 - 0.240 1.100

5 % Vim 6.000 0.367 0.240 1.100

Preparation of ß-TCP Phase pure ß-TCP was prepared by calcination using a calcium

deficient hydroxyapatite with 3 wt% calcium hydrogen phosphate (CHP) at 1000 °C for 2 h.

CHP was added to adjust the Ca/P ratio to exactly 1.5. Chemical and phase purity was

confirmed by X-ray fluorescence (XRF) spectroscopy and X-ray diffraction (XRD)

spectroscopy.

Preparation of microgel- ß-TCP composite material Various concentrations of microgel

dispersions were mixed with an appropriate amount of ß-TCP and either directly freeze-dried

or spray-dried (Mini Spray Dryer B-290, Büchi) at 220 °C to yield granulates with a microgel

content of 10 or 5 wt%. Before spray-drying, the suspensions were milled on a roller platform

with zirconia milling balls (in each case 1 kg of 1 cm and 2 cm in diameter) for 1 day. After

the addition of 1 to 2 drops CONTRASPUM CONC., the suspensions were sieved with a

125 µm sieve.

149

10.2.3 Results and Discussion

Microgels with 0 and 5 mol-% Vim (henceforth called PVCL-Vim) were synthesized. AFM

images show the formation of monodisperse, spherical particles (see Figure 10.19). The

microgel suspensions were mixed with ß-TCP to obtain composite materials with 5, 10, and

15 wt-% microgel content (henceforth called e.g. C10-5% meaning the composite containing

10 wt-% microgel in the suspension with a Vim content of 5 wt-%).

Figure 10.19 (a) AFM image of 5 wt-% Vim microgel; (b) FTIR spectrum of PVCL-Vim

microgels, pure ß-TCP, and the composite C10-5%.

The interaction between the microgel network and β-TCP was investigated with FTIR.

Therefore, FTIR spectra of PVCL-Vim microgels, pure β-TCP, and the composite C10-5%

were recorded (see Figure 10.19). Prominent peaks for β-TCP are at ~ 900 – 1200 cm-1 (PO43-

stretching mode) and 553 and 606 cm-1 (both PO43-

vibration mode). PVCL-Vim microgels show

characteristic peaks at 2921 cm-1 (C-Hring stretching mode), 2856 cm-1 (C-Hstretching), 1633 cm-1

(amide I), 1485 cm-1 (C-Nstretching), 1437 cm-1 and 1418 cm-1 (both C-Hdeformation bands), 1264

cm-1 (C-Hring in-plane bending and C-Nring stretching mode), 1230 cm-1 (C-Hring in-plane bending and C-Nring

stretching mode), and 975 cm-1 (ring deformation mode) [32]. For the amide I band of PVCL, a shift

from 1633 cm-1 to 1618 cm-1 can be detected in the composite spectrum. This indicates that

the C=O group of PVCL interacts with the water molecules on the surface of β-TCP. Further

possible interactions between Vim and β-TCP cannot be detected due to the low concentration

of Vim in the sample. It is likely, however, that the electron pair of nitrogen in Vim interacts

with Ca2+ (see Figure 10.20).

To confirm this data, electrophoretic measurements were performed (see Figure 10.21a).

Uncharged PVCL microgels show no pH dependency. A slight negative charge is caused by

the initiator and results in the colloidal stabilization of the particles. PVCL-Vim microgels

a b

150

with 5 % Vim exhibit a positive charge over a broad range of pH due to the protonation of

Vim. The particles become neutral at the isoelectric point of ~ 9.5. β-TCP is neutral at pH < 5

Figure 10.20 Interactions between PVCL-Vim microgel and β-TCP.

and becomes negative with increasing pH. Measurements below pH 5 were not possible due

to aggregation of β-TCP. A mixture of 5 wt-% microgel in the suspension containing 5 wt-%

Vim has a pH of ~ 7. Thus, at pH 7, a strong interaction between positively charged microgels

and negatively charged β-TCP can be assumed in the composite.

Figure 10.21 (a) Electrophoretic mobility with varying pH. Blue curve: 5 wt-% Vim

microgel; black curve: uncharged PVCL microgel (reference); green curve: pure β-TCP. (b)

XPS spectra of pure β-TCP (green curve), 5 wt-% Vim microgel (blue curve), and composite

material C10-5% (red curve).

XPS measurements reveal the chemical composition of the composite (Figure 10.21b). The

microgel sample with 5 wt-% Vim shows signals at 282 eV (C 1s), 396.5 eV (N 1s) and 528.5

eV (O 1s). Signals in the XPS spectrum of β-TCP at 130 eV and 344 eV were assigned to Ca

2p and P 2p, respectively. The spectrum of the composite C10-5% exhibits signals that are a

combination of both. This further supports the assumption of a successful incorporation of the

microgel into the composite.

a b

151

An important property of a composite that is a possible substitute for bone material is a high

and fast adsorption of water while at the same time it has to be ensured that the material stays

stable and does not decompose. Therefore, the water uptake through the increase in the

sample weight as a function of environmental humidity was investigated using a humidity-

controlled thermomechanical analyzer. The shape of the resulting adsorption isotherms gives

useful knowledge of the microstructure of materials [33].

Figure 10.22 Water sorption for (a) 0 wt-% PVCL-Vim microgel (red curves) compared to

composite C10-0% (blue curves); (b) 5 wt-% PVCL-Vim microgel (red curves) compared to

composite C10-0% (blue curves). Graphs show adsorption and desorption isotherms; (c)

water sorption in porous materials.

Figure 10.22 gives the adsorption and desorption isotherms for pure β-TCP, 0 and 5 wt-%

PVCL-Vim microgels, and their respective composites C10-0% and C10-5%. Measurements

were performed at 37 °C to mimic body temperature. Pure 0 and 5 wt-% PVCL-Vim

microgels show a high water uptake of 32 and 34 %, respectively. The amount is slightly

higher for microgels containing Vim due to the increased hydrophilicity. In the respective

composites, the water sorption is distinctively lower, it decreased to 4 % for both 0 and 5 wt-

a b

c

152

% PVCL-Vim microgels. The isotherms of pure β-TCP are given as a reference to illustrate

the poor water sorption of the material.

For many industrial materials, S-shaped (type II) isotherms are measured (see Figure 10.22c)

[33]. These curves give information about the type of bound water in the porous material. At

low pressures, water is tightly bound by the material. Between the first and second inflection

point, water is loosely bound, while at high pressures, the water molecules are able to move

freely.

In the graphs measured for the microgels and the composites, no sigmoidal curve can be

observed. Instead, only two areas can be distinguished that resemble the areas characteristic

for loosely bound and free water. This can be explained by the lyophilization of the microgel

before the measurement. Through handling at ambient conditions, the microgel adsorbs water

from the air, thus tightly bound water is already present prior to each measurement. If further

water is tightly bound in the microgel network at the beginning of each measurement, the

amount is assumed to be so small that the first inflection is not or only barely visible.

Furthermore, the BET surface area can be calculated from these measurements. The BET

surface area was developed by Brunauer, Emmett, and Teller in 1938 [34] and is an important

factor since the surface area of the pores influences for instance the rate of biodegradation and

chemical reactions [33]. The prerequisite for this is that at low pressures, i.e. p/p0 = 0.05 – 0.35

(with p = pressure at equilibrium; p0 = vapor pressure at saturation), the sorption is strictly

reversible meaning a free movement of the adsorbate on the surface of the adsorbent. Further,

it has to be ensured that no chemical reaction takes place.

The theory is based on the assumption that the adsorption of e.g. gas or liquids on a surface

takes place beginning with a single layer of adsorbate molecules advancing to multiple layers

of adsorbate at saturation [33]. The measurement of grams of absorbate molecules that form a

monolayer, called the monolayer capacity, allows the determination of the number of

molecules and thus the surface area. The actual value of the surface area is dependent on the

type of molecule used for the measurement. For instance, the water molecule is smaller in size

than a nitrogen molecule, therefore water is better able to penetrate into pores and the

resulting surface area is bigger.

The ratio p/p0 can be obtained by the division of RH (in %) by 100%. The change in weight

wt-% is converted into the adsorbed water volume V as follows:

𝑉 = 𝑡𝑜𝑡𝑎𝑙 𝑤𝑒𝑖𝑔ℎ𝑡−𝑑𝑟𝑦 𝑚𝑎𝑠𝑠

𝑑𝑟𝑦 𝑚𝑎𝑠𝑠 (1)

153

The total weight is determined at each point of RH, while the dry mass was determined after

lyophilization.

Plotting p/p0 against (p/p0) / V(1-p/p0), the intercept and the gradient of the resulting graphs

can be used to calculate the BET surface area (see Figure 10.23):

𝐵𝐸𝑇 =(

1

𝑖𝑛𝑡𝑒𝑟𝑐𝑒𝑝𝑡+𝑔𝑟𝑎𝑑𝑖𝑒𝑛𝑡)

𝑀𝑊∗ 𝑁 ∗ 𝐴 (2)

with MW = MW of adsorbate (i.e. H2O, 18.01 g/mol); N = Avogadro’s number ( 6.022 * 1023);

A = molecular area adsorbate (10.78 Å² for water [35]). The BET surface areas are given in

Table 10.4.

Figure 10.23 Graphs for the calculation of the BET surface area.

Table 10.4 BET surface areas for microgel samples and composite material.

Sample 0 wt-% Vim µ-gel 5 wt-% Vim µ-gel C10-0% C10-5%

BET [m²/g] 163 204 18.5 18.2

Pure microgels have an approximately tenfold higher surface area than the composite

material. BET surface areas values for pure β-TCP are given in literature as 1-2 m²/g [6][36].

Therefore, the incorporation of microgels into the composite material greatly influences the

surface area values and changes the textural properties of β-TCP.

The water sorptions for the microgels and their respective composite materials as a function

of time are given in Figure 10.24. The sorption rates for all samples decreased with increasing

humidity, i.e. a longer time was needed to reach equilibrium. Microgel samples containing

5 wt-% Vim have a longer sorption time than uncharged microgels due to the higher

hydrophilicity of the positively charged microgels. Furthermore, in accordance with Figure

10.22, it can be seen that pure microgel samples take up higher amounts of water than their

respective composite material.

154

Figure 10.24 Sorption rates of pure β-TCP, microgel samples and their respective composite.

The thermochemical stability of the microgels as well as the composites was analyzed (see

Figure 10.25). The graph for pure β-TCP is shown as reference to demonstrate that β-TCP is

stable until temperatures to 850 °C. For pure microgels, the first weight loss occurs at around

100 °C resulting from the loss of water. In the range between 150 and 350 °C, acrylic groups

are decomposed. The third step at 441 °C is due to the decomposition of the polymer

backbone.

Figure 10.25 Thermogravimetrical analysis of 5 wt-% PVCL-Vim microgel, pure β-TCP and

composite material with various microgel content in suspension.

The composite materials show the same decomposition behavior. The polymer decomposition

is shifted slightly to 450 °C indicating an improved thermal stability. At 600 °C, 96.4 %,

90.7 %, and 87.8 % of remaining β-TCP were recorded for the samples C5-5%, C10-5%, and

C15-5%, respectively. This data further indicates that the desired amount of microgel was

assured in the suspension.

Further testing whether the composites are suitable material for bone tissue repairing, 3 D

powderbed printing is a convenient tool to create samples that can be tested regarding their

155

mechanical strength. To ensure that the composite material is applicable for 3 D powderbed

printing, knowledge about the morphology and particle size distribution is essential.

Figure 10.26 SEM images of composites (a) C10-5%; (b) C5-5%; and (c) pure β-TCP; all

after spray-drying.

Figure 10.26 shows that the composites consist of granulates (a,b). The granulate size of

composite C5-5% is bigger compared to C10-5%. The spherical form of granulates as well as

the grain size (< 1µm) makes it difficult to distinguish between β-TCP and PVCL-Vim

microgels. β-TCP on the other hand forms only few granulates, while the main material

consists of single grains with an average size of 1.46 µm (c). This supports the assumption

that microgels act as glue for the grains to form granulates. The printed sample C10-5% can

be seen in Figure 10.27.

Figure 10.27 Printed sample C10-5%.

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10.2.4 Summary

This work aimed at the preparation and characterization of β-TCP/microgel composites. For

this, cationic PVCL/AAEM/Vim microgels were used because of the possible interactions

that arise from the carboxylic group of PVCL as well as the free electron pair of the nitrogen

of Vim and Ca2+ and PO43- in β-TCP. The microgels were further chosen as binding material

between β-TCP particles because of their high water content. This could be proven with FTIR

spectroscopy and TEM. TGA measurements confirmed that the sintering process pyrolyzes

the microgels and leaves pure β-TCP. It was shown, however, that the presence of microgels

facilitates the pressing of the composites and the consequent spray-drying process that was

not discussed in this chapter. Thermoanalytical measurements demonstrated that the

composite material absorbs a higher amount of water due to the excellent water absorption

capability of microgels. The BET surface, i.e. the surface area of pores inside the material, is

also increased by a factor of 10 in comparison to pure β-TCP. A poor stability of the material

in water, however, suggests further improvement of the material with regard to possible

application as bone scaffolds. In future, the microgels are also applicable to be loaded with

osteogenic or osteoinductive drugs to further enhance bone regeneration.

157

10.2.5 Literature

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[2] K. A. Hing, Philos. Trans. A. Math. Phys. Eng. Sci. 2004, 362, 2821–50.

[3] J. M. Rueger, Orthopade 1998, 27, 72–79.

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[5] S. M. Schnürer, U. Gopp, K.-D. Kühn, S. J. Breusch, Orthopade 2003, 32, 2–10.

[6] K. Adamska, M. Woźniak, A. Voelkel, Ann. UMCS, Chem. 2010, 65, DOI

10.2478/v10063-010-0004-4.

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158

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Heng, et al., Ann. Rheum. Dis. 2013, 1, 1–11.

[28] A. G. Sullad, L. S. Manjeshwar, T. M. Aminabhavi, Polym. Bull. 2014, 72, 265–280.

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Pharm. Pharm. Sci. 2014, 6, 94–100.

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Mater. 2005, 280-283, 1613–1618.

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159

11 Coating of Surfaces: Protein-repellant Surfaces with Zwitterionic Microgels

11.1 Introduction

A material that comes in contact with the human body triggers a biological response that can

lead to an immune reaction, thrombosis or embolism [1][2]. The design of so-called non-

fouling, hemocompatible materials is essential and required in the biomedical field, especially

for the development of contact lenses, implants, prostheses, drug delivery carriers, and

sensors [3][4]. In general, a non-fouling material should be hydrophilic, overall electrically

neutral and preferably a hydrogen bond acceptos [5]. Many authors agree that a hydrophilic

surface minimizes protein adhesion[2][1][3]. The surface should be electrically neutral so no

electrostatic interactions between the surface and the protein occur. The absence of hydrogen

bond donors might reduce the amount of hydrogen bonds [3].

The exact process of protein adhesion onto a surface is not yet fully understood. The

hydration state of a surface, however, has a great influence on the adhesion process of

proteins onto a surface. K. Ishihara et al. describe this phenomenon as follows [6]: The

adsorption of proteins onto a polymer surface in aqueous media results in the loss of bound

water by the protein and thus a conformational change. This causes irreversible adsorption of

the protein. If the water state on the surface, however, is similar to the surrounding aqueous

solution, no water needs to be released by the protein and no conformational change takes

place. Thus, proteins can reversibly come into contact with a surface.

Not only coating with proteins leads to fouling, but also the coating with bacteria. Here, too,

the exact adhesion process is unclear. G. Cheng describes the process of the creation of an

undesired biofilm. Bacteria deposit non-specifically and reversibly on the surface [7].

Subsequently, bacteria begin to produce exopolysaccarides (EPS) that enclose the bacteria

film. Due to the charged and neutral polysaccharide groups, the EPS is able to filter nutrients

from the surrounding liquid medium and further protect the bacteria film from the body’s

immune response.

Betaines are known for their ultra low-fouling properties. Zhang et al. used carboxybetaine

(CB) to polymerize nanogels that encapsulate dextran as a model drug and Fe3O4 as imaging

reagents for magnetic resonance imaging (MRI) [8]. They could show in their study that there

is a low uptake amount of macrophage cells indicating little interactions with the immune

system. CB is therefore a promising candidate for non-fouling materials and a possible

substitute for prevalent polyethylene glycol (PEG) and phosphorylcholine (PC). These

materials are widely used for e.g. marine coatings, but offer various disadvantages: PEG, for

instance, is prone to oxidation, and both PEG and PC are short of functional groups that might

160

immobilize ligands [4]. Even before the use of PEG and PC, neutral poly-2-hydroxyethyl

methacrylate (PHEMA) was used for the coating for non-fouling materials, but several

authors reported the considerably decreased anti-fouling properties when in contact with

human blood serum and plasma [9][10][11]. S. Chen et al. interpreted the high resistance of

zwitterions to protein adhesion as the result of the strong hydration through ionic solvation.

They prepared zwitterionic hydrogels and showed that the adhesion of both IgG and

fibrinogen is very low under physiological conditions [4]. Z. Zhang et al. confirm that the

tightly bound water around the negatively charged pendant group is the driving factor behind

the non-adhesion behavior [12]. Especially in zwitterionic hydrogels, the ratio between free and

bound water is higher as compared to other hydrophilic polymers. Y.-J. Shih and Y. Chang

found out that the non-fouling behavior of SB can be tuned by the molecular weight of the

polymer [3]. A polySB polymer with a molecular weight of 135 kDa exhibited both the

strongest hydration capability for binding water and subsequently the most effective non-

fouling behavior.

11.2 Experimental Part

For the coating of surfaces, zwitterionic microgels from my diploma thesis were used. They

have a zwitterionic content of 0, 2, and 5 wt-%.

The coating procedure is described in the Experimental Part in chapter 4.

161

11.3 Results and Discussion

Zwitterionic microgels with a varying amount of phosphobetaine (PB) as well as pure PVCL

microgels were coated on Si-wafers after plasma activation to study the interfacial properties

of the microgels. Si-wafers were used because of the facile deposition of a variety of

polymers, their clear chemical structure, and their wide applications as various biomaterials.

Figure 11.1 Schematic representation of zwitterionic microgels on hydrophilic plasma-treated

Si-wafer.

AFM images show the surface topography before and after the coating. The images reveal

that betaine-free microgels form a homogeneous coating layer with equal distances between

the particles and no gaps or voids (see Figure 11.2). Microgels with 2 wt-% PB also form a

monolayer with particles closer to each other. The smaller distance is due to the zwitterionic

nature of particles and charges that attract each other. Particles with 5 wt-% PB form a

thorough multilayer with very small interparticle distances.

The roughness of the surfaces was analyzed quantitatively by determining the rms roughness.

According to Y. Xu, the protein repellence properties of a surface are directly linked to its

roughness [13]. A surface is considered to be smooth when it has an rms below 0.5 µm [14]. The

native wafer (not shown) has a very low rms of 7 nm. The values for the surface roughness

decrease slightly from microgels with 0 wt-% to 2 wt-% and 5 wt-% PB and are 19.3 ±

5.8 nm, 14.0 ± 5.1 nm, and 10.5 ± 3.7 nm, respectively. A comparison of those values with

the PDIs of the microgels (0.048, 0.031, and 0.029, respectively) indicates that there is a

direct correlation between the polydispersity of the sample and the surface roughness.

Another explanation is the structure of the particles. All rms are still below the microgel

particle sizes by a factor of roughly 10 meaning that the surface looks smooth on a length

scale of the particle size. On a molecular level, the microgels have a “hairy” surface with

loose ends that form loops and dangling ends that might lead to a higher surface roughness.

An increased content of zwitterionic PB, however, leads to the formation of inter- and

intramolecular ionic bridges between the negatively charged quaternary ammonia group and

162

the positively charged phosphonium group, thus “gluing” the tangling loose ends to the

particle surface.

Figure 11.2 Coating of Si-wafer with (a) pure PVCL microgels; (b) 2 wt-% PB; (c) 5 wt-%

PB. Insets show the respective contact angle measured in air at room temperature.

Furthermore, the contact angle of the coatings was determined to investigate the wettability of

the microgel-coated surfaces. Hydrophilic surfaces have a contact angle of 0 – 6 ° and

hydrophobic surfaces of 50 – 70 ° [15]. The contact angle was measured immediately after the

deposition of the water droplet and again after 2 min. A disparity in the contact angle over

time can be attributed to two factors: swelling of the dried microgels and reorientation of

functional groups on the surface. Table 11.1 gives the values for all coated surfaces. The

contact angle of uncoated Si-wafer is given in literature as ~ 11 ° [14]. All coated surface have

a significant higher contact angle than uncoated Si-wafer due to the hydrophobic backbone of

both PB and PVCL. For the coated surfaces, it is apparent that the surface becomes more

hydrophilic with increasing PB content in the microgels due to the zwitterionic nature of PB.

Table 11.1 Contact angles of PB microgels with varying PB content on Si-wafer in air at

room temperature. The values are the average of three separate drops on different areas.

PB content / % Contact angle immediately

after deposition / °

Contact angle after 2 min

of deposition / °

0 56.4 ± 3.10 49.8 ± 0.35

2 50.1 ± 4.32 36.5 ± 4.95

5 31.2 ± 5.64 16.5 ± 2.24

PB microgels coated on Si-wafer were exposed to BSA BODIPY FL conjugate protein to test

the effect of zwitterionic coatings on protein adsorption. BSA was chosen due to its well-

known properties (IEP ~ 5) and its wide-ranged use as a model protein. Measurements were

carried out in PBS buffer (pH ~ 7.4) to mimic typical physiological condition. Figure 11.3

shows fluorescence microscopy images after the incubation with BSA BODIPY FL

a b c

163

conjugate. Betaine-free PVCL microgels are shown on the respective left side as a reference.

High amounts of protein adsorb to the surface as indicated by the light green coloring. BSA

will bind to the hydrophilic silicon oxide surface though BSA has almost no net charge at pH

~ 5 [16]. Still, the local positive charges of BSA are attracted by the slightly negatively charged

oxide [17].

Figure 11.3 Images of protein BSA adhesion on Si-wafers coated with (a) 2 wt-% PB; (b)

5 wt-% PB; and (c) 10 wt-% PB. The reference PVCL microgels containing no PB is given on

the respective left side. (d) Determination of cell adhesion with human dermal fibroblasts on

coated Si-wafer.

The coating with zwitterionic microgels greatly affects the adhesion of proteins. The light

green color vanishes increasingly with a growing content of PB in the microgel. Since

electrostatic attraction is one of two main factors for protein adhesion (besides hydrophobic

interactions), a decrease in protein adhesion on zwitterionic surfaces confirms the

electroneutrality of the surface layer. These results are in agreement with earlier observations

made by E. Hatakeyama et al. [18]. They compare quaternary phosphonium polymers with

PEG-based polymers and state that some analogues have the same or even better protein-

repellency than PEG-based polymers. These results are also in agreement with the results

Reference Reference

Reference

a b 2 wt-% PB 5 wt-% PB

c 10 wt-% PB d

164

given by E. F. Murphy et al. [17]. They found that the outer layer of 25 Å of a thin layer of

phosphoryl choline on a surface consisted of approximately 86 % water. Therefore, it can be

assumed that an increase of PB leads to an increasing steric hindrance that hinders protein

deposition. Because methacrylate groups increase the adhesion of proteins on a surface [17],

this suggests further that the zwitterionic compound in the microgel is located on the particle

surface.

E. Ostuni et al. describe how the adsorption of proteins entails cell and bacteria adhesion [19].

A surface that is capable to prevent protein adsorption should thus be able to suppress cell

adhesion. Therefore, cell tests with human dermal fibroblasts were performed. Figure 11.3d

presents the results after 24 h of incubation. Pure PVCL microgels as well as PVCL microgels

with a low amount of PB exhibit the same high cell adhesion behavior as the uncoated

surface. In contrast, the cell adhesion decreases significantly with increasing PB content.

After coating, 73% less cell adsorption was observed in comparison to the surface coated with

the betaine-free or low amount of PB coated surface.

Both the reduced cell and protein adhesion can be attributed to an enhanced hydration of the

surface through the zwitterionic PB.

In a next step, samples with a very high content of betaine are examined. The microgels were

synthesized via emulsion polymerization (described in chapter 5). A sulfobetaine sample with

a monomer ratio of VCL:SB = 1:1 as an exemplary sample was coated on a glass surface and

dried at air overnight. Similar to the results discussed above, the sample was exposed to BSA

tetramethylrhodamine conjugate protein and analyzed with a light microscope as well as

contact angle measuring device.

Figure 11.4a shows the improved protein repellency of microgels with a high betaine content

of ~ 50 mol-%. No protein can be seen on the coated surface, while the reference surface

coated with pure PVCL microgels shows a bright red indicating a complete protein coating.

The protein-repellency is greatly improved compared to surfaces coated with zwitterionic

microgels with a low betaine content (see Figure 11.3). The surfaces are further more

hydrophilic with a contact angle of 9 ± 2 °.

165

Figure 11.4 (a) Protein-repellency of zwitterionic microgels with a monomer ratio of

VCL:SB = 1:1. (b) Contact angle measurement.

It is obvious that for a possible application for protein-repellent catheters in the medical field,

it has to be ensured that zwitterionic microgels are biocompatible. Zwitterionic microgels

(VCL:SB = 1:1) as well as pure PVCL microgels as reference were tested with regard to their

cytotoxicity. A negative test, however, states only that a material contains no harmful

substances and causes no harmful effects, it can therefore only be understood as an indication

whether a material is biocompatible, not as a final proof.

In the following, the influence of coated glass disks on cell adhesion during osteogenic

differentiation was analyzed for three different types of cells: human mesenchymal stem cells

(hMSC), diploid fibroblast cells (dFb), and mouse fibroblast cells (L929). Fibroblasts and

mesenchymal stem cells are the natural precursor cells of bone tissue [20]. Fibroblasts are cells

of connective tissue that differentiated from mesenchymal stem cells and form collagen to

strengthen the extracellular matrix. Mesenchymal cells are cells from bone marrow that are

still undifferentiated and are able to differentiate in various cell types such as bone or

cartilage. Cytotoxicity experiments were conducted by cand. med. P. Kandt and S. Neuss-

Stein of the Institute of Pathology and at the Biointerface Group of Helmholtz Institute for

Biomedical Engineering (RWTH Aachen, University Hospital) in the framework of the ERS

Project “Antimicrobial and tissue regenerating nanogels for implant coating”. Representative

images are given in Figure 11.5 for the microgel sample VCL:SB = 1:1 prepared via inverse

mini-emulsion (see Chapter 5) and for pure PVCL microgels as a reference.

9 ± 2 ° b

166

Figure 11.5 Fluorescent (FDA/PI) live/dead staining after osteogenic differentiation of (a) +

(d) dFb; (b) + (e) hMSC; and (c) + (f) L929. (a) – (c) PVCL microgels; (d) – (f) Microgels

prepared via inverse mini-emulsion with a monomer ratio of VCL:SB = 1:1. The scale bars

represent 75 (a,c,d,f) and 100 (b,e) µm. Images were kindly provided by cand. med. Pierre

Kandt.

Cells show either typical flat and spindle-shaped (dFb and hMSC) or spherical morphology

(L929). The ratio between living (green) and dead (red) cells indicates whether the surface is

cytotoxic. According to ISO 10993, microgels are not considered cytotoxic if not more than

5 % of dead L929 cells are present. This is true for both microgel samples and both pure

PVCL and zwitterionic microgels are therefore not cytotoxic. Images show that the surfaces

coated with zwitterionic microgels display an improved cell response. Figure 11.5c reveals

that a surface coated with PVCL exhibits more dead cells than a surface coated with SB

(Figure 11.5f). Furthermore, SB coated surfaces are better coated than PVCL coated surfaces.

a b c

d e f

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11.4 Summary

This chapter showed the possible application of zwitterionic microgels as coating material in

the biomedical field. Coating experiments on Si-wafer as a model surface resulted in

homogeneous layers without gaps and voids. Contact angle measurements showed that coated

surfaces are less hydrophilic than uncoated surfaces due to the hydrophobic polymer

backbone. Still, an increase in PB leads to a decrease in the contact angle. Amongst others, K.

Ishihara et al. demonstrated that a high uptake of water is a prerequisite for successful protein

repellence [6]. Fluorescence active BSA was used to test the protein-repellent properties. Data

show that an increase in PB content results in a decrease in protein adhesion. This is due to

the improved hydration of the surface. An improvement in lower cell adhesion further

demonstrates the possible potential of zwitterionic microgels for protein and cell repellent

surfaces. Experiments were repeated for microgels samples with a high SB content that were

prepared via inverse mini-emulsion. Their protein-repellent properties are increased compared

to PB microgels with a low betaine content.

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11.5 Literature

[1] Y. Chang, W.-Y. Chen, W. Yandi, Y.-J. Shih, W.-L. Chu, Y.-L. Liu, C.-W. Chu, R.-C.

Ruaan, A. Higuchi, Biomacromolecules 2009, 10, 2092–100.

[2] Y. Chang, W. Chang, Y. Shih, T. Wei, G. Hsiue, Appl. Mater. Interfaces 2011, 3,

1228–1237.

[3] Y.-J. Shih, Y. Chang, Langmuir 2010, 26, 17286–94.

[4] S. Chen, S. Jiang, Adv. Mater. 2008, 20, 335–338.

[5] E. Ostuni, R. G. Chapman, R. E. Holmlin, S. Takayama, G. M. Whitesides, Langmuir

2001, 17, 5605–5620.

[6] K. Ishihara, H. Nomura, T. Mihara, K. Kurita, Y. Iwasaki, N. Nakabayashi, J. Biomed.

Mater. Res. 1998, 39, 323–30.

[7] G. Cheng, Z. Zhang, S. Chen, J. D. Bryers, S. Jiang, Biomaterials 2007, 28, 4192–9.


[9] C. Zhao, L. Li, Q. Wang, Q. Yu, J. Zheng, Langmuir 2011, 27, 4906–13.

[10] B. Mrabet, M. N. Nguyen, A. Majbri, S. Mahouche, M. Turmine, A. Bakhrouf, M. M.

Chehimi, Surf. Sci. 2009, 603, 2422–2429.

[11] C. Yoshikawa, A. Goto, Y. Tsujii, T. Fukuda, T. Kimura, K. Yamamoto, A. Kishida,

Macromolecules 2006, 39, 2284–2290.

[12] Z. Zhang, T. Chao, L. Liu, G. Cheng, B. D. Ratner, S. Jiang, J. Biomater. Sci. Polym.

Ed. 2009, 20, 1845–59.

[13] Y. Xu, M. Takai, K. Ishihara, Biomaterials 2009, 30, 4930–8.

[14] R. S. Faibish, W. Yoshida, Y. Cohen, J. Colloid Interface Sci. 2002, 256, 341–350.

[15] G. Kissinger, W. Kissinger, Phys. Status Solidi 1991, 123, 185–192.

[16] Y. Mukai, E. Iritani, T. Murase, J. Memb. Sci. 1997, 137, 271–275.

[17] E. F. Murphy, J.R. Lu, J. Brewer, J. Russell, J. Penfold, Langmuir 1999, 15, 1313–

1322.

[18] E. S. Hatakeyama, H. Ju, C. J. Gabriel, J. L. Lohr, J. E. Bara, R. D. Noble, B. D.

Freeman, D. L. Gin, J. Memb. Sci. 2009, 330, 104–116.

[19] E. Ostuni, R. G. Chapman, M. N. Liang, G. Meluleni, G. Pier, D. E. Ingber, G. M.

Whitesides, Langmuir 2001, 17, 6336–6343.

[20] D. Duarte, C. Panfil, K. Raupach, S. Neuss, M. Weber, C. Otten, U. Reisgen, H.

Fischer, Biomed. Eng. / Biomed. Tech. n.d., 40.

169

12 Summary

The aim of this work was the synthesis of polyampholyte microgels and the understanding of

the influence of distribution of ionizable groups on the microgels’ properties, their

interactions with proteins and possible applications.

In the first part, both zwitterionic and ampholyte microgels based on poly(N-

vinylcaprolactam) (PVCL) and poly(N-isopropylacrylamide) (PNIPAm) were prepared and

the ionizable groups were incorporated either statistically, as core-shell or Janus-like.

Chapter 5 dealt with the synthesis and characterization of zwitterionic particles. In the first

part, particles with a low amount of betaine (i.e. up to 10 wt-%) were prepared with

surfactant-free free-radical precipitation polymerization in water. Particles containing

sulfobetaine (SB) have a hydrodynamic radius of ~ 200 nm independent on the amount of SB

incorporated. Since zwitterionic microgels are able to crosslink even without the presence of a

classical crosslinker such as N,N’-methylenebis(acrylamide) (BIS) due to the formation of

intra- and intermolecular ionic bridges between the sulfo group and the quaternary ammonium

group, microgels without the use of BIS (“non-crosslinked”) are prepared and compared to

classically crosslinked particles (“crosslinked”). The softness of the particles was investigated

with AFM. It could be shown that crosslinked particles are stiffer than non-crosslinked

particles. This was attributed to the increase in inter- and intramolecular ionic bridges. The

degradation of crosslinked and non-crosslinked particles after the addition of salt is studied

with FTIR, DLS, and FESEM spectroscopy. It could be seen that particles degrade completely

in 1 M of NaCl. DLS measurements show that the particle size decreases from ~ 200 nm to ~

20 nm. Microscopy images confirm that the previous monodisperse, spherical particles

degrade to non-defined polymer aggregates.

Since it is shown that the incorporation of amounts of betaines higher than 10 wt-% is not

possible using free-radical precipitation polymerization, particles with a higher amount of SB

are prepared using free-radical inverse mini-emulsion in the second part of chapter 5. The

focus of particle characterization was on the temperature-sensitivity of the particles due to an

effect not seen for microgels with a low betaine content: beginning from a monomer ratio of

VCL:SB = 1:1, microgels not only exhibited an LCST, but also a UCST. The first is

generated by the collapse of PVCL polymer chains, the latter by the collapse of polySB

chains. The position of the phase transition temperatures is strongly dependent on the amount

of SB incorporated. An increasing amount of SB shifted both UCST and LCST to higher

temperatures. This behavior was both monitored with DLS and UV-Vis measurements.

Chapter 6, 7, and 8 discussed the synthesis of ampholyte microgels with itaconic acid (IA)

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and vinylimidazole (Vim) with different architectures: statistically, core-shell, and Janus-like,

respectively. The amounts of ionizable groups was varied and verified with FTIR

spectroscopy. The varying distributions of ionizable groups were confirmed with TEM after

staining of the carboxylic groups with U(Ac)3.

Microgels with a statistical distribution of ionizable groups were obtained by mixing all

monomers in water at 70 °C. DLS and electrophoresis showed that particles swell at high and

low pH, while they are in a collapsed state at the particles’ isoelectric point (IEP). It is

possible to tune the position of the IEP as well as the degree of swelling by the ratio between

ionizable groups. Particles with a symmetric ratio of ionizable groups have a typical V-shaped

curve displaying the hydrodynamic radius as a function of pH. In-situ AFM measurements in

water further affirmed that particles are stiffer at pH ~ IEP due to the collapse of the microgel

network compared to particles at low and high pH.

Core-shell microgels were prepared in a two-step synthesis wherein the shell was synthesized

after the complete synthesis of the core. DLS measurements showed that the location of the

negative charge in the core or in the shell greatly influences the swelling behavior of the

particles in different pH. Particles with IA in the core and Vim in the shell behaved like

microgels with a statistical distribution of ionizable groups, i.e. the hydrodynamic radius

exhibited a typical V-shaped curve with varying pH, though the degree of swelling is much

less pronounced at high pH than at low pH. An increase in shell thickness, however, led to an

intensified swelling of the shell at high pH. Particles with a reverse location of ionizable

groups, i.e. Vim in the core and IA in the shell, do not swell at high pH. This was attributed to

the fact that the shell is too “weak” to counteract the collapse of the particle core.

In chapter 8, the polymerization of NIPAm with either Vim or IA as comonomer was closely

investigated with calorimetry to obtain information about particle formation and monomer

reactivities. For the synthesis of ampholyte Janus-like particles, both microgel dispersions

(NIPAm-IA and NIPAM-Vim) were initiated separately and mixed after a defined amount of

time. TEM measurements confirmed the formation of Janus-like particles if the dispersions

were mixed after 1 min after both polymerizations were initiated.

The second part of this thesis looked closely at the influence of different microgel architecture

on the uptake and release of proteins. The uptake and release of cytochrome c (cyt-c) as a

model protein is investigated in chapter 9. Since cyt-c is positively charged over a broad range

of pH, while ampholyte microgels have an IEP of ~ 6.5, experiments were conducted at

different pH where microgel and protein have opposite or like charges. The kinetic of release

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was monitored with UV-Vis spectroscopy as well as optically due to the red color of the

protein’s heme group. Different triggers such as pH, temperature and salt solution were tested

for a complete or partial release of protein. SLS and DLS measurements showed the influence

of protein uptake on the microgels’ hydrodynamic radius and molecular weight.

The third part studied possible applications of the microgels prepared in the previous chapters.

Chapter 10 dealt with the use of charged microgels in biohybrid systems. The first part of the

chapter focused on zwitterionic microgels based on PVCL and phosphobetaine (PB) as

possible reaction vessels for the controlled growth of CaCO3 crystals. Two different synthesis

routes were tested to ensure crystal growth within the microgel particle. Following, the

maximum capacity of CaCl2 uptake was investigated gravimetrically as well as with ITC. As

expected, there was a higher capability to bind Ca2+ with a higher amount of PB. The

properties of CaCO3 were studied with XRD and FTIR. It could be shown that the common

calcite is the only crystal structure formed. SEM and TEM measurements optically confirmed

the formation and location of crystals and demonstrated that only specific synthesis

parameters lead to a crystal growth within the microgel network. Heating of the microgel

dispersion released the CaCO3 crystals as was shown with TEM and F-FFF.

The second part looked at the application of positively charged microgels based on PVCL and

Vim as bone substitute material in combination with β-tricalcium phosphate (β-TCP). In

collaboration with the university hospital, this work focused on the chemical properties of the

microgels with or without the presence of β-TCP, so the best parameters for the formation of

green bodies were identified. Here, FTIR spectroscopy was used for the confirmation of the

successful incorporation of Vim, while electrophoresis, TGA, and XPS measurements

confirmed the incorporation of Vim-microgels in the hybrid material. Water sorption

measurements were conducted to study the amount as well as the kinetics of moisture uptake

as well as determine the BET surface of the composite. SEM completed the characterization

of the biohybrid material.

Chapter 11 had a closer look on the properties of zwitterionic microgel coatings on Si-wafer.

In a first step, the right parameters for a complete formation of mono- or thin poly-layer were

investigated via AFM. Contact angle measurements delivered information of the

hydrophilicity of the coated surfaces. An increase in the betaine amount leads to a decrease in

hydrophilicity. Protein-repellency was examined with the help of fluorescence microscopy.

Here, surfaces coated with zwitterionic microgels showed great improvement over surfaces

coated with non-zwitterionic microgels.

172

13 Nomenclature

AAEM 2-(Methacryloyloxy)ethyl acetoacetate

AFM Atomic Force Microscopy

AGET ATRP Activators generated by electron transfer atom transfer radical polymerisation

AIBN Azobisisobutyronitrile

AMPA 2,2-Azobis(2-methylpropionamidine) dihydrochloride

APS Ammonium persulfate

APTMS (3-Aminopropyl)trimethoxysilane

BET Brunauer-Emmett-Tellett

BIS N,N’-Methylenebis(acrylamide)

BSA Albumin from borine serum

β-TCP β-tricalcium phosphate

CB Carboxybetaine

δ Chemical Shift

Ð Dispersity

DLS Dynamic Light Scattering

DSC Differential Scanning Calorimetry

EM Electrophoretic mobility

EPS Exopolysaccharides

F-FFF Flow Field-Flow Fractionation

FTIR Fourier-Transformed Infrared Spectroscopy

HAp Hydroxylapatite

HEMA 2-hydroxyethyl methacrylate

IA Itaconic Acid

IEP Isoelectric point

ITC Isothermal Titration Calorimetry

KPS Potassium persulfate

λ Wavelength

LCST Lower Critical Solution Temperature

MADIX Macromolecular Design via the Interchange of Xanthates

MMA Methylmethacrylate

Mn Number average molar mass

MRI Magnetic Resonance Imaging

NIPAm N-isopropylacrylamide

NMR Nuclear Magnetic Resonance

PB Phosphobetaine

PBS Phosphate Buffered Saline

PC Phosphorylcholine

PDI Polydispersitätsindex

PDMS Polydimethylsiloxan

PEG Polyethylene glycol

PHEMA Poly-2-hydroxyethyl methacrylate

PMMA Polymethylmethacrylate

PS Polystyrene

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PVCL Poly-N-Vinylcaprolactam

PNIPAm Poly-N-isopropylacrylamide

RAFT Reversible Addition-Fragementation Chain Transfer Polymerization

RH Hydrodynamic radius

rpm Rounds per minute

SAM Self-assembled monolayer

SB Sulfobetaine

SDS Sodium dodecyl sulfate

SEM Scanning Electron Microscope

SLS Static Light Scattering

Span 80 Sorbitan monooleate

T Temperature

TEM Transmission Electron Microscope

TEMED N,N,N’,N’-tetramethylethylenediamine

TGA Thermogravimetric Analysis

Tween 80 PEG 20-Sorbitan monooleate

UCST Upper Critical Solution Temperature

VCL N-Vinylcaprolactam

Vim 1-Vinylimidazole

VPTT Volume Phase Transition Temperature

wt-% Weight percent

XRD X-Ray Diffraction

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14 Appendix

Chapter 5.1 – Non-crosslinked zwitterionic microgels via precipitation polymerization

Figure 14.1 FTIR spectra of non-crosslinked zwitterionic microgels prepared via precipitation

polymerization. Inset shows zoom-in of peak at 1050 cm-1 belonging to the SO3- group of SB.

For determination of SB content, the calibration curve in Figure 14.2 was used.

Chapter 5.2 – Zwitterionic microgels via emulsion polymerization

Figure 14.2 (1) Calibration curve for determination of sulfobetaine (SB) amount in PVCL-SB

microgels via FTIR spectra. (2) FTIR spectra of zwitterionic microgels with SB prepared with

emulsion polymerization. Inset shows peaks at 1051 cm-1 (SB) and 1635 cm-1 (PVCL) that

were used for generation of calibration curve.

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Figure 14.3 Change of hydrodynamic radius RH of emulsion droplets over time with varying

amount of surfactant. Measurements were conducted at 70 °C. Inset shows the point of time

when the emulsion breaks.

Figure 14.4 Change of hydrodynamic radius RH of emulsion droplets over time with

surfactant concentrations below the cmc. Measurements were conducted at 70 °C. The PDI of

the droplets fluctuates between 0.7 and 1.0.

Chapter 7 - Core-Shell microgels

Figure 14.5 Colloidal stability of core-shell microgels at 20 °C, pH 7.

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Figure 14.6 FTIR spectra for core-shell microgels with increasing shell thickness.

Figure 14.7 RH as a function of temperature for core and core-shell microgels and their

respective transition temperatures. Measurements were conducted in pH ~ 6.0 phosphate

buffer.

Chapter 9 - Interactions with proteins

jkkjhkjhkhjkj hjjj

Figure 14.8 UV-Vis spectra of (a) pure cytochrome C and (b) pure microgel sample

(VCL:Vim:IA = 80:10:10) at pH 3, 6.2, and 8. Measurements were conducted at T = 25 °C.

a b

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Figure 14.9 Change of absorbance with dialysis time for ampholyte microgel with (a)

statistical distribution of ionizable groups (VCL:Vim:IA = 80:10:10); and core-shell

structures: (b) cVCL-/sNIPAm+ (1:1); (c) cVCL+/sNIPAm-; (d) cVCL-/sNIPAm+ (1:5).

Black curve: pH 3; red curve: pH 6.2; blue curve: pH 8.

Chapter 10.1 - Biomineralization

Figure 14.11 FTIR spectrum of microgels with 10 wt-% PB and varying CaCO3 content,

synthesized via gas diffusion. (Red) Pure CaCO3 (calcite); (green) Unmineralized microgel

with 10 wt-% PB; (black) Microgel with 13 wt-% CaCO3; (blue) Microgel with 28 wt-%

CaCO3; (turquois) Microgel with 67 wt-% CaCO3.

a b

c d

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Table 14.1 Assignment of XRD peaks of calcite.

2Θ / ° Peak

29.4 (104)

36.1 (110)

39.3 (113)

43.7 (202)

47.6 (018)

48.4 (116)

57.6 (122)

60.8 (214)

64.8 (300)

Figure 14.10 XRD diffraction pattern of microgel with 2 wt-% PB and 13 wt-% CaCO3 via

gas diffusion: (a) with pre-dialysis; (b) without pre-dialysis; (c) high concentration of

microgel. Black lines indicate the peak positions for calcite as found in literature (JCPDS: 86-

2334).

b

c

a

179

Danksagung

Ich möchte mich herzlich bei denjenigen bedanken, die mir während meines Studiums und

meiner Dissertation beigestanden haben.

Zuerst möchte ich Prof. Dr. Andrij Pich für die interessante Themenstellung und für sein

fortwährendes Interesse an meiner Arbeit danken. Des Weiteren bedanke ich mich für die

Freiheit, meine Arbeit zu großen Teilen selbst gestalten zu können, sowie die Möglichkeit, an

nationalen und internationalen Konferenzen und Fortbildungen teilzunehmen.

Für die finanzielle Unterstützung danke ich dem Sonderforschungsbereich SFB 985.

Prof. Dr. Walter Richtering, Prof. Dr. Igor Protemkin, Andreas Schmid, Andrey Rudov und

Arjan Gellissen danke ich für die gute Zusammenarbeit und die vielen fruchtbaren Treffen im

Rahmen des SFB985.

Zusätzlich bin ich auch Pierre Kandt für die gute Zusammenarbeit im Rahmen des Projektes

„Antimicrobial and tissue regenerating nanogels for implant coating“ dankbar und für seine

geduldigen Erklärungen zum Thema Zellwachstum.

Weiterhin möchte ich Prof. Dr. Andrew Lyon dafür danken, dass ich 5 Monate in seinem

Arbeitskreis forschen durfte und für seine Zeit und Interesse, die er meiner Arbeit und meinen

Ergebnissen entgegengebracht hat.

Natürlich bin ich auch dankbar für die Hilfe der Studenten, die ihre Forschungs- und

Bachelorarbeit bei mir erstellt sowie als Hilfskraft gearbeitet haben. Dazu gehören Keti

Piradashvili, Alexander Töpel, Helmi Maria Ala-Mutka, Artur Gantarev, Julia Blöhbaum und

Miriam Al Enezy.

Des Weiteren danke ich meinen Kollegen und Freunden innerhalb des Arbeitskreises für die

wirklich tolle Atmosphäre und den sehr guten Zusammenhalt während der letzten Jahre.

Hierbei sei besonders Labor 3.04 und 3.05 hervorgehoben (Christian Willems, Dominic

Kehren, Andreea Balaceanu, Jason Zografou, Thorsten Palmer).

Zu guter Letzt bedanke ich mich bei meiner Familie, die mich während des gesamten

Studiums unterstützt hat.

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Schlusserklärung

Hiermit erkläre ich, dass ich die vorliegende Arbeit selbstständig verfasst und keine anderen

als die hier angegebenen Quellen und Hilfsmitteln benutzt habe.

Ferner erkläre ich, dass ich nicht anderweitig mit oder ohne Erfolg versucht habe, eine

Dissertation einzureichen oder mich einer Doktorprüfung zu unterziehen.

Aachen, den 03.07.2015

………………………………………………………………………….

Ricarda Schröder

ISTRIBUTION OF IONIZABLE GROUPS - RWTH Publications

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