Hybrid mesoporous silica nanoparticles

Elizabete Coutinho

e-mail: elizabete.coutinho@ist.utl.pt

CQFM – Centro de Química-Física Molecular, Instituto Superior Técnico, 1049-001 Lisboa, Portugal.

ABSTRACT: A decrease in the side effects of a drug can be obtained if it is delivered in

an effective and timely manner to the needed location. Therefore, systems are needed to

improve the transport of the drug and the location of the target cells, resulting in an increase in

drug efficiency and decreased toxicity to healthy tissues. Nanoparticles have gained much

importance in medicine due to the fact that they have high area to volume ratio and, after

appropriate functionalization, the ability to recognize target sites.

In this work, hybrid spherical mesoporous silica nanoparticles (MSNs) were synthesized

with a core-shell design. The mesoporous silica core has a well-defined morphology with pores

that can incorporate molecules. The MSNs are characterized by ordered pores with diameters

arround 2-2.5 nm and volumes above 1 mL / g. RAFT polymerization (Reversible Addition-

Fragmentation chain Transfer), associated with the grafting-from method, were used to grow a

biocompatible polymer shell with temperature conformation dependence. This conformational

change from extended to collapsed can be used to control the release of molecules

incorporated in the pores, providing a convenient platform for controlled release of drugs. The

MSNs have diameters between 140 and 220 nm. We prove the successful release of molecules

from the MSNs pores through thermal stimulation of the polymeric shell. These nanoparticles

open new pathways in the development of controlled release systems.

KEYWORDS: mesoporous silica nanoparticles, core-shell nanoparticles, controlled

release, functionalization, RAFT.

1. INTRODUCTION

The hydrophobic nature of most

chemotherapeutic agents makes them poorly

soluble in water and therefore limits the

administration in higher doses. Thus, systems

that improve the transport of the drug and its

delivery to the target cells are required,

resulting in enhanced drug efficacy and

decreased toxicity to healthy tissues.These

ideas continue to be a priority in cancer

therapy. Mesoporous silica nanoparticles

(MSNs) have a mesoporous structure with

well-defined pore diameters between 2 and 4

nm, and volume exceeding 1 mL/g pore.1 In

this structure, the mesopores are aligned and

appear in form of honeycombs making it very

interesting and important for many

applications. The pores act as individual

reservoirs without links between them. These

internal structures can be controlled by the

initial reagents or the surfactant used. The

mesoporous silica nanoparticles have

diameters of 40 to hundreds of nanometers

and are a biocompatible three-dimensional

network with silanol groups (≡Si-OH) on the

surface.2

The main objective of this work is to

develop hybrid MSNs with a thermoresponsive

polymer shell to obtain a smart release control

mechanism, able to accommodate large drug

loads and deliver their cargo on demand to a

desired location.3,4,5

The hybrid MSNs were

prepared by incorporating a perylenediimide

(PDI) derivative in the silica network (MSN-PDI)

for bioimaging. The surface of MSNs were

funcionalized with (3-aminopropyl)

triethoxysilane (APTES). The RAFT

polymerization method associated with the

grafting from method was used to growth the

polymeric shell.6,7

A fluorescent probe,

Sulforhodamine B (SRB), was incorporated into

the hybrid MSNs in order to study the controlled

release using fluorescence techniques.

2. MATERIALS AND METHODS

2.1. Materials

Absolute ethanol (EtOH, 99,9% Scharlau),

hydrochloric acid (HCl, 37% Panreac),

ammonium hydroxide solution (NH4OH, 25%

Fluka), tetraethyl orthosilicate (TEOS, 98%

Aldrich), N-cetyltrimethylammonium bromide

(CTAB, 99% Sigma), (3-aminopropyl)

triethoxysilane (APTES, 98% Sigma-Aldrich), N-

(3-dimethylaminopropyl) carbodiimide (EDC,

98% Sigma-Aldrich) were used without further

purification. The RAFT agent, 3-

(benzylsulfanylthiocarbonylsulfanyl) propionic

acid was used for RAFT polimerization, 2-(2-

methoxyethoxy)ethyl methacrylate (MEO2MA,

95% Sigma-Aldrich, Mn=188,22 g mol-1

) and

poly(ethylene glycol) methyl ether methacrylate

(OEGMA, 98% Sigma-Aldrich, Mn=475 g mol-1

),

2,2-azobis (2-methylpropionitrile) (AIBN, 99%

Sigma-Aldrich) were used without further

purification. Commercial toluene and

dichloromethane were distilled over calcium

hydride prior to use. The Perylenediimide

derivative was synthetized according to the

literature.8

2.2. Synthesis of the MSNs

The perylenediimide-functionalized mesoporous

silica nanoparticles (MSN-PDI) were prepared

by a sol-gel modified process.9 In a

polypropylene flask CTAB (0.113 g) was

dissolved in 58.7 mL of 0.5 M NH4OH at 50ºC.

2.5 mL of a TEOS solution (0.2 M in ethanol),

0.5 mL of a PDI solution (6 mg PDI and 15 mL

EtOH) and 9.2 mL of absolute ethanol were

added to the surfactant solution dropwise while

stirring. The stirring was continued for 5 h. TEOS

solution (2.5 mL, 0.2 M in ethanol) and PDI

solution (0.5 mL) were added in the mixture and

kept stirring for 1 h. The mixture was then aged

at 50º C for 24 h. The nanoparticles were

recovered by centrifugation at 19118 g for 20

min each cycle. The MSNs were washed 2 times

with ethanol/water and 3 times with ethanol. The

MSNs were dried at 50ºC. The amino

modification of the silica surface was performed

by dispersing the obtained nanoparticles in a

solution of APTES in dry toluene. The ratio of

the reaction mixture used was 0.2 g MSNs: 10

mL toluene: 0.468 mL APTES. The reaction

occurs at 125ºC under reflux and argon

atmosphere for 24 h. Finally, the nanoparticles

were centrifuged for three cycles (19118 g, 20

min), redispersed in absolute ethanol and finally

dried overnight at 50º C. To remove the

surfactant, the nanoparticles were placed in a

polypropylene flask with an acidic ethanol

solution (0.5 M HCl, 20 mL of this solution for

each 500 mg of particles) and stirred for 2 h at

40º C. The nanoparticles were centrifuged three

times and redispersed in ethanol (19118 g, 10

min each cycle) and subsequently dried

overnight at 50° C.

2.3. Synthesis of RAFT Agent Functionalized

MSNs (MSN-RAFT)

To immobilize the RAFT agent on the

nanoparticles surface, the EDC and dry

dichloromethane were added to the MSN-PDI-

APTES and RAFT agent. The mixture was

stirred under an argon atmosphere at room

temperature for 24 hours. The nanoparticles

functionalized with RAFT agent (MSN-PDI-

RAFT) were recovered by centrifugation in 3

cycles (19118 g, 20 min each cycle), and

redispersed in absolute ethanol.

2.4. Synthesis of MSN-Poly

The polymer shell was synthesized by

introducing MSN-PDI-RAFT, OEGMA, MEO2MA

and ethanol in the schlenk tube. The mixture

was placed in an ultrasonic bath for 30 minutes

and stirred for 45 minutes at room temperature

under argon atmosphere. A solution of AIBN (~8

mg, 10 mL ethanol) was stirred for 45 min at

room temperature under argon atmosphere.

After, the schlenk tube was placed in an oil bath

at 70° C and 30 µl of the AIBN solution was

added to the mixture. The reaction mixture was

maintained under argon atmosphere for 24

hours. At the end of the reaction, was

centrifuged in 3 cycles (8497 g, 10 min each

cycle) and redispersed in absolute ethanol.

2.5. Loading and Release of Sulforhodamine

B

For loading hybrid nanoparticles with

sulforhodamine B (MSN-POLY-SRB) a solution

of SRB (pH 7, 4.47 × 10-3

M) was prepared. First,

3 mg of MSN-POLY were added to 3 mL of the

SRB solution and the mixture was stirred

overnight at 20° C. The temperature was

changed to 50° C and mantained during 3 hours.

1 mL of this dispersion was centrifuged in 3

cycles (8497 g, 10 min each cycle, 40° C), and

redispersed in phosphate buffer to remove the

SRB which was not loaded in the MSNs.

Supernatants were stored to measure the SRB

which wasn´t incorporated in MSN-POLY. The

amount of SRB loaded into MSN-POLY was

calculated from the difference between the

concentration of SRB solution used in the loading

and the concentration the separated medium

after centrifugations. For the release experiment,

200 µL of the dispersion with MSN-POLY and

SRB were placed in a dialysis tube and 3.5 mL of

phosphate buffer were introduced in the cell with

a magnetic stirrer. A kinetic study was performed

by changing the temperature between 20° C and

50° C, each 20 minutes, during for 4 hours.

3. Methods

TEM images were obtained in a Hitachi

transmission electron microscope (model H-

8100 with a LaB6 filament) with an accelerator

voltage of 200 kV. One drop of particles

dispersion in ethanol was placed on a carbon

grid and dried in air before observation. Images

were processed with the Fiji software. Zeta

potential (ξ) and hydrodynamic diameter (Dh)

measurements of the particles were obtained

using a Zetasizer Nano ZS, model ZEN3600.

The absorption spectra were recorded on a

Jasco V-660 spectrophotometer with a Peltier

temperature control. Fluorescence emission and

excitation spectra were recorded on a Horiba-

Jobin Yvon Fluorolog 3 spectrofluorimeter and

Right Angle geometry was used. All spectra

were recorded using 1 cm x 1 cm quartz cells.

4. RESULTS AND DISCUSSION

The fluorescent MSNs were obtained through a

sol–gel modified procedure, using CTAB as

template and ethanol as solvent. The excitation

and emission spectra of MSN-PDI dispersed in

ethanol are similar to the spectra of free PDI in

ethanol (Figure 1) meaning that the

incorporation of the PDI in the MSNs does not

affect the fluorescence properties of the dye.

Figure 1 - Normalized excitation (dashed lines, λemi =

560 nm) and emission (solid lines, λexc = 500 nm) for

PDI (green) and MSN-PDI 7a (blue).

The surface of the MSNs was modified with

APTES to incorporate primary amino groups on

the external surface.10

Then, the template was

removed using an acidic ethanol solution

allowing the pores to be completely available.

The synthesis of the polymer shell starts with the

anchoring of the RAFT agent on the external

surface of the MSN-PDI particles. The amino

groups on the external surface reacted with the

RAFT agent to obtain MSN-PDI-RAFT. These

nanoparticles were used on the RAFT synthesis

of the PEG-acrylate monomers, to obtain core-

shell silica-polymer nanoparticles (MSN-POLY)

(Figure 2).

Figure 2 - Different steps involved in the preparation of

hybrid MSNs coated with a thermoresponsive

biocompatible PEG-acrylate copolymer, incorporating a

PDI dye in the silica network.

The diameters of the mesoporous silica

nanoparticles were measured using TEM (DTEM)

and DLS (Dh). Table 1 summarizes the average

diameters obtained for the different synthesis

with both techniques. As seen in Table 1, the

hydrodynamic diameter is higher than the

diameter obtained by TEM, which shows the

tendency of nanoparticles to aggregate in

dispersion. However, the Dh is usually higher

than DTEM due to the hydration layer around the

nanoparticle in the particles dispersion.

0

0,2

0,4

0,6

0,8

1

1,2

400 500 600 700

Inte

nsi

ty (

a.u

)

Wavelength(nm)

Table 1 - Average diameters and standard deviations

obtained by DLS and TEM for the core of MSNs at 25º

C.

Sample Dh (nm) DTEM (nm)

MSN-PDI 2a 210±7 150±32

MSN-PDI 3a 220±5 160±35

MSN-PDI 3b 180±14 170±27

MSN-PDI 5b 170±2 160±34

MSN-PDI 7a 160±2 140±38

MSN-PDI 7b 170±10 160±44

From the TEM images of MSN-PDI (Figure 3), it

is possible to observe the spheroid shape and

the mesopores network along the nanoparticles

(Figure 3 C). The RAFT polymerization was

conducted to synthesize a copolymer formed

from a mixture of two PEG-acrylates. The

thermossensitive behavior of the polymer shell

was tested by analyzing the Dh of the hybrid

nanoparticles at 25 and 40º C, for different

samples (Table 2).

Table 2- Average hydrodynamic diameters of different

samples of hybrid nanoparticles dispersed in water at

25 and 40° C.

The average Dh for sample MSN-POLY 3b was

290 ± 5 nm at 25ºC, decreasing to 180 ± 10 nm

at 40ºC, providing the temperature-switchable

collapsed/expanded conformation behavior. The

average hydrodynamic diameter for sample

MSN-PDI 3b at 25ºC (Table 1) was 180 ± 14

nm. This value is equal to that obtained for the

MSN-POLY at 40ºC, which indicates that at this

temperature the chains of polymer shell are

collapsed onto the silica surface.

Figure 3 - Transmission electron microscope images of

MSN-PDI, A and B showing the particle morphology

and C showing the structure mesoporous.

The Dh of MSN-POLY 7a and MSN-POLY 7a

at 40ºC are lower than Dh of MSN-PDI,

because the silica nanoparticles (MSN-PDI)

are more likely to aggregate than the silica

nanoparticles with polymer shell (MSN-POLY)

which are more stable and so are better

Sample Dh (nm) 25ºC Dh (nm) 40ºC

MSN-POLY 2a 270±21 180±26

MSN-POLY 3a 210±11 150±7

MSN-POLY 3b 290±5 180±10

MSN-POLY 5b 190±2 140±1

MSN-POLY 7a 310±16 180±12

MSN-POLY 7b 190±5 150±3

A

B

C

dispersed. The TEM images for MSN-POLY 7b

(Figure 3 A) clearly show the polymer shell

around the silica nanoparticles. We also

measured the Zeta potencial in the different

steps of the synthesis of the nanoparticles

(Figure 4). The zeta potential of the silica

nanoparticles with polymer (MSN-POLY)

increased compared with the RAFT agent

nanoparticles (MSN-PDI-RAFT), suggesting

the surface modification of the nanoparticles

with the polymer.11

Figure 4- Average of Zeta potential of the different steps

of the synthesis of MSNs. The nanoparticles were

dispersed in water and measured at 25° C.

Figure 5 – Dinamic light scattering (DLS) of MSN-POLI

2a (A) and and MSN-POLI 7a (B) with LCST at 36,3ºC

and 35,6ºC, respectively.

The LCST of the polymer shell in MSN-POLY

was characterized by DLS, by measuring de Dh

from 20ºC to 50ºC (Figure 5). The polymer shell

present a LCST arround 36°C.12

To determine

the hysteresis we perform rapid cooling and

heating cycles (Figure 6). At temperatures below

the transition temperature (20°C), the diameter

was 218 nm, and above this temperature (50°C)

was 116 nm. When MSN-POLY was cooling, the

diameter was 114 nm at 50° C and 208 nm at

20°C, which indicates that the polymer chains

expanded at low temperature, but collapse onto

the silica surface at high temperature.13

Figure 6 - Transmission electron microscope image of

MSN-POLY 7b (A) showing a core-shell nanoparticle

and representation of the hydrodynamic diameters at

different temperature (B). The figure present a heating

cycle (20 - 50ºC, black) and followed by a cooling cycle

(50 - 20ºC, gray).

100

150

200

250

20 30 40 50

Dia

me

ter

(n

m)

Temperature ( °C)

A

B

A

B

3.2. Release of sulforhodamine B

The MSNs coated with a polymeric shell were

tested as controlled release containers for

sulforhodamine B (SRB) molecules. The

emission (λexc = 520 nm) and excitation (λemi =

620 nm) spectra were recorded at 20° C and 50°

C (Figure 6), because this temperature will be

used in the release experiment. In figure 6, we

can observe that the fluorescence intensity at 50

° C is lower than at 20° C. This happens due to

the increase in the non-radiactive components

with temperature leading to a decrease in the

quantum yield. A ratio of fluorescence

intensities, RF = IF (50ºC) / IF (20ºC), was used to

correct the fluorescence intensities in the

release experiments. The SRB was loaded into

MSN-POLY and the study of release were

performed by switching the temperature

between 50 and 20° C, every 20 minutes. The

emission of free SRB and SRB loaded MSN-

POLY was followed for 4 hours. The kinetics

started at 50°C because at this temperature the

polymer shell was collapsed and the MSNs were

loaded. The fluorescence intensity obtained at

50ºC for the SRB and MSN-SRB were

normalized with the ratio RF (Figure 7).

Figure 7 - Excitation (dashed lines, λemi = 620 nm) and

emission (solid lines, λexc = 520 nm) spectra of SRB at

20° C (red) and 50° C (blue).

Figure 8 - Fluorescence intensity obtained for SRB

(blue) and MSN-SRB (green), measured continuously

during 4 hours at λemi = 585 nm and λexc = 565 nm. The

fluorescence intensities measured at 50° C for SRB

(red) and MSN-SRB (purple) were normalized with the

RF ratio.

The release profiles of MSN-POLY can be

observed by applying heating - cooling cycles

to the nanoparticles. The values at each

plateau (20ºC and 50ºC) were fitted with a 1st

order equation (Figure 9). The slope of the

different temperature for SRB and MSN-SRB

are compiled in Figure 10. The slopes of MSN-

SRB at 20°C are identical over time because

at this temperature the polymer chains are

hydrated and expanded allowing SRB

molecules to flow from the core to the shell but

forcing them to remain there due to the

electrostatic interations with the polymer

chains. The slopes for MSN-SRB at 50ºC are

higher than for free SRB at 50ºC, because by

increasing the temperature to 50ºC, the

extended polymer chains of MSN-SRB shrink

rapidly, releasing the SRB molecules that were

in the polymer shell and thus increasing the

fluorescent intensity in the cell bottom

compartment. The slopes decrease over time,

because the amount of SRB released

decreases as the particle load is depleted.

Figure 9- Fluorescence intensities obtained at 20 ° C for

SRB (red) and MSN-SRB (blue) and normalized

fluorescence intensities obtained for the SRB at 50º C

(green) SRB and MSN-SRB at 50° C (purple). The

fluorescence intensities were measured to λemi= 585 nm

and λexc = 565 nm.

Figure 10 - Representation of the slopes obtained

from the adjustments made to the variations of the

fluorescence intensity of SRB (gray) and MSN-SRB

(blue) at 20° C, and SRB (yellow) and MSN-SRB

(orange) at 50° C.

The representation of the slopes versus time

(Figure 10) shows the free SRB slopes at 20°C

are similar over time (due to the slow diffusion of

free SRB molecules in solution by the

membrane). The free SRB slopes at 50°C not

significantly changed (although, the slopes were

higher than slopes at 20°C, due to the increased

porosity of the membrane with the temperature).

Figure 11 - Schematic representation of the behavior

of MSN-POLY loaded with sulforhodamine B. The

polymer at 50º C (a) is collapsed closing the pores.

Decreasing the temperature to 20ºC (b), the SRB

molecules flow from the pores to the hydrated

expanded polymer shell, remaining there duo to

electrostatic interations. Upon increasing the

temperature to 50ºC (c), the polymer shell collapsed

squeezing the SRB molecules.

The proposed mechanism of release is

presented in Figure 11. Initially, at 50ºC, the

MSNs were fully loaded and the polymer chains

are collapsed closing the pores entrance (Figure

11 a). By decreasing the temperature to 20°C,

SRB molecules flow to the polymer shell and

remain there (Figure 11 b). Increased the

temperature to 50°C, the polymer chains

collapsed and SRB is released (Figure 11 c). By

decreasing the temperature to 20°C again, a

new cycle starts (Figure 11 d), with the system

behaving as a nano-pump for the drug model.

CONCLUSIONS

A novel drug delivery carrier based on a

mesoporous silica core and a stimui-responsive

polymer shell has been successfully prepared.

The MSNs have incorporated in their structure a

0

500

1000

1500

2000

2500

3000

0-2

0

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47

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90

-10

0

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42

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0-1

60

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0-1

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19

0-2

00

Slo

pe

Time (min)

fluorescent PDI derivative for bioimaging, and

after template removal the pores are completely

available to accommodate molecules. The

characterization by TEM shows that the

nanoparticles are mesoporous, monodispersed

and they have a spheroid shape. DLS

measurements showed the termoresponsive

behavior of the polymer shell. The release of a

model molecule (SRB) from the MSN-POLY

could be controlled by adjusting the

temperature. This hybrid nanocontainer have

potential applications as a nano-pump drug

delivery system controlled by temperature.

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