New Generation Nanomedicines Constructed from …...weight loss of 20% compared with the weight...

Therapeutics, Targets, and Chemical Biology

New Generation Nanomedicines Constructedfrom Self-Assembling Small-Molecule ProdrugsAlleviate Cancer Drug ToxicityHangxiang Wang1, Zhongjie Lu1, Lijiang Wang2, Tingting Guo1, Jiaping Wu1,Jianqin Wan1, Liqian Zhou1, Hui Li1, Zhen Li3, Donghai Jiang1, Penghong Song1,Haiyang Xie1, Lin Zhou1, Xiao Xu1, and Shusen Zheng1

Abstract

The therapeutic index for chemotherapeutic drugs is deter-mined in part by systemic toxicity, so strategies for dose intensi-fication to improve efficacy must also address tolerability. Inaddressing this issue, we have investigated a novel combinatorialstrategy of reconstructing a drug molecule and using sequentialdrug-induced nanoassembly to fabricate supramolecular nano-medicines (SNM). Using cabazitaxel as a target agent, we estab-lished that individual synthetic prodrugs tethered with polyun-saturated fatty acids were capable of recapitulating self-assemblybehavior independent of exogenous excipients. The resultingSNM could be further refined by PEGylation with amphiphilic

copolymers suitable for preclinical studies. Among these cabazi-taxel derivatives, docosahexaenoic acid–derived compound 1retained high antiproliferative activity. SNM assembled with com-pound 1 displayed an unexpected enhancement of tolerability inanimals along with effective therapeutic efficacy in a mouse xeno-graft model of human cancer, compared with free drug adminis-tered in its clinical formulation. Overall, our studies showed howattaching flexible lipid chains to a hydrophobic and highly toxicanticancer drug can convert it to a systemic self-deliverablenanotherapy, preserving its pharmacologic efficacy while improv-ing its safety profile. Cancer Res; 77(24); 6963–74. �2017 AACR.

IntroductionChemotherapy remains amajor treatment regimen for themost

devastating cancers, but the known toxicity of many existingchemotherapeutics stresses the need for new drug candidates(1). For example, cabazitaxel, a newly developed semisynthetictaxane, was of particular interest due to its therapeutic potential tocircumvent the obstacles of the current taxane-based therapeuticparadigms (e.g., the clinical use of paclitaxel and docetaxel)because of its low affinity for multidrug resistance proteins anddrug efflux avoidance (2, 3). Unfortunately, high systemic toxicityof cabazitaxel was observed in phase I studies, establishing itsMTDasonly 25mg/m2 in a 1-hour infusion every 3weeks (4). The

toxicity profile of cabazitaxel is significantly lower than those forpaclitaxel and docetaxel (175 and 60–100 mg/m2 for paclitaxeland docetaxel every 3 weeks, respectively, approved by the FDA;ref. 5). Neutropenia was found to be a major clinical concernassociatedwith cabazitaxel, hindering its dose intensification and,therefore, efficacy (6). Currently, cabazitaxel is only approved bythe FDA as a second-line chemotherapeutic for patients withmetastatic castration-resistant prostate cancer (2). Nonetheless,innovative strategies are desperately desired to construct prom-ising agents with the aim of achieving optimal therapeutic out-comes and good safety profiles.

Supramolecular chemistry enables noncovalent assembly ofindividual molecular building blocks (e.g., small-moleculeprodrug entities, sensing molecules), thus providing innovativeapproaches to the creation of fascinating nanostructures forbiomedical application (7–14). For example, Hamachi andcolleagues developed a novel sensing mechanism for selectiveprotein detection based on the self-assembly of small amphi-philic probes (9, 10, 15). In their system, the signal is turned offdue to self-assembled nanoaggregation of probes but can beturned on in response to molecular disassembly induced byprotein recognition. On the other hand, such supramolecularnanoassemblies were extended to the creation of novel drugdelivery systems for future cancer therapy (16). However, mosthydrophobic chemotherapeutics alone cannot self-assembleinto stable nanoprecipitates in aqueous media without thehelp of adjuvant matrices. By exploiting the innate hydropho-bicity of therapeutics, various amphiphilic prodrugs covalentlycoupled with hydrophilic motifs [e.g., oligo(ethylene glycol)(17–19), polypeptides (20, 21), and copolymers (22–25)] havebeen leveraged to mimic the self-assembly behavior that ubiq-uitously occurs in nature. More intriguingly, several groups,

1The First Affiliated Hospital; Collaborative Innovation Center for Diagnosis andTreatment of Infectious Diseases; Key Laboratory of Combined Multi-OrganTransplantation, Ministry of Public Health, School of Medicine, Zhejiang Univer-sity, Hangzhou, Zhejiang Province, P.R. China. 2Zhejiang-California InternationalNanosystems Institute, Zhejiang University, Hangzhou, P.R. China. 3School ofPharmacy, Dalian Medical University, Dalian, P.R. China.

Note: Supplementary data for this article are available at Cancer ResearchOnline (http://cancerres.aacrjournals.org/).

Corresponding Authors: Hangxiang Wang, Key Lab of Combined Multi-organTransplantation,Ministry of Public Health, Key LabofOrganTransplantation, TheFirst Affiliated Hospital, Zhejiang University School of Medicine, 79 QingchunRoad, Hangzhou, Zhejiang 310003, P.R. China. Phone: 86-571-88208152; E-mail:[email protected]; and Shusen Zheng, Key Lab of Combined Multi-organTransplantation, Ministry of Public Health. Key Lab of Organ Transplantation,The First Affiliated Hospital, Zhejiang University School of Medicine, 79 Qing-chun Road, Hangzhou, Zhejiang 310003, P.R. China. Phone: 86-571-87236570;Fax: 86-571-87236884; E-mail: [email protected]

doi: 10.1158/0008-5472.CAN-17-0984

�2017 American Association for Cancer Research.

CancerResearch

www.aacrjournals.org 6963

on April 27, 2020. © 2017 American Association for Cancer Research. cancerres.aacrjournals.org Downloaded from

Published OnlineFirst October 20, 2017; DOI: 10.1158/0008-5472.CAN-17-0984

http://crossmark.crossref.org/dialog/?doi=10.1158/0008-5472.CAN-17-0984&domain=pdf&date_stamp=2017-12-5

http://cancerres.aacrjournals.org/

including us, have recently demonstrated that the attachmentof lipophilic moieties to anticancer agents conferred the pro-drug amphiphiles with the ability to form colloidally stablenanoaggregates rather than precipitants, despite the enhancedoverall lipophilicity of prodrugs (26–31). The above strategy,which is capable of recapitulating the self-assembly process, isunprecedented and should be practical for the facile fabricationof useful nanotherapeutics. However, challenges with respect tothe engineering of hydrophobic therapeutics and the relatednanoassemblies, especially for highly hydrophobic drugs, havenot been well explored to date.

Motivated by this need, we sought to develop a strategy forhydrophobic and toxic anticancer therapeutics that can bereformed and supramolecularly assembled into nanoparticles,thereby yielding remarkably improved in vivo performance(e.g., toxicity profile and anticancer efficacy; Fig. 1A and B).Relying on chemical derivation of target therapeutics (e.g.,cabazitaxel), several prodrug amphiphiles that spontaneouslyformed nanocrystals were obtained upon blending with aque-ous solutions. We further screened their antiproliferative activ-ities using in vitro tests, allowing for the identification of hitsthat are well suited for preclinical studies (Fig. 1A). Further-more, in vivo studies demonstrated that the systemic toxicity ofcabazitaxel prodrug-assembled PEGylated nanoparticles wassubstantially reduced, enabling dose intensification for futureclinical studies. Although there have been extensive studiesfocusing on the fabrication of nanotherapeutics, the workdescribed here represents a simple approach to generate newgeneration nanomedicines that alleviate drug toxicity whilepreserving pharmacologic efficacy.

Materials and MethodsSynthesis of cabazitaxel-based prodrugs

A small panel of compounds containing six cabazitaxel pro-drugs was synthesized and characterized by 1H NMR and high-resolution mass spectrometry. The synthetic procedures aredescribed in detail in the Supplementary Materials and Methods.

Preparation of prodrug-assembled SNMs and PEGylated SNMsCabazitaxel prodrug-assembled supramolecular nanomedi-

cines (SNM) were prepared using a reprecipitation method.Briefly, cabazitaxel prodrugs (10 mg) were dissolved in DMSO(1mL), and the solution was then rapidly injected into deionized(DI) water (9 mL) using a pipette. For PEGylation of SNMs,prodrugs were premixed with DSPE-PEG2000 at a ratio of 10:1(w/w) in DMSO, and the solutions were then injected into DIwater. For in vitro and in vivouse, the solutions containing SNMsorPEGylated SNMs (termed pSNMs) were dialyzed against DI waterfor 6hours at 4�C to removeDMSO. The dialysis tubes (Spectrum,molecular weight cutoff of 3.5 kDa) were continuously stirred,and the drug concentration was determined by analytic high-performance liquid chromatography (HPLC).

Hydrolysis rates of cabazitaxel prodrugsThe in vitro hydrolysis of the cabazitaxel prodrugs in the

presence or absence of human serum albumin (HSA) wasevaluated by HPLC analysis. Briefly, 10 mL aqueous solutionsof prodrugs 1, 3, and 5 (50 mmol/L) were incubated with orwithout HSA (10 mg/mL) at 37�C. At various time intervals,aliquot samples (50 mL) were collected and subjected to ana-

lytic RP-HPLC at a flow rate of 1.0 mL/minute using a gradientof 70% to 100% acetonitrile in water as the mobile phase. UVdetection was at a wavelength of 220 nm. The amounts ofcompounds were calculated by establishing standard curves.The hydrolysis rate of the prodrugs was calculated as a functionof the incubation time.

Stability of prodrug-assembled SNMs and pSNMsThe SNMs and pSNMs (0.2 mg/mL, cabazitaxel equivalent) in

PBS (pH 7.4) or supplemented with 20% (v/v) FBS were incu-bated at 37�C. Dynamic light scattering (DLS) analysis wasutilized to determine particle sizes after incubation for an extend-ed period of time.

Cell lines and cell cultureHuman breast cancer cell line Bcap37 was kindly provided by

Dr. Donghai Jiang and Prof. Weimin Fan (Medical University ofSouth Carolina, Charleston, SC; refs. 32, 33). Human triple-negative breast cancer (TNBC; MDA-MB-231), prostate cancer(DU145), and colorectal carcinoma (LoVo) cell lines were pur-chased from the cell bank of the Chinese Academy of Sciences(Shanghai, China) between 2012 and 2017 and were authenti-cated using short tandem repeat DNA fingerprinting. Bcap37 andLoVo cellsweremaintained inRPMI1640medium.MDA-MB-231cellswere cultured in L-15, andDU145 cellswere cultured inMEMmedium. Media were supplemented with 10% FBS, penicillin(100 U/mL), and streptomycin (100 mg/mL). Cells were culturedin a humid atmosphere at 37�C and 5% CO2.

In vitro cytotoxicity assaysIn vitro cytotoxicity of the nanodrugs and free cabazitaxel was

measured by MTT (3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyl-tetrazolium bromide) assay (34). Cells were seeded in 96-wellplates (5,000 cells/well) and incubated at 37�C for 24 hours.The cells were treated with serial dilutions of the drugs andfurther incubated at 37�C for 72 hours. At the end of theexposure, 30 mL of MTT solution (5 mg/mL in PBS) was addedto each well. After further incubation for 4 hours, the MTTsolution was removed from the wells, and the purple MTT-formazan crystals were dissolved by the addition of 100 mL ofDMSO. The absorbance of each individual well was determinedat 490 nm using a microplate reader (Multiskan FC, ThermoFisher Scientific). The viability of untreated cells was adjustedto represent 100%, and the concentrations of various drugsrequired to inhibit cell growth by 50% (IC50) were extrapolatedfrom the dose–response curves.

Evaluation of in vivo drug toxicityAll studies involving animals were approved by The Animal

Care and Use Committee of Zhejiang University. They werehoused under aseptic conditions and given an autoclaved rodentdiet and sterile water. We evaluated in vivo toxicity of free caba-zitaxel and cabazitaxel prodrug-assembled pSNMs by measuringthe body weight change of healthy ICRmice (4–5weeks old). Theanimals were randomized into four groups (n¼ 10/group). Micewere intravenously injected with pSNM 1 (10, 20, 30, 40, 50,60, and 70 mg/kg) three times on days 0, 3, and 6. Saline andfree cabazitaxel in its clinical formulation (10, 20, 30, 40, and50 mg/kg) were injected as controls. The body weights of themiceweremonitored and recorded every 3 days. TheMTDand thedeath of mice were defined as the allowance of a mean body

Wang et al.

Cancer Res; 77(24) December 15, 2017 Cancer Research6964




weight loss of 20% compared with the weight before drug treat-ments and as resulting in no death due to toxic effects.

To assess the side effects of nanodrugs, such as neutropenia,healthy ICR mice were intravenously injected with pSNM 1, andfree cabazitaxel (20 and 40 mg/kg, cabazitaxel equivalence) twotimes (on days 0 and 3). Blood samples were collected andperipheral white blood cell (WBC) counts were measured on day0 before drug injection and on days 3, 7, and 10 after adminis-tration of pSNMs and free cabazitaxel.

In vivo pharmacokinetic properties of pSNMsThe plasma concentrations of cabazitaxel prodrug-assem-

bled pSNM 1 (at a cabazitaxel-equivalent dose of 20 mg/kg)were compared with free cabazitaxel (20 mg/kg) in SD rats(�250 g, n ¼ 5 in each group). After a single intravenousinjection of drugs, blood samples were collected at 5, 15, and30 minutes and 1, 2, 4, and 7 hours and were then immediatelysubjected to centrifugation for 10 minutes at 3,000 � g, withthe resulting plasma samples collected in microfuge tubes. Theconcentrations of cabazitaxel and prodrugs were analyzed byRP-HPLC.

In vivo antitumor activityThe human breast carcinoma cell line Bcap37 was grown to

80% confluency in 90-mm tissue culture dishes. After cell harvest,theywere resuspended in PBS at 4�C to afinal concentration of 2.5� 107 cells/mL. The right flank of 5-week-old Balb/c nude micewas subcutaneously injected with 200 mL of cell suspensioncontaining 5 � 106 cells in a disposable syringe. After the tumorreached approximately 50mm3 in volume at day 14 after implan-tation, the animals were randomized into five groups (n ¼ 7 ineach group). The mice were intravenously injected with cabazi-taxel prodrug-assembled pSNMs (10, 20, and 40mg/kg) a total ofthree times ondays 0, 3, and6. Saline and cabazitaxel (formulatedin polysorbate 80/ethanol, 1:1 v/v, 10mg/kg) were intravenouslyinjected as controls. Tumor growth and body weight were mon-itored and recorded every 3 days. The length (L) andwidth (W) oftumors were measured with calipers and tumor volume wascalculated using the following formula: V ¼ (L � W2)/2, withW being smaller than L. Mice were sacrificed by CO2 inhalation atthe endpoint of the study.

Histologic analysisFor histologic analysis, the excised tumors and organs were

fixed in 4% paraformaldehyde in phosphate buffer, embedded inparaffin, and sectioned into 5-mm–thick slices. The tumor andorgan sections were stained with hematoxylin and eosin (H&E,Sigma). For the terminal deoxynucleotidyl transferase–mediateddUTP nick end labeling (TUNEL) assay, the dewaxed and rehy-drated tumor sections were incubated with proteinase K for 15minutes at 37�C, rinsed with PBS twice, and rinsed with theTUNEL In Situ Cell Death Detection Kit according to the manu-facturer's protocol (Sigma-Aldrich). The TUNEL-stained cellswerecounterstained with DAB (DAKO) and visualized by opticalmicroscopy in 10 random fields for each group.

Statistical analysisAll quantitative data are presented as the mean � SD. The

statistical significance between measurements was assessed usingan unpaired Student t test. A P value less than 0.05was consideredstatistically significant, whereas a P value less than 0.01 was

considered highly significant. Survival studies were analyzedusing Kaplan–Meier plots.

ResultsRational design of cabazitaxel-based prodrugs

To demonstrate the validity of our concept, we used theanticancer drug cabazitaxel. Accordingly, a small panel of syn-thetic cabazitaxel derivatives was constructed, primarily employ-ing polyunsaturated fatty acids [PUFA; e.g., docosahexaenoicacid (DHA), a-linolenic acid, and linoleic acid], monounsatu-rated fatty acids (e.g., oleic acid), and saturated fatty acids (e.g.,stearic acid, heptanoic acid) as modifiers (Fig. 1C). Betweencabazitaxel's two free hydroxyl groups, the group at the 20

position is exposed outside and is more reactive than the other,making it accessible to selective chemical derivatization (35).Therefore, we molecularly edited this potent agent via esterifi-cation as the ester bond is susceptible to hydrolysis or esterases,thereby making it capable of releasing active therapeutics intheir native forms in the body (36, 37). Simple esterification inthe presence of the coupling reagents (EDC/DMAP) producedgood yields of the corresponding cabazitaxel derivatives (58%–

91%; Fig. 1C; ref. 38). The resulting compounds 1–6 wereunambiguously characterized by 1H NMR and high-resolutionmass spectrometry (see Supplementary Material).

Prodrug self-assembly in aqueous solution andcharacterization

Having these compounds in hand, we next investigated theself-assembly behavior of the cabazitaxel derivatives by quicklyinjecting a solution of compounds in DMSO into aqueoussolution (i.e., water). This screening procedure allowed therapid identification of the prodrugs 1–5 that were capable offorming nanoaggregates (designated as SNMs 1–5) in DI waterat final concentrations of 1 mg/mL. Transmission electronmicroscopy (TEM) visualization showed the formation of uni-formly spherical nanostructures, which was observed for all ofthe prodrug suspensions (Fig. 2A; Supplementary Fig. S1);however, SNMs 4 and 5 were observed to have a tendency toself-aggregate (Supplementary Fig. S1). DLS analysis revealedthat the hydrodynamic diameters (DH) were approximately 120nm for SNMs 1 and 3, being smaller than others, and that thepolydispersity was low (Supplementary Fig. S1). Thus, theSNMs tended to swell in the solution state, leading to anobserved diameter larger than those obtained from the TEMimaging that was conducted in the dry state on carbon-coatedcopper grids. In contrast, neither derivative 6 nor the parentcabazitaxel molecule has the ability to self-assemble in aqueousmedia, exclusively forming large precipitates upon rapid aque-ous mixing.

To reduce clearance by the reticuloendothelial system(RES) and prolong blood circulation when administeredin vivo (39), we PEGylated these SNMs (designated as pSNMs)by blending the prodrugs with 1,2-distearoyl-sn-glycero-3-phosphoethanolamine-N-[methoxy (polyethylene glycol)2000] (DSPE-PEG2000) at a 10:1 (w/w) ratio. The amphiphilicDSPE-PEG2000, certified by the FDA for clinical use, is misciblewith many hydrophobic drugs to form nanoassemblies andhas been extensively exploited for in vivo drug delivery (40).Intriguingly, the mean DH of these fabricated pSNMs 1–5 uponPEG cloaking distinguishably decreased compared with the

Design of Potent Prodrug Nanoassemblies for Cancer Therapy

www.aacrjournals.org Cancer Res; 77(24) December 15, 2017 6965




bare SNMs (DLS data, Fig. 2A; Supplementary Fig. S1). Notably,the cloaking of DSPE-PEG2000 yielded nanoparticles with excel-lent water dispersibility as evidenced by TEM images (Fig. 2A;Supplementary Fig. S1). The stability of these as-preparednanoassemblies was tested using bare and PEGylated SNMs1 and 3 as model scaffolds. We found that all bare andPEGylated SNMs were thermodynamically stable in buffered

solutions, showing no visible precipitates after one week at4�C. However, PEGylated SNMs 1 and 3 exhibited variationsin particle size after 2 days in the presence of 20% (v/v) FBS(Fig. 2B). This could be attributed to the presence of relativelylarge nanoaggregates after long-term incubation with FBS orpotentially due to the formation of the protein corona onthe pSNM surface (41). Notably, under the present mixingratio, prodrug/DSPE-PEG2000 10:1 (w/w), the drug loadingsare exceptionally high at 66.3%, 69.3%, 69.2%, 69.1%, and68.9% (at a cabazitaxel-equivalent loading) for PEGylatedSNMs 1, 2, 3, 4, and 5, respectively.

In vitro cytotoxicity assaysTo demonstrate the general applicability of cabazitaxel-assem-

bled SNMs, we next evaluated the cytotoxicity using a panel ofcancer cell lines (i.e., human breast cancer Bcap37 and TNBCMDA-MB-231 cells, prostate cancerDU145 cells, and colon cancerLoVo cells) by assessing cell viability with the MTT assay. In thisexperimental setting, free cabazitaxel administered in DMSOwasincluded as a reference. The results are shown in Fig. 3A and B;Supplementary Fig. S2, and Table 1. The IC50 values of freecabazitaxel were 1.5 � 0.2, 66.6 � 4.5, 2.0 � 0.4, and 20.9 �2.2 nmol/L in Bcap37, MDA-MB-231, DU145, and LoVo cells,respectively, and these values are in agreement with previousresults across different cell types (4). Treatment with SNM 1 for72 hours was observed to induce potent apoptosis in four celllines, and the IC50 values were in the same range compared withthat of free cabazitaxel. However, compared with free cabazitaxel,slightly less cytotoxicity was observed for bare and PEGylated

Figure 1.

Rational prodrug design and screening for generating systemically injectableand better tolerated cytotoxic nanomedicines. A, In vitro and in vivoscreening process for the optimization of prodrug candidates. Prodrugderivatives are first constructed to validate the ability to self-assembleto form SNM in aqueous media. Cell-based in vitro studies are nextperformed to identify promising prodrug candidates. Finally, only those hitsshowing potent antiproliferative activity are subjected to in vivoevaluation. B, Self-assembly of individual prodrugs into colloidally stablenanoaggregations (i) and PEGylation of these SNMs using amphiphiliccopolymers for preclinical studies (ii). C, Chemical structures of cabazitaxelderivatives 1–6 tethered with fatty acids via a hydrolytic ester bond.

Figure 2.

Characterization of small-molecule prodrug-assembled nanoparticles. A,Transmission electron microscopy images of SNM 1 and PEGylated SNM 1stained with 2% uranyl acetate. Scale bars, 200 nm. Insets, distribution ofhydrodynamic diameters measured by DLS. B, Stability of SNMs andPEGylated SNMs in PBS (pH 7.4) with or without 20% (v/v) FBS at 37�C. Thedata are presented as the means � SD (n ¼ 3).

Wang et al.





SNM 1 in all tested cell lines. The IC50 values for SNM 1 werehigher than free cabazitaxel, ranging from 1.8- to 7.2-foldenhancement (Table 1). Moreover, pSNM 1 displayed 3.2-, 1.4-,and 1.2-fold higher potency than bare SNM 1 in Bcap37, MDA-MB-231, and LoVo cells, respectively. Unexpectedly, markedreduction of cytotoxicity was observed for other SNMs. In partic-ular, the IC50 values ranged from 4.5 to 80.0 mmol/L for bare orPEGylated SNMs 3, 4, and 5 in Bcap37, MDA-MB-231, and LoVocell lines, approximately three orders of magnitude less effectivethan SNM 1.

To elucidate the mechanism underlying the distinct cytotox-icity possessed by the cabazitaxel prodrug-assembled SNMs, weexplored the kinetic aspects of ester prodrug hydrolysis underphysiologic conditions. Exploiting cabazitaxel-based prodrug1, 3, and 5 as model compounds, we investigated the hydrolyticevents of the ester bond by performing HPLC analysis toevaluate the decay of intact prodrugs and the concomitantgeneration of active cabazitaxel. The hydrolytic products werecabazitaxel molecules, as confirmed by the HPLC chart, sup-porting the identical mode of action with free cabazitaxel when

Figure 3.

In vitro cytotoxicity of various nanodrugs in comparison with free cabazitaxel (CTX) against human cancer cell lines. Human breast cancer Bcap37 cells (A)and human prostate cancer DU145 cells (B) were tested. Left, cabazitaxel-assembled bare SNMs 1–5; right, PEGylated SNMs 1-5. Free cabazitaxeldissolved in DMSO was used as a reference. The cells were treated with various drugs for 72 hours, and cell viabilities were determined by the MTT assay.The data are presented as the means � SD (n ¼ 4).

Table 1. In vitro cytotoxicity of SNMs assembled in water as compared with free cabazitaxel dissolved in DMSO after 72-hour incubation (expressed as IC50� SD innmol/L)a

IC50 (nmol/L)Cell line Free CTX 1b 2 3 4 5

Bcap37 Bare 1.5 � 0.2 10.8 � 1.4c 535.5 � 21 19,058 � 1,616 24,106 � 1,506 36,961 � 2,930PEGylated 3.4 � 0.4c 168 � 10 4,518 � 530 8,148 � 847 26,225 � 2,711

MDA-MB-231 Bare 66.6 � 4.9 386.5 � 18.6c 742.7 � 128.5 21,012 � 1,001 25,863 � 1,333 42,515 � 3,651PEGylated 278.5 � 35.4c 460.4 � 41.2 12,930 � 1,113 23,660 � 1,368 39,357 � 2,216

DU145 Bare 2.0 � 0.4 6.2 � 0.9d 317.7 � 25.6 1,873 � 243 2,832 � 232 3,637 � 217PEGylated 7.7 � 0.9d 182.1 � 10.6 267 � 43 428 � 37 1,536 � 161

LoVo Bare 20.9 � 2.2 42.8 � 4.4c 1819 � 136 35,049 � 1,863 28,070 � 1,087 79,953 � 5,883PEGylated 36.7 � 3.0c 1172 � 84 25,336 � 1,113 34,107 � 1,035 70,154 � 2,008

Abbreviation: CTX, cabazitaxel.aDetermined by MTT assay.bStatistical analysis of IC50 values of SNM 1 or pSNM 1 as compared with that of free cabazitaxel.cP < 0.001.dP < 0.01.






using SNMs as therapeutics (data not shown). Surprisingly,prodrug 1 showed faster hydrolysis kinetics than othersin HEPES buffer (pH 7.4), which was further accelerated bythe presence of HSA (10 mg/mL of HSA in pH 7.4 HEPESbuffer; Supplementary Fig. S3). Thus, the distinct cytotoxicity ofthe ester prodrug 1 tethered to DHA could be attributable tothe fact that it undergoes more rapid hydrolysis to releasetherapeutically active components, resulting in a higher poten-cy in vitro.

In vivo systemic toxicityThe clinically used cabazitaxel causes severe neutropenia, a

frequently observed side effect of taxanes, in up to 94% oftreated patients at the 25 mg/m2 dose (42). Neutropenia, whichis characterized by reduced WBC counts, is a significant dose-limiting effect. Our approach presented here may be able topartially overcome the systemic toxicity of the formulated drugs.To verify our assumption, we used healthy ICR mice to carefullyevaluate whether the current delivery technologies have an effectin the prevention of neutropenia, which additionally should be

promising for dose escalation in future studies. Prior to pre-clinical studies, the as-prepared pSNMs containing cabazitaxelprodrugs were dialyzed against DI water to remove the organicsolvent DMSO, and the drug concentration was determined byanalytic HPLC. Because of its higher in vitro efficacy, PEGylatedSNM 1 was selected for in vivo studies. Figure 4A shows the WBCcounts in mice that received intravenous injections of pSNM 1(20 or 40 mg/kg, cabazitaxel equivalence) compared with thosethat received the same dosage of free cabazitaxel (as formulatedin polysorbate 80 and ethanol, 1:1 v/v). A significant reductionin WBC counts was induced by the cytotoxic cabazitaxel by day3, reduced to just 39% compared with the WBC counts on day 0.When we increased the free cabazitaxel dose to 40 mg/kg, theWBC counts were substantially reduced to 45%. Moreover, thereduction of WBC is persistent, and almost no restoration wasobserved. Very strikingly, the mice that received pSNM 1 at bothdoses (i.e., 20 and 40 mg/kg, cabazitaxel equivalence) did notexhibit the myelosuppression during the observation period,indicating that the generated nanodrugs exhibit a low incidenceof neutropenia.

Figure 4.

In vivo toxicity of PEGylated SNM 1 compared with free cabazitaxel (CTX) in ICR mice. A and B, Reduced neutropenia (A) and maximum body weight changes(B) observed for 18 days post intravenous administration of drug formulations. The data are presented as the means � SD (n ¼ 10). C, Survival ofhealthy ICR mice following intravenous injection of cabazitaxel-loaded pSNM 1 (at cabazitaxel-equivalent doses). The mice receiving the same dose offree cabazitaxel were used as controls. The mice were defined to be dead when the body weight loss exceeded 20%.

Wang et al.





We then investigated the MTD of the SNMs in animalscompared with free cabazitaxel in a dose escalation manner.Healthy ICR mice (n ¼ 10, five females and five males in eachgroup) were intravenously injected with 10, 20, 30, 40, 50, 60,and 70 mg/kg (cabazitaxel equivalent dose) of pSNM 1 threesuccessive times every 3 days. As comparisons, saline and freecabazitaxel administered in its conventional form wereinjected. The MTD was estimated on the basis of the thresholdat which the body weight loss exceeded 20%. Only a dose of 10mg/kg of free cabazitaxel was tolerated by the mice, whereashigher doses (i.e., 20 mg/kg) led to significant weight loss(Fig. 4B; Supplementary Fig. S4). Specifically, the cabazitaxeldose of only 20 mg/kg was sufficient to cause death in the mice(Fig. 4C). Very encouragingly, intravenous administration ofpSNM 1 (up to 70 mg/kg at a cabazitaxel-equivalent dose)did not induce obvious weight variations or the incidence ofdeath. The pSNMs that were fabricated with prodrug/DSPE-PEG2000 at a 10:1 (w/w) ratio were saturated in DI water ata cabazitaxel-equivalent concentration of approximately 9mg/mL, making further dose escalation inapplicable. However,we can define the MTD as at least 70 mg/kg, which represents a7-fold higher tolerability compared with that of free cabazitaxelin its conventional form.

Pharmacokinetics and biodistribution of pSNM 1To further explore the advantage of our SNM technology to

extenddrug circulation in theblood, and themechanism involvedin low systemic toxicity, we performed pharmacokinetic analysisof pSNM 1 and compared the results with those of free cabazitaxelin Sprague–Dawley (SD) rats. After a single intravenous injectionof drug formulations (at a 20 mg/kg cabazitaxel equivalence),bloodwas drawn at predetermined time intervals. The cabazitaxelmolecules in serum were then extracted and subsequently sub-jected to HPLC analysis. The plasma concentration of total caba-zitaxel versus time and related pharmacokinetics parameters wereshown in Fig. 5A; Supplementary Table S1, respectively. Freecabazitaxel exhibited rapid clearance from the blood, whereasprodrug-assembled pSNM 1 substantially prolonged drug reten-tion in vivo. When we quantified the amounts of nonhydrolyticintact prodrugs and released free cabazitaxel separately, we foundthat only a small portion of free cabazitaxel was hydrolyzed frompSNM 1 at an initial time point of 7 hours postadministration(Supplementary Fig. S5), indicating that the pSNM scaffoldinhibited the release of free drug even in the presence of abundantmouse serum.

We further investigated the behaviors of clearance and tumoraccumulation using the pSNM platform in breast carcinoma(Bcap37) xenograft-bearing Balb/c nude mice. To allow real-timetracking of pSNM 1 in animals, a near-infrared fluorescent probe(DiR)was encapsulated in nanoparticles (termedDiR@pSNM 1).DiR that was dissolved together with free cabazitaxel in polysor-bate 80/ethanol was included as a reference. Following intrave-nous injection, whole-body fluorescence imaging was performedusing an in vivo imaging system. As shown in Fig. 5B, the fluo-rescent signals were eliminated quickly in mice that received DiRformulated in polysorbate 80/ethanol, whereas the fluorescencein mice receiving DiR@pSNM 1 was maintained for up to 48hours postinjection. Noticeably, the DiR fluorescent signal waspredominantly localized to the tumor regionwhenDiR@pSNM 1was injected (Fig. 5B). Mice were sacrificed after 48 hours, andex vivo imaging of tumors and major organs was performed

(Fig. 5B, right). The results revealed that DiR was significantlyaccumulated in the tumors of DiR@pSNM 1–administered micethan those treatedwith freeDiR (Fig. 5C).We further validated thedrug tissue distribution over time by conducting HPLC analysisafter intravenous injection of drugs. Systemic administration ofpSNM 1 led to persistent retention of drug in tumors (Supple-mentary Fig. S6). In contrast, injection of free cabazitaxel resultedin notably reduced drug amounts in tumors at 6 hours postad-ministration, but showed high accumulation in liver and kidney.Our pSNM scaffold has increased drug accumulation in tumors,with lower toxicity to normal tissues, making it a promisingcandidate as a nanotherapeutic.

In vivo antitumor efficacyThe potent in vitro activity and alleviated drug toxicity of

SNM prompted us to further examine the therapeutic potentialin Balb/c nude mice subcutaneously bearing Bcap37 xenografts.Because 1-assembled SNM was the most effective based onextrapolation from the in vitro dose–response curves, PEGylatedSNM 1 was evaluated in terms of the therapeutic efficacy com-pared with free cabazitaxel. As indicated in Fig. 6A and B, admin-istration of free cabazitaxel at a 10 mg/kg dosage led to a delay intumor growth but additionally caused significant weight loss(�23%, and warranting sacrifice at day 12), suggesting severesystemic toxicity in Balb/c nude mice. In comparison, a compa-rable therapeutic effect was achieved by administering pSNM 1 ata dose of 10 mg/kg (Fig. 6A); however, the weights of the miceremained unaffected (Fig. 6B). The improved tolerability ofassembled prodrugs in animals inspired us to attempt an inten-sified regime (20or 40mg/kg cabazitaxel equivalence). As a result,we observed strikingly improved outcomes, with tumor shrinkageor even disappearance in pSNM-treated groups after injection oftolerable doses of 20 and 40mg/kg (Fig. 6A, C, and D). Althoughthe mice receiving pSNMs at a dose of 40 mg/kg had an initialbody weight loss, gradual restoration of weight occurred afterstopping injections (Fig. 6B).

Cabazitaxel was observed to exhibit high activity against arange of different tumors in phase I studies (3). We thereforeassessed whether our nanodrugs are efficacious to represstumor growth in additional models. For this purpose, twonude mouse models were established: human TNBC MDA-MB-231 and human prostate cancer DU145 xenograft. Forthese experiments, we reduced the dose of free cabazitaxel to7.5 mg/kg as systemic administration of 10 mg/kg caused deathin Bcap37 tumor xenograft-bearing nude mice. Intravenousadministration of pSNM 1 at 7.5 mg/kg (cabazitaxel equiva-lence) resulted in a superior antitumor effect in MDA-MB-231xenograft-bearing mice compared with free cabazitaxel (Sup-plementary Fig. S7). For DU145 xenograft-bearing mice, com-parable tumor suppression was observed for pSNM 1 and freecabazitaxel at the same dose of 7.5 mg/kg (Supplementary Fig.S8). Significantly, treatment with pSNM 1 at 15 mg/kg led tosubstantial inhibition or shrinkage of tumors in both models.Importantly, no obvious loss of body weight was observed inmice treated with all dosages of pSNM 1, whereas although nomice receiving free cabazitaxel at 7.5 mg/kg died, they did showa decrease in body weight that exceeded 20%.

A TUNEL assay of the excised Bcap37 tumors revealed thatefficient and extensive intratumoral apoptosis was induced bycabazitaxel agents after intravenous injection of SNMs (Fig. 6E),which was well correlated with the hematoxylin and eosin (H&E)






staining (Supplementary Fig. S9). These data support the excellentin vivo anticancer efficacy of the SNM platforms. To examine thetoxicity of the drugs, we performed H&E staining of other majororgans frommice after they received three injections. As illustratedin Supplementary Figs. S10–S14, we confirmed that free cabazi-taxel (10 mg/kg) induced damage to major organs in mice,particularly in the lungs. Conversely, no damage was observedin saline- or pSNM-treated groups, even when the dose was

increased to 40 mg/kg. Together, these results indicate that theSNM platform is extremely well tolerated by animals and is notassociated with systemic toxicity.

DiscussionBecause of its poor water solubility, cabazitaxel is currently

formulated in polysorbate 80 and ethanol (1:1 v/v) for its clinical

Figure 5.

In vivo pharmacokinetics andbiodistribution studies of PEGylatedSNM 1. A, Time-dependent in vivononinvasive near-infrared (NIR)fluorescence images of mouse wholebody. To monitor nanoparticles, DiRprobe (0.8 mg/kg weight) that wasencapsulated within pSNM 1 wasintravenously injected into Bcap37tumor-bearing nude mice via the tailvein. Free DiR (0.8 mg/kg weight)that was dissolved in polysorbate80/ethanol was used as a control.Typical ex vivo fluorescence imagesof the tumor and major organsexcised from the mice at 48 hourspostadministration (right). B,Quantitative analysis of fluorescentintensities in the region of tumorsand major organs at 48 hourspostadministration. The data arepresented as the means � SD (n ¼ 5).�� , P < 0.001. C, In vivo plasmaconcentration–time profile of thedrugs following intravenous injectionof pSNM 1 in SD rats. The total amountof cabazitaxel (CTX) molecules,including intact or free cabazitaxel,in the plasma was extracted anddetermined by HPLC. The data arepresented as the means � SD (n ¼ 5).

Wang et al.





Figure 6.

Therapeutic effects of pSNM 1 against Bcap37 xenograft tumors were evaluated in a nude mouse model. A, Comparative efficacy study using PEGylated SNMs.pSNM 1 was injected three successive times. Saline and cabazitaxel (CTX; 10 mg/kg) were additionally intravenously injected as controls. Arrowsrepresent the intravenous injections. The data are expressed as the means � SD (n ¼ 7). � , P < 0.05; �� , P < 0.01. B, Relative body weight changes of themice receiving various drugs. C, Tumor weights in each group at the endpoint of the study. D, Representative images of the excised tumors at the endpointfrom the groups treated with saline and SNMs. E, Representative TUNEL analysis of the excised tumors from the treated groups on day 2 postinjection of thedrugs. The image magnification is �400. Potent induction of apoptosis was observed in the slices, which is consistent with the tumor inhibition results.






use. However, the use of polysorbate 80 inevitably will result inlowdrug loadings and cause excipient-associated side effects (43).In this study,we show that synthetic cabazitaxel prodrugs tetheredwith PUFAs are able to recapitulate self-assembly behavior inaqueous solutions independent of excipients. PUFAs, which arestructurally analogous to the squalenemoiety that was developedfor drug conjugation byCouvreur and colleagues (28, 44), exhibitseveral unique features as follows: (i) they have possible p–pstacking between prodrugmolecules that renders nanoassembliessufficiently stable in vivo; (ii) they are commercially available andinexpensive; and (iii) these entities play an essential role inbiological functions and are also abundant in the human body,thereby obviating concerns over excipient-associated side effects(45). These features promise PUFAs as useful skeletons for thegeneration of oncology drugs.

In a previous study, a hydrophobic vitamin E motif wasexploited to construct paclitaxel-based prodrugs, which facilitatedthe amphiphilic self-assembly of drugs into nanoparticles (46). Inthat study however, the insertion of a disulfide bond within themolecular construct was required, which inevitably led to atedious synthesis process. Indeed, cabazitaxel should exhibithigher hydrophobicity relative to paclitaxel due to the methyla-tion of hydroxyl group in the 7 position of cabazitaxel. Thus, ourresults demonstrate a simple approach to generate highly hydro-phobic cabazitaxel amphiphiles, while minimizing syntheticefforts and offering attractive properties, such as supramolecularnanoassembly in aqueous media, for potentiating clinicaltranslation.

In addition, SNM assembled with 1 was administered tohealthy ICR mice via the tail vein to assess the tolerability. Thisnanodrug displayed an unexpectedly higher safety profile thanfree drug in animals, as indicated by continued increase in bodyweight and a low incidence of neutropenia. Detailed pharma-cokinetics and tissue distribution studies indicated that thedrastically alleviated toxicity could be explained by the factthat pSNM reservoir can inhibit the premature release ofcytotoxic cabazitaxel during systemic circulation. Nonetheless,alleviating the systemic toxicity of anticancer cabazitaxel agentfacilitated the dose intensification and thereby improved effi-cacy. On the other hand, we can expect that once accumulatedwithin cancerous tissues via the enhanced permeability andretention (EPR) effect (47–49), the pSNM scaffold may prolongthe exposure of tumor cells to cabazitaxel at therapeuticallyrelevant concentrations.

The therapeutic activity of using 1-assembled pSNM wasconducted in three different tumor xenograft-bearing micemodels. These in vivo studies obviously demonstrate thatalthough pSNM 1 showed slightly less cytotoxicity than freecabazitaxel in cell-based in vitro tests, this nanodrug exhibitedcomparable potent antitumor efficacy. Correlating with in vivopharmacokinetic analysis using SD rats, these therapeuticresults strongly support the hypothesis that individual cabazi-taxel prodrugs can be constrained within the core nanostruc-tures by being less hydrolyzed during systemic circulation.Moreover, the PEG cloaking on particles' surfaces could pro-long SNMs' persistence in the blood, thus making the nano-

drugs preferentially accumulate in tumor lesions via the EPReffect.

In conclusion, through exploitation of a combinationalstrategy that involved a structure-guided drug reconstructionand a sequential prodrug-induced self-assembly process, wehave developed a practical and expedient route through whichto construct clinically potent and safe nanotherapeutics. Severaladvantages are combined in this scaffold, including (i) excep-tionally high drug loading, for example, close to 69% forcabazitaxel; (ii) the ability to self-assemble into well-definednanostructures independent of exogenous excipients; (iii) com-posite composition of purely FDA-approved materials; and (iv)feasibility of the fabrication of sub-100 nm nanodrugs that aresuitable for deep tumor penetration. The simple process of drugformulation via self-assembly makes this strategy greatly ben-eficial for scaling up; (v) remarkable alleviation of systemictoxicity with a high safety margin as compared with that of freecabazitaxel in its clinical formulation. Of note, cabazitaxelprodrug candidates were demonstrated to possess significantlyreduced systemic toxicity, while reserving therapeutic efficacyand prolonged in vivo pharmacokinetics, relative to free caba-zitaxel. Given the intrinsic feature of cabazitaxel in circumvent-ing drug resistance (50), further work is being conducted toevaluate the anticancer potential of these nanoassemblies indocetaxel-resistant malignancies.

Disclosure of Potential Conflicts of InterestNo potential conflicts of interest were disclosed.

Authors' ContributionsConception and design: H. Wang, Z. Li, X. Xu, S. ZhengDevelopment of methodology: H. Wang, H. Xie, L. ZhouAcquisition of data (provided animals, acquired and managed patients,provided facilities, etc.): H. Wang, Z. Lu, L. Wang, T. Guo, J. Wu, J. Wan,L. Zhou, H. Li, Z. LiAnalysis and interpretation of data (e.g., statistical analysis, biostatistics,computational analysis): H. Wang, L. Wang, T. Guo, J. Wu, Z. Li, D. Jiang,P. Song, H. Xie, L. Zhou, X. XuWriting, review, and/or revision of the manuscript: H. Wang, S. ZhengAdministrative, technical, or material support (i.e., reporting or organizingdata, constructing databases): H. Wang, L. Wang, T. Guo, J. Wu, D. Jiang,P. Song, H. Xie, X. XuStudy supervision: H. Wang, S. Zheng

Grant SupportThis work was supported by grants from the National Natural Science

Foundation of China (grants 81571799 and 81773193 to H. Wang;31671019 to H. Xie; 81421062 and 91542205 to S. Zheng) and theNatural Science Foundation of Zhejiang Province (grant LY14H180005to L. Wang).

The costs of publication of this article were defrayed in part by thepayment of page charges. This article must therefore be hereby markedadvertisement in accordance with 18 U.S.C. Section 1734 solely to indicatethis fact.

Received April 7, 2017; revised August 21, 2017; accepted October 13, 2017;published OnlineFirst October 20, 2017.

References1. Holohan C, Van Schaeybroeck S, Longley DB, Johnston PG. Cancer drug

resistance: an evolving paradigm. Nat Rev Cancer 2013;13:714–26.2. Galsky MD, Dritselis A, Kirkpatrick P, Oh WK. Cabazitaxel. Nat Rev Drug

Discov 2010;9:677–8.

Wang et al.





3. Mita AC, Figlin R, Mita MM. Cabazitaxel: more than a new taxane formetastatic castrate-resistant prostate cancer? Clin Cancer Res 2012;18:6574–9.

4. Mita AC, Denis LJ, Rowinsky EK, DeBono JS, Goetz AD, Ochoa L, et al.Phase I and pharmacokinetic study of XRP6258 (RPR 116258A), anovel taxane, administered as a 1-hour infusion every 3 weeksin patients with advanced solid tumors. Clin Cancer Res 2009;15:723–30.

5. Eniu A, Palmieri FM, Perez EA. Weekly administration of docetaxel andpaclitaxel in metastatic or advanced breast cancer. Oncologist 2005;10:665–85.

6. Bracarda S, Gernone A, Gasparro D, Marchetti P, Ronzoni M, Bortolus R,et al. Real-world cabazitaxel safety: the Italian early-access program inmetastatic castration-resistant prostate cancer. Future Oncol 2014;10:975–83.

7. Cabral H, Nishiyama N, Kataoka K. Supramolecular nanodevices: fromdesign validation to theranostic nanomedicine. Acc Chem Res 2011;44:999–1008.

8. Sosnik A. Drug self-assembly: a phenomenon at the nanometer scale withmajor impact in the structure–biological properties relationship and thetreatment of disease. Prog Mater Sci 2016;82:39–82.

9. Takaoka Y, Sakamoto T, Tsukiji S, Narazaki M, Matsuda T, Tochio H, et al.Self-assembling nanoprobes that display off/on F-19 nuclear magneticresonance signals for protein detection and imaging. Nat Chem 2009;1:557–61.

10. Mizusawa K, Ishida Y, Takaoka Y, Miyagawa M, Tsukiji S, Hamachi I.Disassembly-driven turn-on fluorescent nanoprobes for selective proteindetection. J Am Chem Soc 2010;132:7291–3.

11. Zhang M, Soto-Rodriguez J, Chen IC, Akbulut M. Adsorption and removaldynamics of polymeric micellar nanocarriers loaded with a therapeuticagent on silica surfaces. Soft Matter 2013;9:10155–64.

12. Zhang M, Xu C, Wen L, Han MK, Xiao B, Zhou J, et al. A hyaluronidase-responsive nanoparticle-based drug delivery system for targeting coloncancer cells. Cancer Res 2016;76:7208–18.

13. Kumar M, Gupta D, Singh G, Sharma S, Bhat M, Prashant CK, et al. Novelpolymeric nanoparticles for intracellular delivery of peptide cargos: anti-tumor efficacy of the BCL-2 conversion peptide NuBCP-9. Cancer Res2014;74:3271–81.

14. Huang Y-H, Peng W, Furuuchi N, Gerhart J, Rhodes K, Mukherjee N, et al.Delivery of therapeutics targeting the mRNA-binding protein HuR using3DNA nanocarriers suppresses ovarian tumor growth. Cancer Res 2016;76:1549–59.

15. Takaoka Y, Sun Y, Tsukiji S, Hamachi I. Mechanisms of chemical pro-tein19F-labeling and NMR-based biosensor construction in vitro and incells using self-assembling ligand-directed tosylate compounds. Chem Sci2011;2:511–20.

16. Pei Q, Hu X, Liu S, Li Y, Xie Z, Jing X. Paclitaxel dimers assemblingnanomedicines for treatment of cervix carcinoma. J Control Release2017;254:23–33.

17. Shen Y, Jin E, Zhang B, Murphy CJ, Sui M, Zhao J, et al. Prodrugs forminghigh drug loading multifunctional nanocapsules for intracellular cancerdrug delivery. J Am Chem Soc 2010;132:4259–65.

18. Wang J, Sun X, Mao W, Sun W, Tang J, Sui M, et al. Tumor redoxheterogeneity-responsive prodrug nanocapsules for cancer chemotherapy.Adv Mater 2013;25:3670–6.

19. Lee Y, Kim H, Kang S, Lee J, Park J, Jon S. Bilirubin nanoparticles as ananomedicine for anti-inflammation therapy. Angew Chem Int Ed2016;55:7460–3.

20. MacKay JA, Chen MN, McDaniel JR, Liu WG, Simnick AJ, Chilkoti A. Self-assembling chimeric polypeptide-doxorubicin conjugate nanoparticlesthat abolish tumours after a single injection. Nature Mater 2009;8:993–9.

21. McDaniel JR, Bhattacharyya J, Vargo KB, Hassouneh W, Hammer DA,Chilkoti A. Self-assembly of thermally responsive nanoparticles of agenetically encoded peptide polymer by drug conjugation. Angew ChemInt Ed 2013;52:1683–7.

22. Cabral H, Murakami M, Hojo H, Terada Y, Kano MR, Chung UI, et al.Targeted therapy of spontaneous murine pancreatic tumors by polymericmicelles prolongs survival and prevents peritoneal metastasis. Proc NatlAcad Sci U S A 2013;110:11397–402.

23. Yen HC, Cabral H, Mi P, Toh K, Matsumoto Y, Liu X, et al. Light-inducedcytosolic activation of reduction-sensitive camptothecin-loaded polymeric

micelles for spatiotemporally controlled in vivo chemotherapy. ACS Nano2014;8:11591–602.

24. Maksimenko A, Bui DT, Desma€ele D, Couvreur P, Nicolas J. Significanttumor growth inhibition from naturally occurring lipid-containing poly-mer prodrug nanoparticles obtained by the drug-initiated method. ChemMater 2014;26:3606–9.

25. Zhang S, Zou J, Elsabahy M, Karwa A, Li A, Moore DA, et al. Poly(ethyleneoxide)-block-polyphosphester-based paclitaxel conjugates as a platformfor ultra-high paclitaxel-loaded multifunctional nanoparticles. Chem Sci2013;4:2122–6.

26. Wang H, Xie H, Wu J, Wei X, Zhou L, Xu X, et al. Structure-based rationaldesign of prodrugs to enable their combination with polymeric nanopar-ticle delivery platforms for enhanced antitumor efficacy. Angew Chem IntEd 2014;53:11532–7.

27. Wang H, Xie H,Wang J, Wu J, Ma X, Li L, et al. Self-assembling prodrugs byprecise programming of molecular structures that contribute distinctstability, pharmacokinetics, and antitumor efficacy. Adv Funct Mater2015;25:4956–65.

28. Maksimenko A, Dosio F, Mougin J, Ferrero A, Wack S, Reddy LH, et al. Aunique squalenoylated andnonpegylateddoxorubicin nanomedicinewithsystemic long-circulating properties and anticancer activity. Proc Natl AcadSci U S A 2014;111:E217–26.

29. Arias JL, Reddy LH, Othman M, Gillet B, Desmaele D, Zouhiri F, et al.Squalene based nanocomposites: a new platform for the design of mul-tifunctional pharmaceutical theragnostics. ACS Nano 2011;5:1513–21.

30. Desmaele D, Gref R, Couvreur P. Squalenoylation: a generic platform fornanoparticular drug delivery. J Control Release 2012;161:609–18.

31. Zhang W, Lin W, Pei Q, Hu X, Xie Z, Jing X. Redox-hypersensitive organicnanoparticles for selective treatment of cancer cells. Chem Mater2016;28:4440–6.

32. Jiang D, Sui M, Zhong W, Huang Y, Fan W. Different administrationstrategies with paclitaxel induce distinct phenotypes of multidrug resis-tance in breast cancer cells. Cancer Lett 2013;335:404–11.

33. Sui M, Huang Y, Park BH, Davidson NE, Fan W. Estrogen receptor a

mediates breast cancer cell resistance to paclitaxel through inhibition ofapoptotic cell death. Cancer Res 2007;67:5337–44.

34. Xie H, Xu X, Chen J, Li L, Wang J, Fang T, et al. Rational design ofmultifunctional small-molecule prodrugs for simultaneous suppressionof cancer cell growth and metastasis in vitro and in vivo. Chem Commun2016;52:5601–4.

35. Bensaid F, Thillaye du Boullay O, Amgoune A, Pradel C, HarivardhanReddy L, Didier E, et al. Y-shaped mPEG-PLA cabazitaxel conjugates: well-controlled synthesis by organocatalytic approach and self-assembly intointerface drug-loaded core-corona nanoparticles. Biomacromolecules2013;14:1189–98.

36. Rautio J, Kumpulainen H, Heimbach T, Oliyai R, Oh D, Jarvinen T, et al.Prodrugs: design and clinical applications. Nat Rev Drug Discov2008;7:255–70.

37. Fang T, Dong Y, Zhang X, Xie K, Lin L, Wang H. Integrating a novel SN38prodrug into the PEGylated liposomal system as a robust platform forefficient cancer therapy in solid tumors. Int J Pharm 2016;512:39–48.

38. WangH, Koshi Y,MinatoD,NonakaH, Kiyonaka S,Mori Y, et al. Chemicalcell-surface receptor engineering using affinity-guided, multivalent orga-nocatalysts. J Am Chem Soc 2011;133:12220–8.

39. Knop K, Hoogenboom R, Fischer D, Schubert US. Poly(ethylene glycol) indrug delivery: pros and cons as well as potential alternatives. Angew ChemInt Ed 2010;49:6288–308.

40. Wei T, Liu J, Ma H, Cheng Q, Huang Y, Zhao J, et al. Functionalizednanoscalemicelles improve drug delivery for cancer therapy in vitro and invivo. Nano Lett 2013;13:2528–34.

41. Sch€ottler S, Becker G, Winzen S, Steinbach T, Mohr K, Landfester K, et al.Protein adsorption is required for stealth effect of poly(ethylene glycol)-and poly(phosphoester)-coated nanocarriers. Nat Nanotechnol2016;11:372–7.

42. Agarwal N, Sonpavde G, Sartor O. Cabazitaxel for the treatment ofcastration-resistant prostate cancer. Future Oncol 2011;7:15–24.

43. Weiszh�ar Z, Cz�ucz J, R�ev�esz C, Rosivall L, Szebeni J, Rozsnyay ZComple-ment activation by polyethoxylated pharmaceutical surfactants: Cremo-phor-EL, Tween-80 and Tween-20. Eur J Pharm Sci 2012;45:492–8.

44. Gaudin A, Yemisci M, Eroglu H, Lepetre-Mouelhi S, Turkoglu OF,Donmez-Demir B, et al. Squalenoyl adenosine nanoparticles provide






neuroprotection after stroke and spinal cord injury. Nat Nanotechnol2014;9:1054–62.

45. Kris-Etherton PM, Taylor DS, Yu-Poth S, Huth P,Moriarty K, Fishell V, et al.Polyunsaturated fatty acids in the food chain in theUnited States. Am JClinNutr 2000;71:179S–88S.

46. Wang Y, Liu D, Zheng Q, Zhao Q, Zhang H, Ma Y, et al. Disulfide bondbridge insertion turns hydrophobic anticancer prodrugs into self-assem-bled nanomedicines. Nano Lett 2014;14:5577–83.

47. Matsumoto Y, Nichols JW, Toh K, Nomoto T, Cabral H, Miura Y, et al.Vascular bursts enhance permeability of tumour blood vessels andimprove nanoparticle delivery. Nature Nanotechnol 2016;11:533–8.

48. Sun Q, Radosz M, Shen Y. Challenges in design of translational nanocar-riers. J Control Release 2012;164:156–69.

49. Prabhakar U, Maeda H, Jain RK, Sevick-Muraca EM, ZamboniW, Farokhzad OC, et al. Challenges and key considerationsof the enhanced permeability and retention effect for nano-medicine drug delivery in oncology. Cancer Res 2013;73:2412–7.

50. Vrignaud P, S�emiond D, Lejeune P, Bouchard H, Calvet L, Combeau C,et al. Preclinical antitumor activity of cabazitaxel, a semisynthetictaxane active in taxane-resistant tumors. Clin Cancer Res 2013;19:2973–83.


Wang et al.




2017;77:6963-6974. Published OnlineFirst October 20, 2017.Cancer Res Hangxiang Wang, Zhongjie Lu, Lijiang Wang, et al. Small-Molecule Prodrugs Alleviate Cancer Drug ToxicityNew Generation Nanomedicines Constructed from Self-Assembling

Updated version

10.1158/0008-5472.CAN-17-0984doi:

Access the most recent version of this article at:

Material

Supplementary

http://cancerres.aacrjournals.org/content/suppl/2017/10/20/0008-5472.CAN-17-0984.DC1

Access the most recent supplemental material at:

Cited articles

http://cancerres.aacrjournals.org/content/77/24/6963.full#ref-list-1

This article cites 50 articles, 12 of which you can access for free at:

Citing articles

http://cancerres.aacrjournals.org/content/77/24/6963.full#related-urls

This article has been cited by 2 HighWire-hosted articles. Access the articles at:

E-mail alerts related to this article or journal.Sign up to receive free email-alerts

Subscriptions

Reprints and

[email protected]

To order reprints of this article or to subscribe to the journal, contact the AACR Publications Department at

Permissions

Rightslink site. Click on "Request Permissions" which will take you to the Copyright Clearance Center's (CCC)

.http://cancerres.aacrjournals.org/content/77/24/6963To request permission to re-use all or part of this article, use this link



http://cancerres.aacrjournals.org/lookup/doi/10.1158/0008-5472.CAN-17-0984

http://cancerres.aacrjournals.org/content/suppl/2017/10/20/0008-5472.CAN-17-0984.DC1

http://cancerres.aacrjournals.org/content/77/24/6963.full#ref-list-1

http://cancerres.aacrjournals.org/content/77/24/6963.full#related-urls

http://cancerres.aacrjournals.org/cgi/alerts

mailto:[email protected]

http://cancerres.aacrjournals.org/content/77/24/6963


New Generation Nanomedicines Constructed from …...weight loss of 20% compared with the weight...

Documents

Transcript of New Generation Nanomedicines Constructed from …...weight loss of 20% compared with the weight...