Biamphiphilic triblock copolymer micelles as a multifunctional platform for anticancer drug delivery

Biamphiphilic triblock copolymer micelles as amultifunctional platform for anticancer drug delivery

Wen Zhu,1 YanLi Li,2 LiXin Liu,2 WenLong Zhang,1 YongMing Chen,1 Fu Xi1

1Laboratory of Polymer Physics and Chemistry, Institute of Chemistry, The Chinese Academy of Sciences,

Beijing 100190, China2College of Life Science, Graduate University of the Chinese Academy of Sciences, Beijing 100049, China

Received 9 June 2010; revised 2 September 2010; accepted 28 September 2010

Published online 29 November 2010 in Wiley Online Library (wileyonlinelibrary.com). DOI: 10.1002/jbm.a.32985

Abstract: Novel micelles from biamphiphilic triblock copoly-

mer poly(ethylene glycol)-b-poly(e-caprolactone)-b-poly(acrylicacid) (PEG-b-PCL-b-PAA) as new multifunctional nanocarriers

to delivery anticancer drugs were evaluated. The well-defined

triblock copolymers prepared by controlled polymerizations

self-assembled into micelles in aqueous solution with a hydro-

dynamic radius of 13 nm as obtained by dynamic light scatter-

ing (DLS) and a low critical micellization concentration of 2.9

� 10�4 g/L. The hydrophobic PCL cores of micelles were

applied to load hydrophobic drug doxorubicin and the func-

tional PAA subcoronas clung to the micellar core were used to

carry cisplatin through covalent interaction. The results indi-

cated that two anticancer drugs had been loaded by different

mechanism either separately or simultaneously. Drug loading

content and efficiency as well as release profiles were eval-

uated. Furthermore, internalization and cytotoxicity of the anti-

cancer nanoparticles against human bladder carcinoma EJ

cells were studied. The biamphiphilic triblock copolymer

micelles provided not only biocompatibility and biodegradabil-

ity, but also abilities for loading single and dual anticancer

drugs, indicating that this was a useful multifunctional plat-

form for anticancer drug delivery. VC 2010 Wiley Periodicals, Inc.

J Biomed Mater Res Part A: 96A: 330–340, 2011.

Key Words: block copolymers, cisplatin, doxorubicin, drug

delivery systems, nanoparticles

INTRODUCTION

Micelles self-assembled from amphiphilic block copolymersused for drug delivery systems have attracted a great inter-est because of their unique advantages.1–5 The core-shellarchitecture of amphiphilic block copolymer micelles sup-plies a hydrophobic core to encapsulate hydrophobic drugsand a hydrophilic corona to stabilize the particles in aque-ous solutions.6 Also critical micellization concentration(CMC) of block copolymer micelles was very low relativelyto that of lipids which is important for stabilization ofmicelles during delivery process.7,8 It was noteworthy thatthe polymer micelles with poly(ethylene glycol) (PEG) coro-nas show a prolonged plasma circulation, a reduced cytotox-icity and a passive targeting to solid tumors through the so-called enhanced permeability and retention (EPR) effect dueto their anti-adhesive shells and nano-scale size which ispreferential for angiogenic tumour vasculature.9–11 In addi-tion, structures and functions of amphiphilic block copoly-mers could be well controlled by the polymerization meth-ods including atom transfer radical polymerization(ATRP),12,13 reversible addition fragmentation transfer(RAFT) polymerization14,15 and ring-opening polymerization(ROP),16,17 which have greatly facilitated the control of com-position, size, and functionality of polymeric micelles usedfor drug delivery systems.18

Diblock copolymer micelles with PEG coronas are thesimplest systems and have been widely applied to load anti-cancer drugs by either physical or chemical interaction.19–21

For the physical interaction to support drugs, micelles ofbiodegradable poly(ethylene glycol)-b-polycaprolactone andpoly(ethylene glycol)-b-polylactide are typical examples thathave been applied for delivering anticancer drugs like dox-orubicin (DOX),22 cisplatin (CDDP),23 paclitaxel (TAX),16,24

and estradiol.25 For the case of chemical loading, drugs havebeen linked to one block copolymer segment via chemicalbonds that may be cleaved to deliver drug molecules undercertain conditions. For example, Bae et al. have attachedDOX onto poly(ethylene glycol)-b-poly(aspartic acid) via apH sensitive bond which may release drugs in acidic lyso-somes.21 Nishiyama et al. have developed CDDP deliverysystems using either poly(ethylene glycol)-b-poly(asparticacid)26 or poly(ethylene glycol)-b-poly(glutamic acid)27 tocomplex the drugs via coordination of carboxylic groupsand platinum (II) atoms. However, the diblock copolymermicelle systems loading drugs by either physical or chemicalinteraction may only deliver single drug for one function.

The use of drug cocktails has become a widely adoptedstrategy in clinical cancer therapy.28 The clinical and pre-clinical studies have shown that cocktail therapy of cancerwith dual anticancer drugs may lead to better tumor

Correspondence to: Y. M. Chen; e-mail: [email protected] or L. X. Liu; e-mail: [email protected]

Contract grant sponsor: National Science Foundation of China; contract grant numbers: 20534010, 20625412

Contract grant sponsor: Knowledge Innovation Program of the Chinese Academy of Sciences; contract grant number: KJCX2-YW-H19

330 VC 2010 WILEY PERIODICALS, INC.

regression compared to either drug alone.29–31 Polymericvehicles in dual-drug delivery systems that combined differ-ent drugs show advantages in medical treatment. Ahmedet al. have developed a TAX/DOX loaded delivery system ofdegradable polymersomes and evaluated pH-triggered drugrelease.32 Wei et al. have reported a dual-drug delivery sys-tem based on hydrogel/micelle composites.33 Lee et al. havestudied polymeric micelles self-assembled from four-armheparinized copolymers to deliver basic fibroblast growthfactor and indomethacin.34 Qiu et al. have reported polye-thylenimine-graft-poly(e-caprolactone) micelles as dual car-riers of genes and DOX.35 Nevertheless, limited researcheson amphiphilic block copolymer micelles of dual-drug deliv-ery systems have reported so far. To obtain a so-called mul-tifunctional delivery system,36–41 we endow the polymermicelles with multifunctional characters by using the amphi-philic triblock copolymers.

Biamphiphilic triblock copolymer is a type of block co-polymer that contains one hydrophobic block between twohydrophilic blocks. Such copolymers may form micelles dueto the amphiphilicity and their hydrophobic cores can beapplied to trap hydrophobic guest molecules and the thirdblock may be designed as a functional block to capture dif-ferent species via chemical interaction. Previously, vesicle ormicelle formation of biamphiphilic triblock copolymers havebeen studied and inorganic nanoparticles have been stabi-lized by the third segments.42,43 Also Eisenberg and co-workers have studied the vesicles of poly(ethylene glycol)-b-poly(e-caprolactone)-b-poly(acrylic acid) (PEG-b-PCL-b-PAA) biamphiphilic triblock copolymers and their interac-tion with bovine serum albumin.44

Herein, biamphiphilic triblock copolymer PEG-b-PCL-b-PAA with low molecular weight distribution was preparedby a combination of ROP and ATRP. We explored the func-tions of PCL and PAA blocks to evaluate the copolymermicelles as a multifunctional anticancer drug delivery vehi-cle that can load not only single drug but also dual anti-cancer drugs. As indicated in Scheme 1, the hydrophilic PEGblocks form a micellar corona and provide a protective coat-ing between the micellar core and the external circumstanceto prevent non-specific protein recognition. The micellarcore formed by hydrophobic PCL blocks is a reservoir ofhydrophobic drugs such as DOX and, moreover, it may de-grade in biologic conditions to facilitate the renal clearanceof the micelles. The PAA blocks with abundant carboxylicfunctional groups form a subcorona layer which is used to

carry different drugs such as CDDP through covalent inter-action. We expected such a delivery system could carry anddeliver DOX and CDDP for chemotherapy of antitumor appli-cation. Drug loading content and efficiency as well as drugrelease behavior and cytotoxicity against human bladdercarcinoma EJ cells have been evaluated. All the three indi-vidual blocks have been approved by the United States Foodand Drug Administration (FDA) for medical uses.

EXPERIMENTAL PART

MaterialsMethoxy poly(ethylene glycol) (PEG-OH, Mn 5000, Alfa) wasdried by azeodistillation with benzene and freeze-dried. e-Caprolactone (e-CL, Aldrich, 99%) and tert-butyl acrylate(tBA, Aldrich, 99%) were dried over CaH2. Lipase PS(enzyme) from Pseudomonas cepacia (Amano Pharma-ceutical) was further purified by freeze-drying; 1,3,4,6,7,8-Hexahydro-2H-pyrimido[1,2-a]-pyrimidine (TBD, Aldrich,98%), N,N,N0,N00,N00-pentamethyldiethylenetriamine (PMDETA,Aldrich, 99%), 2-bromoisobutyryl bromide (Aldrich, 98%), 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyl tetrazolium bromide(MTT, Duchefa), CDDP (Boyuan, China, >99%) and trifluoro-acetic acid (TFA, Merck-Schuchardt, 99%) were used withoutfurther purification. Toluene was distilled from sodium/ben-zophenone before use. Dialysis bags with molecular weightcutoffs (MWCO) of 3500 and 14,000 Da, respectively wereobtained from Viskase. DOX-HCl (Haizheng, China) was depro-tonated at pH 9.6 to obtain the hydrophobic DOX.22 All otherchemicals and solvents were of analytical grade and wereused as received.

Synthesis of ATRP macroinitiator PEG-b-PCL-BrDiblock copolymer PEG-b-PCL with a hydroxyl end group ofthe PCL block was synthesized by ring-opening polymeriza-tion according to the method developed by Pratt et al.45 e-CL (9.1 g, 80 mmol) was added to a solution of TBD (27.8mg, 200 lmol) and PEG-OH (10.0 g, 2 mmol) in dried tolu-ene (125 mL). The solution was then stirred for 22 h at am-bient temperature and quenched by addition of benzoic acid(35 mg). The product was obtained by precipitating twice indiethyl ether and dried under vacuum. The monomer con-version evaluated by precipitation was 70%. The terminalhydroxyl end groups of PCL blocks were modified with 2-bromoisobutyryl bromide to obtain the macroinitiator PEG-b-PCL-Br as a white powder according to the literature.12

SCHEME 1. Micelle structure of PEG-b-PCL-b-PAA triblock copolymer as a multifunctional platform for anticancer drug delivery. [Color figure

can be viewed in the online issue, which is available at wileyonlinelibrary.com.]

ORIGINAL ARTICLE

JOURNAL OF BIOMEDICAL MATERIALS RESEARCH A | FEB 2011 VOL 96A, ISSUE 2 331

1H NMR (400 MHz, CDCl3) d 1.38 (m, 56H, CH2CH2CH2,CL unit), 1.64 (m, 112H, CH2CH2CH2, CL unit), 1.94 (s, 6H,AC(¼O)C(CH3)2ABr), 2.31 (t, 56H, CH2C(¼O)O, CL unit),3.65 (s, 456H, AOCH2CH2OA, EG unit), 4.08 (t, 56H,AC(¼O)OCH2, CL unit).

Synthesis of triblock copolymer PEG-b-PCL-b-PAAPEG-b-PCL-b-PtBA was first synthesized by ATRP usingPEG-b-PCL-Br as macroinitiator and CuBr/PMDETA as cata-lyst. PEG114-b-PCL28-Br (2.30 g, 0.27 mmol) and CuBr (20mg, 0.14 mmol) were introduced into a dried Schlenck flaskfilled with nitrogen. A cycle of evacuation under vacuumand backfilling with nitrogen was repeated three times toremove oxygen. Anisole (5.5 mL), tBA (3.5 g, 27 mmol), andPMDETA (30 lL, 0.14 mmol) were deoxidized and added tothe Schlenck flask via a gastight syringe. The mixture wasdegassed three times by freeze-pump-thaw cycles. Polymer-ization was performed under nitrogen flow at 60�C. Thepolymers formed were then dissolved in CHCl3 and passedthrough a short alkaline alumina column to remove the cat-alysts. The triblock copolymer PEG-b-PCL-b-PtBA was recov-ered by precipitating twice into hexane and dried in vacuumat room temperature. Then, PEG-b-PCL-b-PtBA was dis-solved in CH2Cl2 and a fivefold molar excess of TFA (withrespect to the amount of tert-butyl groups in the tBA units)was added. The reaction mixture was stirred at room tem-perature for 48 h and the hydrolysate was precipitatedtwice in diethyl ether and dried in vacuum at roomtemperature.

1H NMR (400 MHz, DMSO-d6) d 1.30 (m, 56H, CH2CH2CH2,CL unit), 1.55 (m, 162H, CH2CH2CH2, CL unit, ACH2CH(COOH)A), 2.27 (m, 81H, ACH2C(¼O)O, CL unit, ACH2CH(COOH)A), 3.51 (s, 456H, AOCH2CH2OA, EG unit), 3.98 (t,56H, AC(¼O)OCH2A), 12.25 (s, 25H, ACH2CH (COOH)A).

Preparation of PEG-b-PCL-b-PAA micelles (P-E)Self-assembly of PEG114-b-PCL28-b-PAA25 was accomplishedby dropping water slowly into a 1 wt % DMF solution ofthe amphiphilic triblock copolymer under vigorous agitationto yield a 0.1 wt % mixed solution. DMF was then taken outof the mixed solution by dialysis (MWCO ¼ 14,000 Da)against water for 3 days. The size of micelles was deter-mined by dynamic light scattering (DLS) after filteringthrough a 0.45 lm Millipore filter in order to remove par-ticles larger than 450 nm in diameter.

Preparation of anticancer drugs loaded nanoparticlesAnticancer drugs, CDDP or/and DOX, were incorporatedinto the micelles to form anticancer nanoparticles by differ-ent ways. As listed in Table I, CDDP was loaded into thecore by hydrophobic interaction (P-C), into PAA corona bycoordination (P-EC) or into both core and corona by twointeractions respectively (P-CC). DOX was loaded into thecore by hydrophobic interaction (P-D). CDDP was loadedinto the PAA while DOX was loaded into the core (P-DC).The detail procedures were described as follows:

P-C. PEG114-b-PCL28-b-PAA25 (10 mg) and CDDP (2 mg)were dissolved in DMF (1 mL) and then 4 mL of water wasadded slowly into the solution under vigorous agitation toform CDDP loaded nanoparticles. The unloaded CDDP andorganic solvents were removed from the mixed solution bydialysis (MWCO ¼ 14,000 Da).

P-D. PEG114-b-PCL28-b-PAA25 (10 mg) and DOX (2 mg)were dissolved in THF/DMF (1 mL, volume ratio ¼ 10) andthen 4 mL of water was added slowly into the solutionunder vigorous agitation to form DOX loaded nanoparticles.The unloaded DOX and organic solvents were removed fromthe mixed solution by dialysis (MWCO ¼ 14,000 Da).

P-EC. According to the literature,46 CDDP (7.4 mg) was sus-pended in 3 mL distilled water and mixed with silver nitrate([AgNO3]/[CDDP] ¼ 1) to form an aqueous complex. The so-lution was kept in dark at room temperature over night.AgCl precipitated after reaction was eliminated by passingthrough a 0.22 lm filter. P-E was added to the CDDP aque-ous solution ([COOH] ¼ 5 mmol/L and [CDDP]/[COOH] ¼1.0) and stirred at 37�C for 72 h. Purification was carriedout by dialysis (MWCO ¼ 14,000 Da) to remove theunreacted CDDP.47

P-CC. P-C and CDDP aqueous complex described abovewere stirred at 37�C for 72 h ([COOH] ¼ 5 mmol/L and[CDDP]/[COOH] ¼ 1.0). Purification was carried out by dial-ysis (MWCO ¼ 14,000 Da) to remove the unreacted CDDP.

P-DC. P-D and CDDP aqueous complex were stirred at 37�Cfor 72 h ([COOH] ¼ 5 mmol/L and [CDDP]/[COOH] ¼ 1.0).Purification was carried out by dialysis (MWCO ¼ 14,000Da) to remove the unreacted CDDP.

The drug loading contents were determined by a UV-1601 UV-Visible Spectrophotometer (Shimadzu). Linear cali-bration curves were obtained for DOX in a mixed solutionof DMSO and CHCl3 (volume ratio ¼ 1) and in distilledwater. The absorbance was measured with a UV-visiblespectrophotometer at 485 nm.22 Quantitative determinationof CDDP was performed in double distilled water.48 Onemilliliter of the sample solution was mixed with 1 mL of o-phenylenediamine (OPDA) solution (1.2 mg/mL in DMF).Then the mixture was placed in a 100�C water bath for 10min and the absorbance of the solution was measured at

TABLE I. CDDP and DOX Supported with Micelles of

PEG114-b-PCL28-b-PAA25 by Different Approaches

DrugCarriers

Drug Loaded intoMicellar Core by

Hydrophobic Effect

Drug Loadedin PAA Block

by Coordination

P-C CDDP –a

P-D DOX –a

P-EC –a CDDPP-CC CDDP CDDPP-DC DOX CDDP

a No drug was loaded.

332 ZHU ET AL. BIAMPHIPHILIC TRIBLOCK COPOLYMER MICELLES FOR DRUG DELIVERY

703 nm. The amount of CDDP in the nanoparticles was cal-culated in reference to standard solutions of free CDDP. Thesizes of the anticancer nanoparticles were determined byDLS after filtering through a 0.45 lm Millipore filter respec-tively of each sample.

The drug loading content and the encapsulation effi-ciency were calculated based on the following formulas:

Loading content ðwt %Þ ¼Amount of drug in anticancer nanoparticles=

Total weight of anticancer nanoparticles� 100%;

Loading efficiency ð%Þ ¼Amount of drug in anticancer nanoparticles=

Total amount of drug used in the preparation of anticancer

nanoparticles� 100%:

Measurement of critical micellizationconcentration (CMC)The CMCs of empty micelles P-E and CDDP loaded P-ECwere determined by an established fluorescence-basedmethod.49 In short, a stock solution of the sample was firstprepared at a concentration of 0.1 g/L and then diluted tonine different concentrations down to 10�7 g/L. Each sam-ple was then prepared by dropping 60 lL of a pyrene solu-tion (2.5 � 10�5 mol/L in acetone) into an empty vial,evaporating the acetone by gentle heating, adding 3 mL ofone copolymer solution, ultrasonic shaking the closed vialsfor 30 min and standing still for 24 h before detection. Thefinal concentration of pyrene in water thus reached 5 �10�7 mol/L, which was slightly below the pyrene saturatedconcentration in water of 6 � 10�7 mol/L at room tempera-ture. Fluorescence spectra of the samples were recordedwith a Cary Eclipse Fluorescence Spectrophotometer (Var-ian) and were determined at kex¼ 333 nm with bandwidthsof 10 nm for excitation and 2.5 nm for emission. Intensityof the bands I1 at 372 nm and I3 at 383 nm was then eval-uated, and their ratio was plotted versus the polymerconcentration.

Enzymatic biodegradationPyrene was used as a probe to determine biodegradation ofthe PEG-b-PCL-b-PAA micelles in Lipase PS aqueous solu-tion. Pyrene loaded micelles were prepared by the sameprocedure used for the CMC experiment and proper amountof Lipase PS aqueous solution was added to start the biode-gradation. The fluorescence intensity profiles were recordedby using a fluorescence spectrophotometer during the enzy-matic biodegradation at 37�C.

In vitro drug releaseIn vitro release of the drugs from the anticancer nanopar-ticles was studied in a 0.01M phosphate buffered saline(PBS, pH 7.4) with 0.16M NaCl and in distilled water. 10 mLof solution of anticancer nanoparticles (0.1 wt %) wascharged into a dialysis bag (MWCO ¼ 3500 Da) which was

then submerged into a beaker containing 400 mL of the cor-responding buffer solution. The beaker was maintained in awater bath of 37�C and was stirred at constant 100 rpm. Atregular time intervals, 0.5 mL of the medium was removedfor UV analysis of its drug content and the same volume offresh buffer solution was added to keep the volume of deliv-ery solution. A profile showing the cumulative amount ofdrug release as a function of time was plotted at eachrelease condition. Each release experiment was repeatedthree times.

Cytotoxicity studyCytotoxicities of drug loaded nanoparticles and free drugswere measured against human bladder carcinoma EJ cellsby MTT assay. All growth media were prepared by supple-menting RPMI 1640 with 5% penicillin-streptomycin, 10%fetal bovine serum, 0.9% NaCl and sterilized with 0.22 lmfilter prior to use. Density of the cell line solution was firstmeasured by a haemocytometer and then the cells wereseeded into 96-well plates at a density of 3000 cells perwell. The plates were incubated in a humidified 37�C envi-ronment with 5% CO2 for 24 h. Then, samples of differentdrug formulations (free CDDP, free DOX, P-E, P-CC, and P-DC) were dissolved in growth media and then seriallydiluted to give a range of final drug concentration from 0 to1000 mg/L. They were then incubated for 24, 48, 72, and96 h respectively before adding 20 lL of MTT solution intothe media of each well. After incubation for another 4 h,0.15 mL of DMSO was added to each well to dissolve theMTT crystals formed. The plates were vigorously shakenbefore measuring by microplate reader (MULTISCAN MK-III,Thermo-Electron). A test wavelength of 570 nm and a refer-ence wavelength of 630 nm were used.50 Cell viability ofsample was expressed as a percentage of the intensity ofthe controls standard deviation. Hundred percent referredto conditions without treatment (control). Each experimentwas repeated four times at each sample concentration.

Cell internalizationDOX accumulated in bladder carcinoma EJ cells was local-ized using a FV1000-IX81 (Olympus) confocal laser scan-ning microscope (CLSM) with a lens of UPLANSAPO100XONA 1.40 (oil). The cells were seeded on cover-slidesfor 24 h and were then treated with free DOX (1 mg/L) anddual-drug loaded P-DC (20 mg/L), respectively before incu-bated in a humidified atmosphere of 5% CO2 at 37�C. After1, 24, 48, and 72 h, cell nuclei were stained with Hoechst33258 (4 mg/L) for 40 min to identify the micelle locationbefore CLSM examination. The media were replaced withPBS before examined. Hoechst 33258 and DOX were excitedat 405 and 559 nm with emissions at 422 and 619 nm,respectively.

Analysis and characterization1H NMR spectrum was obtained on a Bruker DMX400 spec-trometer. CDCl3 and DMSO-d6 were used as solvents andTMS as an internal standard.

ORIGINAL ARTICLE


Apparent molecular weights, Mn, and molecular weightdistributions, Mw/Mn, of the polymers were determined bysize exclusion chromatography (SEC) equipped with aWaters 515 pump, a Waters 2414 refractive index detector,and a combination of column Styragel HT2, HT3, HT4; theeffective molar mass ranges were 100–10,000, 500–30,000and 5000–600,000 respectively. Linear polystyrene stand-ards were applied for calibration. The eluent was THF at aflow rate of 1 mL/min at 35�C.

Dynamic light scattering (DLS) was performed on a laserlight scattering spectrometer (ALV/DLS/SLS-5022F)equipped with a multi-s digital time correlator (ALV5000)and a cylindrical 22 mW UNIPHASE He-Ne laser (k0 ¼632.8 nm). Temperature was kept at 25�C, and scatteringdata were collected at 90�.

Fourier transform infrared (FT-IR) spectra of the sam-ples were obtained on a Nicolet Avatar 330 FT-IR spectrom-eter at frequencies ranging from 500 to 3800 cm�1.

RESULTS AND DISCUSSION

The synthesis of PEG114-b-PCL28-b-PAA25 biamphiphilic tri-block copolymer was carried out by four steps as shown inScheme 2. Firstly, the diblock copolymer PEG114-b-PCL28-OHwas synthesized by a ROP of e-caprolactone in whichPEG114-OH was used as the initiator and TBD as the cata-lyst. The ROP took place under a mild condition with highactivity as reported previously.45 Secondly, PEG114-b-PCL28-OH was acetylated with 2-bromoisobutyryl bromide toobtain the macroinitiator PEG114-b-PCL28-Br. Thirdly, themacroinitiator was used for synthesis of the third block byATRP using tBA as the monomer and CuBr/PMDETA as thecatalyst. Finally, PEG114-b-PCL28-b-PtBA25 was convertedinto PEG114-b-PCL28-b-PAA25 by hydrolysis with fivefoldmolar excess of TFA (with respect to the amount of tert-butyl groups in the side chains). Molecular weight distribu-

tions of the block copolymers were shown in Table II andthe SEC curves were collected in Figure 1. The biamphi-philic triblock copolymer PEG114-b-PCL28-b-PtBA25 showeda low molecular weight distribution of 1.07 and no macroi-nitiator left according to the SEC curve, which indicatedhigh efficiency of the acetylation. The degree of polymeri-zation (DP) of PCL block was estimated through compa-rison of the peak areas of 1H-NMR spectrum (Fig. 2) of3.65 ppm (ACH2CH2O of the PEG block) and 3.98 ppm(AC(¼O)OCH2A of the PCL block). The DP of PAA blockwas determined in the same way by comparing the peakareas of 3.65 ppm (ACH2CH2OA of the PEG block) and2.27 ppm (ACH2CH (COOH)A of the PAA block). The hydro-lysis of PtBA was also confirmed by IR analysis that thepeaks of tert-butyl group (asymmetric doublet at 1365 and1395 cm�1) were disappeared and the peak of carbonylgroup decreased at 1735 cm�1. The stretching band ofcarbonyl at 1735 cm�1 after hydrolysis was the signal ofcarbonyl groups of the PCL backbone.

Self-assembled micelles of PEG114-b-PCL28-b-PAA25 andanticancer nanoparticles were all obtained by dissolving theblock polymers (and anticancer drugs) in DMF and then dia-lyzing against water to induce micellization followed by

SCHEME 2. Synthetic route for PEG-b-PCL-b-PAA triblock copolymer.

TABLE II. Characteristics of Block Copolymers during

Their Preparation

BlockCopolymera Mn (NMR)a Mn (SEC)b Mw/Mnb

PEG114-b-PCL28-OH 8200 12600 1.07PEG114-b-PCL28-b-PtBA25 11600 16200 1.07PEG114-b-PCL28-b-PAA25 10200 11000 1.15

a Composition and number average molecular weights Mn of the

block copolymers were estimated from 1H-NMR spectra.b Mn and Mw/Mn (where Mw is the weight average molecular

weight) of the block copolymers were determined by SEC.


removal of organic solvent and unloaded drugs. The hydro-dynamic radius of 0.1% (w/w) solutions of the emptymicelles P-E was 13 nm as measured by DLS [Fig. 3(a)].Drug loading had little influence in hydrodynamic radius ofthe micelles, which was similar to the literature in the samedrug loading content level.27 The hydrodynamic radii ofdrug loaded micelles P-C, P-D, P-EC, P-CC, and P-DC were 13nm [Fig. 3(b)], 12 nm [Fig. 3(c)], 12 nm [Fig. 3(d)], 15 nm[Fig. 3(e)], and 13 nm [Fig. 3(f)], respectively. In all cases,some large particles ranging in �90 to 200 nm showing aslow relaxation mode were observed probably due to theassociation of small individual micelles.51,52

CMC of the present triblock copolymer was determinedon the basis of fluorescence measurement with pyrene as ahydrophobic probe. Figure 4(a) gave a CMC of 2.9 � 10�4

g/L, which was comparable with that of PEG-b-PCL diblockcopolymers reported in literature.53 The CMC of P-EC con-tained CDDP was evaluated in the same way and the valuewas 3.2 � 10�4 g/L as shown in Figure 4(b), indicating that

the coordination of CDDP had little influence in stabilizingthe micelles and the hydrophobic effect of PCL block shouldbe the driving force of the self-assembly. Such a low micelli-zation concentration is important to stabilize the drug deliv-ery nanocargoes during in vivo application.54

It is known that PCL is a biodegradable polymer anddegradation of the empty micelles P-E of triblock copoly-mers in the presence of lipase PS was estimated by fluores-cence measurement with pyrene as probe. It is known thatpyrene shows higher fluorescence intensity in nonpolarenvironment (PCL core) because of its long lifetime (�400ns) and high quantum yield. In contrast, it has a lower fluo-rescence intensity in water due to the shorter lifetime(�200 ns) and lower quantum yield. The initial concentra-tion of P-E was 5 � 10�2 g/L that was much higher than itsCMC, so the hydrophobic pyrene was loaded into the PCLcore before biodegradation. As shown in Figure 5, the fluo-rescence intensity of pyrene in micelles P-E kept mostlyunchangeable when there was no lipase in the solution. Incontrast, when the initial concentration of Lipase PS was 6� 10�3 g/L, the fluorescence intensity gradually decreasedas time increased, indicating that pyrene was released fromPCL cores to water because of the degradation. According tothe literature, PEG-b-PCL nanoparticles with a PCL coredegraded by Lipase PS followed a one-by-one fashion andthe biodegradation rate of the present micelles wascomparable.53

Table III listed drug loading contents and loading effi-ciencies of the samples prepared by different approaches. P-C and P-D loaded CDDP and DOX within the micellar corerespectively. CDDP loading content of P-C was 8.5 wt % andloading efficiency was 46.4%; DOX loading content of P-Dwas 11.3 wt % and loading efficiency was 65%. CDDP wasalso loaded into sub-corona of PAA by substituting its chlo-ride ligands with carboxylic ligands to give P-EC. CDDPloading content of P-EC was 10.0 wt % and loading effi-ciency was 17.0%. As for P-CC, CDDP was first loaded intothe PCL core by hydrophobic interaction and then was sup-ported into the PAA sub-corona by coordination. Due to thelost CDDP during second step of loading, it is hard to know

FIGURE 1. SEC curves of starting PEG114-OH and block copolymers in

THF.

FIGURE 2. 1H NMR spectrum of PEG114-b-PCL28-b-PAA25 triblock co-

polymer in DMSO-d6.

FIGURE 3. DLS curves of (a) micelles P-E and drug carries of (b) P-C,

(c) P-D, (d) P-EC, (e) P-CC, and (f) P-DC.

ORIGINAL ARTICLE


the CDDP contents loaded in every step. Owing to the stabil-ity of CDDP coordination in solutions of low salinity, weinferred that the coordinated loading content of CDDP in P-CC should be similar to that of P-EC, which was about 10wt %. Anyway, the whole CDDP loading content of P-CCreached 15.4 wt %, which is pretty high. Therefore, CDDPdrug molecules could be supported into the micelles ofbiamphiphilic triblock copolymer by different mechanisms.To fulfill loading dual-drug, i.e., CDDP and DOX into thesame micellar carrier, DOX was first supported into the PCLcore by hydrophobic interaction and then CDDP was loadedin the PAA subcorona of the micelle. In this recipe, loadingcontents of DOX and CDDP were 6.3 wt % and 10 wt %,respectively. Comparing with that of P-D, DOX loading con-tent dropped from 11.3 wt % to 6.3 wt % probably due tothe drug lost during the procedure to load CDDP. Aboveresults indicated that the present triblock copolymer micelleis a flexible carrier to load different drugs either by hydro-phobic interaction or by coordination interaction.

In vitro release of anticancer drugs from the nanopar-ticles of biamphiphilic block copolymers was carried out in0.01M PBS (pH 7.4) containing 0.16M NaCl at 37�C in order

to mimic biologic condition and in distilled water. Figure 6exhibited CDDP release curves of P-C, P-EC, and P-CC. CDDPloaded in P-C was attributed to hydrophobic effect, and therelease was found to follow a diffusing way that more than70% of CDDP was released during the first 40 h no matterin PBS containing 0.16M NaCl or in distilled water [Fig.6(a)]. CDDP loaded by coordination in P-EC exhibited a sus-tained release during 250 h with 55% of CDDP released inPBS containing 0.16M NaCl [Fig. 6(b)]. In contrast, nearlyno CDDP was released in distilled water, indicating that thepresence of Cl� was essential for ligand substitution ofCDDP from the carboxyls of PAA blocks.55 CDDP loaded inP-CC by both hydrophobic effect and coordination releasedapproximately 65% in PBS containing 0.16M NaCl during250 h. But only 30% of total CDDP released in distilledwater attributed to the hydrophobic loading [Fig. 6(c)]. Thisresult proves that CDDP was indeed trapped into the P-CCsystem by two ways.

Figure 7 showed the drug release curves of dual-drugsystem, P-DC, in PBS containing 0.16M NaCl. It is observedthat over 70% of DOX released during 80 h [Fig. 7(a)] and65% of CDDP released during 265 h [Fig. 7(b)]. The releaseof DOX followed a diffusing approach whereas that of CDDPfollowed a ligand exchange way. Therefore, two kinds ofdrugs were released from their carriers separately.

Figure 8 showed the in vitro cytotoxicity of the micellesof PEG114-b-PCL28-b-PAA25 copolymers in human bladdercarcinoma EJ cells. The cell viabilities were above 68% forthe copolymer concentrations investigated (125–1000 mg/L)

FIGURE 4. CMCs determined by fluorescence measurements of (a) P-E and (b) P-EC.

FIGURE 5. The fluorescence intensity of pyrene in micelles P-E (5 �10�2 g/L) without and with Lipase PS (6 � 10�3 g/L) during biodegra-

dation at 37�C.

TABLE III. Drug Loading Contents and Loading Efficiencies of

the Samples in Table I

Sample

Loading Content(wt %)

Loading Efficiency(%)

CDDP DOX CDDP DOX

P-C 8.5 – 46.4 –P-D – 11.3 – 65P-EC 10.0 – 17.0 –P-CC 15.4 – 21.3 –P-DC 10.0 6.3 18.3 37.6


and that of 24 h was higher than the case of 48 h for eachcopolymer concentration. Thus the PEG114-b-PCL28-b-PAA25

triblock copolymer showed no significant toxicity to humanbladder carcinoma EJ cells even in a high concentration of1000 mg/L.

To evaluate and compare the in vitro cytotoxicities ofanticancer nanoparticles P-CC and P-DC, we fixed the wholedrug concentration on the same value of 10 mg/L to obtaincell viabilities (%) during different incubated times. Asshown in Figure 9, free CDDP and DOX exhibited an instantcytotoxicity in the first 48 h as the cell viabilities of themdropped to 35.5% and 16.8%, respectively, and thendecreased slowly. In contrast, the cell viabilities of P-CC andP-DC decreased less than 10% from 24 h to 48 h, whichwas due to the time-consuming drug release from anti-

cancer nanoparticles. During 96 h, both of two free drugskilled 99% of the cells and drug loaded nanoparticles killedonly 75% of them. It indicated that P-CC and P-DC did showcytotoxicity to the tumor cells but slow release of drugsfrom nanoparticles reduced the cytotoxicity to a certainextent.

The concentrations that 50% of cells were killed (IC50)in different incubated times of free CDDP, free DOX, P-CC,and P-DC were listed in Table IV. The IC50s of P-CC and P-DC were not obtained in the experimental concentrations(5–500 mg/L) at 24 h due to two reasons. Firstly, IC50s offree drugs at 24 h were relatively high, which were 44.7mg/L and 7.8 mg/L for CDDP and DOX, respectively, andIC50s of P-CC and P-DC should be higher in view of thedrug loading contents. Secondly, the drug release was a

FIGURE 6. CDDP release curves of (a) P-C, (b) P-EC, and (c) P-CC in 0.01M PBS (pH 7.4) containing 0.16M NaCl (l) or in distilled water (o) at

37�C.

FIGURE 7. Release curves of (a) DOX and (b) CDDP from the P-DC dual-drug system in 0.01M PBS (pH 7.4) containing 0.16M NaCl at 37�C.

ORIGINAL ARTICLE


time-consuming process according to the release curves, soless than 20% of the drug released from the anticancernanoparticles at 24 h, which led to a high IC50. As timeincreased, the cytotoxicity of dual-drug loaded P-DCincreased faster than single-drug loaded P-CC. Further incu-bated from 48 h to 96 h, the IC50 of P-DC decreased sharplyfrom 177.4 mg/L to 13.4 mg/L and that of P-CC decreased

FIGURE 8. In vitro cytotoxicity of PEG-b-PCL-b-PAA triblock copoly-

mer. The cell viabilities were given relatively to the control and were

expressed as a function of the copolymer concentration in human

bladder carcinoma EJ cells.

FIGURE 9. Cytotoxicity study of free anticancer drugs and anticancer

nanoparticles in human bladder carcinoma EJ cells. The whole drug

concentration of each sample was 10 mg/L. Standard deviation for

each data point was averaged over four samples (n ¼ 4).

TABLE IV. IC50 (mg/L) of the Free Drugs and Anticancer

Nanoparticles in Different Incubated Times

Time (h) Free CDDP Free DOX P-CC P-DC

24 44.7 6 3.2 7.8 6 0.4 N/A N/A48 5.9 6 0.3 0.2 6 0.01 91.4 6 5.8 177.4 6 7.672 1.9 6 0.4 0.1 6 0.01 68.0 6 1.2 57.1 6 1.296 0.5 6 0.04 <0.1 46.4 6 2.6 13.4 6 0.8

FIGURE 10. CLSM images of human bladder carcinoma EJ cells incubated with free DOX for 1 h (a), 24 h (b), 48 h (c), and 72 h (d), and with dual-drug

loaded P-DC for 1 h (e), 10 h (f), 24 h (g), and 48 h (h). For each panel, images from left to right show the cells with nuclear staining by Hoechst 33258, with

DOX fluorescence and overlays of both images. [Color figure can be viewed in the online issue, which is available at wileyonlinelibrary.com.]


slowly from 91.4 mg/L to 46.4 mg/L. Apparently, differenttendencies of the cell viabilities indicated a potentialadvantage of dual-drug loaded anticancer nanoparticles.

CLSM was used to observe the internalization of P-DCdual-drug system and free DOX was used for comparison.Cell nuclei were stained in order to determine whether DOXentered the nuclei and the difference between free DOX andP-DC was discussed. For most particulate carriers, it is gen-erally assumed that the triggering mechanism must occur inthe endosome to release the drugs in the cytoplasm.56,57 Asshown in Figure 10, the red fluorescence and blue fluores-cence represented the localization of DOX and nuclei of thecells, respectively. DOX intracellular distribution for the P-DC was quite different from that of free DOX. After 1 h ofincubation with free DOX, the fluorescence was observed incell nuclei and very weak in cytoplasm. Incubation with freeDOX for 24, 48, and 72 h, the stronger DOX fluorescencewas observed in the nuclei, and still no obvious fluores-cence was detected in the cytoplasm. In contrast, the DOXfluorescence for the P-DC system was observed only in cyto-plasm rather than the cell nuclei after incubation for 1 and24 h, indicating that P-DC nanoparticles were taken up fromextracelluar fluid into cells by endocytosis. It is notable thatafter 48 h of exposure to P-DC, DOX fluorescence wasobserved in the nuclei and after 72 h of exposure, DOX fluo-rescence in the nuclei was even stronger, owing to the diffu-sion of released DOX from the P-DC into the nuclei. Delayedfluorescence accumulation of DOX from cytoplasm to cellnuclei implied that the DOX-loaded P-DC nanoparticles wereinternalized into the cell and released DOX intracellularly.58

CONCLUSION

Micellization of biamphiphilic triblock copolymer PEG114-b-PCL28-b-PAA25 was studied and the novel micelles as anti-cancer drug delivery vehicles were evaluated. We haveshown that the PCL cores and PAA subcoronas of triblockcopolymer micelles provided two reservoirs respectively toload different anticancer drugs through different mecha-nism. The PCL core may trap hydrophobic drugs such asDOX whereas the PAA may load CDDP by coordination. Thein vitro release data indicated that the drugs loaded by dif-ferent manners exhibited different release mechanisms. It isnoteworthy that two drugs may be loaded either individu-ally or simultaneously. CLSM studies showed that the drug-loaded nanoparticles (P-DC) were internalized by cells andDOX was released in the cytoplasm and then entered thecell nuclei, which was very different from the localization offree DOX. Noticeably, dual-drug loaded nanoparticles P-DCdisplayed superior tumor cell growth inhibition to single-drug CDDP loaded nanoparticles P-CC, which indicatedpotential advantage of dual-drug loaded delivery system. Inprinciple, the PAA block can be loaded with other drugs andimaging agents that react with carboxyl groups, while thePCL block can trap different hydrophobic drugs. By takingPEG corona and degradability of PCL into consideration, thepresent biamphiphilic triblock copolymer micelles may be aversatile platform for anticancer drug delivery application.

REFERENCES1. Adams ML, Lavasanifar A, Kwon GS. Amphiphilic block copoly-

mers for drug delivery. J Pharm Sci 2003;92:1343–1355.

2. Jones MC, Leroux JC. Polymeric micelles—A new generation of

colloidal drug carriers. Eur J Pharm Biopharm 1999;48:101–111.

3. Rosler A, Vandermeulen GWM, Klok HA. Advanced drug delivery

devices via self-assembly of amphiphilic block copolymers. Adv

Drug Deliv Rev 2001;53:95–108.

4. Mikhail AS, Allen C. Block copolymer micelles for delivery of can-

cer therapy: Transport at the whole body, tissue and cellular lev-

els. J Control Release 2009;138:214–223.

5. Nishiyama N, Kataoka K. Current state, achievements, and future

prospects of polymeric micelles as nanocarriers for drug and

gene delivery. Pharmacol Ther 2006;112:630–648.

6. Kwon GS, Forrest ML. Amphiphilic block copolymer micelles for

nanoscale drug delivery. Drug Develop Res 2006;67:15–22.

7. Park JH, Lee S, Kim JH, Park K, Kim K, Kwon IC. Polymeric nano-

medicine for cancer therapy. Prog Polym Sci 2008;33:113–137.

8. Torchilin VP. Structure and design of polymeric surfactant-based

drug delivery systems. J Control Release 2001;73:137–172.

9. Maeda H, Greish K, Fang J. The EPR effect and polymeric drugs:

A paradigm shift for cancer chemotherapy in the 21st century.

Adv Polym Sci 2006;193:103–121.

10. Duncan R. The dawning era of polymer therapeutics. Nat Rev

Drug Discov 2003;2:347–360.

11. Torchilin VP. Micellar nanocarriers: Pharmaceutical perspectives.

Pharm Res 2007;24:1–16.

12. Zhang Q, Remsen EE, Wooley KL. Shell cross-linked nanoparticles

containing hydrolytically degradable, crystalline core domains. J

Am Chem Soc 2000;122:3642–3651.

13. Narrainen AP, Pascual S, Haddleton DM. Amphiphilic diblock, tri-

block, and star block copolymers by living radical polymerization:

Synthesis and aggregation behavior. J Polym Sci Part A: Polym

Chem 2002;40:439–450.

14. Stenzel MH, Barner-Kowollik C, Davis TP, Dalton HM. Amphiphilic

block copolymers based on poly (2-acryloyloxyethyl phosphoryl-

choline) prepared via RAFT polymerisation as biocompatible

nanocontainers. Macromol Biosci 2004;4:445–453.

15. ten Cate MGJ, Rettig H, Bernhardt K, Borner HG. Sequence-

defined polypeptide-polymer conjugates utilizing reversible addi-

tion fragmentation transfer radical polymerization. Macromole-

cules 2005;38:10643–10649.

16. Kim JH, Emoto K, Iijima M, Nagasaki Y, Aoyagi T, Okano T,

Sakurai Y, Kataoka K. Core-stabilized polymeric micelle as poten-

tial drug carrier: Increased solubilization of taxol. Polym Advan

Technol 1999;10:647–654.

17. Tian HY, Deng C, Lin H, Sun JR, Deng MX, Chen XS, Jing XB.

Biodegradable cationic PEG-PEI-PBLG hyperbranched block copol-

ymer: Synthesis and micelle characterization. Biomaterials 2005;

26:4209–4217.

18. Xu FJ, Neoh KG, Kang ET. Bioactive surfaces and biomaterials via

atom transfer radical polymerization. Prog Polym Sci 2009;34:

719–761.

19. Kwon GS, Kataoka K. Block-copolymer micelles as long-circulat-

ing drug vehicles. Adv Drug Deliv Rev 1995;16:295–309.

20. Kwon GS. Polymeric micelles for delivery of poorly water-soluble

compounds. Crit Rev Ther Drug Carrier Syst 2003;20:357–403.

21. Bae Y, Fukushima S, Harada A, Kataoka K. Design of environ-

ment-sensitive supramolecular assemblies for intracellular drug

delivery: Polymeric micelles that are responsive to intracellular

pH change. Angew Chem Int Edit 2003;42:4640–4643.

22. Shuai XT, Ai H, Nasongkla N, Kim S, Gao JM. Micellar carriersbased on block copolymers of poly(e-caprolactone) and poly(eth-ylene glycol) for doxorubicin delivery. J Control Release 2004;98:415–426.

23. Xu PS, Van Kirk EA, Murdoch WJ, Zhan YH, Isaak DD, Radosz M,Shen YQ. Anticancer efficacies of cisplatin-releasing pH-respon-sive nanoparticles. Biomacromolecules 2006;7:829–835.

24. Shuai XT, Merdan T, Schaper AK, Xi F, Kissel T. Core-cross-linked

polymeric micelles as paclitaxel carriers. Bioconjug Chem 2004;

15:441–448.

25. Soo PL, Lovric J, Davidson P, Maysinger D, Eisenberg A. Polycap-

rolactone-block-poly(ethylene oxide) micelles: A nanodelivery sys-

tem for 17 beta-estradiol. Mol Pharm 2005;2:519–527.

ORIGINAL ARTICLE


26. Nishiyama N, Kato Y, Sugiyama Y, Kataoka K. Cisplatin-loaded

polymer-metal complex micelle with time-modulated decaying

property as a novel drug delivery system. Pharm Res 2001;18:

1035–1041.

27. Nishiyama N, Okazaki S, Cabral H, Miyamoto M, Kato Y,

Sugiyama Y, Nishio K, Matsumura Y, Kataoka K. Novel cisplatin-

incorporated polymeric micelles can eradicate solid tumors in

mice. Cancer Res 2003;63:8977–8983.

28. Chiu GNC, Wong MY, Ling LU, Shaikh IM, Tan KB, Chaudhury A,

Tan BJ. Lipid-based nanoparticulate systems for the delivery of

anti-cancer drug cocktails: Implications on pharmacokinetics and

drug toxicities. Curr Drug Metab 2009;10:861–874.

29. Harasym TO, Tardi PG, Harasym NL, Harvie P, Johnstone SA,

Mayer LD. Increased preclinical efficacy of irinotecan and flox-

uridine coencapsulated inside liposomes is associated with tu-

mor delivery of synergistic drug ratios. Oncol Res 2007;16:

361–374.

30. Flavell DJ, Noss A, Pulford KAF, Ling N, Flavell SU. Systemic

therapy with 3BIT, a triple combination cocktail of anti-CD19, -

CD22, and -CD38-saporin immunotoxins, is curative of human B-

cell lymphoma in severe combined immunodeficient mice. Cancer

Res 1997;57:4824–4829.

31. Frassineti GL, Zoli W, Silvestro L, Serra P, Milandri C, Tienghi A,

Gianni L, Gentile A, Salzano E, Amadori D. Paclitaxel plus doxoru-

bicin in breast cancer: An Italian experience. Semin Oncol 1997;

24:19–25.

32. Ahmed F, Pakunlu RI, Srinivas G, Brannan A, Bates F, Klein ML,

Minko T, Discher DE. Shrinkage of a rapidly growing tumor by

drug-loaded polymersomes: pH-triggered release through copoly-

mer degradation. Mol Pharm 2006;3:340–350.

33. Wei L, Cai CH, Lin JP, Chen T. Dual-drug delivery system based

on hydrogel/micelle composites. Biomaterials 2009;30:2606–

2613.

34. Lee JS, Bae JW, Joung YK, Lee SJ, Han DK, Park KD. Controlled

dual release of basic fibroblast growth factor and indomethacin

from heparin-conjugated polymeric micelle. Int J Pharm 2008;346:

57–63.

35. Qiu LY, Bae YH. Self-assembled polyethylenimine-graft-poly (epsi-

lon-caprolactone) micelles as potential dual carriers of genes and

anticancer drugs. Biomaterials 2007;28:4132–4142.

36. Nasongkla N, Bey E, Ren JM, Ai H, Khemtong C, Guthi JS, Chin

SF, Sherry AD, Boothman DA, Gao JM. Multifunctional polymeric

micelles as cancer-targeted. MRI-ultrasensitive drug delivery sys-

tems. Nano Lett 2006;6:2427–2430.

37. Blanco E, Kessinger CW, Sumer BD, Gao J. Multifunctional micel-

lar nanomedicine for cancer therapy. Exp Biol Med 2009;234:

123–131.

38. Hong GB, Yuan RX, Liang BL, Shen J, Yang XQ, Shuai XT. Folate

functionalized polymeric micelle as hepatic carcinoma-targeted.

MRI-ultrasensitive delivery system of antitumor drugs. Biomed

Microdevices 2008;10:693–700.

39. Rapoport N, Gao ZG, Kennedy A. Multifunctional nanoparticles

for combining ultrasonic tumor imaging and targeted chemother-

apy. J Natl Cancer Inst 2007;99:1095–1106.

40. Bae Y, Jang WD, Nishiyama N, Fukushima S, Kataoka K. Multi-

functional polymeric micelles with folate-mediated cancer cell tar-

geting and pH-triggered drug releasing properties for active

intracellular drug delivery. Mol Biosyst 2005;1:242–250.

41. Huang CK, Lo CL, Chen HH, Hsiue GH. Multifunctional micelles

for cancer cell targeting, distribution imaging, and anticancer

drug delivery. Adv Funct Mater 2007;17:2291–2297.

42. Liu FT, Eisenberg A. Preparation and pH triggered inversion of

vesicles from poly(acrylic acid)-block-polystyrene-block-poly(4-

vinyl pyridine). J Am Chem Soc 2003;125:15059–15064.

43. Azzam T, Bronstein L, Eisenberg A. Water-soluble surface-anch-

ored gold and palladium nanoparticles stabilized by exchange of

low molecular weight ligands with biamphiphilic triblock copoly-

mers. Langmuir 2008;24:6521–6529.

44. Wittemann A, Azzam T, Eisenberg A. Biocompatible polymer

vesicles from biamphiphilic triblock copolymers and their interac-

tion with bovine serum albumin. Langmuir 2007;23:2224–2230.

45. Pratt RC, Lohmeijer BGG, Long DA, Waymouth RM, Hedrick JL.

Triazabicyclodecene: A simple bifunctional organocatalyst for acyl

transfer and ring-opening polymerization of cyclic esters. J Am

Chem Soc 2006;128:4556–4557.

46. Ye HF, Jin L, Hu RZ, Yi ZF, Li J, Wu YL, Xuguang XG, Wu ZR. Pol-

y(gamma,L-glutamic acid)-cisplatin conjugate effectively inhibits

human breast tumor xenografted in nude mice. Biomaterials

2006;27:5958–5965.

47. Nishiyama N, Kataoka K. Preparation and characterization of size-

controlled polymeric micelle containing cis-dichlorodiamminepla-

tinum(II) in the core. J Control Release 2001;74:83–94.

48. Schechter B, Rosing MA, Wilchek M, Arnon R. Blood-levels and

serum-protein binding of cis-platinum(II) complexed to carboxy-

methyl-dextran. Cancer Chemother Pharmacol 1989;24:161–166.

49. Colombani O, Ruppel M, Schubert F, Zettl H, Pergushov DV, Mul-

ler AHE. Synthesis of poly(n-butyl acrylate)-block-poly(acrylic

acid) diblock copolymers by ATRP and their micellization in

water. Macromolecules 2007;40:4338–4350.

50. Seow WY, Xue JM, Yang YY. Targeted and intracellular delivery

of paclitaxel using multi-functional polymeric micelles. Biomateri-

als 2007;28:1730–1740.

51. Allen C, Yu YS, Maysinger D, Eisenberg A. Polycaprolactone-b-

poly(ethylene oxide) block copolymer micelles as a novel drug

delivery vehicle for neurotrophic agents FK506 and L-685,818. Bio-

conjug Chem 1998;9:564–572.

52. Giacomelli C, Borsali R. Morphology of poly(ethylene oxide)-

block-polycaprolatone block copolymer micelles controlled via the

preparation method. Macromol Symp 2006;245:147–153.

53. Gan ZH, Jim TF, Li M, Yuer Z, Wang SG, Wu C. Enzymatic biode-

gradation of poly(ethylene oxide-b-epsilon-caprolactone) diblock

copolymer and its potential biomedical applications. Macromole-

cules 1999;32:590–594.

54. Zhang GD, Zhang R, Wen XX, Li L, Li C. Micelles based on biode-

gradable poly(L-glutamic acid)-b-polylactide with paramagnetic gd

ions chelated to the shell layer as a potential nanoscale IVIRI-visi-

ble delivery system. Biomacromolecules 2008;9:36–42.

55. Nishiyama N, Yokoyama M, Aoyagi T, Okano T, Sakurai Y, Kataoka

K. Preparation and characterization of self-assembled polymer-

metal complex micelle from cis-dichlorodiammineplatinum(II) and

poly(ethylene glycol)-poly(alpha,beta-aspartic acid) block copoly-

mer in an aqueous medium. Langmuir 1999;15:377–383.

56. Lackey CA, Press OW, Hoffman AS, Stayton PS. A biomimetic pH-

responsive polymer directs endosomal release and intracellular

delivery of an endocytosed antibody complex. Bioconjug Chem

2002;13:996–1001.

57. Lo CL, Huang CK, Lin KM, Hsiue GH. Mixed micelles formed from

graft and diblock copolymers for application in intracellular drug

delivery. Biomaterials 2007;28:1225–1235.

58. Wang F, Wang YC, Yan LF, Wang J. Biodegradable vesicular nano-

carriers based on poly(epsilon-caprolactone)-block-poly(ethyl eth-

ylene phosphate) for drug delivery. Polymer 2009;50:5048–5054.


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Biamphiphilic triblock copolymer micelles as a multifunctional platform for anticancer drug delivery