dg

laee, WI 53SA

Received 6 June 2009Accepted 24 July 2009Available online 12 August 2009

Keywords:Gold nanoparticlespH-sensitive

molecules and can be further functionalized in various ways toimprove their drug delivery functionalities. For example, Corbierreet al. reported polystyrene-functionalized Au NPs by the covalent

excellent stability in physiological conditions because PEG hasexcellent solubility in water [8]. The PEG shell can also greatlyreduce the interaction between the Au NPs and the plasma protein,thereby greatly minimizing the uptake of the Au NPs by the retic-uloendothelial system (RES), which can lead to a much longercirculation time in the bloodstream. This will allow the Au NPs topreferentially accumulate in the tumor sites through the leakytumor neovasculature by the enhanced permeability and retention(EPR) effect. In addition, due to their intense light scattering power,

* Corresponding author. Department of Mechanical Engineering, University ofWisconsin-Milwaukee, Milwaukee, WI 53211, USA. Tel.: 1 414 229 5946; fax: 1414 229 6958.

Contents lists availab

Biomat

journal homepage: www.elsev

Biomaterials 30 (2009) 60656075E-mail address: sgong@uwm.edu (S. Gong).1. Introduction

Gold (Au) NPs have received considerable attention during thepast decade due to their potential applications in catalysis, chem-ical sensing, electronics, optics, and biology [1]. Particularly,monolayer-protected Au NPs have recently emerged as an attrac-tive candidate for delivering various therapeutic agents such asdrugs, peptides, proteins, and nucleic acids into their targets [2,3].Recent progress in monolayer-protected Au NPs involves usingthiolated compounds to stabilize Au NPs through thiol linkages [2].The monolayer ranges from small organic compounds to macro-

attachment of thiol-terminated polystyrene prepared by anionicpolymerization [4]. Au NPs with tetra(ethylene glycol)ylatedcationic ligands and uorogenic ligands were synthesized forhydrophobic drug delivery [5]. Thomas and Klibanov synthesizedAu NPs with branched polyethylenimine monolayer to providehybrid Au NP-polymer transfection vectors [6]. Wang et al. recentlyfabricated effective binders for DNA by anchoring b-cyclodextrin onthe periphery of oligo(ethylenediamino)-modied Au NPs [7].

Monolayer-protected Au NPs are one of the most promisingcandidates for cancer treatment because of their functional versa-tility. Functionalized Au NPs with an outer PEG shell can offerDrug deliveryTumor-targetedCellular uptakeCytotoxicity0142-9612/$ see front matter 2009 Elsevier Ltd.doi:10.1016/j.biomaterials.2009.07.048rubicin)-b-poly(ethylene glycol) copolymer (Au-P(LA-DOX)-b-PEG-OH/FA) was synthesized as a tumor-targeted drug delivery carrier. The Au-P(LA-DOX)-b-PEG-OH/FA NPs consist of an Au core, a hydrophobicpoly(L-aspartate-doxorubicin) (P(LA-DOX)) inner shell, and a hydrophilic poly(ethylene glycol) andfolate-conjugated poly(ethylene glycol) outer shell (PEG-OH/FA). The anticancer drug, doxorubicin(DOX), was covalently conjugated onto the hydrophobic inner shell by acid-cleavable hydrazone linkage.The DOX loading level was determined to be 17 wt%. The Au-P(LA-DOX)-b-PEG-OH/FA NPs formed stableunimolecular micelles in aqueous solution. The size of the Au-P(LA-DOX)-b-PEG-OH/FA micelles weredetermined as 2452 and 1025 nm by dynamic light scattering (DLS) and transmission electronmicroscopy (TEM), respectively. The conjugated DOX was released from the Au-P(LA-DOX)-b-PEG-OH/FAmicelles much more rapidly at pH 5.3 and 6.6 than at pH 7.4, which is a desirable characteristic fortumor-targeted drug delivery. Cellular uptake of the Au-P(LA-DOX)-b-PEG-OH/FA micelles facilitated bythe folate-receptor-mediated endocytosis process was higher than that of the micelles without folate.This was consistent with the higher cytotoxicity observed with the Au-P(LA-DOX)-b-PEG-OH/FA micellesagainst the 4T1 mouse mammary carcinoma cell line. These results suggest that Au-P(LA-DOX)-b-PEG-OH/FA NPs could be used as a carrier with pH-triggered drug releasing properties for tumor-targeteddrug delivery.

2009 Elsevier Ltd. All rights reserved.Article history: Gold (Au) nanoparticles (NPs) stabilized with a monolayer of folate-conjugated poly(L-aspartate-doxo-a r t i c l e i n f o a b s t r a c tGold nanoparticles with a monolayer ofblock copolymer for tumor-targeted dru

Mani Prabaharan a, Jamison J. Grailer b, Srikanth PilaDepartment of Mechanical Engineering, University of Wisconsin-Milwaukee, MilwaukbDepartment of Biological Sciences, University of Wisconsin-Milwaukee, Milwaukee, WcDepartment of Materials, University of Wisconsin-Milwaukee, Milwaukee, WI 53211, UAll rights reserved.oxorubicin-conjugated amphiphilicdeliverya, Douglas A. Steeber b, Shaoqin Gong a,c,*

I 53211, USA211, USA

le at ScienceDirect

erials

ier .com/locate/biomateria ls

monolayer-protected Au NPs targeted to cancer tissuemay improvesurgeons ability to identify metastatic lesions. Specically, thetunability of Au NPs optimal scattering wavelength (plasmonresonance) may provide a means to dramatically enhance theapparent optical contrast of lesions relative to neighboring tissue.For instance, the optical properties of the Au NPs can greatlyenhance the contrast of X-ray computed tomography (CT) imaging[9]. Finally, monolayer-protected Au NPs can be used to destroy thetumor cells without damaging the surrounding tissue by photo-thermal therapy since they can efciently convert the absorbedlaser light energy into localized heat [10].

Au NPs with an end-functionalized monolayer, regarded asa macroinitiator, can be used to prepare Au NPs covalently conju-gated with polymer monolayer using surface-initiated polymeri-zation. We recently synthesized amphiphilic Au NPs witha polycaprolactone-methoxy poly(ethylene glycol) monolayer bythe ring-opening polymerization of 3-caprolactone using mercap-toundecanol stabilized Au NPs as the macroinitiator, followed byesterication with carboxyl terminated methoxy poly(ethyleneglycol) as the drug deliver carrier [11]. In this work, we describe thesynthesis of folate (FA)-conjugated amphiphilic Au NPs witha poly(L-aspartate-doxorubicin)-b-poly(ethylene glycol) monolayer(Au-P(LA-DOX)-b-PEG-OH/FA) (cf. Fig. 1) by the ring-openingpolymerization of b-benzyl L-aspartate N-carboxyanhydride (BLANCA) using amine functionalized Au NPs as a macroinitiator,followed by coupling reactions with a-hydroxy-u-carboxyl poly-(ethylene glycol) (HOPEGCOOH) and subsequently folate for

molecules exhibit excellent in vivo stability that is independent ofthe polymer concentration or some other environmental uctua-tion such as temperature or pH due to its covalent nature [12].

Due to the presence of FA, Au-P(LA-DOX)-b-PEG-OH/FA NPsoffer active tumor-targeting ability in addition to their passive-targeting ability, which is attributed to the EPR effect exhibited bythe tumor tissue [13,14]. Conjugation of DOX onto the NPs isexpected to improve the drug loading level and greatly reduce thechance of premature drug release during circulation in the blood-stream. Furthermore, since the hydrazone linkage between theDOX and polymer is prone to hydrolysis in an acidic condition, therelease rate of DOX from the NPs will increase in the acidic envi-ronment of the endosomal intracellular compartments after theAu-P(LA-DOX)-b-PEG-OH/FA NPs are internalized by the tumorcells via the folate-mediated-endocytosis, thereby providinga sufcient concentration of DOX in the tumor cells within a shortperiod of time (cf. Fig. 1) [1517]. The structure of the Au-P(LA-DOX)-b-PEG-OH/FA NPs was characterized by 1H NMR spectros-copy. The micellar properties of the Au-P(LA-DOX)-b-PEG-OH/FANPs were determined by dynamic light scattering (DLS) andtransmission electron microscopy (TEM) analyses. The in vitro DOXrelease studies were performed at pH 5.3, 6.6, and 7.4 at 37 C. Thecellular uptake and cytotoxicity of the Au-P(LA-DOX)-b-PEG-OH/FAmicelles against 4T1 mouse mammary carcinoma cells wereassessed using confocal laser scanning microscopy (CLSM), owcytometry, and the MTT assay.

H

M. Prabaharan et al. / Biomaterials 30 (2009) 606560756066tumor-targeted drug delivery. The DOXmolecules were conjugatedto the hydrophobic poly(L-aspartate) segment by pH-sensitivehydrazone linkage. Au-P(LA-DOX)-b-PEG-OH/FA NPs can formstable unimolecular micelles in aqueous solutions due to theirunique amphiphilic multi-arms and globular architecture. Incontrast to conventional polymeric micelles that require theaggregation of multiple linear amphiphilic molecules to achieve themicellar behavior above the critical micelle concentration, unim-olecular micelles formed by individual multi-arm amphiphilic

O

O

OO

O

NH2OH

H3C

OH

OCH3

OH

OH

NHNO

DOX conjugated via

a pH-sensitive hydrazone

bond

Hydrophobic poly(L-aspartate) segment

Hydrophilic PEG segment

Au core

[H+]Fig. 1. Schematic illustration of the Au-P(LA-DOX)-b-P2. Materials and methods

2.1. Materials

Tetrachloroauric acid (HAuCl4), 2-aminoethanethiolHCl (AETHCl), b-benzylL-aspartate (BLA), and triphosgene were purchased from SigmaAldrich. Anhydroushydrazine, sodium borohydride (NaBH4), 4-dimethylamino pyridine (DMAP),N-hydroxy succinimide (NHS) and 1,3-dicyclohexylcarbodiimide (DCC) werepurchased from Acros and used without further purication. HOPEGCOOH withan Mw of 3000 was purchased from RAPP Polymere GmbH, Germany. FA wasobtained from bio-WORLD, USA. Triethylamine (Alfa Aesar) was distilled before use.

DOX conjugated by hydrazone bond

FA ligand

+OH

O

O

O

O

NH2OH

H3C

OH

OCH3

OH

OH

O

DOXNPEG-OH/FA NP and its pH-triggered drug release.

Anhydrous DMF was purchased from Fisher Scientic Company. The model drug,doxorubicinHCl (DOXHCl), was supplied by Tecoland Corporation, USA, and usedas supplied. NIH-3T3 and 4T1 cell lines were purchased from ATCC, USA. All otherchemicals used were of analytical reagent grade.

2.2. Synthesis of amine functionalized Au NPs

Fig. 2 shows the reaction scheme for the synthesis of Au-P(LA-DOX)-b-PEG-OH/FA. Au NPs were rst synthesized by the NaBH4 reduction method [18]. In a typicalexperiment, 100 mL of 104 M aqueous solution of HAuCl4 was reduced by 0.01 g ofNaBH4 under vigorous stirring at room temperature in the dark. After 12 h ofreaction, the excess amount of NaBH4 was removed by dialysis against distilledwater for 4 h using a dialysis tubing (molecular weight cut-off of 2 kDa). The numberof Au NPs in aqueous solution was determined as 5.811015 g/L by atomicabsorption spectroscopy (AAS). Thereafter, 2.41108 M of AETHCl was treatedwith 100 mL of aqueous solution containing the Au NPs at room temperature inorder to functionalize 25 molecules of AETHCl per Au NP surface. After 6 h, theresulting thiol stabilized Au NPs were treated with 2.41108 M of triethylamine for30 min followed by dialysis against deionized water for 24 h to obtain the aminefunctionalized Au NPs.

2.3. Synthesis of BLANCA

13 g (60 mmol) of BLA was suspended in 100 mL of anhydrous THF. With thissolution, 9 g (30 mmol) of triphosgene in 10 mL anhydrous THF was slowly addedover a period of 30 min. Then, the mixture was stirred at 55 C under nitrogen forabout 3 h until a clear solution was observed. Thereafter, the solvent was removed

under vacuum and the product was puried by recrystallization using a mixedsolvent of THF and hexane. The formation of BLANCA was conrmed by 1H NMR(300 MHz, CDCl3): 7.457.3 (5H, m, Ar-H), 6.2 (1H, s, NH), 5.2 (2H, s, CH2), 4.61 (1H, t,CH) and 2.85 (2H, t, CH2) ppm.

2.4. Synthesis of Au-poly(b-benzyl L-aspartate) (AuPBLANH2)

1 g of BLANCA (4.016 mmol) was dissolved in 50 mL of anhydrous DMF understirring at room temperature. Then, 100 mg of amine functionalized Au NPs in 10 mLof DMF was added to the BLANCA solution. Thereafter, the reaction mixture wasstirred at 55 C in a steam of dry nitrogen. After 48 h, the reaction mixture waspoured into a 10-fold volume of diethyl ether at 0 C, and a precipitate was collectedby ltration and washed with diethyl ether, followed by drying in vacuum.

2.5. Synthesis of Au-poly(b-benzyl L-aspartate)-b-poly(ethylene glycol) (Au-PBLA-b-PEG-OH/FA)

Au-PBLA-b-PEG-OH/FAwas prepared using a two-step reaction procedure. First,Au-PBLA-b-PEG-OHwas prepared by reacting 0.5 g of AuPBLANH2with 1 g of HOPEGCOOH (0.33 mmol) using DCC (0.07 g, 0.33 mmol) and NHS (0.04 g, 0.33 mmol)as the condensing agents in 50 mL of anhydrous DMF, as shown in Fig. 2. Thereactionwas allowed for 12 h at room temperature under constant stirring. After theby-product, dicyclohexylurea, was removed by ltration, the product Au-PBLA-b-PEG-OH was dialyzed against deionized water using a dialysis tubing (molecularweight cut-off of 12 kDa) for 48 h and freeze-dried. Thereafter, 0.5 g of Au-PBLA-b-PEG-OH reacted with 1.77 mg of FA in presence of DCC and DMAP as the catalysts inanhydrous DMF for 24 h to obtain the product Au-PBLA-b-PEG-OH/FA.

Au

Gold NPs

BLA-NCA

OO

HN

OO

O

HN

H

O

OO

nSNH

NHS/DCC

HOOCO

OHm

HN

O

OO

nSNH OC

OOHm

HN

O

DOX

AuAu

HAuCl4NaBH4 S

NH2Au25

2525

HS-CH2-CH2-NH3Cl

TEA

M. Prabaharan et al. / Biomaterials 30 (2009) 60656075 6067HNN N

NNH

NH2

COOH

COOHN

O

H

O

FA

DMAP/DCC

O

O

SNH

HN

O

NHNH2

O

nSNH OC

OOH/FAm

Au

Au

25Fig. 2. Reaction scheme for the synthesn OCO

OH/FAm NH2NH2

HN

O

O

nS NH OCO

OH/FAm

OH

O

OO

O

NH2OH

H3C

OH

OCH3

OH

OH

NHN

Au-P(LA-DOX)-b-PEG-OH/FA

Au

25

25is of Au-P(LA-DOX)-b-PEG-OH/FA.

ater2.6. Synthesis of Au-P(LA-DOX)-b-PEG-OH/FA

Au-P(LA-DOX)-b-PEG-OH/FA was synthesized by using a two-step reactionprocedure. In the rst step, the benzyl groups of Au-PBLA-b-PEG-OH/FA weresubstituted with hydrazide groups by reacting 0.5 g of Au-PBLA-b-PEG-FA withexcess of anhydrous hydrazine (20 mg) in 50 mL of dry DMF at 40 C for 24 h.Thereafter, the formed product, Au-P(LA-hydrazide)-b-PEG-OH/FA, was dialyzedagainst distilled water for 48 h and followed by freeze-drying. In the next step,0.25 g of Au-P(LA-hydrazide)-b-PEG-OH/FA was dissolved in 25 mL of DMF, and anexcess amount of DOXHCl (75 mg, 0.137 mmol) was added. The mixed solutionwasstirred at room temperature for 24 h. Thereafter, the reaction mixture was dialyzedagainst alkaline water (pH 8) for 48 h to obtain Au-P(LA-DOX)-b-PEG-OH/FA. Afterfreeze-drying, the product was puried using LH20 gel column to completelyremove the unreacted DOX. The absence of free DOX in the product was conrmedby thin layer chromatography (TLC).

2.7. Preparation of micelles from the Au-P(LA-DOX)-b-PEG-OH/FA NPs

Au-P(LA-DOX)-b-PEG-OH/FA micelles were prepared using the membranedialysis method. Briey, 50 mg of Au-P(LA-DOX)-b-PEG-OH/FA was dissolved in5 mL of DMF under stirring. With this solution, 15 mL of Millipore water was addeddropwise. Thereafter, the polymer solution was dialyzed against Millipore waterusing a dialysis tubing (molecular weight cut-off 2 kDa) for 96 h followed by freeze-drying. In order to determine the drug conjugation level, aweighed quantity (25 mg)of Au-P(LA-DOX)-b-PEG-OH/FA was treated with 0.1 N HCl solutions at roomtemperature for 48 h under uniform stirring. After centrifugation, the DOX in thesupernatant was assayed by UVvisible spectrophotometer at a wavelength of485 nm. All the experiments were carried out in triplicate.

2.8. Characterization

1H NMR spectrum of the samples was recorded on a Bruker DPX 300 spec-trometer using CDCl3 and DMSO as the solvents at 25 C. Absorbance measurementswere carried out using Varian Cary 100 Bio UVvisible spectrophotometer. Thecalibration curve of absorbance against different concentrations of DOXwasmade at485 nm. The particle size was determined by DLS using a Beckman Coulter PCSsubmicron particle size analyzer with angle detection at 90 . For TEM studies, a dropof particle or micelle solutions (0.05 mg/mL) containing 0.8 wt% phosphotungsticacid was deposited onto a 200 mesh copper grid coated with carbon and dried atroom temperature. The shape and size of the particles were observed at 75 kV witha Hitachi H-600 TEM.

2.9. Drug release

The release studies were performed in a glass apparatus at 37 C in acetatebuffer (pH 5.3 and 6.6) and phosphate buffer (pH 7.4) solutions. First, 50 mg of Au-P(LA-DOX)-b-PEG-OH/FA micelles was dispersed in 5 mL of medium and placed ina dialysis bag with a molecular weight cut-off of 2 kDa. The dialysis bag was thenimmersed in 95 mL of the releasemedium and kept in a horizontal laboratory shakermaintaining a constant temperature and stirring (100 rpm). Samples (2 mL) wereperiodically removed and the volume of each sample was replaced by the samevolume of fresh medium. The amount of released DOX was analyzed with a spec-trophotometer at 485 nm. The drug release studies were performed in triplicate foreach of the samples.

2.10. Cellular uptake and cytotoxicity

The cellular uptake experiments were performed using ow cytometry andCLSM. For ow cytometry, 4T1 cells (1106) were seeded in 6-well culture platesand grown overnight. The cells were then treated with free DOX (34 mg/mL) or Au-P(LA-DOX)-b-PEG-OH/FA or Au-P(LA-DOX)-b-PEG-OH (i.e., both FA-conjugated andFA-free) micelles (DOX concentration 34 mg/mL) for 30 and 120 min. Thereafter,the cells were lifted using Cellstripper (Media Tech, Inc) and washed, and the DOXuptake was analyzed using a FACSCalibur ow cytometer (BD Biosciences). Aminimum of 2104 cells was analyzed from each sample with uorescence inten-sity shown on a four-decade log scale. For CLSM studies, 4T1 cells (1106) wereseeded onto 22 mm round glass coverslips, placed in 6-well plates, and grownovernight. Cells were treatedwith free DOX (34 mg/mL) or Au-P(LA-DOX)-b-PEG-OH/FA or Au-P(LA-DOX)-b-PEG-OH micelles (DOX concentration 34 mg/mL) for 2 h.Then, the cells were washed and xed with 1.5% formaldehyde. Coverslips wereplaced onto glass microscope slides and DOX uptake was analyzed using a Leica TCSSP2 Confocal System installed on an upright compound microscope (Leica, Wetzler,Germany). Digital monochromatic images were acquired using Leica ConfocalSoftware (Version 2.61). To determine the DOX distribution in the nucleus andcytoplasm of the 4T1 cells, the DOX mean uorescence intensity (MFI) in the CLSMimages was measured in a 4 mm2 area located in the nucleus or cytoplasm (n 10cells) for each sample using ImageJ software (http://rsb.info.nih.gov/ij).

M. Prabaharan et al. / Biom6068The cytotoxicity of Au-P(LA-DOX)-b-PEG-OH/FA and Au-P(LA-DOX)-b-PEG-OHmicelles against NIH-3T3 and 4T1 cells was assessed using the MTT assay [19]. Thecells were cultured in FA-free RPMI medium supplemented with 10% fetal calfserum, penicillin/streptomycin, and L-glutamine (complete media, all from Invi-trogen Corp., Grand Island, NY) at 37 C with 5% CO2. Then, 4T1 cells were culturedand lifted as above before being seeded (104) into 96-well plates and incubated for24 h. The medium was then replaced with fresh medium containing the free DOX(34 mg/mL), Au-P(LA-DOX)-b-PEG-OH/FA and Au-P(LA-DOX)-b-PEG-OH micelles(DOX concentration 34 mg/mL) and incubated for 48 h. Thereafter, the wells werewashed three times with warm phosphate buffer solution and incubated again foranother 4 h with FA-free RPMI containing 250 mg/mL of MTT. After discarding theculture medium, 150 mL of DMSO was added to dissolve the precipitates, and theresulting solution was measured for absorbance at 570 nm with a reference wave-length of 690 nm using a microplate reader (Molecular Devices).

3. Results and discussion

3.1. Synthesis and characterization of Au-P(LA-DOX)-b-PEG-OH/FA

The reaction scheme for the synthesis of Au-P(LA-DOX)-b-PEG-OH/FA NPs is shown in Fig. 2. The amine functionalized Au NPswere rst synthesized by using a two-step procedure. In the rststep, Au NPs were prepared by reducing the Au salt, HAuCl4, usingNaBH4 in aqueous solution at room temperature. In the second step,Au NPs were functionalized with the amine groups by reactingAETHCl on the monolayer of Au NPs through thiol linkers in thepresence of triethylamine. The number of amine functionality onthe Au NPs was maintained as 25 by controlling AETHCl mole ratioto the number Au NPs during the reaction. Thereafter, AuPBLANH2 was prepared by the ring-opening polymerization of BLANCAusing amine functionalized Au NPs as an initiator in anhydrousDMF. The formation of AuPBLANH2 was conrmed by the

1HNMR spectrum, as shown in Fig. 3A. The peaks at 7.3 ppm and5.1 ppm are ascribed to the protons of benzyl and methylenegroups in the PBLA side chains, respectively. The signal at 2.7 ppmcorresponds with the methylene group of the side chain, whichconnects the main chain of PBLA (CHCH2COO), was alsoidentied. The peaks at 2.9 and 3.0 ppm were observed due to themethylene protons of AETHCl. These results conrm the formationof AuPBLANH2.

To obtain Au-PBLA-b-PEG-OH, the amino end groups of AuPBLANH2 were coupled with the carboxylic acid terminal groupsof HOPEGCOOH by the amide linkage. The coupling reaction wascarried out in dry DMF at room temperature in the presence of DCCand NHS as the activating agent for the carboxylic group. In the 1HNMR spectrum of Au-PBLA-b-PEG-OH (Fig. 3B), in addition to thecharacteristic peaks of PBLA, the peak at 3.6 ppmwas observed dueto the methylene protons of oxyethylene units of PEG. These resultsclearly indicate the formation of Au-PBLA-b-PEG-OH. From the 1HNMR spectra (Fig. 3B), the number average molecular weight (Mn)of the PBLA-b-PEG-OH segment was determined as 4975 bycalculating the relative intensity ratio of the methylene proton ofthe PEG chain (OCH2CH2, d 3.6) and the methylene proton nearthe benzyl group of the PBLA chain (COOCH2C6H5, d 5.1).

Au-PBLA-b-PEG-OH/FA was synthesized by reacting g-carboxylgroup of FAwith some of the terminal hydroxyl groups of Au-PBLA-b-PEG-OH by ester forming reaction using DMAP and DCC as thecatalysts. The presence of FA in the product, Au-PBLA-b-PEG-OH/FA,was conrmed by the appearance of weak signals at 6.78.7 ppm,which corresponded with the aromatic protons of FA (Fig. 3C). Toconjugate DOX onto Au-PBLA-b-PEG-OH/FA, the benzyl groups ofAu-PBLA-b-PEG-OH/FA were rst substituted with hydrazidegroups byesteramide exchange aminolysis reaction. The hydrazidegroups of resultingproductwere then conjugatedwithDOX throughan acid-sensitive hydrazone bond to obtain Au-P(LA-DOX)-b-PEG-OH/FA, as shown in Fig. 2. The absence of aromatic protons peak at7.3 ppm and benzyl methylene proton at 5.1 ppm in the 1H NMRspectrum of Au-P(LA-DOX)-b-PEG-OH/FA (Fig. 3D) proved the

ials 30 (2009) 60656075complete debenzylation of the poly(L-aspartate) (PLA) segments of

Fig. 3. 1H NMR spectrum of (A) Au-PBLA-NH2; (B) Au-PBLA-b-PEG-OH; (C) Au-PBLA-b-PEG-OH/FA; and (D) Au-P(LA-DOX)-b-PEG-OH/FA.

M. Prabaharan et al. / Biomaterials 30 (2009) 60656075 6069

Au-P(LA-DOX)-b-PEG-OH/FA. Moreover, the conjugation of DOXwas conrmed by the presence of characteristic DOX peaks at 5.4,5.3, 4.0, 2.1,1.9, and 1.2 ppm in addition to the characteristic peaks ofPLA, PEG, and FA. The substitution level of DOXon the PLA backbonewas determined as 17 wt% by UVvisible spectrophotometer anal-ysis at 485 nm, which corresponds with 47 DOX molecules per Au-P(LA-DOX)-b-PEG-OH/FA NP on average.

The formation of Au, amine functionalized Au, and Au-PBLA-b-PEG-OH/FA NPs was conrmed by UVvisible spectroscopyanalysis, as shown in Fig. 4. The UVvisible spectra of Au, aminefunctionalized Au, and Au-PBLA-b-PEG-OH/FA NPs showed char-acteristic surface plasmon resonance (SPR) bands at 512, 525, and565 nm, respectively. These results indicate the formationand existence of Au NPs [20,21]. The width of the absorption bandand the position of the maximum absorption peak depend on themorphology of the particles (size, shape, and uniformity), coagu-lation among the particles, and the dielectric environment [22,23].The signicant red shift in SPRwith peak broadening of Au-PBLA-b-PEG-OH/FA and amine functionalized Au NPs compared to the AuNPs suggests a linear increase in particle size consequent to thesurface modication [24].

the size distribution was relatively broad, ranging from 24 to52 nm, and the average micelle size was 34 nm with a poly-dispersity index of 0.029 (Fig. 5B). The increased size and sizedistribution of micelles formed from Au-P(LA-DOX)-b-PEG-OH/FANPs might be due to the presence of a fairly thick P(LA-DOX)-b-PEG-OH/FA monolayer on the surface of Au NPs. These resultsclearly indicate that the Au-P(LA-DOX)-b-PEG-OH/FA NPs couldform core/shell unimolecular micelles and that the monolayer ofamphiphilic P(LA-DOX)-b-PEG-OH/FA copolymer stabilizes the AuNPs in aqueous solutions.

The size and morphology of amine functionalized Au and Au-P(LA-DOX)-b-PEG-OH/FA NPs were further evaluated by TEM. Asshown in Fig. 6A, amine functionalized Au NPs are spherical with anaverage diameter of 6 nm. Furthermore, it was observed that aminefunctionalized Au NPs are well-separated from each other (i.e., noaggregates), indicating AET effectively stabilize the Au NPs. Fig. 6Bshows a TEM image of the micelles made from Au-P(LA-DOX)-b-PEG-OH/FA NPs. The morphology of these micelles appeared aswell-dened Au core and P(LA-DOX)-b-PEG-OH/FA shell structureswith a diameter in the range of 1025 nm. In addition, no aggre-

-10 0 10 20 30 40 50 60 70 80 900

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Fig. 5. Size distribution of (A) amine functionalized Au NPs; and (B) Au-P(LA-DOX)-b-PEG-OH/FA NPs.

M. Prabaharan et al. / Biomater60703.2. Particle size analysis

For drug nanocarriers, the size and stability are importantproperties that inuence their in vivo performance. These twofactors will directly affect the biodistribution and circulation timeof the carriers. Stable and smaller particle sizes (

observed in the TEM image, conrming that the monolayer of

Au

aterials 30 (2009) 60656075 6071amphiphilic P(LA-DOX)-b-PEG-OH/FA copolymer on the Au coreprevents the NPs from aggregation. The diameter of micelles madefrom the Au-P(LA-DOX)-b-PEG-OH/FA NPs observed by TEM issmaller than their diameter obtained from the DLS experiment. Thelarger value from the DLS measurement relative to TEM is mostlikely due to the existence of a swollen PEG corona around the Aucore. The size range of Au-P(LA-DOX)-b-PEG-OH/FA micellesdetermined by the DLS and TEM experiments could be suitable foran injectable anti-cancer drug delivery carrier because it allows forextravasation at tumor sites due to the EPR effect, and the FA-receptor-mediated endocytosis process that leads to the preferredaccumulation of drug-conjugated micelles within tumors [26].Fig. 6. TEM images of (A) amine functionalized

M. Prabaharan et al. / Biom3.3. Drug release

The drug release behavior of the Au-P(LA-DOX)-b-PEG-OH/FAmicelles was investigated under a simulated physiological condi-tion (PBS, pH 7.4) and in an acidic environment (acetate buffer, pH5.3, and 6.6) at 37 C to assess the feasibility of using Au-P(LA-DOX)-b-PEG-OH/FA NPs as an anticancer drug delivery carrier. Asshown in Fig. 7, the rate and amount of DOX release from the Au-P(LA-DOX)-b-PEG-OH/FA NPs were strongly dependent on the pHof the medium. Au-P(LA-DOX)-b-PEG-OH/FA NPs showed a muchfaster DOX release at pH 5.3 and 6.6 than at pH 7.4. At pH 5.3 and6.6, Au-P(LA-DOX)-b-PEG-OH/FA micelles released out about 34and 27%, respectively, of the conjugated DOX for the rst 2 h, andliberated 94 and 83%, respectively, after 45 h; however, at pH 7.4,the micelles released only 5% of DOX for 2 h and less than 15% after45 h. This result shows that the release of DOX from the Au-P(LA-DOX)-b-PEG-OH/FA NPs in an acidic environment was governed bythe acid-cleavable characteristic of the hydrazone linkage betweenthe DOX molecules and the NPs. An acid-cleavable hydrazonelinkage can undergo hydrolysis under acidic conditions; thus,unmodied DOX can be released from the Au-P(LA-DOX)-b-PEG-OH/FA NPs by hydrolysis of the hydrazone linkage at the 13-ketoposition of the DOX molecules (cf. Fig. 1). The DOX release prolesfrom the Au-P(LA-DOX)-b-PEG-OH/FA NPs agree with thosereported previously with DOX-conjugated polymeric micelles[27,16].The strong, pH-dependent DOX release prole is highly desir-able for effective treatment of cancer. The very slow DOX releaserate observed at pH 7.4, mimicking the physiological conditions ofthe bloodstream, ensures minimal DOX release during bloodcirculation. It is expected that the Au-P(LA-DOX)-b-PEG-OH/FAmicelles will accumulate in the tumor tissue preferentially throughthe EPR effect. Once in the tumor tissue, these Au-P(LA-DOX)-b-PEG-OH/FA micelles will be internalized by the tumor cells, largelyvia folate-receptor-mediated endocytosis, and will be located in theacidic endosomal compartments where DOX could be cleaved fromthe Au NPs and subsequently diffuse into the cytosol and later intothe nucleus [28,29]. Therefore, the much higher release rateobserved at pH 6.6 and 5.3, mimicking the physiological environ-ments of the tumor tissue and the endocytic compartments of thetumor cells, respectively, can help to quickly provide a sufcient

NPs; and (B) Au-P(LA-DOX)-b-PEG-OH/FA NPs.level of DOX in the tumor tissue/cells, thereby greatly enhancingthe efcacy of cancer therapy.

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Fig. 7. DOX release prole from the Au-P(LA-DOX)-b-PEG-OH/FA NPs at 37 C.

3.4. Cellular uptake

The cellular uptake of Au-P(LA-DOX)-b-PEG-OH/FA micelles bythe folate receptor positive cell line, 4T1 cells, was studied by ow

cytometry and compared with free DOX and FA-free Au-P(LA-DOX)-b-PEG-OH micelles to understand the effect of FA on thecellular uptake. Since DOX itself is uorescent, it was used directlyto measure cellular uptake without additional markers. Therefore,

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M. Prabaharan et al. / Biomaterials 30 (2009) 60656075607220 m0

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H, and (C) Au-P(LA-DOX)-b-PEG-OH/FA micelles at 37 C for 2 h. (D) DOX mean uo-X, FA-targeted and non-targeted unimolecular micelles shown in (A), (B) and (C). (DOX

the uorescence intensity is proportional to the amount of DOXinternalized by the cells. Fig. 8 shows the ow cytometry histo-grams of cell-associated DOX uorescence for 4T1 cells. Cellswithout any DOX treatment were used as a negative control andshowed only the auto-uorescence of the cells. For 4T1 cells incu-bated with equivalent DOX concentration in each formulation andincubated for the same time, the Au-P(LA-DOX)-b-PEG-OH/FAmicelles showed higher uorescence intensity than Au-P(LA-DOX)-b-PEG-OH micelles. This result directly conrms that the cellularuptake of the micelles can be enhanced by attaching FA on theirsurface and the Au-P(LA-DOX)-b-PEG-OH/FA micelles were trans-ported into the 4T1 cells by a FA-receptor-mediated endocytosisprocess. The cellular uptake of free DOX was greater than that ofAu-P(LA-DOX)-b-PEG-OH and Au-P(LA-DOX)-b-PEG-OH/FA NPs,indicating that free DOX was rapidly transported into cells viaa passive diffusion mechanism [30].

The cellular uptake of free DOX, Au-P(LA-DOX)-b-PEG-OH, andAu-P(LA-DOX)-b-PEG-OH/FA micelles by 4T1 cells were compara-tively estimated by a CLSM, as shown in Fig. 9. As such, 4T1 cellstreated with free DOX for 2 h showed that DOX was predominantlyaccumulated in the nucleus (Fig. 9A and D, p< 0.0005). The intenseDOX accumulation in the nucleus for free DOX occurred becauseintracellular free DOX molecules in the cytosol were rapidlytransported to the nucleus and avidly bound to the chromosomalDNA [31].

In the case of the DOX-conjugated micelles, less intense reduorescencewas observed in the cytoplasm and nucleus, indicatingthat the DOX-conjugated micelles were initially located within theendosomal intracellular compartments, releasing cleaved DOX inthe cytosol region in a sustained manner. However, consistent tothe ow cytometry ndings, the internalization of the Au-P(LA-DOX)-b-PEG-OH/FA micelles was much higher compared with theAu-P(LA-DOX)-b-PEG-OH micelles, resulting in a stronger uores-cence intensity. In addition, there was signicantly more DOXuorescence in the nucleus compared to the cytoplasm in the Au-P(LA-DOX)-b-PEG-OH/FA treated cells (Fig. 9C and D, p< 0.0005),while the Au-P(LA-DOX)-b-PEG-OH treated cells had uniformDOX uorescence in the nucleus and cytoplasm (Fig. 9B and D,p 0.47). These results clearly indicate that the cellular uptake ofthe Au-P(LA-DOX)-b-PEG-OH/FA micelles were facilitated bya folate-receptor-mediated endocytosis process while the Au-P(LA-DOX)-b-PEG-OH micelles were transported into cells througha non-specic endocytosis mechanism.

3.5. Cytotoxicity

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1. Control2. Au-P(LA-DOX)-b-PEG-OH3. Au-P(LA-DOX)-b-PEG-OH/FA4. Free DOXFig. 10. Cytotoxicity of free DOX, Au-P(LA-DOX)-b-PEG-OH, and Au-P(LA-DOX)-b-PEG-OH/FA NPs against (A) NIH-3T3 and (B) 4T1 cells (incubation time, 48 h).1 2 3 40

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1. Control2. Au-P(LA-DOX)-b-PEG-OH/FA(FA free medium)3. Au-P(LA-DOX)-b-PEG-OH/FA(Free FA, 0.5 mg/mL)4. Au-P(LA-DOX)-b-PEG-OH/FA(Free FA, 1 mg/mL)The cytotoxic effects of free DOX, Au-P(LA-DOX)-b-PEG-OH, andAu-P(LA-DOX)-b-PEG-OH/FA micelles against the cultured NIH-3T3and 4T1 cells were evaluated using the MTT assay [19]. NIH-3T3cells are healthy mouse embryonic broblast cells without over-expressed FA receptors; 4T1 cells are mouse mammary carcinomacells with over-expressed FA receptors. Cells were treated with freeDOX or DOX-conjugated micelles with an equivalent concentrationof DOX (34 mg/mL). Fig. 10A shows the NIH-3T3 cell viability in thepresence of free DOX, Au-P(LA-DOX)-b-PEG-OH, and Au-P(LA-DOX)-b-PEG-OH/FA micelles. The results showed no signicantdifference in the viability of NIH-3T3 cells when cultured in thepresence of Au-P(LA-DOX)-b-PEG-OH and Au-P(LA-DOX)-b-PEG-OH/FAmicelles, which indicates the cytotoxicity of DOX-conjugatedmicelles with or without FA was similar against NIH-3T3 cells. Thisobservation clearly conrms that the FA molecule present on thesurface of the DOX-conjugated micelles does not have any effect onthe NIH-3T3 cellular uptake.

Fig.10B shows the cytotoxic effect of free DOX, Au-P(LA-DOX)-b-PEG-OH, and Au-P(LA-DOX)-b-PEG-OH/FA micelles against 4T1cells. The cell viability in the presence of Au-P(LA-DOX)-b-PEG-OH/FA micelles was lower than that in the presence of Au-P(LA-DOX)-Fig. 11. Effect of free FA on the viability of 4T1 cells incubated with Au-P(LA-DOX)-b-PEG-OH/FA NPs for 48 h (DOX concentration: 34 mg/mL).

Nano Letter 2002;2:41921.[21] Bahadur KCR, Aryal S, Bhattarai N, Kim HY. Ceramic modication of N-acylated

chitosan stabilized gold nanoparticles. Scripta Materialia 2006;54:202934.

aterb-PEG-OH micelles. This result indicates that FA-targeting ligandspresent on the surface of Au-P(LA-DOX)-b-PEG-OH/FA micellesplayed an important role in enhancing the cytotoxic effect bybinding the micelles with the over-expressed receptor located onthe surfaces of the 4T1 cells and subsequently increasing theirintracellular uptake as a result of the receptor-mediated endocy-tosis. In the presence of free DOX, the cell viability of the cultured4T1 cells dramatically decreased, indicating that the cytotoxicity offree DOX was much higher than that of the DOX-conjugatedmicelles. As previously discussed, the fast diffusion of free DOX intothe cell nuclei might be the reason for this observation.

The presence of free FA in the mediummay hamper the bindingbetween FA-conjugated micelles and folate receptors throughcompetitive interaction. To estimate the role of free FA on thecellular uptake of Au-P(LA-DOX)-b-PEG-OH/FA micelles, 4T1 cellswere incubated with Au-P(LA-DOX)-b-PEG-OH/FA micelles (DOXconcentration: 34 mg/mL) in a culture medium containingincreasing concentrations of free FA. The cell viability in the pres-ence of Au-P(LA-DOX)-b-PEG-OH/FA micelles increased withincreasing free FA concentration, indicating that the cytotoxicity ofAu-P(LA-DOX)-b-PEG-OH/FA micelles against 4T1 cells was inhibi-ted by excess free FA (Fig. 11). The cell viability of Au-P(LA-DOX)-b-PEG-OH/FA micelles was approximately 10% in the presence ofFA-free medium, but it was about 28 and 45% in the presence ofmedium containing 0.5 and 1 mg/mL free FA, respectively. Theseresults conrm that free FA molecules inhibited the cellular uptakeof the Au-P(LA-DOX)-b-PEG-OH/FAmicelles by competitive bindingto the folate receptors on the cancer cell surface.

4. Conclusions

FA-conjugated amphiphilic Au-P(LA-DOX)-b-PEG-OH/FA NPswas synthesized as a tumor-targeted, anticancer drug deliverynanocarrier. The anticancer drug DOX was conjugated onto thehydrophobic inner shell of the NPs via an acid-labile hydrazonelinkage. The amount of DOX-conjugated onto the NPs was found tobe 17 wt%. Due to its amphiphilic multi-arm and globular structure,Au-P(LA-DOX)-b-PEG-OH/FA NPs formed stable unimolecularmicelles composed of an Au core, P(LA-DOX) inner shell, and PEG-OH/FA outer shell in aqueous solution. The size of micelles madefrom the Au-P(LA-DOX)-b-PEG-OH/FA NPs was determined as 2452 and 1025 nm by DLS and TEM, respectively. The release of DOXfrom the Au-P(LA-DOX)-b-PEG-OH/FA NPs depended strongly onthe pH values of themedium. Itwas found that DOX released rapidlyat acidic pHs such as those encountered in tumor tissue and theendocytic compartments of the tumor cell due to the hydrolysis ofhydrazone linkage. Flow cytometry and confocal image analysisrevealed that the extent of 4T1 cellular uptake for Au-P(LA-DOX)-b-PEG-OH/FA micelles was greater than FA-free Au-P(LA-DOX)-b-PEG-OH micelles due to the folate-receptor-mediated endocytosismechanism. The cytotoxicity of Au-P(LA-DOX)-b-PEG-OH/FAmicelles to 4T1 cells was higher than that of FA-free Au-P(LA-DOX)-b-PEG-OHmicelles, indicating that the FA-conjugatedmicelles havethe ability to selectively target to cancer cells. These results indicatethat the Au-P(LA-DOX)-b-PEG-OH/FA NPs could be a promisinganticancer nanomedicine to achieve better efcacy for chemo-therapy. Finally, the Au-P(LA-DOX)-b-PEG-OH/FA NPs potentiallycan be used for photothermal cancer therapy and contrast agent forvarious imaging modalities (e.g., computed tomography (CT)imaging), thereby making targeted cancer theranostics possible.

Acknowledgement

We acknowledge the nancial support from the University of

M. Prabaharan et al. / Biom6074Wisconsin-Milwaukee.[22] Liz-Marzan LM. Nanometals: formation and color. Materials Today 2004;7:2631.

[23] Link S, El-Sayed MA. Optical properties and ultrafast dynamics of metallicnanocrystals. Annual Review of Physical Chemistry 2003;54:33166.

[24] Daniel MC, Astruc D. Gold nanoparticles: assembly, supramolecular chemistry,quantum-size-related properties, and applications toward biology, catalysis,and nanotechnology. Chemical Review 2004;104:293346.

[25] Schmalenberg KE, Frauchiger L, Nikkhouy-Albers L, Uhrich KE. Cytotoxicity ofAppendix

Figures with essential colour discrimination. Certain gures inthis article, in particular Figs. 1 and 9, are difcult to interpret inblack and white. The full colour images can be found in the on-lineversion, at doi:10.1016/j.biomaterials.2009.07.048.

References

[1] Luo S, Xu J, Zhang Y, Liu S, Wu C. Double hydrophilic block copolymermonolayer protected hybrid gold nanoparticles and their shell cross-linking.Journal of Physical Chemistry B 2005;109:2215966.

[2] Ghosh P, Han G, De M, Kim CK, Rotello VM. Gold nanoparticles in deliveryapplications. Advanced Drug Delivery Reviews 2008;60:130715.

[3] Tom RT, Suryanarayanan V, Reddy PG, Baskaran S, Pradeep T. Ciprooxacin-protected gold nanoparticles. Langmuir 2004;20:190914.

[4] Corbierre MK, Cameron NS, Sutton M, Mochrie SGJ, Lurio LB, Ruhm A, et al.Polymer-stabilized gold nanoparticles and their incorporation into polymermatrices. The Journal of American Chemical Society 2001;123:104112.

[5] Hong R, Han G, Fernandez JM, Kim BJ, Forbes NS, Rotello VM. Glutathione-mediated delivery and release using monolayer protected nanoparticlecarriers. The Journal of American Chemical Society 2006;128:10789.

[6] Thomas M, Klibanov AM. Conjugation to gold nanoparticles enhances poly-ethylenimines transfer of plasmid DNA into mammalian cells. Proceedings ofNational Academy of Science United States of America 2003;100:913843.

[7] Wang H, Chen Y, Li XY, Liu Y. Synthesis of oligo(ethylenediamino)-betacyclo-dextrin modied gold nanoparticle as a DNA concentrator. Molecular Phar-maceutics 2007;4:18998.

[8] Cheng Y, Samia AC, Meyers JD, Panagopoulos I, Fei B, Burda C. Highly efcienthotodynamic drug delivery with gold nanoparticle vectors for in vivo therapyof cancer. The Journal of American Chemical Society 2008;130:106437.

[9] Kim DP, Lee JH, Jeong YY, Jon S. Antibiofouling polymer-coated gold nano-particles as a contrast agent for in vivo X-ray computed tomography imaging.The Journal of American Chemical Society 2007;129:76615.

[10] Jain PK, El-Sayed IH, El-Sayed MA. Au nanoparticles target cancer. Nano Today2007;2:1829.

[11] Aryal S, Pilla S, Gong S. Multifunctional nano-micelles formed by amphiphilicgold-polycaprolactone-methoxy poly(ethylene glycol) (AuPCLMPEG) nano-particles for potential drug delivery applications. Journal of Nanoscience andNanotechnology 2009;9:18.

[12] Prabaharan M, Grailer JJ, Pilla S, Steeber DA, Gong S. Amphiphilic multi-arm-block copolymer based on hyperbranched polyester, poly(L-lactide) andpoly(ethylene glycol) as a drug delivery carrier. Macromolecular Bioscience2009;9:51524.

[13] Prabaharan M, Grailer JJ, Steeber DA, Gong S. Thermo-sensitive micelles basedon folate-conjugated poly(N-vinylcaprolactam)- block-poly(ethylene glycol)copolymers for tumor-targeted drug delivery. Macromolecular Bioscience,doi:10.1002/mabi.200800366.

[14] Chen S, Zhang XZ, Cheng SX, Zhuo RX, Gu ZW. Functionalized amphiphilichyperbranched polymers for targeted drug delivery. Biomacromolecules2008;9:257885.

[15] Prabaharan M, Grailer JJ, Pilla S, Steeber DA, Gong S. Folate-conjugatedamphiphilic hyperbranched block copolymers based on boltorn H40, poly-(L-lactide) and poly(ethylene glycol) for tumor-targeted drug delivery.Biomaterials 2009;30:300919.

[16] Lee Y, Park SY, Mok H, Park TG. Synthesis, characterization, antitumor activityof pluronic mimicking copolymer micelles conjugated with doxorubicin viaacid-cleavable linkage. Bioconjugate Chemistry 2008;19:52531.

[17] Guan H, McGuire MJ, Li S, Brown KC. Peptide-targeted polyglutamic aciddoxorubicin conjugates for the treatment of avb6-positive cancers. Bio-conjugate Chemistry 2008;19:181321.

[18] Selvakannan PR, Mandal S, Phadtare S, Pasricha R, Sastry M. Capping of goldnanoparticles by the amino acid lysine renders them water-dispersible.Langmuir 2003;19:35459.

[19] Mosmann T. Rapid colorimetric assay for cellular growth and survival: appli-cation to proliferation and cytotoxicity assays. Journal of ImmunologicalMethods 1983;65:5563.

[20] Wyrna D, Beyer N. One-dimensional arrangements of metal nanoclusters.

ials 30 (2009) 60656075a unimolecular polymeric micelle and its degradation products. Bio-macromolecules 2001;2:8515.

[26] Yuan F, Dellian M, Fukumura D, Leunig M, Berk DA, Torchilin VP, et al. Vascularpermeability in a human tumor xenograft: molecular size dependence andcutoff size. Cancer Research 1995;55:37526.

[27] Yoo HS, Lee EA, Park TG. Doxorubicin-conjugated biodegradable polymericmicelles having acid-cleavable linkages. Journal of Controlled Release2002;82:1727.

[28] Gillies ER, Frechet JMJ. pH-responsive copolymer assemblies for controlledrelease of doxorubicin. Bioconjugate Chemistry 2005;16:3618.

[29] Lee CC, Cramer AT, Frechet JMJ. An intramolecular cyclization reaction is responsiblefor the invivo inefcacyandapparentpHinsensitivehydrolysiskineticsofhydrazonecarboxylate derivatives of doxorubicin. Bioconjugate Chemistry 2006;17:13648.

[30] Yoo HS, Park TG. Folate-receptor-targeted delivery of doxorubicin nano-aggregates stabilized by doxorubicinPEGfolate conjugate. Journal ofControlled Release 2004;100:24756.

[31] Gabbay EJ, Grier D, Fingerie RE, Reimer R, Levy R, Pearce SW, et al. Interactionspecicity of the anthracyclines with DNA. Biochemistry 1976;15:206270.

M. Prabaharan et al. / Biomaterials 30 (2009) 60656075 6075

Gold nanoparticles with a monolayer of doxorubicin-conjugated amphiphilic block copolymer for tumor-targeted drug deliveryIntroductionMaterials and methodsMaterialsSynthesis of amine functionalized Au NPsSynthesis of BLA-NCASynthesis of Au-poly(beta-benzyl l-aspartate) (Au-PBLA-NH2)Synthesis of Au-poly(beta-benzyl l-aspartate)-b-poly(ethylene glycol) (Au-PBLA-b-PEG-OH/FA)Synthesis of Au-P(LA-DOX)-b-PEG-OH/FAPreparation of micelles from the Au-P(LA-DOX)-b-PEG-OH/FA NPsCharacterizationDrug releaseCellular uptake and cytotoxicity

Results and discussionSynthesis and characterization of Au-P(LA-DOX)-b-PEG-OH/FAParticle size analysisDrug releaseCellular uptakeCytotoxicity

ConclusionsAcknowledgementAppendixReferences