This article appeared in a journal published by Elsevier. The attachedcopy is furnished to the author for internal non-commercial researchand education use, including for instruction at the authors institution

and sharing with colleagues.

Other uses, including reproduction and distribution, or selling orlicensing copies, or posting to personal, institutional or third party

websites are prohibited.

In most cases authors are permitted to post their version of thearticle (e.g. in Word or Tex form) to their personal website orinstitutional repository. Authors requiring further information

regarding Elsevier’s archiving and manuscript policies areencouraged to visit:

http://www.elsevier.com/copyright

Author's personal copy

Polyethylene glycol-conjugated hyaluronic acid-ceramide self-assemblednanoparticles for targeted delivery of doxorubicin

Hyun-Jong Cho a, In-Soo Yoon a, Hong Yeol Yoon b, Heebeom Koo b, Yu-Jin Jin a, Seung-Hak Ko c,Jae-Seong Shim c,d, Kwangmeyung Kimb, Ick Chan Kwon b, Dae-Duk Kim a,*

aCollege of Pharmacy and Research Institute of Pharmaceutical Sciences, Seoul National University, Seoul 151-742, Republic of KoreabCenter for Theragnosis, Biomedical Research Institute, Korea Institute of Science and Technology (KIST), Hwarangno 14-gil 6, Seongbuk-gu, Seoul 136-791, Republic of KoreacBiogenics Inc., Daejeon 305-510, Republic of Koread Skin & Tech Inc., Seongnam 461-713, Republic of Korea

a r t i c l e i n f o

Article history:Received 4 October 2011Accepted 22 October 2011Available online 9 November 2011

Keywords:DoxorubicinHyaluronic acid-ceramide-polyethyleneglycolProlonged circulationTheranostic nanoparticleTumor-targeting

a b s t r a c t

Polyethylene glycol (PEG)-conjugated hyaluronic acid-ceramide (HACE) was synthesized for the prepa-ration of doxorubicin (DOX)-loaded HACE-PEG-based nanoparticles, 160 nm in mean diameter witha negative surface charge. Greater uptake of DOX from these HACE-PEG-based nanoparticles wasobserved in the CD44 receptor highly expressed SCC7 cell line, compared to results from the CD44-negative cell line, NIH3T3. A strong fluorescent signal was detected in the tumor region upon intrave-nous injection of cyanine 5.5-labeled nanoparticles into the SCC7 tumor xenograft mice; the extendedcirculation time of the HACE-PEG-based nanoparticle was also observed. Pharmacokinetic study in ratsshowed a 73.0% reduction of the in vivo clearance of DOX compared to the control group. The antitumorefficacy of the DOX-loaded HACE-PEG-based nanoparticles was also verified in a tumor xenograft mousemodel. DOX was efficiently delivered to the tumor site by active targeting via HA and CD44 receptorinteraction and by passive targeting due to its small mean diameter (<200 nm). Moreover, PEGylationresulted in prolonged nanoparticle circulation and reduced DOX clearance rate in an in vivo model. Theseresults therefore indicate that PEGylated HACE nanoparticles represent a promising anticancer drugdelivery system for cancer diagnosis and therapy.

� 2011 Elsevier Ltd. All rights reserved.

1. Introduction

Doxorubicin (DOX) is an anthracycline antibiotic widely used forcancer chemotherapy. Its indications include cancers of the thyroid,breast, stomach, lung, ovaries, and bladder; soft sarcomas; multiplemyeloma; leukemias; and Hodgkin’s lymphoma. However, becauseof severe side effects including cardiomyopathy and congestiveheart failure, tumor-targeting delivery vehicles have been investi-gated [1e3]. Unfortunately, most of the formulations have notshown beneficial effects in clinical trials.

An approach that could be useful for future clinical applicationsis the development of nano-sized vehicles for anticancer drugdelivery [4e6]. Most of the nanoparticles could passively accu-mulate in tumors by an enhanced permeability and retention (EPR)effect [7]. The highly permeable vascular structure of the neoplasmwhich is induced by rapid and abnormal angiogenesis as well asa dysfunctional lymphatic drainage system can result in the

retention of nanoparticles and consequent release of the drug at thetumor site [8]. Nevertheless, passive targeting methods lackselective cellular uptake strategies, and therefore cannot guaranteecomplete cancer cell-targeted delivery. The incorporation ofvarious targetingmoieties including peptides, antibodies, and smallchemicals (i.e., folic acid) into the nanoparticles could, however,confer active targeting properties.

Hyaluronic acid (HA) is one of the highly efficient targetingmolecule that can bind to the CD44 receptor, which is overex-pressed in many types of cancer cells [9]. Due to its biocompati-bility, HA has been the focus of several anticancer drug targetingstudies as previously reported [10e12]. Efficacies have beenconfirmed in an amphiphilic HA derivative-based nanoparticlesystems using CD44 receptor targeting. In our previous report [13],hyaluronic acid-ceramide (HACE) was synthesized as an amphi-philic HA derivative, and nanoparticles based on HACE andPluronic�were developed for tumor-targeted delivery of docetaxel.Tumor-targeting via HA and CD44 receptor interaction and theability of docetaxel-loaded nanoparticles to overcome multidrugresistance were also verified.

* Corresponding author. Tel.: þ82 2 880 7870; fax: þ82 2 873 9177.E-mail address: ddkim@snu.ac.kr (D.-D. Kim).

Contents lists available at SciVerse ScienceDirect

Biomaterials

journal homepage: www.elsevier .com/locate/biomateria ls

0142-9612/$ e see front matter � 2011 Elsevier Ltd. All rights reserved.doi:10.1016/j.biomaterials.2011.10.064

Biomaterials 33 (2012) 1190e1200

Author's personal copy

Numerous theranostic nanoparticles that make concurrentcancer diagnosis and therapy have been developed and evaluated[14,15]. There has also been considerable progress in terms of thedevelopment of HA derivative-based nanoparticles for cancerdiagnosis [10]. In this investigation, an attempt has been made toincrease the circulation time of the HACE system in the blood-stream by conjugation with polyethylene glycol (PEG). Nano-particles based on polymers and lipids modified with PEG havebeen reported where an improved in vivo performance wasdemonstrated [16e18]. PEGylation was shown not only to provideprolonged in vivo circulation, but also lower clearance of the loadeddrug. Therefore, hereinwe describe the physicochemical propertiesof DOX-loaded, self-assembled, HACE-PEG-based nanoparticles.The in vitro DOX release pattern from these nanoparticles will beinvestigated. In particular, CD44-mediated cellular uptake effi-ciency of DOX from developed nanoparticles will be evaluated inSCC7 (CD44 receptor-positive cell line) and NIH3T3 (CD44 receptor-negative cell line). Moreover, the in vivo antitumor efficacy of DOX-loaded, HACE-based nanoparticles tested in the tumor xenograftmouse model will be reported.

2. Materials and methods

2.1. Materials

HA oligomer (4.7 kDa) and DS-Y30 (ceramide 3B; mainly N-oleoylphytos-phingosine) were purchased from Bioland Co., Ltd. (Cheonan, Korea) and DoosanBiotech Co., Ltd. (Yongin, Korea), respectively. Chloromethylbenzoyl chloride, tetra-n-butylammonium hydroxide (TBA), N-(3-dimethylaminopropyl)-N0-ethyl-carbodiimide hydrochloride (EDC), adipic acid dihydrazide (ADH), 1-hydroxybenzotriazole (HOBt), methoxypolyethylene glycol amine (mPEG-NH2,MW: 5000), and pyrene were obtained from SigmaeAldrich Co. (St. Louis, MO).Cyanine 5.5 (Cy5.5) was purchased from Amersham Biosciences (Piscataway, NJ).Doxorubicin hydrochloride (DOX HCl) was purchased from Boryung PharmaceuticalCo., Ltd. (Seoul, Korea). Dulbecco’s modified Eagle’s medium (DMEM), RPMI 1640(developed by Roswell Park Memorial Institute), penicillin, streptomycin, and heat-inactivated fetal bovine serum (FBS) were obtained from Gibco Life Technologies,Inc. (Grand Island, NY). All other reagents were of analytical grade and were ob-tained from commercial sources.

2.2. Synthesis and characterization of HACE-PEG

HACE was synthesized as described in our previous report [13]. Briefly, HA(12.21 mmol) and TBA (9.77 mmol) were added to 60 mL of double distilled water(DDW) and stirred for 30 min. Activated HA-TBA was obtained after freeze-drying.To synthesize the DS-Y30 linker, DS-Y30 ceramide (8.59 mmol), triethylamine(9.45mmol) in 25mL of tetrahydrofuran (THF), and 4-chloromethylbenzoyl chloride(8.59 mmol) in 10 mL of THF were mixed. After stirring for 6 h at 60 �C, the DS-Y30-containing linker was obtained by concentration and recrystallization. HA-TBA(8.10 mmol) and the DS-Y30-containing linker (0.41 mmol) were then solubilizedin amixture of THF and acetonitrile (4:1, v/v) and stirred for 5 h at 40 �C. SynthesizedHACE was obtained following elimination of impurities and organic solvent. PEGwas conjugated to HACE by amide bond formation between the amine group ofmPEG-NH2 and the carboxyl group of HA under EDC and NHS-mediated reaction.HACE (80 mmol) was dissolved in 75 mL of DDW. HOBt (200 mmol) solubilized in7 mL of methanol was added to the HACE solution. EDC (200 mmol) and sulfo-NHS(200 mmol) solution in DDW (15 mL) was added to the HACE and HOBt solution for1 h. After stirring for 4 h, mPEG-NH2 solution (20 mmol) in DDW (20 mL) was slowlyadded to the stirred solution. This mixture was stirred for 1 day at room tempera-ture. The resulting solution was then loaded into a dialysis bag (MWCO; 6e8 kDa)and dialyzed against the methanol and DDW mixture (1:1, v/v) for 2 days. Afterfreeze-drying, a yellowish white powder was obtained as a product. The introduc-tion ratio of PEG to HACE was measured by 1H-NMR analysis. HACE and HACE-PEGwere dissolved in D2O for analysis by 1H-NMR (500 MHz).

2.3. Preparation and characterization of DOX-loaded, HACE-PEG-basednanoparticles

DOXHCl (100mg) was dissolved in 10mL of DMSO, and 0.12mL of triethylamine(TEA) was added. After 12 h of stirring at room temperature, the solutionwas freeze-dried and desiccated to obtain the base form of DOX which was loaded into the self-assembled nanoparticles.

Self-assembled nanoparticles were prepared by the following procedures:10 mg of HACE or HACE-PEG with 1.5 mg of DOX were solubilized in 1 mL of DMSO

and DDW mixture (v/v, 1:1). The solvent was removed under a gentle stream ofnitrogen gas at 70 �C for 4 h. Then, 1 mL of DDWwas added to the tubes, followed byvortexing for 2 min to prepare self-assembled nanoparticles. It was then filteredthrough a syringe filter with a 0.2-mm pore size (Minisart RC 15; Satorius StedimBiotech GmbH, Göttingen, Germany) to eliminate insoluble drug.

Particle size, polydispersity index, and zeta potential were measured usinga light-scattering spectrophotometer (ELS-Z; Otsuka Electronics, Tokyo, Japan)according to the manufacturer’s protocol. To measure encapsulation efficiency, self-assembled nanoparticles were disrupted with 100� the volume of DMSO, and thedrug content was analyzed by high performance liquid chromatography (HPLC).DOX was quantitatively analyzed with a Waters HPLC system (Waters Co., Milford,MA) equipped with a reversed-phase C-18 column (Xbridge�, RP-18, 250 � 4.6 mm,5 mm;Waters Co.), a separation module (Waters e2695), and a fluorescence detector(Waters 2475). The mobile phase consisted of 0.1 M sodium acetate buffer (pH 4.0adjusted with acetic acid) and acetonitrile (71:29, v/v), and the eluent was moni-tored at excitation and emission wavelengths of 470 nm and 565 nm, respectively,with a flow rate of 1.0 mL/min. The injection volume for drug analysis was 20 mL. Thelower limit of quantification (LOQ) was 50 ng/mL. The inter- and intraday variance ofthis HPLC method were within the acceptable range.

The morphology of DOX-loaded nanoparticles was observed by transmissionelectron microscopy (TEM). Nanoparticle suspensions were stained with 2% (w/v)phosphotungstic acid. They were then placed on copper grids with films, air-driedfor 10 min, and observed by TEM (JEM 1010; JEOL, Tokyo, Japan). The length ofthe scale bar in figures is 100 nm at 200,000� magnification.

2.4. In vitro release test of DOX from nanoparticles

Aliquots (150 mL) of DOX-loaded HACE or HACE-PEG nanoparticle formulations(polymer:drug ¼ 10:1.5) was loaded into Mini-GeBAflex tubes, which had a molec-ular weight cutoff of 12e14 kDa (Gene Bio-Application Ltd., Kfar Hanagide, Israel).These tubes were then immersed in phosphate buffered saline (PBS, pH 7.4) ina shaking bath at 37 �C. The dissolution medium (10 mL) was rotated at 50 rpm.Aliquots of medium (0.2 mL) were collected at determined times (1, 2, 3, 4, 6, 9, 12,24, 48, 72, and 96 h), and replaced with an equivalent volume of fresh medium(37 �C). The released amounts of DOX were analyzed by the previously describedHPLC method.

2.5. In vitro cytotoxicity tests of blank nanoparticles

NIH3T3 and SCC7 cell lines were purchased from Korean Cell Line Bank (KCLB,Seoul, Korea) and American Type Culture Collection (Rockville, MD), respectively.NIH3T3 cells were cultured with DMEM supplemented with 10% FBS, 100 U/mLpenicillin, and 100 mg/mL streptomycin. SCC7 cells were cultured with RPMI 1640including 10% FBS, 100 U/mL penicillin, and 100 mg/mL streptomycin in a 5% CO2

atmosphere and 95% relative humidity at 37 �C.Cytotoxicities of HACE and HACE-PEG for NIH3T3 and SCC7 cells were evaluated

by MTS-based assay. After NIH3T3 and SCC7 cells achieved 70e80% confluency, thecells were trypsinized and seeded onto 48-well plates at a density of 2.0 � 104 cellsper well. After 24 h of incubation, the cell culture media were removed. Cytotoxicitywas measured after 12 and 24 h of incubation with various concentrations ofpolymers at 37 �C under a 5% CO2 atmosphere and 95% relative humidity. After theincubation period, cells were treated with MTS-based CellTiter 96� AQueous OneSolution Cell Proliferation Assay Reagent (Promega Corp., WI) at 37 �C for 4 haccording to the manufacturer’s protocol. The absorbance was read at a wavelengthof 490-nm with an EMax Precision Microplate Reader (Molecular Devices, Sunny-vale, CA).

2.6. Cellular uptake study

Cellular uptake and distribution of DOX from developed nanoparticles wereobserved by confocal laser scanning microscopy (CLSM). After NIH3T3 and SCC7cells achieved 70e80% confluency, the cells were trypsinized and seeded ontoculture slides (BD Falcon, Bedford, MA) at a density of 1.0�105 cells per well (surfacearea of 1.7 cm2 per well, 4 chamber slides) and incubated for 24 h at 37 �C. DOX(50 mg/mL), alone or entrapped in nanoparticles, was added and incubated for 2 h at37 �C. After incubation, all reagents were removed. Cells were washed with PBS (pH7.4) at least 3 times and fixed with 4% formaldehyde solution for 10 min. The liquidcontent was then dried completely. VECTASHIELD mounting medium with 40 ,6-diamidino-2-phenylindole (DAPI) (H-1200; Vector Laboratories, Inc., Burlingame,CA) was added to prevent fading and to stain nuclei. They were observed by CLSM(LSM 510; Carl-Zeiss, Thornwood, NY).

2.7. Tumor-targeting properties of nanoparticles observed by in vivo imaging

For the animal experiments, HACE and HACE-PEG were labeled with the near-infrared fluorescence (NIRF) dye, Cy5.5, to monitor in vivo biodistribution accord-ing to the method reported previously [10,13]. Briefly, ADH-modified Cy5.5 wasconjugated with each polymer in a 1:25 weight ratio in the presence of EDC and

H.-J. Cho et al. / Biomaterials 33 (2012) 1190e1200 1191

Author's personal copy

HOBt. The Cy5.5 content in HACE and HACE-PEG was fixed by the absorbance at680 nm determined using an UV/vis spectrophotometer.

Female BALB/c nude mice (5 weeks of age; Charles River, Wilmington, MA) wereused to generate the tumor xenograft mouse model. The SCC7 cell suspension(1�106 cells in 0.1 mL of cell culture medium) was injected subcutaneously into thebacks of the mice. Injection of Cy5.5-labeled nanoparticles was initiated 14 daysafter injection of cancer cells when the tumor volume reached approximately200 mm3. The tumor size was measured using Vernier calipers, and the tumorvolume (mm3) was calculated using V ¼ 0.5 � longest diameter � shortest diam-eter2. Cy5.5-labeled HACE and HACE-PEG-based nanoparticle formulations withequal fluorescence were injected into the tail vein of the tumor xenograft mice. Thetumor accumulation profiles were assessed using the eXplore Optix system (ARTAdvanced Research Technologies Inc., Montreal, QC, Canada). Laser power and counttime settings were optimized at 3 mW and 0.3 s per point, respectively. The laserdiode at a wavelength of 670 nmwas used to excite the Cy5.5-conjugated polymericnanoparticles. NIRF emission at 700 nm was collected and detected through a fastphotomultiplier tube (Hamamatsu, Japan) and a time-correlated single-photoncounting system (Becker and Hickl GmbH, Berlin, Germany), respectively. Thetumor-targeting properties of the nanoparticles were assessed by measuring theNIRF intensity in the whole body and tumor region (50 mm2). All data were calcu-lated using the region of interest (ROI) function of the AnalysisWorkstation software(ART Advanced Research Technologies Inc.).

The tumors were dissected from the mice 24 h after intravenous injection ofCy5.5-conjugated HACE-based nanoparticles. NIRF images of organs and tumorswere observed using a 12-bit CCD camera (Kodak Image Station 4000 MM; Kodak,New Haven, CT) equipped with a special C-mount lens and Cy5.5 bandpass emissionfilter (680e720 nm; Omega Optical, Brattleboro, VT). The NIRF intensities in severalorgans and tumors were calculated in the ROI (n ¼ 3).

2.8. In vivo pharmacokinetic study in rats

The femoral artery and vein were cannulated with a polyethylene tube (PE-50;Clay Adams, Parsippany, NJ) under ketamine anesthesia (50 mg/kg, intramuscularinjection). DOX solution and HACE/DOX and HACE-PEG/DOX at a dose of 4 mg/kgwere administered to rats via the femoral vein over 1 min (total injection volume ofapproximately 0.6 mL). Blood samples of approximately 300 mL were collected viathe femoral artery at 0 (control), 1 (at the end of infusion), 5, 15, 30, 60, 120, 180, 240,and 360 min after intravenous administration of DOX solution and formulations.After centrifugation of blood samples, 150 mL aliquots of plasma were storedat �70 �C (Model DF8517; Ilshin Laboratory Company, Seoul, Korea) until HPLCanalysis of DOX. Approximately 0.3 mL of heparinized 0.9% sodium chloride-injectable solution (20 U/mL) was used to flush the cannula immediately afterobtaining each blood sample to prevent blood clotting.

The concentrations of DOX in the above samples were determined by the HPLCmethods reported previously with slight modifications [19,20]. A 150 mL plasmasample was deproteinized with 300 mL of acetonitrile and 100 mL of methanolcontaining 1 mg/mL of propranolol (internal standard) was added. After vortexingand centrifugation at 16,000 � g for 10 min, 400 mL of the supernatant was trans-ferred to a clean tube and evaporated under a gentle stream of nitrogen gas at roomtemperature. The residue was reconstituted in 60 mL of mobile phase for plasmasampling. After vortexing and centrifugation, a 25 mL aliquot was injected ontoa reversed-phase (RP) HPLC column (C18). The mobile phases, 10 mM KH2PO4 (pH4.0):acetonitrile:methanol (70:25:5, v/v/v) with 0.1% (v/v) TEA for in vivo studies,were run at a flow rate of 1 mL/min. The column effluent was monitored witha fluorescence detector set at excitation and emission wavelengths of 470 nm and575 nm, respectively. The retention times of DOX and propranolol were approxi-mately 3.5 and 6 min, respectively. The lower limit of quantitation (LLOQ) value ofDOX in rat plasma was 0.01 mg/mL. The inter- and intraday coefficients of variationwere <12.1%.

The following pharmacokinetic parameters were calculated using WinNonlin�

(Pharsight, Mountain View, CA): total area under the plasma concentrationetimecurve from time zero to time infinity (AUC), terminal half-life (t1/2), time-averagedtotal body clearance (CL), apparent volume of distribution at steady state (Vss),and mean residence time (MRT).

2.9. In vivo antitumor efficacy test

Female BALB/c nude mice (5 weeks of age; Charles River) were used to preparethe tumor xenograft mousemodel. Mice were maintained in a light-controlled roomkept at a temperature of 22 � 2 �C and relative humidity of 55 � 5% (Animal Centerfor Pharmaceutical Research, College of Pharmacy, Seoul National University, Korea).The experimental protocols used in the animal studies were approved by the AnimalCare and Use Committee of the College of Pharmacy, Seoul National University. TheSCC7 cell suspension (1 � 106 cells in 0.1 mL of cell culture medium) was subcuta-neously injected into the backs of the mice. Tumor treatment was started 10 daysafter injection, when the tumor volume reached approximately 50e100 mm3. Thetumor size was measured using Vernier calipers, and the tumor volume (mm3) wascalculated using V ¼ 0.5 � longest diameter � shortest diameter2. Experimentalgroups were as follows: control, DOX solution, HACE:DOX (10:1.5), and HACE-

PEG:DOX (10:1.5). DOX (1.75 mg/kg dose) was injected intravenously into eachmouse on days 7, 9, 11, and 14. Tumor volumes and body weight were measured for21 days.

2.10. Statistical analysis

All experiments in this study were performed at least three times, and the datawere expressed as the means � standard deviation (SD). Statistical analyses wereperformed by analysis of variance (ANOVA). In all analyses, p < 0.05 was taken toindicate statistical significance.

3. Results and discussion

3.1. Preparation and characterization of HACE-PEG-basednanoparticles

HA has been widely investigated as a targeting moiety in anti-cancer drug delivery because it binds with the CD44 receptor,which is overexpressed in several types of tumor tissue. However,its hydrophilic nature impedes its application to the delivery ofhydrophobic drugs. Thus, amphiphilic HA derivatives, as modifiedHA structures, were developed for highly efficient incorporation ofdrugs into the hydrophobic core of self-assembled nanoparticles[10,12,13]. In particular, conjugation of HA-based nanoparticleswith PEG to enhance the retention time of nanoparticles in thebloodstream was reported previously [11].

In the present study, PEG was conjugated to HACE to prolong itscirculation time in the bloodstream and to control DOX releasefrom the nanoparticles (Fig. 2a). In our previous report [13],ceramide was successfully conjugated to HA oligomers to create anamphiphilic HA derivative (Supplemental Fig. S1). Activated HA-TBA was first synthesized, and the ceramide, including the linker,was then attached. Chloromethylbenzoyl chloride, which was usedas a linker (L1), was conjugated to ceramide by esterification, andlinker-ceramide was conjugated to HA-TBA by ether bond forma-tion. Based on the HACE conjugate, HACE-PEG was synthesized viaan EDC-mediated amide coupling reaction between the aminegroup of mPEG-NH2 and the carboxyl group of HA (Fig. 1a). Fig. 1bshows the 1H-NMR spectra of HACE and HACE-PEG. As reportedpreviously [13], the peaks for the methyl group of ceramide(0.9 ppm), the N-acetyl group (1.8 ppm) and methylene andhydroxyl groups at the sugar unit (3.3e4.0 ppm) of HA, and thearomatic ring of the linker (7.5e8.0 ppm) were confirmed in 1H-NMR spectra, indicating successful synthesis of HACE. Conjugationof PEG to HACE was confirmed by the peak at 3.5e3.6 ppm(CH2eCH2eO) [11]. The degree of substitution (DS) of PEG wascalculated by the integration ratio between the peaks for HA(1.9e2.0 ppm) and PEG (3.6e3.7 ppm) in 1H-NMR spectra (Fig. 1b).The DS of PEG to HACE was 3.37% in this investigation. CAC wasdetermined from fluorescence intensity measurements in thepresence of pyrene as a probe. The fluorescence intensity ratio (I373/I383, I1/I3) according to the HACE-PEG concentration was plotted,and the CAC was estimated from the threshold concentration ofself-assembled nanoparticles (Supplemental Fig. S2). The CAC valueof HACE-PEG from fluorescence intensity measurements was0.035 mg/mL. CAC of HACE-PEG was slightly lower than that ofHACE conjugate (0.042 mg/mL) reported previously [13]. This valuewas significantly lower than those of other low molecular weightdetergents reported previously [21,22]. A low CAC value implies themaintenance of a stable micellar structure following dilution ina large volume of body fluid after intravenous injection.

For encapsulation of DOX into nanoparticles, a solvent evapo-ration method was used in this study. This method has severaladvantages, including short preparation period and drug encap-sulation efficiency comparable with that of the dialysis method[13]. As shown in Table 1 and Fig. 2b, DOX-loaded nanoparticles

H.-J. Cho et al. / Biomaterials 33 (2012) 1190e12001192

Author's personal copy

Fig. 1. Synthesis and characterization of HACE-PEG. (a) Synthesis of HACE-PEG. (b) 1H-NMR spectra of HACE and HACE-PEG. They were solubilized in D2O for 1H-NMR analysis(500 MHz). Peaks 1 and 2 indicate HA and PEG for calculating the degree of substitution (DS), respectively.

H.-J. Cho et al. / Biomaterials 33 (2012) 1190e1200 1193

Author's personal copy

with a narrow size distribution (0.2 polydispersity index) wereprepared. The mean diameter of nanoparticles is known to bea critical factor in determining their in vivo fate, including bothretention time and biodistribution. The mean diameters of DOX-loaded HACE and HACE-PEG nanoparticles were 114.13 and159.30 nm, respectively. Although the incorporated drug type anddrug and polymer concentrations were slightly different, the meandiameters of the nanoparticles were in a similar range comparedwith our previous findings [13]. In particular, the mean diameter ofHACE-PEG-based nanoparticles was higher than that of HACE

nanoparticles (Table 1). This may have been caused by the presenceof a hydrophilic PEG shell structurewith water intake. Regardless ofthe increase in mean diameter due to the conjugation of PEG, theparticle size of DOX-loaded HACE-PEG nanoparticles was<200 nm,which implied low susceptibility to reticuloendothelial system(RES) clearance [23]. TEM images also revealed their sphericalshapes and similar mean diameters as measured using a light-scattering system (Fig. 2b). The zeta potential values of bothnanoparticles were negatively charged (�11.60 to �14.10 mV)because the ionized carboxylic group of HAwas located in the shell,

Fig. 2. Characterization of DOX-loaded HACE-based nanoparticles. (a) Schematic illustration of HACE-PEG-based nanoparticles. (b) Size distribution and TEM images of DOX-loadednanoparticles. The length of the scale bar in TEM images is 100 nm (�200,000).

H.-J. Cho et al. / Biomaterials 33 (2012) 1190e12001194

Author's personal copy

and these values may have prevented the aggregation of nano-particles by electrostatic repulsion.

3.2. In vitro release tests

The in vitro DOX release profiles are presented in Fig. 3. Theeffects of PEGylation of nanoparticles on drug release were inves-tigated in this study. DOX base was incorporated and sink condi-tions were adequately maintained in the release test. As shown inFig. 3, amounts of DOX release (%) within 12 h from HACE andHACE-PEG nanoparticles were 17.22 � 2.69% and 13.38 � 1.37%,respectively. Amounts of DOX release were 35.17 � 1.45% and29.17 � 3.34% for the two groups after 96 h, respectively. Althoughthe drug release rate decreased slightly with increasing incubationtime, a sustained drug release patternwas observed in both groups.In this investigation, DOX release from HACE-PEG nanoparticleswas delayed compared with that from HACE-based nanoparticles.Many previous reports have indicated sustained drug releasepatterns from PEGylated nanoparticle systems [24,25]. The PEGshell may delay drug diffusion and dissolution from the hydro-phobic core. The larger mean diameter of HACE-PEG-based nano-particles compared with HACE-based nanoparticles may alsoinfluence the drug release pattern. As discussed in our previousreport [13], the drug loading method, type of drug incorporated,amount of drug loading, conjugation ratio of PEG, mean diameter,surface charge of nanoparticles, and other factors are thought todetermine the drug release profile from nanoparticles.

3.3. In vitro cytotoxicity studies of HACE and HACE-PEG

NIH3T3 and SCC7 cells were selected for cytotoxicity tests ofHACE and HACE-PEG. NIH3T3 is a mouse embryonic fibroblast cell

line used as a CD44-negative cell line [26], and SCC7 is a mousesquamous cell carcinoma cell line that shown high levels of CD44expression [10]. Further determination of CD44 expression levels inboth cell lines was not performed because it has been reportedpreviously. Cell viability (%) was measured after incubation withHACE and HACE-PEG for 12 and 24 h in the two cell lines. The cellviabilities (%) were >80% with concentrations of 100e500 mg/mL inboth cell lines (Fig. 4). The cytotoxicities of HACE in U87-MG, MCF-7, andMCF-7/ADR cells were shown to be negligible in our previousstudy [13]. The results of cytotoxicity tests in NIH3T3 and SCC7 cellswere similar with those of our previous study and suggested thatHACE-based nanoparticles can be used as a drug delivery vehiclewithout serious cytotoxicity.

3.4. In vitro cellular uptake studies

The cellular uptake efficiency of DOX from nanoparticles wasevaluated by CLSM. NIH3T3 and SCC7 cells were used as CD44-receptor-negative and CD44-positive cell lines, respectively. Asshown in Fig. 5, the intracellular distribution of DOX was evaluatedby CLSM with DAPI staining of nuclei. The fluorescence intensity ofDOX from nanoparticles in SCC7 cells was stronger than that inNIH3T3 cells. It seemed that drug from HACE-based nanoparticleswas introduced principally by receptor-mediated endocytosis,specifically HA and CD44 receptor interaction, which was identifiedin our previous study [13]. Therefore, it was not further investigatedin this study. Moreover, red fluorescence of DOX was observed inthe nuclei of SCC7 cells after 2 h incubation with HACE-basednanoparticles, suggesting that it can interrupt DNA replicationand is involved in cell death. In addition, there was no significantdifference in fluorescence intensity between free DOX solution andDOX-loaded nanoparticle groups. This may be related to thedifference in free DOX uptake mechanism, i.e., passive diffusion, asreported previously [27,28]. It was also noted that the amount ofcellular DOX uptake was not significantly influenced by PEGylationof nanoparticles. In summary, the cellular uptake of DOX fromHACE-based nanoparticles was improved in SCC7 cells comparedwith that in NIH3T3 cells, and its cellular distribution due toreceptor-mediated endocytosis was observed.

3.5. In vivo tumor-targeting observed by NIRF imaging

The in vivo biodistribution and tumor-targeting efficiency ofHACE-based nanoparticles were evaluated in a SCC7 tumor xeno-graft mouse model. For real-time monitoring of nanoparticles inthe body, the NIRF dye Cy5.5 was conjugated to HACE and HACE-PEG. As shown in Fig. 6a, a fluorescent signal was observed in thewhole bodies of mice. Interestingly, both HACE and HACE-PEG-based nanoparticles seemed to be excreted via the kidney afterinjection without accumulation in other non-target tissues andorgans. Although fluorescence intensity was observed in the kidneyfor 24 h, the fluorescence signal intensity in the kidney wasdecreased while that in the tumor region was intensified. In

Time (h)

0 20 40 60 80 100 120

Re

le

as

ed

a

mo

un

ts

o

f D

OX

(%

)

0

10

20

30

40

50

60

HACE:DOX = 10:1.5

HACE-PEG:DOX = 10:1.5

Fig. 3. In vitro release test of DOX from nanoparticles. Data are expressed asmeans � SD (n ¼ 3).

Table 1Characterization of DOX-loaded HACE-based self-assembled nanoparticles.

Composition Mean diameter (nm) Polydispersity index Zeta potential (mV) Encapsulation efficiency (%)a Drug content (%)b

HACE:DOX (10:1.5) 114.13 � 7.86 0.19 � 0.03 �14.10 � 1.37 73.09 � 0.60 9.36 � 0.83HACE-PEG:DOX (10:1.5) 159.30 � 8.75 0.21 � 0.06 �11.60 � 1.40 90.98 � 3.64 12.01 � 0.42

Values are mean � standard deviation (SD) (n ¼ 3).

a Encapsulation efficiencyð%Þ ¼ actual amount of doxorubicin in formulationinput amount of doxorubicin in formulation

� 100.

b Drug Contentð%Þ ¼ actual amount of doxorubicin in formulationamount of doxorubicin� loaded formulation

� 100.

H.-J. Cho et al. / Biomaterials 33 (2012) 1190e1200 1195

Author's personal copy

particular, the fluorescence signal intensity of the HACE-PEG groupin the tumor region was stronger than that of the HACE groupduring the entire monitoring period (Fig. 6b and c). These obser-vations suggested that PEGylation increased the circulation time of

nanoparticles in the bloodstream and resulted in highly selectiveaccumulation at the tumor site, as reported previously [11].

The biodistribution of HACE-based nanoparticles in tumorxenograft mice was also evaluated by ex vivo imaging. Mice were

Fig. 5. In vitro cellular uptake studies of DOX in NIH3T3 and SCC7 cells. DOX solution and DOX-loaded nanoparticles based on HACE and HACE-PEG were incubated for 2 h. Cellularuptake and distribution of DOX were examined by CLSM. Red and blue colors indicate DOX and DAPI, respectively. The length of the scale bar is 10 mm. (For interpretation of thereferences to colour in this figure legend, the reader is referred to the web version of this article.)

Polymer concentration (mg/mL)

0.0 0.1 0.2 0.3 0.4 0.5 0.6

NIH

3T

3 cell viab

ility (%

)

0

20

40

60

80

100

HACE (12h)

HACE-PEG (12h)

Polymer concentration (mg/mL)

0.0 0.1 0.2 0.3 0.4 0.5 0.6

NIH

3T

3 cell viab

ility (%

)

0

20

40

60

80

100

120

HACE (24h)

HACE-PEG (24h)

Polymer concentration (mg/mL)

0.0 0.1 0.2 0.3 0.4 0.5 0.6

SC

C7 cell viab

ility (%

)

0

20

40

60

80

100

120

HACE (12h)

HACE-PEG (12h)

Polymer concentration (mg/mL)

0.0 0.1 0.2 0.3 0.4 0.5 0.6

SC

C7 cell viab

ility (%

)

0

20

40

60

80

100

120

HACE (24h)

HACE-PEG (24h)

a

b

Fig. 4. In vitro cytotoxicity tests of HACE and HACE-PEG in (a) NIH3T3 and (b) SCC7 cells for 12 and 24 h. Cell viability was measured by MTS-based assay according to variouspolymer concentrations. Data are expressed as means � SD (n ¼ 4).

H.-J. Cho et al. / Biomaterials 33 (2012) 1190e12001196

Author's personal copy

sacrificed at 24 h postinjection, and ex vivo images of organs andtumors were obtained. As shown in Fig. 7, in the case of the HACE-PEG group, the strongest fluorescence intensity was observed intumors among all dissected tissues and organs. Interestingly, thefluorescence intensity in tumors in the HACE-PEG group wassignificantly stronger than that in kidneys (p < 0.05). In addition, itwas assumed that the relatively strong fluorescence intensity in thekidneys cannot induce serious toxicity because the kidney isa representative organ for elimination, and continuous washoutover 24 h was observed (Fig. 6a). The prolonged circulation time of

PEGylated nanoparticles is thought to be related to the serumstability provided by the PEG shell. Many nanoparticles wereshown to mainly accumulate in the organs related with RES such asliver, and spleen [29]. However, the amounts of HACE-basednanoparticles in the liver and spleen were relatively lowcompared to those of the tumor (Fig. 7). It seems that avoidance andreduction of HACE-based nanoparticle accumulation in RES organshas been accomplished. Thus, the developed nanoparticles showsuperior tumor-targeting capability with negligible accumulationin non-tumor regions.

Fig. 6. In vivo NIRF imaging in SCC7 tumor-bearing mouse after intravenous injection of DOX-loaded HACE and HACE-PEG nanoparticles. In vivo time-dependent whole body (a) andtumor (b) images of mice bearing SCC7 tumors are presented. (c) Time-dependent profiles of fluorescence intensity in the tumor region (per 50 mm2) are shown. Error bars indicateSD (n ¼ 3).

H.-J. Cho et al. / Biomaterials 33 (2012) 1190e1200 1197

Author's personal copy

3.6. In vivo pharmacokinetic studies

The plasma concentrationetime profiles of DOX after 1 min ofintravenous infusion of DOX solution and DOX-loaded nano-particles (HACE and HACE-PEG) at a dose of 4 mg/kg to rats areshown in Fig. 8, and relevant pharmacokinetic parameters are listedin Table 2. Biexponential declines in plasma DOX concentrationswere observed over 6 h after dosing with DOX-loaded nanoparticleformulations. The pharmacokinetic parameters of DOX solution(4 mg/kg) in the present study were consistent with those reportedin a previous study on the pharmacokinetics of intravenous DOX at16 mg/kg, suggesting linear pharmacokinetics of intravenous DOXwithin the dose range [19]. Compared with the DOX solution, theHACE and HACE-PEG-based nanoparticles provided significantlyhigher AUC (1.89- and 3.61-fold, respectively), terminal t1/2 (1.45-and 1.99-fold, respectively), and MRT (1.57- and 2.87-fold, respec-tively) and lower CL (1.93- and 3.71-fold decrease, respectively) ofDOX. These in vivo pharmacokinetic properties of DOX from HACE-based nanoparticles were thought to be related to its in vitro

sustained DOX release pattern. Interestingly, the HACE-PEG-basednanoparticles achieved further prolonged systemic exposure ofDOX compared with the HACE-based nanoparticles. The decreasedclearance of DOX in plasma, i.e., increased retention time, in theHACE-PEG-based nanoparticle group compared with the HACE-based nanoparticle group may be explained by the in vitro sus-tained drug release (shown in Fig. 3) and in vivo enhanced circu-lation of nanoparticles based on HACE-PEG. As shown in the NIRFimaging study (Figs. 6 and 7), the prolonged circulation of PEGy-lated nanoparticles is also relevant to the alteration of pharmaco-kinetic parameters of DOX. Moreover, the results of thisinvestigation could be explained by previous reports describing thelonger circulation time of released drugs attained by PEGylatednano-sized delivery vehicles [17,30].

3.7. In vivo antitumor efficacy test

The in vivo antitumor efficacy of DOX-loaded HACE-basednanoparticle formulations was evaluated in the SCC7 tumor-

Fig. 7. Ex vivo fluorescence imaging 1 day postinjection of nanoparticles. (a) Optical and Cy5.5 filtered images of excised organs and tumors are shown. (b) Fluorescence intensityvalues are presented. Error bars indicate SD (n ¼ 3). *p < 0.05 compared with the fluorescence intensity of the kidney in the HACE-PEG group.

H.-J. Cho et al. / Biomaterials 33 (2012) 1190e12001198

Author's personal copy

bearing mouse model. DOX solution and DOX-loaded nanoparticleformulations, based on HACE and HACE-PEG, were intravenouslyinjected on days 7, 9, 11, and 14, and tumor growth and body weightwere monitored for 21 days (Fig. 9). Tumor volumes of DOX solu-tion, HACE/DOX, and HACE-PEG/DOX groups on day 21 were 48.7%,32.7%, and 19.2% of that in the control group, respectively(p < 0.05). The most efficient inhibition of tumor growth wasobserved in the HACE-PEG/DOX-treated group. This can beexplained by the results of in vivo NIRF imaging and pharmacoki-netic studies. Accumulation in the tumor site and circulation timeof HACE-PEG-based nanoparticles were improved compared tothose of HACE-based nanoparticles (Figs. 6 and 7). In addition,clearance of DOX fromHACE-PEG nanoparticles was also decreasedcompared with that of HACE nanoparticles (Table 2). The tumor-targeting and prolonged circulation time of DOX-loaded HACE-PEGnanoparticles were thought to contribute to the enhanced in vivoantitumor efficacy in the tumor xenograft mousemodel. The lack ofsignificant change in body weight during the 21-day observationperiod also confirmed the lack of serious systemic toxicity of thedeveloped nanoparticles (Fig. 9b). After 21 days, the tumor wasdissected from mice and histopathologically stained. The results ofTUNEL assay shown in Supplementary Figure S3 indicated induc-tion of apoptosis. In particular, the strongest brown color in theHACE-PEG/DOX group as a result of diaminobenzene stainingsuggested continuous and efficient delivery of DOX to the tumor

region and pharmacological efficacy among all experimentalgroups.

4. Conclusions

In summary, PEG-modified HACE was synthesized, and self-assembled nanoparticles based on HACE-PEG were prepared fortumor-targeted delivery of DOX. In vitro DOX release from HACE-based nanoparticles was sufficiently sustained, and efficientcellular uptake of DOX via HA and CD44 interaction was alsoexhibited in the CLSM study. Improved retention time in thebloodstream and accumulation of HACE-PEG nanoparticles at thetumor site were confirmed in the tumor xenograft mouse model.Reduced clearance of DOX from HACE-PEG nanoparticles wasrevealed in a pharmacokinetic study in rats. Improved half-life ofDOX in the HACE-PEG/DOX group led to the antitumor efficacy inthe tumor xenograft mice.

Acknowledgments

This work was supported by the National Research Foundationof Korea (NRF) grant funded by the Korean government (MEST) (No.2011-0030635).

Time (min)

0 60 120 180 240 300 360

Pla

sm

a c

on

ce

ntra

tio

n o

f D

OX

( g

/m

L)

0.01

0.1

1

10

100

DOX solution

HACE/DOX

HACE-PEG/DOX

Fig. 8. In vivo pharmacokinetic profiles after intravenous injection of DOX solution andDOX-loaded nanoparticle formulations (HACE:DOX ¼ 10:1.5 and HACE-PEG:DOX ¼ 10:1.5) in rats at a dose of 4 mg/kg. Points represent the means � SD(n ¼ 3).

Table 2Pharmacokinetic parameters of DOX after 1-min intravenous infusion of DOXsolution and formulations (HACE/DOX, and HACE-PEG/DOX) at a dose of 4 mg/kg torats.

Parameter DOX solution HACE:DOX¼ 10:1.5

HACE-PEG:DOX¼ 10:1.5

AUC (mg min/mL) 65.18 � 16.47 123.37 � 22.53a 235.61 � 25.93a,b

Terminal t1/2 (min) 76.37 � 8.83 110.51 � 17.34a 152.61 � 17.63a,b

CL (mL/min/kg) 63.76 � 14.18 33.11 � 5.64a 17.20 � 2.02a,b

Vss (mL/kg) 1189.49 � 204.99 850.59 � 113.58 948.99 � 384.82MRT (min) 18.82 � 1.14 29.52 � 5.94a 54.04 � 15.88a,b

Data present as mean � SD (n ¼ 3).a p < 0.05 compared to DOX solution group.b p < 0.05 compared to HACE:DOX ¼ 10:1.5 group.

Time (day)

0 5 10 15 20 25

Tu

mo

r v

olu

me

(m

m3)

0

1000

2000

3000

4000

5000

6000

7000

Control

DOX solution

HACE/DOX

HACE-PEG/DOX

*

*, #

*, #, +

a

Time (day)

0 5 10 15 20 25

Bo

dy

w

eig

ht (g

)

10

15

20

25

30

35

Control

DOX solution

HACE/DOX

HACE-PEG/DOX

b

Fig. 9. In vivo antitumor efficacy of DOX-loaded nanoparticles in the SCC7 tumor-bearing mouse model. (a) Tumor volume (mm3) profiles according to time (days) areshown. DOX solution and DOX-loaded nanoparticles (HACE:DOX ¼ 10:1.5 and HACE-PEG:DOX ¼ 10:1.5) were injected on days 7, 9, 11, and 14. Points indicate themeans � SD (n ¼ 4e5). (b) Body weight (g) was also measured with tumor sizemeasurement for 21 days. Points indicate the means � SD (n ¼ 4e5). *p < 0.05compared with the control group; #p < 0.05 compared with the DOX solution group;þp < 0.05 compared with the HACE/DOX group.

H.-J. Cho et al. / Biomaterials 33 (2012) 1190e1200 1199

Author's personal copy

Appendix. Supplementary data

Supplementary data associated with this article can be found, inthe online version, at doi:10.1016/j.biomaterials.2011.10.064.

References

[1] Nukolova NV, Oberoi HS, Cohen SM, Kabanov AV, Bronich TK. Folate-decoratednanogels for targeted therapy of ovarian cancer. Biomaterials 2011;32:5417e26.

[2] Smith LA, Cornelius VR, Plummer CJ, Levitt G, Verrill M, Canney P, et al. Car-diotoxicity of anthracycline agents for the treatment of cancer: systemicreview and meta-analysis of randomised controlled trials. BMC Cancer 2010;10:337e50.

[3] Upadhyay KK, Bhatt AN, Mishra AK, Dwarakanath BS, Jain S, Schatz C, et al. Theintracellular drug delivery and anti tumor activity of doxorubicin loadedpoly(gamma-benzyl L-glutamate)-b-hyaluronan polymersomes. Biomaterials2010;31:2882e92.

[4] Lu RM, Chang YL, Chen MS, Wu HC. Single chain anti-c-met antibody conju-gated nanoparticles for in vivo tumor-targeted imaging and drug delivery.Biomaterials 2011;32:3265e74.

[5] Meng H, Xue M, Xia T, Ji Z, Tarn DY, Zink JI, et al. Use of size and a copolymerdesign feature to improve the biodistribution and the enhanced permeabilityand retention effect of doxorubicin-loaded mesoporous silica nanoparticles ina murine xenograft tumor model. ACS Nano 2011;5:4131e44.

[6] Salva R, Taratula O, Garbuzenko O, Minko T. Tumor targeted quantum dot-mucin 1 aptamer-doxorubicin conjugate for imaging and treatment ofcancer. J Control Release 2011;153:16e22.

[7] Matsumura Y, Maeda H. A new concept for macromolecular therapeutics incancer chemotherapy: mechanism of tumoritropic accumulation of proteinsand the antitumor agent smancs. Cancer Res 1986;46:6387e92.

[8] WangM, ThanouM. Targeting nanoparticles to cancer. Pharm Res 2010;62:90e9.[9] Orian-Rousseau V. CD44, a therapeutic target for metastasising tumours. Eur J

Cancer 2010;46:1271e7.[10] Choi KY, Chung H, Min KH, Yoon HY, Kim K, Park JH, et al. Self-assembled

hyaluronic acid nanoparticles for active tumor targeting. Biomaterials 2010;31:106e14.

[11] Choi KY, Min KH, Yoon HY, Kim K, Park JH, Kwon IC, et al. PEGylation ofhyaluronic acid nanoparticles improves tumor targetability in vivo. Bioma-terials 2011;32:1880e9.

[12] Saravanakumar G, Choi KY, Yoon HY, Kim K, Park JH, Kwon IC, et al. Hydro-tropic hyaluronic acid conjugates: synthesis, characterization, and implica-tions as a carrier of paclitaxel. Int J Pharm 2010;394:154e61.

[13] Cho HJ, Yoon HY, Koo H, Ko SH, Shim JS, Lee JH, et al. Self-assembled nano-particles based on hyaluronic acid-ceramide (HA-CE) and Pluronic� fortumor-targeted delivery of docetaxel. Biomaterials 2011;32:7181e90.

[14] Jeong H, Huh M, Lee SJ, Koo H, Kwon IC, Jeong SY, et al. Photosensitizer-conjugated human serum albumin nanoparticles for effective photodynamictherapy. Theranostics 2011;1:230e9.

[15] Sanson C, Diou C, Thévenot J, Ibarboure E, Soum A, Brûlet A, et al. Doxorubicinloaded magnetic polymersomes: theranostic nanocarriers for MR imaging andmagneto-chemotherapy. ACS Nano 2011;5:1122e40.

[16] Al-Jamal WT, Al-Zamal KT, Cakebread A, Halket JM, Kostarelos K. Bloodcirculation and tissue biodistribution of lipid-quantum dot (L-QD) hybridvesicles intravenously administered in mice. Bioconjug Chem 2009;20:1696e702.

[17] Kim JY, Kim JK, Park JS, Byun Y, Kim CK. The use of PEGylated liposomes toprolong circulation lifetimes of tissue plasminogen activator. Biomaterials2009;30:5751e6.

[18] Lim SM, Kim TH, Jiang HH, Park CW, Lee S, Chen X, et al. Improvedbiological half-life and anti-tumor activity of TNF-related apoptosis-inducingligand (TRAIL) using PEG-exposed nanoparticles. Biomaterials 2011;32:3538e46.

[19] Lee HJ, Paik WH, Lee MG. Pharmacokinetic and tissue distribution changes ofadriamycin and adriamycinol after intravenous administration of adriamycinto alloxan-induced diabetes mellitus rats. Res Commun Mol Pathol Pharmacol1995;89:165e78.

[20] Son HK, Choi JS. Effects of morin on the bioavailability of doxorubicin for oraldelivery in rats. J Kor Pharm Sci 2009;39:243e8.

[21] Kratohvil JP, Hsu WP, Kwok DI. How large are the micelles of di-a-hydroxybile salts at the critical micellization concentrations in aqueous electrolytesolutions? Results for sodium taurodeoxycholate and sodium deoxycholate.Langmuir 2002;2:256e8.

[22] Phillips JN. The energetics of micelle formation. J N Trans Faraday Soc 1995;51:561e9.

[23] Gaucher G, Dufresne MH, Sant VP, Kang N, Maysinger D, Leroux JC. Blockcopolymer micelles: preparation, characterization and application in drugdelivery. J Control Release 2005;109:169e88.

[24] Bhadra D, Bhadra S, Jain S, Jain NK. A PEGylated dendritic nanoparticulatecarrier of fluorouracil. Int J Pharm 2003;257:111e24.

[25] Kumar PV, Agashe H, Dutta T, Jain NK. PEGylated dendritic architecture fordevelopment of a prolonged drug delivery system for an antitubercular drug.Curr Drug Deliv 2007;4:11e9.

[26] Somasunderam A, Thiviyanathan V, Tanaka T, Li X, Neerathilingam M,Lokesh GL, et al. Combinatorial selection of DNA thioaptamers targeted to theHA binding domain of CD44. Biochemistry 2010;49:9106e12.

[27] Misra R, Sahoo SK. Intracellular trafficking of nuclear localization signalconjugated nanoparticles for cancer therapy. Eur J Pharm Sci 2010;39:152e63.

[28] Shuai XT, Ai H, Nasongkla N, Kim SJ, Gao JM. Micellar carrier based on blockcopolymers of poly(ε-caprolactone) and poly(ethylene glycol) for doxorubicindelivery. J Control Release 2004;98:415e26.

[29] Lankveld DP, Oomen AG, Krystek P, Neigh A, Troost-de Jong A,Noorlander CW, et al. The kinetics of the tissue distribution of silver nano-particles of different sizes. Biomaterials 2010;31:8350e61.

[30] Zhu S, Hong M, Tang G, Qian L, Lin J, Jiang Y, et al. Partly PEGylated poly-amidoamine dendrimer for tumor-selective targeting of doxorubicin: theeffects of PEGylation degree and drug conjugation style. Biomaterials 2010;31:1360e71.

H.-J. Cho et al. / Biomaterials 33 (2012) 1190e12001200