Facile PEGylation of Boltorn® H40 for pH-responsive drug carriers

Polymer 54 (2013) 2020e2027

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Facile PEGylation of BoltornÒ H40 for pH-responsive drug carriers Chunlai Tu a,1, Lijuan Zhu a,1, Feng Qiu a, Dali Wang a, Yue Su a, Xinyuan Zhu a, b, **, Deyue Yan a, * a

School of Chemistry and Chemical Engineering, State Key Laboratory of Metal Matrix Composites, Shanghai Jiao Tong University, 800 Dongchuan Road, Shanghai 200240, People’s Republic of China b Instrumental Analysis Center, Shanghai Jiao Tong University, 800 Dongchuan Road, Shanghai 200240, People’s Republic of China

a r t i c l e i n f o

a b s t r a c t

Article history: Received 5 October 2012 Received in revised form 21 November 2012 Accepted 10 December 2012 Available online 17 December 2012

A pH-responsive drug delivery system was developed through facile PEGylation of commercial aliphatic dendritic polyester BoltornÒ H40 (H40) by forming the acetal linkages. Benefiting from its amphiphilic structure, the star copolymer (H40-star-MPEG) with a hydrophobic H40 core and many hydrophilic poly(ethylene glycol) (MPEG) arms was able to self-assemble into stable micelles in aqueous solution. Moreover, the size of self-assembled micelles could be easily tailored by only changing the grafting ratio of MPEG chains. The degradable behavior of H40-star-MPEG was measured by NMR technique under neutral and acidic deuterium aqueous medium. The experimental results showed that a fast hydrolysis of H40-star-MPEG occurred at a low pH solution, resulting from the cleavage of acetal linkages. The low cytotoxicity of H40-star-MPEG micelles was confirmed by cell evaluation. As a model anticancer drug, the hydrophobic drug doxorubicin (DOX) was encapsulated into the self-assembled H40-star-MPEG micelles. In an acidic environment, the drug release of DOX-loaded micelles was accelerated greatly. The DOXloaded micelles could be internalized by the cancer cells efficiently, resulting in the effective inhibition to cancer cell proliferation. All of these results suggest that facile PEGylation of H40 with acetal linkages provides a promising pH-responsive drug carrier for cancer therapy. Ó 2012 Elsevier Ltd. All rights reserved.

Keywords: PEGylation Acetal linkage pH-responsive

1. Introduction The development of polymeric drug delivery systems (PDDSs) has attracted great attention due to the structural designability of polymers and the promising clinical applications [1e6]. Safety and efficiency are two key points in the design of polymeric carriers. For safety, different strategies including PEGylation, biocompatible units and biodegradable linkages, have been introduced [7e13]. For efficiency, both stimuli-responsive ability and targeting activity are required. Thus, a number of biocompatible polymeric systems have been developed, which respond to different stimuli such as temperature [14,15], light [16e19], glutathione (GSH) [20e22] or pH value [23e29]. Among various kinds of stimuli-responsive materials, pH-sensitive systems are of particular interest for the in vivo application due to the existing pH gradients between the normal tissues and the tumors as well as endosome and lysosome. Hydrolysis of the pH-sensitive linkages can be stimulated by change of pH

* Corresponding author. Tel.: þ86 21 34205699; fax: þ86 21 34205722. ** Corresponding author. School of Chemistry and Chemical Engineering, State Key Laboratory of Metal Matrix Composites, Shanghai Jiao Tong University, 800 Dongchuan Road, Shanghai 200240, People’s Republic of China E-mail addresses: [email protected] (X. Zhu), [email protected] (D. Yan). 1 These authors are joint first authors. 0032-3861/$ e see front matter Ó 2012 Elsevier Ltd. All rights reserved. http://dx.doi.org/10.1016/j.polymer.2012.12.029

to disrupt the carriers, then the bioactive cargoes will be released in targeted tissues. Therefore, acid-labile acetals, firstly exploited by Fréchet and coworkers, are now extensively studied and applied in developing pH-sensitive delivery vehicles [26,30e38]. pH-sensitive biomaterials can be utilized for rational design of safe and efficient delivery systems. It’s well-known that polyesters are widely used for biomedical applications owing to their biodegradability and biocompatibility. Especially, dendritic polyesters have provided a fruitful strategy for drug delivery because of its interior cavities suited for encapsulating drugs, as well as numerous modifiable terminals [39e42]. By introducing acid-labile acetal linkages into dendritic polymers, Fréchet and coworkers developed a pHresponsive linear-dendritic copolymer comprising poly(ethylenen glycol) (PEG) and polyester dendron, and the periphery of dendritic polymer was attached by hydrophobic groups with cyclic acetals [35,43]. The resultant copolymer formed stable micelles in aqueous solution at neutral pH but disintegrated into unimers at mildly acidic pH. Apparently, the hydrolysis of acetals in the micelle core resulted in the controlled release of payloads. Haag and coworkers also constructed an amphiphilic dendritic polyglycerol by using acetal/ketal linkages for the pH-dependent delivery [44,45]. These dendritic nanocarriers could selectively release the encapsulated guest molecules in a physiologically relevant pH range, but tedious synthesis was adopted.

C. Tu et al. / Polymer 54 (2013) 2020e2027

In this study, we developed a facile PEGylation approach to a commercially available (Perstorp Co.) dendritic polyester BoltornÒ H40 (H40) with cyclic acetal linkages. The neighboring hydroxyl terminals of H40 could be easily cyclized with aldehyde terminal group of poly(ethylene glycol) monomethyl ether (MPEG). Benefiting from its amphiphilic structure, the resultant star copolymer (H40star-MPEG) was able to self-assemble into stable micelles with pHresponsiveness in aqueous solution. Moreover, the size of selfassembled micelles could be easily tailored by only changing the grafting ratio of MPEG chains, enhancing their passive targeting ability via the enhanced permeability and retention (EPR) effect [46,47]. The biological evaluation showed that these PEGylated micelles with acetal linkages provided a promising pH-responsive drug carrier for cancer therapy. 2. Experimental part 2.1. Materials BoltornÒ H40 (H40) was obtained from Perstorp Polyols AB Co., Sweden. Poly(ethylene glycol) monomethyl ether (MPEG, Mn ¼ 2000) was obtained from Aldrich Chemical Co p-Toluenesulfonyl chloride (TsCl, 99%) was purchased from Acros Chemical Co Dichloromethane (DCM) and cyclohexane were refluxed with calcium hydride and distilled before use. 4-Hydroxybenzaldehyde (PHBA, 98%), potassium carbonate (K2CO3), anhydrous sodium sulfate (Na2SO4), tetrahydrofuran (THF), triethylamine and indium(III) chloride (InCl3) were supplied by Sinopharm Chemical Reagent Co Dulbecco’s modified Eagle’s medium (DMEM), fetal bovine serum (FBS), and phosphate buffered solution (PBS) were purchased from PAA Laboratories GmbH. 3-(4,5-Dimethyl-thiazol2-yl)-2,5-diphenyl tetrazolium bromide (MTT) and HOECHST 33342 were from SigmaeAldrich. Doxorubicin hydrochloride (DOX$HCl) was purchased from Beijing Huafeng United Technology Corp. All the other chemicals were used as received without any further purification. Distilled water was used in all experiments. Clear polystyrene tissue culture treated 6-well, 24-well and 96-well plates were obtained from Corning Costar. 2.2. Purification of H40 Commercially available H40 (8 g) was dissolved in acetone (100 mL). Ether (100 mL) was slowly added to the above solution under vigorous stirring. After 12 h, the precipitates were filtered and washed twice with an acetone/ether mixture (v/v, 1:1). The purified H40 was dried under vacuum (4.2 g, yield 53%). 2.3. Synthesis procedure of H40-star-MPEGs MPEG with 4-hydroxybenzaldehyde end-group (MPEGePHBA) was synthesized according to our previous report [48]. Typically, in a 250 mL three-neck flask round bottom equipped with water segregator and condenser pipe, MPEGePHBA (1.0 g) and dendritic aliphatic polyester H40 (0.5 g) were dissolved in THF (15 mL). Subsequently, both InCl3 (3 mg, catalyst) and cyclohexane (100 mL) were added. The mixture was refluxed under vigorous stirring. After 24 h reaction, the solvent was removed and the product was dissolved in THF (20 mL). The resulting solution was purified by neutral alumina column chromatography with THF as the eluent. The obtained product was partially evaporated and then precipitated in diethyl ether (150 mL) to yield white powder (1.14 g, yield 76.5%). 1 H NMR (CDCl3, 298K) d ¼ ppm: 0.99e1.55 (m, H40), 3.37 (s, e OCH3), 3.63 (m, eOCH2CH2Oe), 3.82 (t, PhenyleOCH2CH2Oe), 4.09

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(t, PheOCH2CH2Oe), 4.26 (br, H40), 5.36 (br, cyclic acetaleCH), 6.84 (m, Phenyl), 7.36 (m, Phenyl). 2.4. Preparation of DOX-loaded micelles DOX-loaded micelles were prepared as follows: Briefly, H40star-MPEG (580 mg) was dissolved in dimethylformamide (DMF) (5 mL), followed by adding a predetermined amount of DOX$HCl and 2 M equivalents of triethylamine and stirred at room temperature for 2 h. Then the mixture was added slowly to deionized water (15 mL). After being stirred for an additional 6 h, the solution was dialyzed against deionized water for 24 h (MWCO ¼ 3500), during which the water was renewed every 4 h. To determine the total drug loading, the DOX-loaded micelle solution was lyophilized and then redissolved in DMF. The UV absorbance at 500 nm was measured to determine the DOX concentration. Drug-loading content (DLC) and drug-loading efficiency (DLE) were calculated according to the following formula:

DLC ðwt%Þ ¼ ðweight of loaded drug=weight of polymerÞ  100% DLE ðwt%Þ ¼ ðweight of loaded drug=weight in feedÞ  100% 2.5. Nuclear magnetic resonance (NMR) spectroscopy 1

H NMR spectra of intermediate products were carried out on Varian Mercury plus 400 NMR spectrometer (400 MHz, 298 K) and deuterated chloroform (CDCl3) as a solvent. 2.6. Fourier transform infrared (FTIR) spectroscopy FTIR spectra were performed on a Bruker Equinox-55 FTIR spectrometer. All sample pellets were prepared by grinding the solid sample with solid potassium bromide (KBr). 2.7. Size-exclusion chromatography (SEC) The molecular weights of the synthesized samples were evaluated by SEC technique. The size-exclusion chromatography e multiangle laser light scattering (SEC-MALLS) system consisted of a Waters 2690D Alliance liquid chromatography system, a Wyatt Optilab DSP differential refractometer detector, and a Wyatt MALLS detector. Two PL mix-D columns (Styragel HR3, HR4) were used in series with 80  C external temperature (column temperature) and 50  C internal temperature (RI temperature). DMF containing 0.5 M LiBr was used as eluent at a flow rate of 1 mL/min. The data were processed with Astra software (Wyatt Technology). 2.8. Transmission electron microscopy (TEM) TEM studies were performed with a JEOL 2010 instrument operated at 200 kV. The samples were prepared by directly dropping micelle solutions onto carbon-coated copper grids and drying at room temperature overnight without staining before measurement. 2.9. Degradation experiments Studies of degradation rate of acetal linkages were carried out on Varian Mercury plus 400 NMR (400 MHz, 298 K) in deuterium water (D2O) with different pH values (pH ¼ 7.4 and 5.3) at 37  C. H40-star-MPEG (10 mg) was dissolved in D2O with pH ¼ 7.4 and 5.3, respectively. 1H NMR data were collected at different time intervals.

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2.10. In vitro cytotoxicity assay The cytotoxicity of H40-star-MPEG against cultured NIH/3T3 cells was evaluated in vitro by MTT assay. The MTT assay is based on the ability of a mitochondrial dehydrogenation enzyme in viable cells to cleave the tetrazolium rings of the pale-yellow MTT and form formazan crystals with dark-blue color. The dependent relationship between the number of surviving cells and the absorbance intensity of the formed formazan determines the cytotoxicity of H40-star-MPEG micelles in aqueous solution. NIH/3T3 cells were seeded into 96-well plates at an initial seeding density of 8.0  103 cells/well in 200 mL medium. After 24 h incubation, the culture medium was removed and replaced with 200 mL medium containing serial dilutions of samples. The cells were grown for another 24 h. Then 20 mL of 5 mg/mL MTT solution in PBS was added to each well. After incubating the cells for another 4 h, the medium containing unreacted dye was removed carefully. The obtained purple formazan crystals were dissolved in 200 mL per well dimethyl sulfoxide (DMSO) and the absorbance was measured in a BioTek Elx800 at a wavelength of 490 nm. 2.11. Drug release experiments The release studies were performed at 37  C in both mimetic physiological and lysosome conditions: pH ¼ 7.4 and 5.3 buffer solutions. Firstly, DOX-loaded H40-star-MPEG (25 mg) was dissolved in medium (2.5 mL) and placed in a dialysis bag with a molecular weight cut-off (MWCO) of 3.5 kDa. The dialysis bag was then immersed in 72.5 mL of the release medium and stirred at a constant temperature. Samples (2 mL) were periodically sucked up and filled the same volume of fresh medium. The amount of released DOX was analyzed with UV/Vis spectroscopy (Lambda20, Perkin Elmer, Inc., USA) at 500 nm. The drug release studies were performed in triplicate for each of the samples. 2.12. Cellular uptake studies 2.12.1. Flow cytometry Flow cytometry was used to provide statistics on the uptake of DOX-loaded H40-star-MPEG into a human cervical carcinoma

Hela cell line. HeLa cells (5.0  105 cells per well) were seeded in six-well culture plates and grown overnight. Then, the DOXloaded H40-star-MPEG dissolved in DMEM culture medium with a polymer concentration of 0.557 mg/mL and the free DOX with concentration of 0.02 mg/mL were added to different wells. The cells were incubated at 37  C for 15 min, 30 min, 1 h, 2 h and 4 h, respectively. After incubation, samples were prepared for flow cytometry analysis by removing the cell growth media, rinsing with cold PBS, and treating with trypsin. Data for 1.0  104 gated events were collected and analysis was performed by means of a BD FACS Calibur flow cytometer and CELLQuest software. 2.12.2. Confocal laser scanning microscopy (CLSM) For the CLSM studies, HeLa cells (2.0  105) were seeded on cell culture coverslips in a 24-well tissue culture plate. After 24 h culture, the DOX-loaded H40-star-MPEG dissolved in DMEM culture medium with a polymer concentration of 0.557 mg/mL and the free DOX with concentration of 0.02 mg/mL were added to different wells. The cells were incubated at 37  C for predetermined time intervals. Then, the cells were washed with PBS and fixed with 4% paraformaldehyde at room temperature, and the slides were rinsed with cold PBS for three times. Finally, the cells were stained with HOECHST 33342 and the slides were mounted and observed with a LSM510 META. 2.13. In vitro anticancer experiments The HeLa cells with a density of 8.0  103 cells/well in 200 mL medium were cultured for 24 h after seeding into a 96-well plate. The culture medium was removed and replaced with 200 mL fresh medium containing serial dilutions of free DOX and DOX-loaded H40-star-MPEG, respectively. The cells were grown for another 48 h. Then, the wells were quickly washed three times with PBS. Thereafter, 20 mL of 5 mg/mL MTT assays stock solution in PBS was added to each well, and incubated again for another 4 h. After discarding the culture medium, the obtained purple formazan crystals were dissolved in 200 mL per well DMSO and the absorbance was measured in a BioTek Elx800 at a wavelength of 490 nm.

Fig. 1. Synthesis route of H40-star-MPEG.

C. Tu et al. / Polymer 54 (2013) 2020e2027 Table 1 Characterization data of H40, H40-star-MPEG-1, H40-star-MPEG-2 and H40-starMPEG-3. Sample

H40/PEG molar ratio

Mn (104 g/mol)

Mw (104 g/mol)

Mw/Mn (PDI)

H40a H40-star-MPEG-1 H40-star-MPEG-2 H40-star-MPEG-3

1:0 1:3.5 1:7 1:14

0.28 1.51 2.07 3.05

0.51 2.28 3.17 5.02

1.80 1.51 1.53 1.81

a

From Perstorp data sheet.

3. Results and discussion

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disappear, while a proton signal at 5.36 ppm ascribed to cyclic acetal can be observed. These results confirm the formation of the acetal linkages and successful synthesis of H40-star-MPEGs. The molecular structure of H40-star-MPEGs was further verified by FTIR studies. As shown in Fig. 3, the strong peak at 1111 cm1 is assigned to the CeOeC stretching vibration of ether in MPEG backbone, indicating the presence of MPEG. The strong peak at 1690 cm1 in Fig. 3(A) is a characteristic absorption of C]O stretching due to the existence of benzaldehyde, which disappears in Fig. 3(B) and (C). In the meantime, two new peaks at 1738 cm1 and 1063 cm1 ascribed to the stretching vibration of ester of H40 and ether of acetal ring appear. These results confirm the formation of H40-star-MPEGs.

3.1. Synthesis and characterization of H40-star-MPEGs 3.2. Physical properties of H40 and H40-star-MPEGs A large number of hydroxyl groups on the periphery of H40 provide an opportunity for the formation of acetal by reacting with aldehyde in the existence of catalyst such as toluenesulfonic acid and indium(III) salts. To improve reaction efficiency, a proper catalyst is important. Ranu and coworkers developed a simple and efficient procedure for acetalization of carbonyl compounds [49]. According to this literature, we chose InCl3 as the catalyst and modified the experimental procedure. As shown in Fig. 1, the benzaldehyde terminal groups of MPEGePHBA reacted with the hydroxyl groups of H40 to form pH-responsive amphiphilic multiarm H40-star-MPEG through the acetal linkages. By changing the H40/MPEGePHBA feeding ratio from 1/3.5 to 1/14, three H40-starMPEG samples were prepared (See Table 1). Since only few PEG chains were conjugated to H40, H40-star-MPEG-1 was hard to dissolve in water and chloroform. After purification and removal of InCl3, the products were analyzed by 1H NMR technique (Fig. 2). Besides the characteristic peaks of H40 at 4.27, 3.46 and 1.16 ppm for the methylene and methyl protons respectively, the peaks at 3.67 and 3.37 ppm belong to the methylene and methyl protons of MPEG. Obviously, the peaks at 9.87 ppm of adehyde signal

Numbereaverage molecular weights and polydispersity index (PDI) of H40-star-MPEGs were determined by the SEC-MALLS technique. The results are listed in Table 1. The numbereaverage molecular weights of H40-star-MPEG-1, H40-star-MPEG-2 and H40-star-MPEG-3 are 1.51  104, 2.07  104 and 3.05  104 with a PDI value of 1.51, 1.53 and 1.81, respectively. These results coincide with the H40/MPEG mole ratios. Due to the poor water-solubility of H40-star-MPEG-1, only H40-star-MPEG-2 and H40-star-MPEG-3 were used in the following measurements. 3.3. Degradation behavior of acetal linkages The acetal linkages in H40-star-MPEGs endowed the polymer with pH-sensitive property. Acetal bonds are stable under the neutral and basic conditions. However, the hydrolysis of acetal bonds is accelerated dramatically in an acidic environment, which eventually leads to the cleavage of acetal linkages. The degradation behavior of H40-star-MPEGs was studied in D2O solutions (pH ¼ 7.4 and 5.3) at 37  C through 1H NMR technique. Fig. 4 (A1 and B1)

Fig. 2. 1H NMR spectra of (A) MPEGePHBA; (B) H40-star-MPEG-2 and (C) H40-star-MPEG-3.

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e0 /(e0 þ e00 )]. The hydrolysis curves in Fig. 4(A2) and 4(B2) show that the hydrolysis ratio of H40-star-MPEG is highly dependent of the pH value. Within the beginning 5 h, the hydrolysis ratio has an initial burst and then slows down to reach a platform. The final hydrolysis ratios of degradation at pH ¼ 7.4 and 5.3 after 75 h are found to be 10.9% and 61.8%, respectively. 3.4. Morphology of DOX-loaded H40-star-MPEGs

Fig. 3. FTIR spectra of (A) MPEGePHBA, (B) H40-star-MPEG-2 and (C) H40-star-MPEG-3.

shows the 1H NMR hydrolysis spectra of H40-star-MPEG under neutral and acidic conditions. It can be found that the proton signals of the benzaldehyde terminal at 9.67, 7.84, and 7.04 ppm (g0 , f0 , and e0 ) increase with time whereas those signals from phenyl acetal decrease (7.26, 6.86, and 5.35 ppm for g00 , f00 , and e00 ) under the acidic medium in Fig. 4(B1). At the meanwhile, there is no obvious variation under the neutral condition, as showed in Fig. 4(A1). These results indicate that the MPEG chains depart from the dendritic core under acidic condition, while no obvious change under mimic neutral physiology condition. Moreover, the quantitative hydrolysis curves of H40-star-MPEG were obtained from the integrated area of benzaldehyde and phenyl acetal [f0 /(f0 þ f00 ) or

It is well-known that drug efficiency is significant related with size of carriers. H40-star-MPEGs have a hydrophobic H40 core and pendent hydrophilic PEG arms. In an aqueous solution, H40-starMPEGs are able to encapsulate the DOX and spontaneously selfassemble into micelles. The TEM images in Fig. 5 show the formation of spherical micelles when DOX is loaded in H40-star-MPEG-2 and H40-star-MPEG-3, respectively. The mean diameter of H40star-MPEG-2 micelles is about 35  5 nm, while that of DOX-loaded H40-star-MPEG-3 is about 15  6 nm. This result indicates that the mean diameter of drug-loaded H40-star-MPEGs decreases with the increase of MPEG chains, because hydrophilic MPEG shell can protect the hydrophobic H40 from aggregation. Thus, the drugloaded micelles with different size can be readily obtained by only controlling the amount of MPEG arms. 3.5. In vitro cytotoxicity The relative cytotoxicity of H40-star-MPEGs micelles was estimated by MTT assay against NIH/3T3 cells. Fig. 6 shows that cell viability after 24 h incubation with H40-star-MPEG-2 and H40-starMPEG-3 up to 1 mg/mL remains above 90% compared to untreated cells. This suggests the low cytotoxicity of H40-star-MPEGs, attributing to low cytotoxicity of MPEG and biodegradable ester groups.

Fig. 4. Hydrolysis of H40-star-MPEG in D2O medium with different pH values (pH ¼ 7.4, 5.3 respectively) at 37  C (400 MHz, D2O, 298 K): 1H NMR spectra of H40-star-MPEG under natural condition (A1) and acidic condition (B1). The signals of g0 , f0 and e0 are related to benzaldehyde whereas g00 , f00 and e00 belong to phenyl acetal. Hydrolysis ratio of H40-starMPEG as determined from f0 /(f0 þ f00 ) or e0 /(e0 þ e00 ) under natural condition (A2) and acidic condition (B2).

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Fig. 5. Transmission electron microscope (TEM) photos of DOX-loaded H40-star-MPEG-2 (A) and H40-star-MPEG-3 (B) (scale bar: 50 nm).

3.6. Drug release behavior Drug-loading content and drug-loading efficiency of DOXloaded H40-star-MPEGs were measured by UV/Vis spectroscopy. The dry H40-star-MPEG micelles were redissolved in DMF. Based on the standard curve of DOX at 500 nm, the drug-loading content was found to be 3.72% with drug-loading efficiency of 37.5%. To access the stimuli-responsive property of H40-star-MPEGs, the drug release behavior of DOX-loaded H40-star-MPEGs at different pH values (simulated physiological condition, PBS, pH ¼ 7.4 and an acidic environment, PBS, pH ¼ 5.3) was observed. The in vitro drug release curves in Fig. 7 show that the medium pH strongly influences the DOX release from the H40-star-MPEG micelles. The final cumulative drug release at pH ¼ 7.4 is about 20% after over 80 h, suggesting that DOX-loaded H40-star-MPEG micelles are stable under physiological condition. However, an initial 50% release arrives only after 12 h at pH ¼ 5.3, and the cumulative release reaches a plateau of almost 70% after 50 h. These results show that the release profile of DOX-loaded H40-star-MPEG has considerable

Fig. 6. Cell viability of H40-star-MPEGs against NIH/3T3 cells after cultured for 24 h with different polymer concentration. NIH/3T3 cells incubated without micelles were used as the control.

pH dependence. The acid-cleavable acetal linkages will undergo hydrolysis under acidic condition and induce the departure of MPEG chains and dissociation of micelles, resulting in the release of drug. 3.7. Cellular uptake of free DOX and DOX-loaded micelles Flow cytometry analysis was performed to evaluate and compare cellular uptake process of DOX-loaded H40-star-MPEG micelles and free DOX by a human cervical carcinoma HeLa cell line. HeLa cells were cultured for predetermined time intervals with DOX concentration of 20 mg/mL. Benefiting from the fluorescence of DOX itself, no extra fluorescent probe needs to be conjugated. Histograms of cell-associated DOX fluorescence of HeLa cells are shown in Fig. 8. Cells with free DOX treatment were served as a negative control. Fig. 8(A) shows that the free DOX can be internalized by HeLa cells slowly, while Fig. 8(B) and 8(C) shows that the intracellular internalization of DOX becomes much faster for DOXloaded H40-star-MPEG groups. The fast intracellular internalization of DOX-loaded micelles compared with the free DOX might be

Fig. 7. Cumulative release curves of DOX from DOX-load H40-star-MPEG-2 under different pH values at 37  C: pH ¼ 7.4 and pH ¼ 5.3 PBS buffer solutions respectively.

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Fig. 8. Flow cytometry histogram profiles of HeLa cells incubated with (A) free DOX, (B) DOX-loaded H40-star-MPEG-2 and (C) DOX-loaded H40-star-MPEG-3 for different time intervals.

related to the endocytosis mechanism of nanoparticles. It should be pointed out that the detected fluorescence by flow cytometry might come from surface-interacting or intracellular DOX. Thus, CLSM was carried out to monitor the intracellular distribution of drug. HeLa cells were incubated with free DOX and DOX-loaded H40star-MPEGs at 37  C for 15 min, 0.5 h and 2 h, respectively, and then fixed with paraformaldehyde. After that, the treated samples were observed directly with CLSM in the fluorescent mode. Fig. 9 gives the DOX fluorescence in cytoplasm after 15 min cell incubation with free DOX. After 0.5 h, the DOX fluorescence can be observed in both cytoplasm and nucleus. As the time goes on, DOX remains in nucleus. For cells incubated with DOX-loaded H40-star-MPEG micelles, a similar cellular uptake behavior with free DOX is observed at the beginning 15 min. The DOX fluorescence remains in

the cytoplasm after 0.5 h incubation and then gradually diffuses to the nucleus after 2 h. The cellular uptake of DOX-loaded H40-starMPEG-3 (S3 in Fig. 9) is more efficient than that of H40-star-MPEG2 (S2 in Fig. 9), which can be explained by the size effect of small micelles. 3.8. Anticancer effect assay The antitumor effect of free DOX and DOX-loaded H40-starMPEG micelles against cultured HeLa cells was evaluated by using MTT assay. Here, free DOX was used as a control. The HeLa cells were treated with drug-loaded H40-star-MPEGs and free drug at different DOX dose from 0.5 to 8 mg/mL for 48 h. The cytotoxicity of free DOX and DOX-loaded H40-star-MPEG micelles against cultured

Fig. 9. CLSM images of HeLa cells incubated with DOX, DOX-loaded H40-star-MPEG-2 (S2) and DOX-loaded H40-star-MPEG-3 (S3) for different time intervals. Free DOX was used as the control. The cell nuclei were stained with HOECHST 33342. (scale bar: 40 mm)

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References

Fig. 10. Cells viability of HeLa cells incubated with free DOX and DOX-loaded H40-starMPEG micelles for 48 h.

HeLa cells is shown in Fig. 10. The results show that the viability of HeLa cells depends on DOX concentration. Similar to free DOX, the DOX-loaded micelles perform potent effect of inhibition to HeLa cancer cell proliferation. The inhibition of cancer cell growth is attributed to the intracellular DOX released from the H40-starMPEG micelles and final entry into the nuclei of HeLa cells. Therefore, H40-star-MPEG micelles can be used as efficient anticancer drug delivery carriers and applied in chemotherapy. 4. Conclusion A PEG-sheddable drug delivery system was readily constructed by facile PEGylation of aliphatic polyester H40 via pH-sensitive acetal linkages, which was stable under physiologic neutral condition and became fast hydrolysis in an acidic environment such as endosome (pH < 6.0). The degradation rate in acidic medium was much faster than that in neutral solution, which was confirmed by NMR analysis. The H40-star-MPEG samples with different amount of MPEG arms exhibited different self-assembly behavior. With the increase of MPEG arms, the self-assembled H40-star-MPEGs micelles became smaller and smaller. In vitro cytotoxicity of H40-star-MPEGs was low. Furthermore, we used these coreeshell amphiphilic micelles to encapsulate hydrophobic anticancer drug DOX. The DOX-loaded micelles could be internalized by HeLa cancer cells efficiently and the drug could be released in cytoplasm, exhibiting good anticancer efficiency. As demonstrated in this work, we developed a simple and effective PEGylation method for expanding the use of dendritic polyester BoltornÒ H40 for the biomedical applications. Acknowledgment This work is sponsored by the National Basic Research Program (2012CB821500, 2013CB834506), National Natural Science Foundation of China (20974062), Shanghai Rising-Star Program (11QH1401500) and China National Funds for Distinguished Young Scientists (21025417).

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