Hyperbranched Polyphosphates for Drug Delivery Application: Design

Apr 5, 2010 - The chlorambucil dose of the conjugate and free drug required for 50% cellular growth inhibition were 75 and 50 μg/mL, respectively, ...
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Hyperbranched Polyphosphates for Drug Delivery Application: Design, Synthesis, and In Vitro Evaluation Jinyao Liu, Wei Huang,* Yan Pang, Xinyuan Zhu, Yongfeng Zhou, and Deyue Yan* School of Chemistry and Chemical Engineering, State Key Laboratory of Metal Matrix Composites, Shanghai Jiao Tong University, 800 Dongchuan Road, Shanghai, 200240, P. R. China Received February 20, 2010; Revised Manuscript Received March 20, 2010

A water-soluble hyperbranched polyphosphate (HPHEEP) was synthesized through the self-condensation ringopening polymerization (SCROP) of 2-(2-hydroxyethoxy)ethoxy-2-oxo-1,3,2-dioxaphospholane (HEEP), and its suitability as a drug carrier was then evaluated in vitro. Methyl tetrazolium (MTT) and live/dead staining assays indicated that HPHEEP had excellent biocompatibility against COS-7 cells. The good biodegradability of HPHEEP was observed by NMR analysis, and the degradation products were nontoxic to COS-7 cells. Flow cytometry and confocal laser scanning microscopy analyses suggested that HPHEEP could be easily internalized by vivid cells and preferentially accumulated in the perinuclear region. Furthermore, a hydrophobic anticancer drug, chlorambucil, was used as a model drug and covalently bound to HPHEEP. The chlorambucil dose of the conjugate and free drug required for 50% cellular growth inhibition were 75 and 50 µg/mL, respectively, according to MTT assay against an MCF-7 breast cancer cell line in vitro. This high activity of the conjugate may be attributed to the biodegradability of HPHEEP so as to release the chlorambucil in cells. Therefore, on the basis of its biocompatibility and biodegradability, HPHEEP could provide a charming opportunity to design some excellent drug delivery systems for therapeutic applications.

Introduction Owing to the poor selectivity of current toxic drugs in chemotherapy, many different strategies have been developed to improve their therapeutic efficacy and selectivity.1 In the past few decades, various polymers have been widely used as carriers to bind drug molecules covalently. Differing from the small drugs easily diffusing into and out of the cell, the polymer-drug conjugates cross the cellular membrane by endocytosis, which is a highly selective uptake of drugs.2-9 Meanwhile, drugs being conjugated to polymers can also improve their water solubility properties, decrease their toxicity, and protect them from possible enzymatic degradation or hydrolysis. To date, a great number of linear polymer-drug conjugates have been investigated for intracellular drug delivery.10 Recently, dendritic polymers including hyperbranched polymers and dendrimers have gained widespread attention for drug conjugate because they can carry multiple copies of drug and/ or targeting/imaging ligands.11 Dendritic polymers are good candidates for drug carriers because of their intrinsic properties, such as branching architecture, 3D globular architecture, a large number of terminal functional groups, lower viscosity and better solubility.12-17 However, dendritic systems often have problems with cytotoxicity or inherent nondegradability retarding the drug release from the conjugates.18-23 Therefore, biodegradable hyperbranched polymers with good biocompatibility have been receiving more and more attention in the application of drug delivery systems. As an important class of eminent biomaterials, polyphosphates have good biocompatibility, biodegradability, and the structural similarity to naturally occurring nucleic and teichoic acids.24-33 They can be degraded naturally into harmless low-molecularweight products through hydrolysis or enzymatic digestion of * Corresponding authors. E-mail: [email protected] (W.H.); dyyan@ sjtu.edu.cn (D.Y.).

phosphate linkages under physiological conditions.34 In recent years, some linear random and block copolymers with phosphate units have been synthesized and applied to drug and gene delivery.35-37 Some biocompatible and biodegradable hydrogels based on polyphosphates have also been produced for cell encapsulations.38-41 A water-soluble hyperbranched polyphosphate, which integrates the advantages of hyperbranched polymers and polyphosphates together, has been reported in our recent communication.42 Because of the large number of surface functional hydroxyl groups, it could be used as a carrier for intracellular drug delivery. In this article, both biocompatibility and biodegradability of HPHEEP were investigated, and then the cellular uptake, the drug conjugate, and the corresponding activity analyses were also discussed.

Experimental Section Materials. Diethylene glycol (Acros, 99%) was purified by distillation under reduced pressure just before use. Tetrahydrofuran (THF) was dried by refluxing with the fresh sodium-benzophenone complex under N2 and distilled just before use. Triethylamine (TEA) was refluxed with phthalic anhydride, potassium hydroxide, calcium hydride in turn and distilled just before use. N,N-dimethylformamide (DMF) was dried over calcium hydride and then purified by vacuum distillation. Acridine orange (AO), ethidium bromide (EB), N-hydroxysuccinamide (NHS), chlorambucil, N,N-dicyclohexylcarbodiimide (DCC), 4-dimethylaminopyridine (DMAP), and 3-(4,5-dimethyl-thiazol-2-yl)-2,5-diphenyl tetrazolium bromide (MTT) were purchased from Sigma and used as received. Clear polystyrene tissue-culture-treated 12-well and 96-well plates were obtained from Corning Costar. HPHEEP was synthesized j n ) 4250 g/mol, PDI ) 2.48).42 All other according to the literature (M reagents and solvents were purchased from the domestic suppliers and used as received. Characterizations. 1H, 13C, and 31P nuclear magnetic resonance (NMR) analyses were recorded on a Varian Mercury Plus 400 MHz

10.1021/bm100188h  2010 American Chemical Society Published on Web 04/05/2010

Hyperbranched Polyphosphates for Drug Delivery spectrometer with deuterium oxide (D2O) and deuterated dimethyl sulfoxide (d6-DMSO) as solvents. Gel permeation chromatography (GPC) measurements were performed on a Perkin-Elmer series 200 system (10 µm PL gel 300 × 7.5 mm mixed-B and mixed-C column, linear polyglycol calibration) equipped with a refractive index (RI) detector. NaNO3 aqueous solution (0.05 M) was used as the mobile phase at a flow rate of 0.6 mL/min at 40 °C. Synthesis of HPHEEP-chlorambucil Conjugate. Under a nitrogen atmosphere, chlorambucil (113 mg, 0.370 mmol), HPHEEP (300 mg, 0.0706 mmol), DCC (84.1 mg, 0.407 mmol), and DMAP (4.53 mg, 0.0370 mmol) were added to a 50 mL flask with 20 mL of dry DMF and stirred at room temperature for 48 h. Then, the reaction mixture was filtered to remove dicyclohexylurea, and the DMF in filtrate was removed by the vacuum distillation. The crude product was purified by dissolving in methanol and precipitated in diethyl ether repeatedly. The pure HPHEEP-chlorambucil conjugate was obtained after drying in vacuo at room temperature for 24 h. Synthesis of RB-Labeled HPHEEP. NHS (85.2 mg, 0.741 mmol) and rhodamine B (RB) (355 mg, 0.741 mmol) were dissolved in 20 mL of dry DMF. Then, DCC (153 mg, 0.741 mmol) and DMAP (90.5 mg, 0.741 mmol) were also added to the mixture and stirred at 50 °C for 12 h. Afterward, HPHEEP (300 mg, 0.0706 mmol) was dissolved in 10 mL of dry DMF and added to the above mixture. Then, the mixture was conducted at room temperature for another 60 h. Some insoluble substance in the mixture was filtered, and the DMF in filtrate was removed by the rotary evaporator. The crude product was dissolved in distilled water, washed with chloroform repeatedly, and dialyzed in distilled water to remove the free RB. The pure RB-labeled HPHEEP was obtained. In Vitro Degradation of HPHEEP. In vitro degradation of HPHEEP was performed at 37 °C in the different phosphate buffered saline (PBS) solutions (pH 10.5, 7.4, and 2.5). The concentrations of HPHEEP were all set at 10 mg/mL. At the predetermined intervals, some samples were taken out, freeze-dried, and analyzed by 1H and 31 P NMR in D2O. Cell Culture. COS-7 (a cell line derived from kidney cells of the African green monkey) and MCF-7 (a human breast adenocarcinoma line) were cultivated in DMEM (Dulbecco’s modified Eagle’s medium) containing 10% FBS (fetal bovine serum) and antibiotics (50 units/ mL penicillin and 50 units/mL streptomycin) at 37 °C under a humidified atmosphere containing 5% CO2. Cytotoxicity Measurements. The relative cytotoxicity of HPHEEP was estimated by MTT viability assay and AO/EB double-staining methods against COS-7 cells. MTT assay: COS-7 cells were seeded into 96-well plates at 8 × 103 cells per well in 200 µL of medium. After 24 h incubation, the culture medium was removed and replaced with 200 µL of medium containing serial dilutions of HPHEEP. The cells were grown for another 24 h. Then, 20 µL of 5 mg/mL MTT assays stock solution in PBS was added to each well. After incubating the cells for 4 h, the medium containing unreacted MTT was carefully removed. The obtained blue formazan crystals were dissolved in 200 µL/well dimethyl sulfoxide (DMSO), and the absorbance was measured in a Perkin-Elmer 1420 Multilabel counter at a wavelength of 490 nm. The MTT assay of the hydrolytic degradation products of HPHEEP was used with the same method as that described above. AO/EB Double Staining. DNA-binding dyes AO and EB were used to detect the morphology of apoptotic and necrotic cells.43 COS-7 cells were seeded in six-well plates at 5 × 105 cells per well in 1 mL of complete DMEM and cultured for 24 h, followed by removing culture medium and adding 1 mL HPHEEP solutions (in DMEM medium) with the concentration of 10 mg/mL. After 24 h of incubation, cells were rinsed by PBS twice and incubated in PBS containing AO (5 µg/mL) and EB (5 µg/mL) at 37 °C in 5% CO2 for 10 min. Live and dead cells were imaged by a Leica DM 4500B fluorescence microscope. Cellular Uptake of HPHEEP. The cellular uptake experiments were performed on flow cytometry and confocal laser scanning microscopy (CLSM). For flow cytometry, COS-7 cells were seeded in six-well

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Scheme 1. Synthesis Route of HPHEEP-Chlorambucil/RB Conjugates

plates at 5 × 105 cells per well in 1 mL of complete DMEM and cultured for 24 h, followed by removing culture medium and adding RB-labeled HPHEEP solutions (1 mL of DMEM medium) at the concentration of 1 mg/mL for predetermined time intervals. Thereafter, culture medium was removed, and cells were washed with PBS three times and treated with trypsin. Subsequently, 2 mL of PBS was added to each culture well, and the solutions were centrifugated for 5 min (1000 rpm). After the supernates were removed, the cells were resuspended in 0.5 mL of PBS. Data for 1 × 104 gated events were collected, and analysis was performed by means of a BD FACSCalibur flow cytometer and CELLQuest software. CLSM Studies. COS-7 cells were seeded in six-well plates at 2 × 105 cells per well in 1 mL of complete DMEM and cultured for 24 h, followed by removing culture medium and adding RB-labeled HPHEEP solutions (1 mL DMEM medium) at the concentration of 1 mg/mL. After incubation at 37 °C for the predetermined intervals, culture medium was removed, and cells were washed with PBS three times. Then, the cells were fixed with 4% formaldehyde for 30 min at room temperature, and the slides were rinsed with PBS three times. Finally, the cells were stained with 2-(4-amidinophenyl)-6-indolecarbamidine dihydrochloride (DAPI) for 10 min, and the slides were rinsed with PBS for three times. The slides were mounted and observed by a LSM 510META. Cell Proliferation Assay. The cytotoxicity of HPHEEP-chlorambucil conjugate against MCF-7 cells was evaluated in vitro by MTT assay using chlorambucil dissolved in DMSO as the control. (The final concentration of DMSO in medium was 0.5% v/v.) MCF-7 cells were seeded into 96-well plates at 8 × 103 cells per well in 200 µL medium. After 24 h of incubation, the culture medium was removed and replaced with200µLofmediumcontainingserialdilutionsofHPHEEP-chlorambucil conjugate. The cells were grown for another 96 h. Then, 20 µL of 5 mg/mL MTT assays stock solution in PBS was added to each well. After the cells were incubated for 4 h, the medium containing unreacted MTT was removed carefully. The obtained blue formazan crystals were dissolved in 200 µL/well DMSO, and the absorbance was measured in a Perkin-Elmer 1420 Multilabel counter at a wavelength of 490 nm.

Results and Discussion Synthesis and Characterization of HPHEEP-Chlorambucil/ RB Conjugates. The synthetic route of HPHEEP-drug conjugates is shown in Scheme 1. First, the HPHEEP was synthesized through the SCROP of HEEP according to our previous report42 with a degree of branching of 0.48 and about 20 terminal

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Figure 2. Cell viability of COS-7 cells against HPHEEP after cultured for 24 h with different polymer concentrations determined by MTT viability assay.

Figure 1. 1H NMR spectra of (A) HPHEEP, (B) chlorambucil, and (C) HPHEEP-chlorambucil conjugate in d6-DMSO.

hydroxyl groups determined by NMR and GPC measurements. (See the Supporting Information.) Then, in the presence of DCC and DMAP, the carboxyls of chlorambucil can be condensed withthehydroxylofHPHEEPtoproducetheHPHEEP-chlorambucil conjugate. Here the condensation reaction was conducted at room temperature in dry DMF for 48 h under nitrogen. The unreacted chlorambucil was removed by dissolving in methanol and precipitating in diethyl ether repeatedly. The resonance at 2.17 ppm assigned to the proton (-CH2CH2COOH) of free chlorambucil disappeared in the 1H NMR spectrum of HPHEEP-chlorambucil conjugate (Figure 1). This indicates the free drug has been removed from the conjugate completely. In addition, the drug content of the resulting conjugate was also calculated according to the 1H NMR spectrum and the graft ratio of chlorambucil/hydroxyl is 64.2%. To study the cellular uptake of HPHEEP, RB was used as a fluorescent probe. We obtained the RB-labeled HPHEEP by using NHS and DCC as activating and coupling agents, respectively. The free RB was removed by dissolving the crude product in distilled water, washed with chloroform repeatedly, and dialyzed in distilled water. 1H NMR analysis reveals that the RB molecules are covalently bound to HPHEEP (1H NMR not shown). In Vitro Cytotoxicity. It is very important to evaluate the potential toxicity of polymeric materials for drug delivery applications. As a class of eminent biomaterials, linear polyphosphates have been extensively investigated in drug delivery systems and shown nontoxicity to various cultural cell lines.36,41 Here the in vitro cytotoxicity of HPHEEP against COS-7 cells was studied using 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. Therefore, the number of surviving cells is directly proportional to the level of formed formazan.44,45 As shown in Figure 2, the cell viability

Figure 3. Fluorescence images of COS-7 cells after incubation (A) without or (B) with 10 mg/mL of the HPHEEP for 24 h. (The cells were stained by AO and EB.)

after 24 h of incubation with HPHEEP up to 10 mg/mL remains nearly 100% compared with the untreated cells. Apparently, this hyperbranched polyphosphate has good biocompatibility to COS-7 cells. Furthermore, the AO/EB double staining results also demonstrate the good biocompatibility of HPHEEP. Generally speaking, healthy cells have green nuclei and uniform chromatin with intact cell membrane. On the contrary, the cells undergoing apoptosis have orange nuclei and condensed chromatin with nuclear shrinkage. What is more, the cells in necrosis or in the late stage of apoptosis have red nuclei with damaged cell membrane.43,46 As shown in Figure 3, the fluorescence of COS-7 cells after incubated with HPHEEP of 10 mg/mL for 24 h is

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almost the same as that of the untreated cells. This result indicates that the cells are viable. In Vitro Degradation. The most important advantage of linear polyphosphates over conventional biocompatible polymers (e.g., PEG) is the biodegradation of them. In addition, their degradation rate could be adjusted by controlling the chemical structure of the backbone and side chain in polyphosphates. For example, Leong and Mao reported that the polyphosphate with the pendant amino groups degraded rapidly in aqueous solution.47 Moreover, the degradation products could have minimal toxic effects and good biocompatibility by choosing the biocompatible building units of polyphosphate. The in vitro degradation behavior of HPHEEP was evaluated in PBS at 37 °C under neutrality (pH 7.4), acidity (pH 2.5), and basicity (pH 10.5) conditions, respectively. The degradation products were lyophilized for NMR analysis in D2O. The 1H NMR spectra of the original HPHEEP and the degradation products of HPHEEP at different time are shown in Figure 4A. With the prolongation of degradation time, the signals assigned to the protons of HPHEEP decline gradually, whereas some new signals appear and escalate, which can be assigned to the protons of the degradation products. Similar results are also observed in their 31 P NMR spectra in Figure 4B. In addition, the content of phosphorus in the residual HPHEEP at the different degradation time is used to describe the degradation profiles of HPHEEP under neutrality (pH 7.4), acidity (pH 2.5), and basicity (pH 10.5) conditions, respectively, and the results are shown in Figure 4C. It is obvious that the degradation of HPHEEP is accelerated under basic or acidic environments. In particular, in the acidic solution, the degradation rate of HPHEEP reaches the maximum. This indicates that the hydrolysis of the phosphate bond in HPHEEP could be catalyzed by base or acid.48 The cytotoxicity of the degradation products is a very important parameter to evaluate the biological safety of materials.49 For example, poly(lactide) and poly(lactide-co-glycolide) have the satisfactory biocompatibility, but their high concentrated degradation products have a toxic influence according to the previous report.50 Here the cytotoxicity of the degradation products of HPHEEP, which was hydrolyzed under neutrality condition (pH 7.4) over 35 days, was determined by MTT viability assay against COS-7 cells. The cell viability after 24 h of incubation with the degradation products at different concentration is shown in Figure 5. Compared with the untreated cells, the cell viability nearly remains 100% after 24 h of incubation with the concentration of degradation products up to 10 mg/mL. This result confirms that the degradation products of HPHEEP also have good biocompatibility to COS-7 cells. Cell Internalization. The cellular uptake of HPHEEP by COS-7 cells was analyzed by flow cytometry. COS-7 cells were cultured for predetermined time intervals with the RB-labeled HPHEEP solution (1 mg/mL) before analysis. The cellular uptake of HPHEEP is expressed as an increase in the fluorescent cell number, and the uptake profile is shown in Figure 6. The fluorescence intensity of cells is increased with the incubation time. After 1 h of incubation, the cellular uptake of HPHEEP by COS-7 reaches a maximum value. According to Rejman’s report, who examined the internalization rate of the fluorescent latex beads by murine melanoma cells, smaller objects should be taken up faster.51 Therefore, we speculate that this efficiency of intracellular uptake can be attributed to the low molecular weight as well as the small hydrodynamic volume of HPHEEP. Moreover, the cellular uptake of HPHEEP by COS-7 cells was also evaluated by CLSM. COS-7 cells were incubated with HPHEEP (1 mg/mL) at 37 °C for the predetermined time

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Figure 4. NMR spectra of HPHEEP in D2O at different degradation times cultured at 37 °C under neutrality conditions (pH 7.4): (A) 1H and (B) 31P spectra. (C) Phosphorus content of HPHEEP as a function of degradation time at neutral (pH 7.4), acidic (pH 2.5), and basic (pH 10.5) conditions.

intervals. As a control, COS-7 cells were also incubated with free RB. The RB fluorescence of the cells was directly observed under CLSM. As shown in Figure 7, the RB fluorescence in all cells pretreated by RB-labeled HPHEEP is accumulated preferentially in the perinuclear region instead of the nucleus (Figure 7A-O). Furthermore, the fluorescence remains in cytoplasm when the incubation time is prolonged. However, the fluorescence is mostly localized in the cell nucleus when cells are pretreated by free RB (Figure 7P-R). These results indicate thatHPHEEPmaybetransportedbytheendocytosismechanism.52,53 Unlike the simple passive diffusion between the extracellular and intracellular milieu of small molecules, the cellular uptake of HPHEEP by the endocytosis processes is unidirectional.54

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Figure 5. Cytotoxicity of the degradation products of HPHEEP to COS-7 cells by MTT viability assay.

Figure 6. Cellular uptake of the RB-labeled HPHEEP by COS-7 cells versus the time by flow cytometry analysis.

Therefore, HPHEEP is not only transported efficiently but also retained at the site of cytoplasm. These characteristics of HPHEEP are very important for its use as a carrier for intracellular drug delivery. Activity Analysis of HPHEEP-Chlorambucil Conjugate. On the basis of the excellent biocompatibility, good biodegradability, and easy cellular uptake of HPHEEP, we further evaluated its potential as an intracellular drug carrier. Chlorambucil, a hydrophobic anticancer drug, was used as a model drug. The HPHEEP-chlorambucil conjugate was prepared by coupling the end hydroxyl groups of HPHEEP with the carboxyl group of chlorambucil in the presence of DCC and DMAP. To investigatethepotentialtherapeuticeffectofHPHEEP-chlorambucil conjugate, in vitro evaluation was performed by MTT assay against an MCF-7 breast cancer cell line. The cytotoxic activity of HPHEEP-chlorambucil conjugate was compared with that of free chlorambucil. The results of MCF-7 cell proliferation are shown in Figure 8. The dose of the conjugated chlorambucil required for 50% cellular growth inhibition (IC50) is 75 µg/ mL. This indicates that the conjugate is able to enter the cell and produce the desired pharmacological action. The IC50 of the free chlorambucil is 50 µg/mL. Compared with the IC50 of the free drug, the potency of HPHEEP-chlorambucil is very

Figure 7. CLSM images of COS-7 cells incubated with RB-labeled (A-O) HPHEEP and (P-R) RB at the different incubation times. (The nucleus was stained with DAPI.)

attractive because polymer-drug conjugates often show significantly less activity than that of the free drug in cells.55 The antiproliferation effect of the conjugate is mostly attributed to the release of drug from the HPHEEP-chlorambucil conjugates in the course of incubation due to the good biodegradability of HPHEEP. Lysosomes in cells are rich in enzymes, which conduce to release free drugs from polymer-drug conjugates.56 Moreover, the release process is also influenced by many other factors including the stability of chemical bonds, the hydrophilicity of surrounding mediums, the type of spacer, and the steric hindrance at the center of reaction.57 Once the HPHEEP-chlorambucil conjugate is endocytosed into the cells, the acidity conditions and the enzymes will accelerate the hydrolysis of phosphate units in HPHEEP and result in the rapid release of free chorambucil correspondingly. The released drugs

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References and Notes

Figure 8. Activity of HPHEEP-chlorambucil conjugate against MCF-7 cells.

eventually reach the nucleus depending on the concentration gradient, which provides the therapeutic effect.58 Further studies are underway to investigate the stability of the polymer-drug conjugate at various pHs and in the presence of enzymes and to understand the intracellular hydrolysis of the conjugate as well as the efficacy in vivo.

Conclusions A water-soluble hyperbranched polyphosphate with a great number of terminal hydroxyl groups was synthesized by the SCROP of HEEP. In vitro MTT and AO/EB staining assays indicated that HPHEEP possessed the excellent biocompatibility against COS-7 cells. The NMR analyses proved that HPHEEP was hydrolytically degradable and the degradation process was accelerated under basic and acidic environments, especially in acidic environments. Furthermore, the MTT assay demonstrated that the degradation products were nontoxic to COS-7 cells. Flow cytometry and CLSM analyses suggested that HPHEEP could be easily internalized by the vivid cells through the endocytosis mechanism and preferentially accumulated in the perinuclear region instead of the nucleus. To evaluate the potential of HPHEEP as carrier for intracellular drug delivery, the HPHEEP-chlorambucil conjugate was synthesized. The IC50 value of the conjugated chlorambucil was found to be 75 µg/mL using methyl tetrazolium assays against an MCF-7 breast cancer cell line in vitro, which was only slightly higher than that of the free chlorambucil (50 µg/mL). The significant activity of the conjugate can be attributed to the biodegradability of HPHEEP, which releases free chlorambucil in cells. The HPHEEP-drug conjugate could be further functionalized by a targeting moiety to deliver the drugs to specific cells in vivo. These results indicate that HPHEEP is an excellent candidate for drug delivery systems and therapeutic applications. Acknowledgment. We gratefully acknowledge the financial supports provided by the National Basic Research Program (2007CB808000, 2009CB930400), National Natural Science Foundation of China (no. 50873058, no.50633010), and Shanghai Leading Academic Discipline Project (no. B202). Supporting Information Available. Synthesis and characterization of HPHEEP are given. This material is available free of charge via the Internet at http://pubs.acs.org.

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