Alendronate-Conjugated Amphiphilic Hyperbranched Polymer Based

Sep 4, 2012 - DOX-loaded micelles were prepared via the encapsulation of hydrophobic DOX into the amphiphilic H40-star-PEG/ALE. The details were as ...
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Alendronate-Conjugated Amphiphilic Hyperbranched Polymer Based on Boltorn H40 and Poly(ethylene glycol) for Bone-Targeted Drug Delivery Hongying Chen,†,# Guolin Li,†,# Huirong Chi,† Dali Wang,‡ Chunlai Tu,‡ Lijie Pan,§ Lijuan Zhu,‡ Feng Qiu,‡ Fulin Guo,*,† and Xinyuan Zhu*,‡ †

Department of Oral and Maxillofacial Surgery and §Department of Preventive Oral Health, The First Affiliated Hospital of Harbin Medical University, 23 Youzheng Street, Nangang District, Harbin 150001, People’s Republic of China ‡ 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 S Supporting Information *

ABSTRACT: A novel type of alendronate(ALE)-conjugated amphiphilic hyperbranched copolymer based on a hydrophobic hyperbranched Boltorn H40 (H40) core with ALE targeting moiety and many hydrophilic poly(ethylene glycol) (PEG) arms was synthesized as a carrier for bone-targeted drug delivery. The star copolymer H40-star-PEG/ALE was characterized using nuclear magnetic resonance (NMR), Fourier transformed infrared spectroscopy (FTIR), and gel permeation chromatography (GPC) analysis. Benefiting from its highly branched structure, H40-star-PEG/ALE could form micelles in aqueous solution, which was confirmed by transmission electron microscopy (TEM) and dynamic light scattering (DLS) techniques. The cytotoxicity and hemolysis of the H40-star-PEG/ALE micelles were evaluated via methylthiazoletetrazolium (MTT) assay against NIH/3T3 normal cells and red blood cell (RBC) lysis assay, respectively. As a model anticancer drug, doxorubicin (DOX) was encapsulated into the H40-star-PEG/ALE micelles. The anticancer activity of DOX-loaded micelles was evaluated by MTT assay against an HN-6 human head and neck carcinoma cell line. The strong affinity of H40-star-PEG/ALE micelles to bone was confirmed by the hydroxyapatite (HA) binding assay. These results indicate that the H40-star-PEG/ALE micelles are highly promising bonetargeted drug carriers for skeletal metastases.

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the enhanced permeability and retention (ERP) effect.1,9−11 In the literature, it is also frequently mentioned as passive targeting. However, the drug dose at carcinoma sites is usually low by the passive targeting, resulting in the poor efficacy for tumor inhibition.12 To further improve the specific targeting and delivery efficiency, active targeting ability of the drug delivery systems is required. Active targeting can be achieved via functionalization of the micelle carriers with targeting ligands such as antibodies, peptides, and small molecules, which can recognize and bind to specific receptors that are unique to carcinoma cells or pathological tissues. 13,14 The drug bioavailability in the body is obviously enhanced and the adverse effects of chemotherapy drugs to normal tissues are enormously reduced.1−5,15,16 It is well-known that bisphosphonates exhibit a high affinity for the bone mineral HA.17,18 For the metastasized and/or invaded bone, the hydroxyapatite exposed in tumor surroundings can be used as the specific receptor for those bisphosphonate-functionalized drug carriers.

INTRODUCTION The skeleton is invariably metastasized or invaded by the malignancy at advanced stages.1,2 In particular, the primary carcinomas of the breast, lung, kidney, and oral cavity have the powerful capacity of metastasis and/or invasion to the bone.1−3 The bone metastasis or invasion is responsible for the disability (pathological fracture, hypercalcemia) and mortality.1−4 During the past decades, the clinical therapies focus mainly on the surgical resection and the aggressive chemotherapy and radiotherapy.5,6 For chemotherapy, the chemotherapeutic agents can inhibit the progression of cancers efficiently, but it is difficult to deliver drugs to the skeletal lesion sites because of the blood-bone barrier.1,7 Futhermore, drugs themselves lack a desired selectivity to target the tumor tissue, leading to severe side effects including the toxicity of cardiac tissues and the inhibition of bone marrow.1,8 Recently, polymeric micelles formed by self-assembly of amphiphilic copolymers in aqueous solution have attracted tremendous attention as a drug delivery vehicle. These micelles can accumulate into tumor tissue because of the abnormal interstices of the vascular endothelium and the absence of lymphatic drainage system of some tumors, which is defined as © 2012 American Chemical Society

Received: June 12, 2012 Revised: August 20, 2012 Published: September 4, 2012 1915

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freeze−drying way after dialysis against deionized water using the membranous tube with the molecular weight cutoff 3.5 kDa for 48 h. Synthesis of H40-CO-NHS. Briefly, 3.3 g (0.2 mmol) H40COOH was dissolved in 20 mL anhydrous CH2Cl2. Then, 3.0 g (1.8 mL) sulfoxide chloride (SOCl2) was to the 100 mL flask in an ice− water bath with stirring for 3 h, and the reaction was run in room temperature for another 24 h. The reaction solution was quickly evaporated under vacuum, and then 1.19 g (10.3 mmol) Nhydroxysuccinamide (NHS) with 20 mL fresh anhydrous CH2Cl2 was added to the flask in an ice−water bath with stirring for 24 h. Subsequently, the resulting mixture was poured into water, extracted with CH2Cl2, and dried by anhydrous MgSO4. The solvent was concentrated by evaporation. At last, the concentrated solution was precipitated by cold diethyl ether, and product was dried under vacuum to a constant weight. Synthesis of H40-star-PEG. Briefly, 0.9 g (0.045 mmol) H40-CONHS was dissolved in 20 mL anhydrous dioxane under moderate stirring at the room temperature. After completely dissolved, 2.88 g (2.88 mmol) PEG-NH2 was quickly added to the flask under the same reaction conditions for 24 h. The crude product formed was precipitated with the cold diethyl ether and dried under the vacuum. Synthesis of H40-star-PEG/ALE. Briefly, 0.5 g (0.012 mmol) H40-star-PEG was dissolved in 20 mL dioxane under moderate stirring at the room temperature. After completely dissolved, 0.123 g (0.378 mmol) ALE was quickly added into the flask. Then, 30.7 mg (0.77 mmol) NaOH was slowly added into the flask to regulate pH = 8.0 under the identical reaction condition for 24 h. The formed crude product was precipitated with dialysis against deionized water using the membranous tube with the molecular weight cutoff 3.5 kDa. Preparation of H40-star-PEG/ALE Micelles and Their CMC. Typically, a total of 20 mg H40-star-PEG/ALE copolymer was dissolved in 2 mL DMF and stirred at room temperature for 3 h. Then, the 6 mL deionized water was slowly added into the copolymer solution under stirring for 2 h, and the solution was transferred into dialysis tubing (MWCO = 3.5 kDa) and dialyzed against the deionized water for 24 h. During the process, the water was renewed at appropriate intervals. To study the CMC of H40-star-PEG/ALE copolymer, DPH was employed as a UV−vis probe to monitor the absorbance at 313 nm. The H40-star-PEG/ALE copolymer solution with series of concentrations (from 5 × 10−6 to 0.5 mg/mL) was prepared, and then methanol solution of DPH was added. The DPH concentration was kept at a constant of 5.0 × 10−6 mol/L. The 313 nm wavelength absorbance of all solutions was recorded on Perkin-Elmer Lambda 20/2.0 UV−vis spectrophotometer.24 Characterization. 1H NMR, 13C NMR, and 31P NMR spectra were recorded on Bruker AVANCEIII 400 spectrometer with DMSOd6, D2O, and CDCl3 as the solvents at 25 °C. Molecular weights and polydispersity index (PDI) of the synthesized samples were measured by GPC. 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 poly(ethylene glycol) 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. FTIR spectra were recorded on a Paragon 1000 instrument by KBr sample holder method. DLS measurements were recorded on a Malvern Zetasizer Nano S device equipped with a 4.0 mW laser operating at λ = 633 nm. All samples of 0.5 mg/mL were tested with a scattering angle of 173° and at 37 °C. TEM studies were performed with a JEOL JEM-100CX-II instrument operated at 200 kV to observe the shape and size of the micelles. Samples were prepared by directly dropping the solution onto carbon-coated copper grids and then airdrying at room temperature overnight before measurement. The UV− vis spectra were measured on a Perkin-Elmer Lambda 20/2.0 UV−vis spectrophotometer. The calibration curve of absorbance measurements against a series of concentration of DOX was measured at 500 nm. The fluorescence spectra were recorded on a QM/TM/RM fluorescence spectrophotometer (Photon Technology International, Inc.) at room temperature. Preparation of DOX-Loaded Micelles. DOX-loaded micelles were prepared via the encapsulation of hydrophobic DOX into the

Miller et al. reported the N-(2-hydroxypropyl) methacrylamide copolymer−paclitaxel−alendronate conjugate as an effective bone-delivery system.2,11,19 Salerno et al. prepared DOX-loaded bone-targeting nanoparticles based on the polymer of poly(lactic-co-glycolide)−alendronate conjugate.1,20 The linear polymers were employed as the backbone of the conjugates in the previous literature.1,2,11,19,20 Comparing to the linear polymers of the same molecular weight and composition, the amphiphilic star-shaped block copolymers exhibit smaller hydrodynamic radius and lower melt and solution viscosities.21 Due to the low critical micelle concentration (CMC) of amphiphilic star-shaped block copolymers, the self-assembled micelles have unique characteristics such as nanosize, high stability, and a core−shell architecture in vivo.21 Thus, it can be imagined that if bisphosphonates are conjugated onto the surface of dendritic micelles, a bone-targeted drug delivery system with high stability might be developed. In this work, a novel bone-targeting micelle with high stability was designed and successfully prepared from H40-starPEG/ALE consisting of an aliphatic biodegradable polyester H40 inner core with alendronate bone-targeting moiety and many hydrophilic linear PEG arms. The biodegradable polyester H40 inner core served as the reservoir for hydrophobic drugs, while the hydrophilic PEG outer shell enhanced the micelle stability and prolonged the circulation time in the bloodstream. In the meantime, the conjugation of bisphosphonates provided strong affinity to the skeleton mineral hydroxyapatite, realizing the targeting of bone tissue. As a model anticancer drug, the hydrophobic DOX was encapsulated into the H40-star-PEG/ALE micelles, and the strong affinity of DOX-loaded H40-star-PEG/ALE micelles to bone tissue was validated by the hydroxyapatite binding assay.

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MATERIALS AND METHODS

Materials. Triethylamine (TEA) and methylene dichloride (CH2Cl2) were dried over calcium hydride for 48 h and then distilled before used. Tetrahydrofuran (THF) was dried by refluxing with the fresh sodium-benzophenone complex (a thick purple color indicating a moisture- and oxygen-free solvent) and distilled just before used. H40 was obtained from Perstorp Polyols AB, Sweden. Aminopoly(ethylene glycol) monomethyl ether (PEG-NH2) with Mn = 1 kDa was purchased from Jiaxing Biomatrix and Biotechnology limited company and used as received. ALE and 1,6-diphenyl-1,3,5-hexatriene (DPH) were used as received without further purification. Doxorubicin hydrochloride (DOX·HCl) was purchased from Beijing Huafeng United Technology Corporation and used as received. Clear polystyrene tissue culture treated 6-well, 12-well, and 96-well plates were obtained from Corning Coastar. Dulbecco’s modified Eagle’s medium (DMEM), fetal bovine serum (FBS), penicillin, and streptomycin were obtained from Biological Industries Ltd. NHydroxysuccinamide (NHS, Acros), polyethylenimine (PEI, water free, Mn = 10 kDa, Aldrich) were used as received. Dialysis tube (MWCO = 3.5 kDa) was purchased from Shanghai Lvniao Technology Corp. Dextran (Mn = 40 kDa) and all other chemical reagents of analytical grade were purchased from Shanghai Chemical Reagent Co. and used as received unless mentioned. Synthesis of H40 with Carboxyl Terminal Group (H40COOH). H40-COOH was prepared by reacting purified H40 with succinic anhydride.22,23 In a typical procedure, 3.0 g (25.6 mmol hydroxyl groups) H40 was dissolved in 75 mL anhydrous THF in a 150 mL flask under moderate stirring for 30 min at room temperature. After completely dissolved, 3.84 g (38.4 mmol) succinic anhydride and 0.75 mL of TEA as the catalyst were quickly added into the flask under the equal reaction condition for 24 h. The crude product formed was precipitated in the cold diethyl ether twice and dried under the vacuum. The purified H40-COOH was obtained by employing the 1916

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Cellular Uptake of DOX-Loaded Micelles by HN-6 Cells. The experiments of intracellular drug release were performed on flow cytometry and confocal laser scanning microscopy (CLSM). Flow Cytometry. HN-6 cells were seeded in 6-well plates at 5 × 105 cells per well in 1.5 mL of complete DMEM and cultured for 24 h. Then, the DOX-loaded H40-star-PEG/ALE micelles dissolved in DMEM culture medium at a final DOX concentration of 5 μg/mL were added to different wells and the cells were incubated at 37 °C for 5 min, 15 min, 0.5 h, and 3 h, respectively. Thereafter, the samples were prepared for flow cytometry analysis by removing the cell growth media, rinsing twice with ice-cold PBS, and treating with trypsin (150 μL). Then, 1 mL of ice-cold PBS was added to each culture well. Data for 1.0 × 104 gated events were collected and analysis was performed by means of a BD FACSCalibur flow cytometer and CELLQuest software. CLSM. Cell uptake was further analyzed by CLSM studies. HN-6 cells were seeded into 6-well plates at 1 × 105 cells per well in 1 mL of complete DMEM and cultured for 24 h, followed by removing the complete medium and incubating with DOX-loaded H40-star-PEG/ ALE micelles (1 mL of DMEM medium) at a final DOX concentration of 5 μg/mL. The cells were incubated at 37 °C for predetermined intervals. Subsequently, the cells were washed twice with ice-cold PBS and then fixed with 4% paraformaldehyde for 30 min at room temperature, and the slides were rinsed with ice-cold PBS for three times. Finally, the cells were stained with Hoechst 33342 for 7 min and the slides were rinsed with PBS three times. The slides were mounted and observed with a LSM 510 META. Activity Analyses. The cytotoxicity of DOX-loaded H40-starPEG/ALE micelles and free DOX against HN-6 cells was evaluated in vitro by MTT assay. HN-6 cells were seeded into 96-well plates with a density 8.0 × 103 cells per well in 200 μL of medium. After 24 h of incubation, the culture medium was removed and replaced with 200 μL of a medium containing serial dilutions of DOX-loaded micelles or free DOX. The cells were grown for another 48 h. Then, 20 μL of 5 mg/mL MTT assay stock solution in PBS was added to each well. After incubating the cells for 4 h, the medium containing unreacted MTT was removed carefully. The obtained blue formazan crystals were dissolved in 200 μL DMSO and the absorbance was measured in BioTek SynergyH4 at a wavelength of 490 nm. HA Binding Assay. First, the DOX-loaded H40-star-PEG/ALE and DOX-loaded H40-star-PEG micelles were counted to contain the equivalent quantity of DOX (1.0 mg). Then, the two samples were dissolved in PBS (pH = 7.4, 5 mg/mL). The solution (4 mL) was incubated with HA powder (270 mg), used to imitate the hard bone tissue, with 9 mL of PBS (pH = 7.4) at 37 °C in a 20 mL flask. As controls, both DOX-loaded H40-star-PEG/ALE and DOX-loaded H40-star-PEG were incubated at the same condition without HA powder. Then, the incubated samples were centrifuged at 5000 rpm for 5 min and a sample from the upper layer (2 mL) was collected after 0, 5, 15, 30, and 60 min, respectively. The UV−vis absorbance at 500 nm wavelength was employed to detect the unbound DOX-loaded micelles of the collected samples. The degree of HA binding (expressed as percent binding) was estimated as mean of three independent experiments according to the following formula:

amphiphilic H40-star-PEG/ALE. The details were as follows: 50 mg H40-star-PEG/ALE was dissolved in 4 mL DMF, followed by adding 5 mg DOX and the theoretical drug loading content was 10 wt %. Then, the mixture was added slowly to 6 mL of deionized water and stirred for another 3 h. Subsequently, the solution was dialyzed against deionized water for 24 h (MWCO = 3.5 kDa), and the deionized water was renewed every 4 h. The DOX-loaded nanoparticle solution was lyophilized and then dissolved in DMF. The DOX concentration was determined by the UV−vis measurements at absorbance wavelength of 500 nm. Drug loading content (DLC) and drug loading efficiency (DLE) were calculated according to the following formula: DLC(wt%) = (weight of loaded drug/weight of polymer) × 100%

DLE(%) = (weight of loaded drug/weight of drug in feed) × 100% In Vitro Drug Release Assay. The drug release assay was carried out in glass bottles at 37 °C in phosphate buffer (pH = 7.4) and acetate buffer (pH = 5.0) solutions. First, 4 mL DOX-loaded micelles were placed in a dialysis tube with a molecular weight cutoff of 3.5 kDa. Then, the dialysis bag was quickly immersed in 50 mL of the release medium in a laboratory shaker keeping the stirring (100 rpm) and a constant temperature (37 °C). At the predetermined intervals, 2 mL sample was withdrawn and the equal volume of fresh medium was replenished. The amount of released DOX was analyzed with the fluorescence measurements at 480 nm of excitation spectrum. The DOX-release studies were performed in triplicate, and the results were expressed as the average data with standard deviations. Cell Culture. NIH/3T3 normal cells (a mouse embryonic fibroblast cell line) and HN-6 cancer cells were cultured in DMEM supplied with 10% FBS, and antibiotics (50 units/mL penicillin and 50 units/mL streptomycin) at 37 °C in a humidified atmosphere containing 5% CO2. Cytotoxicity Measurements of H40-star-PEG/ALE Micelles. The relative cytotoxicity of H40-star-PEG/ALE micelles against NIH/ 3T3 cells was estimated by MTT viability assay. In the MTT assay, NIH/3T3 cells were seeded into 96-well plates at a seeding density of 1.0 × 104 cells per well in 200 μL of complete medium. After 24 h of incubation, the culture medium was removed and replaced with 200 μL of a medium containing serial concentration of the H40-star-PEG/ ALE micelles. The cells were grown for another 48 h. Then, 20 μL of 5 mg/mL MTT assay stock solution in PBS was added to each well. After incubating the cells for 4 h, the medium containing unreacted dye was removed carefully. The obtained blue formazan crystals were dissolved in 200 μL DMSO and the absorbance was measured with BioTek SynergyH4 at a wavelength of 490 nm. Red Blood Cells (RBC) Lysis Assay. Ten mL of blood was obtained from the ear artery of a male New Zealand white rabbit. The 2% w/v RBC solution was acquired. Briefly, the blood was centrifuged at 2000 rpm for 10 min at 4 °C. Then, the supernatant was removed, and the erythrocytes were resuspended with ice-cold PBS (pH = 7.4). The cells were again centrifuged at the same condition. The process must be repeated more than four times to ensure the removal of any released hemoglobin. Finally, the supernatant was carefully removed, and the cells were resuspended in ice cold PBS to get RBC solution (2% w/v).2,19,25 The copolymers and reference polymers (Dextran and PEI) were also prepared at serial concentration (0.02, 0.1, 0.2, 1, and 2 mg/mL) with PBS (pH = 7.4). Then, 2 mL of the H40-star-PEG/ALE copolymer and reference polymers (Dextran and PEI) were added to 2 mL of the 2% w/v RBC solution in 5 mL centrifuge tubes and incubated for 1 h at 37 °C. Dextran and PBS were used as negative controls, while PEI and 1% v/v solution TritonX-100 (100% lysis) were used as positive controls. After 1 h of incubation, the samples were centrifuged at 2000 rpm for 10 min, and the supernatants (200 μL) were transferred to 96-well plates. The absorbance of released hemoglobin was tested with a BioTek SynergyH4 at a wavelength of 545 nm.

[(DOX‐loaded micelles concentration without HA) − (DOX‐loaded micelles concentration with HA)] /(DOX‐loaded micelles concentration without HA) × 100%

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RESULTS AND DISCUSSION Synthesis and Characterizations of H40-star-PEG/ALE. Bisphosphonates such as ALE exhibit strong affinity to the skeleton mineral HA. If they are conjugated onto the surface of micelles, an efficient drug delivery system with high stability and active targeting ability could be obtained. The construction and application of such micelles for bone-targeting drug delivery are illustrated in Scheme 1. First, the amphiphilic 1917

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shown in Figure S2 and Figure S3 in the Supporting Information. As shown in Figure S2, the typical signals of H40-star-PEG/ALE can be detected at 3.57 (a), 3.25 (b), 3.06 (c), 2.54 (d), 2.43−2.46 (e), 2.23 (f), 1.79−1.85 (g), and 1.66 (h) ppm. Based on 1H NMR of H40-star-PEG/ALE in Figure S2, about 22% of the −OH groups of H40 were functionalized by ALE and 31% of the −OH groups of H40 was grafted with PEG. The percentage of weight of ALE and PEG in the H40star-PEG/ALE copolymer is 15% and 67%, respectively. The obvious shift of phosphate group of copolymer can be observed compared to the spectrum of ALE in Figure S3. The 13C NMR spectra of H40 and H40-star-PEG/ALE are shown in Figure S4 in the Supporting Information. The typical signals of H40-starPEG part can be observed at 33.2, 34.2, 38.9, 58.08, 65.35− 71.00, and 181.3 ppm. The typical signals from the ALE moiety can be detected at 23.8, 32.4, 50.8, and 183.2 ppm. The appearance of these peaks confirms the successful conjugation. Hence, the given NMR data confirm that the H40-star-PEG/ ALE is successfully synthesized. The structures of all intermediates and H40-star-PEG/ALE were further analyzed by FTIR. The representative FTIR spectra are given in Figure S5 in the Supporting Information. In the spectra of H40 (Figure S5A) and H40-COOH (Figure S5B), the sharp peaks at 1733 cm−1 confirm the existence of carbonyl groups. After ligation with PEG-NH2 and ALE, the FTIR spectrum of H40-star-PEG/ALE in Figure S5C differs obviously from that of H40 and H40-COOH. The peak at around 1733 cm−1 is weakened greatly, indicating that the terminal carboxyl groups have been coupled with the primary amines of PEG-NH2 and ALE. In the meantime, the appearance of two strong bands at 1112 and 952 cm−1 can be attributed to the C−O−C and −HPO3 stretching vibration of PEG and ALE, respectively. FTIR observation further supports the formation of H40-star-PEG/ALE. The molecular weights and their polydispersities of the synthesized products were characterized by GPC, and the results are shown in Table 1. It can be found that the numberaverage molecular weight (Mn) of H40-star-PEG/ALE is 1.41 × 104 with a PDI of 1.30.

Scheme 1. Construction and Application of H40-star-PEG/ ALE Micelles for Bone-Targeted Drug Delivery

hyperbranched copolymer H40-star-PEG/ALE was prepared through four-step reactions, the detailed synthetic route of the polymer is given in Scheme 2, and the chemical structural formula of H40 is also shown in Figure S1 in the Supporting Information. With the help of TEA catalyst, the carboxyl groups are introduced into the surface of H40 to produce H40-COOH via the reaction of H40 with the succinic anhydride.24,25 After the reaction between H40-COOH and NHS, H40-CO-NHS is obtained. Based on the coupling reaction of H40-CO-NHS and PEG-NH2, the hydrophilic PEG segments are grafted onto the hydrophobic H40 core to form H40-star-PEG. To accomplish the bone targeting property, ALE is further incorporated to H40-star-PEG via the reaction between the residual NHS groups of H40-star-PEG and the primary amine of ALE to obtain the final product H40-star-PEG/ALE. The 1H NMR and 31 P NMR spectra of the copolymer H40-star-PEG/ALE are Scheme 2. Synthetic Route of H40-star-PEG/ALE

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absorbance intensity of DPH strengthens exponentially with the concentration. The two different states can be reflected by two fitting lines in Figure 2A. The intersection of the horizontal line and the rapidly rising line forms the turning point, which is the CMC value of the H40-star-PEG/ALE copolymer (0.002 35 mg/mL). The detected low CMC value confirms the formation of the H40-star-PEG/ALE micelles with high stability in aqueous solution. Meanwhile, H40-star-PEG/ALE micelles can remain stable in vivo because of the low CMC value. Stability of H40-star-PEG/ALE Micelles in PBS (pH = 7.4) Buffer. The enhanced stability of star copolymer micelles were evaluated in PBS (pH = 7.4) at different time intervals by DLS. As shown in Figure 2B, the size variation of the micelles is very slight in 96 h, and the average diameter is around 16 nm. Therefore, the stability of prepared micelles offers the possibility for introduction to the body with the potential of therapeutics delivery. In Vitro Cytotoxicity of H40-star-PEG/ALE Micelles. The cytotoxicity of H40-star-PEG/ALE micelles was assessed in vitro by MTT assay against the NIH/3T3 normal cells. The MTT test is based on the capacity of a mitochondrial dehydrogenation enzyme in living cells to cleave the tetrazolium rings of the pale yellow MTT and form formazan crystals with dark-blue color.29 Figure 3A gives the relative cell viability after 48 h incubation with the H40-star-PEG/ALE copolymers with a series of concentrations. The result illustrates that no distinct cytotoxicity against NIH/3T3 cells is observed even the concentration of copolymers is up to 1 mg/mL. Accordingly, these copolymers show low cytotoxicity to NIH/3T3 normal cells. Hemolysis Experiment. The biocompatibility of H40-starPEG/ALE copolymer under physiological conditions was

Table 1. GPC Results of H40, H40-COOH, and H40-starPEG/ALE

a

sample

Mn

Mw

Mw/Mn

H40a H40-COOH H40-star-PEG/ALE

2800 6100 14100

5100 7800 18300

1.80 1.28 1.30

From perstorp data sheet.

Characterization of Micelles. The size is an important parameter for micelles to be a drug delivery system, because the small-sized micelles (