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Contents lists available at ScienceDirect

International Journal of Pharmaceutics journal homepage: www.elsevier.com/locate/ijpharm

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Pharmaceutical nanotechnology

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In vitro and in vivo evaluation of APRPG-modified angiogenic vessel targeting micelles for anticancer therapy

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Pan Guo, Shuangshuang Song, Zhao Li, Ye Tian, Jiatong Zheng, Xinggang Yang, Weisan Pan * Department of Pharmaceutics, Shenyang Pharmaceutical University, 103 Wenhua Road, 110016 Shenyang, China

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A R T I C L E I N F O

A B S T R A C T

Article history: Received 25 August 2014 Received in revised form 27 February 2015 Accepted 30 March 2015 Available online xxx

The study was aimed to evaluate the antitumor potential of the Ala-Pro-Arg-Pro-Gly (APRPG)-modified angiogenic vessel targeting drug delivery system using paclitaxel (PTX) as a model drug. In this study, an angiogenesis homing peptide APRPG was conjugated to the amphipathic copolymer PLGA–PEG and the synthesized copolymer APRPG–PEG–PLGA was used to prepare PTX encapsulated micelles (APRPG–PEG-Mic). The micelles were uniform spherical and exhibited a unimodal particle size distribution and a slight negative zeta-potential. The in vitro drug release result demonstrated a significant sustained release property of APRPG–PEG-Mic. Compared to Taxol1 and Cont-PEG-Mic, APRPG–PEG-Mic showed a stronger cytotoxicity against two cancerous cell lines. In the cell uptake studies, the APRPG-modified micelles enhanced intracellular fluorescent intensity in EA.hy926 cells. The biodistribution study revealed the accumulation of APRPG–PEG-Mic in tumor tissues as a result of passive accumulation and active targeting. In comparison with Taxol1 and Cont-PEG-Mic, APRPG–PEGMic reduced the tumor volume more significantly and prolonged the survival time of tumor-bearing mice, indicating a higher antitumor efficacy and lower systematic side effects of APRPG–PEG-Mic. The results indicated that APRPG-modified micelles could be an efficient target-delivery method to angiogenic vessels and a highly promising therapeutic system in anticancer therapy. ã 2015 Published by Elsevier B.V.

Keywords: Anticancer therapy Paclitaxel Angiogenic vessel targeting Micelle Long circulation Tumor targeting

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1. Introduction

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In recent years, anticancer therapy is expected to be safe, effective, and specific to solid tumors. Meanwhile, the anticancer drugs are desired to be suitable for a broad spectrum of cancers. But as it is known, anticancer drugs and ordinary chemotherapies may injure normal tissues as well as tumor tissues. To overcome the severe side effects and poor clinical outcomes of traditional chemotherapy, anticancer therapy is expected to be more effective and specific to tumor tissues. The targeting drug delivery systems which can enhance tumor targeting efficacy and reduce the systemic toxicity by delivering anticancer drugs specifically to the tumor sites became more and more important in anticancer therapy (Rösler et al., 2012; Kedar et al., 2010). Scientists make various kinds of attempt to achieve these goals, and among which, the angiogenic vessel targeting drug delivery system shows significant effects and attractive potentials. The growth of tumors

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* Corresponding author. Tel.: +86 24 23986313. E-mail address: [email protected] (W. Pan).

is highly dependent on sufficient supply of oxygen and nutrients to tumor cells by abundant vascular (Yousefi et al., 2014). The generation of new blood vessels is regulated by a balance between pro- and anti-angiogenic molecules in normal tissues, but this process is out of control in tumor (Conway et al., 2001). These provide us a new way to fight against tumors. On one hand, the angiogenesis is a critical element for the maintenance, proliferation and metastasis of tumors (Weis and Cheresh, 2011), and on the other hand, the efficient blood supply given by neovascular makes it possible to deliver anticancer drugs to all regions of a solid tumor in effective quantities (Carmeliet and Jain, 2000). In addition, since angiogenic endothelial cells have growing character, these cells may also be damaged by cytotoxic anticancer drugs as well as tumor cells, resulting in an enhanced damage to tumor tissues by damaging the newly formed vessels along with the inhibition effects to tumor cells by anticancer drugs (Oku et al., 2003). It is reported that Oku et al. isolated a 5-amino acid peptide Ala-Pro-Arg-Pro-Gly (APRPG) using an angiogenesis mouse model to identify a targeting ligand for angiogenic vessel-specific drug delivery. As a result, APRPG was identified as a novel peptide homing to angiogenic vessels (Murase et al., 2010). In previous

http://dx.doi.org/10.1016/j.ijpharm.2015.03.067 0378-5173/ ã 2015 Published by Elsevier B.V.

Please cite this article in press as: P. Guo, et al., In vitro and in vivo evaluation of APRPG-modified angiogenic vessel targeting micelles for anticancer therapy, Int J Pharmaceut (2015), http://dx.doi.org/10.1016/j.ijpharm.2015.03.067

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study, the APRPG showed specific binding to rhVEGF-stimulated endothelial cells and accumulated in angiogenic sites in vitro, and the adriamycin liposome modified with APRPG suppressed tumor growth in vivo (Maeda et al., 2004). The APRPG-modified nanoparticle was developed for delivering TNP-470 to ovarian cancer (Wang et al., 2014), and the siRNA/PEI–PEG–APRPG complex also exhibited high efficiency as antitumor therapy with regard to tumor growth, microvessel density, and VEGF protein levels (Lu et al., 2011). The above studies indicated that the peptide became a useful probe for angiogenesis and has been used for Q3 targeting neovascular in anticancer therapy. Paclitaxel (PTX), a naturally diterpenoid, is a mitotic inhibitor widely used in cancer chemotherapy. PTX exhibits a significant activity against a broad spectrum of cancers, such as lung, ovarian, breast, colon, bladder, head and neck carcinomas (Zhang et al., 2013; Bu et al., 2014). However, the main drawback for PTX is the low solubility in water and most pharmaceutical solvents (Zhou et al., 2013). Considering this problem, the commercial brand Taxol1, formulated with high concentration of Cremophor EL and dehydrated alcohol (1:1 v/v), was developed to increase the solubility of PTX. Taxol1 needed to be diluted 5–20 folds with saline or 5% dextrose solution before use, bringing inconvenience in clinical application and disobedience of patients (Crown et al., 2004). In addition, it has been found that Cremophor EL has severe adverseness, including hypersensitivity reactions, nephrotoxicity, cardiotoxicity and neurotoxicity (Gelderblom et al., 2001). Moreover, other serious side effects such as hypersensitivity, fluid retention, neutropenia and nail toxicity are also observed during clinical treatments, which is mainly due to the low selectivity to tumors, hence resulting in high toxicity to normal tissues (Marupudi et al., 2007). Therefore, a novel PTX formulation strategy with low adjuvant content, specific tumor targeting features, and high therapeutic efficacy is preferred. In this study, biocompatible and biodegradable amphipathic copolymer PLGA–PEG was used to prepare PTX-loaded micelles, and the angiogenesis targeting peptide APRPG was conjugated with the copolymer for delivering the drug to tumor sites specifically. It was found that the amphipathic copolymer could spontaneously form micelles in aqueous solvent. The hydrophobic PLGA inner-core was capable of carrying insoluble drug PTX with a higher loading capacity, while the hydrophilic PEG outer-shell provided steric protection to avoid the opsonization and the phagocytosis by reticuloendothelial system (RES) (Adams et al., 2003; Pasut and Veronese, 2009), resulting in the reduced systemic clearance rates and the prolonged circulation half-life in vivo. Moreover, the peptide APRPG is combined with the amphipathic copolymer by an amidation reaction. After intravenous injection, the drug loaded micelles accumulated in the tumor tissues as a result of the passive and active targeting effects, including the enhanced permeability and retention (EPR) effect (Maeda et al., 2000; Fang et al., 2011), the long-circulation character of micelles, and the specific binding of APRPG peptide to angiogenic vessels of tumor. Afterwards, the drug would release from micelles into tumor tissues, and then expressed antitumor effects by damaging the newly formed blood vessels and inhibiting or killing the tumor cells. Herein, the in vitro and in vivo experiments are carried out to evaluate the applicability of APRPG-modified PLGA–PEG micelles for anticancer therapy.

Ltd. (Beijing, China). Carboxyl group terminated poly(D,L-lactic-coglycolic acid) (PLGA-COOH, L:G molar ratio of 50:50, molecular weights 5 kDa) and methoxy poly(ethylene glycol)-poly(D,L-lacticco-glycolic acid) (mPEG–PLGA) were purchased from Jinan Daigang Biomaterial Co., Ltd. (Jinan, China). Peptide APRPG (purity > 98%) was synthesized by TeraBIO Technology Co., Ltd. (Guangzhou, China). N-hydroxysuccinimide (NHS), N,N-dimethylaminopropyl carbodiimide hydrochloride (EDC
HCl), and N,N-diisopropylethylamine (DIEA) were purchased from Shanghai Medpep Co., Ltd. (Shanghai, China). Paclitaxel and docetaxel were offered by Jiangsu Hengrui Pharmceutical Co., Ltd. (Jiangsu, China). Paclitaxel injection (Taxol1) was obtained from Bristol-Myers Squibb (Princeton, UK). Kolliphor1 HS 15 was a kind gift from BASF Co., Ltd. (Ludwigshafen, German). All other chemicals and solvents were of reagent grade or higher, obtained commercially.

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2.2. Animals and cell lines

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Male Kunming mice (20–25 g) were from Shenyang Pharmaceutical University Animal Institution, and were housed under standard conditions. All animal experiments were carried out in accordance with the Rules of Shenyang Pharmaceutical University Bioethics Committee and in compliance with the Guide for the Care and Use of Laboratory Animals of the national laws. Human endothelial cell line EA.hy926, derived by a fusion of human umbilical vein endothelial cells (HUVECs) with a human lung carcinoma A549, was purchased from the Type Culture Collection of the Chinese Academy of Sciences (Shanghai, China). Mouse connective tissue fibroblast cells (L929), human lung carcinoma cells (A549), and sarcoma-180 cells (S-180) were obtained from Nanjing Keygen Biotech Co., Ltd. (Nanjing, China). All four cell lines were cultured in Dulbecco-Modified Eagle’s Medium (DMEM) supplemented 10% fetal bovine serum (FBS), penicillin/streptomycin and L-glutamine (complete medium) at 37  C in a humidified atmosphere containing 5% CO2.

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2.3. Synthesis of APRPG–PEG–PLGA

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2. Materials and methods

PLGA–PEG–COOH was synthesized by a standard EDC/NHS mediated chemistry (Betancourt et al., 2009; Townsend et al., 2007; Zhang et al., 2007), using PLGA terminated in carboxyl groups and a hetero-functional PEG, NH2–PEG–COOH (molecular weights 2 kDa, 4 kDa and 8 kDa) as shown in Fig. 1. Briefly, 1 mmol PLGA–COOH was dissolved in 20 mL of dichloromethane (DCM) and stirred for 4 h at room temperature in the presence of 10 mmol EDC and 10 mmol NHS. Thereafter, the reaction mixture was extracted by double-distilled water to remove unreacted EDC and NHS. The trace water in DCM solution was removed using anhydrous MgSO4, followed by being dried for 2 h under vacuum to obtain polymer PLGA–NHS. PLGA–NHS was redissolved in 20 mL of DCM together with 1.25 mmol HCl
NH2–PEG–COOH, where DIEA was also added to provide alkaline environment, and then reacted overnight under gentle stirring. After reaction, the solution was concentrated and the copolymer was purified by silica gel column chromatography using DCM and methanol (7:1, v/v) as eluent. The resultant PLGA–PEG–COOH was dried under vacuum for 2 h, and stored at 20  C until use. PLGA–PEG–COOH was again activated by EDC/NHS method as described above. The obtained NHS-activated copolymers were conjugated with the amino of peptide APRPG, and the final APRPG–PEG–PLGA was gained.

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2.1. Materials

2.4. Characterization of copolymers

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a-Amino-v-carboxyl poly(ethylene glycol) hydrochloride salt (HCl
NH2–PEG–COOH, molecular weights 2 kDa, 4 kDa and 8 kDa) was purchased from Beijing Kaizheng Biotech Development Co.,

PLGA–PEG–COOH and APRPG–PEG–PLGA copolymers were characterized by 1H NMR spectra using D-CHCl3 at a concentration of 5–10 mg mL1. The 1H NMR spectra was recorded on a Bruker

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Fig. 1. Schematic representation of the amphiphilic copolymer APRPG–PEG–PLGA. Carbodiimide chemistry was used to conjugate PLGA to heterofunctional PEG (NH2– PEG–COOH) and link a pentapeptide Ala-Pro-Arg-Pro-Gly to the amphiphilic PLGA– PEG–COOH copolymer. Carboxyl groups of PLGA or copolymer were activated with EDC and NHS to form an NHS-ester that reacts with terminal amino groups on PEG or peptide to form a stable amide linkage. Abbreviations: NHS, N-hydroxysuccinimide; EDC, N,N-dimethylaminopropyl carbodiimide; DIEA, N,N-diisopropylethylamine.

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0.5% (w/v) Kolliphor1 HS 15 under gentle stirring. Stirring was continued for 6 h at 40  C to allow the evaporation of the organic phase. Afterwards, the obtained APRPG–PEG–PLGA micelles dispersion was filtered through a 0.45 mm cellulose acetate filter and lyophilized (Eyela FDU-1100, Prkakikai, Tokyo, Japan) using 5% (w/v) trehalose as lyoprotectants. Three kinds of copolymer with different PEG molecular weights were used to prepare APRPGmodified PTX-loaded micelles (APRPG–PEG-Mic), and the nonmodified micelles (Cont-PEG-Mic), using mPEG–PLGA instead of APRPG–PEG–PLGA, or blank micelles (Blank-PEG-Mic), with the exception of adding PTX, were prepared in the same way as described above. The lyophilized solid was stored at 4  C and redissolved in water before use.

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2.6. Characterization of micelles

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2.6.1. Transmission electron microscopy (TEM) TEM (JEOL JEM-1200EX, Tokyo, Japan) was employed to observe the morphology of APRPG–PEG-Mic. A drop of micelles dispersion was put onto a 300-mesh Formvar-coated copper grid. After 30 min incubation, the excess liquid was blotted away by filter paper, followed by negatively stained for 10 min at room temperature with freshly prepared phosphotungstic acid buffer 0.3%. Finally, the grid was air-dried prior to imaging.

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2.6.2. Measurement of encapsulation efficiency (EE) and drug loading capacity (DL) PTX-loaded micelles were separated from the unentrapped drug using a Sephadex G-50 column (2.5 cm  1.0 cm) (Song et al., 2014). Briefly, preprocessed Sephadex G-50 was loaded into a 2.5 mL syringe and centrifuged at 2000 rpm for 3 min to obtain a dehydrated column. Then, 200 mL of PTX-loaded micelle dispersion was added into the column and centrifuged at 2000 rpm for 2 min. Subsequently, the column was washed for 3 times with 200 mL of distilled water as eluent. Afterwards, 200 mL of the eluants were mixed with 2.8 mL of acetonitrile, followed by sonication for 10 min to destroy the micelles. After centrifuging at 4000 rpm for 10 min, the content of PTX in the supernatant was detected. In order to measure the total amount of PTX, 50 mL of micelle dispersion was mixed with 2.95 mL of acetonitrile, followed by sonication for 10 min and centrifugation for 10 min. The content of PTX was determined by HPLC using a LC-ATvp pump and SPD-10 Avp ultraviolet light detector (Shimadzu, Kyoto, Japan). HPLC conditions were as follows: a Diamasil1 C18 column (200 mm  4.6 mm, 5 mm, Dikma, Tianjin, China) was used. The mobile phase consisted of acetonitrile and water (52:48, v/v) delivered at a flow rate of 1.0 mL min1. The wavelength was set at 228 nm and the injection volume was 20 mL. The calibration curve was linear in the range of 0.1–50 mg mL1 with a correlation coefficient of r2 = 0.9999. EE and DL of drug-loaded micelles was calculated according to the following equations:

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EE ð%Þ ¼

Amount of drug encapsulated in micelles  100 Total amount of drug

Amount of drug encapsulated in micelles  100 Weight of the carrier materials

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ARX-300 instrument (300 MHz, Bruker Corporation, Fällanden, Switzerland).

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2.5. Preparation of micelles

DL ð%Þ ¼

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PTX-loaded micelles were prepared by a solvent evaporation method with a slight modification (Musumeci et al., 2006; Zhang and Feng, 2006). Given amounts of PTX together with `APRPG–PEG–PLGA and mPEG–PLGA (with a molar ratio of 1:5) were dissolved in 2 mL of acetone. Then the organic phase was slowly added dropwise to 10 mL of aqueous solution containing

2.6.3. Particle size, particle size distribution and zeta-potentials The particle size (diameter, nm), polydispersity index (PI), and surface charge (zeta-potential, mV) of micelles were determined by dynamic light scattering (DLS) using a Zetasizer Nano SZ

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(Malvern Instruments Ltd., UK) at a scattering angle of 90 and at room temperature. 2.6.4. In vitro drug release The in vitro drug release behavior was tested by a dialysis method. Briefly, 1 mL of Taxol1 or PTX-loaded micelle dispersion was sealed in a dialysis bag (molecular weight cutoff: 10 kDa) and placed in 100 mL of phosphate buffer solution (PBS, pH 7.4) containing 0.5% (w/v) Tween1 80, and stirred gently for 72 h at 37  C in a water bath. As the solubility of PTX in PBS (pH 7.4) was about 7 mg mL1, Tween1 80 was used as a solubilizer to improve the solubility of released PTX. Given the amount of PTX in present work, we used 0.5% Tween1 80 which did not damage the structure of micelles. At predetermined time intervals, aliquots of 100 mL were withdrawn and replaced by an equal volume of fresh release medium. The sample was centrifuged at 8000 rpm for

10 min. The amount of PTX in the supernatant was determined by HPLC as described previously. The accumulative drug release was plotted as a function of time. All operations were carried out in triplicate.

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2.7. In vitro cell viability

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The security of newly synthesized APRPG–PEG–PLGA copolymers for intravenous injection was detected in this study. Free copolymer solutions were used to assess the cytotoxicity to L929 normal cell line by MTT assay. The same method was also performed to evaluate the cell viability of EA.hy926 and A549 cells incubated with commercial PTX solution (Taxol1), Cont-PEG-Mic and APRPG–PEG-Mic with different PEG molecular weights. Briefly, the three kinds of cells were cultured under the condition described in Section 2.2, and then were seeded in a sterilized

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Fig. 2. Proton NMR spectra of APRPG–PEG–PLGA copolymers prepared by conjugation of peptide APRPG, carboxyl group terminated PLGA and hetero-functional PEG. (A) Chemical structure of the copolymer and assignment of proton labels. 1H NMR spectrum of (B) PLGA–COOH, (C) NH2–PEG–COOH, (D) PLGA–PEG–COOH, and (E) APRPG–PEG– PLGA copolymers dissolved in deuterated chloroform.

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(the COU6 concentration was 10 mg mL1), respectively. After incubating for 2 h, the cells were washed three times with cold PBS, following by observed under fluorescent inverted microscope. Further more, the cellular uptake mechanism was investigated by the blocking experiment using free APRPG (10 mg mL1) and low temperature test at 4  C.

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2.9. Tissue distribution in tumor bearing mice

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Twenty-seven male Kunming mice weighing at 20–25 g were used in tissue distribution study. Sarcoma-180 (S-180) cell suspension (2  215;106 cells in 0.2 mL of saline) was implanted subcutaneously into the armpit to generate the tumor-bearing mouse model. The length of the longest tumor axis (L, mm) and the shortest axis (S, mm) were measured with a slide caliper, and the tumor volume was calculated using the following equation.

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V¼

Fig. 3. TEM images of paclitaxel-loaded micelles. Abbreviations: TEM, Transmission electron micrographs. 258 259 260 261 262 263 264 265 266 267 268 269 270 271 272 273

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96-well plate at the density of 5  103 cells per well and subsequently cultured overnight. After attachment, the culture medium was removed and the cells were washed three times with cold PBS. Subsequently the cells were incubated with various concentrations of different PTX formulations, respectively. After cells were incubated for an additional 24, 48 and 72 h, 20 mL of MTT solution (3-(4,5-dimethyl-thiazol-2-yl)-2,5-diphenyl-tetrazolium, 5 mg mL1) was added in the medium, and then the cells were incubated for another 4 h at 37  C. After that, the medium was removed and 200 mL of DMSO was added to dissolve the formazan crystals formed in the live cells. The absorbance of each well was then measured at 570 nm using a microculture plate reader (Model ER-8000, Sanko Junyaku Tokyo, Japan). As the growth inhibitory concentration for 50% of the cell population (IC50) is widely used to indirectly reflect the cytotoxicity against cancerous and normal cells, the results were also expressed as IC50 values. 2.8. Cell uptake studies The cellular uptake efficiency of different PTX formulations was evaluated by fluorescent inverted microscope (IX71, Olympus Inc., U.S.). In place of PTX, the insoluble fluorescent dye Coumarin-6 (COU6) was encapsulated into none-modified or APRPG-modified micelles with the same method as PTX micelles. EA.hy926 cells or A549 cells were seeded overnight in a 24-well plate at 37  C in a humidified atmosphere containing 5% CO2. After cell attachment, the medium was replaced by serum-free culture medium containing free COU6 solution, COU6-Mic or APRPG–COU6-Mic

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When the tumor volume reached about 500 mm3, the S-180 tumor-bearing mouse were fasted overnight and randomly divided into three groups of nine mice each. Taxol1, Cont-PEG-Mic and APRPG–PEG-Mic were injected intravenously at a dose of 10 mg kg1 through the tail vein. At 0.5, 2, and 6 h after injection, every three mice of each group were euthanized by cervical dislocation. Then the heart, lung, liver, spleen, stomach, kidney and tumor tissue were collected immediately, washed with saline, and accurately weighed. All tissue samples were homogenized with saline at a concentration of 0.5 g mL1 and stored at 20  C for further analysis. 200 mL of tissue homogenate together with 10 mL of docetaxel solution (100 mg mL1) as the internal standard were added into a 1.5 mL EP tube and vortexed for 1 min. Then 1 mL of acetonitrile was added to the mixture and vortexed for 10 min to precipitate proteins, followed by centrifugation for 10 min at 10,000 rpm. The supernatants were dried under reduced pressure and redissolved with 100 mL of mobile phase for HPLC analysis as described in Section 2.6.2.

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Sarcoma-180 cell were subcutaneously implanted into the armpit of mice as described in Section 2.9. When the tumor volume reached about 100 mm3, the tumor-bearing mice were allocated to six groups of six mice each for the following experiment. The mice were injected intravenously with Taxol1, blank PEG-Mic (mPEG–PLGA micelles without PTX), blank APRPG–PEG-Mic (APRPG–PEG–PLGA micelles without PTX), Cont-PEG-Mic APRPG –PEG-Mic and saline at day 1, 4, 7, 10 and 13 at a dose of 6 mg kg1, respectively. The volumes of the tumor and the body weight of each mouse were monitored every two days after the first administration. After a 21-day treatment, the mice were euthanized, and the tumors were excised and weighed. The tumor

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Table 1 The physicochemical characterization of micelle formulations. Formulations

Particle size (nm)

PI

Zeta potential (mV)

DL (%)

EE (%)

Blank PEG-Mic Cont-PEG-Mic APRPG–PEG2000-Mic APRPG–PEG4000-Mic APRPG–PEG8000-Mic

70.2  5.7 104.0  9.0 109.7  16.1 117.6  13.3 119.9  10.2

0.19  0.03 0.20  0.06 0.19  0.04 0.18  0.04 0.17  0.05

3.94  1.18 6.82  1.17 9.37  0.85 7.06  1.14 5.14  0.45

– 2.37  0.04 2.39  0.04 2.30  0.04 2.35  0.05

– 90.17  1.38 91.47  1.71 88.20  1.30 90.93  1.16

Notes: Parameters represent the mean  S.D. (n = ). Abbreviations: PI, polydispersity index; DL, drug loading capacity; EE, encapsulation efficiency; Blank PEG-Mic, blank micelles without paclitaxe; Cont-PEG-Mic, nonmodified paclitaxel-loaded micelles; APRPG–PEG-Mic, APRPG-modified paclitaxel-loaded micelles.

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Q6 Fig. 4. In vitro release profiles of PTX from commercial Taxol1 (blue square), ContPEG-Mic (red circle) and APRPG–PEG-Mic at different molecular weights (PEG2000: green triangle; PEG4000: orange triangle; PEG8000: purple rhombus). Release experiments were carried out in isotonic PBS (pH 7.4) at 37  C. Each point represents the mean value of three different experiments  S.D. (n = 3). (For interpretation of the references to color in this figure legend, the reader is referred to the web version of this article.) Abbreviations: PTX, paclitaxel; Cont-PEG-Mic, non-modified micelles; APRPG–PEGMic, APRPG-modified micelles; PBS, phosphate buffer solution. 329

inhibition rate (TIR) was calculated using the following equation. W ðsÞ  W ðtÞ  100 TIR ð%Þ ¼ W ðsÞ

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where W(t) was the tumor weight of tested group and W(s) was the tumor weight of saline group.

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The mice were given subcutaneous injections of S-180 cell as described in Section 2.9. When the tumor volume reached about 100 mm3, they were divided into four groups of 10 mice each and injected intravenously with Taxol1, Cont-PEG-Mic APRPG–PEG-

Fig. 5. Cell viability of synthesized APRPG–PEG–PLGA copolymers with different molecular weights in L929 cells (n = 3). The copolymers were added as free solutions. Abbreviations: L929, mice fibroblasts cell line.

Mic and saline every 3 days at a dose of 6 mg kg1, respectively. The time of death and the remaining number of animals were recorded. The graphic was drawn with the number of animals against time. The mortality data were subjected to Kaplan–Meier survival analysis to prepare survival plots.

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2.12. Statistics analysis

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Results were given as mean  standard deviation (S.D). Student ’s two-sample t-test and one way ANOVA for multiple groups were used for statistical evaluation and P < 0.05 was used to indicate statistical significance.

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3. Results and discussion

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3.1. Characterization of copolymers

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We successfully synthesized PLGA–PEG–COOH copolymers by conjugation of PLGA to the amino of NH2–PEG–COOH using an EDC/NHS mediated chemical reaction. Peptide APRPG was conjugated to the PLGA–PEG–COOH by the same reaction. Fig. 2A exhibits the chemical structure of the copolymer and assignment of proton labels. In comparison with the 1H NMR spectrum of PLGA–COOH (Fig. 2B) and NH2–PEG–COOH (Fig. 2C), Fig. 2D indicated the synthesis of PLGA–PEG–COOH. The peak at 3.6 ppm was attributed to the methylene protons of PEG chain and the signals at 1.5, 4.8 and 5.2 ppm revealed characteristic peaks of PLGA, corresponding to lactide methyl doublets, glycolide protons and lactide methine quartets, respectively. (Wang et al., 2011; Jeong et al., 2000) The characteristic peaks of PEG and PLGA in the spectrum demonstrated the structure of PLGA–PEG–COOH. From Fig. 2E we could see the resonances at 1.1 and 1.6–1.8 ppm which assigned to the peptide APRPG. The above results suggested the successful synthesizing of copolymer APRPG–PEG–PLGA.

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3.2. Preparation and characterization of micelles

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In this paper, the PTX-loaded micelles were successfully prepared using the solvent evaporation method. In order to store the PTX-loaded micelles for a long-term, we lyophilized the micelles and investigated the protective effect of different cryoprotectants, including lactose, sucrose, dextran, mannitol, maltose and trehalose, on the appearance, particle size distribution, zeta-potential and EE of redissolved micelles. To the point, the lyophilized powder with trehalose (5% w/v) was satisfactory in appearance and had a similar result in physiochemical properties as micelles before lyophilization (data not shown). The formation of PTX-loaded micelles was further confirmed by TEM image. From Fig. 3 we can see that the micelles were uniform spherical with a light outer shell and a dark inner core. The physicochemical properties and loading parameters of blank and PTX-loaded micelles are summarized in Table 1. It can be seen that the particle size of Blank-PEG-Mic was 70.2  5.7 nm, and that of PTX-loaded micelles increased with the increase of PEG molecular weights, ranging from 104 nm to 119 nm. All kinds of micelles had a unimodal particle size distribution and a little negative zetapotential. The EE of PTX-loaded micelles were around 90% and the DL were 2.30–2.40% and showed no significant differences between various formulations, indicating that the encapsulation ability of the micelles did not vary with the changing of copolymer molecular weights. The in vitro release behavior of different formulations presented as the cumulative release percentage is shown in Fig. 4. Taxol1 released more than 95% of PTX within initial 12 h, while only 49.4%, 47.9%, 55.0% and 60.0% of drug was released from Cont-PEG-Mic, APRPG–PEG2000-Mic, APRPG–PEG4000-Mic and

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Table 2 Cytotoxicity of PTX formulations against EA.hy926 and A549 carcinoma cell lines, expressed as IC50 (nmol) and PF values. Formulations

Taxol1 Cont-PEG-Mic APRPG–PEG2000-Mic APRPG–PEG4000-Mic APRPG–PEG8000-Mic

EA.hy926

A549

24 h

48 h

72 h

PF

24 h

48 h

72 h

PF

342.5  42.7 65.9  5.8 40.2  3.8 45.9  4.0 50.3  6.4

73.5  13.9 22.4  3.4 9.8  2.0 12.6  3.2 13.2  3.3

39.9  7.1 14.3  2.8* 4.7  0.4** 4.8  0.6** 6.9  1.3**

– 2.8 8.5 8.4 5.8

375.2  53.7 76.1  12.7 58.4  7.7 62.9  6.5 68.6  9.8

98.0  15.3 44.5  6.2 33.7  5.7 32.6  4.1 40.4  4.8

42.3  8.3 25.6  6.9* 18.2  4.3* 22.8  3.7* 25.9  3.5*

– 1.7 2.3 1.9 1.7

Q7 Significant differences are indicated as follows: *P < 0.05, **P < 0.01 and ***P < 0.001. Abbreviations: IC50, the growth inhibitory concentration for 50% of the cell population; PF, potentiating factor; EA.hy926, a fusion of human umbilical vein endothelial cells (HUVECs) with a human lung carcinoma A549; A549, human lung cancer cell line; Cont-PEG-Mic, non-modified paclitaxel-loaded micelles; APRPG–PEG-Mic, APRPGmodified paclitaxel-loaded micelles.

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APRPG–PEG8000-Mic during this time, respectively. For these PEGylated micelles, less than 60% PTX was released after 48-h experiment. Especially for APRPG–PEG2000-Mic only 45% of drug was released, showing a sustained release behavior. It can be seen that the release behavior of Cont-PEG-Mic was similar to that of APRPG–PEG2000-Mic indicating that the modification of APRPG to the PEG–PLGA micelles had no effect on the in vitro release behavior. Moreover, the release rate of PTX from PEGylated micelles increased with the increase of PEG molecular weight. This may be because the mechanism of drug release is the diffusion of drug molecules through the copolymer carriers and the hydration process of the hydrophilic group, and the loose structure of micelles with higher molecular weight would lead to accelerating drug release (Li et al., 2011).

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3.3. In vitro cell viability

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The cytotoxicity of mPEG–PLGA and synthesized APRPG–PEG– PLGA copolymers to L929 normal cell line and PTX-loaded micelles to EA.hy926 and A549 cancerous cell lines were assessed using MTT assay (Scherließ, 2011). Fig. 5 shows the viabilities of L929 cells after incubated with APRPG–PEG–PLGA copolymer solution for 72 h. We could see the viabilities (%) were all over 90% at experimental concentrations (1–20 mg mL1). This indicated

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that the mPEG–PLGA and synthesized APRPG–PEG–PLGA copolymers displayed no conspicuous cytotoxicity to normal cells. The IC50 value can indirectly reflect the cytotoxicity of different formulations, so the IC50 values of Taxol1, Cont-PEG-Mic and APRPG–PEG-Mic to EA.hy926 and A549 cancerous cells are summarized in Table 2. It was found that the IC50 values of Cont-PEG-Mic (P < 0.05), APRPG–PEG2000-Mic (P < 0.01), APRPG– PEG4000-Mic (P < 0.01) and APRPG–PEG8000-Mic (P < 0.01) were all significantly lower than that of Taxol1 in the two kinds of cancerous cell lines during 72 h incubation, indicating the encapsulation of PTX into mPEG–PLGA or APRPG–PEG–PLGA micelles could enhance the cytotoxicity of drug. This may be due to the increased cell uptake by cancer cells. That is to say, the Cont-PEG-Mic and APRPG–PEG-Mic could achieve the same effect as commercial PTX solution at a lower dosage. We can also find that there is significant difference between the IC50 values of APRPG– PEG-Mic and Cont-PEG-Mic. To accurately evaluate the cell growth inhibition effect of different PTX-loaded micelles, we defined potentiating factor (PF) as the ratio of IC50 values between Taxol1 and PTX-loaded micelles after 72 h incubation (Ungaro et al., 2012). It is interesting to note that the PF values of APRPG–PEG2000-Mic APRPG–PEG4000-Mic and APRPG–PEG8000-Mic were 8.5, 8.4 and 5.8, which were 3.1, 3.0 and 2.1-fold higher than Cont-PEG-Mic in EA.hy926 cells, respectively, indicating the modification of APRPG

Fig. 6. Cell uptake and the fluorescent intensity of COU6, COU6-Mic and APRPG–COU6-Mic in EA.hy926 cells (A) and A549 cells (B) in 37  C, 4  C or in the presence of free APRPG. The magnification was 10  40. Abbreviations: EA.hy926, a fusion of human umbilical vein endothelial cells (HUVECs) with a human lung carcinoma A549; A549, human lung cancer cell line; COU6, coumarin-6 solution; COU6-Mic, non-modified micelles loading coumarin-6; APRPG–COU6-Mic, APRPG-modified micelles loading coumarin-6.

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Fig. 7. The mean concentrations of PTX in different tissues of S-180 tumor-bearing mice after intravenous administration of Taxol1 (blue bar), Cont-PEG-Mic (red bar) and APRPG–PEG-Mic (yellow bar) at 0.5 h (A), 2 h (B) and 6 h (C) (n = 3). Significant differences are indicated as follows: *P < 0.05 and **P < 0.01. (For interpretation of the references to color in this figure legend, the reader is referred to the web version of this article.) Abbreviations: PTX, paclitaxol; Cont-PEG-Mic, non-modified micelles; APRPG–PEG-Mic, APRPG-modified micelles.

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could enhance the cytotoxicity of drug in EA.hy926 cells. However, in A549 cells, the PF values of the three APRPG–PEG-Mic were only 1.4, 1.3 and 1.0-fold higher than Cont-PEG-Mic where the enhancement of inhibition effect was not obvious. Since the EA.hy926 was a confluent cell line and maintains the differentiated properties of vein endothelium cells, (Baranska et al., 2005; Swiatkowska et al., 2001) the enhancement effect was probably attributed to the specific binding of APRPG to vein endothelium cells. It can be also found from Table 2 that the PF value of APRPG– PEG2000-Mic was higher than that of APRPG–PEG4000-Mic and APRPG–PEG8000-Mic. That may be because the overfolding of PEG chain, if it was too long, covered the APRPG and hindered the binding to vein endothelium cells. It was reported that the evaluation of the behavior in biologically relevant conditions of micelles with different architectures was of utmost relevance to the behavior in vivo. (Ostacolo et al., 2010) As a result, we considered the APRPG–PEG2000-Mic to be the proper formulation in the following in vivo testing.

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3.4. Cell uptake studies

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To investigate the cellular uptake mechanism of APRPG–PEGMic human endothelial confluent cell EA.hy926 and A549 cell were incubated with COU6 solution or COU6-loaded micelles and then

observed under fluorescent inverted microscope. According to the result of preliminary experiment, APRPG–PEG2000-Mic showed better cellular affinity than APRPG–PEG4000-Mic and APRPG– PEG8000-Mic. The similar tendency with cell viability study may be explained by the overfolding of long PEG chain as well, which would block the coordination site of peptide. Therefore, we used APRPG–PEG2000-Mic in cell uptake studies. All the images were taken under the same imaging parameters to better compare the intensity of fluorescence between different COU6 formulations. The Image-Pro Plus was also used to make a quantitative analysis on the pictures taken in this study and the value of mean optical density was considered to represent the intracellular fluorescent intensity. As shown in Fig. 6A, fluorescent signals of EA.hy926 cells incubated with COU6 solution and COU6-loaded micelles were all visualized in the cytoplasm (the dark parts in the cell were cell nucleus). As expected, the fluorescent intensity of APRPG–COU6Mic group reached 0.052, which was significantly higher than COU6-Mic and COU6 solution group. Moreover, when EA.hy926 cells were incubated with free APRPG (10 mg mL1) before COU6 formulations, the intracellular fluorescent intensity of APRPG–COU6-Mic was substantially reduced to 0.027, while as for COU6-Mic and COU6 solution, there was no significant change in fluorescent intensity in the same condition. The difference may be explained by the specific binding of APRPG in EA.hy926 cells. When

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Fig. 8. Therapeutic efficacies of different formulations on tumor-bearing mice. S-180-bearing mice (n = 6 for each group) were injected i.v. Taxol1 (blue square), Cont-PEG-Mic (red circle), APRPG–PEG-Mic (green triangle), blank Cont-PEG-Mic (cyan rhombus), blank APRPG–PEG-Mic (pink cross) or saline (purple inverted triangle), for five times at day 1, 4, 7, 10 and 13 after the tumor volume reached 100 mm3. Injected solutions of each formulation were adjusted to be 6 mg kg1 as PTX concentration in each administration. The body weight (A) and the size of the tumor (B) of each mouse were monitored every two days. Arrows indicate the days of treatment. The tumor weight (bar) after 21 days treatments was weighed and the TIF value (sign and line) was calculated (C). Data were presented as the mean value and S.D. bars. Significant differences are indicated as follows: *P < 0.05, **P < 0.01 and ***P < 0.001. (For interpretation of the references to color in this figure legend, the reader is referred to the web version of this article.) Abbreviations: Cont-PEG-Mic, non-modified paclitaxel micelles; APRPG–PEG-Mic, APRPG-modified paclitaxel micelles; blank Cont-PEG-Mic, non-modified micelles without paclitaxe; blank APRPG–PEG-Mic, APRPG-modified micelles without paclitaxe; S-180, sarcoma-180 cells; i.v., intravenous administration; PTX, paclitaxol; TIF, tumor inhibition rate.

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free APRPG peptide competitively combined with the ligands, the APRPG–COU6-Mic could not specifically combine with EA.hy926 cells, resulting in the decreased cell uptake. In A549 cells (Fig. 6B), as for APRPG–COU6-Mic the fluorescent intensity was weaker than that of EA.hy926 cells treated with APRPG–COU6-Mic. More than that, the intracellular fluorescent intensity of APRPG–COU6-Mic showed no significant difference when incubated with free APRPG (10 mg mL1) before. The remarkable contrast between the EA.hy926 and A549 cells was possibly because the former expressed the character of endothelial cells, including the specific ligands of APRPG. As for COU6 solution and COU6-Mic the fluorescent signals of in A549 cells showed similar result as EA.hy926 cells. In addition, we can also find that the cell uptake of APRPG–COU6-Mic and COU6-Mic was an energydependent process, as evidenced by the substantially reduction in

fluorescent intensity at 4  C. The value reduced to 0.28–0.29 in EA.hy926 cells and 0.30–0.32 in A549 cells, respectively, so we could hardly observe the fluorescent signal for the two COU6 micelles. In contrast, the fluorescent intensity of COU6 solution was not obviously inhibited in the same condition because of passive diffusion. These results demonstrated that APRPG–COU6-Mic can specifically bind to endothelial confluent cells EA.hy926. But similar specific association was not observed in A549 cells, since APRPG has not affinity to non-endothelial cells (Maeda et al., 2003).

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3.5. Tissue distribution in tumor-bearing mice

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To further verify the specific targeting of APRPG–PEG-Mic to tumor xenografts in vivo, we investigated the tissue distribution of

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different PTX formulations in tumor-bearing mice. Given the sustained drug release property and a better inhibition effect to cancerous cells, the APRPG–PEG2000-Mic was used in the in vivo biodistribution experiment instead of APRPG–PEG4000-Mic and APRPG–PEG8000-Mic. As most of the solid tumors were rich of neovascular, S-180 tumor-bearing mice were used as xenograft models in the following in vivo studies. The contents of PTX in various tissues at 0.5 h, 2 h, and 6 h after intravenous injection of different formulations were depicted in Fig. 7. Compared to Taxol1 group, the amounts of drug in Cont-PEG-Mic and APRPG–PEG-Mic groups were less in heart and kidney, implying a lower toxicity to heart and slower elimination in kidney. While the concentration of PTX in liver, lung and spleen in Cont-PEG-Mic and APRPG–PEG-Mic groups was higher than that in Taxol1 group, which may be due to the phagocytose by RES. As expected, the concentration of PTX in the tumor tissues was in the following order at three time points: APRPG–PEG-Mic > Cont-PEG-Mic > Taxol1. The amounts of PTX in Taxol1 group reduced rapidly, and that in Cont-PEG-Mic group decreased in a slower rate, which was probably due to the longcirculating characteristics of PEG chain and EPR effect. It is interesting that, for APRPG–PEG-Mic group, the accumulation of drug in tumors first increased in initial 2 h and subsequently decreased in 6 h. The increase in the first 2 h may be because the long hydrophilic PEG chain provided the protection from the opsonization, hence resulted in a prolonged circulating time. Furthermore, compared to Cont-PEG-Mic the APRPG–PEG-Mic increased the PTX accumulation in tumor by 1.1, 1.7 and 2.0-fold in 0.5, 2 and 6 h, respectively, indicating the modification of APRPG could enhance the accumulation in tumor tissues in vivo. This was mainly attributed to the specific binding of APRPG to neovascular endothelial cells. 3.6. Antitumor efficacy Since PTX-loaded micelles, especially APRPG–PEG-Mic were highly accumulated in tumor tissues in tumor-bearing mice, we carried out the antitumor experiment to investigate if the APRPG– PEG-Mic could exhibit a good therapeutic effect in vivo. Taxol1, Cont-PEG-Mic, APRPG–PEG-Mic and saline were administrated intravenously for five times at day 1, 4, 7, 10 and 13 after the tumor volume reached about 100 mm3, with a dose of 6 mg kg1 as PTX concentration in each administration. From other studies on new drug carriers containing PTX, it is well known that even unloaded carriers may have antitumor effects (Schmitt-Sody et al., 2003); therefore, the blank PEG-Mic and blank APRPG–PEG-Mic were also investigated in this study. From the tumor volumes shown in Fig. 8A, we can see the APRPG–PEG-Mic suppressed tumor growth most effectively (P < 0.01), following with Cont-PEG-Mic (P < 0.01) and Taxol1 (P < 0.05). After a 7-day treatment, the growth rate of tumors in the saline group was regarded as the normal growth rate without any treatments. Compared with saline group, we could see that blank PEG-Mic had no effect on tumor growth (P > 0.05), whereas interestingly, blank APRPG–PEG-Mic showed a slight tumor inhibition effects, which may due to the specific targeting to tumor neovascular and might further enhance the antitumor effect of APRPG-modified micelles. As expected, the tumor volume in APRPG–PEG-Mic group was only 22.0%, 37.2% and 43.6% in comparison with saline, Taxol1 and Cont-PEG-Mic group, respectively, after a 21-day treatment. The significant increase of antitumor efficacy was mainly due to the cooperative effects of passive targeting by long-circulation and EPR effect and the active targeting by the specific binging of APRPG to tumor neovascular, which not only increased the concentration in tumor tissues but also bring indirect lethal damage to tumor cells by the destroy of newly formed blood vessels. As shown in Fig. 8B, we can easily see that the body weight in Taxol1 group decreased significantly

Fig. 9. Kaplan–Meier survival plot of S-180 tumor-bearing mice treated with Taxol1 (blue), Cont-PEG-Mic (red), APRPG–PEG-Mic (green) or saline (purple). Significant differences are indicated as follows: *P < 0.05, **P < 0.01 and ***P < 0.001. (For interpretation of the references to color in this figure legend, the reader is referred to the web version of this article.) Abbreviations: Cont-PEG-Mic, non-modified paclitaxel micelles; APRPG–PEG-Mic, APRPG-modified paclitaxel micelles; S-180, sarcoma-180 cells.

compared to saline group (P < 0.001), which was probably because the repeated injection of high concentration of Cremophor EL and the non-selectively distribution of PTX induced the toxicity to normal tissues. It could be also seen that the increase in body weight of blank APRPG–PEG-Mic and blank PEG-Mic group had no significant difference with saline group (P > 0.05), which indicates a reduction in side effects and systematic toxicity to normal tissues. This result could be further verified by the slightly increase in ContPEG-Mic and APRPG–PEG-Mic groups after a 21-day treatment. In addition, as presented in Fig. 8C, the TIR value in APRPG–PEG-Mic group was calculated to be 70.7%, which was 1.46-fold and 2.10-fold higher than that in Cont-PEG-Mic and Taxol1 group, respectively. The results above demonstrate that APRPG–PEG-Mic showed excellent antitumor activity and decreased the systematic toxicity to normal tissues at the same time.

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The survival rate of treatment groups was considered as the primary parameter in evaluating antitumor efficacy as this parameter is definitive in antitumor studies animal models upon tumor induction (Hoang et al., 2014; Palma et al., 2014). As seen in Kaplan–Meier plot from Fig. 9, all treatment groups were followed up until day 50 and it is evident that the APRPG–PEG-Mic group resulted in the highest survival rate among all groups with the largest number of animals alive for the longest time period (P < 0.001 to saline group and P < 0.05 to Cont-PEG-Mic and Taxol1 group). 60% of tumor-bearing mice were sill alive after 50 days, while the number reduced to 0% in the other three groups. In saline group, all the mice died within 25 days. Obviously, Cont-PEG-Mic and Taxol1 could prolong the survival time slightly. But due to the systemic toxicity of Taxol1 and the none selectivity of Cont-PEGMic their mean survival time were only 25 days. As respect, the APRPG–PEG-Mic group exhibited an obvious prolonged survival time, which may attribute to the targeting profile to tumors and reduced toxicity to normal tissues.

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Please cite this article in press as: P. Guo, et al., In vitro and in vivo evaluation of APRPG-modified angiogenic vessel targeting micelles for anticancer therapy, Int J Pharmaceut (2015), http://dx.doi.org/10.1016/j.ijpharm.2015.03.067

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4. Conclusions

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In this study, we successfully conjugated the peptide APRPG to the amphiphilic copolymer PEG–PLGA and prepared paclitaxelloaded micelles APRPG–PEG-Mic where peptide APRPG was used for active targeting to angiogenic vessels and PEG was used for endowing long-circulation property. PEGylated micelles displayed a sustained release behavior in vitro. Compared to commercial Taxol1 and Cont-PEG-Mic, APRPG–PEG-Mic exhibited higher cytotoxicity and specific association to confluent endothelium cells EA.hy926, achieved higher accumulation in tumor tissues, effectively reduced tumor growth of tumor-bearing mice, decreased systemic toxicity and prolong the survival time of tumor bearing mice. Therefore, the synthesized amphiphilic copolymer APRPG–PEG–PLGA and its application in micelle formulations may be an effective method for delivery of antitumor drugs to angiogenic vessels of tumor tissues and have promising potential for efficient tumor therapy.

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Acknowledgements

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This research was supported by the National Science Foundation (No. 81273447)and the Joint Specialized Research Fund for the Doctoral Program of Higher Education, MOE & Department of Education of Liaoning Province (20102134120002).

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Please cite this article in press as: P. Guo, et al., In vitro and in vivo evaluation of APRPG-modified angiogenic vessel targeting micelles for anticancer therapy, Int J Pharmaceut (2015), http://dx.doi.org/10.1016/j.ijpharm.2015.03.067

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