A safe, simple and efficient doxorubicin prodrug hybrid micelle for overcoming tumor multidrug resistance and targeting delivery

    A safe, simple and efficient doxorubicin prodrug hybrid micelle for overcoming tumor multidrug resistance and targeting delivery Yuling Bao, Mingxing Yin, Xiaomeng Hu, Xiangting Zhuang, Yu Sun, Yuanyuan Guo, Songwei Tan, Zhiping Zhang PII: DOI: Reference:

S0168-3659(16)30360-1 doi: 10.1016/j.jconrel.2016.06.003 COREL 8312

To appear in:

Journal of Controlled Release

Received date: Revised date: Accepted date:

19 February 2016 1 May 2016 2 June 2016

Please cite this article as: Yuling Bao, Mingxing Yin, Xiaomeng Hu, Xiangting Zhuang, Yu Sun, Yuanyuan Guo, Songwei Tan, Zhiping Zhang, A safe, simple and efficient doxorubicin prodrug hybrid micelle for overcoming tumor multidrug resistance and targeting delivery, Journal of Controlled Release (2016), doi: 10.1016/j.jconrel.2016.06.003

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A Safe, Simple and Efficient Doxorubicin Prodrug Hybrid

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Targeting Delivery

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Micelle for Overcoming Tumor Multidrug Resistance and

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Yuling Bao 1, Mingxing Yin 1, Xiaomeng Hu 1, Xiangting Zhuang 1, Yu Sun 1, Yuanyuan Guo 1, Songwei Tan 1,2,*, Zhiping Zhang 1,2,3,*

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Tongji School of Pharmacy

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National Engineering Research Center for Nanomedicine

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Hubei Engineering Research Centre for Novel Drug Delivery System

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Huazhong University of Science and Technology, Wuhan 430030, China

* Corresponding authors:

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Prof. Dr. Zhiping Zhang, E-mail: [email protected]

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Dr. Songwei Tan, E-mail: [email protected]

Address: Tongji School of Pharmacy, Huazhong University of Science and Technology, 13 Hangkong Road, Wuhan, China

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ACCEPTED MANUSCRIPT Abstract

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A pH-sensitive prodrug, TPGS-CH=N-DOX, was introduced by conjugating anticancer drug, doxorubicin (DOX), onto D-α-tocopherol polyethylene glycol 1000 succinate (TPGS) via a cleavable Schiff base linkage. The prodrug was mixed with a PEGylated lipid to form a simple but multifunctional hybrid micelle system, which can realize high drug loading capability and biocompatibility, extended blood circulation time, inhibited drug resistance in cancer cells, improved therapeutic response, reduced side effects, and easy functionalities for targeting delivery. The hybrid micelles exhibited in vitro pH-sensitive drug release, enhanced cellular uptake and strengthened cytotoxicity on both drug-sensitive human breast cancer MCF-7 and resistant MCF-7/ADR cells. P-glycoprotein functional inhibition and mitochondria-associated cell apoptosis induced by TPGS were thought to play an important role in overcoming the multidrug resistance. As a result, the hybrid micelles demonstrated good anticancer efficacy in MCF-7/ADR xenograft model. Additionally, after modifying with a tumor-specific targeting peptic ligand, cRGD, the tumor growth/metastasis inhibition was further evidenced in integrin receptor overexpressed melanoma cancer B16F10 and even murine hepatocarcinoma H22 models. This TPGS-based pH-sensitive prodrug provides a safe and “Molecular economical” way in the rational design of prodrugs for overcoming multidrug resistance and targeting delivery, which can improve the potency for clinical use.

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Keywords: D-α-tocopherol polyethylene glycol 1000 succinate, pH-sensitive, targeting, drug resistance, prodrug

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Chemical compounds studied in this article D-alpha-tocopherol succinate (PubChem CID: 20353); RGD peptide (PubChem CID: 104802); Doxorubicin hydrochloride (PubChem CID: 443939); 4-Formylbenzoic acid (PubChem CID: 12088); DSPE-mPEG-2000 (PubChem CID: 86278269)

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ACCEPTED MANUSCRIPT 1. Introduction

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Doxorubicin (DOX), one of the leading anticancer drugs, has been extensively used in oncology with a broad spectrum of activity against various solid and hematologic malignancies. However, its clinical application is hindered because of the limited efficacy and severe toxicity to normal tissues, especially cardiotoxicity and nephrotoxicity [1]. Moreover, in the mid-late stage of chemotherapy, the intracellular level of DOX can be reduced dramatically induced by the drug resistance which may lead to chemotherapy failure [2]. In the past decades, to improve anticancer efficiency and minimize side effects, various nanocarriers of DOX have been designed, including liposomes, micelles, dendrimers, solid-lipid nanoparticles, and polymeric nanoparticles [3-7]. However, the progress on translating nanomedicine into clinic still keeps great challenge. The successful clinic translation of liposome, e.g. DOXIL®, was also subjected to some limitations such as relative low drug payload, lack of tunable triggers for drug release, poor storage stability and drug leakage during circulation [8, 9].

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Conjugating anticancer agent on the functional polymer is an alternative approach to encapsulate drug by covalent technique, which can probably avoid untimely drug release [10]. One of the major strategies for drug modification is polymer-drug conjugation, which modulates drug on a molecular level to increase the solubility and stability in blood circulation, thus change the biodistribution, improve the pharmacokinetics (PK) and pharmacodynamics (PD), increase therapeutic effects and reduce side effects [11, 12]. This method seems like a promising way to introduce prodrugs to clinical application since some polymeric prodrugs, such as poly-glutamic acid (PGA)-SN38 (NK012; Nippon Kayaku, Co.) [13], N-[2-hydroxylpropyl] methacrylamide (HPMA)-doxorubicin (PK1/FCE28068; Pfizer Inc.) [14], Adagen® (Adenosine deaminase), Onscaspar® (l-asparaginase) and Pegasys® (PEGylated IFN-α-2a) have been approved by FDA in clinical trial or already on the market. These FDA approved polymers, including PGA, PEG and dextran, have been widely used to develop polymer-drug conjugates (prodrugs) [15-17]. In cancer therapy, the therapeutic efficacy of nanomedicine depends not only on nanoparticles localization at the tumor tissue but also on the amount of drug that can take effect [18]. To achieve the fast release of drugs in designed location (e.g. tumor), polymeric prodrugs can be designed to response to the intrinsic stimuli of tumor microenvironment, such as acidic pH, specific enzymes, and redox potential or to realize “active targeting” of tumor cells through selectively overexpressed surface receptors [19-21]. For example, the extracellular pH of solid tumor is more acidic (pH ~6.8) in comparison to normal tissue because of the increased aerobic glycolysis in cancer cells [22, 23], and the endo/lysosomes of cancer cells are even more acidic (pH < 6) [24, 25]. The utilization of sharply changed pH value in different tissue and cellular compartments can be considered as a robust and simple strategy to achieve broad tumor applicability. Thus pH-sensitive prodrugs were usually designed to realize the controllable drug release. The design of an ideal anticancer nanomedicine should prefer to take use of nontoxic 3

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and biocompatible material as the matrix, entrap more payloads in the nanocarrier to decrease excessive carrier material burden and achieve high drug loading efficiency for future clinical application, provide an improved therapeutic effect with enhanced drug distribution and penetration in tumor, reduce the undesired adverse reactions, and so on [26, 27]. The balance between nanotechnology and clinical application is to reject useless procedure, simplify and better the production. D-α-tocopherol polyethylene glycol 1000 succinate (TPGS), a PEGylated vitamin E, is a derivative of the natural Vitamin E which has been approved by FDA as a pharmaceutic adjuvant and used in nanocarriers widely [28, 29]. Those TPGS-based drug delivery systems exhibited high drug encapsulation efficiency, desired drug release kinetics, increased cellular uptake and enhanced therapeutic effects. More importantly, as a P-glycoprotein (P-gp) inhibitor, TPGS has shown great potential in overcoming multidrug resistance (MDR) [30, 31]. Our previous work has exhibited that introducing TPGS to chemotherapeutic nanomedicine could significantly strengthen their P-gp inhibition capability [20, 32], which are in accordance with the works of other research groups [33, 34]. So TPGS can be a suitable candidate for the DOX conjugation and delivery. In the case of MDR tumor, such TPGS-based prodrug can avoid administration of excessively high amount of carrier material or co-administration with resistance inhibitor. The internalized TPGS could also be a functional component rather than exogenous stress in a ‘Molecular Economy’ way-high conjugation of payload, easy fabrication, and acting as a functional component with synergistic antitumor effects (Scheme 1).

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In this work, we designed an endosomal pH-sensitive DOX conjugate based on TPGS. DOX was chemically conjugated to the backbone of the polymer via acid-labile Schiff base linker, which is sufficiently stable at pH 7.4 but readily cleavable in an acidic environment [35, 36]. To increase the stability and functionality of the prodrug system, a PEGylated lipid (another pharmaceutic adjuvant) was introduced. The self-assembly behavior, pH-triggered structure change and environment-sensitive drug release of the prodrug hybrid micelle were executed here. The in vitro cytotoxicity and the related mechanism of overcoming MDR of the prodrug hybrid micelles were studied on DOX-sensitive human breast cancer MCF-7 and -resistant MCF-7/ADR. The therapeutic outcomes were further investigated in MCF-7/ADR xenograft model. Furthermore, through modifying tumor specific ligand of cRGD with the prodrug hybrid micelles, the self-assembled prodrug formulation can be also applied for improving antitumor efficacy on the therapeutic and metastatic models of murine melanoma via targeting delivery.

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Scheme 1. Cancer cell-killing action scheme of TPGS-CH=N-DOX prodrug hybrid micelles in a “Molecular Economy” way.

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2.1 Materials

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2. Materials and Methods

Doxorubicin hydrochloride was obtained from Beijing Huafeng United Technology Co., China. The cRGD peptide, cyclo(Arg-Gly-Asp-D-Phe-Cys) was synthesized by Bioyeargene Biosciences, China. 1,2-distearoyl-sn-glycero-3-phosphoethanolamine-N-[methoxy(polyethylene glycol)-2000] (DSPE-PEG2000) and 1,2-distearoyl-sn-glycero-3-phosphoethanolamine-N-[maleimide(polyethylene glycol)-2000] (DSPE-PEG2000-Mal) were purchased from Avanti Polar Lipids, AL, USA. TPGS, 4-formylbenzoic acid (TPA), dicyclohexylcarbodiimide (DCC) and Trypsin-EDTA were purchased from Sigma Aldrich, USA. 4-Dimethylamino pyridine (DMAP) was purchased from Aladdin, China. RPMI-1640 medium, penicillin-streptomycin, fetal bovine serum (FBS) and trypsin without EDTA were purchased from Hyclone, USA. MTT (3-(4,5-dimethyl-thiazol-2-yl)-2,5-diphenyl tetrazolium bromide) and DAPI were purchased from Biosharp, South-Korea. Lysotracker Green DND26 was obtained from Thermo Fisher, USA. The solvents including dimethyl sulfoxide (DMSO), anhydrous N, N-dimethylformamide (DMF), anhydrous methanol, ethanol, diethyl ether and chloroform were of analytical grade and purchased from Sinopharm, China. 5

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Female C57BL/6 mice and male Sprague–Dawley (SD) rats were purchased from the Experimental Animal Center of Wuhan University, China. Female nude mice were purchased from HFK bioscience Co, LTD (Beijing, China). All animal procedures were performed under the approval of the Ethics Committee of Huazhong University of Science and Technology, China.

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2.2 Preparation and Characterization of TPGS-CH=N-DOX Prodrug Hybrid Micelles

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TPGS-CH=N-DOX prodrug hybrid micelles (TD) were prepared by a simple solvent-evaporation method. Briefly, the prodrug and DSPE-PEG (5:1, w/w) were dissolved in acetone and then added dropwise into aqueous phase under magnetic stirring. After stirred overnight, the resulting mixture was filtered through 0.45 μm syringe filter. To prepare cRGD conjugated micelles (RGD-TD), the equivalent molar amount of DSPE-PEG-Mal was added instead of DSPE-PEG in the above procedure and the obtained micelles were incubated with cRGD peptide at RT for 12 h. After that, the resulting mixture was placed in a Millipore micro-dialyzer (MWCO 10kDa) and centrifuged at 3,000 rpm for 40 min to concentrate the solution and remove free cRGD.

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The particle size, size distribution and ζ potential of the hybrid micelles were determined by the dynamic light scattering (DLS, Zeta Plus, Brookhaven, USA). The surface morphology of the micelles was observed by transmission electron microscope (TEM, JEM-1230, Japan). The stability of hybrid micelles in PBS and FBS was monitored by measuring the size change. TD was diluted in 90% FBS with a final micelles concentration of 1 mg/mL and the particle size was measured at designed time intervals to monitor the particle interaction with protein in the presence of FBS. The pH sensitivity of TD was investigated by testing the particle size distribution in the simulated endo/lysosome conditions (pH = 5.0) at desired time intervals (0 min, 10 min, 20 min, 30 min, 1 h, 2 h, 3 h and 4 h). 2.3 In Vitro Drug Release Kinetics The in vitro DOX release of TD was determined at 37 °C using a dialysis method described previously [37]. The DOX release study was conducted at three different buffer solutions of pH 5.0 (acetate buffer solution) or pH 7.4 and 6.8 (phosphate buffer saline, PBS). DOX concentration was measured using fluorescence spectrophotometer (F-4600, Shimadzu, Japan) with a slit width of 5 nm and excitation/emission wavelength at 479 nm/590 nm. Experiments for all samples were performed three times at each pH value. 2.4 Cell Culture MCF-7 cells, from the American Type Culture Collection (ATCC, Manassas, VA, USA), were cultured in RPMI 1640 medium containing 10% FBS, 100 U/mL penicillin G sodium, and 100 μg/mL streptomycin sulfate (complete 1640 medium). 6

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2.5 Drug Accumulation and Retention of Hybrid Micelles

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MCF-7/ADR cells, donated by Dr. Yaping Li (Shanghai Institute of MateriaMedica, Chinese Academy of Sciences), were cultured in complete 1640 medium with 1 µg/mL DOX. Murine melanoma B16F10 cells, from Chinese Academy of Sciences Cells Bank of China, were cultured in complete 1640 medium. Cells were maintained at 37 °C in a humidified and 5% CO2 incubator.

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MCF-7 or MCF-7/ADR cells were seeded into 6-well plate at a density of 2×105 cells per well. After 24 h incubation, the cells were treated with TD solution or free DOX for different durations (i.e. 0.5, 1, 2, 4, 6, 12 and 24 h) at an equivalent DOX concentration of 5 µg/mL. Afterwards, the cells were trypsinized, washed twice with cold PBS, and resuspended in 300 µL of PBS. The cellular fluorescence intensity of DOX was measured using flow cytometer (Becton Dickinson, San Jose, CA) and the data were analyzed using Cell Quest software. For in vitro cellular pharmacokinetics, the cells were lysed with lysis buffer containing phenylmethanesulfonyl fluoride (PMSF). DOX concentration in cell lysates was measured by HPLC and normalized to the total cellular protein content. To investigate the drug efflux and retention, MCF-7 and MCF-7/ADR cells were treated with free DOX, TD, a mixture of free DOX with 10 mM verapamil (DOX+Verapamil) and a mixture of free DOX with the equivalent free TPGS released from prodrug (DOX+TPGS) for 4 h, respectively. After washing twice with PBS, the cells were further incubated with blank medium for 2 h at 37 °C. Then, the cells were collected and washed with PBS to determine the DOX retained in the cells by flow cytometer.

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2.6 Cellular Distribution of Hybrid Micelles

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MCF-7, MCF-7/ADR and B16F10 cells were separately seeded into 24-well plate at a density of 1.0×104 cells per well. After the cells reached 70% confluence, the medium was replaced with free DOX, TD or RGD-TD at a concentration of 5 μg DOX/mL at 37 °C. At determined time interval, cells were treated with 200 nM Lysotracker Green DND-26 for 20 min (with or without), followed by exposure to 4% paraformaldehyde for 15 min. The cells were further washed with cold PBS, stained with DAPI for 10 min, and then mounted on a glass slide for observation by confocal laser scanning microscopy (CLSM, 710META, Zeiss, Germany). 2.7 In Vitro Cytotoxicity and Apoptosis Analysis The cytotoxicity of hybrid micelles was evaluated in MCF-7, MCF-7/ADR and B16F10 cells, in comparison to free DOX. Briefly, cells were seeded in 96-well plates at a density of 5.0×103 cells per well followed by overnight attachment. The cell medium was then replaced with hybrid micelles with DOX concentration ranging from 0.02 to 20 µg/mL and then incubated for 24, 48 and 72 h, relatively. The cell viability was determined by MTT assay and the half-maximal inhibitory concentrations (IC50) were calculated by SPSS software. The nuclei morphology of MCF-7/ADR cells treated with free DOX and TD was further observed using a 7

ACCEPTED MANUSCRIPT fluorescence inversion microscope (Olympus IX71, Tokyo, Japan). 2.8 The Synergistic Antitumor Function of TPGS

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2.8.1 ROS Production

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MCF-7/ADR cells cultured on 6-well plate at 2.0×105 cells/well were treated for 6 h with serum-free culture medium (control), Rosup (positive control), free DOX, TD and corresponding TPGS, respectively with DOX concentration of 5 μg/mL. Then, the cells were collected, suspended, and incubated with DCFH-DA (10 μM) at 37 °C for 20 min. After rinsing with cold PBS twice, the DCF fluorescence of cells was detected by flow cytometer.

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2.8.2 Mitochondrial Depolarization

2.8.3 ATP Level

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Mitochondrial membrane potential (ΔΨm) was determined by the percentages of the cationic lipophilic fluorochrome JC-1 in red and green fluorescence intensities [38]. The MCF-7/ADR cells seeded on 6-well plate at 2.0×105 cells per well were incubated for 6 h with serum-free culture medium (as control), free DOX, TD and corresponding TPGS, respectively. Then, the cells were trypsinized, harvested and stained with a JC-1 mitochondrial transmembrane potential assay kit (Beyotime, China) according to the manufacturer’s protocol. The FL1 and FL2 fluorescence intensities of each sample were determined using flow cytometer.

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MCF-7/ADR cells seeded into 12-well plate (5.0×104 cells/well) were treated with free DOX, TD and TPGS with the DOX concentration of 1 μg/mL. Intracellular ATP level was determined using the luciferin-luciferase-based ATP luminescence assay kit (Beyotime Institute of Biotechnology, China). 2.9 In Vivo Pharmacokinetics and Biodistribution TD and free DOX were administered intravenously (i.v.) to SD rats at a dose of 10 mg DOX/kg, respectively (n = 4). At predetermined time of 30 min, 1 h, 2 h, 4 h, 8 h, 12 h, and 24 h, blood samples were obtained using a heparinized tube. Plasma samples were isolated by centrifuged at 3,000 rpm for 10 min, mixed with an equal volume of methanol and extracted with 1 mL chloroform by vortex for 5 min. DOX concentration was measured by HPLC with the detector operated at 470 nm/585 nm (excitation/emission), using a Restek C18 reverse phase column (5 µm, 150 mm×4.6 mm). KH2PO4 buffer (1/15 mM, pH 4.21, adjusted with H3PO4) and acetonitrile (75:25 v/v) were used as the mobile phase with a flow rate of 1.0 mL/min. The calibration curve was linear between the concentration ranges of 25 ng/mL to 25 µg/mL in plasma (R2 = 0.9999). Pharmacokinetic parameters such as half-life (t1/2), mean retention time (MRT), area under the curve (AUC), and clearance (CL) were calculated by the drug and statistics (DAS) software (version 2.1.1, Mathematical 8

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For biodistribution study, free DOX, TD and RGD-TD were i.v. injected into B16F10 tumor-bearing mice at a dose of 5 mg DOX/kg, respectively (n = 3). At time intervals of 1, 4, 8 and 24 h after injection, the major organs (heart, liver, spleen, lung and kidney) and tumors were collected from these mice. The tissues were then washed with saline, weighed and homogenized with PBS. The DOX content was measured in a similar way as pharmacokinetics study. The % injected dose per gram tissue was calculated using the following equations:

2.10 In Vivo Tumor Growth Inhibition

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MCF-7/ADR, aggressive murine hepatocarcinoma (H22) and B16F10 models were respectively used to evaluate the therapeutic effect of hybrid micelles.

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In the breast xenograft tumor model, 5×106 MCF-7/ADR cells in 200 µL PBS were inoculated subcutaneously (s.c.) at the right flank of female BALB/c nude mice (4-6 w, 18-20 g). Various treatments were started when tumor reached the volume around 50 mm3. Then, mice were randomly divided into four groups (n = 6) and received i.v. administration of free DOX (5 mg/kg), TD (5 mg DOX/kg), TD (10 mg DOX/kg) and normal saline (N.S.), respectively on day 6, 10, and 14. Tumor sizes were monitored with a digital caliper and calculated based on the formula: (L×W2 )/2, where L and W are the length and width of tumor. In H22 tumor model, 5×106 H22 cells were inoculated s.c. at the right flank in female Kunming mice (4-6 w, 18-20 g). Treatment was initiated when tumor reached a volume around 50 mm3 and the mice in randomly divided groups (n = 7) were treated respectively with N.S., free DOX, TD and RGD-TD at an equivalent DOX dose of 5 mg/kg i.v. on day 1, 3, 5 and 7. Tumor size and body weight were monitored every day. In B16F10 tumor model, 2×105 cells were inoculated s.c. at the right flank of female C57 mice (6-8 week, 14-18 g). When the tumor volume reached approximately 50 mm3, mice were similarly treated as above mentioned groups (n = 8) on day 9, 13, and 17. Tumor size and body weight were monitored every other day. At the end of experiment, all the mice were sacrificed. The collected tumor tissues were analyzed by hematoxylin–eosin (H&E) staining assay and immunofluorescence analysis against CD31 antibody, cleaved PARP antibody/TUNEL or frozen in the dark at -80 °C overnight, and further prepared to frozen sections for observation by CLSM. 9

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The metastasis model was established to determine the effect of the micelles on tumor metastasis using female C57 mice (6-8 week, 14-18 g). B16F10 cells suspension (5×105 cells in 0.1 mL of saline) were injected i.v. to the mice. The mice were randomly divided into 4 groups and treated with above mentioned formulations (n = 5) on day 2, 5, and 8. Twenty days later, animals were euthanized with CO2. The lungs and kidneys were excised by gross anatomy and photographed. Organs containing metastasis were fixed in Bovin fixative solution, and sections were stained with H&E. The number of metastatic lung nodules (pigmented nodules at the surface of the lung) was counted.

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2.12 In Vivo Safety Evaluation

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2.13 Statistics

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To evaluate the safety of hybrid micelles compared to free DOX, healthy Kunming mice (4-6 w, 18-20 g) were randomly divided into 5 groups (n = 4). Mice were treated according to the above-described procedure with an additional group of administrating an equivalent TPGS-TPA amount of the micelles. Blood samples were drawn from the ophthalmic vein on day 9, and alanine aminotransferase (ALT), aspartate transaminase (AST) and blood urea nitrogen (BUN) were measured according to the manufacture's instruction (Nanjing Jiancheng Bioengineering Institute, China). Organs (heart, liver, spleen, lung, and kidney) were also fixed in 4% paraformaldehyde solution, stained with H&E and photographed by optical microscopy.

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Statistical analysis was conducted by using a two-tailed Student’s t-test with p < 0.05 as statistically significant difference. 3. Results and Discussions 3.1 Preparation and Characterization of the Hybrid Micelles To achieve good stability, long circulation and easy surface functionalities for targeting delivery of DOX, the hybrid micelles of prodrug TPGS-CH=N-DOX (TD) were fabricated by a simply process with lipid DSPE-PEG as shown in Figure 1A. Although TPGS-CH=N-DOX prodrug can self-assemble to micelles with size above 300 nm, the micelles were easily aggregated within 24 h (data not shown). The rigid plane of Schiff base linker between TPGS and DOX may limit the degree of freedom of the PEG segment as reported before [39]. So the TPGS-CH=N-DOX prodrug micelle may be with a loose/large core and difficult to maintain the core-shell structure due to the folded thin PEG shell. This unstable micelle structure may be not good enough for realizing the long circulation in blood and good in vivo stability. In order to get an intense core-shell structure, DSPE-PEG2000 containing a strong hydrophobic lipid moiety and a long PEG chain was chosen [21]. The resulted hybrid 10

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micelle was with a decreased diameter of ~150 nm and a near spherical uniform shape (Figure 1B), which may be attributed to the hydrophobic interactions of two kinds of amphiphilic polymers. Thus, we envisioned that this hybrid micelle with small size may substantially have advantages in achieving tumor penetration and accumulation of drugs [40]. The stability of hybrid micelles was conducted in physiologically simulated pH7.4 PBS and FBS (Figure 1C). After incubated in PBS at 37 °C for 5 days, there was no significant size change of the hybrid micelles. This phenomenon was also observed when the dispersing media was changed to FBS. The structural integrity of this hybrid micelle in the bloodstream may realize the tumor passive targeting via enhanced permeation retention (EPR) effect and could lay a basis for long-term circulation and subsequent antitumor activity. The reproducible particle size with a simple method under non-strict conditions makes the TD preparation robust enough for large-scale production.

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The pH-sensitive behavior of the hybrid micelles was further investigated. To demonstrate the cleavage of Schiff base-linked prodrug micelles in the acidic environment, we incubated TD in pH 5.0 buffer solution for simulating endo/lysosomal acidic environment. At the predetermined time points of 10 min, 20 min, 30 min, 1 h, 2 h, 3 h and 4 h, the particle size and size distribution were detected as shown in Figure 1D. The status of TD micelles was original in narrow size range with low polydispersity index (Figure 1D.a). Then the particles size as well as the proportion of expanded particle gradually increased (Figure 1D.b-d). The broken of Schiff base linker resulted in the accumulation of the micelles and the formation of visible large precipitation. Meanwhile a population of particles with size less than 10 nm was appeared after 1 h incubation that may be related to the formation of small sized micelles by dissociated TPGS (Figure 1D.e-h). The above results confirmed the acid-sensitivity of the Schiff base in TD. It is not difficult to infer that the hybrid micelles could be broken quickly under acidic endo/lysosome condition of cancer cells, while remained stable under normal physiological conditions. To determine the DOX release profile from TD, the hybrid micelles were diluted in buffer solution with pH 5.0, 6.8 and 7.4, respectively, and then dialyzed at 37 °C for 168 h. As shown in Figure 1E, TD exhibited obvious pH-related release behavior. At the acidic pH 5.0, about 60% of DOX was released after 24 h incubation. It was almost 4-fold or 2.2-fold higher than that of DOX released at pH 7.4 or 6.8, implying that the hybrid micelles presented the acidic pH-sensitive DOX release property. In contrast, the cumulative release of DOX from the micelles with a succinate linker was both less than 10% at pH 5.0 and pH 7.4 after 24 h of incubation [28]. The results demonstrated that the Schiff base linker has a good ability to adjust and control the profile of pH-triggered DOX release.

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Figure 1. Characterization of TPGS-CH=N-DOX prodrug hybrid micelles. (A) Schematic illustration of the formation of hybrid micelles (TD). (B) TEM image of TD. (C) Time-dependent colloidal stability of TD in PBS and FBS at 37 oC. (D) Change in particle size distribution of hybrid micelles at pH 5.0 in different time intervals (a-h represent 0 min, 10 min, 20 min, 30 min, 1 h, 2 h, 3 h and 4 h, respectively). (E) Cumulative release of DOX from hybrid micelles at different pH values.

3.2 DOX Cellular Uptake and Retention

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The premise of anticancer drug to take effect is first uptaken by tumor cell and then accumulated in efficient concentration for cytotoxicity. The DOX-sensitive MCF-7 cells and resistant MCF-7/ADR cells were employed as in vitro models. Figure 2A showed the cell uptake efficiency determined by flow cytometer on TD as comparison with the pristine DOX with the equivalent DOX concentration of 5 µg/mL. For MCF-7 cells, the high cellular accumulation of free DOX with time dependence was attributed to its passive diffusion. The intracellular DOX intensity of TD was slightly weaker than that of free DOX. It may be attributed to that the micellar association or complexation limited its diffusion ability compared with small molecular drug [41]. When it came to MCF-7/ADR cells (Figure 2B), the internalization of free DOX was extremely low even incubation for 6 h owing to the DOX-resistant property [42]. However, TD exhibited higher intracellular accumulation than free DOX with around 2.3-fold mean fluorescence intensity (MFI) after incubation for 4 h. The prominent inhibiting effect of TPGS on P-gp may be mainly responsible for this phenomenon. The internalization of TD like other nanocarriers also counteracted the effect of P-gp mediated drug efflux. In order to confirm the impact of TD over free DOX in dug accumulation, in vitro cellular pharmacokinetics was performed. After incubating for different time intervals, the intracellular drug accumulation was determined by analysis of DOX concentration 12

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in the cell lysates, which was normalized to total cellular protein content (Figure 2C and 2D). The intracellular concentration of free DOX in MCF-7/ADR cells was only 5.87 ± 0.65 μg/mg protein after 24 h of incubation, which was significantly lower than that in MCF-7 cells (13.32 ± 0.80 μg/mg protein). When incubated with TD, a much higher intracellular accumulation of DOX was observed in MCF-7/ADR cells. The cellular amount of DOX in MCF-7/ADR cells was increased rapidly with the extension of incubation time, reaching 11.83 ± 1.34 μg/mg protein within 4 h, which was about 2-fold of the value of free DOX treatment. Furthermore, the cellular DOX accumulation of TD showed no significant difference in MCF-7/ADR cells and MCF-7 cells, and the value was almost the same as free DOX in MCF-7 cells at similar conditions, which indicated the great potential of TD in overcoming MDR.

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In drug efflux assay, the cells were first treated with free DOX and TD for 4 h, and then incubated with fresh medium for 2 h. The fluorescent intensity of DOX retained in cells was determined by flow cytometer as shown in Figure 2E and F. For MCF-7 cells, free DOX exhibited remarkably higher fluorescence intensity and no significant difference in intensity change after subsequent 2 h incubation with free medium (Figure 2E). However, the content of DOX was very low in free DOX treated MCF-7/ADR cells and decreased 50% within the subsequent incubation as the result of efflux pump of P-gp. When DOX was combined with verapamil (a classical P-gp inhibitor), its concentration in cells was significantly increased. Once verapamil was removed, DOX was pumped outside the cells so an obvious decrease of MFI was emerged. Interestingly, the decrease was prevented by conjugating DOX to TPGS via the pH-sensitive Schiff base linker. While treating MCF-7/ADR cells with TD, the intracellular MFI of DOX was improved and the efflux was depressed. The uptake and retention trend was similar to that of the cells treated with the admixture of DOX and the according amount of TPGS in TD (the group of DOX+TPGS) (Figure 2F). This might be attributed to the dissociated TPGS from the quick hydrolysis of the Schiff base linked prodrug within 4 h exhibited the P-gp inhibition effect. These results indicated that the hybrid micelles can overcome the efflux of P-gp and deliver DOX into MDR cells successfully. This may be in associated with the internalization of micelles and the P-gp inhibiting ability of TPGS. 3.3 Sub-Cellular Localization of Micelles The localization of DOX in cells was qualitatively examined by confocal microscope. In MCF-7 cells (Figure 2G), the red fluorescence intensity in cells incubated with free DOX was comparable to that exposed to TD, which was in accordance with the quantitative experiment results. For TD, the representative red fluorescence was localized in cytoplasm around the nucleus after 4 h incubation, while free DOX could easily enter into nucleus after short time incubation. The phenomena were different in MCF-7/ADR cells (Figure 2H). Free DOX showed faint red fluorescence signals around the nucleus even for 12 h incubation, inferred to be caused by the low drug accumulation as presented in last section. However, much stronger fluorescence signal 13

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was observed in the cytosol when the cells were incubated with TD. This also corresponded well with the results mentioned above where TD exhibited good capability to be retained in MCF-7/ADR cells. More importantly, the red fluorescence could be found in the nucleus of MCF-7/ADR cells after extending incubation time of TD for 24 h, while the cells treated with free DOX only showed slightly increased cytoplasmic fluorescence. The confocal microscope results demonstrated that TD had good capability of overcoming drug resistance as the enhanced drug distribution intracellularly on MCF-7/ADR cells compared with free drug [33].

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For the internalization induced by nanoparticles, the main mechanism was endocytic pathway which could bypass the efflux protein on the membrane and subsequently increase cellular drug accumulation [43]. The image shown in Figure 2I revealed that the red fluorescence was mainly localized in the acidic organelles (green) after 2 h incubation of TD, indicating that the endocytic pathway was involved. The micelles exhibited some endo/lysosomal escape with dominant red fluorescence and the separated green fluorescence after incubation up to 4 h as shown in Figure S5. With further incubation for 8 h, some of the red fluorescence was found to be co-localized with blue fluorescence, indicating the released DOX existed in the nucleus. The co-localization with acidic organelles would trigger the burst release of DOX from this pH-sensitive prodrug hybrid micelles and play an important role in enabling TD to be an efficient formulation for killing MDR cells [44].

Figure 2. In vitro drug uptake/retention and subcellular localization of hybrid micelles. (A-D) The accumulation of DOX in MCF-7 cells (A and C) and MCF-7/ADR cells (B and D) after incubation with DOX and TD for different time. A and B were results analyzed by flow cytometer. C and D were DOX concentration in cells analyzed by HPLC. (E and F) The efflux of DOX from MCF-7 cells (E) and MCF-7/ADR cells (F). Cells were first treated with DOX and TD for 4 h, and then incubated with fresh medium for 2 h. (G, H and I) Confocal microscopic images of MCF-7 14

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The in vitro cytotoxicity and apoptosis induced were further conducted on free DOX and prodrug hybrid micelles. The cytotoxicity profiles on MCF-7 and MCF-7/ADR cells were shown in Figure 3A and the relative IC50 values were also calculated as Table 1. In MCF-7 cells, DOX could be internalized easily and accumulated in cell nuclei spontaneously, thereby resulting in obvious cytotoxicity. The conjugation of DOX to TPGS did not result in the superior cytotoxicity during 72 h incubation. Due to the inordinate efflux of DOX in MCF-7/ADR cells, the IC50 of free DOX was above 500 µg/mL after incubation for 48 h, which was at least 2100-fold resistant to DOX in comparison with the parent MCF-7 cells (IC50 0.23 µg/mL). After conjugating DOX to TPGS with a pH-sensitive bond, the cytotoxicity of TD against MCF-7/ADR cells was remarkably increased by 820-fold, which may be caused by the increased cellular uptake and inhibited effusion from TD. To further present MDR reversing efficiency of TD, resistance index (RI) was calculated as the IC50 ratio of MDR cells to sensitive cells for the same formulations (i.e. IC50(MCF-7/ADR)/IC50(MCF-7)) [32]. The RI of TD was only 2.47 at 72 h, which was 89-fold lower than that of DOX (219.88), indicating the efficient MDR overcoming capability of TD.

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Table 1. IC50 values of DOX and TD against MCF-7 and MCF-7/ADR cells determined by MTT assay. IC50 values (µg/mL) MCF-7

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58.61

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0.23

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>500

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The apoptosis was reported to develop as first mild convolution, condensation of cytoplasm, chromatin compaction and segregation observed, following by nucleus fragmentation, blebbing and apoptosis bodies produced, and ending with phagocytosis. To confirm that the inhibition of cancer cells was a consequence of apoptosis, the nucleus morphology was observed by fluorescence microscope. As presented in Figure 3B, MCF-7/ADR cells exposed to free DOX just showed sporadic apoptosis characters after 24 h incubation. In contrast, TD significantly increased the apoptotic effect of DOX and most of cells treated with TD appeared typical morphology of apoptosis including crescent-shaped chromatin and membrane blebbing. 15

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As a novel group of “mitocans”, α-tocopherol succinate (α-TOS) was reported to be an anticancer agent by targeting and destabilizing mitochondria with the elevated intracellular ROS generation, mitochondrial depolarization and functional degradation [45]. The influence of the hybrid micelles on intracellular ROS production in MCF-7/ADR cells was evaluated using a DCF fluorescence method (Figure 3C). Compared to the control and free DOX, the DCF intensity was remarkably increased after exposure to TD and free TPGS, respectively. It may be attributed to that the α-TOS component in TD and TPGS can promote the intracellular ROS production. It should be mentioned that there was an obvious gap between TD and free TPGS in the amount of ROS production. Considering that the glycation products could inhibit the viability and induce oxidative stress in cells, the high ROS level in the hybrid micelles may be caused from the synergetic effect of DOX and TPGS.

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The accumulation of intracellular ROS could induce some pro-apoptotic proteins to form channels through the mitochondrial outer membrane which leads to mitochondria depolarization characterized by the mitochondria membrane potential (ΔΨm) loss. The decrease of ΔΨm is generally accepted as an indicator of cellular apoptosis [46]. The ΔΨm loss was assessed by a switch of JC-1 dye from red to green. In Figure 3D, there was no distinct decrease of ΔΨm in free DOX treated cells as compared to the control. On contrast, TD and free TPGS both exhibited remarkable decrease of ΔΨm up to 70.7 ± 5.9% and 44.7 ± 4.1%, respectively (Figure 3E), indicating that the hybrid micelles had great potency to induce mitochondrial depolarization.

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Since ATP deficiency is among the most direct evidence for functional impeding of mitochondria, the intracellular ATP level was further evaluated on MCF-7/ADR cells treated with DOX, TD and free TPGS. As shown in Figure 3F, the ATP production were decreased to 55.8 ± 4.2% and 28.5 ± 3.8% for TD and free TPGS, respectively, whereas no significant change was observed in DOX compared to the control. It suggested that the downregulation of ATP level in MCF-7/ADR cells was mainly attributed to TPGS. It is known that mitochondria were considered as 'cellular power plants', playing crucial roles in regulating cell energy metabolism and activating related apoptotic pathways. The results exhibited here strengthened the previous assumption that the micelles containing TPGS could realize P-gp inhibition by cutting off the energy supply of mitochondria, so as to increase drug accumulation in MDR cells. As the exhibited results above, it is clear that compared to the control and free DOX, once exposure to TPGS with or without DOX conjugated, series of changes related to mitochondria had taken place in MCF-7/ADR cells, including the promoted intracellular ROS accumulation, decreased mitochondrial membrane potential, and restrained ATP generation. In a word, the TPGS-conjugated DOX micelles can initiate the mitochondria dependent apoptotic pathways and result in cancer cell apoptosis [34, 16

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38]. The hybrid micelles would also present synergistic effect to overcome the drug resistance in MCF-7/ADR cells, thereby achieving the enhanced cytotoxic efficacy based on the function of TPGS.

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Figure 3. In vitro antitumor efficacy of hybrid micelles. (A) Viability of MCF-7 cells and MCF-7/ADR cells after treatment with DOX and TD at different concentrations for 24, 48 and 72 h, respectively. Data are mean ± SD (n = 5), *p < 0.05 compared with DOX. (B) The nuclei morphology of MCF-7/ADR cells after treated with a) free medium, b) DOX, and c) TD for 24 h. (C) Representative flow cytometry profiles for intracellular ROS production. Green, blue, red, orange and purple exhibited the control, positive control, DOX, TPGS and TD, respectively. (D) Change of mitochondrial membrane potential (ΔΨm) and (E) percentage of cells with decreased ΔΨm. (F) Intracellular ATP level after treated with DOX, TPGS and TD in MCF-7/ADR cells. *p < 0.05.

3.5 In Vivo Pharmacokinetics The long circulation property will promote the delivered drug to accumulate in tumor via EPR effect and thus enhance the antitumor efficacy. As described previously, the core-shell structure with appropriate PEG density made the micelles stable in both PBS and FBS. Here, the in vivo pharmacokinetics of free DOX and TD were studied in SD rats after i.v. administration of 10 mg/kg equivalent DOX (Figure 4) and the pharmacokinetic parameters were summarized in Table 2. Compared to free DOX, TD demonstrated significant improvement in pharmacokinetic profiles. Free DOX was quickly removed from the circulation system after administration with a short half-life (t1/2 = 0.53 h). However, TD exhibited a better pharmacokinetic behavior in 17

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comparison with free DOX. Specifically, the AUC, t1/2 and MRT0-24 for TD were 42.675 ± 9.434 mg·h/L, 2.553 ± 0.683 h and 2.976 ± 0.235 h, respectively, which were up to 7.76-, 4.82-, and 1.34-fold over those of free DOX, respectively. Accordingly, the CL of TD was 7.78-fold lower than that of free DOX. The prolonged drug presence in circulation may be attributed to the conjugation of DOX to the flexible polymer TPGS and the introduction of lipid DSPE-PEG with widely recognized long-circulating characters in the hybrid micelles.

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Figure 4. In vivo pharmacokinetics. Plasma concentration-time profiles of DOX in rats after i.v. administration of various drug formulations at the dose of 10 mg DOX/kg. Data are shown as mean ± SD (n = 4) Table 2. Pharmacokinetic parameters of DOX after i.v. administration of free DOX and hybrid micelles to rats at the dose of 10 mg DOX/kg. Unit

TD

DOX

AUC[a]

mg/L·h

42.675 ± 9.434

5.500 ± 0.257

MRT(0-t)[b]

h

2.976 ± 0.235

2.224 ± 0.018

t1/2[c]

h

2.553 ± 0.683

0.529 ± 0.004

CL[d]

L/h/kg

0.234 ± 0.054

1.821 ± 0.083

V[e]

L/kg

0.877 ± 0.405

1.389 ± 0.072

Tmax[f]

h

0.50

0.50

Cmax[g]

mg/L

11.585 ± 2.453

1.796 ± 0.048

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[a] Area under the curve, [b] Mean retention time, [c] Half-life, [d] Clearance, [e] Apparent volume of distribution, [f] Maximum drug concentration time, [g] Maximum drug concentration.

3.6 In Vivo Antitumor Efficacy of TD in MCF-7/ADR The ability of TD to reverse MDR was evaluated in drug-resistant (MCF-7/ADR) tumor model in nude mice. As shown in Figure 5A, 5C and 5D, free DOX barely showed tumor growth inhibition at a dosage of 5 mg/kg, which was consistent with 18

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the drug-resistant nature of this tumor model [30]. However, TD with the equivalent DOX dosage exhibited significant tumor growth inhibition compared with free DOX. A higher TD dosage (10 mg/kg) led to a further improvement of the antitumor efficacy. The tumor inhibition rates of DOX, TD (5 mg/kg) and TD (10 mg/kg) were 25.4%, 69.4% and 84.3%, respectively. The results confirmed the overcoming MDR property of TD. Meanwhile, both micellar formulations in routine dose were well tolerated as compared to the significant weight loss observed in free DOX-treated mice (Figure 5B). For H&E staining and TUNEL assay (Figure 5E, 5F and S7), drug treated groups exhibited varying degrees of apoptosis. The N.S. group displayed minor necrosis, compact structure and dense tumor cells in tumor tissue. Free DOX showed slightly necrosis. In contrast, TD in 5 mg/kg displayed a much higher necrotic level where apoptotic morphological characteristics appeared, such as nuclear pyknosis and karyorrhexis. The largest numbers of apoptosis cells were found in high dose TD group. These results suggested that our prodrug hybrid micelles were capable of overcoming the drug resistance. It provided us an accessible way to overcome MDR by delivering chemotherapy drug via a micelle system with the polymer acting as a P-gp inhibitor and the cleavable linkage achieving pH-triggered drug release.

Figure 5. In vivo antitumor efficacy of different formulations in BALB/c nude mice implanted with MCF-7/ADR cells. (A) Tumor volume and (B) body weight change during treatment. (C) Image and (D) weight of tumors separated from mice at the end of experiment, bar = 2 cm. (E) H&E staining and immunofluorescence analysis of tumor tissue. Tumor blood vessels, 19

ACCEPTED MANUSCRIPT apoptotic cells and cell nuclei were stained by antiCD31 (red), TUNEL (green) and DAPI (blue), respectively. DOX dosage was 5 mg/kg or 10 mg/kg per administration on days 6, 10 and 14. The data are mean ± SD (n = 6), *p < 0.05.

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3.7 Improving Antitumor Efficacy of TD by Integration of Tumor-Specific Peptidic Ligand 3.7.1 Tumor Growth Delay

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The favorable stability of the prodrug hybrid micelles makes it a promising candidate for further preclinical evaluation. Targeting delivery of nanomedicine to tumor based on the overexpressed receptors on the tumor cells has attracted more attention in improving antitumor efficacy [47]. TD has already demonstrated the superior antitumor effect against drug resistant tumor. Introducing DSPE-PEG in micelles also provided a good capability for surface functionalities, such as conjugating a tumor-specific ligand onto the surface of micelles.

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As a proof-of-concept, we used the cyclic peptide, cRGD (c(RGDfC)), as the targeting ligand to investigate the targeting delivery of this prodrug hybrid micelle system. As shown in Figure 6A, cRGD with a reactive cysteine residue was used to conjugate with DSPE-PEG-Mal in the hybrid micelles via the maleimide-thiol coupling to obtain cRGD-decorated TD (noted as RGD-TD). This fabrication strategy could avoid organic solvent exposure, accordingly protecting the shape of peptide and facilitating the ligand displayed on the micelle surface. In this study, B16F10 cell line was selected as the widely evidenced high integrin receptors level [48]. In vitro results (Figure 6B) showed that RGD-TD exhibited more intense red fluorescence compared to TD, indicating that the receptor mediated endocytosis and thus enhanced the cellular uptake of the modified micelles. The endo/lysosome escape of DOX formulated in RGD-TD was also verified in B16F10, indicating the burst drug release property of TD was kept in RGD-TD (Figure S6). Subsequently, a much higher cytotoxicity was achieved compared with that of TD treatment group (Figure 6C). The in vivo DOX biodistribution was further evaluated in tumor bearing mice as shown in Figure 6D. Compared to free DOX, enhanced accumulation of DOX in tumors was demonstrated in both types of DOX micelles, which may be attributed to the EPR effect of micellar formulations. The results also suggested a more effectively drug accumulation of RGD-TD in tumor compared to TD. Moreover, with the decoration of cRGD ligands, RGD-TD exhibited decreased accumulation in normal tissues which was in line with the previous work about nanoformulations of DOX[49, 50]. The tumor drug retention in 24 h of RGD-TD was about 2.15-fold than TD, which may lead to an improved antitumor response. To further investigate the therapeutic activity of RGD-TD, the antitumor efficacy on B16F10 tumor-bearing mice was evaluated after i.v. administration of free DOX, TD and RGD-TD at a dose of 5 mg/kg, respectively. The targetable micelles with cRGD decoration outperformed TD in retarding tumor growth, suggesting that the incorporation of tumor-specific ligand may help to improve the performance of our TPGS-based prodrug micelles (Figure 20

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6E). In order to reveal the mechanism of enhanced tumor therapy efficiency, immunofluorescence analysis was performed. As shown in Figure 6G, the blood vessel, the cleaved PARP and the nucleus were specifically stained by anti-CD31 antibody (green), anti-PARP antibody (red) and DAPI (blue), respectively. Compared to the control and free DOX, the most severe tumor cell apoptosis was observed in the group treated with RGD-TD. The cleaved PARP was detected more frequently nearby the CD31 marked area especially in TD and RGD-TD, which may attribute to the EPR effect of the micelles. The improved antitumor efficiency may be due to that the acidic pH triggered the rapid cleavage of Schiff base-linked drug, and consequently led to a rapid and burst drug release and improved drug penetration into the tumor center.

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The therapeutic effect of hybrid micelles was also evaluated on H22-bearing mice. H22 is an aggressive murine hepatocellular carcinoma model [51, 52]. As depicted in Figure S8A, rapid and unrestrained growth of tumor was observed in mice treated with N.S. Hybrid micelles TD showed a moderate level of activity in delaying the tumor growth, which was comparable with free DOX, and the therapeutic efficacy was promoted in the RGD-TD treated group (Figure S8A-C). Furthermore, at the end of treatment, tumor tissues of mice treated with DOX or hybrid micelles were fixed and observed by frozen section to evaluate the drug retention effects of hybrid micelles (Figure S8D). Slight red fluorescence was observed in the images of free DOX, which indicated that small amount of residual DOX was observed in solid tumors. However, in TD, especially RGD-TD, stronger red fluorescence intensity was detected. This may be attributed to the long circulation and more effective retention in tumor from the prodrug hybrid micelles. These results appeared that incorporation of cRGD motif into hybrid micelles led to the improvement in antitumor activity which may be due to the targeting effect of cRGD leading to more drug delivered to tumor tissue in vivo [53, 54]. This hybrid micelle system could further improve the potential of this prodrug in targeted antitumor treatment.

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Figure 6. Improved in vivo antitumor activity of TD by incorporation of a tumor-specific targeting ligand, cRGD. (A) Schematic illustration of combination of TPGS-CH=N-DOX prodrug with cRGD-modified DSPE-PEG-Mal for fabrication of targetable micelles (RGD-TD). (B) Confocal microscopic images of B16F10 cells after incubated with TD and RGD-TD for 4 h. (C) Viability of B16F10 cells after treated with different formulations for 24 h. (D) DOX biodistribution profiles in B16F10 tumor bearing mice receiving i.v. administration of different formulations at the dose of 5 mg DOX/kg. (*, p < 0.05 vs DOX; #, p < 0.05 vs TD, n = 3) (E) Tumor growth and (F) body weight of mice bearing B16F10 tumor. (G) Immunofluorescence analysis of tumor tissue. The inserted pictures presented tumor blood vessels (antiCD31, green), cleaved PARP (red) and nuclei (DAPI, blue). All groups (n = 8) of mice were i.v. injected with various formulations at dose of 5 mg DOX/kg on day 9, 13 and 15. The data are means ± SD, *p < 0.05.

3.7.2 Metastasis Inhibition The B16F10 bloodstream metastasis model was constructed to further determine whether RGD-TD could prevent the establishment of tumor metastasis in the advanced cancer and fulfill the “Molecular Economy” insight. TD suppressed tumor metastasis to lung, which was observed from the pictures of the groups (Figure 7A), the count of surface metastatic nodules (Figure 7B) and the histological examination of the lung and kidney tissues (Figure 7C). TD treatment reduced the pulmonary metastatic nodules by 88% and 79% compared with N.S. and free DOX, respectively. The RGD-TD showed improved metastasis inhibition as reduced tumor nodules compared with TD. These results were consistent with previous works, which reported nanoparticles containing TPGS could be an effective, tumor-targeted drug delivery system to be used as a promising prophylactic therapeutic agent against 22

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metastasis of breast cancer [55]. Although more studies are needed to better understand the underlying mechanism, the TPGS-based pH-triggered DOX prodrug could also be a potential agent to reduce the possibility of tumor metastasis in advanced cancer treatment.

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Figure 7. The inhibition effects of DOX formulations on lung metastasis in the B16F10 bloodstream transfer model. (A) Lungs were harvested for imaging. (B) Quantitative analysis of the pulmonary metastatic nodules from each group. (C) Representative H&E staining sections showing the metastases of lungs and kidneys. (*, p < 0.05 vs N.S.; #, p < 0.05 vs DOX, n = 5)

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3.8 In Vivo Safety Evaluation To evaluate the systemic toxicity, different formulations were injected to healthy mice. The H&E staining of major organs including heart, liver, spleen, lung and kidney was displayed in Figure S9A, and the levels of ALT, AST and BUN were tested (Figure S9B). Blood biochemical test did not show any changes compared to healthy mice. However, compared with N.S., obvious abnormal area was observed in hearts of mice treated with free DOX in histopathological analysis. In contrast, no acute pathological change was detected in micelles treated groups, demonstrating that micelles could reduce the cardiactoxicity. Other organ sections did not show significant difference between any groups. According to the results mentioned above in the therapeutic study, all of the mice treated with free DOX lost their weight because of systemic toxicity. On contrary, the micelle treated groups exhibited reduced side effects as the body weight changed slightly during the course of therapy. A higher DOX dosage (10 mg/kg) was further used in TD group to confirm the biocompatibility and tolerance of this prodrug hybrid micelle in animals (Figure 5B and 6F). Based on these results, we concluded that the 23

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In summary, a simple but powerful TPGS-based pH-sensitive DOX prodrug has been developed. As a safe and efficient hybrid micelle system, it was demonstrated to combine the advantages of (1) extended blood circulation time, enhanced long-term stability in physiological environment and improved tumor accumulation via EPR effect by introducing a PEGylated lipid, (2) increased cellular uptake and accelerated drug release through the cleavage of pH sensitive Schiff base linkage, (3) promoted translocation from cytoplasm into cell nucleus in drug resistant cancer cells through the P-gp inhibition ability of TPGS, (4) improved treatment effect against MDR tumor with the incorporation of mitochondria-associated apoptotic pathways. Moreover, the amphiphilicity of prodrug and commercialized lipid DSPE-PEG facilitated easy modifiable functionalities. The improved antitumor efficacy was verified on tumor-bearing mice by decorating tumor-specific ligand (cRGD) with hybrid micelles. The platform presented here has advantages in terms of easy synthesis, economical fabrication, and convenient large-scale of production. Our results provided a new insight of “Molecular Economy” to relieve the carrier burden and maximize the therapy efficiency of nanomedicine in rational prodrug design. This might have great potential to develop a series of functional prodrugs in practical antitumor therapy.

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Acknowledgement

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This work was supported by National Basic Research Program of China (973 Program, 2012CB932501), the National Natural Science Foundation of China (21204024 and 81373360), Natural Science Foundation of Hubei province (2015CFB492) Fundamental Research Funds for the Central Universities (2016YXY138), Chutian Scholar Award and 2013 Youth Scholar Award of HUST. We thank the Analytical and Testing Center of HUST for NMR, TEM and CLSM measurements. References

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TPGS-CH=N-DOX prodrug hybrid micelles were developed with synergistic effect of pH-triggered burst release behavior and additional bioactivity of drug carrier to overcome MDR and inhibit the potential metastasis of tumor.

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