Tumor pH-responsive Release of Drug-conjugated Micelles from Fiber

and eosin (H&E) and observed with a light microscope (Nikon Eclipse E400, Japan). To investigate the proliferation and apoptosis of tumor cells, IHC s...
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Tumor pH-responsive Release of Drug-conjugated Micelles from Fiber Fragments for Intratumoral Chemotherapy Nan He, Zhoujiang Chen, Jiang Yuan, Long Zhao, Maohua Chen, Tao Wang, and Xiaohong Li ACS Appl. Mater. Interfaces, Just Accepted Manuscript • DOI: 10.1021/acsami.7b09519 • Publication Date (Web): 06 Sep 2017 Downloaded from http://pubs.acs.org on September 6, 2017

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Tumor pH-responsive Release of Drug-conjugated Micelles from Fiber Fragments for Intratumoral Chemotherapy

Nan He, Zhoujiang Chen, Jiang Yuan, Long Zhao, Maohua Chen, Tao Wang, Xiaohong Li*

Key Laboratory of Advanced Technologies of Materials, Ministry of Education, School of Materials Science and Engineering, Southwest Jiaotong University, Chengdu 610031, P.R. China

* Corresponding Author. E-mail: [email protected]. Tel: +8628-87634068, Fax: +8628-87634649.

KEYWORDS: Injectable fiber fragment; Micelle-releasing fiber; Tetraphenylethylene-derived micelle; Acid-liable fiber; Glutathione-sensitive micelle

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ABSTRACT: The tumor accumulation of micelles is essential to enhance the cellular uptake and extend the release of chemotherapeutic agents. In the previous study camptothecin (CPT)-conjugated micelles (MCPT) were constructed with disulfide linkages and folate moieties for reduction-sensitive release and cell-selective uptake. This study proposes a strategy to integrate the promicelle polymers (PMCPT) into fiber fragments for intratumoral injection, realizing acid-liable release of PMCPT in response to acidic tumor microenvironment and spontaneous self-assembly into MCPT. Acid-liable 2propionic-3-methylmaleic anhydride (CDM)-linked poly(ethylene glycol) initiates the ring-opening polymerization of DL-lactide as the fiber matrix. There is no apparent burst release of MCPT from fiber fragments and around 80% of accumulated releases after incubation in pH 6.5 buffers for 40 days. Compared to MCPT freshly prepared via solvent evaporation, the micelles released from fiber fragments reveal similar profiles, such as folate-mediated cellular uptake and glutathione-sensitive drug release. Taking advantage of the aggregation-induced emission (AIE) effect of tetraphenylethylene (TPE) derivatives, TPE-conjugated micelles (MTPE) have been successfully been used to track the selfassembly into micelles after release from fibers and subsequent cell internalization into cytosol. The self-assembly-induced fluorescence light-up was also detected after intratumoral injection of fiber fragments. Compared with CPT-loaded fiber fragments and intratumoral or intravenous injection of free MCPT, the sustained release from fiber fragments and high accumulation of micelles in tumors results in significantly higher inhibition of tumor growths, prolongation of animal survivals and induction of tumor cell apoptosis. Thus, the integration of double targeting and double stimuliresponsiveness into fragmented fibers provides a feasible strategy to realize the sustained micelle release from fibers and promote the therapeutic efficacy.

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1. INTRODUCTION Chemotherapeutic drugs generally suffer from poor pharmacokinetics, inappropriate biodistribution in neoplastic tissues, and dose-limiting toxicities associated with the high systemic exposure. The past decades have seen great progress in the understanding of cancer biology, and these advances have been translated into targeted delivery strategies to improve the chemotherapeutic outcome.1 Nanocarriers with stimuli-responsive and tumor cells-targeting properties are currently being explored based on the tumor microenvironment, as well as membrane receptors and intracellular signals of tumor cells.2 As one of the major characteristics of solid tumors, the dysregulated pH in extracellular tumor (pHe) is between 6.0 and 7.0, due to the accumulation of lactic acid as a result of anaerobic glycolysis.3 Various acid-labile groups such as acetal, orthoester, hydrazone, imine and cis-aconityl, have been employed to create polymer architectures or polymer-drug conjugates.4 Typical examples are pH-sensitive polymeric nanoparticles whose physical and chemical properties such as surface charges or exposure of target ligands can be altered by local pHe-triggered cleavage of pH-labile groups.5 Once entering a cell, drug-loaded nanoparticles have to reach the cytoplasm and release the drug in order to achieve the desired therapeutic effect. In addition to endo/lysosomal pH and enzymes, cytoplasmic glutathione (GSH) has been explored for the construction of stimuli-responsive carriers.6 GSH is produced intracellularly and maintained at mM levels in the cytosol and subcellular compartments, whereas a rapid enzymatic degradation limits GSH concentrations to µM levels in plasma. Disulfide linkages are most often incorporated into polymer-drug conjugates to offer structural integrity in the plasma but undergo intracellular disintegration in response to the elevated GSH levels.7 Currently various carriers, such as micelles, hydrogels, liposomes, nanoparticles and dendrimers, have been developed for tumor targeting and stimuli-triggered drug release. Micelles self-assembled from amphiphilic copolymers are in general more stable than liposomes and emulsions constructed from conventional surfactants,8 and their self-assembling structure lead to higher stimuli-

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responsiveness than polymers and solid lipid nanoparticles.9 Micelles with loaded paclitaxel (GenexolPM) and doxorubicin (NK911) are in the final stages of commercialization.10 Even though benefited from the EPR effect and active targeting mechanism, only a small percentage of administered drugs can reach tumor cells. For example, for the commonly investigated micelles, as well as liposomes and nanoparticles, over 95% of the administered dose is known to accumulate in organs other than tumors, particularly in liver, spleen, and lung.11 In addition, it is difficult to prevent the premature drug release due to the disintegration of micelles after dilution in blood circulation and in the presence of blood components.12 To address these challenges, micelles are encapsulated into microspheres and hydrogels to provide additional barriers for prolonged drug release and reduce administration frequency. Zhang et al. prepared an in situ gelling system by entrapping curcumin-loaded micelles into thermosensitive hydrogels to increase the concentration and residence time of curcumin in the abdominal cavity. Compared with free drug and curcumin-loaded micelles, the in situ gel led to lower proliferation activities, more apoptotic cells, and fewer microvessels for the treatment of colorectal peritoneal carcinomatosis.13 Alternatively, drug-releasing implants such as electrospun fibers have displayed great potential for postoperative local chemotherapy, due to their desirable features such as high drug encapsulation efficiency and localized drug doses, as well as tunable fiber degradation rate and drug release profile. Yang et al. prepared core-sheath fibers by coaxial electrospinning using a mixture of doxorubicin-loaded micelles and poly(vinyl alcohol) as the cores and gelatin as the sheaths, capable of maintaining therapeutic drug levels at the tumor site for extended periods of time and potent antitumor efficacy.14 Liu et al. constructed poly(lactic-co-glycolic acid) composite nanofibers by encapsulating paclitaxel- and brefeldin A-loaded micelles via emulsion-electrospinning, exhibiting controlled dual release of hydrophobic drugs and strong therapeutic efficacy against HepG2 cells.15 These micelle-encapsulated nanofibers have accomplished sustained and localized drug delivery against solid tumors due to additional barriers provided by fiber matrices, but some unaddressed issues still remain. The first issue is the release form definition. Micelles with drug loaded by physical ACS Paragon Plus Environment

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entrapment were encapsulated in the fibers, thus the separate release of free drugs and carriers resulted in rare self-assemblies into drug-loaded micelles. Even if the assembly of the released drugs and carriers occurred, the assembled micelles should be quite different from the freshly prepared ones. The second issue is the release rate control. Micelles were encapsulated in the fiber cores, thus the release of micelles, if it is, should be minimal until significant degradation or collapse of fiber matrix due to the large size of micelles. The third issue is the release item monitoring. Up to now no attempt has been made to distinguish between the release of drug-loaded micelles and free drugs from fibers either in vitro or in vivo. However, it should be noted that the self-assembly into drug-loaded micelles after release from fibers is essential for efficient cellular uptake and intracellular stimuli-responsive release of drugs. In the previous study, camptothecin (CPT)-conjugated micelles (MCPT) were constructed from multiarmed amphiphilic copolymers with folate conjugation as targeting groups and CPT conjugation through disulfide linkages (Figure 1a).16 MCPT has shown advantages over drug-entrapped micelles in alleviating the premature drug release during blood circulation, relieving the systemic toxicity and promoting the therapeutic efficacy.17 In addition, to eliminate the invasive surgery for implantation of electrospun fibers, intratumoral injection of drug-loaded fiber fragments has been developed, indicating high accumulation and spatial distributions of drugs within tumors.18 In the current study, the promicelle polymers of MCPT (PMCPT) were electrospun into fiber matrices containing 2-propionic-3methylmaleic anhydride (CDM) linkages for intratumoral injection. As shown in Figure 1c, with the fiber degradation in response to the tumor pHe, PMCPT were gradually released from fiber fragments and spontaneously self-assembled into MCPT, followed by selective micelle internalization into cancer cells via folate receptor (FR)-mediated endocytosis and on-demand CPT release in response to cytosol GSH. Taking advantage of the aggregation-induced emission (AIE) effect of tetraphenylethylene (TPE) derivatives,19 (TPE)-conjugated micelles (MTPE) were employed to elucidate in situ micelle formation via self-assembly after release from fibers both in vitro and in vivo. The released micelles revealed ACS Paragon Plus Environment

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similar profiles, such as their morphology, reductive sensitivity, cellular uptake and cytotoxicity, in comparison with freshly prepared micelles via solvent evaporation. The sustained release of micelles from fiber fragments resulted in dramatic inhibition of tumor growths, prolongation of animal survivals and induction of cell apoptosis in tumor tissues, in comparison with intratumoral injection of fresh micelles and CPT-loaded fiber fragments.

2. EXPERIMENTAL SECTION Materials. Triton X-100, cis-aconitic anhydride (CA), propidium iodide, trypsin, GSH, pyrene, dialysis bags (MW cutoff: 3.5 kDa), and 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) were procured from Sigma (St. Louis, MO). Rabbit anti-mouse antibodies of caspase-3 and Ki67, goat anti-rabbit IgG–horseradish peroxidase (HRP) and 3,3-diaminobenzidine (DAB) developer were purchased from Biosynthesis Biotechnology Co., Ltd. (Beijing, China). All other chemicals were of analytical grade and obtained from Changzheng Regents Company (Chengdu, China), unless otherwise indicated. Anhydrous chloroform and toluene were dried over CaH2 and distilled under reduced pressure prior to use. Preparation of Acid-sensitive Polymers. Figure 1b summarizes the synthesis route for acidsensitive polymers. Two ends of PEG were blocked with CDM, following by conjugation of another two PEG molecules to obtain CDM/PEG. The synthesis and characterization of CDM (Scheme S1 and Figure S1) and CDM/PEG (Figure S2 and S3) are included in the Supporting Information. Copolymers of CDM/PEG with poly(DL-lactide) (CDM/PELA) was prepared by the ring-opening polymerization of DL-lactide, using CDM/PEG as an initiator and stannous chloride as a catalyst (Figure 1b).20 Briefly, CDM/PEG (0.5 g), lactide (2.0 g), and stannous octoate (30.0 mg) were mixed into 20 mL of anhydrous toluene. After toluene removal under reduced pressure, the mixture was incubated at 140 °C for 8 h, followed by dissolution in anhydrous dichloromethane, precipitation in diethyl ether, filtration

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and vacuum drying to obtain CDM/PELA-4. Yield: 2.27 g (91%). The 1H NMR spectrum was recorded on a Bruker AM 400 apparatus, using tetramethylsilane (TMS) as the internal reference. 1H NMR (CDCl3, ppm, δ): 1.57 (d, 3H, -CHCH3), 2.10 (s, 3H, -C-CH3), 2.73 (m, 2H, -CH2CH2CO-), 2.86 (m, 2H, -CH2CH2CO-), 3.65 (large s, H-PEG), 5.09 (q, 1H, -CHCH3) (Figure S4a). Similarly, the weight proportion of DL-lactide to CDM/PEG was adjusted to 5/1 and 6/1, and CDM/PELA-5 and CDM/PELA-6 were obtained. The molecular weight of CDM/PELA was detected by gel permeation chromatography (GPC, Waters 2695 and 2414, Milford, MA) using tetrahydrofuran as the eluent and polystyrene as standard. Preparation of Fiber Fragments with Loaded PMCPT. Fiber fragments with loaded PMCPT were prepared by cryocutting of aligned electrospun fibers as described previously with some modifications.18 Briefly, PMCPT were synthesized as described previously,16 and blend solutions CDM/PELA (0.8 g) and PMCPT (0.2 g) in acetone (4 mL) were transferred to a syringe and then pumped at 0.4 mL/h using a microinject pump (Zhejiang University Medical Instrument Co., Hangzhou, China). A high voltage difference of 20 kV/15 cm was applied between the syringe nozzle and a grounded collector through a high voltage statitron (Tianjing High Voltage Power Supply Co., Tianjing, China). Aligned fibers were collected on an aluminum foil wrapped on a grounded rotating mandrel at a linear rate of around 15 m/s. The fibrous mat was folded at about 1 cm of intervals perpendicularly to fiber alignment and embedded into Cryo-OCT compound (Thermo Fisher Scientific Inc., Waltham, MA). The solidified blocks were sectioned at a thickness of 20 µm using a cryostat microtome (Microme HM550 OMC, Thermo Fisher Scientific Inc., Waltham, MA), followed by ultrasonication in water and collection via centrifugation. CDM/PELA-4, CDM/PELA-5 and CDM/PELA-6 were used to prepare PMCPT-loaded fiber fragments as F4/MCPT, F5/MCPT and F6/MCPT, respectively. For comparison, F4, F5 and F6 fiber fragments were obtained without inoculation of PMCPT. CPT-loaded fiber fragments (F4/CPT, F5/CPT and F6/CPT) were also prepared with an equivalent CPT to MCPT-loaded fibers. ACS Paragon Plus Environment

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Acid-liability of CDM/PEG and CDM/PELA Fibers. The morphology of fiber fragments was observed by scanning electron microscope (SEM, FEI Quanta 200, The Netherlands) equipped with a field-emission gun (20 kV) and a Robinson detector after 2 min of gold coating to minimize the charging effect. The acid liability of CDM/PEG and CDM/PELA fiber fragments was determined after incubation at 37 °C in pH 5.5, 6.5 and 7.4 buffers. At predetermined intervals, CDM/PEG and CDM/PELA were retrieved and dissolved into tetrahydrofuran, and the molecular weight and the distribution were investigated by GPC as above. The 1H NMR spectra of the retrieved CDM/PELA fiber fragments were recorded as above. In Vitro Micelle Release from Fiber Fragments. The release of micelles from fiber fragments were conducted in dialysis bags, which were suspended into the release media. Briefly, 20 mg of fiber fragments with loaded MCPT or MTPE and 10 mL of release media were put into dialysis bags. The dialysis bag was immersed in 30 mL of release media in a 50 mL-conical tube, which was kept in a thermostated shaking water bath that was maintained at 37 °C. At a specified time interval, 1.0 mL of release media was withdrawn from both inside and outside of the dialysis bag and an equal volume of release media was added for continuing incubation. The concentration of MCPT and MTPE micelles in the dialysis bag and free CPT in the conical tube was detected by a fluorescence spectrophotometer (Hitachi F-7000, Japan) at the excitation/emission wavelengths of 365/430 nm, using a standard curve from known concentrations of micelles or CPT.17 The micelles were retrieved from dialysis bags after 3 days for morphological measurement and reductive sensitivity detection. The morphology was observed by transmission electron microscope (TEM, JEM-2100F, JEOL Ltd., Japan) after dropping onto a copper grid and staining with phosphotungstic acid. The average size and its distribution were measured by dynamic light scattering (DLS, Nano-ZS90, Malvern Ltd., UK). The CPT release and size changes in responsive to reductive

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signals were determined after incubation in phosphate buffered saline (PBS) containing 2 µM and 10 and 40 mM GSH. Cellular Uptake of Micelles Released from Fiber Fragments. The cellular uptake of micelles was estimated after incubation of fiber fragments with loaded MCPT and MTPE in culture media of pH 7.4 and 6.5. HepG2 cells were from American Type Culture Collection (Rockville, MD) and cultured in pH 7.4 RPMI 1640 (Gibco BRL, Rockville, MD) containing 10% fetal bovine serum (FBS, Gibco Invitrogen, Grand Island, NY). The pH of culture media was adjusted to pH 6.5 with 0.1 M HCl, and no considerable pH drift in the culture media was observed during the cytotoxicity tests.21 Briefly, HepG2 cells were seeded into 6-well tissue culture plates (TCP) at a density of 1.0 × 106 cells per well and incubated for 24 h. The cells were treated with free CPT, MCPT and MTPE micelles, micelles-loaded fiber fragments (F4/MCPT, F5/MCPT and F6/MCPT), and CPT-loaded fiber fragments (F4/CPT, F5/CPT and F6/CPT) at a final equivalent CPT concentration of 3 µg/mL. After incubation for 24 h, cells were washed with PBS, harvested by trypsin digestion and lysed by 500 µL of 0.5% Triton X-100 for 2 h at 4 °C. Then, the fluorescence intensity of the cell lysate was estimated by a fluorescence spectrophotometer as above. In the meantime, HepG2 cells were washed, fixed with 4 % formaldehyde solution, stained with propidium iodide for 30 min, and observed by confocal laser scanning microscope (CLSM, Leica TCS SP2, Germany). In Vitro Cytotoxicity and Apoptosis Assay of Micelles-loaded Fiber Fragments. The in vitro cytotoxicity and apoptosis were evaluated on HepG2 cells after culture in pH 7.4 and 6.5 media as above. Briefly, HepG2 cells were seeded in 96-well TCPs at a density of 5 × 103 cells per well and incubated overnight before drug treatment. The cells were treated for 72 h with a series of concentrations of free CPT, MCPT and MTPE micelles, micelles-loaded fiber fragments (F4/MCPT, F5/MCPT and F6/MCPT), and CPT-loaded fiber fragments (F4/CPT, F5/CPT and F6/CPT). The amount of fiber fragments was confirmed as the equal amount of micelles or CPT release during 72 h of

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incubation at each pH value. The media were then removed and cells were washed with PBS, followed by the addition of 200 µL of fresh culture media and 20 µL of MTT into each well. After incubation for 4 h, the media was removed and dimethyl sulfoxide (DMSO) was added. The absorbance of each well was measured at 490 nm using Quant microplate spectrophotometer (Elx-800, Bio-Tek Instrument Inc., Winooski, VT). The apoptosis of HepG2 cells was quantified by an Annexin V-fluorescein isothiocyanate (FITC) apoptosis detection kit (Beijing 4A Biotech Co., Beijing, China) according to the manufacturer’s instructions. Briefly, HepG2 cells were seeded in 6-well TCPs at a density of 5 × 105 cells per well and treated as above. After incubation for 72 h, the floating and attached cells were harvested with 0.25% trypsin, washed with PBS, and suspended in 500 µL of binding buffer. After the addition of 5 µL Annexin V-FITC and 5 µL propidium iodide, the sample were gently mixed and incubated at 37 °C for 15 min before analysis with flow cytometry (BD Accuri C6, Franklin Lakes, NJ). Data analysis was performed using BD FACSuite™ (BD Biosciences, CA). Micelle Release in Tumor Tissues from Fiber Fragments. The tumor model was established by subcutaneous injection of murine hepatoma H22 cells as described previously,21 and all animal procedures were approved by the University Animal Care and Use Committee. Briefly, H22 cells were kindly gifted by the State Key Laboratory of Biotherapy of Sichuan University (Chengdu, China), and female Kunming mice weighing 24 ± 2 g were supplied by Sichuan Dashuo Biotech Inc. (Chengdu, China). H22 cells were maintained by transplanting them into the peritoneal cavities of mice for serial subcultivation. Then the mice with viable H22 ascites were sacrificed, and the ascites were withdrawn and diluted with physiological saline to modulate the cell density at l × 107 cells/mL. The cell suspension was subcutaneously inoculated into the right armpit region of each animal at a dose of around 10 µL/g body weight. Tumors were allowed for growth for 7 days to reach about 500 mm3 in volume. The tumor-bearing mice were randomly divided into four groups and treated by intravenous

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injection of MTPE, intratumoral injection of MTPE or F4/MTPE fiber fragments, using without treatment as control. After treatment for 1, 3, 7 and 14 days, animals were sacrificed by cervical dislocation to retrieve tumor tissues. Tumor samples were frozenly sectioned with a thickness of 5 µm, followed by CLSM observation at the excitation/emission wavelengths of 370/443 nm. Tissue sections were kept moist enduring detection, and on the blank tissue section was added dropwise PMTPE in DMSO/H2O mixed solution (1/1, v/v). Antitumor Efficacy of Micelles-loaded Fiber Fragments. The antitumor efficacy was evaluated with respect to the tumor volume, body weight and animal survival. Briefly, the tumor-bearing mice were randomly divided into five groups with 8 mice per group. Mice were treated by intratumoral injection of F4/MCPT, F4/CPT fiber fragments and mixtures of MCPT and F4 fiber fragments, using intravenous injection of MCPT and PBS as control. The CPT dose was equivalent to the total amount of 4.0 mg/kg body weight. The body weights, tumor volumes, and survival rate of animals were monitored every other day after treatment. The length of the major axis (longest diameter) and minor axis (perpendicular to the major axis) of the tumor were measured with a vernier caliper, and the tumor volume was calculated as described previously.18 The number of live animals at each time point was plotted in Kaplan−Meier survival curves, and the 50% mean survival time was obtained for each group. Histopathological and immunohistochemical (IHC) Examination of Tumors Retrieved. After 21 days of treatment, tumors were collected from randomly chosen mice for each group. After fixation in 10% neutral buffered formalin, the tissues were processed routinely, and sectioned at a thickness of 4 µm. To evaluate the cell morphology and tissue necrosis, tumor sections were stained with hematoxylin and eosin (H&E) and observed with a light microscope (Nikon Eclipse E400, Japan). To investigate the proliferation and apoptosis of tumor cells, IHC staining of Ki-67 and caspase-3 was performed on tumor sections as described previously.22 The slides were counterstained with hematoxylin, followed by dehydration with sequential ethanol for sealing and microscope observation. A minimum of five individual microscopic images were randomly selected, and the expression of caspases-3 and Ki-67 ACS Paragon Plus Environment

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proteins were quantified by comparing the positively stained cells with the total number of cells in these areas. Statistical Analysis. Data are expressed as mean ± standard deviation (SD). Whenever appropriate, comparisons among multiple groups were performed by analysis of variance (ANOVA), while a twotailed Student’s t-test was used to discern the statistical difference between two groups. A probability value (p) of less than 0.05 was considered to be statistically significant.

3. RESULTS AND DISCUSSION Characterization of Acid-liable CDM/PEG. Figure 1b summarizes the synthesis route for CDM/PEG. As shown in Figure S2c, the appearance of peaks at 2.1, 2.73 and 2.86 ppm in the 1H NMR spectrum of CDM/PEG indicated successful conjugation of CDM to the backbone of PEG via amide bonds. For comparison, CA linkers were used to link PEG molecules, and the inoculation of CA led to an absorption peak at 6.5 ppm in the 1H NMR spectrum of CA/PEG (Figure S2d). GPC analysis revealed a single peak at Mw of around 6350 for CDM/PEG (Figure S3a, Supporting Information), while CA/PEG eluted as a major peak at Mw of around 6350 and a minor one at 4210 corresponding to di- and mono-CA capped PEG, respectively (Figure S3b). The acid liability of CDM/PEG and CA/PEG was investigated by incubation in pH 7.4, 6.5 and 5.5 buffers for 48 h. As shown in Figure S3a and S3b, no significant degradation of both CDM/PEG and CA/PEG occurred in the neutral environment. In contrast, the incubation in pH 6.5 and 5.5 buffers led to an obvious shift toward lower molecular weight at around 2000. Figure S3c summaries Mw changes after incubation of CDM/PEG and CA/PEG into different buffers. After incubation in pH 6.5 media for 48 h, CDM/PEG was completely disintegrated, while only around 74% of CA/PEG was degraded into PEG fragments.

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pHe-responsive carriers have been widely used for targeted release of chemotherapeutics owing to the acidic tumor extracellular matrix. However, the development of pHe-responsive carriers are particularly arduous because of the delicate difference in pH between normal (pH 7.2−7.4) and tumor tissues (pH 6.2−6.9).23 Maleic acid amide derivatives can be degraded at mildly acidic pH values due to the internal attack of the amide carbonyl group by the β-carboxylate.24 Deng et al. designed the βcarboxylic amides-functionalized micelles that sharply converted from negative to positive charge at pH 6.0, due to the hydrolysis of β-carboxylic amides and exposure of positive-charged amines in the acidic environment of tumors. This response not only resulted in a rapid drug release in acidic conditions, but also effectively enhanced the cellular uptake by electrostatically absorptive endocytosis.25 In the current study, bifunctional linkers of the maleic acid amide derivatives CDM and CA were used for construction of fiber matrix polymers. As indicated above, compared to CA/PEG, CDM/PEG had a more prompt pH response and was utilized for the preparation of CDM/PELA copolymers. Characterization of pHe-Responsive CDM/PELA Fiber Fragments. Figure 1b summarizes the synthesis route for CDM/PELA. As shown in Figure S4a (Supporting Information), peaks at 2.1, 2.73 and 2.86 ppm were assigned to CDM, and those at 1.57 and 5.09 ppm belonged to lactide, while PEG indicated a peak at 3.65 ppm. The integral ratios for methyl groups of lactide (1.57 ppm), methylene groups of CDM (2.73 ppm) and PEG (3.65 ppm) were used to determine the number-average molecular weights (Mn) of CDM/PELA. The Mn of CDM/PELA-4, CDM/PELA-5 and CDM/PELA-6 were determined as 33.7, 41.6 and 48.2 kDa (Figure S4b), which were close to those from GPC analysis at 38.2, 46.3 and 57.8 kDa, respectively (Figure S4c). Figure 2a shows SEM images of MCPT-loaded fiber fragments, indicating uniform morphologies with smooth surface. F4/MCTP, F5/MCTP and F6/MCTP fiber fragments had diameters of 1.43 ± 0.14, 1.54 ± 0.13 and 1.70 ± 0.11 µm, respectively, and their average lengths were 20.7 ± 2.5 µm. The loading

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amounts of PMCPT in fiber fragments was 20.0 % and the CPT loading content in MCPT was around 7.1%,16 resulting in around 1.4 % of CPT loading amount in the fiber fragments. The acid sensitivity of fiber fragments was investigated with respect to the mass loss and Mw reduction after incubation in pH 7.4, 6.5 and 5.5 buffers. As shown in Figure 2b, almost no mass loss was detected for all the fibers after incubation in pH 7.4 buffers. In comparison, after 10 days of incubation, the mass losses of F5 and F6 in pH 6.5 buffers were 27.8% and 22.1%, respectively, while F4 showed the highest mass loss of around 33.5% and 45.4% in pH 6.5 and 5.5 buffers, respectively. Figure 2c displayed the Mw reduction after degradation of fiber fragments, indicating a changing profile similar to that of mass loss. There were no apparent changes under neutral condition, whereas around 74.8%, 77.2% and 79.0% of Mw reductions were observed for F4, F5 and F6, respectively, after incubation in pH 6.5 buffers for 10 days. The acid-labile degradation led to apparent morphological changes, such as swollen and curly compared with original formation. Figure S5 (Supporting Information) shows 1H NMR spectra of F4 fiber residuals after incubation for 7 or 21 days at pH 6.5 as well as the detailed assignment of each peak. Compared with that before degradation, the integral area of the peak at 3.65 ppm (PEG) decreased after degradation for 7 days, and disappeared after 21 days. Thus, it was indicated that the degradation of CDM/PELA in acidic conditions resulted from the acidlabile CDM/PEG segments, followed by breakage of ester bonds in poly(DL-lactide) segments. In Vitro Release of MCPT from Fiber Fragments. We assume that PMCPT could be released from fiber fragments by pHe-triggered fiber degradation and simultaneously assembled into MCPT. The MCPT release from fiber fragments and the simultaneous CPT release from the self-assembled MCPT were investigated within and outside the dialysis bags at pH 6.5 and 7.4. The MCPT release was further compared with CPT release from CPT-loaded fiber fragments at both pH values. As shown in Figure 3a and 3b, the release of MCPT from PMCPT-loaded fibers was less than 10% at pH 7.4 and over 60% at pH 6.5, while approximately 40% and 80% of CPT was released after 40 days from CPT-loaded fibers at pH 7.4 and 6.5, respectively. Therefore, PMCPT-loaded fibers displayed a more prompt response to ACS Paragon Plus Environment

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pHe and more suitable for pHe-triggered release. In addition, CPT-loaded fibers showed an initial burst release in 72 h, followed by a slow and sustained release for 40 days at both pH 7.4 and 6.5 (Figure 3b). The initial burst release could be attributed to the enriched drug molecules close to the fiber surface during electrospinning.26 The burst release of 42.3%, 39.3% and 38.3% for CPT-loaded fibers (F4/CPT, F5/CPT and F6/CPT) at pH 6.5 were more significant than those at pH 7.4 (23.9%, 22.7% and 20.5%), due to the rapid CPT diffusion after degradation of fiber matrices (Figure 2). In comparison, MCPT could be continuously released from PMCPT-loaded fibers (F4/MCPT, F5/MCPT and F6/MCPT) for 6 weeks without apparent burst release and the cumulative release reached 70.6%, 63.9% and 58.3% respectively on day 40 (Figure 3a), achieving a constant diffusion of PMCPT along with the degradation of fiber fragments. The significantly higher MCPT release from F4/MCPT than those of the other fiber fragments (p < 0.05) was due to the higher degradation rate of F4 fiber fragments (Figure 2). As shown in Figure 3c, less than 5% of CPT was released at pH 6.5 and almost no release was detected at pH 7.4 after 40 days. It was indicated that MCPT was not sensitive to the acidic media and the higher CPT release was due to the higher MCPT release from fiber fragments in pH 6.5 buffers. The self-assembled micelles released from F4/MCPT fiber fragments were collected to investigate their morphology and reductive sensitivity with respect to the size change and CPT release. TEM images showed that PMCPT released from F4/MCPT fibers self-assembled into spherical micelles with the size of around 40 nm (Figure 3d). The DLS measurement indicated the released micelles had an average size of 120 nm, which was close to that of freshly prepared MCPT via solvent evaporation.16 Figure 3e shows the size changes of the retrieved micelles in the presence of 2 µM and 10 and 40 mM GSH. There were no apparent size changes without or in the presence of 2 µM GSH, whereas the retrieved micelles swelled from around 120 nm to 440 and 570 nm after incubation for 12 h in PBS containing 10 and 40 mM GSH, respectively. Figure 3f shows the reduction-sensitive release of CPT from the retrieved micelles under different conditions. There was around 20% CPT release after incubation in PBS and PBS containing 2 µM GSH for 100 h. Significantly higher CPT releases of 55% ACS Paragon Plus Environment

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and 60% were detected after incubation in media containing 10 and 40 mM GSH, respectively, indicating the reductive sensitivity of self-assembled micelles released from MCPT-loaded fiber fragments. In Vitro Cell Viability, Apoptosis and Micelle Uptake after Treatment with Fiber Fragments. To determine the therapeutic effectiveness of MCPT-loaded fiber fragments, the cellular behaviors including cellular uptake, cytotoxicity and cell apoptosis were investigated on HepG2 cells. HepG2 cells were selected, since folate grafts of MCPT targeted the over-expressed FR on their surfaces.27 The cellular uptake of MCPT- and CPT-loaded fiber fragments was investigated after incubation with HepG2 cells in pH 7.4 and 6.5 media, and compared with free MCPT and CPT with a final CPT concentration of 3 µg/mL (Figure 4a). Free MCPT and CPT indicated similar uptake efficiency at both pH 7.4 and 6.5. The acid-sensitive release of MCPT or CPT from fiber fragments led to significantly higher uptake efficiencies after incubation in pH 6.5 media than those at pH 7.4. The uptake efficiencies of MCPT released from F4/MCPT, F5/MCPT and F6/MCPT in pH 6.5 media were around 34.2%, 32.5% and 31.4%, respectively, which were around 4.0−5.5 folds higher than those after incubation in pH 7.4 media. MCPT exhibited significantly higher uptake efficiencies than CPT at around 41.2% and 40.8%, respectively (p < 0.05), which was consistent with the previous study.16 The intracellular distribution of micelles was observed by CLSM (Figure 4b). CPT released from the intracellular micelles or diffused into cells could be distributed into nuclei, as seen from the partial co-localization of CPT and nuclei staining dye propidium iodide. Additionally, F4/MCPT exhibited significantly stronger fluorescence intensities after incubation in pH 6.5 media than that of pH 7.4. The cytotoxicities of MCPT-loaded fiber fragments were investigated using MTT assay after treatment for 72 h, and compared with those of CPT-loaded fiber fragments, free micelles and CPT. As shown in Figure 4c, compared with the treatment at pH 7.4, a remarkably higher cytotoxicity was observed for fiber fragments with loaded MCPT or CPT at pH 6.5. Figure 4d summarizes the half

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maximal inhibitory concentration (IC50) of each treatment. MCPT-loaded fiber fragments indicated 8−10 folds lower IC50s after incubation in pH 6.5 media than those of pH 7.4. The apoptosis induced by fiber fragments was determined by flow cytometry after Annexin V-FITC and propidium iodide staining. Figure S6 (Supporting Information) summarizes the flow cytometry images of cells after exposure under both pH 7.4 and pH 6.5. As shown in Figure 4d, compared with the treatment at pH 7.4, treatment of F4/MCPT, F5/MCPT and F6/MCPT at pH 6.5 enhanced the apoptotic effects with 31.1%, 29.6% and 28.3 % of cells in the early and late apoptosis stages. Therefore, MCPT-loaded fiber fragments elicited superior cytotoxicities and apoptotic effects in a slightly acidic microenvironment than in neutral conditions, showing the potential for tumor-triggered drug release. Among the PMCPTloaded fiber fragments under investigation, F4/MCPT showed the best performance in the cellular uptake (Figure 4a,b), inhibition of tumor cell growth and induction of cell apoptosis (Figure 4c,d), which were used for evaluation of in vivo antitumor efficacy. In Vitro Release and Cellular Uptake of MTPE from Fiber Fragments. The release of PMCPT from fiber matrices can be determined from the CPT content, but it is challenging to visualize in situ micelle formation of promicelle polymers after release from fiber fragments both in vitro and in vivo. In the previous study, micelles with physical entrapment of drugs were included in hydrogels or fibers to modulate the release, but the exact release mechanism remains to be elucidated, as well as the question whether or not the released drug circulates in micelles in vivo.28 To address these issues, MTPE was initially used in this study to monitor the micelle release from fibers and distribution in tumor tissues. The AIE based fluorogens are non-luminescent in the solution state but become highly emissive by aggregate formation.29 Thus, the promicelle polymers of MTPE (PMTPE) should generate luminescence for the direct visualization only after self-assembly into MTPE micelles. The synthesis and characterization of PMTPE are included in the Supporting Information (Scheme S2 and Figure S7). As shown in Figure 5a, PMTPE tended to self-assemble in aqueous solution into MTPE

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with hydrophilic PEG shells and hydrophobic cores of TPE and poly(ε-caprolactone) segments. Figure 5b indicates a typical TEM image of MTPE, revealing a spherical morphology with the size of around 42 nm. The DLS measurement showed an average size of 125 nm, which was close to that of MCPT. The critical micelle concentrations (CMCs) were determined as 0.51 and 0.48 µg/mL based on two protocols using pyrene as a fluorescent probe and the AIE phenomenon, respectively (Figure S8, Supporting Information). MTPE-loaded fiber fragments (F4/MTPE, F5/MTPE and F6/MTPE) were fabricated according to the similar protocol for the preparation of MCPT-loaded fiber fragments. As shown in Figure 5c, MTPEloaded fiber fragments exhibited pH-dependent micelle release profiles, similar to MCPT-loaded fiber fragments (Figure 3a). There was around 80% release of MTPE from F4/MTPE after incubation in pH 6.5 buffers for 40 days, while around 10% release was detected at pH 7.4. The released MTPE was observed by TEM, indicating the formation of spherical micelles with an average size of 45 nm through molecular self-assembly (Figure 5b). MTPE and MTPE-loaded fiber fragments were nontoxic to HepG2 cells, according to the cell viability assay (Figure S9, Supporting Information). Thus, F4/MTPE has similar pH-dependent properties (i.e. the micelle release profile and the size and morphology of the released micelles) to F4/MCPT, indicating a feasible model to monitor the self-assembly process of PMCPT released from fibers. The intracellular location of MTPE was observed by CLSM. As shown in Figure 5d, clear blue fluorescence was observed in the cytoplasm of HepG2 cells after treatment with F4/MTPE fiber fragments, similar to that of free MTPE micelles, indicating the cellular uptake of the self-assembled micelles released from fiber fragments. In addition, higher fluorescence intensities of MTPE were detected after incubation in pH 6.5 buffers than those of pH 7.4, showing the acid-sensitive release of MTPE from F4/MTPE fiber fragments. MTPE Release from Fiber Fragments in Tumor Tissues. In order to confirm that the self-assembly into micelles occurred spontaneously in vivo once promicelle polymers released from fiber fragments, ACS Paragon Plus Environment

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tumors with various treatments were retrieved and sectioned for CLSM studies (Figure 6a-e), using tumor slices without any treatment as the control. The dropping of PMCPT in DMSO/H2O solutions on the tissues showed no fluorescence emission (Figure 6a), whereas strong blue fluorescence was detected after intratumoral injection of mixtures of MCPT and F4 for 1 day (Figure 6b), since PMCPT polymers were non-emissive in good solvent of DMSO/H2O but became highly fluorescent after micelle formation. The fluorescent signals decayed rapidly and almost disappeared after 3 days due to the micelle clearance from tumors (Figure 6b). It should be noted that almost no fluorescence was detected after intravenous injection of MTPE (Figure 6c). This might be due to the rapid clearance from the circulation and phagocytosis into liver and other tissues.30 As shown in Figure 6d, the intratumoral injection of F4/MTPE exhibited strong fluorescence signals during 14 days, though the fluorescence intensity reduced at the later periods. It demonstrated that PMTPE could sustainably release from fiber fragments and spontaneously self-assembled into micelles, which were efficiently internalized into tumor tissues. In Vivo Antitumor Efficacy of MCPT-loaded Fiber Fragments. To evaluate the antitumor activities of MCPT-loaded fiber fragments, F4/MCPT, F4/CPT, mixture of MCPT and F4 were intratumorally injected at a CPT dosage of 4 mg/kg into H22 tumor-bearing mice with an average tumor volume of about 500 mm3. MCPT with an equivalent CPT amount were also injected intravenously for comparison. According to the tumor growth curves in Figure 7a, the tumor volumes for the groups of F4/MCPT, F4/CPT, mixture of MCPT and F4, and MCPT were around 29.1%, 37.7%, 66.8% and 74.8% of the saline group on day 21 respectively. F4/MCPT and F4/CPT exhibited superior tumor inhibitory effects due to the sustained release of MCPT and CPT from fiber fragments. Moreover, F4/MCPT resulted in stronger suppression of tumor growth than F4/CPT, probably due to two properties of MCPT, i.e. the FRmediated cellular uptake and redox-responsive drug release. Figure 7b shows the visual images of tumors retrieved after treatment for 21 days, indicating the greatest potency of F4/MCPT for tumor growth inhibition. Figure 7c summarizes the survival rates of tumor-bearing mice in a Kaplan−Meier ACS Paragon Plus Environment

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plotting, showing that 50% mice died after the treatment of MCPT and the mixture of MCPT and F4 for 16 and 15 days, respectively. The intratumoral injection of F4/MCPT extended 50% mean survival time to 32 days, significantly higher than that of 21 days for F4/CPT, indicating the high therapeutic efficacy and low systemic toxicity of MCPT-loaded fiber fragments. Histological Evaluation of Tumors Retrieved. Tumors were retrieved after 21 days of treatment for histopathological and IHC staining. Figure 7d summarizes H&E staining results to reveal the cell morphology and tissue necrosis in tumors. Tumor cells with normal morphology and large amount of chromatin were observed for the saline group. All therapeutic groups exhibited various degrees of tissue necrosis along with the occurrence of pyknotic or missing nuclei (absence of purple staining), while the F4/MCPT treatment led to the highest level of tumor necrosis along with apparent vacuolus degeneration of tumor cells. To further investigate the antitumor activity, IHC staining of caspase-3 (Figure 7e) and Ki-67 (Figure 7f) was performed on tumor sections, and Figure S10 (Supporting Information) summarizes the counting results of positively stained cells after each treatment. A remarkably higher amount of apoptotic cells were detected after intravenous and intratumoral injection of MCPT, at 28.6 ± 2.1% and 39.6 ± 2.6% respectively (p < 0.05), compared with 7.8 ± 1.1% of apoptotic cells for saline group. In addition, the intratumoral administration of F4/MCPT led to the highest apoptosis level at 87.3 ± 4.4% (p < 0.05). It is generally known that Ki-67 is an antigen that corresponds to a nuclear non-histone protein expressed by cells in the proliferative phases, and a higher index of Ki-67 means a faster proliferation in tumor.31 As shown in Figure 7f, the lowest degree of tumor proliferation was detected after F4/MCPT treatment. The counting result of positively stained cells after F4/MCPT treatment was 14.6 ± 1.2%, which was significantly lower than those of MCPT and F4/CPT (p < 0.05), at 37.5 ± 2.3% and 17.1 ± 1.5%, respectively (Figure S10). Intratumoral injection offers benefits to those unresectable or inoperable solid tumors in minimizing their sizes without invasive surgery. As indicated above, the intratumoral administration of MCPTloaded fiber fragments showed the most significant antitumor efficacy with regard to the tumor growth ACS Paragon Plus Environment

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inhibition, animal survival rate, as well as tumor cell apoptosis induction. Compared with conventional intravenous injection, the intratumoral administration offered an advantage of high drug accumulation in tumor tissues. But drugs and micelles still easily migrated away from the tumor and then went into the blood cycle of normal tissues on account of the leaky vascular wall.32 Thus, the intratumoral injection of mixture of MCPT and F4 fails to exert significant inhibition of tumor growth in a manner equivalent to the intravenous injection of MCPT (Fig. 7). It is indicated that the intratumoral injection of short fibers with average lengths of around 20 µm is advantageous in promoting the drug retention and distribution in tumors.18 The sustained release of drugs or micelles from fiber fragments in response to pHe (Figure 3) could localize high drug doses at the tumor site for an extended period of time while limiting the systemic drug exposure to normal tissues. Thus, the intratumoral administration of F4/CPT or F4/MCPT fragmented fibers showed higher antitumor efficacy than others (Figure 7). It has been proved that the poor cellular internalization as well as insufficient intracellular drug release always limited the dosages of anticancer drugs to the level below the therapeutic window, which hampered the efficacy of cancer chemotherapy.33 As shown in Figure 7, F4/MCPT had more potent antitumor effects than F4/CPT, most likely due to the enhanced cell internalization of the released micelles via FRmediated endocytosis (Figure 4) and prompt intracellular drug release in response to elevated GSH levels. Thus, F4/MCPT realized effective micelle concentrations in tumor tissues and elevated intracellular drug levels via dual targeting mechanisms (i.e. localized intratumoral injection of fiber fragments and FR-mediated cellular uptake of micelles) and dual stimuli-responsiveness (i.e. pHe- and GSH- triggered micelle and drug releases respectively).

4. CONCLUSION This study proposes a strategy to load promicelle polymers into fiber fragments by blend electrospinning. The acid-liable breakdown of fiber matrices leads to the release of promicelle

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polymers and spontaneous assembly into micelles in the tumor extracellular matrix, followed by FRmediated cellular internalization and reduction-sensitive intracellular release of drugs. The micelles released from fiber fragments reveal similar profiles to those freshly prepared via solvent evaporation, such as the average size and morphology, FR-mediated cellular uptake and GSH-sensitive drug release. By using the AIE effect of MTPE micelles, we demonstrate that the spontaneous assembly of PMTPE into micelles once they release from fibers, followed by subsequent cell internalization into cytosol. The in situ micelle assembly in response to tumor acidic pH microenvironment is also confirmed from selfassembly-induced fluorescence light-up inside tumor tissues after intratumoral injection of F/MTPE fiber fragments. Compared with F4/CPT fiber fragments and free MCPT, the sustained release of micelles from F4/MCPT results in significantly higher antitumor efficacy. Therefore, intratumoral injection of MCPT-loaded fragmented fibers realizes in situ formation of active targeting and redox-sensitive micelles in response to pHe, representing a powerful strategy for dual targeted delivery and dual stimuli-responsive releases of drugs. Supporting Information available: The synthesis and characterization of CDM and CDM/PEG, 1H NMR spectra of CDM/PELA and the degradation products, GPC elution profiles of CDM/PEG and CA/PEG copolymers, synthesis and characterization of MTPE, CMC determination of MTPE micelles, cytotoxicity of MTPE and MTPE-loaded fiber fragments, cell apoptosis analyses, and the percentages of Ki-67 or caspase-3-positive cells after treatment with fiber fragments were included in the Supporting Information. ACKNOWLEDGMENTS This work is supported by National Natural Science Foundation of China (31771034, 31470922 and 21274117) and Fundamental Research Funds for the Central Universities (2682016YXZT08).

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Figure Legends: Figure 1. (a) Structure of PMCPT amphiphilic copolymers, which are constructed from 4-arm-PEG with folate conjugation onto two arms as targeting groups and ε-caprolactone is copolymerized with another two arms, followed by CPT conjugation through disulfide linkages. (b) Synthesis routes for CDM/PELA copolymers. The two ends of PEG are blocked with CDM, following by conjugation of other PEG molecules to obtain CDM/PEG. CDM/PELA copolymers are prepared by the ring-opening polymerization of DL-lactide, using CDM/PEG as an initiator and stannous chloride as a catalyst. (c) Schematic drawing of the micelle delivery and drug release process. CDM/PELA fiber fragments are degraded and release PMCPT in response to pHe, followed by self-assembly into MCPT with folate ligands and CPT conjugates. MCPT is endocytosed into tumor cells mediated by folate ligands, followed by intracellular release of CPT in response to cytosol GSH. Figure 2. (a) SEM morphologies of F4/MCPT, F5/MCPT and F6/MCPT fiber fragment. (b) The mass loss and (c) molecular weight reduction of F4, F5 and F6 fiber fragment after incubation in pH 7.4, 6.5 and 5.5 buffers at 37 °C for 10 days (n = 3). Figure 3. Micelle and CPT releases from fiber fragments. (a) Percent release of MCPT from F4/MCPT, F5/MCPT and F6/MCPT fiber fragments, (b) percent release of CPT from F4/CPT, F5/CPT and F6/CPT fiber fragments and (c) from F4/MCPT, F5/MCPT and F6/MCPT fiber fragments after incubation into pH 7.4 and 6.5 buffers at 37 °C (n = 3). (d) Typical TEM image of collected micelles after release from F4/MCPT fiber fragments. (e) Typical DLS images of collected micelles after incubation in PBS and PBS containing 2 µM and 10 and 40 mM GSH at 37 °C for 12 h. (f) Percent release of CPT from collected micelles after incubation in PBS and PBS containing 2 µM and 10 and 40 mM GSH at 37 °C (n = 3). Figure 4. Cellular uptake and cytotoxicities of MCPT released from fiber fragments. (a) The uptake efficiency of CPT by HepG2 cells after incubation with MCPT-loaded fiber fragments (F4/MCPT, F5/MCPT and F6/MCPT) and CPT-loaded fiber fragments (F4/CPT, F5/CPT and F6/CPT) in pH 7.4 and ACS Paragon Plus Environment

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6.5 media, compared with free MCPT and CPT with a final CPT concentration of 3 µg/mL (n = 5). (b) Typical CLSM images of cells, counterstained by propidium iodide (red) after incubation with F4/MCPT fiber fragments in pH 7.4 and 6.5 media, compared with free MCPT and CPT. (c) The cytotoxicity to HepG2 cells of MCPT-loaded and CPT-loaded fiber fragments with the equal amount of CPT released to free MCPT and CPT after incubation in pH 7.4 and 6.5 media (n = 5). (d) Summary of the apoptosis rate and IC50 values after incubation with MCPT-loaded and CPT-loaded fiber fragments in pH 7.4 and 6.5 media, compared with free MCPT and CPT. Figure 5. (a) Illustration of the formation of MTPE micelles. (b) Typical TEM images of MTPE freshly prepared and released from F4/MTPE fiber fragments after incubation in pH 6.5 buffers. (c) Percent release of MTPE from F4/MTPE, F5/MTPE and F6/MTPE fiber fragments after incubation into pH 7.4 and 6.5 buffers (n = 3). (d) Typical CLSM images of HepG2 cells, counterstained by propidium iodide (red) after incubation with F4/MTPE fiber fragments in pH 7.4 and 6.5 media, compared with fresh MTPE. Figure 6. MTPE in tumor tissues. (a) Typical CLSM images of tumor tissue sections after dropping of PMTPE in DMSO/H2O mixed solutions, (b) retrieved after intratumoral injection of mixtures of MTPE and F4 fiber fragments for 1 and 3 days, (c) after intravenous injection of MTPE, and (d) after intratumoral injection of F4/MTPE fiber fragments for 1, 3, 7 and 14 days, using blank tissue section as control (e). Figure 7. In vivo antitumor efficacy against H22 tumors. (a) Tumor growth, (b) typical images of tumors retrieved, (c) overall survival of tumor-bearing mice, (d) Typical H&E staining images (“N” represents necrotic area, “T” represents tumor mass), (e) IHC staining images of caspase-3 and (f) Ki67 of tumors retrieved on day 21 after intratumoral injection of F4/MCPT, F4/CPT fiber fragments and mixtures of MCPT and F4 fiber fragments, and intravenous administration of MCPT at a dose of 4.0 mg CPT/kg body weight, using saline injection as control.

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Figure 1. (a) Structure of PMCPT amphiphilic copolymers, which are constructed from 4-arm-PEG with folate conjugation onto two arms as targeting groups and ε-caprolactone is copolymerized with another two arms, followed by CPT conjugation through disulfide linkages. (b) Synthesis routes for CDM/PELA copolymers. The two ends of PEG are blocked with CDM, following by conjugation of other PEG molecules to obtain CDM/PEG. CDM/PELA copolymers are prepared by the ring-opening polymerization of DL-lactide, using CDM/PEG as an initiator and stannous chloride as a catalyst. (c) Schematic drawing of the micelle delivery and drug release process. CDM/PELA fiber fragments are degraded and release PMCPT in response to pHe, followed by selfassembly into MCPT with folate ligands and CPT conjugates. MCPT is endocytosed into tumor cells mediated by folate ligands, followed by intracellular release of CPT in response to cytosol GSH. 141x134mm (300 x 300 DPI)

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Figure 2. (a) SEM morphologies of F4/MCPT, F5/MCPT and F6/MCPT fiber fragment. (b) The mass loss and (c) molecular weight reduction of F4, F5 and F6 fiber fragment after incubation in pH 7.4, 6.5 and 5.5 buffers at 37 °C for 10 days (n = 3). 128x110mm (300 x 300 DPI)

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Figure 3. Micelle and CPT releases from fiber fragments. (a) Percent release of MCPT from F4/MCPT, F5/MCPT and F6/MCPT fiber fragments, (b) percent release of CPT from F4/CPT, F5/CPT and F6/CPT fiber fragments and (c) from F4/MCPT, F5/MCPT and F6/MCPT fiber fragments after incubation into pH 7.4 and 6.5 buffers at 37 °C (n = 3). (d) Typical TEM image of collected micelles after release from F4/MCPT fiber fragments. (e) Typical DLS images of collected micelles after incubation in PBS and PBS containing 2 µM and 10 and 40 mM GSH at 37 °C for 12 h. (f) Percent release of CPT from collected micelles after incubation in PBS and PBS containing 2 µM and 10 and 40 mM GSH at 37 °C (n = 3). 80x43mm (300 x 300 DPI)

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Figure 4. Cellular uptake and cytotoxicities of MCPT released from fiber fragments. (a) The uptake efficiency of CPT by HepG2 cells after incubation with MCPT-loaded fiber fragments (F4/MCPT, F5/MCPT and F6/MCPT) and CPT-loaded fiber fragments (F4/CPT, F5/CPT and F6/CPT) in pH 7.4 and 6.5 media, compared with free MCPT and CPT with a final CPT concentration of 3 µg/mL (n = 5). (b) Typical CLSM images of cells, counterstained by propidium iodide (red) after incubation with F4/MCPT fiber fragments in pH 7.4 and 6.5 media, compared with free MCPT and CPT. (c) The cytotoxicity to HepG2 cells of MCPT-loaded and CPTloaded fiber fragments with the equal amount of CPT released to free MCPT and CPT after incubation in pH 7.4 and 6.5 media (n = 5). (d) Summary of the apoptosis rate and IC50 values after incubation with MCPTloaded and CPT-loaded fiber fragments in pH 7.4 and 6.5 media, compared with free MCPT and CPT. 139x130mm (300 x 300 DPI)

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Figure 5. (a) Illustration of the formation of MTPE micelles. (b) Typical TEM images of MTPE freshly prepared and released from F4/MTPE fiber fragments after incubation in pH 6.5 buffers. (c) Percent release of MTPE from F4/MTPE, F5/MTPE and F6/MTPE fiber fragments after incubation into pH 7.4 and 6.5 buffers (n = 3). (d) Typical CLSM images of HepG2 cells, counterstained by propidium iodide (red) after incubation with F4/MTPE fiber fragments in pH 7.4 and 6.5 media, compared with fresh MTPE. 92x57mm (300 x 300 DPI)

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Figure 6. MTPE in tumor tissues. (a) Typical CLSM images of tumor tissue sections after dropping of PMTPE in DMSO/H2O mixed solutions, (b) retrieved after intratumoral injection of mixtures of MTPE and F4 fiber fragments for 1 and 3 days, (c) after intravenous injection of MTPE, and (d) after intratumoral injection of F4/MTPE fiber fragments for 1, 3, 7 and 14 days, using blank tissue section as control (e). 60x24mm (300 x 300 DPI)

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Figure 7. In vivo antitumor efficacy against H22 tumors. (a) Tumor growth, (b) typical images of tumors retrieved, (c) overall survival of tumor-bearing mice, (d) Typical H&E staining images (“N” represents necrotic area, “T” represents tumor mass), (e) IHC staining images of caspase-3 and (f) Ki-67 of tumors retrieved on day 21 after intratumoral injection of F4/MCPT, F4/CPT fiber fragments and mixtures of MCPT and F4 fiber fragments, and intravenous administration of MCPT at a dose of 4.0 mg CPT/kg body weight, using saline injection as control. 106x75mm (300 x 300 DPI)

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