Acta Biomaterialia 23 (2015) 189–200

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Spatial distribution and antitumor activities after intratumoral injection of fragmented fibers with loaded hydroxycamptothecin Jiaojun Wei, Xiaoming Luo, Maohua Chen, Jinfu Lu, Xiaohong Li ⇑ Key Laboratory of Advanced Technologies of Materials, Ministry of Education of China, School of Materials Science and Engineering, Southwest Jiaotong University, Chengdu 610031, PR China

a r t i c l e

i n f o

Article history: Received 12 January 2015 Received in revised form 10 May 2015 Accepted 18 May 2015 Available online 23 May 2015 Keywords: Fragmented fiber Electrospinning Intratumoral injection Antitumor activity Spatial distribution

a b s t r a c t There was only a small percentage of drug delivered to tumors after systemic administration, and solid tumors also have many barriers to prevent drug penetration within tumors. In the current study, intratumoral injection of drug-loaded fiber fragments was proposed to overcome these barriers, allowing drug accumulation at the target site to realize the therapeutic efficacy. Fragmented fibers with hydroxycamptothecin (HCPT) loaded were constructed by cryocutting of aligned electrospun fibers, and the fiber lengths of 5 (FF-5), 20 (FF-20), and 50 lm (FF-50) could be easily controlled by adjusting the slice thickness. Fragmented fibers were homogeneously dispersed into 2% sodium alginate solution, and could be smoothly injected through 26G1/2 syringe needles. FF-5, FF-20 and FF-50 fiber fragments indicated similar release profiles except a lower burst release from FF-50. In vitro viability tests showed that FF-5 and FF-20 fiber fragments caused higher cytotoxicity and apoptosis rates than FF-50. After intratumoral injection into murine H22 subcutaneous tumors, fragmented fibers with longer lengths indicated a higher accumulation into tumors and a better retention at the injection site, but showed less apparent diffusion within tumor tissues. In addition to the elimination of invasive surgery, HCPT-loaded fiber fragments showed superior in vivo antitumor activities and fewer side effects than intratumoral implantation of drug-loaded fiber mats. Compared with FF-5 and FF-50, FF-20 fiber fragments indicated optimal spatial distribution of HCPT within tumors and achieved the most significant effects on the animal survival, tumor growth inhibition and tumor cell apoptosis induction. It is suggested that the intratumoral injection of drug-loaded fiber fragments provided an efficient strategy to improve patient compliance, allow the retention of fragmented fibers and spatial distribution of drugs within tumor tissues to achieve a low systemic toxicity and an optimal therapeutic efficacy. Ó 2015 Acta Materialia Inc. Published by Elsevier Ltd. All rights reserved.

1. Introduction Cancer remains a major leading cause of death worldwide although diagnosis and treatment methods have recently been improved to reduce the mortality considerably. After surgery, chemotherapy is the most commonly used treatment strategy for most cancers. However, the conventional systemic cancer chemotherapy suffers from poor pharmacokinetics and inappropriate biodistribution, and only a small percentage of administered drugs reach the target tissues and organs [1]. For example, the low molecular weight of anticancer agents leads to a rapid removal from systemic circulation through renal filtration and a limited accumulation in tumors and tumor cells. In addition, highly hydrophobic drug molecules often have a large volume of ⇑ Corresponding author. Tel.: +86 28 87634068; fax: +86 28 87634649. E-mail address: [email protected] (X. Li). http://dx.doi.org/10.1016/j.actbio.2015.05.020 1742-7061/Ó 2015 Acta Materialia Inc. Published by Elsevier Ltd. All rights reserved.

distribution, tending to accumulate in and cause toxicities toward many healthy tissues [2]. Thus, cancer chemotherapy usually indicates severe limitations in the safety and effectiveness, such as systemic toxicity, immunogenic injury, and significant morbidity, which have heavy impacts on the quality of life of patients and hamper its wide clinical application. Therefore, many attempts have been made to develop multifunctional carriers to achieve a selective accumulation of chemotherapeutic agents in tumor tissues, cells and subcellular organelles, and subsequently to have an effective anticancer effect with a sufficient therapeutic index [3]. Targeted drug delivery systems have been proposed to overcome biological barriers, intelligently respond to disease environment and release therapeutic agents. One of the targeting mechanisms is passive targeting through drug accumulation into tumors with leaky vasculature and insufficient lymphatic system, referred as the enhanced permeation and retention (EPR) effect

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[4]. This strategy primarily includes long-circulating liposomes, nanoparticles, and micelles, but over 95% of the administered dose is known to accumulate in organs other than tumors, particularly in liver, spleen, and lungs [5]. Alternatively, active drug targeting involves the cell recognition and uptake enhancement by such ligands as folate, transferrin and galactosamine, taking advantage of the overexpression of tumor cell surface receptors. However, the ligand–receptor interactions occur only on the tumor cell surface and do not have the ability to guide themselves to a target, thus the primary mode of tumor localization still relies on EPR-mediated passive extravasation [6]. Previous studies have shown that the presence of tumor-targeting ligands does not always result in an increased accumulation of drug-loaded nanoparticles in tumors [7]. Florence et al. summarizes the current unmet needs and challenges in targeted drug delivery, and raises awareness on the exaggerated claims of the nanoparticle-based drug targeting [8]. Therefore, given that systemic chemotherapy does not have entire ability to guide themselves to a target, it is very difficult to achieve therapeutic levels of drugs within or adjacent to the tumor tissues. Another strategy to overcome these challenges is known as localized drug delivery, where drug release is limited to the tumor site to maintain therapeutic concentrations of drugs. This makes therapies more efficient with minimal side effects for patients, capabilities of which cannot be achieved by conventional systemic administration of drugs [9]. Over the past decades, implantable drug-releasing systems have become more sophisticated and implants based on silicone rubber or polymers have extensively been used for the administration of steroid hormones, anesthetic agents, antibiotics, anticancer drugs, and insulin [10]. Drug-loaded wafer [11], film composites [12] and fibrous mats [13] have been implanted directly into a tumor or at the site of tumor resection. But the physical implants require direct accessibility through surgical procedures, having a large invasiveness. Alternatively, local injection of nanoparticles, microparticles and in situ forming precipitates into tumors or along the perimeter of tumors was developed for cancer treatment. Benny et al. injected microspheres with entrapped antiangiogenic agents into intracranial glioma tumors in nude mice, causing a remarkable reduction in the tumor volume with a significant decrease in angiogenesis and an increase in apoptosis [14]. Zhao et al. evaluated paclitaxel-loaded nanoparticles on A-549 tumor-bearing mice via intratumoral injection, showing an effective inhibition on the tumor growth and a higher cytotoxicity than commercial paclitaxel formulation Taxol [15]. But the injection of microsphere or nanoparticle suspensions indicates a quick migration from the administration site [16]. Another injectable implant is syringeable liquid formulations, which are injected intramuscularly or subcutaneously and solidified in situ to form solid or semi-solid drug depots. One commercialized product was based on poly(lactide-co-glycolide) (PLGA) solutions in water-miscible organic solvents containing leuprolide acetate, which were subcutaneously injected for the treatment of prostate cancer. The diffusion of organic solvent toward the surrounding aqueous environment led to PLGA precipitation and formation of an implant system for a sustained release of leuprolide acetate over 6 months [17]. As carrier vehicles, several water-miscible organic solvents, such as N-methyl-2-pyrrolidone (NMP) and dimethyl sulfoxide (DMSO) are the most preferred, but their use is restricted owing to controversial reports regarding the toxicity [18]. Alternatively, DuVall et al. evaluated the thermogelling of polylactide–poly(ethylene glycol) copolymers (PELA) at physiological temperatures to sustain the release of paclitaxel after intratumoral injection. Phase II clinical trials have revealed a successful tumor growth inhibition, good tolerability and low systemic exposure [19]. However, the injectability and gelation process should be balanced in the in situ forming hydrogels in response to changes in environmental

temperature and pH [20]. It should be noted that the major limitation of these injectable implants was the extensive burst release rightly after injection into tumors, due to the lag time between the injection of the system and the precipitation/gelation of the polymer. Sometimes the drug levels were higher than the recommended safety margin, which is obviously more problematic for those drugs that have a narrow therapeutic window [21]. In this view, fragmented fibers with hydroxycamptothecin (HCPT) loaded were proposed in the current study for cancer treatment after intratumoral injection. Fragmented fibers not only retain the advantages of continuous fibers such as large specific surface areas, localized and controlled delivery, but also have the ability to reduce the invasion of surgical implantation of fiber mats into tumors. Fragmented fibers were constructed by cryocutting of aligned electrospun fibers, and the lengths could be conveniently controlled by adjusting the slice thickness to achieve a good injectability and tissue remaining after local injection. The in vivo distribution of HCPT released from fiber fragments was investigated in tumors, blood and other tissues, compared with free HCPT and fiber mats. The spatial distributions of HCPT and fiber fragments of different lengths in tumor tissues were also determined from serial tissue sections. The antitumor efficacy was evaluated on H22-tumor bearing mice with respect to tumor growth inhibition, animal survival, histopathological and immunohistochemical (IHC) analysis of tumors retrieved. 2. Materials and methods 2.1. Materials PELA (Mw = 50 kDa, Mw/Mn = 1.23) containing 10% of poly(ethylene glycol) (PEG) was prepared by bulk ring-opening polymerization of lactide/PEG using stannous chloride as the initiator [22]. HCPT with purity of over 98% was from Junjie Biomedical Ltd. (Shanghai, China), and collagenase IV, trypsin, and DMSO were obtained from Sigma–Aldrich (St. Louis, MO). Rabbit anti-mouse antibodies of caspase-3 and Ki-67, goat anti-rabbit IgG-horseradish peroxidase (HRP), and 3,30-diaminobenzidine (DAB) developer were purchased from Biosynthesis Biotechnology Co., Ltd. (Beijing, China). All other chemicals and solvents were of reagent grade or better and purchased from Changzheng Regents Co. (Chengdu, China), unless otherwise indicated. 2.2. Preparation of HCPT-loaded fiber fragments HCPT-loaded fiber fragments were prepared from aligned electrospun fibers (EF) by cryocutting, and aligned fibers were obtained after collecting on a rotating mandrel as described elsewhere [23]. Briefly, 10 mg HCPT was dissolved in 80 ll of DMSO, while 500 mg of PELA was dissolved in 3.0 ml of chloroform/dimethyl formamide (5/1, v/v). The blend of above solutions was transferred to a 5-ml syringe and then pumped at 1.6 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). Fibers were deposited on an aluminum foil wrapped on a grounded rotating mandrel at a linear rate of around 15 m/s. After vacuum drying overnight to remove residual solvents, the fibrous mat was folded at about 1 cm of intervals perpendicularly to fiber alignment and was completely soaked with distilled water. The folded fibrous mat was placed vertically in a plastic embedding cryomold, followed by the addition of Cryo-OCT compound (Thermo Fisher Scientific Inc., Waltham, MA) and freezing at 70 °C for 5 min. The solidified block of gels with fibrous mats

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embedded therein were sectioned using a cryostat microtome (Microme HM550 OMC, Thermo Fisher Scientific Inc., Waltham, MA) at 20 °C. The section thickness was controlled at 5, 20, and 50 lm, which were used to prepare fragmented fibers of FF-5, FF-20, and FF-50, respectively. The fiber bundles were harvested in water and dispersed into individual fiber fragments after ultrasonication for 5 min. The fragmented fibers were collected by centrifugation, freeze-dried and stored at 4 °C, away from light, for further detection. 2.3. Characterization of HCPT-loaded fiber fragments The morphology of electrospun fibrous mats and fragmented fibers was observed by a 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 charging effect. The alignment of electrospun fibers was determined from SEM images by measuring the individual orientation and angular deviation of fibers, and the orientation degree indicated the percentage of fibers distributed from 10° to 10° with respect to the total fibers counted as described previously [23]. The diameter and length of fragmented fibers were measured from SEM images to generate an average value by using the tool of Photoshop 10.0 edition [12]. HCPT is highly fluorescent, and the encapsulation of HCPT in fragmented fibers was examined by a fluorescence microscope (Olympus IX51-FL, Japan). The loading amount and encapsulation efficiency of HCPT were determined after extracting from fiber fragments [24]. Briefly, a known amount of fragmented fibers (ca. 2 mg) were dissolved in 1.0 ml of chloroform and extracted three times with 20.0 ml of pH 7.4 phosphate buffer saline (PBS). The extracted solution was measured by a fluorospectrophotometer (Hitachi F-7000, Japan) with the excitation wavelength of 380 nm and the emission wavelength of 550 nm. The HCPT concentration was obtained using a standard curve from known concentrations of HCPT solutions. The extraction efficiency was calibrated by adding a certain amount of HCPT into polymer/chloroform solution along with the same concentration as above and extracted using the above-mentioned process. The loading amount of HCPT indicated the amount (in milligrams) of HCPT encapsulated per 100 mg of fiber fragments. 2.4. In vitro drug release of HCPT from fragmented fibers In vitro HCPT release was determined from fiber fragments of different lengths, using fiber mats as the control. Briefly, HCPT-loaded fiber mats or fragmented fibers corresponding to around 120 lg HCPT were exactly weighed and put into dialysis bags (3.5 kDa cutoff). To ensure a sink condition, dialysis bags were incubated in 40 ml of PBS and kept in a thermostated shaking water bath that was maintained at 37 °C and 100 r/min. At specified time intervals, 1.0 ml of release media was withdrawn and an equal volume of release media was added for continuing incubation. The HCPT concentration in the release media was detected by a fluorospectrophotometer as mentioned above. 2.5. In vitro cytotoxicity and apoptosis assay of HCPT-loaded fiber fragments HCPT-loaded fiber fragments were sterilized by electron-beam irradiation using a linear accelerator (Precise™, Elekta, Crawley, UK) with a total dose of 80 cGy. The cell viability and apoptosis were evaluated on HepG2 cells after treatment with fragmented fibers of different lengths, compared with free drug and drug-loaded fiber mats. Briefly, HepG2 cells from the American Type Culture Collection (Rockville, MD) were cultured in RPMI 1640 (Gibco BRL, Rockville, MD) containing 10% fetal bovine serum

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(FBS, Gibco BRL, Rockville, MD). Cells were seeded in a 48-well tissue culture plate (TCP) at a density of 1  105 cells per well and allowed to attach overnight before drug treatment. HCPT stock solutions were diluted in RPMI 1640, and drug-loaded fiber fragments releasing equivalent amount of HCPT during 72 h (from in vitro release data) were applied. After incubation for 24, 48, and 72 h, the fiber-containing media were removed, followed by the addition of 200 ll of fresh culture media and 20 ll of Cell Counting Kit-8 (CCK-8) reagent (Dojindo Molecular Technologies, Inc., Kumamoto, Japan) into each well. After incubation for 2 h according to the reagent instruction, 100 ll of culture media was pipetted into another 96-well TCP and the absorbance of each well was measured at 450 nm using a lQuant microplate spectrophotometer (Elx-800, Bio-Tek Instrument Inc., Winooski, VT). The same volumes of culture media and CCK-8 reagent were incubated without cells as the background. 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 a 12-well TCP at a density of 4  105 cells per well and allowed to attach overnight, followed by exposure to drug-loaded fiber fragments or fiber mats for 48 h. The attached and floating cells were harvested with 0.25% trypsin, washed twice with PBS, and suspended in 500 ll binding buffer. After the addition of 5 ll Annexin V-FITC and 5 ll propidium iodide, the samples were gently mixed and incubated at room temperature for 15 min before analysis with flow cytometry (BD Accuri C6, Franklin Lakes, NJ). Data analysis was performed using BD FACSuite™ (BD Biosciences, CA). 2.6. Tumor treatment by intratumoral injection of HCPT-loaded fiber fragments The tumor model was established subcutaneously by injection of murine hepatoma H22 cells as described previously with some modifications [12], 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 22 ± 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 tumors were sacrificed, and the ascites was 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 ll/g body weight. Tumors were allowed for growth for 10 days to reach about 500 mm3 in volume, and the tumor-bearing mice were randomly divided into seven groups with 8 mice per group. The animals were anaesthetized by intraperitoneal injection of pentobarbital at 25 mg/kg. HCPT-loaded FF-5, FF-20, and FF-50 fiber fragments were dispersed homogeneously into sodium alginate solution in saline (2%, w/v), and animals were treated with a single intratumoral injection with 100 ll of fiber fragment suspensions through 26G1/2 needles, using saline injection as control. Free HCPT was formulated into saline containing DMSO (5%, v/v), and each animal was administrated about 0.2 ml into the tumor. HCPT-loaded fiber mats were inserted into the tumor after making a small incision on the skin to expose the tumor as described previously [12]. The HCPT dose was equivalent to the total amount of 4.0 mg/kg body weight. 2.7. In vivo antitumor efficacy of HCPT-loaded fiber fragments The body weights, tumor volumes, and survival rates of animals were monitored after different treatment. The lengths of the major

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axis (longest diameter) and minor axis (perpendicular to the major axis) of a tumor were measured with a vernier caliper, and the tumor volume was calculated as described previously [12]. 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 comparison of treatment efficacy. 2.8. Distribution of HCPT and fragmented fibers in tissues and tumors The distribution of HCPT was determined in tumor and other tissues at different time points as described previously with some modifications [25]. Briefly, animals were treated as above, and sacrificed by cervical dislocation after treatment for 1, 7, and 14 days. Blood samples were collected via eye sockets and immediately centrifuged at 3000 r/min for 10 min to obtain plasma. The heart, liver, spleen, lung, kidney and tumors were collected, cut into small pieces and washed with ice-cold saline to remove bloodstains. The tissue pieces were homogenized in saline, then DMSO was added in the tissue homogenate for continuing homogenization. The above mixtures were vortexed for 3 min and centrifuged for 5 min at 3000 r/min. The amount of HCPT in the supernatant and plasma was measured by a fluorospectrophotometer as above. The amount of HCPT in a tissue was obtained using a standard curve from known concentrations of HCPT in the homogenate of this tissue from untreated mice. The percentage of injected dose (ID%) indicated the ratio of actual amount of drug in a tissue to the total amount of injected drug, and the per cent dose rate (ID%/g) was used to represent the HCPT accumulation in per gram of a tissue [26]. In another group of experiment, tumor tissues were collected for analysis of the spatial distribution of HCPT and fragmented fibers as described previously with some modifications [27]. Briefly, the tumor was retrieved, embedded in OCT compound and sectioned into 50 lm-thick sections in a frozen cutting machine at 20 °C. Each section was incubated in solution containing collagenase IV, trypsin and calcium chlorate to digest the tissues [28]. After centrifugation, the supernatant was collected for the detection of HCPT contents at different locations of tumors. The residue was collected and HCPT was extracted after the addition of DMSO to detect the retention of drug-loaded fiber fragments after injection into tumors. The spatial distribution of HCPT and HCPT-loaded fiber fragments indicated the amount of HCPT (lg) in per gram of tissue pieces at different locations in tumors away from the injection site. 2.9. Histopathological and immunohistochemical evaluation of tumors retrieved After 14 and 30 days of treatment, animals of each group were randomly chosen and euthanized to retrieve tumors. The excised tumors were washed by saline, dried with filter paper, and weighed before fixation in 10% neutral buffered formalin. The tissues were processed routinely, and sectioned at a thickness of 4 lm. To evaluate the cell morphology and tissue necrosis, tumor sections were stained with hematoxylin and eosin (HE) 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 [29]. 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 proteins were quantified by comparing the positively stained cells with the total number of cells in these areas [29].

2.10. Statistics analysis Data are expressed as mean ± standard deviation (SD). The statistical significance of the data obtained was analyzed by the Student’s t-test. Probability values of p < 0.05 were interpreted as denoting statistical significance. 3. Results and discussion 3.1. Characterization of HCPT-loaded fiber fragments HCPT-loaded fiber fragments were obtained from cryocutting of electrospun fibers with a high alignment. HCPT was dissolved in DMSO and blended in the electrospinning solution. DMSO is a relatively safe solvent and widely used in vitro as cryoprotectant of cells and in vivo as water-miscible organic solvents for polymer injection [18]. Fig. 1a shows the SEM image of aligned fibers with uniform morphologies, indicating an average diameter of 0.92 ± 0.15 lm and an orientation degree of 93.8%. Fragmented fibers of different lengths were obtained by controlling the section thickness of cryocutting process. Fig. 1b shows SEM images of the fiber fragments obtained. FF-5, FF-20 and FF-50 fiber fragments indicated lengths of 7.2 ± 2.7, 23.1 ± 3.7 and 56.6 ± 3.0 lm, respectively. To achieve a smooth injection, HCPT-loaded fiber fragments were dispersed into sodium alginate solution in saline (2%, w/v), indicating a homogeneous dispersion profile (Fig. 1c), and the fiber fragment dispersion could be smoothly injected through a 26G1/2 syringe needle. HCPT can emit fluorescent light spontaneously, and Fig. 1d shows the fluorescence images of HCPT-loaded fiber fragments, indicating the presence and well distribution of HCPT in fragmented fibers. Efficient drug encapsulation was one of the advantages of electrospun fibers [30], and the HCPT loading of 2.0% was detected in fiber mats. The loading amounts of HCPT in FF-5, FF-20 and FF-50 fiber fragments were about 1.8%, 2.0% and 2.0%, respectively. There was no apparent drug loss during the cryocutting process, and the slight lower drug content in FF-5 fiber fragments was due to the drug loss from cross sections. 3.2. In vitro drug release of HCPT-loaded fiber fragments The drug release was determined from fragmented fibers of different lengths, compared with fiber mats. Fig. 2 shows similar release profiles of HCPT from these fiber fragments. There were around 20% of initial releases of HCPT from FF-5 and FF-20 fiber fragments during 24 h incubation, followed by a sustained release for 3 weeks. A significantly lower burst release of around 10% was detected from FF-50 fiber fragments (p < 0.05), and the accumulated release of HCPT during 3 weeks was around 50–60% from FF-5, FF-20 and FF-50 fiber fragments. However, the fiber mats indicated less than 5% of burst release and around 35% of accumulated release during 3 weeks. It was indicated that the HCPT release was significantly higher for fragmented fibers than fiber mats, due to the increased release from cross sections of fiber fragments. For these extremely hydrophobic drugs, regulation of the release rate from polymer matrices is one of the challenges to maintain suitable drug concentrations within a therapeutic window for enough exposure time [31]. Generally speaking, if not reached the level of drug toxicity, initial burst release was actually necessary to promptly inhibit the tumor cell growth for cancer treatment. In addition, the intratumoral injection of HCPT-loaded fiber fragments could maintain higher drug concentrations in tumors, while the sustained HCPT release could alleviate the outflow of HCPT into normal tissues.

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Fig. 1. SEM morphologies of (a) electrospun aligned fibers and (b) FF-5, FF-20 and FF-50 fiber fragments. (c) Dispersion profiles of FF-5, FF-20, and FF-50 fiber fragments in 2% (w/v) sodium alginate solution. (d) Fluorescence images of FF-5, FF-20, and FF-5 fiber fragments. Bars represent 50 lm.

Fig. 2. Per cent release of HCPT from fiber mats, FF-5, FF-20, and FF-50 fiber fragments after incubation at 37 °C in PBS (n = 3).

there were significant differences among HCPT-loaded fiber fragments of different lengths and fiber mats (p < 0.05). Annexin V-FITC/propidium iodide staining was used to quantify the percentages of apoptotic cells after incubation with HCPT-loaded fiber fragments for 48 h, compared with HCPT-loaded fiber mats and free HCPT. As shown in Fig. 3b, free HCPT induced the most significant apoptosis, and the percentage of total apoptotic cells, including early and late apoptotic cells was around 22%. In comparison with around 10% of apoptotic cells after treatment with FF-50 fiber fragments, FF-5 and FF-20 indicated a higher apoptosis rate of over 15%. The lower release of HCPT from fiber mats led to a significantly lower apoptosis rate at around 6%. The lactone ring of HCPT shows a pH-dependent equilibrium with an open carboxylate form, which indicates less anti-tumor activity and several unpredictable side effects [32]. In addition, this equilibration is affected by the preferential binding of serum albumin to the carboxylate form, resulting in more rapid opening of the lactone ring under a physiological environment [33]. Considering that an equivalent amount of HCPT was dosed in the cell viability and apoptosis tests, the significantly higher cytotoxicity and apoptosis rate of drug-loaded fiber fragments of shorter lengths should rely on the sustained release profiles.

3.3. Cell viability and apoptosis after treatment with HCPT-loaded fiber fragments

3.4. In vivo distribution of HCPT release from fragmented fibers

The cytotoxicity of HCPT-loaded fiber fragments was determined after incubation with HepG2 cells for 24, 48, and 72 h, compared with HCPT-loaded fiber mats and free HCPT. In the current study, equivalent amount of HCPT with concentrations of 10.0 lg/ml was dosed for each kind of drug-loaded fibers corresponding to that released during 72 h. As depicted in Fig. 3a, the treatment with free HCPT for 24 h led to a significantly lower cell viability at 42.4 ± 3.8%, compared with HCPT-loaded fiber fragments and fiber mats (p < 0.05). However, after incubation with free HCPT for 72 h, the cell viability of around 30% was significantly higher than those of HCPT-loaded fiber fragments (p < 0.05), which may be attributed to the sustained release of its active form of HCPT from fibers [31]. The cell viability decreased along with the incubation time, and the decrease in the cell viability was more remarkable for drug-loaded fiber fragments, at 20.8 ± 1.1%, 24.8 ± 1.3%, 27.9 ± 1.4% after incubation for 72 h with FF-5, FF-20, and FF-50 fiber fragments, respectively (Fig. 3a). A higher cytotoxicity was observed for drug-loaded fiber fragments of shorter lengths, and

To gain a deep insight into the in vivo behavior of HCPT released from fragmented fibers in tumors, a biodistribution study was conducted on H22-tumor bearing mice. HCPT-loaded fiber fragments were intratumorally injected at a dose of 4.0 mg/kg, using free HCPT and intratumoral implantation of fiber mats as control. Fig. 4 summaries the per cent injected dose of HCPT in heart, liver, spleen, lung, kidney, and tumor tissues after treatment for 1, 7, and 14 days. After treatment with free HCPT, there was only around 14.3% of injected dose detected after 1 day, and HCPT was not detectable in above tissues after 7 and 14 days. After intratumoral injection, HCPT easily escaped from the leaky vascular wall in the tumor and then went into the blood cycle of normal tissues, which was eliminated during several hours [34]. It should be noted that microfiber fragments have advantages over nanofiber ones in relieving the burst release and enhancing the tissue retention of drugs, resulting in a less systemic toxicity and a higher local treatment efficacy. Tumor vasculature was relatively leaky, allowing the diffusion across the vessel of particles up to 400 nm in diameter

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Fig. 3. (a) The cytotoxicity to HepG2 cells after incubation with free HCPT, HCPT-loaded fiber mats, FF-5, FF-20, and FF-50 fiber fragments for 24, 48, and 72 h (n = 5; *p < 0.05). (b) Flow cytometry analysis of HepG2 cells after incubation for 48 h with free HCPT, HCPT-loaded fiber mats, FF-5, FF-20, and FF-50 fiber fragments, compared with cells without treatment as the control. Lower left of each image, living cells; lower right, early apoptotic cells; upper right, late apoptotic cells; upper left, necrotic cells. Inserted numbers in each area indicate the percentage of cells present in this area.

[35]. So the fragmented fibers were retained in tumor tissues, and HCPT released from fiber fragments was delivered to most of organs after the first day post-treatment. As shown in Fig. 4a, over

73.4% of the injected dose was accumulated in tumors for FF-20 fiber fragments, and most of the released HCPT was distributed in liver, spleen, and blood at 8.8%, 0.5%, and 10.6% of the injected

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Fig. 4. (a) The percentage of injected dose (ID%) and (b) the per cent dose rate (ID%/g tissue) of HCPT in heart, liver, spleen, lung, kidney, tumors, and blood of H22-tumor bearing mice on days 1, 7, and 14 after intratumoral implantation of HCPT-loaded fiber mats and intratumoral injection of free HCPT, FF-5, FF-20, and FF-50 fiber fragments at a dose of 4.0 mg HCPT/kg body weight (n = 4).

Fig. 5. (a) The contents of HCPT and (b) HCPT-loaded fiber fragments at different locations from the injection site toward the edge of tumor tissues on days 1, 7, and 14 after intratumoral injection of FF-5, FF-20, and FF-50 fiber fragments at a dose of 4.0 mg HCPT/kg body weight.

dose, respectively. The HCPT levels in tumor tissues were 49.0% ID/ g tissue, which was 7.1, 12.6, and 14.4-fold higher than those in liver, spleen, and blood, respectively (Fig. 4b). During the following incubation, there were no significant changes in the tissue distribution of HCPT and the HCPT levels in the tissues after administration for 7 days, compared with those after 1 day. However, after 14 days of treatment with FF-20 fiber fragments, there was still 34.9% of the injected dose detected in the tumor tissues, and much less HCPT was found in normal tissues, such as liver, spleen, and kidney (Fig. 4a). Tumor tissues indicated HCPT levels at 25.9% ID/g tissue, which was significantly higher than those in liver, spleen, and blood at 15.6, 4.4, and 7.1% ID/g tissue, respectively (Fig. 4b). As indicated in Fig. 4, fragmented fibers of different lengths and fiber mats showed a similar tissue distribution profile except that a

higher HCPT level in tumors was determined after injection of fragment fibers of larger lengths and implantation of fiber mats. The above results proved that fragment fibers possessed a good retention after intratumoral injection and the drug steadily released from fibers into tumor tissues. 3.5. Spatial distribution of HCPT and drug-loaded fiber fragments in tumors The HCPT-loaded fiber fragments were injected into the center of a tumor tissue; therefore, the distribution of HCPT released from fragmented fibers was expected to be radial, while greater concentrations could be found at the injection site with gradual decrease toward the tumor border. The retrieved tumors were cut into serial

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Fig. 6. (a) Tumor growth and (b) typical HE staining images (‘N’ represents necrotic area, ‘T’ represents tumor mass, and bars represent 50 lm) of tumors retrieved on day 14, after intratumoral implantation of HCPT-loaded fiber mats and intratumoral injection of free HCPT, FF-5, FF-20, and FF-50 fiber fragments, compared with saline treatment as the control. *p < 0.05.

sections, and the contents of HCPT and HCPT-loaded fiber fragments were determined separately in each section. Fig. 5a summarizes HCPT contents at different locations from the center toward the edge of tumor tissues over the distance in millimeters. The maximal HCPT

concentration was found close to the injection site at around 18.2, 19.2, and 14.3 lg/g tissue after 1 day subsequent to the injection of FF-5, FF-20, and FF-50 fiber fragments, respectively. The HCPT content decreased gradually to around 1 lg/g tissue at a distance

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of 5 mm from the injection site for all the fragmented fibers, which was still considered above the therapeutic level according to the effective concentration for in vitro cytotoxicity to HepG2 cells [12]. As shown in Fig. 5a, there was a remarkable decrease in the HCPT concentration at the injection site along with the treatment time, at around 4.2, 5.4, and 4.0 lg/g tissue after treatment for 14 days with FF-5, FF-20, and FF-50 fiber fragments, respectively. The HCPT levels at the tumor border were higher than 1 lg/g tissue after intratumoral injection of FF-5 and FF-20 fiber fragments. In addition, FF-20 fiber fragments kept higher HCPT concentrations in every tumor section than FF-50, reflecting the effect of drug release profiles on the spatial distribution of HCPT in tumor tissues. Fig. 5b shows the retention of drug-loaded fiber fragments at different locations after intratumoral injection. In the first day, the HCPT loaded in fragmented fibers at the injection site was much higher than other parts of the tumor tissue, at around 700–800 lg/g tissue for all the groups, indicating that fragmented fibers were detained at the injection site. There were apparent decreases in the HCPT contents along with the treatment time, due to the release of HCPT from fiber fragments. FF-20 and FF-50 fiber fragments indicated around 45% and 37% of dropping in the HCPT contents loaded in fibers after 14 days post-treatment, at around 420 and 510 lg/g tissue, respectively. However, the decrease in the HCPT contents at the injection site was more significant after injection of FF-5 fiber fragments for 7 and 14 days, at around 200 and 80 lg/g tissue, respectively. As shown in Fig. 5b, an apparent diffusion of drug-loaded FF-5 fiber fragments after intratumoral injection for 7 and 14 days, leading to significant decreases in the HCPT-loaded fiber fragments. Tumor tissue was very crumbly, and fragmented fibers of shorter lengths easily moved to other part of the tissue. In addition, cells likely phagocytized microparticles with size of less than 5 lm [36], leading to the diffusion of FF-5 fiber fragments from the injection site. Therefore, fragmented fibers of longer lengths indicated higher accumulation into tumors and better retention at the injection site after intratumoral injection, and the diffusion within tumor tissues was more apparent for fragmented fibers of shorter lengths.

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FF-20 fiber fragments for 30 days, respectively, indicating significant differences compared with other groups (p < 0.05). The antitumor efficacy was also evaluated by HE staining of tumor tissues retrieved on day 14 post-treatment. As shown in Fig. 6b, there were still a large amount of living cells in tumors after treated with saline, and the tumor cells showed obvious nucleolus cleavage and high extent of malignant. However, large necrotic areas can be obviously seen in tumor tissues after treatment with free drug, HCPT-loaded fiber fragments and fiber mats. Necrosis within tumors represents a significant prognostic factor of tumor shrinkage after chemotherapy [37]. In addition, the necrotic region in the group of HCPT-loaded fiber fragments was larger than other groups, along with apparent vacuolus degeneration of tumor cells. 3.7. Animal survival after treatment with HCPT-loaded fiber fragments The diffusion of HCPT from tumors to other tissues may cause significant non-specific toxicity. Therefore, the survival rate of tumor-bearing animals reflected not only the antitumor activity but also the toxicity of different treatment. Fig. 7a summarizes the survival rates of tumor-bearing mice after treatment with HCPT-loaded fiber fragments and fiber mats and free HCPT. The intratumoral injection of free HCPT resulted in a survival rate close to that of saline treatment after 30 days, and 50% of mice died within 5 and 17 days for free HCPT and saline treatment, respectively, in line with the toxicity of free HCPT. After intratumoral implantation of HCPT-loaded fiber mats, half of the animals survived for at least 20 days. The 50% survival rates were 18, 25,

3.6. Tumor growth inhibition of HCPT-loaded fiber fragments The antitumor efficacy was evaluated after intratumoral injection of HCPT-loaded fiber fragments and free HCPT and after intratumoral implantation of HCPT-loaded fiber mats, using saline treatment as control. The treatment was performed on H22 tumor bearing mice with a tumor volume of about 500 mm3, and Fig. 6a summarizes the changes in tumor volumes after 30 days of treatment. The saline treatment had no substantial effect on the tumor growth inhibition, and the tumor volume showed an over 9-fold increase after 30 days. The treatment with free HCPT was effective in inhibiting tumor growth during the initial 1 week compared with saline treatment. But there was no apparent effect during the following time period, reaching a tumor volume of around 4000 mm3 after 30 days. By comparison, a significantly lower tumor volume of around 2770 mm3 was observed after intratumoral implantation of HCPT-loaded fiber mats for 30 days (p < 0.05), due to the sustained release of HCPT from fibers and the retention of structural integrity of HCPT released from fibers [31]. As shown in Fig. 6a, the intratumoral injection of FF-50 fiber fragments led to a slightly lower tumor growth rate, reaching a tumor volume of around 2450 mm3 after 30 days, and there was no significant difference compared with that after intratumoral implantation of fiber mats (p > 0.05). The most significant tumor growth inhibition was determined after treatment with FF-5 and FF-20 fiber fragments. The average tumor volume reached only 1750 and 1400 mm3 after intratumoral injection of FF-5 and

Fig. 7. (a) Survive curves and (b) body weight changes of H22-tumor bearing mice after intratumoral implantation of HCPT-loaded fiber mats and intratumoral injection of free HCPT, FF-5, FF-20, and FF-50 fiber fragments, compared with saline treatment as the control.

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Fig. 8. (a) Typical IHC staining images of capase-3 and (c) Ki-67 of tumors retrieved on day 14 after intratumoral implantation of HCPT-loaded fiber mats and intratumoral injection of FF-5, FF-20, and FF-50 fiber fragments. Bars represent 50 lm. (b) The number of capase-3 and (d) ki-67-positive cells among the total number of cells in IHC staining images of tumors retrieved on days 14 and 30 after intratumoral administration of free HCPT, HCPT-loaded fiber mats, FF-5, FF-20, and FF-50 fiber fragments, compared with saline treatment as the control. *p < 0.05.

and 19 days for animals after intratumoral injection of FF-5, FF-20, and FF-50 fiber fragments, respectively. The improved survival rates of animals may be resulted from the superior antitumor effect and reduced side effect of locally sustained release of HCPT from fiber fragments. The animal body weight changes were monitored to show the toxicity of different treatment. Fig. 7b summarizes the variations in body weights of tumor-bearing mice, indicating less than 20% of body weight increases during 25 days of treatment. The average body weight of saline-treated mice was higher than other treatment groups, which was due to the increase in the tumor volume. Among the test groups, the least increase in the body weight after 30 days of treatment was observed for animals treated with free HCPT, due to the toxicity to normal tissues after diffusion out of tumors. During the initial 10 days after treatment with HCPT-loaded fiber mats, animals indicated a less increase in the body weight, due to the surgical intervention of intratumoral implantation. The mice treated with intratumoral injection of HCPT-loaded fiber fragments kept a vigorous and healthy appearance throughout the full experiment, and there was around 15% of body weight gains after 30 days, resulting from a low systematic toxicity of the locally and gradually released HCPT from fibers. 3.8. Tumor cell proliferation and apoptosis after treatment with HCPT-loaded fiber fragments In order to further investigate the antitumor efficacy, the cell proliferation and apoptosis were examined on tumor tissues retrieved

after treatment for 14 and 30 days with HCPT-loaded fiber fragments and fiber mats. It was known that chemotherapy-induced apoptosis by DNA-damaging drugs is thought to be generally dependent on the release of cytochrome c and the subsequent activation of caspase-9 and caspase-3 [38]. Fig. 8a shows typical IHC staining images of caspase-3 in tumor tissues after 14 days of treatment, indicating stronger expressions in the groups of HCPT-loaded fiber fragments and fiber mats and free HCPT than saline treatment. The positive cells were counted from five different areas for each sample, and Fig. 8b summarizes the results after treatment for 14 and 30 days. Compared with around 44.8% of apoptotic cells after intratumoral implantation of HCPT-loaded fiber mats for 30 days, significantly higher amount of apoptotic cells were determined after intratumoral injection of FF-5, FF-20, and FF-50 fiber fragments (p < 0.05), at around 72.1%, 88.4%, and 67.4%, respectively. In the meantime, the treatment with free HCPT and saline led to around 13.0% and 8.6% of apoptotic cells in tumor tissues. In addition, FF-20 fiber fragments induced a significantly higher amount of apoptotic cells than FF-5 and FF-50 (p < 0.05). The released HCPT can maintain an effective curial concentration in tumor tissues, and so induced more necrosis and apoptosis, showing better antitumor efficacy. 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 [39]. Fig. 8c shows typical IHC staining images of murine Ki-67 in tumor tissues after 14 days of treatment, and Fig. 8d summarizes the counting results of positively stained cells. As expected, a significantly lower

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number of Ki-67-stained cells were observed in tumors treated with HCPT-loaded fiber fragments and fiber mats for 30 days (p < 0.05), compared with 86.4% and 90.9% of proliferative cells in tumors after free HCPT and saline treatment, respectively. In addition, a lower proliferation index was determined for tumors after treatment with FF-20 fiber fragments at 12.8%, compared to 19.1% and 25.8% for tumors from FF-5 and FF-50-treated mice, respectively. As indicated above, FF-20 fiber fragments indicated the most significant effects on the tumor growth inhibition, cell apoptosis induction and cell proliferation suppression, compared with FF-5 and FF-50. This should be related with the HCPT accumulation in tumor tissues and the spatial distribution of HCPT within tumors. A higher amount of HCPT was detected in tumor tissues after intratumoral injection of FF-20 fiber fragments than that of FF-5 (Fig. 4), and FF-20 fiber fragments kept higher HCPT concentrations in every tumor section than FF-50 (Fig. 5a). In addition, compared with intratumoral implantation of drug-loaded fiber mats, the injection of fragmented fibers avoided the invasive surgical intervention and achieved more significant antitumor efficacy. Thus, local administration of fragmented fibers was able to overcome these barriers of solid tumors and allow drug accumulation at the target site, and FF-20 fiber fragments could achieve an optimal spatial distribution of HCPT within tumors and a promoted therapeutic efficacy. 4. Conclusion HCPT-loaded fiber fragments were prepared by cryocutting of aligned electrospun fibers, and the fiber length could be easily controlled by the section thickness. FF-5, FF-20 and FF-50 fiber fragments indicated similar release profiles except a lower burst release was detected for FF-50. In vitro cell viability tests showed that FF-5 and FF-20 fiber fragments caused higher cytotoxicity and apoptotic rate than FF-50. Fragmented fibers of longer lengths indicated a higher accumulation into tumors and a better retention at the injection site after intratumoral injection, but showed a less apparent diffusion within tumor tissues. Intratumoral injection of HCPT-loaded fiber fragments showed superior in vivo antitumor activities and fewer side effects than intratumoral implantation of drug-loaded fiber mats. Compared with FF-5 and FF-50 fiber fragments, FF-20 indicated an optimal spatial distribution of HCPT within tumors and achieved the most significant effects on the animal survival rate, tumor growth inhibition and tumor cell apoptosis induction. It is suggested that the intratumoral injection of fragmented fibers provided an efficient strategy to avoid surgical invasiveness, allow the retention of fiber fragments and the release of HCPT within tumor tissues and achieve an optimal therapeutic efficacy. Acknowledgements This work was supported by National Natural Science Foundation of China (21274117 and 31470922), Specialized Research Fund for the Doctoral Program of Higher Education (20120184110004), Scientific and Technical Supporting Programs of Sichuan Province (2013SZ0084), and Construction Program for Innovative Research Team of University in Sichuan Province (14TD0050). Appendix A. Figures with essential color discrimination Certain figures in this article, particularly Figs. 1, 3, 6–8, are difficult to interpret in black and white. The full color images can be found in the on-line version, at doi: http://dx.doi.org/10.1016/j.actbio.2015.05.020.

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