Chitosan Nanosupensions Provide ... - ACS Publications

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Paclitaxel/chitosan nanosupensions provide enhanced intravesical bladder cancer therapy with sustained and prolonged delivery of paclitaxel Yongjia Liu, Ruibin Wang, Jingwen Hou, Binbin Sun, Bangshang Zhu, Zhiguang Qiao, Yue Su, and Xinyuan Zhu ACS Appl. Bio Mater., Just Accepted Manuscript • DOI: 10.1021/acsabm.8b00501 • Publication Date (Web): 26 Nov 2018 Downloaded from http://pubs.acs.org on December 1, 2018

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ACS Applied Bio Materials

Paclitaxel/chitosan

nanosupensions

provide

enhanced intravesical bladder cancer therapy with sustained and prolonged delivery of paclitaxel Yongjia Liu†, Ruibin Wang†, Jingwen Hou†, Binbin Sun†, Bangshang Zhu†,‡,*,Zhiguang Qiao†,*, Yue Su†, Xinyuan Zhu† †School

of Chemistry and Chemical Engineering, Instrumental Analysis Center, Shanghai Key

Laboratory of Orthopaedic Implants, Department of Orthopaedics, Ninth People’s Hospital, School of Medicine, State Key Laboratory of Metal Matrix Composites, Shanghai Jiao Tong University, 200240 Shanghai, China ‡State

Key Laboratory for Modification of Chemical Fibers and Polymer Materials, Donghua

University, 201620 Shanghai, China

* Corresponding author：Prof. Bangshang Zhu and Dr. Zhiguang Qiao, Shanghai Jiao Tong University, 800 Dongchuan Road, Shanghai 200240, P. R. China, Tel.: +86-21-34206995, Fax: +86-21-34205722, E-mail address: [email protected]; dr_ [email protected]

ACS Paragon Plus Environment

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ABSTRACT Bladder cancer (BC) is a very common cancer. Non-muscle-invasive bladder cancer (NMIBC) is the most common type of bladder cancer. After postoperative tumor resection, the chemotherapy intravesical instillation is recommended as a standard treatment to significantly reduce recurrences. Nanomedicines mediated delivery of chemotherapeutic agent targeting cancer could provide a solution to obtain longer residence time and high bioavailability of anti-cancer drug. The approach described here provides a nanomedicine with sustained and prolonged delivery of paclitaxel and enhanced therapy of intravesical bladder cancer, which is paclitaxel/chitosan (PTX/CS) nanosupensions (NSs). The positive charged PTX/CS NSs exhibited a rod-shaped with mean diameter about 200 nm. They have good dispersivity in water without any protective agents, and the positively charged properties make them easy to be adsorbed on the inner mucosa of bladder through electrostatic adsorption. PTX/CS NSs also had a high drug loading capacity and can maintain sustained release of paclitaxel which could be prolonged over 10 days. Cell experiments in vitro demonstrated that PTX/CS NSs had good biocompatibility and effective bladder cancer cell proliferation inhibition. The significant anticancer efficacy against intravesical bladder cancer was verified by in situ bladder cancer model. The paclitaxel/chitosan nanosupensions could provide sustained chemotherapeutic agents delivery with significant anti-cancer efficacy against intravesical bladder cancer.

KEYWORDS: paclitaxel, chitosan, nanosupensions, drug release, intravesical instillation, bladder cancer therapy


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INTRODUCTION Bladder cancer (BC) is a kind of urological tumor that threatens the health and life quality of patients.1-3 Nearly 70% of BC is non-muscle-invasive bladder cancer (NMIBC) which is a highly prevalent disease with a large number of morbidity, mortality and cost.4-6 BC can be treated by surgery,

chemotherapy

and

radiation.

For

NMIBC,

maintenance-based

intravesical

chemotherapy is the primary method for preventing recurrence and progression after transurethral resection of bladder tumors (TURBT).7,8 Intravesical instillation chemotherapy could reduce the recurrence rate of NMIBC by 15-20%, and reduce the long-term recurrence rate by about 5% .9,10 At present, intravesical instillation chemotherapy can be directly injected into bladder through the urethra, and the retention time is usually 0.5-2h.11,12 The anticancer drugs were directly used in the bladder to kill cancer cells.13 In order to obtain a therapeutic effect, a large dose and a high frequency instillation are usually performed.14,15 The shortcomings of local anticancer drugs delivery are obvious, it not only produces some toxic and side effects but also fails to maintain residence and drug efficacy in a long time.16 Large dose intravesical instillation would cause chemical cystitis, which showed a certain degree of hematuria, urinary frequency, urgency and urine pain.17-19 Frequency intravesical instillation would bring pains to patient. Therefore, it is essential to develop a new drug delivery system for intravesical instillation with high drug loading efficiency and long residence time.20,21 Meanwhile, the adverse reactions and drug toxicity caused by systemic drugs should be avoided or reduced.22 Many nanomaterialbased drug delivery carriers worked successfully for intravesical instillation, such as polymer nanoparticles, liposomes and biomolecules.23-26 Paclitaxel (PTX) is one of the most commonly used first-line clinical anticancer drugs for the treatment of various cancers.27 PTX-loaded polymeric nanoparticles were proved effective in treating bladder cancer.28-30 The chitosan (CS),


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a kind of natural carbohydrate macromolecules, is considered as a suitable candidate carrier. The intravesical application of chitosan can cause urothelial cell desquamation, and the mechanism of desquamation is still unclear.31 CS causes a decrease in the function of the urethral epithelial barrier, and the drug can penetrates into the deeper cell layer.32 In our previous work, through a simple co-assembly method, we successfully prepared the PTX/CS core-shell nanofibers with high drug loading efficiency.33 In our experiment, we found that the long PTX/CS nanofibers are very easy to be cut into short rods by ultrasound, so nanosuspension is obtained. Drug nanocrystal techniques with low-cost equipment and easy to scale up are considered as promising method.34,35 The poorly soluble drugs can be formulated as nanosuspensions in liquid system through these methods.36,37 The high-frequency ultrasonic wave can produce shear force to get soft sample shredded. The ultrasonic force is used to produce injection of suspension preparation for small and even drug naonsuspensions.38 In our experiment, we found that the long PTX/CS nanofibers could be easily cut into short nanorods by ultrasonic wave and obtain naonsuspensions. In this paper, we explore the possibility of PTX/CS nanosuspension for intravesical instillation therapy. The PTX/CS NS drug delivery system prepared for intravesical instillation was shown in Scheme 1. To verify drug efficiency, PTX release, cell uptake and cytotoxicity were evaluated in vitro. Furthermore, the bladder retention time and anti-cancer efficacy against intravesical bladder cancer of PTX/CS NSs were evaluated by animal experiments in vivo. EXPERIMENTAL SECTION Materials. Paclitaxel was bought from Shanghai Boshi Biotechnology Co., Ltd. Chitosan (average molecular weight, 144kDa; deacetylation degree 79%), dehydrated alcohol (EtOH),


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acetic acid (HAc), fluoresceinisothiocyanate isomer-I (FITC), 3-(4, 5-dimethylthiazol-2-yl)-2, 5diphenyltetrazoliumbromide (MTT), Hoechst 33342 were bought from Sigma-Aldrich. The reagents were bought from domestic suppliers. All experiments were used the ultrapure water. Preparation of PTX/CS NSs. The synthesis steps used in this study were recently described and validated by ourselves.35 The 2 mg/mL PTX/EtOH solution was added into the same volume 2 mg/mL CS/1% HAc (HAc/water, V/V) solution, then treated by sonication system for 20 min. The obtained PTX/CS nanofibers solution was then put in probe-sonication system for 20 min to get the PTX/CS NSs. FITC labeled CS (FITC-CS) was used to prepare PTX/FITC-CS NSs by using the same method above. The synthesis route of FITC-CS was described in the supporting information. The PTX/CS blends were obtained by adding 2 mg/mL PTX/EtOH solution into the same volume 2 mg/mL CS/1% HAc solution. Water removal. The obtained PTX/CS NSs and PTX/CS blends solutions were concentrated using ultrafiltration device (Amicon Ultra-15 Centrifugal Filter Unit, 10 kDa molecular weight cut-off (MWCO), cutoff). The concentrated solutions were dialyzed for 120 h by using the 3.5 kDa MWCO dialysis tubing in order to remove EtOH and HAc to a very low residual content. Finally, PTX NSs were harvested by freeze-drying. PTX loading content and efficiency. The freeze-dried PTX/CS NSs was dissolved in EtOH/1% HAc (V/V = 1:1). The PTX concentration was quantitatively determined with the ultraviolet-visible spectrophotometry (UV-Vis, ThermoFisher EV300, USA) using a calibration curve method (λabs = 227 nm, Figure S2). The calibration curve used for drug-loading characterization was established with different PTX concentrations in EtOH/1% HAc (V/V = 1:1) solution. The PTX loading content (wt %) = (Weight of PTX in PTX/CS NSs)/(Weight of


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PTX/CS NSs) × 100%, The PTX loading efficiency (wt %) = (Weight of PTX in PTX/CS NSs)/(Total weight of feeding PTX) × 100%. Characterizations and mucoadhesive properties. The morphology of PTX/CS NSs was observed by using a scanning electron microscopy (SEM, FEI Sirion 200, USA). The samples were sputter coated with a conductive thin gold film prior to SEM examination. The Zeta potential of PTX/CS NSs in the ultrapure water was evaluated by a Zeta potential analyzer (Malvern ZS90, UK). The mucoadhesive properties of CS, PTX and PTX/CS NSs were measured by using mucin-particle method. The mucin and samples were suspended in a phosphate buffered saline solution (PBS) at pH 6.5 with a concentration of 1% W/V, then were mixed with equal volumes. The mixtures were incubated at 37 °C for 1 h prior to the test. The particle sizes were detected with a laser particle analyzer (LPSA, Microtrac S3500, USA). The characteristic functional group imaging of PTX/CS NSs were analyzed by nanoscale infrared (Nano-IR). The differential scanning calorimetry (DSC) and X-ray diffraction (XRD) was used to investigate the thermogram properties and the crystalline structure of PTX/CS NSs. Drug release in vitro. Added 1 mg lyophilized PTX/CS NSs to 1 mL of PBS (pH 4.5, 6.5 or 7.4) solution. Then placed the PTX/CS NSs solution in a dialysis bag (3.5 kDa MWCO) and dialyze at 29 mL PBS solution which shaking at 37 °C with a speed of 120 r/min. At specific time intervals, removed the entire volume of PBS and replaced with fresh PBS. The PTX in the obtained buffer solution was extracted with dichloromethane (DCM), dissolved in 4 mL of EtOH /1% HAc (V/V = 1:1) solvent, and measured at a wavelength of 227 nm by a UV-Vis spectrophotometer.


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Cellular uptake. The intracellular uptake behavior of PTX/CS NSs was observed by confocal laser scanning microscopy (CLSM, Leica TCS SP8 STED 3X, Germany). The T24 cells (human bladder cancer cell line) were seeded onto coverslips in 6-well plate at a density of 1.5 × 105 cells. The cells were maintained in Dulbecco's Modified Eagle Medium (DMEM) containing fresh fetal bovine serum (FBS) (10% V/V). The culture medium was contained 100 U/mL penicillin G, and 100 µg/mL streptomycin. The plates were incubated for 24 h at 37 °C in a humidified atmosphere containing 5% carbon dioxide (CO2). The PBS (control) and PTX/FITCCS NSs (PTX concentration 1.25 μg/mL) were added in the culture media. After 2 h, the cells on coverslips were then rinsed with PBS and stained with 4% paraformaldehyde for 20 min. Then the fixed cells were stained with Hoechst 33342 for 15 min followed by further washing with PBS for 3 times. MTT assay. The in vitro cytotoxicity of PTX/CS NSs, PTX/CS blends and PTX were determined by the MTT assay. The NIH/3T3 cells (mouse embryonic fibroblast cell line) or T24 cells were seeded in 96-well plates at 1.0 × 104 cells/well. The culture medium was 200 μL/well. After 24 h incubation, the culture medium was removed and replaced. Then the PTX/CS NSs, PTX/CS blends or PTX of different concentrations (PTX concentration as a benchmark) were added in cells for 48 h. After that the medium were removed and 20 μL of 5 mg/mL MTT was added to each well. Cells were incubated for 4 h and culture media were remove gently with pipe-gun and replaced with 200 μL dimethylsulfoxide (DMSO). The plates were slightly shaken until the crystals were fully dissolved at room temperature. A plate reader (BioTek Synergy H4, USA) were used to measure the OD (optical density) of each well at a wavelength of 490 nm. ROS levels and cell cycle analysis. The intracellular reactive oxygen species (ROS) activity of NIH/3T3 cells and T24 cells after incubating with PTX/CS NSs, PTX/CS blends and PTX (1.25


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mg/mL PTX concentration as a benchmark) was detected. The cells were seeded at a density of 1 × 105 cells/well in 12-wells plate and incubated with PTX/CS NSs, PTX/CS blends or PTX for 48 h, respectively. The cells treated by PBS solution were the negative control group. Then the CellROX® Green Reagent was added to the cells at a final concentration of 5 μM and incubated at 37 °C for 30 min. The flow cytometry (FCM, BD Accuri C6, USA) was used to measure the intracellular ROS fluorescence intensity. The ROS levels were calculated by the collected fluorescence intensity fold of control group. For cell cycle analysis, T24 cells were cultured and exposure to 2 μg/mL PTX, PTX/CS blends and PTX/CS NSs for 24 h. After washing the cells with PBS, the cells were trypsinized and centrifuged at 1000 r/min for 10 min. Then cells was washed with PBS and centrifuged. The cold ethanol was slowly added dropwise to the cells and the cells were fixed in at -20 °C for 1 h. The fixed cells were washed with cooled PBS and centrifuged. A cell cycle assay kit (Bestbio BB4104, China) was used to stain the fixed cells. PI/RNase staining solution is used for FCM analysis (BD FACSCalibur, USA) of DNA content in the cell cycle. The propidium iodide (PI) can bind to DNA and RNA, necessitating treatment with RNase to bind RNA. The cells were first stained with RNase solution for 30 min in a 37 °C water bath. Then cells was washed with PBS and stained with PI, incubating in 4 °C for 30 min without light. The experiment was repeated three times. All analyses were performed using FlowJo software. In situ bladder cancer model induction. The female BALB/c mice (6 weeks old) were bought (Shanghai Slac Laboratory Animal Co. LTD). Before treatment, all animals received care for two weeks under the guidelines of Shanghai Jiao Tong University Animal Care and Use Committee. The female BALB/c mice were anaesthetized with pentobarbital (50 mg/kg intraperitoneally). We disinfected the urethra, inserted a needle tube through the urethra. The


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needle was penetrated deep into the bladder and the bladder was forced to empty the urine. When the needle resistance became large, we stopped and injected 50 μL GFP/luciferase-expressing T24 (GFP/luc-T24) cells suspension (2 × 106/mL). After GFP/luc-T24 cells were injected, the urethra was closed by suture for two hours. In vivo bioluminescence imaging of PTX/CS NSs. The BALB/c mice were randomly divided into three groups (PBS, PTX/CS blends and PTX/CS NSs, n=6). At day 7 after tumor inoculation, the mice were treated with 50 μL PBS (control), PTX/CS blends or PTX/CS NSs (PTX dose 6 mg/kg). The mice were given intravesical instillation treatment once a week. 5 min prior to imaging, the luciferin solution (150 mg/kg) was injected into the abdominal cavity of mice on 7, 14 and 28 days. Animals examined for quantiﬁcation of GFP/luc-T24 tumor growth were imaged from the supine position with a NightOWL LB983 imaging system (Berthold, Bad Wildbad, Germany). The tumor bioluminescence of each mouse was normalized tot the initial flux (day 7). In vivo antitumor efficacies and histological analyses of PTX/CS NSs. On day 7 and day 14, two mice in each group were sacrificed, and after 28 days of treatment, all left mice in each group were humanely killed. The tumor were measured by digital caliper and the volume by the formula V = D (the longest diameter) × d2 (the shortest diameter) /2. All tumor samples were fixed in formalin and embedded in paraffin blocks. The 5 μm sections of the paraffin blocks were cut, stained with H&E (hematoxylin and eosin), and scanned on a microscope imaging workstation (BLISS, Bacus Laboratories Inc., USA). Systemic toxicity, liver and renal function assessment. To monitor systemic toxicity, another set of 60 mice were randomly divided into control groups (PBS and PTX/CS blends) and


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sample group (PTX/CS NSs). All the mice had in situ bladder cancer tumors and received intravesical instillation therapy. The body weight of the mice was recorded every four days during the treatment. Survival rate was estimated every week till the end of the experiment. At the end of the intravesical instillation treatment, the blood samples were collected into tubes with heparin. Blood samples were obtained by centrifugation at 3900 r/min for 10 min and stored at 20 °C till biochemistry assay measurements. The alanine aminotransferase (ALT) and aspartate aminotransferase (AST) indicates liver damage, serum creatinine (Cr) and blood urea nitrogen (BUN) are the biochemical parameters of renal. Statistical analyses. The test was repeated at least three times and the results were expressed as mean ± standard deviation (SD). Significance analysis was analyzed by two-sided t-tests, with *P

Chitosan Nanosupensions Provide ... - ACS Publications

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