br However these drugs are
However, these drugs are only moderately effective in the treatment of major solid tumors because of chemoresistance and the adverse effects of these drugs.  With the development of combination drug therapy, nanoformulations, and nanoparticulate-based drug delivery systems, recent studies have explored the feasibility of dual delivery of biological therapeutics to overcome the current limitations associated with cancer therapy [4–8]. However, conventional administration techniques such as oral administration and intravenous injection limit the bioavailable drug concentration at the tumor site. Therefore, various local drug delivery systems (LDDS) and combination therapies using more than one medication may effectively overcome drug resistance and reduce adverse effects associated with conventional chemotherapy by in-creasing the concentration of the anti-tumor drugs at the tumor site. Furthermore, local accumulation of anti-tumor drugs at the tumor site
will reduce systemic concentrations of these drugs, reducing adverse effects on other organs. Finally, accumulation of drugs at the tumor site may aid in prevention of recurrence of tumor growth following surgical resection . In this study, we developed an acellular submucosa-based porcine jejunum patch loaded with the potent anti-tumor drugs 5-FU and ra-pamycin. We used a nude mouse model of colon cancer to investigate the therapeutic efficacy, systemic toxicity, and applicability of this biological patch for cancer treatment. These patches could also be used as a scaffold for mechanical buttressing of tissue following surgical resection. We demonstrated that this novel biodegradable patch was capable of providing effective sustained release of 5-FU and rapamycin over 2 weeks in vitro and could be easily applied to the exact tumor site, resulting in reduced local growth and recurrence of tumor growth in vivo.
2. Materials and methods
2.1. Biological patch loaded with chemotherapeutic drug
The acellular tissue matrix biological patch was purchased from Cook Medical Co. 5-FU, rapamycin, and chitosan were purchased from Sigma-Aldrich (St. Louis, MO, USA). Polylactic Erastin (PLA) was pur-chased from Beijing XUEBIO Biochemical Technology Co. 5-FU and rapamycin were dissolved in DMSO, and this mixture was subjected to ultrasound emulsification with chitosan and PLA for one hour, resulting in the formation of 5-FU and rapamycin nanoparticles (5-FU-RAPA-PLA-NP). The 5-FU-RAPA-PLA-NP were smeared onto the biological patch. The patch was then vacuum freeze-dried, vacuum-sealed (–760 mmHg, 50 °C) in hermetic packaging, sterilized by gamma irra-diation, then stored at −80 °C until use. Patches loaded with no-drug, 0.1 mg, 0.5 mg, and 1 mg of 5-FU-RAPA-PLA-NP per square centimeter were produced.
2.2. Release profile of the drug-loaded patch
The concentration of 5-FU and rapamycin released from the patch into PBS was determined using HPLC. (Agilent 1260 Infinity II HPLC, Santa Clara, CA US).
Sw480 cells (cryopreserved by our laboratory) were cultured in ATCC-formulated Leibovitz's L-15 medium supplemented with 10% fetal bovine serum (FBS), and 1% penicillin, and streptomycin. LoVo cells (obtained from ATCC) were grown in ATCC-formulated F-12 K Medium with 10% FBS, and 1% penicillin, and streptomycin. All cells were maintained at 37 °C in a 5% CO2 atmosphere.
2.4. Cell growth inhibition assay
Cell growth was evaluated using Cell Counting Kit-8 (CCK-8; Dojindo Laboratories, Japan). Cells were plated at a density of 1 × 104 cells/well in 96-well plates with three replicates for each treatment condition. The medium was replaced with infusion solution after 12 h. Then, 100 μl of serum-free cell culture medium containing 10 μl of WST-8 reagent was added to each well every 24 h, and the plates were incubated at standard conditions for 1 h. Optical absorbance was measured at 450 nm and 630 nm using a microplate reader (Bio-Rad Laboratories, USA). Three independent experiments were performed for quantification.
2.5. Detection of apoptosis by flow cytometry
The proportion of apoptotic cells was detected using Annexin V-APC/PI staining. Cells were plated at a density of 1 × 106 cells/well on Biomedicine & Pharmacotherapy 117 (2019) 109048
6 cm plates and grown overnight. On the next day the cells were treated with the patch infusion solution. Controls received DMSO (negative). After 72 h, cells were harvested, counted, transferred into flow tubes, pelleted, and resuspended in 100 μl of fresh 1× Annexin binding buffer (0.01 M HEPES pH 7.4; 0.14 M NaCl; 2.5 mM CaCl2) containing Annexin V-APC and PI. After staining, samples were analyzed by flow cytometry within 1 h using a FACSCalibur flow cytometer (BD). Annexin V+/PI ± were considered apoptotic cells. The proportion of apoptotic cells was expressed as a percentage of the total cell number, excluding debris based on analysis using a BD FACSDiva instrument and FlowJo software. Each treatment was performed in triplicate, and three replicate experiments were performed.