br Implantation of patches and in vivo
2.11. Implantation of patches and in vivo anti-tumor efficacy
When xenograft tumor volumes reached 100 − 200 mm3, mice were divided into three groups (5 animals per group). Patches (no drug or loaded with 5-FU-RAPA-PLA-NP) were affixed to the bottoms of sub-cutaneously grafted colon tumors in athymic mice. We made an incision near the inoculation site, inserted the patch under the solid tumor, and immobilized the patch by suturing. The other group was injected with 5-FU-RAPA-PLA-NP via the tail vein. All surgical procedures were performed in a specific pathogen-free room. The mice were sacrificed, and the organs and tumors were removed and fixed overnight with 4% paraformaldehyde. The tumors were then embedded in paraffin and cut into sections for H&E and IHC analyses.
Fig. 1. Calculation of combination index (CI) of combined treatment of 5-FU and rapamycin in colon cancer cell lines. A. Dose-effect curve for LoVo cells. B. Fa-Cl plot for LoVo cells. C. Dose-effect curve for sw480 cells. D. Fa-Cl plot for sw480 cells.
Fig. 2. Synergistic effect of combination of 5-FU and rapamycin on colon cancer cell lines. A, C. Flow cytometry analysis of the DNA dye showed that LoVo cells were arrested at G0/G1 phase. B, D. Flow cytometry analysis of the cell cycle showed that sw480 cells were arrested at G0/G1 phase. E, F. The expression of cyclin D1, cyclin E, CDK1, and p21 was evaluated by real-time quantitative PCR and western blotting. * P < 0.05, ** P < 0.01.
Fig. 3. Synergistic effect of combination of 5-FU and rapamycin on colon cancer cell lines in inducing apoptosis. A, C. Flow cytometry analysis of apoptosis in LoVo cells. B, D. Flow cytometry analysis of apoptosis in sw480 cells. E, F. The expression of caspase-3, caspase-9, survivin was evaluated by real-time quantitative PCR and western blotting. * P < 0.05, ** P < 0.01.
Fig. 4. Characterization of drug-loaded path. A. Cumulative rapamycin release profiles for 0.1 mg/cm2, 0.5 mg/cm2, and 1 mg/cm2 patches over 9 days. B. Cumulative 5-FU release profiles for 0.1 mg/cm2, 0.5 mg/cm2, and 1 mg/ cm2 patches for 9 days. C. Photographs presenting the flexible and stretchable properties of patch. Scalebar 2 mm. D, E. SEM images of surface of patches. Scalebar 500 μm. F. SEM images of drug-loaded patch. Scalebar 500 μm.
2.12. Statistical analysis
Statistical analysis of cell growth inhibition assay data was per-formed using Student’s t-test analysis with Prism software (GraphPad-7). All experiments were performed in triplicate. In this study, a p-value less than 0.05 was considered to be statistically significant.
3.1. 5-FU and rapamycin synergistically inhibited colon cancer cell proliferation
Previous studies have demonstrated that 5-FU and rapamycin in-dividually inhibited cancer cell growth. [10–14] However, no studies have evaluated the efficacy of combined treatment with 5-FU and ra-pamycin against colon cancer. To study the synergistic effects of 5-FU and rapamycin on LoVo cells, we analyzed the inhibition rates of dif-ferent drug concentration of 5-FU or rapamycin alone or the combi-nation of 5-FU and rapamycin. The results showed that 5-FU and ra-pamycin had great synergistic effect in wide range of concentration in LoVo cells (Fig. 1A and B) and sw480 cells (Fig. 1C and D).
Both 5-FU and rapamycin have been reported to induce G1 cell cycle stage arrest, resulting in the inhibition of tumor growth. We evaluated whether combined treatment with 5-FU and rapamycin in-duced additive or greater inhibitory effects on the cell cycle. Thus, we performed flow cytometry to determine the cell cycle distribution of cells co-treated with 5-FU and rapamycin. The results indicated that co-treatment with 5-FU and rapamycin resulted in a higher percentage of cells at G1 than that with 5-FU or rapamycin alone (Fig. 2A–D). In addition, we determined the expression of G1 stage-related genes cyclin D1, cyclin E, CDK1, and P21 were significantly inhibited following treatment with 5-FU and/or rapamycin (Fig. 2E and F).
3.2. Co-treatment with 5-FU and rapamycin induced cell apoptosis
Apoptosis is a common consequence of cell cycle inhibition. To further characterize the effects of co-treatment with 5-FU and rapa-mycin on tumor cells, we analyzed apoptosis rates in LoVo cells and sw480 cells following treatment with 5-FU or rapamycin alone, or 5-FU combined with rapamycin. The results demonstrated that 5-FU and
rapamycin had a combined effect on LoVo and sw480 apoptosis (Fig. 3A–D). In addition, we evaluated the expression of apoptosis-re-lated genes, which showed that caspase-3 and caspase-9 expression was increased, and survivin expression was significantly inhibited following treatment with 5-FU and/or rapamycin (Fig. 3E and F).
3.3. Characterization of 5-FU and rapamycin of drug-loaded patches
Our previous studies demonstrated that co-treatment with 5-FU and rapamycin had a coordinated inhibitory effect on colon cancer cells in vitro. In this study, we used ultrasonic emulsification technology to generate nanoparticles from a mixture of 5-FU and rapamycin. The resultant 5-FU-RAPA-PLA-NP was loaded onto an acellular tissue matrix (ACTM) biological patch. We then tested the release kinetics of 5-FU and rapamycin from these patches. As shown in Fig. 4A and B, this novel biological patch exhibited sustained released of 5-FU and rapa-mycin in vitro. We then performed SEM to further characterize the drug-loaded patch (Fig. 4C–F).