In Situ Administration Of Temperature-Sensitive Hydrogel Composite Loading Paclitaxel Microspheres And Cisplatin For The Treatment Of Melanoma

Objective: Construction of an injectable hydrogel system based on paclitaxel microspheres and cisplatin for in situ treatment of melanoma;development of a dual drug slow-release system for sustained delivery in a single dose to enhance drug accumulation at the lesion site while reducing chemotherapy side effects and improving anti-tumor efficacy.Methods: In this study system,firstly,porous PLA microspheres(PPMSs)loaded with PTX were fabricated using a modified double emulsion-solvent evaporation method(W1/O/W2),while the general morphology and distribution of porous PLA microspheres were observed by scanning electron microscopy(SEM),the porosity of the porous microspheres was measured using the mercury-pressure method,and the specific surface area of the porous microspheres was measured by physical adsorption.The drug loading and encapsulation rates of the drug-laden porous microspheres were also analysed using high performance liquid chromatography(HPLC).The dried PELE polymer was dissolved in deionized water and incubated to form a homogeneous and stable hydrogel,and PPMSs and DDP were added sequentially to construct a temperature-sensitive hydrogel system(PPMSs/DDP@Gel)for local injection.The temperature sensitivity of the blank hydrogel and the drug-loaded hydrogel(PPMSs/DDP@Gel)was then analysed for rheological properties and sol-gel transition behaviour.The in vitro cytotoxicity of blank PLA microspheres(Blank-MSs)and microsphere hydrogel system(PPMSs/DDP@Gel)was investigated by MTT colorimetric method,and the in vitro drug release characteristics of paclitaxel-loaded microspheres(PPMSs)and drug-loaded hydrogel system(PPMSs/DDP@Gel)were analysed by dialysis,and different The induction of apoptosis in B16 melanoma cells by different drug treatment groups and the specific effects of paclitaxel in the G2/M phase of the cell cycle by different drug treatment groups were analyzed by flow cytometry.The anti-tumor effects of different treatment groups on the migration of B16 melanoma cells were compared and analysed by scratch assay.In the in vivo study,the C57BL/6 male mouse B16 melanoma model was divided into five groups:(1)saline group(NS);(2)blank microsphere gel group(Blank-MSs@Gel);(3)free paclitaxel and cisplatin group(Free PTX/DDP);(4)paclitaxel-laden microsphere and cisplatin group(PPMSs/DDP)(5)double drug-laden water After the treatment period,three mice from each group were randomly selected to examine the glucose metabolism of their tumor tissues using a small animal PET/CT instrument to assess the anti-tumor effects of the different drug groups.Three mice from each group were randomly euthanized and their heart,liver,spleen,lung,kidney and tumor tissues were stained for HE to assess the safety and biocompatibility of the drug.The PELE hydrogel was injected subcutaneously into the dorsal surface of C57BL/6 mice,and the mice were executed at the time point and the injection site was dissected for observation and photography.Results: In this study,porous paclitaxel-loaded PLA microspheres were successfully prepared by a modified double-emulsion-solvent evaporation method,and both the unloaded blank microspheres and the drug-loaded PPMS showed uniform and well-dispersed morphology with good through-pore structure as observed by SEM.The PPMSs with a theoretical loading of 10%had a high drug loading(8.24±0.09%)and encapsulation(82.4±0.87%).The temperature sensitivity of the hydrogels was demonstrated by test tube inversion and rheological performance analysis.The cytotoxicity assay demonstrated that the blank PLA microspheres had no significant toxic effect on B16 cells,indicating that the carriers used in the experiments had good cytocompatibility.The inhibition of B16 cells increased with increasing concentration and time of PPMSs/DDP@Gel action,indicating that the growth inhibition of B16 cells by this system was concentration-dependent and timedependent.The results of in vitro release assay showed that PTX and DDP had good dual slow-release effect.Cell cycle assays demonstrated that paclitaxel acts specifically in the G2/M phase of the cell cycle.Apoptosis assays demonstrated a stronger pro-apoptotic effect in the PPMSs/DDP@Gel group compared to other groups.Scratch assays showed that the sustained release of the drug in the PPMSs/DDP@Gel group significantly enhanced its ability to inhibit cell migration.In vivo experiments showed that the PPMSs/DDP@Gel group had a stronger anti-tumor effect than the other groups and had no effect on the growth and development of mice,and the overall survival of this group was significantly longer compared to the other groups.PET/CT of mice showed that the PPMSs/DDP@Gel group had the lowest level of glucose metabolism in the tumor tissue.Immunohistochemistry results showed that the PPMSs/DDP@Gel group had the lowest Ki67 positivity rate compared to the other groups,and TUNEL staining showed the highest rate of apoptosis in this group.Conclusion: In this study,porous PLA microspheres loaded with PTX were prepared using a modified double emulsion-solvent evaporation method.the PPMSs still maintained the spherical morphology and porous structure of the PLA microspheres.In addition,an injectable PPMS/DDP@Gel hydrogel composite was constructed to deliver PPMS and DDP for local chemotherapy in a mouse B16 melanoma model.PPMS/DDP@Gel showed enhanced cytotoxicity in vitro and inhibited tumor cell migration as it prolonged the release of PTX and DDP.In vivo anti-tumor assays confirmed that mice treated with PPMS/DDP@Gel exhibited significant inhibition of tumor growth and metabolism without significant systemic side effects.