Injetable Thermosensitive PLGA-PEG-PLGA Hydrogel As Multi-agents Chemotherapeutics And Gene Co-delivery For Osteosarcoma Treatment

Osteosarcoma is the most common primary malignant bone cancer in childrenand adolescents. On average, the annual incidence rate is10-26per1000000, withapproximately400new cases of osteosarcoma diagnosed in the United States eachyear. Osteosarcoma pathologically arises from mesenchymal cells, characterized byspindle cells and aberrant osteoid formation. Typically, the primary sites ofosteosarcoma are the distal femur, the proximal tibia, and the distal humerus, with64%originating from the knee. Typically, osteosarcoma occurs in the metaphysis inproximity to the growth plate, where experience rapid cell division to facilitate theincreased growth velocity during adolescence. The pubertal growth may represent aninciting event in the oncogenesis of osteosarcoma. Local pain, swelling, andlimitation of motion are typical signs and symptoms of osteosarcoma. Currently, thestandard treatments of osteosarcoma comprise surgery and chemotherapy regimens(neoadjuvant and adjuvant chemotherapy). As a result, the long-term survival ofunmetastasis patients has dramatically increased to60%, while the metastasispatients is as low as30%. In this study, we developed a strategy by localizedco-delivery of plasmid DNA and multiple chemotherapeutics drugs usingbiodegradable, thermosensitive PLGA-PEG-PLGA hydrogels for treatment ofosteosarcoma. The target gene therapy on PLK1and multiple chemotherapeuticsagents based on PLGA-PEG-PLGA hydrogels as the co-delivery system exhibit ansynergistic effect on osteosarcoma treatment, with reduced systemic side-effects.The PLGA-PEG-PLGA triblock copolymer was synthesized via ring-openingcopolymerization. The PLGA-PEG-PLGA solution was thermosensitive, undergoing a sol-gel transition with the increasing temperature, and the transition temperaturewas dependent on the hydrophobicity of the PLGA chain and the polymerconcentration. PLGA-PEG-PLGA triblock copolymers tend to self-assemble intomicelles with hydrophobic PLGA cores and hydrophilic PEG shells in aqueoussolution at lower temperatures. The micelle formation and aggregation behavior areclosely associated with the molecular structure and the hydrophobic-hydrophilicbalance of PLGA-PEG-PLGA triblock copolymers. With increasing the temperature,the aggregation number of the micelles increases abruptly and inter-micellaraggregation forms, due to the increase in the hydrophobic interactions of PLGAblocks and partial dehydration of PEG shell. When the polymer concentration is highenough, a sol-to-gel phase transition occurs. However, further increase intemperature to higher region results in serious dehydration of the PEG shell andmarked shrinkage of the micelles, causing the precipitation of the copolymer fromthe solution and a gel-to-sol phase transition. Moreover, PLGA-PEG-PLGAexhibited an excellent property of biocompatibility and biodegradability in vitro andin vivo. As an injectable material, PLGA-PEG-PLGA may be promising candidatesfor applications as gene and drug co-delivery system for localized therapy.The PLGA-PEG-PLGA triblock copolymer was synthesized by thering-opening copolymerization and used as multiple chemotherapy regimensdelivery system. The DOX、CDDP and MTX were co-loaded into the hydrogels byphysical mixing method, which underwent a sol-gel phase transition as a function oftemperature. The release profiles of the drugs from the hydrogels were investigatedin vitro， sustained released from the hydrogel for over12day. Triblock PLGA-PEG-PLGA thermosensitive hydrogel can be used as an excellent drugs carrier forthe osteosarcoma treatment. The multi-drug co-loaded hydrogels exhibitedsignificantly synergistic effects on the anti-proliferation efficacy and expressions ofapoptosis genes against osteosarcoma Saos-2and MG-63cells in vitro. After thesingle peritumoral injection of the drug-loaded hydrogels into a nude mice modelbearing human osteosarcoma Saos-2xenografts, the hydrogels co-loaded with DOX, CDDP and MTX displayed the highest tumor suppression efficacy in vivo for up to16days, as well as led to enhanced tumor apoptosis and increased regulation of theexpressions of apoptosis-related genes. Moreover, the monitoring on the mice bodychange and the ex vivo histological analysis of the key organs indicated that thelocalized treatments caused low systemic toxicity and no obvious damage to thenormal organs. Therefore, the approach of localized co-delivery of DOX, CDDP andMTX by the thermosensitive hydrogels may hold potential for enhancedosteosarcoma treatment.The PLGA-PEG-PLGA triblock copolymer was synthesized by thering-opening copolymerization and used as gene and drug co-delivery system. TheDOX and PLK1shRNA/PEI-Lys were co-loaded hydrogels by the physical mixmethod, which underwent a sol-gel phase transition with temperatureincreasing.Therelease profiles of plasmid DNA from the hydrogels were investigated in vitro, andsustained released from the hydrogel for over7day. Incubated with osteosarcomaSaos-2and MG-63cells, the hydrogel containing PLK1shRNA/PEI-Lys and DOXdisplayed significant synergistic effects inpromoting the apoptosis of osteosarcomacellsin vitro. After subcutaneous injection of the hydrogel containing PLK1shRNA/PEI-Lys and DOX beside the tumors of nude mice bearing osteosarcoma Saos-2xenografts, the hydrogels exhibited superior antitumor efficacy in vivo compared tothe hydrogels loaded with PLK1shRNA/PEI-Lys or DOX alone. It is noteworthy thatthe combination treatment in vivo led to almost complete suppression of tumorgrowth up to16days, significantly enhanced PLK1silencing, higher apoptosis oftumor masses, as well as increased cell cycle regulation. Additionally, ex vivohistological analysis of major organs of the mice indicated that the localizedtreatments showed no obvious damage to the organs, suggesting lower systemictoxicity of the treatments. Therefore, the strategy of localized, sustained co-deliveryof PLK1shRNA and DOX by using the biodegradable, injectable hydrogel may havepotential for efficient clinical treatment of osteosarcoma.In conclusion, PLGA-PEG-PLGA triblock thermosensitive hydeogel is an excellent injectable bimomaterials with good biodegradability and biocompatibility,and hold potential for the osteosarcoma treatment.