Construction Of A Reduction-Responsive Cationic Mirna Carrier And Its Application For Osteosarcoma Gene Therapy

Osteosarcoma(OS)is the most common primary malignant tumor in bone.The primary OS mainly occurs in children and adolescents with high disability and mortality ratios.Many challenges,such as tumor resistance,metastasis,and tumor recurrence under the current treatment mode,still need to be solved urgently.Gene therapy shows great potential in treating tumors.In the process of gene therapy,miRNAs play a very important role in the occurrence and development of OS.The functional levels of many miRNAs are out of balance during the development of OS.Among them,the expression levels of miR-22 and miR-30 d are significantly downregulated.Therefore,these two miRNAs are promising as therapeutic targets for OS.In the process of gene therapy,the delivery carrier is a key factor.Polycations gene carriers have received widespread attention from researchers due to their low immunogenicity and flexible molecular structure design.However,polycations also have the problem of poor degradation performance and greater cytotoxicity.In response to this problem,a polyhydroxy reducing-responsive cationic polymer TGIC-CA(TC)was developed in this dissertation,which was prepared by ring-opening reaction through isocyanuric acid triglyceride(TGIC)and cystamine(CA),and then end-capped by ethylenediamine.In the first part,TC was synthesized to deliver miR-22 in combination with the anticancer drug,Volasertib,for the treatment of OS.TC was prepared by the ring-opening reaction between amino and epoxy groups,which had the diameter of ～200 nm and the zeta potential of ～30 m V.After reduction,the TC nanoparticles were broken,and the zeta potential was decreased to 5 m V.The agarose gel electrophoresis experiment showed TC could form complex with miRNA at the weight ratio of 1.5,and the weight ratio was significantly increased after reduction,which proved the redox responsiveness.The GSH concentration inside OS cells(Saos-2)was higher than that inside osteoblast cells(MC3T3-E1),which lead to a two-magnitude higher level of transfection in Saos-2 cells.TC/miR-22 complexes showed a good tumor suppression effect and the combination treatment group had the best performance.In vivo experiments were performed in an orthotopic OS mouse model and two PDX OS models(chemo-resistant and chemo-intolerant),and TC/miR-22 not only successfully inhibited tumor proliferation,but also reduced the chance of tumor lung metastasis,and also overcame the problem of drug resistance.In the second part,TC was used to deliver miR-30 d to treat orthotopic OS mouse model.In the in vitro experiments,TC/miR-30 d have achieved good anti-tumor effect,which suppressed proliferation and migration of Saos-2 cells and enhanced cell apoptosis.TC/miR-30 d showed preliminary therapeutic effect in the in vivo experiments.The present work is the first study on miR-30 d for treatment of OS,which expanded the application of miRNA in the field of gene therapy for OS.In summary,based on disulfide bonds,a reduction-responsive cationic gene carrier,TC,was constructed and achieved good treatment effects of OS by delivering miR-22 and miR-30 d,respectively,which provided a new idea for the treatment of OS.