Preparation Of Dually Sensitive Polymer-micelles And Its Application Of Synergistic Therapy In Glioma

Glioblastoma,a malignant tumor of central nervous system,has strong infiltration ability,which makes it difficult to eradicate it by surgery.In the presence of blood-brain barrier and multidrug resistance,traditional chemotherapeutic methods can’t achieve the ideal therapeutic effect.As one of the important milestones in modem biological engineering,RNA interference technology has been widely used in the treatment of tumors.It is a new strategy to study the combination of drugs and anti-cancer.Polymer micelle is a kind of carrier which could co-delivery chemotherapy drugs and siRNA.It has high biocompatibility and biological safety.Restricted by the characterization of the structure,the drug release profile of polymer micelles is suboptimal.Utilizing the concentration of the reducing GSH inside lysosome is about 500-1000 times higher than in the extracellular environment,redox-sensitive micelles could be designed.Futhermore,the pH value of the lysosomes(pH 4.5-5.5),endosomes(pH5.5-6.5)are different the normal tissues and bloodstream,there are many pH sensitive micelles.In this study,we designed and synthesized a dually sensitive micelles:hydrophobic core with the terdanry-animo is pH sensitive,the redox-sensitive cationic interlayer could complex siRNA,the outermost hydrophilic end is consisted of PEG,which could help to realize the long circulation of the carrier in vivo.This micelles could stably load the drugs,but release them rapidly in certain conditions.The study mainly consists of the following three parts:Part one:Preparation,characterization and self-assembly of the dually-sensitive random terpolymer PSD and the pH sensitive random terpolymer PND.Take advantages of RAFT(Reversible Addition Fragmentation Chain Transfer Polymerization),we have prepared two kinds of random terpolymer,one contains disulfide bonds,the other doesn’t.The composition and molecular weight of PSD and PND have been characterized and compared by GPC,1H-NMR,and FITC.The results showed that the two kinds of random terpolymers had similar structures.Finally,the CAC(critical aggregation concentration)of PSD and PND were detected by pyrene as fluorescence probe,the results showed that the CAC of PSD was 0.01349(mg/mL),the CAC of PND was 0.002992(mg/mL).Part two:preparation and characterization of drug-loaded micelles.DOX-loaded micelles were prepared by dialysis method,the maximum encapsulate efficiency(EE%)and drug loading(DL%)of DOX were 80.1%and 17.6%,which were detected by ultraviolet spectrophotometry at 490nm.Results of agarose gel electrophoresis showed that,siRNA could be condensed completely by DOX-PSD and DOX-PND at N/P 4.5 and 6.0.In vitro drug release assay improved that,DOX-PSD and DOXPSD/siRNA were pH sensitive and redox-sensitive.In the condition of pH 5.0 and 10 mM GSH,after 72 h,the cumulative drug release ratio of DOX in DOX-PSD and DOX-PND were 81.4%and 47.1%,the stimulus responsive drug release of siRNA were also obvious.Part three,study of the anti-glioma efficiency of DOXPSD/siRNA&DOXPND/siRNA in vitro and in vivo.Cell uptake efficiency,cellular distribution and cellular DOX release were detected by flow cytometry and laser scanning confocal.Gene silencing efficiency of PSDsiRNA and PNDsiRNA were detected by q-rtPCR.The in vitro anti-tumor efficiency were detected by MTT assay.Finally we constructed a subcutaneous tumor model to detect the in vivo anti-tumor efficiency of DOX-PSD,DOX-PND,DoxPSD/siRNA and DoxPND/siRNA.The results has shown that,the prepared DoxPSD/siRNA and DoxPND/siRNA could co-delivery DOX and siRNA into the cytoplasm,decrease the level of YAP-mRNA.Results of MTT assay have shown that co-delivery YAP-siRNA and DOX could decrease the IC50 of DOX(PSD-DOX and PND-DOX),DOX-PSDsiRNA has a better anti-tumor efficiency than DOX-PNDsiRNA.In vivo distribution of DOX-PSDCy5.5-siRNA and DOX-PNDCy5.5-siRNA have improved that micelles for co-delivery could circulate in the blood and accumulate in the tumor tissue,tumor suppression assay in vivo showed that,co-delivery DOX and YAP-siRNA could increase the anti-tumor efficiency of DOX-PSD and DOX-PND,compared with DOXPND/siRNA,DOX-PSD and DOX-PND,DOXPSD/siRNA exhibited the best anti-tumor efficiency.