SiRNA-loaded Reduction-responsive Polymersomes For Orthotopic Glioblastoma Targeted Gene Therapy

Polo-like kinase 1(PLK1)is a key mitosis-related protein widely existing in all types of cancers,whereas normal cells are non-addictive to PLK1.Therefore,we can arrest cancer cells at mitosis and subsequently induce apoptosis without causing severe toxicity by using anti-PLK1 siRNA(siPLK1).siPLK1-loaded liposomes(TKM-080301)for multiple types of advanced solid tumors have entered clinical trials.However,most siRNA-containing nanomedicines under clinical trials are based on cationic polymers or lipid-based nanoparticles,which have poor stability,severe toxicity as well as immune stimulation.Furthermore,RNA interfering(RNAi)therapy for orthotopic glioblastoma are greatly limited owing to the existence of blood brain barrier(BBB).Here,we report on the preparation of brain targeting,disulfide crosslinking,biodegradable chimaeric polymersomes for efficient siPLK1 loading and targeted gene therapy of orthotopic glioblastoma.In Chapter 1,we reviewed the clinical status of glioblastoma therapy,the development of RNAi,vehicle types for siRNA loading and orthotopic glioblastoma targeting strategies,followed by the design and significance of this thesis.In Chapter 2,we designed and prepared the amphiphilic triblock copolymers of poly(ethylene glycol)-b-poly(trimethylene carbonate-co-dithiolane trimethylene carbonate)-b-polyethylenimine(PEG-P(TMC-co-DTC)-PEI))and angiopep-2(ANG)-functionalized poly(ethylene glycol)-b-poly(trimethylene carbonate-co-dithiolane trimethylene carbonate)(ANG-P(TMC-co-DTC).By self-assembly and self-crosslinking with simultaneous loading of siPLK1,the biodegradable,reduction-responsive,brain targeting and siPLK1-loaded chimaeric polymersomes(ANG-CP-siPLK1)were prepared for targeted gene therapy of orthotopic glioblastoma.ANG-CP-siPLK1 show great loading efficiency(94.5%)for siPLK1 as a result of the strong charge interactions between PEI moieties and siPLK1.Furthermore,siPLK1 was stably encapsulated in aqueous lumen of polymersomes owing to the crosslinking via dithiolane of DTC,which prolongs the blood circulation time of siPLK1 by 12-fold.Based on the simultaneous high expression of LRP-1 on both BBB and U-87 MG glioblastoma cells,ANG-CP-siPLK1 can target orthotopic glioblastoma in a "killing two birds with one stone" way via LRP-1 mediation.After uptake by U-87 MG cells,ANG-CP-siPLK1 rapidly escapes(about 4 h)from endosomes,releases the cargo siPLK1 via the decrosslinking of disulfide bonds under intracellular reductive condition,and silences the target gene eventually.Results of quantitative real-time polymerase chain reaction(qRT-PCR)and Western blot indicated that ANG-CP-siPLK1 knockdowns 76%of PLK1 mRNA and 89%of PLK1 oncoprotein,respectively,at the cellular level.In vivo fluorescence imaging further demonstrated that 83%of the bioluminescence of the reporter gene luciferase was silenced by ANG-CP-siPLK1.Subsequently,the in vivo anti-gliblastoma results proved that ANG-CP-siPLK1 significantly inhibits the development of orthotopic glioblastoma,and thus,doubles the survival time of tumor bearing animals without causing obvious toxicity.In Chapter 3,to further improve the targeting capability of siRNA-loaded poymersomes for orthotopic glioblastoma,the tandem dimmer of the domain of apolipoprotein E(denoted as ApoE)targeting multiple low-density lipoprotein receptor(LDLR)superfamily members(including LDLR,LRP-1 and LRP-2)is conjugated to polymersomes.In addition,the natural spermine is chosen as the short hydrophilic moieties of polymersomes,we finally prepared ApoE-CP-siPLK1(44 nm±0.13)with enhanced brain targeting and biocompatibility,which encapsulates siPLK1 at an efficiency of 81%with extremely low N/P of 0.38.Remarkably,ApoE-CP-siPLK1 knockdowns 86%of PLK1 mRNA and significantly extends the survival time of glioblastoma bearing mice from 21 days to 46 days.In Chapter 4,we made a brief summary of this thesis,followed by a short perspective.