Construction And Evaluation Of Novel Macromolecular Vectors For Gene Delivery

Gene therapy has attracted wide attentions as an efficient and low side-effect treatment for cancer disease.The key of its clinical application is availability of safe and efficient gene vectors.Vectors for gene delivery can be categorized into viral vectors and nonviral vectors,and nonviral vectors mainly include cationic lipids,cationic polymers and inorganic nanoparticles.Cationic dendrimers,which have exact structure,controllable size and abundant functional groups on the surface,play a unique role in the delivery systems of cationic polymers.Polyacylthiourea(PATU),which has the surface of double-bond and inner skeleton of anti-tumor activity,was designed and efficiently-synthesized in the preliminary work.Copper-depletion of thiourea group was one of the confirmed anti-tumor mechanisms.Therefore,inherently anti-tumor gene carrier was constructed to realize synergetic therapy where the indirect anti-tumor mechanism would overcome the problem of cytotoxicity-induced low transfection efficiency in traditional drug-gene delivery system.In the first part of thesis,N,N-dimethylcysteamine modified PATU(PATU-DMCA)with different generations was synthesized through thiol-ene Michael addition reaction.Generation 2-Generation 5(G2-G5)PATU-DMCA could package DNA to form nanoparticles with the size of(60-100)nm and zeta potential of(10-20)mV at low N/P ratios.The luciferace transfection efficiency of PATU-DMCA in medium without serum was an order of magnitude higher than traditional dendrimer polyamide amine(PAMAM)in human cervical carcinoma HeLa cells.The subcellular distribution demonstrated that nanoparticles could escape from lysosome via 'proton sponge effect',which facilitated transfection efficiency.In HeLa peritoneal tumor models,G4 PATU-DMCA exihibited equivalent luciferase transfection efficiency to PAMAM and PATU-DMCA/TRAIL could inhibit the growth of tumors with the inhibition of 54.9%(P<0.001).Under the current experimental conditions,PATU-DMCA group exhibited no anti-tumor activity and PATU-DMCA/TRAIL nanoparticles didn't show the expected synergistic effect.From the copper-depletion process of PATU-PEG,copper of binding protein existed in abdominal tumor tissue was not easy to sequester compared with ceruloplasmin and PATU-DMCA/Cu complex might not be cleared quickly as did in i.v.injection.Cationic lipids/polymers play a dominant role in non-viral carriers and low transfection efficiency is the main problem for its application.Cationic carriers/nucleic acid nanocomposites based on strong electrostatic interaction couldn't release nucleic acid efficiently.Besides,cationic carriers would inevitably adsorb plasma protein,thus downgrading transfection efficiency.Shielding layer and environment-responsive design were attempted to reduce adsorption and accelerate intracellular release of nucleic acid in large number of studies,while non-cationic carriers were rarely attempted.In the second part of thesis,non-electrostatic siRNA delivery system was designed based on the boronic ester bond between phenylboronic acid and diol structure at the 3' end of the siRNA.Poly(ethylene glycol)-block-poly(acrylamidobenzoxaborole)copolymer(PEG-PBO)was synthesized through reversible addition-fragmentation chain transfer polymerization(RAFT).PEG-PBO and siRNA could self-assemble into cross-linked system with the size of 90 nm and was further condensed into CaP core-PEG shell nanoparticles upon adding calcium and phosphate ions,where PEG could efficiently control crystal growth.The formed PEG-PBO/siRNA/CaP nanoparticles exhibited high siRNA loading efficiency of 94%,low cytotoxicity and excellent colloidal stability at the neutral pH.PEG-PBO/siRNA/CaP nanoparticles showed significantly higher gene silencing efficacy than lipofectamine 2000/siRNA lipoplex in multiple cancer cells.Moreover,the nanoparticles packaged with siBCL-2 exhibited a significant apoptotic promoting effect.Endocytosis inhibition experiments showed that the nanoparticles were mainly internalized into cells via clathrin-mediated endocytosis.In acidic endosomes/lysosomes,the boronic esters easily broke,and nanoparticles quickly dissociated.Therefore,siRNA rapidly released and escaped from lysosomes via calcium-triggered osmotic swelling,which facilitated subsequent post-transcriptional gene silencing in cytoplasm.PEG-PBO/siRNA/CaP system was attempted for in vivo application,and results demonstrated that nanocomposites had fast plasma clearance and couldn't accumulate in tumors.The size of PEG-PBO/siRNA/CaP increased quickly in the buffer containing glucose.It was speculated that sugar in the serum would complex with PEG-PBO competitively after nanocomposites were i.v.injected,which led to easy disassociation and fast clearance of nanocomposites.Moreover,benzoxaborole also has a strong affinity to CaP nanoparticles.Thus,PEG-PBO was found to stabilize CaP nanoparticles and formed nanoparticles of 200 nm and zeta potential of(-6?-4)mV.PEG-PBO/DNA/CaP nanocomposites showed low cytotoxicity in A549 and HeLa cells in the medium containing FBS.At the optimal Ca/P ratio of 20,the transfection efficiency in serum-free medium was equal to that of PEI,and the transfection efficiency in medium with serum was 2-3 orders of magnitude higher than that of the standard PEI.Different from most cationic vectors,the transfection efficiency of nanocomposites in FBS containing medium was higher than the value in FBS-freee medium.The characteristic of serum resistance was showed in PEG-PBO/siRNA/CaP system as well.The particle size of PEG-PBO/DNA/CaP increased in 48 hours,which showed lower stability than borate ester bonding based siRNA delivery system.In conclusion,novel macromolecular gene carriers-polyacylthiourea and PEG-PBO stabilized CaP nanoparticles-were explored and some positive results were achieved while exerting intrinsic anti-tumor activity of polyacylthiourea and increasing stability need to be explored in the future study.