Responsive Charge-reversal Polymers For Cancer Gene Delivery And EPR Effect In PDX Model Tumor

Gene therapy can deliver the therapeutic gene into tumor cells to aim at the tumor pathogenesis by turning on the killer gene or silencing the mutant gene,which had more advantages over other therapies on safety and efficacy.At present,cationic polymers,as main non-viral vectors,are widely used and studied in gene therapy.However,compared with viral vectors and chemotherapeutics,lower transfection efficiency and poor transport efficiency were still the main bottleneck restricting the application of cationic polymers.Charge-reversal cationic polymers brought new strategies to overcome the biological barrier in gene delivery,which contained electropositive to electrically neutral/electronegative conversion and electrically neutral/negative to the positive transformation.Charge-reversal strategies can be used to transform the stability of complexes to release nucleic acid quickly and responsively for improving gene transfection in tumor cells,as well as to transform the surface properties of complexes adaptively in systemic administration to facilitate blood circulation,tumor penetration and cellular uptake for improveing gene delivery efficiency.In this thesis,aiming at overcoming the bottlenecks in non-viral cancer gene delivery,two charge-reversal cationic polymers,poly[(2-acryloyl)ethyl(p-methylphenoxy)-2,4-dinitrobenzene)diethylammonium chloride](MDBP)and N-[3-((4-capryloyl benzyl)oxy)-3-oxopropyl]-N-methyl-quatemized LPEI(L4)were designed and developed.These two cationic polymers can respond to GSH and esterase in tumor cells respectively to realize charge-reversal process,which can transform the surface charge,particle size and stability of polyplexes for overcoming some biological barriers to improve the delivery efficiency and gene transfection.In the first experimental section,we developed a novel glutathione-responsive charge-reversal polymer for efficient gene delivery with low toxicity.The polymer can be degraded into negative poly(acrylic acid),leading to rapid DNA release and without interference with gene translation under GSH conditions especially in tumor cells.The plasmid DNA could be well condensed by MDBP to form the MDBP/DNA polyplex with diameters of 60-80 nm and potential of 20-25 mV at N/P ratio of 10,exhibiting excellent transfection efficiency in vitro.MDBP/DNA achieved 80-1350 times higer gene transcription efficacy than its analogues incapable of charge-reversal in HeLa,A549 and HepG2 tumor cells.The uptake of MDBP/DNA can be finished quickly by clathrin and caveolae mediated endocytosis pathway among 1 h.For in vivo application,a PEG-functionalized lipid envelop was incorporated to form the liposome-coating MDBP/DNA(LMDBP/DNA)for improving their stability in biological fluids(i.e.blood and ascetics fluid).In 100%serum condition,the gene transfection efficiency of LMDBP/DNA was 3-72 times higher than that of PEI/DNA and Lipo2000/DNA.Furthermore,the LMDBP/pTRAIL had long blood circulation,well accumulation in tumor and enhanced permeability.After intraperitoneal or intravenous administration,the polyplexes resulted in elevated TRAIL expression and caspase-3 activation in tumors,thus eliciting potent tumor suppression similar with the first-line chemotherapy drug paclitaxel,but much slighter adverse effects.Therefore,GSH-responsive charge reversal polymer MDBP was a novel and efficient non-viral gene vector.In the second experimental section,a fusogenic-mimetic charge-reversal polymer L4 was developed for efficient gene delivery.A library of cationic polymers with several alkyl sidechains were designed by high-throughput parallel synthesis,L4 was screened as the best candidate owing to its best structure-activity ability capble of both charge-reversal and membrane fusion.L4 could condense plasmid DNA into 70 nm diameter polyplex and deliver the DNA to nucleus via fusogenic-mimetic pathway,which was able to make DNA avoid falling into lysosomal trap.Actually,L4/DNA polyplex was found to achieve charge reversal by esterase hydrolysis,and then realseseing free DNA was ejected into cytoplasm and nucleus for gene transfection,while the polymer steadily stayed on cytomembrane.The transfection efficiency of L4/DNA was 20 times higher than that of PEI/DNA,reaching 3 orders of magnitude higher than the analogue without esterase-responsive function.Furthermore,an anionic?-PGA polymer was incorporated to form ?PGA-coating L4/DNA(PL4/DNA)to shield positive charges and improve their stability in biological fluids.PL4/DNA could keep fusogenic-mimetic celluar uptake pathway and demonstrated well stability and higher gene transfection in serum condition.Furthermore,after condesening suicide gene pTRAIL,PL4/pTRAIL could validly inhibit tumor growth and induce cell apoptosis.After intraperitoneal administration,PL4/pTRAIL exhibited better anti-tumor effect in than the first-line drug paclitaxel(PTX)and showed lower side effects.Therefore,esterase-triggered charge-reversal polymer L4 with fusogenic-mimetic celluar uptake pathway was a novel and efficient non-viral gene carrier.In the third experimental section,aiming at responding the doubts of Enhanced Permeability and Retention(EPR)effect caused by the poor efficiency and high failure rate of nanomedicines in clinical translational research,the models of patient-derived tumor xenograft(PDX)and cancer cell line-derived xenograft(CDX)were carried out to estimate the universality and potency of EPR effect in clinic.Poly(ethylene glycol)-block-polystyrene(PEG-PS)micelles with diameters of 30 nm,60 nm,100 nm and 200 nm,were used to study the difference of EPR effect between PDX and CDX.The results were summarized as three conclusions:1)EPR effect existed both in PDX and CDX tumor models,in which the distribution trends of PEG-PS micelles with different size were simlar.With the decreasing size of micelles,the tumor accumulation enriched and the vascular permeability enhanced;2)Large-size PEG-PS nanomicelles(>60 nm)had the same ERP effect both in CDX and PDX models.However,smaller size micelles(?30 nm)performed better ERP effect in CDX model with 40%increment than that in PDX;3)The vesicle-vacuole organelles(VVOs)existed in tumor vascular walls were the primary reason for the higher EPR effect of small size micelles in CDX than that in PDX model.These investigations illustrated the similarity and difference of EPR effects between CDX and PDX,responding to the doubts about EPR effect in clinical translational of nanomedicines.Hence,our research will provide important theoretical guidance and scientific basis for the design and clinical translational of nanomedicines to achieve more efficient anticancer effect in the future.