Studv On Hvdrophobic Targeting Ligand Modified Low Molecular Weight PEIs Vector Delivery System For Tumor Gene Therapy

Cationic polymers are widely used in the gene delivery field,owing to their strong stability,low immunogenicity and controllable structures.However,cationic polymer delivery systems can be hardly applied in vivo,due to the poor specificity to tumor cells,severe cytotoxicity caused by their inherent positive charge,and limited transfected efficiency.Thus,cationic polymers with more efficient efficiency,low toxicity and high tumor selectivity are needed.In virtue of its high transfection efficiency,high molecular weight PEI(25K)is conducted as a non-viral gene vector gold standard,but its toxicity limits its application in vivo.Low molecular weight PEI(1.8K)has lower toxicity,but its transfection efficiency is much lower than that of 25K PEI’s.We proposed that the transfection efficiency of low molecular weight PEI can be improved by conjugating small targeting molecules,such as folic acid and glycyrrhetinic acid.In addition,the targeting molecule grafted polymers can specifically identify cancer cells which overexpress folate receptors(FR)or glycyrrhetinic acid receptors(GA-R).Based on these two hypotheses,we synthesized three kinds of grafted 1.8KPEIs.We first designed FA ligand grafted PEIs.The performance of the modified PEIs depends on the characteristics and extent of substituents.The optimal percentage of grafting can achieve the desired polyplex properties for efficient gene delivery.PEI-FA0.65(x represents the average number of FA ligand molecules grafted to a 1.8KPEI molecule)polymer efficiently compacts pDNA into small nanoparticles with a spherical particle size around 40 nm。Its surface charge density in terms of zeta potential is 12.8 mV at weight ratio 3.PEI-FA0.65 has lower toxicity with significantly difference compared to 25KPEI.In the absence of serum conditions,the transfection efficiency was 100 times higher than 1.8KPEI polyplex,equal to or slightly higher than 25K PEI polyplex.But we found that PEI-FA0.65-mediated gene delivery was related to folate receptor-mediated endocytosis,and probably charge-mediated nonspecific uptake played a major role.The high efficient transfection mediated by PEI-FA0.65 polymer was suggested to go through caveolae-mediated endocytosis(CvME)and PEI mediated proton-sponge escaping from lysosomes.In the second part of this thesis,glycyrrhizinic acid and glycyrrhetinic acid ligands were grafted to 1.8KPEI.Due to the strong hydrophobic interaction of glycyrrhetinic acid,PEI-GAx showed improved antiserum activity with the increase of glycyrrhetinic acid grafting.PEI-GA0.75 and PEI-GL10.62 polyplexes also showed high selectivity to HepG2 cells,which overexpress glycyrrhetinic acid receptor.The transfection efficiency of PEI-GA0.75 polyplex or PEI-GL10.62 polyplex was four times or nine times higher than that of 25KPEI polyplex in HepG2 cells,but only equal to or slightly higher than that of 25K PEI polyplex in SW480 cell line.We found that PEI-GA0.75 and GL10.62-mediated gene delivery was related to the overexpressed level of glycyrrhetinic acid receptor on the cell surface of HepG2 cells.Results from the endocytosis inhibition assay,time-dependent transfection and intracellular inspection by confocal laser scanning microscopy,could indicate that the high transfection efficiency of PEI-GA0.75 and GL10.62-mediated gene delivery proceeds via caveolae-mediated endocytosis(CvME)pathway and bypass entry into lysosomes.In a HepG2 intraperitoneal tumor model,PEI-GA0.75 and PEI-GL10.62 carrying luciferase reporter gene gained high gene transfection in vivo,suggesting the potential of application in vivo.In the third part of this thesis,we investigated the lactobionic acid grafted PEIs.We found that PEI-LB Ax could not condense DNA even through at very high N/P ratios.And then,we designed glycyrrhetinic acid and lactobionic acid co-grafted PEIs by two-step synthesis.Even though lactobionic acid was grafted to PEI-GA0.75,PEI-GA0.75-LBA1.25 could still specifically bind to glycyrrhetinic acid receptors.We found that the hydrophilic ligands played an important role in reducing cytotoxicity and improving transfection efficiency in the presence of 10%serum.