Synthesis And Biological Application Of Liver Targeted Gene Vectors With Polyethylenimine As A Backbone

Hepatocellular carcinoma?HCC?is one of the lethal tumors in the world,and it is also one of the most occurred tumors in China.Gene therapy has evolved into a promising treatment for the curative of HCC.Successful gene therapy depends not only on the therapeutic gene but also on the delivery vector.Polyethylenimine?PEI?is one of the most effective cationic polymers for gene delivery which contains lots of amino groups.It can electronically bind to the pDNA and condense it to protect the gene from degradation.Moreover,PEI may facilitate endosomal escape to increase the gene transfection efficacy.Therefore,BPEI25 kDa and LPEI22 kDa were widely studied and considered“the golden standard”of polycation polymers.It has shown that the gene transfection capacity of PEI was molecular weight dependent.PEIs with higher relative molecular weight increase gene transfection efficiency as well as the cytotoxicity.Though low relative molecular weight PEIs are less toxic,they do not display effective transfection property.Therefore,conjugation low relative molecular weight PEI with biodegradable linkers to obtain higher relative molecular weight PEI may increase the gene transfection capacity as well as maintain its low toxicity.In this article,a novel copolymer consisting of branched low relative molecular weight PEI?MW 800 Da?cross-linked by the biocompatible molecule inositol with biodegradable carbamate linkers was synthesized,named PI and the physiochemical properties and biological functions of PI/pDNA were studied.The polymer PI with mole ratio of 1:3 for PEI and inositol monomers which has a relative molecular weight of 8149,could condense plasmid to form compact nanoparticles.It had the highest gene transfection efficiency at N/P 40 which was higher than the control BPEI25 kDa,and also showed low toxicity in HepG2 and HeLa cells.The in vivo gene expression and tissue distribution of the nanoparticles showed that with the intraperitoneal injection of PI/pluc,bioluminescent signals accumulatively appeared in liver areas of mice on day two.The polymer PI as a liver target gene delivery vector with high transfection efficiency and low toxicity warrants further study.Although the toxicity of PI was lower than BPEI25 kDa,it should be decreased further for in vivo gene delivery.In contrast to BPEIs,the LPEI polymers have lower toxicity which could substitute their branched ones.In this article,LPEI2500 was used to be crosslinked by inositol with biodegradable linkers to prepare LPEI-inositol?LPI?,the physiochemical properties and biological functions of LPI/pDNA polyplexes were studied.It showed that the gene transfection efficiency of LPI polyplexes were higher than LPEI25 kDa with lower toxicity.Notablely,the toxicity of LPI was much lower than PI.The in vivo experiment showed that the LPI polyplexes has the optimum luciferase expression on day one which aggregated in the liver of mice.Nanoparticles must circulate in the blood stream for a long time to deliver adequate concentrations of systemically administered therapeutics to target tissues.Coating the surface of nanoparticles with polyethylene glycol?PEG?,or“PEGylation”,is a commonly used approach to shield the surface from aggregation,opsonization,and phagocytosis,which could prolong systemic circulation time.In this article,different mole ratios of mPEG were grafted to the polymer PI to form new polymers PPI.It showed that the polyplexes formed by polymers with low mPEG graft ratios had similar particle sizes and zeta potentials as PI,while particle sizes were increased and the zeta potentials were decreased with higher mPEG graft ratios.Even with the minimal graft ratio of mPEG,the polyplexes showed excellent stability in salt solution.With the mPEG covering,the gene transfection was significantly decreased especially with high mPEG graft ratio,which hardly showed any transfection capacity.The in vivo gene distribution experiment with the optimal vector P_1.3PI showed that with mPEG graft,the polyplexes were mostly distributed to the tumor.PEG covering increased the circular time of polyplexes while hindered the cell uptake which decreased the gene transfection efficiency.Another strategy to improve gene delivery is to conjugate polymers with ligands which could increase cell uptake through ligand-receptor interactions.Galactose is the most used ligand for liver-directed gene delivery because its receptor,asialoglycoprotein?ASGPR?,is exclusively expressed on the surface of hepatocytes and hepatoma cells with high density.ASGPR can recognize terminal?-D-galactose residues with high specificity and efficiency.In this article,the lactobionic acid-modified PEG?LA-PEG?was grafted to the polymer PI to form a new gene vector LA-PegPI.The optimal vector was screened out with the component ratio of LA:PEG:PI=0.06:0.15:1.LA-PegPI could condence pDNA compactly with good stability in salt solution.The polyplexes fomed by LA-PegPI/pluc had high gene transfection efficiency in ASGPR-positive HepG2 cells but not in ASGPR-negative HeLa cells.The LA-PegPI had low toxicity in both HepG2 and HeLa cells.Moreover,the luciferase gene expression in LA-PegPI/pluc-treated mice showed a significant aggregation trend in the liver region on day 3 post treatment and penetrated to the parenchyma of the liver.Therefore,polyplexes formed by LA-PegPI were liable to realize gene delivery to the liver tumor,which should be worthy of application in HCC gene therapy.Finally,we used LA-PegPI as a gene vector to deliver plasmid IL15 for immunotherapy.IL15 is an immunomodulatory cytokine with significant potential for stimulating antitumor T cells and NK cells.Intraperitoneal injection of LA-PegPI/pIL15resulted in significant inhibition of tumor growth and prolonging the survival times in a dose-dependent manner.The antitumor effect correlated with the activation and proliferation of T cells and NK cells,and the increase in cytokines in vivo.In addition,the immunohistochemistry analysis of tumor showed that LA-PegPI/pIL15 could directly inhibit the tumor growth and increase the apoptosis which increased the tumor inhibition capacity of polyplexes.Interestingly,the LA-PegPI/pluc had moderate tumor inhibition effects and prolonged survival times which may due to the stimulation of NK cells proliferation and tumor infiltration.Therefore,LA-PegPI was a good gene vector with immunoadjuvanticity ability.In summary,LA-PegPI/pIL15 is an excellent immunogene therapy agent which warrants further study.