Preparation And Evaluation Of A Novel Modified Polyethylenimine Material And As Oligonucleotide Deltevery System

Nearly half a century, with the development of economy and society, the mostdeadly infectious diseases has been effectively controlled, however, at the same timethe spectrum of human disease also undergone tremendous change. Many diseaseshave been a serious threat to human health, such as cardiovascular disease,respiratory disease, cancers and so on. According to the statistics, death rate ofChina from cancers presents a clear upward trend in the past30years, and1/4ofthe deaths died of cancers,which had become the first reason for death of urban andrural residents. Oligonucleotide,which can specifically regulate gene expression,has been widely used in therapeutic areas of oncology drug development and studiesof gene function, including siRNA, antisense oligonucleotides and aptamers.Antisense oligonucleotides and aptamers have been into the clinical practice,and theclinical trials of siRNA are underway. Nevertheless, various reasons,including theinstability of oligonucleotide itself in vivo environment, the barriers to reach thelesion site, limit its huge potential as therapeutic agents..We only have to establish abetter targeted delivery methods to smoothly deliver the oligonucleotide to the cellsand tissues, so as to give full play to the advantages of oligonucleotides as genetargets for drugs with a low dose, high-efficiency, low-cost, reduced toxicity.PEI-LA was synthesized by N-acylation of PEI-800,combined with linoleoylchloride, which hydrophobically modified PEI.The preparation process of the PEI- LC: the ethanol solution of PEI-LC(10ug/ul)10ul added in90ul HEPES(pH7.4),and suspended quickly10s in the swirl mixing instrument, and then sonicatedultrasonic instrument20s, to obtain a PEI-LC drug-loaded system,with280nmparticle size and34V zeta potential.PEI-LC which combined with LOR-2501, formed PEI-LC/ASO complexdelivery systems,and LOR-2501is a20-mer phosphorothioate ASO targeting the R1subunit of ribonucleotide reductase, an enzyme associated with drug resistance. LOR-2501has shown potent antitumor activities in murine xenograft tumors of the lung,the liver, the ovary, the brain, the breast, and the pancreas.In this experiment, PEI-LCand LOR-2501formed electrostatic copolymer by electrostatic force.PEI-LA wasable to completely retard LOR-2501at N/P above8and the charge of polymers waspositive at N/P above8.Dynamic light scattering revealed the successful formation ofPEI-LA/LOR-2501particles of under200nm in diameter at N/P above8. Nosignificant cytotoxicity was observed with formulations at the tested dosage levels.Observations by confocal microscopy and flow cytometry revealed successfuldelivery to the cytosolic site of action. Subcellular distribution of the PEI-LA/ASOwas analyzed. ASO was localized strong in the cytoplasm but not in the nucleus, nopolymer in the outside of the cell. Compared with free ASO, PEI-LC/cy3-LOR-2501polymer greatly improved cell uptake, and when the N: P ratio of PEI-LC/cy3-LOR-2501and PEI800/cy3-LOR-2501was the same, the cell uptake of the former isalmost twice the latter.All of this proved that PEI-LC transfected the ASO to theright active site. Functional delivery of LOR-2501with PEI-LA was confirmed byreal-time PCR analysis, which revealed PEI-LA/LOR-2501induced downregulationof R1mRNA expression. Much greater downregulation of51%to64%were encountered for PEI-LA/LOR-2501complexes at N/P ratios of6and10, respectively.Free LOR-2501showed only a slight capacity for mRNA downregulation. Westernblot analysis was conducted to determine the effect of PEI-LA/LOR-2501on R1protein level. At higher N/P ratios, the PEI-LA/LOR-2501complexes showedsignificant downregulation activity. From N/P6to10protein downregulationincreased from59%to70%. Finally, internalization inhibitor experiments showedthat clathrin-mediated endocytosis was the principle mechanism of entry whilemacropinocytosis took a secondary role and lipid raft/caveolae mediated endocytosisonly played a minor role in cellular uptake.Nevertheless,PEI-LC polymer was a good drug-delievered system with lowtoxicity, high transfection, but PEI-LC/ASO complex delivery system carried apositive charge, and there were no modified group on his surface,so they might beeasily combined with negatively charged proteins, enzymes or be degraded, resultingin the low transfection efficiency and poor stability. Therefore, we have prepared amore stable folate receptor-targeted liposomes with PEI-LC as material.According tothe literature, folic acid is a small vitamin molecule,and folate receptor wereoverexpressed in most tumor cells, typically20-200times higher than normal cells,so ligands of folic acid has get more attention in targeting system. In the test, weprepared three liposomes,which are folate targeted liposome with antisenseoligonucleotide F-of GSH-L-ASO, F-L-ASO and no folate targeting L-ASO, all ofthem had on apparent toxicity on cells. the F-GSH-L-ASO and F-L-ASO had a traceamount of positive charge on their surface, so it is difficult to be combined with anegatively charged proteins, enzymes,and the folic acid as a ligand can targetliposomes to tumor cells with high expression of the folate receptor, and then exert its antineoplastic activity. The fluorescence of ASO was mainly distributed in the cellsurface and cytoplasm, both F-GSH-L-ASO and F-L-ASO can transfect ASO into theactive sites. Functional delivery of LOR-2501with F-GSH-L-ASO and F-L-ASOwas confirmed by real-time PCR analysis.F-GSH-L-ASO and F-L-ASO induceddownregulation of R1mRNA expression were71%and68%.Compared with thesingle-PEI-LC material, both folate-targeted liposomes had a better effect oftransfection. F-GSH-L-ASO, compared with F-L-ASO, regardless of its physicalproperties (potential and particle size), cellular uptake, transfection or R1mRNAexpression levels were maintained better stability after keeping six days, because F-GSH-PEG-DSPE with GSH has a greater hydrophilicity than F-PEG-DSPE and dueto the GSH moiety in the F-GSH-PEG-DSPE, which is ionized and increases thedistance between the folate molecules on the liposome surface, thus preventing thefolate moiety from seclusion and self-association. So F-GSH-PEG-DSPE liposomesare more stable in solution.Finally, PEI-LC as the material,we prepared AS-ODN microspheres complexesby using method of multiple emulsion/solvent evaporation and then studied theirphysical properties, morphology, and release properties. The study found the size ofmicrosphere,drug loading were increased, the encapsulation efficiency wasdecreased and the overall release rate become faster and faster with the increasingamount of AS-ODN complexes.In summary, polythene imine modified materials PEI-LC can be used as anovel transfection reagent, which overcomes the shortcomings of high cytotoxic ofhigh molecular weight polythene imine (PEI25kDa), of low cell transfection of lowmolecular weight polythene alkyleneamine (PEI800Da),which was capable of forming a relatively stable polymer with antisense nucleotide, and pass to the cellactive sites. Meanwhile, F-GSH-L-ASO liposome is a more stable drug deliverysystem,which had a trace amount of positive charge on their surface, so it is difficultto be combined with a negatively charged proteins, enzymes,and the folic acid as aligand can target liposomes to tumor cells with high expression of the folatereceptor, and then exert its antineoplastic activity.