Study On The Novel IL-18 Loaded Multipolyplex Nanoparticles As Gene Delivery System

The completion of human genome project boosts the discovery of functionalgenes and disease-related genes. Human being has been getting more andmore understandings of us in medicine and physiology, which booms thedevelopment of gene therapy. Gene therapy has become the most activeresearch area in biomedicine in 21st century.With the more and more understanding of the molecular mechanism of tumors,it is recognized that the form and development process of tumors is verycomplex, which includes many factor, steps and genes. Cytokine is a kind ofproteins or polypeptides that produced by immune cells (lymphocyte andmonocyte/macrophage) and relative cells (fibroblast and endothelium cells).Itis of high activity and various functions, which can stimulate and enhance theantitumor immune reaction in body, in order to cure the cancer. Until now,cytokine has been widely used in tumor immune gene therapy in laboratory.But serious immunogenicity of exogenous protein leads much toxicity. DNAvaccine was composed of plasmid DNA encoding different functional protein.Once be transfected in body, plasmid can expressed into protein and inducecorresponding mild immune reaction. It merits includes:①no danger of the"reduced toxic" vaccine related virus,②easy large-scale production with lowcost,③store in dry powder,④low dose of proper gene construction couldinduce protective immunity,⑤Once administration could produce long termimmunity.In spite of the remarkable progress of gene therapy in recent 20 years, manytechnological problems are hindering the further success of gene therapy, and one of them is the efficient delivery of therapeutic genes. Current genedelivery systems include viral vectors and non-viral delivery systems. Viralvectors hold high transfection efficiency and poor biosafety. In contrast,non-viral delivery systems are much safer and easy to large-scale production,among which liposomes and macro-polymer developed best. Both of themhave wide application in pharmaceutics. Liposome is made of lecithin, withgood compatibility. It encapsulates genes interest inside without limitation ofsequence, and is uptaked into cells by endocytosis and amalgamation.Accordingly, transfection of liposomes is relatively higher, but the weakstability and positive charged surface of liposomes limited its application insystemic administration. One the other hand, macro-polymer gene deliverysystem has satisfied biocompatibility, stability and easy modificationcharacters, which has attracted more attention to.This project included the optimization of the formulation and processcondition of multi-polyplex gene delivery system—PPD (PELGE-PLL-DNA),the evaluation of transfection efficiency in vitro and in vivo as well asphysicochemical properties. The mice lung metastasis tumor models wereestablished in order to examine the therapeutic efficacy of lyophilizedmIL-18-PPDs in vivo individually and combined with cisplatin (DPP). Basedon these results, we modified the PPDs with folate to endow the targetingproperty for this delivery system.In the first step, E. Coli (DH 5-α) was employed to amplify the reporter genesand therapeutic genes for our experiments. Plasmid DNA was isolated andpurified from the cells using the Qiagen Giga Endo-free plasmid purificationkit. DNA concentration and purity were quantified by UV absorbance at 260nm and 280 nm. The structural integrity and topology of purified DNA wasanalyzed by agarose gel electrophoresis. Cytotoxicity of series ofbiodegradable material PELGE was studied by MTT assay on various cells.Results show nanoparticles has best compatibility on blood endothelium cells, and the hepatocytes Chang cells took the second place. Considering thehemocompatibility of these series PELGE, low toxic PELGE73 was selectedfor further research.A simple and sensitive assay method for quantitative determination of DNA inPPDs was developed by fluorescent spectrophometer with Hoechst 33258 asthe fluorechrome. The optimal ratio (1:1, w/w) and solvent (glucose injection)for mixing cationic peptide PLL and plasmid DNA was determined by zetapotential and gel electrophoresis to form PD (PLL/DNA) mixture. And thenPD was encapsulated in biodegradable monomethoxy (poly ethyleneglycol)-poly(lactide-co-glycolide)-monomethoxy (poly ethylene glycol)(PELGE) to form core-shell nanoparticles as a novel multi-polyplex genedelivery system—PPD(PELGE-PLL-DNA). Nanoparticles were prepared by adouble emulsification-solvent evaporation technique using F68 (Pluronic F68,namely Poloxamer 188) as surfactant. The preparation process was optimizedafter single factor test using size, polydispersity index and encapsulationefficiency as index. The resulting PPDs were regularly spherical, with thediameter of 150.92±9.04 nm with zeta potential of-27.31±2.02 mV andencapsulation efficiency of 87.99±1.61%.The effects of monomethoxypoly (ethyleneglycol) (mPEG) segments andpolylysine in the polymer on particle size, zeta potential, DNA integrity andtheir merit for escaping from lysosome were investigated. It was found thatthe introduction of hydrophilic mPEG segments in the copolymer chainscould improve the biocompatibility of copolymer and still maintain theproperty for lysosomal escape, while PLL protected DNA from beingdestroyed in ultrasonic and acidic microenvironment during formulation anddegradation. Thus DNA loaded nanoparticles with smaller particle size, betterprotecting property and escaping character were obtained by combined use ofPPD as a new gene delivery system.The in vitro transfection ability of PPDs was evaluated using two kinds of reporter genes, pORF-LacZ genes were transferred into HepG2 and Hela cellsusing PPDs. Theβ-Gal assay qualitatively showed that LacZ-PPDs couldtransfect these cells much efficiently compared with naked plasmid DNA andnanoparticles with out PLL.Quantitative transfection efficiency was determined by measuring theluciferase activity using luciferase assay system. In the case of thePELGE-NPs without PLL, very low luciferase activity was observed and theactivity was similar to the control group, where naked pDNA was used. Whiletransfection of PPDs showed about 3 orders of magnitude higher luciferaseprotein levels compared to the NPs without PLL or the naked DNA for thesame dose of nanoparticles.Taking morphology, color and redispersibility as index chose the formulationof lyophilized PPDs. The resulting lyophilized PPDs were obtained asexpected. The stability tests results showed no obvious change in morphology,particle size, zeta potential and encapsulation efficiency when compared withthat of PPDs before lyophilization. It could also protect DNA fromdegradation by nuclease.We established mice lung metastasis tumor models and examined thetherapeutic efficacy of lyophilized IL-18-PPDs and the combined therapeuticefficacy of chemotherapy and gene therapy. The experiment results showedthat the life spans of tumor-bearing mice in IL-18-PPDs group weresignificantly longer than that of the mice in PBS group, empty nanoparticlesgroup and positive control group. In addition, the average life span of themice in the combined therapeutic groups was much longer than any otherones, which indicated the success of combined therapy. The results ofpathology and histology suggested that the index of lungs and the number ofmetastasis of the mice in IL-18-PPDs combined therapeutic groups weresignificantly less than those in other groups. Based on these results, we modified the PPDs with folate to endow thetargeting property for this delivery system. Fluorescein sodium andpolylysine-condensed DNA (PD) loaded PELGA nanoparticles (PELGA-NPs)and folate modified nanoparticles (PELGA-F-NPs) were prepared,respectively. Effects of the folate conjugation on the uptake of the fluorescentNPs was qualified by fluorescent invert microscopy and quantified byspectrofluorometric measurement, while gene expression was measured byX-gal staining and luciferase assay, both using Hela cells as an in vitro model.Results showed cellular uptake of NPs was enhanced by association ofPELGA-F, while in transfection activity, luciferase expression and X-galstaining also confirmed the increased uptake, which was blocked byco-incubation with free folic acid in medium.As stated above, the combination of polycationic peptides and biodegradablepolymer could be a promising carrier for non-viral gene therapy and couldprovide new strategy in this field.