Study On DNA/Polycation Nano-complexes Used Microneedles Drug Delivery System

DNA vaccination is a technique to inoculate directly eukaryotic expression plasmid containing encoding antigenic gene into body and then the plasmid express the related antigen to produce an immunological response for protecting an organism against disease. It is vaccine of third generation on the basis of gene therapy and transgenic technology, which is a revolution for vaccine. But the DNA vaccine drug delivery system is single relatively. The major is plasmid freeze-dried powder injection which was given by intramuscular injection to delivery DNA into rhagiocrine cell, especially antigen-presenting cells (APCs) including dendritic cells(DCs).Because of the low transfection efficiency, the degree of antigen presentation was low and the immunity effect is not satified. There are a lot of dendritic cells(DCs)(Langerhans cells, 500-1000 cells mm^-2 ) , a high-performance antigen presenting cell(APCs), which have strong immunological effect, so activity cuticular layer refers to the best part to inoculate vaccine. But hydrophilia macromolecule drug like DNA vaccine can not penetrate the keratoderma and enzyme system in skin will degrade DNA vaccine so that antigen presenting cell can not catch DNA vaccine. According that, we designed.Microneedles refers to a needle-shaped microstructure the needles on which have the length of more than 100μm and micrometer size. It fabricated by micro-electro-mechanical systems (MEMS) technology using different material, including silicon, polymer or metal. There has a broad use in the range of biomedicine for microneedle wjich as the drug delivery system have the advantage of exact dosage, high performance, painless and convenient use. The microneedle drug delivery system can be used to activitily target to the definite depth of skin, the Langerhans cell in active cuticular layer. The genetic carrier is synthesized on the base of PEI to enhance the stability DNA in vivo. The Pluronic and Solutol with amphipathic properties are chosed to modify the chain of PEI with high molecular weight in order to enhance the stability of PEI/DNA compounds and shield the positive charge on the surface of compound to diminish the toxicity. Besides, because the hydrophobic group of Pluronic and Solutol have a certain affinity to the cell, the transfection efficiency of DNA vaccine won't be lowered in vivo and vitro. Using the hepatitis DNA vaccine as model drug, to investigate the immunity effect of mouse in vivo after giving the compound of cationic polymer and DNA vaccine by transdermal application of microneedle. In the first part, succinimide ester was used to activation the terminal hydroxyl of Pluronic or solutol, nonionic wetting agent,to get a product with high activation efficiency and activity series. The activation compound was modified with PEI in the different proportion. After the purification through polydextran gel, the product was ultrafiltrated to get rid of salt and then cryodesiccated. According to the above process, we successfully got Graft-PEI compound with a series of different degree of modification per one PEI macromolecule at 1,2,5,10 and 20 Pluronic or Solutol. The result of ¹H-NMR showed the cationic polymerization synthesized by this method had a high graft ratio and PEI can be modified quantitatively by controlling administration dosage molar ratio of which had a satisfactory linear relationship with graft ratio. All that demonstrated that that synthetic method was stability, safety, controllability and had a high yield. There is no other impunity peak, so the purity of these cationic polymerization was high.In the second part, the related property of catiomc grafte polymer was investigated. By analysis blockage of electrophoresis, the N/P ratios need to completely block DNA were increased with the increment of modified degree of PEI, and decreased with the diminished molecular of reactor. The diameter of PEI/DNA and graft-PEI/DNA compound prepared were between 60-400nm. As the N/P ratios increased, the diameter of compound was decreased. The diameter of compound was obviously increased if it modified by macromolecular PEI at high modified degree. The zeta potential of compound with the same N/P ratios decreased with the increase of grafting amount of surfactant. If the modified degree and N/P ratio are same, zeta potential of compound increased a litter with the decrease of molecular of surfactant. According to the result of MTT method to test cytotoxicity, using surfactant to modify PEI can decrease the cytotoxicity in some degree. In the low concentration, the cytotoxicity can be decreased obviously if every PEI was grafted a surfactant, while in the high concentration, the cytotoxity won't decrease unless every PEI was grafted more than five surfactant.In the third part, cationic graft polymer to DNA compound was evaluated for transfection in vivo and vitro. Enhanced green fluorescent protein(GFP) plasmid and Luciferase plasmid was selected as the report genes, respectively. In order to enhance the expression of Luciferase, CMV promoter was cloned into thepGL3-basic Luciferase Reporter vector to construct recombinant plasmid pGL3-CMV Luciferase Reporter vector. In vitro transfection experiment of cationic graft polymer/DNA compound on Hela cell showed the PEI modified by low moleculae L61 and solutol did not significantly decreas the transfection efficiency, and the best transfection effect of PEI modified by low P123 is similar with that of PEI unmodified. As the increasing molecular of surfactant, grafting ratio rise and the decrease of the best transfection efficiency was followed. Expect bellows, investigate the express of Luciferase in all organ of ICR mouse who were injected compound through vena caudalis. The best transfection efficiency of low modified degree is better than that of PEL.In the forth part, the mechanical strength of SU-8 polymer microneedle prepared by MEMS was investigated to indicated that the microneedle array can tresis the aluminium foil and ex vivo skin easily and is difficult to break into two with certain toughness and hardness. This part was also to investigate the promotion of transdermal permeation of different kinds of drag. At first, three micromolecule drag, ketoprofen, methocarbamol and Ganciclovir were selected because of their different octanol-water partition coefficient. The transdermal permeation rate of ketoprofen increase by 4.77 times, methocarbamol 6.06 and Ganciclovir 12.49, which demonstrated that logP is an important fact which can influence the transdermal permeation rate of drag and the promotion of transdermal permeation of water-solubility drag is better than liposolubility drag, with the help of microneedle. Then four macromolecule drag having different molecular weight, insuline, lysozyme, human serum albumin and ovi albumin albumin, were investigated to get the result that macromolecule drag can permeate the skin treated with microneedle array on the lever of the transdermal permeation rate of micromolecule drag while it can not permeate the intact skin and the molecular weight of biomacromolecule drag is inversely proportional to transdermal permeation rate.The fifth part of this paper was to investigate the immune effect of DNA vaccine in vivo. The cello-phagocytosis test verificated that the phagocytosis of PEI modified remained. The anti-DNAase protection test showed the compound can protect the cell from degradation. Microneedle array can prick mouse' skin so that there were apertures about fifth micrometers in mouse' epidermis and these apertures provided passages through which compound can penetrate cuticle. Using hepatitis DNA vaccine as a model drag ,we investigated immunity effect in vivo by determining antibody titer in blood serum, cytokine excreted by spleen cell (γ-INF, IL-4) and proliferation of spleen cell. Compared with injecting cationic graft polymer to DNA compound intramuscularly and injecting hepatitis DNA vaccine aqueous solution intramuscularly, cationic polymerization/DNA vaccine compound was given by transdermal application of microneedle,after four weeks, antibody titre in blood serum and immunity effect were significantly enhanced ,strong immune response was emerged. The sixth part of this paper was to investigate constitution irritation of cationic polymerization/DNA vaccine compound. Constitution irritation was discovered after cationic graft polymer to DNA compound was given by intramuscular injection and transdermal application of microneedle. Inflammatory reaction of intramuscular injection group was more severe than microneedle group, which illustrated cationic polymerization/DNA vaccine compound had well physiological adaption.