Application And Studies On Gene Transfer System Mediated By PEI-cholesterol Lipopolymer With Lipid Microbubbles

Gene therapy is an approach for human disease treatment by inserting therapeutic gene into cells in order to produce desired protein with specific functions and inhibit expression of abnormal genes. The clinical success of gene therapy depends on the development of suitable efficient and targeted gene transfer system. Gene delivery systems are classified into viral and non-viral system. Non-viral vector win us high favor for its several advantages including low immunogenicity and power of carrying high molecular weight of DNA.Poly(ethyleneimine)(PEI) has emerged as a good candidate in the cationic Polymers. PEI(Polyethylenimine) is cationic polymer as non-viral gene delivery vectors used widely in research. PEI is linear or branch polymer. There is high positive charge on its surface, so PEI can condense DNA with negative charge.There are lot of protonate nitrogen atom, endosome and lysosome of PEI and DNA complex swell and rupture(proton sponge effect), DNA release and go into nucleus then express.The molecular weight of PEI varies greatly.The molecular weight of PEI varies from 5000-25000Da for gene delivery vectors. Transgene efficieney of high molecular weight PEI is high, but toxicity is also high.The toxicity of low molecular weight PEI is very low, but generally the transgene efficiency is very low. PEI can make erythrocyte aggregate and bind non-specificly with albumin in blood.Transgene efficieney is low when it was applied in vivo.Therefore, PEI need to be modified. The main purposes of PEI modify are to reduce the toxicity of PEI, improve stability and targeting of PEI/DNA complexes, reduce non-specific binding of albumin, extend their haft-time in the body.Objective:To investigate the toxicity and properties of different kind of PEI-Chol which modified with cholesteryl, a series of PEI-Chol polymers with different rates of amino-modified were synthesized with low and high molecular weight of PEI Screening of suitable PEI-Chol lipopolymers are carried by their cytotoxicity, condensing capacity of DNA, buffer ability, anti-nuclease degradation. Neutral lipid microbubbles was constructed by lipid modified with polyethylene glycol and the bubble/PEI-Chol/DNA complexes was constructed by static self-assembly technique. Ultrasound localization was used to target objective DNA. A novel low toxicity of gene delivery system mediated by PEI-cholesterol lipopolymer was offered to the clinical treatment.Methods and contents:four parts.One part:Synthesis and characterization of PEI-Chol lipopolymersPEI-Chol lipopolymer was synthesized by linking cholesteryl chloroformate to the amino groups of branched poly(ethylenimine) (PEI). Their buffering capacity was calculated by Acid-base titration with formulaβ=dc(HCl)/dpH. DPH as a fluorescent probe was used to determinate the critical micelle concentration of lipopolymers. And MTT assay was used to evaluate the cytotoxicity of the lipopolymers.Two part:Preparation and biophysical properties of PEI-Chol/DNA complexesUsing alkaline lysis method, through different kind of processes include preparation of Escherichia coli cells, re-proliferation of pEGFP-Cl which first insert to coli cells, and extraction of plasmid pEGFP which purity and size was identified by agarose gel. The particle size and Zeta potential of PEI-Chol/DNA and PEI/DNA complexes were measured by Laser Particle Size Determination. The binding ability of condense DNA and against RNase I degrate with PEI-Chol were ivestigated by agarose gel electrophoresis. Morphology of PEI-Chol/DNA complexed was observed under scanning electron microscope.Three part:The Construction and charateristic of Lipid microbubbles/PEI-Chol/DNA self-assembled systemsMicrobubbles prepared by optimizing conditions which selected by central composite experimental design (CCD) and response surface methodology (RSM).The microbubble concentration was used as an index to explore the effect of molecular weight of PEI-Chol and temperature of integration on the microbubble concentration. The security of Lipid bubble/PEI-Chol/DNA complexes was checked in following aspects:effect of ultrasound on the integrity of naked DNA, on the integrity of the microbubble and effect of different polymers on cytotoxicity.Four part:In vitro and in vivo transfection experiment of lipid microbubbles/PEI-Chol/DNA gene transfer system.The gene transfer efficiency in vitro with different PEI, PEI-Chol polymer and microbubble-mediated gene delivery system were presented. Using PEI-Chol25-5 as an example to investigate the effect of fusion temperature, fusion time, N/P ratio and ultrasonic time on the transfer efficiency. The protective effect of MIF siRNA transferred by Lipid bubbles/PEI-Chol/DNA deliver system on acute lung injury induced by lipopolysaccharide (LPS) in mice was investigated.Results:1. IR spectrum indicate the cholestreol was sucessed to link with PEI throught the typical carbonyl peak of 1700cm-1. According to 1H-NMR spectra of PEI-Chol, amine modification rate, composition and molecular weight can be calculated. DSC profiles show the stiffness of PEI was increased after cholesterol was connected..2. PEI-Chols synthesized with same molecular weight of PEI, their critical micelle concentration (CMC) decreased gradually with the increasing of the graftting cholesterol level.3. Different molecular weight of PEI, their buffer capacity enhanced with the increases of molecular weight. But PEI-Chols synthesized with the same molecular weight of PEI, their buffer capacity decrease with the increase of molecular weight.4. The effect of polymers on the relative growth rate of Hela, A549, MCF-7 show that PEI 0.6K and PEI 1.8K did not show significant toxicity, but with the increase of the molecular weight of PEI, their cytotoxicity increased. The grafting of cholesterol can reduce the cytotoxicity of PEI, and the cytotoxicity decreased more significantly with the increasing of the amount of cholesterol.5. Purity and size of pEGFP-Cl prepared by alkaline lysis and extracted by reagent was fit the experiment request.The result is A260/A280=1.89+0.025, plasmid molecular weight of 4.7kb, concentration iof 279.37±0.731μg/ml.6. Low molecular weight of PEI would form more large size of DNA complexes, but the grafting of cholesterol can increase their condense ability and anti-enzyme ability. Zeta potential enhanced with the increasing of molecular weight of PEI and decreased with the increasing of the numbers of cholesterol groups. The PEI/DNA and PEI-Chol/DNA complexes with higher ratio of N/P possessed the smaller size and higher Zeta potential. Scanning electron microscope showed PEI-Chol and DNA can form nano-complexes.7. Microbubbles prepared by optimizing conditions which selected by central composite experimental design (CCD) and response surface methodology (RSM) exhibited uniform particle size distribution, lower Zeta potential(-8.3mV) and can keep stable for two weeks. The optimum preparation conditions are 40mg of DPPC (X1),7.5mg of PEI-Chol (X2) and 4.5mg of DSPE-PEG2000 (X3). The concentration of microbubble is 8.22×109/ml.8. The microbubbles concentration made with PEI-Chol (1.8-1) and PEI-Chol (25-5) is higher than those made by PEI-Chol (10-1) and PEI-Chol (25-1). The Zeta potential of bubble/PEI-Chol/DNA complexes is lower than that of PEI-Chol/DNA complexes.9. Ultrasonic conditions, frequency 2MHz under 120s MI 1.0, do not have a significant effect on survival rate of A549 cells, MCF-7 cells, and on the integration of DNA.10. The optical transfection conditions are as follows:fusion temperature of 55℃, time of 60min, ultrasonic emission parameters:continuous wave, frequency 2 MHz, the depth of 60 mm, mechanical index (mechanical index, MI) 1.0, time of 60s. Cell transfection experiments found that:the grafting of cholesterol to the amino of PEI1.8k has improved significantly the cell transfection efficiency, if combination with lipid microbubbles, transfection rate and the ability of anti-serum were enhanced.11. Results show that acute lung injury animal model caused by LPS stimulation in mice was successful, all of four groups:the normal group, LPS stimulation group, PC+LPS+MIF siRNA treatment group, WP+LPS+MIF siRNA treatment group, were compared in following aspect, such as the pathological changes of lung tissue and release of inflammatory mediators. The results show that pathological changes in lung tissue of model group was significantly, while the PC+LPS+MIF siRNA treatment group is no therapeutic effect, but the WP+LPS+MIF siRNA treatment group can improve effectively the pathological changes, and can effectively reduce the secretion of inflammatory mediators TNF-a, IL-lb, IL-6.The effect of gene transfection clarify this delivery system is feasible and effective.Conclusions:1. PEI-Chol polymers have the following physical characteristics:when the amount of cholesterol group linked to the amino of PEI is appropriate, less impact on the buffer capacity of PEI. And the introduction of cholesterol group can change the hydrophilic and hydrophobic propertise of PEI, that contribute to the formation of micelles. All of these provide us evidence to select the appropriate PEI-Chol.2. PEI-Chol polymers have the following physiological characteristics:the grafting of cholesterol group into PEI can reduce the cytotoxicity of PEI, and effectively improve the DNA condensing ability of low molecular weight PEI, enhanc its role of anti-Rnase and protect DNA.3. Bubble/PEI-Chol/DNA gene delivery system is stable and the ultrasound for its application is less effect on the relative growth rate of cell and the integrity of the DNA.4. Gene transfer system mediated by PEI-cholesterol lipopolymer with lipid microbubbles provided good transfer efficiency with other desirable characteristics such as against-precipitation of plasma proteins low toxicity and ultrasonic targeting release. In vitro it can transfer pEGFP-Cl in A549 and MCF-7 cells, in vivo it can transfer MIF siRNA and offer protective effect in mice with acute lung injury.