Research On DNA/Cationic Liposomes Complexes For Magnetic Targeting Gene Therapy On Unstable Atherosclerotic Plaque

Atheroaclerotic diseases are one of the most harmful diseases with high lethality and disability rate.No proper therapy was developed to cure atherosclerosis at present.It's important to recognize unstable atherosclerotic plaque and give early clinic detection to enhance the stability of the unstable plaque.Gone therapy is a promising approach for the stabilization of unstable plaque.Recent researches have suggested that the expression of apolipoprotein-AI(Apo-AI) and scavenger receptor class B typeⅠ(SR-BI) both exhibited protection for atherosclerosis.So this research took Apo-AI and S--BI as theoretical gene for the therapy of unstable atherosclerotic plaque.Gene vectors for gene therapy are divided into two major types currently:virus vectors and non-virus vectors.The cationic liposomes(CL) is one of the most extensively researched non-virns vectors at present.Cationic liposomes have many advantages such as low toxicity/immunogenicity and potential for oncogenicity,size independent delivery of nucleic acids(from oligonucleotides to artificial chromosomes),ease of preparation,and quality control.But the primary disadvantages are the poor efficiency of transfection and failure of delivering therapeutic nucleic acids to their desired site of action in cells.Moreover,slow vector accumulation and consequently low vector concentration at target tissues have been identified as simple but strong barriers to effective gene delivery.So it is important to improve the target delivering of CL.Magnetic drug targeting(MDT) exploits paramagnetic particles as drug carriers,guides their accumulation in target tissues with local strong magnetic fields,and has been used with some success in the treatment of cancer patients.In this study,the principle of MDT was applied to gene vectors aiming to improve gene transfection efficiency and target selectivity and the applicability to gene delivery with magnetic cationic liposomes in vitro and in vivo was demonstrated.In this study,Apo-AI gene of rat,SR-BI gene of rat and the luciferase report gene was cloned into pcDNA 3.1(+) vector to construct recombinant plasmids pcDNA respectively 3.1(+)/APO-AI,pcDNA 3.1(+)/SR-BI and pcDNA-CMV.These recombinant plasmids can efficiently express in the mammal cell.Construction was confirmed by PCR,enzyme digestion and DNA sequencing.The iron content of the lipoplexes was determined by a Ferrozin-based spectrophotometric assay The standard curve was linear and the regression equation is: A=0.0198C-0.0072,r=0.9999,linear range:5-30μg/mL.The recovery and precision of low,middle and high concentration are accord with methodological request Using DOPE and DC-Chol as lipid materials we prepared magnetic cationic liposomes(MCL) by reverse-phase evaporation method.And on this base we made MCL/DNA complexes.The basic formulation of MCL has been determined by single factor investigation.The preparation conditions were:mixed solvent of chloroform and ether (1:9,V/V),the volume ratio of oil to water was 4:1,the disperse medium was phosphate buffer(pH for 7.4) and the amount of VE was 1%.The MCL suspension was sterilized by radiation sterilization.The content of plasmid DNA was measured by UV sepectrophotometry method.The linear relation between A and concentration of pDNA were good and the regression equation is:A=0.0207C+0.0009,r=0.9994, the linear range was 1.25-20μg/mL.The entrapment efficiency of pDNA was determined by high speed and cold centfifugation and the recovery of low,middle and high concentration are accord with methodological request.These indicated that the system was stable and identical with expectation.The lowest value of DC-Chol/DNA weight ratio was 2.5 to form stable complexes by using agarose gel electrophoresis analysis.The effect of DC-Chol/DNA weight ratio value on the mean particle sizes,zeta potential and encapsulation efficiency of DNA of the lipoplexes were also investigated.And the results suggested that lipoplexes prepared with DC-Chol/DNA weight ratio value around 9 showed smaller particle sizes,higher zeta potential and entrapment efficiency of DNA.The investigation of physico-chemical propertities of the lipolexes:The shape,size and zeta potential of the lipoplexes were characterized by TEM and laser particle size analyzer, respectively.The lipoplexes were relatively and regularly spherical in shape and the magnetic materials were encapsulated in the lipoplexes.The mean particle size,polydispersity index and zeta potential analyzed by photon correlation spectroscopy and laser dopper anemometry were 343.4 nm,0.231 and +37.5 mV,respectively. Investigation on the response of lipoplexes to the magnetic field indicated that lipoplexes showed good responsible property to magnetic field.Cytotoxicity of the MCL with different iron content and CL to the human normal cells(THLE-3) was investigated by MTT assay.Results indicated that MCL and CL showed low cytotoxicity to THLE-3 cells when the weight ratio between DC-Chol/DNA was≤5 and relatively higher cytotoxicity when the ratio≥15.In this research,the transfection conditions of MCL and CL on THLE-3 cells were optimized by using pcDNA-CMV plasmid which contained a luciferase reporter gene.Optimized transfection conditions for MCL were DC-Chol/DNA wt ratio 5, incubated with the cells for 8h and the magnetic induction time 30min.Optimized transfection conditions for CL were DC-Chol/DNA wt ratio 2.5,incubated with the cells for 8h.Results suggested that transfection efficiency between MCL without a magnetic induction and CL exhibited no significant difference.But the transfection efficiency of MCL with an extra magnetic induction was increased by 2.6 times (P＜0.01) as compared with CL under the optimized transfetion conditions.A preliminary study on the targeting distribution action and pharmacodynamic of MCL/DNA complexes in rats were carried out in this research.Gene expression in the organs were examined 24h after intravenous injection of complexes into rats via tail vein(with or without an external magnet fixed on the liver of the rats).Results of gene expression in livers suggested that gene expression transfected with MCL/DNA complexes with magnetic induction(+) were enhanced by 1.5 times(P＜0.01) and 2.7 times(P＜0.01) as compared with that transfected without magnetic induction(-) and CL/DNA complexes,respectively.Using APO-AI and SR-BI as therapy gene and MCL as gene vector with an external magnetic induction,gene therapy was carried out on ATHSC rat models.After 6 weeks of gene therapy,the TC,TG and LDL in gene therapy groups were significantly decreased(P＜0.01)while the HDL was significantly increased(P＜0.01).The atherosclerotic plaque was diminished in each gene therapy group,part of the vessel walls became soft and recovered flexibility;,the endomembrane of lumina became smooth;the vessel walls were not thick obviously.The gross pathologic stained by oil red O showed us that:the irregular red eminentia on the surface of endangium was diminished;the rate of lipid fringe area and fibrous plaque area were 9.21%and 1.16%in group 1,9.20%and 1.18%in group 2,respectively.Hyperplasia endothelialis was observed weakened under the microscope;there were manipulus foam cells aggregated under the fibrous membrane.In the control group,the vessel walls were still stiff and thicken obviously.Many irregular red eminentia on the surface of endangium were observed;the rate of lipid fringe area and fibrous plaque area were 32.25%and 1.66%.Hyperplasia endothelialis was obviously observed under the microscope;a great quantity of foam cells aggregated under the fibrous membrane;the plaque was outstanding to the lumina.These consequences were typical appearance of the fragile plaque.These results indicated that targeting gene therapy on unstable atherosclerotic plaque by using MCL/DNA complexes was effective.