Preparation Of Biomagnetic Nanoparticles And In Vitro Experimental Study Of Magnetic Controlled Gene Therapy On Large Intestine Cancer

Part 1: Preparation and Acute Toxicology of nano-Fe₃O₄ MagneticFluidSummary The nano-Fe₃O₄ magnetic fluid was prepared by chemical coprecipitation method and its acute toxicology was investigated. The acute toxic reaction and the main viscera pathological morphology of mice were evaluated after administered the nano-Fe₃O₄ magnetic fluid by oral, intravenous and intraperitoneal respectively with different doses. The results showed that LD₅₀ (%50 lethal dose)>2104.8mg/kg, ED₀ (maximum non-effect dose) =320.10mg/kg by oral; LD₅₀>438.50mg/kg, ED₀=160.05mg/kg by intravenous; and LD₅₀>1578.6mg/kg, ED₀=320.10mg/kg by intraperitoneal. Degeneration and necrosis of viscera were not found. So the nano-Fe₃O₄ magnetic fluid, of which toxicity is very low, may be used as base material of drug carrier. Part 2: Preparation and Characteristics of Dextran-Coated IronOxide NanoparticlesSummary Magnetic ferrofluid, which has great fluidity and good magnetic property, has been thought in the area of biotechnology. However, it needs lower toxicity and better biocompatible. In order to improve the magnetic particles' properties, some kinds of organic materials were selected to modify the particles. Dextran, a kind of polysaccharide with many significant biological advantages such like biodegradable, biocompatible and bioactive, and some chemical properties such like poly-cationic, hydrophilic and poly-hydroxyl (-OH), can be used as a base material for magnetic carriers. In this study, dextran-coated iron oxide nanoparticles (DCIONPs) were prepared by ultrasonic and chemical coprecipitation method and the characteristics were evaluated. The size and shape of the particles were verified by scanning electron microscope (SEM), the size and Zeta potential were determined by particle size analyzer, the morphology was investigated by atomic force microscope (AFM), and the magnetic property was evaluated by magnetometer. Most of the DCIONPs showed good shape of sphere or oval. The effective diameter was 93.1± 2.2nm and the half width was 26.7±1.3nm, as indicated that the DCIONPs were tiny and in conform size. The Zeta potential was positive. The saturation magnetization was 26.0 ±1.1 emu/g in a magnetic field of 10⁴Oe and the remanence was zero without it, which showed the nanoparticles' superparamagnetism. In a conclusion, the DCIONPs prepared by ultrasonic coprecipitation method had the advantages of small size, narrow distribution and good magnetic property so that they might be used as the magnetic carrier for drug or gene target delivery. Part 3: Synthesis and Characteristics of Magnetite-Gene ComplexSummary Whether or not the biomagnetic material could combine with the nucleic acid for gene transfection was studied in the experiment. The DCIONPs prepared before mixed with the plasmid DNA-pGenesil, which could express enhanced green fluorescent protein (eGFP), adequately in variable quantity ratio. PGenesil-1 was used as the control group. The binding ability was assessed by electrophoresis and BANDSCAN software. Using different gene carrier, like Lipofactamine2000, Sofast and DCIONPs, to transfect the large intestine cancer cells-Lovo cell line in vitro with or without magnetic field. The expression of eGFP was observed in fluorescence microscope and the transfection efficiency was compared. The results showed when the quantity ratio ascended from 1 to 5, the binding ability of pGenesil-1 and DCIONPs strengthened dominantly. However, the binding rate increased indistinctively when the quantity ratio continued ascending. The expression of eGFP showed that the early transfection rate could be improved with magnetic field. In a conclusion, the DCIONPs we synthesized were able to carry plasmid DNA, and to enhance the early transfection efficiency with magnetic field. Part 4: Inhibition of c-Myc Expression in Colorectal Cancer by Magnetic Controlled RNA InterferenceSummary The expression of mRNA, protein of c-myc, and the apoptosis of colorectal cancer cells were evaluated after transfected by magnetic controlled RNA interference. The dextran-coated iron oxide nanoparticles (DCIONPs) synthesized before were used as magnetic gene carriers. The cultured Lovo cells were transfected by plasmid DNA (pGenesil-1-X1, pGenesil-1-X2, pGenesil-1-HK respectively) delivered by DCIONPs in the magnetic field. The expression of c-myc was detected by reverse transcription-polymerase chain reaction (rt-PCR) and western-blot. Flow Cytometer (FCM) and transmission electron microscope (TEM) assessed the cell apoptosis. The c-myc mRNA's level lowered 82% and c-myc protein's expression lowered 85% after magnetofection of pGenesil-l-X2; the apoptosis rate was 23.7% and the apoptosis cells were observed by TEM. In a conclusion, the expression of c-myc in Lovo cells can be inhibited by magnetic controlled RNA interference.