Preparation And Performance Of Starch/DNA Delivery System

As a new novel and promising treatment, gene therapy is presumed a revolution ofmedical science and pharmacy and recognized as a milestone during the progress ofbiomedicine. During gene transmission and transfection, gene delivery carriers determine thetargeting and protection of the gene, therefore, to achieve successful gene therapy,development of proper gene delivery systems could be one of the most important factors.Designed by natural renewable material, non-viral gene delivery systems have been highlyrecommended because of its good biocompatibility, biodegradatibility, free viral toxicity. As apolysaccharide from natural resources and excellent in biocompatibility, starch has adistinctive molecular structure and is easy to modification which can endue starch moleculemore combining site with DNA and can form three dimensional structure to protect DNA andadjust the release of DNA. Therefore, the construction of a new starch non-viralvector-mediated gene delivery system will have very academic significance and applicationvalue.According to the combination mechanism between starch and gene molecule,quaternary-ammonium cationic starches with different molecular weight and different cationiccharge for non-viral gene delivery carrier were designed through enzymolysis method and drycation-modify method. The molecular weight and molecular weight distribution of thecationic starch non-viral gene delivery carrier were determined by gel-permeationchromatography-multi angle light scattering method and the introduced cation groups to thestarch molecule were analysised by Fourier Transform Infrared Spectroscopy. The resultsshowed that the degree of quaternary-ammonium cationic substitution increased as theamount of starch, ratio between alkali and etherifying agent and reaction time increased andthen decreased as these factors further increased. The FTIR spectra of cationic starch arecharacterized by the three important bands: a C-N vibration band of quaternary ammoniumgroup at1385cm^-1, C-H bending vibration band of CH3, CH2in quaternary ammonium groupat1489cm^-1, and a antisymmetry stretching vibration band of quaternary ammonium group at1602cm^-1which showed quaternary ammonium group was successed introduced to the starchmolecule. The weight average molecular mass of all the cationic starches was from1.055×104 g/mol to5.527×104g/mol. Moreover, the particle size and zeta-potential of cationic starchgene delivery carriers with different DS and different molecular weight were investigated byusing dynamic light scattering method at different pH. The results showed that the particlesize and zeta-potential of cationic starch gene delivery carriers were distinctly affected by thepH change of the simulated endosome condition. In acidity environment, cationic starch genedelivery carriers showed the largest mean size about500nm at pH4.0and the smallest meansize about100nm at pH7.0. Cationic starches showed positive charge at the simulatedendosome condition with different pH which ensure the cationic starch and gene molecule cancombine through the electrostatic interaction.High purified plasmid DNA （pAcGFP1-C1） extracted via kit method was selected as themodel gene and was interacted with cationic starch of different structural characteristics toprepare cationic starch/DNA complex delivery system. The effect of pH of the simulatedendosome condition and N/P ratio between cationic starch and DNA on the performance ofcationic starch/DNA complex delivery system was studied. The results showed that cationicstarch and DNA interacted closely as the N/P ratio rised and cationic starch/DNA complexdelivery system could stay stable at a lower pH environment. The interaction between cationicstarch and DNA was also related to the zeta-potential of cationic starch solution and as thezeta-potential increased the interaction between cationic starch and DNA became closer. Theaverage mean size of cationic starch/DNA complex delivery systems was below300nm bylaserlight scattering analysis and the mean size of cationic starch/DNA complex deliverysystem decreased as the zeta-potential increased. Observed from the AFM and TEM, cationicstarch/DNA complex delivery systems were nonapaticles with different particular morphologylike regular sphere, irregular polyhedron or sphere particle with one or more protrusion atvarious N/P ratios which revealed the relationship between the molecular state and number ofcationic starch in solution and the morphous and property of cationic starch/DNA complexes.Furthermore, the effect of molecular weight, the degree of cationic substitution and the N/Pratio on the protection of DNA and the stability of cationic starch/DNA complex deliverysystem in phosphate buffer and lysozyme solution was investagated. Afer the treatment ofstrong anion reagent heparin sodium to let cationic starch/DNA complexes release DNA, theresults of DNA migration retardation from the agarose gel electrophoresis showed that the cationic starch/DNA complex delivery systems were more compacted and the DNA moleculeswere well procted by cationic starch as the molecular weight of the cationic starch increased.Meanwhile, as the molecular weight and the degree of cationic groups increased the DNase Iand lysozyme resistance of cationic starch/DNA complex delivery systems were stronger andthe cationic starch/DNA complex nano-delivery systems were more stable and more difficultto aggregate in simulated body fulids.This research focused on the key scientific problems of the construction of relatednon-viral gene delivery systems. According to the molecular structure, charge status and theelectrostatic interaction with DNA of cationic starch, the starch molecular weight and thedegree of cationic groups were controlled and the cationic starch/DNA complex nano-deliverysystems with different structure were established. The results confirmed that the effect ofmolecular weight, DS of cationic starch, pH of simulated endosome condition and the N/Pratios between cationic starch and DNA on the DNA loaded performance, average mean sizeand the stability of the cationic starch/DNA complex nano-delivery system. This work willnot only contribute to the development of starch-based non-viral gene delivery carriers forhigh gene transmission but also offer technical support to develop non-viral nano-deliverysystem for the requitments of gene therapy.