Effects Of The Nanostucture Changes Of Starch-based Gene Carriers/pDNA Complexes On Their Gene Transfection Properties

One of the key issues in gene therapy is to obtain effective gene carriers suitable for genetansfection. An ideal starch based gene carrier should have suitable binding capacity withgene and can form complex with certain aggregation structure which is responsive to thecellular environment. All these properties can be determined by the chemical structure,molecular molar mass and aggregation behavior of the molecular chain of the gene carrier.Besides, the degree of responsiveness to environmental changes of the aggregation structureof modified starch formed through helix can be controlled by starch modification whichmakes it feasible to regulate the structure of modified starch/gene complexes by adjusting thechain structure of starch molecule and its aggregation structure, thus achieving controlledgene release and high gene transfection efficiency.To meet the demand of intracellular gene trafficking and transfection, starch moleculewith different chain length, molecular size, charge and aggregation structure were obtained bydegradation and the introduction of cationic amino groups. Specifically, acidolysis, dry-heatdegradation and enzymolysis were applied to degrade starch molecules. Afterwards, starchbased gene carriers with different molar mass, various pH buffering capacity and goodbiocompatibility were selected for further study. Small Angle Light Scattering (SAXS)analysis revealed that the internal orderly aggregates of all the selected starch-based genecarriers displayed the mass fractal structure. Besides, the internal orderly aggregates instarch-based gene carrier with the smallest molar mass was determined to be dumbbell-shapedwhile others’were all almost oblate ellipsoid with small subunits and the RMS radius (rg) andmaximum geometric dimension (Dmax) went up as the molar mass (Mw) of starch based genecarriers increased,Plasmid pAcGFP1-C1（pDNA）which is able to express GFP was chosen as the modelgene material and formed complexes with starch based gene carriers with different molarmass and DS of cationic amino groups. By simulating the intracellular pH changes, theparticles size, zeta potential of starch/pDNA complexes and the shape of internal orderlyaggregates were investigated. The effect of molecular structure of starch based gene carriersand the structural changes (including the particle size, charge and shape etc) of the complexeson the transfection efficiency of HepG2cells were studied by using inverted flurescencemicroscopy and flow cytometry. Results indicated that complex formed by M2-DS3starchbased gene carrier showed the highest gene transfection efficiency. The average particle sizeof the complex was180.6nm, the zeta potential was5.48mV, and the shape of the internal orderly aggregates was globular. Starch-based gene carriers with a small molar mass were lesseffective in intertwining pDNA and the exposure of pDNA would lead to low transfectionefficiency. While as the molar mass of starch-based gene carrier was higher, the interactionbetween starch and pDNA became stronger that the structure of the complex was too compactto release pDNA, and the transfection result was unsatisfactory as well. It was concluded thatstarch-based gene carriers with suitable molar mass could interact with pDNA effectively andthe structure of the complexes with suitable aggragation structure was beneficial for pDNAreleasing which resulted in high gene transfection efficiency.This study paid special attention to the responsiveness of structural changes in starchmolecules and the interaction between starch and pDNA towards the cellular environmentalchanges during endocytosis. Starch/pDNA complexes with various structural properties forgene delivery system were developed by adjusting the molecular molar mass, DS of cationicamino groups and the weight ratios between starch and pDNA. Furthermore, the relationshipbetween the gene transfection efficiency and the molecular structure of starch-based genecarriers as well as the nano-structural changes of starch/pDNA complexes under pH changesduring the endocytosis were established. Starch-based gene carriers with substantial genetransfection efficacy were obtained and all these results will promote the potential applicationof starch-based gene carriers in gene therapy.