Study Of G5.NH₂-Grafted Electrospun PLGA Nanofibers Substrate-Mediated Gene Delivery Systems

As a new treatment of disease,Gene therapy is by manipulating the genetic materials to interfere with the development of disease.However,the main obstacle of gene therapy is lack of safe and efficient carriers.Because naked gene therapy drugs?specific expression plasmid DNA?are susceptible to nuclease degradation in tissues or cells.the repulsion between the negative charge of pDNA and the electronegativity of the cell membrane,leading to endocytosis with the cells and the poor ability to escape from endosomes.As a consequence,it is difficult see the effect of gene therapy.Electrospinning can continuous prepare superfine fiber efficiently,using electrospinning method to produce electrospun matrix with the uniform porous structure and high specific surface area give its unique 3D structure can well mimic the natural extracellular matrix,provide a good microenvironment for cells to exchange of nutrients and reproductive.In the past,the universal method of electrospinning technology to make gene carrier is mixing the biocompatible polymer solution and gene drug directly with spinning,gene drugs must be exposed to high voltage electric field and organic solvent environment during spinning process,which is bound to have some impact on the biological activity of gene drugs.In addition,pure blended the drug in the electrospun fiber carrier with spinning may cuase a burst effect when in vitro release,due to delivery of drug is not well distributed and encapsulated.Dendrimer is one of the new nanoscale functional polymers with high branching,symmetry and radial.Because of its high positive charge density,it can effectively compress gene therapy drugs and has strong proton buffering capacity in cell connotations,which has been used as a typical carrier of non viral gene delivery.Considering the problem existing in electrostatic mediated gene transfer system,combined with dendrimers as gene carriers,this experiment firstly proposed the N-terminal fifth generation polyamidoamine dendrimers?G5.NH2?immobilized on poly?lactic-co-glycolic acid??PLGA?nanofiber surface to in situ compression enhanced green fluorescent protein?EGFP?reporter gene,and to fix the nano G5.NH2/pEGFP composite particles to fiber matrix as scaffolds,constructing an in situ transfection,local composite gene delivery system.First of all,this paper adopts self-restraint electrostatic spinning device to research the influence of PLGA concentration and spinning velocity on the properties of nanofiber,when the concentration of PLGA was 25 wt%,spinning velocity was 1 ml/h and the voltage of 20kV that can spin out the fibers of uniform thickness,smaller diameter and considerable morphology.Then,via the layer by layer self-assembled?LBL?method,immobilized G5.NH2 on the surface of PLGA nanofibers to form a fiber based composite gene delivery system.Scanning electron microscope?SEM?showed that the surface morphology of the fiber and the three-dimensional porous structure did not change obviously,but the average fiber diameter increased;X ray photoelectron spectroscopy?XPS?confirmed that after the modification of G5.NH2,obvious characteristic peaks of nitrogen appeared.The appearance of nitrogen showed that G5.NH2 was successfully modified to the surface of fibers,and the modification efficiency increased with the increase of the number of PDADMAC/PAA assembly layers;Hydrophobicity test shows that the hydrophilic property of the material is improved obviously after the material is assembled and modified;The subsequent atomic force microscopy?AFM?test showed that the addition of G5.NH₂ could greatly improve the surface roughness of PLGA nanofibers.It also confirmed the improvement of hydrophilic properties of PLGA nanofibers due to the increase of roughness.Finally,we chose fibroblast NIH 3T3 as model cell,and plasmid DNA carrying enhanced green fluorescent protein?EGFP?reporter gene as a model gene drug.To evaluate the in situ gene transfection efficiency of the nanofibers loaded with the model gene.Fluorescence microscopy and flow cytometry were used to qualitatively and quantitatively analyze the transfection efficiency of the composite nanofibrous matrix,which indicated that the PLGA composite nanofibers based on two layers of PDADMAC/PAA had higher gene transfection efficiency,running up to 3.76%.