Polymer Modified Silicon Nanowire Arrays Assisted Gene Delivery

Gene therapy,a gene level technique which delivers exogenous genes/plasmids into diseased cells to exert a therapeutic effect,has opened up a whole bright perspective for the treatment of many difficult-to-cure diseases.The key step in gene therapy is how to deliver genes into target cells safely and effectively.Traditional transfection methods rely mostly on solution-mediated gene delivery,which greatly limits the efficiency of gene transfection,because genes are less efficient to enter cells by endocytosis and easily degraded.Surface-mediated gene delivery method,compared with conventional transfection methods,can effectively transfer the plasmids into target cells due to its long-term stability and in situ localization.Among many biomaterials,vertical aligned silicon nanowire arrays(SiNWAs,SN)have received great attention due to their good biocompatibility.Lots of studies have found that SN of appropriate length can pierce cell membranes without affecting cell viability.Utilizing this property of SN,foreign biomolecules such as peptides,DNA and RNA etc.can be transferred into cells to exert their effects.However,an oxide layer existing on the surface of SN limits the amount of DNA loading on SN.In order to improve the transfection efficiency of SN,we chemically modified the surface of SN,and meanwhile supplemented with several means that could increase DNA loading ability and promote gene expression.The main studies are as follows:1.We grafted 6-O-sulfonated chitosan on the surface of SN and combined with Ca2+ which can interact with DNA to form nanoparticles to improve DNA loading efficiency and gene transfection efficiency.In this transfection system,we made full use of the ability of SN to penetrate cell membrane and the advantages of Ca2+-related transfection to achieve efficient transfection of HeLa cells.Meanwhile,the good biocompatibility of sulfonated chitosan also makes the system less cytotoxic.We explored the factors affecting the transfection efficiency of the system and found that the transfection efficiency was the highest when the grafting concentration of sulfonated chitosan was 1 mg/mL,the concentration of Ca2+ was 100 mM and the complexing time for Ca2+and DNA was 10 min.Although this transfection method can improve SN transfection efficiency significantly while maintaining low cytotoxicity,there are still some problems to be solved,such as the DNA loading efficiency is low and the transfection effect on GFP is unsatisfactory2.In order to construct a safer and more efficient transfection system,and increase the efficiency of plasmid expression in the nuclei,we prepared a surface with plenty of amino groups by modifying SN with ethanolamine-functionalized poly(glycidyl methacrylate)(PGEA).At the same time,based on this surface,the Ca2+ transfection system supplymented with a small amount of Zn2+ was introduced.Due to the addition of Zn2+,on the one hand,the particle size of the complex formed by Ca2+and DNA(Ca2+@DNA)is well controlled and the stability of the nanoparticle is improved;on the other hand,Zn2+,a significant trace element in living organisms which plays an important role in metabolism and growth,can effectively promote gene expression.The results indicate that the addition of Zn2+could dramatically improve the transfection efficiency by as much as 7-fold.This is mainly because the addition of Zn2+ can effectively increase the release efficiency of the plasmid in the nucleus.Meanwhile,since each component in this transfection system has good biocompatibility,the whole system exhibits low cytotoxicity.In addition,we studied the factors affecting the transfection efficiency of the system.The experimental results showed that when the concentration of Zn2+is 0.2 mM,the concentration of Ca2+ is 110 mM and the surface grafting time is 12 h,the transfection efficiency of the system is the highest.This new surface-mediated gene delivery method is much more efficient and safer and will have great potential in the field of medical therapy3.To explore the effect of polymer-modified SN on protein delivery,the mutated inorganic pyrophosphatase(PPase)was used as a model protein to study the adsorption and release behavior of proteins on pH-responsive surfaces.pH-responsive polymer poly[N,N-(dimethylamino)ethyl methacrylate](PDMAEMA)was grafted onto the surface of SN to prepare a pH-responsive surface(SN-PDMAEMA)by surface-initiated atom transfer radical polymerization(SI-ATRP),and PPase was combined with polymethacrylic acid(PMAA)by disulfide bond to form a protein conjugate,PPase-PMAA.The electrostatic interaction between the polymers and the hydrophobic interaction between the molecules can be used to control the interaction between SN-PDMAEMA and PPase-PMAA by adjusting the pH of the system to change the charge of PMAA and PDMAEMA.The water contact angles,SEM and TEM were used to characterize the surface property and the results indicated the success of surface modification.Adsorption of PPase-PMAA on SN-PDMAEMA at different pH was tested to find out the optimum pH.These biomaterials which utilize interaction between polymer chains to realize the regulation of protein adsorption and release have broad application prospects in the control of protein binding on biomaterial surfaces.In summary,by comprehensively utilizing the ability of SN to penetrate cell membranes and the different physicochemical properties of polymers,grafting different polymers on the surface of SN can endow modified SN with different biological functions to realize the effective delivery and release of different exogenous biomolecules such as DNA,RNA and proteins.It is believed that with the in-depth study of SN and the continuous development of surface modification technology,drug delivery/release based on modified SN will make more progress in the field of disease treatment.