Cationic Polymers Modified SN For Gene Transfection

Gene transfection has been used to treat or alleviate a variety of diseases, and has become an indispensable medical treatment in clinical and basic research. The key factor in the success of gene transfection is to develop much safer and more effective gene vectors and transfection methods in order to directly introduce the nucleic acids into the nucleus and acquire high expression, which is also the bottleneck of gene transfection technology. Non-viral gene vectors can improve the efficiency and security of gene transfection to a certain extent, among which the cationic polymers have many advantages, such as easy synthesis and being easily modified to carry more DNA into the cells and so on, therefore they have been widely used as gene vectors. However, cationic polymers cannot assist DNA directly get into the nuclei, the transfection efficiency cannot be further improved. Meanwhile, the cationic polymers tend to aggregate in vivo which could affect the normal cellular metabolism and thus induce high cytotoxicity. These disadvantages strongly limit their use in the clinic trials. The silicon nanowire arrays(SN) can penetrate cell membrane, in particular the cell nuclei, while maintaining the normal cell viability so that it can carry many biological effectors into cells and the cell nuclei. SN also has good biocompatibility and can be used to support a variety of cells. However, the transfection efficiency of SN alone is quiet low caused by the silicon oxide layer on unmodified SN and the restricted DNA loading capacity, which limit its application in gene transfection.This thesis proposes a novel gene transfection strategy by using cationic polymers modified SN as material platforms for gene transfection combining with approaches which can improve the DNA loading capacity and expression efficiency, aiming at the comprehensive utilization of the direct penetration of SN to cell nuclei and efficient DNA loading and release ability of cationic polymers to get enhanced transfection efficiency while minimizing cytotoxicity. The detailed works are introduced as follows.Firstly, a series of cationic polymers modified SN were constructed: SN was modified with branched polyethyleneimine(PEI, 25 k Da) and poly(dimethylaminoethyl methacrylate)(PDMAEMA) by chemical modification. To further increase the DNA loading and release ability of the surface and achieve cells adhesion and detachment, poly(N- isopropylacrylamide)(PNIPAAm) segment was introduced to PDMAEMA modified SN(SN-PDM). The water contact angles, scanning electron microscopy(SEM), transmission electron microscopy(TEM) and so on were used to characterize the surface and prove the success of the modification. Besides, the influence of these platforms on cell growth was further investigated, the results demonstrated that more cells were attached on SN-PEI(PEI modified SN) and SN-PDM, cells on SN-PDM can maintain normal morphology and cell proliferation, indicating the biocompatibility of SN was improved after modifying with PEI and PDMAEMA and can be used as a efficient platform for gene transfection. The cells on Poly(N-isopropylacrylamide-codiethylaminoethyl methacrylate)(PNIPAAm-co-PDM) modified SN(SN-PNIPAAmPDM) cannot maintain good viability which makes it not suitable to be used as a platform for gene transfection.Based on these, we proposed a novel transfection system involving the use of efficient nucleic acid vector PEI modified silicon nanowire arrays as a platform, supplemented with the complexes of biocompatible PEI(2 k Da) and DNA to increase DNA loading capacity of the surface and promote the efficiency and safety of gene transfection. The impact of SN-PEI surface amino density, the incubation time of DNA on SN-PEI and N/P ratio(the molar ratio of PEI nitrogen to DNA phosphate) for free PEIDNA complexes made of low-molecular-weight b PEI on gene transfection efficiency were investigated in detail, and the results demonstrated that transfection efficiency was optimized on SN-PEI with a PEI grafting time of 3 h, an incubation time of 10 min for tethered PEI-DNA complexes consisting of high-molecular-weight b PEI grafted onto SN, and with an N/P ratio of 80 for free PEI-DNA complexes made of low-molecular-weight b PEI. This strategy effectively improve the transfection efficiency and lower cytotoxicity of the system, but there are some problems to be solved, such as the potential cytotoxic caused by high molecular weight PEI and also the DNA loading capacity is not sufficient needing an extra amount of free PEI-DNA complexesIn order to build a more efficient and safer gene transfection system, we further proposed a transfection system using PDMAEMA modified SN as a platform, combined with the traditional calcium dependent transfection system, in order to make full use of the advantages of the platform and calcium dependent transfection system to get feasible and effective gene transfection. PDMAEMA has better biocompatibility than PEI, traditional calcium dependent transfection system can promote the intracellular release and expression of DNA. The effects of calcium concentration, the polymerization time of PDMAEMA on SN, the incubation time of Ca2+@DNA(the mixture of calcium and DNA) on SN-PDM on the transfection efficiency were also investigated,the results show that a complex containing Ca2+(100 m M) and DNA incubated on SN-PDM with a polymerization time of 24 h for 20 min exhibited the highest transfection and gene expression. This integrated strategy provides a new idea for gene delivery and is expected to be widely used in medical research and clinical treatment.In summary, cationic polymers(efficient gene vector, PEI and PDMAEMA) modified biocompatible SN which can penetrate cell nuclei directly are efficient platform for gene transfection. The platform can not only supports the adhesion and proliferation of cells, but also assist a variety of traditional gene transfection and ultimately achieve efficient, integrated and safe gene transfection and expression. Such hybrid gene transfection system using cationic polymer modified SN as a platform is expected to be helpful to provide efficient platform and new ideas for the construction of new gene transfection materials and drug delivery systems.