Construction Of Glutathione-Sensitive Surface-Mediated Gene Delivery System

Gene therapy has been widely studied for the treatment of various incurable diseases,especially for congenital genetic diseases and tumors.This special therapy obtains the curative effect by repairing genetic defects in human body,in which the edited gene molecules are delivered to target cells via vectors.Therefore,accurate and efficient gene delivery to target cells is a key part of the therapy.Over the last decades,the continuous innovation of viral and non-viral vectors holds promise for the safety and high efficiency of gene delivery.However,due to the vectors are susceptible to the proteins in the serum or cleared by the immune system,the traditional in vivo delivery methods often lead to low gene concentration at the focus which results in a significant reduction in clinical effects.Currently,surface-mediated gene transfection is used as an effective way to address this issue.The special topology and abundant chemical modification strategies of the material surface give more choice for the gene delivery.Moreover,compared with the conventional solution transfection,surface-mediated gene transfection has the following advantages:(1)it can be combined with biomedical materials to achieve in situ gene therapy at the focus;(2)it can help high-throughput cell transfection in vilro.In recent years,the general cell engineering platforms with versatile functions,including surface-mediated transfection,cell harvesting and surface recycling have been favored by researchers.In this work,we designed a series of glutathione-sensitive surface-mediated platforms for high efficient gene transfection based on the intracellular high-concentration reduced glutathione(GSH)environment.This work provides a brand new idea for the combination of surface modification strategy and surface topology.Specific studies are as follows:1.Silicon nanowire arrays(SiNWAs)can achieve intimate,non-toxic interactions with mammalian cells.Simultaneously,the unique one-dimensional nanostructure of SiNWAs,silicon nanowire,is able to partially penetrate into cells or even the nuclei.This property is of great value for the gene delivery of hard-transfected cells.It has been reported that the cationic polymer modified SiNWAs can effectively increase the loading of plasmids and improve the transfection efficiency.However,in these cases,the long-term cell toxicity of cationic polymers and the length of modified SiNWAs are not considered.Building on the above problems,in the first part,we prepared a series of SiNWAs modified by cationic polymers.Among them,the disulfide-linked PEI not only had lower cytotoxicity,but also could be triggered by the high concentration of GSH in the cytoplasm.Beyond that,we explored the effect of SiNWAs length on cell activity.The results showed that the surface of SiNWAs in a certain length range could maintain good cell activity,except that the SiNWAs with large length was easy to break during the modification process.2.S-S bonds can be triggered in response to the high concentration of GSH in the cytoplasm.This intelligent responsiveness is widely used in the design of drug delivery and non-viral vectors.However,limited by the planar structure of the material surface,the glutathione-sensitive surface-mediated gene transfection is rare reported.In the second part,we creatively combined the cell pierced properties of SiNWAs with degradable S-S bonds to prepare an intelligent cell engineering platform(SN-PEICBA)that responded to the intracellular GSH environment for efficient transfection and rapid cell harvesting.The platform was able to achieve high efficient gene transfection for a variety of cells,including mouse embryonic fibroblasts,in the presence or absence of serum.Cells harvested from SN-PEICBA showed high viability and the platform surface can be recycled within three cycles by S-S bonds replacement.This platform had great potential in assisting in vitro cell therapy and other biomedical applications.3.In the third part of the work,we prepared a series of disulfide-linked PNIPAm nanogels.It is found that the suitable size of the nanogels can form a stable modified surface,which can change the cell adhesion and desorption before and after the break of the S-S bonds inside the nanogels,and construct a general coating which can quickly respond to the GSH environment in vitro to realize the efficient collection of transfected cells.