Construction And Evaluation Of Bionic Gene Delivery System Based On Cell Membrane Coating

As a new therapeutic strategy,gene therapy has been widely studied and concerned for its remarkable therapeutic effect on gene-related diseases.However,the lack of safe and efficient gene vectors has been one of the main bottlenecks restricting the development of gene therapy.Viral vector has the advantages of large loading capacity and high gene transfection efficiency,but its clinical application is limited due to severe immunogenicity and tumorigenicity.Non-viral vectors have advantages such as low toxicity and low immunogenicity and have more potential.Polyethylenimine(PEI)is an outstanding representative of non-viral gene vector.Thanks to the abundant amino content,PEI shows excellent DNA aggregation ability and significant “proton sponge” effect.However,the excessive positive charge on PEI surface makes the PEI/DNA complexes susceptible to electrostatic interactions with electronegative biomolecules in vivo,leads to poor stability in vivo,low efficiency of gene delivery,and cationic cytotoxicity.To solve this problem,the existing methods are mainly grafting strategy.The toxicity of PEI can be reduced by grafting PEI to neutral molecules,but part of the amino group of PEI can be consumed in this process,which reduces its electrostatic binding ability to DNA and reduces the efficiency of gene delivery.Therefore,based on the principle of bionics,we proposed a cell membrane “coating” strategy derived from the concept of “learning from nature”.In view of the high toxicity and non-targeting of PEI as a gene vector,the surface of PEI/DNA complexes was coated by using the cell membrane to prepare the CM/PEI/DNA capsule(CPDc for short)bionic gene delivery system,so as to achieve safe gene delivery;at the same time,the vector is “coated” by the cell membrane homologous with the target cell to achieve targeted gene delivery.According to the above ideas,this project attempts to construct and evaluate the gene delivery system based on the cell membrane “coating”,the specific contents are as follows:1.The cytotoxicity of PEI/DNA complexes was reduced and the gene delivery efficiency was improved by cell membrane coating strategy.A series of293T-CM/PEI30k/DNA capsule(293T-CP30 Dc for short)and 293T-CM/PEI70k/DNA capsule(293T-CP70 Dc for short)complexes with mass ratio gradients were synthesized by coating the PEI30k/DNA and PEI70k/DNA complexes with different masses of 293 T cell membranes.The characterization results showed that the complex exhibited a “core-shell”structure of about 200 nm,which proved its successful synthesis.The cell membrane has a high encapsulation rate for PEI/DNA complexes and can protect DNA from nuclease hydrolysis.Safety results showed that the safety of 293T-CP30 Dc and 293T-CP70 Dc complexes was significantly improved compared with PEI/DNA complexes.The efficiency of cell uptake was effectively improved by cell membrane “coating”,when the mass ratio was 2/0.75/1,the uptake efficiency of 293T-CP70 Dc complexes was the highest(93.4%),which was much higher than the PEI70k/DNA complexes;at the same time,293T-CP70 Dc complexes showed the highest transfection efficiency at 76.2%,which was about 10%higher than PEI70k/DNA complexes.These results indicate that the 293T-CP70 Dc complexes has the potential to be developed as a safe and efficient non-viral gene vector.2.By using the cell membrane coated PEI70k/DNA complexes homologous to the target cell,gene targeted delivery was achieved.The 293T-CP70 Dc 、Hep G2-CM/PEI70k/DNA capsule(Hep G2-CP70 Dc for short)and He La-CM/PEI70k/DNA capsule(He La-CP70 Dc for short)complexes with the same mass ratio were prepared by extrusion using 293 T 、 Hep G2 and He La cell membranes.In vitro cell uptake and transfection results showed that all three complexes exhibited the highest uptake and transfection efficiency in homologous cells,but relatively low uptake and transfection efficiency in heterologous cells.Safety results showed that the three compounds exhibited low levels of cytotoxicity in all three types of cells,and showed a certain degree of safety advantage in their homologous cells.In addition,Extracellular matrix(ECM)of three types of cells were extracted to investigate their effects on complex targeting.The results showed that the cell uptake and transfection efficiency of the three complexes in the homologous cells were further improved compared with the other two heterologous cells after ECM was added.In conclusion,the PEI/DNA complexes “coated” with cell membrane is expected to be developed as a safe and efficient gene delivery system with targeted advantages.