Biomimetic Non-viral Gene Delivery Systems Fabricated By Supramolecular Assembly

The development of gene technology and the completion of a working draft of the human genome have made the clinical use of gene medicines definitely possible. Gene therapy involves the treatment of genetic or infectious disease by introducing new genetic material to appropriate cells. Non-viral gene complexes fabricated by supramolecular assembly showed great potential in gene therapy due to the suitable nanometer size, controllable structure and excellent biocompatibility. Improving the stability and transfection efficiency of non-viral gene delivery systems is one of the key points. Virus is a kind of nature supramolecular assembly, enclosed infective nucleic acid in the capsid of proteins. The virus could keep stable in extracellular environment and dis-assembly of the protein shell inside the cells. Following the inspiration of viral structure, biomimetic non-viral gene delivery systems were constructed. The relation between the structure of gene assembly and transfection efficiency was also explored.Polyethylenimine (PEI) was common used as non-viral vector due to its proton-sponge effect. But the stability of PEI_25k/DNA complexes was quite poor in physiological condition. PEGylated polyplexes were fabricated to solve the problem. In this research, PEGylated gene assembly was fabricated by adding the PEG-containing amphiphilic polymer, such as poly(ethylene glycol) cholesterol ether (CPEG). It was of interest to find the addition sequence of CPEG have great effect on the stability and transfection efficiency of the gene complexes. The addition of CPEG into PEI_25k/DNA polyplexes had no effect to improve the stability and transfection efficiency. Whereas the PEI_25k/CPEG/DNA polyplexes, which was formed by adding CPEG and PEI_25k mixture to the DNA solution, showed excellent anti-aggregation effect in physiological salt condition and enhanced transfection efficiency. The influence of CPEG addition sequence on stability may be explained by the possibility of entrapping the CPEG into polyplexes. By adding CPEG and PEI_25k mixture to the DNA solution, charge-neutralized hydrophobic core was formed due to the electrostatic attraction between PEI_25k and DNA. At the same time, cholesterol group was entrapped into the polyplexes driven by the hydrophobic interaction between the cholesterol group of CPEG and hydrophobic core. The PEG chain on the surface sterically hindered the complexes from approaching each other to improve thestability of PEI_25k/CPEG/DNA complexes in physiological salt condition. pEGFP was used to evaluate the transfection efficiency of gene delivery. The polyplexes were prepared to incubate for a period of time in physiological condition and then transfected to HEK293T cells. Fluorescence spectroscopy and flow cytometry was used to evaluate the transfection efficiency. With enhanced stability in physiological salt concentration, PEI_25k/CPEG/pEGFP polyplexes showed improved transfection. In conclusion, the PEGylated polyplexes could be constructed to improve the stability and transfection by entrapment of PEG-containing amphiphilic polymer. It provided a simple and facile approach to prepare the PEGylated polyplexes.The contradiction between tight complex formation outside cells and the easy dissociation of complexes inside cells is a big challenge of non-viral vectors research. Virus could keep stable in extracellular environment and dis-assembly inside the cell. Following the inspiration, a series of cross-linked polyethylenimine (CLPEI) was specially designed via the cross-linking reaction between the low molecular weight polyethylenimine (PEI₁₈₀₀) and dimethyl 3.3'-dithiopropionimidate dihydrochloride (DTBP). The disulfide bonds can be broken at the intracellular concentration of the reductive glutathione (GSH). By selecting the proper condition, glutathione-sensitive gene delivery system was successfully fabricated. The result indicated that CLPEI_50% showed best DNA condensation ability. At a pH range from 7.4 to 5, CLPEI_50% still possessed efficient buffer capacity, which was suggested to increase endosomal release of DNA complexes into the cytoplasm. At pH value of 6.0 and NaCl concentration of 20 mM, CLPEI_50%/DNA polyplexes were about 150 nm in diameter. The polyplexes were unpacked due to the cleavage of the disulfide bonds in CLPEI_50% at intracellular GSH concentration. This kind of biomimetic cross-linking gene vector significantly reduced the cytotoxicity and showed effective transfection.Cross-linking of protein macromonomers accompanies the assembly of viral particles, which provides the viruses the high stability in the host. Following the inspiration, caged polyplexes via biomimetic cross-linker were fabricated to improve the stability and transfection. Thiolated PEI (HS-PEI) was synthesized and then complexed with DNA. In situ shell-cross-linking polyplexes were prepared by the oxidation of sulphydryl in aerial condition; Au nanoparticle (AuNP) cross-linked polyplexes (Au-S-PEI/DNA) were also constructed. Ethidium bromide (EtBr) exclusion assay indicated that thiolated PEI showed sufficient DNA condensation ability to protect the bioactivity of DNA at N/P ratio of 10. The mean diameter ofshell-cross-linking polyplexes was about 150 nm at N/P ratio of 10, pH value of 6.0 and NaCl concentration of 20 mM. AuNP particles were obviously observed at the surfaces of Au-S-PEI/DNA polyplexes by TEM pictures. Its content was increased with the addition dosage of AuNP. Caged polyplexes improved the stability in physiological salt concentration, and shown GSH-sensitive property. The in-vitro transfection experiment indicated that by selecting the proper preparation condition, caged non-viral gene delivery systems were successfully constructed with high stability in physiological condition and transfection efficiency.Combining the best current of non-viral and viral vectors, undoubtedly the artificial virus gene delivery systems represented the future of the gene vector for human gene therapy. This study provided some facile approach to prepare the biomimetic non-viral gene delivery systems by supramolecular assembly. It may have great potential in non-viral gene vector research and application. With the development of effective, safe and targeting gene delivery systems, gene therapy will be widely used in clinic, and bring more hopes to human being.