Preparation Of Functional Generation Five Dendrimer-Entrapped Gold Nanoparticles For Gene Delivery Applications

With the increase of the incidence of cancer and genetic diseases, the gene therapy of cancer and genetic diseases aroused more and more attention. Gene therapy is the technology to transfer exogenous normal gene into the target cells to correct or compensate disease caused by genetic defects and anomalies, so as to achieve the aim of treating disease. The key of gene therapy is to find carriers that can deliver therapeutic genes into target cells safely and efficiently. The vectors of gene transfection can be divided into viral vectors and non-viral vectors. Although viral vectors own high transfection efficiency, their high immunogenicity, carcinogenic and cell toxicity limits their application in gene therapy in vivo. In recent years, researchers set out to explore the use of non-viral vectors for gene therapy applications.In this study, we report two types of new non-viral gene delivery vectors based on amine-terminated generation5poly (amidoamine)(PAMAM) dendrimers (G5.NH2):partially acetylated dendrimer-entrapped gold nanoparticles (Au DENPs-Ac) and partially PEGylated dendrimer-entrapped gold nanoparticles (Au DENPs-PEG). The characterization of the vectors was carried out by1H NMR, UV-Vis spectrometry and TEM. To evaluate the ability of different vectors to condense pDNA, siRNA, ssDNA at different N/P ratios, agarose gel retardation assay was performed. DLS and zeta potential measurements were used to characterize the formed polyplexes. For pDNA gene delivery, the expression of luciferase (Luc) and EGFP gene was selected. For siRNA gene delivery, western blot analysis technology was used to evaluate if the expression of Bcl-2protein was disturbed by Bcl-2-siRNA. For ssDNA gene delivery, we evaluated the gene delivery efficiency by the change of expression of GFP protein in Hela cells. Confocal microscopy and flow cytometry technology were also applied to study intracellular uptake capacity of the polyplexes.The results indicated that with the comparable gene delivery efficiency to the non-acetylated Au DENPs and the less cytotoxicity, the partially acetylated Au DENPs should hold great promise for a variety of different gene delivery applications. PEGylated Au DENPs are able to condense pDNA, siRNA and ssDNA to form polyplexes, which is desirable for gene delivery applications. Under the same Au salt/dendrimer molar ratio, methoxy-terminated polyethylene glycol with lower molecular weight (mPEG, molecular weight is2000)-modified Au DENPs have a higher pDNA delivery efficiency than higher molecular weight of mPEG (molecular weight is5000)-modified Au DENPs. Different gene delivery efficiency assays for nucleic acid (pDNA and siRNA) showed [(Au0)50-G5.NH2-mPEG2k10] could be used as a new non-viral vector for gene therapy at the N/P ratio of5. What is more, PEGylated Au DENPs also have potential application value for ssDNA delivery. The research laid a solid foundation for the application of the functional Au DENPs in the field of gene delivery.