(628b) Nanoparticles Facilitate Gene Delivery in Microorganisms Via An Electrospray Process

Authors: 
Wu, B., Washington University
Lee, Y., Washington University
Chen, D., Washington University
Tang, Y. J., Washington University


Gene delivery, the process of introducing foreign genes into living cells, is a crucial technique in fields such as metabolic engineering and gene therapy. In this study, we developed a novel gene delivery technique, in which electrospray had been employed to introduce plasmid DNAs (pET30a-GFP) into non-competent E. coli cells by nontoxic carriers (e.g., nanoparticles). During the electrospray process, highly charged, monodisperse nano-droplets (a mixture of plasmid DNAs and nanoparticles) were accelerated and uniformly sprayed on a thin layer of bacterial cells which were freshly deposited on a piece of membrane. PCR amplification and restriction enzyme analysis confirmed that pET30a-GFP plasmids had successfully been delivered into the cells. The transformation efficiencies of pET30a-GFP into E. coli were optimized under different electrospray conditions. Comparing the transformation efficiencies using different buffer solutions, CaCl2 (0.01M) was found to be the ideal electrospray solution for gene delivery. Furthermore, gold nanoparticles (NPs) significantly improved plasmid transformation efficiency. For example, when E. coli cells approached the late exponential phase, the transformation efficiency could reach 2×103 cells/ng plasmid, almost tenfold of that in the absence of gold NPs. The transformation efficiency improved with an increase of gold NP size from 20 nm to 50 nm, further increasing NP size to 100 nm could not improve transformation efficiency. Among the tested NP amounts, the optimal transformation efficiency was achieved with a NP amount of 9×105. Electronic microscopy images and gel electrophoresis showed that the morphology of plasmid DNAs remained unchanged during electrospray process. On the other hand, SEM images revealed that the cellular membrane integrity was partially ?damaged? after being electrosprayed with gold NPs, and thus creating channels to allow plasmid DNAs cross cell wall/membrane. Such temporary damage could be repaired after incubation of cells in a rich medium for a few hours. This gene delivery technique can potentially conquer problems which the traditional gene delivery methods cannot. For example, normal cells rather than competent cells are used as plasmid acceptors in the electrospray method, which avoid complicated pretreatment process to prepare competent cells. The novel gene delivery method has its potential to work universally for both bacterial and yeast cells.