Physiological and microdevice effects on electric field and gene delivery in electroporation.

摘要

Gene therapy protocols have been actively seeking new gene delivery techniques for two decades. Successful gene therapy has carried our hope for a cure to thousands of diseases that currently plague mankind including Parkinson's, Alzheimer's, Diabetes, and even cancer. Much effort has been put into using viral vectors to deliver genes and drugs, and laboratory studies had shown viral vectors to be extremely efficient as a gene delivery technique, however new delivery techniques including electroporation are being sought to overcome some safety concerns associated with viral methods.;Electroporation has predominantly been an in vitro delivery method. The main advantage of electroporation is in its ability to process mass quantities of cells in a very short time frame, however it has low cell viability and transfection efficiency. The main problems that hinder the use of electroporation in gene therapy are the use of very high electric fields (>1000V/cm) and non-uniform electric field distribution among cells leading to low cell viability as well as an incompatibility of this process with cell lines important to gene therapy protocols such as stem cells.;We used a novel approach that employs an optical tweezers with a fluidic electroporation chip and propidium iodide dye to study the relationships between cell size and membrane breakdown, as well as cell-cell interactions and their effects on the electroporation process. Experimental results showed that each cell line has a characteristic critical electric field at which the cell begins to electroporate, and it is not dependent on the cell size. We were also able to show through both modeling and experimental results that cell interactions can significantly enhance or reduce the electric field and have shielding effects in certain orientations that may interfere with gene or drug delivery.;Next, we studied a microdevice that used a single micropore to trap and electroporate a cell through electric field focusing effects. A relationship between pore size and electric field using both experimental and modeling techniques, as well as possible delivery mechanisms in micropore electroporation were estabished. Based on experimental results we found that micropore electroporation delivery is controlled by diffusion processes with enhancements from the electric field focusing effects.;The micropore electroporation device was expanded into an array of micropores to study its ability to deliver genes to mass numbers of cells. Both cleanroom and non-cleanroom fabrication methods were compared. We found that the micropore array was able to provide enhanced uniformity, reduced the electric field, and achived a high transfection efficiency through pGFP reporter gene transfection analysis when compared with bulk electroporation.;A new electroporation technique called membrane sandwich electroporation (MSE) was developed and tested using reporter genes pSEAP and pGFP. This microdevice used two porous membranes to achieve a lower electric field that enhanced cell viability, and provided gene confinement near the cells to enhance gene delivery. Transfection results indicated an improvement over bulk and single membrane systems. MSE membranes are commercially available on a mass scale and can provide a more economically viable approach to gene delivery microdevices.