(596ax) PEI/Nucleic Acid Polyplex Preparation for the Optimized Size, Gene Transfection, and Cytotoxicity: Implications in Nonviral Gene Delivery

The “bottom-up” approach of biological nanostructure fabrication has been commonly utilized in the development of non-viral gene carrier systems. The design and use of self-assembling molecules has created a library of different polymeric gene delivery systems that has seen varied levels of successes both in vitro and in vivo.  Among these, polyethyleneimine (PEI) is a highly cationic polymer that holds a prominent position in the area of nonviral gene delivery, lending to its exceptional ability to complex nucleic acids via electrostatic interactions and promoting endosomal escape inside the cell. Unfortunately, the use of PEI polyplexes has produced large variations in transfection efficiencies and toxicities depending on numerous variables such as cell type, presence of serum, incubation duration and polymer to nucleic acid ratio (represented by amines in the polymer to phosphates in nucleic acids, N/P ratios), which have been well documented. Contrarily, formulations of these PEI/nucleic acid polyplexes may also play important roles in both the stability and transfection efficiencies; and to our best knowledge, remain unclear. Polyplex formulations are generally controlled kinetically by numerous variables ranging from extensive pipetting, vortexing, and sonication, followed by a period of incubation. The complexation behavior of polyplexes is dependent on several major characteristics such as molecular weight, charge density, structure of the polymer, ionic strength of buffers, and N/P ratios. In addition, we found that the sequence of addition of components during polyplex formation significantly influences the resulting polyplex size as well as the transfection efficiency.

            In this study, reversing the adding sequence of DNA and PEI was observed to result in significant difference in polyplex formation. It was found that adding PEI to DNA solution (PtoD) exhibited much larger polyplexes than those formed via adding DNA to PEI solution (DtoP) polyplexes. It was hypothesized that a single copy of DNA is complexed into a polyplex when DNA is added to PEI solutions, whereas multiple copies of DNA is incorporated with PEI in the opposite mixing order. PEI/DNA polyplexes prepared by two different mixing orders were incubated with NIH 3T3 cells to assess the transfection efficiency and cell viability. The transfection rate of the cells incubated with the polyplexes prepared by PtoD formulation was slightly higher than that those prepared by DtoP formulation. However, a significant increase in gene expression level was observed with the cells incubated with the polyplexes prepared by PtoD formulation, which implicates the hypothesis of varying copy numbers of complexed DNA in the polyplexes via different formulations. Further polyplex characterizations using HPLC, TEM, and AFM will also be presented.