Efficient transfer of reporter gene-loaded nanoparticles to bone marrow stromal cells (D1) by reverse transfection.

Nucleic acids can be complexed with cationic polymer to form DNA nanoparticles (polyplex) which are then immobilized on the surface coated extracellular matrix protein (ECM), the process termed as reverse transfection. ECM-containing proteins provide a surface for cell attachment and sustain the release of polyplexes from their surface, thereby inducing transgene expression for prolonged period of time. Consequently, long-term expression of the desired protein can be achieved with the smaller amount of required DNA, as compared to bolus delivery. First of all, we investigated the different ECM components as a coating material and the range of optimal coating density in different ECM was examined for enhanced transfection to neighboring cells. Reporter genes such as luciferase (luc) and enhanced green fluorescent protein (eGFP) were initially used to quantitate transfection efficiencies from polyplex from the coated ECMs of Collagen type I (Col I), fetal bovine serum protein (FBS), bovine serum albumin (BSA). DNA was complexed with positively charged polyethyleneimine (PEI) at N/P ratio 9. Our initial work exhibited that, in the case of both NIH/3T3 cell line and bone marrow stromal (D1) cell line, Col I facilitated the greatest cell adhesion compared to the other coating proteins and 0.5 microg/cm2 of Col I coating density resulted in highest transfection efficiency. On the other hand, comparison of reverse delivery system with atelocollagen-I have shown that reverse delivery system to yield ten times higher transfection efficiency than atelocollagen-PEI/DNA delivery system and one hundred times higher than atelocollagen-naked plasmid delivery system. Moreover, the amount of DNA used for reverse delivery system was much lower than the other systems. This methodology would be applied to induce cellular differentiation in 3-dimensional scaffold after coating scaffolds with genes inducing the differentiation in the nanoparticle formulation. Our final goal is to search for the optimal conditions for the differentiation of stem cells to specific cell types.