Non-Viral Gene Delivery in Human Embryonic Palatal Mesenchymal Stem Cells

Introduction of growth factors into clinical practice has improved the prognosis of complex clinical scenarios but with a high degree of variability. This, in part, is due to a lack of a continual supply of these proteins for a prolonged period of time. One method to overcome this drawback is gene therapy. Objective: To develop and test a non-viral gene delivery system that can be employed to deliver the genes of interest into pre-osteoblasts. Methods: Stable polyethylenimine (PEI) – plasmid DNA (cDNA encoding firefly luciferase reporter gene driven by cytomegalovirus promoter/enhancer) nanoplexes were fabricated using an established protocol. Briefly, PEI in differing amounts, corresponding to various N/P ratios (amine/phosphate) to be tested, was added to the desired pDNA solution. The transfection mixture was then vortexed for 20s and incubated for 30 min at room temperature before adding to the cells. The particles synthesized were then characterized for size and surface charge using Malvern Nano-ZS.  The toxicity and transfection efficiency of the synthesized nanoplexes were evaluated in human embryonic palatal mesenchymal stem cells(HEPM 1486). The effect of N/P ratio on the biocompatibility and transfection efficiency of PEI in HEPM cells was evaluated.  The transfection efficiency of PEI-pDNA nanoplexes were evaluated using a standard luciferase assay system. Results: The PEI-pDNA nanoparticles over the whole range of N/P ratios were spherical in shape and found to be < 200 nm in diameter with a small polydispersity index (<0.3), indicating monomodal homogenous size distribution. At the concentrations used for transfection, the nanoplexes displayed low toxicity when evaluated using the MTS assay. An N/P ratio of 10 was found to have the maximum transfection efficiency with acceptable cytotoxicity. Conclusion: PEI-pDNA nanoplexes are an effective vector for delivering target genes to HEPM cells. Future experiments will explore the non-viral delivery of multiple genes for bone regeneration