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Characterization and evaluation of the efficacy of cationic complex mediated plasmid DNA delivery in human embryonic palatal mesenchyme cells
Author(s) -
D'Mello Sheetal,
Salem Aliasger K.,
Hong Liu,
Elangovan Satheesh
Publication year - 2016
Publication title -
journal of tissue engineering and regenerative medicine
Language(s) - English
Resource type - Journals
SCImago Journal Rank - 0.835
H-Index - 72
eISSN - 1932-7005
pISSN - 1932-6254
DOI - 10.1002/term.1873
Subject(s) - transfection , polyethylenimine , microbiology and biotechnology , gene delivery , chemistry , cytotoxicity , reporter gene , calcium , dna , cell culture , in vitro , biophysics , biochemistry , gene , biology , gene expression , genetics , organic chemistry
The purpose of this study was to develop and test a non‐viral gene delivery system that can be employed to deliver genes of interest into a pre‐osteoblastic cell line. Human embryonic palatal mesenchymal (HEPM 1486) cells were transfected with vector‐plasmid DNA (pDNA) complexes. We explored calcium phosphate and polyethylenimine (PEI) as non‐viral vectors and compared their respective in vitro transfection efficacies. Plasmid DNA encoding luciferase protein (LUC) was complexed with PEI (with differing N:P ratios) and calcium phosphate (with differing Ca:P ratios), using established protocols. The complexes prepared were then characterized for size and surface charge, using a Malvern Zetasizer Nano‐ZS. The transfection efficiency and cytotoxicity of the prepared complexes were evaluated in HEPM cells. The PEI–pDNA complexes over the whole range of N:P ratios were found to be < 160 nm in size, while the calcium phosphate–pDNA complexes were relatively bigger. The PEI–pDNA complexes prepared at a N:P ratio of 10 were found to have maximum transfection efficiency at 4 h of treatment, with minimal cytotoxicity. The highest transfection efficiency obtained with calcium phosphate–pDNA complexes (Ca:P 200) was nearly 12‐fold lower than that obtained with PEI–pDNA complexes (N:P 10). Following this, transgene expression in the HEPM cells treated with complexes prepared at a N:P ratio of 10 was further examined, using pDNA coding for enhanced green fluorescent protein (EGFP‐N1) or therapeutically relevant platelet‐derived growth factor B (PDGF‐B). In conclusion, PEI was a more effective vector for delivering genes of interest to pre‐osteoblasts than calcium phosphate. Copyright © 2014 John Wiley & Sons, Ltd.