Gene delivery through cell culture substrate adsorbed DNA complexes

Abstract

Efficient gene delivery is a fundamental goal of biotechnology and has numerous applications in both basic and applied science. Substrate-mediated delivery and reverse transfection enhance gene transfer by increasing the concentration of DNA in the cellular microenvironment through immobilizing a plasmid to a cell culture substrate prior to cell seeding. In this report, we examine gene delivery of plasmids that were complexed with cationic polymers (polyplexes) or lipids (lipoplexes) and subsequently immobilized to cell culture or biomaterial substrates by adsorption. Polyplexes and lipoplexes were adsorbed to either tissue culture polystyrene or serumadsorbed tissue culture polystyrene. The quantity of DNA immobilized increased with time of exposure, and the deposition rate and final amount deposited depended upon the properties of the substrate and complex. For polyplexes, serum modification enhanced reporter gene expression up to 1500-fold relative to unmodified substrates and yielded equivalent or greater expression compared to bolus delivery. For lipoplexes, serum modification significantly increased the number of transfected cells relative to unmodified substrates yet provided similar levels of expression. Immobilized complexes transfect primary cells with improved cellular viability relative to bolus delivery. Finally, this substrate-mediated delivery approach was extended to a widely used biomaterial, poly(lactide-coglycolide). Immobilization of DNA complexes to tissue culture polystyrene substrates can be a useful tool for enhancing gene delivery for in vitro studies. Additionally, adapting this system to biomaterials may facilitate application to fields such as tissue engineering.

Surface polyethylene glycol enhances substrate-mediated gene delivery by nonspecifically immobilized complexes

“…2b). These amounts are similar to, or much greater than, previous reports of lipoplexes binding to SAMs [15,22], but are consistent with binding densities for PEI-DNA complexes on hydrophilic, serum-coated polystyrene substrates [13].…”

“…Poly(L-lysine) (PLL) and polyethylenimine (PEI), modified with biotin residues, were complexed with DNA and bound to a neutravidin substrate [7,8], resulting in 100-fold increased transgene expression from the immobilized complexes relative to bolus delivery of complexes [7]. Plasmid DNA or DNA complexed with cationic polymers or lipids can also interact with substrates through nonspecific mechanisms [10][11][12][13][14][15][16][17][18]. Polyplexes and lipoplexes nonspecifically immobilized to substrates enhanced the extent of transgene expression in both cell lines and primary human-derived cells, along with an increased cellular viability [13].…”

“…Plasmid DNA or DNA complexed with cationic polymers or lipids can also interact with substrates through nonspecific mechanisms [10][11][12][13][14][15][16][17][18]. Polyplexes and lipoplexes nonspecifically immobilized to substrates enhanced the extent of transgene expression in both cell lines and primary human-derived cells, along with an increased cellular viability [13]. This enhancement was dependent on the properties of both the complex (e.g.…”

“…The greatest amounts of binding and transfection were observed on surfaces presenting charged, hydrophilic groups, suggesting that electrostatic interactions allow for reversible interactions between the substrate and complexes and result in efficient gene delivery [22]. Hydrophobic substrates bound similar quantities of DNA as the hydrophilic surfaces, yet transfection was significantly reduced, suggesting the conformation of the DNA complexes may be altered upon binding to hydrophobic surfaces and result in low transfection levels [13,15].…”

“…However, as in traditional gene delivery approaches, further improvements are still needed for substrate-mediated gene delivery to address issues that limit gene transfer, including complex size stability, complex aggregation and strong interactions between the surface and complexes [9,13]. Polyethylene glycol (PEG), which has the monomeric repeat unit [-CH 2 -CH 2 -O]-, is widely used in drug and gene delivery and has been incorporated into DNA complexes of several cationic polymers, including polymethacrylate [23], PEI [24][25][26][27][28][29][30][31][32], PLL [33][34][35] and poly(amidoamine)s [36].…”