Abstract
Substrate mediated gene delivery (SMD) is a method of immobilizing DNA complexes to a substrate via covalent attachment or nonspecific adsorption, which allows for increased transgene expression with less DNA compared to traditional bolus delivery. It may also increase cells receptivity to transfection via cell-material interactions. Substrate modifications with poly(acrylic) acid (PAA) brushes may improve SMD by enhancing substrate interactions with DNA complexes via tailored surface chemistry and increasing cellular adhesion via moieties covalently bound to the brushes. Previously, we described a simple method to graft PAA brushes to Ti and further demonstrated conjugation of cell adhesion peptides (i.e., RGD) to the PAA brushes to improve biocompatibility. The objective of this work was to investigate the ability of Ti substrates modified with PAA-RGD brushes (PAA-RGD) to immobilize complexes composed of branched polyethyleneimine and DNA plasmids (bPEI-DNA) and support SMD in NIH/3T3 fibroblasts. Transfection in NIH/3T3 cells cultured on bPEI-DNA complexes immobilized onto PAA-RGD substrates was measured and compared to transfection in cells cultured on control surfaces with immobilized complexes including Flat Ti, PAA brushes modified with a control peptide (RGE), and unmodified PAA. Transfection was two-fold higher in cells cultured on PAA-RGD compared to those cultured on all control substrates. While DNA immobilization measured with radiolabeled DNA indicated that all substrates (PAA-RGD, unmodified PAA, Flat Ti) contained nearly equivalent amounts of loaded DNA, ellipsometric measurements showed that more total mass (i.e., DNA and bPEI, both complexed and free) was immobilized to PAA and PAA-RGD compared to Flat Ti. The increase in adsorbed mass may be attributed to free bPEI, which has been shown to improve transfection. Further transfection investigations showed that removing free bPEI from the immobilized complexes decreased SMD transfection and negated any differences in transfection success between cells cultured on PAA-RGD and on control substrates, suggesting that free bPEI may be beneficial for SMD in cells cultured on bPEI-DNA complexes immobilized on PAA-RGD grafted to Ti. This work demonstrates that substrate modification with PAA-RGD is a feasible method to enhance SMD outcomes on Ti and may be used for future applications such as tissue engineering, gene therapy, and diagnostics.
Highlights
Nonviral gene delivery is the delivery of exogenous genetic material to cells or tissues, generally to produce a therapeutic protein, with applications in gene therapy, tissue engineering and regenerative medicine, and biomedical implants
After the addition of OptiMEM, poly(acrylic) acid (PAA) brushes swelled to an average thickness of 23 ± 3.0 nm, which is similar to the swelling in 0.1 M phosphatebuffered saline (PBS) reported in our previous study (Rosenthal et al, 2018)
We found that substrates modified with PAA brushes adsorb more overall mass, which may be attributed to immobilization of free and complexed branched polyethylenimine (bPEI), as measured with spectroscopic ellipsometry
Summary
Nonviral gene delivery is the delivery of exogenous genetic material to cells or tissues, generally to produce a therapeutic protein, with applications in gene therapy, tissue engineering and regenerative medicine, and biomedical implants. SMD has been shown to limit complex aggregation and require a lower dose of DNA, as well as increase transgene expression and the number of transfected cells by increasing the local concentration of DNA within the microenvironment around the cell and overcoming a mass transport barrier to gene delivery efficiency (Pannier and Shea, 2004; Bengali et al, 2005, 2007; Pannier et al, 2005, 2008; Rea et al, 2009a; Wang et al, 2010; Pannier and Segura, 2013). For example titanium (Ti) is one of the most commonly used biomaterials (Elias et al, 2008), with many applications that could benefit from nonviral SMD such as enhancing the integration of bone implants by delivering genes to increase osseointegration (Wang et al, 2015; Zhang et al, 2015), geneeluting stents to accelerate re-endothelialization (Sharif et al, 2012), or developing implantable sensors protected by the local delivery of anti-inflammatory and anti-fibrosis genes (Klueh et al, 2014), but to date there have been few studies published using SMD on Ti
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