The grafting of polymer brushes to substrates is a promising method to modify surface properties such as wettability and the affinity toward proteins and cells for applications in microelectronics, biomedical devices, and sensors. Poly(acrylic) acid (PAA) brushes are of high interest because of their stimuli-responsive behavior and the presence of carboxy (COOH) groups, which allow for immobilization of bioactive molecules. The "grafting-to" approach results in homogeneous and well-defined polymer brushes, but, although grafting-to has been demonstrated with PAA brushes on silicon (Si) substrates, it has not been performed on biocompatible materials such as titanium (Ti). Here, we have described a facile method to modify biocompatible Ti substrates with PAA brushes to amplify their substrate functionality. The grafting-to PAA "pseudo" brushes were successfully grafted to Ti substrates and retained their pH-dependent swelling behavior. An RGD peptide was covalently bound to COOH groups of the PAA brushes (PAA-RGD) as a model bioactive group. While NIH/3T cell adhesion was significantly decreased on PAA-functionalized Ti substrates, PAA-RGD on Ti had cell adhesion comparable to that of flat Ti at 24 and 48 h, with significantly more cells adhered to PAA-RGD compared to PAA on Ti at 48 h.

Gene delivery is the transfer of exogenous genetic material into somatic cells to modify their gene expression, with applications including tissue engineering, regenerative medicine, sensors and diagnostics, and gene therapy. Viral vectors are considered the most effective system to deliver nucleic acids, yet safety concerns and many other disadvantages have resulted in investigations into an alternative option, i.e. nonviral gene delivery. Chemical nonviral gene delivery is typically accomplished by electrostatically complexing cationic lipids or polymers with negatively charged nucleic acids. Unfortunately, nonviral gene delivery suffers from low efficiency due to barriers that impede transfection success, including intracellular processes such as internalization, endosomal escape, cytosolic trafficking, and nuclear entry. Efforts to improve nonviral gene delivery have focused on modifying nonviral vectors, yet a novel solution that may prove more effective than vector modifications is stimulating or “priming” cells before transfection to modulate and mitigate the cellular response to nonviral gene delivery. In applications where a cell-material interface exists, cell priming can come from cues from the substrate, through chemical modifications such as the addition of natural coatings, ligands, or functional side groups, and/or physical modifications such as topography or stiffness, to mimic extracellular matrix cues and modulate cellular behaviors that influence transfection efficiency. This review summarizes how biomaterial substrate modifications can prime the cellular response to nonviral gene delivery (e.g. integrin binding and focal adhesion formation, cytoskeletal remodeling, endocytic mechanisms, intracellular trafficking) to aid in improving gene delivery for future therapeutic applications. Impact statement This review summarizes how biomaterial substrate modifications (e.g. chemical modifications like natural coatings, ligands, or functional side groups, and/or physical modifications such as topography or stiffness) can prime the cellular response to nonviral gene delivery (e.g. affecting integrin binding and focal adhesion formation, cytoskeletal remodeling, endocytic mechanisms, and intracellular trafficking), to aid in improving gene delivery for applications where a cell-material interface might exist (e.g. tissue engineering scaffolds, medical implants and devices, sensors and diagnostics, wound dressings).

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.

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