Nanoscale Cell Adhesion Ligand Presentation Regulates Nonviral Gene Delivery and Expression

Abstract

It is hypothesized that the nanoscale organization of cell adhesion ligands in a synthetic ECM regulates non-viral gene delivery. This hypothesis was examined with pre-osteoblasts cultured on substrates which present varied density and spacing of synthetic adhesion ligands. The levels of gene transfer and expression were increased with the density of adhesion ligands, but decreased with the spacing of ligands, due to changes in the cell growth rate. This study provides a material-based control point at the nanometer scale for non-viral gene based therapies. Keywords RGD peptides; plasmid DNA; gel matrix; proliferationNon-viral gene vectors [e.g., plasmid DNA (pDNA)] are being increasingly used in a variety of therapeutic applications 1-3 because of safety concerns related to viral vectors. 4 One of the major challenges with the use of non-viral gene vectors is the very limited range of transgene expression level, owing to the poor efficiency of gene transfer. 5 Therefore, there have been extensive efforts to design novel gene carriers to improve the levels of gene transfer and expression using lipids, polycations, dendrimers, nanorods, and oligopeptides 5-7 and efforts to spatially concentrate the pDNA. 8 In contrast, the design of gene delivery systems has largely ignored the importance of the cellular microenvironment in gene uptake and expression, although target tissues for in vivo gene delivery present varying cellular microenvironments.Gene transfer is related to the rate of cell proliferation, 9 and the chemistry and mechanics of the ECM to which a cell adheres regulates many aspects of cell phenotype, including proliferation, apoptosis and differentiation. 10-14 These findings suggest that alteration of the ECM may allow one to regulate a cell's ability to take up and express exogeneous genes. Recently, we have demonstrated that the mechanical rigidity of synthetic ECM used as adhesion substrates plays a critical role in modulating gene transfer and expression. 15 In this study, we demonstrate a new material-based control point to regulate non-viral delivery by engineering the nanoscale organization of cell adhesion molecules in the synthetic ECM. The underlying mechanism of this regulation appears to be related to the material control over cell proliferation. The spatial distribution of cell adhesion molecules at the nanometer scale was controlled using a gel matrix formed from cross-linked alginate molecules containing covalently bound synthetic oligopeptides containing the Arg-Gly-Asp (RGD) sequence (RGD peptides) which mediate cell adhesion. 16 Because unmodified alginate molecules are highly inert to cell adhesion and protein adsorption, 17 cellular adhesion to gels functionalized with RGD peptides is solely attributed to the bonding between cell receptors and RGD peptides.The overall density of RGD peptides (N RGD ) and the distance between islands of RGD peptides (d RGD ), which are known to regulate cell proliferation 11 , were used as variables to control the spatial organization of RGD ...

Active scaffolds for on-demand drug and cell delivery

“…Plasmid DNA (Aldevron) was combined with linear PEI (Fermentas) to form a polyelectrolyte complex as described in ref. 53. The ratio of the number of amine groups in PEI to the number of phosphate groups (N/P ratio) in DNA was kept constant at 7.…”

Design of Biodegradable Hydrogel for the Local and Sustained Delivery of Angiogenic Plasmid DNA

“…The gWiz vector containing the gene encoding for luciferase (Aldevron) was combined with linear poly(ethyl- Oxidized blocks eneimine) [PEI, MW∼22,000 g/mol] (Fermentas) to form a polyelectrolyte complex as previously described (33). The ratio of the number of amine groups in PEI and the number of phosphate groups (N/P ratio) in pDNA was kept constant at 7.…”

Engineering Integrative Stem Cell and Biomaterial Therapies for Peripheral Artery Disease

“…For PEG gels, a highly utilized sequence is the arginine-glycine-aspartic acid (RGD), which has been shown in several studies to enhance cell viability, growth and differentiation into preferred cell types [101,106]. Further adjustments in the quantity of RGD peptide were shown to influence cell sensitivity to matrix stiffness [107], and spatially organized RGD (via altered PEG molecular weight and fabrication methods) could organize the encapsulated cells into microstructures more representative of the tissue they are recreating [108]. This method has been extended to include whole recombinant protein oligopeptides and biodegradable units (portions of the gel that can be degraded by MMPs) [109].…”