(16e) Design of a Multivalent Binding Platform for Enhanced Non-Viral Gene Transfer to αvβ3 Expressing Cells
- Conference: AIChE Annual Meeting
- Year: 2008
- Proceeding: 2008 Annual Meeting
- Group: Nanoscale Science and Engineering Forum
- Time: Monday, November 17, 2008 - 9:50am-10:10am
Non-viral gene delivery has been widely investigated over the past decade as a means to guide tissue regeneration, treat disease and study gene function. However, low efficiencies of gene transfer and inability to target desired cell populations have limited the use of this approach. Although current non-viral delivery approaches have utilized receptors that are uniquely expressed at the cell surface for targeting and to enhance gene transfer, strategies that target the density of receptors at the cell surface have not been explored. We believe that by targeting multiple ligands simultaneously (multivalent binding), we can enhance targeting of the desired cell type and enhance gene transfer through the engagement of biological pathways that are unique to clustered receptors. Interestingly adenovirus type 2 and 12 utilize this approach to enhance cellular internalization through proteins at their surface that contain spatially constrained integrin binding peptides that can interact with multiple integrin receptors simultaneously. In our present study, we investigated the effect spatially constrained Arg-Gly-Asp (RGD) peptides on the surface of DNA/polyethylene imine (PEI) polyplexes have on the efficiency of gene transfer to cells that have different densities of αvβ3 integrin receptors. RGD peptides were spatially constrained through immobilization to nanoparticles (nano-RGD) and the resulting nano-RGD nanoparticles were used to decorate the surface of DNA/PEI polyplexes. 5-nm nano-RGDs were synthesized with a theoretical density of 15 RGD peptides per particle and were shown to require 5 orders of magnitude lower concentration of RGD to prevent HeLa cell adhesion to vitronectin coated plates compared to free RGD peptide. This indicates that multivalent integrin binding is more effective in preventing cell adhesion than monomeric binding. The effect of nano-RGD on the ability of DNA/PEI polyplexes to transfect HeLa cells was studied for nano-RGD nanoparticles that were either covalently or electrostatically immobilized to the polyplex. Further, the effect on non-viral gene transfer efficiency of αvβ3 integrin density on the cell surface was studied using HeLa cells that were trypsin treated (low integrin density) or not (high integrin density). Polyplexes that were modified with nano-RGD resulted in a 5.4-fold or 37-fold increase in non-viral gene transfer efficiency over unmodified polyplexes for low or high integrin density cells respectively. This indicates that the presentation of the RGD peptides on the surface of DNA/PEI polyplexes affects cells with a high density of αvβ3 more than those that have low αvβ3 density. Further, nano-RGD was able to enhance gene transfer only when the nanoparticles were covalently bound to the DNA/PEI polyplexes. Polyplexes modified with nano-RGD through electrostatic interactions resulted in little to no enhancement of transgene expression compared to unmodified DNA/PEI polyplexes. Interestingly, experiments show that the degree of internalization for nano-RGD modified polyplexes and unmodified polyplexes are similar despite the increase in gene expression, suggesting that the clustered RGD has an effect on cellular trafficking rather than internalization alone. Experiments exploring the optimal concentration of nano-RGDs to enhance gene transfer resulted in an optimal concentration of 10 x 1013 nano-RGD particles, indicating that the density of nano-RGD clusters on the surface of the polyplex affects gene transfer. The use of platforms that can engage multiple receptors at the cell surface may not only enhance the targeting but also activate intracellular pathways that are unique in clustered receptors and result in enhanced gene transfer.