Design and Characterization of Micro-Porous Hyaluronic Acid Hydrogels for Non-Viral DNA Delivery

Vascularization within tissue-engineered constructs remains the primary cause of construct failure following implantation. We are currently investigating two hypotheses to enhance hydrogel scaffold vascularization, both long-term mechanical support and cell-demanded release of non-viral DNA nanoparticles. Our preliminary in vivo studies show that after subcutaneous implantation for 3 weeks enzymatically degradable hyaluronic acid (HA) hydrogels have cellular infiltration only at the periphery of the hydrogel, while hydrogels with micron sized interconnected pores (micro-pore) are extensively infiltrated. Significant positive staining for endothelial markers (PECAM) was also found for micro-pore implants and not for nano-pore implants, even in the absence of pro-angiogenic factors. We believe that an open pore structure will increase the rate of vascularization through enhanced cellular infiltration and that the added delivery of DNA encoding for angiogenic growth factors will result in long lasting angiogenic signals. We have thusfar designed and characterized various pore size (30, 60, and 100 micron) micro-pore HA hydrogels loaded with a high concentration of non-viral DNA/PEI polyplexes, using a previously developed caged nanoparticle encapsulation (CnE) technique. These hydrogels allowed for long-term sustained transfection and transgene expression of incorporated mMSCs in vitro. For all investigated pore sizes, encapsulated DNA polyplexes were released steadily starting by day 4 for up to 10 days. Likewise, transgene expression was sustained over this period, although significant differences between different pore sizes were not observed. Cell viability was also shown to remain high over time, even in the presence of high concentrations of DNA polyplexes. Presently, we are using the knowledge acquired through this in vitro model to design and better predict scaffold-mediated gene delivery for local gene therapy in both subcutaneous implant and wound healing mouse models. We believe the proposed hydrogel system has applications for the controlled release of various DNA particles and other gene delivery vectors for in vivo tissue engineering and blood vessel formation.