Design and Characterization of Porous Hyaluronic Acid Hydrogels for in vitro and in vivo Non-Viral DNA Delivery
Natural wound healing and angiogenesis are a result of a cascade of bioactive signals being delivered at specified times in response to local biological cues. However, for ischemic wounds which cannot heal naturally, external therapies are required. We are interested in engineering hydrogel scaffolds for cell-demanded release of non-viral DNA nanoparticles to more efficiently guide blood vessel formation in such tissues. Vascularization within tissue-engineered constructs still remains the primary cause of construct failure following implantation. While a myriad of approaches to enhance the vascularization of implants are being investigated none have completely solved the problem. We investigated two hypotheses to enhance scaffold vascularization, both long-term mechanical support and DNA delivery. Our preliminary in vivo studies showed that after subcutaneous implantation for three weeks enzymatically degradable hydrogels had cellular infiltration only at the periphery of the hydrogel, while hydrogels with micron sized interconnected pores (µ-pore) were extensively infiltrated. Significant positive staining for endothelial markers (PECAM) was also found for µ-pore implants and not for nano-pore implants, even in the absence of pro-angiogenic factors. We hypothesized 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 would result in long lasting angiogenic signals.
To test these hypotheses, two approaches to make DNA-loaded enzymatically degradable µ-pore hydrogel scaffolds were developed. In the first approach, polystyrene nanoparticles, similar in size to DNA polyplexes, were immobilized to the hydrogel pore surface through protease sensitive peptide tethers. Enzymatically degradable tethers have been utilized for the immobilization and release of growth factors and small drugs, which are only liberated by cleavage caused by cell secreted proteases, such as matrix metalloproteinases (MMPs) or plasmins, during local tissue remodeling. These proteases are known to be up-regulated during wound healing, microenvironment remodeling, and in diseased states and can, therefore, serve as triggers for bioactive signal delivery. The goal was to use peptide sequences that have been shown to degrade at different rates through the action of MMPs to achieve temporally controlled nanoparticle internalization by cells that overexpress MMPs. Cellular internalization of the peptide-immobilized nanoparticles was shown to be a function of the peptide sensitivity to proteases, the number of tethers between the nanoparticle and the biomaterial and the MMP expression profile of the seeded cells. By immobilizing nanoparticles through protease sensitive peptide tethers, release was tailored specifically for an intended cellular target, which over-expresses such proteases.
Alternatively, in the second approach, µ-pore hyaluronic acid-MMP (HA-MMP) hydrogels were used to encapsulate a high concentration of DNA/poly(ethylene imine) polyplexes using a previously developed caged nanoparticle encapsulation (CnE) technique. Porous hydrogels provide the additional advantages of being able to effectively seed cells in vitro post scaffold fabrication and allow for cell spreading and proliferation without requiring extensive degradation. Thus, release of encapsulated DNA polyplexes was assessed in the presence of mMSCs in hydrogels of various pore sizes (30, 60, and 100 µm). Steady release was observed starting by day four for up to ten days for all investigated pore sizes. Likewise, transgene expression in seeded cells 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. Combined these results suggested that DNA nanoparticle internalization and subsequent transgene expression could be controlled by both the protease expression profile of the seeded or infiltrating cells as well as the structural properties of the hydrogel.
Using the knowledge acquired through these in vitro models, 100 and 60 µm porous and nano-pore HA-MMP hydrogels were used to study scaffold-mediated gene delivery for local gene therapy in both a subcutaneous implant and wound healing mouse models. Hydrogels with encapsulated pro-angiogenic (pVEGF) or reporter (pGFPluc) plasmids were tested for their ability to induce an enhanced angiogenic response by transfecting infiltrating cells in vivo. GFP expression in control hydrogels was used to track transfection at one, three, and six weeks in the subcutaneous implant study. While GFP-expressing transfected cells were present inside all hydrogel samples over the course of the study, transfection levels peaked around week three for 100 and 60 µm porous hydrogels. Transfection in nano-pore hydrogels continued to increase over time corresponding with continued gel degradation. Transfection levels of pVEGF, however, did not seem to be high enough to enhance angiogenesis by increasing vessel number, maturity, or size. Only in 60 µm porous hydrogels did the VEGF expression play a role in preventing vessel regression and helping to sustain the number of vessels present from three to six weeks. Regardless, pore size seemed to be the dominant factor in determining the angiogenic response with 60 µm porous hydrogels having more vessels present per area than 100 µm porous hydrogels at the initial onset of angiogenesis at three weeks. Increased pore rigidity may have been a key factor.
The effect of porosity on wound healing was even more pronounced than what was observed in the subcutaneous implants. 100 and 60 µm porous hydrogels allowed for significantly faster wound closure than nano-pore hydrogels, which did not degrade and essentially provided a mechanical barrier to closure. Interestingly, total porosity and not specific pore size seemed to be the dominant factor in determining the wound closure rates. GFP-expressing transfected cells were present throughout the newly formed granulation tissue surrounding all hydrogel samples at two weeks. Transfection levels of pVEGF, however, did not seem to be high enough to enhance angiogenesis within the granulation tissue by statistically increasing vessel number, maturity, or size. Although the anticipated enhancement in angiogenesis in either model was not shown, the presence of transfected cells shows promise for the use of polyplex loaded porous hydrogels to produce a response in vivo.
With further optimization we believe the proposed hydrogel system(s) has applications for controlled release of various DNA particles and other gene delivery vectors for in vivo tissue engineering and blood vessel formation.