Therapeutic strategies that can deliver bioactive signals at different times during tissue formation are essential for the regeneration of complex tissues such as a mature vasculature. During normal wound healing, the events that lead to mature blood vessel formation result from a series of tightly regulated events, which occur sequentially upon environmental changes. As a result, for the generation of mature and stable blood vessels more than one bioactive signal is needed and these signals are needed at different times. This proposal focuses on the design, synthesis and testing (in vitro and in vivo) of a non- viral gene delivery strategy that can deliver multiple DNA sequentially. In our approach, a two component, enzymatically degradable hydrogel composed of a micro porous (5-pore) slow degrading hydrogel and nano-porous (n-pore) fast degrading hydrogel will be used deliver encapsulated DNA nanoparticles at different times. Aim 1 will explore the design and synthesize two component hydrogel scaffolds that can release DNA nanoparticles at two different rates in vitro and in vivo. Aim 2 will explore the ability of the optimized two-component hydrogels to result in temporally controlled gene transfer in vitro and in vivo. Aim 3 will explore the hypothesis that within the wound-healing environment our two-component hydrogel system can release the encapsulated pro-angiogenic polyplexes and growth factors at different rates and result in enhanced angiogenesis and subsequent wound healing. PUBLIC HEALTH RELEVANCE: Angiogenesis, the formation of new blood vessels, represents a pressing clinical need for the treatment of ischemic wounds and is a major obstacle in the translation of tissue-engineered constructs. One major limitation in the generation of mature blood vessels is the inability to deliver therapeutic molecules at the necessary times. This proposal aims to design a gene delivery strategy that can deliver DNA (the therapeutic) at the required times for angiogenesis to take places by using hydrogel scaffolds that are degraded at different rates.