SUMMARY The delayed healing observed in chronic wounds is exacerbated by persistent microbial infections and non- resolving inflammation. Furthermore, the emergence of antibiotic-resistant bacteria has limited the use of these agents for treating infected wounds. Adding to the complexity of chronic wound treatment, infection is usually not the sole cause of wound chronicity. Underlying diseases such as diabetes leave individuals prone to infection by affecting the host immune responses, including inflammatory cell migration, cell signaling, and effector function. An ideal wound healing therapeutic must thus address the impairment of the host immune response while also possessing antibacterial activity. Due to the high prevalence of chronic wound-related amputations and mortality, the need for such a multi-action therapeutic is urgent. Nitric oxide (NO) is an endogenous signaling molecule that represents an attractive, alternative therapeutic for treating chronic wounds due to its innate antibacterial and immunomodulatory function in human physiology. We have pioneered the development of macromolecular NO donor systems that store and spontaneously release NO in dissolved form (i.e., not as a gas) at therapeutically relevant levels. We now aim to develop NO-releasing glycosaminoglycan biopolymers (GAGs) as wound healing therapeutics. GAGs are naturally occurring biopolymers that are immunomodulatory and known to be involved in wound healing physiology. We hypothesize that combining the multi-faceted roles of GAGs and NO will allow for a therapeutic that effectively: 1) eradicates wound pathogens; 2) modulates inflammation; and, 3) promotes re-epithelialization to facilitate timely wound closure. The objective of this project is to define the roles of GAG molecular weight, sulfation patterns and NO-release properties as they related to antibacterial and pro-wound healing activities. In developing a new class of wound- healing therapeutics, we will characterize cell proliferation, adhesion, and migration as a function of NO payloads and GAG structure using cell culture assays and a three-dimensional human skin tissue model. We will evaluate the effect of NO-releasing GAGs on innate immune cell plasticity using primary human cell systems. We will then determine the therapeutic efficacy of the most promising NO-releasing GAG derivatives on antibacterial action, inflammation, and wound closure as a function of infection and diabetes. An iterative approach will be taken to determine the optimal dose, time, and frequency of therapeutic intervention. A systems biology approach will be used to elucidate mechanisms of efficacy and failure, which will inform clinical translation of these therapeutic approaches. This new research program will allow us to build upon our previous successes in developing NO-releasing macromolecular scaffolds, but now with a focus on wound healing. Our goal is to develop a therapeutic that treats infection and promotes wound heali...