Project Summary Following injury, tendons heal via a fibrotic scar-tissue response that impedes full functional restoration. Translation of pharmacotherapies to enhance tendon healing has been hampered by a combination of limited tendon targeting of systemic treatments, and insufficient identification of biologically informed therapeutic targets. In this high-risk high-reward study we will address both of these critical knowledge gaps. We have recently identified genetic knockdown of S100a4 as a novel model of functionally-enhanced tendon healing, thereby identifying S100a4 as a novel therapeutic target to improve tendon healing. Moreover, we have used spatial transcriptomic profiling to define the spatially distinct molecular processes that dictate the fibrotic tendon healing process. Using this approach we defined a macrophage-rich cluster located between the highly reactive tendon stubs at the injury site. This cluster was defined by enriched expression of Acp5, the gene encoding for TRAP (Tartrate resistant acid phosphatase). Our preliminary data further demonstrate regions of robust TRAP activity in the healing tendon. Here, we will capitalize on this exciting finding by leveraging our work using a TRAP binding peptide (TBP) conjugated nanoparticle (NP) drug delivery system. We have demonstrated enhanced homing and retention of TBP-NPs at sites of high TRAP activity including the bone fracture callus and during pathologic bone remodeling. Here, we will test the central hypothesis that TRAP binding peptide loaded nanoparticles (TBP-NPs) efficiently home to the healing tendon, are taken up by macrophages and that TBP-NP delivery of an S100a4 inhibitor enhances tendon regeneration compared to control TBP-NPs. In Aim 1 we will track the systemic and tendon-specific localization and retention of systemically administered fluorescently labelled TBP-NPs compared to scrambled control peptide-NPs. In addition, we will use a combination of cell-type specific fluorescent reporter mouse models to define the specific cell populations that uptake TBP-NPs during tendon healing. In Aim 2 we will define the loading and release profile of an S100a4 inhibitor on TBP-NPs and define the efficacy of TBP-NP drug delivery, compared to free drug and control NPs, to inhibit S100a4 expression and enhance the tendon healing process. Successful completion of these studies will establish a novel nanoparticle-mediate delivery system to target the healing tendon with high efficiency and efficacy, thereby substantially enhancing the translational feasibility of pharmacologically mediating improved tendon healing.