Trainee Speakers

With the discovery that mitochondria can be actively transferred from one cell type to another and promote wound healing, new therapeutic approaches have been developed to harness the benefits of this phenomena. To date, in vivo mitochondria delivery has been limited to the injection of free mitochondria. While increased therapeutic efficacy has been observed, there are distinct limitations to this approach. First, uptake of free mitochondria is particularly inefficient and second, the lack of a delivery vehicle does not capitalize on integrating time-dependent release based on the pathogenesis of the injury or disease. To this end, this study demonstrates that coupling polymer-based surface modification of mitochondria and mitochondria encapsulation in matrix metalloproteinase (MMP) cleavable hydrogel microparticles leads to both an increase in mitochondria uptake by recipient cells and stimuli-responsive release respectively. More specifically, the inclusion of transactivator of transcription (TAT) peptides at the mitochondrial surface leads to a >7-fold increase in free-mitochondria uptake by myoblasts and osteoprogenitor cells. Furthermore, increased mitochondria transfer demonstrated a significant increase in the bioenergetic state of the recipient cell, as characterized by the oxygen consumption rate. Finally, due to a shift in the energetic state of the recipient cells following mitochondria transfer, cellular processes such as proliferation, myogenic differentiation, and osteogenic differentiation were all increased. Myogenic differentiation was characterized through myosin heavy chain immunostaining and osteogenic differentiation was characterized by assessing both calcium deposition and alkaline phosphatase activity. We note that while these results are most directly applicable for muscle and bone regeneration, on-demand mitochondrial release for localized cellular uptake has applications in a wide range of regenerative medicine applications. 

Immune checkpoint blockade therapies hold transformative potential for cancer treatment but face limited efficacy in many patients due to an immunosuppressive tumor microenvironment (TME). Regulatory T cells (Tregs) are key mediators of this suppression, dampening cytotoxic T lymphocyte activity and enabling immune evasion. Although Treg-targeting therapies, such as CTLA4 inhibitors, show promise, their use is constrained by systemic toxicity, underscoring the need for innovative and selective approaches to modulate Treg activity. Our recent findings reveal that activating the Stimulator of Interferon Genes (STING) pathway in Tregs induces a phenotypic shift, reprogramming them toward an anti-tumor, effector T-cell-like state. To harness this phenomenon, we developed a novel protein-based nanocarrier for Treg-specific delivery of STING agonists. This bispecific nanobody-STING agonist conjugate (biNSC) integrates a CTLA4-binding domain for Treg specificity with an albumin-binding domain to enhance pharmacokinetics and circulation time. This dual functionality addresses key pharmacological limitations of STING agonist delivery, enabling targeting and prolonged action within the TME. Our results demonstrate efficient Treg binding, internalization, and controlled drug release, leading to effective STING pathway activation and subsequent phenotypic reprogramming. We demonstrate the potential of this strategy to promote tumor rejection in vivo. This innovative, protein biomaterials-based approach offers significant insights into the design of advanced drug delivery systems for precision immunomodulation and represents a promising strategy to boost cancer immunotherapy outcomes. 

In the inflammatory phase of wound healing, reactive oxygen species (ROS) are produced to fight microorganisms, prevent infection, and promote immune cells. However, hyperglycemia in diabetic wounds impairs this function by sustaining oxidative stress, preventing resolution of inflammation, and blocking the transition to later stages of wound healing.

The objective of this work was to develop a hybrid shear-thinning hydrogel as a bioresorbable and injectable therapeutic delivery platform which can provide controlled delivery of small molecule PHD2 inhibitors through incorporation of ROS-reactive nanoparticles with the biological polymer, hyaluronic acid (HA), to achieve accelerated repair of chronic diabetic skin wounds.

To investigate the impact of polysulfide chemistry on the antioxidant function of resulting hydrogels, different polysulfide monomers were incorporated into nanoparticles which were mixed with HA to yield polysulfide hydrogels possessing a range of ROS-reactivities.

Evaluation of hydrogels in excisional wounds of diabetic mice identified polysulfide chemistries which resulted in sustained release of small molecule PHD2 inhibitors, reduced levels of oxidative stress and inflammatory macrophages, and possessed potent antioxidant functions. These studies ultimately resulted in the identification of polysulfide hydrogels which demonstrate promise as a therapeutic delivery platform for improving outcomes in chronic diabetic wounds.

The following work not only provides a comprehensive analysis on the structure-function relationship of polysulfide chemistry within the hydrogel system but also produced an entire library of hydrogels which possess a range of sensitivities to ROS and resulted in the development of a unique technology that may be used in additional tissue repair and regeneration applications. 

Responsive materials that can leverage biological signals have been of increased interest in regenerative medicine applications. Newly designed synthetic polymers containing oxidation-sensitive thioketal (TK) linkers have shown promise as degradable tissue engineering materials as they are stable in aqueous conditions but selectively broken down by cell-generated reactive oxygen species (ROS). This behavior allows for cell-mediated biomaterial resorption in vivo. However, recent reports have shown current TK-based systems demonstrate insufficient sensitivity to physiological doses of ROS in certain applications.

In this work, we reengineered the structure of the traditional TK bond to improve its responsiveness to ROS. Modified TK materials were first evaluated via NMR degradation studies which showed a correlation between material hydrophilicity and responsiveness. Newly synthesized TK linkers were incorporated into tissue engineering scaffolds where they were stable in aqueous conditions but demonstrated a range of responsiveness to ROS based on the TK bond chemistry. All materials demonstrated limited cytotoxic effects. Next, porous scaffolds were implanted subcutaneously in Sprague Dawley rats and evaluated for biodegradation over 4 weeks. In scaffolds constructed with more hydrophilic TK linkers, tissue area significantly increased, and scaffold area significantly decreased while more hydrophobic formulations had minimal changes over time.

We demonstrated that more hydrophilic TK linkers degrade at lower levels of ROS while more hydrophobic linkers have decreased sensitivity to oxidative degradation both in vitro and in vivo. Engineering the structure of ROS-degradable TK linkers to vary material hydrophilicity is shown to yield biomaterials that are more/less sensitive to oxidative degradation and will expand the potential for cell-degradable biomaterials.