Of all the tools employed by the body’s defence mechanisms, oxidative stress appears to be the most ubiquitous, broad spectrum, and self-injuring. When oxidative mechanisms have been induced (e.g., the leukocyte respiratory burst), it can result in a degenerative cycle of chronic inflammation and cell death, that further stimulates the release of more harsh oxidants. However, under mild conditions, this oxidative stress stimulates tissue regeneration and cellular upregulation of protective phenotypes, improving the overall viability and prognosis of tissue health. As a result, the body maintains a delicate homeostasis of pro-oxidant generation with antioxidant mechanisms. By designing materials that can actively participate in this process in oxidative stress signally, we are able to better design biomaterials for a range of therapeutic applications, from oral mucositis, tissue engineering to cancer therapy. Here we present how an example of how polythiolated poly(beta-amino ester) polymers can be designed as redox responsive hydrogels for drug delivery applications.

Gene therapy is not yet clinically approved for cardiac applications because of the dangerously high dose required to reach efficacy. We have developed an adjuvant particle, “enhancer polymer (ePL),” which increases heart uptake and transduction, thereby enabling cardiac gene therapy. ePL is a cargo-less, spherical particle composed of poly (lactic-co-glycolic acid) (PLGA). Screening a series of particles resulted in the following key characteristics: a diameter of 200 nm, a viscosity of 0.6 dL/g, a 65/35 lactic-to-glycolic acid ratio, and 100% of lactic acid being the L enantiomer of the monomer. The particles are synthesized by nanoprecipitation using poly (vinyl acetate) (PVA) as a surfactant. ePL is not a component of any gene therapy particle and does not alter existing gene therapy technology; rather, it is a separate, adjuvant particle that increases heart delivery and decreases liver delivery. ePL improves heart-targeting of adeno-associated virus (AAV). We measured the uptake of serotypes AAV1 and AAVrh74 carrying the transgene for common biomarkers with and without ePL in vivo. ePL increased heart-targeting of both serotypes, shown by PCR and Western blot (AAVrh74: 14-fold change, AAV1: 1.5-fold change). Further, ePL decreased the amount of dose delivered to the liver. This encompasses the overall effect of ePL: AAV is redirected from the liver to the heart. The mechanism by which ePL does this is by temporarily overwhelming the liver, thereby blocking its uptake of AAV. This increases the circulation half-life of AAV, allowing it to be taken up by the heart. Further, our data shows that ePL induces the release of cell-signaling factors that directly increase uptake of AAV in the heart. Overall, ePL has the potential to enable cardiac gene therapy by improving delivery and reducing harmful off-targeting.

Chemotherapeutic resistance across many types of cancer is one of the greatest complications to cancer therapy and results in few treatment options for patients plagued with this disease. The vital demand for innovative anti-cancer drugs and drug delivery systems arises from these issues, revealing the importance that research on anti-cancerous compounds has on effective care for patients. Endometrial cancer is the most common gynecological cancer worldwide and is commonly treated using carboplatin, a platinum-based drug. Carboplatin resistance and unequitable access to treatment for endometrial cancer patients validates the need for a non-invasive form of endometrial cancer therapy. It is proposed that copper oxide nanoparticles can be used as an alternative to carboplatin when encapsulated in a polymeric nanoparticle for use as a drug delivery system. Copper oxide nanoparticles pose the potential as an anti-neoplastic compound due to Cu2+ ions demonstrating the ability to induce reactive oxygen species leading to double stranded DNA breaks ultimately resulting in apoptosis. Through viability assays, endometrial cell lines all showed sensitivity to copper oxide nanoparticles; however, trends were not identified across all cell lines that were tested. Using this information, Western Blot analysis was used to identify specific expression levels of apoptotic proteins, DNA repair proteins, and protein markers when DNA is damaged. Western Blots involved the treatment of cells in 24-well plates with varying concentrations of copper oxide nanoparticles for 24 hours. Gel electrophoresis separates proteins and primary antibody complexes. The differences in the expression levels of these proteins after cells are exposed to copper oxide nanoparticles can indicate the mechanism of toxicity within the different cell lines, ultimately explaining the differences in viability.

It is increasingly appreciated that mechanics plays a central role in many soft tissue disease processes. Similarly, as efforts to replace tissues have advanced, it is clear that materials must be considered not only in terms of chemical and biological compatibility, but also in terms of their mechanical integration with cells and tissues. In the 21st century the diversity of biomaterial options designed for soft tissue settings has expanded greatly as the field has moved more towards designing materials for clinical needs as opposed to adopting industrial materials. Thermoplastic elastomers that are amenable to solvent processing to achieve desired structure-function relationships comprise a material class that is rapidly expanding and finding new applications. Polyurethane ureas incorporating labile soft-segments have been processed into interconnected porous scaffolds or microfibrillar materials that can serve as temporary functional cardiovascular tissues (e.g. valves, arteries), mechanical supports (e.g. epicardial patches) or scaffolds to generate transferrable tissue units (e.g. a pedicled muscle free flap). An important aspect of this material approach is the ability to tune the desired behavior through both the synthesis and processing steps. An example of this tuning comes with the formation of bio-hybrid materials with integrated bioactive components. Examples of this chemistry, processing and in vivo applications will be discussed.

Cardiovascular complications are one of the leading causes of death in patients with diabetes or kidney disease. Among these complications is vascular calcification, defined as an active process where vascular smooth muscle cells (VSMCs) will phenotypically switch to osteoblast-like cells. This results in hydroxyapatite crystal deposition into arterial tissue which leads to hypertension, atherosclerotic plaque burden, and decreased elastance. Previously our group has shown high levels of phosphate in human aortic vascular smooth muscle cells (HAVSMCs) cultured in calcification media that lead to the over-expression of osteogenic markers such as the runt-related transcription factor 2 (RUNX2) in VSMCs. This was done using an in vitro model using HAVSMCs 3 mmol inorganic phosphate to induce the calcification. Once calcified, the cells showed a decrease in α-smooth muscle actin activity and an increase in RUNX2 that shows the potential of an osteogenic switch. In this previous study, Sclerostin (a potent WNT antagonist) was used to prevent Frizzled and LRP 5/6 co-receptors from attaching to the WNT proteins and thus preventing the up-regulation of calcification. Sclerostin showed potential to regulate vascular calcification and down-regulate RUNX2. RUNX2 is directly linked to the wingless/integrated (WNT) signaling pathway that activates the regulation of bone production. Current experiments seek to examine the role of mechanotransduction in WNT activation for VSMC calcification. Using a FlexCell BioFlex tension system, we are simulating hypertension in VSMCs and identifying any biomarkers related to WNT activation and the osteogenic switch of VSMCs.

Type 2 diabetes mellitus (T2DM) is a major public health concern with significant cardiovascular complications (CVD). Despite extensive epidemiological data, the molecular mechanisms relating hyperglycemia to CVD remain incompletely understood. Since this has huge implications in tissue engineering and regenerative medicine approaches, we here investigate the impact of chronic hyperglycemia on SMC phenotype and function using human aortic smooth muscle cells (HASMCs) cultured under varying glucose conditions in vitro, mimicking normal (5 mM/L), pre-diabetic (10 mM/L), and diabetic (20 mM/L) conditions, respectively. Patient-derived T2DM-SMCs were included for comparative analysis cultured for up to 21 days. Results showed distinct morphological changes with significant increases in cell area, perimeter and F-actin expression in SMCs treated at higher glucose levels. Cell shape index was 0.23 ± 0.01 in SMCs exposed to 10 and 20 mM/L glucose, and 0.48 ± 0.03 for T2DM cells and normal SMCs (5 mM/L) (p < 0.001). AFM analysis showed significant reduction in the Young’s modulus, membrane tether forces, membrane tension, and surface adhesive forces in SMCs with increasing glucose levels. In all these cases, T2DM cells exhibited levels noted in 20 mM/L glucose conditioned cells. A 5 to 6 -fold increase in cell proliferation was observed in SMCs treated with 20 mM/L glucose and in T2DM-SMCs, at day 7, compared to their original seeding density. T2DM-SMCs exhibited elevated levels of pro-inflammatory markers such as IL-6, IL-8, and MCP-1 compared to glucose-treated SMCs. Conversely, growth factors like FGF-2 and TGF-β were higher in SMCs exposed to moderate glucose but lower in T2DM-SMCs. Pathway enrichment analysis showed a significant increase in the expression of inflammatory cytokine-associated pathways, especially involving IL-10, IL-4 and IL-13 signaling in genes that are upregulated at higher glucose levels. Differentially regulated gene (DGE) analysis showed that, compared to SMCs cultured with normal glucose, 513 genes were upregulated, and 590 genes were downregulated in T2DM-SMCs; however, fewer genes (16 – 23) were differentially expressed in SMCs receiving 10 or 20 mM/L glucose. We identified the altered genes involved in ECM organization, elastic fiber synthesis and formation, and ECM proteoglycans, highlighting the role of hyperglycemia in CVD progression.