(357y) Microfluidic Approach to Dampen Stochasticity in Crystalline Drug Release and Cellular Dynamics of Senescent Mesenchymal Stem Cells

Small fluctuations in a cell’s oxidative environment can lead to drastic changes in cell behavior. This is particularly true for cells entering into a senescent state and has become a major research focus in the field of tissue engineering and regenerative medicine. With more than 26 million people in the United States suffering from cartilage loss or damage, the use of mesenchymal stem cells to differentiate into chondrocytes is a common approach to regenerate cartilage. Furthermore, more recent therapeutic approaches involve direct delivery of the MSC secretome harvested in bioreactors for anti-inflammatory, pro-angiogenic and trophic effects. Although promising, cells entering a senescent state with prolonged in vitro culture is a major limitation. Moreover, it is well established that even a small degree of senescence in MSC clusters can propagate to healthy MSCs and drastically impair the therapeutic potential. As such, an easy to implement additive to culture conditions that can reverse and/or prevent the senescent state in MSCs would be greatly impactful to preserve their therapeutic potential. We propose that microfluidic assembled monodisperse antioxidant crystals would be an appropriate additive to culture media that can help regulate the oxidative state of MSCs, minimize senescence, and preserve secretome functionality.

N-acetylcysteine (NAC) is a known antioxidant that has been shown to regulate the intracellular oxidative environment of cells, through both direct scavenging of ROS and stimulation of endogenous antioxidant enzyme production. A major limitation to NAC however is its short lifetime in the soluble form. As such, prolonged release of NAC would facilitate a less frequent dosing regimen. Polymer-directed assembly of small molecule antioxidants has attracted attention as a way to control drug release and efficacy. These systems usually rely on a polymeric excipient that can help form amorphic solid dispersions that decrease the crystallinity of hydrophobic antioxidants resulting in improved bioavailability. However, for hydrophilic drugs such as NAC, an increase in crystallinity is desired for prolonged release and redox control. In our system, to further prolong NAC release we designed a polymer excipient with thermodynamically favorable interactions with NAC that can form stabilized colloidal antioxidant crystals. Herein we report a unique antioxidant crystal that dissolves sustainably through polymer-stabilization using sodium hyaluronate conjugated with dopamine (HA-dopa). Furthermore, rather than using conventional batch crystallization, we adopted a microfluidic platform that can synthesize highly monodisperse NAC crystals decorated with stabilizing HA-dopa. We propose that crystal monodispersity is essential for minimal fluctuations in NAC release and would lead to both proper control of ROS homeostasis and minimal changes in desired MSC protein expression.

In this study we induced senescence in human MSCs via hydrogen peroxide exposure and then characterized the degree of senescence, intracellular ROS level, exosome secretion level, and gene expression for VEGF, IGF, and IL-10 following treatment with either bulk assembled NAC crystals or microfluidic assembled NAC crystals. The results from this study illustrate that NAC crystals with narrow crystal size distributions can revert the senescent state, restore ROS-homeostasis, preserve VEGF, IGF, and IL-10 gene expression, and minimize the coefficient of variation in all reported parameters compared to NAC crystals with large crystal size distributions. We propose that these results would be impactful for culture of engineered cell systems that may be impacted by oxidative stress.

Research Interests:

While my presentation only highlights one of my projects while obtaining my Ph.D., my other projects also revolve around antioxidant drug delivery to control the oxidative environment of cells and mitigate disease pathogenesis. Reactive oxygen species are a useful probe for understanding the current disease state and can also be simultaneously used to influence drug release kinetics. As such, my research interests extend to the design of transient or stimulus responsive drug release platforms with this core principle in mind. Furthermore, as drug delivery approaches become more advanced and sometimes patient specific, the use of novel microfluidic approaches to fabricate advanced materials for drug delivery or diagnostics is also a major interest of mine. Another approach I have been implementing in my current research is to not only engineer new drug delivery and targeted approaches, but to also use them to explore and build upon our understanding of disease progression. Answering fundamental biological questions pertaining to the dynamics of disease states is essential to advancement in therapeutic approaches, and as such would be at the core of my future research initiatives.