(4ej) Engineered End Fate of Artificially Transferred Mitochondria

My future research will be focused on the design of bio-inspired sequential therapeutic interventions with a specific emphasis on artificial mitochondria transfer. Analogous to natural cell-cell communication, artificial mitochondria transfer will synthesize the following processes in a progressive manner: (1) Probe the extracellular environment (2) Remediate the extracellular environment to preserve the viability of delivered mitochondria (3) Initiate the release of mitochondria (4) Manipulate recipient cell uptake of mitochondria and (5) End fate control of exogenous mitochondria. As such the objective of the proposed approach is to PRIME the local stem cell niche to achieve maximal tissue regeneration.

Previous Accomplishments

Artificial Mitochondria Transfer. Stem cell derived mitochondria are a promising therapeutic in regenerative medicine but also a very recent therapeutic consideration. As such, delivery of mitochondria has been limited to the injection of unpackaged mitochondria. To merge conventional drug-delivery concepts with artificial mitochondria transfer, I designed and characterized for the first time an artificial mitochondrial-transfer delivery system that incorporates peptide-driven uptake of exogenous mitochondria and MMP-responsive release from hydrogel microparticle vehicles. This work not only explored a new form of artificial mitochondria transfer, but also resulted in improved myogenic and osteogenic differentiation compared to the conventional free delivery approach in vitro. This work is now at the stage of in vivo validation in a critical-sized calvarial defect rat model.

[1] R.C. Miller, J.S. Temenoff, Microfluidic Assembly of Mitochondria-Loaded Microparticles for On-Demand Delivery, Tissue Engineering and Regenerative Medicine Conference Seattle, WA, June 2024.

Antioxidant Crystal Platforms for Oxidative Stress Control. The overproduction of reactive oxygen species (ROS) and the onset of oxidative stress in cells and tissue has been shown to have major implications in abnormal cell behavior and the pathogenesis of disease. Furthermore, in the field of regenerative medicine and biologics manufacturing, oxidative stress control is essential for the production of useful therapeutics. As such, antioxidant formulations are used to neutralize overproduced ROS. Polymer-directed crystallization of hydrophilic antioxidants has attracted attention as a way to control drug efficacy however limitations still exist with achieving extended release and minimal release variation. As result, ROS-homeostasis is rarely achieved. As such, I developed an advanced antioxidant crystal system that can overcome these drug delivery constraints and control the oxidative environment in injured cells and tissues. In my first publication, I detail the material design of hyaluronate-dopamine stabilization of N-acetylcysteine crystals with an emphasis on optimizing both drug–polymer and polymer–polymer interactions. Applications of this work were extended to addressing silver ion induced oxidative stress in cardiac muscle and daphnia magna. My follow-up work addressed release variation concerns using drop-microfluidics to assemble highly monodisperse crystals while overcoming limitations of crystallization efficiency in micro-drops. The application of the crystals was to control the senescent state in mesenchymal stem cells to improve biologics manufacturing for regenerative medicine. I further refined the crystal design to involve crystal encapsulation in hydrogel microparticles and this work is currently in preparation.

[1] R.C. Miller, J. Lee, Y.J. Kim, H.S. Han, H.J. Kong. In-drop thermal cycling of microcrystal assembly for senescence control (MASC) with minimal variation in efficacy. Advanced Functional Materials, 33 (37) (2023) 2302232

[2] R.C. Miller, Y. Kim, C.G. Park, C. Torres, B. Kim, J. Lee, D. Flaherty, H.S. Han, Y.J. Kim, H.J. Kong. Extending the bioavailability of hydrophilic antioxidants for metal ion detoxification via crystallization with polysaccharide dopamine. ACS Applied Materials and Interfaces, 14 (35)(2022) 39759–39774

The goal of my future research lab is to prioritize the design of artificial mitochondria transfer technologies while leveraging my thesis work on driving ROS-homeostasis. The relationship between mitochondria dysfunction and oxidative stress has been well documented and therefore I propose the co-delivery of antioxidant therapeutics with mitochondria to hopefully document a synergistic therapeutic effect when treating a range of inflammatory diseases and injuries. Since the field of artificial mitochondria transfer is new, I will divide my lab into two main thrusts: (1) the development of mitochondria transfer technology platforms and (2) the development of an organoid-based platform for screening parameters involved in mitochondria transfer.

Thrust 1: Design of artificial mitochondria transfer technology

Motivation. As detailed above, the PRIME approach will be the conceptual framework of designed artificial mitochondria technologies. In principle a dual-gel system can be employed to stagger the release of multiple therapeutic agents. The core gel will contain mitochondria where the gel shell will contain antioxidants. Since ROS can depolarize the mitochondrial membrane and reduce the viability, a ROS-liable gel shell will be designed to respond and release the antioxidant cargo during high levels of extracellular ROS. The goal is that ROS-homeostasis is achieved prior to the release of the mitochondria by a secondary trigger. Mitochondria will be functionalized with specific peptides to drive the endocytosis of the exogenous mitochondria and co-delivered mitochondria fusion proteins (e.g. OPA1) will be used to promote the incorporation the exogenous mitochondria. This type of system will be explored in the context of multiple injury models ranging from musculoskeletal to neurological.

Thrust 2: Organoid-based platform to screen tissue level changes following mitochondria transfer

Motivation. Mitochondria are known to have their own DNA (mtDNA). As such, mitochondria proteins are encoded both in the mitochondria as well as in the nucleus. Therefore, mitochondria are highly heterogeneous across cell type. Furthermore, mitochondria are fundamental to eukaryotic cell metabolism as well as many different cell-type specific processes. Due to the dynamic nature of mitochondria, the resulting efficacy following mitochondria transfer might look very different depending on recipient cell/tissue type. With all of this in mind, a microfluidic platform that can deliver mitochondria to multiple organoid models would be invaluable when screening for optimized mitochondria donor cells and understanding fundamental therapeutic mechanisms.

Teaching Interests

As a faculty member, I will dedicate my teaching to three main areas (1) research mentorship (2) classroom-based education and (3) scientific outreach. Throughout my graduate and postdoctoral positions, I have had the opportunity to develop my teaching skills in all three categories. As a graduate student and postdoctoral fellow, I have actively mentored 4 undergraduate students and 2 high school students in the areas of research. While all students had unique backgrounds, by the end of their training they had become capable of independently designing, performing, and analyzing an experiment. All students have had the opportunity to present their work at local symposia and receive feedback from peers and professors in the field. This method of active learning deemed very beneficial and will remain part of my teaching efforts. While in graduate school I had the opportunity to co-teach a biotransport course with my PI. I learned how to design curriculum, teach long-form and short-form lectures, and design homework and test questions. This process was very rewarding since this same type of education is what led me to pursue a higher education. Finally, I have engaged with multiple scientific outreach initiatives, but I would like to put an emphasis on the Expanding Career, Education, and Leadership (EXCEL) program at Georgia Tech. As an EXCEL mentor, I actively mentored a student with intellectual and developmental disabilities on developing skills that can translate to “lab technician-like” roles in academia or industry. This experience touches a personal chord as my cousin has autism and although his intellectual capabilities are high, I know it will be difficult for him to hold a job that requires communication skills. This program has been established at Georgia Tech for many years and has seen great success. I would love to provide a similar opportunity in my lab whether that be through a departmental program or a personal initiative.