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Dissertation Defense: The Presence and Bioactivity of Connexin-43 and Pannexin-1 in Milk-derived Small Extracellular Vesicles

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Md Ruhul Amin
Dissertation Defense

The Presence and Bioactivity of Connexin-43 And Pannexin-1 in Milk-Derived Small Extracellular Vesicles

Md Ruhul Amin

Graduate Student, Translational Biology, Medicine, and Health
Graduate Research Assistant, Gourdie Lab, Fralin Biomedical Research Institute at VTC
Sept. 18, 2026, at 2 p.m.
Room G101 A/B, 4 Riverside Circle
Visit the Gourdie Lab

About this Dissertation

Milk-derived small extracellular vesicles (mEVs) are an emerging platform for oral delivery of peptide therapeutics, but their potential as both an engineerable delivery vehicle and a naturally bioactive agent in their own right remains incompletely understood. This dissertation investigates the presence, delivery utility, and endogenous bioactivity of two channel-forming membrane proteins, Connexin 43 (Cx43) and Pannexin-1 (Panx1), in bovine mEVs, and tests the premise that channel-protein cargo defines functionally distinct, bioactive subpopulations within an otherwise heterogeneous mEV pool. First, Pannexin-1 and Cx43 are shown to be present in a defined subpopulation of bovine mEVs, using western blotting, single-molecule localization microscopy, and flow cytometry-based single-vesicle detection, demonstrating mEV cargo heterogeneity. Secondly, an esterification-based strategy is developed for loading the Cx43 mimetic peptide αCT11 into mEVs. Orally administered, αCT11-loaded mEVs reduced infarct size by approximately 60% and preserved cardiac function in a murine model of ischemia-reperfusion injury, with efficacy retained when treatment was delayed by up to two hours after reperfusion. Finally, returning to the native, unloaded mEV population, a subpopulation intrinsically enriched in endogenous Cx43 is isolated using collagen-binding capacity as an indirect, function-based isolation strategy. This subpopulation was enriched approximately 22-fold in Cx43 relative to the unselected mEV pool and conferred significant radioprotection in a normal mammary epithelial cell model, exceeding that of unselected mEVs. Together, these findings establish milk EVs as both a natural signaling reservoir, whose bioactivity is concentrated in cargo-defined subpopulations, and an engineerable oral delivery platform for Cx43-targeted peptide therapeutics. This dissertation further establishes collagen-affinity selection as a generalizable, peptide-free strategy for isolating functionally defined extracellular vesicle subpopulations that lack a directly targetable surface marker, with potential applications extending beyond this specific system.

More About the Candidate and Project

Education

Virginia Tech, Translational Biology, Medicine, and Health, Ph.D. Candidate

BRAC University, M.Sc., Biotechnology

University of Dhaka, Bachelor of Medicine and Surgery (MBBS)

Training

Graduate Research Assistant, Gourdie Lab, Fralin Biomedical Research Institute at VTC

Intern Doctor, Mymensingh Medical College Hospital, Dhaka, Bangladesh

Mentors

Robert Gourdie, Ph.D., FAHA, AIMBE, Heywood Fralin Professor, Commonwealth Research Commercialization Fund Eminent Scholar in Heart Reparative Medicine Research, Interim Director, Cancer Research Center — Roanoke

Committee Members

Publications

Amin MR, Lindsay S, Beard C, Marsh SR, Heredia S, Athanasiadi I, Stout RF, Payne LB, and Gourdie RG. A Collagen-Binding Subpopulation of Milk-Derived Small Extracellular Vesicles is Enriched in Endogenous Connexin-43 and Confers Radioprotection In Vitro. (In preparation).

Marsh SR, Toldo S, Amin MR, Beard C, Dogan AB, Mezzaroma E, Andres E, Stout RF, Bannon MS, Kelly J, Toldo M, Gourishankar AS, Payne LB, Abbate A, Sassi Y, Letteri RA, and Gourdie RG. Oral connexin43 -peptide-loaded milk extracellular vesicles enable delayed, injury-targeted cardioprotection via ATP-primed uptake. Nature Biomedical Engineering (In Revision).

Marsh SR, Beard C, Sheng Z, Bannon MS, Amin MR, Letteri RA, Athanasiadi I, Gourdie RG. Oral connexin43–peptide–loaded milk extracellular vesicles mitigate lethal radiation injury while preserving tumour radiosensitivity. Cancer Letters. 2026:218686. doi: https://doi.org/10.1016/j.canlet.2026.218686. 

Amin, M.R., Marsh, S.R., Beard, C. et al. Pannexin-1 is present in a subpopulation of bovine milk–derived small extracellular vesicles. Cell Tissue Res 403, 13 (2026). https://doi.org/10.1007/s00441-026-04044-x

Dogan AB, Marsh SR, Tschetter RJ, E. Beard C, Amin MR, Jane Jourdan L, Gourdie RG. Stabilizing milk-derived extracellular vesicles (mEVs) through lyophilization: a novel trehalose and tryptophan formulation for maintaining structure and Bioactivity during long-term storage. Journal of Biological Engineering. 2025;19(1):4. doi: 10.1186/s13036-024-00470-z.

Amin MR, Marsh S, Beard C, Jourdan J, Gourdie R. Abstract Tu005: Small Extracellular Vesicles Derived from Bovine Milk Contain an Endogenous Carboxyl Terminal Polypeptide of The Gap Junction Protein Connexin-43. Circulation Research. 2024;135(Suppl_1):ATu005-ATu. doi: 10.1161/res.135.suppl_1.Tu005. 

Sujon H, Sarker MHR, Uddin A, Banu S, Islam MR, Amin MR, Hossain MS, Alahi MF, Asaduzzaman M, Rizvi SJR, Islam MZ, Uzzaman MN. Beyond the regulatory radar: knowledge and practices of rural medical practitioners in Bangladesh. BMC Health Services Research. 2023;23(1):1322. doi: 10.1186/s12913-023-10317-w. 

Hossain MU, Bhattacharjee A, Emon MTH, Chowdhury ZM, Ahammad I, Mosaib MG, Moniruzzaman M, Rahman MH, Islam MN, Ahmed I, Amin MR, Rashed A, Das KC, Keya CA, Salimullah M. Novel mutations in NSP-1 and PLPro of SARS-CoV-2 NIB-1 genome mount for effective therapeutics. Journal of Genetic Engineering and Biotechnology. 2021;19(1):52. doi: https://doi.org/10.1186/s43141-021-00152-z.

Moniruzzaman M, Hossain Mohammad U, Islam MN, Rahman MH, Ahmed I, Rahman Tahia A, Bhattacharjee A, Amin MR, Rashed A, Keya Chaman A, Das Keshob C, Salimullah M. Coding-Complete Genome Sequence of SARS-CoV-2 Isolate from Bangladesh by Sanger Sequencing. Microbiology Resource Announcements. 2020;9(28):10.1128/mra.00626-20. doi: 10.1128/mra.00626-20.

Chowdhury FR, Hoque A, Chowdhury FUH, Amin MR, Rahim A, Rahman MM, Yasmin R, Amin MR, Miah MT, Kalam MA, Rahman MS. Convalescent plasma transfusion therapy in severe COVID-19 patients- a safety, efficacy and dose response study: A structured summary of a study protocol of a phase II randomized controlled trial. Trials. 2020;21(1):883. doi: 10.1186/s13063-020-04734-z

Oral Presentations

Endogenous and Exogenous Cx43 Sequences in Milk Extracellular Vesicles in Cardiac Ischemia. Mid-Atlantic Nexin Group Organization (MANGO) symposium, UVA, 2025.

The Potential Presence of Endogenous Carboxyl Terminal Polypeptides of Connexin-43 in Small Extracellular Vesicles Derived from Bovine Milk. Fralin Biomedical Research Institute Retreat, 2024.

Poster Presentations

Amin MR, Lindsay S, Beard C, Marsh SR, Heredia S, Athanasiadi I, Stout RF, Payne LB, and Gourdie RG. The presence and bioactivity of Connexin-43 in milk-derived small extracellular vesicles. Vascular and Heart Research Symposium at Fralin Biomedical Research Institute, 2026.

Amin MR, Marsh SR, Toldo S Beard C, Gourdie RG. Bovine milk-derived small extracellular vesicles loaded with Connexin-43 peptides provide cardioprotection after ischemia-reperfusion injury. American Association of Extracellular Vesicles Annual Meeting, Salt Lake City, UT, 2025.

Amin MR, Marsh SR, Beard C, Payne LB, Gourdie. A Subpopulation of Small Extracellular Vesicles Derived from Bovine Milk Contain Endogenous Carboxyl Terminal Polypeptides of the Gap Junction Protein Connexin-43. 8th Annual Mid-Atlantic Advanced Biomanufacturing Symposium, 2025, UVA.

Amin MR, Marsh SR, Beard C, Jourdan J, Gourdie RG. Small Extracellular Vesicles Derived from Bovine Milk Contain Membrane Channel Proteins Pannexin-1 and Connexin-43. International Gap Junction Conference, Washington DC, 2024.

Amin MR, Marsh SR, Beard C, Jourdan J, Gourdie RG. Small Extracellular Vesicles Derived from Bovine Milk Contain an Endogenous Carboxyl Terminal Polypeptide of The Gap Junction Protein Connexin-43. Basic Cardiovascular Sciences Scientific Sessions, Chicago, 2024.

Amin MR, Marsh SR, Beard C, Jourdan J, Gourdie. Small Extracellular Vesicles Derived from Bovine Milk Contain Membrane Channel Proteins Pannexin-1 and Connexin-43. Vascular and Heart Research Symposium at Fralin Biomedical Research Institute, 2024.

Amin MR, Marsh SR, Beard C, Jourdan J, Gourdie. Small Extracellular Vesicles Derived from Bovine Milk Contain Membrane Channel Proteins Pannexin-1 and Connexin-43. Virginia Drug Discovery Consortium symposium, Virginia Beach, 2024.

Amin MR, Marsh SR, Beard C, Jourdan J, Gourdie RG. The potential presence of a novel and endogenous truncated isoform of Cx43 in milk-derived small extracellular vesicles. Vascular and Heart Research Symposium at Fralin Biomedical Research Institute, 2023.

  • Dr. Ray A. Gaskins Health Sciences Graduate Fellowship (2025-26)
  • Best Poster (Third Place): International Gap Junction Conference, 2024
  • Best Poster (Third Place): Vascular and Heart Research Symposium at Fralin Biomedical Research Institute, 2024
  • Served as a judge in the Roanoke County Science Fair, 2024
  • Served as a mentor in the Translational Biology, Medicine, and Health Graduate Program mentor-mentee program, 2023
  • Served as an IP (Intellectual Property) consultant for a team in the Translational Biology, Medicine, and Health Shark Tank competition, 2022