Optimizing genetic engineering approaches for protein loading into bacterial extracellular vesicles for vaginal drug delivery.

Abstract

There is a critical gap in the development of new therapeutic platforms designed to treat gynecologic and obstetric diseases. Compared to systemic drug delivery, vaginal administration of nanoparticle formulations limits off-target side effects while increasing therapeutic concentration in target tissues, showing promise for clinical translation. However, these formulations suffer from limited scalability, high-cost reagents, and long optimization timelines. Recent work highlights the potential of bacterial extracellular vesicles (bEVs) as a low-cost, tunable platform for therapeutic applications. Here, we evaluate bEVs as a therapeutic carrier for vaginal drug delivery. We demonstrate the loading of the model protein moxNeonGreen into Escherichia coli Nissle 1917-derived bEVs. By optimizing growth parameters, we increase protein loading into bEVs. We evaluate the effect of bEVs on the vaginal microenvironment, and observe no negative impact on vaginal epithelial cells, endocervical cells, or vaginal bacteria in vitro. Additionally, we observe the retention of bEVs in the murine female reproductive tract for more than six hours. This study provides a framework for using genetically engineered bEVs to rapidly generate customizable therapies for a range of gynecologic and obstetric conditions, addressing longstanding challenges in women's health therapeutics.Copyright © 2026 The Authors. Published by Elsevier B.V. All rights reserved.

Authors

Darby Steinman, Varunaa Sri Hemanth Kumar, Ryan A McIlvaine, Pranshu Tyagi, Hahnbit Kang, Raifah Alam, Anguo Liu, Christopher M Jewell, Amy Plotkin, Irina Burd, Sara Molinari, Hannah C Zierden

Fischell Department of Bioengineering, University of Maryland, College Park, MD 20742, USA; Robert E. Fischell Institute for Biomedical Devices, University of Maryland, College Park, MD 20742, USA; Department of Chemical & Biomolecular Engineering, University of Maryland, College Park, MD 20742, USA; Department of Obstetrics, Gynecology and Reproductive Sciences, University of Maryland School of Medicine, Baltimore, MD 21201, USA. Electronic address: hzierden@umd.edu.