Poster M20 - Stabilizing Milk-Derived Extracellular Vesicles (mEVs) through Lyophilization

Introduction: Extracellular Vesicles (EVs) are widely investigated for their implications in cell-cell signaling, immune modulation, disease pathogenesis, cancer, regenerative medicine, and as a potential drug delivery vector; however, maintaining integrity and bioactivity of EVs between Good Manufacturing Practice (GMP) separation/filtration and end-user application remains a consistent bottleneck towards commercialization. Milk-derived EVs (mEVs), separated from raw bovine milk, provide a relatively low-cost, scalable platform for large scale EV production; however, the reliance on cold supply chain for storage remains a logistical and financial burden for biologics that are unstable at room temperature.

Herein, we aim to characterize and engineer a freeze-dried, mEV formulation that can be stored at room temperature without sacrificing bioactivity and can be reconstituted before delivery. By adding appropriate excipients (e.g., crystallizing sugars like trehalose) we can create a solid formulation that preserves mEV bioactivity during long-term, room temperature storage.

Materials and

Methods: mEVs were isolated from raw bovine milk according to a previously established protocol. Briefly, raw milk is skimmed, defatted, chelated with EDTA, and subsequently filtered via Tangential Flow Filtration (TFF). Once isolated, mEVs were classified via Zeta Potential, to measure surface charge, Nanoparticle Tracking Analysis (NTA), to estimate particle size distribution, Transmission Electron Microscopy (TEM), to visualize mEV vesicles, and confocal fluorescent microscopy to measure mEV’s ability to retain loaded dye (Calcein-AM/Cell tracker deep red). Electric cell-substrate impedance sensing (ECIS) was used to monitor human dermal fibroblast (HuDF) migratory response after an induced wound, similar to a traditional scratch-assay, which provides insights to the mEV’s bioactivity. Lyophilization of mEVs was performed after samples were frozen on dry ice for 5 minutes and placed on maximum vacuum (20 mTorr), standard shelf temperature (20 ºC), and minimum condenser temperature (-96 ºC). Differential Scanning Calorimetry (DSC) was used to observe the heat transfer changes of powdered samples, specifically glass transition temperature (Tg) and membrane melting onset (Tm).

Results, Conclusions, and Discussions: We have shown that mEVs do not remain stable at room temperature over time, resulting in a significant loss in bioactivity after 1 month. Even in cold storage conditions, we found that mEVs lose structural integrity over time (at 4ºC and -80ºC) and lyophilized mEVs without any excipient are destroyed in the process of sublimation. However, the addition of 50mM Trehalose showed to retain mEV particle size and bioactivity in vitro in a concentration-dependent manner. Lyophilized samples with Trehalose generated a fine, white powder with a Tg well over room temperature (50-60 ºC). Furthermore, a composite mixture of Trehalose and additional membrane stabilizing excipients showed even better retention of structure and bioactivity post-lyophilization, which was comparable to freshly-isolated mEVs.

Therapeutically, mEVs have the potential for being a versatile therapeutic and drug delivery vector; however, large-scale GMP production and storage remains a significant bottleneck. Herein, we show that mEVs can be synthesized from raw bovine milk and can be stored at room temperature for extended periods of time without sacrificing gross structure and bioactivity, paving the way for the broader adoption and commercialization of mEV-based therapies, offering a scalable, cost-effective solution for drug delivery, and beyond.

Acknowledgements (Optional): The authors would like to thank Dr. Thomas Staley and the Materials Science and Engineering Department at Virginia Tech for use of their facilities and the Red Gates Foundation for funding this study.