Advancements in Lyophilized Exosomes for Enhanced Stability and Therapeutic Applications

Introduction:

Extracellular Vesicles (EVs), such as exosomes, exhibit significant potential across various pathological conditions and the global EV market is expected to exceed $500 million by 2026.  The clinical applicability of these models is, however, limited by their preservation and storage at -80°C. Lyophilization is a great method to achieve a stable biopharmaceutical product and this technology could be explored as a novel strategy for EV preservation. Understanding the interplay between cryoprotectants and lyophilization could accelerate EV research and further their therapeutic potential.

Methods:

We conducted freeze-thaw studies on EVs with five different buffers. EVs were then lyophilized using ten cryoprotectant (CPA) combinations, with subsequent evaluation of particle concentration and size via Nanoparticle Tracking Analysis (NTA). Finally, four rehydration buffers were tested to ensure isotonicity, with the most effective selected for downstream assays. To evaluate functionality of lyophilized EVs, we performed cell migration assays and lipopolysaccharide (LPS) challenge assays using primary human skin cells (dermal fibroblasts and keratinocytes) and monocytes (ThawReady™ THP-1 NF-kB-Luc2), respectively.  

Results:

Freeze-thaw (FT) studies were performed on EVs prior to lyophilization to select the most effective EV buffer. Among the 5 buffers tested, HEPES Buffered Saline (HBS) showed a significantly higher EV particle concentration with no particle loss, and it efficiently maintained EV particle size up to 3 FT cycles. Ten different CPA combinations consisting of either single or combination of CPAs were tested and evaluated for EV particle concentration and particle size by NTA. We identified a formulation that resulted in a more consistent particle size and concentration as compared to the non-lyophilized control. To maintain isotonicity, 4 different rehydration buffers (RHBs) were tested. The RHB that maintained the most effective tonicity was chosen for use in downstream assays. Functional assays confirmed that lyophilized EVs exhibit both pro-migratory and anti-inflammatory properties, which are dependent on the cryoprotectant and RHB.  These findings underscore the critical role of selecting appropriate CPAs and RHBs for maintaining the functional integrity of lyophilized EVs. 

Conclusion:

Overall, the present study clearly demonstrates that successful lyophilization of EVs can be achieved by optimizing the combinations of cryoprotectants and rehydration buffers. Importantly, our EV lyophilization process preserved the functional integrity of EVs, demonstrating their potential for long-term stable formulations.