Optimized lyophilization protocol for better efficacy and storage capabilities of probiotic extracellular vesicles used in gastrointestinal disease treatment

Abstract

The Nanoparticle drug delivery system is a current research area that many are working on to explore the possible therapeutic capability for treatment. With the usage of nanoparticles, we are hopeful of better drug delivery capabilities and a higher rate of treatment success. In this study, we look at a specific type of nanoparticle, the cell-derived nanovesicles, and specifically for this project, the Extracellular Vesicles (EVs) that are derived from the Lactobacillus rhamnosus GG(LGG) probiotic. These would then be used in the treatment of Inflammatory Bowel disease which comes in the form of either ulcerative colitis or Crohn’s disease. However, the storage capability of such a novel treatment method is greatly compromised by the delicate and intricate properties of the EVs which require it to be stored in specific conditions of 81 degrees Celsius. Therefore, lyophilization has been used to aid in this process. These EVs that would be used in the drug formulation would then be treated with cryoprotectants, the disaccharides of either sucrose or trehalose in the ratio of (1:100). These would then be put to a storage test by leaving them in 2 different temperature conditions of 4℃, 25℃. Respectively to study the effects of different storage environments and the viability of EVs. After which efficacy tests were carried out to verify the effectiveness of these EVs being stored in different conditions. This includes TEER and QPCR tests to determine whether the structural integrity is retained or compromised, followed by detecting if there are immune response and immunomodulatory effects. Results show that lyophilization with cryoprotectants was able to maintain the structural integrity of the EVs while enhancing the storage capability of it. Sucrose specifically showed better results in general compared to trehalose, which can translate to being the better lyophilization protocol to be used to treat the probiotic EVs. For the aspect of therapeutic effects, sucrose-treated EVs performed better in enduring a tighter barrier, while trehalose showed better results in eliciting an immune response. The reason for such may be due to sugar toxicity which can be further studied