Ascorbic acid formulation using milk-derived extracellular vesicles: a stepwise process evaluation

Labile hydrophilic compounds, such as ascorbic acid (AA), present challenges for nanoscale formulation due to their chemical instability and strong tendency to recrystallize during processing and storage. Although various nanocarrier-based formulation approaches have been developed, achieving physical stability while maintaining biological compatibility remains difficult. In this study, we aimed to develop a practical and biocompatible formulation approach using milk-derived extracellular vesicles (mi-EVs) and to establish a solvent-free formulation strategy through stepwise evaluation of processing conditions. An AA formulation based on mi-EVs (milkCsome) was developed through sequential evaluation of key processing parameters. Sequential physical treatment—sonication followed by rotation—improved the visual homogeneity of the formulation, with no visible precipitation observed after 16 h of storage at 4 °C under the selected conditions. Under the selected processing conditions, milkCsome achieved approximately 16% AA retention after purification and retained antioxidant activity. Biological evaluations showed no detectable reduction in cell viability under the tested conditions, and the formulation was classified as non-irritant in a reconstructed human skin model. Overall, this study presents a simple, solvent-free process for preparing a milk-derived extracellular vesicle-based AA formulation with antioxidant activity and biological compatibility under the conditions tested.