(275e) Stimulative Piezoelectric Nanofibrous Scaffold (PES) for Enhancing the Biogenesis of Small Extracellular Vesicles

Small extracellular vesicles (sEVs) are promising nanocarriers for drug delivery. However, there are still challenges to efficiently produce sEVs, which hinder their clinical applications. To help facilitate this challenge, we developed a stimulative 3D culture platform for enhancing sEV production, consisting of a piezoelectric nanofibrous scaffold (PES) activated through the application of low-frequency acoustic waves to produce cell stimulation in a 3D biomimetic microenvironment (Fig A). By combining cell stimulation and 3D culture in our PES platform, we demonstrated a 49-fold increase in production rate per cell in hepatocellular carcinoma cells, and an 11-fold enhancement in mouse 3T3 fibroblast cells with minimal deviation in particle size and protein composition compared to standard 2D culture (Fig B). During our investigation, we discovered a 16% increase in ATP levels, and a 1.5-fold higher concentration of calcium ions in cells cultured in the stimulative PES platform compared to 2D culture (Fig C-D). We found that the enhanced sEV production is attributed to the activation and upregulation of crucial sEV production steps regulated by metabolites, and secondary messengers, by the synergetic effect of stimulations and 3D microenvironment. Moreover, cell morphology changes that lead to cytoskeleton redistribution due to cell-matrix interactions in 3D culture facilitate intracellular sEV trafficking and thus impact the production rate (Fig E). Overall, our work provides a promising 3D cell culture platform based on piezoelectric biomaterials to enhance sEV production and accelerate the potential use of sEVs for drug delivery and broad biomedical applications.

Abstract Figure: A. Schematic of the stimulative PES for enhanced sEV production. B. The measured EV production rate per cell using ExoELISA. C. ATP levels for all groups at various cell counts using Celltiterglo. D. The relative Ca²⁺ concentrations of all groups measured by Fura-2AM. E. The correlation between the roundness and the sEV production rate and fold change in production rate post stimulation.