Researcher(s)
- Hashim Kenyi, Chemical Engineering, University of Delaware
Faculty Mentor(s)
- Catherine Fromen, Chemical and Biomolecular Engineering, University of Delaware
Abstract
Authors: Hashim Kenyi, Dhana Mahadik, Catherine A. Fromen*
Previous work from the Fromen Laboratory has shown that nanoparticle uptake by macrophages can increase extracellular vesicle (EV) production. EVs are lipid-bound particles secreted by cells that mediate intercellular communication and contribute to immune function. However, their therapeutic potential is limited by the lack of standardized storage methods that preserve EV structure and cargo. This project examined nanoparticle (NP) uptake by macrophages and evaluated lyophilization as an alternative EV storage strategy.
RAW264.7 macrophages were dosed with poly(ethylene glycol) diacrylate NPs formulated with either negatively charged 2-carboxylethyl acrylate (CEA) or positively charged 2-aminoethyl methacrylate (AEM) to evaluate phagocytic uptake. NP size, polydispersity index (PDI), and zeta potential were characterized by dynamic light scattering (DLS), while NP concentration was evaluated using thermogravimetric analysis (TGA). Uptake was visualized over time using Cytation fluorescence imaging and quantified by flow cytometry based on NP-associated fluorescence.
Following NP dosing, EVs were isolated by ultracentrifugation. The laboratory’s current storage standard is PBS at 4 °C. To evaluate an alternative for room-temperature storage, EVs were lyophilized in PBS, PBS with 5% w/v sucrose, or PBS with 1% w/v sucrose and 4% w/v mannitol, then reconstituted in DI water after two weeks. EV stability was assessed using nanoparticle tracking analysis (NTA) and DLS. The PBS–sucrose–mannitol formulation produced the highest recovered particle concentration and demonstrated a lower average size and PDI than the other formulations.
Together, these findings advance understanding of charge-dependent NP–macrophage interactions and support lyophilization with sucrose and mannitol as a promising strategy for preserving EVs beyond conventional refrigerated storage.