Poster CC14 - PEGDA NP Internalization by Bone-Marrow Derived Macrophages Drives Extracellular Vesicle Secretion, Inducing a Pro-Survival Response

Introduction: Nanoparticle (NP) carriers provide a multi-functional, engineered, base platform for novel immunotherapies targeting antigen-presenting cells (APCs). Researchers have shown these APCs have intrinsic cellular responses following the internalization of these NPs, often focusing on the immunological activation of macrophages or dendritic cells (DCs). However, the intrinsic cellular response of NPs when it relates to extracellular vesicle (EV) secretion and functionality remains limited in scope.

Materials and

Methods: Herein, we report the use of a poly (ethylene glycol) diacrylate (PEGDA) NP chemistry, previously shown to have minimal effect on the phenotype of bone marrow-derived macrophages (BMM), to elucidate changes to BMM secretion of EV population. Using differential ultracentrifugation and nanoparticle tracking analysis, we characterized a dose-dependent relationship between NP dosed and EV secreted in BMM and RAW 264.7, an engineered macrophage cell line. We then used brightfield microscopy and image analysis to quantify changes in culture population following EV dosing, while using luminescence-based metabolic assays to measure as a secondary measure. Furthermore, we used flow cytometry to characterize changes to BMM phenotype following dosing with different stimuli-derived BMM EVs. Flow Cytometry was then further used to measure the internalization of delivered PEGDA NP through a Cy5-tag.

Results, Conclusions, and Discussions: Following NP internalization, BMM and RAW 264.7 cultures have a positive correlation between NP internalization and EV secretion, up to an 8-fold increase in total EV secretion. Furthermore, within the population of secreted EVs, CD63+ EVs showed encapsulation of PEGDA fragments. We go on to show EVs derived from NP-dosed BMMs promote survival and increased metabolic activity in naive BMM cultures. To our knowledge, these results are the first to show a correlation between hydrogel NPs and EV secretion. These findings reveal a new response to particle internalization with ramifications across the immune system and point to the potential engineering for this EV secretion response as a method for in situ EV-mediated transfer of NP therapeutics.

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