(M1530-03-16) On-Demand Endogenous Nanoparticle Based Cargo Loading of Extracellular Vesicles for Therapeutic Delivery

Purpose: Current cancer therapeutics typically have off-target effects, hindering their efficiency. Extracellular vesicles (EVs) are an emerging alternative therapeutic delivery platform, due to their high biocompatibility and long circulation times. However, low efficiency of EV cargo loading for large scale production remains a substantial gap for clinical utility. Herein, we developed a “smart” polymeric micelle as the first nanoparticle-based cellular loading tool (ExoPack) to enhance the site-specific packaging of cargos into EVs. The functionalized polymeric micelles were designed to carry therapeutic peptides for a photo-controlled, on-demand release into the EV biogenesis pathway. The photo-controlled release strategy enables specific cargo loading following cellular endocytosis, which is a precursor to EV packaging and secretion, all while retaining native EV properties. The ExoPack nanoparticles are also tagged with specific cellular receptor ligands for rapid cellular uptake and internationalization, leading to high-efficient cargo loading.

Methods: The ExoPack nanoparticles were self-assembled using a nanoprecipitation method from two polymeric components. The nanoparticle micelles were then incubated with mature dendritic cells and exposed to near-UV LED light for cargo release. Nanoparticle cellular uptake and cellular cytotoxicity were determined using flow cytometry. EVs were then isolated from the nanoparticle treated cell culture media using differential ultracentrifugation. The EVs were characterized by nanoparticle tracking analysis for size, zeta potential, and particle number, and by TEM for morphological determinations. Then, EV cargo loading efficiency following the endogenous ExoPack loading approach was measured using a custom ELISA kit. Finally, following the incubation of ExoPack based gp100 peptide loaded EVs with OT-1 T cells, the resulting T cell proliferation was determined through a CFSE dilution assay and assessments of T cell activation were performed using Granzyme B and perforin labeling.

Results: The characterization of ExoPack properties, cellular internalization, EV cargo loading efficiency, and T cell activation ultimately demonstrate the effective production of cargo loaded EVs following endogenous ExoPack based loading. Upon incubation with cells, ExoPack nanoparticle uptake increases steadily over time, while retaining significant cell viability (Fig. 1). Following nanoparticle cellular uptake, UV based cargo release, and endogenous EV loading, the resulting cellularly secreted EVs retained a size, zeta potential, concentration, and morphology similar to native EVs. However, the ExoPack treated EVs displayed enhanced peptide cargo loading (Fig. 2). These ExoPack based cargo loaded EVs were then able to enhance T cell proliferation and activation (Fig. 3). The upregulated T cell activation capabilities of ExoPack EVs compared to control native EVs and peptide alone establishes them as a promising cancer immunotherapy treatment strategy. Further investigation on ExoPack based peptide loaded EV immune response functionality in vivo through T cell activation and tumor elimination is currently underway.

Conclusion: Compared to other cellular engineering approaches, our ExoPack cargo loading strategy can be employed endogenously, without the need of genetic modifications of cells, therefore retaining the natural properties of cellularly secreted EVs. The endogenous ExoPack based cargo loading method also promotes the cellular production of therapeutic cargo loaded EVs, meeting demands for the large-scale, high-efficient production of well-designed therapeutic EVs. The resulting EVs have immunostimulatory functionality, which potentially opens a new avenue for precision cancer immunotherapy.

Acknowledgements: The authors would like to thank the Interdisciplinary Center for Biotechnology Research (ICBR) core facility at the University of Florida. We acknowledge funding support from NIH 1R35GM133794 and NIH TARGETED Challenge to Dr. Mei He. The authors claim no conflict of interest.


Cellular biocompatibility and uptake rates of ExoPack nanoparticles over time following treatment with mature dendritic cells.


Characterization of native EVs and EVs following endogenous ExoPack nanoparticle treatment in terms of EV size, concentration, zeta potential, morphology, and cargo loading concentrations.


T cell proliferation and activation following treatments of EVs, peptide, and peptide loaded EVs.