Targeted delivery of efficacious drug levels with minimal off-target effects remains a major pharmacological challenge as exemplified by the poor delivery of proteins, antisense oligonucleotides (ASO), and other therapeutics to may organs. Drug delivery platforms that allow for controlled pharmacokinetic profile and cell targeting, including nanoparticles, liposomes and cell-derived extracellular vesicles (EV), are thus aggressively pursued by biopharma. However, several key gaps remain in engineering the appropriate size and surface composition of drug carrier nanoparticles, which dictates their biodistribution. Despite their favorable immunogenicity profile, EVs generated from human stem cells or immortalized cell lines invariably show entrapment by reticuloendothelial (RES) cells in liver, spleen, and bone marrow following intravenous (IV) dosing. In contrast, the persistently high plasma level of endogenous, organ derived EVs likely reflects reduced clearance and longer circulation times, both properties important for allowing for efficient payload delivery to multiple target tissues. Also, surface molecular signatures comprised of exposed proteins and lipids, which vary across plasma EV subsets, are likely to dictate their tropism to specific organs or cells. This research will capitalize on the naturally engineered properties of endogenous EVs to develop the first human plasma derived drug delivery product. We have developed a novel and sensitive multiplexed immunoassay, suitable for the use with unprocessed plasma, to characterize plasma EV subsets based on specific profiles of surface-exposed proteins. This method enabled us to discover of a novel endogenous subset of nano-EVs (nEV), whose small diameter (10-40 nm) contrasts sharply with the 50-200 nm diameter reported for most EVs. The nEVs contain protein and RNA markers of cells representing several organs as well as surface lipid and protein features to reduce RES clearance. We hypothesize that this newly discovered EV subset is naturally adapted for long-range inter- organ communications, and thus ideally suited for drug delivery. This project will develop a robust and scalable method for isolation of large amounts of nEVs from commercially acquired human plasma. We will also quantitatively characterize in vivo nEVs PK and biodistribution using in vivo positron emission tomography (PET) and fluorescence imaging. The following specific aims will be pursued in the current proposal: Aim 1: Nano-EV production: scale-up and characterization. Aim 2: Analysis of nEV biodistribution and plasma half-life. By demonstrating that nEVs have improved biodistribution properties over traditional EVs and that they can be easily isolated in large amounts from human plasma then phase 2 of our SBIR will focus on optimizing drug loading and demonstrating efficacy in disease models.
Public Health Relevance Statement: NARRATIVE Extracellular vesicles (EVs) are membrane-bound nanoparticles released by all cell types that transfer proteins and RNA between cells and tissues; however, most attempts to implement EVs for drug delivery utilized production in cell culture and show no significant improvement over artificial liposomes, likely due to low representation of EV subpopulation suitable for long-range transport. Here we propose drug delivery using plasma EVs, specifically a newly identified smaller plasma nanoEVs, which offer potential advantages due to improved safety, biodistribution and potential brain penetration.
Project Terms: Blood; Blood Reticuloendothelial System; Bone Marrow; Bone Marrow Reticuloendothelial System; Brain; Brain Nervous System; Encephalon; Cell Culture Techniques; cell culture; cell cultures; Cell Line; CellLine; Strains Cell Lines; cultured cell line; Cells; Cell Body; Ion-Exchange Chromatography Procedure; Ion Exchange Chromatography; Communication; Drug Carriers; Pharmaceutical Preparations; Drugs; Medication; Pharmaceutic Preparations; drug/agent; Engineering; Half-Life; Human; Modern Man; Immunoassay; Lipids; Liposomes; Liposomal; Liver; hepatic body system; hepatic organ system; Methods; Drug Kinetics; Pharmacokinetics; Plasma; Blood Plasma; Plasma Serum; Reticuloendothelial System, Serum, Plasma; Blood Plasma Volume; Plasma Volume; Positron-Emission Tomography; PET; PET Scan; PET imaging; PETSCAN; PETT; Positron Emission Tomography Medical Imaging; Positron Emission Tomography Scan; Rad.-PET; positron emission tomographic (PET) imaging; positron emission tomographic imaging; positron emitting tomography; Production; Proteins; Research; RNA; Non-Polyadenylated RNA; RNA Gene Products; Ribonucleic Acid; Safety; Spleen; Spleen Reticuloendothelial System; Testing; Tissues; Body Tissues; Measures; Antisense Oligonucleotides; Anti-Sense Oligonucleotides; Antisense Agent; anti-sense agent; anti-sense oligo; antisense oligo; Drug Delivery; Drug Delivery Systems; customs; Custom; Organ; improved; Procedures; Surface; Clinical; Penetration; Phase; Tropism; Therapeutic; Intravenous; Autologous; cell type; experience; Membrane; membrane structure; cell immortalization; novel; Disease model; disorder model; Reporting; Property; RNA marker; Size Exclusion Chromatography; Molecular Sieve Chromatography; Molecular Interaction; Binding; Dose; Molecular Profiling; Molecular Fingerprinting; molecular profile; molecular signature; Reproducibility; in vivo; Small Business Innovation Research Grant; SBIR; Small Business Innovation Research; Radiolabeled; radiolabeling; radiologically labeled; nano; human stem cells; immunogenicity; nano particle; nano-sized particle; nanosized particle; nanoparticle; Biodistribution; site targeted delivery; targeted delivery; scale up; fluorescent imaging; fluorescence imaging; multiomics; multiple omics; protein markers; protein biomarkers; extracellular vesicles; delivery vehicle; delivery vector; pharmacologic; Circulation; Diameter
