Project Grant R43GM150371

NATURAL PLASMA NANO-EVS FOR DRUG DELIVERY - ABSTRACT 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.