Exosomes are the nanoscopic lipid bi-layered extracellular vesicles with the potential to be utilized as targeted therapeutics. In our investigation, we compared three major exosome isolation techniques that were Total Exosome Isolation reagent (TEI), Protein organic solvent precipitation (PROSPR) and differential ultracentrifugation (UC) based on the biophysical and physicochemical characteristics of exosomes isolated from COLO 205 and MCF-7 cancer cell's conditioned media with an aim to select a suitable method for translational studies. 3D image analysis and particle size distribution of exosomes from their HRTEM images depicted the morphological differences. Molecular and analytical characterization of exosomes using western blotting, Raman and ATR-FTIR spectroscopy and the multivariate analysis on the spectral data obtained, assessed for better molecular specifications and purity of particle. TEI method isolated exosomes with higher exosomal yield, purity, and recovery directly translatable into drug delivery and targeted therapeutics whereas ultracentrifuge had good recovery of particle morphology but showed particle aggregation and yielded exosomes with smaller mean size. PROSPR technique isolated a mixture of EVs, showed lower protein recovery in PAGE and western blotting but higher spectroscopic protein to lipid ratio and distinguishable EV population in multivariate analysis compared to exosomes isolated by TEI and UC. This comparative study should help in choosing a specific exosome isolation technique required for the objective of downstream applications.

Exosomes are the nanoscopic lipid bi-layered extracellular vesicles that can deliver molecular medicine in the form of targeted therapeutics with great fidelity demonstrating enhanced permeability and high retention effect. In our investigation, we focused on the comparison of three major exosome isolation techniques based on the biophysical and physicochemical characteristics of exosomes isolated from COLO 205 and MCF-7 cancer cell's conditioned media. Commercially available Total Exosome Isolation reagent (TEI), Protein organic solvent precipitation (PROSPR) and differential ultracentrifugation are the three methods used for isolation with an aim to select a suitable method for clinical translation.HRTEM images of exosomes are subjected to 3D image analysis and particle size distribution of exosomes depicted the morphological differences. Molecular and analytical characterization of exosomes using western blotting, Raman and ATR-FTIR spectroscopy and the multivariate analysis on the spectral data obtained, assessed for better molecular specifications and purity of particle. TEI method isolated exosomes with higher exosomal yield, purity, and recovery directly translatable into drug delivery and targeted therapeutics whereas ultracentrifuge had good recovery of particle morphology but showed particle aggregation and yielded exosomes with smaller mean size. PROSPR technique isolated a mixture of EVs and showed the least recovery of exosomes in particle size distribution. This comparative study should help in choosing a specific exosome isolation technique required for the objective of downstream applications. IntroductionExosomes are the minuscule ultrafine bioparticles, the lipid vesicles with a potential to carry cellular cargo from one cell to another 1,2 . They are involved in cell-cell communication playing a major role in growth and development, immune response and even in regulating the tumor microenvironment and disease progression. Exosomes are efficient and robust in their function and have versatile cargo composed of uniquely sorted material carrying it from the cytosol of parent cell to the target cell throughout the body 3 . They are highly efficient due to their nonimmunogenic delivery, their ability to easily fuse with the cell membrane, avoid phagocytosis and circumventing the lysosomes via bypassing the engulfment 4,5 .Exosomes are the ideal drug delivery system with the applications of targeted therapeutics, prognosis and diagnostics of the diseases 6 . This is because the synthetic lipid nanoparticles like lipoplexes can only be used in vitro and not in vivo as a delivery system for gene therapy and transfection 7-9 . Also, in animal models, these lipoplexes elucidate a strong immune response with increased toxicity levels and accumulation in the liver, hindering the drug activity in the target 10 .The efficiency, biocompatibility, low accumulation of exosomes in organs and tissues with low toxicity levels are been harnessed for multiple novel therapeutic strategies [11][12][13] .During the dev...