Isolation and Characterization of Exosomes with HSV Glycoprotein D for Targeted Delivery into White Blood Cells

Abstract

Exosomes are naturally occurring extracellular vesicles with diameters varying from 30nm to 150nm that are enclosed by a lipid bilayer membrane like that of a human cell membrane. They are secreted by different types of cell lines and cells such as neural, hematopoietic, fibroblast, epithelial, muscle, stem cell, and tumor cells, and have been found in surrounding biological fluids. These nanoscale exosomes efficiently transport a variety of cargo, including lipids, metabolites, functional proteins, and nucleic acids, to the target cell, where they play a crucial role in intra- and intercellular communication. Their natural delivery capability makes them an ideal candidate for therapeutics delivery systems compared to synthetic counterparts, which often exhibit limitations such as immunogenicity, cytotoxicity, biocompatibility, and adverse side effects. Both natural exosomes and those that have been modified with other substances to increase their target delivery ability have been used. This study has focused on natural delivery vehicles such as exosome-based targeted drug delivery vehicles that provide unique opportunities for the delivery of therapeutics across specific physical barriers and address challenges posed by synthetic drug delivery systems. In this study, exosomes were modified for targeted therapeutics delivery by surface engineering with the peptide of Herpes simplex virus Glycoprotein D that binds to the herpes virus entry mediator (HVEM) receptor. The cell-targeting function of these engineered exosomes was introduced by expressing HSV glycoprotein D in HEK293T cell and followed by isolation of exosome from supernatant of engineered cells. Characterization using vi scanning electron microscopy confirmed the cup-shaped morphology and size of the exosomes, and the presence of the target gene was validated through western blotting. These engineered exosomes exhibit selective binding to white blood cells via the HVEM receptor that is present in blood cell populations such as white blood cells (Natural killer cells, monocytes, T cells, B cells), epithelial cells, and endothelial cells, offering potential application in treating leukemia (, B-cell leukemia, T-cell leukemia, Myeloid leukemia), autoimmune diseases, immunodeficiency diseases, and tumor, etc. by loading therapeutic agents into them