Engineering Natural Killer Cell Derived Extracellular Vesicular Nanodrug for Tumor Targeted Bioimaging and Therapy

Natural Killer (NK) cells are large granular lymphocytes that belong to the innate immune system, and their major function is to provide host defense against microbial infections and tumors by immune surveillance of the cell surface to find the abnormal expression of major histocompatibility complex (MHC) class I molecules and cell stress markers. NK cells target cancer cells directly via inhibitory and activating receptors on cell surfaces and can kill cancer cells without prior sensitization, therefore NK cells are now at the forefront of the cell-based preclinical studies. However, their limited numbers (5–10% of peripheral blood lymphocytes) and their lower isolation yield (~2-5% yield) hinder their use in cell-based therapy. To overcome these limitations, high-yield cellular products such as extracellular vesicles (EVs) are of great interest because a single cell produces thousands of EVs equipped with parent cell-specific cargos of proteins, lipids, and genetic materials, which can be selectively taken up by neighboring or distant target cells. Within this background, we hypothesized that precision drug delivery will be enhanced when guided by EVs derived from NK cells that naturally seek cancer. Recently, the PI demonstrated experimental variabilities that affect EVs production and proteomic cargos using mouse macrophage and breast cancer cell-derived EVs. These EVs are heterogeneous in size and were found unstable for longer periods when stored at +4 to -80 °C. However, when engineered with liposomes, they are monodispersed and exhibit a higher order of colloidal stability and enhanced tumor delivery. Engineered EVs showed differential targeting and uptake against normal and cancerous cells, putting them in the group of potential tumor-targeted drug delivery candidates. These outstanding findings from engineered EVs further support the reported evidence that EVs carry functional properties from their parent cells, which are instrumental in target recognition. For example, EVs secreted by macrophages target tumors. Lesson learned from the EVs’ biology and our experience in liposomes, we further hypothesized that engineering EVs with liposomes will overcome their respective shortcomings, such as size heterogeneity and instability in EVs and the lack of targeting biomarkers in liposomes, to efficiently navigate complex biological system to deliver drugs to their targets. Aligned with this hypothesis, we propose to engineer hybrid EVs (HEVs) generated from the engineering of NKEVs with liposomes containing clinically used anticancer doxorubicin and near-infrared dye for image-guided downstream therapeutic analysis. Our specific aims are: Aim 1. To engineer NKEVs with liposomes as a nanodrug HEVs and study their physicochemical properties. Aim 2. To elucidate the biocompatibility and cellular specificity of HEVs. Aim 3. To evaluate the biodistribution and therapeutic efficiency of HEVs. We will develop two molecularly different; MCF-7 (ER/PR positive and HER2 negative) and MDA-MB-231 (triple negative, ER/PR/HER2 negative); human breast cancer xenografts and study biodistribution and therapeutic efficiency. The research team anticipates the results will assist drug delivery system designs in accelerating the precision imaging and therapy of cancer.