Development of Lymphatic‐Targeted Fluorescent Organic Nanoparticles to Label Cells in vitro

Abstract

We have developed a new class of hyaluronic acid (HA)‐conjugated fluorescent organic nanoparticles. These conjugates are designed to specifically target lymphatic endothelial cells (LECs) based on the ligand‐receptor relationship between HA and lymphatic vessel endothelial hyaluronan receptor 1 (LYVE‐1). The lymphatic vasculature undergoes dramatic remodeling during inflammation, wound healing, and in the tumor microenvironment, and is a significant route of metastasis. The long term goal of this project is to develop new tools for pre‐clinical and clinical imaging of pathological lymphatic vessel remodeling signatures that are indicative of the presence of early malignancy. Our work here has focused on 10 kDa and 100 kDa HA‐dye conjugates; future translational experiments will test nanoparticles with a paramagnetic core. The primary objective here was to optimize the dosage for delivery of HA‐conjugated fluorescent dyes to immortalized mouse and primary human LECs for detection by fluorescence microscopy. This included identification of LYVE‐1 expressing experimental cell lines, analysis of cell death, and dose‐dependent HA‐dye in vitro labeling studies. The toxicity of the HA‐conjugated dye was determined using a caspase 3/7 glo luminescence assay that measured the amount of cleaved caspase 3/7 in HA‐treated mouse and human lymphatic cells. The dye was found to be non‐toxic to both the mouse and human lymphatic cells at all dilutions tested including the working staining dilution as compared to staurosporine control used to induce apoptosis. LYVE‐1 expression in human and mouse LECs was confirmed by western blotting and immunofluorescence microscopy techniques including epifluorescence and spinning disk confocal. Fluorescence microscopy analysis showed a dose‐dependent increase in HA‐conjugate dye labeling of LECs. We have shown that HA‐conjugated dyes are non‐toxic to both mouse and primary human LECs, and that this conjugate can experimentally label cells by fluorescence microscopy. Ongoing tubulogenesis and migration experiments are designed to determine if LEC function is impaired with the HA‐conjugate dyes bound. Co‐culture and corneal explant assays will confirm specificity of HA‐conjugate dye for LECs.Support or Funding InformationIn collaboration with Dr. Aaron M. Mohs and Denis Svechkarev at the University of Nebraska Medical Center and bio‐systems networks & translational research (BioSNTR)This abstract is from the Experimental Biology 2019 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.