UC Riverside

ABSTRACT OF THE DISSERTATION

Red-Cell-Derived Contrast Delivery Vehicles for Biomedical Imaging

by

Jack Tang

Doctor of Philosophy, Graduate Program in Bioengineering

University of California, Riverside, December 2019

Dr. Bahman Anvari, Chairperson

Red blood cells (RBCs) have potential as drug carriers for the extended delivery of diagnostic and therapeutic cargos due to the presence of immunomodulatory proteins on their membrane surface. RBC carriers can be used to deliver a variety of diagnostic and/or therapeutic payloads, including small molecule drugs, macromolecular therapeutics, and contrast agents. We encapsulate the FDA-approved NIR-fluorescent dye, indocyanine green (ICG), within the membranes of RBC ghosts and evaluate the potential of such a construct for biomedical fluorescence imaging. These ICG-loaded, RBC-derived constructs are hereon referred to as NIR erythrocyte-mimicking transducers (NETs).

First, we characterize the material properties of micron-sized (µNETs) and nano-sized NETs (nNETs), which have respective relevance in vascular imaging, and cancer imaging. We demonstrate that their absorption and fluorescence characteristics can be optimized for specific imaging system specifications by adjusting the concentation of ICG used to fabricate them. We maximized the fluorescence signal of µNETs and nNETs by loading them using a hypotonic solution containing 20 µM ICG. Our data also provide some insight regarding the localization of ICG to the phospholipid membrane of NETs. We then demonstrate the fluorescence stability of NETs at specific temperatures that mimic storage (4°C) and usage conditions (37°C).

We further investigate the effect of frozen storage (-20°C) on extruded nano-sized NETs. We observed that size, zeta potential, absorption, and fluorescence properties of extruded nNETs did not change upon thawing after eight weeks of frozen storage. We then tested their uptake in SKOV3 cancer cells, and their biodistribution in Swiss Webster mice and found similar performance between frozen and non-frozen nNETs.

Lastly, we determine that NETs display phosphatidylserine (PS), a known marker of senescence, on their membrane surface. Presence of surface-exposed PS can potentially reduce the circulation time of NETs. We then introduce a new method to potentially increase the circulation half-life of NETs in vivo. We demonstrate partial confinement of PS to the inner leaflet of NETs by enriching their membranes with cholesterol. We demonstrate reduced uptake of cholesterol-enriched NETs by macrophages, which can potentially translate to extended circulation time. We then demonstrate that cholesterol enrichment can prolong the circulation half-life of micron-sized NETs, and discuss the implications of cholesterol-enrichment on nano-sized NETs.