Erythrocyte Carriers: Tool Development for Erythrocyte Mediated Diagnostic and Drug Delivery Public Deposited

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  • March 19, 2019
  • Oien, Nathan
    • Affiliation: Eshelman School of Pharmacy, Division of Chemical Biology and Medicinal Chemistry
  • Carrier erythrocytes have become increasingly popular in literature and in the clinical setting due to their low immunogenic response, ease of use, and well defined structure and physiology. Small molecules, therapeutic proteins, and optical biosensors have each successfully been loaded into erythrocytes, establishing a strong methodological foundation for our work that utilizes erythrocytes as "carriers" of fluorescent biosensors and therapeutic agents. The initial stage of our research focused primarily on optimization of erythrocyte loading procedures using exogenous fluorophore labeled peptides and proteins (the term `loading' refers to addition of exogenous material to the cytosol of erythrocytes). We visualized loading using widefield and confocal fluorescence microscopy. Upon our establishment of an optimized erythrocyte loading method, we used photo responsive cassettes to investigate whether erythrocytes can be manipulated to contain the cassette, and that upon microscope-mediated photolysis a fluorescence increase (without significant leakage) is observed. With the successful development of our erythrocyte loading method, we next sought to determine whether such innovative technology could be utilized to monitor endogenous kinase activity. Erythrocytes possess highly complex biochemical pathways, which include enzymes important in glycolysis and maintenance of extracellular homeostasis. One of the most prominent enzymes in cells, including enucleated erythrocytes, is cAMP dependent protein kinase (PKA). PKA in red blood cells is particularly relevant to the study of Malaria as it is a vital player in the protozoan parasite invasion of erythrocytes.[1] Thus, we proposed developing a method to monitor endogenous PKA activity in erythrocytes. This ability to monitor the endogenous PKA activity will be beneficial in assessing erythrocyte response during exogenous stimuli, serving as both a biological sensor and as a diagnostic tool to detect malarial invasion. We first aimed to monitor PKA activity through the development of a bi-molecular assay. Our innovative work demonstrates that the interaction between a positively charged peptide that is covalently bound to a far-red fluorophore and a negatively charged quencher that interacts with our labeled-peptide results in quenched fluorescence. More importantly, our work demonstrates that after phosphorylation, the quencher is displaced and a fluorescence increase is observed. Upon verification of optimization and reproducibility, our carefully designed continuous assay was then used to monitor real-time endogenous PKA activity in erythrocyte lysates and within intact erythrocytes, the latter of which is possible through our established erythrocyte loading method. With the completion of both our erythrocyte loading method and novel real-time detection of endogenous PKA activity, our next objective was to determine whether we could utilize erythrocytes for spatial and temporal- specific delivery of therapeutics using cobalamin-drug conjugates, where cobalamin acts as a light responsive photoprotecting group. In this instance, as opposed to intracellular loading, we use lipids to anchor the drug conjugates to the exterior surface of the erythrocyte membrane. To test drug release from membranes, we utilized near-infrared (NIR) light to release anti-inflammatory therapeutics from the vitamin B12 derivative, hydroxyl-cobalamin. Cobalamin is an organometallic compound containing a cobalt center surrounded by a corrin ring. The cobalt axial bound ligand is readily cleaved using light. Interestingly, cobalamin also contains a modifiable hydroxyl sugar. We show by lipidating the hydroxyl sugar and attaching therapeutics to the cobalt axial bond, that we could anchor the complex to erythrocyte membranes. Furthermore, we demonstrated photo-release of axially bound therapeutics using 525 nm light. Lastly, we were able to extend the wavelengths of light-mediated delivery into the NIR region by introducing lipidated NIR fluorophores to the erythrocyte membranes. Excitation of the lipidated fluorophore led to photocleavage of the cobalt small molecule bond. Using erythrocytes as carriers represents a simple and attractive method as a delivery tool. Our work demonstrates the utility of erythrocytes as carriers for delivering diagnostic tools and therapeutics that respond to light resulting in spatial and temporal control of delivery and activation.
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  • In Copyright
  • Liu, Jian
  • Lawrence, David
  • Jacobson, Ken
  • Bear, James
  • Frye, Stephen
  • Doctor of Philosophy
Degree granting institution
  • University of North Carolina at Chapel Hill Graduate School
Graduation year
  • 2014
Place of publication
  • Chapel Hill, NC
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