Seeing What Sticks: Anchoring Capabilities of Moieties for Use in Cell-Conveyed Therapeutics

Red blood cells (RBCs) have been employed to convey and deliver a variety of therapeutic agents, from small molecules to proteins. The therapeutics are typically installed within the RBC interior via a pore-forming process that results in membrane disruption and a partial loss of hemoglobin. An alternative approach, namely appending therapeutics to the RBC surface, has received significantly less attention. Here we focus on the characterization of an array of membrane anchoring modalities (noncovalent, reversible covalent, and covalent). Surface modification is experimentally simpler and structurally less invasive than its membrane disruptive counterpart. This panel is designed, synthesized and assessed with respect to RBC loading capacity, retention, and rate of transfer to other cell populations. The cell surface anchors are appended to a structural scaffold (cobalamin) that can house and deliver therapeutic agents. Imaging studies for a series of representative derivatives reveal that these species are not internalized by the RBCs, consistent with the absence of an active endocytic pathway in mature RBCs. Furthermore, enzymatic digestion of the glycocalyx failed to impair loading or retention, suggesting that the derivatives are likely anchored to the RBC membrane. The structural motifs identified in this study provide a template for the development of membrane tethered therapeutics that are specifically designed to be transported to diseased sites by RBCs.