(Revised Abstract) DESCRIPTION (provided by applicant): Nearly all synthetics - including nano-scale liposomes, particles, or our recently developed polymer vesicles - are limited in in vivo performance due largely to phagocytes (eg. RES macrophages) which recognize all as foreign. To delay such recognition, the last decade has seen widespread efforts to effectively mimic the cell's glycocalyx with hydrophilic polymers such as PEG. However, at least 50% of PEG-modified STEALTH liposomes - as a prime example - still end up being phagocytosed in the liver and spleen (even when a tumor is present elsewhere to take up vesicles through enhanced permeability. While circulation times of our pure polymer vesicles with their denser and more stable PEG brushes are longer in rats than STEALTH liposomes, the vesicles still circulate much less than the organism's own blood cells. Inhibiting clearance of the body's own cells are specific mechanisms of protein-protein interaction that involve the ubiquitous membrane protein CD47: knockout mouse studies together with our own work on yeast-displayed human CD47 suggest 'marker of self' CD47 transiently binds to phagocyte receptors and turns off motile processes. We propose to engineer particles - primarily our polymer vesicles and more recent worm micelles - to display recombinant mouse or rat CD47 and demonstrate truly long-circulating, phagocyte-inhibiting particles in mice. Beyond this R21 proposal, our long-term objectives are to exploit such hybrid systems for controlled degradation/release carriers of drugs and imaging agents. One therapeutic direction envisioned is the delivery of various antioxidants targeted to the endothelium of oxidant stressed lung. This is a difficult-to-target tissue that is often inflamed (i.e. rich in activated leukocytes). In addition to providing a means of avoiding phagocytosis, CD47 could prove therapeutically useful in any such inflamed tissue by passivating accumulated leukocytes. Our newest biocompatible and hydrolysable PEO-PLA copolymers were synthesized crudely but already demonstrate kinetic control of degradation/release; these and related copolymers will be re-fined, made in larger quantities, and modified for protein attachment. Separate studies with a recombinant -100-aa extracellular immunoglobulin domain of human CD47 already demonstrate phagocyte inhibition in vitro, but the mouse protein must be expressed, purified, attached to particles, and also tested in vitro. Finally, we propose assessing the pharmacokinetics of CD47-displaying particles - primarily our own PEG-based copolymer vesicles and micelles -by IV injection into mouse and following circulation time, biodistribution, among other key responses.
NIH ExPorter
NIH ExPorter