Interaction of spherical particles with cells and within animals has been studied extensively, but the effects of shape have received little attention. Here we use highly stable, polymer micelle assemblies known as filomicelles to compare the transport and trafficking of flexible filaments with spheres of similar chemistry. In rodents, filomicelles persisted in the circulation up to one week after intravenous injection. This is about ten times longer than their spherical counterparts and is more persistent than any known synthetic nanoparticle. Under fluid flow conditions, spheres and short filomicelles are taken up by cells more readily than longer filaments because the latter are extended by the flow. Preliminary results further demonstrate that filomicelles can effectively deliver the anticancer drug paclitaxel and shrink human-derived tumours in mice. Although these findings show that long-circulating vehicles need not be nanospheres, they also lend insight into possible shape effects of natural filamentous viruses.
Foreign particles and cells are cleared from the body by phagocytes that must also recognize and avoid clearance of “self” cells. The membrane protein CD47 is reportedly a “marker of self” in mice that impedes phagocytosis of self by signaling through the phagocyte receptor CD172a. Minimal “Self” peptides were computationally designed from human CD47 and then synthesized and attached to virus-size particles for intravenous injection into mice that express a CD172a variant compatible with hCD47. Self peptides delay macrophage-mediated clearance of nanoparticles, which promotes persistent circulation that enhances dye and drug delivery to tumors. Self-peptide affinity for CD172a is near the optimum measured for human CD172a variants, and Self peptide also potently inhibits nanoparticle uptake mediated by the contractile cytoskeleton. The reductionist approach reveals the importance of human Self peptides and their utility in enhancing drug delivery and imaging.
Phagocytosis of foreign cells or particles by macrophages is a rapid process that is inefficient when faced with “self” cells that display CD47—although signaling mechanisms in self-recognition have remained largely unknown. With human macrophages, we show the phagocytic synapse at cell contacts involves a basal level of actin-driven phagocytosis that, in the absence of species-specific CD47 signaling, is made more efficient by phospho-activated myosin. We use “foreign” sheep red blood cells (RBCs) together with CD47-blocked, antibody-opsonized human RBCs in order to visualize synaptic accumulation of phosphotyrosine, paxillin, F-actin, and the major motor isoform, nonmuscle myosin-IIA. When CD47 is functional, the macrophage counter-receptor and phosphatase-activator SIRPα localizes to the synapse, suppressing accumulation of phosphotyrosine and myosin without affecting F-actin. On both RBCs and microbeads, human CD47 potently inhibits phagocytosis as does direct inhibition of myosin. CD47–SIRPα interaction initiates a dephosphorylation cascade directed in part at phosphotyrosine in myosin. A point mutation turns off this motor's contribution to phagocytosis, suggesting that self-recognition inhibits contractile engulfment.
Phagocytes engulf foreign cells but not ‘self’ in part because self cells express CD47 as a ligand for signal regulatory protein SIRPα, which inhibits phagocytosis. Motivated by reports of upregulation of CD47 on both normal and cancerous stem cells [1] and also by polymorphisms in SIRPα [2], we show here that inhibition of engulfment correlates with affinity of CD47 for SIRPα – but only at low levels of CD47. One common human polymorph of SIRPα is studied and binds more strongly to human-CD47 than to mouse-CD47 (Kd ≈ 0.12 μM and 6.9 μM, respectively) and does not bind sheep red blood cells (RBC) – which are well-established targets of human macrophages; in comparison, a common mouse polymorph of SIRPα binds with similar affinity to human and mouse CD47 (Kd ≈ 0.22 μM). Using immunoglobulin (IgG)-opsonized particles with varying levels of either human- or mouse-CD47, the effective inhibition constants Ki for blocking phagocytosis are then determined with both human- and mouse-derived macrophages. Only human phagocytes show significant differences in man versus mouse Ki's and only at CD47 levels below normal densities for RBCs. While phospo-signaling through human-SIRPα shows similar trends, consistent again with the affinity differences, saturating levels of CD47 (> Ki) can signal and inhibit phagocytosis regardless of man versus mouse. Quantitative analyses here prompt more complete characterizations of both CD47 levels and SIRPα polymorphisms when attempting to study in vivo effects of these key proteins in innate immunity.
CD47 is a transmembrane protein that is a marker of “self”. CD47 binding to its cognate receptor in leukocytes and macrophages, signal regulatory protein alpha (SIRPα), causes inhibition of inflammatory cell attachment. We hypothesized that immobilization of recombinant CD47 on polymeric surfaces would reduce inflammation. Recombinant CD47 was appended to polyvinyl chloride (PVC) or polyurethane (PU) surfaces via photoactivation chemistry. Cell culture studies showed that CD47 immobilization significantly reduced human neutrophil (HL-60) and human monocyte derived macrophage (MDM) (THP-1) attachment to PVC and PU respectively. A neutralizing antibody, directed against SIRPα, inhibited THP-1 and HL-60 binding to PU and PVC surfaces respectively. This antibody also increased the level of SIRPα tyrosine phosphorylation, thereby indicating a direct role for SIRPα mediated signaling in preventing inflammatory cell attachment. Studies using human blood in an ex vivo flow-loop showed that CD47 modified PVC tubing significantly reduced cell binding and neutrophil activation compared to unmodified tubing or poly-2-methoxy-ethylacrylate (PMEA) coated tubing. In ten-week rat subdermal implants, CD47 functionalized PU films showed a significant reduction in markers of MDM mediated oxidative degradation compared to unmodified PU. In conclusion, CD47 functionalized surfaces can resist inflammatory cell interactions both in vitro and in vivo.
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