Nanomedicine, Volume IIA: Biocompatibility
© 2003 Robert A. Freitas Jr. All Rights Reserved.
Robert A. Freitas Jr., Nanomedicine, Volume IIA: Biocompatibility, Landes Bioscience, Georgetown, TX, 2003
220.127.116.11.4 Inhibit Phagocytic Engulfment
Even if a medical nanorobot has been recognized and has attached to the phagocyte outer surface (typically across a ~20 nm gap bridged by ~12 nm strands ), the device can still prevent complete engulfment from taking place. Macrophages challenged with a particular type of target usually bind many more targets than they ingest . Fortunately, internalization is a relatively slow process and most particles that become bound to the phagocyte surface are not ingested . On rare occasions, phagocytosed particles are actually expelled [2870, 3400]. Indeed, a recent study  found that the homophilic ligation of CD31 (also known as platelet-endothelial cell adhesion molecule-1, or PECAM-1)  on viable leukocytes promoted their detachment under low shear, leading to active repulsion of viable cells from macrophages, whereas such CD31-mediated detachment was disabled in apoptotic leukocytes, promoting capture and tethering, tight binding, and macrophage ingestion of dying cells. Hence CD31 is an example of a cell-surface molecule that promotes tethering of dying cells to phagocytes during apoptosis, but prevents phagocyte ingestion of closely apposed viable cells by transmitting “detachment” signals .
Phagocytosis is an uptake of large particles governed by the actin-based cytoskeleton. It is a dynamic process including actin polymerization around the particle for internalization, with phagosome maturation governed by a complex mix of proteins including actin, the Arp2/3 complex, Rho-family GTPases, filament-capping proteins, tropomyosin, Rho kinase and myosin II [6062-6064]. Complement-opsonized (CO) and antibody-opsonized (AO) particles are phagocytosed differently by macrophages [3401, 3402] – CO particles sink into the cell, whereas AO particles are engulfed by lamellipodia that project from the cell surface. During the ingestion of CO particles, punctate structures rich in F-actin, vinculin, alpha-actinin, paxillin, and phosphotyrosine-containing proteins are distributed over the phagosome surface . These foci can be detected underneath bound CO particles within 30 seconds of cell activation, and their formation requires active protein kinase C. Complement receptor-mediated internalization requires intact microtubules and is accompanied by the accumulation of vesicles beneath the forming phagosome . By contrast, during the ingestion of AO particles (Fcgamma receptor mediated phagocytosis), all proteins are uniformly distributed on or near the phagosome surface. Ingestion of AO beads is blocked by tyrosine kinase inhibitors (e.g., which could be released from, or tethered to, medical nanorobots), whereas the phagocytosis of CO particles is not .
Phagocytic particle ingestion can require actin assembly and pseudopod extension, two cellular events that may coincide spatially and temporally but may use distinct signal transduction events or pathways . Medical nanorobots that have become bound to the extracellular phagocyte surface may attempt to inhibit either or both of these signal transduction pathways.
For example, during actin assembly, engagement of particle-bound immunoglobulin IgG ligands by receptors for the Fc portion of IgG results in receptor aggregation and recruitment of cytosolic tyrosine kinase, especially Syk . The onset of uptake is accompanied by tyrosine phosphorylation of several proteins, which persists for up to 3 minutes, is concentrated at phagocytic cups and nascent phagosomes, and is correlated with the accumulation of actin filaments . (Later, during phagosome maturation, tyrosine phosphorylated proteins and microfilaments disappear from the periphagosomal regions .) Phosphorylation of tyrosine residues occurs within immunoreceptor tyrosine activation motif (ITAM) consensus sequences found in FcgammaR subunits, which allows further recruitment and activation of Syk . Syk tyrosine kinase activity is required for FcgammaR-mediated actin assembly, which is controlled by several GTPases, including Rac1 and CDC42 . Rac1 and CDC42 (two Rho proteins involved in the signal transduction through the FcRs) are required (1) to coordinate actin filament organization and membrane extension to form phagocytic cups, (2) to allow particle internalization during FcR-mediated phagocytosis, and (3) to enable the phosphotyrosine dephosphorylation required for particle internalization .
Actin assembly can be inhibited by Clostridium difficile toxin B, which is a general inhibitor of Rho GTP-binding proteins . Inhibition of Rac1 or CDC42 severely inhibits particle internalization but not F-actin accumulation . In laboratory tests with cells, inhibition (via knockout of gene expression in a mutant line) of CDC42 function results in pedestal-like structures with foreign particles at their tips on the phagocyte surface. Inhibition of Rac1 results in particles bound at the surface that are enclosed within thin unfused membrane protrusions . This demonstrates that Rac1 and CDC42 have distinct functions and may act cooperatively in the assembly of the phagocytic cup . Phagocytic cup closure and particle internalization has also been blocked when phosphotyrosine dephosphorylation is inhibited by treatment of the phagocytic cells with phenylarsine oxide, an inhibitor of protein phosphotyrosine phosphatases . Ceramide also inhibits tyrosine phosphorylation in human neutrophils .
During pseudopod extension, phosphatidylinositol 3-kinase (PI3K) is recruited to the plasma membrane, triggering exocytosis from an internal membrane source as is required for pseudopod extension . (Macrophage spreading on opsonized surface is accompanied by insertion into the plasma membrane of new membrane from intracellular sources .) One or more isoforms of PI3K are required for maximal pseudopod extension, though not for phagocytosis per se. PI3K is required for coordinating exocytic membrane insertion and pseudopod extension .
Pseudopod extension may be partially inhibited using wortmannin (WM) or LY294002, which are two inhibitors of PI3K . Both of these specifically inhibit phagocytosis without inhibiting Fcgamma receptor-directed actin polymerization, by preventing maximal pseudopod extension. Decreasing the size of test beads, and hence the size of pseudopod extension required for particle engulfment, de-inhibited phagocytosis (in presence of these inhibitors) by up to 80% at the very smallest submicron particle sizes. For larger (nanorobot-sized) foreign particles, phagocytosis is blocked before phagosomal closure. Both compounds also inhibit macrophage spreading on opsonized surfaces (e.g., on substrate-bound IgG) .
Amphiphysin II associates with early phagosomes in macrophages and participates in receptor-mediated endocytosis by recruiting the GTPase dynamin to the nascent endosome. There is a signaling cascade in which PI3K is required to recruit amphiphysin II to the phagosome, after which the amphiphysin II in turn recruits dynamin to the phagosome . A modified amphiphysin II molecule with its dynamin-binding site ablated away inhibits phagocytosis at the stage of membrane extension around the bound foreign particles . Both phenylbutazone and chloramphenicol also have shown an inhibitory effect on the engulfment stage of phagocytosis of yeast particles by cultured human monocytes . Of course, it will be important to identify substances that produce minimal effects on other cells or cellular functions.
As might be expected, bacteria already employ a wide variety of strategies to avoid engulfment when physically contacted by host phagocytes . Some of these strategies could in principle be mimicked by medical nanorobots. Most commonly, many important pathogenic bacteria bear substances on their surfaces that inhibit phagocytic adsorption or engulfment. Resistance to phagocytic ingestion is usually due to an antiphagocytic component of the bacterial cell surface, such as:
(1) Cell Wall Substances – polysaccharide surface slime (alginate slime  and biofilm polymers) produced by Pseudomonas aeruginosa [3302, 3411]; O antigen associated with LPS of E. coli (smooth strains) ; and K antigen (acidic polysaccharides) of E. coli or the analogous Vi (K) antigen (microcapsule) of Salmonella typhi .
(2) Fimbriae and M Protein – fimbriae in E. coli , and M protein and fimbriae of Group A streptococci [3302, 3303]. For example, Streptococcus pyogenes has M protein, a fibrillar surface protein whose distal end bears a negative charge that interferes with phagocytosis . Enterococci also have antiphagocytic surface proteins  such as M protein.
(3) Capsules – polysaccharide capsules of S. pneumoniae (unless antibody is present), Treponema pallidum, Klebsiella pneumoniae, Bacteroides fragilis, and Clostridium perfringens, and the Enterococci inhibit engulfment [3301-3303]. Haemophilus influenzae expresses a mucoid polysaccharide capsule of thickness ~1 microbial diameter which prevents digestion by host phagocytes, although many of these bacteria remain susceptible to opsonization [3302-3304]. The protein capsule on cell surface of Yersinia pestis resists engulfment .
Macrophages can also bind and engulf a variety of particles in the absence of specific opsonins, a process known as nonspecific phagocytosis , nonopsonic phagocytosis , or opsonin-independent phagocytosis . Polystyrene microspheres are often used to demonstrate this . For instance, during patocytosis (Section 18.104.22.168) of hydrophobic >0.5-micron particles by phagocytes, actin-independent capping of hydrophobic polystyrene microspheres on the plasma membrane precedes actin-dependent uptake of the microspheres into the surface-connected compartments . Microsphere transport from plasma membrane invaginations into spaces created by unfolding stacks of internal microvilli are inhibited by administering primaquine . Studies of non-specific endocytosis and binding of liposomes by mouse peritoneal macrophages also found that particle internalization declined markedly after anchorage of the cells to polystyrene substrate . Inhibitors are potentially available to medical nanorobots to halt these processes too. For example, staurosporine selectively inhibits nonspecific phagocytosis while having no effect on receptor-mediated phagocytosis .
Last updated on 30 April 2004