© 2003 Robert A. Freitas Jr. All Rights Reserved.

Robert A. Freitas Jr., Nanomedicine, Volume IIA: Biocompatibility, Landes Bioscience, Georgetown, TX, 2003

15.4.3.6.3 Avoid Phagocytic Binding and Activation

Once physical contact with a phagocyte has occurred, particle binding to specific cell surface receptors is the first step in the phagocytosis of a medical nanorobot. Receptors able to mediate phagocytosis are expressed almost exclusively in neutrophils, monocytes and macrophages, and receptor clustering is thought to occur upon particle binding which in turn generates a phagocytic signal, activating the phagocytic process [3345].

Several pathways of phagocytic signal transduction have been identified [3345], including the activation of tyrosine kinases or serine/threonine kinase C, leading to phosphorylation of the receptors and other proteins which are recruited at the sites of phagocytosis. Monomeric GTPases of the Rho and ARF families which are engaged downstream of activated receptors, in cooperation with phosphatidylinositol 4-phosphate 5-kinase and phosphatidylinositol 3-kinase lipid modifying enzymes, can modulate locally the assembly of the submembranous actin filament system leading to particle internalization [3345, 5261]. It may be possible for nanorobots to affirmatively influence, modulate, or even extinguish the phagocytic activation signal by physical, chemical, or other means, perhaps using GTPase or kinase inhibitors [3346-3353] such as genistein (50 µM) [3346, 3352], herbimycin (17 µM) [3346], staurosporine and trifluoperazine [3349]. In many cases there are two or more pathways that must be simultaneously inhibited, although in a few cases these pathways may share a common inhibitor [3348]. For instance, CNI-1493 is a potent macrophage deactivator or “pacifier” [2593-2595].

Binding of particles to phagocytes may also be directly inhibited. Phagocytosis requires the internalization of a significant fraction of the plasma membrane, which results in the intracellular deposition of large particles [3354]. But this internalization does not diminish the number of receptors on the cell surface and has no effect on receptor-mediated uptake [3354].

In the case of receptor-mediated phagocytic binding, dansylcadaverine, amantadine, and rimantadine induce inhibition of endocytosis of complement-coated zymosan particles by human peripheral PMN leukocytes. These drugs block receptor-mediated endocytosis, possibly by their actions on phospholipid metabolism [3355] or by covalent coupling to cellular membranes [3356]. Cell-bound or soluble protein A produced by Staphylococcus aureus [1728] attaches to the Fc region of IgG and blocks the cytophilic (cell-binding) domain of the antibody. Thus the ability of IgG to act as an opsonic factor is inhibited, and opsonin-mediated ingestion (“opsonophagocytosis” [3303]) of the bacterium is blocked.

In the case of nonreceptor phagocytic binding, medical nanorobots could emit or expose on their surfaces chemical surfactants which would repel the lipid bilayer wall, e.g., by reducing the nanorobot’s coefficient of adhesion to very low or even to negative values (Section 9.2.3).

Many other substances that inhibit phagocytosis (keeping in mind the cross-talk between phagocytic receptors, the multiple signaling domains within these receptors, and the many downstream effector pathways leading to actin polymerization and particle internalization [3357]) could be further investigated for their suitability in this nanomedical context, including:

(1) Opioids and Anesthetics. Chemokine-induced phagocytosis is inhibited in the presence of mu-opioid receptor agonists such as morphine, DAMGO, methadone, and endomorphine [3315] in murine macrophages [3358] and rat splenic macrophages [3359]. Lidocaine at 30 mM significantly inhibits phagocytosis of latex particles in bovine monocytes [3360]. The membrane-active drug procaine inhibits the phagocytosis of latex particles by normal monocytes and the proliferation of lymphocytes in an allogeneic mixed leukocyte culture [3361]. Finally, while the phagocytosis of inert latex particles by human blood monocytes is not affected by the presence of ethanol [3362], the phagocytosis of opsonized red cells by Kupffer cells is slightly impaired by ethanol [3363].

(2) Hormones. Vasoactive intestinal polypeptide (VIP) inhibits alveolar macrophage phagocytosis of polystyrene beads, with maximum inhibition of 46% at 0.1 µM concentration [3316]. Dexamethasone inhibits phagocytosis by human trabecular meshwork (eye) cells in vitro, with polystyrene particle uptake reduced from 3.5 beads/cell to 1.5 beads/cell, a 57% reduction [3364]. Two cholecystokinin octapeptides (CCK-8s and CCK-8) significantly inhibit neutrophil ingestion of latex beads. This inhibitory effect is maximized at 0.1 nM concentration [3365], and inhibition of neutrophil mobility and phagocytosis “could be carried out through an increase of the intracellular cAMP levels” [3319, 3365]. Gastrin-17 and gastrin-34, maximally at 0.1 nM, inhibit the ingestion of latex beads in human peripheral blood neutrophils [3319]. Prostaglandins also inhibit particle ingestion [3366].

(3) Toxins. Pertussis toxin decreases the phagocytosis of IgG-opsonized Staphylococcus aureus pathogens by human granulocytes [3367]. Many bacterial exotoxins that are adenylate cyclases such as anthrax toxin edema factor [3368] and pertussis toxin [3369] decrease phagocytic activity. The ability of rat alveolar macrophages to phagocytose Saccharomyces cerevisiae and Staphylococcus aureus microbes was significantly reduced in vitro in the presence of T-2 toxin exceeding ~0.1 µM concentration [3370]. Mycotoxins such as aflatoxin B1 [3371] significantly impair Kupffer cell phagocytosis, although aflatoxin is a known genotoxin and thus would not be ideal in this application.

(4) Bacterial Factors and Enzymes. PMN-inhibitory factor (PIF) extracted from B. pertussis cells showed little cytotoxicity and inhibited phagocytosis to opsonized targets by PMNs [3291]. Phagocytic activity of neutrophils was reduced by a staphylococcus aggressin [3588], and extracellular slime produced by Staphylococcus epidermidis interferes with human neutrophil functions in vitro, including degranulation and phagocytosis [3373]. In the presence of 12 µg/ml of S. aureus lipase, almost no killing of the microbe by human granulocytes occurs, mostly due to a lack of bacterial uptake [3290]. YopH [3374] and other Yersinia Yop proteins [3375] inhibit the phagocytosis of Yersinia enterocolitica by human granulocytes [3374] and macrophages [3375].

(5) Antibiotics. Phagocytosis is diminished or suppressed by erythromycin, roxithromycin, cefotaxime, tetracyclines, ampicillin, gentamicin, and bacitracin [3293, 3294].

(6) Mechanical. Colchicine (at 150 mg/kg) and cytochalasin B (at 0.15 mg/kg) greatly depress pulmonary macrophage endocytosis or “particle uptake” in hamsters [3376], e.g., by inhibiting cytoskeletal actin microfilament polymerization. Cytochalasin B inhibits phagocytosis [3366]; Cytochalasin D, a drug that affects actin polymerization and particle internalization, also inhibits the binding stage of phagocytosis [3388, 3389]. Eicosapentaenoic acid rigidifies the plasma membrane of human neutrophils, leading to reduced phagocytosis [3286]. Macroscale physical shock [3377, 3378] or surgical manipulation [3379] can depress phagocytic function in Kupffer cells. A 5% cyclical strain applied to cultured peritoneal mouse macrophages at 1 Hz for 24-48 hours partially suppresses their phagocytosis of latex particles [5331].

(7) Other. Ammonium acetate reduces (in a concentration-dependent manner) the phagocytic uptake of mannosylated latex microspheres by human microglia and astroglioma cells [3380] – the threshold for action is >~2 mM for microglia [3381]. A variety of dissolved metal ions such as Ni⁺⁺ and Cr⁺⁺⁺ suppress phagocytic efficiency. For example, increasing Ni from 29 ppm to 58 ppm depresses phagocytic efficiency in cultured rabbit alveolar macrophages by 78% [234, 3382], and GdCl₃ can prevent surface attachment of particles to Kupffer cells [3383]. However, such metal ions are probably genotoxic or cytotoxic at the concentrations necessary to suppress phagocytic efficiency. A heat-stable inhibitor of neutrophil phagocytosis has been demonstrated in sarcoma cells [3281]. Liposome particle uptake by liver endothelial and Kupffer cells is inhibited by poly(inosinic acid) and other anionic macromolecules [3384]. Kupffer cell phagocytic activity is also reduced by methylpalmitate [3385] and silica [3386]. Short-chain ceramides inhibit IgG-mediated phagocytosis by PMNs [3387]. The glycoprotein horseradish peroxidase (HRP) inhibits the binding stage of phagocytosis [3388]. Monoclonal IgM cold agglutinins (~1 mg/ml) impair various phagocytic functions of human phagocytes including adhesiveness, phagocytosis, phagocytic index and intracellular bactericidal activity of PMNs [3390]. Decreasing the pH of influent perfusate through liver RES (e.g., hepatic sinusoids) increases the uptake of carbon particles, so pH gradient across the liver lobule may be involved in the regulation of particle uptake at the sublobular level [3391]. Amantidine, an adamantane-based drug, weakly inhibits phagocytosis in PMNs [5545]. Phagocyte adhesion might also be reduced by using hydrophilic or anionic surfaces [5507]. Once again, it is important to note that many of these substances may have effects on other cells and cellular functions, suggesting caution when choosing a particular antiphagocytic substance for use in medical nanorobot designs.

Care must also be taken to avoid the use of nanorobot coatings which possess, or may induce, fusogenic conformations [3392-3396], in which case specific fusogen inhibitors [3393-3398] might need to be simultaneously deployed.

Last updated on 30 April 2004