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
22.214.171.124.1 Avoid Phagocytic Contact
One simple avoidance method employed by a few pathogens that may occasionally be practical for medical nanorobots is to confine all activities to regions of the human body that are inaccessible to phagocytes. For example, certain internal tissues such as the lumens of glands, the urinary bladder and kidney tubules, and various surface tissues such as the skin are not regularly patrolled by phagocytes . The heart and muscle tissues also are relatively macrophage-poor .
If reliable methods can be found for the remote (noncontact) detection of nearby phagocytes, akin to the detectability of bacterial metabolic chemical plumes (Section 8.4.3), then most motile nanorobots should also be able to outrun any “pursuing” phagocytes. For example, activated phagocytes emit various telltale substances  such as cytokines [3228-3234], enzymes [3235-3237], histamine , taurine , and so forth. Lipoxins  recruit healthy macrophages to phagocytose apoptotic neutrophils, another cautionary chemical plume to avoid. Of course, nanorobots must be able to distinguish all these emissions locally from background concentrations normally present.
If remote phagocyte detection methods* cannot be made reliable, then nonmotile nanorobots must employ contact avoidance techniques. One potentially useful approach is to make use of the natural mediators of cellular chemotaxis (movement along a spatial gradient or directed cell locomotion) and chemokinesis (general random movement or nondirected cell locomotion) [3241, 3242]. Specific chemicals are known to be chemorepellents, chemotaxis antagonists, chemotactic factor enzymes or antibodies, or negative chemokinesis agents for various cell types. Alternatively, emission of decoy chemoattractants followed by a quick course change by nanorobots could also frustrate phagocytic pursuit.
* Interestingly, it should be possible to detect from the outside of a phagocytic cell that the cell has already ingested even a chemically inert nanorobot. For example, the decrease in capacitance of a cell that has ingested latex particles has been measured experimentally – a change of -250 fF/particle for 0.8-micron latex beads and -480 fF/particle for 3.2-micron beads . Monocytes that have ingested latex microspheres also display different surface markers .
Repelling pathogens from normal cells is of great medical interest, so it is not surprising that a great deal of research has been done on inducing negative chemotaxis in pathogenic microbes, which we shall now briefly summarize. Among the bacteria, E. coli moves away from chemorepellent molecules produced by stimulated phagocytic leukocytes including peroxide, hypochlorite, and N-chlorotaurine . Chemorepellents indole and benzoate induce motor-direction switching , and lipophilic weak acids, decreases in extracellular pH, and nigericin also induce chemorepellent response . Short-chain alcohols or DMSO are chemorepellents for the Gram-negative bacterium Myxococcus xanthus , and phenol is a chemorepellent for the flagellate bacterium Vibrio alginolyticus . Known chemorepellents for the bacterium Bacillus subtilis include chlorpromazine (a CNS depressant), local anesthetics, and tetraphenylboron (a lipophilic anion) . Of course, some of these substances are toxic to human cells and thus would not be appropriate chemorepellent molecules for medical nanorobots.
Among the protozoans, Trichomonas vaginalis exhibits negative chemotaxis to peroxide , with significant chemorepulsion by the spermicide Nonoxynol-9 and by nitroimidazoles such as metronidazole . Some chemorepulsion has also been seen in response to the antifungal imidazoles such as ketoconazole and miconazole . Lysozyme is a chemorepellent for Paramecia at 0.5-1.0 µM [3252, 3253] and also for the unicellular eukaryotic ciliated protozoan Tetrahymena thermophila . Pituitary adenylate cyclase activating peptide (PACAP-38) is a peptide hormone chemorepellent for Tetrahymena with an EC50 at 10 nM concentration [3253, 3255], and leukocyte N-t-BOC-Norleucine-Leu-Phe (maximized at ~1 pM) is also chemorepulsive to Tetrahymena . Other nontoxic chemorepellents to Paramecia, effective in nM to µM concentrations, include GTP, the oxidants NBT and cytochrome c, the secretagogues alcian blue and AED, and the dye cibacron blue ; all but AED and cytochrome c are chemorepulsive to Tetrahymena .
Chemorepellents are known for neural cells and include semaphorins [3257-3264], netrins [3263-3266], slit ligand [3267, 3268], and other neural factors [3269, 3270]. Chemotaxis of murine spleen cells was decreased in the presence of the lipoxygenase inhibitors azelastine and ketotifen . Interestingly, negative necrotaxis (movement away from dead cells [3272-3275]) has been observed in the motile unicellular green algae Euglena gracilis . The colorless cryptomonad Chilomonas paramecium and the ciliate Tetrahymena pyriformis exhibit negative necrotaxis following lysis of same-species cells or of Euglena cells, and the cellular content of Euglena lysed by laser irradiation heating or by mechanical means acts as a chemorepellent to intact Euglena cells .
What about chemorepellents for phagocytes? Neutrophils can respond to spatial concentration gradients as small as 1% , and some research has been done on inhibiting chemotaxis in phagocytic cells. For example, monocyte migratory inhibition factor (MIF)  inhibits macrophage migration, with a maximum inhibitory effect at 1 ng/ml for both unchallenged and particle-challenged macrophages [3276, 3277]. Human alveolar macrophages can release a noncytotoxic factor that inhibits neutrophil chemotaxis and random migration . Excess zinc immobilizes macrophages , and mononuclear cells cultured from hyperimmunoglobulin-E (HIE) patients produced a ~61 kD protein factor that nontoxically inhibited normal neutrophil and monocyte chemotaxis  while serum from those patients contained a 30-40 kD inhibitor of PMN and monocyte chemotaxis . A heat-stable inhibitor of neutrophil chemotaxis was demonstrated but not chemically isolated in 1975 , and it is now known that phospholipase A2 inhibitors  and a ubiquitin-like peptide  inhibit PMN chemotaxis. Lymphocyte-specific protein 1 (renamed leukocyte-specific protein 1 or LSP1) is a negative regulator of neutrophil chemotaxis . Polyamines such as putrescine at 1 mM and spermidine at 0.1-0.5 mM inhibit chemotaxis (but not phagocytosis or engulfment) by PMNs in vitro . PMN locomotion is also inhibited by diclofenac sodium, a nonsteroidal anti-inflammatory agent, at concentrations below 10 µg/ml , and eicosapentaenoic acid somewhat rigidifies the plasma membrane of human neutrophils, leading to reduced chemotaxis . Chemotaxis of PMNs is suppressed with IV concentrations of gamma globulin >~3.0 mg/ml, although adhesiveness to microbes is simultaneously enhanced .
Much phagocyte chemorepellent research occurs in the context of elucidating bacterial avoidance strategies (such as might be mimicked by medical nanorobots). Some bacteria or their products inhibit phagocyte chemotaxis. For example, Streptococcal streptolysin O (which also kills phagocytes) is a true chemotactic repellent [3302, 3288], even in very low concentrations. Staphylococcus aureus produces toxins that inhibit the movement of phagocytes ; granulocytes are almost immobilized when administered 12 µg/ml of purified S. aureus lipase . Pertussis toxin, produced by the bacterium Bordetella pertussis, inhibits chemotaxis of neutrophils and other phagocytes ; a PMN-inhibitory factor (PIF) extracted from B. pertussis cells shows little cytotoxicity and inhibits chemotaxis of PMNs . Fractions of Mycobacterium tuberculosis inhibit leukocyte migration . The Clostridium perfringens phi toxin inhibits neutrophil chemotaxis , and other “specific antigen” can suppress basophil chemotaxis . Phagocyte chemotaxis is generally reduced by antibiotics such as cefotazime, rifampin, and teicoplanin . Rifampin and tetracyclines inhibit granulocyte chemotactic activity . Leukocyte, lymphocyte and monocyte chemotaxis is inhibited by methylprednisolone and azathioprine, whereas only lymphocytes are chemotactically inhibited by cyclosporine .
Phagocyte chemoattractants that serve specific signaling purposes can be counteracted by specific inhibitors. For example:
(1) Formyl peptides such as fMLP (n-formyl Met-Leu-Phe) are commonly produced by bacteria and thus serve as neutrophil chemoattractants. Numerous inhibitors of fMLP chemoattraction are known: (a) recombinant human tumor necrosis factor-alpha suppresses PMN chemotaxis toward fMLP by 80% ; (b) uteroglobin (a steroid-dependent secretory protein) inhibits human phagocyte chemotaxis in response to formyl peptide attractants with half-maximal inhibition at 1.2 µM ; (c) monoclonal antibody to the alpha chain of the CD11b/CD18 complex inhibits PMN chemotactic response to fMLP ; (d) anti-integrin-associated protein antibodies inhibit phagocyte chemotaxis in PMN and monocytes ; (e) synthetic cannabinoid CP55,940 induces significant inhibition of both chemokinesis and fMLP-induced chemotaxis in rat peritoneal macrophages (typical dose ~0.4 mg/kg) ; and (f) human recombinant granulocyte-macrophage colony-stimulating factor inhibits human neutrophil chemotaxis towards both fMLP and the complement split product C5a, without itself having any chemotactic or chemokinetic activity .
(2) Complement factor C5a (Section 126.96.36.199) enhances chemotaxis, but inhibitors are known. For example, C5a-mediated granulocyte migration towards Streptococcus pyogenes is inhibited by solubilized fragments of C5a peptidase , which is released by a cysteine proteinase produced by the bacterium [3305-3307]. Also, a new complement receptor antagonist (the cyclic peptide Phe-[Orn-Pro-D-Cyclohexylalanine-Trp-Arg])  inhibits C5a-induced neutrophil chemotaxis.
(3) Sense-antisense methodology has been used to design novel complementary peptides as inhibitors of N-acetyl-PGP neutrophil chemoattractant .
(4) Chemotaxis by human neutrophils toward several common chemoattractants was inhibited by 80-95%, maximally at a concentration of ~50 µM of the protein kinase inhibitor 1-(5-isoquinolinesulfonyl) piperazine, without affecting the random migration of the neutrophils .
(5) Chemokine-induced chemotaxis was generally inhibited in monkey leukocytes in the presence of mu-opioid receptor agonists such as morphine, DAMGO, methadone, and endomorphine .
(6) Vasoactive intestinal polypeptide (VIP) inhibited alveolar macrophage chemotaxis to endotoxin-activated rat serum, with maximum inhibition of 46% at 0.1 µM concentration .
(7) Various bacterial endotoxins inhibited neutrophil chemotaxis to chemokine IL-8 without themselves being chemotactic for neutrophils , and leukocyte migration was inhibited by a staphylococcal aggressin .
(8) A specific chemoattractant for neutrophils was completely blocked in vitro, and 40% blocked in vivo, using an antagonist to the chemoattractant receptor; the antagonist itself had no chemotactic activity .
(9) Gastrin-17 and gastrin-34, maximally at 0.1 nM, inhibit cell mobility in human peripheral blood neutrophils . The inhibitory effect of gastrin is similar to that obtained with EGTA, a well-known calcium chelating compound.
General-purpose chemoattractant inhibitors also are known or possible. For instance, alpha1-proteinase inhibitor induces chemotaxis and chemokinesis at low concentrations of 0.02-2 mg/ml (normal alveolar surface-fluid concentrations in the lung) but inhibits chemotaxis of PMNs to known chemoattractants, at higher concentrations of 2-10 mg/ml (corresponding to inflammatory blood levels) . And semaphorins, originally described as neuronal chemorepellents, have now been identified in the immune system . (Human CD100 is a leukocyte semaphorin [3320, 3321], although as of this writing no chemorepulsive activity has been experimentally confirmed for CD100.) Semaphorins are also found on the surfaces of murine lymphocytes , and may be present on human lymphocyte surfaces  and on human monocytes , though again there is as yet no confirmed evidence of chemorepulsive activity. It is important to note that these chemotactic inhibitors may have significant effects on other cells and on cellular activity, thus precluding their use with nanorobots.
More research is required to select, or more likely to design, an ideal chemorepellent agent that might be secreted (perhaps at nM concentrations, ~1 molecule/micron3 or less) by, or surface-tethered to, medical nanorobots seeking to avoid contact with phagocytes. Note that bioactive substances released locally by nanorobots can later be retrieved by similar means, thus avoiding nonlocal accumulations of these substances following nanomedical treatment.
Last updated on 30 April 2004