Any invading microbe that readily
attracts white cells (phagocytes) capable of eating it, and then allows itself
easily to be ingested and killed, is generally unsuccessful as a parasite.
That’s why most successful bacteria interfere to some extent with the
activities of phagocytes or find some way to avoid their attention [1].
Bacterial pathogens have devised numerous diverse strategies to avoid
phagocytic engulfment and killing, mostly aimed at blocking one or more of the
steps in phagocytosis, thereby halting the process [1].
Similarly, natural phagocytic cells
presented with any significant concentration of medical nanorobots [2] also may
attempt to internalize these nanorobots. How often will such an opportunity
arise? There may be an average of one ~730 micron3 granulocyte (e.g., neutrophil) in
every ~3 x 105 micron3 of human blood, one ~1525 micron3 monocyte in every ~2 x 106 micron3 of blood, and one >1525 micron3 macrophage in every ~2 x 105 micron3 of human tissues. By random thermal
motions in a quiet fluid, a 2-micron nanorobot would trace out a volume
containing one neutrophil in ~70 sec at 310 K ([2], Eqn. 3.1), or would diffuse
the ~40 micron mean free distance ([2], Eqn. 9.72) between nanorobot and the
nearest macrophage in quiet watery tissue in ~4000 sec ([2], Eqn. 3.1). In a
small (1 mm diameter) artery with blood flowing at 100 mm/sec, each 2-micron
nanorobot, in a total bloodstream population of 1012 such nanorobots, would collide with a
white cell once every ~3 seconds near the periphery of the vessel but only once
every ~300 seconds near the center of the vessel ([2], Section 9.4.2.2), a
rheological disparity that will be amplified by phagocyte margination ([2],
Section 9.4.1.3). Studies of macrophage particle-ingestion kinetics show that
the number of particles ingested by each phagocytic cell may rise tenfold as
the local particle concentration rises from 5 particles per cell to 150
particles per cell [3].
From these crude estimates, it becomes
apparent that virtually every medical nanorobot placed inside the human body
will encounter phagocytic cells many times during its mission. Thus all
nanorobots which are of a size capable of ingestion by phagocytic cells must
incorporate physical mechanisms and operational protocols for avoiding and
escaping from phagocytes. Ingestion may require from many tens of seconds to
half an hour to go to completion, depending upon the size of the internalized
particle, so medical nanorobots should have plenty of time to detect and to
actively prevent this process. The initial strategy for medical nanorobots is
first to avoid phagocytic contact or recognition, and if this fails, then to
avoid nanorobot binding to the phagocyte surface, and phagocytic activation.
One simple avoidance method employed by
a few pathogens that may occasionally be practical for medical nanorobots is to
confine 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 [1]. 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 ([2], Section 8.4.3), then
most motile nanorobots should be able to outrun any “pursuing” phagocytes.
If remote phagocyte detection methods
cannot be made reliably available, and for nonmotile nanorobots, other contact
avoidance techniques must be employed. 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) [4]. Specific chemicals are known to
be chemorepellents, chemotaxis antagonists, chemotactic factor enzymes or
antibodies, or negative chemokinesis agents for various cell types.
For example, monocyte migratory
inhibition factor inhibits macrophage migration, with a maximum inhibitory
effect at 1 ng/ml for both unchallenged and particle-challenged macrophages
[5]. Excess zinc immobilizes macrophages [6], and mononuclear cells cultured
from hyperimmunoglobulin-E (HIE) patients produced a ~61 kD protein factor that
nontoxically inhibited normal neutrophil and monocyte chemotaxis [7] while
serum from those patients contained a 30-40 kD inhibitor of granulocyte and
monocyte chemotaxis [8]. Phospholipase A2 inhibitors and a ubiquitin-like
peptide [9] inhibit neutrophil chemotaxis, leukocyte-specific protein 1 (LSP1)
is a negative regulator of neutrophil chemotaxis [10], and polyamines such as
putrescine at 1 mM and spermidine at 0.1-0.5 mM inhibit chemotaxis (but not
phagocytosis or engulfment) by neutrophils in vitro [11]. Granulocyte
locomotion is also inhibited by diclofenac sodium, a nonsteroidal
anti-inflammatory agent, at concentrations below 10 micrograms/ml [4], and
eicosapentaenoic acid somewhat rigidifies the plasma membrane of human
neutrophils, leading to reduced chemotaxis [12]. In other experiments,
chemotaxis by human neutrophils toward several common chemoattractants was
inhibited by 80%-95%, maximally at a concentration of ~50 microM of the protein
kinase inhibitor 1-(5-isoquinolinesulfonyl) piperazine, without affecting the
random migration of these white cells [13].
Much phagocyte chemorepellent research
occurs in the context of elucidating bacterial avoidance strategies —
strategies that 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 chemotactic repellent [1], even in very
low concentrations.Staphylococcus aureus produces toxins that inhibit the
movement of phagocytes; granulocytes are almost immobilized when administered
12 micrograms/ml of purified S.
aureus lipase [14]. Pertussis
toxin, produced by the bacterium Bordetella
pertussis, inhibits chemotaxis of neutrophils and other phagocytes; a
PMN-inhibitory factor (PIF) extracted from B.
pertussiscells showed little cytotoxicity and inhibited chemotaxis of
neutrophils [15]. Fractions of Mycobacterium
tuberculosis inhibit
leukocyte migration [1], the Clostridium
perfringens phi toxin
inhibits neutrophil chemotaxis [1], and other “specific antigen” can suppress
basophil chemotaxis. Phagocyte chemotaxis is generally reduced by antibiotics
such as cefotazime, rifampin, and teicoplanin [16]. 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. More research is
required to select, or more likely to design, the 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 during nanomedical treatment.
Chemorepulsion is adequate for a few
devices on simple missions of limited duration, but large numbers of medical
nanorobots on longer more complex missions will inevitably come into physical
contact with a phagocyte. The least disruption to normal immune processes is
achieved if the nanorobot surface can deny recognition to the inquiring
phagocyte at the moment of physical contact. Surface-bound moieties are
generally preferable to free-released molecules when large numbers of in vivo
nanorobots are involved. For example, each nanorobotic member of an internal
communication network ([2], Section 7.3.2), stationed perhaps ~100 microns
apart throughout the tissues, must continuously avoid being ingested by passing
phagocytes. An approach that relies primarily on antiphagocytic chemical
releases risks extinguishing all phagocytic activity throughout the body,
severely compromising the natural immune system.
By 2001, “long-circulating”
phagocytosis-resistant particles [17] and stealth drug carriers [18] have
become the objects of active and extensive research. It is well-known that
nanoparticle adsorption and internalization by phagocytes are inhibited by the
presence of a coating of polysaccharide (e.g., heparin or dextran) chains in a
brush-like configuration, or by very hydrophilic coatings. Low phagocytic uptake
is achieved using a surface concentration of 2%-5% by weight of PEG, giving
efficient steric stabilization (e.g., a distance of ~1.5 nm between two
adjacent terminally-attached PEG chains in the covering brush) and avoiding
uptake by neutrophils [19]. Experiments by Davis and Illum [18] suggest that
polystyrene particles sterically stabilized with adsorbed poloxamer polymer
could achieve an extrapolated zero phagocytic uptake using a ~10 nm thick
coating on 60 nm diameter particles or a ~23 nm thick coating for 5.25 micron
diameter particles, thus eliminating nonspecific phagocytosis. Another study
found that pegylated sheep red blood cells (RBCs) were ineffectively
phagocytosed by human monocytes, unlike untreated sheep RBCs. Electrical
characteristics also are important. Phagocytosis of polystyrene beads (as
measured by cellular oxygen consumption) appears strongly dependent on surface
potential and thus upon fixed surface charge, and surface charge heterogeneity
across domains as small as 1-4 microns can greatly affect phagocytic ability.
Rather than coatings which phagocytes
cannot recognize at all, medical nanorobots alternatively could carry surfaces
that phagocytes will recognize as “friendly.” For example, coatings that mimic
natural immune-privileged cells could be used. Nanorobot exteriors could be
covalently bound with essential erythrocyte coat components — a simulated RBC
surface could be useful in the bloodstream, but might provoke a response in the
tissues. Similarly, fibroblast-like surface might be useful in the tissues, but
is not normally seen in the bloodstream and phagocytes might respond to its
presence there. Simulated neutrophil or monocyte surfaces would be better,
since these cells normally migrate from blood to tissues, hence the immune
system expects to see these surfaces virtually everywhere; lymphocytes are
likewise normally present in both blood and tissues but are also adept at
passing through the endothelial lining, the lymphatic processes, and the lymph
nodes without being detained or trapped, eventually returning to the arterial
circulation. The ideal solution may be for the medical nanorobot to display a
specific set of self-markers at its surface, perhaps including moieties such as
CD47. CD47 is a surface protein present on almost every cell type that provides
an explicit phagocytic inhibitor signal to NK cells and to macrophages [20].
Microbial pathogens employ similar
strategies to create antiphagocytic surfaces that avoid provoking an
overwhelming inflammatory response, thus preventing the host from focusing the
phagocytic defenses [1]. Enveloped viruses and some bacterial pathogens can
cover their external cell surface with components that are seen as “self” by
the host’s phagocytes and immune system, a strategy that hides the true
antigenic surface. Phagocytes then cannot recognize the bacteria upon contact
and the possibility of opsonization by antibodies to enhance phagocytosis is
minimized [1]. For example, Group A streptococci can synthesize a capsule
composed of hyaluronic acid, the “ground substance” (tissue cement) found in
human connective tissue. The streptococcal hyaluronic acid capsule is
nonantigenic and thus very effective in preventing attachment of the organism
to the macrophage [21]. Additionally, the cytoplasmic membrane of Streptococcus pyogenes contains antigens similar to those
found on human cardiac, skeletal and smooth muscle cells, on heart valve
fibroblasts, and in neuronal tissues, resulting in molecular mimicry and an
immune tolerance response by the host [22]. Other examples include pathogenic Staphylococcus aureus that produces cell-bound coagulase
which clots fibrin on the bacterial surface [1], the syphilitic agent Treponema pallidum that binds human fibronectin to its
surface [1], and a variety of bacteria that cause meningitis that avoid
phagocytosis either by preventing deposition of complement by sialic acid on
the surface or by modification of lipopolysaccharide (LPS). Haemophilus influenza expresses a mucoid polysaccharide
capsule that prevents digestion by host phagocytes; a few strains resist
opsonization and have become serum resistant by modification of their LPS
O-antigen side chains, rendering them “invisible” to host immune defenses.
What if the nanorobot has been
recognized as foreign by a white cell? As the next line of defense, medical
nanorobots can directly inhibit phagocytic binding and activation. In the case
of receptor-mediated binding, dansylcadaverine, amantadine, and rimantadine
induce inhibition of endocytosis of complement-coated zymosan particles by
human granulocytes.
These drugs block receptor-mediated
endocytosis, possibly by their actions on phospholipid metabolism [23],
although dansylcadaverine is not an endocytosis inhibitor in cells lacking
transglutaminase activity. Cell-bound or soluble protein A produced by Staphylococcus aureus [24] 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
of the bacteria 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 negative values ([2], Section
9.2.3).
Phagocyte activation can also be
directly inhibited. Several pathways of phagocytic signal transduction have
been identified [25], 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 that leads to particle
internalization. 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 [26] such
as genistein (50 microM), herbimycin (17 microM), staurosporine and
trifluoperazine; 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. CNI-1493 is a potent and well-known macrophage deactivator or
“pacifier” [27].
Acknowledgements
The author thanks C. Christopher Hook,
M.D., and Stephen S. Flitman, M.D., for helpful comments on an earlier version
of this paper.
Copyright 2001 Robert A. Freitas Jr.
All Rights Reserved
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Note: An article by Robert A Freitas Jr. on the potential
applications of advanced nanotechnology to dental care appeared
applications of advanced nanotechnology to dental care appeared
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