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
184.108.40.206.1 Clearance of Particles from Tissues
Immobile nanorobots found in extravascular tissues are susceptible to being phagocytized either by resident tissue macrophages and other phagocytic cells such as fibroblasts, or by newly arriving phagocytes such as neutrophils and monocytes that have immigrated from the blood by passing through blood vessel walls via diapedesis (Section 220.127.116.11) into the adjoining tissue. Sell  notes that if particles are injected into connective tissue, the local phagocytes will ingest them; if particles are injected into the brain, the microglia will absorb them.
For example, macrophages patrol or readily enter the tissues of the peritoneum  and thorax . The capture of polystyrene particles 0.3-3 microns in diameter by rat peritoneal macrophages was studied in vitro . The most efficient accumulation by the macrophages was of 0.6-micron particles, yielding an endocytic index of 4.56 micron3/cell-sec. Hydrophilized (via hydroxymethylation) 3-micron particles had a tenfold higher rate of capture, an endocytic index of 37.9 micron3/cell-sec . Upon injection into rat peritoneum, 3-micron particles showed selective accumulation in the omentum whereas 0.8-micron particles were better able to leave the peritoneal compartment . After 5 hours, most particles (72-86%, depending on particle type) still remaining in the peritoneum had been endocytosed by cells .
Other similar experiments found that:
(1) 1- to 5-micron poly(lactic/glycolic acid) microspheres were efficiently taken up by macrophages both in culture and after intraperitoneal injection into mice, with saturation of phagocytosis after 3 hours ;
(2) peritoneal phagocytes from striped bass ingested ~3-micron latex beads during a 30-minute incubation time, giving a phagocytic capacity of ~4 beads/phagocyte ;
(3) sterically stabilized (coated) polystyrene microspheres with thicker coatings are decreasingly phagocytosed by mouse peritoneal macrophages ;
(4) 30- to 120-micron microspheres injected intraperitoneally in rats were large enough to be retained more or less permanently in the peritoneal cavity, whereas microspheres with diameters <24 microns were cleared from the peritoneal cavity through fenestrations in the diaphragm, and eventually were observed in the lymphatic system ;
(5) for peritoneally-injected microspheres in mice, 1.4- and 6.4-micron PMMA particles and 1.2- and 5.2-micron polystyrene particles were engulfed by macrophages, but 12.5-micron polystyrene particles were not ;
(6) fused aluminosilicate microparticles injected into beagle dog peritoneal cavities were translocated to mesenteric, left sternal and right sternal lymph nodes, with a small percentage also going to the left tracheobronchial lymph node ; and
(7) inert tungsten particles instilled into the pleural space of dogs were translocated to the thoracic lymph nodes in 1-7 days .
Colloidal carbon particles injected intravitreously into chicken eyes were actively ingested by hyalocytes (the resident macrophages) by the second day, without significant leukocyte recruitment . Noted the researchers: “In the second stage (at 7-14 days), a large number of macrophages infiltrated the ciliary body and emigrated into the vitreous chamber. In the third stage (at 30 days), the infiltration by macrophages into the ciliary body was complete. The carbon-laden macrophages disappeared from the vitreous body but accumulated on the pecten oculi and retina. They were exclusively drained through the scleral venous sinus in the iridocorneal angle.” Another experiment in which 0.02- to 0.07-micron carbon particles were injected into the vitreous humor of monkeys produced cellular proliferation of mononuclear phagocytes and inflammatory cells after 1 week, continued macrophagic response along with fibrovascular proliferation into the vitreous after 3 weeks, deposition of extracellular fibrous material and traction retinal detachment after 4-5 weeks, and carbon-laden macrophages aggregated over the optic disk and fovea, along with prepapillary neovascularization and cystoid macular edema after 10 weeks .
But there are other phagocytic cells in the eye besides macrophages. The trabecular meshwork is a specialized tissue in the anterior chamber of the eye that regulates aqueous humor outflow and pressure . Meshwork cells are actively phagocytic and may operate to keep the drainage pathways free of cellular debris, pigment, and other particulate material [3009, 3010]. When meshwork cells are exposed to latex microspheres, within 4 hours the cells exhibit a short-term loss of cell-matrix adhesiveness and an increase in cellular migratory activity, returning to normal after 24 hours . Ingestion rates are 3-4 beads per phagocytic cell . However, Buller et al  reported that the presence of a foreign particle does not always induce a phagocytic response by human trabecular cells, because free particles were observed in the intertrabecular spaces and in Schlemm’s canal. Latex microspheres injected into rabbit corneal stroma were endocytosed by keratocytes (corneal fibroblasts) and stored for >800 days in the keratocyte cytoplasm .
As another example, consider the phagocytes in brain tissue. One experiment  demonstrated the ability of rat astrocytes to ingest 0.05- to 0.2-micron fluorescent polystyrene microspheres. In another experiment , colloidal carbon injected into the cerebral cortex of neonatal rats was ingested in membrane-bound vacuoles and sequestered in lysosomes of young astrocytes (phagocytic star-shaped neuroglial cells with many branching processes). Carbon-laden astrocytes were seen in the immediate vicinity of the site of the injection after 4 days (and in abundance after 10-21 days), in the surrounding (apparently normal) neuropil, and in the perivascular regions. This showed that young astrocytes could engulf foreign particles injected into the developing brain.
In adult brains, however, it appears that astrocytes are involved in phagocytosis  of cell debris and foreign particles only as a second line of defense . The microglia [3016-3021] appear to be the first line of defense, distributed, unlike astrocytes, throughout the brain in non-overlapping territories . Microglia belong to the RES and are the resident macrophages in brain tissue, in the spinal cord, and in the retina . In one experiment  involving implantation of polystyrene microspheres in rat brain, both microglial cell and nonspecific astrocytic (proliferative) brain tissue reactions were seen in the first few days, similar to that found after damage to the CNS. Some foreign-body giant cells (Section 18.104.22.168) were also observed. After 9 months, the microspheres appeared to be engulfed by histiocytic cells, with microsphere clusters surrounded by a non-necrotic sheath of collagen and astrocytic cells . Additional phagocytic cells found in the brain include macrophages [3023, 3024] such as pericytes , perivascular [3026, 3027] cells, and meningeal  cells. Researchers also have studied: (1) the passive displacement of 6- to 10-micron microspheres throughout the brain parenchyma ; (2) the drainage of particles in cerebrospinal fluid directly from the subarachnoid space into the nasal lymphatics in the rat  and in man [3030-3032] (up to 20-30% of CSF may drain by this route ); and (3) the intracellular transport of latex microspheres inside peripheral nerve cells in both anterograde and retrograde directions .
Phagocytes which have ingested foreign particulate matter present in tissues may persist at the site of ingestion if they are unable to solubilize the material. Indeed, some cells have been shown to persist for years at the site of particulate insoluble foreign bodies . Tattoos  are an excellent example of this persistence. In one experiment , the fate of India ink particles and of polystyrene latex beads injected into the murine dermis and subcutis of the skin of the auricle and back was observed with the naked eye and by light microscopy and electron microscopy. Ink particles injected as tattoo patterns remained essentially unchanged for life, to the naked eye. Microscopic examination revealed that both India ink particles and latex beads were endocytosed by fibroblasts and macrophages in the dermis and subcutis. In fibroblasts, numerous ink particles or small latex beads (0.22 micron in diameter) were packed into vacuoles 0.1-10.0 micron in diameter, occupying a large volume of the cytoplasm of the cell body and pseudopods. In macrophages, numerous particles and larger beads (both 0.22 micron and 2.0 microns) were taken up into the cell body. Dermal and subcutaneous fibroblasts that take up and store ink particles and latex beads move poorly after particle ingestion, and thus are almost fixed in the connective tissue, lending persistence to tattoos. The researchers  note that this represents “a specific non-inflammatory defense mechanism that protects the living body, without immune reactions, against injuries and invasions by non-toxic foreign agencies.” This description would likely apply equally well to inert medical nanorobots.
Last updated on 30 April 2004