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

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

15.4.3.6.6 Induce Exocytosis of Phagosomal Vacuole

Consider a medical nanorobot that has become trapped inside an intracellular vacuole or phagosome that has pinched off and now resides entirely within the intracellular space of the phagocytic cell. Without leaving the phagosome and prior to its merger with a lysosome, the nanorobot may escape by redirecting the phagosomal transport destination back to the plasma membrane, where the nanorobot can then be exocytosed, whole, from the cell.

To accomplish this, existing centripetal (endocytic) targeting proteins first must be removed from the vacuolar wall, and then new centrifugal (exocytic) targeting proteins must be embedded on the external phagosomal surface to redirect the vacuole back to the plasma membrane. One example of plasma membrane targeting (i.e., regulated exocytosis [3437-3440], reverse endocytosis [3441], or related processes [3442]) is the synaptic vesicle targeting protein synaptobrevin (VAMP-1 and -2). This protein binds only to the neuron-specific plasma membrane proteins syntaxin 1A and 1B, thus ensuring proper vesicle docking and fusion exclusively to the neuron plasma membrane from intracellular origins [3443]. (Fusion of two distinct lipid bilayers is energetically unfavorable in the absence of such specialized targeting proteins.) Regulated exocytosis is well known in neurons and in endocrine and exocrine cells [3438], and even in conventional lysosomes in response to rises in the intracellular free Ca⁺⁺ concentration [3444]. Similar centrifugal transport pathways have been identified in phagocytes [3372, 3445], wherein intracellular vesicles are targeted exclusively to plasma membrane surface receptors (e.g., CD11b, CD18) [3372]. Other possible mediators of secretory vesicle regulated exocytosis [3446-3448] are being investigated. Vacuolar retargeting strategies are employed by bacteria, as for example Legionella pneumophila, which, once internalized into a vacuole, evidently redirects its transport to the endoplasmic reticulum [3389]. The recently-discovered actin-based motility of bead-containing macrophage phagosomes [6064] might also be purposefully manipulated. More research is required to identify specific proteins and mechanisms to aid nanorobots in escaping phagosomes.

Inert phagocytosed particles can be rapidly exocytosed by phagocytes. In one experiment [3449], ~50% of an ingested load of inert oil emulsion particles was released from rabbit neutrophils in 2400 seconds at 37 ^oC. Electron microscopy confirmed an exocytic release process. Particles were extruded through a network of processes often accompanied by membranous vesicles. Neutrophils undergoing particle exocytosis remained intact. By feeding the cells differently labeled particles, the investigators showed that phagocytosis and exocytosis of the same particles can occur concurrently, and that particle ingestion can accelerate particle release [3449].

One less-well-targeted approach is for medical nanorobots to induce their own degranulation from the phagocyte by releasing secretagogues for that cell. For example, IgA and granulocyte-macrophage colony-stimulating factor (GM-CSF) are the two most potent secretagogues for human eosinophils, and IL-5, IL-3, TNFalpha and RANTES also induce eosinophil degranulation [3450, 3455]. C3b, IL-1, IL-6, fMLP, the divalent calcium ionophore A23187, and GM-CSF are secretagogues for human neutrophils [3451-3455]. Elevated levels of intracellular free Ca⁺⁺ can stimulate exocytosis, and can also inhibit endocytosis that has been evoked by dynamin I vesiculation, dynamin II GTPase activity, or receptor mediation [3431].

It is also possible that cell eversion and extrusion of contents might be triggered chemically. For instance, the nucleus of an oocyte can be ejected if the cell is treated with etoposide and cycloheximide (chemical enucleation) [3456-3458]. Microtubule poisons such as colchicine, colcemid and vinblastine cause extrusion of cellular nuclei [3459-3461], and EDDF is involved in erythroid cell denucleation [3462]. There are also a few older reports of nuclear extrusion in lymphocytes [3463, 3464], cell enucleation [3465], extreme nuclear convolution [3466] and nuclear blebbing [3467], though R. Bradbury notes that normal failures of the cell division process can result in the production of micro- or satellite- nuclei, which are not “normal” processes that can be biochemically invoked but rather are pathological situations that develop in pre-cancerous or cancerous cells. It’s also important to note that the goal of nanorobot escape should not come at the cost of the destruction of the phagocyte.

More selective induction of localized non-nuclear cytoplasmic extrusions by medical nanorobots may be possible. Such controlled extrusions might be functionally similar to the production of lamellipodia or pseudopodia (Section 15.4.3.6.4) or a long list of related structures including giant granules [3468]; tubular vermipodia [3469]; cytoplasmic bulbous protrusions [3470]; hairy cell leukemic irregular cytoplasmic projections [3471-3474]; cytoplasmic membrane blebbing (zeiosis [3475]); and arborization during (1) apoptosis [3476] (whether chemically [3477-3482] or biologically [3483-3487] elicited), (2) cytotoxic T cell attack [3488], (3) viral budding [3489-3491], (4) nonlethal bacterial challenge [3492], (5) chemical induction [3493-3497], (6) mechanical trauma [3498, 3499], (7) locomotion [3500, 3501], or (8) vesicular release [3502, 3503]. Such extrusions would result in the ejection of a bleb of cytoplasm containing the trapped nanorobot into the extracellular space with relatively little loss of material, or diminution of viability, of the phagocytic cell. A similar process of extrusion is employed by the intracellular bacteria Shigella and Listeria (see below).

In some circumstances, efficiency may be gained if the medical nanorobot first escapes from the phagosome in which it is trapped – possibly via reverse cytopenetration (Section 9.4.5) – before pursuing its ultimate exit from the cell. Such escape is not difficult and has been mastered by many species of intracellular pathogens. For example, Listeria monocytogenes relies on several molecules for quick lysis of the phagosome – listeriolysin O (a pore-forming hemolysin toxin) [3504, 3505] and two forms of phospholipase C [3506, 3507]. Listeria ivanovii employs several phospholipases to similar effect [3508]. Once free in the cytoplasm, Listeria induces its own movement via a remarkable process of host cell actin polymerization and formation of microfilaments within a comet-like tail [3509-3512] (Section 9.4.6). Another intracellular bacterium, Shigella flexneri, also lyses the phagosomal vacuole and escapes [3513], then induces cytoskeletal actin polymerization for the purpose of intracellular movement [3514] and, eventually, cell-to-cell spreading [3515]. The bacterial factor used by Shigella to breach the vacuolar membrane is IpaB [3513], one of the secreted invasin proteins it uses to invade cells [3517-3519]. Rickettsia also enters host cells inside phagosomes but are detected free in the cytoplasm a short time later [3520], starting in as little as 30 seconds [3302]. In one experiment [3521], half of the phagocytosed bacteria were freed from the phagosome after only 12 minutes. A bacterial enzyme, phospholipase A2, and other hemolysins and toxins seem to be responsible for dissolution of the phagosome membrane by Rickettsia [3522, 3523]. The intracellular protozoan parasite Trypanosoma cruzi also can escape from phagocytic vacuoles into the cytoplasm [3524, 3525], assisted by a hemolysin [3527] and a phospholipase C [3528]. Experiments show that ~70% of the parasites are free, just 2 hours after infection [3526]

Once the medical nanorobot is free in the cytosol, it has several options for final escape from the phagocyte. First, it can use presentation semaphores (Section 5.3.6) to display on its surface all necessary targeting proteins to allow it eventually to be naturally exocytosed from the cell. Second, the nanorobot, if still motile, can locomote (Section 9.4) to the cytosolic face of the plasma membrane, then directly undertake cytopenetration (Section 9.4.5), perhaps assisted by fusogens (Section 9.4.5.4) or membrane fluidizers such as n-butanol [3296]. Third, it can re-enter an intracellular vesicle already targeted for exocytosis and “ride” the vesicle out of the cell.

Once again, the bacteria have a lesson to teach. Usually when intracellular pathogens have actively replicated inside the host cell, the cell dies, often by lysis, releasing the pathogens extracellularly [3389]. However, a few intracellular bacteria that can quickly escape phagosomal confinement and enter the host cytosol can achieve cell-to-cell spreading without ever leaving the cytosolic compartments of adjacent cells. Shigella and Listeria, upon reaching the plasma membrane, induce the formation of plasma membrane protrusions that invaginate into the neighboring cell, resulting in the creation of a double-membraned vacuole containing the bacterium, whose walls are subsequently lysed, releasing the bacterium into the neighboring cell [3389]. Shigella flexneri requires the cell adhesion molecule E-cadherin during this process [3516] and other mediators are being studied intensively [3515, 3529]. A medical nanorobot could use similar means to extrude itself into adjacent cells, or into the extracellular spaces, thus escaping the phagocyte.

Last updated on 30 April 2004