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IMM Report Number 38: Nanomedicine


In conjunction with Foresight Update 51

Will Intracellular Medical Nanorobots Disrupt the Cytoskeleton (Part I)?

By Robert A. Freitas Jr.
Research Scientist, Zyvex Corp.

Robert A. Freitas  Jr.
Robert A. Freitas Jr.

Active medical nanorobots [1, 2] maneuvering inside living cells could disturb or disrupt any of the many functions of the cytoskeleton. The cytoskeleton is the internal structural framework of a cell, consisting of several different types of filaments (see below). The functions of the cytoskeleton include: (1) mechanical support, such as cell movement, adhesive interaction with the extracellular matrix (ECM) and neighboring cells, and stabilization of cell shape including cellular “tensegrity”; (2) integration of diverse cellular activities, such as intracellular movement including transport and positioning to the appropriate locations of organelles, intracellular particles, RNA and proteins; and (3) intracellular signal transduction, which includes structural information regarding cellular origin and differentiation state [3]. In diverse cell types, microtubule and actin filament networks cooperate functionally during many processes, such as vesicle and organelle transport, cleavage furrow placement, directed cell migration, spindle rotation, and nuclear migration [4]. In principle, nanorobots could mechanically disrupt any or all of these functions during nanorobotic intracellular locomotion or manipulation of cell structures, especially if cytoskeletal/membrane links are disturbed.

Two classes of danger seem most significant. First is the risk of direct mechanical cytoskeletal reorganization, the subject of Part I of this article. The second is the risk of possible disruption of vesicular transport and related molecular motor diseases, the subject of Part II (next time). In both cases, it appears likely that potential problems can be avoided by conservative design.

Generalized disruption of the cytoskeleton can be very harmful to living cells. Disorganization of the cytoskeletal architecture has been associated with diseases as diverse as heart failure, rotavirus infection, sickle cell anemia, lissencephaly (smooth brain, lacking convolutions), and Alzheimer’s disease. A “collapse transition” of neurofilament sidearm domains may contribute to amyotrophic lateral sclerosis (ALS) and to Parkinson’s disease. Stress-related cytoskeletal fracture can be caused by 1-Hz cyclic forces imposed by a mechanical probe on isolated rat ventricular myocytes [5]. Cancer cells forced through 5- to 12-micron pores in polycarbonate membrane suffer traumatic spatial dissociation among components of the cell periphery, the cytoskeleton, and nucleus, inducing a ~1-week dormant state in the cells due to the mechanical trauma [6].

Nanorobots could induce various cell pathologies by mechanically disrupting specialized cytoskeletons consisting of cytoplasmic networks of ~6-nm diameter actin microfilaments, ~10-nm intermediate filaments, ~25-nm microtubules, or their many associated proteins ([1], Section 8.5.3.11), with effects similar to those of chemical disruption. Functions of these specialized cytoskeletons that could be disturbed include mechanical integrity and wound-healing in epidermal cells, cell polarity in simple epithelia, contraction in muscle cells, hearing and balance in the inner ear cells, axonal transport in neurons, and neuromuscular junction formation between muscle cells and motor neurons [7].

As a nanorobot enters a cell through the plasma membrane, the first risk is transmembrane linkage disruptions. Muscular dystrophy may be caused by disorganization of links between the intracellular cytoskeleton and the ECM [10], and the disruption of proper adhesive interactions with neighboring cells can lead to fatal defects in extracellular tissue architecture [11]. Epithelial cells subjected to mechanical strain may release in vivo proteases to cut intercellular adhesions [12]. Looking inward, cellular mechanoprotective adaptations involve a coordinated remodeling of the cell membrane and the associated cytoskeleton [13]. For example, the breakage of major cytoskeletal attachments between the plasma membrane and peripheral myofibers in cardiac myocytes predisposes the cell to further mechanical damage from cell swelling or from ischemic contracture [14]. As another example, attacking fungus cells (that are infecting parsley cells) extend a penetrating hypha through the cell membrane, eliciting a defensive cytoskeletal reorganization [15]. A local mechanical stimulus produced by a needle of the same diameter as the fungal hypha inserted through the host cell wall similarly induces the translocation of cytoplasm and nucleus to the site of stimulation, the generation of intracellular reactive oxygen intermediaries, and the expression of some elicitor-responsive genes. Without the mechanical stimulation, the morphological changes are not detected [15].

Mechanical disruption of cytoskeleton associated proteins by passing nanorobots could produce various cytopathologies. For instance, plectin is a 580 kD intracellular protein that links intermediate filaments with actin microfilaments, microtubules, and plasma membrane. Widespread disruption of plectin function results in severe skin blistering and muscular degeneration, consistent with plectin’s role in stabilizing cells against external mechanical forces [16] and as a regulator of intracellular actin dynamics [17]. Disturbance of centrosomes or other in cyto fixed polarity markers could result in developmental or morphogenetic defects during subsequent cell division. Mechanical disturbance of cytoskeleton associated proteins could also alter the mechanical properties of cells, such as the ability of the cytoskeleton to deform and flow.

Actin microfilaments might be disrupted by the mechanical activities of medical nanorobots. In the simplest case, endothelium exposed to shear stress undergoes cell shape change, alignment, and microfilament network remodeling in the direction of flow, though these changes can be blocked with nocodazole. Glomerular distention is also associated with cellular mechanical strain. A contractile cytoskeleton in mesangial cells, formed by F-actin-containing stress fibers, maintains structural integrity and modulates glomerular expansion. Mesangial cells have a cytoskeleton capable of contraction that is disorganized in long-term diabetes. Disorganization of stress fibers may be a cause of hyperfiltration in diabetes. Cutting the actin lattice may diminish both cell contractility [18] and mechanical signal transduction into the nucleus [19]. Care must also be taken to ensure that the surfaces or activities of intracellular nanorobots do not provide unplanned foci for actin polymerization, given that the kinetics of actin polymerization is autocatalytic and that the actin-based motility of functionalized microspheres can be reconstituted in vitro from only five pure proteins. Widespread actin disruption might produce symptoms analogous to elliptocytosis (abnormally large number of oval-shaped red cells in the blood) and other inherited hemolytic disorders that are caused by disorganization of the subsurface spectrin-actin cell cortex in the erythrocyte [20].

Intermediate filaments might also be disrupted mechanically. Perturbations in the architecture of the intermediate filament cytoskeleton in keratinocytes and in neurons can lead to degenerative diseases of the skin, muscle cells, and nervous system [7-9]. Knockout of the extensive keratin filament network jeopardizes the mechanical integrity of the epidermal cell, producing cell fragility and cytolysis manifesting as blistering skin disorders. Tissues lacking intermediate filaments fall apart, are mechanically unstable, and cannot resist physical stress, which leads to cell degeneration [21]. Perinuclear clumping of fragmented keratin intermediate filaments accompanies many keratin disorders of skin, hair, and nails. In active muscle, intermediate filaments play an important role in the organization and stabilization of myofibril-membrane attachment sites. Their disruption can eliminate the deep membrane invaginations that are normally present in the healthy sarcolemma (the membrane surrounding striated muscle fiber) [8]. Neuronal intermediate filaments are normally anchored to actin cytoskeleton. If this anchoring fails, the cell displays short, disorganized and unstable microtubules that are defective in axonal transport. Neuronal survival requires viable interconnects among all three cytoskeletal networks [9]. Impairment of normal axonal cytoskeletal organization in Charcot-Marie-Tooth disease results in distal axonal degeneration and fiber loss.

The microtubule cytoskeleton could become disorganized due to careless intracellular operations by nanorobots, possibly: (1) simulating congenital brain malformation; (2) giving results similar to treatment with vincristine, a microtubule depolymerizing drug that produces peripheral neuropathy in humans accompanied by painful paresthesias and dysesthesias (abnormal sensations of numbness, tingling, prickling, or burning); or (3) giving results similar to treatment with ethanol, leading to oxidative injury producing a loss of gastrointestinal barrier integrity. Mechanical disturbance of the microtubule cytoskeleton induces electrophysiological modification of cell-cycle-dependent EAG potassium channels in mammalian tissue cells, and mechanical strain can induce a major decline in tubulin production in osteoblasts [22]. Nanorobot mechanical operations could also induce buckling and loop formation of tubulin fibers, as has been observed [23] inside shrinking vesicles when the surface tension of the shrinking bubble overcomes the Euler buckling strength of the fibers — intracellular tubulin twisted into 5-micron tennis-racquet shapes has also been observed [24].

Microtubules allowed to form under microgravity conditions show almost no self-organization and are locally disordered, unlike microtubules formed in 1-g conditions. Nanorobotic manipulations of cytoskeletal elements that offset, reduce, or cancel the stimulative effects of normal gravity could produce the same sort of cellular architectural disorganization as observed under microgravity conditions ([1], Section 4.4.2) that alters the pattern of microtubular orientation.

A nanorobot with sharp edges that cuts a microtubule probably cleaves the hydrogen bonds between the alpha and beta monomers, rather than the covalent bonds within the monomers. This creates a new “plus” and “minus” end for the microtubule. In most cases this would not be fatal for the cell and in fact normally would have little impact because large-scale microtubule network patterns (e.g., asters, whorls, and interconnected pole networks) are self-assembling and are motor-molecule concentration-dependent [25]. Nevertheless, in cyto nanorobots should avoid physically severing cytoskeletal elements whenever possible. Simple estimates of mechanical strength ([1], Table 9.3) applied to typical fiber diameters suggest tensile failure strengths of ~170 pN for actin microfilaments, ~300-500 pN for microtubules and ~20,000 pN for intermediate filaments. Nanorobots should avoid applying local forces of these magnitudes or larger in the vicinity of such fibers.

Force thresholds for cellular activation ([2], Section 15.5.4.1) may be considerably less than the indicated tensile failure strengths. By the end of 2002, the absolute force thresholds for failure, the range of mechanical frequency responses, and the threshold fraction of disturbed cytoskeleton required to elicit cellular response all had yet to be precisely determined. For example, during mitosis a force of 15-20 pN is required to detach microtubule-bound chromosomes [26] but a tensile force of up to 210 pN is required to detach a microtubule from a kinetochore [27]. Moreover, a nanorobot presenting a 1-micron2 forward surface during intracellular locomotion through a (20 micron)3 tissue cell intercepts only ~0.25% of the entire cytoskeleton during each 20-micron of transcellular travel. In cyto medical nanorobots may be restricted to speeds of ~10 microns/sec while traversing intracellular clear paths ([1], Section 8.5.3.12) and ~1 micron/sec during transfilamentary intracellular locomotion, with progressive resealing of cytoskeletal elements that must be temporarily severed to allow the nanorobot to pass ([1], Section 9.4.6). Intranuclear locomotion conservatively should progress no faster than natural chromosomal dragging rates during mitosis [26], or ~0.1 micron/sec, applying forces of at most ~50 pN ([1], Section 9.4.6).

Copyright 2003 Robert A. Freitas Jr. All Rights Reserved

References

1. R.A. Freitas Jr., Nanomedicine, Volume I: Basic Capabilities, Landes Bioscience, Georgetown, TX, 1999; http://www.nanomedicine.com

2. R.A. Freitas Jr., Nanomedicine, Volume IIA: Biocompatibility, Landes Bioscience, Georgetown, TX, 2003; http://www.nanomedicine.com

3. D. Broekaert, “Cytoskeletal polypeptides: cell-type specific markers useful in investigative otorhinolaryngology,” Int. J. Pediatr. Otorhinolaryngol. 27(May 1993):1-20.

4. B.L. Goode et al., “Functional cooperation between the microtubule and actin cytoskeletons,” Curr. Opin. Cell Biol. 12(February 2000):63-71.

5. G.C. Bett, F. Sachs, “Whole-cell mechanosensitive currents in rat ventricular myocytes activated by direct stimulation,” J. Membr. Biol. 173(1 February 2000):255-263.

6. H. Gabor, L. Weiss, “Mechanically induced trauma suffered by cancer cells in passing through pores in polycarbonate membranes,” Invasion Metastasis 5(1985):71-83.

7. E. Fuchs, “The cytoskeleton and disease: genetic disorders of intermediate filaments,” Annu. Rev. Genet. 30(1996):197-231.

8. R.B. Cary, M.W. Klymkowsky, “Disruption of intermediate filament organization leads to structural defects at the intersomite junction in Xenopus myotonal muscle,” Development 121(April 1995):1041-1052.

9. Y. Yang et al., “Integrators of the cytoskeleton that stabilize microtubules,” Cell 98(23 July 1999):229-238.

10. R.D. Cohn, K.P. Campbell, “Molecular basis of muscular dystrophies,” Muscle Nerve 23(October 2000):1456-1471.

11. C. Hagios et al., “Tissue architecture: the ultimate regulator of epithelial function?” Philos. Trans. R. Soc. Lond. B. Biol. Sci. 353(29 June 1998):857-870.

12. H.L. Kain, U. Reuter, “Release of lysosomal protease from retinal pigment epithelium and fibroblasts during mechanical stresses,” Graefes Arch. Clin. Exp. Ophthalmol. 233(April 1995):236-243.

13. K.S. Ko, C.A. McCulloch, “Partners in protection: interdependence of cytoskeleton and plasma membrane in adaptations to applied forces,” J. Membr. Biol. 174(15 March 2000):85-95.

14. M.D. Sage, R.B. Jennings, “Cytoskeletal injury and subsarcolemmal bleb formation in dog heart during in vitro total ischemia,” Am. J. Pathol. 133(November 1988):327-337.

15. S. Gus-Mayer et al., “Local mechanical stimulation induces components of the pathogen defense response in parsley,” Proc. Natl. Acad. Sci. (USA) 95(7 July 1998):8398-8403.

16. P.G. Allen, J.V. Shah, “Brains and brawn: plectin as regulator and reinforcer of the cytoskeleton,” Bioessays 21(June 1999):451-454.

17. K. Andra et al., “Not just scaffolding: plectin regulates actin dynamics in cultured cells,” Genes Dev. 12(1 November 1998):3442-3451; G. Wiche, “Role of plectin in cytoskeleton organization and dynamics,” J. Cell Sci. 111(September 1998):2477-2486.

18. J. Pourati et al., “Is cytoskeletal tension a major determinant of cell deformability in adherent endothelial cells?” Am. J. Physiol. 274(May 1998):C1283-C1289.

19. F. Guilak, “Compression-induced changes in the shape and volume of the chondrocyte nucleus,” J. Biomech. 28(December 1995):1529-1541.

20. Z. Zhang et al., “Dynamic molecular modeling of pathogenic mutations in the spectrin self-association domain,” Blood 98(15 September 2001):1645-1653.

21. M. Galou et al., “The importance of intermediate filaments in the adaptation of tissues to mechanical stress: evidence from gene knockout studies,” Biol. Cell 89(May 1997):85-97.

22. M.C. Meazzini et al., “Osteoblast cytoskeletal modulation in response to mechanical strain in vitro,” J. Orthop. Res. 16(March 1998):170-180.

23. M. Elbaum et al., “Buckling microtubules in vesicles,” Phys. Rev. Lett. 76(20 May 1996):4078-4081.

24. D. Kuchnir Fygenson et al., “Mechanics of microtubule-based membrane extension,” Phys. Rev. Lett. 79(1997):4497-4500.

25. T. Surrey et al., “Physical properties determining self-organization of motors and microtubules,” Science 292(11 May 2001):1167-1171.

26. A.J. Hunt, J.R. McIntosh, “The dynamic behavior of individual microtubules associated with chromosomes in vitro,” Mol. Biol. Cell 9(October 1998):2857-2871.

27. R.B. Nicklas, “Measurements of the force produced by the mitotic spindle in anaphase,” J. Cell Biol. 97(1983):542-548.

More on the medical applications of nanotechnology at:
http://www.foresight.org/Nanomedicine/index.html
Also, please check out http://www.nanomedicine.com/. Robert Freitas has acquired ownership of the domain, and has put up a cleaner and very extensively internally-linked version of Nanomedicine Vol. I at http://www.nanomedicine.com/NMI.htm

IMM would appreciate learning your thoughts on the above article.