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 Nanorobotic Leukocytolysis

Nanorobotic leukocytolysis – the mechanical fragmentation of white cells by individual nanorobots or by nanoaggregates – is a less serious concern than hemolysis and thrombocytolysis because there is only 1 white cell in normal human blood for every 740 red cells and 36 platelets. Nevertheless, mechanical white cell fragmentation has been seen in at least one patient who was using single-lumen subclavian hemodialysis catheters [4071]. A leukemic patient [4114] who underwent therapeutic mechanical leukapheresis suffered white cell fragmentation with complications including renal failure and disseminated intravascular coagulation (DIC) (Chapter 17). In another case [4115], the blood of a patient with extreme leukocytosis was found to have large numbers of platelet-sized particles, originally counted as platelets, but which post-mortem immunological analysis revealed to be leukocyte cell fragments at a particle concentration ~24 times the normal white cell count. In yet another study [4063], blood smears from half of all patients with septic shock or DIC showed leukocytic fragments, always associated with fragmented erythrocytes, all of which cases (of leukocytolysis) later proved fatal. The appearance of leukocytic fragments in such cases is believed to be related to mechanical shearing through microangiopathic fibrin strands which may also cause erythrocyte fragmentation [4063], though in DIC death is the result of the concomitant thrombus formation and hemorrhaging.

The responses of white cells to mechanical stress are well known. For example, the sudden imposition of ~0.04 N/m2 fluid shear stress induces adherent leukocytes to retract their pseudopods, a process involving the breakdown of F-actin and which eventually causes the cell to round and detach from a glass surface [4116]. Raising the peak fluid stress to 0.07 N/m2 does not increase the rate of pseudopod retraction [4116]. Pseudopod retraction serves a useful biological purpose: to minimize leukocyte entrapment in capillaries. Interestingly, these effects of shear stress may be overridden by integrin-mediated membrane adhesion [4117]. That is, the ability of shear stress to inhibit pseudopod formation may be counteracted by stimulatory agents.

A threshold shear stress above 0.04 N/m2 is required to support rolling of leukocytes on selectin-coated surfaces [4118] (Section In one experiment [4119], shear forces on leukocytes rolling on adhesion-molecule-coated surfaces ranged from 0.2-1.5 N/m2 for VCAM-1 and up to 3-4 N/m2 for selectins. In another experiment [4120] involving leukocytes rolling on endothelium, the equilibrium force that would balance fluid shear stresses on the leukocyte and the attachment forces at its site of contact with the endothelium spanned 0.11-7.61 nN for wall shear stresses ranging from 0.2-2.5 N/m2 in venules 23-49 microns in diameter in cat mesentery. Another experiment [4121] found that adherent human PMNs (polymorphonuclear leukocytes such as neutrophils) are virtually all detached from human umbilical vein endothelial monolayers at a shear stress of ~1 N/m2. However, leukocytes remain rolling at up to 65 microns/sec and attached to endothelium via ~200 selectin binding sites/micron2 at shear stresses up to at least 3.2 N/m2 – a neutrophil 8.5 microns in diameter has ~240 microvilli/cell with ~260 L-selectin molecules per microvillus and crawls using up to 9 microvillus tether “feet” during rolling [4122]. The nature of the attachment surface is critical. For instance, half of all adhered human fibroblasts will detach from an FEP-Teflon surface at a fluid shear stress of 2 N/m2 but more than half of similar cells that are adhered to a glass surface will require 35 N/m2 to detach, after “rounding” (i.e., assuming a spheroidal shape) [4123].

As for cell lysis, critical shear stress levels have been defined [4124] for the viability, morphology, size, and lysis of adherent mammalian cells between 1-2.5 N/m2. For example, neutrophils adherent to cardiovascular device material subjected to shear stress above 0.6 N/m2 for 1 hour undergo complete apoptosis, displaying irreversible cytoplasmic and nuclear condensation while maintaining intact membranes [4125]. Leukocyte suspensions exposed to higher shear stress are subject to cell swelling as well as lysis [4059] and, in T lymphocytes, a depression of the proliferative response [4126]. In other studies, neutrophils exposed in vitro to shear stress of 7.5-15 N/m2 for 10 minutes will release enzymes both from azurophilic and specific granules [4127]. The number of ruptured leukocytes rises significantly at these levels of mechanical trauma. At 15 N/m2, the remaining intact cells display morphological changes including clublike cytoplasmic protrusions, spherical shape, and a circumferential distribution of cytoplasmic granules [4127]. Degranulation of cytoplasmic alkaline phosphatase granules begins to appear [4059]. The frequency of disrupted leukocytes increases with shear stress above 15 N/m2 [4059]. Human neutrophils undergo homotypic aggregation in the physiological range of fluid shear stress of 1.2-3 N/m2, along with an increase in intracellular Ca++ concentration [4128], but aggregates of disrupted cells disappear after exposure to 45 N/m2 for 10 minutes [4059]. At still higher shear stresses of 60 N/m2, cell destruction is marked [4127]. Intact PMNs contain fewer cytoplasmic granules, a large number of vacuoles, and condensed nuclear chomatin [4127]. A 10-minute shear stress of 60 N/m2 destroys 25% of human leukocytes [4059]. On the other hand, non-shear hydrostatic pressures of 10-50 N/m2 have no measurable influence on the shear stress response of leukocytes [4116]. During micropipette aspiration leukocytes neither retract nor project pseudopods in response to purely hydrostatic pressures of 100 N/m2 or above [4116]. Interestingly, the bursting strength of whole mammalian hybridoma cells has been measured [4129] by squashing them between two parallel plates. The required bursting force is ~2000 nN (~6400 N/m2) for 10-micron diameter cells and ~4500 nN (~3600 N/m2) for 20-micron cells. From this data, we conclude that mechanical interactions between leukocytes and nanorobots or nanorobotic organs imposing shear forces exceeding ~1 N/m2 for more than an hour may induce white cell apoptosis, or rupture at 10-50 N/m2 – an important limitation in medical nanorobot mission design.

Shear forces arising from collisions between bloodborne leukocytes and individual free-floating nanorobots are at most <0.1 N/m2 (Section, but may be far less for encounters involving co-vasculomobility or histonatation where the speed of interaction is substantially lower. While it is possible that individual medical nanorobots may unintentionally induce pseudopod retraction or other minor physiological changes in white cells, this possibility may be greatly reduced, given an appropriate device configuration and mission design.


Last updated on 30 April 2004