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 Cytopuncture and Membrane Resealing

The routine successful transplantation of cell nuclei by microbiologists using micropipettes demonstrates that cells can naturally recover from extreme membrane and cytoplasmic trauma. As noted in Section, it is not uncommon to observe rapid natural resealing of plasma membranes with little loss of intracellular contents [4239, 4240]. In one experiment, tissue cell plasma membranes were punctured using 2- to 3-micron diameter micropipettes and a 300-millisec transit (wounding) time, and the torn plasma membrane spontaneously resealed in 10-30 sec with relatively little visible loss of injected dye [4239]. We can estimate (Section that a cytopenetrating 1-micron nanorobot with a 10-millisec transit time may allow cytosolic leakage of only 0.006-0.03 micron3, or ~0.0001-0.0004% of typical tissue cell volume, per nanorobot transit.

Interestingly, Maroto and Hamill [5642] point out that most animal cell types [5643] naturally release ATP (or UTP) into the extracellular medium, whereupon these external molecules, at µM concentrations [5650], “act on ATP receptors that regulate diverse functions, including pain and touch sensation, smooth muscle contractility, synaptic transmission, platelet aggregation, epithelial fluid secretion, and endothelial release of vasorelaxants [5649-5653]; abnormalities in ATP release may contribute to specific human diseases, most notably cystic fibrosis [5647, 5654].” ATP release is often mechanosensitive and appears to arise through mechanical stimulation of brefeldin A sensitive membrane trafficking of ATP containing transport vesicles [3973, 5642-5649]: a Xenopus oocyte releases ATP at a basal rate of ~1.3 ATP molecules/µm2-sec, but even gentle mechanical stimulation can dramatically increase this to ~6700 ATP molecules/µm2-sec (assuming 1.2-mm diameter oocytes) [5642]. Care must be taken in medical nanorobot design and mission specification to avoid activities which may elicit elevated pathological ATP releases.

Microelectrodes traditionally used for intracellular injection had 0.5-micron diameter tips, beveled over a 1-2 micron length, and used very high fluid injection pressures of 0.3-1.5 atm [4235]. “Stab” microinjection at high pressure (0.1-0.2 atm) can be problematic in small cells (2-15 microns in diameter) because the nucleus-to-cytoplasm ratio is higher for these cells, hence the nucleus is more likely to be damaged during the stab. In one experiment [4236], less than 5% of neutrophils survived the high-pressure stab intact, but a low-pressure (~0.01 atm) injection through a lipid bridge produced a ~100% survival rate. Optical fiber tips ~0.1 micron in diameter or “optodes” have been poked through a cellular plasma membrane to measure cytoplasmic pH and the concentrations of other intracellular analytes, making a penetration volume of just a few micron3 in single cells and in single rat embryos, without ill effect on these cells [4237, 4238]. Membrane resealing after electroporation has also been studied [5981].

Exocytosis-based resealing [4240-4242] of a microneedle puncture through the fibroblast plasma membrane occurs in 5-10 sec [4242], but a second puncture at the same site heals faster than the initial wound [4241]. At first wounding, the cell uses existing endocytotic compartment to add membrane necessary for resealing. But Ca++ entry at the first wound stimulates vesicle formation from the Golgi apparatus, resulting in more rapid resealing of the second membrane disruption [4241]. Plasma membrane disruptions are resealed by changes in the cellular cytoskeleton (partial disassembly) [4243] and by an active molecular mechanism thought to be composed of, in part, kinesin, CaM kinase, snap-25, and synaptobrevin [4244]. Transmission electron microscopy [4244] reveals that vesicles of a variety of sizes rapidly (in seconds) accumulate in large numbers within the cytoplasm surrounding the disruption site, and that microvilli-like surface projections overlie this region. Tufts of microvilli rapidly appear on wounded cells. A local exocytosis is induced, rather than global exocytosis, in response to wounding. One or more internal membrane compartments accumulate at the disruption site and fuse there with the plasma membrane, resulting in the local addition of membrane to the surface of the mechanically wounded cell [4244]. As an existence proof for membrane-patching medical nanorobots, specialized membrane-patching organelles are known in some species. For example, “reserve granule” or “yolk granule” fusion-competent cytoplasmic organelles in sea urchin eggs allow Ca++-regulated fusion with a rapid (t1/2 < 1 sec) response capable of erecting large (>1000 micron2) continuous membrane boundaries [4242]. The cells of many species of fungi cells have a specialized peroxisomal plasma resealing organelle called the Woronin body [4245]. In some circumstances, cells can re-seal themselves even after major dissections, and survive. For example, a rapidly vibrating (100 Hz) micropipette with a <1-micron tip diameter has been used to completely sever individual dendrites from single neurons without damaging cell viability [4246].


Last updated on 30 April 2004