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

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

15.5.5.4 Viability of Confined, Pressurized, or Desiccated Cells

The effects of prolonged mechanical pressure between nanorobotic surfaces and biological tissues has already been addressed (Section 15.5.3.4.1). But in some applications [4609] it will be necessary to tightly confine individual cells in partially or completely enclosed containers for transport, storage, diagnosis or repair.

Cells confined on lithographically produced 2-dimensional islands of similar surface area to the cell typically may attach and generate normal secretion products, whereas larger islands will promote cell spreading instead and still larger islands may promote cell growth or replication (Section 15.2.2.3). Cells placed on square pedestals of roughly their own size will take up a square shape (Section 15.2.2.3). Cells including platelets, fibroblasts, osteoblasts and macrophages deposited or grown on diamond surfaces maintain their integrity, showing normal cell adhesion and no evidence of cytotoxicity (Section 15.3.1.2). Endothelial cells cultured on pyrolytic carbon or LTIC show no change in cell adherence with well-spread (not rounded) cells on this surface (Section 15.3.3.2). As for Teflon: monocytes and macrophage adhere weakly to Teflon surfaces, showing no obvious structural or functional defects; leukocytes and lymphocytes are not activated by Teflon in vitro, though platelets may be (Section 15.3.4.2); and endothelial, epithelial, neural and bacterial cells attach poorly to Teflon (Section 15.3.4.2). Fibroblasts and epithelioid cells adhere well to sapphire and experience no cytotoxic or antiproliferative effects, and osteoblasts show normal biochemical and biological functions on sapphire (Section 15.3.5.4).

Will full 3D confinement – cell containerization – cause cells to become apoptotic, or accelerate cell mitosis or proliferation? It is well known that a loss of contact with ECM by tissue cells, mediated by transmembrane proteins [4952], can induce apoptosis (Sections 10.4.1.1 and 15.5.4.2). Forcing cells to adopt an unnatural square shape by culturing them on square-shaped planar adhesive islands enhances the rate of apoptotic cell death, “as indicated by an accelerated permeabilization of the outer mitochondrial membrane, loss of the mitochondrial inner transmembrane potential, and an increased frequency of nuclear apoptosis” [4943]. This outcome may be avoided by inserting inhibitors of apoptosis [4914] or IAPs (Section 10.4.1.1) into the containerized cell during the period of containerization, then withdrawing the IAPs from the cytoplasm using molecular sorting rotors, after cell decontainerization but prior to final release, or by other means (Section 15.5.7.6).

A wide range of evidence [4944-4951] supports the conclusion that mechanical stress can induce cellular hypertrophy, mitosis and proliferation of cells. Thus it may also be advisable to introduce mitotic inhibitors into the cell (Section 10.4.1.3) to suppress mitosis during containerization, if containerization is found to produce unacceptable mechanical stress on the cell. These inhibitors can later be withdrawn and the cell should then resume its normal functioning. During containerization the cellular mRNA translation machinery could be temporarily shut down almost completely (and there are also selective inhibitors of transcription [4919-4921]). For instance, a number of ribosomal inhibitors are known – e.g., aminoglycosides inhibit translation in bacteria by binding to the A site in the ribosome [4915], and an anti-RNA antibody is an inhibitor of ribosome-associated GTP hydrolysis [4916] – and other fully reversible inhibitors of transcription are known, such as the eIF4E-binding proteins (4E-BPs) which interdict translation initiation by preventing recruitment of the translation machinery to mRNA [4917]. It should be possible to recognize and selectively extract (using molecular sorting rotors or adhesion antennae; Sections 9.3.2 and 10.4.2.5.2) specific mRNA strands or various intracellular messenger molecules that would otherwise transduce the mitotic signal. This should be possible because anti-mRNA can be targeted to inhibit a single species of mRNA molecule within cells [4918] and because nanorobots should be able to intercept and alter intracellular messages as described in Section 7.4.5.4. However, R. Bradbury notes that mRNA molecules may have very directed routes from the nucleus where they are generated to the ribosome locations where they are utilized and that it could take longer to “read” an mRNA molecule than to sample the surface of a simple protein messenger molecule, looking for a unique amino acid tag – and as a result, cell functioning might be slightly altered by the delays the sampling process introduces.

Although red cells in vivo are often confined to tightly-packed rouleaux (Section 9.4.1.2), the biological effects of van der Waals adhesion between cell plasma membranes and tightly confining diamondoid walls are presently unknown and should be investigated further. For example, the consequences of physically preventing surface fluctuations on the plasma membranes of red cells (Section 15.5.5.1.2) or white cells (Section 15.5.5.3.2) have not yet been studied. In this example, it should be possible to ameliorate some negative effects by interposing thin compliance-matching coatings at the cell-nanorobotic wall interface, possibly combined with cytoplasmic biochemical amendments.

Cells can tolerate static pressures of 300-1000 atm without exhibiting altered physiology (Section 10.3.3, Table 10.3), but compression of cells to force them through holes or tubes, or from one tight compartment to another, must not apply shear forces sufficient to induce biological response (Section 15.5.4.1). For instance, >0.04 N/m² shear stress (16 pN over a (20 micron)² cell membrane area) can induce adherent leukocytes to retract their pseudopods, eventually rounding and detaching from a glass surface [4116]. The minimum force required to initiate signal transduction may be as low as ~10 pN (Section 9.4.3.2.1). If these values must be exceeded during cell transport, then the cell’s responses must be actively disabled as described above or as described in Sections 7.4.5.4, 9.4.7.4, and 9.4.7.6, unless it is determined that the effects are reversible after the stress is removed.

While the partial dehydration of cells to reduce their volume in order to permit more convenient transport in vivo by specialized carrier nanorobots [4609] might possibly trigger a response from cellular stretch-activated channels (Section 15.5.3.1), Owen Hamill notes that this is unlikely because cell inflation rather than cell shrinkage is generally assumed to activate these channels. In the case of bone cells, these responses are stimulated at linear strains of just >0.15% (Section 9.4.3.2.1). For stretch-sensitive cell types, such responses must be actively disabled as described above. But many mammalian cells can provably survive the loss of 50% of their water [4922]. Fibroblasts (e.g., mouse L-929 cells) have survived from 45% [4923] to 65% [4924] decrease in total cell volume (the latter representing 85% water loss by volume [4925]) via dehydration, and erythrocytes have survived 73% volume reduction by dehydration [4926]. Other cells, particularly bacteria [4927-4930], can tolerate significant shrinkages without loss of viability.* However, even desiccation of microbes leads to dramatic lipid phase changes wherein carbohydrates, proteins and nucleic acids initially suffer spontaneous, reversible low activation energy Maillard reactions forming products that more slowly re-arrange, cross-link, etc. to give non-native states [4931]. So while initial products may spontaneously reverse to native states when water is restored, later products only do so through energy consumption and enzymatic activity, e.g., repair [4931].

* The record-holder may be the El Tor microbial strain of Vibrio cholerae, which according to Rita R. Colwell of the University of Maryland Biotechnology Institute [4932] can shrink itself 150- to 300-fold when plunged suddenly into cold salt water (e.g., cold shock [5425]), becoming the size of a large virus without loss of viability.

According to G. Fahy [4933], in the 1980s LifeCell Corp. devised a technique by which cells were “vitri-dried”: cells were cooled so rapidly that ice either doesn’t form or forms such small crystals that they don’t damage the cells, after which space-quality vacuums were used to distill off the water at very low temperatures [4934]. After vitri-dried cells were stored at room temperature for a short time, they could be rehydrated and, allegedly, recovered life functions, but were not able to divide [4934]. Experiments by others in the 1990s found that human mesenchymal stem cells that are air-dried and stored under vacuum are still viable when rehydrated ~12 hours later [4935]. Vacuum greatly enhances the ability of cultured human cells to withstand desiccation. Cells dried slowly in such a way that cellular structures are maintained and stored under vacuum in darkness can withstand desiccation even in the absence of added carbohydrates or polyols [4936]. Anhydrobiotic organisms such as the tardigrades [4937] survive practically complete drying in the absence of freezing, a feat made possible by the presence of a sugar, trehalose, whose geometry can support membrane structure against collapse by substituting for water at the polar head groups of the lipids [4930, 4938]. One research group reports that “introduction of the genes for trehalose biosynthesis allowed human cells in culture to be reversibly desiccated for up to 5 days” [4936]; and that human primary fibroblasts expressing trehalose and containing no detectable water could be maintained in the dry state for up to five days [4940]. However, another group [4939] reports that trehalose does not improve desiccation tolerance in mouse cells. More research is required to resolve this issue.

Last updated on 30 April 2004