Nanomedicine, Volume I: Basic Capabilities
© 1999 Robert A. Freitas Jr. All Rights Reserved.
Robert A. Freitas Jr., Nanomedicine, Volume I: Basic Capabilities, Landes Bioscience, Georgetown, TX, 1999
10.4.1.4 Chemical Poisoning
There are a great many (often inefficient) ways to poison a cell or virion simply by releasing chemical agents near, on, or within it, but if done incautiously this approach may lead to necrotic, rather than apoptotic, cell death. Here are a few examples of some well-known classes of biocidal agents, many of which may be inappropriate for use by in vivo medical nanorobots:
1. Phagosomal Biochemicals -- Both types of phagocytic cells (e.g., neutrophils and macrophages) contain specialized organelles that fuse with newly formed phagocytic vesicles (phagosomes), exposing phagocytosed microorganisms to a barrage of enzymatically produced, highly reactive molecules of superoxide (O2-) and hypochlorite (HOCl, the active ingredient in bleach), called the "oxidative burst" that punctures cell walls, and to a concentrated mixture of lysosomal hydrolases.531 Human neutrophils also use antimicrobial peptides such as the serprocidins (e.g., proteinase 3, azurocidin, and cathepsin G, a metabolic inhibitor) against fungi and bacteria.2132
2. Cytolytic Enzymes -- Lysozymes, present in tears, nasal mucus and sputum, destroy the cell walls of many airborne Gram-positive bacteria by catalyzing the hydrolysis of b-1,4 linkages between N-acetyl muramic acid and N-acetyl glucosamine (peptidoglycan degradation) in bacterial cell walls996-- causing the bacteria to burst open, spilling their contents. Lysozyme, zymolase, glucalase and lyticase are frequently used with bacteria and yeast cells to dissolve coats, capsules, or capsids. Granulysin released by cytolytic T lymphocytes directly kills intracellular Mycobacterium tuberculosis by altering the membrane integrity of the bacillus.2165
3. Organelle Poisons -- Certain drugs and other substances, if injected into the cytoplasm, may interfere with the workings of specific organelles or cell subsystems. For example, the fungal metabolite drug brefeldin A disrupts the Golgi, causing it to collapse back into the ER.2347,3441,3442 Colchicine,939 vinblastine and vincristine936 bind to tubulin, causing microtubules to disassemble, griseofulvin (an antifungal agent) prevents microtubule assembly,996 and taxol inhibits microtubule depolymerization, while cytochalasin B inhibits the polymerization of cytoskeletal actin microfilaments.939 Adociasulfate-2 is a kinesin motor inhibitor.2390 High concentrations of vitamin A weaken the lysosomal membrane. Proteasome inhibitors such as vinyl sulphone2911 are usually lethal for the eukaryotic cell. Oligomycin inhibits the F0F1-adenosine triphosphatase (ATPase) proton pump of the mitochondrial inner membrane, and cyanide poisons mitochondrial oxidative phosphorylation.2073 Nitric oxide (conc. ~60 nanomolar) inhibits mitochondrial respiration,2918,2919 specifically by inhibiting cytochrome oxidase by outcompeting oxygen at the oxygen binding site;2920 NO also prevents viral replication by inactivating a crucial protease.2969 Antipsychotic drugs such as chlorpromazine, thioridazine, and fluphenazine are potent peroxisome inhibitors.3067 The nucleus of an oocyte is ejected from a cell treated with etoposide and cycloheximide (chemical enucleation),3719,3720 microtubule poisons such as colchicine, colcemid and vinblastine cause extrusion of nuclei,3721 and EDDF is involved in erythroid cell denucleation;3722 there is at least one report of nuclear extrusion in lymphocytes.3723 Selected organelle populations (e.g., mitochondria) could also be ubiquinitized.
4. Antibiotics -- Drugs that prevent bacteria from multiplying (bacteriostatic) or that kill bacteria outright (bactericidal) are called antibiotics; in 1998, ~100 such drugs were FDA approved for U.S. use. Major groups include the aminoglycosides, cephalosporins, macrolides, penicillins, polypeptides, quinolones, sulfonamides, and tetracyclines;2119 many are naturally-derived products. All act by interfering with protein synthesis, cell wall construction, or DNA replication. For example, gonococci isolated in the preantibiotic (pre-resistance) era were inhibited by benzylpenicillin (C16H18N2O4 S, MW = 334 daltons) in concentrations as low as ~7 molecules/micron3;2135 in the 1990s, some highly resistant bacterial isolates required ~1000-fold higher concentrations. Vancomycin derivatives are active against Gram-positive bacteria at 10-100 molecules/micron3.3227 Antibiotics are available with protein/RNA synthesis inhibition activity against either prokaryotic or eukaryotic cells (Table 10.5).
5. Bactericidal Phages -- Bacteriophages (viruses that infect bacteria) are capable of penetrating bacterial membranes and delivering foreign DNA which can take control of all metabolic processes; in ~700 sec after penetration, the first complete virion particles begin appearing in the cytoplasm; at ~1500 sec, the bacterium bursts, necrotically liberating ~200 virus particles. These viruses are often very host-specific. Still, it may be possible to design artificial general-purpose bacteriophages that are capable of disabling or destroying all bacterial DNA, or are capable of replicating more bacteriophage particles to which only the targeted bacterial cells are susceptible (see also Section 10.4.1.1). Engineered macrophages or artificial neutrophils might also be deployed to achieve targeted bacterial digestion. Reaching intracellular parasites such as mycoplasmas and rickettsias might be one of the biggest challenges for such biorobots.
6. Bacteriocins -- Probably 99% of all bacteria generate at least one bacteriocin, small proteins that function as narrow-spectrum antibiotics that may have developed as poisons to kill competing bacteria.2121,3724-3726 In 1998, ~80 bacteriocins were known, most of them produced by fermentation microbes, including nisin which is used in 45 countries including the U.S. as a commercial food additive for pasteurized egg products. Bacteriocins seem to work by entering the outer membrane of a susceptible bacterium, congregating in groups, and forming pores that allow the unregulated outflow of essential ions. The target bacterium begins breaking down ATP in a vain attempt to produce enough new protons to recharge the membrane, a futile cycle that quickly results in exhaustion of bacterial ATP. Bacteriocins are most effective in acid and least effective in salty environments. Bacteriocins of Gram-positive bacteria such as Listeria and Clostridium botulinum are ineffective against Gram-negative bacteria such as E. coli and Salmonella which have a protective double-walled outer membrane. In 1998, several Gram-negative bacteriocins were known. One example is the colicins, water-soluble cytotoxins secreted by and active against E. coli, that form voltage-sensitive ion channels in the bacterial inner membrane that kill the cell by selectively siphoning out key cell nutrients2150,2151 and inhibit protein synthesis.3190
7. Porins and Superporins -- C. Sublette notes that it should be possible to design and insert into a cell a piece of RNA or DNA designed to produce high levels of a "superporin" protein that migrates to the cell membrane, then self-assembles into a large channel in the membrane several orders of magnitude larger than an ion channel, allowing a large, lethal ion influx or outflux. For example, normal cytosolic Ca++ concentration ranges from 60-3000 ions/micron3;531 blood plasma concentration is ~106 ions/micron3 (Appendix B), but even cytosolic concentrations as low as ~104 ions/micron3 may be toxic (Section 220.127.116.11). (See also Section 10.4.2.1.) Possible templates for such subunits are the bacterial porins,2133 minus their charged amino acid residues lining the inner passage which produce nonspecific aqueous diffusion channels across the outermost LPS bacterial membrane, and granulysin,2165 perforin and members of the amoebapore family2166,2167 which are thought to damage target cell membranes by inducing formation of microscopic pores.
8. Ionophores -- Ionophores are small hydrophobic molecules that dissolve in lipid bilayers, thus increasing ion permeability of cell membranes.531 Most are synthesized by microorganisms, presumably as biological weapons to weaken their competitors. There are two classes of ionophores -- (highly temperature-sensitive) mobile ion carriers, and channel formers -- both operating by shielding the charge of the transported ion so that it can penetrate the hydrophobic interior of the lipid bilayer.531 (Ionophores permit net movement only down their electrochemical gradients, since no energy sources are available.) Valinomycin, a ring-shaped polymer that increases K+ permeability of membranes, is an example of a mobile ion carrier. Another example is A23187, which acts as an ion-exchange shuttle, carrying two H+ out of the cell for every divalent cation (such as Ca++ and Mg++) carried in. Transport rates through a mobile carrier are ~2 x 104 ions/sec.531 A channel-forming ionophore is gramicidin (C148H210N 30O26, MW = 2822 daltons), a 15-residue linear polypeptide with all hydrophobic side chains. Two such molecules come together in the bilayer to form a transmembrane channel that selectively allows monovalent cations (most readily H+, K+ somewhat less readily, Na+ still less readily) to flow down their electrochemical gradients at a rate of ~2 x 107 cations/sec.531 The dimers are unstable, constantly forming and dissociating, with average channel open time ~1 sec.531 Gramicidin is produced by Bacillus brevis and is active against Gram-positive cocci and bacilli. A 5 microgram dose kills, in vitro, 109 pneumococci or group A streptococci in 2 hours at 30°C,751 giving a fatal dose of ~1000 molecules/micron3 assuming a ~1 cm3 culture volume in this experiment.
9. Channel Blockers -- Cells import and export nutrients, ions, water, and wastes through a variety of gated channels (Section 3.3.3) and transporter molecular pumps (Section 3.4.1). If these channels or pumps are permanently blocked, cytocide may ensue. Blocking the ~103/micron2 cellular transport systems embedded in a typical eukaryotic cellular membrane requires ~103/micron2 "steric plugs" that permanently jam in the throat of critical ion channels or pumps. For example, the acetylcholine receptor channel narrows to ~0.65 nm at its waist, so an efficiently designed steric plug could have a volume of ~1 nm3 or ~10-21 gm. (By comparison, a single molecule of 318-dalton tetrodotoxin, or puffer fish toxin, has mass ~0.53 x 10-21 gm; apparently, saxitoxin (shellfish toxin, human IV lethal dose ~68 micrograms) and palytoxin are slightly smaller, whereas botulin toxin is a fairly high-mass zinc metalloprotease protein that inhibits release of acetylcholine at the neuromuscular junction.) Plugging all channels in the surface of a (2 micron)3 bacterial cell (~10 micron2) or a (20 micron)3 tissue cell (~1000 micron2) requires ~104 or ~106 plugs, respectively, a nanorobot-dispensed ~10-5 micron3 or ~10-3 micron3 dosage of plugs at the target cell, assuming ~100% efficiency. A mean migration distance per plug of ~1 micron gives a Brownian diffusion time of ~1 millisec, so a micron-scale nanorobot with 0.1 micron3 of onboard storage carries sufficient plug-doses to incapacitate ~104 bacterial cells in ~10 sec or ~102 tissue cells in ~0.1 sec, ignoring cell-to-cell travel time. Assuming a mean interbacterial separation of ~100 microns (an implied pathogen number density of ~106/cm3 in the tissue) and a maximum nanodevice intercellular travel speed of ~100 micron/sec (Section 18.104.22.168), a nanorobot spends ~1 sec in transit between neighboring bacterial cells, ~0.002 sec in cell-type recognition (Section 22.214.171.124), and ~0.001 sec killing the bacterium once the nanorobot arrives.
10. Channel Destroyers -- Some smaller number (perhaps as few as one) of an enzyme-like toxin molecule capable of destroying either the steric or the electrochemical specificity of each ion channel by permanently altering the physical structure of that channel. For example, in the case of the acetylcholine receptor channel, an artificial enzyme possibly could be designed to alter the ring of negatively charged residues at the entrance to the receptor, allowing anions to enter the pore and ultimately depolarizing the cell. Enzymes typically operate at ~1000 Hz and the diffusion time across ~20 nm between adjacent ion channels is ~0.5 microsec, so a single enzyme neutralizes all 104 channels in a bacterial cell in ~10 sec and ~1000 artificial enzymatic molecules would incapacitate the cell in ~10 millisec.
11. Complement -- The complement system is a group of ~20 soluble serum-resident proteins acting in combination with specific cytolysin antibodies in amplification cascades along two separate pathways to initiate reactions to foreign antigens (Chapter 15). Complement proteins are enzymes that act on foreign cells by punching holes in their membranes by inserting lipid-soluble pores (the membrane attack complex, C5-C9), causing necrotic osmotic cytolysis. In addition, complement plus antibody designates cells to be engulfed by phagocytic cells.
12. Animal Venoms and Toxins -- The bufagin from the Bufo arenarum toad of Argentina (arenobufagin, C25H34O6, MW = 430 daltons) has a mean lethal toxicity of 92 microgram/kg,585 the equivalent of ~106 bufagin molecules per 20-micron tissue cell (~100 molecules/micron3). Most reptile venoms have lethal toxicities of 200-1000 micrograms per kg of body weight.585 Sea anemone granulitoxin (4958 daltons) has a mouse LD50 of 400 micrograms/kg (~49 molecules/micron3);3458 another sea anemone protein extract has a mouse LD50 of 40 micrograms/kg.3459 Maurotoxin, a 34-residue scorpion venom (Scorpio maurus), has an intracerebroventricular mouse LD50 of ~80 nanograms,3460 or ~0.4 molecules/micron3. The LD50 value to mice of Vespa luctuosa hornet venom is 1600 micrograms/kg, the most lethal known wasp venom.3457 Venoms and toxins may not be cytocidal against all cell types -- some act by interfering with nerve transmission or by paralyzing muscles.
13. Plant and Microbial Toxins -- A variety of microbes manufacture neurotoxins or neurolysins that destroy the ability of human ganglion and cortical neural cells to function.3461 A single molecule of some neurotoxins can incapacitate a cell, though the rate of action tends to be very slow. For example, botulin toxin is the second deadliest poison known (e.g., median human lethality ~5-50 nanograms/kg).3462 The toxin acts at cholinergic nerve terminals to prevent acetylcholine release, a permanent physiologic denervation that causes muscle paralysis, often resulting in death by respiratory system failure in 2-8 days. Recovery requires the sprouting of new axon twigs and the formation of new myoneural junctions,2122 which is why the recovery time for sub-lethal botulin poisoning is very long, several months to a year or more. Botulin is a ~150,000-dalton protein comprised of 1285 amino acid,2171,3462 which implies a lethal concentration of 0.2-2 molecules per 20-micron tissue cell. Other toxins are capable of killing a cell outright with just a few molecules, such as ricin: mouse and rabbit IV LD50 ~400-4000 nanograms/kg or 30-300 molecules per 20-micron tissue cell,3463-3465 possibly due to lower specificity in binding to cells and other pharmacokinetic effects. Ricin is a phytotoxic enzymatic 65,750-dalton protein3466 taken from the seeds of the castor oil plant that cleaves the ribosome complexes, shutting down protein manufacturing. This damage is normally irreversible, killing the organism in ~4 days at minimum dosage and ~8 hours at the highest dosages, so a reasonable lower limit for killing an individual cell is probably ~104 sec.
14. Antifungals -- Antifungal agents such as amphotericin B or fluconazole are fungistatic. For example, the growth of Candida and many other fungi is inhibited by amphotericin B (C47H73NO17, MW = 924 daltons) concentrations of 30-1000 micrograms/cm3 in vitro.2119 A number of natural antimicrobial peptides are induced in epithelial cells at sites of inflammation; for example, lingual antimicrobial peptide (LAP), a member of the b-defensin class, has been isolated from bovine tongue and exhibits both antifungal (e.g., Candida) and antibacterial (e.g., E. coli) activity2129 at concentrations as low as ~16 microgram/cm3. In 1998, antifungals were known to target membrane function, metabolism, cell wall synthesis, protein and ergosterol synthesis, nuclear division, and nucleic acid synthesis and function.2131 In those circumstances where fungal cells have learned to communicate to effect mating or cooperative behaviors, such communication may be disrupted to their detriment. If the genetic pathways involved in these behaviors include primitive forms of apoptosis, these could be exploited as well.
15. Antivirals -- Antiviral drugs may work by interfering with viral replication processes, including cell attachment, cell uptake, viral coat removal, and viral DNA or RNA replication by the cell, and are primarily virostatic. For example, acyclovir (MW = 225 daltons) in concentrations of 0.01-13.5 micrograms/cm3 inhibits by 50% the growth of herpes simplex virus in vitro.2119 In 1998, ~20 antivirals were FDA approved for U.S. use. Antibodies may also serve a virostatic function. For example, the rabies-like vesicular stomatitis virus (VSV) rhabdovirus has a surface envelope with ~1200 identical glycoprotein molecules that form a regular and densely ordered pattern of spike tips.2139 Rhabdoviruses are neutralized if they cannot dock with their cellular receptors; this requires a minimum of 200-500 IgG antibody molecules bound per virion.2140 Another challenge for medical nanorobots will be the retroviruses, which insert their genome into the host DNA, thus will require chromosomal editing or replacement (Chapter 20) to remove.
16. Iodine-Based Microbicides (iodophors) -- In tincture of iodine, all of the iodine is in the free form at ~10% by weight and is readily available for instantaneous reaction and killing of bacteria in a few seconds.360 The minimum lethal iodine dose for a 2-micron bacterium (assuming complete exhaustion of a 0.1-micron perimicrobial iodophor layer) may be of order ~0.001 picomoles or ~0.1 picogram iodine (~108 molecules), which is ~3% of the mass of a bacterium -- not terribly efficient, as expected for a broad-spectrum agent. Iodoacetate inactivates most cytoplasmic enzymes and blocks anaerobic metabolism, or glycolysis (fluoride is also a glycolytic poison).758 Yodoxin (64% organically bound iodine) is amoebicidal.2119 The broadest-known spectrum iodine-based microbicide is betadine (povidone-iodine) 10% solution,2119 employed in surgical scrub and many other general antiseptic uses. Betadine kills most bacteria in 15-30 sec, and also kills viruses, fungi, yeasts and protozoa; no bacterial resistance has been reported.
17. Silver -- Silver foil has been used in bacteriocidal wound dressings,2158 and silver metal has been employed for centuries to purify water. In 1998, there were more than 600 silver-based antibacterial products available in Japan including silver-impregnated pens, floppy disks, calculators, ATM machines, floor tiles, plastic food wrap, socks, shirts, public park sand, and toilet seats.2126 At least 20 companies in the U.S. offer silver/copper-based systems for swimming pool sanitation and commercial air conditioning cooling towers. The passive dissociation of silver from the metallic phase into a wound with antimicrobial results2123 and the antiseptic action of silver compounds2124 are well known. A quantitative study of the metal's antimicrobial properties in vitro found that a silver ion concentration of ~25 micrograms/cm3 (~105 ions/micron3) reduced the number density of Staphylococcus and Pseudomonas bacterial cells and Candida fungal cells by a factor of 1-10 million relative to microbial control aliquots.2125
18. Platinum, Bismuth, and Other Elements -- Platinum-based cisplatin (PtCl2H6 N2, MW = 300 daltons) is an antitumor and bacteriostatic drug that interacts with tumor-cell DNA by forming a Pt-GG intrastrand crosslink as the critical lesion leading to cytotoxicity.2136 A typical ~100 mg whole-body dose2119 produces a mean concentration of ~2000 molecules/micron3. Colloidal bismuth subcitrate exerts a direct antimicrobial effect against H. pylori (gastritis) at concentrations of 4-25 micrograms/cm3 (6000-38,000 molecules/micron3), with treated bacteria showing deposits of bismuth on their surface and internally.2137 Mercury (chronic toxicity in blood ~600 atoms/micron3), lead (toxic in blood at ~2900 atoms/micron3), and arsenic (toxic in blood at ~4800 atoms/micron3) are general poisons (Appendix B) that have been used for medicinal purposes. Indirect poisons such as iron and copper catalyze free-radical formation. Interestingly, bacterial growth is constrained by essential nutrients, and human immune cells scavenge and sequester iron when faced with a bacterial threat; nanorobotic "deferritization" of a bacterium could kill the organism.
19. Sugar -- Medical lore has it that sugar, and especially honey2158,2334 (Section 126.96.36.199), makes an excellent antibacterial disinfectant. Blood glucose levels of 0.01-0.07 M in untreated diabetics cause tissue and cellular damage, at least partly via osmotic cellular dehydration (honey and NaCl salt have a similar effect), and protein glycosylation and Maillard reaction (Section 188.8.131.52) which diminish enzyme function; ~15% cytosolic glucose concentrations are toxic.
20. Hydrogen-Bond and Disulfide-Bond Breaking Agents -- At high concentrations, guanidine hydrochloride disintegrates the bacterial Slayer coat which is held together only by noncovalent forces.525 Local heating (hyperthermia; Section 10.4.2.3) also disrupts H-bonds. Other agents are available to break disulfide bonds, disrupting many classes of proteins.
21. Nuclear Alkalination and Other Nucleic Disruption -- RNA breaks down in slightly alkaline environments (e.g., pH >~ 8.0;1591 to increase the pH of a typical cellular nucleus from 7.2 to 8.0 would require the injection of ~105 OH- ions. RNA is also broken down by RNases; DNA is broken down by DNases, or by using restriction enzymes.
22. Tissue Liquefaction -- A 2-hour exposure to 0.25% trypsin enzyme solution digests and breaks up the extracellular matrix,570 turning an organized tissue into a jumble of individual cells.
23. Other Poisons -- Cyanide causes cells to swell and lyse at concentrations of ~0.002 M (~1 x 106 ions/micron3).758 Assuming serum reference levels, ethanol should be cytotoxic at ~0.09 M (~50 x 106 molecules/micron3).1604 Ethylene oxide (C2H4O) gas is a microbial sterilant and fumigant; chlorine dioxide (chlorite ion) is an antimicrobial sometimes found in mouthwashes. Chlorine and bromine are used as antibacterials in swimming pools and hot tubs. Most chemotherapy agents also kill cells. Common lab detergents such as Triton X-100 and tributyl phosphate readily destroy bacteria and the majority of viruses sheathed in protein envelopes.3252
Nanorobot biocidal delivery vehicles can be used to present the chemical agent on a cell-by-cell basis. For example, a major whole-body infection involving ~1012 pathogens (e.g., ~106 bacteria/cm3) with each microbe capable of being killed by ~105 precisely-delivered biocidal molecules each of molecular weight ~1000 daltons requires a minimum treatment dose of ~1017 biocidal molecules or ~0.1 mm3 of material. This dose could be carried and dispensed in ~1000 sec by one billion ~1 micron3 "pharmacyte" nanorobots (total volume of therapeutic nanorobots ~0.001 cm3) assuming each nanorobot has ~0.1 micron3 of internal storage for the biocidal agent and assuming as before a mean ~1 sec cell-to-cell transit time.
Last updated on 16 April 2004