The Future of Nanofabrication and Molecular Scale Devices in Nanomedicine
Research Scientist, Zyvex Corp.
published July 2002
The original version of this paper was submitted for publication in September 2000 but was not published until July 2002, as Chapter 4 in the book: Renata G. Bushko, ed., Future of Health Technology, IOS Press, Amsterdam, The Netherlands, 2002, pp. 45-59. The abstract is indexed in Medline as: Robert A. Freitas Jr., “The future of nanofabrication and molecular scale devices in nanomedicine,” Studies in Health Technology and Informatics 80(2002):45-59, and this is the correct literature citation for the paper. The document reprinted here differs slightly from the published original.
Nanotechnology is engineering and manufacturing at the molecular scale, and the application of nanotechnology to medicine is called nanomedicine. Nanomedicine subsumes three mutually overlapping and progressively more powerful molecular technologies. First, nanoscale-structured materials and devices that can be fabricated today hold great promise for advanced diagnostics and biosensors, targeted drug delivery and smart drugs, and immunoisolation therapies. Second, biotechnology offers the benefits of molecular medicine via genomics, proteomics, and artificial engineered microbes. Third, in the longer term, molecular machine systems and medical nanorobots will allow instant pathogen diagnosis and extermination, chromosome replacement and individual cell surgery in vivo, and the efficient augmentation and improvement of natural physiological function. Current research is exploring the fabrication of designed nanostructures, nanoactuators and nanomotors, microscopic energy sources, and nanocomputers at the molecular scale, along with the means to assemble them into larger systems, economically and in great numbers.
“There is a growing sense in the scientific and technical community that we are about to enter a golden new era,” announced Richard E. Smalley, winner of the 1996 Nobel Prize in Chemistry, in recent Congressional testimony . On June 22, 1999, Smalley spoke in support of a new National Nanotechnology Initiative before the Subcommittee on Basic Research of the U.S. House Science Committee in Washington, DC. “We are about to be able to build things that work on the smallest possible length scales, atom by atom,” Smalley said. “Over the past century we have learned about the workings of biological nanomachines to an incredible level of detail, and the benefits of this knowledge are beginning to be felt in medicine. In coming decades we will learn to modify and adapt this machinery to extend the quality and length of life.” Smalley founded the Center for Nanoscale Science and Technology at Rice University in Texas in 1996. But he became personally interested in the medical applications of nanotechnology in 1999, after he was diagnosed with a type of non-Hodgkin’s lymphoma (the same sort that killed King Hussein of Jordan). Smalley then endured an apparently successful course of chemotherapy that caused all the hair on his head to fall out.
“Twenty years ago,” Smalley continued, “without even this crude chemotherapy I would already be dead. But twenty years from now, I am confident we will no longer have to use this blunt tool. By then, nanotechnology will have given us specially engineered drugs which are nanoscale cancer-seeking missiles, a molecular technology that specifically targets just the mutant cancer cells in the human body, and leaves everything else blissfully alone. To do this, these drug molecules will have to be big enough – thousands of atoms – so that we can code the information into them of where they should go and what they should kill. They will be examples of an exquisite, human-made nanotechnology of the future. I may not live to see it. But, with your help, I am confident it will happen. Cancer – at least the type that I have – will be a thing of the past.”
The term “nanotechnology” generally refers to engineering and manufacturing at the molecular or nanometer length scale. (A nanometer is one-billionth of a meter, about the width of 6 bonded carbon atoms.) The field is experiencing an explosion of interest. Nanotechnology is so promising that the U.S. President, in his January 2000 State-of-the-Union speech, announced that he would seek $475 million for nanotechnology R&D via the National Nanotechnology Initiative, effectively doubling federal nanotech funding for FY2001. The President never referred to “nanotechnology” by name, but he gushed about its capabilities, marveling at a technology that will someday produce “molecular computers the size of a tear drop with the power of today’s fastest supercomputers.”
After the President’s speech, Walter Finkelstein, president and CEO of NanoFab Inc. in Columbia, MD, agreed that it was conceivable that the technology could be used to develop computers chips so small that they could be injected into the bloodstream – “Fantastic Voyage-like,” he said – to locate medical problems. In February 2000, John Hopcroft, dean of the College of Engineering at Cornell University, announced plans for a new 150,000-square-foot nanotechnology research center. The facility already has $12 million per year of earmarked funding and is expected to support 90 local jobs and approximately 110 graduate students. “The implications of this research are enormous,” Hopcroft asserted, and include “the development of mechanical devices that can fight disease within the human body.
In May 2000, the National Cancer Institute signed an agreement with NASA, the U.S. space agency, to study the medical potential of nanoparticles. Nanoscience has also attracted the attention of the U.S. National Institutes of Health (NIH), which hosted one of the first nanotechnology and biomedicine conferences in June 2000. In July, the National Science Foundation (NSF) announced a Nanoscale Science and Engineering Initiative to provide an estimated $74 million in funding for nanotechnology research. Northwestern University in Evanston, Illinois will spend $30 million on a new nanofabrication facility of its own, joining existing operations such as the Stanford Nanofabrication Facility (started in 1985 with $15 million of backing from 20 industrial sponsors) and the Cornell Nanofabrication Facility, expected to attract 450 researchers in 2000, half of them visiting scientists. Cornell is spending $50 million on a new building for the Facility, and has just won a $20 million, five-year grant from the NSF to operate a new nanobiotechnology center which will make nanoscale tools available to biologists.
Burgeoning interest in the medical applications of nanotechnology has led to the emergence of a new field called nanomedicine [2, 3]. Most broadly, nanomedicine is the process of diagnosing, treating, and preventing disease and traumatic injury, of relieving pain, and of preserving and improving human health, using molecular tools and molecular knowledge of the human body.
It is most useful to regard the emerging field of nanomedicine as a set of three mutually overlapping and progressively more powerful technologies. First, in the relatively near term, nanomedicine can address many important medical problems by using nanoscale-structured materials that can be manufactured today. This includes the interaction of nanostructured materials with biological systems – in June 2000, the first 12 Ph.D. candidates in “nanobiotechnology” began laboratory work at Cornell University. Second, over the next 5-10 years, biotechnology will make possible even more remarkable advances in molecular medicine and biobotics (microbiological robots), some of which are already on the drawing boards. Third, in the longer term, perhaps 10-20 years from today, the earliest molecular machine systems and nanorobots may join the medical armamentarium, finally giving physicians the most potent tools imaginable to conquer human disease, ill-health, and suffering.
The initial medical applications of nanotechnology, using nanostructured materials, are already being tested in a wide variety of potential diagnostic and therapeutic areas.
For example, fluorescent tags are commonplace in medicine and biology, found in everything from HIV tests to experiments that image the inner functions of cells. But different dye molecules must be used for each color, color-matched lasers are needed to get each dye to fluoresce, and dye colors tend to bleed together and fade quickly after one use. “Quantum dot” nanocrystals have none of these shortcomings. These dots are tiny particles measuring only a few nanometers across, about the same size as a protein molecule or a short sequence of DNA. They come in a nearly unlimited palette of sharply-defined colors, can be excited to fluorescence with white light, and can be linked to biomolecules to form long-lived sensitive probes to identify specific compounds. They can track biological events by simultaneously tagging each biological component (e.g., different proteins or DNA sequences) with nanodots of a specific color.
Quantum Dot , the manufacturer, believes this kind of flexibility could offer a cheap and easy way to screen a blood sample for the presence of a number of different viruses at the same time. It could also give physicians a fast diagnostic tool to detect, say, the presence of a particular set of proteins that strongly indicates a person is having a heart attack. On the research front, the ability to simultaneously tag multiple biomolecules both on and inside cells could allow scientists to watch the complex cellular changes and events associated with disease, providing valuable clues for the development of future pharmaceuticals and therapeutics. In mid-2000, Genentech began evaluating the dots for commercial utility in a variety of cellular and molecular assays. A related technology called PEBBLES (Probes Encapsulated by Biologically Localized Embedding) , pioneered by Raoul Kopelman at the University of Michigan, allows dye-tagged nanoparticles to be inserted into living cells to monitor metabolism or disease conditions.
Another early goal of nanomedicine is to study how biological molecular receptors work, and then to build artificial binding sites on a made-to-order basis to achieve specific medical results. Buddy D. Ratner at the University of Washington in Seattle has researched the engineering of polymer surfaces containing arrays of artificial receptors. In a recent series of experiments , Ratner and his colleagues used a new radiofrequency-plasma glow-discharge process to imprint a polysaccharide-like film with nanometer-sized pits in the shape of such biologically useful protein molecules as albumin (the most common blood protein), fibrinogen (a clotting protein), lysozyme and ribonuclease (two important enzymes), and immunoglobulin (antibodies). Each protein type sticks only to a pit with the shape of that protein. Ratner’s engineered surfaces may be used for quick biochemical separations and assays, and in biosensors and chemosensors, because such surfaces will selectively adsorb from solution only the specific protein whose complementary shape has been imprinted, and only at the specific place on the surface where the shape is imprinted. The RESIST Group at the Welsh School of Pharmacy at Cardiff University  and others have looked at how molecularly imprinted polymers could be medically useful in clinical applications such as controlled drug release, drug monitoring devices, and biological and antibody receptor mimics.
Dendrimers represent yet another nanostructured material that may soon find its way into medical therapeutics. Starburst dendrimers are tree-shaped synthetic molecules with a regular branching structure emanating outward from a core. Dendrimers form nanometer by nanometer, so the number of synthetic steps or “generations” dictates the exact size of the particles in a batch. Each molecule is typically a few nanometers wide but some have been constructed up to 30 nanometers wide, incorporating more than 100,000 atoms. The peripheral layer of the dendrimer particle can be made to form a dense field of molecular groups that serve as hooks for attaching other useful molecules, such as DNA, which hunker down amongst the outermost branches.
In 1998, James R. Baker Jr. co-founded the Center for Biologic Nanotechnology at the University of Michigan to bring together doctors, medical researchers, chemists and engineers to pursue the use of dendrimers as a safer and more effective genetic therapy agent . For Baker, these nanostructures are attractive because they can sneak DNA into cells while avoiding triggering an immune response, unlike viral vectors commonly employed today for transfection. The dendrimer molecule is decorated with specific snippets of DNA, then injected into biological tissue. Upon encountering a living cell, dendrimers of a certain size trigger a process called endocytosis in which the cell’s outermost membrane deforms into a tiny bubble, or vesicle. The vesicle encloses the dendrimer which is then admitted into the cell’s interior. Once inside, the DNA is released and migrates to the nucleus where it becomes part of the cell’s genome. The technique has been tested on a variety of mammalian cell types , and Baker hopes to begin clinical human trials of dendrimer gene therapy in 2001. Donald Tomalia, another co-founder of the Center for Biologic Nanotechnology, recently reported using glycodendrimer “nanodecoys” to trap and deactivate influenza virus particles . The glycodendrimers present a surface that mimics the sialic acid groups normally found in the mammalian cell membrane, causing virus particles to adhere to the outer branches of the decoys instead of the natural cells.
Medical nanomaterials also may include “smart drugs” that become medically active only in specific circumstances. A good example is provided by Yoshihisa Suzuki at Kyoto University, who has designed a novel drug molecule that releases antibiotic only in the presence of an infection . Suzuki started with the common antibiotic molecule gentamicin and bound it to a hydrogel using a newly developed peptide linker. The linker can be cleaved by a proteinase enzyme manufactured by Pseudomonas aeruginosa, a Gram-negative bacillus that causes inflammation and urinary tract infection, folliculitis, and otitis externa in humans. Tests on rats show that when the hydrogel is applied to a wound site, the antibiotic is not released if no P. aeruginosa bacteria are present. But if any bacteria of this type are present, then the proteolytic enzyme that the microbes naturally produce cleaves the linker and the gentamicin is released, killing the bacteria. “If the proteinase specific to each bacterium [species] can be used for the signal,” writes Suzuki, “different spectra of antibiotics could be released from the same dressing material, depending on the strain of bacterium.” This specificity of action is highly desirable because the indiscriminate prophylactic use of antibiotics is associated with the emergence of strains of drug-resistant bacteria, and most antibiotics apparently have at least some toxicity for human fibroblasts.
Immunotoxins are another class of smart drugs, in this case activating only in the presence of cancer cells. An immunotoxin molecule is an engineered hybrid of functional protein modules fabricated from two different types of proteins: a toxin and an antibody. Toxin proteins are normally produced and released by infectious bacteria. The protein binds to the surface of a host cell, penetrates it, and kills it. Toxin molecules are so potent that just a few of them can kill a cell. Antibodies are proteins produced by the immune system to recognize and bind to specific foreign materials. An immunotoxin molecule is made by fusing a part of the gene encoding a toxin with a part of the gene encoding an antibody that recognizes surface features on cancer cells. This creates a novel gene that can be used to express a new synthetic protein molecule. This new molecule will bind only to a cancer cell (via a module from the antibody protein), then penetrate it and kill it (via modules from the toxin protein). The first experiments with mice showed that these engineered proteins successfully eliminated certain tumors. Then early in 2000, National Cancer Institute researchers confirmed that an immunotoxin made from a truncated form of Pseudomonas exotoxin was cytotoxic to malignant B-cells taken from patients with hairy cell leukemia . A second clinic trial at the Universitaet zu Koeln in Germany also found that a ricin-based immunotoxin had moderate efficacy against Hodgkin’s lymphoma in some patients .
Mauro Ferrari, director of the Biomedical Engineering Center at Ohio State University and chairman of the BioMEMS Consortium on Medical Therapeutics, with Tejal Desai has created what could be considered one of the earliest therapeutically useful nanomedical devices . Ferrari and his collaborators at the Biomedical Microdevices Center at the University of California at Berkeley employed bulk micromachining to fabricate tiny cell-containing chambers within single crystalline silicon wafers. The chambers interface with the surrounding biological environment through polycrystalline silicon filter membranes which are micromachined to present a high density of uniform nanopores as small as 20 nanometers in diameter. These pores are large enough to allow small molecules such as oxygen, glucose, and insulin to pass, but are small enough to impede the passage of much larger immune system molecules such as immunoglobulins and graft-borne virus particles. Safely ensconced behind this artificial barrier, immunoisolated encapsulated rat pancreatic cells may receive nutrients and remain healthy for weeks, happily secreting insulin back out through the pores, while the immune system remains blissfully unaware of the foreign cells which it would normally attack and reject.
Ferrari and Desai believe that microcapsules containing replacement islets of Langerhans cells – most likely easily-harvested piglet islet cells – could be implanted beneath the skin of some diabetes patients. This could temporarily restore the body’s delicate glucose control feedback loop without the need for powerful immunosuppressants that can leave the patient at serious risk for infection. Supplying encapsulated new cells to the body could also be a valuable way to treat other enzyme or hormone deficiency diseases, including encapsulated neurons which could be implanted in the brain and then be electrically stimulated to release neurotransmitters, possibly as part of a future treatment for Alzheimer’s or Parkinson’s diseases.
The flow of materials through nanopores can also be externally regulated. The first artificial voltage-gated molecular nanosieve was fabricated by Charles R. Martin and colleagues  at Colorado State University in 1995. Martin’s membrane contains an array of cylindrical gold nanotubules with inside diameters as small as 1.6 nanometers. When the tubules are positively charged, positive ions are excluded and only negative ions are transported through the membrane. When the membrane receives a negative voltage, only positive ions can pass. Future similar nanodevices may combine voltage gating with pore size, shape, and charge constraints to achieve precise control of ion transport with significant molecular specificity. In 1997, an exquisitely sensitive ion channel switch biosensor was built by an Australian research group . The scientists estimated that their sensor could detect a minute change in chemical concentration equivalent to a single sugar cube tossed into Sidney harbor, or roughly one part in a billion billion.
Daniel Branton at Harvard University has conducted an ongoing series of experiments using an electric field to drive a variety of RNA and DNA polymers through the central nanopore of an alpha-hemolysin protein channel mounted in a lipid bilayer similar to the outer membrane of a living cell . As early as 1996, the researchers had determined that the individual nucleotides comprising the polynucleotide strands must be passing single-file through the 2.6 nanometer-wide nanopore, and that changes in ionic current could be used to measure polymer length. By 1998, Branton had shown that the nanopore could be used to rapidly discriminate between pyrimidine and purine segments (the two types of nucleotide bases) along a single RNA molecule. In 2000, the scientists demonstrated the ability to distinguish between DNA chains of similar length and composition that differ only in base pair sequence. A similar research effort at the University of California at Santa Cruz has produced nanopore devices with read rates potentially up to 1000 bases per second . Because nanopores can rapidly discriminate and characterize DNA polymers at low copy number, future refinements of this experimental approach may eventually provide a low-cost high-throughput method for very rapid genome sequencing.
Biotechnology originally contemplated the application of biological systems and organisms to technical and industrial processes, but in recent times the field has expanded to include genetic engineering and the emerging fields of genomics, proteomics, transcriptomics, gene chips, artificial chromosomes, and even biobotics. Biotechnology now takes as its ultimate goal no less than the engineering of all biological systems, even completely designed organic living systems, using biological instrumentalities or “wet” nanotechnology. There are many good summaries of biotechnology elsewhere, so here we focus on efforts to engineer natural nanomachines to create new cellular devices.
During the 1990s, bioengineered viruses of various types and certain other vectors routinely were being used in experimental genetic therapies as “devices” to target and penetrate certain cell populations, with the objective of inserting therapeutic DNA sequences into the nuclei of human target cells in vivo. Retrovirally-altered lymphocytes (T cells) began to be injected into humans for therapeutic purposes. Another example was the use, by Neurotech (Paris), of genetically modified cerebral endothelial cell vectors to attack glioblastoma. This was the first therapeutic use of genetically engineered endothelial cells in humans; Phase I/II clinical studies were underway in 2000.
Engineered bacteria were also being pursued by Vion Pharmaceuticals in collaboration with Yale University. In their “Tumor Amplified Protein Expression Therapy” program , antibiotic-sensitive Salmonella typhimurium (food poisoning) bacteria were attenuated by removing the genes that produce purines vital to bacterial growth. The tamed strain could not survive very long in healthy tissue, but quickly multiplied 1000-fold inside tumors which are rich in purines. The engineered bacteria were available in multiple serotypes to avoid potential immune response in the host, and Phase I human clinical trials were underway in 2000 using clinical dosages. The next step would be to add genes to the bacterium to produce anticancer proteins that can shrink tumors, or to modify the bacteria to deliver various enzymes, genes, or prodrugs for tumor cell growth regulation.
In 1998, Glen Evans, then at the University of Texas Southwestern Medical Center, described the possible construction of synthetic genomes and artificial organisms. His proposed strategy involved determining or designing the DNA sequence for the genome, synthesizing and assembling the genome, then introducing the synthetic DNA into an enucleated pluripotent host cell to create an artificial organism. Genome engineers could modify an existing microbe by adding a biochemical pathway borrowed from other organisms, though this remains a difficult task because tailoring an existing system to match unique requirements demands detailed knowledge about the pathway. But ultimately, says Adam P. Arkin at Lawrence Berkeley National Laboratory, “we want to learn to program cells the same way we program computers.” Some genome engineers have started by building the biological equivalent of the most basic switch in a computer – a digital flip-flop. “Cells switch genes on and off all the time,” observes MIT’s Thomas F. Knight, Jr., who has pioneered some of this research. A cellular toggle switch, made of DNA and some well-characterized regulatory proteins, might be devised to turn on a specific gene when exposed to a particular chemical. These could be used in gene therapies – implanted genes might be controlled with single doses of specially selected drugs, one to switch the gene on, another to switch it off.
Arcady Mushegian of Akkadix Corp.  has looked at the genes present in the genomes of fully sequenced microbes to see which ones are always conserved in nature. He concludes that as few as 300 genes are all that may be required for life, constituting the minimum possible genome for a functional microbe. An organism containing this minimal gene set would be able to perform the dozen or so functions required for life – manufacturing cellular biomolecules, generating energy, repairing damage, transporting salts and other molecules, responding to environmental chemical cues, and replicating. The minimal microbe – a basic cellular chassis – could be specified by a genome only 150,000 nucleotides bases in length. Glen Evans, now at Egea BioSciences, can already produce made-to-order DNA strands that are 10,000 nucleotide bases in length  and is striving to increase this length by at least a factor of ten. The engineered full-genome DNA, once synthesized, would then be placed inside an empty cell membrane – most likely a living cell from which the nuclear material had been removed. These artificial biobots could be designed to produce useful vitamins, hormones, enzymes or cytokines in which the patient’s body was deficient, or to selectively absorb and metabolize into harmless endproducts harmful substances such as poisons, toxins, or indigestible intracellular detritus, or even to perform useful mechanical tasks.
Besides their direct medical applications, biobots might be employed in molecular construction. Gerald J. Sussman at MIT notes that when computer parts are reduced to the size of single molecules, engineered microbes could be directed to lay down complex electronic circuits. “Bacteria are like little workhorses for nanotechnology; they’re wonderful at manipulating things in the chemical and ultramicroscopic worlds,” he says. “You could train them to become electricians and plumbers, hire them with sugar and harness them to build structures for you.”
The third major branch of nanomedicine – molecular nanotechnology (MNT) or nanorobotics [2, 22] – takes as its purview the engineering of all complex mechanical medical systems constructed from the molecular level. Just as biotechnology extends the range and efficacy of treatment options available from nanomaterials, the advent of molecular nanotechnology will again expand enormously the effectiveness, comfort and speed of future medical treatments while at the same time significantly reducing their risk, cost, and invasiveness. MNT will allow doctors to perform direct in vivo surgery on individual human cells.
The first and most famous scientist to voice these possibilities was the late Nobel physicist Richard P. Feynman, who worked on the Manhattan Project at Los Alamos during World War II and later taught at CalTech for most of his professorial career. In his remarkably prescient 1959 talk “There’s Plenty of Room at the Bottom,” Feynman proposed employing machine tools to make smaller machine tools, these to be used in turn to make still smaller machine tools, and so on all the way down to the atomic level . Feynman prophetically concluded that this is “a development which I think cannot be avoided.” Such nanomachine tools, nanorobots and nanodevices could ultimately be used to develop a wide range of atomically precise microscopic instrumentation and manufacturing tools – that is, nanotechnology.
Feynman was clearly aware of the potential medical applications of the new technology he was proposing. After discussing his ideas with a colleague, Feynman offered  the first known proposal for a nanomedical procedure to cure heart disease: “A friend of mine (Albert R. Hibbs) suggests a very interesting possibility for relatively small machines. He says that, although it is a very wild idea, it would be interesting in surgery if you could swallow the surgeon. You put the mechanical surgeon inside the blood vessel and it goes into the heart and looks around. (Of course the information has to be fed out.) It finds out which valve is the faulty one and takes a little knife and slices it out. Other small machines might be permanently incorporated in the body to assist some inadequately functioning organ.” Later in his historic lecture in 1959, Feynman urged us to consider the possibility, in connection with biological cells, “that we can manufacture an object that maneuvers at that level!”
Extending nanomedicine to molecular machine systems will probably require, among many other things, the ability to build precise structures, actuators and motors that operate at the molecular level, thus enabling manipulation and locomotion. For example, in 1992 K. Eric Drexler of the Institute for Molecular Manufacturing theorized that an efficient nanomechanical bearing could be made by bending two graphite sheets into cylinders of different diameters, then inserting the smaller one into the larger one . By 2000, John Cumings and Alex Zettl at U.C. Berkeley had demonstrated experimentally that nested carbon nanotubes do indeed make exceptionally low-friction nanobearings .
But early mechanical nanorobots might be made, at least in part, of DNA. The idea of using DNA to build nanoscale objects has been pioneered by Nadrian Seeman at New York University . Two decades ago, Seeman recognized that a strand of DNA has many advantages as a construction material. First, it is a relatively stiff polymer. Its intermolecular interaction with other strands can be readily predicted and programmed due to the base-pair complementarity of nucleotides, the fundamental building blocks of genetic material. DNA also tends to self-assemble. Arbitrary sequences are readily manufactured using conventional biotechnological techniques, and DNA is readily manipulated and modified by a large number of enzymes. During the 1980s, Seeman worked to develop strands of DNA that would zip themselves up into more and more complex shapes – first tiny squares, then three-dimensional stick-figure cubes comprised of 480 nucleotides each, then a truncated octahedron containing 2550 nucleotides. By the mid-1990s, Seeman could fabricate nanoscale DNA stick figures of almost any regular geometric shape, by the billions per batch.
In 1999, Seeman reported yet another breakthrough – the construction of a mechanical DNA-based device that might serve as the basis for a nanoscale robotic actuator . The mechanism has two rigid double-stranded DNA arms a few nanometers long that can be made to rotate between fixed positions by introducing a positively charged cobalt compound into the solution surrounding the molecules, causing the bridge region to be converted from the normal B-DNA structure to the unusual Z-DNA structure. The free ends of the arms shift position by 2-6 nanometers during this fully reversible structural conversion, like a hinge opening and closing. “It’s a very simple nanomachine,” admits Seeman, “but in the scheme of molecular devices it’s huge because it generates more than four times the amount of movement produced by typical molecular devices.” A large version of the device might function as an elbow, while smaller devices could serve as finger joints.
Bernard Yurke at Bell Laboratories and Andrew Turberfield at the University of Oxford synthesized another DNA actuator using three single strands of artificial DNA which, when placed together, find their complementary partners and self-assemble to form a V-shaped structure . The open mouth of this nanotweezer can be made to close by adding a special “fuel” strand which binds to the single-stranded DNA dangling from the ends of the arms of the tweezers and zips them closed. A special “removal” strand, when added, binds to the fuel strand and pulls it away, opening the nanotweezers again. The cycle may then be repeated.
In 1999, Philip Kim and Charles Lieber at Harvard University created the first general-purpose nanotweezer . Its working end is a pair of electrically controlled carbon nanotubes made from a bundle of multiwalled carbon nanotubes. To operate the tweezers, a voltage is applied across the electrodes, causing one nanotube arm to develop a positive electrostatic charge and the other to develop a negative charge. The attractive force can be increased or decreased by varying the applied voltage – 8.5 volts completely closes the arms, while lower voltages give different degrees of grip. Using the tool, Kim and Lieber have successfully grasped 500-nanometer clusters of polystyrene spheres, about the same size scale as cellular substructures. They were also able to remove a semiconductor wire 20 nanometers wide from a mass of entangled wires. At present, each of the tweezer’s arms is about 50 nanometers wide and 4 microns long. But by growing single-walled nanotubes directly onto the electrodes, the researchers hope to produce nanotweezers small enough to grab individual macromolecules.
Other researchers are developing nanomotors for future nanorobots. Most notably, Carlo Montemagno at Cornell University has modified a natural biomotor to incorporate nonbiological parts, creating the first artificial hybrid nanomotor . Montemagno started with natural ATPase, a ubiquitous enzyme found in virtually every living organism and which helps to convert food into usable energy in living cells. The moving part of an ATPase molecule is a central protein shaft (or rotor, in electric-motor terms) that rotates in response to electrochemical reactions with each of the molecule’s three proton channels (comparable to the electromagnets in the stator coil of an electric motor). ATP (adenosine triphosphate) is the fuel that powers the molecular motor’s motion.
Using the tools of genetic engineering, Montemagno added metal-binding amino acid residues to the ATPase. This allowed each motor molecule to bind tightly to nanoscale nickel pedestals prepared by electron beam lithography. Properly oriented motor molecules 12 nanometers in diameter were then attached to the pedestals with a precision approaching 15 nanometers, and a silicon nitride bar a hundred nanometers long was bound to the rotor subunit of each motor molecule, all by self-assembly. In a microscopic video presentation, dozens of bars could be seen spinning like a field of tiny propellers. The group’s first integrated molecular motor ran for 40 minutes at 3-4 revolutions per second. Subsequent motors have been operated for hours continuously by feeding them plenty of ATP. Montemagno has been measuring things like horsepower and motor efficiency, simple tests that would be familiar to any mechanical engineer inspecting a car engine. Montemagno is also trying to build a solar-powered, biomolecular motor-driven autonomous nanodevice, wherein light energy is converted into ATP which then serves as a fuel source for the motor. “We think we’ll be able to make autonomous devices that are powered by light on a scale of 1 micron or less, smaller than bacteria,” he says.
Montemagno is developing a chemical means of switching his hybrid motors on and off reliably. By engineering a secondary binding site tailored to a cell’s signaling cascade, he plans to use the sensory system of the living cell to control nanodevices implanted within the cell. Montemagno envisions tiny chemical factories operating inside living cells. He speculates that these nanofactories could be targeted to specific cells, such as those of tumors, where they would synthesize and deliver chemotherapy agents. Within three years he expects to have a motor assembled within a living cell, with the cell’s physiology providing the energy to run it. “My 10-year goal is to make a device that harvests single molecules within a living cell, maybe a cellular pharmacy that produces a drug, stores it within the cell, and then based upon some signal, releases it,” Montemagno said in 2000. “For a technology that wasn’t expected to produce a useful device before the year 2050, I think we’ve made a pretty good start. But we have a long way to go before it’s safe to turn these little machines loose in the human body.”
Nanomotor research is progressing in other laboratories as well. For instance, a 78-atom chemically-powered rotating motor was synthesized in 1999 as a proof of principle by chemist T. Ross Kelly at Boston College . Ben Feringa at the University of Groningen in the Netherlands has built an artificial 58-atom motor molecule that spins when illuminated by solar energy . Another potential nanorobot power source is a modified microbial fuel cell – laboratory demonstrations of such cells contain captive bacteria or immobilized enzymes  which, when fed organic material, convert chemical energy into electricity that could be used to power tiny motors.
Truly effective medical nanorobots may require onboard computers to allow a physician to properly monitor and control their work. Molecular electronics or “moletronics” is a hot research topic in nanotechnology right now. For example, in 2000, a collaborative effort between UCLA and Hewlett Packard produced the first laboratory demonstration of completely reversible room-temperature molecular switches that could be employed in nanoscale memories, using mechanically interlinked ring molecules called catenanes . Two independent companies – Molecular Electronics Corp. in Texas and California Molecular Electronics Corp. in California – have sprung up with the explicit goal of building the first commercial molecular electronic devices including memories and other computational components of computers, possibly in the next few years, using techniques of self-assembly.
As machine structures become more complex, getting all the parts to spontaneously self-assemble in the right sequence will be increasingly difficult. To build such complex structures, it makes more sense to design a mechanism that can assemble a molecular structure by what is called positional assembly – that is, picking and placing molecular parts exactly where you want them. A device capable of positional assembly would work much like the robot arms that manufacture cars on automobile assembly lines in Detroit, or which insert electronic components onto computer circuit boards with blinding speed in Silicon Valley. Using the positional assembly approach, the robot manipulator picks up a part, moves it to the workpiece, installs it, then repeats the procedure over and over with many different parts until the final product is fully assembled.
One of the leading proponents of positional assembly at the molecular scale is Zyvex Corp., a privately-held nanotechnology research and development corporation headquartered in Richardson, Texas . Zyvex is the first engineering company with the explicit goal of creating a molecular assembler that uses positional assembly to manufacture atomically precise structures. As a first step toward this goal, in 1998 Zyvex demonstrated the ability to use three independently-controlled inch-long robotic arms to manipulate tiny carbon nanotubes in three dimensions, under the watchful eye of a scanning electron microscope that can monitor objects and motions as small as 6 nanometers at near-video scan rates. Zyvex still has a very long way to go before it can assemble nanoscale parts into useful machines, but its work is a small step in the right direction and the research continues today. Zyvex engineers are also conceiving and testing various manufacturing architectures that may someday enable massively parallel, or exponential, construction of large batches of identical molecular machines simultaneously. This might allow vast numbers of nanodevices – ultimately including medical nanorobots – to be produced relatively inexpensively and to molecular specifications.
The idea of placing autonomous self-powered nanorobots inside of us might seem a bit odd, but actually the human body already teems with such nanodevices. For instance, more than 40 trillion single-celled microbes swim through our colon, outnumbering our tissue cells almost ten to one. Many bacteria move by whipping around a tiny tail, or flagellum, that is driven by a 30-nanometer biological ionic nanomotor powered by pH differences between the inside and the outside of the bacterial cell. Our bodies also maintain a population of more than a trillion motile biological nanodevices called fibroblasts and white cells such as neutrophils and lymphocytes, each measuring perhaps 10 microns in size. These beneficial natural nanorobots are constantly crawling around inside of us, repairing damaged tissues, attacking invading microbes, and gathering up foreign particles and transporting them to various organs for disposal from the body.
The greatest power of nanomedicine will emerge in a decade or two when we learn to design and construct complete artificial nanorobots using nanometer-scale parts and subsystems including sensors, motors, manipulators, power plants, and molecular computers. If we make the reasonable assumption that we will someday be able to build these complex medical nanorobots, and build them cheaply enough and in sufficiently large numbers to be useful therapeutically, then what are the medical implications? We have space here to describe only a few of the many possibilities [2, 35-39].
One thing that would change dramatically is clinical diagnostics and treatment. Consider a patient who goes to his doctor with a mild fever, nasal congestion, discomfort, and cough. In the nanomedical era, taking and analyzing microbial samples will be as quick and convenient as the electronic measurement of body temperature using a tympanic thermometer in a late 20th-century clinical office or hospital. The physician faces the patient and pulls from her pocket a lightweight handheld device resembling a pocket calculator. She unsnaps a self-sterilizing cordless pencil-sized probe from the side of the device and inserts the business end of the probe into the patient’s opened mouth in the manner of a tongue depressor. The ramifying probe tip contains billions of nanoscale molecular assay receptors mounted on hundreds of self-guiding retractile stalks. Each assay receptor is sensitive to the chemical signature of one of thousands of specific bacterial coats or viral capsids.
The patient says “Ahh,”  and a few seconds later a three-dimensional color-coded map of the throat area appears on a display panel held in the doctor’s hand. A bright spot on the screen marks the exact location where the first samples are being taken. Underneath the color map scrolls a continuously updated microflora count, listing in the leftmost column the names of the ten most numerous microbial and viral species that have been detected, key biochemical marker codes in the middle column, and measured population counts in the right column. The number counts flip up and down a bit as the physician directs probe stalks to various locations in the pharynx to obtain a representative sampling, with special attention to sores or exudate. After a few more seconds, the data for two of the bacterial species suddenly highlight in red, indicating the distinctive molecular signatures of specific toxins or pathological variants. One of these two species is a known, and unwelcome, bacterial pathogen. The diagnosis is completed and the infectious microbe is promptly exterminated using a patient-inhaled aerosol of mobile nanorobots which the physician has programmed to seek out and destroy that one microbial strain. After a few minutes the nanorobots have finished their work and are retrieved by the doctor. A resurvey with the diagnostic probe reveals no evidence of the pathogen.
Another major change that nanomedicine will bring is the ability to dramatically extend natural human capabilities. As a simple example, a few years ago I designed an artificial mechanical red cell called a “respirocyte” . Still entirely theoretical, the respirocyte measures 1 micron in diameter and just floats along in the bloodstream. It is a spherical nanorobot made of 18 billion atoms precisely arranged in a diamondoid structure to make a tiny pressure tank that can be pumped full of up to 3 billion oxygen (O2) and carbon dioxide (CO2) molecules. Later on, these gases can be released from the tank in a controlled manner using tiny molecular pumps. Gases are stored onboard at pressures up to about 1000 atmospheres.
Respirocytes mimic the action of the natural hemoglobin-filled red blood cells. Gas concentration sensors on the outside of each device let the nanorobot know when it is time to load O2 and unload CO2 (at the lungs), or vice versa (at the tissues). Each respirocyte can store and transport 236 times as much gas per unit volume as a natural red cell. So the injection of a 5 cc therapeutic dose of 50% respirocyte saline suspension, a total of 5 trillion individual nanorobots, into the human bloodstream can exactly replace the gas carrying capacity of the patient’s entire 5.4 liters of blood. But up to 1 liter of respirocyte suspension could safely be added to the bloodstream, which could keep a patient’s tissues safely oxygenated for up to 4 hours in the event a heart attack caused the heart to stop beating. Or it would enable a healthy person to sit quietly at the bottom of a swimming pool for four hours, holding his breath, or to sprint at top speed for at least 15 minutes without breathing.
Similarly, an artificial mechanical platelet or “clottocyte”  could make possible complete hemostasis in just 1 second, even for moderately large wounds, a response time 100-1000 times faster than the natural system. The basic clottocyte is conceived as a serum oxygen/glucose-powered spherical nanorobot, 2 microns in diameter, that contains a compactly-folded fiber mesh. Upon command from its control computer, the device unfurls its mesh packet in the vicinity of an injured blood vessel – following, say, a cut through the skin. Soluble thin films coating certain parts of the mesh dissolve upon contact with plasma water, revealing sticky sections (e.g., complementary to blood group antigens unique to red cell surfaces) in desired patterns. Blood cells are immediately trapped in the overlapping artificial nettings released by multiple neighboring activated clottocytes, and bleeding halts at once. While up to 300 natural platelets might be broken and still be insufficient to initiate a self-perpetuating clotting cascade, even a single clottocyte, upon reliably detecting a blood vessel break, can rapidly communicate this fact to its neighboring devices , immediately triggering a progressive carefully-controlled mesh-release cascade. Clottocytes may perform a clotting function that is equivalent in its essentials to that performed by biological platelets, but at only 0.01% of the bloodstream concentration of those cells or about 20 nanorobots per cubic millimeter of serum. Hence clottocytes appear to be about 10,000 times more effective as clotting agents than an equal volume of natural platelets.
Medical nanorobots will also be able to intervene at the cellular level, performing in vivo cytosurgery. The most likely site of pathological function in the cell is the nucleus – more specifically, the chromosomes. In one simple cytosurgical procedure, a nanorobot controlled by a physician would extract existing chromosomes from a diseased cell and insert new ones in their place. This is called chromosome replacement therapy. The replacement chromosomes will be manufactured to order, outside of the patient’s body in a laboratory benchtop production device that includes a molecular assembly line, using the patient’s individual genome as the blueprint. The replacement chromosomes are appropriately demethylated, thus expressing only the appropriate exons that are active in the cell type to which the nanorobot has been targeted. If the patient chooses, inherited defective genes could be replaced with nondefective base-pair sequences, permanently curing a genetic disease. Given the speed with which nanorobots can be administered and their potential rapidity of action, it is possible that an entire whole-body procedure could be completed in one hour or less. Robert Austin at Princeton University has also begun early thinking along these lines, hoping someday to design a nanoprobe capable of identifying biological markers that are specific for targeted diseases. “Then you just pop open the cells, remove the bad DNA from that cell, and repair it on a single-cell level,” he says. “That’s a long way down the road, but it will happen.”
In the first half of the 21st century, nanomedicine should eliminate virtually all common diseases of the 20th century, and virtually all medical pain  and suffering as well. Only conditions that involve a permanent loss of personality and memory information in the brain – such as an advanced case of Alzheimer’s disease or a massive head trauma – may remain incurable in the nanomedical era. Because aging is believed to be the result of a number of interrelated molecular processes and malfunctions in cells, and because cellular malfunctions will be largely reversible, middle-aged and older people who gain access to an advanced nanomedicine can expect to have most of their youthful health and beauty restored. And they may find few remaining limits to human longevity in this wonderfully vigorous state. It is a bright future that lies ahead for medicine, but we shall all have to work very long and very hard to bring it to fruition.
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Robert A. Freitas Jr. is the author of Nanomedicine, the first book-length technical discussion of the potential medical applications of molecular nanotechnology and medical nanorobotics. Volume I was published in October 1999 by Landes Bioscience while Freitas was a Research Fellow at the Institute for Molecular Manufacturing in Palo Alto, California. Freitas is completing Volumes II and III as a Research Scientist at Zyvex Corp., a nanotechnology R&D startup company headquartered in Richardson, Texas, and is also consulting on molecular assembler design at Zyvex.
Last updated by the author on 19 May 2003