ABSTRACT:- 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. In the relatively near term, nanomedicine can address many important medical problems by using nanoscale-structured materials and simple nanodevices that can be manufactured today, including the interaction of nanostructured materials with biological systems.In the mid-term, biotechnology will make possible even more remarkable advances in molecular medicine and biobotics, including microbiological biorobots or engineered organisms. In the longer term, perhaps 10?0 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 aging.

INTRIDUCTION TO NANOMEDICINE: Robert A. Freitas, Jr. is Senior Research Fellow at the Institute for Molecular Manufacturing(IMM) in Palo Alto, California, and was a Research Scientist at Zyvex Corp.(Richardson, Texas), the first molecular nanotechnology company, during 2000-2004.He received B.S. degrees in Physics and Psychology from Harvey Mudd College in 1974 and a J.D. from University of Santa Clara in 1979.Freitas co-edited the 1980 NASA feasibility analysis of self-replicating space factories and in 1996 authored the first detailed technical design study of a medical nanorobot ever published in a peer-reviewed mainstream biomedical journal.More recently, Freitas is the author of Nanomedicine, the first book-length technical discussion of the potential medical applications of molecular nanotechnology and medical nanorobotics; the first two volumes of this 4-volume series were published in 1999 and 2003 by Landes Bioscience. His research interests include: nanomedicine, medical nanorobotics design, molecular machine systems, diamond mechanosynthesis (theory and experimental pathways), molecular assemblersand nanofactories, and self-replication in machine and factory systems.He has published 25 refereed journal publications and several contributed book chapters, and most recently co-authoredKinematic Self-Replicating Machines (2004), another first-of-its-kind technical treatise.

Annual U.S. federal funding for nanotechnology R&D exceeded $500 million in 20021 reaching $849 million in FY 20042 and could approach $1 billion in next year budget.The European Commission has set aside 1.3 billion euros for nanotechnology research during 2003? 2006,3 with annual nanotechnology investment worldwide reaching approximately $3 billion in 2003.The worldwide market for nanoscale devices and molecular modeling should grow 28%/year, rising from $406 million in 2002 to $1.37 billion in 2007, with a 35%/year growth rate in revenues from biomedical nanoscale devices.4 In December 2002 the U.S. National Institutes of Health announced a 4-year program for nanoscience and nanotechnology in medicine.3 Burgeoning interest in the medical applications of nanotechnology has led to the emergence of a new field called nanomedicine.3_ 5?2 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.5 The NIH Roadmap new Nanomedicine Initiatives, first released in late 2003, nvision that this cutting-edge area of research will begin yielding medical benefits as early as 10 years from now?and will begin with stablishing a handful of Nanomedicine Centers staffed by a highly interdisciplinary scientific crew including biologists, physicians, mathematicians, engineers and computer scientists gathering extensive information about how molecular machines are built?who will also develop new kind of vocabulary exicon to define biological parts and processes in engineering terms?13 Even state-funded programs have begun, such as New York Alliance for Nanomedical Technologies.14 In the relatively near term, over the next 5 years, nanomedicine can address many important medical problems by using nanoscale-structured materials and simplenanodevices that can be manufactured today (Section 2). This includes the interaction of nanostructured materials with biological systems.7 Over the next 5?0 years, biotechnology will make possible even more remarkable advances in molecular medicine and bioboticsicrobiological robots or engineered organisms (Section 3).In the longer term, perhaps 10?0 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 aging.

Nanopores: Perhaps one of the simplest medical nanomaterials is a surface perforated with holes, or nanopores.In 1997 Desai and Ferrari created what could be considered one of the earliest therapeutically useful nanomedical devices,15 employing 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, secreting insulin back out through the pores while the immune system remains unaware of the foreign cells which it would normally attack and reject.Microcapsules containing replacement islets of Langerhans cells most likely easily-harvested piglet islet cellsould be implanted beneath the skin of some diabetes patients.16 This could temporarily restore the body 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,17 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 or Parkinson diseases. The flow of materials through nanopores can also be externally regulated.18 The first artificial voltage-gated molecular nanosieve was fabricated by Martin and colleagues19 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.Martin?s recent efforts20 have been directed at immobilizing biochemical molecular-recognition agents such as enzymes, antibodies, other proteins and DNA inside the nanotubes as active biological nanosensors,21 to perform drug separations,22_ 23 and to allow selected biocatalysis.23 Others are investigating synthetic nanopore ion pumps,24 voltage-gated nanopores embedded in artificial membranes,25 and an ion channel switch biosensor that detects changes in chemical concentration of ~10 Molecular dynamics theoretical studies of viscosity and diffusion through nanopores are in progress. Finally, Daniel Branton team 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.29 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.Current research is directed toward reliably fabricating pores with specific diameters and repeatable geometries at high precision,30 understanding the unzipping of double-stranded DNA as one strand is pulled through the pore31 and the recognition of folded DNA molecules passing through the pore,32 experiments with new 3?0 nm silicon-nitride nanopores,32 and investigating the benefits of adding electrically conducting electrodes to pores to improve longitudinal resolution ossibly to the single-base level for DNA?32 Nanopore-based DNAsequencing devices could allow per-pore read rates potentially up to 1000 bases per second,33 possibly eventually providing a low-cost high-throughput method for very rapid genome sequencing.

Artificial Binding Sites and Molecular Imprinting: 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.Molecular imprinting34_ 35 is an existing technique in which a cocktail of functionalized monomers interacts reversibly with a target molecule using only noncovalent forces.The complex is then cross-linked and polymerized in a casting procedure, leaving behind a polymer with recognition sites complementary to the target molecule in both shape and functionality.Each such site constitutes an induced molecular emory,?capable of selectively binding the target species.In one experiment involving an amino acid derivative target, one artificial binding site per (3.8 nm)3 polymer block was created. Chiral separations, enzymatic transition state activity, and high receptor affinities have been demonstrated. Molecularly imprinted polymers could be medically useful in clinical applications such as controlled drug release, drug monitoring devices, quick biochemical separations and assays,36 recognition elements in biosensors and chemosensors,37 and biological and receptor mimics including artificial antibodies (plastibodies) or biomimicking enzymes (plastizymes).37 But molecularly imprinted polymers have limitations, such as incomplete template removal, broad guest affinities and selectivities, and slow mass transfer.Imprinting inside dendrimers (Section 2.7) may allow quantitative template removal, nearly homogeneous binding sites, solubility in common organic solvents, and amenability to the incorporation of other functional groups. Quantum Dots and Nanocrystals: 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.uantum 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.The y come in a nearly unlimited palette of sharply-defined colors which can be customized by changing particle size or composition.P articles can be excited to fluorescence with white light, can be linked to biomolecules to form long-lived sensitive probes to identify specific compounds up to a thousand times brighter than conventional dyes used in many biological tests, and 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 Corp. (www.qdots.com), 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 or to detect known cellular cancer markers.38 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.Quantum dots are useful for studying genes, proteins and drug targets in single cells, tissue specimens, and living animals.39 Quantum dots are being investigated as chemical sensors,40 for cancer cell detection,38 gene expression studies,41 gene mapping and DNA microarray analysis,42 immunocytochemical probes,43 intracellular organelle markers,44 live cell labeling,45_ 46 medical diagnostics and drug screening,47 SNP (Single Nucleotide Polymorphism) genotyping,48 vascular imaging,49 and many other applications.50_ 51 Quantum dot physics has been studied theoretically52 and computationally using time-dependent density functional theory53 and other methods. Researchers from Northwestern University and Argonne National Laboratory have created a hybrid anodevice?composed of 4.5-nm nanocrystals of biocompatible titanium dioxide semiconductor covalently attached with snippets of oligonucleotide DNA.57 Experiments showed that these nanocomposites not only retain the intrinsic photocatalytic capacity of TiO2 and the bioactivity of the oligonucleotide DNA, but more importantly also possess the unique property of a light-inducible nucleic acid endonuclease (separating when exposed to light or x-rays). For example, researchers would attach to the semiconductor scaffolding a strand of DNA that matches a defective gene within a cell, then introduce the nanoparticle into the cell nucleus where the attached DNA binds with its defective complementary DNA strand, whereupon exposure of the bound nanoparticle to light or x-rays snips off the defective gene.Other molecules besides oligonucleotides can be attached to the titanium dioxide scaffolding, such as navigational peptides or proteins, which, like viral vectors, can help the nanoparticles home in on the cell nucleus. This simple nanocrystal nanodevice might one day be used to target defective genes that play a role in cancer, neurological disease and other conditions, though testing in a laboratory model is at least two years away.

Fullerenes and Nanotubes: Soluble derivatives of fullerenes such as C60 have shown great utility as pharmaceutical agents.These derivatives, many already in clinical trials (www.csixty.com), have good biocompatibility and low toxicity even at relatively high dosages.Fullerene compounds may serve as antiviral agents (most notably against HIV,59 where they have also been investigated computationally60_ 61), antibacterial agents (E. coli,62 Streptococcus,63 Mycobacterium tuberculosis,64 etc.), photodynamic antitumor65_ 66 and anticancer67 therapies, antioxidants and anti-apoptosis agents which may include treatments for amyotrophic lateral sclerosis (ALS or Lou Gehrig disease)68 and Parkinson disease.Single-w alled69_ 70 and multi-walled71?3 carbon nanotubes are being investigated as biosensors, for example to detect glucose,72_ 74 ethanol,74 hydrogen peroxide,71 selected proteins such as immunoglobulins,70 and as an electrochemical DNA hybridization biosensor.

Nanoshells and Magnetic Nanoprobes: Halas and West at Rice University in Houston have developed a platform for nanoscale drug delivery called the nanoshell.75_ 76 Unlike carbon fullerenes, the slightly larger nanoshells are dielectric-metal nanospheres with a core of silica and a gold coating, whose optical resonance is a function of the relative size of the constituent layers.The nanoshells are embedded in a drug-containing tumortargeted hydrogel polymer and injected into the body.The shells circulate through the body until they accumulate near tumor cells.When heated with an infrared laser, the nanoshells (each slightly larger than a polio virus) selectively absorb the IR frequencies, melt the polymer and release their drug payload at a specific site.Nanoshells offer advantages over traditional cancer treatments: earlier detection, more detailed imaging, fast noninvasive imaging, and integrated detection and treatment.77 This technique could also prove useful in treating diabetes.Instead of taking an injection of insulin, a patient would use a ballpoint-pen-size infrared laser to heat the skin where the nanoshell polymer had been injected.The heat from nanoshells would cause the polymer to release a pulse of insulin.Unlik e injections, which are taken several times a day, the nanoshell-polymer system could remain in the body for months. Nanospectra Biosciences (www.nanospectra.com), a private company started by Halas and West, is developing commercial applications of nanoshell technology.Nanospectra is conducting animal studies at the MD Anderson Cancer Center at the University of Texas, specifically targeting micrometastases, tiny aggregates of cancer cells too small for surgeons to find and remove with a scalpel.The company hopes to start clinical trials for the cancer treatment by 2004 and for the insulindelivery system by 2006.In mid-2003, Rice researchers announced the development of a point-of-care whole blood immunoassay using antibody-nanoparticle conjugates of gold nanoshells.78 Varying the thickness of the metal shell allow precise tuning of the color of light to which the nanoshells respond; near-infrared light penetrates whole blood very well, so it is an optimal wavelength for whole blood immunoassay.79 Successful detection of sub-nanogram-per-milliliter quantities of immunoglobulins was achieved in saline, serum, and whole blood in 10?0 minutes.78 An alternative approach pursued by Triton BioSystems (www.tritonbiosystems.com) is to bond iron nanoparticles and monoclonal antibodies into nanobioprobes about 40 nanometers long.The chemically inert probes are injected and circulate inside the body, whereupon the antibodies selectively bind to tumor cell membranes.Once the tumor (whether visible or micrometastases) is covered with bioprobes after several hours, a magnetic field generated from a portable alternating magnetic field machine (similar to a miniaturized MRI machine) heats the iron particles to more than 170 degrees, killing the tumor cells in a few seconds.80 Once the cells are destroyed, the body excretion system removes cellular residue and nanoparticles alike.T est subjects feel no pain from the heat generated.80 Triton BioSystems plans to start designing human tests and ask the FDA for permission to begin human clinical trials in 2006. Mirkin group at Northwestern University uses magnetic microparticle probes coated with target proteinbinding antibodies plus 13-nm nanoparticle probes with a similar coating but including a unique hybridized arcode?DNA sequence as an ultrasensitive method for detecting protein analytes such as prostate-specific antigen(PSA).81 After the target protein in the test sample is captured by the microparticles, magnetic separation of the complexed microparticle probes and PSA is followed by dehybridization of the bar-code oligonucleotides on the nanoparticle probe surface, allowing the determination of the presence of PSA by identifying the bar-code sequence released from the nanoparticle probe.Using polymerase chain reaction on the oligonucleotide bar codes allows PSA to be detected at 3 attomolar concentration, about a million times more sensitive than comparable clinically accepted conventional assays for detecting the same protein target.

Targeted Nanoparticles and Smart Drugs: Multisegment gold/nickel nanorods are being explored by Leong group at Johns Hopkins School of Medicine82 as tissue-targeted carriers for gene delivery into cells that an simultaneously bind compacted DNA plasmids and targeting ligands in a spatially defined manner?and allow recise control of composition, size and multifunctionality of the gene-delivery system.?The nanorods are electrodeposited into the cylindrical 100 nm diameter pores of an alumina membrane, joining a 100 nm length gold segment and a 100 nm length nickel segment.After the alumina template is etched away, the nanorods are functionalized by attaching DNA plasmids to the nickel segments and transferrin, a cell-targeting protein, to the gold segments, using molecular linkages that selectively bind to only one metal and thus impart biofunctionality to the nanorods in a spatially defined manner.Leong notes that extra segments could be added to the nanorods, for example to bind additional biofunctionalities such as an endosomolytic agent, or magnetic segments could be added to allow manipulating the nanorods with an external magnetic field. Targeted radioimmunotherapeutic agents83 include the FDA-approved ancer smart bombs?that deliver tumorkilling radioactive yttrium (Zevalin) or iodine (Bexxar) attached to a lymphoma-targeted (anti-CD20) antibody.84 Other antibody-linked agents are being investigated such as the alpha-emitting actinium-based anogenerator?molecules that use internalizing monoclonal antibodies to penetrate the cell and have been shown, in vitro, to specifically kill leukemia, lymphoma, breast, ovarian, neuroblastoma, and prostate cancer cells at becquerel (picocurie) levels,85 with promising preliminary results against advanced ovarian cancer in mice.86 However, drug specificity is still no better than the targeting accuracy of the chosen antibody, and there is significant mistargeting, leading to unwanted side effects. Enzyme-activated drugs, first developed in the 1980s and still under active investigation,87 separate the targeting and activation functions.F or instance, an antibodydirected enzyme-triggered prodrug cancer therapy is being developed by researchers at the University of Gottingen in Germany.88 This targeted drug molecule turns lethal only when it reaches cancer cells while remaining harmless inside healthy cells.In tests, mice previously implanted with human tumors are given an activating targeted enzyme that sticks only to human tumor cells, mostly ignoring healthy mouse cells.Then the antitumor molecule is injected.In its activated state, this fungal-derived antibiotic molecule is a highly-strained ring of three carbon atoms that is apt to burst open, becoming a reactive molecule that wreaks havoc among the nucleic acid molecules essential for normal cell function.But the molecule is injected as a prodrugn antibiotic lacking the strained ring and with a sugar safety-catch.Once the sugar is clipped off by the previously positioned targeted enzyme, the drug molecule rearranges itself into a three-atom ring, becoming lethally active.Notes chemist Philip Ball:89 he selectivity of the damage still depends on antibody ability to hook onto the right cells, and on the absence of other enzymes in the body that also activate the prodrug.? A further improvement in enzyme-activated drugs are mart drugs?that become medically active only in specific circumstances and in an inherently localized manner. Yoshihisa Suzuki at Kyoto University has designed a novel drug molecule that releases antibiotic only in the presence of an infection.90 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.T ests 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,?wrote Suzuki,90 ifferent spectra of antibiotics could be released from the same dressing material, depending on the strain of bacterium.?In subsequent work an alternative antibiotic release system triggered by thrombin activity, which accompanies Staphylococcus aureus wound infections, was successfully tested as a high-specificity stimulus-responsive controlled drug release system.91 Other stimulus-responsive mart?hydrogels are being studied, including a hydrogelcomposite membrane co-loaded with insulin and glucose oxidase enzyme that exhibits a twofold increase in insulin release rate when immersed in glucose solution, demonstrating hemically stimulated controlled release?andhe potential of such systems to function as a chemicallysynthesized artificial pancreas.?2 Nanoparticles with an even greater range of action are being developed by Raoul Kopelman group at the University of Michigan.Their current goal is the development of novel molecular nanodevices for the early detection and therapy of brain cancer, using silica-coated iron oxide nanoparticles with a biocompatible polyethylene glycol coating.93 The miniscule size of the particles?0?00 nanometershould allow them to penetrate into areas of the brain that would otherwise be severely damaged by invasive surgery.The particles are attached to a cancer cell antibody or other tracer molecule that adheres to cancer cells, and are affixed with a nanopacket of contrast agent that makes the particles highly visible during magnetic resonance imaging (MRI).The particles also enhance the killing effect during the subsequent laser irradiation of brain tissue, concentrating the destructive effect only on sick cells unlike traditional chemotherapy and radiation which kills cancerous cells but also destroys healthy cells.Nanoparticles allow MRI to see a few small brain tumor cells as small as 50 microns depending on the cancer type, tumor cells can range from 5?0 microns each and may grow in locations separate from the tumor site, hence are sometimes not visible to surgeons.Fei Yan, a postdoc in Kopelman lab, is working on these nanodevices, called the Dynamic Nano-Platform (Fig.1), now being commercialized as therapeutic anosomes?under license to Molecular Therapeutics (www.moleculartherapeutics.com). According to the company, he nanosome platform provides the core technology with interchangeable components that provide ultimate flexibility in targeting, imaging and treatment of cancer and cardiovascular disease indications.?

Dendrimer-Based Devices: Dendrimers94 represent yet another nanostructured material that may soon find its way into medical therapeutics.95 Starburst dendrimers are tree-shaped synthetic molecules with a regular branching structure emanating outward from a core that form nanometer by nanometer, with the numberof synthetic steps or enerations?dictating the exact size of the particles, typically a few nanometers in spheroidal diameter.The peripheral layer can be made to form a dense field of molecular groups that serve as hooks for attaching other useful molecules, such as DNA, which can enter cells while avoiding triggering an immune response, unlike viral vectors commonly employed today for transfection.Upon encountering a living cell, dendrimers of a certain size trigger a process called endocytosis in whichthe cell outermost membrane deforms into a tiny bubble, or vesicle.The vesicle encloses the dendrimer which is then admitted into the cell interior.Once inside, the DNA is released and migrates to the nucleus where itbecomes part of the cell genome.The technique has been tested on a variety of mammalian cell types96 and in animal models,97_ 98 though clinical human trials of dendrimer gene therapy remain to be done.Glycodendrimer anodecoys?have also been used to trap and deactivate some strains of influenza virus particles.99_ 100 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.In July 2003,Starpharma (www.starpharma.com) was cleared by the U.S. FDA for human trials of their dendrimer-based anti- HIV microbicide.Their product has been successful in preventing simian-HIV.Computational simulations have also been done on some dendrimer-based nanoparticles. James Baker group at the University of Michigan is extending this work to the synthesis of multi-component nanodevices called tecto-dendrimers built up from a number of single-molecule dendrimer components.102?06 Tecto-dendrimers have a single core dendrimer surrounded by additional dendrimer modules of different types, each type designed to perform a function necessary to a smarttherapeutic nanodevice (Fig.2).Bak er group has built a library of dendrimeric components from which a combinatorially large number of nanodevices can be synthesized. The initial library contains components which will perform the following tasks: (1) diseased cell recognition,(2) diagnosis of disease state, (3) drug delivery, (4) reporting location, and (5) reporting outcome of therapy.By using this modular architecture, an array of smart therapeutic nanodevices can be created with little effort. For instance, once apoptosis-reporting, contrast-enhancing, and chemotherapeutic-releasing dendrimer modules are made and attached to the core dendrimer, it should be possible to make large quantities of this tecto-dendrimer as a starting material.This framework structure can be customized to fight a particular cancer simply by substituting any o ne of many possible distinct cancer recognition or argeting?dendrimers, creating a nanodevice customized to destroy a specific cancer type and no other, while also sparing the healthy normal cells.In three nanodevices synthesized using an ethylenediamine core polyamidoamine dendrimer of generation 5, with folic acid, fluorescein, and methotrexate covalently attached to the surface to provide targeting, imaging, and intracellular drug delivery capabilities, the argeted delivery improved the cytotoxic response of the cells to methotrexate 100-fold over free drug.?05 At least a half dozen cancer cell types have already been associated with at least one unique protein which targeting dendrimers could use to identify the cell as cancerous, and as the genomic revolution progresses it is likely that proteins unique to each kind of cancer will be identified, thus allowing Baker to design a recognition dendrimer for each type of cancer.106 The same cell-surface protein recognition-targeting strategy could be applied against virus-infected cells and parasites.Molecular modeling has been used to determine optimal dendrimer surface modifications for the function of tecto-dendrimer nanodevices and to suggest surface modifications that improve targeting. NASA and the National Cancer Institute have funded Baker lab to produce dendrimer-based nanodevices that can detect and report cellular damage due to radiation exposure in astronauts on long-term space missions.107 By mid-2002, the lab had built a dendrimeric nanodevice to detect and report the intracellular presence of caspase-3, one of the first enzymes released during cellular suicide or apoptosis (programmed cell death), one sign of a radiation-damaged cell.The device includes one component that identifies the dendrimer as a blood sugar so that the nanodevice is readily absorbed into a white blood cell, and a second component using fluorescence resonance energy transfer (FRET) that employs two closely bonded molecules.Before apoptosis, the FRET system stays bound together and the white cell interior remains dark upon illumination.Once apoptosis begins and caspase-3 is released, the bond is quickly broken and the white blood cell is awash in fluorescent light.If a retinal scanning device measuring the level of fluorescence inside an astronaut body reads above a certain baseline, counteracting drugs can be taken.

Radio-Controlled Biomolecules: While there are already many examples of nanocrystals attached to biological systems for biosensing purposes,the same nanoparticles are now being investigated as a means for directly controlling biological processes.Jacobson and colleagues108 have attached tiny radio-frequency antennas?.4 nanometer gold nanocrystals of less than 100 atomso DNA.When a ~1 GHz radio-frequency magnetic field is transmitted into the tiny antennas, alternatingeddy currents induced in the nanocrystals produce highly localized inductive heating, causing the doublestranded DNA to separate into two strands in a matter of seconds in a fully reversible dehybridization process that leaves neighboring molecules untouched. The long-term goal is to apply the antennas to living systems and control DNA (e.g., gene expression, the ability to turn genes on or off) via remote electronic switching.This requires attaching gold nanoparticles to specific oligonucleotides which, when added to a sample of DNA, would bind to complementary gene sequences, blocking the activity of those genes and effectively turning them off.Applying the rf magnetic field then heats the gold particles,causing their attached DNA fragments to detach, turning the genes back on.Such a tool could give pharmaceutical researchers a way to simulate the effects of potential drugs which also turn genes on and off.109 Says Gerald Joyce:110 ou can even start to think of differential receiversifferent radio receivers that respond differently to different frequencies.By dialing in the right frequency, you can turn on tags on one part of DNA but not other tags.? The gold nanocrystals can be attached to proteins as well as DNA, opening up the possibility of future radio frequency biology electronically controlling more complex biological processes such as enzymatic activity, protein folding and biomolecular assembly.In late 2002,Jacobson announced that his team had achieved electrical control over proteins as well.111 The researchers separated an RNA-hydrolyzing enzyme called ribonucleaseS into two pieces: a large protein segment made up of 104 amino acids and a small 18-amino-acid strand called the S-peptide.The RNAase enzyme is inactive unless the small strand sits in the mouth of the protein.Jacobson?s group linked gold nanoparticles to the end of S-peptide strands and used the particles as a switch to turn the enzyme on and offn the absence of the rf field, the S-peptides adopt their usual conformation and the RNAase remains active, but with the external rf field switched on, the rapidly spinning nanoparticles prevented the S-peptide from assembling with the larger protein, inactivating the enzyme.

MICR OSCALE BIOLOGICAL ROBOTS: One convenient shortcut to nanorobotics is to engineer natural nanomachine systemsicroscale biological viruses and bacteriao create new, artificial biological devices. Efforts at purely rational virus design are underway but have not yet borne much fruit.F or example, Endy et al.112 computationally simulated the growth rates of bacteriophage T7 mutants with altered genetic element orders and found one new genome permutation that was predicted to allow the phage to grow 31% faster than wild type; unfortunately, experiments failed to confirm the predicted speedup.Better models are clearly needed.Nevertheless, combinatorial experiments on wild type T7 by others have produced new but immunologically indistinguishable T7 variants which have 12% of their genome deleted and which replicate twice as fast as wild type.117 The Synthetic Biology Lab at MIT (syntheticbiology. org) is building the next generation T7, a bacteriophage with a genome size of about 40 Kbp and 56 genes.Considerations in the redesign process include: dding or removing restriction sites to allow for easy manipulation of various parts, reclaiming codon usage, and eliminating parts of the genome that have no apparent function.?Young and Douglas118 have chemically modified the Cowpea chlorotic mottle virus (CCMV) viral protein cage surface to allow engineering of surface-exposed functional groups.This includes the addition of lamanin peptide 11 (a docking site for lamanin-binding protein generously expressed on the surface of many types of breast cancer cells) to the viral coat, and the incorporation of 180 gadolinium atoms into each 28-nm viral capsid, allowing these tumor-targeting particles to serve as tumor-selective MRI contrast agents.119 The researchers have investigated re-engineering the artificial virion to make a complete tumor-killing nanodevice, exploiting a gating mechanism that results from reversible structural transitions in the virus.120 The natural viral gate of CCMV has been reengineered to allow control by redox potential; cellular interiors have a higher redox potential than blood, so viral capsids could be shut tight in transit but would open their redox-controlled gates after entering targeted cancer cells, releasing their payload of therapeutic agents.In principle, the four capabilities of the engineered capsids?high-sensitivity imaging, cell targeting, drug transport, and controlled deliveryepresent a potentially powerful, yet minimally toxic, way to fight metastasized cancer. The rational design and synthesis of chimeric viral replicators is already possible today, and the rational design and synthesis of completely artificial viral sequences, leading to the manufacture of completely synthetic viral replicators, should eventually be possible.In a three-year project121 culminating in 2002, the 7500-base polio virus was rationally manufactured rom scratch?in the laboratory by synthesizing the known viral genetic sequence in DNA, enzymatically creating an RNA copy of the artificial DNA strand, then injecting the synthetic RNA into a cell-free broth containing a mixture of proteins taken from cells, which then directed the synthesis of complete (and fully infectious) polio virion particles. Engineered bacterial iorobots?are also being pursued. Mushe gian122 concludes that as few as 300 highly conserved 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 lifeanufacturing cellular biomolecules, generating energy, repairing damage, transporting salts and other molecules, responding to environmental chemical cues, and replicating.Thus a minimal synthetic microbe a basic cellular chassisould be specified by a genome only 150,000 nucleotide bases in length.Used in medicine,these artificial biorobots could be designed to produce useful vitamins, hormones, enzymes or cytokines in which a patient body was deficient, or to selectively absorb and metabolize into harmless end products harmful substances such as poisons, toxins, or indigestible intracellular detritus, or even to perform useful mechanical tasks. In November 2002, J.Craig Venter, of human genomesequencing fame, and Hamilton O.Smith, a Nobel laureate, announced123 that their new company, Institute for Biological Energy Alternatives (IBEA), had received a $3 million, three-year grant from the Energy Department to create a minimalist organism, starting with the Mycoplasma genitalium microorganism.W orking with a research staff of 25 people, the scientists are removing all genetic material from the organism, then synthesizing an artificial string of genetic material resembling a naturally occurring chromosome that they hope will contain the minimum number of M. genitalium genes needed to sustain life.The artificial chromosome will be inserted into the hollowed-out cell, which will then be tested for its ability to survive and reproduce.T o ensure safety, the cell will be deliberately hobbled to render it incapable of infecting people, and will be strictly confined and designed to die if it does manage to escape into the environment. In 2003, Egea Biosciences (www.egeabiosciences.com) received he first [patent]124 to include broad claims for the chemical synthesis of entire genes and networks of genes comprising a genome, the perating system?of living organisms.?Egea proprietary GeneWriter?and Protein Programming?technology has: (1) produced libraries of more than 1,000,000 programmed proteins, (2) produced over 200 synthetic genes and proteins, (3) produced the largest gene ever chemically synthesized of over 16,000 bases, (4) engineered proteins for novel functions, (5) improved protein expression through codon optimization, and (6) developed custom genes for protein manufacturing in specific host cells.Egea?s software allows researchers to author new DNA sequences that the company hardware can then manufacture to specification with a base-placement error of only ~10-4, which Egea calls ord processing for DNA?125 The patent recites one preferred embodiment of the invention as the synthesis of gene of 100,000 bp_ _ _ from one thousand 100-mers.The overlap between airs?of plus and minus oligonucleotides is 75 bases, leaving a 25 base pair overhang.In this method, a combinatorial approach is used where corresponding pairs of partially complementary oligonucleotides are hybridized in the first step.A second round of hybridization then is undertaken with appropriately complementary pairs of products from the first round.This process is repeated a total of 10 times, each round of hybridization reducing the number of products by half. Ligation of the products then is performed.?The result would be a strand of DNA 100,000 base pairs in length,long enough to make a very simple bacterial genome.

MEDICAL NANOROBOTICS: The third major development pathway of nanomedicine molecular nanotechnology (MNT) or nanorobotics5 7 126 takes as its purview the engineering of complex nanomechanical systems for medical applications.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, precision 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 ability to design, construct, and deploy large numbers of microscopic medical nanorobots will make this possible.

Early Thinking in Medical Nanorobotics: In his remarkably prescient 1959 talk here Plenty of Room at the Bottom,?the late Nobel physicist Richard P.Fe ynman 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.127 Feynman was clearly aware of the potential medical applications of the new technology he was proposing.After discussing his ideas with a colleague,Feynman offered127 the first known proposal for a nanomedical procedure to cure heart disease: 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.Y ou 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, hat we can manufacture an object that maneuvers at that level. The vision behind Feynman remarks became a serious area of inquiry two decades later, when K.Eric Drexler, while still a graduate student at the Massachusetts Institute of Technology, published a technical paper128 suggesting that it might be possible to construct, from biological parts, nanodevices that could inspect the cells of a living human being and carry on repairs within them.This was followed a decade later by Drexler seminal technical book126 laying the foundations for molecular machine systems and molecular manufacturing, and subsequently by Freitas?technical books5 7 on medical nanorobotics.

Nanocomputers: Truly effective medical nanorobots may require onboard computers to allow a physician to properly monitor and control their work.In 2000, a collaborative effort between UCLA and Hewlett Packard produced the first laboratory demonstration of completely reversible roomtemperature molecular switches that could be employed in nanoscale memories, using mechanically interlinked ring molecules called catenanes,145 and there has been much recent progress with nanotube- and nanorod-based molecular electronics.146 147 Several private companies are pursuing the first commercial molecular electronic devices including memories and other computational components of nanocomputers using techniques of self-assembly, and there is also the possibility of low-speed biology-based digital nanocomputers.

Assembly and Molecular Manufacturing: As machine structures become more complex, getting all the parts to spontaneously self-assemble in the right sequence is increasingly difficult.T o build complex nonperiodic structures, it makes more sense to design a mechanism that can assemble a molecular structure by what is called positional assemblyhat is, picking and placing molecular parts.A device capable of positional assembly would work much like the robot arms that manufacture cars on automobile assembly lines.In this 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. (www.zyvex.com),the first engineering firm to espouse an explicit goal of using positional assembly to manufacture atomically precise structures, or more specifically, user-controlled fabrication tool capable of creating molecularly precise structures with 3-dimensional capability in an economically viable manner.?Zyvex has already demonstrated the ability to positionally assemble large numbers of MEMSscale parts, and has 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.Agilent Laboratories has created an ultra-high-precision micromover platform229 capable of providing linear two-dimensional movement in steps of 1.5 nanometers, the width of about 9 bonded carbon atoms.The core of the micromover is a stepper actuator or linear motor that does not rotate, but instead steps right to left or front to back.The platform can travel a total of 30 micrometers in each direction in 2.5 milliseconds; since each micrometer is made up of 1,000 nanometers, the micromover would take approximately 20,000 steps to traverse 30 micrometers, a distance which is about half the width of a single human hair.Martel?s group at MIT has worked on a similar nano-positioning device called the NanoWalker. Kim and Lieber231 created the first general-purpose nanotweezer whose working end is a pair of electrically controlled carbon nanotubes made from a bundle of multiwalled carbon nanotubes.T o 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.Kim and Lieber have successfully grasped organelle-sized 500-nanometer clusters of polystyrene spheres, and have removed a semiconductor wire 20 nanometers wide from a mass of entangled wires, using tweezer arms about 50 nanometers wide and 4 microns long, but the technique creates a large electric field at the tweezer tips which can alter the objects being manipulated.In 2001, Boggild group232 used standard micromachining processes to carve from a tiny slab of silicon an array of cantilevered micro-pliers which could be opened and closed electrically.Boggild then used an electron beam to grow a tiny carbon nanotweezer arm from the end of each cantilever, angled so that the tips were only 25 nanometers apart, making a better-controlled nanotweezer.233 Nanotube-based nanotweezers have since been reported by others. Precise positional covalent attachment of molecules to surfaces is also being pursued.Blackledge et al.236 used a palladium-coated SFM (Scanning Force Microscope) tip to chemically modify terminal functional groups on an organosiloxane-coated surface to create biotin-streptavidin assemblies in patterns with minimum 33 nm line widths.Diaz et al.237 employ redox probe microscopy (RPM) in which an SFM tip is modified with redox-active materials,whereupon the interactions between tip and an adsorbate or between tip and a surface are modulated by the electrode potential.This system has also been used as a microtweezer to manipulate and position objects.Hla and Rieder238 239 have reviewed recent progress in using scanning tunneling microscopy (STM) to manipulate and synthesize individual molecules. The ultimate goal of molecular nanotechnology is to develop a manufacturing technology that can inexpensively manufacture most arrangements of atoms that can be specified in molecular detailncluding complex arrangements involving millions or billions of atoms per product object, as in the hypothesized medical nanorobots(Section 4.5). This will provide the ultimate manufacturing technology in terms of its precision, flexibility, and low cost.T wo central mechanisms have been proposed to achieve these goals at the molecular scale: programmable positional assembly including fabrication of diamondoid structures using molecular feedstock (Section 4.4.1), and massive parallelism of all fabrication and assembly processes.

Medical Nanorobot Designs and Scaling Studies: 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.F or instance,more than 40 trillion single-celled microbes swim through our colon, outnumbering our tissue cells almost ten to one.5 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.5 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. There are ongoing attempts to build MEMS-based microrobots intended for in vivo use.F or example, the R-Sub?project of the NanoRobotics Laboratory of Ecole Polytechnique in Montreal will use a Magnetic Resonance Imaging (MRI) system as a means of propulsion for a microrobot in the blood vessels.279 In this approach,a variable MRI magnetic field would generate a magnetic force on a robot containing ferromagnetic particles, providing a miniaturized system of propulsion able to develop sufficient power to direct a small device through the human body.Applications of the first generation prototype might include targeted drug release, the reopening of blocked arteries, or taking biopsies.The project is currently gathering necessary information to define design rules for this type of microrobot, with a long-term goal o further miniaturize the system and to create a robot made up of nanometric parts,?making it ossible to carry out medical applications in the blood vessels which are still inaccessible.?Other approaches to MEMS-based microrobots intended for in vivo use have been described in the literature,280 including the magnetically-controlled ytobots?and aryobots?proposed by Chrusch et al.281 for performing wireless intracellular surgery. There are preliminary proposals for hybrid bionanorobots that could be constructed using currently foreseeable technologies.F or example, Montemagno282 283 plans to use his modified ATPase motors to create a nanorobot that acts as a harmacy in a cell?by entering a cell, grabbing proteins produced by the cell that will not be used, and storing them until they are needed later by the patient.The device would consist of a tiny nickel drum, attached to the ATP-powered biological motor, which is coated with antibodies that adsorb the target molecules, whereupon an electric field pulls the molecules to a storage chamber and holds them in place. The greatest power of nanomedicine will emerge in a decade or two when we learn to design and construct complete artificial nanorobots using diamondoid nanometerscale 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 diamondoid medical nanorobots (Sections 4.2 and 4.4.1), and to build them cheaply enough and in sufficiently large numbers to be useful therapeutically (Section 4.4.2), then what are themedical implications? There are many possibilities5? 284?88 but the development pathway will be long and arduous.First, theoretical scaling studies are used to assess basic concept feasibility.These initial studies would then be followed by more detailed computational simulations of specific nanorobot components and assemblies, and ultimately full systems simulations, all thoroughly integrated with additional simulations of massively parallel manufacturing processes from start to finish consistent with a design-for-assembly engineering philosophy.Once molecular manufacturing capabilities become available, experimental efforts may progress from component fabrication and testing, to component assembly, and finally to prototypes and mass manufacture, ultimately leading to clinical trials.In 2004,progress in medical nanorobotics remains largely at the concept feasibility stageince 1998, the author has published four theoretical nanorobot scaling studies,285?88 two of which are summarized briefly below.Note that these studies are not intended to produce an actual engineering design for a future nanomedical product.Rather , the purpose is merely to examine a set of appropriate design constraints, scaling issues, and reference designs to assess whether or not the basic idea might be feasible, and to determine key limitations of such designs.Issues related to biocompatibility of medical nanorobots are extensively discussed elsewhere.

Respirocytes: The artificial mechanical red blood cell or espirocyte?85 is a bloodborne spherical 1-micron diamondoid 1000- atmosphere pressure vessel (Fig.8) with active pumping powered by endogenous serum glucose, able to deliver 236 times more oxygen to the tissues per unit volume than natural red cells and to manage carbonic acidity.The nanorobot is made of 18 billion atoms precisely arranged in a diamondoid 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 the same molecular pumps.Respiroc ytes 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) (Fig.9).An onboard nanocomputer and numerous chemical and pressure sensors enable complex device behaviors remotely reprogrammable by the physician via externally applied acoustic signals. 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 entire 5.4 liters of blood. If up to 1 liter of respirocyte suspension could safely be added to the human bloodstream,7 this could keep a patient tissues safely oxygenated for up to 4 hours in the event a heart attack caused the heart to stop beating, even in the absence of respiration.Primary medical applications of respirocytes will include transfusable blood substitution; partial treatment for anemia, perinatal/neonatal and lung disorders; enhancement of cardiovascular/neurovascular procedures, tumor therapies and diagnostics; prevention of asphyxia; artificial breathing; and avariety of sports, veterinary, battlefield and other uses.

References: 1. National Nanotechnology Initiative: Research and Development FY(2002); http://www.nano.gov/2002budget.html. 2. M.C.Roco, ational Nanotechnology Investment in the FY, 2004;Budget Request,?AAAS Report XXVIII: Research & Development FY (2004); http://www.aaas.org/spp/rd/04pch25.htm. 3. anomedicine: Grounds for Optimism, and a Call for Papers,ancet 362 (2003):673; http://www.alphagalileo.org/nontextfiles/P_5258_14984_1.pdf. 4. B-162 Biomedical Applications of Nanoscale Devices,?Business Communications Company, Inc., 25 Van Zant Street, Norwalk, CT 06855 (2003); http://www.bccresearch.com/editors/RB-162.html. 5. R.A.Freitas, Jr., Nanomedicine, Volume I: Basic Capabilities, Landes Bioscience, Georgetown, TX (1999); http://www.nanomedicine.com/NMI.htm. 6. R.A.Freitas, Jr., J. Amer. Dent. Assoc. 131, 1559 (2000); http://www.rfreitas.com/Nano/Nanodentistry.htm. 7. R.A.Freitas, Jr., Nanomedicine, Volume IIA: Biocompatibility,Landes Bioscience, Georgetown, TX (2003); http://www.nanomedicine.com/NMIIA.htm. 8. D.O.W eber, Health Forum J. 42, 32 (1999). 9. R.A.Freitas, Jr., Stud. Health Technol. Inform. 80, 45 (2002). 10. K.Bogunia-K ubik and M.Sugisaka, Biosystems 65, 123 (2002). 11. C.A.Haberzettl, Nanotechnology 13, R9 (2002). 12. D.F .Emerich and C.G.Thanos, Expert Opin. Biol. Ther. 3, 655(2003). 13. IH Roadmap: Nanomedicine,?National Institutes of Health(2003); http://www.nihroadmap.nih.gov/nanomedicine/index.asp. 14. lliance for Nanomedical Technologies,?http://www.research.cornell.edu/anmt/. 15. T.A.Desai, W.H.Chu, J.K.T u, G.M.Beattie, A.Hayek, and M.Ferrari, Biotechnol. Bioeng. 57, 118 (1998). 16. L.Leoni and T.A.Desai, IEEE Trans. Biomed. Eng. 48, 1335(2001). 17. S.L.T ao and T.A.Desai, Adv. Drug Deliv. Rev. 55, 315 (2003). 18. S.B.Lee and C.R.Martin, J. Am. Chem. Soc. 124, 11850 (2002). 19. M.Nishiza wa, V.P .Menon, and C.R.Martin, Science 268, 700(1995). 20. C.R.Martin and P.K ohli, Nature Rev. Drug Discov. 2, 29(2003).

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