Prosthesis And Medical Applications Of Polymers

Gizem Sagay

Nanomachınes and bıomaterıals

1. Nanotechnology

The prefix "nano-" (from the Greek root nanos, or dwarf) means one-billionth (10^-9–comparison of length scales are shown in Fig.1 ) of something. The term "nanotechnology" refers most generally to technology on the scale of a billionth of a meter, or a nanometer (a nanometer is ~6 carbon atoms wide). Similarly, the words "nanomachine," "nanorobot," "nanomotor" and "nanocomputer" may refer to complex engineered objects fabricated by positioning matter with molecular control.

This emerging field of research is aimed at increasing control over material structures of nanoscale size (1 to 100 nanometres) in at least one dimension. Nanotechnology, therefore, comprises a cluster of emerging techniques combined from physics, chemistry, biology, engineering and microelectronics that are capable of manipulating matter at the minutest levels of detail - the nanoscale. This discipline will, over the coming decades, result in the development of new scientific knowledge and new technologies in areas ranging from ICT to medicine and biotechnology.

Fig. 1 Comparison of length scales

1.1. NANOMACHINES

Human molecules are nanomachines. The greatest example of that power at present is in every living thing. Every blood cell for example is full of thousands of "little machines" that move around in the liquid world of the cell doing the business of life -- enzymes, hormones, RNA, and DNA. These little machines are molecules. They range in size from about one to several tens of nanometers across - they are like nanomachines.

A typical medical nanodevice might be a minute robot assembled from nanoscale parts. A typical future nanomedical treatment (e.g. to combat a bacterial or viral infection) could consist of an injection of nanorobots suspended in fluid. Some nanorobots would be intended to travel through the bloodstream to their target; others would be nonbloodborne tissue-traversing nanorobots, alimentary or bronchialtravelling nanorobots. Each species of medical nanorobot would be designed to accomplish a specific task, and many shapes and sizes would be possible. As a syringe is today used to inject medication into the patients' bloodstream, tomorrow, nanorobots could transport and deliver chemical agents directly to a target cell. Nanorobots could find and repair damaged organs, detect and destroy a tumour mass. They would be able to communicate their positions, operational statuses, and the success or failure of the treatment as it progresses. They would tell you their physical coordinates in the body, so you know where they are. They would tell you how many cancer cells they have encountered and inactivated.

The ability to swim or crawl. Some medical nanodevices may have mobility - the ability to swim through the blood, or crawl through body tissue or along the walls of arteries. Others could have different shapes, colours, and surface textures, depending on the functions they would be intended to perform. They would have different types of robotic manipulators, different sensor arrays. Each medical nanodevice could be designed to do a particular job extremely well, and would have a unique shape and behaviour. They would have only temporary residence in the body, and the immune system response (biocompatibility) would in principle be no more difficult to manage than for medical implants generally. There are many useful power sources for the nanodevices readily available in the human body and many different ways to communicate with the machines as they do their work. Each nanorobot would have its own computer and sensor to receive the physician's

messages and compute and implement the appropriate response. And a navigational network could be installed in the patient's body, with stationkeeping navigational elements providing high positional accuracy to all passing nanorobots

that interrogate them, wanting to know their location.

Nanoscale materials technology has already found widespread use in medicine, including biocompatible materials and analytical techniques, surgical and dental practice, nerve cell research using intracellular electrodes, biostructures research and biomolecular research using near-field optical microscopy, scanning-probe microscopy and optical tweezers, and vaccine design, and also many 20th century bulk chemical and biochemical manufacturing techniques along with much of classical pharmacology.

As for "biotechnology," the original meaning of this word contemplated "the application of biological systems and organisms to technical and industrial processes". In recent times, the field has expanded to include genetic engineering and now takes as its ultimate goal no less than the engineering of all biological systems, even completely artificial organic living systems, using biological instrumentalities.

The third branch, molecular nanotechnology, takes as its purview the engineering of all complex mechanical systems constructed from the molecular level potentially offering new tools for medical practice, the principal subject of this book. Observes G.M. Fahy, "the difference between nanotechnologists and biotechnologists is that the former do not restrict themselves to the biological limitations of the latter, and they are much more ambitious about the kinds of accomplishments that they want to achieve."

It is possible to imagine a molecular nanotechnology that uses mechanical nanorobotic systems. Such systems will have many constitutional differences from biological-based systems. For instance, mechanical systems will transport parts, materials, energy and instructions via fixed channels, whereas most (but not all) biological systems operate by diffusion. Mechanical systems will have structures constrained by specific geometries, whereas biological systems have structures defined by patterns of containment and interconnection the shape of a membrane compartment in a cell matters less than its continuity and the contents of the volume it defines. Mechanical systems will be deterministically manufactured by operations analogous to manual construction, whereas engineered ribosomes will self-assemble via diffusion and stochastic matching of complementary parts; in other words, biology uses recipes, while mechanical systems use blueprints. Cells grow, with their parts adapting to one another; mechanical nanorobots may be constructed from parts of fixed structure. Biology uses self-repair; mechanical systems generally do not largely because self-repair (by component exchange) will not be needed in molecular mechanical systems, whose designs may be made simpler by relying upon high component redundancy. These and other differences imply a number of important advantages that mechanical-based medical systems will enjoy over biological-based medical systems, which, taken together, strongly suggest that predominantly mechanical nanosystems may be the approach of choice for a mature medical nanotechnology.

2. BRIEF HISTORY OF NANOMACHINES

Nanotechnology was first proposed by Nobel physicist Richard Feynman in December 1959, in a talk in which he also issued a seemingly "impossible" challenge to build a working electric motor no larger than a 1/64thinch (400micron) cube, backed by a $1000 prize to spur interest in the new field. Just 11 months later, engineer William McLellan had constructed a 250microgram 2000rpm motor out of 13 separate parts and collected his reward. (McLellan's entire motor is only as big as the period at the end of this sentence.) In 1995, a $250,000 Feynman Grand Prize was made available, this time sponsored by theForesight Institute, for any engineer who could build the firstprogrammable nanometerscale robotic arm.

In the 1980s and 1990s, a handful of authors began speculating about the physical forms that future medical nanorobots might take. A few created artist's conceptions of their devices. During this time, only the broadest analyses of the missions and capabilities that might be desired had been attempted. Detailed technical and engineering studies, in many cases, still lay years in the future.

Images are provided from both the mechanical and biological traditions of medical nanotechnology, although there is a primary emphasis on the former. Two examples are shown below.

T4 Bacteriophage

Cell Repair Machines I

Progress is being made on Feynman's topdown approach in a relatively new engineering field known as Micro ElectroMechanical Systems or MEMS, originally an extension of chipetch technology rather than micromanipulation. Conventional purelyelectronic devices fabricated on silicon chips with ~0.2 micron features must be coupled to external systems to produce mechanical effects, but MEMS devices integrate mechanical components directly withelectrical circuitry. Thus while microelectronic chips merely route electrons, microelectromechanical systems give the electronicsimmediate access to control applications, enabling single microdevices to interact directly with the physical world.

Today, the proposed research is aimed at developing a new class of biologically inspired robots that exhibit much greater robustness than today’s robots for performing in unstructured environments. This new class of robots will be substantially more compliant and stable than current robots, and will take advantage of new developments in materials, fabrication technologies, sensors and actuators. Applications will include autonomous or semi-autonomous tasks such as reconnaissance and de-mining for small, insect-like robots and human interaction tasks at a larger scale. The research involves a close collaboration among robotics and physiology.

3. Technical Description Of the New Class of Biologically Inspired Robots

Consider a crab scuttling through the surf zone and grabbing some prey. Although the crab has a primitive brain and motor control system it accomplishes these tasks robustly, and in an extremely challenging environment that would quickly damage most robots, or at least leave them stymied. The point here is that if we are to develop robots that can survive in unconstrained environments such as the surf zone for de-mining beaches, or the ocean floor for retrieving wreckage, we must adopt some of the same strategies that crabs and other animals employ for survivability and robust task execution.

In contrast to the crab, today's robots are built like miniature Behemoths, with metal links, ball bearing joints and steel cables powered by DC motors. The mechanisms are stiff and very under damped. Much of the control effort is expended on achieving precise endpoint position or force control, using a small number of high-accuracy sensors. As a result, robots can surpass human performance for highly constrained tasks but cannot function in unconstrained environments as well as the simplest of animals.

Self-stabilization by limb morphology. Full (1989) has shown that the most successful animals on Earth – the arthropods - are actually “soft”. Both the number and arrangement of segments as well as segment stiffness appear to play a role. Rigid segment, statically stable models simply can’t explain agile running, turning, climbing, and recovery from perturbations.

Fig.2

It is a fact that many-legged, sprawled postured animals are highly statically stable (Ting et al., 1994). Most possess a wide base of support and a low center of mass. They often have at least three legs on the ground. Yet, the locomotion of polypedal, sprawled postured animals must be considered a rapid, repetitive, gross behavior. Therefore, the dynamics found in nature is the greatest inspiration source in making a design.

3.1. Is Molecular Manufacturing Possible?

Most contemporary industrial fabrication processes are based on "topdown" technologies, wherein small objects are sawn or machined from larger objects, or small features are imposed on larger objects, in either case by removing unwanted matter. The results of such processes may be small, such as micronfeatured integrated circuits, or very large, such as jet aircraft, but in most cases the material is being processed in chunks far larger than molecular scale.

Molecular manufacturing, on the other hand, represents a "bottomup" technology. Desired products will be built directly by "assembler" machines, molecule by molecule, making larger and larger objects with atomic precision. The results of such processes may also be very small or very large, much as biology builds both micronsized bacteria and 100meter tall sequoia trees. However, since assemblers add matter only where it is intended, little need be removed and hence there may be minimal waste during the process. By guiding with precision the assembly of molecules and supra-molecular structures, such a manufacturing system could construct an extraordinarily wide range of products of unprecedented quality and performance.

4. NANOTECHNOLOGY AND BIOMATERIALS

The last decade has seen the thrilling development in material science in the nanometer scale. Nano- sized semi-conductors with well-tailored electrical, optical or mechanical properties have been synthesized. Recently, people start to realize the exciting potential of biomolecules in nanotechnology. Characterized by their high binding affinity and specificity, biomolecules are ideal for providing control in organizing technologically important (non-biological) objects into functional material. Theoretical calculations, which have been used extensively in the studies of biomolecules and inorganic materials separately, are expected to play a leading role in the understanding of such issues. Computational work can also predict or help us to understand the dynamical, optical and other properties of those hybrid, which potentially lead to the design of new materials.

A confluence in scientific advancements associated with molecular biology and nanofabrication technology now offer, for the first time, the potential of engineering functional hybrid organic/inorganic nanomechanical systems. Below, examples of the newest achievements are explained in detail.

5. DISCUSSIONS ABOUT NANOROBOTS

As their production and design are very recent, researchers have some concerns about this new invention. Some of them are discussed below.

5.1. Will Serum Proteins Stick to Nanorobot Surfaces?

When an artificial nanoorgan is implanted in the body, it may be desirable to promote rapid adhesion to cells and tissues. But for medical nanorobots floating or swimming in the circulation such adhesion will not normally be desirable. Thus one preliminary question is whether sticky biological "gunk" will adhere to the surface of a diamondoid nanorobot when it is placed in the bloodstream, and if so, what can be done about it?

When a foreign material is implanted into a host tissue, the first event to occur at the tissue-material interface which dictates biocompatibility is the noncovalent adsorption of plasma proteins from blood onto the surface. Protein adsorption is much more rapid than the transport of host cells to foreign surfaces. Once proteins have adsorbed to the surface of the foreign material, host cells no longer see the underlying material, but only the protein-coated surface overlayer. This adsorbed protein overlayer -- rather than the foreign material itself - then mediates the types of cells that may adhere to the surface, which ultimately can determine the type of tissue that forms in the vicinity. Thus the type and state of adsorbed proteins, including their conformational changes, will be critical determinants of biocompatibility , including nanorobot-cell interactions and nanorobot surface fouling.

An effective way to create nonadhesive nanorobot surfaces may be the biomimetic approach. For example, the external region of a cell membrane, known as the glycocalyx, is dominated by glycosylated molecules, which direct specific interactions such as cell-cell recognition and contribute to the steric repulsion that prevents undesirable non-specific adhesion of other molecules and cells. Scientists have modified a pyrolytic graphite surface by attaching oligosaccharide surfactant polymers which, like a glycocalyx, provides a dense and confluent layer of oligosaccharides that mimics the non-adhesive properties of a glycocalyx. The surfactant polymers consist of a flexible poly(vinyl amine) backbone (MW ~ 6000 daltons, diameter 0.25 nm) with multiple randomly-spaced dextran (MW ~ 1600 daltons, diameter ~0.9 nm) and alkanoyl (hexanoyl or lauroyl) side chains which constrain the polymer backbone to lie parallel to the substrate. Solvated dextran side chains protrude into the aqueous phase, creating a glycocalyx-like monolayer coating 0.7-1.2 nm thick as measured by tapping-mode AFM. Dextran has a stable helical structure; steric repulsion between adjacent dextrans is believed to produce a brush-like conformation. In vitro experiments show that the resulting biomimetic surface, which the authors assert undergoes spontaneous adsorption on diverse hydrophobic surfaces, is effective in suppressing at least ~90% of all plasma protein adsorption from human plasma protein solution

5.2. Nanomedicine: Is Diamond Biocompatible With Living Cells?

The exteriors of many medical nanorobots and nanorobot aggregates may be made of diamond. That's why the biocompatibility of diamond surfaces and diamond particles is of considerable interest in nanomedicine. Some nanomedical applications will demand a nonadhesive interface, while other applications may require complete tissue integration with the nanodevice, using biocompatible surfaces of engineered bioactivity, probably including nanostructured materials able to promote and stabilize cell attachment. Atomically-precise diamond surfaces aren't readily available, so cell responses cannot yet be seriously investigated. However, the biocompatibility of comparatively rough, bulk manufactured diamond surfaces has been addressed experimentally by a handful of researchers — for example, in connection with diamond-coated orthopedic prostheses already proposed, developed, or in clinical use.

In biomaterials research, it has been found that even though a bulk material may be well-tolerated by the body, finely divided particles of the same material can often lead to severe and even carcinogenic complications in test animals. Differences in particle size influence histological reaction and cytokine production. Many nanomedical applications will involve "particle" sized diamondoid objects (e.g., micron-scale individual medical nanorobots), so it is of great interest to review the experimental data relating to the reactions of specific cells to the presence of diamond particles. We already know that finely divided carbon particles are well-tolerated by the body — the passive nature of carbon in tissue has been known since ancient times, and both charcoal and lampblack (roughly spherical 10-20 nm particles) were used for ornamental and official tattoos.

Thus it appears that diamond is extremely — indeed outstandingly — biocompatible with living cells.

5.3. Could Medical Nanorobots Be Carcinogenic?

Biocompatibility is an important property that must be carefully engineered into all medical nanorobots used in nanomedicine . One key aspect of biocompatibility is whether implanted nanoorgans, or in vivo medical nanorobots, might induce undesirable genetic changes as a side effect of their presence or activities inside the human body. Such undesirable changes might take many forms. For instance, mutagenicity is the production of inheritable coding flaws in chromosomes that otherwise may retain much genetic functionality. (All carcinogens are mutagens but not vice versa – a mutation may be lethal to a cell, may prevent cellular replication, or may not affect metabolic or growth processes sufficiently to produce malignant behavior .) Genotoxicity is a more serious injury to the chromosomes of the cell, such that when the cell divides, fragments of chromosomes and micronuclei remain in the cytoplasm. Teratogenicity is the ability of a foreign material (or a fetotoxic agent) to induce or increase the risk of developing abnormal structures in an embryo, or birth defects. Carcinogenicity is the ability to produce or increase the risk of developing cancer – materials may be directly carcinogenic or may potentiate other agents. Tumorigenic materials tend to induce neoplastic transformations, especially malignant tumors.

Direct experimental exploration of the carcinogenicity of likely nanorobot building materials has barely begun, but information available to date appears guardedly optimistic. For example, diamond (DLC) coatings exhibit low mutagenicity toward human fibroblasts in vitro and there are no reports of diamond carcinogenicity or tumorigenesis. Alumina (sapphire) produces no mutagenic or carcinogenic effects on cultured human osteoblasts or when used as a blood-contacting material in a centrifugal blood pump ; while aluminum ion leached from sapphire at the highest plausible concentrations (~10^-5 M; ) might inhibit eukaryotic transcription, experiments suggest that the mutagenicity, carcinogenicity, and teratogenicity of aluminum is low . Teflon particles appear to be noncarcinogenic , even though tetrafluoroethylene (a monomer used in Teflon manufacture) is hepatocarcinogenic after long-term inhalation by mice. There are no reports of carcinogenicity from pyrolytic carbon, graphite, or pure India ink in humans .In rodents, the inhalation of carbon black particles can produce pulmonary neoplasms and lung carcinoma and particle-elicited macrophages and neutrophils can exert a mutagenic effect on in vitro rat epithelial cells .

Medical nanorobots normally will have chemically-inert nonleachable surfaces, but designers should ensure that all possible nanorobot effluents are noncarcinogenic

6. RECENT ACHIEVEMENTS

Selected examples of the newest interesting achievements are explained below which are molecular motors, ATP-driven rotary biomolecular nanodevice for arterial blood clot removal and constructing biological motor powered nanomechanical devices.

6.1. MOLECULAR MOTORS

The final aim for the researches in this field is to produce extremely small, self-propelled bionic machines that do their builders' bidding in plant and animal cells, including those in humans.

Such machines could travel through the body, functioning as mobile pharmacies, for example, dispensing precise doses of chemotherapy drugs exclusively to cancer cells.

The engineers' breakthrough is in integrating a living molecular motor with a fabricated device at the "nano" scale, a few billionths of a meter in size. The first integrated molecular motor, a molecule of the enzyme ATPase coupled to a metallic substrate with a genetically engineered "handle," ran for 40 minutes at 3 to 4 revolutions per second, Carlo Montemagno and George Bachand report in the September issue of the journal Nanotechnology (Vol. 10, No.3).

Molecular motors are hardly new. For billions of years they have been nature's way of accomplishing life's essential tasks at the atomic level. The ATPase molecular motors are found in the membranes of mitochondria, the microscopic bodies in the cells of nearly all living organisms, as well as in chloroplasts of plant cells, where the enzyme is responsible for converting food to usable energy. The moving part of an ATPase 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).

The ATPase molecules were produced by Escherichia coli bacteria that were genetically engineered to include a gene sequence from the thermophilic bacterium Bacillus PS3. With further genetic manipulation, engineers expect E. coli to turn out ATPase molecules with tiny propellers -- making each a kind of nano-motorboat -- as well as other useful structures, predicts Montemagno, an assistant professor of agricultural and biological engineering.

ATP (adenosine triphosphate) is the fuel for the molecular motor's motion. Energy becomes available when atomic bonds between phosphate atoms are broken during hydrolysis to convert ATP into ADP (adenosine diphosphate). During ATP hydrolysis, the tail rotates in a counterclockwise direction; it rotates clockwise during ATP synthesis from ADP.

Engineers tagged the ATPase molecule's rotor with fluorescent microspheres that are 1 micron (1 millionth of a meter) in diameter and observed microsphere movement with a differential interferometer and with a CCD (charge-coupled device) kinetics camera.

The "handle" for attaching the ATPase motor to the nanofabricated metallic substrates is a synthetic peptide composed of histidine and other amino acids. The histidine peptide allows the molecular motors to adhere to nanofabricated patterns of gold, copper or nickel -- the three standard contact materials in integrated circuits. The patterned metal substrates were created by evaporative deposition which specializes in creating structures a few nanometers in size.

With the integration of biomolecular motor devices and cell-signalling systems -- by engineering a secondary binding site tailored to a cell's signalling cascade cascade -- researchers plan to use the cell's sensory system to control nanodevices implanted in living cells.

Besides demonstrating that an organic molecular motor and an inorganic, nanofabricated device can be integrated, the system provides a platform for studying fundamentals of ATPase's operation, which is not fully understood. The device will have more brawn than brains until the molecular motors are attached to more advanced nanofabricated devices that can provide directions.

Integration of biological motors and NEMS

Platforms for the production of both biomolecular motors and NEMS must be established in order to integrate these technologies and produce hybrid systems. Initial research efforts, therefore, have focused on the development of these platforms. In addition, we also have begun evaluating the engineering properties of F₁-ATPase.

6.2. Constructing Biological Motor Powered Nanomechanical Devices

INTRODUCTION

Scientific advancements in both molecular biology and nanofabrication technology now provide the potential of engineering functional hybrid organic/inorganic nanomechanical systems. Scientists have been studying a wide range of organic molecular motors for some time. Concurrently, inorganic, primarily silicon based, micromechanical devices have been pursued as useful devices. Only very recently has the size scale of nanofabricated inorganic mechanical devices approached a size scale that could conceivably be compatible with the force production and dimensions of molecular motors.

The rotary motion of the ỵ subunit of F₁-ATPase in response to the synthesis/hydrolysis of ATP has been previously demonstrated (Noji et al., 1997). The force generated by this motor protein was >100 pN, which is among the greatest of any known molecular motor. With a calculated no-load rotational velocity of 17 r.p.s. and a diameter of less than 12 nm (Sabbert et al., 1996; Yashuda et al., 1997), the F₁-ATPase protein is a tailor made nano-motor. These properties, coupled with the fact F₁-ATPase is automatically synthesized using the machinery of life, opens the door to the potential for creating chemically powered nanomechanical devices. Integration of nanoscale biocompatible lithographic processes with biological molecular motors may provide the means for creating a transparent interface between the organic/inorganic world. Insertion and self-assembly of hybrid ATPase-powered NEMS in host cells may be possible by taking advantage of cell physiological processes. In addition, host cells may provide power for the device in the form of ATP, as well as maintain a system for replacing the molecular motors when function ceases.

ATPase is a ubiquitous enzyme that is found in virtually every living organism. It consists of two separate portions: (1) F₀, the hydrophobic, membrane-bound portion that is responsible for proton translocation, and (2) F₁, the hydrophilic portion that is responsible for ATP synthesis and hydrolysis. As protons flow through the F₀, the y subunit of the F₁-ATPase rotates clockwise and ATP is synthesized. Hydrolysis of ATP results in counterclockwise rotation of the y subunit, and drives the reverse flow of protons. The a, b, and c subunits of the F₀-ATPase form the channel which allows protons to flow through the membrane. The nucleotide binding and catalytic sites are located on the three a and three b subunits of the F₁-ATPase, respectively (Kinosita et al., 1998). The y subunit is centrally located within the hexamer, and rotates as a function of ATP synthesis/hydrolysis.

ATPase molecule and membrane

During hydrolysis, counterclockwise rotation of the y subunit provides interaction with all three forms of the y subunits in the order: AMP-PNP > ADP > empty form. The exact mechanism of interaction has yet to be determined. Crystallization of the F₁-ATPase has revealed that all three sites must contain bound nucleotides in order for rotation of the y subunit to occur (Bianchet et al., 1998). Further, the y subunit is displaced from its central axis during rotation.

counterclockwise rotation of the gamma subunit

Despite the superb performance of the F₁-ATPase motor protein, little is truly known about how this enzyme generates rotary motion. Neither the useful life of the motor nor the impact of local environmental variables such as pH and temperature on enzyme activity has been determined. The impact of motor generated waste products (i.e., protons and heat), as well as the effects of load on the performance and life of the motor need to be identified. A rigorous evaluation of the engineering properties of the F₁-ATPase motor protein necessitates the development of assays that will provide consistent measurements of the performance of the F₁-ATPase motor protein under different operating conditions. Our current research effort has focused on integrating the F₁-ATPase with NEMS specifically designed to evaluate motor performance. We will present the results of this effort to construct a hybrid organic/inorganic nanoscale system that both provides insight into the basic mechanics of motor protein motion and establishes a technological foundation for functionally integrating these molecules with manufactured devices.

Integration of Biological Motors and NEMS

Attachment of Biological Molecules to Nanofabricated Substrates

In order to integrate biomolecular motors into NEMS, procedures for the specific attachment and positioning of these motors is essential. Therefore, the objective of this experiment was to evaluate the binding of biological molecules to nanofabricated substrates. Electron beam lithography was utilized to etch an array pattern on a 25 mm coverslip that had been coated with a resist bilayer. Coverslips then were patterned with metal substrates using evaporative deposition of gold, copper, or nickel. Subsequently, the bilayer was removed to expose the array.

A six His-tag peptide was covalently coupled to carboxylate-modified 1 mm fluorescent microspheres using a water-soluble carbodiimide. The His-tagged microspheres were allowed to attach to gold-, copper-, and nickel-coated coverslips for 15 minutes at room temperature. Unattached microspheres were removed through a series of washes, and coverslips were observed using fluorescence microscopy. His-tagged microspheres attached to all three substrates; however, attachment was most frequently observed with nickel-coated coverslips.

To test the strength of attachment, laser tweezers were used to remove the microspheres from the substrate. The laser tweezers, however, were unable to remove microspheres from any of the three substrates suggesting that the bonding strength was greater than 600 pN. Further attempts to remove the microspheres with high velocity flow suggest that the bonding strength increases from gold to unoxidized copper to nickel. Oxidized copper does not serve as a suitable surface for binding of His-tagged microspheres. These experiments demonstrate a chemical mechanism for protein binding and positioning to engineered structures that are compatible with current nanofabrication technologies. Using this knowledge in conjunction with standard e-beam lithographic methods (Craighead and Mankiewich, 1982; Lercel et al., 1995; Lercel et al., 1996; Carr and Craighead, 1997) we can now attach individual motor protein molecules with a precision greater than 30 nm.

Attachment and Movement of Individual Biomolecular Motors

Although the biological and chemical aspects of F1-ATPase have been studied, relatively little is know about the engineering properties of this enzyme. The objectives of this experiment were to: (i) attach F₁-ATPase to a nanofabricated substrate, and (ii) measure the rotational velocity and angle of deformation of the y subunit. Analysis of crystallized F₁-ATPase suggests that the y subunit is displaced from the central axis during rotation a distance >20 Å (Bianchet et al., 1998). By attaching a 1 mm microsphere to the y subunit, the displacement and angle of deformation of the y subunit can be determined by measuring the radial displacement of the microsphere. The angle of deformation will provide valuable insight on the mechanism behind rotation of the y subunit.

The y subunit of the recombinant F₁-ATPase was specifically biotinylated through disulfide linkage to the yCys. The biotinylated protein then was attached to an array of 30 nm gold dots deposited on a coverslip. Fluorescent 1 mm microspheres coated with streptavidin were allowed to bind to the biotinylated y subunits. Subsequently, unattached microspheres were removed through a series of washes. Rotation of the y subunit was initiated by the addition of 2 mM Na₂ATP in presence of 4 mM MgCl₂. Movement of the microsphere was measured using a differential interferometer (Denk and Webb, 1990; Stelick et al., 1998). Images of microsphere movement were also captured at 1 msec intervals using the CCD kinetics camera.

Conclusions

Biological and nanofabrication platforms have been established for the production of organic/inorganic hybrid NEMS. Because of its size and force generation, F₁-ATPase serves as an excellent model system for evaluating the use of biomolecular motors in these hybrid devices. The biological platform that has been established allows us to: (1) easily manipulate the coding sequence, (2) produce large quantities of protein, and (3) place specific "handles" on the enzyme for the precise attachment to nanofabricated devices and substrates. Moreover, the presented nanofabrication platform permits both the construction of chemically active sites that are consistent with the size of the protein and the development of devices that are capable of translating the energy of a biomolecular motor into useful work. These results represent the enabling technologies necessary for integrating NEMS into living organisms.

Further investigation of the engineering properties and motor performance are necessary for the production of functional nanomechanical devices powered by F₁-ATPase. For example, the impact of waste products such as heat and protons on motor performance must be evaluated. Motor performance must be evaluated as a function of heat, pH, load, and local environmental conditions. Moreover, dissecting the interaction between the subunits of the complex may allow us to specifically engineer the protein for increased performance as a biomolecular motor. These efforts will provide a significant step toward the seamless integration of nanoscale technologies into living system, and are central to the creation of organic/inorganic intelligent systems.

ATP-driven Rotary Biomolecular Nanodevice for Arterial Blood Clot Removal

Recent developments in nanotechnology have uncovered the possibility of creating a nanoscale device that may be able to mechanically grind out arterial clots caused by atherosclerosis. Several naturally-occurring biomolecular motors have been studied. These motors are powered by ATP, which is completely safe for the body, and they are extremely efficient. The different kinds of motors are discussed below. In addition, new developments in machining nanoscale devices create seemingly endless possibilities for applying these nanomotors.

Types of Biomolecular Motors

Biomolecular nanomotors are devices constructed on a nanoscale that convert free biochemical energy into mechanical work. Two main types of biomolecular motors are the actin-based or microtubule-based linear motors, and rotary motors.

The actin-based sliding motor is the mechanism used in skeletal muscle to produce movement. A simplified model of this motor involves two types of filaments, actin and myosin, that slide over one another. The thin actin filaments are composed of protein monomers linked together to form chains. The thicker myosin filaments contain many active head units that interact with actin to form cross-bridges. The myosin motor molecules in the head groups are energized by the dephosphorylation cycle of ATP, shown in Figure 2, and work as a team to pull the actin rope. More specifically, ATP fuel packets are loaded onto the myosin molecules, P_i is cleaved from the ATP, and mechanical motion of the myosin results. The myosin then reattaches to another ATP molecule, and the cycle is repeated (Patterson). A summary of the movement of myosin heads on actin filaments is illustrated in figure 3.

Figure2: ATP cycle of myosin heads binding to actin.

Figure 3: The Movement of Myosin Heads

The second type of linear motor, made of kinesin and dynein, is found in all eukaryotic cells. Both kinesin and dynein facilitate movement of organelles along microtubules during cell division; dynein also facilitates locomotion of entire cells with cilia and flagella (Alberts, p. 813). Microtubules are straight, hollow rods which serve as the tracks along which molecular motors can move (Campbell, p. 120). Kinesin and dynein motors carry out ATP dependant movement along microtubules, the former away from the centrosome (anterograde) and the latter towards it (retrograde) (Lodish).

Kinesin proteins have two copies each of a heavy and light chain. Each heavy chain includes a globular ATP binding motor domain at the N-terminus and C-terminus implicated in binding to cargo proteins. The activation of kinesin is promoted by phosphorylation, of ATP, followed by the binding of a cargo vessel to kinesin through a scaffolding protein (see figure 4). The kinesin then slides along the microtubule. The kinesin reaction cycle differs from that of myosin in that kinesin binds tightly to microtubules when it has bound ATP, while myosin dissociates from actin upon binding ATP (Lodish).

Fig. 4: The Activation of Kinesin and the Binding of Cargo Vesicles for Travel on Microtubules

Device Summary

The device designed in this project consists of a liposome embedded with six motor-nanotube-blade assemblies and with multiple KGD sensor sequences, as shown in figure 5. The sensors are used to bind the device to a clot so that grinding can be targeted. A motor-nanotube-blade assembly is shown in figure 6. A nickel blade connected to a carbon nanotube shaft with benzene gears is rotated by six ATP synthase complexes, all attached to carbon nanotubes with benzene gears via biotin-streptavidin linkages. The motor-nanotube-blade assemblies are held together with in a graphite plate to add structural stability. To improve biocompatibility, the nickel blade is coated with titanium, and all parts are coated with PEG and superoxide dismutuse.

Figure5. A Cross-section of the Complete Nanogrinder

Figure 6: The Motor-Nanotube-Blade Assembly

The Biomolecular Motor – ATP synthase

Adenosine triphosphate is the biochemical compound responsible for storing chemical energy in a fashion so that the stored energy is readily available to power cellular functions. The energy stored in ATP is released by the hydrolysis reaction below:

ATP + H₂O Þ ADP + P_i

The above reaction, which uses water to cleave a phosphate from ATP to form adenosine diphosphate, releases 30 kJ/mol of energy under standard biochemical conditions (Nelson, p. 679). Since the forward reaction is so favored in terms of Gibbs free energy, the conditions necessary to produce ATP for cellular use should be excessively harsh. The body cannot function at extreme conditions, so a catalytic pathway using coupled reactions is used to produce ATP instead of harsh conditions. ATP synthase is the fifth and final component of the body’s catalytic coupled reaction pathway. Figure 7 below shows the ATP producing complex found in the inner membranes of the cell’s mitochondria.

Overview of Oxidative Phosphorylation

Fig. 7: The Body’s ATP producing system

7. FUTURE

Plans for commercializing nanotechnology range from the development of nanoscale electronics and computing applications to the creation of molecular machines and manufacturing capabilities at the nanometer level. But most companies fall in the materials area, producing organic, inorganic, and metal nanomaterials.

Anyone who cares about the future should buy Nanosystems, for this is the basic premise of nanotechnology: the future matters and is ours to create. Whether we create well or badly, we and our heirs must then live in our creation. To create what is desirable we must understand what is achievable. Nanosystems gives us a clear and in-depth preview of a rich new vein of the achievable.

8. REFERENCES