New Technology and Clinical Applications of Nanomedicine

Med Clin N Am 91 (2007) 845–862

New Technology and Clinical Applications of Nanomedicine Lian Zuo, PhDa, Wenchi Weib, Michael Morrisb, Jinchi Weib, Mikhail Gorbounovb, Chiming Wei, MD, PhD, FACC, FAHA, FAANb,* a

Department of Cardiology, Emory University School of Medicine, 101 Woodruff Circle, WMB319, Atlanta, GA 30322, USA b Department of Surgery, Johns Hopkins University School of Medicine, 600 N. Wolfe Street/Harvey 606, Baltimore, MD 21205, USA

Nanomedicine is the use of nanotechnology to achieve innovative medical breakthroughs. Although the term ‘‘nanotechnology’’ refers to a wide range of scientific projects involving phenomena or properties of the nanometer scale (w 0.1–100 nm), nanomedicine involves the application of these concepts and tools in clinical and basic medical sciences. We should not use too restrictive a definition of nanotechnology, and that a synergy exists between technology on the nano scale and processes at the micro and macro levels [1]. Although true, such usage may err in the direction of being too inclusive and, in effect, defining nothing. Accepting the premise that manipulations on the nanoscale level must clearly be a major component of the definition, nanotechnology can be understood as embracing the assembly, manipulation, and construction of nanostructures and their practical application to a range of scientific, commercial, and engineering issues. Nanotechnology is already an established discipline, but nanomedicine, with its broad range of ideas, hypotheses, concepts, and undeveloped clinical devices, is still in its early stage. Freitas [2] defines nanomedicine as ‘‘the monitoring, repair, construction and control of human biological systems at the molecular level, using engineered nanodevices and nanostructures.’’ Nanomedicine therefore adopts the concepts of nanoscale manipulation and assembly to clinical applications in medical sciences. Many nanodevices, such as quantum dots, are widely known and broadly marketed but have yet to find their way into a wide range of clinical devices. This situation * Corresponding author. E-mail address: [email protected] (C. Wei). 0025-7125/07/$ - see front matter Ó 2007 Elsevier Inc. All rights reserved. doi:10.1016/j.mcna.2007.05.004 medical.theclinics.com

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is a consequence of the extremely complex and demanding requirements of clinical trials, which may run for years before a product can make the long leap from a concept in the laboratory to a medical device in the clinic. Most accounts of the history and origins of nanotechnology begin in 1959 with Feynman’s [3] discussion outlining the idea of building tiny robots for constructing smaller and smaller machines. This brilliant suggestion did not receive much attention until the mid-1980s, when Drexler [4] published ‘‘Engines of Creation,’’ a popular treatment of the promises and potentials of nanotechnology. Drexler [4] envisioned a discipline of molecular nanotechnology that would allow manufacturers to fabricate products from the bottom up with precise molecular control. With this technology every molecule would be inserted in its specific place, so the manufacturing processes would be clean, efficient, and highly productive. These design and assembly systems would maintain much higher throughputs than modern manufacturing techniques, which use macroscale manipulators to fabricate products. Although a number of nanoassembly processes are in use today, many experts foresee large-scale molecular nanotechnology being used on a large scale between 2010 and 2020 [5]. Although many nanomedicine technologies are in the marketplace now, they are used principally for the detection of particles, in drug delivery systems, as emulsions and carriers for delivering vaccines, and for nanofabricated biomaterials that have unusual properties of strength, hardness, reduced friction, and improved biocompatibility. More exotic concepts, such as nanomachines that could move through the body, troubling-shooting and repairing tiny brain and cardiovascular lesions, lie in the future. Long-term goals of nanomedicine Although exciting in its own right, the present status of nanomedicine is only a milestone on the road to introducing truly innovative technologies. These advances will come about only over a period measured in decades, because of the complexity of clinical trials and the conservatism with which radical technologies are adopted. To move nanomedicine forward, the National Institutes of Health (NIH) has proposed an ‘‘NIH Roadmap’’ to address the barriers and gaps in knowledge that currently constrain progress in biomedical research [6]. The Roadmap will bring together many NIH Institutes and Centers to focus and consolidate research and development in nanomedicine. NIH Roadmap funding will be allocated for three programs: (1) New Pathways to Discovery, a research program that will pursue a comprehensive understanding of the body’s cells and tissues and the operation of complex biologic systems, using the combined tools of structural biology, molecular libraries, imaging, bioinformatics,and computational biology; (2) Research Teams of the Future, a program focusing on interdisciplinary, high-risk research through public–private partnerships; and (3) Re-engineering the

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Clinical Research Enterprise, a program advancing these discoveries into the clinical sphere [7]. The Roadmap concepts were developed by the NIH in consultation with its professional staff and the public to identify and prioritize the most pressing problems facing medical research today. The initiatives were selected because of their potential for having the most significant impact on the progress of medical research. Through the NIH Roadmap, the NIH aims to accelerate the application of new knowledge to the development of new prevention strategies, diagnostics, and treatments and transfer these developments into the public domain. Specifically [8]  Research centers on nanomedicine will help scientists construct synthetic biologic devices, such as miniature, implantable pumps for drug delivery or sensors to scan for the presence of infectious agents or metabolic imbalances that could signify disease.  Nanomedical approaches will be used to quantify better clinically important symptoms and outcomes, including pain, fatigue, and quality of life, that now are difficult to measure. New technologies will be developed to measure these self-reported health states and outcomes across a wide range of illnesses and disease severities.  A cadre of NIH clinical research associates will be established, composed of community-based practitioners who will receive specialized training in clinical research. These individuals will aim to advance the discovery process and to disseminate research findings to the community.  A standardized data system, the National Electronic Clinical Trials and Research network, will be developed to facilitate the sharing of data and resources and to augment research performance and analysis. The NIH Roadmap builds on the progress in biomedical research achieved through the recent doubling of the NIH budget. It reflects a shift to adaptive management of the NIH portfolio to enable rapid responses to emerging needs and opportunities that do not fit clearly within the mission of the traditional grouping of Institutes and Centers. A unique aspect of the NIH Roadmap is the NIH Director’s Pioneer Awards that will enable highly talented, ingenious scientists to pursue ‘‘high-risk–high-reward’’ research. The review process for this new grant mechanism will emphasize the creativity and scientific potential of the person, rather than the project, thus providing a new way of supporting individuals who show the most promise for making seminal contributions to medical research [9,10]. Basic nanomedicine for cellular and molecular dynamics in living cells During the past few years, fluorescent semiconductor nanocrystals (also known as quantum dots) have been tested in most biotechnologic applications

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that use fluorescence, including DNA array technology, immunofluorescence assays [11], and cell and animal biology. Quantum dots tend to be brighter than dyes because of the compounded effects of extinction coefficients that are an order of magnitude larger than those of most dyes [12,13]. Their main advantage resides in their resistance to bleaching over long periods of time (minutes to hours), allowing the acquisition of images that are crisp and well contrasted. This increased photostability is especially useful for three-dimensional optical sectioning, in which bleaching of fluorophores during acquisition of successive z-sections compromises the correct reconstruction of three-dimensional structures. In addition, submicrometer studies of cell ultrastructure have been performed with scanning electron microscopes, transmission electron microscopes, and atomic-force microscopes. Living cells cannot be examined with the first two instruments, however, because those systems require cell fixation and observation in vacuum. Images of living cells obtained with the atomic force microscope may be compromised by direct contact between the cantilever and the sample-deforming soft tissue. High-resolution analysis of activities of live cells is limited by the need to use noninvasive methods. Apparatuses such as scanning electron microscopes, transmission electron microscopes, and atomic force microscopes are not practicable because the necessary processing or the harsh contact with the system probe disturbs or destroys the cell. Optical methods are noninvasive, but they usually are diffraction limited, so their resolution is limited to approximately 1 mm. To overcome these restrictions, the scanning near-field optical microscope (SNOM) was developed for the study of membrane activity in a live cell sample [14]. A near-field optical microscope can detect tiny vertical movements on the cell membrane in the range of 1 nm or less, about three orders of magnitude better than with conventional optical microscopes. It is a purely noninvasive, noncontact method, so the natural life activity of the sample is unperturbed. This methodology will open a new approach to investigate live samples. The extreme sensitivity of SNOM and specialized quantum dots allows measurements that are not possible with any other method on living biomaterial, paving the way for a broad range of novel studies and applications.

Experimental nanomedicine for in vitro and in vivo studies Efficient and selective delivery of genes or drugs to the vasculature Efficient and selective delivery of genes or drugs to the vasculature of tumors is a desirable therapeutic goal. Gene therapy requires the engineering of vehicles that can protect DNA from degradation, exhibit prolonged circulation times, bind efficiently to target cells, and deliver DNA to the nucleus. Synthetic nanoparticles have the potential to meet these criteria, although, currently, nonspecific tissue sequestration of nanoparticles often

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results in toxic side effects and significantly reduces their circulation time [15,16]. A recent report, however, has described the use of novel avb3-integrin–targeted nanoparticles to deliver a cytotoxic gene to tumor blood vessels in mice, with high selectivity and efficacy [17]. Hood and colleagues [17] have described a novel approach to targeted gene therapy of tumor endothelium. In humans, the toxicologic consequences of nonspecific tissue uptake of synthetic nanoparticles can be a problem [16]. Gene therapy with the gene encoding ATPm-RAF has the potential advantage that nonspecific expression should have low toxicity. A thorough characterization of the uptake of avb3-NP/RAF and the associated toxicity in a broad range of tissues is needed. Nanoparticle-mediated delivery of genes or drugs to human tumor endothelium will require refinements in particle chemical engineering and administration protocols to ensure that they are safe, exhibit prolonged circulation times, and have a maximal chance of reaching the target tissue [15,16]. Furthermore, efficient drug delivery will require target antigens that are highly and selectively expressed in tumor endothelium and that undergo a high rate of cell surface turnover. In addition, the choice of gene or drug to be delivered must be optimized to ensure maximal efficacy with minimal side effects. Finally, physiologic differences between species demand that caution be exercised in interpreting the results of animal experiments. Despite these provisos, Hood and colleagues [17] present a promising beginning for this novel anti-angiogenic approach. Pharmacokinetic and toxicologic analyses to determine its potential as a treatment for human cancers are awaited. avb3-Dependent uptake of nanoparticles in vitro and efficacy of nanoparticles in vivo avb3-Dependent uptake of nanoparticles in vitro Hood and colleagues [17] synthesized cationic lipid-based nanoparticles of 40 nm diameter with a synthetic ligand incorporated at the surface. This ligand selectively binds avb3 integrin, and uptake of avb3-NP is predominantly avb3-integrin specific. Efficacy of nanoparticles in vivo Hood and colleagues [17] subcutaneously injected the avb3-negative cell line M21-L into mice to establish small tumors, and the animals subsequently were injected intravenously with avb3-NP coupled to a luciferase reporter plasmid so that the reporter can be selectively targeted to the tumor. Engineering nanomedicine to develop nanodevices, nanobiosensors, nano-electrical mechanical system, nanotubes, and nanowires for biologic application Nanomaterials are exquisitely sensitive chemical and biologic sensors. Nanosensors with immobilized bioreceptor probes that are selective for

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target analyte molecules are called ‘‘nanobiosensors.’’ They can be integrated into other technologies such as ‘‘lab-on-a-chip’’ to facilitate molecular diagnostics. Their applications include detection of micro-organisms in various samples, monitoring of metabolites in body fluids, and detection of tissue pathology such as cancer. Their portability makes them ideal for point-ofcare applications, and they can be used in the laboratory setting as well. Because their surface properties are easily modified, nanowires can be enhanced with virtually any potential chemical or biologic molecular recognition unit, making the wires themselves analyte independent. The nanomaterials transduce the chemical binding event on their surface into a change in conductance of the nanowire in an extremely sensitive, realtime, and quantitative fashion. Boron-doped silicon nanowires (SiNWs) have been used to create highly sensitive, real-time electrically based sensors for biologic and chemical species [18]. Biotin-modified SiNWs were used to detect streptavidin in a concentration at the picomolar range. The small size and capability of these semiconductor nanowires for sensitive, label-free, real-time detection of a wide range of chemical and biologic species could be exploited in array-based screening and in vivo diagnostics. Nanowires and nanotubes carry charge and excitons efficiently and therefore are potentially ideal building blocks for nanoscale electronics and optoelectronics [19,20]. Carbon nanotubes already have been exploited in devices such as field-effect [21,22] and single-electron [23,24] transistors, but the practical utility of nanotube components for building electronic circuits is limited, because it is not yet possible to grow semiconducting or metallic nanotubes selectively [25,26]. The electrical properties of the assembly of functional nanoscale devices are controlled by selective doping. Gatevoltage–dependent transport measurements demonstrate that the nanowires can be synthesized predictably as either n- or p-type. These doped nanowires function as nanoscale field-effect transistors and can be assembled into crossed-wire p–n junctions that exhibit rectifying behavior. Significantly, the p–n junctions emit light strongly and perhaps are the smallest lightemitting diodes that have yet been made. Finally, it has been shown that electric field–directed assembly can be used to create highly integrated device arrays from nanowire building blocks. Diagnostic nanomedicine for biomarker research Nanomolecular diagnostics is the use of nanobiotechnology in molecular diagnostics [27,28]. Numerous nanodevices and nanosystems for sequencing single molecules of DNA are feasible. Given the inherent nanoscale of receptors, pores, and other functional components of living cells, the detailed monitoring and analysis of these components will be made possible by the development of a new class of nanoscale probes. Nanobiotechnologies have the potential for being incorporated in clinical laboratory diagnosis. Nanotechnologies enable diagnosis at the single-cell

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and molecule level, and some of these techniques (eg, biochips) can be incorporated in the current molecular diagnostics. Nanoparticles, such as gold nanoparticles and quantum dots, are the most widely used techniques. The nanotechnology on which nanoscale chips are based is related to nanomanipulation. The droplets used in nanomanipulation are 1 billion times smaller in volume than those used in conventional methods. The levitated particles can be manipulated and positioned with accuracy of up to 300 nm. Using this technology on a ‘‘lab-on-a-chip’’ would refine the examination of fluid droplets containing trace chemicals and viruses. The NanoPro system (BioForce Nanosciences Inc., Ames, IA) embodies proprietary instrumentation and methods for creating a broad spectrum of biologic tests based on the NanoArray (BioForce Nanosciences, Inc., Ames, Iowa) device. This device places molecules at defined locations on a surface with nanometer spatial resolution. Ultraminiaturized tests based on this technology have applications in many areas. Commercial development is targeted toward applications in proteomics/genomics and diagnostics such as virus detection and immunodiagnostics. The NanoReader (BioForce Nanosciences, Inc.) is a customized atomic force microscope optimized for reading NanoArray chips. Using the atomic force microscope as a readout method optimizes analysis. There is no need for secondary reporter systems such as fluorescence, radioactivity, or enzyme-linked detection. Less material is needed because several thousand molecules can be covered with one test. As such, these technologies will extend the limits of current molecular diagnostics and enable point-of-care diagnosis as well as the development of personalized medicine. Although the potential diagnostic applications are unlimited, the most important currently foreseen applications are in the areas of biomarker research, cancer diagnosis, and detection of infectious micro-organisms. Genetic nanomedicine for gene detection and gene delivery Gene delivery is an area of considerable current interest. Genetic materials (DNA, RNA, and oligonucleotides) have been used in molecular medicine and are delivered to specific cell types to inhibit some undesirable gene expression or to express therapeutic proteins. To date, most gene therapy systems are based on viral vectors delivered by injection to the sites where the therapeutic effect is desired. Viral genetransfer techniques can deliver a specific gene to the nucleus of a cell for expression, through integration into the geneome or as episomal vectors. In other systems naked DNA, RNA, or modified nucleic acids, all of which produce many undesirable side effects that can compromise the treatment of patients, have been injected directly into the blood stream. Viral vectors can have potentially dangerous side effects because the unintended integration of the viral DNA into the host genome can result in the incorporation

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of the virus into the host immune system; these techniques therefore have been less successful than originally hoped. Liposome-based gene transfer has relatively low transfection rates, is difficult to produce in a specific size range, can be unstable in the blood stream, and is difficult to target to specific tissues [29]. Injection of naked DNA, RNA, and modified RNA directly into the blood stream leads to clearance of the injected nucleic acids with minimal beneficial outcome [30]. Nonviral vectors, because of their lack of immunogenicity and their easy production, represent a good alternative to viral vectors, but most nonviral vectors have lacked the high transfection efficiency obtained with viral vectors. Existing gene-delivery systems have a variety of limitations. These systems are designed to eliminate an infection by transferring a therapeutic gene to host cells, but they have been largely unsuccessful because only low doses of genetic material can reach the specifically targeted infected cell types. Side effects are increased by the nonspecific targeting of noninfected cells with genes and by host cells’ reaction to the carrier molecules associated with gene delivery. A gene-delivery system that has minimal side effects but high potency and efficiency is needed. The possibility that nanosystems might have unique physical and biologic properties that could be used to overcome the problems of gene and drug delivery has gained interest in recent years. Nanosystems can be designed with different compositions and biologic properties. Some of these systems (eg, nanoparticles, dendrimers, nanocages, micelles, molecular conjugates, and liposomes, among others) have been investigated extensively in drug- and gene-delivery applications [31]. Self-assembled nanoparticles coated with targeting biomolecules [32], for example, use a nanoparticle platform for diagnostic probes and effective targeted therapy [33]. To demonstrate clearly the therapeutic potential of this novel gene-delivery system, some other aspects need to be clarified, mainly, the long-term toxicity of nanoparticles. Nonetheless, the demonstration that nonviral vectors can deliver genes to brain cells effectively, with none of the toxicity associated with viral vectors, gives renewed hope to the field of gene therapy and its practitioners. These gene–nanoparticle complexes might make it possible some day actually to repair the neurologic damage inflicted by strokes and other brain disease.

Clinical nanomedicine: future therapeutic approaches One aspect of clinical nanomedicine is the therapeutic approach (also called the ‘‘therapeutics of nanomedicine’’ or ‘‘nanotherapeutics’’). Nanotherapeutics involves the treatment of disease using techniques and technologies developed on scale raging from the macroscopic to the subcellular level. By improving the bioavailability of drugs and DNA plasmids,

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nanotherapeutics enables specific targeting of medical treatments to tissue, cell, and intracellular compartments. Three main areas of nanotherapeutics are under study and development: drug therapy, gene therapy, and immunotherapy. In drug therapy, nanotechnology can improve the therapeutic potential of many water-insoluble and unstable drugs dramatically, through either size reduction or encapsulation of the drug particles. In gene therapy, polymers and lipids can condense DNA into nanoparticles that can be internalized by cells, followed by delivery of the DNA into the nucleus. The DNA-nanoparticles can deliver functional genes to correct genetic disorders such as hemophilia, cystic fibrosis, and muscular dystrophy. In addition, lipid-based nanosystems, such as nanoemulsions, lipid-core micelles, small unilamellar vesicles, and variations thereof, have long been in existence, and some have long been used to improve patient’s lives. Indeed, lipid-based nanoformulations are among the most attractive candidates for improving drug solubility and for site-specific targeting following parenteral administration. Specifically, great strides are being made with such complexes and nanosystems in combating the growth and spread of cancerous tissues (eg, through exploitation of angiogenic tumor vasculature, combination chemotherapy, and endogenous triggered activation and release of encapsulated lipid pro-drugs), treatment of macrophage infections (through exploitation of macrophage clearance mechanisms), gene transfer (by breaching the endo-lysosomal barrier with cationic lipid vectors), and stimulation of immune responses to antigens (with the aid of vesicular systems and lipid complexes with self-adjuvanting properties). Although lipid-based nanocarriers may overcome solubility or stability issues for the drug and minimize drug-induced side effects through favorable pharmacokinetic profiles and site-specific targeting, there are significant toxicity issues with the carriers themselves that need to be addressed. Nanotherapeutics, however, provides a broad sample of the state of the art of nanotechnology. Oncologic nanomedicine in the early diagnosis and early treatment of cancer Emerging biomicronanotechnologies have the potential to provide accurate, real-time, high-throughput screening of tumor cells without the need for time-consuming sample preparation. These rapid nano-optical techniques may play an important role in advancing early detection, diagnosis, and treatment of disease. Recently, many nanotechnology tools have become available that enable clinicians to detect tumors at an early stage. The potential ability of nanostructures to enter a single tumor cell can help improve the current limit of detection by imaging techniques. Gourley and colleagues [34] shows that laser scanning confocal microscopy can be used to identify a previously unknown property of certain cancer cells

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that distinguishes them, with single-cell resolution, from closely related normal cells. This property is the correlation of light scattering and the spatial organization of mitochondria. In addition, the new technology of nanolaser spectroscopy using the biocavity laser can characterize the unique spectral signatures of normal and transformed cells. These optical methods are powerful new tools that hold promise for detecting cancer at an early stage and may help reduce delays in diagnosis and treatment. Nanotechnology can help diagnose cancer by using dendrimers and, by using nanovectors for the tumor-selective delivery of genes, can kill tumor cells without harming normal healthy cells. These and other technologies currently are in various stages of discovery and development. Targeting and local tumor delivery are the key challenges in the diagnosis and treatment of cancer. Cancer therapies are based on a better understanding of the disease at the molecular level. Nanobiotechnology is being used to refine the discovery of biomarkers, molecular diagnostics, drug discovery, and drug delivery, which are important basic components of personalized medicine and are applicable to the management of cancer as well. Examples are the application of quantum dots, gold nanoparticles, and molecular imaging in diagnostics and in combination with therapeutics, another important feature of personalized medicine. Nanobiotechnology is expected to facilitate the early detection of cancer and more effective and less toxic treatment, increasing the chances of cure. Pharmacologic nanomedicine for drug delivery and drug design The application of nanotechnology in drug design and drug delivery is receiving a great deal of attention. Nanoparticles and nanodevices such as nanobiosensors and nanobiochips are being used to improve drug discovery and development. Nanoscale assays can reduce costs in screening campaigns. In addition, some nanosubstances (such as fullerenes) could be potential drugs in the future. Fullerene molecules have numerous points of attachment, allowing precise grafting of active chemical groups in three-dimensional orientations. This attribute, the hallmark of rational drug design, allows positional control in matching fullerene compounds to biologic targets. Together with other attributes, namely the size of the fullerene molecules, their redox potential, and their relative inertness in biologic systems, it is possible to tailor fullerene-based compounds to the requisite pharmacokinetic characteristics and to optimize their therapeutic effect [35]. Fullerenes have potential applications in the treatment of diseases in which oxidative stress plays a role in the pathogenesis (eg, neurodegenerative diseases). Another possible application of fullerenes is in nuclear medicine, as an alternative to chelating compounds that prevent the direct binding of toxic metal ions to serum components. This application could increase the therapeutic potency of radiation treatments and decrease their

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adverse effects, because fullerenes are resistant to biochemical degradation within the body. Many drugs discovered in the past could not be used in patients because a suitable method of drug delivery was lacking. Nanotechnology also is used to facilitate drug delivery. A product incorporating the NanoCrystal technology of Elan Drug Delivery (King of Prussia, Pennsylvania), a soliddose formulation of the immunosuppressant sirolimus, was approved by the Food and Drug Administration in 2000 [36]. There is a drug approved for the treatment of breast cancer after the failure of combination chemotherapy for metastatic disease or after relapse within six months of adjuvant chemotherapy, containing paclitaxel as albumin-bound particles in an injectable suspension. It is based on nanoparticle technology that integrates biocompatible proteins with drugs to create the nanoparticle form of the drug (with a size of 100–200 nm) to overcome the problems of insolubility encountered with paclitaxel. Now, the trend is to consider drug-delivery issues at the earlier stages of drug discovery and design. A carrier nanoparticle can be designed simultaneously with the therapeutic molecule. Although the in vivo use of nanoparticles might involve some safety concerns, studies are in place to determine the nature and extent of adverse events. Future prospects for the application of nanotechnology in health care and for the development of personalized medicine seem to be excellent. Cardiovascular nanomedicine for heart and vascular diseases Cardiovascular disease remains the leading cause of death in the United States: one out of every four Americans has cardiovascular disease, and one person dies from heart disease every 30 seconds. Although significant advances have been made in the management and treatment of this disease, the effectiveness of early detection and treatment in preventing heart attacks is still questionable, because few heart attacks can be predicted by the physicians. One of the fundamental and unresolved problems in cardiovascular biology is the in vivo detection of atherosclerotic disease and the evaluation of atherosclerotic disease activity. Current technology limits clinicians to diagnostic techniques that either image or functionally assess the significance of large obstructive vascular lesions. Techniques have been developed recently that achieve molecular and cellular imaging with most imaging modalities, including nuclear, optical, ultrasound, and MRI. In addition, current imaging modalities are not capable of imaging atherosclerotic disease at its earliest stages, nor do available techniques allow routine assessment of atherosclerotic lesions susceptible to rupture and/or thrombosis. This is of particular clinical significance given that myocardial infarctions and other sequelae of atherosclerotic disease are just as likely to occur from small non-obstructive coronary artery disease based on the degree of luminal obstruction is fundamentally flawed. Newer technologies must be developed

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that can identify earlier atherosclerotic lesions as well as atherosclerotic lesions that are active or unstable. The role of nanotechnology in cardiovascular diagnosis is expanding rapidly. Nanosystems have been applied to the area of atherosclerosis, thrombosis, and vascular biology. The technologies for producing targeted nanosystems are numerous and in many cases reflect end uses. The results to date indicate a rapid growth of interest and capability in the field. Cardiovascular diagnosis already is being affected by nanosystems that can both diagnose pathology and treat it with targeted delivery systems. Advanced imaging methods and new, targeted, nanoparticle contrast agents for early characterization of atherosclerosis and cardiovascular pathology at the cellular and molecular levels might represent the next frontier for combining imaging and rational drug delivery to facilitate personalized medicine. The rapid growth of nanotechnology and nanoscience could expand the clinical opportunities for molecular imaging greatly.

Neurologic nanomedicine for neuroscience research Nanotechnologies exploit materials and devices with a functional organization that has been engineered at the nanometer scale. In neuroscience, nanotechnology entails specific interactions with neurons and glial cells. Nanomaterials and nanodevices that interact with neurons and glia at the molecular level can be used to influence and respond to cellular events. Nanotechnology can be used to limit and/or reverse neuropathologic disease processes at a molecular level or to facilitate and support other approaches with this goal. Applications of nanotechnology in basic neuroscience investigate molecular, cellular, and physiologic processes in three specific areas: 1. Nanoengineered materials and approaches for promoting neuronal adhesion and growth to understand the underlying neurobiology of these processes or to support other technologies designed to interact with neurons in vivo (eg, coating of recording or stimulating electrodes) [37] 2. Nanoengineered materials and approaches for directly interacting, recording, and/or stimulating neurons at a molecular level [38] 3. Imaging applications using nanotechnology tools, in particular those that focus on chemically functionalized semiconductor quantum dots [39] Applications of nanotechnology in clinical neuroscience include research aimed at limiting and reversing neuropathologic disease states. Nanotechnology approaches are designed to support and/or promote 1. The functional regeneration of the nervous system [40] 2. Neuroprotective strategies, in particular those that use fullerene derivatives [41]

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3. Nanotechnology approaches that facilitate the delivery of drugs and small molecules across the blood–brain barrier [42] Nanotechnology can be used to limit and reverse neurologic disorders by promoting neural regeneration and achieving neuroprotection. The development of nanoengineered scaffolds that support and promote neurite and axonal growth are evolving from tissue-engineering approaches based on the manipulation of bulk materials. Applications of nanotechnologies for neuroprotection have focused on limiting the damaging effects of free radicals generated after injury, which is a key neuropathologic process that contributes to central nervous system ischemia, trauma, and degenerative disorders [43]. Applications of nanotechnology in neuroscience already are having significant effects, which will continue in the foreseeable future. Short-term progress has benefited in vitro and ex vivo studies of neural cells, often supporting or augmenting standard technologies. These advances contribute both to the basic understanding of cellular neurobiology and neurophysiology and to the understanding and interpretation of neuropathology. Applications of nanotechnology in basic and clinical neuroscience are in only the early stages of development, partly because of the complexities of interacting with neural cells and the mammalian nervous system. Nonetheless, an impressive body of research is emerging that hints at the potential contributions these technologies could make to neuroscience research.

Dermatologic nanomedicine for skin research Dermatology is the visual medical specialty par excellence. Although improvements in visual imaging technology are advancing the biologic sciences at the bench, they ultimately are enhancing analysis and diagnosis at the clinical level. In the practice of dermatology, precise and thorough visualization of the skin is a key to a comprehensive examination and accurate diagnosis. The traditional biopsy of the skin is invasive, uncomfortable, and inconvenient. Thus, the prospect of a noninvasive, immediate, in vivo examination is attractive to both patient and dermatologist. Advances in digital dermoscopy, microscopy, imaging, and photography have formed an impressive arsenal with which dermatologists can offer state-of-the-art patient care while streamlining clinical practice and improving academic and research capabilities. Much recent literature addresses the use of advanced digital dermoscopy and microscopy systems in examining and diagnosing pigmented skin lesions and neoplasms. In addition to this practice, dermatologists also are examining and analyzing other skin pathologies, using the same emerging technologies that have made breakthroughs in melanoma diagnosis. These technologies offer enhanced clinical examination and improved methods of analyzing, grading, and standardizing the results of clinical research trials.

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Epiluminescent microscopic dermoscopy and confocal microscopy are imaging technologies that now are popular in clinical practice. The improved resolution and quality of these technologies may minimize the use of other imaging techniques such as optical coherence tomography, commonly used in ophthalmology, and the ubiquitous ultrasound and MRI methods. Other types of advanced imaging are used in the biologic sciences at the bench (eg, two-photon laser microscopy, which has been used in vitro). Several nanoparticles are used in molecular imaging: gold nanoparticles, quantum dots, and magnetic nanoparticles. Gold nanoparticles are particularly good labels for sensors because a variety of analytic techniques can be used to detect them, including optical absorption, fluorescence, Raman scattering, atomic and magnetic force, and electrical conductivity. Gold particles can be used to detect micro-organisms and could replace the polymerase chain reaction and fluorescent tags used currently. Quantum dots are nanoscale crystals of semiconductor material that glow or fluoresce when excited by a light source such as a laser. Quantum dots have fairly broad excitation spectradfrom ultraviolet to reddthat can be tuned depending on their size and composition. At the same time, quantum dots have narrow emission spectra, making it possible to resolve the emissions of different nanoparticles simultaneously and with minimal overlap. Quantum dots are highly resistant to degradation, and their fluorescence is remarkably stable. Bound to a suitable antibody, magnetic nanoparticles are used to label specific molecules, structures, or micro-organisms. Magnetic immunoassay techniques have been developed in which the magnetic field generated by the magnetically labeled targets is detected directly with a sensitive magnetometer. As a visualization-intensive discipline, the practice of dermatology can benefit significantly from advances in noninvasive imaging of the skin. High-resolution dermoscopy, microscopy, and spectrometry now offer dermatologists the opportunity to advance diagnostic and therapeutic modalities to fit a specialty in which physical examinations appreciate the gestalt of a disease or condition. Nanotoxicology in health and environmental research The science of toxicology always has provided the foundation for understanding the interactions between chemistry and biology. Although the use of nanomaterials in commercial products is relatively new, the philosophical basis for performing a toxicologic evaluation of these materials is not expected to differ from that for other materials. A basic tenet of any study designed to develop an understanding of the toxic effects of a material on biologic systems is to understand the physicochemical properties of that material. Consequently, the unique physical and chemical characteristics of engineered nanomaterials that lead to their distinctive properties probably also

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will contribute to the hazards associated with these materials. The safety of these materials will be investigated best by multidisciplinary teams. The toxicologic evaluation and characterization of the safety of nanomaterials is still at an early stage. At present there are few data on the safety of nanomaterials, but this situation is changing rapidly. There also is little debate that, even though nanomaterials exhibit unique properties that clearly distinguish them from their bulk counterparts, many of the methods, tests, assays, and principles that have been the cornerstones of traditional approaches to safety assessment can be applied to the design of the studies characterizing nanomaterial safety. There is a good foundation on how to proceed with characterizing the safety of nanomaterials that has been developed through several years of studying materials such as fine and ultrafine particles, increasing understanding of the lungs and the skin as portals of entry and as potential target organs, and improving approaches to characterize the important roles played by absorption, distribution, metabolism, and excretion. It is generally thought that it is unlikely that nanomaterials will manifest new toxic manifestations despite their unique physical and chemical properties. Therefore, many traditional methods and approaches probably will be applicable to studies of nanomaterials, especially if it is acknowledged that not all nanoscale materials are the same and that potential impacts resulting from the unique properties of these materials are to be expected. Toxicologists must apply the very best science in characterizing the safety of nanomaterials. The development of a comprehensive strategy to address nanomaterial safety must integrate the state-of-the-science for in vitro methods and must include a consideration of mechanisms of action at the onset. Understanding of the mechanisms of action for nanomaterials must be developed at the same time that information is compiled about the toxicity profiles. This approach is in contrast to the retrospective approach that traditionally has been taken by toxicologists and/or risk assessors. It also seems clear that a diverse toxicologic strategy that covers a continuum of effects from short-term in vitro tests to subchronic and chronic studies needs to be considered when evaluating nanomaterials. Whether this strategy can be used as a tiered approach is being debated. There is, however, no debate over the conclusion that no one study should be interpreted as definitive, and that the ultimate approach to the consideration of nanomaterial safety will depend on a consideration of the weight of evidence. Future directions in nanomedicine Nanotechnology is beginning to change the scale and methods of vascular imaging and drug delivery. Indeed, the Nanomedicine Initiatives of the NIH Roadmap envisage that nanoscale technologies will begin yielding more medical benefits within the next 10 years. These benefits are expected to include the development of nanoscale laboratory-based diagnostic and

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