Nanotechnology for the biologist

Scott E. McNeil1 SAIC-Frederick, Nanotechnology Characterization Laboratory, Frederick, Maryland

Abstract: Nanotechnology refers to research and technology development at the atomic, molecular, and macromolecular scale, leading to the controlled manipulation and study of structures and devices with length scales in the 1- to 100-nanometers range. Objects at this scale, such as “nanoparticles,” take on novel properties and functions that differ markedly from those seen in the bulk scale. The small size, surface tailorability, improved solubility, and multifunctionality of nanoparticles open many new research avenues for biologists. The novel properties of nanomaterials offer the ability to interact with complex biological functions in new ways— operating at the very scale of biomolecules. This rapidly growing field allows cross-disciplinary researchers the opportunity to design and develop multifunctional nanoparticles that can target, diagnose, and treat diseases such as cancer. This article presents an overview of nanotechnology for the biologist and discusses “nanotech” strategies and constructs that have already demonstrated in vitro and in vivo efficacy. J. Leukoc. Biol. 78: 585–594; 2005.
Key Words: nanoparticle anticancer drug drug delivery polyethylene glycol

INTRODUCTION
Nanotechnology has achieved the status as one of the critical research endeavors of the early 21st century, as scientists harness the unique properties of atomic and molecular assemblages built at the nanometer scale. Our ability to manipulate the physical, chemical, and biological properties of these particles affords researchers the capability to rationally design and use nanoparticles for drug delivery, as image contrast agents, and for diagnostic purposes. The confluence of these newly acquired capabilities, coupled with advances in imaging, bioinformatics, and systems biology, holds tremendous promise for answering some of biology’s most challenging biochemical and genetic questions. Although biology today is benefiting from a host of technological developments, few may eventually have the paradigmchanging impact on basic research, drug development, and clinical medicine as nanotechnology. By operating in the nanoscale realm, at the very scale of biomolecules, nanotechnology offers a wide range of tools and applications (see Table 1). Near-term applications include drug-delivery platforms [1], enhanced image contrast agents [2], chip-based nanolabs ca0741-5400/05/0078-585 © Society for Leukocyte Biology

pable of monitoring [3] and manipulating individual cells [4], and nanoscale probes that can track the movements of cells [5] and individual molecules [6] as they move about in their environment. Such an unprecedented ability to observe and influence complex systems in vivo and in real time provides detailed information about the fundamental mechanisms and signaling pathways involved in the progression of disease and greatly extends the existing toolset for drug delivery and noninvasive drug monitoring. By providing constructs capable of combining multiple functionalities into a single nanoscale entity, nanotechnology also offers the opportunity to monitor and detect molecular and cellular changes associated with disease states [7]. Given this multifunctional capability, one can imagine building a nanoparticle that can target a specific tissue or cell type, delivering a contrast agent that allows for noninvasive imaging and a therapeutic payload to the target. A nanoparticle might even contain a reporter, such as an apoptotic marker, which signals that the payload has been delivered and is having the desired therapeutic effect. Such combinatorial nanostructures may eventually provide the means to achieve “personalized medicine” by tailoring drug delivery to individual response. Although this may seem futuristic, several groups have already created multifunctional nanodevices and are testing them in in vitro and in vivo systems [1, 8 –18]. One cannot effectively discuss the relevance of nanotechnology to biologists without first discussing terminology. For the purposes of this review, I will follow the definition endorsed by the National Nanotechnology Initiative (NNI), which defines nanotechnology as research and technology development at the atomic, molecular, or macromolecular scale, leading to the controlled creation and use of structures, devices, and systems with a length scale of 1–100 nanometers (nm). Such constructs must also have novel properties and functions because of their small size. For example, carbon nanotubes and gold nanoshells, two different types of nanomaterials, have physical properties different from carbon [19] or gold [20] on the macro scale. Other examples of nanotechnology include dendrimers [21], liposomes [22], and semiconducting quantum dots [23]. In contrast, particles such as DNA, bacteriophage, and monoclonal antibodies (mAb) may have nanometer-sized dimensions but would not be considered examples of nanotechnology for the purposes of this review. Nanotechnology manifests itself in a wide range of materials that can be useful to the biologist [24], a sample of which is

Correspondence: SAIC-Frederick, Nanotechnology Characterization Laboratory, 1050 Boyles St., Frederick, MD 21702-1201. E-mail: mcneils @ncifcrf.gov Received February 4, 2005; revised April 27, 2005; accepted April 28, 2005; doi: 10.1189/jlb.0205074.

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Fig. 1. Multifunctional nanoparticle. The nanoparticle’s “corona” can be functionalized with hydrophilic polymers, targeting molecules, therapeutic drugs, and image contrast agents. The interior core can be solid (e.g., quantum dots) or liquid (e.g., liposomes). Molecules are not shown to scale. PEG, Polyethylene glycol.

listed below (see Table 1). Virtually all of these materials have been designed with chemically modifiable surfaces to attach a variety of ligands that can turn these nanomaterials into biosensors, molecular-scale fluorescent tags, imaging agents, targeted molecular delivery vehicles, and other useful biological tools (Fig. 1). The unprecedented freedom to design and modify nanomaterials to target cells, chaperone drugs, image biomolecular processes, sense and signal molecular responses to therapeutic agents, and guide surgical procedures is the fundamental capability offered by nanotechnology, which promises to impact drug development, medical diagnostics, and clinical applications profoundly (Fig. 2).

NANOTECHNOLOGY APPLIED TO BIOLOGICAL SYSTEMS Size matters
An obvious advantage of nanotechnology as it relates to biological systems is the ability to control the size of the resulting

particles and devices. Nanoscale devices and components are of the same basic size as biological entities, as shown in Figure 3. Nanoscale constructs are smaller than human cells (10,000 –20,000 nm in diameter) and organelles and similar in size to large biological macromolecules such as enzymes and receptors; hemoglobin, for example, is 5 nm in diameter, and the lipid bilayer surrounding cells is on the order of 6 nm thick. Nanoparticles smaller than 20 nm can transit through blood vessel walls. Magnetic nanoparticles, for instance, can image metastatic lesions in lymph nodes because of their ability to exit the systemic circulation through the permeable vascular epithelium [25]. Nanoparticles also offer the ability to penetrate the blood-brain barrier or the stomach epithelium [14, 26 –29]— barriers that make it difficult for legacy therapeutic and imaging agents to reach their intended targets. To be suitable as a drug-delivery platform, the size of nanoparticles must be small enough to avoid rapid filtration by the spleen, with filaments spaced at roughly 200 nm [30], which serve as a meshwork for phagocytotic cells [31]. Similarly, to traverse the liver, the particles must be small enough to pass through the organ’s 150 –200 nm-sized fenestrae and avoid the Kupffer cell-lined sieve plates [32]. Drug-carrying liposomes are believed to have increased lifespans, related in part to their ability to extravasate through splenic and liver fenestrae [33]. The size of nanoscale devices also allows them to interact readily with biomolecules on the cell surface and within the cell, often in ways that do not alter the behavior and biochemical properties of those molecules [34]. Such ready access to the interior of a living cell affords the opportunity for unprecedented gains on the clinical and basic research frontiers. The ability to interact with receptors, nucleic acids, transcription factors, and other signaling proteins at their own molecular scales should provide the data needed to better understand the complex regulatory and signaling networks and transport processes that govern the behavior of cells in their normal state [35] and as they undergo the changes that transform them during the disease process [36]. In particular, nanotechnology will provide an important role in integrating efforts in proteomics (identifying and measuring key cellular proteins and peptides) with systems biology (the integration of cellular pathways and networks) and other scientific investigations into the molecular nature of disease [37– 39]. Nanoscale particles and devices are already proving they

Fig. 2. Medical applications of nanotechnology. The size and tailorability of nanoparticles may lead to their widespread use in a variety of medical applications.

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Fig. 3. Relative size of nanoparticles compared with familiar items.

can deliver therapeutic agents that can act where they are likely to be most effective, that is, within the cell or even within specific organelles. By virtue of their size, nanoparticles such as quantum dots can be endocytosed and used for intracellular imaging [40 – 42]. Despite their small size, nanoparticles can accommodate tens of thousands of atoms or small molecules, such as the magnetic resonance imaging (MRI) contrast agent gadolinium [2], creating the opportunity for improved detection sensitivity of diseases such as cancer in its earliest stage.

Solubility matters
To fully appreciate the powerful use of nanotechnology—and nanoparticles in particular— one must understand the surface chemistry of the particles. Modification of the nanoparticle’s outer layer allows a large variety of chemical, molecular, and biological entities to be covalently or otherwise bound to it (Fig. 1). Manipulation of this corona confers advantageous properties to the particle, such as increased solubility and biocompatibility. Attaching hydrophilic polymers to the surface, such as PEG, greatly increases the hydration (i.e., solubility) of the nanoparticles and can protect attached proteins from enzymatic degradation when used for in vivo applications [43]. This advantage is perhaps most applicable in drug discovery and delivery. A common screening method during drug discovery and profiling is to score new compounds based on “Lipinski’s Rule of Five” [44]. This evaluation filter ranks “chemical leads” based primarily on the compound’s predicted solubility, as molecules with low solubility generally have poor absorption and decreased metabolic stability in vivo [45]. High-throughput screening (HTS) methods, conversely, will often yield lipophilic compounds with high therapeutic activity. The probability of a novel compound being assessed as efficacious and soluble by these filters is therefore quite low and is further reduced by safety and toxicity screening during

development. Indeed, the odds of a newly synthesized compound progressing to development are estimated to be one in 100,000 [46]. Nanoparticles with hydrophilic polymers such as PEG attached to their surface can act as a platform for lipophilic molecules and overcome the solubility barrier. Insoluble compounds can be attached, adsorbed, or otherwise encapsulated in the hydrated nanoparticles [47, 48]. Solubility of the composite entity subsequently becomes a function of the nanoparticle carrier rather than being strictly dependent on the drug itself. New chemical entities (NCEs) shown to be effective under HTS methods, but then discarded as a result of insolubility, may now be resurrected for development as a result of nanotechnology [49] (Fig. 4). Liposomes are perhaps the most common example of this application, and the U.S. Food and Drug Administration (FDA) now approved for market several new molecular entities and applications [43]. The surface addition of PEG (“pegylation”) and other hydrophilic polymers also increases the in vivo compatibility of nanoparticles. When injected intravascularly, uncoated nanoparticles are cleared rapidly from the bloodstream by the reticuloendothelial system (RES) [50]. Nanoparticles coated with hydrophilic polymers have prolonged half-lives, believed to result from decreased opsonization and subsequent clearance by macrophages [51]. This represents a slight paradigm shift from classical pharmacology; plasma protein binding (e.g., to albumin and 1-acid glycoprotein) can be a desired attribute for traditional therapeutic drugs, as it serves to increase bioavailability by limiting first-pass hepatic extraction. Blood components implicated in the RES clearance of nanoparticles include fibronectin, C3, albumin, fibrinogen, immunoglobulin G (IgG), Ig light chains, and the apolipoproteins (apo)A-I and apoE [52–54]. Several groups have also extended the ability to tailor surface chemistry to the nanoparticle’s potential or overall
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Figure 4. The drug-development pipeline. Although a large pharmaceutical company may synthesize several million compounds, only a fraction of those NCEs becomes “leads” for preclinical development as a result of issues associated with their solubility (e.g., Lipinski’s Rule of Five). Nanotechnology allows insoluble compounds to be attached or encapsulated in highly soluble nanoparticles, offering the potential to expand the number of drugs introduced into clinical trials.

charge by derivatizing the surface with cationic or anionic species. The particle’s charge can influence its biocompatibility and ability to traverse biological barriers [28, 55, 56]. In the case of dendrimers, for example, particles displaying amine (cationic) groups on their outer surface were significantly more cytotoxic than carboxylic-terminated (anionic) dendrimers [57]. The latter also had increased circulation times (20 – 40% recovered dose in blood at 1 h) compared with the cationic species ( 2% recovered dose in blood at 1 h) when injected intravenously into rats. Follow-up work improved this strategy by synthesizing an acetylated dendrimer with a physiologically neutral surface, which showed no deleterious effects in mice after 12 weeks [58]. Surface charge has also been implicated in the ability of nanoparticles to cross the blood-brain barrier [26] and their distribution between the vascular and extravascular compartments of tumors [59].

Targeting matters
One of the earliest examples of applying nanotechnology to solving problems in biology was the use of liposomes as drug-

delivery vehicles [60]. A liposomal formulation of the potent but toxic antifungal agent amphotericin B has revolutionized the treatment of life-threatening, systemic, fungal infections in immune-compromised patients by allowing patients to receive normally lethal doses of amphotericin B with minimal risk of toxicity [61]. The liposomes, 50 –70 nm in diameter, are taken up rapidly by macrophages, which then carry the liposome and drug to the site of fungal colonization. Cancer therapy has benefited from the use of liposomal doxorubicin, a formulation that again increases the therapeutic index of the active agent through a combination of passive tumor targeting and reduced toxicity [62]. In this case, coating the liposome with PEG significantly decreases uptake by macrophages and allows the liposomes to concentrate in tumors by escaping from the leaky vasculature surrounding solid tumors [63] through a phenomenon known as the enhanced permeation and retention (EPR) effect (Fig. 5) [64 – 66]. Size alone is not the only way that nanotechnology can enable targeting. Because of the highly tailorable surfaces of nanoscale constructs, it has become a relatively simple matter

Fig. 5. Active and passive targeting. (Upper left, Passive Targeting) The EPR effect. Tumor tissues (upper right, Passive Targeting) are known to have leaky vasculature (red) and decreased lymphatics (blue) compared with normal histology (upper left). This phenomenon results in a passive accumulation of nanoscaled particles (yellow) and is referred to as EPR. (Lower left) Passive targeting to the RES. Drugs bound to nanoparticles can passively target tissues of the RES. The net effect is a discrete distribution pattern (shown in blue) compared with legacy drugs and presumably decreased adverse side-effect. (Upper right, Active Targeting) Nanoparticles with ligands or molecules attached to their surface can target tumor cells preferentially over healthy cells.

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to “decorate” the nanoparticle surface with a variety of targeting agents. As a result, the targeted delivery of drugs [67], nucleic acids [68], and other molecules using nanoparticles is the focus of significant research and development— one that is expected to significantly improve the way clinicians treat cancer and other diseases. Nanoscale devices should soon serve as customizable, targeted drug-delivery vehicles capable of ferrying large doses of chemotherapeutic agents into malignant cells while sparing healthy cells and reducing the deleterious systemic responses to these drugs. Already, a variety of nanoscale drug-delivery devices, including dendrimers [8], ceramic nanoparticles [69], and lipid-encapsulated perfluorocarbon nanoemulsions [70], can actively and passively target cancer cells. Similarly, nanotech strategies that target cells of the immune system may offer new approaches to treat infectious diseases such as human immunodeficiency virus and Leishmaniasis. Targeted delivery of nanoparticles can be accomplished by attaching a mAb or cell-surface receptor ligand that binds specifically to molecules found on the surfaces of targeted cells, be they cancer cells or the angiogenic microcapillaries growing around malignant cells. Targeting molecules that have been used successfully include folate [1], luteinizing hormonereleasing hormone (LH-RH) [71], thiamine [26], receptor-specific peptides [72, 73], aptamers [74], and a wide variety of mAb directed against cell-surface markers, such as integrins [15]. It is interesting to note that these functionalized nanoparticles have been demonstrated to have high avidity for their target cells, believed to be the result of their multivalent interaction [1, 10]. Once bound to the target cell, the nanoparticles are readily internalized by receptor-mediated endocytosis [1, 11, 28, 75]. The multifunctional nature of nanoparticles also offers a novel approach to monitor drug pharmacodynamics and pharmcokinetics in real time. In the late 1960s, researchers began to investigate radiolabeling of chemotherapeutic agents to monitor efficacy at the target site—in this case, the tumor [76]. By demonstrating a correlation between the amount of radiolabel drug (18F-5-fluoro-uracil) imaged in the tumor and its responsiveness to treatment, these pioneers established the field of “noninvasive studies of pharmaceutical drug products” [77]. The pharmacodynamics, pharmacokinetics, and metabolism of stable, isotope-labeled drugs can now be monitored at the site of action in real time by magnetic resonance spectroscopy (MRS) [78]. A significant limitation of MRS, however, is that it requires high drug concentrations and complete labeling of the compound to achieve adequate signal-to-noise ratios (SNR). Functionalized nanoparticles have the potential to greatly increase the SNR of drugs and allow for real-time pharmocodynamic studies. By manipulating the surface chemistry of the nanoparticle, researchers can now attach imaging, targeting, and therapeutic components to the same particle (Fig. 1). Tens of thousands of gadolinium atoms, for instance, can be colocated with the drug on a single nanoparticle to greatly increase the signal strength. As the distance between the drug and contrast agent (a few nanometers) is orders of magnitude less than the spatial resolution of the imaging instrument (hundreds of ), the SNR becomes a function of the abundant contrast agent and does not require labeling the drug itself. This approach offers clinicians a noninvasive tool to concurrently

monitor particle targeting, drug efficacy, and dose-ranging. Liposomes and dendrimers have been functionalized with a therapeutic, targeting component, and contrast agent within the same particle and have demonstrated initial success in animal models [79, 80]. The combinatorial possibilities of these “modular” platforms are truly remarkable, but realization of their clinical use will be contingent on further rigorous pharmaco- and toxicological characterization. Additionally, the chemical complexity of multifunctional nanoparticles does not often lend itself to consistent batch-to-batch synthesis or ease of “scale-up.” These developmental hurdles must be overcome to meet FDA’s current good manufacturing practice regulatory provisions prior to clinical application [81– 83]. On a more unconventional front, efforts are focused on engineering robust nanoconstructs, which will eventually be capable of detecting malignant cells in vivo, pinpointing their location in the body, killing the cells, and reporting back that their payload has done its job. The operative principles driving these current efforts are modularity and multifunctionality, i.e., creating functional building blocks that can be integrated to meet the particular demands of a given clinical situation. A good comparison from the biological world is a viral particle made from a limited set of proteins, each with a specific function, which comes together to create a multifunctional, nanoscaled delivery vehicle for genetic material. Such multifunctional nanodevices may enable new types of therapeutic approaches or broader application of existing approaches to killing malignant cells. For example, silica-coated lipid micelles containing LH-RH as a targeting agent have been used to deliver iron oxide particles to LH-RH receptorpositive cancer cells [71]. Once these particles have been taken up by the target cell, they cannot only be imaged using MRI but also turned into molecular-scale wrecking balls: Applying a rapidly oscillating magnetic field causes the Fe2O3 particles to align and oscillate with enough force to kill the cell [84].

APPLICATION OF NANOTECHNOLOGY: IN VIVO IMAGING
A variety of nanoscale particles have already demonstrated use in imaging tumors and the tumor microenvironment in animal models and human clinical trials, as listed in Table 1. Some of the most advanced work in this area uses dextran-coated, ultra-small superparamagnetic iron oxide (USPIO) nanoparticles to image lymph nodes containing micrometastases in patients with prostate cancer [103]. Other studies have used paramagnetic, gadolinium-labeled, nanoparticulate dendrimers to image lymphatic micrometastases in a mouse breast cancer model [104]. The operative principle here is that normal lymph nodes rapidly accumulate these MRI-dense particles. Tumor cells (i.e., the micrometastases) do not take up the particles and create a “signal void” against a high proximal concentration of superparamagnetic material. When imaged using a standard 1.5 Tesla MRI instrument, normal lymph nodes appear shadowed, and those loaded with metastatic cells or
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TABLE 1. Nanoparticle Dendrimers Ceramic nanoparticle Lipid-encapsulated perfluorocarbon nanoemulsions Magnetic nanoparticles LH-RH-targeted silica-coated lipid micelles Thiamine-targeted nanoparticles Liposomes Nanoparticle-aptamer bioconjugate Anti-Flk antibody-coated 90Y nanoparticles Gold nanoshells Anti-HER2 antibody-targeted gold/silicon nanoparticles CLIO paramagnetic nanoparticles Quantum dots Silicon-based nanowires Carbon nanotubes Transfersomes

Examples of Nanoparticles Used in Biological Research Application Targeting of cancer cells, drug delivery, imaging, boron neutron capture therapy Passive targeting of cancer cells Passive targeting of cancer cells Specific targeting of cancer cells, tissue imaging Specific targeting of cancer cells Directed transfer across Caco-2 cells Specific targeting of cancer cells, gene therapy, drug delivery Targeting of prostate cancer cells Antiangiogenesis therapy Tissue imaging, thermal ablative cancer therapy Breast cancer therapy Imaging of migrating cells Tissue imaging Real-time detection and titration of antibodies, virus detection, chip-based biosensors Electronic biosensors Noninvasive vaccine delivery, drug delivery Reference [1, 8, 85–87] [69] [70] [71, 88] [71] [29] [73, 89, 90] [74] [15] [91–93] [93] [94] [5, 95, 96] [97–99] [100] [101, 102]

HER2, Human epidermal growth factor receptor-2; CLIO, cross-linked iron oxide.

portions of lymph nodes with micrometastases appear enhanced. In a recent clinical trial, Harisinghani et al. [103] administered USPIO nanoparticles to 80 patients scheduled to undergo surgical resection of prostate tumor and/or pelvic lymph nodes. Although 71% of pelvic nodes containing metastases did not meet computed tomography imaging criteria for malignancy, MRI with the superparamagnetic nanoparticles correctly identified every patient with metastatic nodes. In addition, suspicious nodes were identified with the superparamagnetic nanoparticles in nine patients, who then underwent more extensive exploratory surgery, which confirmed the presence of distant metastases. The authors further reported that the nanoparticles were able to occasionally identify micrometastases less than 2 mm in diameter in normal-sized nodes, well below the typical limit of detection of positron-emission tomography (PET), which is considered the most sensitive imaging technique in several clinical scenarios. Gold nanoshells, coated with cancer cell-specific ligands, can act as molecularly targeted contrast agents for optical imaging. Using a variety of ligands, including mAb and aptamers, the nanoshells have been targeted to three different markers overexpressed on cancer cells: epidermal growth factor receptor, matrix metalloproteases, and oncoproteins associated with human papillomavirus infection, the primary cause of cervical cancer [91]. Although optical imaging does have limitations as a result of tissue absorption and reflection of light, the inherent low cost of optical imaging compared with more complex imaging modalities such as MRI and PET has attracted increasing interest from cancer researchers [105], particularly with the development of inexpensive, fiber-optic, confocal imaging systems capable of being fed through ducts and capillaries [106]. In addition, the development of gold nanoshells, which are optically responsive in the near infrared
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(NIR), provides the opportunity to image biological targets through as much as 10 cm tissue, making it possible to image virtually any tissue in the body without requiring fiber optics [92]. NIR imaging of targeted gold-coated nanoparticles containing a dielectric silicon core shows promise as a means to detect cancer’s molecular signatures. By varying the coating’s thickness and the dielectric core’s diameter, it is possible to tune the particle’s optical absorption and scattering spectra from the ultraviolet (UV) to the mid-IR portion of the spectra [107]. This is an example of one of the appealing characteristics of nanotechnology: Specific physical properties can be engineered into the nanodevice. The surface chemistry of the nanoshells can be modified with PEG to increase biocompatibility and with sulfide-based linkers to allow the particles to be functionalized with targeting ligands [104]. Animal experiments demonstrated that gold/silicon nanoparticles coated with antibodies to the breast carcinoma marker Her-2 were able to detect the tumors successfully in vivo. Overexpression of this cell-surface protein is associated with aggressive forms of breast cancer and occurs in 25–30% of patients who respond poorly to standard chemotherapeutic regimens. Once the particles were localized to tumor tissue, increasing the power of the NIR beam for 4 min increased the particle’s temperature by an average of 37.4°C, enough to induce irreversible heat damage in the carcinoma cells [93]. Subsequent experiments demonstrated that tumorbearing mice subjected to this combination of detection and treatment experienced a marked increase in lifespan compared with control animals [108]. CLIO nanoparticles are also being used to track the movement of individual cells throughout the body. For example, CD8 cytotoxic T lymphocytes were labeled with CLIO paramagnetic nanoparticles and injected into mice. The labeled cells were detectable using standard MRI with a detection
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threshold of three cells per imaging point (voxel) in vivo. The labeled T cells were found to migrate to intact tumors [94]. Quantum dots have also been used to label migrating cells. Tumor cells labeled with quantum dots were imaged easily in vivo using emission spectrum multiphoton-scanning microscopy, providing a novel method for monitoring tumor cell extravasation [5]. It should be possible to use differential labeling, a capability offered by nanoparticles such as quantum dots, to track multiple cell types as they interact with one another in vivo.

APPLICATION OF NANOTECHNOLOGY: ENHANCED IN VITRO DIAGNOSTICS
The impact of nanotechnology on biology is certainly not limited to applications within the body. Indeed, the development and use of nanoscale analytical tools are the most promising areas of immediate benefit. Many of the efforts in developing nanoscale in vitro or ex vivo measurement and molecular detection systems rely on the methods being developed to construct nanoscale electronic circuits. For example, 1–2 nmwide, boron-doped silicon nanowires laid down on a silicon grid can be coated with antigens to provide real-time detection and titering of antibodies [97]. Antibody binding to immobilized antigen produces an immediate, measurable change in conductance at antibody concentrations below 10 nm. In the same study, nanowires derivatized with the calcium-binding protein calmodulin provided real-time measurements of calcium ions at physiologic levels. More recently, investigators have developed methods for chemically modifying lithographically etched silicon nanostructures to enable attachment of a wide range of molecules as the first step for creating versatile, chip-based biosensors [98]. Silicon-based arrays made of antibody-conjugated nanowire field effect transistors have also been multiplexed to simultaneously detect single copies of multiple viruses [99]. Functionalized carbon nanotubes can also function as highly specific electronic biosensors [100]. Individual nanoscale particles can also provide remarkable analytical sensitivity in a variety of in vitro assays, in real time and without the use of radioisotopes. Semiconducting quantum dots labeled with tumor marker antibodies, such as anti-Her-2, can greatly reduce the time needed to assess biopsy tissue by eliminating most of the steps needed to prepare a sample for analysis. The labeled quantum dots can simply be added to the biopsy tissue and the results viewed using fluorescence microscopy [109]. Moreover, by controlling the diameter of the particle during synthesis, quantum dots can be engineered to fluoresce at wavelengths ranging from UV to NIR. It is therefore possible to create multiplexed assays using differently colored quantum dots, each labeled with a different substrate [95]. Similarly, gold nanoshells, whose optical properties also depend in a predictable manner on size, have also been derivatized with a variety of polymers and biomolecules. These conjugates have been used to detect picogram-per-milliliter quantities of antibodies and other biomolecules in whole blood. Quantitative measurements can be obtained within 30 min [96].

A discussion of in vitro and in vivo applications of nanotechnology would not be complete without recognition of the instrumentation used to characterize these multifunctional entities. In addition to the traditional spectroscopic methods used to analyze functionality, purity, and molecular weight of chemical molecules, nanoparticles are often characterized by gel electrophoresis, electron microscopy, and the noteworthy capabilities of atomic force microscopy (AFM) [110, 111], which uses a nanoscale probe attached to a cantilever to sense the inter-atomic interactions between the probe and the substrate, analogous to the stylus of a phonograph sensing the grooves on a vinyl record. This instrument is exquisitely sensitive, often achieving spatial resolution of a few angstroms. Although initially a workhorse for the semiconductor industry, AFM imaging is now widely used in biological applications for elucidating biomolecular structures under physiological conditions [112, 113], determining the binding properties of antibody antigens and receptor ligands [114], observing the surface topology of viruses [115], and molecular-scaled imaging of pathologies [116] and histological features [117, 118]. Additional methods to characterize the physical properties of nanoparticles, such as purity, size, size distribution, and hydrodynamic radius, include high-performance liquid chromatography (HPLC), gel permeation chromatography (GPC) or size exclusion chromatography, capillary electrophoresis, and field flow fractionation (FFF). GPC relies on a size-exclusion column that separates the sample into discrete components and determines molecular weight or size distribution [119]. GPC is especially practical for analysis of organic nanoparticles, such as polymers and dendrimers. Traditional liquid chromatography, such as HPLC, uses pressure-driven flow to separate molecules based on their interaction with the stationary phase. FFF, conversely, relies on the differential retention of particles flowing through an electronic field or gradient applied at right angles to a capillary column [120]. The “nontortuous” path offered by FFF is therefore more compatible in fractionating less robust nanoparticles, such as liposomes, and those that may interfere with a traditional chromatographic column, such as metal particles. GPC and FFF use optical methods that detect the eluted peaks by absorbance, refractive index, and light scattering to measure the particle’s hydrodynamic size.

CONCLUDING REMARKS
Although there is a general sense that the technological base in nanotechnology is developed sufficiently to enable biologists to make ready use of these tools and materials, there are still fundamental questions about these materials that must be answered if nanotechnology is ultimately to have a significant impact extending beyond the laboratory and into the clinic. For example, there is a need for better characterization of nanotechnology constructs and production of “reagent-grade” nanomaterials, which permit comparisons between researchers. “Standardized” assays also need to be developed that facilitate rigorous evaluation of nanomaterials in terms of their toxicity and efficacy. The field would also benefit from increased interdisciplinary and international collaborations to verify more
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quickly and extend results to rapidly emerging areas of interest. These areas include nanomaterial development, production, environmental and health impact, bioinformatics, and modeling and simulation tools. Along with the prospects that nanotechnology holds for medical innovation comes the caveat that this is uncharted scientific territory and may have potential risks and hazards. There is evidence that certain nanoscale particles can have detrimental effects on living organisms. Carbon nanoparticles, for instance, have been shown to induce lipid peroxidation in the brain cells of fish and pulmonary inflammation in rats [121, 122]. The Royal Society and The Royal Academy of Engineering, a group that actively monitors this technology, has published a critical review of best practices for nanoparticle risk assessment [123]. On another front, the NNI has set aside $106 million in funding for research into the ethical, environmental, and health implications associated with nanoparticles [124]. Whether actual or perceived, the potential health risks associated with the manufacture, distribution, and use of nanoparticles must be balanced by the overall benefit that nanotechnology has to offer biomedical science, such as the therapeutic and diagnostic applications described in this article. Although nanotechnology is a relatively young field, it is developing rapidly, thanks to a strong foundation of material science and engineering. Biologists are using this innovative technology to overcome boundaries common to cell biology and clinical medicine. As more biologists learn about the capability of nanotechnology and develop cross-disciplinary collaborations with physicists, engineers, and material scientists, these breakthroughs will undoubtedly increase in magnitude and quantity.

ACKNOWLEDGMENTS
This project has been funded in part with federal funds from the National Cancer Institute, National Institutes of Health, under Contract No. N01-C0-12400. The content of this publication does not necessarily reflect the views or policies of the Department of Health and Human Services, nor does mention of trade names, commercial products, or organization imply endorsement by the U.S. Government. The author thanks Dr. Joe Alper for his considerable assistance during the preparation of this review and Allen Kane for designing the figures.

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