THE NOTION OF nanotechnology has evolved since its inception as a fantastic conceptual idea to its current position as a mainstream research initiative with broad applications among all divisions of science. In the first part of this series, we reviewed the structures and principles that comprise the main body of knowledge of nanoscience and nanotechnology (58). This article reviews and discusses the applications of nanotechnology to biological systems that will undoubtedly transform the foundations of disease diagnosis, treatment, and prevention in the future. Specific attention is given to developments in diagnostics and imaging at the nanoscale level. The use of nanoparticles and nanomaterials as biodetection agents for deoxyribonucleic acid and proteins is presented. In addition, nanodevices, such as nanowires, nanotubes, and nanocantilevers, can be combined with nanoarrays and nanofluidics to create integrated and automated nanodetection platforms. Molecular imaging modalities based on quantum dots and magnetic nanoparticles are also discussed. This technology has been extended to the imaging of intracranial neoplasms. Further innovation within these disciplines will form the basis for the development of mature nanomedicine. The final article of the series will focus on additional advancements in nanomedicine, namely nanotherapy and nanosurgery, and will cover the innovations that will lead to the eventual realization of nanoneurosurgery.
Arevolution in nanoscience and nanotechnology has occurred in the past decade. Because of the enormous potential of nanotechnology, the federal government created the National Nanotechnology Initiative (NNI) in 2000 (71). Federal funding for nanotechnology research and development has increased more than eightfold, from an initial $116 million in 1997 to a proposed $982 million in 2005. Currently, the NNI funds more than 100 nanoscience and technology centers and networks of excellence for individuals and institutions.
In June of 2000, the National Institutes of Health (NIH) held its third Bioengineering Consortium (BECON), entitled “Nanoscience and Nanotechnology: Shaping Biomedical Research” (4). One of the goals of the symposium was to “develop a better understanding of nanotechnology as it pertains to biological and medical applications.” The discussions addressed eight contemporary areas of nanoscience and nanotechnology that were most pertinent to biomedicine:  synthesis and use of nanostructures,  applications of nanotechnology to therapy,  biomimetic nanostructures,  biological nanostructures,  electronic/biology interface,  devices for early detection of disease,  tools for the study of single molecules,  nanotechnology and tissue engineering.
In 2002, the NIH announced a 4-year program for the application of nanoscience and nanotechnology in medicine. Most recently, in May of 2004, the NIH announced the launch of the NIH Nanomedicine Roadmap Initiative (70), which is designed to obtain a comprehensive set of measurements on molecules and assemblies of molecules, and use those measurements to understand molecular pathways and networks, and use that knowledge to drive the design and development of new nanomachines and technologies to improve human health.
In support of this initiative, the NIH has sponsored approximately 20 Nanomedicine Development Centers, including the Emory University Center for Development of Biological Nanosensors and the Nanomedicine Center in Membrane Signaling at the University of California, Berkeley, among others.
Nanomedicine, as defined by the NIH, refers to highly specific medical intervention at the molecular scale for curing disease or repairing damaged tissues. This article reviews and discusses the applications of nanoscience and nanotechnology to biological systems that will, undoubtedly, transform the foundations of disease diagnosis, treatment, and prevention in the near future. This medical revolution will usher in the era of mature nanomedicine and will be instrumental in the eventual emergence of nanosurgery and nanoneurosurgery. Mature nanomedicine will involve the design and fabrication of molecular devices with anatomic precision, and then using them in patients to establish and maintain health. Nanoneurosurgery will use elements and capabilities of nanotechnology to diagnose and treat potential, or existing, central and peripheral nervous system diseases (58).
Medical Diagnostics at the Nanoscale level: Utilization of Nanoparticles and Nanomaterials
There are several advantages to using nanostructures as agents in biological detection. In certain cases, assays based upon nanomaterials have shown greater sensitivity, selectivity, and practicality when compared with conventional biodiagnostic systems (80). Characteristics that make nanomaterials attractive as probe candidates are their small size (1–100 nm), which corresponds to a high surface-to-volume ratio; their chemically tailorable physical properties, which are a direct result of the particles' size, composition, or shape, their unusual target binding properties, and their considerable structural stability. For example, a target-binding event involving a nanomaterial can have a significant effect on its physical and chemical properties given its small size, whereas the same binding event in a bulk material may have a negligible effect. The resultant alteration in the properties of the nanomaterial signals that the binding event of interest has occurred.
Current fabrication and synthesis protocols for nanomaterials use the purposeful modulation of their size, shape, and composition, as a means of acquiring superb control of their properties. In fact, the ability to carefully tailor the physical properties of nanomaterials is the foundation of their functionality in biodetection and medical diagnostics (Table 1) (1). Of note, the sizes, shapes, and compositions of quantum dots and metal nanoparticles can be systematically varied to produce materials with specific emissive, absorptive and light-scattering properties (Fig. 1), thereby making these materials ideal for multiplexed analyte detection (9, 30, 44, 89). By virtue of multiplexing, multiple targets can be detected with a single assay.
Nucleic acid sequences unique to every living organism and pathogen provide obvious targets for the identification and diagnosis of various diseases. With the advent of sequencing capabilities based on the polymerase chain reaction (PCR), nucleic acid sequence information is now widely available for many diseases. Despite representing the gold standard in terms of sensitivity (82), PCR analysis has a number of drawbacks, namely cost, sensitivity to contamination, complexity, time-consumption, lack of portability, and poor multiplexing capabilities (50). These drawbacks prohibit moving nucleic-acid based detection from the laboratory to point-of-care settings, including the outpatient clinic, the Third World, and first responder sites in the case of bioterrorism defense. In these settings, rapid, inexpensive, disposable detection assays are needed that do not require extensive processing or user experience.
Abnormal concentrations of certain proteins often signal the presence of specific diseases or cancers. Unfortunately, current protein detection schemes tend to identify abnormalities only after protein levels have exceeded critical threshold concentrations, usually after the underlying disease or cancer is significantly advanced. More sensitive detection methods are needed that would allow for earlier identification of a disease process and, hence, earlier treatment. The current gold standard in protein diagnostics is the enzyme-linked immunosorbent assay (ELISA), which relies heavily on organic fluorophore labeling. Molecular fluorophores also have many drawbacks, including susceptibility to photobleaching, broad absorption and emission bands, and a reliance on expensive, non-portable equipment (80). Again, these properties limit the use of the ELISA to the laboratory setting and preclude use in point-of-care settings. Currently, there is extensive interest in the use of nanomaterials in assays for nucleic acids and protein markers for many diseases, with the hope that the advantages of nanomaterials will eventually overcome the limitations imposed on biodiagnostics by PCR, ELISA, and molecular fluorophores.
Detection of DNA using Nanoparticles
An early indication of the potential of nanoparticles as biodetection agents was reported in 1996, when it was observed that oligonucleotide-modified gold nanoparticles (only 13 nm in size) would aggregate when placed in solution with complementary target deoxyribonucleic acid (DNA) (64). The aggregation process caused the solution to change from red to blue, as shown in Figure 2, and provided a simple and inexpensive way to diagnose disease (18). This technique offered several advantages when compared with conventional fluorophore assays in that it displayed high sensitivity and could detect single-base pair mismatches, it was rapid and easy, its optical read-out was straightforward and did not require expensive equipment, and it had the potential for multiplexing (8). Despite improvements, this assay was limited by its sensitivity, which was not as good as conventional fluorophore assays. However, it inspired others to explore the potential for nanomaterials in biodiagnostic applications.
Nanoparticles have been added to several conventional DNA assays in order to improve sensitivity. For example, surface plasmon resonance (SPR) is a technique used to detect real-time DNA hybridization on targets. When modified with gold nanoparticle tags, more than a 1000-fold increase in sensitivity was observed when compared with the unamplified binding event (61). Recently, an ultrasensitive DNA assay was developed using silica nanoparticles (110). The silica nanoparticles, sized between 2 and 100 nm, have a silica matrix that entraps a large number of fluorophores, a process known as 'doping.' These nanoparticles were functionalized with oligonucleotides and used as labels for chip based “sandwich” DNA analysis. This method results in a notable detection limit of approximately 1 femtomolar (1 × 10−15 M) and is able to discriminate one-base mismatched DNA sequences.
Another highly sensitive chip-based system, termed the “scanometric” assay, is composed of an oligonucleotide-modified glass slide, a gold nanoparticle probe, and the target DNA, as developed by Mirkin et al. (64). After capture of the target DNA between the capture strand and the nanoparticle probe (capture-strand/target/nanoparticle “sandwich”), catalytic reduction of silver onto the gold nanoparticles results in significant amplification of the target signal (Fig. 3A). This amplification allows for the use of a conventional flatbed scanner as a reader (hence the term “scanometric”), and detects target concentrations as low as 50 femtomolar (50 × 10−15 M), more than a 100-fold increase in sensitivity compared with traditional fluorescent assays. This assay can also detect single base-pair mutations with high specificity. Modifications to this technique have recently demonstrated the detection of the MTHFR gene from only a 20 μg sample of human genomic DNA, as well as the mecA gene from methicillin-resistant Syaphylococcus aureus (the mecA gene confers resistance to the antibiotic methicillin) (87). In addition to maintaining sensitivity on the order of 50 femtomolar levels, the target and probe hybridization process was reduced to a single step requiring only 1 hour to complete. Furthermore, a simplified optical detection system (Fig. 3B) was found to be practical and feasible, indicating that complex instrumentation is not required to achieve extremely high sensitivity using nanoparticle probe technology. Moreover, this study demonstrates the ability to detect genomic DNA from infectious agents and human genetic disease predispositions without the need for PCR amplification, and at concentrations relevant to real medical diagnostic applications.
A recent assay, which couples silver enhancement with an additional indirect target amplification method, pushes nanoparticle-based detection limits to levels previously attained only by using PCR. This assay, called bio-bar-code amplification (BCA), uses oligonucleotides that act as bar codes for target DNA (67). This assay relies on two nanoparticles: gold nanoparticles modified with both target capture strands and bar code strands that are subsequently hybridized to bar code DNA and magnetic microparticles modified with target capture strands. In the presence of target DNA, the gold nanoparticles and the magnetic microparticles form sandwich structures that are magnetically separated from solution and washed to remove the hybridized bar code DNA. The bar codes (hundreds to thousands per target) are detected using the scanometric approach, resulting in detection limits as low as 500 zeptomolar (500 × 10−21 M), which corresponds to only approximately 10 target strands in the entire 30 microliter sample (Fig. 4). This method provides high selectivity with a sensitivity that is comparable to PCR-based approaches, without the need for costly, complex, and time-consuming enzymatic amplification. In fact, one advantage of the DNA bio-bar-code assay is that the entire assay can be carried out in 3 to 4 hours, regardless of target concentration. Additionally, it is well suited for multiplexing as bar codes can be synthesized for virtually any target of interest.
DNA assays based on nanoparticle probes coupled with electrochemical reactions have also been reported (15, 74). If oligonucleotide capture strands are immobilized in the gap between two electrodes, and a sandwich assay using gold nanoparticles similar to the scanometric approach is performed, DNA can be detected as a measure of the change in electrical current or resistance between the two electrodes. In the absence of target DNA, there is no current flow across the electrode gap. In the presence of target DNA, the associated nanoparticle probes, and the catalytically deposited silver, current can flow between the electrodes (Fig. 5). This method has sensitivity in the femtomolar range, but, more impressively, demonstrates a mutation selectivity factor of 100,000:1, which is notable considering that an analogous experiment performed using a molecular fluorophore produces a mutation selectivity factor of just 2.6:1. This type of electrical detection method offers the possibility of portability, which could translate into use in a variety of point-of-care settings.
Detection of Proteins using Nanoparticles
Detection strategies for proteins using nanoparticles typically rely on the specific interactions between nanoparticle bound antibodies with the target protein (80). Binding events will generally have an effect on the optical signal of the nanoparticles. There are numerous ways to conjugate antibodies to the surface of nanoparticles, an aspect that makes these assays quite versatile. One recent approach uses antibodies conjugated to the surface of gold nanoshells to detect proteins in saline, serum, and whole blood (34). Nanoshells are a layered, spherical nanoparticles consisting of a dielectric core (silica) surrounded by a thin metal shell (gold or silver), and possess optical absorption and scattering properties. By varying the thickness of the core and the shell layers, the optical properties can be precisely controlled (101) (Fig. 6). After binding with the target protein, the antibody-functionalized nanoshells aggregate, causing a change in their emission spectra that can be detected optically. This assay is simple to perform and rapid, producing results within 10 to 30 minutes, and detects proteins in the range of 88 to 0.8 ng/ml, which is within the range of ELISA. An important aspect of this assay is that no sample preparation is required, and it can detect proteins in serum and whole blood, which is important for an assay designed to function in non-laboratory sites where sample preparation and purification is limited.
The BCA method used for DNA (Fig. 4) has also been used for protein detection, and is unparalleled in terms of assay sensitivity with respect for protein markers. The methodology is similar to that used in the detection of DNA, with two nanoparticle components. In an assay designed to detect prostate specific antigen (PSA), the protein target was detected at 30 attomolar concentration, approximately six orders of magnitude better than what is possible with conventional ELISA (68). With this method, protein markers that signal the presence of various diseases, such as prostate and breast cancer, Alzheimer's disease (AD), and acquired immune deficiency syndrome can be detected at levels that are currently unattainable. Obviously, earlier detection should lead to earlier treatment and, hopefully, improved patient outcomes.
In fact, the bio-bar-code assay was recently used to measure the concentration of amyloid-beta-derived diffusible ligands (ADDLs), which are suspected to be a pathogen marker for AD, within the cerebrospinal fluid (CSF) of 30 individuals (24). Although elevated levels of ADDLs have been found in the brains of AD patients (26), proof of their existence within the CSF has not been shown previously. It is possible that the concentration of ADDLs within the CSF is so low during the early stages of the disease that they cannot be detected with conventional ELISA or blotting assays. Using the principles of the bio-bar-code assay as previously discussed, this study showed that the levels of ADDLs for individuals diagnosed with AD were consistently higher than those of control individuals (P›0.0001). The high sensitivity of the BCA assay enabled measurement of ADDL levels of approximately 200 attomolar (200 × 10−18 M) in normal patients, and higher levels in AD patients. This study shows that the ultrahigh sensitivity bio-bar-code assay can be used to measure the concentration of ADDL in CSF at clinically relevant concentrations. This advance opens up the opportunity to study a wide range of potential pathogenic markers in CSF, which could lead to informative diagnostic tools relative to neurological diseases. Importantly, this study marks one of the first real applications of nanotechnology to medical diagnostics to date.
The Role of Nanodevices in Medical Diagnostics
1. Nanotubes and Nanowires
Carbon nanotubes and nanowires are being explored as signal transducers in the electrical detection of gases (54, 66), small molecules (96), proteins (11, 12, 75, 98), and DNA (29, 105). Similar to nanoparticles, nanotubes and nanowires can be functionalized, or covalently modified with biomolecules to detect targets of interest and to probe biological systems at the nanometer scale (7, 104).
Hahm and Lieber (29) reported on silicon nanowire electronic devices that function as ultrasensitive and selective detectors of DNA. The surfaces of the silicon nanowire devices were functionalized with peptide nucleic acid (PNA) receptors designed to recognize wild type versus a mutation site in the cystic fibrosis transmembrane receptor gene (29). The nanowire sensor device consisted of a silicon nanowire bridging two electrodes and a microfluidic channel, as shown in Figure 7. Introduction of target DNA into the assay resulted in a rapid and immediate change in conductance, while there was a negligible effect upon introduction of the mutant DNA. The target DNA could be detected at concentrations as low as 10 femtomolar (10 × 10−15 M).
Patolsky's (75) group has also modified nanowire arrays with antibodies for influenza A virus, which showed discrete conductance changes after binding and unbinding in the presence of influenza A, but not other viruses (e.g., adenovirus or paramyxovirus), shown in Figure 8. Furthermore, studies of nanowire devices modified with antibodies specific for either influenza or adenovirus show that multiple viruses can be detected in parallel. The possibility of large-scale integration of these nanowire devices suggests potential for simultaneous detection of a large number of distinct viral agents.
Although carbon nanotubes and nanowires are not as easily modified or functionalized as spherical nanoparticles, they provide the distinct advantage of rapid, real-time, label-free detection. With continued research into methods of surface modification, alignment, and integration with microelectrode devices, nanowire and nanotube systems may become viable options as nanostructured biodiagnostic devices with implications in genomics, proteomics, biomedical diagnostics, drug discovery, genetic screening, and biothreat detection (80).
2. Functionalized Nanocantilevers
As previously discussed in Part I of this series, nanocantilivers have been fabricated with enormous sensitivity as mass sensors (38). In addition, they can also be functionalized with antibodies or other biomolecules to operate as highly sensitive, label-free, real-time detectors of genes, messenger ribonucleic acid (mRNA), proteins, bacteria, and viruses (3, 21, 28). In fact, using this technology, sensitivity for bacteria and viruses has been reported at the single cell level (27, 39, 40). Biomolecular binding of the target of interest (DNA, protein, bacteria, etc.) to the antibody-treated regions of the nanocantiliver sensor alters the total mass of the mechanical oscillating cantilever, changing its natural resonant frequency, which is then detected mechanically or electrically (Fig. 9). One study demonstrated that microcantilevers could be used to detect prostate specific antigen (PSA) at clinically relevant concentrations, making this methodology a useful diagnostic assay for prostate cancer (106).
3. Microarrays and Nanoarrays
The use of miniaturized, chip-based, array detection methods, known as “microarrays,” has been prevalent in almost all areas of health-related research for some time (17). This type of biomolecular assay allows for extensive parallel processing of a variety of targets in a small area, as well as a reduction in processing times. Such high throughput detection systems have been remarkably valuable in genomics and proteomics research (62, 84, 95). An example of a protein detection microarray is shown in Figure 10. To fabricate this array, a robot is used to deliver nanoliter volumes of protein samples to a microscope slide, resulting in spots approximately 150 to 200 μm in diameter, and yielding 1600 spots per cm2 (62).
Additional miniaturization and fabrication of nanoarrays would generate many orders of magnitude increase in multiplexed detection in the same area as a microarray. In addition, nanoarrays should allow for significantly smaller sample volumes and possibly lower detection limits. A variety of methods have been developed to pattern biomolecules, such as DNA and proteins, onto surfaces within the nanoscale range. One of the most intriguing is the process of dip-pen nanolithography (DPN), which can be used to directly control the patterning of oligonucleotides and proteins onto surfaces, with a spot size as small as 15 nm (14, 60). An entire array fabricated using DPN would result in 100,000,000 spots occupying the same area as a single 200 × 200 μm2 spot in a conventional microarray, as shown in Figure 11 (25). Using this technology, it is conceivable that someday the entire human genome could be screened for single-nucleotide polymorphisms on a single chip (56). Such an application would require more than 10,000 of today's state-of-the-art chips, yet would be feasible with a single 2 × 2 cm2 chip using nanoarray technology with a spot size of 150 nm (25). Recently, DPN was used to generate nanoarrays with monoclonal antibodies against human immunodeficiency virus-1. These arrays were coupled to nanoparticle probes and demonstrated the detection of human immunodeficiency virus from samples of human plasma with a detection sensitivity that exceeded that of conventional ELISA by more than 1000-fold (59).
4. Microfluidics and Nanofluidics
Although microfluidic biotechnology has existed for some time, multilayer elastomer microfluidics is a powerful new technology that integrates many pumps, valves, and channels within an easily fabricated microchip (37). The development of alternative fabrication methods using soft lithography with silicone rubber has enabled this technology to perform multiple operations in parallel, such as cell sorting, DNA purification, and single-cell genetic profiling (22, 36, 97). In addition, this technology offers large-scale multiparameter analysis, with several potential applications including single cell dissection and analysis (e.g., from needle biopsies) and multiparameter disease detection from tissues or blood (Fig. 12).
Currently, truly integrated microfluidic systems that process only nanoliters of sample material are emerging, that can be termed “nanofluidic systems” (35). A recently fabricated microfluidic chip demonstrated automated nucleic acid purification from small numbers of bacterial or mammalian cells. All the steps in the process, such as cell isolation, cell lysis, DNA or mRNA purification, and recovery, were carried out on a single microfluidic chip in nanoliter volumes without any pre- or postsample treatment. Measured amounts of mRNA were extracted from a single mammalian cell and recovered from the chip (Fig. 13). The achievement of extensive analysis on a single chip represents significant progress toward realization of the “lab-on-a-chip,” where a complete analysis system is fully integrated, automated, and portable.
Integration of Nanodevices and Nanoparticles in Medical Diagnostics
Nanowires, nanotubes, and nanocantilevers have all been functionalized in similar ways and incorporated into nanoelectromechanical transistors and biosensors (11, 12). In addition, these forms of nanotechnology can be fabricated as arrays, enabling multiple detection assays to be performed in parallel (63). Furthermore, these technologies can be coupled with nanoparticle probes to increase sensitivity (88), and can be integrated with elastomer micro- and nanofluidics to create miniaturized and automated microfluidics/nanotechnology platforms, as shown in Figure 14 (72). In this particular example, the microfluidic channel provides a means of sample delivery to the array of nanowire sensors. It is likely that these types of platforms will emerge within the next few years with the ability to integrate multiple operations, such as cell sorting and serum purification, as well as the ability to detect and quantify 5 to 10 biomarkers from single cells or from very small sample fluid volumes (37).
Medical and Molecular Imaging with Nanotechnology
A number of nanoparticles have been explored as potential imaging agents (Table 2). Two of the best studied are quantum dots and magnetic nanoparticles.
1. Quantum Dots
By way of review, quantum dots (qdots) are nanometer sized semiconductor nanocrystals, made of cadmium selenide (CdSe), cadmium sulfide (CdS), or cadmium telluride (CdTe), surrounded by a polymer coating that is inert. The core of the nanocrystal is chosen depending on the emission wavelength range that is being targeted: CdS for ultraviolet blue, CdSe for the majority of the visible spectrum (Fig. 1), and CdTe for the far-red and near-infrared spectrum. The specific size of the particle determines the exact color emission for any given quantum dot, a feature that can be precisely controlled during dot synthesis.
Qdots absorb photons from white light within their core then re-emit essentially monochromatic light at a specific wavelength a few nanoseconds later with extremely bright fluorescence (30). In fact, qdot fluorescence is so bright that the detection of a cell carrying only a single crystal is possible (65). Furthermore, because qdots absorb light over a very broad spectral range, it is possible to excite many dots with a single light source (each emitting a different color), thereby allowing the detection of multiple markers simultaneously (100). The polymer coating provides a safeguard against the well-known inherent toxicity of cadmium, but also enables the attachment of a number of targeting molecules, such as monoclonal antibodies against a particular target of interest (10). Qdots can be targeted by both passive and active means. In passive targeting, qdots injected systemically reach their targets by exploitation of biophysical properties that lead to passive accumulation in a target organ. In active targeting, qdots injected systemically reach their targets by exploitation of molecular recognition (receptor-ligand type interactions) to accumulate at the target site (73).
Because of their intriguing optical properties, qdots have been used as fluorescence labels for biological staining experiments for several years. They have found application as labels of cellular structures and receptors (Fig. 15) (13, 107), can be incorporated within living cells (10), and can be used to track the path and fate of individual cells after loading them with qdots (Fig. 16), which is important for many biological, but also medical, questions (16, 43, 76).
Of particular importance is the use of qdots as contrast agents for imaging purposes in medical diagnostics. In one study, water soluble qdots (CdSe-ZnS nanocrystals encapsulated by an amphiphilic polymer) were injected into the tail veins of live mice (57). Using multiphoton microscopy, the qdots were visualized dynamically through the skin in capillaries hundreds of micrometers deep after the dots had labeled the vasculature in a manner analogous to angiography. Using this method, the researchers were able to easily measure blood flow velocity and to clearly detect the heart rate from temporal undulations of the capillary wall, using noninvasive images obtained directly through the skin (Fig. 17).
Qdots have also been extensively studied as potential probes and labels in cancer targeting and imaging. In one study, qdots were used to label the breast cancer marker Her2 on the surface of both fixed and live cancer cells (107). Her2 is a cancer marker that is overexpressed on the surface of some breast cancer cells. Pathologists also use Her2 to identify tumors that are likely to respond to a specific chemotherapeutic agent, however, current testing procedures miss some tumors that would be sensitive to the drug (85). This study also demonstrated the ability to double label live cells, which has significant implications in terms of multiplexing (Fig. 18).
In a recent report, researchers from the Nie group described the development of multifunctional bioconjugated qdot probes for cancer targeting and imaging in live animals (23). They have also integrated a whole-body macro-illumination system with spectral imaging that enables ultrasensitive and multiplexed imaging of molecular targets in vivo. Core-shell CdSe-ZnS qdots were protected by sophisticated surface coating composed of an amphiphilic polymer and an additional protective ligand. The dots were also functionalized with a monoclonal antibody against a human prostate cancer antigen (PSMA) to provide active tumor targeting. Human prostate cancer cells were implanted subcutaneously into live mice and after a period of tumor growth, bioconjugated quantum dots were injected systemically into the tail vein. The qdots selectively labeled the tumors and generated intense signals that were detected with a novel imaging system (Fig. 19).
2. Magnetic Nanoparticles
Magnetic nanoparticles are becoming important versatile tools in medical and biological diagnostics. Although assays based on magnetic nanoparticles have been used to detect DNA, protein, enzymes, and viruses (77, 79, 109), they have become particularly useful as contrast agents in magnetic resonance imaging (MRI) (6, 86). Superparamagnetic iron oxide nanoparticles (SPIOs), also known as monocrystalline iron oxide nanoparticles (MIONs), consist of an inorganic core of iron oxide surrounded by a polymer coating of dextran or polyethylenglycol (PEG) molecules. The iron oxide crystal core is superparamagnetic, becoming magnetized when placed in an external magnetic field. The average size of the nanoparticles is 25 to 30 nm. Commercially available forms currently include Lumirem (iron oxide nanoparticles coated with silicon), Endorem (magnetite nanoparticles coated with dextran), and Combidex (iron oxide nanoparticles coated with dextran), among others (81). The surface polymer coating allows the nanoparticles to stay in circulation for prolonged periods of time. Eventually, they are taken up avidly by lymph nodes where they are internalized by macrophages (108).
Recently, lymphotrophic SPIOs were used to detect clinically occult lymph-node metastases in patients with prostate cancer (31). This study was particularly interested in patients with clinically occult disease such that the metastases had not caused an increase in the size of the lymph nodes, as these are not detectable with conventional imaging such as contrast enhanced MRI, computed tomography, or positron emission tomography. Current guidelines classify lymph nodes larger than 10 mm as cancerous, whereas normal-sized lymph nodes are generally classified as normal. Clinical studies have shown that SPIOs will only accumulate in non-cancerous lymph nodes and can, therefore, facilitate differentiation between cancerous and non-cancerous nodes (5, 93). Areas in lymph nodes replaced by metastatic tumor will not accumulate the SPIO nanoparticle contrast agent; therefore, affected nodes are characterized by failure of signal dropout on MRI scans (remain bright on MRI scans). Normal nodes avidly accumulate the SPIOs and demonstrate signal dropout (dark appearance on MRI scans). On a patient-by-patient basis, this study showed that the addition of lymphotrophic SPIOs increased the sensitivity of MRI scanning from 45 to 100%, with a specificity of 95.7%. Unexpectedly, even very small metastases (diameter,›2 mm) were occasionally identified within normal-sized lymph nodes (Figs. 20 and 21).
Similar lymphotrophic magnetic resonance imaging techniques were recently used to detect lymph node metastases in patients with colorectal and breast cancers (32). The use of lymphotrophic magnetic nanoparticles accurately distinguished metastatic from normal nodes with an overall sensitivity of 98% and specificity of 92%, and provided further evidence for the feasibility of highly sensitive nodal cancer staging by noninvasive imaging (Fig. 21E). In fact, these agents have been investigated as markers of metastatic disease in a variety of cancers, including breast, prostate, renal, bladder, uterine, rectal, lung, head, and neck cancers to date (2, 33, 45, 47, 52, 53).
Second generation superparamagnetic nanoparticles are known as cross-linked iron oxides (CLIOs) that are produced by cross-linking the dextran surface coating. This generates a more stably coated nanoparticle that can be functionalized by conjugating proteins, antibodies, and oligonucleotides for active-targeting in vivo. This gives rise to biomolecule-nanoparticle conjugates with unique biological properties (46, 78). In addition, the conjugation process is very versatile and even allows for the attachment of additional reporter labels, such as fluorescent tags, thereby creating a single contrast agent with both magnetic and optical properties.
Magnetic nanoparticles are finding widespread application in medical diagnostics and the biological sciences. In addition to their use as biosensors and as molecular markers of cancer, magnetic nanoparticles have been investigated as imaging agents to detect atherosclerotic lesions in patients with symptomatic carotid disease (41, 55, 91), and as markers of inflammation (48) and angiogenesis (102). Modified CLIO nanoparticles were recently developed to function as probes for apoptotic cells (83), as markers of in vivo gene expression (99), and as probes for the detection of dynamic molecular interactions inside living cells (103).
Of particular interest is the use of magnetic nanoparticles for the imaging and labeling of intracranial neoplasms (19, 20). One interesting study investigated the use of MIONs as intraoperative and postoperative imaging agents (51). In this study, malignant glioma cells were stereotactically implanted into the basal ganglia of male rats. Two weeks later, MIONs were injected intravenously and MRI scans were obtained that demonstrated large intracranial neoplasms. After open craniotomy with intentional partial resection of the tumor, postoperative MRI scans revealed the expected residual tumor signal. Significantly, however, no new hyperintensities to indicate surgically induced phenomena were observed. By way of comparison, after the administration of gadolinium contrast, intense contrast enhancement that would have prevented the distinction between residual tumor and surgically induced contrast enhancement was observed (Fig. 22). It is possible that the use of MIONs might be useful as intraoperative contrast agents as they offer a means of detecting residual tumor without superimposed, confusing, surgically induced contrast enhancement that is problematic with conventional paramagnetic contrast agents (gadolinium).
A multimodal nanoparticle was also recently explored as a preoperative MRI contrast agent and intraoperative optical probe (49). A CLIO nanoparticle was functionalized on its surface with Cy5.5, an optically detectable fluorescent label. In the experimental model, a 9L rat gliosarcoma cell line was transfected to express green fluorescent protein (GFP) and was surgically implanted in the intracranial space of rats. After a period of tumor growth, the rats were intravenously injected with the CLIO-Cy5.5 nanoparticle, and MRI scans were obtained that clearly demonstrated the presence of an intracranial tumor. As shown in Figure 23, a craniotomy was then performed and the brain tissue overlying the tumor was removed. Noninvasive optical imaging was performed using a custom built imaging system that included separate channels for white light (as seen by the unaided eye), a filter set for GFP imaging, and a filter set for Cy5.5 imaging. It was demonstrated that Cy5.5 fluorescence clearly visualized the tumor, as indicated by the correlation of tumor extent as determined by GFP fluorescence (the gold standard of tumor extent in this model). It should be noted that near-infrared-fluorescent imaging permitted the visualization of the nanoparticle probe within the glioma through several millimeters of overlaying tissue, i.e., before complete tumor dissection had been performed. This study indicated the feasibility of using a multimodal nanoparticle as an intraoperative imaging agent capable of providing a strong localizing signal under real-time conditions.
An ultrasmall superparamagnetic iron oxide nanoparticle (USPIO) was recently evaluated as an imaging agent in patients with malignant brain tumors both pre- and postoperatively (69). This viral-sized nanoparticle is coated with dextran and is known to show enhancement of malignant intracranial tumors (92). All patients harbored tumors that enhanced on initial MRI scans. But, importantly, in several of the patients, there were areas that demonstrated enhancement with the nanoparticle agent, but not with gadolinium (Fig. 24). In fact, the nanoparticle agent showed either extended regions of the main tumor mass or new lesions altogether. Furthermore, in one patient, images were obtained with only a 0.15 Tesla intraoperative MRI scan that also clearly showed a nanoparticle-enhancing lesion that was not detected with gadolinium. In follow-up images obtained 5 months later, gadolinium enhancement was detected at the sites where only nanoparticle enhancement had been noted previously. This study again confirmed that USPIO nanoparticles could be used to identify residual tumor postoperatively, without the need to readminister the contrast agent. The mechanism for prolonged MRI signal change of USPIOs seems to result from a long plasma half-life (24–30 h) to breach the blood-brain barrier, followed by internalization within reactive cells (astrocytes and microglia) in and around the tumor (92).
One of the limitations of multimodal nanoparticle probes has been internalization within reactive cells, rather than within the glioma cells themselves. Recently, a multimodal nanoprobe capable of targeting glioma cells directly was fabricated. This probe was created by coating iron oxide nanoparticles with polyethylene glycol (PEG) polymers that were subsequently functionalized with two moieties: chlorotoxin (Cltx), a glioma-targeting molecule, and Cy5.5, a near-infrared fluorescent tag (94). This multimodal nanoprobe is multifunctional because MRI scans, as well as fluorescence imaging, can detect it. 9L glioma cells were cultured with nanoparticle probes lacking the Cltx molecule (control) and with nanoparticle probes functionalized with the Cltx molecule, as shown in Figure 25. This study showed that the nanoparticle-Cltx conjugates targeted the glioma tumor cells and that the cellular uptake of the nanoparticle probes could be visualized by fluorescence imaging at the cellular level.
The use of nanoparticles, nanomaterials, and nanodevices is finding broad application in the burgeoning field of nanomedicine. The advantages of using these devices in medical diagnostics and imaging cannot be ignored. Newer and more sophisticated applications are continually being developed. As nanomedicine moves from the research arena to mainstream clinical use, a veritable medical revolution will occur. The ability to diagnose disease via highly developed assays and imaging based on nanotechnology will forever change the practice of medicine.
Part III in this series will focus on additional developments in nanomedicine, namely, nanotherapy, nanosurgery, and nanoneurosurgery.
Bleaching: The loss of fluorescence usually owing to photochemical reactions.
Cantilever: A beam-shaped device that resembles a diving board and is a form of MEMS (microelectromechanical systems). These devices have the ability to detect extremely small displacements and are frequently used as highly sensitive mass sensors.
Lab-on-a-Chip Devices: Multicomponent analysis microchips, also known as micro total analysis systems (microTAS) or sentinel sensors. These sensor devices will contain all the necessary components (pumps, valves, detectors, etc.) to run an entire analysis, from sample preparation, through analysis, to final detection. These devices will be scaled down in size into hand-held, portable, ‘point-of-care’ sensors.
Magnetic Nanoparticles: Nanoparticles with magnetic properties. Subtypes include superparamagnetic iron oxide nanoparticles (SPIOs) and monocrystalline iron oxide nanoparticles (MIONs), both of which consist of an inorganic core of iron oxide with a surrounding polymer coating. The iron oxide core becomes magnetized when placed in an external magnetic field.
Multiplexed Analysis: The ability to detect multiple targets simultaneously with a single assay.
Nanoarray: An ultrasensitive array with dimensions on the nanoscale. Used for biomolecular analysis.
Nanobiotechnology: Investigations that consider biological events on the scale of individual molecules. Used to describe assay systems and manipulations of the physical world at the nanometer and micrometer scale with the intent of detecting, separating, analyzing, manipulating, and characterizing cells or biological molecules of interest.
Nanocrystal: A single crystal, usually inorganic in nature, in the size range of 1 to 100 nm. Used interchangeably with quantum dot.
Nanofluidic Systems: Controlling nanoscale amounts of fluids.
Nanomedicine: Nanotechnology applied to medicine is the controlling of biologically relevant structures with molecular precision. Mature nanomedicine will involve the design and fabrication of molecular devices with anatomic precision, and then employing them in patients to establish and maintain health.
Nanometer: 10−9 meters, a billionth of a meter
Nanoneurosurgery: The application of nanotechnology to the spectrum of nervous system diseases. Nanoneurosurgery will use elements and capabilities of nanotechnology to diagnose and treat potential, or existing, central and peripheral nervous system diseases.
Nanoparticles: A generic term that refers to a structure with a diameter in the 1 to 100 nm range, which is generally functionalized as a drug delivery system or a targeting agent.
Nanoscale: Characterized by length scales ranging from 1 to 100 nm.
Nanoscience: The science relating to and pertaining to events in the nanoscale realm.
Nanoshell: Nanoscale layered metal spheres that can absorb and scatter light at multiple wavelengths. Nanoshells consist of a silica core surrounded by a thin metal shell, usually gold or silver.
Nanotechnology: Defined by NASA as: “The creation of functional materials, devices and systems through control of matter on the nanometer length scale (1–100 nm), and exploitation of novel phenomena and properties (physical, chemical, biological) at that length scale.” The application of nanoscience in order to control processes on the nanometer scale, i.e. between 0.1 nm and 100 nm.
Nanotubes: Exquisitely thin, long macromolecules composed of carbon, and as regular and symmetric as crystals.
Nanowires: Rod-like structures with diameters in the range of nanometers, usually from 20 to 50 nm. Nanowires are available in metallic, semiconductor, magnetic, oxide and polymer varieties and are useful as components in MEMS or NEMS, and function as chemical or biological sensors and electrical transistors.
Nanocantilever: A cantilever with dimensions in the nanoscale.
Quantum Dots: Nanometer-sized fluorescent semiconductor crystals, or electrostatically confined electrons. Something (usually a semiconductor island) capable of confining a single electron, or a few, and in which the electrons occupy discrete energy states just as they would in an atom (quantum dots have been called “artificial atoms”).
Let's face it: “modern” neurosurgery is still pretty crude. We leave holes in things (take out a brain tumor, leave a hole; remove a ruptured disk, leave a hole, etc.) or fix things like we fix buildings. An unstable spine? Screw in a bunch of metal plates like sister ribs in the hull of a rotting schooner. Parkinson's disease? Stick in brain electrodes to shock the bejaysus out of the subthalamic nucleus, but do nothing for the withering cells in the substantia nigra. The vast majority of our therapies are based on empiricism with a fuzzy understanding of the basic disease process and initiated without correcting the root cause of the pathology. And the root cause is usually dysfunction of a tissue because of pathology on a cellular or subcellular level. But, correcting cellular or subcellular pathology in specific cells without clobbering all of the normal neighboring cells has been a pipe dream. Why? Because we have not had the specific tools to kill or alter the function of these cells in vivo or, in most cases, to even identify them. Perhaps, in the near future, we will have these tools thanks to nanotechnology and techniques that will evolve from having these tools.
This article is a primer on nanotechnology. It shows that there is a lot happening. These new technologies will probably transform not just neurosurgery, but medicine in general, in ways that we cannot possibly predict, nor even conceive. But, that's what happens with technology: things developed to solve problems in one field can, in time, result in quantum advances in other fields. For example, denizens of the early 18th century could not have predicted that Alessandro Volta's voltaic pile would evolve into batteries that now power everything from personal computers to space stations. Or that his electrophosorus would become the spark plug that made internal combustion engines possible. When the tools are there, mankind will figure out new ways to use and modify them and solve new problems as science defines them. Technological advancement in one field can enrich progress in many others. In medicine in general and neurosurgery in particular, nanotechnological tools will foster future therapies that will be based, not on empiricism, but on a scientific understanding of the subcellular biochemical processes driving the pathology.
Patrick J. Kelly
New York, New York
Nanomedicine has arrived. The opportunity to utilize nanomachines and nanotechnologies to affect disease and patient care has been, until recently, just a thought, or rather, merely a dream. It is now an impending reality. In this article, Leary et al. have portrayed and presented the evidence that supports the notion that nanotechnology can indeed be melded with biological systems, hence the emergence of nanomedicine. They have covered the field from molecular biological applications to the development and implementation of nanodevices. And, in so doing, they have provided a wonderful introduction to a new field that spans the physical and biological sciences: nanomedicine.
Edward C. Benzel
Part II of this series describes the current uses of nanoparticles for the detection of incredibly small amounts of biological material with the promise of fast, sensitive, and specific assays to replace current difficult and temperamental procedures. The impact of being able to use such technology to image changing receptors and other structures in living cells is phenomenal. The evident potential for use of nanoparticles linked with antibodies opens the door for disease specific imaging with potent diagnostic and therapeutic possibilities in patients that will not depend on a differential effect for expression, but rather will be unique for the disease process under consideration. There can be little doubt that this technology will have a major impact on a variety of types of diagnostic testing.
Charles J. Hodge, Jr.
Syracuse, New York
This work represents the much anticipated sequel to the introduction to nanoscience and nanotechnology published by the authors in Neurosurgery (1). After reading Part I, we were impressed by the tremendous breadth and intricacy of the burgeoning field of nanotechnology. However, at that time, it seemed as though practical application of nanotechnology to clinical medicine was a somewhat distant reality. In Part II, we now see quite clearly that we were mistaken. Nanoscience is emerging as a robust field with numerous applications to both medicine and surgery. Accordingly, we sit on the cusp of a new era in neurosurgery where nanotechnology stands to alter the way we may deliver care to our patients. Examples cited within this exposé include the use of nanotechnology in molecular diagnostics, advanced imaging, therapeutic delivery of active agents, and cellular manipulations. It is noteworthy that nanotechnology seems particularly well suited to neurosurgical practice. The future applications seem to align perfectly with many of the goals of neurosurgical progress. For example, in neurosurgical oncology, precise molecular diagnosis, molecular imaging, and targeted delivery of chemotherapeutics are important potential applications of nanotechnology. Cerebrovascular neurosurgeons will eagerly await an enhanced ability to manipulate endothelial biology with nanoparticles or to improve the structural integrity of vascular channels through reconstruction processes using nanowires. Sterotactic and functional neurosurgeons will revel at the opportunity to manipulate neuronal cell bodies and axons to treat neurodegenerative diseases such as Parkinson's disease. Some would argue that we, as neurosurgeons, have not kept pace with the growing nanotechnology field around us. However, articles such as the present one by Leary et al. do much to raise our awareness of the future of neurosurgery which is fast upon us.
James T. Rutka
The authors have provided a sweeping, insightful, and provocative review of nanotechnology applications in neurosurgery. This evolution of ideas should be no surprise to anyone in our field, but may stimulate all of us with the possibilities for clinical neuroscience applications. The manipulation of molecular particles in biological systems provides much promise for delivering therapeutics at a cellular level. But, this technology should be viewed simply as a tool and not an end to achieve the diagnosis of disease, repair of neurons, and therapies designed to restore cellular health. I think the first success for neurosurgical applications is close. Combined with the potential utility of robotics and computers, nanosurgery on neurons and axons may provide utility to peripheral nerve repair. Sretavan et al. (1) have beautifully shown how these microscale surgery technological advances in the lab may yield clinical applications. Next, I foresee, molecular imaging using fluorescent nanoparticles of particular utility to our field. Veiseh et al. (2) have some worthy insights into the application of optical and magnetic resonance imaging multifunctional nanoprobes for targeting glioma and medulloblastoma. The ability to see tumor margins at a more cellular level would add another order of resection, compared with intraoperative magnetic resonance, in surgically amenable tumors. This advance, coupled with the ability to detect live and dead tumor on magnetic resonance with nanoparticles, may further champion this technology in our field. The future of biomedical/nanoengineering in neurosurgery seems promising.
Richard G. Ellenbogen