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1. © 2006 Nature Publishing Group Nanodevices and nanomaterials can interact with bio- logical systems at fundamental, molecular levels with a high degree of specificity. By taking advantage of this unique molecular specificity, these nanotechnologies can stimulate, respond to and interact with target cells and tissues in controlled ways to induce desired physio- logical responses, while minimizing undesirable effects. Applications of nanotechnology in basic and clinical neuroscience are only in the early stages of development, partly because of the complexities associated with inter- acting with neural cells and the mammalian nervous system. Despite this, an impressive body of research is emerging that hints at the potential contributions these technologies could make to neuroscience research. This review discusses the basic concepts associated with nanotechnology and its current applications in neuroscience. The first section attempts to answer the questions concerning what nanotechnology is and what it encompasses. The second section gives an overview of the main areas of neuroscience nanotechnology research. Specifically, it discusses neuronal adhesion and growth, interfacing and stimulating neurons at a molecular level, imaging and manipulating neurons and glia using functionalized quantum dots, approaches for functional neural regeneration, approaches for neu- roprotection and nanotechnologies for crossing the blood–brain barrier (BBB). The third section discusses some of the unique challenges encountered when apply- ing nanotechnology to neural cells and the nervous system, and the tremendous impact this technology might have on neuroscience research. The fourth sec- tion attempts to define the roles of neuroscientists in advancing neuroscience nanotechnology. The final Departments of Bioengineering and Ophthalmology, and the Neurosciences Program, University of California, San Diego, UCSD Jacobs Retina Center 0946, 9415 Campus Point Drive, La Jolla, California 92037- 0946, USA. e-mail: firstname.lastname@example.org doi:10.1038/nrn1827 Self-assembly The self-organization of molecules into supermolecular structures. Self-assembly is triggered by specific chemical or physical variables, such as a change in temperature or concentration, and reflects an energy minimization process. section speculates on the results that might derive from current applications of nanotechnology and the types of applications that might have the earliest impact in neuroscience. What is nanotechnology? Nanotechnologies are technologies that use engineered materials or devices with the smallest functional organi- zation on the nanometre scale (that is, one billionth of a metre) in at least one dimension, typically ranging from 1 to ~100 nanometres. This implies that some aspect of the material or device can be manipulated and controlled by physical and/or chemical means at nanometre resolu- tions, which results in functional properties that are unique to the engineered technology and not shown by its constituent elements. For example, DNA self-assembly can yield DNA nanotube supermolecular structures that form electrically conducting nanowires that have potential for use in nanoelectronic devices1 . In addition, boron-doped silicon nanowires can function as real-time ultrahigh sensitive detectors for biological and chemical factors2 : nanowires modified with biotin specifically and accurately detect picomolar concentrations of streptavidin. Nanotechnologies are therefore primarily defined by their functional properties, which determine how they interact with other disciplines. Although the chemical and/or physical make up of a nanomaterial or device is important in the overall technological proc- ess, it is secondary to their engineering and functional properties. Considering the two examples described above, DNA does not have the intrinsic capacity to function as an electrically conducting nanowire, and neither boron nor silicon can detect specific chemical Neuroscience nanotechnology: progress, opportunities and challenges Gabriel A. Silva Abstract | Nanotechnologies exploit materials and devices with a functional organization that has been engineered at the nanometre scale. The application of nanotechnology in cell biology and physiology enables targeted interactions at a fundamental molecular level. In neuroscience, this entails specific interactions with neurons and glial cells. Examples of current research include technologies that are designed to better interact with neural cells, advanced molecular imaging technologies, materials and hybrid molecules used in neural regeneration, neuroprotection, and targeted delivery of drugs and small molecules across the blood–brain barrier. REVIEWS NATURE REVIEWS | NEUROSCIENCE VOLUME 7 | JANUARY 2006 | 65
© 2006 Nature Publishing Group Physical and/or chemical triggers (for example, changes in pH, concentration, temperature) Processes such as etching and nanolithography, starting from bulk material Bottom-up approaches (for example, self-assembly, molecular patterning) Top-down approaches (for example, lithography) a b Bottom-up technologies Materials or devices engineered from constituent elements such as specific molecules that are organized into higher-order functional structures. Top-down technologies Materials or devices that are engineered from a bulk material. The various forms of lithography are examples of top-down engineering approaches. Lithography The process of producing patterns in bulk materials. The most common forms of lithography are those associated with the production of semiconductor integrated circuits. species. However, DNA nanowires and boron-doped silicon have gained these novel functional properties. These technologies are described by the new functional outcomes of bringing these components together, not because they had to contain DNA, or boron or silicon. Other materials or devices can be made using the same building blocks with different functional or engineered outcomes, and both electrically conducting nanowires and high-sensitivity chemical sensors can be produced using other chemistry and synthetic approaches. In this functional definition of nanotechnology, it is implicit that this is not a new area of science per se, and that the interdisciplinary convergence of basic fields (such as chemistry, physics, mathematics and biology) and applied fields (such as materials science and the vari- ous areas of engineering) contributes to the functional outcomes of the technology. In this framework, nano- technology can be regarded as an interdisciplinary pursuit that involves the design, synthesis and character- ization of nanomaterials and devices that have the types of property discussed above. In particular, this engineer- ing definition of nanotechnology is what sets it apart from chemistry. Chemistry is an integral component of nanotechnology, but the two are not synonymous, a point that is a potential source of confusion. Chemistry involves the manipulation of matter at nanometre scales resulting in chemical products with specific intrinsic properties (for example, a defined melting point, pKa or charge distribution) that affect how they interact with their environments. As emphasized above, nanotechno- logies are primarily defined in terms of their extrinsic engineered functional properties (of which the intrinsic chemistry could be one of many contributory factors that define these properties). From the neuroscientist’s perspective, the most important aspect of nanotechnologies is the applica- tion of these technologies to neuroscience questions and challenges3,4 . There are two key types of nanotechnology application in neuroscience: ‘platform nanotechnolo- gies’ that can be readily adapted to address neuroscience questions; and ‘tailored nanotechnologies’ that are speci- fically designed to resolve a particular neurobiological issue. This is also the case for other areas of biology and medicine. Platform nanotechnologies use materials or devices with unique physical and/or chemical proper- ties that can potentially have wide-ranging applications in different fields. These technologies have considerable promise, and scientists and engineers are constantly looking for new ways to explore the potential of plat- form nanotechnologies. Tailored nanotechnologies begin with a well-defined biological question, and are developed to specifically address that issue. Owing to the inherent complexity of biological systems in gen- eral, and the nervous system in particular, the tailored approach often results in highly specialized technologies that are designed to interact with their target systems in sophisticated and well-defined ways, and so will be better suited to tackle the particular problem than a generic platform technology. However, because tailored nanotechnologies are highly specialized, their broader application to other biological systems could be limited. In many cases, the application of particular nanotechno- logies in neuroscience has been derived from what was originally a platform technology, and some tailored technologies can be modified to address other (but usually related) scientific questions. From a synthesis standpoint, nanotechnologies can be classified as vari- ations of bottom-up technologies, such as self-assembly, or top-down technologies, such as lithographic methods (BOX 1). Many nanotechnologies combine aspects of both strategies. Examples of current work This section reviews applications of nanotechnology in basic and clinical neuroscience (TABLE 1). It is organized according to the type of application, and compares dif- ferent nanotechnology approaches applied to similar or related neurobiological or physiological objectives. In general, the discussion provides more breadth than depth to give the reader a broad sense of ongoing research. The section is divided into two subsections: appli- cations of nanotechnology in basic neuroscience; and applications of nanotechnology in clinical neuroscience. Applications of nanotechnology in basic neuroscience includethosethatinvestigatemolecular,cellularandphys- iological processes (FIG. 1). This first subsection discusses three specific areas. First, nanoengineered materials and Box 1 | Synthetic approaches in nanotechnology Different approaches used in the synthesis of nanomaterials and nanodevices can accommodate solid, liquid, and/or gaseous precursor materials. In general, most of these techniques can be classified as bottom-up and top-down approaches, and strategies that have elements of both. Bottom-up approaches (panel a) start with one or more defined molecular species, which undergo certain processes that result in a higher-ordered and - organized structure. Examples of bottom-up approaches include systems that self- assemble, a process that is triggered by a local change in a chemical or physical condition. Related techniques include templating and scaffolding methods, such as biomineralization, which rely on backbone structures to support and guide the nucleation and growth of a nanomaterial. Top-down approaches (panel b) begin with a bulk material that incorporates nanoscale details, such as nanolithography and etching techniques. Specific examples include dip-pen nanolithography (in which specific molecules are deposited into desired configurations) and electrostatic atomic force nanolithography (in which molecules are moved around to form desired structures). In all cases, the resultant structures have novel engineered chemical and/or physical properties that the original constituent materials do not have. These emergent properties allow controlled interactions of the nanomaterial or device with its target system. REVIEWS 66 | JANUARY 2006 | VOLUME 7 www.nature.com/reviews/neuro
© 2006 Nature Publishing Group Avd Btn GABAb Patterned molecular interfaces for stimulating and responding a Quantum-dot imaging and molecular tracking Quantum dots c Engineered materials with nanoscale physical features d Patterned neuronal adhesion and growth molecules Cad Fbn PA2 Lam Cad Fbn PA2 Lam γ-Aminobutyric acidGABA BtnAvd Cadherin Fibronectin Phospholipase A2 Laminin Avidin Biotin approaches for promoting neuronal adhesion and growth to understand the underlying neurobiology of these pro- cesses or to support other technologies designed to inter- actwithneuronsin vivo(forexample,coatingofrecording or stimulating electrodes)5–10 . Second, nanoengineered materials and approaches for directly interacting, record- ing and/or stimulating neurons at a molecular level11–13 . Third, imaging applications using nanotechnology tools, in particular, those that focus on chemically functional- ized semiconductor quantum dots14–19 . Applications of nanotechnology in clinical neuroscience include research aimed at limiting and reversing neuropathological disease states (FIG. 2). This subsection discusses nanotechnology approaches designed to support and/or promote the functional regeneration of the nervous system20,21 ; neuro- protective strategies, in particular those that use fullerene derivatives22–26 ; and nanotechnology approaches that facilitate the delivery of drugs and small molecules across the BBB27–38 . As with any classification scheme, there might be a sense of forced categorization when classifying nano- technologies, as some nanotechnologies can be used in more than one area. For example, all three of the basic nanotechnology applications can also contribute to an understanding of neuropathophysiology; and all three of the clinical nanotechnology applications can also increase our understanding of basic molecular and cellular neurobiology. But, for the most part, basic neuro- science applications primarily concern themselves with understanding basic molecular and cellular mechanisms without necessarily considering their potential clinical implications, whereas clinical neuroscience applications are designed to primarily target disease events, and make use of basic molecular and cellular neurobiology only when necessary. Applications in basic neuroscience. Molecular deposition and lithographic patterning of neuronal-specific mole- cules with nanometre resolutions5–10 are an extension of micropatterning approaches39–44 . The deposition of proteins and other molecules that promote and support neuronal adhesion and growth on surfaces that do not support these processes enables the geometric selective patterning and growth of neurons (for example, control- led neurite extension). This allows the study of cellular communication and signalling, and provides a test sys- tem for investigating the effects of drugs and other mol- ecules. The ability to control this process at nanometre as Table 1 | Applications of nanotechnologies in neuroscience Nanotechnology Applications Refs Basic neuroscience Molecular deposition and lithographic patterning of neuronal-specific molecules with nanometre resolution Study of cellular communication and signalling; test systems for drugs and other molecules 5–10 Atomic force microscopy (measures molecularly functionalized surfaces) Interact, record and/or stimulate neurons at the molecular level 11–13 Functionalized quantum dots High-resolution spatial and temporal imaging; molecular dynamics and tracking 14–19 Clinical neuroscience Self-assembling peptide amphiphile nanofibre networks Neuronal differentiation from progenitor cells; neural regeneration 20 Derivatives of hydroxyl- functionalized fullerenes (fullerenols) Neuroprotection mediated by limiting the effects of free radicals following injury 23–26 Poly(ethylene glycol) and polyethylenimine nanogels; poly(butylcyanoacrylate) nanoparticles Transport of drugs and small molecules across the blood–brain barrier 27–36 Figure 1 | Applications of nanotechnologies in basic neuroscience. Nanomaterials and nanodevices that interact with neurons and glia at the molecular level can be used to influence and respond to cellular events. In all cases, these engineered technologies allow controlled interactions at cellular and subcellular scales. a | Chemically functionalized fluorescent quantum dot nanocrystals used to visualize ligand–target interactions. b | Surfaces modified with neurotransmitter ligands to induce controlled signalling. For example, GABA (γ-aminobutyric acid) was immobilized, via an avidin–biotin linkage, to different surfaces to stimulate neurons in predictable (that is, patterned) ways. c | Engineered materials with nanoscale physical features that produce ultrastructural morphological changes. d | Surfaces and materials functionalized with different neuronal-specific effector molecules, such as cadherin and laminin, to induce controlled cellular adhesion and growth. REVIEWS NATURE REVIEWS | NEUROSCIENCE VOLUME 7 | JANUARY 2006 | 67
© 2006 Nature Publishing Group c Functional nanoparticles for delivery across the blood–brain barrier Astrocyte endfoot Nanoparticles Endothelial cells O2 •– • OH ONOO– Nanoparticles Nanoscale scaffold a Functional nanoparticles for free radical neuroprotection b Bioactive nanoscale scaffold materials for neural regeneration Spinal cord Atomic force microscopy (AFM). Scanning probe microscopy that uses a sharp probe moving over the surface of a sample to measure topographic spatial information. opposed to micron resolutions enables the investigation of how neurons respond to anisotropic physical and chemical cues. Micron-scale patterning can provide a functional boundary for controlling and influencing cellular behaviour, but ultimately the neuron detects a stimulus (or stimuli in the case of multiple signals) that, because of the (relatively large) micron-scale resolution of the patterning, is a homogeneous bioactive signal that is averaged over the entire cell. Nanotechnology approaches present subcellular stimuli that can vary from one part of the neuron to another. For example, photolithography andlayer-by-layerself-assemblyhavebeenusedtopattern phospholipase A2 , which promotes neuronal adhesion, on a background of poly(diallyldimethylammonium chloride) (PDDA)8 . This approach facilitates nanoscale patterning at resolutions that can yield complex func- tional architectures that are tailored to the needs of a particular experiment. Layer-by-layer self-assembly has also been used on silicon rubber to pattern alternating laminin and poly-d-lysine or fibronectin/poly-d-lysine ultrathin layers, which are 3.5–4.4 nm thick, that support the growth of cerebellar neurons5 . These studies suggest that bioactive ultrathin layers could coat electrodes designed for long-term implants to promote cell adhesion and limit immune responses. In a different approach, electrodes coated with nanoporous silicon increased neurite outgrowth from PC12 cells, which are clonally derivedneuronalprecursorcells,comparedwithuncoated electrodes, and decreased glial responses, thereby limiting the insulating effects of the glial scar10 . Coating electrodes with ultrathin bioactive layers might have other advan- tages, for example, limiting the increase in the thickness of the electrodes and thereby minimizing local trauma due to their insertion and resultant cellular responses. Other research has shown the effects of nanoscale physical features on neuronal behaviour. Substantia nigra neurons cultured on silicon dioxide (SiO2 ) surfaces with different nanoscale topographies had differential cell adhesion properties6 . Neurons cultured on surfaces with physical features (that is, surface roughness) between20–70nmadheredandgrewbetterthanneurons cultured on surfaces with features <10 nm or >70 nm. These neurons also had normal morphologies and normal production of tyrosine hydroxylase, a marker of metabolic activity. Another emerging area of neuroscience nanotechno- logy is materials and devices that have been designed to interact, record and/or stimulate neurons at the mole- cular level11–13 . Recent research has demonstrated the feasibility of functionalizing mica or glass tethered with the inhibitory neurotransmitter GABA (γ-aminobutyric acid) and its analogue muscimol (5-aminomethyl- 3-hydroxyisoxazole) through biotin–avidin binding interactions12,13 (FIG. 1). The functional integrity of the bound version of the neurotransmitter, which in vivo functions not as a bound ligand but as a diffusible messenger across the synaptic cleft, was shown electro- physiologically in vitro by eliciting an agonist response to cloned GABAA (GABA type A) and GABAC recep- tors in Xenopus oocytes12 . Such sophisticated systems, although still in the early conceptual and testing phases, may provide powerful molecule-based platforms for testing drugs and neural prosthetic devices. At present, all neural prostheses (including neural retinal prostheses) rely on micron-scale features and cannot interact with the nervous system in a controlled way at the molecular level, which is a significant disadvantage. Other research is focusing on achieving nanoscale measurements of cellular responses. Atomic force microscopy (AFM) has been used to measure local nanometre morphological responses to micro-electrode array stimulation of neuro- blastoma cells11 . AFM is a technique that, among other capabilities, allows the measurement of height changes in the topography of a surface (for example, a living cell or a synthetic material) with nanometre resolution45,46 ; in essence, it is a nanoscale cantilever that measures surface topologies at the atomic level. This technique can meas- ure cross-sectional changes in cell height (between 100 and 300 nm), which are produced by biphasic pulses at a frequency of 1 Hz, thereby providing information on ultrafine morphological changes to electrical stimuli in neurons that cannot be achieved by other technologies. Figure 2 | Applications of nanotechnology in clinical neuroscience. Nanotechnology can be used to limit and/or reverse neuropathological disease processes at a molecular level or facilitate and support other approaches with this goal. a | Nanoparticles that promote neuroprotection by limiting the effects of free radicals produced following trauma (for example, those produced by CNS secondary injury mechanisms). b | The development and use of nanoengineered scaffold materials that mimic the extracellular matrix and provide a physical and/or bioactive environment for neural regeneration. c | Nanoparticles designed to allow the transport of drugs and small molecules across the blood–brain barrier. REVIEWS 68 | JANUARY 2006 | VOLUME 7 www.nature.com/reviews/neuro
© 2006 Nature Publishing Group a b 10 8 6 4 2 0 0 500 1,500 2,500 3,500 Maximum measured intensity Quantumdotcounts c d x direction ydirection Photobleaching The progressive loss of fluorescence signal intensity due to exposure to light. This can result in a decreased signal-to-noise ratio. An area of nanotechnology that holds significant promise for probing the details of molecular and cellular processes in neural cells is functionalized semiconduc- tor quantum dot nanocrystals14–19 (FIG. 1). Quantum dots are nanometre-sized particles comprising a heavy metal core of materials such as cadmium–selenium or cad- mium telluride, with an intermediate unreactive zinc sulphide shell and an outer coating composed of selective bioactive molecules tailored to a particular application. The physical nature of quantum dots gives them unique and highly stable fluorescent optical properties that can be changed by altering their chemistry and physical size. Quantum dots can be tagged with fluorescent proteins of interest using different chemical approaches similar to fluorophore immunocytochemistry. However, quan- tum dots have significant advantages compared with other fluorescent techniques. Quantum dots undergo minimal photobleaching and, because they have broad absorption spectra but narrow emission spectra47–50 , can have much higher signal-to-noise ratios, which result in dramatically improved signal detection. In addition, quantum dots can be used for single-particle tracking of target molecules in live cells, such as tracking lig- and–receptor dynamics in the cell membrane51–53 (FIG. 3). Quantum dot labelling of both fixed and live cells is well established, and has been used in a wide variety of cell types — mostly in vitro or in situ54–64 , with some examples in vivo65,66 . Despite the growing literature on the uses of quantum dots in the study of various cell types, their application in the labelling of neurons14–19 and glia19 has been slower to develop, and care must be taken to validate labelling methods that are specific for neural cells, as methods for labelling other cell types are not necessarily suitable for neural cells19 . Recent research has illustrated the potential of this technology in neu- roscience. The real-time dynamics of glycine receptors in spinal neurons have been tracked and analysed using single-particle tracking over periods of seconds to min- utes15 . These investigators characterized the dynamics of glycine receptor diffusion, which differed as a function of the spatial localization of the receptors relative to the synapse depending on whether they were in synaptic, perisynaptic or extrasynaptic regions. In another example, immobilized quantum dots that were conjugated with β-nerve growth factor (βNGF) were shown to interact with TrkA receptors in PC12 cells and to regulate their differentiation into neurons in a controlled way18 . This could provide new tools for studying neuronal signal- ling processes. Although most applications of quantum dots in neuroscience have taken place ex vivo, in vivo microangiography of mouse brains has been achieved using serum that has been labelled with quantum dots14 . New functionalization and labelling methods of quan- tum dots have been developed and subsequently tested using labelled AMPA (α-amino-3-hydroxy-5-methyl-4- isoxazole propionic acid) neurotransmitter receptors16 , and by measuring the cytotoxicity of hippocampal neurons17 . Although quantum-dot nanotechnology, when used correctly, has little cytotoxic effects on cells in vitro (so that experimental results are not affected), their in vivo applications present different challenges due to the possibility of local and systemic toxicity. To address these issues, the safety of using quantum dots with both neural cells and other cell types is an active area of research67–69 . Applications in clinical neuroscience. Applications of nanotechnology that are intended to limit and reverse neurological disorders by promoting neural regenera- tion and achieving neuroprotection are active areas of Figure 3 | The quantum dot toolbox. Fluorescent quantum dots are nanoscale particles that can be chemically functionalized by attaching a large variety of biological molecules to their outer surface (for example, antibodies, peptides and trophic factors). This allows specific molecular interactions in both live and fixed target cells, which can be visualized at high resolutions by taking advantage of the unique optical properties of quantum dots, such as their prolonged photo-stability (that is, minimal photobleaching), large excitation absorption spectra and extremely narrow emission spectra; specific examples of applications of quantum dots in neuroscience can be found in REFS 14–19. a | Primary rat cortical neurons labelled with conjugates of quantum dots and anti-β-tubulin III antibody. β-tubulin is a neuronal-specific intermediate filament protein and so serves as a neuronal marker. b | Primary rat astrocytes labelled with quantum dot–anti-glial fibrillary acidic protein (GFAP) antibody conjugates. GFAP is a glial specific intermediate filament protein. c | One of the main advantages of quantum dot nanotechnology is that qualitative observations as well as quantitative data can be obtained, which provide detailed molecular and biophysical information about the biological system being investigated. For example, by using computational and morphometric tools, individual quantum dots can be counted across a sample image to yield information on the distribution and number of ligand–target interactions. This graph shows the number of quantum dots with a particular intensity. Such quantitative measures could be used in measuring the expression level of cellular markers, such as β-tubulin in a or GFAP in b, which are conjugated to the quantum dots. d | Quantum dots can also be used to carry out single-particle tracking of ligand–target pairs, such as tracking the motion of a receptor in a cell membrane. Illustration of the trajectory of a field of 55 quantum dots undergoing Brownian diffusion, with individual trajectories corresponding to individual quantum dots. The small size, photochemistry and bioactivity of functionalized quantum dots provide an extensive new toolbox for investigating molecular and cellular processes in neurons and glia. Images and data courtesy of the Silva Research Group, University of California, San Diego, California, USA. REVIEWS NATURE REVIEWS | NEUROSCIENCE VOLUME 7 | JANUARY 2006 | 69
© 2006 Nature Publishing Group H+ OH– Amphipathic molecules a b c Absorption spectra The range of wavelengths over which a molecule, such as a fluorophore, or a nanoparticle, such as a quantum dot, are energetically excited. research. The development of nanoengineered scaffolds that support and promote neurite and axonal growth are evolving from tissue engineering approaches based on the manipulation of bulk materials. Examples of micron-scale tissue engineering include poly-(l-lactic) acid (PLLA) and other synthetic hydrogels that have engineered microscale features, and scaffolds derived from naturally occurring materials such as collagen70–79 . One example of a nanoengineered system derived from this type of work is PLLA scaffolds with an ultrastructure consisting of cast PLLA fibres, which have diameters of 50–350 nm and porosity of ~85%20 . The scaffolds were constructed using liquid–liquid phase separation by dissolving PLLA in tetrahydrofuran (THF) rather than casting them on glass. When cultured in the scaffolds, neonatal mouse cerebellar progenitor cells were able to extend neurites and differentiate into mature neurons. A fundamentally different approach for the development of a nanomaterial that promotes and supports neural regeneration is the self-assembly of nanofibre networks composed of peptide-amphiphile molecules21 (FIG. 4). On exposure to physiological ionic conditions, peptide- amphiphile molecules, which consisted of a hydrophobic carbon tail and a hydrophilic peptide head group, self- assembled into a dense network of nanofibres. This trapped the surrounding water molecules and formed a weak self-supporting gel at the macroscopic level. The hydrophilic peptide head groups, which formed the outside of the fibres, consisted of the bioactive laminin- derived peptide IKVAV, which promotes neurite sprout- ing and growth80–83 . Encapsulation of neural progenitor cells from embryonic mouse cortex in the nanofibre net- works resulted in fast and robust neuronal differentiation (30% and 50% of neural progenitor cells differentiated into neurons at 1 and 7 days in vitro, respectively), with minimal astrocytic differentiation (1% and 5% of neural progenitor cells differentiated into astrocytes at 1 and 7 days in vitro, respectively). This approach could there- fore promote neuronal differentiation at an injury site while potentially limiting the effects of reactive gliosis and glial scarring, which are ubiquitous neuropathologi- cal disease processes. Applications of nanotechnologies for neuroprotec- tion have focused on limiting the damaging effects of free radicals generated after injury, which is a key neuropathological process that contributes to CNS ischaemia, trauma and degenerative disorders84–88 . Fullerenols, which are derivatives of hydroxyl-func- tionalized fullerenes (molecules composed of regular arrangements of carbon atoms89–93 ), have been shown to have antioxidant properties. They also function as free radical scavengers, which can lead to a reduction in the extent of excitotoxicity and apoptosis induced by glutamate, NMDA (N-methyl-d-aspartate), AMPA and kainate23–26 . Fullerenol-mediated neuroprotection has been shown in vitro and in vivo. Fullerenol limits exci- totoxicity and apoptosis of cultured cortical neurons in vitro, and delays the onset of motor degeneration in vivo in a mouse model of familial amyotrophic lat- eral sclerosis. The neuroprotective effect of fullerenols might be partly mediated by inhibition of glutamate receptors, as they had no effect on GABAA or taurine receptors. They also lowered glutamate-induced eleva- tions in intracellular calcium, which is an important mechanism of neuronal excitotoxicity23–26 . Another clinically relevant area of intense research is the design of functionalized nanoparticles that can be administered systemically and deliver drugs and small molecules across the BBB27–36 . This is a major clinical objective for the treatment of a wide range Figure 4 | Example of an engineered nanomaterial for neural regeneration. Engineered nanomaterials enable the highly specific induction of controlled cellular interactions that can promote desired neurobiological effects. a | Peptide-amphiphile molecules, which consist of a hydrophilic peptide head group (green circles) and a hydrophobic carbon tail (white circles) joined by a peptide spacer region (yellow circles), can be coaxed to self-assemble into elongated micelles to produce a dense nanofibre matrix99,100 . Under physiological conditions, the self-assembly process traps the surrounding aqueous environment and macroscopically produces a self- supporting gel in which neural progenitor cells and stem cells can be encapsulated. In this way, the growth and differentiation of neural progenitor cells and stem cells can be controlled. b | An example of a peptide-amphiphile nanogel on a 12 mm glass coverslip. c | The surface of the nanofibres consists of laminin-derived, neuronal- specific pentapeptides, which are encountered by the encapsulated cells at high concentrations, resulting in robust differentiation into neurons while suppressing astrocyte differentiation21 . The cells are stained for the neuronal marker β-tubulin III (green) and the astrocyte marker glial fibrillary acidic protein (none is present). All nuclei were stained with a nonspecific nuclear Hoescht stain. Images courtesy of the Stupp laboratory, Northwestern University, Evanston, Illinois, USA. REVIEWS 70 | JANUARY 2006 | VOLUME 7 www.nature.com/reviews/neuro
© 2006 Nature Publishing Group Emission spectra The range of wavelengths over which a molecule, such as a fluorophore, or a nanoparticle, such as a quantum dot, emit light. Synaptic, perisynaptic or extrasynaptic regions Areas where neurotransmitter receptors cluster at, near, or outside the synapse, respectively. TrkA receptors A family of proto-oncogene receptors found throughout the central and peripheral nervous system that bind β-nerve growth factor, which results in downstream signalling effects. of neurological disorders. To achieve this, various materials and synthetic approaches are being investi- gated. Oligonucleotides have been delivered in gels of crosslinked poly(ethylene glycol) and polyethylen- imine28 . Charge differences in the electrostatic forces between the gel and spontaneously negatively charged oligonucleotides provide a reversible delivery mecha- nism that can be used for shuttling molecules across the BBB and then releasing them from the delivery system. Neuropeptides (such as enkephalins), the NMDA recep- tor antagonist MRZ 2/576, and the chemotherapeutic drug doxorubicin have been absorbed onto the surface of poly(butylcyanoacrylate) nanoparticles coated with polysorbate 80 (REFS 31,33,34,37,38). The polysorbate on the surface of the nanoparticles adsorbs apolipo- protein B and apolipoprotein E from the blood, and the nanoparticles are taken up by brain capillary endothelial cells via receptor-mediated endocytosis38 . Nanoparticles that target tumours in the CNS may be a particularly important application of this technology due to the high morbidity and mortality associated with often aggres- sive neoplasms in the physically confined spaces of the cranium and spinal canal. Challenges and opportunities The challenges associated with nanotechnology appli- cations in neuroscience are numerous, but the impact it can have on understanding how the nervous system works, how it fails in disease and how we can intervene at a molecular level is significant. Ultimately, the chal- lenges and opportunities presented by nanotechnology stem from the fact that this technology provides a way to interact with neural cells at the molecular level, which has both positive and negative aspects. The abil- ity to exploit drugs, small molecules, neurotransmitters and neural developmental factors offers the potential to tailor technologies to particular applications. For example, neural developmental factors, such as the cadherins, laminins and bone morphometric protein families, as well as their receptors, can be manipulated in new ways. Nanotechnology offers the capacity to take advantage of the functional specificity of these molecules by incorporating them into engineered materials and devices to have highly targeted effects. The laminins, for example, are large multi-domain trimeric proteins composed of α, β and γ chains, of which 12 isoforms are known82,83,94,95 . The isoforms con- tain different bioactive peptide sequences, which have varying affinities for specific cell types and can induce different effects. For example, the laminin 1 isoform, which is the most studied laminin, contains at least 48 different short peptide sequences that promote neuro- nal adhesion and neurite outgrowth, and some of these peptides (25 of 48 tested) have such effects on specific types of neuron82 . This degree of molecular specificity, which is conferred not only by laminins but by many other signalling molecules that are important in the development and function of the nervous system, can be used to design highly selective nanotechnologies. Indeed, any desired cellular signalling pathway can be targeted using this approach. This discussion also suggests the main technical chal- lenges that are encountered when using nanotechnology applications in neuroscience: the need for greater spe- cificity; multiple induced physiological functions; and minimal side effects. Greater specificity of interactions with target cells and tissues will result in more signifi- cant and specific physiological effects, which should also reduce undesirable and deleterious side effects that are induced by the technology. Another important chal- lenge is the requirement for technologies that are able to multitask, carrying out a diverse set of specific cellular and physiological functions, such as targeting multiple receptors or ligands. This is particularly important when attempting to address multi-dimensional CNS disorders that are the result of numerous interdependent molecular and biochemical events (for example, secondary injury following traumatic brain injury or spinal cord injury). At present, synthetic and engineering processes are not advanced enough to allow nanotechnologies that have been designed to interact with the nervous system to fully meet these criteria. From a biological perspective, the most significant successes of nanotechnology applications in neuro- science will be those that appreciate a detailed under- standing of neurobiology and take advantage of the known (and unknown) molecular details. As suggested above, the main challenge is the ability to design and use more sophisticated technologies that are able to carry out highly targeted and specific functions while minimizing nonspecific interactions. To achieve this, both the design and engineering aspects of nanotechnology as well as our understanding of the underlying neurobiology are cru- cial. This, in turn, will require more interaction between neuroscientists and physical scientists such as chemists and materials scientists. This is not a trivial issue as the scientific language and culture between different disciplines can vary considerably. This is an increasing challenge for interdisciplinary science, which requires people with different training, skills and conceptions of how science should be conducted coming together first to understand and agree on a common challenge or problem, and then to agree on how to address that issue. The next section discusses further the role of neuroscientists in the development and application of nanotechnology. The above discussion pertains to all applications of nanotechnology to neuroscience. Applications of nanote- chnology to the nervous system in vivo present additional challenges. In particular, the inherent complexity of the CNS, as well as its difficult and anatomically restrictive nature, poses a unique set of obstacles. Cellular hetero- geneity and multi-dimensional cellular interactions (for example,spatialandtemporal summationof postsynaptic potentials) underlie the nervous system’s anatomical and functional ‘wiring’ that is the basis of its extremely com- plex information processing. Nanotechnologies designed to interact with CNS cells and processes in vivo must take this complexity into consideration, if only to avoid dis- rupting it. Failure to do so may result in unforeseen and unacceptable ‘side effects’ in the nervous system and/or other physiological systems. A significant challenge in REVIEWS NATURE REVIEWS | NEUROSCIENCE VOLUME 7 | JANUARY 2006 | 71
© 2006 Nature Publishing Group in vivo applications of nanotechnology is that they are designed to physically interact with neural cells at cellu- lar and subcellular levels, but ultimately aim at engaging functional interactions at a systemic level, which usually involves large groups of interacting neurons and glia. At present, there are only a few applications of this type; nonetheless, although technically and conceptually chal- lenging, these types of application could have a significant impact on clinical neuroscience. However, there is still a tremendous amount of (exciting) work to be done. Apart from physiological complexity, the second main consideration for in vivo applications of nanotech- nology is that they must consider the highly anatomically restrictive nature of the CNS. The structures of the CNS are well protected from mechanical and physical injury, and are immunologically privileged behind the BBB and blood–retina barrier, which have unique molecular and cellular environments. Nanotechnologies designed for in vivo applications must be efficiently delivered with minimal disruption to these structures before it can carry out its primary function. This will surely present signifi- cant technical challenges. Similarly, extreme care must be taken to understand and avoid potential safety pitfalls, including both systemic and local side effects associated with the delivery and primary function of the applied technology — an issue that is unique to in vivo nanotech- nology96–98 . As mentioned above, investigating the safety of nanotechnologies is an active and important area of research67–69 . Despite all these challenges, the applica- tions of nanotechnology both in vivo and ex vivo offer tremendous opportunities for understanding normal physiology and for developing therapies. The role of the neuroscientist Neuroscientists have a unique role in developing nano- technologies. Neuroscientists — both researchers and clinicians — need to identify potential applications of nanotechnology in neuroscience and neurology to maxi- mize their impact. Scientists with other specialties can develop powerful platform technologiesandevenprovide neuroscience-specific examples, but it is only with direct input from and in partnership with neuroscientists that broad neurophysiological and clinical applications can be properly formulated and addressed. This requires highly interdisciplinary collaborations with consideration of the requirements of both parties. Some neuroscientists develop neuroscience nanotechnologies for their particu- larpurposes,mostlikelygearedtowardsspecificquestions or objectives for their research. But most neuroscientists find that they lack the expertise and resources needed to design, synthesize and characterize sophisticated nano- engineered materials or devices. It would be unrealistic to assume that we as neuroscientists have knowledge and skills in these areas that are equivalent to those of chemists or materials scientists who have devoted their careers to the synthetic aspects of these technologies. However, chemists and material scientists do not have the comprehensive training in neurobiology, neurophysiol- ogy and neuropathology required to fully appreciate and exploit the potential of nanotechnology in neuroscience. Therefore, it is crucial that different disciplines are able to communicate with each other using a common tech- nical language, which is not a trivial issue — a nucleus means different things to a physicist and a cell biologist. To this end, it is important for neuroscientists who wish to pursue the development of nanotechnology to educate themselves across disciplines. To envision new applica- tions of nanotechnology, it is necessary to understand what has already been accomplished and what can be achieved with this technology. Future directions Applications of nanotechnology to neuroscience are already having significant effects, which will continue in the foreseeable future. Short-term progress has ben- efited in vitro and ex vivo studies of neural cells, often supporting or augmenting standard technologies. These advances contribute to both our basic understanding of cellular neurobiology and neurophysiology, and to our understanding and interpretation of neuropathology. 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