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Neuroscience nanotechnology: progress, opportunities and challenges






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Neuroscience nanotechnology: progress, opportunities and challenges Document Transcript

  • 1. REVIEWS 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. Self-assembly Nanodevices and nanomaterials can interact with bio- section speculates on the results that might derive from The self-organization of logical systems at fundamental, molecular levels with a current applications of nanotechnology and the types molecules into supermolecular high degree of specificity. By taking advantage of this of applications that might have the earliest impact in structures. Self-assembly is unique molecular specificity, these nanotechnologies neuroscience. triggered by specific chemical or physical variables, such as a can stimulate, respond to and interact with target cells change in temperature or and tissues in controlled ways to induce desired physio- What is nanotechnology? concentration, and reflects an logical responses, while minimizing undesirable effects. Nanotechnologies are technologies that use engineered energy minimization process. Applications of nanotechnology in basic and clinical materials or devices with the smallest functional organi- neuroscience are only in the early stages of development, zation on the nanometre scale (that is, one billionth of a partly because of the complexities associated with inter- metre) in at least one dimension, typically ranging from acting with neural cells and the mammalian nervous 1 to ~100 nanometres. This implies that some aspect of system. Despite this, an impressive body of research is the material or device can be manipulated and controlled emerging that hints at the potential contributions these by physical and/or chemical means at nanometre resolu- technologies could make to neuroscience research. tions, which results in functional properties that are This review discusses the basic concepts associated unique to the engineered technology and not shown by with nanotechnology and its current applications in its constituent elements. For example, DNA self-assembly neuroscience. The first section attempts to answer the can yield DNA nanotube supermolecular structures questions concerning what nanotechnology is and what that form electrically conducting nanowires that have it encompasses. The second section gives an overview potential for use in nanoelectronic devices1. In addition, of the main areas of neuroscience nanotechnology boron-doped silicon nanowires can function as real-time research. Specifically, it discusses neuronal adhesion ultrahigh sensitive detectors for biological and chemical and growth, interfacing and stimulating neurons at a factors2: nanowires modified with biotin specifically molecular level, imaging and manipulating neurons and and accurately detect picomolar concentrations of Departments of glia using functionalized quantum dots, approaches for streptavidin. Nanotechnologies are therefore primarily Bioengineering and functional neural regeneration, approaches for neu- defined by their functional properties, which determine Ophthalmology, and the roprotection and nanotechnologies for crossing the how they interact with other disciplines. Although the Neurosciences Program, University of California, blood–brain barrier (BBB). The third section discusses chemical and/or physical make up of a nanomaterial or San Diego, UCSD Jacobs some of the unique challenges encountered when apply- device is important in the overall technological proc- Retina Center 0946, ing nanotechnology to neural cells and the nervous ess, it is secondary to their engineering and functional 9415 Campus Point Drive, system, and the tremendous impact this technology properties. Considering the two examples described La Jolla, California 92037- 0946, USA. might have on neuroscience research. The fourth sec- above, DNA does not have the intrinsic capacity to e-mail: gsilva@ucsd.edu tion attempts to define the roles of neuroscientists in function as an electrically conducting nanowire, and doi:10.1038/nrn1827 advancing neuroscience nanotechnology. The final neither boron nor silicon can detect specific chemical NATURE REVIEWS | NEUROSCIENCE VOLUME 7 | JANUARY 2006 | 65 © 2006 Nature Publishing Group
  • 2. REVIEWS Box 1 | Synthetic approaches in nanotechnology resulting in chemical products with specific intrinsic properties (for example, a defined melting point, pKa or a b 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 Processes such as etching chemistry could be one of many contributory factors and nanolithography, that define these properties). Physical and/or chemical starting from bulk material From the neuroscientist’s perspective, the most triggers (for example, changes in pH, concentration, important aspect of nanotechnologies is the applica- temperature) 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- Bottom-up approaches Top-down approaches (for example, self-assembly, (for example, lithography) fically designed to resolve a particular neurobiological molecular patterning) issue. This is also the case for other areas of biology and Different approaches used in the synthesis of nanomaterials and nanodevices can medicine. Platform nanotechnologies use materials or accommodate solid, liquid, and/or gaseous precursor materials. In general, most of these devices with unique physical and/or chemical proper- techniques can be classified as bottom-up and top-down approaches, and strategies that ties that can potentially have wide-ranging applications have elements of both. Bottom-up approaches (panel a) start with one or more defined in different fields. These technologies have considerable molecular species, which undergo certain processes that result in a higher-ordered and - promise, and scientists and engineers are constantly organized structure. Examples of bottom-up approaches include systems that self- looking for new ways to explore the potential of plat- assemble, a process that is triggered by a local change in a chemical or physical form nanotechnologies. Tailored nanotechnologies condition. Related techniques include templating and scaffolding methods, such as begin with a well-defined biological question, and are 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 developed to specifically address that issue. Owing to bulk material that incorporates nanoscale details, such as nanolithography and etching the inherent complexity of biological systems in gen- techniques. Specific examples include dip-pen nanolithography (in which specific eral, and the nervous system in particular, the tailored molecules are deposited into desired configurations) and electrostatic atomic force approach often results in highly specialized technologies nanolithography (in which molecules are moved around to form desired structures). In all that are designed to interact with their target systems cases, the resultant structures have novel engineered chemical and/or physical in sophisticated and well-defined ways, and so will be properties that the original constituent materials do not have. These emergent properties better suited to tackle the particular problem than a allow controlled interactions of the nanomaterial or device with its target system. generic platform technology. However, because tailored nanotechnologies are highly specialized, their broader application to other biological systems could be limited. species. However, DNA nanowires and boron-doped In many cases, the application of particular nanotechno- silicon have gained these novel functional properties. logies in neuroscience has been derived from what was These technologies are described by the new functional originally a platform technology, and some tailored outcomes of bringing these components together, not technologies can be modified to address other (but because they had to contain DNA, or boron or silicon. usually related) scientific questions. From a synthesis Other materials or devices can be made using the same standpoint, nanotechnologies can be classified as vari- Bottom-up technologies building blocks with different functional or engineered ations of bottom-up technologies, such as self-assembly, Materials or devices outcomes, and both electrically conducting nanowires or top-down technologies, such as lithographic methods engineered from constituent and high-sensitivity chemical sensors can be produced (BOX 1). Many nanotechnologies combine aspects of elements such as specific using other chemistry and synthetic approaches. In this both strategies. molecules that are organized into higher-order functional functional definition of nanotechnology, it is implicit structures. that this is not a new area of science per se, and that Examples of current work the interdisciplinary convergence of basic fields (such This section reviews applications of nanotechnology in Top-down technologies as chemistry, physics, mathematics and biology) and basic and clinical neuroscience (TABLE 1). It is organized Materials or devices that are engineered from a bulk applied fields (such as materials science and the vari- according to the type of application, and compares dif- material. The various forms of ous areas of engineering) contributes to the functional ferent nanotechnology approaches applied to similar or lithography are examples of outcomes of the technology. In this framework, nano- related neurobiological or physiological objectives. In top-down engineering technology can be regarded as an interdisciplinary general, the discussion provides more breadth than depth approaches. pursuit that involves the design, synthesis and character- to give the reader a broad sense of ongoing research. Lithography ization of nanomaterials and devices that have the types The section is divided into two subsections: appli- The process of producing of property discussed above. In particular, this engineer- cations of nanotechnology in basic neuroscience; and patterns in bulk materials. ing definition of nanotechnology is what sets it apart applications of nanotechnology in clinical neuroscience. The most common forms of from chemistry. Chemistry is an integral component Applications of nanotechnology in basic neuroscience lithography are those associated with the production of nanotechnology, but the two are not synonymous, a include those that investigate molecular, cellular and phys- of semiconductor integrated point that is a potential source of confusion. Chemistry iological processes (FIG. 1). This first subsection discusses circuits. involves the manipulation of matter at nanometre scales three specific areas. First, nanoengineered materials and 66 | JANUARY 2006 | VOLUME 7 www.nature.com/reviews/neuro © 2006 Nature Publishing Group
  • 3. REVIEWS Table 1 | Applications of nanotechnologies in neuroscience aimed at limiting and reversing neuropathological disease states (FIG. 2). This subsection discusses nanotechnology Nanotechnology Applications Refs approaches designed to support and/or promote the Basic neuroscience functional regeneration of the nervous system20,21; neuro- Molecular deposition and Study of cellular communication and 5–10 protective strategies, in particular those that use fullerene lithographic patterning of signalling; test systems for drugs and other derivatives22–26; and nanotechnology approaches that neuronal-specific molecules molecules facilitate the delivery of drugs and small molecules across with nanometre resolution the BBB27–38. Atomic force microscopy Interact, record and/or stimulate neurons 11–13 As with any classification scheme, there might be (measures molecularly at the molecular level a sense of forced categorization when classifying nano- functionalized surfaces) technologies, as some nanotechnologies can be used Functionalized quantum dots High-resolution spatial and temporal 14–19 in more than one area. For example, all three of the imaging; molecular dynamics and tracking basic nanotechnology applications can also contribute Clinical neuroscience to an understanding of neuropathophysiology; and all Self-assembling peptide Neuronal differentiation from progenitor 20 three of the clinical nanotechnology applications can amphiphile nanofibre cells; neural regeneration also increase our understanding of basic molecular and networks cellular neurobiology. But, for the most part, basic neuro- Derivatives of hydroxyl- Neuroprotection mediated by limiting the 23–26 science applications primarily concern themselves with functionalized fullerenes effects of free radicals following injury understanding basic molecular and cellular mechanisms (fullerenols) without necessarily considering their potential clinical Poly(ethylene glycol) and Transport of drugs and small molecules 27–36 implications, whereas clinical neuroscience applications polyethylenimine nanogels; across the blood–brain barrier are designed to primarily target disease events, and make poly(butylcyanoacrylate) nanoparticles use of basic molecular and cellular neurobiology only when necessary. Applications in basic neuroscience. Molecular deposition approaches for promoting neuronal adhesion and growth and lithographic patterning of neuronal-specific mole- to understand the underlying neurobiology of these pro- cules with nanometre resolutions5–10 are an extension cesses or to support other technologies designed to inter- of micropatterning approaches39–44. The deposition of act with neurons in vivo (for example, coating of recording proteins and other molecules that promote and support or stimulating electrodes)5–10. Second, nanoengineered neuronal adhesion and growth on surfaces that do not materials and approaches for directly interacting, record- support these processes enables the geometric selective ing and/or stimulating neurons at a molecular level11–13. patterning and growth of neurons (for example, control- Third, imaging applications using nanotechnology tools, led neurite extension). This allows the study of cellular in particular, those that focus on chemically functional- communication and signalling, and provides a test sys- ized semiconductor quantum dots14–19. Applications of tem for investigating the effects of drugs and other mol- nanotechnology in clinical neuroscience include research ecules. The ability to control this process at nanometre as a Quantum-dot imaging and molecular tracking Quantum dots c Engineered materials with d Patterned neuronal nanoscale physical features adhesion and growth b Patterned molecular GABA molecules interfaces for stimulating Btn and responding Cad Fbn PA2 Lam Avd Avd Avidin Btn Biotin Cad Cadherin Fbn Fibronectin PA2 Phospholipase A2 Lam Laminin GABA γ-Aminobutyric acid 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. NATURE REVIEWS | NEUROSCIENCE VOLUME 7 | JANUARY 2006 | 67 © 2006 Nature Publishing Group
  • 4. REVIEWS a Functional nanoparticles the growth of cerebellar neurons5. These studies suggest for free radical neuroprotection that bioactive ultrathin layers could coat electrodes O2•– ONOO– designed for long-term implants to promote cell adhesion Spinal cord and limit immune responses. In a different approach, • OH Nanoparticles electrodes coated with nanoporous silicon increased neurite outgrowth from PC12 cells, which are clonally derived neuronal precursor cells, compared with uncoated electrodes, and decreased glial responses, thereby limiting b Bioactive nanoscale scaffold materials for the insulating effects of the glial scar10. Coating electrodes neural regeneration with ultrathin bioactive layers might have other advan- tages, for example, limiting the increase in the thickness Nanoscale scaffold 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) c Functional nanoparticles for delivery across between 20–70 nm adhered and grew better than neurons the blood–brain barrier cultured on surfaces with features <10 nm or >70 nm. Astrocyte These neurons also had normal morphologies and endfoot 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 Endothelial cells interact, record and/or stimulate neurons at the mole- Nanoparticles cular level11–13. Recent research has demonstrated the feasibility of functionalizing mica or glass tethered with Figure 2 | Applications of nanotechnology in clinical neuroscience. the inhibitory neurotransmitter GABA (γ-aminobutyric Nanotechnology can be used to limit and/or reverse neuropathological disease acid) and its analogue muscimol (5-aminomethyl- processes at a molecular level or facilitate and support other approaches with this 3-hydroxyisoxazole) through biotin–avidin binding goal. a | Nanoparticles that promote neuroprotection by limiting the effects of free interactions12,13 (FIG. 1). The functional integrity of the radicals produced following trauma (for example, those produced by CNS secondary bound version of the neurotransmitter, which in vivo injury mechanisms). b | The development and use of nanoengineered scaffold functions not as a bound ligand but as a diffusible materials that mimic the extracellular matrix and provide a physical and/or bioactive environment for neural regeneration. c | Nanoparticles designed to allow the messenger across the synaptic cleft, was shown electro- transport of drugs and small molecules across the blood–brain barrier. 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, opposed to micron resolutions enables the investigation may provide powerful molecule-based platforms for of how neurons respond to anisotropic physical and testing drugs and neural prosthetic devices. At present, chemical cues. Micron-scale patterning can provide a all neural prostheses (including neural retinal prostheses) functional boundary for controlling and influencing rely on micron-scale features and cannot interact with cellular behaviour, but ultimately the neuron detects a the nervous system in a controlled way at the molecular stimulus (or stimuli in the case of multiple signals) that, level, which is a significant disadvantage. Other research because of the (relatively large) micron-scale resolution of is focusing on achieving nanoscale measurements of the patterning, is a homogeneous bioactive signal that is cellular responses. Atomic force microscopy (AFM) has averaged over the entire cell. Nanotechnology approaches been used to measure local nanometre morphological present subcellular stimuli that can vary from one part responses to micro-electrode array stimulation of neuro- of the neuron to another. For example, photolithography blastoma cells11. AFM is a technique that, among other and layer-by-layer self-assembly have been used to pattern capabilities, allows the measurement of height changes phospholipase A2, which promotes neuronal adhesion, in the topography of a surface (for example, a living cell on a background of poly(diallyldimethylammonium or a synthetic material) with nanometre resolution45,46; in chloride) (PDDA)8. This approach facilitates nanoscale essence, it is a nanoscale cantilever that measures surface Atomic force microscopy patterning at resolutions that can yield complex func- topologies at the atomic level. This technique can meas- (AFM). Scanning probe tional architectures that are tailored to the needs of a ure cross-sectional changes in cell height (between 100 microscopy that uses a sharp particular experiment. Layer-by-layer self-assembly has and 300 nm), which are produced by biphasic pulses at probe moving over the surface of a sample to measure also been used on silicon rubber to pattern alternating a frequency of 1 Hz, thereby providing information on topographic spatial laminin and poly-d-lysine or fibronectin/poly-d-lysine ultrafine morphological changes to electrical stimuli in information. ultrathin layers, which are 3.5–4.4 nm thick, that support neurons that cannot be achieved by other technologies. 68 | JANUARY 2006 | VOLUME 7 www.nature.com/reviews/neuro © 2006 Nature Publishing Group
  • 5. REVIEWS a b 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 c d in dramatically improved signal detection. In addition, 10 quantum dots can be used for single-particle tracking 8 of target molecules in live cells, such as tracking lig- Quantum dot counts and–receptor dynamics in the cell membrane51–53 (FIG. 3). y direction 6 Quantum dot labelling of both fixed and live cells is well established, and has been used in a wide variety 4 of cell types — mostly in vitro or in situ54–64, with some 2 examples in vivo65,66. Despite the growing literature on the uses of quantum dots in the study of various cell 0 types, their application in the labelling of neurons14–19 0 500 1,500 2,500 3,500 Maximum measured intensity x direction and glia19 has been slower to develop, and care must be taken to validate labelling methods that are specific for Figure 3 | The quantum dot toolbox. Fluorescent quantum dots are nanoscale neural cells, as methods for labelling other cell types are particles that can be chemically functionalized by attaching a large variety of not necessarily suitable for neural cells19. Recent research biological molecules to their outer surface (for example, antibodies, peptides and has illustrated the potential of this technology in neu- 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 roscience. The real-time dynamics of glycine receptors unique optical properties of quantum dots, such as their prolonged photo-stability in spinal neurons have been tracked and analysed using (that is, minimal photobleaching), large excitation absorption spectra and single-particle tracking over periods of seconds to min- extremely narrow emission spectra; specific examples of applications of quantum utes15. These investigators characterized the dynamics of dots in neuroscience can be found in REFS 14–19. a | Primary rat cortical neurons glycine receptor diffusion, which differed as a function labelled with conjugates of quantum dots and anti-β-tubulin III antibody. β-tubulin of the spatial localization of the receptors relative to the is a neuronal-specific intermediate filament protein and so serves as a neuronal synapse depending on whether they were in synaptic, marker. b | Primary rat astrocytes labelled with quantum dot–anti-glial fibrillary perisynaptic or extrasynaptic regions. In another example, acidic protein (GFAP) antibody conjugates. GFAP is a glial specific intermediate immobilized quantum dots that were conjugated with filament protein. c | One of the main advantages of quantum dot nanotechnology is β-nerve growth factor (βNGF) were shown to interact that qualitative observations as well as quantitative data can be obtained, which provide detailed molecular and biophysical information about the biological system with TrkA receptors in PC12 cells and to regulate their being investigated. For example, by using computational and morphometric tools, differentiation into neurons in a controlled way18. This individual quantum dots can be counted across a sample image to yield information could provide new tools for studying neuronal signal- on the distribution and number of ligand–target interactions. This graph shows the ling processes. Although most applications of quantum number of quantum dots with a particular intensity. Such quantitative measures dots in neuroscience have taken place ex vivo, in vivo could be used in measuring the expression level of cellular markers, such as microangiography of mouse brains has been achieved β-tubulin in a or GFAP in b, which are conjugated to the quantum dots. d | Quantum using serum that has been labelled with quantum dots14. dots can also be used to carry out single-particle tracking of ligand–target pairs, New functionalization and labelling methods of quan- such as tracking the motion of a receptor in a cell membrane. Illustration of the tum dots have been developed and subsequently tested trajectory of a field of 55 quantum dots undergoing Brownian diffusion, with using labelled AMPA (α-amino-3-hydroxy-5-methyl-4- individual trajectories corresponding to individual quantum dots. The small size, photochemistry and bioactivity of functionalized quantum dots provide an isoxazole propionic acid) neurotransmitter receptors16, extensive new toolbox for investigating molecular and cellular processes in neurons and by measuring the cytotoxicity of hippocampal and glia. Images and data courtesy of the Silva Research Group, University of neurons17. Although quantum-dot nanotechnology, California, San Diego, California, USA. 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 An area of nanotechnology that holds significant due to the possibility of local and systemic toxicity. To promise for probing the details of molecular and cellular address these issues, the safety of using quantum dots processes in neural cells is functionalized semiconduc- with both neural cells and other cell types is an active tor quantum dot nanocrystals14–19 (FIG. 1). Quantum dots area of research67–69. Photobleaching are nanometre-sized particles comprising a heavy metal The progressive loss of core of materials such as cadmium–selenium or cad- Applications in clinical neuroscience. Applications of fluorescence signal intensity due to exposure to light. This mium telluride, with an intermediate unreactive zinc nanotechnology that are intended to limit and reverse can result in a decreased sulphide shell and an outer coating composed of selective neurological disorders by promoting neural regenera- signal-to-noise ratio. bioactive molecules tailored to a particular application. tion and achieving neuroprotection are active areas of NATURE REVIEWS | NEUROSCIENCE VOLUME 7 | JANUARY 2006 | 69 © 2006 Nature Publishing Group
  • 6. REVIEWS a 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 Amphipathic composed of peptide-amphiphile molecules21 (FIG. 4). molecules On exposure to physiological ionic conditions, peptide- amphiphile molecules, which consisted of a hydrophobic carbon tail and a hydrophilic peptide head group, self- H+ 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 OH– 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 c cells from embryonic mouse cortex in the nanofibre net- works resulted in fast and robust neuronal differentiation b (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. Figure 4 | Example of an engineered nanomaterial for neural regeneration. Applications of nanotechnologies for neuroprotec- Engineered nanomaterials enable the highly specific induction of controlled cellular tion have focused on limiting the damaging effects interactions that can promote desired neurobiological effects. a | Peptide-amphiphile of free radicals generated after injury, which is a key molecules, which consist of a hydrophilic peptide head group (green circles) and a neuropathological process that contributes to CNS 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 ischaemia, trauma and degenerative disorders 84–88. nanofibre matrix99,100. Under physiological conditions, the self-assembly process traps Fullerenols, which are derivatives of hydroxyl-func- the surrounding aqueous environment and macroscopically produces a self- tionalized fullerenes (molecules composed of regular supporting gel in which neural progenitor cells and stem cells can be encapsulated. In arrangements of carbon atoms89–93), have been shown this way, the growth and differentiation of neural progenitor cells and stem cells can to have antioxidant properties. They also function as be controlled. b | An example of a peptide-amphiphile nanogel on a 12 mm glass free radical scavengers, which can lead to a reduction coverslip. c | The surface of the nanofibres consists of laminin-derived, neuronal- in the extent of excitotoxicity and apoptosis induced by specific pentapeptides, which are encountered by the encapsulated cells at high glutamate, NMDA (N-methyl-d-aspartate), AMPA and concentrations, resulting in robust differentiation into neurons while suppressing kainate23–26. Fullerenol-mediated neuroprotection has astrocyte differentiation21. The cells are stained for the neuronal marker β-tubulin III been shown in vitro and in vivo. Fullerenol limits exci- (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 totoxicity and apoptosis of cultured cortical neurons Stupp laboratory, Northwestern University, Evanston, Illinois, USA. 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 research. The development of nanoengineered scaffolds receptors, as they had no effect on GABAA or taurine that support and promote neurite and axonal growth receptors. They also lowered glutamate-induced eleva- are evolving from tissue engineering approaches based tions in intracellular calcium, which is an important on the manipulation of bulk materials. Examples of mechanism of neuronal excitotoxicity23–26. Absorption spectra micron-scale tissue engineering include poly-(l-lactic) Another clinically relevant area of intense research The range of wavelengths over acid (PLLA) and other synthetic hydrogels that have is the design of functionalized nanoparticles that can which a molecule, such as a fluorophore, or a nanoparticle, engineered microscale features, and scaffolds derived be administered systemically and deliver drugs and such as a quantum dot, are from naturally occurring materials such as collagen70–79. small molecules across the BBB27–36. This is a major energetically excited. One example of a nanoengineered system derived from clinical objective for the treatment of a wide range 70 | JANUARY 2006 | VOLUME 7 www.nature.com/reviews/neuro © 2006 Nature Publishing Group
  • 7. REVIEWS of neurological disorders. To achieve this, various This discussion also suggests the main technical chal- materials and synthetic approaches are being investi- lenges that are encountered when using nanotechnology gated. Oligonucleotides have been delivered in gels applications in neuroscience: the need for greater spe- of crosslinked poly(ethylene glycol) and polyethylen- cificity; multiple induced physiological functions; and imine28. Charge differences in the electrostatic forces minimal side effects. Greater specificity of interactions between the gel and spontaneously negatively charged with target cells and tissues will result in more signifi- oligonucleotides provide a reversible delivery mecha- cant and specific physiological effects, which should also nism that can be used for shuttling molecules across the reduce undesirable and deleterious side effects that are BBB and then releasing them from the delivery system. induced by the technology. Another important chal- Neuropeptides (such as enkephalins), the NMDA recep- lenge is the requirement for technologies that are able to tor antagonist MRZ 2/576, and the chemotherapeutic multitask, carrying out a diverse set of specific cellular drug doxorubicin have been absorbed onto the surface and physiological functions, such as targeting multiple of poly(butylcyanoacrylate) nanoparticles coated with receptors or ligands. This is particularly important when polysorbate 80 (REFS 31,33,34,37,38). The polysorbate attempting to address multi-dimensional CNS disorders on the surface of the nanoparticles adsorbs apolipo- that are the result of numerous interdependent molecular protein B and apolipoprotein E from the blood, and the and biochemical events (for example, secondary injury nanoparticles are taken up by brain capillary endothelial following traumatic brain injury or spinal cord injury). cells via receptor-mediated endocytosis38. Nanoparticles At present, synthetic and engineering processes are not that target tumours in the CNS may be a particularly advanced enough to allow nanotechnologies that have important application of this technology due to the high been designed to interact with the nervous system to morbidity and mortality associated with often aggres- fully meet these criteria. sive neoplasms in the physically confined spaces of the From a biological perspective, the most significant cranium and spinal canal. successes of nanotechnology applications in neuro- science will be those that appreciate a detailed under- Challenges and opportunities standing of neurobiology and take advantage of the The challenges associated with nanotechnology appli- known (and unknown) molecular details. As suggested cations in neuroscience are numerous, but the impact above, the main challenge is the ability to design and use it can have on understanding how the nervous system more sophisticated technologies that are able to carry out works, how it fails in disease and how we can intervene highly targeted and specific functions while minimizing at a molecular level is significant. Ultimately, the chal- nonspecific interactions. To achieve this, both the design lenges and opportunities presented by nanotechnology and engineering aspects of nanotechnology as well as our stem from the fact that this technology provides a way understanding of the underlying neurobiology are cru- to interact with neural cells at the molecular level, cial. This, in turn, will require more interaction between which has both positive and negative aspects. The abil- neuroscientists and physical scientists such as chemists ity to exploit drugs, small molecules, neurotransmitters and materials scientists. This is not a trivial issue as and neural developmental factors offers the potential the scientific language and culture between different to tailor technologies to particular applications. For disciplines can vary considerably. This is an increasing example, neural developmental factors, such as the challenge for interdisciplinary science, which requires cadherins, laminins and bone morphometric protein people with different training, skills and conceptions families, as well as their receptors, can be manipulated of how science should be conducted coming together in new ways. Nanotechnology offers the capacity to first to understand and agree on a common challenge take advantage of the functional specificity of these or problem, and then to agree on how to address that molecules by incorporating them into engineered issue. The next section discusses further the role of materials and devices to have highly targeted effects. neuroscientists in the development and application of Emission spectra The laminins, for example, are large multi-domain nanotechnology. The range of wavelengths over which a molecule, such as a trimeric proteins composed of α, β and γ chains, of The above discussion pertains to all applications of fluorophore, or a nanoparticle, which 12 isoforms are known82,83,94,95. The isoforms con- nanotechnology to neuroscience. Applications of nanote- such as a quantum dot, emit tain different bioactive peptide sequences, which have chnology to the nervous system in vivo present additional light. varying affinities for specific cell types and can induce challenges. In particular, the inherent complexity of the Synaptic, perisynaptic or different effects. For example, the laminin 1 isoform, CNS, as well as its difficult and anatomically restrictive extrasynaptic regions which is the most studied laminin, contains at least 48 nature, poses a unique set of obstacles. Cellular hetero- Areas where neurotransmitter different short peptide sequences that promote neuro- geneity and multi-dimensional cellular interactions (for receptors cluster at, near, or nal adhesion and neurite outgrowth, and some of these example, spatial and temporal summation of postsynaptic outside the synapse, peptides (25 of 48 tested) have such effects on specific potentials) underlie the nervous system’s anatomical and respectively. types of neuron82. This degree of molecular specificity, functional ‘wiring’ that is the basis of its extremely com- TrkA receptors which is conferred not only by laminins but by many plex information processing. Nanotechnologies designed A family of proto-oncogene other signalling molecules that are important in the to interact with CNS cells and processes in vivo must take receptors found throughout the development and function of the nervous system, can this complexity into consideration, if only to avoid dis- central and peripheral nervous system that bind β-nerve be used to design highly selective nanotechnologies. rupting it. Failure to do so may result in unforeseen and growth factor, which results in Indeed, any desired cellular signalling pathway can be unacceptable ‘side effects’ in the nervous system and/or downstream signalling effects. targeted using this approach. other physiological systems. A significant challenge in NATURE REVIEWS | NEUROSCIENCE VOLUME 7 | JANUARY 2006 | 71 © 2006 Nature Publishing Group
  • 8. REVIEWS in vivo applications of nanotechnology is that they are develop neuroscience nanotechnologies for their particu- designed to physically interact with neural cells at cellu- lar purposes, most likely geared towards specific questions lar and subcellular levels, but ultimately aim at engaging or objectives for their research. But most neuroscientists functional interactions at a systemic level, which usually find that they lack the expertise and resources needed to involves large groups of interacting neurons and glia. At design, synthesize and characterize sophisticated nano- present, there are only a few applications of this type; engineered materials or devices. It would be unrealistic nonetheless, although technically and conceptually chal- to assume that we as neuroscientists have knowledge lenging, these types of application could have a significant and skills in these areas that are equivalent to those of impact on clinical neuroscience. However, there is still a chemists or materials scientists who have devoted their tremendous amount of (exciting) work to be done. careers to the synthetic aspects of these technologies. Apart from physiological complexity, the second However, chemists and material scientists do not have the main consideration for in vivo applications of nanotech- comprehensive training in neurobiology, neurophysiol- nology is that they must consider the highly anatomically ogy and neuropathology required to fully appreciate and restrictive nature of the CNS. The structures of the CNS exploit the potential of nanotechnology in neuroscience. are well protected from mechanical and physical injury, Therefore, it is crucial that different disciplines are able and are immunologically privileged behind the BBB and to communicate with each other using a common tech- blood–retina barrier, which have unique molecular and nical language, which is not a trivial issue — a nucleus cellular environments. Nanotechnologies designed for means different things to a physicist and a cell biologist. in vivo applications must be efficiently delivered with To this end, it is important for neuroscientists who wish minimal disruption to these structures before it can carry to pursue the development of nanotechnology to educate out its primary function. This will surely present signifi- themselves across disciplines. To envision new applica- cant technical challenges. Similarly, extreme care must tions of nanotechnology, it is necessary to understand be taken to understand and avoid potential safety pitfalls, what has already been accomplished and what can be including both systemic and local side effects associated achieved with this technology. with the delivery and primary function of the applied technology — an issue that is unique to in vivo nanotech- Future directions nology96–98. As mentioned above, investigating the safety Applications of nanotechnology to neuroscience are of nanotechnologies is an active and important area of already having significant effects, which will continue research67–69. Despite all these challenges, the applica- in the foreseeable future. Short-term progress has ben- tions of nanotechnology both in vivo and ex vivo offer efited in vitro and ex vivo studies of neural cells, often tremendous opportunities for understanding normal supporting or augmenting standard technologies. These physiology and for developing therapies. advances contribute to both our basic understanding of cellular neurobiology and neurophysiology, and to our The role of the neuroscientist understanding and interpretation of neuropathology. Neuroscientists have a unique role in developing nano- Although the development of nanotechnologies designed technologies. Neuroscientists — both researchers and to interact with the nervous system in vivo is slow and clinicians — need to identify potential applications of challenging, they will have significant, direct clinical nanotechnology in neuroscience and neurology to maxi- implications. Nanotechnologies targeted at supporting mize their impact. Scientists with other specialties can cellular or pharmacological therapies or facilitating develop powerful platform technologies and even provide direct physiological effects in vivo will make significant neuroscience-specific examples, but it is only with direct contributions to clinical care and prevention. The rea- input from and in partnership with neuroscientists that son for the tremendous potential that nanotechnology broad neurophysiological and clinical applications can be applications can have in biology and medicine in general properly formulated and addressed. This requires highly and neuroscience in particular stems from the capacity interdisciplinary collaborations with consideration of of these technologies to specifically interact with cells at the requirements of both parties. Some neuroscientists the molecular level. 1. Liu, D., Park, S. H., Reif, J. H. & LaBean, T. H. DNA 6. Fan, Y. W. et al. Culture of neural cells on silicon wafers interface. IEEE Trans. Biomed. Eng. 51, 881–889 nanotubes self-assembled from triple-crossover tiles with nano-scale surface topograph. J. Neurosci. (2004). as templates for conductive nanowires. Proc. Natl Methods 120, 17–23 (2002). A good example of electrode modification using Acad. Sci. USA 101, 717–722 (2004). 7. Kim, D. H., Abidian, M. & Martin, D. C. Conducting nanoengineered materials to improve neuronal 2. Cui, Y., Wei, Q., Park, H. & Lieber, C. M. 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