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Surgical Neurology 63 (2005) 301 306 www.surgicalneurology-online.

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Nanotechnology approaches for the regeneration and neuroprotection of the central nervous system
Gabriel A. Silva, MSc, PhD*
Departments of Bioengineering and Ophthalmology, Whitaker Institute for Biomedical Engineering, and Neurosciences Program, University of California, San Diego, CA 92037-0946, USA Received 16 June 2004; accepted 28 June 2004

Abstract

Nanotechnology is the science and engineering concerned with the design, synthesis, and characterization of materials and devices that have a functional organization in at least 1 dimension on the nanometer (ie, one-billionth of a meter) scale. The ability to manipulate and control engineered self-assembling (ie, self-organizing) substrates at these scales produces macroscopic physical and/or chemical properties in the bulk material not possessed by the constituent building block molecules alone. This in turn results in a degree of functional integration between the engineered substrates and cellular or physiological systems not previously attainable. Applied nanotechnology aimed at the regeneration and neuroprotection of the central nervous system (CNS) will significantly benefit from basic nanotechnology research conducted in parallel with advances in cell biology, neurophysiology, and neuropathology. Ultimately the goal is to develop novel technologies that directly or indirectly aid in providing neuroprotection and/or a permissive environment and active signaling cues for guided axon growth. In some cases, it is expected that the neurosurgeon will be required to administer these substrates to the patient. As such, in order for nanotechnology applications directed toward neurological disorders to develop to their fullest potential, it will be important for neuroscientists, neurosurgeons, and neurologists to participate and contribute to the scientific process alongside physical science and engineering colleagues. This review will focus on emerging clinical applications aimed at the regeneration and neuroprotection of the injured CNS, and discuss other platform technologies that have a significant potential for being adapted for clinical neuroscience applications. D 2005 Elsevier Inc. All rights reserved.
Nanotechnology; Neuroprotection; Neuroregeneration; CNS; Bioengineering; Stem cells

Keywords:

1. Introduction Nanotechnology is in a broad sense the science and engineering concerned with the design, synthesis, characterization, and application of materials and devices that have a functional organization in at least 1 dimension on the nanometer (ie, one-billionth of a meter) scale, ranging from a few to about 100 nm. A nanometer is 3 orders of magnitude smaller than a micrometer, 109 vs 106, respectively, and is roughly the size scale of molecules
* University of California, San Diego, Jacobs Retina Center 0946, 9415 Campus Point Dr, La Jolla, CA 92037-0946. Tel.: +1 858 822 4591; fax: +1 858 534 7985. E-mail address: gsilva@ucsd.edu. 0090-3019/$ see front matter D 2005 Elsevier Inc. All rights reserved. doi:10.1016/j.surneu.2004.06.008

(eg, a DNA molecule is about 2.5 nm long, whereas a sodium atom is about 0.2 nm). In particular, the potential impact of self-assembling nanotechnology on science, custom-made molecules that self-assemble or self-organize into higher-ordered structures in response to a defined chemical or physical cue, including applications to biology and medicine, stems from the fact that these nanoengineered materials and devices exhibit bulk mesoscale (ie, midrange scale between micro and macro) and macroscale chemical and physical properties not possessed by the constituent nanoscale building block molecules on their own. This is an emergent property because engineering materials and devices at the nanometer scale imply controlled manipulation of the individual constituent (nanoscale) units and thus (at least in part) control over their molecular synthesis

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and assembly. As such, applications of nanotechnology to medicine and biology allow the interaction and integration of cells and tissues with nanoengineered substrates at a molecular (ie, subcellular) level with a very high degree of functional specificity and control. This article represents the second in a series in Surgical Neurology dedicated to reviewing emerging applications of nanoscience and nanotechnology to clinical neuroscience. The first article (Surg Neurol. 2004;61:216-220) provided an introduction to nanotechnology, an overview of synthesis approaches, some of the main technical challenges associated with developing nanotechnology applications, and a discussion of applications geared toward different areas of medicine and biology. This article will describe emerging applications of nanotechnology aimed at the neuroprotection and functional regeneration of the CNS after traumatic or degenerative insults. In addition, other developing platform technologies are discussed, which may prove to have broad applications to medicine and physiology, including some being developed for rescuing or replacing anatomical and/or functional CNS structures. 2. Applications of nanotechnology to the central nervous system Clinical nanotechnology applications to the central nervous system (CNS) are further behind applications to other areas of medicine and biology, such as, for example, orthopedic applications, DNA/genomic sensors, and novel drug and gene delivery approaches (see, eg, Refs. [1,14,26,27,40,41,45,52,72,73,87]). (The one exception to this are approaches for drug delivery across the blood-brain barrier [20,35,36,44], which will be the subject of the next paper in this series.) This is in part because of some of the unique challenges imposed by the CNS such as restricted and difficult anatomical access, an extremely heterogeneous cellular and molecular environment, and the complexities of the systems anatomical and functional bwiringQ and associated information processing. Despite these challenges, the potential benefits of nanotechnologies for the treatment of both peripheral and CNS disorders are tremendous and may eventually offer the patient and clinician novel therapeutic choices that simply do not exist today. True to the highly interdisciplinary nature of this area of research, it is important that technological advancements occur in conjunction with basic and clinical neuroscience advancements. Therefore, three things must occur in parallel for nanotechnology applications in neurology and neurosurgery to come to fruition: (1) advancements in chemistry and materials science that produce ever more sophisticated synthetic and characterization approaches; (2) advancements in the molecular biology, neurophysiology, and neuropathology of the nervous system; and (3) the design and integration of specific nanoengineered applications to the nervous system which take advantage of the first two points. As these areas develop in an integrated and parallel

fashion, nanotechnology-based applications for nervous system disorders should start to reach the clinic. 3. Neuroprotection Free radical mediated injury is known to play a major role in the disease process of ischemic, traumatic, and degenerative disorders in the CNS [8,19,43,46,49,60,64,82,85]. Chemical species such as superoxide (O2 ), hydroxyl ( OH), peroxynitrite (ONOO), and peroxide (H2O2) can produce a host of oxidative mediated deleterious changes in cells, including DNA fragmentation, peroxidation of cell membrane lipids, decreased mitochondrial energy production, and transporter protein inactivation [16,46,64,85]. One approach being developed to deal with this is the development of carbon-60 fullerene-based neuroprotective compounds [15-17,28]. Fullerenes are molecules composed of large 3-dimensional arrays of evenly spaced carbon atoms, similar to the pattern produced by the rhombuses on a soccer ball. Fullerenols are hydroxyl (ie, OH) functionalized fullerene derivatives (Fig. 1) and have been shown to possess antioxidant and free radical scavenger properties that are able to reduce glutamate-, NMDA-, AMPA-, and kainite-induced excitotoxic and apoptotic cell death (Table 1) [15-17,28]. The mechanism of fullerenol-mediated neuroprotection is due at least in part to inhibition of glutamate channels, since neither GABA(A) or taurine receptors were affected. In addition, fullerenols appear to lower glutamate-induced increases of intracellular calcium concentrations, a critical mechanism of excitotoxicity in neurons [15-17,28]. Other approaches for neuroprotection, including pharmacological, gene therapy, and physiological, continue to be very active areas of research with significant clinical potential [2,24,25,30,32,33,51,53,57,61,70,79]. Fullerenols compliment these approaches by representing novel synthesis methods for the development of new neuroprotective compounds based on the controlled chemical manipulation and functionalization of these highly ordered synthetic

Fig. 1. Representative structure of a neuroprotective fullerene derivative functionalized with carboxylic acid groups attached to the cyclopropane carbons of the C60 molecule. Adapted from Dugan et al. Reprinted with permission from Parkinsonism Relat Disord. 2001;7:243-246.

G.A. Silva / Surgical Neurology 63 (2005) 301306 Table 1 Protective effects of C60 derivatives in biological model systems Compound In vitro Carboxyfullerene C3, D3 Fullerenols C3 Fullerenols C3 C3 C3 System Cortical neuronal cultures Same Injury condition Excitotoxicity: NMDA and AMPA Apoptosis-induced serum by deprivation Ab 1-42 toxicity Oxygen-glucose deprivation Apoptosis after NMDA receptor blockade MPP+ Results 60%-90% A in death References [2], Fig. 2A [3] [2] [3] [3] [3]

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50% A in death

Same Same Same

Complete protection 80% A in death 50% A in death

C3

C3 C3 C3 and D3 C3 C3

In vivo

Mesencephalic dopaminergic neuronal cultures Same Cerebellar granule neuronal cultures Hepatoma cells Epithelial cells FALS mice (with SOD1 G93A mutation)

40% A in death

Kim-Han J.S. and Dugan L.L., unpublished data [7], Fig. 2B

C3

Rats

6-Hydroxydopamine Apoptosis induced by NGF withdrawal TGFb-induced death Radiation Progressive motor deterioration/death produced by overexpression of mutant protein 6-OHDA intrastriatal lesioning (systemic C3) Iron-induced striatal dopamine depletion (intrastriatal C3)

Complete protection Partial protection Partial protection Partial protection Improved motor performance, 9to 12-day increased survival Preserved dopaminergic terminals and behavior Partial preservation of striatal dopamine

[7], Fig. 2B [1] [5] [10] [2,4]

[8]

C3

Rats

[6]

AMPA indicates a-amino-3-hydroxy-5-methyl-4-isoxazolepropionic acid; FALS, familial amyotrophic lateral sclerosis; MPP, massive periretinal proliferation; NGF, nerve growth factor; NMDA, N-methyl-D-aspartate; OHDA, Hydroxypamine; SOD1, copper/zinc superoxide dismutase; TGF, transforming growth factor. Adapted and reprinted with permission from Dugan et al. Parkinsonism Relt Disord 2001;7:243-6.

structures. Given the proposed mechanism of fullerenolinduced neuroprotection, it will be interesting to see what clinical applications develop for secondary injury following traumatic CNS disorders. 4. Neuroregeneration The authors postdoctoral work in collaboration with colleagues focused on nanotechnology approaches for CNS regeneration after spinal cord injury and neural retinal degeneration [71,76-78]. Peptide amphiphile molecules, that is, peptide-based molecules with a hydrophobic tail and hydrophilic head group, were designed to self-assemble into a network of nanofiber scaffolds only when present in physiological ionic conditions [22,23,54]. The surface of the nanofibers, made up of the hydrophilic head groups of aligned peptide amphiphile molecules, consisted of physiologically active peptide sequences designed to engage in cell signaling by acting as ligands for cell surface receptors. In our case the main peptide sequence we explored was the neuron-specific extracellular matrix laminin-derived sequence isoleucine-lysine-valine-alanine-valine (IKVAV) [76-78], which is known to promote the growth and

development of neurites [11,12,29,39,47,59,81,84,86]. The peptide amphiphile molecules existed in a solution of water until they encountered physiological concentrations of cations such as calcium, which triggered their self-assembly into nanofibers that bheldQ the water molecules in place, macroscopically forming a gel-like substrate. Because of this unique property, we were able to encapsulate neural progenitor cells or neural retinal cells in these gels by mixing cell culture suspensions with peptide amphiphile solutions, trapping the cells in the interior of the gels. Because the functional peptide sequence formed the outer surface of the nanofibers, it allowed the functional signaling of encapsulated cells in three dimensions. The results were quite dramatic (Fig. 2), and in the case of encapsulated neural progenitor cells included faster and more robust differentiation into mature neuronal phenotypes compared with controls. By 1 and 7 days in vitro, 30% and 50%, respectively, of the neural progenitor cells expressed the mature neuron marker b-tubulin III [76]. We were also surprised to observe almost a complete exclusion of astrocyte development in these cultures (less then 1% and 5% at 1 and 7 days in vitro, respectively [76]) despite the multipotent nature of the progenitor cells. This suggests

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Fig. 2. Immunofluorescence of mouse neural progenitor cells at 7 days in vitro encapsulated in a peptide amphiphile nanofiber network labeling for neurons (b-tubulin III, green), astrocytes (glial fibrillary acidic protein, orange), or nonspecifically for all cells (Hoechst stain, blue). A: Dissociated neural progenitor cells. Note the 3-dimensional nature of the peptide amphiphile gel: blurred cells are on a different plane of focus than cells in focus. B: Undissociated neurosphere (ie, groups of cells derived from the cellular proliferation of cultured neural progenitor cells). Note the extent of neurite development in b-tubulin positive (green) neurons. In both panels, note the almost negligible degree of astrocyte differentiation and development.

novel approaches for limiting the effects of reactive gliosis and glial scarring after traumatic or degenerative events by transplanting donor cells in vivo with the peptide amphiphile substrates. Similar results were observed with encapsulated retinal cells. The ultimate vision is for the peptide amphiphile solutions to be (minimally invasively) injected stereotaxically in combination with donor cells, have the nanofiber network form in situ and in vivo, and provide functional cellular signaling to both donor and host cells while at the same time limiting the effects of reactive gliosis. This nanofiber system is currently being explored for spinal cord injury, stroke, and degenerative retinal disorders including age-related macular degeneration. 5. Platform technologies In addition to nanotechnologies aimed specifically at replacing or rescuing CNS cells, which is still quite limited, a broader group of technologies being developed can be classified as platform technologies that have the potential for both basic neurobiology and clinical neurology/neurosurgery applications. One area that has received considerable attention is the growth and selected patterning of neural cell types on patterned surfaces. Still being explored on surfaces with microscale physical features or patterns of deposited macromolecules [9-11,84], this approach has recently been extended to the nanoscale by photolithography etching on SiO2-coated Si wafers characterized using atomic force and scanning electron microscopy [18] (see also Ref. [75]). These authors determined that the ability of mixed primary cultures derived from the substantia nigra to grow and survive on surfaces with nanoscale features were closely linked to the physical dimensions of surface roughness, with optimal cell growth falling within a narrow window; features less than 10 nm or greater than 70 nm were associated with decreased cell adherence.

Other applications are taking advantage of some of the unique properties offered by carbon nanotubes to improve chronic CNS electrical stimulation. Clinically, functional electrical stimulation implants are being used more and more to treat intractable pain [34,37,62,69,74] and are also gaining momentum for the treatment of other disorders such as urinary incontinence and parkinsonian-related disorders [36,38,58], in most cases involving neurosurgical intervention. In addition, both stimulating and recording CNS electrodes are important neurophysiological and neuropathological experimental tools [4-6,13,21,42,55,56,63,65-69,80]. One of the most significant problems associated with the development of recording or stimulating chronic CNS electrodes is device failure associated with the fibrotic response mediated by glial and immune cells [3,6,7,31,50]. To address this, some groups are focusing on the development of novel carbon nanofiberbased electrode arrays for CNS neuronal stimulation either on their own using compressed carbon nanofiber structures [48] or in combination with other materials such as using them to reinforce polyurethane composites [48]. They are also in the earliest stages of being developed for a retinal prosthesis [83]. In all cases, the idea is to utilize the unique conductive electrical properties of carbon nanofibers for highly controlled focal stimulation. 6. Conclusions Nanotechnology research aimed at the regeneration and neuroprotection of the CNS will significantly benefit from continued nanotechnology research in parallel with advances in cell biology, neurophysiology, and neuropathology. Ultimately, the goal is to develop technologies that directly or indirectly aid in providing a permissive environment and spatial and temporal cues for guided axon growth after degeneration or secondary injury mechanisms. In some cases, it is expected that the neurosurgeon will be required

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to provide or administer the nanoengineered substrate to the patient. As with all therapeutic strategies for CNS disorders, there is the issue of getting the material, device, or drug to the site where it is needed in the CNS itself. For example, the delivery of peptide amphiphile molecules for nanofiber network formation in vivo in rat models of acute compression spinal cord injury requires a laminectomy and stereotaxic injection. As such, in order for nanotechnology applications directed toward neurological disorders to develop to their fullest potential, it will be important for neurosurgeons, neurologists, and neuroscientists to participate and contribute to the scientific process alongside physical science and engineering colleagues. References
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