Bio-inspired nano tools for neuroscience

Accepted Manuscript Title: Bio-inspired nano tools for neuroscience Author: Suradip Das Alejandro Carnicer-Lombarte James W. Fawcett Utpal Bora PII: DOI: Reference:

S0301-0082(15)30105-2 http://dx.doi.org/doi:10.1016/j.pneurobio.2016.04.008 PRONEU 1442

To appear in:

Progress in Neurobiology

Received date: Revised date: Accepted date:

21-12-2015 14-4-2016 15-4-2016

Please cite this article as: Das, Suradip, Carnicer-Lombarte, Alejandro, Fawcett, James W., Bora, Utpal, Bio-inspired nano tools for neuroscience.Progress in Neurobiology http://dx.doi.org/10.1016/j.pneurobio.2016.04.008 This is a PDF file of an unedited manuscript that has been accepted for publication. As a service to our customers we are providing this early version of the manuscript. The manuscript will undergo copyediting, typesetting, and review of the resulting proof before it is published in its final form. Please note that during the production process errors may be discovered which could affect the content, and all legal disclaimers that apply to the journal pertain.

-inspired Nano tools for Neuroscience Suradip Das1, Alejandro Carnicer-Lombarte2, James W. Fawcett2, Utpal Bora1,3* [email protected] [email protected] 1

Bioengineering Laboratory, Department of Biosciences and Bioengineering, Indian Institute of Technology Guwahati, Guwahati – 781039, Assam, India 2 John Van Geest Centre for Brain Repair, Department of Clinical Neurosciences, University of Cambridge, Robinson Way, Cambridge - CB2 0PY, United Kingdom 3 Mugagen Laboratories Private Limited, Technology Incubation Complex, Indian Institute of Technology Guwahati, Guwahati – 781039, Assam, India *Corresponding author at: Department of Biosciences and Bioengineering, Indian Institute of

Technology Guwahati, Guwahati – 781039, Assam, India.

0|P a ge

Abstract Research and treatment in the nervous system is challenged by many physiological barriers posing a major hurdle for neurologists. The CNS is protected by a formidable blood brain barrier (BBB) which limits surgical, therapeutic and diagnostic interventions. The hostile environment created by reactive astrocytes in the CNS along with the limited regeneration capacity of the PNS makes functional recovery after tissue damage difficult and inefficient. Nanomaterials have the unique ability to interface with neural tissue in the nano-scale thereby influencing the function of a single neuron. The ability of nanoparticles to transcend the BBB through surface modifications has been exploited in various neuro-imaging techniques and for targeted drug delivery. The tunable topography of nanofibers provides accurate spatiotemporal guidance to regenerating axons. This review is an attempt to comprehend the progress in understanding the obstacles posed by the complex physiology of the nervous system and the innovations in design and fabrication of advanced nanomaterials drawing inspiration from natural phenomenon. We also discuss the development of nanomaterials for use in Neuro-diagnostics, Neuro-therapy and the fabrication of advanced nano-devices for use in opto-electronic and ultrasensitive electrophysiological applications. The energy efficient and parallel computing ability of the human brain is inspiring the design of advanced nanotechnology based computational systems. However, extensive use of nanomaterials in neuroscience is raising serious toxicity issues as well as ethical concerns regarding nano implants in the brain. In conclusion we summarize these challenges and provide an insight into the huge potential of nanotechnology platforms in neuroscience.

1

1.0 Introduction The attempt to understand the brain and its functions is quite old, starting from the Egyptians who believed the brain to be mere “cranial stuffing” and assumed the heart to be the seat of consciousness and intelligence. This concept was challenged by Greek philosophers like Alcameon of Croton (470B.C.) and Hippocrates (400 B.C) who hypothesized the brain to be the centre of human intellect. The importance of the brain in governing all aspects of human intelligence and sensation has been mentioned in ancient Vedic scriptures (1000B.C.).

Detailed anatomical study of the human brain was first conducted by Greek anatomist Galen and later carried forward and published by Thomas his book Cerebri anatome (1664). His work lead to the beginning of a new branch of medicine called Neurology. Galvani’s experiments with the frog (1786), Golgi’s staining procedure in the late 1890s and Cajal’s study of the neuron are some of the pivotal events that laid the foundation for modern neuroscience. The anatomical location of the brain and spinal cord along with the delicate nature of the nervous tissue has always been the major challenge in exploring the nervous system. Initial indirect routes of measuring brain activity comprised of thermal recordings and estimating cerebral blood flow. The advancement of technology and advent of noninvasive imaging techniques like computerised tomography (CT), positron emission tomography (PET) and magnetic resonance imaging (MRI) has made possible challenging projects like the human brain mapping which was nearly inconceivable a few decades ago.

The quest for exploring the sub-microscopic elements of the nervous system and developing theranostic technologies capable of interacting at the sub-neuronal level (molecular neuroscience) have led to the application of nanotechnology in neurosciences. It is noteworthy that the technology to develop nanomaterials and manipulate material properties in the atomic scale has existed for thousands of years.

Nanoparticle loaded drug delivery dates back to the Charak Samhita (100 BC) that describes unique metallic-mineral formulations called “Bhasmas” which have now been reported to be biologically prepared nanoparticles (Chaudhary, 2011; Pal et al., 2014). One of the best examples of carbon nanotubes is the Damascus steel made by Indian craftsmen from Wootz steel more than 2000 years ago. The swords made from this steel had cementite nanowires encapsulated by carbon nanotubes which lead to its exquisite sharpness and ultralight weight (Reibold et al., 2006). 2

Materials engineered to the nanometer scale exhibit high surface-to-volume ratio along with unique optical, magnetic, and electrical properties. Nanotechnology is being extensively employed in various disciplines ranging from computer and electronics to chemistry and biomedical applications. In this review, we have focused on the impact nanotechnology has had on the progress of neurological sciences through its role in neuro-imaging techniques, drug delivery platforms and providing nano scale topographical guidance to regenerating nerve tissues. We also attempt to elucidate how the barriers posed by the complex physiology of the nervous system have been overcome by the advancement of nanotechnology through development of bio-inspired and biocompatible nanomaterials. The nano-dimension in perspective of the nervous system is shown in Figure 1A. The overall concept of the present article is summarized in Figure 1B. 2.0 Nanoneurotechnology: Neuro challenges and Nano solutions A search query with the term “nanoneurotechnology” in Web of Science points to a review article by Kanwar et al in 2012 describing nanoparticles as an “untamed dragon” that has the “fire power to heal” several neurological disorders (Kanwar et al., 2012). Although it might be the first time the term “nanoneurotechnology” was reported in a peer reviewed journal, the concept of using nanomaterials in clinical neurology dates back to the late 80s and early 90s. During that period super-paramagnetic particles like iron oxide particles were extensively studied and used as contrast agents in magnetic resonance imaging (MRI) (Renshaw et al., 1986; Stark et al., 1988). In order to escape the reticuloendothelial system (RES) and accumulate in the target tissue, the contrast agents had to be less than 50 nm in size, with a negative surface charge and high surface area to bind with biomolecules. In consideration of these requirements, a Swiss group developed a polypeptide coated magnetite nanoparticle that could be conjugated with a tumor specific antibody. The iron-oxide nanoparticles were able to evade the RES leading to high circulation time and target tissue specific accumulation (Tiefenauer et al., 1993). The study for the first time showed the potential of magnetic nanoparticles to be used as MRI contrast agents. Such efforts fuelled the application of nanomaterials in neuro-diagnostics and neuro-therapy. However, initial attempts to blend the two disciplines were to encounter formidable neurological challenges. Extensive research in nano-science in the last two decades has helped to overcome most of these barriers leading to breakthrough technological developments in nano-neurotechnology. 3.0 Accessing the Brain 3

The complex architecture of the nervous system, its unique anatomy and physiology poses major obstacles for any molecule designed to target and interface with it. The three major neurological challenges which nanotechnology has exhibited the potential to overcome are the highly protected anatomy of the nervous system, the blood brain barrier and the limited regeneration capability of the nervous tissue. 3.1 Biology of the Blood-Brain Barrier (BBB) – The presence of a barrier limiting the flow of molecules between the brain and blood was first realised when renowned scientist Paul Ehrlich intravenously injected a dye and found that the brain remained unstained (Ehrlich P, 1885). Later Ehrlich’s student Edwin Goldmann found that by injecting the dye within the CSF, the brain could be stained (Edwin E Goldmann, 1912; F.W. Mott, 1913). The seminal experiments suggested the presence of a physiological barrier present around the capillaries of the brain which was later confirmed with the advent of the electron microscope. The blood brain barrier (BBB) is a selective physical barrier located at the neurovascular junction in the CNS that controls molecular traffic across the brain and the systemic circulation in the capillary endothelium. The BBB is constituted by several junctions across the neurovascular unit that limit paracellular transport and forces molecules to take the transcellular route (Barbu et al., 2009). The lumen of the cerebral capillary is enclosed by a single brain micro-vascular endothelium cell (BMEC). These endothelial cells are different from the endothelia present in the peripheral system due to the presence of continuous tight junctions and absence of fenestrations. Such structure, combined with an almost absent spontaneous vesicular transport through the BMEC, makes this system impermeable to most substances.. Closely associated with the abluminal surface of endothelia are phagocytic cells of mesodermal origin called pericytes. Although there is limited knowledge about the function of pericytes in the BBB, they have been reported to secrete angiopoeitin which stimulates the production of a major tight junction protein called occludin (Hori et al., 2004). The pericytes and BMEC are ensheathed by a 30-40 nm thick basal lamina composed of various extracellular matrix proteins like laminin, fibronectin, and collagen IV (Farkas and Luiten, 2001). The basal lamina is contiguous with astrocyte endfeet which completes the neuro-vascular complex of the blood brain barrier. Although the role of astrocytes in the BBB is still under debate, there are reports suggesting that perivascular astrocytes might have an important role in maintaining the structural integrity and functioning of the BBB. The astrocytic endfeet contain a high density of intra-membranous particles organized in orthogonal arrays containing Aquaporin water channel and Kir 4.1 potassium channel that 4

regulate the flow of ions (Abbott et al., 2006). The polarity of the astrocyte regulated by such channels (Aquaporin and Kir 4.1) has been shown to affect the expression of agrin, a heparin sulphate proteoglycan responsible for BBB tightening (Verkman, 2002; Wolburg, 2007). In vitro studies have shown that perivascular astrocytes also influence the expression of localized transporters as well as specific enzymes which form a transport and metabolic barrier (Haseloff et al., 2005; Hayashi et al., 1997; McAllister et al., 2001; Sobue et al., 1999). The tight junctions, adherens junction and to a lesser extent the comparatively more permeable gap junctions are the main structural components of the BBB that are responsible for restricting flow of molecules in the brain inter-endothelial cleft. Both tight and adherens junction contain trans-membrane proteins that associate with adjacent cells as well as cytoplasmic proteins which are connected to the actin filaments of the cytoskeleton. Adherens junction or zonula adherens are cell-cell junctions like the desmosomes that are responsible for adhesion of endothelial cells. These junctional complexes also mediate cell polarity and permeability, as well as regulate vascular growth by contact inhibition (Bazzoni and Dejana, 2004). One of the principal proteins of the adherens junction is the Ca2+ regulated vascular endothelial (VE) cadherin. The VE-cadherins form complexes with catenins - cytoplasmic plaque proteins - thereby coupling to the actin cytoskeleton (Vincent et al., 2004). On the other hand, the extracellular domains of the cadherins on adjacent cells assemble by homophilic interaction to form the adherens junction (Lampugnani et al., 1995). The tight junction or zonula occludens is a belt like region comprising of transmembrane and cytoplasmic proteins that span the inter-cellular cleft between adjacent endothelial and epithelial cells in the brain. These junctions appear as a network of strands which regulates paracellular flux across the BBB (Hawkins and Davis, 2005). This “fence” limits molecular trafficking, restricting the movement of even small ions like Na2+ and Ca2+ across the interendothelial space. This leads to a very high trans-endothelial electrical resistance (TEER) of over 1000 ohm.cm2 in the brain, as compared to a resistance of 2 - 20 ohm.cm2 in peripheral capillaries (Butt et al., 1990). The tight junction transmembrane proteins include occludin, claudins and junctional adhesion molecules (JAM). These trans-membrane proteins are associated with several cytoplasmic proteins like ZO-1, ZO-2, ZO-3 and membrane associated guanylate kinase-like proteins (MAGUKS) which link the trans-membrane proteins with the actin filaments of cytoskeleton. Heteropolymers of occludin and claudin form intramembranous fibrils that contain ion-channels allowing selective diffusion of 5

molecules. The role of the various transmembrane and cytoplasmic proteins associated with the junctional complexes of the BBB in molecular transport is described in brief in Figure 2. The BBB acts as both the first line of defence and an inconvenient barrier for treatment delivery to the CNS. Moreover, the structural integrity and the functioning of the BBB has been reported to be compromised in various neurological disorders making it pertinent to develop novel drug delivery platforms that can target the CNS crossing the BBB. 3.2 Nanotools for diagnostics and treatment in Neurology Nanotechnology has had a profound impact on improving the diagnosis of neurological disorders both by developing more sensitive and specific assays for early stage detection of neurodegenerative diseases as well as enhancing the specificity and clarity of neuro-imaging techniques. Although nano-scale imaging techniques like electron microscopy and atomic force microscopy can detect protein misfolding associated with neuro-degenerative diseases, it is usually only possible at very severe stages of the disease (Lyubchenko et al., 2010). Most of the biomarkers for such diseases appear at young age, prior to the appearance of any clinical symptoms, and hence early detection of these biomarkers is essential. Nanomaterials with their high sensitivity and specificity have been integrated into biosensors which can detect ultra-low concentrations of these biomarkers in a non-invasive, inexpensive and rapid manner. Nanomaterial surfaces coated with biomolecular coronas help in lowering the surface energy and facilitate its dispersion (Salvati et al., 2012). The biochemical composition of this corona plays a crucial role in determining its self-life in vivo, its biodistribution and the ultimate fate of the nanomaterials (Mahmoudi et al., 2015). Bioinspired surface modifications of nanomaterials for regulating the specific composition of the outer corona of nanocarriers can enable them to permeate the BBB (predominantly by receptor mediated endocytosis) and deliver their payload into the brain tissue. The ability of nanoparticles to penetrate the BBB has been exploited to develop therapeutic solutions against a variety of neuro-pathological conditions ranging from brain tumors (its delineation and enhanced radio-sensitization) to neurodegenerative diseases like AD, PD, cerebral palsy, and Huntington’s disease. Although the development of nano based platforms to target molecules into the central nervous system is a recent phenomenon, nano sized viral pathogens have been successfully crossing the BBB and localizing in the brain tissue since ages. For example, HIV particles with an average size of 120nm are internalized through the BBB by adsorptive transcytosis (Banks et al., 2001; Mishra et al., 2011).The stealth mechanism adopted by viruses like HIV to cross the BBB have inspired scientists to develop nano6

vehicles functionalized with cell penetrating TAT peptides (derived from HIV). These were found to successfully deliver the drug ritonavir through the BBB within brain parenchyma (Figure 3A) (Rao et al., 2008). Another biological phenomenon which have been exploited in several nanoparticle based delivery strategies is the trans-endothelial migration of leukocytes caused due to increased permeability and hypertrophy of brain endothelia upon secretion of cytokines like TNF-α, interleukins, interferon- γ etc (Persidsky et al., 1997; Williams et al., 2012). Utilizing this ability of leukocytes, nanoporous silicon particles were coated with purified cellular membranes of leukocytes. These were able to efficiently transport and deliver a payload across the endothelium through receptor-ligand interactions. (Figure 3B) (Parodi et al., 2013). The presence of specific receptors like GLUT-1 (Morgello et al., 1995) on the luminal surface of brain endothelium cells have also been exploited for targeted drug delivery by functionalizing nanoparticles with ligand molecules. Gold nanoparticles of 4nm size coated with glucose were reported to be engulfed by receptor mediated transcytosis pathway. The nanoparticles were able to interact with GLUT-1 receptors present on the endothelial surface and cross the BBB at a rate of 10μm/hour (Figure 4) (Gromnicova et al., 2013). 3.2.1 Nano-Neuro Imaging: Nanotools for Neuro-imaging Visualizing the mind at work and acquiring knowledge about its functioning has always been the ultimate aim of neurologists. However, the anatomical and physiological barriers have always remained a major challenge. Fuelled by the strong desire to understand the nervous system, indirect methods to estimate brain activity were developed. The technique of cerebral thermometry, mastered by Angelo Mosso, was widely employed by 19th century neurologists to determine brain activity upon performing cognitive tasks by measuring minute rises in brain temperature. Mosso also worked simultaneously on studying how cerebral blood volume changed upon mental activity (Mosso A, 1881). Mosso’s methods of recording cerebral pulsations and intracranial pressure, and the ingenious equipments designed by him to measure said parameters were still popular in the early 20 th century. His pioneering experiments on cerebral circulation gradually obscured primitive thermo-electric methods and laid the foundation for research on spatial resolution of brain activity by monitoring blood flow in specific regions – the basis for today’s emission tomography and magnetic resonance imaging techniques. During the mid 20th century, American scientist Seymour Kety’s experiments with diffusible gaseous radioactive tracers and intravenous injections of radio-isotopes (85Kr) into carotid artery by Lassen and Ingvar enabled imaging 7

of brain sections by autoradiography methods (Lassen and Ingvar, 1961). A few decades later, these auto-radiographic measurements of cerebral blood circulation led to the invention of computerised tomography (CT) by Hounsfield and positron emission tomography (PET) by Kuhl. These techniques enabled three dimensional image reconstruction of the brain by trans-axial slicing of tissue. While CT scanning gave a more detailed anatomical view, PET could provide higher resolution images of soft tissue and in situ metabolic activity by monitoring glucose uptake (by FDG - Fluorinated deoxyglucose). The principles of nuclear magnetic resonance (NMR) were successfully translated into medicine by Paul Lauterbur who developed magnetic resonance imaging (MRI) in 1973 which could generate crosssectional images of soft tissue with a higher resolution than PET (μm range). The most advanced MRI technique - functional MRI (fMRI) - is also based on a principle established decades ago by Pauling and Coryell that alteration in the percentage of oxygen in haemoglobin can be measured accurately by changes in the magnetic properties of haemoglobin. Functional MRI along with nano based contrast agents have greatly contributed to various projects aimed at mapping the brain and aid in diagnosis of neurodegenerative diseases. 3.2.1.1 Magnetic nanoparticles - Liposomes and iron oxide nanoparticles are the most used nanomaterials in MRI because of their ability to cross the BBB and tunable magnetic properties, respectively. Like most other biomedical innovations, the use of superparamagnetic nanoparticles as contrast agents for neuro-MRI was inspired by the physiology of living beings. It was back in the late 80s and early 90s that the role of iron imbalance in neurodegenerative diseases was first observed. In the body, iron remains in nano-form as ferritin: comprising of a 1 nm thick protein coating called apoferritin and a 25 nm iron oxide core (EC, 1987). A separate investigation revealed that Parkinson’s patients exhibited an increased ferritin level corresponding to enhanced MR signal in the substantia nigra region of the brain (Gorell et al., 1995). Although ultra small iron oxide particles (USPIO) were already used in MR studies of liver, kidney and respiratory diseases; the correlation of ferritin levels with Parkinson’s disease lead to an exponential increase in research activity involving application of USPIO in neuro-MRI. These USPIOs possessed long circulating life and were found to accumulate at the margins of brain tumor leading to improved delineation of tumors in MR images (Enochs et al., 1999). However, their limited ability to cross the BBB as well as toxicity concerns was a major impediment towards the use of USPIOs in therapeutic applications and intracellular imaging in the brain. Once again, the solution lay with nature 8

herself. Taking cue from viruses like HIV which can easily cross the BBB, a Harvard group conjugated USPIOs with HIV derived Tat peptide (responsible for cellular uptake of HIV). They further coated the nanoparticles with dextran thereby making them more biocompatible (Josephson et al., 1999). The high degree of cellular uptake by lymphocytes encouraged them to develop USPIO based multifunctional systems. They further conjugated the dextran-coated USPIO-Tat complex with near-infrared fluorescent probes (NIRF) like Cy5.5 expanding the versatility of the system. Such a system could be used in preoperative MRI to delineate tumor margins as well as in intra-operative imaging of brain tumors (Kircher et al., 2003). The enhanced cellular uptake of surface-functionalized iron oxide nanoparticles encouraged studies using USPIOs as vehicles for drug delivery to brain tumors (Chertok et al., 2008). Further, glioma specific USPIOs were also designed by conjugation with chlorotoxin, a peptide having high affinity and specificity for matrix-metalloproteinase (MMP-2) expressing tumors of neuro-ectodermal origin (Sun et al., 2008). Conjugating such-target specific ironoxide nanoparticles with NIRF probe Cy5.5 enabled MRI as well as intra-operative pathology detection at the cellular level (Veiseh et al., 2005). A Chinese team designed and reported similar glioma specific super-paramagnetic iron oxide nanoparticles (SPIONs) by conjugating these to transferrin, which can bind the transferrin receptor over-expressed in glioma cells. MR imaging even 2 days post-administration of SPIONs revealed significant contrast enhancement on T2-weighed images of glioma, and Prussian blue staining of tumor indicated enhanced retention of nanoparticles inside the tumor cell cytoplasm (Jiang et al., 2012). Enhanced permeability and retention (EPR) of such magnetic nanoparticles were obtained by temporary disruption of BBB by focused ultrasound prior to injecting the nanoparticles. The technique was found to be useful in delivering macromolecular chemotherapeutic agents inside the CNS (Liu et al., 2010). The advent of several commercial varieties of SPIOs like Feridex (Ferimoxides by AMAG Pharma), Resovist (Ferucarbotran by Bayer Healthcare), Combidex (Ferumoxtran-10 by AMAG Pharma) and Clariscan (PEGylated SPIO suspended in glucose) led to their comparison with conventional gadolinium (Gd3+) based MRI contrast enhancers. Gadolinium based agents suffer low circulation time and fast diffusion from cells with low target tissue specificity, as well as certain side effects (Jiang et al., 2012). Studies comparing commercial SPION - based contrast enhancers and gadolinium reported identical efficiency of both as MRI contrast enhancers. Notably, the SPIONs were found to accumulate along the periphery of tumor and hence were primarily effective in determining the tumor margins (Manninger et al., 2005; Varallyay et al., 2002). A separate study by the same group with Ferumoxtran-10 9

(Combidex), a dextran-coated SPION, showed TI and T2 MR signals even in very low magnetic field of up to 0.15 Tesla, which did not enhance with gadolinium. Further, Ferumoxtran-derived T1 signal enhancement was persistent for a longer duration (2 - 5 days) which could be advantageous for post-operative imaging (Neuwelt et al., 2004). Although many SPION-based commercial contrast enhancers were approved in the past, most were banned or got discontinued at a later stage. Hence, presently, gadolinium chelate based complexes remain the preferred MRI contrast enhancers. Recent reports of application of SPIONs in monitoring cerebral blood volume (CBV) have been encouraging. Due to their long half-life and enhanced sensitivity, USPIO nanoparticles can play an important role in estimating CBV through CBV weighted functional MRI (fMRI). The CBV being a critical indicator of tissue viability and vascular activity, fMRI involving USPIOs have been reported to provide enhanced spatial specificity and better monitoring of neurological changes induced upon functional activity and pharmacological interventions (Kim et al., 2013). 3.2.1.2 Liposomes - Liposomes are microscopic artificial phospholipids which have been recognized as promising vehicles for delivery of therapeutic and diagnostic agents to various tissues in the body. Moreover, the ability of such colloidal particles to cross the BBB and accumulate in the CNS along with their easily tunable surface properties makes liposomes an attractive vehicle for neuro-diagnostic applications. In the early 1990s a group of radiologists from Connecticut Health Centre, USA experimented with lipid coated microbubbles (air filled bubbles which were efficient reflectors of sound) as contrast agents for neurosonography. They observed that the lipid coated microbubbles were stable for over 6 months in vitro with an in vivo half-life of 20 hours (Simon et al., 1990). Upon direct injection of these microbubbles as contrast agents into brain parenchyma it was possible to detect growing lesions in 40% less time than without the microbubbles (D’Arrigo et al., 1991). Further, using these lipid coated microbubbles, they were also able to directly correlate signal intensity from the accumulated microbubbles in the glioma with the rate of tumor enhancement, thereby providing a quantitative assessment of tumor progression (Simon et al., 1992). The unique ability of liposomes to trap any drug or contrast agent within its aqueous core or within its membranes has drawn special attention from radiologists. Although these artificial phospholipids have been useful in imaging various vital organs across different imaging modalities (Kabalka et al., 1991; Passariello et al., 1990; Rozenberg et al., 1985; Schwendener et al., 1990) their use in CNS imaging was limited until the late 1980s (Turski et al., 1988). The application of liposomes as contrast agents in gamma and magnetic resonance imaging until the early 1990s as well as the various methods available to 10

design target specific liposomes by surface modifications were reviewed in detail by another radiologist and biochemist from Harvard Medical School, USA (Torchilin, 1996, 1997). Delivery of therapeutic or diagnostic agents in the CNS especially in brain tumors is largely limited by poor diffusion of molecules through tumor and brain interstitium. Although smaller molecules like nanoparticles have higher diffusivity, their therapeutic efficacy is largely diminished due to loss via capillaries. In order to overcome this challenge, a team of neurosurgeons from NIH, USA came up with the concept of using fluid convection or bulk flow to deliver molecules in the brain instead of diffusion. They were able to enhance the distribution of large and small molecules by maintaining a pressure gradient during infusion through brain interstitium (Bobo et al., 1994). This technique of convection-enhanced delivery (CED) of molecules into the brain was later used to target chemotherapeutic agents like Taxol into brain tumors and its response monitored by diffusion weighted MRI (Mardor et al., 2001). Liposomes were also eventually administered into rodent brains and brain tumor models using convection enhanced delivery. In comparison to direct injection method, CED was able to achieve extensive distribution of liposomes loaded with gadodiamide and fluorochromes or gold nanoparticles in brain tissue as monitored by MRI and fluorescence microscopy respectively (Mamot et al., 2004; Saito et al., 2004). Similar results were obtained in primate (monkey) models by real-time monitoring of liposomal distribution by MRI (Saito et al., 2005). Extensive in vivo studies in rodent and non-human primate models subsequently led to the clinical application of CED-based administration of liposomes. At present, convection based delivery of liposomes loaded with chemotherapeutic drug Irinotecan and its monitoring by MRI in recurrent high grade glioma is under Phase 1 clinical trials (“A Phase I Study of Convection-Enhanced Delivery of Liposomal-Irinotecan Using Real-Time Imaging With Gadolinium In Patients With Recurrent High Grade Glioma, ClinicalTrials.gov Identifier: NCT02022644,” n.d.). 3.2.1.3 Quantum dots - Real-time imaging of brain tumor under intra-operative conditions is a much needed utility in a neuro-surgeon’s arsenal. Although intra-operative MRI and ultrasound using USPIO nanoparticles have been reported in some cases, their clinical relevance is largely limited by huge costs and difficult data interpretation techniques. Optical microscopy-based imaging provides a cheaper and simpler alternative. However, tagging nanoparticles with fluorescent dyes does not appear to be particularly helpful under the highly lit conditions of operating room which results in rapid photobleaching of fluorophores. Seminconductor nanocrystals like Quantum dots (QDs) have been proved to have excellent fluorescence quantum yield, higher brightness and are resistant to photobleaching as 11

compared to traditional fluorophores. Quantum dots administered in sufficient doses in rat glioma model were found to be sequestered by macrophages and microglia which infiltrated the tumor tissue. The deep red fluorescence of QDs could thereby be easily detected optically outlining the brain tumor and augmenting surgical resection and biopsy of the tumor (Jackson et al., 2007). These findings were extensively analysed along with the QDs toxicity concerns and methods to improve its biocompatibility, in order to accurately assess their potential as in vivo optical imaging agents for management of brain tumors (Popescu and Toms, 2006). QDs engineered to emit fluorescence in the NIR region (750 nm) were able to achieve higher tissue penetration of up to 5 mm from the skull corresponding to the hypothalamic region of mouse brain. The fluorescence signal peaked within 3 hours of QD administration and gradually decreased over 3 days (Youn et al., 2008). Further, to increase their stability and BBB permeability, QDs were incorporated within the core of PEG-polylactic acid (PLA) nanoparticles conjugated with lectin protein. These nanoprobes were reported to be stable, hydrophilic and could efficiently deliver higher amount of payload specifically into the brain (Gao et al., 2008). Novel surface modification strategies for increasing the biocompatibility and bio-inertness of QDs have resulted in its successful application for in vitro and in vivo tumor imaging. The biofunctionalization of CdSe/CdS/ZnS quantum dots and iron oxide nanocrystals enabled MRI imaging of tumors. Surface coating of the nanoprobes with PEGpolyisoprene complex was reported to enhance its stability, bio-inertness and tumor specificity (Pöselt et al., 2012). Although not applied to date in neurological cases, such platforms can enable quantum dot-based MRI imaging of brain tumors. Conventional imaging techniques like X-ray, ultrasound, MRI, CT and PET were all developed for imaging tissue and organs. The advent of nanomaterials like QDs have developed the field of fluorescence based in vivo optical imaging which can now detect tumor progression at the cellular level. In vitro studies with human glioblastoma cells (U87) proved that QDs can indeed be used to specifically label cancer cells by conjugating it with transferrin (Tf). As tumor cells highly overexpressed Tf receptor, the QD-Tf complex could selectively bind to malignant cells with very high efficiency and prolonged red fluorescence signal for two days (Ryoko Tsukamoto, 2013). Similar studies have also been reported to use QDs for tracking single particle-like neurotransmitters in astrocyte promoting a better understanding of neuronastrocyte communication (Arizono et al., 2014). 3.2.1.4 Gold nanoparticles - Elements with high atomic number (Z) like Iodine have been used for long as radiosensitizing agents in X-ray-based radiotherapy and imaging due to their ability to locally absorb X-rays. Progress in nanotechnology has led to the use of metallic 12

nanoparticles like gold nanoparticles (GNP) as X-ray contrast agents exhibiting better contrast as well as higher tumor localisation than Iodine-based agents. Although several groups have contributed in the progress of GNP in X-ray-based therapy and imaging, the work done by Nanoprobes Inc for over a decade stands out. The research and innovationdriven company formed by scientists of renowned US laboratories first reported the use of GNP as a novel X-ray contrast agent. As a prelude to the important innovation, the group previously proved that enhancement of radiation dose in tumor bearing mice could be achieved with high spatial specificity by using GNPs which had a propensity to localize in high number within tumors (Hainfeld et al., 2004). The team later went on to report that administration of GNPs in high concentration had no toxic effect in tumor bearing mice. High resolution imaging of tumors, kidneys and even tumor vasculature was possible by X-ray using GNP as contrast agent (Hainfeld et al., 2006). Recently, they further used GNPs in micro-CT imaging and radiotherapy for management of brain tumors. The results indicated that GNPs were able to selectively localize in tumors over normal tissue leading to high resolution micro-CT imaging of tumor as well as a prolonged tumor-free survival (Figure 5) (Hainfeld et al., 2013). Plasmonic nanoparticles like GNPs have the capacity to track events at the molecular and cellular level. A detailed kinetic analysis of gold nanocages in a brain tumor model in vitro and in vivo provided a quantitative understanding of the distribution and clearance of the nanoparticles. Two-photon microscopy and photo-acoustic microscopy techniques were used to study the distribution of nanoparticles in vitro and in vivo respectively. It was found that 10-20% of initial gold was cleared by the 6th day of administration (Cho et al., 2013). Similarly, using GNPs in synchrotron-based computed tomography provided high resolution cellular level images of brain tumor (Schültke et al., 2014). Conjugating GNPs with FePt nanoparticles and chelating with gadolinium agents enables the tracking of the nanoprobes by MRI. Such heterogenous nanocomplexes have been shown to be water stable, biocompatible and provide high resolution MRI of brain tumor with accurate location of particles enabling determination of optimal dose of irradiation (Choi et al., 2006; Miladi et al., 2014). 3.2.1.5 Nanoplatforms for multimodal neuro-imaging – With increasing expertise in nanomaterial

synthesis,

chemical

modification

and

compatibility

with

biological

environments, simple nanoparticles have now given way to multifunctional nano-complexes formed of more than one type of material which can be tracked simultaneously by different imaging modalities. Such advanced nano-platforms provide images with higher spatial and temporal resolution giving physicians a better picture of disease prognosis as well as enable 13

theranostic applications. A multifunctional nanoprobe developed in a collaborative work between US and Chinese scientists demonstrates their remarkable expertise in designing such complex molecules. Using a dendrimer as the core molecule, they conjugated this with one NIR (Cy5.5) and a fluorescent dye (rhodamine) for enabling both in vivo and ex vivo optical imaging. It was further functionalised with Gd3+ - DOTA to provide MR visibility. Linking this complex to Angiopep-2 and RGD peptides rendered the nanoprobe more permeable to the BBB and tumor vasculature, thereby better delineating tumor margins for accurate resection and biopsy (Yan et al., 2012). Similar complexes with two order imaging modalities were designed using upconversion nanoparticle conjugated with gadolinium and RGD peptide to facilitate fluorescence guided brain tumor resection as well as MR imaging (Jin et al., 2013). Lanthanide ion-doped gadolinium nanoparticles (NaGdF4:Ho 3+) were also applied as MRI/optical probe. Conjugating with chlorotoxin rendered nanoprobes specific to gliomas leading to significant contrast enhancement and tumor delineation (Deng et al., 2014). Another group designed nano-complexes comprising of a gold core, coated with Raman molecular tag, trans-1,2-bis(4-pyridyl)-ethylene, silica and conjugated with gadolinium. This allowed detection by three imaging modalities: MRI, photo-acoustic imaging and Raman imaging (Kircher et al., 2012). A separate group also developed a similar triple modality imaging of brain tumor. They performed multimodal imaging of brain tumors in mice by bioluminescence-fluorescent diffuse optical tomography-MRI using lipid nanoparticle loaded with NIR dye (Fortin et al., 2012). The application of inorganic and organic nanoparticles in MRI for theranostic purposes in neuro-oncology and other CNS pathologies has been extensively reviewed previously (Cormode et al., 2010; Na et al., 2009; Weinstein et al., 2010). The different bio-inspired modifications of nanomaterials performed for efficient neuro-imaging is summarised in Table 1. 3.2.2 Nano-Neuro Diagnostics: Nanotools for Neuro-diagnostics Alzheimer’s disease (AD) is the most common form of dementia, affecting millions of people worldwide every year. Several hypotheses have been put forward in an attempt to explain the disease mechanism. According to the most common hypothesis, amphipathic peptides called amyloid beta (Aβ) polymerize, aggregate, accumulate and deposit in Alzheimer’s affected brains, forming protofibrils and fibrils. These amyloid fibrils are highly neurotoxic inducing neuronal cell death possibly by triggering oxidative stress. Another hypothesis suggests that such oxidative stress can lead to hyper-phosphorylation of a microtubule-associated protein 14

called tau. Hyperphosphorylated tau-protein results in destabilization of microtubules which in turn compromises synaptic connections. Another possible factor in AD pathogenesis is thought to be amyloid derived diffusible ligands (ADDL) which are short oligomers derived from Aβ monomers. ADDL has been shown to be more toxic than Aβ fibrils even at nanomolar concentrations. The initial steps towards developing an assay for AD detection were taken by scientists from Northwestern University, USA. They were able to study the binding kinetics of ADDL and anti-ADDL antibody using silver nanotriangles and localised surface plasmon resonance (LSPR) (Haes et al., 2004). The study was extended further to detect ADDL in the CSF of patients using a gold nanoparticle based bio-barcode assay. The assay could detect below femtomolar concentrations of ADDL rapidly, thereby providing the first significant example of nano-based diagnostic assay for early detection of AD (Georganopoulou et al., 2005). Nanoparticle based plasmonic sensors for detection of Aβ peptide aggregates can be a potent device for early detection of AD. Gold nanoparticle coated with anti-Aβ antibody have been reported to detect ultra-low concentrations of amyloid-beta peptide across various platforms like surface plasmon resonance, dot blot assay and even by naked eye (J. H. Lee et al., 2009; SAKONO et al., 2012; Wang et al., 2012). Further, the application of anti-Aβ antibody-labelled quantum dots enabled real time imaging of protein aggregation, oligomerization and fibrillation (Tokuraku et al., 2009). Similar biosensors were developed using gold nanoparticle to detect ultra-low concentrations of other AD biomarkers like tau-protein and acetylcholine esterase (AChE) in CSF using two-photon Rayleigh scattering property and colorimetric assay techniques, respectively (Liu et al., 2012; Neely et al., 2009). Multifunctional nanoparticles capable of detecting as well as inhibiting Aβ fibril formation have been designed using gold and iron oxide for potential theranostic applications in AD (Choi and Lee, 2013; Skaat et al., 2013). Like AD, another neurodegenerative disease primarily affecting the aged - Parkinson’s disease (PD) - is also characterised by abnormal protein accumulation. The deposition of high amounts of alpha-synuclein protein leads to formation of Lewy bodies inside neurons. However, the major cause of PD is thought to be the drastic depletion of neurons in the substantia nigra region of the brain primarily involved in secretion of a catecholamine neurotransmitter called dopamine (DA). Tyrosine hydroxylase, a critical enzyme in catecholamine biosynthesis pathway, was monitored by gold-nanoparticle-based DNA barcode assay. Its quantification provided a direct indication of the levels of dopamine and hence detect the onset of PD (An et al., 2013, 2012). Similar attempts at measuring dopamine were made by other groups using different transducer platforms. For example, a single walled 15

carbon nanotube-based voltammetric biosensor was designed to simultaneously detect DA and Adenosine (metabolic indicator of neuronal activity) with very low detection limit (34.7 μM for adenosine, 7 μM for dopamine) (Goyal and Singh, 2008). Another team reported a voltammetric biosensor designed to detect three potential PD biomarkers – Uric Acid (UA), DA and Ascorbic Acid (AA). The biosensor was fabricated with ZnO nanowire arrays on 3D graphene foam and was capable of detecting all three analytes with high sensitivity and accuracy from clinical samples of PD patients (Figure 6) (Yue et al., 2014). Recent attempts to detect PD biomarkers in exhaled breath using nanotechnology platforms are undergoing clinical trials (Rambam Health Care Campus and Technion, n.d.). The need for a cost-effective, rapid and highly sensitive diagnostic platform for early detection of biomarkers related to neurodegenerative diseases prompted the European Union to initiate a multi-institute collaborative initiative for development of nanosystems for early diagnosis of neurodegenerative diseases (NADINE) in 2010. 3.2.3 Nano-Neuro Therapy: Nanotools for Neurotherapy The complex physiology of the BBB and the restricted neuroanatomical access poses a major challenge to surgical intervention and drug delivery in the nervous system. An ideal platform for delivering therapeutics across the BBB should have the following properties – a) The delivery should specific and reach the intended site of action without damaging the BBB. b) Sufficient circulation time of drug. c)

It should be able to transport sufficient amount of the drug to maintain therapeutic efficacy inside the brain.

However, the rapid progress in fabrication of nanomaterials has provided solutions to many of the neurological challenges as discussed in earlier sections. Glioblastoma multiforme - primary brain tumors arising from astrocytic cell types - is the most aggressive form of intracranial malignancies. It’s a unique form of malignancy which displays polymorphism by gross examination, at the microscopic level as well as at the genetic level with a variety of genetic mutations contributing to disruption of multiple biochemical and cell-cycle pathways (Holland, 2000). The BBB and the Blood-Brain Tumor Barrier (BBTB) poses the major hurdles for drug delivery inside the tumor depending on the stage of malignancy. At the initial stages of the disease, the tumor cells rely on the normal 16

BBB for supply of nutrients and benefits from the high TEER of the endothelial cells which restricts drug transport. However, at advanced stages of malignancy, when tumor cells invade normal tissue, neo-vasculature is formed with continuous fenestrated microvessels allowing only molecules less than 12 nm to pass through (Liu and Lu, 2012). Thus, strategies targeting therapeutic agents inside brain tumors have mainly evolved using dual targeting nanoparticles functionalised with molecules specific for tumor cells as well as tumor neo-vasculature. Over the years, Chinese groups have reported extensively on a variety of functional coatings on polymeric nanoparticles to obtain dual targeting of brain tumors. Nanoparticles were functionalized with Interleukin-13 peptide (Gao et al., 2014) and lactoferrin (Miao et al., 2014) to enhance uptake by tumor cells. These were further decorated with tumor neovasculature targeting RGD moieties (Gao et al., 2014) and tLyp-1 peptides (Miao et al., 2014) to cross the BBTB. In some cases, lipoprotein receptor protein binding molecules like Peptide-22 (Zhang et al., 2013) and Angio-pep-2 (Xin et al., 2012) could individually achieve efficient delivery of paclitaxel by dual targeting of neo-vascualture and malignant cells. In another study, the same team managed to decorate the polymeric nanoparticles with aptamers specific for nucleolin which is over-expressed in both cancer cells and tumor vasculature (Guo et al., 2011). Nano-formulations of naturally occurring anti-cancer compounds like reservatol was delivered by lipid based nanocapsules and were reported to inhibit glioma growth in vitro and in vivo (Figueiró et al., 2013). Another group demonstrated the efficiency of polymeric nanoparticles as vehichles for targeted gene delivery inside gliomas. The chlorotoxin-conjugated PEG-PLA nanoparticles could efficiently cross the BBB and reach the perinuclear region, thereby facilitating the delivery of GFP-encoding DNA inside tumor cells (Kievit et al., 2010). In order to obtain sustained release of drugs in situ, scaffolds in the form of nanofibers were found to hold potential as implants for glioma therapy. PLGA nanofibers loaded with paclitaxel exhibited sustained release of drug over 60 days in vitro on cultures of C6 glioma cell line (Xie and Wang, 2006). Alzheimer’s disease (AD) is thought to be mainly caused by aggregation and accumulation of Aβ fibrils in the brain tissue. Hence most of the therapeutic strategies developed for AD have focussed on inhibition of amyloidogenesis and destruction of Aβ fibrils by targeted irradiation methods and localised delivery of anti-amyloid molecules. Metal ions like Cu2+, Fe3+ and Zn2+ tend to accumulate in ageing brain and have been shown to interact with and stabilize Aβ fibrils thereby facilitating progression of AD. By using Cu2+-chelator- Dpenicillamine as a conjugate with nanoparticles, scientists observed that it was possible to resolubilize Aβ1-42 aggregates (Cui et al., 2005). This showed that nanoparticle based 17

platforms could be used to reduce metal ion accumulation, potentially reversing many neurodegenerative diseases. Enhanced delivery of natural anti-oxidant and anti-amyloid compounds like curcumin can be achieved by employing biocompatible nanomaterials tagged with moieties that direct the nanoprobes into neuronal cells. Studies with curcumin-loaded polymeric nanoparticles conjugated with tetanus toxin derived Tet-1 peptide as well as enhanced delivery of curcumin into mouse brain by PBCN-NP are some examples (Mathew et al., 2012; Sun et al., 2010). Metallic nanoparticles like gold nanoparticles have been reported to inhibit Aβ fibril formation as well as aid in targeted photothermal (Triulzi et al., 2008) and microwave irradiation (Araya et al., 2008) for Aβ aggregates. Surface modification of GNPs with COOH groups provided a net negative charge which was found to inhibit and redirect Aβ fibrillation (Liao et al., 2012). Apart from GNPs, Quantum dots (QDs) have also been conjugated with anti-amyloid molecules like N-acetyl-L-cysteine (NAC) which can inhibit fibrillation by quenching the nucleation and elongation processes (Xiao et al., 2010). Nano-formulations of polymeric materials like PEGylated poly-lactic acid (PLA) and polysaccharide nanogels (cholesterol bearing pullulan) also exhibited potential as platforms for targeting amyloid plaques (Zhang et al., 2014) and as chaperones inducing conformational change in Aβ fibrils respectively (Ikeda et al., 2006). Similarly, estradiolloaded polymeric nanoparticles were given orally in rat AD model, keeping in mind the enhanced risk of the disease in post-menopausal women due to reduced estrogen levels. Nanoparticle-mediated delivery aided in brain localisation of estradiol increasing bioavailability and therapeutic efficiency at lower drug concentration (Mittal et al., 2011). In a separate approach towards AD treatment an Italian group delivered acetylcholinesterase inhibitor drug Tacrine via albumin nanoparticle. The intranasal delivery of the drug through nanocarrier was expected to increase bioavailability, enhance permeation and circulation time of the active ingredient (Luppi et al., 2011). The second most prevalent neurodegenerative disorder after AD is Parkinson’s disease (PD). Although the pathophysiology of PD has been extensively studied, the disease etiology largely remains unknown (idiopathic) till date. PD is primarily associated with loss of dopaminergic neurons in the substantia nigra leading to deficiency of the neurotransmitter dopamine. In order to combat this deficiency, Levo-dopa (L-DOPA) which is a biological precursor of dopamine is generally administered to PD patients. The low bioavailability of LDOPA upon systemic or oral administration has necessitated nanotechnology based delivery platforms to ensure efficient transport of dopamine across the BBB and maximum 18

localization of the molecule in the brain. Initial attempts for site-specific delivery of dopamine to minimise its peripheral side effects were focussed on designing and biometric simulation of a prototype nano-enabled scaffold device (NESD) comprising of an alginate scaffold embedded with dopamine-loaded cellulose acetate phthalate (CAP) nanoparticles (Pillay et al., 2009). The device was implanted in the parenchyma of the frontal lobe of rats and was found to deliver dopamine over 30 days with 10 fold more dopamine in CNS as compared to systemic concentration. Similar attempts for dopamine delivery were also made with chitosan nanoparticles (De Giglio et al., 2011). Intraperitoneal administration of dopamine loaded chitosan nanovectors were found to induce dose-dependent increase of dopamine presence in the striatum as observed through in vivo brain microdialysis experiments. Other neuro-protecting molecules like the corticotropin-releasing hormone urocortin and genes encoding growth factors like GDNF also arrests development of Parkinsonism when administered through polymeric nanoparticles conjugated with lactoferrin (Hu et al., 2011; Huang et al., 2010). Similarly, angiopep conjugated dendrimers have been used to transport human GDNF genes into the affected areas of the brain for recovery of dopaminergic neurons and restore locomotory activity in Parkinson’s induced animal models (Huang et al., 2013). Oxidative stress is one of the main causes leading to loss of dopaminergic neurons in PD. Hence, attempts to specifically deliver antioxidants into the substantia nigra of diseased brain have also been reported. Cationic block copolymer polyethyleneimine-PEG (PEI-PEG) of 60 – 100 nm size were loaded with catalase enzyme to form a nanozyme complex which exhibited longer stability in vitro and in vivo with 0.6% of the injected dose reaching the brain (Batrakova et al., 2007). Besides dopamine deficiency, another pathophysiology associated with PD is the accumulation of α-synuclein protein forming inclusions called Lewy bodies which are found distributed throughout the diseased brain. Gold nanoparticles were observed to act as chaperones, preventing the misfolding of αsynuclein and hence can be used as a therapeutic to sequester and regulate α-synuclein homeostasis (Yang et al., 2014, 2013). These pathophysiologies are ultimately responsible for deterioration of electrical activity in the thalamic, subthalamic nucleus, globus pallidus and medial globus pallidus region of the parkinsonian brain. Temporal administration of magneto-electric nanoparticles facilitates non-invasive stimulation of these regions thorugh an external magnetic field thereby improving the brain’s electrical activity (Yue et al., 2012). The different bio-inspired modifications of nanomaterials performed for efficient neurotherapy is summarised in Table 2. 19

4.0 Neuroregeneration 4.1 Biology of Neuroregeneration In a pioneering experiment on split brain research, famous neurobiologist Dr. Robert Sperry pulled out an eye from a frog, rotated it 1800 and put it back in. The animal was able to repair and rewire its neural circuits but had an inverted vision. As a result, when an insect was kept above, the frog shot its tongue downward to catch it. Similar results were obtained even after the optic nerve was cut. The experiment proved the ability of neurons to regenerate and rewire neural circuits and Dr Sperry was conferred with the Nobel Prize in 1981. Such remarkable neural regeneration and plasticity is found in many other animals like salamanders, lampreys, and gold fish which can regenerate entire limbs. Unfortunately, humans don’t possess such ability. The number of neurons we are born with are essentially all that we will ever have in our lifetime. However, adult stem cells called neural stem cells isolated from specific areas of the brain (olfactory bulb, dentate gyrus, and cerebellum) and spinal cord have been shown to differentiate into neural cells in vitro. This ability is somewhat more limited in vivo, with new neurons being actively generated only in the subventricular zone and the dentate gyrus. The former migrate to the olfactory bulb, becoming there interneurons, while the latter contribute to memory formation in the hippocampus (Ming and Song, 2005). Many neuronal cell types can also regenerate their axons if these are damaged. Axon regeneration should allow neurons to re-connect to their target, restoring any function loss caused by the damage. However, axon regeneration in vivo is very much limited by local inhibitory signals in the central nervous system (Sandvig et al., 2004). The major source of these inhibitory signals is the glial scar formed by the glial cells of the CNS (mostly astrocytes) around the region of injury. The reactive astrocytes along with myelin and cellular debris forms the glial scar which acts as an impenetrable barrier inhibiting the regenerating neurons from reaching the synaptic target. The unfavourable cellular milieu for neural regeneration makes even minor injuries/tissue damage in the CNS fatal. However, the glial cells in the peripheral nervous system (PNS) i.e. the Schwann cells play a much more constructive role in nerve regeneration than their CNS counterparts. Upon nerve damage, the Schwann cells shed their myelin sheath and release cytokines which causes macrophages and monocytes to rush to the site of injury and clear the debris. Phagocyte infiltration is aided by the easy physiological access to the peripheral nerves as compared to the heavily shielded CNS. At the proximal ends of the cut, axons sprout growing ends from a nearby node of 20

Ranvier. These axon sprouts extend to form new axons. Axon guidance is mediated by neurotrophins secreted by Schwann cells and the extracellular matrix sheaths that surrounded axons prior to damage, which allows regeneration to occur at a rate of 2 – 5 mm/day (Schmidt and Leach, 2003; Williams et al., 1983). Therefore, injuries resulting in small gaps in the peripheral nerves can be healed by regeneration. However, in case of gaps larger than 6 mm, surgical intervention becomes necessary. 4.2 Nanostructures facilitating Neuroregeneration Through the use of structures which actively encourage and guide axons to reconnect with their targets, nanotechnology is gradually revolutionising the field of regenerative neuroscience. Some examples of different types of synthetic and natural polymers used to produce nanofibers as well as the fabrication techniques utilised are listed in Table 3. Although axonal regrowth in the PNS occurs much more efficiently than in the CNS, reinnervation and functional recovery are often poor. This is particularly the case for lesions in which large spaces between nerve stumps are created. This gap, lacking the Schwann cells necessary for axonal regrowth, poses a major obstacle for regenerating axons. In these cases, the golden standard for treatment of PNS damage has been the use of autologous nerve grafts to bridge the two ends. However, these grafts pose several disadvantages, requiring surgical intervention on a second nerve to harvest the necessary tissue (with the associated morbidity). Instead, nanotechnology may hold the answer for the development of alternatives to these autologous grafts, in the form of tissue engineered nerve grafts (TENGs). In the most classical sense a TENG consists of a hollow tube connecting the two nerve stumps. Examples of this are NeuraGenTM (Integra LifeSciences) (Archibald et al., 1995) and NeuroMendTM (Stryker Orthopaedics): collagen-based nerve conduits which are currently available for clinical use. Despite their simple design, collagen conduits guide Schwann cells and regenerating axons through the lesion, while restricting access of cell types foreign to the nerve. More recently developed TENGs rely on a combination of physical and biochemical cues to actively facilitate the growth of axons through it. These cues are often introduced in the form of nanofibers. Nanofibers possess a number of advantages for their use as scaffolds for neural regeneration, since (1) their physical properties can be tailored to specifically meet the requirements of a particular type of lesion; (2) their large surface area – to – volume ratio enhances the presentation of biochemical cues; and (3) they can be fabricated from a number of biocompatible and degradable materials. 21

Electrospinning is one of the most common methods for the fabrication of nanofibers. This technique allows for the production of fibers with a wide range of dimensions, which can be easily regulated through variations in the polymer solution, electric field strength, and field pattern used in the fabrication process. Synthetic materials, including poly(ε-caprolactone) (PCL) (Panseri et al., 2008), poly(ε-caprolactone-co-ethyl ethylene phosphate) (PCLEEP) (Chew et al., 2007), and poly(L-lactic acid) (PLLA) (Yang et al., 2005); are the most common components used for the fabrication of electrospun nanofibers for nerve regeneration. Figure 7 demonstrates the effect of topographical alignment of nanofibers as well as sustained delivery of growth factors on nerve regeneration and myelination. Kim et al. (2008), for example, used poly(acrylonitrile-co-methylacrylate) (PAN-MA) fibers (with a diameter of 400 – 600 nm) to bridge a 17 mm rat tibial nerve lesion gap. They observed that, following a 16 week recovery period, these rats exhibited a sensory and motor recovery on par with that of rats with autologous grafts. Notably, this effect was only seen in animals with PAN-MA fibers aligned with the axis of axonal regeneration, suggesting that the topographical cues provided by the nanofibers are a crucial component of their regenerative potential (Kim et al., 2008). Similarly, in a recent study Jiang et al. (2012) implanted either PCL microfibers (981 ± 83 nm diameter) or nanofibers (251 ± 32 nm diameter) into a 15 mm rat sciatic nerve lesion. Regeneration through the graft, as seen by retrograde labelling, was much greater in nanofiber than in microfiber grafts; which highlights the importance of nanofiber scale in their properties as regenerative scaffolds (Xu et al., 2014). Naturally occurring materials, such as chitosan (Wang et al., 2008), have also been employed as nanofiber scaffolds. Although these materials more closely resemble the tissue they are intended to replace, potentially exhibiting greater biocompatibility, their weak mechanical properties limit their use as nerve graft scaffolds. The major alternative to electrospinning for the fabrication of nanofibers is self-assembly. This technique involves the design of molecules, particularly peptides, capable of assembling by noncovalent bonding into polymeric structures directly in their target areas (Zhang, 2003). Self-assembled peptide nanofibers (SAPNs) are characterised by a very small diameter (5-10 nm) in contrast to electrospun fibres, which often are orders of magnitude larger. These dimensions closely resemble those of extracellular matrix (ECM) proteins, making SAPNs a close imitation of the natural cellular environment. Additionally, these fibers break down into L-amino acids which, being common in all organisms, are non-toxic.

Several self-

assembling peptide (sapeptide) sequences have been used for the formation of SAPN scaffolds. Arginine-alanine-aspartate sequences (RAD16), for example, have been shown to 22

support adhesion and facilitate neurite outgrowth of primary neurons and neuronal cell lines (PC12) in vitro (Holmes et al., 2000). Sapeptides can also be modified so as to include certain motifs, introducing functional properties in the final SAPNs which may be desirable in thescaffold. For example, Gelain et al. (2006) studied the effect of functionalisation of RADA16 (arginine-alanine-aspartate-arginine) sapeptides with bone marrow-homing motifs. Functionalised SAPN scaffolds were found to support neural stem cell proliferation to a greater degree than pure RADA16 SAPN scaffolds (Gelain et al., 2006).

Axon regeneration in the CNS often fails, due to a combination of inhibitory environment and lack of growth-supporting factors. Although these limiting factors have often been addressed individually, no therapy currently exists to treat CNS lesions in humans. It is likely that, given the multimodal nature of the problem, the issues limiting regeneration in the CNS will have to be addressed simultaneously to achieve successful regeneration. Given the wide range of unique properties that they can exhibit, nanotechnologies are thus an attractive platform for the development of these therapies. As in the PNS, nanofiber scaffolds have been used to promote the regeneration of axons through lesions in the CNS. Although electrospunfibers have sometimes been used for this purpose (Meiners et al., 2007), SAPNs are more commonly employed due to their ability to polymerise in situ, minimising the need for surgical intervention. Tysseling‑Mattiace et al. (2008) injected laminin motif-derived IKVAV (isolucine-lysine-valine-alanine-valine) sapeptides into mouse thoracic (T10) spinal cord compression injuries. These mice not only had reduced glial scar formation, but also exhibited enhanced functional recovery 9 weeks after injury (Tysseling-Mattiace et al., 2008). RADA16 sapeptide scaffolds have also been used in in vivo CNS regeneration studies, in both spinal cord (Guo et al., 2007) and optic tract (Ellis-Behnke et al., 2006) lesion models. The efficacy of SAPN based scaffolds in repairing of animal brain is illustrated in Figure 8. Nanoscale structures applied in neuronal regeneration are not limited to fibres. Membrane buckling has been used to produce very small microchannels – with a diameter similar to that of individual axons – on to nano-thin silicon membranes (Cavallo et al., 2014). These channels can guide neurite outgrowth in in vitro neuronal cultures, and may be engineered with designs to promote network formation.

23

More complex approaches to CNS regeneration have also been taken. One such example is the combination of nanofiber scaffolds with stem cell therapy. Neural stem cells (NSCs) can aid regenerating axons through the formation of a growth-supportive microenvironment, and are considered to hold great therapeutic potential for the treatment of CNS injury and disease (Martino and Pluchino, 2006). Poly(lactic-co-glycolic acid) (PLGA) scaffolds containing pores for axonal guidance, and an underlying layer seeded with NSCs, were implanted in rats with spinal cord hemisection lesions (Teng et al., 2002). Implantation of this scaffold led to axonal regeneration and a functional recovery which was superior to that seen in rats implanted with the scaffold or NSCs alone. HPMA (2-hydroxypropyl methacrylamide) hydrogels functionalised with RGD peptides and seeded with mesenchymal stem cells have also been used in spinal cord regeneration studies. When used to bridge a compression injury in rat spinal cord, axons grew through the porous hydrogel structure, resulting in improved sensory and motor recovery after 6 months (Hejcl et al., 2010). The conductive properties of certain nanomaterials also make them well suited to combine neural scaffolding with electrical stimulation. Although studies have been mainly limited to in vitro testing, neuronal cultures have been reported to exhibit increased neurite outgrowth in response to electrical stimulation (Kimura et al., 1998). Electrospun nanofibers coated with electrically conductive polymers have been studied by several groups as potential platforms for the fabrication of regenerative scaffolds. Lee et al. (2009) cultured PC12 neuronal cells on a mesh of poly(lactic-co-glycolic acid) (PLGA) nanofibers coated with a nano-thick layer of electroconducting polypyrrole (PPy). Not only did the nanofibers support the extension of neurites from the cells, but neurite length and number was greatly increased when cells were electrically stimulated (J. Y. Lee et al., 2009). Carbon nanotubes also hold great potential for the development of multifunctional scaffolds. Although they are non-degradable; their size (~1 nm diameter), electrical conductivity, and excellent mechanical properties make them good candidates for their use as scaffolds for neural regeneration. Moreover, in vivo studies in rat SCI lesion models have shown that carbon nanotube scaffolds, functionalised with polyethylene glycol, are capable of supporting axonal regeneration through the lesion and promote functional recovery (Roman et al., 2011). In the peripheral nervous system, carbon nanotube and glass microfiber conduits have been used to promote axon regrowth and muscle reinnervation in sciatic nerve lesions (Ahn et al., 2015). Limited in vivo studies with nanoparticles has been the major hindrance in establishing long term safety and efficacy of using synthetic nanoparticles in fabricating nerve conduits. To address this need our group fabricated silk-gold nanocomposite nerve conduits and monitored functional and 24

morphological regeneration of sciatic nerve for 18 months after neurotmesis-grade injury in rats (Das et al., 2015). The nanocomposite conduits pre-seeded with rat Schwann cells SCTM41 were observed to achieve near normal values of nerve conduction velocity (50 m/sec), compound muscle action potential (29.7 mV) and motor unit potential (133 μV) after 18 months. Finally, more traditional treatments like the direct delivery of growth-supportive compounds to lesions have also benefitted from nanotechnology. Although agents such as epidermal growth factor receptor (EGFR) inhibitors have been shown to reduce the axon growthinhibitory effect of chondroitin sulphate proteoglycans and myelin on axon regeneration (Koprivica et al., 2005), sustained delivery is necessary for their therapeutic effects to be seen. Since systemic administration is not a desirable delivery route due to associated sideeffects, consequence of the role played by EGFR in other tissues (Fakih and Vincent, 2010), a nanotechnology platform was developed for the sustained, local delivery of EGFR inhibitors. PLGA microspheres and nanospheres loaded with the EGFR inhibitor AG1478 were administered to the eye of rats following injury of the optic nerve.

Although both

microspheres and nanospheres caused axons to regenerate through the site of injury 2 weeks later, survival of these axons at 4 weeks post-lesion was only observed in nanosphere-injected animals. This observation was attributed to the ease of administration and more stable drug release profile of nanospheres compared to their larger counterparts, and serves as an example of the advantages that nanotechnology can have over seemingly similar approaches to axonal regeneration (Robinson et al., 2011).

5.0 Nano-neuroelectronics The progress of nanotechnology has brought about remarkable miniaturisation of electronic systems upto the nanodimensions. Such nanoelectronic systems are better suited for biological applications since they can communicate not only on cellular levels but with specific molecules. In neuroscience these nano-electronic interfaces are being widely used to study signal transduction at single ion channel level (Marshall and Schnitzer, 2013) as well as develop advanced probes for recording minute electrical signals for early detection and treatment of neurological disorders. Further the nano-scale precision of such systems has been exploited to study the intricate process of myelination and has also shown the potential

25

to revolutionize the field of neurosurgery (Guo et al., 2008; Lee et al., 2012; Yanik et al., 2004). 5.1 Nano-electrodes for Neural interface Neural electrodes are generally used for stimulating and recording the electrical signals generated by a particular nerve or brain region. These electrodes are conventionally made of metals and can be used externally or if required implanted inside the body to record electrical signals or stimulate host tissue. However such neural electrodes are largely challenged by their size, mechanical properties as well as their tendency to elicit inflammatory responses in the host tissue over time. Transforming such electrodes to the nano dimension or even surface patterning by coating with nanomaterials enhances their sensitivity, biocompatibility and minimises tissue damage. The topography of such nano-electrodes mimics the native neural architecture and helps in reducing glial scar upon implantation in the CNS and guides regenerating axons when intended to interface with injured peripheral nerves. One of the most used varieties of nanomaterials for fabrication of neural electrodes are carbon nanotubes (CNTs). The excellent mechanical and electrical properties of CNTs reduces glial scarring while its high surface area increases charge injection capacity (Wang et al., 2006) and decreases interfacial impedance with neurons (Cui et al., 2003). Both single walled (SW) and multi walled (MW) CNTs have been used to develop multielectrode arrays for recording and stimulating neurons (Nguyen-Vu et al., 2006; Wang et al., 2006; Whitten et al., 2007). Vertically aligned carbon nanofibers (VACNFs) have conical structures with tiny tips that allow it to penetrate through the glial layer into the tissue thereby enabling simultaneous neuronal stimulation and recording of electrical and chemical responses (Zhang et al., 2012). The 3D brush like structure of VACNFs promote proliferation of PC12 cells, while coating it with polypyrrole resulted in improved capacity of neural stimulation (de Asis et al., 2009; Nguyen-Vu et al., 2007, 2006). CNF-based neural chips can also stimulate and record electrophysiological signals from brain tissue slices in vitro (Yu et al., 2007). In a separate study, VACNFs were integrated on thin film transistors. The setup acted as a multi electrode array (MEA) platform which enhanced electrical selectivity of cells as well as enabled intracellular sensing (Park et al., 2009). Crystalline Silicon based nanowires (NW) have been widely reported for developing Field effect Transistors (FETs) with 104 – 109 times higher sensitivity than conventional planar FETs (Sant et al., 2004; Schöning and Poghossian, 2002; Soldatkin et al., 2003). Coating such NW-FETs with a combination of cell adhesive poly-l-lysine and cell repellent 26

fluorinated silane promotes neurite outgrowth in a specific direction thereby enabling the formation of interconnected neural networks with axons and dendrites crossing arrays of NW devices (Patolsky et al., 2006). The nanotopography of Silicon NW-FETs further allows highly localized stimulation of neurons which can be operated as “artificial synapses” (Patolsky et al., 2006). The nanowire-based devices were found to generate somatic action potentials upon application of biphasic excitatory signals. A linear array of 4 NW-FETs and 5 NW-FETs separated by a gap was reported to be able to successfully monitor axon/dendrite signal propagation in rat cortical neurons in vitro (Patolsky et al., 2006). Although for fabrication of FETs silicon-based nanowires remain the preferred material, there have been attempts to develop NWs from other materials which are bio-inert and biocompatible. In such a study, gallium phosphide nanowires (GaP NW) were found to promote neuronal adhesion and axonal outgrowth resulting in better cell survival than planar surfaces (Hällström et al., 2007). Hundreds of NWs were observed to penetrate the cells, making such devices a potential platform for intracellular recording (Figure 9). 5.2 Nanostructured opto-electronic devices The tunable optical properties of nanomaterials as well as their scope of miniaturisation makes photoactive nanostructured devices an ideal platform for designing optoelectronic implants which can interface with neural tissue. Such technologies hold great promise in developing artificial retinas and similar devices to restore or improve vision in patients with ophthalmic disorders. Initial attempts at fabricating sucha bio-nano device were aimed at exploiting the quantum confinement effects of semiconductor nanoparticles. Thin films of HgTe nanoparticles were produced by layer by layer assembly and neuronal cells were allowed to proliferate on it. The cells could then be stimulated by the photocurrents generated in these films (Figure 9) (Pappas et al., 2007). Further improvements on such prototypes were achieved when a group from Israel in collaboration with Newcastle University, UK developed a wireless version of similar thin film based optoelectronic devices. They reported wireless

photostimulation

of

retina

using

a

nanorod-nanotube

hybrid

platform.

Semiconductor nanorods of CdSe/CdS core shell architecture were adhered to carbon nanotubes via plasma polymerised acrylic acid mid-section to enable efficient charge transfer through the layers. The device was found to successfully stimulate neurons on chick embryonic retina following pulsed photstimulation for different time intervals and varying intensities of violet light (405 nm) (Bareket et al., 2014).

27

The variety of nanomaterials used in neurological electrodes and opto-electronic devices are illustrated in Figure 9. 5.3 Brain inspired computing The above examples illustrate the rapid advancement of nano scale electronic platforms and how it has revolutionized neurological sciences through development of ultra-sensitive neural electrodes and innovative opto-electronic devices for restoring vision. However, the human brain still remains a source of inspiration behind most of the present developments in electronics. Although present day supercomputers far exceed the brain in terms of storage capacity and computational power, the human brain is yet to be surpassed in terms of energy efficiency and its massive capacity for parallel computing. Hence to address future requirements of energy efficient and parallel computing it is essential to learn from the brain through brain inspired computing. Nanoscale electronic devices made of phase-changing materials like chalcogenide glass (GST) are being used as building blocks for brain inspired computing (Kuzum et al., 2012). The device exhibits spike timing dependent plasticity (STDP) which is an electronic analogue of a biological synapse. Multinational companies like IBM are already engaged in extensive research to develop the next generation electronic chipsets based on brain inspired computing. Their latest TrueNorth chipset attempts to simulate the ultra high density of neuronal network by packing “5.4 billion transistors wired into an array of 1 million neurons and 256 million synapses.” The current chip also consumes 100 times less energy than previous generation chipsets (Merolla et al., 2014). 6.0 Conclusions In the present review we have attempted to study the applications of nanotechnology in clinical neurology as well as in basic neuroscience with a special focus on the impact of bioinspired

modifications

of nanomaterials

and

their

efficacy

in

overcoming

the

neurophysiological barriers. It is evident that most of the promising nanotech-based therapeutic/diagnostic/imaging solutions have their roots in natural phenomena like transBBB movement of HIV-TAT peptide, activated leukocytes, receptor based translocation via GLUT-1 receptor and others. Moreover the review also showcases how progress in the field of nanoelectronics is influencing neuroscience by facilitating cellular level electrical stimulation and signal recording as well as developing novel opto-electronic devices to aid the visually challenged. More recently, the highly energy efficient functioning of the human

28

brain and its exceptional ability to perform multiple tasks in a parallel fashion have motivated scientists to develop brain-inspired computational systems. In the midst of widespread research in nano-neurotechnology, the ethical and toxicological concerns pertaining to the application of nanomaterials in neuroscience cannot be ignored. Neurotoxicicty of nanomaterials have been extensively reviewed by several groups (Cupaioli et al., 2014; Suh et al., 2009). The biomolecular composition of the nanomaterials corona plays a crucial role in determining their toxicity in vivo. Neurotoxicity caused by nanomaterials are mainly attributed to their size, reactive oxygen species (ROS) produced by them and the intrinsic toxicity of the bare material (Krol et al., 2013). Long duration toxicology and biocompatibility studies in vitro and in vivo are essential to establish the safety of nanomaterials before they are actually used in patients. Further, brain implants using nanomaterials spark ethical concerns about the ability of a patient suffering from a brain disorder to provide an educated consent during clinical trials. However, such challenges should not hinder innovation. Nanotechnology, if used safely and ethically, has the potential to help elucidate the intricate mechanisms involved in neuro-pathophysiology and lead to the development of highly efficient theranostic solutions to address the challenges of the human nervous system. Acknowledgements SD and UB would like to thank and acknowledge Central Silk Board, Ministry of Textiles, Bangalore, Govt of India for providing partial funding support for this work. SD would like to thank IIT Guwahati and Ministry of Human Resource Development (MHRD), Govt of India for fellowship. ACL and JWF would like to thank NIHR (National Institute for Health Research), UK, Cambridge Biomedical Research Centre, Christopher and Dana Reeve Foundation and Engineering and Physical Sciences Research Council (EPSRC) for funding.

29

References A Phase I Study of Convection-Enhanced Delivery of Liposomal-Irinotecan Using Real-Time Imaging With Gadolinium In Patients With Recurrent High Grade Glioma, ClinicalTrials.gov Identifier: NCT02022644 [WWW Document], n.d. URL https://clinicaltrials.gov/ct2/show/NCT02022644 Abbott, N.J., Rönnbäck, L., Hansson, E., 2006. Astrocyte–endothelial interactions at the blood–brain barrier. Nat. Rev. Neurosci. 7, 41–53. doi:10.1038/nrn1824 Ahn, H.-S., Hwang, J.-Y., Kim, M.S., Lee, J.-Y., Kim, J.-W., Kim, H.-S., Shin, U.S., Knowles, J.C., Kim, H.-W., Hyun, J.K., 2015. Carbon-nanotube-interfaced glass fiber scaffold for regeneration of transected sciatic nerve. Acta Biomater. 13, 324–34. doi:10.1016/j.actbio.2014.11.026 An, J.H., Kim, T.-H., Oh, B.-K., Choi, J.W., 2012. Detection of Dopamine in Dopaminergic Cell Using Nanoparticles-Based Barcode DNA Analysis. J. Nanosci. Nanotechnol. 12, 764–768. doi:10.1166/jnn.2012.5403 An, J.H., Oh, B.-K., Choi, J.W., 2013. Detection of tyrosine hydroxylase in dopaminergic neuron cell using gold nanoparticles-based barcode DNA. J. Biomed. Nanotechnol. 9, 639–43. doi:10.1166/jbn.2013.1525 Araya, E., Olmedo, I., Bastus, N.G., Guerrero, S., Puntes, V.F., Giralt, E., Kogan, M.J., 2008. Gold nanoparticles and microwave irradiation inhibit beta-amyloid amyloidogenesis. Nanoscale Res. Lett. 3, 435–443. doi:10.1007/s11671-008-9178-5 Archibald, S.J., Shefner, J., Krarup, C., Madison, R.D., 1995. Monkey median nerve repaired by nerve graft or collagen nerve guide tube. J. Neurosci. 15, 4109–4123. Arizono, M., Bannai, H., Mikoshiba, K., 2014. Imaging mGluR5 dynamics in astrocytes using quantum dots. Curr. Protoc. Neurosci. 1–18. doi:10.1002/0471142301.ns0221s66 Banks, W.A., Freed, E.O., Wolf, K.M., Robinson, S.M., Franko, M., Kumar, V.B., 2001. Transport of human immunodeficiency virus type 1 pseudoviruses across the blood-brain barrier: role of envelope proteins and adsorptive endocytosis. J. Virol. 75, 4681–91. doi:10.1128/JVI.75.10.4681-4691.2001 Barbu, E., Molnar, E., Tsibouklis, J., Gorecki, D.C., 2009. The potential for nanoparticle-based drug delivery to the brain: overcoming the blood¡Vbrain barrier. Expert Opin. Drug Deliv. 6, 553– 565. Bareket, L., Waiskopf, N., Rand, D., Lubin, G., David-Pur, M., Ben-Dov, J., Roy, S., Eleftheriou, C., Sernagor, E., Cheshnovsky, O., Banin, U., Hanein, Y., 2014. Semiconductor nanorod-carbon nanotube biomimetic films for wire-free photostimulation of blind retinas. Nano Lett. 14, 6685– 92. doi:10.1021/nl5034304 Batrakova, E. V, Li, S., Reynolds, A.D., Mosley, R.L., Bronich, T.K., Kabanov, A. V, Gendelman, H.E., 2007. A macrophage-nanozyme delivery system for Parkinson’s disease. Bioconjug. Chem. 18, 1498–1506. doi:10.1021/bc700184b Bazzoni, G., Dejana, E., 2004. Endothelial cell-to-cell junctions: molecular organization and role in vascular homeostasis. Physiol Rev. 84, 869–901. doi:10.1152/physrev.00035.2003 Bobo, R.H., Laske, D.W., Akbasak, A., Morrison, P.F., Dedrick, R.L., Oldfield, E.H., 1994. Convection-enhanced delivery of macromolecules in the brain. Proc Natl Acad Sci U S A 91, 2076–2080. doi:10.1073/pnas.91.6.2076 Butt, a M., Jones, H.C., Abbott, N.J., 1990. Electrical resistance across the blood-brain barrier in anaesthetized rats: a developmental study. J. Physiol. 429, 47–62. doi:10.1113/jphysiol.1990.sp018243 Cavallo, F., Huang, Y., Dent, E.W., Williams, J.C., Lagally, M.G., 2014. Neurite guidance and threedimensional confinement via compliant semiconductor scaffolds. ACS Nano 8, 12219–27. doi:10.1021/nn503989c Chaudhary, A., 2011. Ayurvedic Bhasma: Nanomedicine of ancient India - Its global contemporary perspective. J. Biomed. Nanotechnol. 7, 68–69. doi:10.1166/jbn.2011.1205 Chertok, B., Moffat, B. a., David, A.E., Yu, F., Bergemann, C., Ross, B.D., Yang, V.C., 2008. Iron oxide nanoparticles as a drug delivery vehicle for MRI monitored magnetic targeting of brain tumors. Biomaterials 29, 487–496. doi:10.1016/j.biomaterials.2007.08.050 Chew, S.Y., Mi, R., Hoke, A., Leong, K.W., 2007. Aligned Protein-Polymer Composite Fibers 30

Enhance Nerve Regeneration: A Potential Tissue-Engineering Platform. Adv. Funct. Mater. 17, 1288–1296. doi:10.1002/adfm.200600441 Cho, E.C., Zhang, Y., Cai, X., Moran, C.M., Wang, L. V, Xia, Y., 2013. Quantitative analysis of the fate of gold nanocages in vitro and in vivo after uptake by U87-MG tumor cells. Angew. Chem. Int. Ed. Engl. 52, 1152–5. doi:10.1002/anie.201208096 Choi, I., Lee, L.P., 2013. Rapid detection of A?? aggregation and inhibition by dual functions of gold nanoplasmic particles: Catalytic activator and optical reporter. ACS Nano 7, 6268–6277. doi:10.1021/nn402310c Choi, J.S., Jun, Y.W., Yeon, S.I., Kim, H.C., Shin, J.S., Cheon, J., 2006. Biocompatible heterostructured nanoparticles for multimodal biological detection. J. Am. Chem. Soc. 128, 15982–15983. doi:10.1021/ja066547g Cormode, D.P., Jarzyna, P. a., Mulder, W.J.M., Fayad, Z. a., 2010. Modified natural nanoparticles as contrast agents for medical imaging. Adv. Drug Deliv. Rev. 62, 329–338. doi:10.1016/j.addr.2009.11.005 Cui, X., Wiler, J., Dzaman, M., Altschuler, R. a., Martin, D.C., 2003. In vivo studies of polypyrrole/peptide coated neural probes. Biomaterials 24, 777–787. doi:10.1016/S01429612(02)00415-5 Cui, Z., Lockman, P.R., Atwood, C.S., Hsu, C.H., Gupte, A., Allen, D.D., Mumper, R.J., 2005. Novel D-penicillamine carrying nanoparticles for metal chelation therapy in Alzheimer’s and other CNS diseases. Eur. J. Pharm. Biopharm. 59, 263–272. doi:10.1016/j.ejpb.2004.07.009 Cupaioli, F.A., Zucca, F.A., Boraschi, D., Zecca, L., 2014. Engineered nanoparticles. How brain friendly is this new guest? Prog. Neurobiol. doi:10.1016/j.pneurobio.2014.05.002 D’Arrigo, J.S., Simon, R.H., Ho, S.Y., 1991. Lipid-coated uniform microbubbles for earlier sonographic detection of brain tumors. J. Neuroimaging 1, 134–9. Das, S., Sharma, M., Saharia, D., Sarma, K.K., Sarma, M.G., Borthakur, B.B., Bora, U., 2015. In vivo studies of silk based gold nano-composite conduits for functional peripheral nerve regeneration. Biomaterials 62, 66–75. doi:10.1016/j.biomaterials.2015.04.047 de Asis, E.D., Nguyen-Vu, T.D.B., Arumugam, P.U., Chen, H., Cassell, A.M., Andrews, R.J., Yang, C.Y., Li, J., 2009. High efficient electrical stimulation of hippocampal slices with vertically aligned carbon nanofiber microbrush array. Biomed. Microdevices 11, 801–808. doi:10.1007/s10544-009-9295-7 De Giglio, E., Trapani, A., Cafagna, D., Sabbatini, L., Cometa, S., 2011. Dopamine-loaded chitosan nanoparticles: Formulation and analytical characterization. Anal. Bioanal. Chem. 400, 1997– 2002. doi:10.1007/s00216-011-4962-y Deng, Y., Wang, H., Gu, W., Li, S., Xiao, N., Shao, C., Xu, Q., Ye, L., 2014. Ho3+ doped NaGdF4 nanoparticles as MRI/optical probes for brain glioma imaging. J. Mater. Chem. B 2, 1521. doi:10.1039/c3tb21613f EC, T., 1987. Ferritin: structure, gene regulation, and cellular function in animals, plants and microorganisms. Annu Rev Biochem 56, 289–315. ussere und innere Sekretion bingen  : Verlag der H. Laupp’schen Buchhandlung. Ehrlich P, 1885. Das Sauerstoff-Bedürfniss des Organismus Eine farbenanalytische Studie. Berlin. Ellis-Behnke, R.G., Liang, Y.-X., You, S.-W., Tay, D.K.C., Zhang, S., So, K.-F., Schneider, G.E., 2006. Nano neuro knitting: peptide nanofiber scaffold for brain repair and axon regeneration with functional return of vision. Proc. Natl. Acad. Sci. U. S. A. 103, 5054–5059. doi:10.1073/pnas.0600559103 Enochs, W.S., Harsh, G., Hochberg, F., Weissleder, R., 1999. Improved delineation of human brain tumors on MR images using a long-circulating, superparamagnetic iron oxide agent (COMBIDEX***). J. Magn. Reson. Imaging 9, 228–232. doi:10.1002/(SICI)15222586(199902)9:2<228::AID-JMRI12>3.0.CO;2-K [pii] F.W. Mott, 1913. THE LATE PROFESSOR EDWIN GOLDMANN’S INVESTIGATIONS ON THE CENTRAL NERVOUS SYSTEM BY VITAL STAINING. Br. Med. J. 2, 871–873. Fakih, M., Vincent, M., 2010. Adverse events associated with anti-EGFR therapies for the treatment of metastatic colorectal cancer. Curr. Oncol. 17 Suppl 1, S18–S30. 31

Farkas, E., Luiten, P.G.M., 2001. Cerebral microvascular pathology in aging and Alzheimer’s disease, Progress in Neurobiology. doi:10.1016/S0301-0082(00)00068-X Figueiró, F., Bernardi, A., Frozza, R.L., Terroso, T., Zanotto-Filho, A., Jandrey, E.H.F., Moreira, J.C.F., Salbego, C.G., Edelweiss, M.I., Pohlmann, A.R., Guterres, S.S., Battastini, A.M.O., 2013. Resveratrol-loaded lipid-core nanocapsules treatment reduces in vitro and in vivo glioma growth. J. Biomed. Nanotechnol. 9, 516–526. doi:10.1166/jbn.2013.1547 Fortin, P.-Y., Genevois, C., Koenig, A., Heinrich, E., Texier, I., Couillaud, F., 2012. Detection of brain tumors using fluorescence diffuse optical tomography and nanoparticles as contrast agents. J. Biomed. Opt. 17, 126004. doi:10.1117/1.JBO.17.12.126004 Gao, H., Yang, Z., Cao, S., Xiong, Y., Zhang, S., Pang, Z., Jiang, X., 2014. Tumor cells and neovasculature dual targeting delivery for glioblastoma treatment. Biomaterials 35, 2374–82. doi:10.1016/j.biomaterials.2013.11.076 Gao, X., Chen, J., Wu, B., Chen, H., Jiang, X., 2008. Quantum dots bearing lectin-functionalized nanoparticles as a platform for in vivo brain imaging. Bioconjug Chem 19, 2189–2195. doi:10.1021/bc8002698 Gelain, F., Bottai, D., Vescovi, A., Zhang, S., 2006. Designer self-assembling peptide nanofiber scaffolds for adult mouse neural stem cell 3-dimensional cultures. PLoS One 1, e119. doi:10.1371/journal.pone.0000119 Georganopoulou, D.G., Chang, L., Nam, J.-M., Thaxton, C.S., Mufson, E.J., Klein, W.L., Mirkin, C. a, 2005. Nanoparticle-based detection in cerebral spinal fluid of a soluble pathogenic biomarker for Alzheimer’s disease. Proc. Natl. Acad. Sci. U. S. A. 102, 2273–2276. doi:10.1073/pnas.0409336102 Gorell, J., Ordidge, R., Brown, G., Deniau, J., Buderer, N., Helpern, J., 1995. Increased iron-related MRI contrast in the substantia nigra in Parkinson’s disease. Neurology 45, 1138–43. doi:10.1212/WNL.45.6.1138 Goyal, R.N., Singh, S.P., 2008. Simultaneous voltammetric determination of dopamine and adenosine using a single walled carbon nanotube - Modified glassy carbon electrode. Carbon N. Y. 46, 1556–1562. doi:10.1016/j.carbon.2008.06.051 Gromnicova, R., Davies, H.A., Sreekanthreddy, P., Romero, I.A., Lund, T., Roitt, I.M., Phillips, J.B., Male, D.K., 2013. Glucose-coated gold nanoparticles transfer across human brain endothelium and enter astrocytes in vitro. PLoS One 8, e81043. doi:10.1371/journal.pone.0081043 Guo, J., Gao, X., Su, L., Xia, H., Gu, G., Pang, Z., Jiang, X., Yao, L., Chen, J., Chen, H., 2011. Aptamer-functionalized PEG-PLGA nanoparticles for enhanced anti-glioma drug delivery. Biomaterials 32, 8010–8020. doi:10.1016/j.biomaterials.2011.07.004 Guo, J., Su, H., Zeng, Y., Liang, Y.-X., Wong, W.M., Ellis-Behnke, R.G., So, K.-F., Wu, W., 2007. Reknitting the injured spinal cord by self-assembling peptide nanofiber scaffold. Nanomedicine 3, 311–21. doi:10.1016/j.nano.2007.09.003 Guo, S.X., Bourgeois, F., Chokshi, T., Durr, N.J., Hilliard, M.A., Chronis, N., Ben-yakar, A., 2008. Femtosecond laser nanoaxotomy lab-on-a- chip for in vivo nerve regeneration studies. Nat. Methods 5, 531–533. doi:10.1038/NMETH.1203 Haes, A.J., Hall, W.P., Chang, L., Klein, W.L., Van Duyne, R.P., 2004. A localized surface plasmon resonance biosensor:  first steps toward an assay for Alzheimer’s disease. Nano Lett. 4, 1029– 1034. doi:10.1021/nl049670j Hainfeld, J.F., Slatkin, D.N., Focella, T.M., Smilowitz, H.M., 2006. Gold nanoparticles: a new X-ray contrast agent. Br. J. Radiol. 79, 248–53. doi:10.1259/bjr/13169882 Hainfeld, J.F., Slatkin, D.N., Smilowitz, H.M., 2004. The use of gold nanoparticles to enhance radiotherapy in mice. Phys. Med. Biol. 49, N309–N315. doi:10.1088/0031-9155/49/18/N03 Hainfeld, J.F., Smilowitz, H.M., O’Connor, M.J., Dilmanian, F.A., Slatkin, D.N., 2013. Gold nanoparticle imaging and radiotherapy of brain tumors in mice. Nanomedicine (Lond). 8, 1601– 9. doi:10.2217/nnm.12.165 Hällström, W., Mårtensson, T., Prinz, C., Gustavsson, P., Montelius, L., Samuelson, L., Kanje, M., 2007. Gallium phosphide nanowires as a substrate for cultured neurons. Nano Lett. 7, 2960– 2965. doi:10.1021/nl070728e Haseloff, R.F., Blasig, I.E., Bauer, H.C., Bauer, H., 2005. In search of the astrocytic factor(s) modulating blood-brain barrier functions in brain capillary endothelial cells in vitro. Cell. Mol. 32

Neurobiol. 25, 25–39. doi:10.1007/s10571-004-1375-x Hawkins, B.T., Davis, T.P., 2005. The Blood-Brain Barrier / Neurovascular Unit in Health and Disease. Pharmacol. Rev. 57, 173–185. doi:10.1124/pr.57.2.4.173 Hayashi, Y., Nomura, M., Yamagishi, S.I., Harada, S.I., Yamashita, J., Yamamoto, H., 1997. Induction of various blood-brain barrier properties in non-neural endothelial cells by close apposition to co-cultured astrocytes. Glia 19, 13–26. doi:10.1002/(SICI)10981136(199701)19:1<13::AID-GLIA2>3.0.CO;2-B Hejcl, A., Sedý, J., Kapcalová, M., Toro, D.A., Amemori, T., Lesný, P., Likavcanová-Mašínová, K., Krumbholcová, E., Prádný, M., Michálek, J., Burian, M., Hájek, M., Jendelová, P., Syková, E., 2010. HPMA-RGD hydrogels seeded with mesenchymal stem cells improve functional outcome in chronic spinal cord injury. Stem Cells Dev. 19, 1535–1546. doi:10.1089/scd.2009.0378 Holland, E.C., 2000. Glioblastoma multiforme: the terminator. Proc. Natl. Acad. Sci. U. S. A. 97, 6242–6244. doi:10.1073/pnas.97.12.6242 Holmes, T.C., de Lacalle, S., Su, X., Liu, G., Rich, a, Zhang, S., 2000. Extensive neurite outgrowth and active synapse formation on self-assembling peptide scaffolds. Proc. Natl. Acad. Sci. U. S. A. 97, 6728–6733. doi:10.1073/pnas.97.12.6728 Hori, S., Ohtsuki, S., Hosoya, K., Nakashima, E., Terasaki, T., 2004. A pericyte-derived angiopoietin1 multimeric complex induces occludin gene expression in brain capillary endothelial cells through Tie-2 activation in vitro. J. Neurochem. 89, 503–13. doi:10.1111/j.14714159.2004.02343.x Hu, K., Shi, Y., Jiang, W., Han, J., Huang, S., Jiang, X., 2011. Lactoferrin conjugated PEG-PLGA nanoparticles for brain delivery: Preparation, characterization and efficacy in Parkinsons disease. Int. J. Pharm. 415, 273–283. doi:10.1016/j.ijpharm.2011.05.062 Huang, R., Ke, W., Liu, Y., Wu, D., Feng, L., Jiang, C., Pei, Y., 2010. Gene therapy using lactoferrinmodified nanoparticles in a rotenone-induced chronic Parkinson model. J. Neurol. Sci. 290, 123–30. doi:10.1016/j.jns.2009.09.032 Huang, R., Ma, H., Guo, Y., Liu, S., Kuang, Y., Shao, K., Li, J., Liu, Y., Han, L., Huang, S., An, S., Ye, L., Lou, J., Jiang, C., 2013. Angiopep-conjugated nanoparticles for targeted long-term gene therapy of parkinson’s disease. Pharm. Res. 30, 2549–2559. doi:10.1007/s11095-013-1005-8 Ikeda, K., Okada, T., Sawada, S.I., Akiyoshi, K., Matsuzaki, K., 2006. Inhibition of the formation of amyloid β-protein fibrils using biocompatible nanogels as artificial chaperones. FEBS Lett. 580, 6587–6595. doi:10.1016/j.febslet.2006.11.009 Jackson, H., Muhammad, O., Daneshvar, H., Nelms, J., Popescu, A., Vogelbaum, M. a, Bruchez, M., Toms, S. a, 2007. Quantum dots are phagocytized by macrophages and colocalize with experimental gliomas. Neurosurgery 60, 524–9; discussion 529–30. doi:10.1227/01.NEU.0000255334.95532.DD Jiang, W., Xie, H., Ghoorah, D., Shang, Y., Shi, H., Liu, F., Yang, X., Xu, H., 2012. Conjugation of functionalized SPIONs with transferrin for targeting and imaging brain glial tumors in rat model. PLoS One 7, e37376. doi:10.1371/journal.pone.0037376 Jin, J., Xu, Z., Zhang, Y., Gu, Y.-J., Lam, M.H.-W., Wong, W.-T., 2013. Upconversion nanoparticles conjugated with Gd(3+) -DOTA and RGD for targeted dual-modality imaging of brain tumor xenografts. Adv. Healthc. Mater. 2, 1501–12. doi:10.1002/adhm.201300102 Josephson, L., Tung, C.H., Moore, A., Weissleder, R., 1999. High-efficiency intracellular magnetic labeling with novel superparamagnetic-tat peptide conjugates. Bioconjug. Chem. doi:10.1021/bc980125h Kabalka, G.W., Davis, M.A., Holmberg, E., Maruyama, K., Huang, L., 1991. Gadolinium-labeled liposomes containing amphiphilic Gd-DTPA derivatives of varying chain length: targeted MRI contrast enhancement agents for the liver. Magn Reson Imaging 9, 373–377. doi:10.1016/0730725X(91)90425-L Kanwar, J.R., Sun, X., Punj, V., Sriramoju, B., Mohan, R.R., Zhou, S.F., Chauhan, A., Kanwar, R.K., 2012. Nanoparticles in the treatment and diagnosis of neurological disorders: Untamed dragon with fire power to heal. Nanomedicine Nanotechnology, Biol. Med. 8, 399–414. doi:10.1016/j.nano.2011.08.006 Kievit, F.M., Veiseh, O., Fang, C., Bhattarai, N., Lee, D., Ellenbogen, R.G., Zhang, M., 2010. Chlorotoxin labeled magnetic nanovectors for targeted gene delivery to glioma. ACS Nano 4, 33

4587–4594. doi:10.1021/nn1008512 Kim, S.-G., Harel, N., Jin, T., Kim, T., Lee, P., Zhao, F., 2013. Cerebral blood volume MRI with intravascular superparamagnetic iron oxide nanoparticles. NMR Biomed. 26, 949–962. doi:10.1002/nbm.2885 Kim, Y., Haftel, V.K., Kumar, S., Bellamkonda, R. V., 2008. The role of aligned polymer fiber-based constructs in the bridging of long peripheral nerve gaps. Biomaterials. doi:10.1016/j.biomaterials.2008.03.042 Kimura, K., Yanagida, Y., Haruyama, T., Kobatake, E., Aizawa, M., 1998. Electrically induced neurite outgrowth of PC12 cells on the electrode surface. Med. Biol. Eng. Comput. 36, 493–498. doi:10.1007/BF02523221 Kircher, M., Mahmood, U., King, R., Weissleder, R., Josephson, L., 2003. A multimodal nanoparticle for preoperative magnetic resonance imaging and intraoperative optical brain tumor delineation. Cancer Res. 63, 8122–8125. Kircher, M.F., de la Zerda, A., Jokerst, J. V, Zavaleta, C.L., Kempen, P.J., Mittra, E., Pitter, K., Huang, R., Campos, C., Habte, F., Sinclair, R., Brennan, C.W., Mellinghoff, I.K., Holland, E.C., Gambhir, S.S., 2012. A brain tumor molecular imaging strategy using a new triple-modality MRI-photoacoustic-Raman nanoparticle. Nat. Med. 18, 829–34. doi:10.1038/nm.2721 Koprivica, V., Cho, K.-S., Park, J.B., Yiu, G., Atwal, J., Gore, B., Kim, J. a, Lin, E., Tessier-Lavigne, M., Chen, D.F., He, Z., 2005. EGFR activation mediates inhibition of axon regeneration by myelin and chondroitin sulfate proteoglycans. Science 310, 106–110. doi:10.1126/science.1115462 Krol, S., Macrez, R., Docagne, F., Defer, G., Laurent, S., Rahman, M., Hajipour, M.J., Kehoe, P.G., Mahmoudi, M., 2013. Therapeutic benefits from nanoparticles: The potential significance of nanoscience in diseases with compromise to the blood brain barrier. Chem. Rev. doi:10.1021/cr200472g Kuzum, D., Jeyasingh, R.G.D., Lee, B., Wong, H.P., 2012. Materials for Brain-Inspired Computing 2179–2186. Lampugnani, M.G., Corada, M., Caveda, L., Breviario, F., Ayalon, O., Geiger, B., Dejana, E., 1995. The molecular organization of endothelial cell to cell junctions: differential association of plakoglobin, beta-catenin, and alpha-catenin with vascular endothelial cadherin (VE-cadherin). J. Cell Biol. 129, 203–217. doi:10.1083/jcb.129.1.203 Lassen, N., Ingvar, D., 1961. The blood flow of the cerebral cortex determined by radioactive krypton. Experientia 17, 42–43. Lee, J.H., Kang, D.Y., Lee, T., Kim, S.U., Oh, B.K., Choil, J.W., 2009. Signal enhancement of surface plasmon resonance based immunosensor using gold nanoparticle-antibody complex for beta-amyloid (1-40) detection. J Nanosci Nanotechnol 9, 7155–7160. doi:10.1166/jnn.2009.1613 Lee, J.Y., Bashur, C. a., Goldstein, A.S., Schmidt, C.E., 2009. Polypyrrole-coated electrospun PLGA nanofibers for neural tissue applications. Biomaterials 30, 4325–4335. doi:10.1016/j.biomaterials.2009.04.042 Lee, S., Leach, M.K., Redmond, S. a, Chong, S.Y.C., Mellon, S.H., Tuck, S.J., Feng, Z.-Q., Corey, J.M., Chan, J.R., 2012. A culture system to study oligodendrocyte myelination processes using engineered nanofibers. Nat. Methods 9, 917–922. doi:10.1038/nmeth.2105 Liao, Y.H., Chang, Y.J., Yoshiike, Y., Chang, Y.C., Chen, Y.R., 2012. Negatively charged gold nanoparticles inhibit Alzheimer’s amyloid-β fibrillization, induce fibril dissociation, and mitigate neurotoxicity. Small 8, 3631–3639. doi:10.1002/smll.201201068 Liu, D., Chen, W., Tian, Y., He, S., Zheng, W., Sun, J., Wang, Z., Jiang, X., 2012. A highly sensitive gold-nanoparticle-based assay for acetylcholinesterase in cerebrospinal fluid of transgenic mice with alzheimer’s disease. Adv. Healthc. Mater. 1, 90–95. doi:10.1002/adhm.201100002 Liu, H.-L., Hua, M.-Y., Yang, H.-W., Huang, C.-Y., Chu, P.-C., Wu, J.-S., Tseng, I.-C., Wang, J.-J., Yen, T.-C., Chen, P.-Y., Wei, K.-C., 2010. Magnetic resonance monitoring of focused ultrasound/magnetic nanoparticle targeting delivery of therapeutic agents to the brain. Proc. Natl. Acad. Sci. U. S. A. 107, 15205–15210. doi:10.1073/pnas.1003388107 Liu, Y., Lu, W., 2012. Recent advances in brain tumor-targeted nano-drug delivery systems. Expert Opin. Drug Deliv. 9, 671–686. doi:10.1517/17425247.2012.682726 Luppi, B., Bigucci, F., Corace, G., Delucca, A., Cerchiara, T., Sorrenti, M., Catenacci, L., Di Pietra, 34

A.M., Zecchi, V., 2011. Albumin nanoparticles carrying cyclodextrins for nasal delivery of the anti-Alzheimer drug tacrine. Eur. J. Pharm. Sci. 44, 559–565. doi:10.1016/j.ejps.2011.10.002 Lyubchenko, Y.L., Kim, B.-H., Krasnoslobodtsev, A. V, Yu, J., 2010. Nanoimaging for protein misfolding diseases. Wiley Interdiscip. Rev. Nanomed. Nanobiotechnol. 2, 526–43. doi:10.1002/wnan.102 Mahmoudi, M., Sheibani, S., Milani, A.S., Rezaee, F., Gauberti, M., Dinarvand, R., Vali, H., 2015. Crucial role of the protein corona for the specific targeting of nanoparticles. Nanomedicine 10, 215–226. doi:10.2217/nnm.14.69 Mamot, C., Nguyen, J.B., Pourdehnad, M., Hadaczek, P., Saito, R., Bringas, J.R., Drummond, D.C., Hong, K., Kirpotin, D.B., McKnight, T., Berger, M.S., Park, J.W., Bankiewicz, K.S., 2004. Extensive distribution of liposomes in rodent brains and brain tumors following convectionenhanced delivery. J. Neurooncol. 68, 1–9. doi:10.1023/B:NEON.0000024743.56415.4b Manninger, S.P., Muldoon, L.L., Nesbit, G., Murillo, T., Jacobs, P.M., Neuwelt, E. a., 2005. An exploratory study of ferumoxtran-10 nanoparticles as a blood-brain barrier imaging agent targeting phagocytic cells in CNS inflammatory lesions. Am. J. Neuroradiol. 26, 2290–2300. doi:26/9/2290 [pii] Mardor, Y., Roth, Y., Lidar, Z., Jonas, T., Pfeffer, R., 2001. Monitoring response to convectionenhanced taxol delivery in brain tumor patients using diffusion-weighted magnetic resonance imaging. Cancer Res. 61, 4971–4973. Marshall, J.D., Schnitzer, M.J., 2013. Optical strategies for sensing neuronal voltage using quantum dots and other semiconductor nanocrystals. ACS Nano 7, 4601–4609. doi:10.1021/nn401410k Martino, G., Pluchino, S., 2006. The therapeutic potential of neural stem cells. Nat. Rev. Neurosci. 7, 395–406. doi:10.1038/nrn1908 Mathew, A., Fukuda, T., Nagaoka, Y., Hasumura, T., Morimoto, H., Yoshida, Y., Maekawa, T., Venugopal, K., Kumar, D.S., 2012. Curcumin loaded-PLGA nanoparticles conjugated with Tet1 peptide for potential use in Alzheimer’s disease. PLoS One 7, 1–10. doi:10.1371/journal.pone.0032616 McAllister, M.S., Krizanac-Bengez, L., Macchia, F., Naftalin, R.J., Pedley, K.C., Mayberg, M.R., Marroni, M., Leaman, S., Stanness, K.A., Janigro, D., 2001. Mechanisms of glucose transport at the blood-brain barrier: An in vitro study. Brain Res. 904, 20–30. doi:10.1016/S00068993(01)02418-0 Meiners, S., Ahmed, I., Ponery, A., 2007. Engineering electrospun nanofibrillar surfaces for spinal cord repair: a discussion. Polym. … 1348, 1340–1348. doi:10.1002/pi Merolla, P.A., Arthur, J. V, Alvarez-Icaza, R., Cassidy, A.S., Sawada, J., Akopyan, F., Jackson, B.L., Imam, N., Guo, C., Nakamura, Y., Brezzo, B., Vo, I., Esser, S.K., Appuswamy, R., Taba, B., Amir, A., Flickner, M.D., Risk, W.P., Manohar, R., Modha, D.S., 2014. A million spikingneuron integrated circuit with a scalable communication network and interface. Science (80-. ). 345, 668–673. doi:10.1126/science.1254642 Miao, D., Jiang, M., Liu, Z., Gu, G., Hu, Q., Kang, T., Song, Q., Yao, L., Li, W., Gao, X., Sun, M., Chen, J., 2014. Co-administration of dual-targeting nanoparticles with penetration enhancement peptide for antiglioblastoma therapy. Mol. Pharm. 11, 90–101. doi:10.1021/mp400189j Miladi, I., Alric, C., Dufort, S., Mowat, P., Dutour, A., Mandon, C., Laurent, G., Bräuer-Krisch, E., Herath, N., Coll, J.L., Dutreix, M., Lux, F., Bazzi, R., Billotey, C., Janier, M., Perriat, P., Le Duc, G., Roux, S., Tillement, O., 2014. The in vivo radiosensitizing effect of gold nanoparticles based mri contrast agents. Small 10, 1116–1124. doi:10.1002/smll.201302303 Ming, G., Song, H., 2005. Adult neurogenesis in the mammalian central nervous system. Annu. Rev. Neurosci. 28, 223–250. doi:10.1146/annurev.neuro.28.051804.101459 Mishra, a., Lai, G.H., Schmidt, N.W., Sun, V.Z., Rodriguez, a. R., Tong, R., Tang, L., Cheng, J., Deming, T.J., Kamei, D.T., Wong, G.C.L., 2011. Translocation of HIV TAT peptide and analogues induced by multiplexed membrane and cytoskeletal interactions. Proc. Natl. Acad. Sci. 108, 16883–16888. doi:10.1073/pnas.1108795108 Mittal, G., Carswell, H., Brett, R., Currie, S., Kumar, M.N.V.R., 2011. Development and evaluation of polymer nanoparticles for oral delivery of estradiol to rat brain in a model of Alzheimer’s pathology. J. Control. Release 150, 220–8. doi:10.1016/j.jconrel.2010.11.013 Morgello, S., Uson, R.R., Schwartz, E.J., Haber, R.S., 1995. The human blood-brain barrier glucose 35

transporter (GLUT1) is a glucose transporter of gray matter astrocytes. Glia 14, 43–54. doi:10.1002/glia.440140107 Mosso A, 1881. Concerning the circulation of the blood in the human brain. Leipzig: Verlag von Viet & Company. Na, H. Bin, Song, I.C., Hyeon, T., 2009. Inorganic nanoparticles for MRI contrast agents. Adv. Mater. 21, 2133–2148. doi:10.1002/adma.200802366 Neely, A., Perry, C., Varisli, B., Singh, A.K., Arbneshi, T., Senapati, D., Kalluri, J.R., Ray, P.C., 2009. Ultrasensitive and highly selective detection of Alzheimer’s disease biomarker using twophoton Rayleigh scattering properties of gold nanoparticle. ACS Nano 3, 2834–2840. doi:10.1021/nn900813b Neuwelt, E. a., Várallyay, P., Bagó, a. G., Muldoon, L.L., Nesbit, G., Nixon, R., 2004. Imaging of iron oxide nanoparticles by MR and light microscopy in patients with malignant brain tumours. Neuropathol. Appl. Neurobiol. 30, 456–471. doi:10.1111/j.1365-2990.2004.00557.x Nguyen-Vu, T.D.B., Chen, H., Cassell, A.M., Andrews, R., Meyyappan, M., Li, J., 2006. Vertically aligned carbon nanofiber arrays: an advance toward electrical-neural interfaces. Small 2, 89–94. doi:10.1002/smll.200500175 Nguyen-Vu, T.D.B., Chen, H., Cassell, A.M., Andrews, R.J., Meyyappan, M., Li, J., 2007. Vertically Aligned Carbon Nanofiber Architecture as a Multifunctional 3-D Neural Electrical Interface. IEEE Trans. Biomed. Eng. Biomed. Eng. 54, 1121–1128. doi:10.1109/TBME.2007.891169 Pal, D., Sahu, C.K., Haldar, A., 2014. Bhasma  : The ancient Indian nanomedicine. J. Adv. Pharm. Technol. Res. 5, 4–12. doi:10.4103/2231-4040.126980 Panseri, S., Cunha, C., Lowery, J., Del Carro, U., Taraballi, F., Amadio, S., Vescovi, A., Gelain, F., 2008. Electrospun micro- and nanofiber tubes for functional nervous regeneration in sciatic nerve transections. BMC Biotechnol. 8, 39. doi:10.1186/1472-6750-8-39 Pappas, T.C., Wickramanyake, W.M.S., Jan, E., Motamedi, M., Brodwick, M., Kotov, N. a., 2007. Nanoscale engineering of a cellular interface with semiconductor nanoparticle films for photoelectric stimulation of neurons. Nano Lett. 7, 513–519. doi:10.1021/nl062513v Park, J., Kwon, S., Jun, S.I., Mcknight, T.E., Melechko, A. V, Simpson, M.L., Dhindsa, M., Heikenfeld, J., Rack, P.D., 2009. Active-Matrix Microelectrode Arrays Integrated With Vertically Aligned Carbon Nanofibers. {IEEE} Electron Device Lett. 30, 254–257. doi:10.1109/LED.2008.2011927 Parodi, A., Quattrocchi, N., van de Ven, A.L., Chiappini, C., Evangelopoulos, M., Martinez, J.O., Brown, B.S., Khaled, S.Z., Yazdi, I.K., Enzo, M.V., Isenhart, L., Ferrari, M., Tasciotti, E., 2013. Synthetic nanoparticles functionalized with biomimetic leukocyte membranes possess cell-like functions. Nat. Nanotechnol. 8, 61–8. doi:10.1038/nnano.2012.212 Passariello, R., Pavone, P., Patrizio, G., Di Renzi, P., Mastantuono, M., Giuliani, S., 1990. Liposomes loaded with nonionic contrast media. Hepatosplenic computed tomographic enhancement. Invest. Radiol. 25 Suppl 1, S92–4. Patolsky, F., Timko, B.P., Yu, G., Fang, Y., Greytak, A.B., Zheng, G., Lieber, C.M., 2006. Detection, stimulation, and inhibition of neuronal signals with high-density nanowire transistor arrays. Science (80-. ). 313, 1100–1104. doi:10.1126/science.1128640 Persidsky, Y., Stins, M., Way, D., Witte, M.H., Weinand, M., Kim, K.S., Bock, P., Gendelman, H.E., Fiala, M., 1997. A model for monocyte migration through the blood-brain barrier during HIV-1 encephalitis. J. Immunol. 158, 3499–510. Pillay, S., Pillay, V., Choonara, Y.E., Naidoo, D., Khan, R. a., du Toit, L.C., Ndesendo, V.M.K., Modi, G., Danckwerts, M.P., Iyuke, S.E., 2009. Design, biometric simulation and optimization of a nano-enabled scaffold device for enhanced delivery of dopamine to the brain. Int. J. Pharm. 382, 277–290. doi:10.1016/j.ijpharm.2009.08.021 Popescu, M.A., Toms, S.A., 2006. In vivo optical imaging using quantum dots for the management of brain tumors. Expert Rev Mol Diagn 6, 879–890. doi:10.1586/14737159.6.6.879 Pöselt, E., Schmidtke, C., Fischer, S., Peldschus, K., Salamon, J., Kloust, H., Tran, H., Pietsch, A., Heine, M., Adam, G., Schumacher, U., Wagener, C., Förster, S., Weller, H., 2012. Tailor-made quantum dot and iron oxide based contrast agents for in vitro and in vivo tumor imaging. ACS Nano 6, 3346–3355. doi:10.1021/nn300365m Rambam Health Care Campus and Technion, I.I. of T., n.d. Exploratory Study to Detect Volatile 36

Biomarkers of Idiopathic Parkinson’s Disease and Parkinsonism From Exhaled Breath Using a Nanomedical Artificial Olfactory System [WWW Document]. URL https://clinicaltrials.gov/ct2/show/NCT01246336 Rao, K.S., Reddy, M.K., Horning, J.L., Labhasetwar, V., 2008. TAT-conjugated nanoparticles for the CNS delivery of anti-HIV drugs. Biomaterials 29, 4429–4438. doi:10.1016/j.biomaterials.2008.08.004 Reibold, M., Paufler, P., Levin, a a, Kochmann, W., Pätzke, N., Meyer, D.C., 2006. Materials: carbon nanotubes in an ancient Damascus sabre. Nature 444, 286. doi:10.1038/444286a Renshaw, P.F., Owen, C.S., McLaughlin, a C., Frey, T.G., Leigh, J.S., 1986. Ferromagnetic contrast agents: a new approach. Magn. Reson. Med. 3, 217–25. doi:10.1002/mrm.1910030205 Robinson, R., Viviano, S.R., Criscione, J.M., Williams, C. a., Jun, L., Tsai, J.C., Lavik, E.B., 2011. Nanospheres delivering the EGFR TKI AG1478 promote optic nerve regeneration: The role of size for intraocular drug delivery. ACS Nano 5, 4392–4400. doi:10.1021/nn103146p Roman, J.A., Niedzielko, T.L., Haddon, R.C., Parpura, V., Floyd, C.L., 2011. Single-walled carbon nanotubes chemically functionalized with polyethylene glycol promote tissue repair in a rat model of spinal cord injury. J. Neurotrauma 28, 2349–62. doi:10.1089/neu.2010.1409 Rozenberg, O.A., Hanson, K.P., Alijakparov, M.T., Davidenkova, E.F., Zerbin, E.A., 1985. [Diagnostic possibilities of the contrast medium verografin in liposomes (animal experiments)]. Radiol. Diagn. (Berl). 26, 285–92. Ryoko Tsukamoto, H.Y., 2013. Quantum Dots Conjugated with Transferrin for Brain Tumor Cell Imaging. J. Cell Sci. Ther. 04. doi:10.4172/2157-7013.1000150 Saito, R., Bringas, J.R., McKnight, T.R., Wendland, M.F., Mamot, C., Drummond, D.C., Kirpotin, D.B., Park, J.W., Berger, M.S., Bankiewicz, K.S., 2004. Distribution of liposomes into brain and rat brain tumor models by convection-enhanced delivery monitored with magnetic resonance imaging. Cancer Res. 64, 2572–9. doi:10.1158/0008-5472.CAN-03-3631 Saito, R., Krauze, M.T., Bringas, J.R., Noble, C., McKnight, T.R., Jackson, P., Wendland, M.F., Mamot, C., Drummond, D.C., Kirpotin, D.B., Hong, K., Berger, M.S., Park, J.W., Bankiewicz, K.S., 2005. Gadolinium-loaded liposomes allow for real-time magnetic resonance imaging of convection-enhanced delivery in the primate brain. Exp. Neurol. 196, 381–9. doi:10.1016/j.expneurol.2005.08.016 SAKONO, M., ZAKO, T., MAEDA, M., 2012. Naked-eye Detection of Amyloid Aggregates Using Gold Nanoparticles Modified with Amyloid Beta Antibody. Anal. Sci. 28, 73. doi:10.2116/analsci.28.73 Salvati, A., Åberg, C., Dawson, K.A., Monopoli, M.P., Åberg, C., Salvati, A., Dawson, K.A., Aberg, C., Salvati, A., Dawson, K.A., 2012. Biomolecular coronas provide the biological identity of nanosized materials. Nat. Nanotechnol. 7, 779–786. doi:10.1038/nnano.2012.207 Sandvig, A., Berry, M., Barrett, L.B., Butt, A., Logan, A., 2004. Myelin-, reactive glia-, and scarderived CNS axon growth inhibitors: expression, receptor signaling, and correlation with axon regeneration. Glia 46, 225–251. doi:10.1002/glia.10315 Sant, W., Pourciel-Gouzy, M.L., Launay, J., Do Conto, T., Colin, R., Martinez, A., Temple-Boyer, P., 2004. Development of a creatinine-sensitive sensor for medical analysis. Sensors Actuators B Chem. 103, 260–264. doi:10.1016/j.snb.2004.04.104 Schmidt, C.E., Leach, J.B., 2003. Neural tissue engineering: strategies for repair and regeneration. Annu. Rev. Biomed. Eng. 5, 293–347. doi:10.1146/annurev.bioeng.5.011303.120731 Schöning, M.J., Poghossian, A., 2002. Recent advances in biologically sensitive field-effect transistors (BioFETs). Analyst. doi:10.1039/b204444g Schültke, E., Menk, R., Pinzer, B., Astolfo, A., Stampanoni, M., Arfelli, F., Harsan, L.-A., Nikkhah, G., 2014. Single-cell resolution in high-resolution synchrotron X-ray CT imaging with gold nanoparticles. J. Synchrotron Radiat. 21, 242–50. doi:10.1107/S1600577513029007 Schwendener, R.A., Wüthrich, R., Duewell, S., Wehrli, E., von Schulthess, G.K., 1990. A pharmacokinetic and MRI study of unilamellar gadolinium-, manganese-, and iron-DTPAstearate liposomes as organ-specific contrast agents. Invest. Radiol. 25, 922–32. Simon, R.H., Ho, S.Y., D’Arrigo, J., Wakefield, A., Hamilton, S.G., 1990. Lipid-coated ultrastable microbubbles as a contrast agent in neurosonography. Invest. Radiol. 25, 1300–4. Simon, R.H., Ho, S.Y., Perkins, C.R., D’Arrigo, J.S., 1992. Quantitative assessment of tumor 37

enhancement by ultrastable lipid-coated microbubbles as a sonographic contrast agent. Invest. Radiol. 27, 29–34. Skaat, H., Corem-Slakmon, E., Grinberg, I., Last, D., Goez, D., Mardor, Y., Margel, S., 2013. Antibody-conjugated, dual-modal, near-infrared fluorescent iron oxide nanoparticles for antiamyloidgenic activity and specific detection of amyloid-?? fibrils. Int. J. Nanomedicine 8, 4063–4076. doi:10.2147/IJN.S52833 Sobue, K., Yamamoto, N., Yoneda, K., Hodgson, M.E., Yamashiro, K., Tsuruoka, N., Tsuda, T., Katsuya, H., Miura, Y., Asai, K., Kato, T., 1999. Induction of blood-brain barrier properties in immortalized bovine brain endothelial cells by astrocytic factors. Neurosci. Res. 35, 155–164. doi:10.1016/S0168-0102(99)00079-6 Soldatkin, a. P., Montoriol, J., Sant, W., Martelet, C., Jaffrezic-Renault, N., 2003. A novel urea sensitive biosensor with extended dynamic range based on recombinant urease and ISFETs. Biosens. Bioelectron. 19, 131–135. doi:10.1016/S0956-5663(03)00175-1 Stark, D.D., Weissleder, R., Elizondo, G., Hahn, P.F., Saini, S., Todd, L.E., Wittenberg, J., Ferrucci, J.T., 1988. Superparamagnetic iron oxide: clinical application as a contrast agent for MR imaging of the liver. Radiology 168, 297–301. doi:10.1148/radiology.168.2.3393649 Suh, W.H., Suslick, K.S., Stucky, G.D., Suh, Y.H., 2009. Nanotechnology, nanotoxicology, and neuroscience. Prog. Neurobiol. doi:10.1016/j.pneurobio.2008.09.009 Sun, C., Veiseh, O., Gunn, J., Fang, C., Hansen, S., Lee, D., Sze, R., Ellenbogen, R.G., Olson, J., Zhang, M., 2008. In vivo MRI detection of gliomas by chlorotoxin-conjugated superparamagnetic nanoprobes. Small 4, 372–379. doi:10.1002/smll.200700784 Sun, M., Gao, Y., Guo, C., Cao, F., Song, Z., Xi, Y., Yu, A., Li, A., Zhai, G., 2010. Enhancement of transport of curcumin to brain in mice by poly(n-butylcyanoacrylate) nanoparticle. J. Nanoparticle Res. 12, 3111–3122. doi:10.1007/s11051-010-9907-4 Teng, Y.D., Lavik, E.B., Qu, X., Park, K.I., Ourednik, J., Zurakowski, D., Langer, R., Snyder, E.Y., 2002. Functional recovery following traumatic spinal cord injury mediated by a unique polymer scaffold seeded with neural stem cells. Proc. Natl. Acad. Sci. U. S. A. 99, 3024–3029. doi:10.1073/pnas.052678899 Tiefenauer, L.X., Kuhne, G., Andres, R.Y., 1993. Antibody Magnetite Nanoparticles - in-Vitro Characterization of a Potential Tumor-Specific Contrast Agent for Magnetic-ResonanceImaging. Bioconjug. Chem. 4, 347–352. Tokuraku, K., Marquardt, M., Ikezu, T., 2009. Real-time imaging and quantification of amyloid-beta peptide aggregates by novel quantum-dot nanoprobes. PLoS One 4, e8492. doi:10.1371/journal.pone.0008492 Torchilin, V., 1996. Liposomes as delivery agents for medical imaging. Mol Med Today 2, 242–249. doi:1357431096888058 [pii] Torchilin, V.P., 1997. Surface-modified liposomes in gamma- and MR-imaging. Adv. Drug Deliv. Rev. 24, 301–313. doi:10.1016/S0169-409X(96)00472-3 Triulzi, R.C., Dai, Q., Zou, J., Leblanc, R.M., Gu, Q., Orbulescu, J., Huo, Q., 2008. Photothermal ablation of amyloid aggregates by gold nanoparticles. Colloids Surfaces B Biointerfaces 63, 200–208. doi:10.1016/j.colsurfb.2007.12.006 Turski, P., Kalinke, T., Strother, L., Perman, W., Scott, G., Kornguth, S., 1988. Magnetic resonance imaging of rabbit brain after intracarotid injection of large multivesicular liposomes containing paramagnetic metals and DTPA. Magn. Reson. Med. 7, 184–196. Tysseling-Mattiace, V.M., Sahni, V., Niece, K.L., Birch, D., Czeisler, C., Fehlings, M.G., Stupp, S.I., Kessler, J. a, 2008. Self-assembling nanofibers inhibit glial scar formation and promote axon elongation after spinal cord injury. J. Neurosci. 28, 3814–23. doi:10.1523/JNEUROSCI.014308.2008 Varallyay, P., Nesbit, G., Muldoon, L.L., Nixon, R.R., Delashaw, J., Cohen, J.I., Petrillo, A., Rink, D., Neuwelt, E. a., 2002. Comparison of two superparamagnetic viral-sized iron oxide particles ferumoxides and ferumoxtran-10 with a gadolinium chelate in imaging intracranial tumors. Am. J. Neuroradiol. 23, 510–519. Veiseh, O., Sun, C., Gunn, J., Kohler, N., Gabikian, P., Lee, D., Bhattarai, N., Ellenbogen, R., Sze, R., Hallahan, A., Olson, J., Zhang, M., 2005. Optical and MRI multifunctional nanoprobe for targeting gliomas. Nano Lett. 5, 1003–1008. doi:10.1021/nl0502569 38

Verkman, A.S., 2002. Aquaporin water channels and endothelial cell function. J. Anat. 200, 617–27. doi:10.1046/j.1469-7580.2002.00058.x Vincent, P. a, Xiao, K., Buckley, K.M., Kowalczyk, A.P., 2004. VE-cadherin: adhesion at arm’s length. Am. J. Physiol. Cell Physiol. 286, C987–97. doi:10.1152/ajpcell.00522.2003 Wang, C., Liu, D., Wang, Z., 2012. Gold nanoparticle based dot-blot immunoassay for sensitively detecting Alzheimer’s disease related β-amyloid peptide. Chem. Commun. 48, 8392. doi:10.1039/c2cc33568a Wang, K., Fishman, H. a, Dai, H., Harris, J.S., 2006. Neural stimulation with a carbon nanotube microelectrode array. Nano Lett. 6, 2043–8. doi:10.1021/nl061241t Wang, W., Itoh, S., Matsuda, A., Aizawa, T., Demura, M., Ichinose, S., Shinomiya, K., Tanaka, J., 2008. Enhanced nerve regeneration through a bilayered chitosan tube: the effect of introduction of glycine spacer into the CYIGSR sequence. J. Biomed. Mater. Res. A 85, 919–28. doi:10.1002/jbm.a.31522 Weinstein, J.S., Varallyay, C.G., Dosa, E., Gahramanov, S., Hamilton, B., Rooney, W.D., Muldoon, L.L., Neuwelt, E. a, 2010. Superparamagnetic iron oxide nanoparticles: diagnostic magnetic resonance imaging and potential therapeutic applications in neurooncology and central nervous system inflammatory pathologies, a review. J. Cereb. Blood Flow Metab. 30, 15–35. doi:10.1038/jcbfm.2009.192 Whitten, P.G., Gestos, A.A., Spinks, G.M., Gilmore, K.J., Wallace, G.G., 2007. Free standing carbon nanotube composite bio-electrodes. J. Biomed. Mater. Res. B. Appl. Biomater. 82, 37–43. doi:10.1002/jbm.b.30702 Williams, D.W., Eugenin, E. a., Calderon, T.M., Berman, J.W., 2012. Monocyte maturation, HIV susceptibility, and transmigration across the blood brain barrier are critical in HIV neuropathogenesis. J. Leukoc. Biol. 91, 401–415. doi:10.1189/jlb.0811394 Williams, L.R., Longo, F.M., Powell, H.C., Lundborg, G., Varon, S., 1983. Spatial-temporal progress of peripheral nerve regeneration within a silicone chamber: parameters for a bioassay. J. Comp. Neurol. 218, 460–470. doi:10.1002/cne.902180409 Wolburg, H., 2007. Blood–Brain Interfaces — from Ontogeny to Artificial Barriers. Wiley-VCH, Weinheim, Germany. Xiao, L., Zhao, D., Chan, W.H., Choi, M.M.F., Li, H.W., 2010. Inhibition of beta 1-40 amyloid fibrillation with N-acetyl-l-cysteine capped quantum dots. Biomaterials 31, 91–98. doi:10.1016/j.biomaterials.2009.09.014 Xie, J., Wang, C.H., 2006. Electrospun micro- and nanofibers for sustained delivery of paclitaxel to treat C6 glioma in vitro. Pharm. Res. 23, 1817–1826. doi:10.1007/s11095-006-9036-z Xin, H., Sha, X., Jiang, X., Zhang, W., Chen, L., Fang, X., 2012. Anti-glioblastoma efficacy and safety of paclitaxel-loading Angiopep-conjugated dual targeting PEG-PCL nanoparticles. Biomaterials 33, 8167–8176. doi:10.1016/j.biomaterials.2012.07.046 Xu, J., Ruifa, M., Ahmer, H., Sing Yian, C., 2014. Nanofibrous nerve conduit-enhanced peripheral nerve regeneration. J. Tissue Eng. Regen. Med. 8, 377–385. doi:10.1002/term.1531 Yan, H., Wang, L., Wang, J., Weng, X., Lei, H., Wang, X., Jiang, L., Zhu, J., Lu, W., Wei, X., Li, C., 2012. Two-order targeted brain tumor imaging by using an optical/paramagnetic nanoprobe across the blood brain barrier. ACS Nano 6, 410–20. doi:10.1021/nn203749v Yang, F., Murugan, R., Wang, S., Ramakrishna, S., 2005. Electrospinning of nano/micro scale poly(L-lactic acid) aligned fibers and their potential in neural tissue engineering. Biomaterials 26, 2603–10. doi:10.1016/j.biomaterials.2004.06.051 Yang, J.A., Johnson, B.J., Wu, S., Woods, W.S., George, J.M., Murphy, C.J., 2013. Study of wildtype α-synuclein binding and orientation on gold nanoparticles. Langmuir 29, 4603–15. doi:10.1021/la400266u Yang, J.A., Lin, W., Woods, W.S., George, J.M., Murphy, C.J., 2014. α-Synuclein’s Adsorption, Conformation, and Orientation on Cationic Gold Nanoparticle Surfaces Seeds Global Conformation Change. J. Phys. Chem. B 118, 3559–3571. doi:10.1021/jp501114h Yanik, M.F., Cinar, H., Cinar, H.N., Chisholm, A.D., Jin, Y., Ben-Yakar, A., 2004. Neurosurgery: functional regeneration after laser axotomy. Nature 432, 822. doi:10.1038/432822a Youn, J., Won, N., Kim, S., Choi, J.H., 2008. Near-Infrared Quantum Dots Imaging in the Mouse Brain, in: Biomedical Optics. OSA, Washington, D.C., p. BSuE2. 39

doi:10.1364/BIOMED.2008.BSuE2 Yu, Z., McKnight, T.E., Ericson, M.N., Melechko, A. V., Simpson, M.L., Morrison, B., 2007. Vertically aligned carbon nanofiber arrays record electrophysiological signals from hippocampal slices. Nano Lett. 7, 2188–2195. doi:10.1021/nl070291a Yue, H.Y., Huang, S., Chang, J., Heo, C., Yao, F., Adhikari, S., Gunes, F., Liu, L.C., Lee, T.H., Oh, E.S., Li, B., Zhang, J.J., Huy, T.Q., Luan, N. Van, Lee, Y.H., 2014. ZnO Nanowire Arrays on 3D Hierachical Graphene Foam: Biomarker Detection of Parkinson’s Disease. ACS Nano 8, 1639–1646. doi:10.1021/nn405961p Yue, K., Guduru, R., Hong, J., Liang, P., Nair, M., Khizroev, S., 2012. Magneto-Electric NanoParticles for Non-Invasive Brain Stimulation. PLoS One 7, 1–5. doi:10.1371/journal.pone.0044040 Zhang, B., Sun, X., Mei, H., Wang, Y., Liao, Z., Chen, J., Zhang, Q., Hu, Y., Pang, Z., Jiang, X., 2013. LDLR-mediated peptide-22-conjugated nanoparticles for dual-targeting therapy of brain glioma. Biomaterials 34, 9171–9182. doi:10.1016/j.biomaterials.2013.08.039 Zhang, C., Wan, X., Zheng, X., Shao, X., Liu, Q., Zhang, Q., Qian, Y., 2014. Dual-functional nanoparticles targeting amyloid plaques in the brains of Alzheimer’s disease mice. Biomaterials 35, 456–465. doi:10.1016/j.biomaterials.2013.09.063 Zhang, H., Yu, M., Xie, L., Jin, L., Yu, Z., 2012. Carbon-Nanofibers-Based Micro-/Nanodevices for Neural-Electrical and Neural-Chemical Interfaces. J. Nanomater. 2012, 1–6. doi:10.1155/2012/280902 Zhang, S., 2003. Fabrication of novel biomaterials through molecular self-assembly. Nat. Biotechnol. 21, 1171–1178. doi:10.1038/nbt874

40

Figures Captions Figure 1: Nano in Neuro – (A) The figure illustrates the nano-scale with respect to the human nervous system. (B) Brief summary of the significant role played by nanotechnology in addressing neurological challenges. Figure 2: Mechanisms of molecular transport across the Blood-Brain-Barrier (BBB). Figure 3: Trans-BBB delivery of nanoparticles inspired by Nature’s Trojan horse – (A) Cell penetrating TAT peptides present in HIV have been used to coat polymeric nanoparticles for efficient delivery into the CNS. Adapted with permission from (Mishra et al., 2011). Copyright, 2011, The National Academy of Sciences of the USA. Adapted with permission from [29]. Copyright, 2008, Elsevier (B) The ability of leukocytes to cross the BBB has been exploited in designing silicon nanoparticles coated with activated monocyte cell membrane. Adapted from (Rao et al., 2008). Copyright 2012 Society for Leukocyte Biology. Reprinted by permission from Macmillan Publishers Ltd: [Nature Nanotechnology] (Parodi et al., 2013), copyright (2012). Figure 4: Receptor mediated endocytosis of nanoparticles – Glucose coated gold nanoparticles were able to cross the BBB binding to GLUT-1 transporter protein present in brain endothelial cells. Adapted with permission from (Gromnicova et al., 2013). Copyright, 2013, Gromnicova et al. Figure 5: Gold nanoparticle imaging and radiotherapy of brain tumors in mice. (i) Irradiation and tumor dissection (A) Irradiation setup showing mouse with a lead collimator. (B) Mouse brain and glioma at necropsy 10 days after tumor initiation and 4 h after gold nanoparticle intravenous injection (4 g Au/kg). The tumor (curved arrow) was removed (place of removal: straight arrow). The removed tumor was black compared with normal brain tissue due to gold nanoparticle uptake. (ii) Microcomputed tomography imaging of brain tumors after intravenous gold nanoparticle injection (A–C) Live mouse microcomputed tomography images of brain tumors 9 days postimplantation and 15 h after intravenous (iv.) gold nanoparticle (AuNP) injection. (A & B) Same mouse (A) before and (B) 15 h after iv. injection (4 g Au/kg). (C) Larger tumor imaged after iv. injection of 1.7 g Au/kg. (D–F) Live mouse microcomputed tomography images of a typical brain tumor 9 days postimplantation and 1 h after iv. AuNP injection (1.7 g Au/kg). (D) Individual blood vessels in the tumor could be discerned. (E) Focal spots of gold were also observed (arrows). (F) AuNP leakage was very irregular in some vessels (arrows). Adapted from (Hainfeld et al., 2013). Copyright 2013, Future Medicine. Figure 6: ZnO nanowires arrays on 3D grapheme foam (NWA/GF) for detection of Parkinson’s disease (i) Structural analysis of the integrated ZnO NWA/GF. (a) Schematic of the ZnO NWA/GF electrode and detection of UA, DA, and AA. (b-e) SEM images of the ZnO NWAs on the 3D GF at different magnifications. Inset: EDX of the ZnO NWAs. (f) SEM images of the height of the ZnO NWAs, ∼2 μm. Inset: diameter of the ZnO NWAs, ∼40 nm. (ii) Electrochemical cell. (a) Schematic of three-electrode electrochemical cell setup. The ZnO NWA/GF on the ITO glass served as the working electrode (WE), coupled 41

with a platinum as the counter electrode (CE) and an Ag/AgCl (sat. KCl) as the reference electrode (RE). (b) Photograph of the electrochemical cell used in the experiment. (Adapted with permission from (Yue et al., 2014). Copyright 2014, American Chemical Society. Figure 7: Poly(ε-caprolactone-co-ethyl ethylene phosphate) (PCLEEP) and aligned GDNF-PCLEEP electrospun nanofiber based conduits. (i) Cross-sectional views of nerve conduits with aligned electrospun fibers (EF), a) Longitudinally aligned (EF-L) and b) circumferentially aligned (EF-C). Insets: higher-magnification views of the cross sections. c) Aligned PCLEEP fibers in nerve guide conduits, GDNF-encapsulated fiber diameter, φ=(3.96 ±0.14) μm and plain PCLEEP fiber φ= (5.08 ± 0.05) μm. d) Inner surface of an empty nerve guide conduit.(ii) TEM images of cross sections of regenerated sciatic nerve, 8–10 mm from the proximal end of nerve conduits. a) In the control group, showing the absence of myelinated axons and the presence of fibrous tissues. b) In the EF-L group, showing the tendency of myelinated axons regenerating in close proximity to PCLEEP fibers (circled). c) In the EF-L-GDNF group, demonstrating the presence of a large number of myelinated axons. (Adapted with permission from Chew et al., 2007). Copyright 2007, John Wiley and Sons. Figure 8: Self-Assembling Peptide Nanofiber Scaffold (SAPNS) mediated repair for the animal brain. (i) (a) Molecular model of the RADA16-I molecular building block. (b) Molecular model of numerous RADA16-I molecules undergo self assembly to form well ordered nanofibers with the hydrophobic alanine sandwich inside and hydrophilic residues on the outside. (c) The SAPNS is examined by using scanning electron microscopy. (Scale bar, 500 nm.) (ii) SAPNS allows axons to regenerate through the lesion site in brain. The darkfield composite photos are parasagittal sections from animals 30 days after lesion and treatment. (a) Section from brain of 30-day-old hamster with 10 μl of saline injected in the lesion at P2. The cavity shows the failure of the tissue healing. The retinal projections, in light green at the top left edge of the cavity, have stopped and did not cross the lesion. Arrows indicate path and extent of knife cut. (b) A similar section from a 30-day-old hamster with a P2 lesion injected with 10μl of 1%SAPNS. The site of the lesion has healed, and axons have grown through the treated area and reached the caudal part of the superior colliculus (SC). Axons from the retina are indicated by light-green fluorescence. The boxed area is an area of dense termination of axons that have crossed the lesion. Arrows indicate path and extent of knife cut. (c) Enlarged view of boxed area in b. The regrown axons, shown in white, were traced with cholera-toxin fragment B labeling by using immunohistochemistry for amplification of the tracer. (iii) Optic tract regeneration and functional return of vision. This SAPNStreated adult animal turns toward the stimulus in the affected right visual field in small steps, prolonged here by movements of the stimulus away from the animal. Each frame is taken from a single turning movement, at times 0.00 (a), 0.27 (b), 0.53 (c), and 0.80 (d) sec from movement initiation. The animal reached the stimulus in the last frame. This is 29% slower than most turns by a normal animal. The recording was made 6 weeks after surgery and treatment when the animal started to show a response.( Adapted with permission from (Ellis-Behnke et al., 2006)). Copyright, 2006, The National Academy of Sciences of the USA. 42

Figure 9: Nano-electronics in neuroscience. (A) Neural interface using vertically aligned multiwalled carbon nanotube (CNT) pillars as microelectrodes; Adapted with permission from (Wang et al., 2006). Copyright, 2006, American Chemical Society. (B) PC12 neural cell (green) spanning collagen-coated carbon nanofibers (CNF) microbundles; Adapted with permission from (Nguyen-Vu et al., 2007). Copyright 2007, John Wiley and Sons. (C) Wellspread non-neuronal cell penetrated by numerous nanowires. C (i) Note bending of the nanowires (white arrow heads). C (ii) Underside of cell body (mechically flipped over at rinsing) penetrated by nanowires. Note membrane adhesion to the nanowires (arrows). Adapted with permission from (Hällström et al., 2007). Copyright 2007, John Wiley and Sons. (D) Schematic of coupling between HgTe nanoparticle and a neuron and (E) Electron microscopy images of one of the cells grown on LBL layers in the early stages of differentiation. Adapted with permission from (Pappas et al., 2007). Copyright 2007, American Chemical Society (F) Schematic representation of the photoactive electrode preparation. Nanorod (NR) conjugation onto a CNT film is based on covalent binding enabled by a ppAA coating of the CNTs. Light is absorbed by the film, followed by charge separation at the NR−CNT interface which elicits a neuronal response. Adapted with permission from (Bareket et al., 2014). Copyright 2014, American Chemical Society.

43

Figure 1

44

Figure 2

45

Figure 3

46

Figure 4

47

Figure 5

48

Figure 6

49

Figure 7

50

Figure 8

Figure 9

51

Tables Table 1: Bio-inspired modifications of nanomaterials for neuro-imaging.

Nanomaterial Magnetic nanoparticles

Bio-inspired modifications USPIOs conjugated with HIV-Tat peptide and dextran to facilitate crossing the BBB and enhance biocompatibility USPIOs conjugated with Tat + Dextran+ Cy5.5 probe for intraoperative imaging of brain tumors Conjugating USPIOs with scorpion venom derived peptide Chlorotoxin to glioma specificity. SPIONs conjugated with Transferrin (bind with the Transferrin receptors over-expressed in glioma cells) for contrast enhancement in MRI. Increased ferritin levels in Parkinson’s patients led to use of USPIOs to estimate CBV for diagnosis of neurological disorders through fMRI.

Liposomes

Air filled bubbles are efficient reflectors of sound. Lipid coated micro-bubbles (liposomes) have been used as contrast agents for rapid detection of tumors through neurosonography.

Quantum dots

QDs were incorporated within the core of PEG-polylactic acid (PLA) nanoparticles conjugated with Lectin protein to enhance uptake by lymphocytes and aid in transport across BBB QDs conjugated with transferrin since tumor cells highly overexpressed transferrin receptor.

References (Josephson et al., 1999) (Kircher et al., 2003) (Sun et al., 2008; Veiseh et al., 2005) (Jiang et al., 2012) (Gorell et al., 1995; Kim et al., 2013) (D’Arrigo et al., 1991; Simon et al., 1992, 1990) (Gao et al., 2008)

(Ryoko Tsukamoto, 2013)

Gold nanoparticles

Exploiting the natural tendency of elements with higher atomic number to efficiently absorb X-Rays, GNPs have been used as contrast agents.

(Hainfeld et al., 2006, 2004)

52

Table 2: Bio-inspired modifications of nanomaterials for neuro-therapy.

Disease

Nanomaterial

Glioblastoma

Polymeric

multiforme

nanoparticles

Alzheimer’s disease Parkinson’s disease

Polymeric nanoparticles

Polymeric nanoparticles

Dendrimer

Bio-inspired modifications

References

Coated with IL-13 peptide (binding with glioma specific IL13Rα-2 receptor) and RGD peptide (binding with αvβ3 which is overexpressed on neovascular endothelial cells) Coated with lactoferrin (lactoferrin receptor is overexpressed in brain endothelial cells and glioma cells). Coated with peptide-22 (special binding efficiency with LDL receptor highly expressed at the BBB) Coated with angio-pep-2, a specific ligand of low density lipoprotein receptor related protein (LRP) which is over-expressed on BBB and glioma cells Coated with aptamers specific for nucleolin which is over-expressed in both cancer cells and tumor vasculature Metal ions (like Cu2+, Fe3+ and Zn2+) interact with and stabilize Aβ fibrils. Hence coating with Cuchelator like D-penicillamine have shown to resolubilize Aβ1-42 aggregates Brain specific delivery of corticotrophins like urocortin and genes encoding growth factors like GDNF through nanoparticles conjugated with lactoferrin. Conjugated with Angiopep to transport human GDNF genes into the affected areas of the brain for recovery of dopaminergic neurons.

(Gao et al., 2014)

(Miao et al., 2014) (Zhang et al., 2013) (Xin et al., 2012)

(Guo et al., 2011) (Cui et al., 2005)

(Hu et al., 2011)

(Huang et al., 2013)

53

Table 3: Nanomaterials and fabrication methods used in neuro-regeneration Fabrication method

Target tissue

References

Poly(lactic-co-glycolic acid) (PLGA)/Poly(ε-caprolactone) (PCL)

Electrospinning

Rat sciatic nerve

(Panseri et al., 2008)

Copolymer of caprolactone and ethyl ethylene phosphate (PCLEEP) with GDNF Poly(acrylonitrile-co-methylacrylate) (PAN-MA)

Electrospinning

Rat peripheral nerve

(Chew et al., 2007)

Electrospinning

Rat tibial nerve lesion

(Yang et al., 2005)

Poly(ε-caprolactone) (PCL) Chitosan

Electrospinning Electrospinning

Rat sciatic nerve Rat sciatic nerve

Polyamide nanofibers bound with tenascin-C Single-walled carbon nanotubes functionalized with polyethylene glycol (SWNT-PEG) PLGA nanospheres

Electrospinning

Rat spinal cord

Purchased

Rat spinal cord

(Xu et al., 2014) (Wang et al., 2008) (Meiners et al., 2007) (Roman et al., 2011)

oil-in-water with cosolvent single emulsion solvent evaporation technique Self assembly

Rat optic nerve

(Robinson et al., 2011)

Mice spinal cord injury

(TysselingMattiace et al., 2008) (Holmes et al., 2000)

Material used

IKVAV (isolucine-lysine-valinealanine-valine) peptide Peptides of Arginine–Alanine– Aspartate (RAD)16-I and RAD16-II

Self assembly

primary neurons and neuronal cell lines (PC12)

54