Nanomedicine: Nanotechnology, Biology, and Medicine11 (2015) 1467–1479
Review Article
Nanometric agents in the service of neuroscience: Manipulation ofneuronal growth and activity using nanoparticles
Pazit Polak, PhD, Orit Shefi, PhD⁎
Engineering, Bar Ilan University, Ramat-Gan, IsraelThe Bar-Ilan Institute of Nanotechnology & Advanced Materials, Bar Ilan University, Ramat-Gan, Israel
Received 7 January 2015; accepted 15 March 2015
nanomedjournal.com
Abstract
Nerve regeneration and recovery could provide great therapeutic benefits for individuals suffering from nerve damage post trauma ordegenerative diseases. However, manipulation of nerves presents a huge challenge for neuroscientists and is not yet clinically feasible. In recentyears, nanoparticles have emerged as novel effective agents for control of neuronal growth and behavior. Nanoparticles may facilitate the needednerve manipulation abilities for therapeutic and diagnostic purposes including within the brain. This review aims at presenting the currentlyavailable literature regarding the interactions between inorganic nanoparticles and neurons. A wide range of nanoparticles are presented, includinggold, iron oxide, cerium oxide, nanotubes and quantum-dots. The nanoparticles enhance neuronal differentiation and survival, direct growth andregulate electrical activity. The studies are summarized in a concise table, arranged by the function and type of nanoparticle. The latest studiespresent a novel interdisciplinary approach, which could be harnessed for clinical applications in nanomedicine.
Nanoparticles are materials with a basic structural unit thathas at least one dimension smaller than 100 nm in length. Due totheir small size, nanoparticles can interact with and affect cellsand tissues at the molecular level. In recent years, nanoparticleshave emerged as a novel effective tool for manipulation ofneuronal behavior, growth and differentiation.1 Control ofneuronal recovery could provide great therapeutic benefits forindividuals suffering from nerve damage post trauma ordegenerative diseases. In the US alone, 250,000-400,000 patientssuffer from a spinal cord injury,2 and 1.4 million sustaintraumatic brain injury3 each year. However, manipulation ofnerve cells, which possess unique complex morphology andelectrical activity, presents a huge challenge for neuroscientists,especially within the central nervous system, and is not yetclinically feasible. Nanoparticles may facilitate the needed nervemanipulation for therapeutic as well as diagnostic purposes.
Please cite this article as: Polak P, Shefi O, Nanometric agents in the servinanoparticles. Nanomedicine: NBM 2015;11:1467-1479, http://dx.doi.org/10.10
Nanoparticles have different characteristics, i.e. the materialthey are made of, their size, shape, electric charge, magnetic andoptical properties. Moreover, nanoparticles can be modified byconjugation of reactive functional groups and cargos. Thesecharacteristics determine the nature of the interactions betweenthe nanoparticles and cells, such as the ability of nanoparticles tobind or penetrate into cells, or to affect biochemical reactions.The nature of the interactions determines the cellular response tothe nanoparticles, as manifested by modifications of cellularmorphology, activity or differentiation.
Nanoparticles can have cytotoxic effects,4,5 likely becausethey induce the formation of reactive oxygen species that causeoxidative stress.6,7 It is important to note, however, that there aresubstantial difficulties in assessing the toxicity of nanomaterialswhen interacting with biosystems, due to the lack of clearcharacterization of the materials when challenged in biologicalstudies.8,9 In the case of cationic nanoparticles, cytotoxicity isfurther enhanced by their ability to induce nanoscale disruptionsin the plasma membranes of cells.10-15 Nanoparticle coatings canalso have cytotoxic effects, e.g. polydimethylamine, a frequentlyused coating for nanoparticles in biomedical applications, wasfound to induce cell death in cortical neurons isolated from chickembryos, by removal of the plasma membrane.16
ce of neuroscience: Manipulation of neuronal growth and activity using16/j.nano.2015.03.005
Table 1The table summarizes the most essential parameters of the studies presented in this review, categorized according to activity, type of nanoparticle, coating, use,type of activity, and toxicity.
Activity Type of nanoparticle(average dry sizein nm)
Functional coating Use Active or mediator? Toxicity? Ref(s)
Differentiationand survival
Quantum dots(15-20)
Conjugated to NGF Labeling NGF for intracellulartracking, understanding the processof differentiation
Mediator No 40
Iron oxide (11) Enhance differentiation of PC12 cells Active Yes (at highconcentration)
41
Gold nanorods(48.6 × 13.8)
Coated withpoly(4-styrenesulfonicacid) or SiO2
Enhance differentiation ofNG108-15 cells
Active No 42
Iron oxide (23) Conjugated to NGF Stabilize NGF and thereby enhanceneuronal differentiation
Gold (5/40) Enhance electrical excitability of neurons Active No 95
Manganeseferrite (6)
Induce electrical activity in cultured neuronsexpressing a temperature sensitive ionchannel, via radio frequency magneticfield heating
Mediator No 102
Carbon nanotubes(film of 50-70)
Enhance electrical excitability of neurons Active No 103
Copper oxide(10-70)
Inhibit electrical excitability of hippocampalneurons
Active No 96
Silver (5, 50-100) Inhibit electrical excitability of neurons Active No 98,99
Carbon black (55) Inhibit electrical excitability of primarymurine cortical networks of neurons andglia cells
Active No 100
Iron oxide (b100) Inhibit electrical excitability of primarymurine cortical networks of neurons andglia cells
Active No 100
Titanium oxide(b100)
Inhibit electrical excitability of primarymurine cortical networks of neurons andglia cells
Active Yes (generatereactive oxygenspecies)
100
Blood brain barrier Gold (30) Conjugated to insulin Cross the blood brain barrier, can bedetected by CT
Active (CT target) andmediator (carrier ofligand for crossingthe blood brain barrier)
No 109
Imaging andtheranostics
Gold (50) Single cell resolution CT imaging ofglioma tumors in rats
Active No 119
Iron oxide (15) Conjugated to afluorescent dye and anantibody againstamyloid-β peptides
Inhibit in-vitro amyloid aggregate formationin PC12 cells. Dye can be detected by MRI
Mediator No 121
Quantum dots(15-20)
Conjugated to BDNFor NGF
Intracellular tracking of single molecules Mediator No 126-128
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Figure 1. Reprinted from Kim et al,41 with permission from Elsevier. (A) TEM image of iron oxide nanoparticles used for neurite outgrowth. Particle size was11 nm. (B) Effect of iron oxide nanoparticles on differentiation efficiency as a function of the dose (5-40 μg/ml) and time (1, 3, and 5 days after the induction ofdifferentiation).
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Massive research has been performed studying the interactionsbetween biological tissue and nanometric elements. Here wefocus on neuronal manipulations and imaging via several typesof inorganic nanoparticles. Many of the reviewed studies involvegold nanoparticles which are highly suited for biomedicalapplications.17-21 We review the literature presenting the effectson neuronal growth and differentiation, survival and regener-ation, activity, drug delivery and imaging. We discusspotential chemical and molecular mechanisms behind theeffects of nanoparticles. The presented studies range frombasic to applicative research. The presented studies aresummarized in a concise table (Table 1), according to thefunction, type of nanoparticle and other characteristics.
Nanoparticles enhance neuronal differentiation?
Neuronal differentiation is well-studied. It involves outgrowthof neuronal branches (neurites) out of the cell body. The neuriteselongate, bifurcate and connect to neighbor cells forming afunctional neural network. Typically, one of the neuritesdifferentiates into an axon, the fiber through which electricalimpulses travel away from the neuron's body to other cells. Theother neurites either differentiate into dendrites, sites at whichinformation is received from other neurons, or fail to becomefunctional and retract. Different parameters have been shown toinduce and affect neurite outgrowth and elongation. These includemolecular cues such as growth factors, neurotransmitters, interac-tion with glial cells or extracellular matrix proteins, topographicalcues, or mechanical tensile forces.22-30 A commonly used, wellcharacterized model for neuronal differentiation is the PC12 cellline, derived from rat adrenal medulla. When exposed to nervegrowth factor (NGF) in vitro, PC12 cells demonstrate a typicalneuronal behavior: they stop proliferation, extend branchingprocesses, become electrically excitable,31,32 express sodiumchannels,33,34 and show changes in cellular composition associated
with neuronal differentiation.35 During this differentiation process,NGF is internalized into the cells via endosomes, from which asignaling cascade is initiated leading to expression of typicalneuronal markers, e.g. GAP4336,37 and b3-tubulin.38,39 Other cellsfrom neuronal lineages also respond to the presence of NGF byproducing neurites.
Nanometric elements have been utilized in studying themechanisms of neuronal differentiation. For example, Cui et alused NGF conjugated to quantum-dots to track the movement ofthe factor in real time in compartmentalized cultures of rat neurons,at the single molecule level.40 Surprisingly, they found that themajority of nerve growth factor-containing endosomes containedonly a single dimer, indicating that a single nerve growth factordimer is sufficient to sustain signaling. Moreover, several attemptshave been made to develop nanoparticle-based treatmentsto promote neuronal differentiation. Park and co-authorsdemonstrated that iron oxide nanoparticles (Figure 1, A) taken upbyPC12 cells enhanced neurite outgrowth, both in terms of percentof neurite-bearing cells and in terms of neurite length.41 The effectwas synergistic with NGF, and occurred in a dose-dependentmanner, up to a point of neurotoxicity (Figure 1, B). In anotherstudy, McArthur and colleagues showed that culturing of theNG108-15 neuronal cell line with gold nanorods that were coatedwith either poly(4-styrenesulfonic acid) or SiO2, resulted in a20-25% increase in differentiation, i.e., the percentage of neuronswith neurites. There was no effect for culturing the cells withuncoated gold nanorods.42
How can nanoparticles affect differentiation? McArthur andcolleagues hypothesized that the nanoparticles trigger the activationof one or more transcription factors which in turn is responsible forthe increased percentage of neurons with neurites.42 Thishypothesis is supported by previous studies on iron oxidenanoparticles. Park and colleagues performed gene expressionanalysis, and found extensive changes in response to the uptake ofiron oxide nanoparticles.41 Particularly, the authors observedchanges in genes related to the cytoskeleton, signaling molecules,
Figure 2. Reprinted fromMarcus et al,56 with permission of The Royal Society of Chemistry. (A) Schematic illustration of NGF-NPs synthesis. (B) SEM imageof PC12 cells three days after induction of differentiation by NGF-NPs. Scale bar = 100 μm. Inset: Fluorescence confocal microscopy image (a single focalplane) illustrating the internalization of NGF-NPs into PC12 cells. NPs are rhodamin labeled (red). Scale bar = 10 μm. (C and D) Effect of NGF-NPs onmorphological parameters of neuronal differentiation at different NGF concentrations, comparing between the three treatments: free NGF (gray), non-conjugatedNPs with free NGF (light blue) and NGF-NPs (orange). (C) Total neurite length per cell. (D) Number of branching points. ANOVA test, ⁎⁎P b 0.01 and⁎⁎⁎P b 0.001.
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receptors for growth hormones and ion channels, all required andexpected to occur during neuronal differentiation. In general, little isknown about the influence of iron oxide nanoparticles onintracellular events. One study reported changes in the expressionof various genes in human fibroblasts in response to iron oxidenanoparticles.43 As with neurons, many of the genes whoseexpression changed in fibroblasts exposed to iron oxide nanopar-ticles, were associated to the cytoskeleton, signaling moleculesrelated to cell movement and cell interactions such as integrins,tyrosine kinases and protein kinase C family members, receptorsfor growth hormones and ion channels, and Ras related proteins.In addition, increases in the levels of expression of genes relatedto cellular rearrangements such as procollagen, collagen, laminin,elastin, and matrix matalloproteinases were reported. One explana-tion for the effect on gene expression is that several inorganicparticles, including iron oxide nanoparticles, have the potential torelease inorganic ions. Metal ions can modulate cell attachmentand affect neuronal differentiation: iron,44 manganese,45,46 cobalt,47
and aluminum.48 In the case of manganese, the ions play animportant role in neuronal differentiation by activating cell
adhesion molecules,49-51 which interact with the extracellularmatrix and direct cell binding and signaling. Neuronal differen-tiation is known to be influenced by the amount and subcellulardistribution of integrin clusters, i.e. cell adhesion.52,53 However,the mechanism by which metal ions affect gene expression is stillunclear. Several hypotheses have been suggested: metal ions mayaffect gene expression via induction of reactive oxygen speciesformation,54 they may directly affect NGF,55 or they may functionas enzyme cofactors.
We have taken a complementary approach, combining theiron oxide nanoparticles effect with the differentiation factor. Wehave covalently conjugated NGF to iron oxide nanoparticles(Figure 2, A), which resulted in slower degradation than freeNGF.56 Treatment of PC12 cells with conjugated NGFsignificantly promoted neurite outgrowth and increased thecomplexity of the neuronal branching trees, compared to freeNGF at the same concentration (Figure 2, B-D).
In addition to simply inducing neuronal differentiation,several groups have reported active manipulations of neuronaldifferentiation, using external forces. Menciassi and coauthors57
Figure 3. Reprinted from Riggio et al,82 with permission from Elsevier. Nerve regeneration mediated by magnetic nanoparticles (MNPs). MNPs bind to theinjured nerve, a magnetic field is thus applied. MNPs create a mechanical tension which stimulates nerve regeneration in the direction imposed by the magneticforce. This physical guidance directs more efficiently the regeneration of the injured nerve from the proximal toward the distal stump.
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used piezoelectric properties of boron nitride nanotubes, which arestructurally analogous to carbon nanotubes but possess superiormechanical, chemical, and electrical properties.58-61 These solubleboron nitride nanotubes were incubated with PC12 or SH-SY5Ycells, and internalized into cytoplasmic vesicles by the cells. Thecells proliferated normally and did not show signs of oxidative stressor apoptosis. Ultrasounds were used to impart mechanical stress tothe nanoparticles. By virtue of their piezoelectric properties, thenanoparticles polarized and conveyed electrical stimuli of severaltens of nA to the cells. As a result of the electric stimulation, both celltypes exhibited greater neurite sprout compared to control cultures.The electric stimulation did not accelerate the differentiationprocess; however the number of neurites per differentiated cellwas higher in the cultures that underwent stimulation. Moreover,cells treated with nanoparticles and ultrasound had neurites withapproximately double the length compared to control cells.Surprisingly, the NGF receptor TrkA was not involved in theelectric stimulation-induced boost in neurite number and length. Theeffect was mediated, at least in part, by calcium channels, asinhibition of calcium channels reduced the number and length ofneurites in electrically stimulated neurons almost to control levels.
In another study, Park and coauthors62 delivered electricalstimulation to PC12 cells via gold nanoparticles attached to asurface of cover glass. A high amount of cell death (more than30%) was observed with constant current stimulation; however,switching to an alternating current resulted in good cell viability.The alternate current electrical stimulation caused a ~6 foldincrease in neurite length compared to unstimulated cultures, aswell as increased expression of differentiation markers. Convec-tion of electrical stimulation, without nanoparticles, has previouslybeen proven as effective also for nerve regeneration, although theexact mechanism is unknown.63-73
Nanoparticles enhance neuronal regeneration and survival
Nerve regeneration following injury is a great challenge forneuroscientists and neurologists. Full recovery of severe neuralinjuries is rare.74 Efforts in the past focused on biochemical cues toexpedite and guide axons, but these are not feasible to deliverinvasively for therapeutic purposes. Recent advances in nanotech-nology pose magnetic nanoparticles as advantageous candidatesfor remotely-guided aiding of neuronal regeneration. Cellularmechanical tension has long been known as a major effector in thedevelopment and morphogenesis of neurons.75-78 Thus, it washypothesized that magnetic nanoparticles incorporated into cells andremotely-guided by magnetic fields could apply tensile forces thatwould stretch neuronal membranes, initiate and elongate axons, andguide them toward their target. Such method would be accurate andminimally invasive. Iron oxide nanoparticles (Fe2O3 or Fe3O4) havethe necessarymagnetic properties and have previously been used forpositioning non-neuronal cells in response to an external magneticfield.79,80 Riggio et al81 succeeded in causing SH-SY5Y andprimary Schwann cell cultures, loadedwith iron oxide nanoparticles,to migrate toward predefined directions without toxic effect on thecells. In a subsequent study,82 they again used magnetic iron oxidenanoparticles, this time carrying NGF in order to trigger neuronaldifferentiation (Figure 3). The nanoparticles were incorporatedinto PC12 cells by endocytosis, and a magnetic field wasapplied. The nanoparticles showed no cytotoxicity, andinduced neurite sprout. Neurite growth was preferentiallyaligned to the direction of the magnetic force (Figure 4).
Our lab is also devoting efforts to finding neuronal regenerativematerials. In a recent study,83 we grew SH-SY5Y cells on surfacescoated with silver nanoparticles (Figure 5), and studied the effecton the development of neurites during initiation and elongation
Figure 4. Reprinted from Riggio et al,82 with permission from Elsevier. Effect of f-MNPs and magnetic field on the orientation of neuronal processes of PC12cells. Four experimental groups were tested: cells treated with both f-MNPs and the magnetic field (f-MNP+, M+), with f-MNPs and a null magnetic field(f-MNP+, M−), without f-MNPs and the magnetic field (f-MNP−, M+) or without f-MNPs and a null magnetic field (f-MNP−, M−). (A) Neurite orientationindex: the box-plot shows the median, interquartile ranges, the max and the minimum value. Dunnett's test ⁎⁎⁎P b 0.001 n = 6. (B) Number of neurites(normalized with respect to the control: f-MNP−, M−) grouped for classes of angles they form with the radial direction outwards. Left panel: Flux density Bdistribution inside the magnetic applicator, calculated from finite element simulation of the NdFeB magnet array. Right panel: graphs of tangential flux density B(T) and derivative dB/dr (Tm−1) along the radial direction inside the applicator. The dotted line is an average value for dB/dr estimated in 46.5 Tm−1.
Figure 5. Reprinted from Alon et al,83 under a Creative Commons Attribution – Non Commercial license. HRSEM images demonstrate the effect of silvernanoparticles on neurite outgrowth. (A) A typical cell grown on silver nanoparticles-coated substrate grows highly straightened neurites in comparison to (B) acell grown on an uncoated control substrate. (C) Neurites of a cell grown on the silver nanoparticles-coated substrate emerging from the cell body and attachingto the silver nanoparticles. (D) The zoomed-in image of (C) reveals nanoscale extensions of the neurites attached to the silver nanoparticles (white arrows).
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growth phases. We found that silver nanoparticles enhancedneurite initiation three-fold compared to cells grown on uncoatedsubstrates, gold or zinc oxide nanoparticles, and two-fold comparedto cells grown on substrates sputtered with a homogenous layer ofsilver. Neurite elongation was also enhanced on silver nanoparti-cles. We showed that the silver nanoparticles were not toxic to thecells, confirming recent work byYen et al84 andRagaseema et al.85
Our results combined with the well-known antibacterial effect of
silver nanoparticles,86-88 suggest the use of silver nanoparticles asan attractive nanomaterial for neuronal repair platforms. Thisnotion is also supported by a previous study which showed that therelease of antibacterial peptides promotes regeneration in an injuredcentral nervous system of a medicinal leech.89
Another approach to increase neural regeneration, taken bythe Margel lab, used conjugation to iron oxide nanoparticles tostabilize and thereby enhance the potency of basic fibroblast
Figure 6. Reprinted from Jung et al,95 under a Creative Commons Attribution license. (A) The representative traces of spontaneous firing from goldnanoparticles-treated and non-treated (No AuNPs) hippocampal CA1 neurons. (B) AuNPs of both sizes significantly increased the rate of spontaneous firing.⁎⁎P b 0.01, Student's t-test, No AuNPs vs. 40-nm AuNPs; ##P b 0.01, Student's t-test, No AuNPs vs. 5-nm AuNPs.
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growth factor.90 This growth factor has high therapeuticpotential for repair and regeneration of damaged tissuesincluding nerve tissue,91 but it has a very short in vivo halflife, only 3-10 minutes.92 Incubation of nasal olfactory mucosacells with growth factor-conjugated nanoparticles resulted insignificantly improved cell outgrowth compared to the sameconcentration, or even five times higher, of the free factor.
Neuronal survival and neuroprotection are also affected byinteraction with nanoparticles. Exposure to cerium oxidenanoparticles enhanced survival of cells isolated from rat spinalcord93: half of the cells received a single dose of nanoparticles atthe time of plating, and cell viability was quantified on days 15and 30. Live-dead cell assays indicated higher cell survival andlower cell death at both time points in nanoparticle-treatedcultures compared to the control cultures. The nanoparticles didnot have cytotoxic effects on the treated cells, as indicated by thepresence of voltage dependent inward and outward currents andaction potentials, similar to that observed for the controls. Theauthors hypothesized that the presence of mixed valence states ofCe3+ and Ce4+ on the surface of the nanoparticles acted as ananti-oxidant that allowed the nanoparticles to scavenge freeradicals from the culture system, thereby promoting cell survival.
Nanoparticles for manipulation of neuronal electrical activity
Since metallic nanoparticles have electric conductivityproperties and are capable of interacting with neurons, a topicof great interest in current research is whether and how theseelectrical properties can affect the electric activity of neurons.Several groups have tested the effects of nanoparticles on theamplitude and time course of the sodium and potassium currentin hippocampal neurons by using the patch–clamp method, with
mixed findings suggesting that some types of nanoparticlesenhance neuronal activity and others inhibit it.
The Zhou group94 showed that when rat hippocampalneurons were incubated with zinc oxide nanoparticles, sodiumand potassium current amplitudes were enhanced. This occurredvia increasing the opening number of sodium channels anddelaying rectifier potassium channels, leading to accumulation ofintracellular sodium and loss of potassium and therebyenhancing the excitability of the neurons. The Jeon groupfound that intracellular gold nanoparticles increased the numberof action potentials, and the intensity of the effect was positivelycorrelated with the size of the nanoparticles (Figure 6).95
However, in another study by the Zhou group,96 copper oxidenanoparticles inhibited delayed rectifier potassium currents. Thisresult is in agreement with a previous study that demonstrated theeffect of free copper on hippocampal neuronal voltage-gatedA-type potassium currents, and found that the currents wereinhibited in the presence of copper. The inhibition wasreversible, time- and dose-dependent.97
Silver nanoparticles also inhibit neuronal activity, bydecreasing the amplitude of sodium currents.98,99 The effect ofthe nanoparticles was not on the voltage sensors, but rather amechanical effect on the ion–channels: the channel conductivitywas decreased, and fewer channels reached an open state.98
Carbon black, iron oxide and titanium oxide nanoparticlesalso inhibit neuronal activity.100 Gramowski et al studied theacute electrophysiological effects of these nanoparticles onprimary murine cortical networks of neurons and glia cells grownon microelectrode array neurochips. Nanoparticles were taken upby the cells, and in response both the number and the frequencyof action potentials decreased in the presence of nanoparticles.This occurred even at very low nanoparticle concentration downto 1 ng/cm2. Carbon nanoparticles were the most destructive for
Figure 7. Reprinted from Schültke et al,119 with permission from the International Union of Crystallography. (A) Local tomography of a part of the primarytumor, synchrotron-based CT. (B) Segmented (a) and reconstructed (b) image of individual gold-loaded cells. Diameters of the cell bodies are between 8 and10 μm. (c) Reconstructed image of GNP-loaded cells in vitro. (C)High-resolution coronal T2
⁎-weighted MR images of the rat brain. Subsequent high-resolutionaxial slices (resolution 60 μm × 60 μm × 60 μm) showing the main bulk of the tumor in the right hemisphere as a dark void caused by the gold nanoparticlesand the small hemorrhages typical for a high grade glioma. A coronal slice of the CT scout image was overlaid with a corresponding MRI slice.
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spike rate and burst rate, followed by iron and titanium. Theresearchers noticed a surprising, unexplained biphasic behaviorof the carbon nanoparticles: at very high concentrations, thesenanoparticles induced an increase in neuronal activity, resem-bling the electrical activity seen in epileptic seizures.101 Reactiveoxygen species were generated only in cells that took up thetitanium nanoparticles, but not iron or carbon nanoparticles, andtherefore reactive oxygen species cannot explain the observedneurotoxic effects.
In addition to studying the effects of nanoparticles onneurons, much effort is devoted to develop nanoparticle-basedtechnologies for active manipulations of neuronal electricalactivity. Huang et al102 used radio-frequency magnetic-fieldheating of manganese ferrite nanoparticles to remotely activatecultured neurons. The neurons expressed the temperature-sensitiveion channel TRPV1, as well as a chimeric membrane proteinwith biotin. The manganese ferrite nanoparticles wereconjugated to streptavidin, and were thus tethered to the targetcells. The local temperature increase generated by thenanoparticles opened the TRPV1 channels and caused aninflux of calcium ions, which resulted in a neuronal depolarizationthat was sufficient to elicit action potentials. No toxic effectswere observed.
In addition to these studies, using single-cell electrophysiologyand electron microscopy, the Ballerini group showed that neuronalcell membranes form tight contacts with carbon nanotubes.Functionalized single- or multi-wall carbon nanotubes weredeposited on glass slides, generating a thin meshwork about50-70 nm thick. The contact of neurons with these carbonnanotubes promoted neuronal activity.103,104 Carbon nanotubesmight favor electrical shortcuts between the proximal and distalcompartments of the neuron, thereby affecting its electricalactivity.103 These effects may also impact differentiation.
In summary, some types of nanoparticles show great promiseas tools for manipulation of neuronal activity (zinc oxide, gold,manganese ferrite, carbon nanotubes), while other types ofnanoparticles have neurotoxic effects and are thus inadequate forthis purpose (copper oxide, silver, carbon black, iron oxide,titanium oxide). However, it is clear that the effects on neuronal
activity must be taken into account when conducting researchinvolving inorganic nanoparticles and neuronal cells.
Nanoparticles across the blood brain barrier
The blood brain barrier is a physical barrier between the bloodand the brain and spinal cord. It guards the nervous system againstfluctuations in blood composition and undesirable traffic ofxenobiotics, and keeps neurotransmitters and other molecules thatact within the central nervous system separate from the rest of thebody. It is formed by endothelial cells, smooth muscle cells,pericytes, microglial cells and astrocytes, which line the walls ofblood vessels and are densely interconnected by tight junctions,allowing passage only to small lipophilic compounds or tosubstrates of active transport such as glucose or nucleotides.105-107
The blood brain barrier is essential for normal nervous systemfunction; however, it blocks drugs that are aimed to treat centralnervous system diseases from reaching their target. In recent yearsmuch effort is devoted to finding drugs that can circumvent thisbarrier and enter into the central nervous system, either on theirown or with the help of carriers. In the past decade, nanoparticleshave gained increasing attention as potential carriers of drugsacross the blood brain barrier. Drug conjugation can be either viaattachment to the surface of the nanoparticles, or encapsulation ofthe drug within a nanoshell.
Onemethod to transport drugs through the blood brain barrier isto transiently disrupt the barrier with localized heat, for example byusingmagnetic resonance. The groups ofMartel andGirouard usedthis method to successfully introduce a control dye through theblood brain barrier of live mice.108 An external apparatus waspositioned near the skull of themouse, and generated an alternatingmagnetic field over a radius of a few millimeters. To our opinion,this preliminary result is encouraging. In order to be clinicallyrelevant, the next stepwill be to focus the hyperthermia to a smallerarea, enough to fit only the drug. This can be achieved for exampleby conjugation of the drug to magnetic nanoparticles (such asFe3O4), and remote activation of hyperthermia at a resonance
Figure 8. Reprinted fromSkaat et al,121 under aCreativeCommonsAttribution–Non Commercial license. Fluorescence imaging of rat brains incubated withamyloid-β 40 (amyloid-β peptides that self-assemble to form neurological toxicaggregates) only (A1), BAM10 (monoclonal antibody against amyloid-b40)-conjugated nanoparticles only (B1), amyloid-β 40 followed by anti-rabbitIgG-conjugated nanoparticles (C1), and amyloid-β 40 followed by BAM10-conjugated nanoparticles (D1). (A2–D2) are the corresponding bright lightimages of the fluorescent images seen in (A1–D1) respectively.
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frequency specific to the nanoparticles, that would not affect theenvironment temperature.
A completely different approach is to link a ligand to thedrug-nanoparticle conjugate, specifically targeting the activetransporters found on the blood brain barrier. Hopefully, this candeceive the transporter allowing the drug-nanoparticle conjugate tocross the barrier. Such approach was taken by Popovtzer andcolleagues, who used gold nanoparticles conjugated to insulin.109
Gold nanoparticles were chosen since they can be quantitativelydetected in the brain ex vivo by atomic absorption methods, and invivo by CT imaging.110 Insulin was chosen as a ligand since highlevels of insulin receptors are present in several regions within thebrain,111,112 and because it was previously shown to enable albuminnanoparticles to cross the blood brain barrier.113 Insulin can beregarded here both as a drug and as a targeting ligand. The outcomeof this study was positives, with 5% of the injected nanoparticlescrossing the barrier, compared to other studies that investigated thepenetration of liposome-based nanoparticles into the brain andfound only up to 0.5% penetration success rate.114-118
In addition to the recent use ofmetallic nanoparticles, manymoreattempts at crossing the blood brain barrier have been reported usingsolid lipid nanoparticles (a type of nanoparticles made of a solidlipid core stabilized by a surfactant), as well as using polymericnanoparticles (made of biocompatible copolymers such as poly(alkylcyanoacrylate), polylactide, or poly(D,L-lactide-co-glycolate)), asreviewed in Franze and Guck75 and Bray.76 Although massiveprogress has been achieved using organic materials, these types ofnanoparticles are not the focus of this review.
Nanoparticles for neuronal imaging and theranostics
Imaging techniques are widely used for diagnostic proceduressuch as tumor detection. Traditional imaging is done via X-rayradiography, MRI, ultrasound, endoscopy, or PET scans. In recentyears, new imaging methods that are more specific, less invasiveand use non-radiative agents are emerging. Nanoparticles are idealcandidates for development of such imaging methods, because oftheir small size, biocompatibility, and accurate targeting abilityusing either remote magnetic positioning or specific ligands.
The same insulin-conjugated nanoparticles that were reportedin the previous section for crossing the blood brain barrier,109
also served as CT contrast agents to highlight specific brainregions in which they accumulated. These regions weredifferentiated from surrounding tissue, as gold induces strongerX-ray attenuation.
Schultke et al also used gold nanoparticles as CT contrastagents, for single-cell resolution imaging of brain tumors.119
Currently, MRI is the imaging method with the highest spatialresolution, of about 1 mm.120 This resolution is insufficient, atleast in the small animal models used for research and drugdevelopment, and naturally, higher resolution would benefithuman medicine as well. In this study, glioma cells (C6) wereloaded with gold nanoparticles, and injected into the rightcerebral hemispheres of adult rats. CT data were merged withMRI scans, to combine the benefits of both methods: thegeometrically correct, single-cell resolution of the CT, and thedetailed representation of soft tissue structure of the MRI. Using
this combination, the tumor was visualized with great detail,down to a ~1 μm resolution (Figure 7).
In another work by theMargel lab, the abilities for drug deliveryand imagingwere combined on one metallic nanoparticle.121 Theydesigned iron oxide nanoparticles conjugated to both a fluorescentdye, and to a monoclonal antibody against amyloid-β peptides thatself-assemble to form neurological toxic aggregates.122,123 Theantibody is well known to recognize the amyloid peptides, preventaggregate formation and reduce the aggregate burden.124,125 Thesenanoparticles inhibited in-vitro amyloid aggregate formationapproximately 5-fold compared to untreated and 2-fold comparedto free monoclonal antibodies. The authors hypothesize thatconjugation to nanoparticles stabilizes the antibodies, therebyprolonging their activity. Cell viability was examined in PC12cells, and increased when the cells were exposed to conjugatednanoparticles (~90%), compared to PC12 cells exposed only toamyloid aggregates (~70%). The conjugated nanoparticles alsospecifically detected aggregates and were successfully used forMRI scan in a rat brain ex-vivo (Figure 8).
As mentioned, nanoparticle-based imaging can also be used forintracellular imaging.40 For example, the Cui group conjugated thegrowth factors brain derived neurotropic factor (BDNF)126 orNGF127,128 to quantum-dots and tracked intracellularly at thesingle molecule level. The observations of these studies revealedthe mechanisms of intracellular movement of BDNF and NGF.Such techniques have the potential to offer important insights intomechanisms of action of many molecules, both for basic researchand theranostics. Notably, Alivisatos and colleagues presented anovel promising method for using nanoparticles to measure alsovoltage in neurons leading to imaging of function.129
Discussion
Here we review the plethora of recent advances in theinterface between neuronal tissue and inorganic nanoparticles(Table 1). Better understanding of how neurons interact with
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nanoparticles could provide critical support for neuronalregeneration and tissue engineering and may contribute to thedevelopment of novel diagnostic and therapeutic approaches.The studies demonstrate the sensitivity of the interactionoutcome to the characteristics of the nanoparticle of interest.Moreover, doses are a critical parameter that may balancebetween a positive therapeutic response and neurotoxicity.
Some studies actively manipulate neuronal parameters, e.g.morphology, activity or drug delivery, while others take a morepassive approach and use nanoparticles mainly for imaging. Inrecent years the trend is to combine the active and passivemodalities, using a single technology both for diagnostics(passive) and regenerative therapeutics (active).
We have presented a wide variety of nanoparticle-basedapproaches to manipulate neuronal growth and regeneration viadirect interactions or asmediators. The location and accessibility oftreatment target should also be taken into account, from theperipheral nerves to the central nervous system, including thebrain. Here we reviewed novel promising methodologies thatare still in their infancy regarding therapeutics. However, amassiveamount of research is devoted to develop ways to direct thenanoparticles and even crossing the BBB, making these novelapproaches clinically relevant. Taking into account the studiesreviewed here, this trend is expected to expand and include furtheractive approaches as in nano-medicine theranostics.
Acknowledgments
We thankmembers of the Shefi lab Shmulik Schwartz, NoaAlonand Michal Bouhnik-Marcus for help reviewing the literature.
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