Letter pubs.acs.org/NanoLett
Nanostructured Antagonist of Extrasynaptic NMDA Receptors Alex Savchenko,†,‡ Gary B. Braun,§ and Elena Molokanova*,∥ †
Department of Bioengineering and Pediatrics, University of California, San Diego, California 92093, United States Stanford University, Stanford, California 94305, United States § Sanford Burnham Prebys Medical Discovery Institute, La Jolla, California 92037, United States ∥ Nanotools Bioscience, Encinitas, California 92024, United States ‡
S Supporting Information *
ABSTRACT: Glutamatergic cytotoxicity mediated by overactivation of N-methyl-D-aspartate receptors (NMDARs) is implicated in numerous neurological disorders. To be therapeutically viable, NMDAR antagonists must preserve physiological role of synaptic NMDARs (sNMDARs) in synaptic transmission and block only excessive pathological activation of NMDARs. Here we present a novel NMDAR antagonist that satisfies this two-fold requirement by exploiting spatial differences in NMDAR subcellular locations. Specifically, we designed a hybrid nanodrug (AuM) to be larger than the synaptic cleft by attaching memantine, NMDAR antagonist, via polymer linkers to a gold nanoparticle. We show that AuM efficiently and selectively inhibited extrasynaptic NMDARs (eNMDARs), while having no effect on sNMDARs and synaptic transmission. AuM exhibited neuroprotective properties both in vitro and ex vivo during such neurotoxic insults as NMDAR-mediated cytotoxicity in cerebrocortical cell culture and oxygen-glucose deprivation in acute hippocampal slices. Furthermore, AuM prevented dendritic spine loss triggered by Aβ oligomers in organotypic hippocampal slices and was more effective than free memantine. Using a novel rational design strategy, we demonstrate a proof of concept for a new class of neuroprotective drugs that might be beneficial for treatment of several neurological disorders. KEYWORDS: Gold nanoparticles, neuron, NMDA receptors, nanostructured antagonist, nanoparticle conjugates, neuroprotection, Alzheimer’s disease, ischemic stroke, extrasynaptic, neurological disorders
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neuroprotective effects.6−8 In contrast, activation of eNMDARs in various pathological conditions was shown to play a major role in triggering apoptotic pathways.5,7,9,10 If the spatial separation of physiological and pathological NMDAR-mediated signaling cascades indeed takes place, then selective eNMDAR antagonists will be expected to act as pathology-specific drugs.11 The task of designing selective eNMDAR antagonists is very challenging. Although NMDARs can comprise multiple subunits with distinct pharmacological properties (e.g., NR2A, NR2B), different subunit types are found both in synaptic and extrasynaptic locations.12,13 Their relative presence in these locations can vary from one neuron to another and change over time as a result of activity- and experience-dependent switches in NMDAR subunit composition.14 In addition, NMDARs with a fixed subunit composition have substantial mobility between synaptic and extrasynaptic locations.12,13 Thus, pharmacological tools relying on a specific subunit composition are not expected to achieve a location-specific inhibition of NMDARs in complex multineuronal networks. Another option is to exploit differences in glutamate exposure patterns: transient high glutamate concentrations in
he N-methyl-D-aspartate receptors (NMDARs) play a critical role in numerous physiological processes, including developmental plasticity, learning, and memory. At the same time, pathological overactivation of NMDARs has been linked to neuronal damage in various neurological disorders from acute hypoxic-ischemic brain injury to chronic neurodegenerative diseases such as Huntington’s, Parkinson’s, and Alzheimer’s diseases, HIV-associated neurocognitive disorders, and amyotrophic lateral sclerosis.1 Therefore, NMDARs are considered important therapeutic targets,2 and several NMDAR antagonists have been tested to evaluate their neuroprotective properties. However, in clinical studies most NMDAR antagonists at therapeutic concentrations exhibited serious psychotomimetic side effects (i.e., hallucinations, drowsiness, and even coma)3 because they inhibited both physiological and pathological NMDAR-mediated brain activity indiscriminately. It has become increasingly clear that to be clinically tolerable and pharmacologically efficient NMDARs antagonists should be highly discriminative in their action: block only excessive, pathological activity while preserving normal synaptic function.4 Interestingly, numerous findings over the past decade suggest that NMDARs situated at different subcellular locations can have different and sometimes opposing impacts on the neuronal fate.5 Generally, activation of sNMDARs tends to stimulate multiple pro-survival pathways, thus providing © XXXX American Chemical Society
Received: May 16, 2016 Revised: July 26, 2016
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Nano Letters the synaptic cleft (sNMDARs) versus tonically elevated glutamate elsewhere (eNMDARs). Indeed, several studies suggest that the clinical tolerability of memantine, a FDAapproved NMDAR antagonist,4,15 is due to its uncompetitive/ fast off-rate mechanism of action, enabling more efficient blocking of tonic rather than high-frequency modes of NMDAR activity.16−18 Unfortunately, memantine inhibits both eNMDARs and sNMDARs, although with somewhat lower affinity toward sNMDARs.19 We decided to pursue an alternative strategy for designing subtype selective NMDAR antagonists. To engineer a drug that would spare sNMDARs and exclusively inhibit eNMDARs, we took into consideration geometrical parameters of their respective subcellular locations and constructed a nanodrug that due to its dimensions would be able to interact with one receptor subtype but not the other. We reasoned that a nanostructure larger than the synaptic cleft should not be able to access receptors inside the synapse (e.g., sNMDARs) but can reach receptors located elsewhere (e.g., eNMDARs) (Figure 1a). The synaptic cleft at common excitatory synapses has an average width of ∼25 nm with the coefficient of variation of ∼26%,20 which means that chemical entities above ∼30 nm in size are precluded from entering the cleft. This size regime is considerably larger than a typical drug molecule but is within the range of synthetic nanoparticles. To implement our rational drug design, we engineered a nanostructure (Figure 1b) comprised from a known NMDAR antagonist (memantine) tethered to the ends of a 3 kDa thiol-PEG-carboxyl PEG that is centrally attached to an inert spherical gold (Au) nanoparticle core 13 nm in diameter (Figure 1c). We also used a shorter inert thiol-PEG-methoxy 2 kDa diluent PEG to form a dense monolayer and passivate the remaining Au surface. The single amine group of memantine was coupled with carboxyls by amide bond formation to create stable conjugates. High-density PEG can diminish affinity of a tethered ligand,21 especially if the linker PEG that carries the ligand is surrounded by PEG strands with higher molecular weight (MW). The final hybrid nanostructure, named AuM, contained ∼50 memantine molecules per nanoparticle and exhibited an overall hydrodynamic size of ∼35 nm (Figure 1d). Au nanoparticles have been increasingly used as in vivo nanocarriers due to their general nontoxic properties and welldefined surface chemistry that forms stable adducts with thiol anchoring ligands.22 Thiol-terminated PEG molecules create a stable brush-like layer on gold nanoparticles, enabling longterm storage of a functional construct.23 This property has led to extensive use of thiol-PEG for in vitro and in vivo applications, particularly when long circulation time is required, because thiol-PEG also has the ability to confer resistance to nonspecific interactions between the gold and proteins present in the biological environment.23 Additionally, thiol coatings are often stable in culture medium and serve as model systems to allow attachment of multiple functional groups, either drugs or for targeting cells, to the outside of nanoparticles.24,25 The thickness of a thiol-PEG monolayer on Au and its resistance to aggregation and fouling can be controlled by the surface density and MW of the PEG;21,26 densely packed 2−5 kDa PEG layers can protect Au nanoparticles while substantially increasing their diameter.21 We characterized the physicochemical properties of AuM by evaluating its stability in various experimental conditions. If AuM formed aggregates, the plasmon peak would broaden and shift to longer wavelengths and the hydrodynamic diameter
Figure 1. Hybrid nanostructured antagonist for exclusive inhibition of eNMDARs. (a) The rational drug design strategy. (b) AuM coupling scheme. Step (i): Au-citrate was coated by functionalized PEG molecules. Step (ii): Memantine was coupled to carboxylates using EDC/NHS, to form an amide bond. (c) Transmission electron microscopy shows the Au core diameter of ∼13 nm. (d) Dynamic light scattering measurements show AuM stability against aggregation. (1) as-synthesized; (2) 5-month storage at 4 °C in PBS; (3) 5-month storage in PBS then incubated with 10% FBS for 1 h. The shoulder of the FBS sample peak is due to the presence of albumin with a monodisperse diameter of ∼8 nm. (e) UV−vis absorption spectrum of AuM after various conditions: (1) as-synthesized; (2) 5-month storage at 4 °C; (3) incubated in PBS at 37 °C overnight; (4) incubated in PBS with 10% FBS at 37 °C overnight.
would increase. In contrast, substantial loss of the PEGMemantine coating would manifest as a decrease in the hydrodynamic diameter toward that of a “naked” Au nanoparticle. We determined that AuM exhibited a stable hydrodynamic diameter through five months storage time (4 °C) and after the addition of 10% fetal bovine serum (FBS) to the buffer, suggesting structural stability of AuM and its PEGMemantine coating, the lack of aggregation, and negligible serum absorption (Figure 1d). The UV−vis spectra of AuM under different experimental conditions (temperature, storage time, and serum exposure) also showed no substantial changes in plasmon peak position or shape, confirming its resistance to aggregation (Figure 1e), as is typical for thiol-PEG gold coatings.23 To qualitatively evaluate the pharmacological activity of AuM, we first investigated its effects on NMDARs in calcium imaging experiments with cultured cerebrocortical neurons. We established that AuM appears to be an effective inhibitor of B
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focused our analysis on NMDAR-mediated component of sEPSCs by implementing the established temporal separation method9 based on distinct activation kinetics of different EPSC components.27,28 We found that regardless of the synaptic activity level in neuronal networks NMDAR-mediated sEPSCs were not affected by AuM at the maximum concentration explored (100 nM), whereas 10 μM memantine nearly completely inhibited synaptic activity (Figure 3b,c). These results were supported by detailed analysis of charge transfer (shaded area in the inset of Figure 3a), amplitude, and frequency of NMDAR-mediated sEPSCs in the presence of AuM and memantine (Figure 3d). While having no effect on sNMDAR-mediated activity, 100 nM AuM blocked 56.31 ± 4.52% (n = 14) of whole-cell ion currents elicited by 200 μM NMDA (Figure 4a). Auglucosamine and Au-PEG750 nanoparticles at 100 nM had no effect on NMDAR-mediated currents (Figure 4b), which remain 97.24 ± 2.63% and 95.63 ± 2.07% of their control values (n = 4), respectively. Taken together, these results indicate that (a) conjugated memantine retains its pharmacological properties (in agreement with previous findings29); (b) flexibility and length of PEG linkers allow conjugated memantine a sufficient degree of freedom to reach and block NMDAR pores; and (c) pharmacological properties of a fully assembled nanostructure are determined solely by conjugated antagonist molecules rather than by Au nanoparticles or their polymer coatings. Because the global population of NMDARs consists from sNMDARs and eNMDARs, and sNMDARs are not susceptible to AuM inhibition, we can conclude that AuM acts as a selective eNMDAR antagonist. Conjugating memantine via its sole primary amine results in a neutral chemical entity, and as expected we found no voltage dependence of NMDAR inhibition with AuM (Figure 4c). We also detected no trapping of memantine from AuM nanoparticles inside NMDARs (Figure 4d), probably because PEG-
NMDARs, as calcium influx triggered by saturating NMDA concentration (200 μM) was partially blocked by both AuM and memantine (Figure 2a). However, spontaneous glutama-
Figure 2. AuM is a selective antagonist of eNMDARs. (a) AuM is an efficient antagonist of NMDARs as demonstrated by representative calcium imaging traces from cerebrocortical neurons activated by 200 μM NMDA and inhibited by 10 nM AuM (left) or 10 μM memantine (right). (b) AuM is not effective in inhibiting glutamatergic synaptic activity as demonstrated by representative calcium imaging traces in spontaneously active neurons in the presence of 10 nM AuM (left) and 10 μM free memantine (right). n > 100 cells in three independent experiments for each condition in panels a and b. AU, arbitrary units. Cartoons on the left depict an experimental scheme with the red color highlighting the areas with activated NMDARs. Application events are marked by bars.
tergic synaptic activity (and associated calcium flux) was inhibited by free memantine, but not by AuM (Figure 2b), indicating pronounced selectivity of AuM block. To quantitatively verify this finding, we monitored spontaneous excitatory postsynaptic currents (sEPSCs) and
Figure 3. AuM does not inhibit spontaneous synaptic activity. (a) Cartoon depicts an experimental scheme with the red color highlighting the areas with activated NMDARs. (b,c) Whole-cell recordings of sEPSCs in neurons in the presence 100 nM AuM (b) and 10 μM free memantine (c). (d) Analysis of charge transfer, amplitude, and frequency of NMDAR-mediated sEPSCs in the presence of AuM or memantine (n ≥ 7 in each case). Inset in (a) shows a typical sEPSC with arrows pointing to the peaks of fast AMPAR- (1), and slow NMDAR-mediated (2) components. Bars, 500 pA and 300 ms. Data are presented as mean ±SEM ***, P < 0.005, unpaired t-test. Application events are marked by bars. C
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Figure 4. Electrophysiological assessment of pharmacological properties of AuM. Cartoons on the left depict an experimental scheme with the red color highlighting the areas with activated NMDARs. (a) NMDAR-mediated currents elicited by application of 200 μM NMDA are blocked by 100 nM AuM. (b) Au nanoparticles coupled to glucosamine (left) or PEG 750 (right) have no effect on NMDAR-mediated currents. Application events in (a,b) are marked by bars. (c) Current−voltage (I−V) relationships for fully activated NMDARs in the absence (squares) and presence (circles) of 100 nM AuM. (d) Memantine molecules conjugated to Au nanoparticles do not get trapped inside blocked NMDARs after NMDA removal, as demonstrated by matching amplitudes of NMDAR-mediated currents marked by asterisks. (e) Dose−response curves for AuM inhibition of fully activated global NMDARs (black, n = 10 cells) and eNMDARs (blue, n = 6 cells) were fitted with a standard Hill equation. Data are presented as mean ± SEM. (f,g) Box plots of unbinding (τoff) time and binding (τon) constants for AuM at different concentrations with the number of cells for each condition shown below the plots. The box represents 50% of the data, the horizontal bar is the median, the closed symbol is the mean, and the error bars indicate the full range. In all electrophysiological studies, each n represents an independent neuron.
found to be concentration-independent (Figure 4g). It is possible that flexible coiled PEG linkers can sporadically affect the time required for efficient positioning of conjugated memantine inside a NMDAR pore, leading to the wide distribution of the τon values and the apparent lack of concentration dependence. An additional confounding factor is that due to the multimolecular nature of AuM, the distribution of memantine in extracellular solution has a highly heterogeneous profile: localized spots of highly concentrated memantine in a vast empty volume. For example, 100 nM of AuM with 50 memantine molecules each is equivalent to the macroscopic memantine concentration of 5 μM. At the same time, 50 memantine molecules conjugated to one nanoparticle have only a small volume (∼2 × 10−20 L) to explore. Under these circumstances, the microscopic (local) concentration of memantine near any given NMDAR interacting with an AuM nanoparticle is ∼5 mM, regardless of macroscopic AuM concentrations. To directly assess the selectivity of AuM with respect to eNMDARs, we implemented the established pretreatment protocol for their pharmacological isolation.7,9 Only the eNMDAR subpopulation remain active in pretreated neurons, and 100 nM AuM nearly completely inhibited NMDARmediated currents when applied on such neurons. Fitting the dose−response curves produced an AuM IC50 of 5.11 ± 0.81 nM for inhibition of eNMDARs with a Hill coefficient of 0.89
conjugated memantine is able to reach only a superficial site, as opposed to a deep binding site, inside a NMDAR pore.18,30 Dose−response curves for AuM inhibition of fully activated NMDARs (Figure 4e) revealed that IC50 for AuM is 4.87 ± 1.28 nM (in nanoparticle molarity units) and the Hill coefficient for AuM is very close to unity (0.94 ± 0.17, n = 10 cells), as is expected for a pore blocker. The high affinity of AuM is attributed to the immediate availability of numerous memantine molecules tethered to the same nanoparticle in the close vicinity to a NMDAR. The cross-linking of multiple eNMDARs with several memantine molecules from one nanoparticle is unlikely to contribute to the increased affinity of AuM, because distances between sparsely distributed eNMDARs13 (∼3 receptors per 1 μm2)31 is much greater than the “reach” of PEG linkers of a single 40 nm nanostructure (∼2.5 × 10−3 μm2). We found that the unbinding time constant (τoff) for AuM was 0.508 ± 0.024 s across the tested concentration range (Figure 4f), which is appears to be faster than for free memantine.17,30 One possible explanation for this difference is that while free memantine can bind to two different binding sites (deep and superficial), PEG-linked memantine can only reach the superficial binding site. This hypothesis is supported by the finding that the time constant of memantine unbinding from its superficial site is comparable to τoff for AuM.30 Surprisingly, the binding time constant (τon) for AuM was also D
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Figure 5. Neuroprotective properties of AuM. (a) AuM prevented NMDA-induced toxicity in cultured cerebrocortical neurons: representative images of neurons stained with calcein AM (green, live cells) and EthD-1 (red, dead cells) after indicated treatments. (b) Quantification of cell death in treatment groups as indicated (n = 5 coverslips for each treatment). (c) AuM diffusion in the brain tissue after the 30 min incubation period: representative histological images of control and AuM-treated slices with autometallographic staining of the gold. When compared with the untreated control slices, the AuM-treated slices appear darkly stained due to the presence of silver-enhanced gold NPs. Arrows indicate the approximate extent of AuM diffusion into the tissue. (d) AuM protected acute hippocampal slices during OGD: normalized levels of LDH released from slices that were treated as indicated under corresponding bars (n = 6 for each treatment). (e) AuM prevented dendritic spine loss mediated by Aβ oligomers in hippocampal slices: representative images of dendritic spines from organotypic hippocampal slices from five YFP transgenic mice for four experimental conditions. (f) Quantification of the number of dendritic spines from neurons treated as indicated under the corresponding bars (n = 7 for each treatment). Data are presented as mean ±SEM *, P < 0.05; **, P < 0.01; ***, P < 0.005, one-way ANOVA with a Bonferroni posthoc test.
± 0.11 (n = 6 cells), closely matching the corresponding values for inhibition of the global NMDAR population in untreated neurons (Figure 4e). These results confirm that AuM is an efficient antagonist of eNMDARs. To evaluate neuroprotective properties of AuM, we induced NMDA-mediated cytotoxicity by 1 h treatment of cerebrocortical neurons with 200 μM NMDA alone or 200 μM NMDA in combination with either 10 μM memantine or 50 nM AuM. We
analyzed the cell death levels 24 h later and determined that AuM nearly completely prevented NMDA-induced toxicity, and AuM was significantly more neuroprotective than free memantine (Figure 5a, b). To examine whether AuM could be useful in vivo in the dense extracellular space of the brain, we directly evaluated the diffusion properties of AuM in the brain tissues (Figure 5c). We prepared 2 mm thick brain slices, immersed them into E
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Nano Letters oxygenated artificial cerebrospinal fluid (ACSF) solution with 400 nM AuM, and then performed autometallographic staining of the gold23 for visualization of AuM by histological analysis. From the brightness profile, we determined the diffusion coefficient (D) for AuM: 1.9 ± 0.4 × 10−8 cm2/s (n = 6) at 34 °C. This value means that AuM diffuses faster than 35 nm quantum dots (D = 0.167 × 10−8 cm2/s) but slower than 70 kDa dextran (14 nm hydrodynamic diameter, D = 6.48 × 10−8 cm2/s), both of which were determined in vivo in rat cortex.32 Efficient diffusion of AuM through the brain tissue is probably related to its dense brush-like PEG coating because PEG-coated nanoparticles as large as 114 nm in diameter can readily diffuse through the interstitial space, resulting in efficient brain penetration.33 In view of these findings, we tested AuM effects ex vivo on acute hippocampal slices under hypoxic−hypoglycemic conditions that simulate ischemic stroke events. Ischemic insults are accompanied by decreased glutamate uptake and reversed glutamate transport, leading to increased glutamate concentrations in the extracellular space. Using oxygen-glucose deprivation (OGD) as an ischemic stroke model, numerous studies established a crucial role of eNMDARs to ischemic cell death,7,8,11 suggesting a neuroprotective role for an eNMDARspecific inhibitor. Under certain experimental conditions, sNMDARs were shown to be also involved in mediating cell death34 but it is not clear whether protocol differences affected the results of these studies.35 To evaluate the contribution of eNMDARs to OGDtriggered injury in acute hippocampal slices in our experimental conditions, one group of slices was pretreated with MK-801 and bicuculline to selectively block sNMDARs7 prior to OGD. Following 45 min OGD treatment and 6 h recovery, we discovered that eNMDARs were responsible for the majority of OGD-induced injury as indicated by the similar levels of lactate dehydrogenase (LDH) released into ACSF from slices with and without pretreatment with MK-801 and bicuculline. AuM (50 nM) dramatically decreased the extent of OGD-induced injury, thus confirming that AuM can reach and inhibit eNMDARs in the brain tissue. Moreover, AuM was more efficient than memantine (10 μM) in providing neuroprotection under hypoxic−hypoglycemic conditions (Figure 5d). Next, we sought to determine how AuM performs during long-term neurotoxic events, similar to the ones that might occur during Alzheimer’s disease (AD). Synaptic loss is the hallmark of AD.36 Numerous in vitro and in vivo studies demonstrated that exposure to soluble Aβ oligomers leads to a pronounced decrease in the density of dendritic spines, the primary postsynaptic sites of excitatory synapses.37−40 Moreover, the decrease in spine density correlates with cognitive impairments in transgenic mouse models of AD.39,41 Because loss of dendritic spines can be considered an important biomarker for AD, various studies of potential treatments for this disease are relying on changes in dendritic spine density to assess the treatment efficiency.37,42,43 Soluble Aβ oligomers are known to trigger signaling pathways, leading to overactivation of eNMDARs,10,44 followed by the decrease in the dendritic spine density.40,42 Inhibition of eNMDARs is expected to disrupt these neurodegenerative pathways and prevent the dendritic spine loss. To test the ability of AuM to preserve dendritic spines from destructive influence of Aβ oligomers, we treated organotypic hippocampal slices from thy1-YFP-H transgenic mice with 250 nM Aβ1−42 oligomers in the presence and absence of 10 μM free
memantine or 50 nM AuM, each for 10 days. We determined that AuM was significantly more effective than free memantine in protecting synaptic dendritic spines from Aβ oligomers (Figure 5e,f). Note that memantine is approved by the FDA only for treatment of moderate-to-severe AD15 when the synaptic loss is very pronounced, and possible detrimental effects of memantine are minimized. In contrast to free memantine, AuM might be beneficial at earlier stages of AD because it does not inhibit sNMDARs, and thus its effects are not dependent on the relative number of synapses. The results of our neurotoxicity experiments suggest that blocking eNMDARs (i.e., by AuM, a selective eNMDAR inhibitor) is sufficient for protecting neurons from NMDARmediated toxicity, whereas blocking sNMDARs concurrently with eNMDARs (i.e., by memantine) can decrease the survival rate. Taken together, these findings provide further support for the concept about the opposing roles of sNMDARs and eNMDARs on neuronal fate.5,11 By taking into consideration relative dimensions of pharmacologically active nanostructures and ease of access to different subcellular locations, we developed a new rational drug design strategy that can enable selective manipulation of extrasynaptic receptors. Particular implementation of this design has resulted in a novel hybrid nanostructured antagonist (AuM) that is capable of inhibiting eNMDARs but has no effect on sNMDARs. The most apparent application is to use AuM as a pharmacological tool to exclusively block eNMDAR-mediated pathways in order to elucidate functional contribution of eNMDARs under various pathological conditions. Better understanding of underlying mechanisms could help to devise more efficient treatment options. Further development of rationally designed eNMDAR antagonists might potentially result in a novel class of neuroprotective drugs. The translational potential of AuM stems from our findings that AuM successfully protected neurons from neurotoxic assaults in three different neurotoxicity models and was significantly more effective than free memantine. We verified that AuM can successfully diffuse through and penetrate the brain tissue, which is the key requirement for antagonists targeting receptors located throughout the brain. Importantly, AuM provided neuroprotection in the brain slices both under hypoxic−hypoglycemic conditions and during exposure to Aβ oligomers, which further confirms efficient brain penetration by AuM and its functionality ex vivo. The specifics of the AuM design (e.g., size and surface coating) play an important role in such positive outcome. For example, 35 nm hydrodynamic diameter of AuM belongs to the favorable range as the gaps in the extracellular space of the brain are usually larger than ∼50 nm.32,33,45 Additionally, PEG-coated nanoparticles of ∼100 nm have been shown to be able to readily diffuse through the interstitial space, resulting in substantial penetration of the brain tissue.33,46 AuM (also PEG-coated, but smaller in size) is even more efficient in overcoming obstacles during its diffusion through the complex organized brain tissue. To further facilitate AuM passage through the brain and aid with its delivery to the brain, AuM can be combined with approved excipients or its extended surface area can be used to conjugate additional specialized targeting ligands. Local, rather than systemic, administration routes would certainly be more efficient for AuM, although nanoparticles of similar size to AuM can pass through the intact blood-brain barrier (BBB) of healthy animals45,47 and even F
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(12) Tovar, K. R.; Westbrook, G. L. Neuron 2002, 34, 255−264. (13) Petralia, R. S.; Wang, Y.-X.; Hua, F.; Yi, Z.; Zhou, A.; Ge, L.; Stephenson, F.; Wenthold, R. J. Neuroscience 2010, 167, 68−87. (14) Paoletti, P.; Bellone, C.; Zhou, Q. Nat. Rev. Neurosci. 2013, 14, 383−400. (15) Danysz, W.; Parsons, C. G. Br. J. Pharmacol. 2012, 167, 324− 352. (16) Chen, H.; Pellegrini, J.; Aggarwal, S.; Lei, S.; Warach, S.; Jensen, F.; Lipton, S. J. Neurosci. 1992, 12, 4427−4436. (17) Chen, H.; Lipton, S. A. J. Physiol. 1997, 499, 27−46. (18) Johnson, J. W.; Kotermanski, S. E. Curr. Opin. Pharmacol. 2006, 6, 61−67. (19) Xia, P.; Chen, H.-S. V.; Zhang, D.; Lipton, S. A. J. Neurosci. 2010, 30, 11246−11250. (20) Rusakov, D. A.; Kullmann, D. M. J. Neurosci. 1998, 18, 3158− 3170. (21) Rahme, K.; Chen, L.; Hobbs, R. G.; Morris, M. A.; O’Driscoll, C.; Holmes, J. D. RSC Adv. 2013, 3, 6085−6094. (22) Doane, T. L.; Burda, C. Chem. Soc. Rev. 2012, 41, 2885−2911. (23) Perrault, S. D.; Walkey, C.; Jennings, T.; Fischer, H. C.; Chan, W. C. Nano Lett. 2009, 9, 1909−1915. (24) Dhar, S.; Daniel, W. L.; Giljohann, D. A.; Mirkin, C. A.; Lippard, S. J. J. Am. Chem. Soc. 2009, 131, 14652−14653. (25) Maus, L.; Spatz, J. P.; Fiammengo, R. Langmuir 2009, 25, 7910− 7917. (26) Jokerst, J. V.; Lobovkina, T.; Zare, R. N.; Gambhir, S. S. Nanomedicine 2011, 6, 715−728. (27) Lester, R.; Clements, J. D.; Tong, G.; Westbrook, G. L.; Jahr, C. E. The time course of NMDA receptor-mediated synaptic currents. In The NMDA receptor; Collingridge, G. L.; Watkins, J. C., Eds. Oxford: New York, 1994; Vol. 11, pp 206−218. (28) Edmonds, B.; Gibb, A.; Colquhoun, D. Annu. Rev. Physiol. 1995, 57, 495−519. (29) Simoni, E.; Daniele, S.; Bottegoni, G.; Pizzirani, D.; Trincavelli, M. L.; Goldoni, L.; Tarozzo, G.; Reggiani, A.; Martini, C.; Piomelli, D.; Melchiorre, C.; Rosini, M.; Cavalli, A. J. Med. Chem. 2012, 55, 9708− 9721. (30) Kotermanski, S. E.; Wood, J. T.; Johnson, J. W. J. Physiol. 2009, 587, 4589−4604. (31) Cottrell, J. R.; Dubé, G. R.; Egles, C.; Liu, G. J. Neurophysiol. 2000, 84, 1573−1587. (32) Thorne, R. G.; Nicholson, C. Proc. Natl. Acad. Sci. U. S. A. 2006, 103, 5567−5572. (33) Nance, E. A.; Woodworth, G. F.; Sailor, K. A.; Shih, T.-Y.; Xu, Q.; Swaminathan, G.; Xiang, D.; Eberhart, C.; Hanes, J. Sci. Transl. Med. 2012, 4, 149ra119−149ra119. (34) Wroge, C. M.; Hogins, J.; Eisenman, L.; Mennerick, S. J. Neurosci. 2012, 32, 6732−6742. (35) McKay, S.; Bengtson, C. P.; Bading, H.; Wyllie, D. J. A.; Hardingham, G. E. Neuropharmacology 2013, 74, 119−125. (36) Palop, J. J.; Mucke, L. Nat. Neurosci. 2010, 13, 812−818. (37) Shankar, G. M.; Bloodgood, B. L.; Townsend, M.; Walsh, D. M.; Selkoe, D. J.; Sabatini, B. L. J. Neurosci. 2007, 27, 2866−2875. (38) Shankar, G. M.; Li, S.; Mehta, T. H.; Garcia-Munoz, A.; Shepardson, N. E.; Smith, I.; Brett, F. M.; Farrell, M. A.; Rowan, M. J.; Lemere, C. A. Nat. Med. 2008, 14, 837−842. (39) Spires-Jones, T.; Knafo, S. Neural Plast. 2012, 2012, 1−10. (40) Wei, W.; Nguyen, L. N.; Kessels, H. W.; Hagiwara, H.; Sisodia, S.; Malinow, R. Nat. Neurosci. 2010, 13, 190−196. (41) Jacobsen, J. S.; Wu, C.-C.; Redwine, J. M.; Comery, T. A.; Arias, R.; Bowlby, M.; Martone, R.; Morrison, J. H.; Pangalos, M. N.; Reinhart, P. H. Proc. Natl. Acad. Sci. U. S. A. 2006, 103, 5161−5166. (42) Shrestha, B. R.; Vitolo, O. V.; Joshi, P.; Lordkipanidze, T.; Shelanski, M.; Dunaevsky, A. Mol. Cell. Neurosci. 2006, 33, 274−282. (43) Smith, D. L.; Pozueta, J.; Gong, B.; Arancio, O.; Shelanski, M. Proc. Natl. Acad. Sci. U. S. A. 2009, 106, 16877−16882. (44) Li, S.; Jin, M.; Koeglsperger, T.; Shepardson, N. E.; Shankar, G. M.; Selkoe, D. J. J. Neurosci. 2011, 31, 6627−6638.
more so when the BBB is pathologically compromised (e.g., as a result of neurological disorders such as Alzheimer’s disease, stroke, and brain trauma48). Importantly, novel drug design strategy utilized here for AuM and eNMDARs can be extended to a variety of nanoparticle core materials and conjugated modulators (agonists or antagonists) targeting a wide range of ion channels and receptors exhibiting a location-specific functional dichotomy.
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ASSOCIATED CONTENT
S Supporting Information *
The Supporting Information is available free of charge on the ACS Publications website at DOI: 10.1021/acs.nanolett.6b01988. Methods and additional information (PDF)
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AUTHOR INFORMATION
Corresponding Author
*E-mail:
[email protected]. Author Contributions
A.S. and G.B.B. contributed equally to this work. Notes
The authors declare no competing financial interest.
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ACKNOWLEDGMENTS We thank Traci Fang Newmeyer for excellent technical assistance with primary neuronal cultures. We thank Brian Lee, Dr. Yuliya Medvedeva, and Dr. Juan C. Piña-Crespo for help with brain slice preparations. We thank Dr. Michael J. Sailor at UC San Diego for use of the dynamic light scattering instrument. G.B.B. was supported by NIH T32 fellowship (CA121949).
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