extfieldsofstudy: Chemistry Medicine

s2fieldsofstudy:
Biology
Materials Science

text:
Metal Nanoclusters with Synergistically Engineered Optical and Buffering Activity of Intracellular Reactive Oxygen Species by Compositional and Supramolecular Design

Metal nanoclusters featuring tunable luminescence and high biocompatibility are receiving attention as fluorescent markers for cellular imaging. The recently discovered ability of gold clusters to scavenge cytotoxic reactive oxygen species (ROS) from the intracellular environment extends their applicability to biomedical theranostics and provides a novel platform for realizing multifunctional luminescent probes with engineered anti-cytotoxic activity for applications in bio-diagnostics and conceivably cellular therapy. This goal could be achieved by using clusters of strongly reactive metals such as silver, provided that strategies are found to enhance their luminescence while simultaneously enabling direct interaction between the metal atoms and the chemical surroundings. In this work, we demonstrate a synergic approach for realizing multifunctional metal clusters combining enhanced luminescence with strong and lasting ROS scavenging activity, based on the fabrication and in situ protection of Ag nanoclusters with a supramolecular mantle of thiolated-Au atoms (Ag/Au-t). Confocal imaging and viability measurements highlight the biocompatibility of Ag/Au-t and their suitability as fluorescent bio-markers. ROS concentration tests reveal the remarkable scavenging activity of Ag-based clusters. Proliferation tests of cells in artificially stressed culture conditions point out their prolonged anti-cytotoxic effect with respect to gold systems, ensuring positive cell proliferation rates even for long incubation time.

However, when overexpressed in stressed conditions (i.e. chemical intoxication, UV exposure, overheating), ROS lead to accelerated cell ageing and, in extreme cases, to premature cellular death 27 . Although the exact mechanism of ROS scavenging by Au clusters is not fully understood and it is likely associated with the ability of the thiol capping ligands to buffer oxygen radicals [28][29][30] , this newfound ability of metal clusters to reduce oxidative stress is particularly beneficial for biomedical theranostics as it opens the way to novel multifunctional optical probes synergistically engineered for diagnostic and, potentially, therapeutic applications.
One possible strategy for achieving higher ROS harvesting performances would be to use clusters of more reactive metals than gold, such as silver. Ag based nanomaterials are known for their antibacterial properties, being their toxicity commonly associated to the release of Ag + , which is at the basis of the strategies for developing new antibiotics [31][32][33][34] . However, the toxicity mechanism of Ag based nanoparticles may not be directly applicable to all biological systems, i.e. in mammaliam cells probably due to the more complex sub-cellular organization. On the other hand, a comparative study of the biologic activity of Ag clusters with reduced (Ag 0 ) or oxidized (Ag + ) metallic core has shown stronger cytotoxicity of the first due to favoured release of Ag + species leading to the expression of intracellular ROS 35 .
One possible limitation to the use of Ag clusters as multifunctional luminescence markers is, however, that the emission efficiency of systems protected with small capping molecules, such as thiols, is typically very low [36][37][38] . In order to address this issue, Ag clusters capped with bulky biomolecules [39][40][41] and polymers 42 , which protect the metal cores from luminescence quenchers 43 , have been realized. The steric encapsulation of the metal cores, however, increases the hydrodynamic size of the clusters, which could limit their permeability in subcellular imaging 44 and, more detrimental for ROS scavenging, prevents direct contact between the cluster and its chemical surroundings.
In order to simultaneously achieve enhanced optical and anti-cytotoxic performances, it is therefore paramount to develop synergistic passivation strategies that protect the excited states of the clusters while ensuring accessibility of ROS-sensitive surface metal core and sulfur functionalitity of the respecive thiol ligands to the intracellular environment. One possible approach could be supramolecular protection of the Ag clusters with a thin mantle of aggregated Au-thiolate complexes, as recently theoretically predicted for Au clusters by H. Hakkinen 45 and validated experimentally by Xie and coworkers 4,46 , who enhanced the luminescence efficiency of gold clusters by surface condensation of aggregated Au-thiolate oligomers. A core-shell approach, where a shell of fluorescent Ag 2 and Ag 3 clusters was added to Au clusters, has also been recently used for obtaining highly fluorescent core-shell particles 47 .
Taking inspiration from these pioneering studies, in this work, we develop a one-pot aqueous route for the synthesis and in situ protection of Ag clusters with Au-thiolate complexes, resulting in enhanced and spectrally pure blue emission and strong ROS scavenging activity. The synthetic rationale exploits the higher reactivity of silver with respect to gold 37 that, when co-added with a reducing agent, drives the fast nucleation of Ag clusters that are successively decorated with Au-thiolate complexes. The progressive shelling of the silver clusters is confirmed by side-by-side structural and optical measurements on bare and Au-complexed Ag clusters (Ag/Au-t) during the synthesis reaction. Confocal imaging and viability measurements on NIH/3T3 fibroblast cells demonstrate the excellent biocompatibility of the Ag/Au-t clusters and their suitability as blue fluorescent markers. ROS concentration tests reveal, for the first time, the strong scavenging activity of both Ag and Ag/Au-t systems and confirm the ROS harvesting property of Au clusters 24 . Remarkably, proliferation tests of cells with artificially accelerated metabolic activity highlight the prolonged ROS buffering effect of Ag and Ag/Au-t clusters with respect to gold systems, ensuring positive and high cell proliferation rate even after 96 hours of incubation in stressed condition.

Results
Synthesis and Structure of complexed Ag clusters. The Ag/Au-t capped with 16-mercaptohexadecanoic acid (MHDA)-tetrabutylammonium (TBA) salt were prepared according to the bottom-up route shown in Fig. 1a, which is based on the chemical reduction of the Ag precursor (AgNO 3 ) in the presence of HAuCl 4 (nominal Ag:Au ratio 1:2), mercapto-palmitic acids and tetrabutyl ammonium salts [48][49][50] . As control materials, we fabricated monometallic clusters by adopting the same reaction conditions as the synthesis of the Ag/Au-t systems, but introducing exclusively Ag or Au precursors in the reaction medium. The molar ratio between the capping ligands and metal precursors, which is a key parameter for tuning the cluster size 1 , has been kept constant for all syntheses. In order to monitor the nucleation of the clusters, we measured the optical absorption of aliquots extracted from the reaction medium over time, as shown in Fig. 1b. The time evolution of the absorption intensity for each system is reported in Fig. 2a. The Ag/Au-t clusters show a narrow absorption spectrum peaked at ~345 nm, whose intensity increases with the reaction time as the cluster population progressively grows. The spectral position of the absorption maximum remains unchanged at all stages of the synthesis, which indicates the formation of clusters with identical dimensions according to the typical size-focused growth of metal clusters [51][52][53] . The absorption spectrum of the Ag/Au-t systems is essentially identical to the Ag clusters, which suggests that both routes lead to the nucleation of Ag cores of comparable size corresponding to ca. three silver atoms 54 . This assignment is confirmed by electrospray-ionization mass spectrometry (ESI-MS) analysis (Fig. S2), which indicates that bare silver clusters are composed of three Ag atoms capped by two MHDA ligands. The experimental and simulated isotopic distribution pattern corresponding to the [Ag 3 (MHDA) 2 (TBA) 4 Fig. 1b (top panel) reports the experimental spectrum of the latter structure together with the corresponding simulated pattern. Notably, no feature observed for the bare Ag sample is found in the spectrum of the complexed clusters (Fig. S2), which indicates that the Ag/Au-t ensemble is composed by nearly monodispersed Ag 3 cores decorated with Au-thiolate units.
The absorption spectrum of the control Au clusters is markedly different from both Ag and Ag/Au systems, with two broader peaks at 390 nm and 422 nm (Fig. S1). The absence of such absorption features in the spectrum of the Ag/Au clusters indicates that the population of Au clusters in the bimetallic sample is negligible. The comparison between the absorption spectra of the three systems suggests that, in the bimetallic reaction, the nucleation of the Ag cores is favoured over the synthesis of the Au clusters. This picture is supported by the comparison of the formation kinetics of the three cluster types shown in Fig. 2a, which we monitored through the time evolution of the respective absorption amplitude. The monometallic Ag clusters show a rapid growth of their absorption spectrum at the very early stage of the synthesis, which reaches saturation in approximately three hours, indicating the conclusion of the nucleation reaction. Conversely, the formation of monometallic Au clusters is much slower, with the characteristic absorption features being detectable only after over 24 hours of reaction. This striking difference between the growth kinetics is ascribed to the lower reduction potential of silver (Ag + /Ag, ~0.8 V) with respect to gold (Au +3 /Au, ~1.0 V), which accelerates the formation kinetics of the Ag clusters with respect to the Au ones 37 . Notably, the synthesis of the Ag/Au systems follows a fast trend similar to the monometallic Ag clusters, suggesting that the bimetallic reaction is initially driven by the nucleation of the Ag cores followed by the preferential incorporation of Au atoms with respect to homonucleation of Au clusters.
In order to gather deeper insights into the mechanism of the bimetallic reaction, we performed X-ray photoelectron spectroscopy (XPS) measurements on two Ag/Au cluster aliquots, taken respectively after 10 hours and after 80 hours of reaction time. In both cases, the samples were thoroughly washed by precipitation prior to the XPS analysis. Figure 2b reports the XPS spectrum of Ag/Au-t clusters showing the binding energies of Au 4 f and Ag 3d electrons. At the initial stage of the reaction, the binding energies obtained for silver are E Ag (3d 5/2 ) = 368.2 eV and E Ag (3d 3/2 ) = 374.2 eV, consistent with the values found for small-sized thiolated-Ag clusters 55 , bimetallic Ag-Au systems 56 , as well as with the optical absorption data in Fig. 1b. For gold, the initial binding energies are E Au (4f 7/2 ) = 84.7 eV and E Au (4f 5/2 ) = 88.5 eV (Fig. 2b), in agreement with the energies of thiolated Au atoms 57 . After 80 hours, the XPS spectrum shows a shift of the binding energies of both metals: The silver energies decrease to E Ag (3d 5/2 ) = 367.5 eV and E Ag (3d 3/2 ) = 373.3 eV, indicating the reduction of the Ag atoms upon removal of thiol ligands from the cluster surface. The Au binding energies increase to E Au (4f 7/2 ) = 85.1 eV and E Au (4f 5/2 ) = 88.8 eV, indicating the oxidation of metallic gold to Au +1 atoms, which is consistent with their final location on the Ag cluster surfaces and with the formation of oligomeric Au-thiolate complexes 57 . This is supported by the binding energies observed in the reference monometallic Ag systems [E Ag (3d 5/2 ) = 367.7 eV and E Ag (3d 3/2 ) = 373.7 eV, Fig. S3], which are very close to those of the Ag/Au-t case, although slightly shifted to higher values due to the presence of the thiol capping on the surface 55 . The binding energies measured for the monometallic Au clusters are significantly lower [E Au (4f 7/2 ) = 84.3 eV and E Au (4f 5/2 ) = 88.5 eV, Fig. S3] 57 , thus confirming the absence of isolated gold clusters at the end of Ag/Au-t clusters synthesis. The gradual complexation of the Ag cores by thiolated-Au species is further confirmed by the quantitative analysis of the XPS data. Specifically, the Ag:Au atomic ratio in the aliquot extracted after 10-hours reaction is 1:0.5, with a concentration of gold atoms four times lower than the nominal feeding ratio of 1:2. This indicates that, at the early stages of the synthesis, most of the gold atoms are not attached to the Ag clusters and are thus washed away during purification. On the other hand, the Ag:Au atomic ratio after 80 hours of reaction is 1:1.5, which is close to the nominal concentration of precursors and indicates that gold atoms have been incorporated in the cluster architecture and thus unaffected by the purification procedure.
Photoluminescence Properties. The progressive condensation of thiolated Au species on the Ag cluster surfaces has beneficial effects on their emission efficiency, as highlighted by side-by-side continuous-wave and time-resolved photoluminescence (PL) measurements performed during the synthesis reaction. Figure 3a shows the evolution of the PL spectrum of Ag/Au-t clusters excited at 355 nm (130 μJ/cm 2 ) as a function of the reaction time, together with the absorption spectrum after 80-hours reaction. The PL spectrum consists of a narrow peak at 435 nm that grows over time without any appreciable spectral modification, with full width at half maximum of ~45 nm at all reaction stages (Fig. 3a).
Importantly, the increase of the PL intensity during the synthesis is not only due to the growing number of clusters in the sample, but also to the progressive enhancement of their PL quantum yield. This effect is shown in Fig. 3b, where we report the evolution of the PL efficiency and sample absorbance over time, which further enables us to monitor the progress of the Au-thiolate passivation process. Specifically, in the first 30 hours of reaction, which correspond to the time required for nearly concluding the synthesis of the bare Ag cores (Fig. 3b triangles and Fig. 2a), the PL efficiency is nearly constant and relatively low, in agreement with previous reports on Ag clusters protected with small thiol ligands [36][37][38] . In this time interval, the growth of the PL signal is therefore due to the increasingly larger number of clusters in solution, consistent with over 10-fold increase of the ensemble's absorbance. After ca. 30-hours reaction, when the absorption trend of Ag/Au-t clusters is markedly slower (3-fold increase until reaction end) and that of the monometallic Ag clusters is essentially constant (Fig. 2a), the PL quantum yield undergoes progressive increase, resulting in ~10-fold enhancement in the final products with PL efficiency ~2% (Fig. 3b). This effect is consistent with the gradual protection of a steady population of Ag clusters by Au-thiolate complexes that passivate non-radiative quenching channels. Accordingly, the PL decay dynamics of Ag/Au-t clusters becomes progressively slower, as shown in Fig. 3c. We notice that other mechanisms could, in principle, lead to enhanced PL, such as the intercalation of Au atoms between the early-formed Ag cores and their capping thiols, or the substitution of Ag atoms with thiolated Au species. Both these mechanisms would, however, result in significant alteration of the electronic structure of the clusters, either due to increase of their size by incorporation of additional atoms 10 , or to the modification of their chemical composition 38 , in disagreement with the optical absorption and PL spectra in Figs 1b and 3a. We note that the final PL quantum yield of our Ag/Au-t clusters is ~2%, which indicates that the shelling is incomplete. This is, however, beneficial for the applicability of Ag/Au-t clusters as multifunctional luminescent probes in cellular theranostics, as it increases the PL efficiency enough for fluorescence imaging, while still allowing direct interaction between the metal cores and the intracellular environment. This enables, as we demonstrate below, to exploit the strong ROS-scavenging activity of the thiolated silver clusters.

Cellular Imaging and ROS essays.
To experimentally validate the applicability of our proof-of-concept Ag/Au-t clusters as fluorescent bio-markers, we performed in vitro imaging experiments on NIH/3T3 fibroblast cells. Figure 4a reports representative RGB (red-green-blue) images of NIH/3T3 cells co-stained with the red emitting dye CyTrak-26 -for selectively marking the nucleus -and with increasing concentration of Ag/Au-t  clusters that disperse in the cytoplasm as they are not functionalized with target-specific ligands. We notice that the Ag/Au-t clusters do not aggregate unless at high concentration (33 μM, obtained with a 1:10 dilution of the mother solution, Fig. 4a right panel). After having confirmed the effectiveness of the supramolecular shelling in rendering Ag clusters suitable as fluorescent bio-probes, we proceeded with the investigation of the effects of Ag/Au-t on the cellular biology in direct comparison with their unshelled counterparts. With this aim, we performed the 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) essay and monitored the evolution of ROS over time. The results of the MTT test in standard culture conditions reported in Fig. 4b indicate that neither Ag nor Ag/Au-t clusters affect the cell viability. The viability tests reveal cell proliferation rates comparable for stained cells with respect to untreated cells for the whole duration of the experiment (96 hours), outlining the high biocompatibility of these material systems. As previously demonstrated 24 , the culture viability is unaffected also by Au clusters, which however, slow down the cell proliferation at long incubation times. The improved cell viability suggests that Ag and Ag/Au-t clusters can also exert a protective role even in standard cell culture conditions when ROS-dependent mechanisms of cell growth regulation occur, as showed by the ROS concentration tests reported in Fig. 4c. The ROS level in a standard cell culture is associated with the acute stress induced by the initial cell detachment from the culture vessel required to perform the test (24 hours to 48 hours) 58 and to the negative regulation of the cell culture proliferation -also known as "contact inhibition" -when grown to confluence (72 hours to 96 hours) 59 . Therefore, this continuous stress condition results in a constant ROS concentration level for the unstained control culture at each time point considered. Conversely, the presence of metal clusters is able to counterbalance the ROS overproduction in stained cultures. In particular, all clusters show comparable ROS-scavenging abilities in the first 48 hours, reducing the level of oxidants agents produced upon cell detachment to similar values. At longer times, when the occurrence of cell confluence induces additional stress, only Ag and Ag/Au-t systems still operate effectively, turning out in a rapid cell growth even at 96 hours from seeding (Fig. 4b). The Au clusters, despite being able to decrease the overall ROS concentration, show a weaker ability to compensate this additional stress with respect to their Ag and Ag/Au-t counterparts, thus causing a slower proliferation (Fig. 4b). Beside confirming recent results on Au clusters 24 , these findings demonstrate, for the first time, the ROS scavenging activity of thiolated Ag clusters. These results are further confirmed by independent experiments using the ROS-Glo ™ H 2 O 2 Assay (Supplementary Fig. S4), which demonstrate that both Ag and Ag/Au-t systems strongly reduce the concentration of H 2 O 2 also in aqueous solution. Importantly, the scavenging activity of Ag clusters is found to be stronger than for Au systems in agreement with the higher reactivity of silver atoms. Specifically, in Au-clusters stained cells, the ROS concentration drops by ~70% in 96 hours, whereas in cells marked with Ag-based clusters over 90% reduction is achieved already after 72 hours. According to the partial passivation effect of Au-thiolate of the Ag cores, which accounts for the enhanced PL efficiency with respect to bare Ag clusters shown in Fig. 3, the ROS scavenging activity of the shelled systems is slightly lower than for the bare Ag clusters, yet still more effective than for the Au clusters. Notably, PL measurements on Ag/Au-t clusters in water as a function of time (Fig. S5) reveal that their emission efficiency is essentially unaffected by ROS concentrations as high as 0.5 mM for over five hours of exposure and undergoes only 40% drop for very high ROS content (5 mM), which suggests that these emitters could be employed for bio-imaging measurements also in harsh conditions.
It is worth pointing out that these encouraging findings cannot be considered as a proof of the cluster scavenging abilities in severe oxidative stress conditions that trigger cell death 60 , since the low levels of intracellular ROS recorded are only symptomatic of signal transduction pathways involving the responses to growth factors, hypoxia, and other receptor-ligand systems 61 . Therefore, in order to verify that this newfound ROS buffering effect of Ag and Ag/Au-t clusters has significant implications, we studied the cellular proliferation in highly stressed conditions upon addition of the metabolic accelerator menadione, which induces intense toxic oxidative stress mimicking the condition of in vivo tissue injury and mitochondrial DNA damage 62 . Figure 4d reports the incremental proliferation ΔP = 100 × (P stain − P CTR )/P CTR of cluster-stained NIH/3T3 fibroblasts (P stain ) in the presence of menadione. ΔP was calculated with respect to the population of the unstained control culture (P CTR ) incubated in identical conditions. In agreement with the stronger ROS scavenging effect of Ag and Ag/Au-t clusters with respect to Au clusters (Fig. 4c), Fig. 4d shows markedly increased proliferation of the respective cell cultures, with ΔP > 200% within the first 48 hours vs. ΔP ~ 25% for cells containing Au clusters. Furthermore, Ag-based clusters show longer lasting ROS buffering effect than Au clusters, resulting in significant cell growth (ΔP > 50%) even after 96-hours incubation, when cells stained with Au clusters show a mild proliferation drop.

Conclusions
In summary, we have demonstrated a synergic strategy for realizing multifunctional metal clusters combining amplified and prolonged anti-cytotoxic activity and spectrally pure photoluminescence for applications in bio-diagnostics and conceivably cellular therapy. The approach consists in the use of Ag clusters with stronger reactivity than Au with oxygen radicals and their partial passivation with a mantle of Au-thiolate complexes that reduce non-radiative luminescence quenching channels, while still preserving sufficient accessibility to enable direct interaction of the thioated Ag core with its chemical environment. As a result, these complexed clusters can be used in cellular theranostics as fluorescence markers and intracellular scavengers of cytotoxic species with beneficial effects on the cellular viability. We note that the reported proof-of-principle Ag/Au-t clusters are not optimized in terms of the gold coating thickness or the choice of the capping ligand, and further improvements in the photoluminescence quantum efficiency and ROS scavenging activity might therefore be expected by adjusting the silver core coverage and functionalization. The strategy demonstrated here for Au-passivated Ag clusters is not metal or size specific and might, in principle, be extended to other compositions, so as to achieve biocompatible multifunctional metal nanoclusters with diagnostic and potentially therapeutic ability.

Materials and Methods
Materials. Typically, 3 mL of 16-mercaptohexadecanoic acid (Sigma Aldrich, 90%, 0.0347 M) (with the necessary volume of tetrabutyl ammonium hydroxide solution, technical, ~40% in H 2 O,until neutralization) were added to a 5.4 mL of ultrapure water under vigorous stirring. Next, 500 µL of HAuCl 4 ·3H 2 O(Sigma Aldrich, 99.999% trace metal bases) solution (0.0147 M) were added to the mixture followed by 250 µL of AgNO 3 (Sigma Aldrich, 99.9999% trace metal bases) solution (0.0147 M) and stirred for five minutes, after which 750 µL of NaBH 4 (Sigma Aldrich, granular, 99,99% trace metal bases) 0.05 M were injected and immediately incubated at 35 °C. In a like manner, silver and gold monometallic clusters were prepared by adding 750 µL of the correspondent metal precursor solution (AgNO 3 and HAuCl 4 ·3H 2 O respectively) in order to maintain the ligand-to-metal ratio constant. The mixtures were left to react until no changes in absorption spectra were observed. Then, the samples were purified by precipitating them with isopropanol 3 times followed by their resuspension in ultrapure water.
Optical Measurements. Absorption spectra were collected with a Cary Varian 50 spectrophotometer at normal incidence in 1 mm quartz Suprasil cuvettes (bandpass 1 nm). PL measurements were performed at room temperature using a Varian Eclipse spectrometer with a Xenon lamp as a continuous wave light source. To measure the PL quantum yield (QY), a solution of 9,10 diphenyl-anthracene in tetrahydrofuran (10 −6 M) was used as fluorescence standard 63 . Time-resolved PL profiles were recorded with an Edinburg Instruments FLS 980 spectrometer using a 3.65 eV EP-LED as excitation source (pulse width 900 ps). All spectra were corrected for the instrumental response. Fluorescence micrographs were collected using a Canon EOS 400D camera coupled to a Nikon Ti-U inverted microscope. Samples were excited with a Xenon lamp whose emission was spectrally filtered with a DAPI excitation filter (320-400 nm).
Scientific RepoRts | 7: 5976 | DOI:10.1038/s41598-017-05156-9 Mass Spectrometry. Electrospray-ionization mass spectrometry (ESI-MS) experiments in positive-ion mode were performed on a hybrid quadrupole/time-of-flight (qTOF) instrument equipped with a nanoelectrospray ion source (AB Sciex, ForsterCity, CA, USA). The samples were centrifuged at 14.000 × g for 5 minutes in order to remove insoluble materials. The resulting supernatants were diluted 1:1 in acetonitrile and infused by borosilicate-coated capillaries of 1 μm internal diameter (Thermo Fisher Scientific, Waltham, MA USA). The main instrumental parameters were: ion-spray voltage 1.1 kV; curtain gas 20 PSI; declustering potential 80 V. The recorded spectra were averaged over 1 minute acquisition time. The simulation of the isotopic distribution was performed by the software IsoPro 3.1 (Software Tools for Mass Spectrometry). X-ray Photoelectron Spectroscopy. XPS measurements were performed using a Kratos Axis Ultra DLD spectrometer with monochromatic Al Kα source operated at 15 kV and 10 mA. The specimen for XPS was prepared by drop casting 200 μl of a clean and concentrated solution onto a silicon wafer. All the analyses were performed over an area of 300 × 700 microns. High-resolution analyses were carried out with a pass energy of 10 eV. In order to minimize the damaging of the organic ligands, fast spectra were collected on several areas of the specimen (acquisition time no longer than 8 minutes on each area) and successively averaged. The Kratos charge neutralizer system was used during data acquisition. Spectra have been charge corrected to the main line of the carbon 1 s spectrum set to 284.8 eV (C-C bond). Spectra were analyzed using Casa XPS software (version 2.3.16). From a quantitative point of view, it is possible to estimate the Au:Ag atomic ratio in the specimen by calculating the ratio between the areas under the Au-4f and Ag-3d profiles, after normalization to the relative sensitivity factors (RSF), which depend on the cross sections for photoemission. For reference RSF (Au-4f) = 6.25 while RSF(Ag 3d) = 5.987. Such procedure yields a gold-to-silver ratio Au:Ag = 18:37 (~0.5:1) and 28:19 (~1.5:1) after 10 hours and 80 hours of reaction, respectively.
Staining and Biocompatibility Tests. NIH/3T3 cells (ATCC ® CRL-1658 ™ ) were thawed and plated on cell culture dish in DMEM high glucose (Gibco) supplemented with 10% FBS (Euroclone) for 48 hours before use. For immunofluorescence experiments, the cells were plated in a 12 multi-well plate at a density of 5 × 10 4 cells/ well; in each well a round cover glass slide was added in order to growth the cells on its surface and then visualise them by confocal microscopy. Before seeding, cells were stained with CyTRAK, following the producer protocol. Briefly, cells were centrifuged twice in serum free medium (400 g, 5 minutes) and then stained for 5 minutes with Dye Solution, as reported in datasheet. Cells were then rinsed twice in complete medium and seeded at the right density after checking fluorescence. After 24 hours the nanoparticles were added to the cells at the concentration indicated and, at 24, 48 and 72 hours, CyTRAK staining was performed in order to visualize cell nuclei and. Cover glass were then mounted with a 1:1 v/v glycerol-PBS solution.
MTT Test. For proliferation experiments, cells were seeded in a 96 multiwell at a density of 3 × 10 3 cells/well in triplicate; after 24 hours nanoparticles were added to the cell medium and at 24, 48 and 72 hours and 96 hours an MTT test was performed (Methylthiazolyldiphenyl-tetrazolium bromide, Sigma). Briefly: a 50 μg/ml MTT solution was added to the samples; after 4 hours of incubation at 37 °C the medium was removed, the converted dye solubilised with DMSO (dimethylsulfoxide, Sigma) and the absorbance measured at 560 nm (GloMax Discover, Promega). Unstained cells in growth medium were used as control condition. The proliferation test under oxidative stress has been performed by adding menadione (Sigma Aldrich, C 6 H 4 (CO) 2 C 2 H(CH 3 ) to prompt cell apoptosis progressively increasing the intracellular level of ROS.

ROS Test.
For the evaluation of the Reactive Oxygen Species (ROS) produced and released in vitro, cells were seeded as described before for the MTT test; the analysis were performed 24, 48, 72 and 96 hours after the addition of clusters in culture medium; a ROS-Glo ™ H 2 O 2 Assay (Promega) was used, following the producer protocol. Unstained cells are reported as negative control. The Non-Lytic assay was applied and the relative luminescence units were measured by a plate reader (GloMax Discover, Promega). For solution studies, triplicate samples of metallic clusters in water (7 μM) were tested using ROS-Glo ™ H 2 O 2 Assay (Promega), following the manufacturers' protocol. An opaque white 96 wells plate was loaded with 50 μl of each sample dispersed in H 2 O 2 (5 mM) saturated water, followed by the incubation with H 2 O 2 substrate solution and ROS-Glo detection solution. After 20 min incubation at room temperature, the luminescence was measure by GloMax Discover (Promega) plate reader. The luminescence was recorded continuously every 15 minutes for 5 hours.


The role of exosomes in the pathogenesis of Alzheimer’ disease

Exosomes are small vesicles secreted by most cell types including neurons that function in intercellular communication through transfer of their cargo or encapsulate and eliminate unnecessary cellular components and therefore have a broad impact on nerve development, activation and regeneration. In addition, exosomes have been observed to be involved in spreading pathological misfolded proteins, thereby leading to the onset and propagation of disease. Alzheimer disease (AD) is the most common form of dementia and characterized by two types of lesions: amyloid plaques and neurofibrillary tangles. Accumulating evidence has demonstrated that exosomes are associated with amyloid precursor (APP) and Tau proteins and play a controversial role in Alzheimer’s disease process. In this review, we will discuss the role of exosomes in the metabolism and secretion of APP and Tau proteins and their subsequent impact on AD pathogenesis.

Background
According to the 2016 World Alzheimer Report, there are 47 million people living with dementia worldwide [1]. It is estimated that the total worldwide cost of dementia is $818 billion (USD) and is expected to reach $1 trillion (USD) by 2018, thus placing a huge burden on individuals, families, and societies [1]. As the leading cause of dementia, Alzheimer's disease (AD) accounts for an estimated 60 to 80% of all cases [2]. It is clinically characterized by cognitive impairment, a variety of neuropsychiatric symptoms and the restriction of daily life activities [3]. AD is pathologically defined by the deposits of the protein fragment beta-amyloid (Aβ plaques) outside neurons and twisted fibers of the protein tau that build up inside neurons (NFTs). The cause for most AD cases is still uncovered except for 1 to 5% of cases which develop as a result of mutations in the presenilin1 (PSEN1), presenilin2 (PSEN2), or amyloid precursor protein (APP) genes [4].
Recently, the role of "Prion-like mechanisms" in the pathogenesis of neurodegenerative diseases has attracted more and more attention. It has been suggested that pathologically misfolded proteins can transfer their conformation to properly folded proteins, thus resulting in the propagation of disease [5]. For instance, plaques and tangles tend to spread through the cortex in a predictable pattern as Alzheimer's disease progresses [6]. While, the mechanisms underlying the spread of misfolded proteins still poorly understood. There are several pathways for signal delivery and material communication between cells, such as synaptic transmission, direct communication trough gap junction and paracrine signaling [7]. Among these hypotheses, accumulating evidence supports the idea that exosomes may play as a messenger to participate in cell communication and contribute to this lesions spreading [8,9].
Exosomes were first reported in reticulocytes and considered to function in the disposal of unnecessary cellular components [10,11]. Exosomes are nanosized extracellular vesicles (generally 50-100 nm diameter) that can be released by nearly all cell types, including neuronal cells [12]. The exosomes' molecular contents include proteins, lipids and genetic material. Exosomes are released in bodily fluids and shuttle molecules for long distances for the purpose of intercellular communication. Exosomes have been reported to implicate in the spread of pathological proteins involved in neurodegenerative diseases, such as AD, Parkinson's disease (PD) and the prion diseases. APP, βsecretase, γsecretase has been detected in exosomes, what's more, exosomal proteins such as Alix and Flotillin were also found to be accumulated in the plaques of AD patient brains [13].
In this review, we will discuss role of exosomes in the metabolism and secretion of APP and Tau proteins and the subsequent impact on AD pathogenesis.

Biogenesis of exosomes
Exosomes are small membrane vesicles that are generated via endocytic pathways [14,15]. Inward budding of the plasma membrane forms small vesicles, which undergo fused together to form the early endosome. Intraluminal vesicles (ILVs) begin to compose through invagination of the limiting endosomal membrane during the maturation process of early endosome. Upon creation, cytoplasmic molecules such as proteins, lipids, and RNAs are encapsuled into the lumen and accumulated within the late endosome, thus forming multivesicular bodies (MVBs). There are two fates for MVBs, some of which transport to lysosomes for degradation (dMVBs), while others fuse with the plasma membrane and release ILVs into the extracellular space as exosomes (sMVBs). Compared with the dMVBs which are enriched in bismonoacylglycerophoshate (BMP, LBPA), the sMVBs contain more of ceramides [16,17]. ILV formation is the key step in exosome biogenesis [18]. The formation of ILVs is mainly regulated by the complex of multi-molecular machinery named Endosomal Sorting Complex Required for Transport (ESCRT) [19,20]. However, studies have shown that depletion of ESCRT subunits does not totally impair the composition of MVBs, which indicate that other mechanisms may exist in the process of ILVs formation [21]. It suggested that proper level of lipids and tetraspanin-enriched micro-domains is needed for MVBs formation [22][23][24][25]. Exosome secretion is also regulated by membrane depolarization.

Molecular contents of exosomes
The molecules within exosomes can be divided into two types: constitutive molecules and cargo molecules. Constitutive molecules are unique to exosomes regardless of the cell type from which they are derived and play an essential role in keeping fundamental structures and functions of exosomes. Cargo molecules, on the other hand, are proteins, lipids and genetic material which are sorted, encapsulated and transported by exosomes. The cargo molecules are variable according to cell origin and the physiological or pathological conditions when exosomes generate. In addition, sorting of molecules into exosomes is thought to be a selective process, since some accumulated factors observed in exosomes are barely detectable in parental cells.
In addition to proteins and lipids, genetic materials are also found in exosomes, such as DNA, mRNA, miRNA, ribosomal RNA (rRNA), circular RNA, and long noncoding RNA (lnRNA) [37][38][39][40][41][42]. Among them, small RNA (<30 nucleotides) account for a large proportion, making up >50% of all exosomal RNA species [38,40,43]. However, a few studies have shown different results in which ribosomal RNA, in particular 28S and 18S rRNA subunits, were found to be the major class of RNA in exosomes [39]. These conflicting results may be due to the purity of the exosome preparation and differences in cell origin. It has been shown that exosome RNA is functional. Valadi and colleagues detected the expression of mouse proteins after transfer of mouse exosomal RNA to human mast cells [43]. What' more previous studies showed that miR-222 transferred through exosome was able to increase tumor malignancy in melanoma through suppression of p27Kip1 expression and induction of the PI3K/AKT pathway [44].

Function of exosomes in the central nervous system (CNS)
Exosomes can be released by most cell types in the CNS, such as neurons, astrocytes, oligodendrocytes and microglia, and participate in regulating neuronal development, regeneration, and modulation of synaptic functions [45][46][47]. The main physiological roles of exosomes include eliminating cellular waste, regulating immune response and communicating between neural cells [20,48,49]. Once released into extracellular space, exosomes act as messengers, can be captured by neighboring cells or internalized by cells with a certain distance, or enter body fluids and taken up by different tissues [50]. There are several ways for signal transduction mediated by exosomes, such as receptor-ligand pathway, endocytosis and phagocytosis [51]. Because of the double membrane structure, exosomes pathway may have a higher efficiency in transfer substance.
In CNS, both glia and neuron secrete exosomes is regulated by glutamate in a certain degree. It has been hypothesized that exosomes can be served as messenger to mediate the communication between neuron and glia. While, as the reported, exosomes derived from neurons can only be captured by neurons, but not glia. It is interesting to note exosomes secreted by neuroblastoma cells can bind with both of neurons and glial cells. It demonstrate that cell communication mediated by exosomes has cell-selectivity [52].
In addition, the function of exosomes may be variable among different cell origins. Evidence shows that exosomes derived from N2a cells or isolated from human cerebrospinal fluid can abolish the synaptic plasticity disruption caused by both synthetic and AD brainderived Aβ [53]. However, Asai and colleagues observed that exosomes derived from microglia can spread tau protein, and inhibiting exosome synthesis significantly reduced tau propagation in vitro and in vivo [47].
Except the physiological function, the role of exosomes in spreading toxic proteins and inducing the propagation of diseases such as AD has been discussed extensively.

Impact of exosomes on amyloidogenic processing of APP
The major component of amyloid deposits is small peptides, 39-43 amino acids in length named Aβ, which is derived from a sequence of successive cleavages of APP [54]. APP is a type-I transmembrane glycoproteins. Three secretases termed α, β and γ-secretases are involved in the metabolism of APP. In the amyloidogenic pathway, upon cleavage by β-secretase (BACE-1) and γ-secretase, a large soluble ectodomain fragment (sAPP-β), membranebound C-terminal fragment (β-CTF), a small APP intracellular fragment (AICD) and Aβ peptides are produced [55,56]. In the non-amyloidogenic pathway, APP is initially cleaved at the α-secretase site, generating sAPP-α and α-CTF. The latter is further processed by the γ-secretase complex, releasing AICD and a p3 peptide [57]. β-cleavage of APP mainly occurs in early endosomes [51,54]. Immunofluorescence experiments in HeLa cells (APP mutant) observed the colocalization of sAPPβ, APP and BACE with early endosomal markers (Rab5) and early endosomal antigen-1 [51]. It has been found that Aβ is accumulated in MVBs and can be released into extracellular space through exosomes [13,58]. Although only a very small portion of Aβ (<1%) is associated with exosomes, APP, βand γsecretase have been detected in exosomes, suggesting that except transport Aβ peptide in the extracellular space, cleavage of APP to generate Aβ could be the main mechanism of spreading lesions [8,48,59].
However, the exact role of exosomes in AD progress is still controversial. Several studies have observed that exosomes play a harmful role. A unique Aβ species, tightly binding with GM1, was found in brains of early pathological stage of AD [60]. Endocytic pathway impairment in neurons, including the enlargement of early endosomes and the up-regulation of Rab5 was observed in the brain of a patient with sporadic AD. This impairment significantly accelerated the release of GM1associated exosomes and induced amyloid fibril formation [61]. Furthermore, exosomes mediate the apoptosis of astrocytes caused by Aβ exposure. Wang and colleagues found that amyloid peptides could activate neutral sphingomyelinase 2 (nSMase2) and induce an increase of PAR-4 and ceramide-containing exosomes secretion in astrocytes. The exosomes were able to be captured by astrocytes and cause apoptosis [62]. Alternatively, fewer amyloid plaques were observed in a mouse model of AD after injection of GW4869, an inhibitor of nSMase2, through prevention of the secretion of exosomes [63]. The protective function of exosomes was also found in various studies. Neuronal exosomes rich in glycosphingolipids could capture Aβ and promote uptake of Aβ by microglia, thus decreasing Aβ and amyloid deposition in APP transgenic mice [64][65][66]. The cellular prion protein (PrP C ), a glycosylphosphatidylinositol (GPI)-anchored surface glycoprotein highly expressed on exosomes, was shown to bind oligomeric Aβ42 with high affinity via its flexible N-terminus and accelerate fibrillization of amyloid beta, thereby reducing the neurotoxic effects imparted by oligomeric Aβ [53,67]. It should be noted that exosomes utilized as protective agents in recent studies almost always come from healthy cells.
In brief, exosomes may serve as a double-edged sword in Aβ metabolism. The imbalanced metabolism of APP may cause accumulation of intracellular Aβ. When beyond the clearance capacity of lysosomes or glial cells, the toxic protein will be released into extracellular space and spread through the brain via the exosome pathway.

Impact of exosomes on tau pathology
Hyperphosphorylated tau proteins are the major components of NFTs [68]. Tau protein is a member of the family of microtubule-associated proteins encoded by the MAPT gene. Because of the alternative splicing of exon 10, there are two major tau isoforms in the adult brain, denoted as 3R and 4R [5,69]. An abnormal 3R/4R balance is thought to impair the function of tau in keeping stabilization of microtubule structure and material transport [70]. Differences in 3R/4R expression also exist among different diseases. For instance, 3R is the main tau isoform in Pick's disease, while the 4R tau isoform is a significant component of inclusions in progressive supranuclear palsy (PSP) and corticobasal degeneration (CBD) [71,72]. In AD, two major tau isoforms are present in the filaments [73]. Tau pathology developed within a definite pattern in AD. The first involved region is entorhinal cortex (Braak stages I-II), then developed to limbic areas (Braak stages III-IV), finally reaches neocortical areas (Braak stages V and IV) [6]. The mechanism of this spreading characteristic of tauopathy throughout human brain has been discussed many years. There is accumulating evidence that tau aggregates spread and replicate in a prion-like manner, with the uptake of pathological tau causing misfolded aggregations of monomeric tau in recipient cells [74,75]. Exosomemediated secretion pathways may play an important role in this progress. Studies showed that tau can be exported via an exosome-mediated mechanism in the M1C neuroblastoma tauopathy model, where it is enriched in a phospho-tau biomarker for early AD (AT270). In addition, exosome-associated tau is also present in human CSF samples [76]. Previous studies discovered that propagation of mutant tau between brain regions depended on the presence of microglia, the resident phagocytes of the brain. Microglia spread tau via exosome secretion and depletion of microglia or inhibition of exosome synthesis significantly reduced tau propagation in vitro and in vivo [47]. Polanco and colleagues detected tau in exosomes from tau transgenic rTg4510 mice, and these vesicles were capable of seeding tau aggregation in a threshold-dependent manner [32].

Conclusions
Increased attention has been paid to the prion-like mechanism involved in the propagation of AD. In this review, we have illustrated the biogenesis and function of exosomes and their impact on amyloidogenic processing and tau pathology. The exosomes pathway may have a "double-edged sword" effect on the process of AD. And the effect is dependent on the cell origins of exosomes and the conditions when exosomes formed. The identification of exosomal pathways could provide not only important insights in the pathogenesis of AD, but due to the tissue-specificity and non-immunogenicity of exosomes, could also serve as an ideal platform for delivery of therapeutic drugs. Furthermore, the molecules packaged in exosomes can be secreted into a variety of bodily fluids, which may serve as biomarkers of disease. Despite the potential benefits of exosomes in diagnosis and therapy, there are some remaining issues, such as making improvements in exosomal isolation techniques and developing a more thorough understanding of the role of exosomes from different cell types under different conditions, which should be the focus of future studies.


s2fieldsofstudy: Biology Medicine

text:
A novel in vitro potency assay of antisera against Thai Naja kaouthia based on nicotinic acetylcholine receptor binding

Snake envenomation is an important medical problem. One of the hurdles in antivenom development is the in vivo assay of antivenom potency which is expensive, gives variable results and kills many animals. We report a novel in vitro assay involving the specific binding of the postsynaptic neurotoxins (PSNTs) of elapid snakes with purified Torpedo californica nicotinic acetylcholine receptor (nAChR). The potency of an antivenom is determined by its antibody ability to bind and neutralize the PSNT, thus preventing it from binding to nAChR. The PSNT of Naja kaouthia (NK3) was immobilized on microtiter wells and nAChR was added to bind with it. The in vitro IC50 of N. kaouthia venom that inhibited 50% of nAChR binding to the immobilized NK3 was determined. Varying concentrations of antisera against N. kaouthia were separately pre-incubated with 5xIC50 of N. kaouthia venom. The remaining free NK3 were incubated with nAChR before adding to the NK3 coated plates. The in vitro and in vivo median effective ratio, ER50s of 12 batches of antisera showed correlation (R 2) of 0.9809 (p < 0.0001). This in vitro assay should be applicable to antisera against other elapid venoms and should reduce the use of live animals and accelerate development of life-saving antivenoms.

Snake envenomation is an important medical problem especially in the developing world. It has been estimated that around 421,000-2.5 million people are envenomed annually with about 20,000-94,000 fatalities 1 . Antivenoms (AVs) are the rationale and the most effective therapy of snake envenomation. However, this serious public health problem has so far been neglected and effective, affordable antivenoms remain unavailable in many parts of the developing world. Recently, efforts from a number of research institutions are underway to solve this problem 2 .
In the development and production of an AV, at least two major steps are involved: an effective immunization program and the pre-clinical testing to assess the neutralizing potential of the AV against the lethal effects of homologous and heterologous venoms. The accepted AV potency assay is the standard murine lethality assay to determine the median lethal dose (LD 50 ) that estimates the lethality of the venom and the median effective dose (ED 50 ) of the AV 3,4 . For this in vivo assay, three to five mice per venom dose are used and the total of about 6 different doses are tested. Thus, about 30 mice are needed for the determination of the LD 50 of a venom. Similarly, about 30 mice are needed for the ED 50 determination of an AV against a venom. Therefore, a large number of mice will be used for the in vivo neutralization assays. For example, the number of mice required by European Pharmacopoeia using this method to test the activity of one European viper venom antiserum (LD 50 and ED 50 tests combined) against five venoms, is 374 mice per batch of antivenom 5 . Using this figure, testing a pan-specific AV against 27 different venoms 6 would require about 2,020 mice. The assay is very costly, laborious and can give highly variable results. Moreover, some lethality studies have been shown to be inconsistent, suggesting that rodent death may not measure relevant efficacy outcomes in humans 7 . Lastly, witnessing the suffering and death of a large number of animals is the most difficult part of the experiment for many. In Buddhist countries like Thailand, most, if not all, laboratory personnel and graduate students refuse to do such experiments. Thus, it is becoming increasingly difficult to perform the in vivo assay for ethical and religious, as well as regulatory reasons.
For the above reasons, various types of in vitro neutralization assays have been developed to be used in place of, or to reduce the number of the in vivo assay. It has been reported that venom toxicity and effectiveness of AV can be studied using the chick biventer cervicis preparation [8][9][10] . This assay was used for screening AVs against the neurotoxic effects of venoms 11 . However, this in vitro assay requires the preparation of chick biventer cervicis muscles and the assay is laborious and time-consuming. In vitro neutralization of some venom enzymatic activities have been studied for use in AV potency assay 12 . It was shown that the neutralization of phospholipase A 2 activities by antivenom against Micrurus nigrocinctus highly correlated with the in vivo neutralization activity. Also, the inhibition of indirect hemolytic activity induced by phospholipase A 2 was also shown to correlate well with the in vivo potency of a polyspecific antivenom 13 . However, these assays are applicable only to antivenoms directed against venoms with enzymatic activities that parallel the lethality of the venoms.
Numerous investigators have studied and reported the use of ELISA for AV potency determinations together with the correlation between the results of the in vitro and in vivo neutralization assays [14][15][16][17][18][19][20] . However, the "neutralizing potency" described as "in vitro ELISA titer" occasionally did not correlate well with the in vivo neutralizing activity. For example, Ibrahim and Farid 21 studied the lethality-neutralizing potency, ELISA antibody level and the avidity indexes of a polyvalent antivenom against seven snake venoms. They showed poor correlation between the in vivo and in vitro assays with the in vitro assays always giving high values. ELISA has at times been criticized on the grounds that the antigen-antibody 'binding' reaction measured cannot be assumed to be the same as the 'neutralization' reaction of the antigen.
Elapid snakes (cobras, kraits and mambas) produce the lethal postsynaptic neurotoxins (PSNTs) which bind specifically and quasi-irreversibly with nicotinic acetylcholine receptor (nAChR) at the muscle end-plate 22,23 . This binding results in the inhibition of neuromuscular transmission which can lead to respiratory arrest and death 23 . Thus, it should be possible to develop an in vitro functional assay to test antivenom against elapid venoms based on the ability of the antivenom antibodies to inhibit the binding of PSNTs to nAChR. Such an in vitro assay would closely mimic the lethality reactions of PSNTs of the elapids in vivo, in particular cobras (Naja sp.). Cobras are in general listed as Category 1 of medically important snakes throughout most parts of Asia and Africa 4,24 .
An in vitro potency assay based on PSNT binding to nAChR was previously studied for the venom of coral snake Micrurus nigrocinctus 12 . It was found that, the ED 50 of the horse antisera against M. nigrocinctus in neutralizing the lethal effect of the venom did not correlate with the antivenom ability to inhibit the nAChR-binding activity but correlated well with the inhibition of the venom phospholipase A 2 activity.
By using a different reaction scheme and conditions from that studied above 12 , we report here the development of a novel in vitro potency assay of monospecific antisera against the venom of the Thai monocled cobra Naja kaouthia based on nAChR binding. The assays gave excellent correlation (R 2 = 0.9809; p < 0.0001) with the corresponding in vivo assay using mice. This in vitro assay should be useful in reducing or partially replacing the in vivo assays used to test antivenoms against N. kaouthia and other elapid venoms.

Results
Development of the in vitro neutralization assay using nAChR-PSNT binding. The optimal concentrations of NK3, nAChR, rat anti-nAChR antibody and goat-anti-rat HRP conjugate in the in vitro potency assay. The optimal concentrations of NK3, nAChR, rat anti-nAChR antibody and goat-anti-rat HRP conjugate used in the in vitro potency assay were studied as described in Materials and Methods (step: pre-incubation 2). The results are shown in Fig. 1. As the concentration of NK3 used to coat the microtiter plate increased, the signal as measured by the OD 450nm increased. This was also observed when the concentration of nAchR used to bind the immobilized NK3 was increased. To economize on the nAChR available while obtaining reasonably high OD 450nm signal, it was decided to use 15 µg/ml of NK3 for coating the plates and 0.707 µg/ml of nAChR for binding to the NK3 coated plate. A 1:1600 dilution of rat anti-nAchR serum and a 1:4500 dilution of goat anti-rat-IgG conjugated HRP were used.
Inhibition of nAChR binding to NK3-coated plate by N. kaouthia venom or N. kaouthia cytotoxin. Crude N. kaouthia venom was used to determine the 50% inhibition of nAChR binding (in vitro IC 50 ). In the first step, crude N. kaouthia venom at various concentrations was incubated with the purified nAChR (0.707 µg/ml) for 1 hr at 25 °C. The solution was then transferred to NK3 coated plates. The concentration of the crude venom that reduced nAchR binding by 50% was defined as the IC 50 . The results (Fig. 2) showed that the IC 50 of N. kaouthia was 0.0281 µg/ml.
To study the specificity of the nAChR binding, N. kaouthia cytotoxin I at various concentrations (0.4875 to 0.0152 µg/ml) was tested as described above. It was shown (Fig. 2) that the cytotoxin had no effect on the nAChR binding. Thus, the inhibition reaction was specific to the postsynaptic neurotoxins.
Neutralization of N. kaouthia venom by horse monospecific antisera as determined by nAChR binding to the NK3-coated plate. Twelve horse monovalent anti-N. kaouthia sera were serially diluted 2-fold from 1:500 to 1:512,000 and the dilutions were separately incubated with 5xIC 50 of N kaouthia venom (1.4029 µg/ml) in the 'Pre-incubation 1' experiment. After ultrafiltration and 'Pre-incubation 2' , the reaction mixtures were added to the NK3-coated plates. The binding of the free nAChR to the plate was measured at OD 450nm and the results are shown in Fig. 3.
The in vitro median effective ratio (ER 50 s), expressed as µg of venom neutralized per µl of antiserum, and the in vivo ER 50 s (mg of venom neutralized per ml of antiserum) of the 12 horse antisera are shown in Table 1. The correlation coefficient, R, between the in vitro ER 50 s and the in vivo ER 50 s was 0.9904, and the coefficient of determination for the regression model was R 2 = 0.9809 (p < 0.0001), as shown in Fig. 4.

Discussion
It is reported here the first successful development of an in vitro potency assay for antiserum against an elapid snake based on nicotinic acetylcholine receptor binding. The reactions employed in the in vitro assay closely mimicked those of the in vivo toxicological reactions of the elapid postsynaptic neurotoxins. Unlike the ELISA which at times gave poor correlation with in vivo assay and has often been criticized in that the antibody binding did not necessarily result in toxin neutralization, the present in vitro assay involved the binding and neutralization by the antisera antibodies of the lethal snake toxins thus preventing them from binding to nAchR. The correlation     between the in vivo assay using mice and the developed in vitro assay was very high as supported by the correlation coefficient of R = 0.9904. An in vitro assay based on PSNT binding to nAchR was studied by Stiles 25,26 and Alape-Giron et al. 12,27 . Using an ELISA format, the bindings of purified nAchR from T. californica to the immobilized long and short PSNTs were shown to be specific. Furthermore, these researchers showed that horse antivenom against M. nigrocinctus nigrocinctus venom contained antibodies that inhibited the binding of the venom α-neurotoxins to purified nAchR, and reversed the binding of the toxins already complexed with the receptor 12 . The antivenom ED 50 in neutralizing the lethal effect of the venom was shown not to correlate with the antivenom's ability to inhibit the nAchR-binding activity (r = 0.34; p > 0.05) but correlated well with the inhibition of phospholipase A 2 activity. From these results, they concluded that the lethality of the venom was the result of the combined actions of various toxins 12 and recent proteomics results have shown that the short-chain α-neurotoxins are likely to play a leading role in the lethality induced by this venom 28 .
The in vitro assay reported here involved reaction schemes that were different from those reported by Alape-Giron et al. 12 . Two salient features of the present assay protocol were as follows.
First, the two crucial reactions i.e., the antibody-venom toxins reaction, and the toxin-nAChR reaction, were carried out in solution rather than on solid surface. This was to allow for total exposure of the reactants' surface residues resulting in more complete binding with their counterparts, and also to avoid any possible steric interactions between the two high molecular weight reactants (IgG and nAchR) which are likely to be more pronounced on solid surface.
Second, after the venom-antibody reaction in 'Pre-incubation 1' , the antibodies, free or toxin bound, were removed from the reaction mixture by ultrafiltration. This was important in that if these antibodies were not removed, any free excess antibody remaining in the 'Pre-incubation 1' reaction could react with the immobilized toxins in the final reaction resulting in reduced binding of nAChR (added in the later step) to the immobilized toxins. Furthermore, since the dissociation constant of toxin-antibody complexes are usually in the micromolar range while the toxin-nAchR dissociation constant is closer to nanomolar range 29 , it is conceivable that, without the ultrafiltration to remove the antibodies, the antibody-bound toxin might dissociate and form tighter complex with nAChR. These reactions would shift the equilibrium of the toxin-antibody reaction, and the measured amount of nAChR bound to the immobilized toxin would also be reduced.
The described assay procedure was thought to improve the reactions involved and to eliminate or minimize any inaccuracy of reactant concentrations measured; leading to highly correlated in vitro and in vivo results.
It should be noted in Fig. 3 that at higher concentrations of the antisera Nk-A Nk-B and Nk-I, the nAChR bindings were decreased. This phenomenon, often observed in immunoassays, is known as the 'prozone phenomenon' or 'hook effect' where, at excess concentration of antibody, immunochemical reactions e.g., hemagglutination, were inhibited or become less pronounce 30,31 .
The in vitro potency assay described here should be applicable to antivenoms against most, if not all, elapids whose venoms contain mainly or exclusively postsynaptic neurotoxins as major lethal components. However, the usefulness of the assay for some elapids producing other lethal toxins e.g., some Bungarus venoms may contain, in addition to PSNTs, presynaptic neurotoxins (β-neurotoxins) which are highly lethal 32,33 . Thus, this in vitro nAchR binding assay which worked well with antisera against the Naja venoms might not work as well with some of the Bungarus venoms, depending on the abundance of the β-neurotoxins present in the particular venom and their role to the overall neurotoxicity.
Since effective, affordable antivenoms against snake venoms remain unavailable in many parts of the world 2 , studies were being made to produce pan-specific antivenoms that cover multiple snake venoms from wide geographical areas 6 . Such a pan-specific antivenom could be produced in large volume and, due to the economy of scale, could be produced at low cost. However, in the development of such pan-specific antivenoms, a large number of mice would be needed to assay its efficacy against many homologous and heterologous venoms, and over the years, the cumulative number of mice used will be even more perturbing considering the need to repeat the assay from batch to batch of antivenom. With the developed in vitro assay described here, the development and production of poly-specific or pan-specific AVs should become easier and simpler. This should eventually result in saving the lives of mice and the victims of snake envenomation.
In conclusion, the assay should reduce the use of mice for potency assays for example, during the immunization program and/or fractionation process of antivenom production. In some cases, it may even replace the in vivo assay. The in vitro assay is less expensive, less biologically variable and could avoid the ethical and religious issues involved. The in vitro assay could facilitate the development and production of new and effective antivenoms, especially the pan-specific antivenoms which usually employ a large number of mice. The availability of new antivenoms combined with the reduction in production cost could, in turn, save the lives of more snakebite victims, which are mostly from the poorer regions of the world 34 .

Materials and Methods
Materials. Electroplaque tissue from Torpedo californica (Pacific electric ray) was obtained from Dr. Charles Winkler, Aquatic Research Consultants (San Pedro, CA, USA). Naja kaouthia (NK, formally known as Naja naja siamensis) venom from pool of several adult snakes of Thai origin and horse monovalent antisera against N. kaouthia was purchased from Queen Soavabha Memorial Institute (QSMI). Benzoquinonium dibromide was purchased from Santa Cruz (Dallas, TX, USA). Goat against rat IgG conjugated with horse radish peroxidase (HRP) was purchased from Abcam (SF, USA). N. kaouthia postsynaptic toxin 3 (NK3, formally known as N. n. siamensis toxin 3) was purified as described by Karlsson et al. 35 . N. kaouthia cytotoxin (CTX-I) was purified as described by Tan et al. 36 . N-hydroxysuccinamide-Sepharose (NHS-Sepharose) was from GE Health Care. All other reagents were from Sigma Chemical, St Louis. Missouri, unless stated otherwise.
SCIenTIFIC REpoRTS | 7: 8545 | DOI:10.1038/s41598-017-08962-3 Methods. Purification of nAChR and production of anti-nAChR antibody in rats. Purification of nAChR from T. californica electroplaque was carried out as described by Lindstorm et al. 37 . The purified receptor (10 µg) in 0.1 ml phosphate buffer saline (PBS) pH 7.4 was emulsified with Complete Freund adjuvant and injected subcutaneously into each of the eleven Wistar rats. The second and third immunizations were carried out using the receptor emulsified in Incomplete Freund adjuvant and alum as the adjuvant, respectively. Blood of each rat was collected from the heart at the end of the experiment.
In vivo neutralizing activity of horse monospecific antisera against N. kaouthia venom. The intravenous median lethal dose, LD 50 , of N. kaouthia venom, 0.18 (0.12-0.27) µg/g, was adapted from Tan et al. 38 of the same laboratory using the same batch of venom as with the current work. Neutralization of lethality was conducted as described by Ramos-Cerrillo et al. 39 . Briefly, a challenge dose of the venom constituting 5 LD 50 in 50 μl saline was pre-incubated at 37 °C for 30 min with varying dilutions of the pooled horse sera in normal saline, to give a total volume of 250 μl. The venom-antiserum mixture was subsequently injected into the caudal vein of the mice. The mice were allowed free access to food and water ad libitum and the number of survival after 48 h was recorded. The effective dose-50 (ED 50 ) was determined as the volume of antiserum that protected 50% of the challenged mice from death using probit analysis. The neutralizing efficacy of the antiserum was also expressed as median effective ratio (ER 50 ± 95% C.I. where C.I. is confidence interval) in mg venom/ml antiserum that gave 50% survival of the mice tested.
Development of the in vitro neutralization assay using nAChR-PSNT binding. Optimal conditions of nAChR, rat anti-nAChR antibody and goat-anti-rat HRP conjugate binding to NK3 coated microtiter plate. This assay was the basic assay format for in vitro binding of solubilized, purified nAChR to the elapid PSNTs immobilized on the microtiter plate. Briefly, purified NK3 at various concentrations were coated to the microtiter wells (Polystylene High Binding 3590, Costar). After washing with 0.05% TWEEN 20 in phosphate buffered saline (PBST), the plate was blocked with 200 µl/well of PBST and 1% BSA for 2 hr. The purified nAChR (in PBS containing 0.05% Tween 20 and 0.15 BSA) at various concentrations were added to bind the immobilized NK3 by incubation at 25 °C for 1 hour. After 3 time washings to remove the unbound nAChR, rat anti-nAChR serum at various concentrations was added and incubated at 25 °C for 1 hr; this was followed by addition of goat-anti-rat-HRP conjugate (ab7097, Abcam) and incubated for 1 hr at room temperature. After 4 washes with PBST, 100 µl/well of freshly prepared substrate solution (0.01% w/w 3,3′,5,5′-tetramethyl benzidine and 0.003% hydrogen peroxide in 0.075 M citrate buffer, pH 5.0) was added. The plate was allowed to stand in the dark for 30 min at 25 °C and the reaction was stopped by adding 25 µl of 4 N sulfuric acid. The absorbance of 450 nm was read against blank using an ELISA reader (Multiskan Go, Thermo Scientific). Optimal concentrations of NK3 (used for coating the plate), nAChR, rat anti-nAChR antibody and goat-anti-rat-HRP conjugate were estimated and used in the experiments that followed.
Inhibition of nAChR binding to the NK3 coated plate by N. kaouthia venom. The ability of an elapid venom (N. kaouthia) which contains PSNTs to inhibit the binding of nAChR to NK3 immobilized plate was studied and was expressed as IC 50 (venom concentration inhibiting 50% of the nAChR binding). In this assay, N. kaouthia crude venom at various concentrations was pre-incubated (25°C for 1 hr) with a fixed and optimal concentration of nAChR before the mixture was added to the NK3-coated plate and incubated at 25°C for 1 hour. This was followed by additions of rat anti-nAChR serum at 1:1600 dilution and incubated at 25°C for 1 hr, followed by 1:4500 diluted goat-anti-rat-HRP conjugate (Abcam) and incubated for 60 min at 25 °C. A parallel experiment using purified NK3 as the reference standard in place of the venom was also carried out. The concentration of the tested venom used in the pre-incubation step that inhibited 50% of the nAChR binding to the immobilized NK3 was the median inhibitory concentration (IC 50 ) of that venom.
Inhibition of the N. kaouthia venom PSNTs from binding to nAChR by horse antisera. Using a format similar to that described above, an in vitro assay of horse antiserum potency (in vitro ED 50 ) was carried out. Horse sera at various amounts (0.94 nl-0.96 µl) were pre-incubated at 37 °C for 1.5 hr with a fixed amount (5 × IC 50 ) of N. kaouthia venom in 137 mM NaCl, 2.68 mM KCl, 8.10 mM Na 2 HPO 4 , 1.47 mM KH 2 PO 4 , 0.05% TWEEN20, 0.1% w/v BSA in a total volume of 480 µl. This was referred to as 'Pre-incubation 1' . The mixture was then filtered through a 100 kDa MWCO ultrafiltration membrane (Amicon ® ) to remove antibody-toxin complexes, free antibodies and some other high molecular weight horse serum proteins. The filtrates (126 µl) containing the remaining free venom PSNTs were then incubated with an optimal amount of nAChR (14 µl in the same buffer) at 25 o C for 1 hr as described above and this was referred to as 'Pre-incubation 2' . The mixtures containing any remaining free nAchR were then added to the microtiter wells immobilized with NK3, followed by the rat anti-nAChR antibody, goat-anti-rat HRP conjugate, etc. The reaction products were then processed as described above. Wells incubated with a non-immune horse serum in place of antisera were included as background control.
The percentage of nAChR binding was then determined using the following formula: %nAChR binding (OD sample OD Ag control) 100 (OD max OD Ag control) = − × − 'OD max' represented the binding of nAChR (optimal amount) which was not pre-incubated with the venom or antiserum.
'OD Ag control' represented the binding of nAChR after being pre-incubated with 5 folds of IC 50 of N kaouthia venom (and without antiserum in 'Pre-incubation 1').
'OD sample' represented the binding of nAChR after nAChR (optimal amount) was pre-incubated with filtrate from 'Pre-incubation 1' (where 5 folds of IC 50 of N. kaouthia venom was pre-incubated with various amount of antiserum).
From the results, dose-response curves of horse sera volumes vs percents of nAChR binding were constructed. The in vitro neutralizing activities (ED 50 s) represented the horse antiserum volumes at which the nAChR binding was inhibited by 50 percent compared to wells incubated with buffer in place of antisera. The in vitro median effective ratio, ER 50 , represented µg venom/µl antiserum that the nAChR binding was inhibited by 50% was calculated. The results of the in vitro study on nAChR binding for every batch of the horse antisera (Nk-A to Nk-L) were presented as means ± S.D. of 4 determinations. Miscellaneous procedures. Protein concentration was determined by the procedure described by Lowry et al. 40 and by BCA Protein assay Kit (Pierce TM ) using bovine serum albumin as the standard. IC 50 and ED 50 values were determined using GraphPad Prism 5.0 program and BioStat 2009 version 5.8.3.0, respectively. The correlation analysis was evaluated by linear regression using GraphPad Prism 5.0 software. In brief, the correlation coefficient R was determined from the linear regression model, and R 2 (coefficient of determination) is the square of the correlation coefficient. An R 2 of 0.8-1.0 indicates that the regression line well fits the data in correlation. The statistical significance of the correlation test was set at p < 0.05.