Disaggregation Ability of Different Chelating Molecules on Copper Ion

22 Jul 2014 - ... Chinese Academy of Sciences, Beijing 100190, P. R. China ..... 405 nm (purple with shadows, right-hand bars) reflecting the aggregat...
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Disaggregation Ability of Different Chelating Molecules on Copper Ion-Triggered Amyloid Fibers Linyi Zhu, Yuchun Han,* Chengqian He, Xu Huang, and Yilin Wang* Key Laboratory of Colloid and Interface Science, Beijing National Laboratory for Molecular Sciences, Institute of Chemistry, Chinese Academy of Sciences, Beijing 100190, P. R. China S Supporting Information *

ABSTRACT: Dysfunctional interaction of amyloid-β (Aβ) with excess metal ions is proved to be related to the etiology of Alzheimer’s disease (AD). Using metal-binding compounds to reverse metal-triggered Aβ aggregation has become one of the potential therapies for AD. In this study, the ability of a carboxylic acid gemini surfactant (SDUC), a widely used metal chelator (EDTA), and an antifungal drug clioquinol (CQ) in reversing the Cu2+-triggered Aβ(1−40) fibers have been systematically studied by using turbidity essay, BCA essay, atomic force microscopy, transmission electron microscopy, and isothermal titration microcalorimetry. The results show that the binding affinity of Cu2+ with CQ, SDUC, and EDTA is in the order of CQ > EDTA > SDUC, while the disaggregation ability to Cu2+-triggered Aβ(1−40) fibers is in the order of CQ > SDUC > EDTA. Therefore, the disaggregation ability of chelators to the Aβ(1−40) fibers does not only depend on the binding affinity of the chelators with Cu2+. Strong selfassembly ability of SDUC and π−π interaction of the conjugate group of CQ also contributes toward the disaggregation of the Cu2+-triggered Aβ(1−40) fibers and result in the formation of mixed small aggregates.

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INTRODUCTION Alzheimer’s disease (AD) is one of neurodegenerative diseases. It is defined by the progressive neuronal cell loss, protein aggregation, and oxidative stress1,2 and characterized by the increased production of amyloid-β peptides (Aβ) and the presence of extracellular Aβ aggregates.3 Aβ peptides are derived from amyloid precursor protein (APP) containing 39− 43 amino acids. Although the pathogenesis of AD has not been entirely understood, the amyloid cascade hypothesis has been the dominant theory to explain the etiology.3 Both Aβ oligomers and fibrils participate in the formation of amyloid deposits, but the former turns out to be more neurotoxic than the latter.4,5 Further studies 6,7 found remarkably high concentrations of Zn2+, Fe3+, and Cu2+ ions in the neuritic plaques of AD brains, which are 3−5 times higher than those in healthy brains. It was reported that Zn2+ ions can accelerate the Aβ aggregation rapidly at physiological pH range, while Cu2+ ions show stronger acceleration effect under slightly acidic condition below pH 6.8.8,9 A growing number of evidence10,11 indicates that the presence of metal ions alters the kinetic pathway of amyloid-β, leading its aggregation away from stable fibrillar structure and resulting in more neurotoxic structures, and the recent evidence has been well reviewed by Hane and Leonenko.12 Furthermore, these metal ions are implicated in the formation of reactive oxygen species (ROS) in Aβ pathology.13−16 Aβ can produce H2O2 in the presence of Cu2+ and Fe3+ ions,17,18 and H2O2 reacts with these metal ions © 2014 American Chemical Society

to produce ROS through Fenton cycling, thus inducing oxidative stress.19,20 Although the detailed interaction mechanism of the metal ions with Aβ is still unclear, it is generally accepted that the metal ions can coordinate with Aβ at Nterminal residues, such as His6, His13, and His14 imidazole and the carbonyl groups, and in turn promote the aggregation of Aβ.13,21−24 Recent studies on the removal of Cu2+ from Cu2+-Aβ aggregates using chelating agents have suggested that the extent of Cu2+-induced Aβ aggregation and ROS production can be minimized by metal chelators.13,25−35 Metal chelators, such as ethylenediaminetetraacetic acid (EDTA),29 diethylenetriaminepentaacetic acid (DTPA),29 N1,N2-bis(pyridine-2-yl-methyl)ethane-1,2-diamine (ENDIP),34 and clioquinol (CQ), can inhibit and reverse Cu2+-triggered Aβ aggregation in vitro.36 EDTA and DTPA can bind to Cu2+ ions through their carboxylates and form complexes. ENDIP seizes Cu2+ ions through forming highly stable tetradentate complexes. CQ binds Cu2+ ions by nitrogen and oxygen donors, and it has shown promising results in early clinical trials of Alzheimer’s patients.33,36 Moreover several groups26,32,35 have worked on structural modification to create bifunctional chelating agents with the abilities of binding amyloid and chelating metal ions. Received: April 2, 2014 Revised: July 20, 2014 Published: July 22, 2014 9298

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For instance, Lim et al.32 synthesized two bifunctional molecules by incorporating nitrogen and/or oxygen donors into Aβ aggregate-imaging probes 125IMPY and p-125I-stilbene and found that they can modulate the generation of Cu2+triggered Aβ aggregates, promote their disaggregation, and finally reduced the toxicity arising from Cu2+-Aβ. Wang et al.35 synthesized two bifunctional platiniferous chelators, PC1 and PC2, taking cyclen as a metal chelating unit and Pt(bipyridine)Cl2 as an Aβ-binding unit. Both PC1 and PC2 show significant inhibition against Aβ aggregation and H2O2 formation. Lincoln et al.37 design a N-heterocyclic amine chelator pyclen, which can not only disaggregate Cu2+-triggered Aβ aggregates but also have antioxidant capacity in vivo and protective capabilities. Given the complexity of the metallobiology in Alzheimer’s disease, it is particularly challenging to design metal-binding agents that can mitigate damaging effects of metals while preserving their beneficial properties.30 For this purpose, Franz et al.30,31 designed and synthesized two prochelators: boronic ester-masked 8-hydroxyquinoline derivative (QBP) and peptide EVNLDAHFWADR (SWH). When QBP and SWH are activated by H2O2 and β-secretase (BACE), respectively, they can convert into 8-hydroxyquinoline (8HQ) and H2NDAHFWADR (CP). Both 8HQ and CP are able to sequester Cu2+ from Aβ and prevent Aβ aggregation or disassemble AβCu2+ aggregates. Because H2O2 and BACE normally exist in brain, this strategy imparts chelation specificity for Cu2+ only when H2O2 and BACE are in excess. The above studies highlight metal sequestration as a promising treatment for AD, indicating the potential requirement of developing new efficient agents to eliminate effects of metals on Aβ aggregation. In previous studies,38,39 we found that cationic gemini surfactant C12C6C12Br2 and tetrameric surfactant PATC (N1,N16-didodecyl-7,10-bis(3-(2-(dodecyldimethylammonio) ethylamino)-3-oxopropyl)-N 1 ,N1 ,N 16 ,N 16 -tetramethyl-4,13dioxo-3,7,10,14-tetraazahexadecane-1,16-diaminium tetrabromide) can disassemble mature Aβ(1−40) fibers through their strong self-assembly ability and electrostatic binding with Aβ(1−40). Previously, we synthesized a carboxylic acid gemini surfactant sodium 4,8-dioctyl-3,9-dioxo-6-hydroxy-4,8-diaza1,11-undecanedicarboxylate (SDUC) and studied its selfassembly in aqueous solution upon the addition of Cu2+ ions.40 Cu2+ ions can significantly affect the self-assembly of SDUC by coordinating its carboxylic groups. It was found that SDUC vesicles undergo a relatively fast fission process when a small amount of Cu2+ is added. So it is expected that the combination of chelating ability and strong self-assembling ability in SDUC can make SDUC effective in disassembling Cu2+-triggered Aβ fibers. Therefore, this work has comparatively studied the effects of SDUC, EDTA, and CQ on Cu 2+-triggered Aβ(1−40) aggregates. The molecular structures of the three chelators SDUC, EDTA, and CQ are shown in Figure 1. SDUC only has half number of carboxylic groups of EDTA. However, the

results show that SDUC can disassemble most of the Aβ(1− 40)/Cu2+ fibers with much higher efficiency than EDTA, though it is not as good as CQ. Moreover, CQ and EDTA have very close binding affinity with Cu2+ ions, but CQ exhibits much stronger ability in disaggregating Cu2+-triggered Aβ(1− 40) fibers than EDTA. So the disaggregation ability of chelators to Cu2+-triggered Aβ(1−40) fibers does not only depend on the binding affinity of the chelators with Cu2+. The possible mechanism will be discussed.

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EXPERIMENTAL SECTION Materials. Amyloid β(1−40) (trifluoroacetate salt) (Aβ(1− 40), code, 4307-v; lot, 590708) was purchased from Peptide Institute Inc. Japan). The purity of Aβ(1−40) was higher than 95%. Carboxylic acid gemini surfactant SDUC was synthesized and purified as described previously.40 The structure of SDUC was characterized by 1H NMR spectroscopy, mass spectroscopy, and the purity was verified by elemental analysis. 1,1,1,3,3,3Hexafluoro-2-propanol (HFIP) was from ACROS. CuCl2· 2H2O, NaCl, ethylenediaminetetraacetic acid (EDTA), clioquinol (CQ), and piperazine-N′ (2-ethanesulfonic acid) (HEPES) were purchased from Alfa Aesar. Glycine (Gly) was from Sigma-Aldrich. The purity of all the reagents was ≥99%. All the stock solutions were filtered through a 0.22 μm filters (Gelman Sciences, Ann Arbor, MI) to remove any particulate matter. All the experiments were carried out in 20 mM HEPES buffer of pH 7.4 with 150 mM NaCl unless stated otherwise. Pure water (18.2 MΩ·cm) was obtained from a Milli-Q equipment. Preparation of Aβ(1−40) Solution. Aβ(1−40) peptide (∼0.5 mg) was first dissolved in 600 μL of HFIP and incubated at room temperature for 1 h in sealed vials. Next, the solution was bath-sonicated for 30 min. Afterward, HFIP was removed by evaporation under a gentle stream of nitrogen. Then an aliquot of Aβ(1−40) was redissolved with 200 μL of DMSO and diluted in 4.5 mL of HEPES buffer. At last the solution was centrifuged at 13 000 rpm for 15 min. The supernatants were taken out to be used in the subsequent experiments. The DMSO concentration is less than 5% in the final Aβ(1−40) solution. The concentration of Aβ(1−40) was determined by Micro BCA protein assay (Pierce) and fixed to ∼25 μM. Turbidity Assays. The mixture solutions of 25 μM Aβ(1− 40) and 25 μM CuCl2 were added to individual wells of a 96well plate and incubated for 2 h at 37 °C with stationary oscillation to form Aβ(1−40)/Cu2+ fibers. After that, a small amount of concentrated SDUC, EDTA, or CQ (prepared in DMSO and the final amount of DMSO is less than 5%) solution was added into the Aβ(1−40)/Cu2+ fibers solution, keeping the final concentration of each chelator at 50 μM, which is lower than the critical micelles concentration (CMC) of SDUC (0.26 mM). Meanwhile the separate addition of HEPES buffer into the Aβ(1−40)/CuCl2 mixture solution was also performed as chelator-free control group. The above mixture solutions were again incubated for 2 h with stationary oscillation. Afterward, the solution turbidity was measured by the absorbance at 405 nm on a plate reader set to collect the data for 4 times with 30 s of shaking between each reading. The turbidity of the solutions was determined by averaging the data after subtracting the Aβ(1−40)-negative matched control from each well. The mixture solutions of 25 μM Aβ(1−40) and 50 μM SDUC as well as the fresh 25 μM Aβ(1−40) as control group were incubated in individual wells of a 96-well plate at 37 °C for 4 h. Then the turbidity was measured as described above.

Figure 1. Molecular structures of SDUC, EDTA, and CQ. 9299

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BCA Assays. BCA protein assay is a detergent-compatible formulation based on bicinchoninic acid (BCA) for the colorimetric detection and quantitation of total protein. 25 μM Aβ(1−40) solution was incubated with 25 μM CuCl2 in individual wells of a 96-well plate for 2 h at 37 °C with steady agitation to form Aβ(1−40)/Cu2+ fibers. After that, 10 μL of 1 mM chelator solution or HEPES buffer for the control group was added in each well with Aβ(1−40)/Cu2+ fibers and incubated for 2 h with constant agitation. The same samples after the turbidity assays were centrifuged for 15 min at 13 000 rpm, and then 20 μL of each supernatant was withdrawn for the Pierce MicroBCA assay by following the kit protocol. The soluble Aβ(1−40) in the solution was colorimetric detected. Isothermal Titration Microcalorimetry (ITC). A TAM 2277-201 isothermal titration microcalorimeter (Thermometric AB, Järfälla, Sweden) with a 1 mL stainless steel sample cell was used to measure the enthalpy change during the binding of the chelators with Cu2+ ions. The reference cell was loaded with buffer. The sample cell was initially loaded with 700 μL of chelator solution. Since Cu2+ ions easily hydrolyze into Cu(OH)2 precipitate at pH 7.4, CuCl2 was mixed with a weak ligand glycine to form Cu(Gly)2 as the titrant.41 Cu(Gly)2 was continuously injected into the stirred sample cell in portions of 5 μL using a 500 μL Hamilton syringe controlled by a Thermometric 612 Lund pump until the desired concentration range was covered. The system was stirred at 60 rpm with a gold propeller. The observed enthalpies (ΔHobs) were obtained by integrating the areas of the peaks in the plot of thermal power against time. All the measurements were conducted at 25.00 ± 0.01 °C. Atomic Force Microscopy (AFM). AFM morphology images were recorded by using a Multimode Nanoscope IIIa AFM (Digital Instruments, Santa Barbara, CA). The samples after the turbidity assays above were observed under AFM, which means the samples of Aβ(1−40)/Cu2+ fibers incubated with chelator solutions or only buffer (control group) for 2 h were used to prepare the AFM samples. To prepare specimens for AFM, 5−8 μL of each sample solution above was deposited onto freshly cleaved mica surface for 5−10 min. The sample was then briefly rinsed with Milli-Q water and dried with a gentle stream of nitrogen gas. All morphology images were recorded using a tapping mode at 512 × 512 pixel resolution and a scan speed of 1.0−1.5 Hz. Analysis of the images was carried out using the Digital Instruments Nanoscope Software (Version 512r2). All the samples were prepared and observed at least five times. Transmission Electron Microscopy (TEM). The same sample solutions imaged by AFM were also observed by TEM. The sample of 5 μL was dropped on glow-discharged grids (formvar/carbon 300 mesh, Electron Microscopy Sciences) and was kept for 2 min at room temperature. Excess samples were removed using filter paper and then washed twice with small amounts of pure water. Each grid was incubated with uranyl acetate (1% in pure water, 5 μL) for 1 min and then was blotted off and dried for 15 min at room temperature. The samples were imaged under FEI Tecnai 20 electron microscope (110 kV, 8000−12 000 magnification).

fraction of Aβ(1−40) which has not aggregated and precipitated from the solution, i.e., the soluble Aβ(1−40). These two methods are complementary and provide reliable results about the disaggregation ability of the chelators to the Cu2+-triggered Aβ(1−40) fibers. Both the turbidity and BCA results are shown in Figure 2.

Figure 2. Soluble Aβ(1−40) peptide percentages (blue, left-hand bars) from BCA assays and the turbidity absorbance values at 405 nm (purple with shadows, right-hand bars) reflecting the aggregate level in Aβ(1−40)/Cu2+ (control), Aβ(1−40)/Cu2+/CQ, Aβ(1−40)/Cu2+/ EDTA, and Aβ(1−40)/Cu2+/SDUC at 4 h time point. Error bars represent ±SD above and below the average absorbance value (determined in triplicate). ap < 0.05 vs Aβ(1−40)/Cu2+ group (4h), bp < 0.05 vs Aβ(1−40)/Cu2+/CQ group, cp < 0.05 vs Aβ(1−40)/Cu2+/ EDTA group, and dp < 0.05 vs Aβ(1−40)/Cu2+/SDUC group. The results of the Aβ(1−40)/Cu2+ control group at 2 h time point are also shown, indicating the initial state of Aβ(1−40) before the disaggregation.

After Aβ(1−40) was incubated with Cu2+ for 2 h at 37 °C with steady agitation, the absorbance value of the Aβ(1−40)/ Cu2+ fiber solution is 0.028 ± 0.010 (not shown) with a big deviation about 36%, which is due to the formation of long fibers and the resultant nonuniform solution. The remaining soluble peptide is ∼20%. Both the turbidity results and soluble peptide suggest Aβ(1−40)/Cu2+ fibers have been formed after 2 h incubation before the additions of SDUC, EDTA, CQ, and buffer (for control group). Incubated for a further 2 h, the absorbance of the Aβ(1−40)/Cu2+ mixture solution without chelators (control group) is 0.026 with a small deviation, which suggests that the Aβ(1−40)/Cu2+ mixture may become more homogeneous or slightly grow with time. The additions of CQ, SDUC, and EDTA reduce the absorbance values to 0.005, 0.012, and 0.022, respectively. This means that the aggregation levels of Aβ(1−40) increase in the order of Aβ(1−40)/Cu2+/ CQ, Aβ(1−40)/Cu2+/SDUC, Aβ(1−40)/Cu2+/EDTA, but their aggregation levels are lower than the Aβ(1−40)/Cu2+ mixture both at 2 and 4 h time point. Meanwhile, the amount of remaining soluble Aβ(1−40) determined by BCA assay decreases in the same order. The soluble Aβ(1−40) decreases to about 20% of original Aβ(1−40) amount after incubated with Cu2+ ions for 2 h, while the additions of CQ, SDUC, and EDTA into Aβ(1−40)/Cu2+ mixture solution enhance the amount of soluble Aβ(1−40) to more than 90%, 62%, and 40%, respectively. Apparently, the disaggregation ability of CQ, SDUC, and EDTA to Cu2+-triggered Aβ(1−40) fibers is in the order of CQ > SDUC > EDTA. In order to know the aggregate transitions of Aβ(1−40) induced by the additions of the chelators, the morphologies of the Aβ(1−40)/Cu2+ fibers incubated with these three chelators for 2 h were characterized by AFM and TEM, and the images are shown in Figure 3. For comparison, the morphologies of Aβ(1−40) with Cu2+ in the absence of chelators are also shown in Figure 3 (A1 and A2). Obviously, the Aβ(1−40)/Cu2+

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RESULTS AND DISCUSSION The disaggregation ability of SDUC, CQ, and EDTA to Cu2+triggered Aβ(1−40) fibers was monitored by turbidity assays and BCA assays. Turbidity assays reflect the aggregation levels of the Aβ(1−40)/Cu2+ mixture, while BCA assays report the 9300

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Figure 3. AFM and TEM images of Aβ(1−40)/Cu2+ fibers itself (A1, A2) and Aβ(1−40)/Cu2+ fibers incubated with CQ (B1, B2), EDTA (C1, C2), or SDUC (D1, D2) for 2 h. All of the AFM images are 5 × 5 μm2 in size and 10 nm in height. The scale bar of TEM indicates 1 μm. A1 and A2 are chelator-free control groups.

Figure 4. Variations of the observed enthalpy against the molar ratio of Cu2+ ions to the chelators and the corresponding thermodynamic fitting curves (red lines) for titrating Cu(Gly)2 into 50 μM CQ (a), EDTA (b), and SDUC (c) in 20 mM HEPES buffer at 25.00 ± 0.01 °C. The ITC data were analyzed with standard Marquardt methods in an ITC package.

mixture without chelator forms long fibers of several micrometers (A1 and A2). The Aβ(1−40)/Cu2+ fibers were even able to be observed by naked eye. After the Aβ(1−40)/Cu2+ mixture being treated with CQ, EDTA, or SDUC, the aggregates show obvious morphology transitions. For the case of Aβ(1−40)/Cu2+ fibers mixed with CQ, both AFM and TEM images (B1, B2) display small spherical aggregates with a diameter of 10−50 nm. Meanwhile, the Aβ(1−40)/Cu2+ fibers treated by EDTA change into a large amount of short and branched fibers (C1, C2), while the Aβ(1−40)/Cu2+ fibers being treated by SDUC (D1, D2) mainly exist as small spherical aggregates. The size of the spherical aggregates of Aβ(1−40)/Cu2+/SDUC is polydisperse, varying from a few nanometers (majority) to almost 100 nm (few). Besides them, there are still a little amount of short fibrils. Clearly, the size and amount of the Aβ(1−40)/Cu 2+ /SDUC aggregates are obviously smaller than those of Aβ(1−40)/Cu2+/EDTA fibers. Comparing to the Aβ(1−40)/Cu2+/CQ aggregates, some aggregates of the Aβ(1−40)/Cu2+/SDUC are much larger, although most of the Aβ(1−40)/Cu2+/SDUC aggregates are smaller, Overall, the visualized results are consistent with the turbidity and BCA results, again proving that the disaggregation ability of the chelators to Cu2+-triggered Aβ(1−40) fibers is in the order of CQ > SDUC > EDTA. As to the effect of CQ on the Cu2+-triggered Aβ(1−40) fibers, the AFM and TEM observations are basically consistent with the results of Lim and co-workers42 but show a little

difference. The results of Lim and co-workers indicate that CQ can only partially disaggregate Cu2+-triggered Aβ(1−40) fibers, and some fibers still exist in the end. However, no fibers were found in the present AFM and TEM images after CQ treatment. This is probably ascribed to the difference of experimental conditions. We performed all the experiments in physiological pH 7.4 instead of pH 6.6, which Lim and coworkers used. As mentioned before, Cu2+ ions accelerate Aβ aggregation more strikingly under weak acidic condition;8,9 thus, more mature fibers should be formed at pH 6.6, at which CQ cannot completely disaggregate all the mature fibers. As to previous consideration, the reason why the chelators show different abilities in disaggregating the Aβ(1−40)/Cu2+ fibers should be directly related with their abilities of seizing Cu2+ ions from the Aβ(1−40)/Cu2+ fibers. Because Cu2+ ions form complexes with Aβ(1−40) in the fibers, if chelators want to seize Cu2+ ions from the complexes, they should possess stronger binding ability with Cu2+ ions than Aβ(1−40). Previous studies32,42,43 reported the structural characterization of the CQ−Cu2+ complexes. CQ captures Cu2+ ions by the functional group OH and deprotonated N on the quinoline ring at a Cu2+/chelator molar ratio of 1:2.32 As to EDTA, it has four carboxy groups and forms planar hybrid coordination compound with a Cu2+ ion.44 SDUC only has two carboxy groups, and the carboxy groups are separated by a spacer group. As conjectured in the previous work,40 the carboxylic groups of each SDUC molecule might form complexes with Cu2+ ions 9301

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Table 1. Binding Stoichiometry (n), Binding Constant (K), and Binding Enthalpy Changes (ΔH) of Cu2+ with CQ and EDTA by Fitting ITC Curves Cu2+/CQ Cu2+/EDTA Cu2+/SDUC Cu2+/Aβ(1−40)a a

n

K

ΔH (kJ/mol)

0.08 ± 0.04 0.28 ± 0.02

(4.24 ± 0.16) × 106 (2.32 ± 0.23) × 106

−27.32 ± 0.17 −12.29 ± 0.15

(2.4 ± 0.2) × 109

From ref 33.

On the other hand, SDUC is a pH sensitive surfactant with two pKa (pKa1 = 6.0, pKa2 = 7.8).39 The protonation degree of SDUC changes with the pH of the solution. At lower pH, SDUC carries less charge, and the electrostatic attraction between SDUC and Cu2+ will be certainly decreased. Besides, the self-assembly behavior of SDUC will also be impacted by the pH. Therefore, if we measure the binding constant under weakly acidic condition, it cannot reflect the binding affinity of SDUC with Cu2+ at pH 7.4. Thus, the interaction of SDUC with Cu2+ ions can be only qualitatively discussed as follows. As described previously,39 when Cu2+ was added in the SDUC solution at pH 7.0, the curvature of the vesicles increased and smaller vesicles were extruded from the larger ones. These results indicate that Cu2+ ions can significantly affect the aggregation of SDUC. Cu2+ cannot interact with the hydrophobic chains of SDUC, so the aggregates transition indicates the existence of the binding interaction between Cu2+ and the hydrophilic headgroup of SDUC. At the present pH 7.4 (very close to the pH in ref 39), according to the pKa values, half number of the carboxylic acid groups of SDUC molecules are protonated and carry negative charges. So the negatively charged carboxylic acid groups of SDUC should interact with positively charged Cu2+ through electrostatic attraction. Meanwhile, as a functional group of many common metal chelators (such as EDTA, CDTA, and DTPA), carboxylate groups are capable of binding Cu2+. Correspondingly, the carboxylate group of SDUC may at least show weak chelating interaction with Cu2+. Overall, the interaction between the headgroups of SDUC and Cu2+ should exist, but the Cu2+/SDUC binding constant might be lower than that of Cu2+/glycine, which is 7.9 × 105 according to the literature.29 Therefore, the binding ability of these three chelators with Cu2+ ions is that CQ is slightly stronger than EDTA, while CQ and EDTA are much stronger than SDUC, i.e., CQ > EDTA > SDUC. Simon and co-workers45 obtained the binding constant of Cu2+ with Aβ(1−40) peptide through titrating Cu(Gly)2 solution into Aβ(1−40) solution at the same experimental conditions to our ITC experiment. The relative binding constant of Cu2+ with Aβ(1−40) is (2.4 ± 0.2) × 109, which is significantly higher than the binding constants of Cu2+ with EDTA, CQ, and SDUC. This shows that the binding affinity of Cu2+ with Aβ(1−40) is much stronger than that of Cu2+ with these three chelators. Therefore, it is not enough to disassemble Aβ(1−40)/Cu2+ fibers only by chelating effect of chelators with Cu2+ ions. According to the present results, it can be proposed that in the disaggregation progress the chelator CQ may interact with Aβ(1−40)/Cu2+ fibers in other ways than just chelating with Cu2+ ions. In particular, the weak binding ability of SDUC with Cu2+ ions manifests that the effective disaggregation ability of SDUC to Aβ(1−40)/Cu2+ fibers must rely on other interactions. If we could perform ITC experiments monitoring the disaggregation procedure of Aβ(1−40)/Cu2+ fibers by the

together with the carboxylic groups from neighboring SDUC molecules. The chelation mechanism of EDTA and SDUC with Cu2+ ions may be the same; i.e., they seize Cu2+ ions by their carboxylic groups. From theoretical analysis, the number of carboxylic groups of each EDTA is twice that of SDUC. So EDTA should possess much stronger chelating ability than SDUC. However, herein EDTA is in an inferior position in disaggregating Aβ(1−40)/Cu2+ fibers relative to SDUC. In order to understand this point, the following ITC experiments were carried out to obtain the binding constants of these chelators with Cu2+ ions. Since Cu2+ ions easily hydrolyze into Cu(OH)2 and precipitate in solution at pH 7.4, a weak ligand Gly was used to stabilize Cu2+ ions by forming Cu(Gly)2. All the ITC curves for titrating Cu(Gly)2 into CQ, EDTA, and SDUC in HEPES buffer are shown in Figure 4. The binding processes of both CQ and EDTA with Cu2+ ions start from large exothermic enthalpy, then abruptly decrease, and finally finish when the enthalpy values are very close to zero. However, the binding process of SDUC with Cu2+ ions just displays constant and very small exothermic enthalpy value. These results indicate that the binding of CQ and EDTA with Cu2+ ions is much stronger than that of SDUC. The ITC curves of CQ and EDTA with Cu2+ ions were analyzed with standard Marquardt methods in an ITC package (supplied by Microcal Inc.) by assuming the binding sites of the chelators with Cu2+ to be identical. The binding stoichiometry (n), binding constant (K), and binding enthalpy changes (ΔH) of Cu2+ with the chelators were extracted from fitting the ITC curves and are summarized in Table 1. This analysis method is described in the Supporting Information. It should be noted that the obtained binding parameters are relative values due to the competition binding of glycine with Cu2+. The relative binding constants of CQ and EDTA with Cu2+ are (4.24 ± 0.16) × 106 and (2.32 ± 0.23) × 106, respectively, which indicates that the binding affinity of CQ with Cu2+ is only slightly stronger than that of EDTA. However, the binding stoichiometry values (n) of Cu2+ with CQ and EDTA are 0.08 ± 0.04 and 0.28 ± 0.02, respectively, which means that EDTA is even able to bind more Cu2+ ions than CQ. For the case of SDUC, the ITC curve does not present the binding process of SDUC with Cu2+. The reason might be that the binding affinity of Cu2+ with SDUC is weaker than with glycine, resulting that Cu2+ ions are still bound with glycine when Cu(Gly)2 is added into the SDUC solution. If by measuring the interaction of SDUC with Cu2+ ions at weakly acidic condition the formation of Cu(OH)2 can be avoided, then Cu(Gly)2 will be not necessary. But the interactions of SDUC with Cu2+ ions at lower pH cannot reflect the interactions at pH 7.4 used in the study. On one hand, the formation of metal complexes with other ligands is often dependent on pH of the solutions because there is a competition between ligand−metal ion and ligand−proton. 9302

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three chelators, the interaction thermodynamic data might help to understand the above results. However, because the Aβ(1− 40)/Cu2+ aggregates grew with time, the aggregate growth might produce incessantly thermal fluctuation after the Aβ(1− 40)/Cu2+ mixture being loaded in the sample cell of calorimeter. Thus, we could not obtain stable and reproducible ITC curves. In the absence of available techniques to study the interactions in the complicated systems, we return to the molecular structures and properties of these three chelators to understand the obtained results. Each Aβ(1−40) molecule has 3.5 metal ion binding sites of various affinities.14,34 In the disaggregation experiment, the molar ratio of Cu2+ and Aβ(1− 40) was kept at 1:1 to incubate Aβ(1−40)/Cu2+ fibers; thus all added Cu2+ have been bound by Aβ(1−40). The Cu2+triggered Aβ(1−40) fibers are very large as shown in Figure 3A, even precipitate from solution. So the chelators need to get close to the binding sites of Aβ(1−40) to seize Cu2+ and affect the aggregation of Aβ(1−40)/Cu2+ mixture, which relies on electrostatic, hydrophilic, and hydrophobic nature of the chelators. CQ has a large hydrophobic conjugate group; thus, it prefers to escape from water environment into the hydrophobic microdomain of Aβ(1−40)/Cu2+ aggregates, which endowing CQ more chances to capture Cu2+. Because the binding affinity of CQ with Cu2+ is weaker than that of Aβ(1−40), CQ molecules may just bind with Cu2+ on Aβ(1− 40) rather than remove Cu2+ from Aβ(1−40). Then π−π interaction of the conjugate groups of CQ with other CQ molecules and with the conjugate groups in Aβ(1−40) may interfere the ordered structure of Aβ(1−40) fibers and lead to the reorganization of the mixtures. Thus, the fibers are disaggregated into small spherical mixed aggregates. However, EDTA is very hydrophilic and favors to stay in water phase; hence, its chances of entering the Aβ(1−40)/Cu2+ aggregates are less. Moreover, EDTA does not have other groups to interact with Aβ(1−40) except the carboxylate groups to chelate Cu2+ ions. Thus, EDTA shows very weak ability to disaggregate the Aβ(1−40)/Cu2+ fibers. As to SDUC, its difference from CQ and EDTA is that it is a gemini surfactant with two long hydrophobic chains and has strong self-assembly ability in solution. The hydrophobic chains of SDUC tend to join the hydrophobic microdomain of Aβ(1−40) aggregates, while the chelation groups of SDUC would like to chelate and/ or electrostatically bind with Cu2+ on Aβ(1−40). Although SDUC cannot separate Cu2+ ions from Aβ(1−40) due to its weak binding ability with Cu2+, SDUC molecules can be anchored on Aβ(1−40) through the binding with Cu2+ ions. The anchored SDUC molecules can self-assemble with each other because of the enhanced local concentration on Aβ(1− 40). Thus, the self-assembly of SDUC may disassemble the Aβ(1−40)/Cu2+ fibers and form mixed small aggregates of Aβ(1−40)/Cu2+/SDUC. The above discussion might raise the question whether SDUC is capable of affecting Aβ(1−40) aggregation in the absence of Cu2+ through its self-assembly ability. Based on our previous study,38 anionic surfactants (SDS and surfactin) cannot disassemble negatively charged Aβ(1− 40) fibers. As an anionic surfactant, SDUC also shows very weak interaction with Aβ(1−40). In the absence of Cu2+, turbidity and BCA assays were used to compare the aggregates level of Aβ(1−40) with or without SDUC. The result (Figure 5) shows the aggregation level of Aβ(1−40) and Aβ(1−40)/ SDUC mixture are equivalent, indicating that SDUC does not affect the aggregation of Aβ(1−40) without the participation of

Figure 5. Soluble Aβ(1−40) peptide percentages (blue, left-hand bars) from BCA assays and the turbidity absorbance values at 405 nm (purple with shadows, right-hand bars) reflecting the aggregate level in Aβ(1−40) and Aβ(1−40)/SDUC solution after incubated for 4 h. Error bars represent ±SD above and below the average absorbance value (determined in triplicate).

Cu2+. Besides, the ITC technique was also used to study the interaction between SDUC and Aβ(1−40), and the results are shown in Figure S1 of the Supporting Information. Comparing with the calorimetric titration curves of C12C6C12Br2 being titrated into Aβ(1−40), which reflects the strong interaction of cationic surfactant with Aβ(1−40), the enthalpy change for the interaction between SDUC and Aβ(1−40) is negligible. Overall, the interaction of SDUC with Aβ(1−40) is too weak to affect the aggregation of Aβ(1−40). Herein, the Cu2+ ions actually provide binding sites for anionic SDUC with Aβ(1− 40) so that the SDUC can stay on Aβ(1−40)/Cu2+ fibers and then affect the aggregation of Aβ(1−40). In summary, besides the chelating interaction with Cu2+ ions, chelators need other synergistic effects to disaggregate Cu2+-triggered Aβ(1−40) fibers. The proposed mechanism of disaggregating Cu2+triggered Aβ(1−40) fibers by the chelators is illustrated by simplified cartoons in Figure 6.

Figure 6. Proposed mechanism of disaggregating Cu2+-triggered Aβ(1−40) fibers by the chelators. CQ may bind with Cu2+ on Aβ(1− 40), and the π−π interaction of the conjugate groups of CQ molecules with the conjugate groups in Aβ(1−40) may interfere with the ordered structure of Aβ(1−40) fibers, leading to the reorganization of the mixtures and the formation of spherical mixed aggregates. SDUC may join the hydrophobic microdomain of Aβ(1−40) aggregates with its hydrophobic chains, while the chelation groups of SDUC may chelate and/or electrostatically bind with Cu2+ on Aβ(1−40) and make SDUC molecules anchored on Aβ(1−40). The enhanced local concentration of SDUC molecules may self-assemble and disassemble the Aβ(1− 40)/Cu2+ fibers, forming mixed small aggregates of Aβ(1−40)/Cu2+/ SDUC. 9303

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CONCLUSIONS In the present work, a carboxylic acid gemini surfactant SDUC, a widely used metal chelator EDTA, and an antifungal drug clioquinol CQ have been systematically compared in the abilities of disassembling Cu2+-triggered Aβ(1−40) fibers. The results show that the binding affinity of Cu2+ with CQ, SDUC, and EDTA is in the order of CQ > EDTA > SDUC, while the disaggregation ability to Cu2+-triggered Aβ(1−40) fibers is in the order of CQ > SDUC > EDTA. The anionic gemini surfactant SDUC can disassemble most of Cu2+ triggered Aβ(1−40) fibers, and it is much more efficient than EDTA, though less effective than CQ. Therefore, the disaggregation ability of chelators to the Aβ(1−40) fibers does not only depend on the binding affinity of the chelators with Cu2+. Besides the chelating interaction with Cu2+ ions, the chelators need other synergistic effects to disaggregate Cu2+-triggered Aβ(1−40) fibers. Strong self-assembly ability of SDUC and π−π interaction of the conjugate group of CQ may also contribute to the disaggregation of the Cu2+-triggered Aβ(1− 40) fibers and result in the formation of mixed small aggregates. Although CQ is very effective agent in disaggregating Cu2+triggered Aβ(1−40) fibers, it has very low solubility in water. However, SDUC can form vesicles at physiological pH; thus, it is a bifunctional amphiphile chelating agent, disassembling Cu2+-triggered Aβ(1−40) fibers and improving the solubility of other hydrophobic therapeutic agents (such as CQ). In conclusion, metal chelators for disassembling Cu2+-triggered Aβ(1−40) fibers can be further optimized by introducing additional amphiphilic nature and conjugate group.

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ASSOCIATED CONTENT

S Supporting Information *

ITC analysis process. This material is available free of charge via the Internet at http://pubs.acs.org.

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AUTHOR INFORMATION

Corresponding Authors

*E-mail: [email protected] (Y.L.W.). *E-mail: [email protected] (Y.H.). Notes

The authors declare no competing financial interest.

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ACKNOWLEDGMENTS We are grateful for financial support from the National Natural Science Foundation of China (21025313, 21003137, and 21321063).

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