DOI: 10.1002/chem.201400079
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& Functionalized Nanoparticles
Inhibition of Amyloid Fibril Growth and Dissolution of Amyloid Fibrils by Curcumin–Gold Nanoparticles Sharbari Palmal,[a] Amit Ranjan Maity,[a] Brijesh Kumar Singh,[b] Sreetama Basu,[b] Nihar R. Jana,*[b] and Nikhil R. Jana*[a]
form, curcumin is water-soluble and can efficiently interact with amyloid protein/peptide, offering enhanced performance in inhibiting amyloid fibrillation and dissolving amyloid fibrils. Our results imply that nanoparticle-based artificial molecular chaperones may offer a promising therapeutic approach to combat neurodegenerative disease.
Abstract: Inhibition of amyloid fibrillation and clearance of amyloid fibrils/plaques are essential for the prevention and treatment of various neurodegenerative disorders involving protein aggregation. Herein, we report curcumin-functionalized gold nanoparticles (Au-curcumin) of hydrodynamic diameter 10–25 nm, which serve to inhibit amyloid fibrillation and disintegrate/dissolve amyloid fibrils. In nanoparticle
Introduction
cles,[13, 15] anionic quantum dots,[16] a-synuclein-functionalized quantum dots,[14] and peptide-functionalized g-Fe2O3 nanoparticles.[17] In contrast, retardation of amyloid fibrillation has been reported for hydrophobic polymer nanoparticles,[19] N-acetyl-lcysteine-capped CdTe quantum dots,[20] peptide-functionalized g-Fe2O3 nanoparticles,[17] dipeptide-conjugated polymer nanoparticles,[18] peptide-capped protein microspheres,[21] thioglycolic acid-capped CdTe quantum dots,[22] dihydrolipoic acidcapped CdSe/ZnS quantum dots,[23] and fetal bovine serumcoated graphene oxide.[24] In addition, dose-dependent acceleration or inhibition of Ab fibrillation has been observed for amine-terminated polystyrene nanoparticles.[25] However, only a few studies have demonstrated the dissolution of preformed fibrils by nanoparticle-based systems.[26–28] For instance, peptide-functionalized gold nanoparticles[26] and thioflavin S-conjugated graphene oxide[27] have been used to dissolve amyloid fibrils under photothermal conditions. Anionic gold nanoparticles have also been shown to induce partial fibril dissociation.[28] These findings suggest that a nanoparticle-based approach might be a promising option for the treatment of various neurodegenerative diseases. To achieve this, the primary need is to develop biocompatible nanoparticle systems that can efficiently inhibit fibrillation and dissolve amyloid fibrils/ plaque. In order to obtain specific interactions with amyloid structures, nanoparticles have been functionalized with molecules of various affinities, such as a peptide,[26] sialic acid,[29, 30] thioflavin,[27] a stilbene derivative,[31] and curcumin.[32–43] Among these, curcumin is one of the most studied molecules and acts as a very effective molecular chaperone in relation to various human neurological disorders and is known to inhibit amyloid fibrillation and to disintegrate amyloid fibrils.[32–43] Curcumin (M.W. 368.38) is a natural polyphenolic compound extracted from the Indian spice turmeric.[44] The chemical name of curcumin is difurylmethane, and it is usually found alongside other
Amyloid protein fibrillation is a generic feature of various human neurological disorders, such as Alzheimer’s, Parkinson’s, and Huntington’s diseases.[1–8] Amyloid fibrils are ordered protein aggregates consisting of crossed b-sheet secondary structures.[1–8] They are highly neurotoxic, causing neuronal cell death. Alzheimer’s disease is the most devastating neurodegenerative disorder affecting millions of people worldwide.[6] Accumulation of fibrillar aggregates in the brain is the pathological hallmark of Alzheimer’s disease. b-Amyloid peptides (Ab1–40, Ab1–42), derived from amyloid precursor protein (APP) through b- and g-secretase-based proteolysis, are mainly responsible for fibrous plaque formation.[1] Currently, no definite diagnostic tool and no proper treatment exist for this disease. We envisage that inhibition of amyloid fibrillation and dissolution of fibrillar aggregates should offer a promising therapeutic approach towards Alzheimer’s disease. Recent reports have shown that nanoparticles can significantly influence fibrillation processes, depending on the particle size and surface functionality.[9–24] For instance, amyloid fibrillation is accelerated by bare carbon nanotubes,[11] bare cerium oxide,[11] bare TiO2 nanoparticles,[12] polymer particles,[11] polymer-coated quantum dots,[11] anionic gold nanoparti-
[a] S. Palmal, A. R. Maity, Dr. N. R. Jana Centre for Advanced Materials Indian Association for the Cultivation of Science Kolkata-700032 (India) E-mail: [email protected] [b] B. K. Singh, S. Basu, Prof. N. R. Jana National Brain Research Centre Manesar, Gurgaon-122050 (India) E-mail: [email protected] Supporting information for this article is available on the WWW under http://dx.doi.org/10.1002/chem.201400079. Chem. Eur. J. 2014, 20, 1 – 9
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Full Paper polyphenol compounds such as demethoxycurcumin, bisdemethoxycurcumin, and so on (see Figure S1 in the Supporting Information).[43, 45] However, curcumin is hydrophobic in nature and insoluble in water, which restricts its therapeutic application.[45] Thus, in vitro and in vivo inhibition of amyloid fibrillation by curcumin have been performed after synthesizing water-soluble curcumin-based conjugates with small molecules, polymers, and nanoparticles.[36, 40–42] However, to the best of our knowledge, there have not hitherto been any reports of the use of curcumin-based nanoparticles or polymers to dissolve amyloid fibrils/plaque, although there are preliminary reports that non-conjugated curcumin can disintegrate amyloid fibrils.[35] In the present study, we have prepared water-soluble curcumin-functionalized gold nanoparticles (Au-curcumin) and have found that they not only inhibit amyloid fibrillation but also disintegrate and dissolve amyloid fibrils without any external agent or force.
Figure 1. a) Optical properties and b) representative TEM image of Au-curcumin. The inset shows an image of a test tube containing an aqueous solution of Au-curcumin. Arrow indicates the curcumin absorption band at 420 nm.
(Figure 1 and Figures S3–S10 in the Supporting Information). Although curcumin is insoluble in water, Au-curcumin becomes water-soluble and displays an absorption band due to curcumin at 420 nm and an Au plasmon band at 535 nm. The high water-solubility of Au-curcumin can be ascribed to the presence of primary and secondary amine groups on the surface of the silica-coated Au nanoparticles as only some of the primary amine groups are used for conjugation. The presence of the curcumin band suggests that the molecule is covalently bound to the Au nanoparticle. The presence of free curcumin in Au-curcumin can be excluded as the samples were extensively washed with ethanol/chloroform (Figure S5). In the FTIR spectrum, the appearance of characteristic curcumin peaks with a C-O-C stretching vibration at around 1020 cm 1 and a C=O/C=C vibration at around 1506 cm 1 also supports the covalent attachment of curcumin-COOH to a gold nanoparticle. A fluorescamine test confirmed the conjugation of a fraction of the amine groups on the surface of the gold nanoparticles with curcumin-COOH (Figure S4). From the absorption band of curcumin and the Au plasmon band seen for Au-curcumin, the number of curcumin molecules linked to each Au nanoparticle has been tentatively estimated as 17 (Figure S6). The synthesis conditions were optimized to prepare Au-curcumin with high water-solubility (Figure S9). The good watersolubility of Au-curcumin is due to the high water-solubility of the silica-coated gold nanoparticles,[46] and causes the curcumin to remain in water. Transmission electron microscopy (TEM) revealed that the size of the core Au nanoparticles was around 3.5 nm, and dynamic light scattering showed the hydrodynamic diameter of Au-curcumin to be 10–25 nm, including the silica shell with attached curcumin moieties (Figure 1 b and Figures S7 and S8). In contrast, the hydrodynamic size of the silica-coated Au nanoparticles was 5–12 nm. The increase in hydrodynamic diameter of Au-curcumin can be attributed to partial crosslinking between particles during the conjugation process, as well as an increased agglomeration tendency of the nanoparticles due to the hydrophobic curcumin. The surface charge of Au-curcumin was determined as positive (+25–30 mV) in the pH range 2–7. This value is similar to that of the silica-coated Au nanoparticle before functionalization,[46]
Results and Discussion Synthesis and characterization of Au-curcumin nanoparticles Gold nanoparticles were selected for this work due to their low toxicity, plasmon-based strong optical properties suitable for detection/imaging, and well-established functionalization methods.[46, 47] The preparation strategy for Au-curcumin is shown in Scheme 1. Curcumin was converted to a monocarbox-
Scheme 1. Synthetic strategy for obtaining curcumin-functionalized Au nanoparticles (Au-curcumin; DMAP = 4-dimethylaminopyridine, EDC = N’-(3dimethylaminopropyl)-N-ethylcarbodiimide). The high water-solubility of Aucurcumin is due to the presence of primary and secondary amine groups on the surface of the silica shell.
ylic acid derivative (curcumin-COOH) at one of its phenolic OH groups[36] and then covalently linked with primary amine-terminated silica-coated Au nanoparticles[46] through conventional EDC coupling. The formation of curcumin-COOH was confirmed by HRMS and other methods (Figures S1 and S2 in the Supporting Information). The Au-curcumin nanoparticles were characterized by UV/Vis absorption and FTIR spectroscopies &
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Full Paper suggesting that a fraction of the amine groups reacted with curcumin and the rest remained free.
by an elongation phase that is extended from one to five days with increasing concentration of Au-curcumin. Fibrils formed from HEWL and Ab1–40 under different conditions were imaged by TEM (Figures 3 and 4). In addition, the average lengths of fibrils formed under different conditions Effect of Au-curcumin on amyloid fibrillation, fibril disintewere measured from the collection of TEM images, and their gration/dissolution, and fibril-induced cytotoxicity length distribution histograms are summarized in Figure 5. The The effect of Au-curcumin on amyloid formation was examined results clearly show that the lengths of the fibrils formed in using hen egg white lysozyme (HEWL) as a widely studied the presence of Au-curcumin were significantly smaller than [48] model amyloid protein as well as Ab1–40 as a real amyloid those of the fibrils formed in the absence of the nanoparticles. [1–8] peptide. In order to investigate the influence on the fibrillaFor example, HEWL fibrils of length 0.4–1.4 mm (average tion process, the monomeric amyloid protein or peptide was 0.8 mm) were formed in the absence of the nanoparticles, but incubated with Au-curcumin under fibril-forming conditions, were restricted to lengths < 0.2 mm (average 0.09 mm) in the and the fibrillation kinetics was monitored by a thioflavin T presence of Au-curcumin. Similarly, Ab1–40 fibrils of length 0.5– (ThT)-based fluorescence assay. ThT is an amyloid-specific ben5 mm (average 2.3 mm) were formed in the absence of the zothiazole dye, the fluorescence of which is enhanced through nanoparticles, but were again restricted to lengths < 0.2 mm binding with amyloid fibrils and is directly correlated with the (average 0.1 mm) in the presence of Au-curcumin. Control ex[49, 50] extent of fibrillation. We observed that Au-curcumin signifperiments were performed using Au nanoparticles without curicantly influenced the fibrillation process for both HEWL and cumin functionalization and using pure curcumin. The results Ab1–40 in a dose-dependent manner (Figure 2 and Figures S11– showed that pure curcumin or Au nanoparticles (without curS23 in the Supporting Information). In general, amyloid protein cumin functionalization) were less active or partially inhibited fibrillation follows a nucleation-dependent pathway that conamyloid fibrillation compared with a similar concentration of sists of three distinct steps, namely i) nucleation, ii) elongation/ Au-curcumin (Figures 3 b–d and 4 b–d). We performed another growth, and iii) equilibration/steady stage, as observed previcontrol fibrillation experiment using a mixture of Au nanoparti[11–24] ously. In HEWL fibrillation, the typical lag time for nucleacles and curcumin and compared the results with those obtion is around 30 min (which does not change in the presence tained using Au-curcumin, Au nanoparticles, and curcumin. of nanoparticles), and this is followed by an elongation phase The Au-curcumin showed a much better inhibitory effect than from 1 to 12 h. However, the rate of elongation becomes the sum of the individual effects of Au nanoparticles and curslower with increasing concentration of Au-curcumin. A similar cumin (Figure S20). These results prove that Au-curcumin is result was also found for Ab1–40. The nucleation process of more efficient than curcumin or Au nanoparticles in inhibiting Ab1–40 fibrillation starts with a lag time of around 12 h (which amyloid fibrillation. does not change in the presence of nanoparticles), followed The most important aspect of Au-curcumin is that it can disintegrate and dissolve amyloid fibrils without any external agent or force. Disintegration and dissolution of HEWL and Ab1–40 fibrils by Au-curcumin was studied by incubating preformed fibrils with Au-curcumin under fibrillation conditions. It was found that the longer fibrils disintegrated and dissolved into smaller fibrils, as evidenced by ThT-based fluorescence assay and TEM study (Figures 2 c, d, 3 f, 4 f). For example, the HEWL fibril length was reduced from 0.4–1.4 mm (average 0.8 mm) to 0.1–0.6 mm (average 0.3 mm) and the Ab1–40 fibril length was reduced from 0.5–5 mm (average 2.3 mm) to < 0.1 mm (average 0.07 mm) after incubating the respective fibrils with Au-curcumin for 24 h (for HEWL) or 7 days (for Figure 2. Kinetics of fibrillation (a, b) and fibril dissolution (c, d) for HEWL (a, c) and Ab1–40 (b, d) as monitored by Ab1–40) (Figure 5). Control experithioflavin T-based fluorescence assay. Details of the conditions are described in the Experimental Section. Chem. Eur. J. 2014, 20, 1 – 9
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Full Paper Western blot analysis was performed to investigate the formation of Ab oligomers and their relative concentration with respect to monomer and fibril[51] (Figure 6). Ab fibrils were grown in the presence of Au/curcumin/ Au-curcumin and used for this study. In addition, Ab monomer, Ab oligomer (partially formed), and Ab fibrils were used as controls. The results showed that the Ab monomer band was more intense for Ab oligomer, Ab monomer, curcumin, and Aucurcumin, compared with that of Ab fibril. In addition, oligomer bands were clearly observed for Ab oligomer and curcumin, smeared oligomer bands were observed for Au-curcumin, and, Figure 3. Fibrillation of HEWL (a–e) and disintegration/dissolution of HEWL fibrils (f) as observed by TEM. Details interestingly, insignificant oligoof the conditions are described in the Experimental Section. a) Long fibril formation in the absence of nanopartimer band was observed for Au. cles or curcumin; b, c) short fibril formation in the presence of Au nanoparticles; d) long fibril formation in the presence of 40 mm curcumin; e) very short and insignificant fibril formation in the presence of Au-curcumin; f) disConsidering that the same conintegration/dissolution of long HEWL fibrils after incubation with Au-curcumin for 24 h. centration of Ab monomer was used for all samples, the strongest Ab monomer band for Aucurcumin indicates a lower population of fibrils or oligomers. The observation of smeared oligomer bands for Au-curcumin and insignificant oligomer band for Au may possibly be ascribed to the binding of Ab oligomers with Au/Au-curcumin and consequent slowing of their movement under electrophoresis. We further investigated the effect of Au-curcumin on the Ab1–40 fibril-induced toxicity towards the neuro2a cell line. Ab1–40 fibrils were prepared separately from Ab1–40 monomer in the presence or absence of Aucurcumin/curcumin. In addition, preformed fibrils were mixed with curcumin/Au-curcumin for Figure 4. Fibrillation of Ab1–40 (a–e)and disintegration/dissolution of Ab1–40 fibrils (f) as observed by TEM. Details of 7 days at 37 8C to disintegrate the conditions are described in the Experimental Section. a) Long fibril formation in the absence of nanoparticles them and then used for cytotoxor curcumin; b, c) long fibril formation in the presence of Au nanoparticles ; d) long fibril formation in the presicity studies. Next, fibrils proence of 40 mm curcumin; e) very short and insignificant fibril formation in the presence of Au-curcumin; f) disintegration/dissolution of long Ab1–40 fibrils after incubation with Au-curcumin for 7 days. duced under different conditions were mixed with neuro2a cells, incubated at 37 8C for 48 h, and cell viability was determined ments showed that Au nanoparticles or curcumin at similar by an MTT (3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium concentrations were completely inactive in dissolving fibrils bromide) assay. The results showed that cell viability was very (Figure 2 c, d and Figure S19). This result confirmed that Aulow (50 %) in the presence of Ab1–40 fibrils, but viability incurcumin is capable of disintegrating and dissolving amyloid fibrils without any external force. creased to 70–90 % in the presence of curcumin or Au-curcu&
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Full Paper Au-curcumin nanoparticles as artificial molecular chaperones
Figure 5. Length distributions of amyloid fibrils for HEWL (a) and Ab1–40 (b) obtained by measuring the fibril lengths from TEM images. Values in brackets indicate the average lengths of the fibrils.
Figure 7. Effect of curcumin/Au-curcumin on Ab1–40 fibril-induced neurotoxicity towards neuro2a cell lines. The first two columns relate to Au/Au-curcumin-induced cell viability, columns 3–5 relate to Ab-induced cell viability, whereby fibrils were prepared in the presence or absence of curcumin/Aucurcumin, and the last three columns relate to cell viability with disintegrated fibrils prepared by the action of curcumin/Au-curcumin. Fibrils were incubated with neuro2a cells for 48 h and cytotoxicity was estimated by MTT assay. The final concentration of Ab1–40 fibrils in terms of monomer was around 1.25 mm, and the final concentration of curcumin in the form of Aucurcumin or as free curcumin was around 2 mm. The final concentration of Au in Au nanoparticles/Au-curcumin was around 0.12 mm.
Figure 6. Western blot analysis of freshly prepared Ab1–40 monomer (lane 1), partially formed Ab oligomer prepared by incubation of monomer for 1 day (lane 2), Ab fibrils prepared by incubation for 7 days (lane 3), Ab fibrils prepared by incubation for 7 days in the presence of Au nanoparticles (lane 4), Ab fibrils prepared by incubation for 7 days in the presence of curcumin (lane 5), and Ab fibrils prepared by incubation for 7 days in the presence of Au-curcumin (lane 6). The concentration of Ab in terms of monomer was kept the same (100 ng) for all the experiments. The weaker band intensity in lanes 3 and 4 can be attributed to the fibrils not entering the gel.
changed in the presence of Au-curcumin, suggesting that this nanoprobe mainly inhibits the fibril elongation/growth phase. At higher concentrations of Au-curcumin, this inhibition is so strong that fibril nucleation is almost stopped. TEM images showed that the Au-curcumin particles were mostly linked with the fibrils, indicating that their attachment to the growing fibril inhibits the protein/peptide self-assembly process. Curcumin is known to inhibit the protein self-assembly process by binding with the oligomer/fibril (but not with the native protein) through aromatic p-stacking interactions.[51–53] Thus, it is expected that Au-curcumin binds with the oligomer/fibril through the curcumin moiety and interferes with the elongation phase of fibrillation. The enhanced inhibitory effect of Aucurcumin compared with free curcumin can be ascribed to its solubility in water and the multiple curcumin moieties present on Au, which give rise to multivalent interaction. Similar multivalent interaction has been observed for peptide-conjugated
min-based inhibited/disintegrated fibrils (Figure 7). Most significantly, cell viability was around 90 % for Au-curcumin compared with 70 % with a similar concentration of curcumin, suggesting that Au-curcumin is more efficient than curcumin towards amyloid detoxification. Similarly, toxicity results with disintegrated fibrils showed that cell viability increased from 50 % to 60 % and 80 % for curcumin and Au-curcumin, respectively. This result suggests that disintegrated fibrils have low toxicity, with Au-curcumin showing a superior performance.
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Considering the prevention and curing aspect of neurodegenerative disease, the nanoparticle probe should have the ability to inhibit the amyloid fibrillation that usually occurs inside cells and the ability to dissolve the amyloid fibrils/plaque inside the brain. In addition, it is preferable that the nanoprobes have optical properties suitable for the detection/imaging of fibrils/plaque, and they should be biocompatible to avoid any adverse side effects. In these respects, the presented Au-curcumin based nanoprobe is biocompatible,[47] has plasmonic properties suitable for detection,[46, 47] and is able to dissolve amyloid fibrils and inhibit fibrillation. Kinetic studies showed that the nucleation/lag time for amyloid fibrillation remained almost un-
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Full Paper protein microspheres in relation to Ab fibrillation.[21] The ability of Au-curcumin to disintegrate fibrillar structures indicates that the anti-amylogenic property of curcumin remains intact after binding with Au nanoparticles, and the enhanced disintegration performance compared with curcumin may possibly arise due to multivalent binding and cooperative interaction. As curcumin is known to have anti-amylogenic properties, we carefully investigated amyloid fibrillation and fibril dissolution in the presence of free curcumin at different concentrations and compared the results with those obtained using Aucurcumin. The effective curcumin concentration in the Au-curcumin used was typically in the range 10–40 mm. However, fibrillation experiments at similar concentrations of free curcumin mostly produced long fibrils and bundles thereof for both HEWL and Ab1–40 (Figures 3 d and 4 d and Figures S13–S18). In addition, ThT-based assay showed that HEWL fibrillation was 40–60 % less with Au-curcumin compared with a similar concentration of free curcumin (Figure S16). Similarly, fibril dissolution studies at equivalent concentrations of curcumin showed that curcumin itself is unable to dissolve amyloid fibrils. These results clearly show that Au-curcumin has much better antiamylogenic properties than free curcumin at a similar concentration.
Experimental Section Reagents Hen egg-white lysozyme (HEWL), amyloid b-protein fragment 1–40 with the sequence Asp-Ala-Glu-Phe-Arg-His-Asp-Ser-Gly-Tyr-Glu-ValHis-His-Gln-Lys-Leu-Val-Phe-Phe-Ala-Glu-Asp-Val-Gly-Ser-Asn-LysGly-Ala-Ile-Ile-Gly-Leu-Met-Val-Gly-Gly-Val-Val, gold(III) chloride, didodecyldimethylammonium bromide (DDAB), 3-(mercaptopropyl)trimethoxysilane (MPS), 2-aminoethyl-aminopropyltrimethoxysilane (AEAPS), tetrabutylammonium borohydride (TBAB), sodium chloride, thioflavin T, curcumin, 4-(dimethylamino)pyridine (DMAP), glutaric anhydride, triethylamine, and N-(3-dimethylaminopropyl)-N’ethylcarbodiimide hydrochloride (EDC) were purchased from Sigma–Aldrich. All reagents were used without further purification.
Preparation of curcumin monocarboxylic acid (curcuminCOOH) A monocarboxylic acid derivative of curcumin was prepared according to the reported method.[36] Briefly, curcumin (500 mg) and DMAP (28 mg) were mixed in tetrahydrofuran (THF; 25 mL) and then triethylamine (0.33 mL) was added. The color of the solution instantaneously changed from yellow to deep-brown. Next, a solution of glutaric anhydride (171 mg) in THF (1.25 mL) was added dropwise to the curcumin solution with stirring, and the resulting solution was heated under reflux under argon atmosphere for 24 h. After completion of the reaction, the THF was removed by using a rotary evaporator. The dried sample was redissolved in ethyl acetate (10 mL) and then dilute aqueous HCl was added with vigorous shaking. The organic phase containing curcumin monocarboxylic acid was extracted with ethyl acetate (four times) and the combined extracts were dried. The product was purified by column chromatography, eluting with a mixture of dichloromethane and methanol (95:5, v/v). The yield of curcumin-COOH was about 45 %.
Conclusion We have synthesized curcumin-functionalized gold nanoparticles (Au-curcumin) and have shown that they inhibit amyloid fibrillation in a dose-dependent manner and are capable of disintegrating/dissolving amyloid fibrils. The superior inhibitory effect towards amyloid fibrillation and the enhanced amyloid fibril dissolution shown by Au-curcumin, compared with similar concentrations of curcumin or Au nanoparticles, can be attributed to the curcumin being rendered soluble in water and the multiple curcumin moieties on each Au nanoparticle. The presented nanotechnology based approach is important, considering the highly interdisciplinary aspect of the prevention and cure of neurodegenerative diseases, which requires in vitro testing of nanoprobes, in vivo targeting by blood–brain barrier crossing, and removal of amyloid plaque. Further work should be directed towards the preparation of similar nanoprobes with in vivo brain-targeting properties that are effective for the removal of amyloid plaque. The Au-curcumin system presented here has two limitations that need to be overcome. Although Au-curcumin itself has high colloidal stability at acidic and neutral pH, the particles are susceptible to precipitation if fibrillation is performed at neutral pH. Thus, all of the reported fibrillation experiments were performed at acidic pH. In addition, it is difficult to attach a greater number of curcumin moieties per nanoparticle, as binding of more curcumin lowers the water-solubility of the Au-curcumin. Hence, future studies should be directed towards the development of water-soluble curcumin-functionalized nanoparticles having a higher density of curcumin. &
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Preparation of silica-coated cationic gold nanoparticles Silica-coated, amine-terminated gold nanoparticles were prepared according to a reported method.[46] Briefly, a solution of 0.01 m AuCl3 and 0.02 m DDAB was prepared in toluene (1 mL). A 0.1 m solution of MPS in toluene (20 mL) was then added. Thereafter, a solution of TBAB (2.5 mg) and DDAB (2.5 mg) in toluene (100 mL) was added under stirring to produce gold nanoparticles. Next, a 0.1 m solution of AEAPS in toluene (100 mL) was added and the mixture was heated at 65 8C. Gold nanoparticles started to precipitate within 5 min of adding AEAPS and heating was continued for a further 5 min. The precipitated particles were washed twice with toluene and twice with ethanol and then dissolved in distilled water.
Preparation of Au-curcumin Curcumin-COOH was covalently conjugated with the amine-functionalized Au nanoparticles by EDC coupling. Curcumin-COOH (4 mg) was first dissolved in ethanol (0.25 mL) and 0.1 m borate buffer (pH 9; 20 mL) was added. A solution of EDC (16 mg) in ethanol (0.25 mL) was then added to the curcumin solution with stirring. A solution of Au nanoparticles in water (1 mL) was then added and the reaction was allowed to proceed for 6 h under stirring. The resulting solution was then centrifuged at 12 000 rpm to precipitate the gold nanoparticle–curcumin conjugate and the precipitate was repeatedly washed with chloroform and ethanol to remove unbound curcumin monocarboxylic acid. Finally, the pre-
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Full Paper water. Next, neuro2a cells were incubated at 37 8C for 48 h after mixing with aliquots (25 mL) of dispersions of Ab1–40 fibrils produced under different conditions, and cell viability was determined by an MTT (3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide) assay. Typically, each well plate containing cells was treated with a freshly prepared MTT solution and incubated for 4 h. The supernatant was then carefully removed leaving violet formazan on the plate. This formazan was then dispersed in DMF/water and its absorbance at 570 nm was measured with a microplate reader. Cell viability was correlated with the absorbance value, assuming 100 % viability for the cells without any fibrils.
cipitated particles were redissolved in distilled water (1 mL) and used for further study.
Amyloid fibrillation study Amyloid fibril formation was studied using HEWL protein and amyloid b-peptide. A 2 mg mL 1 HEWL protein solution was prepared by dissolving HEWL powder in 12 mm hydrochloric acid (2 mL) of pH 2.0 containing 140 mm NaCl and 2.7 mm KCl. The protein solution was then magnetically stirred at 60 8C for 24 h. Similarly, an amyloid b-peptide solution (25 mm) was prepared by first dissolving the lyophilized peptide in DMSO and then diluting with aqueous HCl of pH 2 containing 140 mm NaCl and 2.7 mm KCl. The peptide solution was then incubated at 37 8C for 7 days.
Instrumentation
Amyloid aggregation kinetics was monitored by a thioflavin T (ThT)-based titration method. Typically, a 10 mm stock solution of ThT was prepared by dissolving thioflavin T powder in PBS buffer of pH 7.4. Aliquots (20 mL) of protein solution were collected at timed intervals and mixed with 1 mL of the ThT solution. In the case of amyloid b-peptide, peptide solution (20 mL) was added to ThT solution (200 mL). After 5 min, the fluorescence of ThT was measured under excitation at 440 nm. In order to study the effect of nanoparticles on the fibrillation kinetics, a nanoparticle solution was mixed with protein/peptide solution and the aggregation kinetics was studied by means of the ThT-based assay. Typically, 250–1000 mL of Au-curcumin solution was mixed with HEWL solution, keeping the final volume at 2 mL for HEWL fibrillation study, whereas 25–100 mL of Au-curcumin solution was mixed with Ab1–40 solution for Ab fibrillation study, keeping the final solution volume at 200 mL. The effect of free curcumin on amyloid fibrillation kinetics was studied using a 1 % ethanolic solution. Curcumin is completely soluble in ethanol in the concentration range 0–60 mm. For Western blot analysis, equal amounts of samples were run in 14 % SDS-PAGE and then transfered to a nitrocellulose membrane. Blots were incubated with 6E10 primary antibodies (which detect Ab), washed three times, incubated with horse radish peroxidase (HRP) conjugated secondary antibodies for 3 h, washed, and developed by an enhanced chemiluminescence (ECL) technique.
All UV/Vis spectra were recorded on a Shimadzu UV-2550 UV/Vis spectrophotometer from sample solutions in a quartz cell of 1 cm path length. Fluorescence spectra were measured with a BioTek Synergy MX microplate reader. NMR spectra were measured on a Bruker F NMR (DPX-500 MHz) instrument. High resolution mass spectra (HRMS) were recorded on a Waters QTOF Micro YA263 spectrometer. Fourier-transform infrared spectra were recorded from samples in KBr pellets on a Perkin-Elmer Spectrum 100 FTIR spectrometer. DLS and zeta potential studies were performed using a NanoZS (Malvern) instrument. TEM was performed with an FEI Tecnai G2 F20 microscope with a field-emission gun operating at 200 kV.
Disaggregation study
Keywords: amyloid beta-peptides · amyloid fibrils · curcumin · fibril dissolution · gold nanoparticles · neurochemistry
Acknowledgements The authors would like to thank the Department of Biotechnology (DBT) and the Department of Science and Technology (DST) of the Government of India for financial assistance. S.P. acknowledges the Council for Scientific and Industrial Research (CSIR), India for providing a research fellowship. A.R.M. acknowledges the DST and the Indian Association for the Cultivation of Science for providing a research fellowship.
About 2 mL of aggregated protein solution was centrifuged at 12 000 rpm for 4 min and the precipitated aggregate was redispersed in aqueous HCl (1 mL) of pH 2. An aliquot of 1 mL of nanoparticle solution was mixed with this aggregated protein solution and the mixture was heated at 60 8C for 24 h with stirring. In the case of amyloid b-peptide, preformed fibrils were incubated with the functionalized nanoparticles at 37 8C for 7 days. Dissolution of the protein/peptide was studied by collecting 20 mL aliquots of the solution at timed intervals and subjecting them to the ThT-based assay as described above.
[1] [2] [3] [4] [5] [6] [7] [8] [9]
Cytotoxicity assay Neuro2a cells were cultured in a tissue culture flask with Dulbecco’s modified Eagle’s (DMEM) medium. The cells were sub-cultured in a 24-well culture plate containing cell culture medium (0.5 mL) and incubated overnight for adherence to the culture plate. Ab1–40 fibrils were prepared separately in the presence or absence of Aucurcumin/curcumin, by incubating Ab1–40 monomer solution at 37 8C for 7 days. Disintegrated fibrils were prepared by incubating preformed fibrils either with or without curcumin/Au-curcumin at 37 8C for 7 days. The fibrils were then isolated as a precipitate by centrifuging the solution, and the precipitate was redispersed in Chem. Eur. J. 2014, 20, 1 – 9
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[10] [11] [12] [13]
[14]
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Nanoparticles in amyloid therapy: Curcumin-functionalized water-soluble gold nanoparticles have been found to efficiently inhibit amyloid fibril growth and to disintegrate preformed amyloid fibrils (see figure). Consequently, they serve to reduce amyloid-induced neurotoxicity.
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S. Palmal, A. R. Maity, B. K. Singh, S. Basu, N. R. Jana,* N. R. Jana* && – && Inhibition of Amyloid Fibril Growth and Dissolution of Amyloid Fibrils by Curcumin–Gold Nanoparticles
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Inhibition of Amyloid Fibril Growth and Dissolution of Amyloid Fibrils by Curcumin–Gold Nanoparticles Sharbari Palmal,[a] Amit Ranjan Maity,[a] Brijesh Kumar Singh,[b] Sreetama Basu,[b] Nihar R. Jana,*[b] and Nikhil R. Jana*[a]
form, curcumin is water-soluble and can efficiently interact with amyloid protein/peptide, offering enhanced performance in inhibiting amyloid fibrillation and dissolving amyloid fibrils. Our results imply that nanoparticle-based artificial molecular chaperones may offer a promising therapeutic approach to combat neurodegenerative disease.
Abstract: Inhibition of amyloid fibrillation and clearance of amyloid fibrils/plaques are essential for the prevention and treatment of various neurodegenerative disorders involving protein aggregation. Herein, we report curcumin-functionalized gold nanoparticles (Au-curcumin) of hydrodynamic diameter 10–25 nm, which serve to inhibit amyloid fibrillation and disintegrate/dissolve amyloid fibrils. In nanoparticle
Introduction
cles,[13, 15] anionic quantum dots,[16] a-synuclein-functionalized quantum dots,[14] and peptide-functionalized g-Fe2O3 nanoparticles.[17] In contrast, retardation of amyloid fibrillation has been reported for hydrophobic polymer nanoparticles,[19] N-acetyl-lcysteine-capped CdTe quantum dots,[20] peptide-functionalized g-Fe2O3 nanoparticles,[17] dipeptide-conjugated polymer nanoparticles,[18] peptide-capped protein microspheres,[21] thioglycolic acid-capped CdTe quantum dots,[22] dihydrolipoic acidcapped CdSe/ZnS quantum dots,[23] and fetal bovine serumcoated graphene oxide.[24] In addition, dose-dependent acceleration or inhibition of Ab fibrillation has been observed for amine-terminated polystyrene nanoparticles.[25] However, only a few studies have demonstrated the dissolution of preformed fibrils by nanoparticle-based systems.[26–28] For instance, peptide-functionalized gold nanoparticles[26] and thioflavin S-conjugated graphene oxide[27] have been used to dissolve amyloid fibrils under photothermal conditions. Anionic gold nanoparticles have also been shown to induce partial fibril dissociation.[28] These findings suggest that a nanoparticle-based approach might be a promising option for the treatment of various neurodegenerative diseases. To achieve this, the primary need is to develop biocompatible nanoparticle systems that can efficiently inhibit fibrillation and dissolve amyloid fibrils/ plaque. In order to obtain specific interactions with amyloid structures, nanoparticles have been functionalized with molecules of various affinities, such as a peptide,[26] sialic acid,[29, 30] thioflavin,[27] a stilbene derivative,[31] and curcumin.[32–43] Among these, curcumin is one of the most studied molecules and acts as a very effective molecular chaperone in relation to various human neurological disorders and is known to inhibit amyloid fibrillation and to disintegrate amyloid fibrils.[32–43] Curcumin (M.W. 368.38) is a natural polyphenolic compound extracted from the Indian spice turmeric.[44] The chemical name of curcumin is difurylmethane, and it is usually found alongside other
Amyloid protein fibrillation is a generic feature of various human neurological disorders, such as Alzheimer’s, Parkinson’s, and Huntington’s diseases.[1–8] Amyloid fibrils are ordered protein aggregates consisting of crossed b-sheet secondary structures.[1–8] They are highly neurotoxic, causing neuronal cell death. Alzheimer’s disease is the most devastating neurodegenerative disorder affecting millions of people worldwide.[6] Accumulation of fibrillar aggregates in the brain is the pathological hallmark of Alzheimer’s disease. b-Amyloid peptides (Ab1–40, Ab1–42), derived from amyloid precursor protein (APP) through b- and g-secretase-based proteolysis, are mainly responsible for fibrous plaque formation.[1] Currently, no definite diagnostic tool and no proper treatment exist for this disease. We envisage that inhibition of amyloid fibrillation and dissolution of fibrillar aggregates should offer a promising therapeutic approach towards Alzheimer’s disease. Recent reports have shown that nanoparticles can significantly influence fibrillation processes, depending on the particle size and surface functionality.[9–24] For instance, amyloid fibrillation is accelerated by bare carbon nanotubes,[11] bare cerium oxide,[11] bare TiO2 nanoparticles,[12] polymer particles,[11] polymer-coated quantum dots,[11] anionic gold nanoparti-
[a] S. Palmal, A. R. Maity, Dr. N. R. Jana Centre for Advanced Materials Indian Association for the Cultivation of Science Kolkata-700032 (India) E-mail: [email protected] [b] B. K. Singh, S. Basu, Prof. N. R. Jana National Brain Research Centre Manesar, Gurgaon-122050 (India) E-mail: [email protected] Supporting information for this article is available on the WWW under http://dx.doi.org/10.1002/chem.201400079. Chem. Eur. J. 2014, 20, 1 – 9
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Full Paper polyphenol compounds such as demethoxycurcumin, bisdemethoxycurcumin, and so on (see Figure S1 in the Supporting Information).[43, 45] However, curcumin is hydrophobic in nature and insoluble in water, which restricts its therapeutic application.[45] Thus, in vitro and in vivo inhibition of amyloid fibrillation by curcumin have been performed after synthesizing water-soluble curcumin-based conjugates with small molecules, polymers, and nanoparticles.[36, 40–42] However, to the best of our knowledge, there have not hitherto been any reports of the use of curcumin-based nanoparticles or polymers to dissolve amyloid fibrils/plaque, although there are preliminary reports that non-conjugated curcumin can disintegrate amyloid fibrils.[35] In the present study, we have prepared water-soluble curcumin-functionalized gold nanoparticles (Au-curcumin) and have found that they not only inhibit amyloid fibrillation but also disintegrate and dissolve amyloid fibrils without any external agent or force.
Figure 1. a) Optical properties and b) representative TEM image of Au-curcumin. The inset shows an image of a test tube containing an aqueous solution of Au-curcumin. Arrow indicates the curcumin absorption band at 420 nm.
(Figure 1 and Figures S3–S10 in the Supporting Information). Although curcumin is insoluble in water, Au-curcumin becomes water-soluble and displays an absorption band due to curcumin at 420 nm and an Au plasmon band at 535 nm. The high water-solubility of Au-curcumin can be ascribed to the presence of primary and secondary amine groups on the surface of the silica-coated Au nanoparticles as only some of the primary amine groups are used for conjugation. The presence of the curcumin band suggests that the molecule is covalently bound to the Au nanoparticle. The presence of free curcumin in Au-curcumin can be excluded as the samples were extensively washed with ethanol/chloroform (Figure S5). In the FTIR spectrum, the appearance of characteristic curcumin peaks with a C-O-C stretching vibration at around 1020 cm 1 and a C=O/C=C vibration at around 1506 cm 1 also supports the covalent attachment of curcumin-COOH to a gold nanoparticle. A fluorescamine test confirmed the conjugation of a fraction of the amine groups on the surface of the gold nanoparticles with curcumin-COOH (Figure S4). From the absorption band of curcumin and the Au plasmon band seen for Au-curcumin, the number of curcumin molecules linked to each Au nanoparticle has been tentatively estimated as 17 (Figure S6). The synthesis conditions were optimized to prepare Au-curcumin with high water-solubility (Figure S9). The good watersolubility of Au-curcumin is due to the high water-solubility of the silica-coated gold nanoparticles,[46] and causes the curcumin to remain in water. Transmission electron microscopy (TEM) revealed that the size of the core Au nanoparticles was around 3.5 nm, and dynamic light scattering showed the hydrodynamic diameter of Au-curcumin to be 10–25 nm, including the silica shell with attached curcumin moieties (Figure 1 b and Figures S7 and S8). In contrast, the hydrodynamic size of the silica-coated Au nanoparticles was 5–12 nm. The increase in hydrodynamic diameter of Au-curcumin can be attributed to partial crosslinking between particles during the conjugation process, as well as an increased agglomeration tendency of the nanoparticles due to the hydrophobic curcumin. The surface charge of Au-curcumin was determined as positive (+25–30 mV) in the pH range 2–7. This value is similar to that of the silica-coated Au nanoparticle before functionalization,[46]
Results and Discussion Synthesis and characterization of Au-curcumin nanoparticles Gold nanoparticles were selected for this work due to their low toxicity, plasmon-based strong optical properties suitable for detection/imaging, and well-established functionalization methods.[46, 47] The preparation strategy for Au-curcumin is shown in Scheme 1. Curcumin was converted to a monocarbox-
Scheme 1. Synthetic strategy for obtaining curcumin-functionalized Au nanoparticles (Au-curcumin; DMAP = 4-dimethylaminopyridine, EDC = N’-(3dimethylaminopropyl)-N-ethylcarbodiimide). The high water-solubility of Aucurcumin is due to the presence of primary and secondary amine groups on the surface of the silica shell.
ylic acid derivative (curcumin-COOH) at one of its phenolic OH groups[36] and then covalently linked with primary amine-terminated silica-coated Au nanoparticles[46] through conventional EDC coupling. The formation of curcumin-COOH was confirmed by HRMS and other methods (Figures S1 and S2 in the Supporting Information). The Au-curcumin nanoparticles were characterized by UV/Vis absorption and FTIR spectroscopies &
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by an elongation phase that is extended from one to five days with increasing concentration of Au-curcumin. Fibrils formed from HEWL and Ab1–40 under different conditions were imaged by TEM (Figures 3 and 4). In addition, the average lengths of fibrils formed under different conditions Effect of Au-curcumin on amyloid fibrillation, fibril disintewere measured from the collection of TEM images, and their gration/dissolution, and fibril-induced cytotoxicity length distribution histograms are summarized in Figure 5. The The effect of Au-curcumin on amyloid formation was examined results clearly show that the lengths of the fibrils formed in using hen egg white lysozyme (HEWL) as a widely studied the presence of Au-curcumin were significantly smaller than [48] model amyloid protein as well as Ab1–40 as a real amyloid those of the fibrils formed in the absence of the nanoparticles. [1–8] peptide. In order to investigate the influence on the fibrillaFor example, HEWL fibrils of length 0.4–1.4 mm (average tion process, the monomeric amyloid protein or peptide was 0.8 mm) were formed in the absence of the nanoparticles, but incubated with Au-curcumin under fibril-forming conditions, were restricted to lengths < 0.2 mm (average 0.09 mm) in the and the fibrillation kinetics was monitored by a thioflavin T presence of Au-curcumin. Similarly, Ab1–40 fibrils of length 0.5– (ThT)-based fluorescence assay. ThT is an amyloid-specific ben5 mm (average 2.3 mm) were formed in the absence of the zothiazole dye, the fluorescence of which is enhanced through nanoparticles, but were again restricted to lengths < 0.2 mm binding with amyloid fibrils and is directly correlated with the (average 0.1 mm) in the presence of Au-curcumin. Control ex[49, 50] extent of fibrillation. We observed that Au-curcumin signifperiments were performed using Au nanoparticles without curicantly influenced the fibrillation process for both HEWL and cumin functionalization and using pure curcumin. The results Ab1–40 in a dose-dependent manner (Figure 2 and Figures S11– showed that pure curcumin or Au nanoparticles (without curS23 in the Supporting Information). In general, amyloid protein cumin functionalization) were less active or partially inhibited fibrillation follows a nucleation-dependent pathway that conamyloid fibrillation compared with a similar concentration of sists of three distinct steps, namely i) nucleation, ii) elongation/ Au-curcumin (Figures 3 b–d and 4 b–d). We performed another growth, and iii) equilibration/steady stage, as observed previcontrol fibrillation experiment using a mixture of Au nanoparti[11–24] ously. In HEWL fibrillation, the typical lag time for nucleacles and curcumin and compared the results with those obtion is around 30 min (which does not change in the presence tained using Au-curcumin, Au nanoparticles, and curcumin. of nanoparticles), and this is followed by an elongation phase The Au-curcumin showed a much better inhibitory effect than from 1 to 12 h. However, the rate of elongation becomes the sum of the individual effects of Au nanoparticles and curslower with increasing concentration of Au-curcumin. A similar cumin (Figure S20). These results prove that Au-curcumin is result was also found for Ab1–40. The nucleation process of more efficient than curcumin or Au nanoparticles in inhibiting Ab1–40 fibrillation starts with a lag time of around 12 h (which amyloid fibrillation. does not change in the presence of nanoparticles), followed The most important aspect of Au-curcumin is that it can disintegrate and dissolve amyloid fibrils without any external agent or force. Disintegration and dissolution of HEWL and Ab1–40 fibrils by Au-curcumin was studied by incubating preformed fibrils with Au-curcumin under fibrillation conditions. It was found that the longer fibrils disintegrated and dissolved into smaller fibrils, as evidenced by ThT-based fluorescence assay and TEM study (Figures 2 c, d, 3 f, 4 f). For example, the HEWL fibril length was reduced from 0.4–1.4 mm (average 0.8 mm) to 0.1–0.6 mm (average 0.3 mm) and the Ab1–40 fibril length was reduced from 0.5–5 mm (average 2.3 mm) to < 0.1 mm (average 0.07 mm) after incubating the respective fibrils with Au-curcumin for 24 h (for HEWL) or 7 days (for Figure 2. Kinetics of fibrillation (a, b) and fibril dissolution (c, d) for HEWL (a, c) and Ab1–40 (b, d) as monitored by Ab1–40) (Figure 5). Control experithioflavin T-based fluorescence assay. Details of the conditions are described in the Experimental Section. Chem. Eur. J. 2014, 20, 1 – 9
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Full Paper Western blot analysis was performed to investigate the formation of Ab oligomers and their relative concentration with respect to monomer and fibril[51] (Figure 6). Ab fibrils were grown in the presence of Au/curcumin/ Au-curcumin and used for this study. In addition, Ab monomer, Ab oligomer (partially formed), and Ab fibrils were used as controls. The results showed that the Ab monomer band was more intense for Ab oligomer, Ab monomer, curcumin, and Aucurcumin, compared with that of Ab fibril. In addition, oligomer bands were clearly observed for Ab oligomer and curcumin, smeared oligomer bands were observed for Au-curcumin, and, Figure 3. Fibrillation of HEWL (a–e) and disintegration/dissolution of HEWL fibrils (f) as observed by TEM. Details interestingly, insignificant oligoof the conditions are described in the Experimental Section. a) Long fibril formation in the absence of nanopartimer band was observed for Au. cles or curcumin; b, c) short fibril formation in the presence of Au nanoparticles; d) long fibril formation in the presence of 40 mm curcumin; e) very short and insignificant fibril formation in the presence of Au-curcumin; f) disConsidering that the same conintegration/dissolution of long HEWL fibrils after incubation with Au-curcumin for 24 h. centration of Ab monomer was used for all samples, the strongest Ab monomer band for Aucurcumin indicates a lower population of fibrils or oligomers. The observation of smeared oligomer bands for Au-curcumin and insignificant oligomer band for Au may possibly be ascribed to the binding of Ab oligomers with Au/Au-curcumin and consequent slowing of their movement under electrophoresis. We further investigated the effect of Au-curcumin on the Ab1–40 fibril-induced toxicity towards the neuro2a cell line. Ab1–40 fibrils were prepared separately from Ab1–40 monomer in the presence or absence of Aucurcumin/curcumin. In addition, preformed fibrils were mixed with curcumin/Au-curcumin for Figure 4. Fibrillation of Ab1–40 (a–e)and disintegration/dissolution of Ab1–40 fibrils (f) as observed by TEM. Details of 7 days at 37 8C to disintegrate the conditions are described in the Experimental Section. a) Long fibril formation in the absence of nanoparticles them and then used for cytotoxor curcumin; b, c) long fibril formation in the presence of Au nanoparticles ; d) long fibril formation in the presicity studies. Next, fibrils proence of 40 mm curcumin; e) very short and insignificant fibril formation in the presence of Au-curcumin; f) disintegration/dissolution of long Ab1–40 fibrils after incubation with Au-curcumin for 7 days. duced under different conditions were mixed with neuro2a cells, incubated at 37 8C for 48 h, and cell viability was determined ments showed that Au nanoparticles or curcumin at similar by an MTT (3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium concentrations were completely inactive in dissolving fibrils bromide) assay. The results showed that cell viability was very (Figure 2 c, d and Figure S19). This result confirmed that Aulow (50 %) in the presence of Ab1–40 fibrils, but viability incurcumin is capable of disintegrating and dissolving amyloid fibrils without any external force. creased to 70–90 % in the presence of curcumin or Au-curcu&
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Figure 5. Length distributions of amyloid fibrils for HEWL (a) and Ab1–40 (b) obtained by measuring the fibril lengths from TEM images. Values in brackets indicate the average lengths of the fibrils.
Figure 7. Effect of curcumin/Au-curcumin on Ab1–40 fibril-induced neurotoxicity towards neuro2a cell lines. The first two columns relate to Au/Au-curcumin-induced cell viability, columns 3–5 relate to Ab-induced cell viability, whereby fibrils were prepared in the presence or absence of curcumin/Aucurcumin, and the last three columns relate to cell viability with disintegrated fibrils prepared by the action of curcumin/Au-curcumin. Fibrils were incubated with neuro2a cells for 48 h and cytotoxicity was estimated by MTT assay. The final concentration of Ab1–40 fibrils in terms of monomer was around 1.25 mm, and the final concentration of curcumin in the form of Aucurcumin or as free curcumin was around 2 mm. The final concentration of Au in Au nanoparticles/Au-curcumin was around 0.12 mm.
Figure 6. Western blot analysis of freshly prepared Ab1–40 monomer (lane 1), partially formed Ab oligomer prepared by incubation of monomer for 1 day (lane 2), Ab fibrils prepared by incubation for 7 days (lane 3), Ab fibrils prepared by incubation for 7 days in the presence of Au nanoparticles (lane 4), Ab fibrils prepared by incubation for 7 days in the presence of curcumin (lane 5), and Ab fibrils prepared by incubation for 7 days in the presence of Au-curcumin (lane 6). The concentration of Ab in terms of monomer was kept the same (100 ng) for all the experiments. The weaker band intensity in lanes 3 and 4 can be attributed to the fibrils not entering the gel.
changed in the presence of Au-curcumin, suggesting that this nanoprobe mainly inhibits the fibril elongation/growth phase. At higher concentrations of Au-curcumin, this inhibition is so strong that fibril nucleation is almost stopped. TEM images showed that the Au-curcumin particles were mostly linked with the fibrils, indicating that their attachment to the growing fibril inhibits the protein/peptide self-assembly process. Curcumin is known to inhibit the protein self-assembly process by binding with the oligomer/fibril (but not with the native protein) through aromatic p-stacking interactions.[51–53] Thus, it is expected that Au-curcumin binds with the oligomer/fibril through the curcumin moiety and interferes with the elongation phase of fibrillation. The enhanced inhibitory effect of Aucurcumin compared with free curcumin can be ascribed to its solubility in water and the multiple curcumin moieties present on Au, which give rise to multivalent interaction. Similar multivalent interaction has been observed for peptide-conjugated
min-based inhibited/disintegrated fibrils (Figure 7). Most significantly, cell viability was around 90 % for Au-curcumin compared with 70 % with a similar concentration of curcumin, suggesting that Au-curcumin is more efficient than curcumin towards amyloid detoxification. Similarly, toxicity results with disintegrated fibrils showed that cell viability increased from 50 % to 60 % and 80 % for curcumin and Au-curcumin, respectively. This result suggests that disintegrated fibrils have low toxicity, with Au-curcumin showing a superior performance.
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Considering the prevention and curing aspect of neurodegenerative disease, the nanoparticle probe should have the ability to inhibit the amyloid fibrillation that usually occurs inside cells and the ability to dissolve the amyloid fibrils/plaque inside the brain. In addition, it is preferable that the nanoprobes have optical properties suitable for the detection/imaging of fibrils/plaque, and they should be biocompatible to avoid any adverse side effects. In these respects, the presented Au-curcumin based nanoprobe is biocompatible,[47] has plasmonic properties suitable for detection,[46, 47] and is able to dissolve amyloid fibrils and inhibit fibrillation. Kinetic studies showed that the nucleation/lag time for amyloid fibrillation remained almost un-
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Full Paper protein microspheres in relation to Ab fibrillation.[21] The ability of Au-curcumin to disintegrate fibrillar structures indicates that the anti-amylogenic property of curcumin remains intact after binding with Au nanoparticles, and the enhanced disintegration performance compared with curcumin may possibly arise due to multivalent binding and cooperative interaction. As curcumin is known to have anti-amylogenic properties, we carefully investigated amyloid fibrillation and fibril dissolution in the presence of free curcumin at different concentrations and compared the results with those obtained using Aucurcumin. The effective curcumin concentration in the Au-curcumin used was typically in the range 10–40 mm. However, fibrillation experiments at similar concentrations of free curcumin mostly produced long fibrils and bundles thereof for both HEWL and Ab1–40 (Figures 3 d and 4 d and Figures S13–S18). In addition, ThT-based assay showed that HEWL fibrillation was 40–60 % less with Au-curcumin compared with a similar concentration of free curcumin (Figure S16). Similarly, fibril dissolution studies at equivalent concentrations of curcumin showed that curcumin itself is unable to dissolve amyloid fibrils. These results clearly show that Au-curcumin has much better antiamylogenic properties than free curcumin at a similar concentration.
Experimental Section Reagents Hen egg-white lysozyme (HEWL), amyloid b-protein fragment 1–40 with the sequence Asp-Ala-Glu-Phe-Arg-His-Asp-Ser-Gly-Tyr-Glu-ValHis-His-Gln-Lys-Leu-Val-Phe-Phe-Ala-Glu-Asp-Val-Gly-Ser-Asn-LysGly-Ala-Ile-Ile-Gly-Leu-Met-Val-Gly-Gly-Val-Val, gold(III) chloride, didodecyldimethylammonium bromide (DDAB), 3-(mercaptopropyl)trimethoxysilane (MPS), 2-aminoethyl-aminopropyltrimethoxysilane (AEAPS), tetrabutylammonium borohydride (TBAB), sodium chloride, thioflavin T, curcumin, 4-(dimethylamino)pyridine (DMAP), glutaric anhydride, triethylamine, and N-(3-dimethylaminopropyl)-N’ethylcarbodiimide hydrochloride (EDC) were purchased from Sigma–Aldrich. All reagents were used without further purification.
Preparation of curcumin monocarboxylic acid (curcuminCOOH) A monocarboxylic acid derivative of curcumin was prepared according to the reported method.[36] Briefly, curcumin (500 mg) and DMAP (28 mg) were mixed in tetrahydrofuran (THF; 25 mL) and then triethylamine (0.33 mL) was added. The color of the solution instantaneously changed from yellow to deep-brown. Next, a solution of glutaric anhydride (171 mg) in THF (1.25 mL) was added dropwise to the curcumin solution with stirring, and the resulting solution was heated under reflux under argon atmosphere for 24 h. After completion of the reaction, the THF was removed by using a rotary evaporator. The dried sample was redissolved in ethyl acetate (10 mL) and then dilute aqueous HCl was added with vigorous shaking. The organic phase containing curcumin monocarboxylic acid was extracted with ethyl acetate (four times) and the combined extracts were dried. The product was purified by column chromatography, eluting with a mixture of dichloromethane and methanol (95:5, v/v). The yield of curcumin-COOH was about 45 %.
Conclusion We have synthesized curcumin-functionalized gold nanoparticles (Au-curcumin) and have shown that they inhibit amyloid fibrillation in a dose-dependent manner and are capable of disintegrating/dissolving amyloid fibrils. The superior inhibitory effect towards amyloid fibrillation and the enhanced amyloid fibril dissolution shown by Au-curcumin, compared with similar concentrations of curcumin or Au nanoparticles, can be attributed to the curcumin being rendered soluble in water and the multiple curcumin moieties on each Au nanoparticle. The presented nanotechnology based approach is important, considering the highly interdisciplinary aspect of the prevention and cure of neurodegenerative diseases, which requires in vitro testing of nanoprobes, in vivo targeting by blood–brain barrier crossing, and removal of amyloid plaque. Further work should be directed towards the preparation of similar nanoprobes with in vivo brain-targeting properties that are effective for the removal of amyloid plaque. The Au-curcumin system presented here has two limitations that need to be overcome. Although Au-curcumin itself has high colloidal stability at acidic and neutral pH, the particles are susceptible to precipitation if fibrillation is performed at neutral pH. Thus, all of the reported fibrillation experiments were performed at acidic pH. In addition, it is difficult to attach a greater number of curcumin moieties per nanoparticle, as binding of more curcumin lowers the water-solubility of the Au-curcumin. Hence, future studies should be directed towards the development of water-soluble curcumin-functionalized nanoparticles having a higher density of curcumin. &
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Preparation of silica-coated cationic gold nanoparticles Silica-coated, amine-terminated gold nanoparticles were prepared according to a reported method.[46] Briefly, a solution of 0.01 m AuCl3 and 0.02 m DDAB was prepared in toluene (1 mL). A 0.1 m solution of MPS in toluene (20 mL) was then added. Thereafter, a solution of TBAB (2.5 mg) and DDAB (2.5 mg) in toluene (100 mL) was added under stirring to produce gold nanoparticles. Next, a 0.1 m solution of AEAPS in toluene (100 mL) was added and the mixture was heated at 65 8C. Gold nanoparticles started to precipitate within 5 min of adding AEAPS and heating was continued for a further 5 min. The precipitated particles were washed twice with toluene and twice with ethanol and then dissolved in distilled water.
Preparation of Au-curcumin Curcumin-COOH was covalently conjugated with the amine-functionalized Au nanoparticles by EDC coupling. Curcumin-COOH (4 mg) was first dissolved in ethanol (0.25 mL) and 0.1 m borate buffer (pH 9; 20 mL) was added. A solution of EDC (16 mg) in ethanol (0.25 mL) was then added to the curcumin solution with stirring. A solution of Au nanoparticles in water (1 mL) was then added and the reaction was allowed to proceed for 6 h under stirring. The resulting solution was then centrifuged at 12 000 rpm to precipitate the gold nanoparticle–curcumin conjugate and the precipitate was repeatedly washed with chloroform and ethanol to remove unbound curcumin monocarboxylic acid. Finally, the pre-
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Full Paper water. Next, neuro2a cells were incubated at 37 8C for 48 h after mixing with aliquots (25 mL) of dispersions of Ab1–40 fibrils produced under different conditions, and cell viability was determined by an MTT (3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide) assay. Typically, each well plate containing cells was treated with a freshly prepared MTT solution and incubated for 4 h. The supernatant was then carefully removed leaving violet formazan on the plate. This formazan was then dispersed in DMF/water and its absorbance at 570 nm was measured with a microplate reader. Cell viability was correlated with the absorbance value, assuming 100 % viability for the cells without any fibrils.
cipitated particles were redissolved in distilled water (1 mL) and used for further study.
Amyloid fibrillation study Amyloid fibril formation was studied using HEWL protein and amyloid b-peptide. A 2 mg mL 1 HEWL protein solution was prepared by dissolving HEWL powder in 12 mm hydrochloric acid (2 mL) of pH 2.0 containing 140 mm NaCl and 2.7 mm KCl. The protein solution was then magnetically stirred at 60 8C for 24 h. Similarly, an amyloid b-peptide solution (25 mm) was prepared by first dissolving the lyophilized peptide in DMSO and then diluting with aqueous HCl of pH 2 containing 140 mm NaCl and 2.7 mm KCl. The peptide solution was then incubated at 37 8C for 7 days.
Instrumentation
Amyloid aggregation kinetics was monitored by a thioflavin T (ThT)-based titration method. Typically, a 10 mm stock solution of ThT was prepared by dissolving thioflavin T powder in PBS buffer of pH 7.4. Aliquots (20 mL) of protein solution were collected at timed intervals and mixed with 1 mL of the ThT solution. In the case of amyloid b-peptide, peptide solution (20 mL) was added to ThT solution (200 mL). After 5 min, the fluorescence of ThT was measured under excitation at 440 nm. In order to study the effect of nanoparticles on the fibrillation kinetics, a nanoparticle solution was mixed with protein/peptide solution and the aggregation kinetics was studied by means of the ThT-based assay. Typically, 250–1000 mL of Au-curcumin solution was mixed with HEWL solution, keeping the final volume at 2 mL for HEWL fibrillation study, whereas 25–100 mL of Au-curcumin solution was mixed with Ab1–40 solution for Ab fibrillation study, keeping the final solution volume at 200 mL. The effect of free curcumin on amyloid fibrillation kinetics was studied using a 1 % ethanolic solution. Curcumin is completely soluble in ethanol in the concentration range 0–60 mm. For Western blot analysis, equal amounts of samples were run in 14 % SDS-PAGE and then transfered to a nitrocellulose membrane. Blots were incubated with 6E10 primary antibodies (which detect Ab), washed three times, incubated with horse radish peroxidase (HRP) conjugated secondary antibodies for 3 h, washed, and developed by an enhanced chemiluminescence (ECL) technique.
All UV/Vis spectra were recorded on a Shimadzu UV-2550 UV/Vis spectrophotometer from sample solutions in a quartz cell of 1 cm path length. Fluorescence spectra were measured with a BioTek Synergy MX microplate reader. NMR spectra were measured on a Bruker F NMR (DPX-500 MHz) instrument. High resolution mass spectra (HRMS) were recorded on a Waters QTOF Micro YA263 spectrometer. Fourier-transform infrared spectra were recorded from samples in KBr pellets on a Perkin-Elmer Spectrum 100 FTIR spectrometer. DLS and zeta potential studies were performed using a NanoZS (Malvern) instrument. TEM was performed with an FEI Tecnai G2 F20 microscope with a field-emission gun operating at 200 kV.
Disaggregation study
Keywords: amyloid beta-peptides · amyloid fibrils · curcumin · fibril dissolution · gold nanoparticles · neurochemistry
Acknowledgements The authors would like to thank the Department of Biotechnology (DBT) and the Department of Science and Technology (DST) of the Government of India for financial assistance. S.P. acknowledges the Council for Scientific and Industrial Research (CSIR), India for providing a research fellowship. A.R.M. acknowledges the DST and the Indian Association for the Cultivation of Science for providing a research fellowship.
About 2 mL of aggregated protein solution was centrifuged at 12 000 rpm for 4 min and the precipitated aggregate was redispersed in aqueous HCl (1 mL) of pH 2. An aliquot of 1 mL of nanoparticle solution was mixed with this aggregated protein solution and the mixture was heated at 60 8C for 24 h with stirring. In the case of amyloid b-peptide, preformed fibrils were incubated with the functionalized nanoparticles at 37 8C for 7 days. Dissolution of the protein/peptide was studied by collecting 20 mL aliquots of the solution at timed intervals and subjecting them to the ThT-based assay as described above.
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Cytotoxicity assay Neuro2a cells were cultured in a tissue culture flask with Dulbecco’s modified Eagle’s (DMEM) medium. The cells were sub-cultured in a 24-well culture plate containing cell culture medium (0.5 mL) and incubated overnight for adherence to the culture plate. Ab1–40 fibrils were prepared separately in the presence or absence of Aucurcumin/curcumin, by incubating Ab1–40 monomer solution at 37 8C for 7 days. Disintegrated fibrils were prepared by incubating preformed fibrils either with or without curcumin/Au-curcumin at 37 8C for 7 days. The fibrils were then isolated as a precipitate by centrifuging the solution, and the precipitate was redispersed in Chem. Eur. J. 2014, 20, 1 – 9
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FULL PAPER & Functionalized Nanoparticles
Nanoparticles in amyloid therapy: Curcumin-functionalized water-soluble gold nanoparticles have been found to efficiently inhibit amyloid fibril growth and to disintegrate preformed amyloid fibrils (see figure). Consequently, they serve to reduce amyloid-induced neurotoxicity.
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S. Palmal, A. R. Maity, B. K. Singh, S. Basu, N. R. Jana,* N. R. Jana* && – && Inhibition of Amyloid Fibril Growth and Dissolution of Amyloid Fibrils by Curcumin–Gold Nanoparticles
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