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Abstract: Inhibition of amyloid fibrillation and clearance of form, curcumin is water-soluble and can efficiently interact
amyloid fibrils/plaques are essential for the prevention and with amyloid protein/peptide, offering enhanced per-
treatment of various neurodegenerative disorders involving formance in inhibiting amyloid fibrillation and dissolving
protein aggregation. Herein, we report curcumin-functional- amyloid fibrils. Our results imply that nanoparticle-based ar-
ized gold nanoparticles (Au-curcumin) of hydrodynamic di- tificial molecular chaperones may offer a promising thera-
ameter 1025 nm, which serve to inhibit amyloid fibrillation peutic approach to combat neurodegenerative disease.
and disintegrate/dissolve amyloid fibrils. In nanoparticle
Chem. Eur. J. 2014, 20, 1 9 1 2014 Wiley-VCH Verlag GmbH & Co. KGaA, Weinheim & &
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suggesting that a fraction of the amine groups reacted with by an elongation phase that is extended from one to five days
curcumin and the rest remained free. with increasing concentration of Au-curcumin.
Fibrils formed from HEWL and Ab140 under different condi-
tions were imaged by TEM (Figures 3 and 4). In addition, the
Effect of Au-curcumin on amyloid fibrillation, fibril disinte- average lengths of fibrils formed under different conditions
gration/dissolution, and fibril-induced cytotoxicity were measured from the collection of TEM images, and their
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
[48] the presence of Au-curcumin were significantly smaller than
model amyloid protein as well as Ab140 as a real amyloid
[18] those of the fibrils formed in the absence of the nanoparticles.
peptide. In order to investigate the influence on the fibrilla-
For example, HEWL fibrils of length 0.41.4 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, Ab140 fibrils of length 0.5
(ThT)-based fluorescence assay. ThT is an amyloid-specific ben-
5 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
[49, 50] (average 0.1 mm) in the presence of Au-curcumin. Control ex-
extent of fibrillation. We observed that Au-curcumin signif-
periments were performed using Au nanoparticles without cur-
icantly influenced the fibrillation process for both HEWL and
cumin functionalization and using pure curcumin. The results
Ab140 in a dose-dependent manner (Figure 2 and Figures S11
showed that pure curcumin or Au nanoparticles (without cur-
S23 in the Supporting Information). In general, amyloid protein
cumin functionalization) were less active or partially inhibited
fibrillation follows a nucleation-dependent pathway that con-
amyloid fibrillation compared with a similar concentration of
sists of three distinct steps, namely i) nucleation, ii) elongation/
Au-curcumin (Figures 3 bd and 4 bd). We performed another
growth, and iii) equilibration/steady stage, as observed previ-
[1124] control fibrillation experiment using a mixture of Au nanoparti-
ously. In HEWL fibrillation, the typical lag time for nuclea-
cles and curcumin and compared the results with those ob-
tion 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 cur-
slower with increasing concentration of Au-curcumin. A similar
cumin (Figure S20). These results prove that Au-curcumin is
result was also found for Ab140. The nucleation process of
more efficient than curcumin or Au nanoparticles in inhibiting
Ab140 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 disin-
tegrate and dissolve amyloid fi-
brils without any external agent
or force. Disintegration and dis-
solution of HEWL and Ab140 fi-
brils by Au-curcumin was stud-
ied by incubating preformed fi-
brils with Au-curcumin under fi-
brillation conditions. It was
found that the longer fibrils dis-
integrated 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
0.41.4 mm (average 0.8 mm) to
0.10.6 mm (average 0.3 mm) and
the Ab140 fibril length was re-
duced from 0.55 mm (average
2.3 mm) to < 0.1 mm (average
0.07 mm) after incubating the re-
spective fibrils with Au-curcumin
Figure 2. Kinetics of fibrillation (a, b) and fibril dissolution (c, d) for HEWL (a, c) and Ab140 (b, d) as monitored by for 24 h (for HEWL) or 7 days (for
thioflavin T-based fluorescence assay. Details of the conditions are described in the Experimental Section. Ab140) (Figure 5). Control experi-
Chem. Eur. J. 2014, 20, 1 9 www.chemeurj.org 3 2014 Wiley-VCH Verlag GmbH & Co. KGaA, Weinheim & &
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cipitated particles were redissolved in distilled water (1 mL) and water. Next, neuro2a cells were incubated at 37 8C for 48 h after
used for further study. mixing with aliquots (25 mL) of dispersions of Ab140 fibrils pro-
duced under different conditions, and cell viability was determined
Amyloid fibrillation study by an MTT (3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bro-
mide) assay. Typically, each well plate containing cells was treated
Amyloid fibril formation was studied using HEWL protein and amy- with a freshly prepared MTT solution and incubated for 4 h. The su-
loid b-peptide. A 2 mg mL 1 HEWL protein solution was prepared pernatant was then carefully removed leaving violet formazan on
by dissolving HEWL powder in 12 mm hydrochloric acid (2 mL) of the plate. This formazan was then dispersed in DMF/water and its
pH 2.0 containing 140 mm NaCl and 2.7 mm KCl. The protein solu- absorbance at 570 nm was measured with a microplate reader. Cell
tion was then magnetically stirred at 60 8C for 24 h. Similarly, an viability was correlated with the absorbance value, assuming 100 %
amyloid b-peptide solution (25 mm) was prepared by first dissolving viability for the cells without any fibrils.
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 All UV/Vis spectra were recorded on a Shimadzu UV-2550 UV/Vis
(ThT)-based titration method. Typically, a 10 mm stock solution of spectrophotometer from sample solutions in a quartz cell of 1 cm
ThT was prepared by dissolving thioflavin T powder in PBS buffer path length. Fluorescence spectra were measured with a BioTek
of pH 7.4. Aliquots (20 mL) of protein solution were collected at Synergy MX microplate reader. NMR spectra were measured on
timed intervals and mixed with 1 mL of the ThT solution. In the a Bruker F NMR (DPX-500 MHz) instrument. High resolution mass
case of amyloid b-peptide, peptide solution (20 mL) was added to spectra (HRMS) were recorded on a Waters QTOF Micro YA263
ThT solution (200 mL). After 5 min, the fluorescence of ThT was spectrometer. Fourier-transform infrared spectra were recorded
measured under excitation at 440 nm. In order to study the effect from samples in KBr pellets on a Perkin-Elmer Spectrum 100 FTIR
of nanoparticles on the fibrillation kinetics, a nanoparticle solution spectrometer. DLS and zeta potential studies were performed
was mixed with protein/peptide solution and the aggregation ki- using a NanoZS (Malvern) instrument. TEM was performed with an
netics was studied by means of the ThT-based assay. Typically, FEI Tecnai G2 F20 microscope with a field-emission gun operating
2501000 mL of Au-curcumin solution was mixed with HEWL solu- at 200 kV.
tion, keeping the final volume at 2 mL for HEWL fibrillation study,
whereas 25100 mL of Au-curcumin solution was mixed with Ab140
solution for Ab fibrillation study, keeping the final solution volume Acknowledgements
at 200 mL. The effect of free curcumin on amyloid fibrillation kinet-
ics was studied using a 1 % ethanolic solution. Curcumin is com- The authors would like to thank the Department of Biotech-
pletely soluble in ethanol in the concentration range 060 mm.
nology (DBT) and the Department of Science and Technology
For Western blot analysis, equal amounts of samples were run in
(DST) of the Government of India for financial assistance. S.P.
14 % SDS-PAGE and then transfered to a nitrocellulose membrane.
acknowledges the Council for Scientific and Industrial Research
Blots were incubated with 6E10 primary antibodies (which detect
Ab), washed three times, incubated with horse radish peroxidase (CSIR), India for providing a research fellowship. A.R.M. ac-
(HRP) conjugated secondary antibodies for 3 h, washed, and devel- knowledges the DST and the Indian Association for the Culti-
oped by an enhanced chemiluminescence (ECL) technique. vation of Science for providing a research fellowship.
Chem. Eur. J. 2014, 20, 1 9 www.chemeurj.org 7 2014 Wiley-VCH Verlag GmbH & Co. KGaA, Weinheim & &
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& & Chem. Eur. J. 2014, 20, 1 9 www.chemeurj.org 8 2014 Wiley-VCH Verlag GmbH & Co. KGaA, Weinheim
FULL PAPER
Nanoparticles in amyloid therapy: Cur- & Functionalized Nanoparticles
cumin-functionalized water-soluble gold
nanoparticles have been found to effi- S. Palmal, A. R. Maity, B. K. Singh, S. Basu,
ciently inhibit amyloid fibril growth and N. R. Jana,* N. R. Jana*
to disintegrate preformed amyloid fibrils && &&
(see figure). Consequently, they serve to
reduce amyloid-induced neurotoxicity. Inhibition of Amyloid Fibril Growth
and Dissolution of Amyloid Fibrils by
CurcuminGold Nanoparticles
Chem. Eur. J. 2014, 20, 1 9 www.chemeurj.org 9 2014 Wiley-VCH Verlag GmbH & Co. KGaA, Weinheim & &