DOI: 10.1002/chem.
201400079 Full Paper
& Functionalized Nanoparticles
Inhibition of Amyloid Fibril Growth and Dissolution of Amyloid
Fibrils by CurcuminGold Nanoparticles
Sharbari Palmal,[a] Amit Ranjan Maity,[a] Brijesh Kumar Singh,[b] Sreetama Basu,[b]
Nihar R. Jana,*[b] and Nikhil R. Jana*[a]
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-functional-
ized gold nanoparticles (Au-curcumin) of hydrodynamic di-
ameter 1025 nm, which serve to inhibit amyloid fibrillation
and disintegrate/dissolve amyloid fibrils. In nanoparticle
Introduction
Amyloid protein fibrillation is a generic feature of various
human neurological disorders, such as Alzheimers, Parkinsons,
and Huntingtons diseases.[18] Amyloid fibrils are ordered pro-
tein aggregates consisting of crossed b-sheet secondary struc-
tures.[18] They are highly neurotoxic, causing neuronal cell
death. Alzheimers disease is the most devastating neurodege-
nerative disorder affecting millions of people worldwide.[6] Ac-
cumulation of fibrillar aggregates in the brain is the pathologi-
cal hallmark of Alzheimers disease. b-Amyloid peptides (Ab140,
Ab142), 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 envis-
age that inhibition of amyloid fibrillation and dissolution of fi-
brillar aggregates should offer a promising therapeutic ap-
proach towards Alzheimers disease.
Recent reports have shown that nanoparticles can signifi-
cantly influence fibrillation processes, depending on the parti-
cle size and surface functionality.[924] For instance, amyloid fi-
brillation 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: camnrj@iacs.res.in
[b] B. K. Singh, S. Basu, Prof. N. R. Jana
National Brain Research Centre
Manesar, Gurgaon-122050 (India)
E-mail: nihar@nbrc.ac.in
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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form, curcumin is water-soluble and can efficiently interact
with amyloid protein/peptide, offering enhanced per-
formance in inhibiting amyloid fibrillation and dissolving
amyloid fibrils. Our results imply that nanoparticle-based ar-
tificial molecular chaperones may offer a promising thera-
peutic approach to combat neurodegenerative disease.
cles,[13, 15] anionic quantum dots,[16] a-synuclein-functionalized
quantum dots,[14] and peptide-functionalized g-Fe2O3 nanopar-
ticles.[17] In contrast, retardation of amyloid fibrillation has been
reported for hydrophobic polymer nanoparticles,[19] N-acetyl-l-
cysteine-capped CdTe quantum dots,[20] peptide-functionalized
g-Fe2O3 nanoparticles,[17] dipeptide-conjugated polymer nano-
particles,[18] peptide-capped protein microspheres,[21] thiogly-
colic acid-capped CdTe quantum dots,[22] dihydrolipoic acid-
capped CdSe/ZnS quantum dots,[23] and fetal bovine serum-
coated graphene oxide.[24] In addition, dose-dependent acceler-
ation 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.[2628] For instance, pep-
tide-functionalized gold nanoparticles[26] and thioflavin S-conju-
gated graphene oxide[27] have been used to dissolve amyloid
fibrils under photothermal conditions. Anionic gold nanoparti-
cles have also been shown to induce partial fibril dissocia-
tion.[28] These findings suggest that a nanoparticle-based ap-
proach might be a promising option for the treatment of vari-
ous 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 struc-
tures, nanoparticles have been functionalized with molecules
of various affinities, such as a peptide,[26] sialic acid,[29, 30] thiofla-
vin,[27] a stilbene derivative,[31] and curcumin.[3243] 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.[3243] Curcumin
(M.W. 368.38) is a natural polyphenolic compound extracted
from the Indian spice turmeric.[44] The chemical name of curcu-
min is difurylmethane, and it is usually found alongside other
1  2014 Wiley-VCH Verlag GmbH & Co. KGaA, Weinheim & &

polyphenol compounds such as demethoxycurcumin, bisde-
methoxycurcumin, 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 applica-
tion.[45] Thus, in vitro and in vivo inhibition of amyloid fibrilla-
tion by curcumin have been performed after synthesizing
water-soluble curcumin-based conjugates with small mole-
cules, polymers, and nanoparticles.[36, 4042] However, to the best
of our knowledge, there have not hitherto been any reports of
the use of curcumin-based nanoparticles or polymers to dis-
solve amyloid fibrils/plaque, although there are preliminary re-
ports 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 exter-
nal agent or force.
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-(3-
dimethylaminopropyl)-N-ethylcarbodiimide). The high water-solubility of Au-
curcumin 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-termi-
nated silica-coated Au nanoparticles[46] through conventional
EDC coupling. The formation of curcumin-COOH was con-
firmed 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
& & Chem. Eur. J. 2014, 20, 1 9 www.chemeurj.org
Full Paper
Figure 1. a) Optical properties and b) representative TEM image of Au-curcu-
min. The inset shows an image of a test tube containing an aqueous solu-
tion of Au-curcumin. Arrow indicates the curcumin absorption band at
420 nm.
(Figure 1 and Figures S3S10 in the Supporting Information).
Although curcumin is insoluble in water, Au-curcumin be-
comes 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 sur-
face 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 exten-
sively 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 nanoparti-
cle. A fluorescamine test confirmed the conjugation of a frac-
tion of the amine groups on the surface of the gold nanoparti-
cles with curcumin-COOH (Figure S4). From the absorption
band of curcumin and the Au plasmon band seen for Au-cur-
cumin, 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-cur-
cumin with high water-solubility (Figure S9). The good water-
solubility of Au-curcumin is due to the high water-solubility of
the silica-coated gold nanoparticles,[46] and causes the curcu-
min 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 hy-
drodynamic diameter of Au-curcumin to be 1025 nm, includ-
ing 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 512 nm. The increase
in hydrodynamic diameter of Au-curcumin can be attributed
to partial crosslinking between particles during the conjuga-
tion 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
(+2530 mV) in the pH range 27. This value is similar to that
of the silica-coated Au nanoparticle before functionalization,[46]
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suggesting that a fraction of the amine groups reacted with
curcumin and the rest remained free.
Effect of Au-curcumin on amyloid fibrillation, fibril disinte-
gration/dissolution, and fibril-induced cytotoxicity
The effect of Au-curcumin on amyloid formation was examined
using hen egg white lysozyme (HEWL) as a widely studied
[48]
model amyloid protein as well as Ab140 as a real amyloid
[18]
peptide. In order to investigate the influence on the fibrilla-
tion process, the monomeric amyloid protein or peptide was
incubated with Au-curcumin under fibril-forming conditions,
and the fibrillation kinetics was monitored by a thioflavin T
(ThT)-based fluorescence assay. ThT is an amyloid-specific ben-
zothiazole dye, the fluorescence of which is enhanced through
binding with amyloid fibrils and is directly correlated with the
[49, 50]
extent of fibrillation. We observed that Au-curcumin signif-
icantly influenced the fibrillation process for both HEWL and
Ab140 in a dose-dependent manner (Figure 2 and Figures S11
S23 in the Supporting Information). In general, amyloid protein
fibrillation follows a nucleation-dependent pathway that con-
sists of three distinct steps, namely i) nucleation, ii) elongation/
growth, and iii) equilibration/steady stage, as observed previ-
[1124]
ously. In HEWL fibrillation, the typical lag time for nuclea-
tion is around 30 min (which does not change in the presence
of nanoparticles), and this is followed by an elongation phase
from 1 to 12 h. However, the rate of elongation becomes
slower with increasing concentration of Au-curcumin. A similar
result was also found for Ab140. The nucleation process of
Ab140 fibrillation starts with a lag time of around 12 h (which
does not change in the presence of nanoparticles), followed
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.
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Full Paper
by an elongation phase that is extended from one to five days
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
average lengths of fibrils formed under different conditions
were measured from the collection of TEM images, and their
length distribution histograms are summarized in Figure 5. The
results clearly show that the lengths of the fibrils formed in
the presence of Au-curcumin were significantly smaller than
those of the fibrils formed in the absence of the nanoparticles.
For example, HEWL fibrils of length 0.41.4 mm (average
0.8 mm) were formed in the absence of the nanoparticles, but
were restricted to lengths < 0.2 mm (average 0.09 mm) in the
presence of Au-curcumin. Similarly, Ab140 fibrils of length 0.5
5 mm (average 2.3 mm) were formed in the absence of the
nanoparticles, but were again restricted to lengths < 0.2 mm
(average 0.1 mm) in the presence of Au-curcumin. Control ex-
periments were performed using Au nanoparticles without cur-
cumin functionalization and using pure curcumin. The results
showed that pure curcumin or Au nanoparticles (without cur-
cumin functionalization) were less active or partially inhibited
amyloid fibrillation compared with a similar concentration of
Au-curcumin (Figures 3 bd and 4 bd). We performed another
control fibrillation experiment using a mixture of Au nanoparti-
cles and curcumin and compared the results with those ob-
tained using Au-curcumin, Au nanoparticles, and curcumin.
The Au-curcumin showed a much better inhibitory effect than
the sum of the individual effects of Au nanoparticles and cur-
cumin (Figure S20). These results prove that Au-curcumin is
more efficient than curcumin or Au nanoparticles in inhibiting
amyloid fibrillation.
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
Ab140) (Figure 5). Control experi-
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Western blot analysis was per-

formed to investigate the forma-
tion of Ab oligomers and their
relative concentration with re-
spect 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 con-
trols. The results showed that
the Ab monomer band was
more intense for Ab oligomer,
Ab monomer, curcumin, and Au-
curcumin, 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 (ae) and disintegration/dissolution of HEWL fibrils (f) as observed by TEM. Details
of the conditions are described in the Experimental Section. a) Long fibril formation in the absence of nanoparti-
interestingly, insignificant oligo-
cles or curcumin; b, c) short fibril formation in the presence of Au nanoparticles; d) long fibril formation in the mer band was observed for Au.
presence of 40 mm curcumin; e) very short and insignificant fibril formation in the presence of Au-curcumin; f) dis- Considering that the same con-
integration/dissolution of long HEWL fibrils after incubation with Au-curcumin for 24 h. centration of Ab monomer was
used for all samples, the stron-
gest Ab monomer band for Au-
curcumin indicates a lower pop-
ulation of fibrils or oligomers.
The observation of smeared oli-
gomer 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 conse-
quent slowing of their move-
ment under electrophoresis.
We further investigated the
effect of Au-curcumin on the
Ab140 fibril-induced toxicity to-
wards the neuro2a cell line.
Ab140 fibrils were prepared sep-
arately from Ab140 monomer in
the presence or absence of Au-
curcumin/curcumin. In addition,
preformed fibrils were mixed
with curcumin/Au-curcumin for
Figure 4. Fibrillation of Ab140 (ae)and disintegration/dissolution of Ab140 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
or curcumin; b, c) long fibril formation in the presence of Au nanoparticles ; d) long fibril formation in the pres- them and then used for cytotox-
ence of 40 mm curcumin; e) very short and insignificant fibril formation in the presence of Au-curcumin; f) disinte- icity studies. Next, fibrils pro-
gration/dissolution of long Ab140 fibrils after incubation with Au-curcumin for 7 days. duced under different conditions
were mixed with neuro2a cells,
ments showed that Au nanoparticles or curcumin at similar incubated at 37 8C for 48 h, and cell viability was determined
concentrations were completely inactive in dissolving fibrils by an MTT (3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium
(Figure 2 c, d and Figure S19). This result confirmed that Au- bromide) assay. The results showed that cell viability was very
curcumin is capable of disintegrating and dissolving amyloid fi- low (50 %) in the presence of Ab140 fibrils, but viability in-
brils without any external force. creased to 7090 % in the presence of curcumin or Au-curcu-

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Figure 5. Length distributions of amyloid fibrils for HEWL (a) and Ab140 (b) obtained by
measuring the fibril lengths from TEM images. Values in brackets indicate the average
lengths of the fibrils.
Figure 6. Western blot analysis of freshly prepared Ab140 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 pre-
pared 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.
min-based inhibited/disintegrated fibrils (Figure 7). Most signif-
icantly, cell viability was around 90 % for Au-curcumin com-
pared with 70 % with a similar concentration of curcumin, sug-
gesting that Au-curcumin is more efficient than curcumin to-
wards amyloid detoxification. Similarly, toxicity results with dis-
integrated 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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Full Paper
Au-curcumin nanoparticles as artificial molecular
chaperones
Considering the prevention and curing aspect of neu-
rodegenerative disease, the nanoparticle probe
should have the ability to inhibit the amyloid fibrilla-
tion 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 bio-
compatible,[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-
Figure 7. Effect of curcumin/Au-curcumin on Ab140 fibril-induced neurotox-
icity towards neuro2a cell lines. The first two columns relate to Au/Au-curcu-
min-induced cell viability, columns 35 relate to Ab-induced cell viability,
whereby fibrils were prepared in the presence or absence of curcumin/Au-
curcumin, and the last three columns relate to cell viability with disintegrat-
ed fibrils prepared by the action of curcumin/Au-curcumin. Fibrils were incu-
bated with neuro2a cells for 48 h and cytotoxicity was estimated by MTT
assay. The final concentration of Ab140 fibrils in terms of monomer was
around 1.25 mm, and the final concentration of curcumin in the form of Au-
curcumin or as free curcumin was around 2 mm. The final concentration of
Au in Au nanoparticles/Au-curcumin was around 0.12 mm.
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. Curcu-
min is known to inhibit the protein self-assembly process by
binding with the oligomer/fibril (but not with the native pro-
tein) through aromatic p-stacking interactions.[5153] Thus, it is
expected that Au-curcumin binds with the oligomer/fibril
through the curcumin moiety and interferes with the elonga-
tion phase of fibrillation. The enhanced inhibitory effect of Au-
curcumin 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 multi-
valent interaction has been observed for peptide-conjugated
5  2014 Wiley-VCH Verlag GmbH & Co. KGaA, Weinheim & &

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 disintegra-
tion 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 dissolu-
tion in the presence of free curcumin at different concentra-
tions and compared the results with those obtained using Au-
curcumin. The effective curcumin concentration in the Au-cur-
cumin used was typically in the range 1040 mm. However, fi-
brillation experiments at similar concentrations of free curcu-
min mostly produced long fibrils and bundles thereof for both
HEWL and Ab140 (Figures 3 d and 4 d and Figures S13S18). In
addition, ThT-based assay showed that HEWL fibrillation was
4060 % less with Au-curcumin compared with a similar con-
centration of free curcumin (Figure S16). Similarly, fibril dissolu-
tion 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 anti-
amylogenic properties than free curcumin at a similar concen-
tration.
Conclusion
We have synthesized curcumin-functionalized gold nanoparti-
cles (Au-curcumin) and have shown that they inhibit amyloid
fibrillation in a dose-dependent manner and are capable of dis-
integrating/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 attrib-
uted to the curcumin being rendered soluble in water and the
multiple curcumin moieties on each Au nanoparticle. The pre-
sented nanotechnology based approach is important, consider-
ing the highly interdisciplinary aspect of the prevention and
cure of neurodegenerative diseases, which requires in vitro
testing of nanoprobes, in vivo targeting by bloodbrain 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.
& & Chem. Eur. J. 2014, 20, 1 9 www.chemeurj.org
Full Paper
Experimental Section
Reagents
Hen egg-white lysozyme (HEWL), amyloid b-protein fragment 140
with the sequence Asp-Ala-Glu-Phe-Arg-His-Asp-Ser-Gly-Tyr-Glu-Val-
His-His-Gln-Lys-Leu-Val-Phe-Phe-Ala-Glu-Asp-Val-Gly-Ser-Asn-Lys-
Gly-Ala-Ile-Ile-Gly-Leu-Met-Val-Gly-Gly-Val-Val, gold(III) chloride, di-
dodecyldimethylammonium bromide (DDAB), 3-(mercaptopropyl)-
trimethoxysilane (MPS), 2-aminoethyl-aminopropyltrimethoxysilane
(AEAPS), tetrabutylammonium borohydride (TBAB), sodium chlo-
ride, thioflavin T, curcumin, 4-(dimethylamino)pyridine (DMAP), glu-
taric anhydride, triethylamine, and N-(3-dimethylaminopropyl)-N-
ethylcarbodiimide hydrochloride (EDC) were purchased from
SigmaAldrich. All reagents were used without further purification.
Preparation of curcumin monocarboxylic acid (curcumin-
COOH)
A monocarboxylic acid derivative of curcumin was prepared ac-
cording 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 solu-
tion 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 mono-
carboxylic 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 dichlorome-
thane and methanol (95:5, v/v). The yield of curcumin-COOH was
about 45 %.
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 so-
lution of MPS in toluene (20 mL) was then added. Thereafter, a solu-
tion 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 fur-
ther 5 min. The precipitated particles were washed twice with tolu-
ene and twice with ethanol and then dissolved in distilled water.
Preparation of Au-curcumin
Curcumin-COOH was covalently conjugated with the amine-func-
tionalized 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 etha-
nol (0.25 mL) was then added to the curcumin solution with stir-
ring. A solution of Au nanoparticles in water (1 mL) was then
added and the reaction was allowed to proceed for 6 h under stir-
ring. The resulting solution was then centrifuged at 12 000 rpm to
precipitate the gold nanoparticlecurcumin conjugate and the pre-
cipitate was repeatedly washed with chloroform and ethanol to
remove unbound curcumin monocarboxylic acid. Finally, the pre-
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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 amy-
loid 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 solu-
tion 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.
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 ki-
netics was studied by means of the ThT-based assay. Typically,
2501000 mL of Au-curcumin solution was mixed with HEWL solu-
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
at 200 mL. The effect of free curcumin on amyloid fibrillation kinet-
ics was studied using a 1 % ethanolic solution. Curcumin is com-
pletely soluble in ethanol in the concentration range 060 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 devel-
oped by an enhanced chemiluminescence (ECL) technique.
Disaggregation study
About 2 mL of aggregated protein solution was centrifuged at
12 000 rpm for 4 min and the precipitated aggregate was redis-
persed in aqueous HCl (1 mL) of pH 2. An aliquot of 1 mL of nano-
particle 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.
Cytotoxicity assay
Neuro2a cells were cultured in a tissue culture flask with Dulbec-
cos modified Eagles (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. Ab140
fibrils were prepared separately in the presence or absence of Au-
curcumin/curcumin, by incubating Ab140 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 www.chemeurj.org
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water. Next, neuro2a cells were incubated at 37 8C for 48 h after
mixing with aliquots (25 mL) of dispersions of Ab140 fibrils pro-
duced under different conditions, and cell viability was determined
by an MTT (3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bro-
mide) assay. Typically, each well plate containing cells was treated
with a freshly prepared MTT solution and incubated for 4 h. The su-
pernatant 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.
Instrumentation
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.
Acknowledgements
The authors would like to thank the Department of Biotech-
nology (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. ac-
knowledges the DST and the Indian Association for the Culti-
vation of Science for providing a research fellowship.
Keywords: amyloid beta-peptides amyloid fibrils curcumin
fibril dissolution gold nanoparticles neurochemistry
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Received: January 8, 2014
Published online on && &&, 0000
8  2014 Wiley-VCH Verlag GmbH & Co. KGaA, Weinheim
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FULL PAPER
Nanoparticles in amyloid therapy: Cur-
cumin-functionalized water-soluble gold
nanoparticles have been found to effi-
ciently inhibit amyloid fibril growth and
to disintegrate preformed amyloid fibrils
(see figure). Consequently, they serve to
reduce amyloid-induced neurotoxicity.
Chem. Eur. J. 2014, 20, 1 9 www.chemeurj.org
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& Functionalized Nanoparticles
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
CurcuminGold Nanoparticles
9  2014 Wiley-VCH Verlag GmbH & Co. KGaA, Weinheim & &