Engineered polymer nanoparticles containing hydrophobic dipeptide for inhibition of amyloid-β fibrillation

Article abstract of DOI:10.1021/bm3011177

Protein aggregation into amyloid fibrils is implicated in the pathogenesis of many neurodegenerative diseases. Engineered nanoparticles have emerged as a potential approach to alter the kinetics of protein fibrillation process. Yet, there are only a few reports describing the use of nanoparticles for inhibition of amyloid-β 40 (Aβ₄₀) peptide aggregation, involved in Alzheimer's disease (AD). In the present study, we designed new uniform biocompatible amino-acid-based polymer nanoparticles containing hydrophobic dipeptides in the polymer side chains. The dipeptide residues were designed similarly to the hydrophobic core sequence of Aβ. Poly(N-acryloyl-l- phenylalanyl-l-phenylalanine methyl ester) (polyA-FF-ME) nanoparticles of 57 ± 6 nm were synthesized by dispersion polymerization of the monomer A-FF-ME in 2-methoxy ethanol, followed by precipitation of the obtained polymer in aqueous solution. Cell viability assay confirmed that no significant cytotoxic effect of the polyA-FF-ME nanoparticles on different human cell lines, e.g., PC-12 and SH-SY5Y, was observed. A significantly slow secondary structure transition from random coil to β-sheets during Aβ₄₀ fibril formation was observed in the presence of these nanoparticles, resulting in significant inhibition of Aβ₄₀ fibrillation kinetics. However, the polyA-FF-ME analogous nanoparticles containing the l-alanyl-l-alanine (AA) dipeptide in the polymer side groups, polyA-AA-ME nanoparticles, accelerate the Aβ₄₀ fibrillation kinetics. The polyA-FF-ME nanoparticles and the polyA-AA-ME nanoparticles may therefore contribute to a mechanistic understanding of the fibrillation process, leading to the development of therapeutic strategies against amyloid-related diseases.

Full text of DOI:10.1021/bm3011177

Article
pubs.acs.org/Biomac
Engineered Polymer Nanoparticles Containing Hydrophobic
Dipeptide for Inhibition of Amyloid‑β Fibrillation
Hadas Skaat,† Ravit Chen,†,‡ Igor Grinberg, and Shlomo Margel*
Department of Chemistry, Bar-Ilan Institute of Nanotechnology and Advanced Materials, Ramat-Gan 52900, Israel
S
* Supporting Information
ABSTRACT: Protein aggregation into amyloid fibrils is
implicated in the pathogenesis of many neurodegenerative
diseases. Engineered nanoparticles have emerged as a potential
approach to alter the kinetics of protein fibrillation process.
Yet, there are only a few reports describing the use of
nanoparticles for inhibition of amyloid-β 40 (Aβ40) peptide
aggregation, involved in Alzheimer’s disease (AD). In the
present study, we designed new uniform biocompatible amino-
acid-based polymer nanoparticles containing hydrophobic
dipeptides in the polymer side chains. The dipeptide residues
were designed similarly to the hydrophobic core sequence of Aβ. Poly(N-acryloyl-L-phenylalanyl-L-phenylalanine methyl ester)
(polyA-FF-ME) nanoparticles of 57 6 nm were synthesized by dispersion polymerization of the monomer A-FF-ME in 2-
methoxy ethanol, followed by precipitation of the obtained polymer in aqueous solution. Cell viability assay confirmed that no
significant cytotoxic effect of the polyA-FF-ME nanoparticles on different human cell lines, e.g., PC-12 and SH-SY5Y, was
observed. A significantly slow secondary structure transition from random coil to β-sheets during Aβ40 fibril formation was
observed in the presence of these nanoparticles, resulting in significant inhibition of Aβ40 fibrillation kinetics. However, the
polyA-FF-ME analogous nanoparticles containing the L-alanyl-L-alanine (AA) dipeptide in the polymer side groups, polyA-AA-
ME nanoparticles, accelerate the Aβ40 fibrillation kinetics. The polyA-FF-ME nanoparticles and the polyA-AA-ME nanoparticles
may therefore contribute to a mechanistic understanding of the fibrillation process, leading to the development of therapeutic
strategies against amyloid-related diseases.
Several studies on the aggregation process of Aβ have
identified the critical peptidic sequence involved in amyloid
aggregates formation.14−18 The hydrophobic core from residues
17−20 of Aβ, LVFF sequence, is crucial for the formation of β-
sheet structures.14−18 It was also demonstrated that this
peptidic region binds to its homologous sequence in Aβ and
prevents its aggregation into amyloid fibrils.19−21 Such
inhibition is mainly based on recognition between the pairs
of phenylalanine residues (FF) through hydrophobic inter-
actions.14−21 This FF motif has served as a key platform for the
development of peptide and peptidomimetic inhibitors of Aβ
fibrillation.17 Although peptide or protein analogues with
specific binding sites may be of primary importance for studies
of neurodegenerative disorders, therapeutic agents have not
been developed within this strategy. The main problems are the
blood-brain barrier (BBB) permeability, the complexity of their
synthesis, and their low in vivo stability and efficacy.22
INTRODUCTION
■
The formation of amyloid aggregates is a pathological hallmark
of many diseases, including Parkinson’s, Huntington’s, mad
cow, prion, and Alzheimer’s disease (AD).1−3 The common
feature of amyloid diseases is the transition of proteins from the
normally soluble form into amyloid fibrils, organized mainly
into cross β-sheets, which accumulate in the extracellular space
of various tissues.4−6 The extracellular deposits that character-
ize AD are composed of fibrils of a small peptide with 39−43
amino acids, the amyloid-β (Aβ) peptide.7 Aβ is continuously
secreted by normal cells in culture and is detected as a soluble
peptide in the plasma and cerebrospinal fluid of healthy
individuals.8,9 In AD patients, Aβ exists as insoluble fibrillar
aggregates.10 It is generally accepted that the Aβ peptides can
self-assemble to form neurological toxic aggregates with various
morphologies, such as dimers, oligomers, protofibrils, and
fibrils, which eventually deposit as insoluble plaques and cause
the death of brain cells.11 Currently, there are no effective drugs
to cure these diseases and treatment options are extremely
limited.12 A coherent pharmacological approach for preventing
amyloid aggregation encompasses agents able to specifically
stabilize the initial conformations through interfering with the
intermediate species and/or destabilizing the β-sheet con-
formations.13
Recent literature concerning novel biomaterials indicates
increasing interest in developing nanoparticles to detect,
prevent, and treat protein-misfolding diseases.23−25 Their
potential influence on protein fibrillation is a function of both
Received: April 8, 2012
Revised: August 17, 2012
Published: August 17, 2012
© 2012 American Chemical Society
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Figure 1. Synthetic route of the polyA-FF-ME nanoparticles.
the surface interfacial properties, including charge, of the
nanoparticle and its enormous surface area to volume ratio.
Increased local protein concentration on the nanoparticle
surface and/or changes in protein conformation upon binding
could promote aggregation while trapping of early intermedi-
ates may inhibit further aggregation.26 Engineered biocompat-
ible nanoparticles offer advantages as therapeutic agents for
amyloid-related diseases since they allow modification of
surface properties so as to control the adsorption and
interaction processes. Moreover, in vitro and in vivo studies
have shown that nanoparticles are capable of overcoming the
difficulty of crossing the BBB and have greater in vivo
stability.27,28
Various nanoparticles such as copolymer particles of N-
isopropylacrylamide/N-tert-butylacrylamide, cerium oxide,
quantum dots (QDs), carbon nanotubes, and titanium oxide
have been reported to promote protein assembly into amyloid
fibrils in vitro, by assisting the nucleation process.29,30 Only a
few studies have been reported on the inhibitory effect of
nanoparticles on the Aβ fibrillation process. Very recently,
Cabaleiro-Lago et al. reported the inhibition of the Aβ40 fibril
formation by copolymer nanoparticles of variable hydro-
phobicity,31 and also demonstrated the dual effect of
commercial polystyrene (PS) nanoparticles with amino
modification toward the Aβ40 and Aβ42 fibril formation.32 Yoo
et al. have shown an inhibition effect of CdTe QDs on Aβ40
fibrillation.33 Fluorinated nanoparticles34 and sulfonated and
sulfated PS nanoparticles35 have been also reported as potential
candidates for the inhibition of Aβ fibril formation. Our
previous work showed that the Leu-Pro-Phe-Phe-Asp (LPFFD)
peptide conjugated iron oxide nanoparticles slightly inhibit the
Aβ40 fibrillation.36
ization behavior, structures, and properties.42−44 Yet, to the best
of our knowledge, there is no report on the preparation of
polymeric nanoparticles from N-acryloyl amino acid mono-
mers.
The present article describes a novel designed synthesis of
uniform biocompatible amino-acid-based polymer nanopar-
ticles containing hydrophobic dipeptides in the polymer side
chains. The dipeptide residues were designed similarly to the
hydrophobic core sequence of Aβ. Poly(N-acryloyl-L-phenyl-
alanyl-L-phenylalanine methyl ester) (polyA-FF-ME) nano-
particles of 57
6 nm were synthesized by dispersion
polymerization of the monomer A-FF-ME in 2-methoxy
ethanol, followed by precipitation of the obtained polymer in
aqueous solution. These nanoparticles were well-characterized
including their cytotoxic effect on different human cell lines, i.e.,
PC-12 (pheochromocytoma cell line) and SH-SY5Y (neuro-
blastoma cell line). Kinetics of the Aβ40 fibrillation process in
the absence and the presence of varying concentrations of the
polyA-FF-ME nanoparticles, and their analogues polyA-AA-ME
nanoparticles containing the L-alanyl-L-alanine (AA) dipeptide
instead of FF in the polymer side chains, have been elucidated.
A significantly slow direct secondary structure transition from
random coil to β-sheets during Aβ40 fibril formation was
observed in the presence of the polyA-FF-ME nanoparticles.
This observation indicates that these nanoparticles significantly
increase the lag time and hence the kinetics of Aβ40 fibrillation.
However, the opposite behavior was observed in the presence
of the polyA-AA-ME nanoparticles, indicating acceleration of
Aβ40 fibrillation kinetics.
MATERIALS AND METHODS
■
Materials. The following analytical-grade chemicals were pur-
chased from commercial sources and used without further purification:
L-phenylalanyl-L-phenylalanine (FF), acetyl chloride, methanol anhy-
drous, acryloyl chloride, triethylamine (TEA), dimethylaminopyridine
(DMAP), dry dichloromethane (DCM), hydrochloric acid (1 M),
hexane, magnesium sulfate, sodium bicarbonate, sodium hydrogen
sulfate, calcium chloride, 2-methoxyethanol, polyvinylpyrrolidone
(PVP, MW 360 000), benzoyl peroxide (BP), amyloid β protein
fragment 1−40 (Aβ40), trifluoroacetic acid (TFA), 1,1,1,3,3,3-
hexafluoro-2-propanol (HFIP), dimethyl sulfoxide (DMSO), and
Thioflavin T (ThT) from Sigma (Israel); L-alanyl-L-alanine methyl
ester (AA-ME) hydrochloride salt from D-Chem (Israel); Dulbecco’s
modified eagle medium (DMEM), fetal calf serum (FCS), L-glutamine,
penicillin, streptomycin, and 2,3-bis-(2-methoxy-4-nitro-5-sulfophen-
Poly(amino acid) nanoparticles have recently attracted great
attention due to their potential nontoxicity, biocompatibility,
nonimmunogenicity, and biofunctionality. These nanoparticles
containing amino acid moieties are potentially useful in many
different biomedical applications, such as drug or gene delivery
agents, tissue engineering scaffolds, and chiral recognition.37−40
The synthesis and radical polymerization of various acryl
monomers having amino acid moieties in the side chain have
been reported.41 Their corresponding polymers, poly(N-
acryloyl amino acids), are expected to be functional materials,
and much attention has been paid to their unique polymer-
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yl)-2H-tetrazolium-5-carboxanilide (XTT) cell viability assay kit from
Biological-Industries (Israel). Water was purified by passing deionized
water through an Elgastat Spectrum reverse osmosis system (Elga,
High Wycombe, UK). The cell lines PC-12 and SH-SY5Y were
purchased from ATCC (USA). The culture medium used in this study
was composed of 90% DMEM, 10% FCS, 2 mM L-glutamine, 50
units/mL penicillin, and 50 μg/mL streptomycin.
was then dried over MgSO4, filtered, and evaporated to produce white
solid. Pure white crystals of N-acryloyl-AA-ME (A-AA-ME) were
obtained by recrystallization of the white solid twice from DCM/
hexane (yield 44%).
1H NMR (CDCl3, 300 MHz): δ 6.6 (broad doublet, J3 = 6.8 Hz,
NH near the double bond, 1H), 6.3 (dd, J2 = 0.6 Hz, J3 = 16.8 Hz, 1H,
CH2CH), 6.28 (broad, NH, 1H), 6.16−6.05 (dd, J3cis = 10.5 Hz,
J3trans = 16.8 Hz, 1H, CH2CH), 5.69 (dd, J2 = 0.6 Hz, J3 = 10.5 Hz,
1H, CH2CH), 4.59 (double-quartet, J3 = 6.8 H, J3 = 7.2 Hz, 1H,
C(O)NHCH), 4.54 (double-quartet, J3 = 6.8 H, J3 = 7.2 Hz, 1H,
C(O)NHCH), 3.76 (s, 3H, OCH3), 1.43 (d, J3 = 6.8 3H, CHCH3),
1.41 (d, J3 = 7.2, 3H, CHCH3). 13C NMR (CDCl3, 200 MHz): δ 173.4
(C(O)NH, 1C), 172.0 (C(O)OCH3, 1C), 165.4 (C(O)CHCH,
1C), 130.6, 127.6 (2C, double bond), 52.9 (1C, OCH3), 49.0, 48.5
(2C, C(O)NHCHCH3), 18.8, 18.5 (2C, CHCH3). TOF MS ES+: 229
(MH+, 12%), 251 (MNa+, 100%). HRMS MALDI: 229.1215 (MH+).
Elemental analysis (calculated): C, 52.62; N, 12.27; H, 7.07. Found: C,
51.96; N, 12.30; H, 7.02.
Synthesis of A-FF-ME. The monomer A-FF-ME was synthesized
by esterification of FF followed by its reaction with acryloyl chloride,
according to a procedure reported previously,45,46 as shown in Figure 1
(steps I−II). Briefly, FF (1 g, 3.2 mmol) was dissolved in a flask
containing anhydrous methanol (50 mL). The flask was immersed in
an ice bath and acetyl chloride (5 mL) was then added dropwise. The
reaction mixture was stirred for 2 h at 0 °C and then stirred at room
temperature overnight. The obtained FF methyl ester (FF-ME)
hydrochloride salt was then isolated by removal under reduced
pressure of methyl acetate and methanol. Pure white brilliant crystals
of FF-ME hydrochloride salt were obtained in quantitative yield
(98%). The obtained FF-ME hydrochloride salt (1.08 g, 3.0 mmol)
was dissolved in dried DCM (45 mL). TEA (890 μL, 6.4 mmol) and
DMAP (40 mg, 0.32 mmol) were then added to the solution. The
reaction mixture was immersed in an ice bath. Acryloyl chloride (410
μL, 5 mmol) dissolved in dry DCM (5 mL) was then added dropwise
to the reaction mixture. The solution was stirred at room temperature
overnight and then diluted with further amount of DCM (40 mL).
The DCM solution was then washed with HCl (1 M) and brine. The
organic phase was then dried over MgSO4, filtered, and evaporated to
produce a white solid. Pure white crystals of N-acryloyl-FF-ME (A-FF-
ME) were obtained by recrystallization of the white solid twice from
DCM/hexane (yield 44%).
Synthesis of PolyA-AA-ME Nanoparticles. The polyA-AA-ME
was prepared by a similar procedure to that described above for the
polyA-FF-ME, substituting the monomer A-FF-ME for the A-AA-ME.
The formed soluble polyA-FF-ME was precipitated as uniform
nanoparticles by adding 100 μL of its solution to 0.5 mL distilled
water. The formed polyA-AA-ME nanoparticles were then washed by
extensive dialysis against water. Dried polyA-AA-ME nanoparticles
were then obtained by lyophilization.
1H NMR (D2O, 300 MHz): δ 4.6−4.2 (broad, C(O)NHCH, 2H),
3.7−3.6 (broad, 3H, OCH3), 2.3−2.1 (broad, 1H, CHCH2), 2.1−1.8
(broad, 2H, CHCH2), 1.5−1.3 (broad 6H, CHCH3).
Cytotoxicity Assay. XTT assay was performed to determine the
cytotoxicity of the polyA-FF-ME nanoparticles on PC-12 and SH-
SY5Y cell lines.47 Cells were seeded in a 96-well plate at a density of 1
× 104 cells/well in 100 μL culture medium and grown in a humidified
1H NMR (CDCl3, 200 MHz): δ 7.26−7.17 (m, 8H, aromatic
hydrogen), 6.95−7.00 (m, aromatic hydrogen, 2H), 6.3 (dd, J2 = 1.6
Hz, J3 = 16.8 Hz, 1H, CH2CH), 6.14 (m, NH, 2H), 6.1 (dd, J3cis =
10.0 Hz J3trans = 16.8 Hz, 1H, CH2CH), 5.65−5.70 (dd, J2 = 1.6
Hz, J3 = 10.0 Hz, 1H, CH2CH), 4.75 (m, 2H, C(O)NHCH), 3.68
(s, 3H, OCH3), 2.93−3.13 (m, 4H, PhCH2). 13C NMR (CDCl3, 200
MHz): δ 171.1 (C(O)NH, 1C), 170.1 (C(O)OCH3, 1C), 165.1
(C(O)CHCH, 1C), 130.2, 135.4 (2C, aromatic quaternary carbon),
130.2, 129.3, 129.1, 128.67, 128.60, 127.29 (aromatic carbon, 10C),
127.14, 127.08 (2C, double bond), 54.0, 53.2 (2C, C(O)NHCH), 52.2
(1C, OCH3), 38.1, 37.8 (2C, PhCH2). TOF MS ES+: 381 (MH+,
100%), 403 (MNa+, 50%). HRMS MALDI: 403.1628 (MNa+).
Elemental analysis (calculated): C, 69.46; N, 7.36; H, 6.36; O,
16.82. Found: C, 68.22; N, 7.21; H, 6.47; O, 16.45.
Synthesis of PolyA-FF-ME Nanoparticles. In a typical experi-
ment, 30 mg of the vinylic monomer A-FF-ME, 10 mg PVP, and 2 mg
of BP were added to 1 mL of nitrogen-bubbled 2-methoxy ethanol.
The mixture was shaken at room temperature to dissolve the solids,
giving concentrations of 3%, 1%, and 0.2% (w/v) A-FF-ME, PVP, and
BP, respectively. The mixture was then shaken at 75 οC for 18 h
(Figure 1, step III). The formed soluble polyA-FF-ME was
precipitated as uniform nanoparticles by adding 100 μL of its solution
to 1 mL distilled water (Figure 1, step IV). The formed polyA-FF-ME
nanoparticles were then washed by extensive dialysis against water.
Dried polyA-FF-ME nanoparticles were then obtained by lyophiliza-
tion.
ο
5% CO2 atmosphere at 37 C. After 18 h at 37 °C, different volumes
of polyA-FF-ME nanoparticles dispersed in water were added to the
cells, giving final concentrations of 0.02 and 0.2 mg/mL per well. After
incubation for 24 or 48 h at 37 °C, 50 μL XTT solution was added to
each well according to the kit manufacturer’s instructions. Absorbance
was read at 480 nm, and the absorbance of corresponding
concentrations of the nanoparticles was subtracted from the reading.
Cell viability was determined as a percentage of the negative control
(cultured cells in medium without nanoparticles).
Preparation of the Aβ40 Fibrils in the Absence and Presence
of the PolyA-FF-ME Nanoparticles or PolyA-AA-ME Nano-
particles. Lyophilized Aβ40, synthesized by Sigma Israel, was stored at
−20 °C immediately upon arrival. To obtain a homogeneous solution
free of aggregates, a variant of Zagorski’s protocol48 was followed, as
discussed in our previous publication.36 Briefly, 0.5 mg of the Aβ40 was
first dissolved with TFA, followed by TFA evaporation with N2. This
process was then repeated two more times. To remove TFA
thoroughly, HFIP was added and then evaporated with N2. This
HFIP treatment was also repeated another two times. The dry aliquot
was completely resuspended in a solution containing 0.5 M DMSO
and 0.1 M PBS (pH 7.4) to a final volume of 2.88 mL, in order to
reach a final Aβ40 concentration of 40 μM. Before use, all solutions
were filtered through 0.20 μm pore size filters. For initiating the Aβ40
fibrillation process, samples of the Aβ40 solutions (40 μM) were
incubated in 1.5 mL eppendorf tubes in a bath heater at 37 °C with
gentle shaking. To monitor the appearance and growth of fibrils,
aliquots from the tubes were taken at different times and added to a
black 96-well plate, and the ThT fluorescence (20 μM ThT added to
each well) was measured at 485 nm with excitation at 435 nm in a
plate reader.49
1H NMR (CDCl3, 200 MHz): δ 7.3−6.8 (broad, aromatic
hydrogens and amide hydrogens, 12H), 4.8−4.3 (broad, C(O)NHCH,
2H), 3.7−3.3 (broad, 3H, OCH3), 3.2−2.7 (broad, 4H, PhCH2), 2.4−
2.1 (broad, 1H, CHCH2), 2.1−1.8 (broad, 2H, CHCH2).
Synthesis of A-AA-ME. The monomer A-AA-ME was synthesized
by the reaction of commercial AA-ME hydrochloride salt with acryloyl
chloride in cold water, according to a procedure that was reported
previously.42 Briefly, the AA-ME hydrochloride salt (1 g, 4.74 mmol)
was dissolved in water (10 mL). NaHCO3 (880 mg, 9.49 mmol) was
then added to the solution. The reaction mixture was immersed in an
ice bath. Acryloyl chloride (470 μL, 5.7 mmol) was then added
dropwise to the solution. The chilled solution was vigorously stirred
for 30 min. The solution was acidified with NaHSO4 (1 M) to pH 3
and was then washed three times with ethyl acetate. The organic phase
A similar process to that described above was also performed in the
presence of different concentrations of the polyA-FF-ME nanoparticles
or polyA-AA-ME nanoparticles. Briefly, different volumes, 2.6−26 μL,
(10−100% (w/wAβ40)) of an aqueous dispersion of nanoparticles (3.4
mg/mL) were added to 0.5 mL of 40 μM Aβ40 PBS solution, as
described above. The formation of the fibrils was then initiated by
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Figure 2. HRSEM image (A) and size histogram (B) of the polyA-FF-ME nanoparticles.
quickly raising the temperature of the aqueous mixture from room
RESULTS AND DISCUSSION
■
temperature to 37 °C at different time intervals.
Designed Synthesis of the polyA-FF-ME Nanopar-
1
Characterization. H and 13C NMR spectra were obtained with a
ticles. Recent progress in elucidating the structural properties
of Aβ fibrils has enabled the design of inhibitors of Aβ fibril
formation.14−21 In the present study, we designed new uniform
biocompatible amino-acid-based polymer nanoparticles for
inhibition of Aβ40 fibrillation process. The FF residues, the
hydrophobic core of residues 19−20 of the Aβ protein, are
well-known as crucial sequences for the formation of β-sheet
structures that trigger the fibrillation process.14−18 It has been
suggested that the Aβ assembly is partially driven by
hydrophobic interactions through recognition between the FF
pairs.14−18 Short peptides homologous to the hydrophobic core
of Aβ, for example, LPFFD, were designed to bind specifically
to full-length Aβ and used as inhibitors of its fibrillation process
via the FF recognition motif.19−21 The rational of our design is
to engineer polymer nanoparticles containing extremely high
concentrations of the FF motif. This motif in the nanoparticles
therefore should act as a recognition motif within Aβ and
interferes in its fibril formation. Figure 1 describes the synthetic
route of the monomer A-FF-ME, followed by its polymer-
ization and precipitation to form the polyA-FF-ME nano-
particles.
Bruker AC 200 or 300 MHz spectrometer. Chloroform-d chemical
shifts are expressed in ppm downfield from tetramethylsilane used as
internal standard. High-resolution mass spectra were obtained on an
AutoFlex III Tof/Tof (Bruker, Germany) in MALDI mode. Elemental
analysis was performed using an elemental analyzer, model
FlashEA1112 (Thermo Electron Corporation). Fourier transform
infrared (FTIR) analysis was performed with a ALPHA FTIR
spectrophotometer equipped with attenuated total reflection (ATR)
model A220/D-01, Bruker. Low-resolution transmission electron
microscopy (TEM) pictures were obtained with a FEI TECNAI C2
BIOTWIN electron microscope with 120 kV accelerating voltage.
Samples for TEM were prepared by placing a drop of diluted sample
on a 400-mesh carbon-coated copper grid. High-resolution scanning
electron microscopy (HRSEM) images were obtained with a FEI
Magellan 400 L electron microscope. Samples for HRSEM were
prepared by spreading a drop of diluted sample on a glass surface and
then drying it at room temperature. The dried sample was coated with
gold in a vacuum for 4 min before viewing under HRSEM. The
average size and size distribution of the dry nanoparticles were
determined by measuring the diameter of more than 100 particles with
the image analysis software AnalySIS Auto (Soft Imaging System
GmbH, Germany). Hydrodynamic diameter and size distribution of
the nanoparticles dispersed in an aqueous phase were measured using
a particle analyzer, model NANOPHOX (Sympatec GmbH,
Germany). The thermal behavior of the nanoparticles was measured
by ThermoGravimetric Analysis (TGA) and differential scanning
calorimetric (DSC), model STAR-1 System, Mettler Toledo. The
analysis was performed with approximately 10 mg of dried samples in a
dynamic nitrogen atmosphere (20 mL/min) at heating rate of 10 °C/
min. Fluorescence intensity and absorbance at room temperature were
measured using a multiplate reader Synergy 4, using Gen 5 software.
Electrokinetic properties (ζ-potential) as a function of pH were
determined by Zetasizer (Zetasizer 2000, Malvern Instruments, UK).
Circular dichroism (CD) spectra were measured on a Jasco J710
spectropolarimeter at 22 °C over the wavelength range 180−260 nm
with 0.2 nm resolution. All measurements in solution were recorded in
a 0.1 cm path length cell. Each spectrum is an average of four
measurements. The spectra were corrected by subtracting the buffer
solution or nanoparticles’ dispersion baseline. All samples of Aβ40
solutions were diluted with phosphate buffer (PB) solution to a final
concentration of 10 μM. Aβ40 solutions sampled at long incubation
times (120 and 336 h) when the fibrils were already formed were
centrifuged prior to scanning.
Characterization of the polyA-FF-ME Nanoparticles.
Figure 2 presents the dry (A) and hydrodynamic (B) diameters
and size distributions of the polyA-FF-ME nanoparticles as
measured by HRSEM images and light scattering, respectively.
The dry diameter is 57
6 nm, while the hydrodynamic
diameter is 200 27 nm. The difference in the diameter
measured by HRSEM and light scattering is due to the fact that
the second method also takes into account the surface-adsorbed
solvent (water) molecules.
FTIR spectra of the monomer A-FF-ME (A) and the polyA-
FF-ME nanoparticles (B) are shown in Figure 3. The IR
spectrum of the monomer A-FF-ME (Figure 3A) indicates
stretching vibrations at 1757 and 1206 cm−1 corresponding to
the methyl ester (ME) groups, and stretching vibrations at
1672, 1647, and 1552 cm−1 corresponding to the amide groups.
The absorbance peak at 1624 cm−1 corresponds to the
stretching vibration of CC bonds. The absorbance peaks
from 2980 to 3070 and from 1456 to 1496 cm−1 are
characteristic of the C−H stretching (aromatic and aliphatic)
and CH2 groups, and peaks at 700 and 3269 cm−1 correspond
to the stretching of C−C and N−H bonds, respectively. The IR
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Figure 3. FTIR spectra of the monomer A-FF-ME (A) and the polyA-
FF-ME nanoparticles (B).
spectrum of the polyA-FF-ME nanoparticles (Figure 3B) is
similar to that of the monomer except for the typical stretching
vibration of CC bonds that is not present in the polyA-FF-
ME nanoparticles. The chemical structures of the monomer A-
FF-ME and its corresponding polymer were also confirmed by
1
1H NMR analysis (in CDCl3), as shown in Figure 4. The H
NMR spectrum of the monomer (Figure 4A) reveals the typical
peaks of the acrylic protons at 6.3−6.1 ppm and 5.70−5.65
ppm, the aromatic protons at 7.2 and 7.0 ppm, the amide
protons at 6.1 ppm, the proton attached to chiral carbon atom
at 4.75 ppm, the methylene protons at 3.1 ppm, and the ME
protons at 3.68 ppm. The 1H NMR spectrum of the polyA-FF-
ME nanoparticles (Figure 4B) reveals the complete absence of
the acrylic protons at 5.5−6.5 ppm and the presence of broad
peaks of the aromatic and amide protons at 7.3−6.8 ppm, the
proton attached to chiral carbon atom at 4.8−4.3 ppm, the ME
protons at 3.7−3.3 ppm, the methylene protons at 3.2−2.7
ppm, and the aliphatic chain protons of the polymer backbone
at 2.4−1.8 ppm.
Figure 4. 1H NMR spectra (in CDCl3) of the monomer A-FF-ME (A)
and the polyA-FF-ME nanoparticles (B).
The thermal behavior of the polyA-FF-ME nanoparticles is
shown in Figure 5. The TGA curve exhibits a steep slope
between 280 and 450 °C, indicating a 68% weight loss due to
the polymer decomposition, leaving residual carbon. The Tm of
the polyA-FF-ME nanoparticles measured by DSC is ca. 165 °C
(data not shown).
To learn about the stability of the polyA-FF-ME nano-
particles in aqueous medium, ζ-potential measurements can be
employed as an indirect indicator of their surface charge. Figure
6 illustrates the ζ-potential curve of the polyA-FF-ME
nanoparticles as a function of pH. The titration curve of the
polyA-FF-ME nanoparticles, obtained by adding HCl to an
aqueous dispersion of the polyA-FF-ME nanoparticles, shows
an increase of the ζ-potential from −13 to 2 mV while changing
the pH from 11 to 2. This change in the surface potential can
be attributed to the surface ME groups of the polyA-FF-ME
nanoparticles. In both basic and acidic environments, the ME
groups undergo partial hydrolysis. In a basic environment, the
surface charge potential is negative due to hydrolysis of ME
groups to CO−O−, whereas in an acidic environment, the
surface charge potential is slightly positive due to the
protonation of the carboxyl groups.50 The measured isoelectric
point of the polyA-FF-ME nanoparticles is at pH 4.2 as shown
in Figure 6.
Figure 5. TGA thermogram of the polyA-FF-ME nanoparticles.
In Vitro Cytotoxicity Study of polyA-FF-ME Nano-
particles. The cytotoxic effect of the polyA-FF-ME nano-
particles on different human cell lines, e.g., PC-12 and SH-
SY5Y, was examined using XTT cell viability assay, as shown in
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Figure 8. Kinetics of the Aβ40 fibrils formation in PBS at 37 °C in the
absence (A) and presence of 10 (B), 20 (C), and 100 (D) % (w/
wAβ40) of the polyA-FF-ME nanoparticles. The kinetics of the Aβ40
fibril formation were measured using ThT binding assay, as described
in the Materials and Methods. The reported values are the averages of
five measurements of each triplicate tested sample. The error bars
indicate the standard deviation.
Figure 6. ζ-potential of the polyA-FF-ME nanoparticles as a function
of pH. The reported values are the average of five measurements. The
error bars indicate the standard deviation.
Figure 7. This figure shows the cell viability % in the presence
of different concentrations of the nanoparticles after incubation
that the stability against agglomeration of these nanoparticles
was found to be dependent at the ionic strength of the PBS,
e.g., in 0.1 M PBS, they are stable, while in 0.5 M PBS, they
aggregate. Due to this instability issue, the kinetics was
accomplished in the presence of 0.1 M PBS as a continuous
phase. In the absence of the nanoparticles (Figure 8A), the
main growth of the Aβ40 fibrils, corresponding to the end of the
lag time, occurred approximately 60 h after initiating the
fibrillation process, and was completed after approximately 120
h. On the other hand, the kinetics of the Aβ40 fibril growth in
the presence of increasing concentrations of the polyA-FF-ME
nanoparticles was significantly delayed (Figure 8B−D). For
example, Figure 8 exhibits that the presence of 10 (B), 20 (C),
and 100 (D) % (w/wAβ40) of the polyA-FF-ME nanoparticles
inhibits the initiation of the fibril formation to be only after 85,
99, and 233 h, respectively. These observations indicate that the
polyA-FF-ME nanoparticles substantially increase the fibrilla-
tion lag time and thereby inhibit the kinetics of the nucleation
and oligomer formation prior to the formation of the Aβ40
fibrils. This inhibitory effect might be explained by the presence
of the pairs of FF residues of these nanoparticles, which are
known for their high affinity to the corresponding residues of
Aβ40 prefibril aggregates, e.g., monomers and oligomers,
through hydrophobic interactions. This high binding affinity
disturbs the monomer-critical nuclei equilibrium by trapping
the monomers and/or blocking the growing oligomer ends on
the surface of the nanoparticles, thereby decreasing their
solution concentration and interfering with their elongation to
form fibrils. However, the ability to distinguish whether these
nanoparticles adsorbed the monomers, or oligomers, or both,
remains to be investigated.
Figure 7. Viability of PC-12 and SH-SY5Y cells exposed to increasing
concentrations of polyA-FF-ME nanoparticles after 24 or 48 h at 37
°C, as determined by the XTT assay described in the Materials and
Methods. The reported values are the average of measurements
performed on at least eight wells for each tested sample of two
independent experiments, and expressed as a percentage of the
negative control (cultured cells in medium without nanoparticles).
The error bars indicate the standard error of the mean.
for 24 or 48 h. After 24 h of exposure of the PC-12 and SH-
SY5Y cells to the nanoparticles, no significant toxicity was
observed for nanoparticle concentration of 0.2 mg/mL
(concentration used for our fibrillation studies) or even ten
times higher (ca. 2.0 mg/mL). Minor toxicity was observed
after 48 h of exposure to nanoparticle concentration of 0.2 mg/
mL for PC-12 only, and to the higher concentration of 2.0 mg/
mL for both cell lines, with approximately 80% cell viability.
Effect of polyA-FF-ME Nanoparticles on the Kinetics
of Aβ40 Fibrillation Process. Kinetics of the Aβ40 fibril
formation in PBS at 37 °C, monitored by the temporal
development of ThT binding,49 in the absence (A) and
presence (B−D) of increasing concentrations of the polyA-FF-
ME nanoparticles are shown in Figure 8. It should be noted
Our results imply that the nucleation process of Aβ40 is
strongly disturbed by the presence of the polyA-FF-ME
nanoparticles assuming a tight interaction between the
nanoparticles and Aβ40 prefibril intermediates. A similar effect
was reported for the fibrillation process of Aβ and islet amyloid
polypeptide (IAPP) proteins in the presence of copolymeric N-
isopropylacrylamide/N-tert-butylacrylamide nanoparticles with
different ratios between these monomers having variable
hydrophobicity.25,26,31 In these studies, the retardation of the
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fibrillation process was also explained by the depletion of free
monomers and oligomers (prefibrils) from solution due to their
adsorption on the nanoparticle surface. This observation led to
an increase in the lag time of the fibrillation process but did not
prevent the fibril formation. It should be noted that, until
recently, it was assumed that Aβ had to be assembled into
amyloid fibrils to exert its cytotoxic effect.14,16,21 However,
recent studies have revealed that the soluble oligomers are
significantly more toxic to neuronal cells than the monomers
and the formed fibrils.17,20,21,25,35 Therefore, there is great
interest in developing nanoparticles that are able to disturb the
monomer-critical nuclei equilibrium by trapping active
monomers and prefibril intermediates on the nanoparticle
surface, thereby delaying further growth, thereby reducing their
toxicity in solution.31,33−35
nanoparticles. Therefore, the increased local concentration of
the Aβ40 prefibrils adsorbed on the polyA-AA-ME nanoparticle
surface promoted their growth into fibrils. It is important to
note that the promoting effect achieved in the presence of the
polyA-AA-ME nanoparticles may also be considered a desired
process. The polyA-AA-ME nanoparticles decrease the
fibrillation lag time, thereby shorting the half-life time of the
Aβ40 prefibrils, thereby decreasing their toxicity in solution.
The secondary conformational change of Aβ40 from α-helix
and/or random coil to β-sheet is probably the key step in the
process to form oligomeric aggregates.11 Hence, CD measur-
ments were performed to determine the secondary structure
changes during the Aβ40 fibril formation in the absence and the
presence of the polyA-FF-ME nanoparticles. Figure 10 presents
Our assumption that the observed inhibitory effect in the
presence of the polyA-FF-ME nanoparticles is due to specific
hydrophobic interactions between the nanoparticles and the Aβ
prefibril aggregates, via the FF recognition motif, was examined
by replacing the polyA-FF-ME nanoparticles by polyA-AA-ME
nanoparticles. The chemical structures of the monomer A-AA-
ME and its corresponding polymeric nanoparticles were
1
confirmed by H NMR analysis, as shown in the Supporting
Information, Figure S1. The dry and hydrodynamic diameters
of the polyA-AA-ME nanoparticles are 52 8 and 214 23
nm, respectively, which are similar to those obtained for the
polyA-FF-ME nanoparticles, as shown in the Supporting
Information, Figure S2. Figure 9 presents typical sigmoidal
Figure 10. CD spectra of the Aβ40 in the absence (A) and the presence
(B) of 100% (w/wAβ40) of the polyA-FF-ME nanoparticles dispersed
in PB solution at different time periods of the fibrillation process.
Figure 9. Kinetics of the Aβ40 fibril formation in PBS at 37 °C in the
absence (A) and presence of 10 (B), 20 (C), and 100 (D) % (w/
wAβ40) of the polyA-AA-ME nanoparticles. The kinetics of the Aβ40
fibril formation was measured using ThT binding assay, as described in
the Materials and Methods. The reported values are the average of five
measurements of each triplicate tested sample. The error bars indicate
the standard deviation.
CD spectra of the Aβ40 in the absence (A) and the presence
(B) of 100% (w/wAβ40) of the polyA-FF-ME nanoparticles at
different time periods during the fibrillation process. In the
absence of the nanoparticles (Figure 10A) before the initiation
of the fibrillation process, the CD spectrum of the freshly
prepared Aβ40 aqueous solution at pH 7.4 shows one strong
minimum at 198 nm indicative of a typical spectrum of a native
protein dominated by random coil conformation, as described
in the literature.36,51,52 By heating the Aβ40 solution to 37 °C
for 120 h, the CD spectrum of the Aβ40 displays a loss of
intensity of the 198 nm peak accompanied by a maximum
ellipticity at 200 nm and a broad minimum ellipticity at 217
nm, which is typical of the presence of extensive β-sheet
structures. Almost the same CD spectra of Aβ40 were observed
in the presence of 100% (w/wAβ40) of the polyA-FF-ME
nanoparticles. It is rather interesting to note that the CD
curves showing the kinetics of the Aβ40 fibrils formation in PBS
at 37 °C in the absence (A) and the presence of 10 (B), 20 (C),
and 100 (D) % (w/wAβ40) of the polyA-AA-ME nanoparticles.
It is rather interesting to note that the observed behavior is the
opposite of the behavior obtained in the presence of similar
concentrations of the polyA-FF-ME nanoparticles. The kinetics
of the Aβ40 fibrils growth in the presence of 10, 20, and 100%
(w/wAβ40) were accelerated, and initiated after 53, 38, and 18 h,
respectively (see Figure 9). This promoting effect probably
results from the absence of the FF interfering moieties in these
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Figure 11. TEM images of the Aβ40 in the absence of the polyA-FF-ME nanoparticles, 120 h after initiation of the fibrillation process (A), and in the
presence of 100% (w/wAβ40) of the polyA-FF-ME nanoparticles dispersed in PBS, 200 h (B), 233 h (C), and 330 h (D) after initiation of the
fibrillation process.
spectrum of the freshly prepared Aβ40 aqueous solution in the
presence of 100% (w/wAβ40) of the polyA-FF-ME nanoparticles
(Figure 10B) exhibits one strong minimum at 198 nm and a
weak maximum at 224 nm, which are stable for more than 233
h of heating. Only beyond that time does the transformation to
β-sheet structure start to occur.
absence of the nanoparticles. These observations indicate the
trapping of amorphous Aβ40 aggregates by their adsorption on
the surface of the polyA-FF-ME nanoparticles through specific
interactions, leading to their partial depletion from solution
which interferes with their elongation into massive fibrils.
To further confirm the achieved inhibitory effect on the Aβ40
fibril formation in the presence of the polyA-FF-ME nano-
particles, TEM images were obtained to detect morphological
changes in Aβ40 solution, as shown in Figure 11. In the case of
control Aβ40 solution, in the absence of nanoparticles (Figure
11A), massive networks of entangled fibrils with microsized
lengths were observed after 120 h of the fibrillation process. On
the other hand, when Aβ40 solution was incubated in the
presence of 100% (w/wAβ40) of the polyA-FF-ME nano-
particles, only free nanoparticles in the background were
observed even after 200 h of the fibrillation process (Figure
11B), indicating that the formation of the fibrils had not yet
occurred. After 233 h (Figure 11C), absorption of amorphous
Aβ40 aggregates on the surface of the polyA-FF-ME nano-
particles was initiated. The internal dark and bright areas
around the nanoparticles (as indicated by white arrows)
illustrate the adsorbed dense and less dense multiple layers of
the amorphous prefibril aggregates. It is further interesting to
note that after 330 h (Figure 11D) the formed fibrillar
networks were much less massive than those observed in the
CONCLUSIONS
■
The present manuscript describes the design, synthesis, and
characterization of novel polyA-FF-ME nanoparticles, by
polymerization of the synthesized monomer A-FF-ME,
followed by precipitation of the soluble polymer in aqueous
solution. These studies demonstrate the significant inhibition of
the Aβ40 fibrillation process in the presence of these
nanoparticles. This inhibition is probably due to the
intermolecular attractive hydrophobic interactions between
the pairs of FF residues of the nanoparticles with the
corresponding residues of the Aβ40 prefibrillar aggregates,
which disrupt the self-assembly of Aβ40 into fibrils. This
observation was confirmed by the promoting effect achieved in
the presence of the polyA-AA-ME nanoparticles.
The present studies were performed using pure Aβ40 without
competition from other proteins for binding to the nanoparticle
surface. These conditions are quite unlike those in any realistic
clinical situation. Still, our studies intend to provide a new
mechanistic insight into amyloidogenic peptide interaction with
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other amyloidogenic proteins under competitive conditions.
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ASSOCIATED CONTENT
■
S
* Supporting Information
1H NMR spectra of the monomer A-AA-ME and the polyA-
AA-ME nanoparticles. TEM image and size histogram of the
polyA-AA-ME nanoparticles. This material is available free of
charge via the Internet at http://pubs.acs.org.
AUTHOR INFORMATION
Corresponding Author
■
*E-mail: Shlomo.margel@mail.biu.ac.il. Tel: 972-3-5318861.
Fax: 972-3-6355208.
Present Address
‡Currently on sabbatical from Israel Institute for Biological
Research, Ness-Ziona 74100, Israel.
Author Contributions
(32) Cabaleiro-Lago, C.; Quinlan-Pluck, F.; Lynch, I.; Dawson, K. A.;
Linse, S. ACS Chem. Neurosci. 2010, 1, 279−287.
(33) Yoo, S. I.; Yang, M.; Brender, J. R.; Subramanian, V.; Sun, K.;
Joo, N. E.; Jeong, S. H.; Ramamoorthy, A.; Kotov, N. A. Angew. Chem.,
Int. Ed. 2011, 50, 5110−5115.
(34) Rocha, S.; Thunemann, A. F.; Pereira, M. C.; Coelho, M.;
Mohwald, H.; Brezesinski, G. Biophys. Chem. 2008, 137, 35−42.
(35) Saraiva, A. M.; Cardoso, I.; Saraiva, M. J.; Tauer, K.; Pereira, M.
†These authors contributed equally to this study.
Notes
The authors declare no competing financial interest.
ACKNOWLEDGMENTS
■
These studies were partially supported by a Minerva Grant
(Microscale & Nanoscale Particles and Films).
C.; Coelho, M. A.; Mohwald, H.; Brezesinski, G. Macromol. Biosci.
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2010, 10, 1152−1163.
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