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ORIGINAL RESEARCH
published: 30 July 2019
doi: 10.3389/fchem.2019.00480
Frontiers in Chemistry | www.frontiersin.org 1July 2019 | Volume 7 | Article 480
Edited by:
Erik Reimhult,
University of Natural Resources and
Life Sciences Vienna, Austria
Reviewed by:
Zuzana Gazova,
Institute of Experimental Physics
(SAS), Slovakia
Anna Igorevna Sulatskaya,
Institute of Cytology (RAS), Russia
Konstantin K. Turoverov,
Institute of Cytology (RAS), Russia
*Correspondence:
Alyona Sukhanova
alyona.sukhanova@univ-reims.fr
Igor Nabiev
igor.nabiev@univ-reims.fr
Specialty section:
This article was submitted to
Nanoscience,
a section of the journal
Frontiers in Chemistry
Received: 06 November 2018
Accepted: 24 June 2019
Published: 30 July 2019
Citation:
Sukhanova A, Poly S, Bozrova S,
Lambert É, Ewald M, Karaulov A,
Molinari M and Nabiev I (2019)
Nanoparticles With a Specific Size and
Surface Charge Promote Disruption of
the Secondary Structure and
Amyloid-Like Fibrillation of Human
Insulin Under Physiological
Conditions. Front. Chem. 7:480.
doi: 10.3389/fchem.2019.00480
Nanoparticles With a Specific Size
and Surface Charge Promote
Disruption of the Secondary
Structure and Amyloid-Like
Fibrillation of Human Insulin Under
Physiological Conditions
Alyona Sukhanova1,2*, Simon Poly3, Svetlana Bozrova2, Éléonore Lambert1,
Maxime Ewald1, Alexander Karaulov4, Michael Molinari1and Igor Nabiev1,2*
1Laboratoire de Recherche en Nanosciences, LRN-EA4682, UFR de Pharmacie, Université de Reims Champagne-Ardenne,
Reims, France, 2Laboratory of Nano-Bioengineering, Moscow Engineering Physics Institute, National Research Nuclear
University MEPhI, Moscow, Russia, 3Department of Membrane Biophysics, Interfaculty Institute of Biochemistry, University of
Tübingen, Tübingen, Germany, 4Department of Clinical Immunology and Allergology, Sechenov First Moscow State Medical
University, Moscow, Russia
Nanoparticles attract much interest as fluorescent labels for diagnostic and therapeutic
tools, although their applications are often hindered by size- and shape-dependent
cytotoxicity. This cytotoxicity is related not only to the leak of toxic metals from
nanoparticles into a biological solution, but also to molecular cytotoxicity effects
determined by the formation of a protein corona, appearance of an altered protein
conformation leading to exposure of cryptic epitopes and cooperative effects involved
in the interaction of proteins and peptides with nanoparticles. In the last case,
nanoparticles may serve, depending on their nature, as centers of self-association
or fibrillation of proteins and peptides, provoking amyloid-like proteinopathies, or as
inhibitors of self-association of proteins, or they can self-assemble on biopolymers as on
templates. In this study, human insulin protein was used to analyze nanoparticle-induced
proteinopathy in physiological conditions. It is known that human insulin may form
amyloid fibers, but only under extreme experimental conditions (very low pH and
high temperatures). Here, we have shown that the quantum dots (QDs) may induce
amyloid-like fibrillation of human insulin under physiological conditions through a
complex process strongly dependent on the size and surface charge of QDs. The
insulin molecular structure and fibril morphology have been shown to be modified at
different stages of its fibrillation, which has been proved by comparative analysis of
the data obtained using circular dichroism, dynamic light scattering, amyloid-specific
thioflavin T (ThT) assay, transmission electron microscopy, and high-speed atomic
force microscopy. We have found important roles of the QD size and surface
charge in the destabilization of the insulin structure and the subsequent fibrillation.
Remodeling of the insulin secondary structure accompanied by remarkable increase in
the rate of formation of amyloid-like fibrils under physiologically normal conditions was
observed when the protein was incubated with QDs of exact specific diameter coated
Sukhanova et al. Nanoparticles Induce Amyloid-Like Protein Fibrillation
with slightly negative specific polyethylene glycol (PEG) derivatives. Strongly negatively
or slightly positively charged PEG-modified QDs of the same specific diameter or QDs
of bigger or smaller diameters had no effect on insulin fibrillation. The observed effects
pave the way to the control of amyloidosis proteinopathy by varying the nanoparticle size
and surface charge.
Keywords: nanomaterials, protein adsorption, quantum dots, proteinopathies, insulin, fibrillation, amyloidosis
INTRODUCTION
Proteinopathies are disorders resulting from changes in protein
conformation and subsequent aggregation of protein molecules
with altered tertiary and quaternary structures, which accumulate
in cells and internal environment of the body (Dobson, 2003).
These aggregates, particularly their small-sized intermediate
forms, have been found to provoke cell membrane oxidation,
interfere with ion homeostasis, the mitochondrion functioning,
and inter- and intracellular signaling, thus inducing apoptosis
(Glabe, 2006).
The protein alteration begins with changes in their secondary
structure (Dobson, 2003). This results from destabilization of
the macromolecule conformation caused by local changes in the
intra- and intercellular media, which may be induced by various
external factors (Shemetov et al., 2012). The structural stability is
regained through the formation of amyloid-like fibrils consisting
of organized associations of modified protein molecules (Go,
1984). This is a two-step process: at the first step, small amyloid
oligomers are formed, and at the second step, they assemble
into fibrils (Chiti and Dobson, 2006; Glabe, 2006). Protein
molecule domains abnormally enriched with β-sheets serve as
the primary foci of aggregation. Several hypotheses have been
suggested about the origin of these foci. First, the domains
that are normally folded, mostly α-helix structures, may be
unfolded due to the influence of external factors, such as the
extremely low pH or high temperature, to fold again into β-
sheets (Nguyen et al., 1995). Second, the protein domains that
are normally unfolded may form β-folds (Pawar et al., 2005;
Abelein et al., 2012). Third, the stage of a completely unfolded
molecule may be skipped, β-sheets arising directly from another
folded conformation. In this case, only small regions of the
macromolecule are unfolded because of local destabilization,
which initially leads to aggregation of the locally altered protein
monomers. This, in turn, results in further conformational
changes and, finally, formation of amyloid-like protofibrils
(Chiti and Dobson, 2009).
It should be noted that precisely the water-soluble oligomeric
pre-fibrillar structures are most prone to trigger the mechanisms
of cell damage and apoptosis (Glabe, 2006), whereas the
protofibrillar and fibrillar structures, being insoluble, exhibit
much less cell and tissue toxicity (DeMarco and Daggett, 2004).
Neurodegenerative disorders, including Parkinson’s and
Alzheimer’s diseases (PD and AD, respectively), have been found
to be proteinopathies. Proteinopathic alterations have been found
in various human and animal tissues, as well as in fungi and
prokaryotes, e.g., Escherichia coli (de Groot et al., 2009). They
may occur in peptides and proteins with different structures
and functions. No amino acid sequence has been identified
as mandatory for a proteinopathy to develop (Pastor et al.,
2007), although larger protein macromolecules have been found
to provide better conditions for the formation of abnormal
β-folded domains and the resultant proteinopathy (Zhang
et al., 2000). Proteinopathies may affect both extracellular and
intracellular proteins. For example, heavy-chain amyloidosis is
accounted for by accumulation of immunoglobulin aggregations,
and PD is related to intracellular aggregation of α-synuclein
(Giasson et al., 2003).
Here, we analyzed human insulin aggregation as a model
of nanoparticle-induced proteinopathy. Insulin is a hormone
secreted by pancreatic β-cells and controlling the blood content
of glucose. It is a relatively low-molecular-weight water-soluble
protein dimer, its monomers attached to each other via two
disulfide bonds. Insulin dimers have been found to associate
with one another under physiologically normal conditions
to form α-helical hexamers capable of binding Zn2+cations
(two or four per hexamer) (Chang et al., 1997; Xu et al.,
2012). Experiments using small-angle X-ray scattering analysis
(Vestergaard et al., 2007) have demonstrated that these three-
dimer associations may give rise to the formation of long fibrils.
The secondary structure of insulin in these fibrils largely varies
depending on the medium composition and other factors, as
evidenced by TEM and FTIR-spectroscopy data. Importantly,
not only β-sheet, but also α-helix may be the predominant
conformation of the fibrillated protein (Nielsen et al., 2001a).
It has been hypothesized (Nielsen et al., 2001b; Ahmad et al.,
2003) that insulin fibrillation starts with dissociation of native
insulin hexamers into monomers, after which equilibrium
is established between unfolded monomers and a partly
folded form, whose subsequent oligomerization results in
fibrillation nuclei. This assumption agrees with the hypothesis
that insulin fibrillation is mediated by its partly unfolded
form (Chiti and Dobson, 2009). Furthermore, recent data
indicate that insulin could form fibril superstructures through
lateral alignment of individual fibrils (Babenko et al., 2011;
Babenko and Dzwolak, 2013).
Amyloid-like fibers of insulin protein have been identified in
type II diabetic patients (Pease et al., 2010). Insulin molecules
also tend to refold and aggregate on arterial walls and membrane
surfaces in vivo. The aggregation of insulin followed by formation
of amyloid-like structures is one of the main problems in insulin
production and storage, as well as delivery (Sluzky et al., 1991;
Nielsen et al., 2001b). Agitation may play the crucial role in this
process (Malik and Roy, 2011).
Frontiers in Chemistry | www.frontiersin.org 2July 2019 | Volume 7 | Article 480
Sukhanova et al. Nanoparticles Induce Amyloid-Like Protein Fibrillation
Nanoparticles, with their unique properties, including bright
and stable fluorescence, small size, and capacity for binding
capture agents, are regarded as promising fluorescent or
magnetic labels to be used for detection of proteinopathies
(Georganopoulou et al., 2005). Nanoparticles have also been
assumed to interact with amyloidogenic peptides and proteins,
thus affecting their fibrillation. This effect is promoted by the
high surface-to-volume ratio of nanoparticles, high free Gibbs
energy of the interaction, and tunable electrical charge. In this
connection, it was suggested that nanoparticles may be used
for preventing the aggregation of amyloid-prone peptides and
proteins or even inducing disaggregation of amyloid fibrils. For
example, maghemite nanoparticles have been found to inhibit
the fibrillation of insulin (Skaat et al., 2009a,b). Nanoparticles
of various types have been tested to select the most promising
candidates, with special focus on a rapid natural clearance after
their interaction with protein aggregates (Xiao et al., 2010). On
the other hand, a number of nanoparticles have been shown
to exert the opposite effect, i.e., facilitate amyloid nucleation
by absorbing peptides and proteins, thereby increasing their
local concentration (Linse et al., 2007). In the latter case, the
nanoparticles may play the role of the centers of protein pseudo-
crystallization provoking their further fibrillation.
Of special interest is to study how semiconductor QDs
affect the insulin structure in a solution under the conditions
corresponding to those in the internal environment of the
human body. Owing to their bright, narrow-band fluorescence
(Vokhmintcev et al., 2016), QDs have been paid special attention
in terms of using them as tags for diagnostic nanoprobes
(Brazhnik et al., 2015) and biosensors (Artemyev et al., 2009)
in various fields of medicine and biology and as carriers for
in vivo targeted delivery in animal experiments (Bilan et al.,
2016; Ramos-Gomes et al., 2018). It has been found that QDs
coated with organic ligands can inhibit the aggregation of
amyloid beta (Aβ) peptides forming amyloid plaques in AD.
Specifically, CdTe QDs with a shells of thioglycolic acid (TGA)
(Yoo et al., 2011) and N-acetyl-L-cysteine (Skaat et al., 2009b)
can suppress the fibrillation of the Aβ(1-40) peptide, and those
with a dihydrolipoic acid shell inhibit the fibrillation of Aβ(1-42)
(Thakur et al., 2011).
Our study has demonstrated important roles of the QD
size and surface charge in the perturbation of the insulin
protein secondary structure and its subsequent fibrillation under
physiological conditions. The remarkable increase in the rate
of amyloid-like fibrillation was observed when the protein was
treated with QDs of 12 nm hydrodynamic diameter coated with
PEG-OH derivative, providing slightly negative charge (−6 mV)
for the QD surface. Strongly negative or slightly positive PEG-
modified QDs of the same specific diameter or QDs of bigger or
smaller diameters had no effect on insulin fibrillation.
Although it is known that human insulin may form amyloid
fibers in vivo and in vitro under extreme environmental
conditions (Bucciantini et al., 2002; Jiménez et al., 2002), the
capability of nanoparticles to induce insulin fibrillation under
physiological conditions was not known up to now. It is
worth mentioning that the mechanism of QD-induced insulin
fibrillation under physiological conditions may differ from those
described for insulin fibrillation under extreme conditions (such
as low pH, high temperatures, or strong agitation) described
earlier. In any case, the important role of insulin in various
biological processes and the presence of this protein in different
biological fluids and tissues emphasize the necessity to identify
the mechanism of its oligomerization and fibrillation in the
presence of nanoparticles in order to find the way to the control of
amyloidosis proteinopathies involving this and similar proteins.
MATERIALS AND METHODS
Human recombinant insulin protein, ThT, methanol,
chloroform, sodium phosphate dibasic, sodium phosphate
monobasic, sodium hydroxide, poly-L-lysine, and DL-cysteine
hydrochloride hydrate were purchased from Sigma-Aldrich,
US. The PEG derivatives HS-C11-EG6, HS-C11-EG6-NH2, and
HS-C11-EG6-OCH2-COOH were purchased from ProChimia
Surfaces Sp, Poland.
Preparation of a Human Insulin Protein
Solution to Study the Fibrillation Process
Dehydrated human insulin protein was dissolved in 0.01 M HCl
to obtain the stock solution with a concentration of 20 mg/ml.
Then, aliquots of the stock solution were diluted with different
buffers to obtain experimental solutions with a concentration of
2 mg/ml (3.44 µM). These human insulin protein preparations
were incubated alone or with CdSe/ZnS QDs at different
temperatures varying from 25 to 50◦C.
Synthesis and Solubilization of CdSe/ZnS
Quantum Dots
CdSe/ZnS QDs were synthesized according to the procedure
adapted from that described earlier (Sukhanova et al., 2004).
Briefly, two solutions were prepared, one containing 10 g
of trioctylphosphine oxide (TOPO, Aldrich) and 5 g of
hexadecylamine (HDA, Fluka), and the second containing 80 mg
of elemental Se and 110 µl of dimethyl cadmium (Strem, 97%)
in 1 ml of trioctylphosphine (TOP, Fluka). The first solution was
dried, degassed under vacuum at 180◦C, purged with argon, and
heated to 340◦C under argon flow. Then, fast (<1 s) injection
of the second solution into the first one yielded CdSe cores
approximately 2 nm in size. Further growth of CdSe cores to
the desired size (and, hence, the desired fluorescence color) was
induced by prolonged refluxing of the solution at 280◦C. After
completion of the process, CdSe cores were precipitated at 60◦C
with methanol, washed twice with methanol, and dried. In order
to grow an epitaxial ZnS shell on the CdSe core, a powder of
CdSe cores was dissolved in a mixture of 10 g of TOPO and 5 g
of HDA. Once again, the mixture was dried and degassed under
vacuum at 180◦C and purged with argon. A solution containing
210 µl of hexamethyldisilthiane (Fluka) and 130 µl of diethyl
zinc (Strem, 97%) in 2 ml of TOPO was added dropwise to this
mixture at 220◦C under argon flow and intense stirring. The
resultant colloidal solution of CdSe/ZnS NPs was slowly cooled
to 60◦C, and QDs precipitated with methanol were washed twice
with methanol and dried. The synthesized QDs contained a
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Sukhanova et al. Nanoparticles Induce Amyloid-Like Protein Fibrillation
CdSe core 2.3 nm (green fluorescence color), 3.1 nm (orange),
or 3.9 nm (red) in diameter and an epitaxial shell of several ZnS
monolayers (Table 1).
The QDs were solubilized in water using a procedure similar
to that published earlier (Sukhanova et al., 2012). Briefly, QDs
were first transferred to water after the attachment of DL-
cysteine (Sigma) to their surface. The resultant water-soluble
QDs displayed a bright green (533 nm), orange (570 nm), or red
(610 nm) photoluminescence (PL) with a quantum yield close
to 40% at room temperature. Then, DL-cysteine on the surface
of QDs was replaced with thiol-containing PEG derivatives with
carboxyl or hydroxyl group at the end of the polymer chain; or
with a mixture of 10% of thiol-containing PEG derivatives with
amino group and 90% of thiol-containing PEG derivatives with
hydroxyl groups. Briefly, 156 µl of a 150 mg/ml hydroxy-PEG
solution in water, or 138 µl of a 150 mg/ml carboxy-PEG solution
in water, or a mixture of 25 µl of a 100 mg/ml amino-PEG and
140 µl of a 150 mg/ml hydroxy-PEG solutions were added to
1 ml of 10 mg/ml preparations of DL-Cys QDs in pure water.
The samples were incubated overnight at +4◦C, pre-cleaned by
centrifugation with Amicon Ultra-15 filter units with a 10 kDa
cut-off (Millipore), and finally purified from excess of ligands
by gel exclusion chromatography on home-made Sephadex-25
(Sigma) columns.
The described procedure have allowed us to prepare
nanoparticles of different diameters and controlled surface
charges that are water-soluble and stable in aqueous buffer
solutions (Table 1).
QD Stability Analysis
QD solutions were prepared in 10 mM sodium phosphate buffers
(pH 6.0, 7.0, or 8.0). These QD solutions were incubated at
different temperatures (25, 37, or 50◦C) and analyzed two times
a day during the next 2 weeks using the dynamic light scattering
analysis, absorption spectral analysis, and fluorescence spectral
analysis techniques.
Insulin Secondary Structure Analysis
Secondary structure analysis was carried out by measuring
CD spectra with a Jasco J815 CD spectrometer at 10◦C. All
measurements were carried out in solutions using a 1 mm path
length cell (Hellma). The samples were diluted ten times in the
initial buffer. Each spectrum is an average of 20 measurements.
UV-Vis Absorption and Fluorescence
Spectroscopy
UV-Vis absorption spectra were recorded with a Jasco V630Bio
spectrophotometer. Fluorescence spectra were obtained using a
Jasco FP6600 spectrofluorimeter. Both UV-Vis absorption and
fluorescence measurements were obtained employing quartz cells
with a 1 cm optical path length.
ThT Fluorescence Assay
A 10 µM solution of ThT (Sigma) was prepared in 10 mM sodium
phosphate buffer at pH 7.0. Thirty microliter aliquots of human
insulin protein were taken after different periods of incubation
and mixed with 300 µl of a 10 µM solution of ThT. The ThT
fluorescence was measured at 482 nm in the semi-micro quartz
cell with a 1 cm optical path length at the excitation wavelength
of 440 nm.
Mean Average Diameter and Zeta Potential
Measurements
QD average hydrodynamic diameters were measured at different
temperatures (25, 37, or 50◦C) using the dynamic light scattering
(DLS) technique by means of a Malvern Nano-ZS device
(Malvern Instrument Ltd., UK). The colloidal stability of the
samples was evaluated as a function of time in a sodium
phosphate buffer solution at different pH values (pH 6.0, 7.0, or
8.0) after incubation of the samples at 25, 37, or 50◦C.
The zeta potential was determined by means of the same
device; it was calculated from the electrophoretic mobility
using the Smoluchowsky relationship and approximation.
The electrophoretic determination of the zeta potential was
made at a moderate electrolyte concentration. Zeta-potential
measurements of QD solutions were carried out at 25, 37, or 50◦C
in a sodium phosphate buffer solution with pH 6.0, 7.0, or 8.0.
Transmission Electron Microscopy
A 10 µl sample was applied onto a copper grid with carbon mesh
200 and pre-treated for 10 min with 1 mg/ml poly-L-lysine. After
that, the grid was placed into a 1.5 ml test tube and centrifuged
for 5 min at 750 g to remove residual solution. Then, the sample
was contrasted with 2% uranyl acetate for 40–50 s. An image was
obtained with a JEOL-2100F transmission electron microscope
(Jeol Ltd., Japan) at an accelerating voltage of 200 kV. The data
were recorded using the DigitalMicrographTM imaging software
(Gatan Inc., USA).
High-Speed Atomic Force Microscopy
Measurements
AFM topography images were recorded using a self-built high-
speed atomic force microscope (HS-AFM) setup based on a
RIBM equipment (Research Institute of Biomolecule Metrology
Co., Ltd, Ibaraki, Japan) (Ando et al., 2001). Experiments were
performed at room temperature in tapping mode using silicon
nitride cantilever with a spring constant of ∼0.2 N m−1 (BL-
AC10DS-A2, Olympus). An amorphous carbon layer was grown
on the original tip through electron-beam deposition and then
sharpened by plasma etching offering an apex of ∼4–5 nm. The
cantilever’s free oscillation amplitude was set at 1–2 nm and the
set-point amplitude was ∼85% of the free oscillation amplitude
so to avoid strong interactions between tip and samples. All
measurements were made in imaging buffer (10 mM sodium
phosphate, pH 7.2). For observations, a droplet of 2 µL was
deposited on freshly cleaved mica. After 5 min of incubation
at room temperature, samples were rinsed with imaging buffer.
Images were treated with the RIBM software with a flatten filter.
Height profiles were used to accurately determine particles size.
The fibril length after each time of incubation was estimated
for nearly 120 objects using segmented line measurement by
means of the ImageJ software (https://imagej.nih.gov/ij/).
Frontiers in Chemistry | www.frontiersin.org 4July 2019 | Volume 7 | Article 480
Sukhanova et al. Nanoparticles Induce Amyloid-Like Protein Fibrillation
TABLE 1 | Physicochemical properties of CdSe/ZnS QDs emitting fluorescence at 530, 570, or 610 nm, coated with PEG derivatives containing terminal groups with
different charges.
SampleaMaximum of emission (nm) Core diameter (nm)bHydrodynamic diameter (nm)cζ-potential (mV)c
QD530-PEG-OH 533 2.3 ±0.2 9.0 ±1.0 −4.2 ±0.2
QD570-PEG-OH 570 3.1 ±0.3 12.0 ±1.5 −6.0 ±0.3
QD570-PEG-COOH 570 3.1 ±0.3 12.0 ±1.5 −36.0 ±2.0
QD570-10%PEG-NH2/90%PEG-OH 570 3.1 ±0.3 12.0 ±1.5 +6.0 ±0.3
QD610-PEG-OH 610 3.9 ±0.3 15.0 ±2.0 −8.9 ±0.4
aFor all measurements, QD concentrations of ∼0.5–1.5 µM were used. In all cases, the absorbance at the first exciton band was <0.10.
bQD core average diameters were calculated using transmission electron microscopy images of the corresponding CdSe cores as an average of diameters of nearly 100 nanoparticles.
cQD average hydrodynamic diameters and zeta potentials were measured using a Malvern Nano-ZS device as described in section UV-Vis Absorption and Fluorescence Spectroscopy.
Presented results are an average of measurements made in triplicate.
RESULTS AND DISCUSSION
Interaction of CdSe/ZnS QDs With Human
Insulin in vitro Followed by Dynamic Light
Scattering Measurements and High-Speed
Atomic Force Microscopy
CdSe/ZnS core/shell QDs with the CdSe cores 2.3, 3.1, and
3.9 nm in diameter (fluorescing at 533, 570, and 610 nm,
respectively) were coated with three-functional PEG derivatives
containing terminal OH- (PEG-OH), COOH- (PEG-COOH),
or NH2- (PEG-NH2) groups (Table 1) as described in section
Materials and Methods. These procedures yielded batches of
well-characterized water-soluble QDs of the same hydrodynamic
diameter (12 nm) with slightly negative (−6 mV, QD570-PEG-
OH), slightly positive (+6 mV, QD570-10%PEG-NH2/90%PEG-
OH), or strongly negative (−36 mV, QD570-PEG-COOH)
surface charges, as well as batches of slightly negative QDs with
smaller (9 nm, QD530-PEG-OH) and larger (15 nm, QD610-
PEG-OH) hydrodynamic diameters (Table 1). These panels of
QDs have been used in our research for studying human
insulin fibrillation induced under physiological conditions by the
nanoparticles with different but precisely controlled sizes and
surface charges.
Dynamic light scattering (DLS) analysis (Figure 1) has shown
that the slightly negative CdSe/ZnS QDs carrying the PEG-OH
derivative on the surface (QD570-PEG-OH, Table 1) remained
preserved during more than a week in a phosphate buffer solution
(pH 7/0) at 37◦C (Figure 1D). Nor did a human insulin solution
in the same buffer exhibit any sign of aggregation during 1 week
at the same temperature and pH (Figure 1E). DLS analysis also
demonstrated that the QDs 570 nm coated with the PEG-COOH
derivative (QD-PEG-COOH) or with a mixture of the PEG-OH
and PEG-NH2derivatives (QD-PEG-OH/PEG-NH2) were also
sufficiently stable (data not shown). The stabilities of QD and
QD-insulin solutions were constantly controlled by recording
their DLS and UV-Vis absorption and fluorescence spectra.
Co-incubation of QD-PEG-OH and recombinant human
insulin was found to induce association between insulin and
QDs: as seen from the DLS spectra of a freshly made QD-
insulin mixture, it contained particles larger than the particles
detected in both original solutions of insulin and QD-PEG-OH
(Figure 1A). The absorption and fluorescence spectra in the
UV-Vis region showed that the QDs in the QD-insulin mixture
also remained stable under physiologically normal conditions
for more than a week. Thus, the QDs with a PEG-OH shell
facilitate the aggregation of human insulin at pH 7.0 and a
temperature of 37◦C, with the aggregate size rapidly increasing,
so that aggregates several micrometers in size appeared within
24 h of co-incubation (Figure 1C). In contrast, co-incubation
of QD-PEG-COOH or QD-PEG-OH/PEG-NH2(Table 1) with
insulin under the same conditions does not cause noticeable
insulin aggregation (data not shown).
Estimation of the changes in the sizes of aggregates in
the QD-insulin mixture showed their rapid growth followed
by the decrease of originally formed smaller complexes.
The QD-induced aggregation of insulin occurred in several
stages. At the early kinetics stage (Figure 1B), the quantity
of partly folded intermediates of the pre-aggregated protein
was decreased and accompanied by formation of some amount
of larger aggregates (200–700 nm). The stage of late kinetics,
which lasted for 24 h, ended in the strong decrease of the
quantity of initial, small intermediates resulted in appearance
of significant quantity of 30-nm, 500–1,000 nm, and even 3-
µm and larger aggregates (Figure 1C). We assume that the
30-nm pre-aggregates were fibrillation nuclei and 500-nm and
bigger particles were amyloid-like fibrils. This assumption agrees
with the pathway of the fibril formation out of globular
proteins described earlier (Chiti and Dobson, 2009), where
partly unfolded forms of proteins aggregated into oligomers,
which underwent structural rearrangements resulting in fibrils.
Human insulin were earlier reported to form amyloid-like
fibrils (Bouchard et al., 2000; Nielsen et al., 2001b), but those
studies were performed under extreme acidity (pH 2.0) and
temperature (60–70◦C) conditions. We have found no available
published data on insulin fibrillation under physiologically
normal conditions. However, our experiments (Figure 1) have
shown that QDs induce the formation of insulin aggregates under
the conditions corresponding to the internal environment of the
human body.
We have also analyzed the time course of QD-induced
aggregation of human insulin using high-speed atomic force
microscopy (AFM) and compared the results with the DLS
data described above. Figure 2 shows AFM images illustrating
the formation of insulin fibrils during 6 h of incubation. The
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Sukhanova et al. Nanoparticles Induce Amyloid-Like Protein Fibrillation
FIGURE 1 | Variation of the size distributions of nanoparticles, human insulin, and nanoparticle–insulin complexes as estimated from the dynamic light scattering (DLS)
spectra. (A) The DLS spectra of insulin protein alone (green), PEG-OH-modified CdSe/ZnS QDs with a core diameter of 3.1nm (Table 1) alone (red), and a freshly
prepared mixture of insulin with QDs (blue) recoded immediately after mixing of insulin and the QDs. Human insulin (2 mg/ml) was incubated in the presence of QDs
(3.44 µM) in a 10 mM so dium phosphate buffer solution (pH 7.0) at 37◦C. (B) The same QD–insulin mixture as in (A) where the DLS spectra were recorded after 0
(red), 5 (green), 10 (blue), 20 (black), and 30 min (magenta) of incubation. (C) The same QD–insulin mixture as in (A) where the DLS spectra were recorded after 0
(red), 12 (green), and 24 h (blue) of incubation. (D) Control experiment: a QD solution (3.44 µM) alone was incubated during 7 days in a 10 mM sodium phosphate
buffer solution (pH 7/0) at 37◦C. DLS spectra were recorded after 0 (red), 1 (green), 2 (blue), 3 (black), 4 (violet), 5 (rose), 6 (brown), and 7 (dark green) days of
incubation. (E) Control experiment: a human insulin solution (2 mg/ml) alone was incubated in a 10 mM sodium phosphate buffer solution (pH 7.0) at 37◦C, and the
measurements were done at the same time points as in (D).
formation of the fibrils was analyzed and estimated after 0.5, 1,
2, 4, and 6 h of incubation. Control samples included insulin
solution in a 10 mM sodium phosphate buffer (pH 7.2) not
containing nanoparticles (Figures 2A, a and d), insulin in a
10 mM sodium phosphate buffer solution (pH 7.2) containing
SH-PEG-OH polymer, the free ligand that is on the QD surface
(Figures 2A, b and e), and CdSe/ZnS-S-PEG-OH QDs in a
10 mM sodium phosphate buffer solution (pH 7.2) without
insulin (Figures 2A, c and f).
We initially estimated the stabilities of CdSe/ZnS-S-PEG-OH
QDs alone and insulin alone in a 10 mM sodium phosphate
buffer solution (pH 7.2) at 37◦C for 6 h. Within this period,
no aggregation of CdSe/ZnS-S-PEG-OH QDs or insulin was
observed. At the initial moment of time (0 h of incubation),
there was no aggregation in the experimental sample, and insulin
remained in the form of monomers 18.4 ±4.56 nm in size. After
30 min of incubation, no noticeable changes occurred in the
experimental samples containing CdSe/ZnS-S-PEG-OH QDs;
aggregation was not observed (Figure 2B, 30 min). However,
after 1 h of incubation of insulin in the presence of CdSe/ZnS-S-
PEG-OH QDs, insulin oligomers 22.4 ±4.46 nm in length were
formed (Figure 2B, 1 h). Later, after 2 h of incubation, the insulin
oligomers were substantially increased in size, reaching 51.74 ±
8.96 nm (Figure 2B, 2 h). During the next 2 h (after a total of 4 h
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Sukhanova et al. Nanoparticles Induce Amyloid-Like Protein Fibrillation
FIGURE 2 | Dynamic AFM imaging of insulin fibrillation in the presence or absence of QDs at physiological conditions. (A) Insulin molecules at the zero point of
measurement (a) and after 6 h of incubation (d); (b, e) insulin molecules in the presence of PEG-OH at the zero point of measurement (b) and after 6h of incubation (e);
CdSe/ZnS-S-PEG-OH QDs at the zero point of measurement (c) and after 6 h of incubation (f). (B) Dynamic AFM imaging of insulin fibrillation in the presence of
CdSe/ZnS-S-PEG-OH QDs under physiological conditions for 0.5, 1, 2, 4, and 6 h incubations.
of incubation), distinct fibrillation was observed, with the length
of the fibrils increased to 133.6 ±17.57 nm (Figure 2B, 4 h),
which indicated a high rate of fibril formation. During the last 2 h
of incubation, large threadlike fibrils with a length of 175–200 nm
were formed (Figure 2B, 6 h).
Figure 3 shows the summary of the time course of the growth
of insulin fibrils in the presence of CdSe/ZnS-S-PEG-OH QDs
under the same conditions as that shown in Figure 2 but followed
by dynamic light scattering measurements. It is worth noting that
no fibrillation was observed in the control insulin solutions not
containing nanoparticles or control QD solutions not containing
insulin, which suggested that the insulin fibril formation was
specifically induced by CdSe/ZnS-S-PEG-OH QDs. The data
on the time course of insulin fibrillation in the presence of
CdSe/ZnS-S-PEG-OH QDs followed by dynamic light scattering
(Figure 3) were closely correlated with the dynamic AFM data
(Figure 2): the hydrodynamic size of insulin aggregates was
changed from 14 to 140 nm during 4 h of incubation and
further to the sizes of nearly 200 nm during the last 2 h of
incubation (Figure 3).
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Sukhanova et al. Nanoparticles Induce Amyloid-Like Protein Fibrillation
FIGURE 3 | Changes in the sizes of aggregates of insulin during its incubation
in the presence of CdSe/ZnS-S-PEG-OH QDs during 0.5, 1, 2, 4, and 6 h of
incubation.
We further analyzed the specific molecular structural
characteristics of the aggregates formed by the human
insulin in the presence of QD-PEG-OH nanoparticles under
physiological conditions.
QD-Induced Insulin Aggregation Is
Structurally Similar to Amyloid Fibrillation
Phenomena
Analysis of the circular dichroism (CD) spectra of insulin
aggregates resulting from QD-PEG-OH nanoparticle interaction
with insulin under the aforementioned conditions indicated
structural alterations of insulin corresponding to amyloid-like
fibrillation. Signs of this process were observed in the far-UV
region of the CD spectrum of insulin (Figure 4). A solution
of pure QD-PEG-OH nanoparticles had no CD activity in the
UV spectral region (data not shown), whereas a pure insulin
solution had a characteristic CD spectrum reflecting the native
α-helix-rich secondary structure of this protein, which remained
unchanged for 24 h of incubation (Figure 4A). The decrease in
the CD intensity at a wavelength of 222 nm upon co-incubation
of insulin with the QDs (Figure 4B) indicates a decrease in its α-
helix-rich secondary structure. We have calculated the content of
insulin secondary structures by deconvolution of its CD spectra
using three different approaches: CONTIN-CD, SELCON3, and
the recently published BeStSell method (Micsonai et al., 2018).
The results obtained by any of these methods did not show
significant alpha-to-beta transition upon QD-induced insulin
fibrillation. Instead, we observed transformation of nearly 10%
of insulin α-helix into unordered or undefined structures. It is
worth mentioning that insulin is a low-molecular-weight water-
soluble protein dimer. Its monomers are attached to each other
via two disulfide bonds and have been found to associate with
one another under physiologically normal conditions to form
α-helical hexamers capable of binding Zn2+cations (two or
FIGURE 4 | Changes in the insulin secondary structure in the presence of
PEG-OH-modified CdSe/ZnS QDs. Circular dichroism spectra of human
insulin (2 mg/ml) were recorded (A) in the absence or (B) in the presence of
QDs (3.44 µM). The QDs were also incubated alone (a control sample), when
they did not display a noticeable CD signal (baselines in B). All solutions were
incubated at 37◦C for 24 h in a sodium phosphate buffer solution (pH 7.0) at
37◦C; the CD spectra were recorded after 0 (blue), 12 (red), and 24 h (green).
four per hexamer) (Chang et al., 1997; Xu et al., 2012). These
three-dimer associations may give rise to the formation of long
fibrils (Vestergaard et al., 2007) where the secondary structure
of insulin largely varies depending on the medium composition
and other factors. That is why not only β-sheet, but also α-helix
or unordered structures may be the predominant conformation
of the fibrillated protein (Nielsen et al., 2001a). Furthermore,
some data indicate that insulin could form fibril superstructures
through lateral alignment of individual fibrils (Babenko et al.,
2011; Babenko and Dzwolak, 2013). Moreover, the mechanism of
QD-induced insulin fibrillation under physiological conditions
may differ from those described for insulin fibrillation under
extreme conditions (such as low pH, high temperatures, or strong
agitations) described earlier. In our case, the insulin CD spectra
do not indicate strong alpha-to-beta transition upon QD-induced
insulin fibrillation under physiological conditions, but show
protein unfolding, which may be an indirect indicator of protein
secondary structure modifications induced by this process.
In order to investigate the effect of QDs on the fibrillation of
human insulin in more specific detail, we monitored the time
course of QD-induced insulin fibrillation using the amyloid-
specific dye ThT. The fluorophore ThT, as well as some
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Sukhanova et al. Nanoparticles Induce Amyloid-Like Protein Fibrillation
FIGURE 5 | Fibrillation kinetics of insulin at 37◦C monitored using the
ThT-based fluorescence assay. Human recombinant insulin (2 mg/ml) was
incubated in the absence (red) or in the presence (blue) of PEG-OH-modified
CdSe/ZnS QDs (3.44 µM) in a 10 mM sodium phosphate buffer solution (pH
7.0) at 37◦C for 30 h.
ThT derivatives, has been found to specifically bind with
amyloid fibrils and is used for in vitro amyloid detection. The
quantum yield of the ThT in an aqueous solution is as low
as 0.01% (Sulatskaya et al., 2010). Although the fluorescence
intensities of ThT bound to amyloid fibrils formed by different
amyloidogenic proteins differ significantly, the conclusion that
the ThT fluorescence quantum yield increases several orders of
magnitude upon dye incorporation into the amyloid fibril is
always reasonable (Sulatskaya et al., 2017). It was shown that, in
the case of insulin fibrils, the binding of ThT is characterized by
the highest binding constants, and the insulin fibril-bound ThT
possesses the highest fluorescence quantum yield, reaching 83%
(Kuznetsova et al., 2012).
Here, we have used the ThT assay to confirm QD-induced
insulin fibrillation in the presence of QD-PEG-OH nanoparticles
with a hydrodynamic diameter of 12 nm (Table 1) under
physiological conditions. Within 25 h of incubation of insulin
(2 mg/ml) in the presence of ThT and without QDs, no
fluorescence was detected at a wavelength of 482 nm under
the standard conditions (pH 7.0 and a temperature of 37◦C)
upon 440-nm excitation (Figure 5). Therefore, no amyloid-
like fibrils were formed. If the mixture contained QD-PEG-
OH nanoparticles, the intensity of ThT fluorescence at 482 nm
started to increase and exhibited an almost linear increase during
24 h of incubation, which proved the formation of amyloid-like
fibrils (Figure 5).
Effects of Temperature and pH on
QD-Induced Insulin Protein Fibrillation
It has been shown earlier that the native insulin protein may
form amyloid fibrils under extreme in vitro conditions, namely
at a temperature of >60◦C and pH <2.37. It is also known
that an increase in the incubation temperature or a decrease
in pH accelerates insulin fibrillation (Bouchard et al., 2000).
Therefore, we analyzed the effects of temperature and pH on the
kinetics of insulin fibrillation in the presence of QD-PEG-OH
nanoparticles with a hydrodynamic diameter of 12 nm (Table 1)
under physiological conditions.
Identical samples of human insulin mixed with QD-PEG-OH
nanoparticles were incubated at pH 7.0 and temperatures of 25,
37, and 50◦C. The ThT fluorescence assay was used to estimate
the influence of temperature variation on the insulin fibrillation
rate. Figure 6A shows that there was no noticeable increase in
the ThT-specific fluorescence signals after the incubation of the
sample at 25◦C for 6 h. A rise of the temperature of incubation
for the QD-PEG-OH/insulin reaction mixture to 37◦C and then
to 50◦C considerably accelerated insulin fibrillation (Figure 6A).
These data show that the QD-induced insulin fibrillation is
temperature-dependent, as was previously reported for amyloid-
like insulin fibrillation in the absence of QDs (Bouchard et al.,
2000; Nielsen et al., 2001b).
It is known that an extremely low pH destabilizes the
human insulin protein structure, provoking the alpha-to-beta
transition of its secondary structure and fibrillation of the
protein (Bouchard et al., 2000). Therefore, we investigated how
variation of pH around its physiological values may influence
QD-induced insulin fibrillation. Reaction mixtures of human
insulin and QD-PEG-OH nanoparticles were prepared in sodium
phosphate buffer solutions with pH 6.0, 7.0, or 8.0 and incubated
at a temperature of 37◦C. QD-induced insulin fibrillation was
analyzed using the ThT fluorescence assay.
Figure 6B shows that the rate of QD-induced insulin protein
fibrillation was pH-dependent: the rate of the increase in the
ThT-specific fluorescence signal was positively correlated with
the increase in pH of the incubation buffer solution. In contrast
to the literature data (Nielsen et al., 2001b), the rate of insulin
fibrillation in the presence of QDs was found to be decreased at
lower pH values, whereas an increase in pH caused an increase in
the insulin fibrillation rate. This may be due to the influence of
pH of the medium on the local ionization of QD surface groups.
This finding supports the hypothesis that the role of QD-PEG-
OH nanoparticles in the induction of insulin fibrillation may
be related to their action as a pH-specific destabilization agent
in a complex manner. The observed specificity of the effect of
pH on the insulin protein and its involvement in QD-induced
fibrillation led to the assumption that this effect is related to the
QD surface charge. Indeed, variation of the local pH affected
the charge at the surface of QDs and the QD-insulin interface.
In order to test this hypothesis, we studied the relationships
between the QD surface charge, QD diameter, and QD-induced
amyloid-like fibrillation of insulin.
Effects of the QD Surface Charge and
Diameter on QD-Induced Insulin Fibrillation
In order to analyze the role of the QD surface charge in
insulin fibrillation, we compared the characteristics of insulin
fibrillation induced by QDs of the same hydrodynamic diameter
(12 nm) but coated with different ligands, namely, PEG-COOH
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Sukhanova et al. Nanoparticles Induce Amyloid-Like Protein Fibrillation
FIGURE 6 | Fibrillation kinetics of insulin at different (A) temperatures and (B)
pH values monitored in the presence of PEG-OH-modified CdSe/ZnS QDs by
means of the ThT-based fluorescence assay. Human recombinant insulin (2
mg/ml) was incubated in the presence of PEG-OH-modified CdSe/ZnS QDs
(3.44 µM) in a 10 mM so dium phosphate buffer solution (pH 7.0). (A) Reaction
mixtures were incubated for 24 h at temperatures of 50◦C (black), 37◦C (red),
or 25◦C (blue). (B) Reaction mixtures were incubated for 12 h at pH 8.0
(black), pH 7.0 (red), or pH 6.0 (blue).
or PEG-OH polymer, or a mixture of PEG-NH2and PEG-OH
polymers (Table 1), using the amyloid-specific ThT assay. Insulin
samples were incubated in the presence of QDs with different
coatings at pH 7.0 and 37◦C for 24 h. It was shown that only
QD-PEG-OH nanoparticles provoked QD-induced fibrillation,
providing highly reproducible results, such as those presented
in Figure 5, whereas the ThT fluorescence was non-detectable
when QD-PEG-NH2or QD-PEG-OH nanoparticles at the same
concentrations were used for incubation with the same quantities
of insulin. Therefore, only the slightly negatively charged QD-
PEG-OH nanoparticles 12 nm in diameter (Table 1) induced
insulin fibrillation.
As can be seen in Supplementary Figure 1, neither QD-
PEG-COOH nor QD-PEG-OH/PEG-NH2nanoparticles affected
the insulin secondary structure, whereas QD-PEG-OH, as it
was noted above, altered it by disordering the insulin α-helix
structure. The zeta potential values measured at pH 7.0 and
37◦C were −6.0 mV for QD-PEG-OH, −36.0 mV for QD-PEG-
COOH, and +6.0 mV for QD-PEG-OH/PEG-NH2nanoparticles
(Table 1). Thus, the nanoparticles with a slightly negative surface
charge most strongly induce human insulin fibrillation. This
finding correlates with the earlier data (Wagner et al., 2010) on
insulin fibrillation induced by gold nanoparticles, which also had
a low negative surface charge.
We have further used QDs of different diameters with the
same coating/charge (PEG-OH) in order to vary the curvature of
the charged surface interacting with human insulin in a solution.
For this purpose, the CdSe/ZnS QDs with hydrodynamic
diameters of 9, 12, and 15 nm were used (Table 1). All these
QDs were solubilized in water and coated with the same PEG-
OH derivative. The zeta potentials of these QDs were measured
by the DLS technique in order to estimate the charge on the
surface of each particular type of QDs, which yielded the values
of −4.2, −6.0, and −8.9 mV, respectively (Table 1). Comparative
analysis of the capacity of these QDs for inducing insulin
fibrillation during co-incubation in a buffer solution at pH 7.0
and 37◦C were analyzed using the ThT amyloid-specific assay.
The data showed that only QD-PEG-OH nanoparticles with a
hydrodynamic diameter of 12 nm induced fibrillation, yielding
highly reproducible results, such as those presented in Figure 5,
whereas the ThT fluorescence was non-detectable when QD-
PEG-OH nanoparticles 9 or 15 nm in hydrodynamic diameter
were incubated with the same concentrations of insulin.
As shown in Supplementary Figure 2, only QD570-PEG-
OH nanoparticles with a hydrodynamic diameter of 12 nm
induced detectable changes in the insulin secondary structure
upon their co-incubation. QD530-PEG-OH (9 nm in diameter)
and QD610-PEG-OH (15 nm in diameter) nanoparticles did not
induce any observable changes in the insulin CD spectra under
the same conditions of incubation; hence, they did not affect
the secondary structure of the insulin protein. The differences
between the spectra shown in Supplementary Figure 2C can
be entirely explained by the noise resulting from the high
optical density explained by the larger size of the QD610-PEG-
OH nanoparticles.
Supplementary Figure 3 confirms the above conclusion and
shows that the kinetics of modification of the insulin secondary
structure in the presence of QD-PEG-OH nanoparticles 12 nm in
hydrodynamic diameter strongly depends on the QD to insulin
molar ratio. Indeed, the data of Supplementary Figure 3 show
that progressive increase in the QD concentration in the reaction
mixtures where insulin was kept at a constant concentration
provokes acceleration of QD-induced modification of the α-
helix-rich insulin secondary structure. It is noteworthy that
analysis of insulin solution incubated under the same conditions
without QD-PEG-OH nanoparticles did not show any signs of
modification of the insulin secondary structure during 24 h of
incubation at a temperature of 37◦C (Figure 4A). Recent data on
the influence of CdTe QDs coated with TGA indicate that these
nanoparticles inhibit the modification of the secondary structure
of amyloidogenic peptide even in the case of a 100-fold excess of
Aβ(1-40) over QDs (Yoo et al., 2011). In contrast, in our study,
QD-PEG-OH nanoparticles with a hydrodynamic diameter of
12 nm promoted modifications of insulin secondary structure
in a concentration-dependent manner. This difference can be
explained by the different origin of the functional groups exposed
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Sukhanova et al. Nanoparticles Induce Amyloid-Like Protein Fibrillation
FIGURE 7 | TEM images of QD–insulin mixtures incubated at 37◦C. Human recombinant insulin (2 mg/ml) was incubated in presence of PEG-OH-modified CdSe/ZnS
QDs (3.44 µM) in a 10 mM sodium phosphate buffer solution (pH 7) at 37◦C. The images were obtained after (A) 0 and (B) 24 h of incubation. (C) shows a magnified
area framed in a white rectangle in (B).
on the QD surface. It is worth mentioning that QDs bearing
terminal carboxylic groups had no effect on the insulin secondary
structure (Supplementary Figure 2) or protein fibrillation under
physiological conditions.
Finally, in order to directly confirm the formation of fibrillary
structures and their association with QDs, we obtained electron
micrographs of the samples before and after 24 h of co-incubation
of insulin and QD-PEG-OH nanoparticles with a hydrodynamic
diameter of 12 nm (Figure 7). Immediately after QD–insulin
mixing, there were no ordered structures in the QD-insulin
solutions (Figure 7A). After 24 h, fibrils with lengths ranging
from 500 to 1,000 nm and an average width of 20–25 nm
were formed in the samples (Figures 7B,C). The formation
of these fibrils is a hallmark of insulin aggregation and was
earlier observed in many studies (Ortiz et al., 2007; Ivanova
et al., 2009). It is important that the presence of nanoparticles
in the fibrils (Figure 7B) was previously observed only in
the case of artificial insulin polymerization in a thin layer
containing magnetic nanoparticles (Andersson et al., 2012). Our
experiments showed association of QDs and nucleation units of
insulin under physiological conditions. These data indicate direct
copolymerization of QDs and insulin, with the particles evenly
distributed along the fibrils. This confirms that QDs provoke the
aggregation of insulin and then remain involved in the process,
forming complexes with the intermediates of aggregation.
CONCLUSION
Our study has demonstrated that QDs with a specific
hydrodynamic diameter (about 12 nm) coated with slightly
negative (zeta potential, −6 mV) PEG-OH derivative (Table 1)
promote the fibrillation of human insulin under physiological
conditions. We have found an important role of the QD size
and surface charge in the destabilization of the insulin protein
structure and the subsequent insulin fibrillation. Our results
confirm that QDs may influence the secondary structure of
human insulin and its behavior in a solution. Strongly negative
(−36 mV) or slightly positive (+6 mV) PEG-modified QDs
(Table 1) of the same hydrodynamic diameter (12 nm), as
well as larger (15 nm) or smaller (9 nm) QDs with the same
strong negative or small positive charges or a small negative
charge (−6 mV) do not promote the fibrillation of insulin under
physiological conditions. In contrast, slightly negative (−6 mV)
QDs with a hydrodynamic diameter of 12 nm bearing hydroxyl
groups on their surface (Table 1) have been shown to strongly
accelerate insulin fibrillation.
The finding that insulin fibrillation depends not only on the
QD charge, but also on the diameter of QDs and, hence, the
size of the charged surface that can interact with a protein
molecule and distribution of the charge over this surface due to
its different curvature may explain some contradictions between
results of similar experiments performed with other proteins and
on different classes of nanoparticles (Jiménez et al., 2002; Wu
et al., 2008). Apparently, the main, if not the only, reason for these
contradictions is that the charge distribution was not taken into
account in those studies.
Although it is known that human insulin may form amyloid
fibers under extreme environmental conditions (Bucciantini
et al., 2002; Jiménez et al., 2002), the capability of nanoparticles to
induce insulin fibrillation under physiological conditions has not
been known until now. It should be noted that the mechanism of
QD-induced insulin fibrillation under physiological conditions
may differ from those described for insulin fibrillation under
extreme conditions (such as low pH, high temperatures, or
strong agitation) described earlier. To determine the protein-
specificity of the effect of the surface charge pattern on QD–
protein interaction, additional studies are required on the
interaction between nanoparticles with different physicochemical
properties and amyloid-prone and non-amyloid-prone proteins.
If the results of these studies confirm our hypothesis that the
surface charge pattern affects the amyloidogenicity of native
proteins, then it may be possible to develop “anti-amyloid”
nanoparticles exposing a specific pattern of electrical charges
on their surface that would modify the in vivo conditions in
such a way that previously unfolded amyloid-prone proteins
refold into their native form. This would not only offer
the possibility to decrease the risk entailed in the use of
Frontiers in Chemistry | www.frontiersin.org 11 July 2019 | Volume 7 | Article 480
Sukhanova et al. Nanoparticles Induce Amyloid-Like Protein Fibrillation
nanoparticles in vivo but also pave the way to develop a
potent nanoparticle-based tools for the treatment of amyloid-
related diseases.
AUTHOR CONTRIBUTIONS
AS and IN proposed the concept and designed this study. AS
prepared the water-solubilized and stabilized nanoparticles of
controlled charge. SP and SB prepared the samples of the insulin-
nanoparticle complexes and performed DLS, fluorometric,
and CD measurements. ÉL, ME, and MM characterized the
complexes and performed high-speed AFM measurements. AS,
IN, and AK performed comparative analysis of the results. IN
and AS co-wrote the manuscript. All authors commented on
the manuscript.
FUNDING
This study was supported by the Russian Science Foundation
(contract no. 17-15-01533).
ACKNOWLEDGMENTS
We are grateful to Dr. Anton Shemetov, Dr. Konstantin
Rumyantsev, and Mr. Vladimir Ushakov for technical assistance.
SUPPLEMENTARY MATERIAL
The Supplementary Material for this article can be found
online at: https://www.frontiersin.org/articles/10.3389/fchem.
2019.00480/full#supplementary-material
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Conflict of Interest Statement: The authors declare that the research was
conducted in the absence of any commercial or financial relationships that could
be construed as a potential conflict of interest.
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