Elucidation of the effect of some cholinium amino acid ionic liquids on the thermal and the conformational stability of insulin

Article abstract of DOI:10.1016/j.molliq.2019.03.074

The major concerns about protein-based drugs are their stability i.e. maintaining the protein in the folded state throughout processing and storage, as well as the preparation of novel formulation. Stabilization of the monomeric form of insulin (In) under the condition of low pH has been a recent challenge. In our earlier investigation, we found that 1-butyl-3-methylimidazolium-based ionic liquids (ILs) containing acetate, trifluoroacetate or dicyanamide anions enhance In thermal stability and prevent protein aggregation. In the present study, six non-toxic ILs containing biocompatible cholinium cation [Chol] and an anion charged amino acid (asparginyl (Asp), glutaminyl (Glu), lysinyl (Lys) and arginyl (Arg)) were synthesized using a two-step procedure. Their effect of the ILs on the In secondary structures was evaluated using FTIR spectroscopy. Rearrangement in the protein molecules, an increase in the beta-structures on behalf of the α-helices, partial denaturation but no aggregation was observed in all In solutions containing ILs. Differential scanning calorimetry was applied to elucidate the effect of ILs on thermal stability of In. Interestingly, in presence of [Chol][Glu] and [Chol]₂[Asp] the denaturation temperature of the In was shifted to higher temperatures with 9.6 and 4.1 °C, respectively.

Full text of DOI:10.1016/j.molliq.2019.03.074

Accepted Manuscript
Elucidation of the effect of some cholinium amino acid ionic
liquids on the thermal and the conformational stability of insulin
M. Guncheva, P. Ossowicz, E. Janus, S. Todinova, D. Yancheva
PII:
S0167-7322(18)36686-8
DOI:
Reference:
https://doi.org/10.1016/j.molliq.2019.03.074
MOLLIQ 10615
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Journal of Molecular Liquids
Received date:
Revised date:
Accepted date:
19 December 2018
20 February 2019
14 March 2019
Please cite this article as: M. Guncheva, P. Ossowicz, E. Janus, et al., Elucidation of the
effect of some cholinium amino acid ionic liquids on the thermal and the conformational
stability of insulin, Journal of Molecular Liquids, https://doi.org/10.1016/
j.molliq.2019.03.074
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ACCEPTED MANUSCRIPT
Elucidation of the effect of some cholinium amino acid ionic liquids on the thermal and the
conformational stability of insulin
M. Guncheva1*, P. Ossowicz2, E. Janus2, S. Todinova2, D. Yancheva1
1Institute of Organic Chemistry with Centre of Phytochemistry, Bulgarian Academy of Sciences,
Sofia, Bulgaria
2Institute of Organic Chemical Technology, Faculty of Chemical Technology and Engineering,
West Pomeranian University of Technology Szczecin, Szczecin, Poland
3Institute of Biophysics and Biomedical Engineering, Bulgarian Academy of Sciences,
Sofia, Bulgaria
Abstract
The major concerns about protein-based drugs are their stability i.e. maintaining the protein in the
folded state throughout processing and storage, as well as the preparation of novel formulation.
Stabilization of the monomeric form of insulin (In) under the condition of low pH has been a
recent challenge. In our earlier investigation, we found that 1-butyl-3-methylimidazolium-based
ionic liquids (ILs) containing acetate, trifluoroacetate or dicyanamide anions enhance In thermal
stability and prevent protein aggregation. In the present study, six non-toxic ILs containing
biocompatible cholinium cation [Chol] and an anion charged amino acid (asparginyl (Asp),
glutaminyl (Glu), lysinyl (Lys) and arginyl (Arg)) were synthesized using a two-step procedure.
Their effect of the ILs on the In secondary structures was evaluated using FTIR spectroscopy.
Rearrangement in the protein molecules, an increase in the beta-structures on behalf of the α-
helices, partial denaturation but no aggregation was observed in all In solutions containing ILs.
Differential scanning calorimetry was applied to elucidate the effect of ILs on thermal stability of
In. Interestingly, in presence of [Chol][Glu] and [Chol]2[Asp] the denaturation temperature of the
In was shifted to higher temperatures with 9.6 and 4.1ºC, respectively.
Keywords: insulin; ionic liquids; cytotoxicity; secondary structure; thermal stability.
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1. Introduction
Insulin (In) is a peptide hormone secreted by the beta-cells of the pancreas, and regulates
the glucose levels in blood. In is a small protein (5800 Da) and consists of two chains A-chain
(21 amino acid residues) and B-chain (30 amino acid residues) that are linked by two disulfide
bridges, additional intrachain cysteine bridge in A-chain stabilize the molecule [1]. The active
form is the monomeric has low stability and is available at 20% acetic acid at pH 1.9. At other
aqueous solution e.g. diluted HCl (pH 1.9), more concentrated solutions, higher pH (2-8) or in
presence of small amounts of phenol exists as a diamer. In presence of bivalent ions (Zn+2, etc.)
dimmers associate in one of the three allosteric hexamers, which differ in their secondary
structure [2].
Insulin is discovered in 1916 by Dr. Banting and Best, and in 1922 for the first time it was
administrated to a young diabetic patient [1]. Hundred years later, insulin is the only alternative
for patients with diabetes I type and applied in some cases of diabetes type II. In the eighties,
insulin was obtained exclusively by extraction from pig or bovine pancreas, and subsequent
enzymatic modification to convert it in the human analogue of insulin. Later on, progress in
insulin production was made applying recombinant DNA techniques [3]. Nowadays, many novel
insulin analogues present on the market e.g. very rapid-acting (onset of action < 15 min, peak:
30-90 min, duration: 3-5 h), rapid acting (onset of action: 30-60, min., peak: 2–3 h, duration 5-8
h), intermediate-acting (onset of action: 1.5-4 h, peak: 4-10 h, duration: 10-16 h) long-acting
(onset of action: 1.5 h, duration: 24 h) insulin analogues [1, 4].
Still, the prescription correct dosage is a challenging task especially, having in mind that the
pharmacokinetics of In depends on many factors e.g. metabolism of each individual and
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absorption rate, which depends on the injection site, injection depth, concentration, massage on
the injection site. Application of higher In dosages may result in weight gain due to anabolic
processes and increase the risk of hypoglycemia, while lower dosages may result in not effective
control of the blood glucose levels and multiple organ failure [1]. On the other hand, in order to
be avoided the traumatic effects of In injections, development of proper non-invasive In drug
delivery systems is of current interest for the pharmaceutical industry.
Another challenge that faces the pharmaceutical industry is related to insulin stability
upon isolation, purification and storage, because the correct folding and structure is a key factor
for the biological activity of proteins. Insulin is prone to fibrillate and aggregate upon contact
with hydrophobic surfaces i.e. Teflon, peristaltic pump tubing, under agitation at pH 7.0 and
37ºC, air-water interface, at sites of the injection (injection-localized amyloidosis) [5, 6]. In
molecules undergo dramatic structural changes upon freeze-drying, prolonged exposure to light,
shaking or stirring, pressure, etc. [7, 8]
Typically, In is stabilized in presence of phenol, m-cresol, methylparabene, glycerol in
solution, and in presence of zinc chloride, sodium chloride, etc. as powder. Zhang et al. observed
a suppressed proteolytic degradation of In in formulations containing cyclodextrine and casein
(or protamine) [9]. In another research, Kraineva et al. have found that only weak interactions
occur between cubic mesoporous lipid systems based on monoolein and In molecules, thus the
structure of the encapsulated in the lipid matrix In is not significantly altered/changed [10].
Rasmussen et al. have shown that in comparison to polysorbate 80, sucrose and human serum
albumin, α-crystalline prevents more effectively In from fibrillation induced by vigorous stirring
[11].
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Ionic liquids (ILs) are salts that are typically composed of organic cations and
organic/inorganic anions; have melting points bellow 100ºC, low vapor pressure, and high
thermal stability [12]. Their physicochemical properties can be tuned by modification or selection
of an appropriate cation and anion, thus they are referred as designer’s solvents. They have
numerous applications in research and industry. Room temperature ionic liquids, particularly
those consisting of biocompatible cations and anions, are of great interest for biotechnology and
protein chemistry as reaction media for biocatalytic reactions aiming to enhance enzyme activity
and selectivity; or as solvents aiming to improve protein solubility, crystallization and stability
and to facilitate protein extraction and separation [13]. Yet, the potential of ILs in stabilization of
protein and enzymes is not fully revealed although there are many research papers that have
shown promising results. For example, Takekiyo et al. have found that 1-butyl-3-
methylimidazolium thiocyanate ([bmim][SCN]), ethylammonium nitrate ([EAN][NO3], and
propylammonium nitrate ([PAN][NO3]) prevent the formation of In amyloid aggregates, while
[bmim][Cl] and [EAN][NO3] are cryoprotectants for lysozyme [14, 15]. Kumar and Venkatesu
showed that In is stabilized in more folded conformation and its denaturation and self-
aggregation is suppressed in presence of ILs containing trimethylammonium cation [16]. It is
noteworthy to be mentioned that choline dihydrogen phosphate ([Chol][dhp]) enhance the storage
stability of cytochrome C (>18 months at room temperature), enhance the thermal stability of
lysozyme and stabilize the structure of human serum albumin [13, 17, 18]. In an in vitro
experiment, Tanner et al. observed that IL based on choline and geranic acid (ratio 1:2) facilitated
the transdermal delivery of In [19].
Conversely, many other researchers reported of detrimental effect of many ILs on protein
stability e.g. imidazolium-and pyridinium based ILs accelerate the amylogenesis in for α-
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synuclein and α-lactalbumin, and triethylammonium-based ILs stimulate the fibrillation and
aggregation of Aβ1-40 peptide [14], butyl-3-methylimidazolium tetrafluoroborate ([bmim][BF4])
denatured human serum albumin [18], while I s containing choline cations and
bis trifluoromethylsulfonyl imide, he anoate, trifluoromethanesulfonate, trimethyllactate anions
decrease the thermal stability of lysozyme [17] .
Interestingly, Rodrigues at al. observed for a choline-based series of ILs that some anions
lactate or dihydrogen phosphate stabilize lysozyme only when added at high concentration
(above 0.5 M, and 1 M, resp.), while lower tested concentrations destabilize the protein [17]. On
contrary, 1-tetradecyl-3-methyl imidazolium bromide added at low concentrations (below critical
micelle concentration) stabilizes bovine serum albumin, while higher concentrations of this IL
denature the protein [13].
In summary, protein–IL interactions are complex and it is not possible to generalize the
observed for one particular system and to transfer it to another. However, biocompatatible ILs
have potential to be applied in preparation of drug delivery systems and formulations of
biotechnological incl. protein-based drugs, thus worth to be further investigated.
Here, we report the synthesis of a series of ionic liquids containing a choline cation (Chol)
and a charged amino acid (Asp, Glu, Arg, Lys) as an anion. Glutaminate and asparginate were
obtained as dicholinium salts as well. The cytotoxicity of the ILs on fibroblasts was evaluated.
Then, the effect of the six choline amino acids on the secondary structure and the thermal
stability of In was evaluated.
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2. Materials and Methods
2.1. Materials
Insulin from porcine pancreas United States Pharmacopeia reference standard, (2-
hydro yethyl trimethylammonium cholinium chloride [Chol][Cl] ≥97% , Dowe Monosphere
550 A UPW (OH form) resin, thiazolyl blue tetrazolium bromide (MTT) (98%), Dulbecco’s
Modified Eagle’s Medium high glucose), L-Glutamine–Penicillin–Streptomycin solution were
purchased from Sigma-Aldrich. L-glutamic acid, L-arginine, L-aspartic acid ≥99% and -lysine
monohydrate ≥98.5% were provided by Carl Roth.
Murine embryotic fibroblast (3T3) cell line was purchased from American Type Culture
Collection (ATCC).
2.2. Synthesis of the choline amino acids [Chol][AA]
Mono- and di- cholinium salts of amino acids were synthesized by two-step procedure (Scheme
1). In the first step, chloride anion in cholinium chloride was exchanged to hydroxide anion using
the ion exchange resin. The content of chloride anions in collected eluates was checked with
AgNO3. The eluates without chloride were used in the second synthesis step. The concentration
of cholinium cation was determined by titration method in biphasic system in the presence of two
indicators (dimidium bromide/disulphine blue) and sodium lauryl sulfate (SLS) as titrant.
In the second synthesis step, amino acid (L-glutamic acid, L-arginine, L-aspartic acid or
L-lysine) was dissolved in the aqueous solution of cholinium hydroxide. Molar ratio of cholinium
hydroxide to amino acid was 1:1 for monosalts synthesis and 2:1 for di- salts. The mixture was
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stirred at room temperature for 24 h, subsequently. The water was evaporated at 60°C under
vacuum by using rotary evaporator. The obtained ionic liquids were dried in vacuum oven at
60°C.
The compounds were identified by 1H NMR. The NMR spectra were recorded in D2O on
a BRUKER DPX-400 spectrometer operating at 400.13 MHz.
Thermal stability was determined based on thermogravimetric analysis carried out using a
termomicrobalance TG 209 F1 Libra® NETZSCH within the temperature range of RT ÷ 800°C
at a constant heating rate of 10°C·min−1, in the air.
The specific rotation [α]20D, for all of the amino acid ionic liquids reported here were
measured in aqueous solutions on AUTOPOL IV Polarimeter (Rudolph Research Analytical).
The concentrations were 0.01 g/cm3.
2.3. Cytotoxicity of [Chol][AA] on fibroblasts
Murine embryotic fibroblast (3T3) (ATCC) were cultured in DMEM high glucose
medium containing L-Glutamine–Penicillin–Streptomycin and 10% fetal bovine serum. at
humified atmosphere, 37○C and 5% CO2. Cells were seeded in a sterile 96-well plate at 1·104
cells per well and incubated for 24 h at 37oС and 5% СО2 for obtaining adherent cell cultures and
good cell spreading. Then, the cells were incubated for additional 48 hours with 0.05, 0.5, and 5.0
mmol/L [Chol][AA] and the effect of the ionic liquids on cell viability was assessed in a
colorimetric assay using 3-[4,5-dimethylthiazol-2-yl]-2,5-diphenyltetrazolium bromide dye
(MTT). All experiments were presented in triplicate [20].
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One-way ANOVA with Tukey–Kramer post-hoc test was applied for the analysis of the
experimental data (Graph Pad Software Inc., San Diego, USA). The values were considered to be
significantly different if the p value was < 0.01.
2.4. Differential scanning calorimetry (DSC)
The changes in the phase transitions of In in presence of the ILs were performed on
DASM 4 (Privalov, BioPribor)-built-in highly sensitive calorimeter with cell volumes of 0.5 ml,
a sensitivity > 0.017 mJ/K and a noise level below 0.05 μW. An over-pressure of 2 atm dry
nitrogen was kept constant over the samples in the cells throughout the scans to avoid any
degassing during heating. A scan rate of 1°C/min from 30 to 110°C with equilibrium period of 30
min was used. Buffer or buffer-IL baseline was established prior to the analysis of the samples.
Then, the transitions were corrected for the difference in heat capacity between the initial and
final state by using the corresponding linear chemical baseline. The temperature at the maximum
of the excess heat capacity curve was taken as the transition temperature (Tm). DSC
measurements with In and In-ionic liquid complexes were carried out in 10 mM KCl/HCl buffer,
pH 2.0 (20ºC). The In-IL complexes were prepared immediately before the measurements at a
In/IL molar ratio of 1.5, the pH was adjusted if necessary. The concentration of insulin was kept
constant (3 mg/mL, 0.517 mM) for all experiments. Assays with higher concentration of the ILs
in this buffer resulted in decrease in the protein solubility and precipitation of the solutions.
Molecular mass of 5 800 Da for In was used in the calculation of its molar concentration. The
van’t Hoff enthalpy ΔHVH) was calculated using the following equation:
,
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where R is gas constant (KJ/K·mol); Tm is the estimated transition temperature (K); Cp is the
specific head capacity KJ/mol.K ; and ΔHcal is the calculated from the experimental curve
enthalpy (KJ/mol). All experiments are conducted in triplicates and data are expressed as means
±the standard deviation (SD).
2.5
Infrared spectroscopy
FTIR spectra of the In (25 mg/mL) dissolved in KCl/HCl (pH 2.0, 10 mM) or in 10 mM
aqueous solution of the ILs (in the same buffer) were recorded on Bruker Tensor 27
spectrometer, equipped with a detector of deuterated triglycine sulphate (DTGS). The FTIR
spectra were collected by direct deposition of the samples on attenuated total reflectance (ATR)
element (diamond crystal) in frequency region 4000 – 600 cm-1 (ATR) with 128 scanning and at
resolution of 1 cm-1. The spectra of the proteins were referenced to the respective spectra of 10
mM KCl/HCl (pH 2.0) or 10 mM solutions of the corresponding ionic liquid the same media in
order to substact their absorptions.
The effect of the [Chol][AA] on In secondary structure was estimated comparing the
obtained second-derivative FTIR spectra in the 1700–1600 cm-1 region (Amide I band) with the
spectrum of In in IL-free medium. In order to describe quantitatively the changes in the In
secondary structure induced by the tested choline amino acids, the ATR-FTIR spectra were
treated in accordance with the established methods given in the literature [21]. ATR-FTIR spectra
were Fourier deconvoluted by Opus software version 5.5 using band width of 14 cm-1, 2.9
resolution enhancement factor, and Lorentzian lineshape. Second derivative spectra were
obtained using the Savitzky-Golay algorithm based on 25 smoothing points. Then, the relative
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contribution of each band component of the Amide I band was determined by curve fitting
following the procedure of OPUS program. In the fitting, the number of components and the
initial values of their position were set as determined from the second derivative spectra. The
initial bandwidth of all components was set to 14 cm-1 and the components were approximated by
mixed Lorentzian/Gaussian functions. The curve-fitting was performed according to the Local
Least Squares algorithm. The assignment of the Amide I band positions to secondary structure
was done according to [22].
3. Results and Discussion
3.1. Synthesis and characterization of the ILs
The four mono- and the two dicholinium salts of charged amino acids i.e. (2-
hydroxyethyl)trimethylammonium
hydroxyethyl)trimethylammonium
hydroxyethyl)trimethylammonium
hydroxyethyl)trimethylammonium
hydroxyethyl)trimethylammonium
L-glutaminate
L-asparaginate
L-argininate
L-lysinate
[Chol][Glu],
[Chol][Asp],
[Chol][Arg],
[Chol][Lys],
(2-
(2-
(2-
bis(2-
bis(2-
L-glutaminate
[Chol]2[Glu]
and
hydroxyethyl)trimethylammonium L-asparaginate [Chol]2[Asp] were synthesized using the
following scheme (Scheme 1). The characterization of the compounds is given as Supplementary.
3.2. Cytotoxicity of the ILs on fibroblasts
Choline is an essential micronutritient participating in many processes related to cell
structure, synthesis of neurotransmitters, and other and choline-deficiency have a negative effect
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on liver structure and function, atherosclerosis and other neurological disorders [23]. On the other
hand, amino acids are essential for protein synthesis, which are crucial for almost all biological
processes in the living organisms [24]. Therefore, the ILs based on choline cation and aminoacid
anions are considered biocompatible and possibly could find a biopharmaceutical application.
All ILs applied at concentration of 0.05 and 0.5 mmol/L had no effect on 3T3 fibroblast
proliferation for 48 hours (Fig. 1.). We found that the most of the tested cholinium amino acid
salts produced weak effects on 3T3 cell growth even at 5.0 mmol/L. Interestingly, we observed a
significant reduction of the cell growth (up to 50%) only with [Chol][Arg] and a moderate anti-
proliferative activity (up to 25%) with [Chol][Glu] in the experiments with the highest tested
concentration.
In addition, Hou et al. reported that [Chol][Lys], [Chol][Arg], [Chol][Asp] and
[Chol][Glu] inhibit acetylcholinesterase with half maximal inhibitory concentration (IC50) values
in the range from 3.4 to 3.8 mmol/L [25]. They also found that more than 87% of [Chol][Asp]
and [Chol][Glu] are biodegraded for 28 days, while the biodegradability of arginate and lysinate
is about 70% for the same period [25].
3.3. Effect of the ILs on insulin thermal stability
The effect of the ILs on thermal unfolding of In was monitored using DSC. We observed
a non two-state thermally induced unfolding of In with a Tm centered at 75.4°C and ∆ Hm 119.9
of kJ/mol in IL-free medium. As can be seen from the DSC curves (Fig. 2), in presence of the ILs
the unfolding occurs as a single transition, except for the sample containing [Chol][Asp].
Interestingly, in this medium the thermal denaturation of In is a more complex process consisting
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of two endothermic events centered at 79.2 and 84.7°C. For In-[Chol][Lys] complexes, the DSC
curve has a relatively symmetric shape, which is an indication of existence of more than one
structural unit. The results from the DSC studies indicate that the thermal denaturation
temperature is lower in presence of [Chol][Arg], [Chol]2[Glu] and [Chol][Lys], and the observed
endothermic peaks are centered at 65.6, 68.6 and 70.0 °C, respectively. On the other hand, in
presence of [Chol]2[Asp] and [Chol][Glu] In denaturation transitions shift to higher temperature –
79.9 and 85.1°C, respectively.
The transition parameters for In thermal unfolding in presence of choline-amino acid ILs are
given in Table 1. Based on the experimental thermodynamic parameters, van’t Hoff enthalpies
ΔHvH) were calculated to evaluate the transition states during the thermal unfolding of In in
presence of ILs. The effect of the media on the cooperatively of the unfolding transitions of In
was evaluated based on the ΔHvH/ΔHcal ratio so called size of the cooperative unit. Interestingly,
only in presence of [Chol]2[Glu] the transition occurs in a two stage model (ΔHvH/ΔHcal ≈1),
which indicates a sharp equilibrium transition from native to denature state. The highest value of
the ΔHvH/ΔHcal ratio (~5.3) was estimated for the In denaturation in presence of monocholine
glutaminate, which suggests the formation of molecule associates (multimers), which probably is
due to strong intermolecular interactions as well as possible partial aggregation. In IL-free
medium and in presence of [Chol][Lys], [Chol][Asp], and [Chol]2[Asp] the ΔHvH/ΔHcal ratio was
in the range between 0.18 and 0.85, which is evidence that in these media the In thermal
unfolding proceeds with formation of one or several stable intermediates.
DSC is sensitive to the conformation of the molecule and any change in the conformation
affects the position and shape of the curves. The results from the calorimetric studies suggest that
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the tested ILs, even added to the media at low concentration have a significant effect on the
In structure.
3.4. Effect of [Chol][AA] on insulin secondary structure.
Typically, the polypeptide chain gives rise of several characteristic infrared absorption
bands amongst which the Amide I band (1700–1600 cm-1) region is the most applied for protein
conformational studies. Amide I band is due mainly to the stretching C=O vibrations and is very
sensitive to changes in the backbone conformation, on the other hand, the band is typically
characterized with higher intensity in comparison to Amide II and Amide III bands [22, 26].
Furthermore, the frequencies of the amide I band components correlate closely to each secondary
structural elements of protein. For the FTIR deconvoluted spectrum of In dissolved in KCl/HCl
(pH 2.0, 10 mM) ten well-defined components in the Amide I band region are observed. (Fig.
S1). The two bands at 1650 cm-1 and 1661 cm-1 are assigned to α-helices and 310-helices,
respectively, while the band at 1942 cm-1 corresponds to random coils and unordered structures.
The peaks for β-sheets (1626 cm-1, 1634 cm-1), β-turns 1673 cm-1, 1683 cm - 1) and anti-parallel
β-sheets or intermolecular aggregates (1616 cm-1, 1690 cm-1) are also clearly distinguished.
Interestingly, a band centered at 1600 cm-1 that is due to Tyr and the other amino acid side-chains
was observed only in the FTIR spectrum of the native In and we assume that in presence of ILs
these residues are less exposed to the solvent. The analysis of the secondary structure of In show
high consistency with the data reported in the literature for the secondary structure of In based on
FTIR spectroscopy and crystallography [27, 28].
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The original, the second derivative and the deconvoluted FTIR spectra in the amide I
region of In in KCl/HCl (pH 2.0, 10 mM) in presence of [Chol][AA] as well as the estimated
peak positions and the relative band areas are given in supplementary (Fig. S1, Table S1). The
percentages of the main secondary structural elements are summarized in Table 2. As can be
seen, the Amide I band region is composed of six to nine components depending on the media
composition. The approximate structural content is varying in the range as follows: 10.6-21.2%
α-helix; 12.4-29.7 % 310-helix; 10.6-21.2% random coils and unordered structures; and 30.0-
39.0% β-structures, and 12.9-18.4% antiparallel β-sheets, which is clear evidence that the ILs
induce conformational changes in the protein molecules. We faced a problem with recording of
the FTIR spectrum of In in presence of [Chol][Asp], which we ascribe to the observed
precipitation of the In-[Chol][Asp] sample or to an insufficient correction of the baseline in this
medium.
In terms of decreasing total helical content (%) of the In samples, the effect of the IL
series
reads:
[Chol][Arg]>[Chol]2[Glu]>[Chol][Lys]>[Chol][Glu]>IL-free
medium>[Chol]2[Asp], which correlates with the observed effect of the ILs on thermal unfolding
of In i.e. expectedly the molecules with the higher helical content were less thermostable.
Interestingly, the most compact structure of In is realized in presence of [Chol]2[Glu],
while the most unfolded structure is obtained in presence of [Chol][Glu]. In addition, the
monocholine glutaminate seems to stimulate In fibrillation and aggregation, which is consistent
with the observed from the DSC experiments.
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In spite of some spectral variations, which are evidence for the rearrangements in the
protein molecules, we observed similar patterns of the Amide I band, which suggests that almost
in all media containing the tested ILs In is near the native conformation.
Conclusion
Despite the observed rearrangements in the In molecule in the presence of the tested cholinium-
based ILs, it remains close to the native structure. The aggregation and fiblilation of the In was
suppressed in presence of the lysinate and the arginate salts. The two ILs, however, exhibited the
highest toxicity and decrease the thermal stability of the In. Choline-based ILs have potential to
stabilize proteins, but better selection or modification of the anion should be done. The results of
this study enrich the knowledge on the protein-IL interactions.
References
[1] A.M. Gualandi-Signorini, G. Giorgi, Insulin formulations – a review, Eur. Rev. Med.
Pharmacol. Sci. 5 (2001) 73–83.
[2] M. J. Maltesen, S. Bjerregaard, L. Hovgaard, S. Havelund, M. van de Weert, H. Grohganz,
Multivariate analysis of phenol in freeze-dried and spray-dried insulin formulations by NIR and
FTIR, AAPS Pharm. Sci. Tech. 12 (2) (2011) 627–636.
[3] D. F. Berensona, A. R. Weissa, Z.-li Wan, M. A. Weiss, Insulin analogs for the treatment of
diabetes mellitus: therapeutic applications of protein engineering, Ann. N. Y. Acad. Sci. 1243
(2011) E40–E54.
15

ACCEPTED MANUSCRIPT
[4] S. Atkin, Z. Javed, G. Fulcher, Insulin degludec and insulin aspart: novel insulins for the
management of diabetes mellitus, Ther. Adv. Chronic. Dis. 6(6) (2015) 375–388.
[5] Q.-X Hua, M. A. Weiss, Mechanism of insulin fibrillation, J. Biol. Chem. 279 (20) (2004)
21449–21460.
[6] V. Sluzky, J. Tamada, A. M. Klibanov, R. Langer, Kinetics of insulin aggregation in aqueous
solutions upon agitation in the presence of hydrophobic surfaces, Proc. Nati. Acad. Sci. USA. 88
(1991) 9377–9381.
[7] J. L. Whittingham, D. J. Scott, K. Chance, A. Wilson, J. Finch, J. Brange, G. Guy Dodson,
Insulin at pH 2: structural analysis of the conditions promoting insulin fibre formation, J. Mol.
Biol. 318 (2002) 479–490.
[8] A. Loksztejn, W. Dzwolak, Vortex-induced formation of insulin amyloid superstructures
probed by time-lapse atomic force microscopy and circular dichroism spectroscopy, J. Mol. Biol.
395 (2010) 643–655.
[9] L. Zhang, H. Jiang, W. Zhu, L. Wu, L. Song, Q. Wu, Y. Ren, Improving the stability of
insulin in solutions containing intestinal proteases in vitro, Int. J. Mol. Sci. 9 (2008) 2376-2387.
[10] J. Kraineva, V. Smirnovas, R. Winter, Effects of lipid confinement on insulin stability and
amyloid formation, Langmuir 23 (2007) 7118-7126.
[11] T. Rasmussen, R. Tantipolphan, M. van de Weert, W. Jiskoot, The molecular chaperone α-
crystallin as an excipient in an insulin formulation, Pharm. Res. 27 (2010) 1337–1347.
[12] R. Hayes, G. G. Warr, R. Atkin, Structure and nanostructure in ionic liquids, Chem. Rev. 5;
115 (13) (2016) 357-6426.
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ACCEPTED MANUSCRIPT
[13] R. Patel, M. Kumari, A. B. Khan, Recent advances in the applications of ionic liquids in
protein stability and activity: a review, Appl. Biochem. Biotechnol. 172 (8) (2014) 3701–3720.
[14] T. Takekiyo, E. Yamaguchi, H. Abe, Y. Yoshimura, Suppression Effect on the formation of
insulin amyloid by the use of ionic liquids, ACS Sus. Chem. Eng. 4 (2) (2016) 422–428.
[15] Y. Yoshimura, T. Takekiyo, T. Mori, Structural study of lysozyme in two ionic liquids at
cryogenic temperature, Chem. Phys. Lett. 664 (2016) 44–49.
[16] A. Kumar, P. Venkatesu, Prevention of insulin self-aggregation by a protic ionic liquid, RSC
Adv. 3 (2013) 362–367.
[17] J. V. Rodrigues, V. Prosinecki, I. Marrucho, L. P. N. Rebelo, C. M. Gomes, Protein stability
in an ionic liquid milieu: on the use of differential scanning fluorimetry, Phys. Chem. Chem.
Phys. 13 (2011) 13614–13616.
[18] T. Takekiyo, Y. Yoshimura, Suppression and dissolution of amyloid aggregates using ionic
liquids, Biophys. Rev. 10 (3) (2018) 853–860.
[19] E. E. L. Tanner, K. N. Ibsen, S. Mitragotri, Transdermal insulin delivery using choline-based
ionic liquids (CAGE), J. Control. Release 286 (2018) 137–144.
[20] T. Mosmann, Rapid colorimetric assay for cellular growth and survival: Application to
proliferation and cytotoxicity assays. J. Immunol. Meth. 65 (1983) 55–63.
[21] A. Natalello, D. Ami, S M. Doglia, Fourier transform infrared spectroscopy of intrinsically
disordered proteins: measurement procedures and data analyses. Methods Mol Biol. 895 (2012)
229–44.
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ACCEPTED MANUSCRIPT
[22] A. Barth, Infrared spectroscopy of proteins, Biochim. Biophys. Acta 1767 (2007) 1073 -
1101.
[23] S. Zeisel, K-A. da Costa, Choline: an essential nutrient for public health, Nutr. Rev. 67(11)
(2009) 615–623.
[24] J. M. Berg, J. L. Tymoczko, L.t Stryer, Chapter 3. Protein Structure and Function, W H
Freeman (Ed.), Biochemistry, 5Ed. New York, 2002.
[25] X.-D. Hou, Q.-P. Liu, T. J. Smith, N. Li, M.-H. Zong, Evaluation of toxicity and
biodegradability of cholinium amino acids ionic liquids, PLOS ONE 8 (3) (2013) e59145.
[26] J. Kong, S. Yu, Fourier Transform infrared spectroscopic analysis of protein secondary
structures, Acta Biochim. Biophys. Sinica 39(8) (2007) 549–559.
[27] L. Xie, C.-L. Tsou, Comparison of secondary structures of insulin and proinsulin by FTIR, J.
Prot. Chem. 12 (4) (1993) 483–487.
[28] J. Wei, Y.-Z. Lin, J.-M. Zhou, C.-L. Tsou, FTIR studies of secondary structures of bovine
insulin and its derivatives, Biochim. Biophys. Acta. 1080 (1991) 29-33.
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List of Figures and Tables
Scheme 1. Synthesis and structures of the cholinium-based amino acid ionic liquids [Chol]n[AA].
Fig. 1. Cell viability of 3T3 cells after 48-hour incubation with [Chol]n[AA].
Fig. 2. DSC thermograms of the native In in KCl/HCl buffer (pH 2.0, 10 mM) (black line); In-ILs
complexes: In-[Chol][Glu] (A); In-[Chol][Asp] (B); In-[Chol][Arg] (C); In-[Chol]2[Glu] (D); In-
[Chol]2[Asp] (E); In-[Chol][Lys] (F). Experiments were performed at a heating rate of 1ºC/min.
In concentration was 3 mg/mL. Molar ratio of In to ILs was 1:1.5.
Table 1. Thermodynamic parameters of the of the thermal unfolding of insulin in aqueous
solutions containing [Chol]n[AA].
# In all experiments the final insulin concentration (3 mg/mL, 0.517 mM). Molar ratio of In to
ILs is 1:1.5. IL-free medium contains only KCl/HCl (pH 2.0, 10 mM).
Table 2. Quantitative estimation of the main secondary structural elements of insulin in KCl/HCl
(pH 2.0, 10 mM) in absence and in presence of choline-based amino acid salts calculated from
their curve fitted FTIR spectra.
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CH3
H3C N+
CH3
H3C N+
CH3
[Chol]n[AA]
CH3
H3C N+
CH3
AAn-
ion exchage resin
AA
OH-
OH
Cl-
OH
- H2O
OH
CH3
n = 1 or 2
Ox
-
-
O-
-
O
NH
O
AA:
O
Ox
2
NH
2
O
NH NH
O
O
O
O
[Lys]
[Arg]
NH
NH
NH
[Glu]
NH2
O
2
2
2
-
[Asp]
x: or H
Scheme 1. Synthesis and structures of the cholinium-based amino acid ionic liquids [Chol]n[AA].
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Fig. 1. Cell viability of 3T3 cells after 48-hour incubation with [Chol]n[AA].
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A
B
C
6
3
0
6
D
E
F
3
0
40
60
80
100
40
60
80
100
40
60
80
100
Temperature (C)
Fig. 2. DSC thermograms of the native In in KCl/HCl buffer (pH 2.0, 10 mM) (black line); In-ILs
complexes: In-[Chol][Glu] (A); In-[Chol][Asp] (B); In-[Chol][Arg] (C); In-[Chol]2[Glu] (D); In-
[Chol]2[Asp] (E); In-[Chol][Lys] (F). Experiments were performed at a heating rate of 1ºC/min.
In concentration was 3 mg/mL. Molar ratio of In to ILs was 1:1.5.
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Table 1. Thermodynamic parameters of the of the thermal unfolding of insulin in aqueous
solutions containing [Chol]n[AA].
In in presence
of ionic liquids
IL-free medium 119.9 ± 1.3 5.6 ± 0.06
[Chol][Glu]
[Chol]2[Glu]
∆ H,
kJ/mol
Cp,
kJ/mol.K
Tm,
oC
T1/2,
oC
∆ HvH,
kJ/mol
47.7
171.2
63.0
75.4 ± 0.2 19.0 ± 0.20
85.1 ± 0.3 4.9 ± 0.05
68.6 ± 0.2 15.2 ± 0.20
32.4 ± 0.3
57.0 ± 0.9
5.2 ± 0.04
3.7 ± 0.04
Cp1=6.1± Tm1=79.2 ±
0.06 0.3
Cp2=6.6± Tm2=84.7±
[Chol][Asp]
76.6 ± 1.5
86.7 ± 1.8
11.8 ± 0.20
0.05
0.2
[Chol]2[Asp]
[Chol][Arg]
[Chol][Lys]
6.2 ± 0.04
101.9 ± 3.2 5.7 ± 0.03
194.4 ± 3.8 6.9 ± 0.07
79.9 ± 0.1
65.6 ± 0.2
70.0 ± 0.4
13.7 ± 0.15
17.0 ± 0.20
27.4 ± 0.30
74.1
53.4
34.7
# In all experiments the final insulin concentration (3 mg/mL, 0.517 mM). Molar ratio of In to
ILs is 1:1.5. IL-free medium contains only KCl/HCl (pH 2.0, 10 mM).
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Table 2. Quantitative estimation of the main secondary structural elements of insulin in KCl/HCl
(pH 2.0, 10 mM) in absence and in presence of choline-based amino acid salts calculated from
their curve fitted FTIR spectra.
Sum of the estimated relative areas, %
Secondary
structural
element
Media containing the corresponding ionic liquid
IL-free
media
[Chol][Glu] [Chol]2[Asp] [Chol]2[Glu] [Chol][Arg] [Chol][Lys]
antiparallel
β-structures
β-turns, β-
sheets
13.1
32.1
18.4
34.2
16.9
39.0
17.4
32.0
14.2
32.8
12.9
29.9
unordered
structures
and
15.4
10.6
12.7
21.2
12.2
18.4
random
coils
helical
structures
α-helices,
310-helices)
side-chains
residues of
the amino
acids
32.3
7.21
36.8
30.8
29.4
40.6
38.7
–
–
–
–
–
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Graphical abstract
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Highlights




Low toxic ionic liquids with cholinium cation and amino acid anion [Chol][AA] were synthesized.
Additives of [Chol][AA] to insulin [In] solutions prevented aggregation.
All [Chol][AA] induce conformational changes in In molecules.
The glutaminate and asparginate based ionic liquids enhanced thermal stability of In.
26