R. Arunkumar et al. / Journal of Molecular Liquids 314 (2020) 113602
3
(
Sigma-Aldrich, 99.7 wt%), sodium acetate (Sigma-Aldrich), potassium
in Fig. 1. Cytotoxicity measurements were made at multiple PIL, solvent
and salt concentrations, and are shown in Figs. 2 to 6. The cell viability
towards the controls of choline chloride, DMSO and ethanol are
shown in Fig. 2. The cell viability for the PILs and salts are grouped rela-
tive to the anion, and are shown in Figs. 3–6 for the acetates, formates,
glycolates and nitrates, respectively. The EC50 values were calculated
from these, as the solvent or salt concentration where there were 50%
viable cells, and these values are provided in Table 1. These toxicity re-
sults are then correlated to the aqueous PIL solution physicochemical
properties of density, surface tension and viscosity.
nitrate (Sigma-Aldrich) and ethanol (Sigma-Aldrich) were also ob-
tained. The following chemicals were supplied from Life technologies,
Dulbecco's Modified Eagle Medium (DMEM), foetal bovine serum
(
FBS), penicillin-streptomycin (10,000 U/mL), and MTT 3-(4, 5-
dimethylthiazol-2-yl)-2, 5-diphenyltetrazolium bromide) assay
reagent.
2
.1. PIL synthesis and characterisation
The PILs were synthesized by slowly adding equimolar amounts of
Choline chloride is considered as a highly biocompatible salt [27],
and DMSO is used routinely in biological applications such as cryopres-
ervation, despite being relatively toxic [37]. Due to their frequent use
with biological molecules, choline chloride and DMSO were used as con-
trols in this study. Similarly, ethanol was included as an amphiphilic
molecular solvent with a similar alkyl chain length to the PIL cations.
The cell viability in these solvents at different solvent concentrations
is provided in Fig. 2, and their EC50 values in Table 1. We observed
that choline chloride had a higher EC50 value of 3.2 mM compared to
the EC50 of 2.8 mM of DMSO, indicating the choline chloride is less toxic.
The cell viability relative to the PIL concentration is provided in
Figs. 3 to 6, for the acetates, formates, glycolates and nitrates, respec-
tively. It is evident from these Figures and Table 1 that the anion had a
significant effect on the toxicity. Of the 10 PILs studied, those with ace-
tate anions in general had the lowest toxicities, followed by the
glycolates. The cell viability versus PIL concentration for the acetate
and glycolate containing PILs are relatively linear, as shown in Figs. 3
and 4, and are similar to the plots observed for choline chloride and
DMSO. EtAA and EAA were identified as the least toxic PILs towards
these cells, with EC50 values of 2.5 mM and 2.4 mM respectively, placing
them as slightly more toxic than choline chloride and DMSO. In contrast,
the formates had an EC50 ~0.4 mM and nitrate containing PILs both had
EC50 well below 0.4 mM, with the nitrates significantly more toxic than
the formates, which can be seen from comparing Figs. 5 and 6. Interest-
ingly, the comparison salt of sodium acetate was more toxic than any of
the acetate containing PILs, whereas the potassium nitrate was less toxic
than either EAN or EtAN. The toxicity of the ethanol was greater than
most of the PILs towards the HaCat cells, with the exception of the
nitrate PILs.
the acid to the base. The solution was continuously stirred, and the tem-
perature maintained below 10 °C using an ice bath. The water content of
the PILs was determined by Karl Fischer Titration, using a Mettler Toledo
DL39 Karl Fischer coulometer, and the PILs all had b2 wt% water after
1
synthesis. Proton nuclear magnetic resonance ( H NMR) was taken of
each PIL on a Bruker 300 MHz instrument, and these are provided in
Fig. S1 of the ESI. The nitrate containing PILs of EAN and EtAN were
not included since the absence of protons on the nitrate anion
prevented confirmation of the stoichiometry. A Kibron EZPi Du Nuoy
surface tensiometer was used to determine the surface tension of the
PILs at room temperature (between 21 and 25 °C). The densities of the
PILs were measured at 20 °C using a vibrating tube Anton Paar density
meter (DMA 4500 M). A SV-A sine wave vibro viscometer was used to
determine the dynamic viscosity of the PILs. The kinematic viscosity
was obtained by dividing the dynamic viscosity by the density of the
respective PILs.
2
.2. Cell line
Human skin keratinocyte (HaCaT) cells were used. Cells were cul-
tured in Dulbecco's Modified Eagle Medium (DMEM), containing 10%
foetal bovine serum (FBS) and 1 vol% penicillin-streptomycin
2
10,000 U/mL). Cells were maintained at 37 °C with 5 vol% CO and
(
9
5 vol% relative humidity.
2
.3. MTT assay
In vitro cytotoxicity of the ILs was assessed via a MTT 3-(4,5-
dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide) assay. Cells
were seeded at a density of 1 × 10 cells/mL by adding 100 μL per well
into 96-well microtiter plates and allowed to incubate for 24 h. The me-
dium was then removed and replaced with 100 μL of IL, salt or molecular
solvent solutions at different concentrations up to 7 mM. After 24 h,
The cations had less of an effect on the toxicity, with changes in the
cations structure not having as dramatic effect on the toxicity. In gen-
eral, the ethanolammonium cation was slightly less toxic than the eth-
ylammonium cation, with the exception of the acetates where they
were similar across the concentration range used. This is evident from
Table 1, and also from the cell viability plots. The effect of multiple sub-
stitution on the cation was explored, taking the least toxic EtAA and
comparing to DEtAA and TEtAA. It can be seen from the EC50 values in
Table 1 and in Fig. 3, that increasing the number of ethylhydroxyl groups
5
1
0 μL MTT reagent (stock solution of 5.0 mg/mL was prepared in phos-
phate buffered saline) was added to each well to give a final concentra-
tion of 0.5 mg/mL, and the plates incubated at 37 °C for 4 h. The culture
medium was then removed by using a pipette and the resulting
formazan crystals dissolved in 100 μL of DMSO. The plates were shaken
for 30 s, and the optical density measured at 570 nm on a Spectramax
Paradigm Molecular devices microplate reader. DMSO and choline chlo-
ride was used as positive controls, the untreated cells were used as a
negative control, and wells containing complete media with ILs, in the
absence of cells, were used as cell-free controls to ensure no interaction
of the ILs with the MTT reagent. The EC50 values were obtained through
linear regression to find the IL, solvent or salt concentrations where
there were 50% viable cells.
100
90
80
70
60
50
40
DMSO
ChCl
Ethanol
3
. Results
30
20
Cytotoxicity measurements for the viability of HaCat cells towards
10
0
the ten protic ionic liquids (PILs), choline chloride, DMSO, potassium ni-
trate, sodium acetate and ethanol were determined. HaCat cells are a ro-
bust commonly used reproducible cell line, and hence they were chosen
to enable comparison to other studies. The chemical structures of the ILs
and other salt and solvents added used in this investigation are shown
0
1
2
3
4
5
6
7
Solvent concentraꢀon (mM)
Fig. 2. Viability of HaCat cells towards choline chloride, DMSO and ethanol.