The structure – Activity correlation in the family of dicationic imidazolium surfactants: Antimicrobial properties and cytotoxic effect

Article abstract of DOI:10.1016/j.bbagen.2020.129728

Background: The development of new effective microbicide surfactants and the search for the structure–biological activity relationship is an important and promising problem. Surfactants containing imidazolium fragment attract attention of researchers in the field of chemotherapy, because these compounds often exhibit high antimicrobial activity. The aim of this work is to identify the newly synthesized surfactants from the viewpoint of their potential usefulness in pharmacology and medicine. For this purpose, a detailed study of antimicrobial, hemolytic and cytotoxic activity of dicationic alkylimidazolium surfactants of the m-s-m (Im) series with a variable length of a hydrocarbon tail (m = 10, 12) and a spacer fragment (s = 2, 3, 4) was carried out. Methods: Aggregation of surfactants in solutions was estimated by tensiometry and conductivity. Antimicrobial activity was determined by the serial dilution technique. Cytotoxic effects of the test compounds on human cancer and normal cells were estimated by means of the multifunctional Cytell Cell Imaging system. Cell Apoptosis Analysis was made by flow cytometry. Results: The test compounds show high antimicrobial activity against a wide range of test microorganisms and do not possess high hemolytic activity. Importantly, some of them display a bactericidal activity comparable to ciprofloxacin fluoroquinolone antibiotic against Gram-positive bacteria, including methicillin-resistant strains of S. aureus (MRSA). The cytotoxicity of the compounds against normal and tumor human cell lines has been tested as well, with cytotoxic effect and selectivity strongly controlled by structural factor and kind of cell line. Superior results were revealed for compound 10–4-10 (Im) in the case of HuTu 80 cell line (duodenal adenocarcinoma), for which IC₅₀ value at the level of doxorubicin and a markedly higher selectivity index (SI 7.5) were demonstrated. Flow cytometry assay shows apoptosis-inducing effect of this compound on HuTu 80 cells, through significant changes in the potential of mitochondrial membrane. Major conclusions: Antibacterial properties are shown to be controlled by alkyl chain length, with the highest activity demonstrated by surfactants with decyl tail, with the length of the spacer fragment showing practically no effect. The results indicate that the mechanism of cytotoxic effect of the compounds can be associated with the induction of apoptosis via the mitochondrial pathway. General significance: Selectivity against pathogenic microorganisms and low toxicity against eukaryotic cells allow considering dicationic imidazolium surfactants as new effective antimicrobial agents. At the same time, high selectivity against some cancer cell lines indicates the prospect of their using as components of new anticancer drugs.

Full text of DOI:10.1016/j.bbagen.2020.129728

BBA-GeneralSubjects1864(2020)129728
Contents lists available at ScienceDirect
BBA - General Subjects
journal homepage: www.elsevier.com/locate/bbagen
The structure – Activity correlation in the family of dicationic imidazolium
surfactants: Antimicrobial properties and cytotoxic effect
Alexandra D. Voloshinaa, Syumbelya K. Gumerovaa, Аnastasiia S. Sapunovaa, Natalia V. Kulika,
⁎
Alla B. Mirgorodskayaa, , Alla A. Kotenkob, Tatiana M. Prokopyevab, Vasilii A. Mikhailovb,
Lucia Ya Zakharovaa, Oleg G. Sinyashina
a Arbuzov Institute of Organic and Physical Chemistry, FRC Kazan Scientific Center, Russian Academy of Sciences, Arbuzov str., 8, Kazan 420088, Russia
b L.M. Litvinenko Institute of Physical Organic Chemistry and Coal Chemistry, 70 R. Luxemburg St., 83114 Donetsk, Ukraine
A R T I C L E I N F O
A B S T R A C T
Keywords:
Background: The development of new effective microbicide surfactants and the search for the structure–-
biological activity relationship is an important and promising problem. Surfactants containing imidazolium
fragment attract attention of researchers in the field of chemotherapy, because these compounds often exhibit
high antimicrobial activity. The aim of this work is to identify the newly synthesized surfactants from the
viewpoint of their potential usefulness in pharmacology and medicine. For this purpose, a detailed study of
antimicrobial, hemolytic and cytotoxic activity of dicationic alkylimidazolium surfactants of the m-s-m (Im)
series with a variable length of a hydrocarbon tail (m = 10, 12) and a spacer fragment (s = 2, 3, 4) was carried
out.
Dicationic imidazolium surfactants
Antimicrobial activity
Cytotoxicity
Hemolysis
Resistance
Apoptosis
Methods: Aggregation of surfactants in solutions was estimated by tensiometry and conductivity. Antimicrobial
activity was determined by the serial dilution technique. Cytotoxic effects of the test compounds on human
cancer and normal cells were estimated by means of the multifunctional Cytell Cell Imaging system. Cell
Apoptosis Analysis was made by flow cytometry.
Results: The test compounds show high antimicrobial activity against a wide range of test microorganisms and
do not possess high hemolytic activity. Importantly, some of them display a bactericidal activity comparable to
ciprofloxacin fluoroquinolone antibiotic against Gram-positive bacteria, including methicillin-resistant strains of
S. aureus (MRSA). The cytotoxicity of the compounds against normal and tumor human cell lines has been tested
as well, with cytotoxic effect and selectivity strongly controlled by structural factor and kind of cell line. Superior
results were revealed for compound 10–4-10 (Im) in the case of HuTu 80 cell line (duodenal adenocarcinoma),
for which IC50 value at the level of doxorubicin and a markedly higher selectivity index (SI 7.5) were demon-
strated. Flow cytometry assay shows apoptosis-inducing effect of this compound on HuTu 80 cells, through
significant changes in the potential of mitochondrial membrane.
Major conclusions: Antibacterial properties are shown to be controlled by alkyl chain length, with the highest
activity demonstrated by surfactants with decyl tail, with the length of the spacer fragment showing practically
no effect. The results indicate that the mechanism of cytotoxic effect of the compounds can be associated with
the induction of apoptosis via the mitochondrial pathway.
General significance: Selectivity against pathogenic microorganisms and low toxicity against eukaryotic cells
allow considering dicationic imidazolium surfactants as new effective antimicrobial agents. At the same time,
high selectivity against some cancer cell lines indicates the prospect of their using as components of new an-
ticancer drugs.
1. Introduction
will form the basis of new drugs. Surfactants containing imidazolium
fragment attract attention of researchers in the field of chemotherapy,
Due to the widespread occurrence of multidrug-resistant infectious
agents, there is a demand to search for safe chemical compounds, which
because these compounds often exhibit high antimicrobial activity
[1–6]. Functionalization of imidazolium surfactants with various
⁎ Corresponding author.
E-mail address: mirgorod@iopc.ru (A.B. Mirgorodskaya).
https://doi.org/10.1016/j.bbagen.2020.129728
Received 8 June 2020; Received in revised form 28 August 2020; Accepted 3 September 2020
Availableonline06September2020
0304-4165/©2020ElsevierB.V.Allrightsreserved.

A.D. Voloshina, et al.
BBA-GeneralSubjects1864(2020)129728
substituents makes it possible to modify their hydrophilic-lipophilic
balance and aggregation properties, as well as to tune their biological
activity. Dicationic imidazolium surfactants are of a particular interest
[7–9]. They represent a large class of geminal surfactants with two
hydrophobic tails and two positively charged head groups covalently
linked by a spacer fragment. Gemini surfactants display superior
properties to corresponding monocationic analogues: they more effec-
tively reduce the surface tension at the phase boundary, have an order
of magnitude lower critical micelle concentration (CMC), possess high
solubility, and high wetting and solubilization effects [10–15]. These
properties of dicationic surfactants predetermine their broad applica-
tion as solubilizers and adjuvants, nanocontainers and nanoreactors, as
well as non-viral vectors for gene delivery into cells of a living or-
ganism. Dicationic surfactants also show antimicrobial effect, which is
maximum in compounds with a tail length C10-C12 [16,17]. In con-
trast, monocationic amphiphiles, whose antimicrobial activity shows a
positive trend with an increasing the carbon chain length, reach max-
imum activity in case of tetra- or hexadecyl compounds, above which a
so-called ‘cut off effect’ is observed [3,4,18,19].
2. Materials and methods
2.1. Alkylimidazoles and bis-imidazolium salts. General remarks
Alkylimidazoles and bis-imidazolium salts were made according to
Scheme 1.
This scheme is known from literature [37,38], but some details look
incomplete or controversial. That is why detailed synthetic description
provided for representative examples. Structure and purity of final and
intermediary compounds were confirmed by 1H NMR and IR spectra,
and appropriate results of elemental analysis.
Step 1. N-Dodecylimidazole.
In a high form beaker (250 ml), imidazole (8.19 g; 0.120 M) was
dissolved in dimethylsulfoxide (50 ml), and coarsely ground potassium
hydroxide (12.89 g; 0.23 M) was added under stirring. Turbid mixture
was heated at 50–60 °C and stirred until sediment became compact.
Dodecyl bromide (25.06 g; 0.1005 M; approx. 24.1 ml) was added from
dropping funnel over approx. Half an hour at 60–65 °C under intense
stirring (at least 300 rpm). Heating was stopped and stirring was con-
tinued for an hour. The mixture was diluted with water up to 150 ml.
Top layer was separated with a funnel, dissolved in methylene
dichloride (50 ml), and washed with cold water (2 × 25 ml). Organic
layer was separated, and volatiles were immediately distilled of on
water bath, and, then, under reduced pressure. The residue was dis-
solved in acetonitrile (50 ml) and filtered. Solvent was removed under
vacuum (20–30 mmHg). The crude product is yellowish oil; yield
23.22 g (9.82 × 10−2 M; 97%). After distillation in vacuo (130–135 °C/
0.1 mmHg) - colorless liquid, solidified at freezing, mp 11–12 °C. 1H
NMR (400 MHz, CDCl3, δ, ppm): 7.45 (s, 1H), 7.05(s, 1H), 6.90 (s, 1H),
3.92 (t, J = 7.1 Hz,2H), 1.80–1.75 (m, 2H), 1.33–1.22 (m, 18H), 0.88
(t, J = 6.8 Hz, 3H).
The structure of head group and spacer fragment plays an important
role in the aggregation behavior and properties of gemini surfactants
[20–22]. There is a large body of recent literature data on the ag-
gregation behavior of imidazolium dicationic surfactants in solutions,
in which CMC values and adsorption parameters are determined
[1,23,24]. There is a few information about their solubilization effect
and complexation with proteins and DNA [1,25–29]. By contrast, an
information on the antimicrobial properties of such compounds is
fragmentary. However, some data on dicationic imidazolium surfac-
tants indicate their significant potential. Thus, the study of 3,3′(α,ω-
dioxaalkyl)-bis(1-alkylimidazolium) chlorides showed the high effi-
ciency of this surfactants against Staphylococcus aureus, Enterococcus
faecalis, Acinetobacter baumannii, Escherichia coli, Klebsiella pneumoniae,
Enterobacter cloacae, Pseudomonas aeruginosa, Candida krusei, and Can-
dida albicans [30,31]. In addition, antimicrobial effect was demon-
strated for a series of new dicationic imidazolium surfactants con-
taining amide group in the spacer fragment, which depends on the
length of hydrophobic tail. High antifungal activity of these compounds
against Candida albicans, one of the main opportunistic pathogens that
cause a wide range of human diseases, should particularly be noted
[32].
Step 2. 1,2-bis(3′-dodecylimidazolium-1′-yl)ethane dibromide.
Freshly prepared solution of N-dodecylimidazole (2.93 g, 1.24
10−2 M) in tetrahydrofuran (2 ml), and 1,2-dibromoethane (0.98 g; 5.2
10−2 M) were placed in closed vessel, and mixture was heated up to
45–50 °C for ten days. After cooling, reaction mixture was diluted with
acetone (25 ml) and thoroughly mashed. Non-dissolved part was fil-
tered, washed with acetone (2 × 5 ml) and dried. White powder was
obtained; yield 2.48 g (5.78 × 10−3 M; 72%). Crude product was re-
crystallized from isopropanol (20 ml). 1H NMR (400 MHz, CDCl3, δ,
ppm): 10.34 (s, 2H), 8.86 (s, 2H), 7.14 (s, 2H), 5.31 (s, 4H), 4.15 (t,
J = 7.5 Hz, 4H), 1.95–1.87 (m, 4H), 1.40–1.24 (m, 36H), 0.88 (t,
J = 6.5 Hz, 6H). Anal.Calcd for C32H60Br2N4: C, 58.18; H, 9.15; N,
8.48; Br 24.19. Found: C, 57.98; H, 9.56; N, 8.39; Br 23.80. IR (KBr, ν,
cm−1): 3400 br, 3020, 2910, 2840, 1570, 1560, 1465, 1445, 1160, 850,
799, 710, 640, 620.
In this regard, the development of new effective microbicides based
on dicationic imidazolium surfactants and the search of structure–-
biological activity relationship is an important and promising problem.
Taking into account the wide use of amphiphilic compounds in cancer
chemotherapy for the preparation of nanoparticles with target agents
and antitumor drugs [33–36], the investigation of antimicrobial prop-
erties of imidazolium surfactants should be extended by their cyto-
toxicity study.
Synthetic procedures, melting points, spectral data for other di-
bromides under investigation summated in Supplementary Info.
The aim of this work is to identify new bioactive surfactants that can
be used in pharmacology and medicine. For this purpose, a detailed
study of antimicrobial, hemolytic and cytotoxic activity of a number of
dicationic alkylimidazolium surfactants of the m-s-m (Im) series with a
variable length of the hydrophobic radical and a varying distance be-
tween the head groups was carried out (Fig. 1).
2.2. Surface tension and conductivity measurements
Surface tension measurements were performed by the ring detach-
ment method using KRUSS K6 tensiometer. Specific conductivities were
measured with Inolab Cond 720 conductometer. Experimental tem-
peratures were maintained at 25
0.1 °C, unless otherwise indicated.
All experiments were accurate within 4%.
2.3. Antimicrobial activity
H2m+1
H
Cm
2m+1
Antimicrobial activity of the bis-imidazolium salts was determined
by the serial dilution technique in Muller Hinton Broth 2 [39]. The
cultures used for testing included Gram-positive bacteria: Staphylo-
coccus aureus АТСС 6538Р FDA 209P, Bacillus cereus АТСС 10702 NCTC
8035; Gram-negative bacteria: Escherichia coli ATCC 25922, Pseudo-
monas aeruginosa ATCC 9027, and fungi: Aspergillus niger BKMF-1119,
Trichophyton mentagrophytes var. gypseum 1773, and Candida albiсans
Cm
N
N
N
CH2
N
s
2Br
m-s-m (Im), where m=10,12; s=2; 3; 4
Fig. 1. The structural formula of dicationic alkylimidazolium surfactants.
2

A.D. Voloshina, et al.
BBA-GeneralSubjects1864(2020)129728
Scheme 1. The synthesis of bis-imidazolium salts
ATCC 10231; methicillin-resistant strains of S. aureus (MRSA) was ob-
tained from hospital patients with chronic tonsillitis in the Republican
Clinical Hospital (Kazan, Russia). The bacterial load was 3.0 × 105 cfu/
ml. The results were recorded every 24 h for 5 days. Cultures were
incubated at 37 °C. The experiment was repeated three times. The di-
lutions of the compounds were prepared immediately in nutrient
media; 5% DMSO was added for better solubility and the test strains
were not inhibited at this concentration. The minimum inhibitory
concentration (MIC) was defined as the minimum concentration of a
compound that inhibits the growth of the corresponding test micro-
organism. The growth of bacteria as well as the absence of the growth
due to the bacteriostatic action of bis-imidazolium salts were recorded.
To determine minimal bactericidal concentration (MBC), an aliquot of
the bacterial culture was transferred onto Mueller-Hinton agar in a 10-
cm Petri dish and incubated for 24 h at 37°С. MBC was the minimal
concentration of the tested compound at which bacterial colonies were
not detected indicating that the bacteria were killed with an efficiency
of > 99.9% [40,41].
normal cells were estimated by means of the multifunctional Cytell Cell
Imaging system (GE Health Care Life Science, Sweden) using the Cell
Viability Bio App which precisely counts the number of cells and
evaluates their viability from fluorescence intensity [42]. Two fluor-
escent dyes that selectively penetrate the cell membranes and fluoresce
at different wavelengths were used in the experiments. DAPI is able to
penetrate intact membranes of living cells and colors nuclei in blue and
Propidium iodide dye penetrates only dead cells with damaged mem-
branes, staining them in yellow. IC50 was calculated using an online
tool: MLA—“Quest Graph™ IC50 Calculator.” AAT Bioquest, Inc., 25
July 2019, https://www.aatbio.com/tools/ic50-calculator. DAPI and
propidium iodide were purchased from Sigma. The M-Hela clone 11
human, epithelioid cervical carcinoma, strain of Hela, clone of M-Hela;
human breast adenocarcinoma cells (MCF-7); human duodenal cancer
cell line (HuTu 80); from the Type Culture Collection of the Institute of
Cytology (Russian Academy of Sciences) and Chang liver cell line
(Human liver cells) from N. F. Gamaleya Research Center of Epide-
miology and Microbiology were used in the experiments. The cells were
cultured in a standard Eagle's nutrient medium manufactured at the
Chumakov Institute of Poliomyelitis and Virus Encephalitis (PanEco
company) and supplemented with 10% fetal calf serum and 1% non-
essential amino acids. The cells were plated into a 96-well plate (Ep-
pendorf) at a concentration of 1 × 105 cells/ml, 150 μl of medium per
well, and cultured in a CO2 incubator at 37 °C. Twenty-four hours after
seeding the cells into wells, the compound under study was added at a
preset dilution, 150 μl to each well. The dilutions of the compounds
were prepared immediately in nutrient media; 5% DMSO that does not
induce inhibition of cells at this concentration was added for better
solubility. The experiments were repeated three times. Intact cells
cultured in parallel with experimental cells were used as a control.
2.4. LIVE/DEAD BacLightTM bacterial viability method
To count live and dead cells, Stapylococcus aureus 209 P (control and
test samples) were stained with a commercial dye mix of the LIVE/
DEAD BacLightTM Bacterial Viability kit for 30 min in the dark. Then, the
suspension was mixed with an equal volume of 0.5% low-melting
agarose. The preparations were analyzed using a Nikon Eclipse Ci-S
fluorescence microscope (Nikon, Japan). Cell counting in a given scan
volume was carried out differentially according to red and green
fluorescence.
2.5. Hemolytic activity
2.7. Flow cytometry assay
Hemolytic activity of the bis-imidazolium surfactants was estimated
by comparing the optical density of a solution containing the test
compound with that of blood at 100% hemolysis. A 10% suspension of
human red cells was used as an object of investigation. Red cells with
heparin was washed three times with physiological saline (0.9% NaCl)
solution, centrifuged for 10 min at 800 rpm, and resuspended in phy-
siological saline (0.9% NaCl) solution to a concentration of 10%. The
concentrations of the compounds that corresponded to the MIC values
for the bacterial test strains were prepared in physiological saline (0.9%
NaCl) solution (supplemented with 5% DMSO), and 4.5 ml of the
compound at the corresponding dilution was added to 0.5 ml of a 10%
suspension of erythrocytes. The samples were incubated for 1 h at 37 °C
and centrifuged for 10 min at 2000 rpm. Release of hemoglobin was
controlled by measuring the optical density of the supernatant on
Microplate reader Iinvitrologic (Russia) at λ = 540 nm. The control
sample corresponding to zero hemolysis (blank experiment) was pre-
pared by adding 50 μl of 10% red blood cell suspension to 450 μl of
physiological saline solution (0.9% NaCl). The control sample corre-
sponding to 100% hemolysis was prepared by adding of 50 μl of 10%
red blood cell suspension to 450 μl of distilled water.
2.7.1. Cell culture
M-Hela cells at 1 × 106 cells/ well in a final volume of 2 ml were
seeded into 6-well plates. After 24 h of incubation, various concentra-
tions of compound 10-4-10 (Im) were added to wells.
2.7.2. Cell apoptosis analysis
The cells were harvested at 2000 rpm for 5 min and, then, washed
twice with ice-cold PBS, followed by resuspension in binding buffer
100 μl. Next, the samples were incubated with 0.35 μl of annexin V-
Alexa Fluor 647 and 0.1 μl of propidium iodide for 40 min at room
temperature in the dark. Finally, the cells were analyzed by flow cy-
tometry (Guava easy Cyte, MERCK, USA).
The experiments were repeated three times.
2.7.3. Mitochondrial membrane potential
The cells were harvested at 2000 rpm for 5 min and, then, washed
twice with ice-cold PBS, followed by resuspension in JC-10 (10 μg/ml)
and incubation at 37 °C for 10 min. After the cells were rinsed three
times and suspended in PBS, the JC-10 fluorescence was observed by
flow cytometry.
2.6. Cytotoxicity assay
2.7.4. Statistical analysis
Cytotoxic effects of the test compounds on human cancer and
IC50 was calculated using an online tool: MLA-“Quest Graph™ IC50
3

A.D. Voloshina, et al.
BBA-GeneralSubjects1864(2020)129728
Table 1
Antimicrobial activity of dicationic alkylimidazolium surfactantsa.
Test compounds
Minimum bacteriostatic concentration (MIC), μM
Minimum fungistatic concentration (MIC), μM
Sa
Bc
Ec
Pa
MRSA-Sa
Tm
Ca
An
10–2-10 (Im)
10–3-10 (Im)
10–4-10 (Im)
Im-10b
0.8
1.5
1.4
0.06
0.1
1.5
1.5
1.4
0.1
0.1
0.1
12.9
6.2
1.0
0.5
0.2
25.8
12.6
12.3
2.2
0.8
0.9
3.1
3.1
6.2
0.1
51.6
51.0
49.5
4.7
4.5
3.8
6.5
0.4
0.3
0.3
206
202
–
18.2
16.8
0.1
0.4
6.2
0.1
3.0
6.2
1000 [3]
500 [3]
23.6
23.1
22.7
81[44]
0.7
500 [3]
23.6
23.1
22.7
12–2-12 (Im)
12–3-12 (Im)
12–4-12 (Im)
12–2-12 (Ме)c
Сiprofloxacin
Norfloxacin
Ketoconazole
5.9
0.4
0.3
0.9
11.8
11.6
11.3
1.1
1.8
23.6
1.7
1.8
1.7
3.0
5.8
5.6
0.1
0.3
0.3
23.6
46.2
182
1.9
3.1
1.9
–
–
–
–
5.8
0.9
0.7
1.6
1.4
23.1
1.7
1.6
11.3
22.7
11.5
9.7 [44]
325[44]
0.7
7.5
0.05
0.5
1.4
0.1
2.1
0.05
0.02
0.7
0.05
1.1
340
29
24.4
4.7
12.1
7.3
0.5
7.3
0.5
Test compounds
Minimum bactericidal concentration (MBC), μM
Minimum fungicidal concentration (MFC), μM
10–2-10 (Im)
10–3-10 (Im)
10–4-10 (Im)
12–2-12 (Im)
12–3-12 (Im)
12–4-12 (Im)
Сiprofloxacin
Norfloxacin
1.5
1.5
3.0
5.9
5.8
11.3
0.7
7.5
0.1
0.1
0.2
0.4
0.3
3.1
0.2
0.2
0.2
25.8
12.6
6.2
2.1
1.1
51.6
12.6
12.3
94.4
23.1
45.4
0.7
4.2
1.2
1.1
7.5
1.8
3.6
0.06
4.2
6.4
6.2
6.2
24.4
23.1
11.4
–
0.5
0.5
0.4
206
202
395
94.4
46.2
182
17.4
15.3
28.1
8.6
6.5
0.4
0.3
0.3
–
–
–
–
–
–
3.1
6.2
3.1
0.4
6.2
11.8
23.1
11.3
1.4
0.9
94.4
23.1
22.7
0.7
8.7
1.9
1.8
23.6
26.2
22.7
1.8
1.8
0.8
1.7
0.7
3.6
3.9
1.9
0.8
1.7
12.4
0.9
0.6
0.1
0.05
2.3
24.4
2.1
24.4
49.0
Ketoconazole
7.3
0.5
7.3
0.5
a
Average of three values measured; standard deviation (SD); − means non-active;
Im-10 - 1-Decyl-3-methylimidazolium chloride;
b
c
12–2-12 (Ме) - ethylene-1,2-bis(dimethyldodecylammonium) dibromide.
Calculator.” AAT Bioquest, Inc., 25 July 2019, https://www.aatbio.
com/tools/ic50-calculator [43].
Table 2
CMC values of gemini imidazolium surfactants investigated by different tech-
niques.
The cytometric results were analyzed by the Cytell Cell Imaging
multifunctional system using the Cell Viability BioApp and Apoptosis
BioApp application. The data in tables and graphs are given as the
Surfactant
CMC, mmol/l
Surface tension method
Conductivity method
mean
standard error.
10–2-10 (Im)
10–3-10 (Im)
10–4-10 (Im)
12–2-12 (Im)
12–3-12 (Im)
12–4-12 (Im)
4.0
4.3
5.0, 3.37a
4.2, 4.50 a
0.55a
5.0, 4.60a
5.6, 5.07a
0.64 a
3. Results and discussion
3.1. Antimicrobial activity of test compounds
0.53, 0.53 a
0.65, 0.72 a
0.70, 0.61a
0.83, 0.73 a
Table 1 summarizes results on the antibacterial activity of geminis
under study. The compounds were tested for antibacterial (bacterio-
static and bactericidal) activity against a number of Gram-positive S.
aureus 209P (Sa), B. сereus 8035 (Вс) and Gram-negative bacteria E. coli
F-50 (Ec), Pseudomonas aeruginosa 9027 (Ра) including methicillin-re-
sistant strains of S. aureus (MRSA-Sa). Antifungal activity was studied
on Trichophyton mentagrophytes var. gypseum 1773, Aspergillus niger
1119 and Candida albicans 10,231.
a
From [1].
makes it possible to conclude that in all cases, the active surfactant
concentration turned out to be lower than CMC; i.e., these substances
are active in non-aggregated state. The dicationic imidazolium surfac-
tants are significantly more active than their monocationic analogues
[3], as well as dicationic surfactants with ammonium head group [45]
(see Table 1).
Typically, the primary information on the antimicrobial properties
of newly synthesized compounds can be derived from their screening
assessment aimed at the determining the MIC value, i.e. the lowest
concentration that completely inhibits the visible growth and pro-
liferation of microorganisms [39,44]. It follows from the data in Table 1
that the compounds have a broad spectrum of antimicrobial activity.
Moreover, imidazolium surfactants with decyl radical showed higher
activity than their dodecyl analogues with respect to all bacterial strains
including MRSA-Sa and the yeast-like fungus C. albicans. It should be
noted that all compounds studied have amphiphilic character and form
aggregates beyond the CMC, with the functional properties of mono-
meric and aggregated molecules markedly differing. To elucidate
whether surfactants in monomer or aggregated form contribute to the
antimicrobial effect, aggregation threshold needs to be determined. To
this end, CMC values were examined by methods of tensiometry and
conductometry (Figs. S1, S2), which are summarized in Table 2 along
with literature data. The comparison of the values in Tables 1 and 2
Although the MIC value is the primary characteristics of anti-
microbial activity, it gives few information, whether this drug belongs
to bacteriostatic or bactericidal agents. Importantly, bacteriostatic and
bactericidal drugs are assumed to differ in mechanism of action [46].
Therefore, minimal bactericidal concentration (MBC) was further de-
termined as the lowest concentration of antimicrobial agents needed to
kill the bacteria. Values MBC and MIC complement each other, and
their relation may serve as a criterion for the attribution of the com-
pound tested to either bacteriostatic or bactericidal agents. Anti-
bacterial agents are usually considered as bactericidal if MBC is not
more than four times higher compared to MIC [46]. This case is ob-
served for the series 10-s-10(Im) and 12-s-12(Im); therefore, these
compounds were attributed to bactericidal agents (the same is observed
in the case of fungicidal activity). Noteworthy, dicationic surfactants of
10-s-10(Im) series are of particular interest from the viewpoint of their
potential as novel antimicrobial agents, since their MBC values in
4

A.D. Voloshina, et al.
BBA-GeneralSubjects1864(2020)129728
relation to Gram-positive bacteria including MRSA and MFC against C.
albicans are markedly lower compared to commercial preparation. All
test compounds did not show high antimicrobial activity against T.
mentagrophytes var. gypseum 1773 and A. niger 1119 (please see
Table 1).
3.3. Effect of compound 10–2-10 (Im) on alteration of cell wall
permeability of S. aureus
It is of special interest to elucidate elementary mechanisms of an-
timicrobial effect of amphiphilic compounds studied. The methods
using fluorescent dyes that selectively penetrate membranes are the
most reliable methodological approaches for identification of viable
bacterial cells in vitro [52]. One of these methods, LIVE/DEAD Ba-
cLightTM Bacterial Viability (Invitrogen) (a method of vital staining of
bacteria), is widely used for qualitative and quantitative analyses of
viable cells in populations. This method indicates the state of the bac-
terial population of Staphylococcus aureus after exposure to anti-
microbial agents. It involves two dyes that emit at different wave-
lengths. The low molecular weight dye SYTO 9 emits in the green
spectral range and could penetrate intact membranes. The high mole-
cular weight dye propidium iodide, which emits in the red spectral
range, penetrates only the cells with damaged membranes. As a result,
living cells are stained in green and dead cells are stained in red.
Fig. 3 shows the images of a bacterial population of Staphylococcus
aureus after exposure to the leader compound 10–2-10 (Im) at bacter-
iostatic and bactericidal concentrations. Using fluorescence micro-
scopy, the ratio of living to dead cells before and after exposure to the
compound was calculated due to selective staining with fluorophores. It
can be seen that the number of dead cells (stained in red) increases
compared to the control with an increase in the concentration of
compounds. Assessment of the quantitative effect of compound 10–2-
10(Im) on the cells is given in Fig. 4. It can be seen that the bacterial
cytoplasmic membrane starts to destabilize upon the cultivation of
bacteria in the presence of the test compound at its MIC; i.e., that is, the
number of cells stained in red increases in the S. aureus population.
Significant changes in the permeability of S. aureus cytoplasmic mem-
branes were observed with an increase in the concentrations of com-
pounds to MBC and higher.
In general, antimicrobial effect of cationic surfactants has a com-
plicated mechanism, with
a
variety of factors contributing
[18,20,47,48]. Both, the structural characteristics of surfactants and
kind and structure of microorganisms should be taken into account.
Two different groups of bacteria, Gram-positive and Gram-negative
demonstrate different sensitivity toward the cationic surfactants. This is
due to the fact that these families of bacteria differ in the structure of
their cell walls. Unlike with Gram-positive bacteria that have inner
cytoplasmic membrane neighboring with cell wall, Gram-negative
bacteria have additional outer membrane, which prevents the access of
foreign membranotropic compounds, thereby increasing the resistance
of cell against antimicrobial agents including surfactants [49].
These structural specificities are responsible for the differences in
the values of MIC and MBC between these two groups of bacteria.
Similarly, for gemini imidazolium surfactants under study, the active
concentration determined for Gram-positive bacteria (S. aureus, B.
Сereus) are few lower compared to those for Gram-negative bacteria (E.
coli F-50, Pseudomonas aeruginosa). Meanwhile, all these values are
comparable with the reference preparations (please, see Table 1).
3.2. Hemolytic activity
An important characteristic in evaluating the biological activity of
new chemical compounds is their mammalian cell cytotoxicity.
Therefore, m-s-m (Im) dicationic alkylimidazolium surfactants were
tested for cytotoxicity against human red blood cells (hemolytic ac-
tivity). Fig. 2 and Table S 1 show the degree of hemolysis of human
erythrocytes caused by the compounds in the concentration range of
0.8–50 μM.
These results suggest that the specific mechanism of action of the
test compounds is associated with the damage to bacterial cytoplasmic
membrane. The hydrophobic part of the molecule (alkyl chain) pre-
sumably assists in the cell membrane penetration resulting in its da-
mage and replacement of extracellular fluid by intracellular, which
leads to cell lysis and death [30]. According to current concepts anti-
microbial activity of cationic surfactants is mainly mediated through
their integration with the lipid membranes [53,54], which results in
anomalous changes in the membrane functions conjugated with their
native permeability, i.e. transport of substances, energy transformation,
etc. Under the higher surfactant concentration exceeding CMC values,
dual effect can occur due to micelle formation. On the one hand, co-
operative positively charged aggregates show enhanced electrostatic
affinity toward cell membrane and may serve as a ‘depot’ maintaining
surfactant concentration needed. On the other, beyond the definite
threshold, solubilization of lipid components of membranes can occur
accompanied by irreversible changing the structure of proteins and
their functionality [48,55–57]. The structure of charged species, i.e.
head groups and counterions influences the antimicrobial activity, e.g.
surfactants with cyclic head groups demonstrate the higher effect
compared to acyclic analogs. However, the key factor is alkyl chain
length responsible for the ‘cut off’ effect assuming the occurrence of the
optimal alkyl chain length. In the case of monocationic imidazole-
containing surfactants, maximal activity is achieved for hexadecyl de-
rivatives [3,4,19]. For gemini surfactants studied herein, decyl deriva-
tives show the highest antimicrobial effect, whereas spacer length plays
the minor role. These dicationic imidazoloim surfactants appeared to
surpass their monocationic counterparts and ammonium analogs in the
activity.
The MIC and MBC values of the test compounds fall within this
range. It follows from these data that surfactants with a decyl hydro-
phobic substituent turned out to be less toxic than their dodecyl ana-
logues. Gramicidin S antibiotic, with a bacteriostatic and bactericidal
effect at 0.8–12.5 μM, was used as a reference preparation. It is known
that Gramicidin S results in erythrocyte hemolysis and thus cannot be
used for intravenous administration. Nevertheless, due to the suffi-
ciently high antimicrobial activity, it is widely used in medicine for
external treatment of purulent wounds and abscesses and as a part of
complex preparations for the treatment of the pharynx and oral cavity
[50,51]. Dicationic alkylimidazolium surfactants in the concentration
range under study possess lower hemolytic activity than Gramicidin S
antibiotic (Fig. 2, Table S2). Hemolysis of m-s-m (Im) compounds at
concentrations of ≤12.5 μM is less than 50% and they may be of in-
terest for further studies with living objects.
Fig. 2. Hemolytic activity of dicationic alkylimidazolium surfactants: 1–10–2-
10(Im), 2–10–3-10(Im), 3–10–4-10(Im), 4–12–2-12(Im), 5–12–3-12(Im),
6–12–4-12(Im).
5

A.D. Voloshina, et al.
BBA-GeneralSubjects1864(2020)129728
Fig. 3. The effect of dicationic surfactant on the permeability and integrity of the cytoplasmic membrane of Staphylococcus aureus 209p using fluorescence micro-
scopy: (A) image of the control sample; (B) image of the test sample treated with compound 10–2-10(Im) at a bacteriostatic concentration of 0.8 μM; (C) image of the
test sample treated with compound 10–2-10(Im) at a bactericidal concentration of 1.5 μM; (D image of the prototype treated with compound 10–2-10(Im) at a
concentration doubled by MBC(2 × MBC), i.e. 3.1 μM.
3.5. Induction of apoptotic effects by test compounds
1.00E+12
1.00E+11
1.00E+10
1.00E+09
1.00E+08
1.00E+07
1.00E+06
One of the main mechanisms of the cytotoxic activity of compounds
is the induction of apoptosis (programmed cell death), which eliminates
significantly damaged or dead cells [59]. Now, apoptosis is one of the
key mechanisms addressed in the development of new antitumor drugs.
With reference to the above mentioned, the study of the apoptosis-
inducing ability of the leader compound (10–4-10 (Im)) with a selective
cytotoxic effect is of a particular interest. Apoptotic effects were eval-
live
dead
uated using the annexin
V kits, which can bind phosphatidy-
lethylserine. There is a minimum content of phosphatidylserine on the
surface of healthy cell membranes; therefore, interaction of annexin V
with these cells is marginal. When there is apoptosis, phosphati-
dylserine molecules appear on the surface of the cells and could interact
with the protein. This interaction results in the increase in the fluor-
escence intensity of apoptogenic cells, which is detected by a flow
cytometer.
Control
0.8
1.5
3.1
Concentrations of 10-2-10 (Im), µ
Fig. 4. Quantitative assessment of the effect of the compound 10–2-10 (Im) on
the permeability of the S. aureus cytoplasmic membrane.
According to the flow cytometry data (Figs. 5, 6), a dose-dependent
induction of apoptosis is observed as a result of 24-h incubation of
HuTu 80 cells with 10–4-10(Im) dicationic surfactant. With an increase
in the concentration of the compound, an increase in the number of
cells at the irreversible late apoptosis stage is observed; 31.37 and
41.89% at the concentrations of the compounds of 5 and 10 μM, re-
spectively. These results are consistent with the literature data, which
show the cytotoxic potential of imidazolium surfactants through in-
duction of apoptosis [60].
3.4. Cytotoxicity of test compounds toward cancer and normal human cell
lines
Due to the fact that amphiphilic compounds have recently been
widely used in cancer chemotherapy for delivery of target agents and
antitumor drugs, we studied the cytotoxic activity of the dicationic
imidazolium surfactants against normal and cancer human cell lines.
Studies of the effect of compounds on normal (non-tumor) cell lines
would assess the safety of the test compounds on the human body,
while discovery of selectivity against cancer cells would enhance cy-
totoxic properties of the delivered antitumor agents. Compounds of the
m-s-m(Im) series were tested for cytotoxicity against normal (Сhang
liver - human normal liver cells) and human tumor cell lines (M-Hela -
epithelioid cervical carcinoma; MCF-7 – human breast adenocarcinoma
cells; HuTu 80 - human duodenal adenocarcinoma). The tested com-
pounds showed high activity against cancer cell lines and moderate
cytotoxicity against normal cells. As can be seen, cytotoxic effect de-
pends both on hydrophobicity of surfactants and spacer length, with the
specificity displayed toward the cell line. The most significant results
were obtained in the case of compound 10–4-10(Im) showing selective
cytotoxicity toward duodenal adenocarcinoma cell line (HuTu 80) and
cervical carcinoma cell line (M-Hela), which were comparable with the
doxorubicin (the reference drug). Selectivity of compounds for cancer
cells is an important criterion to assess the cytotoxic effect. For this
purpose, the selectivity index (SI) was calculated as the ratio between
the IC50 value for normal cells and the IC50 value for cancer cells. The
values for the test compounds are given in Table 3.
3.6. Effects on the mitochondrial membrane potential (Δψm) by the lead
compound
There are two mechanisms for the induction of apoptosis: the ex-
trinsic pathway, through death receptors, and the internal pathway
mediated by mitochondria. The extrinsic pathway triggers apoptosis in
response to external stimuli, during which specific ligands bind at
‘death’ receptors on the surface of the cell membrane. These receptors
are typically members of the Tumor Necrosis Factor Receptor (TNFR)
gene family, such as TNFR1 or FAS. TNFR-associated death domain
(TRADD) and Fas-associated death domain (FADD) trigger the activa-
tion of the zymogenic forms of caspases −8 and − 10, which leads to
downstream caspases activation and the formation of the so-called
death-inducing signal complex (DISC). As a result of these processes,
the essential substrates for cell viability are cleaved, thereby causing
cell death. [61].
In case of mitochondrial apoptotic pathway, cell death occurs as a
result of irreparable DNA damage. For this reason, the cell triggers an
intrinsic apoptotic cascade. The internal pathway of apoptosis induc-
tion is followed by a disruption of the mitochondrial membrane, which
leads to a decrease in its potential, which is a key indicator of the state
of cells [62].
The compounds with SI ≥ 3 are usually highly selective [58]. In this
regard, it can be considered that compound 10–4-10(Im) are highly
selective toward HuTu 80 and M-Hela cell lines. The SI values for these
lines were 7.3 and 3.9, respectively. Doxorubicin and tamoxifen re-
ference drugs were far inferior to the leading compounds in selectivity.
6

A.D. Voloshina, et al.
BBA-GeneralSubjects1864(2020)129728
Table 3
In vitro cytotoxic effects (μM) and selectivity index values (SI) of dicationic alkylimidazolium surfactantsa.
Test compound
Cancer cell lines
Normal cell lines
M-Hela
MCF7
IC50
HuTu 80
IC50
Chang liver
IC50
SI
SI
SI
10–2-10 (Im)
10–3-10 (Im)
10–4-10 (Im)
12–2-12 (Im)
12–3-12 (Im)
10–4-12 (Im)
Doxorubicin
Tamoxifen
41.6
37.4
6.8
3.3
3.1
0.6
1.1
3.9
1.2
1.2
1.0
1.0
1.5
25.7
25.5
25.3
25.7
19.2
25.2
3,0
2.3
2.1
2.4
2.2
1.6
2.1
1.0
1.5
1.0
0.6
0.7
0.5
1.0
1.7
8.4
8.5
3.6
16.6
6.3
10.8
3.0
–
0.5
3.0
4.6
7.3
1.0
2.0
1.2
1.0
25.5
39.1
26.4
16.7
12.6
12.9
3,0
2.1
3.2
2.5
1.4
1.1
1.2
0.7
0.2
0.5
13.6
12.8
12.6
3,0
1.2
1.2
1.0
1.4
0.4
0.8
0.1
0.1
0.2
2.2
0.2
28
2.5
25
42.1
3.5
a
The experiments were repeated for three times. The results are expressed as the mean
standard deviation (SD).
The possibility of apoptosis through the mitochondrial pathway was
assessed by flow cytometry using the JC-10 fluorescent dye (in the
Mitochondria Membrane Potential Kit). In normal cells, JC-10 is accu-
mulated in the mitochondrial matrix, where it forms red-fluorescent
aggregates. However, in apoptotic and necrotic cells, JC-10 diffuses
from mitochondria, turns into a monomeric form, and emits green
fluorescence, which is detected by a flow cytometer [62].
A decrease in the mitochondrial membrane potential of HuTu 80
cells was detected after treatment with compound 10–4-10 (Im), which
become more marked with an increase in its concentration. It is clear
from the results in Fig. 7, that the number of green-emissive cells in-
creases with an increase in the concentration of the test compound, and
vice versa, the content of red-emissive aggregates decreases (Fig. 8).
Thus, the data analysis showed that the leader compound (10–4-
10(Im)) has apoptogenic activity against the cancer cell line (HuTu 80)
and the induction of apoptosis most likely occurs through the internal
mitochondrial pathway.
Fig. 6. Representative histograms for the number of cells (% of the total) at the
early and late stages of apoptosis for the control and experimental groups after
treatment with 10–4-10(Im). The values are presented as mean
SD (n = 3):
(*) P > 0.01 compared to control.
fluoroquinolone antibiotic including methicillin-resistant strains of S.
aureus, while fungicidal activity against C. аlbicans was comparable
with Ketoconazole. The mechanism of the antibacterial effect of the
dicationic surfactants is associated with damage to the cytoplasmic
membrane. The highest antimicrobial activity has been observed in the
case of surfactants containing decyl hydrophobic tail.
4. Conclusion
The antimicrobial, hemolytic and cytotoxic activities of dicationic
imidazolium surfactants with a variable length of a hydrophobic group
and spacer fragment have been studied. It has been shown that the
nature of a hydrophobic tail is the main structural factor affecting the
antimicrobial activity of the compounds, while the length of the spacer
fragment has practically no effect on their biological properties. The
compounds have displayed high antimicrobial activity against a wide
range of test microorganisms and did not have high hemolytic activity.
In the case of Gram-positive bacteria, compounds of the 10-s-10 (Im)
series have shown bactericidal activity at the level of the ciprofloxacin
Cytotoxicity study of dicationic imidazolium surfactants against
normal and tumor human cell lines has demonstrated a selective cy-
totoxic effect of compound 10–4-10 (Im) against HuTu 80 duodenal
adenocarcinoma, the mechanism of which is presumably apoptosis in-
duction via the mitochondrial pathway.
Importantly, MBC and MFC of the leader compounds are markedly
Fig. 5. Apoptosis induction in HuTu 80 cells incubated with 5 and 10 μM of compound 10–4-10 (Im) for 24 h. Quantification of dot plots expressed as percentage of
total cells (mean SD, n = 3).
7

A.D. Voloshina, et al.
BBA-GeneralSubjects1864(2020)129728
Fig. 7. Flow cytometry analysis of M-Hela cells treated with 10–4-10 (Im).
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The authors declare that they have no conflict of interest.
Acknowledgements
This work is supported by the Russian Science Foundation (grant №
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Appendix A. Supplementary data
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