Deep eutectic solvent an efficient reaction medium for the synthesis of chromeno pyrazolo and indazolo phthalazine derivatives

Article abstract of DOI:10.1002/jhet.3713

The volatile, harmful and hazardous solvents have impact on human health, all organisms and environment. To reduce the use of hazardous volatile solvents, there is need to develop the alternative green reaction medium for organic transformations and is th

Full text of DOI:10.1002/jhet.3713

Received: 23 June 2019
Revised: 10 September 2019
Accepted: 22 September 2019
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DOI: 10.1002/ardp.201900180
F U L L P A P E R
Arginolipid: A membrane‐active antifungal agent and its
synergistic potential to combat drug resistance in clinical
Candida isolates
Mrunal Patil1
Preeti Mehta2
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Shashir Wanjare2
Pradeep Vavia1
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Vivek Borse3
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Rohit Srivastava3
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1Department of Pharmaceutical Sciences and
Technology, Institute of Chemical Technology,
Mumbai, India
Abstract
Antifungal drug resistance exhibits a major clinical challenge for treating nosocomial
fungal infections. To find a possible solution, we synthesized and studied the
antifungal activities of three different arginolipids (Nα‐acyl‐arginine ethyl ester)
against clinical drug‐resistant isolates of Candida. The most active arginolipid, oleoyl
arginine ethyl ester (OAEE) consisting of a long unsaturated hydrophobic chain, was
tested for its mode of action, which revealed that it altered ergosterol biosynthesis
and compromised the fungal cell membrane. Also, OAEE was found to exhibit
synergistic interactions with fluconazole (FLU) or amphotericin B (AmB) against
planktonic Candida cells, wherein it reduced the inhibitory concentrations of these
drugs to their in vitro susceptible range. Studies conducted against the C. tropicalis
biofilm revealed that the OAEE+AmB combination synergistically reduced the
metabolic activity and hyphal density in biofilms, whereas OAEE+FLU was found to
be additive against most cases. Finally, the evaluated selective toxicity of OAEE
toward fungal cells over mammalian cells could establish it as an alternative
treatment for combating drug‐resistant Candida infections.
2Department of Microbiology, Seth G.S.
Medical College and K.E.M. Hospital, Mumbai,
India
3Department of Biosciences and
Bioengineering, Indian Institute of
Technology‐Bombay (IIT‐B), Mumbai, India
Correspondence
Prof. Pradeep R. Vavia, Department of
Pharmaceutical Sciences and Technology,
Institute of Chemical Technology, N.P. Marg,
Matunga (E), Mumbai, Maharashtra 400019,
India.
Email: pr.vavia@ictmumbai.co.in
Funding information
Department of Science and Technology,
Ministry of Science and Technology, India,
Grant/Award Number: Inspire Fellowship IF
130868
K E Y W O R D S
arginolipids, biofilms, Candida, drug resistance, membrane disruption, synergism
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1
INTRODUCTION
standard”[5] for antifungal treatment, its use has been limited consider-
ing its nephrotoxic adverse effect and recent reports of resistance.[6,7]
Incidences of fungal infections are expected to increase in the foresee-
able future due to increased numbers of patients with immunodefi-
ciency conditions.[1] Among existing fungal infections, Candida is the
most common cause of opportunistic fungal disease and the fourth
common cause of nosocomial bloodstream infections.[2] Over the
decades, fluconazole (FLU) and amphotericin B (AmB) are being used
as the drugs of choice in the management of candidiasis.[3] Though FLU
has an excellent efficacy–toxicity profile, concomitant with its wide-
spread use and owing to its fungistatic nature, clinical failures
correlating with its elevated minimum inhibitory concentrations (MIC)
have been reported in the past.[4] Conversely to AmB being the “gold
Additionally, the inhibitory concentrations of antifungal drugs have
upsurged due to the unique ability of the Candida species to switch
morphology and form biofilms. These biofilms are inherently tolerant to
high antifungal doses and the host defense mechanism.[8] As a result,
medical practitioners are provoked to use high doses of antifungal
agents or opt for new drugs like echinocandins and second‐generation
triazoles,[9] but unfortunately, high cost, differential pharmacokinetics,
unique predisposition of drug–drug interactions and the unusual
toxicities associated with long‐term therapy restricted their use.[10]
Escalating toxic side effects and evolutionary pressure have incited an
urgent need for developing new antifungal agents, preferentially, a
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Arch Pharm Chem Life Sci. 2019;e1900180.
wileyonlinelibrary.com/journal/ardp © 2019 Deutsche Pharmazeutische Gesellschaft
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PATIL ET AL.
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chemosensitizing agent, which augments the intracellular concentration
of conventional antifungal drugs in the fungal cells, thereby making
them susceptible even at low concentrations. One such category of the
antifungal agent is cationic amino acid conjugated amphiphiles, that has
gained popularity among researchers as next‐generation therapeutic
agents, primarily due to their notable antifungal activity.[11–13] As
studied, the antifungal activity of these amphiphiles is a consequential
outcome of their interaction with a negatively charged fungal cell
membrane, which results in enhanced permeability.[14,15] These agents
have advantages over other synthetic amphiphiles in the context of
their ease of synthesis, low toxicity, biodegradability, and multifunction-
ality and are renewable sources of raw materials, which makes them a
suitable choice for industrial scale‐up.[16,17] Cationic amino acid‐based
amphiphiles especially arginine‐based fatty acids (arginolipids) have
been studied for their antifungal application in biomedical,[18] derma-
tological,[19] and food products[20,21]; however, till date, their proficiency
to overcome drug resistance and their interaction with conventional
antifungal drugs still remains unexplored. We, thereby, hypothesize that
using arginolipid in combination with conventional antifungal drugs
could enhance their potency, which may in turn aid in combating drug
resistance. Hence, to study our hypotheses, we considered synthesizing
three arginolipids (Nα‐acyl‐arginine ethyl ester), namely, decanoyl
arginine ethyl ester (DAEE; Figure 1a), lauroyl arginine ethyl ester
(LAEE, Figure 1b) and oleoyl arginine ethyl ester (OAEE; Figure 1c).
These arginolipids were prepared by conjugating L‐arginine ethyl ester
with three different fatty acid chains. Arginine (Arg) as the head group
was considered in the first place owing to its ability to delocalize the
cationic charge within its guanidinium group and retain it regardless of
being partially buried within fungal membrane protein.[22,23] Addition-
ally, with a perspective of maintaining an optimum balance between
polarity and hydrophobicity, three different fatty acids having a
different chain length and the presence of unsaturation were considered
for conjugating with Arg and the impact of their corresponding
hydrophobicity on antifungal efficacy was evaluated.
separately was evaluated against clinical planktonic Candida cells and
preformed C. tropicalis biofilms.
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2
RESULT AND DISCUSSION
Synthesis
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2.1
Arginolipids were synthesized and characterized using a previously
published protocol by our research group.[24] These synthesized
arginolipids consisted of a single L‐arginine ethyl ester moiety
conjugated to a lipophilic fatty acid. Fatty acids having a variable
chain length and presence of the unsaturation state, namely,
decanoic acid (C10, saturated), lauric acid (C12, saturated), and oleic
acid (C18, unsaturated), were used to yield DAEE, LAEE, and OAEE,
respectively, and the effect of their resulting hydrophobicity on
antifungal activity was evaluated.
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2.2
Antifungal activity of arginolipids
The MIC90 values of arginolipids tested against clinical drug‐resistant
Candida albicans and non‐albicans isolates were found to be
substantially diverse (Table S1). A possible reason for all arginolipids
to exhibit low to moderate antifungal activity could be anticipated due
to the presence of the cationic L‐arginine head group. As studied
previously,[25,26] the cationic guanidinium residues of Arg form stable
electrostatic and hydrogen‐bonding interactions with the anionic
phosphodiester phospholipid content of fungal cell membranes, which
results in pulling the phosphodiester phospholipid through the
hydrophobic part of the membrane, simultaneously dragging along
hydrogen‐bonded water molecules to create a permeable toroidal
pore, thus perturbing the fungal membrane. However, our studies
suggest that the cationic group (polar group) alone is not responsible
for the antifungal activity. The hydrophobicity imparted by the carbon
chain length plays a major role in deciding the potency of antifungal
activity. As observed, MIC90 of OAEE (15.25–62.5 µg/ml) were found
to be comparatively lower than that of moderately active LAEE
(31.25–250 µg/ml) and less‐active DAEE (125–500 µg/ml). Apart from
having a higher alkyl chain length (C18), lower MIC of OAEE to some
The present study explores the antifungal activity of the
synthesized arginolipids on clinical drug‐resistant isolates of Candida
and defining the mechanism of action of the most active arginolipid
(OAEE). Furthermore, the interaction of OAEE with FLU and AmB
FIGURE 1 Chemical structures of the synthesized arginolipids

PATIL ET AL.
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extent could be ascribed to the presence of kinking (fixed bend) within
its hydrophobic carbon chain, which leads to formation of a different
molecular conformation that occupies a greater cross‐section due to
increased motion freedom as it gets inserted into the fungal lipid
bilayer, thereby, easily compromising membrane integrity.[27] On the
contrary, LAEE showed comparatively low MIC than DAEE, possibly
due to the presence of lauric acid (C12), which is said to have the best
balance between hydrophobic and hydrophilic groups, permitting it to
reach sufficient concentrations to interact with acyl chains of
membrane phospholipids.[24] Additionally, Paul and Jeffrey[28] showed
that the critical micelle concentration (CMC) of an amphiphile is
correlated with its antimicrobial property, as near CMC, an equilibrium
is established between the lipid bilayer of cell membrane and a
coexisting micellar pseudo phase in the aqueous medium, which
results in dissolution of several components of the lipid bilayer into the
formed micelles leading to obliteration of cell membrane integrity.
Considering the given report, we investigated CMC values of
arginolipids using the Nile Red encapsulation method (Supporting
Information, Section S.3) and found them to be correlatable with their
respective MIC. The low MIC exhibiting arginolipid, OAEE showed the
lowest CMC (178 µM), followed by LAEE (261 µM) and DAEE
(694 µM).
compromised. Upon entering the cell, PI intercalates itself within
the nucleic acid and emits a fluorescence signal.[31] In the absence of
OAEE (Figure 2c‐i), the percentage of C. albicans SP306 cells with PI
fluorescence was only 0.3%, indicating an intact cell membrane.
After treatment with OAEE: MIC × 0.25, MIC × 0.5 and MIC, the PI
fluorescence was found to be 20.15% (Figure 2c‐iii), 70.6% (Figure 2c‐iv)
and 83.5% (Figure 2c‐v), respectively, signifying dose‐dependent mem-
brane damage. Also, as observed at MIC, OAEE caused membrane
disruption equivalent to that caused by 0.1% of Triton X‐100
(Figure 2c‐ii), which resulted in 86.9% PI‐stained cells.
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2.4
Combination of OAEE with antifungal drugs
against planktonic Candida cells
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2.4.1
Checkerboard and time‐kill assay
The fractional inhibitory concentration index (FICI) calculated from
the checkerboard assays is listed in Table 1. OAEE exhibited
synergistic interaction with both AmB and FLU against all clinical
Candida isolates. No indifferent or antagonist interactions were
observed.
The time‐kill curves (Figure 3) were studied to confirm synergistic
interaction between OAEE and antifungal drugs. It was observed that
the combinations (OAEE+FLU and OAEE+AmB) were relatively
analogous in C. albicans and non‐albicans isolates. Post 8 hr of
incubation, OAEE+FLU resulted in >2 log10 CFU (colony‐forming
units)/ml decrease as compared to FLU alone in test isolates
suggesting a synergistic interaction. Thereafter, at 12 hr, OAEE
+FLU caused 3.029 and 3.134 log10 CFU/ml reduction as compared
to FLU alone in C. albicans SP306 and C. glabrata BC199, respectively.
A reduction of >3 log10 CFU/ml observed with OAEE+FLU combina-
tion indicated that OAEE converted fungistatic activity of FLU to
fungicidal. It was worth noting that unlike OAEE+FLU, the combina-
tion of OAEE+AmB displayed synergistic activity in both the isolates
precisely after 4 hr of incubation (>3 log10 CFU/ml). The OAEE+AmB
combination caused 3.71 and 3.05 log10 CFU/ml reduction as
compared to AmB alone in C. albicans SP306 and C. glabrata
BC199, respectively. It can be inferred that the fungicidal activity
of AmB was dramatically enhanced by the addition of OAEE.
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2.3
Mechanism of action for OAEE
OAEE being the most active antifungal arginolipid, we speculate that it
interacts with fungal cell membranes and might also contribute to
altering the normal sterol biosynthetic pathway. To investigate our
hypothesis, the sterol content of four clinical isolates subjected to
different concentrations (MIC, MIC × 0.5, and MIC × 0.25) of OAEE was
determined spectrometrically.
A typical ultraviolet (UV) spectrum
representing the sterol content of C. albicans SP360 upon treatment
with different concentrations of OAEE is shown in Figure 2a (inset). The
four‐characteristic peak of spectra represents the presence of ergosterol
and 24(28)‐dehydroergosterol (24(28)‐DHE). A dose‐dependent decrease
in height of the absorbance peaks with an increase in the concentration
of OAEE indicated an alteration in the ergosterol biosynthesis path-
way.[29,30] At MIC, a flat spectral curve was obtained, which signified the
absence of detectable ergosterol in extracts. Similar results were
observed among non‐albicans species (Figure 2a). Hence, it can be
interpreted that OAEE exhibits a dose‐dependent effect on fungal sterol
biosynthesis, which may conceivably alter membrane permeability.
To affirm the role of OAEE in membrane perturbation 3,3′‐
dipropylthiadicarbocyanine iodide (DiSC3(5)), a membrane potential‐
dependent probe that rapidly accumulates into the bilayer of
polarized cells causing its fluorescence to quench, was used. As
observed from Figure 2b, addition of OAEE to C. albicans SP306 cell
suspension resulted in rapid release of dye into the medium, causing
an increase in fluorescence intensity. The response indicates that
OAEE depolarizes the fungal membrane in a dose‐dependent manner.
We then performed the propidium iodide (PI) dye exclusion assay to
further support our hypothesis. PI is a membrane‐impermeant dye that is
generally excluded from viable cells unless their cell membrane is
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2.4.2
Molecular dynamic simulation (MDS) studies
Stability of interactions between OAEE+AmB and OAEE+FLU
complexes was assessed by running their simulation trajectories
and analyzing root mean square deviation (RMSD) and root mean
square fluctuation (RMSF) parameters. It was observed that OAEE
+AmB and OAEE+FLU complexes exhibited a hydrogen‐bonding
interaction along with hydrophobic and the van der Waals
interactions. As analyzed, the OAEE+AmB complex showed two
hydrogen‐bonding interactions (Figure 4a): (a) between AmB’s
carbonyl oxygen (C═O) of the carboxylic acid with amine
functionality (–NH–) of the amide group of OAEE, (b) between
AmB’s hydroxyl group (–OH–) of carboxylic acid and amine
functionality (–NH2–) of guanidine in OAEE. In the case of OAEE

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FIGURE 2 (a) Bar graph representing percentage reduction in ergosterol content of treated samples in comparison with control cells
(physiological saline solution treated) expressed as mean standard deviation. The inset figure represents a typical UV spectrophotometrically
obtained sterol profile of Candida albicans SP306. (b) Depolarization of C. albicans SP306 cell membrane using DiSC3(5) assay. (c) Flow cytometric
graph and confocal laser‐scanning microscopy image representing membrane permeabilization of C. albicans SP306 using the propidium iodide dye
exclusion assay for (i) untreated cells, (ii) 0.1% Triton X‐100, (iii) OAEE–MIC × 0.25 (7.81 µg/ml), (iv) OAEE–MIC × 0.5 (15.62 µg/ml), and (v)
OAEE–MIC (31.25 µg/ml) treated cells. For flow cytometry, the data was collected from 25,000 to 35,000 cells. The scale bar = 5 µm. DISC3(5),
3,3′‐dipropylthiadicarbo‐cyanine iodide; MIC, minimum inhibitory concentrations; OAEE, oleoyl arginine ethyl ester; UV, ultraviolet
+FLU, hydrogen‐bonding interaction was seen between the nitrogen
(–N–) of the triazole ring with amine functionality (–NH2–) of
guanidine in OAEE (Figure 4b). Also, as analyzed, the average RMSD
values for OAEE+AmB and OAEE+FLU complex were found to be
5.05 and 6.64 Å, respectively, whereas the average RMSF values for
OAEE+AmB and OAEE+FLU complex were found to be 7.32 and
8.33 Å, respectively. As evident, these values were considerably
lower, which indicated that the complexes so formed are stable.[32]
Interestingly, though an equilibration stage of both the complexes
was achieved smoothly within 2 ns, fluctuations were more evident

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TABLE 1 Minimum inhibitory concentrations and in vitro interactions between OAEE and antifungal drugs (FLU and AmB) against drug‐
resistant clinical isolates of Candidaa
MIC90 of antifungal agents expressed in µg/ml
MIC tested alone
MIC in combination (OAEE + FLU)
MIC in combination (OAEE + AmB)
Isolate
Species
number
FLU
AmB
OAEE
OAEE
FLU
FICI
OAEE
AmB
FICI
Candida albicans
SP237
SP190
SP306
M180
BC372
128
64
1
2
2
4
1
31.25
62.5
3.90
7.81
7.81
3.90
1.95
16
2
0.25 (S)
0.31 (S)
0.37 (S)
0.25 (S)
0.18 (S)
3.90
3.90
3.90
7.81
1.95
0.25
0.25
0.25
0.5
0.37 (S)
0.12 (S)
0.18 (S)
0.25 (S)
0.18 (S)
128
16
31.25
62.5
8
2
64
31.25
8
0.12
Candida tropicalis
BC321
SP258
SP411
M206
M280
32
64
64
64
64
2
0.25
2
0.5
4
31.25
62.5
31.25
62.5
1.95
15.62
1.95
15.62
3.90
2
2
16
8
0.12 (S)
0.18 (S)
0.28 (S)
0.37 (S)
0.31 (S)
3.90
15.62
1.95
7.81
1.93
0.12
0.01
0.25
0.06
0.25
0.12 (S)
0.31 (S)
0.18 (S)
0.25 (S)
0.18 (S)
15.25
4
Candida
BC379
BC324
BC405
BC380
32
64
64
64
8
8
8
8
31.25
62.5
62.5
62.5
15.62
15.62
15.62
15.62
2
2
8
4
0.31 (S)
0.31 (S)
0.31 (S)
0.37 (S)
7.81
7.81
3.90
7.81
1
0.37 (S)
0.2 (S)
haemulonii
1
1
0.18 (S)
0.18 (S)
0.5
Candida glabrata
BC194I
BC194II
BC460
BC426
BC126
BC199
BC571
128
128
64
1
1
4
2
1
4
1
62.5
62.5
31.25
62.5
62.5
62.5
31.25
31.25
7.81
3.90
15.62
15.62
7.81
16
16
4
0.37 (S)
0.25 (S)
0.18 (S)
0.37 (S)
0.18 (S)
0.25 (S)
0.18 (S)
15.62
15.62
7.81
7.81
7.81
7.81
7.81
0.12
0.12
0.5
0.5
0.12
0.5
0.37 (S)
0.37 (S)
0.25 (S)
0.37 (S)
0.25 (S)
0.25 (S)
0.25 (S)
64
8
128
128
64
16
16
4
3.90
0.12
Abbreviations: AmB, amphotericin B; FICI, fractional inhibitory concentration index; FLU, fluconazole; MIC, minimum inhibitory concentrations; OAEE,
oleoyl arginine ethyl ester; S, synergism.
aMIC represented are an average of at least two independent experiments and each experiment was performed in triplicate.
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Atomic force microscopy (AFM)
in OAEE+FLU as compared to OAEE+AmB complex (Figure 4d), thus
suggesting that the OAEE+AmB complex had better stability in
comparison with OAEE+FLU. Hence, it could be inferred that the
stable hydrogen bond and van der Waals interactions between
OAEE and antifungal could be one of the possible reasons for their
enhanced antifungal effect in combination.
2.4.3
Morphological damage was assessed by comparing the diameter versus
height plot of control and treated cells. Roughness expressed as the root
mean square (RMS) value was analyzed using AFM. Control C. albicans
SP306 cells (Figure 5a) displayed a smooth and uniform surface
FIGURE 3 Time‐kill curves of (a) Candida albicans SP306 and (b) C. glabrata BC199 isolate obtained after treating with OAEE, AmB, and
FLU alone and in combination. The concentrations tested against C. albicans SP306 isolate were FLU (64 µg/ml), AmB (1 µg/ml), OAEE
(15.62 µg/ml), and their respective combinations. For C. glabrata BC199 the concentration tested were FLU (64 µg/ml), AmB (2 µg/ml), OAEE
(31.25 µg/ml), and their respective combinations. The experiments were performed three times. Data were expressed as mean standard
deviation. AmB, amphotericin B; CFU, colony‐forming units; FLU, fluconazole; OAEE, oleoyl arginine ethyl ester

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FIGURE 4 (a) Hydrogen‐bonding interactions (yellow line) between OAEE–AmB. (b) Hydrogen‐bonding interactions (yellow line) between
OAEE+FLU. (c) RMSD plot and (d) RMSF plot of OAEE+AmB and OAEE+FLU complexes. AmB, amphotericin B; FLU, fluconazole; OAEE, oleoyl
arginine ethyl ester; RMSD, root mean square deviation
(RMS = 228.7 nm). Cells exposed to a subinhibitory concentration of
OAEE (Figure 5b), FLU (Figure 5c), and AmB (Figure 5d) exhibited
significant (p < 0.05) increase in surface roughness RMS = 300.6, 272.5,
and 335.03 nm, respectively, which suggested minor to moderate surface
deformation (wrinkle formation). However, cells exposed to OAEE+FLU
(Figure 5e) and OAEE+AmB (Figure 5f), displayed an enormous (p < 0.01)
increase in surface roughness RMS = 595.2 and 423.1 nm, respectively as
compared to control, signifying complete deformation of surface integrity.
These deformations are the consensual outcome of the collapsed cell
membranes due to leakage of internal cell content.
and that for AmB was found to be 4–16 μg/ml. The MBEC value of OAEE
ranged between 250 and 1,000 μg/ml.
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2.5.2
Checkerboard assay
The combination of OAEE+FLU exhibited synergistic interactions only
among three out of five preformed biofilms of C. tropicalis isolates. It was
worth noting that despite the synergism, the combinatorial MIC of FLU
(MIC of FLU obtained in combination study with OAEE) ranged between
32 and 128 μg/ml which, as per CLSI M27‐A2 guidelines,[35] falls under
non‐susceptible (i.e dose‐depended/resistant) concentration.[35] There-
fore, the OAEE+FLU combination failed to reduce the MIC of FLU to a
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2.5
Activity of OAEE and its combinations on
susceptible range. Conversely, OAEE+AmB exhibited
a synergistic
preformed C. tropicalis biofilm
interaction among all tested isolates and the combinatorial MIC of
AmB was found to be <1 µg/ml (susceptible range), excepting for C.
tropicalis M280 (4 μg/ml).
C. tropicalis has been recognized as a biofilm producer, surpassing C.
albicans.[33] The ability of C. tropicalis to survive in high salt concentration
and form a low‐carbohydrate‐containing extracellular matrix (ECM)
makes them more resistant toward detachment from the surface.[34]
Thus, OAEE and its combination with antifungal agents were investigated
on the preformed biofilm of drug‐resistant clinical isolates of C. tropicalis.
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2.5.3
Atomic force microscopy
Morphological changes within the treated C. tropicalis M206 biofilms
were analyzed by comparing their resulting roughness (RMS) and
cellular build‐up (maximum height, hmax) against that of control
biofilms. The three‐dimensional (3D) AFM topographical images of
control biofilms (Figure 6a) revealed a dense and heterogeneous
network of yeast, pseudo‐hyphae, and hyphae having RMS = 564.9
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2.5.1
Minimal biofilm eradication concentration
(MBEC)
The preformed biofilms of clinical C. tropicalis isolates were found to be
resistant to FLU and AmB (Table 2). A significant eradication of biofilm
was obtained with FLU, only at high concentration, that is, >512 μg/ml
nm and
hmax = 6.786 µm. Biofilms treated with a subinhibitory
concentration of OAEE (RMS = 609.7 nm and hmax = 6.564 µm) and

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FIGURE 5 AFM tapping mode images of Candida albicans SP306 upon treatment with (a) physiological saline (control), (b) OAEE (15.62 µg/
ml), (c) FLU (64 µg/ml), (d) AmB (1 µg/ml), (e) OAEE+FLU (15.62 + 64 µg/ml) combination, and (f) OAEE+AmB (15.62 + 1 µg/ml) combination. The
data exhibited consist of two‐ and three‐dimensional images along with the height versus diameter curve of the marked cell (black dotted lines).
AFM, Atomic force microscopy; AmB, amphotericin B; FLU, fluconazole; OAEE, oleoyl arginine ethyl ester
FLU (RMS = 591.7 nm and hmax = 6.507 µm) showed few surface
irregularities but no significant reduction in their cellular densities
was observed (Figure 6b and Figure 6c, respectively). Interestingly, a
significant (p < 0.05) decrease in the maximum height of biofilms was
observed (RMS = 400.6 nm, hmax = 5.932 µm) with a subinhibitory
concentration of AmB (Figure 6d), hence, signifying minor sensitiza-
tion to the subjected dose. Biofilms subjected to a combination of
OAEE + FLU (Figure 6e) and OAEE+AMB (Figure 6f) exhibited a
significant (p < 0.01) increase in surface roughness (RMS = 712.0 and
651.7 nm, respectively) and a drastic reduction (p < 0.01) in maximum
height (hmax = 3.406 and 3.120 µm, respectively) was observed.
Though both combinations exhibited a flattened and furrowed
surface; it was OAEE–AMB that distinctively reduced cell density
as compared to OAEE+FLU (visually confirmed).

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TABLE 2 Minimum biofilm eradication concentration (MBEC50) and in vitro interaction between OAEE and antifungal drugs against
preformed biofilms of Candida tropicalis
MIC50 of antifungal agents expressed in µg/ml
MBEC50 tested alone
MBEC50 in combination (OAEE + FLU)
MBEC50 in combination (OAEE + AmB)
Candida tropicalis
species
OAEE
250
FLU
128
256
512
256
512
AmB
8
OAEE
31.25
125
FLU
64
FICI
OAEE
62.5
250
AmB
1
FICI
BC321
SP258
SP411
M206
M280
0.37 (S)
0.62 (I)
0.75 (I)
0.5 (S)
0.25 (S)
0.37 (S)
0.37 (S)
0.25 (S)
0.31 (S)
0.37 (S)
1,000
500
4
128
256
64
0.5
1
8
125
62.5
62.5
62.5
250
4
62.5
62.5
0.25
4
500
16
128
Abbreviations: AmB, amphotericin B; FICI, fractional inhibitory concentration index; FLU, fluconazole; I, indifference; MIC, minimum inhibitory
concentrations; OAEE, oleoyl arginine ethyl ester; S, synergism.
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2.6
Cytotoxicity studies
reducing biofilm density. Additionally, our studies revealed that
OAEE is less cytotoxic and could be considered for its use to combat
drug resistance. However, in the case of antibiofilms therapy, the
application of OAEE could only be extended to implantology as its dose
in combination was still found to be comparatively high.
To justify the selectivity of the OAEE toward fungal cells in
comparison to mammalian cells, we evaluated its cytotoxicity, against
HepG2 cells, an immortalized human liver hepatocellular carcinoma
cell line, and HEK 293 cells, an immortalized human embryonic
kidney cell line. It was observed that the GI50 value of OAEE against
HEK 293 and Hep2G (Figure S14) was found to be 44.3 and >80 µg/
ml, whereas, LC50 values for both the cell lines were found to be
>80 µg/ml, which is approximately more than 1.5 to 2‐fold higher
than the MIC values of OAEE obtained against planktonic clinical
isolates of Candida (15.25–62.5 µg/ml). Presumably, this effect could
be attributed to the fundamental differences between the plasma
membrane of the mammalian and fungal cells. The fungal cell plasma
membrane contains ergosterol, which has a higher percentage of
negatively charged lipids such as phosphatidylinositol, which are
known to interact with antifungal cationic molecules.[11] Hence, it can
be said that OAEE was selectively toxic toward fungal cells over
mammalian cells due to its interaction with ergosterol containing
fungal cells than cholesterol containing mammalian cells.
Briefly, considering the amphiphilic nature of OAEE, we intend to
explore its potential role in entrapping and formulating antifungal
drugs into pharmaceutical emulsions in the near future. Also, we
presume that the OAEE‐assisted polymeric or lipidic pharmaceutical
formulation could further minimize the cytotoxicity profile of OAEE.
Thus, it is expected that this unique combination involving
arginolipids and antifungal agents could serve an alternative to
replace conventional monotherapies and would widen the potential
treatment choice against drug‐resistant Candida species.
|
4
EXPERIMENTAL
General
|
4.1
Roswell Park Memorial Institute medium (RPMI‐1640; with L‐glutamine,
2% glucose with 0.165 mol/l 3‐morpholinopropanesulfonic acid, pH
7.0 0.1), DiSC3(5), resazurin sodium salt, PI, 2,3‐bis(2‐methoxy‐4‐nitro‐
5‐sulfophenyl)‐2H‐tetrazolium‐5‐carboxanilide salt (XTT), FLU, and AmB
was purchased from Sigma‐Aldrich (St. Louis, MO). Sabouraud dextrose
agar (SDA) was purchased from Hi‐Media (India). All other inorganic
chemicals were purchased from Merck (India). Water used was obtained
from the Milli‐Q system, Millipore Corporation. Clinical isolates of
Candida were obtained from routine specimens taken from in‐house
patients of K.E.M. Hospital, Mumbai. These specimens were collected,
isolated, identified, and coded (according to their source of isolation, viz.,
BC: blood culture, SP: sputum, and M: miscellaneous were used as a
prefix) at the Department of Microbiology, K.E.M Hospital and G. S. Seth
Medical College, Mumbai (ethical approval no. EC/OA‐60/2014). Isolates
used in the present study were drug‐resistant against FLU/AmB or both.
Drug resistance was screened using the CLSI M27‐A2 microbroth
dilution protocol.[35] The list of drug‐resistant isolates with their
respective MIC is given in Table 1.
|
3
CONCLUSION
As evident from our experimental work, OAEE exhibited superior
antifungal activity not only due to its cationic head group but also
due to the presence of a higher alkyl chain (lipophilicity) consisting of
a structural kink. However, the role of structural kinking in antifungal
activity needs further investigation. OAEE compromised the fungal
cell membrane by affecting its content. This mechanism indeed was
responsible for OAEE exhibiting synergistic interaction with tested
antifungal drugs. Studies conducted against clinical drug‐resistant
planktonic Candida isolates and the preformed C. tropicalis biofilm
suggested that OAEE fluidizes the fungal cell membrane, permitting
easy entry of antifungal agents, thereby potentially reducing the MIC
of AmB and transforming the fungistatic nature of FLU to fungicidal.
These results were supported by atomic force microscopy analysis,
which revealed that OAEE and antifungal drug combination together
caused cellular content leakage in both planktonic Candida cells and
biofilms. The combination of OAEE+AmB was more effective in
The InChI codes of the investigated compounds together with the
biological activity data are provided as Supporting Information.

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FIGURE 6 AFM tapping mode images of Candida tropicalis M206 biofilms upon treatment with (a) physiological saline (control), (b) OAEE
(125 µg/ml), (c) FLU (128 µg/ml), (d) AmB (2 µg/ml), (e) OAEE+FLU (125 + 128 µg/ml) combination, and (f) OAEE+AmB (125 + 2 µg/ml)
combination. The data exhibited consist of two‐ and three‐dimensional images along with height versus diameter curve of the cell
(black dotted lines)
|
|
Antifungal activity of arginolipids
4.2
Synthesis
4.3
The syntheses of arginolipids, namely DAEE, LAEE, and OAEE, were
carried out using the Schotten Baumann reaction protocol previously
published by our research group.[24] The synthesized arginolipids were
characterized using spectrometric analytical methods (Supporting
Information, Section S.1) and their CMC was determined using the
Nile Red encapsulation method (Supporting Information, Section S.2).
The MIC90 of synthesized arginolipids was determined against drug‐
resistant clinical isolates of Candida using the CLSI M27‐A2 broth
microdilution protocol.[35] Briefly, the primary stock solution of
synthesized arginolipids was appropriately diluted to two‐fold
strength (<1% dimethyl sulfoxide [DMSO]) of the desired concentra-
tions (7.812–1000 µg/ml) using RPMI‐1640 medium. An aliquot

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PATIL ET AL.
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(100 µl) of prepared dilution was added to wells of a sterile, flat‐
bottomed 96‐well microtitration plate (Hi‐Media), separately fol-
lowed by the addition of 100 µl of a freshly prepared mid‐exponential
phase Candida cell suspension (2.5 × 103 CFU/ml). Media and growth
control were included in all plates, and they were incubated in a
shaker incubator operated at 200 rpm at 35°C for 24 hr. Post
incubation, 20 µl of resazurin solution (0.015%w/v) was added to
each well followed by incubation at 35°C for 30 min. The MIC90
values were then determined by measuring fluorescence (emission
590 nm and excitation 560 nm) using a multimode microplate reader
(BioTek Synergy™ H1).[36,37]
(final concentration equivalent to its MIC, MIC × 0.25, and MIC × 0.5)
was added to wells, separately and the fluorescence intensity was
measured every minute for 30 seconds. Untreated and Triton X‐100
(0.1%)‐treated cells served as negative and positive controls.
|
4.4.3
PI dye exclusion assay
An aliquot (100 µl) of OAEE concentration equivalent to its MIC,
MIC × 0.25, and MIC × 0.5 was added to 900 µl of C. albicans SP306
cell suspension (1 × 107 cells/ml) prepared in 5 mM HEPES and 5 mM
glucose (pH 7.4) and incubated at 35°C for 60 min. Cells were
harvested and resuspended in 900 ml of 5 mM HEPES solution. An
aliquot of 100 µl of PI solution (1 mg/ml) was added to resuspended
cells and incubated for another 15 min at room temperature in the
dark. Data was collected using a flow cytometer instrument (Beck-
man Coulter XL‐MCL) equipped with a 15 mV argon laser from
25,000 to 35,000 cells at excitation of 488 nm and emission of 614/
650 nm.[38] Data analysis was performed using FlowJo 8.7 software.
Untreated cells and 0.1% Triton X‐100 (membrane lytic agent)‐[39]
treated cells served as the negative and positive control, respectively.
|
4.4
Mechanism of action for OAEE
|
4.4.1
Sterol quantification method (SQM)
OAEE being the most active arginolipid, its sensitivity towards sterol
biosynthesis process was determined using the SQM protocol
reported by Arthington‐Skaggs et al.[29] This method gives an
absolute in vitro measurement of steady‐state amounts of membrane
ergosterol following the addition of various concentrations of OAEE,
that is, MIC, MIC × 0.5 and MIC × 0.25 to cell suspension of Candida
isolates. Briefly, 24 hr incubated OAEE and physiological saline
solution treated (control) cells were harvested (3,000g, 1 min at 4°C),
weighed and digested with 25%w/v ethanolic potassium hydroxide
solution at 85°C for 1 hr. Post digestion, the non‐saponifiable
lipid content was extracted by the addition of a n‐heptane/sterile
water (3:1) mixture. The n‐heptane layer was then separated and
analyzed using a UV/Visible spectrophotometer (Jasco) for the
presence of ergosterol and the late sterol intermediate 24(28)‐
DHE. The ergosterol content was calculated as a percentage of the
wet weight of cells using the following equations:
The cells were photographed using
a confocal laser‐scanning
microscope (Zeiss LSM780; Germany) and images were processed
on zen black and zen blue software.
|
4.5
Combination of OAEE with antifungal drugs
against planktonic Candida cells
|
4.5.1
Checkerboard assay
The in vitro interaction of OAEE with FLU and AmB, separately
against clinical drug‐resistant isolates of Candida was tested using
a two‐dimensional (2D) broth microdilution checkerboard techni-
que. A four‐fold strength of the desired antifungal drugs and OAEE
test concentrations were prepared in RPMI‐1640 (<1%v/v DMSO).
An aliquot (50 µl) of each antifungal drug (FLU/AmB) concentra-
tion was added to columns 1 to 8 of 96‐well plate, separately
followed by addition of 50 µl of each concentration of OAEE to
%Ergosterol + %24(28)‐DHE = [(A281.5/290) × F]
/pellet weight,
%24(28)‐DHE = [(A230/518) × F]/pellet weight,
%Ergosterol = [%Ergosterol + %24(28)‐DHE] − %24(28)‐DHE,
rows
A to H. Freshly prepared Candida cell suspension
where F is the factor for dilution in ethanol and 290 and 518 are the E‐
values (%/cm) determined for ergosterol and 24(28)‐DHE, respectively.
(2.5 × 103 CFU/ml) was added to each well excluding the media
control wells. Plates were incubated at 35°C for 24 hr. The MIC90
values were determined by using the resazurin assay. The data
obtained from the spectrometric method were analyzed nonpar-
ametrically with the Loewe additivity (LA) model. FICI was
calculated using the following equation.
|
4.4.2
Depolarization of fungal cell membrane
C. albicans SP306 cell suspension (1 × 107 cells/ml) was prepared in
5 mM 4‐(2‐hydroxyethyl)‐1‐piperazineethanesulfonic acid (HEPES),
5 mM glucose and 5 mM KCl solution (1:1:1). To the prepared cell
FICI = Ccomb/MICAalone + Ccomb/MICBalone
,
)
(
)
(
A
B
suspension, DiSC3(5) dye was added to achieve
a 2‐µM final
concentration. An aliquot (190 µl) of suspension containing dye was
then transferred to a black 96‐well plate and preincubated for
20 min. The quenched fluorescence (due uptake of dye in bilayers of
the cell) was measured at excitation, 622 nm and emission, 670 nm,
using a multimode microplate reader. Furthermore, 10 µl of OAEE
where CAcomb and CBcomb are the concentrations of drugs A and B at
isoeffective combinations respectively. MICAalone and MICBalone are the
MIC values of drugs A and B when acting alone. FICI of ≤0.5
represented synergy, values between >0.5 and
4 represented
indifference, and values ≥4 represented antagonism.[40]

PATIL ET AL.
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4.5.2
Time‐kill assay
AC40 TS (BioLever Mini); small nitride cantilever silicon tip f = 110 kHz
and k = 0.09 N/m (Oxford Instruments, UK). Images were processed
and roughness analysis expressed as RMS (i.e., the standard deviation
of the distribution of heights over a 20 × 20‐μm2 imaged area) was
calculated using Igor Pro software (WaveMetrics, Inc).[43]
Time‐kill curves were studied to confirm synergism and fungicidal activity
of combinations. Time‐kill assay of OAEE in the presence and absence of
FLU/AmB was evaluated against one of C. albicans (SP306), and the one
of C. non‐albicans (C. glabrata BC199) species. Candida inoculums
(5 × 105 CFU/ml) were prepared in RPMI‐1640 medium and subjected
to OAEE concentration equivalent to its (0.5 × MIC) alone, FLU/AmB
(0.5 × MIC) alone and combinations, separately. DMSO comprised <1% of
the total test volume. These cultures were then incubated at 35°C with
continuous shaking at 200 rpm. An aliquot of 100 µl was removed at
predetermined time intervals (0, 4, 8, 12, and 24 hr), harvested, washed
three times with sterile physiological saline (PS) and serially diluted in
RPMI‐1640 medium. An aliquot of 20 µl from each dilution was spread
onto the SDA plates and incubated at 35°C for 48 hr to determine the
number of CFU/ml. Synergism and indifference were defined as a
decrease of ≥2 log10 and <2 log10 CFU/ml with respect to the most active
drug, and antagonism was defined as an increase of ≥2 log10 CFU/ml with
respect to the least active drug.[41,42]
|
4.6
Activity of OAEE and its combinations on
preformed C. tropicalis biofilm
|
4.6.1
Minimal biofilm eradication concentration
(MBEC)
An in vitro 96‐well static microplate model was used to prepare
preformed biofilms.[44] Briefly, 100 µl of fresh C. tropicalis suspension
(1 × 106 CFU/ml) prepared in RPMI 1640 medium was introduced in each
well of sterile, polystyrene, flat‐bottomed 96‐well plates (Hi‐Media).
Plates were kept stationary and incubated at 35°C for 24 hr to facilitating
adherence. After 24 hr, the medium was carefully aspirated and
nonadherent cells were removed by washing twice with sterile PS
solution. Each well was replenished with 100 µl of fresh RPMI 1640
medium consisting of a range of test concentrations of FLU (8–1,024 μg/
ml), AmB (1–128 μg/ml), and OAEE (15.625–2,000 μg/ml), separately.
Plates were sealed using parafilm and incubated for 24 hr at 35°C. Post
incubation, plates were washed twice with PS solution and incubated
with 100 µl of XTT‐menadione solution (1 µM prepared in 10‐mM
menadione–acetone solution) in the dark at 35°C for 2 hr. An aliquot of
90‐µl solution from each well was transferred to a fresh 96‐well plate and
the absorbance was recorded at 492 nm.[45] The concentration producing
a 50% reduction in XTT readings, as compared to the drug‐free control,
|
4.5.3
Molecular dynamics simulation studies
To understand molecular level interactions between OAEE–AmB and
OAEE–FLU, molecular simulation studies were carried out using the
Desmond Molecular Dynamics system (Version 2.4; D. E. Shaw
Research, New York, NY, 2010). A Maestro module 2D sketcher in
Schrodinger software was used to draw structures of OAEE, AmB, and
FLU and the structure refinements were done by ligand preparation
(Ligprep module) module. Energy minimization of complexes of OAEE
with AmB and FLU was performed using Macromodel module. The
energy minimized complexes were immersed in the SPC water system
in an orthorhombic box in system builder panel of Desmond. The
system was relaxed before MDS using the default relaxation protocol.
NPT ensemble was used at 300 K and the simulation was run for 10 ns.
Different molecular interactions, as well as their stability over a 10‐ns
period, were studied from stored trajectories by calculating the
parameters such as RMSD and RMSF.
was marked as MIC50
.
|
4.6.2
Checkerboard assay
A 2D (8 × 8) checkerboard microdilution method was used to determine
the interaction of OAEE in combination with antifungal drugs (FLU and
AmB) on prebiofilm. The choice of an appropriate range of drug
concentration was based on MIC findings of individual antifungal agent.
Briefly, 50 µl of two‐fold OAEE concentration ranges were added
vertically (A–H) and 50 µl of two‐fold FLU or AmB dilutions were added
horizontally (1–8) to the preformed biofilms in a 96‐well plate. The
interaction was quantified using XTT–menadione reduction solution. FICI
was calculated and data were interpreted using the LA model.
|
4.5.4
Atomic force microscopy
OAEE and its combination induced morphological alteration were
evaluated using AFM. Briefly, an aliquot of C. albicans SP306 cell
suspension with a cell density of 1 × 105 CFU/ml was introduced in a
six‐well plate (Hi‐Media). The concentrations equivalent to OAEE
(31.25 µg/ml), FLU (64 µg/ml), and AmB (1 µg/ml) were added
separately and in combination (OAEE+FLU and OAEE+AmB) to the
well. PS‐treated cells served as a control. A sterile round glass cover
slide was placed in each well, and the plate was incubated for 6 hr at
35°C. Later, the suspension from each well was carefully aspirated and
the cover slides were washed twice with PS solution. The cover slides
were then air‐dried and fixed using a double‐sided tape to the
microscope holder. AFM (MFP‐3D‐BIO version‐15.112, Asylum
Research, Santa Barbara) was operated in tapping mode using a BL‐
|
4.6.3
Atomic force microscopy
Flat sterile medical‐grade silicone disks, with a total surface area of
314.159 mm2, were placed in a 12‐well, flat‐bottom polystyrene plate
(Hi‐Media). A C. tropicalis M206 cell suspension (3 ml) containing
1 × 106 CFU/ml was pipetted into each well and incubated at 35°C for
24 hr. Disks were washed thrice with 5 ml of PS solution transferred to
another 12‐well microtiter plate containing 3 ml of test concentration;
OAEE (125 µg/ml), FLU (128 µg/ml), and AmB (2 µg/ml) and their
combinations, separately. The disks were incubated for 24 hr at 35°C.
Post incubation, the disks were washed and dried at room

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temperature for 1 hr. AFM analysis was conducted, as mentioned
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4.7
Cytotoxicity studies
In vitro cytotoxicity of OAEE was evaluated against HEK 293 and
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B
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Statistical analysis
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ACKNOWLEDGMENTS
[24] K. Patel, M. Tyagi, J. Monpara, L. Vora, S. Gupta, P. Vavia, J. Nanopart.
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This study was financially supported by the Department of Science and
Technology, India under the Inspire fellowship (IF 130868) received by
Mrunal Patil. Authors are grateful to the SAIF, CRNTS, and the
Department of Biosciences and Bioengineering, IIT, Mumbai, for
providing microscopy analysis facility and expertise. The authors
acknowledge the Advanced Center for Treatment Research and
Education in Cancer (ACTREC), Mumbai, India for the SRB assay.
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CONFLICT OF INTERESTS
The authors declare that there are no conflict of interests.
[34] M. Cavalheiro, M. C. Teixeira, Front. Med. 2018, 5, 28.
[35] Clinical Laboratory Standards Institute, NCCLS Document M27‐A2,
22, 2002, p. 1.
ORCID
Mrunal Patil
http://orcid.org/0000-0002-5722-6567
http://orcid.org/0000-0001-8568-7857
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Pradeep Vavia
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SUPPORTING INFORMATION
Additional supporting information may be found online in the
Supporting Information section.
https://doi.org/10.1002/ardp.201900180