Eudesmic acid-polyoxomolybdate organo-conjugate as novel anticancer agent

Article abstract of DOI:10.1016/j.molstruc.2021.130612

In this work, trimethylated gallic acid (Eudesmic acid, EU) was selected for the synthesis of an organo-conjugate (EU₂POMo) from TRIS modified Anderson-type manganese polyoxomolybdate (POMo) for the first time. EU₂POMo was synthesized through amide bonding between POMo and EU using carbodiimide coupling strategy. Some of the quantum chemical properties of POMo and EU₂POMo beside the DFT and TD-DFT calculations were done using the Gaussian program. The cytotoxicity was studied on breast cancer cell lines (MCF-7 and MDA-MB-231) comparing the Human Umbilical Vein Endothelial Cell line (HUVEC) using the MTT method. The cellular uptake was determined using the ICP-MS method, and the apoptosis value was checked by the flow cytometry technique on the MDA-MB-231 cell line. The structure was approved by FTIR, NMR spectroscopy as well as elemental analysis. Quantum chemical calculations proposed better stability and lower chemical potential for EU₂POMo, and internal energy and dipole moment were higher in the EU₂POMo. Both POMo and EU₂POMo showed reasonable anti-cancer effects on breast cancer cell lines (MCF-7 and MDA-MB-231), and the results were somewhat in favor of POMo. Interestingly, EU₂POMo showed no significant cytotoxicity on the HUVEC and was safer than POMo. Cellular uptake (33.5% versus 29.2%) and apoptosis value (28% versus 15%) in the case of EU₂POMo were slightly better than POMo. In conclusion, this study aimed to introduce a novel, potent and safe anti-cancer Anderson type polyoxometalate to cancer studies. Based on results, this conjugate has sufficient potential for further cancer chemotherapy assessments, specifically breast cancer.

Full text of DOI:10.1016/j.molstruc.2021.130612

Journal of Molecular Structure 1240 (2021) 130612
Contents lists available at ScienceDirect
Journal of Molecular Structure
journal homepage: www.elsevier.com/locate/molstr
Eudesmic acid-polyoxomolybdate organo-conjugate as novel
anticancer agent
Maryam Ramezani-Aliakbaria, Azim Soltanabadib, Hojjat Sadeghi-aliabadia,
Jaleh Varshosazc, Bahram Yadollahid, Farshid Hassanzadeha, Mahboubeh Rostamie,∗
a Department of Medicinal Chemistry, School of Pharmacy and Isfahan Pharmaceutical Sciences Research Center, Isfahan University of Medical Sciences,
Isfahan, Iran
b Department of Physical Chemistry, Faculty of Chemistry, Razi University, P.O. Box: 6714967346, Kermanshah, Iran
c Novel Drug Delivery Systems Research Center and Department of Pharmaceutics, School of Pharmacy and, Isfahan University of Medical Sciences, Isfahan,
Iran
d Department of Chemistry, University of Isfahan, Isfahan, Iran
e Novel Drug Delivery Systems Research Center and Department of Medicinal Chemistry, School of Pharmacy and Pharmaceutical Sciences and, Isfahan
University of Medical Sciences, Isfahan, Iran
a r t i c l e i n f o
a b s t r a c t
Article history:
In this work, trimethylated gallic acid (Eudesmic acid, EU) was selected for the synthesis of an organo-
conjugate (EU2POMo) from TRIS modified Anderson-type manganese polyoxomolybdate (POMo) for the
first time.
Received 10 February 2021
Revised 28 April 2021
Accepted 29 April 2021
Available online 8 May 2021
EU2POMo was synthesized through amide bonding between POMo and EU using carbodiimide coupling
strategy. Some of the quantum chemical properties of POMo and EU2POMo beside the DFT and TD-DFT
calculations were done using the Gaussian program. The cytotoxicity was studied on breast cancer cell
lines (MCF-7 and MDA-MB-231) comparing the Human Umbilical Vein Endothelial Cell line (HUVEC) us-
ing the MTT method. The cellular uptake was determined using the ICP-MS method, and the apoptosis
value was checked by the flow cytometry technique on the MDA-MB-231 cell line.
Keywords:
Anderson type manganese
polyoxomolybdate (POMo)
Organo-conjugate
Eudesmic acid (EU)
Apoptosis
The structure was approved by FTIR, NMR spectroscopy as well as elemental analysis. Quantum chemical
calculations proposed better stability and lower chemical potential for EU2POMo, and internal energy and
dipole moment were higher in the EU2POMo. Both POMo and EU2POMo showed reasonable anti-cancer
effects on breast cancer cell lines (MCF-7 and MDA-MB-231), and the results were somewhat in favor
of POMo. Interestingly, EU2POMo showed no significant cytotoxicity on the HUVEC and was safer than
POMo. Cellular uptake (33.5% versus 29.2%) and apoptosis value (28% versus 15%) in the case of EU2POMo
were slightly better than POMo.
Anticancer agent
Quantum chemical calculations
In conclusion, this study aimed to introduce a novel, potent and safe anti-cancer Anderson type polyox-
ometalate to cancer studies. Based on results, this conjugate has sufficient potential for further cancer
chemotherapy assessments, specifically breast cancer.
© 2021 Elsevier B.V. All rights reserved.
1. Introduction
and the best applicable method for treatment in this regard. How-
ever, the general toxicity and side effects of non-selective function
Cancer is an abnormal growth and uncontrollable cell division
in a specific part of the body that rapidly spreads and hurts other
organs and other tissues [1]. Breast cancer is one of the most com-
monly diagnosed cancers in women impacting 2.1 million women
each year and causes the most considerable number of cancer-
related deaths among them. Chemotherapy is the most interesting
in these drugs (such as cisplatin, doxorubicin, etc.), drastically re-
duce patients’ quality of life [2,3]. So the researchers always try
to introduce new drugs with better potencies and minimized side
effects on healthy cells.
Polyoxometalates (POMs) with well-documented activities in
cancer treatment have gained significant interest in this context.
POMs are a class of anionic clusters composed of transition metals
in the highest oxidation state (W(VI), Mo(VI), V(V)), and oxygen
atoms [4]. These metal-oxo clusters with variable physicochem-
∗
Corresponding author.
E-mail address: m.rostami@pharm.mui.ac.ir (M. Rostami).
https://doi.org/10.1016/j.molstruc.2021.130612
0022-2860/© 2021 Elsevier B.V. All rights reserved.

M. Ramezani-Aliakbari, A. Soltanabadi, H. Sadeghi-aliabadi et al.
Journal of Molecular Structure 1240 (2021) 130612
–
ical features have emerged in different fields such as catalysis,
nanoscience, macromolecular crystallography, and medicine [5,6].
The simple and inexpensive chemical preparation and modifi-
cation make POMs valuable candidates as anti-cancer agents. Since
Yamase’s first study in 1988 [7] on the anti-cancer effects of POMs,
to date, many research and review articles [8–13] have confirmed
the anti-cancer activities of these compounds. Therefore, these
compounds appear to have the potential to be considered as anti-
cancer drugs clinically. However, one of the biggest problems in
entering POMs into clinical evaluations is the high intrinsic toxicity
of these compounds to normal cells and healthy tissues. Although
this toxicity is still lower than many other anti-cancer drugs, the
high IC50 about mM in POMs has made it difficult for these com-
pounds to enter the clinic. Therefore, researchers in this field have
always tried to reduce IC50s besides lowering the toxic effects of
these compounds.
tetrahydrate, glacial acetic acid (GAA), N Hydroxy succinimide
(NHS), Tris(hydroxymethyl)aminomethane, 3-(4,5-dimethylthiazol-
2-yl)−2,5-diphenyl tetrazolium bromide (MTT), 1-Ethyl-3-(3-
dimethylaminopropyl) carbodiimide hydrochloride (EDC) were
purchased from Sigma-Aldrich Company (Germany). The MCF-7,
MDA-MB-231, and HUVEC cell lines were supplied by the Pasteur
Institute (Iran). Trypsin/EDTA, Streptomycin/penicillin, FBS and
RPMI 1640 were purchased from PAA Company (Australia). All
of the other necessary materials and solvents were provided by
similar sources and used without further purification.
2.2. Instrumentation
(1H &13C) NMR spectra were recorded on NMR (Bruker Biospin
AC-80, 400 MHz, Germany) spectrometer with deuterated DMSO
as solvents at 25 °C and chemical shifts have been recorded in
ppm units relative to tetramethylsilane (TMS). Fourier-transform
infrared (FT-IR) spectra (KBr pellet) were recorded on a FT-IR
Gallic acid (GA), as a naturally occurring phenolic structure,
is well known for the high biological activity, such as antiviral
and anti-cancer effects [14]. The cytotoxic effects of GA and its
derivatives on various cancerous cell lines were approved [15]. Eu-
desmic acid (EU), the tri-methoxylated form of GA, as presents
in colchicine (a tubulin inhibitor anti-tumor drug), is the most-
studied mimetic moiety to dedicate the anti-cancer activity to a
chemical structure. Notably, 3,4,5 tri methoxyphenyl (TMP) is a
chief moiety in tubulin inhibitor anti-cancer drug structures, which
induces intense anti-cancer activity [16].
(6300, JASCO, Japan) instrument in the range of 350–7800 cm−1
.
UV-Visible spectral analysis was carried out by use of UV-mini-
1240, (Shimadzu, Kyoto, Japan) spectrophotometer. The absorbance
of each well in MTT assay was measured using a microplate reader
of Awareness Statfax 2100.
The cells were assessed using BD FACSCalibur flow-cytometer
(Becton Dickinson, USA) and the FlowJo-V10 software was used to
analysis of data. Elemental analyses (C, H and N) were conducted
on a Elementar, Vario EL (III) CHNSO elemental analyzer, for this
the sample was dried at room temperature under a vacuum of
10−4 mmHg overnight.
Molecular hybridization has been well experienced as an effec-
tive strategy in medicinal chemistry [17], in most cases, the hybrid
molecules emerge integrated biological effects of their sub-units.
In this regard, different pharmacophores, organic or inorganic moi-
eties, could be hybridized rationally to get the desired effects. It
seems the hybridization could be an ideal strategy to control the
inherent cytotoxicity of POMs [18,19] and make them more selec-
tive.
Mn, and Mo were determined by an inductively coupled plasma
ICP-OES spectrometer (Perkin Elmer, Optima 7300DV). To deter-
mine the molybdenum content in cellular uptake, study was again
carried out by Agilent 7800 ICP-MS.
During the last decades, there are valuable reports on the anti-
cancer activity of organic hybrid POMs [8,9] both in vitro and in
vivo.
2.3. Methods
2.3.1. Chemical synthesis of EU2POMo conjugate
Some of these hybridization approaches, such as binding to
amino acids, Biotin [20,21], tocopherol succinate [22], peptides
[23], bisphosphonate [9], etc. have been pioneers in this regard. It
should be noted that the modification through the covalent bond-
ing is more interesting than physical or ionic hybridization because
of better biological stability and more selectivity [8,19] of POMs
conjugates.
Synthesis of (TBA)4[α-Mo8O26]: Firstly, Sodium molybdate dihy-
•
drate (Na2MoO4 2H2O) (5 g, 20.7 mmol) was dissolved in 12 mL
distilled water, and the solution was acidified to pH 3 by HCl (6 N).
The reaction mixture was stirred vigorously for several minutes
then a solution of TBAB in water (3.34 g, 10.4 mmol) in 10 mL
water was added to it. After stirring for 10 min, the white pre-
cipitate was filtered and washed successively with distilled wa-
ter, ethanol, and diethyl ether. The product, a white powder, was
dissolved in acetonitrile, and the colorless cubic crystals were ob-
tained by keeping them at −10°C overnight. The product was dried
under vacuum and stored for the final step (C) [24]. The elemental
analysis was used beside the FTIR analysis for structure approval.
Elemental analysis: calculated for C64H44N4Mo8O26: C, 35.70; H,
6.74; N, 2.60; Mo, 35.64. Found: C, 35.62, H, 6.81; N, 2.56; Mo,
35.63.
Following our recent studies and interests, we aimed to evalu-
ate the synergistic effect of the EU on the cytotoxicity of an Ander-
son type polyoxomolybdate (POMo). As explained, Eudesmic acid
is found in the chemical structure of anti-cancer compounds, es-
pecially tubulin inhibitors, and therefore we expected that binding
of EU to the POMo structure could enhance the anti-cancer effects
of the hybrid conjugate. So, EU2POMo conjugate was synthesized
using an amide bonding strategy, and the cytotoxicity of this novel
conjugate was studied on two types of cancerous cell lines beside
the HUVEC normal cells by MTT assay. Furthermore, the value of
the apoptosis pathway in cytotoxicity effect was studied quantita-
tively. As a complementary evaluation, to compare quantum chem-
ical properties and stability of prepared conjugate (EU2POMo) with
the initial POMo, quantum chemical calculations and TD-DFT were
done using the Gaussian program.
Synthesis of Mn (CH3COO)3.2H2O:
A
solution of Mn
(CH3COO)2•2H2O in GAA (0.16 g/mL) was heated to 110°C,
and 0.68 g of KMnO4 was added in small portions for 20 min.
The reaction mixture was cooled, poured in water, and left to
crystallize overnight. The brown product was air-dried and stored
for the final step (C) [25].
Synthesis of [N(C4H9)4]3[MnMo6O18 {(OCH2)3CNH2}2](POMo): At
the final step to get the POMo, a mixture of (TBA)4[α-Mo8O26
]
2. Experimental section
(8 g, 3.7 mmol), Mn (CH3COO)3 .2H2O (1.49 g, 5.6 mmol), and TRIS
(1.56 g, 12.8 mmol) in150 mL acetonitrile was refluxed for 16 h.
The orange solution was filtered to eliminate any precipitates, the
large orange crystals were obtained by ether diffusion of filtrate for
a long time and dried under vacuum (8). The chemical structure
was studied and approved by FTIR, and 1H NMR spectroscopies as
2.1. Materials
Sodium molybdate (Na2MoO4•2H2O), Tetrabutylammonium
bromide (TBAB), Eudesmic acid (EU), Manganese (II) acetate
2

M. Ramezani-Aliakbari, A. Soltanabadi, H. Sadeghi-aliabadi et al.
Journal of Molecular Structure 1240 (2021) 130612
well as by the elemental analysis by CHNS and ICP-OES methods
[20]. The elemental analysis was used beside the 1H NMR, FTIR,
and UV–visible analysis for structure approval. Elemental analysis:
calculated for C56H124MnMo6N5O24: C 35.73%; H 6.64%; N 3.72%,
Mn 2.92%, Mo 30.59%; found experimental C 35.73%; H 6.73%; N
3.62%, Mn 2.86%, Mo 30. 45%.
and incubated for 24 h at 37 °C. POMo and EU2POMo solutions
with a 200 μg/mL concentration were treated on cells, and incu-
bation was followed for the next 24 h at the same condition. After
that, the cells were washed three times with cold PBS and then
harvested using Trypsin. The apoptosis rate was detected accord-
ing to the operation steps of the Annexin V-FITC PI (Propidium Io-
dide) apoptosis assay kit (IQ product, Netherlands) in the dark for
15 min at room temperature. The apoptosis level was determined
using FACS Calibur flow cytometer, and just the single cells were
gated for fluorescence analysis [35].
Synthesis of EU2POMo: EU (0.4 g, 2 mmol) was activated by
EDC/NHS (0.41 g, and 0.23 g respectively, 2 mmol) in dried ace-
tonitrile for 24 h under N2 atmosphere at room temperature. The
resulting solution was filtered and was added into the solution
of POMo (1.88 g,0.001 mol) in dried acetonitrile, and the reaction
mixture was stirred for 24 h at room temperature to get EU2POMo
[26]. The final solution was first dried under reduced pressure to
get the product and then washed thoroughly with the cold ethanol
to get the purified product. The chemical structure was studied
and approved by FTIR and (1H, 13C) NMR, UV-visible spectroscopy
and complete elemental analysis by CHNS and ICP-OES methods.
Elemental analysis: calculated for C76H146 MnMo6N5O32 (Mol.Wt:
2272.56 g/mol): C, 40.17; H, 6.48; Mn, 2.42; Mo, 25.33; N, 3.08;
found experimental C, 40.08; H, 6.97; Mn, 2.47; Mo, 25.49; N, 3.18;
2.3.6. Cellular uptake analysis
The cellular uptake of EU2POMo in comparison to POMo was
checked on the MDA-MB-231 cell line by ICP-MS. The MDA-MB-
231 breast cancer cells were grown in flasks at 37 °C in a 5%
CO2 atmosphere until at least 70% of confluency. The medium was
removed and replaced with 14 mL RPMI-15% FBS and 1% DMSO
containing 90 μg/mL concentration of POMo, and EU2POMo. Af-
ter 24 h incubation time at mentioned condition, the medium was
removed, and the cells were washed with 10 mL PBS then de-
tached by trypsinization and re-suspended in 10 mL PBS (pH 7.4).
The suspension of cells was centrifuged (2000 rpm, 5 min), and
the isolated pellets were washed twice with ice-cold PBS. The iso-
lated pellets were re-suspended in 500 μL deionized water (cells
were counted before lysis) and the cells lysed using a sonotrode.
For protein determination by the Bradford method, an aliquot of
each lysate samples was stored at −20 °C. The lysates were stabi-
lized by the addition of 15 μL of HNO3 (10%)–HCl (30%) and 15 μL
of Triton X-100 (1%) for their molybdenum content determination
by ICP-MS. The cellular concentration of Mo was expressed as μg
per each mg of cellular protein [12, 36].
2.3.2. Computational method
The quantum chemistry calculations have been used to deter-
mine the geometry optimization and electronic structure of POMo
and EU2POMo. The geometry optimizations were performed at the
B3LYP [27,28] level of theory. The 6–31 G basis set used for all
atoms, and DGDZVP [29] for the Mn and Mo. All calculation calcu-
lated by Gaussian 09 program.
2.3.3. Stability of EU2POMo
For this purpose, a solution of EU2POMo (40 μg / mL) were pre-
pared at pH=7.4 in PBS-1% DMSO. Upon mixing with PBS, the clear
solution was retained. The solutions were scanned immediately, af-
ter 72 and after 96 h by UV–Vis spectrophotometer [30,31].
2.3.7. Statistical analysis
All data were analyzed using one-way ANOVA using SPSS soft-
ware (the version of 21) followed by student t-test to evaluate the
difference between groups or by Post Hoc LSD test for more than
two groups. The p-value lower than 0.05 was considered as a sig-
nificant difference between averages.
2.3.4. In vitro cell viability assay
Two types of breast cancer cell lines comprising MCF-7 and
MDA-MB-231 beside the normal cell line (HUVEC) were selected
for in vitro cytotoxicity evaluations. The cells were cultured at
the standard condition of 10% fetal bovine serum (FBS) and 1%
penicillin-streptomycin at 37 °C in an atmosphere of 5% CO2, then
were retained in RPMI-1640 (GIBCO) medium for following cyto-
toxicity evaluation using MTT protocol [32]. Typically, stock solu-
tions were prepared at a 500 μg/mL concentration in phosphate-
buffered saline (PBS). Cells at a density of 5 × 103 per well were
seeded, then incubated at the same condition as culturing step
for 24 h. Various concentrations of the test groups (POMo, and
EU2POMo) ranging from 25, 50, 100, 200 μg/mL were treated reg-
ularly on MCF-7, MDA-MB-231, and HUVEC Cells plates. After in-
cubation for 72 h, the medium was removed, and 20 μL of MTT
solution (5 mg/mL) was added to each well, and incubation was
continued for 4 h. After that, the medium was replaced with 150
μL DMSO to solubilize the purple formazan precipitates, and the
absorbance was read using a microplate reader at 570 nm. The cell
viability was calculated using the following equation [33];
3. Results and discussion
3.1. Synthesis of EU2POMo
Eudesmic acid is known as the trimethylated derivative of gal-
lic acid, which has been considered a significant component in the
structure of tubulin inhibitor compounds in the field of medici-
nal chemistry [14]. This structural component is found in known
molecules such as combretastatin A4, podophyllotoxin, colchicine,
and phenstatin, which can bind to the colchicine acceptor site [15].
For this reason, the participation of this structural part in molec-
ular designs based on the structure-reactivity relationship in the
tubulin polymerization inhibition strategy is well known and usu-
ally leads to the improvement of anticancer activities in the final
compounds. Therefore, the binding of this moiety to the Anderson-
type manganese polyoxomolybate seemed to be a powerful strat-
egy in designing a cytotoxic agent.
A − A
(
(
)
)
t
b
b
Cell Survival % =
× 100
(1)
A − A
c
In recent years, Anderson-type polyoxometalate compounds
have received much attention with the possibility of inserting
TRIS-like groups into them [37], which can provide suitable func-
tional groups for modifying the structure of polyoxmetalates.
Among these, Anderson-type manganese polyoxomolybdate has
been considered by many research groups for different purposes.
This polyoxomolybdate has shown good anticancer properties, and
in numerous evaluations, its hybrid structures have provided new
efficiencies and capabilities.
in which At, Ab and Ac represent mean absorbance of the treat-
ment, blank and negative control, respectively [34]. For each treat-
ment, the average of 9 runs were considered, and results were
given as Mean
SD.
2.3.5. Flow cytometry analysis of cell apoptosis
To verify the apoptosis pathway, briefly, MDA-MB-231 cells
were seeded in a 12-well plate with a density of 104 cells/well
3

M. Ramezani-Aliakbari, A. Soltanabadi, H. Sadeghi-aliabadi et al.
Journal of Molecular Structure 1240 (2021) 130612
Fig. 1. Chemical procedure for synthesis of EU2POMo from Na2MO4.
With these explanations and following our recent studies
[22,38], we aimed to achieve synergistic effects by synthesis a
new cytotoxic compound from hybridization eudesmic acid with
Anderson-type manganese polyoxomolybdate (POMo).
For this purpose, [TBA]4[α-Mo8O26] was synthesized simply
according to the earlier reports as the essential core of POMo.
After the addition of Mn(OAc)3 and TRIS to [TBA]4[α-Mo8O26],
[TBA]3[MnMo6O18 {(OCH2)3CNH2}2](POMo) was isolated as orange
crystals in 80% yield [39]. The reaction of POMo with 2 moles of EU
gives in hand the final conjugate EU2POMo as a pale orange pow-
der (Fig. 1). Based on available sources, in the chemical scaffold
of manganese Anderson type polyoxomolybdate, six edge-sharing
octahedral MoO6 units arrange themselves around a core of the
MnO6, build an Anderson type structure. The TRIS moieties are in-
serted into the scaffold via its alkoxy groups on the Mn (III) ion;
in this manner, two amine groups of TRIS are oriented outwards
and are available for further modification of POMo. The organic
molecules can cover both sides of the planar hexagon polyoxo-
molybdate structure via the chemical bonding with amine groups
of TRIS symmetrically or unsymmetrically [39].
Fig. 2. The optimized geometries of a: POMo and b: EU2POMo.
As shown in Fig. 1, we utilized both amine groups of POMo
for the conjugation to the EU. The amidation reaction between EU
and POMo was carried out through the carbodiimide strategy us-
ing EDC/NHS reagents [40]. Purification through precipitation and
recrystallization yields the pure final product in an almost quanti-
tative yield.
Table 1
The Thermodynamic energies ꢀU° ꢀH° and ꢀG° of
f,
f
f
POMo and EU2POM species at B3LYP/6–31G-DGDZVP level
of theory (6–31 basis set for C, N, O, atoms and
DGDZVP basis set for Mn & Mo atom).
G
H
Compounds
ꢀU° / eV
ꢀH° / eV
ꢀG° / eV
f
f
f
3.2. Computational assessments
POMo
−375.321
−646.204
−376.749
−648.856
−355.991
−610.923
EU2POMo
The chemical structure of POMo and EU2POMo conjugate was
optimized using Gaussian software protocols, and also, some
physicochemical properties were studied using quantum chemical
calculations. These results helped us to have a prediction on some
of the structural features and reactivity.
The electric dipole moment (μ0) is an essential molecular pa-
rameter that plays an essential role in dipole-dipole interactions
and other applications in solids and liquids. Table 2 shows the cal-
culated values of μ0 (in Debye) for POMo and EU2POMo; accord-
ing to Table 2, the dipole moment of EU2POMo is more signifi-
cant than that of POMo. The higher dipole moment in EU2POMo
causes EU2POMo to dissolve more in polar solvents and interact
more with water and polar liquids.
The optimized geometries of EU2POMo and POMo, obtained by
the Gaussian 09 program, are presented in Fig. 2. The geometry op-
timizations were performed at the DGDZVP level for Mo atoms and
B3LYP/6–31 G level for all others atoms. The obtained ꢀU° values
were compared to examine the stability of POMo and EU2POMo re-
lated structures. The more negative the ꢀU° level of a compound,
the more stable it is. Table 1 shows the ꢀUf°, ꢀHf° and ꢀGf° for
POMo and EU2POMo, as can be seen, the internal energy is in
the order EU2POMo < POMo. Therefore, the EU2POMo conjugate
is about twice more stable than the initial POMo; these results are
in good agreement with those obtained by Schönweiz et al. [41], at
least in the case of POMo.
The molecular orbital theory is used routinely to determine the
stability and activity of molecules. Various values such as HOMO-
LUMO gap (Egap), global hardness (or chemical) (η), chemical po-
tential (μ) and electrophilicity (ω) can be calculated in this frame-
work [42]. These values can be calculated from the following equa-
tions using the highest occupied molecular orbital (HOMO) and the
4

M. Ramezani-Aliakbari, A. Soltanabadi, H. Sadeghi-aliabadi et al.
Journal of Molecular Structure 1240 (2021) 130612
Table 2
Energies of HOMO and LUMO, HOMO-LUMO energy gap (Egap), chemical hardness (η), chemical
potential (μ), electrophilicity (ω) and dipole moment (μ0).
Compounds
E
HOMO/eV
ELUMO/eV
Egap/eV
η/eV
μ/eV
ω/eV
μ0 / Debye
POMo
1.169
0.791
3.925
3.539
2.755
2.748
1.377
1.374
−2.547
−2.165
4.468
3.222
1.569
4.196
EU2POMo
Fig. 3. Topology of HOMO & LUMO orbitals for POMo and EU2POMo.
lowest unoccupied molecular orbital (LUMO).
samples [38, 39]. Spectral data for [TBA]4[α-Mo8O26] and POMo
are provided as follow:
[TBA]4[α-Mo8O26]; FTIR (KBr): ν (cm−1) (500–1000 cm−1), 954
(m), 942 (m), 923, (Mo=O, vs), 912 (vs), 906 (s), 852 (m), 807 (vs),
664 (vs), 560 (m).
E
LU MO − EHOMO
(
)
η =
(2)
(3)
2
E
HOMO + ELU MO
(
)
μ = −
[TBA]3[MnMo6O18 {(OCH2)3CNH2}2] (POMo); FTIR (KBr):
ν
2
(cm−1), 3448 (N H str., br), 2960 (C Hstr., s), 2934 (C Hstr., s), 1608
–
–
–
μ2
2η
–
(N Hdef., w), 1479 (s), 1381 (m), 1040 (s), 939 (s, Mo=O), 917 (s,
ω =
(4)
Mo=O), 900 (s, Mo=O), 663 (s, Mo-O-Mo); 1H NMR (400 MHz,
DMSO-d6): 0.94 (t, 36 H), 1.32 (m, 24 H), 1.57 (m, 24 H), 3.12 (m,
24 H), 61.8 (s, 12 H); UV–Vis. (CH3CN): λmax, 216 nm (O2− to Mo6+
charge transfer) [23].
Table 2, shows the calculated HOMO, LUMO, η, μ and ω for
POMo and EU2POMo. According to this table, the energy of the gap
(HOMO-LUMO) in EU2POMo is slightly larger than that of POMo.
Also based on the Table 2, the chemical potential of EU2POMo
is greater than that of POMo which would be a great prediction
for reactivity of EU2POMo compare to the POMo. The spatial dis-
tributions of HOMO and LUMO molecular orbitals for POMo and
EU2POMo, besides the calculated energy levels, are shown in Fig. 3.
According to these shapes, we find that the shape of these orbitals
is almost the same for both compounds, and that in both com-
pounds, the major orbital density is concentrated on the central
core of the POMo.
The structure of EU2POMo was characterized by FTIR spec-
troscopy (Fig. 4-left), UV-visible spectroscipoy (Fig. 5-down), 1H
NMR spectroscopy (Fig. 6-up), 13C NMR (Fig. 6-down), and com-
plete elemental analysis (using CHNS & ICP-OES), the results are as
follow:
EU2POMo conjugate; FTIR (KBr): ν (cm−1) 3324 (w, br), some
medium weight bands below 3000 cm−1, 1632.38 (m, amide C=O
of EU), 1479 (m), 1041 (s), 939 (s, Mo=O), 917 (s, Mo=O), 900 (s,
Mo=O), 663 (s, Mo-O-Mo). 1H NMR (400 MHz, DMSO-d6): 1.07
(t, 36 H, POMo), 1.33–1.35 (m, 24 H, POMo), 1.5–1.8 (m, 24 H,
POMo), 3.23 (m, 24 H, POMo), 7.33 (d, J = 4.4, H Ar. EU), 8.53
(bs, 2H, newly formed amide NH), 62.20 (s, 12 H, POMo); 13C
NMR (100 MHz, DMSO-d6): 175.37 (C=O), 152.75 (=C-OMe, meta-
position), 143.06 (=C-OMe, para-position), 137.64 (=C-C=O), 123.17
In the following section, the characterization of the EU2POMo
structure will present beside the predicted data to approve the
successful chemical synthesis of EU2POMo.
The structure of POMo has been constructed in two steps, as
described earlier and due to the existence of numerous valid re-
ports for this structure, only FTIR, 1H NMR, and elemental analy-
sis results were checked and verified with comparison to standard
+
–
–
(=C-H), 84.99 (-CH2 O (TRIS)), 74.32 (N C(TRIS), 60.19 (-N -CH2-),
5

M. Ramezani-Aliakbari, A. Soltanabadi, H. Sadeghi-aliabadi et al.
Journal of Molecular Structure 1240 (2021) 130612
Fig. 4. Experimental FTIR (Left) & Predicted IR (Right) spectrums for POMo & EU2POMo.
greater intensities. The POMo has a maximum absorption at the
frequency of 705 cm−1, but the EU2POMo has maximum absorp-
tion at a wavenumber of 709 cm−1; these wavelengths are related
to the Mo-O bond stretching frequencies. Adding the aromatic
rings causes the absorption to shift to the higher wavenumbers
and the intensity of absorption increases. The change in higher
wavelengths and increasing absorption intensity for EU2POMo is
logical because of the addition of aromatic rings, increases the μ0
(dipole moment), and the intensity of absorption per frequency is
proportional to the μ changes. Due to the more significant dipole
moment (μ) in the EU2POMo, this compound has a higher adsorp-
tion intensity than the POMo. As will discuss in the following sec-
tion, there is a similar pattern for experimental conditions for com-
paring POMo and EU2POMo IR spectra.
With an in-depth look at the experimental FTIR spectrum
(Fig. 4-left) of the final conjugate compare to the POMo, we find
–
some changes after the conjugation. For example, N H stretch-
ing frequency has somewhat decreased from 3448 cm−1 to 3324.7
cm−1, the carbonyl group stretching frequency has been added to
spectra with a change in location from 1719 cm−1 in EU to the
lower wavenumbers around 1632–1680 cm−1 in EU2POMo due to
the conjugation, this spectral area is the characteristic feature of
aromatic amide conjugates. Furthermore, those characteristic fea-
tures of EU moiety, specifically benzene ring (CH stretching vibra-
tion), has emerged their properties in the FTIR in the region of
>3000 cm−1. Similarly, an increase in the intensity of bands be-
fore 3000 cm−1, which belongs to CH3 group stretching modes,
again approves the presence of EU moiety. Finally, those vibrations
around 1390 and 1470 cm−1 could be related to the CH3 group vi-
brations; furthermore, νC–O around 1030 cm−1 attest to the bind-
ing of the EU ligand to the POMo core. Furthermore, we have some
additional bands related to EU around the 1500–1650 cm−1 region
–
(stretching and deformation modes of C=C, N H bonds vibrations).
Fig. 5. Experimental (down) & Predicted UV spectrum (up) for POMo & EU2POMo.
Furthermore, we can see the characteristic bands of Anderson-
type POMo at proper regions after conjugation with EU, these
uniquely strong representative bands for Mo6O18 core were located
in 1041, 939, 917, and 663 cm−1, the νMo=O band at 663 cm–1, and
νMo–O–Mo at 939, 917 cm–1 are characteristic of the Anderson struc-
ture [39]. These IR proofs, which are in excellent agreement with
calculated spectra (Fig. 4-right), undoubtedly convinced us of the
correct amide formation between EU and POMo.
56.10 (=C–OCH3), 24.84 (CH2, TBA), 20.72 (CH2, TBA), 13.89 (CH3,
TBA). UV-Vis. (CH3CN): 214 (λmax), 235 nm.
Infra-red spectra: The theoretical IR spectra for two compo-
nents POMo and EU2POMo conjugate, were predicted from the
above-mentioned Gaussian calculations and shown in Fig. 4 (right).
Compare to the POMo, EU2POMo has additional bands related to
the aromatic segments above 3000 cm−1 and in the range of 1000–
1800 cm−1 beside the original POMo characteristic bands with
UV-visible absorption spectrum and TD-DFT calculations; the
TD-DFT calculation was performed using Gaussian (the B3LYP / 6–
6

M. Ramezani-Aliakbari, A. Soltanabadi, H. Sadeghi-aliabadi et al.
Journal of Molecular Structure 1240 (2021) 130612
Fig. 6. 1H NMR spectra (up) & 13 C NMR spectra (down) for EU2POMo conjugate.
31 G method) to estimate the electronic transitions of POMo and
EU2POMo as UV spectra, and the results are shown in Fig. 5(up).
According to the calculated UV–Vis spectrum, which corresponds
to both compounds (POMo & EU2POMo), a doubled broad peak
is seen at a distance of 300–600 nm in the gas phase studies.
These peaks consist of the overlap of two peaks, the maximum
of which occurs between 365 (2.9 eV) and 425 nm. These peaks
can be related to the electronic transition from the HOMO to
LUMO and HOMO-1 to LUMO+1. Due to the small distance be-
tween these levels (Table 2), these peaks have overlapped. The first
peak at λmax = 365 nm is comparable to the difference gap be-
tween HOMO and LUMO (HOMO-LUMO = 2.75 eV) and probably
belongs to a charge transfer from O to Mo.
The experimental UV-visible spectrums of EU2POMo and POMo
were recorded in acetonitrile as the solvent, and results have been
provided in Fig. 5-down besides the theoretical spectrums.
As it can be seen, POMo has a significant absorption at 216 nm,
and in this regard, as the Figure depicts, there are some slight
changes in the general profile of EU2POMo. For EU2POMo, the ba-
sis band for charge transfer from O2− to Mo6+has been retained
with a slight displacement, and another apparent broadband re-
lated to the aromatic segment (π to π∗) which could be reason-
ably related to EU moiety, has been added to the diagram around
240 nm [24,43]. Ventura et al. reported the same results for their
Bombesin-analog peptide conjugate with POMo [23]. By compar-
ing the calculated UV-visible spectrum with that obtained exper-
7

M. Ramezani-Aliakbari, A. Soltanabadi, H. Sadeghi-aliabadi et al.
Journal of Molecular Structure 1240 (2021) 130612
Table 3
imentally, some results are obtained. Firstly, the electronic transi-
tions in the gas phase occur in longer wavelengths than the solu-
tion phase (experimental results); secondly, some electronic tran-
sitions seem to be mixed and altered to have resulted in spectral
appearance changes in the solution phase. It is important to note
that this comparison should be made with great caution, as LUMO
and HOMO molecular orbitals, which are our benchmarks for elec-
tron transfers, are located on the inorganic core based on DFT cal-
culations (Fig. 5-up). So, specifically for EU2POMo, it is needed to
have more information to conclude about electron transfers, and
transfers between other orbitals should be considered carefully. It
should be noted that in all theoretical calculations toward predict-
ing the IR, UV, and NMR, the TBA cations have not been consid-
ered.
NMR studies; the (1H, 13C) NMR experimental spectra of
EU2POMo conjugate are the best complementary data. Fig. 6-up
shows the 1H NMR of the EU2POMo conjugate as it can be seen all
fundamental signals of TBA (signals between 1.0–3.5 ppm, 1, 1.3,
1.6, 3.2, and 3.9 ppm)), and TRIS (62–62.5 ppm) in the POMo scaf-
fold are relocated intact in EU2POMo conjugate. As shown by Mar-
coux et al. [39], due to the strong electron-withdrawing identity
of POMo, its methylene protons (belong to TRIS) have appeared at
a highly downfield area around 63 ppm in 1H NMR with the cor-
rect signal ratio. Furthermore, the related signals of EU were cor-
rectly appeared in the correct regions, as shown in Fig. 6-up about
Comparative cytotoxicity of POMo & EU2POMo on MCF-7 & MDA-MD-231 cell
lines in different times.
Name of sample
POMo
Cell line
MCF-7
Time incubation (h)
IC50(μg/mL)
48
72
48
72
48
72
48
72
303.82
161.5
2.14
1.25
2.31
MDA-MB-231
MCF-7
338.53
194.33
330.43
194.7
1.02
1.89
EU2POMo
0.98
1.67
1.13
MDA-MB-231
346.89
218.6
in EU2POMo conjugate, signal ratios in 1H NMR spectra, elemental
CHNS analysis beside ICP-OES data approved this fact. The results
of all the characterization techniques convinced us that the pro-
posed conjugate structure was synthesized correctly.
3.3. Stability of EU2POMo conjugate
The covalentley modified organo-POMs conjugates either
through the in situ formation or post- functionalization, offers un-
questionable benefits comprising; promising synergistic effects and
better long-term stability both in solid and in aqueous media. In
this regard, the stability of EU2POMo conjugate before any further
study and application should be analyzed [48, 49].
–
7.3 ppm (d, J = 4.4 Hz, HC), and 8.5 ppm (N H amide) with correct
signal ratio to the other parts of spectra. Based on the 1H NMR re-
The UV/vis spectrum of EU2POMo in PBS (Fig. 7) clearly indi-
cates its stability around neutral pH conditions through monitoring
of the characteristic POMo absorption bands, i.e. the characteristic
EU2POMo absorption bands did not undergo significant changes at
any wavelength over a period of 4 days. These results agree well
with the previously observed stability which was reported by Geis-
berger et al. [36].
–
sults, a newly appeared signal of amide (N H, 2H) around 8.5 ppm,
besides the correct ratio of aromatic signals of EU (7.3 ppm, 4H) to
the TRIS around 62.2 ppm (12 H), confirmed the conjugation of 2
molecules of EU to the POMo.
Additional confirmatory information was extracted from the
13C NMR spectrum. In the carbon-13 spectrum shown in Fig. 6-
down, four groups of signals are of great importance, the signals
of the TBA cations appearing in the aliphatic carbons range and
below the 60 ppm region. Aromatic signals appear in the area be-
tween 120 and 153 ppm, TRIS signals with two types of carbons
appearing in the range of 74–85 ppm, and finally, the carbon amide
signal appearing in the 175 ppm region. The two main changes ex-
pected in the spectrum are the addition of aromatic carbon signals
to the related region and the emergence of carbon amide signal in
the downfield area around 175 ppm.
3.4. In vitro cytotoxicity assessments (MTT assay)
To study the effect of EU conjugation on the cytotoxicity profile
of POMo in final product, two cancer cell lines comprising MCF-7
and MDA-MB-231 were selected. The cells were treated with dif-
ferent concentrations ranging from 25 to 400 μg/mL of both POMo
and EU2POMo in the initial evaluation trial at different time of in-
cubation (Table 3). Comparing the results in Table 1 shows that
with increasing the incubation time, the cytotoxicity of POMo and
EU2POMo increases in both cell lines. For this reason, 72 h of incu-
bation time, and concentrations range from 25 to 200 was chosen
for further evaluation.
The type of carbons and the intrinsic symmetry of the molecule
are well inferred from the observed spectral design, as previously
reported by Zhang et al. [44] for the chalcone amide conjugate,
Sullivan et al. [45], and Li et al. [46] for related polyoxomolybdate
conjugates.
The results of in vitro cytotoxicity for the EU2POMo in compar-
ison to the POMo, on the MCF-7, and MDA-MB-231 was provided
in Fig. 8. Based on this diagram, and cytotoxicity data which are
provided in Table 3, the cytotoxicity profile is fully time and dose
responsive (p<0.05 for each comparing). Eventually, Fig. 8c repre-
sents the comparative cytotoxicity of POMo and EU2POMo on the
HUVEC normal cells.
By examining the predicted magnetic resonance spectrum of
the EU2POMo conjugate via quantum chemical calculations us-
ing the GIAO protocol and comparing the results with the re-
sults of the experimental section, RMSD values higher than five
were obtained. Such results are always expected because studies
run in the gas phase, and the solution phase parameters are not
included in the calculations. So, the fundamental differences be-
tween predicted and experimental NMR data arising from differ-
ences between the gas and solution effective parameters. Further-
more, these differences may cause by differences in the structural
constraints in two different phases [47].
Previous reports have repeatedly referred to the potential ac-
tivity of EU as Gallic acid trimethyl ether derivative to introduce
anti-cancer properties in those structures that bearing this moiety.
In this regard, the synergistic effects of the EU for inducing the cy-
totoxic properties in tubulin polymerase inhibitors [15] have been
studied several times. So it seemed that the EU could be the right
candidate for enhancing the anti-cancer properties of POMo.
As can be deduced from Fig. 8a, POMo and EU2POMo exhib-
ited considerably better growth inhibition effects on MCF-7 cell
line. The IC50 of the POMo and EU2POMo on MCF-7 cell line were
146.38 μg/mL, and 194.68 μg/mL respectively. On the other hand,
both of POMo and EU2POMo showed somewhat less cytotoxic ef-
fects on MDA-MB-231 cell line (Fig. 8b), the IC50s were respectively
In general, from comparing the predicted and experimental
spectra, a suitable consistency is inferred between the two experi-
mental parts and the theoretical calculations.
Finally, the complete elemental analysis data for the percent-
age of carbon, hydrogen, nitrogen, manganese and molybdenum
obtained using CHNS and ICP-OES techniques showed an excellent
agreement with the predicted cases for conjugate chemical struc-
ture. Two molecules of EU indeed have been attached to the POMo
8

M. Ramezani-Aliakbari, A. Soltanabadi, H. Sadeghi-aliabadi et al.
Journal of Molecular Structure 1240 (2021) 130612
Fig. 7. UV–Vis. Spectra for EU2POMo during stability assessments after 30 min. 48 h, and 96 h follow up.
Table 4
Cellular uptake quantity of POMo and EU2POMo on MDA-MB-231 cell line based on Mo content determined by ICP-MS method.
The total quantity of
sample in contact with the
cells
Mo total content in 90 μg
Uptake percent regarding
total Mo content
Sample
Total protein(mg)
of sample
Mo(μg/mg protein)
POMo
90 μg/mL
90 μg/ mL
–
4.6
4.1
5.0
28.09
23.26
0
8.2
7.8
0
29.2%
33.5%
0
EU2POMo
Control
156.80 μg/mL and 225.71 μg/mL for POMo and EU2POMo in MDA-
MB-231 cell line. The lower toxicity of the POMo and EU2POMo on
the MDA-MB-231 cell line can be attributed to the specific condi-
tions and different intracellular pathways of this cell line and per-
haps to the higher resistance of the MDA-MB-231 cell line to vari-
ous therapies. Based on the results, the addition of the EU moiety
to the POMo structure could not help improve the toxicity effects
of this compound; however, the obtained IC50 is still in the range
of reported values for this type of compound [10].
and 8.2 μg per each mg of protein, respectively for POMo and
EU2POMo. On the other hand, the total quantity of Mo in POMo
and EU2POMo are different based on the structural formula and
are 28.09 and 23.26 mg for POMo, and EU2POMo respectively.
Considering this fact, we can conclude that the cellular uptake
of EU2POMo is reasonably higher than the POMo. It seems that
adding EU to the structure can improve the cell interactions and
facilitates the entry [18].
The second hypothesis of this study was to reduce the cytotox-
icity effects on HUVEC normal cells, which is confirmed by the re-
sults of normal cell line (Fig. 8c). The cytotoxicity of EU2POMo and
POMo were evaluated on the HUVEC cells at the concentrations
ranging from 25 to 200μg/mL. The EU seems to have protective
effects and help to reduce the toxicity on the normal cell line. In-
terestingly, we did not get any considerable cytotoxicity on HUVEC
for EU2POMo compared to the POMo at the same concentrations.
At concentrations above 50 μg/ ml, EU2POMo is significantly safer
than POMo, which can even alleviate the problem of higher IC50 of
this compound. It seems that we can introduce EU2POMo as a safe
cytotoxic organo-polyoxometalate, the same results were reported
for other hybrid organic-inorganic POMs [18,22].
3.6. Apoptosis quantification using flow cytometry protocol
To quantify the cell apoptosis, MDA-MB-231 cells (this cell line
is routinely resistant to therapy) were treated with the same con-
centration of POMo and EU2POMo (200 μg/mL). After incubation
for 24 h, the cells were analyzed by Annexin V/propidiumiodide
(PI) kit to determine the apoptosis quantity based on the proto-
col provided by the company. The Annexin V binds to cells in the
early apoptosis stage, which can be used as a very specific apop-
totic marker, and PI stains cells in late apoptosis and dead cells
[35].
The results have been presented in Fig. 9; the upper left quad-
rant shows the percent of necrosis in cell death, the upper right
shows late apoptotic cells, the lower left shows normal alive cells,
and the lower right quadrant shows the cells in the early apopto-
sis stage. The results showed that the proportion of apoptotic cells
(in the early and late phase) increased with POMo and EU2POMo
to 15% (total early and late apoptosis), and 28% (total early and
late apoptosis) and 28%, respectively. The proportion of early apop-
totic cells induced by the EU2POMo is significantly higher than the
POMo and more profoundly increased regarding the control group.
So, it can be concluded that the conjugation of EU to the POMo
improved the apoptosis value in cytotoxicity effect on the MDA-
MB-231 cell line.
3.5. Cellular uptake study
The Cellular uptake of POMo and EU2POMo was evaluated com-
paratively on MDA-MB-231 cells by ICP-MS analysis (Table 4) based
on the procedure reported by Geisberger in 2013 [36]. Control ex-
periments without POMo did not display any Mo above the analyt-
ical detection limits, i.e., background concentrations in cells can be
excluded.
After exposing to 90 mg/mL of POMo and EU2POMo, however,
molybdenum contents of MDA-MB-231 cells sharply raised to 7.8
9

M. Ramezani-Aliakbari, A. Soltanabadi, H. Sadeghi-aliabadi et al.
Journal of Molecular Structure 1240 (2021) 130612
Fig. 8. The comparative cytotoxicity of EU2POMo and POM on the a) MCF-7 & b) MDA-MB-231 cancerous cell lines and c) HUVEC normal cell line
∗
∗∗
∗∗∗
In each diagram refers to significant difference p<0.05,
refers to significant difference p<0.01, and refers to significant difference p<0.001.
Fig. 9. Annexin V/PI analysis of apoptosis in MDA-MB-231 cancer cells induced by (A) Control; (B) POMo & (C) EU2POMo.
The question may arise as to why EU2POMo had less cytotoxic
almost in the range of other POMs, it interestingly has better cell
entry and apoptosis value. A more accurate assessment of the anti-
cancer effects of EU2POMo appears to be necessary.
effects than POMo, given the higher cellular uptake and a higher
percentage of apoptosis. It seems important to note that the higher
percentage of cells in the early apoptotic stage cannot be related to
more or less toxicity. According to the available literature [50], dif-
ferent pathways may take from the onset of apoptosis to the stage
of complete cell death. Furthermore, the uptake and clearance of
apoptotic cells by macrophages may be more complex than a sim-
ple removal of cell debris. Hoeppner et al. [51] have demonstrated
that blocking engulfment genes in C. elegans embryos improves
the cell survival when cells are subjected to weak pro-apoptotic
signals. In our study, while EU2POMo has less cytotoxicity effect
4. Conclusion
Achieving new cytotoxic agents with improved effects com-
pared to previous generations has always been the focus of
chemists in the field of medicine. Polyoxometalates are considered
as the next generation of inorganic anticancer drugs. So design-
ing and synthesis of hybrid conjugates of these compounds using
organic molecules that have special properties can be a promis-
10

M. Ramezani-Aliakbari, A. Soltanabadi, H. Sadeghi-aliabadi et al.
Journal of Molecular Structure 1240 (2021) 130612
ing path for further development. In this study, a new generation
of polyoxomolybdate hybrid conjugate was evaluated. Eudesmic
acid organo-conjugate of Anderson type manganese polyoxomolyb-
date (EU2POMo) was synthesized and evaluated for its cytotoxic-
ity in vitro. Preliminary in vitro cytotoxicity results showed that
EU2POMo has comparable cytotoxicity on cancer breast cell lines
(MCF-7 & MDA-MB-231) and interestingly lower cell toxicity on
HUVEC normal cells. Furthermore, it seems that the EU can facili-
tate the entry of POMo into the cell and also emerges more apop-
tosis value in POMo.
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The authors declare that they have no conflicts of interest.
CRediT authorship contribution statement
Maryam Ramezani-Aliakbari: Conceptualization, Methodology,
Validation, Formal analysis, Investigation, Data curtion, Writing
– original draft, Writing – review & editing. Azim Soltanabadi:
Methodology, Validation, Formal analysis, Investigation, Data cur-
tion, Supervision. Hojjat Sadeghi-aliabadi: Methodology, Valida-
tion, Investigation, Data curtion, Supervision. Jaleh Varshosaz: Val-
idation, Investigation. Bahram Yadollahi: Validation, Data curtion.
Farshid Hassanzadeh: Resources. Mahboubeh Rostami: Conceptu-
alization, Methodology, Validation, Formal analysis, Investigation,
Resources, Project administration, Funding acquisition, Data cur-
tion, Writing – original draft, Writing – review & editing, Super-
vision.
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We acknowledge the financial support of novel drug delivery
systems research center at Isfahan University of Medical Sciences,
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