New nanodrug design for cancer therapy: Its synthesis, formulation, in vitro and in silico evaluations

Article abstract of DOI:10.1002/ardp.202000137

The aim of this study was to develop a novel nanosize drug candidate for cancer therapy. For this purpose, (S)-methyl 2-[(7-hydroxy-2-oxo-4-phenyl-2H-chromen-8-yl)methyleneamino]-3-(1H-indol-3-yl)propanoate (ND3) was synthesized by the condensation reacti

Full text of DOI:10.1002/ardp.202000137

Received: 30 April 2020
Revised: 22 June 2020
Accepted: 11 July 2020
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DOI: 10.1002/ardp.202000137
F U L L P A P E R
New nanodrug design for cancer therapy: Its synthesis,
formulation, in vitro and in silico evaluations
Yasemin Budama‐Kilinc1 | Serda Kecel‐Gunduz2 | Burak Ozdemir1 | Bilge Bicak2
Gizem Akman3 | Busra Arvas4 | Feray Aydogan4 | Cigdem Yolacan4
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1Department of Bioengineering, Yildiz
Technical University, Davutpasa Campus,
Abstract
Istanbul, Turkey
The aim of this study was to develop a novel nanosize drug candidate for cancer
therapy. For this purpose, (S)‐methyl 2‐[(7‐hydroxy‐2‐oxo‐4‐phenyl‐2H‐chromen‐8‐
yl)methyleneamino]‐3‐(1H‐indol‐3‐yl)propanoate (ND3) was synthesized by the
condensation reaction of 8‐formyl‐7‐hydroxy‐4‐phenylcoumarin with L‐tryptophan
methyl ester. Its controlled release formulation was prepared and characterized by
different spectroscopic and imaging methods. The cytotoxic effects of ND3 and its
controlled release formulation were evaluated against MCF‐7 and A549 cancer cell
lines, and it was found that both of them have a toxic effect on cancer cells. For drug
design and process development, the molecular docking analysis technique helps to
clarify the effects of some DNA‐targeted anticancer drugs to determine the inter-
action mechanisms of these drugs on DNA in a shorter time and at a lower cost. By
using the molecular docking analysis and DNA binding assays, the interaction
between the synthesized compound and DNA was elucidated and non‐binding
interactions were also determined. To predict the pharmacokinetics, and thereby
accelerate drug discovery, the absorption, distribution, metabolism, excretion and
toxicity values of the synthesized compound were determined by in silico methods.
2Department of Physics, Science Faculty,
Istanbul University, Istanbul, Turkey
3Department of Biology, Science Faculty,
Istanbul University, Istanbul, Turkey
4Department of Chemistry, Yildiz Technical
University, Davutpasa Campus, Istanbul,
Turkey
Correspondence
Cigdem Yolacan, Department of Chemistry,
Yildiz Technical University, Davutpasa
Campus, 34010 Esenler, Istanbul, Turkey.
Email: cigdemyolacan@hotmail.com
Funding information
TUBITAK, Grant/Award Numbers: 115S132,
117S097; Yildiz Technical University,
Grant/Award Number: FBA‐2017‐3168
K E Y W O R D S
anticancer, coumarin derivative, DNA binding, molecular docking, nanoparticle
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1
INTRODUCTION
difficulty in cancer treatment. Due to the side effects and MDR of the
current chemotherapeutic agents,[2] the development of effective
Cancer is a common fatal disease caused by the uncontrolled growth
of cells that have been altered by some effects. It is an important
health problem that is considered the second most common cause of
death after cardiovascular diseases all over the world. Surgery,
chemotherapy and radiotherapy are the main therapeutic ap-
proaches to systematic cancer treatment. However, the treatment
outcome with these methods is generally poor. Thus, it is very im-
portant to find an effective alternative treatment for cancer.[1] Var-
ious chemotherapeutic drugs for cancer treatment have been
developed, but these drugs severely affect healthy tissues such as the
hematopoietic system, bone marrow, gastrointestinal epithelial cells
and hair follicles. Multidrug resistance (MDR) is another important
anticancer drugs with promising bioactivity and important ther-
apeutic effects[3] and limited toxicity[4] is still a great struggle and
urgent need for medicinal chemists.
Chemotherapeutic drugs are systemically active and cannot target
cancer cells. High dosage of chemotherapeutic drugs causes severe
side effects like the destruction of bone marrow cells, which impairs
the erythrocytes' production, cardiotoxicity, nephrotoxicity, hepato-
toxicity and hematotoxicity. To overcome these problems, nano-
particles (NPs) are used in cancer treatment. NPs provide several
advantages in cancer diagnosis and treatment by targeting antigens or
biomarkers that are specific to cancer cells.[5–8] An NP‐mediated drug
delivery system can eliminate drug or drug carrier side effects
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Arch Pharm. 2020;e2000137.
wileyonlinelibrary.com/journal/ardp © 2020 Deutsche Pharmazeutische Gesellschaft
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significantly. In addition, the design of NPs may result in increased
drug effectiveness at lower doses and reduce multiple drug resistance
effects. The major advantage of nanotechnology is targeted drug de-
livery to the site of the disease.[9] Encapsulation of drugs in NPs has
gained great attention as an important approach to the controlled
delivery of the active drug to the target organs. The size, encapsulation
efficiency and release kinetics are important features of these carrier
NPs. The biocompatibility of these systems is another important factor
to allow their use. If the interaction of the material with the body
without any induction of toxicity and immunogenic, thrombogenic and
carcinogenic responses occurs, it has high biocompatibility and can be
used safely as a drug delivery system.[10] Polycaprolactone (PCL) is one
of the certain polymers of the American Food and Drug Administra-
tion that have been approved for drug use. The hydrophobic structure
of micro‐ and nanosized particles produced with PCL prevents the
drug or active substances from dissolving and dispersing in body fluids.
It is frequently used in tissue engineering applications, in particular,
and drug production.[11] In addition, as the drug is loaded, nanosized
PCL spheres follow selective targeting; they carry drugs to only the
diseased region in the body.[12]
For the synthesis of new drugs with improved selective activity
and more clinical effectiveness, determining the interaction between
synthesized molecules and DNA is crucial for rational drug design.
Plant‐derived polyphenolic compounds such as coumarin have a large
number of biological and pharmacological properties. To reveal the
interaction between the newly synthesized compound, which is
coumarin derivative, and DNA, the DNA binding mode has been
indicated by using various experimental and theoretical techniques
such as DNA binding assays and molecular docking analysis. Ultra-
violet (UV)–visible (Vis) absorbance spectra and molecular docking
analysis show the interaction between the synthesized compound
and DNA. The ADME (absorption, distribution, metabolism, excre-
tion) calculations allow to understand and predict the pharmacoki-
netic properties of the synthesized compound with its molecular
structure and properties using computer modeling.[24–26] ADME
properties, which are essential to ensure drug‐like pharmacokinetic
profiles of the synthesized compound, were also calculated using
Molinspiration, SwissADME and PreADMET servers.
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RESULTS AND DISCUSSION
Coumarins that contain benzopyran‐2‐one ring system are an
important class of heterocyclic compounds. Natural and synthetic
coumarins have crucial pharmaceutical properties such as anticancer,
antiviral, antibacterial, antifungal, antioxidant and anti‐inflammatory
effects.[13–18] Coumarin derivatives can be synthesized and functio-
nalized to add desired properties by easy synthetic procedures. Due
to their potential applications in cancer therapy, many studies have
been reported on the design and synthesis of coumarin derivatives to
improve their anticancer potential and investigation of their antic-
ancer effects on various human tumor cell lines.[19–22] There are also
some papers on the activity mechanism of coumarin derivatives on
cancer cells.[23] The design and synthesis of new coumarin derivatives
with a promising effect on cancer cells has been attracting the re-
searchers, due to the encouraging improvements in the activity.
Coumarins have low biodistribution due to their low solubility in the
aqueous solution. Nowadays, nanoformulations are used to over-
come solubility limitations.
2
In this study, (S)‐methyl 2‐[(7‐hydroxy‐2‐oxo‐4‐phenyl‐2H‐chromen‐
8‐yl)methyleneamino]‐3‐(1H‐indol‐3‐yl)propanoate (ND3) was pre-
pared by the condensation reaction of 8‐formyl‐7‐hydroxy‐4‐phe-
nylcoumarin (2) with L‐tryptophan methyl ester (Scheme 1). The
structure of the compound was determined by its spectral data, and
it was concluded that they are in accordance with the structure.
Then, its DNA‐binding activity was investigated via experimental and
theoretical methods.
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2.1
HOMO–LUMO and UV–Vis analysis results
HOMO and LUMO are molecular orbital types that give the ioniza-
tion potential and the electron affinity of the molecule, which are
called “highest occupied molecular orbital” and “lowest unoccupied
ND3
SCHEME 1 The synthesis of ND3

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FIGURE 1 The energy gap and the highest
occupied molecular orbital (HOMO) and
lowest unoccupied molecular orbital (LUMO)
energies of ND3
molecular orbital,” respectively. The energy difference between the
HOMO and the LUMO, which is an important indicator of stability
and also reflects the chemical activity, generally corresponds to the
lowest energy electronic excitation possible in a molecule. Molecules
with a small HOMO−LUMO energy gap are defined as soft mole-
cules, whereas larger ones are defined as hard molecules.[24,25] The
energy gap and HOMO and LUMO energies were determined using
TD‐DFT/B3LYP 6‐311++G(d,p), and the pictorial illustrations of the
frontier molecular orbitals are shown in Figure 1 and tabulated in
Tables 1 and 2. The HOMO was located over the indol ring and
LUMO was presented over iminocoumarin ring, and the HOMO–-
LUMO transition stands for an electron density transfer from in-
dolylethyl group to the iminocoumarin ring with 3.48 and 3.57 eV, for
vacuum and dH2O medium, respectively. The synthesized compound
with a small HOMO–LUMO energy gap has soft molecule properties
with high chemical reactivity, low kinetic stability and high polar-
ization ability. The ionization potential I(−EHOMO) = 5.74 and
5.90 eV, electron affinity A(−ELUMO) = 2.26 and 2.32 eV, the elec-
tronegativity χ = (I + A)/2 = 4 and 4.11 eV, chemical potential
μ = −(I + A)/2 = −4 and −4.11 eV, and hardness η = (I− A)/2 = 1.74 and
1.78 eV values were also calculated for the synthesized compound, as
shown in Table 2 for vacuum and dH2O medium, respectively.
Absorption wavelength λ (nm) and excitation energies E (eV) of ND3
obtained computationally in the dH2O solution and vacuum medium
are tabulated in Table 3.
For the design of new anticancer drugs, DNA binding assay is a
useful method to understand the mechanism of drug interaction with
DNA.[27,28] UV–Vis spectroscopy is the most common instrumental
method for DNA binding assay. This method is based on monitoring
the change of absorption spectra of the drug in increasing con-
centrations of DNA, and the noncovalent interactions between the
drug and DNA take place in three different ways: electrostatic,
intercalation and groove binding.[28,29]
Figure 2 presents the obtained absorption spectra of ND3 with
the addition of the different CT‐DNA concentrations, and a change in
absorbance intensity with hypochromism at 295 and 356 nm was
determined. It was observed that the 14% hypochromism and 5‐nm
bathochromic shift (red shift) occurred at 295 nm, and 15% hypo-
chromism and 4‐nm bathochromic shift (red shift) occurred at
356 nm.
The hypochromic effect and red shift are observed as a result of
binding of drug active substances to DNA by intercalation.[27,30]
TABLE 1 Calculated molecular orbital energies (eV) and energy differences of ND3
TD‐B3LYP/6‐311++G(d,p)
ELUMO+1
ELUMO
EHOMO
EHOMO−1
ΔEHOMO−LUMO
ΔE(HOMO)−(LUMO+1)
ΔE(HOMO−1)−(LUMO)
Vacuum
dH2O
−2.00
−2.26
−5.74
−6.17
3.48
3.73
3.90
−2.02
−2.32
−5.90
−6.34
3.57
3.87
4.02
Abbreviations: HOMO, highest occupied molecular orbital; LUMO, lowest unoccupied molecular orbital.

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TABLE 2 The calculated values of ionization potential, electron affinity, electronegativity, and chemical hardness for the ND3 molecule
Vacuum
TD‐DFT/6‐311++G(d,p)
Energy (AU)
Energy (eV)
Vacuum
HOMO energy
EHOMO
−0.21116
−0.08316
0.21116
0.08316
0.14716
−0.14716
0.064
−5.74596
−2.26290
5.74596
2.26290
4.00443
−4.00443
1.74153
3.48306
LUMO energy
ELUMO
Ionization potential
Electron affinity
Electronegativity
Chemical potential
Chemical hardness
ΔΕ (gap)
I = −EHOMO
A = −ELUMO
χ = (I + A)/2
μ = −(I + A)/2
η = (I − A)/2
ELUMO − EHOMO
0.128
dH2O
HOMO energy
LUMO energy
EHOMO
−0.21685
−0.08538
0.21685
−5.90079
−2.32331
5.90079
2.32331
4.11205
−4.11205
1.78874
3.57748
ELUMO
Ionization potential
Electron affinity
Electronegativity
Chemical potential
Chemical hardness
ΔΕ (gap)
I = –EHOMO
A = –ELUMO
χ = (I + A)/2
μ = –(I + A)/2
η = (I – A)/2
0.08538
0.151115
−0.151115
0.065735
0.13147
ELUMO − EHOMO
Abbreviations: HUMO, highest occupied molecular orbital; LUMO, lowest unoccupied molecular orbital; TD‐DFT, time‐dependent density functional
theory.
The red shift of the absorption spectrum indicates that the difference
of the HOMO and LUMO energy levels decreases, and the drug
molecule interacts with DNA.[31] The drug molecules, intercalating
and/or groove binding with DNA, are important anticancer and an-
timicrobial agents for clinical applications.[32] In this study, after
considering the results of DNA binding assay and in silico calcula-
tions, the anticancer efficiency of ND3 was investigated using in vitro
cell culture against two different cancer cell lines.
41 µg/ml (88 µM) for the A549 cell line and 72 µg/ml (155 µM) for
MCF‐7 cell line, according to MTT assay.
Considering the in vitro cell results, ND3's nanoformulation was
produced to provide slow and controlled release in the cancer area.
Bioavailability of ND3 was aimed to increase by preparing of
nano‐formulation.
ND3‐loaded PCL NPs were prepared following the double‐
emulsion technique. The desired drug concentration can be main-
tained for a long time by encapsulation of drugs, and therefore the
encapsulation efficiency and loading capacity are important terms
in nanoformulation production.[33] The standard curve of ND3
(Figure 4) was used to determine the encapsulation efficiency and
loading capacity. The encapsulation efficiency was calculated as 93%
by using Equation 1. Loading capacity was calculated as 13.8% using
In vitro cell culture studies were performed against MCF‐7 and
A549 cell lines, and 3‐(4,5‐dimethylthiazol‐2‐yl)‐2,5‐diphenylte-
trazolium bromide (MTT) assay was used to evaluate the cytotoxicity
of ND3. As seen in Figure 3, ND3 increased the cytotoxicity in dose‐
dependent manner on both cell lines as compared with control
groups (p < .05). The average IC50 values were determined as
TABLE 3 Calculated absorption wavelengths λ (nm), excitation energies E (eV) and oscillator strengths (f) of ND3 along with transition levels
and assignments in dH2O and vacuum medium
TD‐B3LYP/6‐311++G(d,p)
E (eV)
λ (nm)
f
Major contributors
Symmetry
dH2O
3.1204
397.33
0.0017
H→L
91%
Singlet‐A
3.3835
3.6078
366.44
343.66
0.0021
0.0067
H→L+1
H−1→L
90%
95%
Singlet‐A
Singlet‐A
Vacuum
3.0100
3.2698
3.4810
411.91
379.18
356.17
0.0017
0.0012
0.0055
H→L
88%
11%
11%
88%
94%
Singlet‐A
Singlet‐A
Singlet‐A
H→L+1
H→L
H→L+1
H−1→L

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The physicochemical properties such as average particle size,
polydispersity index and ζ potential are crucial parameters for nano‐
formulated drugs, as they affect the drug distribution, cellular uptake,
and toxicity. In this study, the average particle size, polydispersity
index (PdI) and ζ potential values of NPs were determined with
dynamic light scattering (DLS) technique. The results of free PCL NPs
are given in Figure 5, and it was observed that the average particle
size and ζ potential value were 206.9 nm and −7.62 mV, respectively.
As seen from the figure, free PCL NPs had a narrow size distribution
with 0.043 of the PdI value.
The DLS results of ND3‐loaded PCL NPs are given in Figure 6.
The average particle size and ζ potential values were 279.4 nm and
−3.41 mV, respectively. It was found that ND3‐loaded PCL NPs also
had a narrow size distribution with 0.210 of the PdI value.
The in vitro ND3 release from PCL NPs was studied at pre-
determined time intervals in a phosphate‐buffered solution (PBS) at
pH 7.2 to simulate the physiological pH. It was found that ND3 re-
lease within the first 24 hr was 44.56% of the actual loading, corre-
sponding to 49.9 μg ND3/mg PCL. It was seen from Figure 7, the
release study was completed within 144 hr and 96.6% of the ND3
was released.
FIGURE 2 Absorption spectra of the compound in the absence
and presence of increasing amounts of calf thymus DNA at room
temperature in Tris‐HCl/NaCl buffer (pH 7.2)
The Ames test is one of the short‐time test systems, and it is a
reliable assay that gives precise results about the mutagenicity of
chemical substances. In this study, the mutagenicity of ND3 on
Salmonella typhymurium TA100 and TA98 strains was determined
with Ames/Salmonella Mutagenicity Assay. According to the results of
the in vitro release study of ND3, five different concentrations of
ND3 (112, 108, 90, 78 and 72 µg/plate) were used in the experiment.
The number of colonies of application concentrations and the num-
ber of colonies of the negative control group were compared to
evaluate the results. The sample, which doubled the number of co-
lonies observed in the negative control, was evaluated as muta-
genic.[34] The data obtained from the experimental and control
groups were compared using the SPSS program, and p < .05 was ac-
cepted as significant in all statistical evaluations. As seen from Ta-
ble 4 and Figure 8, it was found that none of the concentrations
Equation 2 by comparing the total amount of encapsulated ND3 in
the synthesized NPs to the total amount of NPs. This means that
each 1 mg ND3‐loaded PCL NPs contained 0.138 mg ND3:
Ai − AS
Ai
(1)
H(%) =
× 100,
where Ai is the absorbance intensity of the substance in free form
and As is the absorbance intensity of the substance after the addition
of DNA at maximum concentration:
Absorbance of experimental group
Absorbance of control group
(2)
Viability % =
× 100.
FIGURE 3 Cell viability results of ND3 on
MCF‐7 and A549 cell lines

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and DG16) and deoxy adenosine (DA17 and DA18), deoxy cytosine
(DC9 and DC11), and deoxy thymine (DT19) base, and they are
shown in Figure 11. As clearly seen from Figure 12, the synthesized
compound was preferentially attached to the DNA. In total, the
oxygen atom of the synthesized ligand formed a stable binding pose
by forming 4 hydrogen bonds with the guanine (DG10, DG16)
residues of DNA. Hydrogen bonds play a very considerable role in
the interaction of the synthesized ligand with DNA, as more hydrogen
bonding interactions (Table 5b) provide a stronger binding at higher
stability and produce lower negative binding energy (−8.8 kcal/mol).
The O1 atom of ligand and DG10 (H3 and H22 atoms) and DG16
(H21 and H22 atoms) of A and B chain of B‐DNA dodecamer formed
hydrogen bonds with 2.5, 3.1, 2.4 and 3.0 Å bond lengths, respec-
tively. The root mean squared deviation (RMSD) values of synthe-
sized compounds bound by B‐DNA dodecamer are also tabulated in
Table 6. The DNA binding activity of the synthesized compound was
evaluated through DNA binding assays and molecular docking
analysis method.
FIGURE 4 The standard curve of ND3
evaluated caused the basepair change or frameshift mutation and
DNA damage (p < .05).
The surface morphology of the ND3‐loaded PCL NPs was de-
termined by SEM. As shown in Figure 9 with different magnification,
SEM images indicated that ND3‐loaded PCL NPs were spherical in
shape, with a uniform distribution, and they had nonaggregated
morphology.
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ADME analysis results
The cytotoxic effect of ND3‐loaded PCL NPs is shown in
Figure 10. All concentrations have a toxic effect on both cell lines as
compared with the control group (p < .05). In addition to this, it was
also determined that ND3‐loaded PCL NP was more effective than
ND3 on A549 cells at 25, 41 and 49 µg/ml concentrations and
41 µg/ml on MCF‐7 cells (p < .05).
2.3
To estimate the pharmacokinetic parameters of the synthesized
compound to be a drug candidate, the ADME profile was calculated
using in silico method, and absorption (A), distribution (D), metabo-
lism (M) and excretion (E) values are given in Tables S1 and S2. The
octanol–water partition coefficient, logP, which defines molecular
hydrophobicity, affects drug absorption, bioavailability, hydrophobic
drug–receptor interactions, metabolism and toxicity of molecules.
The molecular polar surface area[35] is defined as the sum of the
surfaces of polar atoms (usually oxygen, nitrogen, and bound hy-
drogen atoms) in a molecule; this value correlates very well with
human intestinal absorption, the permeability of the Caco‐2 mono-
layers, and blood–brain barrier penetration. Molecular volume is
another feature that is related to the transport properties of mole-
cules, such as intestinal absorption or blood–brain barrier
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2.2
Molecular docking analysis results
The molecular docking analysis revealed that the synthesized com-
pound binds with the B‐DNA dodecamer in nine different con-
formations, and the most stable binding pose is at −8.8 kcal/mol
energy with the largest negative binding energy (Table 5 and
Figure 11). The close interactions between DNA and the synthesized
compound were formed with deoxy guanosine (DG10, DG12, DG14
FIGURE 5 Dynamic light scattering results of free polycaprolactone nanoparticles: (a) average particle size and (b) ζ potential graphics

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FIGURE 6 Dynamic light scattering results of ND3‐loaded polycaprolactone nanoparticles: (a) average particle size and (b) ζ potential
graphics
penetration and is often used to model biological activity. The rule
given by Lipinski,[36] expressed as the 5's rule, defines the probability
of molecules to be drug candidates: the molecular weight of ≤500,
the number of hydrogen bond donor ≤5, the number of hydrogen
bond acceptor ≤10, and the calculated octanol/water distribution
coefficient ≤5. The ADME, drug‐likeness and toxicity properties of
the synthesized compounds are evaluated in Table S1 and S2.
effect. Also, it was presented that ND3‐loaded PCL NPs had high
drug‐loading capacity, good morphological characteristics, and par-
ticle size distributions in the nano range, with a low polydispersity.
To reveal the potential use of the synthesized compound as an
anticancer drug, DNA binding was indicated by experimental and in
silico studies, considering that many antitumor drugs show their ef-
fects by binding to DNA. In addition, the ADME parameters, drug‐
likeness and toxicity properties of the synthesized compound were
calculated for the first time in this study.
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3
CONCLUSION
In conclusion, we believe that this study presents novel per-
spectives for the development of high drug‐loading capacity in the
nanomedicine field.
Conventional drug treatments are generally direct administrations.
However, drug spreads all over the body in these types of adminis-
tration and bioavailability is not in the desired amount. Also, a certain
concentration of drug is required to achieve a therapeutic effect.
In this study, experimental and in silico studies were presented
for drug design on cancer therapy. Our aim was to synthesize a new
coumarin‐based compound and the design of its nanodrug formula-
tion for cancer treatment. It was found that the ND3 had an antic-
ancer effect on MCF‐7 and A549 cell lines, and it had no mutagenic
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4
EXPERIMENTAL
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4.1
Materials and equipment
For the synthesis of ND3, resorcinol, ethyl benzoylacetate and
L‐tryptophan methyl ester hydrochloride were purchased from Aldrich.
FIGURE 7 In vitro release profile of ND3
from polycaprolactone nanoparticles

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TABLE 4 Mutagenicity results of ND3 on
Salmonella typhimurium (n = 3 for each set of
conditions)
Number of revertant colony/plate
Treatment
Concentration (µg/plate)
TA98 (mean SD) TA100 (mean SD)
ND3
112
108
90
51.5 10.14
65
122 3
3
127.5 2.51
134 6.24
138.5 22.5
135 8.08
63.5 3,6
58.5 3.51
59.5 8.18
78
72
Positive control
NPD
SA
10
1
804 60.17*
–
–
965 24.33*
Negative control (DMSO)
Spontaneous control
–
–
52 2.08
129.5 0.5
47.5 2.51
144.5 20.25
Abbreviations: DMSO, dimethyl sulfoxide; NPD, 4‐nitro‐o‐phenylenediamine; SA, sodium azide; SD,
standard deviation.
*Mutagen, the number of revertants compared with negative control is significant at the level of
p < .05 (Tukey's test).
Reagent quality solvents were used without further purification.
Column chromatography was conducted on silica gel 60 (40–63 μM;
Merck). Thin‐layer chromatography (TLC) was carried out on alumi-
num sheets precoated with silica gel 60F254 (Merck). Infrared spectra
were determined on a Thermo Fisher Scientific NICOLET IS10
spectrometer. Nuclear magnetic resonance (NMR) spectra were re-
corded on Bruker Avance III 500‐MHz spectrometer. Chemical shifts
δ are reported in ppm, with tetramethylsilane as the internal stan-
dard and the solvents are CDCl3. Liquid chromatography–mass
spectrometry (LC–MS; quadrupole time‐of‐flight [QTOF]) spectra
were obtained on Agilent G6530B model TOF/QTOF mass spec-
trometer. Optical rotations were measured with Bellingham Stanley
ADP‐410 polarimeter. The synthesis of 8‐formyl‐7‐hydroxy‐4‐phe-
nylcoumarin (2) was carried out according to the literature procedure.[37]
Spectroscopic data of this compound were in accordance with its
structure and literature.[38] The InChI code of ND3 is given in the
Supporting Information.
acid monohydrate (C6H8O7·H2O), potassium phosphate, sodium
dihydrogen phosphate monohydrate (NaH2PO4·H2O), Rosewell Park
Memorial Institute 1640 (RPMI‐1640) medium, trypsin and fetal
bovine serum (FBS) were obtained from Gibco. Dulbecco's modified
Eagle's medium (DMEM) high‐glucose, MTT, sodium hydroxide
(NaOH), sodium chloride (NaCl), magnesium used in mutagenicity
studies, chloride hexahydrate (MgCl2·6H2O), disodium hydrogen
phosphate dihydrate (Na2HPO4·2H2O), potassium chloride (KCl),
L‐histidine, D‐biotin, sodium ammonium phosphate tetrahydrate
(NaHNH4[PO4·4H2O]) and 4‐nitro‐o‐phenylenediamine, which is
used as a mutagen, were purchased from Sigma‐Aldrich. Penicillin
and streptomycin were obtained from I. E. Ulagay. Dichloromethane
(DCM; >99.5%), dimethyl sulfoxide, Tris base, ethylenediaminete-
traacetic acid, NaCl, hydrochloric acid and NaOH were purchased
from Merck Millipore (Darmstadt, Germany). All other chemicals
used in this study were of analytical grade. Ultrapure water from
Millipore Milli‐Q Gradient System was used to prepare the solutions.
Nutrient agar, sodium azide, (SA; NaN3) and nutrient broth were
purchased from Merck Millipore. Two strains of bacteria, TA98 and
Poly(vinyl alcohol) PVA (MW = 31,000–50,000, 87–89%), etha-
nol, CT‐DNA, magnesium sulfate heptahydrate (MgSO4·7H2O), citric
FIGURE 8 Mutagenity results of ND3 on Salmonella typhimurium: (a) TA98 (p < .05) and (b) TA100 (p < .05)

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FIGURE 9 Scanning electron microscopy images of ND3‐loaded polycaprolactone nanoparticles
TA100 of Salmonella typhimurium, were used as the test micro-
organisms to reveal genotoxicity and were obtained from Xenome-
trix. All the chemicals and solvents were of analytical grade.
J = 3.0 Hz, 1H, ArH), 7.07–7.10 (m, 1H, ArH), 7.14–7.17 (m, 1H, ArH),
7.34 (bd, J = 7.5 Hz, 1H, ArH), 7.38–7.40 (m, 2H, ArH), 7.50–7.52 (m,
3H, ArH), 7.53–7.55 (m, 1H, ArH), 7.57–7.60 (bd, J = 8.0 Hz, 1H), 8.66
(s, 1H, CH═N) and 10.6 (bs, 1H, NH); 13C NMR (125 MHz, CDCl3, δ):
29.8 (CH2), 52.6 (OCH3), 70.2 (CH), 106.0 (C3), 109.0 (Caro), 110.1
(Caro), 111. 3 (CaroH), 111.9 (CaroH), 115.7 (CaroH), 118.4 (CaroH),
119.7 (CaroH), 122.3 (CaroH), 123.2 (CaroH), 127.0 (Caro), 128.0
(CaroH), 128.2 (CaroH), 129.0 (CaroH), 129.6 (CaroH), 131.5 (Caro),
135.4 (Caro), 136.2 (Caro), 155.2 (C4), 156.5 (Caro–OH), 161.0
(C═O) and 170.8 (C═O); FTIR (ATR): ν = 3,349 (w), 3,058 (w), 2,957,
2,925 (w), 1,730 (s), 1,626 (m), 1,582 (m), 1,477 (w), 1,378 (m) and
|
4.2
Synthesis of (S)‐methyl 2‐[(7‐hydroxy‐2‐oxo‐4‐
phenyl‐2H‐chromen‐8‐yl)methyleneamino]‐3‐(1H‐
indol‐3‐yl)propanoate (ND3)
L‐Tryptophan methyl ester hydrochloride (1 mmol) in absolute etha-
nol (5 ml) and triethylamine (0.5 ml) suspension was stirred at room
temperature for 1 hr, and then 8‐formyl‐7‐hydroxy‐4‐phenylcou-
marin (2, 1 mmol) dissolved in absolute ethanol was added. The
mixture was refluxed for 2 hr. The reaction was monitored by TLC.
Then alcohol was evaporated, and the crude product was purified by
column chromatography on silica gel (ethyl acetate/hexane 2:3).
Yellow viscous oil, yield 40%. [α]20D = + 79.5 (c = 1.00, CHCl3). 1H
NMR (500 MHz, CDCl3, δ): 3.34 (dd, J = 14.5, 9.0 Hz, 1H, CH2), 3.59
(dd, J = 15.0, 5.0 Hz, 1H, CH2), 3.78 (s, 3H, CH3), 4.39–4.42 (m, 1H,
CH), 6.10 (s, 1H, ═CH), 6.74 (d, J = 9.5 Hz, 1H, ArH), 7.05 (bd,
;
1,193 (m) cm−1 LC–MS (ESI–QTOF): m/z [M+H]+ calcd. for
C28H23N2O5, 467.1617; found, 467.1598.
|
4.3
HOMO–LUMO analysis
The HOMO–LUMO analysis and theoretical UV–Vis spectra were
performed using the Gaussian 09 software program,[39] employing
the time‐dependent density functional theory (TD‐DFT) approach

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FIGURE 10 Cell viability results of ND3‐
loaded polycaprolactone nanoparticles against
MCF‐7 and A549 cell lines
based on the B3LYP/6‐311++G(d,p) level to determine the bandgap
(ΔE), which means the chemical stability, the ionization potential (I),
the electron affinity (A), the absolute electronegativity (χ), chemical
potential (µ) and the absolute hardness (η) values of the synthesized
molecule were also calculated.
streptomycin (100 µg/ml) and 10% FBS (Gibco Lab), and it was
incubated at 37°C with 5% CO2. Also, 3 × 104 cells were seeded in a
96‐well cell culture plate, with 200 µl/well, for the MTT assay.
|
4.6
Cytotoxicity evaluation with MTT assay
|
4.4
DNA binding assay
Cellular viability was assessed by reduction of MTT to formazan.
Briefly, MTT was dissolved in PBS and 40 µl was added to each well
at a final concentration of 5 mg/ml. Cells were incubated for 4 hr at
37°C. After the incubation period, 160 µl DMSO was added to wells,
followed by overnight incubation. The measurement was performed
using an ELISA reader at 450–690 nm. The results are expressed
relative to the control value.
The interaction between ND3 and calf thymus DNA (CT‐DNA) was
investigated by DNA binding assay. Briefly, Tris‐HCl/NaCl buffer was
used for the experiment. During the analysis, the concentration of
ND3 was kept constant (36 µM), and the concentration of CT‐DNA
ranged from 0 to 72 µM. Changes in absorbance intensity can be
calculated with percentage ratios. A formula (Equation 1) has been
developed to calculate these changes.
|
4.7
Preparation of ND3‐loaded PCL NPs
|
4.5
In vitro cell culture
ND3‐loaded PCL NPs were prepared according to the double‐emul-
sion precipitation method. Briefly, 300 mg of PCL was dissolved in
20 ml of DCM, and then 6 mg of ND3 was dissolved in 2 ml of DMSO
and added to 2 ml of PCL solution. They were homogenized by ap-
plying sonication under 70 W energy for 5 min, and emulsion (w/o)
was formed. Furthermore, 20 mg PVA was dissolved in 100 ml of
distilled water. The (w/o) emulsion was pulled with a syringe and then
added dropwise into the PVA solution with continuous stirring. Then,
the mixture was homogenized by applying sonication under 70 W
energy for 5 min, and double emulsion (w/o/w) was formed. The
mixture was left overnight with continuous stirring. The obtained
Human MCF‐7 cell line (ATCC® HTB‐22™) was used for in vitro
cytotoxicity experiments. Briefly, MCF‐7 cells (105 cells/ml) were
cultured in DMEM (Sigma‐Aldrich), high‐glucose medium, penicillin
(100 U/ml), streptomycin (100 µg/ml) and 10% FBS (Gibco Lab) at
37°C and 5% CO2. Cells were passaged twice a week by trypsin, and
the cells were seeded at a density of 2 × 104 cells/200 µl per well in a
96‐well cell culture plate for the MTT assay.
Human A549 cell line (ATCC® CCL‐185™) was cultured in RPMI
1640 (Gibco Lab) medium supplemented with penicillin (100 U/ml),
TABLE 5a The binding affinity, close
interactions, and hydrogen bonding
ND3
Affinity (kcal/mol)
Close interactions
Hydrogen bonding (Å)
interaction of ND3 bound by B‐DNA
dodecamer
−8.8
DG10, DG12, DG14, DG16, DA17, DA18, DC9,
DC11, DT19
DG10, DG16

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FIGURE 11 The best‐docked pose of ND3 with the B‐DNA
|
Determination of encapsulation efficiency and
loading capacity
ND3‐loaded PCL NPs were centrifuged at 10,000 rpm for 45 min and
washed five times to remove any residual organic solvent. To prepare
blank PCL NPs, the remaining steps were performed for equal
amounts without ND3, as described above. Finally, blank and ND3‐
loaded PCL NPs were freeze‐dried for further characterization and
others analysis.
4.9
The concentration of free ND3 in the supernatant was determined by
the equation of ND3 standard curve (Figure 4), and the encapsulation
efficiency and loading capacity were calculated using the following
equations:
Encapsulation efficiency(%)
|
4.8
Preparation of the standard curve
Total ND3 amount − Free ND3 amount
(3)
(4)
=
× 100,
Total ND3 amount
In total, eight different concentrations of ND3 (0.195313, 0.390625,
0.78125, 1.5625, 3.125, 6.25, 12.5 and 25 µg/ml) were prepared by
serial dilution. The UV absorbance value of each concentration was
determined at 231.5 nm by UV–Vis spectrometer. Then the standard
curve was obtained (Figure 4), and the curve equation was used to
determine the encapsulation efficiency and loading capacity.
Encapsulated ND3
Loading capacity(%) =
× 100.
Total NPs weight

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FIGURE 12 The hydrogen bonding interactions between ND3 and the B‐DNA
TABLE 5b The atoms of close interactions between ND3 and
|
4.10
DLS analysis
B‐DNA dodecamer
Atom
The physicochemical properties of NPs were analyzed by using the DLS
technique. The average particle size, polydispersity index and ζ potential
values of NPs were determined by Zetasizer Nano ZS (Malvern
Instruments Ltd., Malvern, UK). The 4‐mW He–Ne laser was used at a
wavelength of 633 nm with a detection angle of 90°. All measurements
were made at a temperature of 25°C, and each sample was prepared
with a PBS solution before filtering with a 0.45‐µm regenerated cellu-
lose membrane (Sartorius, Germany). The particle size, ζ potential and
PdI were reported as the mean of at least 10 measurements.
number of
residues
of DNA
DNA
chain
Residues
of DNA
Atom of
conformer‐1 Interaction (Å)
A
A
B
B
DG10
DG10
DG16
DG16
H3
O1 (ND3)
O1 (ND3)
O1 (ND3)
O1 (ND3)
2.5
3.1
2.4
3.0
H22
H22
H21
|
4.11
Scanning electron microscopy (SEM) analysis
TABLE 6 The root mean squared deviation (RMSD) values of ND3
bounded by B‐DNA dodecamer
The morphology of the ND3‐loaded PCL NPs was revealed using
SEM (Zeiss Supra 50 V). Briefly, the samples were diluted with
ethanol and placed directly in an ultrasound bath for 15 min. Then,
the samples were prepared by dropping 40 µl of ND3‐loaded PCL
NPs on the SEM stubs, which were covered with aluminum foil, and
they were dried for 1 day at room temperature. The SEM images
were obtained at ×100.00 k magnification, 5.00 kV electron high
tension and 9.0 mm working distance with an in‐lens detector.
Affinity
Distribution from
RMSD l.b.
Best mode
RMSD l.b.
Mode (kcal/mol)
1
2
3
4
5
6
7
8
9
−8.8
−8.6
−8.6
−8.5
−8.3
−8.2
−8.2
−8.0
−7.8
0.000
1.205
1.911
2.245
2.601
2.445
2.335
12.339
3.307
0.000
1.668
3.443
3.900
9.064
5.051
9.150
13.747
9.719
|
4.12
In vitro release study
ND3‐loaded PCL NPs were dispersed in 2 ml distilled water and
placed in a dialysis capsule to determine the in vitro release profile of

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ND3. Phosphate buffer (PBS) at pH 7.4 was used as the release
medium. In vitro release study was performed at time intervals 0, 0.5,
1, 2, 3, 4, 5, 6, 7, 8, 24, 48, 72, 96, 120 and 144 hr. Incubation of
samples was achieved in a shaking water bath at 37°C under gentle
agitation. At each time intervals, a 1‐ml sample was taken from the
release medium, and fresh release medium of the same volume was
added instead. The samples were analyzed by UV–Vis spectrometer,
and the amount of ND3 released from the PCL NPs, depending on
time, was obtained by using the following equation:
Z = 40 Å with 0.375‐nm grid spacing. AutoDockVina 1.1.2 program[42]
was implemented for docking analysis that was used to define the most
favorable binding affinities and RMSD values for the synthesized com-
pound. The close interactions, binding affinities, and RMSD were calcu-
lated as a result of the study. Energy‐scoring function is used to
determine the best ligand–DNA pose. PyMol 2.2.3[43] and Auto-
DockTools1.5.6[44] programs were used to observe close interactions and
hydrogen bonding interactions.
Released ND3
Total ND3
(5)
|
4.15
Statistical analysis
Release(%) =
× 100.
|
4.13
Ames/Salmonella mutagenicity assay
For the statistical analysis of Ames/Salmonella mutagenicity assay, the
data obtained from the experimental and control groups were com-
pared using the SPSS program (version 24.0; SPSS Science, Chicago, IL).
Significant differences are indicated by a–b (p < .05, Tukey's honestly
significant difference test) among doses in the same dose column. The
p < .05 was accepted as significant in all statistical evaluations.
For statistical analysis of the cytotoxicity experiments, one‐way
analysis of variance test were used to analyze experimental groups
followed by post‐hoc Tukey multiple comparisons. A p < .05 was
considered statistically significant (Prism Version 7.0; GraphPad
Software, Inc.).
The TA98 and TA100 strains of S. typhimurium were used to determine
the frameshift and basepair change mutation of the ND3. Experiments
were performed as described by Maron and Ames.[40,41] Briefly, the
concentrations of ND3 used in the experiment were determined de-
pending on the in vitro release profile of ND3 (five different con-
centrations: 72, 78, 90, 108 and 112 µg). To ensure the reliability of the
experiment, first, it was checked whether the test strains had original
mutations. Therefore, histidine requirement, presence of R factor, rfa
mutation and uvrB mutation of the test strains were controlled before
starting the study. Before performing the experiment, a single fresh
colony of standard strains of S. typhimurium TA98 and TA100 was in-
oculated in nutrient broth and incubated for 10–12 hr at 37°C in an
incubator. Each strain of S. typhimurium was grown separately in Erlen-
meyer flasks. Autoclaved distilled water was used as a negative control,
and sodium azide (1 µg/plaque) and 4‐nitro‐o‐phenylenediamine
(10 µg/plaque) were used as positive controls for TA98 and TA100
without S9 metabolic activation. 2‐Aminofluorene (5 µg/plaque) was
prepared for TA98 and TA100 metabolic activation. For mutagenity
study, 222 µL of histidine‐biotin solution, 500 µL of sodium‐phosphate
buffer, 100 µL of sample and controls, and 100 µL of bacterial culture
were added to 2 ml top agar kept at 43°C and top agar was poured into
minimal glucose agar (MGA). Then, they were mixed gently and poured
into MGA plaque. These plaques were incubated at 37°C for 48 hr.
Spontaneous revertant colonies (His + revertants) were counted at the
end of the incubation.
ACKNOWLEDGMENTS
The authors would like to thank TUBITAK for its support through the
Yildiz Technical University Scientific Research Foundation (project
number FBA‐2017‐3168). In this study, the infrastructure of Applied
Nanotechnology and Antibody Production Laboratory established with
TUBITAK support (project numbers: 115S132 and 117S097) was used.
CONFLICTS OF INTERESTS
The authors declare that there are no conflicts of interests.
ORCID
Cigdem Yolacan
http://orcid.org/0000-0003-4221-3592
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