Chitosan nano-vehicles as biocompatible delivering tools for a new Ag(I)curcuminoid-Gboxin analog complex in cancer and inflammation therapy

Article abstract of DOI:10.1016/j.ijbiomac.2020.10.153

A novel anticancer and anti-inflammatory agent based on hybrid curcuminoid-Gboxin analog (FLLL49-GbA) and its macromolecular silver(I) complex (Ag(I)FLLL49-GbA) have successfully synthesized. In addition, chitosan nanoparticles (CNPs) were used to encapsu

Full text of DOI:10.1016/j.ijbiomac.2020.10.153

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Chitosan nano-vehicles as biocompatible delivering tools for a
new Ag(I)curcuminoid-Gboxin analog complex in cancer and
inflammation therapy
Serag Eldin I. Elbehairi, Lamia A. Ismail, Mohammad Y. Alfaifi,
Reda F.M. Elshaarawy, Hani S. Hafez
PII:
S0141-8130(20)34799-1
DOI:
https://doi.org/10.1016/j.ijbiomac.2020.10.153
BIOMAC 17043
Reference:
To appear in:
International Journal of Biological Macromolecules
Received date:
Revised date:
Accepted date:
12 August 2020
6 October 2020
20 October 2020
Please cite this article as: S.E.I. Elbehairi, L.A. Ismail, M.Y. Alfaifi, et al., Chitosan nano-
vehicles as biocompatible delivering tools for a new Ag(I)curcuminoid-Gboxin analog
complex in cancer and inflammation therapy, International Journal of Biological
Macromolecules (2018), https://doi.org/10.1016/j.ijbiomac.2020.10.153
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Title page
Chitosan Nano-vehicles as biocompatible delivering tools for a new Ag(I)curcuminoid-Gboxin
analog complex in cancer and inflammation therapy
Serag Eldin I. Elbehairia,b, Lamia A. Ismailc, Mohammad Y. Alfaifia, Reda F.M. Elshaarawyd,e,* , Hani
S. Hafezf,*.
a Biology Department, Faculty of Science, King Khalid University, 9004 Abha, Saudi Arabia
b
Cell Culture Lab, Egyptian Organization for Biological Products and Vaccines (VACSERA
Holding Company), Giza 12311, Egyptc Faculty of Science, Zoology Department, Suez University,
43533 Suez, Egypt.
c Department of Chemistry, Faculty of Science, Port Said University, Port Said, Egypt.
d
Institut für Anorganische Chemie und Strukturchemie, Heinrich-Heine Universität Düsseldorf,
40204 Düsseldorf, Germany.
e Faculty of Science, Chemistry Department,Suez University, 43533 Suez, Egypt.
f Faculty of Science, Zoology Department, Suez University, 43533 Suez, Egypt.
Corresponding author:
Email: reda.elshaarawy@suezuniv.edu.eg.
reel-001@hhu.de
Orcid ID: 0000-0003-2100-8928
Email: hani.hafez@suezuniv.edu.eg
Abstract
A novel anticancer and anti-inflammatory agent based on hybrid curcuminoid-Gboxin analog
(FLLL49-GbA) and its macromolecular silver(I) complex (Ag(I)FLLL49-GbA) have successfully
synthesized. In addition, chitosan nanoparticles (CNPs) were used to encapsulate this macromolecular
complex, targeting enhancing its therapeutic effect and minimizing its side impacts. The encapsulated
Ag(I) complex was significantly triggered apoptosis (P<0.05) with much more rapidly release of
Ag(I)FLLL49-GbA from the CNPs at pH 5.3 than at pH 7.4, which is beneficial for cancer-targeted
drug delivery. Free complex showed promising ability in preventing glucose uptake and lactate
production coupled with cellular ATP depletion in cancer cells. Additionally, there was significant
decrease in the inflammatory cytokines in breast cancer (MCF-7) and lung cancer (A549) cells with
values of P<0.01 and P<0.001 after 24 h incubation. Furthermore, the death-inducing proteins have
been significantly up-regulated (P<0.01 to P<0.001) after 36 h incubation of cancer cells.
Consequently, the novel curcuminoid macromolecule showed significant feasibility in triggering the
high expression of apoptotic caspases caspase 3, caspase 8, P53, and Bax (P<0.01 to P<0.001) after 48
h of chemotherapy. Noteworthy, the cytotoxicity of Ag(I)FLLL49-GbA was significantly increased

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toward cancer cells (MCF-7 > A549), while, reduced toward normal cells (HeLa) after loading on
chitosan Nano-vehicles.
.
Keywords: Ag(I)-curcuminoid macromolecule-loaded Chitosan Nano-vehicles; Anticancer and Anti-
inflammatory; Structure-function relationship.
1. Introduction
Cancer cells adjust their metabolism to generate all molecules and energy sources required to help
the tumor to regulate its survival environment. Such metabolic features of cancer cells are believed to
be a result of mutations in oncogenes and tumor suppressors that manage the cellular metabolism [1].
Natural therapeutics have been used for long periods for their persuasive role to inhibit cancer cell
survival and progression. Amongst natural chemotherapeutics, curcumin (Cur) (1,7-Bis(4-hydroxy-3-
methoxyphenyl)-hepta-1,6-dien-3,5-dione) has been demonstrated as one of the most anticancer
agents with different therapeutic benefits against multiple chronic diseases [2]. Great efforts have
been exerted to enhance the anticancer activity of Cur through the chemical refinement of its structure
[3,4], to create many Cur derivatives, analogs, and conjugates [5,6]. Noteworthy that sustaining α,β-
unsaturated carbonyl segment in the Cur derivatives is an important issue, as it plays a pivotal role in
the anticancer action of such compounds [7,8]. Among these derivatives, 1,7-Bis(3,4-
dimethoxyphenyl)-4-cyclohexyl-hepta-1,6-dien-3,5-dione (FLLL32) and 1,7-Bis(4-hydroxy-3-
methoxyphenyl)-4-cyclohexyl-hepta-1,6-dien-3,5-dione (FLLL49) (Fig. 1) were categorized as
promising highly active anticancer agents [9,10]. These compounds have a great capacity to inhibit
the Signal Transducer and Activator of Transcription 3 (STAT3) protein phosphorylation [9,10,11]
which plays a significant role in the progression, proliferation, migration, invasion, metastasis, and
survival of tumor cells [12,13,14]. Additionally, FLLL32 can reduce STAT3-DNA binding affinity
and expression leading to, Osteosarcoma cell lines apoptosis [9]. However, unfortunately, the poor
aqueous-solubility and rapid clearance of the bioactive Cur and Cur-based compounds prevent from
reaching their maximum potential. Many strategies have been employed to tackle these challenges,
particularly, coordination to metal ions which can be also used to mitigate the inherent drawbacks of
Cur, such as promoted tolerance under physiological conditions. Moreover, metal complexes of Cur
and its derivatives expand their potential applications, as compared to parent ligand, in cancer therapy
[15]. Most notably, silver-based Cur and its derivatives that were emphasized to have multidiscipline
biological applications such as; anticancer and anticancer drug delivery (Ex. ST06-AgNPs and Cur-
AgNPs) [16,17,18], antiviral (Ex. Cur-AgNPs) [19], anti-biofilm (Ex. CurNPs-AgNPs) [20], wound
dressing and antibacterial (Ex. Cur-AgNPs and Ag(I)Cur complex) [21,22].
Nanoparticles-based drug delivery systems (DDS) have gained extensive attention for their ability
to improve the therapeutic index of drugs (bioavailability, chemical stability, selectivity) and reduce
their adverse side effects [23]. Among DDS, polymeric nanoparticles (PNPs), particularly chitosan
nanoparticles (CNPs), have categorized as promising drug carriers due to the non-toxicity,
biocompatibility, biodegradability, and sustainable drug release [24,25]. To the name few, CPNs were

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assigned as effective nanocarriers for versatile drugs including anticancer [25,26], antibiotics [25,27],
anti-HIV medications [28], and…..etc. In addition, the CNPs have also been successfully used for the
encapsulation of curcumin targeting diverse therapeutic applications [29,30].
Therefore, inhibition of glycolysis severely impairs the ATP generation and consequently
prevents cancer cells to be highly dependent on this metabolic pathway for survival. Thus, great
efforts in cancer therapy have targeted the glycolysis process as a therapeutic strategy to prevent the
proliferation and survival of cancer cells, whereas inhibition of mitochondrial oxidative
phosphorylation (OXPHOS) has remained largely unexplored. It noteworthy that a novel small
molecule, 2-ethyl-1-(2-(((1R,3R,5R)-3-isopropyl-5-methylcyclohexyl)oxy)-2-oxoethyl)-3-methyl-1H-
benzo[d]imidazol-3-ium chloride (Gboxin, Gb) (Fig. S1) was explored recently and assessed as a
mitochondrial OXPHOS inhibitor [31].
Motivated by these aforesaid prominent facts and follow-up to our constant pursuit in exploring
novel chemotherapeutic candidates [32,33,34]; the main objective of the current work is to fabricate
novel anti-inflammatory and anticancer agents based on Gboxin analog (GbA) and curcuminoid
derivative (FLLL49), (FLLL49-GbA conjugate) and its Ag(I) complex. In addition, the capacities of
these compounds to induce apoptosis in breast and lung cancer cells will be addressed. Moreover,
exploiting the beneficial effects of CNPs as a sustainable delivering system for encapsulating
Ag(I)LL49-GbA for cancer therapeutics.
2. Material and methods
Chemicals and starting materials used for the synthesis of our targeted materials (FLLL49, GbA,
and CNPs) and different instrumental techniques utilized for full characterization of the fabricated
materials were given in the electronic supplementary information (ESI†).
2.1. Chemistry
2.1.1. Synthesis of the FLLL49-GbA conjugate
A solution of FLLL49 (0.87 g, 2.0 mmol) and GbA (1.96 g, 5.0 mmol) in anhydrous DCM (100
mL) was treated with 1-ethyl-3-(3-dimethylaminopropyl) carbodiimide (EDC) (1.15 g, 6 mmol) and
4-dimethylaminopyridine (DMAP) (0.73 g, 6 mmol) under stirring at room temperature (RT) and N2
atmosphere. After stirring overnight at RT, a saturated aqueous NaHCO3 solution (50 mL) was added
to the reaction mixture. Then, the DCM layer was separated, and the aqueous phase was extracted
with additional amounts of DCM (3 × 25 mL). The organic phases were collected and dried with
anhydrous sodium sulfate. After evaporation to dryness under vacuum, the residue was subjected to
column chromatography on silica gel (200–400 mesh) with a mixture of ethyl acetate/ n-hexane (1:2
V/V) as an eluent. The collected product was further recrystallized in a methanol-acetonitrile mixed
solvent to afford pure compound FLLL49-GbA. Yield 78%, mp 124-125°C. FTIR (KBr, cm-1): 3027

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(m, sh), 2995 (m, sh), 2877 (m, sh), 1734 (s, sh), 1674 (s, sh), 1580, 1534, 1466 (s, sh), 1379 (m, sh),
1150 (s, sh), 967(m, sh), 736 (m, sh). 1H NMR (200 MHz, DMSO-d6) 8.52 (d, J = 7.3, 2H), 8.37 (t, J
= 7.2, 2H), 7.79 (dt, J = 4.6, 1.9 Hz, 2H), 7.62 – 7.51 (m, 4H), 7.11 (dd, J = 15.5, 1.9 Hz, 2H), 6.97 –
6.76 (m, 4H), 6.73 – 6.58 (m, 2H), 5.27 (s, 4H), 3.93 (s, 6H), 3.74 (s, 6H), 3.71 – 3.62 (m, 1H), 2.54
(q, J = 7.3 Hz, 4H), 1.99 – 1.76 (m, 6H), 1.86 (s, 1H), 1.42 (t, J = 7.3 Hz, 6H), 1.25 – 1.01 (m, 4H).
13C NMR (125 MHz, DMSO-d6) δ (ppm): 201.34, 168.21, 157.14, 161.88, 143.41, 141.67, 132.16,
131.78, 131.24, 127.02, 126.72, 126.54, 124.83, 122.30, 113.74, 113.49, 111.63, 56.62, 51.91, 40.02,
39.83, 25.97, 23.61, 19.09, 15.34, 14.17. ESI-MS: in positive mode peaks at m/z 874.45
([C50H54ClN4O8]+, M – Cl‒) a.m.u. and 419.1 ([C50H54ClN4O8]2+, M – 2 Cl‒) a.m.u. Anal. Calcd. for
C50H54Cl2N4O8 (M = 909.90 g/mol): C, 66.00; H, 5.98; N, 6.16 %. Found: C, 65.94; H, 6.02; N, 5.98
%.
2.1.2. Synthesis of Ag(I)FLLL49-GbA complex
A solution of silver nitrate (0.50 g, 3.0 mmol) in acetonitrile (3 mL) was added dropwise with
stirring to a solution of FLLL49-GbA (0.91 g, 1 mmol) in THF (30 mL) at room temperature. After
stirring this mixture overnight at RT, the resulting black solid was collected by filtration through a
celite pad, washed several times with MeOH followed by diethyl ether, and dried in vacuo at RT.
Yield 56%. FTIR (KBr, cm-1): 3432 (s, br), 3054 (m, sh), 2997 (m, sh), 2883 (m, sh), 1768 (s, sh),
1660 (s, sh), 1585, 1539, 1466 (s, sh), 1386 (m, sh), 1153 (s, sh), 956 (m, sh), 742 (m, sh) ), 526 (w,
sh). Anal. Calcd. for C50H54AgCl2N5O11 (M = 1079.77 g/mol): C, 55.62; H, 5.04; N, 6.49 %. Found:
C, 54.98; H, 5.12; N, 6.38 %.
2.1.3. Synthesis of (Ag(I)FLLL49-GbA)CNPs
Initially, a clear chitosan (CS) solution (0.2% w/v) was prepared by dissolving CS in acetic acid
(1% v/v), thereafter, a certain amount of Ag(I)FLLL49-GbA complex was added portion wise into
20 mL CS solution; the final concentration of Ag(I)FLLL49-GbA in CS solution is 0.5 mg/mL. After
complete dispersion, the pH of mixture was adjusted to 6.0 by 0.25 M NaOH. After stirring for
30 min, a solution of a cross-linker sodium tripolyphosphate (TPP) (0.1% w/v) was added dropwise to
this mixture under constant stirring (1000ꢀrpm) at room temperature to achieve final mass ratios of
2.5:1 (CS:TPP). The suspension obtained was further stirred (1000ꢀrpm) at the same temperature for
60 min and then centrifuged at 16000 rpm under ambient conditions for 30 min to isolate the
nanoparticles ((Ag(I)FLLL49-GbA)CNPs) and supernatant. The isolated nanoparticles were washed
thrice by deionized water and then dried by a further freeze-drying process in presence of 3%
mannitol as a lyophilized protecting agent. The blank CNPs were prepared by a similar protocol but
without the addition of Ag(I)FLLL49-GbA complex.
2.1.4. Determination of Ag(I)FLLL49-GbA loading and encapsulation efficiency of CNPs
The Ag(I)FLLL49-GbA complex loading and encapsulation efficacy may be calculated using two

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previously reported methods [26,35], with slight modification. In the first indirect method, the un-
loaded Ag(I)FLLL49-GbA was measured, in the supernatant collected after centrifugation, using UV-
vis spectrophotometer at 432 nm. On the other hand, the second experiment based on direct estimation
of the Ag(I)FLLL49-GbA in the Nano-formulation ((Ag(I)FLLL49-GbA)CNPs) through re-
dispersion of this nanocomposite in milli-Q water (5 mL) and 40 mL of 0.1 M HCl solution under
ultrasonic irradiation for 5 min. Thereafter, this mixture solution was layered with 40 mL of acetic
ether twice. After layering, the content was evaporated under reduced pressure and the obtained
residue was re-dissolved in DMSO (25 mL) for determination of Ag(I)FLLL49-GbA content using
UV-vis spectrophotometer at 432 nm (with DMSO as blank control). The encapsulation and loading
efficacies are calculated using equations (1, 2);
푊푇퐶 − 푊푈퐶
(
)
Encapsulation efficacy % =
x 100
(ퟏ)
(ퟐ)
푊푇퐶
푊푇퐶 − 푊푈퐶
(
)
Loading efficacy % =
x 100
푊
퐶푁푃푠
where WTC is the total weight of Ag(I)FLLL49-GbA complex added to the system; WUC weight of
un-encapsulated complex;, WCNPs is the weight of chitosan nanoparticles.
2.1.5. In vitro release of Ag(I)FLLL49-GbA from chitosan nanoparticles (CNPs).
The Ag(I)FLLL49-GbA-loaded chitosan Nano-formulation ((Ag(I)FLLL49-GbA)CNPs) (0.5 mg)
was dispersed in phosphate-buffered saline (PBS) (pH 7.4) or acetate buffer (ABS) (pH 5.3) (3 mL)
and subsequently dialyzed into a dialysis bag (cut-off 5 kDa) against 40 mL of a mixture of 30%
ethanol and 70% ABS pH 5.3 or PBS 7.4 as a release medium. The content was stored in a 37 °C and
150 rpm shaker incubator. At fixed time intervals, 1 mL samples were drawn and replaced with fresh
medium by equal volume. Each sample was supplemented with 4 mL ethanol and analyzed at 432 nm
against null medium UV spectrophotometers for measuring Ag(I)FLL49-GbA release concentration.
The complex discharge fraction was determined based on the initial amount of Ag(I)FLL49-GbA with
nanoparticles. The following equation was used to estimate the cumulative release of Ag(I) complex
from CNPs. The cumulative release of Ag(I) complex was calculated using equation (3);
퐸
(
)
Cumulative release % =
푥 100
(ퟑ)
퐸표
Where E° and E are the initially loaded and release amount of Ag(I)FLL49-GbA from the
encapsulated form in medium, respectively.
2.2. Anticancer study
2.2.1. Cell culture
The human breast (MCF-7) and lung (A549) cancer cells were provided by VACSERA Tissue
Culture Unit. The cell lines were cultivated in Dulbecco's Modified Eagle Medium (DMEM, Gibco)
and supplemented with a fetal bovine serum (FBS, 10%) (TBD Science), all the contents have been

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incubated overnight at 37°C under 5% CO2 atmosphere.
Additionally, the biological assessments of the curcumiod derivatives activity against tumor cells
have been carried out using their fee form. While, encapsulated complex nano-chitosan form
((Ag(I)FLLL49-GbA)CNPs) activity has been tested against their action for inducing different
apoptosis stages using the DNA binding dyes Acridine orange (AO) and Ethidium bromide (EtBr).
MTT assay
MTT assay has been carried out to testify the cell viability of the treated breast carcinoma (MCF-
7) and lung carcinoma (A549) cells using colorimetric activities of the yellow MTT (3-(4,5-
dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide), each experiment was carried out using 5
replicates. 2 x 103 cells were implanted into a 96-well DMEM-containing platform, and 10% FBS was
implanted in a humidified atmosphere of 5% CO2 at 37 °C for 24 h. The culture medium was replaced
with 200 μL treating medium containing serial concentrations of tested substances and incubated
overnight, followed by addition of 20 μL (5 mg/mL, Sigma) of MTT solution. Afterward, the medium
has been carefully discarded and followed by the addition of 200 μl DMSO and agitated for 10 mins
to be ready for colorimetric reading at 490 nm using a microplate reader (Multiskan-Thermo
Scientific).
2.3. Glucose uptake measurement
The estimation of glucose uptake by cancer cells (MCF-7 and A549) has been detected through
the enzymatic NADPH-amplifying machine for 2-deoxyglucose. Briefly, cells have been seeded into
5-well plates and incubated overnight in Gibco™ RPMI 1640 Medium (cat no# 11875101)
supplemented with 11mM glucose, 1% penicillin/streptomycin (Mediatech), and 10% fetal bovine
serum (Gibco™ 26400044) at 37 °C for 12 h under 5% CO2 atmosphere. Samples had been analyzed
spectrophotometrically at excitation and emission wavelengths of 535 nm and 587 nm, respectively. A
reference curve has generated using 2-deoxyglucose-6-phosphate without cells. RPMI-1640
supplemented with 10% fetal bovine serum
2.4. Lactate Assay
The detection of the lactate amount produced has been carried out using 25 µL of the collected
medium of RPMI 1640 Medium and combined with NADH solution (100 µL, 0.03% of reduced NAD
in phosphate buffer) and pyruvate solution (25 µL. 22.7 mM pyruvic acid in phosphate buffer) at RT.
The amount of consumed NADH was quantified for each 2 min via measuring the absorbance at 340
nm. The mean absorbance was calculated as a percent (%) of the cell-induced via 500 uM NMDA.
Each treatment was examined with five repeats and replicates.
2.5. ATP Assay
The estimation of produced cellular ATP has been carried out using a luciferase-based ATP kit

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(Abnova™ ATP) to detect the activity of the curcuminoid derivatives in energy depletion in the
cancer cells. The principle of the assay depending upon the phosphorylation of glycerol and
fluorometric estimation at (Ex/Em = 535/587 nm).Cells were washed twice with ice-cold PBS followed
by 2.5% (w/v) trichloroacetic acid. Then, cells were collected and centrifuged at 9,000 rpm for 5 min
°
at 40 C, forming supernatant which was diluted and neutralized using tris-acetate buffer (pH 7.7).
The ATP content was calculated in the curcuminoid-treated cancer cells in comparison to ones
supplemented with a standard ATP inhibitor (oligomycin, 1µm). Analysis has been carried out using a
microplate reader (Multiskan-Thermo Scientific).
2.6. Western blot analysis
The current study investigated the expression of proteins in a time-dependent manner after 36 h
for (TNR-F1, DR4, DR5, and TRAIL) and 48 h for (Caspase 8, Caspase 3, P53, Bax) after the
execution of the treatments with FLLL49-GbA and Ag(I)FLLL49-GbA. Cells were cultured in
DMEM medium (Gibco™#31053028) containing the IC50 concentration values of FLLL49-GbA and
Ag(I)FLLL49-GbA in each cell type (MCF-7 and A549). After 24 h incubation, the adherent and
floating cells were collected and lysed in RIPA lysis buffer on ice for 30 min. Samples were
centrifuged at 12,000 rpm at 4 °C for 10 min. The amount of protein was measured by the BCA
protein assay kit (cell signaling technology# #7780). The same amounts of protein were separated
using 12.5% SDS polyacrylamide gels and transferred to PVDF membrane (IPVH00010, Merck
Millipore). The membrane was blocked with 5% skim milk in PBST (PBS with 0.05% Tween-20) for
30 min at room temperature and then probed with primary antibody dilution overnight at 4 °C. TNF-
R1 (D3I7K) (1:000; Cell Signaling Technology #13377), Anti-DR4 antibody (1:1000, Genetex#
GTX66667), anti-DR5 (1:500; MyBioSource# MBS820154), TRAIL Polyclonal antibody (1:500
MyBioSource# MBS8525702), caspases 8 (1:1000; Bioss# bsm-33190M), anti-P53 (1:1000;
Diagenode#C15410083), and anti-Bax (1:500; Biorbyt#orb224395). After blotting the PVDF
membrane was washed with PBS and covered with the specific secondary antibody for 2 h at room
temperature. After three washes, protein bands were captured by a chemiluminescence (ECL) kit
(Thermo Scientific (Cat# 31000). All expressions have been carried out in comparison to β actin
expression.
2.7. Detection of early and late apoptotic cells
DNA binding dyes Acridine orange (AO) and Ethidium bromide (EtBr), were used for the
morphological detection of viable, apoptotic, and necrotic cells. AO is taken up by both non-viable
and viable cells that emit green fluorescence when intercalated into DNA. EtBr is taken up only by
nonviable cells whereas; it is excluded by viable cells and emits red fluorescence by intercalation into
DNA. Cells were seeded on the cover slides inside a six-well plate. Cells were incubated in a CO2
°
incubator (5% CO2) at 37 C for 24 h, then treated with IC50 concentration of each compound and

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incubated for 48 h. Cells were washed with cold 1X PBS for three times. Cells were stained with a
mixture of AO (100 μg/ml)/ EB (100μg/ml) in PBS (1x) with 10% FBS for each well and then
incubated for 5 min at RT. The cover slides with cultured stained cells were transferred immediately
to new slides and the cells were ready to be visualized by the blue filter of the fluorescence
microscope [36,37]. Additionally, the encapsulated Ag(I)FLLL49-GbA complex with CNPs has only
been detected for its ability to induce apoptosis.
2.8. Anti-inflammatory activity of the novel curcuminoid derivatives
The anti-inflammatory activity of novel compounds was assayed by the method described in the
commercial TNF-α, IL-6, and IL1β ELISA kit purchased from Ray Biotech, USA. The expression
level was measured by the enzyme-linked immunosorbent assay (ELISA), using their specific pre-
coated microplates (12 × 8 microwell strips). Absorbance was measured immediately at 450 nm
against blank using an ELISA reader (RayBiotech, Canda). The study of the anti-inflammatory
cytokines was carried out in the time interval basis from 3 - 24 h after treatments.
2.9. Statistical Analysis
All data between two groups were analyzed using Student's t-test and more than two groups have
been conducted using a one-way analysis of variance (ANOVA) followed by Bonferroni tests.
Additionally, all data have been expressed as means ± standard errors (SEs), and P < 0.05 was
considered significant.
3. Results and Discussion
3.1. Chemistry
The novel conjugate (FLLL49-GbA) and its silver (I) complex (Ag(I) FLLL49-GbA) were
synthesized in a three-phases protocol. The first phase involves the preparation of a key starting
material, 1,7-Bis(4-hydroxy-3-methoxyphenyl)-4-cyclohexyl-hepta-1,6-dien-3,5-dione (FLLL49),
starting from curcumin (Cur) through three consecutive chemical reactions (Scheme 1A). Initially,
esterification using acetic anhydride was employed to protect the terminal hydroxyl groups of Cur
molecule, afterward, cycloalkytion of the methylene group in heptadione fragment by reaction
with1,5-diiodopentane in presence of anhydrous K2CO3 to produce diacetoxy derivative of FLLL49
(Ac2FLLL49), eventually, basic hydrolysis of Ac2FLLL49 to afford free FLLL49. The second phase
dedicated to the preparation of 1-(carboxymethyl)-2-ethyl-3-methyl-1H-benzo[d]imidazol-3-ium
chloride (Gboxin-analogue, GbA), a key bioactive reagent used for FLLL49 structural refinement.
Where 2-Ethylbenzimidazole (EBI) was used as a substrate for the preparation of GbA via N-
methylation followed by quaternization reactions using methyl iodide and chloroacetic acid,
respectively. In the final phase, carboxy terminal of GbA was coupled with the hydroxy terminals of
FLLL49 using 1-Ethyl-3-(3`-dimethylaminopropyl) carbodiimide (EDC) and 4-dimethylamino-

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pyridine (DMAP) as a mixed-coupling agent to form FLLL49-GbA conjugate (Scheme 1B).
Scheme 1: Stepwise protocol for the synthesis of; (A) 1,7-Bis(4-hydroxy-3-methoxyphenyl)-4-cyclohexyl-
hepta-1,6-dien-3,5-dione (FLLL49), (B) Gboxin analogue (GbA)-based FLLL49 conjugate (FLLL49-GbA) and
its silver(I) complex (Ag(I)FLLL49-GbA).
Finally, Ag(I)FLLL49-GbA complex was prepared simply by the reaction of FLLL49-GbA with
AgNO3 under the ambient conditions. On the other hand, the physical cross-linking protocol was used
+
to convert cationic chitosan (has NH3 groups that formed in acidic medium) into chitosan
nanoparticles (CNPs). In this context, CNPs were prepared by the ionic gelation of cationic chitosan
with polyanionic sodium tripolyphosphate (TPP) at room temperature [38]. The ionic gelation occurs
through the mutual electrostatic interactions between the cationic and anionic charges of chitosan and
TPP, respectively. In our work, we have performed the ionic gelation experiment in the presence of
Ag(I)FLLL49-GbA complex which results in the encapsulation of complex by CNPs (see Scheme 2).
This to take the full therapeutic benefit of the Ag(I)FLLL49-GbA complex and to overcome its side

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effects to magnifying its usage pharmacological applications.
Scheme 2: One step effective encapsulation of Ag(I)FLLL49-GbA complex with chitosan nanoparticles (CNPs)
3.2. Structural characterizations
3.2.1. Fourier-transform infrared (FTIR) spectroscopy
The changes in the FTIR spectral features of FLLL49-GbA in comparison to the starting materials
(FLLL49 and GbA) (Fig. 1A) could provide preliminary spectral evidence for its successful synthesis.
To name a few, the coexistence of the common absorption bands characteristic for both FLLL49
(3027 cm-1, aromatic C-H; 2995 and 2877 cm-1, CH3 and CH2; 1674 cm-1, C=O) and GbA (1580, 1534
and 1386 cm–1, C=N and C=C groups of benzimidazolium moiety) in the FTIR spectrum of FLLL49-
GbA confirms its formation.
Fig. 1: FTIR spectra of (A) key starting materials; (a) 1-(carboxymethyl)-2-ethyl-3-methyl-1H-
benzo[d]imidazol-3-ium chloride (Gboxin-analogue, GbA); (b) 1,7-Bis(4-hydroxy-3-methoxyphenyl)-4-
cyclohexyl-hepta-1,6-dien-3,5-dione (FLLL49); (c) FLLL49-GbA conjugate; and (d) Ag(I)FLLL49-GbA
complex. (B) (1) chitosan (CS); (2) chitosan nanoparticles (CNPs); (3) Ag(I)FLLL49-GbA complex; and (4)
encapsulated Ag(I)FLLL49-GbA complex ((Ag(I)FLLL49-GbA)CNPs).

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Meanwhile, the disappearance of the broad vibrational band centered at ~3400 cm-1 (attributable
to the vibration phenolic and carboxylic OH groups in FLLL49 and GA) along with the emergence of
a new stretch at 1734 cm-1 (ascribed to C=O group of ester fragment) prove the coupling of FLLL49
with GA through an ester bond. Importantly, the most significant spectral changes observed in the
spectrum Ag(I)FLLL49-GbA as compared to that of parent FLLL49-GbA are; (i) the negative shift of
the carbonyl stretch (Δν = -14 cm-1) coupled with its weakness, attributable to its participation in
chelation of Ag(I) ion. (ii) The emergence of a set of new absorption bands at 1767, 1585, 1539, and
–
1386 cm-1 confirm the involvement of nitrate anion (NO3 ) in the coordination sphere of Ag(I) ion.
–
Since the frequency gap between the vibration modes ν4 (1585cm-1) and ν1 (1386 cm-1) of NO3 group
–
is ~200 cm-1, thus, NO3 is coordinated to Ag(I) ion as a bidentate chelating ligand (Scheme 1B) [39].
3.2.2. Nuclear magnetic resonance (NMR) spectroscopy
1H NMR spectral data GbA, FLLL49, and FLLL49-GbA provide several strong pieces of
evidence for the successful coupling between GbA and FLLL49 to form FLLL49-GbA conjugate. To
name a few, the emergence of two sets of proton signals distinctive for GbA and FLLL49 in the
spectrum of FLLL49-GbA (Fig. 2) offers the first evidence; (i) The foremost set was observed at
chemical shifts of (ppm) 1.42 (3H), 2.54 (2H), 3.96 (3H), 5.27 (2H), and the range of 7.11-8.51 (2H)
attributable to the protons nuclear resonances of ethyl, methyl, methylene, and aromatic fragments of
the Gboxin analog (GbA) molecule [53].
Fig. 2: 1H NMR spectra of (a) 1-(carboxymethyl)-2-ethyl-3-methyl-1H-benzo[d]imidazol-3-ium chloride

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(Gboxin-analogue, GbA); (b) 1,7-Bis(4-hydroxy-3-methoxyphenyl)-4-cyclohexyl-hepta-1,6-dien-3,5-dione
(FLLL49); and (c) FLLL49-GbA in d6-DMSO.
(ii) The second group of proton signals which are characteristic for FLLL49 molecule was
observed at the chemical shift values (ppm): 1.42 – 1.86, 3.74, 6.58-8.52 ppm assignable for the
protons nuclear resonances of cyclohexyl, methoxy, olefinic and aromatic fragments of FLLL49.
Meanwhile, in comparison to the 1H NMR spectra of GbA and FLLL49 (Fig. 3), the disappearance of
the carboxylic COOH phenolic OH protons in the spectrum of FLLL49-GbA conjugate is indicative
of the involvement of these groups in the covalent binding of GbA with FLLL49 through an ester
bond.
3.2.3. Ultraviolet-Visible (UV-Vis) spectroscopy
The successful formation of Ag(I)FLLL49-GbA along with its stability profile under
physiological conditions could be examined based on UV-visible spectroscopy. Where the UV-vis
spectrum of FLLL49-GbA (10-3 M) shows two characteristic peaks centered at 262 and 419 nm. On
the other hand, four absorption peaks and shoulders were observed in the spectrum of Ag(I)FLLL49-
GbA at wavelengths 264, 405, 432, and 457 nm (see Fig S2, ESI†). Noteworthy, the higher energy
peak distinctive to the -* transition for a β-dicarbonyl group of FLLL49-GbA was slightly blue-
shifted (419 to 405) as a consequence of its coordination to metal ion [40]. To assess the stability of
Ag(I)FLLL49-GbA under physiological parameters, the UV-Vis absorption spectrum of a stocked
solution of Ag(I)FLLL49-GbA (10-5 M) in PBS-DMSO buffer (pH = 7.4) was monitored at
physiological temperature (37 °C) over a period of 72 h. As shown in Fig S2, the storage time has no
significant effect on the UV-Vis spectra recorded for the stock solution of Ag(I)FLLL49-GbA with
time, supporting the great stability of Ag(I)FLLL49-GbA complex under physiological conditions.
3.3. Ag(I)FLLL49-GbA-loaded chitosan nanoparticles
3.3.1. Structural and morphological characterization
The FTIR spectra of CS, CNPs, Ag(I)FLLL49-GbA and Ag(I)FLLL49-GbA-loaded chitosan
nanoparticles are depicted in Fig. 1B. As evident in Fig. 1B (2), three main peaks were observed at
3465, 1646, and 1543 cm−1, distinctive for CS segment (see Fig. 1B, (1)) [41,42], along with notice of
new vibration bands 1235 and 1159 cm−1, characteristic for TPP cross-linker [43]; indicating the
successful formation of CNPs. The FTIR spectrum of Ag(I)FLLL49-GbA-loaded chitosan
nanoparticles (Fig. 1B, (4)) shows the combined absorptions bands of the native Ag(I)FLLL49-GbA
complex, chitosan, and TPP cross-linker, however, with some changes in their respective shape,
position and intensity due to the physicochemical interactions occurred between these components. To
the name few, the characteristic main peaks of the Ag(I)FLLL49-GbA complex 2943, 1734, 1660,

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1585, and 1386 cm-1 (Fig. 1B, (3)) were observed in the spectrum of its encapsulated form with some
distortions, red shifts, and/ or decrease in their intensities. Further evidence for the encapsulation of
Ag(I)FLLL49-GbA by CNPs is an observation of a new peak around 750 cm-1 in the spectra of
encapsulated Ag(I)FLLL49-GbA (Fig. 1B, (4)) (characteristic for the out-of-plane bending vibration
mode of the imidazolium ring) [44], however, this peak has slightly shifted and decreased in intensity
than that in free Ag(I)FLLL49-GbA spectrum (Fig. 1B, (3)). Meanwhile, a set of peaks observed at
3461, 1645, 1539, 1234, and 1157 cm−1 (distinctive for CNPs) were also observed in the spectrum of
encapsulated Ag(I)FLLL49-GbA with slight alterations in their sites and/ or intensities (Fig. 1B, (2)
and (4)). In general, the FTIR results confirm the successful interaction between the Ag(I)FLLL49-
GbA and CNPs constituents (CS and TPP) and corroborate the involvement of this silver(I) complex
in the nanoparticle formulation.
The surface morphologies of CNPs and Ag(I)FLLL49-GbA-loaded CNPs were investigated based
on their respective SEM and TEM images that are given in Fig. 3 (A-E). The SEM image of CS (Fig.
3A) revealed a smooth texture with lamellar shape surface. However, the SEM image of CNPs (Fig.
3B) shows the spongy nature throughout the surface, providing them accessible cavities available for
encapsulation of Ag(I)FLLL49-GbA.

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Fig. 3: (A-C) Representative SEM images of CS, CNPs, and Ag(I)FLLL49-GbA-loaded CNPs, respectively. (D,
E) Representative TEM images of CNPs and (Ag(I)FLLL49-GbA)CNPs; inset graphs (F, G) are DLS-based
particle size distribution. (H, I) Zeta potential analysis of CNPs and (Ag(I)FLLL49-GbA)CNPs, respectively.
On the other hand, the SEM micrograph of Ag(I)FLLL49-GbA-loaded CNPs (Fig. 3C) displays a
fibrous surface with some aggregated structures. Meanwhile, the TEM images (Fig. 3D, 3E) showed
mono dispersive and homogenous spherical shapes of CNPs and Ag(I)FLLL49-GbA-loaded CNPs,
with very few agglomerations in case of (Ag(I)FLLL49-GbA)CNPs. The particle size distributions of
native CNPs and Ag(I)FLLL49-GbA-loaded CNPs were determined by dynamic light scattering
(DLS) (Fig. 3F, 3G; inset graphs in 3D, 3E, respectively). The DLS results indicated that the
diameters of CNPs were in the range of 54.51–112.25 nm with polydispersity indices (PDI) 0.29–
0.35, whereas, the diameters Ag(I)FLLL49-GbA-loaded CNPs were ranged from 79.92 nm (PDI 0.32)
to 205.63 nm (PDI 0.45). Noteworthy, the low poly-dispersion indices obtained for both nanoparticle
formulations are indicative of the narrow size distribution of the nanoparticles in their respective
suspension, and consequently formed homogeneous dispersion. This fact could offer plausible
explanation for increasing in size, entrapment efficiency, and zeta potential of nanoparticles when
they were loaded with Ag(I)FLLL49-GbA as compared to the neat CNPs. The Zeta potentials (ZP)
free CNPs and (Ag(I)FLLL49-GbA)CNPs were found to be 29.8±0.3 and 53.5±1.2 mV, respectively
(Fig. 3H, 3I), which in turns mean that these nanoparticles have enough surface charge to stabilize
them against agglomeration. This increasing in zeta potentials of may be attributed to the
spreading of cationic imidazolium groups onto the barrier interface of Ag(I)FLLL49-GbA-
loaded chitosan nanoparticles [45].
3.3.2. Ag(I)FLLL49-GbA loading and encapsulation efficiency
Based on the previous studies related to curcumin-loaded CNPs [29,30], 0.5 mg/mL of
Ag(I)FLLL49-GbA was used as an optimum initial concentration to get Ag(I)FLLL49-GbA-loaded
CNPs with maximum loading and encapsulation efficiency. The loading and encapsulation efficiency
of CNPs for 0.5 mg/mL Ag(I)FLLL49-GbA was found to be 17.9% and 83.5%, respectively. The
elevated loading and encapsulation efficacy of CNPs may be due to the spongy nature of their texture
which may have helped up-taking a large number of Ag(I)FLLL49-GbA molecules in the nanoparticle
core.
3.3.3. Ag(I)FLLL49-GbA release kinetics.
The Ag(I)FLLL49-GbA complex-encapsulated CNPs displayed an increased level of complex
release from the nanoparticles with time, as the complex was released in the treating medium by 5.8%
after 1 h of treatment till reached 45.6% after 24 h of the initial concentration under acidic conditions
(ABS, pH 5.3). While under neutral physiological conditions (pH 5), the released concentration was

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lowered to start with 2.9% after 1 h till 18.5% after 24 h as shown in Fig. 4. Therefore, the acidic pH
(5.3) is considered an ideal medium for releasing of the curcuminoid complex form is encapsulated
Nano-formulation ((Ag(I)FLLL49-GbA)CNPs) with high activity and bioavailability for its activity in
defending and curbing the tumor distribution. The present results were consistent with Popat et al. in
their effort to enhance the bioavailability of the curcumin derivatives by encapsulation with
nanoparticles; that increase their weak water solubility and rapid intestinal metabolism, ameliorated
long-term half-life in circulation which may lead to impressive outcomes for overcoming cancer cell
[46].
Fig. 4. In vitro Ag(I)FLLL49-GbA release profiles from CNPs at physiological and acidic conditions (pH 7.4,
pH 5.3) at 37 °C. Each point represents the mean ± SE (p ≤ 0.05, statistically significant).
Therefore, chitosan nanoparticles were considered as efficient drug delivery carriers for the silver
complex owing to their preferable release for this anticancer agent (Ag(I)FLLL49-GbA) in the acidic
medium that chracteristic for the extracellular environment of cancer cells, thus CNPs increase the
specifity and activity of Ag(I) complex for hampering cancer cell spreading [47].
3.4. Anticancer study
In vitro cytotoxic analysis test of the curcuminoid conjugate (FLLL49-GbA) and its silver(I)
complex (Ag(I)FLLL49-GbA) was carried out to detect their safe dose in comparison with a clinical
drug (cisplatin (CDDP)), using two human cell lines carcinoma MCF-7 and A549, and normal human
cell Hela using a 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) protocol. The
cell viability data were graphically represented in Fig. 5 and the IC50 values of the used compounds
were tabulated in Table 1 (Fig. 5). Generally, the parent conjugate presented less cytotoxic action
than its Ag(I) complex (Ag(I) FLLL49-GbA) toward all tested cell lines. Just to name a few, FLLL49-
GbA and Ag(I)FLLL49-GbA can reduce the viability of MCF-7 cells with a dose-dependent
performance (Fig. 5) and their respective IC50 values of 164.73 (±3.08) and 5.30 (±0.21) µg/mL,
respectively. therefore, it was obvious to state that the mean activity of Ag(I)FLLL49-GbA against
MCF-7 was not only higher than that of FLLL49-GbA with 31-times but also higher than CDDP
activity (IC50 = 10.29 (±0.25) µg/mL) with about 16 times.

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Table 1
IC50 (µg/mL)
Compd.
MCF-7
164.73±3.08
5.30±0.21
2.25±0.23
10.29 ±0.25
A549
Hela
FLLL49-GbA
Ag(I)FLLL49-GbA
(Ag(I)FLLL49-GbA)CNPs
CDDP
13.66±1.38 256.43±2.57
5.61±0.38
2.53±0.35
8.71±0.43
81.04±1.48
123.33±1.25
10.07 ±0.36
Fig. 5: Cell viability of (A) MCF-7; (B) A549; (C) Hela after 24 h of incubation in presence serial
concentrations of FLLL49-GbA, Ag(I)FLLL49-GbA, Ag(I)FLLL49-GbA complex-encapsulated CNPs, and
cisplatin (CDDP). Table 1: IC50 values (µg/mL) of the tested curcuminoid derivatives in comparison to a
clinical drug (CDDP).
To validate the cytotoxicity of the Ag(I)FLLL49-GbA complex-loaded chitosan nanoparticles
toward tested cell lines (MCF-7, A549, and HeLa), was in vitro MTT assay against these cells was
conducted. Interestingly, as revealed from the cell viability counts (Fig. 5), the cytotoxicity of
encapsulated Ag(I) complex has a 1.5-fold increased toward the cancerous cell lines (MCF-7 and
A549), contrary, the cytotoxic effect of Nano-formulation has 2-fold diminished against normal cells
(HeLa) in comparison to the un-encapsulated complex (p ≤ 0.05). For instance, at the end of 24 h of
treating with 10 mg/mL of free/ encapsulated complex; the still survival MCF-7 and A549 cells were
around 49%/ 27% (lower 1.8-time in case Nano-formulation) and 60%/ 35% (lower 1.7-time in case
Nano-formulation), respectively. In contrast, the HeLa cells have increased from 46% to 79% after
remediation with the free and encapsulated complex, respectively. Thus, the cytotoxicity of
Ag(I)FLLL49-GbA complex was significantly increased toward cancer cells (MCF-7 and A549),
while, it has reduced toward normal cells (HeLa) when loaded on chitosan Nano-vehicles. This
increase in cytotoxicity of the encapsulated Ag(I) complex by CNPs could be ascribed to the
enrichment in the internalization of complex-loaded CNPs by an endocytosis mechanism of the Ag(I)
complex into the cancerous cells, enhancing its uptake by cancer cells [26]. In addition, chitosan-
encapsulated Ag(I)FLLL49-GbA Nano-formulation showed ~2.3-times decrease in IC50 values as

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compared to free Ag(I)FLLL49-GbA after 24 h incubation (Table 1).
Although the exact mechanism for the promoted anti-proliferative activity of Ag(I) complexes in
comparison to the parent ligands are not well established, it has been proposed that the coordinated
silver(I) ion can exert its cytotoxic effect through two possible different mechanisms. The first
mechanism involves the binding of Ag (I) ion to thiol groups of proteins and interaction with DNA,
inducing apoptosis [48,49]. Additionally, it can interfere with mitochondrial function in cancer cells
through interaction of Ag(I) complex with mitochondria and thus stimulating the mitochondrial
apoptotic pathway [49]. Furthermore, the greater lipophilicity of Ag(I) complex (Clog P = -0.41) as
compared to the parent curcuminoid analog (Clog P = -2.33) increase the extent of binding to the
extracellular proteins of cancer cells and enhance its reaction kinetics inside them.50 Noticeably that
the anti-proliferation activity of Ag(I)FLLL49-GbA against MCF-7 cells (IC50 = 5.30±0.21 µg/mL) is
comparable to that toward A549 cell lines (IC50 = mL 5.61±0.38 µg/), however, it is ~2 and ~1.5
times higher than the activity of CDDP toward MCF-7 (IC50 = 10.29 ±0.25 µg/mL) and A549 (IC50 =
8.71 ±0.43 µg/mL), respectively. On the other hand, CDDP is more cytotoxic for normal Hela cell
lines (IC50 = 10.07 ±0.36 µg/mL) than Ag(I)FLLL49-GbA (IC50 = 81.04±1.48 µg/mL). Therefore, the
new chemotherapeutic candidate (Ag(I)FLLL49-GbA) is more effective than the clinical drug
(CDDP) in triggering caspases apoptosis in breast and lung cancer cells to prevent their proliferation
without health risks.
3.5. Performance of new curcuminoid derivatives as apoptosis triggering agents
AO/EtBr staining produced four types of cells that revealed the activity of the conjugate and the
complex in inducing apoptosis. As, the normal green nucleus represented the living cells, bright green
nucleus with fragmented chromatin represented the cell that are in early apoptotic, orange-stained
nuclei with chromatin condensation or fragmentation demonstrated the cells in late apoptotic and
necrotic cells were uniformly orange-stained cell nuclei (see Fig. 6). On one hand, FLLL49-GbA
strongly induced an early apoptotic programmed cell death in A549, MCF7, and HeLa cells with
preferable activity toward MCF-7, more than Ag(I)FLLL49-GbA complex, as revealed from high
population of the bright green nucleus. On the other hand, Ag(I)FLLL49-GbA is more effective in
inducing the late apoptotic pathway with an activity sequence against A549 > HeLa > MCF-7 cells.
Additionally, necrotic cells appeared only in the case of treating MCF-7 cells with Ag(I)FLLL49-
GbA complex. Moreover, the encapsulated Ag(I)FLLL49-GbA complex with chitosan nanoparticles
has induced late apoptotic activity with more effective action than other compounds in the three used
cell lines with P<0.05 as shown in Fig. 6.

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Fig. 6: The morphological detection of viable, apoptotic and necrotic cells stained with Acridine orange (AO)
and Ethidium bromide (EtBr). A and B) show the effect of FLLL49-GbA, Ag(I)FLLL49-GbA, and encapsulated
complex with chitosan nanoparticles on A549 tumor cells and their activity to induce early and late apoptotic
cell; C and D) show the effect of FLLL49-GbA, Ag(I)FLLL49-GbA, and encapsulated complex with chitosan
nanoparticles on MCF-7 tumour cells and their activity to induce early and late apoptotic cell; E and F) show the
effect of FLLL49-GbA, Ag(I)FLLL49-GbA, and and encapsulated complex with chitosan nanoparticles on
Hela cells and their activity to induce early and late apoptotic cell The present of late apoptotic cells; nuclei
condensation, as demonstrated by acridine orange/Ethidium bromide staining at 200X. Yellow arrows indicate
live cell, pink arrows indicate early apoptotic Red arrows indicate necrotic and blue arrows indicate late
apoptotic cells.
3.6. Inhibitory effects of FLLL49-GbA and Ag(I)FLLL49-GbA on energy indices
The present study attempted to provide a model for controlling the malignancy among cancer cell
lines by attacking their proliferation with new curcuminoid derivatives. These compounds have been
designed to induce energy depletion in cancer cells that may enhance the constringency for the
essential sources of their vitality and proliferation. Therefore, the inhibitory effects of FLLL49-GbA
and Ag(I)FLLL49-GbA on energy indices (glucose uptake, lactate production, and intracellular
ATP)have been studies. As shown in Fig. 7A, the glucose uptakes were significantly decreased in

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both cancer cell lines (MCF-7 and A549) after 24 h of treatment with FLLL49-GbA (P< 0.05), in
comparison to control cells. On the other hand, the Ag (I)FLLL49-GbA complex induced a high
significant sever decrease in glucose uptakes with P< 0.001 (for MCF-7 cells) and P< 0.01 (for A549
cells). Moreover, both curcuminoid derivatives have significantly decrease effects on the level of
glucose uptake in both cancer cell types than the CDDP treatment (P< 0.05) as shown in Fig. 7A.
Thus, these novel compounds may induce severe blockage for the glucose transporters that
responsible for enhancing the uptake of the glucose molecules. The obtained results have supported
the suggestion of Cho et al. who suggested that the up-regulation of glucose transporters (GLUT)
would contribute to increased glucose uptake that was considered one of the most prominent
characters of serous ovarian carcinoma [51].
Additionally, as lactate is considered one of the most important requirements of cellular energy
production for all living organisms and act as a functional initiator for protein, lipids, and nucleic acid
that enhances the cellular biosynthesis with ATP generation. The present study discussed the role of
the treatments of the novel synthesized curcuminoid derivatives on the produced lactate level that is
can be used as another source of the energy required for the proliferation of cancer cells. Additionally,
lactate rates of the tumor were associated with the increased metastasis and recurrence of the tumor
with poor outcome and stimulation of the inflammatory mediators and angiogenesis [52]. The activity
of FLLL49-GbA and Ag(I)FLLL49-GbA showed a significant decrease in lactate production in both
remediated cancer cells as compared to control cells with p<0.01 and P<0.001, respectively as shown
in Fig 7B. Moreover, their activity in inhibition lactate production was higher than the cisplatin
(P<0.05). Interestingly, these results were considered as supportive with previous research
documented by Elshaarawy et al.53 who revealed that the activity of Pd(II) complex of Gboxin analog-
chitooligosaccharides conjugate (Pd(II)COS@GbA) had an effective role in the depletion of energy
production through its activity in the disruption of the ATPase expression.
Prominently, Ag(I)FLLL49-GbA has promising effects in inducing glucose uptake reduction
(49% and 80% reduction among MCF-7 and A549 cell lines, respectively) and decreasing lactate
production, an essential glycolysis product, (decrease by 61% and 56% for MCF-7 and A549,
respectively).
Noteworthy, the positively-charged ends of new curcuminoid derivatives could act as active sites
to specifically bind and block the negatively-charged lactate anions which highly secreted across the
plasma membrane of proliferated cancer cells as a result of increased glycolysis process [54]. So, the
positive charge can offer the specificity and sensitivity of these compounds toward the cancer cells
more than normal cells.
As the main aim of this study was to explore novel anticancer candidates able to induce
malfunction of energy supply sources of the cancerous cell, that help them to sustain their
proliferation and disrupt the inflammatory system.. So, the capacity of the new curcuminoid
derivatives to inhibit energy production expressed as intracellular ATP was assessed in comparison

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with a standard ATP-production inhibitor (oligomycin, 1µm). On one hand, as shown in Fig. 7C,
oligomycin, and cisplatin treatment have inhibited the intracellular ATP production with a significant
level of P<0.01 and P<0.05, respectively.. On the other side, FLLL49-GbA and Ag(I)FLLL49-GbA
showed a significant decrease in the produced ATP with P<0.01 and P<0.001, respectively to both
cancer cell lines (MCF-7 and A549). Additionally, it was obvious that the novel compounds have an
effective role in the deprivation of the possible sources of energy production and decreased level of
ATP which was correlated with the decreased glucose uptake and lactate production by the cancer
cell. Furthermore, the present study revealed that the novel curcuminoid derivatives enhanced the
inhibition of the energy sources through glycolysis that may trigger the eradication of tumor cell
proliferation. Additionally, the novel synthesized compounds triggered the apoptotic actions in the
form of early, late apoptosis, and necrosis in both cell types as shown in Fig. 6.
Fig.7: The role of novel synthesized FLLL49-GbA and Ag(I)FLLL49-GbA and cisplatin (CDDP) on the level
of glucose uptake, lactate production, and ATP produced on MCF-7 and A549 tumor cell lines: A) relative
ration of glucose uptake; all cultural media has been supplemented with 11mM glucose B) Relative ratio of
lactate production; C) relative ration of intracellular produced ATP; Error bars represent ±S.E., *p < 0.05, **p <
0.01, Student’s t test. All experiments were repeated at least 5 times.
Therefore, our results were mostly agreed with Shiratori et al., Eguchi et al., and Nicotera et al.
who reported that cell death-induction is associated with changes in mitochondrial produced ATP
levels [55,56,57], and this incidence can occur in various ways based on the ATP level decrease in the
cell that has been shown in several cell types to trigger apoptosis [53,58].
3.7. The anti-inflammatory activity of FLLL49-GbA and Ag(I)FLLL49-GbA.
For more profound details of the mechanistic role of the novel synthesized compounds in the
treatment of the cancer cell and metastasis; the present study showed that the enhancement of the anti-
inflammatory activities was important for controlling the cancer cell proliferation to fence the body
homeostasis. Moreover, the present study proposed that the inflammatory stress in the cancer cells
and the role of the treatment in the down-regulation of the inflammatory mediators were in time-
dependent action. Also, the release of various pro-inflammatory, oncogenic mediators like IL-1β,
interleukin-6, and tumor necrosis factor-α (TNF-α) facilitate the elucidation of the tumor
microenvironment. Firstly, Ag(I)FLLL49-GbA complex showed a highly significant effect on the

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produced pro-inflammatory cytokines and oncogenic mediators such as TNF-α, IL1β, and IL6 as
illustrated in Fig. 8. It was revealed that A549 cells are more sensitive than MCF-7 cells; Ag(1)
complex have induced significant decrease for TNF-α in comparison to untreated cells and cisplatin
treatment for both cell lines (MCF-7 and A549) after the 3rd hour of the treatments with P<0.01 to
P<0.001, respectively, as shown in Fig 8A.
Secondly, interleukin-6 (IL-6) is considered as one of the most pro-inflammatory cytokines
characterizing the disturbance of inflammation in cancerous cells and playing a central role in the
expansion and differentiation of the tumor. The treatment with both new compounds for MCF-7 and
A459 cancer cells has a significant putative inhibitory effect on the pro-inflammatory factor IL-6 with
P<0.001 6 h post-treatment (Fig 8b) in comparison to cisplatin that induced down-regulation for the
IL6 with P< 0.05 for MCF-7 from the 3rd hour to 24 h; and P<0.01 for the 6th hour of treatment that
increased their down-regulation activity by the 24th hour for A549 cell lines. Thirdly, Fig. 8C revealed
that cisplatin did not effect on the IL1β expression level after 3 h of treatment for MCF-7 and 6 h for
A549 cell lines.
Moreover, on one side it was obvious that the treatment with both compounds showed a high
inhibitory effect for IL1β expression from the 12th hour of treatment with P<0.001 for both MCF-7
and A549 cell lines. Nevertheless, FLLL49-GbA induced down-regulation of the expression after 12th
hour with a value of P<0.01 and P< 0.001 for MCF-7 and A549 cell lines, respectively, while Ag(I)
induced reduction effect by P<0.001 for both carcinoma cell lines. Additionally, the overlapping
effect was obvious between the treatment with cisplatin and FLLL49-GbA with P<0.01 for A549 cell
lines from the 3rd hour till the 24th hour (Fig. 8C).
Fig. 8: The quantitative anti-inflammatory activity of FLLL49-GbA and Ag(I)FLLL49-GbA and reference drug
cisplatin by ELISA on both tumour cell types MCF-7 and A549. A) Demonstrate the effect on TNF α
expression; B) IL6 expression; C) IL1β expression. Error bars represent ±S.E., *p < 0.05, **p < 0.01, Student’s t
test. All experiments were repeated at least 5 times.
3.8. Putative role of FLLL49-GbA and Ag(I)FLLL49-GbA on activation of death receptors
Caspase activation introduces a dynamic proteolysis cascade in which upstream caspases are
exclusively cleaving peptide caspases, which have been progressively further used to facilitate cell

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destruction through substrates cleavage [59,60]. Concerning the importance of apoptosis mechanisms
in cancer cells, particular attention was paid to the rate of death receptors expression 36 h post-
treatment for both cancer cells with the novel synthesized compounds. As death receptors and their
ligands are considered important mediators for inducing apoptosis and regulation of tumor
proliferation that can be a pivotal therapeutic target for tumor therapy.
Interestingly, in the first 36 h of treatment, the Ag(I) complex has significantly down-regulated
the TNF-R1 expression of MCF-7 cells (P<0.01), more than the action of both the parent ligand
(FLLL49-GbA) and cisplatin CDDP (P<0.05). Additionally, the activity of the novel curcuminoid
derivatives showed significant up-regulation for the TNF-R1 receptor among A549 cell lines. This
may reflect the high activity of Ag (I) complex on the breakage of the death domain-containing
receptors that allowing the protein-protein interaction leading to inhibiting the TNF-α signaling
cascade and its anti-inflammatory action [61]. Therefore, the TNF-α/ TNF-R1 interaction has shown
close relation to caspases cascade pathways and apoptosis through their antagonistic effects that
inhibit or trigger the signaling cascade of the inflammation among cancer cell lines [61].
Additionally, DR4 and DR5 have been significantly up-regulated after 36 h of MCF-7 cell lines
treatment. The treatment with FLLL49-GbA conjugate induced significant up-regulation for DR4 and
DR5 with a value of P<0.01 and P<0.05, respectively; more than the effect of treatment of
Ag(1)FLLL49-GbA complex; while the Ag(I) complex has significant increase for TNF-R1 and
TRAIL expressions; both treatments were more effective than the cisplatin treatment (Fig. 9A,B).
Through reporting its capacity in cancer cell death induction, TRAIL (TNF-related Apoptosis-
Inducing Ligand) has developed a growing interest in oncology and tumorgenesis. Mérino et al.
proposed that TRAIL and TRAIL derivatives were already have been considered as molecules with
substantial anti-tumor action leading to cell death in diverse cancer cells without in vivo massive
consequence in vivo [62]. So, our results concerning TRAIL expression was valuable for the treatment
with the synthesized compounds, as Ag(1) complex induced highly significant expression level than
the ligand and cisplatin with significant value of P<0.01 for MCF-7 cells and P<0.001 for A549 cells
as shown in Fig 9B,C. Additionally, the Ag(1) complex showed effective up-regulation for the death
receptors and TRAIL proteins in A549 cell lines more than that in MCF-7 as follows P<0.01, P<0.01,
P<0.001 for Dr4, DR5, TRAIL, respectively Fig. 9B,C. DI Paolo et al. showed that up-regulating
TRAIL expression can be used as a factor for tumor regression among cyclophosphamide treated
mice [63], and additionally, Quast et al. reported the necessary role of TRAIL expression in triggering
the apoptotic mechanisms dependently on Bax protein expression [64].
3.9. The potential activity of FLLL49-GbA and Ag(I)FLLL49-GbA on activation of the tumor
suppressor proteins P53 and Bax
Moreover, through the obtained results that reflect the significant impact of the new curcuminoid

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derivatives on the tested cancerous cells; so, it was worth studying the effects of these compounds on
caspases activation mechanisms and apoptotic proteins, which are considered as the most important
mechanisms that explain the role of safe chemotherapy. So, the caspases group (8 and 3) and the
apoptotic markers (P53 and Bax) have been estimated 48 h post-treatment with FLLL49-GbA and Ag
(1) FLLL49-GbA to study the final effect of these compounds in triggering the apoptosis and
inhibition of cell proliferation. The slightly weak expression of caspase 8 proteins in the untreated
control cells of MCF-7 and A549 was parallel with the expression of caspases 3, P53, and Bax.
Besides, the Ag(I)FLLL49-GbA induced highly significant up-regulation for caspase 3, tumor
suppressor proteins P53, and Bax with a value of P<0.001 within the 48 h post-treatment of cancer
cell lines (MCF-7 (Fig. 9A,B) and A549 (Fig. 9A,C) in comparison to control untreated cells as
shown in Fig. 9A. Additionally, caspases activation for apoptosis has been reported with various
triggering structural cascade in form of effector caspases that applied in form of caspases 8 that
enhance the extrinsic apoptotic [65,66] and caspases 9 to promote intrinsic apoptotic pathways [67].
Besides, McComb et al. reported that the caspases 3 and 7 revealed an important role with complete
expression integrity for stimulation of the internal and external apoptosis pathways [68]. The down-
regulation has contributed to the decreasing level of the expressional pathway of both caspases 8 and
9 that appeared to be coincident with delayed cytochrome c secretion that may affect the apoptosis
feedback action.
The present study revealed that Bax protein and P53 were highly decreased in their expression in
both types of carcinoma cells. On the contrary, Perego et al. have shown that cisplatin-induced
reduction in Bax protein expression that was closely linked to drug resistance in ovarian carcinoma
cells treatment [69]. So, our results revealed valuable results for the treatment of both cancer cell
types and avoiding the problem of the drug resistance that always appears among cisplatin-treated
carcinoma. Besides that, the novel synthesized compounds were associated with the apoptotic Bax
protein expression up-regulation [70,71].
Fig. 9: Western blot analysis in MCF-7 and A549 treated with FLLL49-GbA and Ag (I) FLLL49-GbA and
reference drug cisplatin after 36hrs and 48hrs of treatments. A) The expression state of TNF-R1, DR4, DR4,
TRAIL, Caspases 8 and 9, P53, Bax; B) MCF-7, C) A549, the densometric analysis of western blotting . Error
bars represent ±S.E., *p < 0.05, **p < 0.01, Student’s t test. All experiments were repeated at least 5 times.

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So, activators of Bax expression can, therefore, be used as substitution drugs that improve their
expression as a support therapy to facilitate the pro-apoptotic pattern with the order to manage the
drug resistance and to increase apoptotic stimuli with tumor reduction [72,73]. Furthermore, an
important implication of these findings is that the novel synthesized curcuminoid conjugate and its
complex had modified the natural defect of the curcumin treatment through up-regulation of their
bioavailability and defending their low aqueous solubility [74]. So, curcumin analogs are considered
as a good approach towards boosting curcumin 's bioavailability, several studies have concentrated on
developing novel supply systems for the improvement of curcumin pharmacokinetics through
encapsulated proteins that had promising anticancer efficiency in MCF-7 cell and high bioavailability
in rats [75].
So, the present study tried to improve the applied therapeutic regimes of curcumin applications by
increasing their rate of bioavailability and action that can be used as a novel anti-tumor drug that is
still under investigation without drug resistance and delude the pleiotropic dysfunction signaling of
apoptosis.
So, the novel synthesized curcuminoid conjugate and its Ag(I) complex represented promising
candidates to be used alone or in combination together with other therapies as an effective anti-cancer
medication. It impacts negatively on numerous signaling pathways and molecular targets that result in
the inhibition of cancer growth and proliferation.
Conclusion
The findings of our research are quite convincing, and thus the following conclusions can be
drawn; 1) the activity of curcumin macromolecule with Gboxin and its complex with Ag (I) have the
ability to control breast and lung cancer cell depending upon their action on the depletion of the
intracellular ATP altogether with the glucose and lactate production. 2) Both derivatives have the
ability to direct cancer cell to apoptotic pathway through time-dependent behavior by inhibition of the
inflammatory cytokines and up-regulation of the death receptors within 36 hours of treatments and
apoptotic caspases such as caspases 3 and caspases 8 with 48 hours of treatment. 3) Additionally, the
findings are quite convincing through the ability of the novel synthesized compounds to induce cancer
cell apoptosis and cell death through P53 and Bax expression up-regulation. 4) Finally, the study
offered a new method to overcome the problems of the natural curcumin characters such as
bioavailability and solubility to ameliorate their activity in defending canner cell proliferation.
Moreover, application of encapsulated Ag(I) complex with chitosan nanoparticles (CNPs)
significantly triggered apoptosis for cancer cells with low cytotoxicity for HeLa normal cells with
released rate at PH medium of 5.3 more than 7.4 with beneficial cancer-targeted drug delivery and
hampering cancer cell spreading. Based on the promising findings presented in this research, it was
valuable to clear out that the curcumin Ag (I) complex has a potential action activity more than the
curcumin conjugate and may provide new perspectives for the development of novel chemotherapies

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with less drug resistance for adjunctive treatments of breast and lung cancer cells.
Appendix A. Supplementary data
Supplementary data (experimental and spectral data) associated with this article will be found, in the
online version, at doi:
Compliance with ethical standards
Conflict of interest all authors contributed equally to the production of the manuscript and show no
conflict of interest.
Funding The authors extend their appreciation to the Dean of Scientific Research at King Khaled
University for supporting this work through general research project fund under grant number (G. R.
P / 124 / 41).
Ethical approval this article does not contain any studies with human participants or animals
performed by any of the authors.
References

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Author statement
Serag Eldin I. Elbehairi; Conceptualization, Methodology, Software, Data curation, Validation,
Visualization, Writing - Original Draft.
Lamia A. Ismail; Conceptualization, Methodology, Software, Data curation, Validation,
Visualization, Writing - Original Draft.
Mohammad Y. Alfaif: Conceptualization, Software, Data curation, Validation, Visualization,
Writing - Original Draft.
Reda F. M. Elshaarawy: Conceptualization, Methodology, Software, Data curation, Validation,
Visualization, Writing - Original Draft, Writing - Review & Editing
Hani S. Hafez: Conceptualization, Methodology, Software, Data curation, Validation, Visualization,
Writing - Original Draft, Writing - Review & Editing
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