Molecular Biology Reports (2023) 50:3503–3513

https://doi.org/10.1007/s11033-022-07667-9

ORIGINAL ARTICLE

Synthesis of Gingerol-loaded Uio-66 nanoparticles and its anti-cancer

effect against gastric cancer cell line (AGS)
Irana Kazazi1 · Fatemeh Ashrafi1 · Maryam Malekloo2

Received: 14 March 2022 / Accepted: 31 May 2022 / Published online: 14 February 2023
© The Author(s), under exclusive licence to Springer Nature B.V. 2022

Abstract
Background Gastric cancer is the world’s fifth most prevalent cancer and its treatments are associated with issues. In this
investigation, a UIO-66 nanoparticle was loaded with Gingerol (UIO-66-Gin) as a great drug carrier vehicle for chemo-
therapy of the AGS cancer cell lines.
Methods and Results UIO-66-Gin characterization was performed using SEM, DLS and FTIR tests. The release profile of
Gin from UIO-66 was also assessed. The cytotoxicity of UIO-66-Gin against AGS cells was assessed using MTT assay.
Caspase3, Caspase9, Bax, and Bcl2 genes expression was evaluated via Real-time PCR and apoptosis rate was performed
using flow-cytometry assay. Size analysis indicated the spherical shape of nano-formulation with the mean size of 174.3 nm.
Release analysis indicated that there was a 50% Gin release from the nanocarrier was reported in roughly 21 h, which
revealed the regulated release of bioactive compound from the UIO-66 formulation in PBS medium. After 48 and 72 h, vari-
ous concentration of both the Gin and UIO-66-Gin started to induce cytotoxicity in cancerous cells. However, the induction
of cytotoxicity was higher in cells treated with UIO-66-Gin. UIO-66-Gin could induce the expression of pro-apoptotic (Bax,
Caspase3, and Caspase9) genes and down-regulate the expression of Bcl2 as anti-apoptotic gene rather than other formula-
tion. Flowcytometry results indicated that the elevation of apoptotic rate in cells treated with UIO-66-Gin was significantly
higher than Gin treated cells.
Conclusions Our investigation revealed the potent anticancer effect and apoptotic induction ability of UIO-66-Gin against
cancerous cells through altering the expression of genes involved in apoptosis.

Keywords Gastric cancer · UIO-66 · Gingerol · Cytotoxicity · Apoptosis

Abbreviations UIO-66 Zirconium 1,4-dicarboxybenzene MOF

Bax  Bcl2 Associated X
Bcl2 B-cell lymphoma 2
AKT serine/threonine-specific protein kinase Introduction
DLS Dynamic Light Scattering
SEM Scanning Electron Microscope Despite the fact that the global incidence of gastric cancer
FTIR Fourier transform infrared has reduced, it is still the world’s fifth most prevalent can-
FITC Fluorescein isothiocyanate cer. In 2018, there were 1,033,701 reported cancer cases and
PS phytosiderophores 782,685 fatalities worldwide[1]. Gastric cancer is caused by
ZIF-8 Zeolitic imidazolate framework-8 a mixture of environmental causative factors and the aggre-
gation of definite genetic changes[2]. H. pylori, incorrect
lifestyle, and a poor socioeconomic position are all key risk
factors for gastric cancer[3]. In the initial stages of gastric
Fatemeh Ashrafi
F_ashrafi@iau-tnb.ac.ir; Mnfa.ashrafi@yahoo.com cancer, surgical intervention is frequently used, but adju-
vant treatments including such radiation therapy or chemo-
1
Department of Biology, Tehran North Branch, Islamic Azad therapy are frequently utilized in the advanced stages[4].
University, 16511-53311 Tehran, Iran Due to various side effects of standard treatments, there is a
2
Department of Biology, Science and Research Branch,
Islamic Azad University, Tehran, Iran

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3504
considerable need for complementary and innovative rem-
edies in cancer therapy.
Now days, natural components and phytochemicals are
becoming trendier owing to their great benefits to human
health and anti-cancer properties. Ginger is an antiquated
herb employed to relieve various diseases due to its anti-
bacterial, antifungal and antioxidant characteristics [5–7].
[6]-Gingerol, a potent phenolic component obtained from
ginger, has medicinal effects such as anti-inflammatory,
antioxidant, and anticancer activity[8]. The capacity of
6-gingerol to alter multiple signaling pathways, particu-
larly nuclear factor-kB (NFkB), AKT, extracellular-signal-
regulated kinase (ERK)1/2, c-Jun N-terminal kinase, and
p53, is largely responsible for its anticancer effects[9–11].
However, 6-gingerol has a number of disadvantages that
limit its potential use, including temperature, pH, and oxy-
gen susceptibility, and low water solubility[12]. As a result,
the advancement of drug delivery carriers for the safer and
more regulated administration of 6-gingerol is critical.
Recently, the establishment of a nanotechnology-based
delivery system for these chemicals has received a lot of
interest. Metal Organic Frameworks (MOFs) have been
designed for biomedical purposes as a composite particles
consisting of crystalline porous materials attributed to their
larger specific surface area and significant porosity in com-
parison to other inorganic materials[13]. The nanometric-
MOFs (NMOFs) demonstrated distinct physicochemical
features, such as a greater specific surface area when com-
pared to the micrometer scale MOFs, resulting in significant
drug loading and therapeutic effectiveness[14]. For drug
delivery applications, NMOFs including such ZIF-8 [15],
Metal–Organic Framework MIL-101 [16], and UIO-66 [17]
have been employed.
UIO-66, a zirconium-based MOF, offers remarkable
features such as strong mechanical capabilities and water
stability, allowing it to be used in biological and medici-
nal applications[18, 19]. Because of their great stability in
aqueous media and bloodstream, as well as their excellent
biocompatibility, UIO-66 MOFs may be a suitable choice
for drug delivery of chemotherapeutics and phytochemical
agents to treat cancers. Furthermore, the existence of open
pores in the UIO-66 MOF matrix resulted in the incorpo-
ration of a high concentration of bioactive compounds. In
addition, UIO-66 MOF might be used as a pH-sensitive
vehicle for the regulated delivery of bioactive compounds
into malignant cells[20]. Therefore, the Gingerol (as anti-
cancer agent) was loaded into UIO-66 MOFs (UIO-66-
Gin) fabricated using the aerogel approach in the current
investigation. DLS, SEM, and FTIR analyses were also
used to analyze the synthesized UIO-66-Gin. Experiments
on therapeutic release were also conducted. Consequently,
the anticancer activity and apoptotic effect of UIO-66-Gin
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Molecular Biology Reports (2023) 50:3503–3513
formulation was tested against AGS (gastric cancer cell
lines) cells using MTT-assy, Real-time PCR technique and
flow-cytometery approach.
Materials and methods
Nanoparticle preparation
All materials were purchased commercially (Sigma-Aldrich
and merck) and employed absolutely. UIO-66(ZrCl4) was
produced using earlier described processes. In a glass scintil-
lation vial, zirconium chloride (ZrCl4) (0.053 g, 0.227mmol)
and terephthalic acid (TPA) (0.038 g, 0.227mmol) were
combined in N,N-dimethylformamide (DMF) (26.5mL,
340mmol). The combination was then heated in an oven at
120 °C for 24 h. Finally, the produced yellow precipitate
was a sign of the synthesis of UIO-66 nanoparticles[21].
To load Gin powder into the UIO-66 structure, 2.5 mg of
Gin powder was dissolved in 10 ml of ethanol, followed by
10 mg of synthesized UIO-66. For 24 h, the current mixture
was stirred. The resultant mixture was then washed three
times with ethanol solvent before being vacuum-pumped
to remove the solvent. Eventually, the precipitate was dried
overnight at 79 ° C and then the formation of UIO-66-Gin
nanoparticles was observed.
Characterization tests and drug release
Scanning electron microscopy (SEM) (TESCAN, VEGA
3SB) and dynamic light scattering (DLS) (Zetasizer Nano
S90) were used to analyze the morphology and particle size
measurements of UIO-66-Gin nano-formulation, respec-
tively. The functional groups of Gin, UIO-66, and UIO-
66-Gin were determined using Fourier-transform infrared
spectroscopy (FTIR) using FT-IR Spectrophotometer
(Model 8300, Shimadzu Corporation, Tokyo, Japan) rang-
ing from 4000 − 400 cm− 1.
To accomplish Gin release from generated nano-formu-
lation, 5 mg UIO-66-Gin were incubated at 37 °C in 2 mL
Phosphate-buffered saline (PBS) before being sealed in a
dialysis bag (molecular cut off 12 kD). The samples were
then immersed in 20 mL of 0.1 M PBS (pH value of 7.4).
For three days, the suspensions were placed in a thermo-
stated shaking water bath (Hidolff) at 37 °C and 100 rpm. At
various intervals (12 h, 24 h, 36 h, 48 h, 60 h, and 72 h), 2.0
mL of releasing solution was removed from the dissolving
media, and 2 mL of new buffered was poured to the incuba-
tion media. UV-Vis spectroscopy was used to predict the
overall quantity of Gin.
Molecular Biology Reports (2023) 50:3503–3513
Table 1 Primer sequences for tested genes
Genes Primer sequences
b-actin Forward: 5’- TCCTCCTGAGCGCAAGTAC-3’
Revers: 5’- CCTGCTTGCTGATCCACATCT-3’
Casp3 Forward: 5’- CATACTCCACAGCACCTGGTTA-3′
Revers: 5’- ACTCAAATTCTGTTGCCACCTT-3’
Casp9 Forward: 5’-CATATGATCGAGGACATCCAG-3
Revers: 5’-TTAGTTCGCAGAAACGAAGC-3’
Bax Forward: 5’-CGGCAACTTCAACTGGGG-3′
Revers: 5’- TCCAGCCCAACAGCCG-3′
Bcl2 Forward: 5’- GGTGCCGGTTCAGGTACTCA-3′
Revers: 5’- TTGTGGCCTTCTTTGAGTTCG-3′
Cell culture and cytotoxicity assay
In this study, gastric cancer cell line (AGS) (human gastric
adenocarcinoma, IBRC C10071) was obtained from the Ira-
nian National Center for Genetic and Biologic Resources
and human foreskin fibroblasts cell (HFF) (as normal cell
line) was purchased from Royan Stem Cell Technology
Co, Iran. The cells were incubated at 37 °C in a humidi-
fied environment of 5% CO2. Then, the cells were cul-
tured in RPMI-1640 media supplemented with 10% fetal
bovine serum (FBS; Sigma-Aldrich), 100 U/l penicillin, and
100 mg/l streptomycin and cells were cultivated to 60–80%
confluence.
To investigate the cytotoxic effect of free Gin, and UIO-
66-Gin (18.75, 37.5, 75, 150 and 300 µM) were cultured for
48 and 72 h with 105 cells in a 96-well plate before being
assessed with the colorimetric MTT test. Following 48 and
72 h of cell seeding in culture, the wells were detached and
washed with PBS prior to incubation for 3 h with 20 mL
of 5 mg mL− 1 MTT solution in PBS. DMSO was used to
disintegrate the resulting formazan crystals. The absorbance
of the resultant collections was evaluated at 570 nm using
an EPOCH microplate spectrophotometer (synergy HTX,
Bio-Tek, USA). The Inhibitory Concentration (IC50) value
of Gin and UIO-66-Gin formulation were predicted via
GraphPad Prism 6[22].
Gene expression evaluation
In order to evaluate the expression of pro-apoptotic and
anti-apoptotic genes, firstly, total RNA was extracted using
RNX-Plus (CinnaGen) kit, based on the manufacturer’s pro-
tocols. The first-strand cDNA was synthesized using Fer-
mentas First-strand cDNA Kit, for a total volume of 20 µL.
The Real-time PCR technique (SYBER Green method)
was utilized to evaluate the expression of Bax, Bcl2, Cas-
pase 3, and Caspase 9 genes in the treated cells with both
Gin and UIO-66-Gin. The β-actin gene was utilized as an
internal control. Each reaction mixture contained 12.5 µL
3505
of Master mix (Bioneer, Korea); 1 µL of generated cDNA
(0.1–1 µg of β-actin and intended genes); 1 µL (10mM) of
primers for each gene (Takapuzist business, Tehran, Iran);
and 9.5 µL of DEPC water. The primer sequences are shown
in Table 1. Each gene’s PCR reaction was done individually
and three times. The real-time PCR conditions were tuned
using the Bioneer Exicycler 96 system and the following
schedule: a 10-minute initial denaturation phase at 95 °C,
followed by 40 cycles at 95 °C for 20 s, 55 °C for 40 s, and
72 °C for 40 s [28]. Rest software was used to calculate gene
expression using the 2−ΔΔCt technique[23].
Apoptosis analysis
Annexin V might be represented as a responsive probe for
the flow cytometric assessment of cells bearing apoptosis,
because of its greater affinity for PS, whenever it interlaced
with FITC. Because of its conjugating characteristic, PI
may also stain DNAs in flow cytometry. Because PI cannot
infiltrate the membranes of living and apoptotic cells, it only
labels dead cells. As a result, this staining can be helpful to
distinguish between necrotic, apoptotic, viable, and killed
cells.
An annexin V-FITC/PI staining kit (Affymetrix biosci-
ences, USA) was performed according to manufactures’
instructions to verify whether IC50 concentration of Gin
and nano-formulation of it can induce apoptosis in AGS cell
lines after 24 h or not. For this, AGS cells (105 cells) were
treated with Gin and UIO-66-Gin formulation for 24 h. After
treatment, the cells were washed with PBS and fixed with
70% ethanol solution. For at least 12 h, the suspension was
kept at 4 °C. After centrifugation, the cells were treated with
propidium iodide (PI, 50 g/mL) and FITC-labeled annexin
V (1 mg/mL) and maintained in the dark for 15 min. The
fluorescence of FITC-labeled annexin V and PI was then
quantified at excitation (495 and 535 nm) and emission (519
and 617 nm) wavelengths using the FACSCalibur flow-
cytometry system (Becton, Dickinson, Franklin Lakes, NJ,
USA)[23].
Statistical analysis
GraphPad Prism 6 software was used for statistical data
analysis, and results were represented as mean ± SD. For
comparing two independent groups, the Student t test was
employed, and for analyzing multiple samples, the ANOVA
test was applied. Significant analysis was defined as a p
value < 0.05.
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3506 Molecular Biology Reports (2023) 50:3503–3513

Fig. 1 (A) DLS graph of UIO-66. (B) DLS graph pf UIO-66-Gin. (C) SEM micrograph of UIO-66-Gin. (D) FTIR spectra of Gin, UIO-66 and
UIO-66-Gin. (E) The release profile of Gin and UIO-66-Gin in PBS medium after 72 h

Results
UIO-66-Gin characterization
Drug loaded MOFs were prepared by the aerogel proce-
dure via UIO-66 as a vehicle. The UIO-66 and UIO-66-
Gin nanoparticles had average diameter in the range of
174.3 nm (PDI = 0.182) and 233.9 nm (PDI = 0.190), respec-
tively, which was confirmed with DLS method (Fig. 1 A and
Fig. 1B). This allows it to cross the blood-brain barrier and
concentrate in bone marrow. SEM of UIO-66-Gin (Fig. 1 C)
indicates the spherical shape with smooth and uniform sur-
face and agglomeration of drug-loaded nanoparticles.
The FTIR pattern for UIO-66-Gin nanoparticles indi-
cates different characteristic peaks of Gin and Zr in the
range of 498–1748 cm− 1. It is representative of Zr–(OC)
asymmetric stretching (468 and 553 cm− 1), O–H bending
and Zr–O modes (670 and 749 cm− 1), C–O root in C–OH of

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Molecular Biology Reports (2023) 50:3503–3513
carboxylic acid (1401 cm− 1), in- and out-of phase stretching
modes of the carboxylate group (1505 cm− 1), and C–C in
the aromatic compound of the organic linker (1583 cm− 1),
which indicates successful synthesized UIO-66 nano-for-
mulation. When Gin joins the UIO-66 structure, the C = O
bond absorption band appears in the 1748 cm− 1, which can
confirm the appropriate loading of Gin into the UIO-66 for-
mulation (Fig. 1D).
Drug release
The dialysis methodology was used to assess in vitro drug
release. Figure 1E depicts the findings of a 72-hour release
profile of Gin from the UIO-66 nanopaticle in PBS pH 7.4
at 37 °C. Figure 1E shows the cumulative release process of
the Gin in the PBS release medium over 72 h. Figure 1E also
indicates Gin release from the nanocarrier (62.21%) was
less than the drug solution (98.59%) over 72 h of release.
We observed 50% of the Gin release into the medium in
less than the first 6 h, while 50% Gin release from nanocar-
rier was recorded in about 21 h. This result represents the
controlled release of Gin from the nanocarrier at PH = 7.4
in PBS medium.
Anticancer activity
The cytotoxicity of the proposed drug delivery system was
evaluated against AGS cells (as cancerous cell line) and
HFF cells (as normal cell line) to confirm the improved anti-
tumor activity of UIO-66-Gin compared to free Gin after
48 and 72 h (Fig. 2 A, Fig. 2B, and Fig. 2 C). Figure 2 A
indicates that higher concentration (62.5, 125 and 250 µg/
ml) of UIO-66-Gin has cytotoxic effects against HFF cells
which it is significantly higher than MOF cytotoxicity alone
(P < 0.001). Figure 2B C demonstrate that both free drug
and prepared nanocarrier could significantly decrease AGS
cells viability in various concentration after 48 and 72 h
(P < 0.05). However, the proliferation inhibition of cancer
cells via both drug and nano-formulation was greatly time-
dependent, as the 72-hour treatment significantly reduced
the survival of the AGS cells treated with different con-
centrations of both Gin alone and Gin loaded in the nano-
formulation compared to the 48-hour treatment (P < 0.05).
Figure 2D and E also show the IC50 level calculated for
Gin and UIO-66-Gin against cancer cells at 48 and 72 h,
which according to this graph, the IC50 level decreases sig-
nificantly after 72 h compared to 48 h (P < 0.05). Altogether,
we illustrate that UIO-66-Gin nanoparticles are more toxic
(especially in higher concentration) against AGS cell lines
compared to free Gin. However, higher concentrations of
3507
Gin-UI0-66 have a significant cytotoxic effect than UIO-66
alone, which suggests that lower concentrations of this for-
mulation should be used to treat cancer cells because lower
concentrations have a potential effect on cancer cells.
Apoptotic genes expression
Apoptotic genes expression in AGS cells treated with
IC50 concentration of Gin and UIO-66-Gin formulation
was assessed using Real-time PCR technique. Figure 3 A,
Fig. 3B C have been demonstrated a substantial up-regu-
lation in Caspase 3, Caspase 9, and Bax genes expression
in both free drug and nano-formulation treated cells com-
pared to internal gene (P < 0.001). This elevation in the
expression of pro-apoptotic genes was remarkably higher in
UIO-66-Gin treated cells rather than free Gin treated cells
(P < 0.001). We also observed (Fig. 3D) a remarkable down-
regulation of the expression of Bcl2 gene in cells treated
with UIO-66-Gin in comparison with cells treated with free
Gin and internal control (P < 0.001). This data revealed the
potent apoptotic activity of UIO-66-Gin compared to free
drug.
Flowcytometry assay
The apoptosis to necrosis ratio in AGS cells was assessed
using flow cytometry technique, where AGS cells were
essentially exposed to various samples (IC50 concentrations
of Gin and UIO-66-Gin) and the cells were then stained
with Annexin V-FITC and PI. Annexin V-FITC can stain
phosphatidylserine, which can move beyond the cell mem-
brane during the early stages of apoptosis; While PI can be
utilized to stain the nucleus during the necrosis. The flow
cytometry outcomes are illustrated in Fig. 4. The Gin can
elicit 19.51% apoptosis (13.7% early apoptosis and 5.81%
late apoptosis) and 4.78% necrosis in AGS cells. In other
hand, UIO-66-Gin has led to 41.7% apoptosis (21.7% early
apoptosis and 20% late apoptosis) and 8.49% necrosis in the
cancerous cells. Figure 4D shows a significant induction in
apoptosis percentage in cells treated with both agents, but
there was significant increment in the percentage of apopto-
sis in AGS cells treated with UIO-66-Gin compared to free
Gin (P < 0.001).
Discussion
Plants are being used as remedies for generations, and the
employment of plant-derived compounds in anticancer
medications has expanded. Gin (5-hydroxydecan-3-one
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3508 Molecular Biology Reports (2023) 50:3503–3513

Fig. 2 (A) Cytotoxic activity of different concentration of UIO-66 tion of UIO-66-Gin against AGS cell line after 48 and 72 h. (D) and
and UIO-66-Gin against HFF cell line for 48 and 72 h treatment. (B) (E) Obtained IC50 concentration for Gin and UIO-66-Gin after 48
Cytotoxic activity of different concentration of Gin against AGS cell and 72 h. P < 0.05 was considered statistically significant (*:P < 0.05,
line after 48 and 72 h. (C) Cytotoxic activity of various concentra- **:P < 0.01, and ***:P < 0.001)

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Molecular Biology Reports (2023) 50:3503–3513 3509

Fig. 3 The fold change of the expression of the (A) Caspase3, (B) Caspase9, (C) Bax, and (D) Bcl2 genes in cells treated with IC50 concentration
of Gin and UIO-66-Gin in AGS cancer cell lines. P < 0.05 was considered statistically significant (***:P < 0.001)

substituted by a 4-hydroxy-3-methoxyphenyl moiety at poorly soluble compounds whereas struggling to overcome

position 1) is an innately existing chemical extracted from
Zingiber officinale that has been shown to have antican-
cer effect against a variety of carcinomas, including but
not restricted to breast cancer and colon cancer. Consider-
ing these beneficial benefits, Gin’s therapeutic spectrum is
restricted due to its poor water solubility and low therapeutic
efficacy[24, 25]. The use of porous materials with positively
charged metal ions and structural tunability, such as MOFs,
which prevents crystallization of the amorphous drug phase
and then discharges the drug on such hydrolytic decom-
position, reflects an innovative strategy for the delivery of
the typical limitation of amorphous drug delivery[26]. In
current investigation, we have synthesized a potent UIO-
66-Gin formulation for evaluating anticancer effects against
AGS cancer cell lines.
The characterization of synthesized UIO-66-Gin was
carried out using DLS, SEM and FTIR techniques. DLS
and SEM analysis indicated the spherical shape of nano-for-
mulation with the mean size of 174.3 nm. The comparison
of FTIR spectra of UIO-66 and UIO-66-Gin formulation
indicated while Gin is introduced to the UIO-66 structure,
the C = O bond absorption band occurs in the 1748 cm-1

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Fig. 4 Apoptosis evaluation via flow cytometry after the treatment of cells. (A) Control, (B) Gin, (C) UIO-66-Gin. (D) The diagram of apoptotic
rate in cancerous cells treated with IC50 concentration of Gin and UIO-66 for 24 h. P < 0.05 was considered statistically significant (***:P < 0.001)

spectrum, proving the correct packing of Gin into the UIO-
66 structure. Drug release analysis indicated that there
was a 50% Gin release from the nanocarrier was reported
in roughly 21 h, which revealed the regulated release of
bioactive compound from the UIO-66 formulation in PBS
medium. The pH sensitivity of the UiO-66 framework indi-
cates that it is durable at physiological pH (P = 7.4), but
degrades at acidic condition. Relying on this characteris-
tic, the UiO-66 framework is employed as a drug-vehicle
to release Gin from the framework in physiological condi-
tion, which explains the reason for the controlled release
of the Gin loaded into the framework under physiological
conditions[27].
The basic mechanism by which Gin regulates a wide
range of cancer-related cell signaling pathways, which
include Nuclear Factors (NF-κB), Signal Transducer and
Activator of Transcription 3 (STAT3), Activator Pro-
tein-1 (AP-1), -catenin, Growth Factors Receptors (EGFR,
VEGFR), Mitogen-Activated Protein Kinases (MAPK), and
pro-inflammatory mediators (TNF-α and COX-2)[24]. MTT
assay after 48 and 72 h demonstrated that various concen-
tration of both the Gin and UIO-66-Gin started to induce
cytotoxicity in cancerous cells. However, the induction of
cytotoxicity was higher in cells treated with UIO-66-Gin.
On the other hand, this formulation only had cytotoxic
effects on the normal cells in higher concentration, which

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Molecular Biology Reports (2023) 50:3503–3513
suggested great biocompatibility for normal cells. These
results also suggested that UIO-66-Gin increase cytotoxic-
ity of Gin against cancerous cells through controlled release.
However, the capability of the framework to induce apop-
tosis was not investigated. In all samples, the Gin-loaded
MOFs have a unique dose-dependent and time-dependent
effect on cell survival. This formulation is clearly biologi-
cally active, and so have the ability to be used as drug deliv-
ery systems. Markopoulou et al. study indicated the delayed
release of doxorubicin from Dox@UiO-66 causes cytotoxic
effects for 12 to 16 h, whereas the quick release of doxorubi-
cin causes considerably increased intranuclear localization
and immediate apoptosis[28].
Apoptosis is a type of regulated cell death that entails
the controlled breakdown of intracellular molecules while
preventing inflammation and impact on adjacent cells. Exe-
cutioner caspases are activated by initiator caspases, who
then coordinate their activity to destroy crucial structural
proteins and stimulate other enzymes activation[29]. DNA
fragmentation and membrane blebbing are morphological
markers of apoptosis. Pro- and anti-apoptotic members of
the BCL-2 protein family also regulate a cell’s capacity to
initiate mitochondrial apoptosis. The balance of pro- and
anti-apoptotic BCL-2 proteins guarantees that programmed
cell death is properly regulated during development[30].
In current investigation, Real time PCR analysis revealed
that both anti-cancer agents could induce the expression
of pro-apoptotic (Bax, Caspase3, and Caspase9) genes
and down-regulate the expression of Bcl2 as anti-apoptotic
gene. However, the UIO-66-Gin had a much more remark-
able effect on the expression of genes involved in apoptosis
rather than Free Gin. Previous investigations demonstrated
that Gin causes cell death principally by inducing caspase-
dependent apoptosis pathway, as indicated by a considerable
elevation in caspase-3 activation within fresh tumors and in
vitro caspase-3 and − 9 cleavage[31]. Gin also can regulate
cell cycle arrest, intracellular Ca2 + increase, and reduce the
growth and invasion of cancer cells by AKT and p38MAPK
inactivation[32]. However, to our knowledge, the present
study is the first to demonstrate the effect of Gin on expres-
sion changes of the Bcl2 family genes. Unfortunately, no
study was found on the effect of a UIO-66 containing an
anticancer agent on the expression of genes involved in
apoptosis. However, it had been revealed that MOF can
dramatically improve cancer treatment by caspase-3 activa-
tion[33]. Sp et al. investigation indicated that Gin caused
G0/G1 cell cycle arrest and mitochondrial programed death
through changing the BAX/BCL-2 ratio and cytochrome c
release[31]. In another study, the Gin’s cytotoxic impact on
the MCF-7 cell line was demonstrated by a considerable rise
in the expression of caspase-3, caspase-8, and caspase-9 as
compared to control cells[34]. Therefore, it was shown that
3511
UIO-66-Gin may induce apoptosis through activation of
Caspase-dependent pathway.
Apoptosis is one of the main leads of cancer cell growth
prevention. Flowcytometry results indicated that apoptotic
rate has been elevated in cells treated with both ant-can-
cer agents, however, the elevation of apoptotic rate in cells
treated with UIO-66-Gin was significantly higher than Gin
treated cells. The flowcytometry data were consistent with
the previous real-time PCR finding, indicating that Gin and
UIO-66-Gin formulation probably enhanced apoptosis via
the activation of pro-apoptotic and anti-apoptotic genes.
The great apoptotic activity of UIO-66-Gin might be attrib-
uted to the optimal drug accumulation within the MOF
porous structure, as well as improved drug discharge in the
tumor microenvironment[35]. Li et al. study indicated that
Gin can decrease the growth of gastric cancer cells in time
and concentration-dependent manner and also demonstrate
that Gin can stimulate the programed cell death in gastric
cancer cells[36]. The flowcytometry analysis of Hu et al.
study demonstrated that Gin administration raised apoptotic
rates in HCT116 cells in a concentration-dependent manner,
which it was attributed to the elevation in the expression
levels of caspase-3 and caspase 8 genes and the reduction
of the expression level of Bcl-2 gene in HCT116 cells[37].
These data revealed the potent apoptotic effects of Gin
against cancerous cells. However, targeted drug delivery of
Gin to the cancer cell through its carrier and its controlled
release in cancerous cells resulted in an increase in the rate
of apoptosis in cells treated with the UIO-66-Gin.
For the first time in the current study, we designed Gin
loaded UIO-66 framework for management and preven-
tion of gastric cancer proliferation. The nano-formulation
was synthesized successfully and was approved with SEM,
DLS and FTIR analysis. Cellular and molecular assays also
revealed the potent anticancer effect and apoptotic induction
ability of UIO-66-Gin against cancerous cells through alter-
ing the expression of genes involved in apoptosis. However,
additional laboratory studies on appropriate animal research
are necessary to confirm the effectiveness and safety of the
discovered UIO-66 formulation over free Gin.
Author contributions All authors contributed to the study conception
and design. Material preparation, data collection and analysis were
performed by Irana Kazazi, Fatemeh Ashrafi and Maryam Malekloo.
The first draft of the manuscript was written by Irana Kazazi and all
authors commented on previous versions of the manuscript. All au-
thors read and approved the final manuscript.
Funding The authors declare that no funds, grants, or other support
were received during the preparation of this manuscript.
Statements and Declarations
Competing Interests The authors have no relevant financial or non-
13
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financial interests to disclose.
Conflict of interest Authors declare that they have no conflict of inter-
est.
Ethical approval This article does not contain any studies with human
participants or animals performed by any of the authors.
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