Delivery of oxaliplatin to colorectal cancer cells by folate-targeted UiO-66-NH2

Article abstract of DOI:10.1016/j.taap.2021.115573

Oxaliplatin is being used in different malignancies and several side effects are reported for patients taking Oxaliplatin, including peripheral neuropathy, nausea and vomiting, diarrhea, mouth sores, low blood counts, fatigue, loss of appetite, etc. Here we have developed a targeted anticancer drug delivery system based on folate-conjugated amine-functionalized UiO-66 for the delivery of oxaliplatin (OX). UiO-66-NH₂ (U) and UiO-66-NH₂–FA(FU) were pre-functionalized by the incorporation of folic acid (FA) into the structure via coordination of the carboxylate group of FA. The FTIR spectra of drug-loaded U and FU showed the presence of new carboxylic and aliphatic groups of OX and FA. Powder X-ray diffraction (PXRD) patterns were matched accordingly with the reference pattern and FESEM results showed semi-spherical particles (115–128 nm). The evaluated amounts of OX in U and FU were calculated 304.5 and 293 mg/g_, respectively. The initial burst release of OX was 15.7% per hour for U(OX) and 10.8% per hour for FU(OX). The final release plateau gives 62.9% and 52.3% for U(OX) and FU(OX). To evaluate the application of the prepared delivery platform, they were tested on colorectal cancer cells (CT-26) via MTT assay, cell migration assay, and spheroid model. IC₅₀ values obtained from MTT assay were 21.38, 95.50, and 18.20 μg/mL for OX, U(OX), and FU(OX), respectively. After three days of treatment, the CT26 spheroids at two doses of 500 and 50 μg/mL of U(OX) and FU(OX) showed volume reduction. Moreover, the oxidative behavior of the prepared systems within the cell was assessed by total thiol, malondialdehyde, and superoxide dismutase activity. The results showed that FU(OX) had higher efficacy in preventing the growth of CT-26 spheroid, and was more effective than oxaliplation in cell migration inhibition, and induced higher oxidative stress and apoptosis.

Full text of DOI:10.1016/j.taap.2021.115573

Toxicology and Applied Pharmacology 423 (2021) 115573
Contents lists available at ScienceDirect
Toxicology and Applied Pharmacology
journal homepage: www.elsevier.com/locate/taap
Delivery of oxaliplatin to colorectal cancer cells by folate-targeted
UiO-66-NH2
,
b
,
c
,
,c,
Alireza Hashemzadeha
1, Forouzan Amerizadehb 1, Fereshteh Asgharzadeha,
Majid Darroudid, Amir Avanb c, Seyed Mahdi Hassanianc, Mohammad Landaranie,
,
Majid Khazaeia
,c,*
a Department of Medical Physiology, Faculty of Medicine, Mashhad University of Medical Sciences, Mashhad, Iran
b Department of Medical Biotechnology, Faculty of Medicine, Mashhad University of Medical Sciences, Mashhad, Iran
c Metabolic syndrome Research center, Mashhad University of Medical Sciences, Mashhad, Iran
d Department of Modern Sciences and Technologies, Faculty of Medicine, Mashhad University of Medical Sciences, Mashhad, Iran
e Faculty of chemistry, Shahid Beheshti University, Evin, 1983963113, Tehran, Iran
A R T I C L E I N F O
A B S T R A C T
Keywords:
Oxaliplatin is being used in different malignancies and several side effects are reported for patients taking
Oxaliplatin, including peripheral neuropathy, nausea and vomiting, diarrhea, mouth sores, low blood counts,
fatigue, loss of appetite, etc. Here we have developed a targeted anticancer drug delivery system based on folate-
conjugated amine-functionalized UiO-66 for the delivery of oxaliplatin (OX). UiO-66-NH2 (U) and UiO-66-
NH2–FA(FU) were pre-functionalized by the incorporation of folic acid (FA) into the structure via coordination of
the carboxylate group of FA. The FTIR spectra of drug-loaded U and FU showed the presence of new carboxylic
and aliphatic groups of OX and FA. Powder X-ray diffraction (PXRD) patterns were matched accordingly with the
reference pattern and FESEM results showed semi-spherical particles (115–128 nm). The evaluated amounts of
OX in U and FU were calculated 304.5 and 293 mg/g, respectively. The initial burst release of OX was 15.7% per
hour for U(OX) and 10.8% per hour for FU(OX). The final release plateau gives 62.9% and 52.3% for U(OX) and
FU(OX). To evaluate the application of the prepared delivery platform, they were tested on colorectal cancer cells
(CT-26) via MTT assay, cell migration assay, and spheroid model. IC50 values obtained from MTT assay were
Metal-organic framework
microporous nanomaterials
smart drug delivery system host-guest
interactions
21.38, 95.50, and 18.20
μ
g/mL for OX, U(OX), and FU(OX), respectively. After three days of treatment, the CT26
spheroids at two doses of 500 and 50
μ
g/mL of U(OX) and FU(OX) showed volume reduction. Moreover, the
oxidative behavior of the prepared systems within the cell was assessed by total thiol, malondialdehyde, and
superoxide dismutase activity. The results showed that FU(OX) had higher efficacy in preventing the growth of
CT-26 spheroid, and was more effective than oxaliplation in cell migration inhibition, and induced higher
oxidative stress and apoptosis.
Abbreviations: dntb, 5,5′-dithiobis(2-nitrobenzoic acid); dmso, Dimethyl sulfoxide; tp, Terephthalic acid; u, Uio-66-nh2; atp, 2-aminoterephthalic acid; micro-
tracbel corp., japan, Belsorp-mini apparatus; crc, Colorectal cancer; ddss, Drug delivery systems; dls, Dynamic light scattering; chn, Elemental analysis; edx, Energy-
dispersive x-ray spectroscopy; edta, Ethylenediaminetetraacetic acid; fesem, Field emission scanning electron microscopy; frs, Folate receptors; fa, Folic acid; ftir,
Fourier transform infrared; frα, Fr alpha; fr+, Fr positive; hcl, Hydrochloric acid fuming 37%; 1hnmr, Hydrogen nuclear magnetic resonance; h2o2, Hydrogen
peroxide; mofs, Metal-organic frameworks; dmf, N,n’-dimethylformamide; hno3, Nitric acid 65%; ox, Oxaliplatin; Pbs, phosphate-buffered saline; pd, Pharmaco-
dynamics; pk, Pharmacokinetic; pxrd, Powder x-ray diffraction; mtt, Pyrogallol, 3-[4,5-dimethylthiazol-2-yl]-2,5-diphenyltetrazolium bromide; sbus, Secondary
building units; naoh, Sodium hydroxide; h2so4, Sulfuric acid 95–98%; tba, Thiobarbituric acid; sncl2.2h2o, Tin chloride dihydrate; tca, Trichloroacetic acid; tris, Tris
(hydroxymethyl)aminomethane hydrochloride; fu, Uio-66-nh2–fa; zrcl4, Zirconium tetrachloride.
* Corresponding author at: Department of Medical Physiology, Faculty of Medicine, Mashhad University of Medical Sciences, Mashhad, Iran.
E-mail address: Khazaeim@mums.ac.ir (M. Khazaei).
1
These two authors contributed equally to this work.
https://doi.org/10.1016/j.taap.2021.115573
Received 2 January 2021; Received in revised form 21 March 2021; Accepted 10 May 2021
Available online 13 May 2021
0041-008X/© 2021 Elsevier Inc. All rights reserved.

A. Hashemzadeh et al.
Toxicology and Applied Pharmacology 423 (2021) 115573
1. Introduction
The 5-year survival rate for colorectal cancer (CRC) patients is
around 90% and 5% for stage I and stage IV, respectively. Many aca-
Nanomedicine is aimed at delivering active agents to the cancer
tissues and reduces the usual doses of medications (Onoue et al., 2014;
Faust, 2015; Lazaro and Forgan, 2019). Chances of survival could be
increased greatly by sustained-release dosage, using novel drug delivery
systems (DDSs). The DDS efficacy (%) is contingent on several factors
including physicochemical properties, pharmacokinetic (PK), pharma-
codynamics (PD) and biocompatibility (Petrak, 2005; Rosenholm et al.,
2010; Marcato, 2014). Improving drug efficacy provides a route ‘to
creative and effective therapies’. Metal-organic frameworks (MOFs),
which were first introduced in the early 1990s, have been recognized
recently as a means to improve the DDSs (Orellana-Tavra et al., 2015;
Wu and Yang, 2017),. The interest was piqued to find more information
about the spongy quality of MOFs on drug adsorption, PK, PD,
biocompatibility, bioavailability, cytotoxicity, stability, and specificity
(Ibrahim et al., 2017; Zangabad et al., 2017; Zhou et al., 2017). The
skillful utilization of the high internal surface area lends interesting
characteristics such as high drug loading to MOFs. The drug penetrates
deep into the pores and displays an effective release profile. The
encapsulation, which is promoted by specific modifications using
physical or chemical means, leads to the elaboration of new release
mechanisms and novel sustained or stimulus-responsive drug releases
(Wu and Yang, 2017; Cai et al., 2019). MOFs are so flexible that can
adapt to any changes required for specific goals, especially smart DDSs.
Adjustments could be made by synthetic procedures, through pre/post-
modifications over secondary building units (SBUs) or polytopic organic
linkers (Cao et al., 2018a; Simagina et al., 2018). The evolution of tar-
geted chemotherapy requires a precisely formulated master plan which
employs overexpressed pathways in cancer cells (Singh et al., 2016; Yu
et al., 2016; Clemons et al., 2018). In active targeting, the conjugated
homing ligands bind to a receptor on cancer cells, devising strategies to
produce the next class of anticancer agents (Kue et al., 2016). These
ligands are used to deliver medications selectively into malignant cells.
In this manner, drug-loaded MOFs conjugated with targeting agents
could have higher drug potency against malignancies, which guide drug
payloads through and diffuse across tumor cell membranes; presumably,
drug discharge after internalization could prevent undesirable drug
release into receptor-negative cells (Dai et al., 2016). Targeting is a
contributing factor in selective internalization of enclosed potent che-
motherapeutics to the specific cells. SDDSs (smart drug delivery sys-
tems) may offer slender hope to patients with high-grade cancers in
third-line therapies and beyond, for instance, for metastatic or drug-
resistant cancers (Moore et al., 2017; Moore et al., 2018). Hence to
maximize the utility of DDSs and to find thoughtful solutions to treat
cancer, an integrated drug-transport system composed of targeting
agents is preferred, which may include monoclonal antibodies, peptides,
aptamers, oligo-saccharides, and vitamins (Fan et al., 2012; Top-
orkiewicz et al., 2015; Wu et al., 2015).
demics have researched the effect of FRα as a major biomarker for
suppressing tumor proliferation in CRC (Zhang et al., 2012; Coppede,
2014; Varshosaz et al., 2014; Bansal et al., 2016). There are real issues of
practical and systematic approaches in high-grade CRC treatment. The
right way to resolve the matter could be FA-conjugated nano-vehicles/
drugs (Boddu et al., 2012; Steichen et al., 2013; Chen et al., 2014;
Khoshgard et al., 2014; Lai et al., 2014; Samadian et al., 2016). The
advantages of FA conjugates are due to the higher cytotoxicity, higher
cellular uptake, and the greater capacity to induce apoptosis. FA-
functionalized MOFs have been also reported to be effective in the
treatment of different cancers in both cellular and animal models (Au
et al., 2016; Chowdhuri et al., 2017a; Chowdhuri et al., 2017b; Liu et al.,
2017; Dong et al., 2018; Nejadshafiee et al., 2019).
In this study, we have shown a preliminary design of a new smart
DDS for oxaliplatin (OX) which have an assembly of MOF features using
an active targeting and the aim was to treat high-grade CRC in the future
treatments. We used UiO-66-NH2(U) and FA-conjugated UiO-66-NH2
(FU) to deliver loaded OX to CT-26 cancer cells. The main goal of this
study was to develop new effective nanodrugs for CRC cancer and
compare the drug efficacy in FU and U in cellular models using the MTT
assay, cell migration assay and spheroid models of cancer, and also their
oxidative attributes.
2. Materials and methods
2.1. Instruments and materials
Energy-dispersive X-ray spectroscopy (EDX), EDX mapping and field
emission scanning electron microscopy (FESEM) was carried out using a
TESCAN Mira 3 LMU. Nitrogen adsorption-desorption isotherms were
obtained with a BELSORP-mini apparatus (MicrotracBEL Corp., Japan).
Fourier transform infrared (FTIR) spectra were obtained on an M-500
Fast-Scan IR spectrometer (Buck Scientific, USA). UV–Vis absorption
measurements were recorded on a Cecil Instruments-9500 CE UV–Vis
spectrophotometer. A Horiba SZ-100-Z was used to conduct dynamic
light scattering (DLS) and measure zeta potentials. Sonorex Digitec DT
510 H was utilized for sonication and a STOE-STADV STAIP diffrac-
tometer for Powder X-ray diffraction (PXRD) measurements over the 2θ
of 3–80◦ and Cu K
α radiation, λ = 1.54060 Å. Hydrogen nuclear mag-
netic resonance (1HNMR)
was taken by Bruker AVANCE3 3-300 MHz. Elemental analysis
(CHN) was measured by a FLASH EA 1112 Series CHN analyzer using
Eager 300 software. The cells were observed by Leica-DMI300B inverted
phase-contrast microscope (Leica Microsystems GmbH, Wetzlar, Ger-
many). Epoch microplate spectrophotometer was used for UV–Vis
absorbance measurements in plate reading. Heidolph homogenizers
SilentCrusher tool was employed for homogenizing. Analysis of the Pt
was carried out by an inductively coupled plasma-optical emission
spectrophotometer (ICP-OES; Spectro Arcos 7600/CRMA, Germany).
Oxaliplatin (OX), zirconium tetrachloride (ZrCL4), terephthalic acid
(TP), tin chloride dihydrate (SnCl2.2H2O), Thiobarbituric acid (TBA),
folic acid (FA), oxaliplatin (OX), agarose gel, and PBS (Phosphate-
Buffered Saline) Tablets were purchased from Sigma–Aldrich. Methanol,
acetone, ethanol, hydrogen peroxide (H2O2), N,N′-dimethylformamide
(DMF), dimethyl sulfoxide (DMSO), hydrochloric acid fuming 37%
(HCl), sodium hydroxide (NaOH), nitric acid 65% (HNO3), sulfuric acid
95–98% (H2SO4), pyrogallol, 3-[4,5-dimethylthiazol-2-yl]-2,5-diphe-
nyltetrazolium bromide (MTT), 5,5′-Dithiobis(2-nitrobenzoic acid)
(DNTB), tris(hydroxymethyl)aminomethane hydrochloride (TRIS), eth-
ylenediaminetetraacetic acid (EDTA) and trichloroacetic acid (TCA)
were obtained from Merck chemical company. All the starting reagents
and solvents were of analytical grade. 2-aminoterephthalic acid (ATP)
was synthesized as described previously (Li et al., 2014).
Cancer might lead to changes in the protein expression depending on
the type and stage of cancer. The changes in the level of expression of
many cellular proteins while transforming to cancer cells promote a
continued growth of cell proliferation and metabolism, which led to
escaping from cell death messages (Bahrami et al., 2018). The favorable
prognosis in most patients is diagnosed with the cancer stage and
choosing the right treatment, hence new precautions are needed at
advanced stages of the disease.
The folate receptors (FRs) are synthesized in abnormally large
amounts in malignancies, especially colorectal, brain, breast, ovarian,
and nervous system cancers (Weitman et al., 1994; Hartmann et al.,
2007; Kalli et al., 2008; Markert et al., 2008; Li et al., 2016a). The FR
alpha (FRα) is a cell membrane-anchored protein that has a high affinity
to bind to folic acid (FA) and its conjugated nanoforms, which can target
FR positive (FR+) cancer cells. The biological significance of FA (known
as vitamin B₉ and folacin) is DNA biosynthesis and methylation (Sudi-
mack and Lee, 2000).
CT26 / CRC cell is a murine colorectal carcinoma cell line which is
2

A. Hashemzadeh et al.
Toxicology and Applied Pharmacology 423 (2021) 115573
from a BALB/c mouse. The cell is a clone of the N-nitroso-N-methylr-
ethane-induced undifferentiated CT26 colon carcinoma cell line. These
cells are adherent and have a fibroblast morphology. They will form
tumors and metastases post implantation into syngenic BALB/c mice or
immunocompromised mice. Cell culture was performed in a T25 flask
using CT26 cell line (from ATCC, Manassas, VA, USA) at 37 ◦C under 5%
CO2 humidified atmosphere using RPMI:DMEM composing of 1%
streptomycin/penicillin and 10% FBS. Eventually, in their exponentially
growing phase, at 70%–80% confluence Routine passaging was
accomplished by using trypsin-EDTA of cells (Hashemzehi et al., 2021).
The oxidative stress was evaluated in the recovered ~3 × 106 CRC cells
in PBS (1 mL, 0.01 M, pH = 7.4). Cells were homogenized on ice and
indexes of malondialdehyde (MDA), total thiol (T-SH) and superoxide
dismutase (SOD) were measured.
autoclave. After cooling to room temperature, the products were
collected by centrifugation (4000 rpm, 5 min) and were washed with
DMF (2 × 30 mL) and methanol (3 × 30 mL). As-synthesized U and FU
were soaked in 20 mL of CH2Cl2, 3 times over 1 h (20 min each),. Then,
were immersed in 20 mL of dry n-hexane over 1 h, followed by solvent
evacuation at 100 ◦C for 24 h. The PXRD analysis showed the FU25 was
not formed and the FU15 would be used from now on, which would be
named FU, in all the experiments. Elemental analysis (wt%): Calc. for U
(Zr6O4(OH)4(ATP)6): C, 32.87; H, 2.28; N, 4.78; found: C, 31.94; H,
3.39; N, 5.20; found for FU(5%): C, 32.87; H, 3.09; N, 5.71, and for FU
(15%): C, 35.34; H, 2.78; N, 7.07.
2.4. Loading Oxaliplatin (OX)
A 30 mg sample of U or FU had dispersed 12 mL of OX solution in
distilled water (c = 5 mg/mL). After stirring for 48 h under dark con-
ditions, the OX-loaded U or FU was centrifuged (4000 rpm, 5 min). After
centrifugation, the obtained products were washed with 15 mL distilled
water several times until the supernatant solution had no signal of OX
using UV–Vis spectroscopy. The OX-loaded U and FU were used for
subsequent in vitro tests against CT-26 cells. To evaluate the OX-loading
efficiency, the supernatant and washed solutions were collected and the
residual OX content (ROX) was measured by using UV measurement at
the wavelength of 252 nm. The loading efficiency % (LE%) of OX can be
calculated as follows: LE% = (OOX - ROX)/OOX) × 100%, in which OOX
is the original OX content and (OOX - ROX) is the mass of OX loaded into
U or FU.
2.2. Synthesis of 2-aminoterephthalic acid (ATP)
A three-necked round-bottom flask was charged with 20 g of TP and
sulfuric acid (60 mL, 95–98%). Then, nitric acid (15 ml, 65%) was added
slowly over an hour with stirring at 0 ◦C. The reaction temperature was
increased gradually until the temperature of 60 ◦C was reached. It was
maintained over 1 h at this temperature, then increased gradually over
80 ◦C in 1 h and held at this temperature overnight. Thereafter, the
mixture was cooled by pouring ice water into the reaction vessel. The
product was filtered and washed with 3 l of distilled water. The filtrate
was recrystallized in 1 l of acetone to obtain 2-nitroterephthalic acid
(NTP). Acetone could dissolve NTP, but not the precursor (TP). The
◦
precipitate was filtered and dried in the oven at 60 C. To reduce the
nitro group, eighty milliliters of hydrochloric acid fuming 37% was
added to a three-necked round-bottomed glass and tin chloride dihy-
drate (30 g) was added gradually to the flask with stirring. Thereafter,
10 g of NTP was added to the suspension and reacted at 80 ◦C for 6 h.
After the reaction, the mixture was filtered with distilled water and the
2.5. In vitro drug release
To study the OX release profile at different time intervals, six samples
of 5 mg drug-loaded of MOF were re-dispersed in 1 ml distilled water
◦
(initial pH = 5.5). The suspensions were incubated at 37 C. UV–Vis
◦
precipitate was dried in an oven for 2 h at 60 C to form ATP. Color:
spectroscopy was used to measure the amount of drug release after 1, 3,
6, 12, 24, and 48 h. After incubation, the suspensions were centrifuged
and the supernatants were used for further analysis.
lemon yellow powder (yield: 9.83 g, 45%); m.p. >300 ◦C; 1H NMR (300
MHz, DMSO-d6) δ = 7.79 (d, 1 H, aromatic CH), 7.41 (d, 1 H, aromatic
CH), 7.04 (dd, 1 H, aromatic CH), 9.68 (s, 2H, COOH).
2.6. Growth inhibition evaluation
2.3. Synthesis of UiO-66-NH2 (U) and FA-UiO-66-NH2 (FU)
The cell growth inhibitory activity of OX, U, FU, U(OX) and FU(OX)
was determined by the MTT assay. In detail, 5106 cells were seeded into
96-well plates and incubated for 24 h. Subsequently, various concen-
The syntheses of U and FU were performed using a solvothermal
process (Katz et al., 2013). Briefly, a 100 mL round-bottom flask was
charged with 0.5 g of ZrCl4, 20 mL of DMF and 4 mL of HCl (37%) and
another flask was charged with 0.536 g of ATP and 40 mL of DMF;the
mixtures then were sonicated for 20 min. In the case of FU, the latter
solution does also include 5, 15, and 25 mol% of FA relative to the
metallic precursor (Table 1). The product respectively would be called
FU5, FU15 and FU25. Thereafter, the ligand solution was added to the
ZrCl4 solution and the mixture was sonicated for an additional 20 min
before being heated at 80 ◦C for a period of 36 h under autogenous
pressure conditions using a 100 mL Teflon-lined stainless steel
trations of U, FU (1–1000
μg/mL), or OX (0.001–1000 mM) were
administered for 24 h to find cell viability. Analyses were performed as
described previously [37].
2.7. Scratch assays
Cell migratory behavior of OX, U(OX) and FU(OX) against CT-26
cells (seeded cells = 1 × 105/well) were evaluated in 24-well plates
and the surface of the plates were marked by a yellow Pipette tip. After
Table 1
Synthesis conditions (amount and concentration of solutes and solvents, and reaction temperature, and reaction time).
2-aminoterephthalic
acid (ATP)
ZrCl4
(mg)
HCl (mL)
DMF (mL)
Folic acid (FA):
ZrC14
Synthesis Temperature
(◦C)
(mg)
mole ratio
UiO-66-NH2
536
500
4
4
4
4
60
60
60
60
–
80
80
80
80
(3.00 mmol)
536
(0.216 mmol)
500
FA5-UiO-66-NH2
0.05:1
0.15:1
0.25:1
(3.00 mmol)
536
(0.216 mmol)
500
FA15-UiO-66-NH2
(FU)
(3.00 mmol)
536
(0.216 mmol)
500
FA15-UiO-66-NH2
(3.00 mmol)
(0.216 mmol)
Reaction time: 36 h.
3

A. Hashemzadeh et al.
Toxicology and Applied Pharmacology 423 (2021) 115573
that, the Cells were treated with 20 μM OX, 500 μg/mL U(OX) and FU
Table 2
Folic acid conjugated MOFs in cancer treatment.
(OX), and were compared with control. ImageJ version 1.52a (National
Institutes of Health, Bethesda, MD, USA) was used to measure changes in
the emerged edges.
MOF
MOF
Type
Drug composition
Cell Line
Ref.
Family
ZIFs
ZIF-8
DOXa@ZIF-8-FA
HepG2 cells
B16F10 cells
MGC803 cells
(Jiang et al.,
2018)
2.8. Multicellular spheroids formation
Quercetin@FAb-
BSAc-CuS-ZIF-8
5-FUd@ FA-CSe-5-
FAMf-ZIF-8
(Chowdhuri
et al., 2017a)
(Gao et al.,
2016)
Spheroids were developed and OX, U(OX) and FU(OX) were treated
against CT-26 cells. Briefly, spheroids with a density of 5 × 104 cells/
well in DMEM/F12 + GlutaMAX-I (1:1) were cultured in agarose-coated
DOX-VERg@ZIF-8-
PEGh-FA
FR-positive
B16F10 &
(Zhang et al.,
2017b)
96 well plates and subsequently treated with 8
μ
g/mL OX, 50 and 500
MCF-7/A cells
HeLa and L929
cells
μ
g/mL of U(OX), and 50 and 500 μg/mL of FU(OX). The cytotoxic effects
5-FU@UCNPi-ZIF-
8-FA
(Chowdhuri
et al., 2016b)
(Gao et al.,
2017)
were assessed for 3 days on the inverted phase-contrast microscope.
Finally, spheroid size changes were analyzed by ImageJ version 1.52a
(National Institutes of Health, Bethesda, MD, USA).
MILs
UIOs
5-FU@MIL-53-
NH2-FA-5-FAM
DOX@UCNP-UIO-
66-NH2-FA
MGC-803 and
HASMC cells
TNBC cells
(MDA-MB-468)
and NIH3T3
cells
UIO-66
(Chowdhuri
et al., 2017b)
2.9. Measurement of oxidant indicator
DCAk@Zr-fum-FA
HeLa and MCF-
7 cancer cells
(Abanades
Changes of MDA concentration, an index of lipid peroxidation, was
measured as described previously(Asgharzadeh et al., 2017). First, HCl
(2 mL,37%), TBA (0.375 g) and TCA (15 g) were brought to a volume of
´
´
Lazaro et al.,
2018a)
5-FU@ppyl -UiO-
66-WP6-PEI ꢀ FA
5-FU@UIO-66-
L02 and HeLa
cells
(Wu et al., 2018)
100 mL by distilled water and a volumetric flask, then, 600 μl was
charged into a suspension of CT-26 cells. Next, the solution was incu-
bated in a water bath for 40 min. Then, it was cooled and centrifuged
(1500 rpm, 5 min). Finally, the absorbance was read at λmax = 535 nm
by a UV–Vis spectrophotometer. The MDA concentration, C(m), was
calculated by the following equation: C (m) = Absorbance / (1.65 ×
105).
HepG-2 cells
(Gao et al.,
2018)
NH2-FA-5-FAM
DCA@UiO-66-FA
´
MCF-7 and
(Abanades
´
HEK293 cells
Lazaro et al.,
2018b)
5-FU@UIO-66-
NH2-FA
HeLa and L929
cells
(Dong et al.,
2018)
UIO-68
IRMOF-
3
DOX@UiO-68-FA
Pacm@IRMOF-3-
FA
HepG-2 cells
HeLa and
(Li et al., 2016b)
(Chowdhuri
et al., 2016a)
(Chowdhuri
et al., 2016c)
IRMOFs
2.10. Measurement of antioxidant indicator
NIH3T3 cells
HeLa and L929
cells
DOX@Fe3O4-
OCMC-IRMOF-3-
FA
2.10.1. Total thiol groups (T-SH)
The antioxidant activity was measured by analyzing T-SH content
5-FU@IRMOF-3-FA
HeLa, A549
and KB cells
TNBC cells
(Yang et al.,
2017)
(Bargi et al., 2017). In detail, 50 μL of the sonicated CT-26 cells were
added to 1 mL TRIS-EDTA (TE) buffer (pH = 8.6). The buffer was made
from TRIS-HCl (3 g), EDTA (0.05 g) in a volume of 100 ml distilled water
using a volumetric flask. Then, the absorbance was read at λmax = 412
nm against TE buffer alone (A1). Next, the homogenized cells were
Curn@IRMOF-3-FA
(Laha et al.,
2019)
Other
MOFs
MOF-
808
5-FU@FA-MOF-
808
HeLa and L929
cells
(Dong et al.,
2018)
Bio-
5-FU@Fe3O4-bio-
MOF-CS-FA
MDA-MB-231
and NIH-3 T3
cells
(Nejadshafiee
et al., 2019)
added to a 20 μL solution of DTNB (0.04 g) in methanol (10 mL). After
MOF
15 min, the absorbance was read at λmax = 412 nm again (A2). The
DTNB absorbance was used as the blank (B). Finally, T-SH content, c(T-
SH), was measured with the following equation: c(T-SH) = (A2 ꢀ A1 ꢀ
B) × 1.07/0.05 × 13.6.
a
DOX (doxorubicin).
FA (folic acid).
b
c
d
e
f
BSA (bovine serum albumin).
5-FU (5-fluorouracil).
2.10.2. Superoxide dismutase (SOD) activity assay
CS (chitosan).
SOD activity was measured according to the reported method by
Madesh and Balasubramanian (Madesh and Balasubramanian, 1998). In
this approach, one unit of SOD was evaluated as the amount of enzyme
required to inhibit the rate of MTT reduction by 50%. First, pyrogallol
(0.001 g) and MTT (0.005 g) solutions in distilled water (10 mL) were
made using a volumetric flask (10 mL). Then, the homogenized cells
were poured into the wells containing the above solution and incubated
at room temperature for 5 min. Then, DMSO stopped the reaction (150
5-FAM (5-Carboxyfluorescein).
VER (verapamil hydrochloride).
PEG (pegylated).
g
h
i
UCNP (up-conversion nanoparticle).
DCA (dichloroacetate).
ppy (polypyrrole nanoparticle).
Pac (Paclitaxel).
k
l
m
n
Cur (Curcumin).
μ
l). Finally, the SOD activity was calculated at λmax = 570 nm using a
including ZIFs, Zr-based MOFs like UiOs and MOF-808, Fe-based MILs,
IRMOFs and Bio-MOFs are used in the design of DDSs which are based
on folic acid as the targeting ligand. The stability and the intrinsic
characteristics of MOFs differ with their composition. As a result, a
collection of MOFs can be used to design different delivery systems with
specific aims. There would be a variety of choices if MOFs were available
for use in biomedical applications. Functionalization of DDSs with tar-
geting ligands could improve the chemical or dispersion stability, cell-
membrane crossing and invisibility to macrophages (Zucolotto, 2013;
UV–Vis spectrophotometer. The results were shown as unit/ml of the
homogenate cells.
3. Result and discussion
MOFs malleability in design, especially tuning the surface properties
and pores, and advantages like high surface area and biocompatibility
may resolve many issues in DDSs such as toxicity, drug loading, and
uncontrolled drug release (Horcajada et al., 2010; Miller et al., 2010; Wu
and Yang, 2017). A list of known MOFs with their drug composition and
targeted cancer cell lines used in folate-based DDSs are presented in
Table 2. In most cases, non-toxic and biocompatible families of MOFs
´
´
Abanades Lazaro et al., 2018b). The developed nanocomposites have
provided a promising platform in DDSs to treat different cancers
(Table 2). Table 2 summarized almost all the folate targeted MOFs in
4

A. Hashemzadeh et al.
Toxicology and Applied Pharmacology 423 (2021) 115573
different cell lines such as liver, melanoma, breast, human gastric can-
cers, etc. The effect of drug delivery by folate conjugated MOFs showed
better performance than drugs. Drug delivery systems via active tar-
geting by folic acid are reliable, efficient and affordable. It appears ZIF-8
and UiO-66 are the most used MOFs that are conjugated with folic acid.
In the case of ZIF-8, the most mechanism used in synthesis is in situ self-
assembly of ZIF-8 with other ingredients such as drugs or targeting
agents. It encapsulates the active ingridients inside and releases by
biodegradation over time. Some MILs, such as MIL-100, could also have
a similar mechanism in the release. On the other hand, MOFs such as
UiO-66 are ideal nano-vehicles for oral delivery due to their slow
degradation and high chemical stability in a phosphate solution, which
works for colon-specific drug delivery. Although targeting agents are
clinically approved in CRC, there are still many challenges toward tar-
geted therapy including diagnostic tools, toxicity and costs The inves-
tigation of new effective SDDSs for CRC patients could improve
treatments. Several studies have used a pool of diverse nanoparticles to
introduce new therapies with a better efficacy, a quicker treatment
period, and ability to reduce the neurotoxicity of OX or other side effects
(Brown et al., 2010; Handali et al., 2019; Nguyen et al., 2019). To the
best of our knowledge, there were no studies to focus on the compre-
hension of the OX encapsulation into MOFs and its related delivery
processes in CRC, which is crucial to be improved. We introduced an
effective DDS to eradicate CRC cells by combining U with folic acid (FA)
and enhancing the internalization process in CRC cells (Fig. 1,A). Zr-
based MOFs such as UiOs have higher stabilities than other MOFs and
are biocompatible and efficient DDSs in cellular or animal models (Bai
et al., 2016). In particular, the ability of UiO-66 to penetrate cell walls
(Orellana-Tavra et al., 2016a, 2016b) together with its degradability in
phosphate medium (Orellana-Tavra et al., 2016a, 2016b) makes it a
drug loading and release due to hydrogen bonding, giving rise to DDSs
with functionalities for biological applications.
FA and FA conjugates could attach to FR that is overexpressed by
CRC CT-26 cells (Fig. 1,A). The aim of active targeting is to use receptor
´
´
internalization and to improve the cellular uptake (Abanades Lazaro
et al., 2018a; Wu et al., 2018). Healthy cells are less prone to FA uptake
than CRC cells, which are FR+. In fact, a subset of CRCs actually have
this characteristic, and overexpression occurs in approximately 30–40%
of human CRC tissues, FA can be used in theses conditions (Shia et al.,
2008; D’angelica et al., 2011). FA is essential in one-carbon (1C)
metabolism and biosynthesis of nucleotides, which are essential for
cancer growth (Sudimack and Lee, 2000). Overexpression of the alpha
isoform of FR (FRα) in cancers, especially CRC cells, is the most
important reason for using folic acid as a targeting agent. Therefore, FU
would have the ability to be internalized after binding to CRC cells via
clathrin-mediated endocytosis. In this strategy, FU could result in the
direct killing of CRC cells, but not the healthy ones.
In this research, a one-pot synthesis for direct incorporation of FA
into U was used. According to the previous reports, free carboxylic acids
´
´
in FA can bind to the [Zr6O4(OH)4] clusters (Fig. 1,B) (Abanades Lazaro
et al., 2018b). Different amounts of FA were used to reach the optimal
composition, and 15% FA doping was selected via PXRD and CHN an-
alyses. CHN analyses showed that the contents of nitrogen and carbon
were increased due to the incorporation of folic acid.
FTIR spectra of the F, OX, U, FU, U(OX) and FU(OX) were shown in
Fig. 2,A. The presence of new peaks demonstrated the changes over FU,
U(OX) and FU(OX) that was compared to U, FA and OX. In FT-IR
spectroscopy, the peak of FA at 3232 cmꢀ 1 was related to NH2 group,
at 1693 cmꢀ 1 was associated with
ν
(COOH), at 1614 and 1622 cmꢀ 1
were ascribed to
ν
(N-H) bending vibrations and at 1483 cmꢀ 1 was
´
´
good candidate for folic acid-based DDSs. For instance, Abanades Lazaro
used a PEGylated analog of UiO-66 (Zr-fum) and UiO-66 against HeLa
and MCF-7 cancer cells, which were well tolerated by HEK293 kidney
cells, J774 macrophages, and human peripheral blood lymphocytes
related to the vibration of the phenyl ring (Thu et al., 2015; Nam et al.,
2016). FU showed the peaks at 3232 and 1639 cmꢀ 1 that corresponded
to amine and carboxylate groups in FA, which could not be observed in
U. Characteristic peaks of OX appeared between 2860 and 2990 cmꢀ 1
´
(Abanades et al., 2018). Moreover, the amine-functionalized UiO-66
that was related to ν(CH2) of oxalate ligand. The other peak at 3087
could be regarded as an even better drug carrier than conventional ones
in which numerous amino groups of U or FU can be used to control over
cmꢀ 1 was associated with
ν(NH2) (Agrahari et al., 2017). Oxalate ligand
also showed characteristic peaks of
ν
(COOꢀ )sym and
ν
(COOꢀ )asym at
Fig. 1. Attachment and internalization of FA-conjugated MOFs to FR
α
that is overexpressed by CRC CT-26 cells in proportion with Free MOF (A). Combining U with
folic acid (FA) as a targeting agent (B).
5

A. Hashemzadeh et al.
Toxicology and Applied Pharmacology 423 (2021) 115573
Fig. 2. FTIR spectra of U, FU, U(OX), and FU(OX) in comparison with FA and OX (A). PXRD of U: UiO-66-NH2, U(OX): loaded U with OX FU: UiO-66-NH2-FA, FU
(OX): loaded FU with OX in comparison with simulated pattern with reference code of RUBTAK02 obtained from the Cambridge structural database (B).
1666 and 1699 cmꢀ 1 (Kazbanov et al., 2001; Ray et al., 2016). The peaks
appeared at 521 and 618 cmꢀ 1 were associated with vibrations of Pt-N
bond (Ray et al., 2016). The observed peaks at 1378 and 1318 cmꢀ 1
obtained and matched the simulated one (Reference code: RUBTAK02)
as displayed in Fig. 2,B (Valenzano et al., 2011). The reflection planes
including (111), (200), (222), (400), (331), (420), (333), (440), (600),
(711), and (731) placed at 2θ of 7.3, 8.6, 14.8, 17.1, 18.6, 19.2, 22.3,
24.3, 25.8, 30.8 and 33.1◦ are associated with the simulated pattern
obtained from crystal information file (CIF). The sharp intensities of
peaks and the position of reflection planes showed the structural
integrity after FA doping. PXRD patterns of U(OX) and FU(OX) proved
that the loading does not influence the integrity of MOF. The FWHMs
were used to obtain the changes in crystallite sizes after drug loading.
The obtained crystallite sizes, which were calculated by the scherrer
were ascribed to
ν
(C-O) and
ν
(C-C). The peaks at 811, 776 and 575 cmꢀ
were ascribed to δ(COO) and Pt-O vibrations or their combination forms
´
(Wysokinski et al., 2006). U(OX) and FU(OX), showed the typical vi-
brations of C-H that appeared between 2860 and 2990 cmꢀ 1 in the FTIR
spectrum of OX (Wang et al., 2017; Cao et al., 2018b). U(OX) and FU
(OX) showed the presence of a new vibration at 1693 and 1694 cmꢀ 1
respectively, which was attributed to
ν
(COOꢀ )sym of OX.
Powder X-ray diffraction (PXRD) patterns of the products were
Fig. 3. Absorbance measurements of OX before and after loading in U and FU by UV–Vis spectrophotometry (A).The drug release measurements from U(OX) and FU
(OX) against time (B).The BET analyses of U, U(OX), FU and FU(OX) (C).
6

A. Hashemzadeh et al.
Toxicology and Applied Pharmacology 423 (2021) 115573
equation, were 30.5, 34.6, 23.09 and 17.6 nm for U, U(OX), FU and FU
(OX) respectively.
mapping confirmed the elemental composition of U(OX) and FU(OX)
without known peak related to any impurities. In the case of U(OX) and
For drug encapsulation, the OX was impregnated using activated
MOF in deionized water. After loading, the prepared nanodrugs were
dispersed in a 5% dextrin solution. For drug loading, the maximum
absorbance at λmax = 252 nm was used to obtain the following equation:
A = 0.0015 c + 0.0111 (A: absorbance and c: concentration, Fig. 3,A).
There is also potential absorbance from both the ligand and folic acid,
hence unloaded MOFs were used and treated in similar conditions as the
baseline to cancel the effect of possible detached molecules from the
MOF under the loading conditions. Therefore, overlapping absorbance
would be minimized in drug loading and preventing the overestimation
of the clinical potential of the DDS. The evaluated amounts for U and FU
were calculated 304.5 mg and 293.0 mg per 1 g respectively, which were
equal to 30.4% and 29.3% of OX loading. ICP-OES of the supernatant
after loading also confirms the above results (32.9% for U and 26.1% for
FU.
FU(OX), Pt transitions including PtMα, PtMβ, and PtLα displayed the
presence of OX (Fig. 4,B-a, c). The spatial distribution of OX was
analyzed by EDX mapping, which showed OX was well dispersed in both
U and FU (Fig. 4B- b, d).
Dynamic light scattering (DLS) was used to analyze the changes in
size after OX loading (Fig. 5, Table 4). 25
μ
g mLꢀ 1 of each sample was
prepared for DLS analyses. The particles were completely dispersible in
water and culture medium, which remain unchanged for at least a week.
According to the results, sizes were increased comparatively in water
and culture medium against dry sizes obtained from FESEM images. It
was reported that hydrodynamic sizes were usually biased towards
larger particles or indicating aggregates (Raj et al., 2016). Corona for-
mation is another reason that led to an increase in size in the culture
medium (Mahmoudi et al., 2011; Monopoli et al., 2011; Yu et al., 2013).
Results showed that hydrodynamic sizes of FU and FU(OX) were
changed dramatically. The size of FU in water was 2.4 times larger than
its solid size, and subsequently 4.3 times in culture medium. A similar
trend was observed for FU(OX) in water and culture medium. It is
important to note that zeta potentials for U and FU were positive as
expected, but they got negative after OX loading. Lower positivity in the
zeta potential of FU (14.6 mV) than U (21.2) might be due to the surface
functionalization of U with FA, which led to less available amino groups
in contact with the solvent. The negativity observed in drug-loaded
DDSs might be caused by the anchored OX on the external surface. In
the growth medium, aggregation is a common phenomenon due to
corona formation in the presence of proteins but it can increase the
colloidal stability (Bellido et al., 2014). Corona formation or ionic
strength could also balance the overall zeta potentials in the culture
medium. The results showed that zeta potentials of the particles in
growth media were slightly negative. This could not affect the func-
tionality of the DDSs in vivo because of the acidic conditions of solid
tumors. In acidic environments, amine groups usually get positive,
leading to better interactions with the negatively charged cell
membranes.
The drug release profiles in 48 h for both U(OX) and FU(OX) were
measured (shown in Fig. 3,B). PBS or culture medium was not used due
to the instability of OX in chlorine-containing solutions (Mehta et al.,
2015). It appears that the hydrophilic nature of both U and OX (Screnci
et al., 2000) could influence the amount of drug loading and release
mechanism (Zhang et al., 2017a). The initial burst releases of OX were
15.7% in U(OX) and 10.8% FU(OX) per hour. The releases have reached
a plateau at 62.9% and 52.3% for U(OX) and FU(OX), respectively. The
incomplete drug release might be due to drug entrapment into the pores,
O… H-N hydrogen bond with O atom of OX and NH2 group on U and FU
or medium conditions.
Brunauer-Emmett-Teller (BET) analyses showed the changes in sur-
face area, pore sizes and total pore volumes (Fig. 3,C, Table 3). Analyses
showed a type II isotherm for curves. After drug loading, surface area
and pore sizes were decreased, indicating that OX could penetrate the
pores.
FESEM images showed the morphological changes at different stages
and were used to analyze the influence of drug loading on U and FU
(Fig. 4,A). It appears that the semi-spherical morphology was main-
tained after loading. Furthermore, the size distribution appeared to be
between ~50 to ~200 nm. According to FESEM images, the changes in
diameter were not significant after loading. The sizes(±S.E.M.) were
estimated as follows: U: 105.39 ± 1.35 nm, U(OX): 128.37 ± 1.89 nm,
FU: 118.83 ± 1.67 nm and FU(OX): 115.15 ± 1.97 nm. FESEM images
(Fig. 4,A) clearly showed that the doping of FA and drug loading did not
change the morphologies significantly. The Full width at half-maximum
(FWHM) displayed that the size distribution was increased for U(OX)
(FWHM = 82.16) in comparison to U (FWHM = 59.93), but the change
in FWHM of FU after loading was not significant. Because of the
appearance of new interactions between oxaliplatin and U (or FU) and
stiring, the transformation of grains to crytalites and the aggregation of
particles may occur, which could cause the broadening of the FWHM
and the increase in the range of the particles. The sizes in FESEM images
showed agglomeration and were more than three times larger than the
crystallite sizes. The boundary images of particles (grain sizes) from
FESEM are usually less than the crystallite size obtained from the scherer
equation using PXRD. The grain size indicated that each particle may
contain at least 3 to 6 crystallites.
The MTT assay was used to predict the cytotoxicy of OX, U(OX), and
FU(OX) on colorectal CT-26 cancer cells. During this process, the cells
were exposed to the increasing concentrations of OX, U(OX), and FU
(OX) (0.001 to 1000 μg/mL) for 24 h (Fig. 6,A). The MTT test high-
lighted that nanoparticles, alone, had no significant cytotoxicity, but for
FU(OX) cell death was significantly increased. To compare the cyto-
toxity of OX, U(OX), and FU(OX), the half-maximal inhibitory concen-
trations (IC50) were calculated at 24 h, which were 21.38, 95.50, and
18.20
μg/mL respectively. The IC50 of U(OX) and FU(OX) with the
amount of oxaliplatin loading were 28.9 and 5.33
μ
g/mL accordingly.
The IC50 of FU(OX) was one-quarter of OX, which shows its efficacy.
The functionalization with FA enhanced the uptake of nanoparticles into
CT-26 cells, hence the particles exhibited higher toxicity. The results
were in line with our hypothesis of increased cytotoxicity by using FA as
a targeting agent.
To investigate more, we used three other tests including migration
assay, spheroid test and oxidative stress to confirm the MTT results. In
the first attempt, a scratch assay was used to find the impression of U
(OX) and FU(OX) on cell migration(Fig. 6,A). The scratched area was
photographed every day, then the changes were calculated by the
ImageJ software, and finally, the groups were compared. The results
showed that the gap between the cells was filled by the migratory cells
after 4 days in the control groups, while this process was not noticeable
in treated groups. FU(OX) was shown to be more efficient in comparison
with other groups (Fig. 6,B). Therefore, the data confirms that FU(OX),
At the same time, energy-dispersive X-ray (EDX) analyzed and EDX
Table 3
Surface area, pore size, and total pore volume of U, U(OX), FU and FU (OX).
Sample
Surface area
Pore size
[Å]
Total pore volume
[m2/g]
[p/p0 = 0.990]
at 500 μg/mL, prevented migration better than U(OX).
Next, a spheroid assay was used to examine the anti-cancer capacity
of U(OX) and FU(OX) in proportion with OX in a three dimensional (3-D)
cellular structure. Although the spheroid size is not an appropriate
measurement for cell proliferation, it was reported because it is
U
714.93
357.74
573.39
376.14
12.2
10.64
9.87
7.38
0.4205
0.2501
0.3381
0.2167
U(OX)
FU
FU(OX)
7

A. Hashemzadeh et al.
Toxicology and Applied Pharmacology 423 (2021) 115573
Fig. 4. FESEM images and size distribution of (a), (b) U with magnifications of 1
μ
m and 200 nm, (c), (d) U(OX) with magnifications of 500 nm and 200 nm, (e), (f)
FU with magnifications of 1 μm and 200 nm, and (g), (h) FU(OX) with magnifications of 1 μm and 200 nm (A), the particle sizes were measured by ImageJ software
and the frequencies (particle size distribution) were analyzed by SPSS software using descriptive statistics. The elemental composition of the nano drugs and platinum
spatial distribution using EDX and EDX mapping of (a), (b) U(OX), and (c), (d) FU(OX) and their corresponding SE images (B) with a magnification of 500 nm by a
TESCAN Mira 3 LMU device.
compatible with the previous results. After spheroid formation, they
were treated with two doses of nanodrugs (500 and 50 μg/mL) and 20
μ
M of OX (Fig. 6,C). After three days, we observed noteworthy differ-
ences between the sizes of the spheroids in treated and control groups.
The obtained results showed that treatments could diminish the prolif-
eration and progression ability of CRC cells in three-dimensional
models. It was also found that FU(OX) was more effective than OX
and U(OX) in inhibiting cell proliferation in the 3D-model of cell culture
(Fig. 6,C). In more detail, the sizes of the spheroids were decreased in the
following manner: FU(OX) at 50
at 500 g/mL ≅ FU(OX) at 500
μ
g/mL < U(OX) at 50
μg/mL < U(OX)
μ
μ
g/mL < OX 20 M < control. It appears
μ
that FU(OX) can compete with OX and displays higher inhibition rates
toward CT-26 cells in both cell migration and spheroid tests, which
could confirm the applicability of the folate conjugated form. It is
possible that FU(OX) would be superior in vivo. It should be noted that
the size of spheroids can be generally reduced over time, but the size
reduction showed the same phenomenon that was observed in the MTT
assay.
Fig. 5. DLS size analyses of U, U(OX), FU and FU(OX) in water and cul-
ture medium.
Moreover, oxidative stress analyses were performed to investigate
ROS production. The oxidative stress in the cancer cells is expressed by
the imbalance in the systemic expression of ROS and the antioxidant
Table 4
Average particle size and zeta potential of U, U(OX), FU and FU(OX).
Particle Size [nm]
Zeta Potential [mV]
PXRD
FESEM
Solid
DLS
Crystallite size
Water
Culture medium
Water
Culture medium
U
30.5
34.6
23.1
17.6
105.3 ± 25.6
128.4 ± 35.1
118.8 ± 33.9
115.1 ± 36.6
157.5 ± 22.4
160.3 ± 81.2
289.5 ± 44.5
297.9 ± 47.6
206.1 ± 27.4
289.9 ± 68.1
519.3 ± 118.0
468.1 ± 86.7
21.2
ꢀ 38
ꢀ 1.3
ꢀ 2.2
ꢀ 3.2
ꢀ 3.5
U(OX)
FU
14.6
ꢀ 22.8
FU(OX)
8

A. Hashemzadeh et al.
Toxicology and Applied Pharmacology 423 (2021) 115573
Fig. 6. FU(OX) and U(OX) reduce cell growth and proliferation in colorectal cancer cells.)Growing inhibitory properties of U(OX), FU(OX), and OX were studied after
24 h in CT26 cells. MTT tests were performed in triplicate ((A). Results of the scratch assay of FU(OX), U(OX), and OX in CT-26 cells. The scratch assay was performed
in triplicate (p < 0.01). Inhibition of cancer cell migration increase statistically in FU(OX) treated cells in comparison to control after 3 days (B). After treatment of
CT26 spheroids with OX, U(OX), and FU(OX) at 5xIC50 concentration. (b) a comparison of spheroid size was shown after 7 days. The growth of spheroids treated with
OX, U(OX), and FU(OX) was decreased in comparison to the control group (C). Data are expressed as Mean ± SEM and differences are considered to be statistically
significant at P < 0.05 using One-way analysis of variance (ANOVA), and LSD multiple comparisons test. All data was obtained from three independent experiments.
defense mechanisms (Soleimani et al., 2019). Oxidative stress and
antioxidant metabolism have critical roles in the progression of CRC
(Asadi-Samani et al., 2019). Most forms of chemotherapy rely on the
generation of oxidative stress, a strategy that can cause cancer cell
death. Several studies have argued that increased levels of ROS can
cause cancer cell apoptosis,therefore can be led to the treatment of
cancer (Asadi-Samani et al., 2019). In the present study, MDA as lipid
peroxides in FU(OX), U(OX), and OX groups was increased compared to
control. During chemotherapy, the key reason for elevated cellular ROS
production is the inhibition of the antioxidant system such as SOD and
SH-group (Afrin et al., 2019). Various studies have also shown that
tumor cell growth is reduced by a decrease in the antioxidant defense
system (Xu et al., 2017), our study also confirms this claim. Among OX
and nanodrugs, the results showed FU(OX) could better induce ROS and
disrupt the antioxidant system, which means, in theory, it should pre-
vent cancer progression better than U(OX) or OX (Fig. 7,A-C).
Fig. 7. Intracellular ROS production after treatment or no treatment with U(OX), FU(OX), and OX were explored by measurements of MDA activity, total thiol group,
and SOD activity in colorectal cancer cells. Data are expressed as Mean ± SEM and differences are considered to be statistically significant at P < 0.05 using One-way
analysis of variance (ANOVA), and LSD multiple comparisons test. All data was obtained from three independent experiments. ***P < 0.001 compared to control and
and ###P < 0.001 compared to OX group.
9

A. Hashemzadeh et al.
Toxicology and Applied Pharmacology 423 (2021) 115573
fumarate MOF for selective cytotoxicity and immune system compatibility in
4. Conclusion
nanoscale drug delivery. ACS Appl. Mater. Interfaces 10, 31146–31157.
´
´
˜
Abanades Lazaro, I., Haddad, S., Rodrigo-Munoz, J.M., Orellana-Tavra, C., del Pozo, V.,
Fairen-Jimenez, D., Forgan, R.S., 2018b. Mechanistic investigation into the selective
anticancer cytotoxicity and immune system response of surface-functionalized,
dichloroacetate-loaded, UiO-66 nanoparticles. ACS Appl. Mater. Interfaces 10,
5255–5268.
In summary, UIO-66-NH2 and FA functionalized UIO-66-NH2 was
realized as a new active drug transport system in oxaliplatin delivery
against colorectal cancer cells. FU(OX) showed excellent receptor-
specific targeting for folate receptor-positive colon cancer cells in
MTT, spheroid model, cell migration and oxidative features. FU(OX) had
a higher inhibitory effect on proliferation compared to OX and U(OX),
which could confirm the applicability of the folate conjugated form. FU
(OX) could induce higher levels of ROS in comparison with oxaliplatin
and U(OX). The results obtained from the spheroid model and the cell
migration have also confirmed the superiority of FU(OX) compared to
oxaliplatin. Such drug delivery systems could be used and verified in
biomedical applications in colorectal cancer animal models, which will
be done in future studies. Moreover, this research could be an initiative
to treat CRC patients using non-toxic and biocompatible MOFs.
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The authors declare that all data supporting the findings of this study
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Author contributions
Dr. Alireza Hashemzadeh was initiated, designed and executed the
project and finally drafted the manuscript. The cellular experiments
were performed and written by Frouzan Amerizadeh. The Oxidation
stress tests were done and written by Fereshteh Asgharzadeh. Moham-
mad Landarani was partially contributed in data analyses. The whole
project including the study and performed experiments was supervised
by Prof. Majid Khazaei. He also provided all the chemicals, equipment
and funds. Dr. Amir Avan, Dr. Seyed Mahdi Hassanian and Dr. Majid
Daroudi were both consultants of this project. Dr. Amir Avan also su-
pervised cellular experiments.
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Bansal, D., Gulbake, A., Tiwari, J., Jain, S.K., 2016. Development of liposomes entrapped
in alginate beads for the treatment of colorectal cancer. Int. J. Biol. Macromol. 82,
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Bargi, R., Asgharzadeh, F., Beheshti, F., Hosseini, M., Sadeghnia, H.R., Khazaei, M.,
2017. The effects of thymoquinone on hippocampal cytokine level, brain oxidative
stress status and memory deficits induced by lipopolysaccharide in rats. Cytokine 96,
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Funding sources
Bellido, E., Guillevic, M., Hidalgo, T., Santander-Ortega, M.J., Serre, C., Horcajada, P.,
2014. Understanding the colloidal stability of the mesoporous MIL-100 (Fe)
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and characterization of folate conjugated nanoparticles of doxorubicin using PLGA-
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This work financially supported by National Institute for Medical
Research Development (NIMAD) through grant # 982641, Iran National
Science Foundation (INSF) through grant # 97017412 and Mashhad
University of Medical Sciences (MUMS) through grant # 971010 as a
research project.
Brown, S.D., Nativo, P., Smith, J.-A., Stirling, D., Edwards, P.R., Venugopal, B., Flint, D.
J., Plumb, J.A., Graham, D., Wheate, N.J., 2010. Gold nanoparticles for the improved
anticancer drug delivery of the active component of oxaliplatin. J. Am. Chem. Soc.
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Author statement
Cai, W., Wang, J., Chu, C., Chen, W., Wu, C., Liu, G., 2019. Metal–Organic Framework-
Based Stimuli-Responsive Systems for Drug Delivery. Adv. Sci. 6, 1801526.
Cao, J., Chen, Z., Chi, J., Sun, Y., Sun, Y., 2018a. Recent progress in synergistic
chemotherapy and phototherapy by targeted drug delivery systems for cancer
treatment. Artif. Cells Nanomed. Biotechnol. 46, 817–830.
All authors are aware of and agree to the content of the manuscript.
Neither the submitted paper, nor any similar article, either in whole or in
part, have been or will be submitted or published in any other scientific
journal. The authors have indicated no potential conflicts of interest in
this study.
Cao, Y., Zhang, H., Song, F., Huang, T., Ji, J., Zhong, Q., Chu, W., Xu, Q., 2018b. UiO-66-
NH2/GO Composite: Synthesis, Characterization and CO2 Adsorption Performance.
Chen, F., Ehlerding, E.B., Cai, W., 2014. Theranostic nanoparticles. J. Nucl. Med. 55,
1919–1922.
Chowdhuri, A.R., Bhattacharya, D., Sahu, S.K., 2016a. Magnetic nanoscale metal organic
frameworks for potential targeted anticancer drug delivery, imaging and as an MRI
contrast agent. Dalton Trans. 45, 2963–2973.
Declaration of Competing Interest
The authors have no conflicts of interest to declare.
Acknowledgments
Chowdhuri, A.R., Laha, D., Pal, S., Karmakar, P., Sahu, S.K., 2016b. One-pot synthesis of
folic acid encapsulated upconversion nanoscale metal organic frameworks for
targeting, imaging and pH responsive drug release. Dalton Trans. 45, 18120–18132.
Chowdhuri, A.R., Singh, T., Ghosh, S.K., Sahu, S.K., 2016c. Carbon dots embedded
magnetic nanoparticles@ chitosan@ metal organic framework as a nanoprobe for
pH sensitive targeted anticancer drug delivery. ACS Appl. Mater. Interfaces 8,
16573–16583.
This study was supported by Elite Researcher Grant Committee
under award number #982641 from the National Institute for Medical
Research Development (NIMAD) and grant number # 97017412 from
Iran National Science Foundation (INSF).
Chowdhuri, A.R., Das, B., Kumar, A., Tripathy, S., Roy, S., Sahu, S.K., 2017a. One-pot
synthesis of multifunctional nanoscale metal-organic frameworks as an effective
antibacterial agent against multidrug-resistant Staphylococcus aureus.
Nanotechnology 28, 095102.
Chowdhuri, A.R., Laha, D., Chandra, S., Karmakar, P., Sahu, S.K., 2017b. Synthesis of
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Clemons, T.D., Singh, R., Sorolla, A., Chaudhari, N., Hubbard, A., Iyer, K.S., 2018.
Distinction between active and passive targeting of nanoparticles dictate their
overall therapeutic efficacy. Langmuir 34, 15343–15349.
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