Journal of Solid State Chemistry 300 (2021) 122259

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Journal of Solid State Chemistry

journal homepage: www.elsevier.com/locate/jssc

Chitosan coated biocompatible zeolitic imidazolate framework ZIF-90 for

targeted delivery of anticancer drug methotrexate
Xiang-Xin Cao a, Shui-Li Liu a, Jing-Sheng Lu a, Zhen-Wei Zhang b, c, Gang Wang a, c,
Qing Chen a, c, *, Ning Lin b, c, **
a
Guangxi Scientific Research Center of Traditional Chinese Medicine, Guangxi University of Chinese Medicine, Nanning, 530200, PR China
b
College of Pharmacy, Guangxi University of Chinese Medicine, Nanning, 530200, PR China
c
Guangxi Zhuang Yao Medicine Center of Engineering and Technology, Nanning, 530200, PR China

A R T I C L E I N F O A B S T R A C T

Keywords: In this work, using zeolitic imidazolate framework (ZIF) as carrier, a new biocompatible anti-cancer drug delivery
Metal-organic frameworks system CS@MTX@ZIF-90 (CMZ) was efficiently synthesized by one-pot method. Methotrexate (MTX), an anti-
Biocompatible materials cancer drug as folic acid analogue was loaded into ZIF-90 through Schiff base reaction between amino group in
ZIF-90
MTX and aldehyde group of imidazole-2-carboxaldehyde ligand with drug loading amount about 250 mg/g. As a
Methotrexate
pH-sensitive biomaterial, chitosan (CS) was modificated by the outer layer of the particles to improve the pH
Controlled release
responsiveness of the composite CMZ during the drug release process. By releasing a large amount of MTX under
acidic conditions (the pH environment around tumor cells), and releasing a substantially lower amount of MTX at
a normal environment, CMZ exhibited a pH-responsive and target-selective behavior. In addition, in vitro cyto-
toxicity and anticancer activity results showed that CMZ can inhibit the growth of liver cancer HepG2 cells,
prostate cancer DU145 cells and gastric cancer SGC7901 cells, with only negligible toxicity to normal EC304 cells.
Based upon the results of corresponding experiments, CMZ exhibited high MTX drug loading, cancer-targeted
release and good biocompatibility, which can be used as a good candidate for anticancer drug delivery system.

1. Introduction
With the rapid development of therapeutic diagnostics, a series of
therapeutic methods have been widely studied in cancer treatment [1,2].
Among them, chemotherapy can be considered as a kind of clinical
application currently, which can treat a variety of cancers [3,4]. Meth-
otrexate (MTX, Fig. S1) is an important kind of chemotherapeutic drug
with good curative effect in treating gastric cancer, breast cancer, liver
cancer and lung cancer [5,6]. However, the traditional anticancer drug
will be degraded before it reaches the tumor area, with low retention rate
in tumor, short blood circulation, poor tissue penetration, and certain
drug resistance and toxicity, which seriously limits the clinical applica-
tion of MTX in tumor treatment [7]. Therefore, in order to improve the
therapeutic efficiency of drugs and overcome the toxic and side effects of
drugs on non-tumor normal tissues, the corresponding drug carrier sys-
tems are urgently needed for the clinical application.
Metal-organic frameworks (MOFs), which are constructed by metal
ions and organic ligands to form two- or three-dimensional coordination
polymer materials, have the advantages of porosity, large surface area,
tunable structure and extensive functions [8–12]. Above advantages
cause MOFs to be applicable to gas storage [13], separation [14–16],
catalysis [17], sensing [18,19] and electrodes [20–22]. In recent years,
some biocompatible MOFs have also been used in the field of biomedi-
cine, especially as applied to drug carriers for smart drug delivery sys-
tems [23–28]. However, most of drug delivery systems obtained from
MOFs still exist some shortcomings such as low biological degradation
rate and unnecessary elemental constituents of the human, cadmium for
example, which resulted in low biosecurity with regard to clinical
application. The more prominent problem is that many drug carriers are
unable to target the delivery of drugs, with the result that tumor tissues
cannot be effectively treated. In addition, some drug carriers only work a
few hours in the drug release process, making them difficult to achieve a
slow, sustained treatment. Therefore, MOF-based carrier materials with
targeting, controlled release and good biocompatibility are urgently
needed to be developed for the current delivery systems in cancer
treatment.

* Corresponding author. Guangxi Scientific Research Center of Traditional Chinese Medicine, Guangxi University of Chinese Medicine, Nanning, 530200, PR China.
** Corresponding author. Guangxi Scientific Research Center of Traditional Chinese Medicine, Guangxi University of Chinese Medicine, Nanning, 530200, PR China.
E-mail addresses: qing0082@163.com (Q. Chen), linninginnanning@163.com (N. Lin).

https://doi.org/10.1016/j.jssc.2021.122259
Received 5 March 2021; Received in revised form 2 May 2021; Accepted 3 May 2021
Available online 14 May 2021
0022-4596/© 2021 Elsevier Inc. All rights reserved.
X.-X. Cao et al.
Among MOFs, zeolitic imidazolate frameworks (ZIFs), formed by
imidazole ligands and tetrahedral metal ions, have been widely studied
for their potential applications as drug carriers due to their large cages,
low toxicity and pH-sensitive dissociation of the coordination framework
[29–32]. Compared with widely studied ZIF-8, ZIF-90 has its own char-
acteristics in structure. Utilizing the aldehyde group on the ZIF-90 ligand,
it can form Schiff base bonds with amino groups, which contributes to
efficiently loading drug molecules using its amino groups. When a
weakly acidic cancerous site is reached, the breakage of the coordination
bond in the ZIF-90 structure leads to the collapse of the skeleton, and the
Schiff base bonds are broken because of the hydrolysis reaction, which
leads to the release of the drug. The above featuring performance make
ZIF-90 a good drug carrier for targeting tumor tissues [33–36].
Chitosan (CS) is a multifunctional natural polysaccharide comprised
of β-(1,4)-linked glucosamine and N-acetyl glucosamine units. This
polymer is highly biodegradable, bioavailable, and non-toxic, which has
many important medicinal properties. In addition, the protonation and
deprotonation of the amino groups in CS can lead to some structural
changes (expansion and contraction), making it pH sensitive. When the
composite particles expand in the lower pH environments, the encapsu-
lated drug is released. Contrarily, when the composite particles are
involved in neutral or alkaline conditions, they will contract to prevent
the leakage of drug molecules, which means that CS can be used to design
pH-responsive drug delivery systems [37–39].
To further enhance tumor-specific releasing characteristic of drug
carrier and avoid drug release in non-tumor sites, based upon our pre-
vious work [40–43], we try to build a dual pH-responsive anticancer drug
delivery system with high drug loading. Using biocompatible material
ZIF-90, modified material CS and anticancer drug MTX, drug-loaded
composite materials MTX@ZIF-90 (MZ) and CS@MTX@ZIF-90 (CMZ)
were successfully synthesized and systematically characterized in this
paper. The drug loading capacity of the MTX was about 250 mg/g.
Meanwhile, the drug release behavior of the CMZ with low pH response
was verified by simulating the tumor microenvironment and normal
tissues in vitro. The MTT experiments of EC304, HepG2, DU145 and
SGC7901 cells were carried out to verify the biocompatibility and tar-
geting characteristics of the new drug-loaded particles. This study pro-
vides an example of effective drug carrier for methotrexate, and provides
a favorable modification strategy to enhance the performance of anti-
cancer drug delivery (Scheme 1).
Scheme 1. Schematic diagram of synthesis of CMZ and pH-triggered MTX release from CMZ inside cells.
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Journal of Solid State Chemistry 300 (2021) 122259
2. Experimental section
2.1. General materials and characterizations
The reagents and solvents were commercially available and were
used as received without further purification. Powder X-ray diffraction
(PXRD) patterns were recorded on a Rigaku Miniflex600 diffractometer
with a scanning angle of 5–45
and a scan rate of 4
/min. Scanning
electron microscope (SEM) micrographs were obtained using a Quanta
250 instrument of 15 kV accelerating voltage. The Fourier transform
infrared spectroscopy (FT-IR) spectra were obtained over the 5004000
cm1 wavenumber range by a Nicolet Nexus 470 FT-IR spectrometer,
using the KBr disks of the samples. High-performance liquid chroma-
tography (HPLC) analysis was conducted using the Agilent 1260 II
chromatographic system. Thermogravimetric analysis (TGA) was


measured by NETZSCH STA 449F5 with a rate heating up of 10 C/min



from 26 C to 800 C. Nitrogen (N2) sorption isotherms were measured
on a Micromeritics ASAP 2020 surface area analyzer at 77 K. Dynamic
light scattering (DLS) analysis was obtained on a Zetasizer Nano ZSE
analyzer. The fluorescence images of cell were collected on a CKX53
inverted microscope from Olympus. The Epoch2 Enzyme labeling in-
strument was used to detect the cell viability of EC304 cells, HepG2 cells,
DU145 cells and SGC7901 cells.
2.2. Preparation of ZIF-90
The ligand imidazole-2-carboxaldehyde (2-ICA, 70 mg, 0.72 mmol)
and Zn(NO3)2⋅6H2O (107 mg, 0.36 mmol) were dissolved with 0.1 mL
trimethylamine (TEA) in a solution of 10 mL methanol. After refluxing
and stirring at 70
C for 20 min, the mixture was stirred at room tem-
perature for 1 h. The pale-yellow precipitates were collected and washed
with methanol to give the pure material ZIF-90. The sample was dried at
room temperature. The yield was 98% based on Zn.
2.3. Preparation of MTX@ZIF-90 (MZ) and CS@MTX@ZIF-90 (CMZ)
The particles of MZ and CMZ were obtained by one-pot method in
which the drug MTX was loaded in the synthesis process of ZIF-90. 2-ICA
(70 mg, 0.72 mmol) was dissolved in 5 mL methanol with 0.1 mL TEA.
Meanwhile, MTX (40 mg, 0.09 mmol) was ultrasonically dissolved in 8
X.-X. Cao et al.
mL water with an appropriate amount of sodium hydroxide, and then
Zn(NO3)2⋅6H2O (107 mg, 0.36 mmol) was added. The above solutions
were mixed and heated slightly. After that, the resulted solution was
stirred at room temperature for 2 h, and the yellow crystalline of MZ was
obtained.
Besides that, during the stirring process at room temperature for
preparing MZ, the prepared 1 %w/v CS solution (1.5 g CS was added to 5
wt% 150 mL acetic acid aqueous solution and stirred at room tempera-
ture for 24 h) was added. The resulted solution was stirred for 12 h. The
crystalline sample was washed with ethanol and water three times
respectively, then dried at room temperature, and the yellow product of
CMZ was obtained.
2.4. Drug loading and release
The MTX release from drug-loaded materials was estimated in PBS
solution with two different pH values (pH 7.4 and pH 5.5) at 37
C. MTX-
loaded samples (10 mg) were soaked in PBS buffer solution (40 mL). At
certain intervals, the resulted solution (0.5 mL) was taken out and the
fresh PBS buffer solution was replaced, then the drug concentration was
measured by HPLC. A FeiniGen C18 reverse-phase column (4.6 mm 
250 mm, 5 μm) was used and the mobile phase was composed of 0.3%
phosphoric acid aqueous solution and acetonitrile (85: 15, v/v). The
injection volume was 10 μL with a flow rate of 0.80 mL min1 at 40
C
and the effluent was measured at 302 nm. The drug-loading is calculated
by the final state of drug release, with the equation as follows: MTX
Loading (%) ¼ (weight of total released MTX/weight of total particles) 
100%. According to the standard curve of MTX solution, the equation
was used to calculate the release efficiency of MTX as follows: MTX
Release (%) ¼ (weight of released MTX/weight of loaded MTX in parti-
cles)  100%.
2.5. Cell cytotoxicity and anticancer activity assay
To evaluate the cytotoxicity and anticancer activity of ZIF-90 and
CMZ, the standard 3-(4,5-dimethylthialzol-2-yl)-2,5-dipHenyltetra-
zolium bromide (MTT) assay was implemented for human vascular
endothelial cell EC304, human hepatoma cell HepG2, prostate cancer cell
DU145 and gastric cancer cell SGC7901. These cells were firstly seeded in
96-well microplate at 5  103 cells per well and then incubated for 24 h
at 37
C. After that, different concentrations of samples in culture me-
dium were added and incubated with cells for another 48 h at 37
C.
Then, 10 μL of MTT (5 mg/mL in PBS) solution was added to each well
before the incubation for 3 h. After the supernatants were removed
carefully, 100 μL of DMSO was added and the 96-well microplate was
shaken for 10 min before the absorbance at 490 nm was recorded by a
microplate reader. Cell viability can be obtained according to the
following formula: cell viability (%) ¼ the absorbance value of the
sample well/the absorbance value of the control well  100%. All ex-
periments were executed in triplicate and the results were averaged.
In order to observe the inhibitory effect of CMZ on the proliferation of
cancer cells more intuitively, the acetoxymethyl (calcein-AM) and pro-
pidium iodide (PI) can be used to differentiate live cells (green, Ex: 495
nm; Em: 515 nm) from dead cells (red, Ex: 528 nm; Em: 617 nm). First,
HepG2 cells with the density of 5  104 cells/well were seeded in six-well
microplates and incubated for 24 h at 37
C. Then, ZIF-90, MTX and CMZ
nanoparticles with the concentration of 5 μg/mL were selected for cell
incubation and incubated with cells for 48 h at 37
C. After the cell
suspension was absorbed, the supernatant was removed by 12,000 rpm
centrifugation, and then fresh PBS solution was added to make dead cell
suspension. The solution was added to the primary hole, and 500 μL of
mixed solution of calcein-AM and PI was added to each hole was incu-
bated for 20 min at 37
C with 5% CO2 incubator. The staining was
observed under inverted phase contrast fluorescence microscope.
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Journal of Solid State Chemistry 300 (2021) 122259
3. Results and discussion
3.1. Construction and characterization
To summarize the previous synthesis methods of ZIF-90 [44–47], it
can be seen that the traditional methods of synthesizing ZIF-90 are sol-
vothermal method and solvent diffusion method, with DMF or water/-
alcohol/PVP as the main reaction solvents. These methods can obtain
products with good crystallinity, but the operations are complicated and
the yield is not high (only about 40%). It was reported recently that a new
solvothermal synthesis of ZIF-90 in methanol, and the sample prepared
by this method showed the highest yield and BET surface area for the
ZIF-90 [47]. In this paper, TEA deprotonated the ligand dissolved in
solvent and initiated frameworks formation. Moreover, imidazole based
ligands could be deprotonated under the action of TEA for solvent syn-
theses of ZIFs, without the formation of unnecessary metal hydroxides.
On the basis of the above method, the synthesis herein has been further
optimized. Specifically speaking, the ratio of the amount of starting
materials has been adjusted, the reaction time has been shortened (only
takes more than 1 h), and the reaction can be carried out at room tem-
perature after the starting materials are dissolved. It can be seen that the
reaction conditions have become more mild and convenient, and the
yield can still be as high as 98%. The improved synthesis method in this
work further improves the preparation efficiency of ZIF-90.
The morphology and SEM images of ZIF-90, MZ and CMZ are shown
in Fig. 1, S2 and S3. The particles of synthesized ZIF-90 are grey-white,
and well distributed with regular cubic crystalline morphology.
Compared with ZIF-90, after MTX loading and CS modification, MZ and
CMZ particles are yellow. The obvious changes in color indicate that MTX
has been loaded into the carriers. In addition, it can be seen from the SEM
figures that MZ and CMZ particles are still uniformly formed, with
obvious change in the overall shape of the particles, and the crystallinity
of the crystals is maintained, with only some slight edge blurring caused
by CS modification.
The PXRD patterns and FT-IR spectra were also used to further
confirm the construction of CMZ. The FT-IR spectra are shown in Fig. 2a
and S4. It can be seen that the characteristic absorption peaks of ZIF-90
appear at 2840 cm1 (C–H bending) and 1681 cm1 (C– – O bending),
which indicate the aldehyde groups in 2-ICA ligand, while the stretching
vibration band of the 2-ICA ligand appears clearly at 1200–1400 cm1
after coordination with Zn2þ ions. The spectra of CMZ show that a new
strong stretching vibration absorption is formed at 1651 cm1(C– –N
bending) and the peak of aldehyde group diminished dramatically, which
indicate that MTX is covalently loaded into ZIF-90 through the Schiff
base reaction between the aldehyde and the amino groups [33]. Mean-
while, there are characteristic peaks of CS at 1080 cm1 and 1210 cm1
in the spectra of CMZ, which further illustrate the successful coating of CS
on ZIF-90 and verify the formation of CMZ.
The structure and purity of as-synthesized ZIF-90 was further
confirmed by the PXRD measurement. As shown in Fig. 2b, the X-ray
diffraction patterns of the as-synthesized ZIF-90 match well with the
simulated patterns, indicating that pure phases of ZIF-90 were obtained.
Meanwhile, it can be seen that when CS is used for surface modification
to form CMZ, the diffraction pattern of CMZ is basically the same as ZIF-
90, indicating that the framework remains stable. In addition, the PXRD
Fig. 1. SEM images of ZIF-90, MZ and CMZ (scale bar ¼ 200 μm).
X.-X. Cao et al. Journal of Solid State Chemistry 300 (2021) 122259

positively charged CS with a large number of amino protonation was

introduced as the MZ layer, which caused the change of zeta potential
from the negative charge of the ZIF-90 and MZ to the positively charged
CS coating. The positive charges on the surface of the particles were also
good for the cellular uptake, as the cell membranes were negatively
charged [48].

3.2. Thermogravimetry and gas adsorption analysis

Thermal stabilities of ZIF-90, MZ and CMZ are also studied by TGA, as

presented in Fig. 4a. For the ZIF-90, there is a sharp weight loss from
room temperature to 70
C, due to the removal of guest methanol mol-
ecules in the pores. In contrast, MZ and CMZ both show a gentle weight
loss in that range, indicating much less solvent molecules adsorbed in the
pores, which should be attributed by the MTX loading. And from 70
C to
240
C, the weight variation between CMZ and MZ is 10%, which is due
to the modification of CS. Besides, ZIF-90, MZ and CMZ all exhibit similar
main decomposition stages after 300
C, suggesting that MTX loading
and CS modification have no obvious impact on the thermal stability of
ZIF-90.
After the synthesized ZIF-90, MZ and CMZ were activated by drying
under vacuum at 50
C, the N2 sorption experiments were conducted to
measure the porosity of them. As shown in Fig. 4b, the permanent
porosity of ZIF-90 was proved by N2 adsorption at 77 K, which showed a
type I isotherm. The isothermals displayed a steep rise under a relatively
low pressure due to the existence of micro pores. Whereas, a hysteresis
loop occurred in the isotherm of ZIF-90 at p/p0 ¼ 0.3, which might be due
to the slight shrinkage of micro pores caused by the existence of
aldehyde-based 2-ICA ligands [49]. It was worth noting that the

Fig. 2. (a) FT-IR spectra of ZIF-90, CS, CMZ and MTX; (b) PXRD patterns of
simulated ZIF-90, ZIF-90, MZ synthesized with different concentrations of MTX
and CMZ.

of the MZ products obtained by using different amounts of MTX was

measured (Fig. 2b). It can be seen that when the amount of the drug is 20,
30 and 40 mg, respectively, the PXRD pattern of the product shows the
formation of stable MZ framework. But when the amount of MTX in-
creases to 50 mg, the PXRD pattern of the product changed, and MZ was
not formed. The possible reason is that too many drug molecules hinder
the formation of the framework. Therefore, the maximum amount of
drug that can be added is 40 mg in this reaction system.
The surface properties of different samples were determined by
measuring the zeta potential in aqueous solution. As shown in Fig. 3, the
zeta potential of ZIF-90 was 4.68 mV, and the value of that slightly
decreased to 18.5 mV after the MTX loaded. Furthermore, it was
observed that the zeta potential changed significantly from 18.5 mV
(MZ) to þ3.5 mV (CMZ) after CS coated for MZ. This demonstrated that

Fig. 4. (a) TGA curves of ZIF-90, MZ and CMZ; (b) N2 adsorption-desorption

Fig. 3. Zeta potential of ZIF-90, MZ and CMZ. isotherms of ZIF-90, MZ and CMZ.

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X.-X. Cao et al. Journal of Solid State Chemistry 300 (2021) 122259

maximum adsorption capacity of the empty ZIF-90 framework was about

400 cm3/g, while the maximum adsorption capacity of MZ was less than
half of that of ZIF-90, which indicated that the pores were occupied by
the drug molecules. The Brunauer-Emmett-Teller (BET) surface area
decreased from 1226.0 m2/g for ZIF-90 to 419.5 m2/g for MZ, respec-
tively. These results further confirmed that the voids available in the
framework of ZIF-90 were occupied by MTX molecules. Additionally, the
loss of hysteresis loop and low nitrogen adsorption after the coating of
chitosan affirmed that the pores of CMZ were blocked by polymer coating
[50].

3.3. Drug loading and release study

According to the drug standard curve (Fig. S6) and drug loading
equation, the amount of loaded drug in the ZIF-90 could be calculated to
be 250 mg/g. The relatively high MTX load was due to the covalent load
of MTX and the framework. This value of the drug loading here is higher
than those of most MTX-loaded MOF carriers, but still lower than the
value of MOF carriers that have large pores or have strong interactions
between the framework and the drug (Table S1).
In order to prove the pH stability of the ZIF-90, the samples were
immersed in PBS solution with different pH for 48 h, and then their PXRD
were measured (Fig. S5). Obviously, the framework of ZIF-90 was more
stable in high pH conditions than lower pH conditions. The framework of
ZIF-90 was unstable at lower pH (5.5–6.5), which was similar to the
environment around tumor site. This meant that the pH had a significant
effect on the stability of ZIF-90. Therefore, as a candidate of drug carrier,
ZIF-90 showed the potential for cancer-targeted delivery of anticancer
drugs due to the collapse of the framework in the environment of the
tumor site.
Drug release studies were assessed under simulated normal physio-
logical and tumor tissue environments (pH 7.4 and pH 5.5). It could be
seen that the MTX-loaded MZ and CMZ exhibited sustained release
characteristics. The drug release curves of MZ only showed slight pH
Fig. 5. The release of MTX from MZ (a) and CMZ (b) in pH 5.5 (red) and 7.4
responsiveness (Fig. 5a). As shown in Fig. 5b, MTX was slowly released
PBS solution. (For interpretation of the references to color in this figure legend,
from the CMZ particles, and it was almost completely released in about the reader is referred to the Web version of this article.)
100 h under the acidic condition (pH 5.5). However, about 60% of total
attached MTX was released within 25 h in the PBS solution with pH 7.4.
Obviously, CMZ had better pH responsiveness in drug release through the exhibited good chemotherapeutic effect, and the inhibitory effect on the
modification of CS. These results revealed that the surface modification growth of cancer cells became more obvious with the increase of the
of CS could improve the pH responsiveness of CMZ during drug release, concentration. Compared with the group of free MTX drug, the drug-
which was related to the inherent properties of CS. At lower pH values, loaded group of CMZ also achieved a higher inhibitory effect on cancer
the CS transformed into a swollen polymeric matrix [51], which exposed cells and showed a similar level of inhibition to the group of free MTX
MZ to a low pH environment, caused the destroy of the MZ framework drug at each concentration. Based upon the comparison between normal
and the release of MTX. On another hand, the CS layer showed and cancer cells in vitro, the composite particle CMZ had obvious tar-
contraction mechanism at pH 7.4, which leads to the limited release of geting effect on cancer cells. Above results showed that the CMZ showed
MTX in a large extent. When CMZ entered into cancer cells, MTX mole- good biocompatibility and low cytotoxicity, so that it could be employed
cules were released due to the expansion of CS in low pH environment. as an efficient anticancer delivery system. The calcein-AM (green) and PI
The released MTX acted as a folic acid antagonist to prevent the normal (red) were used for double staining of living cells and dead cells to further
synthesis of cancer cell genetic material, thereby achieving the effects of detect the effect of apoptosis (Fig. 7). The fluorescence images showed
inhibiting cancer cell proliferation. that there were more PI stained cells and more cancer cell death in MTX
and CMZ groups, while there were less cell death and lower cytotoxicity
3.4. In vitro cytotoxicity and anticancer activity in ZIF-90 group, indicating that the anticancer delivery system of CMZ
can effectively induce apoptosis of cancer cells.
It is necessary to study the toxic properties of drug carriers. At first the
standard MTT assay against the EC304 human vascular endothelial cell 4. Conclusions
was selected to evaluate the cytotoxic activity of ZIF-90 and CMZ. As
shown in Fig. 6a, even at the concentration of 20 μg/mL, the cell viability In conclusion, we have successfully synthesized a new targeted anti-
of ZIF-90 and CMZ were higher than 80% and 75%, respectively, sug- cancer drug delivery system of CMZ by using a one-pot method. MTX was
gesting the negligible cytotoxicity of ZIF-90 and CMZ. To further evaluate selected as a model of anticancer drug and loaded into the particles with
the in vitro anticancer activity of CMZ, as can be seen from Fig. 6b, c and the drug loading efficiency as high as 250 mg/g. The synthetic method of
6d, different concentrations of human hepatoma HepG2 cells, prostate ZIF-90 has been optimized, using milder reaction conditions and shorter
cancer DU145 cells and gastric cancer SGC7901 cells administration reaction time. So the product can obtain a yield of up to 98%, which
groups (MTX, ZIF-90 and CMZ) were being set. Nearly half of viable providing a more efficient preparation method for MOF-based composite
cancer cells treated with MTX and CMZ at 20 μg/mL were killed after 48 materials. After the surface modification of drug-loaded particles with
h of incubation, both of free MTX and MTX-loaded CMZ particles CS, the pH responsiveness of the drug delivery system was successfully

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X.-X. Cao et al. Journal of Solid State Chemistry 300 (2021) 122259

Fig. 6. (a) Relative viabilities of CE304 cells incubated with ZIF-90 and CMZ at different concentrations; Relative viabilities of HepG2 cells (b), DU145 cells (c) and
SGC7901 cells (d) incubated with ZIF-90, MTX and CMZ at different concentrations. GraphPad Prism 6.0 was used to assess statistical significance: nsp > 0.05, *p <
0.05, **p < 0.01, ***p < 0.001.

Fig. 7. The fluorescence photos of HepG2 cells dual-stained by calcein-AM (green) and PI (red) with multiple therapies (Scale bar ¼ 100 μm). (For interpretation of
the references to color in this figure legend, the reader is referred to the Web version of this article.)

improved. In addition, the composite particle CMZ has little toxicity to
normal cells EC304, but it is more toxic to cancer cells, showing an
obvious targeted inhibitory effect on cancer cells. In summary, the
preparation of CMZ with desirable drug-loading capabilities, controlled
drug delivery properties and low toxicity provides an excellent candidate
for the anti-cancer drug delivery system.
CRediT authorship contribution statement
Xiang-Xin Cao: Methodology, Data curation, Writing – original draft.
Shui-Li Liu: Methodology, Resources. Jing-Sheng Lu: Methodology.
Zhen-Wei Zhang: Guidance and, Formal analysis, of High-performance
liquid chromatography. Gang Wang: Guidance and, Formal analysis,

6
X.-X. Cao et al.
of potential experiment. Qing Chen: Conceptualization, Resources,
Writing – review & editing, Visualization, Supervision, Project adminis-
tration, Funding acquisition. Ning Lin: Investigation, Project adminis-
tration, Funding acquisition.
Declaration of competing interest
The authors declare that they have no known competing financial
interests or personal relationships that could have appeared to influence
the work reported in this paper.
Acknowledgements
This research was supported by National Natural Science Foundation
of China (21861009), Guangxi Natural Science Foundation under Grant
No. 2018GXNSFAA281317, Thousands of Young and Middle-aged
Backbone Teachers Training Project of Guangxi Colleges and Univer-
sities (Gui-Jiao 2020-58) as well as University innovation foundation of
Guangxi University of Chinese Medicine (YCSY2020077).
Appendix A. Supplementary data
Supplementary data to this article can be found online at https://do
i.org/10.1016/j.jssc.2021.122259.
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