Microporous and Mesoporous Materials 343 (2022) 112194

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Microporous and Mesoporous Materials
journal homepage: www.elsevier.com/locate/micromeso

Zinc forms of faujasite zeolites as a drug delivery system for
6-mercaptopurine
Marcel Jakubowski a, Malgorzata Kucinska b, Maria Ratajczak c, Monika Pokora d,
Marek Murias b, Adam Voelkel a, Mariusz Sandomierski a, *
a

Institute of Chemical Technology and Engineering, Poznan University of Technology, Berdychowo 4 Str., 60-965, Poznan, Poland
Department of Toxicology, Poznan University of Medical Sciences, Dojazd 30 Str., 60-631, Poznan, Poland
Institute of Building Engineering, Poznan University of Technology, Piotrowo 5 Str., 60-965, Poznan, Poland
d
Center for Advanced Technologies, Adam Mickiewicz University, Poznan, Uniwersytetu Pozna´
nskiego 10 Str., 61-614, Poznan, Poland
b
c

A R T I C L E I N F O

A B S T R A C T

Keywords:
6-mercaptopurine
Drug delivery system
Zeolites
Ion exchange

Zn2+

6-Mercaptopurine (MERC) is a chemotherapeutic drug with varying activity depending on the dose. MERC has
been used to treat various diseases such as blood cancer, inflammatory bowel disease, or Crohn’s disease. Un­
fortunately, current methods of administering this drug are characterized by poor bioavailability (about 16%). In
this work, carriers for mercaptopurine based on X and Y-type zinc zeolites were developed for the first time. The
prepared carriers were well characterized by various research techniques (SEM/EDS, FTIR, and Elemental
analysis). The research confirms that the drug was trapped on the surface through coordination interactions
between zinc cations and the sulfur and nitrogen atoms of the mercaptopurine molecule. The drug release profile
particularly evidences this. “Burst release” of the drug from the carrier was not observed during the first hours of
release. Instead, 30% of the drug was released from both carriers in the first 10 h. The rest were released in about
20 h. Both carriers were also characterized by a large amount of drug released (78% and 88%). The cytotoxicity
study of the MCF-7 cell line at different concentrations for 72 h showed that MERC could be effectively released
from materials. Moreover, both free-form zeolites did not affect cell viability and thus might be considered
biocompatible carriers.

1. Introduction

One of the main reasons for this is the poor solubility of
6–mercaptopurine monohydrate (0.170 μg/ml), which is used in the
commercially available form of this drug [7,8]. The next problem is the
short half-life in plasma, ranging from about 1 to 3 h, unlike its active
metabolites, where this time varies from 3 to 13 days. This is because the
renal system rapidly eliminates mercaptopurine. This drug has unde­
sired side effects, such as bone marrow suppression and hepatotoxicity
[2,9]. Controlled release of drugs is one of the pioneering fields of sci­
ence that includes a multidisciplinary scientific approach contributing
to health protection. Designing suitable vehicles for drug delivery is a
challenge for biomedical scientists [10]. Considering the things
mentioned above, there should be a need to discover a promising drug

delivery system for mercaptopurine. Some exist, but most are based on
creating disulfide bonds between the carrier and the drug. The problem
with this drug delivery system (DDS) is that the drug can be released
only if there is enough glutathione (GSH) concentration in the cell. For
example, Gong et al. designed a system based on UiO – 66 – (SH)2

6–mercaptopurine (MERC) is a purine analog, a drug with antiinflammatory, immunosuppressive, and cytotoxic properties. The ac­
tivity of this compound is dependent on the dose. It will work as an
anti–inflammatory drug in small doses, but in higher doses, it will have
immunosuppressive and cytotoxic properties [1,2]. One of the most
serious diseases in which this drug finds application is Acute Lympho­
blastic Leukemia (AAL), which is used especially as a very important
agent in maintenance therapy [3,4]. The use of 6-mercaptopurine is not
limited to the treatment of leukemia. It has several applications in many
serious diseases, such as other hematological malignancies, inflamma­
tory bowel disease, Crohn’s disease, systemic lupus erythematosus, and
rheumatoid arthritis. Moreover, MERC is an important immunomodu­
lating agent to prevent transplant rejection [5,6]. However, one of the
main problems during the 6-mercaptopurine therapy is a low bioavail­
ability, ranging from 10% to 50%, with an average value of 16% [1].
* Corresponding author.
E-mail address: (M. Sandomierski).

/>Received 3 March 2022; Received in revised form 11 August 2022; Accepted 18 August 2022
Available online 23 August 2022
1387-1811/© 2022 The Authors. Published by Elsevier Inc. This is an open access article under the CC BY license ( />

M. Jakubowski et al.

Microporous and Mesoporous Materials 343 (2022) 112194


zinc ions can be exchanged by Na+ and K+ ions present in human
plasma. After that, 6–mercaptopurine will lose its interaction with the
carrier, and the drug will be intelligently released. The research scheme
is shown in Fig. 1. Prepared ion-exchanged zeolites were characterized
with various methods to confirm the successful ion exchange and drug
adsorption on its surface. The sorption capacity and release of the drug
were examined for all prepared materials. To our best knowledge, it is
the first time using zinc exchanged zeolites as a mercaptopurine drug
delivery system.
2. Experimental
2.1. Materials
Sodium zeolite X and Y, zinc nitrate hexahydrate, 6-mercaptopurine
(MERC), tris (hydroxymethyl) aminomethane (TRIS), (99.8%), sodium
chloride (99%), sodium bicarbonate (99%), sodium sulfate (99%), po­
tassium phosphate dibasic trihydrate (99%), potassium chloride (99%)
were purchased from Sigma-Aldrich (St. Louis, MO, USA). Hydrochloric
acid (36–38%) was purchased from Avantor Performance Chemicals
(Gliwice, Poland). The materials were used without further purification.
Reagents used for in vitro experiments, such as Dulbecco’s Modified
Eagle Medium (DMEM), fetal bovine serum (FBS), phosphate-buffered
saline (PBS), trypsin-EDTA, L-glutamine, penicillin, and streptomycin
solution, dimethyl sulfoxide (DMSO), 3-(4, 5-dimethylthiazol-2-yl)-2,5diphenyltetrazolium bromide (MTT) were obtained from Sigma Aldrich
(St. Louis, MO, USA). CellTiter-Glo® One solution was obtained from
Promega (Madison, WI, USA). The DMSO for dissolving formazan
crystals was obtained from Avantor Performance Materials (Gliwice,
Poland).

Fig. 1. The scheme of the research presented in this work.


metal-organic framework. In their study, the drug was only released
when the glutathione was presented in the solution [11]. The systems
with a different type of release were also created; for instance, Kaur et al.
synthetized a system based on Zeolitic imidazolate framework (ZIF)
nanoparticles with 6–mercaptopurine encapsulated inside the particle.
However, release in this system is only controlled by the dissolution of
ZIF particles [12]. Considering that, there should be a carrier that would
release the drug under the influence of body fluid. That carrier should be
biocompatible and release medicine gradually to avoid the toxic effects
on healthy cells. Zeolites could be ideal for these applications; to our best
knowledge, they have never been used to deliver mercaptopurine. Ze­
olites are biocompatible aluminosilicate materials containing micropo­
rous structure [13]. They have many industrial applications, such as
molecular sieves for water remediation and catalysis [14–16]. They also
have biomedical applications such as the separation of biomolecules,
drugs, and genes delivery or construction of biosensors. The use of ze­
olites in biomedical applications is possible due to their stability in
human body fluids [17]. The stability in the environment of body fluids
has been proven for type X and Y zeolites, which confirms their potential
as a carrier in drug delivery [18,19]. Zeolites are composed of MO4
tetrahedrons, where M stands for Al or Si. During the crystallization of
this material, the building blocks are linked together through an oxygen
bridging atom, which creates a negative charge. Exchangeable cations
balance this negative charge. In natural zeolites, it could be, for
example, Na+, K+, Ca2+, or Mg2+. But that cation can be exchanged, for
example, on Zn2+, Cu2+, and many other metal cations [20–22]. Type A
and FAU zeolites with high ion exchange capacity are the
best-characterized zeolites. Type A zeolites have pores around 4 Å. In
this work, we want to focus on FAU zeolites: X and Y. The pore size of
these zeolites is 6–8 Å. Due to the pore size, FAU zeolites have a greater

sorption potential for MERC than type A zeolites. Both FAU zeolites are
composed of a sodalite cage but have different Si/Al ratios. In the X type
zeolite Si/Al = 1–1.5 and for the Y type Si/Al = 2.4–2.7 [23,24]. Dif­
ferences in silica to alumina ratio influence the cation exchange [25].
Those zeolites were previously used as drug delivery systems for many
medicaments, for example, ketoprofen and cyclophosphamide [26,27].
In our previous work, we proved that it is possible to use Ca2+ exchanged
zeolites A and X, as a drug delivery system for anti–osteoporotic drugs
(Bisphosphonates), with prolonged-release [22]. We want to use the fact
that 6–mercaptopurine has few binding sites from sulfur and nitrogen
atoms that can form complexes with transition metals [28,29]. In this
work, we prepare Zn2+ exchanged FAU zeolites that can adsorb drug on
its surface, unlike unexchanged forms. Under the influence of body fluid,

2.2. Ion exchange
Ion exchange was carried out by mixing a 50 ml of 0.5 M solution of
zinc nitrate with 2 g of X or Y zeolite. Zeolites were mixed with the
solution for 24 h and then centrifuged. This process was repeated three
times. Subsequently, the material was washed with distilled water three
times and dried in an oven for 24 h at 100 ◦ C.
The materials after ion exchange were named Zn-X and Zn–Y.
2.3. Characterization methods
2.3.1. Scanning electron microscopy (SEM) and energy dispersive
spectroscopy (EDS)
SEM images were recorded using a scanning electron microscope
VEGA 3 (TESCAN, Czech Republic). The SEM toll was equipped with an
EDS analyzer (Bruker, UK). EDS was used to conduct the elemental
analysis of the samples. The final concentration of each element was
obtained by taking the average of measurements at ten different spots.
2.3.2. Nitrogen Adsorption/Desorption measurements

The nitrogen adsorption isotherm technique determined the BET
surface area and pore parameters of obtained zeolites using an ASAP
2420 analyzer (Micromeritrics). Before experiments, the samples were
outgassed at 200 ◦ C in a vacuum chamber.
2.3.3. Fourier-transform infrared spectroscopy
FT-IR analysis of all materials was performed using a Vertex70
spectrometer (Bruker Optics, Germany). The IR spectra were recorded in
a KBr pellet. The tests were carried out in the spectral range of
4000–600 cm− 1 with a resolution of 4 cm− 1 and 32 scans for signal
accumulation.
2.3.4. Elemental analysis
Measurements were performed on the FLASH 2000 elemental
analyzer. The samples were weighed in tin capsules (approximately 2
2


M. Jakubowski et al.

Microporous and Mesoporous Materials 343 (2022) 112194

mg) and introduced into the reactor using an autosampler together with
an appropriate, precisely defined portion of oxygen. After combustion at
a temperature of 900–1000 ◦ C, the flue gases were transported in helium
flow to the second furnace of the reactor filled with copper, and then
through a water trap to the chromatographic column, which separates
the individual products from each other. The separated gases were
detected by a thermal conductivity detector.
2.3.5. UV–vis spectroscopy
UV–Vis spectrophotometer UV-2600 (Shimadzu, Japan) was applied
to determine mercaptopurine concentration during sorption and release

process. Measurements were made in the range of 300–400 nm (λ max =
320 nm).
The amount of MERC retained on the zeolite was calculated from the
amount of drug remaining in the starting solution using the following
formula:
The amount of drug in the starting solution (0.015 mg/ml) - The
amount of the drug in the solution after sorption ¼ The amount of drug
retained by the carrier.
The amount of drug retained was tested using a calibration curve
prepared in a Tris-HCl solution.
The amount of released drug was tested using a calibration curve
prepared in SBF.
2.4. Drug sorption and release

Fig. 2. SEM images for zinc zeolite X and Y before and after sorption of MERC.

Drug sorption was initiated by introducing 20 mg of zinc zeolite
samples into polypropylene tubes. Each tube was filled with 30 ml of
MERC solution with a concentration of 0.015 mg/ml (the drug was
dissolved in 0.1 M TRIS-HCl buffer at pH = 7.4). The sorption process
was based on the following steps: samples were shaken on a laboratory
rotator mixer (speed 50 rpm) for 24 h at room temperature, then the
samples were centrifuged (10 min with 4000 rpm). 1 ml of the solution
was taken from the sample and analyzed by UV–Vis spectroscopy. After
analysis, the solution was placed back into the tube. The polypropylene
tube was then shaken to distribute the carrier in the solution and placed
on the rotator for 24 h. All steps were repeated on consecutive days. The
entire sorption lasted one week. Six repetitions were made.
The materials were named by combining the names of the carrier
(Zn-X or Zn–Y) and drug -MERC).

The same procedure was used for sodium zeolites, but the drug was
not retained, and its results are not presented.
Drug release was initiated by introducing carriers after drug sorption
in a vial with 1 ml of simulated body fluid (SBF) with a pH of 7.4. The
amount of drug released was measured after each hour for up to 30 h
using UV-VIS spectroscopy. Each time, the samples were centrifuged
(10 min with 4000 rpm). The solution was taken from the sample and
analyzed by UV–Vis spectroscopy. The drug carrier was flooded with a
new portion of SBF (1 ml) to provide a new portion of ions. The release
was performed at human body temperature.

materials. In this case, the cell culture medium was used as a control.
Cells were incubated with MERC and zeolites for 72 h under cell culture
conditions. After incubation, the CellTiter-Glo® assay was performed
according to the manufacturer’s protocol. The luminescence was
measured using an opaque white clear-bottom plate with a Tecan
Infinite M Plex microplate reader (Mă
annedorf, Switzerland).
The MTT assay used in our preliminary experiments was performed
according to our well-established protocol [30]. MCF-7 cells were
washed twice with PBS, and MTT (0.59 mg/mL) was added to each well.
After incubation lasting 1.5 h, the formazan crystals were dissolved in
200 μL of DMSO, and the absorbance was measured at 570 nm with a
plate reader (Biotek Instruments, Elx-800, Highland Park, Winooski,
Vermont, USA).
Each experiment was performed with six replicates, four times (for
MERC) and three times for zeolite-based materials.
The statistical analysis was performed using GraphPad Prism®8
(GraphPad Software, Inc., La Jolla, CA, USA). One-way ANOVA with
post-hoc Tukey’s test was used to determine the significance; p < 0.05

was considered significant.
3. Results and discussion
Scanning electron microscopy images show no significant differences
in the zinc zeolites X and Y morphology before and after drug sorption
(Fig. 2). This shows that the drug does not precipitate on the surface of
the zeolites. Additionally, we can see that the molecules do not
agglomerate or aggregate, which is important as this would prevent
their use as drug carriers. The lack of crystallized substance on the
surface of the carriers also proves that the drug was retained in the form
of a monolayer by means of coordination bonds between Zn2+ and free-

2.5. Biological activity
The human breast carcinoma MCF-7 cell line was purchased from the
European Collection of Authenticated Cell cultures (ECACC, Salisbury,
UK). MCF-7 cells were maintained in DMEM supplemented with 10% (v/
v) FBS, 1% (v/v) L-glutamine (200 mM), 1% (v/v) 10 000 penicillin units,
10 mg/mL streptomycin solution. Cells were cultured at 37 ◦ C, with 5%
CO2 and 95% humidity.
MCF-7 cells were seeded at a density of 10 × 103 cells/well in a 96well plate. In the case of free drug, MERC was tested at a concentration
range of 0.3 μM, 0.6 μM, 1.2 μM, 2.5 μM, 5 μM, and 10 μM. DMSO was
used as a control, and the concentration did not exceed 0.1%. Materials
were tested at a concentration of 5 μM, 1.2 μM, and 0.3 μM in a cell
culture medium. To determine the potential cytotoxic activity of zeo­
lites, MCF-7 cells were treated with free and encapsulated drug

Table 1
The content of elements in the tested materials based on the EDS [wt %].
N
S
Zn

Si
Al
Na

3

Zn-X

Zn-X-MERC

Zn–Y

Zn–Y-MERC

0
0
11.41 ± 2.2
24.79 ± 0.74
13.93 ± 0.56
2.67 ± 0.47

1.93 ± 0.62
0.19 ± 0.19
9.65 ± 1.47
29.26 ± 4.74
16.35 ± 2.99
2.62 ± 0.33

0
0

3.53 ± 1.60
37.28 ± 4.83
9.66 ± 1.31
1.64 ± 0.43

2.31 ± 0.57
0.21 ± 0.2
3.26 ± 1.17
38.65 ± 8.02
9.54 ± 1.65
1.52 ± 0.47


M. Jakubowski et al.

Microporous and Mesoporous Materials 343 (2022) 112194

Fig. 3. SEM images of carriers (first row). Elemental mapping of the same regions indicating the spatial distribution of zinc (second row), nitrogen (third row) and
sulfur (fourth row).

electron pairs from nitrogen and sulfur atoms in mercaptopurine [28,
29]. However, differences in size can be observed when comparing the X
and Y zeolites. Those for zeolite Y are significantly smaller. The smaller
particles can affect the sorption of the drug because the surface is more
easily accessible to the drug.
Using energy-dispersive X-ray spectroscopy (EDS), it was possible to
confirm the effectiveness of the ion exchange process, the sorption of the
drug on the material surface, and whether the ion exchange took place
on the entire surface of the material or only at points.
The exact EDS results are shown in Table 1.

We can conclude that the ion exchange process was successful basing
on the obtained results. This is evidenced by the higher content of zinc
ions in relation to the amount of sodium ions in the tested material. The
results also show that a much larger amount of Zn2+ ions is in the X
zeolite than in the Y. Type X zeolite has lower silicon to the aluminum
ratio in its structure, which is consistent with literature reports on this
subject. As a result, zeolite X has many more active sites capable of ion
exchange. For this reason, the zinc ion content is more significant in the
zeolite X than in the Y zeolite [23,24]. As mentioned previously, this
technique was also used to confirm the sorption of the drug on the
surface. The effectiveness of sorption is evidenced by the appearance of
new elements - nitrogen and sulfur, indicating that the drug was retained
on the surface and in the pores of the material. In addition, higher
percentages of these elements on the Y zeolite indirectly suggest that it is
the material with more of the drug retained. The EDS analysis made it
possible to perform a surface mapping that allows seeing the distribution
of the content of a given element on the surface (Fig. 3). The mapping
made in terms of the content of zinc, nitrogen, and sulfur ions shows that

Fig. 4. Pore size distribution in the range of 20–200 Å.

the drug was sorbed over the entire surface of the material, and not just
pointwise in some places. Even distribution and non-agglomeration of
the drug are very important because, in such a case, a given amount of
carrier will consistently deliver the same drug dose. This will counteract
the occurrence of local toxic reactions.
The Nitrogen Adsorption/Desorption results also demonstrate the
drug sorption efficacy. Zeolite Y has a larger specific surface area than
4



M. Jakubowski et al.

Microporous and Mesoporous Materials 343 (2022) 112194

Fig. 5. Structure of mercaptopurine and IR spectra for drug and carriers before and after sorption.

zeolite X. The specific surface area drops significantly after drug sorp­
tion (about 40%) for both zeolites. The decrease is due to the surface
being covered with the MERC layer. However, considering the exact
value of the decrease in the specific surface area, the decrease was
greater for zeolite Y (260.59 m2 g− 1) than for X (228.65 m2 g− 1). This
may indirectly mean that more drug has been adsorbed on zeolite Y. As
can be seen, the number of micropores is several times greater for all
types of materials than the number of other pores. After drug sorption,
the following decrease in micropore area was observed for zeolite X
(210.28 m2 g− 1) and Y (244.32 m2 g− 1). These values are close to the
total surface change indicating that the drug is retained mainly in the
micropores, which is not surprising since the X and Y zeolites mostly
have this type of pores. Apart from micropores, mesopores also occur in
materials, but their volume is small. Fig. 4 shows the distribution of
mesopores. The results are similar to those obtained by other research
teams for zeolite X and Y [31,32]. Both materials contain mesopores
with a diameter of about 40 Å. As shown in Fig. 5, a slight decrease in the
mesopore content is visible for zeolite X, while is practically not for Y.
Large changes are noticeable in the volume of micropores. For zeolite X,
the volume of micropores decreased by 0.103 cm3⋅g− 1, while for zeolite
Y, it decreased by 0.119 cm3⋅g− 1. This information may also indirectly
mean that more drug has been adsorbed on zeolite Y.
Another analysis that confirms that the drug has been retained on the

surface is FTIR spectroscopy, which allows the identification of func­
tional groups. As can be seen in Fig. 5, the spectrum obtained before
drug sorption comprises different bands. In both zeolites, there is a wide
band with a peak at the wavenumber of about 3600 cm− 1 and a band at
the wavenumber of 1637 cm− 1 and 1635 cm− 1 for zeolites X and Y,
respectively. The bands can be attributed to the stretching and bending
vibrations of the hydroxyl group and the water adsorbed on the zeolite
surface. The bands in the range 1250 cm− 1 – 600 cm− 1 belong to the
zeolite aluminosilicate network [33]. As seen in the presented spectra,
new bands appear after drug sorption, confirming the drug’s effective
sorption on the zeolite surface. In both cases, the band assigned to O–H
stretching vibrations is shifted to a wavenumber of about 3450 cm− 1.
New bands also appeared in the spectrum. The bands at wavenumber
3230 cm− 1 and 3229 cm− 1 can be assigned to N–H stretching vibrations
for the X and Y zeolite, respectively. Both spectra after drug sorption also

Fig. 6. Sorption of mercaptopurine in zinc X and Y carriers after 1, 2, 3, and 7
days determined using UV–Vis spectroscopy.

show bands that can be attributed to the stretching vibrations of the C–H
at the wavenumber around 3000 cm− 1. Mercaptopurine can exist in
– S group is trans­
different tautomeric forms, in one of them, the C–
formed into a C–S–H group. The vibrations of the S–H group can be seen
at the wavenumber around 2600 cm− 1. Significant changes can also be
seen in the range of 1750–1250 cm− 1. Three bands at the wavenumber,
approximately 1525 cm− 1 can be assigned to N–H bending vibrations.
The last new band visible in both samples at the wavenumber 1297 cm− 1
and 1295 cm− 1 for zeolite X and Y, respectively, can be attributed to
– S group vibrations [34–36].

C–
Another study that was carried out to confirm the effective sorption
of the drug on the surface of the material is the elemental C, H, and N
analysis, which allows determining the percentage of these elements in
the tested samples. The results of this study are summarized in Table 3.
5


M. Jakubowski et al.

Table 2
Characteristics of
measurements.

Microporous and Mesoporous Materials 343 (2022) 112194

materials

BET surface area [m2⋅g− 1]
t-Plot Micropore Area [m2⋅g− 1]
Total pore volume [cm3⋅g− 1]
t-Plot micropore volume
[cm3⋅g− 1]

based

on

nitrogen


adsorption/desorption

Zn-X

Zn-XMERC

Zn–Y

Zn–YMERC

568.46
501.65
0.312
0.246

339.81
291.37
0.198
0.143

659.32
602.80
0.338
0.295

398.73
358.48
0.209
0.176


Table 3
Elemental analysis of carrier before and after drug sorption.
Zn-X
Zn-X-MERC
Zn–Y
Zn–Y-MERC

N

C

0
1.19 ± 0.17
0
1.43 ± 0.12

0.03
5.72
0.03
6.43

± 0.01
± 0.06
± 0.01
± 0.12

Fig. 8. Total release of mercaptopurine from the zinc X and Y zeolite under the
influence of SBF.

cannot penetrate deep inside the material. Closure of the entrances to

the pores is also indicated by the fact that in zeolite Y, the volume of the
pores decreased more than in zeolite X. The reduction in pore size
greatly affects sorption since the drug size is 6.5 Å (Fig. 5), and the FAU
pore size is 6 - 8 Å. The situation is the opposite for zeolite Y, which has a
lower ion exchange capacity, so its pores remain larger, and drug mol­
ecules can penetrate deeper into its structure [28,29,37]. The amount of
ions is smaller, meaning less drug is retained at the entrance to the pores.
A schematic drawing of the pores clogging is shown in Fig. 7.
Drug release was checked using UV-VIS spectroscopy (Fig. 8). As can
be seen, during the first 10 h, the drug was released gradually from both
materials prepared in almost identical amounts (up to about 30%). The
release of mercaptopurine then accelerated for zeolite Y. For both car­
riers, there is no “burst release” that often occurs. The drug is released
slowly in small doses. This is likely because the drug is not physically
adsorbed in the pores but, as previously mentioned, is adsorbed via
coordination interactions between the drug and zinc ions [28,29]. In
both cases, it was also possible to obtain large amounts of drug release
from the carrier. 78% of the drug was released from zeolite X after 31 h,
and 88% was released from zeolite Y after 30 h.
Comparing the materials prepared by us to other carriers for 6mercaptopurine, we find that they focus mainly on the release of the
drug under the influence of various factors, e.g., pH or GSH. For
example, Gong et al. prepared a system based on the metal-organic UiO66 network. The drug was released only when the fluid used for the
release contained GSH [11]. On the other hand, the system prepared by
our team enables the release of the drug in all conditions, and thus its use
in treating other diseases, not only cancer. Furthermore, our system
allows the release of zinc ions, which may help in the fight against
leukemia because people suffering from blood cancer have a reduced
concentration of zinc, which affects the outcome of the fight against the
disease [38].
6-Mercaptopurine is a well-known prodrug belonging to the thio­

purine family that works via conversion to the cytotoxic 6-thioguanine
nucleotides (6-TGN) [39]. Mercaptopurine has been used in treating
acute lymphoblastic leukemia for over 50 years [39]. However, MERC is
also a potential candidate for treating different cancers, such as breast or
ovarian tumors, mainly as a combinatorial treatment [40,41]. As
described by Singh et al. MERC might be a promising approach for
treating triple-negative breast tumors [42]. The cytotoxic and immu­
nosuppressive effects of MERC are achieved through the different mo­
lecular modes of action [43]. Several mechanisms have been proposed,
such as inhibition of de novo purine synthesis, decreased DNA methyl­
ation, and incorporation of thioguanosine nucleotides into the DNA
resulting in induction of the mismatch repair system and apoptosis [43].

Fig. 7. Potential interactions between zinc ions and mercaptopurine in the
pores of the zeolite X and Y.

We can see that the carbon and nitrogen content increases significantly
after the drug sorption process. The appearance of nitrogen, absent in
the test sample before the drug loading process, is particularly impor­
tant. The higher content of each element on zeolite Y suggests that more
drug is adsorbed on this material.
As mentioned, the drug sorption study was carried out using UV–Vis
spectroscopy (Fig. 6). During the first 2 days, no significant differences
in the amount of drug retained were noticed. Noticeable differences are
after day 3 and week. Drug loading was found to be more effective on Ytype zeolite. During sorption, the carriers retained 0.27 mg of the drug
for zeolite X and 0.29 mg of the drug for zeolite Y. Based on these results,
the sorption/encapsulation efficiency is approximately 0.014 mg of drug
per mg of Zn-X carrier, and 0.015 mg of drug per mg of Zn–Y carrier.
The quantitative and surface analysis studies have shown that more
significant amounts of the drug are retained in Y zeolite. The obtained

results may be surprising due to the over 3 times higher content of zinc
ions in the X-type zeolite after ion exchange, thanks to which the drug
was adsorbed on the surface. One explanation may be that zeolite Y has
smaller particles, which increases sorption. It may also be because the
pores have become significantly smaller after ion exchange. In partic­
ular, the results from EDS show how much more zinc is present in zeolite
X compared to Y. Reports from the literature show that the ion exchange
of zeolites with zinc ions causes the pore volume to decrease. It can be
guessed that the more zinc is exchanged for sodium, the greater the
reduction of pores will be. As previously described, the lower surface
area and pore volume of zeolite X was confirmed by Nitrogen Adsorp­
tion/Desorption analysis (Table 2). This situation causes the entrances
of the pores in zeolite X to be quickly clogged, and the drug molecules
6


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Microporous and Mesoporous Materials 343 (2022) 112194

using the CellTiter-Glo® One Solution Assay. The principle of this test is
based on the measurement of adenosine triphosphate (ATP), a widely
used cell viability marker [49]. The ATP-luminescent assay is more
sensitive than other viability assay methods, such as MTT [49].
Noticeably, it was found that MERC treatment decreased the intracel­
lular ATP concentration, resulting in the activation of AMPK [50]. As a
result, several AMPK downstream targets are inhibited, affecting glucose
and glutamine metabolism [50]. Our preliminary experiments showed
the difference in MERC treatment between the luminescent ATP and
MTT assays (Fig. S1). MTT is a colorimetric assay based on the con­

version of MTT substrate into formazan crystals by mitochondrial de­
hydrogenase enzyme present only in the viable cells [51]. Thus, cell
viability (more appropriate metabolic activity) correlated with mito­
chondrial function. Based on these experiments, ATP measurement
could better reflect the in vitro activity of MERC. The most significant
difference was observed for a lower concentration of MERC, at a con­
centration of 3 μM cell viability was 34%, and 64% for ATP and MTT
assay, respectively. Based on the preliminary study, the ATP-based assay
was selected to evaluate the tested materials’ anticancer activity.
First, we defined the IC50 value for the MERC after 72 h treatment.
The pure 6-mercaptopurine (dissolved in DMSO) decreased the cell
viability in a dose-dependent manner, with an IC50 value of 1.92 μM
(Fig. 9). Based on these experiments, the doses for further studies were
selected.
It should be emphasized that the empty materials without the
attached drug did not significantly reduce cell survival (Fig. 10).
Therefore, these results indicated that both zeolites might be considered
non-toxic materials. Our experiments showed that tested materials
released the MERC and affected cancer cell viability, as it is presented in
Fig. 10. The Zn–Y-MERC and Zn-X-MERC at a 5 μM significantly reduced
cell viability to approximately 50% and 60% compared to control cells,
respectively (Figs. 10–12). These results are consistent with release ex­
periments. The zeolite Y released the drug (88%) more efficiently than
zeolite X (78%) after incubation lasting 30 h.
Zeolites exerted lower activity than pure MERC, which decreased cell
viability to approx. 30% at a dose of 5 μM (Fig. 10). In general, it is
commonly observed that modified drugs designed for controlled release
exhibited less cytotoxicity than parent drugs [52,53]. The difference in
activity might be related to the delayed release of the drug and the
different solubility of the incorporated agent compared to the free form.

However, the lower activity of the compound against cancer cells might
decrease the cytotoxic effect on normal cells and thus reduce the risk of
side effects. Kong et al. tested the dual turn-on fluorescence signal-based
controlled release system for Doxorubicin (CDox) [52]. In this study, the
IC50 value of CDox was 4.3 μM, 2.34 μM, and 4.51 μM for human cervical
adenocarcinoma (HeLa), human hepatocellular carcinoma (HepG2),
and murine mammary carcinoma (4T-1) cell lines, respectively. On the
other hand, free Dox was more cytotoxic with IC50 values of 1.11 μM,
0.84 μM, and 1.28 μM for HeLa, HepG2, and 4T-1, respectively [52]. In
the study presented by Yang, three homodimeric doxorubicin prodrugs
were synthesized using a thioether bond (DSD NP), disulfide bond
(DSSD NPs), or trisulfide bond (DSSSD NPs) as linkers to provide
nanoassemblies for efficient and more selective Doxorubicin delivery to

Fig. 9. Cytotoxicity of free MERC against MCF-7 cells. Cells were treated
with MERC at a concentration of 10 μM, 5 μM, 2.5 μM, 1.2 μM, 0.6 μM, and 0.3
μM for 72 h. The effect of MERC was measured using a luminescence-based
assay. Data are expressed as the mean ± SD from four independent experi­
ments. The representative images were taken with a DS-SMc digital camera
attached to a Nikon Eclipse TS100 microscope. The scale bar corresponds to
100 μm.

The 6-TGN is incorporated into the DNA due to its structural similarity
to endogenous purine-based guanine. In general, thiopurines exerted the
delayed cytotoxic effect due to the requirement for passing at least one S
phase of the cell cycle to allow the incorporation of 6-TG into DNA [39].
Besides several advantages, there are some limitations to using MERC,
and one important problem lies in pharmacokinetics. MERC has poor
bioavailability due to its weak aqueous solubility, rapid metabolism, and
short half-life of 1.9 ± 0.6 h [44]. Thus, huge efforts have been made to

overcome the drawbacks mentioned above, e.g., numerous systems were
designed to improve the pharmacokinetic profile, reduce side effects,
and potentiate the activity [35,45–48].
In the presented work, we tested novel zeolite-MERC materials to
evaluate their potential as drug carriers for controlled release. The
cytotoxic effect of tested materials and the free drug was determined

Fig. 10. The activity of Zn–Y-MERC, Zn–Y-MERC,
and their free forms against MCF-7 cells. Panel A
presents the cytotoxic activity of Zn–Y and Zn–YMERC, while panel B presents the results for Zn-X and
Zn-X-MERC. Cells were treated for 72 h with tested
materials to achieve the MERC concentration of 5 μM,
1.2 μM, and 0.3 μM. The cell viability was measured
using a luminescence-based assay. Data are expressed
as the mean ± SD from three independent experi­
ments. Statistical significance between groups was
assessed by Tukey Multiple Comparison Test (**p <
0.01, ***p < 0.001; ****p < 0.0001).
7


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Microporous and Mesoporous Materials 343 (2022) 112194

Fig. 11. Morphological assessment of MCF-7 cells following Zn–Y and Zn–Y-MERC treatment. The right bottom panel presents the cell viability (presented as a
% of control) after exposition to the tested material. The images were taken with a DS-SMc digital camera attached to a Nikon Eclipse TS100 microscope. The scale
bar corresponds to 100 μm.

cancer cells. The authors found that two tested nanostructure DSSD NPs

and DSSSD NPs had lower cytotoxicity on cancer cells than Doxorubicin,
and these results might be a consequence of the delayed release of the
active drug from prodrug nanoassemblies [53]. In summary, the results
of our research provide new and relevant information on the use of
zeolite as a drug carrier. Moreover, these data confirmed that zeolite
drug conjugates are the potential system for controlled drug release.

4. Conclusions
This work demonstrates using two Zn2+ exchanged zeolite materials
as a carrier for the mainly leukemia drug - mercaptopurine. Using the
performed analyses, i.e., SEM/EDS, FTIR, and elemental analysis, it was
possible to confirm the efficiency of ion exchange and the retention of
the drug on the surface of the material. Studies have confirmed that the
drug does not precipitate on the surface but is retained through
8


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Microporous and Mesoporous Materials 343 (2022) 112194

Fig. 12. Representative images of MCF-7 after treatment with Zn-X and Zn-X-MERC. The right bottom panel presents the cell viability (presented as a % of
control) after exposition to the tested material. The images were taken with a DS-SMc digital camera attached to a Nikon Eclipse TS100 microscope. The scale bar
corresponds to 100 μm.

coordination interactions with zinc cations. Based on the SEM images, it
was possible to establish that both ion exchange and drug sorption did
not cause aggregation of carrier particles. Based on EDS mapping, it was
also possible to confirm that ion exchange and drug sorption occur
evenly over the entire material surface. This is evidenced by the even

distribution of zinc, nitrogen, and sulfur on the surface. Y-type zeolite
has been shown to retain more drug than zeolite X, possibly due to
clogging of the pores in zeolite X. The drug was released from both

materials for approximately 30 h 78% of the drug was released from the
X-type zeolite and 88% from the Y-type zeolite. The release profile
shows that the drug is released gradually from both carriers in a
controlled manner. The cytotoxicity studies show that both materials
effectively release drug and affect the viability of cancer cells. The
presented approach could unlock new ways to design potential strate­
gies for controlled drug release; however, further studies are needed to
better describe zeolites as drug carriers.
9


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Microporous and Mesoporous Materials 343 (2022) 112194

CRediT authorship contribution statement
[13]

Marcel Jakubowski: Writing – original draft, Investigation. Mal­
gorzata Kucinska: Writing – review & editing, Methodology, Investi­
gation. Maria Ratajczak: Investigation. Monika Pokora: Investigation.
Marek Murias: Supervision. Adam Voelkel: Writing – review & edit­
ing, Writing – original draft, Supervision, Resources. Mariusz Sando­
mierski: Writing – original draft, Visualization, Methodology,
Investigation, Formal analysis, Data curation, Conceptualization.


[14]
[15]
[16]
[17]

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.

[18]
[19]

Data availability

[20]

All data generated or analyzed during this study are included in this
published article.

[21]

Acknowledgements

[22]

This research was funded by the Ministry of Education and Science
(Poland).

[23]


Appendix A. Supplementary data

[24]

Supplementary data to this article can be found online at https://doi.
org/10.1016/j.micromeso.2022.112194.

[25]
[26]

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