Zinc forms of faujasite zeolites as a drug delivery system for 6-mercaptopurine

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).

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 (http://creativecommons.org/licenses/by/4.0/)

Zinc forms of faujasite zeolites as a drug delivery system for

6-mercaptopurine

Marcel Jakubowskia, Malgorzata Kucinskab, Maria Ratajczakc, Monika Pokorad,

Marek Muriasb, Adam Voelkela, Mariusz Sandomierskia,*

aInstitute of Chemical Technology and Engineering, Poznan University of Technology, Berdychowo 4 Str., 60-965, Poznan, Poland

bDepartment of Toxicology, Poznan University of Medical Sciences, Dojazd 30 Str., 60-631, Poznan, Poland

cInstitute of Building Engineering, Poznan University of Technology, Piotrowo 5 Str., 60-965, Poznan, Poland

dCenter for Advanced Technologies, Adam Mickiewicz University, Poznan, Uniwersytetu Pozna´nskiego 10 Str., 61-614, Poznan, Poland

A R T I C L E I N F O

Keywords:

6-mercaptopurine

Drug delivery system

Zeolites

Ion exchange

Zn 2+

A B S T R A C T 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

6–mercaptopurine (MERC) is a purine analog, a drug with anti-

inflammatory, 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]

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

* Corresponding author

E-mail address: mariusz.sandomierski@put.poznan.pl (M Sandomierski)

Contents lists available at ScienceDirect Microporous and Mesoporous Materials

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https://doi.org/10.1016/j.micromeso.2022.112194

Received 3 March 2022; Received in revised form 11 August 2022; Accepted 18 August 2022

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,

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%), popo-tassium 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)-2,5- diphenyltetrazolium 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)

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

Fig 1 The scheme of the research presented in this work

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

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

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 96-

well 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

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 (M¨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-

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

Table 1

The content of elements in the tested materials based on the EDS [wt %]

Zn 11.41 ± 2.2 9.65 ± 1.47 3.53 ± 1.60 3.26 ± 1.17

Si 24.79 ± 0.74 29.26 ± 4.74 37.28 ± 4.83 38.65 ± 8.02

Al 13.93 ± 0.56 16.35 ± 2.99 9.66 ± 1.31 9.54 ± 1.65

Na 2.67 ± 0.47 2.62 ± 0.33 1.64 ± 0.43 1.52 ± 0.47

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

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

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)

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

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

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 different tautomeric forms, in one of them, the C––S group is trans-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 C––S group vibrations [34–36]

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

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

Fig 6 Sorption of mercaptopurine in zinc X and Y carriers after 1, 2, 3, and 7

days determined using UV–Vis spectroscopy

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 Y-

type 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

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 6- mercaptopurine, 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 UiO-

66 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]

Table 2

Characteristics of materials based on nitrogen adsorption/desorption

measurements

Zn-X Zn-X- MERC Zn–Y Zn–Y- MERC BET surface area [m 2⋅ g − 1 ] 568.46 339.81 659.32 398.73

t-Plot Micropore Area [m 2⋅g − 1 ] 501.65 291.37 602.80 358.48

Total pore volume [cm 3 ⋅g − 1 ] 0.312 0.198 0.338 0.209

t-Plot micropore volume

[cm 3 ⋅g − 1 ] 0.246 0.143 0.295 0.176

Table 3

Elemental analysis of carrier before and after drug sorption

Fig 7 Potential interactions between zinc ions and mercaptopurine in the

pores of the zeolite X and Y

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

influence of SBF

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

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

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–Y- MERC, 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)

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

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

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

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

CRediT authorship contribution statement

Marcel Jakubowski: Writing – original draft, Investigation

Mal-gorzata Kucinska: Writing – review & editing, Methodology,

Investi-gation Maria Ratajczak: InvestiInvesti-gation Monika Pokora: InvestiInvesti-gation

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

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

Data availability

All data generated or analyzed during this study are included in this

published article

Acknowledgements

This research was funded by the Ministry of Education and Science

(Poland)

Appendix A Supplementary data

Supplementary data to this article can be found online at https://doi

org/10.1016/j.micromeso.2022.112194

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