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).

Microporous and Mesoporous Materials 343 (2022) 112194 Contents lists available at ScienceDirect 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 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 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 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 to h, unlike its active metabolites, where this time varies from 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: mariusz.sandomierski@put.poznan.pl (M Sandomierski) https://doi.org/10.1016/j.micromeso.2022.112194 Received 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 (http://creativecommons.org/licenses/by/4.0/) 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 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 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 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 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 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 (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− with a resolution of cm− 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 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 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 with 4000 rpm) 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 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 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 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 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, 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 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 The content of elements in the tested materials based on the EDS [wt %] N S Zn Si Al Na Zn-X Zn-X-MERC Zn–Y Zn–Y-MERC 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 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 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 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 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 M Jakubowski et al Microporous and Mesoporous Materials 343 (2022) 112194 Fig 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 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− 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− and a band at the wavenumber of 1637 cm− and 1635 cm− 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− – 600 cm− 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− New bands also appeared in the spectrum The bands at wavenumber 3230 cm− and 3229 cm− can be assigned to N–H stretching vibrations for the X and Y zeolite, respectively Both spectra after drug sorption also Fig Sorption of mercaptopurine in zinc X and Y carriers after 1, 2, 3, and 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− 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− Significant changes can also be seen in the range of 1750–1250 cm− Three bands at the wavenumber, approximately 1525 cm− can be assigned to N–H bending vibrations The last new band visible in both samples at the wavenumber 1297 cm− and 1295 cm− 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 M Jakubowski et al Table 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 Elemental analysis of carrier before and after drug sorption Zn-X Zn-X-MERC Zn–Y Zn–Y-MERC N C 1.19 ± 0.17 1.43 ± 0.12 0.03 5.72 0.03 6.43 ± 0.01 ± 0.06 ± 0.01 ± 0.12 Fig 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 - 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 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 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 days, no significant differences in the amount of drug retained were noticed Noticeable differences are after day 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 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 M Jakubowski et al 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 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 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 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 Cytotoxicity of free MERC against MCF-7 cells Cells were treated with MERC at a concentration of 10 μM, 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 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) M Jakubowski et al 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 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 M Jakubowski et al 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 M Jakubowski et al 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] References [27] [1] M Zacchigna, F Cateni, G Di Luca, S Drioli, A simple method for the preparation of PEG-6-mercaptopurine for oral administration, Bioorg Med Chem Lett 17 (2007) 6607–6609, https://doi.org/10.1016/j.bmcl.2007.09.064 [2] J.E Polifka, J.M Friedman, Teratogen update: azathioprine and 6-mercaptopurine, Teratology 65 (2002) 240261, https://doi.org/10.1002/tera.10043 [3] J.P Bă okkerink, E.H Stet, R.A De Abreu, F.J Damen, T.W Hulscher, M.A Bakker, J.A van Baal, 6-Mercaptopurine: cytotoxicity and biochemical pharmacology in human malignant T-lymphoblasts, Biochem Pharmacol 45 (1993) 1455–1463, https://doi.org/10.1016/0006-2952(93)90045-x [4] S Bhatia, W Landier, M Shangguan, L Hageman, A.N Schaible, A.R Carter, C L Hanby, W Leisenring, Y Yasui, N.M Kornegay, L Mascarenhas, A.K Ritchey, J N Casillas, D.S Dickens, J Meza, W.L Carroll, M.V Relling, F.L Wong, Nonadherence to oral mercaptopurine and risk of relapse in hispanic and nonhispanic white children with acute lymphoblastic leukemia: a report from the Children’s Oncology Group, J Clin Oncol 30 (2012) 2094–2101, https://doi.org/ 10.1200/JCO.2011.38.9924 [5] O.H Nielsen, B Vainer, J Rask-Madsen, Review article: the treatment of inflammatory bowel disease with 6-mercaptopurine or azathioprine, Aliment Pharmacol Ther 15 (2001) 1699–1708, https://doi.org/10.1046/j.13652036.2001.01102.x [6] S Sahasranaman, D Howard, S Roy, Clinical pharmacology and pharmacogenetics of thiopurines, Eur J Clin Pharmacol 64 (2008) 753–767, https://doi.org/ 10.1007/s00228-008-0478-6 [7] J.-R Wang, X Yu, C Zhou, Y Lin, C Chen, G Pan, X Mei, Improving the dissolution and bioavailability of 6-mercaptopurine via co-crystallization with isonicotinamide, Bioorg Med Chem Lett 25 (2015) 1036–1039, https://doi.org/ 10.1016/j.bmcl.2015.01.022 [8] A.B Talib, M.H Mohammed, Preparation, characterization and preliminary cytotoxic evaluation of 6-mercaptopurine-coated biotinylated carbon dots nanoparticles as a drug delivery system, Mater Today Proc (2021), https://doi org/10.1016/j.matpr.2021.06.341 [9] P Podsiadlo, V.A Sinani, J.H Bahng, N.W.S Kam, J Lee, N.A Kotov, Gold nanoparticles enhance the anti-leukemia action of a 6-mercaptopurine chemotherapeutic agent, Langmuir 24 (2008) 568–574, https://doi.org/10.1021/ la702782k [10] M Prabaharan, J.F Mano, Chitosan-based particles as controlled drug delivery systems, Drug Deliv 12 (2005) 41–57, https://doi.org/10.1080/ 10717540590889781 [11] M Gong, J Yang, Y Li, J Gu, Glutathione-responsive nanoscale MOFs for effective intracellular delivery of the anticancer drug 6-mercaptopurine, Chem Commun 56 (2020) 6448–6451, https://doi.org/10.1039/D0CC02872J [12] H Kaur, G.C Mohanta, V Gupta, D Kukkar, S Tyagi, Synthesis and characterization of ZIF-8 nanoparticles for controlled release of 6-mercaptopurine [28] [29] [30] [31] [32] [33] [34] [35] [36] [37] [38] 10 drug, J Drug Deliv Sci Technol 41 (2017) 106–112, https://doi.org/10.1016/j jddst.2017.07.004 Purnomo, P.H Setyarini, D Sulistyaningsih, Zeolite-based biomaterials for biomedical application: a review, AIP Conf Proc 1977 (2018), 030013, https:// doi.org/10.1063/1.5042933 M Pan, H.M Omar, S Rohani, Application of nanosize zeolite molecular sieves for medical oxygen concentration, Nanomater Basel Switz (2017) E195, https:// doi.org/10.3390/nano7080195 X.-H Fang, F Fang, C.-H Lu, L Zheng, Removal of Cs+, Sr2+, and Co2+ ions from the mixture of organics and suspended solids aqueous solutions by zeolites, Nucl Eng Technol 49 (2017) 556–561, https://doi.org/10.1016/j.net.2016.11.008 M Dusselier, M.E Davis, Small-pore zeolites: synthesis and catalysis, Chem Rev 118 (2018) 52655329, https://doi.org/10.1021/acs.chemrev.7b00738 O Martinho, N Vilaỗa, P.J.G Castro, R Amorim, A.M Fonseca, F Baltazar, R M Reis, I.C Neves, In vitro and in vivo studies of temozolomide loading in zeolite structures as drug delivery systems for glioblastoma, RSC Adv (2015) 28219–28227, https://doi.org/10.1039/C5RA03871E T Ceyhan, M Tatlier, H Akỗakaya, In vitro evaluation of the use of zeolites as biomaterials: effects on simulated body fluid and two types of cells, J Mater Sci Mater Med 18 (2007) 1557–1562, https://doi.org/10.1007/s10856-007-3049-y A Petushkov, J Freeman, S.C Larsen, Framework stability of nanocrystalline NaY in aqueous solution at varying pH, Langmuir 26 (2010) 6695–6701, https://doi org/10.1021/la9040198 L Bacakova, M Vandrovcova, I Kopova, I Jirka, Applications of zeolites in biotechnology and medicine – a review, Biomater Sci (2018) 974–989, https:// doi.org/10.1039/C8BM00028J M Sandomierski, M Zieli´ nska, A Voelkel, Calcium zeolites as intelligent carriers in controlled release of bisphosphonates, Int J Pharm 578 (2020), 119117, https://doi.org/10.1016/j.ijpharm.2020.119117 F Morante-Carballo, N Montalv´ an-Burbano, P Carri´ on-Mero, N Espinoza-Santos, Cation exchange of natural zeolites: worldwide research, Sustainability 13 (2021) 7751, https://doi.org/10.3390/su13147751 M.D Oleksiak, J.A Soltis, M.T Conato, R.L Penn, J.D Rimer, Nucleation of FAU and LTA zeolites from heterogeneous aluminosilicate precursors, Chem Mater 28 (2016) 4906–4916, https://doi.org/10.1021/acs.chemmater.6b01000 C Schroeder, M.R Hansen, H Koller, Ultrastabilization of zeolite Y transforms brønsted-brønsted acid pairs into brønsted-lewis acid pairs, Angew Chem., Int Ed Engl 57 (2018) 14281–14285, https://doi.org/10.1002/anie.201808395 X Xu, J Wang, Y Long, Zeolite-based materials for gas sensors, Sensors (2006) 1751–1764, https://doi.org/10.3390/s6121751 M.G Rimoli, M.R Rabaioli, D Melisi, A Curcio, S Mondello, R Mirabelli, E Abignente, Synthetic zeolites as a new tool for drug delivery, J Biomed Mater Res A 87A (2008) 156–164, https://doi.org/10.1002/jbm.a.31763 C.V Uglea, I Albu, A Vatajanu, M Croitoru, S Antoniu, L Panaitescu, R M Ottenbrite, Drug delivery systems based on inorganic materials: I Synthesis and characterization of a zeolite-cyclophosphamide system, J Biomater Sci Polym Ed (1995) 633–637, https://doi.org/10.1163/156856294X00572 D Dorniani, M.Z bin Hussein, A.U Kura, S Fakurazi, A.H Shaari, Z Ahmad, Preparation and characterization of 6-mercaptopurine-coated magnetite nanoparticles as a drug delivery system, Drug Des Dev Ther (2013) 1015–1026, https://doi.org/10.2147/DDDT.S43035 A.A Sharfalddin, A.-H Emwas, M Jaremko, M.A Hussien, Transition metal complexes of 6-mercaptopurine: characterization, Theoretical calculation, DNABinding, molecular docking, and anticancer activity, Appl Organomet Chem 35 (2021), e6041, https://doi.org/10.1002/aoc.6041 M Kucinska, A Plewinski, W Szczolko, M Kaczmarek, T Goslinski, M Murias, Modeling the photodynamic effect in 2D versus 3D cell culture under normoxic and hypoxic conditions, Free Radic Biol Med 162 (2021) 309–326, https://doi.org/ 10.1016/j.freeradbiomed.2020.10.304 Q Al-Naddaf, S Lawson, A.A Rownaghi, F Rezaei, Analysis of dynamic CO2 capture over 13X zeolite monoliths in the presence of SOx, NOx and humidity, AIChE J 66 (2020), e16297, https://doi.org/10.1002/aic.16297 O.S Travkina, M.R Agliullin, N.A Filippova, A.N Khazipova, I.G Danilova, N G Grigor’eva, N Narender, M.L Pavlov, B.I Kutepov, Template-free synthesis of high degree crystallinity zeolite Y with micro–meso–macroporous structure, RSC Adv (2017) 32581–32590, https://doi.org/10.1039/C7RA04742H H Faghihian, N Godazandeha, Synthesis of nano crystalline zeolite Y from bentonite, J Porous Mater 16 (2009) 331–335, https://doi.org/10.1007/s10934008-9204-0 A.J Viudez, R Madue˜ no, T Pineda, M Bl´ azquez, Stabilization of gold nanoparticles by 6-mercaptopurine monolayers Effects of the solvent properties, J Phys Chem B 110 (2006) 17840–17847, https://doi.org/10.1021/jp062165l G.P Kumar, J.S Sanganal, A.R Phani, C Manohara, S.M Tripathi, H L Raghavendra, P.B Janardhana, S Amaresha, K.B Swamy, R.G.S.V Prasad, Anticancerous efficacy and pharmacokinetics of 6-mercaptopurine loaded chitosan nanoparticles, Pharmacol Res 100 (2015) 47–57, https://doi.org/10.1016/j phrs.2015.07.025 A Sirivat, N Paradee, Facile synthesis of gelatin-coated Fe3O4 nanoparticle: effect of pH in single-step co-precipitation for cancer drug loading, Mater Des 181 (2019), 107942, https://doi.org/10.1016/j.matdes.2019.107942 S Chen, J Popovich, W Zhang, C Ganser, S.E Haydel, D.-K Seo, Superior ion release properties and antibacterial efficacy of nanostructured zeolites ionexchanged with zinc, copper, and iron, RSC Adv (n.d.) 37949–37957 https ://doi.org/10.1039/c8ra06556j A Morshed, I Soma, T.R Choudhury, S Islam, M Salim, A Matin, M.S Hasan, Nutritional assessment and serum zinc level in children with acute lymphoblastic M Jakubowski et al [39] [40] [41] [42] [43] [44] [45] [46] Microporous and Mesoporous Materials 343 (2022) 112194 [47] S.H Mosavi, R Zare-Dorabei, Synthesis of NMOF-5 using microwave and coating with chitosan: a smart biocompatible pH-responsive nanocarrier for 6-mercapto­ purine release on MCF-7 cell lines, ACS Biomater Sci Eng (2022) 2477–2488, https://doi.org/10.1021/acsbiomaterials.2c00068 [48] L Wang, C Dai, Y Fang, X You, J Wu, A drug/carrier dual redox-responsive system based on 6-mercaptopurine dimer-loaded cysteine polymer nanoparticles for enhanced lymphoma therapy, Nano Res 15 (2022) 4544–4551, https://doi org/10.1007/s12274-021-4037-0 [49] T.L Riss, R.A Moravec, A.L Niles, S Duellman, H.A Benink, T.J Worzella, L Minor, Cell viability assays, in: G.S Sittampalam, A Grossman, K Brimacombe, M Arkin, D Auld, C.P Austin, J Baell, B Bejcek, J.M.M Caaveiro, T.D.Y Chung, N.P Coussens, J.L Dahlin, V Devanaryan, T.L Foley, M Glicksman, M.D Hall, J V Haas, S.R.J Hoare, J Inglese, P.W Iversen, S.D Kahl, S.C Kales, S Kirshner, M Lal-Nag, Z Li, J McGee, O McManus, T Riss, P Saradjian, O.J Trask, J R Weidner, M.J Wildey, M Xia, X Xu (Eds.), Assay Guid Man., Eli Lilly & Company and the National Center for Advancing Translational Sciences, Bethesda (MD), 2004 http://www.ncbi.nlm.nih.gov/books/NBK144065/ (Accessed May 2020) accessed [50] A.A Fern´ andez-Ramos, C Marchetti-Laurent, V Poindessous, S Antonio, P Laurent-Puig, S Bortoli, M.-A Loriot, N Pallet, 6-mercaptopurine promotes energetic failure in proliferating T cells, Oncotarget, 8, https://doi.org/10.186 32/oncotarget.17889, 2017, 43048-43060 [51] S Kamiloglu, G Sari, T Ozdal, E Capanoglu, Guidelines for cell viability assays, Food Front (2020) 332–349, https://doi.org/10.1002/fft2.44 [52] X Kong, B Dong, X Song, C Wang, N Zhang, W Lin, Dual turn-on fluorescence signal-based controlled release system for real-time monitoring of drug release dynamics in living cells and tumor tissues, Theranostics (2018) 800–811, https:// doi.org/10.7150/thno.21577 [53] Y Yang, B Sun, S Zuo, X Li, S Zhou, L Li, C Luo, H Liu, M Cheng, Y Wang, S Wang, Z He, J Sun, Trisulfide bond–mediated doxorubicin dimeric prodrug nanoassemblies with high drug loading, high self-assembly stability, and high tumor selectivity, Sci Adv (2020), https://doi.org/10.1126/sciadv.abc1725 eabc1725 leukaemia, Bangladesh J Child Health 40 (2016) 12–16, https://doi.org/10.3329/ bjch.v40i1.31548 P Karran, N Attard, Thiopurines in current medical practice: molecular mechanisms and contributions to therapy-related cancer, Nat Rev Cancer (2008) 24–36, https://doi.org/10.1038/nrc2292 C Roberts, V.Y Strauss, S Kopijasz, C Gourley, M Hall, A Montes, J Abraham, A Clamp, R Kennedy, S Banerjee, L.K Folkes, M Stratford, S Nicum, Results of a phase II clinical trial of 6-mercaptopurine (6MP) and methotrexate in patients with BRCA-defective tumours, Br J Cancer 122 (2020) 483–490, https://doi.org/ 10.1038/s41416-019-0674-4 S Nicum, C Roberts, L Boyle, S Kopijasz, C Gourley, M Hall, A Montes, C Poole, L Collins, A Schuh, S.J Dutton, A phase II clinical trial of 6-mercaptopurine (6MP) and methotrexate in patients with BRCA defective tumours: a study protocol, BMC Cancer 14 (2014) 983, https://doi.org/10.1186/1471-2407-14-983 B Singh, V.N Sarli, A Lucci, Inhibition of resistant triple-negative breast cancer cells with low-dose 6-mercaptopurine and 5-azacitidine, Oncotarget 12 (2021) 626–637, https://doi.org/10.18632/oncotarget.27922 S.A Coulthard, S McGarrity, K Sahota, P Berry, C.P.F Redfern, Three faces of mercaptopurine cytotoxicity in vitro: methylation, nucleotide homeostasis, and deoxythioguanosine in DNA, Drug Metab Dispos 46 (2018) 1191–1199, https:// doi.org/10.1124/dmd.118.081844 G.L Chan, G.R Erdmann, S.A Gruber, A.J Matas, D.M Canafax, Azathioprine metabolism: pharmacokinetics of 6-mercaptopurine, 6-thiouric acid and 6-thio­ guanine nucleotides in renal transplant patients, J Clin Pharmacol 30 (1990) 358–363, https://doi.org/10.1002/j.1552-4604.1990.tb03606.x Y Zou, D Mei, J Yuan, J Han, J Xu, N Sun, H He, C Yang, L Zhao, Preparation, characterization, pharmacokinetic, and therapeutic potential of novel 6-mercap­ topurine-loaded oral nanomedicines for acute lymphoblastic leukemia, Int J Nanomed 16 (2021) 1127–1141, https://doi.org/10.2147/IJN.S290466 J.-R Wang, X Yu, C Zhou, Y Lin, C Chen, G Pan, X Mei, Improving the dissolution and bioavailability of 6-mercaptopurine via co-crystallization with isonicotinamide, Bioorg Med Chem Lett 25 (2015) 1036–1039, https://doi.org/ 10.1016/j.bmcl.2015.01.022 11 ... J.S Sanganal, A. R Phani, C Manohara, S.M Tripathi, H L Raghavendra, P.B Janardhana, S Amaresha, K.B Swamy, R.G.S.V Prasad, Anticancerous efficacy and pharmacokinetics of 6-mercaptopurine loaded... D Dorniani, M.Z bin Hussein, A. U Kura, S Fakurazi, A. H Shaari, Z Ahmad, Preparation and characterization of 6-mercaptopurine- coated magnetite nanoparticles as a drug delivery system, Drug Des... S Bhatia, W Landier, M Shangguan, L Hageman, A. N Schaible, A. R Carter, C L Hanby, W Leisenring, Y Yasui, N.M Kornegay, L Mascarenhas, A. K Ritchey, J N Casillas, D.S Dickens, J Meza, W.L Carroll,