Zinc Zeolite as a Carrier for Tumor Targeted and pH-responsive Drug Delivery

In this work, for the first time, a material was prepared that releases the drug in a controlled manner under the influence of the pH of cancer environment. The material is zinc zeolite, which only releases the drug at an acidic pH. The release of the drug in an acidic environment indicates a very high potential of the synthesized material in the treatment of cancer. This material does not release the drug at pH 7.4 even for more than 100 h which proves that healthy organs will not be affected. The advantage of this material over those previously described in the literature is that it releases the drug very quickly under the influence of the cancerous environment (88% of the drug during approx 2 h), as well as the fact that it does not release the drug in a different environment. Materials with both features have not been previously described, there are only works on materials having one of these features. Moreover, this material releases zinc ions which additionally act on cancer cells by inducing apoptosis of cancer cells by increasing the intracellular production of reactive oxygen species.

obtain the appropriate drug concentration at the site of the tumor. It is also known that the pH in the tumor microenvironment is lower than under normal conditions [10,11]. Zeolites seem to be an ideal candidate for use as a carrier for zoledronate. It is because they are biocompatible materials with lots of biomedical applications, for example: drugs and genes delivery or construction of biosensors [12,13]. The structure of these materials is made of MO4 tetrahedrons, where M stands for Al or Si. The difference in valence of these two elements causes that the lattice has a negative charge that must be compensated by cations. These cations are exchangeable [10,14]. There are different type of zeolites but in our work we want to use zeolite X. These type of zeolite is characterized by Si/Al ratio ranging from 1 to 1,5 and pore sizes from 6Å-8Å [15]. In the previous work we proved that zeolites with substituted calcium ions can be used as carriers of risedronate, which belongs to the group of bisphosphonates. Which indicates that bisphosphonates have great affinity to divalent cations [16]. In this paper, we would like to propose a zeolite X-based zoledronate delivery system with Zn 2+ ions that could release the drug under the influence of the tumor's acidic microenvironment. In addition, it was proved that zinc ions in appropriate concentrations can induce apoptosis of cancer cells by increasing the intracellular production of reactive oxygen species [17]. In our opinion, such a composition will allow for the synergistic anti-cancer effect of zinc ions and zoledronate. This carrier should allow the drug to be delivered to the site of action without releasing the drug to healthy tissue. The scheme of the described studies is shown in Fig. 1.

Ion Exchange
Ion exchange was carried out by mixing a 20 ml of 0.5 M solution of the zinc chloride with 1 g of sodium zeolite for 24 h. Then zeolite was centrifuged. This process was repeated three times. After that, zeolite was washed with distilled water to remove excess zinc chloride three times and dried in an oven for 24 h at 100 °C.
The material after ion exchange was named Zn-X.

Drugs Sorption
Zoledronate sorption was initiated by introducing 20 mg of zinc zeolite into falcon polypropylene tubes. Each tube was filled with 15 ml of zoledronate solution with a concentration of 0.2 mg ml − 1 . The samples were shaken for one week at room temperature then the samples were centrifuged. The amount of drug adsorbed on the carrier was determined by the difference between the amount of drug in the starting solution and the amount of drug remaining after sorption. Six repetitions of drug sorption were performed to determine the reproducibility of this process. The material after drug sorption was named Zn-X-ZOL. The same procedure was used for Na-X zeolite, but the drug was not retained, and results for it are not presented.

Drug Release
Carrier after drug sorption was placed in 5 ml of simulated body fluid (SBF) or 5 ml of acetate buffer at 36.6 °C [18,19]. The amount of drug released was measured using UV-VIS spectroscopy. Each time, the samples were centrifuged (5 min with 4500 rpm) and the fluid was replaced. Six repetitions of drug release were performed to determine the reproducibility of this process.
In the case of SBF, the samples were placed in solution for 100 h and the amount of drug released was measured every hour, then every 8 h, and then every 24 h. Due to the fact that the drug was not released during the entire process, the results were not presented.
In the case of the acetate buffer, the samples were placed in the solution for 15 min, after which the amount of released drug was tested. A new solution was then added and the amount of drug released was checked again after 15 min. The carrier was flooded a total of 9 times because no more drug was released after this time. The total release time was 135 min.

X-ray Diffractometry (XRD)
Samples were examined by X-ray diffraction using a D8 Advance diffractometer (Bruker) with a Johanson monochromator and a LynxEye detector. The measurement step was between 0.002 and 0.03, while the time for each step was between 2 and 3 s. Poly(methyl methacrylate) cuvettes were used during the tests. λCu Kα1 = 1.54 Å.

Scanning Electron Microscopy (SEM) and Energy Dispersive Spectroscopy (EDS)
SEM images were recorded with the use of scanning electron microscope VEGA 3 (TESCAN, Czech Republik). 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 4 different spots.

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.

Fourier-Transform Infrared Spectroscopy
FT-IR analysis of all materials was performed using a Ver-tex70 spectrometer, Bruker Optics. All materials were studied using a single reflection diamond ATR crystal. 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.

Elemental Analysis
Measurements were performed on the FLASH 2000 elemental analyzer. Zeolite before and after sorption were weighed in tin capsules (approximately 2 mg) and introduced into the reactor by means of 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.

Inductively Coupled Plasma mass Spectrometry (ICP-MS)
Measurements were carried out on a mass spectrometer with induction induced plasma ICP-MS NexION 300d by PerkinElmer. Liquid samples were subjected to quantitative analysis. For this purpose, a calibration curve was made and a zinc concentration was determined based on it. The analysis was performed in the KED system to avoid the matrix affecting the zinc content. The results were obtained in ppm.

Results
The first step in this work was to determine the effect of ion exchange on the faujasite structure. This was tested using XRD and the results are presented in Fig. 2. The shape of the Zn-X does not differ from Na-X zeolite. These results confirm that there is no influence of ion exchange with Zn 2+ The efficiency of ion exchange in sodium zeolite was confirmed by EDS analysis ( Table 1). Comparison of ion content with the Na-X zeolite (Na ~ 11.3% wt %) shows that the ions only exchanged and did not e.g. precipitated out in a different form [23]. After ion exchange, no chloride is present in the zinc zeolite. EDS analysis, in addition to the effectiveness of ion exchange, also confirms the effectiveness of drug sorption. Effectiveness of sorption is demonstrated by the increase in the amount of phosphorus and nitrogen. Both these elements are not present in the surface layer before sorption of the drug and significant amounts can be observed after that process.
The distribution of the drug on the carrier surface was performed using EDS mapping (Fig. 4). As can be seen from the distribution of all elements, zoledronate is visible on the entire surface of the carrier. Drug adsorption is proved by the presence of phosphorous and nitrogen, which are present in the structure of zoledronate.
The Nitrogen Adsorption / Desorption results are shown in Table 2. After ion exchange with zinc ions, a decrease in the value of the specific surface area and the pore volume can be seen. This is a typical phenomenon and has already been described in the literature [24,25]. The specific surface area of carrier drops significantly after drug sorption (about 24%). The decrease is due to the surface being covered with the ZOL layer. As can be seen, the number of micropores on the crystal structure of zeolite X. The lack of changes is important because in this work we want to prove that the incorporation of the zinc ion, and not the change of the zeolite structure, affects the sorption and release of the drug. In addition, it was checked whether the sorption of the drug affects the change in the crystalline properties of the material. As can be seen from Fig. 2, the results for Zn-X-ZOL are the same as for the other materials. This indirectly indicates that the drug did not crystallize, but was adsorbed through interactions with zinc ions.
Na-X zeolite particles used in this work have an average size of 2 μm. As can be seen in all SEM images, this value occurs also for Zn-X, both before and after zoledronate sorption (Fig. 3). The particles are very similar in size and appearance. This confirms that the incorporation of zinc ions and zoledronate does not affect the particle size and shape of zeolite [22]. The carrier after sorption of the zoledronate do not agglomerate. It indicates that the drug is probably present on the surface and in the pores of the zinc zeolite, rather than precipitated.   [30,31].
The results of the elemental analysis also confirm the effectiveness of drug sorption in the prepared materials ( Table 3). This is evidenced by the increase in the amount of carbon and nitrogen compared to zeolites prior to sorption.
The exact amount of drug retained on the carrier was determined using UV-Vis analysis (Fig. 6). Approximately 1.1 mg of the drug was retained in 20 mg of carrier. The amount of drug retained on the carrier was determined based on the amount of drug remaining in the starting solution. The 5% drug content on the carrier surface can be considered high content. But of course it is more important whether such a carrier-drug system is stable and the drug is not released into healthy human tissues. This feature can only be proven when released in an appropriate environment: in SBF or in the cancer environment.
The amount of drug retained on the carrier surface is very important. However, more important is whether the drug is released, because only then can it act on diseased cells. Only one release profile can be seen in the presented chart (Fig. 7). This is because the drug is not released under the influence of SBF. This proves the assumption presented in this paper. We have prepared a material that will not release the drug under the influence of body fluids, and thus will not affect healthy organs. We studied drug release under SBF for over 100 h and no drug release was observed. This proves high stability and the fact that our system is not toxic and affects only cancer cells in the human body. Such material can be safely removed from the body without adversely affecting other organs. This is because zeolite X (FAU) is considered biocompatible. Lutzweiler et al. proved that magnesium and calcium zeolites do not have cytotoxic properties [32]. Cytotoxicity studies were also carried out for the zinc zeolite described in this paper. The absence of zeolite toxicity was proven using MCF-7 cells [33].
is several times greater for Zn-X material than the number of other pores. After drug sorption, the following decrease in micropore area was observed for zeolite Zn-X (207.38 m 2 ·g − 1 ). Large changes are also noticeable in the volume of micropores. For zeolite Zn-X, the volume of micropores decreased by 0.102 cm³·g − 1 . The obtained results confirm the effectiveness of zoledronate sorption on zinc zeolite.
Another technique used in the characterization of the materials was IR spectroscopy (Fig. 5). The spectrum after ion exchange is typical of FAU zeolite. The first two bands at 3400 cm − 1 and 1640 cm − 1 are assigned to the absorbed water and hydroxyl groups on the surface of the zeolite. The main confirmation of the FAU structure are the bands in the range of 1200 − 600 cm − 1 . The strong and broad band in the range 1200 − 900 cm − 1 is assigned to the asymmetric internal T-O stretching vibrations of the TO 4 tetrahedra. The two bands between 800 and 650 cm − 1 are assigned to the stretching vibration of Al-O in the Si-O-Al bonds [26,27]. New bands characteristic for organic compounds are visible after sorption of the zoledronate. Bands in the range 1580-1320 cm − 1 can be assigned to C = C and C = N stretch vibrations [28,29]. These bands are derived from the  Table 3 Elemental analysis of carrier before and after drug sorption C N Zn-X 0.10 ± 0.01 0 Zn-X-ZOL 2.80 ± 0.13 0.64 ± 0.10 During the research, the amount of zinc released from the carrier was also determined (Fig. 8). As in the case of the drug, zinc ions are quickly released under the influence of an acidic environment. This is desirable because the large amount of ions released in the environment of the cancer can affect the induction of cancer apoptosis cells by increasing the intracellular production of reactive oxygen species [17]. The release of drug and ions at the disease site indicates a double therapeutic effect of the prepared material.

Conclusion
Zinc form of zeolite X was used as carrier for the zoledronate. The results presented in this study confirm the effectiveness of ion exchange and drug sorption. The morphological properties of the material after sorption did not change, which proves that the drug is attached to zinc ions in the zeolite structure, and not precipitated. The sorption efficiency was confirmed by the presence of nitrogen and phosphorus ions in the analyzed samples. The carrier is capable of retaining about 5% of the drug, based on the weight of the carrier. Most importantly, the drug is released only in an acidic environment (cancerous environment). At a pH of 7.4, the drug is not released at all, which confirms that it will not affect healthy human cells. A comparison of Importantly, our material releases the drug in contact with the cancer environment in about 2 h. Looking at the profile, it is obvious that the material proposed in this paper targets the cancer environment. As can be seen in the first 15 min, the dose is lower. This is only because the acidic environment must reach the active sites in the zeolite structure. Importantly, during about 2 h, the carrier released almost 88% of the drug.
Zoledronate is released only in cancer environment from zinc zeolites because zoledronate have a nitrogen atom in structure with a free electron pair, which can potentially bind with zinc ions, which slows down the release [34][35][36].
Comparing our material to those known by us and presented in the literature, it can be concluded that the material presented in this work has a greater application potential. For example, Cai et all. presented the material -hydroxyapatite loaded polymeric nanoparticles and investigated the effect of pH on zoledronate release [37]. These studies determined that release was influenced by pH, but drug was released at both pH 7.4 and pH 5.0. Which proves that the drug will reach both healthy and affected cells. Another example is calcium zoledronate nanoscale Metal-Organic Frameworks prepared by Au et all. [5]. This interesting study confirmed that the creation of MOFs allows more drug to be released in an acidic environment. However, the release lasted for 24 h which is a long time for the target drug release. In addition, 5% of the drug was also released at pH 7.4 within 24 h, so this material was not totally nontoxic to healthy tissues. Xiao et all. obtained hybrid polymeric nanoparticles with high zoledronic acid loading [38]. The production of this material made it possible to increase the release in an acidic environment. However, as in the previous examples, the drug was also released at pH = 7.4. Benyettou et all. prepared a magnetic nanoparticle-based drug delivery system [39]. The our material with literature reports shows its great potential. To our knowledge, no material has yet been developed that would release the drug in a very short time and only in an acidic environment. Moreover, this material releases zinc ions which additionally act on cancer cells by induction apoptosis of cancer cells by increasing the intracellular production of reactive oxygen species [17]. This is the first report on this type of material. In the next stages of research in the future, we will focus on the preparation of zoledronate retained zinc nanozeolites and determine their effect on cancer cells in in vivo studies.