Zeolitic Imidazolate Framework‑8 (ZIF-8) modified titanium alloy for controlled release of drugs for osteoporosis

The aim of this work was to prepare a biocompatible implant material that enables the release of drug for osteoporosis—risedronate. To achieve this goal, a titanium implant coated with a biocompatible Zeolitic Imidazolate Framework 8 (ZIF-8) layer was prepared that promotes osseointegration at the bone-implant interface. The modifications of the titanium alloy as well as sorption and desorption processes were confirmed using a variety of methods: SEM, EDS XPS, and FT-IR imaging (to determine surface modification, drug distribution, and risedronate sorption), and UV–Vis spectroscopy (to determine drug sorption and release profile). Both the ZIF-8 layer and the drug are evenly distributed on the surface of the titanium alloy. The obtained ZIF-8 layer did not contain impurities and zinc ions were strongly bounded by ZIF-8 layer. The ZIF-8 layer was stable during drug sorption. The drug was released in small doses for 16 h, which may help patients recover immediately after surgery. This is the first case of using ZIF-8 on the surface of the titanium alloy as carrier that releases the drug under the influence of body fluids directly at the site of the disease. It is an ideal material for implants designed for people suffering from osteoporosis.

Osteoporosis is the most common bone disease, affecting more than 200 millions of people worldwide 1,2 . The prevalence of osteoporosis increases with age and, as a result, more and more people suffer from it due to aging of the population 3 . Currently used drugs for osteoporosis can be divided into two main groups. The first includes antiresorptive drugs (inhibiting osteoclasts) while the second bone-forming drugs (stimulating osteoblasts) 4 . The most popular antiresorptive drugs used for osteoporosis are bisphosphonates (BPs) 5,6 . Such a wide application of BPs is related to their high selectivity to the bone 7 . Bisphosphonates are ideally suited for the treatment of bone disease because they have a high affinity for hydroxyapatite crystals 8,9 . Most often, bisphosphonates are taken orally during treatment (in tablet form). Less popular methods of their administration are injections, infusions, and intranasal or transdermal applications. Each of the BP delivery methods used so far has disadvantages. When the drug is administered orally, only a small part of the drug (1-5%) goes to the circulatory system 10 . Oral administration is also associated with many side effects. Some of the many are: heartburn, nausea, irritation of the esophagus, and gastric ulcer. Flu-like symptoms are the main side effects of intravenous administration. Transdermal drug delivery results in local toxicity caused by the release of too much drug over a short period of time 11 . Overall, these drugs are very effective, but better delivery methods are required. One of the directions of new drug release materials for this drug should be implants because osteoporosis is a disease that is often diagnosed too late, so parts of the bone must be replaced 12 . These should be implants that will release the drug in a controlled manner without 'burst release' to cause local toxicity.
Titanium alloys are commonly used materials in implantology 13 . One of the most widely used is Ti6Al4V, which has very good biocompatibility and low elastic modulus comparable to human bone 14 . Despite the fact that titanium alloys are ideal materials for implant production, it is not possible to use them alone in controlled release of drugs. To produce a material with such properties, the surface of the implant must be previously modified. One type of promising modification is the formation of a biocompatible Zeolitic Imidazolate Framework-8 (ZIF-8) on their surface 15 .
Zeolitic imidazolate frameworks (ZIFs) are a subclass of metal organic frameworks (MOFs) 16 . ZIFs consist of tetrahedral metal ions (specifically Zn and Co) bridged by imidazolate ligands, and have the advantages of both  [17][18][19][20] . These properties indicate a potential application for ZIFs in catalysis, adsorption, and separation [21][22][23] . Moreover, its potential for use in biomedical applications is also attracting increasing attention [24][25][26] . For example, Liu et al. prepared catechol-chitosan-ZIF-8 hydrogels that promote implantation stability, angiogenesis, and osteogenesis for bone regeneration applications 27 . ZIF nanoparticles were also used as anti-inflammatory and antibacterial platforms to treat periodontitis 28 . In another publication, Sun et al. have reported a pH-sensitive drug delivery system based on ZIFs 29 . ZIFs can also be used in the modification of titanium alloys. Porous titanium modified with nanoscale ZIF-8 coating has been shown to enhance osteogenic and antibacterial activity, increase extracellular matrix mineralization and promotes alkaline phosphatase activity 30 . Subsequent research shows that the ZIF-8 layer enhanced cell bioactivity and also promoted osseointegration at the bone-implant interface 15 . All this information indicates a very high potential for titanium modification with ZIF-8 and the use of this modification in endoprostheses.
The materials considered during the selection of a carrier in the controlled release of bisphosphonates should have divalent cations in their structure, as we have proven in previous work 31 . This is caused by strong interactions between divalent cations and the phosphonium groups of bisphosphonates 32 . Due to this, a thin layer of ZIF-8 on the surface of the titanium alloys appears to be ideal for the targeted release of bisphosphonates in a place where a bone fragment was needed to be removed. The research scheme of this work is shown in Fig. 1.
In this work, the biocompatible Zeolitic Imidazolate Framework-8 (ZIF-8) was obtained on the Ti6Al4V alloy surface and used as a risedronate carrier. Two types of alloy modification were used, one previously described and one proposed for the first time in this work. This is the first case of using the Zeolitic Imidazolate Framework

Preparation of ZIF-8 on the surface of the alloy modified with alkali heat treatment (Ti-AHT-ZIF-8).
The methodology of this modification was carried out in accordance with that carried out by Zhang et al. 15 . The titanium discs were sanded, washed with water, ethanol, and acetone. Alkali heat treatment (AHT) of Ti6Al4V was performed according to the procedure described by Chosa et al. 33 . The polished titanium discs were soaked in a 5 M sodium hydroxide aqueous solution at 60 °C for 24 h, followed by washing in distilled water and drying in an oven at 100 °C for 24 h. Subsequently, the discs were heated to 600 °C at a rate of 5 °C/min in an electrical furnace and kept at 600 °C for 1 h. Coating with a ZIF-8 layer on the AHT titanium surface was carried out using a simple and environmentally friendly hydrothermal method. In the first stage of creating the ZIF-8 layer zinc nitrate hexahydrate (0.11 g) and MeIm (2.27 g) were dissolved in 40 ml of deionized water, and stirred for 20 min. The resulting solution was diluted eight times. The solution was then transferred to an autoclave in which the AHT titanium discs were placed and heated at 37 °C for 6 h. Finally, ZIF-8 modified titanium discs were obtained by rinsing with deionized water and drying at 37 °C for 24 h. The resulting material was named Ti-AHT-ZIF-8.

Preparation of ZiF-8 on the surface of the alloy modified with zinc titanate (Ti-ZnTit-ZIF-8).
The formation of the ZIF-8 layer on the surface of the titanium alloy presented in this section has not been previously described in the literature. The titanium discs were sanded, washed with water, ethanol, and acetone. Ti6Al4V plates were placed in 5 M sodium hydroxide aqueous solution at 60 °C for a 24 h 34 . The alloy was then washed with distilled water and dried in an oven for 24 h at 100 °C (Ti-NaTit). After drying, the material was placed in a 0.5 M aqueous solution of zinc nitrate for 24 h at room temperature. This process was repeated 3 times. After that, the material was washed with distilled water 3 times and dried in an oven for 24 h at 100 °C (Ti-ZnTit). In the next step, the material was immersed in the MeIm solution for one hour and rinsed again with water. The next steps are analogous to the procedure for Ti6Al4V-AHT-ZIF-8. In the first stage of creating ZIF-8 zinc nitrate hexahydrate (0.11 g) and MeIm (2.27 g) were dissolved in 40 ml of deionized water, and stirred for 20 min. The resulting solution was diluted eight times. The solution was then transferred to an autoclave in which the zinc titanate modified titanium discs were placed, and heated at 37 °C for 6 h. Finally, ZIF-8 modified titanium discs were obtained by rinsing with deionized water and drying at 37 °C for 24 h. The resulting material was named Ti-ZnTit-ZIF-8.  Table 1. The amount of drug released was measured after each 1 h for up to 16 h using UV-Vis spectroscopy. Each time the SBF was replaced with a new portion. Three repetitions were made for both materials.  UV-Vis spectroscopy. UV-Vis spectrophotometer UV-2600 (Shimadzu, Japan) was applied to determine the concentration of risedronate during the sorption and release process. Measurements were made in the range of 240-305 nm (λ max = 262 nm).

Results
The first technique to confirm the effectiveness of surface modification with ZIF-8 was SEM. As can be seen in Fig. 2, ZIF-8 was produced on the titanium surface after the AHT process. Many particles can be seen on the surface, and these results are consistent with those obtained by Zhang et al. 15 . The surface of Ti-AHT-ZIF-8 was compared with the surface after drug adsorption (Ti-AHT-ZIF-8-RSD). There are no more ZIF-8 crystals on the Ti-AHT-ZIF-8-RSD surface, which proves that they were washed away during the sorption process. This is negative information because it indicates that the drug could not be retained on the surface for controlled release.
Using the SEM analysis, the samples produced by the method proposed by our team were also characterized (Fig. 3). The surface of the alloy after alkaline treatment (Ti-NaTit) is very porous, which is consistent with reports from the literature 35,36 . No significant changes were noticed after ion exchange (Ti-ZnTit). Visible changes occur after the formation of the ZIF-8 layer (Ti-ZnTit-ZIF-8). The surface of the alloy is no longer porous, and a very irregular coating is visible on its entire surface. The layer obtained differs significantly from that produced by the first methodology (Ti-AHT-ZIF-8). The structure of this layer is similar to the ZIF-8 layer obtained on Al plates by the ligand-assisted solvothermal conversion of ZnAl-CO3 layered double hydroxide by Zhang et al. 37 . The occurrence of a "crack" in the sample is related to the fact that there was a larger scratch in this place, formed during grinding. This scratch was not completely covered by the ZIF-8 layer. As can be seen on the sample, after sorption of the drug, such a crack does not occur. Importantly, the layer produced by the method proposed by our team also occurs after drug adsorption (Ti-ZnTit-ZIF-8-RSD). The differences between Ti-ZnTit-ZIF-8 and Ti-ZnTit-ZIF-8-RSD are likely due to the drug on the surface Ti-ZnTit-ZIF-8-RSD.
The distribution of the layer on the surface of the alloy was also performed using EDS mapping (Fig. 4). As can be seen from the zinc distribution, this layer is visible on the entire surface of the alloy for both Ti-ZnTit-ZIF-8 The presence of zinc in both samples was also confirmed by XPS analysis (Fig. 5). The Zn binding energy range shows peaks at 1023.2 eV that correspond to Zn 2p 3/2 , and peaks at a higher binding energy of 1045.9 eV associated with Zn 2p 1/238 . The ~ 23.1 eV difference between Zn 2p 3/2 and Zn 2p 1/2 indicates the oxidation state + 2 for Zn ions in both samples and this was not changed after drug sorption 39,40 . The presence of nitrogen on the surface of the Ti-ZnTit-ZIF-8 sample indicates the effectiveness of the ZIF-8 layer formation. A narrow symmetric peak in the N 1 s XPS spectra of ZIF-8 indicates that there is only one form of nitrogen in the framework. The presence of only one peak indicates that there are no defects in the obtained layer and that all rings are coordinated by zinc 41 . Furthermore, the peak for nitrogen shifts to a higher binding energy than that for nitrogen in the free molecule, also indicating that it is bound to the metal 42,43 . Two nitrogen peaks are visible after drug sorption. One can be assigned to the ZIF-8 layer and the other to the drug. These peaks indicate the presence of C = N/C-N bonds 44,45 . The peak for P observed in the XPS spectra of Ti-ZnTit-ZIF-8-RSD clearly indicates the effectiveness of drug sorption (Fig. 6). The deconvolution of high-resolution P 2p spectra for Ti-ZnTit-ZIF-8-RSD shows a characteristic asymmetric P 2p 1/2 and P 2p 3/2 , confirming the presence of risedronate on the surface 46,47 . For Ti-ZnTit-ZIF-8, these peaks do not exist.
The distribution of the ZIF-8 layer, and drug was determined using FT-IR imaging. As can be seen in Fig. 6, both are present over the entire surface of the material. The bands for which the ZIF-8 and drug distribution were determined are not present in Ti-Zn-Tit material. The imaging of the ZIF-8 layer was performed for bands in the range 1486.3 and 1381.3 cm −1 that can be assigned to the imidazole ring stretching 48,49 . The imaging of the drug distribution was performed for P-O band in the range 1177.7 and 982.1 cm −1 , which clearly confirms the presence of the drug 50 . These bands also occur after drug sorption, which indirectly means that the ZIF-8 layer has not been removed. The even distribution of the ZIF-8 and risedronate that has been achieved on the surface of this material is very important because the drug should be delivered from the surface of the endoprosthesis to the same extent at each point of contact with the tissues.
UV/Vis spectroscopy was used to determine the exact amount of drug retained by the modified alloys. The results are presented in Fig. 7. The titanium alloy modified with the method proposed in this study retained more than 10 times more drug. www.nature.com/scientificreports/ The potential reason for the high effectiveness of the second material is presented in Fig. 8. Probably, there are only single ZIF-8 crystals on the surface of the Ti-AHT-ZIF-8 material that was visible in the SEM analysis. Because of the low availability of zinc ions in such large crystals, the drug has limited ability to attach. On the other hand, an evenly distributed layer of ZIF-8 is formed on the surface of the entire Ti-ZnTit-ZIF-8 material.
Because of this, it is possible to attach a lot more drug to its surface. In addition, the ZIF-8 layer is connected to the alloy surface by zinc ions from zinc titanate, which contributes to its stability on the surface.
To explain in more detail why the modification presented in this article allows for the creation of a ZIF-8 layer that is more stable and distributed over the entire surface, one should look at the process scheme shown in Fig. 9. In the first stage, a typical modification of a titanium alloy was used, in which sodium titanate is produced on its surface under the influence of sodium hydroxide. In the second stage, sodium ions are exchanged for zinc ions in the process of ion exchange. The exchange with zinc ions was carried out because they participate in the crystallization of ZIF-8 and it was assumed that they could contribute to greater adhesion of the produced www.nature.com/scientificreports/ ZIF-8 layer to titanium. In the next step, the plates were placed in the MeIm solution. This step was to create a thin MeIm layer, which was also to contribute to increasing the adhesion between the alloy and the ZIF-8 layer. Figure 10 shows that the ZIF-8 structure (which is shown in Fig. 3) is not yet formed on the alloy surface after this step. There are some changes compared to zinc titanate layer, but they are minor. This layer initiates the build-up of ZIF-8 on the surface of the titanium alloy. The final step was to place the plate in a solution of MeIm and zinc ions. All of these steps contributed to the creation of a layer that is more stable and allows the retention of an incomparably greater amount of the drug.
Retaining the desired amount of drug on the surface is the first step in the preparation of the drug carrier and is not the most important step. More important is how much medicine is released and at what time. The release results are presented in Fig. 11. Drug release from the Ti-AHT-ZIF-8-RSD surface was very short and a small  The surfaces of endoprostheses are several hundred times larger, which will allow the selection of the appropriate dose of the drug for a given application. Importantly, the drug was released in small doses, which proves that the alloy will not cause toxic reactions. On the basis of the results obtained, it can be assumed that the presented material is suitable for supporting the recovery of people suffering from osteoporosis immediately after surgery.

Conclusions
In the presented work, we created a stable and biocompatible layer of ZIF-8 on the surface of a titanium alloy by modifying the previously described method 15 . We attached a drug for osteoporosis to the created layer. Both the ZIF-8 layer obtained with our method and the drug on its surface were evenly distributed across the entire surface of the alloy, which was confirmed by EDS mapping, and FTIR imaging. The results of the XPS analysis show that the layer obtained by our method has no defects or impurities in the form of unbound imidazole rings. Comparing our material to that described in the literature, it was found that it retains 10 times more of the drug. Additionally, our material releases a much larger dose of the drug within 16 h. Due to its biocompatibility and enhanced osseointegration, the material described in this work is a potential material for use as an element of endoprostheses. Due to the fact that it will also release the drug after the operation, it will increase  www.nature.com/scientificreports/ the chances of a quick recovery of the patient. Furthermore, the proposed implant also has zinc ions that have antibacterial properties.

Data availability
The datasets generated during and/or analysed during the current study are available from the corresponding author on reasonable request.