Synthesis, Characterization, and Cytotoxicity Evaluations of Silver–Zeolite Nanocomposite

: Zeolites of natural origin are materials exhibiting many positive effects on the human body. Silver-modified zeolites have already been introduced as bactericidal agents, although studies dealing with their toxicity are insufficient. This work describes the synthesis of activated and silver-loaded Bulgarian zeolite using a simple wet impregnation method. Morphological characteristics and compositions of natural zeolite, activated zeolite, and Ag-nanocomposites were studied by the X-ray diffraction (XRD), scanning electron microscopy (SEM), and transmission electron microscopy (TEM) methods. Silver loading is approximately 13 wt. %, with mean Ag particle size around 19 nm. Analyses of the samples included antioxidant activity assays based on ABTS radical scavenging ability and in vitro cytotoxicity tests with human normal fibroblasts and three adenocarcinoma cell lines. The experiments were performed with natural, activated, and Ag-modified zeolite in comparison to two commercial food supplements. Our results indicated moderate antioxidant activity of the tested samples. Silver-modified zeolite demonstrated cytotoxic effects against both tumor cells and normal fibroblasts, but the detected levels of inhibition were stronger against the adenocarcinoma cells, suggesting anti-tumor potential. The present article indicates a new aspect of Bulgarian natural zeolite and Ag-loaded zeolite biological activity. It highlights the need for detailed toxicity evaluations of Ag-nanocomposites prior to healthcare applications.


Introduction
Natural zeolites (NZs) are well-known, extensively examined, and still-developing materials with various applications.Currently, they are beneficial for application in industry, agriculture, environmental protection, biological technologies, and biomedicine [1].These hydrated aluminosilicate minerals with a porous structure exhibit valuable physicochemical properties such as cation exchange ability, molecular sieving, catalysis, great adsorption capacity, and reusability [2].Furthermore, zeolites are considered stable at ambient conditions [3], possibly non-toxic, and even edible.For example, clinoptilolite is used as a food supply that delivers essential minerals to organisms, reduces the pH of the body, improves tissue oxygenation, stimulates skin regeneration, and eliminates noxious substances [4].In the field of biomedical applications, it is also mentioned as an anti-diarrheal drug, anti-cancer agent, tocopherol absorbent, carrier of ibuprofen, sulfamethoxazole, and aspirin, and as a provider for the release of diclofenac in bone tissue engineering [5].
Among 70 types of NZ registered, clinoptilolite is the most abundant in nature and is broadly applicable worldwide [6].The crystalline structure of clinoptilolite is composed of tetrahedral SiO 4 and AlO 4 frameworks, connected by oxygen atoms [7].These negatively charged channels are stabilized by cations-Na + , K + , Ca 2+ , and Mg 2+ .The location, coordination mode, charge, size, and ionization potential of these ions affect the ion exchange capability of the zeolite and its ability to trap positively charged toxic substances [8].This exclusive feature of clinoptilolite has been utilized in ammonium ion removal, heavy metal removal [9], organic contaminant removal, CO 2 post-combustion adsorption, etc. [10].The adsorption efficiency is frequently limited by impurities in NZ composition.To reduce their impact, mechanical, chemical, or thermal treatments are usually employed.Desired specific properties for the adsorbents are high sorption capacity, enhanced ion exchange, high surface area, mechanical strength, thermal stability, etc. [7].
Simultaneously, another interesting option given by the ion exchange ability is the development of new modified materials.Zeolites supporting "antibiotic metal cations" (Ag + , Zn 2+ , Cu 2+ ) are already used for industrial applications, medical device coatings, air pollution prevention, and food packaging [11].Recent water disinfection methods have involved bactericidal clinoptilolites impregnated with metal ions of Hg, Sn, Pb, Bi, Cd, Cr, and Ti [12].
Among the broad spectrum of antibacterial agents, silver ions, metal silver, and nanoparticles are commonly considered alone or loaded on different carriers as bacterial growth inhibitors.Silver is regarded as non-toxic, biocompatible, resistant to sterilization, etc.The non-ionized form of the metal does not exhibit any bactericidal effect, but when exposed to an aqueous environment, a release of effective antimicrobial Ag + is evident.Nano-sized silver also represents a powerful bactericidal tool [11,13].Various physical, chemical, and biological routes for the synthesis of silver nanoparticles (AgNPs) have already been established.They include thermal decomposition, UV-irradiation, pyrolysis, sonochemical and microwave-assisted synthesis, direct chemical reduction, and others [14].
To reduce costs and to avoid the use of harmful substances in the above-mentioned applications, new "green" methods are being developed.Thus, non-toxic and efficient reducing and capping agents, and also stabilizers from natural origin are involved in nanoparticle synthesis.Different plant extract-supported investigations have been completed [15].Other successful routes utilize marine macroalgae (Ulva fasciata Delile), bacteria (Pseudomonas aeruginosa), and brown algae (Cystoseira crinita).All of these methods aimed to improve biological effects (antioxidant, anti-inflammatory, and/or antimicrobial) and to define new applications of the obtained nanosilver [16].
Despite all the advantages of nanoparticles described, there is evidence of harmful effects caused by these materials.Nanosilver has been shown to manifest toxicity to mouse embryonic fibroblasts [17], rat germline cells, rat hepatocytes, alveolar macrophages, and peripheral blood mononuclear cells [18].In animal experimental models, accumulation of AgNPs in the brain [19] and testes was observed [20].Studies with cell lines derived from different organs of the human body show liver, lung, reproductive, and other tissue-specific toxicity.Oxidative stress, inflammation response, DNA and molecular damage, growth inhibition, mitochondrial disruption, and cell morphology changes can be caused by the penetration of nanosilver in cells.A relationship between the surface area of nanosized Ag and the dose response has also been established [21].The final properties of nanoparticles are strongly dependent on their size, shape, and concentration [22].The available data indicate that the growing use of Ag species should be carefully monitored.
Chemical production of AgNPs performed on a matrix plays a key role in the prevention of aggregation and regulation of controlled particle release for a long time period.Thereby, the reduction in chemical and antimicrobial activity caused by the instability of nanosilver can be avoided [23,24].
Coatings 2024, 14, 681 3 of 14 The availability, low cost, chemical resistance, thermal stability, and non-toxicity of natural minerals make them preferred as carriers in the synthesis of composite materials.Kaolinite, diatomite, mesoporous silica, montmorillonite, sepiolite, and zeolite are mainly applicable in nanoparticle aggregation prevention.Their ability to assist the photocatalytic activity of nanosilver in the formation of reactive oxygen species (ROS) or photocatalytic degradation of bacteria is also established [25].Clinoptilolite is the most investigated among all natural zeolites as a silver deposition matrix and for bactericidal properties.The ability of Ag + to move out of the network into aqueous media preserves the effective concentration for a long time [11].Nevertheless, the toxicity effects induced by the exposure to Ag-zeolite nanocomposites are still under examination.
The aim of the present investigation was to impregnate natural Bulgarian clinoptiloliterich rock with nanosized silver, and to estimate the morphology and biological activity of the obtained composite material in comparison to zeolite-based food supplements available on the local pharmaceutical market.

Preparation of Ag-Zeolite Composite
The experimental procedures of the current project are schematically represented in Figure 1.
Coatings 2024, 14, x FOR PEER REVIEW 3 of 15 Thereby, the reduction in chemical and antimicrobial activity caused by the instability of nanosilver can be avoided [23,24].The availability, low cost, chemical resistance, thermal stability, and non-toxicity of natural minerals make them preferred as carriers in the synthesis of composite materials.Kaolinite, diatomite, mesoporous silica, montmorillonite, sepiolite, and zeolite are mainly applicable in nanoparticle aggregation prevention.Their ability to assist the photocatalytic activity of nanosilver in the formation of reactive oxygen species (ROS) or photocatalytic degradation of bacteria is also established [25].Clinoptilolite is the most investigated among all natural zeolites as a silver deposition matrix and for bactericidal properties.The ability of Ag + to move out of the network into aqueous media preserves the effective concentration for a long time [11].Nevertheless, the toxicity effects induced by the exposure to Ag-zeolite nanocomposites are still under examination.
The aim of the present investigation was to impregnate natural Bulgarian clinoptilolite-rich rock with nanosized silver, and to estimate the morphology and biological activity of the obtained composite material in comparison to zeolite-based food supplements available on the local pharmaceutical market.

Preparation of Ag-Zeolite Composite
The experimental procedures of the current project are schematically represented in Figure 1.In our research, clinoptilolite-rich rocks from the region of Bulgarian East Rhodopes were used.This natural material was first crushed, milled, and sieved.The pore size of the laboratory sieve used was 50 µm.The chemical composition and the theoretical cation exchange capacity of Rhodopean zeolite were already reported in [26].
To increase the ionic exchange efficiency, the mineral was activated [27].It was transformed in sodium form using 1 M NaOH (solid-to-liquid ratio 1:5) for 2 h at 80 ± 2 °C with stirring.The obtained samples were thoroughly washed to pH = 7 and dried in an oven for 2 h at 140 ± 2 °C.
Silver impregnation was performed by placing the material in contact with 0.1 M AgNO3 solution (solid-to-liquid ratio 1:20) for 5 h at room temperature with stirring.The production of AgNPs performed chemically on a matrix can be done without reducing agents [28].The obtained suspension was vacuum-pumped and washed multiple times In our research, clinoptilolite-rich rocks from the region of Bulgarian East Rhodopes were used.This natural material was first crushed, milled, and sieved.The pore size of the laboratory sieve used was 50 µm.The chemical composition and the theoretical cation exchange capacity of Rhodopean zeolite were already reported in [26].
To increase the ionic exchange efficiency, the mineral was activated [27].It was transformed in sodium form using 1 M NaOH (solid-to-liquid ratio 1:5) for 2 h at 80 ± 2 • C with stirring.The obtained samples were thoroughly washed to pH = 7 and dried in an oven for 2 h at 140 ± 2 • C.
Silver impregnation was performed by placing the material in contact with 0.1 M AgNO 3 solution (solid-to-liquid ratio 1:20) for 5 h at room temperature with stirring.The production of AgNPs performed chemically on a matrix can be done without reducing agents [28].The obtained suspension was vacuum-pumped and washed multiple times until a negative reaction for Ag + occurred.The as-prepared samples of Ag-loaded composite were dried in dark conditions overnight and then calcinated for 2 h at 200 ± 2 • C in a furnace.
In order to compare the biological properties of silver-loaded NZ, two different nutritional supplements of zeolite origin (marked as ZD and ZX in our study) were purchased from the local pharmacy.The main chemical composition of the supplements, designated by the distributor, consists of SiO 2 , NaOH, CaO, and KOH.The composition of the first product is also denoted by the presence of Gymnema sylvestre and Cinnamomum verum.

Characterization of Zeolite Samples
X-ray powder diffraction data were collected with PANalytical Empyrean (Malvern PANalytical, Almelo, The Netherlands).The patterns were obtained between 5 and 70 • 2θ using Cu/Ni radiation.
The structure and morphology of the Ag-loaded clinoptilolite were investigated using scanning electron microscopy (Prisma TM E SEM by Thermo Fisher Scientific, with an attachment for element analysis-Energy Dispersive X-ray (EDX) analyzer, Waltham, MA, USA).
The distribution, shape, and size of the deposited silver particles were studied by transmission electron microscopy (TEM) (Talos F200C Thermo Fisher Scientific, Waltham, MA, USA).Suspension from the material was added dropwise onto a formvar/carboncoated copper grid, and then the TEM observation of the samples was performed at different magnifications and an operating voltage of 200 kV.

Antioxidant Activity Evaluations of Zeolite Composites
The ABTS radical-scavenging activity method was used to analyze the antioxidant activity of zeolite samples [29].The method is based on the ability of the tested sample to scavenge 2,2 ′ -azino-bis(3-ethylbenzothiazoline-6-sulfonic acid) diammonium salt cation radical (ABTS + /).ABTS + was prepared by mixing 1/1 (v/v) 7 mM ABTS (Roche Diagnostics, Switzerland) stock solution and 2.45 mM potassium persulfate (Merck KGaA, Darmstadt, Germany).The resulting mixture was incubated for 4 to 16 h until the reaction was complete and the absorbance at 730 nm was verified (0.7 ± 0.05).For the spectrophotometric reading, 900 µL ABTS + solution was mixed with 100 µL test sample (10 mg/mL zeolite composite suspended in deionized water).After 10 min absorbance was read at 730 nm using a SpectraMax i3x spectrophotometer (Molecular Devices, San Jose, CA, USA).Ascorbic acid was used as a positive control and assayed in different concentrations (500, 250, 100, 50, 25 µg/mL).All samples were run in triplicates.
The percent antioxidant activity for each tested extract, reflecting its ability to scavenge the ABTS + cation radical, was calculated using the formula: ((Ac − At)/Ac) × 100, where Ac is the absorbance of the control sample containing only ABTS + radical cation and distilled water, and At is the absorbance of the test sample.
The MTT [3-(4,5-dimethylthiazol-2-yl)-2,4-diphenyl tetrazolium bromide] assay was used to assess cytotoxicity and antitumor potential of zeolite samples.For these experiments, cell suspensions with a concentration of 1 × 10 5 cells/mL were seeded on 96-well plates (TPP, Trasadingen, Switzerland) (100 µL/well) and cultured under standard condi-tions for 24 h.The cells were then treated with different concentrations of zeolite composites (30,60,125,250, and 500 µg/mL) for a test-period of 24 h.For this aim, 1 mg/mL zeolite suspensions were prepared in DMEM, and the appropriate amount of test sample was added to the corresponding wells on the culture plates.All samples were set to a final volume of 200 µL.Mitomycin C (MMC) was used as a positive control for cytotoxicity and was assayed at the same concentrations as the test samples.In addition, a control of untreated cells cultured for the same time period in standard growth medium was analyzed in all experiments.
At the end of the 24 h test period, MTT (Merck KGaA, Darmstadt, Germany) solution was added to the cell culture medium to a final concentration of 0.5 mg/mL.The plates were incubated for 2 h in the dark at 37 • C, 5% CO 2 , and high humidity.The culture medium was then removed and the cells were washed with DPBS.Accumulated formazan in MTTtreated cells was solubilized using 100 µL/well dimethyl sulfoxide (DMSO, Merck KGaA, Darmstadt, Germany).Then, the culture vessels were incubated at room temperature under continuous mild shaking for 15 min, after which absorbance was measured at 570 nm using a SpectraMax i3x spectrophotometer (Molecular Devices, San Jose, CA, USA).Percent inhibition of cell viability and metabolic activity was calculated based on data obtained for treated cells and control cells cultured under standard conditions without addition of test sample to the growth medium.The 50% inhibiting concentration (µg/mL) (IC 50 ) was calculated for the samples that induced higher than 50% inhibition of cellular metabolic activity and viability.

Statistics
Statistical significance of the data was determined by the Student's t-test using the StatView software (version 5.0) (SAS Institute Inc., Cary, NC, USA).Values of p lower than 0.05 were considered statistically significant.

Results and Discussion
All sets of mineral samples used in the current study and the corresponding characteristic features are presented in Table 1.2-sample NZ).The intensities and the positions of the reflections are in accordance with PDF 98-001-0145.The recorded patterns of commercial zeolite supplements (Figure 2-samples ZD and ZX) are indistinguishable from the other samples, so it can be assumed that they originate from the same type of zeolite.
The observations of pre-treated samples and natural zeolite show minor changes in the intensities and the positions of all peaks.This is probably caused by the presence of impurities or by the removal of some species during processing.The activation step has a negligibly weak impact on the crystal structure of the zeolite.Complete transformation of clinoptilolite occurs at NaOH concentration in the range 2.0-4.0M. Simultaneously, even at low concentrations, the specific surface area and pore volume can be affected [30].Inspection of the XRD data confirms that the main tuff composition is clinoptilolite with characteristic peaks observed at 9.75°, 11.12°, 22.35°, and 30.02° 2θ (Figure 2-sample NZ).The intensities and the positions of the reflections are in accordance with PDF 98-001-0145.The recorded patterns of commercial zeolite supplements (Figure 2-samples ZD and ZX) are indistinguishable from the other samples, so it can be assumed that they originate from the same type of zeolite.
The observations of pre-treated samples and natural zeolite show minor changes in the intensities and the positions of all peaks.This is probably caused by the presence of impurities or by the removal of some species during processing.The activation step has a negligibly weak impact on the crystal structure of the zeolite.Complete transformation of clinoptilolite occurs at NaOH concentration in the range 2.0-4.0M. Simultaneously, even at low concentrations, the specific surface area and pore volume can be affected [30].
The XRD data of Ag-NZ are practically identical to those of the NZ, which is in good agreement with previous study [31].The structure of the clinoptilolite is preserved despite the activation procedure involving sodium hydroxide and subsequent silver deposition.According to [27], the intensity variation of the peaks recorded at 9.75° and 11.12° 2θ are mainly associated with the change in channel content and the formation of extra-framework species.Treatments with silver, sodium carbonate, ammonium, and iron solutions are reported to affect these peaks.The same effect is also obvious in the XRD pattern of our silver-loaded zeolite.Such intensity decrement due to Ag impregnation is previously observed in [32].Therefore, the absence of strong changes in the X-ray intensity distribution indicates that nanoparticles do not fill zeolite channels and are mainly located on the surface.In the XRD spectra of the Ag-NZ sample, no characteristic silver signals were observed, which may be attributed to the limitations of the detection method employed.The formation of large particles and high silver loading are typically associated with the appearance of silver species diffraction maximums [27,33].Simultaneously, in [33][34][35], it is outlined that the absence of characteristic peaks does not negate Ag potential presence, as the analysis is constrained by the particle size and concentration.Furthermore, the color change observed from white or pale green (for NZ) to grey (for Ag-NZ) has also been identified as an indicator of metallic silver formation [36].In light of the aforementioned statements and the observations obtained via SEM and TEM (given below), it is probable that different silver species, including Ag cations, Ag clusters, and nanoparticles are present in the final Ag-NZ composite system.The XRD data of Ag-NZ are practically identical to those of the NZ, which is in good agreement with previous study [31].The structure of the clinoptilolite is preserved despite the activation procedure involving sodium hydroxide and subsequent silver deposition.According to [27], the intensity variation of the peaks recorded at 9.75 • and 11.12 • 2θ are mainly associated with the change in channel content and the formation of extraframework species.Treatments with silver, sodium carbonate, ammonium, and iron solutions are reported to affect these peaks.The same effect is also obvious in the XRD pattern of our silver-loaded zeolite.Such intensity decrement due to Ag impregnation is previously observed in [32].Therefore, the absence of strong changes in the X-ray intensity distribution indicates that nanoparticles do not fill zeolite channels and are mainly located on the surface.In the XRD spectra of the Ag-NZ sample, no characteristic silver signals were observed, which may be attributed to the limitations of the detection method employed.The formation of large particles and high silver loading are typically associated with the appearance of silver species diffraction maximums [27,33].Simultaneously, in [33][34][35], it is outlined that the absence of characteristic peaks does not negate Ag potential presence, as the analysis is constrained by the particle size and concentration.Furthermore, the color change observed from white or pale green (for NZ) to grey (for Ag-NZ) has also been identified as an indicator of metallic silver formation [36].In light of the aforementioned statements and the observations obtained via SEM and TEM (given below), it is probable that different silver species, including Ag cations, Ag clusters, and nanoparticles are present in the final Ag-NZ composite system.

SEM and TEM Observations
Scanning electron micrographs and spectra presented in Figure 3 reveal similar morphology and chemical composition of NZ, Na-NZ, and the commercially available zeolite supplements.
In our measurements, Na + ions were not detected in the natural zeolite (NZ).Expectedly, after the activation process of the natural zeolite, they emerged in all recorded spectra.A typical one is shown in Figure 3-sample Na-NZ.In contrast, Na + appears in the composition of commercial zeolites (ZD, ZX).
EDX data, collected via three map analyses of each sample, are shown in Table 2.The amount of the principal components of natural zeolite remains unchanged after the activation process (sample Na-NZ) and silver deposition (sample Ag-NZ).A decrease in Na + amount as a consequence of the silver loading indicates that sodium ions are predominantly exchanged.These ions are preferable for Ag + due to the comparable dimensions of their ionic radii, the location, and the zeolite channel dimensions [37].The silver deposits are measured at approximately 13 wt.%.In our measurements, Na + ions were not detected in the natural zeolite (NZ).Expectedly, after the activation process of the natural zeolite, they emerged in all recorded spectra.A typical one is shown in Figure 3-sample Na-NZ.In contrast, Na + appears in the composition of commercial zeolites (ZD, ZX).
EDX data, collected via three map analyses of each sample, are shown in Table 2.The amount of the principal components of natural zeolite remains unchanged after the activation process (sample Na-NZ) and silver deposition (sample Ag-NZ).A decrease in Na + amount as a consequence of the silver loading indicates that sodium ions are predominantly exchanged.These ions are preferable for Ag + due to the comparable dimensions of their ionic radii, the location, and the zeolite channel dimensions [37].The silver deposits are measured at approximately 13 wt.%.    4. The silver immobilization does not affect the size and form of the zeolitic particles.In the modified samples, nanosized Ag species and agglomerates with irregular shapes are uniformly dispersed on the rough zeolite surface.Such clusters, formed of layered particles, are typical for silver nanocomposites [8].These results are also confirmed by observations at higher magnifications (Figure 4).
The morphological characteristics of nanoparticles exert a major impact on biocompatibility, genotoxicity, and cytotoxicity [38].Although the formation of AgNPs is commonly attributed to the presence of reducing agent or thermal reduction at high temperatures, nanosized silver as a result of wet impregnation at low temperatures (close to 200 • C) has been previously reported [31].
Coatings 2024, 14, 681 8 of 14 SEM images of composite structures are presented in Figure 4.The silver immobilization does not affect the size and form of the zeolitic particles.In the modified samples, nanosized Ag species and agglomerates with irregular shapes are uniformly dispersed on the rough zeolite surface.Such clusters, formed of layered particles, are typical for silver nanocomposites [8].These results are also confirmed by observations at higher magnifications (Figure 4).The morphological characteristics of nanoparticles exert a major impact on biocompatibility, genotoxicity, and cytotoxicity [38].Although the formation of AgNPs is commonly attributed to the presence of reducing agent or thermal reduction at high temperatures, nanosized silver as a result of wet impregnation at low temperatures (close to 200 °C) has been previously reported [31].
To determine the distribution, shape, and size of the obtained Ag particles, transmission electron microscope observations at different magnifications were conducted.As can be seen from Figure 5, most of the individual nanoparticles are spherical.Furthermore, detailed observations of the obtained nanoparticles reveal an interplanar spacing of 0.235 To determine the distribution, shape, and size of the obtained Ag particles, transmission electron microscope observations at different magnifications were conducted.As can be seen from Figure 5, most of the individual nanoparticles are spherical.Furthermore, detailed observations of the obtained nanoparticles reveal an interplanar spacing of 0.235 nm (as measured by inverse Fourier transform) corresponding to the d-spacing of (111) plane of FCC AgNPs (PDF 00-04-0783).The potential mechanisms of their formation can be attributed to the reduction of Ag + in air conditions [31], or under the influence of light or heat [39].Additionally, an auto-reduction process of silver-exchanged zeolites has been previously described [40].
The silver particles obtained in this study appear in a variety of sizes as measured by imaging software (Image J 1.54i) and shown in Figure 5.The dominant nanoparticle dimensions are between 5 and 20 nm.The distribution histogram showed a mean size of around 19 nm (Figure 6).Larger species and aggregations are also present.The highest diameter of a single particle measured in our observations is around 60 nm.Taking into account the dimensions of the zeolite channels, described in detail in [31], it can be concluded that the obtained nanoparticles are located on the surface of the crystals.nm (as measured by inverse Fourier transform) corresponding to the d-spacing of (111) plane of FCC AgNPs (PDF 00-04-0783).The potential mechanisms of their formation can be attributed to the reduction of Ag + in air conditions [31], or under the influence of light or heat [39].Additionally, an auto-reduction process of silver-exchanged zeolites has been previously described [40].The silver particles obtained in this study appear in a variety of sizes as measured by imaging software (Image J 1.54i) and shown in Figure 5.The dominant nanoparticle dimensions are between 5 and 20 nm.The distribution histogram showed a mean size of around 19 nm (Figure 6).Larger species and aggregations are also present.The highest diameter of a single particle measured in our observations is around 60 nm.Taking into account the dimensions of the zeolite channels, described in detail in [31], it can be concluded that the obtained nanoparticles are located on the surface of the crystals.

Biological Activity Evaluations
Antioxidative properties have been reported for different zeolite samples [5].Therefore, our investigations were continued with an analysis of the ability of NZ, activated NZ, Ag-NZ, ZD, and ZX to scavenge the ABTS radical-a commonly used assay to determine antioxidant activity [29].The results presented on Figure 7 indicate a strong antiox-

Biological Activity Evaluations
Antioxidative properties have been reported for different zeolite samples [5].Therefore, our investigations were continued with an analysis of the ability of NZ, activated NZ, Ag-NZ, ZD, and ZX to scavenge the ABTS radical-a commonly used assay to determine antioxidant activity [29].The results presented on Figure 7 indicate a strong antioxidative potential for ZD, which could be attributed to the herbal component of this pharmaceutical zeolite product.The natural zeolite sample showed moderate radical scavenging activity that was affected by the modification process.Activated NZ had significantly higher antioxidant activity, while nanosilver incorporation resulted in a tendency for reduced ABTS radical scavenging capacity of the zeolite Ag-NZ.However, it is evident that the Ag-NZ sample possesses antioxidative properties that are superior to the ZX pharmaceutical product.

Biological Activity Evaluations
Antioxidative properties have been reported for different zeolite samples [5].Therefore, our investigations were continued with an analysis of the ability of NZ, activated NZ, Ag-NZ, ZD, and ZX to scavenge the ABTS radical-a commonly used assay to determine antioxidant activity [29].The results presented on Figure 7 indicate a strong antioxidative potential for ZD, which could be attributed to the herbal component of this pharmaceutical zeolite product.The natural zeolite sample showed moderate radical scavenging activity that was affected by the modification process.Activated NZ had significantly higher antioxidant activity, while nanosilver incorporation resulted in a tendency for reduced ABTS radical scavenging capacity of the zeolite Ag-NZ.However, it is evident that the Ag-NZ sample possesses antioxidative properties that are superior to the ZX pharmaceutical product.The potential toxicity of Ag-modified NZ was analyzed using normal (HFFC fibroblasts) and adenocarcinoma (lung A549, cervical HeLa, colon HT-29) human cells.Hence, we were also able to determine antitumor activity of the studied zeolites.The results from these experiments are displayed in Figure 8.Interestingly, we detected significant antitumor effects of NZ and the activated Na-NZ (Figure 8A,B).The metabolic activity and viability of all three tumor cell lines was negatively influenced by NZ and Na-NZ added The potential toxicity of Ag-modified NZ was analyzed using normal (HFFC fibroblasts) and adenocarcinoma (lung A549, cervical HeLa, colon HT-29) human cells.Hence, we were also able to determine antitumor activity of the studied zeolites.The results from these experiments are displayed in Figure 8.Interestingly, we detected significant antitumor effects of NZ and the activated Na-NZ (Figure 8A,B).The metabolic activity and viability of all three tumor cell lines was negatively influenced by NZ and Na-NZ added to the culture medium in concentrations higher than 100 µg/mL.On the other hand, NZ and Na-NZ did not affect HFFC fibroblasts even when the cells were treated with high test-sample concentrations (500 µg/mL).This result also indicates good biocompatibility properties of the natural and activated zeolite samples.Similar data were obtained for the ZX; only colon HT-29 adenocarcinoma cells were not affected by this sample (Figure 8E).The ZD commercial product showed significant inhibitory activity specifically against cervical cancer cells (Figure 8D; Table 3).Our data support previous findings for antitumor activity [5] and potential application as antitumor drug carriers of zeolite nanomaterials [41].However, Ag-loaded NZ demonstrated high cytotoxicity (Figure 8C; Table 3).Normal fibroblasts were affected to a lower extent compared to the tumor cell lines, but even at low concentrations (30 µg/mL), Ag-NZ induced more than 40% inhibition of cellular metabolic activity and viability.Undoubtedly, the deposition of nanosized silver species on the zeolite composite surface affects its biological activity and is associated with increased cytotoxicity of the sample.This could influence the therapeutic potential of the nanocomposite.The antitumor and other beneficial effects (i.e., antimicrobial properties) of the sample could be exerted together with significant negative impacts on normal cells of the organism which are in close proximity to the site of Ag-zeolite nanocomposite application.Our data do not correspond to the findings of Kaur et al., who showed that Ag-modified synthesized ZSM-3 zeolite had no toxicity against RAW264.7 mouse cells [42].Possible reasons for the different activity could be the applied methodology for zeolite modification with silver.An important aspect is the tested cell type and the different mode of response by mouse RAW264.7 macrophages in comparison with human fibroblasts.Thus, biocompatibility evaluations should be performed with a panel of cell lines with accent on human cells types in order to clarify potential biomedical applications.As evidenced by the present study, the toxicity of Ag-NZ nanocomposites against normal cells should be thoroughly analyzed prior to their biomedical application.To improve biocompatibility properties, different silver surface modifications could be per-formed [43].In general, toxicity of AgNPs depends on their size, shape, surface charge, and modification [44], which determine the mode of interaction with cells and different biomolecules.For instance, smaller Ag species are more toxic than Ag particles with larger size [45], possibly due to the mechanism by which they pass through the cell membrane and act in the cells.On the other hand, small-sized particles have larger specific surface area providing higher reactivity that could also be associated with stronger toxic potential.Hence, the toxic potential of silver particles could be affected by size, shape, and surface properties, and their careful modification represents the main route to reduce Ag-NZ nanocomposite cytotoxicity.

Conclusions
The attention to nanosized materials in all scientific fields is growing due to the diversity of their unique properties.The purpose of our research was to investigate the possible use of Bulgarian natural zeolite as unconventional support for the production of nanosized silver.The combination of the worldwide manifested health-boosting effects of NZ and the high capability of Ag against different bacterial strains offers a great perspective for the development of new treatment procedures, water disinfection methods, etc.At the same time, safety to humans must be guaranteed.The results reported in the present article include morphological and biological features of Ag-modified NZ.Ag-loading with particle dimensions larger than zeolite channels are evinced.The silver surface saturation affected the survival levels of adenocarcinoma and nontumor human cells.The inhibitory activity of Ag-NZ against normal fibroblasts was significantly lower compared to tumor cell lines which indicated antitumor properties.However, a negative influence on normal fibroblasts was also evident, demonstrating the need for thorough toxicity evaluation of Ag-modified NZ prior to its biomedical applications.NZ and activated NZ also demonstrated antitumor potential similar to commercial zeolite-based food supplements.Although their effects were milder compared to Ag-NZ, unmodified NZ and Na-NZ showed good biocompatibility and no harmful effects against normal human cells.

Figure 1 .
Figure 1.Preparation procedures of silver-modified natural zeolite samples.

Figure 1 .
Figure 1.Preparation procedures of silver-modified natural zeolite samples.

Figure 2 .
Figure 2. XRD diffraction patterns of the studied zeolites.

Figure 2 .
Figure 2. XRD diffraction patterns of the studied zeolites.

Coatings 2024 ,
14, x FOR PEER REVIEW 7 of 15 3.1.2.SEM and TEM Observations Scanning electron micrographs and spectra presented in Figure 3 reveal similar morphology and chemical composition of NZ, Na-NZ, and the commercially available zeolite supplements.

Figure 5 .
Figure 5. TEM images at different magnifications of nanosized Ag.

Figure 6 .
Figure 6.Particle size distribution of Ag-NZ composite.

Figure 6 .
Figure 6.Particle size distribution of Ag-NZ composite.

Table 1 .
Tested samples and abbreviations used.Inspection of the XRD data confirms that the main tuff composition is clinoptilolite with characteristic peaks observed at 9.75 • , 11.12 • , 22.35 • , and 30.02 • 2θ (Figure