Abstract
This work reports, the obtainment and stabilization of organic nanoparticles (γ-sitosterol) and Ag cations on the external area of Ca-clinoptilolite zeolite (Z17) are presented. The novelty of the present study lies in the formation and stabilization of nanoparticle organic molecules (γ-sitosterol) and inorganic cations (Ag) on the external area of Ca-clinoptilolite zeolite. The γ-sitosterol nanoparticles were obtained using Tournefortia hirsutissima and were later stabilized on the external area of Ca-clinoptilolite. The Ca-clinoptilolite-rich mineral as well as silver zeolitic minerals was characterized by using X-ray diffraction (XRD) and scanning electron microscopy (SEM); elementary composition of the zeolitic mineral was determined by microanalyses (EDS) and high-resolution adsorption (HRADS) using N2 adsorption as a probe molecule. The EDS results about the exchange with Ag indicate the following sequence: Z17Ag03 > Z17Ag-09 > Z17Ag-06 > Z17Ag-01. The XRD studies indicate that only in the sample Z17Ag-03 there is an average crystal size of nanoparticles formed of 45.387 nm as determined by Sherrer’s equation. SEM images of clinoptilolite showed the presence of crystals with irregular and poorly defined box shapes that are typical for this type of zeolites. The nanoporosity of natural zeolites was studied by N2 adsorption; external areas were estimated through the De Boer and BET equations. The specific surface area calculated by BET equation, , presents the following sequence: γ-SZ17 > Z17 > Z17Ag09 > Z17Ag03 > Z17Ag01 > Z17Ag06, while for the values of the external area obtained by the method, the sequence is : γ-Z17 > Z17 > Z17Ag03 > z17Ag09 > Z17Ag06 > Z17Ag01. For its part, the pore size distributions tell us that the sample Z17 exhibits a sharp multimodal distribution with pore size maxima happening at 1.262, 1.933, 3.824, and 4.282 nm. Z17Ag01 exhibits a bimodal distribution with pore size maxima occurring at 5.45 and 5.533 nm. The sample Z17Ag06 exhibits a sharp bimodal distribution with pore size maxima happening at 3.666 and 7.021 nm, and the sample Z17Ag09 exhibits a sharp trimodal distribution with pore size maxima happening at 1.853, 4.275 and 5.967 nm. Antibacterial activities of the exchanged samples with Ag on the external surface area were measured as a function of exchange level against Gram-positive bacteria (P. aureoginosa, K. pneumonie, and A. baumannii) and Gram-negative bacteria (E. coli, S. saprophyticcus, S. aureus, and E. feacalis); the selectivity sequence of the clinoptilolite and antibacterial activity results are presented. Z17Ag09 clinoptilolite seemed to be a promising antibacterial material.
1. Introduction
Diabetes mellitus (DM) is a disease known for its multiple complications, one of them being the diabetic foot [1]. There is a great interest in the development of materials with reliable and environmentally friendly microbicidal action to counteract the effects of this disease. Metals and their cations such as silver (Ag), zinc (Zn), copper (Cu), mercury (Hg), cadmium (Cd), chromium (Cr), and lead (Pb) can be used in a variety of ways as antimicrobial agents. Among these metals, Ag and Zn have long been used in medicines. The antibacterial activity of Ag is well known, it has an inhibitory effect on the growth of bacteria and retains its activity for a long time; also, there is no risk of developing bacterial resistance [2]. With the development of fabrication strategies for novel nanomaterials and nanostructures, Ag-loaded nanocomposites have been regarded as a very broad-spectrum antimicrobial agent, which can encourage their potential applications in various fields excelling in very specific areas such as biomedicine, food preservation, and water purification [3]. Among the diverse metal nanoparticles, it is silver nanoparticles (AgNPs) that have become very important and most frequently used [4]. The biocidal effect of AgNPs mainly depends on the type of bond that exists and the subsequent release of silver ions. To enhance their bactericidal properties, AgNPs require suitable substrates that are compatible with their bactericidal properties. The resulting AgNPs agents exhibit excellent antibacterial properties when chosen well. It is noted that their antimicrobial activities increase with decreasing particle size. The mechanism of action is explained by damage to the membrane of the bacterial cell, which causes leakage of the cytoplasm. In addition, two types of action mechanisms are considered, i. e., contact action and release of Ag+, which are responsible for the antimicrobial effect of AgNPs [5, 6]. In order to enhance their bactericidal effect, AgNps require suitable carriers that are compatible with their properties. Therefore, the design and fabrication of novel Ag-nanostructures suitable for such purposes are necessary to open up new possibilities with this type of cations and Ag nanoparticles and to obtain complex materials that act as an effective antibacterial agent. To date, various nanocomposite materials have been proposed, into which Ag is being incorporated; among them, nanocomposite Ag-graphene, nanocomposites based on Ag-oxide, Ag-macromolecules, and Ag-zeolites (AgZ) are known [7]. The nanoporous lattice of the zeolite allows metal cations to move freely, and this apparently is responsible for their activity toward microorganisms [8]. One of the best known and most frequently used zeolites to perform ion exchange treatments with bactericidal cations is clinoptilolite. Clinoptilolite (framework-type HEU) is a hydrophilic zeolite and has a three-dimensional three-channel system, filled by cations and water molecules (Figure 1) [9]. Natural zeolites are complex adsorption systems, since in addition to micropores, they also have mesopores and macropores [10]. In similar samples, A. Semra and U. Semra [11] studied the bactericidal activity of different types of exchangeable cations in a clinoptilolite. For his part, Dimova et al. [12] have studied exchanged Ag-clinoptilolite through Rietveld structural refinement and three independent Ag sites were located in channels of the clinoptilolite structure. At the same time, Concepcion-Rosabal et al. and Rodriguez-Fuentes et al. discussed the characteristics of Ag-clinoptilolites (natural and synthetic) containing Ag in various states. Many of the results obtained using modified zeolites have been applied and published in available patents [13, 14]. Also, Bogdanchikova et al. [15, 16] have presented a literature review regarding the reexamination of the structure and composition of Ag-clinoptilolite and its applications as antibacterial agent.
Pavelic et al. [17] analyzed the scientific literature on the health effects and safety of various clinoptilolite-based materials for medical use; they have proposed some complex, science-based hypotheses about possible biological mechanisms underlaying the observed effects on the health and body homeostasis. Colella [18, 19] reviewed the biomedical and veterinary applications of natural zeolites. Cerria et al. [20] carried out very relevant works on zeolites exchanged with Zn and their applications in antiacne therapies. On the other hand, another very important compound is γ-sitosterol, which is used to accelerate the healing process of the diabetic foot, as it reduces hyperglycemia, increases insulin secretion, and inhibits glucogenesis [21]. Many authors propose to develop a protein antidiabetic drug. Oral administration of γ-sitosterol in induced diabetic rats resulted in a significant decrease in blood glucose and glycosylated hemoglobin with a significant increase in plasma insulin level, body weight, and food intake [21, 22].
The purpose of this work is to obtain a group of materials based on γ-sitoesterol-clinoptilolite (γ-SZ17) and Ag-clinoptilolite (Z17AgX) with a number of emerging pores created during the ion exchange process. To do this, the γ-SZ17 will be mixed in alcoholic solutions with Z17Ag, previously prepared at 3 different concentrations; thereafter, specific ion exchange time protocols have been followed. The experimental techniques employed to characterize Z17AgX clinoptilolite susbtrates include X-ray diffraction (XRD), scanning electron microscopy (SEM), energy dispersive X-ray spectroscopy (EDS), and high-resolution adsorption (HRADS).
2. Experimental Section
2.1. Materials
γ-Sitosterol-clinoptilolite (γ-SZ17) was synthesized, purified, and characterized in a manner similar to that previously reported [23]. The Mexican zeolitic mineral clinoptilolite from San Gabriel Chilac (Z17), Puebla, Mexico, was used in this work. The mineral clinoptilolite was powdered and sieved. Exchanged Ag-clinoptilolite samples Z17AgX were prepared from Z17 precursor and exchanged either , , , or times with 0.01 N solutions of the corresponding cation salts (AgNO3) at 298 K for 6 h. ROH, AgNO3, and HCl of a commercial analytical grade were used without further purification. N2 and He ultrahigh-purity gases (>99.999%, INFRA Corp.) were employed for the technique characterization of natural and chemically treated samples.
2.2. Methodology
The exchanged zeolite (Z17AgX) was dried at 423 K for 12 h. Subsequently, a mixture of ethanol was prepared with γ-SZ17 and Z17AgX in a 4 : 1 ratio, under rigorous stirring, and 373 K heat treatment was given for 12 h in order to evaporate the solvent.
2.3. Experimental Techniques
2.3.1. X-Ray Diffraction (XRD)
Phase analysis by X-ray diffraction (Siemens D-500 X-ray Diffractometer) of samples was carried out at room temperature using CuKα as the radiation source at a scan speed of 0.5/min and a step scan of 0.02. The crystalline phase compositions were identified with reference to standard JCPDS cards available in the system software. The mean average crystalline size of exchanged Ag-clinoptilolite samples Z17AgX was calculated using Debye-Scherrer’s equation [24].
2.3.2. Fourier Transform Infrared Spectroscopy (FTIR)
To obtain the infrared spectra, the infrared spectroscopy method with attenuated total reflectance accessory (ATR) was used. This method involves placing a sample on top of a crystal with a high refractive index. An infrared beam from the instrument is passed into the accessory and up into the crystal. It is then reflected internally in the crystal and back towards the detector which is housed in the instrument. When the beam is reflected within the crystal, it penetrates into the sample by a few microns.
2.3.3. Scanning of Electron Microscopy (SEM)
Photomicrographs of samples under study were obtained with a Tescan Vega, model JSM-5300 scanning electron microscope. The samples were mounted on aluminium stubs and subsequently coated with Au/Pd using a sputter coater (Polarons SC 7610, Fision Instruments)
2.3.4. Energy Dispersive Spectroscopy (EDS)
The chemical analyses were performed through EDS.
2.3.5. N2 Adsorption
The N2 sorption isotherms were measured at the temperature of 77 K (boiling temperature at altitude conditions of the City of Puebla, Mexico) in an automatic volumetric sorption instrument Quantachrome Autosorb-AS1. Isotherms were determined in the range of relative pressures ; the saturation pressure, , was recorded continuously during the course of the adsorption measurements. Before starting the adsorption run, 60–80-mesh particles of each substrate were thoroughly degassed at 623 K for 20 h at a pressure lower than 10−6 mbar which was provided by the turbopump of the instrument. Textural results (surface areas and pore volumes) were obtained from pertinent analyses of the N2 isotherms at 77 K; the following approaches were chosen to calculate the desired textural properties: (i) the BET equation, (ii) the Langmuir equation, (iii) the -plots for determining the external surface area, and (iv) the Gursvich rule to assess the total pore volume. The pore size distributions of the samples under study were evaluated from data of N2 desorption isotherm by means of the BJH equation [25].
2.4. Antimicrobial Activity
As previously described [26], the Z17AgX antimicrobial activity was studied by performing an agar well diffusion method. This method was done against different bacterias, such as Gram-negative bacteria (E. coli ATCC 25922, S. saprophyticcus ATCC savage, S. aureus ATCC 29331, S. epidermidis ATCC 12228, and E. feacalis ATCC 29211) and Gram-positive bacteria (P. aureoginosa ATCC 27853, K. pneumonie ATCC 700603, and A. baumannii). Each sample was arranged by preparing, sterilizing, and solidifying each Mueller-Hinton agar plates. For every single bacterial strain, the samples were uniformly swabbed by sterile cotton swabs on the individual petri plates. By carrying out gel puncture, it was possible to prepare different wells of determined size. Into these wells, distinct concentrations of Z17AgX were filled up. The choosen concentrations were 0.010, 0.03, 0.06, and 0.09). Following this process, the plates were incubated for 24 h at 37°C. Subsequently, each plate was examined for a zone of incubation in millimeter.
3. Results and Discussion
3.1. XRD
The X-ray diffraction patterns of the samples obtained, Z17, Z17AgX, and the γ-sitoesterol-clinoptilolite (γ-SZ17) are presented in Figure 2. From this figure, different peaks are observed for each particular material. In the diffraction pattern corresponding to Z17Ag-03, signals located at °, 35.38°, 35.50°, 42.15°, and 42.20° stood out. These peaks can be attributable to the Ag incorporation into the zeolite framework [27, 28]. The average mean size of nanoparticles (AgNPs) was determined using Debye-Sherrer’s equation. The size was estimated to be approximately 45.387 nm from the breadth of the reflection principal. As the size is considerably low, the AgNPs must be polycrystalline, and the single crystal shows higher size range [29, 30]. A signal in ° is attributable to extract with γ-sitoesterol for sample γ-SZ17. This mark suggesting the bioorganic phase crystallization occurs on the surface of the silver nanoparticles. Similar results were found in silver nanoparticles synthesized by using edible mushroom extract [31, 32]. The signals corresponding to sample Z17 are located at the following °, 11.06°, 13.03°, 14.82°, 16.86°, 17.2°, 19.04°, 22.35°, 25.04°, 28.09°, 31.71°, and 32.67° (Figure 2). From this figure, it is observed the clinoptilolite signals prevail. The signals existing in the majority fundamentally correspond to the clinoptilolite zeolite (JCPDS 3 0427). Followed by small amounts of impurities such as montomorillonite (JCPDS 29-1498), quartz (2θ~27°, JCPDS 3-0427), and in some trace cases of mica, orthoclase, cristobalite, and magnesium calcite. These results were obtained by processing the diffraction patterns and the atomic coordinates of the phases present in each sample studied, using the High Score Plus 3.0e computer program, Table 1.
(a)
(b)
(c)
(d)
3.2. EDS
From the sequence of the zeolites studied, it is observed that in all the zeolites studied, they present a Si/Al molar . Based on this aspect, it can be assumed that this type of zeolites is the product of sediments from saline lakes and seas of these characteristics. It has been shown that, among zeolites of this type with a high amount of Si and with a molar ratio greater than 4, K-clinoptilolite zeolite predominates. This is due to the dominant rocks that are formed in deep water areas. However, Na-clinoptilolite and Ca-clinoptilolite zeolites can form under a wide group of parameters and appear in hydrothermal active systems of volcanic rocks, as well as in fractures and in internal cavities of these rocks. On the other hand, impurities associated with Fe3+, which resides in the tetrahedral sites of this structure, can be associated with hematite impurities [33].
From the results reported in Table 2, the following sequences can be established: γ-SZ17: Ca > Na > Fe > K > Mg > Ti; Z17: Ca > Na > K > Fe > Mg > Ti > S; Z17Ag01: Ca > Na > Ag > K > Fe > Mg > Ti; Z17Ag03: Ag > Ca > K > Fe > Na > Mg > Ti; Z17Ag06: Ca > Ag > Na > K > Fe > Mg > Ti; and Z17Ag09: Ca > Ag > Na > K > Fe > Mg >Ti. It is observed from these results that for the samples exchanged with Ag, the following sequence is established: Z17Ag03>z1709>Z17Ag06>Z17Ag01 and Si/Al ratio es: Z17Ag09>Z17>γ-SZ17>Z17Ag06>Z17Ag01>Z17Ag03.
3.3. SEM
SEM images of clinoptilolite crystals at different approximations are shown in Figure 3. This figure shows the presence of crystals with irregular and poorly defined box shapes typical for this type of zeolites. Also, from this figure, the presence of a group of lamellar pores can be observed. On the other hand, at very low concentrations, the presence of Ag on Z17 (Ca-clinoptilolite) is not detected by SEM. However, this presence can be quantified through XRD and EDS (results presented in Table 2 and Figure 2). This effect has already been previously reported [34].
3.4. Adsorption of N2
In Figure 4, the adsorption isotherms of N2 at 77 K in the studied samples are observed (volume adsorbed, , vs. relative pressure, ). This figure shows the formation of type II-IV isotherms within the IUPAC classification [25], for samples that are subjected to chemical treatments with AgNO3. However, there is the formation of a stepped type VI isotherm for sample Z1709. The formation of a type H3 hysteresis cycle associated with the presence of pores in the form of sheets and/or lamellars is observed. The experimental points corresponding to the adsorption branch in areas of -0.25 were evaluated by the Langmuir and BET equations. In the area of -0.8, the experimental points were evaluated with the De Boer equation in order to evaluate the external area, . At , the total pore volume, , was evaluated by means of Gursvitch’s rule. On the other hand, the experimental points of desorption branches were considered for the evaluation of the pore size distribution using the BJH equation. The results of these estimates are reported in Table 3.
(a)
(b)
(c)
From this table, it is observed that there is a decrease in the specific surface estimated by BET equation in the zeolite with chemical modification. This behavior of the chemically modified zeolites is associated with the existence of a blockage of the pores or channels. This blockage is caused by the organic γ-sitosterol (γ-SNPs) and inorganic silver nanoparticles (AgNPs), since their diameters and/or sizes are superior to the channels of the zeolite structure. On the other hand, it is observed that the values of the external area are considerable. The values of the external area are very important since it is in that area of the structure where the nanoparticles of γ-SNPs and AgNPs will be formed and stabilized. Figure 5 shows the curves calculated by the BJH method. In this figure, Z17 exhibits a sharp multimodal distribution with pore size maxima happening at 1.262, 1.933, 3.824, and 4.282 nm. For its part, Z17Ag01 exhibits a bimodal distribution with pore size maxima occurring at 5.45 and 5.533 nm. For Z17Ag03, representative pore size results could not be obtained by the BJH method because of the flat aspect of the resultant curve. In contrast, Z17Ag06 exhibits a sharp bimodal distribution with pore size maxima happening at 3.666 and 7.021 nm. Finally, the sample Z17Ag09 exhibits a sharp trimodal distribution with pore size maxima happening at 1.853, 4.275, and 5.967 nm, and these values are in the last column of Table 3.
3.5. Infrared Spectroscopy FTIR
In Figure 6, the IR spectra of the natural zeolite Z17; the zeolite treated with γ-sitosterol and γ-SZ17, and of the zeolites exchanged with Ag and Z17AgX are shown. Common bands are observed for all the samples studied. The wideband around 3496 cm-1 and 1630 cm-1can be assigned to water molecules physisorbed in zeolites and for an internal tetrahedral asymmetric stretching O-H bending. The narrow band around 1450 cm-1 determined in the Ag-exchanged clinoptilolites is attributed to the presence of nitrates, which are located in the external area of the lattice corresponding to the exchanged clinoptilolite zeolites. The strong band 1050 cm-1 assigned to external tetrahedral linkage asymmetric stretching depends on Ag concentration on the external surface area of exchanged zeolites. The weak bands located at 870 and 795 cm-1 corresponds to external tetrahedral linkage symmetric stretching. These bands correspond to the Al-O-Si bonds present in the exchanged clinoptilolite zeolites [35].
3.6. Antibacterial Effect
The bacterecidal effect of the prepared samples (Z17AgX) were measured on Gram-negative and Gram-positive strains, Table 4, using disk diffussion method. The results obtained were compared among the samples prepared with Ag at different concentrations because both the Z17 precursor sample and the one γ-SZ17 gave an adverse result to this effect. The disk difusion method shows the magnitude of the susceptibility of the pathogenic microorganisms (Figures 7 and 8). The mean of three replicates of the diameter (in millimeters) of inhibition zones containing Z17AgX suspension is presented in Table 4. Among the Gram-positive strains, the samples Z17AgX show a bactericidal effect; however, the one with the greatest bactericidal effect is sample Z17Ag09. The results of this behavior are presented in Table 4. From this table, the following sequence is established for Gram-positive strains present: A. baumannii (17 mm) > P. aureoginosa (15 mm) > K. pneumonie (11 mm). On the other hand, the results corresponding to the Gram-negative strains present the following sequence: S. aureus (15 mm) > S. saprophyticcus (13 mm) > E. feacalis (12 mm) = E. coli (12 mm). The results shown for the Z17AgX indicate that they are effective as antibacterial agents. Ag particles damage the cell walls and membranes of pathogenic bacteria, inhibiting enzymatic activity and bacterial replication. Ag cations are microbicidal at low concentrations and do not have major side effects in humans [2].
(a)
(b)
(c)
(a)
(b)
(c)
(d)
4. Conclusions
Clinoptilolite-rich minerals from San Juan Gabril Chilac, Puebla, Mexico, and those of modified forms were characterized by using XRD, SEM, EDS, and nitrogen adsorption analysis techniques. Based on the X-ray analysis, the purity percentage of the crystalline phases constituting the original zeolitehas been established. For Z17: Ca-clinoptilolite (~80) > quartz, calcite (~12.6) > mordenite (~8.8). Additionally, X-ray analysis of the zeolite samples has shown that the modification of clinoptilolites with a solution of AgNO3 and γ-sitosterol does not lead to significant structural changes. The organic (γ-sitosterol) and inorganic (Ag+1) nanoparticles form nanoparticles in the external area of the clinoptilolite zeolite. Their degree of dispersion and stabilization is a function of the external area developed, as well as the zeolite degree of mesoporosity. From the results shown above, it can be concluded that AgZ1709 exchanged zeolite is the most promising material as a microbicidal agent, which in combination with γ-SZ17 would be recommended for its application in diabetic foot treatments.
Data Availability
No data were used.
Conflicts of Interest
The authors declare no conflict of interest.
Authors’ Contributions
All authors discussed and agreed upon the idea and made scientific contributions: writing and original draft preparation, MAH; experiment designing, MA and GIH; experiment performing, RP; data analysis, ER; writing, review, and editing, MAS and VP. All authors have read and agreed to the published version of the manuscript.
Acknowledgments
This work was supported by the VIEP and the Academic Body “Investigación en zeolitas” (CA-95) (PROMEP-SEP).