Halloysite nanotubes (HNTs; Al2Si2O5(OH)4⋅nH2O) with a hollow tubular morphology (named by mineralogist M. Berthier in 1826) are aluminosilicates of the kaolin group. Recently, experiments have been conducted to create green nanocomposites with antimicrobial activity by incorporating several antimicrobial compounds into HNTs (Massaro et al., Reference Massaro, Lazzara, Noto and Riela2020). Significant advantages of incorporating HNTs into nanocomposites include improved thermal and mechanical stability of antimicrobial nanocomposites as well as extending their lifespan and release of antimicrobial chemicals (Saadat et al., Reference Saadat, Pandey, Tharmavaram, Braganza and Rowtani2020).
One of the most pressing health issues in the world is microbial infections, which are on the rise as a result of the planet's rapidly increasing human population. These infections have been treated using a number of conventional chemical-based antibacterial agents. The emergence of resistant pathogens, which can result in illnesses that are challenging or impossible to treat, is a key drawback of using such medications. Additionally, the hazardous by-products from making these chemical compounds can cause health issues and pollution of the environment (Haiwei et al., Reference Haiwei, Hanjun and Xiaogang2016).
Today, studies are being carried out to develop new nanocomposites with antimicrobial properties enhanced with support materials such as HNTs and montmorillonite (MMT; Ding et al., Reference Ding, Wang, Chen, Liu and Zhang2014; Duan et al., Reference Duan, Zhao, Liu and Zhang2015; Shu et al., Reference Shu, Yi, Ouyyang and Yang2017; Srivastava & Dwivedi, Reference Srivastava and Dwivedi2018). Zhao et al. (Reference Zhao, Wan, Fu, Meng, Ou and Zhong2019) have shown that halloysite has very few side effects on the development of Caenorhabditis elegans and that it is only effective on the growth of C. elegans offspring. It has been reported that clay nanotubes can be used as a drug to alleviate colitis, as a nanocarrier targeting the inflamed colon by electrostatic adsorption or as an interfacial stabilizer for emulsions (Feng et al., Reference Feng, Luo, Li, Fan, Wang, He and Liu2023a).
Numerous products and formulations of quaternary ammonium compounds (QACs) are commercially available and widely used in industry. QACs are cationic surfactants (positively charged surfactants) that affect microbial cell walls and membranes. These positively charged compounds show antimicrobial effects by easily binding to the negatively charged surfaces of many microorganisms (Chauret, Reference Chauret, Batt and Tortorello2014).
HNTs and organoclays are used in the cosmetics (Kamble et al., Reference Kamble, Ghag, Gaikawad and Panda2012) and food industries (Deshmukh et al., Reference Deshmukh, Kumar and Gaikwad2023; Kumar et al., Reference Kumar, Deshmukh, Hakim and Gaikwad2024), as well as in the chemical industry as templates or nanoreactors for biocatalysts (Shchukin et al., Reference Shchukin, Price, Sukhorukov and Lvov2005) and in the pharmaceutical industry for encapsulation and controlled drug release (Meirelles et al., Reference Meirelles and Raffin2017; Silva et al., Reference Silva, Lima, Honório, Trigueiro, Osajima, Lobo and Filho2019). For this reason, scientific studies aimed at expanding and improving these usages by increasing the antimicrobial properties of clays and their derivatives have attracted much recent attention. Liu et al. (Reference Liu, Huang, Huang, Vithanage, Lazzara and Rajapaksha2023) reported that biocompatible HNT–polymer nanocomposites can be used in drug delivery and are promising for other biomedical applications such as tissue engineering, cancer diagnosis/treatment, wound dressing and as biosensors. Therefore, they suggested that the biological effects and toxicology of HNTs should be comprehensively investigated (Liu et al., Reference Liu, Huang, Huang, Vithanage, Lazzara and Rajapaksha2023). Industrial products based on HNTs are developing rapidly due to their good mechanical, thermal and biological properties.
Clay and clay-containing materials can be used in the development of many new industrial products due to their unique mechanical, thermal and biological properties. Because of this, our team has previously conducted physicochemical and mechanical studies of Cloisite 10A (C10A), Cloisite 15A (C15A) and halloysite (Çankaya & Sahın, Reference Çankaya and Sahın2019; Çankaya et al., Reference Çankaya, Azarkan and Özek2021a, Reference Çankaya, Vurgun and Yalçın2021b). In addition, in our studies, it was determined that the anticancer effect of C10A (half-maximal inhibitory concentration (IC50): 3.5 μg mL–1) against HeLa cancer cells was greater than that of halloysite (Çankaya et al., Reference Çankaya, Vurgun and Yalçın2021b). Determining the antibacterial, antifungal and antioxidant properties of C10A, C15A and halloysite clays was the goal of the current investigation.
Experimental
C10A clay (particle size <15 μm; Nanoclay 1-135, modified with dimethyl, benzyl, hydrogenated tallow, quaternary ammonium chloride), C15A organoclay (particle size <20 μm; Nanoclay 1-140, modified with dimethyl, dihydrogenated tallow, quaternary ammonium chloride) and halloysite clay (particle size <10 μm) were purchased from Esan-Eczacıbaşı (Çankaya & Sahın, Reference Çankaya and Sahın2019; Çankaya et al., Reference Çankaya, Azarkan and Özek2021a). X-ray fluorescence (XRF) chemical analyses of the C10A and C15A organoclays and the HNT clay provided by Esan-Eczacıbaşı are given in Table 1, and their X-ray diffraction (XRD) traces are given in Fig. 1. Although the interlayer distance value of MMT clays is ~15 Å, it can reach 38–40 Å for C10A and C15A when organic modification is performed. Scanning electron microscopy (SEM) and transmission electron microscopy (TEM) images of the HNT clay provided by Esan-Eczacıbaşı are given in Figs 2 & 3. For HNT, the Brunauer–Emmett–Teller (BET) specific surface area is 128 m2 g–1, maximum moisture is 5.43%, average nanotube length is 1.2 μm, average inner diameter is 20 nm and average outer diameter is 40 nm. The distance between layers is 7 Å for dihydrate and 10 Å for hydrate.
XRF analysis of the C10A, C15A and HNT clays.
XRD traces of (a) C10A, (b) C15A and (c) halloysite clays.
SEM images of HNT.
TEM images of HNT.
The bacteria (S. aureus ATCC 25923, C. glabrata ATCC 90030, B. subtilis ATCC 6051, E. faecalis ATCC 551289, E. coli ATCC 25922, K. pneumoniae NRLLB4420, P. aeruginosa ATCC 27853, L. monocytogenes ATCC 1911) and fungi (Trichoderma longibrachiatum MK910052.1, Fusarium solani KT876631.1, Mucor plumbeus MH870585, Trichoderma viride MH398583.1, Neocosmospora falciformis MH2444 10.1, Penicillium glabrum MK910045.1) used in the study were obtained from Usak University Vocational School of Health Services.
Antibacterial and antifungal activity test
The antibacterial effect was assessed using the disc diffusion method. In this study, a qualitative approach was preferred over a quantitative minimum inhibitory concentration (MIC) approach as the clay samples were suspended in a broth medium. Briefly, after 0.1 g of C10A, C15A and halloysite clays were dissolved in 1 mL of dimethyl sulfoxide (DMSO), 20 μL of each sample was absorbed into empty antibiotic discs under aseptic conditions. Test strains were incubated for 24 h. The bacterial suspension at 0.5 McFarland turbidity was spread on the Mueller–Hinton agar (MHA) surface with a sterile swab. Paper discs (6 mm) impregnated with halloysite and organoclay were placed on the agar surface. The test solution preparation agent – DMSO – served as a negative control. Petri plates were incubated at 37°C for 24 h. The zone of inhibition around the disc is usually taken as the diameter, which corresponds to the sharpest edge of the region where growth is inhibited from edge to edge and can be measured in millimetres using a calliper. The antimicrobial effect against each test strain was evaluated by comparing C10A, C15A and halloysite inhibition zones with the control inhibition zone (Biemer, Reference Biemer1973; Isik & Özdemir-Kocak, Reference Isik and Özdemir-Kocak2009).
To assess antifungal efficacy, the well diffusion method was used. Briefly, 0.1 g of C10A, C15A and halloysite were used in the antifungal activity experiments after they were dissolved in 1 mL of deionized water. Wells of 6 mm were opened in the middle of Petri dishes containing nutrient agar medium, and 20 μL of a sample was inoculated into each well. Then, 20 μL of deionized water was inoculated into the wells as a control to determine whether there was an effect stemming from the sample and solvent. Spot planting of fungal isolates, which were previously defined at the species level via molecular analysis, was carried out at equal distances from the well. After 7 days of incubation in the dark at 27 ± 2°C, the distances from the fungal colony to the well were measured. The percentage of inhibition was calculated by comparing these distances with that from the control group (Korcan et al., Reference Korcan, Kayhan, Ünal and Jahan2021).
Determination of antioxidant activity using DPPH (free radical-scavenging effect)
The antioxidant activity of the samples was assessed using the 2,2-diphenyl-1-pixyl-hydrazil (DPPH) test, as presented previously (Villano et al., Reference Villano, Fernandez-Pachon, Moya, Troncoso and Garcıa-Parrilla2007), with some modifications. In brief, 150 μL of each sample was mixed with 5850 μL of DPPH solution. This was then incubated at 27°C for 1 h in the dark. Absorbance was measured at 515 nm with a Shimadzu brand UV-1800 spectrophotometer. The positive control was gallic acid. Equation 1 was used to assess each sample's capacity to scavenge the DPPH radical:
where A control is the absorbance of the control without the sample and A sample is the absorbance of the mixture containing the sample.
Results and discussion
Antibacterial and antifungal activity test results
Table 2 provides the findings obtained regarding the antibacterial effect using the disc diffusion method. C10A appeared to have an antimicrobial effect on all tested microorganisms. The microorganisms for which C10A showed the greatest antimicrobial effect were L. monocytogenes (30 mm), E. faecalis and S. aureus (27 mm). Among the test materials, only C10A was found to have an antimicrobial effect on C. glabrata (the only yeast species tested), at 18 mm. Compared with the control group, C10A showed greater antimicrobial activity against P. aeruginosa. C15A showed an antimicrobial effect only on S. aureus (6 mm) and K. pneumoniae (15 mm). Halloysite, on the other hand, showed antimicrobial activity against P. aeruginosa (10 mm), E. faecalis (9 mm) and S. aureus (7 mm; Figs 4 & 5 & Table 2).
Antimicrobial effect results determined using the disc diffusion method.
C = chloramphenicol; E = erythromycin; P = penicillin; TE = tetracycline; VA = vancomycin.
Bacterial inhibition zones formed around clays on agar. (a) K. pneumoniae, (b) B. subtilis, (c) L. monocytogenes, (d) P. aeruginosa, (e) E. coli, (f) S. aureus, (g) E. faecalis, (h) C. glabrata.
Comparison of the antibacterial properties of the studied clays.
There have been many studies conducted on the antibacterial properties of natural and chemically modified synthetic clay minerals (Tong et al., Reference Tong, Yulong, Peng and Zirong2005; Haydel et al., Reference Haydel, Remenih and Williams2008; Williams et al., Reference Williams, Haydel, Giese and Eberl2008). Cerium-loaded halloysite prevents DNA damage, apoptosis, keratosis and abnormal hyperplasia caused by ultraviolet light, thus preventing malignant transformation. It has also been reported that cerium-loaded halloysite prevents biofilm formation by causing oxidative stress to bacterial membranes (Feng et al., Reference Feng, Zhang, Chen, Zhou and Liu2023b). In addition, gold-loaded halloysite–chitin composite hydrogels have been reported to exhibit high antibacterial activity (Zhao et al., Reference Zhao, Feng, Zhou, Tan and Liu2023). Today, French clays rich in smectite are used in the treatment of necrotizing fasciitis ulcers, the causative agent of which is Mycobacterium ulcerans. Although the mineralogy, crystal size and major element chemistry of the clays used previously are similar, it has been found that they have different antimicrobial properties. Therefore, it is assumed that the reasons for these differences in antibacterial mechanisms are not physical but result from chemical transfers or reactions. Williams & Haydel (Reference Williams and Haydel2010) demonstrated in their cation-exchange experiments that exchangeable cations play a role in the antibacterial process. They reported that the pH and oxidation state buffered by clay mineral surfaces play key roles in controlling the chemistry of the solutions and the redox-related reactions occurring in the bacterial cell wall. We believe that the antimicrobial effect in this study is due to the alteration and disruption of the cytoplasmic membrane permeability of bacteria. However, as stated previously in the literature (Williams et al., Reference Williams, Haydel, Giese and Eberl2008), considering that the clay species used retain metal ions from the environment, the mechanism of antimicrobial action of the clay changes depending on the metal retained.
As a result of the antifungal activity experiments, it was determined that the test substances showed antifungal activity (Table 3).
Inhibitory effect of the studied clays on fungal mycelium growth.
ND = not detected.
Except for F. solani, C10A was found to be the type of clay with the most effective antifungal activity against the other fungi tested. C10A inhibited the mycelial growth of T. viride by 85.32% and that of T. longibrachiatum by 85.00%. C15A was the least inhibiting clay type (14.29%) of N. falciformis mycelial growth. Halloysite did not have an inhibitory effect on P. glabrum mycelial growth. We determined that C15A inhibited the mycelial growth of both T. viride and M. plumbeus by 66.67% (Figs 6 & 7 & Table 3).
Inhibition of fungal mycelial growth by the studied clays.
Comparison of the antifungal properties of the studied clays.
Some researchers have found that natural clay minerals do not show antibacterial action. There are a few reports of clay materials having an antimicrobial effect when antimicrobial agents are added. Yue et al. (Reference Yue, Yang, Li, Zhang, Qin and Han2019) reported that pure halloysite was ineffective against Aspergillus niger, Penicillium citrinum and T. viride. This suggests that halloysites show biocidal activity only when impregnated with oligodynamic metals (Yue et al., Reference Yue, Yang, Li, Zhang, Qin and Han2019). The clays we used in this study contained metals such as Al, Si, Na, Fe, Ti, Ca, Mg and K, and it can be thought that these metals caused the observed antifungal activity. For example, MMTs carrying cetylpyridinium or modified MMTs with copper and silver ions have bacteriostatic and bactericidal activities (Carcelli et al., Reference Carcelli, Mazza, Pelizzi, Pelizzi and Zani1995; Uchida, Reference Uchida1995; Hu & Xia, Reference Hu and Xia2006; Jo et al., Reference Jo, Rim, Park, Yuk and Lee2007; Wang et al., Reference Wang, Li, Ren, Zhao, Li and Leng2007). Meng et al. (Reference Meng, Zhou, Zhang and Shen2009) prepared a new polydimethyloxane/MMT–chlorhexidine acetate (PDMS/OMMT) nanocomposite film by adding chlorhexidine acetate solution to MMT. The addition of OMMT increased the thermal stability of the composite. In addition, when the antimicrobial activity of PDMS/OMMT was evaluated, it was found that it showed strong activity against E. coli and S. aureus (Meng et al., Reference Meng, Zhou, Zhang and Shen2009). In another study, it was determined that a multifunctional MMT-based composite material containing 5-fluorocytosine, antibacterial metal copper ions and quaternized chitosan showed in vitro antibacterial activity against E. coli, S. aureus and Candida albicans (Sun et al., Reference Sun, Xi, Wu, Song, Huang and Chu2019).
In this study, it was determined that the antifungal properties of the organoclays were higher than that of halloysite. The most effective antifungal clay type was determined to be C10A.
Jennings et al. (Reference Jennings, Minbiole and Wuest2015) reported that the differences in the antimicrobial activity of QACs are due to the differences in their molecular structure. For example, phenyl (6-phenylnaphthalene) substitution into QACs has been found to significantly increase their antibacterial activity (Zhang et al., Reference Zhang, Giurleo, Parhi, Kaul, Pilch and LaVoie2013; Jennings et al., Reference Jennings, Minbiole and Wuest2015). The results of this study support the notion that adding a phenyl group to a clay increases its antimicrobial effect, similar to the results of Jennings et al. (Reference Jennings, Minbiole and Wuest2015). The ionic interaction of QACs with the bacterial cell membrane disrupts it, allowing proteins, nucleic acids and internal low-molecular-weight materials to flow out, which causes rapid cell lysis (Maillard, Reference Maillard2002; Chapman, Reference Chapman2003; Gilbert & Moore, Reference Gilbert and Moore2005). The C10A organoclay has a phenyl ring, unlike the C15A organoclay. The QAC side-chains pierce the bacterial intramembrane region as a result of the electrostatic interaction between the positively charged QAC and the negatively charged bacterial cell membrane. This causes cytoplasmic material to leak from the cell and/or the cell to break down (Denyer, Reference Denyer1995).
Although they display notably greater activity against Gram-positive bacteria, QACs can be regarded as broad-spectrum antibiotics due to their ability to affect the bacterial cell membrane. Gram-positive bacteria possess a single phospholipid cell membrane and a thickened peptidoglycan cell wall, whereas Gram-negative bacteria are enclosed by two cell membranes and a relatively thin coating of peptidoglycan. This second membrane means that QACs and other membrane-targeting antiseptics often display an eightfold reduction in activity against Gram-negative pathogens. QACs have also been reported to be potent antifungal agents (Vieira & Carmona-Ribeiro, Reference Vieira and Carmona-Ribeiro2006).
Antioxidant activity test results
When the results of the DPPH experiments were examined, it was determined that the saturated solutions of all test substances presented antioxidant activity (Fig. 8 & Table 4).
Changes of colour after reduction by the studied clays.
DPPH radical-scavenging activities of the studied clays.
Ying (Reference Ying2006) reported that most QACs contain chloride or bromide anions. These structures allow QACs to easily adsorb negatively charged substances (Ying, Reference Ying2006). Additionally, the inner structure of the HNT is positively charged with Al–OH charges, whereas the outer surface is negatively charged because it contains Si–O–Si bonds (Massaro et al., Reference Massaro, Lazzara, Noto and Riela2020). This structure causes chemically positively and negatively charged ions to be attached inside or outside the halloysite, respectively. In particular, the positively charged inner surface provides a large area for negatively charged free radicals to attach. However, this adhesion can be increased or reversed by changing the ambient pH and adding different radicals/antioxidants into the system. Therefore, composites with enhanced antimicrobial and antioxidant properties can be formed by adding antioxidants such as ascorbic acid (Baschieri et al., Reference Baschieri, Amorati, Benelli, Mazzocchetti, D'Angelo and Valgimigli2019), fatty acids (Lee et al., Reference Lee, Kim and Park2018) or plant extracts (Nastasi et al., Reference Nastasi, Kontogiorgos, Daygon and Fitzgerald2022) to the structure of the clays (Zhong et al., Reference Zhong, Lin, Liu, Jia, Luo and Jia2017).
Conclusions
The modified C15A used in this study has two long-chain alkyl groups attached, whereas the modified C10A has a single long-chain alkyl group and a benzyl group attached. It can be said that the differences in the observed antibacterial effect are due to these structures of the molecules. The Cl found in organoclays C10A and C15A and in the quaternary ammonium cations in its structure can be separated from the organoclay by microbial enzymes. Although this causes an increase in antimicrobial activity, the free radicals replacing Cl could contribute to increased antioxidant activity. In addition, it is known that the alkyl chains and benzyl groups in the structures of QACs have antimicrobial properties. Therefore, more than one mechanism might play a role in the observed antimicrobial effect. Halloysite exhibits antioxidant activity by being positively charged inside the nanotube, interacting with negatively charged ions physicochemically and, as a result, trapping the negatively charged ions inside the tube. Studies conducted by adding various antioxidants to the clay structure found that doing so can increase the antioxidant/antimicrobial activity, helping to more fully explain the underlying antioxidant mechanism. According to these results, organoclays as green source materials could have some applications in industry. For example, antimicrobial polymers are used as additives and coatings in food processing, biomedical devices, filters and antifouling paints. Creating composites by adding the C10A organoclay, which we found to have antimicrobial and antioxidant properties, to plastic materials used in the health, food and biomedical fields can make these materials antiseptic. According to our literature review, this study will provide a significant scientific contribution regarding the antimicrobial and antioxidant properties of C10A, C15A and halloysite. When C10A, C15A and halloysite clays are combined with materials with known antimicrobial and antioxidant properties, these properties can be increased. Future studies should focus on the development of new and effective antimicrobial/antioxidant clay composites.
Conflicts of interest
The authors declare none.
