Anti-inflammatory, anti-bacterial, and cytotoxic activity of fibrous clays

Colloids and Surfaces B: Biointerfaces 129 (2015) 1–6

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Anti-inflammatory, anti-bacterial, and cytotoxic activity of fibrous clays Javiera Cervini-Silva a,b,c,∗ , Antonio- Nieto-Camacho d , María Teresa Ramírez-Apan d , Virginia Gómez-Vidales e , Eduardo Palacios f , Ascención Montoya f , Elba Ronquillo de Jesús a a

Departamento de Procesos y Tecnología, Universidad Autónoma Metropolitana, Unidad Cuajimalpa, México, Mexico Earth Sciences Division, Lawrence Berkeley National Laboratory, Berkeley, CA, USA c NASA Astrobiology Institute, Mountain View, CA, USA d Laboratorio de Pruebas Biológicas, Instituto de Química, Universidad Nacional Autónoma de México, Ciudad Universitaria, México, Mexico e Laboratorio de Resonancia Paramagnética Electrónica, Instituto de Química, Universidad Nacional Autónoma de México, Ciudad Universitaria, México, Mexico f Direccion de Investigación y Posgrado, Instituto Mexicano del Petróleo, Mexico b

a r t i c l e

i n f o

Article history: Received 12 October 2014 Received in revised form 5 March 2015 Accepted 6 March 2015 Available online 16 March 2015 Keywords: Early anti-inflammatory response Frequency of inversion sites Silanol groups

a b s t r a c t Produced worldwide at 1.2 m tons per year, fibrous clays are used in the production of pet litter, animal feed stuff to roof parcels, construction and rheological additives, and other applications needing to replace long-fiber length asbestos. To the authors’ knowledge, however, information on the beneficial effects of fibrous clays on health remains scarce. This paper reports on the anti-inflammatory, antibacterial, and cytotoxic activity by sepiolite (Vallecas, Spain) and palygorskite (Torrejon El Rubio, Spain). The anti-inflammatory activity was determined using the 12-O-tetradecanoylphorbol-13-acetate (TPA) and myeloperoxidase (MPO) methods. Histological cuts were obtained for quantifying leukocytes found in the epidermis. Palygorkite and sepiolite caused edema inhibition and migration of neutrophils ca. 68.64 and 45.54%, and 80 and 65%, respectively. Fibrous clays yielded high rates of infiltration, explained by cleavage of polysomes and exposure of silanol groups. Also, fibrous clays showed high inhibition of myeloperoxidase contents shortly after exposure, but decreased sharply afterwards. In contrast, tubular clays caused an increasing inhibition of myeloperoxidase with time. Thus, clay structure restricted the kinetics and mechanism of myeloperoxidase inhibition. Fibrous clays were screened in vitro against human cancer cell lines. Cytotoxicity was determined using the protein-binding dye sulforhodamine B (SRB). Exposing cancer human cells to sepiolite or palygorskite showed growth inhibition varying with cell line. This study shows that fibrous clays served as an effective anti-inflammatory, limited by chemical transfer and cellular-level signals responding exclusively to an early exposure to clay, and cell viability decreasing significantly only after exposure to high concentrations of sepiolite. © 2015 Elsevier B.V. All rights reserved.

1. Introduction Produced worldwide at 1.2 m tons per year, fibrous clays are used for the production of pet litter, animal feed stuff to roof parcels, construction and rheological additives, and other applications needing to replace long-fiber length asbestos. Yet, information on

∗ Corresponding author at: Departamento de Procesos y Tecnología, Universidad Autónoma Metropolitana, Unidad Cuajimalpa, Av Vasco de Quiroga 4871, Cuajimalpa de Morelos, Santa Fe Cuajimalpa, Mexico, D.F. C.P. 05348, Mexico. Tel.: +52 55 26 36 38 00x3827. E-mail address: [email protected] (J. Cervini-Silva). http://dx.doi.org/10.1016/j.colsurfb.2015.03.019 0927-7765/© 2015 Elsevier B.V. All rights reserved.

the beneficial effects of fibrous clays on health applications remains scarce. Most commonly, fibrous clays have been used as support for bioactive substances such as povidone-iodine (PVP-I)/sepiolite or Cu2+ or Ag+ /sepiolite formulations for killing bacteria (e.g., E. coli, S. aureus; [1,2]) or adsorbing odors [2]). Sepiolite and palygorskite contain ribbons of 2:1 phyllosilicates, also referred as “polysomes” [3], which extend parallel to the caxis of the fiber and present periodical inversions of apical oxygens in the tetrahedral sheet. Such inversions take place every six and four atoms of Si (three and two tetrahedral chains) for sepiolite and palygorskite, respectively. Stemming from the aforementioned structure is the porosity resulting from (i) the inter-fibers/bundles ˚ and (ii) the spaces, with a pore-effective diameter (pd ) >15 A;

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˚ Tunnels have repeated inversions of the silicate layer (pd < 15 A). been recognized as an important parameter determining the transfer to and from solutes. Sepiolite owns a higher number of adsorption sites than palygorskite (Table 1A; [4]), along with larger tunnels, allowing for greater diffusion rates [5–8]. This paper reported on the anti-inflammatory, antibacterial and cytotoxic activity by two-well characterized fibrous clays (Tables 1A and 2A), one sepiolite (Vallecas, Spain) and one palygorskite (Torrejón El Rubio, Spain [9]). Obtained results were compared to those reported for tubular halloysite [10]. This study addressed the response of two inflammatory cells, macrophages and neutrophils, presenting multiple pathways of interaction with pathogens. Reportedly, macrophages interact with pathogens via phagocytosis or by destroying them after secretion of enzymes and/or toxic biochemicals [11]. In addition, macrophages can recruit other types of inflammatory and immune effector cells, such as neutrophils [11] that, after the skin, constitute the primary protection for organisms against pathogens. Actually, various human diseases are characterized by the massive migration of neutrophils during acute inflammatory responses in the colon, stomach, lung, urinary tract and skin [12].

2. Materials and methods 2.1. Clay specimens Sepiolite (Vallecas, Spain) and palygorskite (Torrejon El Rubio, Spain), and halloysite (CLA site in Maralinja Tjarutja, Barton locality, South Australia) subject of study were kindly provided as a gift by Dras. Emilia García-Romero (Departamento de Cristalografía y Mineralogía, Facultad de Ciencias Geológicas, Universidad Complutense, Madrid, Spain) and Mercedes Suárez (Departamento de Geología, Universidad de Salamanca, Salamanca, Spain), and Dr. John Keeling (Resource Evaluation and Planning Section, Geological Survey, Minerals and Energy Resources, Primary Industries and Resources, South Adelaide, Australia). Sepiolite and palygorskite [5,10] and tubular halloysite [13] were 100% and 95% pure. Fibres ranged from <1 ␮m to centimeters in length. 2.2. Surface characterization The structural and compositional properties of sepiolite and palygorskite were listed in Tables 1A and 2A as reported elsewhere [3,4,9]. Electrophoretic mobility determinations were conducted using a Zetasizer Nano ZS ZEN3600 (Malvern Instruments, UK). The suspensions were adjusted to 20% in weight and pH 7.4 using a phosphate buffer. Clay specimens were prepared for microscopic analysis by placing a subsample on an Al plate covered with carbon tape. To induce conductivity, the sample was covered with a 10–15 nm film of Au. Scanning Electron Microscopy was conducted using a Nova-200 Nanolab dual beam scanning electron microscope with an amplification range from 30× through 250,000, with a resolution of up to 1.1 nm, coupled with an X-ray Si (Li) ultrathin window-energy dispersive spectrometer. 2.3. Biological tests Fibrous clays (sepiolite or palygorskite) were screened in vitro against human cancer cell lines (Table 3A). Cell lines were supplied by the National Cancer Institute (USA). The cytotoxicity was determined using the protein-binding dye sulforhodamine B (SRB) in microculture assay to measure cell growth according to the

National Cancer Institute [14] (Supplementary information section) [35]. Adult male Wistar rats (200–250 g) approved by the Animal Care and Use Committee (NOM-062-ZOO-1999) were provided by the Instituto de Fisiología Celular, UNAM. They were maintained at 25 ◦ C on a 12/12 h light–dark cycle with free access to food and water. Determinations of the mouse ear edema using 12-Otetradecanoylphorbol-13-acetate (TPA) as inflammatory agent were conducted according to methods described elsewhere [15] (Supplementary information section). Estimations of myeloperoxidase (MPO) enzymatic activity are used in inflammation models as an enzymatic marker specific for migration and cellular infiltration. This is particularly the case for neutrophils (also known as polymorphonuclear leukocytes, or PMNs) bearing high contents of myeloperoxidase [16] (Supplementary information section). Upon application of TPA, infiltration and pro-inflammatory effects in neutrophils are temporarily shuffled [17]. Thus, the activity of sepiolite and palygorskite with incubation time (t, h) was assessed. Histological cuts were obtained as described elsewhere [18]. Briefly, ear specimens were fixed in solution of 10% formalin. Ears were dehydrated, embedded in paraffin, and sectioned. Five␮m sections were stained with hematoxylin–eosin. Infiltration of leucocytes was evaluated in selected areas (10× objective). The quantification of leukocytes found in the epidermis was conducted counting cells per field. For each experimental condition (basal, TPA, sepiolite, and palygorskite; see Graphical abstract), four distinct histological sections were selected and analyzed in four different fields. 3. Results and discussion 3.1. Surface composition High-resolution micrographs confirmed that fibers flocking into groups composed both sepiolite and palygorskite (Fig. 1). Fibers were cylindrical and elongated, with an increased curvature toward the ends. Reported variability for length and thickness [19] did not allow for drawing conclusive values for either average length or porosity. As evidenced by EDS analysis sepiolite and palygorskite were composed by Si, Mg, and Al; and Si, Mg, Al, and Fe, respectively (Table 2A; [9]). Both clays contained small amounts of TiO2 , NaO, K2 O, and CaO (≤1%). 3.2. Cell viability Murine macrophages (ca. 10 × 104 cells mL−1 ; cell line no. RAW 264.7, ATTC) were exposed to clay for 24 h (Table 3A). Aminoguanidine was used as standard for inhibiting nitric oxides. Solid concentrations were 10, 31, 100, and 310 ␮g mL−1 . Cell viability before and after adding 1 ␮g mL−1 LPS in sepiolite and palygorskite suspensions was: 85.8 and 100%, 79.2 and 94.7%, 75.7 and 86.5%, and 60.7 and 64.1%; and 100 and 98.3%, 98.1 and 95%, 96.5 and 96.8%, and 94.1 and 93.2%, correspondingly. Exposing cancer human cells to sepiolite or palygorskite showed growth inhibition varying with cell line (Table 3A). Nevertheless, both clays caused a similar extent of cytotoxicity in U251. In contrast, exposing PC-3 cells to fibrous clays showed comparable mortality rates. K-562, HCT-15, and MCF-7 cells showed high growth inhibition if exposed to sepiolite but not to palygorskite. Most notably, SKLU-1 cells showed mortality rates seven times higher in the presence of palygorskite. In all, cell growth inhibition increased after exposure to fibrous clays compared to reference compounds, FeSO4 or AAPH, or other inorganic compounds (e.g., Pb as PbO2 or Pb(II); [20]), and was highly-specific.

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Fig. 1. HRSEM of (a) sepiolite (Vallecas, Spain) and (b) palygorskite (Torrejón El Rubio, Spain).

Experiments were conducted from 5 to 10 times (Figs. 2 and 3 and Table 4A). In all experiments the dose corresponded to 1 mg ear−1 . Data corresponded to average values ± standard error. Obtained results were analyzed using the Dunnett’s test. Values

with p ≤ 0.5(* ) and p ≤ 0.01 (** ) were considered to differ statistically from the TPA group. Edema inhibition (EI) and MPO content inhibition (CI) are expressed in percentage (%). Infiltration shortly after exposure depended on the structure of the fibrous clay, with palygorkite inducing early edema inhibition (Fig. 2). As determined from accompanying histological cuts at 4 and 24 h (Figs. 4 and 5), sepiolite and palygorskite caused decreases on the number of infiltrated cells per field (Fig. 6). At 4 h, edema inhibition by palygorskite (SBET = 105 m2 g−1 ) surpassed that by sepiolite (SBET = 308 m2 g−1 ); however, at 24 h edema inhibition values compared (Fig. 2). EI (%) was defined as EI (%) = EI (%, 4 h)–EI (%, 24 h) (Table 4A). EI (%) positive values showed a higher activity at t = 4 h than at 24 h, while negative ones did otherwise. For palygorskite EI was +13.6, denoting a pronounced decrease in activity with time. For sepiolite the opposite appeared to be true, and EI was −9.45, showing an increased activity with time. Clearly, the mechanism of edema inhibition by sepiolite and palygorskite differed, and was not accounted for by the number of surface sites available.

Fig. 2. Anti-inflammatory properties of sepiolite and palygorskite, and tubular halloysite as determined by the ear-edema test after 4 and 24 h. Data values corresponded to mean ± S.D (5 ≤ N ≤ 10). Data analysis was conducted using ANOVA and Dunnett’s tests. Significance levels (p ≤ 0.05 (*); p ≤ 0.01 (** )) were compared against TPA control group.

Fig. 3. The effect of TPA, sepiolite, palygorskite, tubular halloysite, and indomethacin on MPO production after 4 or 24 h. Data values corresponded to mean ± S.D (5 ≤ N ≤ 10). Data analysis was conducted using ANOVA and Dunnett’s tests. Significance levels (p ≤ 0.05 (*); p ≤ 0.01 (** )) were compared against TPA control group.

As evidenced by experiments conducted with murine macrophages (RAW264.7) stimulated with 1 ␮g mL−1 lipopolysaccharides from E. coli, sepiolite and palygorskite did not inhibit the production of nitric oxide, regardless of concentration (10–310 ␮g mL−1 ; not shown). On the other hand, a different scenario held true for cell viability. The effect of sepiolite on cell viability depended on concentration, 85.8% at 10 ␮g mL−1 and 60.7% at 310 ␮g mL−1 . In contrast, cell viability was reduced only by 20% after adding 310 ␮g mL−1 of palygorskite. Overall, fibrous clays showed a more important effect on cell viability compared to tubular halloysite (75.5%; [10,21,22]), soluble Ca (94.2%), TiO2 (90.8%), SHCa-1 (89.1%), MgO (86.4%), soluble Mg (86.3%), or CaO (83%; [23]). 3.3. Infiltration and migration of neutrophils vs fibrous clays

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Fig. 4. Representative histological sections from mice ears exposed to TPA (4 h) and stained with hematoxylin-eosin treated with 1 mg ear−1 sepiolite or palygorskite for 4 or 24 h (20× Scale = 100 ␮m). Arrows indicate the infiltrated leukocytes.

Contrary to infiltration, myeloperoxidase content inhibition showed a different trend in reactivity (Figs. 2 and 3). At 4 h, palygorskite inhibited the migration of neutrophils to a higher extent compared to sepiolite, with corresponding myeloperoxidase content inhibition values ca. 80 and 65%. At 24 h, however, myeloperoxidase content inhibition values were 9 and 3%, respectively. Fibrous vs tubular clays. We compared the anti-inflammatory activity by fibrous [sepiolite (SBET = 308 m2 g−1 ) and palygorskite (SBET = 105 m2 g−1 )] vs tubular clays [halloysite (SBET = 74.6 m2 g−1 )] (Figs. 2 and 3). The role of clays on infiltration depended on

structure and exposure time. At 4 h, edema inhibition decreased according to palygorskite > sepiolite > halloysite, whereas at 24 h it followed the order halloysite > sepiolite ∼ palygorskite. Clays owing a high frequency in the periodic inversions of the apical oxygen of the tetrahedral sheet and increased length (i.e., palygorskite) favored infiltration shortly after exposure. Electrophoretic mobility (em) determinations for sepiolite, palygorskite, and halloysite were −1.44 ± 0.155, −1.24 ± 0.114, and −4.055 ± 0.18 ␮m cm V−1 s−1 , respectively, with corresponding conductivity values 17.46 ± 0.53, 17.16 ± 0.53, and 17.1 ± 0.8 mS cm−1 . Our data showed a slight negative relation

Fig. 5. Representative histological sections from mice ears exposed to TPA (24 h) and stained with hematoxylin-eosin treated with 1 mg ear−1 sepiolite or palygorskite for 4 or 24 h (20× Scale = 100 ␮m). Arrows indicate the infiltrated leukocytes.

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4. Conclusions The outcome of this study showed that fibrous clays acted as anti-inflammatory. Palygorkite and sepiolite caused edema inhibition and migration of neutrophils ca. 68.64 and 45.54%, and 80 and 65%, respectively. Unlike tubular clays (e.g., halloysite), fibrous clays inhibited edema shortly after exposure, favored by the cleavage of polysomes and subsequent exposure of silanol groups. Fibrous clays did not affect cell viability, except when exposed to high concentrations of sepiolite. Acknowledgements

Fig. 6. Number of infiltrated cells per field after exposure to TPA, sepiolite, palygorskite, and indomethacin. Data values corresponded to mean ± S.D (5 ≤ N ≤ 10 animals). Data analysis was conducted using ANOVA and Dunnett’s tests. Significance levels (p ≤ 0.05 (*); p ≤ 0.01 (** ) were compared against TPA control group.

between early inhibition in the contents of myeloperoxidase and surface sites available. Fibrous clays showed high inhibition of myeloperoxidase contents shortly after exposure, but decreased sharply afterwards. In contrast, tubular clays caused an increasing inhibition of myeloperoxidase with time (Fig. 3). Thus, clay structure restricted the kinetics and mechanism of myeloperoxidase inhibition. The mechanism(s) of interaction between cells and silicon dioxide particles [24–26] and fibrous clays differed. Silicon dioxide has been proposed as an effective insecticide. For instance, exposing anthropods to silicon dioxide caused death, explained by lipid adsorption from their cuticle, and subsequent desiccation [24,27]. The insecticidal activity of silicon oxide particles has been related to surface properties [24–26] such as adsorption capacity [28], chemical composition [25], particle size [29], and specific surface [30]. Evidence presented herein, using the TPA methods, aimed to unveil acute inflammatory responses, showed otherwise. Infiltration by fibrous or tubular clays (Figs. 2 and 3) appeared to be limited by chemical transfer. Palygorskite owns a higher frequency in the periodic inversion of the apical oxygen of the tetrahedral sheet (Table 1A). In turn, a higher extent of edema inhibition by palygorskite vs sepiolite observed shortly after exposure was attributed to an early dissolution following a more abundant cleavage of polysomes, leaving a higher number of silanol groups exposed [31]. On the other hand, a 29 NMR report evidenced increases in the electron density at (SiO4 )+ tetrahedrons at external surfaces, strongly suggesting their role as adsorption and reaction sites [32]. Current work aims to unveil how clay hydration relates to the inhibition of edema and migration of neutrophils. Neutron diffraction analysis of palygorskite confirmed a smaller tunnel structure relative to sepiolite [33]. Reportedly, structural H2 O in palygorskite, comparable to O10 in sepiolite, formed hydrogen bonds with two zeolitic H2 O molecules, at 2.55 and 2.85 A˚ [33,34]. Unlike in sepiolite, zeolitic H2 O molecules in palygorskite appeared to bind more strongly to the structural framework. The lack of such additional H-bonds explained albeit partially water loss after exposing sepiolite to vacuum or gentle heating [34].

The authors thank María del Rocío Galindo Ortega and Carolina López Pacheco (UAM-Cuajimalpa), and Daniela Rodríguez ˜ (Unidad de Histología, Instituto de Fisiología Celular, Montano UNAM) for technical assistance; and Drs. Georgios D. Chyssikos (Theoretical and Physical Chemistry Institute, National Hellenic Research Foundation, Athens, 11635, Greece), Vassilis Gionis (Institute of Materials Science, N.C.S.R. “Demokritos”, 15310, Aghia Paraskevi, Attiki, Greece); and Stephan Kaufhold (BGR Bundensansaltfür Geowissenschaften und Rohstoffe, Hannover, Germany) for providing insightful comments during the preparation of this manuscript. This project was supported in part by Universidad Autónoma Metropolitana Unidad Cuajimalpa (Grant No. 33678). Appendix A. Supplementary data Supplementary data associated with this article can be found, in the online version, at http://dx.doi.org/10.1016/j. colsurfb.2015.03.019. References [1] S. Ohta, K. Nagai, M. Maruyama, H. Inoue, Y. Katsuyama, O. Atobe, H. Ogata, S. Yamato, E. Tachikawa, H. Zenda, Biol. Pharm. Bull. 22 (1999) 42. [2] N.C. Yalin, B. Benli, Proceedings of NANOCon2013, October 16–18, Brno, Czech Rep, 2013. [3] M.P.S. Krekeler, S. Guggenheim, Appl. Clay Sci. 39 (2008) 98. [4] J.M. Serratosa, Proceedings of the 6th International Clay Conference, Oxford UK, Elsevier, Amsterdam, 1979, pp. 99–109. [5] M. Sánchez del Río, P. Martinetto, C. Reyes-Valerio, E. Dooryhée, M. Suárez, Archaeometry 48 (2006) 115. [6] C. Dejoie, P. Martinetto, E. Dooryhée, R. Brown, S. Blanc, P. Bordat, P. Strobel, P. Odier, F. Porcher, M. Sánchez del Río, E.V. Eslande, P. Walker, M. Anne, MRS Proc. 1319 (2010). [7] S. Ovarlez, F. Giulieri, A.-M. Chaze, F. Delamare, J. Raya, J. Hirschinger, Chem. Eur. J. 15 (2009) 11326. [8] R. Giustetto, O. Wayhyudl, Microporous Mesoporous Mater. 142 (2011) 221. [9] M. García-Romero, M. Suárez, Clays Clay Miner. 58 (2010) 1. [10] H. Cornejo-Garrido, A. Nieto-Camacho, V. Gómez-Vidales, M.T. Ramírez-Apan, P. del Angel-Vicente, J.A. Montoya, M. Domínguez-López, D. Kibanova, J. Cervini-Silva, Appl. Clay Sci. 57 (2012) 10. [11] G.W. Hunninghake, J.E. Gadek, H.M. Fales, R.G. Crystal, J. Clin. Investig. 66 (1980) 473. [12] A.C. Chin, C.A. Parkos, Ann. Rev. Pathol. Mech. Dis. 2 (2007) 11. [13] P. Pasbakhsh, G.J. Churchman, J.L. Keeling, Appl. Clay Sci. 74 (2013) 47. [14] A. Monks, D. Scudiero, P. Skehan, R. Shoemaker, K. Paull, D. Vistica, C. Hose, J. Lanfley, P. Cronise, A. Vaigro-Wolff, M. Gra-Goodrich, H. Campbell, J. Mayo, M. Boyd, J. Natl. Cancer Inst. 83 (1991) 757. [15] M. Merlos, L.A. Gomez, M. Giral, M.L. Vericat, J. Garcia-Rafarell, J. Forn, Br. J. Pharmacol. 104 (1991) 990. [16] W.F. Bradley, Am. Mineral. 25 (1940) 405. [17] L.M. De Young, J.B. Kheifets, S.J. Bailaron, J.M. Young, Agents Actions 26 (1989) 335. [18] A. Mescher, Junqueira’s Basic Histology: Text and Atlas, Thirteenth Edition, McGraw Hill Lange, New York, 2013, 520 pp. [19] M. Suárez, E. García-Romero, Appl. Clay Sci. 67–68 (2012) 72. [20] H. Cornejo-Garrido, D. Kibanova, A. Nieto-Camacho, G. Guzmán, M.T. Ramírez˜ J. Cervini-Silva, Chemosphere 84 Apán, P. Fernández-Lomelín, M.L. Garduno, (2011) 1329. [21] J. Cervini-Silva, A. Nieto-Camacho, E. Palacios, A. Montoya, V. Gómez-Vidales, M.T. Ramírez-Apan, Colloids Surf. B 111 (2013) 651. [22] J. Cervini-Silva, A. Nieto-Camacho, M.T. Ramírez-Apan, The anti-inflammatory properties of different naturally-occurring halloysites, in: P. Pasbakhsh, J.

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