vermiculite and release study

    Preparation of antibacterial chlorhexidine/vermiculite and release study Magda Saml´ıkov´a, Sylva Holeˇsov´a, Marianna Hund´akov´a, Erich Pazdziora, ˇ Luboˇ s Jankoviˇc, Marta Val´asˇkov´a PII: DOI: Reference:

S0301-7516(16)30270-8 doi:10.1016/j.minpro.2016.12.002 MINPRO 2995

To appear in:

International Journal of Mineral Processing

Received date: Revised date: Accepted date:

30 September 2014 12 November 2016 6 December 2016

Please cite this article as: Saml´ıkov´a, Magda, Holeˇsov´ a, Sylva, Hund´ akov´a, Mariˇ anna, Pazdziora, Erich, Jankoviˇc, Luboˇ s, Val´ aˇskov´a, Marta, Preparation of antibacterial chlorhexidine/vermiculite and release study, International Journal of Mineral Processing (2016), doi:10.1016/j.minpro.2016.12.002

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ACCEPTED MANUSCRIPT Preparation of antibacterial chlorhexidine/vermiculite and release study Magda Samlíkováa,b,*, Sylva Holešováa,b, Marianna Hundákováa,b, Erich Pazdziorac, Ľuboš

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Jankovičd, Marta Valáškováa,b

Nanotechnology Centre, VŠB – Technical University of Ostrava, 17.listopadu 15/2172, 708 33

Ostrava – Poruba, Czech Republic

IT4Innovations Centre of Excellence, VŠB – Technical University of Ostrava, 17.listopadu

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15/2172,708 33 Ostrava – Poruba, Czech Republic

Institute of Public Health Ostrava, Centre of Clinical Laboratories, Partyzánské náměstí 7, 702 00

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Ostrava, Czech Republic

Institute of Inorganic Chemistry, Slovak Academy of Sciences, Dúbravska cesta 9, 845 36 Bratislava,

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Slovak Republic

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Corresponding author: Magda Samlíková, E-mail address: [email protected]

Tel: +420 597 329 357, Fax: +420 597 321 640

ACCEPTED MANUSCRIPT Abstract The antibacterial chlorhexidine/vermiculite (CA/Ver) was successfully prepared through the

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intercalation process and the stability of CA on the vermiculite matrix and was investigated

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by stirring in aqueous solutions under the influence of different pH and temperature. The

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content of CA was determined by total organic carbon (TOC) analysis before and after stability tests. The structure of all samples was characterized by X-ray powder diffraction (XRD) and Fourier–transform infrared spectroscopy (FTIR). The antibacterial activity of

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prepared CA/Ver samples was evaluated by finding a minimum inhibitory concentration

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(MIC) against Enterococcus faecalis, Staphylococcus aureus, Escherichia coli and Pseudomonas aeruginosa. The content of chlorhexidine ranged from 209 to 231.6 mg of CA in 1 g of the whole sample after the intercalation process. After stability study, only a slight

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outflow of CA from the Ver matrix (< 5%) was noted. The antibacterial test confirmed that the outflow of CA was negligible.

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After 30 min of exposition the MIC of organovermiculite samples before and after stability test were the same for Staphylococcus aureus and Escherichia coli with value 3.33 (%; w/v)

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and the MIC decreased to 0.014 (%; w/v) with longer time of exposition (120 h). A small difference was observed at Enterococcus faecalis where MIC was 10 (%; w/v) after 30 min of exposition for the sample after stability test in neutral pH. However, after 24h of treatment the MIC value decreased to 0.014 (%; w/v). And finally, bacterial strain Pseudomonas aeruginosa showed a great resistance against antibacterial organovermiculite samples and MIC did not decreased under 10 (%; w/v) even after 5 days of exposition.

Keywords: vermiculite, chlorhexidine diacetate, antibacterial activity, stability test

ACCEPTED MANUSCRIPT 1

Introduction Clay minerals with a layered structure show some structural characteristics that are useful

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for biological, pharmaceutical, cosmetic and medical applications (Choy et al., 2007).

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Modified clay minerals are often employed as carriers of drug, proteins, and in general of

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active products (Fong et al., 2010; Nien et al., 2011; Perioli et al., 2011). The possibility to incorporate some antibacterial organic or inorganic compounds to the layered structure predetermines the clay minerals to be a desired materials in medicine. The intercalation of

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organic compounds into layered inorganic clays provides a useful and suitable route to

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prepare organic–inorganic hybrids that contain properties of both the inorganic host and organic guest in a single material.

Among the natural clay minerals, montmorillonites are often used for the preparation of

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antibacterial materials. Many scientific research teams all over the world are interested in the study of synthesis, characterization and controlled release of antibacterial organic compounds

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using montmorillonite; for example chlorhexidine acetate (He et al., 2006; Meng et al., 2009; Yang et al., 2007) and cetylpyridinium chloride (Özdemir et al., 2010; Özdemir et al., 2013).

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The organoclay samples prepared using chlorhexidine diacetate are often predetermined to dental usage whereas the cetylpyridinium chloride adsorbed on montmorillonite facilitates the compatibility of montmorillonite with textiles, paper, paints and polymers which can have the potential to be used in different antibacterial applications. Also the preparation of antibacterial materials with inorganic cation are very frequent. The heavy metal cations as Ag+ (Magaña et al., 2008; Xu et al., 2011), Cu2+ (Drelich et al., 2011) or Zn2+ (Malachová et al., 2011) were loaded with montmorillonite or vermiculite by the cation exchange procedure. Antibacterial materials were tested against Gram-positive and Gram-negative bacterial strains. Vermiculite is another phyllosilicate mineral with a structure consisting of 2:1 layers composed from two silica tetrahedral sheets attached to a central magnesium octahedral sheet.

ACCEPTED MANUSCRIPT The net negative layer charge arises from the substitution of Si4+ located in the tetrahedral for Al3+ or by substitution of Al3+ located in octahedral for lower charge cations like Mg2+ or Fe2+

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and from the presence of vacancies. The negative layer charge is balanced by the positively

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charged interlayer material (Valášková et al., 2012).

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A novel antibacterial organovermiculites with different mass ratios of chlorhexidine diacetate were prepared by ion exchange reactions. These organovermiculites strongly inhibited the bacterial growth against gram-positive and gram-negative bacteria. Antibacterial

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activity of resultant organoclays strongly depends on the content of CA (Holešová et al.,

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2010).

A new form of mucoadhesive oral film from carmellose with intercalated chlorhexidine in vermiculite was also researched. This innovative biomaterial is able to release the drug

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directly to the targeted area and fight with oral infection. The antimicrobial and antimycotic activity of prepared films with aim of finding the most suitable composition for clinical

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application were evaluated. The results demonstrated the suitability of the prepared formulation for clinical use (Gajdziok et al., 2015).

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This study follows the previously published results aimed at the evaluation of toxicological properties of vermiculite nanocomposites. Clay mineral vermiculite was subjected to in vivo toxicological analysis and its influence on gastrointestinal tract was investigated. The samples of tissue were subjected to histological examination. Neither systemic nor local reactions were observed. Therefore the toxicity of vermiculite to a mammal model organism can be excluded (Holešová et al., 2014). The release of the organic substances from the drug carrier is very important information, and was intensively studied (Farkas et al., 2007; Leung et al., 2005; Verraedt et al., 2010; Wu et al., 2013; Young et al., 2008).

ACCEPTED MANUSCRIPT Chlorhexidine diacetate is a cationic chlorophenyl bisbiguanide that binds well to negatively charged surfaces, and according to the concentration used shows bacteriostatic and

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bactericidal effect against a wide variety of gram–positive and gram–negative bacterial

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strains. The antibacterial activity of CA is due to the positively charged parts of the CA

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molecule, which reacts with the phosphate groups of lipopolysaccharides in the bacterial cell wall (Attin et al., 2008).

The main objective of this work was the comparative study of the stability of prepared

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antibacterial chlorhexidine/vermiculite samples (CA/Ver) before and after stirring in aqueous

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solutions under the influence of pH values 2 and 7 and different temperatures. The procedure should simulate a local inflammation in the human body with an acidic character and elevated temperature (Xu et al., 2013). The structure of organovermiculite samples before and after

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stability tests was characterized using the X-ray powder diffraction (XRD) and Fourier transform infrared spectroscopy (FTIR). The content of CA was determined by total organic

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carbon (TOC) analysis. The antibacterial effects were tested against gram-positive (Enterococcus faecalis, Staphylococcus aureus) and gram-negative (Escherichia coli,

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Pseudomonas aeruginosa) bacteria strains.

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Experimental methods

2.1

Materials

Natural vermiculite (abbreviated Ver) from Santa Lucía, Brazil (supplied by Grena, Co., Czech Republic) was ground in a vibrating mill for 3 min. The sieved fraction under the 40 m was used for the experiment. The cation exchange capacity (CEC) of Ver was 1.06 meq/g. The crystallochemical formula of Ver was calculated per formula units from the results of the elemental composition of Ver determined by X-ray fluorescence elemental analysis (Perioli et al., 2011).

ACCEPTED MANUSCRIPT (Si6.16Al1.84)(Al0.08Fe3+0.74 Fe2+0.04Mg5.02Ti0.12)O20(OH)4(Ca0.13K0.36Na0.16) Chlorhexidine diacetate (abbreviated CA, C22H30N10Cl2.2C2H4O2, from Sigma Aldrich,

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Co., Czech Republic) and ethanol (C2H5OH, from Vitrum VWR, Co., Czech Republic) as a

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solvent were used for the preparation of CA/Ver. For the determination of cation exchange

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capacity the cadmium nitrate (Cd(NO3)2, from Vitrum VWR, Co., Czech Republic) and nitric acid (HNO3, from Vitrum VWR, Co., Czech Republic) were used. The Cation Exchange Capacity assessment

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2.2

The cation exchange capacity (CEC) of Ver was determined by cation exchange with Cd2+.

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Ver was mixed with aqueous solution of Cd(NO3)2, suspension was shaken for 20 min and then centrifuged. The procedure was repeated five times always with fresh solution of

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Cd(NO3)2. Solid phase was filtered, washed with deionized water and dried at 40°C.

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Subsequently dried sample was dissolved in HNO3 and shaken for 24 hours. After filtration

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the Cd2+ in solution were analyzed using an atomic absorption spectrometry (AAS). The CEC

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of Ver was determined to be 1.06 meq/g (Klika et al., 2016).

Sample preparation

The ethanolic solution of CA (1.33g CA, 70 ml ethanol) was prepared in the concentration corresponding to the 1 x CEC value of Ver. The ethanolic solution of CA and the 2g of vermiculite dispersed in 30 ml of demineralized water were stirred together for 6h at 75°C. After centrifugation (3800 rpm, 5 min.) a wet solid sample was obtained, the CA/Ver was dried at 80°C overnight.

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The stability test

CA/Ver sample was dispersed in 100 ml of demineralized water with a pH of 2 or 7

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(maintained using HCl during the whole experiment) and stirred for 24h under different

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temperatures (20°C and 40°C). After centrifugation (3800 rpm, 5 min.) the samples were

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dried at 80°C overnight and labelled as CA/Ver/2_20, CA/Ver/2_40, CA/Ver/7_20 and CA/Ver/7_40. Characterization methods

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The crystallochemical formula of Ver was calculated per formula units from the results of

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the elemental composition of Ver determined by X-ray fluorescence elemental analysis on an energy dispersive spectrometer SPECTRO X LAB with Rh X-ray lamp and secondary and

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polarization targets.

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X-ray diffraction (XRD) patterns were recorded using the diffractometer RIGAKU Ultima

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IV (reflection mode, Bragg-Brentano arrangement, CuKα radiation) in an ambient atmosphere under constant conditions (2-60° 2θ, scan speed 2°/min, 40kV, 40mA).

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The IR spectra were obtained using the Fourier transformation (FTIR) spectrometer NEXUS 470 (ThermoNicolet, USA) on solid samples prepared using KBr pressed-disc technique. The spectrometer was equipped with Globar IR source, KBr beam splitter, and DTGS detector. For each spectrum, 128 scans were obtained with resolution of 4 cm-1 in the range of measurements 4000 – 400 cm-1. The content of total organic carbon (TOC) was analysed by TOC analyser HORIBA EMIA-320V2 in oxygen atmosphere at 3 000°C for 60 s. 2.6

Antibacterial test

Antibacterial activity of the prepared samples was determined by minimum inhibitory concentration (MIC) which is the lowest concentration of a sample that completely inhibits

ACCEPTED MANUSCRIPT bacterial growth. The dilution and cultivation were conducted on the microtitration plate with 96 wells where each of the wells contained 100µl of pure glucose broth. To the first set of

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wells on the plate 50µl of 10% (w/v) organovermiculite in water dispersion was added. These

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dispersions were further diluted by a threefold diluting method in glucose stock in such

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manner, that the second to the seventh set of wells contained a sample dispersed in concentrations of 3.33%, 1.11%, 0.37%, 0.12%, 0.041% and 0.014%. The eighth set of wells contained pure glucose stock as a check test.

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A volume of 1µl of glucose suspensions with S. aureus CCM 3953 (1.1x109cfu ml-1), E. faecalis CCM 4224 (1.2x109cfu ml-1), E. coli CCM 3954 (1.2x109 cfu ml-1) or P. aeruginosa

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CCM 1960 (1.4x109 cfu ml-1), provided by a Czech collection of microorganisms (CCM, http://www.sci.muni.cz/ccm/), was put into all the wells. After the elapsed time of exposition

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of antibacterial samples (0.5, 1, 1.5, 2, 3, 4, 5 hours, and then after 24 hour intervals for 5 days) bacterial suspensions were transferred from each well to 100 µl of the fresh glucose

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stock and incubated in thermostat at 37°C for 24 hours. After this time the results were obtained. If there is a pure suspension in the well, tested sample had antibacterial effect. On

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the other side, cloudy solution means that tested sample did not prevent the bacterial growth (Kneiflová, 1988).

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Results and discussion

3.1

X-ray powder diffraction

The XRD patterns of natural Ver, organovermiculite CA/Ver and samples after stability tests are shown in Fig. 1. The basal reflections of Ver (Fig. 1a) with the interlayer values d001 = 2.30 nm, d002 = 1.43 nm and d003 = 1.26 nm correspond to the interstratified layered structure with the mixture of the layered hydrate domains and hydrated phases with two and one water layers (Marcos et al., 2009; Walker, 1956).

ACCEPTED MANUSCRIPT The XRD pattern of CA/Ver (Fig. 1b) shows the basal reflections of intercalated layers with the interlayer values d = 3.04 nm, d = 2.23 nm, d = 1.87 nm and d = 1.11 nm.

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The interlayer space of sample CA/Ver/7_20 (Fig. 1c) after stability test in comparison to

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CA/Ver changed to the lower values d = 2.90 nm, d = 2.03 nm, d = 1.51 nm and d = 1.01 nm.

interlayer space (Simha Martynková et al., 2007).

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The decrease of interlayer space value may correspond to the reorganization of material in the

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Influence of temperature (40°C) was observed mainly in the sample CA/Ver/7_40 (Fig. 1d) where interlayer distance decreased from d = 3.04 nm to d = 2.72 nm. Similarly, the basal

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reflection d001 = 3.04 nm in CA/Ver (Fig. 1b) has shifted to the lower value d001 = 2.81 nm in CA/Ver/2_20 (Fig. 1e).

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According to the XRD patterns, samples CA/Ver after stability test showed slightly lower

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interlayer space value. This change occurred probably due to the reorganization of material in the interlayer space of vermiculite. The TOC analysis (see chapter 3.3, Table 1) showed a

Fourier-transform infrared spectroscopy

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decrease in the content of carbon in the structure CA.

The IR spectrum of parent natural Ver (Fig. 2a) shows a band at 3674 cm-1 in the OH stretching region attributed to the Mg3OH unit. Together with absorption at 684 cm-1, which belongs to OH bending vibration, these bands suggest the trioctahedral character of vermiculite (Farmer, 1974). Absorption band at 3405 cm-1 corresponds to the OH stretching vibration of adsorbed water and the band at 1646 cm-1 can be assigned to OH bending vibration of adsorbed water. Finally, the intensive band at 1004 cm-1 was assigned to Si-O stretching vibration together with Si-O bending vibration at 450 cm-1 (Farmer, 1974). The IR spectrum of CA (Fig. 2b) shows bands at 3461, 3327, 3175 and 3129 cm-1 corresponding to the NH stretching vibrations of secondary amine and imine groups. A very weak band at 3014

ACCEPTED MANUSCRIPT cm-1 belongs to C-H stretching vibration of aromatic rings. Two bands at 2936 and 2860 cm-1 are assigned to asymmetric and symmetric C-H stretching vibrations of methylene groups

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(Silverstein et al., 1991; Socrates, 2001). The C=N stretching vibration of the imine group

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appears at 1641 cm-1. The bands that occurred in the 1580-1412 cm-1 region are due to NH

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deformation vibrations of secondary amine and imine groups and C=C stretching vibrations of aromatic rings. Absorptions at 1250, 1091, 823 and 724 cm-1 belong to the C-N stretching vibration of secondary aromatic amines, C-Cl stretching vibration of aromatic halogen

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compounds, C-H out-of-plane deformation vibration of aromatic ring and C-H rocking

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deformation vibration of methylene group, respectively (Silverstein et al., 1991; Socrates, 2001). After intercalation of CA into Ver the well-resolved bands in O-H and N-H stretching region are replaced by weak, broad, new bands at 3350 and 3218 cm-1 (Fig. 2c). These

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changes might indicate that OH groups of Ver are interacting through hydrogen bonds with some of the NH groups of CA.

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The IR spectra of organovermiculite samples, after stability test, are shown in Fig. 3a), b). In the Fig. 3a) curve 1 belongs to CA/Ver/7_20 and curve 2 is CA/Ver/7_40; in Fig. 3b) curve

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3 is CA/Ver/2_20 and curve 4 is CA/Ver/2_40. It can be seen there are no changes in positions of characteristic bands compare to the same bands in IR spectrum of CA/Ver (Fig. 2b). Thus we assume that there are no chemical and/or structural changes after acid and/or heat treatment. 3.3

TOC analysis

Stability of CA on the Ver matrix was determined by the stability test (see chapter 2.3). The corresponding content of carbon was obtained by averaging the contents of the three measurements for each sample (Table 1). An average amount of carbon in organovermiculite CA/Ver was 11.58 ± 0.07 %. This matches to 231.6 mg of CA in 1 g of whole sample. The

ACCEPTED MANUSCRIPT CA was intercalated from 58.1%. So only half of the used dosage was utilized during intercalation.

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Except for one, other samples after stability test contained nearly identical amounts of CA.

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See table 1 for results. The loss of CA in samples CA/Ver/2_20, VCA/Ver/2_40 and

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CA/Ver/7_20 was about 4% in comparison to CA/Ver. The largest decrease was observed in sample CA/Ver/7_40 under conditions of pH 7 and temperature 40°C. Intercalated amount of CA was 52.4% which corresponded to 209 mg of CA in 1 g of samples. We can estimate that

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this larger loss of content of CA (9.76 %) after stability test is due to combination of higher

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temperature and also pH. However, the decrease of active component (CA) from Ver matrix is in the order of tens of milligrams, which is a negligible amount. Moreover, as we can see in the next chapter (3.4) this loss does not have any significant effect on the antibacterial activity

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of the sample. These positive results suggest that the prepared organovermiculite CA/Ver sample is very stable after 24h intervals of testing. Compared to the literature these results are

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very low, pointing to a decisive role of preparation method. Xu et al. published 58% release of intercalated CA after 24 h at 7.4 pH. They used the same composite, CA/Ver, but different

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preparation method. The preparation of CA/Ver was carried out via ultrasonication in deionised water for 30 minutes (Xu et al., 2013). The higher amount of CA release from organoclay was also observed in the study of Meng et al. Although, they used montmorillonite instead of vermiculite they reported a 24% release of intercalated CA from CA/MMT after 24 h. They used very similar preparation method as we did in this study with one difference. They dissolved CA in water instead of ethanol (Meng et al., 2009). The higher amount of released CA could be also influenced by different bonds between CA and montmorillonite compared to bonds between CA and vermiculite. These findings are pointing out that the preparation method used in this study could be effectively used for the preparation of CA clays with long lasting release of CA.

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Antibacterial assessment

An antibacterial activity of samples was monitored on the gram-positive (Enterococcus faecalis, Staphylococcus aureus) and gram-negative (Escherichia coli, Pseudomonas

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aeruginosa) bacteria strains during the time periods: 0.5 h, 2 h, 4 h, 24 h and 120 h. The

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results of antibacterial studies (Table 2) confirmed that prepared organovermiculite sample CA/Ver displayed antibacterial effect. The intercalation of chlorhexidine into vermiculite is supported by the results of infrared spectroscopy (Fig. 2) where we can see characteristic

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vibrations of chlorhexidine. Stability of intercalated CA was studied based on the TOC analysis. Because only intercalated chlorhexidine contained carbon. So this technique help us

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to determine the content of carbon which was recalculated to an intercalated amount of chlorhexidine. The content of carbon in CA/Ver sample was 11.58% which is 231.6 mg CA in

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organovermiculite sample. Intercalated amount was 58.1%. There was not a sharp decrease of

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content of CA after the stability test. The samples CA/Ver/2_20, CA/Ver/2_40 and

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CA/Ver/7_20 contained 222 mg of CA in average. The outflow of CA was around 4% for each sample which is around 10 mg of CA in 1 g of sample. The higher loss of CA had only sample CA/Ver/7_40. There was 9.76% loss of CA from the sample. We can presume that

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higher value of pH and temperature 40°C had negative influence on releasing the CA from the vermiculite matrix. Although the loss of 9.76% after stability test corresponded to 20 mg of the loss of CA. These findings led to antibacterial activities of the samples CA/Ver/2_20, CA/Ver/2_40, CA/Ver/7_20 and CA/Ver/7_40 which were almost the same as of CA/Ver before the stability testing. This indicates that the amount over 200 mg of CA in 1 g of sample is sufficient for the inhibition of bacterial growth and small deviations of CA content are negligible. The lowest MIC values 0.014 (%, w/v) for CA/Ver (Table 2) revealed high efficiency of the sample after 24 hours which continues for the next period of observation until 5 days. This indicates that the antibacterial activity of prepared organovermiculite last for long period of time which is very important for the application of these antibacterial

ACCEPTED MANUSCRIPT materials (He et al., 2006). On the other hand, the high MIC value 10 (%, w/v) for the very resistant P. aeruginosa were observed even after 120 hours. According to literature, this

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occurs with all gram-negative bacteria, especially in case of P. aeruginosa, which has an

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overall outer-membrane permeability that is ~ 12 – 100-fold lower than for Escherichia coli

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(Hancock, 1998). The resistance of the outer membrane limits the movement of CA molecules into the cell. To the best of authors’ knowledge, no other research group conducted the same tests. Nevertheless, similar findings were published by other groups. Even though a different

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antibacterial test method was used (inhibitory zone test) the results support our findings. CA

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vermiculite showed high antibacterial activity against Escherichia Coli and Staphylococcus Aureus as well. On the other hand, CA montmorillonite displayed higher antibacterial activity against Staphylococcus Aureus but lower antibacterial activity against Pseudomonas

al., 2009; Xu et al., 2013).

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Aeruginosa, proving the Pseudomonas Aeruginosa being resistant bacterial strain (Meng et

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Based on the results obtained in this study it seems that this material could be used as a functional nanocomposite part of mucoadhesive oral film in dentistry thanks to its long-term

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antibacterial activity. The mucoadhesive oral film will help to treat a common oral cavity ailments as infectious stomatitis is (Gajdziok et al., 2015).

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Conclusions In this study the stability of prepared antibacterial organovermiculite CA/Ver was

observed. The XRD pattern showed that CA was successfully intercalated into the interlayer space of natural Ver. The FTIR spectra confirmed a presence of CA in organovermiculite samples. Stability of CA/Ver was determined by the process of releasing of CA from vermiculite matrix in aqueous solutions. Special conditions (pH 2 and 7, temperature 20°C

ACCEPTED MANUSCRIPT and 40°C) were used for the simulation of situation when human body is fighting with some kind of inflammation. There were no significant changes in antibacterial action after stability

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tests. All the samples had very good antibacterial effect against E. faecalis, S. aureus and E.

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coli, especially after 24 h and longer exposures. An exception are the poor results of

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antibacterial activity of prepared samples against the very resistant P. aeruginosa bacterial strain. The TOC analysis and XRD measurements confirmed only a slight outflow of CA from the vermiculite matrix after stability test. The largest decrease of content of CA was

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noticed in sample CA/Ver/7_40. However, this loss is in the order of tens of milligrams of

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active substance (CA), which is a negligible amount. Obtained results imply that the amount of CA in nanocomposite CA/Ver did not rapidly change after the stability test and suggest that

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the prepared sample could be used as functional part of nanocomposite materials.

Financial

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Acknowledgment support

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the

IT4Innovations

Centre

of

Excellence

project

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no.cz.1.05/1.1.00/02.0070; Ministry of Educations, Youth and Sports of the Czech Republic,

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project SP2013/78 and Czech Grant Agency, projects GA ČR 210/11/2215 are gratefully acknowledged. The authors gratefully acknowledge the assistance of Daniel Casten for polishing the writing style and English text.

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Figures captions Fig. 1. The XRD patterns of samples: a) Ver, b) CA/Ver, c) CA/Ver/7_20, d) CA/Ver/7_40, e) CA/Ver/2_20 and f) CA/Ver/2_40.

Fig. 2. IR spectra of: a) Ver, b) CA and c) CA/Ver.

Fig. 3. IR spectra of: a) CA/Ver/7_20 (curve 1), CA/Ver/7_40 (curve 2) and b) CA/Ver/2_20

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(curve 3), CA/Ver/2_40 (curve 4).

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Fig. 1.

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Fig. 2.

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Fig. 3.

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Intercalated amount of CA (%)

CA/Ver

11.58 ± 0.07

231.6

58.1

CA/Ver/2_20

11. 11 ± 0.01

222.2

CA/Ver/2_ 40

11. 08 ± 0.02

221.6

CA/Ver/7_20

11.16 ± 0.01

223.2

CA/Ver/7_ 40

10.45 ± 0.04

209.0

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Content of C Average of 3 measuring (%)

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55.7

Loss of CA after stability test (%) 4.06

55.6

4.32

55.9

3.63

52.4

9.76

ACCEPTED MANUSCRIPT Table 2 MIC values of organovermiculite samples Staphylococcus aureus MIC (%, w/v)

0.5 h > 10

2h > 10

4h > 10

24 h > 10

120 h > 10

0.5 h > 10

2h > 10

4h > 10

24 h > 10

120 h > 10

CA/Ver

3.33

3.33

3.33

0.014

0.014

3.33

3.33

3.33

0.014

0.014

CA/Ver/2_20

3.33

3.33

3.33

3.33

0.014

3.33

1.11

1.11

0.014

0.014

CA/Ver/2_40

3.33

10

10

1.11

0.014

1.11

1.11

1.11

CA/Ver/7_20

10

10

10

3.33

0.014

3.33

3.33

3.33

CA/Ver/7_40

10

10

10

3.33

0.014

1.11

1.11

3.33

4h > 10

24 h > 10

120 h > 10

CA/Ver

3.33

3.33

3.33

0.014

0.014

CA/Ver/2_20

3.33

3.33

1.11

0.014

0.014

CA/Ver/2_40

3.33

3.33

1.11

0.014

CA/Ver/7_20

3.33

3.33

3.33

0.014

CA/Ver/7_40

3.33

1.11

1.11

0.014

0.014

0.014

0.014

0.5 h > 10

2h > 10

4h > 10

24 h > 10

120 h > 10

> 10

> 10

10

10

10

10

> 10

10

10

10

0.014

> 10

> 10

10

10

10

0.014

10

10

10

10

10

0.014

> 10

> 10

> 10

> 10

> 10

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0.014

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2h > 10

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0.5 h > 10

CE P

0.014

Pseudomonas aeruginosa MIC (%, w/v)

Ver

AC

0.014

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Escherichia coli MIC (%, w/v)

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Ver

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Enterococcus faecalis MIC(%, w/v)

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Antibacterial nanocomposite was successfully prepared Stability of chlorhexidine diacetate in vermiculite matrix was investigated Prepared nanocomposites showed good antibacterial effect against bacterial strains CA/Ver has the potential to be used in long lasting antibacterial applications

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