Preparation of novel organovermiculites with antibacterial activity using chlorhexidine diacetate

Journal of Colloid and Interface Science 342 (2010) 593–597

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Journal of Colloid and Interface Science www.elsevier.com/locate/jcis

Preparation of novel organovermiculites with antibacterial activity using chlorhexidine diacetate Sylva Holešová a,*, Marta Valášková a, Eva Plevová b, Erich Pazdziora c, Katerˇina Mateˇjová c a

Nanotechnology Centre, VŠB-Technical University of Ostrava, 17.listopadu 15/2172, 708 33 Ostrava-Poruba, Czech Republic Institute of Geonics AS CR, Studentská 1768, 708 00 Ostrava-Poruba, Czech Republic c Institute of Public Health Ostrava, Centre of Clinical Laboratories, Partyzánské námeˇstí 7, 702 00 Ostrava, Czech Republic b

a r t i c l e

i n f o

Article history: Received 20 August 2009 Accepted 21 October 2009 Available online 25 October 2009 Keywords: Chlorhexidine diacetate Organovermiculite Structure evaluation Antibacterial materials

a b s t r a c t The novel antibacterial organovermiculites with different mass ratios of chlorhexidine diacetate (CA) were successfully prepared by ion exchange reactions. The resultant organovermiculites were characterized using X-ray diffraction (XRD), Fourier transform infrared spectroscopy (FTIR) and thermal properties of prepared organovermiculites were investigated by simultaneous thermogravimetry (TG) and differential thermal analysis (DTA). The antibacterial activity of prepared organovermiculites against Enterococcus faecalis, Escherichia coli and Pseudomonas aeruginosa was evaluated by finding minimum inhibitory concentration (MIC). Antibacterial studies showed that the organovermiculites strongly inhibited the growth of variety of microorganisms. Ó 2009 Elsevier Inc. All rights reserved.

1. Introduction Clay minerals have continuously attracted attention for the possibility of modifying their layered structure by intercalation. The development of new materials with ability to inhibit bacterial growth is a field of growing interest due to the worldwide concern of public health. In recent years, syntheses and applications of antibacterial compounds anchored on solid supports, e.g. mesoporous silica [1], hydroxyapatite [2] and clay mineral matrices [3–8], have been studied. Antibacterial compounds supported on the clay mineral matrix are generally known as inorganic and/or organic antibacterial materials. In first case, the most used are inorganic cations of heavy metals, e.g. Ag+, Cu2+, Zn2+ [9–16]. The clay-based inorganic materials have advantage such as high thermal stability, but on the other hand there are more disadvantages like an accumulation of the harmful heavy metals, mostly in the pseudo-hexagonal cavities of the silicate layers resulting in a decrease of the antibacterial activity. The claybased organic materials in spite of low thermal stability show many advantages compared to inorganic materials, mainly display organophilicity, so it is easy to adhere and exterminate bacteria. Montmorillonite is the most commonly used smectitic clay mineral for the preparation of antibacterial materials. Vermiculite is other clay mineral which in comparison with smectites has greater layer charge originated from tetrahedral substitution. The charge density in the silicate layers is expressed by the cation exchange capacity (CEC) and varies between 120 and 200 cmol(+)/ * Corresponding author. Fax: +420 597 321 640. E-mail address: [email protected] (S. Holešová). 0021-9797/$ - see front matter Ó 2009 Elsevier Inc. All rights reserved. doi:10.1016/j.jcis.2009.10.051

kg in dry vermiculite. Vermiculites most commonly contain Mg in the interlayer position, and minor amounts of Ca, Na, and K. The interlayer water consists of two discrete sheets and the water molecules within each sheet are arranged in a near hexagonal array. The Mg ions lie between the water sheets and are octahedrally coordinated with the water molecules. Chlorhexidine is substituted biguanidine with strong basic character that forms stable salts with several acids, e.g. chlorhexidine diacetate (CA). Chlorhexidine salts are antibacterial agents, used for human and animal disinfection [17,18], with very wide range of antimicrobial activity, being effective either against Gram-positive and Gram-negative microorganisms. Syntheses of novel organovermiculites with antibacterial activity were performed on the Na-vermiculite (NaVER) and chlorhexidine diacetate (CA). The prepared organovermiculites (NaVER_CA) were studied using the powder X-ray diffraction (XRD) and Fourier transform infrared analysis (FTIR). Thermal properties were investigated by simultaneous thermogravimetry (TG) and differential thermal analysis (DTA). The antibacterial activity was evaluated by finding the minimum inhibition concentrations (MIC) of NaVER_CAs against the Gram-positive bacteria Enterococcus faecalis and Gram-negative bacteria Escherichia coli and Pseudomonas aeruginosa. 2. Materials and methods 2.1. Materials The natural Mg-vermiculite from a deposit Letovice in the Czech Republic was selected as starting material. The original powder

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sample was ground in a planetary mill for 20 min, and then passed through a 0.045 mm sieve and fraction <40 lm was utilized for experiment. Its crystallochemical formula calculated from the results of the elemental chemical analysis was: (Si3.13Al0.86Ti0.02) (Mg2.53Fe0.45Al0.02)O10(OH)2(Mg0.19K0.01Ca0.02) per O10(OH)2 and the cation exchange capacity (CEC) was 140 cmol(+)/kg. The chemicals used for the sample preparation were chlorhexidine diacetate (C22H30N10Cl22C2H4O2, Sigma Aldrich) and ethanol as a solvent. 2.2. Vermiculite modifications The Mg-vermiculite was converted into its fully saturated Nafrom (NaVER) using cation exchange procedure with 1.0 M aqueous NaCl solution. Five solutions of CA in ethanol were prepared in the different concentrations CA accordant with the CEC of VER: 0.2, 1.0, 2.0, 3.0 and 4.0, and then stirred and heated with NaVER suspended in water. After centrifugation, solid products were dried and samples for the experiment were named NaVER_0.2CA, NaVER_1.0CA, NaVER_2.0CA, NaVER_3.0CA and NaVER_4.0CA, respectively. 2.3. Analytical methods and equipment The XRD patterns were recorded using the X-ray diffractometer INEL equipped with a curved position-sensitive detector CPSD 120 (reflection mode, Ge-monochromatized Cu Ka1 radiation). Diffraction patterns of the samples were taken in ambient atmosphere under constant conditions (2000 s, 35 kV, 20 mA). The samples were located in a flat rotation holder; the measurement was repeated three times. The XRD measurement was performed at normal laboratory conditions (25 °C, 43% of humidity). The mid-infrared spectra were obtained on a Perkin Elmer 2000 Fourier transform infrared spectrometer. For each sample, 32 scans were recorded in the 4000–400 cm1 spectral range with a resolution of 4 cm1 at room temperature using the KBr pressed disc technique (0.8 mg of sample and 290 mg of KBr). Thermal properties were investigated by simultaneous thermogravimetry and differential thermal analysis using a thermal analyzer (Setaram TG/DTA 12). The TG/DTA curves were recorded in a static air atmosphere at a heating rate 20 K min1 to the final temperature 1200 °C. Measurement of each sample was carried five times to get predicative results.

tion of 3.33%, 1.11%, 0.37%, 0.12%, 0.041% and 0.014%. The eight set of hollows contained pure glucose stock as check test. A volume of 1 l1 of glucose suspensions of E. faecalis CCM 4224 (9.3  108 cfu ml1), E. coli CCM 3988 (1.1  109 cfu ml1) and P. aeruginosa CCM 1960 (6.3  108 cfu ml1), provided by Czech collection of microorganisms (CCM), was put into hollows. Bacterial suspensions was after the elapse of 30, 60, 90, 120, 180, 240 and 300 min and then during 6 days always in 24 h interval transferred from each hollow to 100 ll of the fresh glucose stock and bacteria were incubated in thermostat at 37 °C for 24 and 48 h [19]. Antibacterial activity was evaluated by turbidity, which is display of bacterial growth. 3. Results and discussions 3.1. X-ray diffraction analysis XRD patterns in Fig. 1 show an intensive basal reflections (0 0 2) of NaVER and (0 0 2)int with (0 0 4)int of organovermiculites. The d(0 0 2) value of 1.198 nm in NaVER can be compared with the d-spacing values ranged from the interval 1.178–1.185 nm reported for monolayer hydrates of NaVER [20–22]. The reflection (0 0 2) of NaVER was observed at all organovermiculites, except the NaVER_2.0CA. The d(0 0 2) spacing close to the 1 nm identified in NaVER_4.0CA (Table 1) corresponded to the dehydrated vermiculite phase [23]. The expansion of the interlayer space d(0 0 2) from 1.198 nm in NaVER to the d(0 0 2)int ranged from 2.006 nm to the 2.146 nm in organovermiculites was adequate to the content of intercalated (int) CA (Table 1). Low concentration of 0.2  CEC CA only weekly intercalated into the NaVER. Alternative broadening and intensity of basal peaks in all others organovermiculites signified that there may be various types of layers present along

2.4. Antibacterial activity test The minimum inhibitory concentration (MIC) of prepared NaVER_CAs was determined by their lowest concentration that completely inhibits bacterial growth. The dilution and cultivation were preceded on the microtitration plate with 96 hollows. The first set of hollows on the plate contained 10% (w/v) organovermiculites water dispersion. This dispersion was further diluted by a threefold diluting method in glucose stock in such manner, that second to seventh set of hollows contained sample dispersed in concentra-

Fig. 1. XRD patterns with the basal reflection (0 0 2) of NaVER (dotted line), and two basal reflections (0 0 2)int and (0 0 4)int of NaVER_0.2CEC (a), NaVER_1.0CEC (b), NaVER_2.0CEC (c), NaVER_3.0CEC (d), and NaVER_4.0CEC (e).

Table 1 The layer spacing values observed for Na-vermiculite and organovermiculites NaVER_CA.

a b

Samples

d(0 0 2) (nm)

d(0 0 2)int (nm)

d(0 0 4)int (nm)

d(0 0 l)intb (nm)

hLciint (nm)

NaVER NaVER_0.2CA NaVER_1.0CA NaVER_2.0CA NaVER_3.0CA NaVER_4.0CA

1.198 1.182 1.178 – 1.168 1.118

– 2.006 2.074 2.099 2.118 2.146

– – 1.068 1.042 1.056 1.083

– – 2.134 ± 0.680 2.109 ± 0.668 2.124 ± 0.500 2.160 ± 0.566

62a 24 34 47 36 37

hLci calculated for non-intercalated NaVER. Average d(0 0 l) value for seven replicates with standard deviation in the round bracket.

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c* direction. The variable non-integral series of peaks and peak broadening indicated an irregular mixed-layer clay mineral structure [24]. The average d values and the standard deviations calculated from seven basal reflections in a sequence on the XRD patterns of NaVER_1.0CA, NaVER_2.0CA, NaVER_3.0CA and NaVER_4.0CA showed non-integral series characteristic for the irregular mixed-layer structures (Table 1). The width of the most intense peak (0 0 2)int supported existence of different crystallite sizes (i.e. effective size of the coherent scattering region normal to the reflecting atomic planes). The mean crystallite sizes hLci calculated using the Scherrer´s equation from the full width at half maximum (FWHM) of the (0 0 2)int were the smallest in NaVER_0.2CA and the biggest in NaVER_2.0CA (Table 1). 3.2. FTIR spectroscopy The mid-IR spectra for NaVER and selected organovermiculites NaVER_0.2CA and NAVER_4.0CA are shown in Fig. 2. The infrared spectrum for NaVER showed the main band at 3447 cm1 attributed to the stretching vibrations of structural OH groups. This stretching band slightly decreased in NaVER_0.2CA and NAVER_4.0CA due to the adsorbing water loss. The difference was ob-

served for the H–O–H bending mode at 1670 cm1 which implies a change in coordinated water in the interlayer of organovermiculites. The bands at 999 and 817 cm1 corresponded to Si–O–Si(Al) stretching and Al–O–H bending vibrations, respectively [25]. The increase of Al–O–H bending vibrations from 817 to 826 cm1 suggested interaction of the basic NH centres of CA with the octahedral Al–O–H of vermiculite. The main vibrational absorptions for original and intercalated chlorhexidine diacetate (CA) are listed in Table 2. New absorptions occurred in organovermiculites spectra about 2930 cm1 and 2860 cm1 were attributed to asymmetric and symmetric C–H stretching bands of CA. In all spectra, some bands are shifted to higher wavenumbers when compared to the original CA spectrum. This behavior suggests ability of hydrogen bond formation between NH groups of CA and structural OH groups of vermiculite. 3.3. Thermal analysis DTA curves of the selected samples NaVER, NaVER_0.2CA and NaVER_4.0CA showed in the low temperature interval 50–250 °C (Fig. 3) endothermic peaks relating to different dehydration of vermiculite at 165 °C in NaVER, 157 °C in NaVER_0.2CA and 144 °C in

Fig. 2. IR spectra for selected samples: NaVER (a), NaVER_0.2CA (b) and NaVER_4.0CA (c).

Table 2 Infrared vibrational assignments for original CA and organovermiculites NaVER_CA. Vibration samples

mN–H, asym. (cm1)

mN–H, sym. (cm1)

mC–H, asym. (cm1)

mC–H, sym. (cm1)

mC=N (cm1)

Original CA NaVER_0.2CA NaVER_1.0CA NaVER_2.0CA NaVER_3.0CA NaVER_4.0CA

3329 – 3344 3342 3341 3337

3133 3225 3214 3209 3210 3199

2936 2937 2935 2934 2934 2934

2861 2861 2860 2860 2859 2860

1645 1647 1647 1646 1647 1647

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Fig. 3. DTA curves for selected samples: NaVER, NaVER_0.2CA and NaVER_4.0CA in temperature range from 50 to 250 °C.

Fig. 5. DTG curves for selected samples: NaVER, NaVER_0.2CA and NaVER_4.0CA.

3.4. Antibacterial assessment

Fig. 4. DTA curves for selected samples: NaVER, NaVER_0.2CA and NaVER_4.0CA in temperature range from 750 to 900 °C.

NaVER_4.0CA. This corresponded to the fact, that water content between the layers decreased related to the increasing content of added organic substance. This interval was also attributed to the loss of some water molecules trapped in the hexagonal holes [26]. At the high temperature interval 750–900 °C (Fig. 4) continued dehydroxylation of vermiculite. In comparison with NaVER endothermic peak at maximum 816 °C, they were in organovermiculites NaVER_0.2CA and NaVER_4.0CA more intensive and shifted to higher temperatures 826 and 830 °C, respectively, accordingly with increasing content of CA. Exothermic effects in NaVER observed in the peaks maxima at 850 °C corresponded to crystallization of enstatite [26–28]. According to DTG curves (Fig. 5) the mass loss in temperature interval (25–550 °C) for NaVER was realized at one step, temperature of maximal reaction velocity was 155 °C. The mass loss of prepared organovermiculites in the uniform temperature interval had three steps. The first step with maximum at 148 °C observed for NaVER_0.2CA and at 126 °C observed for NaVER_4.0CA corresponded to the water release. In second step, the peaks with maximum about 240 °C conformed to the evaporation of adsorbed CA, while peaks in third step with maximum about 450 °C were assigned to the oxidation of residual CA with the organovermiculite [7].

Table 3 presents the MICs of parent NaVER which didnot have any antibacterial activity and MICs of prepared NaVER_CA organovermiculites. These results confirmed considerable antibacterial effect at low concentrations against Gram-positive E. faecalis bacteria and sensitive Gram-negative fermented E. coli sticks. The growth of very resistant microbe P. aeruginosa was successfully inhibited by samples NaVER_2.0, 3.0 and 4.0 with MIC values from 0.37 to 0.041 (% w/v). Noteworthy fact is that samples with CA concentration P2.0  CEC showed bacteriostatic effect as early as 30 min and even after 240 min they had bactericidal effect. It is known, that antibacterial effect of organic materials depends on their sorption ability to bacteria, so the increased content of CA in resultant organovermiculites had influence to their properties. It caused increasing hydrophobicity of organovermiculites, which improved the affinity between organovermiculites and bacteria [4]. Also electrostatic forces between negatively charged bacteria and organovermiculites increased due to higher CA content, so bacteria were immobilized onto organovermiculites surface [29]. This interaction could change the permeability of the microorganism’s cell membrane, caused a leakage of intracellular components and following by death of the cell. It was emerged that the choice of the type of an inorganic matrix is very important. In comparison with the work aimed with chlorhexidine diacetate (CA) on montmorillonite matrix [7] the novel CA-vermiculite composite has already been effective in low CA concentration (0.2  CEC) and completely inhibited bacterial growth of higher bacteria load of the E. faecalis (9.3  108 cfu ml1), E. coli (1.1  109 cfu ml1) and P. aeruginosa (6.3  108 cfu ml1) in comparison with CA incorporated in mesoporous silica [1], where was bacteria load of E. coli and/or S. auereus 106 cfu ml1.

Table 3 MICs (% w/v) of NaVER and organovermiculites against the different microorganisms. Sample

Enterococcus faecalis MIC

Escherichia coli MIC

Pseudomonas aeruginosa MIC

NaVER NaVER_0.2CA NaVER_1.0CA NaVER_2.0CA NaVER_3.0CA NaVER_4.0CA

– 0.37 0.12 0.014 0.014 0.014

– 0.014 0.014 0.014 0.014 0.014

– >10 >10 0.37 0.12 0.041

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4. Conclusions Novel antibacterial organovermiculites that combined the benefit of organic material (Chlorhexidine diacetate) with the excellence of inorganic material (Vermiculite) were prepared via intercalation technique. The XRD patterns, IR spectra and TG analysis confirmed that CA has been intercalated into the interlayer space of vermiculite. The XRD analysis proved the best agreement of CA within the interlayer of the NaVER_2.0CA. Antibacterial test for all organovermiculites prepared at low concentrations showed good activity against Gram-positive E. faecalis and Gram-negative E. coli bacteria, compared to test against P. aeruginosa bacteria, where MIC values were higher. Samples NaVER_2.0, 3.0 and 4.0CA contained sufficient amount of CA for bactericidal effect. The used of vermiculite inorganic matrix had very important role for antibacterial activity. The results from this study may be used for future development of new types of nanocomposite biomaterials with antibacterial activity. Acknowledgments This work was supported by the Czech Grant Agency (Project GACR No. 205/08/0869), and by the Ministry of education, youth and sports of the Czech Republic (MSM 6198910016). References [1] I. Izquierdo-Barba, M. Vallet-Regí, N. Kupferschmidt, O. Terasaki, A. Schmidtchen, M. Malmsten, Biomaterials 30 (2009) 5729. [2] F. Chai, J.-C. Hornez, N. Blanchemain, C. Neut, M. Descamps, H.F. Hildebrand, Biomol. Eng. 24 (2007) 510.

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