Influence of the powder dimensions on the antimicrobial properties of modified layered double hydroxide

Applied Clay Science 75–76 (2013) 46–51

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Influence of the powder dimensions on the antimicrobial properties of modified layered double hydroxide Valeria Bugatti a, Luigi Esposito a, Laura Franzetti b, Loredana Tammaro a,⁎, Vittoria Vittoria a a b

Department of Industrial Engineering, University of Salerno, Via Ponte Don Melillo, 84084 Fisciano (SA), Italy DISTAM — Università di Milano, Via Celoria 2, 20133 Milano, Italy

a r t i c l e

i n f o

Article history: Received 28 May 2012 Received in revised form 26 February 2013 Accepted 28 February 2013 Available online 1 April 2013 Keywords: Layered double hydroxide High energy ball milling Powder dimensions Antimicrobial activity Controlled release

a b s t r a c t Nano-hybrids of layered double hydroxides (LDHs), or hydrotalcite-like compounds, containing the antimicrobial anions of o-hydroxybenzoate (o-BzOH), also known as salicylate, were synthesized by a direct coprecipitation method. Elemental analysis indicated the chemical formula [Zn0.65Al0.35(OH)2] (C7H5O3)0.35 ∗ 0.7 H2O with a value of the molar fraction x = MIII/MIII + MII of 0.35. FT-IR spectroscopy and X-ray diffractograms confirmed the presence of o-hydroxybenzoate anions into the clay galleries, and indicated the presence of small size and disordered crystallites. The initial powder dimensions, reported as “surface weighted mean”, D[3,2], were reduced by using high energy ball milling (HEBM) technique for 1 min, 2 min, and 5 min. Also the morphology, investigated by SEM, was influenced by HEBM: big agglomerated particles were found for the pristine samples, while well-defined platelets with uniform and thinner size were present in the milled samples. The release of the o-hydroxybenzoate anion into a saline solution was measured for the initial powder with 25.0 μm of D[3,2] and for the powder with the smallest dimensions (D[3,2] = 8.2 μm). It was observed that the lower the powder dimensions the higher the percentage of active anions released into the saline solution. Also the diffusion parameter was found higher for the lower dimension powders. The antimicrobial activity against Escherichia coli was found much more effective for the sample with the lower powder dimensions, and even a slow but progressive reduction was noted after 24 h. In the presence of particles of 8.2 μm the microbial growth after 96 h of incubation is not evident. © 2013 Elsevier B.V. All rights reserved.

1. Introduction Great attention has recently emerged around the hybrid organic– inorganic systems and in particular to those in which layered fillers are modified by substituting the inorganic ions between the lamellae by organic ions (Carrizo et al., 2007; Evans and Duan, 2006; Rives, 2001; Winter et al., 2005). Such nano-hybrids firmly bind the organic molecules with ionic bonds, and can be considered as a reservoir where guest species are stored, protected from oxidation and photolysis, and released on demand by a chemical signal, that is by de-intercalation process. For these reasons the research in intercalation chemistry has shifted the attention from the study of the insertion mechanism to the preparation of new materials, with very specific properties, not obtainable through other synthetic procedures. Materials, that are cheap and naturally occurring, as natural clays or synthetic layered phosphates acquire a great added value after intercalation of molecules with special functionalities. Layered double hydroxide (LDHs), or hydrotalcite-like compounds, have received considerable attention as active molecular ion delivery ⁎ Corresponding author. Fax: +39 089 964057. E-mail address: [email protected] (L. Tammaro). 0169-1317/$ – see front matter © 2013 Elsevier B.V. All rights reserved. http://dx.doi.org/10.1016/j.clay.2013.02.017

vehicles, because of their anion exchange properties (Cavani et al., 1991; Vaccari, 1999). They are practically the only class of lamellar hosts consisting of positively charged layers balanced by exchangeable anions. Therefore functional organic species with anionic groups can be intercalated, via anion exchange process only in this class of layered hosts. These compounds have the general formula: h

MðIIÞð1−xÞ MðIIIÞx ðOHÞ2

i  Ax=n ⋅mH2 O

where M(II) is a divalent cation such as Mg, Ni, Zn, Cu or Co and M(III) is a trivalent cation such as Al, Cr, Fe or Ga with An− an anion of charge n such as CO32−, Cl−, NO3−or organic anion. Modified LDHs can be prepared with simple procedures, at high level of purity (Miyata, 1975; O'Hare, 2002). The new trend of the research is based on the fact that the active molecules, fixed by ionic bonds to the inorganic lamellae can carry out the specific activity being anchored to the lamellae, or being slowly released in particular environments (Sammartino et al., 2005; Tammaro et al., 2005, 2007, 2009). The active component may be released via a de-intercalation process, occurring because of anion exchange or displacement reactions and therefore the release rate is also dependent on the rate of the de-intercalation process, and this in

V. Bugatti et al. / Applied Clay Science 75–76 (2013) 46–51

turn depends on the electronic and steric structure of the guest species. Among the different specialties, pharmaceutical formulations are being intensively studied. Indeed the current trend of pharmaceutical technology requires formulations that are able to maintain pharmacologically active drug levels for long periods, avoiding repeated administrations or to localize the drug release in its pharmaceutical target. Hydrotalcite layered solids are the most suitable to provide this idea, due to their bio-compatibility as well as their ability to intercalate active species. One of the most important parameters to be studied, when LDHs intercalated with active molecules are used in powder form, is the powder diameter dimension, that can determine different threshold values to fulfill the specific activity (Herrero et al., 2009; Zhao et al., 2002). In previous papers we reported the preparation and the characterization of nano-hybrids, based on ZnAl-LDH intercalated with antimicrobial species (Bugatti et al., 2010, 2011; Costantino et al., 2009). In this paper we studied the dependence of the antimicrobial activity of one of these nano-hybrids on the powder dimension distribution. The antimicrobial molecule o-hydroxybenzoate (o-BzOH) was chosen and the powders, as obtained by the synthetic path, were ground in a high energy ball milling (HEBM) equipment, obtaining progressively decreasing dimensions. The strong influence of the powder dimensions on the antimicrobial activity is shown. 2. Experimental section 2.1. Materials Zn(NO3)2 ∗ 6H2O, Al(NO3)3 ∗ 9H2O, NaOH and sodium ohydroxybenzoate (sodium salicylate) were purchased from SigmaAldrich (Italy). 2.1.1. Preparation of ZnAl-o-BzOH by coprecipitation method 30 mL of an aqueous solution of Zn(NO3)2 ∗ 6H2O (12.9 g, 43.4 mmol) and Al(NO3)3 ∗ 9H2O (8.14 g, 21.7 mmol) were added to 30 ml of a sodium salicylate solution (5.9 g, 36.9 mmol) under stirring and under nitrogen flow. The pH slowly reached the value of 7.5 by adding 1 M NaOH. At the end, the precipitate was washed with distilled water and left in oven at 50 °C for 24 h, under vacuum (Frunza et al., 2008). 2.2. Methods of investigation Elemental analyses for the detection of Zn and Al atoms were carried out by an atomic absorption spectrophotometer (Model AAnalyst 100, Perkin Elmer) using solutions prepared by dissolving the samples in concentrated nitric acid. The C and H atoms were analyzed by an elemental analyzer CHNS/O (Model Flash EA 1112, Thermo), equipped by a thermoconductivity detector (TCD). X-ray powder diffraction (XRPD) patterns were taken, in reflection, with an automatic Bruker diffractometer, using nickel-filtered CuKα radiation (λ = 1.54050 Å) and operating at 40 kV and 40 mA, step scan 0.05° of 2θ, and 3 s of counting time. Fourier transform infrared (FT-IR) absorption spectra were obtained by a Perkin–Elmer spectrometer, model Vertex 70 (average of 32 scans, at a resolution of 4 cm−1). Thermal analysis (TGA/DTA) was carried out in air atmosphere with a Mettler TC-10 thermobalance from rt to 800 °C at a heating rate of 5 °C/min. The particle size distribution was measured using a Mastersizer 2000S (MalvernInst) Laser particle size analyzer (measurement range: 0.020 μm–2000 μm). The samples were dispersed in degassed and deionized water, as dispersant, under stirring at 3000 rpm. All samples were sputter coated with gold (Agar Automatic Sputter Coater Mod.B7341, Stansted, UK) at 30 mA for 180 s and the

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morphology of the powders analyzed by a scanning electron microscope (SEM Mod. LEO 420, Assing, Italy). The release kinetics of the active molecule in a fixed volume of a saline solution (0.9% w/v) were obtained by ultraviolet spectrometric measurement at ambient temperature, using a Spectrometer UV-2401 PC SHIMADZU. The antimicrobial activity of powders against Escherichia coli , characterized by different dimensions, was tested. Solutions of 0.4 g of ZnAl-o-BzOH powder in flask, containing 10 ml of Tryptic Soy Broth (4% w/v), were prepared and in each solution a E. coli suspension was added, so that the final concentration was 1.0 × 101 CFU/ml. The flasks were incubated at 30 °C under stirring to favorite the contact powder-microbial cells. At established times (0, 24 h, 48 h and 96 h) and in sterile conditions, the count of vital cells was performed, according to tenfold progressive dilutions in 0.85% sterile trypton salt solution. The count was effected in the selective differential medium, TBX (International Standard Organization, 2005) spread plates, and incubated at 37 °C for 24 h. 2.3. Dimension reduction of ZnAl-o-BzOH by milling procedure Powders of LDH modified with o-hydroxybenzoate (ZnAl-o-BzOH) (vacuum dried for 24 h) were milled, at room temperature (rt) in a Retsch (Haan, Germany) centrifugal ball mill (model S100). The powders of ZnAl-o-BzOH were milled in a cylindrical steel jar of 50 cm3 with five steel balls of 10 mm of diameter (Bugatti et al., 2010; Costantino et al., 2009). The rotation speed used was 580 rpm, and the milling time was: 1 min, 2 min and 5 min. In the following, the powders by milling will be coded as: ZnAl-o-BzOHmn, where n is the milling time. 3. Results and discussion 3.1. Elemental analysis The values of relative percentages and molar ratio of Zn, Al, C and H are reported in Table 1. The Zn/Al molar ratio of the starting solution of the Zn and Al nitrates was confirmed. The chemical formula obtained from the elemental analysis was the following: [Zn0.65Al0.35(OH)2] (C7H5O3)0.35 ∗ 0.7 H2O with value of the molar fraction x = MIII/MIII + MII of 0.35 and molecular weight of 146.52 g/mol; the amount of ortho-hydroxybenzoate intercalated in ZnAl-o-BzOH is 32.7 wt.% of the total weight. Therefore almost all the aluminum is co-precipitated with the zinc ions to obtain a solid with the stoichiometry of two Zn(II) atoms for each Al(III) atom. This corresponds to an ideal arrangement of the brucite-like sheet with each aluminum atom surrounded by six zinc atoms (Oswald and Asper, 1977). 3.2. Structural and thermal characterization FT-IR absorption spectroscopy of the ZnAl-o-BzOH sample gives information on the presence of intercalated benzoate anion and the brucite-type layer (see Fig. 1). The broad band in the 3750–2500 cm −1 region results from an overlapping of hydrogen vibrations: stretching vibration of structural hydroxyl units bound to the divalent cations and the trivalent cation

Table 1 Values for elemental analysis of ZnAl-o-BzOH.

% Grams Moles Molar ratio

Zn

Al

C

H

25.5 25.5 0.39 1.86

5.7 5.7 0.21 1.0

17.66 17.66 1.47 7.0

3.15 3.15 3.15 15.0

V. Bugatti et al. / Applied Clay Science 75–76 (2013) 46–51

together with the hydroxyl stretching bands of physically adsorbed water. We observed at least three water OH stretching vibrations at around 3060, 3450 and 3580 cm−1. The shoulder at about 3060 cm−1 can be assigned to solvation water molecules highly condensed into the microporosity, the band at around 3450 cm−1 is attributed to hydrogen bonded water. The band at around 3580 may be due to adsorbed water. (Chatelet et al., 1996; Frost et al., 2003; Kloprogge and Frost, 2001). The sharp and intense bands at 1610 and 1363 cm−1 are ascribable to the asymmetric and symmetric stretching vibrations of the C\O bonds of COO − groups, confirming the presence of o-hydroxy-benzoate anions into the inorganic galleries. Between 1000 and 700 cm − 1 broad bands due to librational modes of the hydroxyl groups and water can be seen, while roughly below 650 cm − 1 the lattice translational modes can be observed. The band at around 450 cm − 1 has been ascribed to a condensed [AlO6] 3 − group or as single Al\O bonds (Kloprogge and Frost, 2001; Valcheva-Traykova et al., 1993). Thermal behavior of the pristine powders of the intercalate ZnAl-o-BzOH, before milling procedure, was investigated and the TGA curve is represented in Fig. 2. As inferred in previous papers (Bugatti et al., 2011; Costantino et al., 2009) the two endothermic weight losses, between 80 and 300 °C, are, very likely, related to the loss of 0.7 mol of co-intercalated water and 1 mol of water derived from dehydroxylation of the inorganic layers. The weight loss of about 20% was in good agreement with the value of about 21% calculated on the basis of the stoichiometric formula [Zn0.65Al0.35(OH)2] (C7H5O3)0.35 ∗ 0.7 H2O. The main weight loss between 330 and 500 °C may be ascribed to endothermic decarboxylation of benzoate and to exothermal combustion of the residual organic part. Over 800 °C there is no more mass loss and the residue is ascribable to the ZnO and ZnAl2O4. 3.3. Particle size distribution Increasing milling times, from 1 min to 5 min, were used to reduce the original powder dimensions. In Fig. 3 we report the particle size distribution of the original powder and of powders at different milling times. We observed that the original powders of ZnAl-o-BzOH show an asymmetric particle size distribution, centered at 227 μm as volume weighted mean D[4,3] extrapolated from the curve (see Table 2), with a shoulder at about 40 μm and a tail for low particle size distribution. The milling process reduces the value of the principal peak and reduces the shoulder as well. The peaks become more symmetrical, even if small peaks at higher dimensions appear, indicating a small re-aggregation of some powders. From these data we derived the “surface weighted

90 80 70 60 50 40 30 20 10 0 100

200

300

400

500

600

800

Fig. 2. TGA curve of ZnAl-o-BzOH. Operative conditions: air flow; heating rate: 5 °C/min.

mean”, D[3,2] that is reported in Fig. 4 as a function of the milling time (Allen, 1992). We observe a rapid decrease up to 2 min of milling, whereas a constant value is observed for the longer time investigated. In any case we succeeded in obtaining powders with different particle dimensions (see Table 2), to investigate the antimicrobial effect as a function of this parameter.

3.4. ZnAl-o-BzOH characterization after milling 3.4.1. X-ray analysis X-ray diffractograms of the pristine powders and the powders milled for 1 min, 2 min and 5 min are shown in Fig. 5. The crystal structure and X-ray identification of layered double hydroxides has been well established so far. Moreover it is well known that many physico-chemical properties of LDHs are dependent on their structural and crystal–chemical features. The pristine powders obtained by co-precipitation show broad reflection for the basal planes, centered at 5.7° and 11.7° of 2θ, indicating very small and disordered crystallites. Moreover the non-basal planes of the family (0kl), in our case the reflection at 34.1° of 2θ, could suggest the presence of stacking faults, as shown in many cases. Considering now the powders milled for 1 min we observe an even increased broadness of the basal reflections, extending toward lower and lower angles, indicating a further reduction of crystallite dimensions and order. Moreover the intensity is very much decreased, if compared to the reflection at 34.1° of 2θ. We can analyze the inverse of the width at half-height, 1/A (θ−1), as an

1363

2,5

6 ZnAl-o-BzOH ZnAl-o-BzOHm1 ZnAl-o-BzOHm2 ZnAl-o-BzOHm5

5

Volume (%)

2,0 1,5 1,0 430

0,5

4 3 2 1

0,0

0 0

4000

700

Temperature (°C)

1610

3,0

Absorbance (a.u.)

100

Residual Weight (%)

48

3500

3000

2500

2000

1500

1000

Wavenumber (cm-1) Fig. 1. Infrared spectrum in absorbance of ZnAl-o-BzOH.

500

250

500

750

1000

1250

1500

1750

2000

Particle Size (micron) Fig. 3. Particle volume (%) versus particle size (micron) of pristine ZnAl-o-BzOH and of the powders at different milling times.

V. Bugatti et al. / Applied Clay Science 75–76 (2013) 46–51

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Table 2 Values of particle size distribution. Volume weighted mean D[4,3] (micron)

ZnAl-o-BzOH 227 ZnAl-o-BzOHm1 54 ZnAl-o-BzOHm2 48 ZnAl-o-BzOHm5 49

d

Surface weighted mean D[3,2] (micron)

Intensity (a.u.)

Sample

25.0 16.0 9.9 8.2

c b

order parameter, and we report this parameter in Fig. 6 as a function of the milling time. We observe a rapid decrease of the order parameter in the first minute of milling, followed by a partial increase, indicative of a re-aggregation on continuing the milling.

a 5

3.4.2. Scanning electron microscopy (SEM) LDHs containing organic anions generally produce materials of varying shapes and sizes (Herrero et al., 2009; Kim et al., 2011; Zhao et al., 2002). The morphology of all samples has been investigated by scanning electronic microscopy (SEM) and micrographs of the LDHs powders were shown in Fig. 7. The pristine sample ZnAl-o-BzOH presents irregular aggregates of plate-like particles. However, significant differences in the shape and size of the particles were observed in the samples treated by milling procedure. While big agglomerated particles were found for the pristine samples, well defined platelets with uniform and thinner size are present in the milled samples. As shown in Fig. 7 the average diameter of the platelets of the milled sample ZnAl-o-BzOHm5 (Fig. 7B) is smaller and considerably more uniform in distribution than the unmilled sample ZnAl-o-BzOH (Fig. 7A). The SEM micrographs revealed the size of particles to be around 25 μm for ZnAl-o-BzOH and in the range 8–10 μm for ZnAl-o-BzOHm5.

15

20

25

30

35

40

2θ (°) Fig. 5. X-ray powder diffraction patterns of: ZnAl-o-BzOH (a), ZnAl-o-BzOH m1 (b), ZnAl-o-BzOHm2 (c), and ZnAl-o-BzOHm5 (d).

3.4.4. Antimicrobial activity The antimicrobial properties of sodium salicylate, a benzoate derivative, (Russell, N. J., Gould, G. W., Eds. Food Preservatives; Springer: Dordrecht, The Netherlands, 2003), is already known, being an inhibitor of Gram-negative bacteria such as E. coli, an important alterative for the fresh foods such as meat, fish and dairy products. It seemed of interest to carry out tests to prove that this property is still present in ZnAl-o-BzOH powder and how different powder dimensions interact with microbial cells and their effect on growth process. The last investigated aspect is the main goal of this study. The antimicrobial power of the ZnAl-o-BzOH powder was investigated by using the indirect viable cell count method, in which each colony that can be counted, called CFU (colony-forming units), is related to the number of viable bacteria in the sample. The tests were performed with the coliform microorganism E. coli (DSM30083T) used as model bacteria. A suspension of E. coli was added to solutions of powder of ZnAl-o-BzOH (see Experimental section). The evolution of E. coli in the presence of 4% (w/v) solution of ZnAl-o-BzOH powders in comparison with the pure culture medium (TSB) is reported in Fig. 9. All microbiological determinations were performed in triplicate, and the numerical values are the results of arithmetic average (International Standard Organization, 2005). Interestingly a proportional correlation with powder dimensions was observed, for almost all cases: the inhibition is higher in the presence of a particle of lower dimensions. Overall, in reducing the size of particles, a lower microbial growth is observed. The particles with lower dimension are able to play a better antimicrobial activity due to the higher contact area. After 24 h, even if slow, a progressive reduction

30 0,28

0,26

20

1/A

Surface Weighted Mean (micron)

3.4.3. Release properties The release of the active molecules from different powder dimensions was investigated by putting the pristine powders of ZnAl-o-BzOH and the milled powders of ZnAl-o-BzOHm5 having the lower particle dimensions, in contact with a saline solution, with chloride ions, as counter-ions, at a concentration of 0.9% w/v. As shown in Fig. 8 we observed different release kinetics, depending on the powder dimensions, as expected. The lower the powder dimensions, the higher the percentage of active molecule released into the saline solution, and the diffusion phenomenon. It is interesting to note that both powder samples seem to show a hysteresis behavior, leveling off at about 70% of the expected release. Sorption hysteresis has been observed in some cases for these hydrotalcite-like compounds (Tronto et al., 2004), and has to be carefully stated if controlled release is investigated.

10

0,24

10 0,22 0 0

1

2

3

4

5

Milling Time (min)

0,2 0

1

2

3

4

Milling Time (min) Fig. 4. Surface weighted mean (micron) versus milling time (minutes) of pristine ZnAl-o-BzOH and milled powders.

Fig. 6. The inverse of the width at half-height as a function of the milling time.

5

50

V. Bugatti et al. / Applied Clay Science 75–76 (2013) 46–51

A

1,0E+09 1,0E+08

Blank 25 micron 16 micron 9,9 micron 8,2 micron

1,0E+07

CFU/g

1,0E+06 1,0E+05 1,0E+04 1,0E+03 1,0E+02 1,0E+01 1,0E+00 1,0E-01

0

24

48

72

96

Time (hours)

B

Fig. 9. Evolution of E. coli in the presence of 4% (w/v) solutions of ZnAl-o-BzOH powders with different dimensions as a function of contact time (hours).

cells. The main result of this study is that the milled particles with dimension smaller than the pristine ZnAl-o-BzOH powder are able to inhibit the microbial growth at all contact times. This effect doesn't last immediately as tested for the pristine powder. 4. Conclusions

Fig. 7. SEM micrographs of the powders: ZnAl-o-BzOH (A); and ZnAl-o-BzOHm5 (B).

Released Fraction (wt/wt %)

was noted. In particular, in the presence of particles of 8.2 μm the microbial growth after 96 h of incubation is not evident. The inhibition behavior of E. coli of the pristine particles (25 μm) is in contrast with the trend of the microbial growth of the milled powders for all the contact time. In fact this sample shows at 24 h a value of CFU lower than the value of the 16 μm particles. In presence of pristine particles (25 μm) after an initial inhibition, the growth shows a trend similar to that of the blank. This behavior can be explained with an initial delayed phase of the cells, that have to adapt to the new environment, and then their growth proceeds more rapidly. The reduction of the size of particles facilities their diffusion in medium and the contact with the

100 ZnAl-o-BzOH ZnAl-o-BzOHm5

80 60 40 20 0 0

12

24

36

48

60

72

84

96 108 120 132 144 156 168

Contact time (hours) Fig. 8. Release of o-BzOH molecules from ZnAl-o-BzOH as released fraction (wt/wt.%) normalized to the expected final concentration versus the contact time (hours).

We have prepared a modified layered double hydroxide with o-hydroxybenzoate anions by a direct coprecipitation method, obtaining a less crystalline structure than that reported in literature with other preparation methods. The mean dimensions of the pristine ZnAl-o-BzOH corresponding to 25 μm were reduced by milling procedure grounding the powders for 1, 2 and 5 min, obtaining dimension of 16.0, 9.9 and 8.2 μm, respectively. SEM investigation shows a morphology with irregular agglomerated particles for the pristine samples and with uniform and smaller platelets for the milled samples. Comparing the release properties of powders with the higher and lower dimensions we found a higher release kinetics for the lower powder dimensions. Also the antimicrobial activity against E. coli was found much more effective for the sample with the lower powder dimensions. These results indicate that it is possible to modulate the release kinetics and the antimicrobial activity of a modified layered double hydroxide depending on the particle size of the powders. References Allen, T., 1992. Particle Size Measurement, 4th edition. Chapman & Hall (ISBN 041235070). Bugatti, V., Costantino, U., Gorrasi, G., Nocchetti, M., Tammaro, L., Vittoria, V., 2010. Nano-hybrids incorporation into poly(ε-caprolactone) for multifunctional applications: mechanical and barrier properties. European Polymer Journal 46, 418–427. Bugatti, V., Gorrasi, G., Montanari, F., Nocchetti, M., Tammaro, L., Vittoria, V., 2011. Modified layered double hydroxides in polycaprolactone as a tunable delivery system: in vitro release of antimicrobial benzoate derivatives. Applied Clay Science 52, 34–40. Carrizo, D., Del Arco, M., Martín, C., Rives, V., 2007. A comparative study between chloride and calcined carbonate hydrotalcites as adsorbents for Cr(VI). Applied Clay Science 37, 231–239. Cavani, F., Trifiro, F., Vaccari, A., 1991. Hydrotalcite-type anionic clays: preparation, properties and applications. Catalysis Today 11, 173–301. Costantino, U., Bugatti, V., Gorrasi, G., Montanari, F., Nocchetti, M., Tammaro, L., Vittoria, V., 2009. New polymeric composites based on poly(ε-caprolactone) and layered double hydroxides containing antimicrobial species. ACS Applied Materials & Interfaces 1, 668–677. Chatelet, L., Bottero, J.Y., Yvon, J., Bouchelaghem, A., 1996. Competition between monovalent and divalent anions for calcined and uncalcined hydrotalcite: anion exchange and adsorption sites. Colloids and Surfaces A: Physicochemical and Engineering Aspects 111, 167–175. Evans, D.G., Duan, X., 2006. Preparation of layered double hydroxides and their applications as additives in polymers, as precursor to magnetic materials and in biology and medicine. Chemical Communications 5, 485–496.

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