Antimicrobial Nanocomposites Prepared from Montmorillonite / Ag + / Quaternary Ammonium Nitrate

1Ministry of Forestry Bioethanol Research Center, Changsha 410004, China 2Hunan Engineering Research Center for Woody Biomass Conversion, Changsha 410004, China 3Bioenvironmental Research Institute, Central South University of Forestry and Technology, Changsha 410004, China 4Transpoints Inc., P.O. Box 141742, Gainesville, FL 32614, USA 5Research Institute of Wood Industry, Chinese Academy of Forestry, Beijing 100091, China

Meanwhile, polymers used in several industries such as food processing, biomedical devices, and filtering are required to have antiseptic ability to minimize the transmission of bacterial infections [19].The dispersibility and compatibility of antimicrobials with polymers is one of the key factors for the preparation of antimicrobial polymers.To improve the compatibility between the antimicrobial and polymer, surface modification of the antimicrobial is required.Furthermore, a lot of researches [20][21][22][23] shown that nanoparticles, such as clay and graphene nanoplatelets which was incorporated in antimicrobial polymer nanocomposites, allowed for the tuning of the release of antimicrobial agents, especially reducing the burst release effect, without hindering the antimicrobial activity of the obtained materials.The aim of this work was to prepare organic antiseptic MMT with good compatibility and dispersibility for use as a nanoadditive in polymers.For this purpose, MMT was modified with Ag + and quaternary ammonium nitrate via a one-step solution-intercalation technique.The structures of different antimicrobial organic MMTs were characterized by X-ray diffraction (XRD), Fourier transform infrared spectroscopy (FTIR), scanning electron microscopy (SEM), transmission electron microscopy (TEM), and energy dispersive spectroscopy (EDS) techniques, and the thermal stability was confirmed by differential scanning calorimetrythermogravimetric (DSC-TG) analysis.The antimicrobial activity of the nanocomposites was evaluated by examining the minimum inhibitory concentration (MIC) and killing rate.

Experimental
2.1.Materials.Sodium MMT used in this study was supplied by Zhejiang Fenghong Clay Chemicals Co., Ltd (China).The cation exchange capacity (CEC) of MMT was 90 meq (100 g) −1 .Silver nitrate (AgNO 3 ) with a purity of 99.8% was provided by Hunan Hipure Chemical Reagent Factory (China).Dimethyl octadecyl hydroxy ethyl ammonium nitrate (DOHEAN) at 50% (w/w) in butanol was provided by Jiangsu Hai'an Petrochemical Plant (China).Other reagents used in this study were of analytical grade.

Synthesis of Antimicrobial
Organoclays. 10 g sodium MMT was dispersed in 200 mL deionized water and stirred at 80 ∘ C. AgNO 3 (equimolar with the CEC) was dissolved in deionized water and then slowly dropped into the MMT sol, which was then kept at 80 ∘ C for 1 h with stirring.An equimolar quantity of DOHEAN was added to the Ag + and MMT sol and kept at 80 ∘ C for 2 h with stirring.The intercalated montmorillonite (Ag-OMMT) was repeatedly washed with deionized water to remove residual AgNO 3 and DOHEAN.This composite was then dried at 100 ∘ C for 24 h and then ground to a size less than 300 mesh.
The preparation of Ag-MMT and OMMT were consistent with the above methods but absented the process of addition AgNO 3 and DOHEAN, respectively.

Measurements.
XRD measurements were performed using a D/Max 2550 diffractometer (Rigaku Electrical Co., Ltd.) with a Cu target and K  radiation ( = 0.154 nm).TG and DSC curves were recorded at 20-800 ∘ C with a heating rate of 10 ∘ C/min under N 2 (Netzsch STA 449 C).FTIR spectra were collected from KBr pressed disks on a Nicolet 380 spectrophotometer.SEM images were recorded with a JEOL JSM-6380LV microscope, and TEM and EDS characterizations were performed on a Tecnai G2 20 FEI AEM.

Evaluation of Antimicrobial Activity. Gram-positive bacteria Staphylococcus aureus, Gram-negative bacteria
Escherichia coli, and fungi Candida albicans were provided by the China Center of Industrial Culture Collection (CICC at Beijing).[24].MIC tests were performed in MHA for the bacteria and fungi.A serial twofold dilution of Ag-OMMT was added to an equal volume of medium to obtain a concentration of 5000 g/mL, which was serially diluted by double technique to achieve solutions of 2500-9.77g/mL.Control dishes containing equal volumes of distilled water were also prepared.After cooling and drying, the plates were inoculated with 2 L of 10 7 CFU/mL strain solutions and incubated aerobically at 27 ∘ C for 16-20 h for bacteria or 72-96 h for fungi.Growth control samples of each tested strain were also included.The MIC was defined as the lowest concentration required to inhibit bacterial growth, that is, the concentration at which <5 microorganism colonies were visible.[24].The microorganism suspension was diluted using 0.9% (w/v) sterile saline water to 10 4 CFU/mL. 1 mL of cell suspension was added to 95 mL of 0.05, 0.025, and 0.0125 mg/mL nanocomposite (Ag-MMT and Ag-OMMT) solutions that had been autoclaved at 121 ∘ C for 20 min.Nanoscale SiO 2 was used as a control.The samples were removed after 2 h shake cultivation.50 L aliquots were spread on nutrient agar plates, which were incubated at 37 ∘ C for 24 h, and the numbers of colonies were counted for each solution.The percent reductions in plate colony counts were calculated by comparing the experiment plates to the control.All presented data were averaged from at least 3 parallel experiments, where the discrepancies among them were <5%.

Structure and Morphology.
The XRD patterns of unmodified MMT, Ag-MMT, OMMT, and Ag-OMMTs (modified with different amounts of Ag + ) are presented in Figure 1.Table 1 shows the -spacing of 001 ( 001 ) for MMT as calculated by Bragg's equation [25].Ag-MMT features a larger basal spacing (1.381 nm) than MMT (1.258 nm), indicating that Ag + was exchanged in the silicate layers.Both OMMT and Ag-OMMT feature wide basal spacings with high  001 values between 1.96 nm and 2.03 nm, indicating that DOHEAN has been intercalated into the MMT layers.
As shown in Figure 1, minimal metallic silver is present in all the Ag-OMMTs (at 2 = 38 ∘ ) but is absent in Ag-MMT.This is also noted in Figure 2, which shows the XRD patterns of Ag-MMT and Ag-OMMTs having different amounts of Ag + after calcination at 750 ∘ C for 2 h.In the presence of easily oxidizable organic cations, Ag + as an oxidant was partly reduced to metallic silver during the preparation process; after high temperature calcination, the reaction of Ag + and DOHEAN was complete.However, in the absence of organic cation, Ag + could not easily be deoxidized, even after calcination at 750 ∘ C.This has also been demonstrated by EDS (Figure 3).
Figure 3 shows the EDS pattern of Ag-OMMT and the morphologies of Ag-MMT and Ag-OMMT.The modified MMTs appear as sandwich-like crystals in the TEM images.Numerous black spots are homogeneously dispersed in the MMT crystals, as shown in Figure 3(b), which are metallic silver nanoparticles as demonstrated by EDS and XRD analyses.These silver nanoparticles are smaller in Ag-MMT (particle diameter within 2-5 nm, Figure 3(a)) than in Ag-OMMT (particle diameter within 10-20 nm, Figure 3(a)) because Ag + is more easily deoxidized in the presence of organic cations; this is in agreement with the XRD results (Figures 1 and 2). Figure 4 presents SEM micrographs showing the morphology change of MMT before and after modification.Unmodified MMT (Figure 4(a)) has a compact and flat surface, while, after modification, the MMT surface becomes crinkled and rough with wide interspacing (Figure 4(b)), which is desirable for use as a nanoadditive.

DSC-TG.
DSC-TG curves of DOHEAN, MMT, Ag-MMT, OMMT, and Ag-OMMT are shown in Figure 5.The TG curve of each sample shows an endothermal peak below 100 ∘ C with a corresponding weight-loss due to the removal of water.The DSC curves of MMT and Ag-MMT both feature a second endothermal peak at 679.2 ∘ C and 674 ∘ C, respectively, corresponding to the loss of hydrated water of the interlayer cations and the structural hydroxyls [26].
The sharp exothermal peak on the DSC curve of DOHEAN represents the evaporation or decomposition of DOHEAN.However, there are two exothermal peaks on the DSC curves of OMMT and Ag-OMMT.The low-temperature exothermal peak corresponds to the evaporation or decomposition of DOHEAN on the silicate plate surfaces, and the other peak represents the evaporation or decomposition of DOHEAN between the silicate plates.The TG curves of OMMT and Ag-OMMT reveal that the evaporation or decomposition of DOHEAN occurs at approximately 220 ∘ C, which is higher than that of pure DOHEAN (160 ∘ C).This indicates that the organic cation has intercalated into the MMT layers, similar to the initial state, and that the silicate platelets have the ability to protect organic molecules from decomposition.[27].A similar behavior was reported by Scaffaro et al. [28] during the preparation of poly(ethyleneco-vinyl acetate) films with two commercial formulations of nisin.

FTIR.
Figure 6 shows the FTIR spectra of MMT, DOHEAN, Ag-MMT, OMMT, and Ag-OMMT.Compared to MMT, OMMT has additional absorption peaks appearing at 2921, 2850, and 1384 cm −1 .The peaks at 2921 and 2850 cm −1 arise from -CH 2 -and -CH 3 stretching vibrations, while the one at 1384 cm −1 belongs to C-H symmetric deformation vibrations [29].This further reveals that DOHEAN has intercalated into the MMT layers.The peak at 3100-3700 cm −1 represents O-H stretching vibrations [30] and the peak at 1638 cm −1 belongs to H-O-H bending vibrations [31].This   OMMT can inhibit the growth of S. aureus; however, its ability to inhibit the growth of E. coli and C. albicans is less pronounced.This phenomenon is due to the different cell structures of these microbes.S. aureus, a Gram-positive bacterium, consists of a thick peptidoglycan layer and a cytoplasmic membrane.Its peptidoglycan layer is extensively crosslinked in three dimensions to form a solid mesh.Despite its thickness, the peptidoglycan layer of Gram-positive bacteria is not a barrier to the diffusion of foreign molecules.Gram-negative bacteria, however, have a small layer of peptidoglycan and an outer membrane made of a toxic liposaccharide layer.Because of this structure, Gram-negative bacteria are unusually permeable to foreign molecules.Therefore, Gram-negative bacteria are generally less susceptible to antibiotics and antibacterial agents than Gram-positive bacteria [32].
Figure 3: EDS pattern of Ag-OMMT and TEM micrographs of Ag-MMT and Ag-OMMT.

Table 1 :
001 of MMT modified with different organic cations.

Table 2 :
MIC of the samples.As shown in Table2, Ag-MMT and Ag-OMMT have obvious antimicrobial activity against a wide variety of microorganisms, including Grampositive bacteria, Gram-negative bacteria, and fungi.They have the same MIC for E. coli and C. albicans, which are 2.5 and 0.625 mg/mL, respectively.In addition, Ag-OMMT has a higher MIC (0.313 mg/mL) for S. aureus than Ag-MMT (1.25 mg/mL).Strong antimicrobial activity was also observed, as outlined in Table3.At a concentration of 0.0125 mg/mL, Ag-OMMT can kill 100% of the S. aureus, E. coli, and C. albicans population in 2 h, and Ag-MMT can kill 99.995% of the S. aureus, 90.15% of E. coli, and 93.68% of C. albicans in 2 h.