Antimicrobial activity of delaminated aminopropyl functionalize

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SCIENCEWORLDLIB.COM Research Paper Antimicrobial activity of delaminated aminopropyl functionalized magnesium phyllosilicates Gayathri Chandrasekaran a, Hyo-Kyung Han b, Geun-Joong Kim c, Hyun-Jae Shin a,⁎ a b c Department of Chemical and Biochemical Engineering, Chosun University, Gwangju 501-759, Republic of Korea College of Pharmacy, Dongguk University, Pil-dong 3-ga, Jung-gu, Seoul, Republic of Korea Department of Biological Science, College of Natural Sciences, Chonnam National University, Gwangju 500-757, Republic of Korea a b s t r a c t Keywords: AMP clay Antimicrobial activity Membrane permeability Intracellular enzymes leakage In this study, dispersed aminopropyl functionalized magnesium phyllosilicates (AMP clay) were tested for antimicrobial activity against various Gram negative and Gram positive bacteria including multidrug resistant bacterial strains and fungi. The AMP clay strongly inhibits the growth of microorganisms such as Escherichia coli, Staphylococcus aureus and Candida albicans. The inhibition of microbial growth by AMP clay is attributed to the amino propyl groups and their charge interactions. The bactericidal effect of AMP clay occurred within h, and 95% killing was observed within h. In addition, AMP clay could kill microbes by disrupting membrane integrity and thus essential components inside the cells. This effect was also evaluated through membrane depolarization assays and measured by the release of intracellular enzymes. The assessment of cell damage by the AMP clay was obtained by scanning electron microscopy (SEM) observation. Moreover, AMP clay activity was stably maintained after a long storage time of up to 30 days at different temperature conditions. The goals of this study were to synthesize the dispersed AMP clay from a commercially available substrate, which can be applied to various biomedical applications and environmental protection. 1. Introduction Clays are used in various scientific and industrial applications because they are present in nature, and they can be modified both chemically and physically (Aguzzi et al., 2007). The difference between the natural and synthetic materials lies in their surface areas, which are proportional to the degree of delamination or exfoliation of the clay layers (Carrado, 2000). Generally, smectite clay is modified organically via organo-cation exchange (Xue and Pinnavaia, 2010). An aminopropyl-functionalized magnesium phyllosilicate (AMP clay) contains a trioctahedral smectite with an organic propylamine group. There have been several recent reports on the synthesis and characterization of organoclay-modified derivatives in the form of nanocomposites for biomolecules encapsulation, immobilization, biosensing devices, nanoreactors and enzyme reactors (Ferreira et al., 2008; Holmström et al., 2007; Johnsy et al., 2009; Patil et al., 2005). Therefore, the interest in the use of organically modified clays has increased with time. In fact, they possess swelling ability, high cation exchange capacity, high layer aspect ratio, high specific surface area, film formation and adsorptive properties. In particular, smectites appear to be good candidates in industry, agriculture, ⁎ Corresponding author. Tel.: + 82 62 2307518; fax: + 82 62 2307226. E-mail address: shinhj@chosun.ac.kr (H.-J. Shin). environmental remediation, catalysis, coatings, ceramics, and polymer materials due to their submicron size (Xue and Pinnavaia, 2010). In addition, various applications of organoclays have been suggested for groundwater purification, hazardous waste landfills and industrial wastewater treatment (Pernyeszi et al., 2006). Clay materials can be used as geotechnical barrier adsorbents for toxic organic chemicals and heavy metals and can also serve as candidates for antibacterial applications (Boyd et al., 1988; Oya et al., 1991). Recently, the antimicrobial effects of nanoclay composite films, which were prepared by organically modified nanoclays have been reported. It has been hypothesized that these properties could result from the quaternary ammonium groups of organically modified clays (Rhim et al., 2009). The use of organically modified clay has greater advantages than clay-based inorganic antibacterial materials. The synthesis of inorganic antibacterial clays using heavy metals is comparably difficult due to serious metal toxicity in humans and the environment. These heavy metals also decrease the antibacterial activity due to the formation of insoluble compounds (He et al., 2006; Pernyeszi et al., 2006). However, the clays that have been reported to date were insoluble, which limits their applications. As far as we know, there has been no report on the antimicrobial activity of AMP clay. To make dispersed clay, AMP clay was synthesized and evaluated for antimicrobial activity against bacteria, fungi and multidrug resistant strains. The mechanism of antimicrobial action was investigated by various parameters, such as membrane depolarization, release of intracellular enzymes, and scanning electron microscopy. Therefore, this dispersed AMP clay may exert antimicrobial activity by a membrane disruption mechanism. (MIC) (Park et al., 2008). The MIC values were calculated as an average of several independent experiments conducted in triplicate. 2. Experimental 2.5. Antifungal activity 2.1. AMP clay synthesis The fungal strains of C. albicans (KCTC 7270) were cultured at 28 °C in YPD media (dextrose 2%, peptone 1%, and yeast extract 0.5%, pH 5.0 to 5.5). Fungal cells (final concentration × 10 CFU/ml) that were grown in 100 μl of YPD media were seeded in each well of a microtiter plate containing 100 μl of twofold serially diluted AMP-clay in buffer I or buffer II, as described above. After incubating for 24 to 30 h at 28 °C, the lowest concentration of AMP clay inhibiting the growth of fungi was microscopically determined to be the MIC (Park et al., 2007). The AMP clay was prepared by sol–gel synthesis (Burkett et al., 1997; Whilton et al., 1998). In brief, magnesium chloride (1.68 g, 7.24 mM) was first dissolved in ethanol (40 g), and the solution was stirred at room temperature. Then, 3-aminopropyltriethoxysilane (2.6 ml, 11.7 mM) was added drop-wise under magnetic stirring. The resulting white suspension that formed after few minutes was stirred overnight. The resulting products were centrifuged, washed in ethanol (3 × 50 ml) and dried at 40 °C. The white solid was ground to produce a powder (Patil et al., 2007). 2.2. AMP clay characterization The microstructure of AMP clay was investigated with Fourier transform infrared spectroscopy (FT-IR), silicon nuclear magnetic resonance ( 29Si NMR), X-ray diffraction (XRD) and transmission electron microscopy (TEM) analysis. The FT-IR spectrum of AMP clay was obtained with KBr pellets at the ratio of 10:90 by weight (NICOLET 6700, Thermoscientific, USA). Solid state Silicon-29-NMR measurements were performed to determine Si associated with different numbers of hydroxyl end groups using an Avance 400 (Bruker, Germany) at room temperature. The powder XRD pattern was obtained with an X-ray diffractometer (XPert PRO, PANalytical B.V., Almelo, Holland) using CuKα radiation at 20 mA and 40 kV. Powder samples were pressed onto a glass sample holder. Scans were recorded between 2° and 70° (2θ) with a step size of 0.05° and a scanning speed of 2°/min. TEM (TECNAI20, EEI, Netherland) imaging was performed on a bright field emission microscope with a Lab electron gun and an accelerating voltage of 200 kV. 2.6. Radial diffusion assay The experiment was performed as described previously by Nordahl et al. (2004) with some modification. Briefly, E. coli and S. aureus isolates were grown for 18 h at 37 °C in 10 ml of media. The underlay consisted of 1% agarose and 1/100 dilution of Luria Bertani (LB) broth in PBS (1% tryptone, 0.5% yeast extract, 1% sodium chloride pH 7.2). The overlay consisted of 6% LB and 1% agarose in distilled water. The bacterial culture × 105 colony forming units (CFU)/ml were mixed with 10 ml of underlay solution, allowed to melt at 48 °C and was then poured into petri dishes. When the agarose was solidified, approximately 4-mmdiameter wells were made, and the wells were filled by AMP clay solutions in various concentrations. Buffer in one well served as a control. To cause AMP clay diffusion on the under layer, the plates were incubated at 37 °C for h. The molten overlay was then poured on the underlay, and the plates hardened. This was followed by incubation at 37 °C for 48 h. Finally, the antibacterial activity was determined by measuring the diameters of the inhibitory zones around the well. The activity was represented in radial diffusion units (RDU) defined as (diameter of clear zone (in millimeters) − 6) × 10. All the experiments were performed triplicate, and the antibacterial activity was expressed as the mean of inhibition diameters (mm). 2.3. Microorganisms 2.7. Trans-membrane depolarization assay Bacterial and fungal cultures used in this present study were obtained from the Korean Collection for Type Cultures (KCTC). Gram positive species were Bacillus subtilis (KCTC 1918), Listeria monocytogenes (KCTC 3710) and Staphylococcus aureus (KCTC 1621) while the Gram negative species were Escherichia coli (KCTC 1682), Salmonella typhymurium (KCTC 1926) and Pseudomonas aerugenosa (KCTC 1637). Candida albicans, a fungal strain, was KCTC 7270. Antibiotic resistant E. coli strains (CCARM 1229 and CCARM 1238) and S. aureus strains (CCARM 3108, CCARM 3089) were obtained from the Culture Collection of Antibiotic-Resistant Microbes (CCARM) at Seoul Women's University in Korea. The trans-membrane depolarizing activity of the AMP clay was performed as described previously (Papo et al., 2002). The experiments were performed for the Gram-positive bacteria (S. aureus CCARM 3108), Gram-negative bacteria (E. coli CCARM 1229) and a fungal strain of C. albicans. The cells of bacteria and fungi were grown to mid-logarithmic phase at 37 °C and 28 °C under shaking at 150 rpm. Then, the cell resuspension OD600 of 0.05 was subjected to washing twice with the buffer (20 mM glucose, mM HEPES, and 0.1 M KCl at pH 7.3). The bacterial and fungal cells without the AMP clay were incubated with μM 3,3′-diethylthiodicarbocyanine iodide (DiSC3-5, which was acquired from Molecular Probes, Eugene, OR, USA) until there was no detection of fluorescent dye for h. Then, AMP clays with various concentrations were added, and the change in intensity of the fluorescence emission of the dye was measured at the excitation and emission wavelength (622 and 670 nm). 2.4. Antibacterial activity Bacterial cells were cultured at 37 °C in appropriate culture media. The antimicrobial activity of AMP clay was determined by the microdilution method. In brief, aliquots of bacterial suspensions (50 μl) in the mid-logarithmic phase at a concentration of × 105 colony forming units (CFU)/ml in an appropriate culture medium were added to each well containing 50 μl of AMP clay solution that had been twofold serially diluted in buffer I (10 mM sodium phosphate buffer, pH 7.2) or buffer II (phosphate buffered saline (PBS)), 1.5 mM KH2PO4, 2.7 mM KCl, 8.1 mM Na2HPO4, and 150 mM NaCl (pH 7.2). The plates containing the bacterial cells were incubated at 37 °C for 24 h. At the end of the incubation, 50 μl of 10-fold diluted samples were plated on appropriate agar plates and were then incubated for 24 h, after which the colonies were counted. The lowest concentration of AMP clay that completely inhibited growth was defined as the minimum inhibitory concentration 2.8. Intracellular enzyme activity Aspartate aminotransferase (AST) and alanine aminotransferase (ALT) activities were measured using an assay kit (Asan Pharmaceutical, Korea) based on the method (Ma et al., 2010; Reitman and Frankel, 1957). 6.25 mg/ml of AMP clay and 1.0 ml bacterial suspension 2×107 CFU/ml were added into the sterile tubes containing the LB broth. Then, the tubes were agitated on an orbital shaker at 150 rpm and cultured for h at 37 °C. This was followed by filtration. Then, the filtered solution was subjected to the enzyme activity assay. Untreated cells were used to determine background lysis, and cells treated with 1% Triton X-100 were Fig. 1. Characterizations of AMP clay structure with FTIR spectrum explains the trioctahedral band at 670 cm− (A), 29Si HPDEC NMR spectrum shows the T3 moiety for silane structures (B), X-ray diffraction pattern characterize 060 reflection at 0.156 nm for the trioctahedral structure confirmation (C), and TEM images of exfoliated sheets of AMP clay in distilled water with a layered edge view (D). used to determine total enzymes (100% lysis). Each experiment was performed in triplicate. 2.9. Scanning electron microscopy (SEM) For SEM analysis, the AMP clay was treated with mid log phase of E. coli, S. aureus and C. albicans in PBS (pH 7.2), and the strains were incubated at 37 °C and 28 °C. After 60 min, the AMP clay treated cells (2 × 10 CFU/ml) were centrifuged at 3000 rpm for min, followed by washing with the same buffer. The resulting supernatant was removed, and the pellets were fixed with 1% glutaraldehyde in 0.2 M sodium-cacodylate buffer (pH 7.4) for h at °C. Controls were the microorganism without AMP clay treatment. After fixation with glutaraldehyde, the samples were extensively washed with same buffer at pH 7.4, subjected to gold coating and were investigated with a scanning electron microscope (Hitachi S-2400N, Japan). 2.10. Determination of bactericidal activity by kinetic study The bactericidal activity of AMP clay on E. coli (CCARM 1229) and Streptococcus strains (CCARM 3108) was studied by a time-kill assay. The bacterial culture of 2×105 colony forming units (CFU)/ml was added to LB media containing AMP clay at 1× MIC and 2× MIC and was incubated at 37 °C. The samples were withdrawn at various time intervals for every half an hour up to h, diluted using PBS buffer and plated on the LB agar plates. The plates were incubated for 24–36 h at 37 °C, and the kinetics of bactericidal activity were determined by plotting the concentration of surviving bacteria against time (Jeong et al., 2010). 2.11. Storage stability of AMP clay The antibacterial activity of AMP clay was determined at different temperatures over several days. Five aliquots of approximately 600 μl Table The MIC values of AMP clay against microorganisms. MIC (mg/ml) Microorganism Gram (−) bacteria E. coli S. typhimurium P. aeruginosa Gram (+) bacteria S. aureus B. subtilis L. monocytogenes Fungal strain C. albicans Resistant strains E. coli CCARM 1229c E. coli CCARM 1238c S. aureus CCARM 3108d S. aureus CCARM 3089d a AMP clay Buffer Ia Buffer IIb 3.12 0.78 3.12 6.25 1.56 6.25 1.56 3.12 3.12 3.12 6.25 6.25 1.56 3.12 3.12 3.12 0.78 0.78 6.25 6.24 1.56 1.56 Buffer 1: 10 mM Sodium phosphate buffer, pH 7.2. Buffer II: phosphate buffered saline (1.5 mM KH2PO4, 2.7 mM KCl, 8.1 mM Na2HPO4, 135 mM NaCl), pH 7.2. c Multidrug-resistant Escherichia coli strains. d Multidrug-resistant Staphylococcus aureus strains. b Fig. 2. Radial diffusion assay: Panel A shows the results of testing at 6.25 and 12.5 mg/ml of AMP clay against E. coli; and Panel B shows the results of testing at 6.25 and 12.5 mg/ml of AMP clay against S. aureus. The control well was the only buffer. The diameter (mm) of the inhibition zone was measured and averaged in triplicate. were withdrawn from the stock solution. The aliquots were further subdivided into 300 μl each and stored at room temperature and °C. After a fixed number of days, the solutions were subjected to antibacterial activity. 3. Results and discussion 3.1. Characterization of AMP clay The synthesis of AMP clay was confirmed by FTIR, 29Si NMR, XRD and TEM. The FTIR spectrum of AMP clay, shown in Fig. 1A, exhibited absorption bands corresponding to terminal NH2 stretching (3600– 3200 cm − 1), NH2 bending (1658 cm − 1), CH2 stretching (2950– 2850 cm− 1), Si\O\Si stretching (1120 cm− 1), and Mg\O\Si bending (670 cm− 1). The latter spectrum explains the surface property of the clays. The 29Si NMR spectrum for AMP clay is shown in Fig. 1B. Three signals at −66.5,−58.6, and−48.4 ppm are associated with R-SiO3-(T3), R-SiO2-OH-(T2) and R-SiO-(OH)2-(T1) in the inorganic–organic backbone structure. The T3 moiety predominates among the silane structures. The XRD pattern of the AMP clay is characterized by a d001 spacing at 1.44 nm due to the presence of the aminopropyl organic chains between layers (Fig. 1C). The position of the 060 peak indicates the presence of a trioctahedral Mg-phyllosilicate clay mineral although the d060 value (0.156 nm) is larger than usually reported (Datta et al., 2007). TEM investigations showed the presence of delaminated 30–150 nm ultrathin sheets and clay sheets edge indicates the layered structure of 20 nm in thickness as shown in Fig. 1D (Ferreira et al., 2008; Holmström et al., 2007; Lee et al., 2010). Overall, AMP clay is hydrophilic and possesses protonated (R-NH2) groups, which create a number of bonding sites for ion exchange within the interlayer spaces and serve as surface group on the lamella, which are driven by electrostatic forces for their antimicrobial effect. 3.2. Antimicrobial activity Antibiotic resistance is increasing at a rate that exceeds by far the development of new types of antibiotic agents. Therefore, there is an increasing need to synthesize novel compounds with antibiotic activity. To our knowledge, the antimicrobial properties of organically modified derivatives of magnesium phyllosilicates have not been reported so far. The AMP clay had MICs of 6.25 mg (both in low and high ionic strength buffer) against many bacterial strains (Table 1). However, this AMP clay showed twofold higher activity against S. aureus and S. typhimurium than other bacteria. In addition, the MICs for resistant E. coli and resistant S. aureus were 3.25 and 1.25 mg, respectively, which were higher than their corresponding normal strains. The AMP clay also exerted antifungal activity against C. albicans cells. In contrast, this clay did not show antifungal activity against plant fungi pathogens (Verticillium sp., Phytophthora sp., and Fusarium wilt) (data not shown). Thus, the antimicrobial activity of this AMP clay clearly suggests that it is significantly selective for most microbial membrane cells except plant pathogens. 3.3. Radial diffusion assay (RDA) To distinguish variable activities of AMP clay towards E. coli and S. aureus, RDA experiments were performed. The results showed more activity on S. aureus but slightly less active against E. coli (Fig. 2). The zone of inhibition of the microorganisms is presented in Table 2. It is proposed that the AMP clay may serve as an antimicrobial agent for the control of pathogenic organisms in the presence of salt conditions. Generally, E. coli and S. aureus are human pathogens because they cause urinary tract infections, respiratory system failure, dermatitis, skin lesions, soft tissue infection and food poisoning. However, the toxicity of AMP in humans must be elucidated in further studies. Hemolytic and cytotoxicity assays have already shown that AMP clay has little or no toxicity against human cells (data not shown). Table Measurements of the zone of the inhibitory effect of AMP clay on microorganisms using radial diffusion assay. Zone of inhibition (units) Microorganism 6.25 mg/ml 12.5 mg/ml E. coli S. aureus 7–8 10–11 Fig. 3. Scanning electron micrographs of untreated and treated of E. coli KCTC 1682 (A), S. aureus KCTC 1621 (B) and C. albicans cells KCTC 7270 (C) untreated cells with normal smooth surfaces, shrunken and deformed cells at 1× MIC, and completely ruptured by AMP clay treated cells at 2× MIC. 3.4. Scanning electron microscopy (SEM) Visual observation by SEM micrographs of E. coli, S. aureus and C. albicans cells before and after the treatment with AMP clay showed extensive cell damage. The untreated E. coli cells had a smooth surface. In contrast, when the AMP clay was applied at 1× MIC, membrane damage was observed in the morphology of the treated bacteria, and, at 2× MIC, the cells were damaged severely, which led to heavy leakage and shape change with fragmentation. The fungal cells treated with AMP clay at 1× MIC level shrank, and, at 2× MIC, degradation Fig. 4. Time course curves of membrane depolarization activities of the AMP clay against intact E. coli KCTC 1682 (A), S. aureus KCTC 1621 (B), and C. albicans KCTC 7270 (C). Membrane depolarization was measured by an increase in the fluorescence of DiSC3-5 (excitation wavelength λex = 620 nm; emission wavelength λem = 670 nm) after the addition of AMP clay at different concentrations: 1/2× MIC (♦), 1× MIC (■), 2× MIC (▲). The fluorescence increase obtained using 0.1% Triton X-100 was taken as 100%. All the experiments were preformed in triplicate, and the mean was taken for statistical analysis. Error bars represent the standard deviation. due to the electrostatic interactions between the cationic aminopropyl groups on the clay lamellar surface and the anionic bacterial membrane (Nagrete-Herrera et al., 2004). There are also reports on the antibacterial effects of organoclays saturated with alkylammonium cations of quaternary amines and quaternary ammonium groups (Malachova et al., 2009; Özdemir et al., 2010, Rhim et al., 2009). Here, it is suggested that cationic (hydrophilicity) property of aminopropyl groups controls the antimicrobial properties of the AMP clay. 3.6. Analyses of leaking enzymes from cells Fig. 5. Percentage leakage of intracellular enzyme AST and ALT of the E. coli KCTC 1682 (black bars) and S. aureus KCTC 1621 (gray bars) microorganisms during the exposure to AMP clay at 6.25 mg/ml. All the experiments were preformed in triplicate, and the mean was taken for statistical analysis. Error bars represent the standard deviation. occurred (Fig. 3). These phenomena suggest that the AMP clay leads to swelling of the cells due to adsorption onto the bacterial cell surface and membrane disruption, which leads to cell death. However, Brown and Melling (1969) reported that Mg, Ca and Zn are present in the cell wall of the bacteria. It is suggested that divalent cations are responsible for the cross-linkages through the phosphate groups present in the lipoprotein and lipopolysaccharides of the cell walls. It therefore appears that this type absorption is closely linked with the initial binding of AMP clay to bacterial membrane. 3.5. Membrane depolarization assay To further determine the manner in which the interaction of this AMP clay with the bacterial plasma membrane results in cell death, the ability of the AMP clay to induce membrane depolarization in both bacteria and fungi was evaluated. Membrane depolarization was measured in intact microbial cells by monitoring the increase of DiSC3-5 fluorescence after addition of the AMP clay. This indicates the dose-dependent dissipation of the membrane potential, probably due to the leakage of protons and small ions (e.g., Na+ and K +) (Fig. 4). There was also a correlation between cytoplasmic membrane depolarization and MIC values, indicating that the AMP clay induced maximum dissipation of the membrane potential. This result indicated that the selected AMP clay has a direct effect on the living (microbial) energized cell membranes. The mechanism depends on the clay minerals involved on the functional groups and the physicochemical properties of the organic compounds. In normal conditions, the cell wall of the bacteria is negatively charged due to the presence of lipoteichoic acid, lipoproteins and lipopolysaccharides. The initial binding of bacteria and clays occurs AST and ALT are members of the transaminase family of enzymes. Except for structural injury of the cell wall or membrane permeability, there were no intracellular enzymes released from the bacterial cell. After treatment of the bacteria with AMP clay, the release rate of intracellular material increased as was shown by determining the cytoplasmic enzyme AST and ALT in the culture media (Fig. 5). This assay can be used as a general indicator of bacterial membrane disruption or injury (Korzeniewski and Callewaert, 1983; Ma et al., 2010). Apparently, this AMP clay interacted with the cell surface and led to penetration into the cytoplasmic barrier, causing larger membrane permeability (leakage of AST and ALT components). Then, it caused bacterial damage, which is consistent with the SEM observations. These results clearly indicate a membranolytic mechanism. This result also suggests that a sequence of steps is occurring at the membrane, beginning with depolarization (small ion leakage), followed by larger components leakage, which accompanies morphological changes in the microbial surface and ultimately cell death (Fig. 3). 3.7. Time killing assay The time-killing assay was performed as a final study to determine whether the antimicrobial action of AMP clay was microbicidal or growth inhibiting. Fig. shows the time-killing studies of AMP clay against E. coli (Fig. 6A) and S. aureus (Fig. 6B). At concentrations equal to or above the MIC, the AMP clay kills the strain significantly faster at 2× compared to 1× MIC concentrations. Also the AMP clay kills Gram positive bacteria slightly more slowly than Gram negative bacteria. This result is consistent with the membrane depolarization experiments described above, which show the immediate effects of this AMP clay on the integrity of the E. coli membrane compared to S. aureus. Variations in the cell wall structure and compositions between the Gram negative and Gram positive bacteria may be attributed to the different damage mechanism of the AMP clay. The AMP clay needs a longer time to reach the plasma membrane of the Gram positive bacteria than the Gram negative species due to their greater peptidoglycan layers. The fungi C. Fig. 6. Kinetics of the bactericidal activity of the AMP clay against E. coli KCTC 1682 (A) and S. aureus KCTC 1621 (B). Bacteria treated with AMP clay were diluted at the appropriate times and then plated on LB agar. The CFU was then counted after 16 h of incubation at 37 °C. White squares and blank squares are and times the MIC value, respectively; cells (2 × 105 CFU/ml) incubated in the absence of AMP clay served as controls. All the experiments were preformed in triplicate, and the mean was taken for statistical analysis. Error bars represent the standard deviation. Fig. 7. Schematic model of the action of AMP clay in bacterial cell membranes. AMP clay amino propyl group interacts electrostatically with the lipid membrane. Then, AMP clay is able to moves to the space between the two bacterial membranes and induces fusion or hemifusion of the inner leaflet of the outer membrane and the inner membrane of bacteria. These membrane fusions promote membrane permeability and leakage of the bacterial content and lead to cell death. albicans required more than h for 80% killing (data not shown), implying that this clay permeabilizes fungal membranes much more slowly than bacterial membranes. 3.8. Stability and storage properties The antibacterial activity of AMP clay did not change after storage for 30 days at room temperature and at °C. These results indicated that the AMP clay was stable in PBS buffer in storage conditions for all temperatures. Thus, the low cost and favorable storage stability AMP clay make it an excellent carrier for antimicrobial agents in a variety of environmental conditions. 4. Conclusions Synthetic AMP clay showed antimicrobial and membrane permeability activities. Based on the results obtained, a schematic representation of the mechanism of action of AMP clay is proposed (Fig. 7). AMP clay was organized in a perpendicular or parallel orientation across the outer leaflet of the outer bacterial membrane. The amino propyl group (positive charges) in the AMP clay interacted electrostatically with the negatively charged lipid membrane when the AMP clay was inserted into the inner leaflet of the bacterial inner membrane. This interaction disrupted the higher tight of lipid bilayer, encouraged membrane fusion events and promoted increased membrane permeability, which culminated with the leakage of the bacterial content. Characterization of the synthesized dispersed AMP clay will necessarily precede the development of therapeutic agents. In particular, the AMP clay may have some utility in dental caries prevention, and these possibilities are currently being investigated. 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Xue, S., Pinnavaia, T.J., 2010. Methylene-functionalized saponite: a new type of organoclay with CH2 groups substituting for bridging oxygen centers in the tetrahedral sheet. Appl. Clay Sci. 48, 60–66. . Paper Antimicrobial activity of delaminated aminopropyl functionalized magnesium phyllosilicates Gayathri Chandrasekaran a , Hyo-Kyung Han b , Geun-Joong Kim c , Hyun-Jae Shin a, ⁎ a Department of. property of aminopropyl groups controls the antimicrobial properties of the AMP clay. 3.6. Analyses of leaking enzymes from cells AST and ALT are members of the transaminase family of enzymes. Except. Gwangju 500-757, Republic of Korea abstract Keywords: AMP clay Antimicrobial activity Membrane permeability Intracellular enzymes leakage In this study, dispersed aminopropyl functionalized magnesium