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Acsami 7b15001
Acsami 7b15001
■ INTRODUCTION
Methicillin-resistant Staphylococcus aureus (MRSA) is an
toxicity to humans, thus enabling its biomedical use.12−14 While
the beneficial microbicidal effects of silver are well charac-
increasingly dangerous and antibiotic-resistant bacterial patho- terized, proposed mechanisms of action vary greatly and
gen.1,2 Infections caused by healthcare-acquired MRSA often include generation of reactive oxygen species (ROS), cell
cause systemic infections, such as bacteremia, pneumonia, or membrane damage, inhibition of respiration, and inactivation of
surgical site infections, and are contracted while the patient is in iron−sulfur clusters of bacterial dehydratases involved in amino
a healthcare setting.3,4 In contrast, community-acquired MRSA acid biosynthesis.15−17 Relevant to aqueous solutions and
(CA-MRSA) mainly causes skin and soft tissue infections, such biological conditions, silver occurs as metallic Ag or ionic silver
as cellulitis or skin abscesses, and its prevalence is rising.5−7 As (Ag+). The antimicrobial activity of silver is related to
the antibiotic-resistance crisis continues to worsen, identifying dissolution and bioavailability of ionic silver.18
complementary therapeutic strategies for combatting CA- In another example, Otto et al.19 have shown that some
MRSA infections is crucial.8 natural clays undergo ion exchange when placed in aqueous
Inorganic antimicrobial materials offer several advantages environments and that the release of metal ions, including iron,
over their conventional organic counterparts, including ease of
preparation, higher chemical and thermal stability, safety, and Received: October 4, 2017
cost effectiveness.9−11 Silver (Ag) exhibits broad-spectrum Accepted: October 27, 2017
antimicrobial activity at low concentrations with relatively low Published: October 30, 2017
copper, zinc, cobalt, and nickel, is responsible for the an aluminosilicate precursor mixture with the composition of
antibacterial properties of clays in vitro. Likewise, exchanging 3.0Na2O:1.0Al2O3: 4.0SiO2:32.4H2O. The mixture was prepared by
native metal ions in zeolites with antimicrobial ions such as Ag+, dissolving 4.555 g of NaOH pellets (Sigma-Aldrich) and 11.711g of
Cu2+, or Zn2+ represents a commonly used strategy for water glass (Sigma-Aldrich) in deionized water (DI water) (8.190 g),
followed by the addition of 5.735 g of metakaolin (MetaMax, BASF).
generating inorganic antimicrobial materials.20−24 Unlike the The mixture was stirred with a mechanical mixer (RW 60 digital mixer,
natural clay, zeolites can be synthesized, allowing control over IKA) at 800 rpm for 40 min, producing a visually homogeneous and
their purity, composition, and crystallinity,25,26 and their free-flowing dispersion. After addition of 15 mL of canola oil (J.M.
interconnected micropore channels release large quantities of Smucker Co., Crisco), the mixture was stirred for another 10 min and
antimicrobial metal ions into the environment to kill then transferred to 50 mL polypropylene tubes. After tightly capping
bacteria.27−30 Kwakye-Awuah et al.9 investigated the antimicro- the tubes, the mixture was heated at 60 °C for 54 h. The product,
bial action of silver-ion-exchanged zeolites, with a faujasite-type exhibiting a paste consistency, was washed with hot DI water multiples
(FAU) structure and particle size ranges from 2 to 9 μm, and times. The final product was collected after vacuum filtration with cold
reported complete bactericidal activity against Escherichia coli, DI water until the pH of the filtrates reached 8 approximately. The
product (nZeo) was then dried in a lab oven at 110 °C overnight and
Pseudomonas aeruginosa, and S. aureus within 1 h. A new
stored in sealed glass vials at room temperature for further use.
procedure capable of loading zeolite A with high concentrations Preparation of Ag-Ion-exchanged Zeolites (Ag−Zeolites).
of silver loading was reported with the resulting silver-loaded Ion exchange was carried out by following a previously reported ion-
zeolite A exhibiting antibacterial activity against E. coli and S. exchange condition for high silver loading.31 Zeolites, nZeo, or
aureus.31 reference microsized zeolite (13X, Sigma-Aldrich; hereafter called
Recently, nanostructured zeolites have attracted significant “mZeo”) (1 g) were suspended in 150 mL of UV-irradiated, nanopure
attention as an effective material in antimicrobial applications.27 water (dH2O), and the pH was adjusted to 5.5 by slowly adding 0.01
The premise is that with significantly shorter diffusion lengths M nitric acid. A silver nitrate solution (0.118 M) was prepared by
and higher external surface area, nanostructured zeolites would dissolving 1 g of AgNO3 (99.9%, Sigma-Aldrich) into 50 mL of dH2O,
be more effective in ion release32 and thus would exhibit greater and the pH was adjusted to around 5.5 by adding 0.01 M nitric acid.
The AgNO3 solution and the zeolite suspension were combined, and
antimicrobial efficacy than conventional microsized zeolites. the mixture was stirred gently for 24 h. The particles were collected by
However, the previous reports have indicated no significant centrifugation, washed with dH2O until the silver-ion concentration of
advantage of nanostructured zeolites or even worse perform- the supernatant became less than the detection limit of a silver-ion-
ance in comparison to conventional microsized zeolites. For selective electrode (10−6 M; Mettler Toledo, Part No. DX308), and
example, Dong et al.10 reported comparable antibacterial dried at 90 °C in a lab oven overnight. Preparation of all silver-
activities of silver-ion-exchanged and reduced silver nano- containing materials was completed in a darkroom to avoid potential
structured zeolites (EMT structure, 10−20 nm) against E. coli. photoreduction of silver ions.
In another study, FAU-type silver-ion-exchanged nanostruc- Characterization of Zeolites and Ag−Zeolites. Powder X-ray
tured zeolites (50−400 nm) killed E. coli and Enterococcus diffraction (PXRD) patterns of the dried samples were collected on
Siemens D5000 powder X-ray diffractometer with Cu Kα radiation
faecalis about 1.3−2 times more slowly than FAU-type with a wavelength of 1.5406 Å at a scan speed of 2.0 degrees/min and
microsized zeolite (3 μm).33 It is well known that the structural a step size of 0.04°. Scanning electron microscopy (SEM) images of
disordering and formation of amorphous surface layer on powdered samples were collected using an XL30 environmental FEG
zeolite surfaces prohibit efficient diffusion of molecules and (FEI) microscope operating at 15 kV acceleration voltage. Trans-
ions, which may be associated with poor performance.34 The mission electron microscopy (TEM) was carried out using a Titan 80-
surface resistance against molecular diffusion becomes domi- 300 FEG-TEM (FEI Co.) operated at 300 kV. The microscope was
nant as the crystal size decreases, already reaching 90% of the equipped with an ultrabright X-FEG electron gun and two biprisms
overall resistance when the particles are in the submicrometer located in the first and second selected area aperture planes, separated
range, far larger than nanocrystallites (<100 nm).35 by an “extra lens”. Both cameras were mounted on the TEM with the
K2 located downstream of the UltraScan. The microscope was
Since previous studies failed to report the kinetics of the Ag-
operated in the “counted mode” under the submit mode to count
ion release from silver-ion-exchanged nanostructured zeolites, it single-electron events. Brunauer−Emmett−Teller (BET) surface areas
is not clear yet if the poor ion release kinetics could be a were estimated with a Micrometrics ASAP 2020 volumetric adsorption
potential origin of inferior antibacterial performance.33 analyzer with nitrogen as the adsorbate at 77 K. Prior to analysis,
Furthermore, there have been no complete studies on the samples (300 mg) were degassed at 300 °C for at least 12 h under
concentration dependence of the antibacterial efficacy together vacuum until a residual pressure of ≤10 μm Hg was reached. Specific
with the cytotoxity of these new materials despite the surface areas were determined from the BET equation. The t-plot
controversies associated with different silver materials and method was used to distinguish the micropores from the mesopores in
eukaryotic cell cytotoxicity.36−41 Herein, comprehensive studies the samples and to calculate the external surface areas. The mesopore
were reported on material characteristics, silver-ion release volumes were calculated after subtracting the micropore volume from
the total pore volume. Mesopore size distributions were obtained
kinetics, and antibacterial performance of silver-ion-exchanged using the Barrett−Joyner−Halenda (BJH) method assuming a
nanostructured zeolites with a high crystallinity to establish cylindrical pore model.42 Elemental compositions of the zeolite
them as an effective antibacterial material. Their inhibitory and samples were determined by combining Rutherford backscattering
bactericidal activities against MRSA were also quantified against (RBS) and particle-induced X-ray emission (PIXE) data. RBS profiles
planktonic and biofilm cells and compared to those of and PIXE spectra were obtained with a 2.0 MeV He2+ particle beam
microsized silver-ion-exchanged zeolites. Cytotoxicity of high produced by a tandem accelerator in a high-vacuum area. RUMP
silver-loaded nanostructured zeolites was also assessed against simulation program was used to fit the RBS data, and GUPIX was used
macrophages, epithelial cells, and fibroblasts. to fit the He PIXE data. The hydrodynamic particle size of the nZeo
■
sample was measured on Malvern Nano-ZS instrument with a helium
neon (HeNe) laser with 4 mW output power at 632.8 nm wavelength
EXPERIMENTAL SECTION at the scattering angle of 173°. The refractive index of the material was
Synthesis of Nanostructured Zeolite. Nanostructured zeolite chosen to be 1.47. After dispersing particles via ultrasonication for 10
samples (hereafter called “nZeo”) were synthesized by first preparing min, samples were diluted and subjected to DLS measurements.
Silver-Ion Release Kinetics. A continuous flow technique was Microdilution Antibacterial Susceptibility Testing. Exponen-
employed to determine silver-ion release from the Ag−zeolites at tial phase MRSA cultures were diluted to 105 CFU/mL. Bacterial
room temperature. The flow system (Figure S1a) consisted of a suspensions (100 μL) in cation-adjusted Mueller Hinton broth
sample holder and a peristaltic pump (Masterflex L/S Pump model no. (CAMHB) were added to wells of 96-well microtiter plates containing
07554-80 with Pump Head no. 07518-00, Cole-Parmer), maintained at Ag−nZeo, Ag−mZeo, or vancomycin (0.25 μg/mL). Minimum
a constant flow rate. A silver-ion-selective electrode was used for a inhibitory concentration (MIC) was determined by measuring the
continuous measurement of silver-ion concentration of the effluent. absorbance at 600 nm after 24 h standing incubation at 37 °C. Cell
The sample holder (Figure S1b), consisting of a polypropylene sample viability was determined by plating duplicate 10-fold serial dilutions for
chamber with a diameter of 6.35 mm, was fitted with two each sample onto Mueller Hinton agar plates and enumerating
polytetrafluoroethylene (PTFE) filters (4 cm in diameter and 0.45 colonies after 16 h incubation at 37 °C.
μm in pore size) on both ends. A sample (0.1 g) was loaded into the Antimicrobial Time-Kill Testing of MRSA USA300 with Ag−
sample chamber, and the chamber was subsequently plugged with nZeo. MRSA USA300 cultures at mid-logarithmic phase were
silica wool on both ends. The front and back silica plugs were 8 and 19 centrifuged, resuspended in 1.1% Na2SO4 (w/v), and diluted to 107
mm thick and weighed 23 and 53 mg, respectively. The influent and CFU/mL. Bacterial suspensions were added to the wells of 96-well
effluent were carried through 3.1 mm (internal diameter) PTFE tubing microtiter plates containing Ag−nZeo or nZeo. The microtiter plate
connected to the peristaltic pump. An unbuffered 0.90 wt % NaNO3 was placed at 37 °C for the short duration of the experiment. Samples
(0.16 M) solution in DI water was passed through the chamber at two of the experimental wells were collected at 3, 7, and 10 min and
different flow rates of 5.0 (fast) and 0.6 (slow) mL/min, and the silver- subjected to 10-fold serial dilutions. At specified times, sodium
ion concentration was measured continuously in the receiving beaker thioglycolate (0.5% final concentration) was added to all experimental
for up to 25 min. The flow rates were constant within 3% throughout wells and included in serial dilutions to neutralize silver and prevent
the experiment. The measurements were carried out multiple times to additional killing. Cell viability was determined by plating duplicate
obtain triplicate data for each sample and for each flow rate. From the samples on TSA and enumerating colonies after 16 h incubation at 37
silver-ion concentration values measured by the silver-ion-selective °C.
electrode during each experiment, the accumulated amount of silver Biofilms. MRSA USA300 biofilms were grown in TSB
ion released at a given time (qt) was calculated by multiplying the supplemented with 1% glucose in MBEC 96-well microtiter plates
volume of the effluent by the silver-ion concentration at the time. The (Innovotech, Edmonton, Canada), which allows biofilm growth on lid
maximum monitoring time periods were limited by the volume of the pegs, for 24 h. During growth, methicillin (2 μg/mL) was added to
receiving beaker and the minimum concentration that the silver-ion- promote biofilm formation,43 and proteinase K (2 μg/mL) was
selective electrode can detect. included to minimize biofilm formation.43 After 24 h of growth, 2-fold
Bacterial Strains and Growth Conditions. MRSA USA300 LAC serial dilutions of Ag−nZeo (128, 256, 512, 1024, and 2048 μg/mL)
(received from Dr. Juliane Bubeck Wardenburg, University of Chicago, were added to the wells of the microtiter plate. A growth control,
Chicago, IL) was grown in trypticase soy broth (TSB) or on trypticase negative control using the nZeo, and positive control of 70% ethanol
soy agar (TSA). Cultures were grown overnight for 17−19 h at 37 °C were included. To determine if soluble silver ion at high
with gentle rotary mixing and subsequently diluted 1:40 into fresh concentrations would affect viability, 0.5% AgNO3 (w/v) was included
medium for growth to mid-logarithmic phase at 37 °C for 2.5 h. as a control. After static incubation at 37 °C for 24 h, biofilms were
Agar Diffusion Assays. The antibacterial activity of the Ag−nZeo thoroughly washed with dH2O and removed from lid pegs by indirect
and Ag−mZeo was determined by agar diffusion assays using 10 mg of sonication (Branson Ultrasonic Bath CPXH 1800 Series) in dH2O for
Ag−nZeo or nZeo and the following antibiotics: doxycycline (30 μg), 10 min. Cell viability was determined by plating duplicate 10-fold serial
amoxicillin with clavulanic acid (20/10 μg), trimethoprim/sulfame- dilutions for each sample onto TSA plates and enumerating colonies
thoxazole (25 μg), and oxacillin (1 μg) (Becton Dickinson, NJ). Wells after 24 h incubation at 37 °C.
(1 mm diameter) were generated in TSA plates by removing agar Eukaryotic Cell Culture. Detroit 551 (ATCC CCL-110) human
bores with the end of a sterile Pasteur pipet. Dry zeolite (10 mg) was skin fibroblasts and WM-115 (ATCC CRL-1675) human skin
subsequently funneled into each well by pouring through a sterile epithelial cells were maintained in Eagle’s Minimum Essential Medium
Pasteur pipet. The plates were incubated at 37 °C after MRSA (EMEM) supplemented with 10% fetal bovine serum (FBS). U-937
inoculation onto the agar surface, addition of the zeolites to the wells, (ATCC CRL-1593.2) human monocytes were maintained in RPMI-
addition of 5 μL of dH2O to respective wells, addition of 10 μL 1640 supplemented with 10% FBS. Cells were incubated at 37 °C with
suspension of zeolite (10 mg) on the agar surface, and placement of 5% CO2.
control antibiotic disks. Zones of inhibition were measured after 20− Cytotoxicity of Ag−nZeo against Eukaryotic Cells. Eukaryotic
24 h. Since the wells, surface suspensions, and antibiotic disks differed cell viability was assessed using a modified resazurin reduction
in diameter, four quadrant radius measurements were recorded and microtiter plate assay (REMA).44 Cells were seeded at 50% confluency
averaged for each zone of inhibition. To determine diameters of in a 96-well microtiter plate. Ag−nZeo were added to the wells at final
inhibition zones, radius measurements were doubled and disk width (6 concentrations of 2, 4, 8, 16, 32, 64, and 128 μg/mL. Growth controls,
mm) was added. All agar diffusion assays were performed at least three nZeo negative controls, and hygromycin B positive controls were
times. Prior to use, all nZeo materials were sterilized by 180 °C heating included in every assay. After adding resazurin (0.025 wt % final
for 2 h. concentration) to wells and incubating for 4 h, fluorescence of
Antibacterial Susceptibility Testing of Ag−nZeo in Suspen- resofurin, the product of resazurin reduction, was measured with a
sion. Exponential-phase MRSA cultures were prepared by diluting Spectramax M2Microplate Reader (Molecular Devices) at Ex/Em
overnight cultures into fresh TSB and continuing growth at 37 °C with 560/590 nm. To establish a baseline level of fluorescence, each
gentle rotary mixing until the cultures reached mid-logarithmic phase experimental well had an adjacent “blank” well, which was subtracted
of growth (∼2.5 h). Cells were then diluted to a concentration of 107 from the total fluorescence value of the experimental well.
CFU/mL (OD600 = 0.08−0.1). Prior to use, the cells were collected by Fluorescence values were compared to those of the growth controls
centrifugation and resuspended with sterile dH2O, followed by a to assess viability. Cytotoxicity was determined by calculating the
second centrifugation and final resuspension in dH2O. Cells were lowest concentration of Ag−nZeo that significantly reduced percent
adjusted to a concentration of approximately 107 CFU/mL. Sterilized viability.
nZeo, Ag−nZeo, mZeo, or Ag−mZeo was added to 1 mL of the initial Statistical Analyses. All biological experiments were performed in
bacterial suspension. Positive controls of bacterial growth without triplicate. Quantitative data were expressed as means ± standard
nZeo were included in each experiment. Cell viability was determined deviation (SD) or standard error of the mean (SEM). Statistical
by plating in duplicate on TSA plates either directly from the analyses were performed using repeated measures of two-way or one-
experimental samples or following appropriate 10-fold dilutions at the way analysis of variance (ANOVA) and Dunnett’s or Tukey’s multiple
specified times. comparisons tests. For interpretation of biofilm data, nonparametric
Figure 1. SEM images of (a) Ag−mZeo and (b) Ag−nZeo; TEM (c) and HRTEM (d) images of nZeo; TEM (e) and HRTEM (f) images of Ag−
nZeo.
one-way ANOVA and Dunn’s multiple comparisons test was used due that the particle size of the nZeo ranges from 100 to 700 nm
to the high level of variability in biofilm generation. Statistical analyses (Figure S2). From the TEM images of both nZeo (Figure 1c)
were performed using GraphPad Prism 6 (GraphPad Software, San and Ag−nZeo (Figure 1e), it was found that the submi-
Diego, CA), and adjusted p values of <0.05 were considered
statistically significant. crometer-sized particles are aggregates of intergrown zeolite
■
nanocrystals with a lateral dimension between 20 and 40 nm
RESULTS AND DISCUSSION (Figure 1c). Textual pores between the aggregated nanocrystals
revealed a hierarchical pore structure with both zeolitic
Morphology of Ag−Zeolites. The morphology of zeolite
micropores and mesopores to the aggregates. This type of
samples was investigated by SEM and TEM. Due to the beam
“nanostructured” zeolite material has gained significant interest
sensitivity of zeolites to the electron irradiation45 as well as the
fact that silver ions are readily reduced by electron beam,46 low- as their unique morphology allows improved molecular
dose TEM imaging mode was applied to obtain the intact diffusion.47−50 The individual nanocrystals also exhibited well-
morphology for the nZeo and Ag−nZeo samples. As shown in established facets and highly ordered lattice fringes in their
the SEM image (Figure 1a), Ag−mZeo exhibited a typical HRTEM image (Figure 1d), indicating a high crystallinity of
isotropic crystal shape of the FAU zeolite with average particle the nZeo. The HRTEM image of Ag−nZeo clearly showed the
sizes of 1−3 μm and sharp crystal facets. In comparison, the crystalline fringes associated with FAU-framework lattices,
Ag−nZeo showed submicrometer-sized particles with highly indicating that crystallinity is largely maintained (Figure 1f). It
textured surfaces (Figure 1b). The DLS measurement revealed is noted that our synthetic method for this type of
39274 DOI: 10.1021/acsami.7b15001
ACS Appl. Mater. Interfaces 2017, 9, 39271−39282
ACS Applied Materials & Interfaces Research Article
nanostructured zeolite is unique, and detailed studies on the respectively, based on the difference in the concentrations of
synthetic conditions and mechanism will be published silver ion in solution before and after the ion exchange. These
separately. The small particle sizes are likely due to the initial values are lower than the highest 34 wt % loading that has been
high molal concentration (10.1 m) of NaOH which is known to reported for zeolite X with a similar Si/Al ratio (1.5) in which
limit the particle growth in zeolite synthesis.51 all Na ions are exchanged by Ag ions.56 This incomplete Ag-ion
Chemical Structure and Ag Content of Ag−Zeolites. exchange in our samples and yet the absence of Na ions from
Zeolite samples were characterized by the powder X-ray the elemental analysis indicates that some Na ions were
diffraction (PXRD) patterns (Figure 2). Both the nZeo and the replaced by protons instead of Ag ions during ion exchange
whereby the pH was maintained around 5.5.25 In any event, it is
noted that the Ag loadings in both Ag−nZeo and Ag−mZeo
were relatively high in comparison to FAU-type zeolites used in
previous antibacterial efficacy studies (Table S1).9,57,58
Pore Characterization of Ag−Zeolites. The mZeo
exhibited a classical type I isotherm of microporous materials,
characterized by immediate uptake at low relative pressure
followed by horizontal adsorption and desorption branches
(Figure 3a). In contrast, the nZeo showed a combination of
type I and type IV isotherms, represented by a large H1-type
hysteresis, indicating the presence of mesopores (Figure 3a).
Figure 2. Powder X-ray diffraction (PXRD) of (a) zeolite and (b) Ag− The BJH desorption pore width distribution of the nZeo clearly
zeolite samples along with theoretical simulated pattern of NaX
revealed the presence of mesopores, whereby the pore width
zeolite. Peak (*) near 25° is due to anatase impurity in metakaolin.
range extends into the macropore region (Figure 3b).59 The
presence of the mesopores and small macropores is attributed
commercial mZeo samples showed diffraction peaks character- to the aggregate morphology of the nZeo observed in TEM
istic of highly crystalline FAU-type zeolite, as referenced to the studies (Figure 1e and 1f). The micropore volume of the nZeo
simulated pattern52 shown in Figure 2a. Given the high was 0.29 cm3/g, which is comparable to that of the mZeo (0.31
crystallinity of nZeo observed in TEM, the peak broadening cm3/g) (Table 1), hence corroborating the high crystallinity of
observed for the nZeo samples allowed us to estimate the
average crystalline domain size to be 24 nm based on the Table 1. Pore Characteristics of Zeolites and Silver-Ion-
Scherrer equation. The crystallite size is consistent with the size Exchanged Zeolites Used in This Study
range of the zeolite particles observed from the TEM studies.
SBET Smicro Vtotal Vmicro Vmeso
The Si/Al ratios of the nZeo and mZeo were 1.47 and 1.06, sample (m2/g) (m2/g) (cm3/g) (cm3/g) (cm3/g)
respectively, based on the unit cell constants with a FAU
mZeo 694 663 0.34 0.31 0.021
structure (a = 24.866 and 24.999 Å, respectively). Both samples
Ag−mZeo 469 405 0.27 0.19 0.09
can be considered as zeolite X based on their low Si/Al ratios.53
nZeo 754 617 0.57 0.29 0.29
After silver-ion exchange, both Ag−nZeo and Ag−mZeo
Ag−nZeo 458 322 0.48 0.15 0.33
samples exhibited PXRD patterns that are in agreement with
previous reports (Figure 2b).54,55 No Bragg peaks associated
with metallic silver were observed in either Ag−nZeo or Ag− the nZeo observed with the PXRD analysis. Assuming a 100%
mZeo (Figure 2b). However, the Bragg peaks were somewhat crystallinity of the mZeo, the crystallinity of the nZeo was 94%
broader for both Ag−nZeo and Ag−mZeo than for the original based on the micropore volume. However, the total pore
zeolite samples, indicating some loss of crystallinity during the volume for the nZeo was 0.57 cm3/g, much higher than 0.34
ion exchange. cm3/g for the mZeo (Table 1).
From the elemental analysis, the silver loadings for Ag−nZeo The micropore volumes of the Ag−zeolites were drastically
and Ag−mZeo were estimated to be 24 and 22 wt %, lower than those of the original zeolites (Table 1; 0.15 cm3/g
Figure 3. (a) Nitrogen sorption isotherms and (b) BJH pore width distributions of the zeolite samples.
Figure 4. (a) Average silver-ion release kinetics curves of Ag−zeolites at fast rate (FR, 5.5 mL/min) and slow rate (SR, 0.6 mL/min). Error bars are
shown in every 3 min. (b) Pseudo-second-order (PSO) linear regression of silver release kinetics data.
for Ag−nZeo and 0.19 cm3/g for Ag−mZeo). Such apparent the ion exchange. The loss of the crystallinity during the ion
decreases in micropore volumes are often associated with exchange was estimated to be only ∼17% for both nZeo and
amorphization of the crystal structure, which is already clear mZeo. However, it is noted that this microporosity-based
from the PXRD patterns of the samples. Since amorphization estimation of crystallinity did not consider the effect of Ag0
adversely affects the ion diffusion and release kinetics of cluster and nanoparticle formation during the preconditioning
zeolites, further examination is required to estimate the degree (degassing) of the Ag−zeolite samples which is onerous to
of the amorphization. Notably, it is important to consider that quantify in our studies.
the apparent loss of micropore volume may not be due to Silver-Ion Release Kinetics. To assess the efficiency of
amorphization. For example, a simple substitution of Na ions silver-ion release by Na-ion exchange, time-dependent silver-
(atomic weight = 22.99 g/mol) with Ag ions (atomic weight = ion release amounts were measured while the samples were in a
107.87 g/mol) in any zeolite would lead to a lower value of continuous flow of a Na-ion solution. In contrast to batch
“gravimetric” micropore volume (micropore volume per unit techniques for kinetics studies, continuous flow techniques are
mass of the sample) even when the corresponding “volumetric” advantageous in that the desorbed species (silver ions in this
micropore volume (micropore volume per unit volume of the case) are continuously removed from the ion exchanger (Ag−
sample) does not change. Furthermore, during degassing of the nZeo and Ag−mZeo), and hence, resorption of the silver ions
samples in the gas sorption studies the silver ions were is prevented.61 Furthermore, the vigorous mixing condition in
noticeably reduced to Ag0 to form clusters or nanoparticles, batch techniques can cause abrasion or disintegration of the
which can also affect the micropore volume results. However, ion-exchanger particles, particularly for the Ag−nZeo, during
the size of the metal ions may not be a significant factor as Na the measurement. For continuous flow techniques, it is
and Ag ions have similar ionic radii (1.02 and 1.00 Å, important to maintain an efficient flow of the effluent during
respectively, with CN = 6).60 measurements. On the basis of their tap densities, Ag−nZeo
In order to examine the effect of Ag ions, the Ag−nZeo and and Ag−mZeo had 81% and 73% volume porosity when
Ag−mZeo were immersed in 3 M NaNO3 in DI water (pH = packed. When packed in the sample chamber, the sample
6.5) overnight, which resulted in a partial exchange of the Ag region was about 7 and 6 mm long for Ag−nZeo and Ag−
ions by Na ions. The micropore volume and surface area of the mZeo, respectively. Lengths did not change noticeably after the
resulting bimetallic Na,Ag−nZeo and Na,Ag−mZeo are listed flow experiment, indicating that the samples maintained
in Table S2 together with those of the original (Na) and Ag− porosity during the continuous flow of the Na-ion solution,
zeolites used for the experiments. The contents of the hence minimizing the possibility of resorption of Ag ions.
remaining Ag ions in the samples were consistent with the qe Indeed, during all measurements, the flow rate remained
values from the kinetics studies (see below). The measured Na constant within 3% of deviation.
and Ag contents indicate that all of the protons in Ag−zeolites The silver-ion release curves for different samples (Ag−nZeo
were exchanged by Na ions, together with significant amounts and Ag−mZeo) at the two different flow rates are shown in
of Ag ions. The stoichiometric presence of the metal ions in the Figure 4. As shown in Figure 4a, most of exchangeable silver
Na,Ag−zeolites allowed us to estimate the effect of the atomic ions were released within 5 min for the fast flow rate (5.5 mL/
weight difference between Na and Ag on the gravimetric min) and 20 min for the slow flow rate (0.6 mL/min). These
micropore volume. If all of the Ag ions in Na,Ag−zeolites were flow rates were chosen because of their relevance to the
simply substituted by Na ions, the resulting hypothetical Na− operating conditions of medical apparatuses, such as intra-
zeolites would have micropore volumes of 0.24 cm3/g for nZeo venous cannulas. Ag−nZeo always released more silver ions
and 0.26 cm3/g for mZeo. These values amount to 77% and than Ag−mZeo in both slow and fast flow rates. After
83% crystallinity for nZeo and mZeo, respectively, in examining various kinetic models, including the zeroth-order,
comparison to the original 94% and 100% crystallinity before first-order, pseudo-first-order (PFO), second-order, pseudo-
39276 DOI: 10.1021/acsami.7b15001
ACS Appl. Mater. Interfaces 2017, 9, 39271−39282
ACS Applied Materials & Interfaces Research Article
Figure 5. Agar diffusion assays showing Ag−nZeo ion diffusion and MRSA inhibition. (A) Ag−nZeo and nZeo or (B) Ag−mZeo and mZeo (1 mg)
were applied (W,S: wet, surface; W,W: wet, well; D,W: dry, well) to the agar as described in the Experimental Section. Diameter measurements
(described in the Experimental Section) were determined from at least three independent experiments using (C) Ag−nZeo and nZeo or (D) Ag−
mZeo and mZeo. Values represent the mean diameter measurements and SEM of three independent experiments. The following antibiotic disks
were used as controls: doxycycline (D-30), amoxicillin with clavulanic acid (AmC-30), trimethoprim/sulfamethoxazole (SXT), or oxacillin (OX-1).
(****) Adjusted p ≤ 0.0001; (**) adjusted p ≤ 0.01; one-way ANOVA with Tukey’s multiple comparisons testing.
second-order (PSO), Elovich, and intraparticle diffusion (IPD) released by the time t and until the equilibrium, respectively,
models (Figure S3 and Table S3), the PSO model was found to and k is the rate constant with the boundary condition qt=0 = 0;
show the best fit with the experimental data (Figure 4b) for all qt=∞ = qe, the differential eq 1 can be transformed into a linear
of the experiments (Table 2). PSO kinetics is usually associated form of the integral eq 2
with chemical reaction and diffusional processes,62 which is
consistent with the silver-ion release process including the ion- dqt /dt = k(qe − qt )2 (1)
exchange reaction and ion diffusion inside pores of the zeolites.
Silver-ion release kinetics using the PSO model is described
t /qt = 1/kqe2 + 1/qe (2)
by eq 1, where qt and qe are the total silver-ion amounts
39277 DOI: 10.1021/acsami.7b15001
ACS Appl. Mater. Interfaces 2017, 9, 39271−39282
ACS Applied Materials & Interfaces Research Article
Table 3. MRSA Minimum Inhibitory Concentration (MIC) and Minimum Bactericidal Concentration (MBC) of Ag−nZeo and
Ag−mZeo, along with the Ag Equivalency for Each Concentration
sample medium time MIC (μg/mL) MIC Ag equiv (μg/mL) MBC (μg/mL) MBC Ag equiv (μg/mL) CFU reduction (%)
Ag−nZeo TSB 2h 8 1.92 >2048 >491 N/A
Ag−mZeo TSB 2h 4 0.88 >2048 >450 N/A
Ag−nZeo TSB 24 h 16 3.84 32 7.68 99.99
Ag−mZeo TSB 24 h 16 3.52 32 7.04 99.93
Ag−nZeo CAMHB 24 h 4 0.96 4 0.96 99.99
Ag−mZeo CAMHB 24 h 2−4 0.44−0.88 8 1.76 99.99
The half-life (t1/2) is calculated using eq 3, a unique PSO Since agar diffusion assays incubate for 20−24 h and Ag−nZeo
parameter to describe the sorption kinetics63 and Ag−mZeo have similar silver loading amounts, rapid silver-
ion release kinetics would not be a major contributing factor in
t1/2 = 1/kqe (3) assessing MRSA inhibition.
Minimum Inhibitory Concentration (MIC) and Mini-
On the basis of the PSO model, the qe values for Ag−nZeo
mum Bactericidal Concentration (MBC) of Ag−nZeo
were ∼0.95 mmol/g for both fast (5.5 mL/min) and slow flow
against MRSA. As a reference to clinical antimicrobial
(0.6 mL/min) rates (Table 2). In contrast, the corresponding
susceptibility testing, MRSA was incubated with 2-fold serial
qe values for Ag−mZeo were only 0.41 and 0.30 mmol/g,
dilutions of Ag−nZeo (0.25−1024 μg/mL) for 24 h in
despite the similar silver loading (Table 2). Furthermore, the
CAMHB. The result gave MIC and MBC values of 4 μg/mL
Ag−mZeo half-lives were more than double those of Ag−nZeo
at both flow rates (Table 2). Although not as drastic as ours, the Ag−nZeo (Ag equivalency of 0.96 μg/mL; Table 3).
improvement in release amounts for nanostructured or Subsequent susceptibility testing of MRSA in TSB revealed
nanosized zeolites has been reported previously for Ag− 24 h MIC and MBC values of 16 and 32 μg/mL, respectively,
LTA.64 The observation for those zeolite materials was for Ag−nZeo or Ag−mZeo (Table 3). While these values are
attributed to their larger surface areas and smaller particle excellent and compatible to the lowest values that have been
sizes which would be beneficial for better release kinetics, as the reported for zeolites with similar silver contents,31 the excellent
diffusion time is quadratically proportional to the diffusion performance was not associated with other components, such
length in an ideal case.64,65 In such a situation, one might as the anatase impurity (1.1 wt %), because control nZeo and
suspect that the total release amount from Ag−mZeo would mZeo did not exhibit antibacterial activity. Potential effects of
eventually reach values for the Ag−nZeo. However, con- Si/Al ratio are worth mentioning, as it has been reported that
vergence of Ag−mZeo and Ag−nZeo total release amounts Si/Al ratios can affect the antibacterial efficacy of FAU zeolites.
would occur very slowly, well beyond our measurement period. For example, AgY (Si/Al = 2.83; 9.7 Ag wt %) showed an MIC
Nonetheless, the probability of Ag−mZeo and Ag−nZeo value of 200 μg/mL against E. coli and Bacillus subtilis in
converging release amounts was not in agreement with the lysogeny broth agar, while AgX (Si/Al = 1.64; 9.8 Ag wt %)
best-fit PSO model where the silver-ion release appears to reach performed worse with MIC = 300 μg/mL.57 While not
its equilibrium during our measurement periods (Figure 4). completely implausible, however, such a Si/Al effect may not be
Alternatively, it is possible that the Ag−nZeo and the Ag− as significant in comparing nZeo and mZeo, as both are zeolite
mZeo have different silver-ion distributions in the FAU X due to their low Si/Al ratios (1.47 vs 1.06).
structure in such a way that more labile silver ions are available It is noted that the Ag−nZeo and Ag−mZeo did not display
in the Ag−nZeo than in the Ag−mZeo. The slight difference in significant differences in broth antimicrobial efficacy against
Si/Al ratio alone does not lead to discernible silver-ion MRSA (Table 3). Given the drastic difference in silver-ion
distributions in the same FAU structure.56,66−68 Unfortunately, release kinetics found for the Ag−nZeo and Ag−mZeo (Table
the peak broadness due to the small crystallite size and the 2), the disparity of the environments under which the release
slight loss of crystallinity does not allow the refinement of silver kinetics and antibacterial efficacy were studied could be
atom positions and their occupancies. contributing factors. While the release kinetics were charac-
Ag−nZeo and Ag−mZeo Inhibit MRSA in Agar terized in 1 h using 100 mg of Ag−nZeo or Ag−mZeo and
Diffusion Assays. To assess MRSA inhibition and ion unbuffered sodium nitrate, the broth antibacterial susceptibility
diffusion characteristics of the silver-ion-exchanged zeolite experiments were performed for 24 h with microgram
particles, agar diffusion assays were performed. Measurements quantities of Ag−nZeo and Ag−mZeo in complex bacterial
of silver-ion inhibition (11−12 mm) revealed that Ag−nZeo growth media. Organic molecules, particularly protein extracts,
and Ag−mZeo particles embedded in agar wells inhibited peptides, and amino acids found in TSB and CAMHB, could
MRSA growth (Figure 5). Both Ag−nZeo and Ag−mZeo bind free silver ions, limiting bioavailability and abrogating
particles that were embedded in agar wells yielded significantly differences in Ag−nZeo and Ag−mZeo silver-ion release
larger zones of inhibition than Ag−nZeo and Ag−mZeo kinetics. Furthermore, chloride anions present in bacterial
aqueous suspension applied to the agar surface (Figure 5). The growth media (86 mM in TSB and 1.3 mM in CAMHB) could
nZeo and mZeo not subjected to silver-ion exchange did not promote silver chloride precipitation, thereby negating any
inhibit MRSA (Figure 5). Both Ag−nZeo in aqueous potential effects of Ag−nZeo rapid silver-ion release kinetics
suspensions and dry Ag−nZeo embedded in agar wells and diminishing the antibacterial activity of free silver ions.
inhibited MRSA similarly, indicating that water present in the Rapid Killing of MRSA USA300 by Ag−nZeo and Ag−
agar is sufficient to initiate silver-ion exchange and diffusion. mZeo. To investigate the correlation between the silver-ion
These inhibition zones demonstrated that Ag−nZeo and Ag− release kinetics and the antibacterial activity of Ag−nZeo and
mZeo similarly inhibit MRSA growth in nutrient-rich TSA. Ag−mZeo, kill curve experiments were performed with MRSA
39278 DOI: 10.1021/acsami.7b15001
ACS Appl. Mater. Interfaces 2017, 9, 39271−39282
ACS Applied Materials & Interfaces Research Article
Cytotoxicity Testing of nZeo. To assess the cytotoxicity antibacterial efficacy and low cytotoxicity of Ag−nZeo
of nZeo against eukaryotic cells, three different human cell lines establishes the material as a new effective antibacterial against
were exposed to 2-fold serial dilutions of Ag−nZeo, ranging MRSA. Future experiments will test the practical use of Ag−
from 2 to 128 μg/mL, and monitored metabolic activity over 4 nZeo as well as their effects in vivo.
days. Ag−nZeo did not elicit cytotoxicity upon exposure to
WM-115 human skin epithelial cells, indicating that the
minimum cytotoxic concentration is >128 μg/mL (Table 4).
■
*
ASSOCIATED CONTENT
S Supporting Information
The Supporting Information is available free of charge on the
Table 4. Ag−nZeo Cytotoxicity against Human Cells for 4 ACS Publications website at DOI: 10.1021/acsami.7b15001.
Days
Tables of elemental compositions of silver-ion-exchanged
cytotoxic conc. Ag−nZeo cytotoxic conc. Ag equiv zeolite X samples and fitting parameters for silver-ion
cells medium (μg/mL) (μg/mL) diffusion kinetics models; photographs of the flow system
Detroit EMEM 64 15.3 and the zero-length column sample chamber; kinetics
551
curves fitting with various kinetics models (PDF)
■
WM-115 EMEM >128 >30.6
U-937 RPMI 64 15.3
1640 AUTHOR INFORMATION
Corresponding Authors
Detroit 551 human skin fibroblasts and U-937 human *E-mail: [email protected].
monocytes exposed to Ag−nZeo (2−128 μg/mL) showed *E-mail: [email protected].
significant reduction in viability at a minimum tested
concentration of 64 μg/mL (Table 4). These results indicated ORCID
that the cytotoxic concentrations are 4−16-times greater than Shaojiang Chen: 0000-0002-6952-1010
the concentrations necessary to inhibit MRSA growth (Table Dong-Kyun Seo: 0000-0001-9201-4079
3). Importantly, cytotoxicity was assessed with extended Notes
incubations over 4 days, which is four times longer than our The authors declare no competing financial interest.
■
longest antimicrobial susceptibility experiments (24 h).
Cytotoxicity of nZeo without silver was performed at 128 ACKNOWLEDGMENTS
μg/mL nZeo with no change in viability, demonstrating that Ag This research was supported by Public Health Service grant
ions are responsible for all cytotoxic activity. The observed
AI121733 awarded to S.E.H. and D.-K.S. from the NIH
cytotoxicity of the Ag−nZeo was consistent with a very recent
National Institute of Allergy and Infectious Diseases. We
report on silver-ion-exchanged nanosized EMT (Ag−EMT).41
gratefully acknowledge the use of facilities within the LeRoy
Our comprehensive studies indicate that Ag−nZeo is an
Eyring Center for Solid State Science at Arizona State
antibacterial material that can rapidly and effectively kill MRSA
University and Gatan, Inc. for the loan of the K2 IS camera
with dosages (0.5−1 μg/mL; 2 h) much lower than
for the TEM studies as well as BASF for their donation of
concentrations and exposure times (64−128 μg/mL; 4 days)
metakaolin.
■
whereby the material becomes cytotoxic.
■ CONCLUSIONS
Comprehensive and comparative studies on porosity, morphol-
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