Novel triclosan-bound hybrid-silica nanoparticles and their enhanced antimicrobial properties

Article abstract of DOI:10.1002/adfm.201101557

The design and synthesis of novel hybrid-silica nanoparticles (NPs) containing the FDA-approved antimicrobial triclosan (Irgasan) covalently linked within the inorganic matrix for its controlled, slow release upon interaction, is reported. The NPs are in

Full text of DOI:10.1002/adfm.201101557

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Novel Triclosan-Bound Hybrid-Silica Nanoparticles
and their Enhanced Antimicrobial Properties
Igor Makarovsky, Yonit Boguslavsky, Maria Alesker, Jonathan Lellouche, Ehud Banin,
and Jean-Paul Lellouche*
proteins.[8–10] The use of silica as a carrier
system has received much attention because
of its physical and chemical versatility (e.g.,
its ability to tune the mesoporous structure
and control specific surface properties) and
its nontoxic nature.[11,12] Indeed, compounds
that can bridge inorganic and organic mate-
rials, such as silane-coupling agents bound
to various functional moieties, are available.
For example, silane-coupling agents can be
used for surface grafting (via free silanol
groups) of antibacterial agents. In order to
increase the content of the deliverable mole-
cules inside the inorganic matrix, one may
The design and synthesis of novel hybrid-silica nanoparticles (NPs) con-
taining the FDA-approved antimicrobial triclosan (Irgasan) covalently linked
within the inorganic matrix for its controlled, slow release upon interaction,
is reported. The NPs are in the range of 130 30 nm in diameter, with a
smooth and spherical morphology. Characterization of the hybrid-silica NPs
containing triclosan, namely T-SNPs, and their appropriate linkers is accom-
plished by microscopic and spectroscopic techniques. Preliminary antimicro-
bial activity is studied through bacterial-growth experiments. The T-SNPs are
found to be superior in killing bacteria, as compared with the free biocide.
incorporate a suitable silane derivative into the sol-gel synthetic
process of the inorganic matrix in situ.[13] The modified Stöber
method can be employed for this purpose.[14]
1. Introduction
In the context of increased bacterial resistance to common antibi-
otic treatments, nanoscale materials offer a unique opportunity to
bring innovative and more-effective solutions to bacterial-disease
control. Currently, several options have already been success-
fully explored using various types of organic/inorganic and
composite nanomaterials. For example, such options include:
i) silver (Ag) nanoparticles (NPs);[1] ii) carbon-based nanomate-
rials like single-walled carbon nanotubes (SWCNTs), C60 fuller-
enes, and graphene oxide;[1–4] iii) FDA-approved bioactive glasses
(SiO2–Na2O–CaO–P2O5) of the 45S5 type;[1] iv) metal oxide (TiO2,
MgO, ZnO) NPs;[1,5] magnesium fluoride (MgF2) NPs;[6] and
v) chemically modified (antibiotic-decorated) gold (Au) NPs.[7]
Each of these optional nanoscale systems clearly possesses its own
specific mechanism of action, cellular target(s), potential delivery
capability, and overall therapeutic advantages and disadvantages.
Inorganic-organic hybrid-silica nanoparticles (NPs), which
are functionalized ceramic materials prepared from silicon
dioxide, otherwise known as silica (SiO2), have been employed
as carrier systems for the controlled delivery of drugs, genes, and
There are several ways to incorporate guest molecules into
a sol-gel system. The first is the physical incorporation of drug
substances, drug loading for example, into sol-gel-derived silica
materials. This method was first introduced in 1983.[15] The
sol-gel method is a simple, versatile and low-temperature way
of preparing porous, amorphous ceramic materials that are
light- and heat-stable without being hazardous to humans or to
the environment.[16] Silica is an essential nutrient and plays an
important role in many functions of living organisms, having
a direct relationship to mineral absorption. Amorphous silica
is nontoxic, biocompatible and biodegradable, freely dispersible
throughout the body and ultimately excreted in the urine.[17] The
feasibility of mesoporous silica as a drug-delivery system has
been documented since the beginning of the millennium and
has been subject to a number of reviews.[14,18–23] These studies
have demonstrated that mesoporous silica can serve as a vehicle
for the successful intracellular delivery of otherwise poorly
soluble or membrane-impermeable agents.[24–26] The ceramic
carrier matrix effectively protects the payload molecules from
enzymatic degradation during delivery, as well as increases the
solubility and permeability of a drug with otherwise poor bio-
availability. Room-temperature-processed mesoporous silica
is rapidly dispersed in water, but, when loaded with a large
amount of guest molecules such as drugs, the aqueous solu-
bility can be adversely affected due to the increased hydropho-
bicity of the pore walls.[17,27] The large adsorption (drug-loading)
capacity is one of the many useful features of mesoporous silica
materials, together with the control of material properties on
the nanometer scale, such as the pore and particle sizes.[28]
An alternative to drug loading is covalently binding the
guest molecules to the ceramic matrix. Although the reloading
I. Makarovsky, Dr. Y. Boguslavsky, M. Alesker, Prof. J.-P. Lellouche
Institute of Nanotechnology and Advanced Materials
Department of Chemistry
Bar-Ilan University
Ramat-Gan 52900, Israel
E-mail: Jean-Paul.M.Lellouche@biu.ac.il
J. Lellouche, Dr. E. Banin
The Mina and Everard Goodman Faculty of Life Science
Institute for Nanotechnology & Advanced Materials
Bar-Ilan University
Ramat-Gan 52900, Israel
DOI: 10.1002/adfm.201101557
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property is thus lost in several cases, the greater stability, along
with other advantages, such as controlled slow release, protec-
tion from degradation and modifiable solubility, are gained.
We have chosen this alternative as a model for our studies
and triclosan (Irgasan) as the guest molecule to be incorpo-
rated inside the inorganic matrix. Triclosan is a well-known,
commercial, Food and Drug Administration (FDA) approved,
synthetic, non-ionic, broad-spectrum antimicrobial agent, pos-
sessing mostly antibacterial, but also some antifungal and anti-
viral properties.[29] Triclosan is fairly insoluble in aqueous solu-
tion, unless the pH is alkaline, and is readily soluble in most
organic solvents. It is chemically stable and can be heated up to
200 °C for up to 2 h.[30] This thermal stability makes it suitable
for incorporation into various reinforced plastic materials.
In 1998 the molecular target of triclosan was finally eluci-
dated. Numerous studies conducted on different bacteria strains
showed that triclosan acts on a defined bacterial target in the
bacterial-fatty-acid biosynthetic pathway, the nicotinamide ade-
nine dinucleotide (NADH)-dependent enoyl-[acyl carrier pro-
tein] reductase (ENR).[31–33] ENR catalyzes the final, regulatory
step in the fatty-acid synthase cycle, the reduction of a carbon-
carbon double bond in the enoyl moiety that is covalently linked
to an acyl carrier protein. The phenol ring of triclosan forms
a face-to-face interaction with the nicotinamide ring, allowing
extensive π–π-stacking interactions. Additional Van der Waals
contacts are made by both phenolic rings, with residues lining
the active site, the substrate-binding pocket of the enzyme and
the nucleotide cofactor. Hydrogen bonds are formed by the
phenolic hydroxyl group of triclosan with the 2′–OH group of
the nicotinamide ribose of the nucleotide and with the phenolic
oxygen of tyrosine 156, which is believed to function as a proton
donor during the catalytic cycle of ENR.[32,34]
Over the last 30 years, triclosan has become the most-potent
and widely used bisphenol.[30] Triclosan is used in many con-
temporary consumer and professional health-care products.
These include hand soaps, surgical scrubs, shower gels, deo-
dorant soaps, health-care-personnel hand washes, hand lotions
and creams, toothpastes, mouthwashes, and underarm deo-
dorants.[29] Triclosan is also incorporated into fabrics and plas-
tics, including children’s toys, toothbrush handles, as well as
surgical drapes, hospital over-the-bed table tops, etc. Triclosan
has been extensively tested in humans and animals. Acute-
toxicity, chronic-toxicity, mutagenicity, reproduction and tera-
tology investigations have been recently reviewed.[29,35] Due to
its poor bioavailability, triclosan-based products usually appear
as formulations in various organic/inorganic matrix-loaded
compounds.
to be broken by enzymes, subsequently releasing the active
triclosan, which will act upon its target inside the cell. Basi-
cally, the enzymes produced by the bacteria themselves will be
responsible for the release of the antimicrobial agent. Such a
covalent linkage ensures the above-mentioned properties and
also prevents leaching, providing an improved mechanism for
controlled release.
Herein, we report the design and synthesis of the innovative
linker, triclosan-(3-(triethoxysilyl)propyl)carbamate (TTESPC),
and the resulting nanosized, silica-based particles. Each particle
contained the FDA-approved antibacterial agent, triclosan, cova-
lently linked within the matrix for its controlled slow release
upon interaction. The particles were prepared according to a
modified Stöber method and the biocide-silanated linker was
incorporated into the silica matrix during the particle-formation
process. We demonstrate, that, for the first time (according
to an extensive literature survey), a covalently bound biocide,
namely triclosan, in an inorganic particulate matrix exerts its
biological activity to the full extent and even exceeds that of the
free biocide.
2. Results and Discussion
2.1. NPs Containing Covalently Bound Triclosan (T-SNPs)
The synthesis of triclosan-(3-(triethoxysilyl)propyl)carbamate
(TTESPC) was accomplished through a direct carbamoylation
of triclosan (Figure 1) with 3-isocyanatopropyltriethoxysilane in
the presence of the Lewis acid, tetraoctyltin,[46] Sn(Oct)4, as out-
lined in Scheme 1, and gave a yield of 64%. This is a new com-
pound designed specifically to introduce the triclosan moiety
inside the inorganic silica matrix. The relatively facile and effi-
cient synthesis allows for an easy scale-up process.
It is a well-known fact that tin-based organometallic com-
pounds catalyze reactions between hydroxyl compounds and
isocyanates very efficiently.[47] The proposed structure of the
TTESPC reagent was confirmed by 1H-NMR, 13C-NMR, IR, and
UV–vis spectroscopy and mass spectrometry (MS) (Figure S1,
Supporting Information). The presence of the aliphatic moieties
upfield on the one hand (δ = 3.81, 3.20, 1.63, and 1.21 ppm) and
the aromatic peaks slightly shifted downfield and more clus-
tered indicate that covalent attachment had indeed occurred.
The broad triplet at 5.3 ppm due to the proton attached to the
carbamate nitrogen further confirmed that the reaction had
succeeded (see the Experimental Section).
Once the TTESPC linker had been synthesized, the next step
was to prepare the corresponding nanospheres with a desirable
diameter of around 100–150 nm. We achieved this goal through
a series of experiments using a modified Stöber method as
Numerous papers have already been published on triclosan
loading into an organic or inorganic matrix (e.g., melt-mixed
into polystyrene for antibacterial efficacy, sustained-released
from TiO2 particles, inclusion into biodegradable β-cyclodextrin
for bacteria-growth resistance, and loaded into poly(D,L-lactide-
co-glycolide) (PLGA), poly(D,L-lactide) (PLA) and poly(vinyl
alcohol) (PVAL) copolymers for periodontal disease, etc.).[36–45]
Triclosan-loaded NPs (TiO2 nanocapsules) have also been pre-
pared, but triclosan has never been covalently bound to an inor-
ganic matrix.[37] In this study we developed a linker that will
allow covalent binding between silica NPs and triclosan. The
covalent bond between the linker and the biocide is designed
Cl
OH
O
Cl
Cl
Figure 1. Chemical structure of triclosan (Irgasan).
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O
OH
(EtO)3Si
N
H
O
O
O
(EtO)3Si
NCO
Sn(Oct)4/Tol.
Cl
Cl
Cl
Cl
Cl
Cl
Triclosan
TTESPC
NH4OH(aq):TEOS:TTESPC(EtOH)/EtOH
600C/24 h
OH
O
Cl
Cl
Cl
NCO
(EtO)3Si
NCO
T-SNPs
Scheme 1. A synthetic pathway for the fabrication of T-SNPs.
mentioned below. We found that a temperature dependence
exists between the amount of linker added to the reaction mix-
ture, the amount of linker found in the formed NPs and their
subsequent relative diameters.
was measured by elemental analysis (Equation 1). The rather-
low final concentration of triclosan in the NPs was probably
due to the relatively high hydrophobicity of the TTESPC, which
hinders the incorporation of this sterically demanding linker
into the midst of the inorganic, hydrophilic silica matrix.
Figure 3 shows scanning-electron-microscopy (SEM) and
transmission-electron-microscopy (TEM) micrographs of the
T-SNPs obtained in a typical experiment at 60 °C with 2.5% (w/v)
of TTESPC. One can appreciate from these micrographs
the smooth, spherical morphology of the NPs. These NPs
were obtained with a narrow size distribution and an average
diameter of 130 30 nm. DLS studies showed a hydrodynamic
diameter of 164.3 nm (Figure S2, Supporting Information),
which is in a good accordance with the actual TEM size of
similar dried particles, when considering the likely adsorption
of water molecules onto the NP surface.
ζ-potential and particle-size titration versus rising pH was
performed in order to estimate the relative stability of the NPs
in aqueous media (Figure S3, Supporting Information). As
one can observe, the ζ-potential increases in absolute value,
from −8 mV at a pH of 3.7 to −36 mV at a pH of 12, which
means that the particles became more stable. The ζ-potential
is ≈−20 mV when the pH reaches a physiological value
(pH = 7.4). Interestingly, the linker molecules, which are
hydrophobic in nature, probably prefer to be oriented towards
the inner part of the particle. Thus, the linker molecules have
minimal influence on the net surface potential of the NPs.
Further evidence of the stability of these particles is the lack
Figure 2A shows a correlation between the average nanopar-
ticle diameter (measured by dynamic light scattering (DLS))
and the TTESPC’s initial concentration. We observed that by
reducing the linker’s initial concentration from 10 to 2.5 wt%,
the particle’s diameter started stabilizing at a 200 nm value
until reaching a final 160 nm one. The optimal compromise
between the final triclosan weight percent inside the NPs and
their corresponding diameter was obtained when an initial con-
centration of 2.5% (w/v) of the linker was added to the reac-
tion mixture. In order to estimate the amount of linker present
in the NPs, a measurable molecular entity could be used as
an internal standard. For this purpose, measurable quantities
of chlorine (Cl) could be directly correlated to the amount of
incorporated triclosan: its quantity was determined by ele-
mental analysis. Figure 2B describes the correlation between
the initial linker concentration and the triclosan weight per-
centage inside the T-SNPs as a function of synthesis tempera-
ture. The dashed curve is for syntheses performed at 25 °C, for
which no dependency of the triclosan content on the TTESPC
concentration was observed. The solid curve relates to syn-
theses performed at 60 °C. One can observe that as the linker
percentage diminishes and reaches a certain value, (2.5% w/w),
the triclosan content inside the T-SNPs rises to 0.79 wt%. The
amount of triclosan was calculated from the Cl quantity that
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A
(a)
(b)
4000
3000
2000
1000
0
B
(a)
(b)
.9
.8
.7
.6
.5
.4
.3
.2
2
4
6
8
10
TTESPC Conc./ %, w/v
Figure 2. A) The effect of the initial TTESPC concentration on the average
particle diameter, as measured by DLS, at 25 °C (dashed line, a) and
60 °C (solid line, b). B) The effect of the initial TTESPC concentration on
the triclosan content, as calculated from elemental-analysis data (dashed
line, a: 25 °C; solid line, b: 60 °C).
Figure 3. A) HR-SEM micrograph of T-SNPs (scale: 100 nm). B) TEM
(scale: 500 nm) micrograph of T-SNPs.
of aggregation over a relatively wide pH-value range, since the
measured average diameters of the T-SNPs remain practically
stable until a pH of 10, disclosing 150–170 nm values (i.e., very
close to their TEM-measured diameter).
of the CO stretching (νC=O) band gives insight into the molecular
interaction occurring in the system under study. In particular, the
shift to lower wavenumbers (“red-shift”) may be attributed to the
presence of hydrogen-bond interactions with the carbamate car-
bonyl inside the inorganic solid SiO2 matrix. The opposite effect,
namely the shift to higher wavenumbers, (“blue-shift”) of the νCH
band of alcohols in polar organic compounds and surfactants
observed for aqueous solutions, has also been observed.[48]
Figure 5 illustrates the thermal analyses performed on the
T-SNPs. Curve a corresponds to the thermogravimetric-analysis
(TGA) thermogram (using a 25–500 °C temperature profile;
10 °C min−1 , N2 atmosphere, 100 mL min−1 ). This thermogram
consists of several slopes, with one main-step slope showing
approximately 3% weight loss (at approximately 100 °C), which
may correspond to a loss of water molecules entrapped in the
inorganic matrix. The second slope, between approximately 100
and 190 °C (approximately 2% weight loss) may correspond to
a loss of the water molecules that participate in the hydrogen
bonding between the silanol groups on the surface and near
the surface[49] and a release of CO gas from the NPs as their
Figure 4 shows the Fourier transform IR (FTIR) spectra of
the linker, TTESPC, and the T-SNPs and reveals the character-
istic peaks of the functional groups present. The IR spectrum
of TTESPC (Figure 4a) reveals absorption peaks at 3320 cm−1 ,
which corresponds to the carbamate NH asymmetric-stretching
band, 2885–2974 cm−1 (alkane CH2 asymmetric stretching
bands), 1717 cm−1 (carbamate C=O stretching band), 1487 cm−1
(aromatic C=C stretching bands), 1280 cm−1 (Si–O–C stretching
bands), 1080 cm−1 (phenolic symmetrical C–O stretching bands),
and 789 cm−1 (C–Cl stretching bands). The IR spectrum of the
T-SNPs (Figure 4b) shows a broad curve with a peak at 3390 cm−1 ,
which corresponds to the carbamate NH and alkane CH2
asymmetric-stretching bands, a peak at 1639 cm−1 (a red-shifted
carbamate C=O stretching band), a broad curve with a peak
at 1108 cm−1 (Si–O–C ether stretching bands, aromatic C=C
stretching bands and phenolic symmetrical C–O stretching
bands), a peak at 949 cm−1 (aromatic C–H stretching bands), and
a peak at 789 cm−1 (C–Cl stretching bands). A wavenumber shift
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The exotherm with its peak at 320 °C may be attributed
to the formation of radicals created as a result of chlorine-radical
combination into Cl2. The same peak appears in the DSC
thermogram of the linker itself but shifted to a much lower
temperature (approximately 190 °C). This can be explained
by the fact that the inorganic matrix shields and protects the
entrapped linker molecules from the external environment,
resulting in a higher degradation temperature. Curves b (dotted
curve) and c (short-dashed curve) correspond to DSC thermo-
grams of triclosan and of the linker, TTESPC, respectively. One
can clearly see one sharp endotherm in each curve, with peaks
corresponding to the melting points of triclosan and TTESPC
(i.e., 56 °C and 83 °C in curves b and c respectively). There is
another endotherm, at approximately 344 °C, corresponding
to the boiling point of triclosan. Beyond this point, the NP
decomposition begins. The lack of the peaks mentioned before
in curve d further emphasizes the covalent attachment of the
linker to the inorganic matrix, as well as the absence of the free
biocide or linker within the inorganic matrix of the T-SNPs.
2.0
1.5
1.0
.5
(a)
1280
1487
1080
1717
3320
789
2885-2974
?
0.0
4000
3500
3000
2500
2000
1500
1000
500
Wavenumber/ cm-1
(b)
1000-1300
?
3
2
1
0
2.2. Preliminary Biological Results
In order to investigate the antimicrobial activity of these NPs,
a series of biological experiments were designed and carried
out. Figure 6 depicts the antimicrobial activity of the T-SNPs
against two common bacterial pathogens, Escherichia coli
(E. coli) (Gram-negative) and Staphylococcus aureus (S. Aureus)
(Gram-positive), in the presence of different concentrations of
T-SNPs. The results presented demonstrate that, for the two
types of bacteria, the T-SNPs caused a reduction in growth in
a dose-dependent manner and the minimal bactericidal con-
centration (MBC) measured was 10 μg mL−1 for both E. coli
and S. aureus. Despite the similar MBCs, we observed a differ-
ence in the sensitivity of these strains to the T-SNPs treatment.
E. coli seems to be more sensitive compared with S. aureus,
and complete killing was observed after 14 h, whereas com-
plete killing of S. aureus was observed only after 22 h.
Unloaded SNPs, and TTESPC (the linker itself) served as con-
trols and no antibacterial activities were observed.
468
949
789
1639
2800-3700
Figure 4. a) FTIR spectrum of the linker, TTESPC. b) FTIR spectrum of
the T-SNPs.
degradation starts. This fact may be corroborated with the DSC
thermogram (see the long-dashed curve d, Figure 5), with two
proximal exothermic peaks between 110 and 200 °C. Afterwards,
the degradation of the organic content begins, as evidenced by
the moderate weight loss in the TGA curve (approximately 6.5%
weight loss) and the moderate exotherm in the DSC thermogram.
Next, we wanted to rule out the possibility of the leaching of
triclosan from the NPs (even without the presence of bacteria),
which would suggest that the T-SNPs are unstable. A high con-
centration of T-SNPs (50 μg mL−1 ) was incubated in a growth
medium (without bacteria) for 24 h (same conditions as for the
killing experiments mentioned below). Then, the NPs were cen-
trifuged, filtered and the supernatant was incubated separately
with E. coli and S. aureus and their growth was monitored. It
can clearly be observed from the plotted graphs (Figure 6A,B,
dashed curves) that the growth rates of the two pathogens were
not impeded and resembled the control. This strongly suggests
that the activity required the presence of bacteria and that either
no triclosan was released from the T-SNPs in the first incubation
step or the amount of released triclosan remained very low and
was not sufficient to impede bacterial growth. To further vali-
date that the antimicrobial properties of the T-SNPs were solely
mediated by the release of triclosan, a second series of
experiments was done. In this experiment, we grew a tri-
closan-resistant E. coli RJH108 strain with the highest tested
100
98
96
94
92
90
88
8
6
d
4
2
0
b
-2
-4
-6
-8
-10
-12
a
c
100
200
300
400
500
Temperature/°C
Figure 5. TGA thermogram of T-SNPs (curve a) and DSC thermograms of
triclosan (curve b), TTEPSC (curve c), and T-SNPs (curve d).
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E. coli
bound triclosan. At this concentration, the free
triclosan only began to impede the bacterial
growth, whereas the T-SNPs killed all of the
bacteria. At this concentration of triclosan
(≈0.04 μg mL−1 , dotted lines in Figure 7a,b),
the T-SNPs afforded an increase of 5–6 log
in killing capacity compared with the free
biocide. It is likely that the strong antimicro-
bial effect stems from the protection of the
harsh chemical and/or biochemical environ-
ment provided by the NPs during delivery.
For example, silica matrices are known to
provide UV protection.[52–54] This means
that these matrices prevent the UV-sensitive
triclosan from UV-induced decomposition
to deleterious dioxins.[55] Furthermore, the
T-SNPs may deliver a local high concentra-
tion of triclosan near the bacteria via mem-
brane attachment, thus increasing its overall
efficacy.
A
14
RJH 108 (50
Supernatant
µ
g mL-1
)
)
12
10
8
0 µ
g mL-1
SNPs (50
µ
g mL-1
)
TTESPC (50
µ
g mL-1
2
µ
g mL-1
g mL-1
6
5
µ
4
10
25
50
µ
µ
µ
g mL-1
g mL-1
g mL-1
2
0
0
2
4
6
8
10 12 14 16 18 20 22 24
Time, hours
S. aureus
B
12
10
8
Supernatant
g mL-1
To examine the possibility that the T-SNPs
intimately interact with the bacteria, TEM
measurements were conducted to investigate
the mode of action of the NPs on the tested
cells (Figure 8). Untreated E. coli and S. aureus
(Figure 8A,B) cells both showed the normal
cell morphology, possessing the distinct cell
walls and membrane structures typical of
Gram-negative and Gram-positive bacteria.
Quite interestingly, the T-SNP-treated sam-
ples of both E. coli and S. aureus (Figure 8C,D)
showed that the interacting T-SNPs were
localized either on the cell surface or within
the cell membrane, disclosing pronounced
damage to the cell walls (Figure 8E,F).
Although no NP internalization was detected,
our imaging results reinforce the impor-
0
µ
SNPs (50
TTESPC (50
mL-1
µ )
g mL-1
µ
g
)
2
5
µ
µ
g mL-1
g mL-1
6
4
10
25
µ
µ
g mL-1
g mL-1
2
50
µ
g mL-1
0
0
2
4
6
8
10 12 14 16 18 20 22 24
Time, hours
Figure 6. A–B) Antimicrobial activity of T-SNPs. Killing curves of E. coli (A) and S. aureus
(B) grown in different concentrations of T-SNPs (2 to 50 μg mL−1 ) are shown. The growth
was determined by the viable counts and is expressed in colony-forming units (CFU).
E. coli RJH108 (dotted-curve line in A) represents the growth of the triclosan-resistant strain tance of direct NP-bacteria interactions for
exposed to the T-SNPs at a concentration of 50 μg mL−1 . The dashed-curve lines refer to
the promoted antibacterial activity. In this
manner, TEM analyses further direct us to a
possible mechanism of action of the T-SNPs,
emphasizing the importance of the role
played by membrane-associated enzymes in
the triclosan-release phase. The presence of
E. coli (A) and S. aureus (B) grown in the supernatant of TSB or TSB-Glu previously incubated
with T-SNPs (50 μg mL−1 ) for 24 h at 37 °C. Untreated bacteria and unloaded NPs (SNPs
50 μg mL−1 ) served as positive and negative controls respectively. The error bars represent the
standard deviation of three independent experiments.
concentration of T-SNPs (50 μg mL−1 ).[33] We reasoned that if
the triclosan does indeed induce the killing of the bacteria, this
strain, being resistant to triclosan, will survive. Indeed, this
strain was not affected by the presence of the T-SNPs (dotted
line in Figure 6a).
Our next step was to compare our T-SNPs with regard
to the activity of free triclosan. The MBC of the free triclosan
that affected the growth of the two types of bacteria was meas-
ured (Figure 7A,B) and found to be 0.1 μg mL−1 . This result
is in accordance with known literature data.[50,51] A similar
killing-curve pattern was observed with the T-SNPs, yet with a
significant distinction. As calculated from the elemental anal-
ysis, the triclosan constituted only 0.79 wt% of the NPs, meaning
that the highest concentration of NPs taken for the experiments,
50 μg mL−1 , encompassed only 0.041 μg mL−1 of covalently
enzymes in general and esterases in particular is known in the
literature.[56]
3. Conclusions
We have designed, synthesized, and fully characterized a novel
and potentially multifunctional nanosized particulate SiO2
matrix that, upon enzymatic degradation (esterases), can release
the FDA-approved antimicrobial agent, triclosan, in a controlled
manner. Triclosan is covalently linked inside the hybrid-SiO2
NPs that are thus intrinsically designed to withhold the release
of the biocide without any prior direct interaction with patho-
genic bacteria. Preliminary biological experiments dealing with
these T-SNPs show an unexpected and remarkably enhanced
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triclosan (Irgasan) (>97%), ethanol (EtOH) (high-
performance liquid chromatography (HPLC)
grade), toluene (99.8%), 3-(triethoxysilyl)propyl
isocyanate (95%), ammonium hydroxide (A.C.S.
reagent grade), and tetraethyl orthosilicate (TEOS)
(99.999%). Tetraoctyltin (for synthesis) was
purchased from Merck. Water was purified by the
passage of deionized water through an Elgastat
Spectrum reverse-osmosis system (Elga Ltd., High
Wycombe, UK).
E. coli
A
14
0 µg mL-1
12
10
8
0.025 µg mL-1
0.04 µg mL-1
0.05 µg mL-1
6
Triclosan-(3-(triethoxysilyl)propyl)carbamate
(TTESPC):
Triclosan
(5-chloro-2-(2,4-
4
dichlorophenoxy)phenol) (1 g, 3.45 mmol, 1 eq)
and dry toluene (5.0 mL) were added to a three-
necked round-bottom flask under a N2 atmosphere,
so as to obtain a 0.7 M solution. 3-(Triethoxysilyl)
propyl isocyanate (1.28 mL, 5.18 mmol, 1.5 eq)
and tetraoctyltin (3.02 mL, 5.18 mmol, 1.5 eq)
were added simultaneously to the reaction mixture,
which was stirred at room temperature until no
progress in the reaction could be observed by thin-
layer chromatography (TLC) (4:1 n-hexane:EtOAc)
n-hexane:ethyl acetate (EtOAc)). Toluene was
evaporated until an off-white oil emerged. Upon
crystallization overnight, white crystals were
obtained. These were filtered with cold n-hexane
to remove traces of the stannane complex and
dried under vacuum to yield 63.5% (1.17 g) of a
white crystalline powder. mp 83–84 °C; 1H NMR
(300 MHz, CDCl3, 25 °C, TMS, δ): 7.43 (d, J = 2.5 Hz,
1H, ClCCHCl), 7.26 (d, J = 2.5 Hz, 1H, ClCCHC(O)
C(O)), 7.15 (dd, J = 8.8, 2.5 Hz, 1H, ClCCHCHC(O)
CCl), 7.13 (d, J = 2.5 Hz, 1H, ClCCHCHC(O)C(O)),
6.87 (d, J = 8.8 Hz, 1H, ClCCHCHC(O)C(O)), 6.83
(d, J = 8.8 Hz, 1H, ClCCHCHC(O)CCl), 5.36 (bt,
1H, NH), 3.81 (q, J = 7.0 Hz, 6H, CH3CH2O), 3.20
(m, 2H, NHCH2), 1.63 (m, 2H, NHCH2CH2), 1.21
(t, J = 7.0 Hz, 9H, CH3CH2O), 0.62 (m, 2H, SiCH2);
13C NMR (75.5 MHz, CDCl3, 25 °C, TMS, δ): 153.2
(1C, NHC = O(O)), 151.4 (1C, CHC(O)C(O)), 147.0
(1C, ClC(O)CH), 142.3 (1C, NHCO2C), 130.2 (1C,
ClCCHCl), 129.3 (1C, ClCCHC(O)), 129.1 (1C,
ClCCHCHC(O)CCl), 128.1 (1C, ClCCHCHC(O)CCl),
126.4 (1C, ClCCHCHC(O)C(O)), 125.6 (1C,
ClCC(O)CH), 124.8 (1C, ClCCHC(O)), 120.4 (1C,
ClCCHCHC(O)CCl), 120.2 (1C, ClCCHCHC(O)
C(O)), 58.5 (3C, CH3CH2O), 43.6 (1C, NHCH2),
22.8 (1C, NHCH2CH2), 18.2 (3C, CH3CH2O), 7.5
(1C, SiCH2); IR (KBr): ν = 3320 (m; νas(NH)),
2974 (m; νas(CH2)), 2927 (m; νas(CH2)), 2885 (m;
0.1 µg mL-1
0.375 µg mL-1
0.5 µg mL-1
1 µg mL-1
2
0
0
2
4
6
8
10 12 14 16 18 20 22 24
Time, hours
S. aureus
B
12
10
8
0 µg mL-1
0.025 µg mL-1
0.04 µg mL-1
0.05 µg mL-1
6
4
0.1 µg mL-1
0.375 µg mL-1
2
0.5 µg mL-1
1 µg mL-1
0
0
2
4
6
8
10 12 14 16 18 20 22 24
Time, hours
Figure 7. A–B) Minimal inhibitory concentration (MIC) of triclosan. Killing curves of E. coli (A)
and S. aureus (B) exposed to variable concentrations of free-triclosan (0.025 to 1 μg mL−1 ) solu-
tions. Untreated bacteria served as a negative control. The error bars represent the standard
deviation of three independent experiments. The dotted 0.04 μg mL−1 killing curves correspond
to the effective T-SNPs concentration mentioned in Figure 6.
antimicrobial activity (an increase of 5–6 log in killing capacity
compared to the free biocide). This is probably due to the con-
trolled, contact-mediated delivery of the antimicrobial particles,
each serving as a lethal “Trojan horse” during the NP digestion/
bioenzymatic degradation (patent design/material),[57] where
the triclosan molecule is presumably released.
νas(CH2)), 1717 (vs; ν(C=O)), 1534 (s), 1487 (s; νas(aromatic C=C)),
1474 (vs), 1389 (w), 1280 (vs; νas(SiOC)), 1250 (m), 1219 (m), 1187 (m),
1080 (vs; νs(phenolic CO)), 956 (m; νas(aromatic CH)), 789 cm−1 (m;
νas(CCl)); UV–vis (EtOH): λmax (ε) = 276 (2822), 230 (17 407), 209 nm
(32 160); CIMS (m/z (%)) 536.08 (M+, 5.48), 489.98 (C20H23Cl3NO5Si•,
100.00); HRMS (ESI, m/z): [M + H]+ calcd for C22H28Cl3NO6Si, 535.905;
found, 536.085.
Further biological studies are currently being undertaken
that will enable us to get a deeper insight into the overall mech-
anism of the biocidal action of these NPs in order to enhance
and control their efficacy even more.
Hybrid-Silica NPs Containing Triclosan (T-SNPs): General Procedure
Using a Modified Stöber Method: 2.4 mL of a 28–30% solution of aqueous
NH4OH and 25 mL of HPLC grade absolute ethanol were added to a
100 mL vial containing a stirrer at a certain temperature (25 or 60 °C,
depending on the experiment). The medium was stirred for 5 min to
obtain a homogeneous clear solution. Meanwhile, TTESPC (72.1 mg,
2.5% n/n) was dissolved completely in an additional 5 mL of ethanol and
added to the previously described solution. The mixture was stirred for
an additional 15 min in order to hydrolyze the silicate groups of the linker
(TTESPC). Finally, 1.2 mL of TEOS were added to the clear solution, which
was then stirred for 24 h at ambient temperature. T-SNPs of various
4. Experimental Section
Chemicals and Reagents: The following analytical-grade chemicals
were purchased from Aldrich and were used without further purification:
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Figure 8. A–F) TEM of T-SNPs-treated and untreated cells. TEM micrographs of E. coli and S. aureus thin sections: untreated E. coli (A) and untreated
S. aureus (B); E. coli exposed to T-SNPs (50 μg mL−1 ) after 2 h (C) and S. aureus exposed to T-SNPs after 4 h of treatment (D). E–F) Magnified images
of the adjacent micrographs (C and D). The arrows indicate the presence of T-SNPs.
sizes, size distributions and stabilities were prepared by changing the
sol-gel process parameters (e.g., linker, base and TEOS concentrations),
as well as the medium temperature. The resulting NPs were washed
with EtOH using sequential centrifugation cycles (13 000 rpm)
until a neutral pH was reached, then were washed twice with H2O.
Finally, the NPs were lyophilized to dryness.
average diameter, particle-size distribution, and surface morphology of
the particles were obtained by SEM or TEM, followed by a statistical
analysis (ImageJ software) by measuring at least 200 particles for each
sample. The SEM samples were coated with a thin layer of gold by a
sputtering deposition technique.
Thermal measurements were performed by TGA and DSC. The TGA
measurements were carried out using a TA Instruments apparatus (1GA
Q500 model) for TGA, and DSC using a METTLER TOLEDO DSC 822e
instrument, with a 25–500 °C temperature profile (10 °C min−1 , N2
atmosphere, 100 mL min−1 ) for both. TGA afforded the temperature
profiles of the hybrid-silica particles versus TTESPC. DSC revealed
the endothermic and exothermic processes involving the hybrid-silica
particles versus the linker during the heating process.
Analytical Methods and Instrumentation: 1H- and 13C-NMR spectra
were obtained using a Bruker DPX 300 MHz spectrometer. The chemical
shifts are expressed in ppm downfield from Me4Si (tetramethylsilane
(TMS)) used as an internal standard. The values are given using the
δ scale. Multiplicities in the 13C-NMR spectra were determined by off-
resonance decoupling. All of the moisture-sensitive reactions were
carried out in flame-dried reactions vessels. The melting points were
determined using an electrothermal digital melting-point apparatus. The
progress of the reactions was monitored by TLC.
Mass spectra (MS) and high-resolution mass spectra (HRMS) were
obtained using a MICROMASS-AutoSpec high-resolution magnetic-
sector mass spectrometer connected to an HP-5890 series II gas
chromatograph (CI+, chemical ionization).
High-resolution-SEM and energy-dispersive-spectroscopy (EDS)
analyses were carried out on a JEOL JSM-7000F instrument. TEM
and HR-SEM analyses enabled the determination of the morphology,
size and size distribution of the particles, while EDS and elemental
analyses provided particle-composition data. The TEM micrographs
were taken using a Tecnai Spirit instrument (120 kV). Samples for TEM
were prepared by placing a drop of the diluted spheres dispersed in an
(50% v/v) ethanol-water solution on 400 mesh carbon-covered Cu grids
Pk/100 (SPI Supplies West Chester, USA) and then air-drying them. The
Elemental (chlorine) analysis of the NPs was performed using a
combination of an oxygen-flask combustion technique and subsequent
ion chromatography (DIONEX). The relative quantity of triclosan was
calculated according to Equation 1:
mol f (Cl)
g
× 289.64
(Mw of triclosan)
mol
3
(1)
T − SNPs
%
th
e
=
triclosan inside
In Equation 1, molf indicates the amount of chlorine in moles found
by elemental analysis. The expression is divided by 3 due to the presence
of 3 chlorine atoms in the triclosan molecule. If one assumes that the
amount of chlorine found relates to an arbitrary 100 mass units, the
expression result indicates percentages.
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The hydrodynamic particle size, size distribution and ζ-potential of
the particles were determined using a Zetasizer Nano series instrument
(Nano-ZS, Malvern Instruments Ltd., UK) equipped with an MPT-2
multi-purpose titrator (Malvern Instruments Ltd., UK). ζ-Potential
titration analyses were used in order to follow-up the colloidal stability of
the hybrid-silica NPs towards aggregation.
Acknowledgements
Financial support of this work from the Israeli Ministry of Industry
(Biofouling Magnet program) is gratefully acknowledged.
Received: July 10, 2011
Published online: September 13, 2011
FTIR-spectroscopy analysis was performed using a Brüker Equinox 55
FTIR spectrometer. The analysis was performed using 13 mm-diameter
KBr pellets that contained 2 mg of the sample and 198 mg of KBr. The
pellets were subjected to 200 scans at a resolution of 4 cm−1
.
A UV-spectrophotometer (CARY 100 Bio UV–vis spectrophotometer)
was utilized to further characterize the TTESPC and the resulting T-SNPs
(due to the presence of the UV-active chromophore).
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covalently linked triclosan in concentrations below the detection limits
of UV spectroscopy, we exposed E. coli and S. aureus in media previously
incubated for 24 h at 37 °C with T-SNPs at a high concentration of
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