Applied Clay Science 195 (2020) 105733

Contents lists available at ScienceDirect

Applied Clay Science

journal homepage: www.elsevier.com/locate/clay

Research Paper

Aminofunctionalized LAPONITE® as a versatile hybrid material for T

chlorhexidine digluconate incorporation: Cytotoxicity and antimicrobial
activities
Gustavo R. Peraro, Eduardo H. Donzelli, Pollyanna F. Oliveira, Denise Crispim Tavares,
⁎
Carlos H. Gomes Martins, Eduardo F. Molina, Emerson H. de Faria
Grupo de Pesquisa em Materiais Lamelares Híbridos, Universidade de Franca, UNIFRAN, Av. Dr. Armando Salles Oliveira, 201, Parque Universitário, 14404-600 Franca,
SP, Brazil

A R T I C LE I N FO A B S T R A C T

Keywords: Aminofunctionalized LAPONITE® RD was employed for efficient chlorhexidine digluconate (CHX-DG) in-
LAPONITE® RD corporation. The structure and properties of the unloaded (Lap-APTES) and loaded (Lap-APTES:CHX-DG) hy-
Drug delivery brids were investigated by powder X-ray diffraction (PXRD), thermal analysis, small angle X-ray scattering
Hybrid material (SAXS), and infrared absorption spectroscopy (FTIR). A typical adsorption experiment was conducted to evaluate
Incorporation
how the parameters contact time and concentration affected CHX-DG incorporation into the clay and showed
Antibacterial activity
Cytotoxicity
that this compound had high affinity for Lap-APTES, adsorption equilibrium was reached at 90 min, with CHX-
DG incorporation of 1402.7 mg/g. FTIR analysis confirm the interaction by hydrogen bonds between the CHX-
DG and APTES amine groups and the laponite OH groups, resulting in materials with potential antibacterial
properties for controlled drug release. Even at low concentrations (0.042 mg/g), Lap-APTES:CHX-DG was ef-
fective against S. pyogenes. The Lap, Lap-APTES, and Lap-APTES:CHX-DG cytotoxicity to GM07492A cells
(human fibroblasts) was investigated by the XTT colorimetric assay, which revealed that CHX-DG incorporation
into Lap-APTES reduced drug cytotoxicity. Drug release experiments demonstrated that a maximum of 10% (m/
m) CHX-DG was released within 24 h and confirmed the higher affinity between Lap-APTES and CHX-DG.

1. Introduction Gómez-Romero et al., 2010; Shapiro et al., 2015).

New drug generations employ various active species with ther-
apeutic value, including antimicrobials, growth factors, and supple-
ments (e.g., vitamin C). The most advanced medicaments include sys-
tems for the controlled and sustainable delivery of active substances at
the target site (Mayr et al., 2018).
Recent technologies demand that the properties of traditional ma-
terials be combined. Such combination can be achieved by developing
organic-inorganic hybrid materials (OIHMs), which can be applied in
many branches of biology and/or chemistry because they are simple to
process and are amenable to design on the molecular scale. The com-
bination of organic and inorganic components at the nanoscale level
provides numerous opportunities in diverse fields and is a viable choice
to prepare multifunctional materials with applications in science and
technology, including catalyst support, biocatalysis, drug delivery sys-
tems, efficient adsorbents, and synthesis of nanocomposites with
polymers (Mitzi et al., 1999; José and Prado, 2005; Avnir et al., 2006;
The specific features of OIHMs based on natural or synthetic clays,
such as their versatile structure, are particularly attractive when it
comes to incorporating species such as drugs, catalysts, and vitamins,
among others, into these hybrids. By combining functional organic and
inorganic moieties with organic molecules, novel materials can be de-
signed, and a certain degree of control over their surface properties can
be gained. Incorporating antimicrobial agents into these hybrid mate-
rials is notably interesting to prevent wound infections. The develop-
ment of OIHMs derived from clay minerals enables desired physical-
chemical properties, like hydrophobicity, mechanical resistance, and
plasticity, to be controlled, thus increasing the potential of nanoclay
mineral-based drug delivery systems (Ávila et al., 2010). Organosilane
functional groups, such as 3-aminopropyl groups, grafted on LAPON-
ITE® have been elected because these groups can bind to contaminants
via hydrogen bonds, whereas the clay can bind to contaminants through
hydrophobic interactions. The covalent bond between the clay mineral
and the alkoxide provides durable immobilization of organic units of

⁎
Corresponding author.
E-mail address: emerson.faria@unifran.edu.br (E.H. de Faria).

https://doi.org/10.1016/j.clay.2020.105733
Received 14 June 2019; Received in revised form 8 June 2020; Accepted 10 June 2020
0169-1317/ © 2020 Elsevier B.V. All rights reserved.
G.R. Peraro, et al.
biological interest, preventing their immediate leaching into the sur-
rounding solutions. Meanwhile, selective adsorption of contaminants
onto silylated products could be improved by introducing special
functional groups (e.g., –NH2, –SH) into these products (Su et al., 2012;
Su et al., 2013).
In this context, knowing how these functional groups affect drug
release from a solid OIHM and how the resulting material behaves
during CHX-DG adsorption/release cycles is important because the
alkoxide must maintain its structural stability during adsorption and
release.
Another relevant factor that justifies the use of grafted clay minerals
as support to immobilize biologically active molecules concerns the fact
that several organic molecules present low or very low solubility in
aqueous solution and physiological media, which results in low bioa-
vailability and diminishes the therapeutic effect (da Silva et al., 2020).
In this scenario, efficient delivery of poorly water-soluble compounds in
the body has been extensively studied by academia and industries
aiming to develop formulation strategies that can improve the dis-
solution rate and/or solubility/apparent solubility of poorly water-so-
luble molecules (da Silva et al., 2020).
LAPONITE® RD (Lap; Na+ 0.7[(Si8Mg5.5Li0.3)O20(OH)4) is a synthetic
hectorite-type nanoclay mineral. Its structure and composition re-
semble the structure and composition of natural clay minerals be-
longing to the smectite group (montmorillonite, for instance). This clay
mineral can be used to synthesize OIHMs containing different organic
molecules, including alkoxides and cationic or anionic species. In
general, Lap presents higher specific surface area (higher than 350 m2/
g) and swells in the presence of an aqueous suspension, to give orga-
nized exfoliated lamellae depending on specific moieties and clay pla-
telet charge (Yang et al., 2007; Daniel et al., 2008; Ley et al., 2015;
Zhang et al., 2019; Colletti et al., 2020; Ding et al., 2020). Each LAP-
ONITE® RD platelet consists of a central sheet of magnesium ions in
octahedral coordination (O) with oxygen anions and hydroxyl groups
and two outer tetrahedral sheets (T) (Fig. 1). In some octahedral sites,
magnesium cations in the octahedral sheet are isomorphically sub-
stituted for lithium, which results in a negative charge that is neu-
tralized by sodium cations located in the interlayer space. The average
LAPONITE® RD layer dimensions (diameter of 25 nm and layer thick-
ness of 0.92 nm) confer a much more higher edge area to surface ratio
than the ratio that is usually found in natural smectites, which makes
LAPONITE® RD suitable for edge modifications. After grafting reactions
Fig. 1. Schematic representation of the chemical structure of LAPONITE® RD.
2
Applied Clay Science 195 (2020) 105733
with organic moieties and surface modifications, this solid can also
exhibit increased interaction with active molecules preferentially via
ion exchange, which affords multifunctional materials. Another char-
acteristic of this class of synthetic layered material is that, at a suitable
concentration, LAPONITE® RD can promote gel formation in aqueous
systems by means of electrostatic interactions between negatively
charged faces and the positively charged nanoplatelet edges, imparting
outstanding suspension, thixotropy, and other unique crucial properties
that are crucial to drug delivery systems. Under such conditions, the
clay mineral is delaminated, leading to the suspension of individual
nanoparticles (Bagchi et al., 2013; Saha et al., 2014).
The LAPONITE® RD was chosen due to its higher cationic exchange
capacity and specific surface area as compared to the natural parent
clay mineral montmorillonite. Another advantage is its white color,
which can facilitate the use of the hybrid material loaded with chlor-
hexidine digluconate in biological applications such as dental products.
Ghadiri et al. (2013) conducted experiments with laponite (syn-
thetic hectorite-type nanoclay mineral) as a carrier for the in situ local
delivery of tetracycline (a widely employed antimicrobial agent that is
effective against both Gram-positive and Gram-negative organisms,
including betalactamase-producing strains) during the treatment of
periodontal disease. To avoid the undesired side effects of the systemic
administration of antibiotics such as gastrointestinal intolerance and
bacterial resistance and to improve drug efficacy, new intra-periodontal
pocket delivery systems have been developed for periodontal disease.
The synthesis and application of OIHMs combined with anti-
bacterial compounds have drawn the attention of both the academia
and the industry due to the worldwide concern about public health.
Owing to their excellent intercalation ability, the smectite clays mon-
tmorillonite, saponite, and hectorite are the most commonly employed
to prepare antibacterial materials (Ley et al., 2015). However, synthetic
clays (e.g., LAPONITE® RD) without physical-chemical treatment do
not have great affinity for organic molecules. In this sense, functiona-
lization could be a strategy to incorporate various compatible (cationic,
anionic, neutral, hydrophobic, and/or hydrophilic molecules) species
into the clay interlayer spaces, which would depend on two main
characteristics: nature of the guest molecule inserted into the clay in-
terlayer (chemical affinity) and clay particle charge. Moreover, clay-
based hybrid materials can attract and decimate bacteria as a result of
the hydrophobicity that the functionalization procedures impart to the
clay particle surface and to the clay interlayer space. Therefore, species
G.R. Peraro, et al.
Fig. 2. Spatial stereostructure form of chlorhexidine digluconate (CHX-DG).
intercalated into hybrid clay materials can be used for long-term ac-
tivities like wound healing, controlled drug delivery, and treatment of
infection-related diseases (Yang et al., 2007; Bagchi et al., 2013; Saha
et al., 2014). Bagchi et al., 2013 showed the potential use of mon-
tmorillonite composites containing intercalated and surface-adsorbed
copper nanoparticles for therapeutic purposes. The composites dis-
played excellent antimicrobial action against Escherichia coli, Staphylo-
coccus aureus, Pseudomonas aeruginosa, and Enterococcus faecalis: the
mortality rates were higher than 90% after 12 h. Recently, our group
described a new biomimetic heterogeneous material based on a tetra-
carboxyphenylporphyrin−kaolinite hybrid as a multifunctional matrix
that could be applied as catalyst in oxidation reactions and as an an-
tibacterial agent (de Carvalho et al., 2014). The antimicrobial tests
against the oral pathogens Bacillus subtilis, Klebsiella pneumoniae, and
Escherichia coli revealed a strong antibacterial effect, which was at-
tributed to synergism between the clay and the porphyrin.
Chlorhexidine digluconate (CHX-DG; (2,2′-(1,6-Hexanediyl)bis(1-
{(E)-amino[(4-chlorophenyl)amino]methylene} guanidine)-D-gluconic
acid) (Fig. 2), a di-guanidine antiseptic, is an effective bacteriostatic
and bactericidal agent. This cationic antiseptic can be employed as a
common disinfectant or as a bactericidal agent in dentistry. CHX-con-
taining formulations are widely used for urinary or central venous ca-
theter impregnation, pharmaceutical purposes, and cosmetics manu-
facture – they can kill bacteria and fungi at elevated rates without being
toxic to mammalian cells. The two symmetrically arranged chlor-
ophenyl guanidine groups present in CHX-DG underlie its antibacterial
activity: these groups allow CHX-DG to penetrate the bacterial cell wall
and irreversibly disrupt the cell membrane, to kill the microorganism
gradually. Unfortunately, the uncontrolled release nature of CHX-DG
formulations restricts their topical application (Arret et al., 1971; Buck
et al., 1999; Estrela et al., 2003; Gomes et al., 2004).
This research aims to study the potential antibacterial properties of
a Lap hybrid functionalized with 3-aminopropyl triethoxysilane
(APTES), which will be designated Lap-APTES hereafter, incorporated
with CHX-DG; it also evaluates the use of Lap-APTES:CHX-DG (obtained
via adsorption; see experimental details) as a new drug delivery system.
We detail the synthesis and characterization of Lap-APTES and Lap-
ATPES:CHX-DG and investigate the efficiency of CHX-DG incorporation
into Lap-APTES by kinetic experiments. Additionally, we assess the
antimicrobial and cytotoxic activities of the solid material Lap-
APTES:CHX-DG obtained after the equilibrium studies against two
Gram-positive bacteria. Formulations based on Lap-APTES-CHX open
up novel opportunities for the topical application of these materials.
2. Experimental
2.1. Materials
LAPONITE® RD (Lap) from Kremer Pigmente (Germany), which was
3
Applied Clay Science 195 (2020) 105733
kindly supplied by Prof. Miguel A. Vicente Rodrigues and by Prof.
Vicente Rives of the University of Salamanca, Spain, was used as re-
ceived. Its cation exchange capacity (CEC) is 60 meq/100 g. LAPON-
ITE® RD is a synthetic trioctahedral hectorite-type clay composed of
two tetrahedral silica sheets and a central octahedral sheet. Its chemical
formula can be expressed as Si8[Mg5.5Li0.4O24.0]−0.6Na0.6+0.6. Disk-
shaped layers with thickness of approximately 0.9 nm and diameter of
25 nm arise when the clay mineral is dispersed in water.
All the other solvents and reagents were of high-purity commercial
grade (Merck and Sigma-Aldrich); most of them were used as received.
2.2. Lap-APTES preparation
Lap was functionalized with APTES by means of a standardized
method. Briefly, 2.00 g of Lap previously heated in an oven at 120 °C for
1 h was mixed with 1.18 cm3 of APTES (5.06 mmol) in 100 cm3 of dry
toluene and subjected to reflux under nitrogen atmosphere for 24 h. The
resulting solid, designated Lap-APTES, was washed in toluene and dried
in an oven at 120 °C. Lap-APTES was then washed with distilled water
several times and dried in an oven at 80 °C for 12 h (Bizaia et al., 2009;
Bandeira et al., 2012; Marçal et al., 2015).
2.3. Chlorhexidine digluconate (CHX-DG) incorporation into Lap-APTES -
adsorption experiments
CHX-DG (Sigma-Aldrich) incorporation was evaluated by the ad-
sorption methodology. For all the adsorption experiments, the CHX-DG
concentration in the solutions was determined by UV–visible spectro-
scopy conducted on a Hewlett-Packard 8453 Diode Array spectrometer.
The absorption was measured at 254 nm, which corresponded to the
maximum CHX-DG absorbance under the adsorption conditions.
Previously, a calibration curve had been constructed for CHX-DG con-
centrations ranging from 1 to 25 mg L−1. At 254 nm, the absorbance
responded linearly throughout the concentration range used in the
experiments, according to the Beer-Lambert law.
Adsorption kinetics experiments were carried out at ~25 °C in glass
vials. For this purpose, a known amount of the adsorbent, typically
50 mg, was shaken with 5.0 cm3 of the CHX-DG solution at the desired
CHX-DG concentration and for the desired time. To this end, 25 mg L−1
CHX-DG, fixed Lap-APTES mass of 50 mg, and contact times varying
from 1 min to 2 h were employed during the kinetics experiments. At
the end of the experiment, the solid material was separated from the
supernatant by centrifugation at 3500 rpm for 15 min. The CHX-DG
concentration that remained in the supernatant was determined by
UV–Vis spectroscopy at 254 nm; the amount of CHX-DG that adsorbed
onto Lap-APTES was calculated by using Eq. (1):
V (Ci − Ce )
qe =
m (1)
G.R. Peraro, et al.
where qe is the amount of adsorbed CHX-DG (mg/g); Ci and Ce (mg L−1)
are the CHX-DG initial and equilibrium liquid-phase concentrations,
respectively; V is the volume of the solution (dm3); and m is the amount
of Lap-ATPES (g).
Adsorption equilibrium experiments were carried out at ~25 °C in
glass vials. For this purpose, a known amount of the adsorbent, typi-
cally 50 mg, was shaken with 5.0 cm3 of the CHX-DG solution at the
desired CHX-DG concentration (between 1 and 25.000 mg L−1) and
fixed Lap-APTES mass of 50 mg for a fixed contact time of 90 min as
established by kinetics experiment. At the end of the experiment, the
solid material was separated from the supernatant by centrifugation at
3500 rpm for 15 min. The CHX-DG concentration that remained in the
supernatant was determined by UV–Vis spectroscopy at 254 nm; the
amount of CHX-DG that adsorbed onto Lap-APTES was calculated by
using Eq. (1), previously described. The maximum CHX-DG adsorbed
during the equilibrium study was named qe (mg/ g) and referred to the
maximum adsorption capacity at equilibrium concentration.
2.4. Release experiments
To study how the CHX-DG loaded on Lap-APTES affected CHX-DG
release from Lap–APTES, an in vitro simulated study was conducted by
using 0.100 g of Lap-APTES-CHX-DG and 1000 mL of deionized water
at pH 7.2 and 25 °C. At pre-determined time intervals, the CHX-DG
concentration was measured by in situ UV–Vis spectroscopy
(λmax = 254 nm). All the experiments were carried out on three in-
dependent samples; the results are the average of the data. The UV–vis
absorption data were recorded between 190 and 500 nm on an Agilent
Technologies Cary 60 dual beam spectrophotometer fitted with a fiber
optic coupler equipped with a solarization-resistant immersion probe. A
2-mm solution thickness can be analyzed with the immersion probe,
which makes the technique sensitive to the CHX-DG molecules deliv-
ered to the aqueous solutions. The acquisition scan rate was
300 nm min−1; a full spectrum was recorded in 60 s. Standard stock
CHX-DG aqueous solutions with different concentrations were mea-
sured and used to determine the cumulative drug release in water
quantitatively. The release study was performed over 24 h, in triplicate.
The results refer to the maximum release (%), which was estimated on
the basis of the initial Lap-APTES-CHX-DG mass.
2.5. Antibacterial activity tests
2.5.1. Microorganisms
The bacterial cultures were obtained from the American Type
Culture Collection (ATCC). Two microbial strains were tested:
Staphylococcus aureus (ATCC 29213) and Streptococcus pyogenes (ATCC
12345). All the strains were kept in the laboratory and cryopreserved at
−86 °C. Before the experiments, the culture medium was inoculated
with the desired microorganism under appropriate atmospheric con-
ditions, to confirm the strain purity.
2.5.2. Inoculum preparation
The bacterial strains mentioned above were incubated in Brain
Heart Infusion (BHI) agar at 37 ± 0.1 °C for 24 h. Each microorganism
suspension was standardized at 625 nm with the aid of a spectro-
photometer, to match the transmittance of 81, which was equivalent to
0.5 McFarland scale (1.5 × 108 CFU/mL).
2.5.3. Antibacterial assay by the agar diffusion (AD) method
The Lap-APTES:CHX-DG antibacterial activity was determined by
the agar diffusion (AD) method; the well technique and the double-
layer agar system were employed (Gomes et al., 2004).
Brain Heart Infusion agar (25 mL) was poured into sterilized Petri
dishes (15 × 90 mm diameter). Next, 12.5 mL of the BHI agar (50 °C)
and 2.5 mL of each test suspension were gently mixed, poured onto a
previously set layer, and homogeneously distributed into Petri dishes.
4
Applied Clay Science 195 (2020) 105733
After solidification, the seed layer was perforated with a sterilized
stainless-steel cylinder (inside diameter = 4 mm), to form the wells.
The latter were located 25 mm away from the plate border and 40 mm
halfway from each other. Wells containing Lap-APTES, distilled water
(negative controls), or an aqueous CHX-DG solution (0.1 mg/mL, po-
sitive control) were also prepared. The plates were kept at room tem-
perature for 2 h, to allow the agents to diffuse through the agar (Gomes
et al., 2004). Afterward, the plates were incubated at 37 °C and under
aerobic condition for 48 h. At the end of the incubation period, the
inhibition zones formed in the media were measured in mm. The in-
hibitory zone was considered the shortest distance (mm) from the
outside margin of the well to the initial point of microbial growth.
Three replicates were accomplished for each bacterium.
2.5.4. Statistical analysis
The results from the biological assays carried out by the AD method
were submitted to analysis by one-way ANOVA. Individual samples
were tested in triplicate. The mean and standard deviation values of all
the experiments were calculated from the diameter, in mm, of the in-
hibition halo (AD).
2.6. Cytotoxicity assay
2.6.1. Cell lines and culture conditions
Human lung fibroblasts (GM07492A) were employed during these
experiments. The cell line was maintained as monolayer in plastic
culture flasks (25 cm2) containing culture medium (HAM-F10:DMEM,
1:1, Sigma-Aldrich) supplemented with 10% fetal bovine serum
(Nutricell), antibiotics (0.01 mg/mL streptomycin and 0.005 mg/mL
penicillin; Sigma-Aldrich), and 2.38 mg/mL HEPES (Sigma-Aldrich) at
37 °C and in a chamber with 5% CO2.
2.6.2. XTT colorimetric assay
The cytotoxic activity was measured by using the in vitro
Toxicology Colorimetric Assay Kit (XTT; Roche Diagnostics) according
to the manufacturer's instructions. Extracellular XTT reduction occurs
on the plasma membrane surface via transmembrane electron transport
(Buck et al., 1999). This reaction was originally described to evaluate
tumor cell line sensitivity to chemicals. This assay is widely used to
quantify cell proliferation or to determine the cytotoxic effects of che-
micals (Estrela et al., 2003). For these experiments, the cells (104 cells/
well) were plated onto 96-well microplates. Each well received 100 mL
of HAM-F10/DMEM medium containing Lap, Lap-APTES, Lap-AP-
TES:CHX-DG, or APTES. The tested concentrations ranged from 2.48 to
5000 μg/mL. The negative (without treatment) and positive (di-
methylsulfoxide, DMSO 25%; Sigma-Aldrich) controls were included.
After incubation at 37 °C for 24 h, the medium was removed, and the
cells were washed with 100 μL of PBS (phosphate buffered saline) and
exposed to 100 μL of HAM-F10 medium without phenol red. Then,
25 μL of XTT was added to each well. The microplates were covered and
incubated at 37 °C for 17 h. The absorbance of the sample was de-
termined by using a multiplate reader (ELISA – Asys – UVM 340/Mi-
crowin 2000) operating at a test wavelength of 450 nm and a reference
wavelength of 620 nm. The cell viability was expressed as a percentage
of untreated cells, which served as the negative control group and was
designated as 100%. In other words, the results were expressed as a
percentage of the negative control. The experiments were performed in
triplicate.
2.7. Characterization techniques
The powder X-ray diffraction (PXRD) patterns of non-oriented
powder samples were obtained on a Rigaku Miniflex II diffractometer
with Ni-filtered Cu Kα radiation, at 30 kV and 15 mA, at a scanning rate
of 2°/min.
The nanostructure of the materials was monitored by small-angle X-
G.R. Peraro, et al.
ray scattering (SAXS) experiments, performed at the D01A-SAXS2
beamline of the National Synchrotron Light Laboratory (LNLS,
Campinas, Brazil) (Project number: SAXS1–17845 and SAXS1–16981).
A vertical position-sensitive X-ray detector and a multichannel analyzer
were used to record the SAXS intensity (I(q)) as a function of the
modulus of the scattering vector (q = (4π/λ) sin(θ/2), where θ cor-
responds to the scattering angle, and λ is the wavelength of the X ray
beam (λ = 0.148 nm)). The distance between the sample and the de-
tector was 700 mm; 3 s was necessary for each data collection. The
beam center was calibrated by using silver behenate with the primary
reflection peak at 1.076 nm−1. Lap and Lap-APTES were analyzed in
the solid sample holder. The data acquisition time was about 30 s de-
pending on the sample concentration and the scattering vector range.
The background scattering of a blank (SAXS sample holder with capton)
was subtracted from the spectrum of each sample before further data
analysis for the Lap systems was carried out.
The FT-IR spectra were recorded from 4000 to 350 cm−1 on a
Perkin-Elmer 1730 infrared Fourier transform spectrometer; the KBr
pellet technique was employed. About 1 mg of the sample and 300 mg
of KBr were used to prepare the pellets.
Thermogravimetric analysis (TGA) and Differential Thermal
Analysis (DTA) were accomplished on a Thermal Analysis TA instru-
ment SDT Q 600, from 25 to 1000 °C, under oxygen flow (30 mL/min)
and at a heating rate of 10 °C/min.
CHX-DG was incorporated into Lap-APTES by the adsorption
methodology. UV–visible spectroscopy was carried out on a Hewlett-
Packard 8453 Diode Array spectrometer; a quartz cell with optical path
length of 1 cm was employed.
In situ UV–vis adsorption experiments: for all the adsorption ex-
periments, the CHX-DG concentration in the aqueous solution was de-
termined by UV–vis spectroscopy, conducted on an Agilent
Technologies Cary 60 dual beam spectrophotometer fitted with a fiber
optic coupler equipped with a solarization-resistant immersion probe.
This arrangement made the technique sensitive to the CHX-DG mole-
cules present in the aqueous solution. The UV–vis absorption data were
recorded between 190 and 500 nm. The acquisition scan rate was
300 nm min−1, which allowed a full spectrum to be recorded within
60 s. All the experiments were performed on two independent samples.
Cytotoxicity was assayed by Elisa-ASYS, MicroWin program 2000.
3. Results and discussion
3.1. Physical-chemical structure evaluation of LAPONITE® RD hybrid
materials
Lap functionalization with APTES was successful and afforded a
well-ordered Lap-APTES hybrid with interlayer space of 17.33 Å. All
the steps involved in the preparation process have been previously
presented by us and described in the literature (Ávila et al., 2010;
Marçal et al., 2015).
As previously discussed by Su et al. (2012) and Su et al. (2013), the
internal surface, external surface, and broken edge of expandable clay
minerals are possible sites for silane grafting. In the case of internal
surface and broken edge grafting, increased basal spacing is expected,
whereas the basal spacing remains unchanged for external surface
grafting. The basal spacing of grafted clay minerals depends on the
arrangement and steric configuration of the silane in the clay mineral
basal spacing, on the amount of silane that is loaded in the interlayer
spaces, and on interactions among the silane molecules (e.g., inter-
molecular hydrogen bonding) and between the clay mineral and the
silane special group (e.g., –NH2 group from APTES) (Su et al., 2012; Su
et al., 2013). Another important factor is the chemical nature of the
solvent that is used in the silylation reaction: the solvent significantly
influences the amount and distribution of the grafted silane and the
structure of the silylated product (Su et al., 2012; Su et al., 2013).
Fig. 3 shows the PXRD patterns of pristine laponite (Lap), Lap-
5
Applied Clay Science 195 (2020) 105733
APTES, and Lap-APTES:CHX-DG. Order around the direction 001 re-
mained in Lap-APTES. The Lap interlayer space changed slightly after
interaction with CHX-DG, as confirmed by the lower intensity of the
peaks corresponding to the interlayer space d001—it decreased from
17.33 to 15.88 Å, which pointed to slight layer stacking in Lap-APTES.
This behavior suggested that CHX-DG adsorbed onto the surface and
into the Lap grafted layer interlayer space.
For the non-hydrolyzed APTES molecule, the main dimension was
close to 8.8 Å, which was compatible with Lap−APTES (basal spacing
of 17.33 Å). However, APTES and CHX-DG might also have adopted an
almost planar configuration, thereby inducing the reduction in the
basal spacing. The variation (decrease) in the basal distances could also
be assigned to the replacement and rearrangement of the aminopropyl
groups attached between the layers and to the cationic exchange in-
duced by CHX-DG binding in the Lap-ATPES interlayer spaces. Upon
exposure to the aqueous solution during batch adsorptions experiments,
the clay hydrated Na+ cations were exchanged with CHX-DG from the
solution, and consequent CHX-DG incorporation resulted in stacked
layers with reduced basal spacing. The locking effect; i.e., when silanes
(organosilanes or oligomers) are simultaneously condensed with two
adjacent clay mineral layers, and the interlayer height can be controlled
by the configuration of the silanes and/or silane oligomer during silane
grafting onto clay minerals belonging to the smectite group (Su et al.,
2012; Su et al., 2013). This results in materials with silane grafted
between the clay mineral layers, which have potential use as supporting
materials (e.g., drug delivery and targeting, controlled release mate-
rials).
The d001 values of the silylated clay mineral prepared in nonpolar
solvent (toluene) decreased from 17.33 Å in Lap-APTES to 15.88 Å after
intercalation with 25.000 mg L−1 CHX-DG. The small reduction in the
basal spacing even after higher CHX-DG concentration was in-
corporated into Lap-APTES differed from what we verified in the case of
pristine Lap. The neighboring LAPONITE® RD layers were «locked»
during the grafting reaction, and the polarity of the employed solvent
significantly affected the silylation reaction and the structure of the
silylated products (Su et al., 2012; Su et al., 2013).
In the case of Lap functionalized with γ-methacryloxy propyl di-
methyl ethoxysilane and γ-methacryloxypropyl trimethoxysilane,
Herrera et al. (2004) reported interlayer spaces of 17 and 13 Å for the
monofunctional and bifunctional alkoxides, respectively, which re-
sembled the interlayer space verified for the trifunctional alkoxysilane
in the case of Lap-APTES. The grafting reaction on the clay surface may
have separated each individual clay layer, while the alkoxide might also
be present at the edge of the clay platelets.
Concerning Lap-APTES:CHX-DG, the organic groups were probably
incorporated into Lap-APTES via a cation exchange mechanism: the
CHX-DG cations substituted the cations that balanced the hybrid clay
electric charge. This mechanism has been reported for other cationic
molecules such as CTA+ and also for CHX (Ho et al., 1996). Here, the
interlayer space of 15.88 Å (gallery height of 0.30 nm) indicated an
arrangement that closely resembled a monolayer where some digluco-
nate groups tethered to the Lap-APTES surface, whereas the CHX chains
were almost parallel to the La-ATPES layers. On the other hand, the
remaining diffraction effects, namely the effects that do not depend on
layer stacking in the c-direction, did not change drastically as compared
to the parent original Lap and to Lap-APTES. All these observations led
to the conclusion that CHX-DG insertion did not affect the Lap in-
dividual layer structure (Marçal et al., 2015).
As discussed by us in a previous work on kaolinite grafted with
amine groups, interaction with large and charged molecules like ir-
onporphyrins produces two opposite effects: clay layer reorganization,
to increase stacking, and clay layer disordering and even exfoliation,
which makes the clay layers less organized (de Carvalho et al., 2014).
Exfoliation is normally induced in charged clays such as Lap because
cationic exchange with one new molecule of distinct size largely re-
duces interaction between each individual clay layer. The higher
G.R. Peraro, et al.
5000
4000
Intensity (c.p.s.)
3000
2000
1000
0
10 20
Fig. 3. XRPD of the original LAPONITE® RD (Lap), the organofunctionalized Lap-APTES, and Lap-APTES after CHX-DG adsorption.
amount of CHX-DG incorporated into Lap-APTES can promote inter-
action of different layers with the same grafted platelet, to cause ex-
foliation.
The Lap SAXS curves (Fig. 4) showed the typical effect of disordered
structures and confirmed the exfoliated nature of the original Lap,
corroborating the XRPD analysis. Regarding the slopes of the Lap and
Lap-APTES curves, they revealed alterations in the Lap structure and
proved that functionalization elicited Lap layer stacking. In both cases,
the samples remained exfoliated, and insertion of large molecules in
aqueous medium induced little stacking.
We also studied the fractal structure of the grafted and nongrafted
LAPONITE® RD by the Small Angle X-ray Scattering technique. In
general, the SAXS curves could be divided into three basic regions:
Guinier, Fractal, and Porod, as shown in Fig. 4 A and B.
The scatter vector from the Guinier to the fractal region is defined as
qG, and the scatter vector from the fractal to the Porod region is defined
as qP. The mean size of the clusters ξ can be defined by 2Π/qG. From qP,
the size of the particles, α, is 2Π/qP. Thus, when a ln versus ln plot of I(q)
and q can be constructed, the slope of the line Dm is equal to -α (at-
tenuation index 1 < α < 3). If 3 < α < 4, the surface fractal
dimensions DS is equal to 6 – α. We observed the following character-
istics: the mean size of the clusters (ξ), which could be related to the
form of the hybrid organic inorganic material, and the fractal dimen-
sion (D with DS and Dm, which respectively represent mass distribution
and surface coarseness/structure compactness). Fig. 4 illustrates all the
SAXS curves of the Lap and Lap-APTES samples. As previously shown
by Seftel et al. (2015) and Fei He et al. (2008), the parameters ξ, α, D
(DS and Dm), and Rg. can be obtained (See Table 1).
The SAXS curves Of Lap and Lap-APTES presented similar slopes in
the same regions (Fractal and Porod). The aminopropyl groups did not
drastically change the LAPONITE® RD nanostructure. In other words,
the fractal parameters were comparable. The ξ, Dm, and Rg values re-
vealed the most important differences due to APTES grafting and con-
firmed the stacking effect, as judged from the increased ξ values, which
attested to the increased particle size along the z axis. Another im-
portant factor was the lower material compactness, as evidenced by the
reduced Dm values of the modified sample. This could be assigned to the
presence of the organic aminopropyl group chains on the LAPONITE®
RD surface and interlayer space, which resulted in mesopores. This
result agrees with the report of Seftel et al. (2015). Therefore, Lap-
Applied Clay Science 195 (2020) 105733
Lap-APTES:CHX-DG
Lap-APTES
Lap
17.33 Å
12.87 Å
15.88 Å
4 6 8 10 12 14
30 40 50 60 70
2θ (Degrees)
APTES stacking could result from coupling between the clay platelets
and the alkoxide rather than from a single component. Such coupling
allowed partial clay orientation, as previously confirmed by XRD, and
could be favored by the higher amount of amine groups from CHX-DG
employed during the incorporation experiments.
Functionalization strongly affects the interlayer distance of pure
cationic clays (without previous functionalization) (Fig. 5). However,
grafting can impart a hydrophobic character to the clay surface, to
cause clay layer stacking during swelling experiments in aqueous
medium. In this context and as previously observed by us (Bizaia et al.,
2009; Marçal et al., 2015), the distance did not change markedly after
the swelling experiments (clay suspension in aqueous medium), and the
alterations perfectly agreed with the PXRD data (Fig. 3). For Lap-
APTES, swelling after CHX-DG incorporation was clear from the
changes in the slope of the SAXS curves, assigned to the large d dis-
tribution and to the smaller stacking number (N) per crystal domain.
Nevertheless, we were not able to estimate the number of stacks per
crystal domain due to the exfoliated nature of both samples.
Fig. 6 and B show the TG and DTG curves of Lap, Lap-APTES, and
Lap-APTES:CHX-DG. Lap presented three mass loss steps. The first step
occurred between 25 and 200 °C (17% mass loss) and corresponded to
removal of adsorbed water molecules. The second step happened be-
tween 200 and 700 °C (3% mass loss) and referred to Lap dehydrox-
ylation and water removal from the interlayer cations. The exothermic
process centered at 750 °C in the Lap DTA curve was due to phase
transition between enstatite (MgSiO3) and forsterite (Mg2SiO4). As for
Lap-APTES (Fig. 6), it underwent the typical four mass loss steps of
grafted clays. The first step took place between 25 and 220 °C (11%
mass loss). The organic moieties of the APTES molecule were removed
between 220 and 670 °C (13% mass loss). This removal was exothermic
and referred to decomposition of the APTES organic fraction, con-
firming that Lap was indeed functionalized with APTES. Lap-ATPES
experienced the same crystallization process as Lap. Overall, Lap and
Lap-ATPES lost 6% and 14% of their masses between 200 and 900 °C,
respectively. We assigned the 8% difference to APTES grafted on the
laponite clay. On the basis of the thermal analyses, we estimated that
the hybrid material synthesized here has the formula Lap-(APTES)0.141.
The solid containing CHX-DG (Fig. 6C) presented about 15% mass loss
between 200 and 900 °C, showing that an increased amount of organic
matter was introduced into Lap-APTES.
6
G.R. Peraro, et al. Applied Clay Science 195 (2020) 105733

Lap
-0.5
Lap-APTES
Linear fit

d =3.2
-1.0 m

log I (q) (arb. units)

dm=2.5 slope = -dm
-1.5 dm=2.6

dm=1.4
-2.0 dm=1.0
dm=2.6
dm=0.5
qG
-2.5 qP
dm=3.0

Guinier range Fractal range Porod range

-3.0
0.0 0.1 0.2 0.3 0.4 0.5 0.6 0.7
-1
(a) log(q) (Å )

7
Lap
Lap-APTES
6 Linear fit

5
ln I(q)

1
0.0 0.1 0.2 0.3 0.4 0.5 0.6
2 -1 2
(b) q (Å )
Fig. 4. A SAXS curves of the original LAPONITE® RD (Lap) and the organofunctionalized Lap-APTES showing the Guinier, Fractal, and Porod regions. B SAXS Ln I(q)
versus q2 (Å)2 curves of the original LAPONITE® RD (Lap) and the organofunctionalized Lap-APTES for calculation of the gyration radius (Rg).

Table 1 where W(200–600°C) corresponds to the mass loss between 200 and
The fitting fractal parameters for Lap and Lap-APTES extracted from the SAXS 600 °C, WSi = W (200–600°C) x (mSi/mC3H8N), mSi = 28.1 g/mol, and
curves. mC3H8N = 58.1 g/mol. We employed this same equation with adapta-
Sample ξ (nm) α (nm) DS Dm Rg (nm) tions to establish the % of chlorhexidine digluconate that was loaded on
Lap-APTES. Considering the molecular formula of CHX-DG as
Lap 21.19 9.36 2.6 3.2 37.5 (C22H30Cl2N10) and m = 505.4 g/mol) (See Table 2).
Lap-APTES 31.81 10.13 3.0 2.5 51.5
FT-IR spectroscopy helped to monitor ATPES incorporation into Lap
through alterations in the bands relative to the Mg-OH hydroxyl groups,
Si-OH, and water molecules in the interlayer space. Fig. 7 depicts the
Considering that APTES was completely hydrolyzed and condensed
FT-IR spectra of Lap, Lap-APTES, and Lap-APTES:CHX-DG. The FTIR
in Lap, that the loaded silane consisted of organic parts (C3H8N) and an
results also helped to follow CHX-DG incorporation into Lap-ATPES
inorganic part (Si), and that the organic parts were totally removed
through changes in the bands due to the OH and NH2 groups.
between 200 and 600 °C, we calculated the silane load by the following
APTES immobilization onto Lap involves a condensation reaction
equation, according to Tao et al. (2014):
between the APTES silanol groups and the hydroxyl groups present on
100 x W(200 − 600°C ) − WSi the Lap surface. As for CHX-DG adsorption onto Lap-ATPES, it involves
Silane load (%) =
100 − W(200 − 600°C ) − WSi (2) formation of hydrogen bonds between the CHX NH groups and the Lap-

7
G.R. Peraro, et al.
Fig. 5. Schematic representation of different possibilities of APTES grafting into the synthetic clay LAPONITE® RD.
APTES NH2 and OH groups, which was evident from the reduced in-
tensity of the band typical of OH groups (Bizaia et al., 2009; Marçal
et al., 2015).
All the samples displayed a large band typical of SieO vibration at
1008 and 450 cm−1. The stretching vibration attributed to the MgeOH
hydroxyl groups was almost invisible for Lap, but it intensified in the
FT-IR spectrum of Lap-APTES, at 3693 cm−1. The same feature arose in
the FT-IR spectrum of Lap-APTES:CHX-DG. The band ascribed to NH2
vibrations near 3360 cm−1 was less intense in the FT-IR spectra of Lap-
APTES and Lap-APTES:CHX-DG because the wide water band masked
it.
The MgeOH hydroxyl group bending band, located at 651 cm−1,
also revealed important differences between the samples. This band
shifted to higher wavenumber in the FT-IR spectrum of Lap-
APTES:CHX-DG, suggesting that the Lap hydroxyl groups also partici-
pated in the hydrogen bonding between the CHX-DG NH groups and the
Lap-APTES OH and NH2 groups (Bandeira et al., 2012).
The large band at ca. 1620 cm−1 corresponded to interlayer water
bending. This band shifted to 1645 cm−1 in the FT-IR spectrum of Lap-
APTES and Lap-APTES:CHX-DG, which indicated alterations in the
bonding of such water molecules in this sample. The bands at 1333,
1479, 1564 cm−1, corresponding to the amine groups in Lap-APTES,
shifted to 1386, 1535 (sharp band), 1617, and 1690 cm−1 in the FT-IR
spectrum of Lap-APTES:CHX-DG, which confirmed the interaction be-
tween the CHX-DG amine groups and Lap-APTES. The band between
2988 and 2920 cm−1 also presented major relative intensity and con-
firmed CHX-DG incorporation. The vibration at 1190 cm−1 confirmed
the presence of reactive silanols in Lap-APTES and Lap-APTES-CHX,
8
Applied Clay Science 195 (2020) 105733
which could refer to structural defects or free APTES Si-OH groups.
These defects and groups could be located at the broken edges of these
stacks, whereas the Mg-OH hydroxyl groups remained in the internal
space of the individual Lap sheets. Given this structural property, silane
coupling agents seem to be grafted on the Lap surface through con-
densation with the edge silanol groups (Marçal et al., 2015).
The typical amine group bands at 1564, 1479, and 1333 cm−1 in the
spectrum of Lap-APTES shifted very little compared to Lap-APTES:CHX-
DG, confirming that the Lap-APTES NH2 and OH groups and the CHX-
DG NH groups established hydrogen bonds. These results agreed with
the previously discussed PXRD data, which had shown changes in the
interlayer space of the layered Lap-APTES induced by interaction with
CHX-DG.
3.2. Chlorhexidine digluconate incorporation study
After Lap functionalization, we studied CHX-DG incorporation into
the hybrid Lap-APTES. We analyzed the supernatants of the kinetic
study between 1 and 90 min of contact time (Fig. 8). The system
reached adsorption equilibrium at 90 min; the percentage of CHX-DG
incorporation was 99%.
The kinetic study clearly showed that the system achieved max-
imum CHX-DG adsorption within 10 min of contact time. Equilibrium
varied very little between 15 and 90 min of contact time. This indicated
that Lap-APTES was in dynamic equilibrium with the CHX-DG mole-
cules, which suggested that the percentages of adsorbed and desorbed
CHX-DG were equivalent.
Table 3 lists the kinetic data for theoretical modeling according to
G.R. Peraro, et al. Applied Clay Science 195 (2020) 105733

102
Lap
100 100
100 Lap-APTES
Lap-APTES:CHX-DG
98 95
95

96

Mass (%)
90

94 90

85
92
85
90 80

88 80
75

86
75 70
200 400 600 800
(a) Temperature (oC)

-0.2
0.0 0.00
0.0
0.1 0.02
Deriv. Mass (dm/dT)

0.2
0.2 0.04
0.4
0.3 0.06
0.6
0.4 0.08
0.8
0.5 0.10
1.0
Lap
0.6 0.12
Lap-APTES
1.2 Lap-APTES:CHX-DG
0.7 0.14
200 400 600 800

(b) Temperature (oC)

Fig. 6. A TG curves of the original LAPONITE® RD (Lap), Lap-APTES, and Lap-APTES:CHX-DG. B DTG curves of the original LAPONITE® RD (Lap), Lap-APTES, and
Lap-APTES:CHX-DG.

Table 2 (Lagergren, 1898).

APTES and chlorhexidine digluconate loaded on LAPONITE® RD.
1 k 1 1
= 1. +
Sample W(200–600 °C) (%) WSi (%) Silane load CHX-DG load qt q1 t q1 (3)
(%) (%)
where q1 and qt (mg/g) are the amounts of CHX-DG adsorbed at
Lap 2.71 1.31 – –
Lap-APTES 12.14 5.87 7.65 –
equilibrium and at time t, respectively, and k1 is the rate constant
Lap-APTES:CHX-DG 11.68 5.64 7.30 12.65 (min−1) of the first-order model for the adsorption process. We calcu-
lated k1 from the slope of the plots of 1/qt versus 1/t (Fig. 7).
Eq. (4) expresses the pseudo second-order kinetic model (Ho, 2004;
the first-order and pseudo second-order kinetic models. Ho et al., 2006).
Knowledge of adsorption kinetics is fundamental during adsorption
t 1 t
studies. Studying the adsorption kinetics is the first step when it comes = +
qt (k2 q22 ) q2 (4)
to investigating adsorption, and this study provides valuable evidence
of the possible technological applications of an adsorbent for a parti- where q2 is the maximum adsorption capacity (mg/g) for the pseudo
cular separation. In this study, we used the first-order and pseudo second-order adsorption, qt is the amount of CHX-DG adsorbed at
second-order kinetic models to elucidate the mechanism of CHX-DG equilibrium (mg/g) at time t (min), and k2 is the rate constant of the
adsorption onto Lap-APTES. Eq. (3) gives the first-order kinetic model pseudo second-order adsorption (g/(mg.min)). We determined k2 and

9
G.R. Peraro, et al. Applied Clay Science 195 (2020) 105733

Lap between Lap-APTES and CHX-DG. CHX-DG is positively charged and

100 Lap-APTES could displace the Lap-APTES interlayer cations. These two forces
20
Lap-APTES:CHX-DG probably stabilized the CHX-DG molecules between the Lap-APTES
interlayer spaces.
Fast CHX-DG removal from the solution to reach the adsorption
80
3740 equilibrium within a short time evidenced that Lap-APTES efficiently
Transmittance (%)

incorporated the CHX-DG molecules. The characteristic pattern ob-

1620
tained for the kinetic curves until the system achieved saturation also
60 651 suggested that a CHX-DG monolayer arose on the Lap-APTES surface or
3740
1645 1333 1008 450 interlayer space.
3360 1479
2988-2920 1564 He et al., 2006 evaluated CHX incorporation into natural mon-
40 651 tmorillonite, the authors verified that CHX incorporation into the clay
3740 1386 required two days, which greatly contrasted with the time that was
3360 1690
2988-2920
1617
1535
1190 450 necessary for CHX-DG to adsorb onto Lap-APTES (15 min). The shorter
651 adsorption time obtained by us is a consequence of Lap functionaliza-
20
1060 450
tion with APTES, which enabled electrostatic interactions and hydrogen
bonding between the adsorbate and the adsorbent and thus elevated
4000 3500 3000 2500 2000 1500 1000 500
CHX-DG incorporation. Moreover, due to the LAPONITE® RD and Lap-
Wavenumber (cm-1) APTES properties that promotes the very fast incorporation of a higher
amount of CHX-DG on the surface and interlayer space of the grafted
Fig. 7. FT-IR spectra of the original LAPONITE® RD (Lap), Lap-APTES, and Lap-
APTES:CHX-DG.
clay, the methodology proposed herein could be a great alternative for
the market regarding the topic applications of CHX-DG derivatives.
We determined the maximum CHX-DG incorporation into Lap-
APTES by using a typical equilibrium experiment (Fig. 9) in which we
10
varied the CHX-DG concentration between 1 and 25,000 mg L−1 while
maintaining fixed time (90 min, obtained from the kinetics experi-
Amount adsorbed qt(mg.g-1)

9 ment), Lap-APTES mass (50 mg), and volume (5 mL) during the ad-
sorption experiment. Maximum CHX-DG incorporation occurred at
higher CHX-DG concentrations, and a maximum of 1402.735 mg/ g
8
CHX-DG was incorporated into Lap-APTES (70% of CHX-DG in-
corporation). The equilibrium profile study revealed a typical Langmuir
7 (L type curve) curve which, according to Giles et al. (1974) evidenced
that adsorption took place as CHX-DG monolayers interacting with the
Lap-APTES NH2 groups. This also attested to the higher affinity be-
6
Experimental tween the adsorbate and the adsorbent. The L-type curves, or the
Pseudo-first order normal or “Langmuir” isotherms, could be assigned to CHX-DG mole-
5
Pseudo-second order cules adsorbed flat on the Lap-APTES surface, or, sometimes, to the
vertical orientation of the adsorbed CHX-DG molecules with particu-
0 20 40 60 80 100 larly strong intermolecular attraction via hydrogen bonds between the
Time (min) APTES NH2 groups and CHX-DG. Subgroup 2 from Langmuir isotherm
revealed that the systems with an adsorbate monolayer reached the
Fig. 8. Kinetic study for incorporation of CHX-DG into Lap-APTES via bath plateau. This point justified the constant values obtained for two of the
adsorption experiments. tested maximum concentrations, 15,000 and 25,000 mg L−1, and also
evidenced that a higher amount of CHX-DG could be incorporated into
Table 3
Pseudo first- and second-order parameters for chlorexidine digluconate ad- 1600
sorption onto Lap-APTES (C0 = 25 mg L−1, T]25 °C).
1400
Pseudo first-order Pseudo second-order
1200
qt (mg.g−1) 9.15 qt(mg.g−1) 9.58
K1 (1/min) 1.64 K2 (g −1mg·min) 0.29 1000
R2 0.53 R2 0.83
X2 X2
qe (mg g-1)

1.48 0.52 800

600
q2 from the plot of t/qt against t.
400
We determined the kinetic parameters for CHX-DG adsorption onto
Lap-APTES from these plots and listed them in Table 3, which also 200
brings the correlation coefficient values (R2) for the first-order and
0 Equilibrium
pseudo second-order kinetic models. The data adjusted much better to
Lap-APTES/CHX-DG
the second-order kinetic model than to the first-order kinetic model, -200
evidencing that CHX-DG adsorption onto Lap-APTES follows a chemi- 0 2000 4000 6000 8000 10000 12000
sorption mechanism. As previously discussed for the FT-IR results, the Ce (mg L-1)
APTES amine group and the CHX-DG NH groups formed strong hy-
drogen bonds due to the number of amine groups present on the Lap- Fig. 9. Equilibrium study for CHX-DG incorporation into Lap-APTES via bath
APTES surface. However, we cannot discard electrostatic interactions adsorption experiment; 5 mL of CHX-DG solution between 1 and 25,000 mg L−1
and 50 mg of Lap-APTES were used.

10
G.R. Peraro, et al. Applied Clay Science 195 (2020) 105733

Yang et al., 2007

Saha et al., 2014

He et al., 2006

Present work
Reference
Time for CHX-DG incorporation (min)

2880
2880

180
90

Fig. 10. CHX-DG release study from Lap-APTES; 1000 mL of deionized water,
Amount of incorporated CHX-DG (mg/g)

100 mg of Lap-APTES:CHX-DG (CHX-DG content = 49.0 mg/ g), pH = 7.0, and

T = 25 °C were used.

Lap-APTES as a result of the higher affinity between Lap-APTES and

CHX-DG. Table 4 compares different experiments involving CHX-DG
incorporation into inorganic layered solids. The data in this table de-
0.043 to 1402.735

monstrate higher incorporation into grafted matrixes containing amine

3.36 to 160.34
5.36 to 189.9

3.75 to 180.15

groups, under mild conditions.

5.36 to 189.9
Maximum chlorhexidine digluconate (CHX-DG) incorporation (mg/g) into clay minerals and the respective conditions.

3.3. In vitro CHX-DG release assay

b
a

The in vitro drug release assays revealed that CHX-DG adsorbed

2 g of MMT, 30 mL of deionized water, 70 mL of ethanol, room temperature, magnetic stirring.

onto the clay matrix galleries since this drug is poorly soluble in water,
2 g of MMT, 30 mL of deionized water, 70 mLof ethanol, room temperature, magnetic stirring.

and the functionalized matrix has hydrophobic character. We measured

1 g of MMT, 30 mL of deionized water, pH = 4.1 (H3PO4), T = 80 °C, magnetic stirring

CHX-DG release at 260 nm, with the aid of a spectrophotometer. Fig. 10

gives the profile of CHX-DG release from Lap-APTES in water at pH near
5.0 mL deionized water, 0.05 g of Lap-APTES, pH = 7.0, 25 °C, magnetic stirring.

7.0 and 25 °C. The release profile depends on the medium. In water, the
CHX-DG release rate was constant up to 8 h (total CHX-DG release
~10%, Fig. 11, squares). A plateau was not reached, which evidenced
the very slow release of the drug within the first 24 h. Therefore, the
inorganic organophilic clay was able to promote controlled drug release
thanks to the hydrogen bonds established between APTES and CHX-DG
present in the very confined space of the clay galleries. Another fun-
damental point of the inorganic organophilic clay was the control of
water access to the clay interlayer space, which resulted in very slow
drug release in static conditions. The drug release profile also depends
on the aqueous medium pH. In the analyzed condition, CHX-DG release
peaked at 10 h (CHX-DG release = 6%). Before this time, there was an
inclination in the release curve, but no plateau was obtained, which
evidenced a constant CHX-DG release up to 24 h, when the total CHX-
DG release reached ~10% (Fig. 10, squares). This result pointed to a
crucial aspect of the organophilic (APTES) groups attached to the
LAPONITE® RD surface: it promoted very strong interactions with CHX-
Experiment condition

DG via hydrogen bonds and prevented CHX-DG release into the

medium.
a = Ca-MMT; b = Na-MMT

3.4. Cytotoxicity assay

Assessment of cytotoxicity against GM07492A cells allowed a pre-

liminary evaluation of the safety of Lap-APTES-CHX-DG for subsequent
Na-MMTb

applications (Table 5). The IC50 value observed when the cells were
Lap-APTES
Ca-MMTa

Ca-MMT
Ca-MMT

treated with Lap-APTES-CHX-DG was significantly higher than the IC50

Table 4

Matrix

value obtained in the treatment with CHX-DG alone. Therefore, CHX-

DG incorporation into Lap-APTES reduced drug cytotoxicity.

11
G.R. Peraro, et al. Applied Clay Science 195 (2020) 105733

Table 5 hydrophobic alkoxide groups on the Lap surface. Such effect did not
IC50 values obtained in human lung fibroblasts (GM07492A emerge in the case of S. pyogenes.
cells) treated with LAPONITE® RD (Lap), APTES, hybrid or- S. aureus and S. pyogenes have many distinct characteristics. First,
ganic-inorganic materials (Lap-APTES), hybrid organic-in- cell division occurs on a single axis and cells grow in pairs or chains in
organic materials adsorbed with CHX-DG (Lap-APTES:CHX-
the case of S. pyogenes, whereas cell division happens in various di-
DG), and chlorhexidine digluconate (CHX-DG).
rections on multiple axes in the case of S. aureus, which grows as grape-
Treatment IC50 (μg/mL) like clusters. Second, S. aureus produces catalase (an enzyme that
cleaves hydrogen peroxide), but S. pyogenes does not produce this en-
Lap > 5000
APTES > 5000
zyme. Third, unlike S. aureus, S. pyogenes requires enriched media for
Lap-APTES 454.7 ± 47.9a,b growth (fastidious). Finally, most staphylococci are found on the skin,
Lap-APTES:CHX-DG 70.3 ± 9.5a,b,c whereas most streptococci are found in the respiratory tract. These key
CHX-DG 24.0 ± 0.3a,b differences help to explain the results obtained in this work. Obviously,
the availability of CHX-DG incorporated into Lap-APTES affects the
Negative control - without treatment, Positive control - di-
antimicrobial activity.
methylsulfoxide 25%.
Values are the mean ± SD, n = 3. As reported by Greatti et al. (2013), CHX-DG at concentrations
a
Significantly different from the Lap or APTES treatment ranging from 1 to 2% display antibacterial activity against S. pyogenes.
(P < .05). Here, we verified this same activity against S. pyogenes at even lower
b
Significantly different from the Lap-APTES treatment CHX-DG concentration because the functionalized matrix Lap-APTES
(P < .05). protected the drug and promoted its controlled released, which boosted
c
Significantly different from the CHX-DG treatment drug efficacy against this pathogen.
(P < .05). The cytotoxicity of CHX-DG solutions at 0.12% and 2% and at 2% in
the presence of fluoride (laboratorial formula) led to the conclusion that
3.5. Evaluation of the antibacterial potential of Lap-APTES:CHX-DG CHX-DG at 0.12% was significantly less toxic against fibroblast cultures
against bacteria that cause dermatitis in vitro as compared to the other assayed formulations. In other words,
CHX-DX was not toxic at concentrations in which it is effective against
CHX presents many advantages: it is slowly released, and studies pathogenic microorganisms.
have confirmed its residual antimicrobial action (Table 6). Authors Stubbs et al. (1996) describes that CHX-DG is the antiseptic that best
(Arret et al., 1971; Buck et al., 1999; D'Arcangelo et al., 1999; Estrela meets these criteria. It represents an alternative for patients that are
et al., 2003) evaluated the antimicrobial effect of 2% CHX on micro- allergic to iodine because it is little adsorbed by the skin, and its per-
organisms by two methods, agar diffusion and direct exposure. 2% CHX cutaneous absorption is insignificant. Holešová et al. (2015) analyzed
displayed antimicrobial action against the biological indicators Sta- adult human samples obtained from individuals that had used topical
phylococcus aureus and Enterococcus faecalis, among other bacteria. CHX for up to 12 months, to find that there were no CHX circulating
Several works (Gomes et al., 2004; Kalender et al., 2013) have levels in these patients.
shown that CHX substantivity underlies its better action as compared to According to Sebben (1983) and Osuna et al. (1990), a topical an-
other antimicrobials. These authors highlighted that CHX is a good tiseptic should have a broad spectrum of antimicrobial activity, rapid
antimicrobial agent due to the absence of toxicity, which enables its use action, residual antimicrobial activity, low incidence of skin irritation,
in allergic patients. low toxicity, and good action in the presence of organic matter. Ob-
On the basis of the obtained results, from concentrations as low as viously, finding such an antiseptic is no easy task and calls for the
19.475 mg/g, CHX-DG incorporated into Lap-APTES was active against search of hybrid materials that display several beneficial characteristics
Staphylococcus aureus ATCC 29213 (Table 6). Lap-APTES:CHX-DG was and which can directly act on the drug to make it safer and potentially
effective against Streptococcus pyogenes ATCC 12345 (Table 6) at all the more active.
tested concentrations. The antibacterial action of CHX-DG was not
immediate because of the strong interactions between Lap-APTES and 4. Conclusion
this drug. CHX-DG incorporation into Lap-APTES may have protected
the drug or made access to CHX-DG difficult, enabling effective control LAPONITE® was functionalized with the alkoxide APTES, as attested
of drug release into the environment. Lap-APTES alone was also active by the characterization techniques (XRPD, FT-IR spectroscopy, SAXS,
against S. aureus. This effect was a consequence of the presence of the and TGA-DTA). FT-IR spectroscopy proved the affinity of Lap-APTES for
the drug CHX-DG—a large amount of CHX-DG adsorbed onto Lap-
APTES within a short time. This technique also indicated the potential
Table 6 use of Lap-APTES:CHX-DG for controlled drug release. Cytotoxicity
Evaluation of the antibacterial activities of Lap-APTES and Lap-APTES:CHX-DG
studies by the XTT technique revealed that Lap-APTES:CHX-DG was not
against Staphylococcus aureus (ATCC 29213) and Streptococcus pyogenes ATCC
cytotoxic, as judged from cell viability measurements. The antibacterial
(12345).
action of Lap-APTES:CHX-DG was tested by the agar diffusion tech-
Sample (mg/g) Mean ± SD* (mm) nique against pathogenic agents that cause dermatitis (Staphylococcus
aureus and Streptococcus pyogenes). Lap-APTES:CHX-DG in the powder
S. aureus 29,213 S. pyogenes 12,345
form was not effective against S. aureus, probably because the lack of
Lap-APTES:CHX-DG moisture prevented diffusion of the hybrid material nanoparticles in the
0.042 0.0 ± 0.0 10.7 ± 0.6 agar. In contrast, even low Lap-APTES:CHX-DG concentrations were
0.525 0.0 ± 0.0 13.7 ± 0.6
effective against S. pyogenes, attesting to the efficacy of CHX-DG ad-
2.070 0.0 ± 0.0 18.0 ± 1.0
4.259 0.0 ± 0.0 0.0 ± 0.0 sorbed onto Lap-ATPES. Drug release profile revealed very slow CHX-
9.433 0.0 ± 0.0 16.0 ± 1.0 DG release: the maximum release in the evaluated condition was 10%
19.475 9.3 ± 0.6 15.7 ± 1.2 (m/m) within 24 h, which evidenced that Lap-APTES can adsorb CHX-
49.264 12.0 ± 1.0 11.0 ± 1.0 DG molecules and control their release during experiments. The novel
Lap-APTES 0.0 ± 0.0 21.0 ± 1.0
Lap-APTES formulations containing CHX-DG have potential application
CHX-DG 14.3 ± 0.5 12.3 ± 0.5
200 mg L−1 or 0.1 mg/mL on pharmaceutical topical applications against many diseases such as
dermatitis, folliculitis, and erysipelas, and among others.

12
G.R. Peraro, et al.
Declaration of Competing Interest
The authors declare no conflict of interest.
Acknowledgments
We acknowledge the support of the Brazilian research funding
agency Fundação de Amparo à Pesquisa do Estado de São Paulo
(FAPESP) (2013/19523-3, 2013/20455-2 and 2017/15482-1). The
nanostructure of the materials was monitored by small-angle X-ray
scattering (SAXS) experiments, performed at the D01A-SAXS2 beamline
of the National Synchrotron Light Laboratory (LNLS, Campinas, Brazil)
(Project number: SAXS1-17845 and SAXS1-16981). We thank K. J.
Ciuffi for help with thermal analysis and UV–Visible spectroscopy
(FAPESP 1998/11022-3). We thank E. J. Nassar for the FTIR mea-
surements FAPESP (2012/11673-3). This study was partially funded by
Coordenação de Aperfeiçoamento de Pessoal de Nível Superior – Brasil
(CAPES) – Finance Code 001. All the authors thank Conselho Nacional
de Desenvolvimento Científico e Tecnológico (CNPq) (311767/2015-0,
306271/2017-6, and 303135/2018-2).
References
Arret, B., Johnson, D.P., Kirshbaum, A., 1971. Outline of details for microbiological as-
says of antibiotics: second revision. J. Pharm. Sci. 60, 1689–1694.
Ávila, L.R., de Faria, E.H., Ciuffi, K.J., Nassar, E.J., Calefi, P.S., Vicente, M.A., Trujillano,
R., 2010. New synthesis strategies for effective functionalization of kaolinite and
saponite with silylating agents. J. Colloid Interface Sci. 341, 186–193.
Avnir, D., Lev, O., Livage, J., 2006. Recent bio-applications of sol–gel materials J. Mater.
Chem. 11, 1013–1030.
Bagchi, B., Kar, S., Dey, S.K., Bhandary, S., Roy, D., Mukhopadhyay, T.K., 2013. In situ
synthesis and antibacterial activity of copper nanoparticle loaded natural mon-
tmorillonite clay based on contact inhibition and ion release. Colloids Surf. B:
Biointerfaces 108, 358–365.
Bandeira, L.C., Calefi, P.S., Ciuffi, K.J., de Faria, E.H., Nassar, E.J., Vicente, M.A.,
Trujillano, R., 2012. Preparation of composites of laponite with alginate and alginic
acid polysaccharides. Polym. Int. 61, 1170–1176.
Bizaia, N., de Faria, E.H., Ricci, G.P., Calefi, P.S., Nassar, E.J., Castro, K.A.D.F., Nakagaki,
S., Ciuffi, K.J., Trujillano, R., Vicente, M.A., Gil, A., Korili, S.A., 2009.
Porphyrin−Kaolinite as efficient catalyst for oxidation reactions. ACS Appl. Mater.
Interfaces 1, 2667–2678.
Buck, R., Eleazer, P.D., Staat, R.H., 1999. In vitro disinfection of dentinal tubules by
various endodontics irrigants. J. Endod. 25, 786–788.
Colletti, C.G., Massaro, M., Lazzara, G., Cavallaro, G., Milioto, S., Pibiri, I., Noto, R., Riela,
S., 2020. Synthesis, characterization and study of covalently modified triazole
LAPONITE® edges. Appl. Clay Sci. 187https://doi.org/10.1016/j.clay.2020.105489.
(In press).
da Silva, A.F., de Padua, G.S., de Araujo, D.T., Vieira, C.A., de Faria, E.H., 2020.
Immobilization of L-Alanine into Natural Kaolinite Via Amidation Catalyzed by Boric
Acid for the Development of Biohybrid Materials. J Solid State Chem. (In press).
Daniel, L.M., Frost, R.L., Zhu, H.Y., 2008. Edge-modification of laponite with dimethyl-
octylmethoxysilane. J. Colloid Interface Sci. 321, 302–309.
D’Arcangelo, C., Varvara, G., Fazio, P., 1999. An evaluation of the action of different root
canal irrigants on facultative aerobic-anaerobic, obligate anaerobic, and micro-
aerophilic bacteria. J. Endod. 25, 351–353.
de Carvalho, A.L., Ferreira, B.F., Martins, C.H.G., Nassar, E.J., Nakagaki, S., Machado,
G.S., Rives, V., Trujillano, R., Vicente, M.A., Gil, A., Korili, S.A., de Faria, E.H., Ciuffi,
K.J., 2014. J. Phys. Chem. C 118, 24562–24574.
Ding, L., Wang, R., Hu, Y., Xu, F., Zhang, N., Cao, X., Wang, X., Shi, X., Guo, R., 2020.
Folic acid-modified Laponite®-stabilized Fe3O4 nanoparticles for targeted T2-
weighted MR imaging of tumor. Appl. Clay Sci. 186https://doi.org/10.1016/j.clay.
2020.105447. (In press).
Estrela, C., Ribeiro, R.G., Estrela, C.R.A., Pécora, J.D., Sousa-Neto, M.D., 2003.
Antimicrobial effect of 2% sodium hypochlorite and 2% chlorhexidine tested by
different methods. Braz. Dent. J. 14, 58–62.
Fei He, X.H., Li, M., Zhang, S., 2008. SAXS investigations of the fractal character of ad-
ditive silica xerogels. J. Ceram. Process. Res. 9, 389–392.
Ghadiri, M., Hau, H., Chrzanowski, W., Agus, H., Rohanizadeh, R., 2013. Laponite clay as
13
Applied Clay Science 195 (2020) 105733
a carrier for in situ delivery of tetracycline. RSC Adv. 3, 20193–20201.
Giles, C.H., Smith, D., Huitson, A.A., 1974. A general treatment and classification of the
solute adsorption isotherm. I. Theoretical. J. Colloid Interface Sci. 47, 755–765.
Gomes, B.P.F.A., Pinheiro, E.T., Gadê-Neto, C.R., Sousa, E.L.R., Ferraz, C.C.R., Zaia, A.A.,
2004. Microbiological examination of infected dental root canals. Oral Microbiol.
Immunol. 19, 71–76.
Gómez-Romero, P., Ayyad, O., Suárez-Guevara, J., Muñoz-Rojas, D., 2010. Hybrid or-
ganic–inorganic materials: from child’s play to energy applications. J. Solid State
Electrochem. 11, 1939–1945.
Greatti, V.R., Weckwerth, P.H., Sabino, I.T., Vivan, R.R., Duarte, M.A.H., Neves, F.T.A.,
2013. Antibacterial activity in vitro of calcium hydroxide paste associated to bio-
products against on Gram-positive and Gram-negative strains. Salusvita 32, 297–305.
He, H., Yang, D., Yuan, P., Shen, W., Frost, R.L., 2006. A novel organoclay with anti-
bacterial activity prepared from montmorillonite and Chlorhexidini Acetas. J. Colloid
Interface Sci. 297, 235–243.
Herrera, N.N., Letoffe, J.M., Putaux, J.L., David, L., Bourgeat-Lami, E., 2004. Aqueous
dispersions of silane-functionalized laponite clay platelets. A first step toward the
elaboration of water-based polymer/clay nanocomposites. Langmuir 20 (5),
1564–1571.
Ho, Y.S., 2004. Citation review of Lagergren kinetic rate equation on adsorption reac-
tions. Scientometrics 59, 171–177.
Ho, Y.S., 2006. Review of second-order models for adsorption systems. J. Hazard.Mater
136 (3), 681–689. https://doi.org/10.1016/j.jhazmat.2005.12.043.
Ho, Y.S., Wase, D.A.J., Forster, C.F., 1996. Kinect studies of competitive heavy metal
adsorption by sphagnum moss peat. Environ. Technol. 17, 71–77.
Holešová, S., Samlíková, M., Ritz, M., Pazdziora, E., 2015. Antibacterial polyethylene/
clay nanocomposites using chlorhexidine as organic modifier. Mater. Today Proc. 2,
246–252.
José, N.M., Prado, L.A.S.A., 2005. Materiais híbridos orgânico-inorgânicos: preparação e
algumas aplicações. Quim Nova 2, 281–288 (in portuguese).
Kalender, A., Orhan, K., Aksoy, U., Basmaci, F., Er, F., Alankus, A., 2013. Influence of the
quality of endodontic treatment and coronal restorations on the prevalence of apical
periodontitis in a Turkish cypriot population. Med. Princ. Pract. 22, 173–177.
Lagergren, S., 1898. Zur theorie der sogenannten adsorption geloester stoffe.
Veternskapsakad. Handl. 24, 1–39.
Ley, C., Brendle, J., Walter, A., Jacques, P., Ibrahim, A., Allonas, X., 2015. On the in-
teraction of triarylmethane dye crystal violet with LAPONITE® clay: using mineral
nanoparticles to control the dye photophysics. Phys. Chem. Chem. Phys. 17,
16677–16681.
Marçal, L., De Faria, E.H., Nassar, E.J., Trujillano, R., Martín, N., Vicente, M.A., Rives, V.,
Korili, S.A., Gil, A., Ciuffi, K.J., 2015. Organically modified saponites: SAXS study of
swelling and application in caffeine removal. ACS Appl. Mater. Interfaces 7,
10853–10862.
Mayr, J., Saldi, C., Dıaz, D.D., 2018. Release of small bioactive molecules from physical
gels. Chem. Soc. Rev. 47, 1484–1515.
Mitzi, D.B., Prikas, M.T., Chondroudis, K., 1999. Thin film deposition of organic−inor-
ganic hybrid materials using a single source thermal ablation technique. Chem.
Mater. 3, 542–544.
Osuna, D.J., de Young, D.J., Walker, R.L., 1990. Comparison of three skin preparation
techniques part 2: clinical trial in 100 dogs. J. Am. Coll. Vet. Surg. 19, 20–23.
Saha, K., Butola, B.S., Joshi, M., 2014. Synthesis and characterization of chlorhexidine
acetate drug–montmorillonite intercalates for antibacterial applications. Appl. Clay
Sci. 101, 477–483.
Sebben, J.E., 1983. Surgical antiseptics. J. Am. Acad. Dermatol. 9, 759–765.
Seftel, E.M., Niarchos, M., Vordos, N., Nolan, J.W., Mertens, M., Mitropoulos, A.Ch.,
Vansant, E.F., Cool, P., 2015. LDH and TiO2/LDH-type nanocomposite systems: a
systematic study on structural characteristics. Microporous Mesoporous Mater. 203,
208–215.
Shapiro, L., Driess, M., Avnir, D., 2015. Dual catalytic activity of palladium doped with a
rhodium complex in a one-pot, four step process. Chem. Cat. Chem. 14, 2033–2037.
Stubbs, W.P., Bellah, J.R., Vermaas-Hekman, D., Purich, B., Kubilis, P.S., 1996.
Chlorhexidine gluconate versus chloroxylenol for preoperative skin preparation in
dogs. Vet. Surg. 25, 487–494.
Su, L., Tao, Q., He, H., Zhu, J., Yuan, P., 2012. Locking effect: a novel insight in the
silylation of montmorillonite surfaces. Mater. Chem. Phys. 136, 292–295.
Su, L., Tao, Q., He, H., Zhu, J., Yuan, P., Zhu, R., 2013. Silylation of montmorillonite
surfaces: Dependence on solvent nature. J. Colloid Interface Sci. 391, 16–20.
Tao, Q., Su, L., Frost, R.L., Zhang, D., Chen, M., Shen, W., He, H., 2014. Silylation of
mechanically ground kaolinite. Clay Miner. 49, 559–568.
Yang, D., Yuan, P., Zhu, J.X., He, H.-P., 2007. Synthesis and characterization of anti-
bacterial compounds using montmorillonite and chlorhexidine acetate. J. Thermal.
Anal. Calorim. 89 (3), 847–852. https://doi.org/10.1007/s10973-006-8318-3.
Zhang, J., Zhou, C.H., Petit, S., Zhang, H., 2019. Hectorite: Synthesis, modification, as-
sembly and applications. Appl. Clay Sci. 177, 114–138.