International Journal of Pharmaceutics 457 (2013) 224–236

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International Journal of Pharmaceutics

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

Pharmaceutical Nanotechnology

Montmorillonite nanodevices for the colon metronidazole delivery

Ilaria Calabrese a , Gennara Cavallaro b , Cinzia Scialabba b , Mariano Licciardi b ,
Marcello Merli c , Luciana Sciascia c , Maria Liria Turco Liveri a,∗
a
Dipartimento di Fisica e Chimica, University of Palermo, Viale delle Scienze Ed. 17, 90128 Palermo, Italy
b
Dipartimento Scienze e Tecnologie Biologiche Chimiche e Farmaceutiche (STEBICEF), University of Palermo, Via Archirafi 32, 90123 Palermo, Italy
c
Dipartimento di Scienze della Terra e del Mare (DiSTeM), University of Palermo, Via Archirafi 36, 90123 Palermo, Italy

a r t i c l e i n f o a b s t r a c t

Article history: The adsorption profiles of the antibiotic metronidazole (MNE) into the K10-montmorillonite (MMT-K10)
Received 5 August 2013 clay and the subsequent release have been investigated as a function of pH and MNE/MMT-K10 ratio,
Received in revised form in order to evaluate the potential of the MNE/MMT-K10 hybrids as controlled drug delivery system. The
11 September 2013
adsorption mechanism has been first elucidated by performing complementary equilibrium and kinetic
Accepted 15 September 2013
studies and through the X-ray diffractometry (XRD) characterization of the obtained composite materials.
Available online 26 September 2013
The gathered results allowed us to propose a mechanism consisting of a multi-step pathway involving the
neutral and the cationic form of the drug, which interact with different sites of the clay surfaces, i.e. the
Chemical compounds studied in this article:
Metronidazole (PubChem CID: 4173)
interlayer region and the faces of the lamella. In a second step the drug release kinetics has been studied
Montmorillonite (PubChem CID: 9942228) under physiological pH mimicking conditions simulating the oral drug administration and delivery. For
the sake of comparison the commercial formulation has also been employed for the release studies. The
Keywords: investigation of the release profiles and the comparison with the commercial formulation of the drug
K10-montmorillonite reveal that the new-tailor made formulation could be fruitful exploited for successfully prolonged the
Metronidazole
action of drug in the desired site.
Adsorption
Drug delivery
© 2013 Elsevier B.V. All rights reserved.

1. Introduction Within the challenging issue of the targeted drug delivery, to

Amoebiasis is an infection of the large intestine caused by a
single celled protozoan parasite, Entamoeba histolytica. The most
preferred choice of drug for intestinal amoebiasis is the antibi-
otic metronidazole (MNE), which has to be delivered directly in
the colon to exert its efficient action. Nevertheless, the pharma-
cokinetic profile of the conventional tablet metronidazole indicates
that the MNE is completely and rapidly absorbed after oral admin-
istration reaching a concentration in plasma of about 10 mg mL−1
approximately 1 h after a single 500 mg dose (Lau et al., 1992). The
dosage form not only provides minimal amount of metronidazole
for local action in the colon, still resulting in the relief of amoebi-
asis, but with unwanted systemic effects. This way, the design of
more effective strategies for the administration of metronidazole,
aiming at minimizing secondary effects, increasing drug bioavail-
ability and stability or even exhibiting high accuracy in reaching
the target has become significant.
∗ Corresponding author at: Università degli Studi di Palermo, Dipartimento di
Fisica e Chimica, Viale delle Scienze – Parco d’Orleans II – Pad. 17, 90128 Palermo,
Italy. Tel.: +39 091 23898970; fax: +39 091 590015.
E-mail address: marialiria.turcoliveri@unipa.it (M.L. Turco Liveri).
specific areas of the body, several systems have been proposed
with the major purpose of achieving a controlled and low release
rate of the drug in order to ensure a constant in vivo drug con-
centration for a longer period of time while preventing harmful
side-effects and drawbacks. These systems allow to both efficiently
control the site and the rate of delivery and obtain more advantages
in the administration of bioactive molecules (Viseras et al., 2008).
Among the studied drug delivery systems those composed by the
inorganic clay montmorillonite (MMT-K10) and the organic drug
have received particular attention. The reason of the considerable
interest in the smectite clays intercalated by drug molecules can be
attributed to the novel physical and chemical properties exhibited
by these hybrids.
The MMT-K10 clay belongs to the smectite group, whose
structure consists of two silica tetrahedral sheets sandwiching
an edge-shared octahedral sheet of aluminum, known as T-O-T
sheets. A general formula for the whole montmorillonite group can
reads as (½Ca,Na)0.5–1 (Al,Mg,Fe)4–6 (Si,Al)8 O20 (OH)4 ·nH2 O. Water
molecules are allocated in the interlayer sheet and are coordinated
by the exchangeable cations. The size of the cations (Ca2+ , Na+ and
even K+ in the MMT-K10) influences the (0 0 1) basal spacing of the
structure. The surface charge of montmorillonite has been inves-
tigated in several studies (Abend and Lagaly, 2000; Bergaya et al.,

0378-5173/$ – see front matter © 2013 Elsevier B.V. All rights reserved.
http://dx.doi.org/10.1016/j.ijpharm.2013.09.017
 I. Calabrese et al. / International Journal of Pharmaceutics 457 (2013) 224–236 225

2006; Janek and Lagaly, 2001; Penner and Lagaly, 2001; Ramos- Recasting of the XRD chemical analysis yields the following com-
Tejeda et al., 2003; Tombácz and Szekeres, 2004), showing that position for the MMT-K10:
the faces of the lamellae of the clay have permanent negative
charges owing to isomorphic substitutions, while pH-dependent (K0.25 Na0.118 Ca0.022 )(Al1.06 Fe0.026 Mg0.166 )(Si7.39 Al0.61 )O20 (OH)4
charges develop on both the edge of the lamella and in the inter-
Stock solutions of all chemicals used were prepared by weight
layer region. Moreover, MMT-K10 has large specific surface area;
before use. Deionized water from reverse osmosis (Elga, model
exhibits good adsorb ability, cations exchange capacity, standout
Option 3), having resistivity higher than 1 M cm, was used to
adhesive ability, and drug-carrying capability. Thus, MMT-K10 is a
prepare all solutions.
common used substance both as the excipient and active ingredient
in pharmaceutical products (Wang et al., 2008). The intercalation
2.2. Sample preparation
of organic species into layered inorganic solids provides a useful
and convenient route to prepare organic–inorganic hybrids that
Aqueous HCl solutions at the desired pH (0.2, 2.5 and 5.0) were
contain properties of both the inorganic host and organic guest
prepared by proper dilution of the standard 1 M HCl solution.
in a single material (Mohanambe and Vasudevan, 2005). Thus, the
MNE aqueous stock solutions were prepared by weighing the
intercalated drug can be released under proper conditions. More-
antibiotic and dissolving it with the aqueous HCl solution at the
over, intercalation of photosensitivity drug into montmorillonite
required pH.
markedly improves the drug photostability and, for this reason, the
MMT-K10 aqueous suspension was prepared by crushing the
matrix has been proposed as an excipient for drug photostability
clay in an agatha mortar and then mixing the needed amount of
enhancement (Ambrogi et al., 2012). It has also been demon-
the powder with the aqueous HCl solution prepared as described
strated that MMT-K10 could effectively protect unstable bioactive
above. The obtained dispersion was stirred for about 2 h before use.
molecules against harsh biological conditions and release them in
In both cases, the pH of the aqueous solution/dispersion was
a controlled manner, which finally contribute to enhance their effi-
measured and, when necessary, the pH was adjusted to the desired
cacy (Zheng et al., 2007; Fudala et al., 1999; Kollár et al., 2003; Lin
value by adding microvolumes of either HCl or NaOH standard
et al., 2002; Park et al., 2008). In addition, it has not to be leaved out
solution.
of consideration the study on the in vitro and in vivo MMT-K10 tox-
It is worth saying that the above reported procedure was neces-
icity. The toxicity of MMT-K10 has been evaluated in human normal
sary because the use of the commercial buffer makes the aqueous
intestinal cells in short- and long-terms as well as in mouse model
MMT-K10 dispersion less kinetically stable as a consequence of the
(Baek et al., 2012). MMT-K10 could cause some cytotoxic effects at
more favorite coalescence process of the particle clays.
high concentration after long-time exposure, while any remarkable
The MNE/MMT-K10 complexes at a given pH were prepared by
toxicity was found in mice receiving up to the highest dose tested,
adding appropriate aliquots of the drug aqueous solution to the
1000 mg kg−1 . MMT-K10 could be absorbed into the body within
aqueous montmorillonite dispersion. The obtained dispersion was
2 h, but it did not significantly accumulate in any specific organ.
stirred for at least 5 h and, then, centrifuged 10 min at 8000 rpm by
From the above reported MMT-K10 peculiar behavior it can be
means of a Centra MP4R IEC centrifuge. This way, the solid, sepa-
deduced that the investigation of clay–drug interaction and the elu-
rated by the surnatant, was either first frozen and after lyophilized
cidation of the release mechanisms is an essential contribution for
by using a Freeze Dry System Labconco or air dried for 2 days and,
the formulation of clay-based drug delivery systems. The possibili-
then, crushed in an agatha mortar. The former treatment allow us
ties of such systems depend on the amount of drug retained by the
to prepare the samples to be used for the release tests while the
clay as well as on the release kinetics and the total amount released
latter one was applied for obtaining the samples to be used for the
with regard to the therapeutic regime.
XRD characterization.
Bearing in mind these consideration within a wide Research
Project aimed at “Designing and in vitro/in vivo testing ad hoc car-
riers for colon targeted metronidazole delivery system” we have 2.3. XRD characterization
undertaken the present study. The work focuses on the release
profile of MNE from MMT-K10/MNE hybrids (prepared on vary- Powder X-ray diffractometry measurements have been per-
ing the internal parameters within the applied protocols, i.e., pH, formed for the MNE/MMT-K10 hybrids, prepared in the whole pH
clay/antibiotic ratio) in both simulated gastric (SGF) and intesti- range and, for the sake of comparison, for the clay in the absence
nal fluids (SIF) pH. Preliminary to the releasing study we have fully of additives, and for the crystalline drug.
characterized the nanocomposite from the physico-chemical point The samples were mounted on aluminum plates and the XRD
of view. In particular, kinetic and equilibrium studies have been patterns were acquired at room temperature with a Philips PW
carried out in order to elucidate the adsorption mechanism of the 1729 system using Ni-filtered Cu K␣ radiation with  1.5406 Å, in
MNE into the MMT-K10 and establish the nature of the interac- the range of scattering angles 2 = 4–40◦ at the rate of 0.01◦ /s.
tions involved in the hybrid MNE/MMT-K10 formation. Moreover,
the sites of interactions of the clay surface, i.e., the interlayer region 2.4. Spectrophotometric measurements
and the faces of the lamella, have been proposed on the bases of the
XRD results. The UV–vis spectra of the MNE aqueous solutions have been
monitored as a function of the antibiotic concentration in order to
estimate the molar absorption coefficients under the experimental
2. Experimental conditions used in the present work.
Moreover, the spectra have been registered for 8 h every 15 min.
2.1. Materials It has been found that the spectra do not show any significant
change neither in their intensity nor in their form as a function
Montmorillonite K10 (MMT-K10), hydrochloric acid (HCl) and of time. These results clearly indicate that the used drug is stable
sodium hydroxide (NaOH) standard solutions were purchased from under the experimental condition applied in the present work.
Sigma Aldrich. Methonidazole (MNE) was purchased from Fluka. A further prove of the MNE aqueous solution stability has been
The commercial formulation of the drug (Vagilen) was bought in provided by the invariance of the spectra after 4 days from prepara-
local Pharmacy. All materials were used as received. tion. Fig. 1 reports a typical UV–vis spectrum of the metronidazole
226 I. Calabrese et al. / International Journal of Pharmaceutics 457 (2013) 224–236
Fig. 1. UV–vis spectra of 25.0 ␮g mL−1 metronidazole aqueous solution at pH = 0.2
(dashed red line, pH = pKa = 2.5 (continuous black line) and pH = 5.0 (dotted blue
line). (For interpretation of the references to color in this figure legend, the reader
is referred to the web version of this article.)
aqueous solution at three different pH values, namely
pH = pKa = 2.5, where both the cationic and the neutral species of
the drug are present, pH = 0.2 and pH = 5.0 where the antibiotic
exists prevalently in its protonated and neutral form, respectively.
It has been found that the NME aqueous solutions at the different
pH used follow the Lambert–Beer law in the concentration range
3–30 ␮g mL−1 .
The absorption wavelength maxima, max , and corresponding
molar absorption coefficient, ε are shown in Table 1.
2.5. Kinetic of adsorption of the MNE into MMT-K10
The kinetic of the process of adsorption of the MNE into
the MMT-K10 has been studied under constant stirred con-
ditions at 25.0 ◦ C by keeping constant the montmorillonite
amount (0.36 mg mL−1 ) over a wide antibiotic concentration range,
3–30 ␮g mL−1 , and pH range, pH 0.2–7.4.
The kinetic measurements were carried out by monitoring the
spectrum evolution of aqueous solutions of MNE in the presence of
MMT-K10 by using a Analytic Jena S600 diode array spectropho-
tometer equipped with compartments for 1 cm × 1 cm × 5 cm
quartz cells and a magnetic stirring apparatus.
The mixtures for the kinetic runs were prepared directly in the
cuvette by adding 1 mL of the MNE aqueous solution, at the desired
concentrations, to 2.5 mL of the MMT-K10 aqueous dispersion. Each
kinetic measurement has been repeated at least three times in order
to check the reproducibility of the experimental results. It has been
found that the obtained rate constants are reproducible within 3%.
The wavelengths corresponding to the maximum absorbance value,
max , for the MNE at the different pH values have been selected and
the time course of the maximum absorbance decreases has been
detected.
For all cases studied the pH of the MNE/MMT-K10 dispersion
has been measured as function of time and it has been found that
in the time frame of 8 h the pH does not significantly change.
Table 1
Spectrophotometric parameters for the MNE aqueous solution as a function of pH.
pH max (nm) ε (mL cm−1 ␮g−1 ) R2
0.2 277.0 0.037 ± 0.001 0.9956
2.5 318.9 0.003 ± 0.001 0.9960
5.0 319.8 0.051 ± 0.001 0.9959
Fig. 2. Typical UV–vis for 25.0 ␮g mL−1 of MNE/MMT-K10 (A) and pure MMT-K10
(B) aqueous suspension at t = 0 (continuous black line) and t = 20,000 s (dashed red
line). T = 37.0 ◦ C, pH = 1.4. (For interpretation of the references to color in this figure
legend, the reader is referred to the web version of this article.)
To verify the stability of the MMT-K10 aqueous dispersion at the
different pH values with respect to coalescence process we have
monitored the UV–vis spectra of the aqueous clay dispersion for
24 h. It has been found that the coalescence depends to a strong
extent on the pH of work. In particular, for pH values below the
pKa the absorbance values slightly decrease on time augmentation
(12 h), while in the pH range 2.5 < pH ≤ 7.4 the coalescence process
reveals to be very considerable. This way, the quantitative analy-
sis of the adsorption kinetic data at pH > 2.5 has been hampered.
Strongly, it has to be underlined that the time scale of the coalesce
process is longer than that of the adsorption one (5 h), nevertheless,
according to a procedure already applied by Sciascia et al. (2011),
for the sake of accuracy, we have preferred to take into account the
small effect of the coalescence on the absorbance kinetic profile
by subtracting from the kinetic profile recorded at the maximum
absorbance that detected at  = 600 nm. This wavelength has been
chosen by considering that the spectrum of metronidazole aqueous
solution does not show any significant peak and only the coales-
cence process is observable. Fig. 2 shows the typical UV–vis spectra
of the drug/clay suspension (Fig. 2a) and the pure MMT-K10 sus-
pension (Fig. 2b) at the beginning (t = 0) and at the end (t = 20,000 s)
of the adsorption process.
2.6. Adsorption isotherms
The adsorption isotherms have been collected by determining
the metronidazole equilibrium concentration (Ce ) in the surnatant,
then, the difference between the initial antibiotic concentration
and the latter was used to estimate the amount of adsorbed drug
(Cs ) onto the MMT-K10.
In order to obtain more performant MNE/MMT-K10 complexes
to be used for the drug delivery in the target point, MNE load-
ing procedure was optimized by using the higher amounts of
both the substances with respect to the conditions used for the
kinetic studies, namely, the MMT-K10 concentration was set at
30 mg mL−1 while that of the MNE was varied over the wide range
0.4–6.4 mg mL−1 . All complexes have been prepared at 25.0 ◦ C at
pH = 0.2, 2.5 and 5.0. All aqueous dispersions have been prepared
accordingly to the procedure above described.
The choice of these experimental conditions was dictated by
the pKa of the metronidazole (pH = 2.5) and it satisfy the need to
have in solution prevalently the cationic or the neutral species
at pH < pKa and pH > pKa , respectively, and both the cationic and
 I. Calabrese et al. / International Journal of Pharmaceutics 457 (2013) 224–236 227

the oral drug administration and the subsequent physiological

release.
The release of MNE from the MNE/MMT-K10 complexes has
been monitored as following: 100 ␮g of the sample were dispersed
in 500 ␮L of HCl 0.1 M and transferred into a dialysis membrane
(Spectrapor Dispodialyzer MWCO 500 with diameter of 5 mm). Fur-
ther 500 ␮L of HCl 0.1 M have been applied to completely transfer
the sample into the membrane. Subsequently, the membrane was
put in a glass cylinder containing 100 mL of HCl 0.1 N solution. The
system was transferred in a Orbital Shaker (Thermo) and main-
tained for 2 h at 37.0 ◦ C. After 2 h the membrane has been taken
away and put in another glass cylinder containing 100 mL phos-
phate buffer at pH 6.8 and the system maintained at 37.0 ◦ C in the
Orbital Shaker for a time period of 6 h.
At scheduled time intervals, 1 mL of the release medium has
been withdrawn and analyzed by HPLC. To keep constant the vol-
ume of the release medium, 1 mL of fresh solution of either 0.1 M
HCl or phosphate buffer at pH 6.8 has been used to replace the
collected one.
Fig. 3. Speciation diagram of the MNE antibiotic. For the sake of comparison, the kinetic of the MNE release from
the commercial formulation has been studied by following the pro-
neutral species at pH = pKa . These conditions are schematically tocol above described for the MNE/MMT-K10 complexes.
shown in the speciation diagram of Fig. 3. Experiments were carried out in triplicate and the results were
Nevertheless, due to the very high absorbance value of the dis- reported as averages values by applying a protocol based on
persion the spectrophotometric method could not be applied to advanced statistical diagnostics.
follow the kinetic of the adsorption process, thus only the equilib-
rium conditions have been detected and this allow us to gather the
3. Results and discussion
adsorption isotherms.
The MNE equilibrium concentration has been estimated by
The main purpose of the present work was to obtain a complete
means of high performance liquid chromatography (HPLC), using
picture of the mechanism of the adsorption and release of the MNE
an Agilent Liquid Chromatography 1100 Series equipped with a
drug in the MMT-K10 clay. In order to accomplish this goal the
UV–vis detector model Kontron 432.
integration of different complementary techniques was required.
For the chromatographic separation it has been used an inverse
First of all the adsorption process has been investigated by means
phase column Gemini C18 110 Å from Phenomenex as stationary
of systematic kinetic and equilibrium studies and through the XRD
phase, a mobile phase composed by a mixture of acetonitrile and
characterization of the obtained MNE/MMT-K10 complexes.
5 mmol L−1 KH2PO4 (pH 3.0) aqueous solution in the ratio 8/92
In a second step the drug release kinetics has been studied under
(v/v), and a flow velocity of 1 mL min−1 . Samples has been mon-
physiological pH conditions simulating the oral drug administra-
itored at 313 nm, which corresponds to the wavelength of the
tion and delivery.
maximum of the metronidazole aqueous solution at pH 3.0 (Gibson
In order to present the data in the most clear and readable
et al., 1984). The validity of the Lambert–Beer law has been verified
form, in the following subsections we will first discuss the results
over a wide range of drug concentration (5 ␮g mL−1 –11 mg mL−1 ).
obtained from the equilibrium data and from the XRD charac-
terization which provided important information to discuss the
2.7. Kinetic of MNE release kinetic data and to propose the mechanism pathways concerning
the adsorption processes.
In vitro kinetic release studies have been performed by selecting
two MNE/MMT-K10 complexes at different drug loading quantities.
The characteristics of the used complexes are shown in Table 2, 3.1. Adsorption isotherms
where the pH refers to the pH values applied for the MNE/MMT-K10
preparation. The equilibrium absorbance values, obtained at three different
The release of the adsorbed MNE has been monitored in media pH values, have been used to construct the adsorption isotherms
which mimic the physiological pH conditions, i.e. HCl 0.1 M and reported in Fig. 4, where the equilibrium amount of antibiotic
phosphate buffer pH = 6.8, at 37.0 ◦ C. These conditions reproduce adsorbed into the clay (Cs , mmol g−1 ) is plotted as a function of
the equilibrium drug concentration in solution (Ce , mmol cm−3 ).
First of all, it can be observed that the amount of adsorbed
Table 2
Amount of MNE loaded into 1 g of MMT-K10, as a function of the pH values applied drug increases on increasing the equilibrium drug concentration
for the MNE/MMT-K10 preparation. and it is considerably lower at the higher pH. The latter result can
be attributed to the minor presence of the H+ ions promoting the
Sample pH of the Adsorbed MNE
hybrids into 1 g intercalation of the drug in the interlayer region (de et al., 2008).
preparation MMT-K10 Both the Langmuir and the Freundlich models have been applied
(mg g−1 ) to analyze the data and the obtained sorption parameters are col-
␣1 0.2 46.5 lected in Table 3.
␣2 27.3
The discrimination between the two models has been per-
␤1 2.5 45.4 formed by means of the statistical criteria described in Merli et al.
␤2 34.0
(2010) based on advanced statistical diagnostics and robust fitting
␥1 5.0 30.0 techniques. A selection of the applied statistical tests are shown in
␥2 11.3 Table 4 while in Fig. 5 the residual plots are reported.
228 I. Calabrese et al. / International Journal of Pharmaceutics 457 (2013) 224–236

Table 3
Sorption parameters of Langmuir and Freundlich models for the adsorption isotherms of MNE onto MMT-K10 performed at the different pH values.

pH Langmuir Cs = qm ·KL ·Ce /1 + KL ·Ce Freundlich Cs = KF · Cen

−1 −1
q m
(mmol g ) 3
KL (cm mmol ) n KF (cm3 g−1 )

0.2 0.3 ± 0.1 90 ± 30 1.0 ± 0.1 0.46 ± 0.03

2.5 0.5 ± 0.3 380 ± 200 0.67 ± 0.04 0.29 ± 0.01
5.0 0.08 ± 0.02 180 ± 30 0.28 ± 0.07 0.37 ± 0.06

Table 4
Selected figures of merit for the Langmuir and the Freundlich models applied to the adsorption isotherms reported in Fig. 3.

Langmuir Freundlich

pH 0.2 2.5 5.0 0.2 2.5 5.0

2 2.8 × 10−4 9.8 × 10−4 1.4 × 10−5 4.9 × 10−5 4.3 × 10−5 4.5 × 10−5
R2 0.9543 0.8487 0.9854 0.9974 0.9924 0.9231
ESS 1.1 × 10−3 3.9 × 10−3 4.3 × 10−5 2.0 × 10−4 1.7 × 10−4 1.3 × 10−4
FANOVA 248 104 519 1415 2431 164
AIC −33 −26 −28 −44 −45 −22

The Freundlich model has proved to be the most reliable for
the experiments carried out at pH = 0.2 and pH = 2.5, thus indicat-
ing that the drug, can interact in parallel with multiple types of
sorption sites (Schwarzenbach et al., 2003), while the Langmuir
model better reproduces the data obtained at pH = 5.0, indicating
that, under these conditions, the clay offers a unique adsorption
site to the neutral form of the MNE.
The Langmuir isotherm, observed at pH = 5.0, can be explained
by considering that, under these experimental conditions, the only
available interaction site is the interlayer region, while the Freund-
lich isotherms, which we recall to correspond to heterogeneous
adsorption, observed under more acidic conditions, indicates that
the drug interacts with both the interlayer region and the nega-
tively charged faces of the lamella.
A comparison between the Freundlich parameters obtained at
pH = 0.2 and pH = 2.5 reveals that higher values of nf and Kf (adsorp-
tion intensity and adsorption capacity, respectively) are obtained
at the lower pH.
Moreover, it can be observed that the Freundlich exponent n
is about 1 at pH = 0.2, which means that the adsorption sites are
almost homogeneous in energy, while the n value less than 1
obtained at pH = 2.5 implies that progressive adsorption of the MNE
modifies the sorbent in a manner that increases its sorption capac-
ity, such as forming new adsorption sites (Gereli et al., 2006; Tsai
et al., 2003; Jiang et al., 2002; Özcan et al., 2004).

Fig. 4. Adsorption isotherms of MNE onto MMT-K10 performed at pH = 0.2 (A),

pH = 2.5 (B) and pH = 5.0 (C). Symbols denote experimental points; line corresponds
to the fit by Langmuir (red line) and Freundlich (blue line) models. (For interpreta- Fig. 5. Residuals plot for the adsorption isotherms performed at pH = 0.2 (A and A ),
tion of the references to color in this figure legend, the reader is referred to the web pH = 2.5 (B and B ) and pH = 5.0 (C and C ) fitted by using the Langmuir (A–C) and the
version of this article.) Freundlich (A –C ) models.
 I. Calabrese et al. / International Journal of Pharmaceutics 457 (2013) 224–236 229

Fig. 7. XRD patterns of the hydrated MMT-K10 (A) and MNE (B). T = 25.0 ◦ C, pH 2.5.

the high pH value this behavior can be easily explained by taking

into account that the MNE exists mainly in the neutral form, there-
fore all the drug enters the clay interlayer through a protonation
(de et al., 2008) rather than a cation exchange mechanism. On the
other hand, at strongly acidic pH, the cation exchange reaction is
hindered due to the competition between the cationic drug and H+
protons, which exist on the MMT-K10 interlayer at low pH value
as the silanol groups on the clay surface get protonated (Seki and
Kadir, 2006).

3.2. XRD analysis

Fig. 6. Dubinin–Radushkevich adsorption isotherms of MNE onto MMT-K10 per- The adsorption site of the drug into the MMT-K10 has been
formed at pH = 0.2 (A), pH = 2.5 (B) and pH = 5.0 (C). Symbols denote experimental
investigated by means of XRD measurements. Powder X-ray
points; line corresponds to the fit by D–R equation.
diffraction measurements have been performed, at different pH
values, for the MMT-K10 clay both in the absence and in the pres-
In a second step, in order to obtain information about the nature ence of the drug, and for the crystalline drug.
of adsorption type, the Dubinin–Radushkevich (DR) equation has Typical XRD patterns of the pure MMT-K10 and MNE are pre-
been applied: sented in Fig. 7.
ln Cs = ln Xm − kε2 As for the XRD pattern of the MMT-K10 hydrated at the desired
pH, in the absence of drug (Fig. 7a), the presence of two charac-
where ε = RT ln(1 + 1/Ce ) is the Polanyi Potentiali, R (kJ mol−1 K−1 ) is teristic peaks has to be highlighted, namely the one at 2 = 8.9◦ ,
the gas constant, T (K) is temperature, Xm is the adsorption capac- which corresponds to an interlayer distance of 9.9 Å, and the less
ity and k is a constant related to adsorption energy through the intense peak at 2 = 5.3◦ (d = 16.6 Å) which, according to Bromberg
following equation (Qadeer et al., 1995): et al. (2011) and Cui et al. (2008) has been ascribed to the interlayer
−0.5 spaces intercalated with water molecules. The comparison with the
E = −(2k)
diffractogram of the pure MNE, reported in Fig. 7b, indicates that
The linear plot of ln Cs versus ε2 (Fig. 6) allowed us to determine there is no significant overlap for the selected peaks.
the values of Xm , k and E reported in Table 5. Fig. 8 shows the X-ray diffraction patterns for the parent MMT-
It is known (Malik et al., 2005; Singh and Pant, 2004) that the K10 and the MNE/MMT-K10 hybrids at three different pH values
magnitude of E is useful for estimating the type of adsorption: (namely pH = 0.2, 2.5, 5.0).
when E is in the range 8–16 kJ mol−1 , it can be assumed that the Perusal of the figure evidences the successful intercalation of
drug is adsorbed through the cation exchange process. The data MNE, highlighted by the appearance of a new peak at 2 = 6.6◦
reported in Table 5 seem to indicate that the ionic exchange pro- (d = 12.2 Å) whose intensity increases on increasing the pH.
cess is favored at pH = 2.5, while at the other pH value a border-line These results could be explained in the light of the equilibrium
situation is observed, and, if any, it occurs only to a low extent. At data which showed that the total amount of adsorbed drug was
lower at high pH, this way, if all the adsorbed drug would be inter-
Table 5
calated in the interlayer, the opposite intensity trend should be
Sorption parameters of the Dubinin–Radushkevich model for the adsorption observed. Therefore, it is reasonable to rule out that the interlayer
isotherms of MNE onto MMT-K10 performed at the different pH values. region is the only available interaction site for the adsorbed drug
pH 0.2 2.5 5.0
and to propose that the drug interacts with different sites of the
Xm (mol g−1 ) 0.36 ± 0.02 0.32 ± 0.01 0.147 ± 0.007 clay surface (i.e. the interlayer region and the negatively charged
k (mol2 kJ−2 ) (7.7 ± 0.3) × 10−3 (4.1 ± 0.2) × 10−3 (8.4 ± 0.5) × 10−3 faces of the lamella).
E (kJ mol−1 ) 8.0 ± 0.3 11.0 ± 0.5 7.7 ± 0.5 On the other hand, since it is well known that at pH = 0.2
R2 0.9010 0.9824 0.9821
and pH = 5.0 the drug are present in solution principally in the
230 I. Calabrese et al. / International Journal of Pharmaceutics 457 (2013) 224–236
Fig. 8. XRD patterns of the parent MMT-K10 (black lines) and MNE/MMT-K10
hybrids (red lines) prepared at pH = 0.2 (A), pH = 2.5 (B) and pH = 5.0 (C). The insets
show the enlargement of the small angle X-ray diffraction patterns. (For interpreta-
tion of the references to color in this figure legend, the reader is referred to the web
version of this article.)
protonated and neutral form, respectively, the trend of the inten-
sity of the peak as a function of pH range brought us to reasonably
assume that the interlayer region is able to accommodate mainly
the neutral species.
Bearing in mind all these considerations it can be proposed that
the drug adsorption onto the clay pathways mainly occurs as fol-
lows:
at strongly acidic pH, where the protonated species strongly pre-
dominate, the great majority of the protonated drug interacts with
the lamella negative faces, while a smaller amount enter the inter-
layer space through cationic exchange reaction.
at pH » pKa , where MNE exists mainly in its neutral form, almost all
the drug enters the clay interlayer thus producing a very intense
peak.
at pH = pKa , where both the species coexist, the neutral form of the
drug is intercalated, while the remaining part of the protonated
drug mostly interacts both with the external lamella faces and
with the interlayer region.
The lower MNE content in the interlayer observed at acidic pH
can be ascribed to the competing reaction between the cationic
MNE species and the H+ . In fact the higher H+ concentration at acid
pH hinders the intercalation of the MNEH+ species.
Fig. 9. Adsorption kinetics of MNE onto MMT-K10 obtained at (A) pH = 0.2 and (B)
pH = 2.5 fitted by pseudo-first order model (red line), pseudo-second order model
(blue line) and DEM order model (green line). (For interpretation of the references
to color in this figure legend, the reader is referred to the web version of this article.)
It is worth to note that the proposed mechanism is able to
account for the different shape of the isotherms reported in the
previous paragraph, as a function of the pH values.
Finally it is possible to observe that the characteristic peak of the
MMT-K10 at 2 = 5.3◦ remains almost unchanged after drug/clay
interaction at pH 0.2 and pH 2.5 whereas it completely disappears
when the drug/clay complex is prepared at pH 5.0. These results
allow us to draw the conclusion that the incoming of the neutral
MNE wholly displaces the intercalated water molecules from the
interlayer spaces, which is, indeed, confirmed by the increase of the
intensity of the peak corresponding to the basal interlayer at 9.9 Å
(Fig. 8c).
3.3. Adsorption kinetics
As already discussed in the experimental section, for pH values
higher than 2.5 the coalescence process have a significant influence
in the absorbance time-course, therefore the kinetic profiles have
been analyzed only in the pH range 0.2–2.5.
In Fig. 9 typical plots of the absorbance versus time at two dif-
ferent pH values are reported along with the correlated models.
Obtained results demonstrated that, for all the pH values inves-
tigated, the absorbance decreases as a function of time, due to the
MNE adsorption on clay, until a constant value is reached.
Three different kinetic models, among the most extensively
used, have been applied (see Fig. 9), i.e. first-order, second-order
and double exponential models (the reader can refer to Sciascia
et al. (2011) and references therein for an extended description of
these models).
Analogously to the procedure applied for the analysis of the
equilibrium data, the discrimination among the different models
has been performed by means of the statistical criteria shown in
 I. Calabrese et al. / International Journal of Pharmaceutics 457 (2013) 224–236 231

Fig. 10. Typical residuals plot for the adsorption kinetics performed at pH = 0.2 (A–C) and pH = 2.5 (A –C ) fitted by using the pseudo-first order (A and A ), the pseudo-second
order (B and B ) and double-exponential (C and C ) models.

Table 6 and the analysis of the residuals (typical plots reported in
Fig. 10).
Moreover it has been observed that the rate constant values are
almost independent of the drug concentration (data not shown for
the sake of clarity) while slightly depend on the pH values (Fig. 11).
According to the literature (Qiu et al., 2009), the DEM model
describes a mechanism consisting of two parallel reactions involv-
ing two spectroscopically distinguishable species, which can be
easily renowned as the neutral and the cationic form of the drug.
Intriguingly the kinetic data seem to demonstrate that the effect
of the presence of the two form of the antibiotic is relevant, not
only at pH = pKa , where both species are significantly present, but
also at strongly acidic pH, where the cationic form sharply predom-
inates. This can be taken as an indication of the high sensitivity of
the kinetic method which allow us to obtain information which
cannot be yielded through equilibrium studies.
As already discussed in the previous paragraphs, the reaction
entailing the neutral species involves the acidic interlayer region
of the clay (Lahav and Chang, 1976) which absorbs the neutral
molecules by following protonation mechanism:
while the adsorption of the protonated form occurs via two differ-
ent pathways, i.e., the cation exchange mechanism:
C-MMT-K10 + nMNEH+ → MNEH–MMT-K10 + Cn+ (R2a)
where Cn+ represents the exchangeable cations of the clay (in the
case of the K10-MMT-K10 employed in the present work Cn+ = K+ ,
Na+ and Ca2+ ), and the electrostatic interaction between the posi-
tively charged drug and the negatively charged faces of the lamella:
MMT-K10(−) + MNEH+ → MNEH–MMT-K10 (R2b)
where MMT-K10(−) indicates the negatively charged faces of the
montmorillonite lamella.
The occurrence of process (R2a) is not only supported by the
literature (Ghanshyam et al., 2009) but is also in good agreement
with our DR sorption parameters reported in Table 5, while the XRD
pattern, obtained at strongly acidic conditions, indirectly proves the
occurrence of reaction (R2b). It is worth to say that, since reactions

H-MMT-K10 + MNE → MNEH–MMT-K10 (R1)

Table 6
Selected figures of merit for the pseudo-first order, pseudo-second order and
double-exponential models applied to the adsorption kinetics reported in Fig. 10.

Pseudo-first Pseudo-second Double-

order model order model exponential
model

pH 0.2
2 9.8 × 10−7 1.33 × 10−6 6.3 × 10−7
R2 0.99682 0.99568 0.99794
ESS 2.5 × 10−4 3.4 × 10−4 1.6 × 10−4
FANOVA 6.0 × 107 4.4 × 107 5.5 × 107

pH 2.5
2 1.10 × 10−5 5.03 × 10−6 2.97 × 10−6
Fig. 11. Rate constant values obtained from the application of the DEM models to the
R2 0.99030 0.99546 0.99731
kinetic adsorption data as a function of the pH of preparation of the MNE/MMT-K10
ESS 1.2 × 10−3 5.4 × 10−4 3.15 × 10−4
complexes. The reported values are averaged over the whole MNE concentration
FANOVA 1.57 × 106 3.4 × 106 3.46 × 106
range.
232 I. Calabrese et al. / International Journal of Pharmaceutics 457 (2013) 224–236
Fig. 12. Chromatogram of 0.01 mg mL−1 of MNE aqueous solution at pH = 2.5 and T = 37.0 ◦ C.
(R2a) and (R2b) involve the same species, the spectrophotometric
method is not able to distinguish between them. Therefore, we have
a situation of two parallel adsorption reactions, which macroscopi-
cally result in a single observable event composed internally of two
individual adsorption rate constants.
In order to associate a kinetic constant value to each adsorption
path, the dependence of the kinetic constant values on the pH has
been analyzed.
Perusal of Fig. 11 indicates that the kinetic constants are almost
independent of the pH for values less than 1.5 and afterwards
the rate constant associated with the faster reaction path starts to
increase while, for the other one a small decrease can be detected.
Since the adsorption process of the neutral species (R1) occurs
through a protonation mechanism, it is reasonable to associate the
rate constant of this step to the decreasing trend of the k values on
increasing the pH. Thus, the lower kinetic constant values should
correspond to the rate through which reaction (R1) takes place.
As a consequence, the higher value of the kinetic constant could
be associated to the adsorption of the cationic species. The increas-
ing trend of the rate constant with pH can be explained by taking
into account that, from the kinetic point of view, the spectro-
photometric observed process is the sum of two different pathways,
namely (R2a) and (R2b). It can be reasonable said that the cationic
exchange capacity of clay minerals decreases on increasing the pH,
which means that the rate constant associated with reaction (R2a)
should decrease on pH increasing, the augmentation of the nega-
tive charges on the faces of the lamella on increasing the pH, will
favor the electrostatic interaction between the drug and the clay,
therefore the rate constant associated to step (R2b) increases on
increasing the pH.
Therefore, the trend of the kinetic constants, not only seems to
indicate that the electrostatic interaction play a relevant role in the
whole adsorption process of the protonated drug, but can also be
taken as a further proof of the occurrence of the three proposed
reaction steps.
3.4. Evaluation of the release capability of the MNE from
MMT-K10
The in vitro kinetic release study of the adsorbed MNE from
a series of complexes MNE/MMT-K10 (prepared under different
experimental conditions) has been undertaken in media which
mimic the physiological pH conditions, i.e., the simulated gastric
fluids pH (HCl 0.1 M; pH = 1.0) and the simulated intestinal fluids
pH (phosphate buffer pH = 6.8) at 37.0 ◦ C. It is worth noting that
the time frame used for studying the kinetic of the drug release,
i.e. 2 h at pH 1 and than 6 h at pH 6.8, corresponds to the residence
times of the drug in the stomach and in the intestine respectively,
mimicking the normal gastro-intestinal transit. Actually, these con-
ditions reproduce the oral drug administration and the consequent
physiological release. For the sake of comparison the commercial
formulation has also been employed for the release studies. Since
our purpose is the developing and production of new dosage for-
mulation the kinetic of the drug release studies reveal to be not only
necessary but also very informative to verify that the drug disso-
lution takes place in an appropriate path. This way, the new-tailor
made drug delivery system could allow to achieve the prefixed goal,
i.e., to use the MNE/MMT-K10 as a reservoir formulation for the
modified drug delivery.
First of all, we would like to point out that in all the performed
experiments the HPLC analysis of the drug, in both physiological
media, reveals that the chromatogram of the metronidazole does
not show any significant changes neither in the form nor in the
shape with respect to that obtained for the pristine drug. As an
example, a typical chromatogram of MNE is shown in Fig. 12.
Moreover, the retention time remains unaltered. These are very
important results which clearly suggests that the kind of interaction
between the montmorillonite and the drug makes the MNE/MMT-
K10 complex very stable and the interaction of the clay with the
MNE does not induce any change in the chemical nature of the
antibiotics. These results could be fruitful exploited for successfully
prolonged the action of drug in the desired site. The converse effect
of a system MMT-K10/digoxin hybrids (Carretero, 2002) has been
observed by other authors.
The obtained cumulative release profiles are reported in Fig. 13.
Obtained data clearly indicates that the kinetic of the release
process is strongly dependent on the type of drug formulation used.
In particular, the commercial tablet is able to sustain a continuous
release of the MNE in both media (pH 1 and 6.8), while for the
Fig. 13. Kinetics of MNE release from MMT-K10. Symbols denote experimental
points; line corresponds to the fit by zero-order model (red line) and first-order
model (black line). (For interpretation of the references to color in this figure legend,
the reader is referred to the web version of this article.)
 I. Calabrese et al. / International Journal of Pharmaceutics 457 (2013) 224–236
MNE/MMT-K10 complexes the experimental conditions applied
for the complexes preparation, i.e. both the pH applied for the
MNE/MMT-K10 preparation and the amount of drug loaded onto
the MMT-K10 play a relevant role and indeed influence the MNE
release profile.
Comparison of the trends obtained for the two types of for-
mulations (the new-prepared ˛, ˇ,  and the commercial one)
reveals that, after a lag time, for the commercial tablet a conspic-
uous amount of MNE, about 40%, is released at pH 1 and after 6 h
almost the 100% of the drug is present in the buffered medium at
pH 6.8 as free drug. Differently, the MNE loaded into the clay is not
easily released at pH 1 within the 2 h of incubation, only about the
10%, and on passing time (6 h) the amount of released antibiotic
notably increases at pH 6.8 till it reaches a plateau value.
Moreover, a further examination of the release profiles evi-
dences that the equilibrium percentage of MNE does not always
reach the 100% and this can be explained by invoking the elec-
trostatic interaction between the positively charged drug and the
anionic charges at surface of the MMT-K10. Analogous explana-
tion has been proposed for the system sertraline/MMT-K10 (Nunes
et al., 2007).
It has to be highlighted that these explanations further corrob-
orate the proposed adsorption mechanism.
The incomplete release of the MNE from the MMT-K10 formu-
lation represents an accomplishment of the prefixed target since it
implies that the MNE/MMT-K10 complex should not be protected
by an enteric coating, usually the Eudragit® , if it is intended to
produce controlled release at the intestine level. Thus, the prepara-
tion of MNE/MMT-K10 formulation would not result neither time
consuming nor waste of money.
Besides, the kinetic profile, obtained for all cases investigated
in the intestinal pH mimicking medium, indicated that the usual
initial burst (Betsiou et al., 2012) is not present with our systems.
This way, the sustained release could be fruitful exploited for the
prolongation of the drug action.
The slow and subsequent sustained release processes observed
in the two physiological conditions may be ascribed to the different
kind of exchangeable cations present in the media, e.g. H+ in the in
the stomach pH mimicking medium and Na+ and K+ in the intestinal
pH mimicking medium. The observed behavior can be attributed to
the different affinity of the MMT-K10 toward the MNE drug and the
different cations which follows the order H+ < MNE < Na+ /K+ .
The less amount of released drug at pH 1, despite the very
high drug solubility at pH < 2 (Wu and Fassihi, 2005) demonstrates
the higher affinity of the cationic drug, wherever it is adsorbed,
i.e. the interlayer and the anionic charged surfaces, for the clay.
Thus, from the dependence of the released amount on the loaded
drug quantity we can draw the conclusion that the adsorbed MNE
species interact in a favorable manner, making the MMT-K10 sur-
face more hydrophobic. As a consequence, the unshackle of the
drug is hampered. Moreover, it has to be also taken into account
that the lower loaded MNE formulation contains a higher concen-
tration of MMT-K10, which reflects into an higher hydrophilicity of
the MNE/MMT-K10 complex. This situation may ensure the pref-
erential formation of surface channel, which, in the case of the
release of the hydrophilic drug favors the transport of drug through
them.
It can be noticed that both in the two simulated situations the
quantity of released MNE from the MMT-K10 slightly depends on
the loaded drug amount, however, the dependence is very unusual.
In fact, at pH 1 the MMT-K10/MNE complex with higher drug con-
tent is able to liberate a greater amount of metronidazole than the
sample having a lower drug content, while the converse occurs at
pH 6.8, i.e., the higher the loaded antibiotic into the MMT-K10 the
lower the amount of free drug in solution.
233
Table 7
Kinetic models applied to the release kinetic profiles reported in Fig. 13.
Zero order Qt = Qe + k0 t
First order Qt = Qe (1 − e−k1 t )
Second order Qt = Qe (Qe kt/(1 + Qe kt))
DEM Qt = Qe − A1 e−k1 t − A2 e−k2 t
√
Higuchi Qt = kH t
Kosmeyer–Peppas Qt = Qe ktn
To better understand the release mechanism of drug molecules
from the different carriers, several kinetic models (Costa and Lobo,
2001) collected in Table 7 have been applied.
Analogously to the procedure applied for the analysis of the
above discussed data, the discrimination among the different mod-
els has been performed by means of the statistical criteria shown
in Tables 8 and 9.
It is worth to say that the attempt to use the double exponen-
tial model for the regression analysis failed because of the high
redundancy of the model parameters.
The results reported in Table 8 indicate that the release of the
MNE in the stomach pH mimicking conditions follows, for all cases
examined, the zero-order rate law.
This result implies that the formulations release the same
amount of drug in the unity of time, which, indeed, represents
the ideal way of drug release in order to realize a pharmacological
prolonged action.
It should be noted that the Kosmeyer–Peppas model also give
good statistical parameters, but, since the obtained exponent value
is close to one, it actually represents a zero-order kinetic model.
In Fig. 14 the zero-order kinetic rate constants for the release
process at pH 1 are reported as a function of the pH of drug loading.
We have also calculated other parameters (Costa and Lobo,
2001) used to characterize the drug release profile, like the tx %,
the sampling time (t%) and the dissolution efficiency (DE).
The t% is the time necessary to the release of a certain percentage
of drug, t% corresponds to the quantity of drug liberated in that time,
the dissolution efficiency (DE) of a pharmaceutical formulation is
defined as the ratio between the area under the dissolution curve
up to a certain time and the area of the rectangle obtained at the
100% dissolution in the same time (see Fig. 15) The value of the
calculated parameters are collected in Table 10.
Comparison of all the releasing parameters obtained for the two
investigated samples reveals that the higher the MNE loaded into
the MMT-K10 the faster the release rate.
Fig. 14. Rate constant values obtained from the application of the zero-order model
to the kinetic release data in the SGF conditions as a function of the pH of preparation
of the MNE/MMT-K10 complexes.
234 I. Calabrese et al. / International Journal of Pharmaceutics 457 (2013) 224–236

Table 8
Selected figures of merit for the different kinetic model applied to the release kinetics in SGF medium reported in Fig. 13.

␣1 ␣2 ␤1 ␤2 ␥1 ␥2

Zero-order model
R2 0.99818 0.99763 0.99988 0.99861 0.99989 0.99893
ESS 0.017241 0.04213 0.0128 0.0397 0.00228 0.008491
FANOVA 2190 16,823 32,673 2866 36,028 985

First-order model
2 0.017109 0.001793 0.00409 0.013 0.00081 0.06692
R2 0.99774 0.99697 0.99988 0.99758 0.99976 0.99449
ESS 0.021327 0.05379 0.01828 0.069 0.00844 0.020077
FANOVA 1758 1423 12,739 1655 10,500 737

Second-order model
2 0.07379 0.01792 0.00849 0.02409 0.00281 0.06851
R2 0.99766 0.99697 0.99983 0.99746 0.99956 0.99436
ESS 0.22136 0.05376 0.02546 0.07226 0.00842 0.20553
FANOVA 1694 1424 23,514 1580 10,792 719

Higuchi model
2 0.04 0.0659 0.07 0.02 0.09 0.07
R2 0.84445 0.88864 0.86288 0.85332 0.87117 0.85753
ESS 0.196 0.264 0.0269 0.0532 0.003 0.069
FANOVA 47 75 55 51 60 53

Kosmeyer–Peppas
2 0.01624 0.00856 0.0055 0.07691 0.00165 0.01767
R2 0.99948 0.99855 0.99989 0.9919 0.99998 0.99855
ESS 0.04873 0.02569 0.01649 0.23073 0.00496 0.05301
FANOVA 7700 2982 36,294 494 171,062 2794

Table 9
Selected figures of merit for the different kinetic model applied to the release kinetics in SIF medium reported in Fig. 13.

␣1 ␣2 ␤1 ␤2 ␥1 ␥2

Zero-order model
R2 0.93315 0.83325 0.92088 0.64754 0.93845 0.95648
ESS 206 408 373 1508 214 204
FANOVA 85 25 59 8 92 110

First-order model
2 0.24709 0.54973 0.11082 2.75223 0.13791 0.73803
R2 0.9996 0.9991 0.99991 0.99807 0.9998 0.99937
ESS 0.98838 1.64919 0.33245 5.50446 0.55165 2.21408
FANOVA 18,939 8516 71,697 3385 45,986 9965

Second-order model
2 42 96.14903 92 415.25046 32 96.80362
R2 0.93207 0.84277 0.92177 0.70889 0.95448 0.91762
ESS 210 384 368 1245 158 387
FANOVA 165 71 127 32 297 112

Higuchi model
2 173 153 332 421 163 534
R2 0.71942 0.67659 0.71766 0.66425 0.76636 0.64901
ESS 1039 988 1662 1915 976 2062
FANOVA 75 69 67 54 111 48

Kosmeyer–Peppas
2 64 101 62 458 53 72
R2 0.87362 0.83441 0.723 0.67836 0.82621 0.93861
ESS 294 405 253 1376 246 288
FANOVA 39 67 54 28 876 150

Table 10 Moreover, they are lower for the sample prepared at strongly
Release parameters in the SGF medium.
acidic pH and result to have nearly the same higher values for the
t120% DE (%) samples prepared at pH = 2.5 and 5.0.
Commercial tablet 42 19 The obtained trends can be explained by taking into account that
␣1 14 7 the cationic form of the MNE has to exchange with the cation H+
␤1 20 12 and the process takes place in the interlayer. Thus, since on increas-
␥1 21 11 ing the pH the amount of drug present in the interlayer increases
␣2 6.0 3.5
(accordingly to the adsorption kinetic and XRD results) the cation
␤2 8.6 4.2
␥2 8.0 3.7 exchange is not only favored but speeded up.
As for the release of the MNE in the intestine pH mimicking
conditions for all cases examined it has been found that it follows
a first-order rate law. This release profile implies that the drug is
 I. Calabrese et al. / International Journal of Pharmaceutics 457 (2013) 224–236 235

negatively charged edge, with respect to that prepared at pH = 5 (␥1,

␥2) respectively. The latter effect, which implies that the release of
the drug through cationic exchange is favored due to the shielded
charges on the clay, is more significant under the stomach pH
mimicking conditions than under the intestine pH mimicking one,
because at higher pH the net negative charge on the faces of the
clay is higher.

4. Conclusions

In the present paper, the employment of a new-tailored made

drug delivery system, obtained by the intercalation of the antibiotic
metronidazole (MNE) into the K10-montmorillonite clay (MMT-
K10), has been proposed.
First of all, a series of MNE/MMT-K10 hybrids have been
Fig. 15. Schematic representation of the dissolution of a drug from a pharmaceutical prepared by varying the pH and the amount of loaded drug.
dosage form. The dissolution efficiency is calculated as: DE (%) = (SA/R) × 100, where The obtained composites have been fully characterized from the
SA is the shaded area and R is the whole rectangle, calculated as y100 × t.
physico-chemical point of view. In particular, kinetic and equi-
librium studies have been carried out in order to elucidate the
adsorption mechanism of the MNE into the MMT-K10 and to
establish the nature of the interactions involved in the hybrid
MNE/MMT-K10 formation. Moreover, the interactions sites of the
clay surface have been proposed on the bases of the XRD results.
The results of these studies allowed us to establish that the
adsorption process strongly depends on the pH conditions and to
propose a multistep adsorption mechanism involving either proto-
nation, ionic exchange and electrostatic interactions.
In a second step, the potential application of the MNE/MMT-K10
hybrid for the in vitro target delivery has been tested by means of
the drug release under physiological pH mimicking conditions sim-
ulating the oral drug administration and delivery. The investigation
of the release profiles and the comparison with the commercial
formulation of the drug reveals that the new-tailor made formu-
lation could be fruitful exploited for successfully prolonged the
action of drug in the desired site. Moreover, the new nanomaterials
present some important advantages, since the very low releasing
Fig. 16. Rate constant values obtained from the application of the first-order model
rate of the MNE from the MMT-K10 formulation in the SGF rep-
to the kinetic release data in the SIF conditions as a function of the pH of preparation
of the MNE/MMT-K10 complexes. resents an accomplishment of the prefixed target since it implies
that the MNE/MMT-K10 complex should not be protected by an
enteric coating if it is intended to produce controlled release at the
liberated in a manner that is proportional to the amount of drug
intestine level.
remaining bond to the clay, in such a way, that the released quantity
The results obtained in the present work imply that the use of
by unit of time diminish.
these nanodevices reduce both time and costs for the drug delivery.
For both kinds of samples the release kinetic constant values
and the release efficiency (Fig. 16 and Table 11) are almost the same
while the released quantities (as discussed above) are higher for the Acknowledgments
sample loaded with lower MNE. Moreover, all the releasing param-
eters vary following the pH of preparation in the order 0.2 < 5 < 2.5. The authors gratefully acknowledge the financial support pro-
This trend can be explained by considering the different interac- vided by the MIUR (PRIN grant 2010EARRRZ 003).
tion between the clay and the drug and by taking into account the
charge present in the MMT-K10 on varying the pH. References
The higher release obtained with the sample prepared at
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