Lipid-core nanocapsules restrained the indomethacin ethyl ester hydrolysis in the gastrointestinal lumen and wall acting as mucoadhesive reservoirs

Article abstract of DOI:10.1016/j.ejps.2009.11.004

The aim of this work was to investigate if the indomethacin ethyl ester (IndOEt) released from lipid-core nanocapsules (NC) is converted into indomethacin (IndOH) in the intestine lumen, intestine wall or after the particles reach the blood stream. NC-IndOEt had monomodal size distribution (242 nm; PDI 0.2) and zeta potential of -11 mV. The everted rat gut sac model showed IndOEt passage of 0.16 μmol m^-2 through the serosal fluid (30 min). From 15 to 120 min, the IndOEt concentrations in the tissue increased from 6.13 to 27.47 μmol m^-2. No IndOH was formed ex vivo. A fluorescent-NC formulation was used to determine the copolymer bioadhesion (0.012 μmol m^-2). After NC-IndOEt oral administration to rats, IndOEt and IndOH were detected in the gastrointestinal tract (contents and tissues). In the tissues, the IndOEt concentrations decreased from 459 to 5 μg g^-1 after scrapping, demonstrating the NC mucoadhesion. In plasma (peripheric and portal vein), in spleen and liver, exclusively IndOH was detected. In conclusion, after oral dosing of NC-IndOEt, IndOEt is converted into IndOH in the intestinal lumen and wall before reaching the blood stream. The complexity of a living system was not predicted by the ex vivo gut sac model.

Full text of DOI:10.1016/j.ejps.2009.11.004

European Journal of Pharmaceutical Sciences 39 (2010) 116–124
Contents lists available at ScienceDirect
European Journal of Pharmaceutical Sciences
journal homepage: www.elsevier.com/locate/ejps
Lipid-core nanocapsules restrained the indomethacin ethyl ester hydrolysis in
the gastrointestinal lumen and wall acting as mucoadhesive reservoirs
Vitória Berg Cattania,1, Luana Almeida Fiela,1, Alessandro Jägerb, Eliézer Jägera,
Letícia Marques Coloméa, Flavia Uchoaa, Valter Stefanib, Teresa Dalla Costaa,
Sivia Stanisc¸ uaski Guterresa, Adriana Raffin Pohlmanna,b,∗
a Programa de Pós-Graduac¸ ão em Ciências Farmacêuticas, Faculdade de Farmácia, Universidade Federal do Rio Grande do Sul,
Avenida Ipiranga, 2752, Porto Alegre, RS, 90610-000, Brazil
b Departamento de Química Orgânica, Instituto de Química, Universidade Federal do Rio Grande do Sul, Av. Bento Gonc¸ alves,
9500, P Box 15003, Porto Alegre, RS, 91501-970, Brazil
a r t i c l e i n f o
a b s t r a c t
Article history:
The aim of this work was to investigate if the indomethacin ethyl ester (IndOEt) released from lipid-core
nanocapsules (NC) is converted into indomethacin (IndOH) in the intestine lumen, intestine wall or after
the particles reach the blood stream. NC–IndOEt had monomodal size distribution (242 nm; PDI 0.2) and
zeta potential of −11 mV. The everted rat gut sac model showed IndOEt passage of 0.16 ␮mol m−2 through
the serosal fluid (30 min). From 15 to 120 min, the IndOEt concentrations in the tissue increased from 6.13
to 27.47 ␮mol m−2. No IndOH was formed ex vivo. A fluorescent-NC formulation was used to determine
the copolymer bioadhesion (0.012 ␮mol m−2). After NC–IndOEt oral administration to rats, IndOEt and
IndOH were detected in the gastrointestinal tract (contents and tissues). In the tissues, the IndOEt concen-
trations decreased from 459 to 5 ␮g g−1 after scrapping, demonstrating the NC mucoadhesion. In plasma
(peripheric and portal vein), in spleen and liver, exclusively IndOH was detected. In conclusion, after oral
dosing of NC–IndOEt, IndOEt is converted into IndOH in the intestinal lumen and wall before reaching
the blood stream. The complexity of a living system was not predicted by the ex vivo gut sac model.
© 2009 Elsevier B.V. All rights reserved.
Received 22 July 2009
Received in revised form
12 November 2009
Accepted 14 November 2009
Available online 22 November 2009
Keywords:
Indomethacin ethyl ester
Fluorescent-labeled polymer
Polyester
Lipid-core nanocapsules
Ex vivo and in vivo intestinal absorption
1. Introduction
sules has been reported asastrategytoreduce their gastrointestinal
toxicity (Ammoury et al., 1993; Chasteigner et al., 1995; Guterres
Polymeric nanoparticles have been proposed to increase the
apparent solubility of lipophilic drugs, protect them from enzy-
matic attack, control drug release, and improve activity or even to
reduce their side effects (Couvreur et al., 2002; Schaffazick et al.,
2003). The term nanoparticles refers to nanospheres or nanocap-
sules that are, respectively, matricial (Pohlmann et al., 2007) and
vesicular structures (Jäger et al., 2007) varying in size from 10 to
1000 nm (Soppimath et al., 2001; Torchilin, 2006). The polymeric
nanoparticles promote drug selective passage through biological
barriers when their distribution sizes range from 100 to 600 nm.
The encapsulation of diclofenac or indomethacin, non-steroidal
anti-inflammatory drugs, in polymeric nanospheres and nanocap-
et al., 1995; Müller et al., 2001). Indomethacin has also been
used as a drug model in several studies concerning nanopartic-
ulated systems (Calvo et al., 1996; Guterres et al., 2000; Kim
et al., 2001; Vinogradov et al., 2002; Pohlmann et al., 2002).
More recently, comparative studies have been performed in order
to propose the nanoencapsulation mechanisms for indomethacin
and its ethyl ester derivative (Pohlmann et al., 2004, 2007,
2008; Cruz et al., 2006a; Poletto et al., 2008), as well as to
determine the different drug release behavior from nanospheres,
nanoemulsion and nanocapsules (Cruz et al., 2006b). The nanoen-
capsulation of indomethacin ethyl ester within semi-crystalline
polyester nanocapsules (NC) has been used to simulate a perfect
sink condition by releasing the drug after its interfacial alka-
line hydrolysis creating a concentration gradient in the system
(Pohlmann et al., 2004). Regarding the biological activity, the
indomethacin-loaded lipid-core nanocapsules (NC–IndOH) and the
co-encapsulation of indomethacin and indomethacin ethyl ester in
nanocapsules showed selective cytotoxicity after treating glioma
cell lines (U138-MG and C6) (Bernardi et al., 2008). Further-
more, after intraperitoneal administration, the NC–IndOH were
∗
Corresponding author at: Departamento de Química Orgânica, Instituto de
Química, Universidade Federal do Rio Grande do Sul, Av. Bento Gonc¸ alves, 9500,
P Box 15003, Porto Alegre, RS, 91501-970, Brazil. Tel.: +55 51 3308 7237;
fax: +55 51 3308 7304.
E-mail address: pohlmann@iq.ufrgs.br (A.R. Pohlmann).
Both authors have contributed equally to this paper.
1
0928-0987/$ – see front matter © 2009 Elsevier B.V. All rights reserved.
doi:10.1016/j.ejps.2009.11.004

V.B. Cattani et al. / European Journal of Pharmaceutical Sciences 39 (2010) 116–124
117
able to increase the intratumoral penetration of the drug reduc-
ing the growth of the glioma (C6) implanted in rats (Bernardi
et al., 2009a). In addition, the effects of systemic treatment
with NC–IndOH were evaluated in rat models of acute and
chronic edema. The drug nanoencapsulation increased the anti-
inflammatory effect in long-term models of inflammation, allied
to an improved gastrointestinal safety (Bernardi et al., 2009b).
Previously, the in vitro release experiment using gastric or intesti-
nal simulated fluids containing enzymes (pepsin and pancreatin)
did not show any indomethacin ethyl ester release or degrada-
tion (Cruz et al., 2006a; Pohlmann et al., 2007). The experiment
showed that polymeric nanocapsules and nanospheres containing
indomethacin ethyl ester are stable in vitro even in the presence of
pepsin and pancreatin. However, indomethacin ethyl ester-loaded
lipid-core nanocapsules (NC–IndOEt) showed antiedematogenic
activity in acute treatment in rats, indicating that the drug was
released in vivo from the nanocapsules (Cruz et al., 2006a).
That in vivo experimental protocol was not designed to show
which substance, indomethacin or indomethacin ethyl ester, was
responsible for the biological effect. In this way, we performed
a pharmacokinetic study using the same formulations to eval-
uate the plasma levels of each substance after i.v. and oral
dosing (Cattani et al., 2008). In the study, we demonstrated that
the indomethacin ethyl ester acts as a pro-drug. However, if
indomethacin ethyl ester is converted into indomethacin after oral
dosing of nanocapsules, either at the intestine lumen, intestine wall
or after the nanocapsules reach the blood stream, remains to be
elucidated.
After oral administration of nano- and microparticles the main
uptake route has been considered the follicle-associated epithe-
lium (FAE) in small intestine due to the presence of gut-associated
lymphoid tissue (Peyer’s patches). There are three possible trans-
port routes for macromolecules, microorganisms and particles in
FAE: (1) paracellular transport; (2) transcellular uptake across
enterocytes by endocytic process; and (3) endocytosis by M cells
(Carreno-Gómez et al., 1999). The literature described several
experimental models to investigate the interaction between parti-
cles and gastrointestinal tract (Hussain et al., 2001; Ponchel and
Irache, 1998). Among the ex vivo models, the everted intestinal
sac model (Wilson and Wiseman, 1954) has been used in the past
50 years to study bioadhesive properties and substances uptake
and transport in the intestine (Chen et al., 2003; Carreno-Gómez
et al., 1999; Mainardes et al., 2006). Furthermore, bioadhesion and
uptake of particles were previously studied in vivo (Jani et al., 1990;
Limpanussourn et al., 1998; Jani et al., 1996; Damge et al., 1996;
Florence and Hussain, 2001) and in vitro (Hussain et al., 1997;
Santos et al., 1999; Santos et al., 2003; Mainardes et al., 2006).
Recently, we prepared lipid-core nanocapsules varying the
concentrations of sorbitan monostearate and oil (capric/caprylic
triglyceride), both constituents of the core, to investigate if the sus-
tained release from those carriers could be a consequence of the
variation of the core viscosity and the particle surface area (Jäger
et al., 2009). The augmentation in the sorbitan monostearate con-
centration enhanced the resistance to the pro-drug diffusion due to
the core viscosity increase. On the other hand, after increasing the
oil concentration, despite the possibility of diminishing the core
viscosity, the pro-drug release was delayed due to the decrease in
the surface area of the colloidal system as a consequence of the
increase in the particle mean size.
Secondly, after oral administration of the pro-drug-loaded lipid-
corenanocapsules torats, the concentrationsofindomethacin ethyl
ester and indomethacin were determined in stomach, small intes-
tine, liver, spleen, as well as in plasma from peripheric and portal
vein blood.
2. Materials and methods
2.1. Materials
Poly(ε-caprolactone)
65,000 g mol−1) was supplied by Aldrich® (Strasbourg, France).
Dicyclohexylcarbodiimide, 4-(N,N-dimethyl)aminopyridine
(PCL)
(Mn = 42,500
and
Mw =
and indomethacin were obtained from Sigma (St. Louis, USA).
Caprylic/capric triglyceride (density 0.94 g ml−1), sorbitan monos-
tearate and polysorbate 80 were acquired from Delaware® (Porto
Alegre, Brazil). TC 199 Earle® intestinal simulated medium was
obtained from Sigma–Aldrich (Strasbourg, France).
o-Aminophenol, 1,2-phenylenediamine, 5-aminosalicylic acid
were acquired from Aldrich® (reagent grade) and used without
purification. Diethyl ␤-ethoxymethylenmalonate and polyphos-
phoric acid were purchased from Acros Chemicals®. Methyl
methacrylate, acquired from Aldrich®, was purified before poly-
merization by passing it through activated alumina (Merck®).
2,2ꢀ-Azo-bis-isobutyrylnitrile (AIBN) was obtained from Merck®
and purified before use by recrystallization from methanol and
maintained under vacuum. Silica gel 60 (Merck®) was used for
preparative chromatographic column separations. All solvents
were used as received or purified using classical standard pro-
cedures. Spectroscopic grade solvents (Merck®) were used for
fluorescence and UV–vis measurements and chromatographic
grade solvents (Merck®) were used for liquid chromatography
(HPLC) measurements.
1H NMR analyses were performed at room temperature on
a Varian® (model VXR-200, 200 MHz) using tetramethylsilane as
internal standard and DMSO-d6 (Aldrich) or CDCl3 (Merck) as sol-
vents. The UV–vis and fluorescence analyses were carried out using
a Shimadzu® UV-1601 PC spectrometer and a Hitachi spectroflu-
orometer (model F-4500), respectively. Fluorescence spectrum
correction was performed to enable measuring a true spectrum
by eliminating instrumental response such as wavelength charac-
teristics of the monochromator or detector using Rhodamine B as
standard (quantum counter).
2.2. Synthesis and nanocapsule preparation
2.2.1. Synthesis of indomethacin ethyl ester
The synthesis of indomethacin ethyl ester (IndOEt) was car-
ried out under argon by preparing a solution of indomethacin
(5 mmol) in ethanol (20 ml) that was added of 4-(N,N-
dimethyl)aminopyridine (0.2 mmol) (Pohlmann et al., 2004). The
solution was stirred for 10 min at 0 ◦C following the addition
of dicyclohexylcarbodiimide (5 mmol). After 30 min at 0 ◦C and
16 h at room temperature, the solvent was evaporated under
reduced pressure. The residue was added of dichloromethane
(30 ml) and the suspension was filtered. The filtrate was extracted
with saturated NaHCO3 aqueous solution (3 × 10 ml) to remove
the unreacted indomethacin. Then, the organic phase was dried
with anhydrous MgSO4, filtered and evaporated. The product was
purified by column chromatography (Silica gel 60, 70–230 mesh)
using ethyl acetate and cyclohexane (1:1, v/v) as eluent. The iso-
lated product was obtained as a solid (80% of yield) presenting a
melting point (uncorrected) of 82–83 ◦C.
Taking those considerations into account, the aim of this work
was to investigate if indomethacin ethyl ester released from the
lipid-core nanocapsules is converted into indomethacin in the
intestine lumen, intestine wall or after the particles reach the blood
stream. First, the everted rat gut sac model was used to verify the
transport of the pro-drug and the bioadhesion of the nanocapsules.
1H NMR 200 MHz (ı, ppm) CDCl3: 7.66 and 7.46 (AB system, 2H
and 2H, ArH p-chlorobenzoyl), 6.97 (d, 1H J = 2.5 Hz, H-4), 6.87 (d,

118
V.B. Cattani et al. / European Journal of Pharmaceutical Sciences 39 (2010) 116–124
1H J = 9.0 Hz, H-7), 6.67 (dd, 1H J = 9.0 and 2.5 Hz, H-6), 4.16 (q, 2H
J = 7.1 Hz, OCH2), 3.84 (s, 3H, OCH3), 3.65 (s, 2H, CH2), 2.38 (s, 3H,
CH3), 1.27 (t, 3H J = 7.1 Hz, CH3CH2O).
acetate solution (2%, w/v). The analyses were performed using a
microscope JEM 1200 Exll, operating at 80 kV (Centro de Microscopia
Eletrônica- UFRGS).
13C NMR 75 MHz (APT, ı, ppm) CDCl3: 170.9 (CO-ester), 168.3
(CO-amide), 156.0, 139.2, 135.9, 134.0, 130.8, 130.7 and 112.7
(7 × Cq), 131.1 and 129.1 (4 × CH p-chlorobenzoyl), 114.9, 111.6
and 101.3 (3 × CH indol), 61.0 (OCH2), 55.7 (OCH3), 30.4 (CH2), 14.2
and 13.3 (CH3 and CH3CH2).
Indomethacin or indomethacin ethyl ester contents in nanocap-
sules were determined by HPLC (PerkinElmer S-200 with injector
S-200, detector UV–vis), a guard-column (C18) and a column
(LiChrospher® 100 C18, 250 mm, 4 mm, 5 ␮m, Merck) using a
mobile phase (1.2 ml min−1) consisted of acetonitrile/water (70:30,
v/v) adjusted to apparent pH 5.0 with 10% (v/v) acetic acid. The
methodology was previously validated (Jäger et al., 2009) showing
linearity between 1.00 and 50.00 ␮g ml−1 (r > 0.9999), inter- and
intraday variability lower than 2.0%, accuracy between 99.42% and
2.2.2. Lipid-core nanocapsules containing indomethacin or
indomethacin ethyl ester
Nanocapsules were prepared by interfacial deposition of pre-
formed polymer as previously described (Jäger et al., 2009). Briefly,
at 40 ◦C, poly(ε-caprolactone) (0.500 g), capric/caprylic triglyc-
eride (1.65 ml), sorbitan monostearate (0.385 g) and indomethacin
or indomethacin ethyl ester (0.050 g) were dissolved in acetone
(135 ml). Then, at room temperature, the solution was injected
into an aqueous solution (265 ml) of polysorbate 80 (0.385 g). After
10 min, acetone was eliminated and the suspension concentrated
under reduced pressure to a final volume of 50 ml (drug or pro-
drug final concentration at 1 mg ml−1). Formulations were made in
triplicate.
104.78% and limit of quantification of 1.00 ␮g ml−1
.
2.4. Ex vivo drug absorption using rat everted gut sac
Male Wistar rats (250–300 g, CREAL/UFRGS, Porto Alegre, Brazil)
were handled in accordance with the provisions of the Guide to
care and use of experimental animals in all experimental proce-
dures (UFRGS Ethics in Research Committee, # 2005403). Animals
were sacrificed and the entire small intestine quickly excised and
flushed through several times with a NaCl solution (0.9%, w/v) at
room temperature. The intestine was immediately placed in warm
(37 ◦C) TC 199 (Earle’s salts), pH 7.4, and, then, gently everted over
a glass rod (2.5 mm diameter). The intestine was filled with fresh
oxygenated medium and clamped at both ends. The resulting large
gut sac was divided into eight small sacs of approximately 2.5 cm in
length using braided silk suture, starting from the end of the duo-
denum to ensure maximal metabolic activity from the upper/mid
jejunum. Each replicate was analyzed using the sacs of the intestine
from one rat.
NC–IndOH and NC–IndOEt suspensions (2 ml for each flask)
were added in flasks containing TC 199 medium (18 ml each) at
37 1 ◦C. At the appropriate time intervals (2, 5, 10, 15, 30, 60,
90 and 120 min) each sac was removed and maintained over the
recipient for 5 s to drip the liquid. Each sac was cut open and the
serosal fluid drained into a tube. The serosal fluid and the sac tissue
were analyzed. The serosal fluid aliquot was added of acetonitrile,
centrifuged (5 min at 5290 × g) and the supernatant was collected,
filtered (0.45 ␮m) and analyzed by HPLC. The sac tissue was macer-
ated with acetonitrile (1 min) and centrifuged (5290 × g) to collect
supernatants, which were filtered (0.45 ␮m) and analyzed by HPLC.
The transport of indomethacin and indomethacin ethyl ester from
the mucosal to the serosal side was determined, considering an
average area of 4.61 0.45 cm2 for the sacs. The integrity of the
sacs was verified by glucose measurement in both the external
medium and the serosal fluids using a glucose meter (Accu-Check,
Roche, Switzerland). As glucose is actively transported by the small
intestine, healthy metabolically active sacs that are not leaking
accumulate glucose in the serosal fluid (Barthe et al., 1999).
2.2.3. Synthesis of the fluorescent-labeled copolymer
The syntheses of the fluorescent-labeled copolymer was per-
formed as previously described (Jäger et al., 2007). The reaction was
based on the radical copolymerization of methyl methacrylate and
the fluorescent benzazole derivative. Briefly, the 5-aminosalicylic
acid was condensed with the 1,2-phenylenediamine. The
2-(4ꢀ-amino-2ꢀ-hydroxyphenyl)benzimidazole
was
iso-
lated and purified by column chromatography. Diethyl
␤-ethoxymethylenmalonate was added to a solution of 2-(4ꢀ-
amino-2ꢀ-hydroxyphenyl)benzimidazole in ethanol. The vinylene
derivative was obtained with satisfactory yield and its chemical
identity was verified by NMR. The copolymer (PMMA-HBN) was
prepared by polymerization of vinylene derivative with methyl
methacrylate in the presence of AIBN. PMMA-HBN was purified by
dissolution in chloroform (1 ml) and precipitation into petroleum
ether (20 ml), as much as any dye was not detected in the filtrate
by UV spectroscopy. Then, PMMA-HBN was characterized by
UV spectroscopy and by size exclusion chromatography (LDC
Analytical Model Constametric 3200).
2.2.4. Fluorescent-labeled nanocapsules
Nanocapsules were prepared using PMMA-HBN synthe-
sized as described above. A solution of PMMA-HBN (0.075 g),
capric/caprylic triglyceride (0.825 ml) and sorbitan monostearate
(0.190 g) in acetone (33.8 ml) was injected into an aqueous solu-
tion (66.2 ml) of polysorbate 80 (0.190 g). Acetone was eliminated
and the suspension concentrated under reduced pressure to a final
volume of 25 ml (copolymer final concentration at 3 mg ml−1). For-
mulations were made in triplicate.
2.5. Ex vivo fluorescent nanocapsules uptake using rat everted
gut sac
2.3. Physicochemical characterization of nanocapsules
Fluorescent nanocapsules uptake was evaluated using the same
procedure described for the indomethacin and indomethacin ethyl
ester ex vivo absorption with some modifications. Briefly, each sac
was placed in flasks containing 19 ml of TC 199 medium at 37 1 ◦C
and added of 1 ml of NC–PMMA-HBN suspension (3 mg ml−1 of
copolymer). At the appropriate time intervals (0, 15, 30, 60, 90,
120 and 150 min), each sac was removed and maintained over
the recipient for 5 s to drip the liquid. Each sac was cut open
and the serosal fluid drained into a tube. The drained serosal
fluid was extracted with chloroform (vortex, 5 min), centrifuged
(5 min at 5290 × g) and the supernatant was collected for analysis.
The pH values were determined at 25 ◦C using a potentiometer
DPMH-2 (Digimed). Size, polydispersity index and zeta potential
were determined using a Zetasizer Nano ZS (Malvern) after dilut-
ing the samples with water (MilliQ®) for the size measurements or
10 mM NaCl aqueous solution for zeta potential analysis. Measure-
ments were made in triplicate.
Transmission electron microscopy (TEM) was carried out
using nanocapsule suspensions diluted in water. The sample was
deposited on specimen grid (Formvar-Carbon support film, Elec-
tron Microscopy Sciences) and negatively stained with uranyl

V.B. Cattani et al. / European Journal of Pharmaceutical Sciences 39 (2010) 116–124
119
where CT is the analite concentration (␮g g−1) in tissue, CS is the
analite concentration in supernatant, VS is the supernatant volume
and P is the tissue weight.
The sac tissue was macerated with chloroform (1 min) and cen-
trifuged (5 min at 5290 × g). The organic phase was collected for
analysis.
To evaluate the possibility of nanocapsule bioadhesion to the
intestinal tissue an additional group was studied. One hour after the
oral administration of NC–IndOEt in a dose of 10 mg kg−1 of drug,
the rats were sacrificed and blood samples, liver, spleen, stomach
and small intestine and their contents were harvested and weighed.
After the content removal, stomach and intestinal tissues were cut
along the longitudinal axis and the intestinal mucosa of the seg-
ments was scrapped with a wet-swab slide. Then, the tissues were
processed as described above.
Blood samples were collected into heparinized reaction tubes
and centrifuged (14,811 × g for 15 min at 21 ◦C) to separate the
plasma. Before the analysis, plasma samples were deproteinized
by adding 200 ␮l of acetonitrile, vortexed for 10 s and centrifuged
at 20,200 × g, 21 ◦C for 10 min. The supernatant was separated to
inject into the HPLC system.
Indomethacin and indomethacin ethyl ester were quantified
in plasma and tissues, using a previously validated HPLC method
with UV detection (Cattani et al., 2008). The HPLC system con-
sisted of a Waters 600 solvent delivery system, a Waters 717
plus auto-injector and UV 2487 (Dual ꢀ Absorbance, Waters)
detector. Chromatographic separation was achieved on a 4 ␮m
Nova-Pak® 150 × 3.9 mm C18 column (Waters®, Dublin, Ireland),
at 45 ◦C, using a mixture of acetonitrile:0.02 M ammonium dihy-
drogen phosphate (70:30, v/v), apparent pH 5, as mobile phase.
The analyses were carried out using a flow rate gradient, starting
with a 0.8 ml/min until 3.5 min, swifting to 1.3 ml/min over 2.5 min
and returning to the initial conditions over 0.5 min. The autosam-
pler was set to inject 50 ␮l sample aliquots and indomethacin
and indomethacin ethyl ester were detected at 267 nm. The assay
for both plasma and tissue samples was linear over a concentra-
tion range of 0.5–40 ␮g ml−1, had a low limit of quantification
(0.5 ␮g ml−1) and the determination coefficients were higher than
0.98. The accuracy of the method was higher than 85%. Intra- and
interday relative standard deviations were less than 11.8% and 9.2%,
respectively.
2.6. Validation of the PMMA-HBN quantification
The quantification of PMMA-HBN extracted from the fluo-
rescent nanocapsules was performed by fluorescence (Hitachi
Spectrofluorometer, model F-4500). To validate the methodology,
different proportions of fluorescent nanocapsules were added into
TC 199 Earle® intestinal simulated medium under magnetic stir-
ring at 37 1 ◦C, providing concentrations of 7.5, 12.5, 25, 50, 75,
100, 125 and 150 ␮g ml−1 of fluorescent copolymer. One milliliter
of each mixture was extracted with 1 ml of chloroform in vor-
tex (5 min). Each organic phase was gently transferred to a quartz
cuvette for analysis. Linear calibration curves (9 curves) showed
correlation coefficients higher than 0.99. Inter- and intraday vari-
ability did not exceed 10.6% and 7.3%, the limit of detection was
3.5 ␮g ml−1 and the accuracy was 97.9%. Considering that a blank
of tissue (intestinal tissue extracted with chloroform) showed flu-
orescent intensity between 40.2 and 44.7 arbitrary units, at each
time interval (0–150 min) a blank of tissue was carried out in
parallel with the tested sample containing NC–PMMA-HBN. The
fluorescence of each blank was subtracted from the fluorescence
of the sample at each time interval.
2.7. In vivo intestinal drug absorption
2.7.1. Animals
Animals in this study were handled in accordance with the
provisions of the Guide to care and use of experimental animals
and all experimental procedures were approved by UFRGS Ethics
in Research Committee (# 2005478). The experiments were per-
formed on male Wistar rats weighing 290–320 g, which were
purchased from Fundac¸ ão Estadual de Produc¸ ão e Pesquisa em Saúde
(Porto Alegre, Brazil) and were kept at constant room tempera-
ture (22 1 ◦C) and 60% relative humidity, under controlled 12 h
light–dark cycle (light on from 7 a.m. to 7 p.m.) during the acclima-
tion period with free access to water and food. For the experiments,
the animals were kept fasten overnight (14–18 h) with free access
to water.
3. Results and discussion
3.1. Preparation and characterization of the nanocapsules
2.7.2. In vivo intestinal absorption after oral administration of
NC–IndOEt
The indomethacin ethyl ester was prepared and isolated with
yields of 80%. In parallel, the fluorescent copolymer (PMMA-HBN)
was successfully obtained presenting Mn of 276,000 g mol−1 and
Mw of 1,603,000 g mol−1. The fluorescent dye was chemically
bound at 558 ␮g/g of copolymer (2.5 mol mol−1).
The intestinal absorption was investigated in male Wistar rats
(n = 4) after oral administration of nanocapsules corresponding to
a 10 mg kg−1 indomethacin ethyl ester dose. After pre-determined
time points (0.25, 1 and 2 h), the rats were sacrificed and liver,
spleen, blood samples (from peripheric and from portal vein),
stomach and small intestine and their contents were harvested,
weighted and quantified. Stomach, duodenum, jejunum and ileum
were isolated and the tissue and the luminal contents were sep-
arated and homogenized with acetonitrile (0.5–2.0 ml) using a
PowerGen125 apparatus for approximately 3 min in an ice bath.
The homogenized samples were transferred to Falcon tubes and
centrifuged at 5292 × g for 20 min. A 100 ␮l aliquot of the super-
natants were treated with acetonitrile (200 ␮l) and analyzed by a
validated HPLC/UV method. When necessary, samples were prop-
erly diluted with the mobile phase considering the range of the
calibration curve. The indomethacin and indomethacin ethyl ester
tissue and luminal content concentrations were calculated by Eq.
(1):
All nanocapsule formulations showed macroscopic homoge-
neous appearance, like a milky white bluish opalescent liquid.
NC–IndOH, NC–IndOEt and NC–PMMA-HBN were directly obtained
by interfacial deposition of pre-formed polymers without any
subsequent step of filtration or centrifugation. The diameters (z-
average) and the polydispersity indexes were 262, 242 and 215 nm
and 0.18, 0.20 and 0.09, respectively. The suspensions were formed
by nanoparticles distributed in one narrow population in each
case. Fig. 1a shows a schematic representation of the nanocap-
sule, composed by a lipid core and a polymeric wall, containing
the drug retained in the particle. Fig. 1b shows the size distri-
butions of three different batches of NC–IndOEt. The lipid-core
nanocapsules are spherical shape as depicted in Fig. 1c. The size
distributions are in accordance with other nanocapsule systems
described in the literature (Chasteigner et al., 1995; Calvo et al.,
1996). The pH values of suspensions were 6.0, 6.0 and 6.1 for
CS · VS
CT
=
(1)
P

120
V.B. Cattani et al. / European Journal of Pharmaceutical Sciences 39 (2010) 116–124
Fig. 2. Amounts of indomethacin, indomethacin ethyl ester and fluorescent copoly-
mer per square meter of intestine as a function of time in the ex vivo rat everted
gut sac experiment after adding the NC formulations: NC–IndOH, NC–IndOEt and
NC–PMMA-HBN (mean + S.D.).
11.93, 27.31 and 32.88 ␮g/g of tissue, respectively. In the serosal
fluid, the indomethacin ethyl ester concentration was below the
pro-drug limit of quantification between 2 and 30 min of experi-
ment. After 30 min, 0.03% of the initial dose was determined in the
serosal fluid corresponding to a passage of 0.16 0.02 ␮mol m−2
.
This value remained constant within 30 and 120 min.
Itis noteworthytomention thatno indomethacin (free acid)was
formed within the period of experiment, indicating that no hydrol-
ysis of indomethacin ethyl ester took place. In a previous work,
the pro-drug release was assayed adding indomethacin ethyl ester-
loaded nanocapsules in simulated gastric fluid containing pepsin,
as well as in simulated intestinal fluid containing pancreatin, and
no hydrolysis was observed independent of the medium (Cruz et
al., 2006a).
Fluorescent-labeled nanocapsules (NC–PMMA-HBN) were
assayed to determine the bioadhesion and if the nanocapsules
were able to cross the gut wall. The physicochemical stability of
the fluorescent dye chemically bound to the polymer wall has
been already demonstrated (Jäger et al., 2007). The encapsulation
of indomethacin ethyl ester in NC–PMMA-HBN was not possi-
ble due to a lack of specificity in the copolymer quantification
by fluorescence spectroscopy. So, placebo NC–PMMA-HBN was
added into the external medium. Regarding the ex vivo uptake of
NC–PMMA-HBN, the serosal fluid showed low fluorescence inten-
sity within 150 min of experiment. The copolymer concentrations
Fig. 1. Schematic illustration of the lipid-core nanocapsules containing
indomethacin ethyl ester (longitudinal cut) (a); size distribution profiles of
three different batches of NC–IndOEt (b); and TEM photomicrograph of
NC–IndOEt formulation.
a
NC–IndOH, NC–IndOEt and NC–PMMA-HBN, respectively. The zeta
potential values were −10.6 0.4, −11.3 1.2, and −10.7 1.2 mV
for NC–IndOH, NC–IndOEt and NC–PMMA-HBN, respectively, as a
consequence of a polymeric wall coated with polysorbate 80. The
negative density of charge is related to the chemical structures
of the polymers (PCL and PMMA-HBN), and the low modulus val-
ues of zeta potential are due to the polysorbate 80 coating, which
stabilizes the nanocapsules by a steric mechanism.
were below the validated limit of quantification (7.5 ␮g ml−1
;
4.7 × 10−6 ␮mol ml−1) for the whole experiment. The intestinal
tissue showed a copolymer bioadhesion of 0.012 ␮mol m−2 (Fig. 2).
After 15, 60 and 120 min, the concentrations of PMMA-HBN in the
tissue were practically constant (43.77, 42.28 and 49.33 ␮g g−1
,
respectively). The main factors which affect the particle bioadhe-
sion and uptake are the average particle size, zeta potential and
hydrophilicity–hydrophobicity balance (Florence, 1997; Ponchel
and Irache, 1998; Hussain et al., 2001; Shakweh et al., 2004).
So, the bioadhesion observed for the lipid-core nanocapsules
(NC–PMMA-HBN) is due to the hydrophobic and nanoscopic
nature of the particles. On the other hand, the formulation showed
a neglected and not quantifiable uptake ex vivo.
In parallel, indomethacin-loaded nanocapsules (NC–IndOH)
were also evaluated in the ex vivo experiment to verify the molec-
ular diffusion through the gut wall. Indomethacin solubilizes at
pH 7.4 by interfacial ionization and desorption from the nanocap-
3.2. Ex vivo drug absorption using rat everted gut sac
Indomethacin ethyl ester was quantified as a function of time
in the serosal fluid and intestinal tissue. The total time of exper-
iment was 120 min, i.e. 7200 s. The intestinal tissue showed an
increase of indomethacin ethyl ester from 0.04% to 0.24% of the
initial dose within 120 min. Indomethacin ethyl ester ranged from
6.13 to 27.47 ␮mol m−2 within 7200 s (120 min) (Fig. 2). After 15, 60
and 120 min, the concentrations of indomethacin ethyl ester were

V.B. Cattani et al. / European Journal of Pharmaceutical Sciences 39 (2010) 116–124
121
Fig. 3. Transport across ex vivo gut sac (mean + S.D.) after adding NC–IndOH or
NC–IndOEt in the external medium.
sules (Pohlmann et al., 2004). Indomethacin ranged from 61.30 to
261.04 ␮mol m−2 within 7200 s (120 min) (Fig. 2), corresponding
to 0.46–1.93% of the drug initial dose. After 15, 60 and 120 min,
the concentrations of indomethacin were 151.90, 216.65 and
289.74 ␮g/g of tissue, respectively.
The profiles of indomethacin and indomethacin ethyl ester
determined in the gut sac as a function of time fit to a power
law model, y = axn (r = 0.9929 and 0.9360, respectively). The n
coefficients were 0.33 for both substances, indicating that both
substances diffuse in the tissue (Fickian diffusion). After the addi-
tion of NC–IndOH in the external medium, the permeation of
indomethacin through the gut sac wall was 0.27–1.69% of the
initial dose (2–120 min of experiment). The cumulative mass
transfer as a function of time showed an indomethacin flux of
0.027 0.004 ␮mol m−2 s−1 (r = 0.9941) (Fig. 3).
Since indomethacin is adsorbed at the polymeric wall of the
nanocapsules, as previously demonstrated (Pohlmann et al., 2004),
and no drug crystal was simultaneously formed with the nanocap-
sules (Pohlmann et al., 2008), the indomethacin transport occurred
by interfacial ionization (nanocapsules/aqueous phase interface),
dissolution (in the aqueous phase) and drug permeation through
the gut wall by molecular diffusion. In the case of indomethacin
ethyl ester, considering its low solubility in the aqueous medium
(less than 1 ␮g ml−1) allied to the lack of interfacial hydrolysis
ex vivo, its release occurred exclusively at the gut sac after the
nanocapsule bioadhesion. Indomethacin was mostly released from
the nanocapsules in the external medium, while indomethacin
ethyl ester, a non-ionizable substance, is exclusively released in
the ex vivo tissue. The different solubility of those substances in
aqueous media can explain the results.
Fig. 4. Indomethacin ethyl ester (top) and indomethacin concentrations (bottom) in
the luminal contents of stomach and small intestine (mean + S.D.) after oral admin-
istration of NC–IndOEt (pro-drug at 10 mg kg−1) (n = 4).
indomethacin ethyl ester concentration reached a maximum
(854 ␮g ml−1) in the content of ileum after 60 min.
After
NC–IndOEt
oral
administration
(10 mg kg−1),
indomethacin was also detected in the contents of the stom-
ach and the small intestine. The indomethacin concentration in
the content of the stomach was lower than 6 ␮g ml−1, whereas
in the contents of the duodenum and jejunum it ranged between
30 and 74 ␮g ml−1 after 15 and 120 min (Fig. 4). In the content
of ileum, the indomethacin concentrations showed a tendency to
increase from 2 to 65 ␮g ml−1 after 15 and 60 min, respectively,
and then a tendency to decrease to 29 ␮g ml−1 after 120 min.
The results demonstrated that indomethacin ethyl ester was
hydrolyzed at least in some extension in the lumen of the small
intestine.
3.3. In vivo intestinal absorption after oral administration of
NC–IndOEt
After NC–IndOEt oral administration (10 mg kg−1), the
indomethacin ethyl ester and indomethacin concentrations
in the gastrointestinal tissues and theirs contents were evalu-
ated. The indomethacin ethyl ester concentration was practically
constant in the content of the stomach ranging between 990 and
1480 ␮g ml−1 within 120 min after oral dosing (Fig. 4). Regarding
the content of duodenum, the indomethacin ethyl ester concen-
trations showed a tendency to increase from 1715 to 2620 ␮g ml−1
after 15 and 60 min of dosing, respectively. After 120 min, it was
observed a tendency to decrease to 940 ␮g ml−1. After the same
period, it showed a tendency to decrease from 2450 (15 min)
to 335 ␮g ml−1 (120 min) in the content of jejunum. Finally, the
The gastrointestinal tissues were analyzed after the NC–IndOEt
oral administration. The indomethacin ethyl ester concentra-
tions in the stomach were practically constant between 15 and
120 min ranging from 603 to 895 ␮g g−1 (Fig. 5). In the duode-
num, the indomethacin ethyl ester concentration rose from 770
to 3020 ␮g g−1 after 15 and 60 min, respectively, and decreased
to 440 ␮g g−1 after 120 min. In the jejunum, the indomethacin
ethyl ester concentration showed an augmentation from 950
to 1530 ␮g g−1 after 15 and 60 min, respectively, followed by
a
decrease to 107 ␮g g−1 after 120 min. In the ileum, the
indomethacin ethyl ester concentrations were very low, showing
a tendency to decrease from 93 to 16 ␮g g−1 from 15 to 120 min.

122
V.B. Cattani et al. / European Journal of Pharmaceutical Sciences 39 (2010) 116–124
Fig. 6. Indomethacin ethyl ester and indomethacin concentrations in the scrapped
tissues for mucus removal, after oral administration of NC–IndOEt (pro-drug at
10 mg kg−1) (n = 4).
where higher concentrations were observed. The indomethacin
ethyl ester tissue concentrations without mucus revels a strong
nanoparticles interaction with the polysaccharide chains of mucus.
The nanoparticles interaction with the mucus gel layer and mucus
membrane is a consequence of the physicochemical interactions
between mucus and non-swellable and hydrophobic polymers,
such as poly(␧-caprolactone). Considering the probable mecha-
nism of indomethacin absorption after NC–IndOEt administration
in vivo, part of the indomethacin ethyl ester released in the mucus is
hydrolyzed by the lumen esterases. Besides, nanocapsules adhered
to the epithelium can likely release the indomethacin ethyl ester,
which penetrates the gastrointestinal tissues to be hydrolyzed to
indomethacin.
After NC–IndOEt oral administration (10 mg kg−1), exclusively
indomethacin was detected in plasma (peripheric and portal vein),
spleen and liver. After 15, 60 and 120 min, indomethacin concentra-
tions in plasma and portal vein were similar (p > 0.05) ranging from
6 to 35 ␮g ml−1 (Fig. 7). The indomethacin concentrations in spleen
were lower (p < 0.05) than in liver at all time points investigated and
increase with time up to 1 h, reaching a plateau, between 1 and
2 h, of 70 and 120 ␮g g−1, for spleen and liver, respectively (Fig.
7). The results showed that indomethacin ethyl ester was totally
hydrolyzed in gastrointestinal lumen, and probably tissue, before
reaching the blood stream.
Fig. 5. Indomethacin ethyl ester (top) and indomethacin concentrations (bottom)
in stomach and small intestine tissues (mean + S.D.) after oral administration of
NC–IndOEt (pro-drug at 10 mg kg−1) (n = 4).
After NC–IndOEt oral administration, the indomethacin concen-
trations in stomach rose from 4 to 102 ␮g g−1 after 15 and 120 min,
respectively (Fig. 5). In the duodenum and jejunum, the concen-
trations were similar rising from 70 and 92 (15 min) to 137 and
107 ␮g g−1 (120 min), respectively. In the ileum, the indomethacin
concentration increased from 9 to 106 ␮g g−1 after 15 and 120 min,
respectively. The hydrolysis of the indomethacin ethyl ester was
likely occurring in the tissue wall.
A parallel experiment was carried out to evaluate the nanocap-
sule adhesion to the gastrointestinal luminal mucus. After 60 min
of NC–IndOEt oral administration, the stomach and small intes-
tine were scrapped for mucus removal before homogenization.
The indomethacin ethyl ester concentrations in the tissues were
much lower than before scrapping. Its concentrations decreased
from 459 to 5 ␮g g−1 from the stomach to the ileum (Fig. 6). On
the other hand, for indomethacin formed by ester hydrolysis, a
slight difference was barely observed before and after scrapping.
Its concentrations in stomach, duodenum, jejunum and ileum were
58, 126, 88 and 56 ␮g g−1, respectively, after 60 min (Fig. 6). The
mucoadhesion probably delayed the ester hydrolysis in the lumen
as previously described for poly(alkyl cyanoacrylate) nanoparticles
(Dembri et al., 2001).
The results showed that the indomethacin ethyl ester is mainly
accumulated in the mucus. The indomethacin ethyl ester uptake
was more intense at the stomach and proximal small intestine,
Fig. 7. Indomethacin concentration–time profiles in peripheric plasma, portal vein
plasma, liver and spleen after oral administration of NC–IndOEt (pro-drug at
10 mg kg−1) (n = 4/time) (mean + S.D.).

V.B. Cattani et al. / European Journal of Pharmaceutical Sciences 39 (2010) 116–124
123
Theoretically, ester pro-drugs could be absorbed and, by first
pass metabolism, be converted in free acid drugs, detected in
plasma. However, in the present work this hypothesis was refused
considering the absence of indomethacin ethyl ester in portal vein
plasma and in liver. Additionally, the liver, despite its high esterase
activity, can be marginally involved in the conversion of ester pro-
drugs after their uptake by the gastrointestinal tract (Braggio et al.,
2002).
Calvo, P., VilaJato, J.L., Alonso, M.J., 1996. Comparative in vitro evaluation of several
colloidal systems, nanoparticles, nanocapsules, and nanoemulsions, as ocular
drug carriers. J. Pharm. Sci. 85, 530–536.
Carreno-Gómez, B., Woodley, J.F., Florence, A.T., 1999. Studies on the uptake of
tomato lectin nanoparticles in everted gut sacs. Int. J. Pharm. 183, 7–11.
Cattani, V.B., Pohlmann, A.R., Dalla Costa, T., 2008. Pharmacokinetic evaluation of
indomethacin ethyl ester-loaded nanoencapsules. Int. J. Pharm. 363, 214–216.
Chasteigner, S., Fessi, H., Cavé, G., Devissaguet, J.P., Puisieux, F., 1995. Gastro-
intestinal study of a freeze-dried oral dosage form of indomethacin-loaded
nanocapsules. STP Pharma Sci. 5, 242–246.
Chen, Y., Ping, Q., Guo, J., Lv, W., Gao, J., 2003. The absortion behavior of cyclosporine
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4. Conclusion
Cruz, L., Schaffazick, S.R., Dalla Costa, T., Soares, L.U., Mezzarila, G., Silveira,
N.P., Schapoval, E.E.S., Pohlmann, A.R., Guterres, S.S., 2006a. Physico-chemical
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nanocapsules by PCS, TEM, SAXS, interfacial alkaline hydrolysis and antiede-
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Cruz, L., Soares, L.U., Dallla Costa, T., Mezzalira, G., Pesce da Silveira, N., Guterres, S.S.,
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Damge, C., Aprahamian, M., Marchais, H., Benoit, J.P., Pinget, M., 1996. Intestinal
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Dembri, A., Duchene, D., Ponchel, G., 2001. The intestinal mucus protects
poly(isobutylcyanoacrylate) nanoparticles from enzymatic degradation. STP
Pharma Sci. 11, 175–180.
The ex vivo experiment showed
a limited passage of
indomethacin ethyl ester from the luminal side through the serosal
fluid. In addition, the lipid-core nanocapsules were stable and
no indomethacin ethyl ester was released in the luminal side.
So, indomethacin was not formed by hydrolysis of the ester
in the external medium. The lipid-core nanocapsules released
indomethacin ethyl ester at the tissue after bioadhesion, but no
enzymatic hydrolysis took place. In this way, indomethacin was not
formed in the tissue. The comparison between the different formu-
lations (NC–IndOEt, NC–IndOH and NC–PMMA-HBN) showed that
indomethacin ethyl ester is absorbed in the tissue by a diffusion
mechanism.
Florence, A.T., 1997. The oral absortion of micro- and nanoparticulates: neither
exceptional nor unusual. Pharm. Res. 14, 259–266.
Florence, A.T., Hussain, N., 2001. Transcytosis of nanoparticle and dendrimer deliv-
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The in vivo experiment showed that the lipid-core nanocapsules
were able to restrain the indomethacin ethyl ester hydrolysis in the
gastrointestinal lumen and wall acting as mucoadhesive reservoir
systems for the pro-drug. Furthermore, after oral dosing of lipid-
core nanocapsules, our study showed that indomethacin ethyl ester
is converted into indomethacin before the pro-drug reach the blood
stream. Lipid-core nanocapsules are stable in the gastrointestinal
tract and can be used for oral delivery of lipophilic drugs.
Finally, distinct results were observed comparing the ex vivo and
in vivo models. The complexity of a living system was not mimic
by the ex vivo experiment, and the gut sac model was not able
to predict the in vivo behavior of indomethacin ethyl ester-loaded
lipid-core nanocapsules.
Guterres, S.S., Fessi, H., Barrat, G., Devissaguet, J.P., Puisieux, F., 1995. Poly(d,l-
lactide) nanocapsules containing diclofenac: formulation and stability study.
Int. J. Pharm. 113, 57–63.
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benzoate as oil core on the physicochemical properties of spray-dried powders
from polymeric nanocapsules containing indomethacin. Drug Deliv. 7, 195–199.
Hussain, N., Jaitley, V., Florence, A.T., 2001. Recent advances in the understanding of
uptake of microparticulates across the gastrointestinal lymphatics. Adv. Drug.
Deliv. Rev. 50, 107–142.
Hussain, N., Jani, P.U., Florence, A.T., 1997. Enhanced oral uptake of tomato lectin
conjugated nanoparticles in the rat. Pharm. Res. 14, 613–618.
Jäger, A., Stefani, V., Guterres, S.S., Pohlmann, A.R., 2007. Physico-chemical charac-
terization of nanocapsule polymeric wall using fluorescent benzazole probes.
Int. J. Pharm. 338, 297–305.
Jäger, E., Venturini, C.G., Poletto, F.S., Colomé, L.M., Pohlmann, J.P.U., Bernardi, A.,
Battastini, A.M.O., Guterres, S.S., Pohlmann, A.R., 2009. Sustained release from
lipid-core nanocapsules by varying the core viscosity and the particle surface
area. J. Biomed. Nanotechnol. 5, 130–140.
Jani, P., Halbert, G.W., Langridge, J., Florence, A.T., 1990. Nanoparticle uptake by
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Acknowledgments
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Kim, S.Y., Lee, Y.M., Shin, H.J., Kang, J.S., 2001. Indomethacin-loaded methoxy
The authors thank Centro de Nanociência e Nanotecnologia
UFRGS, CNPq/Brasilia/Brazil, FINEP/MCT, Universal/CNPq,
Rede Nanocosméticos CNPq/MCT, IBSA/CNPq, CAPES and
CAPES/COFECUB.
–
poly(ethylene
glycol)/poly(epsilon-caprolactone)
diblock
copolymeric
nanosphere: pharmacokinetic characteristics of indomethacin in the normal
Sprague–Dawley rats. Biomaterials 22, 2049–2056.
Limpanussourn, J., Simon, L., Dayan, A.D., 1998. Transepithelial transport of large
particles in rat: a new model for the quantitative study of particle uptake. J.
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