LPDT_A_938857-2014

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ISSN: 1083-7450 (print), 1097-9867 (electronic)
Pharm Dev Technol, Early Online: 1–9
! 2014 Informa Healthcare USA, Inc. DOI: 10.3109/10837450.2014.938857
RESEARCH ARTICLE
Elucidation of intestinal absorption mechanism of carvedilol-loaded
solid lipid nanoparticles using Caco-2 cell line as an in-vitro model
Mansi K. Shah, Parshotam Madan, and Senshang Lin
College of Pharmacy and Health Sciences, St. John’s University, Queens, NY, USA
Abstract
Enhanced oral bioavailability of poorly aqueous soluble drugs encapsulated in solid lipid
nanoparticles (SLNs) via lymphatic delivery has been documented. Since no in-vitro lymphoid
tissue is currently available, human excised Caco-2 cell monolayer could be alternative tissue
for development of an in-vitro model to be used as a screening tool before animal studies are
undertaken. Therefore, optimized carvedilol-loaded SLNs (FOPT-SLNs) were prepared,
characterized, and evaluated using Caco-2 cell line as an in-vitro model. Physical mixture of
components of FOPT-SLNs (FOPT-PM) and carvedilol solution were used as control groups.
From the studies of effect of SLNs concentration and cells incubation time, suitable carvedilol
concentration and incubation time were selected for the model in which cells were subjected
to five pretreatments for 24 h or 1 h of cell incubation and then followed with treatment of
FOPT-SLNs, FOPT-PM or 100 mg/mL solution of carvedilol, for additional 24 h of cell incubation.
The results obtained in this model suggest that main absorption mechanism of FOPT-SLNs
could be endocytosis and, more specifically, clathrin-mediated endocytosis. When TranswellÕ
permeable supports were used for the cells, carrier-mediated mechanism for FOPT-SLNs and
passive absorption mechanism (transcellular and paracellular) for FOPT-PM and drug solution
were concluded.
Keywords
Absorption mechanism, Caco-2 cells,
carvedilol, cellular uptake, intestinal
absorption, solid lipid nanoparticles
History
Received 18 February 2014
Revised 21 June 2014
Accepted 23 June 2014
Published online 29 July 2014
Introduction
Solid lipid nanoparticles (SLNs) have emerged as a novel
approach for oral controlled drug delivery system. SLNs are
comprised of a biodegradable solid lipid core stabilized by a
surfactant interfacial region. This delivery system combines the
advantages of nanoparticle systems (solid core) and emulsion
systems (surfactant interfacial region) to overcome the temporal
and stability issues that other drug delivery systems exhibit. SLNs
have been evaluated and reported as a potential delivery system
for several administration routes1
including the parentral, ocular2
and oral3–7
routes. Improvement in oral bioavailability of many
poorly aqueous soluble drugs encapsulated in SLNs as the
delivery system has been documented in the literature8–15
.
Several mechanisms have been reported to enhance the oral
bioavailability of drug incorporated in SLNs. These include
dissolution/solubilization of the co-administered lipophilic
drug due to presence of lipids and stimulation of lymphatic
transport16–18
. Following oral administration, substances absorbed
across the membranes of the small intestines enter either systemic
circulation or lymphatic system. In general, the small molecular
weight hydrophilic substances enter the systemic circulation via
hepatic portal vein by passive absorption mechanism; while the
large molecular weight lipophilic substances such as SLNs can
enter the lymphatic system via mesenteric lymph duct and
subsequently appear in systemic circulation. Substances via the
mesenteric lymph duct, in contrast to the hepatic portal vein, enter
the systemic circulation directly without first passing through the
liver19–21
. Therefore, intestinal lymphatic absorption of drug-
loaded SLNs has potential advantage of avoiding the first-pass
metabolism and thereby reducing the required drug dose and its
side effects.
Although evidence of intact SLNs entering the lymphatic
system following intra-duodenal administration of SLNs to the
rats has been reported22
, the mechanistic understanding of access
of SLNs to the lymph remains relatively poor. Till date, the
in-vitro method for elucidating the absorption mechanism of
SLNs administered orally as well as differentiating endocytic
process for lymphatic delivery from the passive absorption has not
been reported. Several researchers have reported methods such as
intra-duodenal administration, ex-vivo permeation study with
cannulation of lymphatic duct, and animal studies in rats23–26
.
However, there is no in-vitro method, which can be used as a
screening tool for the lymphatic delivery of SLNs before
performing in-vivo experiments27
. Thus, the availability of
reliable high throughput screening methods for rapid evaluation
and prediction of the absorption mechanism of SLNs formulation
is needed. Since there is no in-vitro lymphoid tissue currently
available for the evaluation of lymphatic delivery, human excised
Caco-2 cell monolayer (a colon rectal adenocarcinoma cell line of
human origin) could be the alternative tissue due to the fact that it
mimics most transport pathways in the gastrointestinal tract and it
has demonstrated to be a particularly valuable tool for estimating
human drug absorption potential28–30
.
Caco-2 cell line of human origin represents a model for
intestinal cells and is recommended by the U.S. Food and Drug
Address for correspondence: Senshang Lin, PhD, College of Pharmacy
and Health Sciences, St. John’s University, Queens, NY, USA. Tel: (718)
990 5344. Fax: (718) 990 1877. E-mail: linse@stjohns.edu
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Administration (FDA) for absorption studies because it spontan-
eously differentiates in culture to form confluent monolayer
which has remarkable morphological and biological similarity to
that of intestinal epithelium. In addition, Caco-2 cells not only
have been widely developed and used, but also are accepted tool
for the investigation of transport across the small intestinal
epithelium and particle uptake into human intestine as an
alternative to the animal models31
. Research has been reported
where biodegradable nanoparticles were bound to intestinal
epithelial cells and were able to stimulate the uptake and transport
of intact nanoparticles suggesting the carrier-mediated transport
mechanism32
. However, the direct relationship of Caco-2 cell line
with the lymphatic absorption has not been evaluated and reported
in the literature. It has been reported that the dietary lipids or
lipid-based formulations are absorbed via the intestinal lymph.
In addition, Caco-2 cell line has been used to determine the
effect of lipid based excipients as formulation variable for
the lymphatic delivery of therapeutic agents33
. Therefore, in this
investigation, Caco-2 cell line was utilized for its application for
the evaluation of intestinal absorption of carvedilol from lipid-
based SLNs.
The efficiency of drug delivered by the SLNs via intestinal
lymphatic absorption is mainly dependent upon the types of lipids
and surfactants used6,16,17
. Recently, carvedilol-loaded SLNs
fabricated to study the effect of lipids and surfactants using
design of experiment approach for optimization of formulation
was reported34
. The recommended dose for carvedilol is approxi-
mately 3.125 to 6.25 mg twice a day for about 7–14 d for the
treatment of hypertension, left ventricular dysfunction or follow-
ing myocardial infarction. Several dose-related side effects, such
as bradycardia, cardiac insufficiency, cardiogenic shock and
cardiac arrest as well as presystemic metabolism in the liver lead
to liver damage preventing its use in patients with impaired
hepatic functions have been reported35–37
. The properties of
carvedilol such as a Biopharmaceutical Classification System
Class II drug, oral bioavailability of about 25% due to high first-
pass metabolism, and repetitive dosing for the treatment make
this drug an interesting candidate for oral administration for
improved bioavailability via alternative absorption mechanism. In
addition, carvedilol possesses fluorescence property which makes
it an ideal candidate to study the absorption mechanism using
fluorescence spectroscopy. In this investigation, the optimized
carvedilol-loaded SLNs (i.e. 7.5% Compritol 888 ATO, 5.0%
Poloxamer188 and 1.11% carvedilol) were prepared, character-
ized, and then evaluated using Caco-2 cell line as in-vitro model
for elucidation of intestinal absorption mechanism of SLNs.
Materials and methods
Materials
Carvedilol and Compritol 888 ATO (COMP) were obtained as a
free sample from Caraco Pharmaceuticals (Detroit, MI) and
Gattafosse (Paramus, NJ), respectively. Monobasic potassium
phosphate, dibasic potassium phosphate, glacial acetic acid,
Poloxamer188 (P-188), polyethylene glycol 400 (PEG), and
HPLC grade methanol were purchased from VWR International
(West Chester, PA). Dialysis membrane (spectra/Por dialysis
membrane MWCO: 6-8000), sodium azide and sucrose were
obtained from Spectrum Labs (Rancho Dominguez, CA). Caco-2
cell line was obtained from American Type Culture Collection
(ATCC, Manassas, VA). Dulbecco’s Modified Eagle’s Medium
(DMEM/F12) with L-glutamine and 15 mM HEPES medium, fetal
bovine serum (FBS), 1% streptomycin, trypsin, Lucifer yellow
and Tween 80 were purchased from Sigma-Aldrich (St. Louis,
MO). All chemicals were of analytical or technical grade and
were used without further treatment.
Analytical methodology
Analysis of carvedilol by HPLC
Carvedilol concentrations were assayed by reverse phase HPLC
method published in the literature38
. Briefly, the assay employed a
HP1100 series (Agilent Technologies, Wilmington, DE) with a
mBondapak 125 A, C18 column (10.0 mm, 3.9 Â 300 mm). The
mobile phase consisted of methanol, 0.0333 M phosphate buffer
and glacial acetic acid in the ratio of 6:4:0.033 (v/v/v). The
injection volume and flow rate were 30 mL and 1 mL/min,
respectively. The UV detector was set at a wavelength of
284 nm with a retention time of 3.9 min. A stock solution of
1000 mg/mL was prepared by dissolving 25 mg of carvedilol
in sufficient methanol to prepare 25 mL of solution.
Standard solution samples were prepared from the stock solu-
tion by serial dilutions (to obtain concentrations in the range of
0.5 mg/mL to 200 mg/mL) to establish the calibration plot of
standards. The carvedilol content of samples was then quantified
by the peak area method from the obtained calibration plot.
No additional peaks were observed during quantification of
carvedilol.
Analysis of carvedilol by fluorescence spectroscopy
A stock solution of 100 mg/mL was prepared by dissolving 10 mg
of carvedilol in sufficient methanol to prepare 100 mL of solution.
A series of carvedilol standard solutions were prepared from this
stock solution to obtain concentrations in the range of 5 ng/mL
to 25 mg/mL using DMEM media without phenol red.
Fluorescence intensity of these solutions was measured at an
excitation wavelength of 254 nm and an emission wavelength
of 356 nm using SpectraMax M5e
(Molecular Devices,
Sunnyvale, CA)39
.
Preparation of optimized carvedilol-loaded SLNs
The optimized carvedilol-loaded SLNs (FOPT-SLNs), concluded
from our previous publication, were used in this investigation34
.
A high shear hot homogenization method was used to prepare the
FOPT-SLNs. Thermal stability of carvedilol was not evaluated
because published reports attest thermal stability of carvedi-
lol40,41
. Briefly, the lipid (7.5% w/v of COMP) was heated to
about 5–10
C above its melting point (70–72
C) and 1.11% w/v
of carvedilol was added and dissolved in it. The aqueous
surfactant solution (5.0% w/v of P-188) was heated separately to
the same temperature and added to the melted lipid. The mixture
was homogenized at 25 000 rpm for 10 min using a high-shear
homogenizer (Sentry Microprocessor, Kent City, MI). The
resulted dispersion was diluted with 10.0 mL of water (final
preparation, 20.0 mL) and allowed to cool at room temperature
with constant stirring.
Characterization of optimized carvedilol-loaded SLNs
Particle size
Photon correlation spectroscopy was employed to measure
particle size distribution of FOPT-SLNs. The formulation was
diluted 10 times using distilled water before the determination.
DelsaÔ Nano Series Zeta Potential and Submicron Particle Size
Analyzer (Brea, CA) were used at a scattering angle of 90
at
room temperature. To ensure low intensity of the particles in
the samples suitable for measurement by the instrument, the
effect of dilution on SLNs was performed. Since there was no
effect of dilution with water on particle size, for the ease and
accuracy of mathematical treatments, the 10 times of dilution was
selected.
2 M. K. Shah et al. Pharm Dev Technol, Early Online: 1–9
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Entrapment efficiency
Although the ultracentrifugation method is more reliable and
faster than the dialysis bag method, ultracentrifugation was not
used because of difficulty in obtaining the pellet of the SLNs and
clear supernatant due to the fact that the prepared SLNs have
density less than that of water. Therefore, entrapment efficiency
was determined using the dialysis bag method which separates un-
entrapped drug from FOPT-SLNs42
. Briefly, to determine un-
entrapped drug, 1.0 mL of the 20.0 mL prepared formulation
was placed in dialysis bag (spectra/Por dialysis membrane
MWCO: 6–8000) at 4
C in a beaker containing 20.0 mL of pH
6.8 phosphate buffer with 30% PEG 400. At predetermined time
intervals, samples (1 mL each) were withdrawn up to 8 h and
immediately replaced with the fresh medium. The collected
samples were diluted with 1.0 mL of methanol and analyzed by
HPLC method described previously. The following equation was
used to calculate entrapment efficiency.
EE% ¼
Dt À Dm
Dt
Â 100: ð1Þ
where, EE% ¼ percent entrapment efficiency, Dt ¼ total amount
of drug initially added, Dm ¼ amount of drug in medium
(un-entrapped).
The FOPT-SLNs dispersion (without un-entrapped drug)
obtained at the end of the above procedure was collected and
solubilized using methanol as the dissolvent to break the SLNs.
Samples were analyzed by the HPLC method described above for
entrapment efficiency and mass balance was studied. The dialysis
membrane used in the present investigation has a molecular cut
off 6000–8000 and it has a pore size smaller than approximately
20 nm. To ensure its filter function, a standard drug solution of
known volume and concentration was added inside the dialysis
bag and drug concentrations from both inside and outside the bag
were measured using the same experimental condition described
earlier. A rapid and complete distribution of drug from inside to
outside of the dialysis bag was observed and reached equilibrium
within 1 h confirming that the dialysis bag functioned as a filter to
separate the non-entrapped drug from the entrapped drug.
In-vitro drug release study
The in-vitro release profiles of FOPT-SLNs were determined
using dialysis bag technique. The dialysis bags were soaked in
distilled water for 12 h before use. FOPT-SLNs (0.5 mL) were
added into the dialysis bag and both ends of the bag were tied with
clamps. The bags were placed in a beaker containing 30.0 mL of
dissolution medium, and the beakers were placed in a thermo-
static shaker (VWR, Shell Lab, Cornelius, OR) and then shaken
horizontally at 100 rpm at 37
C. To simulate the environment of
jejunum and ileum, phosphate buffer solution (pH 6.8) containing
1% v/v of Tween 80 was used. Aliquots (0.5 mL) were withdrawn
at predetermined time intervals and the withdrawn volume was
immediately replaced with the fresh medium. The samples were
analyzed by HPLC method to determine the drug content. All
studies were carried out in triplicate.
In-vitro model to elucidate the absorption mechanism
Cellular uptake of optimized carvedilol-loaded SLNs
Effect of number/concentration of SLNs. The obtained Caco-2
cells were cultured in T-75 tissue-culture flasks with growth
media containing Ham’s F-12 K medium (Kaighn’s modification
of Ham’s F-12 containing 2 mM of L-glutamine and 1500 mg of
NaHCO3 per liter) supplemented with 10% FBS, heparin (100 mg/
mL), ECGS (40 mg/mL), and 1% penicillin–streptomycin solution.
Based upon the cell count obtained from cell culture prepared,
Caco-2 cells were seeded in 48-well plates, at the density of
50 000 cells per well, with complete fresh growth medium until
cells were confluence. To determine the effect of number/
concentration of SLNs on cellular uptake, dilutions were made in
terms of carvedilol concentration due to the limitations of
calculating the number/concentration of SLNs. Therefore, the
stock suspension of FOPT-SLNs in a DMEM/F12 media without
serum and antibiotics was prepared and dilutions were made
representing the decrease in number/concentration of SLNs
accordingly. In order to differentiate the cellular uptake mechan-
ism of FOPT-SLNs from that of passive absorption of released
carvedilol form FOPT-SLNs, carvedilol solution was prepared as
a control group to study passive absorption. In addition, for better
understanding of the role of SLNs as a carrier for carvedilol via
cellular uptake mechanism, the lipid (COMP) and surfactant
(P-188), used for the preparation of FOPT-SLNs, were presented
as physical mixture with same carvedilol concentration (FOPT-
PM). Therefore, carvedilol solution and FOPT-PM in a DMEM/
F12 media having respective concentration of FOPT-SLNs were
prepared as the control groups.
To initiate the experiment, the growth medium from the
previously seeded 48-well plates was replaced with the FOPT-
SLNs, FOPT-PM or carvedilol solution with different concentra-
tions of carvedilol. These plates were incubated at 37
C for 3 h.
At the end of the incubation period, the FOPT-SLNs, FOPT-PM
or carvedilol solution was removed from the wells. Furthermore,
to remove residuals of FOPT-SLNs, FOPT-PM, carvedilol
solution and dead cells, if any, from the wells, the cell monolayer
was rinsed and washed three times with PBS. For the cell lysate
and solubilization, 0.1 mL of 1% Triton X-100 and 0.2 N NaOH in
a 1:1 ratio was added to each well. To this, 0.4 mL of methanol
was added and samples were centrifuged at 15 000 rpm for 10 min
to extract the carvedilol from cells. The supernatant was collected
and analyzed using previously described HPLC method for
carvedilol concentration. In a preliminary experiment, cytotox-
icity study was performed and the cell death was observed when
the SLNs concentration was 4500 mg/mL. Therefore, concentra-
tion of SLNs upto 200 mg/mL was evaluated. In addition, before
and after each experiment, TEER value and Lucifer yellow
permeability was also measured to ensure intact cell monolayer
integrity.
Effect of incubation time. In order to evaluate the effect of
incubation time required for the completion of cellular uptake of
SLNs, similar studies with incubation time longer than 3 h such as
6 h and 24 h were performed.
Uptake mechanism elucidation using pretreatment design
For better understanding the role of SLNs as carriers, Caco-2 cells
in the wells were pretreated with different treatments such as
incubating without carriers (drug solution), with the carriers
(blank SLNs), or with the drug-loaded carriers (FOPT-SLNs).
The hypothesis is that if the SLNs are being uptaken by
transporters of Caco-2 cells, then pretreating the cells with
blank SLNs would occupy those transporters by blank SLNs.
Therefore, reduction in the cellular uptake of FOPT-SLNs would
be expected upon further incubation of the same cells with FOPT-
SLNs. However, in the case of carvedilol solution, which is
expected to exhibit passive absorption mechanism, upon addition
to carriers (SLNs) or by inhibiting the transporters, the amount of
carvedilol absorbed should not be reduced.
Based upon the hypothesis described above, the cells in the
wells were pretreated with the growth media (pretreatment A) and
blank SLNs (pretreatment B) for 24 h as control groups. In order
to study the effect of carrier competition between blank SLNs and
DOI: 10.3109/10837450.2014.938857 Intestinal absorption of carvedilol-loaded solid lipid nanoparticles 3
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FOPT-SLNs, cells were pretreated with blank SLNs together with
FOPT-SLNs in 1:1 ratio (pretreatment C) for 24 h. In addition,
cells were pretreated with sodium azide (0.1%w/v) as a specific
known pharmacological endocytosis inhibitor (pretreatment D)
for 1 h43,44
. To further understand the role of clathrin on the
cellular uptake of SLNs, cells were pretreated for 1 h with sucrose
(0.45 M) which creates hypertonic environment disrupting the
formation of clathrin-coated vesicles (pretreatment E)45
. At the
end of the incubation period (24 h for pretreatments A, B and C
while 1 h for pretreatments D and E), the medium was aspirated
and cells were rinsed and washed twice with cold PBS to remove
the medium completely as well as any dead cells formed during
the incubation. Thereafter, FOPT-SLNs, FOPT-PM, or carvedilol
solution was added to all six groups (including one group without
any pretreatment) and the cells were incubated for 24 h. At the end
of the second incubation period, cells in the wells were lysed and
carvedilol content was determined by the HPLC method as
described previously. A total of 24 experiments were performed in
quadruplicate. All the pretreatments were prepared in growth
media.
Since carvedilol was present in the pretreatment C, additional
set of pretreatment C was performed. At the end of the incubation
period of pretreatment, cells in the wells were lysed and carvedilol
content was determined as described earlier.
Absorption mechanism of optimized carvedilol-loaded SLNs
During the experiments of cellular uptake of carvedilol-loaded
SLNs, the Caco-2 cells were grown on 48-well plates where there
are no permeable supports (e.g. filtration inserts). There were no
resulting apical (donor) and basolateral (receptor) compartments
from the formation of cell monolayer after incubation. Therefore,
it would be difficult to differentiate whether the carvedilol present
in the cells was due to cellular uptake of SLNs or passive
absorption mechanism of carvedilol. In an attempt to resolve this
issue, studies were designed and performed using TranswellÕ
permeable supports with 0.4 mm polycarbonate membrane having
diameter of 6.5 mm, pore size of 0.4 mm and cell growth area of
0.33 cm2
. The use of permeable support system provided
convenient, independent access to apical and basolateral com-
partments since the cells were grown on permeable supports.
Therefore, the free drug which has undergone the passive
absorption could be determined from the basolateral side and
cellular uptake of SLNs could be determined from the cells as
described previously.
Seeding of Caco-2 cells using TranswellÕ
permeable
supports. Caco-2 cells suspension was seeded at the density of
5000 cells/well on each TranswellÕ
permeable support. The
volume of cell suspension in complete growth media used was
0.1 mL. In the basolateral side, 0.5 mL of complete growth media
was added. Growth media from both sides were removed and
replaced every 2 d for 20 d until cells were confluent. Higher
seeding density to minimize the number of days to reach
confluence was not used because high seeding density tends to
form multilayers instead of confluent monolayer.
Cell monolayer integrity and development of tight
junctions. The integrity of the cell monolayer was checked
once every week starting at day 4 and then at day 21 post seeding,
as well as at the beginning and the end of each absorption
experiments by measuring the transepithelial electrical resistance
(TEER) using EVOM2 (World Precision Instrument, Sarasota,
FL). In addition, absorption of Lucifer yellow across the cell layer
was determined at the end of each experiment as a control.
Absorption mechanism: passive absorption versus cellular
uptake. Absorption mechanism studies were performed after
21 d of post seeding. Before the experiments, the cells-seeded
TranswellÕ
permeable supports were rinsed with sterile cold PBS
to remove dead cells and TEER was measured. Experiments were
initiated by adding 0.1 mL of FOPT-SLNs, FOPT-PM or
carvedilol solution prepared in media (free from serum, anti-
biotics and phenol red) at apical side and 0.5 mL of same media
without any formulation in basolateral side. These TranswellÕ
permeable supports were incubated at 37
C for 24 h. At the end
of incubation period, samples were withdrawn from basolateral
side and apical side to determine the carvedilol content
representing passive absorption of carvedilol and unabsorbed
carvedilol, respectively. The cells-seeded polycarbonate mem-
brane were washed three times with cold PBS and then cells were
lysed by adding 0.1 mL of 1% Triton-X 100 and 0.2 N NaOH in a
1:1 ratio for carvedilol content which represents absorbed
carvedilol from cellular uptake of SLNs. Samples for the transport
studies were analyzed using fluorescence spectroscopy method as
described previously.
Results and discussion
Analytical methodology
Analysis of carvedilol by HPLC
The peak area value was found to increase linearly with the
concentration of carvedilol within the measured concentration
range with a regression coefficient (r2
) value of 0.9998. The
results indicated that the HPLC method, adopted for the detection
and quantification of carvedilol, is a reliable and robust method.
Analysis of carvedilol by fluorescence spectroscopy
The fluorescence intensity of carvedilol increases with the increase
in concentration with a linear regression (r2
) of 0.9952. The results
indicated that the fluorescence method, adopted for the detection
and quantification of carvedilol, is a reliable and robust.
Characterization of optimized carvedilol-loaded SLNs
The particle size of FOPT-SLNs was 161 ± 13 nm and the entrap-
ment efficiency was 94.8 ± 3%. Although about 48.6 ± 4.7% of
carvedilol was released from FOPT-SLNs at the first 12 h,
only 1.9 ± 0.2% additional carvedilol was released at the end of
24 h experiment34
. Thus, entrapping carvedilol into a lipid by
FOPT-SLNs formulations was able to retain about 50% of
carvedilol before the uptake of carvedilol-loaded SLNs from the
region of small intestine.
Cellular uptake of optimized carvedilol-loaded SLNs
Effect of number/concentration of SLNs
The effect of number/concentration of SLNs on their cellular
uptake by Caco-2 cells, determined by carvedilol concentration
absorbed, after 3, 6 and 24 h of cell incubation, respectively, is
shown in Figure 1. Upon increasing the concentration of
carvedilol in FOPT-SLNs, FOPT-PM and carvedilol solution,
the amount of carvedilol found in the Caco-2 cells was observed
to increase in all cases. Cells incubated for 3 h exhibited increase
in carvedilol amount in the cell with increasing carvedilol
concentration in all formulations. As shown in Figure 1(a), at
the end of 3 h incubation, increasing the carvedilol concentration
from 25 mg/mL to 200 mg/mL, the carvedilol amount in cells
increased from 5.99 mg to 40.33 mg, 1.88 mg to 23.35 mg, and
2.35 mg to 19.63 mg for FOPT-SLNs, FOPT-PM and carvedilol
solution, respectively. In addition, increase in cellular uptake with
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increase in carvedilol concentration was also observed when cells
were incubated for 6 h (Figure 1b) and 24 h (Figure 1c). In the
case of FOPT-SLNs, since the dilutions were made in terms of
carvedilol concentration, it was found that as the carvedilol
concentration was increased, the number/concentration of SLNs
also increased. This increase was much more dramatic than that
seen in FOPT-PM or carvedilol solution. Thus, for FOPT-SLNs,
the carvedilol amount in Caco-2 cells was also dependent on the
concentration of carvedilol and hence on the number/concentra-
tion of SLNs in the medium.
Effect of incubation time
The effect of incubation time on the cellular uptake of carvedilol
from FOPT-SLNs, FOPT-PM and carvedilol solution is shown in
Figure 2. In the case of FOPT-SLNs (Figure 2a), upon increasing
the incubation time from 3 h to 24 h, the amount of carvedilol
found in Caco-2 cells increased from 40.33 mg to 192.77 mg at
carvedilol concentration of 200 mg/mL. Similar trend was
observed in the case of FOPT-PM (Figure 2b) and carvedilol
solution (Figure 2c) indicating that increasing the incubation time
led to increased carvedilol uptake by Caco-2 cells. It is interesting
to note that in the case of carvedilol solution and FOTP-PM, when
the incubation time was increased form 3 h to 6 h, there was no
significant change in the amount of carvedilol in the cells. The
possible reason for this observation could be the lack of sufficient
time for the uptake process to be significant. Therefore, for future
experiments, cells were incubated for longer incubation period
(b) FOPT-PM
0 50 100 150 200
CarvedilolamountobtainedinCaco-2cells(µg)
0
20
40
60
80
100
(c) Carvedilol solution
Carvedilol concentration (µg/mL)
0 50 100 150 200
0
20
40
60
80
100
(a) FOPT-SLNs
0 50 100 150 200
0
50
100
150
200
250
Incubation time: 3 h
Figure 2. Effect of incubation time of SLNs on their cellular uptake by
Caco-2 cells, determined by carvedilol concentration absorbed, from
(a) FOPT-SLNs, (b) physical mixture of components of FOPT-SLNs
(FOPT-PM) and (c) carvedilol solution (data represent mean ± standard
deviation, n ¼ 4).
(b) Incubation time: 6 h
0 50 100 150 200
0
20
40
60
80
100
(a) Incubation time: 3 h
0 20 40 60 80 100 120 140 160 180 200
0
20
40
60
80
100
FOPT-SLNs
FOPT-PM
Carvedilol solution
(c) Incubation time: 24 h
Carvedilol concentration (µg/mL)
0 50 100 150 200
0
50
100
150
200
250
Figure 1. Effect of number/concentration of SLNs on their cellular
uptake by Caco-2 cells, determined by carvedilol concentration absorbed,
from FOPT-SLNs, physical mixture of components of FOPT-SLNs
(FOPT-PM) and carvedilol solution at (a) 3 h, (b) 6 h and (c) 24 h
incubation time (data represent mean ± standard deviation, n ¼ 4).
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(i.e. 24 h) to eliminate the effect of incubation time and for better
understanding of the cellular uptake mechanism from the
formulations.
Effect of formulation
It is evident from Figures 1 and 2 that, irrespective of the number/
concentration of SLNs and incubation time, higher amount of
carvedilol was found from FOPT-SLNs as compared to that from
the drug solution and FOPT-PM. Moreover, similar amount of
carvedilol was observed between the drug solution and
FOPT-PM. These results suggest the crucial role of SLNs for
the cellular uptake of carvedilol. The possible explanation for this
observation could be the carrier effect of SLNs for preferential
cellular uptake leading to the higher amount of carvedilol as
compared to the carvedilol solution and FOPT-PM, which tend to
follow passive absorption mechanism. However, at the concen-
tration range studied, the saturation of carrier was not observed to
conclude the carrier-mediated transport mechanism for
FOPT-SLNs. Thus, to understand and elucidate the cellular
uptake mechanism further in detail, cells were subjected to a
pretreatment design.
Uptake mechanism elucidation using pretreatment design
As shown in Table 1 and Figure 3, when Caco-2 cells were
pretreated with growth media (pretreatment A), the amount of
carvedilol absorbed by cells was found to increase from 76.12 mg
(without any pretreatment) to 86.55 mg for FOPT-SLNs (pretreat-
ment A). Similar trend was observed for FOPT-PM (19.23 mg
versus 41.20 mg) and carvedilol solution (22.58 mg versus
30.00 mg). The higher uptake in the case of FOPT-PM compared
to the carvedilol solution could be attributed to the presence of
lipid and surfactant. The increased amount of carvedilol absorbed
from all three formulations in pretreatment A was expected, since
the total number of cells might have increased due to the presence
of growth media for cell growth during 24 h of incubation. More
cells indicate more cellular uptake of carvedilol. Since all the
samples were prepared in the growth media, the data obtained
from pretreatment B and pretreatment C were compared with that
from pretreatment A. When cells were pretreated with blank
SLNs (pretreatment B), the carvedilol absorbed by cells was
found to reduce from 86.55 mg to 41.50 mg upon treatment with
the FOPT-SLNs. This obtained reduction could be attributed to
the already occupied transporters of cells by blank SLNs during
the pretreatment period. Subsequently, fewer transporters were
available for FOPT-SLNs to be absorbed. Thus, reduction in
carvedilol absorption was observed upon the addition of FOPT-
SLNs after pretreatment B. Furthermore, reduction in carvedilol
absorbed was not observed from FOPT-PM (41.20 mg versus
39.20 mg) and carvedilol solution (30.00 mg versus 31.20 mg). This
finding indicates that the amount of carvedilol uptaken from
FOPT-PM and carvedilol solution could not be attributed to the
carrier-mediated transport mechanism.
When FOPT-SLNs formulation was coupled with pretreatment
C in which cells were pretreated with FOPT-SLNs together with
blank SLNs (1:1 ratio), the total amount of carvedilol found in the
cells was 78.09 mg. Since carvedilol uptaken at the end of the
pretreatment period was 40.00 mg, the amount of carvedilol
absorbed from the addition of FOPT-SLNs after the pretreatment
was calculated to be 38.09 mg (78.09 mg–40.00 mg), which was
smaller than that of the pretreatment A (86.55 mg) but was similar
Figure 3. The in-vitro model in which Caco-2
cells were subjected to pretreatments A, B
and C for 24 h and pretreatments D and E for
1 h of cell incubation, respectively, and then
followed with treatment of FOPT-SLNs,
FOPT-PM or carvedilol solution (containing
100 mg/mL carvedilol) for additional 24 h of
cell incubation for the uptake mechanism
elucidation of FOPT-SLNs (data represent
mean ± standard deviation, n ¼ 4).
FOPT-SLNs FOPT-PM Carvedilol solution
0
20
40
60
80
100
WIthout any pretreatment
Pretreatment A:Growth media
Pretreatment B:Blank SLNs
Pretreatment C:Blank and FOPT-SLNs
Pretreatment D:Sodium azide
Pretreatment E:Sucrose
Table 1. Comparison of carvedilol observed in Caco-2 cells which were
subjected to pretreatments A, B and C for 24 h and pretreatments D and E
for 1 h of cell incubation, respectively, and then followed with treatment
of formulation (FOPT-SLNs, FOPT-PM or carvedilol solution) containing
100 mg/mL of carvedilol, for additional 24 h of cell incubation (data
represent mean ± standard deviation, n ¼ 4).
Carvedilol amount obtained in
Caco-2 cells (mg)
Cells pretreatment FOPT-SLNs FOPT-PM
Carvedilol
solution
Without any treatment 76.12 ± 12.37 19.23 ± 4.99 22.58 ± 0.52
Pretreatment A: Growth
media
86.55 ± 0.87 41.20 ± 4.23 30.00 ± 4.32
Pretreatment B: Blank
SLNsa
41.50 ± 2.30 39.20 ± 1.22 31.20 ± 3.31
Pretreatment C: Blank
and FOPT-SLNsb
78.09 ± 6.14 62.79 ± 5.54 59.06 ± 4.75
Pretreatment D: Sodium
azidec
27.61 ± 4.21 25.04 ± 3.43 21.89 ± 3.25
Pretreatment E: Sucrosed
28.69 ± 1.53 18.01 ± 3.75 23.16 ± 3.73
a
Caco-2 cells were pretreated blank SLNs for carrier effect.
b
Blank SLNs together with FOPT-SLNs for effect of carvedilol.
c
Sodium azide as an endocytosis inhibitor.
d
0.45 M sucrose as an inhibitor for clathrin-mediated endocytosis.
Forpersonaluseonly.

to that of the pretreatment B (41.05 mg). This similarity in
carvedilol uptaken from FOPT-SLNs coupled with pretreatment C
or pretreatment B suggests that the number of transporters
occupied was similar between pretreatment C and pretreatment B
despite their difference in terms of carvedilol loading on SLNs.
This finding confirms the crucial role of SLNs as carriers for the
carvedilol absorption. In contrast, with the same analysis
approach, since the carvedilol uptaken after the pretreatment C
was 40.00 mg, the amount of carvedilol absorbed after the addition
of FOPT-PM or carvedilol solution was 22.79 mg (62.79 mg–
40.00 mg) and 19.06 mg (59.06 mg–40.00 mg), respectively. This
obtained carvedilol absorption was decreased as compared to their
respective pretreatments A and B. The possible reason could be
that the already absorbed carvedilol during pretreatment period
may have resulted in the decreased concentration gradient.
Therefore, the major mechanism of carvedilol absorption from
FOPT-PM and carvedilol solution, respectively, coupled with the
pretreatments B or C would be passive transport of free
carvedilol. This finding further confirms that the amount of
carvedilol absorbed was dominated from the cellular uptake of
SLNs by Caco-2 cells.
In order to further confirm the cellular uptake mechanism of
carvedilol from FOPT-SLNs, specific active absorption inhibitors
such as sodium azide (0.1% w/v) and sucrose (0.45 M) were used.
Since cells were incubated only for 1 h with pretreatments D and
E, the data were compared to that obtained without any
pretreatment. When the endocytosis process was hampered by
sodium azide (pretreatment D), the amount of carvedilol uptaken
from FOPT-SLNs was reduced drastically from 76.12 mg to
27.61 mg. However, the reduction was not significant as compared
to that with any pretreatment from FOPT-PM (25.04 mg versus
19.23 mg) and carvedilol solution (21.89 mg versus 22.58 mg),
respectively. The reduction of carvedilol amount from FOPT-
SLNs therefore indicates endocytosis as the major mechanism
for the cellular uptake of FOPT-SLNs by Caco-2 cells.
Furthermore, sucrose (pretreatment E), a specific inhibitor of
clathrin-dependent endocytosis, significantly affected the FOPT-
SLNs uptake. The amount of carvedilol absorbed reduced from
76.12 mg to 28.69 mg with sucrose without any inhibitors.
Therefore, it can be concluded that the main absorption mech-
anism of carvedilol-loaded SLNs is endocytosis and specifically,
clathrin-mediated endocytosis. In contrast, after pretreatment E,
the amount of carvedilol obtained in Caco-2 cells for FOPT-PM
and carvedilol solution remained relatively similar to that of
without any pretreatment (18.01 mg versus 19.23 mg and 23.16 mg
versus 22.58 mg, respectively).
In summary, for FOPT-SLN, the amount of carvedilol uptaken
reduced significantly when the cells were pretreated with pretreat-
ment C (blank SLNs and FOPT-SLNs) as compared to pretreat-
ment A. The reduction as compared to without any pretreatment
was also observed when cells were pretreated with pretreatment D
(0.1%w/v sodium azide) and pretreatment E (0.45 M sucrose).
Thus, reduction in carvedilol absorption due to already occupied
transporters or inhibition of transporters indicates the carrier-
mediated transport mechanism of FOPT-SLNs by Caco-2 cells.
However, in similar comparison, the reduction in carvedilol
absorption was not observed from FOPT-PM and carvedilol
solution. In other words, upon addition of SLNs or transporter
inhibitors did not affect the amount of carvedilol uptaken by Caco-
2 cells. Therefore, the passive absorption mechanism of carvedilol
from drug solution and FOPT-PM was concluded.
Absorption mechanism of optimized carvedilol-loaded SLNs
Cell monolayer integrity and development of tight
junctions. TEER values greater than 400 V were used for
absorption mechanism study. At the end of each experiment,
transport of luciferase yellow was found to be less than 1% in all
the cases and confirm that the monolayer integrity of Caco-2 cells
was maintained over the course of each experiment.
Absorption mechanism: passive absorption versus cellular
uptake. According to the design of TranswellÕ
permeable
supports, it is expected that drug permeated by passive absorption
through transcellular or paracellular mechanisms can be observed
in the basolateral compartment and drug absorbed through
carrier-mediated transport mechanism may remain within the
cells over the filter support. When cells were incubated with
FOPT-SLNs, majority of the carvedilol (60.57 mg) was found in
the Caco-2 cells and about 23.65 mg of carvedilol permeated to the
basolateral compartment (Figure 4). The amount of carvedilol
absorbed in Caco-2 cells can be attributed to the carrier-mediated
transport mechanism. On the other hand, for carvedilol solution,
about 22.24 mg of carvedilol was found in Caco-2 cells and
majority of the carvedilol (52.66 mg) was obtained in the
basolateral compartment. From the cellular uptake study
Figure 4. Comparison of carrier-mediated
absorption mechanism of FOPT-SLNs and
passive absorption mechanism of FOPT-PM
and carvedilol solution (containing
100 mg/mL carvedilol) using Transwell
permeable supports for Caco-2 cells
following 24 h of cell incubation (data
represent mean ± standard deviation, n ¼ 4).
FOPT-SLNs FOPT-PM Carvedilol solution
Amountofcarvedilol(µg)
0
20
40
60
80
100
Transcellular absorption
Carrier mediated absorption
Passive transcellular absorption
Passive paracellular and transcellular absorpti
53.22
22.6723.65
76.12
60.57
19.23 22.58
22.24
52.66
Forpersonaluseonly.

described previously, passive absorption mechanism for FOPT-
PM and carvedilol solution was concluded. Thus, the carvedilol
obtained from Caco-2 cells can be attributed to the passive
transcellular absorption mechanism for FOPT-PM and carvedilol
solution. However, the carvedilol obtained in the basolateral
compartment may have been due to the combination of passive
transcellular and paracellular absorption mechanisms in all three
formulations studied. In addition, similar results were obtained for
carvedilol solution and FOPT-PM. Therefore, when carvedilol
was present in the physical mixture of COMP and P-188, it
behaved similar to the carvedilol solution. In contrast, the
obtained higher carvedilol amount in Caco-2 cells from FOPT-
SLNs as compared to that from FOPT-PM confirms the role
of SLNs as carrier for cellular uptake of carvedilol instead of
lipid (COMP) and surfactant (P-188) present in the physical
mixture.
In order to get the mass balance and ensure that the carvedilol
did not bind to the TranswellÕ
permeable supports used,
carvedilol in the apical compartment was measured at the end
of the experiments. The unabsorbed amounts remained in the
apical compartment were 7.65 mg, 19.05 mg and 21.24 mg for
FOPT-SLNs, FOPT-PM and carvedilol solution, respectively.
Therefore, approximately 91.87 mg (60.57 + 23.65 + 7.65 mg),
94.94 mg (22.67 + 53.22 + 19.05 mg) and 96.14 mg (22.24 +
52.66 + 21.24 mg) from the total of 100 mg were accounted for
from the FOPT-SLNs, FOPT-PM and carvedilol solution,
respectively.
For the differentiation of results obtained from the evaluation
of absorption mechanism of optimized carvedilol-loaded SLNs,
the amount of carvedilol obtained in Caco-2 cells during the
cellular uptake study (Figure 1c) and carvedilol obtained due to
passive absorption mechanism using TranswellÕ
permeable
supports were compared as shown in Figure 4. The amount of
carvedilol obtained in the Caco-2 cells during cellular uptake
study (76.12 mg, 19.23 mg, 22.58 mg for FOPT-SLNs, FOPT-PM
and carvedilol solution, respectively) can be attributed to the
transcellular absorption mechanism. In order to differentiate the
transcellular absorption further from active to that of passive
mechanism, study was performed using TranswellÕ
permeable
supports. Based upon the results obtained with pretreatments
(Table 1), the carvedilol absorbed in Caco-2 cells (60.57 mg) could
be attributed to the active absorption and endocytosis in case of
FOPT-SLN. However, since reduction in carvedilol absorption
was not observed in case of FOPT-PM and carvedilol solution,
carvedilol obtained in Caco-2 cells can be attributed to the passive
transcellular absorption mechanism. Moreover, the amount
of carvedilol obtained in the basolateral side for FOPT-SLNs,
FOPT-PM and carvedilol solution (23.65 mg, 53.22 mg, 52.66 mg,
respectively) could be due to the combination of passive
paracellular and transcellular absorption mechanisms. Thus, the
data obtained from both studies are in accordance with each other
and suggest the carrier-mediated mechanism for SLN and passive
absorption mechanism (transcellular and paracellular) for the drug
solution and FOPT-PM.
Conclusion
The higher amount of carvedilol absorbed by Caco-2 cells,
irrespective of carvedilol concentration and incubation time
studied was obtained from FOPT-SLNs as compared to that
from FOPT-PM and carvedilol solution. The data obtained from
the model using pretreatment design, endocytosis and specif-
ically, clathrin-mediated endocytosis, of FOPT-SLNs was
concluded. Studies using TranswellÕ
permeable supports for the
cells, not only the carrier-mediated mechanism for FOPT-SLNs
was further confirmed but also passive absorption mechanism
(transcellular and paracellular) for FOPT-PM and drug solution
were concluded.
Acknowledgements
The authors acknowledge St. John’s University for providing financial
assistance and research facilities to carry out this research.
Declaration of interest
The authors declare no conflict of interest (monetary or otherwise) in
conducting this research. The authors alone are responsible for the content
and writing of the article.
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