Caco-2 Cell Conditions Enabling Studies of Drug Absorption from Digestible Lipid-Based Formulations
Caco-2 Cell Conditions Enabling Studies of Drug Absorption from Digestible Lipid-Based Formulations
Janneke Keemink 0 1 2
Christel A. S. Bergström 0 1 2
0 ABBREVIATIONS FaSSIF Fasted state simulated intestinal fluid FFA Free fatty acid HBSS Hank's Balanced Salt Solution LBF Lipid-based formulation LC Long-chain LFCS Lipid formulation classification system MC Medium-chain P
1 Department of Pharmacy, Uppsala Biomedical Center, Uppsala University , P.O. Box 580, SE-751 23 Uppsala , Sweden
2 Christel A. S. Bergström
Purpose To identify conditions allowing the use of cell-based models for studies of drug absorption during in vitro lipolysis of lipid-based formulations (LBFs). Methods Caco-2 was selected as the cell-based model system. Monolayer integrity was evaluated by measuring mannitol permeability after incubating Caco-2 cells in the presence of components available during lipolysis. Pure excipients and formulations representing the lipid formulation classification system (LFCS) were evaluated before and after digestion. Porcine mucin was evaluated for its capacity to protect the cell monolayer. Results Most undigested formulations were compatible with the cells (II-LC, IIIB-LC, and IV) although some needed mucin to protect against damaging effects (II-MC, IIIB-MC, ILC, and IIIA-LC). The pancreatic extract commonly used in digestion studies was incompatible with the cells but the Caco2 monolayers could withstand immobilized recombinant lipase. Upon digestion, long chain formulations caused more damage to Caco-2 cells than their undigested counterparts whereas medium chain formulations showed better tolerability after digestion. Conclusions Most LBFs and components thereof (undigested and digested) are compatible with Caco-2 cells. Pancreatic enzyme is not tolerated by the cells but immobilized lipase can be used in combination with the cell monolayer. Mucin is beneficial for critical formulations and digestion products.
Caco-2 cells; digestion; intestinal absorption; lipid-based formulation
Drug dissolution in gastrointestinal fluids is crucial for drug
absorption. However, approximately 70% of new drug
candidates show insufficient solubility to allow intestinal absorption
). Therefore, formulation strategies have been developed to
improve bioavailability. Of these, lipid-based formulations
(LBFs) often provide a means to deliver highly lipophilic,
poorly water-soluble compounds at concentrations high enough to
support absorption. These formulations consist of various
mixtures of oils, surfactants, and co-solvents and are classified
according to their composition and physical characteristics in
the lipid formulation classification system (LFCS) (
Ultimately, LBFs are employed to keep the compounds in
solution during their transit in the gastrointestinal tract and
expose the absorptive site to drugs in a solubilized and/or
However, upon oral administration, many of the
formulation components undergo lipolysis which changes the
solvation capacity of the medium keeping the drugs in solution. To
simulate and study this digestion process in vivo, an in vitro
lipolysis model is commonly used (
). This model allows
estimation of (i) the extent of digestion, and (ii) the drug
distribution between the three phases present in the gastrointestinal
tract (i.e. oil, aqueous and solid/precipitated drug, phases).
Unfortunately, these studies do not quantitatively predict the
in vivo performance of the drugs. Rather, they provide rank
order correlations, and it has been speculated that this is due
to the absence of an absorptive sink (
). During in vitro
lipolysis experiments, the drug is not transported away from the
solution as would be the case if it was absorbed. This artifact
drives higher supersaturation levels leading to precipitation
in vitro that would not occur in vivo (7).
Previous work has addressed this issue. For example,
biopharmaceutical modeling has been used to predict intestinal
absorption using in vitro lipolysis data (
). Permeation studies
have also been performed on intestinal tissue of rats with
predigested LBFs (
). Both methods seem promising but do
not capture the dynamics of the in vivo processes. Recently,
Crum et al. suggested a new animal-based model coupling in
situ intestinal perfusion in rats to the in vitro digestion of LBFs
). This method provides real time observations, but is time
consuming and, since it is animal-based, is mainly suited for
mechanistic studies rather than routine screening.
The use of Caco-2 cells in-line with lipolysis would offer an
easier and faster approach than in situ animal studies. Caco-2
cells are a human colon carcinoma cell line considered the
gold standard for the assessment of oral drug absorption
). Differentiated Caco-2 cells resemble the
epithelium of the human intestine and allow the prediction of drug
transport mediated by different pathways, e.g., passive and
active transport via the para- and transcellular routes (
However, digestion media used in lipolysis experiments have
been shown to damage Caco-2 cells (
). The aim of this
study was therefore to evaluate compatibility between Caco-2
cells and individual components present during in vitro
digestions to identify conditions under which Caco-2 cells can be
used in a new in vitro model that simultaneously investigates
digestion and absorption of compounds present in LBFs. A
protective layer of mucin was used to increase biorelevance
and generate Caco-2 compatibility.
MATERIALS AND METHODS
All culture media and supplements were purchased from
Invitrogen AB (Sweden). [14C]-mannitol was purchased from
PerkinElmer Sverige AB (Sweden); Novozym® 435
(immobilized lipase) was obtained from Strem chemicals
(France); and fasted state simulated intestinal fluid (FaSSIF)
powder was obtained from biorelevant.com (UK).
Trismaleate, CaCl2.2H2O, NaCl, NaOH, oleic acid, caprylic
acid, mucin from porcine stomach type III, Soybean oil,
Cremophor EL, Tween 85, PEG400, Carbitol, and porcine
pancreatin extract (8× USP specifications activity) were
purchased from Sigma-Aldrich (USA). Maisine 35–1 was a
kind gift from Gattefossé (France), and Captex 355 and
Capmul MCM were kind gifts from Abitec (USA). Excipient
details can be found in Table I.
Nine formulations were chosen to represent the four LFCS
); these contained long-chain (LC) or
mediumchain (MC) glycerides, surfactants, and co-solvents
(Table II). The LBFs resemble formulations that were
previously used to develop a standardized in vitro lipolysis
), and were herein selected to allow future
comparisons between this standardized method and a potential
digestion-absorption method to be developed based on the
results obtained in the current study. Formulations were
prepared as described previously (
). Briefly, excipients
were pre-heated (37°C, except for Maisine 35–1 70°C) and
weighed into glass vials according to predefined fractions (%
w/w; Table II). Subsequently, vials were sealed, vortex
mixed and placed on a shaker (300 rpm), at 37°C for 24 h.
Preparation of Pancreatic Extract
Pancreatic extract was prepared by mixing 0.6 g pancreatin
powder with 3 mL of lipolysis buffer containing 2 mM
TrisTable II Composition of Investigated LBFs, Representing All Classes of the
Lipid Formulations Classification System
maleate, 1.4 mM CaCl2, and 150 mM NaCl (pH 6.5)
followed by centrifugation for 15 min at 21,000 g and 5°C (
Subsequently, the extract was diluted with digestion medium
(lipolysis buffer supplemented with FaSSIF powder to obtain
sodium taurocholate concentrations of 3.0 mM and lecithin
concentrations of 0.75 mM) to yield a lipase activity of 900
USP units (USPU/mL).
In Vitro Lipolysis
In vitro lipolysis was carried out as described previously with
minor modifications (
). LBF was weighed directly into a
thermostat-jacketed glass vessel (Metrohm, Switzerland)
before digestion medium was added (final concentration of
LBF was 2.5% (w/v)). The formulation was dispersed for
10 min in the digestion medium using a propeller stirrer
(450 rpm). During the dispersion phase, the pH was manually
adjusted to pH 6.5 ± 0.05. The digestion was initiated by
addition of lipase. A pH-stat (Metrohm 907 Titrando) was used
to maintain a pH of 6.5 through titration with 0.2 M (LC- and
IV LBFs) or 0.6 M (MC-LBFs) NaOH. Samples were taken
after 60 min of digestion and treated with 5 μL/mL lipase
inhibitor (0.5 M 4-bromophenyl boronic acid in methanol)
to inhibit further lipolysis.
Digestion was performed with pancreatic extract and
immobilized lipase (Novozyme® 435) in order to (i) compare
the extent of digestion and (ii) select the concentration of
immobilized lipase for performing in vitro lipolysis assays.
Type IIIB-MC and IIIB-LC formulations were selected as
representatives of the MC- and LC-LBFs, respectively. Both
formulations were digested according to the standardized
protocol, using 900 USPU/mL of pancreatic extract, and with
different concentrations of immobilized lipase (125, 250 or
750 PLU/mL). The extent of digestion was determined by
plotting the free fatty acid (FFA) release against time. The
presence of pancreatic extract resulted in a more extensive
digestion than the presence of immobilized lipase.
Immobilized lipase concentrations above 125 PLU/mL
resulted in a limited increase in FFA liberation (Fig. S1).
However, higher concentrations (>125 PLU/mL) impeded
stirring and homogenous sampling. Therefore, a
concentration of 125 PLU/mL was selected to digest all LBFs for
Caco-2 cells, obtained from American Type Culture
Collection (Manassas, Virginia), were cultivated as described
previously in an atmosphere of 90% air and 10% CO2 (
Briefly, Caco-2 cells (passage 95 to 105) were seeded on
permeable polycarbonate filter supports (0.45 μm pore size,
12mm diameter; Transwell Costar, Sigma-Aldrich) at a density
of 44,000 cells/cm2 in Dulbecco’s modified Eagle’s medium
supplemented with 10% fetal calf serum, 1% minimum
essential medium nonessential amino acids, penicillin (100 U/mL),
and streptomycin (100 μg/mL). Monolayers were used for
experiments between day 21 and 26 after seeding.
Caco-2 cells were incubated with components present during
in vitro lipolysis. The conditions are presented in Table III. The
selected concentrations represent ‘worst-case’ scenarios; while
the in vivo situation is dynamic, these experiments were
performed under static conditions with a high concentration of
the test component for a relatively long time (2 h). Digestion
medium was used as a control in all studies.
TEER measurements were used to identify cell monolayers
that were suitable for transport studies. Before and after all
HBSS (pH 6.5)
Pancreatic enzyme (900 USPU/mL)
Immobilized enzyme (125 PLU/mL)
Simulated intestinal fluid containing bile salts and phospholipids
Highest concentration of a single excipient used during in vitro lipolysis (Table II)
Common concentration of single excipient used during in vitro lipolysis (Table II)
Common concentration of single excipient used during in vitro lipolysis (Table II)
Common LBF concentration used during in vitro lipolysis (
), formulations describe in Table II
Common concentration of pancreatic enzyme added to in vitro lipolysis experiments (
Concentration of immobilized enzyme required for digestion of LBF
Caprylic acid (87 mM) Highest concentration of MC FFA released in previous digestions (
Oleic acid (37.5 mM) Highest concentration of LC FFA released in previous digestions (
LBF digested with immobilized enzyme Common concentration of LBF and time span of in vitro lipolysis experiments digested with an enzyme concentration
for 60 min compatible with Caco-2 cells
permeability experiments, cells were washed with pre-warmed
(37°C) Hank’s balanced salt solution (HBSS; 7.4) and
equilibrated with HBSS for 15 min. Subsequently, the confluence
and integrity of the cell monolayers were assessed by
measuring TEER. Only monolayers with initial TEER values greater
than 250 Ω.cm2 were used for compatibility studies.
In all the transport experiments, 600 μL samples were
removed from the basolateral chamber after 30, 60 and 120 min,
and replaced with fresh HBSS. The samples were analyzed in
a liquid scintillation counter (1900CA TriCarb; PerkinElmer
Life Sciences). The apparent permeability coefficient (Papp)
was calculated according to the following equation:
The hydrophilic paracellular marker mannitol was used as a
model compound to investigate the effect of the test
components on the integrity of the Caco-2 monolayers. All solutions
were pre-warmed to 37°C. After, equilibrating cells with
HBSS for 15 min, the buffer was removed and the filters with
the cell monolayers were transferred to wells containing
1.2 mL of fresh, pre-warmed HBSS (pH 7.4).
Transport studies were initiated by adding 400 μL of
digestion medium spiked with [14C] mannitol and components
present during in vitro lipolysis (Table III) to the apical
chamber. For components that were not compatible with the cells,
the impact of mucin as a protective barrier was evaluated;
mucin from porcine stomach type III (50 or 150 mg/mL)
was dissolved in digestion medium and 100–200 μL was
added to the monolayers. The monolayers were then
incubated at 37°C for 10 min before initiating the transport
experiment by adding the tests solutions containing [14C]-mannitol
(final volume 400 μL in the apical chamber).
P app ¼
A x C donor
where Q is the [14C] mannitol appearing in the acceptor
compartment as a function of time (t), A is the surface area of the Transwell
membrane (1.12 cm2), and Cdonor is the initial [14C] mannitol in
the donor compartment. Papp values below 0.5 × 10−6 cm/s were
defined as reflecting confluent monolayers, whereas values
between 0.5 and 1.0 × 10−6 cm/s or >1.0 × 10−6 cm/s reflected
intermediate and high incompatibility, respectively.
Progesterone was used to investigate the impact of mucin as a
diffusion barrier to lipophilic compounds. Similarly to
mannitol, transport studies with progesterone (Cdonor 25 μM) were
performed in the absence and presence of mucin. Samples
were analyzed using a HPLC (Agilent Technologies 1290
Infinity) with a Zorbrax Eclipse XDB-C18 column (4.6 ×
100 mm) (Agilent Technologies). The injection volume was
20 μL. The mobile phase consisted of acetonitrile:sodium
acetate buffer (pH 5) at 85:15 (v/v) and was used at an isocratic
flow rate of 1 mL/min. The retention time of progesterone
was 1.98 min.
Data are presented as mean values with standard deviation
(n = 3). Statistical analysis was performed using one-way
ANOVA followed by a Dunnett’s test. P-values of less than
0.05 were considered statistically significant.
The assessment of the digestion medium on the monolayer
showed that it was compatible with the Caco-2 model as it
showed sufficiently low Papp values for mannitol (<0.5 ×
10−6 cm/s). In addition, Papp values were similar to values
obtained during incubation with HBSS (pH 6.5; Fig. S2)
during an incubation of 2 h. The application of a protective
mucin layer, either in a low or high concentration, did not
considerably affect the permeability of mannitol (a hydrophilic
model compound) or progesterone (a lipophilic model
compound), showing that mucin could be used as protective
barrier without compromising permeation (Fig. S3).
Caco-2 cells were exposed to the single excipients used in the
LBFs for 2 h (Fig. 1). Triglycerides were compatible with
Caco-2 cells at all concentrations tested. The mixed glycerides
showed a concentration-dependent incompatibility. Maisine
35–1 (mono-, di- and tri- LC glycerides) damaged the
monolayer at a concentration of 1.25% (w/v) and Capmul MCM
(mono-, di- and tri- MC glycerides) was incompatible already
at low concentrations ≥ 0.625% (w/v). Cremophor EL and
Carbitol showed intermediate tolerability at all concentrations
whereas Tween 85 and PEG400 were compatible (Fig. 1).
Type II-LC and IV formulations were tolerated by the cells
upon immediate exposure in relevant concentrations of 2.5%
(w/v), i.e., no mucin was required to protect the monolayers
(Fig. 2). For type IIIB-LC LBF Papp values indicated
intermediate tolerability. The cell monolayer integrity was maintained
by adding a low concentration of mucin (100 μL of 50 mg/
mL) together with the type I-LC formulation whereas a higher
concentration (200 μL of 150 mg/mL) was required to protect
against the IIIA-LC. MC-LBFs were generally not compatible
with Caco-2 monolayers (Fig. 2). However, the high mucin
concentration (200 μL of 150 mg/mL) protected the
monolayers against the damaging effects of the II-MC and IIIB-MC
The standardized in vitro method to assess lipolysis of LBFs,
suggested by the LFCS consortium, uses an extract from
porcine pancreas (
). However, this concentration of pancreatic
extract (Table III) disrupted Caco-2 monolayers, even in the
Fig. 2 Effect of undigested LBFs on apical to basolateral transport of mannitol
across Caco-2 monolayers. Bars represent average Papp values ± SD (n = 3).
The black, dark gray, and light gray bars indicate the presence of no mucin,
100 μL of 50 mg/mL mucin, and 200 μL of 150 mg/mL mucin, respectively.
Red, yellow and green regions represent conditions that were not,
intermediately and well tolerated. The control was digestion medium.
presence of mucin (Fig. 3). Therefore, the use of recombinant
lipase immobilized on polymeric beads (Novozym® 435) was
evaluated for the digestion of LBFs. A considerable release of
FFA was observed during the digestion of all LBFs with 125
PLU/mL immobilized lipase (Fig. 4). Immediate exposure to
this concentration of the enzyme was tolerated well by the cells
Caco-2 cells were exposed to caprylic and oleic acid
representing digestion products released from MC- and
LC-formulations, respectively. Mucin protected the cells
against caprylic acid in a concentration-dependent
manner. The highest concentration of mucin was required to
shield the cells completely from its damaging effects. For
the oleic acid, the lower concentration of mucin was
already sufficient to protect monolayers against disruptive
effects (Fig. 5).
Digested Lipid-Based Formulations
LBFs were digested for 60 min to obtain media containing the
excipients, immobilized lipase, and digestion products (Fig. 4).
The MC formulations proved to be less damaging upon
digestion than when administered in their undigested form (Figs.
2 and 6). Only intermediate monolayer damage was observed
for the digested type II-MC and the IIIB-MC formulations. In
contrast, all LC formulations and the type IV formulation
exerted disruptive effects upon digestion whereas damaging
effects were only observed for highly concentrated, undigested
I-LC and IIIA-LC formulations (Figs. 2 and 6). Only the high
mucin concentration (200 μL of 150 mg/mL) was evaluated
for protection of digested LBFs since the low concentration
(100 μL of 50 mg/mL) has shown incomplete protection in
previous experiments with single components of LBFs. In the
presence of high mucin concentrations, all digested
formulations were compatible with the Caco-2 cells (Fig. 6).
Recent studies have identified the absence of an absorption
compartment in the current in vitro lipolysis setup as a major
reason for the poor prediction of the in vivo performance of
). Therefore, the present study evaluated the
compatibility between Caco-2 monolayers—the gold
standard for intestinal absorption in vitro studies (
components present during digestion studies. A number of
excipients, LBFs, and digestion products, were shown to be
tolerated by the cells at concentration levels relevant for
evaluation of LBF performance. The pancreatic enzyme
commonly used in standard lipolysis was found to be
incompatible under all conditions tested, but immobilized
lipase was endured by the Caco-2 monolayers at a
concentration able to digest LBFs representing all classes of the
LFCS (Figs. 3 and 4, respectively). Of particular interest is
the fact that LBF concentrations used in digestion
experiments could be applied without the need for dilution. Cells
tolerated these concentrations during 2 h of incubation,
which is significantly longer than a typical lipolysis
experiment (30–60 min) (
). This opens up the possibility of
coupling the lipolysis setup to an absorption chamber
consisting of Caco-2 cells to perform lipolysis and
absorption studies simultaneously.
Papp and TEER measurements have been used extensively
to evaluate monolayer integrity of Caco-2 cells (
11, 12, 19,20
These parameters correlated well in this study (i.e. a decrease
in TEER corresponded to an increase in Papp of mannitol, Fig.
S3 and S4). However, TEER measurements must be
interpreted with caution. Variability in TEER can be
introduced by fluctuations in temperature, medium formulation,
passage number, and even the positioning of the electrodes.
The most widely used system for measuring TEER consists
of a pair of electrodes (known as chopsticks) and the
electrodes only determine TEER locally (21). We therefore use
the TEER measurements to identify cell monolayers that
are suitable for transport studies, whereas we use a
permeation marker such as mannitol or lucifer yellow to study
monolayer integrity (
). When incubations resulted in
mannitol Papp values below 0.5 × 10−6 cm/s, components
were considered compatible with Caco-2 cells. However,
some conditions resulted in Papp values that were below
values obtained in the control condition (only digestion
medium). A possible explanation is that some excipients
Fig. 4 Apparent titration of FFA
release during in vitro lipolysis (n =
1). (a) MC LBFs (b) LC-LBFs and
including lipids can induce structural changes in the mucin,
possibly resulting in decreased permeation (
).Vors et al.
previously performed lipolysis experiments on emulsions
followed by incubations on differentiated Caco-2 cells
). However, to maintain the monolayer integrity they
had to significantly dilute the digestion medium (1:20). Bu
et al. exposed cells to 0.5% (v/v) LBFs and concluded that
compatibility was influenced by the maturity of
monolayers; differentiation was required for 21 days to optimize
the survival rate (
). Both studies used LBF
concentrations that were much lower than concentrations used in
in vitro lipolysis experiments. Recently, Sadhukha et al.
demonstrated the compatibility of Caco-2 cells with a selection
of undigested LC formulations at concentrations relevant
for in vitro lipolysis experiments. In agreement with the
findings in the current study, they found that digested LC
formulations showed significantly more incompatibility, i.e.,
lower TEER- and higher Papp values, than the
corresponding undigested LBFs (15).
Cremophor EL and Tween 85 have previously been
applied on differentiated Caco-2 cells as single excipients. In
those experiments, a 2 h incubation of Cremophor EL
(0.5% (v/v)) resulted in a slight decrease in cell viability
according to a MTT test, but viability was still around 80% (
Acceptable tolerance upon exposure to the Cremophor EL
was also observed in our study, even after adding relatively
high concentrations (0.625%–1.25% (w/v), Fig. 1). Up to 5%
(w/v) Tween 85 was previously shown to be tolerated well by
Caco-2 cells (
). This corroborates our observations as no
damaging effects were observed at concentrations between
0.125 and 1.25% (w/v) (Fig. 1).
We demonstrated that the compatibility of LBFs and
Caco-2 cells was highly influenced by formulation
composition. A connection was observed between single excipient
incompatibility and the effect of undigested LBFs on the cells.
Undigested LBFs containing Capmul MCM, the excipient
that caused severe integrity loss of the monolayers, were
clearly more disruptive to the cells than other formulations
(Table II and Fig. 2). Moreover, cells exposed to the IIIB-MC
formulation, which contained the lowest fraction of Capmul
MCM, performed better than the cells exposed to other
MCLBFs containing higher concentrations of this excipient. This
is in agreement with previous data from Bu et al. who detected
a decrease in toxicity when formulations contained higher
amounts of Captex 355 in favor of Capmul MCM. In
addition, they observed that formulations consisting of mixtures of
mono-, di-, triglycerides and surfactants were better tolerated
by monolayers than the single lipids or surfactants (
A clear connection between Caco-2 compatibility and the
digestion process was detected. Overall, undigested MC
formulations appeared to be more damaging to the cells than
their digested counterparts (Figs. 2 and 6, respectively).
Given that the critical micelle concentration (CMC) decreases
with increasing chain length of the hydrophobic tails (
free concentrations of glycerides in dispersions of undigested
MC-LBFs is relatively high. These free MC glycerides may
insert into, and disrupt, membranes leading to lipid-induced
rupture of the cells (
). Upon digestion, cells will be
exposed to MC digestion products (MC FFA). Absorption
enhancing effects of MC FFAs have been shown to occur in the
vicinity of the CMC (19). Therefore, caprylic acid exhibited
limited effects on membrane integrity when a much lower
concentration (87 mM) than the CMC (225 mM) was applied
(Fig. 6) (
).This may also provide an explanation for the
mild membrane interactions observed for the digested
MCLBFs (Fig. 6), which when digested contained between 8.8 and
24.0 mM of ionizable FFA. On the contrary, components
present in undigested dispersed LC-LBF will form micelles
at relatively low concentrations resulting in limited exposure
of glycerides to the Caco-2 monolayers. However, oleic acid
(37.5 mM) as well as digested LC-LBFs severely damaged
Caco-2 monolayers (Figs. 5 and 6, respectively). Upon
digestion, released LC FFAs will be incorporated into the cell
membrane and destabilize its lamellar phase through decreased
transition temperature and the formation of inverted
hexagonal phases (26). Oleic acid in particular, has been shown to
affect membranes at mole fractions down to 0.025. Assuming
(i) that the surface area occupied per phospholipid is about
64 Å2 (value for phosphatidylcholine) and (ii) the entire apical
surface of the culture insert is covered by the monolayer (i.e.
no paracellular transport route), only 0.005% of the LC FFA
explored here would have to be inserted to exert a harmful
Mucus, secreted by goblet cells, provides a protective
barrier towards harmful endogenous and foreign substances
in vivo. However, as Caco-2 cells originate from a colon cancer
cell line, they do not always replicate the physiology of in vivo
tissue and, for example, lack a mucus layer (
). Caco-2 cells
could therefore be co-cultured with human mucus-producing
cells to establish a mucus layer containing glycoproteins that
mimic the protective barrier in the human gut (
these co-cultures are relatively difficult to maintain and mucus
layers can be easily removed during washing steps. Therefore,
monolayers were in this study shielded against harmful effects
by applying a protective porcine derived mucin layer on top of
the cell barrier. This strategy has been used previously by
Wuyts et al. who applied a barrier of mucin onto Caco-2 cells
to protect them against fasted state human intestinal fluids
). The concentration they used in their study was
insufficient to completely protect the cells against some of the
damaging effects, and so a larger amount of mucin was applied in
this study (200 μL of 150 mg/mL mucin). Mucin has
previously been found to significantly obstruct the absorption of
lipophilic drugs in co-cultures of Caco-2 cells with
mucinproducing HT29-MTX cells (
). Indeed, Papp of the
lipophilic model compound progesterone was reduced in the presence
of the low concentration of mucin but no statistical significant
difference in Papp value was observed between the low and
high mucin concentration. In both conditions, the transport
was still considerable and the diffusion through the mucin
layer was high (Fig. S5). Papp values obtained after the
addition of mucin onto the Caco-2 cells while studying permeation
of a selection of compounds (including lipophilic compounds)
strongly correlated with Papp values obtained in the absence of
mucin and with fractions absorbed in humans (
we do not expect the mucin to limit the usefulness of Caco-2
cells in combination with mucin in an absorption chamber for
drug permeation studies.
Despite the protective barrier, Caco-2 cells were not able to
tolerate the pancreatic extract. An alternative could therefore
be to use artificial membranes to evaluate absorption of
compounds during digestion of LBFs with the extract. For
example, the biomimetic barrier Permeapad has been shown to
maintain its permeation properties during the digestion of a
type IIIA-LC LBF with the extract (
). However, we initially
targeted the development of a cell-based model since Caco-2
cells enable both active and passive transport mechanisms to
be explored (
). Although compounds formulated in LBFs
typically cross the intestinal barrier through passive diffusion,
other compounds, including bile salts and FFA, are substrates
of transporters (
). Transporter-mediated uptake of these
components changes the composition of the digestion medium
and consequently its solvation capacity. It is therefore to be
expected that cell-based systems more accurately capture the
dynamics of the solubilizing intestinal lipoidal structures than
e.g., artificial membranes.
In order to use Caco-2 cells during digestion studies, we
found that the lipids need to be digested with immobilized
lipase (Novozym® 435) instead of pancreatic extract.
Novozym® 435 is a recombinant lipase B originating from
Candida Antarctica that is immobilized on a macroporous
polyacrylate resin. A modified in vitro digestion model
employing this enzyme has been developed previously,
showing that a similar extent of digestion could be obtained
for the digestion of Captex 355 and Tricaprylin provided
that the digestion lasts long enough. The activity of the
immobilized lipase was shown to be independent of buffer
or pH, enabling its use in protocols mimicking lipid
digestion in different segments of the gastrointestinal tract (
Other advantages are that immobilized lipase (i) enables
easy separation from the digestion medium (ii) is reusable
and, (iii) shows increased thermal stability. The activity of
immobilized lipase is however slightly different to that of
pancreatic extract (Fig. S1). This difference may be due to
the specificity and affinity of the enzymes. The immobilized
lipase consists of only one kind of lipase, whereas pancreatic
extract contains a mixture of enzymes including
phospholipase A2, colipase, and pancreatic lipase-related protein
). In addition, access of the active site of immobilized
lipase to triglycerides might be limited. Due to their low
solubility in the aqueous phase triglycerides will mainly
reside in the oil droplets and digestion needs to occur at the
droplet interface. As immobilized lipase is confined to
polymeric beads, it is likely that steric hindrance slows down
digestion by this enzyme while pancreatic lipase that is
dispersed freely in the digestion medium has easier access to
the droplet interface. In contrast to human and porcine
pancreatic lipase, the immobilized lipase does not display
interfacial activation; i.e. a conformational change in the
presence of a hydrophobic surface, resulting in exposure
of the active site to the solvent. However, the active site is
composed of the same catalytic triad consisting of serine,
aspartic acid and histidine (
). As pancreatic extract in a
concentration of 90 USPU/mL was shown to be
compatible with Caco-2 cells in the presence of high mucin
concentrations (Fig. S6A), the concomitant effects of 125 PLU/mL
immobilized lipase and 90 USPU/mL of pancreatic extract
on the digestion of IIIB-MC was evaluated (Fig. S6B).
Unfortunately, combinations of the enzymes were
incompatible with the cells (Fig. S6A) and had only limited effects
on the extent of digestion (Fig. S6B).
Several studies have been undertaken to increase the
physiological relevance of the in vitro lipolysis setup. For instance, as
lipolysis is initiated in the stomach, a gastric lipolysis phase has
been added (
). To capture the dynamics of the intestinal
processes occurring after administration of a lipid-based drug
delivery system an absorption sink needs to be added. As
pancreatic extract is not compatible with Caco-2 cells (Fig. 3) we
suggest to use immobilized lipase in the development of such a
digestion model including absorption.
In the present study, pre- and post-digestion conditions
tolerated by Caco-2 cells were identified in spite of the studies
being designed as a ‘worst-case’ scenario with high
concentration of natural and digested excipients being in contact with
the cells for as long as 2 h. Hence, Caco-2 cell monolayers
seem as a promising approach to study absorption
simultaneously with lipolysis during performance testing of LBFs.
We here demonstrated that Caco-2 monolayers are a
promising tool in the development of a new method to couple in vitro
lipolysis to an absorption compartment. The pancreatic
extract typically used during lipolysis was damaging for the
Caco-2 cell monolayers and so an immobilized lipase was used
instead; it successfully digested the LBFs and was tolerated by
the cell monolayers. Caco-2 cells, in combination with a
protective mucin barrier, withstood all the undigested and
digested LBFs explored herein except the undigested type
IMC and IIIA-MC formulations. These studies were
performed in a ‘worst case’ scenario where the Caco-2 cells were
exposed to high concentrations of all components for two
hours. Digestion studies typically run for 30–60 min during
which the condition is dynamic and we therefore expect that
the Caco-2 cell model will perform even better under such
ACKNOWLEDGMENTS AND DISCLOSURES
This work has received support from the European Research
Council Grant 638965 and the Swedish Research Council
Open Access This article is distributed under the terms of the
Creative Commons Attribution 4.0 International License
permits unrestricted use, distribution, and reproduction in any
medium, provided you give appropriate credit to the original
author(s) and the source, provide a link to the Creative
Commons license, and indicate if changes were made.
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