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Cell-based in vitro models for predicting drug permeability

Article in Expert Opinion on Drug Metabolism & Toxicology · March 2012

DOI: 10.1517/17425255.2012.673586 · Source: PubMed

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 Review

Cell-based in vitro models for

predicting drug permeability
Bruno Sarmento†, Fernanda Andrade, Sara Baptista da Silva,
1. Introduction Francisca Rodrigues, José das Neves & Domingos Ferreira
†
Department of Pharmaceutical Technology, LTF/CICF, Faculty of Pharmacy, University of Porto,
2. General drug absorption
Portugal
mechanisms
3. In vitro cell models Introduction: In vitro cell models have been used to predict drug permeation in
4. Conclusions early stages of drug development, since they represent an easy and reproducible
Expert Opin. Drug Metab. Toxicol. Downloaded from informahealthcare.com by University of Calgary on 05/24/12

method, allowing the tracking of drug absorption rate and mechanism, with an
5. Expert opinion
advantageous cost--benefit ratio. Such cell-based models are mainly composed
of immortalized cells with an intrinsic ability to grow in a monolayer when
seeded in permeable supports, maintaining their physiologic characteristics
regarding epithelium cell physiology and functionality.
Areas covered: This review summarizes the most important intestinal, pulmo-
nary, nasal, vaginal, rectal, ocular and skin cell-based in vitro models for pre-
dicting the permeability of drugs. Moreover, the similitude between in vitro
cell models and in vivo conditions are discussed, providing evidence that
each model may provisionally resemble different drug absorption route.
Expert opinion: Despite the widespread use of in vitro cell models for drug
permeability and absorption evaluation purposes, a detailed study on the
For personal use only.

properties of these models and their in vitro--in vivo correlation compared

with human data are required to further use in order to consider a future
drug discovery optimization and clinical development.

Keywords: cell models, in vitro models, in vitro--in vivo correlation,

intestinal mucosa, permeability

Expert Opin. Drug Metab. Toxicol. (2012) 8(5):607-621

1. Introduction

In vitro models that closely mimic conditions in the human mucosa yield absorption
data that can help selecting compounds to take forward into clinical development.
The popularity of these models stems from their potential for high throughput,
cost-effectiveness and adequate predictability of absorption potential in humans.
In some cases, the data are sufficiently predictive to support regulatory filing of a
new drug application. Cell culture models offer the advantage of a highly defined
tool, in which the parameters and conditions can be easily changed. In addition,
the use of human cell lines avoids the kind of problems that arise when using animal
tissue for in vitro experiments. Moreover, in vitro techniques for permeability assess-
ment are less laborious, less cost-intensive and offer benefits in terms of ethical
considerations as compared with in vivo animal studies [1].
For many reasons, but particularly because of financial and ethical considerations,
in vitro approaches have been pursued in this area over the last decades. However,
one universal issue with all in vitro systems is that the effect of physiological factors
like diseases, age, hepatic or renal dysfunctions or environment features cannot be
incorporated in the results interpretation. Also, the use of in vitro methods inherently
creates questions about the validity of extrapolating to the in vivo situation [2].
In vitro--in vivo correlation must allow the prediction of the in vivo pharmacokinetic
of a drug based on the in vitro drug permeation profiles. To develop an effective
correlation, the physicochemical and biopharmaceutical properties of the drug as

10.1517/17425255.2012.673586 © 2012 Informa UK, Ltd. ISSN 1742-5255 607

All rights reserved: reproduction in whole or in part not permitted
 B. Sarmento et al.
Article highlights.
. The mechanisms and factors affecting the absorption of
drugs are of great importance in the development
of medicines.
. In vitro models can be used as alternative methodologies
to ex vivo or in vivo models to evaluate the ability of
new drugs to permeate human mucosas.
. Intestinal, pulmonary, nasal, vaginal, rectal, ocular and
skin are among the absorption routes that have been
simulated in cell-based in vitro models, with several
experiments correlating their permeation rates
with in vivo human absorption performance for
Expert Opin. Drug Metab. Toxicol. Downloaded from informahealthcare.com by University of Calgary on 05/24/12
well-known drugs.
. Co-culture models comprising different cells present in
the mucosal epithelia have been proposed to more
closely representing the heterogeneity of human organs
responsible for the absorption of drugs.
.
Particular attention has been focused on the
development of in vitro intestinal models, mostly
because this represents the preferential route of
administration of drugs.
. Skin cell models have also been developed in ready-
to-use commercial kits, with the advantage of requiring
minor conditions of cell maintenance as compared with
other mucosal in vitro cell models.
. The increase in social and scientific concerns about the
animal well care, the needs for rapid and consistent
For personal use only.
bioavailability models by the pharmaceutical companies
and the demands for economical and low time
consumption methodologies give reason for developing
in vitro models with appropriate correlation between
results of drug absorption in vivo and in vitro.
This box summarizes key points contained in the article.
well as the physiological environment in the body must be taken
into consideration. Key factors include drug solubility, acid dis-
sociation constant (pKa), drug permeability, octanol--water par-
tition coefficient (logP) and pH of the environment [3].
The successful application of in vitro models to predict
drug absorption depends on how closely the in vitro model
mimics the characteristics of the in vivo barrier. Although,
it is very difficult to develop a single in vitro system that
can simulate the human in vivo setting, various in vitro sys-
tems are used routinely as decision-making tools in early
drug discovery [4] and the combination of more than one
type of cells (co-cultures) are now a common practice. Epi-
thelial cell monolayers possess the valuable property of grow-
ing on semi-permeable membranes in well-established
models and have the potential to illustrate much of the com-
plexity of the processes involved in drug absorption, while
allowing for routine procedures, which may at least partially
reflect the need of timely information [5]. The complexity
should reflect the variety of membrane transport systems,
metabolism pathways, a polarized cell layer and apical as
well as basolateral compartments [6] and the composition
of the aqueous solution on each side of the cell membrane.
608 Expert Opin. Drug Metab. Toxicol. (2012) 8(5)
Due to poor physical--hemical properties of most drug sub-
stances regarding solubility, the ultimate aim during the drug
development process is to increase epithelial membrane per-
meability, and to achieve significant absorption. Also, regard-
less of the route of administration, epithelial permeation is
required for a drug molecule to reach the systemic circulation.
Even after intravenous, intramuscular or any other parenteral
route of administration, the drug can permeate in the extracel-
lular space via paracellular route, but those drugs targeting
within cell membranes must cross the cell membrane barriers
in order to reach its biological target. Therefore, in vitro per-
meability studies also play a key role in determining a formu-
lation strategy in order to assist solubility, dissolution and
stability [7].
In this paper, we go through the most important
intestinal, pulmonary, nasal, vaginal, rectal, ocular and skin
cell-based in vitro models for predicting the absorp-
tion of drugs. Also, we provide a critical analysis on the
rationale behind the development of these models and,
when the pertinent animal/human data are available, their
in vitro--in vivo correlation.
2. General drug absorption mechanisms
Drug absorption across biological membranes is a complex
multi-pathway process. Drugs can be absorbed passively
through the cell membrane of epithelial cells (transcellular
route) or via the junctions between the cells (paracellular
route) or actively through carrier-mediation via an active (or
secondary active process) or by facilitated diffusion. Various
efflux transporters, such as P-glycoprotein (P-gp), are also
functional, which can limit the absorption [8]. Epithelial
enzymes can be involved in metabolizing drugs to alternate
moieties, which may further be absorbed. Finally, receptor-
mediated endocytosis can also play a role. Although the
review of the mechanisms of drug absorption is out of the
scope of the present review, some general aspects involving
the passage of drugs through epithelia toward blood circula-
tion may help understanding the use of cell models for screen-
ing and preclinical purposes. And because of the multivariate
processes involved in absorption of drugs, it is often difficult
to use a single model to accurately predict in vivo permeability
characteristics [9].
Among the several routes the molecules follow to cross bio-
logical membranes, diffusion through lipid- and carrier-
mediated transport are particularly important regarding the
pharmacokinetic mechanisms [9]. The preferred pathway for
absorption or transport of specific drugs depends on their
physicochemical characteristics as well as the biological mem-
brane features. In general, lipophilic drugs cross the biological
membrane by the transcellular pathway while hydrophilic
drugs tend to cross the membrane paracellularly, although
hydrophilic drugs also cross via transporters.
Transport across epithelia via the transcellular route
comprises the majority of the drugs that passively cross any

epithelium, although the exact mechanism that renders epi-
thelial membranes permeable is most of the times unknown.
Moderate and highly lipophilic molecules are those that pref-
erentially diffuse by the transcellular pathways. In the case of
these last, it is noteworthy that there are molecules like surfac-
tants, medium chain triglycerides and fatty acids which may
enhance permeability across biological membranes.
Transport across epithelia via the paracellular route is addi-
tionally reduced due to the presence of tight junctions
between adjacent cells. Therefore, this route is generally
restricted to small hydrophilic molecules. Also in this absorp-
tion pathway, molecules such as junctional modulators may
Expert Opin. Drug Metab. Toxicol. Downloaded from informahealthcare.com by University of Calgary on 05/24/12
enhance the permeability of the paracellular route and offer
possibilities as absorption enhancers.
A significant contribution of passive mechanisms to overall
absorption requires appropriate molecular size, charge, lipophi-
licity and hydrogen bond potential, that is, appropriate physio-
chemical properties to allow passage through both the apical
and basolateral plasma membranes of the epithelial cells.
In contrast to the properties relevant for passive absorption,
compounds that use specific transporter systems tend to be
hydrophilic and are recognized as substrates along with
nutrients and micronutrients [10]. Active transporters may
generate gradients across barriers with various energy-
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coupling mechanisms. Secondary active transport is indirectly
coupled to the energy of ATP, perhaps through the Na+ elec-
trochemical gradient generated by the ubiquitous Na+/K+-
ATPase, and proton gradient, contrasting with the primary
active transport which directly utilizes ATP during the trans-
port cycle. Compounds that cross the epithelia, including
those which are absorbed passively, may be recognized as sub-
strates for apical active efflux transporters. These efflux trans-
porters will limit drug absorption by transporting compounds
back into the apical epithelium side. Alternatively, com-
pounds may be metabolized following uptake into the epithe-
lial cells by a variety of enzymes, such as those of the
cytochrome P450 family (CYP), particularly CYP3A4/5 in
the intestine. Metabolism may be followed by transfer to the
circulation or excretion back through the epithelial surface
facing the lumen.
Thus, understanding and anticipating the mechanisms
and factors affecting the absorption of drugs are of utmost
importance in the development of medicines.
3. In vitro cell models
3.1 Intestinal models
Drug delivery through the oral route is considered as the pre-
ferred route of administration, mainly due to patient’s com-
pliance to therapeutics. It is user-friendly and reduces the
risk of infection, as well as the pain associated with other inva-
sive routes, and the need for administration by specialized
medical personnel. However, the transport of drugs via the
intestinal membrane is a complex and dynamic process that
includes the passage of compounds across several functional
Expert Opin. Drug Metab. Toxicol. (2012) 8(5)
Cell-based in vitro models for predicting drug permeability
pathways in parallel. There are various functional influx and
efflux mechanisms that define the permeability of com-
pounds. Moreover, several different pathways are available
via which molecules can travel from the lumen into the
systemic circulation.
The intestinal mucosa is characterized by the presence of
villi that constitute the anatomical and functional unit for
nutrient and drug absorption and provides a massive sur-
face area for absorption. The mucosa consists of the epithe-
lial layer, the lamina propria (collagen matrix containing
blood and lymphatic vessels) and the muscularis mucosa.
Thus, any drug entering the bloodstream has to go through
the epithelial layer, part of the lamina propria, and the wall
of the respective blood capillary. The epithelial layer is con-
sidered the limiting hurdle for intestinal drug permeation,
being thus the histological section with most interest for
in vitro simulation.
As far as primary cultures of enterocytes are concerned,
these cells are generally unable to form an organized epithelial
monolayer and therefore do not display an apical and basolat-
eral surface. Thus, various immortalized cell cultures have
been used to investigate transport of drugs through the intes-
tinal barrier [11]. Currently, there are a number of available cell
culture-based in vitro approaches to predict the intestinal
absorption of chemical entities under development (Table 1).
Caco-2 cell monolayers have been, by far, the most used
cell model to simulate the intestinal epithelium monolayer
and predict the flux drugs across human small intestinal tis-
sue [6,12]. This cell line derives from a human colorectal carci-
noma, and large information on intestinal drug absorption
has been obtained using this model. After reaching confluent,
Caco-2 cells differentiate both structurally and functionally
into cells resembling mature enterocytes, including the typical
membrane transporters and tight junctions between adjacent
cells as well P-gp, and have also been shown to express several
transport systems of different molecules [5]. Caco-2 cells grow
as a monolayer and differentiate on a semi-permeable mem-
brane. Thus, it is possible to separate the apical compartment
from the basolateral compartment, which corresponds to the
intestinal lumen side and the serosal side, respectively.
Comparison of drug transport in Caco-2 monolayers with
intestinal drug transport in vivo indicates that the monolayers
can be used to predict drug transport by different pathways
across the intestinal epithelium, in high correlation to the
in vivo situation obtained for drugs that are transported by
the passive transcellular route [13]. The passive paracellular
route is less permeable in the cell monolayers than in vivo,
but experimental data indicate that the selectivity of this
pathway is comparable with the in vivo situation [13].
One of the most disadvantageous drawbacks associated with
Caco-2 cells is that culture-related conditions influence the per-
formance of the cell model, in part due to the intrinsic hetero-
geneity of the parental cell line, leading to selection of
subpopulations of cells becoming prominent in the culture [14].
In addition, several clonal cell lines have been isolated from the
609
 B. Sarmento et al.
Table 1. Cell lines (and their co-culture) used for intestinal permeability assessment of drugs.
Cell line Origin
Caco-2 Human colorectal carcinoma cells
TC-7 Caco-2 subclone
HT29-MTX Human colorectal carcinoma cells
MDCK Dog kidney epithelial cells
IEC-18 Small intestinal crypt-derived rat cells
Expert Opin. Drug Metab. Toxicol. Downloaded from informahealthcare.com by University of Calgary on 05/24/12
Caco-2/HT29 Human colorectal carcinoma cells
co-culture
Caco-2/Raji B Human colorectal carcinoma cells/
co-culture lmphocytes
Caco-2/HT29/Raji Human colorectal carcinoma cells/
B co-culture lmphocytes
MDCK: Madin-Darby canine kidney.
For personal use only.
parental line, exhibiting in general a more homogeneous
expression of differentiation traits, while not always expressing
all characteristics of the parental line [14].
A subclone of Caco-2 cells, TC-7 cells, has also been used
for permeability screening [15], displaying higher enzyme con-
tent compared with native Caco-2 cells [16], like CYP3A, and
lower levels of P-gp. The TC-7 clone exhibited similar cell
morphology to Caco-2 cells, displaying the presence of
brush-border membrane and microvilli, and the formation
of tight junctions. The correlation of permeability values of
passively transcellularly absorbed drugs obtained in
TC-7 clone corresponded to that generated in parental
Caco-2 cells with respect to the extent of absorption in
humans [16]. However, this cells also have transporters and
some paracellular mechanisms.
Another interesting human adenocarcinoma cell line is the
HT29, in particular the HT29-MTX model, derived from the
parental cell line HT29 adapted to a medium containing
methotrexate in order to acquire the morphological and
mucin producing characteristics of goblet cells [17,18], thus
forming a native human mucus layer [19]. These enterocyte
cells may be useful tools to evaluate the mucoadhesion of car-
rier systems, once they can be grown into monolayers and
used for drug permeation studies [20], although its monocul-
ture would not reflect the physiologic proportion of goblet
cells in the human intestine, usually ranging from 10 to
25% of the endothelial cells [21]. HT29 cells have been mostly
used in co-culture cell models with Caco-2, as mentioned
below. The permeability rate at each cell model has been dem-
onstrated to be different for the same drug, depending on the
degree of tightness of the cells. For example, one can easily
610 Expert Opin. Drug Metab. Toxicol. (2012) 8(5)
Main characteristics Ref.
Polarized cells; produce P-gp and establish tight junctions [6,11-13]
in vitro; differentiate and express several relevant efflux
transporters
Similar to Caco-2 cells but with higher brush-border [15,16]
enzymatic content
Mucin-producing cells [17-20]
Kidney cells from dog, with properties similar to Caco-2 [25-27]
cells
Size-selective barrier for paracellularly transported [22,23]
compounds
Mimics the small intestinal epithelial layer containing [20,21] [27,28]
both mucus-producing (HT29) and the columnar absorptive
Caco-2 cells
Simulates the human follicle-associated epithelium; [31-33,36]
Useful for nanoparticle internalization studies through
M cells
Mimics different absorption pathways in the same model; [37-39]
useful for mucoadhesive nanoparticle internalization studies
through M cells and mucin-producing cells
understand that the degree of tight junctions between Caco-
2 cells will result in a lower permeation rate compared with
HT29 cells, without such cell interactions, prevailing
paracellular permeation.
IEC-18 cells, a small intestinal crypt-derived rat cell line,
are another epithelial cell type used in the study of the trans-
port of drugs. Monolayers of IEC-18 cells have been mainly
investigated for screening the passive transcellular and paracel-
lular transport of hydrophilic macromolecules [22,23]. These
cells have a natural ability for being transfected and thus it
is possible to study the effect of enzymes and receptors on
the permeability of drugs [24]. IEC-18 cells are less well differ-
entiated than the Caco-2 cells, and little is known about the
expression of the carrier-mediated transport systems in this
cell line. In addition, IEC-18 cells simulate the small intes-
tine, which could indicate a comparable leakiness/
tightness of the paracellular pathway as observed in human
intestinal tissue [23].
The Madin-Darby canine kidney (MDCK) is an alternative
cell culture model to Caco-2 cells, being first described in
1989 [25]. Monolayer models of MDCK cells are highly corre-
lated with the Caco-2 model, especially for passively absorbed
drugs [26]. Whereas Caco-2 cells are derived from human
colon carcinoma cells, MDCK cells are derived from dog kid-
ney cells, and thus the expression levels of some transporters
may be different in these two cell lines. Indeed, the non-
human and non-intestinal origin of the MDCK cells may be
considered as a disadvantage. They have low expression levels
of transporter proteins and low metabolic activity as com-
pared with Caco-2. These differences in substrate activity
could result from differences in the relative expression levels

of total P-gp in Caco-2 and MDCK cells and differences in
the partitioning of substrates across the cell membrane
bilayers [27].
Co-culture models comprising different cells present in the
intestinal epithelium have been proposed, to more closely rep-
resenting the heterogeneity of the intestinal epithelium at a
cellular level [5]. Many of the co-cultured cell systems may
be of greater interest in academic research but are still not
common in industry. The majority of co-culture models are
based on modifications and improvements of the Caco-
2 cell model. One example is the evaluation of cell monolayer
models based on mixtures of Caco-2/HT29 co-culture for
Expert Opin. Drug Metab. Toxicol. Downloaded from informahealthcare.com by University of Calgary on 05/24/12
their use in screening of drug absorption and intestinal perme-
ability in comparison with the properties of the respective
monocultures [21]. The Caco-2/HT29 model offers the possi-
bility of studying absorption and the effect of goblets cells
simultaneously in drug transport and absorption enhance-
ment, being well correlated with in vivo data [28] and reveals
a paracellular permeability closer to the in vivo human situa-
tion [20]. Moreover, it is possible to modify the permeability
barrier of the cell monolayers both with respect to paracellular
resistance and secretory transport via P-gp [21], allowing more
flexibility in adapting the in vitro system to the in vivo situa-
tion as compared with the monocultures. Another clear
For personal use only.
advantage of this co-culture model is the evaluation of absorp-
tion enhancers and mucoadhesive systems on the permeability
of drugs and its high correlation with the in vivo situation, as
recently reported by our group [29,30].
Another promising co-culture monolayer model based on
Caco-2 cells and human Raji B lymphocytes has been devel-
oped to mimic the human follicle-associated epithelium [31-33].
Raji B lymphocytes are able to convert enterocytes into M
cells [34]. The Caco-2/Raji B co-culture model appears to be
valuable for studying the absorption of large molecules and
nanoparticles containing drugs, due to the high transcytotic
capacities of M cells and their ability to transport a broad range
of materials, including nanoparticles [33,35]. In fact, the impor-
tance of M cells on nanoparticle transcytosis and drug
absorption was demonstrated using this co-culture model [36].
Recently, the co-cultivation of Caco-2/HT29/Raji B cells
allowed to reconstitute a cell culture monolayer system that
better mimics the intestinal barrier, especially to investigate
its interaction with nanoparticles [37]. Results demonstrated
that the Caco-2/HT29/Raji B triple co-culture model may
be reliable in obtaining a more physiological, functional and
reproducible in vitro model of the intestinal barrier in order
to study drug absorption both in solution and when
associated with nanocarriers (Figure 1) [38,39].
3.2 Vaginal and rectal models
The use of in vitro cell models for studying rectal absorption of
drugs has relied mainly on the Caco-2 monolayer model, which
has been detailed above [40,41]. The similarity between these
colon-derived cell monolayers and the epithelial layer at the
colorectum provides the rationale for this option, although
Expert Opin. Drug Metab. Toxicol. (2012) 8(5)
Cell-based in vitro models for predicting drug permeability
the permeability features of drugs in the rectum are different
from the intestine. Experimental work showed analogous
results for the permeability of a low molecular weight (MW)
heparin for the Caco-2 model and after rectal administration
to rabbits (as analyzed by drug-response profiles) in the pres-
ence or absence of permeability enhancers [42,43]. Moreover,
the comparison of permeability studies using Caco-2 cell
monolayers and excised human colorectal tissues showed
good correlation between the two models for different low
MW molecules (e.g., atenolol or dexamethasone), proteins
and peptides [44]. Thus, the Caco-2 monolayer model seems
suitable for the preliminary assessment of rectal drug
absorption, in particular for screening and mechanistic studies.
Different models have been proposed for studying drug per-
meability/absorption on vaginal administration. The most
extensively studied in vitro model has been developed by
Gorodeski et al. [45,46]. It is based on the harvesting of cervical-
vaginal cells from human subjects undergoing hysterectomy,
which are subsequently grown on collagen-coated ceramic-based
filters and differentiated into multilayered squamous stratified
epithelium (typically 5 -- 12 cell layers) [46]. The membranes
retain most phenotypical and biological characteristics of the
human cervical--vaginal epithelium [46-48]. Cervical cell lines,
which are typically simpler to maintain and readily available,
have also been tested as alternatives to primary cells obtained
from hysterectomy [49]. For example, the CaSki endocervical
cell line was shown to form cell monolayers or bi/trilayers,
depending on cell seeding density, and has been shown useful
for studying transport mechanisms [50-52]. Cell-covered filters
are typically mounted on Ussing chambers in order to proceed
with permeation experiments. Interesting findings using this
model include the substantial importance of tight junctions
and its modulation on transepithelial electrical resistance
(TEER) [45], the increase of TEER by seminal fluid [51] and the
modulation of the permeability to pyranine (used as a drug
model undergoing paracellular transport) by factors such as
estrogen and aging (due to changes in the resistance of both
the lateral intercellular space and the intercellular tight
junctions) [53-55], or extracellular ATP and Ca2+ (due to effects
at the tight junctions level) [56,57].
Other cervical--vaginal models have also been described. For
instances, Kaushal et al. used C-33A cervical cell monolayers
grown in Transwell
supports to study the permeability of
b-lactamase, used as a model protein, as mediated by a geneti-
cally engineered Lactococcus lactis strain containing a coding
gene for b-lactamase [58]. The model was shown successful in
detecting differences in b-lactamase permeability when delivered
in the free form or by the modified Lactococcus lactis. Another
interesting model is the commercially available vaginal--ectocer-
vical tissue-like model (EpiVaginal
, MatTek Corp., Ashland,
MA, USA), which has been widely used for testing vaginal
microbicides. The model is based on a 3D culture of non-trans-
formed human vaginal--ectocervical epithelial cells grown on
polycarbonate cell culture tissue inserts. Differentiation is
obtained using a proprietary serum-free medium by submerging
611
 B. Sarmento et al.

xy view

xz view
A. C.
Enterocytes D.

30 Insert membrane
30 *
*
xy view

xz view
B.
Expert Opin. Drug Metab. Toxicol. Downloaded from informahealthcare.com by University of Calgary on 05/24/12

xz view
xy view

xz view
E.

Cell apical
pole
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xz view xz

Figure 1. Display of Raji cells within co-cultures by confocal analysis. Enterocyte actin was stained with rhodamine-
phalloidin (red) and Raji cells were CFSE-labeled (yellow--green). Inverted (B)--(D) and normally oriented co-cultures (E) were
fixed after 5 days of incubation with CFSE-labeled Raji cells. Monocultures were used as control (A) and (D). White arrows
indicate the Raji localization in the cell monolayers. To confirm the correlation between the yellow--green fluorescence and
the presence of Raji cells in the co-cultures, TOTO-3 (blue color), labeling the cell nuclei, were added in the mounting medium
(C). Turquoise blue color resulted from the merge of blue and yellow--green fluorescence (white star). Monocultures were
used as controls (A). Grey lines indicate where, within the cell monolayers, the pictures were taken and analyzed.
Reprinted from [33], Copyright (2007), with permission from Elsevier.

the inserts for 4 days, followed by 7 days at the air--liquid inter-
face [59,60]. Although typically explored for studying pathogen
transmission prevention [61,62] and tissue toxicity [60], this model
shows interesting organotypic features, such as differential
multi-layer structure, possibility of containing non-epithelial
cell elements which compose the cervical--vaginal mucosa (e.g.,
fibroblast-containing lamina propria, dendritic cells) and natu-
ral metabolism [59,60], making it a promising option for perme-
ability studies. The main morphophysiological features of this
model are presented in Figure 2. In one recent study, Fatakda-
wala and Uhland studied the transport of bovine insulin across
the EpiVaginal model (without lamina propria and dendritic
cells) using hydrogen peroxide as a permeability enhancer [63].
Combined permeability, TEER and fluorescence microscopy
with immunostaining for occludin, a component of tight junc-
tions, allowed providing evidence of the enhanced permeability
of insulin in the presence of hydrogen peroxide by reversible
disruption of tight junctions. Even if no ex vivo or in vivo
experiments were performed in order to corroborate the
obtained results, this study provides good prospects for further
use of this model. In another report, Clark et al. used the EpiVa-
ginal model comprising the vaginal--ectocervical epithelial cells
and a fibroblast-containing lamina propria for studying the per-
meability of the microbicide candidate UC781 formulated as
gels [64]. When using two different forms of the drug (micron-
ized and non-micronized), results showed higher permeability
for the micronized form (roughly twofold). However, in vivo
pharmacokinetic experiments in rabbits failed to corroborate
the small but significant differences obtained in vitro.
3.3 Pulmonary and nasal models
The study of the respiratory tract as a route for systemic
administration of drugs and vaccines has increased over the
last years. The efficacy of an inhaled drug is dependent on
its absorption through the lung and nasal epithelium and on
the anatomic region where absorption preferably takes place,
reason why it is important to perform permeation studies
during drug development. Both immortalized cell lines [65]

612 Expert Opin. Drug Metab. Toxicol. (2012) 8(5)

 Cell-based in vitro models for predicting drug permeability

A.
Apical layer
Glycogen-filled layers

Suprabasal layers
Basal layer
Microporous
membrane substrate
B.

Apical layer
Glycogen-filled layers
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Suprabasal layers
Basal layer

Firbroblasts in collagen
gel matrix

C. Apical layer

Glycogen-filled layers

Suprabasal layers
For personal use only.

Basal layer

Lamina propria
containing fibroblasts
and other cells

Figure 2. H&E stained cross-sections of: (A) partial thickness (i.e., epithelial layers without fibroblast-containing lamina
propria), EpiVaginal
VEC-100 tissue, (B) full-thickness EpiVaginal VEC-100-FT tissue with epithelial and lamina propria layers
and (C) native human explant vaginal tissue. The epithelium in all tissues contains nucleated basal and suprabasal cell layers.
As the apical surface is approached, the cells lose their nuclei and become filled with glycogen. In the full thickness and
explant tissues, a lamina propria layer consisting of collagen gel matrix containing vaginal fibroblasts is observed.
Reprinted from [59], Copyright (2010), with permission from Elsevier.

and primary cell cultures [66] have been used to study the per-
meation of drugs through the respiratory epithelium and its
underlying mechanisms. Although the characteristics of pri-
mary cell culture are closer to the native epithelia, they are
less reproducible, more expensive, difficult and time-
consuming to maintain in culture and present reduced life-
span, which explains the preferential use of immortalized
cell lines in permeation studies [67,68].
With respect to human bronchial epithelial cell lines, Calu-
3 and 16HBE14o- cell monolayers have been used as models
for airway epithelium due to their morphological characteris-
tics, barrier properties and expression of drug transport sys-
tems that are also present in vivo [65,69,70]. Different research
groups showed that the culture conditions affect the morpho-
logic characteristics and the TEER of the monolayers as well
as the pattern of drug permeation [71,72]. When grown at the
air--liquid interface, Calu-3 cells present lower TEER values
(Figure 3) and are more permeable to fluorescein-sodium,
dextran [72] and dipeptides, being the absorption carrier-medi-
ated, probably by hPEPT1 transporter [71]. For others com-
pounds the permeability differs between the two monolayer
culture models and are not correlated with TEER values [71].
Due to its ultrastructure, secretory component and integrity,
Calu-3 cell monolayers cultivated at the air--liquid interface
better resemble the in vivo conditions and are more suitable
as a model of the tracheobronchial epithelium than liquid-
covered cultures [72]. In another study, the permeation
through 16HBE14o- cell monolayers showed to correlate
well with the absorption rates of low MW compounds when
using the isolated perfused rat lung (r2 = 0.78), but presenting
lower correlation with the absorption rates of the rat lung
in vivo (r2 = 0.68) [73].
In an interesting study, a modified multistage liquid
impinger with associated Calu-3 cell monolayer was devel-
oped [74]. This in vitro system allowed studying both the
deposition and permeation of particles, two of the critical

Expert Opin. Drug Metab. Toxicol. (2012) 8(5) 613

 B. Sarmento et al.
A. 1000 AIC conditions
800
Teer (ohm.cm2)
600
400
* *
200
0 0
7 10 14 16
Days in culture
Expert Opin. Drug Metab. Toxicol. Downloaded from informahealthcare.com by University of Calgary on 05/24/12
Figure 3. TEER values of Calu-3 monolayers through the culture period. (A) air interfaced culture and (B) liquid-
covered culture.
Reprinted from [71], Copyright (2011), with permission from Elsevier.
parameters in the efficacy of an inhalable formulation. Both
normal oriented air--liquid interface culture and inverted cul-
ture presented similar morphologic characteristics and appar-
ent permeability coefficients (Papp) for fluorescein-sodium
used as a permeability marker. Moreover, when using air
interface cultures, similar absorption rates for three commer-
cial formulations of a two-drug association (salbutamol and
budesonide) were obtained for each drug, thus demonstrating
For personal use only.
their bioequivalence and the usefulness of this system in per-
meation studies [74]. Another research group developed a
Pharmaceutical Aerosol Deposition Device on Cell Cultures
(PADDOCC) containing Calu-3 cell monolayers grown at
the air--liquid interface. The PADDOCC demonstrated to
be reproducible when studying the deposition and absorption
of commercially available dry powder inhalers containing
salbutamol sulfate or budesonide [75].
Regarding the alveolar region, A549 cells have been used as
model for studying the absorption of peptides and pro-
teins [76]. However, some researchers defend that this is not
a good model due to its non-alveolar type I morphology
and low capacity to form tight monolayers with consequent
high permeability, being necessary to resort to primary cul-
tures of human alveolar epithelial cells (hAEpC) [66,67,77].
Monolayers of primary alveolar epithelial cells from animal
sources such as pigs also demonstrated suitability as in vitro
models for drug permeation studies [78].
Recently, the development of co-cultures [77,79] and modified
impinger systems with integrated cell monolayers [74,80,81] have
been proposed in order to better mimic the in vivo conditions
of the alveolar region. A three-dimensional (3D) triple
co-culture monolayer composed by A549 epithelial cells, blood
monocyte-derived macrophages and dendritic cells was pro-
posed as a more realistic simulator of the air--blood barrier of
the lungs [77,79]. The co-cultures expressed E-cadherin, zonula
occludens (ZO)-3, occludin and claudin-2, proteins involved
in cell--cell interactions and tight junction formation [79]. In
contrast with the co-cultures of hAEpC or 16HBE14o- cells,
the co-cultures presenting A549 cells do not regularly express
ZO-1 [77]. It was possible to observe the existence of tight
614 Expert Opin. Drug Metab. Toxicol. (2012) 8(5)
B. 1000 LCC conditions
800
Teer (ohm.cm2)
600
* 400
*
200
17 7 10 14 16 17
Days in culture
junctions by transmission electron microscopy as well as the
presence of macrophages in the basal side and dendritic cells
in the apical side of the epithelial cells. The presence of macro-
phages and dendritic cells reduces the integrity of the triple
co-culture monolayer, with less impact in hAEpC. In the
same way as for co-cultures of 16HBE14o- cells, the A549
cell-based co-cultures resemble the in vivo architecture of the
human airway epithelial barrier, presenting a cuboidal cell mor-
phology. In order to mimic the alveolar epithelial barrier with
squamous cells, it has been necessary to use co-cultures includ-
ing hAEpC cells. This system holds promise in studying the
mechanisms underlying the interaction of particles with the
epithelium and the absorption of drugs [77]. It is also possible
to mimic in vitro the alveolar--endothelial barrier using
co-cultures of lung epithelial cell lines such as NCI-H441
cells [82] or primary human type II alveolar epithelial cells
(HATII) [83], and primary human pulmonary microvascular
endothelial cells (hPMEC). These last co-cultures present the
morphologic and histological characteristics of the alveolocapil-
lary barrier [82,83]. Currently, there are also commercially avail-
able 3D systems based on human-derived tracheal/bronchial
epithelial cells (EpiAirway
, MatTek Corp., Ashland, MA,
USA) [84], but the application of this model for drug absorption
screening is still limited.
In the case of nasal permeation studies, monolayers
obtained with primary cultures of human nasal epithelial cells
have been extensively used [85-88]. Both cells recovered from
normal tissues and polyps biopsies present comparable expres-
sion of organic cationic transporters and absorption of sub-
strates for those transporters, showing that polyps biopsies
may be useful for in vitro studies [85]. When studying the
absorption of anti-allergic drugs such as dexamethasone, tri-
amcinolonacetonide and budesonide through liquid-covered
cell monolayers a log-linear relationship between the drug
logP and the Papp where verified [88]. Although the permeabi-
lity of lipophilic compounds was similar in monolayers grown
at the air--liquid interface and liquid-covered culture condi-
tions, hydrophilic compounds permeated more extensively
when liquid-covered cultures were used due to the lower

TEER values presented by these monolayers. Thus, air--liquid
interface culture conditions seem to be an appropriate and
more reliable in vitro model for nasal epithelial permeation
studies [89]. Besides primary cultures of human nasal epithelial
cells, cell lines such as RPMI 2650 (human), BT (bovine) and
NAS 2BL (rat) have been used in nasal drug development [68].
Calu-3 cells have also been proposed in models of the nasal
epithelium, showing to be suitable in the prediction of nasal
absorption of analgesic drugs [90]. Recently, co-cultures have
been developed for nasal permeation studies composed by
a collagen matrix with embedded human nasal fibro-
blasts covered by a RPMI 2650 epithelial cell layer [91]. This
Expert Opin. Drug Metab. Toxicol. Downloaded from informahealthcare.com by University of Calgary on 05/24/12
system resembled a non-pseudostratified, non-ciliated epithe-
lium with permeation barrier properties comparable with
excised nasal mucosa. Moreover, permeability using this
co-culture model was lower than when using RPMI 2650 cells
alone. On the disadvantage side, the co-culture system is more
complex to handle and grow than the RPMI epithelial
model [91].
3.4 Ocular models
In the last years, numerous cell culture models of the ocular
barriers have been established [92-95]. These in vitro tools play
a fundamental role in studying the ocular barriers, namely their
For personal use only.
function and regulation. In addition, ocular cell culture models
have been used for numerous other purposes, such as studying
passive and active transport of drugs and endogenous substan-
ces, cellular physiology, metabolism and protein expression,
development of delivery systems for genes and antisense oligo-
nucleotides and cytotoxicity studies [95]. These models may also
be useful in identifying compounds and formulations with
favorable pharmacokinetic properties and evaluate metabolism
and drug absorption--structure relationships. Since the pre-
ferred absorption route for small molecules is the cornea and
for biologics including genes is conjunctiva, ocular models
may resemble different ocular structures [95].
The eyeball comprises the anterior segment, in which the
cornea and conjunctiva are the main prominent structures,
and the posterior segment, in which the retina plays the
most important functions [96]. In the anterior segment of
the eye covering both corneal and conjunctival surfaces is
the tear film secreted by the goblet cells of the conjunctiva.
The lachrymal film plays a multifunctional role, since it
hydrates, cleanses, lubricates and serves as a defense against
the pathogens. It also presents an additional obstacle to drug
penetration, because the lachrymal film undergoes a constant
renewal and therefore limits the time of residence of drugs at
the surface of the eye [97]. The cornea is a transparent and
avascular hydrophobic barrier and the primary route of drug
entry into the eye following topical administration, but
administered drugs also reach intraocular tissues by the
conjunctiva-scleral pathway [98]. The human corneal epithe-
lium HCE-T cell model represents a standard tool for
drug permeation, bioavailability pre-screening and toxicity
assessment [99].
Expert Opin. Drug Metab. Toxicol. (2012) 8(5)
Cell-based in vitro models for predicting drug permeability
The conjunctiva lines the inner surfaces of the eyelids and
folds back to cover the front surface of the eyeball, except
for the central portion of the outer eye [100]. It is a highly vas-
cularized, thin/semi-transparent, elastic and heterogeneous
tissue [98]. The IOBA-NHC cell line, a non-transfected, spon-
taneously immortalized epithelial cell line derived from
human conjunctiva, demonstrated high proliferative ability
in vitro and typical epithelial morphology. Conjunctiva epi-
thelial cells are an important source of cytokines and chemo-
kines that are regulated by proinflammatory cytokines and
may play an important role in ocular surface inflammation [92].
In a recent study, a monolayer model based on a immortalized
rat conjunctiva epithelial cell line was developed in order to
study transepithelial antigen delivery [93]. Conjunctiva epithe-
lial cells from Fischer 344 rats were immortalized with
pSV3-neo vector resulting in two distinct cell lines: CJ4.1A
and CJ4.3C [93]. In other study, Chang conjunctiva epithelial
cell and primary human conjunctiva-derived epithelial cell
(HCDEC) models were used for studying the efficiency of a
novel carrier system, bacterial ghosts (BGs), which are empty
bacterial cell envelopes of Gram-negative bacteria, for target-
ing human conjunctiva epithelial cells. Also, these models
were used to test the endocytic capacity of conjunctiva cells
after co-incubation with BGs generated from different bacte-
rial species, and to foreclose potential cytotoxic effects caused
by BGs [101].
The retina is a thin, transparent, highly organized structure
of neurons, glial cells and blood vessels [96]. Of all structures
within the eye, the neurosensory retina has been the most
widely studied. Retina is organized into neural retina and ret-
inal pigment epithelium [100], being the first involved in signal
transduction, leading to vision. Immortalized human
ARPE-19 cells have been fully characterized regarding their
morphology, the expression of retina-specific markers
(CRALP and RPE65) and their barrier properties [95,102].
Monolayers of ARPE-19 cells are a well-established in vitro
model of the outer blood--retinal barrier (BRB). This model
has been used for the development of targeted drug delivery
systems to the posterior segment of the eye [95]. Moreover,
monolayers of ARPE-19 cells have been used by researchers
for a variety of other in vitro experiments, including studies
of regulation of gene expression, polarized distribution and
secretion of proteins, delivery of genes and antisense oligonu-
cleotides, for toxicity studies, and as models of retinal
diseases [95,103,104].
3.5 Skin models
Reconstructed skin models that simulate human skin are now-
adays widely used for efficacy pre-screening of different com-
pounds. Moreover, they are of growing interest for regulatory
purposes in the framework of alternatives to animal test-
ing [105]. In vivo skin absorption measurements are rare, due
to ethical, economical and analytical concerns [106]. According
to the EU and Regulation on Registration, Evaluation,
Authorization and Restriction of Chemicals (REACH)
615
 B. Sarmento et al.
guidelines, animal testing for skin studies should only be
performed if all other approaches have failed to provide a con-
clusive result. Thus, there is a particular demand for animal-
free methods, namely in vitro models for studying drug
permeation/absorption. A number of culture-derived skin
equivalents such as living skin equivalent models (LSEs) and
human reconstructed epidermis (HRE) have been used to
measure percutaneous absorption [107]. In order to develop
alternative methods to animal studies, commercially available
in vitro human skin models comprising epidermal cells have
been the most developed and used, including Episkin
(Epis-
kin SNC, Chaponost, France), EpiDerm
(MatTek Corp.,
Expert Opin. Drug Metab. Toxicol. Downloaded from informahealthcare.com by University of Calgary on 05/24/12
Ashland, MA, USA) and SkinEthic
(Laboratoire SkinEthic,
Nice, France). Reconstructed human skin models are able to
mimic human skin to a large extent, contrasting with the lim-
itations of classical cell monolayers. These 3D models are gen-
erated by growing keratinocyte cultures at the air--liquid
interface on various substrates, and enable the topical applica-
tion of either neat or diluted test materials. Efforts have been
made to develop methods for quantification of skin perme-
ability, validation of diffusion cell setups and correlation of
in vitro data with the in vivo situation [106]. Most publications
in the field of skin permeation research report studies carried
out using a large variety of setups and experimental protocols
For personal use only.
varying from laboratory to laboratory. This raises questions of
standardization and regularization [106].
The EpiSkin model is one of the most used in skin perme-
ation/absorption studies [108]. EpiSkin comprises a human
collagen (types III and I) matrix, representing the dermis, cov-
ered with a film of type IV human collagen, on which
stratified differentiated epidermis derived from human kerati-
nocytes is grown. The model utilizes cell viability as the mea-
sured end point. The mode of application (topical) of the test
material mimics the route of human exposure. To produce a
more suitable model for studying drug penetration, the kera-
tinocytes are cultured for 20 days prior to transfer to the
collagen substrate.
The EpiDerm skin model is mechanistically and function-
ally related to EpiSkin. The model consists of normal, human
epidermal keratinocytes which have been grown in chemically
defined medium to produce a stratified, highly differentiated,
organotypic tissue model of the human epidermis [109]. The
EpiDerm tissue consists of metabolically and mitotically
active cells which are organized into a basal, spinous and gran-
ular layer along with a multi-layered stratum corneum. The
dermal penetration and metabolism of drugs can be easily pre-
dicted by using EpiDerm. As an example, the penetration of
EpiDerm by two hair dye ingredients, p-aminophenol (PAP)
and p-phenylenediamine (PPD), resulted in the biotransfor-
mation of these two aromatic amines via N-acetylation [109].
In this particular study, EpiDerm tissue was exposed to PAP
or PPD either topically or from the bottom of the tissue via
the culture media.
The SkinEthic model is obtained by growing normal
human keratinocytes on polycarbonate culture inserts lifted
616 Expert Opin. Drug Metab. Toxicol. (2012) 8(5)
to the air--liquid interface, and a stratum corneum grown
at the air--liquid interface. Several studies examined com-
pound transport through the SkinEthic model. In one case,
Gabbanini et al. tested the permeation of (D)-a-tocopherol,
retinyl acetate, ascorbic acid and pyridoxine through Ski-
nEthic and synthetic polyethersulfone and polycarbonate
membranes. Results indicated that SkinEthic offered a sig-
nificantly higher barrier to the penetration of vitamins, as
expected for native human epidermis [110]. Additionally,
due to the limited dispersion of experimental kinetic results,
the use of SkinEthic favorably compares with the large vari-
ability normally found with native human epidermis, while
overcoming the problems of limited availability [110].
4. Conclusions
One of the most important tasks presently facing the drug
market is to develop high-throughput, cost-effective and
highly predictive screening models of drug permeability or
absorption that can be used in the early stages of drug discov-
ery [111]. In vitro cell culture-based models have demonstrated
to be extremely useful for permeability screening assessment.
In this regard, in the present review several cell models which
have been tested to mimic as close as possible different
biological human barriers were present. Due to the obvious
interest and application, intestinal models have been the
most studied, being possible, nowadays, to use different cells
in order to study the permeation/absorption of drugs using
specific mechanism. In vitro cell models have been optimized,
becoming more and more complex and incorporating more
than one type of cells in order to more closely resemble
in vivo tissues. Other mucosal tissues have also been emulated
in vitro using cells, namely the bronchial, alveolar, rectal, vag-
inal and ocular regions. Skin cell models have also been widely
used to screen the penetration/permeability of drugs. The
development of ready-to-use commercial skin models pre-
sented the advantage of requiring minor conditions of cell
maintenance as compared with other mucosal in vitro cell
models. Despite the need of further optimization and stan-
dardization of cell growth conditions and accurate correlation
with in vivo absorption profiles, in vitro cell culture models
may be considered one of the most useful techniques to better
predict the permeability/absorption of drugs. In special, the
correlation between any in vitro model and in vivo conditions
must be clearly quantified, ideally employing validates math-
ematical functions, providing algorithms for interlaboratory
reproducibility. Nonetheless, these models are easy to manip-
ulate and avoid the ethical concerns related with the use of
animals in early preclinical stages of drug development.
5. Expert opinion
It is clear that the development of models, methodologies and
generation of appropriate in vitro data to try to simulate the
in vivo behavior, in order to better mimic the biological

barriers and all the mechanisms involved, is crucial for drug
development. Also, there is still a need to continue the
research on the several different methods described above.
In vitro cell culture-based models have been demonstrated
to be very valuable for absorption and permeability screening.
In special, models mimicking the gastrointestinal barrier,
which represent the majority of and most desired route of
administration of drugs due to its convenience. Cell line mod-
els have the potential to illustrate much of the complexity
involved in drug absorption and consequent bioavailability.
Another pragmatic reason to support the development and
use of in vitro cell models is the social and scientific concerns
Expert Opin. Drug Metab. Toxicol. Downloaded from informahealthcare.com by University of Calgary on 05/24/12
about the animal well care as well as the need for rapid and
consistent bioavailability models by the pharmaceutical com-
panies and the demands for economical and low time con-
sumption methodologies. However, additional regulatory
aspects on the different cell culture systems must be released
since this aspect is indeed important and remains a limitation
in a generalized use of cell culture systems.
Despite the widespread use of the cell models, they suffer
from significant shortcomings that render it less than optimal
as a one-stop cell-based permeability-screening tool. They
usually are homogeneous cell system whereas the human
absorption organs are composed of different cell types.
For personal use only.
Thus, in vitro cell models are possible to be established in
the near future in a more robust manner, becoming more
complex and incorporating different cells in order to mimic
as close as possible the in vivo tissues, which give reason for
developing in vitro models with appropriate correlation
between results of drug absorption in vivo and in vitro.
The main advantage of in vivo compared with in vitro
models to establish the bioavailability of drugs is the integra-
tion of dynamic components of the blood circulation, the
tissue layers and all the other factors that can influence
Expert Opin. Drug Metab. Toxicol. (2012) 8(5)
Cell-based in vitro models for predicting drug permeability
absorption. On the other hand, the disadvantage of in vivo
models is that it is impossible to separate the variables
involved in the process of absorption, not allowing the
identification of individual rate-limiting factors.
Whichever type of permeability in question, an ideal in vitro
cell model must be able to determine and to compare the rela-
tionship between in vivo absorption of drugs and the apparent
permeability coefficients obtained in vitro on cell cultures and
even on isolated ex vivo tissue. An ideal cell model, already
studied or improved must be established regarding the higher
drug absorption rate maintaining its physiological integrity.
The mechanisms behind drug absorption must be clearly
highlighted through different methodologies to be employed
directly on the cells, as well as the relationship between the
drug carriers and the efficacy of absorption.
It is clear that for timely and effective drug development, it is
important to gain early insight into the potential of lead com-
pounds. This will allow appropriate optimization of absorption
process to achieve adequate bioavailability resulting in effective
systemic concentrations. Such time-consuming and costly pro-
cedures should be restricted, as far as practical, to later stages of
drug development. Also, the information based on permeabil-
ity is nowadays provided into predictive PBPK models which
are available as off the shelf software packages, which must
be consider.
Declaration of interest
This work was supported by Fundação para a Ciência
e Tecnologia (FCT), Portugal (PTDC/SAU-FCT/70651/
2006/ FCOMP-01-01214-FEDER-007500 as well as grants
SFRH/BPD/35996/2007, SFRH/BD/43393/2008, SFRH/
BD/73062/2010, SFRH/BD/61423/2009 and SFRH/BDE/
51385/2011).
617
 B. Sarmento et al.
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Affiliation
Bruno Sarmento†2,3, Fernanda Andrade1,
Sara Baptista da Silva1, Francisca Rodrigues1,
José das Neves1,2 & Domingos Ferreira1
†
Author for correspondence
1
Department of Pharmaceutical Technology,
LTF/CICF, Faculty of Pharmacy, University of
Porto, Portugal
Tel: +351 222 078 949;
Fax: +351222 003 977;
E-mail: bruno.sarmento@ff.up.pt
2
CICS, Department of Pharmaceutical Sciences,
Instituto Superior de Ciências da Saude -- Norte,
Gandra, Portugal
3
INEB -- Instituto de Engenharia Biomédica,
Porto, Portugal
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