Adapted from (Flaten et al., 2015).
In vitro membrane
models
In order to overcome the disadvantages of ex vivo human and
animal models, mainly related with the availability and the ethical
problems, numerous efforts have been done to develop alternative skin
equivalents based on in vitro approaches. In vitro skin
alternative models may consist either on mimetic membranes (non-lipid
and lipid systems) or cell cultures (Abd et al., 2016, Flaten et al.,
2015, Berben et al., 2018, Sinkó et al., 2014, Faller, 2008). The
existent models and their applications will be reviewed in the following
sections.
The first studies regarding the development of in vitro skin
equivalents developed in mid 70s included the use of normal human
keratinocytes (NHKs) as a model for skin irritancy (Rheinwald, 1989).
The cell culture of NHKs begins with a small piece of human skin (about
0.5-1 cm2) obtained from surgery after separation and
specific treatment, that grows easily in culture medium. It allows the
use of a large number of cells, leading to the opportunity for wide
ranging toxicity screening tests with many substances. This model shows
good ability for testing hydrophilic compounds however presenting less
capability for the evaluation of poorly water soluble compounds and
complex formulations (reviewed in (Ponec, 1992)).
The model was improved with its application in membranes, which support
the NHKs during growing and forming the called reconstructed human
epidermis. More complex models mimicking the full thickness skin
consisting of the fibroblast populated collagen matrices (dermis
equivalent) and an epidermal cover composed by NHKs (Van Gele et al.,
2011).
Nowadays there are different commercially available models of the human
epidermis or full thickness skin. These models will be reviewed with
more detail in section 3.2.3.
Additionally, other acellular in vitro models have been
described: a) non-lipid-based models like the silicone membranes or
poly(dimethylsiloxane) (PDMS) models (Oliveira et al., 2011) and b)
lipid-based models like Parallel Artificial Membrane Permeability Assay
(PAMPA) (Sinko et al., 2012, Sinkó et al., 2014) and Phospholipid
Vesicle-based Permeation Assay (PVPA) systems, as simpler alternative
approaches (Engesland et al., 2013).
Non-lipid-based models
The first studies regarding the use of non-lipid-based skin models date
from 1970 and report the use of silicone membranes to study the release
of salicylic acid (Nakano and Patel, 1970). Later, some studies reported
the use of different microporous membranes based on pure cellulose
acetate, cellulose and polysulfone to investigate the permeation of
hydrocortisone from two commercial creams (Shah et al., 1989). Other
synthetic membranes considering polysulfone, cellulose mixed esters,
polytetrafluoroethylene and polypropylene in their composition were
described and used to study the nitroglycerin release from commercial
ointments (Wu et al., 1992). More recently, some studies describe the
interaction of many drugs and vehicles with skin models of PDMS or with
silicone membranes skin mimetic models (Dias et al., 2007, Watkinson et
al., 2009a, Watkinson et al., 2009b, Watkinson et al., 2011, Oliveira et
al., 2011, Oliveira et al., 2010, Oliveira et al., 2012).
These skin equivalents are simple models with great applicability to
test a basic diffusion mechanisms however they present some
disadvantages namely the lack of similarity with the human skin and they
are not much useful in the study of the permeability of hydrophilic
compounds despite the good results obtained for lipophilic drugs (Abd et
al., 2016, Miki et al., 2015).
Another study reports a new and improved model based on a membrane
impregnated with a polymer of PDMS and PEG 6000 and some preliminary
results elucidating the permeation of drugs in the aqueous solutions
were described by Miki and co-workers (Miki et al., 2015).
Lipid-based models
Parallel Artificial
Membrane Permeability Assay
The lipid-based skin equivalents appeared as valuable alternatives to
non-lipid-based models, improving its complexity and the ability to
mimic the human skin, namely the SC layer.
A first report dates from 1989 and describes the preparation of model
membranes, using SC lipids, to study the permeability of drugs in
the skin. The authors consider ceramides, cholesterol, FFA and
cholesteryl sulphate in its composition (Abraham and Downing, 1989). The
selected lipid composition agrees to those found in the human SClayer and thus it constitutes an advantage for this model since it
better represents the layer in terms of its lipid composition. However,
the most consistent studies started in 1998 when Kansy and co-workers
reported the first PAMPA model as a tool for rapid determination of
passive membrane permeability of drugs (Kansy et al., 1998). This system
included a mimetic membrane from hydrophobic filter coated with
phosphatidylcholine dissolved in n -dodecane as a membrane
barrier, which differentiates the donor and acceptor parts. Even though
this model was oriented for testing the transcellular intestinal
permeability, it was the precursor of the following models, especially
those developed to mimic the human skin.
The first PAMPA model for skin penetration estimation was published by
Ottaviani and co-workers in 2006 (Ottaviani et al., 2006) however this
proposal is, in fact, a non-lipid-based model which incorporates
silicone oil and isopropyl myristate as membrane components. This model
was used to study the permeation of a large variety of drugs and
vehicles (Dobričić et al., 2014, Markovic et al., 2012, Karadzovska and
Riviere, 2013, Karadzovska et al., 2013).
In the same year, Loftsson and co-workers described a novel skin
substitute membrane based on a hydrated semi-permeable cellophane
membrane and a lipophilic membrane of pure n -octanol in a
nitrocellulose matrix. This model was used to study the permeation of
different cyclodextrin formulations and the results have shown that the
drug permeation patterns of the different formulations were similar to
those previously observed for biological membranes (Loftsson et al.,
2006).
In 2012, Sinko et al . designed the skin-PAMPA (Sinko et al.,
2012), which consist of ceramides analogues called the synthetic
ceramides or certramides, as substitution for the native ceramides found
in SC . Certramides are cheaper alternatives to natural ceramides
with the potential to prolong the storage time. This skin-PAMPA model
was used in the skin permeability studies and exhibited poor correlation
with skin epidermis, however it presented a good correlation with full
thickness skin (Sinko et al., 2012). Even though its composition intents
to simulate mainly the SC , in some cases, skin-PAMPA can even be
a valuable alternative to replace models that use real human skin.
Moreover, it constitutes an easy, quick and cost-effective research tool
to study the permeation of pharmaceutical and cosmetic ingredients.
More recently, a modified version of the skin-PAMPA was reported
(Tsinman and Sinkó, 2013) and used to evaluate the skin permeation of
different some ibuprofen-containing formulations. The developed system
was able to distinguish between the different types of formulations the
results correlate well with those found in permeation studies using
human epidermis as a mimetic system.
Several other reports can be found in the literature reporting the use
of PAMPA models to investigate drugs’ permeability in the skin (Markovic
et al., 2012, Dobričić et al., 2014, Faller, 2008, Köllmer et al., 2019,
Alvarez-Figueroa et al., 2011, Vizserálek et al., 2015, Wu et al., 2019,
Luo et al., 2016, Zhang et al., 2019, Balázs et al., 2016). The vast
number of reports considering the use of this type of mimetic models to
study the interaction and permeation of many different bioactive
ingredients highlights the large spectra of applicability of PAMPA
approaches and points out the ability of these barriers to predict the
permeation of drugs in the human skin, despite of the more simplistic
composition of these systems.
Phospholipid
Vesicle-based Permeation Assay
The original PVPA (PVPAo) was introduced as a model for
screening the intestinal permeability and is composed of a consistent
coat of liposomes deposited on a filter support acting as biological
barrier (Flaten et al., 2006b, Flaten et al., 2006a). Later, by changing
the lipid composition of the liposomes used to produce the permeation
barrier, a new PVPA model was developed aiming to mimic the SCbarrier of the skin (Engesland et al., 2013). In this model, liposomes
are located within the pores and on the surface of the membranes (Flaten
et al., 2006a). Thereafter, other modified versions have been reported
(Berben et al., 2018, Engesland et al., 2013, Engesland et al., 2015,
Engesland et al., 2016, Ma et al., 2017, Palac et al., 2014, Zhang et
al., 2016).
Mainly, two skin PVPA models, presenting different lipid composition,
for estimating skin penetration have been described: a) PVPAc -
liposomes made of egg phosphatidylcholine (EPC) (77.1%, w/w) and
cholesterol (22.9%, w/w) (Engesland et al., 2013) and b) PVPAs - lipid
mixture of EPC (50%, w/w), ceramide (27.5%, w/w), cholesterol (12.5%,
w/w), FFA (7.5%, w/w) and cholesteryl sulfate (2.5%, w/w) (Engesland
et al., 2013).
The permeability of different compounds was evaluated in these PVPA skin
models and the results were compared with reported permeabilities using
animal skin models (rat, cattle, dog and pig) and with estimatedin silico values. The PVPA permeability data mainly corresponded
with the literature values of the animal skin penetration assays and thein silico values, with the exception of flufenamic acid that
showed a relatively lower permeation (Engesland et al., 2013).
Later, the PVPAs model was examined with diverse formulations made of
different types of liposomes containing diclofenac sodium salt. The
results showed a rising permeation ranking of diclofenac sodium from
liposomal formulations correlating with the physicochemical parameters
of the liposomal vehicle. The permeability of diclofenac increased in
the availability of the penetration enhancers (Palac et al., 2014). The
PVPA model was further optimized considering a complex skin PVPA
containing all the classes of lipids found in the SC , and the
penetration enhancing effect of menthol was investigated for a set of
active compounds with different physicochemical properties (Ma et al.,
2017).
The previously described PVPA skin models were studied in comparison
with a reconstructed human skin model (EpiSkin®). The
permeability results indicate that the PVPA model has the ability to
distinguish between the liposomal formulations and drug solutions, as
opposed to EpiSkin®. PVPA models were better than
EpiSkin® concerning their potential to determinate the
influence of the formulations on the drug permeability which could be
used in drug development at early stage. Moreover, PVPA barriers
revealed straightforward, effectiveness, economical and long storable
properties (Engesland et al., 2015).
More recently, other two lipid-based models have been reported as skin
mimetic systems and they were used for the study of the effect of a set
of synthetic surfactants on the skin. The new models proposed contain
DPPC and cholesterol in a molar ratio of 7:3 or a mixture of ceramide,
stearic acid and cholesterol in a molar ratio of 14:14:10 (Jurek et al.,
2019).
Recently, our research group recently developed and characterized a
cheaper and simpler alternative SC mimetic model (Shakel et al.,
2019) that simulates this human skin layer and can allow the screening
of drug candidates. The design of this new model was inspired on PVPA
approaches and comprises a lipid composition which closer resembles that
found in the human SC layer, namely in the percentage of
ceramides considered in its constitution. Thus, the novel human SC PVPA
model is made of liposomes composed by ceramide (50% w/w), EPC (25%
w/w), cholesterol (12.5% w/w), free fatty acids (10% w/w) and
cholesteryl sulphate (2.5% w/w). This model presents some advantages
since it can be stored up to 2 weeks at -20 °C, without losing their
integrity.
Cell-based skin models
The complexity of the skin mimetic models has been increasing along the
time, starting from in vitro non-lipid-based to lipid models and
later to models comprising simple or more complex cell cultures. Single
or multi-cell type 2D cultures, in which cells are grown as a monolayer
disposed on solid flat surfaces, such as polystyrene or glass, are some
of the mostly used cell-based approaches due to their relative
simplicity and cost-effectiveness (reviewed in (Randall et al., 2018)).
The first studies regarding 2D skin cell mimetics were performed by
Rheinwald and Green and consist in the growing of human keratinocytes
monolayer culture (obtained from foreskin of newborns) deposited on
plastic culture plates (Rheinwald and Green, 1977). As a further
advance, the authors included a primary fibroblast cell line (3T3) in
the keratinocyte cultures in order to close resemble the skin.
Furthermore, fibroblasts are important for the growth of keratinocyte
cultures, namely by the secretion of extracellular matrix components,
like collagen (Rheinwald and Green, 1975).
The successfully use of 2D skin mimetic models has been reported in many
studies for skin irritation and drug development, as referred in (Ponec,
1992, Amelian et al., 2017). Silva and co-workers established anin vitro epidermal monolayer using human keratinocytes (HaCaT
cells) differentiated in a high calcium concentration medium, and
applied it in the evaluation of drug delivery systems, namely
lipid-based nanocarriers (Silva et al., 2017).
Despite the good performance of 2D skin mimetic models, 3D cellular
models represent more precisely the human skin due to complexity and
stiffness. Bioengineered skin can be obtained by the building of
reconstructed skin models which constitutes artificially fabricated skin
mimetics. 3D (single or multi-cell type) reconstructed skin models
consist in the deposition of different layers of human cells in culture
on a polymeric matrix, thus conducing to the production of a more
complex and interactive system. In some recent studies, the
incorporation of melanocytes (Min et al., 2018, Dai et al., 2018a),
adipocytes (Klar et al., 2017) and endothelial cells (Dai et al., 2018b)
in 3D skin mimetic models has already been reported.
The main differences between 2D and 3D skin models and the advantages of
each class of models are depicted in Table 2.
Table 2 – Main characteristics of 2D and 3D cell-based skin
mimetic models.