In vitro safety assessment of papain on human skin : A qualitative Light and Transmission Electron Microscopy (TEM) study

Revista Brasileira de Ciências Farmacêuticas
Brazilian Journal of Pharmaceutical Sciences
vol. 44, n. 1, jan./mar., 2008
In vitro safety assessment of papain on human skin: A qualitative Light and
Transmission Electron Microscopy (TEM) study
Patrícia Santos Lopes1, Gabriele Wander Ruas2, André Rolim Baby1,*, Claudinéia Aparecida Sales
de Oliveira Pinto1, Ii-sei Watanabe3, Maria Valéria Robles Velasco1, Telma Mary Kaneko1
1Department of Pharmacy, School of Pharmaceutical Sciences, University of São Paulo,2Faculty of Pharmaceutical
Sciences Oswaldo Cruz, 3Institute of Biomedical Sciences, University of São Paulo
Papain is a thiol proteolytic enzyme widely used in dermatology
Uniterms
• Transmission electron
that found applications in wound treatment. Recently, papain was
• microscopy/ qualitative
also used as absorption enhancer which can modify the peptide/
• analysis
protein material in the bilayer domain. We investigated papain safety
• Light microscopy/
*Correspondence:
A. R. Baby
using human skin that was exposed to papain in vitro at different
• qualitative analysis
Laboratory of Cosmetology
times: 4, 24 and 48 hours. The samples were examined using Light
• Stratum corneum
Department of Pharmacy
• Papain/ cosmetic use
School of Pharmaceutical Sciences
and Transmission Electron Microscopy (TEM) to study of the
• Human skin/ cosmetology
University of São Paulo
mechanisms involved in enhancer-skin interaction. After 24 hours,
Prof. Lineu Prestes, n.580 - Blocos 13/15
changes occurred in corneosomes. However, samples of 48 hours
Cidade Universitária,
05508-900 - São Paulo - SP, Brazil
did not show major changes in agreement with the control. These
E-mail: andrerb@usp.br;
findings indicated that papain could be used safely onto the skin.
andre_rolim@uol.com.br
INTRODUCTION
lamellar disks that are extruded from the uppermost cells of
the stratum granulosum (Bergh et al., 1999).
Human skin, and specifically the stratum corneum, as
Enzymes can interfere on the percutaneous absorption
an effective and selective barrier, provides the major control
of drugs by two ways: like a penetration enhancer or
to chemical permeation (Barry, 2001; Elias, 2005; Baby et
producing a delayed absorption. Papain is a thiol proteolytic
al., 2006). The penetration process presents three potential
enzyme widely used in dermatology that has found
pathways to the viable tissue – through hair follicles with
application in wound treatment. Recently, papain was also
associated sebaceous glands, via sweat ducts or across
used as absorption enhancer which can modify the peptide/
continuous stratum corneum. The last one is the choice of
protein material in the bilayer domain (Fein et al., 2005;
most of the compounds, which can bypass through the
Pinto et al., 2007).
corneocytes (transcellular microroute) or through regions
The stratum corneum contains several proteases that
of the semicrystalline gel composed of multiple liquid
may play a central role in the desquamation process. The first
bilayers of ceramides, fatty acids, cholesterol and
enzyme discovered was Stratum Corneum Chymotryptic
cholesterol esters (intercellular microroute) (Barry, 2001).
Enzyme (SCCE ) followed by Stratum Corneum Tryptic
The stratum corneum is regarded as a heterogeneous two-
Enzyme (SCTE). Both of them may be involved in this
compartment system composed of keratin-filled
complex process (Marzulli, Maibach, 1996; Caubet et al.,
corneocytes, embedded in a lipid-enriched intercellular
2004; Brattsand et al., 2005). Bernard and co-workers
matrix. The lipid lamellae are formed by rearrangement of
(2003) have described a thiol protease, probably expressed

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152
P. S. Lopes, G. W. Ruas, A. R. Baby, C. A. S. O. Pinto, I. Watanabe, M. V. R. Velasco T. M. Kaneko
,
as a pro-enzyme, in the lower layers of the epidermis and in
a JEOL 1010 transmission electron microscope and
part activated by an unidentified mechanism in the upper
photographed with Kodak Electron Microscope – Estar
layers during differentiation of the keratinocytes. Therefore,
thick base 4489, 100, 8.3 x 10.2 cm and stored in the
papain, a thiol protease, could be a safe penetration enhancer
laboratory computer. The protocols for the sample
candidate (Bernard et al., 2003).
preparations were the same used in the Anatomy
The aim of this research work was to study the
Department of Biomedical Institute of Sciences – ICB III.
possible behavior by which papain could act as a safe
The experiments have been carried out under approval
penetration enhancer using Light and Transmission
of Ethic Committee of Department of Pharmacy at
Electron Microscopy (TEM).
University of São Paulo, Brazil (CEP/ FCF/USP n º 229).
MATERIAL AND METHODS
RESULTS AND DISCUSSION
Human skin, obtained from breast plastic surgery,
According to the results, corneosomes (or corneo-
was cleaned and cut in small pieces measuring 2 cm2 and
desmosomes) were affected by papain when compared to
placed in 60 mm culture dishes, processed the same day of
control. Figure 1 illustrates the untreated epidermis
the surgery. Subcutaneous fat was removed and the skin
(control). Corneosomes in untreated epidermis was
cleaned with saline solution. A solution of 0.2% (w/v)
characterized by an extracellular electron dense plug,
papain + 0.04% (w/v) cystein in KGM (keratinocytes
which merged imperceptibly with the outer lipid envelope
growth medium) was applied to the stratum corneum (1.96
leaflets of corneocytes (Figure 1B).
cm2 – steel rings) for different periods of contact: 4, 24 and
Corneodesmosomes are intercellular junctions of the
48 h. A control was run at the same conditions, without
tissue from the molecular modification of desmosomes and
papain and cystein.
persist in the stratum corneum up to its surface (Haftek et
Skin samples were removed immediately following
al., 1999). The desmosome or macula adherens forms
the application period and were fixed in 5 mL of
plaque-like structures that, in thin sections, have an
Karnovsky’s fixative solution overnight at 25 °C. Samples
appearance similar to the zonula adherens, but the
were washed in phosphate-buffered saline (PBS) pH 7.4 for
desmosome contains a definite intermediate dense line that
three times and post-fixed in 2% osmium tetroxide for 2 h
lines within the intercellular space and parallels the cell
at 4 °C. A third fixative was used, 0.5% uranyl acetate, for
surface (Bozzola, Russel, 1992).
36 h. Following fixation, the specimens were washed and
The stratum corneum proteins are extensive
dehydrated in a range of ethanol solutions (70, 80, 90, 95,
crosslinked with both cell envelope and intracellular
100%), changed in propylene oxide and embedded in 1 part
proteins. Therefore, increased diffusion across the
of propylene oxide and 1 part of Spurr’s resin. During
corneocytes should have resulted from alterations in its
infiltration, the vials of tissue were gently shaken on a
structure. A decrease in crosslinking density at the cell en-
turntable that positioned the vials at about 30° from the
velope would allow diffusivity enhancement through cell
vertical, and slowly rotated the tissue. This mixture was left
(Suhonen et al., 1999).
overnight and changed for Spurr’s resin for 4 h, changed
Proteolytic digestion, after 24 h papain 0.2% (w/v)
again and left for 1 h more at 37 °C. The final step in
contact, induced dramatic changes (Figure 2). A large
embedding was curing the tissues blocks by polimerization
amount of the intercellular material was lost in the first
of the epoxy mixture. The resin was placed in polyethylene
layer of stratum corneum. Corneosomes, identified by
capsules, and the tissue cube was placed into the resin to
rudiments of their attachment plaques, were devoid of their
gradually sink in the bottom for 5 days at 60 C. Thin
plug material, most of the extracellular components were
sections were done with ultramicrotome (Leica Ultracut®
digested and corneocytes were separated (Figure 2B).
UCT), using glass lamina followed by fixing with
Details of corneodesmosomes released are showed in Figu-
chloroform which was evaporated in hot plate. Then, the
re 3, where it can be seen the amount of tonofilaments,
sections were stain with toluidine blue and they were
intermediate filaments usually composed of keratin, that
observed and photographed in the light microscope to de-
make hairpins turns at the junctional density (Bozzola,
termine which ultrathin sections should be done. Ultrathin
Russel, 1992).
sections (60 nm), obtained with diamond lamina, were
The ultra structural appearance of corneodesmosomes
stained with 4% uranyl acetate alcoholic solution for 30-40
suggested that they were modified during the transition
min and washed with ethanol, water, and next with 0.4%
between viable epidermis and cornified epidermal layers.
lead citrate for 3 min. Grids of 200 mesh were examined in
Part of this change may be explained to the incorporation of

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In vitro safety assessment of papain on human skin
153
FIGURE 1 - (A) Light microscope micrograph of human epidermis. (B) Transmission electron micrograph of control,
untreated epidermis. The corneosomes are characterized by an extracellular electron dense plug (black arrow).
FIGURE 2 - (A) Light microscope micrograph of human epidermis after 24h papain treatment. (B) Transmission electron
micrograph of 24h enzymatic treatment disrupts the corneosome bond (arrow) and modified the superficial stratum
corneum layer.
proteins. One of this is corneodesmosin, a 52-kD protein that
were leached, but the cell adhesion was still maintained by
is specifically expressed in cornified epidermal layer. It is
corneosomes (Figure 4A). The first layer remained
found intracellularly in association with lamellar bodies, in
unscathed although the next ones showed lower contact
the stratum granulosum and in the transition zone between
(Figure 4B). TEM micrographs of epithelia treatment
the stratum granulosum and the stratum corneum, coinciding
during 48 h (Figure 4B), showed that the upper layer of the
with the change in ultrastructural appearance of the
stratum corneum had no changes, while those on keratin
desmosomes, when corneodesmosin is translocated to the
cytoesqueletal appeared only in the second layer. As well,
extracellular parts of desmosomes and is continuously
it was possible to see the corneodesmosomes maintenance.
degraded to smaller constituents of the stratum corneum
The stratum corneum recovery happened after 48 h of
(Marzulli, Maibach, 1996).
treatment, as is shown in Figure 5, where it is possible to note
After 48 h, the contents of outer edges corneocytes
the existence of a major number of cells in each layer.

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P. S. Lopes, G. W. Ruas, A. R. Baby, C. A. S. O. Pinto, I. Watanabe, M. V. R. Velasco T. M. Kaneko
,
transform infrared spectroscopy (ATR-FTIR), photoacustic
spectroscopy (PAS) and Fourier transform Raman
spectroscopy (FT-Raman) (data not showed).
The desquamation process is controlled by specifics
enzymes, which hydrolyzed corneodesmosomes releasing
the dead cells in skin surface. It is precisely controlled by
a cascade of events, a molecular mechanism that is not
completely known (Bernard et al., 2003). It has been shown
that these specific enzymes are produced in keratinocytes.
The thiol group present in the active site of these enzymes
is responsible for cleaving the peptide bond, more
efficiently than triglycolic acid and cystein (Egelrud, 2000).
Papain, as a thiol proteolytic enzyme, could be acting in the
same way that these enzymes.
According to the results, TEM micrographs of
epithelia after treatment during 24 h using papain, showed
changes in the stratum corneum cohesion, this structures
lost integrity (Figure 3). On the other hand, treatment
FIGURE 3 - Transmission electron micrograph of human during 48 h showed no changes in the upper layer of the
skin after treatment during 24 hours using papain 0.2% (p/
stratum corneum (Figure 4).
v). Arrow indicates an amount of tonofilaments.
CONCLUSION
The cause of these in vitro changes is unknown, but
Papain 0.2% (w/v) promoted a proteolytic digestion
they may reflect activation of endogenous mechanisms that
of stratum corneum first layers and induced a decreased
could neutralize or recover the corneocytes adhesion. These
crosslinking in corneosomes region. In the treated samples,
results were substantiated with cell culture and corroborated
after 48 h, the stratum corneum was recovered. These
data from biophysics techniques, like differential scanning
results suggested that papain could be used safely used due
calorimetry (DSC), attenuated total reflectance Fourier
to reversibility of barrier function of stratum corneum.
FIGURE 4 - (A) Light microscope micrograph of human epidermis after 48h papain treatment. (B) Transmission electron
micrograph of stratum corneum first layers after 48 hours. Corneosomes bonds (black arrow) appeared recovered.

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In vitro safety assessment of papain on human skin
155
cutânea e a pele. Após 24 horas de contato entre a pele a
solução de papaína, mudanças ocorreram nos
corneossomos, no entanto, as amostras em contato por 24
horas não evidenciaram alterações relevantes compara-
das com o controle. Os resultados indicaram que a
papaína poderia ser seguramente aplicada sobre a pele.
UNITERMOS: Microscopia eletrônica de transmissão/
análise qualitativa. Microscopia óptica/análise qualitati-
va. Extrato córneo. Papaína/ uso cosmético. Pele huma-
na/cosmetologia.
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Recebido para publicação em 06 de julho de 2007.
Aceito para publicação em 23 de novembro de 2007.

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