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The involvement of protein denaturing activity in the effect of surfactants on skin barrier function

  • Sanyo Women's College


Abstract Background/purpose: Detailed information on the mechanism by which surfactants affect the skin barrier function is still scarce. We investigated the contribution of protein denaturation to the effect of surfactants on barrier function. Methods: The Transmission Index method, which evaluates the actual effect of sur- factants on barrier function, was combined with a microplate assay measuring pro- tein denaturation activity. The correlation between the TI value and the reciprocal of the median effect concentration (1/EC50) was analyzed for 19 surfactants. The contribution of protein denaturation to the effect of surfactants was discussed based on the 1/EC50 per TI value. Results: A few surfactants showed high TI value. Nonionic surfactants had no effect. The EC50 varied without certain trend. For amino acid-based surfactants, there was a gradual inverse correlation between the TI value and the 1/EC50. Conclusion: The difference in the alkyl structure and the ion source affected the skin barrier function. Protein denaturing activity of the surfactant was not a critical factor. This suggests that the effect on intercellular lipids was the major factor. However, the magnitude of the contribution of protein denaturation activity varied depending on the surfactant, suggesting that each surfactant has a different mechanism of influ- ence on skin barrier function.
Skin Res Technol. 2020;00:1–8.
Surfactants are used in many daily necessities and pharmaceuti-
cal excipients bec ause of their excellent functions such as strong
washing action and emulsifying action based on amphiphilicity.
Emulsifiers, solubilizers, stabilizers, and other functions of cos-
metics and pharmaceuticals are one of the most important roles
of surfactant s.1,2 They are known to affect skin barrier function3-5
and in recent years have attracted attention for their function as
transdermal absorption enhancers. This function is made possible by
the formation of surfactant-based drug carriers and their effects on
skin barrier function.6-8 Goto's group established a solid-in-oil (S/O)
nanocarrier technology using a surfactant for transdermal deliver-
ing substances with high physiological activity, such as nucleic acids
and hormones, into the body.9 However, surfactants can damage the
skin.10,11 Therefore, many novel "low-irritant" surfac tants have been
proposed by using polymerized or highly biocompatible ion source
Received: 1 June 2020 
  Accepted: 20 J une 2020
DOI : 10.1111 /sr t.12 939
The involvement of protein denaturing activity in the effect of
surfactants on skin barrier function
Koji Kubota1,2 | Mana Okasaka2,3| Asami Kano2| Sadaki Takata2,3
© 2020 John Wiley & Son s A/S. Published by J ohn Wiley & Sons Ltd
1Depar tment of Pharmac y, Faculty of
Pharma cy, Iryo Sosei University, Iwaki-Cit y,
2Depar tment of Fas hion and B eauty
Science s, Facult y of Liberal Ar ts, Osaka
Shoin Wome n's Univer sity, Higashi-Osaka-
City, Japan
3Divisio n in Fashion and Beaut y Studies,
Gradu ate School of Human Sciences , Osaka
Shoin Wome n's Univer sity, Higashi-Osaka-
City, Japan
Depar tment of Pharmac y, Faculty of
Pharma cy, Iryo Sosei University, 5-5-1,
Chuo-dai Iino, Iwaki-city, Fukushima 970-
8551, Japan.
Funding information
This work w as suppo rted by JSPS KA KENHI
Grant Number JP19K14014.
Background/purpose: Detailed information on the mechanism by which surfactants
affect the skin barrier function is still scarce. We investigated the contribution of
protein denaturation to the effect of sur factants on barrier function.
Methods: The Transmission Index method, which evaluates the actual effect of sur-
factants on barrier function, was combined with a microplate assay measuring pro-
tein denaturation activity. The correlation between the TI value and the reciprocal
of the median effect concentration (1/EC50) was analyzed for 19 surfactants. The
contribution of protein denaturation to the effect of surfactants was discussed based
on the 1/EC50 per TI value.
Results: A few surfactants showed high TI value. Nonionic surfactants had no effect.
The EC50 varied without cer tain trend. For amino acid-based surfactants, there was
a gradual inverse correlation between the TI value and the 1/EC50.
Conclusion: The difference in the alkyl structure and the ion source affected the skin
barrier function. Protein denaturing activity of the surfactant was not a critical factor.
This suggests that the effect on intercellular lipids was the major factor. However, the
magnitude of the contribution of protein denaturation activity varied depending on
the surfactant, suggesting that each surfactant has a different mechanism of influ-
ence on skin barrier function.
adsorption property, alkyl structure, amino acid-base surfactant, counter ion, ionic source,
keratinocyte intercellular lipids
   KUBOTA eT Al.
The mechanism of action by which surfac tants affect skin bar-
rier function is not simple and is thought to be a result of two
primar y processes: disruption of the keratinoc ytes and removal of
intercellular lipids from the stratum corneum.10,11 The keratino-
cytes are enucleated at the final stage of skin tissue cell differen-
tiation, leaving the cytoskeletal protein keratin. In the Brick and
Mortar model, the keratinocytes form a tight struc ture with the
intercellular lipids.15-17 The effects of surfactant s on skin barrier
function can be observed using numerous methods, for example,
cell or tissue toxicity test and measuring of transepidermal water
loss (TEWL).15,18 ,19 These factors vary, and it is likely that they do
not function independently.
Previously, we developed the Transmission Index (TI) method,
which evaluates changes in the skin penetration rate of drugs as a
comprehensive measure of the effects of surfactants on the skin.20
The TI value, which comprehensively represents the degree to which
the surfactant effects skin barrier function, is high for surfactants
that are considered to result in "strong skin irritation." Interestingly,
while amino acid surfactants are considered to have low cytotoxicity
and low irritation, we previously repor ted that the TI value of the
glutamate surfactant was relatively high. We have presumed that
the glutamate surfactants have a strong affinity with the stratum
corneum of the skin sur face to reduce the skin barrier function by
adsorbing to the skin.20 In addition, Japan Ministr y of Economy,
Trade and Industry mentioned the anti-virus activity of quaternary
ammonium salt type surfactant at April 202021; however, the effect
on the skin barrier function of them was not considered. Hence, we
investigate the effect of quaternary ammonium salt type surfactant
on skin barrier function.22 In this report, we studied the mecha-
nism in addition to the evaluation of the effects on the skin barrier
In this study, we measured the contribution of protein denatur-
ation to clarify the effects of surfactants on skin barrier function.
The protein denaturing activity was measured by calculating the
50% effective concentration (EC50) by a microplate assay using
a serially diluted surfactant. In this study, we selected hemoglo-
bin, a pigmentary protein, as the target for protein denaturation
activity and measured the change in absorbance upon addition of
various concentrations of surfactant.23 This experiment was per-
formed on several cationic, amphoteric, and nonionic surfactants,
in addition to 12 anionic surfactants whose TI values were pre-
viously reported. The sur factants were also evaluated based on
the TI method. Following calculation of the EC50, the correlation
between the TI value and the ef fect of the surfactant on protein
denaturation was determined. The experiments were conducted
with due consideration of the elimination of pain of laboratory
animals and reduction of use of them. The mice used in the ex-
periments were carefully bred and properly euthanized by animal
husbandry experts. Extraction of skin samples was done carefully
and reliably by experts in animal surgery. Experiment samples
were split to maximize performance from the smallest laboratory
animal samples. Individual and parts differences were statistically
verified to ensure scientific accuracy.
2.1 | Transmission index method
The TI method, which we reported previously,20 was used to
evaluate the effect of two cationic surfactants, three amphoteric
surfactant s, and two nonionic surfactant s on the skin barrier
function. Dimethyldistearylammonium chloride (Sanyo Chemical
Industries, Ltd.) and trimethylstearylammonium chloride (Toho
Chemical Industry Co., Ltd.) were prepared as cationic sur-
factants. Sodium lauroamphoacetate (Toho), lauryl dimethylami-
noacetic acid betaine (Toho), and cocamidopropyl betaine (BASF
Japan) were prepared as amphoteric surfactants. PEG-60 glyc-
eryl isostearate (Nihon Emulsion Co., Ltd.) and PEG/PPG-25/30
copolymer (ADEKA Corporation) were prepared as nonionic
surfactant s. Each surfac tant was dissolved or diluted in deion-
ized water to a final concentration of 1% to form a homogene-
ous solution. Laboskin® (Hoshino Laboratory Animals, Inc), the
back skin from seven-week-old male Hos:HR-1 hairless mice, was
used as the skin sample. The supplier of the laboratory animals,
Hoshino Laboratory Animals, Inc, is the right supplier, who is
certified by Japanese Society for Laboratory Animal Resources.
Skin permeability testing and concentration measurements of
the permeated drug were evaluated by high-performance liquid
chromatography (HPLC) method according to the Transmission
Index method described previously, and statistical analysis (stand-
ard error, Pearson's square test, Welch's t test) was per formed.
Methylparaben was used as a standard for measuring the time-
dependent rate of change of chemical concentrations. The steady-
state skin permeation rate (Fluxss) was represented by (Formula 1),
where Q is the concentration of methyl paraben that permeated
through the skin and t is the time in hours.
The Transmission Index was determined using (Formula 2) by si-
multaneously comparing the Fluxss of surfactant-treated skin with
the Fluxss of control (water)-treated skin.
2.2 | Evaluation of the effect of surfactants
on proteins
To evaluate the effect of sur factants on proteins, 0.2 g of the
chromoprotein hemoglobin (Sigma-Aldrich Co. LLC.) was dissolved
in pH 7.4 phosph ate -buffered saline (PBS) without Mg2+/C a2+ (Wako
Pure Chemical Industries, Ltd.) for a final weight of 10 g (2% hemo-
globin). This solution was diluted 4 0-fold with cold PBS (4°C) to give
a final concentration of 0.05% hemoglobin. These solutions were
used immediately after preparation.
ss =
Transmission Index
Nineteen surfactants were prepared and classified according
to ionic proper ties. Sodium laurate (NOF CORPOR ATION), sodium
lauryl sulfate (Kao Corporation), sodium polyox yethylene lauryle-
ther sulfate (Kao), sodium lauroyl glutamate (Ajinomoto Co., Inc),
sodium lauroyl methyl taurate (Nikko Chemicals Co., Ltd.), sodium
cocoyl alaninate (Ajinomoto), sodium cocoyl sarcosinate (Nikko),
sodium cocoyl glutamate (Ajinomoto), potassium cocoyl glutamate
(Ajinomoto), triethanolamine cocoyl glutamate (Ajinomoto), sodium
cocoyl methyltaurate (NOF), and sodium taurine cocoyl methyltau-
rate (NOF) were prepared as anionic surfactants. As cationic surfac-
tants, dimethyldistearylammonium chloride (Sanyo Chemical) and
trimethylstearylammonium chloride (Toho Chemical) were prepared.
As amphoteric surfactants, sodium lauroamphoacetate (Toho), lau-
ryl dimethylaminoacetic acid betaine (Toho), and cocamidopropyl
betaine (BASF) were prepared. PEG-60 glyceryl isostearate (Nihon
Emulsion) and PEG/PPG-25/30 copolymer (ADEKA) were prepared
as nonionic surfactant s. Each was diluted with deionized water to
2.5, 0.5, 0.1, 0.02, 0.004, and 0.0008% (w/w) solutions.
A 100 μL of the 0.05% hemoglobin solution was added to a 96-
well microplate (polystyrene, flat bottom, 400 μL/ well, Fukae Kasei
Co., Ltd.). Then, 100 μL of the serially diluted surfactant sample
solutions was added, diluting the final surfactant concentration 1:2
(1.25, 0.25, 0.04, 0.01, 0.002, 0.0004%). After allowing the reaction
to stand for 10 minutes, the mixture was stirred for 20 seconds using
a BIO RAD iMark microplate reader (Bio-Rad), and the optical den-
sity (OD) at 405 nm was measured. The measured value from which
the absorbance of the blank was subtracted was fitted to a Rodbard
function (Formula 3) using ImageJ software (National Institutes of
Health, Bethesda, MD, USA) to obtain parameters affecting the in-
fluence curve described in (Formula 3).
The median between the upper and lower limits of the plateau
of the measured values was determined as the median effect, and
the concentration at the median effect was determined as the 50%
effect concentration (EC50). The experiment was performed four
times and analyzed using statistical methods.
3.1 | Evaluation of surfactant effects on skin barrier
function by TI method
The effect on skin barrier function was evaluated by the TI method.
The previously reported TI values of the 12 kinds of anionic sur-
factants20 and four kinds of quaternary ammonium salt type sur-
factants22 are shown in Figu re 1, alo ngsid e th e TI valu es of the other
surfactant s measured in this study. The initial 12 surfactants were
arranged on the basis of the alkyl structure: (1) sodium laurate, (2) so-
dium laur yl sulfate, (3) sodium polyoxyethylene laurylether sulfate,
(4) sodium lauroyl glutamate, (5) sodium lauroyl methyl taurate, (6) so-
dium cocoyl alaninate, (7) sodium cocoyl sarcosinate, (8) sodium co-
coyl glutamate, (9) potassium cocoyl glutamate, (9) potassium cocoyl
FIGURE 1 The Fluxss of the surfactant-treated samples (gray bar) and the control samples (white bar). Each value represents the
mean ± SE calculated for at least four experimental replicates. P < .01 is indicated with (*), and P > .1 is indic ated with (†) calculated with
Welch's t test against the value of each control sample
   KUBOTA eT Al.
glutamate, (10) triethanolamine cocoyl glutamate, (11) sodium cocoyl
methyltaurate, and (12) sodium taurine cocoyl methyltaurate. The
subsequent 7 surfactants were arranged by sur fact ant properties:
cationic surfactants, (13) dimethyldistearylammonium chloride, and
(14) trimethylstearylammonium chloride; amphoteric surfactants,
(15) sodium lauroamphoacetate, (16) lauryl dimethylaminoacetic acid
betaine, and (17) cocamidopropyl betaine; and nonionic surfactants,
(18) PEG-60 glyceryl isostearate and (19) PEG/PPG-25/30 copoly-
mer. Skin was washed with either a surfactant or the control (deion-
ized water). Then, methyl paraben was added to the skin, and the
amount of methyl paraben was measured for 1-6 hours. Since the
permeation of methylparaben through the skin was constant (linear)
from 1-6 hours (dat a not shown), the permeation amount per unit
time was determined as Fluxss. Fluxss (control), which was measured
simultaneously with Fluxss (surfactant), was subjected to a signifi-
cant difference test using Welch's t test. Treatment with surfactant
resulted in significant differences (P < .05) in Fluxss for many of the
surfactant s. Treatment with (5) sodium lauroyl methyl taurate or
(15) sodium lauroamphoacetate did not significantly impact Fluxss.
Surfactants marked with (†) had almost no significant difference
with respect to Fluxss (control) (P > .1). Table 1 shows the TI values.
It was shown that (1) sodium laurate and the amphoteric surfactants
(14) trimethylstearylammonium chloride and (16) lauryl dimethylami-
noacetic acid betaine had a par ticularly strong effect on skin barrier
3.2 | Evaluation of protein denaturation activity
The OD 405 nm of the hemoglobin solution treated with (6) sodium
cocoyl alaninate is shown in Figure 2. At the highest concentrations
of sodium cocoyl alaninate (0.00 04% and 0.002%), the OD 405 nm
was measured as the upper limit of the denaturation plateau, the
same level as the surfactant blank; thus, no denaturation in hemo-
globin was observed. At the concentrations of 0.05%, 0.25%, and
1.25%, the OD 405 nm was equivalent to the value of the lower
plateau; thus, hemoglobin denaturation was saturated. Fitting
this measurement to a Rodbard function (Formula 3) using ImageJ
Software provided the parameters; a = 0.824, b = 11.02, c = 0.0022,
and d = 0.530. The correlation coefficient was 0.99. The median ef-
fect was determined as the median between the upper and lower
plateaus. The EC50 was obtained by substituting the intermediate
value of the effect into Y in (Formula 3). Other surfactants were
similarly fitted to the Rodbard function, and all were well-correlated.
TABLE 1 TI value, EC50, and SBF-PDA Index of each surfactant
Surfactant Ionic type
Amino acid
ammonium salt TI ± SE
Protein denaturing
EC50 ± SE (%)
1Sodium laurate Anionic 2. 61 ± 0.19 0.0108 ± 0.0014 35.5
2Sodium lauryl sulfate Anionic 1.41 ± 0.07 0.0022 ± 0.0000 32 7. 3
3Sodium laureth sulfate Anionic 1.48 ± 0.10 0.0051 ± 0.0002 13 0.9
4Sodium lauroyl glutamate Anionic *1.91 ± 0.18 0.0397 ± 0.0074 13.2
5 Sodium lauroyl methyl taurate Anionic * 1.20 ± 0.08 0.0175 ± 0.0005 4 7. 5
6Sodium cocoyl alaninate Anionic *0.99 ± 0.06 0.0108 ± 0.0002 93.5
7Sodium cocoyl sarcosinate Anionic *0 .95 ± 0.07 0.0118 ± 0.0004 89.4
8Sodium cocoyl glutamate Anionic * 1.80 ± 0.25 0.0119 ± 0.0001 46.6
9Potassium cocoyl glutamate Anionic *1.59 ± 0.22 0.0239 ± 0.0011 26.3
10 Triethanolamine cocoyl
Anionic *1. 51 ± 0.11 0.0121 ± 0.0001 54.7
11 Sodium cocoyl methyl taurate Anionic *1.17 ± 0 .11 0.0087 ± 0.0003 98.9
12 Sodium t aurine cocoyl methyl
Anionic *1.17 ± 0.09 0.0154 ± 0.0005 55. 8
13 Dimethyldistearylammonium
Cationic 1.31 ± 0.23 ND (>0.05) N/A
14 Trimethylstearylammonium
Cationic 3.12 ± 1.12 0.0 030 ± 0.0001 107.4
15 Sodium lauroamphoacetate Amphoteric 1. 51 ± 0.13 0.0323 ± 0.0014 20.5
16 Lauryl dimethylaminoacetic
acid betaine
Amphoteric 3.18 ± 0.15 0.0238 ± 0.0007 13.2
17 Cocamidopropyl betaine Amphoteric 1.29 ± 0.11 0.0190 ± 0.0003 40.9
18 PEG-60 glyceryl isostearate Nonionic 1.05 ± 0.11 0.0165 ± 0.0008 5 7. 0
19 PEG/PPG-25/30 copolymer Nonionic 0.85 ± 0.07 ND (>1.25) N/A
Note: Values are writ ten as the mean ± the standard error calculated for four experimental replicates.
(19) PEG/PPG-25/30 copolymer did not show hemoglobin dena-
turing ac tivity in the concentration range of this study. Therefore,
we designated the EC50 as >1.25. With (13) dimethyldistearylam-
monium chloride, precipitation occurred at concentrations of 0.25%
or more, so accurate absorbance could not be obtained and the
EC50 could not be calculated. If the absorbance at a concentration
of 0.05% or less was equivalent to the denatured protein, then the
EC50 was designated as at least 0.05% or more. Table 1 shows the
EC50 of each surfactant. Each experiment was performed four times
and analyzed using statistical methods.
The 1/EC50 was determined to ensure proportionality between
the protein denaturing activity and the TI value. Figure 3 shows a
scatter plot of the TI value and 1/EC50. The anionic surfactant so-
dium laur yl sulfate (SDS) was characterized by a relatively small TI
value bu t a lar ge 1/EC50. In contras t, the am photeric surfac ta nt la u-
ryl dimethylaminoacetate betaine had a relatively high TI value and a
low 1/EC50. The TI value and 1/EC50 of anionic surfactant s varied,
and no correlation was found between the values. For the cationic
surfactant, the TI value and the 1/EC50 of dimethyldistearylammo-
nium chloride (for which 1/EC50 could be calculated) both showed
relatively high values. The three amphoteric surfactants had a range
of TI values, but the 1/EC50 values were similar and tended to be
relatively low. PEG-60 glyceryl isostearate, for which 1/EC50 could
be calculated with a nonionic surfactant, exhibited low values for
both TI and 1/EC50.
Figure 4 shows a scatter plot of the TI values and the 1/EC50
of the amino acid-based (including β-amino acid/aminosulfate) an-
ionic surfactants. Among the amino acid surfactants, sodium lauroyl
glutamate had the greatest effect on the skin barrier function, and
sodium cocoyl methyl taurine had the strongest protein denaturing
activity. There was a gradual inverse correlation (R = .557) between
the magnitude of the ef fect on the skin barrier function and the pro-
tein denaturing activity of all nine amino acid surfactants.
We have previously shown that the practical effect of surfactants
on skin barrier function can be assessed by the TI method. In this
study, the effect of surfactants other than the previously reported
anionic surfactants on skin barrier func tion was evaluated by the TI
method. It was confirmed that the TI method is scientifically reliable
and technically stable method for assessing the effect of surfactants
other than anionic surfactants on skin penetration. Although the
number of other than anionic surfactants used on the market is not
as large as the number of anionic sur fact ants, the result s are still
meaning ful for a large number of products. The TI method is excel-
lent in that the effect of a surfactant on the skin barrier function is
evaluated as a practical phenomenon by using the skin permeation
rate of a drug as an index. This method is also applicable to artifi-
cially cultured skin tissue samples. The information obtained by this
method can be used as a substitute sample or as a basis for in silico
simulation. Therefore, it can be applied not only to the development
FIGURE 2 A representative graph depicting the change in
absorbance at 405 nm of surfactant (Sodium cocoyl alaninate)
concentration on the horizontal axis. Measurements were
carried out 4 times. Error bars indicate the standard deviation in
absorbance at each concentration
FIGURE 3 Scatter plot with the TI
value on the horizontal axis and 1/EC50
on the vertical axis. The meaning of the
symbols in the figure is shown in the
legend. The names of the surfactants
mapped to characteristic locations are
shown in the figure
   KUBOTA eT Al.
of transdermal preparations in the pharmaceutical field, but also to
the development of prescriptions for cosmetics.
Of the sur factants evaluated for their effects on skin barrier
function, the TI values of the cationic surfactant (14) stear yltrime-
thylammonium chloride and the amphoteric surfactant (16) lauryldi-
methylaminoacetate betaine were the highest. In particular, (16) had
the greatest effect on the skin barrier function among the surfac-
tants investigated so far. However, the number of moles should be
considered when evaluating the TI value. For example, of the cat-
ionic surfactants (13) distearyldimethylammonium chloride and (14)
stearyltrimethylammonium chloride, one of the methyl groups of
trimethylglycine (betaine) chloride was replaced with stearic acid
in (14), and two were replaced in (13). The molecular weight of (13)
is 586.5, and the molecular weight of (14) is 348.1 (PubChem). The
molar ratio is (13):(14) = 1:1.7; therefore, given equivalent weights
of the two compounds, the molar concentrations are different. The
ratio of the TI values of (13):(14) was 1:2.1, which is very close to
the molar ratio. Therefore, it may be appropriate to compare the
effects of surfactants on skin barrier function on a per number of
molecule basis. However, some surfactant s have polymers with
varying degrees of polymerization or hydrophilic polymers added in
an indefinite form, making it difficult to evaluate the exact number of
molecules. Therefore, the evaluation based on weight concentration
is realistic and practical for functional studies.
In this study, we obtained important information by combining
the TI method with a measurement of protein denaturing activity.
Although the protein denaturing activity of surfactants is not a criti-
cal factor for their effec t on skin barrier function, it is not completely
negligible. For cert ain surfac tants, it may even be a major factor. In
the correlation scatter plot of the TI value and 1/EC50 shown in
Figure 3, when both the TI value and the 1/EC50 value are high, it is
possible that the protein denaturing may increase drug permeability.
As a whole, if the scatter plots show a strong direct correlation, the
effect of the surfactant on the skin barrier function is likely to be due
in some part to the protein denaturing activity. However, there was
no clear general correlation between the TI value and the 1/EC50
value for the 19 surfactants in this study. At least in the surfactants
used in this study, there was no clear relationship between protein
denaturing activity and the effect on skin barrier function as a gen-
eral trend.
Both of the two nonionic surfactants (18 and 19) had low TI val-
ues and low 1/EC50 values, suggesting that no factor affects skin
barrier function other than protein denaturation. On the other hand,
for the cationic surfactant (13), both the TI value and the 1/EC50
value were high, suggesting that protein denaturing activity affected
the skin barrier function in some way. Of interest are sur factants
that exhibit (A) relatively high TI and low 1/EC50 values and (B) rel-
atively low TI and high 1/EC50 values. An inverse correlation be-
tween TI value and 1/EC50 value suggests that protein denaturing
activity is not a factor affecting skin barrier func tion. (1) and (14)
show tendency (A), and (2) shows tendency (B). We speculated that
(1) and (14) act on element s other than proteins to affect skin barrier
function. It was shown that protein denaturing activity does not af-
fect the enhancement of skin permeability of the drug for (2). A sim-
ilar tendency was observed in the correlation between the TI value
of the amino acid surfactant and the 1/EC50 value (Figure 4). In
the amino acid surfactant group, the TI value and the 1/EC50 value
showed a relatively strong negative correlation. This indicates that
for amino acid-based surfactants, protein denaturing activity is not a
factor affecting skin barrier function.
Therefore, a measure combining both the 1/EC50 and the TI was
defined as the Skin Barrier Function-Protein Denaturing Activity
Index (SBF-PDA Index). It was calculated as 1/(EC50 * TI value)
(Table 1). The relative magnitude of this value indicates the mag-
nitude that the protein denaturing activity impacts the skin barrier
function. Some surfactants showed extremely high SBF-PDA Index
values, such as (2), while others exhibited very low values, such as
(4) and (16). In the pairs (4) and (8) and the pairs (5) and (11), which
have the same structural and chemical properties other than their
alkyl groups, the laur yl group surfactant had a lower SBF-PDA Index
value. This suggests that differences in the alkyl structure affect the
SBF-PDA Index value and that the effect of the lauryl group on the
protein denaturing activity is relatively weak.
The effect of the ion source on the SBF-PDA Index value was
attributed to a combination of the sodium salt derived from the lau-
ryl group ([1], [2], [3], [4], and [5]) and the sodium salt derived from
cocoyl group ([6], [7], [8], [11], and [12]). When comparing (1), (2),
(3), (4), and (5), the SBF-PDA Index value of (2) is remarkably high. It
was shown that protein denaturation by (2) played an import ant role
in the effect on skin barrier function. Since (2) has a lauryl group,
the sulfate ion was considered to be a major effective factor.24 On
the other hand, the SBF-PDA Index value of (4) was low. As in the
case of (4), since (7) had the lowest SBF-PDA In dex value in the com-
parison between (6), (7) and (8), it is assumed that the influence of
protein denaturing activit y on the skin barrier function of the an-
ionic surfactant using glutamic acid as the ion source is relatively
small. Conversely, SBF-PDA Index values tended to be higher for
FIGURE 4 Plots of amino acid-based surfactants extracted
from Figure 3. The dot ted line indicates the approximate curve
obtained from all plots. R indicates the correlation coefficient of the
approximate curve
alanine, sarcosine, and taurine-based surfactants that had lower TI
values than glutamic acid sur factants. Differences in the ion source
of amino acid-based sur factants can lead to differences in the mech-
anisms of their effec ts on skin barrier function. In a previous report,
we mentioned that glutamate-based surfactants may affect skin bar-
rier function by adsorbing to skin surface proteins. The results of
this study further demonstrate the particularity of glutamate-based
We compared counter ion differences on SBF-PDA Index values
in (5), (6), and (7). In this comparison, the effect of potassium ion
was the smallest, and the effects of sodium ion and triethanolamine
were comparable. The lack of difference between SBF-PDA Index
values for surfactants with counter cations that were either inor-
ganic or organic for anionic surfactants indicates that the identity
of the counter cation may not affect the SBF-PDA Index. SBF-PDA
Index values are also different in comparisons bet ween sur factants
using sodium ions as counter ions; we hypothesize that there is no
relationship between the presence of sodium ions and the relation-
ship between protein denaturation and skin barrier function.
In the case of quaternary ammonium salt type surfactants, there
was no common point among the three parameters: TI value, EC50,
and SBF-PDA. There were groups with low TI values: (13) and (17),
and groups with high TI values (14) and (16). As mentioned above,
these must be considered in terms of molarity. On the other hand,
SBF-PDA Index value of (13) was significantly higher than the other
three. This indicates that the mechanism of action of (13) on the skin
barrier function is likely to be protein denaturing ac tivit y. The strong
protein denaturing activity may effectively function for the outer
coat of the virus, but at the same time, it should be noted that the
strong ef fect on the skin barrier function.
Although the structural and chemical characteristics of the sur-
factant af fec t the SBF-PDA Index value, no decisive fac tor affecting
the SBF-PDA Index value was found. The protein denaturing activity
of the surfactant was not a major factor in the effect of surfactants
on skin barrier function and was not solely determined by struc-
tural and chemical characteristics. It is extremely difficult to infer
the effects of a given surfactant on skin barrier function from the
structural and chemical properties of a surfactant. Therefore, it is
necessary to investigate the factors affecting skin barrier function
for each surfactant.
This study found that the protein denaturing activity of sur fac-
tants was not a critical factor on their effect on skin barrier func-
tion. This suggests that there is a possibility that the influence of
surfactant s on another element constituting skin barrier function,
such as the intercellular lipids, is stronger. However, because the
mechanisms of skin barrier function are complex, other fac tors may
conceivably impact its function. For example, molecular biological
effects on skin tissue differentiation and intercellular lipid secretion
may be relevant. Examination using the TI method will be effective
for clarif ying these mechanisms in future studies. Another limitation
of this study is the use of hemoglobin as the target for protein de-
naturation. While hemoglobin allowed easy detection of denatur-
ation, the main protein constituting skin barrier function is keratin.
Therefore, there is a possibilit y that the protein denaturation effect
on actual skin may not have been accurately evaluated. In addition,
our study was performed on skin samples extracted from mice, and
species differences between humans and mice cannot be ignored.
Also, the possibilit y of residue on the skin surface due to adsorption
remains. In the future, it is necessary to establish a method for more
accurately evaluating the effect of protein denaturation on skin bar-
rier function and to clarify the role of adsorption. Furthermore, the
next challenge is to investigate the effec ts of sur factants on lipids
and skin barrier function.
TI values representing the ac tual degree of the effect on the skin
barrier function were determined for a total of 19 types of ani-
onic, cationic, amphoteric, and nonionic surfactants. The EC50
for hemoglobin protein denaturation was determined using a se-
rially diluted surfactant, and the correlation with TI values was
examined. For amino acid surfactants, there was a negative cor-
relation between the effect on skin barrier function and the pro-
tein denaturing activity. For other sur factants, there was no clear
correlation between the TI value and the EC50. This suggests
that the protein denaturing activity of the surfactant was not a
major factor affecting skin barrier function. Among the effects
on skin barrier function, the magnitude of the effect by protein
denaturation was evaluated by defining a Skin Barrier Function-
Protein Denaturing Activity Index (SBF-PDA Index). As a result,
it was suggested that the effects of the alkyl structure and the
ion source may affect protein denaturation. It is presumed that
the effect of surfactants on skin barrier function is greater on
lipids. Therefore, in the future, we plan to investigate the effects
of surfactants on lipids.
Miss Asami Kano played a crucial role in this study. Unfortunately,
she passed away suddenly on April 12, 2019. Our research could not
be accomplished without her, so we express our deep appreciation
and respect for her valuable work and contribution to science and
list her as important coauthor. We would like to thank Editage (www.
edita for English language editing.
No potential conflict of interest was repor ted by authors.
Koji Kubota
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How to cite this article: Kubota K, Okasaka M, Kano A,
Takata S. The involvement of protein denaturing activity in
the effect of surfactants on skin barrier function. Skin Res
Technol. 2020;00:1–8. htt ps:// .1111/srt .12939
Background: Healthcare workers (HCWs) wash their hands with tap water (TW) and soap. However, hard TW causes dermatitis. Objectives: The present study aimed to compare the effects of ultra-pure soft water (UPSW) with those of TW on the hands of HWCs. Methods: The present study was a prospective randomized trial with a crossover design. All the nurses in the neonatal intensive care unit (NICU) at the study center were divided into Sequence 1 (UPSW to TW) or 2 (TW to UPSW) and washed their hands with TW or UPSW in alternating four-week periods with a four-week washout period. Trans-epidermal water loss (TEWL) and stratum corneum hydration (SCH) were evaluated. Skin condition was self-assessed. Results: Twenty-one and 22 nurses were assigned to Sequence 1 and Sequence 2, respectively. USPW increased SCH to a significantly greater degree than TW (mean: 26.3 μS ± 12.3 S.D.; 95% confidence interval: 1.12 to 51.54; p= .041) although it did not affect TEWL. UPSW use significantly improved the subjects' skin condition, as reflected in an overall increase in the assessment scores. Conclusions: UPSW improved SCH and the condition of hand skin. Prolonged USPW use may increase nurses' comfort during work and hand hygiene compliance. This article is protected by copyright. All rights reserved.
The packing structure of intercellular lipids in skin forms an important biological barrier but little is known about the changes in packing structure caused by surfactant-induced lipid effluence. In this study, to examine the effect of the decrease in lipids in the lipid model (LM) on the packing structure, a single component was reduced from stratum corneum intercellular LM comprising ceramide ADS, cholesterol, and palmitic acid. The packing structure was assessed using differential scanning calorimetry, Raman spectroscopy, and powder X-ray diffraction. Surfactants were applied to LM to investigate lipid elution and changes in the packing structure. The results showed that the decrease in cholesterol caused disorder in the lipid structure and the amount of cholesterol was strongly inversely correlated with the hexagonal structure ratio (RHex/Ort) in the packing structure. Furthermore, the RHex/Ort values were highly correlated with the amount of lipids eluted by surfactants, making it possible to measure lipid elution using RHex/Ort values. The methods used in this study are a useful alternative to native intercellular lipids for elucidating mechanisms underlying skin irritation due to surfactants.
3D printing hydrogel is attractive to fabricate biomimetic scaffolds for skin repair. However, hydrogels that can be directly 3D printed with excellent mechanical properties are still limited. Here, a dual crosslinked hydrogel based on dynamic oxime crosslinking and hydrophobic interaction is developed for direct extrusion printing. We apply aminooxy terminated Pluronic F127 (AOP127) and oxidized dextran (ODex) as materials. At a lower temperature (ca. 16 °C), AOP127 and ODex form hydrogel with oxime binding for extrusion printing. At higher temperature (37 °C), the PPO segments of AOP127 physically associate, forming the second crosslinking to toughen the hydrogel. The hydrogel exhibits excellent thermosensitivity and self-healability. By 3D printing of AOP127-ODex-15% hydrogel, the obtained scaffolds show high toughness and excellent cytocompatibility. After coated with fibroin, the composite constructs showed adhesion and proliferation of skin cells. The present hydrogel is promising for fabrication of tough scaffold with natural polysaccharides and synthetic polymers.
Aggregation of new multifunctional amphiphilic complexes of 1-alkyl-4-aza-1-azoniabicyclo[2.2.2]octane bromides (Alk = CnH2n+1, n = 14, 16, 18) with lanthanum nitrate, their solubilization activity and complexation ability toward biopolyanions, as well as antimicrobial properties have been studied to establish the influence of the structure of these compounds on the functional activity. The critical micelle concentration, aggregation numbers and size characteristics of metallosurfactant in aqueous solutions, complexation of metallosurfactants with oligonucleotide, DNA and bovine serum albumin (BSA) have been determined by fluorescence spectroscopy, spectrophotometry, dynamic and electrophoretic light scattering, and crcular dichroism techniques. The results have been compared with the properties of metal-free surfactant (ligand), as well as the composition of ligand with inorganic salt. Beneficial effect of the metallosurfactants hydrophobicity on the solubilizing ability of metallomicellar solutions with respect to the water-insoluble dye Orange OT and the high complexing ability of metallomicelles with respect to the DNA decamer (oligonucleotide), DNA and BSA have been established. Metallocomplex bearing hexadecyl ligand effectively interacted with BSA and bound to the tryptophan amino acid residue of the polypeptide.
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Anionic surfactants are often used for cleaning and pharmaceutical purposes because of their strong surfactancy and foaming property. However, they are rarely ingested orally, the skin is a part of the human body most affected by surfactants. Barrier function of the skin is very strong, but the anionic surfactants can cause serious damages to it. Recently, amino acid-based surfactants have attracted attention as a safer option owing to their biocompatibility. Cytotoxicity examinations revealed that the amino acid-based surfactants are superior to sulfate-based surfactants. However, a systematical and comprehensive study related to the effect of these surfactants on skin barrier function has not yet been reported. In this work, skin permeation test using the skin of hairless mice and HPLC method is carried out. The material transmission speed through skin in a steady state was different between each surfactant treatment. We performed a comprehensive analysis of the effect of surfactants on skin barrier function and defined Transmission Index as an index for the degree of effect of surfactants. Glutamate series amino acid-based surfactant were effective to Transmission Index and we guessed the cause was due to adsorption. Based on the finding this study, we suggest using adsorptive property as a measure to the effect on the skin barrier function.
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The skin barrier function has been attributed to the stratum corneum and represents a major challenge in clinical practice pertaining to cutaneous administration of drugs. Despite this, a large number of bioactive compounds have been successfully administered via cutaneous administration because of advances in the design of topical and transdermal formulations. In vitro and in vivo evaluations of these novel drug delivery systems are necessary to characterize their quality and efficacy. This review covers the most well-known methods for assessing the cutaneous absorption of drugs as an auxiliary tool for pharmaceutical formulation scientists in the design of drug delivery systems. In vitro methods as skin permeation assays using Franz-type diffusion cells, cutaneous retention and tape-stripping methods to study the cutaneous penetration of drugs, and in vivo evaluations as pre-clinical pharmacokinetic studies in animal models are discussed. Alternative approaches to cutaneous microdialysis are also covered. Recent advances in research on skin absorption of drugs and the effect of skin absorption enhancers, as investigated using confocal laser scanning microscopy, Raman confocal microscopy, and attenuated total reflectance Fourier-transform infrared spectroscopy, are reviewed. © 2016, Faculdade de Ciencias Farmaceuticas (Biblioteca). All rights reserved.
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Transdermal administration of drugs has advantages over conventional oral administration or administration using injection equipment. The route of administration reduces the opportunity for drug evacuation before systemic circulation, and enables long-lasting drug administration at a modest body concentration. In addition, the skin is an attractive route for vaccination, because there are many immune cells in the skin. Recently, solid-in-oil nanodisperison (S/O) technique has demonstrated to deliver cosmetic and pharmaceutical bioactives efficiently through the skin. S/O nanodispersions are nanosized drug carriers designed to overcome the skin barrier. This review discusses the rationale for preparation of efficient and stable S/O nanodispersions, as well as application examples in cosmetic and pharmaceutical materials including vaccines. Drug administration using a patch is user-friendly, and may improve patient compliance. The technique is a potent transcutaneous immunization method without needles.
Anionic surfactants compromise skin's barrier function by damaging stratum corneum lipids and proteins. The objective of this study was to examine anionic surfactant-induced changes in the skin's polar and transcellular pathways and the resulting impact on surfactant penetration into the skin. Three anionic surfactant formulations and one control formulation were each applied to split-thickness human cadaver skin in vitro for 24 h. Electrical conductivity of the skin, determined using a four-terminal resistance method, and water permeation across the skin, determined using a radiolabeled water tracer, were simultaneously measured at several points over the experimental period. Surfactant permeation across the skin was similarly measured using a radiolabeled sodium dodecyl sulfate tracer. Anionic surfactants rapidly enhanced skin electrical conductivity and water permeability in the excised human skin, resulting in nonlinear enhancements in surfactant permeation across the skin over time. Surfactant penetration into the skin was found to increase linearly with increasing surfactant monomer concentration. Surfactant zeta potential was found to correlate well with skin conductivity, water permeation across the skin, and surfactant permeation across the skin, particularly with long surfactant exposures. Micelle charge is a significant predictor of anionic surfactant-induced damage to the human skin, with more highly charged surfactants inducing the most damage.
Bronchopulmonary dysplasia (BPD) is a complex disorder with multiple factors implicated in its etiopathogenesis. Despite the scientific advances in the field of neonatology, the incidence of BPD has remained somewhat constant due to increased survival of extremely premature infants. Surfactant deficiency in the immature lung, exposure to invasive mechanical ventilation leading to volutrauma, barotrauma and lung inflammation are some of the critical contributing factors to the pathogenesis of BPD. Hence, strategies to prevent BPD in the postnatal period revolve around mitigation of this injury and inflammation. This article reviews the progress made in the last 5 years in the development of new preparations of surfactant, use of corticosteroids and non-invasive ventilation in the prevention of BPD. Emerging techniques of surfactant delivery through minimally invasive and non-invasive routes are also discussed.
Background: Daily skin washing routines can promote undesirable effects on skin barrier function. The stratum corneum (SC) lipid matrix is crucial for skin barrier function. Skin cleansing products are mostly composed of surfactants: surface-active molecules that interact with skin lipids in several ways. The main aim of this work was to investigate the effect produced by surfactants on skin barrier permeability. Porcine skin is a well-accepted and readily available model of the human skin barrier. The effect of two cleansing formulations (based on different surfactant mixtures) on the barrier properties of mammalian skin were evaluated. Methods: Water sorption/desorption (DVS) experiments were used to measure skin permeability. Attenuated total reflectance-Fourier transform infrared (ATR-FTIR) spectroscopy and confocal Raman were useful to study SC lipid organization. Results: The results showed that while anionic surfactants (SLS) had a negative impact on the skin barrier, with a clear increase of alkyl chain disorder; co- surfactants present in the shampoo formulation diminished the detrimental effect of their primary ionic surfactant, inducing less modification on lipid intramolecular chain disorder. Conclusions: The obtained results confirmed that the mild cleansing formulations studied had gentle interaction with skin. The capacity to discriminate between detergent systems was clearly established with both DVS and spectroscopy techniques.
Objective: Surfactants are major ingredients of body soaps and cleansers. Among them, harsh ones have been demonstrated to damage the skin. Stratum corneum (SC), the outermost barrier of skin layer, is rich in intercellular lipids. This lipid structure can be disrupted by surfactants, impairing the barrier function of the skin. Thus, we investigated the surfactant-induced disruption of the intercellular lipid structure of human SC at the molecular level using synchrotron X-ray diffraction. Methods: SC samples from the breast of female Caucasians were treated with sodium dodecyl sulfate (SDS) and analyzed by small-angle and wide-angle X-ray diffraction. Results: We found that an aqueous SDS solution affected the long lamellar structure, which became disorganized. The final disordered lipid state was reached through two or more types of structural change. We propose that the disordered lipid state results from incorporation of SDS into the long lamellar structure. In contrast, the lattice constants in the short lamellar and the hydrocarbon-chain packing structures remained almost unchanged after SDS treatment. Conclusion: We conclude that the disruption of the long lamellar structure plays a key role in the damage to the SC caused by detergents. To our knowledge, this is the first report to clarify the details of the disorganization of the intercellular lipid structure upon surfactant application. The knowledge obtained herein may allow the development of skin restoration methods and cleanser products that do not affect skin barrier functions. This article is protected by copyright. All rights reserved.
Due to the ease of skin accessibility, a large variety of invasive and noninvasive in vitro and in vivo methods have been developed to study barrier function. The measurement of the transepidermal water loss (TEWL) is most widely used in clinical studies. The different methods of determining TEWL, as well as skin hydration, skin pH, tape stripping and other modern less widely used methods to assess skin barrier function, are reviewed, including Raman spectroscopy and imaging methods such as optical coherence tomography and laser scanning microscopy. The modern imaging methods are important developments in the last decades which, however, determine the structure and, hence, cannot replace the measurement of TEWL in questions related to function.