Colloids and Surfaces B: Biointerfaces 179 (2019) 242–249

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Colloids and Surfaces B: Biointerfaces

journal homepage: www.elsevier.com/locate/colsurfb

Evaluation of the biocompatibility and skin hydration potential of vitamin E- T

loaded lipid nanosystems formulations: In vitro and human in vivo studies
⁎
S. Vaza, R. Silvab, M.H. Amarala, E. Martinsb, J.M. Sousa Loboa, A.C. Silvaa,c,
a
UCIBIO, REQUIMTE, Laboratory of Pharmaceutical Technology/Centre of Research in Pharmaceutical Sciences, Faculty of Pharmacy, Porto University, Porto, Portugal
b
UCIBIO, REQUIMTE, Laboratory of Toxicology, Department of Biological Sciences, Faculty of Pharmacy, Porto University, Porto, Portugal
c
FP-ENAS (UFP Energy, Environment and Health Research Unit), CEBIMED (Biomedical Research Centre), Faculty of Health Sciences, Fernando Pessoa University, Porto,
Portugal

A R T I C LE I N FO A B S T R A C T

Keywords: Lipid-based nanosystems, such as nanostructured lipid carriers (NLC) and nanoemulsions (NE) have been de-
Lipid-based nanosystems scribed as promising alternatives to conventional formulations for increase skin hydration. Besides, these sys-
Nanostructured lipid carriers tems have been used as efficient vehicles for lipophilic molecules that improve skin properties (e.g. vitamin E).
Nanoemulsions In this study, we performed comparative investigations between hydrogels formulations containing vitamin
Vitamin E
E-loaded NLC (HG-NLCVE) and vitamin E-loaded nanoemulsion (HG-NEVE). The experiments started with par-
Cutaneous application
Hydrogel
ticle size measurements, which showed no significant differences between nanoparticles/nanodroplets sizes after
Biocompatibility incorporation in the hydrogel net (386 nm vs. 397 nm for HG-NLCVE and 402 nm vs. 514 nm for HG-NEVE).
Skin hydration Afterwards, in vitro biocompatibility studies in human keratinocytes were carried out, being observed that the
lipid-based nanosystems were more cytotoxic for the cells before incorporation in the hydrogel. Finally, the
formulations hydration potential and sensory attributes for skin application were evaluated by in vitro occlusion
tests and in vivo human experiments. The results showed that the HG-NLCVE exhibited the best occlusive
properties, whereas the HG-NEVE performed a faster skin hydration effect. Furthermore, the latter was selected
as the most attractive for skin application, although the HG-NLCVE was described as more suitable to obtain a
long-lasting effect. This study demonstrated the in vitro and in vivo safety and hydration potential of hydrogels
containing vitamin E-loaded lipid-based nanosystems. These results establish a basis to assess the cutaneous use
of these systems, despite more in vivo experiments, for longer periods and in more volunteers, are required before
commercialization.

1. Introduction
The skin plays a vital role in body physiology, being constituted by
dermis, epidermis and ending with the stratum corneum that acts as a
barrier to the exterior [1]. Thereby, in a healthy skin, the stratum cor-
neum hinders the entry of cosmetic actives and drugs, and several
strategies have been proposed to circumvent this limitation. In addi-
tion, other important function of the stratum corneum is the main-
tenance of the skin hydration that is related to the presence of the
natural moisturizing factors and lipids, which originate an occlusive
effect that prevents the transepidermal water loss (TEWL), supporting
the skin water content. Under dry-skin conditions, named xerosis, the
stratum corneum barrier function changes and several undesired con-
ditions might occur, starting with superficial dehydration, release of
inflammatory mediators, hyperproliferation of keratinocytes and dis-
ruption of the epidermal differentiation. Further changes on lipid
structure and enzymatic activity also occur. To overcome this problem,
moisturizing formulations (e.g. creams, ointments, gels and lotions)
containing humectants, occlusives and emollients are topically applied.
In this area, the development of alternative formulations containing
nanosystems, which can improve skin hydration for longer periods and/
or recover its barrier, has been extensively studied. Among these, the
use of lipid nanosystems (e.g. nanoemulsions and lipid nanoparticles)
seems promising, due to the lipid nature of the stratum corneum and the
high biocompatibility of these systems [2–6].
The interest on evaluating the skin hydration status has been
growing, to recognize the normal hydration levels or evaluate the ef-
fectiveness of moisturizing formulations. In this sense, some researchers
have suggested the use of different in vitro and in vivo methods. For
example, Müller and co-workers [7,8] carried out in vitro experiments
to predict the occlusion effect of lipid nanoparticles formulations on the
skin. Nonetheless, despite these tests minimize the required number of

⁎
Corresponding author.
E-mail address: ana.silva@ff.up.pt (A.C. Silva).

https://doi.org/10.1016/j.colsurfb.2019.03.036
Received 19 November 2018; Received in revised form 14 March 2019; Accepted 15 March 2019
Available online 03 April 2019
0927-7765/ © 2019 Elsevier B.V. All rights reserved.
S. Vaz, et al. Colloids and Surfaces B: Biointerfaces 179 (2019) 242–249

animal and human experiments, in vivo studies are always necessary. containing vitamin E, and suggested its suitability for use in cosmetics
Common in vivo skin hydration tests are non-invasive and consist of or dermatological products [6]. In this work, we continue these in vitro
electrometric techniques based on the measurement of skin surface studies, performing comparative investigations between hydrogel for-
parameters, after applying an electrical charge. The hydration state of mulations containing vitamin E-loaded NLC and vitamin E-loaded na-
the skin, which provides information about the stratum corneum barrier noemulsion. Afterwards, the skin hydration potential of both formula-
function, can be evaluated by corneometry, measuring the skin surface tions was evaluated, by in vitro occlusion tests and in vivo human
capacitance, and by the assessment of the TEWL, measuring the gra- experiments. Finally, the formulation with the best sensory attributes
dient of water evaporation from the skin. These methods can also be for skin application was assessed. Thereby, the main objective of this
employed to fast measure the moisturizing efficacy of topical for- work was to improve the knowledge about the in vivo behavior of vi-
mulations [9,10]. tamin E-loaded lipid-based nanosystems formulations, regarding the
Nanoemulsions are colloidal systems typically formed by oil dro- hydration potential and sensory attributes.
plets dispersed in water (oil-in-water, O/W) and stabilized by one or
two surfactants, despite water droplets dispersed in oil (water-in-oil, 2. Materials and methods
W/O) might be used. Generally, nanoemulsions droplets range from few
nanometers up to 200 nm and, sometimes, up to 1000 nm. The ex- 2.1. Materials
cipients used in nanoemulsions are biocompatible and biodegradable,
which means that an absence of toxicity is expected. Depending on their Precirol® ATO 5 (glyceryl palmitostearate) and Miglyol® 812
composition, nanoemulsions have low viscosity and transparent or (medium chain triglycerides of caprylic and capric acids) were acquired
opaque appearance. Although the first term used to refer these systems from Gattefossé (France), Cetrimide® (trimethyltetradecylammonium)
was “microemulsions”, due to the nanometric size of the oil droplets, was obtained from José M. Vaz Pereira, SA (Portugal), Tween® 80
current researchers usually prefer to use the term “nanoemulsions” (polysorbate 80) and alfa tocopherol acetate (vitamin E) were pur-
[4,11–13]. According to their advantageous characteristics over tradi- chased from Acofarma (Spain). For the hydrogels preparation, Guinama
tional systems and other colloidal carriers, the use of nanoemulsions (Spain) provided the gelling agent PFC® (carbomer 2001) and trietha-
has been proposed for improve drugs and cosmetics delivery, particu- nolamine was purchased from Acofarma (Spain). The water used in all
larly for lipophilic molecules that can be incorporated and protected in experiments was purified, obtained from a Direct-Q® Ultrapure Water
the inner phase (i.e. oil droplets) of O/W nanoemulsions. Furthermore, Systems, Merck Millipore (Germany). Reagents used in cell culture,
these systems can be prepared by means of different formulations (e.g. including Dulbecco's modified Eagle's medium (DMEM) with 4.5 g/L
gels, creams, foams and sprays), improving sensorial attributes and glucose and GlutaMAX™, fetal bovine serum (FBS), 0.25% trypsin/
facilitating their topical application [11,14]. 1 mM EDTA and antibiotic (10,000 U/mL penicillin, 10,000 μg/mL
Lipid nanoparticles (solid lipid nanoparticles, SLN and nanos- streptomycin) were purchased from Gibco Laboratories (Alfagene,
tructured lipid carriers, NLC) arise from the concept of O/W nanoe- Invitrogene, Portugal). Dimethyl sulfoxide (DMSO) was obtained from
mulsions, replacing the oil by a solid lipid. In the case of SLN, the oil is Sigma–Aldrich, Inc. (St. Louis, MO, USA). Hank's balanced salt solutions
totally changed by a solid lipid, whereas for the NLC only part of the oil (HBSS) with and without calcium and magnesium [HBSS (+/+) and
is substituted. The matrix of SLN is composed by one solid lipid, (−/−), respectively] were purchased from Biochrom GmbH (Berlin,
whereas a mixture of a solid lipid with a liquid lipid composes the NLC Germany).
matrix. Nonetheless, the amount of liquid lipid existing in the NLC is
always lower than the amount of solid lipid, which provides a solid
2.2. Methods
consistency to the final nanoparticle. Thus, the difference between
these two types of lipid nanoparticles is in the inner structure of the
2.2.1. Preparation of vitamin E-loaded NLC and nanoemulsion
solid matrix. Compared to nanoemulsions and other colloidal carriers,
The vitamin E concentration was selected from screening studies
the advantages of using lipid nanoparticles are related to the higher
with mixtures containing different proportions of solid lipid (Precirol®
protection and controlled release effect of the incorporated molecules
ATO 5) – liquid lipid (vitamin E): 50-50; 60-40; 70-30; 80-20 and 90-10
and the increased long-term stability. Over the years, when compared
[10]. The final solid mixture with higher amount of liquid lipid (70:30)
to SLN, NLC has been showing improved properties, being, therefore,
was selected to prepare the lipid nanosystems.
preferred [5,15]. The cutaneous application of lipid nanoparticles for-
Vitamin E-loaded NLC dispersion and vitamin E-loaded nanoemul-
mulations has been showing promising results for improve the delivery
sion (Table 1) were prepared from the method previously employed by
of cosmetics and drugs. Besides, regarding skin delivery, local appli-
Silva et al. [20]. In brief, lipids and aqueous phase were heated at
cation has been showing better results over transdermal, which might
be related to the lipid nanoparticles size that is usually over 100 nm,
Table 1
avoiding reaching the deeper skin layers and remaining in the stratum
Composition (%, w/w) of the tested formulations: NLC-loaded vitamin E
corneum and superior epidermis [5].
(NLCVE), nanoemulsion-loaded vitamin E (NEVE), hydrogel based on NLC-
Vitamin E has been used for long time in skin formulations, due to loaded vitamin E (HG-NLCVE) and hydrogel based on NE-loaded vitamin E (HG-
its high antioxidant and protecting activity, being applied in several NEVE).
photo-aging cosmetics, and suggested for the treatment of cancer and
NLCVE NEVE HG-NLCVE HG-NEVE
improvement of the skin barrier [6,16–18]. Regarding the lipophilic
character of the vitamin E molecule, it has been transported in lipid (%, w/w)
nanoparticles for improve topical delivery. The investigations showed
that these systems increase skin photo-protection, occlusion, and per- Precirol® ATO 5 7.0 – – –
Miglyol-812® – 7.0 – –
meation of vitamin E, without originating toxicity and irritation.
Vitamin E 3.0 3.0 – –
However, there is a lack of toxicological and human studies for evaluate Cetrimide® 0.5 0.5 – –
more efficiently the safety and effectiveness of these formulations. In Tween® 80 2.5 2.5 – –
fact, a recent review reports only one study with human experiments. Purified water 87.0 87.0 – –
Furthermore, the need of increase the investigations with semi-solid NLCVE – – 99.5 –
NEVE – – – 99.5
formulations based on vitamin E-loaded lipid nanoparticles has been
PFC® – – 0.50 0.5
pointed [19]. In our previous research, we demonstrated the in vitro Triethanolamine – – q.s. pH 6.5 q.s. pH 6.5
biocompatibility and non-irritant potential of a hydrogel based on NLC

243
S. Vaz, et al.
5–10 °C above the solid lipid melting point. Afterwards, the aqueous
phase was added to the melted lipids and homogenized by high-speed
stirring, using an Ultra-Turrax® T25 (IKA Labortechnik, Staufen, Ger-
many), at 13,500 rpm, for 5 min. The obtained oil-in-water (O/W)
emulsion was immediately placed under a probe sonicator (Vibro Cell
VCX 130, 6 mm probe, Sonics & Materials, Newtown, CT, USA), with a
power-output amplitude of 70%, for 15 min. The formed hot O/W na-
noemulsion was transferred to glass vials and drastically cooled down.
2.2.2. Preparation of the hydrogels containing vitamin E-loaded NLC and
vitamin E-loaded nanoemulsion
The composition of the hydrogels based on vitamin E-loaded NLC
and vitamin E-loaded nanoemulsion is shown in Table 1 and was se-
lected from our previous study [6]. For the hydrogels preparation, the
gelling agent PFC® was first powdered in a porcelain mortar, and the
NLC or the nanoemulsion was added, followed by neutralization with
triethanolamine to increase consistency.
2.2.3. Size measurements
Mastersizer 3000 (Malvern, United Kingdom), using laser dif-
fractometry (LD) technique, performed particle/droplet size measure-
ments. The used indexes were particle refractive of 1.4 and absorption
of 0.001, and water dispersant refractive of 1.33, being applied the
Mie's theory.
To check alterations on particle/droplet dimensions after hydrogel
incorporation, size measurements were performed before and after the
hydrogels preparation. For the latter, the hydrogel network was de-
stroyed by dilution with purified water, following by 5 min of sonica-
tion. The particle/droplet size was assessed by the values of volume
distribution (50 and 90%), indicating the particles/droplets with dia-
meter size equal or lower the given percentages. All the results were
reported as the mean ± standard deviation (SD) of five replicates
(n = 5).
2.2.4. Biocompatibility studies
For the evaluation of the formulations biocompatibility, im-
mortalized human keratinocytes (HaCa T cells) were used. The cells
were routinely cultured in 75 cm2 flasks and maintained in a 5%
CO2–95% air atmosphere, at 37 °C. Dulbecco's Modified Eagle's Medium
(DMEM) supplemented with 10% heat inactivated fetal bovine serum
(FBS), 100 U/mL penicillin and 100 μg/mL streptomycin was used, and
the medium was changed every 2 days. Cultures were passaged weekly
by trypsinization (0.25% trypsin/1 mM EDTA). The cytotoxicity of the
developed formulations (NLCVE, HG-NLCVE, NEVE and HG-NEVE) was
evaluated, 24 h after exposure, by the 3-(4,5-dimethylthiazol-2-yl)-2,5-
diphenyltetrazolium bromide (MTT) reduction and by the neutral red
(NR) uptake assays. The concentrations tested (0–100 μg/mL) were
selected from the estimated medium value (2 mg/cm2) for the amount
of semi-solid formulation that should be applied to the skin [21]. In all
experiments, Triton™ X-100 (1%) was used as positive control.
2.2.4.1. MTT reduction assay. The cytotoxicity of the developed
formulations was evaluated by the MTT reduction assay in which the
mitochondrial activity can be used to estimate cell viability [22–24].
The cells were trypsinized, re-suspended in cell culture media, counted
and seeded in 96-well plates at a density of 20,000 cells/well. One day
after seeding, the cells were exposed to the tested formulations
(0–100 μg/mL) in fresh cell culture medium. Twenty-four hours after
exposure, the cell culture medium was removed, followed by the
addition of fresh cell culture medium containing 0.5 mg/mL MTT and
incubation, at 37 °C, in a humidified 5% CO2–95% air atmosphere, for
1 h. The cell culture medium was then removed and the formed
formazan crystals dissolved in 100% DMSO. The absorbance was
measured at 550 nm in a multi-well plate reader (PowerWave-X,
BioTek Instruments, Vermont, USA). The percentage of MTT
reduction relative to that of the control cells (0 μg/mL) was used as
244
Colloids and Surfaces B: Biointerfaces 179 (2019) 242–249
the cytotoxicity measure.
2.2.4.2. Neutral red (NR) uptake assay. The cytotoxicity of the
developed formulations was also evaluated by the NR uptake assay,
which is based on the ability of viable cells to incorporate and bind the
supravital dye NR in the lysosomes, providing a quantitative estimation
of the number of viable cells in a culture [25,26]. Briefly, the cells were
seeded in 96-well plates at a density of 20,000 cells/well and, 24 h
later, exposed to the tested formulations (0–100 μg/mL) in fresh cell
culture medium. After 24 h of exposure, the cell culture medium was
removed and the cells incubated with NR (50 μg/mL in cell culture
medium), at 37 °C, in a humidified, 5% CO2–95% air atmosphere, for
90 minutes. After this incubation period, the cell culture medium was
removed, the dye absorbed only by viable cells extracted (with absolute
ethyl alcohol/distilled water (1:1) containing 5% acetic acid), and the
absorbance measured at 540 nm in a multi-well plate reader
(PowerWave-X, BioTek Instruments, Vermont, USA). The percentage
of NR uptake relative to that of the control cells (0 μg/mL) was used as
the cytotoxicity measure.
2.2.5. Evaluation of the hydration potential of the hydrogels containing
vitamin E-loaded NLC and nanoemulsion
2.2.5.1. In vitro occlusion test. An in vitro occlusion test was performed
to estimate the occlusive properties of the hydrogels containing vitamin
E-loaded NLC and vitamin E-loaded nanoemulsion [27]. For the
experiments, a beaker containing 50 ml of water was sealed with
filter paper. Afterwards, 1.5 g of formulation was spread above the
filter paper and the beaker was stored at 32 ± 1 °C for 24 h. A sealed
beaker filled with water and without applied formulation was used as
reference. To evaluate the water loss by evaporation, the beakers were
weighted after 24 h of storage. Each test was performed in triplicate
(n = 3) and the results were presented as mean ± SD.
The occlusion factor (F) was calculated according to the following
equation:
(A − B ) ⎤
F (%) = ⎡ *100
⎣ A ⎦
where A is the water loss from the reference beaker, and B is the water
loss from the beaker with formulation.
2.2.5.2. In vivo human experiments
2.2.5.2.1. Design of the study. A single-blinded controlled study was
carried out with twenty-five healthy-skin Caucasian volunteers, of both
sexes, aged between 19 and 39 years. Before the trials, all the
participants read and signed the informed consent, which
accomplishes the ethical principles of the Declaration of Helsinki for
research involving human subjects [28]. The name of the study
(Sensorial evaluation and assessment of the skin hydration potential
of semi-solid formulations based on lipid nanosystems) and the name of
the investigator responsible for the experiments were presented in the
top of the informed consent. The date and signatures of the volunteer's
and the responsible for the study were placed in the bottom of the
declaration.
2.2.5.3. Assessment of skin hydration. On the day of the study, the
volunteers did not applied any product on both forearms, being the
exclusion criteria the existence of damage at the tested skin local.
Prior to measurements, the volunteers rest for at least 30 minutes in
a room with controlled temperature (20 ± 1 °C) and relative humidity
(60 ± 1%). The procedures started by defining three application sites
of 4 cm2 on each volunteer forearm, with a distance of 5 cm above the
wrist and 5 cm below the elbow, followed by the measurement of the
skin hydration and skin-barrier function (T0). Afterwards, 20 μl of each
tested formulation was applied in two of these locals, performing
twenty clockwise movements, being the third used as negative control.
S. Vaz, et al.
One hour after this application, the immediate hydration effect was
evaluated by measuring the skin hydration and skin-barrier function
(T60).
For the assessment of skin hydration, a Multi Probe Adapter System
was used (Courage + Khazaka Electronics, Cologne, Germany), which
is connected to a probe used for the determination of the skin surface
hydration (Corneometer® CM 825), and a probe (Tewameter® TM 210)
used for the evaluation of the skin-barrier function, by measurement of
the transepidermal water loss (TEWL). All the measurements were
performed in triplicate (mean ± SD, n = 3).
2.2.5.4. Sensory analysis. Sensory analysis was carried out to evaluate
which of the tested hydrogel formulation presents the best attributes for
skin application. The followed procedures were based on the guidelines
of the ASTM E1490-03 (Standard Practice for Descriptive Skinfeel
Analysis of Creams and Lotions) [29]. The study was single-blinded and
the panel consist of twenty-five volunteers, of both sexes, with ages
ranging from 19 to 39 years. The evaluations were carried out in a room
with controlled temperature (20 ± 1 °C) and relative humidity
(60 ± 1%).
The study consisted on evaluating the skinfeel properties of the
hydrogels containing vitamin E-loaded NLC and vitamin E-loaded na-
noemulsion, using a descriptive analysis method that quantifies several
sensory attributes: spreadability, adhesiveness, ease of removing from
the container, moisture, greasiness and glossiness. The scores scale
ranged from 0 (minimum) to 10 (maximum).
2.2.6. Statistical analysis
All statistical calculations were performed with the GraphPad Prism
version 6.00 for Windows (GraphPad Software, San Diego, CA, USA).
Normality of the data distribution was assessed by three different tests:
KS normality test, D’Agostino & Pearson omnibus normality test and
Shapiro–Wilk normality test. For data with parametric distribution,
statistical comparisons were made using the parametric method of one-
way ANOVA, followed by the Dunnett's multiple comparisons test. For
data with only two groups (e.g. occlusion factor), statistical compar-
isons between groups were estimated using the unpaired t-test. In ex-
periments with two variables, statistical comparisons between groups
were made using two-way ANOVA, followed by the Sidak's multiple
comparisons post hoc test. For the biocompatibility studies, con-
centration–response curves were fitted using least squares as the fitting
method, and the comparisons between curves (LOG IC50, TOP,
BOTTOM, and Hill Slope) were made using the extra sum-of-squares F-
test. Details of the performed statistical analysis are described in each
figure legend. In all cases, p values lower than 0.05 were considered
significant.
3. Results and discussion
3.1. Particle size
The results of LD measurements are presented in Table 2, where
could be observed that all the formulations have 90% of particles/
droplets with sizes in the nanometer range. Moreover, the inclusion of
the NLCVE and NEVE in the hydrogel net did not originated significant
size changes, as already observed in other work [30]. Although the
Table 2
LD diameters of NLC-loaded vitamin E (NLCVE), nanoemulsion-loaded vitamin E (NEVE), hydrogel based on NLC-loaded vitamin E (HG-NLCVE) and hydrogel based on
NE-loaded vitamin E (HG-NEVE), measured on the production day (mean ± SD, n = 5).
NLCVE NEVE
(%) D50 D90 D50 D90
Size (μm) 0.116 ± 0.000 0.386 ± 0.000 0.103 ± 0.004 0.402 ± 0.011
245
Colloids and Surfaces B: Biointerfaces 179 (2019) 242–249
differences between the values are statistically significant (data not
shown), they are not relevant for skin use. Regarding the proposed local
cutaneous application of the tested formulations, these results are sa-
tisfactory, since is desired that nanoparticles/nanodroplets remain in
the upper skin layers avoiding systemic absorption [5,6,31].
3.2. Biocompatibility studies
The biocompatibility of the developed formulations was assessed by
the MTT reduction and NR uptake assays, in HaCaT cells, 24 h after
incubation. In agreement with our previous studies [6], the MTT re-
duction assay demonstrated to be more sensitive for the evaluation of
the cytotoxicity of the developed formulations, when compared to the
NR uptake assay (Supplementary Figure 4).
With the data obtained in the MTT reduction assay, con-
centration–response curves were fitted and a concentration-dependent
effect on cell viability was observed for all the tested formulations, 24 h
after exposure. Furthermore, the NLCVE and NEVE formulations de-
monstrated to be slightly more cytotoxic to HaCaT cells, when com-
pared to their corresponding hydrogel-based formulations, as observed
by the rightwards shifts of the HG-NLCVE and HG-NEVE con-
centration–response curves, when compared to the NLCVE and NEVE
concentration–response curves, respectively (Fig. 1). The IC50 values
were used for statistical comparison and, as observed in Table 3, the
IC50 values of the concentration–response curves of the HG-NLCVE and
HG-NEVE formulations significantly increased, when compared to the
corresponding NLCVE and NEVE formulations, demonstrating a reduced
cytotoxicity of the hydrogel-based formulations towards HaCaT cells.
Indeed, the IC50 value significantly increased from 14.38 μg/mL in
NLCVE to 28.74 μg/mL in HG-NLCVE, and from 16.10 μg/mL in NEVE to
37.49 μg/mL in HG-NEVE. These results may be related to a smaller
ability of the hydrogel-based formulations for penetrate the cells, since
the nanoparticles/nanodroplets are entrapped and have limited diffu-
sion throughout the hydrogel net. Moreover, the cationic surfactant
Cetrimide® provides positive charge to the nanoparticles/nanodroplets
surface, which could originate an additional electrostatic interaction
with the negative charged polymer (PFC®). Besides, it is important to
keep in mind that, during the in vitro experiments, the cell exposure to
formulations is higher when compared to the exposure after skin ap-
plication, since only a small portion of the formulation reaches the
deeper epidermis, bypassing the stratum corneum [6,32]. Some re-
searchers reported results of in vitro studies with vitamin E-loaded lipid
nanosystems for different applications. For example, Bonferoni et al.
[33] observed a stimulating effect on the keratinocytes proliferation
after exposure to vitamin E nanoemulsion, demonstrating its suitability
for use as antioxidant agent for topical wound healing. Oliveira et al.
[34] observed that the use of SLN co-loaded with doxorubicin and vi-
tamin E significantly increased the cytotoxicity against cancer cell lines.
From their findings, the authors proposed the use of this system for
improve cancer therapy. In another study, Abla and Banga [35] re-
ported the achievement of 92.7% of cell viability after exposure to vi-
tamin E-loaded NLC in contrast to 5% of viability for the positive
control. Furthermore, these researchers also observed the maintenance
of the stratum corneum structure after treatment with vitamin E-loaded
NLC, compared to the disruption observed with the positive control.
These results evidenced the safety of using NLC for the topical delivery
HG-NLCVE HG-NEVE
D50 D90 D50 D90
0.097 ± 0.001 0.397 ± 0.021 0.109 ± 0.001 0.514 ± 0.021
S. Vaz, et al. Colloids and Surfaces B: Biointerfaces 179 (2019) 242–249

Fig. 1. (A) NLCVE and HG-NLCVE concentration–response (cell

viability) curves. Results are presented as mean ± SEM from
4 independent experiments (performed in triplicate).
Concentration–response curves were fitted using least squares
as the fitting method and the comparisons between NLCVE and
HG-NLCVE curves (LOG IC50, TOP, BOTTOM, and Hill Slope)
were made using the extra sum-of-squares F-test. Statistical
comparisons were made using two-way ANOVA, followed by
the Sidak's multiple comparisons post hoc test [*p < 0.05;
**p < 0.01; ****p < 0.0001 NLCVE vs. and HG-NLCVE]. (B)
NEVE and HG-NEVE concentration–response (cell viability)
curves. Results are presented as mean ± SEM from 4 in-
dependent experiments (performed in triplicate).
Concentration–response curves were fitted using least squares
as the fitting method and the comparisons between NEVE and
HG-NEVE curves (LOG IC50, TOP, BOTTOM, and Hill Slope)
were made using the extra sum-of-squares F-test. Statistical
comparisons were made using two-way ANOVA, followed by
the Sidak's multiple comparisons post hoc test [*p < 0.05;
**p < 0.01; ****p < 0.0001 NEVE vs. and HG-NEVE].

Table 3 Fig. 2. From the results, it could be observed that, after 24 h, the HG-
IC50 (half-maximum-effect concentrations), TOP (maximal effect), BOTTOM NEVE presents a lower F (67%), when compared to the HG-NLCVE
(baseline) and Hill Slope values of the formulations concentration–response (80%). Therefore, we predict that the HG-NLCVE exerts a higher oc-
curves. clusive effect than HG-NEVE. This occurrence was already observed by
NLCVE HG-NLCVE NEVE HG-NEVE Teeranachaideekul et al., who suggested that lipid nanoparticles form a
film on the surface of the filter paper that avoids water evaporation,
IC50 (μg/mL) 14.38 28.74*** 16.10 37.49**** which is related to the presence of the solid lipid matrix [36]. Müller
TOP 96.49 97.89 98.53 100.3
BOTTOM 9.728 3.934 5.438 1.899e−10
et al. described this phenomenon as one of the mechanisms of lipid
Hill Slope −1.645 −1.309 −1.442 −1.399 nanoparticles for improved skin hydration, avoiding the TEWL
Curve p value (Comparison < 0.0001 < 0.0001 [5,37,38].
between the fitted curves)

Concentration–response curves were fitted using least squares as the fitting

method and the comparisons between curves were made using extra sum-of-
squares F test. In all cases, p values < 0.05 were considered statistically sig-
nificant [***p < 0.001 NLCVE vs. and HG-NLCVE; ****p < 0.0001 NEVE vs.
and HG-NEVE].
of vitamin E.
3.3. In vitro occlusion test
The calculated values of the occlusion factor (F) are presented in
3.4. In vivo human experiments
3.4.1. Assessment of skin hydration
Fig. 3A shows the hydration values at the volunteers skin surface
(i.e. the stratum corneum), before and after the application of the tested
formulations.
From Fig. 3A it could be observed that the HG-NEVE significantly
(p < 0.05) improved the skin hydration, whereas only a slight increase
was detected for the HG-NLCVE. This outcome could be related to the
reinforcement of the stratum corneum lipid barrier. Furthermore, due to
its moisturizing properties, the presence of vitamin E could promote
this effect. According to the lipid-based nanosystems structure, it is

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S. Vaz, et al. Colloids and Surfaces B: Biointerfaces 179 (2019) 242–249

Fig. 2. Occlusion factor (F) of the hydrogel based on NLC-loaded vitamin E (HG-NLCVE) and hydrogel based on NE-loaded vitamin E (HG-NEVE), after 24 h
(mean ± SD, n = 3). Statistical comparisons were made using the Unpaired t test [**p < 0.01 for HG-NLCVE vs. HG-NEVE].

Fig. 3. (A) Values of skin surface hydration, before (T0) and

1 h after (T60) the application of the hydrogel based on NLC-
loaded vitamin E (HG-NLCVE) and hydrogel based on NE-
loaded vitamin E (HG-NEVE). Results are presented as
mean ± SD from 25 independent experiments. Statistical
comparisons were made using two-way ANOVA, followed by
the Sidak's multiple comparisons test [****p < 0.0001 for
each formulation or control, 0 vs. 60 min; ###p < 0.001 and
####
p < 0.0001 for comparisons between formulations or
between formulation and control, at 60 min]. (B) Evaluation
of the skin water barrier function by measurement of the
TEWL, before (T0) and 1 h after (T60) the application of the
hydrogel based on NLC-loaded vitamin E (HG-NLCVE) and
hydrogel based on NE-loaded vitamin E (HG-NEVE). Results
are presented as mean ± SD (n = 3) from 25 independent
experiments. Statistical comparisons were made using two-
way ANOVA, followed by the Sidak's multiple comparisons
test [****p < 0.0001 for each formulation, 0 vs. 60 min;
####
p < 0.0001 for comparisons between formulations or
between formulation and control, at 60 min].

247
S. Vaz, et al.
Table 4
Evaluation of the skinfeel attributes of the hydrogel based on NLC-loaded vi-
tamin E (HG-NLCVE) and hydrogel based on NE-loaded vitamin E (HG-NEVE).
The scores scale ranged from 0 (minimum) to 10 (maximum). The results are
presented as mean score ± SD from 25 independent experiments. Statistical
comparisons were estimated using two-way ANOVA, followed by the Sidak's
multiple comparisons test [*p < 0.05 for HG-NLCVE vs. HG-NEVE].
Attribute HG-NLCVE HG-NEVE
Spreadability 8.04 ± 2.14 8.44 ± 1.79
Adhesiveness 7.36 ± 1.65 7.24 ± 1.90
Ease of removing from the container 8.40 ± 2.06 9.12 ± 1.24
Moisture 7.20 ± 1.30 7.84 ± 1.32
Greasiness 4.00 ± 2.61 4.16 ± 2.87
Glossiness* 3.68 ± 2.05 5.48 ± 2.58
expected that the nanoemulsion release the vitamin E faster, when
compared to the NLC, which has a solid matrix that delays this process
[5,11,19].
Fig. 3B presents the data obtained from the evaluation of the skin
water barrier, where could be noticed that the HG-NLCVE significantly
(p < 0.05) reduced the skin TEWL. This occurrence might be related to
the formation of an occlusive film on the skin surface, which prevents
the water loss by evaporation [5]. In contrast, a non-increase of the
TEWL has been associated to the maintenance of the normal skin bar-
rier function [40]. Similar results, showing an increased skin occlusive
effect performed by vitamin E-loaded lipid nanoparticles, were reported
in other works [7,19]. For example, Abla and Banga [35] carried out
skin permeation studies with vitamin E-loaded NLC and vitamin E-
loaded nanoemulsion and observed that the amount of compound that
reaches the epidermis was higher for the NLC, suggesting that the skin
occlusive effect produced by the NLC increases the permeation of vi-
tamin E. Nonetheless, for the HG-NEVE, was observed a small increase
of the skin TWEL. These results are in agreement with the in vitro oc-
clusion tests, being the lower TEWL observed for the HG-NLCVE, which
has the higher F value. This suggests that the in vitro occlusion test
could be used to predict the in vivo occlusive effect of lipid-based na-
nosystems. Montenegro et al. also suggested this correlation [39].
Thereby, the use of the HG-NLCVE seems more attractive over the HG-
NEVE, which improves the robustness of the biocompatibility studies
results.
The differences observed for the occlusion and skin hydration values
from T0 to T60 are rather small, which is probably related to the
duration of the tests. Nonetheless, the achievement of these small
variations was expected, since our goal was the evaluation of the im-
mediate hydration effect of the formulations on the skin.
3.4.2. Sensory analysis
Table 4 shows the score results for the skinfeel attributes (spread-
ability, adhesiveness, ease of removing from the container, moisture,
greasiness and glossiness) of the tested HG-NLCVE and HG-NEVE for-
mulations.
From Table 4, it could be observed that the volunteers classified
higher the HG-NEVE for the attributes of spreadability, ease of removing
from the container, moisture, greasiness and glossiness. Accordingly,
the HG-NEVE seems more attractive for cutaneous application, being
these properties described as desirable for skin products containing O/
W nanoemulsions [11]. In contrast, the higher adhesiveness was la-
belled for the HG-NLCVE, which means that it could perform a long-
lasting effect.
4. Conclusion
In this study, we demonstrated the in vitro biocompatibility and in
vivo hydration potential of two hydrogel formulations containing vi-
tamin E-loaded lipid-based nanosystems (HG-NLCVE and HG-NEVE). The
248
Colloids and Surfaces B: Biointerfaces 179 (2019) 242–249
results showed that the incorporation of the nanosystems in the hy-
drogel net reduced their cytotoxicity. In addition, it was observed that
the formulation containing NLCVE showed improved occlusive and ad-
hesive properties, whereas the one with NEVE displayed a faster hy-
dration effect and more suitable sensorial properties for skin applica-
tion. However, more in vivo experiments, for longer periods and with
more volunteers, are required before commercialization.
Several researches have been suggesting the advantages of using
formulations containing lipid-based nanosystems for skin application.
In this area, different drugs and cosmetics have been tested, and a lack
of in vivo experiments has been pointed. In particular, the performance
of human studies to evaluate the effectiveness of semi-solid formula-
tions based on vitamin E-loaded lipid nanoparticles formulations has
been missing. Thereby, we consider that our studies accomplish these
requirements, since we performed experiments in 25 volunteers, eval-
uating the hydration potential and carrying out sensory analysis of
hydrogel formulations containing vitamin E-loaded NLC and vitamin E-
loaded nanoemulsions.
The methods and results of these experiments can be employed to
evaluate the feasibility of semi-solid formulations based on lipid na-
nosystems containing vitamin E for dermatological and cosmetic use,
and constitutes an improvement in this research field.
Acknowledgments
Research work funded by national funds provided by FCT –
Fundação para a Ciência e a Tecnologia, in the scope of FCT Project
UID/Multi/04546/2013.
Appendix A. Supplementary data
Supplementary data associated with this article can be found, in the
online version, at https://doi.org/10.1016/j.colsurfb.2019.03.036.
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