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Open Access Full Text Article O r i g in a l R e s e a r c h

Design, characterization, and clinical evaluation

of argan oil nanostructured lipid carriers
to improve skin hydration
International Journal of Nanomedicine downloaded from https://www.dovepress.com/ on 10-Nov-2023

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International Journal of Nanomedicine
11 August 2014
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Deise Michele Tichota 1 Abstract: Given its advantages in skin application (eg, hydration, antiaging, and protection),
Ana Catarina Silva 2,3 argan oil could be used in both dermatological and cosmetic formulations. Therefore, the prepa-
José Manuel Sousa Lobo 2 ration of nanostructured lipid carriers (NLCs) using argan oil as a liquid lipid is a promising
Maria Helena Amaral 2 technique, since the former constitute well-established systems for dermal delivery. The aim
For personal use only.

of this work was to develop a topical formulation of argan oil NLCs to improve skin hydra-
1
Federal University of Rio Grande
do Sul, Porto Alegre, 2Laboratory tion. Firstly an NLC dispersion was developed and characterized, and afterward an NLC-based
of Pharmaceutical Technology/ hydrogel was prepared. The in vivo evaluation of the suitability of the prepared formulation
Centre of Research in Pharmaceutical for the proposed application was assessed in volunteers, by measuring different skin-surface
Sciences, Faculty of Pharmacy,
University of Porto, 3Faculty of Health parameters for 1 month. An argan oil NLC-based hydrogel formulation was successfully prepared
Sciences, Fernando Pessoa University, and characterized. Moreover, the entrapment of the NLCs in the hydrogel net did not affect
Porto, Portugal
their colloidal sizes. Additionally, it was observed that this formulation precipitated an increase
in skin hydration of healthy volunteers. Therefore, we concluded that the preparation of NLC
systems using argan oil as the liquid lipid is a promising strategy, since a synergistic effect on
the skin hydration was obtained (ie, NLC occlusion plus argan oil hydration).
Keywords: argan oil, nanostructured lipid carriers, NLC, hydrogels, skin hydration

Introduction
The skin is the major and outermost organ of the body, and performs several impor-
tant physiological functions. This structure is formed by two layers: the epidermis
and dermis. The former is more external and ends with the stratum corneum (SC),
which plays an important barrier function, protecting the body inside from the external
environment.1,2
The SC surface displays a hydrolipidic film composed of water, hygroscopic com-
pounds (natural moisturizing factors), and lipid compounds that produce an occlusive
effect. Both natural moisturizing factors and lipids form a barrier that has the ability
to prevent water loss by evaporation, helping to maintain normal skin water content.
The normal functioning of the SC can be disturbed under dry-skin conditions. When
Correspondence: Ana Catarina Silva/ this occurs, the effectiveness of the SC-barrier function stops and a cycle of events
Maria Helena Amaral initiates, such as superficial dehydration of the SC, subsequent release of inflamma-
Laboratory of Pharmaceutical
Technology/Centre of Research in tory mediators, induction of epidermal keratinocyte hyperproliferation, and disruption
Pharmaceutical Sciences, Faculty of of epidermal cellular differentiation.1 Accordingly, the evaluation of skin hydration
Pharmacy, University of Porto, 228 Rua
Jorge Viterbo Ferreira, Porto 4050-313, has gained a growing interest in recent years, particularly in the field of experimen-
Portugal tal dermatology. Several in vivo and in vitro methods have been proposed for the
Tel +351 22 042 8624
Fax +351 22 200 3977
determination of skin hydration. Nonetheless, in vivo methods provide more realistic
Email [email protected]/[email protected] information. Among these, electrometric techniques have been the most applied.

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These are based on the determination of electrical changes the proposed application was assessed in volunteers by mea-
(impedance, resistance, and capacitance) that are detectable suring different skin-surface parameters for 1 month.
at the skin surface, by applying different electrical currents.
The skin parameters evaluated more often are SC hydration, Materials and methods
sebum content, microrelief, and transepidermal water loss Materials
(TEWL).3 The measurement of SC hydration gives infor- Argan oil, the gelling agent PFC® (carbomer 2001) and
mation about the amount of water present in this layer. The triethanolamine were purchased from Acofarma (Madrid,
sebum is composed of a lipid mixture produced by sebaceous Spain). Precirol ® ATO5 (glyceryl palmitostearate) and
glands, which has an important role in the maintenance of Apifil® (polyethylene glycol-8 beeswax) were kindly pro-
the SC-barrier function.4 Skin microrelief is used to evaluate vided by Gattefossé (Saint-Priest, France). Witepsol® E 85
skin-hydration efficacy or the antiaging effects of cosmet- (hydrogenated cocoglycerides), Dynasan ® 114 (glyceryl
ics, and could be assessed by measuring the parameters of trimyristate) and Softisan® 142 (hydrogenated cocoglycer-
roughness, scaling, smoothing, and wrinkling.5,6 TEWL is ides) were gifts from Sasol (Witten, Germany). Cetrimide
indicative of dehydration processes occurring, which could and Tween® 80 (polysorbate 80) were obtained from JM Vaz
compromise the effectiveness of the SC-barrier function.7 Pereira (Sintra, Portugal) and Guinama (Valencia, Spain),
The use of moisturizers influences the skin-barrier function respectively. Purified water (Milli-Q® Plus) was obtained
by reducing TEWL. Moreover, this influence depends on from EMD Millipore (Billerica, MA, USA).
the composition of the moisturizer.8 An efficient moisturizer
formulation reduces dry skin and irritation, avoiding the Screening of lipid excipients
conditions that can lead to skin disease.9 When developing an NLC dispersion, the choice of the most
Nowadays, nanostructured lipid carriers (NLCs) are suitable solid–liquid lipid combination, which leads to the
well-established systems that have been successfully used formation of an appropriate solid nanoparticle matrix, is
for dermal delivery of cosmetics and drugs. These carrier fundamental.13,14 Accordingly, the miscibility of argan oil
systems consist of aqueous dispersions of solid nanopar- with five different solid lipids (Apifil, Dynasan 114, Precirol
ticles, composed of a mixture of solid and liquid lipids, and ATO5, Softisan 142 and Witepsol E85) was evaluated in
stabilized by one or two surfactants. The excipients used in increasing proportions, ranging from 50:50 to 90:10 (solid
NLC systems are generally recognized as safe substances, lipid:liquid lipid). For this, various physical mixtures of
which predicts an absence of toxicity for topical application. lipids were heated until 100°C±1°C for 1 hour, with stirring
Moreover, NLCs have been described as efficient systems (200 rpm). Afterward, the mixtures were cooled until room
to improve skin hydration, due to their physiological lipid temperature (20°C±1°C) for solidification. The existence/
composition and occlusive effect properties. Typically, NLC absence of miscibility between the two lipids was analyzed by
dispersions present a low viscosity, which is not advanta- placing a portion of each solidified mixture on a filter paper,
geous for topical application, because it decreases the time followed by visual observation, to verify the presence of oil
of permanence at the application site. To avoid this, NLCs drops, which would be indicative of a lack of miscibility
can be incorporated into traditional semisolid systems (eg, between lipids.15
hydrogels [HGs]), increasing the consistency of final formu-
lations and also the long-term stability of the incorporated Preparation of NLC dispersions
nanoparticles.10,11 The NLC dispersions were prepared according to the method
Argan oil is a natural oil that has been applied in cosmet- previously employed by Silva et al.16 Briefly, the lipids were
ics, because of its antioxidant, hydration, antiaging, and pro- heated at 5°C–10°C above the melting point of the solid lipid
tection properties on the skin.12 Based on the aforementioned (56°C). At the same time, the aqueous phase, composed
properties, the preparation of NLC systems using argan oil as of surfactant, preservative, and purified water, was heated
the liquid lipid is a promising technique. Therefore, the aim at the same temperature. After melting of the lipid phase,
of this work was to develop a topical formulation of argan the aqueous phase was added to the former and homo­
oil NLC to improve skin hydration. For this, firstly an NLC genized under high-speed stirring, using an Ultra-Turrax®
dispersion was developed and characterized, and afterward T25 (IKA, Staufen, Germany), at 8,000 rpm for 5 minutes.
an NLC-based HG was prepared (HG-NLC). The in vivo The pre-emulsion obtained was placed under a probe
evaluation of the suitability of the prepared formulation for sonicator (Sonic & Materials, Newtown, CT, USA), with a

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 Dovepress Clinical evaluation of NLCs for skin hydration

power-output amplitude of 70% for 15 minutes, to allow for particles maintained their nanometric sizes after integration
oil-droplet breakdown. Subsequently, the hot nanoemulsion within the HG structure. The network of the HG was previ-
formed was transferred to glass vials and cooled to room ously destroyed by dilution with purified water and vortexing,
temperature to form the solid nanoparticles, ie, the NLCs. and Z-ave, ZP, and PI were evaluated as described previously
for the NLC dispersions alone.
Preparation of hydrogels In order to estimate the long-term stability of all the
For the preparation of the HG-NLC, the gelling agent PFC prepared systems, the size of the nanoparticles was analyzed
was first powdered in a porcelain mortar. Afterward, the after storage at room temperature (20°C±1°C) and in the
NLC dispersion was added to the mortar and the polymer refrigerator (5°C±1°C) for 90 days.
was neutralized to pH 7 with triethanolamine, to allow for the
formation of the HG. A control HG without NLCs was also Cryo-scanning electron microscopy
prepared to compare the results of all performed studies. The structure of the NLC dispersions alone and after
incorporation in the HG was observed by cryo-scanning
Particle-size, polydispersity-index, electron microscopy (cryoSEM). For this, the samples were
and zeta-potential measurements mounted on metal stubs, rapidly frozen with slush nitrogen
The diameter (Z-ave) and particle-size distribution (evalu- until -210°C, sublimated at -90°C for 90 seconds, and
ated by the polydispersity index [PI]) are parameters that coated with a mixture of gold and palladium under vacuum.
have a direct impact on the physical stability of a dispersion. Subsequently, the samples were fractured, transferred to the
Photon correlation spectroscopy, also known as dynamic chamber, and examined using SEM (JSM-6301F; JEOL,
light scattering (DLS), and laser diffractometry (LD) are the Tokyo, Japan)/INCA Energy 350 (Oxford Instruments,
most widely used techniques for measuring the size and dis- Abingdon, UK)/ALTO 2500 (Gatan, Pleasanton, CA, USA).
tribution of particles in colloidal dispersions.17 Furthermore, The observations were done at -150°C.
during the optimization of an NLC dispersion, it is important
to verify the presence/absence of microparticles that are not pH analysis
detectable by common DLS equipment.16 Accordingly, the The determination of the pH of a formulation intended for
particle-size measurement by LD using a Mastersizer 2000E cutaneous application is extremely important, since it must
(Malvern Instruments, Malvern, UK) was first performed. be compatible with the pH of the application site. The natural
For this purpose, volume distribution of 10%, 50%, and 90% pH of the skin comes from the secretions of sweat and seba-
was measured, which referred to particles with diameters ceous glands, and lactic acid production, which leads to the
equal or lower than the given values. Afterward, to confirm formation of a protective film over the entire skin surface,
the presence of particles in the colloidal size range, the Z-ave designated hydrolipidic film. The skin normally has an aver-
and PI of the prepared NLC dispersions were measured on age pH of 5.5, although this may vary slightly depending on
the production day with DLS (ZetaPALS; Brookhaven Instru- the area of the body.19
ments, Holtsville, NY, USA). Before the measurements, the The evaluation of the pH was performed in all prepared
dispersions were diluted (1:200) with ultrapure water, to HGs on days 7 and 30 after storage at different temperatures.
avoid the light multiscattering related to a high concentra- For this, a glass pH electrode (Basic 20; Crison Instruments,
tion of particles. Barcelona, Spain) was directly dipped in each semisolid
The zeta potential (ZP) refers to the total surface charge formulation. All analyses were performed in triplicate
that a particle acquires in a given environment, and can be (means ± SD).
indicative of good long-term stability of the dispersions.17,18
ZP measurements were performed using the ZetaPALS Texture analysis
apparatus. For this, the dispersions were previously diluted The evaluation of texture parameters, such as adhesiveness
with ultrapure water to a suitable concentration. All the DLS and firmness, of semisolid formulations provides informa-
and ZP experiments were performed at room temperature tion about their mechanical properties. Texture analyses
(20°C±1°C), and the results presented are average values of were performed using a probe that dips in the formulation,
six measurements (n=6) plus standard deviation (SD). with a defined velocity and force. The results of force
The mean particle size of the NLCs was also evaluated versus distance were plotted, allowing the calculation of
after the preparation of the HG, in order to verify if the adhesiveness (negative area) and firmness (maximum force)

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of the formulation. In more detail, adhesiveness is related of NLC incorporation and storage conditions on the flow
with bioadhesion and is a measure of the force required to properties of the HGs.
overcome the attractive forces between the surfaces of the
sample and the probe. On the other hand, firmness is related Color measurements
to the ease of product application on the skin.20–22 A texture The detection of changes in the color of the semisolid
analyzer (TA-XT2i; Stable Micro Systems, Godalming, UK) formulations can indicate the occurrence of degradation on
was used to carry out the texture analysis. The compression their components. In addition, it is well known that lipids
mode was applied to perform a penetration test using a load may undergo degradation resulting from oxidation reactions
cell of 5 kg, a trigger force of 0.05 N, a cylindrical probe that may occur during thermal processing and/or storage.
(25 mm diameter), a penetration depth of 5 mm, and a test Typically, lipid-oxidation reactions generate colored com-
speed of 3 mm⋅s-1. All the experiments were done in triplicate pounds. Therefore, the assessment of the occurrence of color
(means ± SD), at room temperature (20°C±1°C), on days 7 changes in the prepared formulations can give information
and 30, after the preparation of the HGs, in order to study about lipid stability.30,31
the effects of NLC incorporation and storage conditions on Color determination based on the color space L*a*b*
their textural properties. was performed using a colorimeter (Chroma Meter CR-500;
Konica Minolta, Tokyo, Japan), with a D65 light source
Rheological measurements and an observation angle of 2°. The L* means the amount
According to the increase effect on the formulation consis- of reflected light, and can range from 0 (black) to 100%
tency, the use of lipid-nanoparticle dispersions by means (white); the a* and b* represent, respectively, the colors from
of semisolid formulations has been presented as a good green to red or blue to yellow, and the values range from –60
alternative to improve their topical application.11,23–25 The (close to green or blue) to +60 (close to red or yellow).
study of the flow properties (ie, consistency) of semisolid Through the values of a* and b*, the chrome parameter (C*)
formulations gives information about their rheological can be calculated, which reveals the color of the formula-
behavior, which allows estimation of their suitability for tion and allows the detection of color changes. C* can be
the proposed application. These studies can be performed obtained by applying the equation C* = ( a *2 + b *2 ). 32,33
in viscometers, and the respective flow behavior can be The parameter C* was evaluated in both HG and HG-NLC
assessed by analysis of the plots of shear stress versus shear formulations, on the production day, and after 30 days of
rate.22,26,27 Nonetheless, the use of mathematical models storage at different temperatures. All determinations were
that fit the rheological data is important to confirm the flow performed in triplicate, and the results were calculated by
behavior graphically observed. Therefore, two models were mean values (n=3± SD).
applied:28,29 power law (Ostwald–de Waele), and power
law with a yield stress (Herschel–Bulkley). The flow index Human in vivo evaluation of skin hydration
value (n) was used to assess the rheological behavior of Design of the study
the formulations. The study was planned as a single-blinded, controlled trial,
Rheological tests were performed on a rotational visco­ and was performed for 1 month in ten healthy-skin Caucasian
meter (Haake Viscotester™ 550; Thermo Fisher Scientific, volunteers of both sexes aged between 21 and 30 years. Prior
Waltham, MA, USA), with an SV/DIN coaxial cylinder sen- to the experiments, all participants were informed about the
sor. The flow behavior of the developed HG was studied as methodologies of testing, and signed the informed consent.
in our previous work,22 by continuous shear investigations, The latter was prepared according to the recommendations of
which were performed in order to evaluate the shear stress the Declaration of Helsinki, which state the ethical principles
(Pa) as a function of shear rate (s-1). The study started with that should be accomplished for research involving human
a shear rate of 1.0⋅s-1, went up to a maximum of 500⋅s-1, and subjects.34 Before the experiments, the ten volunteers were
then back to 1.0⋅s-1, and the resulting shear stress was mea- randomly divided in two groups. Group 1 consisted of five
sured. To reduce the influence of temperature on the rheologi- volunteers who applied the HG-NLC formulation. Group 2
cal behavior of the HG, a thermostatic water bath was used consisted of five volunteers who applied the HG formulation.
to accurately maintain the sample temperature (20°C±1°C) The exclusion criterion was the existence of skin damage on
during all experiments. The rheological measurements were both forearms of the volunteers. On the first day of the study,
performed on days 1 and 30, in order to assess the effects volunteers could not apply any product on both forearms.

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 Dovepress Clinical evaluation of NLCs for skin hydration

Table 1 Composition of the prepared nanostructured lipid carrier (α=0.05) and Tukey posttests, using SPSS version 21.0
(NLC) dispersions (IBM, Armonk, NY, USA). A P-value 0.05 was considered
Composition (% w/w) NLC1 NLC2 statistically significant.
Precirol ATO5
®
7.00 –
Softisan® 142 – 7.00 Results and discussion
Argan oil 3.00 3.00
Tween® 80 2.50 2.50 Screening of lipid excipients
Cetrimide 0.50 0.50 The results of the miscibility tests demonstrated that both
Purified water 87.00 87.00 Precirol ATO5 and Softisan 142 were suitable for the prepa-
ration of lipid nanoparticles containing argan oil. It was found
that these solid lipids exhibited a miscibility with argan oil at
Prior to the measurements, the volunteers were made to a proportion of 70:30 (solid lipid:argan oil), while the other
rest for at least 30 minutes in a room with both controlled lipids tested were miscible only at the proportions of 80:20
temperature (20°C±1°C) and relative humidity (45%±1%). and 90:10 (data not shown). Therefore, we selected these two
The procedures were determination of the application site, lipids to prepare NLC dispersions (Table 1).
with a distance of 5 cm above the wrist and 5 cm below the
elbow, and measurement of skin hydration, sebum, TEWL, Particle-size, polydispersity-index,
scaliness, smoothness, roughness, and energy parameters. and zeta-potential measurements
Volunteers were instructed to apply the formulation once a According to the LD measurements, both NLC dispersions
day for 1 month. The forearm with no formulation application (with Precirol ATO5 or Softisan 142) revealed the presence
was used as a control. All the measurements were performed of 90% of particles with sizes in the nanometer range (data
in triplicate (means ± SD) on day 0 and 30. not shown). Nonetheless, the NLC1 dispersion had smaller
and more homogeneous nanoparticles. Therefore, we selected
Assessment of skin hydration Precirol ATO5 to prepare the NLCs used for the subsequent
For the assessment of skin hydration, a Multi Probe Adapter studies.
System was used (Courage + Khazaka Electronics, Cologne, Table 2 presents the results of Z-ave, PI, and ZP of the
Germany), which consists of a basic device connected to dif- NLC1 dispersion, measured on the production day and after
ferent probes that allows for the determination of different storage at different temperatures. From Table 2, we can
skin parameters: surface hydration (Corneometer® CM 825), see that all dispersions revealed the presence of nanosized
and lipid content (Sebumeter® SM 810). For microrelief anal- particles (100–200 nm), PI values lower than 0.300, and ZP
ysis the Visioscan® VC 98 (Courage + Khazaka Electronics), higher than |30| mV, which have been described as acceptable
was used to evaluate the following quantitative topographic for topical delivery. Moreover, after storage, no significant
characteristics: scaliness, smoothness, roughness, and energy. variations on these values were observed, indicating good
The skin-barrier function was evaluated by TEWL measure- long-term stability of dispersions.10,35
ment using the Tewameter® TM 210 (Courage + Khazaka The composition of the prepared HGs (HG-NLC and
Electronics). HG) is shown in Table 3. Table 4 presents the NLC Z-ave
after the preparation of the HG. From Table 4, we can
Statistical analysis observe that regarding Z-ave and PI values, no relevant
Statistical analysis of the results of skin-hydration evalua- alterations occurred. Therefore, we concluded that the NLC
tions was performed applying one-way analysis of variance dispersions remained stable after incorporation in the HG

Table 2 Mean diameter (Z-ave), polydispersity index (PI), and zeta potential (ZP) values of the NLC1 dispersions, measured on the
production day and after 90 days of storage, at room temperature (20°C±1°C) and in the refrigerator (5°C±1°C)
Day NLC1
20°C±1°C 5°C±1°C
Z-ave (nm) PI ZP (mV) Z-ave (nm) PI ZP (mV)
0 137.70±0.70 0.21±0.01 53.37±0.92 – – –
90 162.10±0.70 0.19±0.01 39.19±0.84 192.00±1.30 0.21±0.01 42.57±1.50
Note: Values are expressed as mean ± standard deviation.
Abbreviation: NLC, nanostructured lipid carrier.

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Table 3 Composition of the prepared hydrogels (nanostructured Moreover, it was observed that the temperature and storage
lipid carrier-based hydrogel [HG-NLC] and control hydrogel time did not influence these values.
[HG])
Formulation (% w/w) Texture analysis
Composition HG-NLC HG The results of texture analysis are shown in Figure 3. From
Precirol® ATO5 7.00 – Figure 3A, we can observe that the presence of NLCs pre-
Argan oil 3.00 –
cipitated a high decrease in the firmness of the HG. The
Tween® 80 2.50 –
Cetrimide 0.50 0.50 maximum force (ie, firmness) of the HG exhibited values
PFC 0.50 0.50 between 0.7 and 0.8 N, while the maximum force of the
Purified water 86.50 99.00 HG-NLC ranged between 0.2 and 0.3 N. In addition, no
Triethanolamine QS QS
significant changes in the firmness of any formulations
Abbreviation: QS, quantum satis.
were observed after storage at different temperatures
over 30 days.
network. Moreover, stability was observed during storage at With regard to adhesiveness (Figure 3B), the presence of
different temperatures. Similar results have been described NLCs also caused a decrease in the values of HG-NLC, when
elsewhere.22,36 In contrast, a high variation in ZP values was compared to the control (HG). The latter had adhesiveness
observed, which could have been related to the negative values between -1.5 and -2.5 N⋅mm. In contrast, for the for-
charge of the carboxylic groups present on the gelling agent mer, the values ranged between -1.0 and -1.5 N⋅mm. Addi-
(PFC). Nonetheless, the values remain acceptable, since they tionally, there were no significant changes in these ­values
are, in absolute value or modulus, higher than 30 mV.35 over the 30 days of storage at different temperatures.
Typically, an adequate topical semisolid formulation
Cryo-scanning electron microscopy should present major mechanical properties, namely high
Figure 1 presents the typical structure of lipid nanoparticles, adhesiveness to prolong contact time, and good firmness to
with almost spherical shape and smooth surface.27,37 After facilitate its local application.40 The decrease in firmness and
90 days of storage at different temperatures, no visible adhesiveness observed for the HG-NLC, could have been
changes on the NLC aspect were observed, which is in agree- related to the disruption of the HG structure, originated by
ment with the results obtained for Z-ave, PI, and ZP. the presence of the NLC dispersion. This phenomenon could
From Figure 2, we can visualize a typical HG network also be observed in cryoSEM images (Figure 2). Similar
(left)38 and the presence of NLC within the HG net (middle results were described by Gonzalez-Mira et al.36
and right) after storage at different temperatures. From the
latter, we conclude that the HG formulation is effective for Rheological measurements
the incorporation of NLCs without changing their stability, Rheological measurements were performed on the HG-
which is in accordance with the results obtained for Z-ave. NLC formulation and compared with the HG in order to
Similar images of lipid-based HG systems were presented study the effect of the presence of lipid nanoparticles on
by Silva et al.22 the flow behavior of the semisolid formulation. In addition,
the effect of storage at different temperatures was evaluated
pH analysis (Figure 4, A and B).
The results obtained reveal pH values between 7 and 8 (data From the analysis of the rheograms (Figure 4, A and B),
not shown), which are acceptable for cutaneous application.39 it can be seen that the formulations showed a non-Newtonian

Table 4 Mean diameter (Z-ave), polydispersity index (PI), and zeta potential (ZP) values of the nanostructured lipid carrier-based
hydrogel (HG-NLC), on the production day and after 60 days of storage, at room temperature (20°C±1°C) and in the refrigerator
(5°C±1°C)
Day HG-NLC
20°C±1°C 5°C±1°C
Z-ave (nm) PI ZP (mV) Z-ave (nm) PI ZP (mV)
0 170.50±1.80 0.27±0.01 -34.15±0.62 190.90±3.00 0.28±0.01 -40.20±0.63
60 155.00±3.40 0.18±0.02 -44.72±0.68 170.00±1.70 0.25±0.01 -38.55±0.91
Note: Values are expressed as mean ± standard deviation.

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Day 0 20°C – day 90 5°C – day 90

1 µm 3 µm 3 µm

Figure 1 Cryo-scanning electron microscopy images of the nanostructured lipid-carrier dispersions on the production day and after 90 days of storage at room temperature
(20°C±1°C) and in the refrigerator (5°C±1°C) (magnification 40,000×).

HG HG-NLC − 20°C HG-NLC − 5°C

30 µm 6 µm 6 µm

Figure 2 Cryo-scanning electron microscopy images of the prepared hydrogels after 60 days of storage (magnification 40,000×).
Note: Left, control hydrogel (HG); middle, nanostructured lipid carrier-based hydrogel (HG-NLC) at room temperature (20°C±1°C); right, HG-NLC in refrigerator (5°C±1°C).

A 0.9
B 0

0.8 HG 20°C HG-NLC 20°C HG 5°C HG-NLC 5°C

–0.5
Negative area (N.mm)

0.7
Maximum force (N)

0.6
–1
0.5
7 days 7 days
0.4 –1.5
30 days 30 days
0.3
–2
0.2

0.1 –2.5

0
HG 20°C HG-NLC 20°C HG 5°C HG-NLC 5°C –3

Figure 3 Texture analysis (A firmness, B adhesiveness) of the prepared hydrogels, performed on days 7 and 30, after storage at 20°C±1°C and 5°C±1°C.
Note: Control hydrogel (HG) and nanostructured lipid carrier-based hydrogel (HG-NLC).

A 700 B 700

600 600
Shear stress (Pa)

Shear stress (Pa)

500 500

400 HG-NLC 7 days 400 HG-NLC 7 days

300 HG-NLC 30 days 300 HG-NLC 30 days

HG 7 days HG 7 days
200 200
HG 30 days HG 30 days
100 100

0 0
0 200 400 600 0 200 400 600
Shear rate (s ) –1
Shear rate (s–1)

Figure 4 Rheological analysis of the prepared hydrogels, performed on days 7 and 30, after storage at 20°C±1°C (A) and 5°C±1°C (B).
Note: Control hydrogel (HG) and nanostructured lipid carrier-based hydrogel (HG-NLC).

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A B
45 60
11
*
40
50
35
Hydration

Hydration
30 40

25
13
* 30
20

15 20
Sample Control Blank Sample Control Blank

Sample Sample

Figure 5 Box-plot graphics of the results obtained for the human in vivo evaluation of skin hydration.
Note: (A) Day 0; (B) day 30 (sample, nanostructured lipid carrier-based hydrogel; control, no formulation application; blank, hydrogel). *Outlier. Values are the volunteer numbers.

behavior. These HGs exhibited a decrease in apparent and storage.31 It should be noted that the presence of argan
viscosity with an increasing shear rate, ie, a plastic or a oil could have contributed to this effect, according to its
pseudoplastic with yield-value behavior. Furthermore, no described antioxidant properties.41
thixotropy was observed, because there was no decrease
in the apparent viscosity of the formulations with time (the Human in vivo evaluation of skin
upper and lower curves of the rheograms overlap).26 These hydration
data corroborate the results found after the application of The most relevant results of the human in vivo evaluation
the rheological models, where the n-value was less than 1, of skin-hydration tests are presented in Figure 5. From the
which indicates a shear thinning behavior.28,29 results, it can be seen that after application of the HG-NLC
From the observation of all rheograms, it can be seen that formulation, for 30 days, there was a significant increase
the HG-NLC showed lower viscosity than the HG, indicat- in the skin hydration (P,0.05) when compared to the HG
ing that the presence of lipid nanoparticles influences the (blank) application. However, the differences between the
consistency of the HG, leading to a decrease in viscosity. hydration of the forearm where was applied the HG-NLC
These results are in agreement with those obtained in the formulation, and the forearm control where not statisti-
texture analysis. Furthermore, it was observed that during cally significant (P.0.05) (data not shown). Besides, for
storage, all formulations underwent a slight decrease in the HG, the differences were not statistically significant
viscosity. (P0.05). Additionally, in the data (not shown) obtained
for the other evaluated skin parameters (lipid content,
Color analysis TEWL, scaliness, smoothness, roughness, and energy),
The values obtained for the chrome parameter (C*) showed there were no statistically significant differences between
that after 30 days of storage, only minor changes were the values (P0.05) obtained before and after 30 days of
detected for the HG-NLC, while the HG maintained its HG-NLC application.
initial values (data not shown). Despite the former formula- The significant increase in skin hydration observed for
tion not exhibiting color changes visible to the naked eye, a the HG-NLC formulation could be attributed to the pres-
slight decrease in the C* values was detected at both storage ence of lipid nanoparticles. The significant increase in skin
temperatures (data not shown). Nonetheless, these varia- hydration observed for the HG-NLC formulation could be
tions were considered statistically irrelevant. Accordingly, attributed to the presence of lipid nanoparticles, which have
it was concluded that there were no changes in the color of displayed a skin-moisturizing effect when incorporated in
the formulation during storage, which could indicate the semisolid formulations.42–44 Besides, this effect could also be
nonoccurrence of lipid degradation (by means of oxidation attributed to the argan oil, which has been described as a
reactions), and reveals lipid stability during thermal process compound with skin-moisturizing properties.12

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 Dovepress Clinical evaluation of NLCs for skin hydration

Conclusion 10. Müller RH, Shegokar R, Keck CM. 20 years of lipid nanoparticles
(SLN & NLC): present state of development and industrial applications.
An argan oil NLC-based HG formulation was successfully Curr Drug Discov Technol. 2011;8(3):207–227.
prepared and characterized. Moreover, the entrapment of 11. Pardeike J, Hommoss A, Müller RH. Lipid nanoparticles (SLN,
the NLCs in the HG net did not affect their colloidal sizes. NLC) in cosmetic and pharmaceutical dermal products. Int J Pharm.
2009;366(1–2):170–184.
Additionally, it was observed that this formulation resulted 12. Guillaume D, Charrouf Z. Argan oil and other argan products:
in an increase in the skin hydration of healthy volunteers. use in dermocosmetology. Eur J Lipid Sci Technol. 2011;113(4):
403–408.
According to the results obtained in this study, we con- 13. Chakraborty S, Shukla D, Mishra B, Singh S. Lipid – an emerging plat-
clude that the preparation of NLC systems using argan oil form for oral delivery of drugs with poor bioavailability. Eur J Pharm
as the liquid lipid is a promising technique, since they could Biopharm. 2009;73(1):1–15.
14. Mendes AI, Silva AC, Catita JA, Cerqueira F, Gabriel C, Lopes CM.
have a synergistic effect on skin hydration (ie, NLC occlu- Miconazole-loaded nanostructured lipid carriers (NLC) for local
sion plus argan oil hydration). Moreover, the solubilization delivery to the oral mucosa: improving antifungal activity. Colloids
Surf B Biointerfaces. 2013;111C:755–763.
of an additional cosmetic active ingredient (eg, an antiaging 15. Hommoss A. Nanostructured Lipid Carriers (NLC) in Fermal and
compound) in this oil would improve interest in the NLC Personal Care Formulations [doctoral thesis]. Nerlin: Freie Universität
system for skin application. Berlin; 2008.
16. Silva A, González-Mira E, García M, et al. Preparation, characteriza-
tion and biocompatibility studies on risperidone-loaded solid lipid
Acknowledgments nanoparticles (SLN): high pressure homogenization versus ultrasound.
Colloids Surf B Biointerfaces. 2011;86(1):158–165.
This work was supported by both the Conselho Nacional 17. Mehnert W, Mäder K. Solid lipid nanoparticles: production, character-
de Desenvolvimento Científico e Tecnológico (CNPq) ization and applications. Adv Drug Deliv Rev. 2001;47(2):165–196.
18. Wu L, Zhang J, Watanabe W. Physical and chemical stability of drug
and the Coordenação de Aperfeiçoamento de Pessoal de
nanoparticles. Adv Drug Deliv Rev. 2011;63(6):456–469.
Nível Superior (CAPES) of Brazil. The authors are thank- 19. Schmid-Wendtner MH, Korting HC. The pH of the skin surface and its
ful to Paralab for providing the Mastersizer for particle- impact on the barrier function. Skin Pharmacol Physiol. 2006;19(6):
296–302.
size analysis, and also to Professor Sallete Reis, from the 20. Jones D, Woolfson AD, Brown A. Textural analysis and flow rheometry
Department of Chemistry and Physics, Faculty of Pharmacy, of novel, bioadhesive antimicrobial oral gels. Pharm Res. 1997;14(4):
450–457.
Porto University, for the use of the ZetaPALS apparatus for
21. Jones DS, Lawlor MS, Woolfson AD. Examination of the flow
particle-size analysis. rheological and textural properties of polymer gels composed of
poly(methylvinylether-co-maleic anhydride) and poly(vinylpyrrolidone):
rheological and mathematical interpretation of textural parameters.
Disclosure J Pharm Sci. 2002;91(9):2090–2101.
The authors report no conflicts of interest in this work. 22. Silva AC, Amaral MH, González-Mira E, Santos D, Ferreira D. Solid
lipid nanoparticles (SLN)-based hydrogels as potential carriers for oral
transmucosal delivery of risperidone: preparation and characterization
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