AAPS PharmSciTech ( # 2017)

DOI: 10.1208/s12249-017-0737-x

Research Article

Topical Formulation Containing Beeswax-Based Nanoparticles Improved

In Vivo Skin Barrier Function

Carla Souza,1 Luis Alexandre Pedro de Freitas,1 and Patrícia Maria Berardo Gonçalves Maia Campos1,2

Received 17 November 2016; accepted 1 February 2017

Abstract. Lipid nanoparticles have shown many advantages for treatment/prevention of

skin disorders with damaged skin barrier function. Beeswax is a favorable candidate for the
development of nanosystems in the cosmetic and dermatological fields because of its
advantages for the development of products for topical application. In the present study,
beeswax-based nanoparticles (BNs) were prepared using the hot melt microemulsion
technique and incorporated to a gel-cream formulation. The formulation was subsequently
evaluated for its rheological stability and effect on stratum corneum water content (SCWC)
and transepidermal water loss (TEWL) using in vivo biophysical techniques. BNs resulted in
mean particle size of 95.72 ± 9.63 nm and zeta potential of −9.85 ± 0.57 mV. BN-loaded
formulation showed shear thinning behavior, well adjusted by the Herschel-Bulkley model,
and a small thixotropy index that were stable for 28 days at different temperatures. BN-
loaded formulation was also able to simultaneously decrease the TEWL and increase the
SCWC values 28 days after treatment. In conclusion, the novel beeswax-based nanoparticles
showed potential for barrier recovery and open the perspective for its commercial use as a
novel natural active as yet unexplored in the field of dermatology and cosmetics for
treatment of skin diseases with damaged skin barrier function.
KEY WORDS: beeswax; clinical efficacy; lipid nanoparticles; rheology; skin barrier function.

INTRODUCTION advantages over other topical delivery vehicles. The small

One of the main skin functions is the protection from the
external environment, called Bskin barrier function,^ where
the stratum corneum (SC) plays a key role. SC prevents
transepidermal water loss (TEWL) and protects the skin from
environmental irritants, sun irradiation, and bacterial infec-
tions (1). The defects may lead to the increased susceptibility
to intoxication by penetration of harmful agent and increased
TEWL, resulting in various skin diseases, such as atopic
dermatitis and psoriasis or even dried skin (2). In mild skin
diseases, the application of skin care creams aiming at
improving the skin barrier function and the stratum corneum
lipid film can be sufficient. But, in chronic skin diseases, the
side effects related to the drugs cannot be avoided, which
limit their use and the patient compliance. Therefore, the
development of novel concepts, to treat such skin disorders
without or at least with less side effects, is highly desired (3,4).
Lipid nanoparticles have shown potential for
dermatological/cosmetic purposes, once they possess many
1 lipsticks, and other cosmetics formulations (16,17). It is
School of Pharmaceutical Sciences of Ribeirão Preto, University of
São Paulo, Av. do Café s/n, Campus USP, 14040-903, Ribeirão Preto,
São Paulo, Brazil.
2
To whom correspondence should be addressed. (e-mail:
pmcampos@usp.br)
particle size ensures a close contact with the SC, increasing
the skin hydration and decreasing the TEWL (5,6), which can
be an interesting property for treatment/prevention of skin
disorders with damaged skin barrier function (2,7). Further-
more, they are able to control drug release and posses low
toxicity and cytotoxicity (6,8,9). Lipid nanoparticles are based
on a natural or synthetic lipid matrix, which is in the solid
state at room and body temperature, with a nanoscale particle
size ranging from about 40 to 1000 nm (5,10,11). Beeswax is a
natural fatty product that has been used for the development
of several drug delivery systems in the pharmaceutical field,
but very little attention has been given to its cosmetic and
dermatological application (8,12–15). Due to the high hydro-
phobicity and excellent moisture resistance, beeswax is a
favorable candidate for the development of nanosystems for
topical applications once it remains intact after incorporation
into an o/w cream, combining the advantages of traditional
cream or other dermatological formulation.
Traditionally, beeswax has been used as a thickener and
a humectant in the manufacture of ointments, creams,
obtained from the honeycombs of bees (Apis mellifera)
containing esters of saturated fatty acids, and long-chain
alcohols (mainly consists of hydrocarbons, monoesters, dies-
ters, triesters, free myristic acid, and unreported amount of

1530-9932/17/0000-0001/0 # 2017 American Association of Pharmaceutical Scientists


hydroxy acids and diols) that are solid at room temperature
(melting point ca. 54°C) and water insoluble (16,18,19).
Considering its high availability and relatively low industrial
cost, new forms of beeswax application becomes of great
interest. Furthermore, in recent years, there has been an
increasing interest in natural lipid-based systems for dermal
drug delivery due to factors such as physical properties,
biodegradability, and skin biocompatibility (5,6,8,19–21).
Thus, the employment of natural lipids in topical formulations
due to their rich chemical composition follows the tendency
to use renewable resources as a sustainable alternative (2,21).
Within this context, the objective of the present investi-
gation was to develop and evaluate the effect of beeswax-
based nanoparticles (BNs) in maintaining skin barrier func-
tion and aiming at dermatologic and cosmetic purposes. BN
was developed by the hot microemulsion method and
formulations containing BN were characterized and ap-
praised for their in vivo effect on stratum corneum water
content (SCWC) and TEWL after topical application by
in vivo skin biophysical techniques.
MATERIAL AND METHODS
Materials
Polaxamer 407 and Tween 80 (polysorbate 80) were
purchased from Via Farma Ltda (São Paulo, SP, Brazil).
Purified beeswax with an esterification index of 70% and
melting point of 54°C (batch number 153475) was supplied by
Labsynth Ltda (São Paulo, SP, Brazil). The following INCI
ingredients, C12-15 alkyl benzoate, caprylic/capric triglycer-
ide, cetearyl alcohol/dicetyl phosphate/ceteth-10 phosphate,
and sodium polyacrylate/ethylhexyl cocoate/PPG-3 benzyl
ether myristate/polysorbate 20, were provided by Croda do
Brasil Ltda (Campinas, SP, Brazil); acrylates/C10-30 alkyl
acrylate crosspolymer were from Lubrizol do Brasil Aditivos
Ltda (São Paulo, SP, Brazil); and butylene glycol, glycerin,
phenoxyethanol/methylparaben/ethylparaben/butylparaben/
propylparaben/isobutylparaben, disodium EDTA, butylated
hydroxytoluene, and aminomethyl propanol were from
Mapric Produtos Farmacêuticos e Cosméticos (São Paulo,
SP, Brazil).
Preparation of the Beeswax-Based Nanoparticles
BNs were prepared by the hot melt microemulsion
technique proposed by Freitas et al. (22). Beeswax (at 2.5,
5.0, or 10% w/v) in association or not with carnauba wax (1:1;
total lipid content 5.0% w/v) were melted in a beaker in a
thermostatic water bath at 60.0 ± 2.0 or 90. 0 ± 2.0°C when
carnauba wax was present. Tween 80 (1.0% w/v) and
Polaxamer 407 (2.0% w/v) were mixed with deionized water
at 90.0 ± 5.0°C and added slowly to the molten lipid under
magnetic stirring until the predetermined volume (q.s. to
100 mL). The resulting mixture was dispersed for 5 min, at
60.0 ± 2.0°C (or 90.0 ± 2.0°C) and 20,000 rpm, using an Ultra-
turrax—T25 Digital homogenizer (IKA Works Inc, EUA).
Finally, the resulting nanoemulsion was rapidly cooled to
room temperature (25.0 ± 1.0°C) in an ice bath and then kept
at 4.0 ± 2.0°C.
Souza et al.
BN Characterization and Morphology
The average size, zeta potential, and polydispersity of the
BN were determined by photon correlation spectroscopy
using a ZetaSizer Nano Series instrument (Malvern Instru-
ments Limited, Worces, UK). After BN preparation, the
samples for these measurements were maintained for 24 h at
4.0 ± 2.0°C. Ultrapure water (Mili Q, Millipore Inc., USA)
was used as the dispersing medium at a 1:3 ratio (BN
dispersion/water). The surface appearance and shape of BN
were determined 24 h after preparation using an atomic force
microscope (AFM) model SPM-9600 (Shimadzu Co., Kyoto,
Japan). The BN dispersion was diluted 1:200 using ultrapure
water (Milli-Q, Millipore Inc., USA) and spread onto thin
mica plates.
In order to assess the physical stability, samples in
triplicate of the BN dispersion were stored at 4.0 ± 2.0, 25.0
± 2.0, and 37.0 ± 2.0°C and the size, zeta potential, and
polydispersity were measured 7, 14, 21, and 28 days after
preparation.
Preparation of Semi-Solid Formulations
Gel-cream formulations, containing or not the BN
dispersion, were prepared using the o/w emulsification
technique, according to Table I. Oil phase ingredients
(cetearyl alcohol/dicetyl phosphate/ceteth-10 phosphate,
C12-15 alkyl benzoate, caprylic/capric triglyceride, butylated
hydroxytoluene, and phenoxyethanol/methylparaben/
ethylparaben/butylparaben/propylparaben/isobutylparaben)
and aqueous phase ingredients (water, disodium ethylenedi-
aminetetraacetic acid, and glycerin) were heated separately at
70°C. Oil phase was added to aqueous phase under contin-
uous stirring (600 rpm, 10 min, Heidolph magnetic stirrer,
Fisaton, SP, Brazil), followed by spraying of acrylates/C10-30
alkyl acrylate crosspolymer and sodium polyacrylate/
ethylhexylcocoate/PPG-3 benzylether myristate/polysorbate
20 and stirring at 1200 rpm for 30 min to form a homogenous
cream. This gel-cream base was cooled under continuous
stirring at room temperature, and its pH was adjusted to 5.6
with aminomethyl propanol. The formulation containing BN
was prepared by replacing 10% (w/w) of the water phase in
the emulsion with the BN dispersion. BN dispersion was
added to the gel-cream at the end of the process under slow
stirring.
Physical Stability of Semi-Solid Formulations
Formulations containing or not BN dispersion also
underwent preliminary stability tests consisting of visual
evaluation for phase separation after three centrifugation
cycles at 604×g for 30 min and pH measurements 24 h and 7,
14, 21, and 28 days after preparation and storage at 4.0 ± 2.0,
25.0 ± 2.0, and 37.0 ± 2.0°C at 75% relative humidity.
Rheological Behavior
Rheological measurements were performed in a model
DV-III rotational rheometer (Brookfield, Stoughton, MA,
USA), equipped with cone-and-plate test geometry and
analyzed with Brookfield software RHEOCALC V1.01. The
Beeswax Nanoparticles Improves Skin Barrier Function

Table I. Composition of the Evaluated Topical Formulations.

Composition (%, w/w)a Vehicle BN-loaded formulation

Cetearyl alcohol, dicetyl phosphate e ceteth-10 phosphate 2.0 2.0

C12-15 alkyl benzoate 3.0 3.0
Caprylic/capric triglyceride 3.0 3.0
Butylated hydroxytoluene 0.02 0.02
Phenoxyethanol, methylparaben, ethylparaben, propylparaben, butylparaben, isobutylparaben 0.8 0.8
Sodium polyacrylate (and) ethylhexyl cocoate (and) ppg-3 benzyl ether myristate (and) polysorbate 20 0.3 0.3
Acrylates/C10-30 alkyl acrylate crosspolymer 0.3 0.3
Glycerin 3.0 3.0
Disodium ethylenediaminetetraacetic acid 0.1 0.1
BN dispersion – 10.0
Water q.s. 100 g 100 g

BN beeswax-based nanoparticles
a
Qualitative composition was reported in accordance with International Nomenclature of Cosmetic Ingredient (INCI)

samples were evaluated 24 h and 7, 14, 21, and 28 days after
preparation and stored at 4.0 ± 2.0, 25.0 ± 2.0, and 37.0 ±
2.0°C. The sample (0.5 g) was applied on the rheometer plate
and the spindle CP51 was adjusted to a 0.5-mm gap between
the cone and plate. Continuous flow measurements were
performed by increasing the rotation speed from 5.0 to
100.0 rpm and the shear rate from 3.84 to 387.8 s−1 over
90 s, followed by a decrease of shear rate from 387.8 to
3.84 s−1 over 90 s.
The software RHEOCALC V1.01 (Brookfield, Stoughton,
MA, USA) was used to evaluate different rheological models
fitting to the data and to characterize the behavior of the
formulation during the storage time. The models fitted were the
Ostwald, Herschel-Bulkley, and Cross and Cross with yield value,
according to Eqs. 1, 2, 3, and 4, respectively (23).
τ ¼ Kγn
τ ¼ σ0 þ Kγn
 
ð η ∞ − η0 Þ
τ ¼ η0 þ γ ð3Þ
1 þ ðK CR γÞn
 
ð η ∞ − η0 Þ
τ ¼ η0 þ γ þ σ0
1 þ ðK CR γÞn
In Vivo Efficacy Study
The Human Experimentation Committee of the School
of Pharmaceutical Sciences of Ribeirão Preto at University of
São Paulo approved the study protocol (CEP/FCFRP n° 315),
which complied with the ethical guidelines of the 1975
Declaration of Helsinki, revised in 1983. Sixteen healthy
female Brazilian volunteers, aged between 20 and 30 years,
with skin phototypes II–IV, were selected for a randomized
and single-blind clinical study, and they signed the written
informed consent form. Three test areas were defined on each
volar forearm of the participants: one control area and two
other areas for the application of each test formulation: BN-
loaded and non-loaded gel-cream formulation. The measure-
ments were performed after 20-min acclimation to controlled
temperature and humidity conditions of 22–24°C and 45–
55%, respectively. The study was conducted in Ribeirão Preto
ð1Þ
(São Paulo, Brazil) in January 2014.
Each formulation was applied randomly to the volar
forearm of each volunteer and measurements were made
before (basal values, T0) and 28 days after daily application
of the formulations under study. The effects of the formula-
ð2Þ tions on the skin was evaluated in terms of transepidermal
water loss (TEWL) and stratum corneum water content
(SCW), using a Tewameter® TM210 and a Corneometer®
CM825 (Courage + Khazaka Electronic GmbH, Cologne,
Germany), respectively. Five measurements for SCW and
three for TEWL were performed in each testing skin area.
The results are expressed as an averaged SCW (a.u.) and
TEWL (g/m2h) after application of the formulations to each
measurement point. During this interval, the participants
were not allowed to either use any other cosmetic product or
undergo dermatological procedures that could directly
interfere in the results. The same operator performed all the
ð4Þ measurements.

where σ0 is the yield stress, ηo is the viscosity, n is the flow
index, K is the consistency index, η∞ is the viscosity in the
infinite, η0 is the initial viscosity, KCR is the relaxation time, τ
is the shear stress, and γ is the shear rate (23). Measurements
were made in triplicate at 25.0 ± 2.0°C.
Statistical analysis
The results of the rheological measurements are
expressed as mean ± standard deviation and were analyzed
statistically by analysis of variance (ANOVA), with the level
of significance set at p < 0.05. Means were compared by the

Tukey test. For the clinical study, all data are expressed as
mean ± standard deviation and were analyzed statistically by
the Wilcoxon test for comparative analysis of the data
obtained in one area over time, with the level of significance
set at p < 0.05, using the software GraphPad Prism v.7.
RESULTS
Characterization and Stability of BN
The particle size, PdI, and zeta potential, determined by
dynamic light scattering 24 h after preparation, and the pH
values are represented in Table II for each formulation
developed. Formulation F1, containing beeswax at 10.0%
(w/v), had the lower particle size, followed by F2 and F3
formulations, containing beeswax at 5.0 and 10.0% (w/v),
respectively. On the other hand, the zeta potential and pH
have not changed significantly with changes in the formula-
tion composition. BN composed only by beeswax at 5.0% (w/
v) was selected to continue the studies.
Figure 1 shows the morphology of the selected BN
formulation determined by atomic force microscopy (AFM)
for a topographic view (Fig. 1a) and a frontal view (Fig. 1b, c),
with particle size ranging from 66 to 118 nm according to the
measurements made by dynamic light scattering. The systems
were physically stable at 4.0 ± 2.0°C, and no significant change
was observed to the size, zeta potential, and pH after 28 days
of storage, as seen in Table III. At 25 and 37°C, the average
particle sizes increased simultaneously with PdI, while zeta
potential and pH remained stable as compared to their values
24 h after preparation.
Three examples of the size distribution obtained in the
present study using dynamic light scattering, can be seen in
Fig. 2a–c. As shown in Fig. 2a, there is only one peak with a
reasonably narrow distribution and particle sizes ranging
from 60 to 300 nm 24 h after preparation. After storage at
37.0 ± 2.0°C, the profile of size distribution may change
considerably, as seen in Fig. 2b, c, which shows BN size
distributions after 7 and 28 days, respectively. In Fig. 2b, there
are two well-defined peaks, or a bimodal distribution: the first
peak with size from 8 to 60 nm and the second one with a
higher peak size from 50 to 500 nm. Figure 2c also shows a
bimodal distribution with particle size ranging from 10 to
700 nm. Since our analysis must be based on the intensity
distribution, only the largest peaks were taken into account
for mean size determination at the situations where there
were two or more peaks, and the results are presented in
Table III.
Characterization and Stability of Semi-Solid Formulations
Twenty-eight days after storage at 25.0 ± 2.0, 4.0 ± 2.0,
and 37.0 ± 2.0°C, visual and smell inspection of formulations,
containing or not BN, did not reveal any signs of instability
related to appearance, color, odor, and phase separation, and
the centrifugation test did not result in phase separation,
creaming, cracking, or precipitation. Freshly prepared formu-
lations were slightly acidic, with pH ranging from 5.3 to 5.8,
and this parameter did not significantly change over storage
time at different temperatures (Fig. 3). These findings
indicate that the two formulations have good physical
Souza et al.
stability, and no changes were observed after BN
incorporation.
Rheological Behavior
Figure 4 shows the rheograms with ascending and
descending curves obtained for the semi-solid formulations
containing or not BN. It was possible to verify that the flow
curves of all studied formulations showed a non-Newtonian
and viscoplastic behavior (n < 1), i.e., shear thinning with
yield value. The rheograms obtained were better adjusted to
the Herschel-Bulkley model showing regression coefficients
(R2) higher than 0.99. The mean values for consistency index
(K), flow index (n), and yield stress calculated according to
this model (Eq. 2) are represented in Tables IV, V, and VI.
The hysteresis loop was calculated based on the area under
the curve and are represented in Table VII. No significant
change suggesting any physical instability was observed in
these rheological parameters after 28 days of evaluation at all
temperatures.
Clinical Efficacy Study
Figure 5 shows the SCWC and TEWL values obtained
after 28 days of application of the studied formulations on the
volar forearm of 16 volunteers. Only the BN-loaded formu-
lation showed a significant decrease of TEWL values and
increase of SCWC values (Wilcoxon test, α = 5%) compared
to basal values, whereas the untreated controls remained
unchanged. It is important to notice that none of the
volunteers reported side effects such as irritation, local
discomfort, and itch during the study period.
DISCUSSION
Physical Stability of BN and Gel-Cream Formulations
The effect of beeswax content in the lipid matrix on the
properties of BN was studied by preparation of nanoparticles
with three beeswax concentrations in the lipid mixture while
other ingredients (Tween 80 and Polaxamer 407) were kept
constant. As shown in Table II, variation of beeswax in the
lipid mixture from 2.5 to 10% (w/v) had a significant effect on
the particle size and PdI. These results indicate that BN
prepared with higher beeswax concentration presented lower
particle size. When carnauba wax was incorporated into the
system, both particle size and PdI increased compared to the
system containing only beeswax at the same content in the
lipid mixture (5.0% w/v). Zeta potential, and pH values of
nanoparticles, did not change considerably upon increasing
the beeswax content and carnauba wax in the lipid mixture.
Similar results were found by Kheradmandnia et al. (13).
They obtained decreased particle size with increasing bees-
wax concentration. However, they found that the addition of
carnauba wax decreased the particle size.
Considering these results, the BN formulation containing
only beeswax at 5.0% (w/v) was chosen to continue the
studies, because it was considered the most appropriate
system for dermatologic/cosmetic purposes compared to the
others regarding particle size and PdI values. The ideal
particle size is quite controversial in the literature; however,
Beeswax Nanoparticles Improves Skin Barrier Function

Table II. Particle Size, PdI, Zeta Potential, and pH Values (Mean ± Standard Deviation) for the Beeswax-Based Nanoparticles 24 h After
Storage at 4.0 ± 2.0°C.

Composition (w/v)a Particle size (nm) PdI Zeta Potential (mV) pH

F1: 2.5%/0 468.0 ± 105.84 0.701 ± 0.12 −10.89 ± 0.44 4.06 ± 0.17
F2: 5.0%/0 95.72 ± 9.63 0.323 ± 0.03 −8.76 ± 0.58 3.76 ± 0.03
F3: 10.0%/0 28.56 ± 1.26 0.278 ± 0.02 −9.85 ± 0.57 4.75 ± 0.23
F4: 2.5%/2.5% 395.3 ± 23.26 0.442 ± 0.04 −9.23 ± 0.33 3.98 ± 0.12
a
Composition of beeswax/carnauba wax in each formulation

most studies have focused on systems with particles ranging
from 100 to 300 nm for topical application. F2 and F3 were
not considered in order to continue the present study because
they showed higher PdI values, which could impair the
physical stability of the system after storage (5, 6, 8, 13).
According to Malvern ZetaSizer (24), high PdI values
may interfere with the reliability of the estimated size of
particles. The Malvern ZetaSizer software applies a cumula-
tive analysis method to calculate the zeta-average (particle
size) and polydispersity. But, when PdI values are above 0.25,
cumulative analysis is not applicable and neither zeta-average
nor PdI values are reliable. Based on this, it is advised to
extract the diameters directly from the frequency distribution
of size, i.e., from the center value of the peak with higher
intensity (8,24). The PdI value obtained in the present study
(0.323 ± 0.03) was calculated by Malvern software using
cumulative analysis and can only be used for qualitative
purposes and not for quantitative comparison. So, the particle
sizes presented were calculated directly from the frequency
distribution of size (8).
AFM analysis are in accordance to the measurements
made by dynamic light scattering and showed that the
particles are spherical in shape and homogeneously distrib-
uted, ranging in size from 66 to 118 nm, which corroborates
with the use of frequency distributions to obtain average
sizes. The optimum size for nanoparticles is quite controver-
sial and depends on the route of administration (11,25,26). In
general, nanoparticles for systemic administration for

Fig. 1. Atomic force microscopy of the beeswax-based nanoparticles: a topographic and b, c frontal view
 Souza et al.

Table III. Particle Size, PdI, Zeta Potential, and pH Values (Mean ± Standard Deviation) for the Beeswax-Based Nanoparticles 28 days After
Storage at Different Temperatures

Temperature Particle size (nm) PdI Zeta potential (mV) pH

4°C 103.36 ± 30.30 0.462 ± 0.02 −10.70 ± 0.88 4.33 ± 0.14

25°C 164.97 ± 26.30 0.484 ± 0.05 −8.32 ± 0.53 3.93 ± 0.23
37°C 269.80 ± 95.29 0.549 ± 0.03 −10.50 ± 0.91 3.93 ± 0.13

therapeutic purposes, e.g., may be in the 2 to 200-nm range
(27), while particles containing curcuminoids with an average
diameter of 210 nm showed satisfactory size for topical
treatment of skin inflammatory reactions (8). Therefore, we
considered the BN particle size obtained to be suitable for
cosmetic purposes.
The particle size and size distribution, along with zeta
potential, are the most significant parameters for the evalu-
ation of the stability of colloidal systems, once it may affect
the physical stability and biological performance of these
systems (10). Surfactants play an important role on theses
parameters (25). In this study, a mix (2:1) of Tween 80 and

Fig. 2. Size distribution by intensity a 24 h and b 7and c 21 days after storage at 37.0 ± 2°C
Beeswax Nanoparticles Improves Skin Barrier Function
Fig. 3. pH values (mean ± standard deviation) obtained for a vehicle
and b BN-loaded formulation over time and after storage in different
temperatures (n = 3). BN-loaded formulation: formulation containing
beeswax-based nanoparticles
Polaxamer 407 was used as surfactant and was suitable to
obtain nanoparticles with good particle size distribution and
stability. These results can be attributed to the hydrophilic-
Fig. 4. Shear stress as a function of shear rate for a BN-loaded formulation and vehicle 24 h after preparation, b BN-loaded
formulation, and c vehicle 28 days after storage at different temperatures (mean values ± standard deviation, n = 3). BN-
loaded formulation: formulation containing beeswax-based nanoparticles
lipophilic balance (HLB) value of surfactants that is required
for stabilizing the lipid core. It is well known that the HLB is
very important during the preparation of nanosystems and
was considered in the execution of the present study.
According to a previous study, HLB value required to
emulsify beeswax is around 10–16. Tween 80 has HLB value
of 15, which is then suitable for dispersion of beeswax in the
aqueous phase (16,28). Tween 80 provides a steric stabiliza-
tion by an interaction between its hydrophilic groups and
water, thus creating a so-called protective water barrier
between the particles that prevents coagulation (25).
Zeta potential values in the −15 to −30-mV range are
common for stabilized nanoparticles (16,25). High values of
zeta potential in module indicate that the electrostatic
repulsion between particles will prevent their aggregation
and thereby stabilize the nanoparticulate dispersion (29). The
zeta potential for the BN obtained in the present study, about
−10 mV, does not provide strong electrical field around the
nanoparticles but still is not critical for their agglomeration.
The low zeta potential values found in this study are
characteristic of nanoparticles with a Tween 80 particle/
emulsion coating, which in turn stabilizes such particles
sterically at the particle/water interface (30). The zeta
potential does not only represent the individual particle
surface charge but its charge in relation to the electric double
layer resulting from surrounding ions in continuous phase
(24). Acidic groups dissociated on nanoparticle surface will
hold a negative surface. The non-ionic structure of Tween 80
or the acidic pH values of the dispersion (pH ≈ 3–4) may also
give negatively charged particle surface (25).
It is also important to highlight the role of temperature in
the development of these systems. In the present study, the
temperature of both melting the beeswax and the aqueous
 Souza et al.

Table IV. Flow Indexes (n) Values (Mean ± Standard Deviation) of the Formulations 24 h (T0) and 28 days (T28) After Storage at Different
Temperatures.

Formulation T0 T28

4.0 ± 2°C 25.0 ± 2°C 37.0 ± 2°C

Vehicle 0.45 ± 0.07 0.32 ± 0.04 0.29 ± 0.02 0.29 ± 0.02

BN-loaded formulation 0.58 ± 0.04 0.46 ± 0.03 0.43 ± 0.06 0.36 ± 0.07

BN-loaded formulation gel-cream formulation containing beeswax-based nanoparticles

phase were maintained at 60°C. In preliminary tests, temper- Rheological Behavior

atures lower than 60°C resulted in big wax flakes produced by
the process. According to Mehnert and Mader (25), higher
temperatures result in lower particle size due to the decreased
viscosity of the inner phase. The temperature chosen herein
showed to be adequate.
After 28 days, there were no significant changes in
pH, particle size, PdI, or zeta potential of BN dispersion
stored at 4°C, showing a relatively long-term stability of
them at this temperature. Upon storage, a slight increase
in particle size was observed after storage at higher
temperatures. At room temperature, the increase in
particle size could be detected only 28 days after BN
preparation, which may be attributed to the aggregation
of lipid particles. Once aqueous BN dispersions are
intended for topical application, they have to be incorpo-
rated into a semi-solid formulation, in order to have a
proper consistency. Previous reports have suggested that
nanoparticles incorporated into a gel-cream are physically
stabilized by the three-dimensional network structures of
the cream (31–33), which could prevent the agglomeration
between the BN after incorporation into the semi-solid
formulation. Jenning et al. (34) reported that the network
of the gel hampered the polymorphic lipid nanoparticle
transitions and thus enhanced their stability. Under these
conditions, the contact of the particle was decreased and
aggregation was avoided (34).
Hence, the obtained BN dispersion was incorporated
into a gel-cream formulation (at 10% v/w), which was
stable during storage at all temperatures evaluated after
28 days. As the skin pH ranges from 4.6 to 5.8, the
formulations’ pH ranging from 5.3 to 5.8 makes them
suitable for topical application. The physical stability test
is important for cosmetic products to guarantee that its
intended efficacy and physical and chemical quality is
maintained over a period of time under certain conditions
(35).
Rheological behavior plays an integral part in the
assessment of the overall efficiency of topical formulation
and became a valuable tool for the determination of quality
of these products. Especially concerning the application and
performance of a formulation on the skin, consistence index
measurements and hysteresis loop provide important infor-
mation regarding consistency, spreadability, and sensory
properties (36–38). For emulsions, the interfacial film formed,
the concentration and kind of emulsifying agents can modify
this rheological behavior, and therefore, it is important to
assess the influence of each additional ingredient on the
rheological parameter of semi-solid formulations (7,39). They
also permit to monitor changes during storage and give
valuable hints on the microstructure of complex systems
(40,41).
Analysis of the rheograms in Fig. 4 reveals that the flow
curves of all formulations showed non-Newtonian behavior
since their viscosity was not constant after shear stress, with
low thixotropy (42). The non-Newtonian behavior of this kind
of preparation is reflected by the power law index (n) (23).
Several models may be used to establish n in different non-
Newtonian systems. The most adequate model for a
viscoplastic fluid depends on the fluid response to deforma-
tion and how well the experimental data fit the model (41,43).
In the present study, the rheograms obtained were better
adjusted to the Herschel-Bulkley model and the mean values
for hysteresis loop, consistency index (K), flow index (n), and
yield stress obtained with this model are represented in
Tables III, IV, V, and VI. Viscoplastic flow characteristics
were also detected for both tested formulations (n < 1), which
agree with the obtained flow curve behavior. Herschel-
Bulkley model means that these systems start flowing after
achieving a yield value and, beyond this value, the viscosity of
the formulations decreases with increasing shear rate (41,44).
Other studies have demonstrated that the flow curves of

Table V. Consistency Index (K) Values (Mean ± Standard Deviation) of the Formulations 24 h (T0) and 28 days (T28) After Storage at
Different Temperatures.

Formulation T0 T28

4.0 ± 2°C 25.0 ± 2°C 37.0 ± 2°C

Vehicle 38.7 ± 15.2 102.4 ± 4.3 115.9 ± 14.2 112.1 ± 3.5

BN-loaded formulation 26.2 ± 8.2 52.8 ± 6.2 52.9 ± 5.6 98.1 ± 6.8

BN-loaded formulation gel-cream formulation containing beeswax-based nanoparticles

Beeswax Nanoparticles Improves Skin Barrier Function

Table VI. Yield Stress (σ0) Values (Mean ± Standard Deviation) of the Formulations 24 h (T0) and 28 days (T28) After Storage at Different
Temperatures.

Formulation T0 T28

4.0 ± 2°C 25.0 ± 2°C 37.0 ± 2°C

Vehicle 439.8 ± 53.3 250.4 ± 34.2 244.9 ± 8.3 331.0 ± 24.6

BN-loaded formulation 432.7 ± 13.4 323.6 ± 21.5 321.2 ± 34.5 369.3 ± 43.6

BN-loaded formulation gel-cream formulation containing beeswax-based nanoparticles

emulsions are fitted by Ostwald and Herschel-Bulkley models
(23,45,46), which agrees with the results of the present study.
Higher n values were observed for BN-loaded formula-
tions. According to Silva et al. (40), higher values of n means
higher yield values, and it has been claimed to be advanta-
geous for the stability of semi-solid formulations during
storage and for easier local application. In this study,
significant difference was not observed between yield values
of the gel-cream formulation containing or not BN.
Viscosity changes over time, indirectly measured by the
K value, are also a way to indirectly monitor chemical
degradation because changes at the molecular level may
cause changes in viscosity (38,41). In the present study, K
values increased over time for either formulations, containing
or not BN, after 28 days of storage at different temperatures,
because of the emulsifying agent used that increases its
viscosity over time. This result means that the incorporation
of BN into the gel-cream formulation did not impair the
physical stability of the cream (Table IV). However, lower K
values were observed for BN-loaded formulations compared
to the vehicle alone. The decrease in viscosity for semi-solid
formulations containing nanocarriers has been previously
reported in the literature (40,44) and is not related with
instability of the formulations.
The hysteresis loop of the vehicle also covered a larger
area than the BN-containing formulation, indicating a greater
thixotropy to the vehicle. This result contrasts with previous
report in the literature, where higher thixotropy was observed
after incorporation of nanoparticles into a semi-solid formu-
lation (40). Liu et al. (47) demonstrated that thixotropy is
related to the regeneration of hydrogen bonding and the time
needed to restructure the three-dimensional network struc-
ture of a gel. Considering that, it is suggested that the
negatively charged BN may affect the recovery of hydrogen
bonding in the gel-cream and it takes longer time to
restructure the three-dimensional network structure of the
formulation.
For optimum efficacy, a topical formulation should have
low viscosity at high shear rates and a low, but not zero,
thixotropy (7,41,42). Additionally, skin diseases vary in the
size of the area affected requiring preparations that have
specific rheological properties tailored for ease of application
(4). So, our results indicated that BN-loaded formulation
possessed favorable thixotropy and viscosity, suggesting that
BN are stable and suitable for topical application to the skin.
The formulation under study showed these important fea-
tures even after 28 days at 37°C.
Clinical Efficacy Study
Clinical efficacy studies are performed in order to
provide evidence supporting the safety and the claims on
the label of a cosmetic product. This study investigated the
effect of BN on skin barrier function after incorporation into
a gel-cream, aiming at cosmetic and dermatologic purposes
for topical treatment/prevention of skin disorders, such as
atopic dermatitis, psoriasis, or even dried skin. In order to
carry out this function properly and maintain the appearance
of the skin, it is important to maintain the balance between
the SCWC, TEWL, and the skin surface lipids, which is the
hydrolipidic balance of the SC (2,48,49).
The Corneometer® and the Tewameter® have been the
most widely used biophysical methods for evaluating the skin
barrier function. Corneometer® evaluates the skin barrier
function through SCWC, while Tewameter® evaluates the
skin barrier function by the measures of TEWL (1,7,49,50).
TEWL and SCW of healthy skin depend on different factors
such as anatomic site, gender, age, ethnicity, and so on. When
the skin barrier is damaged, TEWL values increase, whereas
healthy skin tends to have low TEWL and high SCW values.
Several studies have demonstrated a direct correlation
between TEWL and the degree of skin water barrier
disruption, in addition to supporting the understanding of
the hydration mechanisms of some formulations. Accordingly,

Table VII. Hysteresis Area Values (Mean ± Standard Deviation) of the Formulations 24 h (T0) and 28 days (T28) After Storage at Different
Temperatures.

Formulation T0 T28

4.0 ± 2°C 25.0 ± 2°C 37.0 ± 2°C

Vehicle 36,265 ± 3819 45,973 ± 3442 41,446 ± 4235 41,667 ± 3276

BN-loaded formulation 15,819 ± 4421 27,831 ± 2126 24,446 ± 3346 41,460 ± 5920

BN-loaded formulation gel-cream formulation containing beeswax-based nanoparticles

 Souza et al.

Fig. 5. Stratum corneum water content (SCWC; a) and transepidermal water loss (TEWL;
b) values of the control area and before and 28 days after daily self-application of vehicle
and BN-loaded formulation on the volunteer’s arm. Statistically significant compared to
baseline values (T0)—Wilcoxon test: *p < 0.1 and **p < 0.01. T0: basal values (before
formulation application). T28: 28 days after treatment with both formulations. BN-loaded
formulation: formulation containing beeswax-based nanoparticles

TEWL and SCW measurements may be considered to be a
powerful non-invasive way to determine the effects of
chemical products on epidermal barrier function, whose
values fit together and allow more dynamic assessment of
the true state of hydration and skin barrier function (1,51).
According to Fig. 5, only the BN-loaded formulation
showed a significant decrease in TEWL values and a
significant increase in SCWC values (Wilcoxon test, α =
5%), which means that application of BN to the skin
helped to improve skin barrier function in a significant
manner. This result could be explained by the small size
of BN particles, which have a larger surface area,
permitting greater adhesive properties of the formulation
to the skin. They are able to form a uniform compact
layer on the skin surface, so that water evaporation from
the SC is reduced in the area treated with BN-loaded
formulation and, consequently, increase the SCWC (5,6).
BN may also led to decreased TEWL values due to the
presence of beeswax, an occlusive agent that is also
capable of forming a film on the skin surface (16).
The discussion about the in vivo occlusive effect of
nanoparticle systems is somewhat controversial. Jenning et al.
(34), for example, did not see a relevant difference in TEWL
values when comparing a gel formulation containing or not
solid lipid nanoparticles at 20.0% (w/v). They attributed this
result to the high standard deviation of TEWL, which may
have masked minor occlusive effects, and also, in part, to the
different composition of the formulations tested. It has been
observed that nanoparticles added to a formulation do not
have an additional occlusive effect when the formulation itself
is already highly occlusive (e.g., using petrolatum or highly
occlusive creams) (26). For this reason, in the present study,
we developed a formulation with low lipidic content, and
even so, it was demonstrated an improvement on the skin
barrier function.
Previous studies have obtained hydration effect on
the skin by applying cosmetic formulations containing
active ingredients such as vitamins, ceramide, panthenol,
and a biotechnological natural extract due to the combi-
nation of humectants and occlusive properties (2,38,50,52–
54), whereas other studies have not shown any effect on
TEWL values, even in the presence of these active
ingredients (55,56). Adversely, conventional creams used
for treat skin diseases suffer from decreased patient
compliance due to oiliness and low spreadability. In the
present study, the ability of the formulation containing BN
Beeswax Nanoparticles Improves Skin Barrier Function
to decrease the TEWL and increase the SCWC without
additional active ingredient was observed. In a previous
study, Kim et al. (2) developed a ceramide-loaded
microparticle and showed that only the system containing
high amounts of ceramide were able to improve damaged
skin barrier function but not empty microparticles. Alter-
natively, it is possible to load BN with an active
ingredient in order to treat specific skin diseases, which
are advantageous, compared to conventional vehicles. It is
well known that nanoparticles are able to control drug
release and increase the skin penetration, reducing the
side effects and toxicity.
CONCLUSION
Considering such results, it is possible to conclude
that the present study permitted the successful production
of beeswax-based nanoparticles by the hot melt
microemulsion technique. BN-loaded formulation signifi-
cantly decreased the TEWL and increased SCWC values
after 28 days of treatment without addition of any active
ingredient. Thus, BN could be suggested as a new, cost
effective, and commercially viable alternative with a
proven effect on skin barrier function. In addition, the
results herein establish the suitability and compatibility of
beeswax as a versatile natural resource that may poten-
tially be applied in the field of cosmetics and dermatology.
Accordingly, challenge is launched for future studies to
assess the efficacy of these beeswax nanosystems incorpo-
rating active ingredients in order to treat specific skin
conditions with damaged skin barrier function.
ACKNOWLEDGMENTS
The authors would like to thank the Coordenação de
Aperfeiçoamento de Pessoal de Nível Superior (CAPES,
Brazil) and the Fundação de Amparo à Pesquisa do Estado
de São Paulo (FAPESP, Brazil) for the financial support to
this study.
COMPLIANCE WITH ETHICAL STANDARDS
Disclosure of Interest The authors declare that they have no
conflicts of interest.
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