FULL PAPER Macromolecular Symposia

Infantile Hemangioma www.ms-journal.de

Development and Characterization of Nanoemulsion

Containing Almond Oil, Biodegradable Polymer and
Propranolol as Potential Treatment in Hemangioma
Daiana S. Coelho, Vania E. B. de Campos, Zaida M. F. Freitas, Eduardo Ricci J

unior,
Michelle B. Caarls, Bruno LourenSc o Diaz, Elisabete P. dos Santos,
and Mariana Sato de S. B. Monteiro*

1. Introduction
Hemangiomas are benign tumors, which cause an abnormal proliferation
Infantile Hemangiomas (IHs) are benign
of endothelial cells. In recent years, propranolol (POP) has been used for
skin lesions caused by proliferating endo-
the therapy of infantile hemangiomas (IH). Topical POP has been thelial cells, which grow after birth and
prescribed instead of systemic steroids to reduce the risk of side effects. usually regress spontaneously. They are the
In order to increase the effectiveness of the POP, nanoemulsions (NEs) most common vascular tumors in children,
were developed as drug delivery carriers for topical delivery. NEs are occurring in 5% to 10% of infants.[1] The
clinical symptoms of hemangioma vary
stable dispersions consisting of oil, water and surfactants. To develop NEs
according to the size of the lesion, location,
water-in-oil, POP was solubilized in two concentrations, at 0.25 wt% and depth of skin penetration and stage of
0.5 wt% in water. To prepare the external phase, the almond oil, a evolution. It can be classified according to
biocompatible vegetable oil, was chosen due its anti-inflammatory proper- the depth of the lesion as superficial, deep
ties. An optimal proportion of surfactant and co-surfactant, Tween 80/ or mixed.[2] The treatment of even small
Span 80, were assessed by pseudo-ternary phase diagram. NEs were hemangioma in the facial area should be
considered, as it is not possible to predict
obtained by ultrasonic processor and the mean droplet size was measured
the outcome. Currently there are not many
by Dynamic Light Scattering (DLS) and it was less than 300 nm. POP was therapeutic options, and drug treatment
included in the best formulation for stability studies, Nuclear Magnetic involves the use of topical or systemic
Resonance (NMR) studies, by the analysis of proton relaxation time, cell agents, for example, corticosteroids and
viability studies and occlusivity test. At the end of the work, the stable propranolol (POP). However, the use of
both drugs may have significant systemic
NEs, containing POP, diluted above 200 fold, did not affect cell viability
adverse effects.[2,3]
and it can be considered as a promising formulation for the treatment of In recent years, some studies have
superficial infantile hemangioma. reported the use of non-selective beta-
blocker, such as POP and timolol maleate,
as a potential new topical agent for the
treatment of superficial hemangioma. The topical use of these
agents is effective, safer and more comfortable for pediatric
patients, compared to the oral route.[1,4]

D. S. Coelho, Z. M. F. Freitas, E. Ricci J

unior, E. P. dos Santos, M. B. Caarls
M. S. de S. B. Monteiro Instituto de Ci^encias Biom
edicas
Faculdade de Farmácia Universidade Federal do Rio de Janeiro
Universidade Federal do Rio de Janeiro Centro de Ci^encias da Sa
ude
Centro de Ci^encias da Sa

ude, Bloco L Cidade Universitária
Cidade Universitária Ilha do Fund~ao, Rio de Janeiro 21945-970, Brazil
Ilha do Fund~ao, CP 68525, Rio de Janeiro 21945-970, Brazil
E-mail: marianasato@pharma.ufrj.br
B. LourenSc o Diaz
V. E. B. de Campos Instituto de Biofísica Carlos Chagas Filho
Instituto de Macromol
eculas Universidade Federal do Rio de Janeiro
Centro de Tecnologia Centro de ci^encias da Sa
ude
Universidade Federal do Rio de Janeiro Cidade Universitária
Ilha do Fund~ao, Rio de Janeiro 21941-598, Brazil Ilha do Fund~ao, Rio de Janeiro 21945-970, Brazil
DOI: 10.1002/masy.201800121

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POP has been used in the treatment of high blood pressure;
however, since 2008, it has also been used off-label for the
treatment of infantile hemangioma.[5] POP is thought to inhibit
the growth of blood vessels and constrict existing blood vessels
within the hemangioma. It acts on beta adrenergic receptors by
decreasing the release of blood vessel growth-signaling
molecules and by triggering programmed cell death.[6,7]
Many studies have shown the efficacy and safety of topical use
of POP in the treatment of superficial IHs.[1,4,5,8] It was observed
that the treatment with 1% w/w of POP cream and 1% w/w of
propranolol hydrophilic ointment were well tolerated without
side effects, which indicates that the topical application is a safe,
effective and cheap therapeutic option for treating superficial
IHs.[2] Moreover, Casiraghi et al (2016) selected a semi-solid
vehicle (e.g. hydrophilic ointment, two lipophilic creams and
hydrophilic cream) for a locally applied drug product containing
1% w/w of propranolol hydrochloride and studied the skin
permeation through human epidermis. It was observed that the
human epidermis was able to be a reservoir compartment of
POP, allowing prolongation of the drug diffusion to the lower
layers of human skin after removal of the topical formulation.
Indeed, the amount of drug retained in the human epidermis
(54.57 μg cm
2) can diffuse to the superficial hemangioma
maintaining a clinical effect. In particular, lipophilic creams may
also take advantage of the occlusive effect and be able to sustain
drug penetration in the human epidermis.[8]
An ideal drug delivery system fulfills the objective of
maximizing therapeutic effect while minimizing toxicity.[9]
Nanoemulsion (NE) is a drug delivery system constituted by
nanodroplets with a size between 20 and 200 nm surrounded by
surfactant. These systems have many advantages compared to
conventional emulsions. Usually, NE can exist as oil-in-water
(O/W) but can also be water-in-oil (W/O) or multiple emulsions
W/O/W.[9,10] Their very large interfacial area positively influences
the drug transport and their delivery to target specific sites. NE
has the ability to protect the drugs from hydrolysis and
enzymatic degradation, making it an ideal drug delivery carrier.
So the major advantages of NEs, as drug delivery system, include
increased drug loading, enhanced drug solubility and bioavail-
ability, reduced patient variability, controlled drug release and
protection from enzymatic degradation.[10–12]
An important fact that distinguished NE from microemulsion
(ME) is the input of external energy that is required to produce a
colloidal dispersion. Generally, equipments such as high
pressure homogeneizer or ultrasonic processor could be an
option to reduce and uniformized the droplet size. NE is usually
prepared with a low surfactant concentration (below critical
micelle concentration).[11,12]
Consequently, during the development of any type of
emulsion-based system, selecting the surfactant or mixture
surfactants is a critical step to obtain the desired formulation,
and it is essential for obtaining systems with long-term stability.
Surfactants are able to form a membrane around droplets of the
dispersed phase and strengthen the interfacial film, ensuring
droplet stability.[13,14]
Almond oil (Oleum amygdalae) is a vegetable oil with
antioxidant activity based on its chemical constitution, because
it presents high concentrations of linoleic acid.[15] Almond oil
has been used in medicine for its numerous health benefits,
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such as: dry skin, psoriasis and eczema.[16] Further, almond oil
seemingly reduces hypertrophic scarring post-operatively and
smoothes and rejuvenates skin. Also, it has emollient and
sclerosant properties.[17] NEs prepared with almond oil were
suitable to delay benzophenone-3 photodegradation under
ultraviolet (UV) radiation.[15] Dermatologists and pediatricians
recommend the use of almond oil for children and babies due to
its properties.[16,17]
Although O/W NE studies have been undertaken in recent
years, relatively few studies on W/O NE currently exist. A
potential transdermal delivery has been studied as a route to
overcome low bioavailability and permeability of thiocolchico-
side using W/O NE.[18] Also, Liu et al. (2016) developed a Panax
notoginseng saponins NE with an average particle size of 28.17 nm
and polydispersity index (PDI) of 0.116.[19]
The objective of this work was to develop (W/O) NE,
containing 0.25 wt% and 0.5 wt% of POP, almond oil and
nonionic surfactant mixtures, Tween 80 and Span 80, to be used
in the treatment of superficial hemangioma. Moreover, the NEs
developed were evaluated by droplet size, polydispersity index,
stability test, low field Nuclear Magnetic resonance (NMR)
measurements, cell viability tests and occlusion measurements.
2. Experimental Section
2.1. Materials
The materials used in this study were: propranolol hydrochloride
purchased from All Chemistry (Brazil), polyoxyethylene sorbitan
monooleate (Tween 80) purchased from Mapric (Brazil), which
contains 20 moles of ethylene oxide, sorbitan monooleate (Span
80) purchased from Sigma–Aldrich (Brazil), Almond oil
purchased from Sarfam (Brazil) and 3-[4,5-dimethylthiazol-2-
yl]-2,5-diphenyltetrazolium bromide, M5655, purchased from
Sigma–Aldrich.
2.2. Propranolol Solubility
POP was subjected to solubility test in water and almond oil. An
excess quantity of POP (50 mg) was added to a defined volume of
water and almond oil (5 mL of each). These solutions were kept
under stirring for 24 h at room temperature (25  C  2  C), and
then a centrifuged at 3000 rpm for 5 min. After that, the
supernatant was removed and filtered through cellulose
membrane. An aliquot of each solution, 1 mL, was diluted with
methanol, and subsequent dilutions were prepared in methanol.
The absorbance was determined using quartz cuvette (1 cm) at
290 nm and a calibration curve was prepared in methanol.[20]
2.3. Determination of the Hydrophile-Lipophile Balance
(HLB)
For the determination of the HLB of almond oil, a series of oil
emulsions was prepared. The water/oil emulsions were
prepared by keeping constant the percentage of purified water
(10%), surfactant mixture (15%) and oil (75%). For the selected
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surfactants, Span 80 (HLB ¼ 4.3) and Tween 80 (HLB ¼ 15), a
series of HLB was calculated from range 5 to 14 using the
Equation 1[21]:
HLBT  T% þ HLBS  S% ¼ HLBmin ð1Þ
where HLBT, HLBs and HLBmix, are the HLB values of Tween 80,
Span 80 and the surfactant mixture, respectively, T% is the mass
percentage of Tween 80 and S% is the mass percentage of Span
80 in the mixture of surfactants, respectively. Initial HLB values
for Tween 80 and Span 80 were adapted from the manufacturer’s
specification.
In HLB assay, 10 g of formulations were developed. The
lipophilic phase was slowly added to the hydrophilic phase,
under constantly magnetic stirring, using a stirring plate (IKA,
model C-MAG HS 7), at 3000 rpm for 5 min at room temperature
(25  C). After 24 h, the best HLB value required to stabilize the
emulsions was characterized macroscopically.
The macroscopic aspects of formulations as well as the variation
in their stability (creaming, coalescence and/or presence of phase
separation) were evaluated by visual inspection.[13,14]
2.4. Construction of Pseudo-Ternary Phase Diagram (PTD)
PTD consisting of oil, water and surfactant mixture were
constructed using the oil method.[22] According to the best HLB
determined previously, the ratio of Span 80/Tween 80 was fixed
at 95:5 on the weight basis. Almond oil constituted the oil phase
constituent and the aqueous phase was composed of recently
distilled and deionized water. Initially the water phase was added
into a mixture of surfactants at the ratios of 1:9, 2:8, 3:7, 4:6, 5:5,
6:4, 7:3, 8:2 and 9:1 (wt%/wt%). The oil phase was titrated to the
water and surfactant mixture at room temperature to total 10 g of
each sample (25  0.2  C). After titration, samples were
homogenized under constantly magnetic stirring, using a
stirring plate (IKA, model C-MAG HS 7), at 3000 rpm for
5 min at room temperature (25  C) to reach an equilibrium. The
Figure 1. Production of Nanoemulsion using ultrsound.
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oil phase concentration yielded 99 formulations in which water/
surfactant mixture ratio ranged from 1:9 to 9:1. Following this,
the final samples with adequate volume of water to solubilize
POP were selected to be processed to obtain NE.[13,14]
The macroscopic aspects of formulations as well as the
variation in their stability (creaming, coalescence and/or
presence of phase separation) were evaluated by visual
inspection and classified according to macroscopic aspects of
formulations as ME (microemulsion), EM (emulsion) and phase
separation. Single-phase and transparent mixtures were
designed as MEs, and this region was plotted on a triangular
graph as ternary or pseudoternary phase diagram using Origin
software (OriginPro8). Opaque mixtures were designated as
emulsions. The compositions in the phase diagram are
expressed in %.[14,23]
2.5. Preparation of Nanoemulsions
NE regions were identified based on phase diagrams. Then they
were processed using an ultrasonic processor (model UP 100 H,
power maximum ¼ 100 W, equipped with a 7-mm-diameter tip,
Dr Hielscher GmbH, Germany), and the input power level was
100% of total input power, which was equal to 100 W, with
continuous cycling. The processing time of NE ranged from
1 min to 10 min. The temperature was kept at 5  C by a cold bath
during the formulation processing. The different processing
times were used to verify the best condition of homogenization
of the formulations. After the determination of the best
processing time, POP was included in the NE. The total amount
obtained for each formulation was 10 g (Figure 1).
Two formulations were developed: one containing 0.25 wt% of
POP and another with 0.5 wt% of POP. The drug was added to
the internal phase of the NE, aqueous phase, with Tween 80.
Then, the oil phase, with Span 80, was slowly poured over to the
aqueous phase while it was being submitted to magnetic stirring,
at 25  C. After that, the NEs with POP were treated for 10 min by
ultrasonic processor (UP 100 H, 60 W, 30 kHz; Hielscher,
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Germany). The total amount obtained for each formulation was
10 g.
2.6. Nanoemulsion Characterization
2.7. Dynamic Light Scattering Experiments (DLS)
Droplet size distribution, mean droplet diameter and polydis-
persity index (PDI) of samples were measured during 3 months
in a commercial DLS instrument (Zetasizer Nano ZS, Malvern
Instruments, Malvern,UK). Measurements were conducted at
room temperature (25  C), and the laser incidence angle in
relation to the sample was 173 , using a 5 mL quartz cuvette. All
values reported correspond to the mean  standard deviation
(SD) of three measurements of each formulation. Polydisper-
sion index (PDI) calculated by the device reflects the
homogeneity profile of each droplet diameter. The measure-
ments were conducted for all samples immediately after
processing and after 30 and 90 days to study formulations’
stability.
2.8. pH Determination
The pH values of the formulations were determined in triplicate
directly from the samples (Oakton Ion6 potentiometer).
2.9. Stability Studies
To verify sample stability, emulsions were stored for 3 months at
5  C  2  C (Consul, model CRM 37/127 refrigerator), 25  -
C  2  C and 40  C  2  C (Brasdonto heater) in sealed glass
bottles for stability studies. At 30 and 90 days, the formulations
were subjected to drop mean diameter analysis by DLS.[24]
2.10. NMR Relaxation Measurements
The POP distribution in NE was evaluated by spin–spin
relaxation time (T2H), in a MARA Ultra low field NMR
spectrometer (Oxford Instruments, Oxford, UK), at 28  C, using
an 18 mm NMR tube, operating at 23 MHz for the hydrogen
nucleus. The pulse sequence used to measure the spin-spin
relaxation time was Carr–Purcell–Meiboom–Gil (CPMG), and
the 90 pulse of 7.5 ms was calibrated automatically by the
instruments software, and τ was 800 ms. The relaxation values
and relative intensities were obtained by fitting the exponential
data with the aid of the WINFIT program. Distribution
exponential fittings were performed using the WINDXP
software. The equipmentś precision is 2%. The relaxation
values were calculated using the following equation (Equation 2).
My ¼ Mo exp
τ=T2 H ð2Þ
where My is a longitudinal component of the magnetization
vectors, Mo is the equilibrium value, τ is time period delay and
T2H is a spin-spin relaxation time.
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2.11. Cell Viability Assay
In this study, HaCaT cell line was used, which is a human
keratinocyte line, and BEND3 cell line, which is a murine
cerebral endothelium line. The cells were cultured in glucose-
rich Dulbecco’s Modified Eagle Medium (DMEM) medium,
supplemented with 10% of fetal bovine serum, 100 U mL
1 of
penicillin and 100 mg of streptomycin and incubated at 37  C
with 5% of CO2.
For the cell viability tests, the cells were plated in 96 well plates
at 3.8  104 cells/well, for HaCaT cell line, and 1.8  104 cells/
well, for BEND3 cell line. Twenty-four hours after, the cells were
incubated with the culture medium or medium containing the
three formulations developed: NE vehicle, NE with 0.25 wt% of
POP and NE with 0.5 wt% of POP, at different dilutions: 100,
200, 400, 800, 1000 and 2000. The formulations were
autoclaved at 120  C for 20 min under high pressure before being
added to the cells.
The determination of cell viability was performed by the
MTT technique (3-[4,5-dimethylthiazol-2-yl]-2,5-diphenylte-
trazolium bromide, M5655, Sigma–Aldrich). This test mea-
sures a reduction of the MTT reagent to formazan and is
directly related to mitochondrial activity, and, consequently, to
cell viability. After 24 h of incubation with the formulations,
the cells were incubated with MTT at the final concentration of
0.5 mg mL
1 for 4 h at 37  C. The formazan crystals formed in
the viable cells were dissolved in Dimethyl sulfoxide (DMSO)
(150 μL well
1), and the absorbance at 562 nm was measured
on Emax Plus plate (Molecular Devices, Sunnyvale, CA).[25]
According to Sklubalová et al. (2018), an aqueous solution of
propranolol hydrochloride was autoclaved at 121  C for 15 min
and remained stable for two weeks, maintaining the drug
content around 100%.[26] Further, the autoclave was chosen as
the sterilization method since some studies used this method to
sterilize W/O NEs without showing a significant change in mean
droplet size and PDI.[27,28]
2.12. Occlusivity Test
In order to evaluate the effects of NEs on the occlusive properties,
an in-vitro occlusion test was performed. Glass bottles (40 mL)
with a diameter of 4.6 cm were filled with 30 g of water and covered
with a filter paper (cellulose filter, 90 mm, Whatman number 6,
cutoff size: 3 mm, USA). 220 mg of the sample was spread
homogeneously with a spatula on the filter surface (13.3 mg cm
2)
and the sample was subsequently stored at 37  C for 48 h in order to
mimic the temperature of the skin surface. The amount of water
remaining in the glass bottles was weighed at 24 and 48 h. The glass
bottles covered with filter paper, soaked with water, were used as
reference.[29] All experiments were done in triplicate (n ¼ 3). The
occlusion factor (F) was computed by Equation 3:
F ¼ ½ðA
BÞ=A  100 ð3Þ
where A stands for the water loss without sample (reference) and
B is the water loss with sample. An F value of 0 indicates no
occlusive effects compared to the reference, while an F value of
100 indicates maximum occlusiveness.
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2.13. Statistical Analysis Table 1. Hydrophile-lipophile balance (HLB) of the systems.

Experimental results were presented as mean  standard % Span 80 % Tween 80 HLB Visual aspects
deviation. Statistical analysis was performed using the One- 10 90 13.93 Phase separation
way Analysis of Variance (ANOVA) with Instat3 software.
20 80 12.86 Phase separation
p > 0.05 was considered statistically significant.
30 70 11.79 Phase separation
40 60 10.72 Phase separation

3. Results and Discussion 50 50 9.65 Phase separation

60 40 8.58 Phase separation
3.1. Propranolol Solubility
70 30 7.51 Phase separation

1 80 20 6.44 Relative homogeneity
POP presented a solubility of 10 mg mL in water and
4.5 mg mL
1 in almond oil. As the drug was more soluble in 90 10 5.37 Homogeneity
water, that solvent was chosen to form the internal phase of NE. 95 5 4.84 Homogeneity

3.2. Determination of the Hydrophile-Lipophile Balance
(HLB)
As mentioned before, a surfactant or mixture of surfactants is a
critical step to obtain the desired emulsion. Surfactants are able
to form a membrane around droplets of the disperse phase and
strengthen the interfacial film, ensuring droplet stability. As it is
known, oils and surfactants exhibit specific HLB values and,
when adding a surfactant or a surfactant mixture with HLB value
corresponding to the oil HLB value, it is possible to maximally
reduce the interfacial tension between oil and aqueous
phase.[13,14]
A pair of surfactants with different values of HLB, one with a
low HLB value and another with high HLB value, can better
stabilize the emulsified system.[30] Surfactant agents act as a
barrier by modifying the coalescence ratio of the disperse phase,
or by creating an interfacial film that produces repulsive
electrostatic forces between the disperse phases.[31] In general, it
is understood that hydrophilic and lipophilic surfactants are
aligned side by side in order to provide higher rigidity and
resistance to the emulsifier film through hydrogen bonds.[12] In
this regard, sorbitan derivates such as the polyoxyethylene
sorbitan monooleate, Tween 80, with HLB value of 15 and
sorbitan oleate, Span 80, with HLB value of 4.3 were used for NE
development. Both surfactants are nonionic have widespread
use in pharmaceutical products due to their biocompatibility,
high stability, low irritation and low toxicity.[13]
All emulsions were prepared in order to determine the critical
HLB of almond oil with the pair of surfactants Span 80 and
Tween 80, as seen in Table 1. According to the results obtained, it
was observed that the HLB of almond oil was 4.84 and the ratio of
the surfactants Span 80 and Tween 80 was 95% and 5%,
respectively. It has been reported that HLB numbers between 4
and 6 are optimum for stabilization of water-in-oil emulsions.[32]
The appearance of this emulsion was homogenous, slightly
yellow, translucent and fluid. This system was more homoge-
nous and limpid than the previous one with 90% of Span 80 and
10% of Tween 80.
Span 80 is a lipophilic emulsifying liquid agent which tends to
form water-in-oil emulsions.[12] It has a strong absorption and
strong affinity (“anchoring”) to the oil surface, because it adopts
a curvature that gives an ultralow interfacial tensions due to its
small molecule.[11] Tween series is a derivative of Span where the
hydroxyl groups on the sorbitan ring are substituted with
polyoxyethylene groups. Tween series is more soluble in water
and widely used as stabilizing agent in oil-in-water emulsion.[11]
Tween 80, a polymeric surfactant, is a homopolymer, which is
formed from the same repeating units. This homopolymer has
little surface activity at the O/W interface, since the homopoly-
mer segments (ethylene oxide) are highly water soluble and have
little affinity to the interface. However, its presence was very
important in the steric hindrance of the droplets, stabilizing
them and avoiding the coalescence and subsequent separation of
phases.[11,12]
In fact, a mixture of surfactants, Span 80 and Tween 80, is
necessary to stabilize the water-in-oil emulsion, reaching an
adequate HLB value. Each surfactant molecule has a specific
conformation at the droplet interface, which contributed to the
stabilization of formulation by distinct mechanism, i.e. Span 80
will be distributed among Tween 80 chains, to fulfill the spaces
and give some interface rigidy and Tween 80 has a high tail
length and causes a steric hindrance of the droplets.[11,12] In this
study, a polymeric surfactant (Tween80) and a small molecule
surfactant (Span 80) were used as surface active agents.
3.3. Pseudo-Ternary Phase Diagram Study
The appropriate ratios among all components were studied by
the ternary diagram method. Using the method described by
Treguier[33] and Lo,[34] the ternary diagram is represented in the
plane as an equilateral triangle, where the three constituents are
symmetrical. Thus, each axis of the triangle corresponds to 100%
of the constituents. In Figure 2, one axis of the pseudo three-
component phase diagram represents the aqueous phase, the
other represents the oil phase and the third represents a mixture
of surfactant at a fixed weight ratio.
In this work, pseudo-ternary diagram was constructed based
on observation and macroscopic identification of the type of
dispersion obtained from the mixtures produced. According to
Figure 2, microemulsions were located in regions of water,
surfactant and oil concentrations ranging from about 5% to 23%,
31% to 82% and 8% to 49%, respectively. All of the icroemulsions
systems were characterized as a homogenous and transparent

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deformation concentration and Gibbs elasticity in the interface.

Thus, in a poor surfactant system, a coalescence rate is
significant. There are not enough amounts to fully stabilize a
new interface. A moderate concentration of surfactant causes a
considerable decrease in the size of the droplets, as this is able to
better stabilize an interfacial area.[36]
It is well known that large amounts of surfactants cause skin
irritation. It is therefore important to determine surfactant
concentration properly and use the optimum concentration of
surfactant in the formulation.[37] From pseudoternary phase
diagrams, the formulations in which the water phase completely
solubilized the drug and which could accommodate the
optimum quantity of surfactant mixture and oil were selected
for study. The composition of the stable formulation was 16% wt,
46.55% wt and 37.45% wt of water phase, oil phase and
surfactant mixture, respectively.

3.4. Nanoemulsion Development

Figure 2. Microemulsion region of pseudo-ternary diagram with
surfactant mixture composed of Span 80/Tween 80 and almond oil as
The processing time of NE ranged from 1 min to 10 min and the
oil phase.
different processing times were used to verify the best condition
of homogenization for formulations in the ultrasonic processor.
systems, containing high amounts of oil and low amounts of Figure 3 and Table 2 show the influence of processing time, in
water.[14] Due to these characteristics, it was more likely that a the ultrasonic processor, on the NE droplet size. It was observed
microemulsion occurred in the region that extends from 82% of that the processing time of 10 min provided smaller droplet
surfactant, and contains low water concentration (less than 23%) sizes, according to size distribution, and a more homogenous
and high oil concentration. system than the other processing times, which was extremely
However, as the amount of water increased and surfactant significant (p < 0.0001). Processing times of less than 10 min did
decreased, the systems lost their transparent appearance and in not show statistical significance (p > 005). After the determina-
this region, milky macroemulsions were obtained.[14] Creamy tion of the best processing time, the POP was included in the NE
water-in-oil macroemulsions were located in regions of water, and processed with this condition.
surfactant and oil concentrations ranging from about 10% to Ultrasonication methods depend on high-frequency sound
55%, 20% to 74% and 8% to 49%, respectively. Creamy oil-in- waves. The ultrasonic waves produce cavitation bubbles which
water macromeulsions were located in regions of water, continue to grow until they implode. This implosion creates
surfactant and oil concentrations ranging from about 49% to shockwaves, which in turn create a jet stream of surrounding
83%, 10% to 28% and 8% to 31%, respectively. liquid, pressurizing dispersed droplets and causing size
Heterogenous systems, characterized as phase separation,
were observed at different points of the diagram, with the water,
surfactant and oil concentrations ranging from about 41% to
53%, 10% to 13% and 34% to 49%, respectively. Other points of
phase separation were observed with the concentrations of 72%
water, 8% surfactant and 20% oil. The occurrence of a phase
separation region suggests that mixtures of surfactants were not
sufficient to disperse and stabilize both phases. In this case, the
formation of multiple layers occurred characterizing the
unstable condition, which caused complete separation of
phases.[14]
In fact, a larger amount of surfactant relative to the internal
phase is required to form a stable emulsified region. This shows
that the amount of surfactant is related to the stability and size of
the emulsion droplets. This can be explained by the Gibbs–
Marangoni effect, which shows that when two newly formed
droplets approach one another, they acquire more surfactants at
their interface and less in the region close to the contact.[35] This
unequal adsorption leads to a surface tension gradient that
attracts more surfactant to this poor region of the substance.
Also, this influx of surfactants stabilizes the droplets against Figure 3. The influence of processing time, in ultrasonic processor, on the
coalescence. The force of the effect is a function of the NE droplet size.

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Table 2. Mean droplet diameter  S.D. (nm) and polydispersity index

(PDI) of NE as a function of processing time.

Sample Processing time Droplet size [nm] PDI

0
NE vehicle 30 551.3  72.9 0.37  0.05
1 min 583.6  59.9 0.40  0.14
3 min 563.3  82.9 0.39  0.02
5 min 586.4  67.5 0.43  0.06
7 min 542.5  45.2 0.37  0.07
10 min 223  10.8 0.28  0.06


The p value is <0.0001, considered extremely significant (10 min compare to other
processing times).

reduction. Investigation into operational parameters has

revealed that droplet size decreases with increasing sonication
time and input power.[10] In our work, the input power remained
constant, at maximum power, and therefore the sonication time
was varied. Consequently, it was observed that the droplet size
decreased with increasing processing time.
3.5. Nanoemulsion Characterization
The NEs developed were liquid homogenous, translucent and
yellow systems. These characteristics make the NEs developed
an attractive candidate for topical application. The NEs were
characterized in terms of droplet size, polydispersity index (PDI)
and pH (Table 3). Figure 4 shows the mean droplet size of NE
vehicle, NE with 0.25 wt% and 0.5 wt% of POP.
It was observed that the mean droplet size of NE vehicle was
223  10.8 nm, the PDI was 0.28  0.06 and pH was 5. Thus, the
system was within the desired range for the intended action:the
treatment of superficial hemangiomas located in the epidermis,
showing a droplet size around 250 nm and reducing the risk of
POP systemic absorption. After the inclusion of 0.25 % wt of
POP, the droplet size (173.6  35 nm) and PDI value
(0.21  0.05) remained constant, without statistical difference
in relation to the vehicle, indicating that this drug concentration
maintained the system stability.
The NE with 0.5 wt% of POP showed the largest droplet size,
around 271.6  15.3 nm, and PDI value around 0.29  0.02,
and pH was 4.5. However, there was a statistical difference
Table 3. Mean droplet diameter  S.D. (nm), polydispersity index
(PDI) and hydrogenionic potential (pH) of three NE containing 16%
wt of aqueous phase, in the presence and in the absence of POP. All
measurements were carried out at 25  C.
Formulation Droplet size [nm] PDI
Figure 4. The droplet size distribution of NE vehicle, NE with 0.25 wt%
and 0.5 wt% of POP.
between NE with 0.25% of POP and 0.5% of POP (p < 0.01). The
higher concentration of the drug in the NE may have saturated
the aqueous phase and contributed to the increased size of the
droplet, due to the increased viscosity. In fact, when relative
viscosity is too high, droplets become resistant to break up.[10] All
formulations had droplets in the nanoscale which is well evident.
The pH remained between 4.5 and 5.0 after POP incorporation,
which is slightly acid, an ideal pH for topical formulations.[38]
3.6. NMR Relaxation Measurements
In spin-spin lattice relaxation, the decay to equilibrium is based
on the spins of the nuclei in neighboring molecules. Because all
nuclei in the sample have varying spin, the spin of the nuclei in
neighboring molecules produce magnetic fields that affect the
spin of the nuclei in other molecules. So, the relaxation time,
T2H, is related to the molecular dynamics of the system. This
means that any change in the molecular mobility in the system
may be reflected in this parameter. Rigid and restricted systems
present a smaller and less intense signal of T2H.[39,40]
The variation of the apparent spin-spin relaxation time (T2H)
of the emulsions is based on the difference of the spin-spin
relaxation time of water molecules inside a water droplet.[41]
Table 4 and Figure 5 show the T2H measurements and
distributions curves for NEs vehicle and with POP, respectively.
The NMR analysis demonstrated that the NEs with almond oil
presented three domains with different T2H values. It can be
correlated to the NEs phases: water, oil and surfactants mixture,
and each phase has an intrinsic relaxation time. It is possible to
pH

NE vehicle 223  10.8 0.28  0.06 5  0.3

Table 4. T2H measurements, by low field NMR
NE 0.25% POP 173.6  35 0.21  0.05 5  0.2
NE 0.5% POP 271.2  15.3 0.29  0.02 4.5  0.1 Sample T2,1 [ms]/% T2,2 [ms]/% T2,3 [ms]/%

NE 34/13% 99/55% 277/31%


The p value is 0.0058 (p < 0.01), compared to NE 0.25% POP, considered very
NE 0.5% POP 28/10% 88/57% 254/32%
significant.

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Figure 5. Distribution curves obtained by low-field nuclear magnetic
resonance of almond oil NE vehicle and with 0.5% of POP.
associate the highest T2,3H value (277 ms) to the hydrogens in
the surfactant chains, Span 80 and Tween 80, the intermediate
T2,2H value (99 ms) to the hydrogens present in the almond oil
structure of the oil phase and the lowest T2,1H value (34 ms) to
the hydrogens present in the water molecules confined into the
nanodroplets.
Usually, the water molecules have T2H above 1 s, however
when water molecules are confined to nanodroplets, as in the
case of NEs developed, the value of T2H is inversely proportional
to the droplet volume. Thus, a small droplet of water has a large
surface area and presents a greater effect of superficial
relaxation, reducing its T2H values. Moreover, the downward
shift of the T2H relaxation time distribution increased with
increasing shear intensity during production, and thus with
decreasing droplet size. Higher T2H values were observed at
lower stirring rates, and lower T2H values were observed with
higher stirring rates.[41,42]
As mentioned before, the NEs were developed using the
ultrasonication method. It is a high-power processor, which
produces violently and asymmetrically imploding vacuum
bubbles, creating micro-jets that break up the original droplets
around nanometer size.[10] This confirmed the lowest T2H value
for the water confined into the nanodroplets which have a mean
diameter around 220 nm.
Moreover, it was possible to guarantee this correlation between
T2H values, domains and NEs components using the area under
the distribution curve. According to LaTorraca et al (1998), the area
under the distribution curve is proportional to the amount of
hydrogen detected.[43] Thus, comparing the amount of each
component in NE, 16% wt of water, 46.55% wt of oil and 37.45% wt
of surfactant mixture with Table 4, it was possible to observe similar
quantities of the hydrogen population detected in T2,3H domain
with surfactants concentration; hydrogen population detected in
T2,2H domain with oil concentration and hydrogen population
detected in T2,1H domain with water concentration.
After the addition of 0.5% of POP into almond oil NE, it was
possible to verify a reduction in all T2H values, demonstrating a
decrease in the molecular mobility and possible hydrogen drug
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Figure 6. Schematic representation of water in oil nanodroplets.
interaction with NE component. Therefore, a slight increase
was observed in the domain corresponding to the water at
28 ms and an enlargement of the peak corresponding to the
surfactants at 265 ms after POP introduction. This showed that
POP was more effectively distributed in the water and in
surfactants. This result was expected since POP is water soluble
drug, and surfactants, as mentioned above, are amphiphilic
molecules.[44]
Using NMR analysis, it was possible to provide a schematic
representation of nanodroplets, indicating the position of each
component in the final structure (Figure 6).
3.7. Stability Studies
The stability of NEs produced was studied over three months at
25  C, 5  C and 40  C. Table 5 shows the results of mean droplet
size and PDI of NEs. The droplet size distribution of NE vehicle,
NE 0.25 wt% of POP, NE 0.5 wt% of POP after 30 and 90 days in
25  C, 5  C and 40  C, demonstrates a good stability with
nanometer sizes.
The statistical analyses were performed while comparing the
temperature of storage and considering the time within the same
group. For example, comparing the NEs with 0.25% of POP at all
temperatures and time analyzed the values did not reach
statistical significance within this group (p > 0.05). These results
showed that these systems remained stable over time.
The NE vehicle 25  C, at 0 h, showed statistical significance
compared to the NE vehicle 25  C, at 90 days (p < 0.01), also to
the NE vehicle 40  C, at 90 days, and NE vehicle 5  C at 30 and
90 days (p < 0.001). The NE vehicle 25  C, at 30 days, showed
statistical significance compared to the NE vehicle 25  C, at

90 days, NE vehicle 40  C, at 90 days, NE vehicle 5 C at 30 and

90 days (p < 0.001) and NE vehicle 40 C, at 30 days (p < 0.05).
The NE vehicle 40  C, at 30 days, showed statistical significance
compared to the NE vehicle 40  C at 90 days (p < 0.01), NE
vehicle 5  C at 30 and 90 days (p < 0.001). The NE vehicle 5  C, at
30 days, showed statistical significance compared to the NE
vehicle 5  C, at 90 days (p < 0.05).
Usually, the increase of temperature changes the viscosity of
oils. According to Singh et al. (2017), the relative viscosities of
two phases, internal or external, has a strong influence on
reducing droplet size.[10] This can explain the decrease in droplet
size when the NE vehicle were stored at 40  C.
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Table 5. Average droplets sizes and PDI of NEs after 30 and 90 days The NE 0.5% POP, 40  C, at 90 days, showed statistical
at 25  C, 5  C and 40  C. significance compared to the NE 0.5%, 5  C, at 30 days (p < 0.05).
The POP showed solubility on both phases, aqueous and oil
Size [nm] and PDI phase. Regarding the decrease of droplet size over time, a
Samples Temperature 0h 30 days 90 days possible explanation is the diffusibility of the drug to an
[ C] interface. Thus, the size of the droplets is reduced by diffusion of
NE Vehicle 25 223  10.8 247.9  5.7 194.6  6.6 the drug from the internal phase, which is aqueous, to the
surfactant interface. According to NMR studies, shown in
0.28  0.06 0.30  0.03 0.30  0.11a)
Figure 6, the drug is also distributed at the interface, confirming
NE Vehicle 40 223  10.8 217.6  17 b)
178.7  16.2c)
a tendency to diffuse. This is also in agreement with the results
0.28  0.06 0.26  0.02 0.31  0.08 of the stability of the NE 0.25% POP, where there was not a
NE Vehicle 5 223  10.8 142.3  14.4d) 112.7  1.7e) statistical difference as a function of time and temperature, since
0.28  0.06 0.15  0.002 0.38  0.07 the drug concentration is not close to saturation, as shown in the
NE 0.25% 25 173.3  35 143  5.9 126.1  5.3
solubility study.
POP Size distribution is an important parameter to prevent Oswald
0.21  0.05 0.20  0.08 0.26  0.11
Maturation, which is the main instability phenomenon. PDI is a
NE 0.25% 40 173.3  35 115.6  7.6 143.7  22.5 dimensionless measure of the width of the size distribution,
POP 0.21  0.05 0.25  0.12 0.29  0.08 calculated from the cumulants analysis ranging from 0 to 1.[12]
NE 0.25% 5 173.3  35 153.7  28.5 142.7  13.6 All the NEs samples, Table 5, presented values between 0.15 and
POP 0.21  0.05 0.24  0.05 0.17  0.05 0.38, indicating a monodisperse distribution over time.
The pH of all NEs remained around 5, slightly acidic, an
NE 0.5% POP 25 271.6  15.3 261.3  55.5 91.4  12.6f)
ideal pH for topical formulations. So, it is expected that the NE
0.29  0.02 0.17  0.05 0.16  0.04
will not irritate the skin by alkalinization or disrupt the skin’s
NE 0.5% POP 40 271.6.  15.3 238.2  18.1 196.1  19.4g) acid mantle. The pH parameter ensures the stability, efficacy and
0.29  0.02 0.20  0.05 0.26  0.09 safety of formulations. Higher systems stabilities are achieved
NE 0.5% POP 5 271.6  15.3 111.9  9.1h) 175.5  17.4i) when they maintain a minimum pH variation.[46]
0.29  0.02 0.15  0.02 0.29  0.12

a)
p-value <0.01 (NE vehicle 25 90 days vs. NE vehicle 25 0 h, NE vehicle 5 30
days); b)p < 0.01 (NE vehicle 40 30 days vs. NE vehicle 40 90 days); c)p < 0.001 (NE
vehicle 40 90 days vs. NE vehicle 25 0 h, NE vehicle 25 30 days, NE vehicle 5 90
days); d)p < 0.001 (NE vehicle 5 30 days vs. NE vehicle 25 0 h, NE vehicle 25
30 days, NE vehicle 25 90 days, NE vehicle 5 90 days, NE vehicle 40 30 days, NE
vehicle 40 90 days); e)p < 0.001 (NE vehicle 5 90 days vs. NE vehicle 25 0 h, NE
vehicle 25 30 days, NE vehicle 25 90 days, NE vehicle 40 30 days, NE vehicle 40
90 days); f)p < 0.001 (NE 0.5% 25 90 days vs. NE 25 0h, NE 25 30 days, NE 40 30
days); g)p < 0.05 (NE 0.5% 40 90 days vs. NE 0.5% 25 0 h, NE 0.5% 5 30 days);
h)
p < 0.001 (NE 0.5% 5 30 days vs. NE 25 0 h, NE 0.5% 25 30 days, NE 0.5% 40 30
days); i)p < 0.01 (NE 0.5% 5 90 days vs. NE 0.5% 25 0 h, NE 0.5% 25 90 days).
It is well known that the solubility of surfactants with
poly(ethylene oxide) is related to the hydration of the oxythylene
groups in water. In fact, hydrogen bonding results in the
bonding of at least two water molecules per EO unit. At low
temperatures, these surfactants are more hydrophilic which
results in a favorable state of energy.[14] So, in the NE vehicle
samples stored at 5  C, the droplets size decreased confirming
the mechanisms described above.
The NE 0.5% POP, 25  C, at 0 h, showed statistical significance
compared to the NE 0.5%, 25  C, at 90 days (p < 0.001), NE 0.5%,
40  C, at 90 days (p < 0.05), NE 0.5%, 5  C, at 30 days (p < 0.001)
and NE 0.5%, 5  C, at 90 days (p < 0.01). The NE 0.5% POP, 25  C,
at 30 days, showed statistical significance compared to the NE
0.5%, 25  C, at 90 days (p < 0.001), NE 0.5%, 5  C, at 30 days
(p < 0.001), NE 0.5%, 5  C, at 90 days (p < 0.05). The NE 0.5% POP,
25  C, at 90 days, showed statistical significance compared to the
NE 0.5%, 40  C, at 30 days (p < 0.001), NE 0.5%, 40  C, at 90 days
(p < 0.01) and NE 0.5%, 5  C, at 90 days (p < 0.05). The NE 0.5%
POP, 40  C, at 30 days, showed statistical significance compared to
the NE 0.5%, 5  C, at 30 days (p < 0.001).
3.8. Cell Viability Assay
The cell viability test was performed to verify if the endothelial
cells or human keratinocytes would be kept alive after being
treated with NEs developed. This test was performed with the
pure NE, NE with 0.25 wt% of POP and 0.50% wt of POP, in
order to verify if the cell viability depends on the drug
concentration. The results of this analysis can be verified in
Figure 7. The controls were the cells not submitted to the
treatment with pure NE, and the parameter of maximum cell
viability was 100%. The results of three treatments at different
dilutions on BEND3 cells are shown in Figure 7A. This
experiment showed statistically significant changes in cell
viability 24 h after treatment with the NE and 0.50% wt of
POP up to 400 fold, and NE with 0.25 wt% of POP up to 200 fold
(p < 0.001). Also, statistically significant results were found in
HACAT cells (Figure 7B), in which viability was reduced after
24h of treatment with the NE and 0.25 wt% of POP up to 200
fold, and NE with 0.5 wt% of POP up to 400 fold (p < 0.001). The
HaCaT cells were more susceptible to the treatment than
BEND3, especially in the more concentrated samples with a
dilution equal to or less than 400 fold. The toxicity results were
considered satisfactory for both strains, where it was observed
more than 50% of cell viability at all concentrations used in the
BEND3 line and at concentrations up to 200 fold dilution in the
HaCaT line. In HaCaT cells, it was observed that in the 200 fold
and 100 fold dilutions, the effect of higher toxicity can be
explained by the presence of the drug, since the higher
concentration of the drug, 0.5 wt%, presented a reduction in
cellular viability compared to the control, NE vehicle, and NE
with 0.25 wt% of POP. In addition, cytotoxicity of the NEs

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Figure 7. MTT cell viability study A) lineage of brain endothelial cells
(BEND3) or B) human keratinocyte lineage (HACAT). Cells were treated
with NE vehicle (NE without POP), 0.25 wt% and 0.50% wt (NE with 0.25
and 0.5% of POP, respectively). The results of three independent
experiments expressed as mean þ S.D. Statistically significant difference
between the groups ( p < 0.001).
formulations appears to be related to dose-dependence, which
showed lower viability at low dilutions.
3.9. Occlusivity Test
The occlusion factor (F) is a parameter that evaluates the ability
of the formulation to maintain skin hydration. Figure 8
compares the result obtained from the evaluation of occlusive-
ness of NE and NE with POP. The occlusion factor of NE vehicle
was similar to the NE with POP at 24 h and 48 h, without
statistical difference (p > 0.05). The occlusion factor is
dependent upon the sample volume, particle size, crystallinity,
lipid concentration and type of colloidal systems.[47] In the
present work, NEs were similar in relation to the particles sizes,
the sample volume, crystallinity, lipid concentration and type of
Macromol. Symp. 2018, 381, 1800121 1800121 (10 of 11)
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Figure 8. Occlusion Factor of NE and NE with POP at 24 h and 48 h after
application. Data give occlusion factor mean values  standard deviation
(SD) (n ¼ 3) and error bars mean S.D.
colloidal system, justifying the maintenance of the occlusion
factor for both formulations around 50%.
The occlusion factor of 50% indicates that the NE has an
occlusive action and forms a barrier on the skin, which
promotes a reduction of water evaporation, and the mainte-
nance of the degree of hydration of the skin.[48] In addition, the
presence of the drug did not present a statistically significant
difference, indicating that the drug does not influence the
hydration test.
These results are in agreement with the study of Montenegro
et al. (2017) where it was observed that the gels containing non-
crystalline nanocarriers without solid lipid, e.g. gel with
nanoemulsion, showed an occlusion factor significantly higher
that the control gel, suggesting that the liquid lipid may
contribute to the occlusive properties of the nanocarriers.[9]
4. Conclusions
POP water-in-oil NEs developed with non-ionic surfactants was
shown for the first time to be a promising formulation for POP
topical delivery. The appropriate concentration of each compo-
nent was obtained by the ternary diagram method. Present
results demonstrate a good stability with nanometer sizes of both
POP concentrations. NMR analysis showed that after POP
inclusion there was a reduction in T2H values, indicating a
possible interaction of the drug with the components of the
system. The feasibility study with the cerebral endothelial cell
line and keratinocytes using the MTT technique showed
satisfactory results and demonstrated higher cell viability at
diluted formulations and also, when a lesser amount of drug,
0.25 wt%, was incorporated. The results showed that the
formulation, with or without POP, had an occlusivity factor
around 50%. The water-in-oil NEs developed in this work have a
potential to be used in HIs treatment.
Acknowledgements
The authors thank to CoordenaSc ~ao de AperfeiSc oamento de Pessoal de
Nível Superior (CAPES), Conselho Nacional de Desenvolvimento
ogico (CNPq) and FundaSc ~ao Carlos Chagas Filho de
Científico e Tecnol

Amparo à Pesquisa do Estado do Rio de Janeiro (FAPERJ) for the financial
support and scholarships.
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Keywords
infantile hemangioma, nanoemulsion, polymeric surfactant, propranolol
[1] S. Tong, D. P. Xu, Z. M. Liu, Y. Du, X. K. Wang, Oncol. Lett. 2016, 12,
1806.
[2] K. Kunzi-Rapp, Pediatr. Dermatol 2012, 29, 154.
[3] T. B. Yang, P. McMahon, D. D. De Leon, J. R. Treat, Pediatr. Dermatol.
2016, 33, 381.
[4] J. Mashiah, A. Kutz, S. H. Rabia, E. B. Ilan, I. Goldberg, E. Sprecher,
A. Harel, Int. J. Dermatol. 2017, 56, 148.
[5] C. Leaute-Labreze, E. Dumas de la Roque, T. Hubiche, F. Boralevi,
J. B. Thambo, A. Taieb, N. Engl. J. Med 2008, 358, 2649.
[6] X. He, Y. Liu, K. Li, A. Yang, R. Wang, S. Liu, Exp. Ther. Med. 2017, 13, 2645.
[7] L. Zhang, H. W. Wu, W. Yuan, J. W. Zheng, Drug. Des. Devel. Ther.
2017, 11, 1401.
[8] A. Casiraghi, U. M. Musazzi, P. Rocco, S. Franze, P. Minghetti, Skin.
Pharmacol. Physiol. 2016, 29, 210.
[9] L. Montenegro, C. Parenti, R. Turnaturi, L Pasquinucci, Pharma-
ceutics 2017, 9, 58.
[10] Y. Singh, J. G. Meher, K. Raval, F. Ali Khan, M. Chaurasia, N. K. Jain,
M. C. Chourasia, J. Control Release 2017, 252, 28.
[11] E. Sigward, N. Mignet, P. Rat, M. Dutot, S. Muhamed, J. M. Guigner,
D. Scherman, D. Brossard, S. Crauste-Manciet, Int. J. Nanomedicine.
2013, 8, 611.
[12] D. J. McClements, Soft Matter 2012, 8, 1719.
[13] V. E. de Campos, E. Ricci-Junior, C. R. Mansur, J. Nanosci.
Nanotechnol. 2012, 12, 2881.
[14] T. N. Barradas, V. E. B. De Campos, J. P. Senna, C. S. C. Coutinho,
B. S. Tebaldi, K. G. H. Silva, C. R. E. Mansur, Colloid Surf. A:
Physicochem. Eng. Aspects 2015, 480, 214.
[15] J. S. Almeida, L. Luciane Jezur, M. C. Fontana, K. Paese, C. B. Silva,
A. R. Pohlmann, S. S. Guterres, R. C. R. Beck, Lat. Am. J. Pharm. 2009,
28, 8.
[16] Z. Ahmad, Complement. Ther. Clin. Pract. 2010, 16, 10.
[17] Y. Sultana, K. Kohli, M. Athar, R. K. Khar, M. Aqil, J. Cosmet. Dermatol.
2007, 6, 14.
[18] D. Kumar, J. Ali, S. Baboota, Drug. Deliv. 2016, 23, 591.
[19] Z. Liu, Q. Zhang, L. Ding, C. Li, Z. Yin, G. Yan, J. Pi, J. Li, N. Li, D. Qi,
Curr. Drug. Deliv. 2016, 13, 600.
[20] J. M. Marques Junior, A. L. Muller, E. L. Foletto, A. B. da Costa,
C. A. Bizzi, E. Irineu Muller, J. Anal. Methods Chem. 2015, 795, 102.
[21] L. Streck, M. M. de Ara
ujo, I. de Souza, M. F. Fernandes-Pedrosa,
E. S. do Egito, A. G. de Oliveira, A. A. da Silva-J
unior, J. Mol. Liq. 2014,
196, 8.
Macromol. Symp. 2018, 381, 1800121 1800121 (11 of 11)
Macromolecular Symposia
www.ms-journal.de
[22] L. Streck, V. H. Sarmento, P. R. Machado, K. J. Farias,
M. F. Fernandes-Pedrosa, A. A. da Silva-Junior, Int. J. Mol. Sci.
2016, 17.
[23] F. Shakeel, S. Baboota, A. Ahuja, J. Ali, S. Shafiq, J. Nanobiotechnol.
2008, 6, 8.
[24] Y. Wei, X. Ye, X. Shang, X. Peng, Q. Bao, M. Liu, M. Guo, F. Li, Colloid
Surf. A Physicochem. Eng. Aspects 2012, 396, 7.
[25] M. S. Jangdey, A. Gupta, S. Saraf, Drug. Deliv. 2017, 24, 1026.
[26] Z. Sklubalová, L. Zahálka, L. Matysová, S. Klovrzová,
M. Petrzelová, P. Horák, K. Kucerová, P. Solich, Eur. J. Hosp.
Pharm. 2018, 25, A27.
[27] K. Prabhakar, S. M. Afzal, G. Surender, V. Kishan, Acta Pharm. Sin. B
2013, 3, 345.
[28] M. Zhang, B. Yang, W. Liu, S. Li, Asian J. Pharm. Sci. 2017, 6, 521.
[29] P. Wavikar, P. Vavia, AAPS. PharmSciTech. 2013, 14, 222.
[30] S. Agatonovic-Kustrin, D. W. Morton, R. Singh, Colloid Surf. A
Physicochem. Eng. Asp. 2012, 415, 9.
[31] S. Tcholakova, N. D. Denkov, A. Lips, Phys. Chem. 2008, 10, 1608.
[32] V. Lemaitre-Aghazarian, P. Piccerelle, J. P. Reynier, J. Joachim,
R. Phan-Tan-Luu, M. Sergent, Pharm. Dev. Technol. 2004, 9, 125.
[33] J. P. Treguier, I. Lo, M. Seiller, F. Puisieux, Pharm. Acta. Helv. 1975,
50, 421.
[34] I. Lo, A. T. Florence, J. P. Treguier, M. Seiller, F. Puisieux, J. Colloid.
Interface. Sci. 1977, 59, 8.
[35] T. J. Wooster, M. Golding, P. Sanguansri, Langmuir 2008, 24, 12758.
[36] T. Tadros, P. Izquierdo, J. Esquena, C. Solans, Adv. Colloid. Interface
Sci. 2004, 108, 303.
[37] K. P. Wilhelm, G. Freitag, H. H. Wolff, J. Am. Acad. Dermatol. 1994,
30, 944.
[38] S. M. Ali, G. Yosipovitch, Acta Derm. Venereol. 2013, 93, 261.
[39] P. S. R. C Silva, L. R. Menezes, M. I. B. Tavares, Mater. Sci. Appl. 2016,
7, 150.
[40] M. R. Tavares, L. R. Menezes, J. C. Dutra Filho, L. M. Cabral, M. I.
B. Tavares, Polymer. Test. 2017, 60, 39.
[41] A. Barrabino, S. Kelesoglu, G. H. Sørlandb, S. Simona, J. Sjöblom,
Colloid Surf. A: Physicochem. Eng. Aspects 2014, 443, 368.
[42] M. Balcaen, L. De Neve, L. Vermeir, T. Courtin, K. Dewettinck,
D. Sinnaeve, P. Van der Meeren, J. Colloid Interface Sci. 2018, 514,
364.
[43] G. A. Latorraca, K. J. Dunn, P. R. Webber, R. M. Carlson, Magn. Reson.
Imag. 1998, 16, 659.
[44] H. Metz, K. Mäder, Int. J. Pharmaceut. 2008, 364, 170.
[45] A. Gupta, H. B. Eral, T. A. Hattona, P. S. Doyle, Soft Matter. 2016, 12,
2826.
[46] S. Wissing, R. Muller, Int. J. Pharm. 2002, 242, 377.
[47] G. Grove, C. Zerweck, T. Houser, A. Andrasfay, B. Gauthier,
C. Holland, D. Piacquadio, J. Drugs Dermatol. 2017, 16, 140.
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