Research Article
Structural Characterization and In Vitro Antioxidant
Activity of Kojic Dipalmitate Loaded W/O/W Multiple
Emulsions Intended for Skin Disorders

Maíra Lima Gonçalez, Diana Gleide Marcussi, Giovana Maria Fioramonti Calixto,
Marcos Antonio Corrêa, and Marlus Chorilli

Department of Drugs and Medicines, School of Pharmaceutical Sciences, UNESP, Rodovia Araraquara-Jaú, Km 1,
Campus, 14801-902 Araraquara, SP, Brazil

Correspondence should be addressed to Máıra Lima Gonçalez; goncalez.m.l@gmail.com
and Marlus Chorilli; chorilli@fcfar.unesp.br

Received 24 November 2014; Accepted 16 January 2015

Academic Editor: Ajit S. Narang

Copyright © 2015 Máıra Lima Gonçalez et al. This is an open access article distributed under the Creative Commons Attribution
License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly
cited.

Multiple emulsions (MEs) are intensively being studied for drug delivery due to their ability to load and increase the bioavailability
of active lipophilic antioxidant, such as kojic dipalmitate (KDP). The aim of this study was to structurally characterize developed
MEs by determining the average droplet size (Dnm) and zeta potential (ZP), performing macroscopic and microscopic analysis
and analyzing their rheological behavior and in vitro bioadhesion. Furthermore, the in vitro safety profile and antioxidant activity
of KDP-loaded MEs were evaluated. The developed MEs showed a Dnm of approximately 1 micrometer and a ZP of −13mV, and
no change was observed in Dnm or ZP of the system with the addition of KDP. KDP-unloaded MEs exhibited “shear thinning”
flow behavior whereas KDP-loaded MEs exhibited Newtonian behavior, which are both characteristic of antithixotropic materials.
MEs have bioadhesion properties that were not influenced by the incorporation of KDP.The results showed that the incorporation
of KDP into MEs improved the safety profile of the drug. The in vitro antioxidant activity assay suggested that MEs presented a
higher capacity for maintaining the antioxidant activity of KDP. ME-based systems may be a promising platform for the topical
application of KDP in the treatment of skin disorders.

1. Introduction

Skin disorders such as skin aging and hyperchromia interfere
negatively in the social behavior of people. These disorders
are mainly caused by reactive oxygen species induced, for
example, by prolonged exposure toUVradiation, establishing
a condition in which the oxidative attack of biomolecules is
increased [1–6].

The current treatments include laser procedures, der-
mabrasion and microdermabrasion, and chemical peels.
Nevertheless, these treatments present several disadvantages
such as persistent erythema, hypopigmentation, keloids, and
hypertrophic scars [7].

Another common treatment modality is through the
use of cosmetics with tretinoin and hydroquinone drugs;

however, many studies indicate that tretinoin can cause skin
irritation (e.g., erythema, stinging, burning, and dryness) and
hydroquinone can have carcinogenic and mutagenic effects
[7].

Therefore, developing new formulations to reduce these
degenerative processes and increase people’s self-esteem is an
ongoing goal. To this end, interest has grown in antioxidant
drugs that interfere with the generation of free oxygen
radicals and the reactions they trigger [8]. Among these
antioxidant drugs, kojic acid (KA) has been highlighted
because it presents antioxidant activity by chelating iron ions.
KA also presents depigmentation activity by chelating the
copper ion present in the active site of tyrosinase, which
mediates the formation of melanin from the amino acid
tyrosine [9–11].

Hindawi Publishing Corporation
BioMed Research International
Volume 2015, Article ID 304591, 8 pages
http://dx.doi.org/10.1155/2015/304591

http://dx.doi.org/10.1155/2015/304591


2 BioMed Research International

O
O

O
O O

O

Figure 1: Structural formula of kojic dipalmitate (KDP) [12].

Nevertheless, KA is unstable at high temperatures (40∘C)
and presents a labile oxidative property against light. There-
fore, KA is used as kojic dipalmitate (KDP) (Figure 1), which
is hydrolyzed by esterases present in skin cells to promote the
in situ release of kojic acid [12].

KDP is a liposoluble white powder that is heat- and light-
stable over a wide pH range [12].

Due to difficulties in solubilizing fat-soluble drugs such as
KDP, a search for strategies to convey the drug is still required.
KDP can be incorporated into colloidal carrier systems, such
as multiple emulsions (MEs), a single colloidal system con-
sisting of both water-in-oil and oil-in-water emulsion system
types. MEs are classified as oil-in-water-in-oil (O/W/O) or
water-in-oil-in-water (W/O/W) depending on the dispersed
and external phase compositions [13].

Therefore, MEs are being intensively studied as a drug
delivery system due to their ability to load lipophilic drugs to
increase their bioavailability and protect such drugs against
biological degradation and oxidation processes. This can
prolong the drug release, which could possibly reduce the
required dosages and application time of the formulation [14–
16].

Thereby, ME can increase the effectiveness of KDP, acting
as a promising drug delivery system to prevent and treat skin
disorders [17].

However, MEs contain large and polydisperse droplets
that are extremely thermodynamically unstable due to their
easy and spontaneous separation into three distinct phases.
MEs also tend to undergo coalescence, flocculation, or
creaming [18]. One way to obtain stable MEs is to reduce the
droplet size through altering the method of preparation and
monitoring the system over time to ensure that the droplet
size does not increase. Another way to obtain the MEs stable
over long periods of time is to choose the correct surfactant
system that conserves the droplet size through changes in the
interfacial tension between internal aqueous and oil phases in
W/O/W emulsions [19].

The aim of this study was to perform the structural
characterization of MEs through determining the average
droplet size and zeta potential (ZP), performingmacroscopic
and microscopic analyses, and evaluating the rheological
behavior and in vitro bioadhesion of the MEs. Furthermore,
the in vitro safety profile and antioxidant activity of ME-
incorporated KDP was evaluated.

2. Materials and Methods

2.1. Materials. For preparing the formulations, Kojic dipal-
mitate (KDP) from Via Pharmaceuticals, Brazil, sorbitan
monooleate (Span 80) from Bold, Brazil, liquid petrolatum

Table 1: Composition (%) of the KDP-unloaded ME formulations
used in this study.

Formulations
Milli-Q
water
(%)

Span
80
(%)

Liquid
petrolatum

(%)

Tween 20
40% (%)

Primary
emulsion

(%)
Primary
emulsion 35 20 45 — —

Multiple
emulsion 10 — — 10 80

from Teclab, Brazil, sorbitan monooleate ethoxylated 20
OE (Tween 20) from Vetec, Brazil, and water (Milli-Q)
were used. In determining the in vitro antioxidant activity
of the formulations, 2,2-diphenyl-1-picrylhydrazyl (DPPH)
purchased from Fluka, Buchs, Switzerland, was employed.

2.2. Methods

2.2.1. ME Development. The ME W/O/W system was devel-
oped by a two-step process. Initially, the primaryW/O emul-
sion composed of 20% Span 80 (hydrophobic surfactant, HLB
(hydrophile-lipophile balance) = 4.3), 45% liquid petrolatum,
and 35% water (Milli-Q) was obtained, in which the liquid
petrolatum and Span 80 were heated to approximately 40∘C
and dispersed in water heated to the same temperature. The
heat was maintained while the mixture was agitated using
magnetic stirrer for 1 minute. The primary emulsion was
dispersed into an aqueous solution of Tween 20 (hydrophilic
surfactant−HLB= 15) to generate theW/O/WMEcomposed
of 80% of the primary emulsion, 10% of the solution in 40%
Tween 20, and 10% water (Milli-Q) [20].

Table 1 shows the components and their concentrations
used in the preparation of the KDP-unloaded MEs. To
obtain KDP-loaded MEs, 1.25% of the total quantity of liquid
petrolatum was reduced from the primary emulsion and an
equivalent percentage of KDP was incorporated.

The particle size distribution of a dispersed system
depends on the speed of agitation between the dispersed and
dispersing phases during the emulsification process and the
rate of addition of one phase over another. These parameters
were adjusted to obtain a proper system [21].

2.2.2. Physicochemical Characterizations of ME

(1) Macroscopic and Microscopic Analysis. The macroscopic
appearance, organoleptic properties, and homogeneity of
the MEs were evaluated after 24 hours of preparation. The
MEs were also observed using an optical Leitz DM RXE
microscope (LeicaTM) at 25±0.5∘C.The samples were placed
on glass slides and covered with a cover slip, and images were
captured at 100x magnification.

( 2) Droplets Distribution and Quantification

(A) Optical Microscope. MEs were placed on a glass slides,
coveredwith a cover slip and observed under a LeitzDMRXE
microscope (LeicaTM) coupled to a camera, which allowed
for the approximate count of droplets of the MEs samples.



BioMed Research International 3

Captured images were scanned and analyzed using the Motic
Images Plus 2.0 software.

(B) Dynamic Light Scattering. The size and distribution
of droplets were determined by an optical particle ana-
lyzer (Zetasizer Nano-ZS ZEN3600, Malvern Instruments)
through the technique of dynamic light scattering. The
samples were diluted in Milli-Q water at a 1 : 20 ratio and
placed in the equipment. The analyses were performed in
triplicate.

(3) Determination of pH. For the determination of pH, 1 g of
formulation was diluted into 10mL of Milli-Q water. Then,
the pH was checked using a digital pH meter (DM-Digimed
23). This assay was performed in triplicate.

(4) Determination of Electrical Conductivity. After the con-
ductivity (DigimedDM-32)was calibratedwithKCl 0.1N, the
electrical conductivity was determined by directly inserting
the electrode into the samples. This assay was performed in
triplicate.

(5) Determination of the Zeta Potential. Zeta potential values
were obtained from the derivation of the electrophoretic
mobility using the Zetasizer Nano-ZS ZEN3600 optical
particle analyzer (Malvern Instruments). The samples were
diluted in distilled water at a 1 : 20 ratio and placed in the
equipment to be analyzed. The analyses were performed in
triplicate.

(6) Rheological Study. Continuous flow was analyzed at 32 ±
0.1

∘Cand in triplicate on a controlled-stressAR2000 rheome-
ter (TA Instruments, New Castle, DE, USA) equipped with
parallel plate geometry (40mmdiameter) and a sample gap of
200𝜇m. Samples of the systems were carefully applied to the
lower plate, ensuring that sample shearing was minimized,
and allowed to equilibrate for 3min prior to analysis.

Continual testing was performed using a controlled shear
rate ranging from 0.01 to 100 s−1 and back. Each stage
lasted 120 s, with an interval of 10 s between the curves. The
consistency index and flow index were determined using the
power law described in (1) for quantitative analysis of flow
behavior:

𝜏 = 𝑘 ⋅ 𝛾

𝜂

, (1)

where “𝜏” is shear stress; “𝛾” is shear rate; “𝑘” is the
consistency index; and “𝜂” is the flow index.

(7) In Vitro Bioadhesion Test. In vitro bioadhesion of the
systems was evaluated using TA.XT Plus texture analyzer
(Stable Micro Systems, Surrey, England). A “Hold Until
Time” test was used to evaluate the force required for the
formulation to leave the animal skin.

The ear skin of domestic pigs was used as an experimental
model. The ear skin of domestic pigs purchased from a local
slaughterhouse was used as experimental model. The skin
was separated from the cartilage with a scalpel and a 500 𝜇m
thick layer stratum corneum and epidermis was separated

from the adipose tissuewith a dermatome (NouvagTCM300,
Goldach, USA), prior to analysis. Before the test, the skins
were maintained in 0.9% NaCl solution for 10 minutes and
were affixed to the probe with the aid of rubber rings.

MEs (7.5 g) were placed in Falcon (BD) centrifuge tubes
with a capacity of 45mL and centrifuged at 3500 rpm (Sorval,
DuPont Model TC 6) for 3 minutes to eliminate air bubbles
and obtain smooth surfaces.The Falcon tube was fixed below
the probe.The sample was placed 10mm below the analytical
probe, which was lowered at a constant speed (1mm/s)
until it reached the sample (detected by a 2mN triggering
force) and remained in contact with the formulation for 60
seconds. Then, the probe was removed slowly at a speed of
0.5mm/s. The force exerted by the probe on the skin surface
was determined using the Texture Exponent Lite software to
obtain a result curve for the bioadhesive force versus time.
Assays were performed in triplicate.

2.2.3. In Vitro Biological Assays

(1) Erythrocyte Hemolysis. The erythrocyte hemolysis assay
was performed using the experimental procedure described
by Jumaa et al., 1999 [22], and Huang et al., 2008 [23].
Briefly, before use, freshly collected human blood (Opositive)
was washed three times with a 7.4 pH solution of 0.01M
Tris-HCl containing 0.15M NaCl (Tris-saline). A suspension
containing 1% (v/v) erythrocytes was prepared with packed
red blood cells resuspended in Tris-saline. KDP-loaded MEs,
KDP-unloaded MEs, and free KDP were dissolved in Tris-
saline to a final concentration of 27𝜇M. As a positive control
(100% lysis), a 1% (v/v) Triton X-100 solution was used. After
incubation for 1 hour at 37∘C, the samples were centrifuged at
3000×g for 2 minutes. Aliquots of 100 𝜇L of the supernatant
were transferred to 96-well microplates and the absorbance
was determined at 405 nm using a BioRad Model 3550-
UV (USA) microplate reader. The assay was performed in
triplicate. The percentage of hemolysis was calculated using
the following equation: % hemolysis = (absorbance of the test
sample/absorbance at 100% lysis) × 100.

(2) In Vitro Nonspecific Cytotoxicity. In vitro testing was
performed using J-774 mouse macrophages as a template
to analyze the safety profile of the formulations. Cells were
seeded in the bottoms of 96-well microplates (Nunclon) at a
density of 2.5–10.0 × 105 cells/well and treated for 48 hours
with different doses of the KDP-unloaded MEs, KDP-loaded
MEs, and free KDP of 1, 5, 10, or 18.6 𝜇M. The cells were
washed with PBS after removal of the compounds and cell
viability was assessed by colorimetry using formazan (MTT).

The method of 3-[4,5-dimethylthiazol-2-yl]-2,5-diphen-
yltetrazolium bromide (MTT) is a simple, reliable, and repro-
ducible colorimetric method for measuring mitochondrial
metabolic reduction of yellow tetrazolium salt to insoluble
formazan crystals in aqueous solutions of viable cells. Cells
and MTT (0.4mg/mL) were incubated at 37∘C for 3 hours.
Subsequently, the supernatant was removed and formazan
crystals were dissolved in DMSO (180mL). The plates were
agitated for 10 minutes and the optical density was measured
using a multiwall spectrophotometer operating at 560 nm.



4 BioMed Research International

The concentrations were tested in triplicate using six addi-
tional controls (cells inmedium). Cell viability was calculated
using the following equation: Cell viability (%) = [OD

560

(sample)/OD
560

(control)] × 100.

2.2.4. In Vitro Antioxidant Activity. Free radical scav-
enging activity was evaluated using the 2,2-diphenyl-1-
picrylhydrazyl (DPPH) test with modifications [24]. One
hundredmicroliters of free KDP,ME formulations, or control
(ethanol; 10–60 𝜇g/mL) was added to 3.9mL of DPPH solu-
tion in ethanol (60 𝜇M). After 30 minutes of storage in a dark
place, the absorbancewasmeasured using a spectrophotome-
ter at 517 nm. All measurements were repeated three times.
Free radical scavenging activity was calculated using the
following formula: % inhibition of DPPH = [(𝐴

0
−𝐴

1
)/𝐴

0
×

100], where𝐴
0
= absorbance of control and𝐴

1
= absorbance

of sample. The IC
50

value was determined by plotting the
concentration of formulations versus the percentage of DPPH
that was maintained at a steady state [24].

2.2.5. Statistical Analyses. Data were analyzed using the
means and standard deviation and compared by analysis of
variance (ANOVA). Tukey’s test was used to assess significant
differences between samples, where values of 𝑃 < 0.05
were considered statistically significant. The Origin 7.0 SRO
program was used for the treatment of the data.

3. Results and Discussion

3.1. Physicochemical Characterization of ME. The MEs were
viscous, white, and opaque with a characteristic odor. By
microscopic analysis (Figure 2), larger droplets of less than
10 𝜇m of diameter were observed. At certain times, smaller
droplets were observed within the larger droplets, character-
istic of ME formulations. Moreover, there was no change in
the appearance of the ME formulations with the addition of
KDP.

Figure 3 shows the size of droplets and the calculated size
distribution of droplets of the KDP-unloaded MEs.

The droplets had a maximum diameter of 12.487𝜇m and
a minimum diameter of 0.056 𝜇m, with an average diameter
of 1.534 𝜇m. 72% of the droplets in the formulation were
observed to have diameters ranging from 0.06 to 1.21 𝜇m.The
addition of KDP did not cause statistically significant changes
in droplet size (𝑃 > 0.05).

Different sizes of droplets can be obtained depending
on the method of emulsification selected, explaining the
method by which the stability of the system is influenced
[25]. Because the droplet size within the emulsion determines
the probability that phenomena such as flocculation and
coalescence will occur, in general, the smaller the size of the
dispersed droplets, the greater the stability of the system [25].

Thedynamic light scattering technique showed that KDP-
unloaded MEs had two populations of droplets of different
sizes. The results showed that 73.3% of the formulation
droplets had an average diameter of 1.016 𝜇mand the remain-
ing 23.7% had an average diameter of 350 nm, as could be
observed morphologically using an optical microscope.

Figure 2: Microscopy of KDP-unloaded MEs (100x) at 25 ± 0.5∘C.

80

85

90

95

100

0
10
20
30
40
50
60
70
80
90

100

Fr
eq

ue
nc

y 
(%

)

Cu
m

ul
at

iv
e f

re
qu

en
cy

 (%
)

0
.0
6

–1
.2
1

1
.2
1

–2
.3
6

2
.3
6

–3
.5
1

3
.5
1

–4
.6
6

4
.6
6

–5
.8
1

5
.8
1

–6
.9
6

6
.9
6

–8
.1
1

8
.1
1

–9
.2
6

9
.2
6

–1
0
.4
1

1
0
.4
1

–1
1
.5
6

1
1

.5
6

–1
2

.7
1

Size distribution of multiple emulsion cells

Feret diameter (𝜇m)

Figure 3: Size distribution of droplets of KDP-unloaded MEs
analyzed by optical microscope.

Through the statistical ANOVA test, the average pH
values of the KDP-unloaded MEs (pH = 4.62) and KDP-
loaded MEs (pH = 4.55) were found to be equal (𝑃 > 0.05),
leading to the conclusion that the incorporation of KDP
induced no significant change in pH.These pH values are well
tolerated by the skin, which presents a slightly acid pH (4.5 to
5.5) [20].

The pH is an important parameter to consider because
large variations in the pHvalues of the formulations can cause
chemical incompatibilities between the ingredients of the
formulation, therefore compromising the efficacy and safety
of the active compounds. In addition to being a parameter
related to the intrinsic stability of the formulation, it is essen-
tial to control the pHof topical application products to ensure
that over time they do not cause changes in the microflora of
the skin or induce irritation or skin sensitization, which could
degrade the product and reduce its activity [21].

Through the statistical ANOVA test, the average electrical
conductivity of KDP-unloadedMEs (34.2 𝜇S/cm) and that of
KDP-loaded MEs (31.90𝜇S/cm) were found to be equal (𝑃 >
0.05), leading to the conclusion that the incorporation ofKDP



BioMed Research International 5

induced no significant change in the electrical conductivity of
the ME system.

In terms of conductivity, the electrical properties of oil
and water are extremely distinct and can be used to distin-
guish whether the ME external phase is aqueous or oily. In
accordance with the results obtained, electrical conductivity
superior to 30 𝜇S/cm, it was confirmed that the external phase
is aqueous and that the ME formulation is a W/O/W type of
emulsion [26].

TheKDP-unloadedMEs showed an averageZPof−13mV.
The addition of KDP did not cause statistically significant
changes in the ZP (𝑃 > 0.05).

The ZP results from the electric charges on the surface
of the particle and is related to the physical stability of the
colloidal system [27]. At greater absolute values (more than
+30mV or less than −30mV), the ZP of a system indicates
the tendency of the particles to repel each other, which may
indicate that the system is stable. If, however, the absolute
values for the ZP are low or zero, the particles tend to
agglomerate and the system can easily flocculate [28]. In this
case, the formulation should bemonitored over time to check
whether the intermediate ZP values (−13mV) indicate an
unstable system.

Semisolids products can exhibit a wide range of rheologi-
cal behaviors. A good understanding of formulation rheology
is very important to the design, selection, and operation of
the equipment involved in evaluating the release performance
and administration characteristics of the drug. Furthermore,
rheological studies can provide useful information on the sta-
bility and microstructure of emulsions. MEs can display flow
characteristics depending on the fraction volume, droplet size
and distribution, and the viscosity of the dispersed phase [29–
32].

The relationship between the shear rate (Pa) and shear
stress (1/s) of MEs is presented in Figure 4.

From the data obtained using (1) and shown in Table 2,
KDP-unloaded MEs were demonstrated to exhibit “shear
thinning” flow behavior (𝑛 < 1), whereas KDP-loaded MEs
exhibited Newtonian behavior (𝑛 = 1).

The KDP-unloaded ME formulation behaved as a pseu-
doplastic shear thinning fluid because its viscosity (𝜂∗)
decreased with increasing stress. This may be due to the
breaking of organized structures that promote the formation
of droplets, which are less organized structures [33].

However, the incorporation of the KDP drug had a strong
influence on the formulation, leading the KDP-loaded ME
formulation to behave as a low-viscosity Newtonian fluid.
This phenomenon is related to the intense reduction in the
droplet size with the introduction of KDP, which thinned the
flow and resulted in a higher diffusion coefficient than when
larger oil droplets are present.

Furthermore, the rheogram showed that the up and down
curves did not overlap. The up curve was below the down
curve, which is typical and characteristic of antithixotropic
materials.

Antithixotropic properties occur because the steady
application of shear stress increases the apparent viscosity,
whereas cessation of the applied stress decreases the viscosity
back to its initial value in a time-dependent manner [30].

0 20 40 60 80 100
0

50

100

150

200

250

300

350

KDP-loaded-ME

KDP-unloaded ME

Shear rate (1/s)

Sh
ea

r s
tre

ss
 (P

a)
,𝜂

∗
(P

a·
s)

𝜂
∗ KDP-loaded ME
𝜂
∗ KDP-unloaded ME

Figure 4: Flow rheograms of KDP-unloaded ME (I) and KDP-
loadedME (◻). Closed symbol represents up curve and open symbol
represents down curve. 𝜂∗ (◊) is viscosity. Standard deviations have
been omitted for clarity; however, in all cases, the coefficient of
variation of triplicate analyses was less than 10%.Data were collected
at 32 ± 0.25∘C.

Table 2: Consistency index (𝑘) and flow index (𝑛) of KDP-loaded
MEs and KDP-unloaded MEs.

Formulations 𝑘 𝑛

KDP-loaded ME 0.63 1.02
KDP-unloaded ME 9.42 0.76

The increase in the apparent viscosity of the ME formu-
lations with time at a constant shear was most likely due
to the reorganization of the droplets by strong forces. The
high viscosity of the MEs could retard the free motion of
the droplets, thus delaying creaming, flocculation, and coa-
lescence of the system [33].The conclusion from these results
is that stable MEs can be obtained with the components used
in this study only under the application of vigorous stirring
during preparation so that viscosity is incorporated and the
destabilization of the system is avoided.

The KDP-unloaded MEs and KDP-loaded MEs were
evaluated for their changes in bioadhesion to determine
whether the addition of KDP alters the bioadhesive ability of
the ME system.

Table 3 shows the bioadhesion test results of KDP-
unloaded MEs and KDP-loaded MEs.

The results demonstrate that the incorporation of KDP
did not influence the bioadhesion of the MEs. Moreover, the
formulations showed values very close to the bioadhesion
of polyacrylic acid bioadhesive hydrogels used in previous
studies [32].

Thus, MEs have properties that favor the interaction
between its components and the epithelial cells of the skin.

The investigation of the strength of bioadhesive pharma-
ceutical formulations for topical use is extremely important



6 BioMed Research International

Table 3: Bioadhesion of KDP-unloadedMEs andKDP-loadedMEs.

Formulations Bioadhesive
work (N⋅s) Bioadhesive force (N)

KDP-unloaded MEs 0.0166 ± 0.0016 0.0103 ± 0.0029
KDP-loaded MEs 0.0163 ± 0.0025 0.0099 ± 0.0006

because bioadhesive formulations remain in contact with
the biological substrate for longer periods of time, which
may lead to a gradient of drug concentration at the site of
action and therefore improve the clinical performance of the
treatment.

3.2. In Vitro Biological Assays. Before exposing users to the
product, the potential irritation of a new topical preparation
should be investigated to avoid inducing skin irritation. For
ethical, legal, and financial reasons, in vivo tests were banned
for testing cosmetics and formulations. Thus, in recent years,
various in vitro assays have been conducted to test the
products prior to realizing there in vivo safety profiles [34].

Erythrocyte-induced hemolysis in vitro is considered to
be a simple and reliablemeasure for estimating themembrane
damage caused in vivo because erythrocytes are easily isolated
by centrifugation [35].

In vitro cytotoxicity assays using the hemolysis of red
blood cells were used to assess the safety profile of the
formulations and they are presented in Figure 5.

Free KDP caused lysis of 4.09 ± 0.13% of the erythrocyte
membranes. KDP-unloaded MEs caused lysis of 1.57% ±
0.47% of the erythrocyte membranes. The incorporation
of KDP in ME was 2.98 ± 1.12% and showed decreased
erythrocyte lysis compared to the free KDP.Thus, all systems
showed a tolerable hemolysis of erythrocytes. The positive
control was represented by 100% hemolysis of erythrocytes
using Triton X-100, a known hemolytic agent. The study
results indicate that the treatment developed using the ME
system showed low toxicity and may therefore be a potential
alternative to current therapeutic applications [23].

In vitro cytotoxicity was performed using J-774 mouse
macrophages as a cellular model. The data are shown as the
percentage of cell viability (Figure 6).

The cell viability assays showed that more than 93% of
the normal macrophages treated with free KDP survived.
Moreover, neither the KDP-unloaded MEs nor the KDP-
loaded MEs showed toxic activity. Therefore, the results
indicate the safety and biocompatibility of the formulations
and free KDP with eukaryotic cells [36–38].

3.3. In Vitro Antioxidant Activity. The formulations with or
without the addition of KDP were evaluated for their in vitro
antioxidant activity over a period of 28 days. The results
obtained are shown in Figure 7.

The antioxidant activity was measured based on the
methodology of Blois, 1958 [24], in which the stable radical
DPPH is reduced by antioxidants. The results of this study
are expressed as the percent inhibition of DPPH (%).

Over the period of 28 days, there was a decrease in
the antioxidant power of all the experimental groups, with

H
em

ol
ys

is 
(%

)

0

5

80

90

100

110

Free KDP
KDP-unloaded ME

KDP-loaded ME
Triton X-100

Figure 5: Percentage of hemolysis of red blood cells after treatment
with free KDP, KDP-unloaded MEs, and KDP-loaded MEs.

86

88

90

92

94

96

98

100

1 5 10 18.6

C
el

lu
la

r v
ia

bi
lit

y 
(%

)

Free KDP
KDP-unloaded ME
KDP-loaded ME

Concentration (𝜇M)

Figure 6: Percentage of cell viability after treatment with free KDP,
KDP-unloaded MEs, and KDP-loaded MEs.

the most marked decrease for the free KDP. Differences
between the samples were significant (𝑃 < 0.05) and the
lesser destabilization of the samples is most likely due to the
increased stabilization of the KDP-loaded ME formulation.

Therefore, the ME formulation maintained the antiox-
idant properties of KDP, which makes MEs a promising
vehicle for the incorporation of KDP to facilitate its topical
application in the treatment of skin aging.

4. Conclusion

Through physicochemical characterization assays, it was
confirmed that the ME system developed was of the W/O/W
type with a Dnm of approximately 1 𝜇m and a ZP of −13mV.
No change in Dnm, ZP, or the stability of the system was
observed after the addition KDP. KDP-unloaded MEs exhib-
ited “shear thinning” flow behavior, and the incorporation of



BioMed Research International 7

0

10

20

30

40

50

60

70

0 10 20 30

D
PP

H
 in

hi
bi

tio
n 

(%
)

Days

Free KDP
KDP-loaded ME
KDP-unloaded ME

Figure 7: Percent inhibition of the 2,2-diphenyl-1-picrylhydrazyl
(DPPH) radical by the formulations over a period of 28 days.

the KDP drug strongly influenced the formulation, leading
the KDP-loaded MEs to behave as a Newtonian fluid. This
phenomenon can be related to the intense reduction in
the droplet size in the presence of KDP, which thinned
the flow. However, both flow types are characteristic of
antithixotropic materials. The properties of MEs favor the
interaction between the ME system and the epithelial cells of
the skin. KDP incorporation did not influence the bioadhe-
sion of the MEs, and KDP-loaded MEs showed low toxicity
and a good antioxidant activity. These results indicated that
this is a promising system for using KDP in the treatment of
skin aging.

Conflict of Interests

The authors declare that there is no conflict of interests
regarding the publication of this paper.

Acknowledgments

This work was financially supported by Fundação de Amparo
à Pesquisa do Estado de São Paulo (FAPESP), Conselho Na-
cional de Desenvolvimento Cient́ıfico e Tecnológico (CNPq),
and Programa de Apoio ao Desenvolvimento Cient́ıfico
(PADC-FCF).

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