International Journal of Pharmaceutics 553 (2018) 428–440

Contents lists available at ScienceDirect

International Journal of Pharmaceutics

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

Anti-inflammatory and anti-cancer activity of citral: Optimization of citral- T

loaded solid lipid nanoparticles (SLN) using experimental factorial design
and LUMiSizer®
Aleksandra Zielińskaa,b, Carlos Martins-Gomesc,d, Nuno R. Ferreiraa, Amélia M. Silvac,d,
Izabela Nowakb, Eliana B. Soutoa,
⁎

a
Department of Pharmaceutical Technology, Faculty of Pharmacy, University of Coimbra (FFUC), Pólo das Ciências da Saúde, Azinhaga de Santa Comba, 3000-548
Coimbra, Portugal
b
Faculty of Chemistry, Adam Mickiewicz University in Poznań, Poland
c
Department of Biology and Environment, University of Trás-os Montes e Alto Douro (UTAD), Quinta de Prados, 5001-801 Vila Real, Portugal
d
Centre for Research and Technology of Agro-Environmental and Biological Sciences (CITAB-UTAD), Quinta de Prados, 5001-801 Vila Real, Portugal

ARTICLE INFO ABSTRACT

Keywords: Essential oils containing monoterpenes are widely used in pharmaceuticals and cosmetic products on account of
Monoterpenes their wide range of bioactive properties (including anti-cancer activity). Two monoterpenes (citral and geraniol)
Citral were firstly tested for their anti-inflammatory activity in a RAW 264.7 cell line, demonstrating citral to have
Geraniol enhanced capacity to inhibit NO production (ca. 84% for citral and 52% for geraniol at the lowest tested con-
Solid lipid nanoparticles
centration of 5 µg/ml). As citral showed higher NO inhibitory activity than geraniol, to measure the level of
Factorial design
Cytotoxicity
cytotoxicity of citral, AlamarBlue reduction assay was run in two cell models (non-tumoral HaCaT and tumoral
A431). Citral exhibited a strong cytotoxic effect in both cell lines, i.e. cell viability lower that 10% after 24 h
exposure at 100 µg/ml of monoterpene. An optimized solid lipid nanoparticles (SLNs) formulation for citral was
further developed by design of experiments (22 factorial design), followed by accelerated stability testing
(LUMiSizer®). An optimal SLN composed of 1 wt% of citral, 4 wt% of lipid and 2.5 wt% surfactant were suc-
cessfully produced by hot high pressure homogenization (hot HPH) showing a mean particle size (Z-Ave) of
97.7 nm and polydispersity index of 0.249. The produced formulations were analyzed in a high-end dispersion
analyzer LUMiSizer® to characterize any demixing phenomena, demonstrating to be long-term stable at room
temperature (25 °C), exhibiting very low instability indices (0.032 after production and 0.042 after one month of
storage).

1. Introduction increasing their interest by a set of industries.

Research focusing on innovative therapeutic agents obtained from
natural sources is increasingly growing (Carbone et al., 2018; Sampaio
et al., 2017). Among them, essential oils, which mainly contain
monoterpenes, are being widely used to prevent and treat human dis-
ease (Pereira et al., 2018; Severino et al., 2015). These volatile com-
ponents show a wide range of biological activities, e.g. as antibacterials,
antivirals or antioxidants (Astani and Schnitzler, 2014; Carbone et al.,
2018). These plant-derived substances are potent anti-cancer agents as
they have the potential to efficiently reduce the tumor volume and
tumor cell proliferation without side effects (Edris, 2007). Mono-
terpenes are also employed as flavors and fragrances in cosmetics,
Citral (C10H16O), also known as 3,7-dimethylocta-2,6-dienal
(Hagvall and Brared Christensson, 2014), is a monoterpene and corre-
sponds to 70–85 wt% of the essential oil of Cymbopogon citratus,
commonly known as lemon grass (Bhalla et al., 2013a; Pereira et al.,
2013). Because of its strong lemon-like aroma, this fragrance mono-
terpene is widely used in cosmetics and as a flavor additive in food
industry (Marcus et al., 2013). Additionally, citral is used in the pro-
duction of vitamin A. Scientific literature attributes to citral a broad
spectrum of therapeutic activities, such as antimicrobial, anti-in-
flammatory and anticancer properties. The monoterpene is chemically
unstable, suffering biodegradation into geranic acid and 6-methyl-5-
heptene-2-on over time in aqueous solutions. Moreover, the high

⁎
Corresponding author at: Department of Pharmaceutical Technology, Faculty of Pharmacy, University of Coimbra (FFUC), Pólo das Ciências da Saúde, Azinhaga
de Santa Comba, 3000-548 Coimbra, Portugal.
E-mail address: ebsouto@ff.uc.pt (E.B. Souto).

https://doi.org/10.1016/j.ijpharm.2018.10.065
Received 1 February 2018; Received in revised form 14 October 2018; Accepted 28 October 2018
Available online 29 October 2018
0378-5173/ © 2018 Published by Elsevier B.V.
A. Zielińska et al.
(a) (E)-3,7-dimetyloocta-2,6- (b) (Z)-3,7-dimethylocta-2,6- (c) (Z)-3,7-dimethyl-2,6-
dienal dienal
Fig. 1. Chemical structure of Geranial (a), Neral (b) ad Geraniol (c).
volatility of citral decreases its pharmacological efficacy. Mono-
terpenes, including citral, have a significant effect in cancer prevention
and treatment (Bhalla et al., 2013b). Moreover, the active ingredients
obtained from aromatic plants, such as citronellal or citral, can mod-
ulate a hepatic monooxygenase activity by interacting with procarci-
nogen xenobiotic biotransformation. Bhalla et al. demonstrated that
essential oils can exhibit antimutagenicity towards mutation caused by
UV light (Bhalla et al., 2013b). Hepatocarcinogenesis in rats was shown
to be inhibited by lemon grass oil due to the high content of citral
(Bhalla et al., 2013b).
From the chemical viewpoint, citral is a mixture of two geometric
isomers: geranial (Fig. 1a) – also called trans confirmation, trans-citral
or citral A (approx. 55–70 wt%), and neral (Fig. 1b) – also called cis
confirmation, cis-citral or citral B (35–45 wt%) (Hagvall and Bråred
Christensson, 2014). Scientific literature reports that both of isomers of
citral are formed during the autoxidation pathway of geraniol-1-hy-
droperoxide (Hagvall et al., 2007; Hagvall and Bråred Christensson,
2014; Rudbäck et al., 2013). The natural form of citral consists of citral
A and citral B in the ratio 3:2. Citral, occurring in many essential oils,
constitutes 70–85 wt% of lemon grass oil (Bhalla et al., 2013b; Pereira
et al., 2013).
Geraniol (C10H18O), chemically recognized as a terpenic alcohol
(Z)-3,7-dimethyl-2,6-octadien-1-ol (Fig. 1c), is a commonly used
monoterpene, occurring mainly in rose oil, palmarosa oil or citronella
oil (Hagvall et al., 2007). Additionally, small quantities of geraniol are
also present in geranium, lemon and many other essential oils. Due to
its strong flower (rose-like) scent, geraniol can be an important in-
gredient in wide range of cosmetic products (Hagvall and Bråred
Christensson, 2014), with particular emphasis on perfumes, and also as
a pharmaceutical intermediates (Weng et al., 2015). Geraniol appears
as a clear to pale-yellow oil and it is insoluble in water, but soluble in
most of organic solvents. Moreover, it has the potential to autoxidize
under air exposure and form highly allergenic compounds, similar to
other fragrance monoterpenes (Hagvall et al., 2007; Hagvall and Bråred
Christensson, 2014).
According to a study conducted by Hagvall and Christensson
(Hagvall and Bråred Christensson, 2014), cis/trans-isomeric aldehydes –
geranial and neral –, formed from an unstable hydroperoxide, geraniol-
1-hydroperoxide, are the major oxidation products in the autoxidation
of geraniol. Geraniol has especially significant properties in a form of
geranial (citral A), useful as a starting material for other chemicals such
as ionones, vitamin A, vitamin E and carotenoids (Weng et al., 2015).
To improve the long-term stability of monoterpenes and prolong
their release profile, and decrease the risk of chemical degradation, the
loading of these active ingredients into nanoparticles has been proposed
(Wang and Wang, 2014). There are only a few example in the literature
reporting the use of nanoparticles for the loading citral. Glyceryl
monostearate SLN stabilized by a mixture of Tween 80 and Span 80 at a
weight ratio of 1:1 were produced by Tian et al. (Tian et al., 2018). Qiu
et al. described the development of starch nanoparticles prepared by
short glucan chains for the loading of citral (Qiu et al., 2017). Both
studies confirmed the enhanced ability of nanoparticles to improve
monoterpene stability. Chitosan nanoparticles were also shown to
429
International Journal of Pharmaceutics 553 (2018) 428–440
octadien-1-ol.
improve the biological activities of lemongrass oil when loaded in the
particles in comparison to the bare oil (Natrajan et al., 2015). Nano-
particles based on lipid raw materials, i.e. solid lipid nanoparticles
(SLN) and nanostructured lipid carriers (NLC) are being exploited both
in pharmacy and cosmetic industries, attributed to their nanometric
size and control release. SLN and NLC are obtained from physiological
and biodegradable lipids, classified as Generally Recognized As Safe
(GRAS) chemical compounds (Sanchez-Lopez et al., 2017). Solid lipid
nanoparticles are mainly composed of solid lipids, such as triglycerides
(e.g. tristearate, tripalmitate), some of glycerol monostearate, saturated
fatty acids (e.g. stearic and palmitic), steroids (cholesterol) or waxes
(e.g. cetyl palmitate), at room and body temperatures (Souto and
Müller, 2006), while NLC additionally contain the liquid lipid (oil)
(Muller et al., 2011). Both types of lipid nanoparticles are relatively
stable and well tolerated by the skin (Müller et al., 2002; Naseri et al.,
2015; Souto and Muller, 2008).
This study aimed to assess the cytotoxicity of citral in two skin cell
models (non-tumoral HaCaT and tumoral A431). The anti-in-
flammatory activity of citral was also evaluated and compared with
geraniol for the selection of the best monoterpene to be loaded into a
SLN formulation (composed of Imwitor® 900 K and Kolliphor® P188)
optimized by factorial design experiments. The optimal citral-loaded
SLN dispersion was analyze in LUMiSizer®, which is connected with
STEP-Technology® (Space- and Time-resolved Extinction Profiles) that
enables the characterization of any demixing phenomena (e.g. sedi-
mentation) for all of samples recorded simultaneously with high accu-
racy.
2. Material and methods
2.1. Materials
Imwitor® 900 K (Glycerol Monostearate, Type II), selected as a solid
lipid containing 40–55 wt% of monoglycerides, was offered by Cremer
Oleo GmbH & Co. KG company (Hamburg, Germany). Poloxamer 188
(trade name: Kolliphor® P188) was used as surfactant. Poloxamer 188,
bought from the company BASF (Ludwigshafen, Germany), is a non-
ionic triblock copolymer composed of central hydrophobic poly-
oxypropylene (poly(propylene oxide) PPOx, where x = 28) chain sur-
rounded by two hydrophilic chains of polyoxyethylene (poly(ethylene
oxide, PEOy, where y = 79). Citral (C10H16O) and geraniol (C10H18O)
were purchased from Sigma Aldrich (Madrid, Spain). Ultra-purified
water was obtained from a Milli-Q® Plus system (Millipore, Germany)
and filtered through a 0.22 μm nylon filter before use. All reagents were
analytically pure and were used without further treatment.
2.2. Cell cultures and maintenance
The effect of monoterpenes on the cell viability was tested in human
skin cellular models i.e. a non-tumoral cell line, HaCaT (human kera-
tinocytes, Cell Line Service (CLS), Eppelheim, Germany, (Boukamp
et al., 1988)) and a tumoral-cell line, A431 (human squamous carci-
noma, Cell Lines Services (CLS), Eppelheim, Germany). Additionally, in
A. Zielińska et al.
Table 1
Initial 2-level factorial design, providing the lower (−1), upper (+1) and
central (0) point level values for each studied variable.
Variables Levels
Low (−1) Central Point (0)
Imwitor® 900 K (wt%) 2 4
Poloxamer 188 (wt%) 1.25 2.5
the anti-inflammatory assay a RAW 264.7 (mouse macrophages,
Abelson murine leukemia virus-induced tumor, Cell Lines Services
(CLS), Eppelheim, Germany) cell line was used. The cell lines used were
maintained in DMEM (Dulbecco’s Modified Eagle Medium) supple-
mented with 10% (V/V) fetal bovine serum (FBS), antibiotics (100 U/
mL of penicillin and 100 μg/mL of streptomycin) and 1 mM L-glutamine
in an atmosphere of 5% (v/v) CO2/95% (v/v) air, at 37 °C with con-
trolled humidity.
2.3. Anti-inflammatory activity
The anti-inflammatory activity of monoterpenes was evaluated on
RAW 264.7 cells, seeded in 96-well microplate. Two sets of experiments
were made incubating citral and geraniol with and without stimulation
with 1 μg/mL lipopolysaccharide (LPS, Sigma-Aldrich, USA). Geraniol
and citral were diluted in DMSO (final solution with 0.4% V/V DMSO).
For each well, a volume of 50 μL of supernatant was transferred into a
new 96-well plate, to which the same volume of Griess reagent [0.1%
(w/V) N-(1-naphtlyl) ethylene diamine dihydrochloride and 1% (w/V)
sulfanilamide prepared in 5% (w/V) H3PO4 (V/V)] (Sigma-Aldrich,
USA) were added. After 10 min of incubation in the dark, the absor-
bance was read at 550 nm using a Multiskan EX microplate reader (MTX
Labsystems, USA). A sodium nitrite (Na2NO3) (Sigma-Aldrich, USA)
standard curve was built (0–100 μL) to allow the quantification of re-
sults which are expressed as percentage of nitric oxide production by
the control cells.
2.4. In vitro cytotoxicity assay
The cytotoxic effect of monoterpenes was assessed in the skin cel-
lular models by the AlamarBlue® reduction method. AlamarBlue® (AB)
is a colorimetry redox indicator. The oxidized form of AB, known as
resazurin, is blue and non-fluorescent (measured at a wavelength of
570 nm) and due to cell metabolism rezasurin is reduced to resofurin,
which is pink and fluorescent (measure at a wavelength of 620 nm). For
the cytotoxicity assay, cells were detached from the culture flaks with
trypsin, counted and seeded into 96-well microplates at a density of
5 × 104 cells/mL (100 μL/well). Monoterpene was diluted in dimethyl
sulfoxide (DMSO) (Sigma-Aldrich, USA) to reach the final concentration
of 0.4% (V/V) of DMSO. The cell culture medium was withdrawn from
each well and 100 μL of the test solutions were added. The cells were
exposed to the test solutions for a 24 h period. After the exposure time
the test solutions were retrieved and 100 μL of AB solution (Invitrogen,
Alfagene, Portugal) 10% (V/V) diluted in DMEM was added. After 5 h
of exposure to AB the absorbance was read in a Multiskan EX micro-
plate reader (MTX Labsystems, USA) at 570 nm and 620 nm. The AB
reduction was calculated using the following equation:
( ox 2)(A 1) ( ox 1)(A 2 )
AB reduction% = ' '
× 100
( red 1 )(A 2 ) ( red 2 )(A 1 )
where, ( ox 1) is the molar extinction coefficient of oxidized AB at
570 nm, ( ox 2 ) is the molar extinction coefficient of oxidized AB at
620 nm, ( red 1) is the molar extinction coefficient of reduced AB at
570 nm, ( red 2 ) is the molar extinction coefficient of reduced AB at
620 nm, (A 1) and (A 2 ) are the absorbance of test wells at 570 and
International Journal of Pharmaceutics 553 (2018) 428–440
620 nm, respectively, ( A' 1) and ( A' 2) are the absorbance of the ne-
gative control wells at 570 and 620 nm, respectively (Al-Nasiry et al.,
2007; Andreani et al., 2014). Results are expressed in terms of cell
viability as percentage of control (untreated cells) and are a mean of 4
independent experiments (n = 4) ± standard deviations. Data were
High (+1) expressed as the arithmetical mean ± standard deviation and analyzed
by ANOVA statistical test followed by a Bonferroni multiple comparison
8
5 post-test. All the graphs and statistical analysis was performed in
GraphPad Prism® version 6.00 software (GraphPad Software, La Jolla,
California, USA). A p-value < 0.05 was consider statistically sig-
nificant.
2.5. Experimental factorial design
A factorial design approach was applied to maximize the experi-
mental efficiency requiring a minimum of experiments to optimize the
SLN production (Fangueiro et al., 2012b). The influence of the surfac-
tant ratio (Poloxamer 188) and lipid ratio (Imwitor® 900 K) on citral-
loaded SLNs was evaluated by using a 22 factorial design composed of 2
independent variables (surfactant and lipid concentrations) which were
set at 2-levels each (−1 versus +1). The dependent variables were
mean particle size (Z-Ave), polydispersity index (PDI) and zeta poten-
tial (ZP). For each factor, the lower and higher values of the lower and
upper levels are represented by (−1) and (+1), respectively. The
central point was replicated three times for estimating the experimental
error and represented by (0) (Table 1). The levels were chosen on the
basis of the tested lower and upper values for each variable, according
to pre-formulation studies as well as literature research. The data were
analyzed by STATISTICA 7.0® (Stafsoft, Inc., Tulsa, Oklahoma, USA)
software. The SLN dispersions were randomly produced and in order to
identify the significance of the effects and interactions between them.
The analysis of variance, ANOVA statistical test, was performed for
each response parameter. A p-value < 0.05 was consider statistically
significant.
2.6. Production of SLN
SLN dispersions were produced by dispersing the lipid phase,
composed of citral (1 wt%) and Imwitor® 900 K (2; 4 or 8 wt%) at the
same temperature (all at 5–10 °C above the melting point of lipid), in an
aqueous solution of Poloxamer 188 (1.25; 2.5 or 5 wt%) using the hot
high pressure homogenization (HPH) technique. Briefly, the pre-emul-
sion was processed in a high-shear mixing Ultra-Turrax® T25 (Ystral
GmbH D-7801, Dottingen, Germany) at 10,000 rpm for 10 min followed
by hot HPH (Emulsiflex-C3, Avestin, Inc., Ottawa, Canada) for 30 min
under a pressure of 300 bar.
2.7. Particle size and zeta potential analysis
Z-Ave and PDI were determined using the Zetasizer Nano ZS
(Malvern, Worcestershire, UK), equipped with a particle size range of
0.3 nm to 10 μm, a laser beam (λ = 633 nm; 4 mW) and a scattered
light detector positioned at an angle of 173° (non-invasive backscatter)
in order to unmask scattered light signals of low intensity originated
from smaller particles. Analysed samples were diluted 100 times in
ultra-purified water and the value of Z-Ave and PDI were measured
three times during one cycle (33 runs in each measurement).
Cumulative method was used for data analysis. Data were expressed as
the arithmetical mean ± standard deviation.
The measurements of zeta potential were performed using the
Zetasizer Nano ZS (Malvern, Worcestershire, UK). Analysed samples
were diluted 100 times in ultra-purified water and the value of ZP was
measured three times during one cycle (30 runs in each measurement).
The principle of method was based on an Electrophoretic Light
Scattering (ELS) technique, in which ZP is calculated by determining
the electrophoretic mobility. The electrophoretic mobility is obtained
430
A. Zielińska et al.
by performing electrophoresis on the sample and measuring the velo-
city of the particles using Laser Doppler Velocimetry (LDV) (Zielińska
and Nowak, 2016). The zeta potential is calculated using the Helm-
holtz-Smoluchowski equation included in the software of the system.
Values are presented as the mean of triplicate runs per sample. Data are
expressed as the arithmetical mean ± standard deviation.
2.8. Accelerated stability analysis
A volume of 1 mL of SLN dispersions was placed in the cell, sub-
mitted to 4000 rpm rotor speed at 25 °C. A total of 750 profiles were
recorded in intervals of 20 s. Instability indices were calculated by the
SEPView® software. LUMiSizer® can record the evolution of the trans-
mission profiles and trace instability phenomena using the SEPView®
software. The software also enables the comparison of different samples
and give information about the instability index of each sample.
3. Results and discussion
For the assessment of the anti-inflammatory activity of mono-
terpenes, RAW 264.7 cells were exposed to citral or to geraniol in the
presence and absence of LPS in order to determine the capacity of the
molecules to induce/inhibit NO production. The results expressed as
inhibition of NO production are presented in Fig. 2.
At the lowest tested concentration (5 μg/mL) citral inhibited the NO
production in 84% (against 52% of geranial at the same concentration),
reaching 99% of inhibition at the highest tested concentrations (20 μg/
mL). As the inhibitory action was shown to be higher for citral in
comparison to geraniol, i.e. anti-inflammatory activity of citral is su-
perior to geraniol, the former was selected for in vitro cytotoxicity
analysis and loading in SLN.
Fig. 3 shows the results of the cytotoxicity testing of citral in HaCaT
and A431 cell lines. At all tested concentrations, after 24 h of exposure
to citral, the viability of HaCaT cells was significantly reduced (p-
value < 0.05). The exposure to the lowest tested concentration (50 μg/
mL) reduced the HaCaT cell viability down to 57%, whereas the highest
tested concentration (400 μg/mL) cell viability is significantly reduced
reaching a minimum of 2% of viable cells. The effect of citral on A431
cells was even more dramatic, suffering a 95% reduction of viable cells
when treated with the lowest tested concentration (50 μg/mL), having a
potent cytotoxic effect significantly reducing the cell viability down to
values of approximately 4% when treated with the highest tested con-
centration. In comparison to the non-tumoral cell line (HaCaT), even at
the lower tested concentration (50 μg/mL) citral induced cytotoxic ef-
fects after 24 h of exposure in the tumoral cell line (A431). The
A B
Fig. 2. Citral (A) and Geraniol (B) inhibition of NO production (% of control) at 4 different concentrations: 5, 10, 15 and 20 μg/mL Results are expressed as
mean ± SD (n = 4). All values are normalized to the control, 100% of NO production (0% of inhibition).
431
International Journal of Pharmaceutics 553 (2018) 428–440
Fig. 3. Cell viability determined by AlamarBlue® reduction method, after ex-
position of HaCaT and A431 skin cell lines to different concentrations of citral.
Results are expressed as a percentage of resazurin reduction by control cells
(non-exposed cells). Data were expressed as arithmetical means ± standard
deviations; (*) p-value < 0.05.
pronounced cytotoxic effect of citral in the tumoral A431 cell line de-
monstrates the skin anti-cancer potential of this monoterpene.
In order to optimize citral-loaded SLN, a 22 full factorial design
study was carried out. Seven SLN formulations were produced by high
pressure homogenization (HPH) with different concentrations of lipid
(Imwitor® 900 K) and surfactant (Poloxamer 188) (Table 2). High
pressure homogenization (HPH) is considered to be the most effective
technique for the production of lipid nanoparticles (SLN, NLC) (Souto
et al., 2011), attributed to its capacity to produce small and uniform
size of dispersed phase of a raw dispersion. High pressure homogenizers
mainly push a liquid with a pressure between 100 and 2000 bar,
through a narrow gap (in the range of a few microns). HPH fulfils not
only the key industrial requirements such as regulatory aspects, but also
it allows a large scale production with a relatively low cost (Yadav
et al., 2013; Zielińska and Nowak, 2016). Hot homogenization is per-
formed at a temperature higher than a melting point of a selected lipid.
So-called pre-emulsion of lipids is obtained by the use of Ultra-Turrax®,
a high rotational speed homogenizer (Doktorovova et al., 2018). To be
dispersed, the substance is sucked up by the sucking attachment of the
homogenizer thanks to the high rotational speed of the rotor and then
the comminute emulsion is pressed through the slits of the system
Rotor-Stator. In turn, the cold homogenization has been prepared to
overcome mainly the temperature-induced active compound degrada-
tion and redistribution into the aqueous phase during homogenization
(Zielińska and Nowak, 2016). After production, SLN were stored at
A. Zielińska et al. International Journal of Pharmaceutics 553 (2018) 428–440

Table 2
Response dependent variables (Z-Ave, PDI and ZP) of the two independent factors presented in Table 1 for all of 7 produced citral-loaded SLN.
Sample name Independent variables Dependent Variables

Imwitor® 900 K [wt%] Poloxamer 188 [wt%] Z-Ave [nm] ± SD PDI [–] ± SD ZP [mV] ± SD

SLN1 2 1.25 106.2 ± 0.1 0.221 ± 0.006 −0.018 ± 0.377

SLN2 8 1.25 441.1 ± 143.6 0.486 ± 0.100 −0.056 ± 0.118
SLN3 2 5 164.4 ± 0.5 0.519 ± 0.003 −0.146 ± 0.506
SLN4 8 5 172.5 ± 0.9 0.380 ± 0.013 −0.013 ± 0.265
SLN5 4 2.5 155.2 ± 1.7 0.519 ± 0.027 −0.066 ± 0.109
SLN6 4 2.5 112.6 ± 0.6 0.266 ± 0.005 0.070 ± 0.255
SLN7 4 2.5 97.7 ± 1.2 0.249 ± 0.013 −0.007 ± 0.196

Z-Ave - mean particle size; PDI - polydispersity index; ZP - zeta potential.

room temperature (25 °C). The mean particle size, polydispersity index
and zeta potential were measured 24 h after production and the results
are presented in Table 2. Dynamic light scattering (DLS) is used to
determine the size of particles from below 5 nm to several microns. It
records the variation in the intensity of the scattered light on the mi-
crosecond time scale. The measuring principle of this technique is based
on the fact that particles in gas or liquid exhibit Brownian motion. The
movement (diffusion) of these particles is described by the Stokes-
Einstein equation. Z-average (Z-Ave) size or average particle size Z (also
called the average cumulative) is the most important and most stable
parameter in the DLS technique. Cumulative analysis gives only two
sizes: the average value for the particle size and the parameter relating
to the width of the graph called dispersity or polydispersity index (PDI)
(Zielińska and Nowak, 2016). The mean particle size varied from
97.7 ± 1.2 nm (SLN7) to 441.1 ± 143.6 nm (SLN2), whereas the PDI
ranged from 0.221 ± 0.006 (SLN7) to 0.519 ± 0.027 (SLN5). ZP was
approximately 0 mV in all formulations since the surfactant used has a
non-ionic nature forming a spherically stabilizing adsorbed polymer
layer in the SLN surface (Shah and Agrawal, 2012).
For each of the three dependent variables, analysis of the variance
(ANOVA) was performed using a confidence level of 95% (p-
value = 0.05). No statistical significance (p-value > 0.05) was re-
ported, which indicates that the variation of Imwitor® 900 K and
Poloxamer 188, as well as their interaction, does not affect the response
values of Z-Ave, PDI and ZP of citral-loaded SLN produced by HPH. The
effect of the different concentrations of both Imwitor® 900 K and
Poloxamer 188, as well as their interaction on Z-Ave, was shown to be
not statistically significant on the response variables (Z-Ave, PDI and
ZP) (p-value > 0.05) (Tables 3–5).
To produce new pharmaceutical formulations it is crucial to perform
an experimental design in order to ascertain the parameters that will
affect some specific characteristics of the final dosage form. The ex-
perimental factorial design method enables the analysis of the effects of
the different variables on the properties of the drug delivery system.
Furthermore, factorial design is a reliable statistical method that pro-
vides the selection of the most optimal experimental conditions, such as
different ratios of surfactants and different amounts of lipids (in-
dependent variables). As a result, the factorial design approach can be
effectively assessed by statistical analysis to estimate the influence of
independent variables on the dependent variables (Z-Ave, PDI, ZP),
which ultimately determine the physicochemical properties of lipid
nanoparticles.
The response coefficients were studied for their statistical sig-
nificance by Pareto chart (Fig. 4). The Pareto charts set the t-value of
effect. Having in mind that the results are not statistically significant for
the confidence level selected, the variation of the low value to a high
value of Poloxamer 188 concentration has a positive effect on the
particle size (t-value = 2.175679) as shown in Fig. 4-A. The similar
effect, but with slight less magnitude was observed in the PDI (Fig. 4-B)
(t-value = 0.7356498). Furthermore, the interaction between the var-
iation of the surfactant and lipid ratio from low to high values exhibited
a similar negative effect on the particle size (t-value = −2.07294), as
well as on PDI (t-value = −1.54142).
The physicochemical properties of citral-loaded SLN were optimal
when the relation between the surfactant and lipid concentration was
shown to be directly proportional, i.e. reducing the lipid concentration
requires the reduction of the surfactant concentration. This finding is
supported by Harms and Müller-Goymann (Harms and Müller-
Goymann, 2011), which report that the lipid and surfactant con-
centrations are directly proportional.
The interactive effects on the three dependent variables (z-AVE, PdI
and ZP) were plotted in three-dimensional (3D) response surface charts
(Fig. 5). The variations in the response values were plotted in the Z-axis
against the levels of the two independent variables (Poloxamer 188 in
X-axis and Imwitor® 900 K in Y-axis). As shown in Fig. 5-A, the sur-
factant concentration in the tested levels can be used to produce citral-
SLN with particle sizes below 400 nm (green zone). The higher the lipid
concentration, the bigger the particles (red zone). Regarding the PDI
(Fig. 5-B), the low levels of surfactant and lipid concentrations pro-
duced citral-loaded SLN with low PDI values (green zone), i.e., the
particle distribution tends to be more homogeneous. The ZP seems to
remain neutral (0 mV) in all tested experiments (Fig. 5-C), indicating
that the independent variables (lipid and surfactant concentrations), as
well as their interaction in the two tested levels, have no influence on
the surface electrical charge of the citral-loaded SLN. The ZP is mainly
affected by the nature of the surfactant. Based on the obtained results,
the optimal SLN dispersion was the one of the triplicated central point
composed of 1 wt% citral, 4 wt% of Imwitor® 900 K and 2.5 wt% of
Poloxamer 188. Therefore, SLN7, with 97.7 nm of Z-Ave, 0.249 of PDI
and 0 mV of ZP was selected for further stability studies.
The evaluation of the separation performance of a dispersion ac-
counts for a vital role in the optimization of processes and their

Table 3
Analysis of the mean particle size (Z-Ave) by ANOVA statistical test.
Evaluated factors and their interactions Sum of squares Degrees of freedom Mean square F-value p-value

(1)Imwitor® 900 K 29423.68 1 29423.68 4.733581 0.117826

(2)Poloxamer 188 11074.05 1 11074.05 1.781556 0.274215
1 by 2 26710.45 1 26710.45 4.297086 0.129876
Error 18647.84 3 6215.95
Total 85856.03 6

ANOVA; Var.: Z-Average; R-sqr = 0.7828; Adj: 0.5656 (Design_FD_Citral.sta) 2**(2-0) design; MS Residual = 6215.946 DV: Z-Average.

432
A. Zielińska et al.
Table 4
Analysis of the polydispersity index (PDI) by ANOVA statistical test.
Evaluated factors and their interactions Sum of squares
(1)Imwitor® 900 K 0.003948
(2)Poloxamer 188 0.009248
1 by 2 0.040602
Error 0.051266
Total 0.105064
ANOVA; Var.: PDI; R-sqr = 0.51205; Adj: 0.0241 (Design_FD_Citral.sta) 2**(2-0) design; MS Residual = 0.0170886 DV: PDI.
products. In this study, LUMiSizer® was used in order to assess the
stability of the produced citral-SLN dispersions. By using a centrifugal
separation analysis (CSA), the variation of transmitted light over time
and space is recorded in transmission profiles and provides information
on the separation process kinetics, allowing the calculation of particle
migration velocity, which is intimately related to the particle size dis-
tribution (Fangueiro et al., 2012a; Fernandes et al., 2017; Shah and
Agrawal, 2012).
Dispersion analyzer known as LUMiSizer® has become an equip-
ment for quick characterization of any demixing phenomena, e.g. se-
dimentation, or flotation. This multisample analytical centrifuge allows
for the calculation of the velocity distribution in the centrifugal field as
well as of particle size distribution (Makinen et al., 2015; Xu et al.,
2017).
LUMiSizer® employs the STEP-Technology®, which allows to obtain
Space- and Time-resolved Extinction Profiles over the entire range of up
to 12 different samples simultaneously at constant or variable cen-
trifugal force up to 2300×g (Caddeo et al., 2013). Parallel near-infrared
(NIR) or blue light illuminates the entire sample cell and the trans-
mitted light is detected by the 2087 sensors of the charge-coupled de-
vice line (CCD-line) detector.
The shape and progression of the transmission profiles contains the
information on the kinetics of the separation process and facilitates
particle characterization. By tracing the variation in transmission at any
part of the sample or by tracing the movement of any phase boundary,
the separation behavior of the individual samples can be compared and
carefully analyzed. Based on the extinction profiles, demixing processes
are quantified regarding clarification velocity, sedimentation and flo-
tation velocity of particles, residual turbidity, separated phase volume
(liquid or solid) (Fernandes et al., 2017). The evolution of the trans-
mission profiles of tested samples enables the analysis of their demixing
behavior and stability, as stable colloidal dispersions allow the forma-
tion of a flatbed under a centrifugal field, while aggregated particles
usually give a step-profile (Fernandes et al., 2017). By using the cen-
trifugal sedimentation method, LUMiSizer® provides a fast stability
ranking and shelf-life estimation of undiluted dispersions at their ori-
ginal concentration, in minutes/hours instead of months/years
(Makinen et al., 2015). Moreover, the centrifugal acceleration causes
different sedimentation velocities of particles with different size ranges.
Instability phenomena is directly related to a migration of particles
(sedimentation, flotation) and to changes in particle size distribution
(e.g. due to particle interaction) followed by particle migration
(Fernandes et al., 2017).
Due to the most successful results obtained during the experimental
Table 5
Analysis of zeta potential (ZP) by ANOVA statistical test.
Evaluated factors and their interactions Sum of squares
(1)Imwitor® 900 K 0.002209
(2)Poloxamer 188 0.001795
1 by 2 0.007276
Error 0.014966
Total 0.026246
ANOVA; Var.: ZP; R-sqr = 0.42978; Adj: 0. (Design_FD_Citral.sta) 2**(2-0) design; MS Residual = 0.0049887 DV: ZP.
International Journal of Pharmaceutics 553 (2018) 428–440
Degrees of freedom Mean square F-value p-value
1 0.003948 0.231033 0.663634
1 0.009248 0.541181 0.515240
1 0.040602 2.375982 0.220873
3 0.017089
6
factorial design, two kind of formulations: empty-SLN and citral-SLN
were prepared for the centrifugal separation reanalysis, based on the
composition of SLN7 dispersion. The empty-SLN dispersion was com-
posed of 5 wt% of Imwitor® 900 K and 2.5 wt% of Poloxamer 188, while
the citral-SLN dispersion was composed of 1 wt% citral, 4 wt% of
Imwitor® 900 K and 2.5 wt% of Poloxamer 188. Given that temperature
and light may affect the SLN storage stability (Freitas and Müller,
1998), this centrifugal separation analysis method was applied to esti-
mate and compare the stability of the produced citral-SLN dispersions
right after production and after 1 month of storage at three different
temperatures: 4, 25 and 40 °C. Applying STEP-Technology®, it was
possible to characterize the SLN dispersions stability (with and without
citral), and the transmission profiles of tested SLNs are shown in
Table 6.
Data presented in Table 6 shows that a creaming separation process
occurred in the empty-SLN that was analysed after production. In the
case of the empty-SLN samples, the dispersion stored for one month at
4 °C showed creaming, even though the separation speed was slower
when compared to the freshly produced sample and analysed at 25 °C.
The separation profiles of the empty-SLN stored at 25 and 40 °C for
1 month revealed a mixed behaviour of creaming and sedimentation, as
well as the existence of different particle size populations. In addition,
the speed of separation increases with temperature, highlighting the
importance of this parameter in terms of physical stability of the dis-
persion. According to these results, the most stable sample of empty-
SLN is the one that was stored for 1 month at 4 °C. In the case of citral-
SLN freshly produced that was analyzed at 25 °C, results show that the
sample is stable during the time course of the experiment, although a
very slowly creaming process occurred. When comparing this sample
with the empty-SLN freshly produced and analyzed at 25 °C, the in-
corporation of the monoterpene tends to increase the overall stability of
the dispersion. In the case of the citral-SLN samples that were stored for
one month at 4, 25 and 40 °C, it is interesting to observe that the most
stable sample is the one that was stored at 25 °C and not the sample
stored at 4 °C, indicating that there are other factors besides the tem-
perature affecting the overall stability of the dispersion. In any case,
every sample that was tested showed a creaming separation process.
The creaming occurred at a faster rate for the sample stored at 4 °C,
while the sample stored at 25 °C had the slower separation rate of the
three. Every sample that was stored for one month before the LUMi-
Sizer® analysis showed a faster separation rate when compared to the
sample that was freshly produced.
In summary, the instability process of creaming occurred over time
for all of drug-loaded SLN. Obtained results suggest that the presence of
Degrees of freedom Mean square F-value p-value
1 0.002209 0.442801 0.553362
1 0.001795 0.359801 0.590899
1 0.007276 1.458517 0.313692
3 0.004989
6
433
A. Zielińska et al.
A A
B
C
Fig. 4. Pareto charts of the analyzed effects of the concentration variation of
the Poloxamer 188 as a solid lipid (2), Imwitor® 900 K as a surfactant (1) and
the interaction of both (1by2) for the Z-Ave (A), PDI (B) and ZP (C).
citral delays the appearance of creaming, therefore improving overall
stability of the nanoparticles.
The use of the LUMiSizer® provides more information beyond the
separation profiles, allowing to obtain quantitative data, namely the
instability index. This parameter can be calculated based on the
434
International Journal of Pharmaceutics 553 (2018) 428–440
B
C
Fig. 5. Surface response chart of the influence of: the effect of the wt% of
Imwitor® 900 K and Poloxamer 188 on the Z-Ave (A), on the PDI (B) and on ZP
(C).
clarification (increase in transmission due to phase separation by se-
dimentation or creaming) at a given separation time, divided by the
maximum clarification. The obtained results are shown in Table 7. The
instability index creates a dimensionless number, ranging from 0 (more
stable) to 1 (more unstable). Therefore, samples with high clarification
rates tend to be more unstable for the same total clarification
(Fernandes et al., 2017).
In this study, the empty-SLN had higher instability index values
 Table 6
STEP Profiles with analysis of phenomena of empty-SLN and citral-loaded-SLN, measured after production and after 1 month stored at 4, 25 and 40 °C.
Sample name Measurement time Temp. [°C] STEP Profile
A. Zielińska et al.

Empty – SLN after production 25

435
Creaming process.
1 month 4

Creaming process.
25
International Journal of Pharmaceutics 553 (2018) 428–440

(continued on next page)

 Table 6 (continued)

Sample name Measurement time Temp. [°C] STEP Profile

A. Zielińska et al.

Mixed creaming and sedimentation process with the presence of different particle sizes populations.

436
40

Mixed creaming and sedimentation process with the presence of different particle size populations.

Citral – SLN after production 25

(continued on next page)
International Journal of Pharmaceutics 553 (2018) 428–440
 Table 6 (continued)

Sample name Measurement time Temp. [°C] STEP Profile

A. Zielińska et al.

437
Very slow creaming process.
1 month 4

Creaming process with the presence of different particle size populations.

25
(continued on next page)
International Journal of Pharmaceutics 553 (2018) 428–440
 Table 6 (continued)

Sample name Measurement time Temp. [°C] STEP Profile

A. Zielińska et al.

Slowly occurring creaming process.

438
40

Slowly occurring creaming process.

International Journal of Pharmaceutics 553 (2018) 428–440
A. Zielińska et al. International Journal of Pharmaceutics 553 (2018) 428–440

Table 7 Table 8
Instability index of empty-SLN and citral-SLN, measured after production and Distribution (D) of particles sedimentation (velocity) of empty-SLN and citral-
after 1 month stored at 4, 25 and 40 °C. SLN, measured after production and after 1 month stored at 4, 25 and 40 °C.
Sample Measurement time Storage Temperature Instability index Sample Measurement time Storage D10% D50% D90%
name [°C] [–] name Temperature [µm/s] [µm/s] [µm/s]
[°C]
Empty – SLN after production 25 0.387
1 month 4 0.151 Empty – after production 25 1.392 3.109 6.949
25 0.367 SLN 1 month 4 0.972 1.919 12.49
40 0.584 25 1.989 11.79 62.20
40 0.272 2.283 55.05
Citral – SLN after production 25 0.032
1 month 4 0.495 Citral – after production 25 1.953 2.082 2.205
25 0.042 SLN 1 month 4 1.262 5.859 22.46
40 0.177 25 0.182 0.610 8.529
40 1.156 2.347 10.69

when compared to the citral-SLN samples (with exception for the citral-
SLN stored for one month at 4 °C), confirming the indications obtained
with the observation of the STEP profiles and reinforcing the idea that
the inclusion of citral has a beneficial effect in the formulation in terms
of physical stability of the dispersion. In fact, the citral-SLN samples
that were analyzed at 25 °C after production and after 1 month of sto-
rage at 25 °C had the lowest instability index (0.032 and 0.042, re-
spectively). These results indicate that 25 °C is the ideal temperature to
keep the dispersion more stable over time. On the other hand, keeping
the samples stored at 4 °C led to a faster separation process and to more
unstable dispersions, with a multimodal particle size distribution, as
highlighted by the STEP profile of this sample. When the sample was
stored at 40 °C an intermediate result was obtained, indicating that
when submitted to extreme temperatures, the citral-SLN dispersion will
be more stable in warmer climates than in colder ones.
Assuming a spherical shape for the particles, the use of the cen-
trifugation process enables the application of the following equation:
k r 2 = vm , which defines a quadratic relation between sedimentation
velocity and the particle radius, and in which k is a constant, r is the
radius of the particle and vm is the sedimentation velocity. In this study,
the sedimentation velocities measured by LUMiSizer® are shown in
Table 8. Obtained results correlate with the previous ones for the in-
stability index and the STEP profiles. Citral-SLN analyzed at 25 °C after
production and after one month of storage at 25 °C exhibited the lower
velocity distribution when compared to the other samples. A narrower
velocity distribution was recorded for the freshly prepared sample
(1.953 µm/s–2.205 µm/s), in comparison to that stored at 25 °C for one
month (0.182 µm/s–8.529 µm/s). This result indicates that the latter
has a wider particle size distribution range attributed to the interaction
between particles during the storage period. An overall analysis of these
results confirm that the loading of citral improves the stability of SLN,
given that distribution velocities are in general smaller and narrower in
the loaded SLN when compared to the empty ones. As the sedimenta-
tion velocity is an indicator of particle size, its lower values that were
obtained for citral-SLN show that these samples have a smaller particle
size (smaller particles migrate at lower speed). Finally, citral-SLN also
showed a narrower velocity distribution (D10%, D50% and D90% va-
lues are closer), meaning that the particle size distribution is more
uniform in these cases when compared to the empty SLN.
In summary, the citral-SLN have lower sedimentation speeds when
compared to the empty ones while citral-SLN kept at 25 °C exhibit the
lowest sedimentation speeds, as well as the narrowest velocity dis-
tribution. These result are in agreement with those obtained for in-
stability index and STEP profile responses.
4. Conclusions
We have demonstrated the enhanced anti-inflammatory activity of
citral over geranial and confirmed the cytotoxic effect of citral in two
distinct cell lines (non-tumoral HaCaT and tumoral A431). Citral-SLN
composed of Imwitor and poloxamer were successfully optimized by
factorial design approach, requiring a minimum of performed experi-
ments. According to the obtained results, the optimal SLN dispersion
consisted on our central established point composed of 1 wt% of citral,
4 wt% of Imwitor® 900 K and 2 wt% of Poloxamer 188. Accelerated
stability studies using the LUMiSizer® confirmed the enhanced physi-
cochemical stability of the optimized formulation when stored at 25 °C.
Acknowledgements
The authors acknowledge the financial support received from
Portuguese Science and Technology Foundation (FCT/MCT) and from
European Funds (PRODER/COMPETE) under the project reference M-
ERA-NET/0004/2015-PAIRED, co-financed by FEDER, under the
Partnership Agreement PT2020.
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