International Journal of Pharmaceutics 518 (2017) 228–241

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International Journal of Pharmaceutics

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

Application of aluminum chloride phthalocyanine-loaded solid lipid

nanoparticles for photodynamic inactivation of melanoma cells
Patrícia L. Gotoa , Marigilson P. Siqueira-Mourab , Antonio C. Tedescoa,c,*
a
Universidade de São Paulo, Faculdade de Ciências Farmacêuticas de Ribeirão Preto, Departamento de Ciências Farmacêuticas, Avenida do Café, s/n , Ribeirão
Preto, SP, 14040-903, Brazil
b
Universidade Federal do Vale do São Francisco, Colegiado Acadêmico de Ciências Farmacêuticas, Avenida José de Sá Maniçoba, s/n , Centro, Petrolina, PE,
56304-205, Brazil
c
Universidade de São Paulo, Faculdade de Filosofia Ciências e Letras de Ribeirão Preto, Departamento de Química, Avenida Bandeirantes, 3900, Ribeirão
Preto, SP, 14040-901, Brazil

A R T I C L E I N F O A B S T R A C T

Article history:
Received 17 October 2016 Cutaneous melanoma is the most aggressive skin cancer and is particularly resistant to current
Received in revised form 16 December 2016 therapeutic approaches. Photodynamic therapy (PDT) is a well-established photoprocess that is
Accepted 2 January 2017 employed to treat some cancers, including non-melanoma skin cancer. Aluminum chloride
Available online 4 January 2017 phthalocyanine (ClAlPc) is used as a photosensitizer in PDT; however, its high hydrophobicity hampers
its photodynamic activity under physiological conditions. The aim of this study was to produce solid lipid
Keywords: nanoparticles (SLN) containing ClAlPc using the direct emulsification method. ClAlPc-loaded SLNs
Solid lipid nanoparticles (ClAlPc/SLNs) were characterized according to their particle size and distribution, zeta potential,
Melanoma
morphology, encapsulation efficiency, stability, and phototoxic action in vitro in B16-F10 melanoma cells.
Aluminum chloride phthalocyanine
ClAlPc/SLN had a mean diameter between 100 and 200 nm, homogeneous size distribution
Photodynamic therapy
Glyceryl behenate (polydispersity index <0.3), negative zeta potential, and spherical morphology. The encapsulation
Direct emulsification efficiency was approximately 100%. The lipid crystallinity was investigated using X-ray diffraction and
differential scanning calorimetry and indicated that ClAlPc was integrated into the SLN matrix. The
ClAlPc/SLN formulations maintained their physicochemical stability without expelling the drug over a
24-month period. Compared to free ClAlPc, ClAlPc/SLN exerted outstanding phototoxicity effects in vitro
against melanoma cells. Therefore, our results demonstrated that the ClAlPc/SLN described in the current
study has the potential for use in further preclinical and clinical trials in PDT for melanoma treatment.
© 2017 Elsevier B.V. All rights reserved.

1. Introduction nevertheless, exposure to UV radiation is the main risk factor

The incidence of skin cancer has increased worldwide and has
affected public health globally (Gandhi and Kampp, 2015; Gordon,
2013). Melanoma is the most aggressive type of skin cancer.
Although it represents only 4% of all new skin cancer cases, this
malignancy is highly metastatic and accounts for approximately
85% of skin cancer deaths (Gordon, 2013; Kolk et al., 2014). The
pathogenesis of this cutaneous carcinoma is multifactorial;
predisposing patients to the disease (Gordon, 2013; Lazareth, 2013;
Narayanan et al., 2010). In general, melanoma affects the
melanocytes, which are responsible for producing melanin, the
pigment that protects the skin against the harmful effects of the
sun. Because melanin absorbs UV radiation, it works as a light
shield (Mundra et al., 2015; Narayanan et al., 2010). When detected
in the early stages, the only curative treatment for cutaneous
melanoma is surgical excision of the tumor, which does not always

Abbreviations: PDT, photodynamic therapy; ClAlPc, aluminum chloride phthalocyanine; SLN, solid lipid nanoparticles; ClAlPc/SLN, ClAlPc-loaded SLN; ROS, reactive
oxygen species; HLB, hydrophilic lipophilic balance; PCS, photon correlation spectroscopy; PdI, polydispersity index; PS, photosensitizer drug; EE, encapsulation efficiency;
TEM, transmission electron microscopy; AFM, atomic force microscopy; ANOVA, analysis of variance; CD, crystallinity degree; DMEM, Dulbecco Eagle’s minimum essential
medium; DSC, differential scanning calorimetry; Et, ethanol; FBS, fetal bovine serum; MTT, 4,5-dimethylthiazol-2-yl)2,5-dyphenyl-tetrazolium bromide; NIR, near infrared;
PBS, phosphate-buffered saline; RCF, relative centrifugal force; SD, standard deviation; STEP, space and time resolved extinction profiles; XRD, X-ray diffraction.
* Correspondence to: Laboratório de Fotobiologia e Fotomedicina (FFCLRP) Universidade de São Paulo (USP) Av. dos Bandeirantes 3900, 14040-901, Vila Monte Alegre,
Ribeirão Preto, SP, Brazil.
E-mail address: atedesco@usp.br (A.C. Tedesco).

http://dx.doi.org/10.1016/j.ijpharm.2017.01.004
0378-5173/© 2017 Elsevier B.V. All rights reserved.
 P.L. Goto et al. / International Journal of Pharmaceutics 518 (2017) 228–241
prevent melanoma progression. Immunotherapy, gene therapy,
chemotherapy, and radiotherapy have been used as adjuvant
treatments (Ingraffea, 2013; Monge-Fuentes et al., 2014). However,
these therapies do not always provide significant and effective
results. In addition, the drugs are highly toxic, and drug resistance
may emerge after a short period of treatment. Considering the
incidence, metastasis, mortality rate, and therapeutic resistance of
melanoma, developing effective therapeutic approaches has
become crucial (Girotti et al., 2014; Kawczyk-Krupka et al.,
2013; Vera et al., 2015).
In this scenario, photodynamic therapy (PDT), which has
already been established as a local therapy for several cancers
and skin disorders (Baldea and Filip, 2012; Davids and Kleemann,
2011; Kawczyk-Krupka et al., 2013; Kolk et al., 2014; Sparsa et al.,
2013), might be a potential tool for melanoma treatment. PDT
combines three essential components: (i) the administration of the
photosensitizer (PS), followed by (ii) PS photoactivation with
visible light at an appropriate wavelength and (iii) PS interaction
with molecular oxygen, generating singlet oxygen and reactive
oxygen species (ROS), which lead to cell death. Furthermore, PDT
can be applied several times, alone or in combination with other
modalities, resulting in low systemic toxicity and excellent
cosmetic results (Agostinis et al., 2011; Babilas et al., 2010). Over
the last decades, in vivo and in vitro studies (Davids and Kleemann,
2011; Mroz et al., 2010; Sharma and Davids, 2012) investigating the
role of PDT in cutaneous melanoma treatment concluded that
pigmented tumors were resistant to PDT. Two possible explan-
ations for this fact have been proposed: (i) melanin competed with
PS for light absorption, and (ii) melanin exhibited high antioxidant
power.
Aluminum chloride phthalocyanine (ClAlPc) is a PS that
contains aluminum (III) as a central metal ion, which can enhance
the photophysical and photochemical properties of this phthalo-
cyanine (Siqueira-Moura et al., 2013). ClAlPc presents a high molar
absorption coefficient between 600 and 800 nm, also known as the
“optical window” of biological tissues, in which light with enough
energy to produce ROS can reach the deeper skin layers with little
interference from the body’s endogenous chromophores, such as
melanin (Debele et al., 2015; Ficheux, 2009; Lucky et al., 2015;
Plaetzer et al., 2008). In fact, it has been shown that ClAlPc is
effective in treating pigmented diseases, and it has been suggested
that ClAlPc could overcome the typical PS–melanin competition
(Allison and Sibata, 2010; Idowu and Nyokong, 2007). ClAlPc has
excellent photochemical and photodynamic activity, however, like
most phthalocyanines, ClAlPc is insoluble in water, which limits its
bioavailability and its administration in a physiological environ-
ment. In an aqueous medium, hydrophobic molecules tend to
aggregate, which directly affects the photophysical and photo-
chemical properties of the PS and dramatically reduces the
photodynamic activity of ClAlPc (Chatterjee et al., 2008), requiring
association to specific drug nanocarriers or conjugation of the PS
with hydrophilic molecules for clinical use. The use of nanotech-
nology-based drug delivery systems could overcome these short-
comings, and provide additional advantages, such as (a) promotion
of controlled PS release, (b) protection from drug degradation, (c)
reduced amounts of PS required to achieve therapeutic effects
(hence diminishing the toxicity effects), and (d) increased PS
stability and bioavailability (Bechet et al., 2008; Lucky et al., 2015;
Paszko et al., 2011).
Solid lipid nanoparticles (SLN) have emerged as promising drug
delivery systems (Mehnert and Mader, 2001). First developed in
the 1990s, these solid colloidal particles consist of a solid lipophilic
matrix at body temperature, in which biologically active sub-
stances can be dissolved or entrapped (Müller et al., 2011; Simões
et al., 2015). They have a mean particle size in the submicron range
between 50 and 1000 nm (Müller et al., 2011; Pardeike et al., 2009).
229
In addition, SLN have many advantages when compared to other
colloidal carriers, such as avoidance of organic solvents, low cost
materials, drug release profile controlled by modification of the
solid matrix, improved stability profile, and the possibility of large
scale production (Kakadia and Conway, 2014; Mehnert and Mader,
2001). This colloidal system can be administered via different
routes, but its topical application on the skin has attracted much
attention because SLN strongly adhere to the stratum corneum, to
form a flexible lipid film on the skin. This occlusion effect keeps the
skin hydrated and improves the percutaneous absorption of the
drug (Souto et al., 2006), aiding the treatment of cutaneous
melanoma. In addition, the nanosize and the lipophilic composi-
tion of this colloidal system contribute to its preferred and passive
accumulation in the tumor tissue due to the enhanced permeabil-
ity retention effect (enhanced vascular permeability and poor
lymphatic drainage) of the abnormal tissue (Debele et al., 2015;
Iyer et al., 2006; Mundra et al., 2015).
In this paper we described the preparation and characterization
of SLN as a controlled drug delivery system for ClAlPc using the
direct emulsification method and tested their phototoxicity effect
in B16-F10 cells in vitro to investigate their efficacy in PDT for
melanoma treatment.
2. Materials and methods
2.1. Materials
The solid lipid glyceryl behenate (Compritol 888 CG ATO,
melting range 69–74  C) was a gift from Brasquim (Porto Alegre, RS,
Brazil). The solid lipid stearic acid (melting range 67–72  C),
aluminum chloride phthalocyanine (ClAlPc, purity = 85%), phos-
phate-buffered saline (PBS, pH 7.2–7.4), trypan blue, and 4,5-
dimethylthiazol-2-yl)2,5-dyphenyl-tetrazolium bromide (MTT)
were purchased from Sigma–Aldrich Co. (St. Louis, MO, USA).
Sorbitan isostearate (Hydrophilic Lipophilic Balance = 4.7) and
Polyoxyethylene-40 hydrogenated castor oil (HLB = 14.1) were
kindly supplied from Croda do Brasil Ltda (Campinas, SP, Brazil).
The murine fibroblast cell lineage (NIH-3T3-CRL1658) and the
murine melanoma cells (B16-F10–CRL 6475) were supplied by
ATCC (Virginia, USA). Dulbecco Eagle’s minimum essential medi-
um (DMEM), RPMI-1640 with and without phenol red, and trypsin
– EDTA (0.25%) were acquired from Gibco (New York, USA). Fetal
bovine serum (FBS) was obtained from Cultilab (São Paulo, SP,
Brazil). Methanol, chloroform, ethanol (99.5%), and dimethyl
sulfoxide were purchased from J.T. Baker (Mallinckrodt Baker,
Phillipsburg, USA). All of the other reagents were of analytical
grade and were used as supplied. Ultrapure water, obtained using
the Direct-Q Water Purification System (Millipore, Darmstadt,
Germany), was used to prepare the colloidal dispersions.
2.2. Ternary phase diagram
To obtain SLN with the best features and component ratio
(Anton et al., 2007), ternary phase diagrams were constructed for
the solid lipid, the surfactant blend, and water. First, the non-ionic
surfactants sorbitan isostearate and polyoxyethylene-40 hydroge-
nated castor oil were mixed at ratio of 33:67 (w/w), as reported
previously by Goto et al. (2012), result in a mixture of surfactants
with an HLBfinal = 11. This mixture should provide oil in water
dispersions. Different ratios of the surfactant blend were used in
the final SLN composition. The same points of the ternary diagram
were reproduced for each solid lipid (glyceryl behenate or stearic
acid). The solid lipid and the surfactant blend were added at weight
percentages ranging from 5 to 20% (w/w). Finally, the aqueous
phase (ultrapure water ranging from 60% to 100%, w/w) was added
to complete the dispersion to 100%. After 24 h, the resulting
230 P.L. Goto et al. / International Journal of Pharmaceutics 518 (2017) 228–241
dispersions were visually inspected; transparency, viscosity, and
uniformity were examined. Liquid and stable dispersions were
further analyzed using Photon Correlation Spectroscopy (PCS),
which will be described in further detail.
The colloidal samples were prepared using the direct emulsifi-
cation method at the ratios suggested in Fig. 1, as previously
described by Goto et al. (2012) and Montenegro et al. (2011). The
aqueous solution was composed of ultrapure water; the oil phase
consisted of solid lipids and surfactants, with or without ClAlPc.
Both the aqueous and oil phases were heated in a water bath at
85  2  C, separately. Then, the aqueous phase was added to the
melted oil phase drop-wise, at a constant temperature and under
magnetic stirring (1000 rpm). The resulting mixture was cooled to
room temperature under continuous agitation.
The solid lipid was chosen based on the results provided by the
ternary phase diagram. Unloaded SLN and ClAlPc/SLN containing
different theoretical concentrations of ClAlPc (from 0.05 to
0.40 mg mL 1) were produced.
2.3. Characterization of SLN
2.3.1. Photon correlation spectroscopy analysis
The mean diameter, given as the Z-average, and size distribu-
tion, obtained as the polydispersity index (PdI), of the obtained
dispersions were assessed using dynamic light scattering con-
ducted on a Zetasizer Nano ZS ZEN3600 (Malvern Instruments,
England, UK), with a He-Ne laser; the scattered light was measured
at an angle of 173 . Before measuring, the samples were diluted in
ultrapure water with a 1:100 vol ratio. The autocorrelation
functions were analyzed using the Zetasizer software provided
by Malvern. Using the same equipment, the zeta potential was
determined by measuring the electrophoretic mobility; the
Smoluchowski approximation was used to calculate the values
(Negi et al., 2013). The samples were diluted as described above
and mixed with 1.0% of PBS (Lobovkina et al., 2011). All analyses
were carried out at 25  C.
2.3.2. Encapsulation efficiency and drug loading
The ClAlPc amount in the SLN samples was determined by
solubilizing a known amount of SLN in 3.0 mL of chloroform and
ethanol (1:1, v/v) solution to a final ClAlPc concentration of
0.8 mg mL 1, followed by sonication for 15 min to solubilize all of
the nanoparticles (Bhalekar et al., 2015). The ClAlPc concentration
in the solution was determined by UV/Vis spectroscopy conducted
on a double-beam Ultrospec 7000 (GE Healthcare, Glattbrugg,
Fig. 1. Ternary phase diagrams showing the macroscopic appearance of the dispersions obtained with water, the surfactants blend, and (A) stearic acid or (B) glyceryl
behenate.
Switzerland) spectrophotometer at a wavelength of 679 nm
(y = 0.424x + 0.0206, r = 0.999). Drug loading corresponded to the
amount of drug that the colloidal system was able to incorporate.
The loading was estimated by comparing the total drug content
found in the ClAlPc/SLN and the initial content that was weighed to
produce the formulations, as shown in Eq. (1).
Drug loading (%) = (total drug content/initial drug content
weighed)  100 (1)
Free ClAlPc was obtained by separating the ClAlPc/SLN from the
aqueous medium by centrifugation in a Centrifuge 5810 R
equipped with a FA-45-6-30 rotor (Eppendorf, New York, USA).
Centrifugal filter tubes Microcon YM-100 (Millipore, Tullagreen,
Ireland) were employed, and the samples were submitted to
12,857  g at 4  C for 1 h (Siqueira-Moura et al., 2013). The non-
encapsulated drug present in the filtrate was quantified on a
spectrofluorimeter Fluorolog-3 (Horiba JobinYvon, Tokyo, Japan)
(y = 1E + 08 x + 2808.3, r = 0.999), using methanol as a solvent, with
the PS excitation fixed at 610 nm and the emission fixed at 675 nm.
The encapsulation efficiency (EE) was calculated as the difference
between the total ClAlPc content in the formulation and the non-
encapsulated ClAlPc (Eq. (2)).
EE (%) = [(total drug content drug content non-encapsulated)/
initial drug content weighed]  100 (2)
The calibration curves were adequately obtained to quantify the
ClAlPc content in the different media by spectrophotometry or
spectrofluorimetry (data not shown).
2.3.3. Transmission electron microscopy (TEM)
SLN particles were visualized with a transmission electron
microscope model 100CXII (JEOL, Tokyo, Japan) equipped with a
digital camera Hamamatsu ORCA-HR (Hamamatsu, Japan) operat-
ing at an acceleration voltage of 70 kV. Prior to analysis, the SLN
samples were diluted in ultrapure water with a 1:100 vol ratio. A
drop of this dilution was placed on a copper grid coated with
carbon; the surplus was removed with filter paper and allowed to
dry at room temperature before analysis.
2.3.4. Atomic force microscopy (AFM)
Images were obtained using the tapping mode technique
performed with a Scanning Probe Microscope SPM-9600 (Shi-
madzu, Kyoto, Japan) controlled by SPM Online software. A droplet
of the SLN sample was deposited on a freshly cleaved mica surface
and dried under argon flow. The images were acquired using the
 P.L. Goto et al. / International Journal of Pharmaceutics 518 (2017) 228–241
tapping mode technique, with a silicon cantilever 124 mm in
length. The resonance frequencies ranged from 324 to 369 kHz, and
the spring constants varied from 34 to 51 N m 1.
2.4. Stability tests
2.4.1. Long-term stability
Unloaded SLN and ClAlPc/SLN samples were stored in sealed
flasks and kept at 4  2  C, in the dark, for 24 months. Certain
macroscopic aspects, such as transparency, viscosity, and unifor-
mity were analyzed and the particle size, the PdI, and the zeta
potential were measured using PCS and electrophoretic mobility.
2.4.2. Forced stability measurements
The stability of the SLN dispersions was analyzed in a dispersion
analyzer LUMiSizer 611 (LUM GmbH, Berlin, Germany) that
employed the STEP Technology (Space and Time resolved
Extinction Profiles). This study simultaneously registered the
intensity of the transmitted light (at 880 nm) as a function of time
and the position over the sample. The measurements were
performed at 25  C; the transmission profile was acquired every
65 s for 476 min at a rotation speed of 3618 rpm. To perform the
analyses, specific cuvettes with a radius position of 129.5 mm were
used. Based on the results, it was possible to predict the shelf life of
the SLN samples by using Eq. (3).
Shelf life (seconds) = Measurement time  Relative Centrifugal
Force (3)
2.5. X-ray diffraction (XRD) analysis
The X-ray diffraction patterns were acquired on an X-ray
diffractometer D5005 (Siemens/Bruker-AXS, Germany) with
monochromatic Cu Ka radiation (l = 1.5406 Å). The measurements
were performed at a current of 30 mA and a voltage of 40 kV. The 2u
values ranged from 2 to 50 ; the step size was 0.02 , and the step
time was 1 s. A thin film of each sample was produced for the
analysis. The bulk lipid, ClAlPc powder, freeze-dried ClAlPc-200-
SLN, and unloaded SLN (after 1 and 24 months of storage) were
evaluated.
2.6. Differential scanning calorimetry (DSC) analysis
The DSC thermograms were obtained on a Jade DSC (Perki-
nElmer, Groningen, Netherlands) controlled by Pyris software. Bulk
lipid, freeze-dried ClAlPc-200-SLN, and unloaded SLN, at 1 and 24
months of storage, were weighed (5 mg) separately in a standard
aluminum pan and hermetically sealed. An empty pan was used as
the reference. The analyses were conducted under inert nitrogen
atmospheric conditions at a flow rate of 30 mL min 1 and at a
scanning rate of 5  C min 1, from 10 to 90  C. The crystallinity
degree (CD) was calculated as shown in Eq. (4) (de Carvalho et al.,
2013; Souto et al., 2006).
CD (%) = (melting enthalpy of SLN/melting enthalpy of the bulk
lipid  lipid concentration in the SLN)  100 (4)
2.7. In vitro cell experiments
2.7.1. Cell culture
The NIH-3T3 (murine fibroblasts) cells were grown in DMEM
medium supplemented with 10% FBS, penicillin (100 U mL 1),
streptomycin (100 mg mL 1), amphotericin (2.5 mg mL 1), and
nonessential amino acids. The B16-F10 (pigmented melanoma)
cells were grown in RPMI-1640 medium supplemented with 10%
231
FBS, penicillin (100 U mL 1), streptomycin (100 mg mL 1), and
amphotericin (2.5 mg mL 1). Both cultures were kept at 37  C in
a humidified atmosphere of 5% CO2.
2.7.2. Biocompatibility test of unloaded SLN
The biocompatibility of unloaded SLN was evaluated by
examining the NIH-3T3 cell viability after direct contact with
the nanoparticles using the MTT assay. Three different passages of
the NIH-3T3 cells were seeded separately at 3.5  104 cells per well
on a 96-well plate. The cells were then incubated overnight to
allow them to adhere. In the treatment step, dilutions of unloaded
SLN were prepared at different concentrations, from 0.1 to 50.0%, in
DMEM supplemented with 3% FBS. The DMEM medium was
replaced with the treatment medium. Each concentration was
applied to the cells in triplicate wells with different passages. For
the negative control, the cells were treated with culture medium
without nanoparticles. The plates were incubated at 37  C for 3 h.
After the treatment, the medium was removed, and the NIH-3T3
fibroblasts were gently rinsed with PBS to wash away the excess
nanoparticles. The cells were incubated with fresh DMEM
supplemented with 10% FBS for 24 h before MTT analysis.
2.7.3. Dark cytotoxicity test of ClAlPc
Before applying the photodynamic therapy, the cytotoxicity of
the ClAlPc-loaded SLN on the cancerous cell line was evaluated
under dark conditions. Three different passages of the B16-F10
cells were seeded separately at 5  104 cells per well onto a 24-well
plate and incubated overnight, to allow the cells to adhere.
Treatment of the pigmented melanoma cells under dark conditions
was comprised of two steps. First, two ClAlPc-loaded SLN with two
different concentrations of ClAlPc were tested at the same dilution
(according to the biocompatibility results) in RPMI-1640 supple-
mented with 3% FBS, which resulted in two different ClAlPc
concentrations: 0.31 and 0.75 mg mL 1. In the second step, a stock
solution of free ClAlPc in ethanol (ClAlPc-Et) was diluted to the
same nontoxic ClAlPc concentration in the ClAlPc-loaded SLN
selected for this study (0.75 mg mL 1). Three control wells were
included, namely the solvent control, which contained culture
medium and ethanol at the same proportion used for free ClAlPc;
the negative control, treated with culture medium only; and the
positive control, to which unloaded SLN was added in the culture
medium at the same dilution as in the case of the ClAlPc-loaded
SLN. The B16-F10 cells were kept in treatment medium in a CO2
incubator for 3 h. After washing with PBS, the wells were filled with
fresh RPMI-1640 medium supplemented with 10% FBS. After 24 h
of incubation, the MTT assay was performed.
2.7.4. Photodynamic therapy efficiency
The same B16-F10 cell lineage was used in this study, and the
cells were seeded as described above. The pigmented melanocytic
cells were divided into four experimental groups: cells treated
with ClAlPc-loaded SLN diluted in culture medium, at 0.75 mg
mL 1 ClAlPc; cells treated with free ClAlPc (ClAlPc-Et diluted in
culture medium to 0.75 mg mL 1 ClAlPc); the negative control; and
the laser control (both in culture medium). Following 3 h of
incubation, the treatment medium was removed, the cells were
washed with PBS, and RPMI-1640 without phenol red was added to
each well. Except for the negative control, the other groups were
irradiated with a diode Eagle laser (Quantum Tech., Brazil) at an
excitation wavelength of 670 nm and operating with an average
power of 0.30 mW and a light radiance of 17 mW cm 2 to
accomplish photodynamic therapy. The B16-F10 cells treated with
ClAlPc were irradiated with 0.5, 1.0, and 2.0 J cm 2 light doses. The
laser control group was irradiated with the highest light dose only.
After exposure to the laser light, the medium was replaced with
fresh RPMI-1640 supplemented with 10% FBS and the cells were
232 P.L. Goto et al. / International Journal of Pharmaceutics 518 (2017) 228–241

incubated for an additional 24 h. The cell viability was measured
using the MTT assay.
2.7.5. MTT assay for cell viability
To examine the effects of each treatment on NIH-3T3 and B16-
F10 cell viability, the MTT assay was performed. This quantitative
method is based on the fact that feasible mitochondria from living
cells reduce the yellow tetrazolium dye to purple formazan
(insoluble in water). Briefly, 100 mL (96-well plate) or 500 mL (24-
well plate) of MTT solution (5 mg mL 1 MTT in the adequate culture
medium, without phenol red, for each cell lineage) was added to
each well, and the cells were incubated for 4 h. The MTT solution
was replaced with 100 mL (96-well plate) or 500 mL (24-well plate)
of 2-propanol, to promote cell lysis and to solubilize the formazan.
The absorbance was recorded at 570 and 690 nm in a plate reader
Safire 2 (TECAN Group, Grödig, Austria) (Siqueira-Moura et al.,
2013). The cell viability was calculated with respect to the negative
control (100% viable cells) and was the average value of the three
plates.
2.8. Statistical analysis
The results were presented as the average of three independent
experiments  standard deviation (SD). The statistical analyses
were performed using GraphPad Prism version 5.0 software. The
average differences were compared using one-way ANOVA
followed by the Tukey or Dunnett post hoc test. Differences were
considered statistically significant when the p-values <0.05.
3. Results and discussion
Previous studies have reported the preparation of SLN as a
nanocarrier for ClAlPc using solvent diffusion and nanoprecipita-
tion methods (Almeida et al., 2012; Siqueira-Moura et al., 2013).
Although successful ClAlPc/SLN were obtained, these methods
present some limitations, such as residual traces from the organic
solvents used in the production process and difficulties in
increasing the scale of the production process (Mehnert and
Mader, 2001). In this paper we prepared ClAlPc/SLN using the
direct emulsification method. This method is a relatively simple,
solvent-free and energy-efficient method, that requires low cost
materials and allows large-scale production.
In this study we first performed a ternary phase diagram using
two different solid lipids to define the best composition for
producing a stable SLN. Once the SLN composition was deter-
mined, we prepared unloaded SLN (particles without PS) and SLN
loaded with seven different ClAlPc concentrations. The stability
and physicochemical properties of each SLN formulation –
unloaded and those loaded with PS – were assessed. In the
following experiments: morphological analyses, X-ray diffractions,
and differential scanning calorimetry, only two groups were
compared – the unloaded SLN and the ClAlPc-200-SLN. The latter
corresponded to a formulation with an intermediate PS concen-
tration (see Table 2), that was chosen for investigating the
influence of ClAlPc in the formulation.
3.1. Ternary phase diagram
To prepare ClAlPc/SLN for clinical trials, it is important to
consider their physicochemical properties, which have been
shown to play a critical role in particle stability, size growth,
and drug retention (Das and Chaudhury, 2010; Müller et al., 2011;
Pardeike et al., 2009). Therefore, it is crucial to take into account
how the choice of lipid and surfactant and the ratio between them,
as well as the preparation process affect these properties to
optimize the formulation.
It has been shown that the type of surfactant and lipid used and
the ratio between them determine the particle size, polydispersity,
and the stability of the nanometric systems (Anton et al., 2008;
Kumar and Randhawa, 2013).
Stearic acid and glyceryl behenate were used as solid lipids for
SLN preparation due to their advantages, such as low cost and
biodegradability. Stearic acid and glyceryl behenate are the most
frequently employed compounds used in SLN production and for
conducting in vitro tests (Doktorovova et al., 2014; Li et al., 2011;
Mehnert and Mader, 2001).
Two surfactants – sorbitan isostearate and polyoxyethylene-40
hydrogenated castor oil – were chosen for preparing the
formulation. In our previous study (Goto et al., 2012) we
demonstrated that a blend of these two surfactants at a fixed
ratio of 33:67 (w/w) was suitable for producing submicron
particles after being submitted to the direct emulsification method
(Anton and Vandamme, 2009). Furthermore, studies have shown
that formulations that combine two surfactants efficiently reduce
particle aggregation, yield a smaller particle size, and are more
stable than samples containing one surfactant only (Mehnert and
Mader, 2001).
The ternary phase diagram was used to establish the
appropriate nanoparticle composition as a function of the lipid/
surfactant/water ratio. Fig. 1 illustrates the macroscopic appear-
ance of the dispersions obtained with stearic acid (Fig. 1A) and
glyceryl behenate (Fig. 1B) in the ternary phase diagram, 24 h after
production.
The stearic acid phase diagram (Fig. 1A) showed that four of the
eight dispersions were solid or creamy, and one dispersion led to
phase separation. The other three dispersions with stearic acid,

Table 1
Characterization by PCS of liquid dispersions produced with glyceryl behenate or stearic acid. Statistical analysis by one-way ANOVA with Tukey posthoc test (p < 0.05),
(average  SD, n = 3).

Solid lipid Diagram points Lipid (%)a Surfactants (%)a Diameter (nm) Polydispersity index Zeta Potential (mV)
Glyceryl behenate 35a 10.0 20.0 124.6 (33.0) 0.399 (0.045) 30.2 (2.8)
36a 10.0 10.0 136.7 (14.4) 0.192 (0.012)** 31.4 (2.4)
38a 7.5 7.5 166.4 (15.8) 0.244 (0.039) 31.5 (0.5)
39a 5.0 10.0 84.9 (13.5)* 0.367 (0.049) 30.9 (1.6)
40a 5.0 5.0 178.8 (35.7) 0.292 (0.049) 33.0 (2.8)
Stearic acid 36b 10.0 10.0 1,002.0 (45.0) N/A N/A
38b 7.5 7.5 2,572.0 (500.7) N/A N/A
40b 5.0 5.0 1,602.1 (500.9) N/A N/A

N/A = Not Available.

a
(w/w).
*
Indicates significant difference with samples 38 and 40 (p < 0.05).
**
Indicates significant difference with samples 35 and 39 (p < 0.05).
 P.L. Goto et al. / International Journal of Pharmaceutics 518 (2017) 228–241
containing the same ratio of the lipid-surfactant blend and up to
20% (w/w) of the oily phase, resulted in liquid dispersions, but with
a non-translucent aspect (not common for smaller sized particles).
The phase diagram obtained with glyceryl behenate (Fig. 1B)
indicated that the 20% (w/w) lipid or high lipid/surfactant ratio
formulations presented a creamy aspect. The formulations
composed of up to 10% (w/w) of lipid or with equal or lower
lipid/surfactant ratios resulted in translucent dispersions with
high fluidity, typical of formulations with submicron particles, as
expected in this study (Lippacher et al., 2001; Negi et al., 2014).
Therefore, these results showed that the type of solid lipid and
the ratio of each component in the formulation may influence the
features of the final dispersion.
The particles in the liquid dispersions obtained with glyceryl
behenate and stearic acid were assessed by PCS; the results are
shown in Table 1.
The macroscopic analysis and the PCS analyses showed that the
liquid and opaque dispersions obtained with stearic acid did not
result in submicron particles (>1000 nm). Therefore, this lipid was
excluded for further analyses. On the other hand, formulations
containing glyceryl behenate provided particles with a mean
diameter ranging from 84.9 to 178.8 nm, showing that the glyceryl
behenate combined with the surfactant blend was suitable for
providing particles with submicron size. Considering the mean
diameter, particles with the smallest size (84.9 nm, corresponding
to point 39a in Table 1) were significantly different from the two
dispersions with the largest particles size (166.4 nm and 178.8 nm,
corresponding to points 38a and 40a, respectively). Taking into
account the polydispersity index, the formulation corresponding
to point 36a was significantly different from those corresponding
to points 35a and 39a. In addition, the latter two formulations
presented PdI values higher than 0.3, which have not been
considered optimum for SLN (Das et al., 2011). All the liquid
samples produced with glyceryl behenate presented high zeta
potential values ensuring the stability of the formulations. Taken
together, these results revealed that the best SLN composition
corresponded to point 36a, which was obtained with glyceryl
behenate. This formulation was chosen to encapsulate ClAlPc and
was used in further studies.
3.2. Physicochemical characterization
Unloaded and ClAlPc/SLN, containing seven different theoreti-
cal concentrations of ClAlPc, were prepared using the direct
emulsification method. Their composition consisted of 10.0%
glyceryl behenate, 3.3% sorbitan isostearate, 6.7% polyoxyethy-
lene-40 hydrogenated castor oil, and 80.0% water (w/w). Each
ClAlPc/SLN formulation (samples with different ClAlPc concen-
trations) was labeled according to the corresponding PS concen-
tration (Table 2).
Table 2
Characterization by PCS of unloaded SLN and ClAlPc/SLN and ClAlPc loading capacity after 1 and 24 months of storage. Statistical analysis by one-way ANOVA with Tukey
posthoc test (p < 0.05), (average  SD, n = 3).
Samples ClAlPc concentration (mg mL 1
) Drug loading (%)
1month
Unloaded SLN – –
ClAlPc-50- SLN 50 80.63
ClAlPc-100-SLN 100 83.73
ClAlPc-200-SLN 200 77.24
ClAlPc-250-SLN 250 76.02
ClAlPc-300-SLN 300 77.54
ClAlPc-350-SLN 350 77.40
ClAlPc-400-SLN 400 93.75
233
The mean diameter of the obtained nanoparticles ranged from
119.4 to 160.9 nm, with a narrow size distribution (PdI <0.3), and
zeta potential values between 34.1 and 25.4 mV. The difference
between the particles was not significant, indicating that the
presence of the PS in the formulations with varying concentrations
did not affect the SLN features.
One month after production, the ClAlPc-loading capacity of SLN
ranged from 77.24% to 93.75%; the drug loading decreased slightly
to 69.43–81.52% after 24 months. Similar drug loading values were
found previously for SLN produced with glyceryl behenate and
loaded with hydrophobic drugs (de Carvalho et al., 2013). Our
results showed that the SLN under investigation were able to retain
the ClAlPc during storage, with a slight decrease in loading capacity
after 24 months. This was a fortunate outcome because drug
expulsion, which depends on the chemical nature of the lipid, its
crystallization state, and polymorphic modifications (Müller et al.,
2000), is a problem frequently associated with SLN, as will be
discussed later.
Regarding the encapsulation efficiency, approximately 100% of
the PS amount present in the sample was successfully encapsulat-
ed in the nanocarriers. This was determined by the spectrofluoro-
metric analyses of the aqueous phase, which did not indicate the
presence of the fluorescence emission characteristic of ClAlPc
(Almeida et al., 2012) (data not shown). It is known that the ClAlPc
exhibits high fluorescence emission with the maximum approxi-
mately 675 nm (excitation wavelength at 610 nm). Therefore, even
traces of the PS in the aqueous phase (the outer side of the
nanocarriers) could be detected after dissolving the sample in
methanol.
Using stearic acid as the solid lipid and the surfactant sodium
lauryl to prepare ClAlPc/SLN via the solvent diffusion method
Almeida et al. (2012) reported EE values in the 71 to 85% range.
These differences in EE measurements could be explained by
differences in the solid lipids used. First, the partition coefficient
must be considered for the incorporation of non-polar drugs into
lipid particles. Due to the high intermolecular forces that bind the
molecules, hydrophobic drugs do not always have high solubility in
all types of lipids (Bunjes, 2010). Second, the chemical properties of
the lipid are of great importance because the more perfect the
crystalline lattice of the solid lipid, the greater the amount of drug
expelled from the lipid matrix (Müller et al., 2000). Based on our
results, glyceryl behenate, which is comprised of a mixture of
mono, di and triglycerides, was more suitable for ClAlPc incorpo-
ration than stearic acid.
Siqueira-Moura et al. (2013) described the preparation of
polymeric nanoparticles loaded with ClAlPc via the nanoprecipi-
tation method and reported EE measurements between 56 and
71%. These differences in EE measurements may be explained by
the type and physical state of the oil phase used. In the case of
polymeric particles, soybean oil was used to produce the particles.
Diameter (nm) Polydispersity index Zeta potential (mV)
24 months
– 119.4 (12.3) 0.179 (0.011) 34.1 (4.3)
77.39 136.9 (22.8) 0.221 (0.015) 30.2 (3.3)
81.52 135.7 (3.3) 0.176 (0.017) 29.8 (2.6)
71.20 139.5 (1.0) 0.177 (0.016) 30.9 (2.9)
71.18 141.9 (12.5) 0.176 (0.071) 26.6 (7.6)
69.43 139.5 (1.7) 0.187 (0.013) 26.2 (2.8)
75.52 154.1 (28.2) 0.188 (0.020) 25.7 (4.9)
76.65 160.9 (47.6) 0.209 (0.063) 25.4 (0.7)
234 P.L. Goto et al. / International Journal of Pharmaceutics 518 (2017) 228–241
In addition, the physical state of the lipid inside the particles also
must be taken into account because the mobility of drug molecules
in a solid lipid are reduced when compared to a liquid oil (Mehnert
and Mader, 2001). Therefore, the use of solid lipid nanoparticles
seems to be a more appropriate vehicle for ClAlPc. To our
knowledge, there have not been any studies that tested the
solubility of ClAlPc in the lipids mentioned above.
The major problems using SLN as a drug delivery system are
related to their stability during storage, which can cause changes in
particle size, crystallinity structure, and polymorphism (Das and
Chaudhury, 2010). Therefore, several analyses were performed to
evaluate whether these instabilities occurred in the SLN in the
current study.
3.3. SLN long-term stability
The long-term stability of SLN during storage is critical as
aggregation may occur increasing the particle size (Freitas and
Muller, 1999). The stability of unloaded SLN and ClAlPc/SLN was
investigated by monitoring the mean size, the PdI, and the zeta
potential values of the SLN during storage at 4  C, protected from
light, for 24 months. The results of all SLN formulations showed a
similar pattern. Fig. 2 shows the PCS data for unloaded SLN and
ClAlPc-200-SLN (Table 2 describes the behavior observed for each
ClAlPc/SLN).
The mean size and PdI of the SLN samples measured showed
some variations over time, with slight increases or decreases in the
values, but they did not change significantly (p > 0.05) over the 24-
month period, as shown in Fig. 2. The zeta potential of the
unloaded SLN showed a small decrease, while the ClAlPc/SLN
formulations (represented by ClAlPc-200-SLN) exhibited a slight
increase in the zeta potential. However, there were no significant
differences in the zeta potential values (p > 0.05) during the 24
months. As will be discussed later, the crystal lattice of the SLN
presented small changes during the 24-month period, although
without significant differences. It may be suggested that the
presence of the PS, dissolved in the lipid matrix, could be
responsible for increasing the zeta potential in the ClAlPc/SLN
formulations.
Therefore, our results demonstrated that the nanostructures
remained physically and chemically stable for 24 months under the
current storage conditions. Studies using glyceryl behenate for SLN
preparation also reported long storage stability. Freitas and Muller
(1999) produced unloaded SLN with glyceryl behenate and
reported long-term stability for three years. Using glyceryl
Fig. 2. PCS data for (A) unloaded SLN and (B) ClAlPc-200-SLN, during 24 months of storage at 4  C, protected from the light. Statistical analysis by one-way ANOVA with Tukey
posthoc test (p < 0.05), (average  SD, n = 3).
behenate to prepare risperidone-loaded SLN, Silva et al. (2011)
showed that the preparations remained stable over a two-year
storage period.
Electrostatic repulsion combined with steric hindrance con-
tributed to the long-term stability of loaded and unloaded SLN
(Freitas and Muller, 1999; Mehnert and Mader, 2001).
3.4. Forced stability measurements
Forced stability is an accelerated analysis that helps to predict
the long-term stability of the formulations (Chiu et al., 2012;
Detloff et al., 2007). Considering that the forced stability analysis
was conducted at 25  C, the results indicated the predicted long-
term stability or shelf life of the samples stored at 25  C or at room
temperature.
Near infrared (NIR) transmission profiles of the unloaded SLN
samples and those with varying ClAlPc concentrations were quite
similar. Fig. 3 shows the NIR transmission profiles of unloaded SLN
(Fig. 3A) and ClAlPc-200-SLN (Fig. 3B), representing all of the
ClAlPc/SLN profiles.
The comparison between the first and the last registered
profiles for both the unloaded SLN and the ClAlPc-200-SLN,
showed that the light transmittance was reduced by less than 10%,
probably due to the accommodation of the nanoparticles during
the experiment. The same behavior was observed in the SLN
formulations with 50, 100, 200, 300, 350, and 400 mg mL 1 of
ClAlPc (data not shown). The NIR transmission did not change
during analytical centrifugation, as monitored in situ using STEP
Technology, which indicated that there was no sign of instability.
Thus, it may be concluded that the nanoparticles in the suspension
retained their homogenous distribution across the sample cell
(Chiu et al., 2012).
Fig. 4 shows the integrated transmission of the SLN samples for
comparison of the results.
The results showed integral profiles that resulted in lines
almost parallel to the X axis (Fig. 4). This suggests that there was no
particle migration due to the centrifuge force or modifications of
the particle size distribution that could disturb the local
concentration of the particles, resulting in altered NIR transmis-
sion (Chiu et al., 2012; Detloff et al., 2007). Therefore, we may
conclude that the SLN formulations remained stable during the
test performed at 25  C.
The analysis at 3618 rpm for 476 min corresponded to a
predicted shelf life of 12 months. Considering that there was no
signal of instability and that the test was performed at 25  C, we
 P.L. Goto et al. / International Journal of Pharmaceutics 518 (2017) 228–241 235

Fig. 3. Normalized NIR transmission profiles obtained with (A) unloaded SLN and (B) ClAlPc-200-SLN by forced stability analysis during centrifugation with LUMiSizer. The
first and the last registered profiles are shown in red and in green, respectively. The cuvette draw scheme could illustrate the SLN sample position. (For interpretation of the
references to color in this figure legend, the reader is referred to the web version of this article.)

Fig. 4. Integral transmission as a function of time of unloaded SLN and loaded SLN with different ClAlPc concentrations, obtained during forced stability analysis by
centrifugation with LUMiSizer.

concluded that each SLN produced herein remained stable for 12
months when stored at 25  C.
3.5. SLN morphological analyses
Unloaded SLN and ClAlPc-200-SLN were analyzed using TEM
(Fig. 5) and AFM (Fig. 6).
The images obtained by TEM showed that the mean size of the
unloaded SLN (Fig. 5A and C) and ClAlPc-200-SLN (Fig. 5B and D)
were approximately 100 and 160 nm, respectively. They presented
a submicron and uniform particle size. The unloaded SLN was
smaller than the ClAlPc-200-SLN.
AFM images of unloaded SLN (Fig. 6A and B) and ClAlPc-200-
SLN (Fig. 6C and D) provided additional evidence for the
nanometer size of the particles. They also showed that the
unloaded SLN smaller (110 nm) than the ClAlPc-200-SLN
(170 nm).
Both microscopic analyses revealed that the investigated
nanostructures had spherical or near-spherical morphology. These
results were consistent with those obtained by PCS (see Table 2),
confirming the narrow polydispersity index, as well as the size
difference of unloaded and PS-loaded particles. The absence of
apparent aggregation could confirm the increased stability of the
particles due to the presence of electrostatic repulsion and steric
236 P.L. Goto et al. / International Journal of Pharmaceutics 518 (2017) 228–241
Fig. 5. TEM images obtained from (A and C) unloaded SLN and (B and D) ClAlPc-200-SLN.
hindrance (Almeida et al., 2012). These results were similar to
those reported previously (Rahman et al., 2010; Silva et al., 2011).
3.6. X-ray diffraction analysis
Fig. 7 shows the results from the analysis of polymorphic
behavior of bulk materials and SLN and their diffraction patterns
after 1 and 24 months of storage.
Bulk glyceryl behenate and pure ClAlPc presented reflections
that matched the crystal structure characteristics of these
materials. Glyceryl behenate crystals have orthorhombic packing
with sharp peaks at 2u = 21.34 and 23.53 , which correspond to
short spacing at 0.42 nm and 0.38 nm, respectively. These peaks are
typical of the b’ polymorph of triglycerides (de Carvalho et al.,
2013; Souto et al., 2006). The XRD patterns of the SLN dispersions
displayed reflections (with lower intensity and width) that
resembled those of the bulk lipid. The particle size and sample
amount may account for these findings (de Carvalho et al., 2013;
Souto et al., 2006). An additional peak emerged at 2u  19.3
(0.46 nm), attributed to the b’ and bi polymorphic forms, with
triclinic and orthorhombic packing, respectively (Jenning et al.,
2000). This less ordered crystal lattice of the lipid in the
nanoparticles facilitated the integration of ClAlPc inside the
matrix. There were no specific reflections corresponding to the
drug in the XRD patterns of ClAlPc-200-SLN, suggesting that ClAlPc
was evenly incorporated into the lipid lattice. The polymorphic
behavior of SLN, with or without the drug, after 1 and 24 months
remained unchanged, as shown in the diffraction pattern of the
dispersions. Thus, the drug loading results revealed slightly
decreased values over time due to the crystal lattice that did
not change significantly during storage, which has been suggested
to prevent drug expulsion (Das et al., 2011; Kumar and Randhawa,
2013; Rahman et al., 2010). Analyses of the dispersion crystallinity
and polymorphism were crucial because these parameters
determine drug incorporation and release rates.
3.7. Differential scanning calorimetry analysis
Thermodynamic changes related to the crystallinity of the SLN
were assessed for the bulk lipid and for the SLN dispersions. Fig. 8
illustrates the calorimetric thermograms. Table 3 summarizes the
data extracted from the thermograms.
Bulk glyceryl behenate exhibited an endothermic peak at
70.9  C, which corresponded to the melting of the b’ polymorph
form (Freitas and Muller, 1999). DSC curves obtained for the
nanoparticles exhibited a less intense and broader endothermic
peak, indicating that the lipid matrix crystalized in a different
polymorphic form that was less organized than the bulk glyceryl
behenate. This was also confirmed by the lower melting temper-
atures of the nanoparticles when compared to those of the bulk
lipid. Interaction with the other components of the formulations
(surfactant blend and drug) and the small particle size of the
colloidal dispersions may have disturbed the lipid lattice and
reduced its crystallinity. Lower energy lipid forms might be
produced, resulting in lower melting temperature (Montenegro
et al., 2012; Souto et al., 2006).
 P.L. Goto et al. / International Journal of Pharmaceutics 518 (2017) 228–241
Fig. 6. Height and tridimensional AFM images obtained from (A and B) unloaded SLN and (C and D) ClAlPc-200-SLN.
Fig. 7. X-ray diffraction patterns of bulk glyceryl behenate, bulk ClAlPc, unloaded
SLN, and ClAlPc-200-SLN. For the SLN dispersions, the X-ray analyses were
performed after 1 and 24 months of storage at 4  C.
237
As seen in Table 3, the degree of crystallinity of the lipid core in
the SLN dispersions was much lower than the bulk glyceryl
behenate (de Carvalho et al., 2013; Freitas and Muller, 1999).
ClAlPc/SLN exhibited a smaller degree of crystallinity than the
unloaded SLN, suggesting that ClAlPc was incorporated into the
lipid structure. The incorporation of PS into the lipid matrix may
have increased the number of lattice defects (an increase in crystal
order disturbance), but it was not enough to modify the
polymorphic behavior of the SLN dispersions (de Carvalho et al.,
2013).
After 1 and 24 months of storage, unloaded and ClAlPc- SLN did
not differ in terms of polymorphic forms, consequently, the
crystallinity degree did not change significantly, which may have
prevented the drug from being expelled during storage (Pardeike
et al., 2009).
The DSC results agreed with the XRD data and showed that the
ClAlPc/SLN did not tend to expel the drug or to form gels. Gelation
is a typical instability phenomena reported in aqueous dispersions
of glyceryl behenate SLN during storage, resulting from changes in
the degree of crystallinity (100–130%) and polymorphic transitions
(Freitas and Muller, 1999). Together, the DSC and XRD results may
238 P.L. Goto et al. / International Journal of Pharmaceutics 518 (2017) 228–241
Fig. 8. DSC curves (heating mode) of bulk glyceryl behenate, unloaded SLN, and
ClAlPc-200-SLN analyzed after 1 and 24 months of storage at 4  C.
suggest that such instability phenomena did not occur in our
experiments.
3.8. In vitro cell experiments
3.8.1. Biocompatibility test of SLN formulations
For preparing nanoparticles as drug delivery systems, it is
essential to assess cell biocompatibility and the potential cytotoxic
effects of the formulations, to ensure nontoxicity, biocompatibility,
and safety (Doktorovova et al., 2014; Zanatta et al., 2008). The NIH-
3T3 cell line has been employed in biocompatibility tests (Liu et al.,
2012; Zanatta et al., 2008) because these cells are quite sensitive to
the irritating components of pharmaceutical and cosmetic
formulations (Maier et al., 1991). For the biocompatibility assays,
NIH-3T3 cells were incubated in culture medium containing
different dilutions of the unloaded SLN for 3 h. As a negative
control, the NIH-3T3 cells were incubated with culture medium
only, without the particles. The MTT assay was performed 24 h
after the treatment step. The viability of the NIH-3T3 cells were
Table 3
Crystallinity degree (CD) and parameters extracted from the DSC thermograms of the bulk glyceryl behenate and the SLN dispersions.
Samples Melting temperature ( C)
Bulk glyceryl behenate 70.9
Unloaded SLN – 1 month 66.9
Unloaded SLN – 24 months 66.7
ClAlPc-200-SLN – 1 month 67.0
ClAlPc-200-SLN – 24 months 66.6
Fig. 9. Cell viability, using MTT assay, of NIH-3T3 cells incubated with a screening of unloaded SLN or with the culture medium only (negative control, NC). The mean value of
cell viability is written above the columns. Statistical analysis by one-way ANOVA with Dunnett posthoc test (p < 0.05), (average  SD, n = 3).
calculated in comparison to the negative control. The results are
shown in Fig. 9.
The fibroblast culture exposed to unloaded SLN sample diluted
at concentrations of 0.10, 0.20, and 0.39% (v/v) showed a cell
viability of over 70%. According to ISO 10993-5:2009 (ISO, 2009), a
reduction in cell viability exceeding 30% is considered a cytotoxic
effect, therefore these dilutions of unloaded SLN formulations
could be seen as cell-friendly. Nevertheless, statistical analysis
revealed that only the 0.10 and 0.20% dilutions did not differ
significantly from the negative control. Therefore, unloaded SLN
formulations up to 0.20% could be considered biocompatible with
NIH-3T3 cells. The latter dilution was used in the ClAlPc colloidal
system in subsequent in vitro tests.
3.8.2. Dark cytotoxicity test of ClAlPc
For assessing the cytotoxicity of the ClAlPc, pigmented
melanoma skin cancer cells were used in the next two experi-
ments. Based on the previous biocompatibility results, the SLN
formulations (loaded or not with PS) were diluted in the culture
medium at 0.20% (v/v). Nontoxicity in dark conditions is an
essential feature for that application of PS in PDT (Bechet et al.,
2008). The toxicity test in the dark was performed in two steps. In
the first step, neoplastic cells were treated with two ClAlPc/SLN
formulations – ClAlPc-200-SLN and ClAlPc-400-SLN – containing
200 and 400 mg mL 1 of ClAlPc, respectively. These formulations
showed a final concentration of 0.31 and 0.75 mg mL 1 of ClAlPc,
respectively, after being diluted in the culture medium. As a
positive control B16-F10 cells were incubated with unloaded SLN
diluted at 0.20% (v/v). As a negative control, B16-F10 cells were
incubated only with culture medium. The results of the MTT
proliferation assay are illustrated in Fig. 10A.
The cell viability values in the positive control groups were
87.83% (8.85), in the group ClAlPc/SLN at 0.31 mg mL 1 ClAlPc
were 83,04% (5,69), and in the ClAlPc/SLN at 0.75 mg mL 1
ClAlPc were 85,57% (8,24). Statistical analysis showed that the
cell viability values for the negative and positive control groups, as
Onset( C) Melting enthalpy (J g 1
) CD (%)
68.7 120.4 100.0
62.5 50.7 84.2
62.1 526 87.3
62.7 49.2 81.7
62.1 46.1 76.6
 P.L. Goto et al. / International Journal of Pharmaceutics 518 (2017) 228–241 239

Fig. 10. Cell viability of B16-F10 cells, using MTT assay, in dark conditions, with (A) ClAlPc/SLN – ClAlPc-200-SLN and ClAlPc-400-SLN – with final concentration of 0.31 and
0.75 mg mL 1 ClAlPc, respectively; (B) free ClAlPc (0.75 mg mL 1) and ethanol control. In both cases, negative control (NC) and positive control (PC) were used. Statistical
analysis by one-way ANOVA with Tukey posthoc test (p < 0.05), (average  SD, n = 3).

well as for both ClAlPc/SLN did not differ significantly (Fig. 10A,
p > 0.05). Therefore, the results suggested that the pigmented
melanoma cells tolerated the ClAlPc/SLN regardless of the ClAlPc
concentration. The formulation with a higher concentration of
ClAlPc (0.75 mg mL 1) was chosen for further analysis in this study.
In the second step of this test, the toxicity of free ClAlPc at
0.75 mg mL 1 (diluted in culture medium from an ethanol stock
solution) was assessed as well. For excluding the potential toxicity
of the organic solvent, B16-F10 cells were incubated in culture
medium containing only ethanol; which was called the ethanol
control group. The amount of ethanol was equivalent to the
amount of the PS stock solution used in the tests. As expected
(Fig. 10B), cell viabilities in the positive control group
(90.87%  7.63), ethanol control group (99.77%  19.49),

Fig. 11. Cell viability of B16-F10 cells, using MTT assay, treated with (A) encapsulated ClAlPc (ClAlPc-400-SLN) and (B) free ClAlPc (ClAlPc-Et) dilutions (0.75 mg mL 1
ClAlPc)
for 3 h, followed by laser irradiation. Statistical analysis by one-way ANOVA with Tukey posthoc test (p < 0.05), (average  SD, n = 3).
240 P.L. Goto et al. / International Journal of Pharmaceutics 518 (2017) 228–241
B16-F10 cells treated with free ClAlPc (84.59%  10.04), and B16-
F10 cells treated with encapsulated ClAlPc (84.94%  6.48) were
not significantly different (p > 0.05). Therefore, the amount of
ethanol used in the test was not toxic to the pigmented neoplastic
cells.
Because PS was shown to be non-toxic in the B16-F10 cells
under dark conditions, the PS at a concentration of 0.75 mg mL 1
was used in subsequent analyses, which included laser light
application (PDT).
3.8.3. Photodynamic therapy efficiency
PDT kills cancer cells by generating a toxicity effect through the
combination of PS, light dose, and oxygen (Plaetzer et al., 2008).
B16-F10 melanoma cells were incubated with culture medium
containing ClAlPc-400-SLN or free ClAlPc (ClAlPc-Et), both at a
concentration of 0.75 mg mL 1 of ClAlPc, for 3 h, this condition did
not result in dark toxicity. For assessing the cellular damage caused
by ClAlPc as a function of laser irradiation, different light doses
were applied on neoplastic cells: 0.5, 1.0, and 2.0 J cm 2. Two
groups were treated with culture medium only: the negative
control group (non-irradiated) and the laser control (irradiated
with the greatest light dose used in this study). The MTT assay was
performed after 24 h of the irradiation step. Fig. 11 shows the cell
viability of the pigmented melanotic cells.
The laser control groups received 2.0 J cm 2 of irradiation. The
results in all groups were not significantly different from the
negative control (p > 0.05). The number of viable cells decreased by
only 10%, showing that the greatest light dose applied herein did
not harm the cells in the absence of the PS.
PDT efficiency was directly related to the energy applied to the
B16-F10 cells and were more effective when these cells were
treated with SLN encapsulated ClAlPc. At the lowest light dose
applied, cell viabilities after treatment with free ClAlPc and ClAlPc/
SLN decreased to 54.1% (5.0) and 64.4% (7.0), respectively,
relative to the negative control (p < 0.05). These results suggested
the pigmented melanoma cells irradiated with 0.50 J cm 2 were
more sensitive to the ClAlPc/SLN treatment (p < 0.001), which
showed a significant phototoxic effect, when compared to the free
PS treatment (p < 0.05). A two-fold increase in the laser light
radiation (1.0 J cm 2) reduced the viability of the cells exposed to
ClAlPc/SLN by more than 50% (26.2%  0.47) but did not alter the
viability of the cells exposed to free PS. Further increases in light
dose (2.0 J cm 2) reduced the viability of the B16-F10 cells treated
with ClAlPc/SLN even further: 15.1% (2.0) vs. 48.9% (19.1) in
the group incubated with free ClAlPc. ClAlPc incorporated into SLN
was clearly more efficient in reducing cell viability than free PS: the
reduction was 3.2 times higher in the ClAlPc/SLN.
Although ClAlPc has a clear cytotoxic effect on B16-F10 cells, the
high hydrophobicity of ClAlPc represents an important challenge
for PDT administration, as in most PS drugs. Therefore, the
development of nanocarriers as a PS delivery system has emerged
as a potential tool for increasing the bioavailability of hydrophobic
PS and for protecting the drug against degradation. The colloidal
system described in the current study may enhance the
phototoxicity effects of ClAlPc playing a crucial role in delivering
ClAlPc in its monomeric form to cells without impairing the PS
photodynamic properties (Bechet et al., 2008; Chatterjee et al.,
2008; Paszko et al., 2011; Zhao et al., 2013).
Furthermore, it has been suggested that the ClAlPc could
overcome competition with melanin for light during PDT. ClAlPc
belongs to the metallophthalocyanine class of second-generation
PS drugs, which have a maximum absorption wavelength between
670 and 680 nm (Siqueira-Moura et al., 2013). Therefore, the ClAlPc
could avoid the light absorption scattering caused by melanin,
which has a maximum absorption at approximately 530 nm
(Castagnos et al., 2014). In this context, the current colloidal system
described as a ClAlPc nanocarrier may be a promising tool for PDT
adjuvant therapy in combating pigmented melanoma, even in the
presence of melanin (Davids and Kleemann, 2011; Sharma and
Davids, 2012).
4. Conclusion
Using the direct emulsification method, we have successfully
prepared solid lipid nanoparticles as nanocarriers for aluminum
chloride phthalocyanine improving its bioavailability during
photodynamic therapy. Two surfactants (sorbitan isostearate
and polyoxyethylene-40 hydrogenated castor oil) and glyceryl
behenate – as the solid lipid – were chosen for preparing the
formulations. The morphological analysis agreed with the PCS
results and showed that the nanoparticles obtained had a spherical
shape and measured between 100 and 200 nm, with a low
polydispersity index (<0.3), and high zeta potential. Both long-
term and accelerated stability studies showed that the SLN
dispersions were stable over at least a 12-month storage period.
XRD and DSC analysis showed that the SLN dispersions presented a
lower degree of crystallinity suggesting that ClAlPc was incorpo-
rated into the SLN lipid matrix and it retained its physicochemical
stability after 24 months, without expelling the drug. Toxicity
studies revealed suitable biocompatibility of ClAlPc/SLN with NIH-
3T3 fibroblast cells and with B16-F10 cells under dark conditions.
ClAlPc/SLN exerted significant phototoxic effects on pigmented
melanoma cells and caused damage in a light dose-dependent
manner. Therefore, all of the results reported herein confirmed that
SLN is a potential lipid nanocarrier for the encapsulation of
hydrophobic photosensitizer drugs to be used in photodynamic
therapy protocols and in further studies on photosensitization of
neoplastic cell lines.
Acknowledgments
A.C.T. thanks the Brazilian Federal Agency for Support and
Evaluation of Graduate Education (CAPES), the State of São Paulo
Research Foundation (FAPESP) Thematic project # 2013/50181-1 &
Project FINEP 01.10.0758.01, and the National Council for Scientific
and Technological Development (CNPq) for their financial support.
P.L.G. thanks CAPES for the scholarship. We also thank the National
Institute of Science and Technology (INCT) of Nanobiotechnology
project 573880/2008-5 for their financial support.
References
Agostinis, P., Berg, K., Cengel, K.A., Foster, T.H., Girotti, A.W., Gollnick, S.O., Hahn, S.
M., Hamblin, M.R., Juzeniene, A., Kessel, D., Korbelik, M., Moan, J., Mroz, P.,
Nowis, D., Piette, J., Wilson, B.C., Golab, J., 2011. Photodynamic therapy of cancer:
an update. CA Cancer J. Clin. 61, 250–281.
Allison, R.R., Sibata, C.H., 2010. Oncologic photodynamic therapy photosensitizers: a
clinical review. Photodiagn. Photodyn. Ther. 7, 61–75.
Almeida, E.D.P., Costa, A.A., Serafini, M.R., Rossetti, F.C., Marchetti, J.M., Sarmento, V.
H.V., Nunes, R.S., Valerio, M.E.G., Araújo, A.A.S., Lira, A.A.M., 2012. Preparation
and characterization of chloroaluminum phthalocyanine-loaded solid lipid
nanoparticles by thermal analysis and powder X-ray diffraction techniques. J.
Therm. Anal. Calorim. 108, 191–196.
Anton, N., Vandamme, T.F., 2009. The universality of low-energy nano-
emulsification. Int. J. Pharm. 377, 142–147.
Anton, N., Gayet, P., Benoit, J.P., Saulnier, P., 2007. Nano-emulsions and nanocapsules
by the PIT method: an investigation on the role of the temperature cycling on
the emulsion phase inversion. Int. J. Pharm. 344, 44–52.
Anton, N., Benoit, J.P., Saulnier, P., 2008. Design and production of nanoparticles
formulated from nano-emulsion templates – a review. J. Control. Release 128,
185–199.
Babilas, P., Schreml, S., Landthaler, M., Szeimies, R.M., 2010. Photodynamic therapy
in dermatology: state-of-the-art. Photodermatol. Photoimmunol. Photomed.
26, 118–132.
Baldea, I., Filip, A.G., 2012. Photodynamic therapy in melanoma – an update. J.
Physiol. Pharmacol. 63, 109–118.
 P.L. Goto et al. / International Journal of Pharmaceutics 518 (2017) 228–241
Bechet, D., Couleaud, P., Frochot, C., Viriot, M.L., Guillemin, F., Barberi-Heyob, M.,
2008. Nanoparticles as vehicles for delivery of photodynamic therapy agents.
Trends Biotechnol. 26, 612–621.
Bhalekar, M.R., Upadhaya, P.G., Nalawade, S.D., Madgulkar, A.R., Kshirsagar, S.J.,
2015. Anti-rheumatic activity of chloroquine-SLN gel on wistar rats using
complete freund’s adjuvant (CFA) model. Indian J. Rheumatol. 10, 58–64.
Bunjes, H., 2010. Lipid nanoparticles for the delivery of poorly water-soluble drugs. J.
Pharm. Pharmacol. 62, 1637–1645.
Castagnos, P., Siqueira-Moura, M.P., Goto, P.L., Perez, E., Franceschi, S., Rico-Lattes, I.,
Tedesco, A.C., Blanzat, M., 2014. Catanionic vesicles charged with
chloroaluminium phthalocyanine for topical photodynamic therapy. In vitro
phototoxicity towards human carcinoma and melanoma cell lines. RSC Adv. 4
(39972-39377).
Chatterjee, D.K., Fong, L.S., Zhang, Y., 2008. Nanoparticles in photodynamic therapy:
an emerging paradigm. Adv. Drug Deliv. Rev. 60, 1627–1637.
Chiu, H.T., Yang, H.M., Liu, C.S., Hsu, H.Y., 2012. Synthesis, stability and properties of
polyurethane/acrylic hybrids using m-TMXDI-based anionic poly(urethane-
urea) dispersion. Polym. Plast. Technol. Eng. 51, 945–953.
Das, S., Chaudhury, A., 2010. Recent advances in lipid nanoparticle formulations
with solid matrix for oral drug delivery. AAPS PharmSciTech 12, 62–76.
Das, S., Ng, W.K., Kanaujia, P., Kim, S., Tan, R.B., 2011. Formulation design,
preparation and physicochemical characterizations of solid lipid nanoparticles
containing a hydrophobic drug: effects of process variables. Colloids Surf. B 88,
483–489.
Davids, L.M., Kleemann, B., 2011. Combating melanoma: the use of photodynamic
therapy as a novel, adjuvant therapeutic tool. Cancer Treat. Rev. 37, 465–475.
de Carvalho, S.M., Noronha, C.M., Floriani, C.L., Lino, R.C., Rocha, G., Bellettini, I.C.,
Ogliari, P.J., Barreto, P.L.M., 2013. Optimization of a-tocopherol loaded solid lipid
nanoparticles by central composite design. Ind. Crops. Prod. 49, 278–285.
Debele, T.A., Peng, S., Tsai, H.C., 2015. Drug carrier for photodynamic cancer therapy.
Int. J. Mol. Sci. 16, 22094–22136.
Detloff, T., Sobisch, T., Lerche, D., 2007. Particle size distribution by space or time
dependent extinction profiles obtained by analytical centrifugation
(concentrated systems). Powder Technol. 174, 50–55.
Doktorovova, S., Souto, E.B., Silva, A.M., 2014. Nanotoxicology applied to solid lipid
nanoparticles and nanostructured lipid carriers – a systematic review of in vitro
data. Eur. J. Pharm. Biopharm. 87, 1–18.
Ficheux, H., 2009. Principes et applications thérapeutiques de la photothérapie
dynamique. Ann. Pharm. Fr. 67, 32–40.
Freitas, C., Muller, R.H., 1999. Correlation between long-term stability of solid lipid
nanoparticles (SLN) and crystallinity of the lipid phase. Eur. J. Pharm. Biopharm.
47, 125–132.
Gandhi, S.A., Kampp, J., 2015. Skin cancer epidemiology, detection, and
management. Med. Clin. North Am. 99, 1323–1335.
Girotti, M.R., Saturno, G., Lorigan, P., Marais, R., 2014. No longer an untreatable
disease: how targeted and immunotherapies have changed the management of
melanoma patients. Mol. Oncol. 8, 1140–1158.
Gordon, R., 2013. Skin cancer: an overview of epidemiology and risk factors. Semin.
Oncol. Nurs. 29, 160–169.
Goto, P.L., Vilela, J.M.C., Andrade, M.S., Santos, O.D.H., 2012. Preparation and
characterization of polymeric nanocapsules produced by in situ polymerization
from nanoemulsions produced by direct emulsification. J. Disper. Sci. Technol.
34, 228–233.
International Organization for Standardization (ISO), 2009. ISO 10993-5:2009
Biological Evaluation of Medical Devices – Part 5: Tests for in Vitro Cytotoxicity.
http://www.iso.org/iso/catalogue_detail.htm?csnumber=36406 (Accessed 17
July 2016).
Idowu, M., Nyokong, T., 2007. Photophysical and photochemical properties of zinc
and aluminum phthalocyanines in the presence of magnetic fluid. J. Photochem.
Photobiol. A: Chem. 188, 200–206.
Ingraffea, A., 2013. Melanoma. Facial Plast. Surg. Clin. North Am. 21, 33–42.
Iyer, A.K., Khaled, G., Fang, J., Maeda, H., 2006. Exploiting the enhanced permeability
and retention effect for tumor targeting. Drug Discov. Today 11, 812–818.
Jenning, V., Schäfer-Korting, M., Gohla, S., 2000. Vitamin A-loaded solid lipid
nanoparticles for topical use: drug release properties. J. Control. Release 66,
115–126.
Kakadia, P.G., Conway, B.R., 2014. Solid lipid nanoparticles: a potential approach for
dermal drug delivery. Am. J. Pharmacol. Sci. 2, 1–7.
Kawczyk-Krupka, A., Bugaj, A.M., Latos, W., Zaremba, K., Sieron, A., 2013.
Photodynamic therapy in treatment of cutaneous and choroidal melanoma.
Photodiagn. Photodyn. Ther. 10, 503–509.
Kolk, A., Wolff, K.D., Smeets, R., Kesting, M., Hein, R., Eckert, A.W., 2014. Melanotic
and non-melanotic malignancies of the face and external ear – a review of
current treatment concepts and future options. Cancer Treat. Rev. 40, 819–837.
Kumar, S., Randhawa, J.K., 2013. Preparation and characterization of Paliperidone
loaded solid lipid nanoparticles. Colloids Surf. B 102, 562–568.
Lazareth, V., 2013. Management of non-melanoma skin cancer. Semin. Oncol. Nurs.
29, 182–194.
Li, S., Ji, Z., Zou, M.J., Nie, X., Shi, Y., Cheng, G., 2011. Preparation, characterization,
pharmacokinetics and tissue distribution of solid lipid nanoparticles loaded
with tetrandrine. AAPS PharmSciTech 12, 1011–1018.
Lippacher, A., Müller, R.H., Mäder, K., 2001. Preparation of semisolid drug carriers for
topical application based on solid lipid nanoparticles. Int. J. Pharm. 214, 9–12.
241
Liu, M., Zhang, Y., Wu, C., Xiong, S., Zhou, C., 2012. Chitosan/halloysite nanotubes
bionanocomposites: structure, mechanical properties and biocompatibility. Int.
J. Biol. Macromol. 51, 566–575.
Lobovkina, T., Jacobson, G.B., Gonzalez-Gonzalez, E., Hickerson, R.P., Leake, D.,
Kaspar, R.L., Contag, C.H., Zare, R.N., 2011. In vivo sustained release of siRNA from
solid lipid nanoparticles. ACS Nano 5, 9977–9983.
Lucky, S.S., Soo, K.C., Zhang, Y., 2015. Nanoparticles in photodynamic therapy. Chem.
Rev. 115, 1990–2042.
Müller, R.H., Mäder, K., Gohla, S., 2000. Solid lipid nanoparticles (SLN) for controlled
drug delivery – a review of the state of the art. Eur. J. Pharm. Biopharm. 50, 161–
177.
Müller, R.H., Shegokar, R., Cornelia, M.K., 2011. 20 years of lipid nanoparticles (SLN &
NLC): present state of development & industrial applications. Curr. Drug Discov.
Technol. 8, 207–227.
Maier, K., Schmitt-Landgraf, R., Siegemund, B., 1991. Development of an in vitro test
system with human skin cells for evaluation of phototoxicity. Toxicol. In Vitro 5,
457–461.
Mehnert, W., Mader, K., 2001. Solid lipid nanoparticles: production, characterization
and applications. Adv. Drug Deliv. Rev. 47, 165–196.
Monge-Fuentes, V., Muehlmann, L.A., de Azevedo, R.B., 2014. Perspectives on the
application of nanotechnology in photodynamic therapy for the treatment of
melanoma. Nano Rev. 5 (24381), 1–14.
Montenegro, L., Sarpietro, M.G., Ottimo, S., Puglisi, G., Castelli, F., 2011. Differential
scanning calorimetry studies on sunscreen loaded solid lipid nanoparticles
prepared by the phase inversion temperature method. Int. J. Pharm. 415, 301–
306.
Montenegro, L., Ottimo, S., Puglisi, G., Castelli, F., Sarpietro, M.G., 2012. Idebenone
loaded solid lipid nanoparticles interact with biomembrane models:
calorimetric evidence. Mol. Pharm. 9, 2534–2541.
Mroz, P., Huang, Y.Y., Szokalska, A., Zhiyentayev, T., Janjua, S., Nifli, A.P., Sherwood, M.
E., Ruzié, C., Borbas, K.E., Fan, D., Krayer, M., Balasubramanian, T., Yang, E., Kee, H.
L., Kirmaier, C., Diers, J.R., Bocian, D.F., Holten, D., Lindsey, J.S., Hamblin, M.R.,
2010. Stable synthetic bacteriochlorins overcome the resistance of melanoma to
photodynamic therapy. FASEB J. 24, 3160–3170.
Mundra, V., Li, W., Mahato, R.I., 2015. Nanoparticle-mediated drug delivery for
treating melanoma. Nanomedicine 10, 2613–2633.
Narayanan, D.L., Saladi, R.N., Fox, J.L., 2010. Review: ultraviolet radiation and skin
cancer. Int. J. Dermatol. 49, 978–986.
Negi, J.S., Chattopadhyay, P., Sharma, A.K., Ram, V., 2013. Development of solid lipid
nanoparticles (SLNs) of lopinavir using hot self nano-emulsification (SNE)
technique. Eur. J. Pharm. Sci. 48, 231–239.
Negi, L.M., Jaggi, M., Talegaonkar, S., 2014. Development of protocol for screening
the formulation components and the assessment of common quality problems
of nano-structured lipid carriers. Int. J. Pharm. 461, 403–410.
Pardeike, J., Hommoss, A., Muller, R.H., 2009. Lipid nanoparticles (SLN, NLC) in
cosmetic and pharmaceutical dermal products. Int. J. Pharm. 366, 170–184.
Paszko, E., Ehrhardt, C., Senge, M.O., Kelleher, D.P., Reynolds, J.V., 2011. Nanodrug
applications in photodynamic therapy. Photodiagn. Photodyn. Ther. 8, 14–29.
Plaetzer, K., Krammer, B., Berlanda, J., Berr, F., Kiesslich, T., 2008. Photophysics and
photochemistry of photodynamic therapy: fundamental aspects. Lasers Med.
Sci. 24, 259–268.
Rahman, Z., Zidan, A.S., Khan, M.A., 2010. Non-destructive methods of
characterization of risperidone solid lipid nanoparticles. Eur. J. Pharm.
Biopharm. 76, 127–137.
Sharma, K.V., Davids, L.M., 2012. Depigmentation in melanomas increases the
efficacy of hypericin-mediated photodynamic-induced cell death. Photodiagn.
Photodyn. Ther. 9, 156–163.
Silva, A.C., González-Mira, E., García, M.L., Egea, M.A., Fonseca, J., Silva, R., Santos, D.,
Souto, E.B., Ferreira, D., 2011. Preparation, characterization and biocompatibility
studies on risperidone-loaded solid lipid nanoparticles (SLN): high pressure
homogenization versus ultrasound. Colloids Surf. B 86, 158–165.
Simões, M.C.F., Sousa, J.J.S., Pais, A.A.C.C., 2015. Skin cancer and new treatment
perspectives: a review. Cancer Lett. 357, 8–42.
Siqueira-Moura, M.P., Primo, F.L., Espreafico, E.M., Tedesco, A.C., 2013. Development,
characterization, and photocytotoxicity assessment on human melanoma of
chloroaluminum phthalocyanine nanocapsules. Mater. Sci. Eng. C Mater. Biol.
Appl. 33, 1744–1752.
Souto, E.B., Mehnert, W., Müller, R.H., 2006. Polymorphic behaviour of
Compritol1888 ATO as bulk lipid and as SLN and NLC. J. Microencapsul. 23, 417–
433.
Sparsa, A., Bellaton, S., Naves, T., Jauberteau, M., Bonnetblanc, J., Sol, V., Verdier, M.,
Ratinaud, M., 2013. Photodynamic treatment induces cell death by apoptosis or
autophagy depending on the melanin content in two B16 melanoma cell lines.
Oncol. Rep. 29, 1196–1200.
Vera, R.E., Lamberti, M.J., Rivarola, V.A., RumieVittar, N.B., 2015. Developing
strategies to predict photodynamic therapy outcome: the role of melanoma
microenvironment. Tumor Biol. 36, 9127–9136.
Zanatta, C.F., Ugartondo, V., Mitjans, M., Rocha-Filho, P.A., Vinardell, M.P., 2008. Low
cytotoxicity of creams and lotions formulated with Buriti oil (Mauritia flexuosa)
assessed by the neutral red release test. Food Chem. Toxicol. 46, 2776–2781.
Zhao, L., Liu, J., Zhang, L., Gao, Y., Zhang, Z., Luan, Y., 2013. Self-assembly properties,
aggregation behavior and prospective application for sustained drug delivery of
a drug-participating catanionic system. Int. J. Pharm. 452, 108–115.