Liposomes containing 3-arylamino-nor-β-lapachone derivative: Development, characterization, and in vitro evaluation of the cytotoxic activity

Article abstract of DOI:10.1016/j.jddst.2021.102348

This study aimed to encapsulate the novel synthetic naphthoquinone ENSJ39 in liposomes, characterize them, and evaluate their in vitro cytotoxic activity in different cancer cell lines. Liposomes were obtained by the dried-lipid film hydration method, and

Full text of DOI:10.1016/j.jddst.2021.102348

Journal of Drug Delivery Science and Technology 62 (2021) 102348
Contents lists available at ScienceDirect
Journal of Drug Delivery Science and Technology
journal homepage: www.elsevier.com/locate/jddst
Research paper
Liposomes containing 3-arylamino-nor-β-lapachone derivative:
Development, characterization, and in vitro evaluation of the
cytotoxic activity
a
a
a
a
´
Luciana V. Rebouças , Fatima C.E. Oliveira , Daniel P. Pinheiro , Maria Francilene S. Silva ,
Vanessa Pinheiro G. Ferreirab, Roberto Nicoleteb, Augusto C.A. Oliveiraa, Renata G. Almeidac,
c
d
d,
Eufranio N. da Silva Júnior , Marcia S. Rizzo , Marcília P. Costa *, Guilherme Zocoloe,
ˆ
f
f
a
´
Fabio O.S. Ribeiro , Durcilene A. da Silva , Claudia Pessoa
a
´
Laboratory of Experimental Oncology, Department of Physiology and Pharmacology, Federal University of Ceara, 60430-275, Fortaleza, CE, Brazil
b
˜
´
´
Fundaçao Oswaldo Cruz Ceara (FIOCRUZ-CE), 61760-000, Eusebio-CE, Brazil, Post-graduate Program Northeast Biotechnology Network (RENORBIO) Itaperi
Campus 60, 714-903, Fortaleza, CE, Brazil
c Institute of Exact Sciences, Department of Chemistry, Federal University of Minas Gerais, 31275-013, Belo Horizonte, MG, Brazil
d Interdisciplinary Laboratory for Advanced Materials, Federal University of Piauí, 64049-550, Teresina, PI, Brazil
e Embrapa Tropical Agroindustry, 60511-110, Fortaleza, CE, Brazil
f Biodiversity and Biotechnology Research Center, Biotec, Federal University of Piauí, 64202-020, Parnaíba, PI, Brazil
A R T I C L E I N F O
A B S T R A C T
Keywords:
This study aimed to encapsulate the novel synthetic naphthoquinone ENSJ39 in liposomes, characterize them,
and evaluate their in vitro cytotoxic activity in different cancer cell lines. Liposomes were obtained by the dried-
lipid film hydration method, and characterizations included thermogravimetric analysis, differential scanning
calorimetry, dynamic light scattering technique, and atomic force microscopy. Simulation of storage conditions,
in vitro cytotoxicity by MTT, Trypan blue exclusion assay, and evaluation of morphological changes in tumor cell
lines were also analyzed. Liposomes containing ENSJ39 (LE39) exhibited an average size of 31 ± 5.81 nm, zeta
potential of +53.5 mV, polydispersity index of 0.24, and high encapsulation efficiency (96.58 ± 0.09%). They
were thermally stable up to 250 ◦C and able to preserve the mass loss of the free drug. After four months of
storage, they maintained excellent physical-chemical characteristics. The encapsulated drug showed less cyto-
toxic activity than the free drug only in HCT-116 and SNB-19 cells and caused cytoplasmic vacuolization plus an
increase in the number of non-viable HCT-116 cells. ENSJ39 encapsulation method was effective in obtaining
stable liposomes that presented substantial cytotoxicity activity against tumor cell lines.
Naphthoquinone
Drug delivery
Nanoencapsulation
Anticancer activity
1. Introduction
the incidence of cancer and death is generally even higher [4].
Despite the considerable arsenal of drugs already available for cancer
Cancer is a worldwide public health problem, characterized by un-
controlled cell proliferation [1]. According to the World Health Orga-
nization (WHO), over 18 million new cancer cases were reported in
2018, and the most prevalent were lung (11.6%), breast (11.6%), col-
orectum (10.2%), and prostate (7.1%) [2]. International estimates from
studies in more than 100 countries around the world indicate cancer as
the first or second leading cause of premature death (in people aged
30–69). These studies predict 29 million cases by 2040, due to global
aging and population growth [3]. In low- and middle-income countries,
treatment, in many cases, therapeutic success is not achieved due to
failures in therapeutic schemes as a result of resistance, reduced patient
survival, high rates of relapse, reduced tumor selectivity, low thera-
peutic efficacy, adverse effects, and unsustainable costs [5]. Thus,
extensive research and investment are focused on developing increas-
ingly potent and less toxic compounds.
Various studies have demonstrated the biological potential of
naphthoquinones. This class of compounds presents microbicidal, try-
panosomicidal, molluscicidal, anti-inflammatory, leishmanicidal,
* Corresponding author. Federal University of Piauí, College of Pharmacy, Health Sciences Center, Piauí, Teresina, Brazil, Ininga, 64049-550,
E-mail address: marciliapc@ufpi.edu.br (M.P. Costa).
https://doi.org/10.1016/j.jddst.2021.102348
Received 27 August 2020; Received in revised form 29 December 2020; Accepted 11 January 2021
Available online 19 January 2021
1773-2247/© 2021 Elsevier B.V. All rights reserved.

L.V. Rebouças et al.
Journal of Drug Delivery Science and Technology 62 (2021) 102348
virucidal, and antivenom properties [6–15]. The antitumor potential of
quinoidal compounds is also widely known and well-studied [16–21].
The antitumor ability of naphthoquinones is generally determined by
their structural framework, in particular their redox system consisting of
carbonyl groups, which is an essential redox center capable of gener-
ating reactive oxygen species (ROS). The generation of ROS is intrinsi-
cally related to the antitumor properties of quinonoid compounds. The
reduction process can be catalyzed biologically through one- or two-
electron reducing enzymes. NADPH-cytochrome P450 reductase and
NAD(P)H quinone oxidoreductase 1 (NQO1) are examples of enzymes
that can act as redox agents [16,22]. The reduction processes are
responsible for generating elevated amounts of ROS, resulting in the
stimulation of oxidative stress and alkylation of cellular nucleophiles
(including DNA, lipids, protein, and other biomolecules). These effects
are described as mechanisms of cytotoxicity, leading to cell damage [23,
24].
research group as ENSJ39, was synthesized from lapachol as previously
described in the literature [16,19] (Fig. 1). Soybean phosphatidylcho-
line (PC), cholesterol (CH), stearylamine (SA), trehalose, and Trypan
blue dye were purchased from Sigma-Aldrich (Missouri, USA). Aceto-
nitrile, chloroform, methanol, and dimethyl sulfoxide (DMSO) were
obtained from Merck (Darmstadt, Germany). All chemicals were of
analytical grade. Fetal bovine serum and Dulbecco’s modified eagle
˜
medium (DMEM) were purchased from Cultilab (Sao Paulo, Brazil).
RPMI 1640 medium, trypsin-EDTA, penicillin, and streptomycin were
purchased from GIBCO (California, USA). Doxorubicin (Doxolem) was
ˆ
purchased from Zodiac Produtos Farmaceuticos S/A (Brazil). The Quick
´
Panoptic kit was obtained from Laborclin (Parana, Brazil).
2.2. Development of liposomal formulations
ENSJ39 liposomes (LE39) were prepared by the dried-lipid film hy-
dration method followed by sonication, as previously reported [45].
Briefly, PC, CH, and SA (7:2:1 M ratio) were dissolved in a mixture of
chloroform and methanol (3:1 v/v) under magnetic agitation. ENSJ39
(1:28.48 drug/lipid molar ratio) was then added to the organic lipidic
phase. Unloaded liposome (LBR) was synthesized in the same way
without the drug (ENSJ39).
A survey of publications in the last 20 years on PubMed and Web of
Science databases, using the descriptors ‘naphthoquinone and cancer’,
revealed a total of 3236 articles, indicating that the number of papers
has quintupled since early 1999. The number of publications related to
this chemical class demonstrates the growing interest in its pharma-
cology and mechanism of action in oncology.
Among naphthoquinones reported in literature, the compound 2,2-
dimethyl-3-((2-methyl-4-nitrophenyl)amino)-2,3-dihydronaphtho(1,2-
b)furan-4,5-dione (ENSJ39), a nor-β-lapachone derivative, demon-
A dried-lipid film was obtained through evaporation of organic sol-
vents under reduced pressure at 38 ± 1 ◦C for 30 min, rotation of 80 rpm,
using an IKA RV10 Rotary Evaporator (Wilmington, USA). The resulting
lipid layer was hydrated with 10 mL of phosphate buffer solution (PBS)
(pH 7.4) with or without trehalose (0.1, 5, and 10%), to obtain multi-
lamellar liposomes in suspension (0.6 mg/mL of ENSJ39). Then, small
unilamellar vesicles were obtained by sonication using a Qsonic probe
(Newtown, USA), operating in pulsate mode with a potency of 125 W
and a frequency of 20 kHz for 300 s (5 s on/2 s off). Finally, LE39 and
LBR were frozen overnight and freeze-dried (MicroModulyo Freeze
Dryer; Thermo, USA) in 200 bars for 24 h and stored at 8 ◦C. Both for-
mulations were also stored at 8 ◦C to be monitored after 24 h of
preparation.
strated potent cytotoxic activity displaying IC50 values less than 2 μM
against several cancer cell lines such as HL-60, MDA-MB-435, HCT-8,
HCT-116, and SF-295 [16,19,25]. This compound induced apoptosis
(mitochondrial pathway) and intracellular ROS generation in HL-60
cells, as well as DNA strand breaks in LNCap (NQO1-) and DU-145
(NQO1+) cells [20,26]. However, at the same time, ENSJ39 has poor
solubility, which makes in vivo administration difficult, interfering with
subsequent studies.
Moreover, previous studies with similar naphthoquinones have
shown that certain compounds from this class are responsible for in vivo
toxic effects, including hemolytic anemia and renal tubular necrosis in
animals [27–31]. Solubility problems have also been reported [27,32,
33].
2.3. Measurement of particle characteristics
Drug delivery strategies to improve bioavailability and reduce
toxicity have been applied to naphthoquinoidal compounds [34–38].
The incorporation of nanotechnology has proved quite promising
compared to traditional medicine. This approach allows the develop-
ment of encapsulated drugs with reduced off-target toxicity, increasing
the concentration of the drug in a particular target, improving the
therapeutic efficacy, and also addressing problems related to the phys-
icochemical properties of the non-encapsulated substance, such as sol-
ubility and stability [39,40].
The particle size, size distribution, and zeta potential of LE39 and
LBR diluted (1:100 w/v) were determined by Malvern Zetasizer Nano-
◦
ZS90 (Worcestershire, UK) at 25 C scattering angle of 90◦. Liposome
samples were diluted with ultrapure water and sonicated before mea-
surement, as required, for a satisfactory particle count. The distribution
and the mean diameter of particles were evaluated as well as their
standard deviation and polydispersity index (PDI). Data were obtained
by averaging ten measurements.
The number of particles per mL of lyophilized liposomes (LE39 and
LBR) were investigated in NanoSight NS500 equipment (Marven, UK).
The LE39 and LBR liposome suspensions, previously diluted 1: 40,000 in
purified water, were injected into the visualization camera. Three
different batches of each sample were used, and the results were
expressed as average.
Liposomes can be used to deliver low molecular weight drugs, large
proteins, and even therapeutic nucleic acid sequences. Constituted by a
bilayer lipid vesicle between 50 nm and 5 μm in size, the similarity of
liposomes to biological membranes endows them with particular prop-
erties, such as biocompatibility and biodegradability. Therefore, lipo-
somes are highly versatile nanostructures for encapsulation and delivery
of bioactive agents in nanomedicine applications [41–44].
Particle morphological analysis was performed using an Atomic
Force Microscopy (AFM) as previously described [46]. Briefly, 10 μL of
Due to the importance of naphthoquinones in the progress of phar-
macological studies focusing on cancer treatment and knowing the ad-
vantages of using liposomes, the present study aimed to evaluate the in
vitro physical-chemical and biological characteristics of liposomes
containing the new synthetic naphthoquinone ENSJ39.
diluted samples (1:10 v/v) was sonicated for 15 min, spread onto freshly
cleaned mica disks, and dried for around 15 min at 36 ◦C. LE39 and LBR
were analyzed using the AFM Workshop TT-AFM microscopy (Califor-
nia, USA). The images were taken in tapping mode using a silicon
cantilever (TAP 300-G10, TED PELLA) with a resonance frequency of
approximately 238 kHz. The images were processed using Gwyddion
2.45 software.
2. Material and METHODS
Thermal analysis of ENSJ39 and lyophilized liposomes (LE39 and
LBR) were investigated by Thermogravimetric Analysis (TGA) and Dif-
ferential Scanning Calorimetry (DSC) performed, respectively, in Perkin
Elmer STA 6000 (Massachusetts, USA) and Shimadzu DSC-60 (Kyoto,
Japan). Measurements were performed at a temperature range from 25
2.1. Material
The chemical compound 2,2-dimethyl-3-((2-methyl-4-nitrophenyl)
amino)-2,3- dihydronaphtho(1,2-b)furan-4,5-dione, named by our
2

L.V. Rebouças et al.
Journal of Drug Delivery Science and Technology 62 (2021) 102348
Fig. 1. Synthetic route to obtain naphthoquinone ENSJ39 [19].
to 400 ◦C (TGA) at a heating rate of 10 ◦C/min under nitrogen flow (50
mL/min) by using a closed aluminum pan, in which approximately 10
mg of the sample was placed. Thermograms obtained by DSC of
lyophilized liposomes and pure drug were performed at a temperature
range from 25 to 240 ◦C at a heating rate of 10 ◦C/min under a dynamic
nitrogen atmosphere using a closed aluminum pan, in which approxi-
mately 10 mg of the sample was placed.
colorimetric MTT (3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyl tetrazo-
lium bromide)) assay, as described by Mosmann [47], with modifica-
tions. Cell lines were maintained in flasks containing RPMI 1640 or
DMEM medium supplemented with 10% fetal bovine serum, 100 U/mL
penicillin, and 100
atmosphere.
μ
g/mL streptomycin at 37 ◦C and 5% CO2
ENSJ39 and liposomes were diluted in culture medium in a serial
dilution of 0.03–3.78
and incubated for 72 h, at the same conditions described above. Doxo-
rubicin (0.04–2.72 g/mL) was used as a positive control. After 72 h
incubation, 100 L of MTT solution (0.5 mg/mL) was added to each
well, and cells were incubated for 3 h. Then, the supernatant was
removed, 100 L of DMSO was added to solubilize the formazan crystals,
μg/mL, added to the cells seeded in 96-well plates
2.4. Encapsulation efficiency
μ
μ
Drug content was determined using the Genesys 10S UV–Vis spec-
trophotometer (Thermo, Massachusetts, USA) at 256 nm. The calibra-
tion curve of ENSJ39 in acetonitrile was accomplished using six
μ
and the absorbance was measured using a microplate spectrophotom-
eter (DTX800 Multimode Detector, Beckman Coulter, California, USA)
at 595 nm. The absorbances obtained were used to calculate the IC50
values by nonlinear regression employing appropriate statistical soft-
ware. Statistical significance was calculated by Analysis of Variance
(ANOVA) followed by Tukey’s test, with a significance level of p < 0.05.
All treatments were performed in triplicate in at least three independent
experiments.
standard solutions in the range of 2–50 μg/mL prepared from a stock
solution (0.2 mg/mL). Then, samples of LE39 and LBR were quantified
as a total fraction and purified liposomal fraction.
To determine the total drug content in the formulations, liposomes
were diluted (1:10 w/v) in acetonitrile, sonicated for 15 min, filtered
through a PTFE membrane (0.22 μm), and analyzed by spectropho-
tometry. To separate the non-encapsulated drug, LE39 was preliminarily
filtered through an Ultrafree® centrifugal filter (Millipore, USA). The
free drug in the supernatant was measured after centrifugation of the
sample at 5000 rpm for 2 h. Then, it was diluted (1:10 w/v) in aceto-
nitrile, sonicated for 15 min, filtered through a PTFE membrane (0.22
2.7. Trypan blue exclusion assay
μ
m), and analyzed by spectrophotometry. Assays were performed in
HCT-116 tumor cells were chosen for this assay and the morpho-
logical analysis described below (topic 2.8), because this cell line has
been used in previous studies carried out by our group to elucidate the
mechanisms of action for ENSJ39 (Unpublished data) [25].
triplicate.
The absorbance value of LE39 was subtracted from the absorbance
value of LBR in each procedure and then used in the equation of the
calibration curve to obtain concentrations of drugs in total and purified
liposomal fraction. Next, the encapsulation efficiency (EE) was calcu-
lated according to Equation (1).
Trypan blue exclusion assay was performed as described by Strober
[48], with modifications. This assay is based on the principle that living
cells possess intact cell membranes that exclude certain dyes, such as
Trypan blue, whereas dead cells do not.
concentration of drug in purified liposom al fraction (
μ
g/m L )
HCT-116 cells were seeded in six-well plates at a seeding density of 5
EE (%) =
× 100
concentration of drug in total fraction (
μg/m L )
× 104 cells/well and treated with ENSJ39 and LE39 (considering the
encapsulation efficiency) at concentrations of 2.32
μ
g/mL and 3.48
μ
g/
g/
(1)
mL. Untreated cells represented negative control, and doxorubicin 1
μ
mL was used as a positive control. After 72 h of treatment, cells were
harvested, centrifuged for 5 min at 1500 rpm, and resuspended in 1 mL
2.5. Evaluation of stability
of PBS. Then, 90 μL of the cell suspension was mixed with 10 μL of 0.4%
Liposomes stability was evaluated using long-term stability testing,
as described by Cavalcanti [38], with modifications. Particle size, zeta
potential, and polydispersity index (PDI) of the diluted liposomal for-
mulations (1:100 w/v) were monitored after 24 h of preparation and
during storage, at predetermined time intervals (1–4 months) at room
temperature (25 ◦C) and under refrigeration (8 ◦C). For long-term sta-
bility evaluation, LE39 and LBR were diluted in purified water at 1/100
(w/v). Measurements were performed using a Malvern Zetasizer
Nano-ZS90 (Worcestershire, UK).
Trypan blue dye for counting viable and non-viable cells in a Neubauer
chamber under an optical microscope (Nikon Eclipse E100, Tokyo,
Japan). Assays were performed in triplicate in at least three independent
experiments.
Data were calculated from mean ± standard deviation (SD) and
compared by Analysis of Variance (ANOVA) followed by Tukey’s test,
with a significance level of p < 0.05, using appropriate statistical
software.
2.8. Morphological analysis of HCT-116 cells
2.6. Cytotoxicity assay
The morphological analysis allowed evaluation of possible modifi-
cations related to cell death patterns after treatment with the tested
samples. HCT-116 cells were seeded in 24-well plates, with circular
coverslips at the bottom of each well, at a seeding density of 5 × 104
cells/well. The next day, cells were treated with ENSJ39 and LE39
The cytotoxicity of ENSJ39 and liposomes were evaluated against
HCT-116 (7 × 104 cells/well), SNB-19 (1 × 105 cell/well), HL-60 (3 ×
105 cells/well), PC-3 (1 × 105 cell/well), B16F10 (7 × 104 cells/well),
and normal murine fibroblast L929 (7 × 104 cells/well) cells by the
3

L.V. Rebouças et al.
Journal of Drug Delivery Science and Technology 62 (2021) 102348
(considering the encapsulation efficiency) at concentrations of 2.32
μ
g/
agglomerates for both ENSJ39-loaded and unloaded liposomes (Fig. 3).
The liposomes were flattened and slightly deformed, probably due to the
adsorption on the mica surface, which can interfere with the average
diameter of particles [58].
mL and 3.48 μg/mL. Untreated cells represented negative control.
After 72 h, the coverslips were removed and stained with the Quick
Panoptic kit, which contains 0.1% triarylmethane solution 1 (fixative),
0.1% xanthene solution 2 (cytoplasm dye), and 0.1% thiazine solution 3
(nucleus dye). After staining, cells were evaluated under an optical
microscope (Nikon Eclipse E200, Tokyo, Japan) coupled to a camera.
The 2D phase image in Fig. 3 illustrates two regions of the liposome:
a central nucleus (yellow arrow) surrounded by a membrane (white
arrow). This confirmed that the formulated nanoparticles had appro-
priate liposomal morphological characteristics compatible with a uni-
lamellar vesicle structure [59].
3. Results and DISCUSSION
The lipid membrane was thicker in LE39 than in LBR (Fig. 3a and b,
white arrow), suggesting that ENSJ39, a lipophilic compound, was
incorporated within the liposomal phospholipid bilayer. This may
contribute to greater stability and compacting of liposomes. A similar
phenomenon occurs when cholesterol is used in the preparation of li-
posomes. Voluminous steroid rings of cholesterol are interspersed
among the phospholipid bilayer and provide rigidity and greater sta-
bility to the nanoparticle [60,61].
3.1. Characterization of LE39 and LBR
Liposomes prepared by the dried-lipid film hydration method ob-
tained satisfactory results, such as small particle size, high zeta potential
values, and low PDI values. LE39 exhibits an average diameter of 31 ±
5.81 nm, the zeta potential of +53.5 ± 1.41 mV, and PDI of 0.24 ± 0.01.
Similar results were obtained for LBR liposomes: particle size of 33 ±
7.71 nm, the zeta potential of +58.3 ± 2.04, and PDI of 0.23 ± 0.02.
LE39 and LBR liposome suspensions contained around 1380 and 579
billion particles/mL, respectively.
In the thermal analysis of ENSJ39 and liposomes, the TGA curve
(Fig. 4a) indicated that LE39 and LBR are thermally stable up to 228 ◦C,
while ENSJ39 degrades at 25 ◦C. This degradation was preserved in the
liposomes. The mass loss for LE39 was lower than that for ENSJ39 (free
drug) up to 328 ◦C. Above this temperature, the degradation of the free
drug remained stable. Rapamycin and paclitaxel were also protected
from degradation when encapsulated in liposomes [61]. Likewise, the
thermogravimetric evaluation of liposomes containing β-lapachone
showed that the encapsulation system prevented degradation as
occurred in free drug [38].
The particle size obtained for liposomes in the present study is within
the definition of lipid nanoparticles [43,49]. Zeta potential values were
close to +60 mV, which are considered optimal and indicate colloidal
stability due to strong electrostatic repulsions between particles and
decreased tendency to aggregate [50,51]. PDI values were less than
0.25, indicating a very narrow distribution, resulting in a homogeneous
system [49,51], which was confirmed by the particle size distribution of
LE39 (Fig. 2a).
The DSC thermogram (Fig. 4b) displays endothermic peaks
compatible with the melting of the material (crystalline to amorphous
state) in ENSJ39, LE39, and LBR samples at 112 ◦C, 164 ◦C, and 177 ◦C,
respectively. The melting peaks of LE39 and LBR showed similar aspects.
The first one suffered a slight broadening at its base and presented in-
termediate melting temperature compared to free drug and unloaded
liposomes.
Processing techniques have been reported in the literature which
ensure that those characteristics are maintained and the half-life of the
formulation increases. The use of freeze-drying is an alternative pro-
cedure, as the dry state has greater stability and allows the formulation
to be reconstituted with a vehicle at the time of administration [53].
During this process, cryoprotectants are commonly employed to avoid
ice crystals that could damage the liposomes. In lipid nanoformulations,
the use of trehalose, glucose, sucrose, or mannitol can form a protective
glassy matrix in the nanoparticles [43,54,55].
A double melting peak in ENSJ39 may indicate polymorphism,
which means the presence of different crystalline morphologies
(Fig. 4b). The disappearance of this peak in the DSC thermogram of LE39
may denote the absence of the drug crystalline state, suggesting that the
drug was entrapped and molecularly dispersed in an amorphous state
into the liposome [35,62,63]. That may have contributed to improving
the solubility of the compound, as amorphous drugs are more soluble
than crystalline ones, resulting in higher bioavailability [62,64]. The
freeze-dried sample of LE39 was easily dispersed in an aqueous vehicle
as opposed to the free drug, which requires an organic solvent like
DMSO.
We evaluated the particle size, zeta potential, and PDI in freeze-dried
formulations. Trehalose at concentrations of 5% and 10% was effective
in maintaining the fundamental characteristics of LE39 (Fig. 2), allow-
ing the preservation and stability of the nanostructures. Trehalose is an
excellent cryoprotectant for the lyophilization process of liposomal
nanosystems [43,56], and its use has already been reported in the
literature, with concentrations ranging from 0.1 to 10% [38,57].
Concerning the morphological aspects, AFM images of the liposomes
showed regular spherical shape, smooth surface, and absence of
Fig. 2. Characterization of LE39 liposomes. Values of particle size and zeta potential (a), and polydispersity index (PDI) (b) of the LE39 suspension and freeze-dried
with three different trehalose concentrations (0.1%, 5%, and 10%).
4

L.V. Rebouças et al.
Journal of Drug Delivery Science and Technology 62 (2021) 102348
Fig. 3. AFM images of liposomes containing ENSJ39 (LE39) (a) and unloaded liposome (LBR) (b). White arrows indicate the membrane of phospholipids, and yellow
arrows indicate the central nucleus. (For interpretation of the references to color in this figure legend, the reader is referred to the Web version of this article.)
3.2. Encapsulation efficiency
lipid molar ratio and PC:CH:SA molar ratio as here. β-lapachone lipo-
somes were resistant to accelerated stability tests and maintained their
initial properties even after 60 days, in suspension form, and up to 1
year, in the freeze-dried form [38].
The data obtained from the analytical calibration curve of ENSJ39
yielded the following linear regression equation (r2 = 0.9984): absor-
bance = 0.0308 x ENSJ39 concentration (μg/mL) + 0.0161. The total
drug content in the liposomes was 82.3% (±0.04). Following this
equation, concentrations of the drug in purified fraction and total lipo-
somal fraction were calculated and applied to Equation (1), resulting in
96.58% (±0.09) of encapsulation efficiency.
3.4. In vitro cytotoxicity
The IC50 values, obtained after 72 h treatment, for ENSJ39 and LE39
are presented in Table 1, along with data obtained for doxorubicin
(positive control).
This method of determining the encapsulation efficiency by UV/
visible spectrophotometry has also been applied in other studies
involving different liposomal nanoformulations [57,65], including li-
posomes containing naphthoquinones [38,66]. The remarkable encap-
sulation ratio of ENSJ39 is in agreement with other efficiency values of
β-lapachone into liposomes, using the same lipid film method and
similar preparation conditions as LE39: 97.09% [38] and 97.4% [66].
The extent of encapsulation efficiency in liposomal systems is generally
higher for non-polar drugs than for polar drugs [59], which corroborates
the rate obtained for LE39, as ENSJ39 is a highly non-polar compound.
Naphthoquinone drugs appear to have better rates when encapsu-
lated in liposomal nanosystems. Previous data showed an encapsulation
efficiency of 41.9% for β-lapachone in PLA-PEG micelles [67], and its
encapsulation in PLGA-microcapsules had a trapping rate of 19.36%
[35].
The IC50 values for ENSJ39 did not undergo significant changes be-
tween tumor cell lines, exhibiting cytotoxic activity for all of them, with
higher cytotoxicity for HL-60 cells. Previous studies for ENSJ39 and
other arylamine analogs revealed potent cytotoxic activity, with some
compounds displaying IC50 values less than 1 μM against the tumor cell
lines HL-60, MDA-MB-435, HCT-8, SF-295, PC-3, and B-16 [16,19,20,
26].
A previous study of the mechanisms of action for ENSJ39 [25] sug-
gested its bioactivation by the NQO1 enzyme (Unpublished data). As
observed in Table 1, ENSJ39 exhibited potent activity in HL-60 tumor
cells, even though this cell line is reported to have reduced expression of
NQO1 [68]. This result corroborates with previous data, in which ary-
lamino-nor-β-lapachone analogs had considerable activity against the
same leukemia cell line, also similar to the effect observed with the
precursor molecule (nor-β-lapachone) [20,26]; thus suggesting that
ENSJ39 bioactivation can be induced by other reductases, in addition to
NQO1. No bioreductive antitumor agent is activated by a single reduc-
tive enzyme [69]. Moreover, some experiments using dicoumarol
(NQO1 inhibitor) and naphthoquinone drugs have shown that despite
the protection of tumor cells overexpressing NQO1, no cell lines have
exhibited significantly reduced expression of this enzyme, as HL-60 cells
[70,71]. In any case, even though studies on the cytotoxicity of ENSJ39
have been conducted, this compound is new; therefore, its exact
mechanism is still being unraveled.
3.3. Stability evaluation of liposomes
◦
ENSJ39 liposomes (LE39) stored at room temperature (25 C) and
◦
under refrigeration (8 C) did not exhibit drug precipitation and lipid
flocculation when evaluated 24 h after preparation (Fig. 5). During the
evaluation period of four months, a small precipitate of easy resus-
pension was observed after one month of storage in both conditions
(Fig. 5).
After four months of storage, zeta potential values decreased (Fig. 6);
however, at 8 ◦C, they remained within a range considered stable (˃ ±
30 mV) [52]. Furthermore, particle size, size distribution, and PDI
values of LE39 stored at 8 ◦C were similar to those in the initial condition
(Fig. 6), suggesting this is a better storage option than 25 ◦C.
These results are in agreement with a similar study of β-lapachone
encapsulated in liposomes by the lipid film method using the same drug:
There was a statistical difference in cytotoxicity between LE39 and
ENSJ39 for HCT-116 and SNB-19 cells (Fig. 7). The encapsulated drug
showed less cytotoxic activity than the unencapsulated one on those
tumor cell lines.
In any case, according to a cytotoxic activity classification reported
´
by Perez-Sacau et al. [72] and Costa et al. [35], both ENSJ39 and LE39
5

L.V. Rebouças et al.
Journal of Drug Delivery Science and Technology 62 (2021) 102348
Fig. 4. Thermogravimetric Analysis (TGA) (a) and Differential Scanning Calorimetry (DSC) (b), ENSJ39 drug, liposomes containing ENSJ39 (LE39), and unloaded
liposome (LBR) curves.
were highly active (IC50 < 1
μ
g/mL) to HL-60 cells and moderately
generate changes in the absorption patterns of the encapsulated drug
and, consequently, influence the bioavailability and the magnitude of its
cytotoxic effects on cells [57,77].
active (1 g/mL < IC50 < 10
μ
μg/mL) to the other tested cell lines. The
cytotoxicity was also evaluated in empty liposomes (LBR), and it did not
demonstrate significative proliferative inhibition (data not shown).
In agreement with our results, other comparative studies evaluating
cytotoxicity of free drugs and their encapsulated form showed differ-
ences in toxicity. The evaluation of free and liposome-encapsulated
doxorubicin showed that cytotoxicity and uptake of the free drug were
higher than in the nanoformulation [73,74]. Stealth liposomes of
isoniazid and rifampicin also had less cytotoxicity than free drugs, even
though both systems had the same cellular uptake [75].
Liposomes have benefits related to a passive delivery in the tumor
environment, which ensures that a higher concentration of drugs rea-
ches the focus of action and with a longer release time [41–43]. More-
over, although a free drug has effective in vitro cellular activity, at in vivo
level, there is no guarantee that large concentrations of the drug will be
able to reach the tumor site. Free doxorubicin, for example, which has
suitable in vitro cellular uptake, has difficulty reaching the tumor site in
vivo, as it disappears by rapid opsonization and is absorbed by the
reticular endothelial system of the liver and spleen [73].
In drug delivery systems, such as liposomes, less cytotoxicity can
occur due to slower and more controlled release of the drug from lipo-
somes compared to free drug, considering the same incubation time [34,
73,74,76]. Furthermore, liposomes can interact with target cells in
different ways depending on their lipid composition, steric stabilization,
particle size, surface charge, and other aspects. Those parameters
The ENSJ39 and LE39 were also analyzed by the Trypan Blue
exclusion assay in HCT-116 cells. Both caused a reduction in the number
of viable cells at 2.32 and 3.48 μg/mL, similar to the positive control
(doxorubicin) (Fig. 8). When evaluating the number of non-viable cells,
significant differences were observed in free and encapsulated ENSJ39
6

L.V. Rebouças et al.
Journal of Drug Delivery Science and Technology 62 (2021) 102348
Fig. 5. LE39 subjected to stability evaluation at 25 ◦C (a) and 8 ◦C (b) for four months. Yellow arrows indicate precipitation. (For interpretation of the references to
color in this figure legend, the reader is referred to the Web version of this article.)
Fig. 6. Stability evaluation of LE39 liposomes. Evaluation of particle size, zeta potential (a), and PDI (b) of LE39 suspension after 24 h of preparation, four months
later at 25 ◦C and 8 ◦C.
Table 1
Cytotoxic activity of ENSJ39, LE39 and Doxorubicin (DOX), as a positive control, evaluated by the MTT assay after 72 h of exposure for different cell lines.
Samples
IC50
(μ
g/mL) (CI 95%)a
HCT-116
PC-3
SNB-19
HL-60
B16F10
L929
ENSJ39
LE39
1.16 (1.08–1.25)
4.46 (3.41–5.83)
0.11 (0.09–0.16)
1.33 (1.19–1.48)
1.53 (1.14–2.05)
0.41 (0.32–0.51)
1.61 (1.40–1.85)
3.98 (3.50–4.52)
1.20 (1.03–1.39)
0.23 (0.20–0.27)
0.58 (0.53–0.64)
0.02 (0.01–0.02)
1.59 (1.52–1.65)
1.68 (1.11–3.31)
0.73 (0.61–0.88)
2.12 (1.92–2.34)
2.53 (2.28–2.80)
0.93 (0.86–1.02)
DOX
a
IC50 values with 95% confidence intervals (CI) were obtained by non-linear regression from at least three independent experiments.
compared to controls. However, no significant differences were
observed between concentrations.
in the number of cells per field, intense cytoplasmic loss, membrane
integrity loss (black arrows), reduction of cell volume, and nuclear
pyknosis (white arrows) (Fig. 9). The cellular damage suggests an
apoptotic process induced by ENSJ39, described by Pinheiro [25] and
Cavalcanti [26] as reduced cell viability and increased number of cells
with apoptotic characteristics (reduced cell volume, peripheral chro-
matin condensation, and apoptotic bodies) associated with reduced
The results obtained in the Trypan Blue assay and the MTT test were
in agreement for the morphological disorders caused in HCT-116 cells
after treatment with ENJ39 and LE39 for 72 h, in both tested concen-
trations (Fig. 9). While the negative control exhibited intact cells with
well-defined cytoplasm and nucleus, ENSJ39 caused a notable decrease
7

L.V. Rebouças et al.
Journal of Drug Delivery Science and Technology 62 (2021) 102348
Fig. 7. IC50 values for free and encapsulated ENSJ39 (LE39) after 72 h treatment in different cell lines. Data are presented as mean ± SD from three independent
experiments performed in triplicate. a = p < 0.05 compared to ENSJ39 treatment by ANOVA followed by Tukey’s test.
Fig. 8. Effect of ENSJ39 and LE39 on the viability
of HCT-116 cells determined by the Trypan Blue
exclusion assay after 72 h of treatment. Data are
presented as mean ± SD from three independent
experiments performed in triplicate. a = p < 0.05
compared to viable cells in the negative control; b
= p < 0.05 compared to non-viable cells in the
negative control; c = p < 0.05 compared to non-
viable cells in the positive control by ANOVA fol-
lowed by Tukey’s test. (For interpretation of the
references to color in this figure legend, the reader
is referred to the Web version of this article.)
membrane integrity.
the drug from degradation. LE39 formulation was capable of encapsu-
lating above 80% of ENSJ39 and can be quickly dispersed in an aqueous
vehicle, unlike the free drug, improving the solubility of ENSJ39. In vitro
cytotoxicity studies indicated that the free drug had higher activity in
HCT-116 and SNB-19 cells, but ENSJ39 and LE39 had similar cytotoxic
activity against other tumor cell lines exhibiting the most potent activity
in HL-60 tumor cells. Morphological features compatible with an
apoptotic cell death process were determined by optical microscopy.
Therefore, those results support that ENSJ39 liposomes are viable and
have a potential application in future in vivo studies, such as xenograft
tumor models. However, only in vivo studies can prove the potential of
these liposomes as a promising drug delivery system for antitumor
therapy.
HCT-116 cells treated with LE39 displayed cell swelling and intense
cytoplasmic vacuolization (red arrows), probably indicating cellular
uptake of the liposomes. Mitotic events were found after LE39 treatment
and negative control (green arrows), but not for the free drug. Cellular
damage caused by the tested compounds was more intense at the highest
concentration (3.48 μg/mL).
4. Conclusion
In this study, ENSJ39 liposomes (LE39) were developed using the
dried-lipid film hydration method. LE39 formulation was characterized
as a homogeneous system, stable after a four-month storage period, due
to the high zeta potential values obtained. Thermal analysis, including
DSC and TGA, indicated the successful coating of liposomes with
ENSJ39 in an amorphous state, and the nanosystem was able to protect
8

L.V. Rebouças et al.
Journal of Drug Delivery Science and Technology 62 (2021) 102348
Fig. 9. Morphological analysis of HCT-
116 cells after 72 h of treatment with
different concentrations of ENSJ39 and
LE39. (a) The negative control received
no treatment; (b) ENSJ39 2.32
(c) ENSJ39 3.48
g/mL (e); and LE39 3.48
μ
g/mL;
g/mL; (d), LE39 2.32
g/mL. Green
μ
μ
μ
arrows indicate mitotic events, black
arrows indicate cytoplasmic loss, yellow
arrows indicate nuclear pyknosis, and
red arrows indicate vacuoles. (For
interpretation of the references to color
in this figure legend, the reader is
referred to the Web version of this
article.)
Author contributions
Júnior, Guilherme Zocolo, Durcilene A. da Silva and Claudia Pessoa;
Supervision: Claudia Pessoa.
Author contributions Conceptualization: Claudia Pessoa; Formal
´
analysis and investigation: Luciana V. Rebouças, Fatima C. E. Oliveira,
Declaration of competing interest
The authors declare that they have no conflict of interest.
Acknowledgments
´
and Daniel P. Pinheiro; Methodology: Fatima C. E. Oliveira, Augusto C.
ˆ
A. Oliveira, Renata G. Almeida, Eufranio N. da Silva Júnior, Marcia S.
´
Rizzo, Marcília P. Costa, Guilherme Zocolo, Fabio O. S. Ribeiro, Durci-
lene A. da Silva, Maria F. S. Silva, Vanessa P. G. Ferreira and Roberto
Nicolete; Writing – original draft preparation: Luciana V. Rebouças;
ˆ
Writing – review and editing: Daniel P. Pinheiro, Eufranio N. da Silva
We thank the National Cancer Institute (Bethesda, MD, USA) for the
donation of tumor cell lines used in this study and thank the Brazilian
Agricultural Research Corporation, called EMBRAPA (Fortaleza, CE,
Júnior, Marcília P. Costa, Guilherme Zocolo, Vanessa P. G. Ferreira and
ˆ
Roberto Nicolete; Resources: Claudia Pessoa, Eufranio N. da Silva
9

L.V. Rebouças et al.
Journal of Drug Delivery Science and Technology 62 (2021) 102348
C. Pessoa, Preclinical genotoxicology of nor-β-lapachone in human cultured
lymphocytes and Chinese hamster lung fibroblasts, Chem. Res. Toxicol. 24 (9)
(2011) 1560–1574, https://doi.org/10.1021/tx200180y 10.1021/tx200180y.
[18] E.N. da Silva Júnior, M.C.B. de Souza, A.V. Pinto, F.R. Maria do Carmo, M.
O. Goulart, F.W. Barros, C. Pessoa, L.V. Costa-Lotufo, R.C. Montenegro, M.O. de
Moraes, V.F. Ferreira, Synthesis and potent antitumor activity of new arylamino
Brazil), for collaboration in necessary analytical measurements.
Appendix A. Supplementary data
Supplementary data to this article can be found online at https://doi.
org/10.1016/j.jddst.2021.102348.
derivatives of nor-β-lapachone and nor-α-lapachone, Bioorg. Med. Chem. 15 (22)
(2007) 7035–7041, https://doi.org/10.1016/j.bmc.200 7.07.043.
[19] E.N. da Silva Júnior, C.F. de Deus, B.C. Cavalcanti, C. Pessoa, L.V. Costa-Lotufo,
R. Montenegro, M.O. de Moraes, M.C.F.R. Pinto, C.A. de Simone, V.F. Ferreira, M.
O.F. Goulart, C.K.Z. Andrade, A.V.C. Pinto, 3-Arylamino and 3-alkoxy-nor-
β-lapachone derivatives: synthesis and cytotoxicity against cancer cell lines,
J. Med. Chem. 53 (1) (2010) 504–508, https://doi.org/10.1021/jm900865m.
[20] B.C. Cavalcanti, I.O. Cabral, F.A.R. Rodrigues, F.W.A. Barros, D.D. Rocha, H.I.
Funding information
The authors are grateful to the Brazilian National Council of Scien-
tific and Technological Development (Conselho Nacional de Desenvol-
˜
F. Magalhaes, D.J. Moura, J. Saffi, J.A.P. Henriques, T.S.C. Carvalho, M.O. Moraes,
´
C. Pessoa, I.M.M. Melo, E.N. da Silva Júnior, Potent antileukemic action of
naphthoquinoidal compounds: evidence for an intrinsic death mechanism based on
oxidative stress and inhibition of DNA repair, J. Braz. Chem. Soc. 24 (1) (2013)
145–163, https://doi.org/10.1590/S0103-50532013000100019.
vimento Científico e Tecnologico-CNPq) for the financial support
granted to C. Pessoa (PQ-1B, Process nº: 303,102/2013-6).
[21] E.H.G. Cruz, C.M.B. Hussene, G.C. Dias, E.B.T. Diogo, I.M.M. Melo, B.L. Rodrigues,
M.G. Silva, W.O. Valença, C.A. Camara, R.N. Oliveira, Y.G. Paiva, M.O.F. Goulart,
B.C. Cavalcanti, C. Pessoa, E.N. da Silva Junior, 1,2,3-Triazole-, arylamino- and
thio-substituted1,4-naphthoquinones: potent antitumor activity,
References
[1] D. Hanahan, R.A. Weinberg, Hallmarks of cancer: the next generation, Cell 144
(2011) 646–674, https://doi.org/10.1016/j.cell.2011.02.013.
electrochemicalaspects, and bioisosteric replacement of C-ring-modified
lapachones, ioorg. Med. Chem. 22 (5) (2014) 1608–1619, https://doi.org/
10.1016/j.bmc.2014.01.033.
[2] World Health Organization (WHO), Cancer fact sheets. https://gco.iarc.fr/today/
data/factsheets/cancers/39-All-cancers-fact-sheet.pdf, 2020. (Accessed 29 April
2020).
[22] J.L. Bolton, M.A. Trush, T.M. Penning, G. Dryhurst, T.J. Monks, Role of quinones in
toxicology, Chem. Res. Toxicol. 13 (3) (2000) 135–160, https://doi.org/10.1021/
tx9902082.
[3] A. Jemal, L. Torre, I. Soerjomataram, F. Bray, The Cancer Atlas, American Cancer
Society, Atlanta, 2019.
[4] World Health Organization (WHO), Key statistics. https://www.who.int/cancer/
resources/keyfacts/en/, 2020. (Accessed 29 April 2020).
[23] J.L. Bolton, T. Dunlap, Formation and biological targets of quinones: cytotoxic
versus cytoprotective effects, Chem. Res. Toxicol. 30 (1) (2017) 13–37, https://doi.
org/10.1021/acs.chemrestox.6b00256.
[5] H. Maeda, M. Khatami, Analyses of repeated failures in cancer therapy for solid
tumors: poor tumor-selective drug delivery, low therapeutic efficacy and
unsustainable costs, Clin. Transl. Med. 7 (11) (2018) 1–20, https://doi.org/
10.1186/s40169-018-0185-6.
[24] Y. Kumagai, Y. Shinkai, T. Miura, A.K. Cho, The chemical biology of
naphthoquinones and its environmental implications, An. Ver. Pharmacol. Toxicol.
52 (2012) 221–247, https://doi.org/10.1146/annurev-pharmtox-010611-134517.
[25] D.P. Pinheiro, Evaluation of the Cytotoxic Potential and Molecular Mechanism of
Synthetic Naphthoquinones ENSJ39 and ENSJ108: in Vitro and in Vivo Studies.
[6] A.L. Lourenço, P.A. Abreu, B. Leal, E.N. da Silva Júnior, A.V. Pinto, M.C. Pinto, A.
M. Souza, J.S. Novais, M.B. Paiva, L.M. Cabral, C.R. Rodrigues, V.F. Ferreira, H.
C. Castro, Identification of nor-beta-lapachone derivatives as potential
antibacterial compounds against Enterococcus faecalis clinical strain, Curr.
Microbiol. 62 (v) (2011) 684–689, https://doi.org/10.1007/s00284-010-9763-6.
[7] C.N. Pinto, A.P. Dantas, K.C. de Moura, F.S. Emery, P.F. Polequevitch, M.C. Pinto,
S.L. de Castro, A.V. Pinto, Chemical reactivity studies with naphthoquinones from
Tabebuia with anti-trypanosomal efficacy, Arzneim Forsch/Drug Res. 50 (12)
(2000) 1120–1128, https://doi.org/10.1055/s-0031-1300337.
´
´
Thesis (Doctoral), Federal University of Ceara, 2017. Ceara/Brazil, http://www.
repositorio.ufc.br/bitstream/riufc/26907/1/2017_tese_dppinheiro.pdf. (Accessed
1 April 2020), 2020.
˜
[26] B.C. Cavalcanti, H. Magalhaes, F. Rodrigues, M.O. Moraes, C. Pessoa, Arylamino-
nor-β-lapachone derivative-induced apoptosis in human prostate cancer cells:
involvement of NAD(P)H:quinone oxidoreductase (NQO1), BMC Proc. 7 (Suppl 2)
(2013) 12, https://doi.org/10.1186/1753-6561-7-S2-P12.
[8] T.P. Barbosa, C.A. Camara, T.M.S. Silva, R.M. Martins, A.C. Pinto, M.D. Vargas,
New 1,2,3,4-tetrahydro-1-aza-anthraquinones and 2-aminoalkyl compounds from
norlapachol with molluscicidal activity, Bioorg. Med. Chem. 13 (23) (2005)
6464–6469, https://doi.org/10.1016/j.bmc.2005.06.068.
[27] V.S. de Sena Pereira, C.B. Silva de Oliveira, F. Fumagalli, F. da Silva Emery, N.B. da
Silva, V.F. de Andrade Neto, Cytotoxicity, hemolysis and in vivo acute toxicity of 2-
hydroxy-3-anilino-1,4-naphthoquinone derivatives, Toxicol. Rep. 3 (2016)
756–762, https://doi.org/10.1016/j.toxrep.2016.09.007.
[9] E.R. Almeida, A.A. da Silva-Filho, E.R. dos Santos, C.A. Lopes, Antiinflammatory
action of lapachol, J. Ethnopharmacol. 29 (2) (1990) 239–241.
[28] B.K. Aithal BK, M.R.S. Kumar, B.N. Rao, R. Upadhya, V. Prabhu, G. Shavi,
K. Arumugam, S.P. Sajankila, N. Udupa, K. Satyamoorthy, B.S.S. Rao, Evaluation of
pharmacokinetic, biodistribution, pharmacodynamic, and toxicity profile of free
juglone and its sterically stabilized liposomes, J. Pharmaceut. Sci. 100 (8) (2011)
3517–3528, https://doi.org/10.1002/jps.22573.
ˆ
[10] M.M. Sitonio, C.H. Carvalho-Júnior, A. Campos-Ide, J.B. Silva, M.C. Lima, A.
´
J. Goes, M.B. Maia, P.J. Rolim-Neto, T.G. Silva, Anti-inflammatory and anti-
arthritic activities of 3,4-dihydro-2,2-dimethyl-2H-naphthol[1,2-b]pyran-5,6-dione
(β-lapachone), Inflamm. Res. 62 (1) (2013) 107–113, https://doi.org/10.1007/
s00011-012-0557-0.
[29] R. Munday, B.L. Smith, C.M. Munday, Comparative toxicity of 2-hydroxy-3-alkyl-
1,4-naphthoquinones in rats, Chem. Biol. Interact. 98 (2) (1995) 185–192, https://
doi.org/10.1016/0009-2797(95)03645-8.
˜
[11] T.T. Guimaraes, M.C.F.R. Pinto, J.S. Lanza, M.N. Melo, R.L. Monte-Neto, I.M.
M. Melo, E.B.T. Diogo, V.F. Ferreira, C.A. Camara, W.O. Valença, R.N. Oliveira,
[30] R. Munday, B.L. Smith, C.M. Munday, Effect of inducers of DT-diaphorase on the
haemolyticactivity and nephrotoxicity of 2-amino-1,4-naphthoquinone in rats,
Chem. Biol. Interact. 155 (3) (2005) 140–147, https://doi.org/10.1016/j.
cbi.2005.06.001.
´
F. Frezard, E.N. Silva-Júnior, Potent naphthoquinones against antimony-sensitive
and -resistant Leishmania parasites: synthesis of novel α- and nor-α-lapachone-
based 1,2,3-triazoles by copper-catalyzed azide–alkyne cycloaddition, Eur. J. Med.
Chem. 63 (2013) 523–530, https://doi.org/10.1016/j.ejmech.2013.02.038.
[31] R. Munday, B.L. Smith, C.M. Munday, Structure-activity relationships in the
haemolytic activityand nephrotoxicity of derivatives of 1,2- and 1,4-
naphthoquinone, J. Appl. Toxicol. 27 (3) (2007) 262–269, https://doi.org/
10.1002/jat.1206.
´
˜
[12] S.L.S. Gomes, G.C.G. Militao, A.M. Costa, C. O Pessoa, L.V. Costa-Lotufo, E.
F. Cunha-Junior, E.C. Torres-Santos, P.R.R. Costa, A.J.M. Silva, Suzuki-miyaura
coupling between 3-Iodolawsone and arylboronic acids. Synthesis of lapachol
analogues with antineoplastic and antileishmanial activities, J. Braz. Chem. Soc. 28
(8) (2017) 1573–1584, https://doi.org/10.21577/0103-5053.20160326.
[13] T.M. Ngoc, N.T.T. Phuong, N.M. Khoi, S. Park, H.J. Kwak, N.X. Nhiem, B.T.
T. Trang, B.H. Tai, J.H. Song, H.J. Ko, S.H. Kim, A new naphthoquinone analogue
and antiviral constituents from the root of Rhinacanthus nasutus, Nat. Prod. Res. 33
(3) (2019) 360–366, https://doi.org/10.1080/14786419.2018.1452004.
[32] R.Y. Yang, D. Kizer, H. Wu, E. Volckova, X.S. Miao, S.M. Ali, M. Tandon, R.
E. Savage, T.C. Chan, M.A. Ashwell, Synthetic methods for the preparation of ARQ
501 (β–lapachone) humam blood metabolites, Bioorg. Med. Chem. 16 (10) (2008)
5635–5643, https://doi.org/10.1016/j.bmc.2008.03.073.
[33] F.C. de Abreu, D.C.M. Ferreira, M.O.F. Goulart, O. Buriez, C. Amarote,
Electrochemical activation of β-lapachone in β-cyclodextrin inclusion complexes
and reactivity of its reduced form towards oxygen in aqueous solutinons,
J. Electroanal. Chem. 608 (2) (2007) 125–132, https://doi.org/10.1016/j.
jelechem.20 07.05.020.
˜
[14] M.A. Strauch, M.A. Tomaz, M. Monteiro-Machado, B.L. Cons, F.C. Patrao-Neto, J.
M. Teixeira-Cruz, M.S. Tavares-Henriques, P.D. Nogueira-Souza, S.L.S. Gomes, P.R.
R. Costa, E. Schaeffer, A.J.M. Silva, P.A. Melo, Lapachol and synthetic derivatives:
in vitro and in vivo activities against Bothrops snake venoms, PloS One 14 (1)
(2019) 1–18, https://doi.org/10.1371/journal.pone.0211229.
[34] K.H. Kim, T. Le, H.K. Oh, B. Heo, J. Moon, S. Shin, S.H. Jeong, Protective
microencapsulation of β-lapachone using porous glass membrane technique based
on experimental optimisation, J. Microencapsul. 34 (6) (2017) 545–559, https://
doi.org/10.1080/02652048.2017.1367850.
˜
[15] V.C. Roa-Linares, Y. Miranda-Brand, V. Tangarife-Castano, R. Ochoa, P.A. García,
´
M.A. Castro, L. Betancur-Galvis, A. San Feliciano, Anti-herpetic, anti-dengue and
antineoplastic activities of simple and heterocycle-fused derivatives of terpenyl-
1,4-naphthoquinone and 1,4-anthraquinone, Molecules 24 (7) (2019) 1–17,
https://doi.org/10.3390/molecules24071279.
[35] M.P. Costa, A.C.S. Feitosa, F.C.E. Oliveira, B.C. Cavalcanti, G.C. Dias, E.W.
S. Caetano, F.A.M. Sales, V.N. Freire, S.D. Fiore, R. Fischer, L.O. Ladeira, E.N. da
Silva Júnior, C. Pessoa, Encapsulation of nor-β-lapachone into poly(D,L)-lactide-co-
glycolide (PLGA) microcapsules: full characterization, computational details and
cytotoxic activity against human cancer cell lines, Med. Chem. Commun. 8 (10)
(2017) 1993–2002, https://doi.org/10.1039/C4MD00371C.
[16] E.N. da Silva Júnior, G.A.M. Jardim, C. Jacob, U. Dhawa, L. Ackermann, S.L. de
Castro, Synthesis of quinones with highlighted biological applications: a critical
update on the strategies towards bioactive compounds with emphasis on
lapachones, Eur. J. Med. Chem. 179 (2019) 863–915, https://doi.org/10.1016/j.
ejmech.2019.06.056.
[36] M.P. Costa, A.C. Feitosa, F.C. Oliveira, B.C. Cavalcanti, E.M. da Silva, G.G. Dias, F.
A. Sales, B.L. Sousa, I.L. Barroso-Neto, C. Pessoa, E.W. Caetano, S. Di Fiore,
R. Fischer, L.O. Ladeira, V.N. Freire, Controlled release of nor-β-lapachone by plga
˜
[17] B.C. Cavalcanti, F.W. Barros, I.O. Cabral, J.R. Ferreira, H.I. Magalhaes, H.V. Júnior,
E.N. da Silva Júnior, F.C. de Abreu, C.O. Costa, M.O. Goulart, M.O. Moraes,
10

L.V. Rebouças et al.
microparticles: a strategy for improving cytotoxicity against prostate cancer cells,
Journal of Drug Delivery Science and Technology 62 (2021) 102348
liposome bilayers, Mol. Pharm. 10 (11) (2013) 4281–4293, https://doi.org/
10.1021/mp400362v.
Molecules 21 (7) (2016) 873, https://doi.org/10.3390/molecules21070873.
[37] T. Arasoglu, B. Mansuroglu, S. Derman, B. Gumus, B. Kocyigit, T. Acar,
I. Kocacaliskan, Enhancement of antifungal activity of juglone (5-hydroxy-1,4-
naphthoquinone) using a poly(d,l-lactic-co-glycolic acid) (PLGA) nanoparticle
system, J. Agric. Food Chem. 64 (38) (2016) 7087–7094, https://doi.org/10.1021/
acs.jafc.6b03309.
[59] V. Nekkanti, S. Kalepu, Recent advances in liposomal drug delivery: a review,
Pharm. Nanotechnol. 3 (1) (2015) 35–55, https://doi.org/10.2174/
2211738503666150709173905.
[60] C. Demetzos, Differential scanning calorimetry (DSC): a tool to study the thermal
behavior of lipid bilayers and liposomal stability, J. Liposome Res. 18 (3) (2008)
159–173, https://doi.org/10.1080/08982100802310261.
[38] I.M. Cavalcanti, E.A. Mendonça, M.C. Lira, S.B. Honrato, C.A. Camara, R.
V. Amorim, J. Mendes-Filho, M.M. Rabello, M.Z. Hernandes, A.P. Ayala, N.
[61] J.O. Eloy, R. Petrilli, J.F. Topan, H. Antonio, J. Barcellos, D.L. Chesca, L.N. Serafini,
D.G. Tiezzi, R.J. Lee, J.M. Marchetti, Co-loaded paclitaxel/rapamycin liposomes:
development, characterization and in vitro and in vivo evaluation for breast cancer
therapy, Colloids Surf. B Biointerfaces 141 (2016) 74–82, https://doi.org/
10.1016/j.colsurfb.2016.01.032.
˜
S. Santos-Magalhaes, The encapsulation of β-lapachone in 2-hydroxypropyl-
β-cyclodextrin inclusion complex into liposomes: a physicochemical evaluation and
molecular modeling approach, Eur. J. Pharmaceut. Sci. 44 (3) (2011) 332–340,
https://doi.org/10.1016/j.ejps.2011.08.011.
[39] F. Farjadian, A. Ghasemi, O. Gohari, A. Roointan, M. Karimi, M.R. Hamblin,
Nanopharmaceuticals and nanomedicines currently on the market: challenges and
opportunities, Nanomedicine 14 (1) (2019) 93–126, https://doi.org/10.2217/
nnm-2018-0120.
[62] V. Sanna, A.M. Roggio, S. Siliani, M. Piccinini, S. Marceddu, A. Mariani, M. Sechi,
Development of novel cationic chitosan-and anionic alginate-coated poly (d, l-
lactideco-glycolide) nanoparticles for controlled release and light protection of
resveratrol, Int. J. Nanomed. 7 (2012) 5501–5516, https://doi.org/10.2147/IJN.
S36684.
[40] J. Hare, T. Lammers, M.B. Ashford, S. Puri, G. Storm, S.T. Barry, Challenges and
strategies in anti-cancer nanomedicine development: an industry perspective, Adv.
Drug Deliv. Rev. 108 (2017) 25–38, https://doi.org/10.1016/j.addr.2016.04.025.
[41] J.O. Eloy, R. Petrilli, F.S.G. Praça, M. Chorillic, Recent advances with targeted
liposomes for drug delivery, in: V. Torchilin (Ed.), Handbook of Materials for
Nanomedicine: Lipid-Based and Inorganic Nanomaterials, Jenny Stanford
Publishing, New York, 2020, pp. 85–122.
[63] A.S. Zidan, O.A. Sammour, M.A. Hammad, N.A. Megrab, M.D. Hussain, M. Khan,
M.J. Habib, Formulation of anastrozole microparticles as biodegradable anticancer
drug carriers, AAPS PharmSciTech 7 (2006) E1–E9, https://doi.org/10.1208/
pt070361.
[64] J.O. Eloy, J. Saraiva, S. de Albuquerque, J.M.M. Marchetti, Solid dispersion of
ursolic acid in gelucire 50/13: a strategy to enhance drug release and trypanocidal
activity, AAPS PharmSciTech 13 (2012) 1436–1445, https://doi.org/10.1208/
s12249-012-9868-2.
[42] Y. Panahi, M. Farshbaf, M. Mohammadhosseini, M. Mirahadi, R. Khalilov,
S. Saghfi, A. Akbarzadeh, Recent advances on liposomal nanoparticles: synthesis,
characterization and biomedical applications. Artif Cells Nanomed, Biotechnol. 45
(4) (2017) 788–799, https://doi.org/10.1080/21691401.2017.1282496.
[43] J. Kalra, M.B. Bally, Lipossomes, in: W.P. Cheng, A. Lalatsa (Eds.), Uchegbu IF,
˜
[65] L.C. Gayoso, A.I. Morenoa, H.M.L. Rolima, R.M. Freitasa, N.S. Santos-Magalhaes,
Antidepressant-like activity of liposomal formulation containingnimodipine
treatment in the tail suspension test, forced swim test and MAOB activity in mice,
Brain Res. J. 1646 (1) (2016) 235–240, https://doi.org/10.1016/j.
¨
Schatzlein AG, Springer Fundamentals of Pharmaceutical Nanoscience, Springer
Science, New York, 2013, pp. 27–63.
brainres.2016.06.004 10.1016/j.brainres.2016.06.004.
[44] G. Bozzuto, A. Molinari, Liposomes as nanomedical devices, Int. J. Nanomed. 10
(2015) 975–999, https://doi.org/10.2147/IJN.S68861.
[66] I.M.F. Cavalcanti, T.G.C. Menezes, L.A.A. Campos, M.S.F. Ferraz, M.A.V. Maciel, M.
˜
N.P. Caetano, N.S. Santos-Magalhaes, Interaction study between vancomycin and
[45] D. Lasic, Liposomes, Am. Sci. 80 (1) (1992) 20–31. www.jstor.org/stable
/29774555.
liposomes containing natural compounds against methicillin-resistant
Staphylococcus aureus clinical isolates, Braz. J. Pharm. Sci. 54 (2) (2018) 1–8,
https://doi.org/10.1590/s2175-97902018000200203.
[46] F.M. Sombra, A.R. Richter, A.R. de Araújo, F.O.S. Ribeiro, J.F.S. Mendes, R.O.
S. Fontenelle, D.A. da Silva, A.C.B. de Paula, J.P.A. Feitosa, F.M. Goycoolea, R.C.
M. de Paula, Nanocapsules of Sterculia striata acetylated polysaccharide as a
potential monomeric amphotericin B delivery matrix, Int. J. Biol. Macromol. 130
(1) (2019) 655–663, https://doi.org/10.1016/j.ijbiomac.2019.02.076.
[47] T. Mosmann, Rapid colorimetric assay for cellular growth and survival: application
to proliferation and cytotoxicity assays, J. Immunol. Methods 65 (1) (1983) 55–63,
https://doi.org/10.1016/0022-1759(83)90303-4.
[67] E. Blanco, E.A. Bey, Y. Dong, B.D. Weinberg, D.M. Sutton, D.A. Boothman, J. Gao,
β-Lapachone-containing PEG–PLA polymer micelles as novel nanotherapeutics
against NQO1-overexpressing tumor cells, J. Contr. Release 122 (3) (2007)
365–374, https://doi.org/10.1016/j.jconrel.2007.04.014.
´
´
´
[68] R.I. Bello, C. Gomez-Díaz, G. Lopez-Lluch, N. Forthoffer, M.C. Cordoba-Pedregosa,
P. Navas, J.M. Villalba, Dicoumarol relieves serum withdrawal-induced G0/1
blockade in HL-60 cells through a superoxide-dependent mechanism, Biochem.
Pharmacol. 69 (11) (2005) 1613–1625, https://doi.org/10.1016/j.
bcp.2005.03.012.
[48] W. Strober, Trypan blue exclusion test of cell viability, Curr. Protoc. Im. 111 (1)
(2015) A3B1–A3B3, https://doi.org/10.1002/0471142735.ima03bs111.
[49] Official Journal of the European Union, Commission recommendation of 18
October 2011 on the definition of nanomaterial (2011/696/EU), 2019 November
08. Available from: https://ec.europa.eu/research/industrial_tech
nologies/pdf/policy/commission-recommendation-on-the-definition-of-nanomater
18102011_en.pdf.
[69] J. Fourie, C.J. Oleschuk, F.J. Guziec, L. Guziec, D.J. Fiterman, C. Monterrosa,
A. Begleiter, The effect of functional groups on reduction and activation of quinone
bioreductive agents by DT-diaphorase, Canc. Chemother. Pharmacol. 49 (2002)
101–110, https://doi.org/10.1007/s00280-001-0395-1.
[70] E.A. Bey, M.S. Bentle, K.E. Reinicke, Y. Dong, C.R. Yang, L. Girard, J.D. Minna, W.
G. Bornmann, J. Gao, D.A. Boothman, An NQO1- and PARP-1-mediated cell death
pathway induced in non-small-cell lung cancer cells by beta-lapachone, Proc. Natl.
Acad. Sci. U.S.A. 104 (28) (2007) 11832–11837, https://doi.org/10.1073/
pnas.0702176104.
[50] V.R. Patel, Y.K. Agrawal, Nanosuspension: an approach to enhance solubility of
drugs, "J. Adv. Pharm. Technol. Research"" (JAPTR)" 2 (2) (2011) 81–87, https://
doi.org/10.4103/2231-4040.82950.
[51] H. Chen, X. Chang, D. Du, W. Liu, J. Liu, T. Weng, Y. Yang, Y. Xu, X. Yang,
Podophyllotoxin-loaded solid lipid nanoparticles for epidermal targeting, J. Contr.
Release 110 (2) (2006) 296–306, https://doi.org/10.1016/j.jconrel.2005.09.052.
[52] S. Bhattacharjee, DLS and zeta potential – what they are and what they are not?
J. Contr. Release 235 (2016) 337–351, https://doi.org/10.1016/j.
jconrel.2016.06.017.
[71] M. Ough, A. Lewis, E.A. Bey, J. Gao, J.M. Ritchie, W. Bornmann, D.A. Boothman, L.
W. Oberley, J.J. Cullen, Efficacy of beta-lapachone in pancreatic cancer treatment:
exploiting the novel, therapeutic target NQO1, Canc. Biol. Ther. 4 (1) (2005)
95–102, https://doi.org/10.4161/cbt.4.1.1382. https://doi.org/10.4161/cbt.4.1
.1382.
˜
´
˜
´
[53] C.M. Batista, C.M.B. Carvalho, N.S.S. Magalhaes, Liposomes and their therapeutic:
[72] E. Perez-Sacau, R.G. Díaz-Penate, A. Estevez-Braun, A.G. Ravelo, J.M. García-
Castellano, L. Pardo, M. Campillo, Synthesis and pharmacophore modeling of
naphthoquinone derivatives with cytotoxic activity in human promyelocytic
leukemia HL-60 cell line, J. Med. Chem. 50 (4) (2007) 696–706, https://doi.org/
10.1021/jm060849b.
state of art applications, Rev. Bras. Ciencias Farm. 43 (2) (2007) 167–179, https://
doi.org/10.1590/S1516-93322007000200003.
[54] Y. Chen, J. Chen, Y. Cheng, L. Luo, P. Zheng, Y. Tong, Z. Li, A lyophilized sterically
stabilized liposome-containing docetaxel: in vitro and in vivo evaluation,
J. Liposome Res. 27 (1) (2017) 64–73, https://doi.org/10.3109/
08982104.2016.1158185.
[73] S.H. Jung, S.H. Jung, H. Seong, S.H. Cho, K.S. Jeong, B.C. Shin, Polyethylene
glycol-complexed cationic liposomes for enhanced cellular uptake and anticancer
activity, Int. J. Pharm. 382 (1) (2009) 254–261, https://doi.org/10.1016/j.
ijpharm.2009.08.002.
[55] N. Changsan, H. Chan, F. Separovic, T. Srichana, Physicochemical characterization
and stability of rifampicin liposome dry powder formulations for Inhalation,
J. Pharmaceut. Sci. 98 (2) (2009) 628–639, https://doi.org/10.1002/jps.21441.
[56] A. Akbarzadeh, R. Rezaei-Sadabady, S. Davaran, S.W. Joo, N. Zarghami,
Y. Hanifehpour, M. Samiei, M. Kouhi, L. Nejati-Koshki, Liposome: classification,
preparation, and applications, Nanoscale Res. Lett. 8 (102) (2013) 1–9, https://doi.
org/10.1186/1556-276X-8-102.
[74] J. Wu, A. Lee, Y. Lu, R.J. Lee, Vascular targeting of doxorubicin using cationic
liposomes, Int. J. Pharm. 337 (1) (2007) 329–335, https://doi.org/10.1016/j.
ijpharm.2007.01.003.
[75] P. Deol, G.K. Khuller, Lung specific stealth liposomes: stability, biodistribution, and
toxicity of liposomal antitubercular drugs in mice, Biochim. Biophys. Acta 1334 (2)
(1997) 161–172, https://doi.org/10.1016/s0304-4165(96)00088-8.
[76] D.C. Drummond, C.O. Noble, M.E. Hayes, J.W. Park, D.B. Kirpotin,
Pharmacokinetics and in vivo drug release rates in liposomal nanocarrier
development, J. Pharmaceut. Sci. 97 (11) (2008) 4696–4740, https://doi.org/
10.1002/jps.21358.
[57] M.C.B. Lira, M.P. Siqueira-Moura, H.M.L. Rolim-Santos, F.C.S. Galetti, A.
R. Simioni, N.P. Santos, E.S.T. do Egito, S.L. Silva, A.C. Tedesco, N.S. Santos-
˜
Magalhaes, In vitro uptake and antimycobacterial activity of liposomal usnic acid
formulation, J. Liposome Res. 19 (1) (2009) 49–58, https://doi.org/10.1080/
08982100802564628.
[58] I. Onyesom, D.A. Lamprou, L. Sygellou, S.K. Owusu-Ware, M. Antonijevic, B.
Z. Chowdhry, D. Douroumis, Sirolimus encapsulated liposomes for cancer therapy:
physicochemical and mechanical characterization of sirolimus distribution within
[77] J.A. Kamps, Assessment of liposome–cell interactions, Methods Mol. Biol. 606
(2010) 199–207, https://doi.org/10.1007/978-1-60761-447-0_15.
11