European Journal of Pharmaceutical Sciences 162 (2021) 105816

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European Journal of Pharmaceutical Sciences

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

New nanotechnological formulation based on amiodarone-loaded lipid core

nanocapsules displays anticryptococcal effect
Natália Kronbauer Oliveira a, 1, Luiza Abrahão Frank b, Eamim Daidrê Squizani a,
Julia Catarina Vieira Reuwsaat a, Bárbara Machado Marques a, Heryk Motta a,
Ane Wichine Acosta Garcia a, Uriel Perin Kinskovski a, Vanessa Abreu Barcellos a,
Augusto Schrank a, d, Adriana Raffin Pohlmann b, c, Charley Christian Staats a, d,
Sílvia Stanisçuaski Guterres b, Marilene Henning Vainstein a, d, Lívia Kmetzsch a, d, *
a
Centro de Biotecnologia, Universidade Federal do Rio Grande do Sul, Porto Alegre, Brazil
b
Programa de Pós-Graduação em Ciências Farmacêuticas, Universidade Federal do Rio Grande do Sul, Porto Alegre RS, Brazil
c
Departamento de Química Orgânica, Instituto de Química, Universidade Federal do Rio Grande do Sul, Porto Alegre RS, Brazil
d
Departamento de Biologia Molecular e Biotecnologia, Universidade Federal do Rio Grande do Sul, Porto Alegre, Brazil

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

Keywords: Cryptococcus neoformans is the etiological agent of cryptococcal meningoencephalitis. The recommended avail­
Cryptococcus neoformans able treatment has low efficiency, with high toxicity and resistance as recurrent problems. In the search of new
Nanoformulation treatment protocols, the proposal of new pharmacological approaches is considered an innovative strategy,
Amiodarone
mainly nanotechnological systems considering fungal diseases. The antiarrhythmic drug amiodarone has
Fluconazole
New strategy
demonstrated antifungal activity against a range of fungi, including C. neoformans. Here, considering the
Lipid-core nanocapsules importance of calcium storage mediated by transporters on cryptococcal virulence, we evaluated the use of the
calcium channel blocker amiodarone as an alternative therapy for cryptococcosis. C. neoformans displayed high
sensitivity to amiodarone, which was also synergistic with fluconazole. Amiodarone treatment influenced some
virulence factors, interrupting the calcium-calcineurin signaling pathway. Experiments with murine cryptococ­
cosis models revealed that amiodarone treatment increased the fungal burden in the lungs, while its combination
with fluconazole did not improve treatment compared to fluconazole alone. In addition, we have developed
different innovative nanotechnological formulations, one of which combining two drugs with different mecha­
nisms of action. Lipid-core nanocapsules (LNC) loaded with amiodarone (LNCAMD), fluconazole (LNCFLU) and
both (LNCAMD+FLU) were produced to achieve a better efficacy in vivo. In an intranasal model of treatment, all the
LNC formulations had an antifungal effect. In an intraperitoneal treatment, LNCAMD showed an enhanced anti­
cryptococcal effect compared to the free drug, whereas LNCFLU or LNCAMD+FLU displayed no differences from the
free drugs. In this way, nanotechnology using amiodarone formulations could be an effective therapy for
cryptococcal infections.

1. Introduction Rajasingham et al., 2017). The number of casualties resulting from

Invasive fungal infections are the major cause of mortality in
immunocompromised individuals, particularly HIV-infected. Although
antiretroviral therapy (ART) has decreased the incidence of systemic
fungal infections in high-income countries, many low-income regions,
such as the sub-Saharan Africa, still present high rates of AIDS-related
mortality (Armstrong-James et al., 2014; Limper et al., 2017;
fungal diseases was estimated at 700,000 annually, mainly due to four
distinct infections: cryptococcal meningitis, Pneumocystis pneumonia,
histoplasmosis and aspergillosis (Denning, 2016). Cryptococcal menin­
goencephalitis is an opportunistic infection in humans caused by the
encapsulated yeast Cryptococcus neoformans; it is one of the main causes
of death in HIV patients. According to 2013 estimates, there are 223,100
cases of cryptococcal meningitis globally, resulting in 181,100 fatalities

* Corresponding author at: Av Bento Gonçalves, 9500, Bloco IV, prédio 43421, sala 222. Porto Alegre, RS, Brazil.
E-mail address: livia.kmetzsch@ufrgs.br (L. Kmetzsch).
1
Present address: Department of Microbiology and Immunology, Stony Brook University, Stony Brook, NY, USA.

https://doi.org/10.1016/j.ejps.2021.105816
Received 22 November 2020; Received in revised form 21 February 2021; Accepted 18 March 2021
Available online 21 March 2021
0928-0987/© 2021 Elsevier B.V. All rights reserved.
N.K. Oliveira et al.
(Rajasingham et al., 2017). C. neoformans cells produce virulence de­
terminants to cope with the host environment, such as a polysaccharide
capsule, melanin, phospholipase B1, and urease; all of which contribute
towards survival and dissemination during infection. These de­
terminants also endow the fungus to evade antimicrobial agents, pro­
vide antioxidant properties, and degrade membranes, promoting
propagation of fungal cells to the brain parenchyma (Emery et al., 1994;
Kwon-Chung and Rhodes, 1986; Olszewski et al., 2004; Santangelo
et al., 2004; Syme et al., 1999).
Cryptococcosis treatment is mainly based on amphotericin B and
fluconazole, however, many countries depend only on fluconazole
treatment, considering the high toxicity and prolonged hospitalization
that results in daily intravenous administration of amphotericin B
(Bicanic et al., 2015). Nevertheless, even for those receiving the rec­
ommended therapy of amphotericin B and fluconazole (flucytosine is
recommended in combination with amphotericin B, but this drug is not
available in several regions), mortality can still reach up to 70% in
low-income countries, highlighting the necessity to develop new ther­
apeutic protocols for improved cryptococcosis treatment (Mourad and
Perfect, 2018; Rajasingham et al., 2017). The control of fungal diseases
is a challenge due to the functional similarities between the fungal and
host cellular machinery, limiting the availability of specific targets for
antifungal drugs with less undesired effects (Armstrong-James et al.,
2014; Idnurm et al., 2005). Nanotechnology applied to drug delivery is
known to alter the physical-chemical properties of drugs (Frank et al.,
2015), offering an interesting therapeutic option for this type of disease.
In addition, the performance of drugs can be altered by encapsulating in
lipid core nanocapsules (Santos-Gandelman et al., 2018; Truong et al.,
2018).
Furthermore, nanotechnology system can help with drug-controlled
release and targeting, besides being considered an innovative system for
the treatment of fungal infections. Dapsone-loaded lipid-core nano­
capsules demonstrated antibacterial and antifungal activity against
isolates of Staphylococcus aureus multidrug-resistant, Aspergillus fumiga­
tus, Aspergillus flavus and Aspergillus niger (Cé et al., 2016). The main
advantages of encapsulating lipophilic drugs are: increased chemical
stability and/or photostability; increased uptake by cells; improved
interaction with tissues, leading to better drug penetration and more
efficient distribution; enhanced efficacy and/or bioavailability; and,
finally, decreased side effects (Couvreur and Vauthier, 2006; Frank
et al., 2015). For instance, the nanoencapsulation of amphotericin B, the
leading compound against fungi infection, decreased the drug’s toxicity,
resulting in higher doses administrated safely and enhanced therapeutic
efficacy (Adler-Moore and Proffitt, 1993). Polymeric nanocapsules are
formed by an oil core surrounded by a polymer wall. When the core is
formed by a dispersion of a solid lipid in an oil, the structure is named
lipid-core nanocapsules (LNC), since this nanocarrier presents two
diffusional barriers to deliver the drug encapsulated within its core
(Jäger et al., 2009). In addition, these systems can be used for drug
co-encapsulation (Frank et al., 2020).
The antiarrhythmic drug amiodarone has demonstrated antifungal
activity against a range of fungi, including C. neoformans (Courchesne,
2002). Evidence in Saccharomyces cerevisiae has shown that amiodarone
disrupts Ca2+ homeostasis, triggering an immediate dose-dependent
calcium influx, followed by continuous calcium release from the vacu­
ole. Amiodarone also caused activation of the calcineurin pathway,
leading to nuclear accumulation of the transcription factor Crz1, regu­
lating Crz1-dependent genes (Gupta et al., 2003; Zhang and Rao, 2007).
Distinct processes in C. neoformans are dependent on the
Ca2+-calcineurin signaling pathway, including virulence and growth at
37◦ C (Cruz et al., 2001; Kozubowski et al., 2011, 2009; Odom et al.,
1997). Different stress conditions lead to increased calcium levels sensed
by calmodulin, which then trigger the calcium-calcineurin signaling
pathway, ultimately resulting in the activation of the transcription factor
Crz1 and expression of calcineurin-responsive genes (Kozubowski et al.,
2009; Lev et al., 2012; Yoshimoto et al., 2002). Calcium pumps,
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European Journal of Pharmaceutical Sciences 162 (2021) 105816
channels and transporters are fundamental in the regulation of the levels
of cytosolic calcium, such as the extracellular calcium uptake channel
Cch1 (Liu et al., 2006), the sarcoplasmic/endoplasmic reticulum
Ca2+-ATPase Eca1 (Fan et al., 2007), and the vacuolar membrane pro­
teins Vcx1 and Pmc1 (Kmetzsch et al., 2013, 2010). PMC1 knockout cells
displayed decreased tolerance to calcium, reduced lung fungal burden
and the absence of brain colonization in murine models of cryptococ­
cosis (Kmetzsch et al., 2013; Squizani et al., 2018). Studies off label have
already shown that amiodarone has a synergistic effect capable of acting
on fungi already resistant to fluconazole (Gamarra et al., 2010). Amio­
darone encapsulation has already been proposed as an antiarrhythmic
drug (Ahmed et al., 2019) and fluconazole encapsulation analyzed to
treat infectious diseases (Bianchin et al., 2019). However, as far as we
know, no work in the literature proposes the use of amiodarone alone or
in combination with fluconazole for the treatment of fungal diseases.
Considering the impact of nanotechnology in the treatment of fungal
diseases, the present study evaluated the effect of an innovative strategy
based on lipid-core nanocapsules (LNC) loaded with amiodarone
(LNCAMD), fluconazole (LNCFLU) and both (LNCAMD+FLU) as anti-
cryptococcal formulations in vivo.
2. Experimental procedures
2.1. Fungal strains and media
C. neoformans serotype A H99, pmc1 mutant and pmc1::PMC1 com­
plemented strains (Kmetzsch et al., 2013) were maintained on YPD
medium plates (1% yeast extract, 2% peptone, 2% dextrose and 1.5%
agar). The media were supplemented with nourseothricin (100 µg/mL)
or hygromycin (200 µg/mL) for selective growth of pmc1 or pmc1::PMC1
cells, respectively.
2.2. Chemicals
Standard amiodarone hydrochloride (AMD; Sigma-Aldrich) and flu­
conazole (FLU; Fagron) were utilized and dissolved in dimethyl sulf­
oxide (DMSO; Sigma-Aldrich) to produce 10 mM stock solutions.
2.3. Antifungal susceptibility testing and drug interaction evaluation
The minimum inhibitory concentration (MIC) of AMD and FLU for
H99, pmc1 and pmc1::PMC1 strains were determined according to the
Clinical and Laboratory Standards Institute (CLSI) document M27-A2
with modifications (National Comitee on Clinical Laboratory Stan­
dards, 2002). Cultures were grown overnight at 30◦ C with shaking, and
cells were adjusted to achieve a final concentration of 5×102 cells/mL in
RPMI 1640 medium (Gibco Life Technologies). Plates were incubated at
37◦ C for 48 h. The OD600 was measured using a plate reader (Spec­
tramax i3, Molecular Devices). AMD-FLU interaction were evaluated by
checkerboard method, in which different ranges of drug concentrations
were combined (Afeltra et al., 2004). The MIC was defined as the lowest
drug concentration showing at least 90% growth reduction compared
with the drug-free control. Drug interaction was determined by the
previously defined FIC index, and was defined as strongly synergistic if
the FIC index was < 0.5, synergistic if the FIC index was < 1, additive if
the FIC index was = 1, no effect if the FIC index was > 1 and antagonistic
if the FIC index was > 2 (Joffe et al., 2017; Mor et al., 2015).
2.4. Phenotypic characterization assays
Cultures of H99 and pmc1 mutant cells were grown overnight in YPD
medium at 30◦ C with shaking for phenotypic characterization. Melanin
production was examined on glucose-free asparagine medium plates, as
previously reported (Kmetzsch et al., 2013), in the presence of
increasing concentrations of FLU and AMD. Capsule formation was
evaluated in cells that were cultivated for 72 h in minimal medium as
N.K. Oliveira et al.
described previously (Kmetzsch et al., 2013) with or without 3.125
µg/mL AMD (the half-MIC value for pmc1) or 0.78 µg/mL AMD and
0.125 µg/mL FLU. Evaluation of urease activity was conducted in Rob­
ert’s urea broth as detailed previously (Squizani et al., 2018) in the
presence or absence of 3.125 µg/mL AMD or 0.78 µg/mL AMD and 0.125
µg/mL FLU.
2.5. Quantitative real-time RT-PCR analysis
Cultures of H99 and pmc1 cells were grown overnight in YPD me­
dium at 30◦ C with shaking for RNA extraction. Subsequently, 5×107
cells of each strain were incubated for 10 min in RPMI 1640 medium at
37◦ C (Gibco Life Technologies) with or without 12.5 µg/mL AMD. RNA
extraction, DNase treatment and reverse transcription reactions were
performed as described (Squizani et al., 2018). Real-time PCR reactions
were performed using the StepOne Real-Time PCR System (Applied
Biosystems). PCR thermal cycling conditions included an initial step at
95◦ C for 10 min, followed by 40 cycles at 95◦ C for 15 s, 55◦ C for 15 s and
72◦ C for 1 min. PCR reactions were performed in a final volume of 20 µL,
containing 4 µL SYBR Green (1:100; Invitrogen), 0.1 µL dNTP (5 mM), 2
µL PCR buffer 10x, 1.2 µL MgCl2, 0.5 U Platinum Taq DNA Polymerase
(Invitrogen) supplemented with 0.4 µL (10 pmol) of each primer and 1
µL of the cDNA template. Each cDNA sample was analyzed in triplicate
with each primer pair and melting curve analysis was performed at the
end of the reaction to confirm the amplification of a single PCR product.
Data were normalized to actin cDNA amplified in each set of PCR ex­
periments and the 2− ΔCt method was used to determine the relative
expression (Livak and Schmittgen, 2001). The primers utilized in these
experiments are listed in Table S1.
2.6. Determination of intracellular calcium levels
The intracellular free calcium concentration was determined using
the acetoxymethyl ester of Fluo-4 (Fluo-4 AM; Thermo Fisher Scientific).
Briefly, H99 and pmc1 cells were cultured in YPD medium overnight
with shaking at 30◦ C. Then, 107 cells of each strain were incubated for 1
h at 37◦ C with shaking in MilliQ H2O with or without 25 µg/mL AMD.
The cells were loaded with 2 µM Fluo-4 AM for 1 h at 37◦ C with shaking.
Fluo-4 AM fluorescence was measured by flow cytometry of 5,000
events (Guava EasyCyte, Millipore) and a histogram was generated for
each strain using FlowJo software (count normalized to Mode X Green-B
fluorescence).
2.7. Production of lipid-core nanocapsules (LNC)
The LNC were produced by the interfacial deposition of preformed
polymer method (Jäger et al., 2009). Briefly, an organic phase consisting
of poly(ε-caprolactone) (PCL; Sigma-Aldrich) (0.1 g), capric-caprylic
triglyceride (CCT; Delaware) (0.165 mL), sorbitan monostearate (Span
60®; Sigma-Aldrich) (0.038.5 g) and dioxane (Vetec Química) (2 mL)
dissolved in acetone (25 mL) was prepared before the addition of 0.002 g
of AMD, FLU or a combination of 0.002 g of each drug. The solution was
maintained under magnetic stirring at 40◦ C, then injected into an
aqueous phase (53 mL) containing polysorbate 80 (Tween 80®; Henri­
farma) (76.8 mg). After 10 min, the translucent solution was evaporated
to 10 mL under reduced pressure in a rotary evaporator at 37◦ C (Rota­
vapor R II, Büchi). The nanocapsules loaded with amiodarone hydro­
chloride, fluconazole and both drugs in combination were called,
respectively, LNCAMD, LNCFLU and LNCAMD+FLU. Alongside, lipid-core
nanocapsules prepared without drugs were similarly produced and
used as controls (LNC).
2.8. Characterization of lipid-core nanocapsules
The lipid-core nanocapsule aqueous dispersions were evaluated
regarding their pH, particle size distribution and zeta potential
3
European Journal of Pharmaceutical Sciences 162 (2021) 105816
immediately after production (Paese et al., 2017). The pH was deter­
mined by potentiometry (B474, Micronal), particle size was assessed by
laser diffraction (Mastersizer 2000, Malvern Panalytical), and dynamic
light scattering (Zetasizer Nano ZS, Malvern Panalytical) was performed
as previously described (Frank et al., 2017). Zeta potential was deter­
mined by electrophoretic mobility (Zetasizer Nano ZS, Malvern Pan­
alytical) as previously described (Frank et al., 2017). The drug content
(n = 3) was determined after the drug extraction from the nanocapsules
by high performance liquid chromatography (HPLC) with ultraviolet
detection (HPLC-UV, Series 200, PerkinElmer). The quantification
method was adapted (Bolderman et al., 2009; Sadasivudu et al., 2009)
and validated according to our purposes. A C18 reversed phase column
(Merck & Co) was used as stationary phase and an acetonitrile:water
(35:65 v/v) mobile phase was used for FLU with detection at 260 nm.
For AMD, acetonitrile:isopropanol:water:ammonia (80:10:10:0.025
v/v) was used as the mobile phase with detection at 260 nm for AMD.
The injection volume was 20 µL and calibration curves (n = 3) were
made to determine the drug concentration showing linearity (r = 0.998)
in the range of 2 to 20 µg/mL.
2.9. In vivo studies
In vivo studies were conducted according to a previously described
intranasal inhalation infection model (Kmetzsch et al., 2010) using five
female BALB/c mice (CEMIB, UNICAMP; ~ 5 weeks old) for each
treatment group and control group. Fungal cells were cultured overnight
in 50 mL of YPD medium at 30◦ C with shaking, washed twice and sus­
pended in PBS. Mice were infected with 105 yeast cells. For the free AMD
analysis, at 24 h post-infection, mice were given single or combined
treatment of AMD (10 mg/kg) and FLU (15 mg/kg, 5 mg/kg or 1 mg/kg)
intraperitoneally. For nanocapsule analysis, mice were given the
following treatment intranasally: AMD (0.5 mg/kg), FLU (0.5 mg/kg),
AMD+FLU (0.5 mg/kg of each drug), LNC (0.5 mg/kg), LNCAMD (0.5
mg/kg), LNCFLU (0.5 mg/kg) and LNCAMD+FLU (0.5 mg/kg of each drug).
In another experimental group, the animals were given the following
treatment intraperitoneally: AMD (1 mg/kg), FLU (1 mg/kg), AMD+FLU
(1 mg/kg of each drug), LNC (1 mg/kg), LNCAMD (1 mg/kg), LNCFLU (1
mg/kg) and LNCAMD+FLU (1 mg/kg of each drug). The treatments were
given once daily for 13 days after the infection. On day 14 post-infection,
animals were euthanized using a lethal injection of sodium thiopental,
and the lungs and brain were aseptically excised. Tissues were macer­
ated in PBS and the resulting suspensions were plated on YPD for colony
forming unit (CFU) determination. Combinatorial drug efficacy was
assessed by C. neoformans organ burden reduction in the treated mice
relative to the untreated controls. Mice were housed in groups of five
and kept in filtered top-ventilated cages with food and water ad libitum.
All efforts to minimize animal suffering were made. Before infection
assays, mice were intraperitoneally anesthetized with 100 mg/kg keta­
mine and 16 mg/kg xylazine. Mice were examined twice daily for any
signs of suffering, defined as weight loss, weakness, or inability to obtain
food or water. At the first signs of suffering, mice were humanely
sacrificed.
2.10. Ethical statement
The use of animals in this work was performed with approval of the
Universidade Federal do Rio Grande do Sul Ethics Committee for Use of
Animals (CEUA Project Number 34285) and comply with ARRIVE
guidelines.
3. Results
3.1. AMD has antifungal activity in vitro and synergy with FLU
The in vitro effect of AMD and FLU against C. neoformans H99 strain,
the pmc1 mutant and the complemented pmc1:PMC1 strains were
N.K. Oliveira et al.
assessed as an initial screening for their fungicidal activity, in order to
develop a nanotechnological formulation and improve the drug efficacy.
Our data indicated MIC defined as 12.5 µg/mL of AMD for H99 and
pmc1::PMC1 strains and 6.25 µg/mL for the pmc1 strain (Fig. 1). In order
to evaluate the synergy between AMD and FLU, a checkerboard assay
was performed. First, MIC values for FLU alone were obtained by testing
different concentrations of drug ranging from 0.125 to 64 µg/mL. All
strains presented a MIC of 0.5 µg/mL. Later, once combined distinct
ranges of concentrations of AMD and FLU, the MIC values consistently
decreased. To categorize the drug interaction, the FIC index was
calculated based on the values for the drugs alone and in combination,
defined as 0.75 for all strains. Consequently, the drugs were considered
synergistic (Table 1).
3.2. Urease activity is affected by AMD
Assays for polysaccharide capsule size formation, melanin produc­
tion, and urease activity in the presence of AMD alone or in combination
with FLU were performed to evaluate the effect of AMD on C. neoformans
virulence factors. The relative capsule size was determined for H99 and
pmc1 strains in minimal medium for 72 h. However, no significant dif­
ferences were observed between the treatment conditions (Fig. 2A).
Melanin formation was analyzed in an asparagine medium plate sup­
plemented with L-DOPA, a natural catecholamine oxidized by the lac­
case enzyme (Idnurm et al., 2005). The same pattern of melanization
was observed on fungal colonies analyzed in the solid medium with or
without the drug (Fig. 2B). The urease activity was increased in the
presence of AMD for both H99 and pmc1 strains. However, when AMD
and FLU were added in lower concentrations, this effect is only observed
for the H99 strain (Fig. 2C). Taken together these results demonstrate
that AMD can affect urease production, an important virulence deter­
minant of C. neoformans.
3.3. Genes related to the Ca2+-calcineurin pathway are differentially
expressed in the presence of AMD
Considering that AMD is a calcium channel blocker, we sought to
determine if the expression of genes related to the Ca2+-calcineurin
signaling pathway could be altered by exposure to the drug. The
expression patterns of genes encoding calcineurin (Cna1), the tran­
scription factor Crz1, and the Cch1, Eca1, Vcx1 and Pmc1 transporters
were evaluated by RT-qPCR in H99 and pmc1 strains. AMD treatment led
to increased expression of CRZ1, VCX1, and PMC1 genes, while levels of
CCH1 were decreased as compared to control conditions in H99 cells
(Fig. 3). In the absence of PMC1, AMD treatment causes an increase of
VCX1 gene expression, suggesting that AMD treatment increases intra­
cellular calcium levels and activates the Ca2+-calcineurin pathway.
However, CNA1 and ECA1 transcript levels were not altered (Fig. 3).
Fig. 1. Minimum inhibitory concentration determination for C. neoformans
strains H99, pmc1 and pmc1::PMC1. The assays were performed in RPMI-1640
medium supplemented with 0.78 – 12.5 µg/mL AMD for 48 h at 37◦ C. Subse­
quently, the optical density was evaluated at 600 nm for yeast growth and
normalized by control samples in RPMI-1640 medium. The experiment was
performed three times with biological triplicates.
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European Journal of Pharmaceutical Sciences 162 (2021) 105816
Table 1
Checkerboard test.
Strain MICa MICa combination FIC indexb Interpretationc
AMD FLU AMD FLU
H99 12.5 0.5 3.125 0.25 0.75 Synergy
pmc1 6.25 0.5 1.56 0.25 0.75 Synergy
pmc1::PMC1 12.5 0.5 3.125 0.25 0.75 Synergy
a
Values in µg/mL
b
Afeltra J, 2004
c
Mor V, 2015 and Joffe L, 2017Abbreviations: AMD – amiodarone; FLU –
fluconazole
3.4. AMD increases the levels of free intracellular calcium
Employing Fluo-4 AM fluorescence analysis, the intracellular cal­
cium cell levels were evaluated. The H99 strain had very low levels of
intracellular calcium in control conditions as compared to the pmc1
strain, consistent with the lack of a transporter to the vacuole, the main
stocking organelle for calcium. However, the addition of AMD to the
cells increased the intracellular uptake as compared to the respective
strain controls (Fig. 4A, B). Considering that an excess of intracellular
calcium is toxic to the cell, the unrestrained increase in calcium uptake
might be the primary mode of action of AMD in yeast cells.
3.5. AMD did not display antifungal effects in murine cryptococcal
infection
To evaluate the antifungal effect of AMD in a murine model, mice
were infected intranasally with the H99 strain and treated for 13 days
using intraperitoneal administration of drugs. After this time point, the
CFU was determined in lung tissues (Fig. 5). Mice treated with 15 mg/
kg, 5 mg/kg and 1 mg/kg of FLU had a dose-dependent decrease in the
lung fungal burden. Conversely, treatment with 10 mg/kg of AMD alone
increased the fungal burden in the lungs. When the FLU and AMD
treatments were combined, the antifungal capacity remained equivalent
to those of FLU alone, indicating that the antifungal effect is due to FLU
activity only. These results suggest that AMD in its free form and in such
high dosage is not efficient against cryptococcosis, even causing
increased fungal burden in some animals.
3.6. Physicochemical characteristics of lipid-core nanocapsules
Since AMD demonstrated in vitro effectiveness against C. neoformans,
we aimed to investigate if innovative nanotechnological formulations
could increase the antifungal effect and decrease the adverse effects of
this drug in vivo. In this context, we prepare lipid core nanocapsules
(LNC) containing amiodarone, fluconazole, or the combination of these
drugs. The analysis of particle size by laser diffraction demonstrated
only populations of nanometric size, as illustrated in a radar chart
(Fig. 6) for all formulations tested. In 8 axes, we observe the mean di­
ameters and diameters of nanocapsules at percentiles 10, 50 and 90
under the size distribution curves determined by volume and by number
of nanoparticles. The polydispersity of diameters (span=[d(90)-d(10)/d
(50)]) lower than 2 for all formulations indicated that they were all
suitable for pharmaceutical applications (Bianchin et al., 2015).
All LNC formulations showed narrow size distributions by dynamic
light scattering analysis, which mean hydrodynamic diameters were
around 200 nm, with similar polydispersity index (PDI ~ 0.22).
Therefore, the incorporation of the drugs into the nanocapsules did not
significantly change its size. For all formulations, the zeta potential was
slight negative close to - 8 mV, taking into account the presence of
polyester (PCL) and non-ionic surfactant (polysorbate 80) at the inter­
face particle/water (Jäger et al., 2009; Paese et al., 2017). All formu­
lations showed a slightly acid pH of 6, considered suitable for nasal and
intraperitoneal use in animal model.
N.K. Oliveira et al. European Journal of Pharmaceutical Sciences 162 (2021) 105816

Fig. 2. AMD affects phenotypic characteristics

of C. neoformans. (A) Polysaccharide capsule
formation was determined in minimal medium
for H99 and pmc1 cells for 72 hours, 37◦ C, 5%
CO2 in the presence or absence of 3.125 µg/mL
AMD or 0.78 µg/mL AMD and 0.125 µg/mL
FLU. Cells were counterstained with India ink
and measured using IMAGEJ software. Bars
indicate mean with SD and individual mea­
surements are shown scattered. (B) Melanin
formation was evaluated for H99 and pmc1 cells
on glucose-free asparagine medium plates sup­
plemented with L-DOPA. Plates were incubated
for 5 days at 37◦ C in the presence or absence of
30 µg/mL AMD or 7.5 µg/mL AMD and 1.5 µg/
mL FLU. (C) Urease activity was evaluated for
H99 and pmc1 cells in Robert’s urea broth for 4
h at 37◦ C, supplemented with 3.125 µg/mL
AMD or 0.78 µg/mL AMD and 0.125 µg/mL
FLU. Subsequent evaluation of OD at 560 nm.
Bars indicate mean with SD and individual
measurements are shown scattered. Statistical
analysis was conducted using Student’s t test. *
p < 0.05, ** p < 0.01. The experiments were
repeated at least two times, with biological
triplicates.

Fig. 3. AMD alters expression of genes involved in the calcium-calcineurin signaling pathway and virulence determinants. Transcript levels of calcineurin (CNA1),
CRZ1 and calcium transporters (CCH1, ECA1, VCX1 and PMC1) were quantified by RT-qPCR. H99 and pmc1 cells were exposed to control conditions or 12.5 µg/mL
AMD for 10 min in RPMI-1640 medium. Data was normalized to actin and bars indicate mean with SD and individual measurements are shown scattered. Statistical
analysis was conducted by one-way ANOVA with Tukey’s post hoc test or Student’s t test. * p < 0.05, ** p < 0.01. The analysis was performed with biological
triplicates.

3.7. LNC improves the AMD antifungal effect
A mice model of cryptococcosis was utilized to test the efficacy of
LNC formulations, as described above. LNCAMD and LNCAMD+FLU altered
the antifungal effect of AMD in comparison to that observed for the free
drug. After 13 days of intraperitoneal treatment with 1 mg/kg of the free
(AMD and FLU) or encapsulated drugs (LNCAMD and LNCFLU), the fungal
burden in lung tissues was measured. Although AMD was not effective in
controlling fungal growth in vivo, LNCAMD significantly reduced the
fungal burden (p < 0.05) (Fig. 7A). The efficacy of FLU was similar to the
combination of FLU+AMD. LNCFLU improved the reduction in CFU
counts, which was similar to the effect of LNCFLU+AMD. Hence, the

5
N.K. Oliveira et al.
Fig. 4. AMD affects intracellular calcium levels in C. neoformans. Intracellular
calcium was evaluated during incubation with 25 µg/mL AMD for 1 h at 37◦ C,
followed by incubation with 2 µM Fluo 4-AM fluorescent marker for 1 h at 37◦ C.
Flow cytometry analysis was performed. (A) Histograms (count normalized to
Mode X Green-B fluorescence) were generated by FlowJo software. Normalized
to Mode = scales as percentages of the maximum count. (B) Analysis of total
fluorescent cell counts. Bars indicate mean with SD and individual measure­
ments are shown scattered. Statistical analysis was performed by Student’s t
test. * p < 0.05, ** p < 0.01, **** p < 0.0001. The analysis was performed
twice with biological triplicates.
Fig. 5. AMD fails to demonstrate an antifungal effect in a murine cryptococ­
cosis model. Female BALB/c mice were challenged with H99 cells intranasally.
At 24 h post-infection, mice were given single or combination treatments of
AMD (10 mg/kg) and/or FLU (15, 5 and 1 mg/kg) intraperitoneally. The fungal
burden was determined after 13 days of treatment in the lungs. Bars indicate
mean with SD and individual measurements are shown scattered. Statistical
analysis was performed by one-way ANOVA with Tukey’s post hoc test
comparing each treatment with the control group. * p < 0.05, *** p < 0.001. In
vivo experiments were performed once with statistically relevant groups.
combination of FLU and AMD with and without nanoencapsulation is
neither additive nor synergistic for C. neoformans in vivo.
In the intranasal treatment performed under the same experimental
6
European Journal of Pharmaceutical Sciences 162 (2021) 105816
conditions, a completely different profile was observed with 0.5 mg/kg
of the drugs administered. The effects of free FLU and AMD had
considerable heterogeneity between animals, not observed in the co-
treatment or in the LCN treated groups. All nanoformulation groups
caused decrease in fungal burden compared to the control group
(Fig. 7B), including the LNC control, indicating a possible antifungal
effect of the formulation itself.
4. Discussion
New pharmaceutical approaches, such as the use of drugs with
known effects in other diseases, have emerged as a promising alternative
to treat C. neoformans infections, considering their established toxi­
cology and pharmacology (Butts and Krysan, 2012). Courchesne et al.
determined a potent fungicidal effect of the antiarrhythmic drug AMD
against C. neoformans and several other fungi in vitro (Courchesne,
2002). The present study further evaluated this antifungal effect, both in
vitro and in vivo against a wild type strain and a mutant with defects in
calcium mobilization. The pmc1 mutant demonstrated a higher sensi­
tivity to AMD compared to the wild-type H99 and reconstituted pmc1:
PMC1 strains. The increased sensibility of pmc1 was associated with an
increase in intracellular calcium caused by AMD. As previously
demonstrated in S. cerevisiae, AMD triggered a dose-dependent elevation
in intracellular calcium (Gupta et al., 2003). Our data obtained with
Fluo-4 AM fluorescence presented the same profile, demonstrating the
undoubted effect of AMD in the homeostasis of calcium and a possible
mechanism of action, considering that the excess of calcium is toxic to
the cell. Furthermore, pmc1 mutant had much higher calcium concen­
tration than H99 in control conditions, probably due to the mutant’s
impaired capacity to direct calcium to the vacuole.
To further explore the effects of AMD in calcium homeostasis, we
analyzed the transcriptional response of genes involved in the Ca2+-
calcineurin pathway, as it has been previously demonstrated that AMD
targets this pathway in S. cerevisiae (Zhang and Rao, 2007). The increase
in CRZ1 transcript levels observed in this study for C. neoformans is in
agreement with the results reported in C. albicans (Gamarra et al., 2010),
as well as in the upregulation of PMC1 when exposed to AMD, which is
consistent with the increase in intracellular calcium levels. There was
also an increase in VCX1 expression, but only in the pmc1 mutant,
possibly due to a compensatory mechanism due to the absence of the
Pmc1 transporter. However, CCH1 was downregulated in H99, contrary
to the upregulation observed in C. albicans (Gamarra et al., 2010), but
consistent with its localization in the extracellular membrane. This
transcriptional profile further reinforces the possible mechanism of
AMD action, based on an increasing intracellular calcium levels and
further activation of the calcineurin pathway, probably leading to high
stress, disruption in nutrient sensing and delaying cell cycle progression
(Zhang and Rao, 2007).
The combination of AMD and FLU were in vitro synergistic against
C. neoformans, as previously demonstrated for S. cerevisiae (Gupta et al.,
2003). C. albicans and Candida tropicalis strains resistant to FLU also
present AMD+FLU synergy, while susceptible strains usually do not (Da
Silva et al., 2013; Gamarra et al., 2010). Nonetheless, AMD and FLU
were already established in the literature as drugs that enhance each
other’s activities. These two drugs also affected the urease activity,
which was increased with the addition of AMD and FLU in both H99 and
pmc1 mutant strains. Despite urease being the main mechanism for
paracellular transmigration, no significant fungal burden was found in
brain tissues of mice treated with AMD (results not shown). Further­
more, recent findings by our group indicate that intracellular calcium
levels might influence urease activity (Squizani et al., 2018).
The antifungal activity of AMD was then evaluated in a murine
model of cryptococcosis, as a possible alternative therapeutic strategy.
However, AMD treatment induced an increase in mice lung fungal
burden, suggesting a possible intensification of the fungal virulence.
Several studies have demonstrated that calcium channel blockers can act
N.K. Oliveira et al.
Fig. 6. Radar chart of Phl-LNC presenting the volume-weighted mean diameters (D[4,3]) and the diameters at percentiles 10, 50 and 90 under the size distribution
curves by volume and by number of particles. The results obtained were the mean of three analyses for each batch. The analysis was performed three times.
Fig. 7. AMD nanocapsules demonstrate enhanced antifungal activity against
C. neoformans. Female BALB/c mice were challenged with H99 cells intrana­
sally. (A) At 24 h post-infection, mice were given 1 mg/kg treatments of AMD,
FLU, AMD+FLU, LNC, LNCAMD, LNCFLU and LNCAMD+FLU intraperitoneally. An
outlier animal was excluded from the LNCAMD group. (B) At 24 h post-infection,
mice were given 0.5 mg/kg treatments of AMD, FLU, AMD+FLU, LNC, LNCAMD,
LNCFLU and LNCAMD+FLU intranasally. One outlier animal was excluded from
the control, LNC, LNCFLU and LNCAMD groups. The amount of LNC-loaded drugs
were adjusted to contain 0.5 or 1 mg/kg of amiodarone and/or fluconazole.
Fungal burden was determined after 13 days of treatment in the lungs. Bars
indicate mean with SD and individual measurements are shown scattered.
Statistical analysis was conducted by one-way ANOVA with Tukey’s post hoc
test comparing each treatment with the control group. * p < 0.05, ** p < 0.01,
*** p < 0.001. In vivo experiments were performed once with statistically
relevant groups.
7
European Journal of Pharmaceutical Sciences 162 (2021) 105816
as immunosuppressants, inhibiting the activation of T cells, mast cells
and macrophages (Liu and Matsumori, 2011). AMD, although not used
in those studies, might have the same effects, hence decreasing the
clearance of the fungus. Also, AMD metabolization generates a diversity
of metabolites, whose antifungal activity is not known (Jeong et al.,
2018). It is known, though, that AMD can cause acute and chronicle lung
toxicity in some patients (Papiris et al., 2010). The potential adverse
effects would have to be taken in consideration regarding doses and
length of treatment.
Considering the benefits of the encapsulation of drugs, such as
increased efficacy, bioavailability and decreased side effects (Frank
et al., 2015), we produced lipid-core nanocapsules containing AMD, FLU
and both drugs in the same nanoparticle. In mice treated intraperito­
neally, LNCAMD enhanced antifungal activity compared with the free
AMD, while the LNCFLU formulation had no effect. Lipid-core nano­
capsules containing FLU were already produced for the treatment of
resistant C. albicans. They presented a high encapsulation efficiency and
antifungal effect, however the drug concentration used was consider­
ably higher (Bianchin et al., 2019). An algorithm that considers the drug
distribution coefficient (log D) to determine its distribution in the
lipid-core nanocapsule could explain the different efficiencies of the
nanocapsules developed (Oliveira et al., 2013). According to the model,
LNCFLU can be categorized as a type II, in which the drug is dispersed on
the surface of the nanocapsule. In contrast, LNCAMD is classified as type
VI, in which the drug is mainly encapsulated at the core, so this differ­
ence in encapsulation may be responsible for the better results with
LNCAMD. Furthermore, nanocapsule agglomeration in the abdominal
cavity after intraperitoneal administration is possible, leading to an
impaired distribution in the organism (Bulcão et al., 2013). Diversely,
the intranasal treatment induced higher homogeneity between animals
and indicated an antifungal effect for all nanocapsule formulations,
which was not observed for the free drugs. The antimicrobial effect of
the LNC prepared without drug has been reported previously, although
the formulation is not exactly the same (Cé et al., 2016; De Marchi et al.,
2017), and indicates that the formulation itself might have antifungal
activity. The effect of LNC in animal models was already demonstrated
(Bueno et al., 2013), so the effect of the nanostructure is not surprising.
In this way, we speculate that the mechanism of action of these
LNC-based formulations would be associated with the higher
N.K. Oliveira et al.
penetration of the drugs inside yeast cells and consequent alteration of
calcium homeostasis (AMD) or proper plasmatic membrane function
(FLU). Moreover, these formulations would be safe for use, as previous
reports from our group demonstrated the safety of LNC-based formula­
tions on non-tumor cell line (Frank et al., 2017), by intraperitoneal in­
jection in a rat model (Bulcão et al., 2013), by intradermal injection in
rat (Bulcão et al., 2014), as well in immune cells (Sandri et al., 2019).
Also, we previously demonstrated that the lipid-core nanocapsules are
formed on the nanoscopic scale as unimodal distributions and that the
mechanism of kinetic degradation of LNCs is mainly based on
non-catalyzed hydrolysis prevailed, which suggest high stability (for at
least 90 days) (Fiel et al., 2013). In the present work, we reveal that the
new pharmaceutical strategy developed based on nanotechnology has
the potential to be used in the treatment of fungal diseases.
5. Conclusion
This study demonstrated that AMD presents an in vitro effect against
C. neoformans and synergy with FLU. While AMD did not demonstrate in
vivo activity against intranasal infection in its free form, the nano­
encapsulation promoted an upgrade in the antifungal effect of the drug.
The use of lipid-core nanocapsules in drug repurposing represents a
viable alternative to improve the control of cryptococcosis infection and
opens the possibility for a possible new treatment of this disease.
Funding
This work was supported by grants from the Brazilian agencies
Conselho Nacional de Desenvolvimento Científico e Tecnológico
(CNPq), Coordenação de Aperfeiçoamento de Pessoal de Nível Superior
(CAPES), and Fundação de Amparo à Pesquisa no Estado do Rio Grande
do Sul (FAPERGS and PRONEX 2014). This study is part of the National
Institute of Science and Technology in Pharmaceutical Nanotechnology:
a transdisciplinary approach (INCT-NANOFARMA), which is supported
by São Paulo Research Foundation (FAPESP, Brazil) Grant #2014/
50928-2 and by Brazilian National Council for Scientific and Techno­
logical Development (CNPq, Brazil) Grant #465687/2014-8.
Statement of Author Contributions
NKO: conceived and designed the analysis, data acquisition, data
interpretation, wrote the manuscript; LAF: conceived and designed the
analysis, data acquisition, data interpretation, critical review of the
manuscript; EDS, JCVR, BMM, HM, AWAC e UPK: contributed in data
acquisition, data interpretation; ARP: conceived and designed the
analysis, financial support, critical review of the manuscript; CCS e SSG:
conceived and designed the analysis, data interpretation, financial
support, critical review of the manuscript; AS e MHV: conceived and
designed the analysis, financial support, critical review of the manu­
script; LK: conceived and designed the analysis, data interpretation,
financial support, critical review of the manuscript.
Declaration of Competing Interest
None.
Supplementary materials
Supplementary material associated with this article can be found, in
the online version, at doi:10.1016/j.ejps.2021.105816.
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