Efficacy and non-toxicity of ciclopirox olamine-loaded liposomes against Cryptococcus neoformans clinical isolates

Abstract

The aim of this study was to evaluate the efficacy and non-toxicity of ciclopirox olamine-loaded liposomes against Cryptococcus neoformans clinical isolates. Initially, 24–1 fractional experimental design was carried out to obtain an optimized formulation of liposomes containing CPO (CPO-LipoC), which were then used to prepare stealth liposomes (CPO-LipoS). Liposomal formulations were characterized by their mean size diameter, polydispersity index (PDI), and drug encapsulation efficiency (EE%). Immunosuppressed mice were exposed to CPO-LipoS at 0.5 mg/kg/day for 14 days to verify possible histopathological alterations in the liver and kidneys. Immunosuppressed mice infected with C. neoformans were treated with CPO-LipoS at 0.5 mg/kg/day for 14 days to quantify the fungal burden in spleen, liver, lungs, and brain. CPO-LipoS presented a mean size diameter, PDI, and EE% of 101.4 ± 0.7 nm, 0.307, and 96.4 ± 0.9%, respectively. CPO-LipoS was non-toxic for the liver and kidneys of immunosuppressed mice. At the survival curve, all infected animals submitted to treatment with CPO-LipoS survived until the end of the experiment. Treatment with CPO-LipoS reduced C. neoformans cells in the spleen (59.3 ± 3.4%), liver (75.0 ± 3.6%), lungs (75.7 ± 6.7%), and brain (54.2 ± 3.2%). CPO-LipoS exhibit antifungal activity against C. neoformans, and the encapsulation of CPO into stealth liposomes allows its use as a systemic drug for treating cryptococcosis.

Introduction

Cryptococcus neoformans is an opportunistic fungal pathogen that causes cryptococcosis. The genus Cryptococcus is composed of ten species, in which most of the isolates of this genus belong to the group Cryptococcus gattii/C. neoformans species complexes and some non-pathogenic species such as Cryptococcus amylolentus, Cryptococcus depauparatus, and Cryptococcus luteus. Cryptococcus neoformans sensu stricto, formerly called the C. neoformans variety grubii, is globally the major cause of systemic cryptococcosis among immunocompromised individuals. Furthermore, this strain is strongly associated with bird excreta, especially pigeon droppings [1, 2]. Cryptococcosis is initiated as a pulmonary infection, and in conditions of immune deficiency, it can disseminate to the blood stream and central nervous system (CNS). Over the last decades, as vulnerable populations have expanded, cryptococcal meningitis has become an infection of global importance, with up to one million new infections annually, and it has significant morbidity and mortality rates, especially among patients who are also infected with the human immunodeficiency virus (HIV) [3, 4].

Amphotericin B (AmB) deoxycholate combined with flucytosine (5-FC) is the main antifungal regimen in the induction therapy of invasive cryptococcosis, considered the severe form of the disease. After induction therapy, treatment is continued with fluconazole in maintenance therapy. However, the limitations of treatment during induction therapy with AmB combined with 5-FC are its nephrotoxicity and hepatotoxicity [5, 6]. Similarly, hydroxypyridone derivatives have been evaluated for use in treating fungal infections. Among these agents, ciclopirox olamine (CPO) [(6-Cyclohexyl-1-hydroxy-4-methylpyridin-2(1H)-one and 2-aminoethanol)] is one of the most widely used in clinical practice because of its broad spectrum, low cost, and high potency. Despite the biological activities of CPO, this molecule presents a number of drawbacks that must be overcome for a future in vivo application, such as its low water solubility (Log P = 2.03), low systemic bioavailability, and short half-life (2.1 h) [7, 8].

The mechanism of action of CPO in fungi is not completely understood [9]. However, CPO has the ability to chelate polyvalent metal cations, such as iron cations, in both oxidation states. This characteristic enables the inhibition of metal-dependent enzymes, such as cytochromes, catalases, and peroxidases present in fungi. Inhibition of these enzymes interferes with mitochondrial function, energy production, and transport across membranes. Furthermore, there are no reports of fungal strains showing resistance to CPO after using this molecule as a fungicide, even after its use in clinical medicine [9, 10].

Recently, drug delivery systems have been developed and successfully used to combat microbial infections. Among these systems, liposomal drug delivery systems have been used to improve the bioavailability of antimicrobial agents with low solubility and reduce their toxicity for treating bacterial and fungal infections [11, 12]. One approach that is currently used to overcome those problems are lipidic formulations of AmB, including conventional liposomes (AmBisome®), which have been developed to enhance the therapeutic index and reduce the nephrotoxicity of AmB, but conventional liposomes are quickly removed from circulation by the mononuclear phagocyte system (MPS) [13]. Thus, the second generation of liposomes includes stealth formulation. In this system, liposomes are coated with hydrophilic molecules, usually polyethylene glycol (PEG), to prevent their uptake by MPS, thus prolonging the circulation of the vesicles and allowing the encapsulated drug to reach its target [12, 14].

Other studies described the encapsulation of CPO in drug delivery systems for the skin delivery of CPO. Verma and Palani [15] proposed the encapsulation of CPO into multilamellar liposomes of phosphatidylcholine and cholesterol incorporated in a blend agar-carrageenan gel. Furthermore, the transdermal potential of ethosomes bearing ciclopirox olamine, which are known to have limited transdermal permeation, was investigated by Girhepunje et al. [16]. The targeting of CPO to the epidermal and dermal sites was verified after the topical administration of CPO-loaded ethosomes in the dorsal skin of nude albino rats.

Thus, the present study aims to develop and characterize CPO-loaded stealth liposomes (CPO-LipoS) to evaluate the in vitro and in vivo antifungal activities of CPO-LipoS against C. neoformans. For studying in vivo systemic cryptococcosis, several studies have previously shown that immunosuppressed mice are susceptible to systemic cryptococcosis due to their capacity to mimic disseminated severe human diseases associated with immune deficiency. Therefore, these animal models allow novel therapeutic alternatives to be evaluated [17, 18]. Finally, the lack of prior studies on the systemic treatment of fungal infections involving CPO-loaded liposomes against C. neoformans makes this a pioneering work in this field.

Material and methods

Reagents and culture medium

Soya phosphatidylcholine (PC) and distearoyl phosphatidyl-ethanolamine-polyethylene glycol 2000 (DSPE-PEG2000) were supplied by Lipoid GmBH (Ludwigshafen, Germany). Cholesterol (Chol), stearylamine (SA), ciclopirox olamine (CPO), Roswell Park Memorial Institute Medium (RPMI), morpholine-propane sulfonic acid (MOPS), Sabouraud dextrose agar (SDA), and chloramphenicol were purchased from Sigma-Aldrich (St. Louis, MO, USA). Dexamethasone was purchased from Teuto (Brazil). Microtiter plates were supplied by TPP (Trasadingen, Switzerland), and 0.22-µm membranes were purchased from Millipore (Bedford, MA, USA).

Fractional experimental design and characterization of CPO-loaded liposomes

A two-level 2^4–1 experimental fractional design was used to study the CPO encapsulation into conventional liposomes and to evaluate the influence that the lipid concentrations and the drug:lipid molar ratio had on the following variables: mean particle size, polydispersity index (PDI), and drug encapsulation efficiency (EE%).

This study was based on an initial liposome formulation consisting of PC 82.32 mM, Chol 23.52 mM, SA 11.76 mM, and CPO 2 mg/mL. The factors were defined for experimental design as follows: factor A = PC (41.16, 61.74, and 82.32 mM); factor B = Chol (11.76, 23.52, and 35.28 mM); factor C = SA (0.0, 5.88, and 11.76 mM); and factor D = CPO (2.0, 2.5, and 3.0 mg/mL). Experiments were randomly assayed to nullify the effect of inappropriate nuisance variables. The central point was performed in triplicate. Next, stealth liposomes (CPO-LipoS) were prepared by replacing 5% of the PC with DSPE-PEG2000.

Ciclopirox olamine encapsulated into conventional (CPO-LipoC) and stealth (CPO-LipoS) liposomes by the hydration of the thin lipid film, followed by sonication. Initially, for preparing CPO-LipoC, Chol, PC, SA, and CPO were solubilized in organic solvent, chloroform, and methanol (3:1, v/v) under magnetic stirring. For the preparation of CPO-LipoS, SA was replaced by DSPE-PEG2000. The solvents were removed by rotary evaporation for 60 min (37 ± 1 °C, 80 rpm), resulting in a thin lipid film formation. This film was then hydrated with 10 mL of pH 7.4 phosphate buffer solution, which resulted in multilamellar liposomes (MLV). Next, the liposomal suspension was sonicated (Vibra Cell, BRANSON, USA) at 200 W and 40 Hz for 300 s to form small unilamellar liposomes (SUV).

The liposomes were characterized by the mean particle size, PDI, and EE%. The particle size and PDI of the liposome dispersions were measured by photon correlation spectroscopy (Beckman Coulter Delsa™Nano S Particle analyzer). EE% was determined by the ultrafiltration/ultracentrifugation technique using Ultrafree® units (Millipore, USA, MW cut-off = 10,000 Da). Samples of liposomes (400 µL) were inserted into the filtration unit and submitted to ultracentrifugation at 8792 g for 1 h in a Kubota KR-20000 T centrifuge (Kubota, Osaka, Japan) fitted with a Kubota RA-1 M rotor. Ten microliters of the sample that passed through the filter (unloaded CPO amount) was diluted in 5 mL of methanol, and the CPO was quantified at 304 nm using a UV spectrophotometer (Ultrospec^@ 300, Armshan Pharmaceutical) [19, 20].

In vitro studies with Cryptococcus neoformans clinical strains

The experimental protocol was approved by the Human Research Ethics Committee of the Federal University of Pernambuco (#080/09, Recife, Brazil). Thirty C. neoformans clinical strains were isolated from cerebrospinal fluid (CSF) that had been obtained by lumbar puncture of immunocompromised patients treated at the Pernambuco Neurological Diagnosis Unit (Recife, Brazil). The clinical samples were processed for mycological diagnosis using standard methods Hoog et al. [21], direct examination with China ink staining and isolation in culture at the Medical Mycology Laboratory, and they were stocked in the URM-UFPE Culture Collection.

Antifungal activity of CPO-LipoC and CPO-LipoS against C. neoformans clinical isolates was assessed according to the Clinical and Laboratory Standards Institute [22]. In order to obtain a yeast inoculum (1.0 to 5.0 × 10⁶ CFU.mL⁻¹), each strain was cultured in a tube containing 20 mL of 4% SDA plus yeast extract at 35 °C for 2 days. After that, yeast suspensions were prepared in sterile physiological solution (0.85%) and adjusted to 90% transmittance at 530 nm. Two serial dilutions from 1:100 and 1:20 were made to obtain a final inoculum containing 0.5 to 2.5 × 10³ CFU.mL⁻¹. The tests were performed in RPMI 1640 medium buffered with 3-(N-morpholino) propanesulfonic acid (MOPS). Microplate wells were inoculated with 100 µL of the previously obtained inoculum. CPO-LipoC and CPO-LipoS were added by serial dilution at concentrations ranging from 0.125 to 512 µg/mL to determine the minimum inhibitory concentration (MIC) and minimum fungicidal concentration (MFC) against thirty C. neoformans clinical isolates. Liposomes without drug (empty liposomes) were used to evaluate any possible effects of the liposome constituents on in vitro fungal growth. The microplates were incubated at 35 °C in a non-CO₂ incubator and visually evaluated 72 h after the incubation. The MICs corresponded to the lowest drug concentration that showed growth inhibition compared to the control wells. After MIC determination, 100 μL from each well was placed on Sabouraud agar and then incubated at 35 °C for 72 h. The MFC was the lowest drug concentration that killed at least 99% of the yeast colonies in comparison with the drug-free control growth [23]. Each experiment was performed in triplicate.

In vivo studies with Cryptococcus neoformans clinical strains

Initially, the animals were immunosuppressed with dexamethasone to mimic a patient with HIV. Posteriorly, these animals were infected with C. neoformans and submitted to a treatment with CPO-LipoS to analyze the fungal burden in organs, including the spleen, liver, lungs, and brain. Additionally, histopathological analyses of the liver and kidneys of the immunosuppressed animals exposed to CPO-LipoS were conducted to investigate the possible toxic effects. The mice were handled according to established experimental procedures after receiving approval from the Animal Ethics Committee of the Federal University of Pernambuco (protocol #23076.003830), in conformity with international laws and policies and complying with ARRIVE guidelines [24].

Twenty-five 5- to 6-week-old male Mus musculus mice (35–40 g) obtained from the Multidisciplinary Center for Biological Research in Science: Laboratory Animals Division (CEMID, Campinas, SP, Brazil) were used in experiments after a 1-week adaptation period in the laboratory. The mice were randomly housed in a cage with sterile bedding and were kept under standard environmental conditions (12-h dark/light cycle), temperature (22 ± 2 °C), and humidity (60–70%). Autoclavable natural polypropylene cages were lined with wood shavings. Mice colonies were monitored every 3 days using a dirty-bedding sentinel routine. Sterilized water and dry food (Presence®, Purina, Brazil) were available ad libitum. The immunosuppression protocol was conducted using dexamethasone (Teuto®, Brazil) at 5 mg/mouse administered intraperitoneally 4 days before C. neoformans infection and once per week after infection, as described by Capilla et al. [15]. The total leukocyte counts in the blood of the immunosuppressed (IS, n = 20) compared to those immunocompetent (IC, n = 5) mice were used to confirm immunosuppression.

Histopathological analysis of the liver and kidneys in immunosuppressed mice after treatment with CPO-LipoS

Five IS mice not infected with approximately 40 g of weight were used for histopathological analysis. The mice were deprived of feed for 12 h but had access to water ad libitum. A volume of 0.2 mL of CPO-LipoS, corresponding to 0.5 mg/kg of CPO, was administered by the intravenous route to the IS mice daily for 2 weeks. The animals were observed over these 14 days for changes in behavior, weight, consumption of food and water, clinical signs of toxicity, and mortality, and these metrics were recorded daily. The organs chosen for this study were the liver and kidneys because CPO is metabolized in these organs [25]. The tissues were fixed in 10% formaldehyde, processed for paraffin embedding, and stained with hematoxylin–eosin for visualization of histological alterations. Histopathological analysis was performed using blind reading of samples by two investigators [26].

C. neoformans infection model and treatment with ciclopirox olamine encapsulated into liposomes

The experimental model of cryptococcosis was used in accordance with Medeiros et al. [23]. Immunosuppressed mice were randomly divided into 3 groups with 5 mice each (n = 15). Initially, the mice were challenged with 0.2 mL of C. neoformans URM5811 (10⁶ cells/mL) by tail vein. Seven days after infection, group 1 of infected mice were daily treated with CPO-LipoS (0.5 mg/kg/iv) for 14 days (n = 5/15); group 2 corresponds to infected and untreated mice (n = 5/15); and group 3 received daily empty liposomes for 14 days (n = 5/15) to evaluate any possible effects of the liposome constituents on in vivo fungal growth. The mice were observed daily for clinical signs and mortality until the end of the experiments in order to obtain the survival curve.

C. neoformans burden examination

The mice were euthanized by inhalation of halothane-saturated air (Halocarbon Laboratories, NJ, USA) in modified bell jars until death. After euthanasia, the spleen, liver, lungs, and brain were removed, weighed, and macerated in PBS (1:10 or 1:100, w/v) to verify C. neoformans burden. All samples were submitted to serial dilutions with PBS, and 100 µL aliquots were stained with China ink for the direct examination of C. neoformans. The 100 µL aliquots were transferred to plates of SDA with chloramphenicol (50 mg/mL) and incubated at 37 ± 2 °C for 48–72 h, and the CFU/g of the organs was counted [26].

Statistical analysis

Statistical analysis of the fractional experimental design was carried out using Tukey’s multiple comparison test, and in vivo data were analyzed by a one-way analysis of variance (ANOVA) with a significance level of p < 0.05, using OriginPro Academic 2015 (Origin Lab., Northampton, USA; Registration ID: KFW-DH3-9H3).

Results

Fractional experimental design of CPO-loaded liposomes

Eleven runs of two-level 2^4–1 fractional experimental design of CPO-LipoC and their responses are shown in Table 1. The variable PC concentration at higher levels (82.32 mM) compared to the lowest (41.16 mM) reduced the mean diameter of the liposomes (runs 2, 4, and 6). Tukey’s test revealed no significant differences (p < 0.05) in the PDI and EE% values. The intermediate Chol concentration (23.52 mM) reduced the mean diameter and PDI of the liposomes and improved the drug EE% (runs 9, 10, and 11). The intermediate SA concentration (5.88 mM) resulted in the lowest mean diameter and PDI of liposomes and the highest CPO EE%. In addition, the absence of SA induced drug precipitation after storing the formulations at 4 °C for 48 h. Finally, the highest CPO concentration (3 mg/mL) reduced the EE% from 96.6 ± 0.3% to 89.8 ± 0.2%.

Table 1.

2^4–1 Fractional experimental design of ciclopirox olamine-loaded liposomes

Liposome formulations (run)	CPO (mg/mL)	Lipids (mM)			Liposome properties
		SA	PC	Chol	Ø (nm)	PDI	CPO EE%
1	2	0.0	41.16	11.76	156.7 ± 3.8	0.359	98.6 ± 0.5
2	3	0.0	82.32	11.76	142.8 ± 3.1	0.310	96.6 ± 0.3
3	3	0.0	41.16	35.28	153.9 ± 3.8	0.319	95.9 ± 0.4
4	2	0.0	82.32	35.28	133.4 ± 0.3	0.472	93.6 ± 1.0
5	3	11.76	41.16	11.76	189.1 ± 4.5	0.481	90.6 ± 0.3
6	2	11.76	82.32	11.76	156.1 ± 1.5	0.297	98.7 ± 0.5
7	2	11.76	41.16	35.28	177.2 ± 2.4	0.557	89.6 ± 0.5
8	3	11.76	82.32	35.28	90.5 ± 1.0	0.435	89.8 ± 0.2
9 (c)	2.5	5.88	61.74	23.52	122.9 ± 1.4	0.274	97.5 ± 1.0
10 (c)	2.5	5.88	61.74	23.52	125.1 ± 1.4	0.301	97.6 ± 0.5
11 (c)	2.5	5.88	61.74	23.52	124.7 ± 0.4	0.264	97.4 ± 0.1

CPO, ciclopirox olamine; SA, stearylamine; PC, soya phosphatidylcholine; Chol, Cholesterol; Ø, mean size diameter; PDI, polydispersity index; EE%, drug encapsulation efficiency; c, central point

Based on the fractional experimental design, the formulations of the central point presented the lowest values for the mean size diameter (122.9 ± 1.4 nm to 125.1 ± 1.4 nm) and PDI (0.264 to 0.301) and a higher drug EE% (97.4 ± 0.1% to 97.5 ± 1.0%) (runs 9, 10, and 11). Thus, this formulation was chosen to prepare CPO-LipoS using DSPE-PEG2000. The CPO-LipoS formulation presented a mean size diameter, PDI, and EE% of 101.4 ± 0.7 nm, 0.307, and 96.4 ± 0.9%, respectively.

No significant differences (p > 0.05) were found in the physicochemical parameters (mean particle size, PDI and drug encapsulation efficiency) of the CPO-LipoC (formulation 11) and CPO-LipoS formulations. This result indicates that the surface pegylation of the liposomes did not affect their physicochemical parameters or CPO encapsulation.

In vitro antifungal activity of CPO-loaded liposomes

The results obtained for the antifungal activity of CPO-LipoC and CPO-LipoS against C. neoformans clinical isolates are shown in Table 2. CPO-LipoC and CPO-LipoS presented fungistatic activity at concentrations ranging from 1 to 2 mg/L and fungicidal activity at concentrations from 1 to 8 mg/L.

Table 2.

Susceptibility testing of ciclopirox olamine-loaded conventional and stealth liposomes against C. neoformans clinical strains

Cryptococcus neoformans clinical strains	CPO-LipoC		CPO-LipoS
	MIC	MFC	MIC	MFC
	mg/L		mg/L
URM 5809	2	8	2	8
URM 5810	1	2	1	2
URM 5811	1	1	1	1
URM 5812	1	2	1	2
URM 5813	1	1	1	1
URM 5814	1	2	1	2
URM 5815	1	2	1	2
URM 5816	1	2	1	2
URM 5818	1	8	1	8
URM 5819	2	8	2	8
URM 5820	1	2	1	2
URM 5821	1	8	1	8
URM 5822	2	8	2	8
URM 5823	2	8	2	8
URM 5824	1	1	1	1
URM 5825	1	1	1	1
URM 117	2	2	2	2
URM 123	1	2	1	2
URM 124	1	1	1	1
URM 125	1	2	1	2
URM 126	1	2	1	2
URM 127	1	2	1	2
URM 128	1	2	1	2
URM 129	2	2	2	2
URM 131	2	2	2	2
URM 132	2	4	2	4
URM 144	2	4	2	4
URM 150	1	2	1	2
URM 151	1	2	1	2
URM 155	1	2	1	2
URM 156	2	2	2	2
URM 157	1	2	1	2

CPO-LipoC, ciclopirox olamine-loaded conventional liposomes; CPO-LipoS, ciclopirox olamine-loaded stealth liposomes; MIC, minimum inhibitory concentration; MFC, minimum fungicidal concentration

Immunosuppression assay

As expected, a decrease in the total and differentiated white blood cell counts was found after 14 days of infection, except for the monocyte counts, which remained the same (p < 0.05) during the first week of the treatment of mice because of the administration of dexamethasone. No statistically significant differences in the cell counts of the tested mice groups were found. The white blood cell counts in the blood of the immunosuppressed mice (IS = 2.49 ± 0.05 × 10³/µL) were drastically diminished compared with that of the immunocompetent mice (IC = 9.52 ± 0.64 × 10³/µL) (p < 0.05).

Histopathological analysis of liver and kidneys in immunosuppressed mice after treatment with CPO-LipoS

All mice survived and seemed healthy after treatment with CPO-LipoS at 0.5 mg/kg for 2 weeks (n = 5/5). No significant changes in the intake of food and water or body weight were observed during the experiments. Moreover, the histological examination of the liver and kidneys of the animals treated with CPO-LipoS exhibited renal, glomerular, and normal tubule architectures (Fig. 1a), and hepatic parenchyma (Fig. 1b) without vascular alterations, indicating the absence of renal and hepatic toxicity of CPO-LipoS at 0.5 mg/kg in all evaluated animals (n = 5/5). Regarding the survival curve, all infected animals submitted to treatment with CPO-LipoS survived until the end of the experiment, while the infected and untreated animals died up to 72 h (Fig. 2).

Fig. 1.

Histopathological analysis of the kidneys (a) and liver (b) of immunosuppressed mice after exposure for 14 days of CPO-LipoS 0.5 mg/kg administered daily by the intravenous route

Fig. 2.

Survival rates of Cryptococcus neoformans infected Mus musculus mice followed by treatment with CPO-LipoS at 0.5 mg/kg/day for 14 days and untreated mice

C. neoformans burden after treatment with CPO-LipoS

Initially, C. neoformans URM 5811 produced a disseminated infection in IS mice that ranged from 10⁴ to 10⁶ CFU/g organ after 14 days of inoculation (p < 0.05). As expected, the mice that received empty liposomes exhibited a fungal burden similar to the untreated group. However, CPO-LipoS treatment reduced C. neoformans cells in the spleen, liver, lungs, and brain after 14 days of treatment compared to the untreated group (Fig. 3). The treatment caused significant decreases in C. neoformans burden, with 59.3 ± 3.4% in the spleen, 75.0 ± 3.6% in the liver, 75.7 ± 6.7% in the lungs, and 54.2 ± 3.2% in the brain (p < 0.05).

Fig. 3.

Cryptococcus neoformans burden in mice treated daily with CPO-LipoS 0.5 mg/kg, iv (dashed bar). After 7 days of mice infection, the treatment regimens were continued for 14 days. The percentage of organ fungal burden was analyzed in comparison with the infected mice without treatment (white bar)

Discussion

Cryptococcosis is one of the most important HIV-related opportunistic fungal infections, and it has high rates of persistence and recurrence. Along with the opportunistic aspect of this fungus, the ability of C. neoformans to induce systemic cryptococcosis raises concern [27, 28]. Thus, the most effective therapy for cryptococcosis is focused on drugs or nanosystems that target drugs to the non-lung targets of C. neoformans, such as the brain [29].

Oliveira et al. [10] determined the antifungal activity of CPO against clinical strains of C. neoformans to verify the applicability of this molecule and its in vitro activity. CPO exhibit MIC ranging from 0.25 to 1 mg/L and MFC from 0.25 to 4 mg/L against these C. neoformans strains. The observation of the in vitro results and the comparison of the results of the encapsulated molecule in vivo, it is possible to observe a maintenance in the inhibition efficiency of CPO against clinical strains of C. neoformans.

Studies have proposed drug combination therapy and encapsulation of toxic antifungal agents, such as CPO, into drug delivery systems, particularly in stealth liposomes, due to their ability to improve the drug therapeutic index and reduce toxicity [29–32]. From the initial formulation of this study, a 2^4–1 fractional experimental design was performed to verify the influence of lipid concentrations and the presence of a positively charged lipid, stearylamine, on the stability and drug entrapment efficiency of liposomes. The fractional experimental design indicated that formulation 11 (PC 61.74 mM, Chol 23.52 mM, SA 5.88 mM, and CPO 2.5 mg/mL) had remarkable result for liposomal formulation, i.e., a lower particle size and PDI and a higher %EE, with an economy of 33% and 50% in the amount of PC and SA, respectively, and an augmentation of 25% in the CPO content. Smaller particle size formulations are interesting for use in intravenous administration because they prevent nanocarriers from clogging vessels and because their low PDI values reflect that the formulation is homogenous in size [20, 21].

Comparing the in vitro antifungal activity of CPO, which has already been reported in the literature [31], with CPO-loaded conventional and stealth liposomes against C. neoformans clinical isolates described in our study, no significant difference was observed. Cavalcanti et al. [33] also observed this phenomenon when they evaluated the in vitro antifungal activity of β-lapachone encapsulated in conventional and stealth liposomes to combat C. neoformans. It can thus be suggested that the composition of liposomes containing CPO or β-lapachone did not influence their in vitro activity [33].

Conventional liposomes are rapidly recognized by the mononuclear phagocyte system acting on the macrophages, liver, and spleen. However, stealth liposomes present polyethylene glycol on their surface, which promotes a sterical stabilization of the vesicles and a long lifetime blood circulation. This stabilization enables the liposomes to have a prolonged circulating time without being opsonized, recognized, and cleared by the MPS, and it consequently improves their therapeutic efficacy, particularly on the brain and lungs, before being removed from the circulation [11,12,34,35,]. Thus, drugs that are encapsulated in stealth liposomes are important for treating cryptococcosis, which can cause fatal meningitis, especially in immunosuppressed patients [29, 33, 36]. In in vivo studies with CPO encapsulated in liposomes, the nanoformulation developed in this study preserved the kidneys and liver of CPO effects, as well as showed the potential of CPO-lipoS to protect mice against death by Cryptococcus infection. Moreover, the treatment with CPO-LipoS at 0.5 mg/kg/day by 2 weeks demonstrated an ability to reduce C. neoformans in the lungs (75.7 ± 6.7%) and brain (54.2 ± 3.2%), suggesting that long-term CPO-LipoS treatment may enhance sterilization in organs. In a previous study using a disseminated Aspergillus fumigatus infection in neutropenic mice, Wallace et al. [37] proposed the use of nystatin encapsulated in liposomes. This liposomal nystatin (2 mg/kg/day) sheltered neutropenic mice against Aspergillus-induced death; reduced the fungal burden in the lungs, spleen, pancreas, kidney, and liver; and did not cause histopathological damage in these organs [37]. Lewis et al. [38] used a higher dose of AmBisome® (> 5 mg/kg/day) in an experimental disseminated Aspergillus terreus infection. Therefore, Lewis et al. [38] demonstrated that a 10 mg/kg daily AmBisome® dose regimen prolonged survival in neutropenic mice and significantly reduced fungal burden in the lung compared to infected mice without treatment. Studies that use liposomes as drug carriers for treating fungal infections are scarce, especially in a cryptococcal infection model, which involves a quantitative analysis of the fungi in organs. Thus, the relevance and novelty of our study were to propose encapsulating CPO in liposomes to treat systemic cryptococcal infections and the use of a low dose of CPO-LipoS treatment regimens (0.5 mg/kg/day), which is sufficient to promote a significant reduction of the fungal burden in a systemic cryptococcal infection model.

Other animal models carried out with rats, guinea pigs, rabbits, and dogs have demonstrated the toxicity of CPO in the liver, lung, and heart [8, 39–41]. Our data showed that CPO-LipoS at 0.5 mg/kg did not cause histopathological alterations in the liver or kidneys of immunosuppressed mice after 2 weeks of daily exposure. These findings indicated that liposomes could prevent the possible side effects of CPO in the main organs responsible for drug metabolization, such as the liver and kidneys [41].

Conclusion

Based on the findings of this study, the ciclopirox olamine-loaded stealth liposomes exhibited in vitro and in vivo antifungal activity without inducing histopathological alterations in the liver and kidneys. Thus, this developed formulation may be considered a promising nanocarrier for further clinical trials of CPO in systemic infections, such as meningeal cryptococcosis, especially in immunocompromised patients.

Author contribution

POK designed the project, executed the laboratorial methodology, analyzed the data, and wrote the article. PHSS, HFSL, SDCJ, and PGC assisted in the laboratory experiments, data analysis, and writing the manuscript. RGLN and RPN performed analysis of antifungal activity. NTPF and JVMLF contributed to in vivo studies. IMFC and NSSM supervised the laboratory experiments and contributed to the critic evaluation of the manuscript. All the authors have read the manuscript and approved its submission.

Funding

The current study was supported by the CNPq [n. 474777/2013–8, n. 484574/2011-6 and n. 477215/2013-0] and by the Science and Technology Support Foundation of Pernambuco State (FACEPE) [APQ-0814–4.03/17 and APQ-0287-4.03/22].

Data availability

All data generated or analysed during this study are included in this published article.

Declarations

Consent to participate

All authors approved the manuscript.

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Conflict of interest

The authors declare no competing interests.