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Antileishmanial and cytotoxic activities of ionic surfactants compared to those of miltefosine
Colloids and Surfaces B: Biointerfaces 183 (2019) 110421
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Antileishmanial and cytotoxic activities of ionic surfactants compared to those of miltefosine
T
Lais Alonsoa, Éder Jeferson Souza Cardosob, Rodrigo Saar Gomesc, Sebastião Antônio Mendanhab, ⁎ Miriam Leandro Dortac, Antonio Alonsob, a
Instituto Federal Goiano, Trindade, GO, Brazil Instituto de Física, Universidade Federal de Goiás, Goiânia, GO, Brazil c Instituto de Patologia Tropical e Saúde Publica, Departamento de Imunologia e Patologia Geral, Universidade Federal de Goiás, Goiânia, GO, Brazil b
A R T I C LE I N FO
A B S T R A C T
Keywords: Surfactant Miltefosine Leishmania Macrophages Membrane fluidity Electron paramagnetic resonance
Using the electron paramagnetic resonance (EPR) of spin-labeled stearic acid and a spin label chemically attached to the membrane proteins, the interaction of miltefosine (MIL) and the ionic surfactants sodium dodecyl sulfate (SDS, anionic), cetyltrimethylammonium chloride (CTAC, cationic) and N-hexadecyl-N,N-dimethyl-3ammonio-1-propanesulfonate (HPS, zwitterionic) with the plasma membrane of Leishmania (L.) amazonensis promastigotes was studied. The spin-label EPR data indicated that the four compounds studied have the ability to increase the molecular dynamics of membrane proteins to a large extent. Compared to the other compounds, SDS produced the smallest increases in dynamics and demonstrated the lowest antileishmanial activity and cytotoxicity to J774.A1 macrophages. The activities of the other three compounds were not different from each other, but CTAC had a stronger activity against L. amazonensis promastigotes at higher cellular concentrations (> 1 × 109 cells/mL) and was the most effective against L. amazonensis-infected macrophages. However, CTAC was also the most cytotoxic to macrophages. By measuring the IC50/CC50 values for assays of different cell concentrations, we estimated the membrane-water partition coefficient (KM/W) as well as the concentrations in the membrane (cm50) and aqueous phase (cw50) of the compounds at their IC50/CC50. Compared to the other compounds, SDS showed the lowest value of KM/W and the highest value of cm50. In all experiments in this study, the data for the zwitterionic molecules HPS and MIL were not significantly different.
1. Introduction Cutaneous leishmaniasis (CL) causes skin lesions and is the most common form of the disease. It is estimated that between 600 000 and 1 million new cases occur worldwide annually; the 10 countries with the highest number of CL cases reported in 2016 were Afghanistan, Algeria, Brazil, Colombia, Iraq, Pakistan, Peru, the Syrian Arab Republic, Tunisia and Yemen, which together account for 84% of globally reported CL incidence [1]. The systemic treatment options for CL are pentavalent antimonial, pentamidine, paromomycin sulfate, miltefosine (MIL) and ketoconazole [2]. MIL is the only oral drug approved for the treatment of leishmaniasis and is an example of successful research and development for a neglected tropical disease that fails to reach the people who need it [3]. The drug was first registered in India in 2002 to treat fatal visceral leishmaniasis and is currently approved in many countries, including the United States (2014) [1]. In Brazil, the Ministry of Health decided to incorporate MIL as a first-line treatment for CL
⁎
within Brazil's Unified Health System (the Sistema Único de Saúde (SUS) (D.O.U., Portaria Nº 56, October 30, 2018)). MIL is an alkylphospholipid that has demonstrated activity against various strains of Acanthamoeba [4], a broad spectrum of pathogenic fungi [5], several Leishmania species [6], Trypanosoma cruzi [7], Streptococcus pneumoniae [8] and various types of tumor cells [9]. MIL also has anti-HIV activity by inhibiting the PI3K/Akt pathway in primary human macrophages, which play a role in vivo as long-lived HIV-1 reservoirs [10]. On the other hand, the oral administration of MIL results in severe toxicity to the epithelial cells of the gastrointestinal tract [11,12] and commonly induces side effects such as anorexia, nausea, vomiting and diarrhea [2]. However, the mechanism of action of MIL is still not well established. Understanding the modes of action of MIL against parasites, fungi and bacteria could help in the rational design of more potent and less toxic analogs. Ionic surfactant molecules bind to proteins through a set of electrostatic and hydrophobic interactions [13]. Accumulating data from in
Corresponding author. E-mail address: [email protected] (A. Alonso).
https://doi.org/10.1016/j.colsurfb.2019.110421 Received 10 April 2019; Received in revised form 8 July 2019; Accepted 2 August 2019 Available online 05 August 2019 0927-7765/ © 2019 Elsevier B.V. All rights reserved.
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concentration of MIL, SDS, CTAC or HPS used, and the IC50 or CC50 values of the compounds were then determined by adjusting the concentration response data to a sigmoid curve.
vitro and in vivo tests have suggested that nonionic surfactants are nontoxic to the skin, whereas cationic surfactants are more toxic than anionic surfactants [14–16]. When a cell membrane is exposed to increasing amounts of surfactant, it initially undergoes non-cooperative interactions where surfactant monomers are incorporated without disrupting the membrane structure; the next phase is cooperative interactions and saturation, where surfactant monomers co-operatively aggregate within the membrane to generate small fragments with concomitant solubilization with the formation of lipid-protein-surfactant complexes [13]. The specific interactions between ionic surfactants and membrane proteins are not yet well understood. Surfactant molecules have been proposed to bind as annular surfactants, forming a micellar belt around proteins with alkyl chains in contact with the highly hydrophobic transmembrane areas of the protein [17]. Spin-label EPR spectroscopy has demonstrated that MIL possesses a detergent-like action on Leishmania and erythrocyte membrane proteins [18–20]. A fatty acid spin label incorporated into the cell membrane in annular or boundary lipid configuration can provide dynamic information on the hydrophobic surface of transmembrane proteins and has demonstrated that MIL can bind in large amounts to membrane proteins, causing dramatic increases in probe mobility. Another spin label preferentially covalently bound to SH groups located at the periphery of the plasma membrane indicated that MIL increases the protein backbone dynamics and causes structural changes to more water-exposed protein conformations. Since the EPR spectrum changes caused by MIL-protein interactions were similar to those observed for interactions of ionic surfactants with BSA [21,22], we decided to carry out a comparative study between MIL and the surfactants SDS, CTAC and HPS with respect to the effect on the cell membranes, the cytotoxicity in macrophages and the antileishmanial activity. Determining biophysical parameters such as the membrane-water partition coefficient of the compounds allowed us to obtain more information regarding the interactions of ionic surfactants with biological membranes and the action of MIL on the Leishmania and macrophage membranes.
2.3. J774.A1 macrophage infection J774.A1 cells were cultured in RPMI 1640 medium (Sigma-Aldrich) supplemented with 10% FCS, 2 mM L-glutamine, 11 mM sodium bicarbonate, 100 U/mL penicillin and 100 μg/mL streptomycin. A total of 4 × 106 cells/mL were infected for 3 h with green fluorescent protein (GFP)-labeled L. amazonensis (IFLA/BR/67/PH8; 5 parasites per cell) on the 6th day of growth. GFP-labeled L. amazonensis promastigotes were cultivated in vitro in Grace’s insect medium supplemented with 10% FCS. Frequently, the GFP-labeled parasites were selected by using 30 μg/mL Hygromycin B [23]. After 3 h of infection, the cells were washed with 1x PBS for removal of non-internalized parasites and cultured for an additional 24 h in the presence of MIL or surfactants at different concentrations. The cells were then collected on ice by the mechanical harvesting of the adherent cells with the aid of cell scrapers (Thermo Scientific). The cells were washed 2 times with PBS, incubated with 1% paraformaldehyde and analyzed by flow cytometry in a BD Accuri™ C6 Flow Cytometry instrument (BD Bioscience, San Jose, CA, USA). Cells were identified by size and complexity (Forward Scatter x Side Scatter), and cellular debris were not analyzed. The data were analyzed using FlowJo software (Tree Star, Ashland, OR, USA). Macrophages were selected by forward versus side scatter (FSC vs SSC), and the percentage of cells expressing GFP+ (infected cells) was evaluated.
2.4. Spin-labeling and EPR spectroscopy To incorporate the lipid spin-label 5-doxyl-stearic acid (5-DSA; Sigma-Aldrich) into the Leishmania membranes, a spin-label film was first prepared on the bottom of a test tube, as described previously [20]. A 1 μL aliquot of a stock solution of 5-DSA (4 mg/mL) in ethanol was transferred to a glass test tube, and after evaporating the solvent, 50 μL Grace’s insect medium containing 1 × 108 cells was added to the spinlabel film, followed by gentle agitation. The spin-labeling of the Leishmania membrane proteins was performed by incubating a cell suspension with 2 mM of the spin label 4-maleimido-2,2,6,6-tetramethylpiperidine-1-oxyl (6-MSL, Sigma-Aldrich) for 90 min at 26 °C. To remove the free spin label, the sample was centrifuged (3,000xg, 4 °C) for 10 min and then resuspended in Grace’s insect medium. This procedure was repeated six times. After the spin-labeling, the samples were treated by adding Grace’s insect medium containing one of the test molecules. For the EPR measurements, the samples were transferred to 1 mm i.d. capillary tubes, which were sealed by flame. An EPR EMX-Plus spectrometer of Bruker (Rheinstetten, Germany) was used to perform the EPR measurements. Spectra were recorded using the following instrumental settings: microwave power, 2 mW; modulation frequency, 100 kHz; modulation amplitude, 1.0 G; magnetic field scan, 100 G; sweep time, 168 s; and sample temperature, 25 °C.
2. Materials and methods 2.1. Cells Promastigotes of Leishmania (L.) amazonensis (MHOM/BR/75/ Josefa) reference strains were grown in 24-well microtiter plates containing 2 mL of Grace’s insect medium (Sigma-Aldrich, St. Louis, MO, USA) supplemented with 20% heat-inactivated fetal bovine serum (FCS) (Corning Life Sciences, Corning, NY, USA), 2 mM L-glutamine, 100 U/mL penicillin G and 100 μg/mL streptomycin (Sigma-Aldrich), as previously described [19,20]. The tests were performed as soon as these parasites reached the logarithmic phase of growth (6th day of growth). The J774.A1 murine macrophage cell line was acquired from the cell bank of Rio de Janeiro (NCE/UFRJ). Cells were maintained in RPMI 1640 medium supplemented with 10% FCS, 2 mM L-glutamine, 100 U/ mL penicillin G and 100 μg/mL streptomycin at 37 °C in a humidified atmosphere (RH ˜95%) with 5% CO2. 2.2. In vitro assays of antiproliferative activity and cytotoxicity in macrophages Parasites or macrophages at several cell concentrations were treated with increasing concentrations of MIL (Avanti Polar Lipids Inc., Alabaster, AL, USA) or surfactants SDS, CTAC and HPS (Sigma-Aldrich) in culture medium supplemented with 10% FCS. After incubation for 24 h in 96-well culture dishes (100 μL for parasites and 200 μL for macrophages) at 26 °C for promastigotes and 37 °C for macrophages, the cell viability was assessed by measuring the reduction in 3-(4,5dimethylthiazol-2-yl)-2,5-diphenyl tetrazolium bromide (MTT; SigmaAldrich) by metabolically active cells, as described previously [20]. The percentage of viable cells relative to the control was calculated for each
2.5. Statistical analysis Data are expressed as the mean ± S.D. of at least three independent experiments. Comparisons between different groups were performed using one-way analysis of variance (ANOVA). Tukey’s test was used to identify significant differences between means among the different treatments. Statistical significance was accepted as p < 0.05.
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membrane proteins [24–26]. Because of these interactions with the transmembrane proteins, 5-DSA can monitor the dynamics at the periphery of proteins into the lipid bilayer. Thus, the changes in 5-DSA spectra caused by surfactants and MIL are mainly associated with changes in the dynamics of the membrane protein component. Previous studies have shown that MIL does not cause changes in model membranes when prepared using a rigorous extrusion process. For instance, the EPR spectra of 5-DSA in extruded vesicles of 1,2-dipalmitoyl-snglycero-3-phosphocholine (DPPC) showed no changes up to 35 mol% MIL [27]. Notably, these experiments were performed using high cell concentrations (2 × 109 cells/mL). In a previous work, for this cellular concentration, the MIL IC50 value increased to approximately 1 mM (0.3 × 109 molecules/cell), and at this MIL concentration, a significant change in the 2A// parameter was already observed, indicating an association between the inhibition of parasitic growth and changes in the protein dynamics of the cell membrane [20]. The EPR spectra from Fig. 1 also show that the protein dynamics increase progressively over a long concentration range of approximately 1 to 20 mM MIL or surfactants, and this result is in accordance with the view that these compounds bind in large amounts to membrane proteins. The EPR spectra of spin-labeled proteins, such as those presented for 6-MSL in Fig. 1B, are generally composed of two spectral components corresponding to two populations of probes differing in motility, which are technically denoted by strongly (S) and weakly (W) immobilized components [28,29]. Although the site-directed spin-labeling method has been widely used to examine the protein structure [30], the origin of the S and W components is still not well understood. As described in previous works [20,22], our interpretation is that the component of greater mobility, W, which presents three sharp resonance lines that are predominant in the EPR spectra from Fig. 1B, has an isotropic hyperfine splitting, 2a0, of 17.1 G, resulting from a fraction of the nitroxide moiety probes dissolved in the buffer. On the other hand, the lower mobility of component, S, whose low and high magnetic field resonance lines were used to measure parameter 2A// (Fig. 1B, control), is due to strong interactions between the nitroxide side chain and the protein backbone. Among these interactions, a hydrogen bond is formed between the nitroxide radical and the lateral polypeptide chain, as deduced from the observed value for the z-component of the 14N-hyperfine tensor, Azz, which may be assessed from the spectral simulation or measured at temperatures less than −140 °C (the value of 2A// tends to 2Azz = ˜70 G). These two components are in thermodynamic equilibrium, and low temperatures favor the formation of the S component. At the instant that the nitroxide side chain is hindered in the protein (S configuration), its mobility reflects the segmental motion of the protein backbone.
Fig. 1. EPR spectra of spin-labeled 5-DSA incorporated in Leishmania amazonensis promastigote membranes (panel A) and 6-MSL covalently bound to the SH groups (panel B) of Leishmania membrane proteins for samples of untreated cells (control) and cells treated with four concentrations of HPS, CTAC or SDS or two concentrations of MIL. The concentrations are expressed as the number of molecules per cell. The values of the EPR parameter 2A// (outer hyperfine splitting), which is given by the separation in magnetic field units between the first peak and the last inverted peak of the spectrum, are also indicated. The estimated experimental error for the 2A// parameter was 0.5 G. The total scan range of the magnetic field in each EPR spectrum was 100 G (X axis), and the intensity is in arbitrary units (Y axis).
3. Results
3.2. Growth at inhibitory and cytotoxic concentrations of the compounds are assay cell-concentration dependent
3.1. MIL and the surfactants SDS, CTAC and HPS increase the molecular dynamics of Leishmania membrane proteins
Surfactants and MIL IC50 values measured for L. amazonensis promastigotes were dependent on the cell concentrations used in the experiments (Fig. 2). These compounds also showed a cell-concentrationdependent behavior for CC50 values measured in the J774.A1 murine macrophage cell line (Fig. 3). Hydrophobic molecules accumulate in the cell membrane and exhibit this type of behavior because they have an inhomogeneous distribution in the cell suspension. Thus, the growth inhibitory or cytotoxicity concentration being measured decreases with the amount of cell membrane in the suspension. The equation describing the variation in the IC50 or CC50 values with the cell concentration in the assay has as covariates the membrane-water partition coefficient (KM/W) of the test molecule as well as the molecule concentrations in the cell membrane (cm50) and aqueous phase (cw50) at the IC50 (or CC50). The derivation of this equation was demonstrated in previous works [20], and in this work, it is re-presented below with some descriptive modifications. At the IC50, the tested molecule in the cell suspension is distributed
Fig. 1 shows the EPR spectra of the spin labels 5-DSA (Fig. 1A) and 6-MSL (Fig. 1B) in Leishmania membranes for the samples untreated and treated with several concentrations of MIL and surfactants. For all the treated samples, the EPR spectra of both spin labels showed large reductions in parameter 2A// (outer hyperfine splitting), indicating remarkable increases in molecular dynamics. For treatment with a concentration of 6 × 109 molecules/cell, the 6-MSL spectra showed a decreased signal-to-noise ratio compared to untreated cells, which was due to the reduction in the nitroxide radical by exposure to the cytoplasm content, indicating cell lysis. The changes in the EPR spectra caused by the surfactants at several concentrations were similar, except for SDS and probe 5-DSA, in which the parameter 2A// showed considerably higher values for SDS at concentrations of 1 and 3 × 109 molecules/cell. In the cell membrane, the probe 5-DSA behaves as annular or boundary lipids that preferentially surround the hydrophobic surface of 3
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membrane, respectively, in the suspension. As KM/W = cm50/cw50, Eq. 1 can be rewritten as follows:
IC50 =
c w50 Vw + KM/W cw50 Vm (2)
Vw + Vm
or
(Vw /Vm) + KM/W ⎤ c w50 . IC50 = ⎡ ⎢ ⎣ (Vw /Vm) + 1 ⎥ ⎦
(3)
Eq. 3 shows that the IC50 values trend towards the cw value for diluted samples (Vw > > Vm) or for low KM/W values (hydrophilic drugs). However, for hydrophobic molecules and higher cell concentrations, IC50 will be much larger than cw50. To calculate Vm, it is necessary to estimate the membrane volume of a single cell (Vmc). In a previous work [20], a Vmc of 8.17 × 10−13 mL was estimated for the Leishmania amazonensis promastigote. In J774.A1 macrophages, a mean cell diameter of 16.5 μm [31] was previously reported. Assuming a cell membrane thickness of 7.8 nm [20], the Vmc estimated for macrophages is 66.7 × 10−13 mL. However, an error in membrane volume proportionally affects the KM/W value with an inverse relationship. In Eq. 3, Vm can be replaced by Vmc multiplied by the number of cells per mL (cc). In a 1-mL suspension, Vw is approximately equal to 1 mL because the Vm is very small (less than 1 × 10−3 mL). Thus, substituting Vw/Vm with (Vmc.cc)-1, we can rewrite Eq. 3 as follows:
Fig. 2. IC50 values of the surfactants SDS, CTAC and HPS for Leishmania amazonensis promastigotes at several cell concentrations used in the beginning of the experiment. The curve for MIL from previous work [20] was plotted for comparison with those from the surfactants. The best-fit curves shown are based on Eq. 4.
(Vm . cc)−1 + KM/W ⎤ c w50 . IC50 = ⎡ −1 ⎢ ⎣ (Vm . cc) + 1 ⎥ ⎦
(4)
In contrast to the IC50 or CC50 values, the cw50 and cm50 are constants whose values are independent of the cell concentration used in the assay. 3.3. Best-fit parameters obtained from fittings using Eq. 4 The parameters KM/W, cm50 and cw50 obtained from the fit curves shown in Figs. 2 and 3 are presented in Table 1. SDS showed lower affinities for L. amazonensis and macrophage membranes as deduced from the lower KM/W values and was the molecule that required higher Table 1 Biophysical parameters associated with surfactants and MIL interactions with plasma membranes of L. amazonensis promastigotes, J774.A1 macrophages and erythrocytes. Compound
SDS CTAC HPS MILc SDS CTAC HPS MIL
Fig. 3. CC50 values of surfactants SDS, CTAC and HPS and MIL in the J774.A1 murine macrophage cell line for the several cell concentrations used to start the assay. The best-fit curves shown are based on Eq. 4.
SDSd CTACd HPSd MILc
between the aqueous phase and the cell membrane. The number of moles in these two environments can be denoted by nw50 and nm50, respectively. Thus, the IC50 value can be expressed as follows:
IC50 =
nw50 + nm50 , Vw + Vm
KM/W (104)a
log KM/W
L. amazonensis promastigotes 3.82 0.7 ± 0.1 (A)b 2.5 ± 0.3 (B) 4.39 8.1 ± 1.2 (C) 4.91 6.8 ± 0.3 (C) 4.83 J774.A1 macrophages 2.6 ± 0.1 (A) 4.41 4.2 ± 0.3 (B) 4.63 5.5 ± 0.2 (C) 4.74 5.4 ± 0.5 (C) 4.73 Erythrocytes 0.7 3.85 6.8 4.83 5.9 4.77 4.8 4.68
cw50 (μM)
cm50 (M)
404.3 ± 14.3 (A) 11.1 ± 0.6 (B) 10.6 ± 2.3 (B) 10.8 ± 3.0 (B)
2.7 0.3 0.9 0.7
± ± ± ±
0.5 0.1 0.3 0.3
(A) (B) (C) (C)
75.0 12.4 39.8 43.2
1.9 0.5 2.2 2.3
± ± ± ±
0.1 0.1 0.1 0.2
(A) (B) (C) (C)
69.2 2.5 2.6 2.3
± ± ± ±
1.0 0.4 1.1 2.2
(A) (B) (C) (C)
0.5 0.2 0.1 0.1
c,d Data (means) reported in references [20,27] and [32], respectively, and are shown for comparison. a Best-fit parameters obtained by fitting of Eq. 4 on the data presented in Figs. 2 and 3; KM/W, membrane-water partition coefficient; cw50 and cm50, molecular concentrations in the aqueous phase and membrane, respectively, at the IC50, CC50 or HC50 values. b Statistical significance: in each column, the data that are not shown with the same capital letter are significantly different at P < 0.05.
(1)
where Vw and Vm are the volumes of the aqueous medium and 4
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4. Discussion In the experiments shown in Fig. 1, the concentration of 1 × 109 molecules/cell corresponds to 3.3 mM, but the cell concentration used was also quite high (2 × 109 cells/mL). This condition was required for the EPR experiments since capillary-conditioned samples have a maximum volume of 50 μL and at least 1 × 108 cells are required to obtain a good signal. The EPR spectra for high concentrations of MIL and surfactants (Fig. 1) allowed the observation of the great alterations that these compounds can cause in the parasite membrane. In a previous work [20], using a high cell concentration (2 × 109 cells/mL) the MIL IC50 in L. amazonensis promastigotes was approximately 1.1 mM, and the EPR experiment showed a significant change in the cell membrane for this MIL concentration. However, previous results have shown that the EPR spectra of 5-DSA do not detect changes in the erythrocyte membrane at the MIL concentration for 50% hemolysis (HC50). For 18% hematocrit (˜2 × 109 cells/mL), ˜1.1 mM MIL was required to observe significant changes in the probe mobility [32], but for this cell concentration, the MIL HC50 was only 260 μM in PBS [33]. These data are consistent with the cm50 values shown in Table 1 for the erythrocyte (cm50 = 0.1 M) and leishmania parasite (cm50 = 0.7 M) and appear to indicate that the hemolytic effect of MIL on the erythrocyte membrane is a punctuated or well-localized alteration, in contrast to the case of the parasite in which the membrane changes appear to be broad and widespread enough to be detected by EPR spectroscopy. According to this interpretation, MIL would have the ability to interact with many sites of the parasite membrane proteins, and therefore, a higher concentration would be required to damage the membrane; however, in the erythrocyte, MIL would accumulate in certain protein regions, causing more localized damage, and lower concentrations would be sufficient to cause hemolysis. As shown in Fig. 1, ionic surfactants and MIL (zwitterionic) strongly interact with membrane proteins, and it is well known that this type of interaction is favored by molecular charges. Thus, the polar head group of the MIL molecule where the electric charges are present should be responsible for the interaction with the proteins, while the hydrophobic chain must interact with the membrane lipids and thus modulate the interaction with the proteins. It seems reasonable to obtain analogs of MIL with the capacity to cause more punctuated damage to the membrane. Perhaps the addition of hydrophobic groups such as aromatic rings at the end of the fatty chain may favor the accumulation of the drug at certain points in the lipidprotein interface. Although probe EPR spectroscopy is a technique of remarkable sensitivity and reproducibility to assess the molecular dynamics in the cellular membrane, in both the lipid and protein components, EPR spectroscopy is not sensitive enough to detect localized changes in the membrane. As a technique using a spin probe with a molecular ratio of less than one for every 120 unlabeled lipid molecules, the probes become spaced apart on the membrane and, in consequence, membrane changes are detected by the EPR signal only when a considerable fraction of the probes is affected. Thus, electrolyte leakages or cell lysis are events that can be detected at lower concentrations of cell-damaging agents. It is generally accepted that MIL has detergent properties and may cause cell lysis at higher concentrations; however, at clinically relevant concentrations, it is assumed that MIL may cause interference in the phospholipid turnover and lipid-based signal transduction pathways that could initiate the apoptotic machinery [34]. However, in vitro assays indicate that MIL inhibits the growth of parasites only at relatively high concentrations. For instance, a MIL IC50 of approximately 14.4 μM in L. amazonensis promastigotes was observed in the assay with 6 × 106 cells/mL (Fig. 2); this concentration corresponds to 1.44 × 109 molecules/cell. On the other hand, for assays with more physiologically relevant cell concentrations, such as 2 × 109 cells/mL (in blood, there are ˜5 × 109 cells/mL), the MIL IC50 rises to ˜1.2 mM (eq. 4), which corresponds to 0.36 × 109 molecules/cell. Assays using this last
Fig. 4. Leishmanicidal action of SDS, CTAC and HPS surfactants on L. amazonensis-infected J774.A1 macrophages. Miltefosine (MIL) was used as a positive control. Statistical significance: *p < 0.05, compared to control; #p < 0.05, surfactant versus MIL, in each concentration.
membrane concentrations to cause rupture than the other molecules, as indicated by the cm50 data (Table 1). The data in Table 1 also indicated a similar performance for MIL and HPS in the interaction with the plasma membranes of the three cell types.
3.4. Effect of surfactants against intracellular parasites compared to MIL To examine whether the SDS, CTAC and HPS surfactants are capable of affecting L. amazonensis infection in J774.A1 macrophages, the cells were infected with L. amazonensis labeled with GFP for 3 h and treated with different concentrations of the compounds for an additional 24 h before being analyzed by flow cytometry. MIL was adopted as a positive control (Fig. 4). The concentrations of each compound required to cause a significant change over the control samples had the following relationship: CTAC < MIL = HPS < SDS.
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concentration of cells (2 × 109 cells/mL) showed at least ˜5% cell lysis in samples treated with MIL at 0.44 × 109 molecules/cell [19]. It is important to mention that EPR spectroscopy did not detect differences in the interaction of MIL with the promastigote and amastigote forms of L. amazonensis. Additionally, the cw50 values for the two forms were not significantly different. MIL demonstrated greater affinity for the amastigote membrane, but it was better tolerated by the membrane (cm50 much larger than that of the promastigote) [20]. The results of this work are consistent with the accumulation of the compounds studied in the parasitic membrane. For instance, when MIL is added to a suspension of L. amazonensis promastigotes at its IC50, it will be distributed in the aqueous and membrane phases at the estimated concentrations of 10.8 μM (cw50) and 0.73 M (cm50), for any cell concentration used in the experiment. Resistance of Leishmania to MIL has been associated with decreased drug internalization due to an increase in its efflux mediated by overexpression of the ABC transporter P-glycoprotein or inactivation of any one of the two proteins responsible for MIL uptake, the MIL transporter LdMT and its beta subunit LdRos3 [35]. We believe that the LdMT-LdRos3-dependent flippase machinery potentiates the activity of MIL against Leishmania parasites, not because it favors drug internalization but because it promotes a rapid distribution of the drug on the two leaflets of the plasma membrane [20]. Similarly, the overexpression of ABC transporters acting as MIL floppases could slow this process, thus reducing the action of MIL on the parasitic membrane. In a previous work [32], the EPR spectroscopy of 5-DSA was used to analyze the effects of the four compounds studied in this work on the presence of albumin in the blood plasma. At a concentration of 2.5 mM, SDS caused a strong reduction in the albumin dynamics, while the CTAC increased the molecular dynamics and the HPS and MIL did not cause change compared to the control. At 10 mM, all the compounds increased the albumin dynamics compared to the control, but SDS caused the smallest increase compared to the other compounds. SDS also caused minor increases in the dynamics of the membrane proteins of Leishmania (Fig. 1A) and erythrocytes [32]. This result is in agreement with the fact that SDS was the compound that presented higher cm50 in both Leishmania and erythrocytes than the other compounds (Table 1). The KM/W values shown in Table 1 indicated that the affinities of the ionic detergents for the studied cell membranes follow a relationship that seems to be associated with the electrical charge of the compound. The zwitterionic compounds, HPS and MIL, presented the highest KM/W values, the anionic compound, SDS, had the lowest KM/W value, and the cationic CTAC presented intermediate values of KM/W for Leishmania amazonensis and J774.A1 macrophages. The cell surface of erythrocytes has a negative charge that occurs due to the presence of the carboxyl group of sialic acids in the cell membrane [36]. Promastigote and amastigote forms of some species of Leishmania also have a negative surface charge, as detected by cationic particles [37,38]. The electric charge interactions could explain the lower affinity of the anionic SDS in relation to the cationic CTAC and suggest that the zwitterionic compounds would more easily accumulate in the membrane because they have a neutral total charge. All three surfactants were able to reduce the parasitic load on macrophages after 24 h of treatment in a dose-dependent manner. Our data demonstrated that the surfactants are able to kill L. amazonensis internalized by macrophages. For J774.A1 macrophages, a MIL cw50 value of 43.2 ± 2.2 μM was found (Table 1), and this value was much lower than the reported value of 132 ± 8 μM found for peritoneal macrophages obtained from BALB/c mice [20]. Thus, using J774.A1 macrophages, the selectivity index (SI = CC50/IC50) among the Leishmania and macrophage cells became poor and the reduction in the percentage of cells expressing GFP + appears to occur at concentrations of MIL that are already cytotoxic to macrophages. In a recent report, cetyl-trimethylammonium bromide (CTAB), an analogue of CTAC, has been included in a study of the antileishmanial activity and cytotoxicity of 30 generally regarded as safe (GRAS)
excipients [39]. For CTAB, the authors found an IC50 of 8.5 μM for L. amazonensis and a CC50 of 94.4 μM for J774 macrophages. While the IC50 value was close to that found by us (11.1 μM, Table 1), the CC50 was quite different (12–40 μM, Fig. 3); perhaps this discrepancy is related to the degree of adherence of the macrophages at the time of treatment. On the other hand, the MIL CC50 value of J774 macrophages found by us (43.2 μM, Table 1) was close to that reported in another study (56.1 μM) [40]. The observed IC50 of MIL on L. amazonensis (10.8 μM, Table 1) was similar to those reported for L. tropica and L. infantum and lower than that for L. major (22 μM) [41,42]. For L. donovani promastigotes, an IC50 of 3.1 μM was observed, suggesting that this Leishmania species is more sensitive to MIL [43]. This work also suggests that the differences in the sensitivity to MIL between the various species of Leishmania may be related to the lipid composition, cholesterol content and characteristics of the protein component of the cell membrane. 5. Conclusions In this work, we determined three biophysical parameters resulting from the interactions of MIL and three ionic surfactants with the membranes of Leishmania and macrophages, which helped to analyze the modes of action of these compounds on these membranes. Compared to the other compounds, SDS was the least aggressive to these membranes and presented the highest cw50 value. This result may be explained by the lower affinity of SDS for these membranes (lower KM/W values) and fewer localized effects on the membranes than those of the other compounds (with lower cm50 values). The lower membrane affinity presented by SDS than the other compounds may be explained by the fact that these cells have negative surface charges, and SDS is anionic. SDS was also the compound that required higher concentrations to increase the molecular dynamics of Leishmania membrane proteins than that of the other compounds. CTAC was the most aggressive compound for the Leishmania and macrophage membranes, with more localized effects on the membrane (lower cm50 values) than those of the other compounds, possibly due to the accumulation of positive charges at specific membrane sites. For all experiments, the results of the zwitterionic compounds HPS and MIL were not significantly different. This comparative analysis of MIL with ionic surfactants contributes to a better understanding of MIL's mechanism of action on Leishmania parasites, which seems to be associated with the fact that the molecule is zwitterionic. This suggests that the primary action of MIL is on the parasite membrane proteins, in which it has the ability to accumulate, causing protein expansion and the consequent impairment of the cell membrane functions. Since CL is the most common form of leishmaniasis, it may be possible to use low-cost zwitterionic (or cationic) surfactants in skin care formulations, with the additional goal of treating CL. Declaration of Competing Interest The authors declare no conflicts of interest. Acknowledgements This study was financially supported by grants from the Brazilian research funding agencies CNPq (445666/2014-5 and 406521/2016-6), CAPES and FAPEG (201210267001110). Lais Alonso is a recipient of a postdoctoral fellowship from CNPq (150369/2018-2). Antonio Alonso received a research grant from CNPq (303829/2016-8). References [1] World Health Organization, Leishmaniasis 14 March 2018, World Health Organization, Geneva, Switzerland, 2018http://www.who.int/news-room/factsheets/detail/leishmaniasis.
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Antileishmanial and cytotoxic activities of ionic surfactants compared to those of miltefosine
T
Lais Alonsoa, Éder Jeferson Souza Cardosob, Rodrigo Saar Gomesc, Sebastião Antônio Mendanhab, ⁎ Miriam Leandro Dortac, Antonio Alonsob, a
Instituto Federal Goiano, Trindade, GO, Brazil Instituto de Física, Universidade Federal de Goiás, Goiânia, GO, Brazil c Instituto de Patologia Tropical e Saúde Publica, Departamento de Imunologia e Patologia Geral, Universidade Federal de Goiás, Goiânia, GO, Brazil b
A R T I C LE I N FO
A B S T R A C T
Keywords: Surfactant Miltefosine Leishmania Macrophages Membrane fluidity Electron paramagnetic resonance
Using the electron paramagnetic resonance (EPR) of spin-labeled stearic acid and a spin label chemically attached to the membrane proteins, the interaction of miltefosine (MIL) and the ionic surfactants sodium dodecyl sulfate (SDS, anionic), cetyltrimethylammonium chloride (CTAC, cationic) and N-hexadecyl-N,N-dimethyl-3ammonio-1-propanesulfonate (HPS, zwitterionic) with the plasma membrane of Leishmania (L.) amazonensis promastigotes was studied. The spin-label EPR data indicated that the four compounds studied have the ability to increase the molecular dynamics of membrane proteins to a large extent. Compared to the other compounds, SDS produced the smallest increases in dynamics and demonstrated the lowest antileishmanial activity and cytotoxicity to J774.A1 macrophages. The activities of the other three compounds were not different from each other, but CTAC had a stronger activity against L. amazonensis promastigotes at higher cellular concentrations (> 1 × 109 cells/mL) and was the most effective against L. amazonensis-infected macrophages. However, CTAC was also the most cytotoxic to macrophages. By measuring the IC50/CC50 values for assays of different cell concentrations, we estimated the membrane-water partition coefficient (KM/W) as well as the concentrations in the membrane (cm50) and aqueous phase (cw50) of the compounds at their IC50/CC50. Compared to the other compounds, SDS showed the lowest value of KM/W and the highest value of cm50. In all experiments in this study, the data for the zwitterionic molecules HPS and MIL were not significantly different.
1. Introduction Cutaneous leishmaniasis (CL) causes skin lesions and is the most common form of the disease. It is estimated that between 600 000 and 1 million new cases occur worldwide annually; the 10 countries with the highest number of CL cases reported in 2016 were Afghanistan, Algeria, Brazil, Colombia, Iraq, Pakistan, Peru, the Syrian Arab Republic, Tunisia and Yemen, which together account for 84% of globally reported CL incidence [1]. The systemic treatment options for CL are pentavalent antimonial, pentamidine, paromomycin sulfate, miltefosine (MIL) and ketoconazole [2]. MIL is the only oral drug approved for the treatment of leishmaniasis and is an example of successful research and development for a neglected tropical disease that fails to reach the people who need it [3]. The drug was first registered in India in 2002 to treat fatal visceral leishmaniasis and is currently approved in many countries, including the United States (2014) [1]. In Brazil, the Ministry of Health decided to incorporate MIL as a first-line treatment for CL
⁎
within Brazil's Unified Health System (the Sistema Único de Saúde (SUS) (D.O.U., Portaria Nº 56, October 30, 2018)). MIL is an alkylphospholipid that has demonstrated activity against various strains of Acanthamoeba [4], a broad spectrum of pathogenic fungi [5], several Leishmania species [6], Trypanosoma cruzi [7], Streptococcus pneumoniae [8] and various types of tumor cells [9]. MIL also has anti-HIV activity by inhibiting the PI3K/Akt pathway in primary human macrophages, which play a role in vivo as long-lived HIV-1 reservoirs [10]. On the other hand, the oral administration of MIL results in severe toxicity to the epithelial cells of the gastrointestinal tract [11,12] and commonly induces side effects such as anorexia, nausea, vomiting and diarrhea [2]. However, the mechanism of action of MIL is still not well established. Understanding the modes of action of MIL against parasites, fungi and bacteria could help in the rational design of more potent and less toxic analogs. Ionic surfactant molecules bind to proteins through a set of electrostatic and hydrophobic interactions [13]. Accumulating data from in
Corresponding author. E-mail address: [email protected] (A. Alonso).
https://doi.org/10.1016/j.colsurfb.2019.110421 Received 10 April 2019; Received in revised form 8 July 2019; Accepted 2 August 2019 Available online 05 August 2019 0927-7765/ © 2019 Elsevier B.V. All rights reserved.
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concentration of MIL, SDS, CTAC or HPS used, and the IC50 or CC50 values of the compounds were then determined by adjusting the concentration response data to a sigmoid curve.
vitro and in vivo tests have suggested that nonionic surfactants are nontoxic to the skin, whereas cationic surfactants are more toxic than anionic surfactants [14–16]. When a cell membrane is exposed to increasing amounts of surfactant, it initially undergoes non-cooperative interactions where surfactant monomers are incorporated without disrupting the membrane structure; the next phase is cooperative interactions and saturation, where surfactant monomers co-operatively aggregate within the membrane to generate small fragments with concomitant solubilization with the formation of lipid-protein-surfactant complexes [13]. The specific interactions between ionic surfactants and membrane proteins are not yet well understood. Surfactant molecules have been proposed to bind as annular surfactants, forming a micellar belt around proteins with alkyl chains in contact with the highly hydrophobic transmembrane areas of the protein [17]. Spin-label EPR spectroscopy has demonstrated that MIL possesses a detergent-like action on Leishmania and erythrocyte membrane proteins [18–20]. A fatty acid spin label incorporated into the cell membrane in annular or boundary lipid configuration can provide dynamic information on the hydrophobic surface of transmembrane proteins and has demonstrated that MIL can bind in large amounts to membrane proteins, causing dramatic increases in probe mobility. Another spin label preferentially covalently bound to SH groups located at the periphery of the plasma membrane indicated that MIL increases the protein backbone dynamics and causes structural changes to more water-exposed protein conformations. Since the EPR spectrum changes caused by MIL-protein interactions were similar to those observed for interactions of ionic surfactants with BSA [21,22], we decided to carry out a comparative study between MIL and the surfactants SDS, CTAC and HPS with respect to the effect on the cell membranes, the cytotoxicity in macrophages and the antileishmanial activity. Determining biophysical parameters such as the membrane-water partition coefficient of the compounds allowed us to obtain more information regarding the interactions of ionic surfactants with biological membranes and the action of MIL on the Leishmania and macrophage membranes.
2.3. J774.A1 macrophage infection J774.A1 cells were cultured in RPMI 1640 medium (Sigma-Aldrich) supplemented with 10% FCS, 2 mM L-glutamine, 11 mM sodium bicarbonate, 100 U/mL penicillin and 100 μg/mL streptomycin. A total of 4 × 106 cells/mL were infected for 3 h with green fluorescent protein (GFP)-labeled L. amazonensis (IFLA/BR/67/PH8; 5 parasites per cell) on the 6th day of growth. GFP-labeled L. amazonensis promastigotes were cultivated in vitro in Grace’s insect medium supplemented with 10% FCS. Frequently, the GFP-labeled parasites were selected by using 30 μg/mL Hygromycin B [23]. After 3 h of infection, the cells were washed with 1x PBS for removal of non-internalized parasites and cultured for an additional 24 h in the presence of MIL or surfactants at different concentrations. The cells were then collected on ice by the mechanical harvesting of the adherent cells with the aid of cell scrapers (Thermo Scientific). The cells were washed 2 times with PBS, incubated with 1% paraformaldehyde and analyzed by flow cytometry in a BD Accuri™ C6 Flow Cytometry instrument (BD Bioscience, San Jose, CA, USA). Cells were identified by size and complexity (Forward Scatter x Side Scatter), and cellular debris were not analyzed. The data were analyzed using FlowJo software (Tree Star, Ashland, OR, USA). Macrophages were selected by forward versus side scatter (FSC vs SSC), and the percentage of cells expressing GFP+ (infected cells) was evaluated.
2.4. Spin-labeling and EPR spectroscopy To incorporate the lipid spin-label 5-doxyl-stearic acid (5-DSA; Sigma-Aldrich) into the Leishmania membranes, a spin-label film was first prepared on the bottom of a test tube, as described previously [20]. A 1 μL aliquot of a stock solution of 5-DSA (4 mg/mL) in ethanol was transferred to a glass test tube, and after evaporating the solvent, 50 μL Grace’s insect medium containing 1 × 108 cells was added to the spinlabel film, followed by gentle agitation. The spin-labeling of the Leishmania membrane proteins was performed by incubating a cell suspension with 2 mM of the spin label 4-maleimido-2,2,6,6-tetramethylpiperidine-1-oxyl (6-MSL, Sigma-Aldrich) for 90 min at 26 °C. To remove the free spin label, the sample was centrifuged (3,000xg, 4 °C) for 10 min and then resuspended in Grace’s insect medium. This procedure was repeated six times. After the spin-labeling, the samples were treated by adding Grace’s insect medium containing one of the test molecules. For the EPR measurements, the samples were transferred to 1 mm i.d. capillary tubes, which were sealed by flame. An EPR EMX-Plus spectrometer of Bruker (Rheinstetten, Germany) was used to perform the EPR measurements. Spectra were recorded using the following instrumental settings: microwave power, 2 mW; modulation frequency, 100 kHz; modulation amplitude, 1.0 G; magnetic field scan, 100 G; sweep time, 168 s; and sample temperature, 25 °C.
2. Materials and methods 2.1. Cells Promastigotes of Leishmania (L.) amazonensis (MHOM/BR/75/ Josefa) reference strains were grown in 24-well microtiter plates containing 2 mL of Grace’s insect medium (Sigma-Aldrich, St. Louis, MO, USA) supplemented with 20% heat-inactivated fetal bovine serum (FCS) (Corning Life Sciences, Corning, NY, USA), 2 mM L-glutamine, 100 U/mL penicillin G and 100 μg/mL streptomycin (Sigma-Aldrich), as previously described [19,20]. The tests were performed as soon as these parasites reached the logarithmic phase of growth (6th day of growth). The J774.A1 murine macrophage cell line was acquired from the cell bank of Rio de Janeiro (NCE/UFRJ). Cells were maintained in RPMI 1640 medium supplemented with 10% FCS, 2 mM L-glutamine, 100 U/ mL penicillin G and 100 μg/mL streptomycin at 37 °C in a humidified atmosphere (RH ˜95%) with 5% CO2. 2.2. In vitro assays of antiproliferative activity and cytotoxicity in macrophages Parasites or macrophages at several cell concentrations were treated with increasing concentrations of MIL (Avanti Polar Lipids Inc., Alabaster, AL, USA) or surfactants SDS, CTAC and HPS (Sigma-Aldrich) in culture medium supplemented with 10% FCS. After incubation for 24 h in 96-well culture dishes (100 μL for parasites and 200 μL for macrophages) at 26 °C for promastigotes and 37 °C for macrophages, the cell viability was assessed by measuring the reduction in 3-(4,5dimethylthiazol-2-yl)-2,5-diphenyl tetrazolium bromide (MTT; SigmaAldrich) by metabolically active cells, as described previously [20]. The percentage of viable cells relative to the control was calculated for each
2.5. Statistical analysis Data are expressed as the mean ± S.D. of at least three independent experiments. Comparisons between different groups were performed using one-way analysis of variance (ANOVA). Tukey’s test was used to identify significant differences between means among the different treatments. Statistical significance was accepted as p < 0.05.
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membrane proteins [24–26]. Because of these interactions with the transmembrane proteins, 5-DSA can monitor the dynamics at the periphery of proteins into the lipid bilayer. Thus, the changes in 5-DSA spectra caused by surfactants and MIL are mainly associated with changes in the dynamics of the membrane protein component. Previous studies have shown that MIL does not cause changes in model membranes when prepared using a rigorous extrusion process. For instance, the EPR spectra of 5-DSA in extruded vesicles of 1,2-dipalmitoyl-snglycero-3-phosphocholine (DPPC) showed no changes up to 35 mol% MIL [27]. Notably, these experiments were performed using high cell concentrations (2 × 109 cells/mL). In a previous work, for this cellular concentration, the MIL IC50 value increased to approximately 1 mM (0.3 × 109 molecules/cell), and at this MIL concentration, a significant change in the 2A// parameter was already observed, indicating an association between the inhibition of parasitic growth and changes in the protein dynamics of the cell membrane [20]. The EPR spectra from Fig. 1 also show that the protein dynamics increase progressively over a long concentration range of approximately 1 to 20 mM MIL or surfactants, and this result is in accordance with the view that these compounds bind in large amounts to membrane proteins. The EPR spectra of spin-labeled proteins, such as those presented for 6-MSL in Fig. 1B, are generally composed of two spectral components corresponding to two populations of probes differing in motility, which are technically denoted by strongly (S) and weakly (W) immobilized components [28,29]. Although the site-directed spin-labeling method has been widely used to examine the protein structure [30], the origin of the S and W components is still not well understood. As described in previous works [20,22], our interpretation is that the component of greater mobility, W, which presents three sharp resonance lines that are predominant in the EPR spectra from Fig. 1B, has an isotropic hyperfine splitting, 2a0, of 17.1 G, resulting from a fraction of the nitroxide moiety probes dissolved in the buffer. On the other hand, the lower mobility of component, S, whose low and high magnetic field resonance lines were used to measure parameter 2A// (Fig. 1B, control), is due to strong interactions between the nitroxide side chain and the protein backbone. Among these interactions, a hydrogen bond is formed between the nitroxide radical and the lateral polypeptide chain, as deduced from the observed value for the z-component of the 14N-hyperfine tensor, Azz, which may be assessed from the spectral simulation or measured at temperatures less than −140 °C (the value of 2A// tends to 2Azz = ˜70 G). These two components are in thermodynamic equilibrium, and low temperatures favor the formation of the S component. At the instant that the nitroxide side chain is hindered in the protein (S configuration), its mobility reflects the segmental motion of the protein backbone.
Fig. 1. EPR spectra of spin-labeled 5-DSA incorporated in Leishmania amazonensis promastigote membranes (panel A) and 6-MSL covalently bound to the SH groups (panel B) of Leishmania membrane proteins for samples of untreated cells (control) and cells treated with four concentrations of HPS, CTAC or SDS or two concentrations of MIL. The concentrations are expressed as the number of molecules per cell. The values of the EPR parameter 2A// (outer hyperfine splitting), which is given by the separation in magnetic field units between the first peak and the last inverted peak of the spectrum, are also indicated. The estimated experimental error for the 2A// parameter was 0.5 G. The total scan range of the magnetic field in each EPR spectrum was 100 G (X axis), and the intensity is in arbitrary units (Y axis).
3. Results
3.2. Growth at inhibitory and cytotoxic concentrations of the compounds are assay cell-concentration dependent
3.1. MIL and the surfactants SDS, CTAC and HPS increase the molecular dynamics of Leishmania membrane proteins
Surfactants and MIL IC50 values measured for L. amazonensis promastigotes were dependent on the cell concentrations used in the experiments (Fig. 2). These compounds also showed a cell-concentrationdependent behavior for CC50 values measured in the J774.A1 murine macrophage cell line (Fig. 3). Hydrophobic molecules accumulate in the cell membrane and exhibit this type of behavior because they have an inhomogeneous distribution in the cell suspension. Thus, the growth inhibitory or cytotoxicity concentration being measured decreases with the amount of cell membrane in the suspension. The equation describing the variation in the IC50 or CC50 values with the cell concentration in the assay has as covariates the membrane-water partition coefficient (KM/W) of the test molecule as well as the molecule concentrations in the cell membrane (cm50) and aqueous phase (cw50) at the IC50 (or CC50). The derivation of this equation was demonstrated in previous works [20], and in this work, it is re-presented below with some descriptive modifications. At the IC50, the tested molecule in the cell suspension is distributed
Fig. 1 shows the EPR spectra of the spin labels 5-DSA (Fig. 1A) and 6-MSL (Fig. 1B) in Leishmania membranes for the samples untreated and treated with several concentrations of MIL and surfactants. For all the treated samples, the EPR spectra of both spin labels showed large reductions in parameter 2A// (outer hyperfine splitting), indicating remarkable increases in molecular dynamics. For treatment with a concentration of 6 × 109 molecules/cell, the 6-MSL spectra showed a decreased signal-to-noise ratio compared to untreated cells, which was due to the reduction in the nitroxide radical by exposure to the cytoplasm content, indicating cell lysis. The changes in the EPR spectra caused by the surfactants at several concentrations were similar, except for SDS and probe 5-DSA, in which the parameter 2A// showed considerably higher values for SDS at concentrations of 1 and 3 × 109 molecules/cell. In the cell membrane, the probe 5-DSA behaves as annular or boundary lipids that preferentially surround the hydrophobic surface of 3
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membrane, respectively, in the suspension. As KM/W = cm50/cw50, Eq. 1 can be rewritten as follows:
IC50 =
c w50 Vw + KM/W cw50 Vm (2)
Vw + Vm
or
(Vw /Vm) + KM/W ⎤ c w50 . IC50 = ⎡ ⎢ ⎣ (Vw /Vm) + 1 ⎥ ⎦
(3)
Eq. 3 shows that the IC50 values trend towards the cw value for diluted samples (Vw > > Vm) or for low KM/W values (hydrophilic drugs). However, for hydrophobic molecules and higher cell concentrations, IC50 will be much larger than cw50. To calculate Vm, it is necessary to estimate the membrane volume of a single cell (Vmc). In a previous work [20], a Vmc of 8.17 × 10−13 mL was estimated for the Leishmania amazonensis promastigote. In J774.A1 macrophages, a mean cell diameter of 16.5 μm [31] was previously reported. Assuming a cell membrane thickness of 7.8 nm [20], the Vmc estimated for macrophages is 66.7 × 10−13 mL. However, an error in membrane volume proportionally affects the KM/W value with an inverse relationship. In Eq. 3, Vm can be replaced by Vmc multiplied by the number of cells per mL (cc). In a 1-mL suspension, Vw is approximately equal to 1 mL because the Vm is very small (less than 1 × 10−3 mL). Thus, substituting Vw/Vm with (Vmc.cc)-1, we can rewrite Eq. 3 as follows:
Fig. 2. IC50 values of the surfactants SDS, CTAC and HPS for Leishmania amazonensis promastigotes at several cell concentrations used in the beginning of the experiment. The curve for MIL from previous work [20] was plotted for comparison with those from the surfactants. The best-fit curves shown are based on Eq. 4.
(Vm . cc)−1 + KM/W ⎤ c w50 . IC50 = ⎡ −1 ⎢ ⎣ (Vm . cc) + 1 ⎥ ⎦
(4)
In contrast to the IC50 or CC50 values, the cw50 and cm50 are constants whose values are independent of the cell concentration used in the assay. 3.3. Best-fit parameters obtained from fittings using Eq. 4 The parameters KM/W, cm50 and cw50 obtained from the fit curves shown in Figs. 2 and 3 are presented in Table 1. SDS showed lower affinities for L. amazonensis and macrophage membranes as deduced from the lower KM/W values and was the molecule that required higher Table 1 Biophysical parameters associated with surfactants and MIL interactions with plasma membranes of L. amazonensis promastigotes, J774.A1 macrophages and erythrocytes. Compound
SDS CTAC HPS MILc SDS CTAC HPS MIL
Fig. 3. CC50 values of surfactants SDS, CTAC and HPS and MIL in the J774.A1 murine macrophage cell line for the several cell concentrations used to start the assay. The best-fit curves shown are based on Eq. 4.
SDSd CTACd HPSd MILc
between the aqueous phase and the cell membrane. The number of moles in these two environments can be denoted by nw50 and nm50, respectively. Thus, the IC50 value can be expressed as follows:
IC50 =
nw50 + nm50 , Vw + Vm
KM/W (104)a
log KM/W
L. amazonensis promastigotes 3.82 0.7 ± 0.1 (A)b 2.5 ± 0.3 (B) 4.39 8.1 ± 1.2 (C) 4.91 6.8 ± 0.3 (C) 4.83 J774.A1 macrophages 2.6 ± 0.1 (A) 4.41 4.2 ± 0.3 (B) 4.63 5.5 ± 0.2 (C) 4.74 5.4 ± 0.5 (C) 4.73 Erythrocytes 0.7 3.85 6.8 4.83 5.9 4.77 4.8 4.68
cw50 (μM)
cm50 (M)
404.3 ± 14.3 (A) 11.1 ± 0.6 (B) 10.6 ± 2.3 (B) 10.8 ± 3.0 (B)
2.7 0.3 0.9 0.7
± ± ± ±
0.5 0.1 0.3 0.3
(A) (B) (C) (C)
75.0 12.4 39.8 43.2
1.9 0.5 2.2 2.3
± ± ± ±
0.1 0.1 0.1 0.2
(A) (B) (C) (C)
69.2 2.5 2.6 2.3
± ± ± ±
1.0 0.4 1.1 2.2
(A) (B) (C) (C)
0.5 0.2 0.1 0.1
c,d Data (means) reported in references [20,27] and [32], respectively, and are shown for comparison. a Best-fit parameters obtained by fitting of Eq. 4 on the data presented in Figs. 2 and 3; KM/W, membrane-water partition coefficient; cw50 and cm50, molecular concentrations in the aqueous phase and membrane, respectively, at the IC50, CC50 or HC50 values. b Statistical significance: in each column, the data that are not shown with the same capital letter are significantly different at P < 0.05.
(1)
where Vw and Vm are the volumes of the aqueous medium and 4
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4. Discussion In the experiments shown in Fig. 1, the concentration of 1 × 109 molecules/cell corresponds to 3.3 mM, but the cell concentration used was also quite high (2 × 109 cells/mL). This condition was required for the EPR experiments since capillary-conditioned samples have a maximum volume of 50 μL and at least 1 × 108 cells are required to obtain a good signal. The EPR spectra for high concentrations of MIL and surfactants (Fig. 1) allowed the observation of the great alterations that these compounds can cause in the parasite membrane. In a previous work [20], using a high cell concentration (2 × 109 cells/mL) the MIL IC50 in L. amazonensis promastigotes was approximately 1.1 mM, and the EPR experiment showed a significant change in the cell membrane for this MIL concentration. However, previous results have shown that the EPR spectra of 5-DSA do not detect changes in the erythrocyte membrane at the MIL concentration for 50% hemolysis (HC50). For 18% hematocrit (˜2 × 109 cells/mL), ˜1.1 mM MIL was required to observe significant changes in the probe mobility [32], but for this cell concentration, the MIL HC50 was only 260 μM in PBS [33]. These data are consistent with the cm50 values shown in Table 1 for the erythrocyte (cm50 = 0.1 M) and leishmania parasite (cm50 = 0.7 M) and appear to indicate that the hemolytic effect of MIL on the erythrocyte membrane is a punctuated or well-localized alteration, in contrast to the case of the parasite in which the membrane changes appear to be broad and widespread enough to be detected by EPR spectroscopy. According to this interpretation, MIL would have the ability to interact with many sites of the parasite membrane proteins, and therefore, a higher concentration would be required to damage the membrane; however, in the erythrocyte, MIL would accumulate in certain protein regions, causing more localized damage, and lower concentrations would be sufficient to cause hemolysis. As shown in Fig. 1, ionic surfactants and MIL (zwitterionic) strongly interact with membrane proteins, and it is well known that this type of interaction is favored by molecular charges. Thus, the polar head group of the MIL molecule where the electric charges are present should be responsible for the interaction with the proteins, while the hydrophobic chain must interact with the membrane lipids and thus modulate the interaction with the proteins. It seems reasonable to obtain analogs of MIL with the capacity to cause more punctuated damage to the membrane. Perhaps the addition of hydrophobic groups such as aromatic rings at the end of the fatty chain may favor the accumulation of the drug at certain points in the lipidprotein interface. Although probe EPR spectroscopy is a technique of remarkable sensitivity and reproducibility to assess the molecular dynamics in the cellular membrane, in both the lipid and protein components, EPR spectroscopy is not sensitive enough to detect localized changes in the membrane. As a technique using a spin probe with a molecular ratio of less than one for every 120 unlabeled lipid molecules, the probes become spaced apart on the membrane and, in consequence, membrane changes are detected by the EPR signal only when a considerable fraction of the probes is affected. Thus, electrolyte leakages or cell lysis are events that can be detected at lower concentrations of cell-damaging agents. It is generally accepted that MIL has detergent properties and may cause cell lysis at higher concentrations; however, at clinically relevant concentrations, it is assumed that MIL may cause interference in the phospholipid turnover and lipid-based signal transduction pathways that could initiate the apoptotic machinery [34]. However, in vitro assays indicate that MIL inhibits the growth of parasites only at relatively high concentrations. For instance, a MIL IC50 of approximately 14.4 μM in L. amazonensis promastigotes was observed in the assay with 6 × 106 cells/mL (Fig. 2); this concentration corresponds to 1.44 × 109 molecules/cell. On the other hand, for assays with more physiologically relevant cell concentrations, such as 2 × 109 cells/mL (in blood, there are ˜5 × 109 cells/mL), the MIL IC50 rises to ˜1.2 mM (eq. 4), which corresponds to 0.36 × 109 molecules/cell. Assays using this last
Fig. 4. Leishmanicidal action of SDS, CTAC and HPS surfactants on L. amazonensis-infected J774.A1 macrophages. Miltefosine (MIL) was used as a positive control. Statistical significance: *p < 0.05, compared to control; #p < 0.05, surfactant versus MIL, in each concentration.
membrane concentrations to cause rupture than the other molecules, as indicated by the cm50 data (Table 1). The data in Table 1 also indicated a similar performance for MIL and HPS in the interaction with the plasma membranes of the three cell types.
3.4. Effect of surfactants against intracellular parasites compared to MIL To examine whether the SDS, CTAC and HPS surfactants are capable of affecting L. amazonensis infection in J774.A1 macrophages, the cells were infected with L. amazonensis labeled with GFP for 3 h and treated with different concentrations of the compounds for an additional 24 h before being analyzed by flow cytometry. MIL was adopted as a positive control (Fig. 4). The concentrations of each compound required to cause a significant change over the control samples had the following relationship: CTAC < MIL = HPS < SDS.
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concentration of cells (2 × 109 cells/mL) showed at least ˜5% cell lysis in samples treated with MIL at 0.44 × 109 molecules/cell [19]. It is important to mention that EPR spectroscopy did not detect differences in the interaction of MIL with the promastigote and amastigote forms of L. amazonensis. Additionally, the cw50 values for the two forms were not significantly different. MIL demonstrated greater affinity for the amastigote membrane, but it was better tolerated by the membrane (cm50 much larger than that of the promastigote) [20]. The results of this work are consistent with the accumulation of the compounds studied in the parasitic membrane. For instance, when MIL is added to a suspension of L. amazonensis promastigotes at its IC50, it will be distributed in the aqueous and membrane phases at the estimated concentrations of 10.8 μM (cw50) and 0.73 M (cm50), for any cell concentration used in the experiment. Resistance of Leishmania to MIL has been associated with decreased drug internalization due to an increase in its efflux mediated by overexpression of the ABC transporter P-glycoprotein or inactivation of any one of the two proteins responsible for MIL uptake, the MIL transporter LdMT and its beta subunit LdRos3 [35]. We believe that the LdMT-LdRos3-dependent flippase machinery potentiates the activity of MIL against Leishmania parasites, not because it favors drug internalization but because it promotes a rapid distribution of the drug on the two leaflets of the plasma membrane [20]. Similarly, the overexpression of ABC transporters acting as MIL floppases could slow this process, thus reducing the action of MIL on the parasitic membrane. In a previous work [32], the EPR spectroscopy of 5-DSA was used to analyze the effects of the four compounds studied in this work on the presence of albumin in the blood plasma. At a concentration of 2.5 mM, SDS caused a strong reduction in the albumin dynamics, while the CTAC increased the molecular dynamics and the HPS and MIL did not cause change compared to the control. At 10 mM, all the compounds increased the albumin dynamics compared to the control, but SDS caused the smallest increase compared to the other compounds. SDS also caused minor increases in the dynamics of the membrane proteins of Leishmania (Fig. 1A) and erythrocytes [32]. This result is in agreement with the fact that SDS was the compound that presented higher cm50 in both Leishmania and erythrocytes than the other compounds (Table 1). The KM/W values shown in Table 1 indicated that the affinities of the ionic detergents for the studied cell membranes follow a relationship that seems to be associated with the electrical charge of the compound. The zwitterionic compounds, HPS and MIL, presented the highest KM/W values, the anionic compound, SDS, had the lowest KM/W value, and the cationic CTAC presented intermediate values of KM/W for Leishmania amazonensis and J774.A1 macrophages. The cell surface of erythrocytes has a negative charge that occurs due to the presence of the carboxyl group of sialic acids in the cell membrane [36]. Promastigote and amastigote forms of some species of Leishmania also have a negative surface charge, as detected by cationic particles [37,38]. The electric charge interactions could explain the lower affinity of the anionic SDS in relation to the cationic CTAC and suggest that the zwitterionic compounds would more easily accumulate in the membrane because they have a neutral total charge. All three surfactants were able to reduce the parasitic load on macrophages after 24 h of treatment in a dose-dependent manner. Our data demonstrated that the surfactants are able to kill L. amazonensis internalized by macrophages. For J774.A1 macrophages, a MIL cw50 value of 43.2 ± 2.2 μM was found (Table 1), and this value was much lower than the reported value of 132 ± 8 μM found for peritoneal macrophages obtained from BALB/c mice [20]. Thus, using J774.A1 macrophages, the selectivity index (SI = CC50/IC50) among the Leishmania and macrophage cells became poor and the reduction in the percentage of cells expressing GFP + appears to occur at concentrations of MIL that are already cytotoxic to macrophages. In a recent report, cetyl-trimethylammonium bromide (CTAB), an analogue of CTAC, has been included in a study of the antileishmanial activity and cytotoxicity of 30 generally regarded as safe (GRAS)
excipients [39]. For CTAB, the authors found an IC50 of 8.5 μM for L. amazonensis and a CC50 of 94.4 μM for J774 macrophages. While the IC50 value was close to that found by us (11.1 μM, Table 1), the CC50 was quite different (12–40 μM, Fig. 3); perhaps this discrepancy is related to the degree of adherence of the macrophages at the time of treatment. On the other hand, the MIL CC50 value of J774 macrophages found by us (43.2 μM, Table 1) was close to that reported in another study (56.1 μM) [40]. The observed IC50 of MIL on L. amazonensis (10.8 μM, Table 1) was similar to those reported for L. tropica and L. infantum and lower than that for L. major (22 μM) [41,42]. For L. donovani promastigotes, an IC50 of 3.1 μM was observed, suggesting that this Leishmania species is more sensitive to MIL [43]. This work also suggests that the differences in the sensitivity to MIL between the various species of Leishmania may be related to the lipid composition, cholesterol content and characteristics of the protein component of the cell membrane. 5. Conclusions In this work, we determined three biophysical parameters resulting from the interactions of MIL and three ionic surfactants with the membranes of Leishmania and macrophages, which helped to analyze the modes of action of these compounds on these membranes. Compared to the other compounds, SDS was the least aggressive to these membranes and presented the highest cw50 value. This result may be explained by the lower affinity of SDS for these membranes (lower KM/W values) and fewer localized effects on the membranes than those of the other compounds (with lower cm50 values). The lower membrane affinity presented by SDS than the other compounds may be explained by the fact that these cells have negative surface charges, and SDS is anionic. SDS was also the compound that required higher concentrations to increase the molecular dynamics of Leishmania membrane proteins than that of the other compounds. CTAC was the most aggressive compound for the Leishmania and macrophage membranes, with more localized effects on the membrane (lower cm50 values) than those of the other compounds, possibly due to the accumulation of positive charges at specific membrane sites. For all experiments, the results of the zwitterionic compounds HPS and MIL were not significantly different. This comparative analysis of MIL with ionic surfactants contributes to a better understanding of MIL's mechanism of action on Leishmania parasites, which seems to be associated with the fact that the molecule is zwitterionic. This suggests that the primary action of MIL is on the parasite membrane proteins, in which it has the ability to accumulate, causing protein expansion and the consequent impairment of the cell membrane functions. Since CL is the most common form of leishmaniasis, it may be possible to use low-cost zwitterionic (or cationic) surfactants in skin care formulations, with the additional goal of treating CL. Declaration of Competing Interest The authors declare no conflicts of interest. Acknowledgements This study was financially supported by grants from the Brazilian research funding agencies CNPq (445666/2014-5 and 406521/2016-6), CAPES and FAPEG (201210267001110). Lais Alonso is a recipient of a postdoctoral fellowship from CNPq (150369/2018-2). Antonio Alonso received a research grant from CNPq (303829/2016-8). References [1] World Health Organization, Leishmaniasis 14 March 2018, World Health Organization, Geneva, Switzerland, 2018http://www.who.int/news-room/factsheets/detail/leishmaniasis.
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