Effect of the Amphotericin B and Its Copper Complex on a Model of the

Subscriber access provided by CORNELL UNIVERSITY LIBRARY

Article

The Effect of the Amphotericin B and its Copper Complex on a Model of the Outer Leaflet of Human Erythrocyte Membrane Marta Arczewska, Grzegorz Czernel, and Mariusz Gagos J. Phys. Chem. B, Just Accepted Manuscript • DOI: 10.1021/acs.jpcb.6b08555 • Publication Date (Web): 10 Oct 2016 Downloaded from http://pubs.acs.org on October 13, 2016

Just Accepted “Just Accepted” manuscripts have been peer-reviewed and accepted for publication. They are posted online prior to technical editing, formatting for publication and author proofing. The American Chemical Society provides “Just Accepted” as a free service to the research community to expedite the dissemination of scientific material as soon as possible after acceptance. “Just Accepted” manuscripts appear in full in PDF format accompanied by an HTML abstract. “Just Accepted” manuscripts have been fully peer reviewed, but should not be considered the official version of record. They are accessible to all readers and citable by the Digital Object Identifier (DOI®). “Just Accepted” is an optional service offered to authors. Therefore, the “Just Accepted” Web site may not include all articles that will be published in the journal. After a manuscript is technically edited and formatted, it will be removed from the “Just Accepted” Web site and published as an ASAP article. Note that technical editing may introduce minor changes to the manuscript text and/or graphics which could affect content, and all legal disclaimers and ethical guidelines that apply to the journal pertain. ACS cannot be held responsible for errors or consequences arising from the use of information contained in these “Just Accepted” manuscripts.

The Journal of Physical Chemistry B is published by the American Chemical Society. 1155 Sixteenth Street N.W., Washington, DC 20036 Published by American Chemical Society. Copyright © American Chemical Society. However, no copyright claim is made to original U.S. Government works, or works produced by employees of any Commonwealth realm Crown government in the course of their duties.

Page 1 of 43

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

The Journal of Physical Chemistry

The Effect of the Amphotericin B and its Copper Complex on a Model of the Outer Leaflet of Human Erythrocyte Membrane

Marta Arczewska1, Grzegorz Czernel1 and Mariusz Gagoś2

1

Department of Biophysics, University of Life Sciences in Lublin, 20-950 Lublin, Poland,

2

Department of Cell Biology, Institute of Biology and Biotechnology, Maria CurieSkłodowska of University, 20-033 Lublin, Poland

Corresponding authors: Marta Arczewska Department of Biophysics University of Life Sciences in Lublin 20–950 Lublin, E–mail: [email protected] Mariusz Gagoś Department of Cell Biology Institute of Biology and Biotechnology, Maria Curie–Skłodowska University, 20–033 Lublin. E–mail: [email protected]

1

ACS Paragon Plus Environment


1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

Page 2 of 43

ABSTRACT

In this work, a comparative study on the effect of an antibiotic amphotericin B (AmB) and its copper complex on the multicomponent monolayers imitating the outer leaflet of human erythrocyte membrane has been performed by means of the Langmuir monolayer technique. The properties of mixed films were analysed by thermodynamic description of the interactions between the molecules, complemented with the morphology of monolayers established by Brewster Angle Microscopy (BAM). The results revealed differences in the molecular organisation of the two antibiotic forms at the air/water interface, which were explained by the different spatial structure of the complex. The lipophilicity of the complex contributed to considerably more effective interactions with the components of the model membrane, compared to pure antibiotic, expressed by negative values of the excess of free energy of mixing ∆Gexc in the whole range of mole fractions. The mixed films with AmB were more stable as the proportion of lipids in the mixture increased. BAM images demonstrated that the addition of antibiotic at high content into the lipid mixture led to the formation of crystallite structures within the film, probably caused by the expelling of AmB molecules from mixed monolayers. These findings could help to explain the mechanism of the haemolytic activity of polyene antibiotics.

2


Page 3 of 43

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60


1. INTRODUCTION

Opportunistic fungal infections arouse considerable interest in the medical world due to several factors, e.g. the growing frequency of diagnosis thereof in patients with a compromised immune system in combination with complex treatment and failure to achieve a therapeutic success directly associated with increased mortality in hospitalised patients1. The therapeutic difficulties stimulate constant search for improved formulations with low toxicity and high safety for treatment of fungal infections. Amphotericin B (AmB) is the most commonly used heptaene antibiotic from the polyene group synthesised by geophillic actinomycetes Streptomyces nodosus. Although it has been recognised in clinical practice as the gold standard for treatment of severe systemic mycoses, AmB is still one of the most toxic pharmaceuticals.2 The toxic action of AmB is assumed to be based on structural and organisational disorders of the lipid membrane, which promote an uncontrolled increase in its permeability to monovalent ions, in particular K+, and small molecules from the cell interior.3 Several studies have reported that the amphiphilic AmB molecules tend to selfassociation in both aqueous and lipid environments, which is largely responsible for the toxicity of the antibiotic.3,

4, 5, 6

Since AmB is not an ideal formulation, the design of its

derivatives with the better safety pharmacokinetic profile is the goal of many institutions, e.g. by modification of its chemical structure to reduce its level of aggregation. Many biologically active compounds used as drugs are known to exhibit modified pharmacological and toxicological potentials when administered in the form of metal complexes. To address this need, we have synthesised an AmB complex with Cu2+ ions (AmB−Cu2+), which is a novel compound with different physicochemical properties7 and altered biological activity, in comparison with the parent compound.8 The results of the 3



1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

Page 4 of 43

research conducted with UV-Vis, CD, FTIR, and Raman spectroscopy methods showed formation of a stable complex of AmB with copper(II) ions in a stoichiometric ratio of 2:1 in the aqueous solution.7 Recent studies have shown more efficient fungicidal activity of the AmB−Cu2+ complex against Candida albicans, in comparison with the pure AmB form and the commercial Fungizone formulation.8 The authors postulated that the increased antifungal activity of the complex is its unique feature of the complex rather than a result of the sum of the toxic activity of AmB and copper ions. The analysis of the expression profiles of genes associated with RPTEC (human renal proximal tubule cells) incubated with the antibiotic revealed two-fold lower toxicity of AmB−Cu2+ compared to AmB.9 It should be mentioned that the strategy of formation of complexes with transition metal ions is also employed in the case of other compounds, e.g. macrocyclic compounds and Schiff bases, thereby enhancing their efficacy.10 Some of polyene antibiotic complexes with metal ions were synthesised and characterized.11 One of the few studies addressing the problem of the interactions between Cu2+ ions and AmB in biological systems.12 The results reported therein indicate a Cu2+ ion-mediated inhibition of erythrocyte lysis processes induced by AmB. It is well known that the blood copper concentration increases in the course of many infections and diseases. Then, Cu2+ ions can become more accessible for coordination with the substantial number of competing ligands, including proteins, nucleic acids, and a number of drugs.13 On the other hand, it has been suggested that complexes between lipids and active metal ions in redox reactions can elevate the lipid oxidation level,14 as in the case of other biomolecules such as DNA or proteins.15 However, the current interest in copper complexes is related to their potential use as antibacterial, antiviral, anti-inflammatory, and anti-cancer compounds.16 Since AmB is administered via intravenous injection, the outer erythrocyte membrane is the first biological system in contact with the antibiotic. Additionally, it was concluded that 4


Page 5 of 43

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60


the water-soluble self-associated forms of AmB are mainly responsible for being toxic to human erythrocytes whereas monomeric form and insoluble aggregates have been considered as non-toxic.6 Some of the experimental work regarding the relationship between increased membrane permeability and the presence of associated species were obtained on human erythrocytes.6, 17, 18, 19 Therefore, we have undertaken investigations focused on comparison of the influence of AmB and its copper complex on a model membrane resembling outer leaflet of human erythrocyte membrane in its lipid composition. More importantly, red blood cell or its model is routinely used in toxicity determination of the polyene antibiotics. The amphiphilic nature of the amphotericin B structure is responsible for its surface activity, which predisposes the compound for formation of monolayers at the air/water interface. The application of the monomolecular layer technique facilitated investigation of the AmB molecular organisation in a lipid environment, as Langmuir films are good in vitro models mimicking biological membranes. The properties of the ternary lipid system composed of egg sphingomyelin (SM), 1-palmitoyl-2-oleoyl-snglycero-3-phosphocholine (POPC), and 1-palmitoyl-2-oleoyl-snglycero-3-phosphoethanolamine (POPE) in relation to the content of AmB and AmB−Cu2+ in the mixture were analysed with the use of parameters calculated directly from the π−A curve. The analysis of the interactions between the mixed film components and morphological features of these films were visualised by using Brewster angle microscope.

5



1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

Page 6 of 43

2. EXPERIMENTAL SECTION 2.1. Materials The antibiotic amphotericin B (AmB) with a purity of 92.6% was purchased from LKT Laboratories, Inc., and copper chloride from POCH SA, (Poland). Membrane lipids: 1palmitoyl-2-oleoyl-sn-glycero-3-phosphocholine (POPC), 1-palmitoyl-2-oleoyl-sn-glycero-3phosphoethanolamine (POPE) and Egg sphingomyelin (SM) were purchased from Avati Polar Lipids Inc. All the lipid compounds exhibited high purity levels (≥99%); they were used without further purification and stored at −76oC. Chloroform, 2-propanol, acetone, and methanol with a purity ≥ 99% purchased from Sigma-Aldrich were used as solvents for cleaning the through and glass. Stock solutions of the lipids were prepared by dissolving lipid compounds in chloroform/methanol 4:1 (v/v) mixture. The AmB and its copper complex solution were prepared following the methodology described elsewhere. 4, 7, 8

2.2. Methods Langmuir Through. The Langmuir film experiments were obtained with the use of KSV NIMA (Biolin Scientific AB, Finland) trough (working surface area = 783 cm2) equipped with two Delrin barriers enabling symmetrical compression of the monolayer. The surface pressure was measured with the accuracy of ±0.1 mN/m using a Wilhelmy plate made of filter paper (Biolin Scientific) as a pressure sensor connected to an electrobalance. During the experiment, the constant temperature (23°C) of the subphase (deionised water, pH 5.8) was controlled by an external circulating water system from Julabo, with an accuracy of ± 0.1 °C. Appropriate volumes of the analysed solutions were deposited on the air/water interface with the application of Hamilton microsyringe (50 µl), precise to within 1.0 µl. In all experiments, 6


Page 7 of 43

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60


the monolayer was left to equilibrate for at least 10 min after deposition of the lipid mixture and another 5 min after application of the AmB or AmB−Cu2+ solution in specific mole fractions until compression was initiated with the barrier speed of 29 cm2/min (ca. 6.67 Å2/molecule). The measurement of the π−A isotherm was repeated at least four times and that with the highest reproducibility was chosen and taken for the calculation of the thermodynamic functions. Deionised water of conductivity ≤ 0.06µS/cm, used as the subphase, was obtained from a HLP 10 system (HydroLab, Poland). Finally, water for each experiment was filtered through a set of membrane filter (Millipore Express® Plus, 0.22 µm). Brewster Angle Microscopy. The morphology of the monolayers was observed by Brewster Angle Microscopy (BAM) using UltraBAM (Accurion GmbH, Germany) with 50mW laser emitting p-polarized light at a wavelength of 658 nm, polarizer and analyser. Images were recorded by a computer-controlled CCD camera with 696×520 pixels through a 10-fold magnification objective. The lateral resolution of the microscope was 2 µm. The microscope and film balance were located on the table with an active vibration isolation system (Halcyonics, Germany) and placed in an acrylic glass cabinet purged with N2 to maintain a relative humidity of 70–75 % and a dust free environment.

7



1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

Page 8 of 43

2.3. Composition of Model Mixed Membranes with AmB and AmB–Cu2+ The model monolayer was composed of major lipid molecules naturally occurring in the outer layer of human erythrocytes, i.e. egg sphingomyelin, (SM), 1-palmitoyl-2-oleoylsnglycero-3-phosphocholine

(POPC)

and

1-palmitoyl-2-oleoyl-snglycero-3-

phosphoethanolamine (POPE) in a percentage ratio of 44.9% POPC, 43.1% SM, and 12% POPE, where the molar ratio for the SM/PC mixture was 0.95 and, for the SM/POPE mixture, it was maintained at a level of 3.6. The mutual proportion of lipids in the system was established based on literature data in a way to be the best reflection of their ratio in the outer layer of the natural erythrocyte membrane.20, 21, 22 AmB or AmB–Cu2+ was incorporated into these multicomponent monolayers in various concentrations (XAmB and XAmB−Cu2+ = 0.1; 0.3; 0.5; 0.7; and 0.9) relative to the pure mixed film, but the phospholipid ratio was maintained at a constant level. In order to simplify the descriptions and for greater legibility of the paper, the following symbolic notations were used: PC/SM/PE for the mixed lipid systems mimicking the outer erythrocyte membrane; AmB/PC/SM/PE for the different AmB mole fractions in the ternary lipid mixture; and AmB–Cu2+/PC/SM/PE for the mixed monolayers differing in the content of AmB–Cu2+.

2.4. Data Analysis The following parameters were calculated based on the recorded π–A isotherms: I. The compression modulus values (Cs−1) defined according to the formula (Eq. (1))23

C s−1 = − A(dπ / dA)

(1)

8


Page 9 of 43

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60


where A is the mean area per molecule value at the indicated surface pressure π. The highest values of the compressibility of the film are the maximum of the Cs−1–π curve, whereas the values of the surface pressure for which Cs−1 = 0 denote the occurrence of collapse πcoll. The compressibility of monolayers has also been of interest for the determination of the changes in the corresponding phase transitions.24 II. By analysis of the π–A isotherms based on calculation of the mean area per molecule (A) determined in the mixed monolayer as a function of its composition at a specified surface pressure, it was possible to investigate the miscibility and interactions between the components. The positive or negative deviation from the ideal system (miscible components) shows the non-linear behaviour of the plotted relationship.24,

25

The positive deviations

indicate existence of repulsive interactions between the film components, whereas the negative deviations are a result of mutual attraction.26 The linearity of these functions indicates either ideal miscibility of the components resulting from lack of interactions between the molecules or their total immiscibility. The values of ideal areas (Aid) were defined according to a general formula for the quaternary system (Eq. (2))24

id

A1234 = A1 23 ( X 1 + X 2 + X 3 ) + A4 X 4

(2)

where A123, A4 are the mean molecular areas of mixed monolayer and single component of AmB (or AmB–Cu2+), respectively, read directly from the isotherms at the same surface pressure and X1→3, X4 are the mole fractions of monolayer components (lipids and antibiotic). III. To get insight into the thermodynamic stability of the investigated mixed monolayers, values of the excess free energy of mixing, ∆Gexc, were calculated using the following equation (Eq. (3)) π

∆G exc = N ∫ Aexc dπ

(3)

0

9



1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

Page 10 of 43

where: N – Avogadro’s number, Aexc – the excess areas per molecule (Aexc) defined by Eq. (4)

Aexc = A1234 − A1234

id

where A1234 is the mean molecular area in the mixed monolayer.24,

(4) 25, 26

Positive values of

∆Gexc indicate that the mutual interactions between components are weaker than the interaction between pure compounds, and the phase separation in the monolayer can be suggested. Negative values of ∆Gexc are considered as standard parameters of monolayer’s stability and indicate the presence of strong mutual attractive (or lower repulsive) interactions corresponding to the formation of a complex between the monolayer components.27

3. RESULTS 3.1. Analysis of the π – A isotherms for pure components of AmB and AmB–Cu2+ The π – A compression isotherms of the monolayer formed by pure AmB and the AmB– Cu2+ complex at the air/water interface are presented in Figure 2A. During the compression of the monomolecular layer of AmB and AmB–Cu2+, the organisation of the molecules on the surface is altered, which is manifested in the occurrence of a characteristic broad plateau. The presence of the plateau is interpreted as progressive changes in the orientation of AmB molecules caused by compression from the horizontal in an expanded state to vertical position in a liquid-condensed phase and also desorption of molecules from the surface.28, 29, 30 It was confirmed that this plateau transition depends on temperature, and the surface pressure value of it decreases with increasing temperature.29 As for the parental molecule AmB, the AmB– Cu2+ complex could assume two different positions at the interface, depending on the orientation of its long polyene-axis: parallel to the interface or perpendicular to it. In the case of the AmB–Cu2+ complex, the plateau is shorter than in the case of the native AmB. The shape of the AmB compression isotherms is very close to the isotherms reported previously.4, 10


Page 11 of 43

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60


28, 31, 32

The Brewster angle microscopy (BAM) method also confirmed the hypothesis of the

existence of a phase transition, leading to re-orientation of AmB molecules via compression. 33

A 3-fold increase in the relative monolayer thickness was observed by Minones et al.31, and

this result was similar to data related to the cross-sectional area of the AmB molecules. On the other hand, Sykora et al.34 have reported that the phenomenon of molecule aggregation may be equally responsible for the existence of the plateau region. During the compression, AmB molecules are anchored with their polar parts in the subphase, and this orientation facilitates chromophore interactions and formation of H-type aggregates (card pack).35 The exact description of the mechanism of AmB molecule changes on the subphase surface has already been presented in our previous work.36 Extrapolation of the linear sections of the π = f(A) isotherm with the surface pressure π = 0 facilitates calculation of limiting molecular areas occupied by molecules in the horizontal and vertical position relative to the subphase surface. The π – A isotherm region, for which a plateau was observed, is in the surface pressure range of 10 – 11 mN/m both for the AmB–Cu2+ complex and for the pure AmB. Evidently, the compression isotherms of AmB and its complex differ in the limiting molecular area for the molecules in the horizontal orientation. This value reaches 144±1.8 Å2 per molecule for the pure AmB and 137±2.1 Å2 per molecule for the AmB–Cu2+ complex. The limiting molecular area of vertically oriented molecules amounted to 32±0.8 Å2 and 25±1.1 Å2, respectively. The differences observed in the occupied areas can be explained by the different molecular organisation at the air/water interface between AmB and its complex. The different organisation of the AmB–Cu2+ complex at the air/water interface relative to AmB is associated with a significant steric difference between the two compounds in the headgroup region. A closer neighbourhood of AmB molecules than in the case of monomers, which retain a certain distance from each other as a result of electrostatic interactions, imposes formation of the AmB–Cu2+ complex. Non-complexed AmB molecules have an almost 11



1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

Page 12 of 43

parallel orientation towards the interface. Due to the selection rule in the circular dichroism spectroscopy, AmB molecules in the complex are aligned at a certain angle that is different from 0o and 180o,7 see Figure 1B. This leads to reduction of the occupied area per molecule of the complex relative to AmB in the horizontal orientation. As far as the lower value for the vertical arrangement of AmB–Cu2+ monolayers is concerned, it seems that the strong chromophore interactions between neighbouring molecules are mainly connected with difficulties to adopt their polyene-axis standing perpendicular to the interface. For direct visualisation of the structure of the monolayers formed by the pure AmB and its complex with the Cu2+ ions, the Brewster angle microscopy (BAM) technique was employed. The BAM images were taken throughout the process of film compression on the water subphase. The results are shown in Figure 2B and 2C at the various surface pressures. In both cases, typical oval and rectangular domain structures can be noted at the low values of the surface pressure π 0.5, this minimum does not differ notably from the composition of the monolayers and exists at practically the same surface pressure as the pure antibiotic. In case of the AmB–Cu2+/PC/SM/PE mixture, the pseudo-plateau is also shifted to higher surface pressure (∼12 mN/m), compared with the transition pressure of the AmB–Cu2+ complex. It is poorly marked in the Cs−1–π curve for the low content of the complex and for XAmB–Cu2+ ≥ 0.7 it appears at a surface pressure almost equal to that of pure AmB–Cu2+. The interactions between the components in the AmB/PC/SM/PE or AmB– Cu2+/PC/SM/PE mixtures were also analysed in terms of their mutual miscibility based on the additivity rule25. For an ideal miscibility system, the molecular areas Aid are defined by Eq. (2). Figures 5A and 5B shows the mean area per molecule (A) as a function of the mole fraction of AmB and AmB–Cu2+. The values obtained directly from the experimental isotherms and compared with the values resulting from the additivity rule were determined at a constant surface pressure. Since experiments carried out successive compression–expansion process showed that AmB monolayers were only very stable in the pre-plateau region,37 we taken into consideration the results at low surface pressure (π = 5, 10 and 15 mN/m) for further analysis. Furthermore, at higher surface pressures the obtained values could be uncertain due to the increased probability of reduced stability of the monolayers in this region as a result of loss of molecules into the subphase. The negative deviations from ideality, which appears in the whole range of both XAmB and XAmB–Cu2+ for the mixed films at low surface pressures (5 and 10 mN/m), could prove the existence of attractive forces between the components under these conditions. In our opinion, the negative deviations in case of AmB are not due to a condensation and intermolecular attractions, but are rather caused by the expelling of molecules from monolayer during 16


Page 17 of 43

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60


reorientation process, which leads to a reduction in the mean area per molecule. Effectively, at π>5 mN/m, AmB molecules were to be found in the oblique form37 and some of the hydrogen bonds were broken, facilitating desorption of antibiotic from the interface.30 For the higher surface pressure value (15 mN/m), only a slight negative deviation from ideality was noted. The experimental points nearly coincide with the values calculated according to the additivity rule (marked as dashed lines) with only a negligible positive deviation for XAmB >0.7. Since it has been postulated that, in the post-plateau region, the antibiotic may to some extent be dissolved in the subphase indicating less marked deviations from the ideal behaviour under these conditions. From a thermodynamic point of view, it is also possible to calculate quantitatively the interactions and stability of the system using the free energy of mixing values ∆Gexc calculated directly from the π – A isotherms based on Eq(3)26. The results of these calculations for the investigated mixtures, in the form of ∆Gexc = f(XAmB/AmB–Cu2+) dependencies are presented in Figures 6A and 6B. As it can be seen, for the AmB/PC/SM/PE mixtures, the ∆Gexc values change from negative (with two minima at XAmB = 0.3 and XAmB= 0.7) to positive, suggesting that at high AmB content, the interactions between film components become less attractive. A different tendency can be observed for the mixtures of AmB–Cu2+ and mixed lipids system; ∆Gexc is negative in the whole range of AmB–Cu2+ contents and becomes progressively more negative as surface pressure increases. The negative values of the ∆Gexc suggest stronger attractions (or weaker repulsion) between the mixed lipids and the AmB complex, compared with those in the mixtures with the pure antibiotic. When AmB and the PC/SM/PE mixture are considered, the most negative values of ∆Gexc with the minima for XAmB = 0.3 and 0.7 are ≈– 0.8 kJ/mol at π=15 mN/m. The most positive ∆Gexc value is achieved at XAmB = 0.9 and reaches ca. 0.1 kJ/mol at 5 mN/m (Figure 7A). This shows a possibility of phase separation at high AmB content at a low 17



1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

Page 18 of 43

surface pressures. It is evident that the AmB–Cu2+ complex interacts more strongly with the PC/SM/PE system at high complex mole fraction and the ∆Gexc value (≈ –2.8 kJ/mol) is ca. three times greater than that observed in the mixture containing AmB under the same conditions.

3.4. Analysis of BAM Images The properties of the investigated films, i.e. the surface morphology and structure of the formed domains, were analysed with the use of Brewster angle microscopy (BAM). Since BAM images for the individual POPC, SM, and POPE components in the lipid mixture had been described and presented in literature reports,41, 42 they are not shown in this paper. After initiation of compression, the ternary lipid monolayer shows foam-like structures, which are characteristic of the coexistence of gas (dark areas) and liquid expanded phases. In the gaseous phase, it is believed that the molecules lie flat on the subphase, which it can be seen as low-density areas of dark parts of the foam. On the other hand, the higher-density liquid regions (bright zones) are related to the vertical orientation of the molecules. With further compression, the monolayers become almost homogeneous in the wide range of surface pressures. There are only tiny bright dots of the condensed phase, and their number and size slightly increase with further compression up to the collapse point, see Figure 7 (first row). The morphology of these films is comparable to that reported previously for the same lipid system.

21

Similar BAM images were recorded for XAmB = 0.1 and the only difference is that

smaller condensed domains are observed and their brightness is less pronounced. The effect of the antibiotic on the structure of the mixed lipid monolayer is only evident for the higher AmB content in the film (XAmB ≥ 0.3). At the low surface pressure values of π = 0.2 mN/m, there are visible circular and oval condensed-phase structures characteristic for AmB, the 18


Page 19 of 43

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60


number of which increases with the content of the antibiotics in the film. The recorded BAM images of the AmB in the pre-plateau region (5 mN/m) show elongated, rectangular domains with varied optical anisotropy and, ultimately, the image for XAmB = 0.9 is analogous to that for the pure antibiotic. The most interesting effect was recorded at π ≈ 10 mN/m, indicating the region of the AmB phase transition. As it can be seen in Figure 7, the gradual ejection of the two-dimensional condensed-phase structures appears as two separate phases in BAM images with the increasing AmB mole fraction. Finally, the crystal-like structures with sharp edges were formed for XAmB = 0.9, see Figure 9. The different tones of these separated structures are due to optical anisotropy as a consequence of different tilt of the AmB molecules in respect to the plane of laser incidence. The size of these structures ranged from 10 to 100 µm. No such structures were observed in BAM images of the pure antibiotic and in the case of the AmB–Cu2+ complex, see Figures 2B and 2C. Above the phase transition of AmB (25 mN/m), all these structures disappear, giving a homogeneous images for the entire range of the antibiotic. Figure 8 presents BAM images taken for the AmB–Cu2+/PC/SM/PE mixtures differing in the content of AmB–Cu2+. In the range of XAmB–Cu2+ = 0.1 to 0.3, BAM images are almost homogeneous in a wide range of surface pressure, exhibiting small circular dots. In contrast to the mixed films containing AmB, distinct, bright domains start appearing for XAmB–Cu2+ = 0.5 with their greatest number at 5 mN/m. Furthermore, in the plateau transition region, the BAM images show only small, bright dots which merge together in ordered stripes in the range of XAmB–Cu2+ = 0.1 to 0.5. Since no phase separation and two-dimensional structures are observed for the mixtures containing the complex, it is evident that the complex and PC/SM/PE show miscibility at air/water interface in the studied composition.

19



1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

Page 20 of 43

4. DISCUSSION In the presented investigations, a relatively simple system resembling erythrocyte membrane in its lipid composition was used in order to explore the interactions of AmB and AmB–Cu2+ with membrane components. Obviously, the Langmuir films analysed here served as a model of the relevant membrane and did not entirely reflect the complex composition and environmental conditions characteristic for a natural system. With this in mind, three most important lipids representing the outer membrane of human erythrocytes were chosen; however, the cholesterol factor, which evidently has great importance in elucidation of the AmB toxicity to human cells, was unfortunately disregarded. According to a concept (the new sterol sponge model) presented by the group of Burke et al.43 the binding of sterol is essential for fungicidal activity of amphotericin B. Moreover recent studies reported by Grudziński et al.44 show that sterols play a key role in determining molecular organization, localization and orientation of amphotericin B in lipid membranes. This, however, was intentional on our work, since cholesterol at physiological concentrations in mammalian membranes (20−45 mol%) is known for its condensing effect on lipid membranes and its ability to form stable different-stoichiometry complexes with PC.45 To present the clearest comparative image of the mechanisms of the AmB and AmB–Cu2+ interactions with the components of the model system, we intended to exclude a possible mutual influence between them. In light of the discussion presented above, we attempted to simplify the model system to the greatest extent possible in order to investigate an undisturbed impact of AmB and AmB–Cu2+ on the structure of the model membrane with the use of thermodynamic analysis of the compression isotherms obtained and visualisation of the morphology of the mixed films in BAM images. The analysis of the π−A compression isotherms of monolayers formed by the pure AmB and AmB–Cu2+ (Figure 2A), in terms of smaller molecular areas occupied by the complex molecules, allows a conclusion that these two antibiotic forms have different surface 20


Page 21 of 43

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60


properties. The differences were also observed in the surface pressure values at which the collapse pressure occurred. This may be associated with the different molecular organisation of the AmB and its complex at the air/water interface, caused by the aggregation state. It is assumed that AmB toxicity in biological systems is associated with formation of aggregate structures. Our previous spectroscopic analyses of AmB–Cu2+ shows changes in CD spectra from the negative to the positive Cotton effect, which is associated with a different orientation of molecules in the aggregate, from the head-to-tail arrangement in AmB to the head-to-head orientation in the AmB–Cu2+ complex.7 Based on the exciton splitting theory, it can be assumed that the complex with 2:1 stoichiometry exhibits a higher degree of monomerisation and a reduced ability to form self-associated forms in the aqueous environment due to the greater distance between the molecule chromophores (5.06 Å for an AmB dimer and 5.43 Å for AmB–Cu2+).7 This effect is also reflected in the differences in the recorded BAM images, visible especially in the region preceding the phase transition of the antibiotic. As shown in Figure 2C, the elongated rice-grain like structures form ordered assemblies for π = 5 mN/m during compression of the monolayer. The shapes of these domains are more regular and uniform than those observed for the pure AmB at the same surface pressure values. On the other hand, the orientation of the zwitterion AmB molecules in the monolayer is also connected with the presence of the protonated N-group and negatively charged carboxyl group as well as –OH groups in the polyhydroxyl part. The ionized –COO− group is subjected to greater exposure to the aqueous phase than –NH3+ due to its stronger hydration, which orients the macrolide ring closer to the polar environment.46 The ionic state of the molecules is an important factor facilitating the aggregation process and if the carboxyl group is blocked as in case of AmB–Cu2+, the process of creating self-associated forms is limited. Furthermore, it has been reported by Cybulska et al.47 that carboxyl-substituted derivatives of heptaenes had a very poor haemolytic activity. Recently, it has reported a synthesis of AmB-silver hybrid 21



1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

Page 22 of 43

nanoparticles in which molecules of the antibiotic, immobilized at the metal surface are not able to form molecular aggregates that is most probably responsible for relatively low cytotoxicity of this new formulation.48 The results obtained for the mixed lipid systems mimicking the outer erythrocyte membrane with incorporated AmB and AmB–Cu2+ indicate thermodynamically more favourable interactions between the AmB complex and the lipid film components. A change in the collapse pressure was noted over the entire mole fractions analysed, which may imply miscibility of the components in this system. The pseudo-plateau transition (M) visible in Figure 4B was shifted towards higher surface pressures (11.6 mN/m), in comparison with the pure complex (10.4 mN/m). On the other hand, a small shift of the transition (M) (11.2 mN/m) relative to the transition of the pure antibiotic (10 mN/m) was also noted for AmB. Nevertheless, based on this small shift in case of the complex, it can be concluded that the components of the mixed lipid film hinder re-orientation of molecules from the horizontal to vertical position. This proves the existence of interactions between the film components. This effect may be associated with the different spatial structure of the AmB–Cu2+ complex with 2:1 stoichiometry. Based on the changes in the spectra recorded with the FTIR spectroscopy technique, it was demonstrated that the ionised carboxyl group located in the AmB molecule is involved in the formation of the AmB–Cu2+ complex. Binding of the Cu2+ ion with the −COO− group is forced by the close proximity of this ion to the amino sugar group of AmB, and the electrostatic interactions between them facilitate mycosamine moiety rotation49. The different spatial configuration of AmB–Cu2+ in comparison with that of AmB was also evidenced by Raman spectroscopy analysis, which revealed a change in the polarizability of the chromophore induced by the Cu2+ ion.7 The mixed AmB–Cu2+/PC/SM/PE monolayers exhibited negative deviations from ideality in the nearly entire mole fraction range, which were the greatest for XAmB– 22


Page 23 of 43

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60


Cu

2+

= 0.5 − 0.9 at the low pressure values. This indicates considerably stronger interactions

between the components in the mixed AmB–Cu2+-containing monolayers, compared with those containing AmB (Figures 6A and 6B). The interactions between molecules in multicomponent systems are known to be a resultant of interactions between hydrophobic tails of molecules and interactions of their hydrophilic groups and depend on both the electrical charge of the film-forming compounds and their structure. It is possible that the presence of unsaturated bonds in the hydrophobic chain of the POPC and POPE lipids, which are components of a mixed monolayer, prevents achievement of the vertical orientation on the surface by AmB molecules; hence, the polar groups of these lipids are less accessible for the antibiotic. Consequently, the negative deviations from ideality in case of AmB do not result from the intermolecular interactions but are caused by expelling of molecules from monolayers in the plateau region. Similar conclusions were formulated by Minones et al., who reported that AmB-phospholipid interactions manifested by formation of complexes were stronger in the case of saturated rather than unsaturated phospholipids.50 As shown in the presented discussion, the mechanism of incorporation of the AmB– Cu2+ complex in the PC/SM/PE monolayer must differ from that of the pure antibiotic form. Due to coordinate bonding, the polarity of the metal ion is reduced by partial division of the positive charge of the ligand. Additionally, the polarizability of the AmB chromophore is changed, leading to an increase in the lipophilicity of the AmB–Cu2+ complex and the ability to interact with lipid hydrocarbon chains. It may seem that the more lipophilic property of the complex is responsible for its easier penetration of monolayers, leading to further fluidity of mixed ﬁlms as shown by results indicating higher antifungal activity of the AmB–Cu2+ complex against Candida albicans in comparison with the pure AmB.8

23



1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

Page 24 of 43

The analysis of the course of the ∆Gexc function (Figures 6A and 6B) has revealed differences in the character and magnitude of interactions between components in the mixed films. The strength of the interactions was expressed by considerably higher negative ∆Gexc values for AmB–Cu2+ in the whole range of mole fractions (XAmB–Cu2+ = 0.3−0.9). Moreover, for the high AmB content, repulsive interactions between components in the mixtures were observed, which was reflected in the positive ∆Gexc values leading to the phase separation and ejection of AmB from monolayers. Interestingly, there were also observed the positive values of ∆Gexc for other lipid system containing AmB in excess.51 The lipophilicity of the complex enhances the possibility of more efficient interactions with the components of the model membrane and contributes to elucidation of the other result obtained in the present work, i.e. the formation of crystalline structures recorded in the BAM images only in the case of high AmB content in the mixed film (Figures 7 and 9). The appearance of two-dimensional structures visualised in the BAM images can be explained in two ways: formation of crystallites in the mixture caused by the limited AmB solubility in the lipid environment and the fact that the lipid composition of the mixture can induce self-aggregation tendency of the antibiotic chromophores. Such a hypothesis has already been put forward in our previous research on the molecular organisation of the iodacetyl AmB derivative (AmB-I), in which we identified a band (1010 cm−1) characteristic for the crystalline structure of the antibiotic using the FTIR and Raman techniques.52 These results allowed a conclusion that, in model lipid systems, AmB forms aggregated structures, identical to those in the crystal, through mutual chromophore interactions. The sharp shape and size of these structures were identical to those described by Jarzembska et al.53 and obtained during the crystallization of AmB-I with different quantities of amphotericin B additives. No such structures were observed upon incorporation of the complex AmB–Cu2+ into the PC/SM/PE films for all the studied mixtures. Additionally, the investigation based on the structural and spectroscopic studies, 24


Page 25 of 43

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60


reported by Umegawa et al.54 showed the formation of AmB aggregates in POPC membranes. Notably, recent study based on solid-state NMR spectroscopy revealed that AmB exists in the form of large, extramembranous aggregates in POPC liposomes system that are more closely associated with water than lipids.55 Moreover, the effect of phase separation and structural alteration in human erythrocyte membrane induced by antibiotic have been investigated by freeze-fracture electron microscopy18. The authors of this work observed the forming wartlike structures depending on the ionic strength in membrane by treatment of erythrocytes with AmB. On the other hand, polyene antibiotics are known to exhibit haemolytic properties17 depending on their concentration and formulation. In the light of the discussion, it seems probable that the AmB complex with Cu2+ ions binds to charged lipid residues present in the erythrocyte membrane more efficiently through ionic interactions. However, the induction of erythrocyte lysis processes is not the target of this new formulation but, as indicated by Charon et al.12, copper ions inhibit AmB-induced erythrocyte destruction. This may also be explained by the fact that unbound Cu2+ ions can interact with the phosphate group of lipids representing the outer erythrocyte membrane. This conclusion was drawn by Suwalski et al.56 who investigated copper ion-induced changes in the structure of lipid bilayers formed with DMPC and DMPE using X-ray diffraction methods. The involvement of the AmB complex in strong interactions with lipid components of mixed film may reduce the concentration of free antibiotic, which causes toxic effect in natural membranes. This explanation had been previously proposed for N-(1-Piperidinepropionyl)amphotericin B methyl ester derivative of AmB.51

25



1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

Page 26 of 43

5. CONCLUSIONS The main purpose of this study was to investigate the interactions of AmB and its copper complex with a class of lipids compositionally reflecting the outer moiety of the erythrocyte membrane. The results obtained on the basic of the Langmuir monolayer technique indicate that the surface activity of the AmB–Cu2+ complex seems to be stronger compared to the pure AmB. It was found that the addition of AmB into the multicomponent monolayers studied herein was thermodynamically unfavourable in comparison with AmB– Cu2+. Furthermore, the mixed monolayers containing the AmB molecules were more stable as the proportion of lipids in the mixture increased. The observed immiscibility of mixtures containing AmB in excess (XAmB>0.5) at relatively low surface pressure indicating the reorientation region of antibiotic could be caused by expelling of molecules in form of crystal-like structures from interfacial film. Generally speaking, the similar thinking might apply to the natural red cell membrane. At substantial accumulation, the antibiotic may induce comparable membrane disturbances in the natural membrane. However, the presence of membrane proteins in erythrocytes gives other possibilities of interaction with antibiotic and therefore the mechanism could be more complex than in the case of simple lipid system. In light of the above, it appeared that the lipid components of monolayers reflecting the outer layer of human erythrocyte membrane might promote to force out of AmB molecules in form of large aggregates from monolayers during the reorientation process what could explain high toxicity of the antibiotic towards biomembranes.

26


Page 27 of 43

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60


Acknowledgment This research was financed by the National Science Centre of Poland based on decision no. DEC-2012/05/B/NZ1/00037. The research was carried out with the equipment (UltraBAM) purchased thanks to the financial support of the European Regional Development Fund in the Framework of the Polish Innovation Economy Operational Program (contract no. POPW.01.03.00-06-009/11). M. A. would like to express gratitude toward Prof. P. Dynarowicz-Łątka from the Department of General Chemistry, Faculty of Chemistry, Jagiellonian University (Kraków, Poland) for helpful advice.

27



1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

Page 28 of 43

REFERENCES (1) Brown, G. D.; Denning, D. W.; Gow, N. A.; Levitz, S. M.; Netea, M. G.; White, T. C. Hidden killers: human fungal infections. Sci Transl Med 2012, 4, 165rv13. (2) Ellis, D. Amphotericin B: spectrum and resistance. J Antimicrob Chemother 2002, 49 Suppl 1, 7-10. (3) Bagiński, M., Cybulska, B., Gruszecki, W. I. Interaction of polyene macrolide antibiotics with lipid model membranes. In A. Ottova-Liu (Ed.), Advances in planar lipid bilayers and liposomes; Elsevier Science Publ., Amsterdam, 2006; Vol. 3, pp 269-329. (4) Arczewska, M.; Gagoś, M. Molecular organization of antibiotic amphotericin B in dipalmitoylphosphatidylcholine monolayers induced by K(+) and Na(+) ions: the Langmuir technique study. Biochim Biophys Acta 2011, 1808, 2706-13. (5) Gagoś, M., Herec, M., Arczewska, M. Czernel, G. Dalla Serra, M. Gruszecki, W. I. Anomalously high aggregation level of the polyene antibiotic amphotericin B in acidic medium: implications for the biological action. Biophys Chem 2008, 136, 44-9. (6) Legrand, P.; Romero, E. A.; Cohen, B. E.; Bolard, J. Effects of aggregation and solvent on the toxicity of amphotericin B to human erythrocytes. Antimicrob Agents Chemother 1992, 36, 2518-22. (7) Gagoś, M.; Czernel, G.; Kamiński, D. M.; Kostro, K. Spectroscopic studies of amphotericin BCu2+ complexes. Biometals 2011, 24, 915-922. (8) Chudzik, B.; Tracz, I. B.; Czernel, G.; Fiolka, M. J.; Borsuk, G.; Gagoś, M. Amphotericin Bcopper(II) complex as a potential agent with higher antifungal activity against Candida albicans. Eur J Pharm Sci 2013, 49, 850-857. (9) Gola, J.; Skubis, A.; Sikora, B.; Kruszniewska-Rajs, C.; Adamska, J.; Mazurek, U.; StrzalkaMrozik, B.; Czernel, G.; Gagoś, M. Expression profiles of genes related to melatonin and oxidative stress in human renal proximal tubule cells treated with antibiotic amphotericin B and its modified forms. Turk J Biol 2015, 39, 856-864. (10) Chandra, S.; Ruchi; Qanungo, K.; Sharma, S. K. New hexadentate macrocyclic ligand and their copper(II) and nickel(II) complexes: Spectral, magnetic, electrochemical, thermal, molecular modeling and antimicrobial studies. Spectrochim Acta A Mol Biomol Spectrosc 2012, 94, 312-7. (11) Beezer, A. E.; O'Brien, P.; Sham, W. L. The synthesis and characterization of polyene complexes with the divalent metal ions: Mg(II), Ca(II), Ni(II),Cu(II) and Zn(II). Inorganica Chimica Acta 1985, 108, 117-122. (12) Cheron, M.; Bolard, J.; Brajtburg, J. Inhibition by Cu2+ of amphotericin B induced lysis of erythrocytes. FEBS Lett 1984, 178, 127-31. (13) Castillo-Blum, S. E.; Barba-Behrens, N. Coordination chemistry of some biologically active ligands. Coordin Chem Rev 2000, 196, 3-30. (14) Poyton, M. F.; Sendecki, A. M.; Cong, X.; Cremer, P. S. Cu(2+) Binds to Phosphatidylethanolamine and Increases Oxidation in Lipid Membranes. J Am Chem Soc 2016, 138, 1584-90. (15) Uriu-Adams, J. Y.; Keen, C. L. Copper, oxidative stress, and human health. Mol Aspects Med 2005, 26, 268-98. (16) Iakovidis, I.; Delimaris, I.; Piperakis, S. M. Copper and its complexes in medicine: a biochemical approach. Mol Biol Int 2011, 2011, 594529. (17) Brajtburg, J.; Elberg, S.; Schwartz, D. R.; Vertut-Croquin, A.; Schlessinger, D.; Kobayashi, G. S.; Medoff, G. Involvement of oxidative damage in erythrocyte lysis induced by amphotericin B. Antimicrob Agents Chemother 1985, 27, 172-6. (18) Meyer, H. W.; Richter, W.; Winkelmann, H. Nystatin- and amphotericin B-induced structural alterations of the erythrocyte membrane: importance of reduced ionic strength. Exp Pathol 1983, 24, 163-6. 28


Page 29 of 43

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60


(19) Szponarski, W.; Bolard, J. Temperature-dependent modes for the binding of the polyene antibiotic amphotericin B to human erythrocyte membranes. A circular dichroism study. Biochim Biophys Acta 1987, 897, 229-37. (20) Yawata, Y. Cell membrane : the red blood cell as a model; Wiley-VCH: Weinheim, 2003. p xvi, 439 p. (21) Wydro, P. The influence of cholesterol on multicomponent Langmuir monolayers imitating outer and inner leaflet of human erythrocyte membrane. Colloids Surf B Biointerfaces 2013, 103, 6774. (22) Virtanen, J. A.; Cheng, K. H.; Somerharju, P. Phospholipid composition of the mammalian red cell membrane can be rationalized by a superlattice model. Proc Natl Acad Sci U S A 1998, 95, 4964-9. (23) Davies, J. T., Rideal, E. K. Interfacial Phenomena; Academic Press, New York and London,1963. p 265. (24) Dynarowicz-Łątka, P., Kita, K. Molecular interaction in mixed monolayers at the air/water interface Advances in Colloid and Interface Science 1999, 79, 1-17. (25) Costin, I. S., Barnes, G. T. Two-component monolayers. II. Surface pressure—area relations for the octadecanol—docosyl sulphate system. Journal of Colloid and Interface Science 1975, 51, 106121. (26) Gaines, G. L. Insoluble Monolayers at the Liquid–Gas Interfaces, Interscience, New York, chapter 6. 1966. (27) Maget-Dana, R. The monolayer technique: a potent tool for studying the interfacial properties of antimicrobial and membrane-lytic peptides and their interactions with lipid membranes. Biochim Biophys Acta 1999, 1462, 109-40. (28) Saint-Pierre-Chazalet, M.; Thomas, C.; Dupeyarat, M.; Gary-Bobo, C. M. Amphotericin BSterol Complex formation and competition with egg phosphatidylcholine: A Monolayer Study. Biochim. Biophys. Acta 1988, 944, 477-486. (29) Seoane, J. R., Vila Romeu, N., Miñones, J., Conde, O., Dynarowicz, P., Casas, M. The behavior of amphotericin B monolayers at the air/water interface Progress in Colloid and Polymer Science 1997, 105, 173-179. (30) Rey-Gomez-Serranillos, I.; Dynarowicz-Łątka, P.; Minones Jr., J.; Seoane, R. Desorption of amphotericin B from mixed monolayers with cholesterol at the air/water interface. J. Colloid Interface Sci. 2001, 234, 351-355. (31) Minones Jr., J.; Carrera, C.; Dynarowicz-Łątka, P.; Minones, J.; Conde, O.; Seoane, R.; Rodriguez Patino, J. M. Orientational Changes of Amphotericin B in Langmuir Monolayers Observed by Brewster Angle Microscopy. Langmuir 2001, 17, 1477-1482. (32) Gruszecki, W. I.; Gagoś, M.; Kernen, P. Polyene Antibiotic Amphotericin B in Monomolecular Layers: Spectrophotometric and Scanning Force Microscopic Analysis. FEBS Lett. 2002, 524, 92-96. (33) Miñones, J., Jr., Miñones, J., Conde, O., Rodriguez Patino, J. M., Dynarowicz-Łątka, P. Mixed Monolayers of Amphotericin B−Dipalmitoyl PhosphaRdyl Choline: Study of Complex FormaRon. Langmuir 2002, 18, 2817-2827. (34) Sykora, J. C.; Neely, W. C.; Vodyanoy, V. Solvent effects on amphotericin B monolayers. J. Coll. Interface. Sci. 2004, 269, 499-502. (35) Gagoś, M.; Koper, R.; Gruszecki, W. I. Spectrophotometric analysis of organisation of dipalmitoylphosphatylcholine bilayers containing the polyene antibiotic amphotericin B. Biochim. Biophys. Acta 2001, 1511, 90-98. (36) Gagoś, M.; Arczewska, M. FTIR spectroscopic study of molecular organization of the antibiotic amphotericin B in aqueous solution and in DPPC lipid monolayers containing the sterols cholesterol and ergosterol. Eur Biophys J 2012, 41, 663-73. (37) Pham, T. T. H.; Barratt, G.; Michel, J. P.; Loiseau, P. M.; Saint-Pierre-Chazalet, M. Interactions of antileishmanial drugs with monolayers of lipids used in the development of amphotericin Bmiltefosine-loaded nanocochleates. Colloid Surface B 2013, 106, 224-233. 29



1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

Page 30 of 43

(38) Dynarowicz-Łątka, P.; Minones, J.; Conde, O.; Casas, M.; Iribarnegaray, E. BAM studies on the penetration of amphotericin B into lipid mixed monolayers of cellular membranes. Appl Surf Sci 2005, 246, 334-341. (39) Huynh, L.; Perrot, N.; Beswick, V.; Rosilio, V.; Curmi, P. A.; Sanson, A.; Jamin, N. Structural Properties of POPC Mono layers under Lateral Compression: Computer Simulations Analysis. Langmuir 2014, 30, 564-573. (40) Seoane, J. R., Miñones, J., Conde, O., Casas, M., Iribarnegaray, E. Interaction between Amphotericin B and Sterols in Monolayers. Mixed Films of Ergosterol−Amphotericin B. Langmuir 1999, 15, 3570–3573. (41) Prenner, E.; Honsek, G.; Honig, D.; Mobius, D.; Lohner, K. Imaging of the domain organization in sphingomyelin and phosphatidylcholine monolayers. Chem Phys Lipids 2007, 145, 106-18. (42) Hąc-Wydro, K.; Lenartowicz, R.; Dynarowicz-Łątka, P. The influence of plant stanol (betasitostanol) on inner leaflet of human erythrocytes membrane modeled with the Langmuir monolayer technique. Colloids Surf B Biointerfaces 2013, 102, 178-88. (43) Anderson, T. M., et al. Amphotericin forms an extramembranous and fungicidal sterol sponge. Nature Chemical Biology 2014, 10, 400-U121. (44) Grudziński, W.; Sagan, J.; Welc, R.; Luchowski, R.; Gruszecki, W. I. Molecular organization, localization and orientation of antifungal antibiotic amphotericin B in a single lipid bilayer. Sci Rep 2016, 6, 32780. (45) Dynarowicz-Łątka, P.; Hąc-Wydro, K. Interactions between phosphatidylcholines and cholesterol in monolayers at the air/water interface. Colloids Surf B Biointerfaces 2004, 37, 21-5. (46) Gagoś, M.; Arczewska, M. Spectroscopic studies of molecular organization of antibiotic amphotericin B in monolayers and dipalmitoylphosphatidylcholine lipid multibilayers. Biochim Biophys Acta 2010, 1798, 2124-30. (47) Cybulska, B.; Bolard, J.; Seksek, O.; Czerwinski, A.; Borowski, E. Identification of the structural elements of amphotericin B and other polyene macrolide antibiotics of the hepteane group influencing the ionic selectivity of the permeability pathways formed in the red cell membrane. Biochim Biophys Acta 1995, 1240, 167-78. (48) Tutaj, K., et al. Amphotericin B-silver hybrid nanoparticles: synthesis, properties and antifungal activity. Nanomedicine 2016, 12, 1095-103. (49) Matsumori, N., Sawada, Y., Murata, M. Mycosamine orientation of amphotericin B controlling interaction with ergosterol: Sterol-dependent activity of conformation-restricted derivatives with an aminocarbonyl bridge. J Am Chem Soc 2005, 127, 10667–10675. (50) Minones, J.; Dynarowicz-Łątka, P.; Conde, O.; Minones, J.; Iribarnegaray, E.; Casas, M. Interactions of amphotericin B with saturated and unsaturated phosphatidylcholines at the air/water interface. Colloid Surface B 2003, 29, 205-215. (51) Hąc-Wydro, K.; Dynarowicz-Łątka, P.; Grzybowska, J.; Borowski, E. N-(1piperidinepropionyl)amphotericin B methyl ester (PAME)-a new derivative of the antifungal antibiotic amphotericin B: searching for the mechanism of its reduced toxicity. J Colloid Interface Sci 2005, 287, 476-84. (52) Gagoś, M.; Kaminski, D.; Arczewska, M.; Krajnik, B.; Maćkowski, S. Spectroscopic evidence for self-organization of N-iodoacetylamphotericin B in crystalline and amorphous phases. J Phys Chem B 2012, 116, 12706-13. (53) Jarzembska, K. N.; Kamiński, D.; Hoser, A. A.; Malińska, M.; Senczyna, B.; Woźniak, K.; Gagoś, M. Controlled Crystallization, Structure, and Molecular Properties of Iodoacetylamphotericin B. Crystal Growth and Design 2012, 12, 2336-2345 (54) Umegawa, Y.; Matsumori, N.; Oishi, T.; Murata, M. Ergosterol increases the intermolecular distance of amphotericin B in the membrane-bound assembly as evidenced by solid-state NMR. Biochemistry 2008, 47, 13463-9.

30


Page 31 of 43

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60


(55) Wilcock, B. C.; Endo, M. M.; Uno, B. E.; Burke, M. D. C2'-OH of amphotericin B plays an important role in binding the primary sterol of human cells but not yeast cells. J Am Chem Soc 2013, 135, 8488-91. (56) Suwalsky, M.; Ungerer, B.; Quevedo, L.; Aguilar, F.; Sotomayor, C. P. Cu2+ ions interact with cell membranes. J Inorg Biochem 1998, 70, 233-8.

31



1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

Page 32 of 43

Figure Captions Figure 1. (A) Chemical structure of amphotericin B, (B) The proposed structure of the AmB−Cu2+ complex without taking into account the full stereochemistry of complex molecule. Figure 2. The surface pressure–area isotherms of AmB (▲) and AmB−Cu2+ (●) formed at the air/water interface (A). Brewster angle microscopy images of AmB (B) and AmB–Cu2+(C) monolayers at the air/water interface taken at various stages of compression. Temperature 23 °C. Figure 3. The surface pressure–area isotherms for the model outer layer of human erythrocyte membranes differing in the content of (A) AmB and (B) AmB–Cu2+ spread at the air/water interface. Temperature 23 °C. Figure 4. The compression modulus (Cs−1) vs. surface pressure (π) curves for (A) AmB, (B) AmB–Cu2+/PC/SM/PE and their mixtures spread at the air/water interface. The minimum A corresponds to the AmB transition, and M to the pseudo−plateau of mixture film (see in the text). Figure 5. Mean molecular area for the AmB/PC/SM/PE and the AmB–Cu2+/PC/SM/PE mixtures as a function of the molar faction of (A) AmB and (B) AmB–Cu2+ at various constant surface pressures. Dashed lines were drawn based on the additivity rule. Figure 6. Excess free energy o mixing (∆Gexc) as a function of composition for mixtures of (A) AmB/PC/SM/PE and (B) AmB–Cu2+/PC/SM/PE at various constant surface pressures.

32


Page 33 of 43

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60


Figure 7. Brewster angle microscopy images of the AmB/PC/SM/PE mixture differing in the content of AmB taken at various stages of compression. Figure 8. Brewster angle microscopy images of the AmB–Cu2+/PC/SM/PE mixture differing in the content of AmB–Cu2+ taken at various stages of compression. Figure 9. A representative Brewster angle microscopy image (432µm x326 µm) of the AmB/PC/SM/PE mixture of XAmB = 0.9 taken at a surface pressure of 10 mN/m. The line segment in (a) is 100 µm long. The image was corrected geometrically using image processing software (Accurion_Image_I.1.3).

Graphic Symbols TOC: Upper left corner – structure of AmB molecule, lower left corner – surface pressure–area isotherms for the lipid model layer of human erythrocyte membranes differing in the content of AmB from 0% (—) to 100% (—), Insert – BAM images of AmB/PC/SM/PE mixture of XAmB = 0.3 (upper image) and 0.9 (lower image). Upper right corner – structure of AmB copper complex, lower right corner - surface pressure– area isotherms for the lipid model layer of human erythrocyte membranes differing in the content of AmB─Cu2+ from 0% (—) to 100% (—), Insert – BAM images of AmB─Cu2+/PC/SM/PE mixture of XAmB = 0.3 (upper image) and 0.9 (lower image).

33



1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

A

B

Figure 1.


Page 34 of 43

Page 35 of 43

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60


Figure 2.



1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

Figure 3.


Page 36 of 43

Page 37 of 43

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60


Figure 4.



1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

Figure 5.


Page 38 of 43

Page 39 of 43

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60


Figure 6.



1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

Page 40 of 43

PC/SM/PE, XAmB = 0

0.1 mN/m

15 mN/m

25 mN/m

43 mN/m

PC/SM/PE + XAmB = 0.1

0.1 mN/m

10 mN/m

25 mN/m

42 mN/m


0.2 mN/m

5 mN/m

9.4 mN/m

25 mN/m


0.2 mN/m

5 mN/m

9.6 mN/m

25 mN/m


0.2 mN/m

5 mN/m

9.7 mN/m

25 mN/m


0.2 mN/m

5 mN/m

10 mN/m

Figure 7.


25 mN/m

Page 41 of 43

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60


Figure 8.



1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

Figure 9.


Page 42 of 43

Page 43 of 43

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60


254x154mm (96 x 96 DPI)


Effect of the Amphotericin B and Its Copper Complex on a Model of the

Recommend Documents