How Does the Antibiotic Amphotericin B Enter Membranes and What Does It Do There?

Amphotericin B is a popular antifungal antibiotic, but the exact way it works is still a matter of debate. Here, we used monolayers composed of phosphatidylcholine with ergosterol as a model of fungal lipid membranes to study drug incorporation from the aqueous phase and analyze the molecular reorganization of membranes underlying the biological activity of the antibiotic. The results show that the internalization of antibiotic molecules into membranes occurs only in the presence of ergosterol in the lipid phase. Comparison of images of solid-supported monolayers obtained by atomic force microscopy and lifetime imaging fluorescence microscopy shows the formation of intramembrane clusters of various sizes in the lipid phase, consisting mainly of antibiotic dimers and relatively large membrane pores (∼15 nm in diameter). The results reveal multiple modes of action of amphotericin B, acting simultaneously, each of which adversely affects the structural properties of the lipid membranes and their physiological functionality.

Ergosterol was obtained from Merck (Germany).Methanol, 2-propanol and chloroform were purchased from POCH (Poland).Water used in experiments was purified by a Milli-Q Millipore system (Merck, Germany).

Preparation of monomolecular layers
Monomolecular layers were formed using a Teflon trough equipped with movable barriers, an injection port, and a magnetic stirrer.An integrated Langmuir and Langmuir-Blodgett system was purchased from KSV NIMA Instruments (Finland).A Wilhelmy tensiometer with an ashless filter paper (Whatman) as a surface pressure sensor was used to monitor surface pressure.Before starting the main part of the experiment, the DOPC:Ergo (7:3, mol:mol) monolayers were formed at the air-water interface and isotherms of compression were recorded (See Supporting Information Figure S5).A monolayer constituents were deposited at the airwater interface in 50 µl of a DOPC and Ergo solution prepared in a chloroform:methanol (9:1, v/v) solvent mixture.The monolayer compression began after 15 min.necessary for solvent evaporation.The barrier speed was constant at 10 mm/min.To examine the penetration of AmB from the water phase into the lipid monolayers, the films were compressed to 25 mN/m, and this surface pressure was stabilized automatically by the computer-controlled system.AmB was injected into the aqueous subphase, beneath the monomolecular layers, as a solution prepared in water/2-propanol mixture (6:4, v/v).A volume of 100 µl of AmB solution was injected into S3 ~300 ml of water subphase.The concentration of AmB solution was adjusted to maintain the 1:1 ratio of molecules of AmB in the subphase and the total number of DOPC and Ergo molecules forming the lipid monolayers.The process of incorporation of AmB into the monolayers was manifested by a computer-controlled decompression of the films to maintain the surface pressure at 25 mN/m.After stabilization of this process (40 min.), the monomolecular layers were transferred to freshly cleaved Mica substrate by means of the Langmuir-Blodgett technique.The same constant surface pressure was automatically maintained by the system, also during the process of deposition of monomolecular films to a solid support.All experiments were performed at 25 °C.Single-component and multicomponent monomolecular layers formed with AmB, lipids, and sterols were characterized in detail in our previous studies. 2

Atomic Force Microscopy
The monolayers were deposited on a freshly cleaved Mica surface at room temperature and then transferred to an AFM microscope.AFM scanning was carried out using JPK Nanowizard 3 system (Bruker, USA) in a non-contact mode (AC mode).RFESPA-190 cantilevers (Bruker, USA) with a nominal elastic constant of 35 N/m and a typical tip radius of 8 nm were used.The nominal resonance frequency of the cantilevers (provided by the manufacturer) was 190 kHz and the typical operating resonance frequency was 157.4 kHz.AFM images were scanned at 512 × 512 or 1024 × 1024 pixel resolution at 0.8 Hz.To avoid defects and imperfections, a regime of weak tip-sample interaction was applied during scanning by monitoring the tip dithering phase shift.AFM images were processed by subtracting the polynomial fit from each scan line independently then the height images were fed to cross sections using JPKSMP data processing software (Bruker, USA).

FLIM measurements
Time-resolved imaging experiments were conducted employing a MicroTime 200 microscope system purchased from PicoQuant (GmbH, Berlin, Germany).The samples were excited with 405 nm solid-state laser (Picoquant) with pulses characterized by a full width at half maximum (FWHM) of less than 90 ps.This wavelength of laser light was chosen to specifically excite the 0-0 vibrational maximum of the main electronic absorption band of AmB.
The laser light beam was directed at the sample through an Olympus 60x objective with a numerical aperture of 1.2.The resulting fluorescence emission was collected by the same objective and transmitted to an avalanche photodiode detector (Excelitas Technologies) configured in a confocal mode.The detection efficiency of the detector was up to 70% at 500 nm, with a timing resolution down to <250 ps (FWHM).A pinhole diameter of 50 µm was utilized, and scattered light underwent filtration through a long-wavelength pass filter HQ430lp followed by a dichromatic mirror ZT405RDC, both sourced from AHF Analysentechnik.
The analysis of fluorescence components was executed utilizing SymPhoTime v.