On the Interaction between Digitonin and Cholesterol in Langmuir

Article pubs.acs.org/Langmuir

On the Interaction between Digitonin and Cholesterol in Langmuir Monolayers Kamil Wojciechowski,*,† Marta Orczyk,† Thomas Gutberlet,‡ Gerald Brezesinski,§ Thomas Geue,∥ and Philippe Fontaine⊥ †

Faculty of Chemistry, Warsaw University of Technology, Noakowskiego 3, 00-664 Warsaw, Poland Jülich Centre for Neutron Science (JCNS) at Heinz Maier-Leibnitz Zentrum (MLZ), Forschungszentrum Jülich GmbH, Lichtenbergstr. 1, 85748 Garching, Germany § Max Planck Institute of Colloids and Interfaces, Am Mühlenberg 1, Potsdam, Germany ∥ Laboratory for Neutron Scattering and Imaging, Paul Scherrer Institute, WHGA/110, 5232 Villigen − PSI, Switzerland ⊥ Synchrotron SOLEIL, L’Orme des Merisiers, Saint Aubin BP48, Gif-sur-Yvette Cedex, France ‡

S Supporting Information *

ABSTRACT: In this article, we describe the effect of a highly hemolytic saponin, digitonin, on model lipids cholesterol and dipalmitoylphosphatidylcholine (DPPC) using a combination of tensiometric (surface pressure and dilatational surface elasticity), spectroscopic (infrared reflection absorption spectroscopy, IRRAS), microscopic (fluorescence microscopy), and scattering techniques (neutron reflectivity, NR, and grazing incidence X-ray diffraction, GIXD). The monolayers of individual lipids and their 10:9 (mol/mol) mixture were exposed to an aqueous solution of digitonin (10−4 M) by subphase exchange using a setup developed recently in our laboratory. The results confirm that digitonin can adsorb onto both bare and lipid-covered water−air interfaces. In the case of DPPC, a relatively weak interaction can be observed, but the presence of cholesterol drastically enhances the effect of digitonin. The latter is shown to dissociate the weak cholesterol−DPPC complexes and to bind cholesterol in an additional layer attached to the original lipid monolayer.

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only cholesterol but also other 3β-hydroxysterols8 and even simple alcohols,5 cholesterol−digitonin complex precipitation remained the basis of a benchmark method of free cholesterol determination in the blood for many years. The method was further modified to account for cholesterol esters and to enable colorimetric analysis (the digitonin−anthrone method).9 Even though currently cholesterol determination in blood is mostly performed with enzymatic assays, the complexation of cholesterol with digitonin-modified gold nanoparticles was recently proposed as an alternative.10 Another recently proposed potential application of the strong affinity of digitonin for cholesterol is the removal of the latter from dairy products using digitonin-modified polymeric material.11

INTRODUCTION

Digitonin (Figure 1) is a steroid saponin with spirostan aglycone belonging to the group of cardiac glycosides, abundant in the Digitalis purpurea plant (foxglove). It is known for its strong hemolytic activity1 and general cytotoxicity and for inducing biological membrane leakage, presumably because of its strong interactions with membrane-bound cholesterol.2,3 An unusual affinity of saponins for cholesterol was noted for the first time at the beginning of the 20th century. The first indirect evidence of their strong association comes from the experiments of Ransom, who noted that the addition of cholesterol reduced the erythrocyte-lysing activity of saponins.4 In 1909, Windaus, the later Nobel Prize laureate, proposed a method of cholesterol quantification based on the cholesterol−digitonin precipitate.5 Subsequently, the high electron density of the cholesterol−digitonin adduct was successfully employed for the localization of cholesterol in tissues using electron microscopy.6,7 Even though digitonin is capable of precipitating not © XXXX American Chemical Society

Received: May 6, 2016 Revised: July 19, 2016

A

DOI: 10.1021/acs.langmuir.6b01737 Langmuir XXXX, XXX, XXX−XXX

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Figure 1. Chemical structures of digitonin (left) and cholesterol (right). pressure vs time measurements of Gibbs layers, the trough was filled with the appropriate digitonin solution in Milli-Q water of a given concentration, and the surface pressure was recorded for 1 h. For the Langmuir monolayer experiments, the subphase was exchanged using a Gilson’s MINPULS 3 peristaltic pump and Teflon tubing immersed in the subphase on opposite sides of the trough. The lipids were spread first on pure water as chloroform solutions. After the evaporation of the solvent, the monolayers were compressed to the initial surface pressure of 32.5 mN/m. Then a concentrated digitonin solution was introduced into the subphase and recirculated at a flow rate of 2 mL/min in order to achieve the final saponin concentration of 10−4 M. The flow and the barrier positions were maintained for 1 h, and the changes in surface pressure, Π(t), induced by the presence of saponins were monitored. The dilatational viscoelasticity modulus, |E(ω)|, is defined as follows

The first report on the interaction of cholesterol with digitonin in Langmuir monolayers comes from Schulman and Rideal,12 who noted an instantaneous increase in surface pressure from 10 to ∼60 mN/m upon introduction of digitonin (1.6 × 10−3 %, pH 7.2) into the subphase beneath a cholesterol monolayer. Soon after, Langmuir et al.13 reported on the deposition of digitonin from solution onto cholesterol predeposited on solid substrates. The thickness of an initially deposited cholesterol monolayer increased from 18 to 36 Å upon contact with aqueous digitonin solution. The authors estimated the stoichiometry of the surface cholesterol/digitonin complex to be 1:0.72.14 Gogelein and Huby showed that the addition of cholesterol (in a 1:2 w/w ratio) to diphytanoylphosphatidylcholine reduces the minimum concentration of digitonin necessary to induce pores in black lipid membranes by a factor of 20. Cholesterol also amplified the surface pressure response in the respective Langmuir monolayers.15 The 2H NMR using mulibilayers of egg yolk lecithin showed that digitonin causes a disordering of lipids in bilayers, which is especially pronounced in the presence of cholesterol.16 In this article, we describe the interaction of one of the most potent membranolytic steroidal saponins, digitonin, with monolayers composed of cholesterol and DPPC using surface pressure vs time curves, surface dilatational rheology, fluorescence microscopy, neutron reflectivity (NR), grazing incidence angle X-ray diffraction (GIXD), and infrared reflection−absorption spectroscopy (IRRAS). The results confirm a strong and selective interaction of digitonin with cholesterol-rich monolayers mimicking the biological membrane’s outer leaflet.

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|E(ω)| = −

dπ A dA

(1)

where A is the molecular area at a given surface pressure π. The viscoelasticity modulus is a complex quantity and consists of two frequency-dependent contributions: storage (E′) and loss (E″) moduli.

E(ω) = E′(ω) + iE″(ω)

(2)

The two components are obtained from the Fourier transform of harmonic oscillations of the surface pressure (Π) in response to the applied strain (area oscillations). Harmonic oscillations of the area per molecule were applied at the end of each Π vs time measurement by moving both barrier positions around the initial location with a frequency of 0.1 Hz and an amplitude of 2%, which is within the viscoelastic linear regime for the system under study. Each measurement consisted of several harmonic compression/decompression cycles during 300 s. Fluorescence Microscopy. The lipid monolayers on the digitonin subphase were observed using an Olympus BX51WI inverted fluorescence microscope. The microphotographs were taken during the subphase exchange, as described for Π(t) measurements. TopFluor PC (1-palmitoyl-2-(dipyrrometheneborondifluoride)undecanoyl-snglycero-3-phosphocholine) was employed as a fluorescent dye (1 mol % vs the lipid). Fluorescence micrographs from the samples were collected using an MPLFN10X objective, and the images were processed using cellSens software. Neutron Reflectivity. In a reflectivity measurement, the beam intensity, R, reflected from a surface, normalized to the incoming intensity at a certain angle of incidence, θ, is recorded. In the case of a neutron reflectivity (NR) experiment, R defined as above is measured as a function of the scattering wave vector, qz, expressed as the angle of incidence, θ, normalized to the corresponding wavelength, λ, as qz = 4π sin θ/λ. NR measurements were performed at the AMOR time-offlight neutron reflectometer (Paul Scherrer Institute (PSI), Villigen, Switzerland).17 The NR experiments at the air/water interface were performed at three angles of incidence (0.25, 0.65, and 1.4°) using a wavelength band from 3 to 12 Å, covering the necessary q range for the experiments of qzmin = 0.01 Å−1 to qzmax = 0.15 Å−1. The resolution was set by a slit system on the incident side and the time-of-flight parameters to Δqz = 0.006 Å−1. A beam of rectangular cross section 1 × 35 mm2 (for low angles) impinged on the samples at the air/water Langmuir trough. The scattered neutrons were recorded with a 3He

EXPERIMENTAL SECTION

For all experiments, digitonin (Mw = 1229 g/mol) purchased from Carl Roth (cat. no. 4005) was used as received. The aqueous solutions of digitonin were prepared by boiling and cooling to room temperature. 1,2-Dipalmitoyl-sn-glycero-3-phosphocholine (DPPC, cat. no. P0736) and cholesterol (cat. no. 26732) were purchased from Sigma-Aldrich. TopFluor PC (1-palmitoyl-2(dipyrrometheneborondifluoride)undecanoyl-sn-glycero-3-phosphocholine, >99% purity) was supplied by Avanti Lipids (cat. no. 810281). Chloroform (98.5% purity) and ethanol (96% purity) were purchased from Chempur (Poland). Milli-Q water (Millipore) was used to prepare all aqueous solutions in protonated water. For experiments with deuterated water, D2O from Armar Chemicals (Switzerland) was used. All glassware was cleaned with acetone and Hellmanex II solution (Hellma Worldwide) and subsequently rinsed with copious amounts of Milli-Q water. Surface Pressure, Subphase Exchange, and Dilatational Rheology. All measurements were carried out with a small KSV NIMA Langmuir−Blodgett trough (77.5 cm2) equipped with two movable barriers. Each measurement was performed at least in duplicate; in case of any significant discrepancies, it was repeated. The surface pressure was measured using a Wilhelmy plate made of filter paper (Whatman Chr1) connected to an electrobalance. The speed of barrier movement was set to 10 mm/min. Temperature was controlled at 21 °C by water circulation from a thermostat. For the surface B


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IRRAS. In an attempt to establish structural information about the DPPC−cholesterol mixed monolayers on pure water and on digitonin solutions, infrared reflection−absorption spectroscopy (IRRAS) was employed. The experiments were performed with a Bruker IFS66 spectrometer attached to an external air/water reflection unit (XA-511 Bruker) and equipped with a liquid-nitrogen-cooled MCT detector. The measurements were performed using a trough with two compartments. One compartment contained the monolayer system under investigation (sample), whereas the other was filled with the pure subphase (reference). The trough was shuttled by a computercontrolled shuttle system to illuminate either the sample or the reference. The angle of incidence was fixed at 40°, and both s- and ppolarized (KRS-5 wire grid polarizer) IR beams were used. In the first step, the spectrum of a pure subphase (water) was recorded (R0), followed by the sample spectrum (R). The reflectance−absorbance (RA) was plotted as −log(R/R0), allowing for the elimination of the water vapor signal. To maintain a constant water vapor content, the whole system was placed into a hermetically sealed box. The resolution and scanner speed in all experiments were 8 cm−1 and 20 kHz.23 The IRRA spectra were recorded for a Gibbs layer of digitonin and for the lipid films compressed to surface pressures (Π) of 2, 5, 10, 15, 20, 25, 30, and 35 mN/m on water. For the study of the interaction of digitonin with the lipids, the monolayers were always compressed to Π0 = 32.5 mN/m prior to the subphase exchange, as described for surface pressure measurements.

single-detector tube in time-of-flight mode requiring typically 8 h of beam time. The experimentally obtained reflectivity curves were analyzed by applying the standard fitting routine using the Parratt32 software.18 It determines the optical reflectivity of neutrons from planar surfaces using a calculation based on Parratt’s recursion scheme for stratified media.19 The reflecting interface is modeled as consisting of layers of specific thickness, scattering length density (SLD), and roughness, which are the fitting parameters. SLD is defined by the sum of the bound coherent scattering length bc of the reflecting material normalized by the volume, v, as SLD = ∑bc/v. The model reflectivity profile is calculated and compared to the measured data, and then the model is recursively adjusted by a change in the fitting parameters to best fit the data. Grazing Incidence X-ray Diffraction (GIXD). The GIXD technique allows us to obtain high-resolution information about the in-plane organization of periodically organized film-forming molecules by means of in-plane diffraction. The scattered X-ray radiation intensity is measured using a position-sensitive detector (PSD) by scanning over a range of horizontal scattering vectors Qxy, defined as

Q xy ≈

⎛ 2Θxy ⎞ 4π sin⎜ ⎟ λ ⎝ 2 ⎠

(3)

where 2θxy is the angle between the incident and diffracted beams projected onto the horizontal plane. Bragg peaks are resolved in the Qxy direction by integrating the scattering intensity over Qz. Lattice vectors a and b can be correlated with the position of the Bragg peaks’ maxima

d=

⎡ h2 ⎤1/2 ⎛ hk ⎞ 2π k2 = ⎢ 2 + 2 − 2⎜ ⎟cos γ ⎥ sin γ ⎝ ab ⎠ Q xy ⎣a ⎦ b

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RESULTS To provide a better understanding of digitonin−cholesterol interaction in biological membranes, in this study we analyze mixed Langmuir monolayers composed of a model phospholipid (DPPC) and cholesterol (Figure 1). It should be stressed at this point that the Langmuir monolayers of pure cholesterol are rather fragile mechanically and not relevant to membranemimicking studies. For this reason, in most experiments we used 10:9 (mol/mol) DPPC/cholesterol mixtures, which reflect the typical phospholipid-to-cholesterol ratio in human erythrocytes.24 For comparison, pure DPPC monolayers were also used in order to assess the specificity of digitonin toward phospholipids. In all experiments, the digitonin concentration was set to 10−4 M, where its detergent activity is still not very pronounced (the surface pressure of the respective Gibbs layer increases only up to 22 mN/m, Figure 2). This concentration is, however, sufficient to achieve the maximum cell membrane permeabilizing and hemolytic activity (100% hemolytic dose for digitonin, HD100 ≈ 10−5 M).1 Surface Pressure and Dilatational Surface Rheology. Because of the non-negligible surface pressure exerted by the Gibbs layers of digitonin at a bulk concentration of 10−4 M, the lipid monolayers could not be spread directly on digitonin solutions. Instead, they were first spread on Milli-Q water compressed to Π0 = 32.5 mN/m, and only then was the subphase exchanged with the stock digitonin solution. The initial surface pressure (Π0) was chosen to mimic the packing of lipid molecules in real biological bilayers, which is believed to resemble that in monolayers compressed to surface pressures of between 30 and 35 mN/m.25 Monitoring the surface pressure during the subphase exchange allowed us to follow the relaxation of DPPC and DPPC/cholesterol monolayers (Π vs t curves, Figure 2). The procedure described in more detail in our previous paper26 assures complete exchange within 15 min without disturbing the monolayer. The introduction of digitonin into the subphase beneath the DPPC monolayer compressed to 32.5 mN/m increases the surface pressure in a manner similar to that for digitonin adsorbing onto the lipid-free surface (Gibbs layer), indicating

(4)

where d is the lattice repeat distance in the 2D lattice, h and k are Miller indices, and γ is the angle between lattice vectors of the given unit cell. For the hexagonal unit cell, γ = 120°; for the rectangular cell, γ = 90°; and for the oblique cell, γ < 120°. Knowing these parameters, the area of the unit cell (A) can be calculated from A = a × b × sin γ. The coherence length, Lxy, i.e., the average range of crystallinity in the monolayer, can be estimated from the Scherrer formula

⎞ ⎛ 2π ⎟ Lxy ≈ 0.9⎜ ⎜ fwhm ⎟ Q xy ⎠ ⎝

(5) 20,21

where fwhmQxy is the full width at half-maximum of the Bragg peak. GIXD experiments were carried out at the SOLEIL synchrotron center (Saint-Aubin, France) on the liquid surface diffractometer at the SIRIUS beamline. The energy of the incoming X-ray beam, 10.5 keV (λ = 0.118 nm), was selected, and the incident beam was deflected downward by a single-mirror setup to an angle incident on the water surface of 1.80 mrad, below the critical angle of total reflection on the air/water interface (2.05 mrad at 10.5 keV). The dedicated Langmuir trough placed in a gastight canister was mounted on the goniometer of the diffractometer. The surface pressure was measured with a Wilhelmy balance (R&K, Germany) equipped with a filter paper strip. The detailed construction of the diffractometer working at the SIRIUS beamline and used in the GIXD experiments is described in ref 22. Monolayers of pure DPPC, pure cholesterol, and their mixtures at molar ratios (DPPC/cholesterol) of 4:1, 1:1, and 1:2 were first spread on distilled water in a Langmuir trough. They were then compressed to a surface pressure of 32.5 mN/m, and their GIXD spectra were recorded. In the next step, a stock digitonin solution was introduced into the subphase to reach a final concentration of 10−4 M, and the spectra were again recorded. Before each experiment, the canister was sealed and flushed with helium to remove oxygen in order to reduce the scattering background and to minimize the beam damage during the experiment. C


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digitonin form a dense structure with a huge capability to both store and dissipate energy provided by dilatation. Neutron Reflectometry (NR). In the next step, the interaction of digitonin with DPPC and DPPC/cholesterol monolayers was investigated using neutron reflectivity. To enhance the scattering contrast of the systems, lipid-chaindeuterated DPPC (DPPC-d62) and protonated cholesterol were used for this purpose. To highlight the changes in the lipid layer induced by digitonin, the scattering-length density (SLD) of the subphase was matched to that of air (so-called null reflecting water, NRW). The reflectivity curves for bare DPPCd62 and DPPC-d62/cholesterol (10:9 mol/mol) monolayers on pure NRW and NRW containing dissolved digitonin (10−4 M) are shown as an inset in Figure 3. For comparison, the

Figure 2. Surface pressure vs time curves for Gibbs layer of digitonin (blue △) as well as for the DPPC (red ○) and DPPC/cholesterol (□) Langmuir monolayers spread on water, subsequently exchanged with digitonin solution after compression of the monolayers to Π0 = 32.5 mN/m. The green dotted line denotes Π0. All experiments were performed at 21 °C for a digitonin concentration of c = 10−4 M.

some extent of monolayer penetration without substantial lipid removal. This observation is in line with earlier suggestions by Nishikawa et al., who showed that digitonin can be inserted into the lipid bilayer even without cholesterol.27 The penetration hypothesis is also supported by a decrease in the surface dilational viscoelasticity modulus, |E|, from 205.4 mN/ m (DPPC on water, E′ = 176.5 mN/m, E″ = 105.0 mN/m) to 58.5 mN/m (after the subphase exchange, E′ = 58.2 mN/m, E″ = 5.8 mN/m). The latter value is significantly higher than 18.1 mN/m characteristic of the Gibbs layer of digitonin on the lipid-free water surface, again suggesting that at least part of the DPPC remained in the monolayer. Nevertheless, it should be noted that for a previously studied mixture of saponins from Quillaja bark (QBS)26 and pure triterpenoid saponins28 an increase in |E| was observed, in clear contrast to the behavior of digitonin in the present study. This suggests that digitonin penetrates the DPPC monolayer, but instead of strengthening the attractive interactions between the DPPC molecules (as for QBS), it weakens them. The lipid monolayer relaxation curve (Π vs t) is very different when DPPC is mixed with cholesterol (10:9 mol/ mol). The surface pressure rise is much more steep and is accompanied by surface pressure oscillations within the first 1000 s, i.e., even before complete subphase exchange. The final surface pressure is higher than the maximum surface pressure that the DPPC/cholesterol mixture can withstand on pure water in our setup (53 vs 40 mN/m). This suggests a strong interaction of digitonin with the monolayer components, especially with cholesterol. After penetration, the surface dilatational viscoelasticity modulus, |E|, reaches a very high value of 421.8 mN/m, with elastic and viscous contributions of comparable magnitude (E′ = 357.3 mN/m and E″ = 224.2 mN/m, respectively). Both moduli are much higher than for the same monolayer on pure water (E′ = 176.5 mN/m, E″ = 105.0 mN/m). Note that for the analogous digitoninpenetrated DPPC monolayer (E′ = 58.2 mN/m, E″ = 5.8 mN/m) and for digitonin’s Gibbs layer (E′ = 18.1 mN/m, E″ = 0.0 mN/m) the surface dilational viscoelasticity modulus is much smaller and predominantly elastic. This comparison suggests that cholesterol-rich monolayers penetrated by

Figure 3. Scattering-length density (SLD) profiles for bare DPPC-d62 and DPPC-d62/cholesterol (10:9 mol/mol) monolayers on pure NRW and NRW containing digitonin (10−4 M) as well as for the Gibbs layer of digitonin on NRW. The inset shows the original NR data (points), with the solid lines corresponding to the best fits from Table 1. All experiments were performed at 21 °C for a digitonin concentration of c = 10−4 M.

corresponding reflectivity curve for the Gibbs layer of digitonin in NRW at the bare NRW/air interface is also shown. The latter clearly proves that digitonin adsorbs to a large extent on the bare water/air interface, in full agreement with the observed increase in surface pressure (Figure 2). The presence of digitonin in the subphase has only a slight effect on the NR curve for the DPPC-d62 monolayer. The changes are far more pronounced for the corresponding mixed DPPC-d62/cholesterol monolayer (Figure 3, inset). To discuss quantitatively the effect of digitonin on the model lipid layers, the reflectivity curves were analyzed using a slab model that assumes the presence of distinct layers stacked parallel to the interface. Each layer is characterized by its thickness, τ, and scattering-length density, SLD. The best-fit parameters obtained by analyzing the data using Parratt32 software are collected in Table 1. The surface roughness, σ, for a given experimental setup is mostly determined by surface tension (hence surface pressure) and was fixed for all measurements at 2 Å. Fitting of the data for the spontaneously adsorbed digitonin layer gives a thickness of 51.6 Å, corresponding well to the picture of adsorbed molecules oriented perpendicular to the surface. This is in good agreement with recent observations of D


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When the subphase NRW is exchanged for the digitonin solution in NRW, the reflectivity curves deviate strongly above q = 0.025 Å−1. The best-fit parameters collected in Table 1 suggest that the scattering ability of the original mixed lipid monolayer decreased and at the same time a new layer beneath developed. Even if the mixed monolayer was completely depleted from cholesterol, SLD could be reduced at its maximum to 2.95 × 10−6 Å−2. The observed reduction of SLD to 1.61 × 10−6 Å−2 indicates that either about half of the deuterated material (DPPC-d62) has been removed from the original DPPC-d62/cholesterol layer or that additional low-SLD material (e.g., protonated molecules) has been incorporated into the layer. The first possibility does not seem very realistic given the fact that DPPC was not removed by digitonin in the absence of cholesterol (Table 1). Thus, the most plausible explanation is that digitonin is squeezed in between the cholesterol and DPPC molecules from the original monolayer. This is also supported by a huge increase in surface pressure observed when digitonin is introduced into the subphase beneath the DPPC/cholesterol monolayers (Figure 2). The fact that the second layer’s SLD is higher than for the digitonin Gibbs layer (0.68 × 10−6 Å2 vs 0.49 × 10−6 Å2) suggests, however, that some DPPC-d62 (and possibly cholesterol) might have been released to the second layer in exchange for digitonin. The thickness of this layer is the same as that for the digitonin Gibbs layer (51.6 Å), and one can speculate that some lipid molecules removed from the first layer (the original lipid monolayer) may form a complex with digitonin in the second layer, attached beneath the first one. A recent X-ray reflectivity and QCM study by Frenkel et al. provides a similar picture for digitonin interaction with mixed cholesterol/SOPC bilayers adsorbed on Si.35 Thus, in addition to penetrating the first layer, digitonin would also pull part of the mixed lipid monolayer toward the aqueous phase and form a complex there, in a similar way to previously described α-hederin.27 Although it is tempting to associate the lipid pulling events with the surface pressure oscillations seen in Figure 2, we believe that the latter are instead simple artifacts due to partial wetting of the Teflon rim resulting from low surface tension. The NR results suggest that digitonin interacts distinctly with cholesterol and DPPC. The existence of a solid digitonin− cholesterol complex was proven more than a century ago.4,5 However, the details of its structure are scarce,5,16,27,36,37 except for the fact that the powder diffraction pattern of the precipitate is different from those of the substrates38,39 and that the molar ratio of digitonin to cholesterol in the precipitate is 1:15 (confirmed also by our own elemental analysis, not shown). On the other hand, both the literature data39 and our own data (not shown) suggest that the IR spectrum of a bulk digitonin− cholesterol precipitate is a simple sum of the IR spectra of the substrates, despite clear evidence of the formation of a new species (precipitation). In our previous study, we successfully applied UV−vis spectroscopy to determine the stability constants for triterpene saponin complexes with cholesterol and DPPC in the bulk.28 However, this approach failed for digitonin because upon mixing the ethanolic solutions of digitonin and cholesterol at millimolar concentrations, a white precipitate appears immediately. At lower concentrations (2 × 10−4 M), the precipitate starts to appear only after about 3 h, but time-resolved UV−vis spectra of the mixture showed no sign of digitonin−cholesterol interaction prior to the macroscopic precipitation (not shown). Any changes in the spectra (originating from light scattering at the growing crystallites) can

Table 1. Best-Fit Parameters from Neutron Reflectivity of Bare DPPC-d62 and DPPC-d62/Cholesterol (10:9 mol/mol) Monolayers on Pure NRW and NRW with Digitonin (10−4 M)a lipid

DPPC-d62 DPPC-d62 DPPC-d62/ cholesterol (10:9) DPPC-d62/ cholesterol (10:9) a

τ/Å

SLD/Å−2

Digitonin/ NRW NRW Digitonin/ NRW NRW

51.6 ± 1.0

(0.49 ± 0.01) × 10−6

26.2 ± 0.5 27.4 ± 0.5

(5.56 ± 0.11) × 10−6 (5.56 ± 0.11) × 10−6

23.8 ± 0.5

(3.02 ± 0.06) × 10−6

Digitonin/ NRW

26.2 ± 0.5 51.6 ± 1.0

(1.61 ± 0.03) × 10−6 (0.68 ± 0.01) × 10−6

subphase

Error values are from fittings using Parratt32.

Golemanov et al., who suggested that monodesmosidic saponins preferentially adsorb in a perpendicular orientation.29 Because of matching the SLD of the water and air, all reflectivity measured for the digitonin Gibbs layer arises from the adsorbed digitonin. Thus, the SLD measured on NRW is a direct measure of the volume fraction of digitonin (φdigitonin) in the adsorbed layer: SLDlayer = SLDdigitonin × φdigitonin. With theoretically calculated SLDdigitonin = 0.96 × 10−6 Å−2, φdigitonin is found to be 0.5, indicating that about half of the volume in the adsorbed layer is occupied with water (NRW). This corresponds to about 70 water molecules hydrating each digitonin molecule, in good agreement with our previous measurements for Quillaja bark saponins (QBS)30 and other triterpene saponins.28 Using the estimated volume fraction of digitonin, one can calculate the adsorbed amount of digitonin, Γdigitonin, from τ Γdigitonin = φ NAv Vdigitonin digitonin (6) where NAv and Vdigitonin are Avogardo’s number and the molar volume of digitonin, respectively. With Vdigitonin = 2040 Å3 (estimated from the molecular weight), Γdigitonin = 2.1 × 10−6 mol/m2. This value corresponds to the area per molecule of 79 Å2, which further supports the perpendicular orientation of the adsorbed digitonin molecules with respect to the surface plane. The DPPC-d62 monolayer compressed to Π = 32.5 mN/m has a thickness, τ, of 26.2 Å and SLD = 5.56 × 10−6 Å−2, in agreement with the literature data.31−33 When digitonin is allowed to adsorb onto such a monolayer, the corresponding NR curve is best fitted using a single-layer model with only a slightly increased layer thickness, τ = 27.4 Å (Table 1).Taking into account the increase in surface pressure and the dilatational surface rheology modulus, this observation can be rationalized as an effect of limited digitonin penetration into the phospholipid layer. The effect of digitonin on NR curves was subsequently assessed in the presence of cholesterol. The experimentally determined SLD for the mixed DPPC-d62/cholesterol monolayer on NRW of 3.02 × 10−6 Å−2 corresponds to about 53% DPPC-d62 and 47% cholesterol (mol/mol). This fully agrees with the 10:9 mol/mol lipid ratio used for the mixed monolayer deposition. It is also worth noticing that the mixed layer is thinner than that consisting of pure DPPC at the same surface pressure and hence less organized. This agrees well with the GIXD and IRRAS results (see below). A similar effect was reported by Wu el al.34 E


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Figure 4. GIXD patterns for DPPC, cholesterol, and their 10:9 (mol/mol) mixed monolayers on the digitonin solution (10−4 M) subphase. All experiments were performed at 21 °C.

Table 2. Positions of the Diffraction Peaks (Qxy), Repeat Distances (d), and Unit Cell Parameters (a, b, Area of Unit Cell, A) for DPPC, Cholesterol, and Their 10:9 (mol/mol) Mixed Monolayers on Pure Water and on Aqueous Digitonin Solutions (10−4 M)a monolayer DPPC

subphase water

cholesterol DPPC/cholesterol (10:9) DPPC cholesterol

DPPC/cholesterol (10:9)

a

digitonin 10−4 M

Qxy/Ǻ −1

d/Ǻ

1.451 ± 0.029 1.361 ± 0.027 1.087 ± 0.022

4.33 ± 0.09 4.62 ± 0.09 5.78 ± 0.12

1.346 1.453 1.373 1.241 1.018 1.461 1.392 1.249 n.m.

± ± ± ± ± ± ± ±

0.027 0.029 0.027 0.025 0.020 0.029 0.028 0.025

4.67 4.32 4.58 5.06 6.17 4.30 4.51 5.03 n.m.

± ± ± ± ± ± ± ±

0.09 0.09 0.09 0.10 0.12 0.09 0.09 0.10

Miller index (0, (1, (1, (0, (1, (0, (1, (1, (1, (0, (1, (1, n.m.

2) 1) (1, 0) (0, 2) 1) (1, 2) 1) (1, −1) 0) (0, 2) 1) (1, −1)

−1) 1)

a, b/Ǻ

A/Ǻ 2

8.66 5.46 6.67 5.39

± ± ± ±

0.17 0.11 0.13 0.13

47.26 ± 0.95

8.65 5.39 10.13 7.79 8.60 5.40 10.06 n.m.

± ± ± ± ± ± ±

0.17 0.11 0.20 0.16 0.17 0.11 0.20

46.64 ± 0.93

38.58 ± 0.77 25.16 ± 0.50

−1) −1) 1) −1)

78.84 ± 1.58 45.61 ± 0.91

n.m., not measured. Error values are from fittings of the GIXD patterns.

observed the formation of DPPC/cholesterol complexes in mixed monolayers. Whether the stoichiometry of these complexes is fixed is still a matter of debate.41,42 In either case, above 20 mol % cholesterol (DPPC/cholesterol ratio of 4:1) the formation of the phospholipid−sterol complex clearly breaks the initial distorted-hexagonal order of DPPC.41 This is evidenced by the disappearance of the two diffraction peaks (1.451, 1.361 Å−1, corresponding to the repeat distances of d(0,2) = 4.33 Å and d(1,1),(1,−1) = 4.62 Å in the liquid condensed (LC) phase of DPPC. (See Figures S1 and S2 in the Supporting Information.) The unit cell area decreases slightly on passing from pure DPPC (47.3 Å2) to 4:1 DPPC/cholesterol (46.1 Å2) and reaches a value of 25.2 Å2 for a phase-separated DPPC in the 10:9 mixture (corresponding to one alkyl chain in the mixed unit cell). At the other extreme (pure cholesterol), the molecules are organized in a hexagonal manner with d = 5.78 Å and the unit area of 38.5 Å2, in very good agreement with ref 41. When digitonin is introduced beneath the precompressed monolayers, significant changes in their GIXD patterns can be noticed. For bare DPPC and at low cholesterol content (below 20 mol %), digitonin displays the same DPPC-disordering activity as cholesterol (Figures S1 and S2 in the Supporting Information), in agreement with the IRRAS results (see below). However, at higher cholesterol content, clearly a new phase is formed that is distinct from those of DPPC and cholesterol alone. The area of the unit cell for the cholesterol monolayer on digitonin (78.84 Å2) confirms the formation of a strong, tightly packed cholesterol−digitonin complex. Given the area of the

be noticed only when the solution becomes increasingly opaque with time. On the other hand, when DPPC is mixed with digitonin, the UV−vis spectra do not change, and even precipitation could not be noticed. In the absence of any clear information on the structure of digitonin−lipid complexes in the bulk, we decided to probe it in situ with surface-specific techniques: grazing incidence X-ray diffraction (GIXD), fluorescence microscopy (FM), and infrared reflection absorption spectroscopy (IRRAS). Grazing Incidence X-ray Diffraction (GIXD). GIXD experiments were performed by collecting diffraction patterns from the respective lipid monolayers compressed to Π0 = 32.5 mN/m before and after exchange of the water from the subphase for the aqueous digitonin solution, analogous to the surface pressure vs time (Π vs t) experiments. To better understand the role of both lipids in the complexation of digitonin, the DPPC/cholesterol ratio was varied between 0 and 1 (0:1, 1:2, 10:9, 4:1, and 1:0). The representative GIXD results for DPPC, cholesterol, and their 10:9 mixture on the digitonin subphase are shown in Figure 4. (See Figures S1 and S2 in the Supporting Information for all of the GIXD diffraction patterns.) The positions of the diffraction peaks, repeat distances (d), Miller indices, and unit cell parameters (a, b, A) for these monolayers on water and digitonin solutions are collected in Table 2. In the absence of digitonin, the effect of cholesterol on DPPC monolayers observed in our study is in line with earlier GIXD data, e.g., those of Ege et al.40 or Ivankin et al.41 who F


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Figure 5. Fluorescence microphotographs of DPPC/cholesterol (1:0, 4:1, 10:9, and 1:2) Langmuir monolayers before (t = 0 min) and at t = 5 and 10 min after the addition of digitonin. The monolayers were initially compressed to 32.5 mN/m. All experiments were performed at 21 °C for a digitonin concentration of c = 10−4 M.

unit cell for cholesterol on water (38.58 Å2), digitonin in the complex would occupy 40.26 Å2. To our knowledge, this is the first direct proof of the formation of a crystalline-like product by the penetration of an amorphous monolayer by digitonin introduced from the subphase. The GIXD diffractograms of DPPC/cholesterol 10:9 and 1:2 mixed monolayers show that the peak pattern is consistent with a sum of the peaks of cholesterol on digitonin (1.241 Å−1, 1.018 Å−1) and DPPC on digitonin (1.453 Å−1, 1.373 Å−1). This would suggest that digitonin helps to release DPPC from its complex with cholesterol, engaging the latter in an ordered cholesterol−digitonin complex. In the digitonin-penetrated mixed monolayers, the released DPPC (possibly partly engaged in a noncrystalline weak complex with excess digitonin) may phase-separate from the digitonin−cholesterol complex crystallites. The correlation length, Lxy, of the latter can be estimated to be about 300 Å. Fluorescence Microscopy (FM). The presence of crystallites in monolayers containing cholesterol is also evident from fluorescence microscopy (FM) studies. The introduction of digitonin underneath the bare DPPC monolayer does not change the FM picture. However, as little as 20 mol % cholesterol is sufficient to produce a mottled texture of irregular brighter spots (Figure 5). The number of these randomly distributed spots (presumably crystallites) is clearly dependent on the cholesterol content. In the presence of cholesterol, a characteristic stripe demixing pattern of domains can be observed before the introduction of digitonin.28 The shape of the pattern suggests that it may originate from very low line tension. The pattern of precipitate formation seems to follow the original pattern of the stripes, suggesting that they may correspond to the phase-separated, cholesterol-rich dark regions and to the domains rich in the DPPC−cholesterol complexes. The mottled pattern seems to appear preferentially within the bright regions, where cholesterol is in a more fluid state and hence more available for complexation (even though it is bound to DPPC). Interestingly, in an analogous study employing the triterpene saponins (ammonium glycyrrhizate, hederacoside C, and α-hederin28 or Quillaja bark saponin, the QBS mixture43), no similar mottled pattern was observed. Instead, in the presence of triterpene saponins, either the circular domains formed or simply the contrast between the dark and bright stripes was enhanced. The interaction of

digitonin with the cholesterol-containing DPPC monolayers is thus clearly different from that with the triterpene saponins. Although we are not aware of similar FM observations for digitonin, a Brewster angle microscopy (BAM) study by Stine et al. on a related steroidal saponin, tomatin, provides a similar crystallite mottled pattern,44 whereas Korchowiec et al. report only a higher brightness of the BAM micrographs of DPPC/ cholesterol monolayers on digitonin.45 It should also be stressed at this point that the formation of the microscopic precipitate observed in FM is a continuous process and is not correlated with the instabilities in surface pressure observed in Figure 2. This strengthens our hypothesis that the instabilities are not related to any chemical processes within the monolayer but are artifacts due to partial wetting of the trough. Infrared Reflection−Absorption Spectroscopy (IRRAS). Although the IR analysis of the bulk precipitate obtained upon mixing cholesterol with digitonin did not show any sign of chemical interaction between the two molecules,39 we attempted to study their possible interaction in Langmuir monolayers in situ using infrared reflection−absorption spectroscopy (IRRAS). In the first step, the individual and mixed lipid monolayers on pure water were analyzed at different surface pressures. It is especially interesting to compare the changes of asymmetric stretching vibration frequencies (νas) of −CH2 groups upon increasing the amount of cholesterol and the surface pressure (Figure 6). At low surface pressures, the addition of cholesterol shifts νas of the methylene groups to lower wavenumbers, indicative of reducing the number of gauche defects in the palmitoyl chains of DPPC. Thus, at low surface pressures the presence of cholesterol increases the ordering of DPPC molecules in the disordered monolayer, helping to stretch them into trans conformers and thus increasing the layer thickness (cholesterol’s condensing effect46). The situation is different at higher surface pressures, where the addition of cholesterol reduces the order in the mixed DPPC/cholesterol monolayers. At lower cholesterol content (4:1 mixture), the phase-transition pressure is almost not affected by the 20 mol % addition of cholesterol. Also, the order in the high-pressure phase is only marginally decreased (slight increase in the wavenumber). At a high DPPC/ cholesterol molar ratio (10:9), the phase transition can be barely detected, and the wavenumbers at high pressures are very similar to the wavenumbers at low pressures (Figure 6). G


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Figure 6. Surface pressure dependence of νas(CH2) for three DPPC/ cholesterol mixtures (1:0, 4:1, and 10:9 molar ratios). All experiments were performed at 21 °C.

Consequently, the phospholipid molecules in the 10:9 mixed monolayer compressed to Π0 = 32.5 mN/m are less ordered than in bare DPPC and in the 4:1 mixture. This conclusion is in perfect agreement with the disordering of DPPC by cholesterol observed by GIXD and with the reduction of monolayer thickness in the NR data. The introduction of digitonin alters the IRRA spectra of DPPC, cholesterol, and their mixture (10:9 mol/mol) to different extents. The −OH as well as −CO and -PO2 stretching regions of the IRRA spectra are shown in Figure 7a,b, respectively. In the absence of cholesterol, the −PO2− group is involved in binding digitonin, as judged from the shift of ν(−PO2−) of DPPC from 1091 to 1087 cm−1 (Figure 7b). This change may be a consequence of breaking the intermolecular bonds between the originally tightly packed DPPC molecules and could explain the worsening of mechanical properties of the digitonin-penetrated DPPC monolayers observed in the surface dilatational rheology results. Interestingly, the characteristic strong band of −C− O−C− motifs of the digitonin’s glycone groups is weak (and probably overlaps with the DPPC’s ν(−C−O−P−O−C−)) in both polarizations of the IRRA spectra for DPPC monolayer on digitonin, even though it is clearly seen for the digitonin Gibbs layer. The fact that the intensity of the −OH band in the DPPC monolayer is practically not changing (Figure 7a) further confirms that the adsorbed amount of digitonin is low. Thus, even though the effect of digitonin is relatively strongly pronounced for the cholesterol-free monolayers (Figure 2), very little digitonin is penetrating the monolayer. The high surface pressure response to tiny changes in the adsorbed amount of digitonin stems from the high slope of the DPPC compression isotherm in this region. The incorporation of digitonin in the DPPC/cholesterol and bare cholesterol monolayers is most evident from the appearance of an intense digitonin −C−O−C band centered at 1072 cm−1 and an increase in the −OH band intensity (Figure 7). The DPPC’s phosphate and carbonyl groups are also affected: the 1226 cm−1 ν(−PO2−) band present in the DPPC/cholesterol monolayer on water splits into 1220 and 1240 cm−1 bands in the presence of digitonin. On the other hand, the intensity ratio for the carbonyl peaks associated with an H-bonded (1725 cm−1) and a free (1739 cm−1) ester group reverses toward the situation observed in pure DPPC, in agreement with the recent observations of Korchowiec et al.45

Figure 7. (a) IRRA spectra (−OH region) for DPPC, DPPC/ cholesterol (10:9), and cholesterol monolayers on water and digitonin. (b) IRRA spectra (1000−1300 and 1650−1800 cm−1 regions) for DPPC, DPPC/cholesterol (10:9), and cholesterol monolayers on water (bottom curves) and on the digitonin solution (top curves) subphase. The monolayers were initially compressed to Π0 = 32.5 mN/m. All experiments were performed at 21 °C for a digitonin concentration of c = 10−4 M.

This is consistent with the dissociation of the DPPC− cholesterol complex driven by the formation of a new digitonin−cholesterol complex and the release of free DPPC back to the LC state. The latter is evident from the shift in νas(CH2) from 2921.8 to 2920.2 cm−1 upon addition of digitonin beneath the 10:9 DPPC/cholesterol monolayer, pointing to the increasing order in the alkyl chain region. This ordering is not due to only the increased surface pressure because the latter does not have a pronounced effect on DPPC ordering above Π = 10 mN/m (Figure 6).

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DISCUSSION The present results provide an improved basis for understanding the effect of digitonin on DPPC, cholesterol, and their mixtures in a confined environment of Langmuir monolayers. Although digitonin interacts with DPPC molecules probably in a simple manner (e.g., by forming H-bonds between its −OH groups and the DPPC’s carbonyl and phosphate groups), the structure of the digitonin−cholesterol complex is still not clear. Glauert et al. explained their electron micrographs of biological tissues stained with saponins by assuming “a two-dimensional micellar-type structure in a lipid monolayer” arrangement.47 However, this particular model was proposed to explain the H


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disorders it, which might be responsible for the observed reduction of the surface elasticity modulus of the monolayer. The present results suggest that the biological activity of digitonin is related to binding the cholesterol present in biological membranes and engaging it in a phase-separated complex. The phospholipid−cholesterol complexes existing in the membrane are then broken, and probably free phospholipids are released. However, the cholesterol−digitonin complex remains attached to the membrane (probably in the form of crystallites) and contributes to its local mechanical properties. The cholesterol-depleted regions then become the weakest part of the lipid network; consequently, the whole membrane becomes mechanically permeable, hence the strong hemolytic activity of digitonin. Other saponins investigated so far in our previous works do not show such a pronounced affinity for cholesterol; consequently, they affect the membrane structure to a much lesser extent than does digitonin.

complex formation in a specific environment of biological tissue treated with saponins, and the same arrangement does not have to apply to other situations. For example, Takagi et al. rejected the model of Glauert et al. on the basis of its incompatibility with their own solubility results in aqueous−ethanol mixtures.36 Instead, they suggested that the cholesterol−digitonin complex might be of the clathrate type, where digitonin would play the role of a host. The hydrophobic interior of the host would accommodate cholesterol molecules (guest). In our view, both models could be valid, depending on the environment and local confinement imposed by the experimental conditions. Moreover, these models are by no means general, so other arrangements are also possible. In our experiments, the lipid molecules are preorganized in a flat monolayer with practically no freedom of movement in a direction perpendicular to the plane of the interface. Digitonin has to tailor its attack strategy to this constraint and probably cannot engage cholesterol in any micellar-type structures. Nevertheless, the similarity of the steroidal part of digitonin with cholesterol, together with H-bonding between the −OH groups of cholesterol and digitonin (note that the latter contains hydroxyl groups in both the glycone and aglycone parts), will probably favor side-by-side stacking between the two molecules. The electrostatic interaction with a positively charged nitrogen atom of the choline group, together with Hbonding of the carbonyl and phosphate groups, might additionally attract a DPPC molecule to such a complex, so a ternary DPPC−cholesterol−digitonin adduct could also be envisaged. Because of geometrical constrains (shape of the molecules), the complex formation may also introduce some local curvature that might be responsible for the observed increase in thickness of the layer, in line with recent suggestions of Lorent et al. concerning the mode of action of α-hederin, a potent hemolytic triterpene saponin.48

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ASSOCIATED CONTENT

S Supporting Information *

The Supporting Information is available free of charge on the ACS Publications website at DOI: 10.1021/acs.langmuir.6b01737. GIXD patterns for DPPC, cholesterol, and their mixed monolayers compressed to Π0 = 32.5 mN/m on water and digitonin (PDF)

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AUTHOR INFORMATION

Corresponding Author

*E-mail: [email protected]. Notes

The authors declare no competing financial interest.

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ACKNOWLEDGMENTS This work was financially supported by the Polish National Science Centre, grant no. DEC-2011/03/B/ST4/00780 and COST CM1101 Action. Ms. Katarzyna Krzeminska is acknowledged for assistance with UV−vis measurements. The work is based on experiments performed at spallation neutron source SINQ, Paul Scherrer Institute, Villigen, Switzerland and at SOLEIL synchrotron, France. This work has been supported by the European Union in the framework of the European Social Fund through the Warsaw University of Technology Development Programme and by the CALIPSO-founded EU program for the experiments performed at SOLEIL.

CONCLUSIONS Digitonin was shown to be capable of complexing both DPPC and cholesterol when they are arranged in monolayers at the water−air interface. However, the strength of interaction and consequent rearrangements in the monolayer are much higher for cholesterol. When both lipids are present in a mixed monolayer, digitonin successfully competes for cholesterol with DPPC, which leads to the formation of strong cholesterol− digitonin complex and the release of DPPC from its weak complex with the sterol. This is evidenced by pronounced changes in grazing incidence X-ray diffraction (GIXD) patterns but also by neutron reflectometry (NR) data and infrared reflection absorption spectroscopy (IRRAS). Also, a strong increase in surface pressure and surface dilatational viscoelasticity for the cholesterol-containing monolayers penetrated by digitonin confirms the competition hypothesis. The phospholipid released from its complex with sterol becomes more ordered (similar to the situation in bare DPPC monolayer), as does the new digitonin−sterol complex. The molecular structure of the digitonin−cholesterol complex remains unclear, but the GIXD data suggest that the two molecules are so complementary that they fit into one crystallographic unit cell. In the absence of cholesterol, digitonin can still penetrate the DPPC monolayer, leading to an increase in surface pressure. However, in contrast to the situation in the cholesterol-enriched monolayers, it is not strongly bound. The IRRAS and GIXD data suggest that it disrupts the H-bonded network of the phospholipid and

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REFERENCES

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On the Interaction between Digitonin and Cholesterol in Langmuir

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