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Disordering Effects of Digitonin on Phospholipid Monolayers Marta Orczyk, Kamil Wojciechowski, and Gerald Brezesinski Langmuir, Just Accepted Manuscript • DOI: 10.1021/acs.langmuir.6b04613 • Publication Date (Web): 23 Mar 2017 Downloaded from http://pubs.acs.org on March 26, 2017

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Disordering Effects of Digitonin on Phospholipid Monolayers M. Orczyk1, K. Wojciechowski1*, G. Brezesinski2

1

Faculty of Chemistry, Warsaw University of Technology, Noakowskiego 3, 00-664 Warsaw, Poland 2

Max Planck Institute of Colloids and Interfaces, Science Park Potsdam-Golm, 14476 Potsdam, Germany

* corresponding author: [email protected]

Abstract Digitonin, a steroidal saponin obtained from the Foxglove plant (Digitalis purpurea), displays a wide spectrum of biological properties and is often used as a model in mechanistic investigations of biological activity of saponins. In the present study, Langmuir monolayers of zwitterionic (DPPC, DMPE, POPC, POPE, DSPC, DSPE, DPPE) and ionic (DPPS, DPPG) phospholipids were employed, in order to better understand the effect of digitonin on the lipid organization. For this purpose, a combination of surface pressure relaxation, infrared reflection absorption spectroscopy (IRRAS) and fluorescence microscopy measurements, was used. The observed increase of surface pressure (Π) suggests that digitonin can adsorb at the air/water interface, both bare and covered with the uncompressed phospholipid monolayers. However, the detailed analysis of IRRAS and fluorescence microscopy data shows that digitonin interacts with the lipid monolayers in a very selective way, and both the headgroup and the lipid tails affect this interaction. Nevertheless, it should be noted that in no case digitonin caused any disruptive effects on the monolayers. The DPPE and DPPS monolayers get disordered by penetration with digitonin, despite an increase of surface pressure, leading to an unprecedented LC-LE transition. Interestingly, the saponin could be easily squeezed out from these monolayers by mechanical compression. Keywords: saponin, surface pressure relaxation, fluorescence microscopy, IRRAS

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Abbreviations: DPPC DPPC-d62 DMPE POPC POPE DSPC DSPE DPPE DPPS DPPG TopFluor PC LC LE PC PE PS PG

1,2-Dipalmitoyl-sn-glycero-3-phosphocholine; 1,2-Dipalmitoyl-d62-sn-glycero-3-phosphocholine; 1,2-Dimyristoyl-sn-glycero-3-phosphoethanolamine; 1-Palmitoyl-2-oleoyl-sn-glycero-3-phosphocholine; 1-Palmitoyl-2-oleoyl-sn-glycero-3-phosphoethanolamine; 1,2-Distearoyl-sn-glycero-3-phosphocholine; 1,2-Distearoyl-sn-glycero-3-phosphoethanolamine; 1,2-Dipalmitoyl-sn-glycero-3-phosphoethanolamine; 1,2-Dipalmitoyl-sn-glycero-3-phosphoserine; 1,2-Dipalmitoyl-sn-glycero-3-phosphoglycerol; 1-Palmitoyl-2-(dipyrrometheneborondifluoride)undecanoyl-sn-glycero-3-phosphocholine; Liquid-Condensed phase; Liquid-Expanded phase; Phosphatidylcholine; Phosphatidylethanolamine; Phosphatidylserine; Phosphatidylglycerol.

1. Introduction Digitonin is a natural steroidal saponin with a spirostan aglycone, belonging to the group of cardiac glycosides (Fig.1.). It is obtained from a perennial plant Digitalis purpurea (Foxglove) [1]. Owing to its unique amphiphilic structure, digitonin demonstrates a number of useful properties typical for saponins, e.g., foaming and emulsifying properties [2-4]. In general, saponins display a wide spectrum of biological properties, e.g., anti-inflammatory, antibacterial, anti-fungal, anti-viral or anti-cancerogenic [5, 6]. For this reason, they are gaining more and more attention from pharmaceutical industry. Nevertheless, it should be emphasized that saponins show great variability of their biological activity and structures (aglycone, number of saccharides, length of the side chains as well as the position of its linkage to the aglycone part, etc.) [6, 7]. Consequently, finding structure-activity correlations is very difficult for saponins [8]. Elucidation of how the biologically active compounds affect their target is a major challenge in drug delivery research. Some of the drugs exert a strong influence on biological membranes, modifying their structure and functions, sometimes causing their lysis, and consequently death of the cell. Others aim at endocellular targets and pass the lipid bilayer with as little damage as possible. In any case, even little changes of the membrane structure resulting from the presence of biologically active molecules can have a great importance for functioning of the cell in every living organism. However, the complexity of biological response of real living objects (like biological cells) precludes any general and unequivocal conclusions concerning their mechanism at the molecular level. Consequently, several mechanistic studies are performed with model cell analogues, in the form of liposomes, 2 ACS Paragon Plus Environment

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supported lipid bilayers or Langmuir monolayers [9-11]. The latter are especially convenient and simple models of individual leaflets of biological membranes, which can be viewed as an assembly of two back-to-back coupled monolayers. The Langmuir trough enables for coupling numerous in situ measurements providing complementary information about the simulated processes occurring at the membrane surface [9, 11, 12]. Until now, most of the research concerning biological activity of digitonin has been focused on formation of insoluble complexes with sterols (mainly cholesterol) [13-15]. Miyamoto et al. showed that permeability of the cells treated with digitonin at low concentrations can be reversible, which might be useful in delivery of e.g. drugs into living cells [16]. However, the information about possible interactions with phospholipids, main components of each biological membrane, is still scarce [17-19]. With this in mind, we have decided to study the influence of digitonin on a series of phospholipids: zwitterionic (DPPC, DMPE, POPC, POPE, DSPC, DSPE, DPPE) and ionic (DPPS, DPPG). The selected phospholipids are characteristic for both the outer and inner leaflets of erythrocytes cells. While the outer leaflet consists mainly of PC lipids, the inner one is dominated by negatively charged ones and PE [20-22]. As shown by Sudji et al., digitonin is capable of migrating through the outer layer of a membrane to form complexes with sterols in the inner layer [14]. Thus, during its journey through the membrane, digitonin may encounter different types of phospholipids and their interaction with the saponin should be assessed individually. In our experiments, the individual phospholipids were spread on water, which after the solvent evaporation was exchanged with digitonin solution, and the changes in the surface pressure over time, Π(t), were recorded. Additionally, the phase behavior of phospholipid monolayers exposed to the action of digitonin was visualized by fluorescence microscopy, and the structural information on digitonin-induced changes in the monolayer was collected using infrared reflection-absorption spectroscopy (IRRAS).

2. Experimental procedures 2.1.

Materials

Digitonin (Mw = 1229 g/mol, Cat. No. 4005) was purchased from Carl Roth and used without any purification. Aqueous solutions of digitonin were prepared by boiling and cooling down to room temperature. The lipids used in our experiments (table 1): POPE, DMPE, DSPE, DPPG, DPPS, Topfluor PC, DPPC-d62 were purchased from Avanti Polar Lipids and DPPC, POPC, DPPE were purchased from Sigma–Aldrich. All lipids were synthetic products 3 ACS Paragon Plus Environment

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of high purity (>99%). Spreading solutions of DPPC, DPPC-d62, DSPC were prepared in chloroform. TopFluor PC, POPC, POPE were dissolved in chloroform/methanol 9:1 (v/v) mixture

and

DPPE,

DPPG,

DMPE,

DSPE,

DPPS

were

prepared

in

chloroform/methanol/water 65:35:8 (v/v/v) mixture. Solvents used in the experiments: chloroform (purity≥ 99.8%) and methanol (purity ≥ 99.9%) were purchased from SigmaAldrich and used without any further purification. Milli-Q water (Millipore) was used for all experiments.

Fig.1. Structure of digitonin Table 1. Lipids used in experiments DPPC

DMPE

POPC

POPE

DSPC

DSPE

DPPS

DPPE


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DPPC-d62

DPPG

TopFluor PC

2.2.

Measurements and instruments

The surface pressure vs. time, Π(t), dependence for lipid monolayers on digitonin aqueous solutions was measured using a home-built Langmuir trough equipped with a Wilhelmy plate made of filter paper (ashless Whatman Chr1) connected to an electrobalance. The subphase temperature of 21 ⁰C was controlled by means of a thermostat. First, a phospholipid solution in an appropriate solvent was deposited onto a Milli-Q water subphase with a Hamilton micro syringe and left for evaporation for 15 min, before the experiment was started. In the second step, the subphase was exchanged with a digitonin solution by a peristaltic pump with a flow rate of 2ml/min. The digitonin final concentration in the subphase was set to 10-4 M, which is below its CMC (about 5·10-4 M) [23]. The surface pressure was monitored for 6000 s. Analogous Π(t) measurements were performed for the aqueous solutions of digitonin in the absence of the spread phospholipid layers (Gibbs layers of digitonin). In order to determine the structural information about the bare monolayers as well as the monolayers exposed to the action of digitonin, infrared reflection-absorption spectroscopy (IRRAS) was employed. The experiments were performed with a Bruker IFS66 spectrometer equipped with a liquid nitrogen cooled MCT detector attached to an external air/water reflection unit (XA-511, Bruker). The angle of incidence used in the experiments was 40°, and the IR beam was s- and p-polarized by a KRS-5 wire grid polarizer. For the s-polarized light, spectra were co-added over 200 scans, and for p-polarized light – over 400 scans. The whole system was placed into a hermetically sealed box to keep the water vapor content constant. During the measurements, the trough was shuttled between two compartments, so that the IR beam illuminated either the sample or the reference. In the first step, a pure subphase (water) spectrum, R0, was recorded, followed by acquisition of the sample spectrum, 5 ACS Paragon Plus Environment

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R. The effect of strong absorption bands of water was eliminated by calculating the reflectance-absorbance, RA, defined as –log(R/R0). In the experiments where pure lipids were characterized, the Langmuir film was symmetrically compressed by two barriers to the desired surface pressure (i.e., 2, 5, 10, 15, 20, 25, 30 and 35 mN/m) and then the spectrum was recorded. In the experiments where the interaction between the lipid monolayer and digitonin was examined, the spectra were recorded for the uncompressed lipid monolayers, and after exchanging the subphase with the digitonin solution. The IRRA spectrum was also recorded for the Gibbs layer of digitonin (104

M). An OLYMPUS BX51WI epifluorescence microscope with U-MWB2 mirror unit

(excitation filter: 460-490 nm; dichromatic mirror: 500 nm) was used for monolayer visualization. The images were acquired, displayed and analyzed with a cellSens software. In order to visualize the changes in morphology of each phospholipid monolayer (with and without digitonin in the subphase) a fluorescent dye (TopFluor PC) was added (0.5% mol/mol).

3. Results 3.1.

The response of uncompressed and compressed phospholipid monolayers

to the presence digitonin The effect of digitonin on a model biological membrane was studied with help of Langmuir monolayers of single phospholipids exposed to the presence of digitonin in the subphase. In our previous study, employing monolayers compressed to 32.5 mN/m as models of a single leaflet of the lipid membrane, we have shown that digitonin cannot penetrate DPPC in the liquid-condensed (LC) phase [24]. For this reason, in the present study we have decided to use uncompressed monolayers in order to facilitate any possible interactions with digitonin in the subphase. The comparison between IRRA spectra of the uncompressed (left panel) and the compressed (32.0 mN/m, right panel) DPPC-d62 monolayer exposed to the same concentration of digitonin (10-4 M) is shown in Fig. 2. A deuterated lipid was used to enable distinction between the -CH2/-CH3 groups of digitonin and the alkyl groups of the deuterated phospholipid, the latter absorbing in the range 2190-2200 cm-1 (for the -CD2 asymmetric stretch) and 2087-2100 cm-1 (for the -CD2 symmetric stretch) [25]. While the Gibbs layer of digitonin in the absence of the monolayer can be formed at the water/air interface (Fig. 2, green line), in presence of DPPC-d62, the -CH2/-CH3 peaks of digitonin can 6 ACS Paragon Plus Environment

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be seen only for the uncompressed monolayer (Fig. 2, red line). This suggests that digitonin molecules can be easily adsorbed at free spaces between the DPPC-d62 molecules in the coexistence region of the liquid-expanded (LE) and the gaseous (G) phase, but cannot overcome the strong van der Waals interactions between the palmitoyl chains in the liquidcondensed (LC) phase, as demonstrated previously in [24]. The strong adsorption of digitonin onto the uncompressed DPPC-d62 monolayer is also confirmed by fluorescence microscopy (see below), as well as by the increase of surface pressure to 28.2 mN/m. For comparison, without the lipid monolayer the surface pressure for digitonin at the same concentration raises only to 22.2 mN/m. Nevertheless, a significant increase of surface pressure upon introduction of digitonin (from 32.0 mN/m to 38.0 mN/m) can be also observed for the initially compressed DPPC-d62 monolayer, suggesting that a small number of digitonin molecules can still fit in the voids of the phospholipid monolayer in the LC phase. Due to the steepness of the compression isotherm in this range, this number is sufficiently high to raise the surface pressure by 6 mN/m. It is, however, too small to de detected by IRRAS, showing that in some cases surface pressure can be a more sensitive probe of adsorption [27].

0.0010

DPPC-d62+digitonin

DPPC-d62

0.0005

Reflectance-Absorbance

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DPPC-d62

0.0000

-0.0005

DPPC-d62+digitonin

digitonin digitonin

-0.0010 3000

2900

2800

2700

3000

2900

2800

2700

-1

Wavenumber, cm

Fig. 2. IRRA spectra of the -CH2/-CH3 stretching vibration region acquired with s-polarized light for DPPC-d62 uncompressed (left panel) and compressed to 32.0 mN/m (right panel) on water (black line), Gibbs layer of digitonin with a concentration of 10-4 M (green line), and DPPC-d62 monolayers exposed to the action of digitonin (red line).


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Having proved that digitonin can easily penetrate the uncompressed monolayer of DPPCd62, we have proceeded with assessing the effect of digitonin on other individual (protonated) phospholipids. The phospholipids were chosen to enable assessment of the role of degree of unsaturation, length of the fatty acid, headgroup-to-hydrocarbon chains size ratio, and the net charge. As biological membranes are built with phospholipids possessing different headgroups, phosphatidylcholine (PC), phosphatidylethanolamine (PE), phosphatidylglycerol (PG) and phosphatidylserine (PS) lipids were chosen. Another factor taken into account when choosing the phospholipids, was the length of alkyl chains, hence lipids with dimyristoyl (DM), dipalmitoyl (DP) and distearoyl (DS) alkyl chains have been used. We have also taken into consideration the unsaturation of phospholipid tails, hence the choice of a lipid with palmitoyl-oleoyl (PO) alkyl chains. As far as the phase behavior is concerned, the published compression isotherm data [26-33] suggest that the chosen phospholipids may exist in the G, LE and LC phases (DPPC and DMPE), the LE phase occurs in POPE and POPC, and the LC phase in DPPS, DPPE, DPPG, DSPE and DSPC at all employed lateral pressures. In the first stage, we have analyzed the surface pressure response induced by introduction of digitonin at 10-4 M concentration into the subphase beneath the monolayers. In every case, introduction of digitonin resulted in an increase of surface pressure to the values exceeding those for the corresponding Gibbs layer of digitonin adsorbing at the free water surface (without any phospholipid monolayer, Π=22.2 mN/m). The values for penetrated monolayers varied from 23.2 to 33.0 after 6000 s (Table 2), indicating that the phospholipid molecules are not displaced by digitonin molecules. Instead, the latter are probably incorporated between the lipid molecules.

3.2.

The effect of digitonin on packing of the phospholipid films

To better understand the processes within the monolayer caused by digitonin penetration, we analyzed the changes in infrared reflection absorption (IRRA) spectra on pure water and on digitonin solutions (10-4 M). First, the spectra were registered for monolayers on pure water compressed mechanically, at eight different surface pressures, up to the highest values achieved by spontaneous penetration of digitonin: 2, 5, 10, 15, 20, 25, 30 and 35 mN/m. The frequency shifts of the symmetric and asymmetric stretching vibrations reflect changes in the number of trans and gauche conformers. The latter, being less ordered and occupying higher area per molecule, absorb IR of higher wavenumbers, while the more ordered trans conformers, absorb at lower wavenumbers. The data obtained by mechanical compression 8 ACS Paragon Plus Environment

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will serve as a reference and will enable us to probe the effect of digitonin on trans-gauche equilibria of different phospholipids, thus on their structure in digitonin-penetrated monolayers. The asymmetric -CH2 stretching vibration wavenumbers for all phospholipids on pure water as a function of applied surface pressure are shown in Fig. 3. For DPPC and DMPE at low surface pressures (1740 cm-1). The analysis of these bands provide important information on changes of the lipid hydration and consequently on interactions between the lipids and digitonin. The latter, being a glucoside, contains five sugar units consisting of xylose, galactose and glucose, which would normally be expected to dehydrate the lipids [37, 38]. However, the increase of the number of more hydrated carbonyl groups accompanying introduction of digitonin suggests that its sugar groups do not dehydrate the PE and PS lipids. This is most clearly visible for DMPE (Fig. 4), where the –C=O band clearly weakens and shifts to lower wavenumbers upon introduction of digitonin into the subphase 11 ACS Paragon Plus Environment

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[39]. Thus, digitonin probably binds to the lipid molecules by forming a hydrogen-bonded network involving its hydration water. In other words, hydrated saponin molecules replace the water molecules originally hydrating the lipids, forming a new structure where water molecules bridge the lipid headgroups and digitonin’s sugar moieties. In agreement with the observations from Table 2, the most pronounced differences between the spectra of monolayers compressed to the same surface pressure on water and on digitonin can be observed for DPPS, DPPE and DMPE. For the dipalmitoyl phospholipids, the difference can be noticed already by comparing the monolayers before (Fig. 4, black line, 3) and after addition of digitonin (Fig. 4, red line, 2) pointing to a fluidizing effect of digitonin. For DMPE, in contrast to mechanical compression, the “chemical” one does not produce any appreciable change in the ester region, despite a clear increase of surface pressure. This is in perfect agreement with the LE state of DMPE at the equilibrium pressure of 33 mN/m due to the penetration of digitonin. The comparison between the spectra obtained at the same surface pressure of 33 mN/m achieved by mechanical and “chemical” compression clearly reveals the difference in the DMPE headgroup region in the presence and absence of digitonin. Mechanical compression leads to the LC state, and the “chemical” compression maintains the LE state even at high lateral pressures. The changes in the ester band for DPPS, DPPE and DMPE confirm that the penetration by the saponin induces changes in the headgroup region - formation of a hydrogen bonded network involving lipid headgroups, digitonin and its hydration water. These changes prevent the dimyristoyl and dipalmitoyl chains from tight packing, hence the LE phase is still present, despite high surface pressure. Similar conclusions can be drawn from the results for ν(-PO2-) and ν(-C-O-C) of the phospholipids and digitonin, respectively, where both peaks were clearly different for DPPE and DPPS than for the other lipids (Fig. S2 in the supplementary materials). On the other extreme, the IRRA spectra (Figs 4 and S2) for the two distearoyl derivatives (DSPE and DSPC) are not affected by the presence of digitonin. Hence, the additional van der Waals interactions provided by two pairs of methylene groups in DS phospholipids (as compared to DP) are sufficient to overcome the stability gain provided by digitonin penetration. The remaining phospholipids (DPPC, POPC, POPE and DPPG) despite having sufficiently short alkyl chains (and/or unsaturated bonds) to lower the van der Waals interactions, show only limited changes in the ester region. This points to the importance of at least two parameters necessary for the appearance of the compression-induced disorganization


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of a phospholipid monolayer: the proper chain length/saturation and the attractive interactions with the headgroup.

0.002 0.001

1

1

1

2 3 4

2

3

2 0.000

3

4

-0.001

4

DMPE

DPPC

POPC

-0.002

Reflactance-Absorbance

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

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0.002

1

1

0.001

1

3

3

0.000

2

2 -0.001

4

POPE

4

DSPC

3

DSPE

4 2

-0.002 0.002

1 2

0.001

1

1 2

2

0.000

3 -0.001

DPPE -0.002 1800

1780

1760

1740

1720

1800

1780

3

3

DPPG

4

DPPS

4 1760

1740

1720

1800

1780

4 1760

1740

1720

-1

Wavenumber, cm

Fig. 4. IRRA spectra in the ester (-C=O) stretching region of the uncompressed phospholipids (black line, 3), Gibbs layer for digitonin (green line, 1), phospholipids exposed to the action of digitonin (red line, 2) and phospholipids on water compressed mechanically (blue line, 4). All spectra are taken with s-polarized light at an angle of incidence of 40°.

3.4.

The reversibility of digitonin penetration into DPPE and DPPS

monolayers The changes in the ester band region of the phospholipids accompanying their “chemical” compression by digitonin can be reversed by further mechanical compression of the monolayers, as shown for DPPE and DPPS in Figs. 5 and 6, respectively. The stepwise recovery of the –C=O band together with its blue shift with increasing surface pressure suggest that the hydrogen-bonded network involving the lipid’s carbonyl groups, digitonin’s sugar groups and water is broken during mechanical compression. The changes in the 13 ACS Paragon Plus Environment

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carbonyl region are accompanied by a red shift and increase of intensity for the asymmetricCH2 peaks, indicative of increasing ordering of the alkyl chains in the monolayer. The most likely origin of this behavior is an expulsion of digitonin, which would then desorb back to the subphase, allowing for reconstruction of the original order and hydration of the lipids. This hypothesis is further confirmed by the fact that the final position of νas-CH2 corresponds well to that in the mechanically compressed monolayers on pure water (Fig. 3). Similar conclusions can be drawn for DPPS, for which also the –CH2 and –C=O reappearance was observed, although the changes were more abrupt (Fig. 6). The digitonin expulsion is complete already between Π=24 mN/m (the equilibrium value after the subphase exchange from water to digitonin) and Π=30 mN/m (the first value achieved by mechanical compression of the digitonin-penetrated monolayer). Further compression does not significantly increase the order in the digitonin-devoid monolayer, in full agreement with the highly ordered nature of this monolayer on pure water (Fig. 3). It is interesting to note that the interaction of digitonin with DPPE seems to be stronger than with DPPS. In both cases, the condensed monolayers are similarly fluidized by digitonin, but in the case of DPPE a much higher mechanical compression (higher lateral pressure) is needed to expel digitonin and

0.003 0.002 0.001

Wavenumber for CH2 as, cm

-1

induce the LE-LC transition.

Reflectance - Absorbance

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0.002

on digitonin

2924 2922 2920

0.001

2918 on water 2916 20 24 28 32 36 40 44 48 Surface pressure, mN/m

0.000 0.000 -0.001 -0.002

Π

-0.001

Π

-0.003 -0.004

-0.002

-0.005 2940

2920

2900

1800

1750 -1

Wavenumber, cm


1700

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Fig. 5. IRRA spectra of the -CH2 stretching region for uncompressed DPPE exposed to the action of digitonin (black line), and afterwards compressed mechanically to 30, 33, 35, 37, 40, 42, 44, 46, 48 mN/m, respectively (left panel). IRRA spectra of the corresponding -C=O stretch (right panel). Inset in the left panel presents changes in the wavenumber for DPPE exposed to the action of digitonin and then compressed mechanically to the respective surface pressure (□), and the analogous values

Wavenumber for CH2 as, cm

-1

obtained for DPPE on pure water (■).

0.003 0.002

Reflectance - Absorbance

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

Langmuir

0.001

0.002

2924 2922 2920 2918

0.001

20 24 28 32 36 40 Surface pressure, mN/m

0.000 0.000

-0.001 -0.002

Π -0.001

Π

-0.003 -0.004 -0.005 2940

2920

-0.002 1800

2900

1750

1700

-1

Wavenumber, cm

Fig. 6. IRRA spectra of the CH2 stretching region for uncompressed DPPS exposed to the action of digitonin (black line), and afterwards compressed to 30, 33, 35, 37, 40 mN/m, respectively (left panel). IRRA spectra of the corresponding -C=O stretch (right panel). Inset in the left panel presents changes in the wavenumber for DPPS exposed to the action of digitonin and then compressed mechanically to the respective surface pressure (□), and the analogous values obtained for DPPS on pure water (■).

Further characterization of the effect of digitonin on morphology of the monolayers was performed using fluorescence microscopy (FM). Since the IRRAS results suggest that digitonin affects directly the headgroup region (and only indirectly the alkyl chains), the probe with a fluorescent tag attached to the fatty acid part (BODIPY – TopFluor PC) was chosen. The dark patches in the microphotographs obtained using this probe correspond to the 15 ACS Paragon Plus Environment

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LC phase in a bright homogeneous LE background, where the probe preferentially partitions. The images were taken for the monolayers compressed “chemically” (on a subphase containing digitonin), and for those compressed mechanically (on water) to the same values as achieved spontaneously by “chemical” compression with digitonin (Fig. 7). Monolayers of all phospholipids except for POPE and POPC showed microscopic patterns in the surface pressure range of 22-33 mN/m achieved spontaneously during the subphase exchange (Table 2). For the unsaturated POPE, the LE-LC phase transition could be noticed only for mechanically compressed monolayers at Π ≈ 40 mN/m, while for POPC no transition could be observed. Thus, since addition of digitonin can raise surface pressure of POPC and POPE monolayers only up to 30-31 mN/m (Table 2), no traces of the LC phase could be observed under a microscope for these monolayers. For the other phospholipids, the presence of digitonin usually leads to an increase of the area of bright spots (increase of the LE phase area). Even for the saturated distearoyl phospholipids (DSPE and DSPC),where the LC phase is well established in a wide range of surface pressures, the microscopic patterns are slightly affected by the presence of digitonin. However, the most pronounced changes can be observed for the intermediate length alkyl chain derivatives (DM, DP) and especially with PE and PS headgroups. For example, DMPE compressed mechanically shows an almost complete transition to the LC phase at Π = 33 mN/m, while in the same monolayer on digitonin, the LE fraction is still well pronounced (Fig. 7). For the compressed DPPE and DPPS monolayers, which are well ordered when compressed mechanically on pure water, the bright spots of the LE phase also become much more pronounced in presence of digitonin. These observations agree well with the increasing disorder observed in the IRRAS results. Also a subsequent mechanical compression of digitonin-penetrated DPPE and DPPS monolayers provides the same conclusions on digitonin expulsion as the IRRAS data (see above). The experiment was performed following the same procedure as described for IRRAS and the corresponding microphotographs are shown as insets in Fig. 7. The disappearance of bright areas characteristic for the LE phase confirms that mechanical compression of digitonin-penetrated DPPE and DPPS monolayers can re-introduce order (LC phase), probably by expelling digitonin.


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DPPC

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Fig. 7. Fluorescence microphotographs of Langmuir monolayers of DPPC, DMPE, DPPS, DPPE, DSPE, DSPC and DPPG phospholipids labeled with 0.5 mol % TopFluor PC, on water (compressed mechanically to the surface pressure values as in Table 2), and on digitonin (compressed to the same values of surface pressure, but “chemically”). Scale bars 20 µm.

4. Discussion The most significant changes in digitonin-penetrated phospholipid monolayers were observed in IRRAS and FM for the phosphatidylethanolamine and phosphatidylserine phospholipids (PE and PS), and the least significant – for the phoshpatidylcholine and phosphatidylglycerol ones (PC and PG). The alkyl chains also play a role in the extent of monolayer rearrangements, but this effect is only related to the strength of the van der Waals interactions between the neighboring chains. These interactions oppose the phospholipid molecule reorientation following incorporation of digitonin into the monolayer. If they are too strong (like for the distearoyl derivatives, DS), packing of the phospholipid molecules is strong and independent of the presence of digitonin. In these cases, the latter adsorbs in areas devoid of the phospholipid molecules, and contributes to the overall surface pressure. If the alkyl chains are too short and unsaturated (like for the palmitoyl-oleoyl derivatives, PO), they cannot pack efficiently, both in the absence and presence of digitonin. The optimum length of the alkyl chain for observation of the effect of interactions between the phospholipid’s headgroup and digitonin, is C14-C16 (DM, DP). Consequently, the most interesting changes were observed for DMPE, DPPE and DPPS. On the headgroup side, the presence of the – NH3+ group seems to be crucial for complexation of digitonin at the interface. The only difference between the PE and PS groups is the presence of additional COO- group in the latter. This greatly affects the geometry and charge distribution of PS phospholipids but does not seem to have any significant effect on complexation of digitonin. On the contrary, the quaternization of the amino group in PC or its replacement with –OH as in PG (see Fig. 1) preclude the effective bonding. The amine group of PS and PE headgroups offers not only the positive charge but also is a good hydrogen donor for H-bonds with the neighboring phospholipid molecules (especially with their phosphate groups), and with digitonin. The latter has no permanent charge, but its glycone part offers as much as 29 H-bond acceptors. This explains the strong preference for PE over PC headgroup, which offers similar positive charge but no H-bond donors in the quaternary ammonium group. In addition, the –N(CH3)3+ 18 ACS Paragon Plus Environment

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group of PC is more hydrophobic than –NH3+ one, and is consequently probably less exposed to digitonin present in the aqueous phase.

5. Conclusions The surface pressure (Π) response to the introduction of digitonin (10-4 M) beneath the uncompressed lipid monolayers provide rather similar picture for all the phospholipids used in this study (DPPC, DMPE, POPC, POPE, DSPC, DSPE, DPPE, DPPS and DPPG). Only the combined use of infrared reflection-absorption spectroscopy (IRRAS) and fluorescence microscopy (FM) provided a clear evidence for headgroup-selectivity of digitonin recognition. The results of the present study can be summarized as follows: 1) The use of uncompressed phospholipid monolayers exposed to the action of steroidal saponin, digitonin, allows for detailed analysis of changes in orientation and packing induced by mechanical and “chemical” compression. The latter was achieved by allowing spontaneous adsorption of digitonin from the subphase onto the monolayer. 2) The surface pressure achieved during exposure of the monolayers to digitonin in each case was higher than that for its Gibbs layers on the bare water-air interface. This confirms that digitonin molecules penetrate the lipid layers, rather than removing them by a simple detergent action. IRRAS and FM results also confirm this conclusion. 3) The latter techniques clearly show that digitonin displays selectivity towards phosphatidylethanolamine and phosphatidylserine phospholipids, although it is capable of increasing surface pressure also for other phospholipids. The necessary condition for observation of changes in IRRAS and FM is an appropriate range of alkyl chains length: C14C16 (dimyristoyl and dipalmitoyl). For longer alkyl chains (distearoyl), van der Waals interactions prevent effective binding of digitonin, which requires some rearrangements of the phospholipid molecule. 4) We postulate that H-bonding between the protonated amine (-NH3+) group of PE/PS phospholipids and glycone (sugar) groups of digitonin are responsible for the observed selectivity. Quaternization of this amine group (as in PC phospholipids) largely reduces chemical interactions between the lipid and the saponin. On the other hand, the proximity of a carboxylic group (as in PS phospholipids), does not prevent digitonin binding. 5) The presence of digitonin, despite increasing surface pressure, induces disordering of the alkyl chains in DMPE, DPPE and DPPS monolayers. For DPPE and DPPS, which form 19 ACS Paragon Plus Environment

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highly ordered (predominantly LC) monolayers on pure water even at the lowest surface pressure, addition of digitonin can even lead to unprecedented LC-LE transition upon increasing surface pressure. 6) The DPPE-digitonin and DPPS-digitonin complex formation within the monolayer can be reversed by further mechanical compression, which allows for squeezing out digitonin, back to the subphase. Release of digitonin re-introduces similar ordering as observed on pure water. 7) The present spectroscopic and microscopic study points to the lack of specific interaction also for DPPC, for which we previously postulated strong interactions, based on surface pressure, surface dilatational rheology, as well as neutron and X-ray scattering studies [24]. Most likely, for phospholipids with other than PE and PS headgroups, digitonin adsorbs non-specifically within the existing LE domains, causing compression of the monolayers, but without specifically affecting the packing of lipid molecules. 8) In numerous previous publications, the high affinity of digitonin to sterols directed most attention to the effect of the former on cholesterol-rich parts of biological membranes. However, the present observations of interaction of digitonin with PE and PS phospholipids prove that cholesterol does not have to be the only target in biological membranes for the strongly membrane-active digitonin. This suggests that the mechanism of membranolytic activity of digitonin (and possibly also for other saponins) might be more complex than simply “seek cholesterol and bind”. Most likely other lipids present in the membrane, especially of PE and PS types, may also be the target for a saponin attack. In future studies we plan to investigate the effect of saponins on lipid monolayers mimicking bacterial and yeast membranes, which are abundant in PE and PS phospholipids.

Acknowledgements This work was financially supported by the Polish National Science Centre, grant no.UMO-2014/13/N/ST4/04122, COST CM1101 Action, Warsaw University of Technology and the European Union in the framework of European Social Fund through the Warsaw University of Technology Development Programme.

Supporting Information Available.


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Figs. S1 and S2 present the -CH2- , PO2- and –C-O-C- fragments of IRRA spectra of the phospholipids in presence and absence of digitonin. This information is available free of charge via the Internet at http://pubs.acs.org/.

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