Vinblastine

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Vinblastine: cholesterol interactions in lipid bilayers Georgios Leonisa, b, Emmanouil C. Semidalasa, Petros Chatzigeorgioub, Evangelos Pollatosc, Charis E. Semidalasa, d, Michael Rappolte, Kyriakos Virasb and Thomas Mavromoustakosa, * a

National and Kapodistrian University of Athens, Department of Chemistry, Laboratory of Organic Chemistry, Panepistimiopolis, Athens 15771, Greece National and Kapodistrian University of Athens, Department of Chemistry, Laboratory of Physical Chemistry, Panepistimiopolis, Athens 15771, Greece c Technological Educational Institute of Ionian Islands, Department of Food Technology, Vergotis Avenue, Argostoli 28100, Kefalonia, Greece d University of West Attica, Department of Food Science and Technology, Laboratory of Organic Chemistry, Egaleo 12210, Greece e School of Food Science & Nutrition, University of Leeds, Leeds LS2 9JT, UK *Corresponding author. E-mail: [email protected] b

Contents 1. Introduction 2. Experimental tools 2.1 Differential scanning calorimetry 2.2 Raman spectroscopy 2.3 X-ray diffraction 2.4 Molecular dynamics simulations 3. Results 3.1 Differential scanning calorimetry 3.2 Raman spectroscopy 3.3 X-ray diffraction 3.4 Molecular dynamics analysis 4. Discussion 4.1 Differential scanning calorimetry 4.2 Raman spectroscopy 4.3 X-ray diffraction 4.4 Molecular dynamics 5. Concluding remarks Acknowledgments References

Advances in Biomembranes and Lipid Self-Assembly, Volume 29 ISSN 2451-9634 https://doi.org/10.1016/bs.abl.2019.01.008

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© 2019 Elsevier Inc. All rights reserved.

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Abstract The anti-mitotic character of vinblastine (VLBS) may relate to its lipophilic nature leading to its hydrophobic interaction with proteins or to its perturbation in lipid bilayers that decreases the lipid fluidity. For these VLBS actions, our work focused on the effects of vinblastine in lipid bilayers. Our aim is to highlight the effects of this highly lipophilic vinca molecule in lipid bilayers in an attempt to use this information in the future for its delivery in liposomal systems avoiding some of its detrimental side effects. Thus, a combination of Differential Scanning Calorimetry (DSC), Raman spectroscopy, X-ray diffraction and Molecular Dynamics (MD) has been applied to study the interactions of antineoplastic VLBS in lipid bilayers. VLBS appears to be distributed to the hydrophilic and hydrophobic segments of lipid bilayers. When cholesterol (CHL) is present in the membrane, VLBS associates not only with the hydrophilic and hydrophobic segments, but also with CHL. This results in the interference of their dynamic effects and causes an antagonistic influence. Thus, in a real biological environment, the interaction between VLBS molecules and cholesterol-rich domains may be disfavored, and a VLBSeCHL separation may occur. Additionally, in our simulations VLBS molecules that were initially placed in the water layer spontaneously entered the lipid bilayer and incorporated into the outer part of the membrane. Finally, we predict that the presence of VLBS favors interdigitation of membrane’s alkyl chains.

1. Introduction Vinca alkaloids, among them vincaleukoblastine (VLBS), commonly known as vinblastine, are lipophilic molecules used to treat cancers. However, they suffer from various side effects including nausea, vomiting, fatigue, headaches, dizziness, peripheral neuropathy, hoarseness, ataxia, dysphagia, urinary retention, constipation, diarrhea and bone marrow suppression [1]. In addition, vinca alkaloids are susceptible to multidrug resistance [2]. To avoid these side effects, different drug delivery strategies have been applied to reduce toxicity and enhance therapeutic efficiency. One of these strategies, proposed by Gregoriades, employed the concept of liposome-entrapped drugs as liposomal formulations to stabilize the drug delivery of vinblastine in vivo [3,4]. It has been also stated that the anti-mitotic character of VLBS may relate to its lipophilic character and to its hydrophobic interaction with proteins or to its effect of decreasing lipid fluidity [5]. These facts triggered our research interest to include vinblastine in the study of the interactions between lipophilic drugs and lipid bilayers. Such studies generally aim to: (a) understand the effects of highly lipophilic molecules on lipid bilayers; (b) use this information in the future for their delivery in liposomal systems avoiding some of their

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detrimental effects; (c) understand the stereoelectronic features responsible for drug action. Previously, several research groups have used various biophysical methods to study vinblastine:membrane interactions. Differential Scanning Calorimetry (DSC) was used to study the thermal effects of antineoplastic vinblastine (VLBS) [6,7], a molecule that is composed of vindoline and catharanthine moieties, and is well known to enter into animal cells by diffusion through the plasma membrane (Fig. 1) [5,8,9].

Figure 1 QM-optimized geometry of VLBS; the atomic partial charges for the catharanthine moiety are displayed.

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It was found that VLBS in high concentration (x ¼ 0.17) in 1,2-dipalmitoyl-sn-glycero-3-phosphatidylcholine (DPPC) increases enthalpy (DH), a property that is also observed for amphipathic and bulky molecules. This increase of DH is interpreted as a possible increase in the van der Waals interactions in the hydrophobic core of the membrane bilayers [6]. The stereoelectronic similarities of VLBS with other bulky amphoteric molecules exerting analogous thermal effects upon lipid bilayers led to the hypothesis that VLBS induces partial interdigitation of the DPPC alkyl chains. This interdigitation was observed to be lipid specific. VLBS was proposed to be located in the vicinity of the polar head-groups and to end at the upper lipophilic part of the lipid chains [6]. The thermal effects of VLBS in DPPC bilayers containing cholesterol (CHL) have been also studied. It was observed that also in this type of lipid bilayers, VLBS increases DH and probably induces interdigitation when the CHL concentration is low. Upon high CHL concentration levels, VLBS is hindered from exerting its partial interdigitation effects. Also, VLBS did not exert any partial interdigitation effect when bilayers contain other phospholipids than DPPC. Interestingly, for partial interdigitation to occur, both the proper length of the alkyl chain and the specific head-group is required [7]. Hence, the obtained results suggested that CHL plays an important biological role not only as a buffer of fluidity of membrane bilayers, but also affects thermal and dynamic properties of pharmaceutical molecules in membrane bilayers [7]. Saraga et al. applied DSC and compared the thermal effects of VLBS with the similar in structure vinca alkaloid vincristine (VCRS) [8]. They showed that VLBS perturbs the DPPC bilayers more strongly than VCRS. Their obtained results led to the suggestion that these two antitumor drugs may also affect the functioning of cell membranes [5,8]. Berleur et al. applied DSC and electron spin resonance spectroscopy to study the effects of VLBS in DPPC bilayers. They showed that in the gel phase, VLBS interacts with DPPC polar heads and induces an important disorganization of the phospholipid bilayers by also decreasing the cooperativity of the main thermal transition. In the liquid-crystalline phase, VLBS is embedded into the DPPC bilayers and induces the formation of domains, which increase thermal stability [9]. In the present work, we review the results obtained from combining Differential Scanning Calorimetry (DSC), Raman spectroscopy, X-ray diffraction and Molecular Dynamics (MD) has been applied to obtain further information on the interactions of vinca alkaloid VLBS with

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CHL-containing DPPC bilayers. The detailed knowledge of VLBS:membrane interactions is beneficial for the research community in the following aspects: (a) to offer insight on the way bulky amphiphilic molecules act on membrane bilayers, and (b) to provide detailed evidence on the role of CHL when coexisting with bulky amphiphilic molecules in lipid bilayers.

2. Experimental tools 2.1 Differential scanning calorimetry For DSC experiments, appropriate amounts of DPPC and VLBS diluted in chloroform were mixed, dried under stream of argon and then stored under high vacuum overnight. Distilled and deionized water was added to the dried mixtures of DPPCevinca alkaloid to produce a 50% (w/w) mixture/water preparation. The samples were transferred to stainless steel capsules obtained from Perkin-Elmer and sealed. Thermal scans were obtained on a Perkin-Elmer DSC-4 instrument (Norwalk, CT, USA). All samples were scanned from 10 to 60  C at least three times until identical thermal scans were obtained using a scanning rate of 2.5  C min1. The temperature scale of the calorimeter was calibrated using indium (Tm ¼ 156.6  C) and DPPC bilayers (Tm ¼ 41.2  C). The following two diagnostic parameters were used for the study of drug to membrane interactions: Tm (maximum position of the recorded phase transition), and DH (the area under the peak, which represents the enthalpy change during the transition). Further experimental details are given in Refs. [6,7]. The drug concentrations used for the different experiments were x ¼ 0.05 (5% mol vinca alkaloid), x ¼ 0.17 (17% mol vinca alkaloid) and x ¼ 0.25 (25% mol vinca alkaloid). The drug concentrations of x ¼ 0.17 and x ¼ 0.25 were used for the preparations containing DPPC:CHL (x ¼ 0.10 or 10% mol).

2.2 Raman spectroscopy Raman spectra were recorded with a Perkin-Elmer GX Fourier Transform spectrometer (Shelton, CT). A diode pumped Nd:YAG laser at 1064 nm (Norwalk, CT, USA) was used as the excitation source. The scattered radiation was collected at an angle of 180 with respect to the incident beam. Spectra were recorded at a laser power of 400 mW on sample with a resolution of 2 cm1. To obtain a good signal-to-noise ratio, 2500 scans were co-added for each spectrum. The temperature was controlled using the high-temperature cell (CAL 3300, Ventacon Ltd., Winchester, UK).

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The intensity of a Raman band was observed over a period of time to ensure equilibration of the sample at the given temperature. Analysis of the spectra was carried out using Spectrum Software Version No. 3.02.01 (PerkinElmer, Norwalk, CT). Raman spectra of the examined samples were obtained in the frequency region of 3500e400 cm1 and in the temperature range of 25e50  C. The following peak height Raman intensity ratios as a function of temperature were used for the study of drug to membrane interactions: I1090/I1130. This ratio allows the direct comparison of the bilayer disordereorder characteristics between bilayers preparations without or with drug incorporation. I2935/I2880: This ratio measures the effects originating from changes both in inter-chain and intra-chain orderedisorder processes in the bilayer acyl chains. I2850/I2880: This ratio describes the main change occurring in the hydrocarbon-chain region of the lipids and corresponds to intermolecular interactions among aliphatic chains [10e13].

2.3 X-ray diffraction The X-ray scattering set-up was the same as described in detail elsewhere [14]. Briefly, time resolved simultaneous small- and wide-angle X-ray scattering (SAXS and WAXS) experiments were carried out at the Austrian SAXS beamline at ELETTRA, Trieste [15,16]. The angular calibration was performed with silver-behenate [17] for the SAXS regime, and for the WAXS regime the diffraction pattern of p-bromobenzoic acid was used as reference [18]. The lipid dispersion was measured in a thin-walled 1 mm diameter quartz capillary in a steel cuvette (Anton Paar, Graz, Austria) and before each temperature scan, the sample was equilibrated for a period of 10 min at 20  C. Static control experiments before and after the scan were taken with an exposure time of 120 s. For the time resolved experiments the samples were heated from 20 to 60  C and back to 20  C with a scan rate of 1  C min1 taking every minute exposures with a duration of 15 s. In the time resolved X-ray scattering experiments the first order d-spacings of the different gel and fluid phases (reflections with the highest intensity) were derived from the SAXS and WAXS diffraction patterns by standard procedures as described in Refs. [19,20]. All Bragg peaks were fitted by Lorentz distributions and carried out with home written procedures running under IDL 5.2 (Research Systems, Inc., USA).

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2.4 Molecular dynamics simulations Molecular dynamics (MD) calculations were performed on two systems, the first one was a homogeneous bilayer, which contained 256 DPPC molecules (128 per leaflet), and the second one consisted of 256 lipid molecules (128 per leaflet), 40 cholesterol (CHL) molecules and 6 VLBS molecules. All simulations were performed with the AMBER14 software package [21]. The GPU implementation of the AMBER14 code was used to run the simulations on NVIDIA GPU cards, thus achieving nearly 10 ns per day [22]. The initial configurations of the homogeneous DPPC bilayer and the heterogeneous DPPC/CHL bilayer were constructed using CHARMM-GUI Membrane Builder [23e26] and were converted to Lipid14 PDB format using the charmmlipid2amber.py script [21]. Both systems were fully hydrated at the relevant experimental hydration level with 50 water molecules per lipid, using the TIP3P model [27,28]. Also, a salt concentration of 0.15 M (MgCl2) was considered for the water layer using suitable AMBER parameters for the ions [29e31]. DPPC and CHL were defined as standard components in the new force field Lipid14 [32]. The VLBS molecule was optimized in the gas phase at the HF/6-31G* level of theory using the Gaussian 09 program package [33], and the atomic point charges were fitted by the RESP procedure [34]. GAFF (General Amber Force Field) atom types [35] were assigned for VLBS with Antechamber [36]. Fig. 1 shows the structure of VLBS and the atomic partial charges assigned for the catharanthine moiety which were obtained from the Merz-Singh-Kollman (MK) scheme [37]. The DPPC/CHL/VLBS system was initially built with three VLBS molecules into the middle of the bilayer, and three other VLBS units were randomly placed in the water phase near the bilayer. Initially, for both systems all-atom minimization was performed for 10000 steps, of which the first 5000 steps used the steepest descent method and the remaining steps used the conjugate gradient method [38]. Afterward, each system was heated from 0 to 100 K at constant volume using the Langevin thermostat [39] with a collision frequency g ¼ 1.0 ps1 for 160 ps. Weak restraints were applied on DPPC and on DPPC/CHL/VLBS systems (force constant 10 kcal mol1 Å2). In the second phase of heating, the volume was allowed to change freely and the temperature increased to 323 K using the Langevin thermostat (g ¼ 1.0 ps1). The pressure was equilibrated at 1 atm using Berendsen’s anisotropic weak coupling barostat [40] for another 400 ps, and the same weak restraint of 10 kcal mol1 Å2 was maintained.

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Before proceeding to the production run, the systems’ periodic boundary condition dimensions and density were equilibrated for 5 ns using the Berendsen barostat. That was necessary in order to eliminate any errors related to the particle mesh Ewald (PME) method [41,42] for calculating the full electrostatic energy of the periodic box, which runs entirely on CUDA-enabled graphics processing units (GPUs) [22,43e45]. The unrestrained, all-atom MD production run was realized at constant pressure (NPT ensemble) on the hydrated DPPC and DPPC/CHL/VLBS bilayers using the GPU implementation of AMBER 14. Bonds involving hydrogen atoms in the TIP3P water model and in the remaining system (DPPC or DPPC/CHL/VLBS) were constrained throughout the simulations by using SETTLE [46] and SHAKE algorithms [47], respectively, thus allowing a relatively large time-step of 2 fs to be used. Electrostatic interactions were treated with the PME method [41,42] using a nonbonded cutoff of 10 Å. The production run was performed at constant pressure of 1 atm and constant temperature of 323 K. Temperature and pressure were controlled by the Langevin thermostat (g ¼ 1.0 ps1) and by the Berendsen barostat, respectively. The MD simulations yielded a 120 ns trajectory for the homogeneous bilayer and a 250 ns trajectory for the heterogeneous bilayer system. The analysis of results (distance, deuterium order parameters, electron density profile, area per lipid and hydrogen bond calculations) was performed on the last 50 ns of the trajectories using the PTRAJ or CPPTRAJ routines [48] and the VMD molecular visualization program [49].

3. Results 3.1 Differential scanning calorimetry In Fig. 2 and Table 1, the thermal scans of lipid bilayers containing ascending concentrations of VLBS are shown. In pure DPPC bilayers (Fig. 2), two characteristic endothermic peaks are visible referring to the pre-transition (Tm ¼ 34.34  C and DH ¼ 0.85 kcal mol1) and main transition (Tm ¼ 42.2  C and DH ¼ 6.95 kcal mol1), respectively. Below the pre-transition temperature, the DPPC molecules form the well-organized lamellar gel phase, Lb’; while above the main transition temperature the fluid lamellar phase, La, is apparent. An intermediate phase, Pb’, is also

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Figure 2 (A) Thermal scans of (A) DPPC bilayers containing (B) 5% mol VLBS (C) 17% mol VLBS and (D) 25% mol VLBS. (B) Thermal scans of (A) DPPC bilayers (E) containing 10% mol CHL (F) 10% mol CHL and 17% mol VLBS (G) 10% mol CHL and 25% mol VLBS.

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Table 1 Thermal parameters of Tm, DH and CU. Sample Tm (oC)

DH (kcal mol1)

CU

DPPC DPPC:VLBS (95:5) DPPC:VLBS (83:17) DPPC:VLBS (75:25) DPPC:CHL (90:10) DPPC:CHL:VLBS (73:10:17) DPPC:CHL:VLBS (65:10:25)

6.950.35 6.990.35 8.120.41 8.290.41 4.720.24 5.320.27 4.360.22

384 116 161 158 e e e

42.200.02 43.310.02 38.640.02 38.540.02 41.500.02 40.120.02 38.690.02

observed, in which the bilayers are modulated by a periodic undulation normally referred to as the ripple phase. These recorded transition temperatures and enthalpies for pure DPPC bilayers are in agreement with literature values [50]. At the low concentration of 5% mol, VLBS abolishes the pre-transition and shifts the main phase transition (Tm) to a higher temperature (Tm ¼ 43.31  C), while it only causes a small increase in DH. In addition, VLBS broadens the main phase transition resulting in an asymmetric peak with various components interpreted to be caused by drug-rich and poor domains with slightly different Tm’s. This highly asymmetric peak with various components may also illustrate that the molecule is embedded at different topographical positions and acts as an impurity. As the concentration increases to 17% mol, VLBS lowers the phase transition (Tm ¼ 38.64  C) and broadens it in a lesser extend compared with the lower concentration of 5% mol. Furthermore, it causes 16% increase in DH. The peak of the main phase transition appears asymmetric and broad. The same thermal scan of DPPC/VLBS for the higher concentration of 25% mol was observed, indicating that drug causes saturation at the concentration of 17% mol. VLBS causes a further small increase in DH at 25% compared to 17% mol. Table 1 shows the values of cooperative units (CUs) [51] for the various concentrations used. The results indicate that samples containing VLBS have smaller CU than that of DPPC bilayers alone. CU parallels the concentration increase from 5% mol to 17% mol and slightly decreases, when the concentration increases from 17% mol to 25% mol. The thermal effects of CHL on DPPC/CHL 10 mol% bilayers are well known to abolish the pre-transition of DPPC bilayers, to broaden the main phase transition and to slightly decrease Tm [52]. Moreover, it causes a significant lowering of 32% in DH (4.72 kcal mol1) (Table 1, Fig. 2B).

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The addition of 17 mol% VLBS in DPPC/CHL sample causes broadening and inhomogeneity in the bilayers with at least two evident components. Furthermore, DH increases by 11% indicating that VLBS exerts a possible partial interdigitation effect at this ratio regardless the CHL content of DPPC bilayers. When 25% mol of VLBS is added, this inhomogeneity is surprisingly less evident. Therefore, the sample containing 25% mol VLBS and the same amount of cholesterol (x ¼ 0.10) shows a distinct thermal profile. This points out that CHL can aid in the increase of incorporation of VLBS. Since DH decreases, it is tempting to believe that VLBS at this higher concentration, and in the presence of the same CHL concentration, does not induce partial interdigitation.

3.2 Raman spectroscopy We have examined the effects of vinca alkaloids at 715 cm1 and will discuss further below the three classical peak height intensity ratios (i) I2850/I2880; (ii) I1090/I1130; and (iii) I2935/I2880. The peak at 715 cm1 is attributed to the symmetric CeN stretching vibration found in the polar region of the phospholipid [53e55]. Thus, this peak can provide information on the effects of drugs with a polar region. DPPC bilayers alone shift the 715 cm1 peak from gel to liquid crystalline phase toward lower frequencies by 2.8 cm1. The presence of 5% mol VLBS shifts the frequency higher by 1.7 cm1. This clearly indicates that VLBS at low concentration considerably affects the polar region. As the concentration increases to 17% mol, VLBS exerts lesser effect by increasing the frequency only by 1.0 cm1. A higher concentration of 25% mol results in an even lesser increase to 0.3 cm1. The presence of CHL causes lowering of the frequency by 1.8 cm1. When 17% or 25% mol VLBS is added, an increase of the frequency by 1.0 cm1 is observed from gel to liquid crystalline phase. Ratio I2850/I2880: The methylene CeH stretching mode in the region 2800e3100 cm1 provides the most intense bands in the Raman spectrum of lipid samples and is commonly used to monitor changes in the lateral packing properties and mobility of the lipid chain in both gel and liquid crystalline bilayer systems. In particular, the intensity ratio I2850/I2880 provides an order parameter of the bilayer core as the intermolecular interactions between the alkyl chains of sn-1 and sn-2 of the same molecule or opposing molecules are depicted. Reduced ratio signifies an increase of intermolecular interactions between chains [10e13]. In Table 2, is shown that this ratio is constant when the concentration of VLBS ranges from 0 to 17% mol and decreases when its concentration

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Table 2 The Raman ratios of I2850/I2880, I2935/I2880, and I1090/I1130. I2850/I2880 I2935/I2880

Samples DPPC DPPC:VLBS (95:5) DPPC:VLBS (83:17) DPPC:VLBS (75:25) DPPC:CHL (90:10) DPPC:CHL:VLBS (73:17:10) DPPC:CHL:VLBS (65:25:10)

Rmin 0.82 0.81 0.76 0.88 0.84 0.87

Rmax 1 0.99 0.94 1 0.91 0.92

DR 0.18 0.18 0.18 0.12 0.07 0.05

0.94

1.01

0.07 0.63

Rmin 0.46 0.42 0.42 0.43 0.51 0.58

I1090/I1130

Rmax 0.73 0.71 0.57 0.6 0.59 0.66

DR 0.27 0.29 0.15 0.17 0.08 0.08

0.74

0.11 0.97

Rmin 0.71 0.79 0.79 0.75 0.78 0.78

Rmax 1.29 1.51 0.99 0.98 0.9 0.97

DR 0.58 0.72 0.2 0.23 0.12 0.19

1.21

0.24

reaches 25% mol. This indicates that VLBS at higher molar ratio enhances the intermolecular interactions between chains. CHL by itself reduces this ratio, signifying the increase of intermolecular interactions. This ratio remains almost constant when 17 or 25% mol VLBS is incorporated in lipid bilayers containing 10% mol CHL. This is an indicative that 10% mol ratio of CHL prohibits high concentrations of VLBS from modifying the intermolecular interactions. Ratio I1090/I1130: The CeC stretching mode in the region 1050e1150 cm1 directly reflects the intramolecular trans:gauche conformational changes within the hydrocarbon chain region of the lipid matrix. More importantly, the temperature profiles of the peak height intensity ratio I1090/I1130 allows the direct comparison of the bilayer disordereorder characteristics. High ratio signifies the disorder and increase of gauche conformers in the alkyl chain [10e13]. The I1090/I1130 ratios for DPPC bilayers containing the vinca alkaloid VLBS are presented in Table 2. This ratio increases when 5% molar ratio is inserted in DPPC bilayers and decreases at higher molar ratios. This signifies that trans:gauche isomerization is concentration dependent. In the same table, the I1090/I1130 ratios for DPPC bilayers containing the vinca alkaloid VLBS in the presence of 10% mol CHL are shown. It is observed that CHL significantly reduces the trans:gauche ratio. The presence of vinca alkaloid results in an increase in trans:gauche ratio. These results demonstrate that VLBS acts antagonistically against CHL. The antagonistic action between VLBS and CHL can be realized, when DR of DPPC:CHL: VLBS (65:10:25) is compared with DPPC:VLBS (75:25) preparation. Both samples have almost equal value.

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Ratio I2935/I2880: The peak height intensity I2935/I2880 ratio constitutes a sensitive probe to monitor the lipid phase transitions despite the fact that the CeH stretching mode region consists of many superimposed vibrational transitions. Thus, this ratio presents the total intra- and inter-molecular interactions [10e13] and is almost constant when the concentration of VLBS reaches 5% mol (Table 2). Above 5% mol, this ratio decreases. This means that at low concentration the total intra and intermolecular interactions are constant and at higher molar ratios are decreased. CHL significantly decreases the total intra and intermolecular interactions, while the presence of 17% or 25% mol VLBS does not affect this ratio at CHL concentration of 10% mol.

3.3 X-ray diffraction Figs. 3e6 provide an overview of all time-resolved SAXS/WAXS experiments. In the contour plots, high scattering intensities are color-coded with red and orange, while lower scattering intensities are given in green and blue. The phase behavior of pure DPPC bilayers and the corresponding structural changes have been widely studied (Fig. 3A) [12,56]. Briefly, from 20 to about 36  C, the lamellar gel phase (Lb0 ) is existent [57]. The chains are tilted with respect to the bilayer plane with about 32 [14] and are packed in an orthogonal lattice [58,59]. The stable ripple phase (Pb0 ) forms at about 36  C [60,61] and at 42  C the fluid lamellar phase is apparent (La) [62]. Note, that the main transition is not reversible: in cooling direction, two

Figure 3 Temperature scan of multilamellar vesicles of (A) DPPC, and of (B) DPPC:CHL (90:10), from 20 to 60  C and back to 20  C with 1  C min1, i.e. at frame number 1 and 80 the sample temperature was 20  C, and the maximum temperature was reached at frame 40.

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Figure 4 Temperature scan of unilamellar vesicles of DPPC:VLBS (75:25) from 20 to 60  C and back to 20  C with 1  C min1, i.e. at frame number 1 and 80 the sample temperature was 20  C, and the maximum temperature was reached at frame 40.

Figure 5 Temperature scan of vesicles of DPPC:VLBS:CHL (65:25:10) from 20 to 60  C and back to 20  C with 1  C min1.

Figure 6 SAXS patterns of DPPC:VLBS:CHL (65:25:10) at 20  C (A), and 40  C (B). Fitting of the first order diffraction peaks was carried out with Lorentz distributions.

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ripple phases form, i.e., the stable and the so-called metastable ripple phase (Pb0 and Pb0 mtstbl) [18e20]. The interaction of CHL with phosphatidylcholine bilayers has also been widely studied (Fig. 3B) [12,50e53]. At 10 mol%, CHL content, a liquid ordered (lo) phase gets induced. In this phase, the lipid chains are ordered by the presence of CHL, but the lipids are still free to laterally diffuse in the membrane leaflet. In contrast, in the La or liquid disordered (ld) phase the lipids display disordered chains and the lipids are free to diffuse laterally. As shown in the SAXS patterns (Fig. 4), VLBS provokes the unbinding of bilayers in the gel and fluid phase regime. In the gel phase regime, this means that the unbinding is caused probably by electrostatic repulsion (membrane undulation usually plays a minor role in the gel phase). In the fluid phase instead, both electrostatic repulsion as well as the onset of membrane undulation contribute to the unbinding of lipid bilayers. In contrast to the effect of VLBS alone (Fig. 4), the incorporation of VLBS together with CHL in the DPPC bilayers, did not favor unbinding (Fig. 5). Furthermore, a closer look to the SAXS patterns reveals that a macroscopic phase separation into probably CHL-rich and poor domains occurs (Fig. 6). Particularly, at 20  C, two lamellar phases with d-spacings of 77 and 72 Å, respectively, can be distinguished. Note that the phase with the smaller repeat distance appears in the diffraction pattern only as a shoulder, but the peak splitting is clearly seen in the second order diffraction peaks. Nevertheless, above Tm these lamellar phases behave differently. While in the first phase the membrane sheets start to unbind, in the second lamellar phase, the bilayers remain at least partially bound (d ¼ 67 Å at 60  C), which is close to the values found for the La phase of pure DPPC (d ¼ 65 Å) as well as DPPC/CHL bilayers (d ¼ 66 Å; cp. Fig. 3B). A possible explanation of the observed events in the DPPC:VLBS:CHL system could be as follows: (i) CHL-rich and poor domains form in the multilamellar vesicles, and further (ii) VLBS gets enriched in the CHLpoor domains. Note that VLBS alone causes the unbinding of membranes in the gel phase, which in turn means that the electrostatic repulsion CHL-poor domains is somewhat counterbalanced by the stable CHL-rich domains. Another explanation could be that the affinity of VLBS for the DPPC/CHL bilayers is reduced compared to the affinity for pure DPPC bilayers. This would also mean that the electrostatic repulsion overall diminishes in the ternary bilayer system. However, once the melting point is crossed and additional membrane undulations are apparent, the VLBSrich domains experience bilayer unbinding, while in the CHL-rich domains

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Figure 7 The WAXS analysis displays the nearest neighbor chain to chain distances derived from the first order diffraction peaks in the gel phase and rough distances determined from the position of the diffuse scattering maximum in the fluid phase in (A) DPPC, (B) DPPC:CHL (90:10), (C) DPPC:VLBS (75:25), and (D) in DPPC:VLBS:CHL (65:25:10) bilayers.

(i.e., VLBS-poor domains), the bilayers remain at least partially correlated with a d-spacing of 67 Å, since here the electrostatic repulsion strongly diminished. The temperature dependent lipid chain packing of the 4 different samples is presented in Fig. 7. The lipid chain packing provides a relatively good indication for the main transition temperature, Tm. The melting point for DPPC alone is determined by the WAXS recordings to be at 43  C, while for all other systems, Tm is approximately 2 lower. This is readily understood, since CHL as well as VLBS can be considered as impurities in the DPPC bilayers, and hence a lowering of the melting point is expected. Another effect can be observed in the packing density of the lipid chains in

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the gel phase. For instance, at 40  C the first order diffraction peak indicates an average nearest neighbor distance of the lipid chains of 4.27 Å for DPPC and DPPC/VLBS bilayers, whereas 10 mol% CHL causes an overall looser chain packing in the gel phase: the d-spacing is found to be 4.33 Å in both the DPPC/CHL and the DPPC/VLBS/CHL bilayer systems. This is anticipated, since it is well known that CHL above a certain threshold (usually > 5 mol%) induces the liquid ordered phase (lo).

3.4 Molecular dynamics analysis Initially, in the heterogeneous bilayer system, three VLBS units were randomly placed in the water phase (VLBS A, VLBS A0 , VLBS A00 , Fig. 8) and three other units into the middle of the membrane (VLBS B, VLBS B0 , VLBS B00 , Fig. 8). It was observed that two out of the three VLBS molecules in the exterior of the bilayer did not interact with the membrane throughout the simulation (VLBS A0 and VLBS A00 ), while VLBS A eventually entered the DPPC bilayer (after approximately 50 ns). Specifically, in the course of the MD run, VLBS A headed from the water phase to the outer leaflet of the bilayer and remained inside throughout the simulation. In a different way, VLBS B, which was initially placed in the middle of the membrane, also moved toward the outer (opposite) leaflet (Fig. 8). This preference of VLBS for the outer surface of the bilayer can be attributed to strong intermolecular interactions between VLBS and DPPC, such as hydrogen bonds (H-bonds), thus causing the molecules to laterally diffuse through the membrane. It was also evident that both VLBS molecules were practically absorbed onto the surface of the bilayer,

Figure 8 Side views of representative poses of the DPPC bilayers (blue)eCHL (orange)eVLBS (magenta) system at the beginning (I) and at the end (II) of the MD simulation. The view in panel (II) is rotated with respect to (I) in order to better display the placement of VLBS A and VLBS B at the end of the simulation.

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forming strong interfacial interactions. However, a part of VLBS B, the vindoline moiety, was oriented toward the water phase. Fig. 8 depicts two representative poses of the DPPC/VLBS/CHL system at the beginning and at the end of the simulation. Despite that the vindoline group of VLBS B was water-exposed, a part of the molecule remained in the outer leaflet during the production run. More specifically, the VLBS’s catharanthine moiety (Fig. 1), and especially rings C and D were surrounded by several polar headgroups of different DPPC molecules, resulting to a dynamic interaction between VLBS and lipids. The positively charged phosphorus atoms of the DPPC headgroups interacted via electrostatic forces with the O1, O2, O3, and C5 atoms of the catharanthine moiety (Fig. 1). Furthermore, two different DPPC molecules interacted through H-bonds with the catharanthine moiety of VLBS B (Fig. 9). The first one formed permanent H-bonds between its sn-1 ester group and the hydroxyl group O1H3 of VLBS B (Fig. 1). The second DPPC formed intermolecular H-bonds between the oxygen atom of its phosphate group and the N2H1 group of VLBS B (ring B in Fig. 1) for w72% of the simulation time. Another noticeable interaction occurred between the N(CH3)3 group of the choline moiety of one DPPC molecule and the negatively charged aromatic ring A of VLBS B (distance: 4.52  0.39 Å). In parallel and in both sides of ring A there were two phosphate groups interacting mainly with atoms C10, C11, C12 and C13 of the ring. It was observed that VLBS B was not in proximity to CHL molecules during the last part of the simulation (wlast 150 ns). On the contrary, the other molecule (VLBS A) entered the bilayer and was eventually situated at the outer part of the leaflet, while it was surrounded by two CHL molecules placed vertically (to DPPC chains). A detailed examination of the intermolecular interactions revealed the presence of several H-bonds between CHL and VLBS A (Fig. 9). More specifically, H-bonds were observed between the hydroxyl group O1H3 of the catharanthine moiety and the sn-1 ester group, which could be regarded as a similar behavior to the catharanthineeVLBS B interactions. There were also H-bonds between the hydrogen atoms of the methylene group (carbon C6 in Fig. 1) of VLBS A and the two ester groups of two different DPPC molecules (85% for sn-2 chain and 74% for sn-1 chain). In addition, the hydroxyl group of the vindoline moiety formed H-bonds with the phosphate oxygen atoms of one DPPC molecule for 67% of the simulation. The other two VLBS molecules that remained in the bilayer (VLBS B0 , VLBS00 , Fig. 8) displayed similar behavior to VLBS B (they were also

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Figure 9 Representative poses of the system showing principal H-Bond interactions. (I) VLBS BeDPPC interactions (II) VLBS AeDPPCeCHL interactions; hydrogen bonds are marked with black dashed lines.

localized in the outer part of the membrane without interacting with CHL), therefore, the analysis of interactions between these molecules and the membrane is not presented. The initial and final positions of all six VLBS molecules are shown in Table 3. The lipid chains of the membrane undergo various dynamic changes, such as rotation around chemical bonds or lipid axis, trans/gauche isomerization and lateral diffusion [63]. The effect of these conformational variations can be estimated using the deuterium order parameter (SCD), which is a common metric for the description of the lipid bilayer’s microscopic

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Table 3 Positions of VLBS molecules at the beginning and at the end of the MD simulation. VLBS Initial topography (0 ns) Final topography (250 ns)

A A0 A00 B B0 B00

Water phase

DPPC head-groups Water phase

Center of Bilayer

DPPC head-groups

structure [64e66]. Specifically, SCD is used to distinguish the lipid-disordered bilayer phase from the liquid-ordered one, as well as to investigate the order of the hydrated lipid bilayer. This quantity can be obtained from 2H NMR or solid-state 1H13C NMR experiments [67e70] and was first used by Stockton and Smith [71], while Hofs€aß et al. [64] expressed it by means of an order parameter for each eCH2 group according to the following equation: SCD ¼

1 2ð3hcos2 q

CD i

 1Þ

(1)

where qCD is the angle between the membrane normal and a CH-bond (a CD-bond in the experiments). Square brackets in Eq. (1) indicate the ensemble averaging over time and over all CD bonds for all lipids. The results of our calculations are shown in Fig. 10 for the two chains (sn-1, sn-2) of a DPPC molecule. Furthermore, the axial diffusion of CHL about its high motional order axis and its vertical arrangement along the bilayer surface have been confirmed experimentally [72]. The steroid ring of CHL is rigid and it is likely that the existence of CHL in the membrane could reduce the gauche-trans isomerizations of the DPPC hydrocarbon chains. During the last 50 ns of the simulation, it was apparent that the all-trans conformation was adopted by the lipid hydrocarbon chains [73]. In addition, it is known that the DPPC/CHL bilayers are characterized by large quadrupolar splitting (plateau) of rather high order parameters of the DPPC hydrocarbon chains. Consequently, the effect of CHL is to amplify all order parameters in the lipid hydrocarbon chains. This increase in motional order induced by CHL can be seen in Fig. 10, and accounts for the match between the fused steroid ring system and the most ordered part of the DPPC hydrocarbon chains (carbon atoms 2e10). The maximum order parameter of

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Figure 10 Deuterium order parameters (SCD) for selected carbon sites at the DPPC tailgroups for the DPPC and DPPC/CHL/VLBS systems.

SCD ¼ 0.43 was reached, which is near the limiting value of SCD ¼ 0.5 for the DPPC all-trans chain that experiences axial rotation about its long axis [72e74]. Our MD results showed that the sample containing VLBS with CHL maintains the high order parameter expected for DPPC/CHL bilayers (Fig. 10). The hydrophobic thickness of the bilayer is defined as the distance between the two peaks in the electron density profile. It was calculated to be 37.6 Å for the DPPC bilayer and 48.0 Å after the addition of CHL and VLBS, thus corresponding to a 22% increase in hydrophobic thickness (electron density profiles are shown in Fig. 11). The experimental hydrophobic thickness of the DPPC bilayer was found by Nagle et al. [75] in the range of 36.4e39.6 Å, which is in agreement with our calculated value. Furthermore, the quality of the simulated electron density profile is comparable to experimental profiles of DPPC/CHL membranes proposed in other studies [76,77]. The area per lipid is a significant structural feature of a bilayer and is calculated as the product of X and Y dimensions of the simulation box, divided by the number of lipids contained in each monolayer (128 lipids in this study). The average area per lipid over the last 50 ns of the simulation was calculated to be 48.5 Å2 for the DPPC/CHL/VLBS system. The corresponding value for the simulated DPPC bilayer was found to be 61.9 Å2 and is in agreement with experimental values (63.1 Å2 [78] and 64.3

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Figure 11 Electron density profile of (A) the hydrated DPPC bilayer, and (B) the hydrated DPPC/CHL/VLBS system.

Figure 12 Area per lipid for (A) the hydrated DPPC bilayer, and (B) the hydrated DPPC: CHL:VLBS system.

Å2 [79]). Therefore, the addition of CHL and VLBS into DPPC bilayers decreases the area per lipid by 22%. Graphs of the area per lipid are presented in Fig. 12.

4. Discussion 4.1 Differential scanning calorimetry DSC results showed that VLBS at low concentration of 5% mol induces significant broadening and inhomogeneity to lipid bilayers and a decrease of CU, while it does not affect DH. As the concentration increases (17%e25% mol), the broadening is not as pronounced. VLBS lowers the

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phase transition and CU and increases DH. Such thermal behavior may reflect on a possible induction of partial interdigitation of lipid bilayers. Thus, the presence of VLBS packs the lipids closer, but on the other hand its presence creates voids in the lipid bilayers as it is a bulky molecule and thus decreases CU. VLBS in DPPC bilayers containing CHL also causes broadening and inhomogeneity in the bilayers. In addition, when CHL is at low concentration (10% mol), it slightly increases DH at 17% mol, while a slight decrease is observed at 25% mol. Thus, its partial interdigitation effect is blocked by CHL. CHL also appears to significantly affect the thermal profiles of DPPC/VLBS. This signifies the role of CHL in lipid bilayers when VLBS is incorporated.

4.2 Raman spectroscopy DPPC bilayers alone shows a lowering of the 715 cm1 peak value (CeN sv of polar choline group) as the temperature increases due to the increase of gauche conformers in the polar choline head-group. VLBS affects the polar region more drastically at low concentration in DPPC bilayers as depicted by the changes of 715 cm1. In particular, Dn is increased by 1.7 cm1 when the drug concentration is 5% mol, and 1 cm1 at 17% and 25% mol ratios. This is in agreement with DSC data, which clearly show that at 5% VLBS molar ratio the main phase peak is significantly broadened and an increased inhomogeneity is observed. When the concentration of the drug is increased, the drug is incorporated more in the lipophilic segment of the lipid bilayers and thus its effect on the polar groups is not as pronounced. DPPC/CHL bilayers show a decrease of 715 cm1 CeN sv of polar choline group by 1.8 cm1 due to the increase of gauche conformers in the polar choline head-group. Notice, that this increase is less pronounced when compared with DPPC bilayers alone, indicating that CHL results in the decrease of trans:gauche isomerization. In the DPPC/CHL bilayers containing 17% mol or 25% mol VLBS an increase of 1.0 cm1 is observed. This is again in harmony with DSC results where broadening and inhomogeneity is increased in the presence of VLBS. It appears that VLBS has an opposite effect in interacting with polar head-groups in comparison with CHL. Essentially, it interferes with the way the polar head-groups of DPPC bilayers interact with each other. VLBS does not affect I2850/I2880 ratio at concentrations of 5%e17% mol, and at 25% mol decreases, thus signifying the enhancement of

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intermolecular interactions. In contrast, CHL decreases this ratio at 10% mol ratio more effectively. The presence of VLBS in preparations containing 17% mol and 25% mol ratio does not further affect this ratio. VLBS at low concentration of 5% mol does not affect the ratio I2935/I2880. At higher concentrations it lowers the ratio, signifying that it increases intra and inter molecular interactions. 10% mol CHL induces more effective lowering and the presence of VLBS does not seem to further affect this. VLBS increases I1090/I1130 or trans:gauche ratio of alkyl chains at low concentration of 5% mol. and decreases it at higher concentrations. This is in agreement with DSC results, which showed that as the concentration of VLBS increases, more drug is embedded in the alkyl chain that hinders trans:gauche isomerization. 10% mol CHL causes more decrease of trans:gauche ratio. When 10% molar ratio VLBS is incorporated in lipid bilayers containing 17% mol and 25% mol CHL, a small increase of the ratio is achieved. DPPC:CHL: VLBS (65:25:10) sample appears to have almost the same DR as DPPC: VLBS (75:25). Thus, VLBS at this particular concentration acts antagonistically and compensates the trans:gauche effect of CHL.

4.3 X-ray diffraction X-ray diffraction results complement and confirm DSC and Raman results: (i) VLBS is more effective in DPPC bilayers than in DPPC bilayers containing CHL, when the diagnostic parameters for the three techniques, i.e., DH, DR, I2850/I2880, I1090/I1130 and I2935/I2880 ratios, and d-spacing are compared. (ii) DSC and Raman experiments showed a strong association of VLBS with the polar region. X-ray diffraction revealed that VLBS induces unbinding of the membranes in the gel phase. (iii) This strong association of VLBS is reduced when the lipid bilayers also contain CHL. (iv) Using X-ray diffraction, the electrostatic repulsion exerted by VLBS is counterbalanced by membrane stack domains that are rich in CHL. (v) DSC and Raman results showed that the presence of VLBS alone or in combination with CHL abolishes the pre-transition, which is further confirmed by the SAXS/WAXS measurements.

4.4 Molecular dynamics The MD calculations confirmed the possibility of inhomogeneous distribution of VLBS in the lipid bilayers, in agreement with the experimental

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results. Both, interfacial and lipophilic segments of lipid bilayers are occupied by VLBS molecules. When VLBS is localized outside the lipid bilayers, it spontaneously diffuses in them. Importantly, VLBS was found to interact with DPPC bilayers and CHL. These interactions in a biological environment possibly cause antagonistic effects as illustrated by DSC, X-ray diffraction and Raman spectroscopy. Thus, VLBS in a real environment may avoid cholesterol drafts although conserving its ordering effect in the fluid bilayers.

5. Concluding remarks The combination of various biophysical techniques with Molecular Dynamics shed some light on the dynamic and thermal properties of VLBS, especially when it coexists with CHL in lipid bilayers. VLBS and CHL interact with each other and their modes of action interfere in an antagonistic way. This significant observation may not be limited to only VLBS but can be a common property of bulky amphipathic molecules. In addition, it was suggested that interdigitation of the DPPC alkyl chains was caused by the presence of VLBS molecules. Molecular Dynamics, DSC, and X-ray diffraction results showed that VLBS at low concentrations is localized at the interface of the lipid bilayer, thus maximizing its amphoteric interactions. When its concentration is increased, VLBS is also embedded deeper in the lipid bilayer where exerts van der Waals interactions with the alkyl chains of the lipid bilayers and CHL. This distribution of VLBS in lipid bilayers is evident by thermal scans where various components are observable. Molecular Dynamics showed that spontaneous incorporation of VLBS from the water phase into the lipid bilayers occurred during the course of the simulations. However, this does not exclude the possibility that VLBS may explore other ways for entering lipid bilayers, that is, other pathways of facilitated transportation.

Acknowledgments The project was supported by Cy-Tera Project (NEA UPODOMH/STPATH/0308/31), which is co-funded by the European Regional Development Fund and the Republic of Cyprus through the Research Promotion Foundation. G. Leonis and E. Semidalas contributed equally. Part of this manuscript is from P. Chatzigeorgiou dissertation.

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