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A comparative study of the effects of cholesterol and sclareol, a bioactive labdane type diterpene, on phospholipid bilayers
Chemistry and Physics of Lipids 133 (2005) 125–134
A comparative study of the effects of cholesterol and sclareol, a bioactive labdane type diterpene, on phospholipid bilayers I. Kyrikoua , A. Georgopoulosb , S. Hatziantonioub , T. Mavromoustakosa , C. Demetzosb,∗ a b
Institute of Organic and Pharmaceutical Chemistry, National Hellenic Research Foundation, Vasileos Constantinou 48, Athens 11635, Greece Department of Pharmaceutical Technology, School of Pharmacy, Panepistimioupolis, Zografou 15571, University of Athens, Athens, Greece Received 22 April 2004; received in revised form 17 September 2004; accepted 21 September 2004 Available online 18 November 2004
Abstract Sclareol (labd-14-ene-8,13-diol) is a highly water-insoluble molecule that belongs to the labdane type diterpenes and is characterized as a biologically active molecule, due to its cytotoxic and cytostatic effects against human leukemic cell lines. A superimposition study between sclareol and cholesterol, based on their corresponding hydrophobic and polar molecular segments calculated from their lipophilic profiles, revealed their spatial similarities. This structural similarity between the two molecules prompted us to compare their effects on the structure and stability of phospholipid dipalmitoylphosphatidylcholine (DPPC) membranes. Differential scanning calorimetry (DSC) was applied to compare the thermal changes caused by either cholesterol or sclareol when are incorporated in DPPC bilayers. The results showed that sclareol is incorporated into phospholipid model membranes and mimics the thermal effects of cholesterol especially at concentrations up to Xsclareol = 9.1 mol%. These effects can be summarized as the abolition of pre-transition, lowering of the main phase transition and reduction of the enthalpy change (H) of the gel to liquid-crystalline phase transition of DPPC bilayers. At concentrations X ≥ 16.7 mol%, sclareol and cholesterol caused different heterogeneity in lipid bilayers or a reversible transition from a vesicular suspension to an extended peak bilayer network. This different fluidization, exerted by the two molecules at high concentration, may be related to their different stability and the z-average mean diameter of the liposomes they form. Small unilamellar vesicles, prepared by the thin film hydration method showed that DPPC bilayers containing a high concentration of sclareol in equimolar ratio sclareol:cholesterol were unstable, in contrast to the ones containing only cholesterol. © 2004 Elsevier Ireland Ltd. All rights reserved. Keywords: Sclareol; Cholesterol; Differential scanning calorimetry; DPPC bilayers; Liposomes
∗
Corresponding author. Tel.: +30 210 7274596; fax: +30 210 7274027. E-mail address: [email protected] (C. Demetzos).
0009-3084/$ – see front matter © 2004 Elsevier Ireland Ltd. All rights reserved. doi:10.1016/j.chemphyslip.2004.09.021
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1. Introduction Sclareol (labd-14-ene-8,13-diol) is a ditertiary alcohol, widely distributed in nature, belonging to the labdane type diterpenes. The interest in studying labdanes is based on their wide range of biological activities (Singh et al., 1999). Sclareol was first isolated from clary sage oil (Salvia sclarea, Labiatae) (Notin, 1972) and it also occurs in many conifers, as well as being isolated from the genus Cistus. It is used as a fragrance in cosmetics and as a synthon for preparation of Ambra odorants in perfumery (Decorzant and Vial, 1987). Sclareol was recently investigated by our group for its cytotoxic and cytostatic effects against leukemic human cell lines and effects on the morphology and the kind of death in cell lines, as well as on the cell cycle progression using flow cytometry (Dimas et al., 2001). It was found that sclareol significantly interferes with the cell cycle and induces apoptosis in leukemic cell lines, while it does not affect normal and PHAstimulated peripheral blood lymphocytes. The expression of the ongogenes c-myc and bcl-2 was studied using a Western blot analysis. The results showed that the c-myc levels were reduced, while bcl-2 expression remained unaffected. Cholesterol is a common constituent of biological membranes. Its incorporation into phospholipid bilayers is well-known to affect the gel to liquid-crystalline state of model membranes (Ladbrooke et al., 1968). The effect of cholesterol on lipid bilayers is attributed to its molecular features. The 3-hydroxyl group is oriented towards the polar headgroup region of the phospholipid bilayer, while the hydrophobic part of the molecule is oriented towards its nonpolar part. The aim of our study was to investigate whether sclareol interacts with lipid bilayers and influences the gel to liquid-crystalline state of dipalmitoylphosphatidylcholine (DPPC) membranes in a similar way to cholesterol. The rationale behind such a comparison is that molecular superimposition studies showed that these molecules, in spite of their apparent structural dissimilarity, have common stereoelectronic features when appropriate equivalent atoms are properly superimposed. These two amphipathic molecules have their lipophilic and hydrophilic segments spatially arranged in a way that can afford excellent superimposition.
In our extensive research experience, molecules that are very similar in chemical structure do not necessarily have correspondingly similar thermal profiles. Geometrical, stereochemical or amphipathic differences between the molecules result in completely different thermal profiles. This has been proved using a combination of biophysical methods to study the effects, in membrane, of a pair of drugs, (−)-8 tetrahydrocannabinol and its ether analog, that differ in their amphipathic properties. The bioactive psychomimetic drug, (−)-8 -tetrahydrocannabinol, anchors itself at the membrane interface, presumambly through hydrogen bonding with the phospholipids ester groups or with water molecules present at the interface. In this location, the drug molecule can induce bilayer perturbations most effectively. Conversely, the almost inactive analog, (−)-O-methyl-8 tetrahydrocannabinol, locates itself deeper in the bilayer away from the interface, a property which accounts for its decreased ability to perturb the membrane (Yang et al., 1992). In addition we have applied different biophysical methods to study the effects of the anaesthetic steroid alphaxalone (5␣-pregnane3␣-ol-11,20-dione) and its structurally similar inactive congener 16 -alphaxalone (5␣-pregn-16-ene-3␣ol-11,20-dione). These steroids showed different dynamic and thermal effects in membrane bilayers, which has been attributed to the different molecular geometry of the D ring between the two molecules (Mavromoustakos et al., 1994) On the other hand, molecules that are dissimilar in chemical structure, like taxol and vinblastine have been shown to have similar thermal profiles and induce inderdigitation, because they are bulkly and amphipathic and, thus, have stereoelectronic similarities (Kyrikou et al., 2004). Similar observations were made in membrane bilayers containing cholesterol or its precursor lanosterol or its derivatives. Miao et al (Miao et al., 2002, see also references therein) showed that the evolution in the molecular chemistry from lanosterol to cholesterol is manifested in the model lipid-sterol membranes by an increase in the ability of the sterols to promote and stabilize a particular membrane phase, the liquidordered phase, and to induce collective order in the acyl-chain conformations of lipid molecules. Thus, cholesterol, with its streamlined molecular structure, interacts more effectively with lipid chains with conformational order and stabilizes the liquid-ordered state
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of the lipid bilayers more effectively than lanosterol. Urbina et al. (1995) (see also references therein) found that ergosterol was more effective at increasing order and restricting the mobility of DMPC than cholesterol, while the opposite was true for POPC bilayers. The trimethylated precursor, lanosterol, was less effective than the other two sterols, with both saturated and unsaturated lipids. These results showed that the interactions between phosphatidylcholines and sterols were complex and depended on the details of sterol structure and the types of acyl chains present in the phospholipids molecules. Ghosh et al. (2001) applied differential scanning calorimetry (DSC) and fluorescence analysis to study the nature of perturbation induced by cationic cholesterol derivatives. The authors found that the nature of perturbation induced by each of these cationic cholesterol derivatives was dependent on the details of their molecular structure and provided significant information on the nature the interaction between these derivatives and phospholipid bilayers. The modifications of the enthalpy as a function of phase transitions, when sclareol is incorporated into lipid bilayers, have never been studied. In order to compare the effects of sclareol and cholesterol in membrane bilayers, we applied DSC, which is a fast and relatively inexpensive technique that allows the study of the thermotropic properties of membranes in the presence of bioactive molecules. Therefore, it is used as a diagnostic technique to investigate the differential effects that may be caused by the incorporation of additives under study (Koynova and Caffrey, 1998). The thermal changes, induced by the presence of different molar concentrations of sclareol and cholesterol on DPPC lipid bilayers, were studied in order to characterize the types and strength of their interactions. In order to investigate the effect of sclareol and cholesterol on the stability properties of liposomes, multilamellar vesicles (MLVs) and small unilamellar vesicles (SUVs) composed of DPPC, were prepared incorporating cholesterol, sclareol and sclareol/cholesterol. Liposomes are considered to be effective and non-toxic carriers for incorporating lipophilic and hydrophilic compounds and are currently in clinical use (Woodle, 1995) Their sizedistribution (z-average mean diameter) and ζ-potential were measured. The obtained results were correlated with those obtained by DSC.
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Our future goal is to design liposomes as carrier systems, used to deliver poorly water-soluble compounds to the target tissue, with appropriate physicochemical properties to improve their in vivo kinetic parameters and their stability. Such liposomal formulations could be appropriate for lipophilic biologically active compounds such as sclareol.
2. Experimental procedures 2.1. Materials Sclareol has been isolated from Cistus incanus subsp. creticus and from its resin Ladano (Demetzos et al., 1999). C. incanus subsp. creticus was collected in the island of Crete, Greece (collector Demetzos, 1990). The isolation procedures, as well as the identification methodology have been described in recently published papers (Demetzos et al., 1990, 1999). DPPC and cholesterol were obtained from Avanti Polar Lipids Inc., USA; the organic solvent used, chloroform (CHCl3 ), was of spectroscopic grade, Merck, USA. 2.2. Methods 2.2.1. Liposome preparation Liposomes were prepared by the thin-film hydration method. The lipid film was prepared by dissolving the appropriate amount of DPPC (cholesterol and/or sclareol) in chloroform. The solvent was evaporated in a flash evaporator and the film was dried under vacuum for 24 h. Subsequently, the film was hydrated with HPLC grade water and heated for 1 h at 50 ◦ C with stirring. The resultant liposomal suspension, composed of MLVs, was freezed and lyophilized for 24 h and then used for DSC experiments. For the preparation of SUVs, the resultant liposomal suspension was subjected to two sonication periods (of 20 min each interrupted by a 5 min resting period) in a water bath at 25 ◦ C, using a probe sonicator (amplitude 100, cycle 0.7 – UP 200S, dr. hielsher GmbH, Berlin, Germany). The resultant vesicles were allowed to anneal for 30 min. Then the liposomal suspension was centrifuged (20.000 rpm, 20 min, 4 ◦ C, Sorval Ultra Pro 80, fixed angle rotor) in order to separate the SUVs from Titanium and the MLVs liposomes.
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The liposome dispersion was diluted 10 times with HPLC grade water (pH 5.6–5.7) and liposome size distribution and ζ-potential were determined using a photon correlation spectrophotometer (Zetasizer 3000, Malvern, UK). Samples were scattered (633 nm) at an angle of 90 ◦ . The data were analysed by the CONTIN method (MALVERN software). 2.2.2. Differential scanning calorimetry (DSC) The DSC technique was applied to the study of MLV samples using a Perkin-Elmer DSC-7 calorimeter. MLVs were obtained by dispersing the dry residues in appropriate amounts of bi-distilled water by vortexing. After dispersion in water (50%, w/w), an aliquot (ca. 5 mg) was sealed into stainless steel capsules obtained from Perkin-Elmer. Prior to scanning, the samples were held above their phase transition temperature at 50 ◦ C to ensure equilibration. All samples were scanned at least twice until identical thermograms were obtained, using a scanning rate of 2.5 ◦ C/min. The temperature scale of the calorimeter was calibrated using indium (Tm = 156.6 ◦ C) as standard sample. 2.2.3. Molecular modeling Computer calculations were performed on a Silicon Graphics O2 workstation using QUANTA 97 of Molecular Simulation Incorporated. Sclareol and cholesterol were first energy minimized using, as starting structures, their X-ray crystallographic ones (Bernardinelli et al., 1988; Shieh et al., 1981). The lipophilic profiles of the molecules were also calculated. By default, the colour mapping of the lipophilic surfaces is relative, not based on absolute values. The colour scale runs from the most lipophilic (brown) to the most hydrophilic (blue) parts of the molecule (Fig. 1). The lipophilicity force field profile is a powerful tool when one seeks to compare the lipophilic molecular potential of different molecules (Croizet et al., 1990). Superimposition between the minimized structures of sclareol and cholesterol was achieved using torsional flexible and rigid body methods. 3. Results and discussion 3.1. Differential scanning calorimetry The DSC scans for the three preparations of DPPC/cholesterol, DPPC/sclareol and DPPC/
cholesterol/sclareol are shown in Fig. 2. For comparative reasons, a preparation of DPPC was scanned alone and is depicted at the top of Fig. 2. Fully hydrated DPPC bilayers show a characteristic thermogram consisting of a broad low enthalpy transition at 35.3 ◦ C and a sharp enthalpy main transition at 41.2 ◦ C. The DPPC bilayer exists in the gel phase (Lβ ) for temperatures lower than 33 ◦ C, and in the liquid crystalline phase for temperatures higher than 42 ◦ C (Lα ). Between 33 and 42 ◦ C, the phospholipid bilayer exists in Pβ or ripple phase. The DSC scans of DPPC/cholesterol bilayers Xcholesterol = 4.8 mol% (phospholipid:cholesterol 100:5 molar ratio) show a decrease of H (Fig. 3) and abolition of the pre-transition, without significantly affecting the Tm (Fig. 4) of the main phase transition. The addition of cholesterol at Xcholesterol = 9.1 mol% results in broadening of the main lipid phase transition and abolition of the pretransitional peak, as well as H decrease (see Table 1). When a higher concentration of cholesterol was used Xcholesterol = 16.7 mol%, a pronounced peak at 42.4 ◦ C with a broad shoulder spanning from 30 to 39 ◦ C was observed. Similar thermal behavior, caused by bioactive molecules, was explained as the “inherent inhomogeneity” of the membrane bilayer. Thus, it contains domains consisting mainly of pure DPPC bilayers and rich in drug (Bruggemann and Melchior, 1983; Estep et al., 1978). At higher concentration, Xcholesterol = 28.6 mol% the fluidity of the bilayer has increased and the thermal effects are enhanced. A very broad phase transition is observed, spanning 35–50 ◦ C. H is significantly lowered. The presence of sclareol in the DPPC bilayers exerts similar thermal changes as those described by cholesterol up to Xsclareol = 9.1 mol%, summarized as a lowering of H (Fig. 3), a broadening of the width of phase transition and a not significant lowering of Tm . (Fig. 4). At Xsclareol = 16.7 mol%, the heterogeneity caused by sclareol or the reversible transition from a vesicular suspension to an extended peak bilayer network is different. No eminent peak at 42.4 ◦ C is observed, indicating that this domain may not be predominant in the case of DPPC/sclareol bilayers at this molar ratio. Instead, four peaks of similar intensity are observed, which may represent a complex pattern of domains in this bilayer. At Xsclareol = 28.6 mol%, the complex pattern is simplified and two peaks are eminent: a sharp peak centered
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Fig. 1. Molecular structures of DPPC, cholesterol and sclareol and their lipophilic profiles. Blue colour represents the polar moiety and browns the hydrophobic segments.
at 26.5 ◦ C and a small broad one centered at 34 ◦ C. H is further lowered with increasing concentration of sclareol in DPPC bilayers, as in the case of cholesterol. To judge the thermal effects of cholesterol and sclareol when co-dissolved in DPPC bilayers experiments were performed using various equimolar concentrations (the total molar ratio of the co-dissolved cholesterol and sclareol is expressed as Xdrugs ). At the low concentration of Xdrugs = 4.8 mol%, the ther-
mogram resembles the ones corresponding to the bilayers DPPC/cholesterol or DPPC/sclareol. When Xdrugs = 9.1 mol%, the sample appears more homogeneous and no shoulder preceeding the main phase transition is observed. The addition of Xdrugs = 16.7 mol% total concentration modifies the heterogeneity of the membrane bilayers or the reversible transition from a vesicular suspension to an extended peak bilayer network. A peak at 37 ◦ C was observed with a shoulder at higher temperature. The increase of the equimo-
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Fig. 2. Normalized DSC thermograms of fully hydrated bilayers of DPPC with varying amounts (in mol%) of cholesterol (panel A), sclareol (panel B) or equimolar amounts of cholesterol and sclareol (panel C): 0 (curve a), 4.8 (curve b), 9.1 (curve c), 16.7 (curve d) and 28.6 (curve e).
lar quantities Xdrugs = 28.6 mol% shows a different and less complicated thermogram. Two domains are dominant at this concentration, one around 23 ◦ C (sharp peak) and another one extended above 30 ◦ C up to 45 ◦ C (broad peak). This thermogram resembles that of DPPC/sclareol, but is markedly differs from that of DPPC/cholesterol.
3.2. Molecular modeling
Fig. 3. H values versus increasing concentration of Xsclareol or Xcholesterol or Xdrugs .
Fig. 4. Tm values versus increasing concentration of Xsclareol or Xcholesterol or Xdrugs .
Superimpositions of the molecules (Fig. 5), using the torsional flexible method, was achieved in order
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Table 1 Temperature of half integrated enthalpy and enthalpy of the studied multilamellar vesicles (MLVs) preparations (n = 3) Sample (mol%)
Tonset (◦ C)
S.D.
Tm (◦ C)
S.D.
Width (◦ C)
S.D.
H (J/g)
S.D.
DPPC DPPC/chol (x = 4.8) DPPC/chol (x = 9.1) DPPC/chol (x = 16.7) DPPC/chol (x = 28.6) DPPC/scla (x = 4.8) DPPC/scla (x = 9.1) DPPC/scla (x = 16.7) DPPC/scla (x = 28.6) DPPC/chol/scla (x = 2.4 + 2.4) DPPC/chol/scla (x = 4.55 + 4.55) DPPC/chol/scla (x = 8.35 + 8.35) DPPC/chol/scla (x = 14.35 + 14.35)
40.08 39.97 39.65 38.76 37.34 38.77 36.01 26.40 25.52 38.78 37.84 33.57 22.25
0.19 0.00 0.08 0.07 0.03 0.01 0.14 0.04 0.04 0.31 0.01 0.07 0.02
41.20 41.48 41.97 44.54 – 41.24 42.23 36.57 29.20 43.29 39.75 37.21 36.95
0.49 0.02 0.05 0.04 – 0.08 0.13 0.18 0.06 0.02 0.01 0.09 0.21
0.50 0.25 0.30 3.10 – 0.50 0.70 2.50 0.40 0.30 0.55 0.40 1.60
0.11 0.05 0.01 0.00 – 0.02 0.01 0.05 0.07 0.04 0.07 0.23 0.07
44.30 39.12 33.96 17.37 3.98 44.07 42.78 41.23 42.33 42.34 38.87 30.86 28.97
0.46 0.17 0.62 0.82 0.27 0.13 0.62 0.27 0.69 0.37 0.51 0.60 0.18
to reveal their stereoelectronic similarities and differences and provide a reasonable explanation for the DSC scans. In this method, the structures are fitted together by suitably modifying each structure’s rotatory torsional angles. The one structure is the working one which undergoes torsional angle modifications while the other is the target and remains fixed during the superimposition process. The equivalent atoms used for the match during the superimposition process were those of the hydroxyl group of cholesterol and the two hydroxyl groups of sclareol (hydrophilic moieties of the two molecules) and the tricyclic segment of cholesterol, with the corresponding bicyclic of sclareol. The resultant structures were energy minimized and then
superimposed, using a rigid body method in which only translations and rotations of the energy minimized molecules are considered. The calculated RMSD (root mean square distance) for the matched atoms was 0.15, the intersection volume was 198.75 and the union volume was 490.62 (sclareol volume 374.62; cholesterol 314.65). 3.3. Liposome preparation The results of the size distribution, ζ-potential and polydispersity index of MLVs and SUVs, respectively, incorporating cholesterol, sclareol and/or cholesterol, are presented in Table 2.
Fig. 5. Superimposition between cholesterol and sclareol using torsional flexible and rigid body methods.
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Table 2 z-Average mean diameter, polydispersity index, ζ-potential and DPPC recovery of MLV and SUV liposomes Sample composition (mol%)
z-Average mean diameter (nm) MLV
DPPC DPPC/chol (28.6) DPPC/scla (28.6) DPPC/chol/scla (14.3 + 14.3)
1323.0 1402.7 846.2 977.3
-Potential (mV) (SUV)
Polydispersity index (SUV)
−14.4 −21.4 −19.7 −17.4
0.24 0.18 0.35 0.19
SUV ± ± ± ±
531.8 454.0 238.4 134.9
90.7 94.8 83.4 85.8
± ± ± ±
18.4 1.6 5.1 25.5
± ± ± ±
1.7 2.5 1.8 3.7
± ± ± ±
0.02 0.02 0.17 0.06
Data are the mean of three independent experiments ± S.D.
z-Average mean diameter: This term is defined as the mean size using the cumulants analysis. The present of cholesterol in DPPC MLVs results in an increase of the z-average mean when compared to DPPC MLVs alone. In contrast, the presence of sclareol in DPPC MLVs results in a decrease of the z-average mean again when compared to DPPC MLVs alone. The presence of equimolar quantities of the two molecules gives a z-average only slightly higher than that observed in DPPC/sclareol, but lower than that of DPPC bilayers alone or DPPC/cholesterol ones. The same trend was also observed in SUV liposomes. ζ-Potential: This term is defined as the charge in colloidal systems and it is used for complete characterization of a liposomal suspension. It is an indicator of its stability. ζ-Potential remained negative and stable for SUVs liposomes, at between −14.4 (DPPC) and −21.4 (DPPC/cholesterol 28.6 mol%) (Table 2). Stability: The physical stability (t = 4 ◦ C, HPLC grade water pH = 5.6) of the SUV liposomes (Table 2) was measured over a period of 1 month. The results showed that the liposomal formulations of DPPC/sclareol 28.6 mol% and DPPC/cholestero/ sclareol 14.3 + 14.3 mol% were unstable and, after a week, sedimentation was observed. Polydispersity index: This term indicates the width of the size distribution of liposomes. The polydispersity index in all preparations indicated a narrow size distribution of the dispersed vesicles (Table 2). 3.4. Discussion Sclareol or cholesterol, when incorporated into DPPC bilayers with a molar ratio less than X = 9.1 mol%, show similar thermal profiles. At a higher concentration of X = 16.7 mol%, both molecules show a thermal scan consisting of more than one peak,
indicating that the heterogeneity caused by sclareol or cholesterol in lipid bilayers, or the reversible transition from a vesicular suspension to an extended peak bilayer network, is different. At X = 28.6 mol%, the thermogram of DPPC/cholesterol shows a broad peak extending from 35 to 50 ◦ C. The thermogram of DPPC/sclareol is totally different. Two peaks are observed, one sharp at about 26.6 ◦ C and one broad at about 35 ◦ C, indicating still an inhomogeneous membrane with distinct domains. The addition of equimolar quantities shows that, at lower concentrations (total Xdrug = 9.1 mol%), the two molecules have the same thermal properties and act almost synergistically (sharp peaks). Co-dissolving at equimolar quantities (total Xdrugs = 28.6 mol%) interestingly shows a thermal scan approaching the one of DPPC/sclareol. This indicates that sclareol governs the thermal effects when is with cholesterol at high concentration and at 1:1 molar ratio. Tm values versus increasing concentration of Xsclareol or Xcholesterol or Xdrugs are shown in Fig. 4. The results show that all preparations which contain X = ≤16.7 mol% have similar Tm . H values versus increasing concentration of Xsclareol , Xcholesterol or Xdrugs (Fig. 3) show that cholesterol exerts the maximum effect in lowering H. This was expected, because cholesterol is the bulkiest molecule with its lipophilic part extended deeper inside the membrane bilayers. The presence of equimolar quantities of cholesterol and sclareol in membrane bilayers has an intermediate effect, as was expected. The lipophilic profiles of the two molecules were calculated and showed that both molecules consist of three areas, corresponding to high (brown); intermediate (green) and low lipophilicity (blue) (Fig. 1). Such similarity prompted us to superimpose the molecules based on their lipophilic features. The superimposition
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(shown in Fig. 5) reveals that the two molecules have an RMSD of 0.15 if their bicyclic moieties are matched. Interestingly, not only the bicyclic part is matched in the superimposition, but also their hydrophilic hydroxyl groups. More specifically, the -OH group of cholesterol lies between the two hydroxyl groups of sclareol. Such superimposition can explain their similar thermal effects at low concentrations. Cholesterol is well known to anchor to membrane bilayers at the interface and its molecular amphipathic features are responsible for its thermal and dynamic effects. Such effects can be mimicked by sclareol, despite its different structure. Liposomes are carrier systems for drug delivery and targeting, and their physicochemical properties are the main determinants of their efficiency. Bioactive compounds, like the promising anticancer agent sclareol, with low water solubility are candidates for incorporation into liposomes in order to improve their pharmacokinetic properties, which influence their pharmacological efficiency. Therefore, the stability of the membrane bilayers containing high lipophilic drugs and their physicochemical properties are important when designing liposomes as drug carriers. From all the preparations under study only the ones containing cholesterol proved stable. This may be related both to the results of the z-mean diameter and DSC scans. DPPC MLVs or SUVs containing 28.6 mol%, have z-mean diameter by 65 and 14%, correspondingly higher than those of DPPC MLVs or SUVs, containing 28.6 mol% sclareol. DPPC MLVs or SUVs, containing 28.6 mol%, cholesterol, have z-mean diameter by 44%, and 10% correspondingly higher than those of DPPC MLVs or SUVs containing equimolar ratio of cholesterol and sclareol (14.3 + 14.3 mol%). DSC scans show that only DPPC/chol. (28.6 mol%) preparation is homogeneous. The other preparations, containing sclareol or an equimolar ratio of cholesterol and sclareol, were characterized by domains which increase the inhomogeneity and deteriorate the stability of the lipid bilayers. SUV liposomes ζ-potential values were measured in all cases (Table 2) and were negative and in a small range (−14.4 to 21.4). The explanation could be that there are no changes in the orientation of the phosphatidylcholine polar head groups, and we may conclude, at this point, that the incorporation of sclareol does not cause essential changes on the surface of liposomes.
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Sclareol is the first representative of labdanes that has been introduced into liposomal technology and the results of this work may be useful in future liposomal studies on structurally similar bioactive labdanes. Summarizing our results we can conclude that biological active lipophilic molecules should be studied in terms of their interaction with model lipid membranes, in order to gain information about the membrane integrity, to predict the best lipid composition, and to achieve the desired physicochemical characteristics for the preparation of effective types of liposomes.
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Singh, M., Mahesh, P., Sharma, R.P., 1999. Biological activity of the ladbane diterpenes. Planta Med. 65, 2–8. Urbina, A.J., Peketar, S., Hong-biao, L., Patterson, J., Montez, B., Oldfield, E., 1995. Molecular order and dynamics of phosphatidylcholine bilayer membranes in the presence of cholesterol, ergosterol and lanosterol: a comparative study using 2 H-, 13 C- and 31 P-NMR spectroscopy. Biochim. Biophys. Acta 1238, 163–176. Woodle, M.C., 1995. Sterically stabilized liposome therapeutics. Adv. Drug. Deliv. Rev. 16, 249–265. Yang, D.P., Mavromoustakos, T., Beshah, K., Makriyannis, A., 1992. Amphipathic interactions of cannabinoids with membranes. A comparison of cannabinoids with membranes. A comparison between 8-THC and its O-methyl analog using differential scanning calorimetry, X-ray diffraction and solid state 2 H-NMR. Biochim. Biophys. Acta 1103, 25–36.
A comparative study of the effects of cholesterol and sclareol, a bioactive labdane type diterpene, on phospholipid bilayers I. Kyrikoua , A. Georgopoulosb , S. Hatziantonioub , T. Mavromoustakosa , C. Demetzosb,∗ a b
Institute of Organic and Pharmaceutical Chemistry, National Hellenic Research Foundation, Vasileos Constantinou 48, Athens 11635, Greece Department of Pharmaceutical Technology, School of Pharmacy, Panepistimioupolis, Zografou 15571, University of Athens, Athens, Greece Received 22 April 2004; received in revised form 17 September 2004; accepted 21 September 2004 Available online 18 November 2004
Abstract Sclareol (labd-14-ene-8,13-diol) is a highly water-insoluble molecule that belongs to the labdane type diterpenes and is characterized as a biologically active molecule, due to its cytotoxic and cytostatic effects against human leukemic cell lines. A superimposition study between sclareol and cholesterol, based on their corresponding hydrophobic and polar molecular segments calculated from their lipophilic profiles, revealed their spatial similarities. This structural similarity between the two molecules prompted us to compare their effects on the structure and stability of phospholipid dipalmitoylphosphatidylcholine (DPPC) membranes. Differential scanning calorimetry (DSC) was applied to compare the thermal changes caused by either cholesterol or sclareol when are incorporated in DPPC bilayers. The results showed that sclareol is incorporated into phospholipid model membranes and mimics the thermal effects of cholesterol especially at concentrations up to Xsclareol = 9.1 mol%. These effects can be summarized as the abolition of pre-transition, lowering of the main phase transition and reduction of the enthalpy change (H) of the gel to liquid-crystalline phase transition of DPPC bilayers. At concentrations X ≥ 16.7 mol%, sclareol and cholesterol caused different heterogeneity in lipid bilayers or a reversible transition from a vesicular suspension to an extended peak bilayer network. This different fluidization, exerted by the two molecules at high concentration, may be related to their different stability and the z-average mean diameter of the liposomes they form. Small unilamellar vesicles, prepared by the thin film hydration method showed that DPPC bilayers containing a high concentration of sclareol in equimolar ratio sclareol:cholesterol were unstable, in contrast to the ones containing only cholesterol. © 2004 Elsevier Ireland Ltd. All rights reserved. Keywords: Sclareol; Cholesterol; Differential scanning calorimetry; DPPC bilayers; Liposomes
∗
Corresponding author. Tel.: +30 210 7274596; fax: +30 210 7274027. E-mail address: [email protected] (C. Demetzos).
0009-3084/$ – see front matter © 2004 Elsevier Ireland Ltd. All rights reserved. doi:10.1016/j.chemphyslip.2004.09.021
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1. Introduction Sclareol (labd-14-ene-8,13-diol) is a ditertiary alcohol, widely distributed in nature, belonging to the labdane type diterpenes. The interest in studying labdanes is based on their wide range of biological activities (Singh et al., 1999). Sclareol was first isolated from clary sage oil (Salvia sclarea, Labiatae) (Notin, 1972) and it also occurs in many conifers, as well as being isolated from the genus Cistus. It is used as a fragrance in cosmetics and as a synthon for preparation of Ambra odorants in perfumery (Decorzant and Vial, 1987). Sclareol was recently investigated by our group for its cytotoxic and cytostatic effects against leukemic human cell lines and effects on the morphology and the kind of death in cell lines, as well as on the cell cycle progression using flow cytometry (Dimas et al., 2001). It was found that sclareol significantly interferes with the cell cycle and induces apoptosis in leukemic cell lines, while it does not affect normal and PHAstimulated peripheral blood lymphocytes. The expression of the ongogenes c-myc and bcl-2 was studied using a Western blot analysis. The results showed that the c-myc levels were reduced, while bcl-2 expression remained unaffected. Cholesterol is a common constituent of biological membranes. Its incorporation into phospholipid bilayers is well-known to affect the gel to liquid-crystalline state of model membranes (Ladbrooke et al., 1968). The effect of cholesterol on lipid bilayers is attributed to its molecular features. The 3-hydroxyl group is oriented towards the polar headgroup region of the phospholipid bilayer, while the hydrophobic part of the molecule is oriented towards its nonpolar part. The aim of our study was to investigate whether sclareol interacts with lipid bilayers and influences the gel to liquid-crystalline state of dipalmitoylphosphatidylcholine (DPPC) membranes in a similar way to cholesterol. The rationale behind such a comparison is that molecular superimposition studies showed that these molecules, in spite of their apparent structural dissimilarity, have common stereoelectronic features when appropriate equivalent atoms are properly superimposed. These two amphipathic molecules have their lipophilic and hydrophilic segments spatially arranged in a way that can afford excellent superimposition.
In our extensive research experience, molecules that are very similar in chemical structure do not necessarily have correspondingly similar thermal profiles. Geometrical, stereochemical or amphipathic differences between the molecules result in completely different thermal profiles. This has been proved using a combination of biophysical methods to study the effects, in membrane, of a pair of drugs, (−)-8 tetrahydrocannabinol and its ether analog, that differ in their amphipathic properties. The bioactive psychomimetic drug, (−)-8 -tetrahydrocannabinol, anchors itself at the membrane interface, presumambly through hydrogen bonding with the phospholipids ester groups or with water molecules present at the interface. In this location, the drug molecule can induce bilayer perturbations most effectively. Conversely, the almost inactive analog, (−)-O-methyl-8 tetrahydrocannabinol, locates itself deeper in the bilayer away from the interface, a property which accounts for its decreased ability to perturb the membrane (Yang et al., 1992). In addition we have applied different biophysical methods to study the effects of the anaesthetic steroid alphaxalone (5␣-pregnane3␣-ol-11,20-dione) and its structurally similar inactive congener 16 -alphaxalone (5␣-pregn-16-ene-3␣ol-11,20-dione). These steroids showed different dynamic and thermal effects in membrane bilayers, which has been attributed to the different molecular geometry of the D ring between the two molecules (Mavromoustakos et al., 1994) On the other hand, molecules that are dissimilar in chemical structure, like taxol and vinblastine have been shown to have similar thermal profiles and induce inderdigitation, because they are bulkly and amphipathic and, thus, have stereoelectronic similarities (Kyrikou et al., 2004). Similar observations were made in membrane bilayers containing cholesterol or its precursor lanosterol or its derivatives. Miao et al (Miao et al., 2002, see also references therein) showed that the evolution in the molecular chemistry from lanosterol to cholesterol is manifested in the model lipid-sterol membranes by an increase in the ability of the sterols to promote and stabilize a particular membrane phase, the liquidordered phase, and to induce collective order in the acyl-chain conformations of lipid molecules. Thus, cholesterol, with its streamlined molecular structure, interacts more effectively with lipid chains with conformational order and stabilizes the liquid-ordered state
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of the lipid bilayers more effectively than lanosterol. Urbina et al. (1995) (see also references therein) found that ergosterol was more effective at increasing order and restricting the mobility of DMPC than cholesterol, while the opposite was true for POPC bilayers. The trimethylated precursor, lanosterol, was less effective than the other two sterols, with both saturated and unsaturated lipids. These results showed that the interactions between phosphatidylcholines and sterols were complex and depended on the details of sterol structure and the types of acyl chains present in the phospholipids molecules. Ghosh et al. (2001) applied differential scanning calorimetry (DSC) and fluorescence analysis to study the nature of perturbation induced by cationic cholesterol derivatives. The authors found that the nature of perturbation induced by each of these cationic cholesterol derivatives was dependent on the details of their molecular structure and provided significant information on the nature the interaction between these derivatives and phospholipid bilayers. The modifications of the enthalpy as a function of phase transitions, when sclareol is incorporated into lipid bilayers, have never been studied. In order to compare the effects of sclareol and cholesterol in membrane bilayers, we applied DSC, which is a fast and relatively inexpensive technique that allows the study of the thermotropic properties of membranes in the presence of bioactive molecules. Therefore, it is used as a diagnostic technique to investigate the differential effects that may be caused by the incorporation of additives under study (Koynova and Caffrey, 1998). The thermal changes, induced by the presence of different molar concentrations of sclareol and cholesterol on DPPC lipid bilayers, were studied in order to characterize the types and strength of their interactions. In order to investigate the effect of sclareol and cholesterol on the stability properties of liposomes, multilamellar vesicles (MLVs) and small unilamellar vesicles (SUVs) composed of DPPC, were prepared incorporating cholesterol, sclareol and sclareol/cholesterol. Liposomes are considered to be effective and non-toxic carriers for incorporating lipophilic and hydrophilic compounds and are currently in clinical use (Woodle, 1995) Their sizedistribution (z-average mean diameter) and ζ-potential were measured. The obtained results were correlated with those obtained by DSC.
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Our future goal is to design liposomes as carrier systems, used to deliver poorly water-soluble compounds to the target tissue, with appropriate physicochemical properties to improve their in vivo kinetic parameters and their stability. Such liposomal formulations could be appropriate for lipophilic biologically active compounds such as sclareol.
2. Experimental procedures 2.1. Materials Sclareol has been isolated from Cistus incanus subsp. creticus and from its resin Ladano (Demetzos et al., 1999). C. incanus subsp. creticus was collected in the island of Crete, Greece (collector Demetzos, 1990). The isolation procedures, as well as the identification methodology have been described in recently published papers (Demetzos et al., 1990, 1999). DPPC and cholesterol were obtained from Avanti Polar Lipids Inc., USA; the organic solvent used, chloroform (CHCl3 ), was of spectroscopic grade, Merck, USA. 2.2. Methods 2.2.1. Liposome preparation Liposomes were prepared by the thin-film hydration method. The lipid film was prepared by dissolving the appropriate amount of DPPC (cholesterol and/or sclareol) in chloroform. The solvent was evaporated in a flash evaporator and the film was dried under vacuum for 24 h. Subsequently, the film was hydrated with HPLC grade water and heated for 1 h at 50 ◦ C with stirring. The resultant liposomal suspension, composed of MLVs, was freezed and lyophilized for 24 h and then used for DSC experiments. For the preparation of SUVs, the resultant liposomal suspension was subjected to two sonication periods (of 20 min each interrupted by a 5 min resting period) in a water bath at 25 ◦ C, using a probe sonicator (amplitude 100, cycle 0.7 – UP 200S, dr. hielsher GmbH, Berlin, Germany). The resultant vesicles were allowed to anneal for 30 min. Then the liposomal suspension was centrifuged (20.000 rpm, 20 min, 4 ◦ C, Sorval Ultra Pro 80, fixed angle rotor) in order to separate the SUVs from Titanium and the MLVs liposomes.
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The liposome dispersion was diluted 10 times with HPLC grade water (pH 5.6–5.7) and liposome size distribution and ζ-potential were determined using a photon correlation spectrophotometer (Zetasizer 3000, Malvern, UK). Samples were scattered (633 nm) at an angle of 90 ◦ . The data were analysed by the CONTIN method (MALVERN software). 2.2.2. Differential scanning calorimetry (DSC) The DSC technique was applied to the study of MLV samples using a Perkin-Elmer DSC-7 calorimeter. MLVs were obtained by dispersing the dry residues in appropriate amounts of bi-distilled water by vortexing. After dispersion in water (50%, w/w), an aliquot (ca. 5 mg) was sealed into stainless steel capsules obtained from Perkin-Elmer. Prior to scanning, the samples were held above their phase transition temperature at 50 ◦ C to ensure equilibration. All samples were scanned at least twice until identical thermograms were obtained, using a scanning rate of 2.5 ◦ C/min. The temperature scale of the calorimeter was calibrated using indium (Tm = 156.6 ◦ C) as standard sample. 2.2.3. Molecular modeling Computer calculations were performed on a Silicon Graphics O2 workstation using QUANTA 97 of Molecular Simulation Incorporated. Sclareol and cholesterol were first energy minimized using, as starting structures, their X-ray crystallographic ones (Bernardinelli et al., 1988; Shieh et al., 1981). The lipophilic profiles of the molecules were also calculated. By default, the colour mapping of the lipophilic surfaces is relative, not based on absolute values. The colour scale runs from the most lipophilic (brown) to the most hydrophilic (blue) parts of the molecule (Fig. 1). The lipophilicity force field profile is a powerful tool when one seeks to compare the lipophilic molecular potential of different molecules (Croizet et al., 1990). Superimposition between the minimized structures of sclareol and cholesterol was achieved using torsional flexible and rigid body methods. 3. Results and discussion 3.1. Differential scanning calorimetry The DSC scans for the three preparations of DPPC/cholesterol, DPPC/sclareol and DPPC/
cholesterol/sclareol are shown in Fig. 2. For comparative reasons, a preparation of DPPC was scanned alone and is depicted at the top of Fig. 2. Fully hydrated DPPC bilayers show a characteristic thermogram consisting of a broad low enthalpy transition at 35.3 ◦ C and a sharp enthalpy main transition at 41.2 ◦ C. The DPPC bilayer exists in the gel phase (Lβ ) for temperatures lower than 33 ◦ C, and in the liquid crystalline phase for temperatures higher than 42 ◦ C (Lα ). Between 33 and 42 ◦ C, the phospholipid bilayer exists in Pβ or ripple phase. The DSC scans of DPPC/cholesterol bilayers Xcholesterol = 4.8 mol% (phospholipid:cholesterol 100:5 molar ratio) show a decrease of H (Fig. 3) and abolition of the pre-transition, without significantly affecting the Tm (Fig. 4) of the main phase transition. The addition of cholesterol at Xcholesterol = 9.1 mol% results in broadening of the main lipid phase transition and abolition of the pretransitional peak, as well as H decrease (see Table 1). When a higher concentration of cholesterol was used Xcholesterol = 16.7 mol%, a pronounced peak at 42.4 ◦ C with a broad shoulder spanning from 30 to 39 ◦ C was observed. Similar thermal behavior, caused by bioactive molecules, was explained as the “inherent inhomogeneity” of the membrane bilayer. Thus, it contains domains consisting mainly of pure DPPC bilayers and rich in drug (Bruggemann and Melchior, 1983; Estep et al., 1978). At higher concentration, Xcholesterol = 28.6 mol% the fluidity of the bilayer has increased and the thermal effects are enhanced. A very broad phase transition is observed, spanning 35–50 ◦ C. H is significantly lowered. The presence of sclareol in the DPPC bilayers exerts similar thermal changes as those described by cholesterol up to Xsclareol = 9.1 mol%, summarized as a lowering of H (Fig. 3), a broadening of the width of phase transition and a not significant lowering of Tm . (Fig. 4). At Xsclareol = 16.7 mol%, the heterogeneity caused by sclareol or the reversible transition from a vesicular suspension to an extended peak bilayer network is different. No eminent peak at 42.4 ◦ C is observed, indicating that this domain may not be predominant in the case of DPPC/sclareol bilayers at this molar ratio. Instead, four peaks of similar intensity are observed, which may represent a complex pattern of domains in this bilayer. At Xsclareol = 28.6 mol%, the complex pattern is simplified and two peaks are eminent: a sharp peak centered
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Fig. 1. Molecular structures of DPPC, cholesterol and sclareol and their lipophilic profiles. Blue colour represents the polar moiety and browns the hydrophobic segments.
at 26.5 ◦ C and a small broad one centered at 34 ◦ C. H is further lowered with increasing concentration of sclareol in DPPC bilayers, as in the case of cholesterol. To judge the thermal effects of cholesterol and sclareol when co-dissolved in DPPC bilayers experiments were performed using various equimolar concentrations (the total molar ratio of the co-dissolved cholesterol and sclareol is expressed as Xdrugs ). At the low concentration of Xdrugs = 4.8 mol%, the ther-
mogram resembles the ones corresponding to the bilayers DPPC/cholesterol or DPPC/sclareol. When Xdrugs = 9.1 mol%, the sample appears more homogeneous and no shoulder preceeding the main phase transition is observed. The addition of Xdrugs = 16.7 mol% total concentration modifies the heterogeneity of the membrane bilayers or the reversible transition from a vesicular suspension to an extended peak bilayer network. A peak at 37 ◦ C was observed with a shoulder at higher temperature. The increase of the equimo-
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Fig. 2. Normalized DSC thermograms of fully hydrated bilayers of DPPC with varying amounts (in mol%) of cholesterol (panel A), sclareol (panel B) or equimolar amounts of cholesterol and sclareol (panel C): 0 (curve a), 4.8 (curve b), 9.1 (curve c), 16.7 (curve d) and 28.6 (curve e).
lar quantities Xdrugs = 28.6 mol% shows a different and less complicated thermogram. Two domains are dominant at this concentration, one around 23 ◦ C (sharp peak) and another one extended above 30 ◦ C up to 45 ◦ C (broad peak). This thermogram resembles that of DPPC/sclareol, but is markedly differs from that of DPPC/cholesterol.
3.2. Molecular modeling
Fig. 3. H values versus increasing concentration of Xsclareol or Xcholesterol or Xdrugs .
Fig. 4. Tm values versus increasing concentration of Xsclareol or Xcholesterol or Xdrugs .
Superimpositions of the molecules (Fig. 5), using the torsional flexible method, was achieved in order
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Table 1 Temperature of half integrated enthalpy and enthalpy of the studied multilamellar vesicles (MLVs) preparations (n = 3) Sample (mol%)
Tonset (◦ C)
S.D.
Tm (◦ C)
S.D.
Width (◦ C)
S.D.
H (J/g)
S.D.
DPPC DPPC/chol (x = 4.8) DPPC/chol (x = 9.1) DPPC/chol (x = 16.7) DPPC/chol (x = 28.6) DPPC/scla (x = 4.8) DPPC/scla (x = 9.1) DPPC/scla (x = 16.7) DPPC/scla (x = 28.6) DPPC/chol/scla (x = 2.4 + 2.4) DPPC/chol/scla (x = 4.55 + 4.55) DPPC/chol/scla (x = 8.35 + 8.35) DPPC/chol/scla (x = 14.35 + 14.35)
40.08 39.97 39.65 38.76 37.34 38.77 36.01 26.40 25.52 38.78 37.84 33.57 22.25
0.19 0.00 0.08 0.07 0.03 0.01 0.14 0.04 0.04 0.31 0.01 0.07 0.02
41.20 41.48 41.97 44.54 – 41.24 42.23 36.57 29.20 43.29 39.75 37.21 36.95
0.49 0.02 0.05 0.04 – 0.08 0.13 0.18 0.06 0.02 0.01 0.09 0.21
0.50 0.25 0.30 3.10 – 0.50 0.70 2.50 0.40 0.30 0.55 0.40 1.60
0.11 0.05 0.01 0.00 – 0.02 0.01 0.05 0.07 0.04 0.07 0.23 0.07
44.30 39.12 33.96 17.37 3.98 44.07 42.78 41.23 42.33 42.34 38.87 30.86 28.97
0.46 0.17 0.62 0.82 0.27 0.13 0.62 0.27 0.69 0.37 0.51 0.60 0.18
to reveal their stereoelectronic similarities and differences and provide a reasonable explanation for the DSC scans. In this method, the structures are fitted together by suitably modifying each structure’s rotatory torsional angles. The one structure is the working one which undergoes torsional angle modifications while the other is the target and remains fixed during the superimposition process. The equivalent atoms used for the match during the superimposition process were those of the hydroxyl group of cholesterol and the two hydroxyl groups of sclareol (hydrophilic moieties of the two molecules) and the tricyclic segment of cholesterol, with the corresponding bicyclic of sclareol. The resultant structures were energy minimized and then
superimposed, using a rigid body method in which only translations and rotations of the energy minimized molecules are considered. The calculated RMSD (root mean square distance) for the matched atoms was 0.15, the intersection volume was 198.75 and the union volume was 490.62 (sclareol volume 374.62; cholesterol 314.65). 3.3. Liposome preparation The results of the size distribution, ζ-potential and polydispersity index of MLVs and SUVs, respectively, incorporating cholesterol, sclareol and/or cholesterol, are presented in Table 2.
Fig. 5. Superimposition between cholesterol and sclareol using torsional flexible and rigid body methods.
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Table 2 z-Average mean diameter, polydispersity index, ζ-potential and DPPC recovery of MLV and SUV liposomes Sample composition (mol%)
z-Average mean diameter (nm) MLV
DPPC DPPC/chol (28.6) DPPC/scla (28.6) DPPC/chol/scla (14.3 + 14.3)
1323.0 1402.7 846.2 977.3
-Potential (mV) (SUV)
Polydispersity index (SUV)
−14.4 −21.4 −19.7 −17.4
0.24 0.18 0.35 0.19
SUV ± ± ± ±
531.8 454.0 238.4 134.9
90.7 94.8 83.4 85.8
± ± ± ±
18.4 1.6 5.1 25.5
± ± ± ±
1.7 2.5 1.8 3.7
± ± ± ±
0.02 0.02 0.17 0.06
Data are the mean of three independent experiments ± S.D.
z-Average mean diameter: This term is defined as the mean size using the cumulants analysis. The present of cholesterol in DPPC MLVs results in an increase of the z-average mean when compared to DPPC MLVs alone. In contrast, the presence of sclareol in DPPC MLVs results in a decrease of the z-average mean again when compared to DPPC MLVs alone. The presence of equimolar quantities of the two molecules gives a z-average only slightly higher than that observed in DPPC/sclareol, but lower than that of DPPC bilayers alone or DPPC/cholesterol ones. The same trend was also observed in SUV liposomes. ζ-Potential: This term is defined as the charge in colloidal systems and it is used for complete characterization of a liposomal suspension. It is an indicator of its stability. ζ-Potential remained negative and stable for SUVs liposomes, at between −14.4 (DPPC) and −21.4 (DPPC/cholesterol 28.6 mol%) (Table 2). Stability: The physical stability (t = 4 ◦ C, HPLC grade water pH = 5.6) of the SUV liposomes (Table 2) was measured over a period of 1 month. The results showed that the liposomal formulations of DPPC/sclareol 28.6 mol% and DPPC/cholestero/ sclareol 14.3 + 14.3 mol% were unstable and, after a week, sedimentation was observed. Polydispersity index: This term indicates the width of the size distribution of liposomes. The polydispersity index in all preparations indicated a narrow size distribution of the dispersed vesicles (Table 2). 3.4. Discussion Sclareol or cholesterol, when incorporated into DPPC bilayers with a molar ratio less than X = 9.1 mol%, show similar thermal profiles. At a higher concentration of X = 16.7 mol%, both molecules show a thermal scan consisting of more than one peak,
indicating that the heterogeneity caused by sclareol or cholesterol in lipid bilayers, or the reversible transition from a vesicular suspension to an extended peak bilayer network, is different. At X = 28.6 mol%, the thermogram of DPPC/cholesterol shows a broad peak extending from 35 to 50 ◦ C. The thermogram of DPPC/sclareol is totally different. Two peaks are observed, one sharp at about 26.6 ◦ C and one broad at about 35 ◦ C, indicating still an inhomogeneous membrane with distinct domains. The addition of equimolar quantities shows that, at lower concentrations (total Xdrug = 9.1 mol%), the two molecules have the same thermal properties and act almost synergistically (sharp peaks). Co-dissolving at equimolar quantities (total Xdrugs = 28.6 mol%) interestingly shows a thermal scan approaching the one of DPPC/sclareol. This indicates that sclareol governs the thermal effects when is with cholesterol at high concentration and at 1:1 molar ratio. Tm values versus increasing concentration of Xsclareol or Xcholesterol or Xdrugs are shown in Fig. 4. The results show that all preparations which contain X = ≤16.7 mol% have similar Tm . H values versus increasing concentration of Xsclareol , Xcholesterol or Xdrugs (Fig. 3) show that cholesterol exerts the maximum effect in lowering H. This was expected, because cholesterol is the bulkiest molecule with its lipophilic part extended deeper inside the membrane bilayers. The presence of equimolar quantities of cholesterol and sclareol in membrane bilayers has an intermediate effect, as was expected. The lipophilic profiles of the two molecules were calculated and showed that both molecules consist of three areas, corresponding to high (brown); intermediate (green) and low lipophilicity (blue) (Fig. 1). Such similarity prompted us to superimpose the molecules based on their lipophilic features. The superimposition
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(shown in Fig. 5) reveals that the two molecules have an RMSD of 0.15 if their bicyclic moieties are matched. Interestingly, not only the bicyclic part is matched in the superimposition, but also their hydrophilic hydroxyl groups. More specifically, the -OH group of cholesterol lies between the two hydroxyl groups of sclareol. Such superimposition can explain their similar thermal effects at low concentrations. Cholesterol is well known to anchor to membrane bilayers at the interface and its molecular amphipathic features are responsible for its thermal and dynamic effects. Such effects can be mimicked by sclareol, despite its different structure. Liposomes are carrier systems for drug delivery and targeting, and their physicochemical properties are the main determinants of their efficiency. Bioactive compounds, like the promising anticancer agent sclareol, with low water solubility are candidates for incorporation into liposomes in order to improve their pharmacokinetic properties, which influence their pharmacological efficiency. Therefore, the stability of the membrane bilayers containing high lipophilic drugs and their physicochemical properties are important when designing liposomes as drug carriers. From all the preparations under study only the ones containing cholesterol proved stable. This may be related both to the results of the z-mean diameter and DSC scans. DPPC MLVs or SUVs containing 28.6 mol%, have z-mean diameter by 65 and 14%, correspondingly higher than those of DPPC MLVs or SUVs, containing 28.6 mol% sclareol. DPPC MLVs or SUVs, containing 28.6 mol%, cholesterol, have z-mean diameter by 44%, and 10% correspondingly higher than those of DPPC MLVs or SUVs containing equimolar ratio of cholesterol and sclareol (14.3 + 14.3 mol%). DSC scans show that only DPPC/chol. (28.6 mol%) preparation is homogeneous. The other preparations, containing sclareol or an equimolar ratio of cholesterol and sclareol, were characterized by domains which increase the inhomogeneity and deteriorate the stability of the lipid bilayers. SUV liposomes ζ-potential values were measured in all cases (Table 2) and were negative and in a small range (−14.4 to 21.4). The explanation could be that there are no changes in the orientation of the phosphatidylcholine polar head groups, and we may conclude, at this point, that the incorporation of sclareol does not cause essential changes on the surface of liposomes.
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Sclareol is the first representative of labdanes that has been introduced into liposomal technology and the results of this work may be useful in future liposomal studies on structurally similar bioactive labdanes. Summarizing our results we can conclude that biological active lipophilic molecules should be studied in terms of their interaction with model lipid membranes, in order to gain information about the membrane integrity, to predict the best lipid composition, and to achieve the desired physicochemical characteristics for the preparation of effective types of liposomes.
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