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Searching for the role of membrane sphingolipids in selectivity of antitumor ether lipid–edelfosine
Colloids and Surfaces B: Biointerfaces 81 (2010) 492–497
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Colloids and Surfaces B: Biointerfaces journal homepage: www.elsevier.com/locate/colsurfb
Searching for the role of membrane sphingolipids in selectivity of antitumor ether lipid–edelfosine ∗ ˛ ˛ Katarzyna Hac-Wydro , Patrycja Dynarowicz-Łatka Faculty of Chemistry, Jagiellonian University, Ingardena 3, 30-060 Kraków, Poland
a r t i c l e
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Article history: Received 7 April 2010 Received in revised form 16 July 2010 Accepted 21 July 2010 Available online 30 July 2010 Keywords: Edelfosine Ganglioside GM1 Sphingomyelin Monolayers Selectivity
a b s t r a c t Edelfosine is a synthetic antitumor lipid of high selectivity. Its activity on membrane level inspired the investigations on edelfosine–lipid interactions to verify, which of the membrane components may be responsible for the selectivity of this drug. Because of overexpression of gangliosides in tumor progression and the ability of edelfosine to insert into membrane rafts, we have chosen two sphingolipids, i.e. sphingomyelin and ganglioside to investigate in mixtures with edelfosine. It was found that edelfosine shows strong affinity to ganglioside in contrast to sphingomyelin. Differences in the interactions of edelfosine with sphingolipids were analyzed from the point of view of the structure and shape of the interacting molecules. The comparison of the results with those previously reported for edelfosine mixed with other membrane components, allowed us to suggest that gangliosides may be considered as target molecules attracting edelfosine to tumor cells. © 2010 Elsevier B.V. All rights reserved.
1. Introduction Edelfosine (ED) belongs to the family of synthetic single-chain ether lipids of antitumor properties [1,2]. Anticancer action of this drug is closely related to its insertion into tumor cell membranes. This is in contrast to conventional chemotherapeutic agents, which bind directly to DNA [1,3]. The action of edelfosine and other synthetic antitumor lipids (miltefosine and perifosine) at the membrane level stimulates the investigations on the effect of these compounds on model membrane systems. It was postulated that the affinity of these drugs to biomembrane is due to their amphipathic character and phospholipids-like molecular structure [4,5]. It was found that edelfosine interacts strongly with cholesterol [6–8] and in combination with this lipid is able to form liposomes [9]. Moreover, edelfosine in liposomal form does not loose its apoptotic properties while its hemolytic activity is abolished [9]. The interactions of ED with membrane phospholipids were found to be much weaker than the affinity of this drug to cholesterol [7,10]. It was also evidenced that stronger interactions occur when edelfosine is mixed with unsaturated phosphatidylcholines (PCs), namely 1,2-dioleoyl-sn-glycero-3-phosphocholine – DOPC, 1-palmitoyl-2oleoyl-sn-glycero-3-phosphocholine – POPC, as compared to those containing fully saturated chains [7,9,10]. Similar tendency as regards the affinity to membrane lipids was also proved for mil-
∗ Corresponding author. Tel.: +48 012 633 20 82; fax: +48 012 634 05 15. ˛ E-mail address: [email protected] (K. Hac-Wydro). 0927-7765/$ – see front matter © 2010 Elsevier B.V. All rights reserved. doi:10.1016/j.colsurfb.2010.07.045
tefosine [11–13], which was found to incorporate easier to model membranes of lower condensation [12,13]. It was suggested that antitumor lipids are able to incorporate into membrane microdomains [8,14,15]. In comprehensive studies performed on POPC/sphingomyelin (Sph)/cholesterol mixtures, mimicking membrane rafts, it was found that edelfosine can accumulate in microdomains at concentration as high as 21.7% of total lipids content in rafts. The insertion of drug molecules into rafts modifies their organization and biophysical properties [8]. The key role of membrane microdomains in edelfosine activity was also suggested in experiments on mouse lymphoma cells [16]. It was found that edelfosine induces apoptosis in S49 mouse lymphoma cells by raft-dependent internalization. On the other hand, a cell line variant (S49AR ), which is unable to synthesize sphingomyelin, is resistant to this drug. It was explained that the lack of sphingomyelin inhibits the formation of Sph/cholesterol domains, which may prevent edelfosine from incorporation into the cell [16]. Edelfosine is characterized by high selectivity that results from inability of normal cells to incorporate enough amounts of ether lipids [1]. Therefore, the membrane of normal cell is considered as a barrier for ED [1]. It is known that the composition of tumor membranes differs from those of normal cells. These differences involve changes in the concentration of major lipids (cholesterol, Sph and PCs) and proportion of saturated to unsaturated acyl chains [17–20]. All these modifications change the properties of tumor membranes, which are more fluid as compared to normal cells. The fact that edelfosine and miltefosine are of higher affinity to unsaturated phospholipids [12,13] and affect more strongly model
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membranes of higher fluidity [21,22] explains an easier insertion of drug molecules into tumor membrane. However, other factors should also be taken into consideration, like the presence of specific molecules in tumor membranes, which are either absent or in trace amounts in normal cells. Therefore, the investigations on the affinity of edelfosine to the respective membrane components may facilitate the selection of molecule(s) responsible for the aforementioned high selectivity of this drug. Since a characteristic feature of various tumor cells lines is the enhanced expression of gangliosides [23–26], we have chosen this lipid for our experiments. As the second membrane component, we have selected sphingomyelin as a crucial element of lipid rafts. In this work we present the results of the experiments performed for mixtures of edelfosine with sphingomelin and ganglioside GM1 in the Langmuir films formed at the air/water interface. The analysis of the interactions between the components of the foregoing model membranes indicates that ganglioside molecules may be considered as target lipids for incorporation of edelfosine into cancer cells.
ues (GExc ) calculated according to Eq. (3) [28,29].
G
Exc
AExc d
=N
(3)
0
Moreover, to conclude on the thermodynamic stability of the mixed systems, the free energy of mixing (GMix ) values were calculated (Eq. (4)) [28]. GM = GExc + Gid
(4)
where Gid = RT (X1 ln X1 + X2 ln X2 )
(5)
R is the universal gas constant and T is the temperature. To compare the state of the monolayers the compression modulus values (CS −1 ) were analyzed. The values of the foregoing parameter were calculated from the isotherm data points, at a given monolayer composition, according to Eq. (6) [30]: CS −1 = −A
2. Material and methods
493
d dA
(6)
wherein A is the area per molecule at a given surface pressure . The investigated compounds were synthetic products of high purity. Egg sphingomyelin (Sph, ≥98%) and monosialoganglioside (GM1, >98%) were supplied by BioChemica. Edelfosine-1O-octadecyl-2-O-methyl-rac-glycero-3-phosphocholine (ED, ≥99.1%) was purchased from Biaffin GmbH&Co KG, Germany. Spreading solutions of the investigated lipids as well as edelfosine were prepared in the mixture of chloroform/methanol (9:1, v/v) (p.a. POCh, Poland). Mixed solutions of desirable compositions were prepared from the respective stock solutions and deposited onto water subphase with the Hamilton micro syringe, precise to 1.0 L. The monolayers were left after spreading for 5 min before the compression was initiated with the barrier speed of 20 cm2 /min. Measurements were performed with the NIMA (UK) Langmuir trough (total area = 300 cm2 ) placed on an anti-vibration table. Surface pressure was measured with the accuracy of ±0.1 mN/m using Wilhelmy plate made of filter paper (ashless Whatman Chr1) connected to an electrobalance. The surface pressure –area A isotherms have been recorded at four temperatures: 10, 20, 30 and 37 ◦ C. The subphase temperature was controlled thermostatically to within 0.1 ◦ C by a circulating water system. The miscibility and interactions between molecules in the investigated mixtures have been analyzed at the surface pressure of = 30 mN/m. This value has been chosen because it is in the range of surface pressures ( = 30–35 mN/m), where monolayer properties can be related to the properties of bilayers [27]. It should be also pointed out that although the experiments were performed at four temperatures, the results obtained at 37 ◦ C are of the highest biological relevance. From the isotherms the values of the following parameters have been determined and analyzed. First, the excess area per molecule (AExc ) values have been calculated according to Eq. (1) [28]: AExc = A12 − Aid 12
(1)
A12 is the mean area per molecule for binary film determined from the –A isotherms. Aid is the mean area per molecule in the case 12 of ideal mixing or complete immiscibility of the monolayer components is expressed by Eq. (2) [28,29]: Aid 12 = A1 X1 + A2 X2
(2)
A1 and A2 are the mean area per molecule for one-component films of the respective molecules at a given surface pressure and X1 and X2 are the mol fractions of components 1 and 2 in the mixed monolayer. The interactions between molecules in the mixed systems were analyzed quantitatively with the excess free energy of mixing val-
3. Results The surface pressure ()–area (A) isotherms for sphingomyelin (Sph), ganglioside (GM1) and edelfosine (ED) as well as for Sph/ED and GM1/ED mixed monolayers were measured at four temperatures (10, 20, 30 and 37 ◦ C). Figs. 1 and 2 present the –A curves recorded for GM1/ED and Sph/ED mixtures, respectively. The shape of the curves for pure GM1 monolayers and their localization at larger areas as compared to Sph films indicates lower ordering and more expanded character of ganglioside monolayers within the range of all investigated temperatures. This results from the structure of GM1 head group, which is larger than the polar moiety of sphingomyelin and, additionally, it is negatively charged (see Section 4). In consequence, the electrostatic repulsions hinder a close packing of GM1 molecules and ordering of the hydrophobic chains in monolayer. Moreover, in the course of the isotherms recorded for both lipids at the investigated temperatures (except for the monolayer formed by sphingomyelin at 37 ◦ C), a phase transition between LE and LC state appears. We have calculated the compression modulus (CS −1 ) values for the monolayers of both lipids at various temperatures. It has been found that the increase of temperature induces the shift of the phase transition to higher surface pressures. Moreover, the maximal CS −1 values below the transition are lower than the values above it, suggesting a lower ordering of lipid acyl chains. With the increase of temperature, the values of the compression modulus at first maximum increase, while those at the second transition decrease systematically. The compression modulus values found at first maximum for GM1 monolayers are: CS −1 = 30, 58, 89 and 97 mN/m, while at the second maximum: CS −1 = 197, 167, 132 and 120 mN/m at 10, 20, 30 and 37 ◦ C. These values are in a good agreement with those obtained by other authors investigating the properties of GM1 films at various experimental conditions [31]. The increase of the compression modulus values below the phase transition and systematic diminishing of the second maximum were explained as being due to the formation of one expanded phase at higher temperatures. Similar tendency is observed here for sphingomyelin monolayers (at first maximum CS −1 = 25, 43 and 98 mN/m and at the second maximum CS −1 = 350, 330 and 130 mN/m at 10, 20 and 30 ◦ C), however, at 37 ◦ C the phase transition is not detectable and only one maximum of CS −1 (130 mN/m) appears. As far as pure edelfosine is concerned, its monolayers are found to be insensitive to the temperature changes. This is consisted with previously published results on edelfosine monolayers formed at various experimental conditions [5].
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Fig. 2. The surface pressure–area curves for sphingomyelin (Sph)/edelfosine (ED) monolayers at different subphase temperatures.
Fig. 1. The surface pressure–area curves for ganglioside GM1/edelfosine (ED) monolayers at different subphase temperatures.
The addition of edelfosine into the lipids monolayers additionally decreases the compression modulus values (Fig. 3). This effect is more pronounced for sphingomyelin films at lower (10 and 20 ◦ C) temperatures. Since it is known that ED incorporates easily into less condensed monolayer, therefore its incorporation into more
ordered (and thus less accessible) sphingomyelin films strongly disturbs the organization of the film, which reflects in drastic decrease of the compression modulus. The effect observed for less ordered GM1 films and Sph monolayers formed at higher temperatures is less drastic. In the course of the isotherms for mixed Sph/ED monolayers at edelfosine mole fraction XED = 0.3 (T = 10, 20 and 30 ◦ C) as well as for 0.1 and 0.5 (at T = 20 ◦ C), a characteristic bend at the surface pressure near to the collapse for pure edelfosine film appears. Similar bend can be observed on the curve for GM1/ED film of XED = 0.7 at T = 20 ◦ C. This may suggest immiscibility between monolayer components of the foregoing composition at higher surface pressures. To compare the effect of the addition of edelfosine on the area per molecules values in both mixed systems and to draw conclusions on the miscibility of the monolayer components, the excess mean molecular areas (AExc ) were calculated and presented in Fig. 4. As it can be seen, at all investigated temperatures, the AExc = / 0 for all the studied systems, meaning that the mixtures behave non-ideally due to the intermolecular interactions. However, the excess area values for Sph/ED mixtures at all temperatures, except for the lowest one (10 ◦ C), are positive, while for GM1/ED monolayer the contraction of area per molecule (reflected in the negative values of AExc ) was found in the whole range of the monolayer composition and temperatures.
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Fig. 3. Percentage decrease of the compression modulus values (CS −1 ) due to incorporation of edelfosine into the monolayers of the respective sphingolipids ( = 30 mN/m).
The affinity of edelfosine to the investigated membrane lipids reflects also in the excess free energy of mixing values (GExc ) (Fig. 5). The negative values of GExc found for GM1/ED mixtures prove that the interactions between the drug molecules and ganglioside are stronger than the interactions between the molecules in their pure films. On the other hand, the incorporation of ED into sphingomyelin monolayer decreases attractive interactions in the mixed system at 20, 30 and 37 ◦ C as compared to the respective onecomponent films. Only at low temperature, GExc reaches negative values. The results of calculation of the free energy of mixing values are presented in Fig. 6. The lack of local minima on the GMix versus composition plots prove that at = 30 mN/m the components are miscible and no phase separation occurs. Negative values of GMix obtained for both mixed systems prove that the mixed state is thermodynamically more favorable than corresponding unmixed state. However, GM1/ED monolayers are much more stable as compared to sphingomyelin-containing films. 4. Discussion The results of the presented herein experiments prove that the components of both Sph/ED and GM1/ED monolayers are miscible at the investigated surface pressure ( = 30 mN/m) and the mixed systems are thermodynamically more favorable than the corresponding unmixed state. However, the incorporation of edelfosine into ganglioside monolayers reflects in more attractive or less repulsive interactions between molecules in the mixed monolayers as compared to the GM1–GM1 and ED–ED interactions in their pure films. On the other hand, the Sph/ED films are thermodynamically less stable than GM1/ED monolayers, and the addition of the drug
Fig. 4. The excess area per molecule (AExc ) vs composition plots (XED ) for mixed monolayers of edelfosine with sphingolipids at constant surface pressure ( = 30 mN/m).
causes that the interactions between molecules in mixtures are less attractive or more repulsive as compared to those existing in one-component films. These differences are discussed below from the point of view of how the structure of monolayer components determines their interactions in the mixed films. Sphingomyelin and gangliosides belong to a group of sphingosine-containing lipids. A sphingoid based skeleton is synthesized de novo and transformed into ceramides, which are precursors of more complex sphingolipids. A ceramide molecule is composed of fatty acid linked to sphingosine (amino alcohol containing long hydrocarbon chain) by its amino group. Further modification of ceramides generates a new classes of sphingolipids, namely phosphosphingolipids or glycosphingolipids. Sphingomyelin is phosphosphingolipid (ceramide phosphocholine) produced by esterification of the alcohol moiety with phosphorylcholine. Gangliosides are complex acidic glycosphingolipids (in contrast to simple glycosphingolipids, named cerebrosides) formed by attaching of oligosaccharide to ceramide and containing sialic acid molecule(s) linked to the sugar residue [32–35]. The investigated herein ganglioside possesses only one sialic acid, and therefore is classified as monosialoganglioside. All the investigated molecules are of amphipathic nature. In sphingomyelin molecules two chains (amide linked and sphingosine base) form apolar part and phosphocholine is the polar head. Oligosaccharide with sialic acid forms polar head of ganglioside GM1, while ceramide (with two tails) is apolar part of the molecule. Edelfosine is an ether phospholipid possessing only
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Fig. 5. The excess free energy of mixing values (GExc ) vs composition plots (XED ) for mixed monolayers of edelfosine with sphingolipids at constant surface pressure ( = 30 mN/m).
one alkyl (C18) chain (hydrophobic part) and phosphocholine polar moiety. At physiological conditions a polar group of GM1 is, in contrast to zwitterionic sphingomyelin, negatively charged [31,36,37]. The foregoing molecular parameters, e.g. bulkier, as compared to sphingomyelin, and charged polar head reflect in a larger area occupied by GM1 molecules in monolayers at the same surface pressure as compared to Sph. The incorporation of edelfosine into the film separates ganglioside molecules and decreases electrostatic repulsions between hydrophilic heads. This thermodynamically favorable effect reflects in negative values of the excess free energy of mixing observed for GM1/ED mixtures. In the case of Sph/ED systems the interactions are much weaker as compared to those between sphingomyelin and edelfosine molecules in their pure film, which results in higher values of GExc than for GM1-containing mixtures. This is the consequence of a weakening of van der Waals interaction between hydrophobic tails after addition of a single-chain ED into double-chain sphingomyelin film. The excess free energy of mixing values are higher (more positive) at higher temperature. With the temperature rise, thermal movements of molecules increase and the hydrophobic tails are less ordered. Since at 10 ◦ C the packing of molecules is the strongest, the favorable separation of the negatively charged GM1 molecules by edelfosine is the most pronounced (the most negative GExc values). In the case of Sph/ED mixtures the separation of sphingomyelin molecules at lower temperature, in highly condensed monolayers by edelfosine reflects in negative values of GExc . At higher temperatures, the GExc values for Sph/ED mixtures were
Fig. 6. The free energy of mixing values (GMix ) vs composition plots (XED ) for mixed monolayers of edelfosine with sphingolipids at constant surface pressure ( = 30 mN/m).
found to be positive. This is the consequence of a further decrease of van der Waals interactions between hydrophobic tails. The structure of a molecule determines its geometry and shape, which in turn correlates with its tendency to form aggregates of a given structure [38]. It has been proposed [38] that the type of the most favorable aggregates formed by molecules can be predicted from the critical packing parameters, calculated on the basis of area occupied by a polar group, volume and critical length of the hydrophobic region. The classification of a tendency of molecules to form certain aggregates in relation to the shape of a molecule can be found in Ref. [38]. In searching for the liposome of optimal composition to reduce hemolytic activity of edelfosine, its combination with phosphatidylcholines (PCs), phosphatidylethanolamine (PE) and various sterols were investigated [7,9]. The best effect (the highest decrease of hemolytic activity without losing the ability to induce apoptosis of leukemia cells) was found when ED was mixed with sterols as well as with 1,2-dioleoyl-sn-glycero-3phosphoethanolamine (DOPE) [7,9]. It was postulated that this is due to complementarity of molecular shapes of mixed molecules (conical shaped sterols and DOPE and inverted conical shaped edelfosine) [7,9]. For unsaturated phosphatidylcholines (POPC and DOPC) of cylindrical shape, the observed effects were weaker. To verify the side-by-side packing and the degree of shape complementarity between the investigated molecules, Perkins et al. have analyzed the changes in mean area per molecule for ED/lipid Langmuir monolayers [7]. The stronger the reduction of the mean area per molecule for lipids mixed with edelfosine (as compared to the values results from additivity rule), the strongest comple-
˛ ˛ K. Hac-Wydro, P. Dynarowicz-Łatka / Colloids and Surfaces B: Biointerfaces 81 (2010) 492–497
mentarity of molecular geometry and the reduction of hemolytic activity were proved [7]. Thus, the strongest deviations from ideality were observed for ED/cholesterol mixed monolayers. It is of interest to analyze our results from the point of view of the shape of interacting molecules and to compare them with those obtained for mixtures of edelfosine with other major membrane lipids. The molecular shapes of considered lipids are as follows. Cholesterol, as the lipid of a small head group as compared to the apolar part of a molecule, is of conical shape [9,38], similarly to phosphatidylethanolamines [7]. Phosphatidylcholines and sphingomyelin are cylinder-like molecules [39,40] due to similar cross-sectional area of their polar and apolar moieties. On the other hand, ganglioside GM1, with larger polar head as compared to its hydrophobic moiety is, similarly to edelfosine, of inverted conical shape [37,41,42]. Analyzing only the shape of mixed monolayers components it can be expected that, due to geometry complementarities, ED should combine most favorably with cholesterol and DOPE, then with PC and Sph, while its packing with ganglioside should be much less favorable. On the other hand, the magnitude of deviations of the mean area per molecule from the values resulting from additivity rule found for various ED/lipid monolayers at temperature 20 ◦ C changes as follows [6,7,10]: cholesterol > GM1 > unsaturated PCs > saturated PCs > Sph. Moreover, the degree of shape complementarity verified from the Langmuir experiments for cholesterol and edelfosine was much better than in the case of DOPE despite similar conical shape of both lipids [7]. Strong deviations from additivity rule were also found for GM1/ED mixtures although both ganglioside and the drug are of inverted conical shape. The results for cholesterol versus DOPE have been explained by Perkins and at [7]. It was suggested that the molecular shape is estimated from the values of critical packing parameter, which does not consider favorable interactions between mixture components. It can thus be concluded that edelfosine molecules pack favorably with GM1 because separating effect of the drug on negatively charged ganglioside prevails on the shape incomplementarity between both molecules. 5. Conclusions The Langmuir monolayer experiments have been performed for mixtures of edelfosine with the most important membrane components, like sterols [6], saturated and unsaturated phospholipids [7,10] and presented herein sphingolipids (sphingomyelin and ganglioside GM1). The obtained results provide important information, which can be considered from the point of view of the role of respective membrane lipids in incorporation of edelfosine into cell membrane and its selectivity. In conclusion, the membrane phospholipids seem to be not important in the above processes, due to their low affinity to edelfosine found in the monolayer experiments. Similar results have been obtained for the investigated here sphingomyelin. This is an important finding since sphingomyelin is a constituent of lipid rafts, in addition to cholesterol. As it has been proved, edelfosine strongly incorporates into membrane microdomains [8] and changes their properties. This seems to be due to its strong interactions with cholesterol [6]. However, cholesterol cannot be considered as a target molecule for edelfosine because of two reasons. Firstly, the lipid rafts are formed in both normal and tumor cellular membranes. This does not explain the selectivity of edelfosine. Secondly, for the majority of cancer cells, the cholesterol content is lower versus normal cells [19,20]. On the other hand, a characteristic feature of tumor cells is the overexpression of gangliosides, the concentration of which at the normal cell membrane is rather low. Interestingly, as it was found in our experiments, strong interactions exist between edelfosine and GM1 molecules. It enables us to hypothesise that gangliosides, which
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Searching for the role of membrane sphingolipids in selectivity of antitumor ether lipid–edelfosine ∗ ˛ ˛ Katarzyna Hac-Wydro , Patrycja Dynarowicz-Łatka Faculty of Chemistry, Jagiellonian University, Ingardena 3, 30-060 Kraków, Poland
a r t i c l e
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Article history: Received 7 April 2010 Received in revised form 16 July 2010 Accepted 21 July 2010 Available online 30 July 2010 Keywords: Edelfosine Ganglioside GM1 Sphingomyelin Monolayers Selectivity
a b s t r a c t Edelfosine is a synthetic antitumor lipid of high selectivity. Its activity on membrane level inspired the investigations on edelfosine–lipid interactions to verify, which of the membrane components may be responsible for the selectivity of this drug. Because of overexpression of gangliosides in tumor progression and the ability of edelfosine to insert into membrane rafts, we have chosen two sphingolipids, i.e. sphingomyelin and ganglioside to investigate in mixtures with edelfosine. It was found that edelfosine shows strong affinity to ganglioside in contrast to sphingomyelin. Differences in the interactions of edelfosine with sphingolipids were analyzed from the point of view of the structure and shape of the interacting molecules. The comparison of the results with those previously reported for edelfosine mixed with other membrane components, allowed us to suggest that gangliosides may be considered as target molecules attracting edelfosine to tumor cells. © 2010 Elsevier B.V. All rights reserved.
1. Introduction Edelfosine (ED) belongs to the family of synthetic single-chain ether lipids of antitumor properties [1,2]. Anticancer action of this drug is closely related to its insertion into tumor cell membranes. This is in contrast to conventional chemotherapeutic agents, which bind directly to DNA [1,3]. The action of edelfosine and other synthetic antitumor lipids (miltefosine and perifosine) at the membrane level stimulates the investigations on the effect of these compounds on model membrane systems. It was postulated that the affinity of these drugs to biomembrane is due to their amphipathic character and phospholipids-like molecular structure [4,5]. It was found that edelfosine interacts strongly with cholesterol [6–8] and in combination with this lipid is able to form liposomes [9]. Moreover, edelfosine in liposomal form does not loose its apoptotic properties while its hemolytic activity is abolished [9]. The interactions of ED with membrane phospholipids were found to be much weaker than the affinity of this drug to cholesterol [7,10]. It was also evidenced that stronger interactions occur when edelfosine is mixed with unsaturated phosphatidylcholines (PCs), namely 1,2-dioleoyl-sn-glycero-3-phosphocholine – DOPC, 1-palmitoyl-2oleoyl-sn-glycero-3-phosphocholine – POPC, as compared to those containing fully saturated chains [7,9,10]. Similar tendency as regards the affinity to membrane lipids was also proved for mil-
∗ Corresponding author. Tel.: +48 012 633 20 82; fax: +48 012 634 05 15. ˛ E-mail address: [email protected] (K. Hac-Wydro). 0927-7765/$ – see front matter © 2010 Elsevier B.V. All rights reserved. doi:10.1016/j.colsurfb.2010.07.045
tefosine [11–13], which was found to incorporate easier to model membranes of lower condensation [12,13]. It was suggested that antitumor lipids are able to incorporate into membrane microdomains [8,14,15]. In comprehensive studies performed on POPC/sphingomyelin (Sph)/cholesterol mixtures, mimicking membrane rafts, it was found that edelfosine can accumulate in microdomains at concentration as high as 21.7% of total lipids content in rafts. The insertion of drug molecules into rafts modifies their organization and biophysical properties [8]. The key role of membrane microdomains in edelfosine activity was also suggested in experiments on mouse lymphoma cells [16]. It was found that edelfosine induces apoptosis in S49 mouse lymphoma cells by raft-dependent internalization. On the other hand, a cell line variant (S49AR ), which is unable to synthesize sphingomyelin, is resistant to this drug. It was explained that the lack of sphingomyelin inhibits the formation of Sph/cholesterol domains, which may prevent edelfosine from incorporation into the cell [16]. Edelfosine is characterized by high selectivity that results from inability of normal cells to incorporate enough amounts of ether lipids [1]. Therefore, the membrane of normal cell is considered as a barrier for ED [1]. It is known that the composition of tumor membranes differs from those of normal cells. These differences involve changes in the concentration of major lipids (cholesterol, Sph and PCs) and proportion of saturated to unsaturated acyl chains [17–20]. All these modifications change the properties of tumor membranes, which are more fluid as compared to normal cells. The fact that edelfosine and miltefosine are of higher affinity to unsaturated phospholipids [12,13] and affect more strongly model
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membranes of higher fluidity [21,22] explains an easier insertion of drug molecules into tumor membrane. However, other factors should also be taken into consideration, like the presence of specific molecules in tumor membranes, which are either absent or in trace amounts in normal cells. Therefore, the investigations on the affinity of edelfosine to the respective membrane components may facilitate the selection of molecule(s) responsible for the aforementioned high selectivity of this drug. Since a characteristic feature of various tumor cells lines is the enhanced expression of gangliosides [23–26], we have chosen this lipid for our experiments. As the second membrane component, we have selected sphingomyelin as a crucial element of lipid rafts. In this work we present the results of the experiments performed for mixtures of edelfosine with sphingomelin and ganglioside GM1 in the Langmuir films formed at the air/water interface. The analysis of the interactions between the components of the foregoing model membranes indicates that ganglioside molecules may be considered as target lipids for incorporation of edelfosine into cancer cells.
ues (GExc ) calculated according to Eq. (3) [28,29].
G
Exc
AExc d
=N
(3)
0
Moreover, to conclude on the thermodynamic stability of the mixed systems, the free energy of mixing (GMix ) values were calculated (Eq. (4)) [28]. GM = GExc + Gid
(4)
where Gid = RT (X1 ln X1 + X2 ln X2 )
(5)
R is the universal gas constant and T is the temperature. To compare the state of the monolayers the compression modulus values (CS −1 ) were analyzed. The values of the foregoing parameter were calculated from the isotherm data points, at a given monolayer composition, according to Eq. (6) [30]: CS −1 = −A
2. Material and methods
493
d dA
(6)
wherein A is the area per molecule at a given surface pressure . The investigated compounds were synthetic products of high purity. Egg sphingomyelin (Sph, ≥98%) and monosialoganglioside (GM1, >98%) were supplied by BioChemica. Edelfosine-1O-octadecyl-2-O-methyl-rac-glycero-3-phosphocholine (ED, ≥99.1%) was purchased from Biaffin GmbH&Co KG, Germany. Spreading solutions of the investigated lipids as well as edelfosine were prepared in the mixture of chloroform/methanol (9:1, v/v) (p.a. POCh, Poland). Mixed solutions of desirable compositions were prepared from the respective stock solutions and deposited onto water subphase with the Hamilton micro syringe, precise to 1.0 L. The monolayers were left after spreading for 5 min before the compression was initiated with the barrier speed of 20 cm2 /min. Measurements were performed with the NIMA (UK) Langmuir trough (total area = 300 cm2 ) placed on an anti-vibration table. Surface pressure was measured with the accuracy of ±0.1 mN/m using Wilhelmy plate made of filter paper (ashless Whatman Chr1) connected to an electrobalance. The surface pressure –area A isotherms have been recorded at four temperatures: 10, 20, 30 and 37 ◦ C. The subphase temperature was controlled thermostatically to within 0.1 ◦ C by a circulating water system. The miscibility and interactions between molecules in the investigated mixtures have been analyzed at the surface pressure of = 30 mN/m. This value has been chosen because it is in the range of surface pressures ( = 30–35 mN/m), where monolayer properties can be related to the properties of bilayers [27]. It should be also pointed out that although the experiments were performed at four temperatures, the results obtained at 37 ◦ C are of the highest biological relevance. From the isotherms the values of the following parameters have been determined and analyzed. First, the excess area per molecule (AExc ) values have been calculated according to Eq. (1) [28]: AExc = A12 − Aid 12
(1)
A12 is the mean area per molecule for binary film determined from the –A isotherms. Aid is the mean area per molecule in the case 12 of ideal mixing or complete immiscibility of the monolayer components is expressed by Eq. (2) [28,29]: Aid 12 = A1 X1 + A2 X2
(2)
A1 and A2 are the mean area per molecule for one-component films of the respective molecules at a given surface pressure and X1 and X2 are the mol fractions of components 1 and 2 in the mixed monolayer. The interactions between molecules in the mixed systems were analyzed quantitatively with the excess free energy of mixing val-
3. Results The surface pressure ()–area (A) isotherms for sphingomyelin (Sph), ganglioside (GM1) and edelfosine (ED) as well as for Sph/ED and GM1/ED mixed monolayers were measured at four temperatures (10, 20, 30 and 37 ◦ C). Figs. 1 and 2 present the –A curves recorded for GM1/ED and Sph/ED mixtures, respectively. The shape of the curves for pure GM1 monolayers and their localization at larger areas as compared to Sph films indicates lower ordering and more expanded character of ganglioside monolayers within the range of all investigated temperatures. This results from the structure of GM1 head group, which is larger than the polar moiety of sphingomyelin and, additionally, it is negatively charged (see Section 4). In consequence, the electrostatic repulsions hinder a close packing of GM1 molecules and ordering of the hydrophobic chains in monolayer. Moreover, in the course of the isotherms recorded for both lipids at the investigated temperatures (except for the monolayer formed by sphingomyelin at 37 ◦ C), a phase transition between LE and LC state appears. We have calculated the compression modulus (CS −1 ) values for the monolayers of both lipids at various temperatures. It has been found that the increase of temperature induces the shift of the phase transition to higher surface pressures. Moreover, the maximal CS −1 values below the transition are lower than the values above it, suggesting a lower ordering of lipid acyl chains. With the increase of temperature, the values of the compression modulus at first maximum increase, while those at the second transition decrease systematically. The compression modulus values found at first maximum for GM1 monolayers are: CS −1 = 30, 58, 89 and 97 mN/m, while at the second maximum: CS −1 = 197, 167, 132 and 120 mN/m at 10, 20, 30 and 37 ◦ C. These values are in a good agreement with those obtained by other authors investigating the properties of GM1 films at various experimental conditions [31]. The increase of the compression modulus values below the phase transition and systematic diminishing of the second maximum were explained as being due to the formation of one expanded phase at higher temperatures. Similar tendency is observed here for sphingomyelin monolayers (at first maximum CS −1 = 25, 43 and 98 mN/m and at the second maximum CS −1 = 350, 330 and 130 mN/m at 10, 20 and 30 ◦ C), however, at 37 ◦ C the phase transition is not detectable and only one maximum of CS −1 (130 mN/m) appears. As far as pure edelfosine is concerned, its monolayers are found to be insensitive to the temperature changes. This is consisted with previously published results on edelfosine monolayers formed at various experimental conditions [5].
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Fig. 2. The surface pressure–area curves for sphingomyelin (Sph)/edelfosine (ED) monolayers at different subphase temperatures.
Fig. 1. The surface pressure–area curves for ganglioside GM1/edelfosine (ED) monolayers at different subphase temperatures.
The addition of edelfosine into the lipids monolayers additionally decreases the compression modulus values (Fig. 3). This effect is more pronounced for sphingomyelin films at lower (10 and 20 ◦ C) temperatures. Since it is known that ED incorporates easily into less condensed monolayer, therefore its incorporation into more
ordered (and thus less accessible) sphingomyelin films strongly disturbs the organization of the film, which reflects in drastic decrease of the compression modulus. The effect observed for less ordered GM1 films and Sph monolayers formed at higher temperatures is less drastic. In the course of the isotherms for mixed Sph/ED monolayers at edelfosine mole fraction XED = 0.3 (T = 10, 20 and 30 ◦ C) as well as for 0.1 and 0.5 (at T = 20 ◦ C), a characteristic bend at the surface pressure near to the collapse for pure edelfosine film appears. Similar bend can be observed on the curve for GM1/ED film of XED = 0.7 at T = 20 ◦ C. This may suggest immiscibility between monolayer components of the foregoing composition at higher surface pressures. To compare the effect of the addition of edelfosine on the area per molecules values in both mixed systems and to draw conclusions on the miscibility of the monolayer components, the excess mean molecular areas (AExc ) were calculated and presented in Fig. 4. As it can be seen, at all investigated temperatures, the AExc = / 0 for all the studied systems, meaning that the mixtures behave non-ideally due to the intermolecular interactions. However, the excess area values for Sph/ED mixtures at all temperatures, except for the lowest one (10 ◦ C), are positive, while for GM1/ED monolayer the contraction of area per molecule (reflected in the negative values of AExc ) was found in the whole range of the monolayer composition and temperatures.
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Fig. 3. Percentage decrease of the compression modulus values (CS −1 ) due to incorporation of edelfosine into the monolayers of the respective sphingolipids ( = 30 mN/m).
The affinity of edelfosine to the investigated membrane lipids reflects also in the excess free energy of mixing values (GExc ) (Fig. 5). The negative values of GExc found for GM1/ED mixtures prove that the interactions between the drug molecules and ganglioside are stronger than the interactions between the molecules in their pure films. On the other hand, the incorporation of ED into sphingomyelin monolayer decreases attractive interactions in the mixed system at 20, 30 and 37 ◦ C as compared to the respective onecomponent films. Only at low temperature, GExc reaches negative values. The results of calculation of the free energy of mixing values are presented in Fig. 6. The lack of local minima on the GMix versus composition plots prove that at = 30 mN/m the components are miscible and no phase separation occurs. Negative values of GMix obtained for both mixed systems prove that the mixed state is thermodynamically more favorable than corresponding unmixed state. However, GM1/ED monolayers are much more stable as compared to sphingomyelin-containing films. 4. Discussion The results of the presented herein experiments prove that the components of both Sph/ED and GM1/ED monolayers are miscible at the investigated surface pressure ( = 30 mN/m) and the mixed systems are thermodynamically more favorable than the corresponding unmixed state. However, the incorporation of edelfosine into ganglioside monolayers reflects in more attractive or less repulsive interactions between molecules in the mixed monolayers as compared to the GM1–GM1 and ED–ED interactions in their pure films. On the other hand, the Sph/ED films are thermodynamically less stable than GM1/ED monolayers, and the addition of the drug
Fig. 4. The excess area per molecule (AExc ) vs composition plots (XED ) for mixed monolayers of edelfosine with sphingolipids at constant surface pressure ( = 30 mN/m).
causes that the interactions between molecules in mixtures are less attractive or more repulsive as compared to those existing in one-component films. These differences are discussed below from the point of view of how the structure of monolayer components determines their interactions in the mixed films. Sphingomyelin and gangliosides belong to a group of sphingosine-containing lipids. A sphingoid based skeleton is synthesized de novo and transformed into ceramides, which are precursors of more complex sphingolipids. A ceramide molecule is composed of fatty acid linked to sphingosine (amino alcohol containing long hydrocarbon chain) by its amino group. Further modification of ceramides generates a new classes of sphingolipids, namely phosphosphingolipids or glycosphingolipids. Sphingomyelin is phosphosphingolipid (ceramide phosphocholine) produced by esterification of the alcohol moiety with phosphorylcholine. Gangliosides are complex acidic glycosphingolipids (in contrast to simple glycosphingolipids, named cerebrosides) formed by attaching of oligosaccharide to ceramide and containing sialic acid molecule(s) linked to the sugar residue [32–35]. The investigated herein ganglioside possesses only one sialic acid, and therefore is classified as monosialoganglioside. All the investigated molecules are of amphipathic nature. In sphingomyelin molecules two chains (amide linked and sphingosine base) form apolar part and phosphocholine is the polar head. Oligosaccharide with sialic acid forms polar head of ganglioside GM1, while ceramide (with two tails) is apolar part of the molecule. Edelfosine is an ether phospholipid possessing only
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Fig. 5. The excess free energy of mixing values (GExc ) vs composition plots (XED ) for mixed monolayers of edelfosine with sphingolipids at constant surface pressure ( = 30 mN/m).
one alkyl (C18) chain (hydrophobic part) and phosphocholine polar moiety. At physiological conditions a polar group of GM1 is, in contrast to zwitterionic sphingomyelin, negatively charged [31,36,37]. The foregoing molecular parameters, e.g. bulkier, as compared to sphingomyelin, and charged polar head reflect in a larger area occupied by GM1 molecules in monolayers at the same surface pressure as compared to Sph. The incorporation of edelfosine into the film separates ganglioside molecules and decreases electrostatic repulsions between hydrophilic heads. This thermodynamically favorable effect reflects in negative values of the excess free energy of mixing observed for GM1/ED mixtures. In the case of Sph/ED systems the interactions are much weaker as compared to those between sphingomyelin and edelfosine molecules in their pure film, which results in higher values of GExc than for GM1-containing mixtures. This is the consequence of a weakening of van der Waals interaction between hydrophobic tails after addition of a single-chain ED into double-chain sphingomyelin film. The excess free energy of mixing values are higher (more positive) at higher temperature. With the temperature rise, thermal movements of molecules increase and the hydrophobic tails are less ordered. Since at 10 ◦ C the packing of molecules is the strongest, the favorable separation of the negatively charged GM1 molecules by edelfosine is the most pronounced (the most negative GExc values). In the case of Sph/ED mixtures the separation of sphingomyelin molecules at lower temperature, in highly condensed monolayers by edelfosine reflects in negative values of GExc . At higher temperatures, the GExc values for Sph/ED mixtures were
Fig. 6. The free energy of mixing values (GMix ) vs composition plots (XED ) for mixed monolayers of edelfosine with sphingolipids at constant surface pressure ( = 30 mN/m).
found to be positive. This is the consequence of a further decrease of van der Waals interactions between hydrophobic tails. The structure of a molecule determines its geometry and shape, which in turn correlates with its tendency to form aggregates of a given structure [38]. It has been proposed [38] that the type of the most favorable aggregates formed by molecules can be predicted from the critical packing parameters, calculated on the basis of area occupied by a polar group, volume and critical length of the hydrophobic region. The classification of a tendency of molecules to form certain aggregates in relation to the shape of a molecule can be found in Ref. [38]. In searching for the liposome of optimal composition to reduce hemolytic activity of edelfosine, its combination with phosphatidylcholines (PCs), phosphatidylethanolamine (PE) and various sterols were investigated [7,9]. The best effect (the highest decrease of hemolytic activity without losing the ability to induce apoptosis of leukemia cells) was found when ED was mixed with sterols as well as with 1,2-dioleoyl-sn-glycero-3phosphoethanolamine (DOPE) [7,9]. It was postulated that this is due to complementarity of molecular shapes of mixed molecules (conical shaped sterols and DOPE and inverted conical shaped edelfosine) [7,9]. For unsaturated phosphatidylcholines (POPC and DOPC) of cylindrical shape, the observed effects were weaker. To verify the side-by-side packing and the degree of shape complementarity between the investigated molecules, Perkins et al. have analyzed the changes in mean area per molecule for ED/lipid Langmuir monolayers [7]. The stronger the reduction of the mean area per molecule for lipids mixed with edelfosine (as compared to the values results from additivity rule), the strongest comple-
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mentarity of molecular geometry and the reduction of hemolytic activity were proved [7]. Thus, the strongest deviations from ideality were observed for ED/cholesterol mixed monolayers. It is of interest to analyze our results from the point of view of the shape of interacting molecules and to compare them with those obtained for mixtures of edelfosine with other major membrane lipids. The molecular shapes of considered lipids are as follows. Cholesterol, as the lipid of a small head group as compared to the apolar part of a molecule, is of conical shape [9,38], similarly to phosphatidylethanolamines [7]. Phosphatidylcholines and sphingomyelin are cylinder-like molecules [39,40] due to similar cross-sectional area of their polar and apolar moieties. On the other hand, ganglioside GM1, with larger polar head as compared to its hydrophobic moiety is, similarly to edelfosine, of inverted conical shape [37,41,42]. Analyzing only the shape of mixed monolayers components it can be expected that, due to geometry complementarities, ED should combine most favorably with cholesterol and DOPE, then with PC and Sph, while its packing with ganglioside should be much less favorable. On the other hand, the magnitude of deviations of the mean area per molecule from the values resulting from additivity rule found for various ED/lipid monolayers at temperature 20 ◦ C changes as follows [6,7,10]: cholesterol > GM1 > unsaturated PCs > saturated PCs > Sph. Moreover, the degree of shape complementarity verified from the Langmuir experiments for cholesterol and edelfosine was much better than in the case of DOPE despite similar conical shape of both lipids [7]. Strong deviations from additivity rule were also found for GM1/ED mixtures although both ganglioside and the drug are of inverted conical shape. The results for cholesterol versus DOPE have been explained by Perkins and at [7]. It was suggested that the molecular shape is estimated from the values of critical packing parameter, which does not consider favorable interactions between mixture components. It can thus be concluded that edelfosine molecules pack favorably with GM1 because separating effect of the drug on negatively charged ganglioside prevails on the shape incomplementarity between both molecules. 5. Conclusions The Langmuir monolayer experiments have been performed for mixtures of edelfosine with the most important membrane components, like sterols [6], saturated and unsaturated phospholipids [7,10] and presented herein sphingolipids (sphingomyelin and ganglioside GM1). The obtained results provide important information, which can be considered from the point of view of the role of respective membrane lipids in incorporation of edelfosine into cell membrane and its selectivity. In conclusion, the membrane phospholipids seem to be not important in the above processes, due to their low affinity to edelfosine found in the monolayer experiments. Similar results have been obtained for the investigated here sphingomyelin. This is an important finding since sphingomyelin is a constituent of lipid rafts, in addition to cholesterol. As it has been proved, edelfosine strongly incorporates into membrane microdomains [8] and changes their properties. This seems to be due to its strong interactions with cholesterol [6]. However, cholesterol cannot be considered as a target molecule for edelfosine because of two reasons. Firstly, the lipid rafts are formed in both normal and tumor cellular membranes. This does not explain the selectivity of edelfosine. Secondly, for the majority of cancer cells, the cholesterol content is lower versus normal cells [19,20]. On the other hand, a characteristic feature of tumor cells is the overexpression of gangliosides, the concentration of which at the normal cell membrane is rather low. Interestingly, as it was found in our experiments, strong interactions exist between edelfosine and GM1 molecules. It enables us to hypothesise that gangliosides, which
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