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Thermodynamic and structural effects of propranolol on DPPC liposomes
Chemistry o f Physics and Lipids, 55 (1990) 331--337 Elsevier Scientific Publishers Ireland Ltd.
331
Thermodynamic and structural effects of propranolol on DPPC liposomes G. A l b e r t i n P , C. D o n a t i b, R . S . P h a d k e c, M . G . P o n z i Bossi b a n d F. Rustichelli b •Dipartimento di Scienze dei Materiali e della Terra, 60131 Ancona, °lstituto di Fisica Medica 60131 Ancona (Italy) and ~Chem. Phys., Tala Institute o f Fundamental Research, Homi Bhabha Road, Bombay 400005 (India) (Received January 15th, 1990; revision received July 2nd, 1990; accepted July 3rd, 1990)
Some thermodynamic and structural aspects of propranoloI-DPPC liposomes interaction were investigated by DSC and X-ray diffraction: the lamellar arrangement of the lipid matrix remains intact even at high concentrations of the drug (until I:l drug/ lipid molar ratio). However, the bilayer thickness increases significantly and the chains become perpendicular to the lamellar planes, for increasing drug content. At still higher propranolol concentrations a hexagonal phase occurs followed by a lamellar phase, in which the liposomes are destroyed. Moreover, the presence of propranolol has been found to impart fluidity to the lipid matrix. Keywords: DSC; X-ray diffraction; liposomes; propranolol; phase transitions.
Introduction
The pharmacological action of a drug is a complex phenomenon, sometimes involving specific binding to receptors as well as aspecific incorporation into lipid matrix. Moreover, the structure and function of membrane proteins and therefore of some receptors seems to depend on the physical properties such as structure, conformation and fluidity, of the surrounding lipidic medium, which in turn depend on the lipiddrug interaction. These facts justify the interest of a systematic investigation of the physical modifications induced by drugs in lipid aggregates. Propranolol, a prototype /3-adrenoreceptor blocking agent is known to regulate cardiac arrhythmias [1,2]. It is also known that 80070 of the administered drug binds aspecifically to membrane lipids leading to local anaesthesia [3,4]. In fact, the antiarrhythmic activity has been shown to be closely related to the anaesth-
Correspondence to: Professor Franco Rustichelli.
etic property rather than to its adrenergic /3receptor blocking activity [5]. It is therefore desirable to investigate in detail the effects of incorporation of the drug in the membrane matrix. Reports are available in the literature describing altered dynamics and local order of the matrix itself [6--8]. The present work reports some thermodynamic and structural aspects of the drug-liposome system based on DSC and Xray diffraction experiments. We have used L-a dipalmitoyl phosphatidyl choline (DPPC) iiposomes as membrane system, as 30070 of lipids in biological membranes are phosphocholines [9] and DPPC liposomes have been reported to be biocompatible drug carrier systems [10], so that the present investigation could possibly be interesting also for the technology of drug vehiculation. In fact it is particularly important in the case of propranolol to have a suitable carrier system as its bioavailibility is notoriously low on account of its high hepatic first-pass metabolism [1 1]. Lastly, the membrane activity of propranolol may be important to study its toxicity following
0009-3084/90/$03.50 © 1990 Elsevier Scientific Publishers Ireland Ltd. Published and Printed in Ireland
332 overdose [12], thus we have reported also the results obtained at high molar ratios, beyond physiological conditions. On the other hand, the high molar ratio results can help to understand where the drug molecule is localized in the bilayer. Experimental
procedures
Ni-filtered Cu-Ka radiation (i = 0.154 nm) was used in any case. Temperature was controlled by a BT300/301 control system supplied by SMC, (Grenoble, France).
Optical microscopy Optical observations were performed using a Leitz Ortolux 2 polarizing microscope equipped with a Mettler FP52 hot stage.
Materials and Methods Results
1, 2 - dipalmitoyl - 3 - s n - phosphatidylcholine (DPPC) was purchased from Sigma (St. Louis, MO) and was used without further purification. P r o p r a n o l o l . HCI was a gift from Cipla, India.
Sample preparations The appropriate quantities o f propranolol were added to weighted amounts o f D P P C in order to get the desired mixtures. The molar ratio R between propranoiol and D P P C used in the experiments ranged from R = 0 to R = 1; furthermore two samples at higher concentration were examined: R = 2.2 and R = 4. The mixtures were first dissolved in chloroform then dried in a slow nitrogen stream and lyophilized in order to evaporate residual solvents. Distilled water was added in a water to D P P C weight ratio x = 3. Liposomes were obtained by equilibrating the aqueous mixtures for at least 4 h at about 328 K with intermittent vortexing during this period.
DSC Calorimetric scans were obtained in the temperature range 290--330 K. D P P C liposomes without the drug (R = 0) showed characteristic peaks at 309 and 314.5 K (Fig. 1, curve a) corresponding to the phase transitions L~, -" P~, and Po, --- Lo (chain melting transition or main transition) [13]. Liposomes containing low concentrations of propranolol (R < 0.08) exhibited similar behaviour but the transitions occurred at lower temperatures (Fig. 1, curve b).
R=.6
E3 Z iii [
~
R=.I
Calorimetry Differential scanning calorimetry experiments were performed by using DSC 2C Perkin Elmer calorimeter with associated processor. Sealed containers having 20 ~! capacity were used as sample holders. The samples were heated or cooled at 2.5 K / m i n scan rate.
R:.0~
!
X-ray diffraction X-ray diffraction studies were performed by using powder diffractometers equipped either with a conventional X-ray source or with a Rigaku Denki RU300 rotating anode generator and also obtaining pictures using rotating anode source in conjunction with a low angle chamber.
R:0 a [
300
'
310 '
I
320
K
Fig. 1. Calorimetric heating scans for R = 0 (a), R = 0.01 (b), R = 0.1 (c) and R = 0.6 (d). The bar in every scan corresponds to 0.025 eal/(g's). Scan rate 2.5 K/rain.
333
Increasing propranolol content further (R > 0.08) resulted in the disappearance of the La, Pa. transition, whereas the main transition peak appears to be broadened and shifted to lower temperatures (Fig. 1, curves c and d ): X-ray diffraction data did confirm that this peak corresponds to the chain melting transition.The full width at half maximum of the main transition peak increases reaching a maximum near R = 0. I, then decreases. Figure 2 depicts the temperatures of the two phase transitions, versus the drug-to-DPPC molar ratio R. The enthalpy per DPPC mole does not diminish in the considered concentration range, increasing in case at molar ratios around R = 0.1, where phase coexistence was observed by using X-ray diffraction, as discussed below. For the two highest concentrations (R = 2.2 and R = 4) no calorimetric peaks were detected in the temperature range 290--330 K.
X-ray diffraction R < l. X-ray diffraction patterns show that mixtures having molar ratio R < 1 are iamellar both in gel and in melted chain phase. This fact is confirmed by the corresponding textures, seen in polarized light microscopy: in fact birefringence patterns characteristic of liposomes [14] or of planar lamellar arrangements [15] are seen. Room temperature. At room temperature, incorporation of low drug content (R < 0.08) does not alter significantly the diffraction pat-
3,5 Y
3,ol
terns with respect to that obtained from pure DPPC liposomes (Fig. 3a and 3b, lower curves); in fact in the low angle region the peak positions in s -~ units are in the ratio 1 : 2 : 3 : 4 . . . typical of a lameilar phase [16]; in the high angle region the well known composite peak appears, characteristic of the hydrocarbon chain two dimensional organization in a distorted hexagonal lattice with tilted chains (L#, phase). At higher drug content (R >1 0.08) the propranoloi induces important alterations (higher curves of Fig. 3a and 3b) and the layer thickness increases from 6.3 ± 0.3 nm (pure DPPC) to 11 ± 0.5 nm (molar ratio R >~ 0.6) (Fig. 4); these values were calculated from the low angle peak positions obtained by the diffractometric and photographic techniques. Coexistence of lameilar phases was observed for molar ratios around R = 0.I and the layers spacings reported in Fig. 4, correspond to the higher diffracted intensities, measured in the Xray pictures. 5
8
3
.... I /~Ii|
T'=303K
.~6 l
~3 •
,
i
.tO S I ~ ) ,
i
T=303K
\
!:
¢
II
'
~
2 S'(nm)
6
¢
305
o I i
i
i
MOLAR RATIO R Fig. 2. Calorimetric peak maximum temperatures vs. the propranolol DPPC molar ratio R. DPPC/water = 1/3 (w/ w). O, higher temperature peak; O, lower temperature peak.
1°
I
3*
I
I
I
5* 20
19" 20°
21°
I
22°
23°
2B Fig. 3. X-ray diffraction patterns from reference sample (R = 0, lower curves) and from R = 0.1 and R = 0.6: (a) low angle and (b) high angle patterns. Lower scale: take off angle 20 ( % Higher scale: s" units (nm). s = 2 * sin (O)/A.
334
.~
.1.2
.~ ~;~TTnl
I= 303 K
12
+
u'l LU Z
1
¥8 LU ~6
C
/.
8 MOLAR RATIO R
/i
Fig. 4. Lamellar repeat spacing vs. molar ratio R at the temperature T = 303 K .
In the high angle region, for molar ratio R >/ 0.08, the two peaks are replaced by one intense peak, which becomes sharper and shifts towards higher diffraction angles for increasing drug content (Fig. 3b). No other peaks were detected for s -t >/ 0.3 (diffraction angle 20 g 30). These facts indicate that the hydrocarbon chains become perpendicular to the lameUar planes and remain still elongated [17,18]. The high angle large peak obtained at R = 0.1 can be related to a coexistence of phases, which is confirmed by low angle data (see above) and by the large calorimetric peak (Fig. 1). Higher temperatures. Figure 5 reports the high angle diffraction patterns obtained at different concentrations at a temperature 2 K below the chain melting transition temperature; in all cases (R ~< l) the two dimensional lattice of the chains is hexagonal [17,18], the lattice parameter / being related to the peak positions by:
R=.I
/ I ~jo
2~
2#
22 = 23 ° 29
Fig. 5. High angle X-ray diffraction patterns from reference sample (P~. phase) and from R = 0.1 a n d R = 0 . 3 at a temperature 2 K below the main transition temperatures. Lower scale: take off angle 20 (o). Higher scale: s-' units
(nm).
angle peak is broad indicating that the chains are melted. The low angle peaks indicate that the phase is lamellar and that moreover the interlayer periodicity increases with propranolol content reaching at R = 0.6 a value which is t(~rn) c
~9
i.n ./.2 1.8
2
Is*V~
I..I /.7
Variations of peak positions in s "~ units (left scale) and of lattice parameter 1 (right scale) with drug content are reported in Fig. 6. At temperatures higher than chain melting temperatures, the diffraction patterns, for molar ratio R g 1 show a iamellar L, phase: the high
6 MOLAR
,9 RA'I'IO
R
Fig. 6. Position of the X-ray high angle peak in s -~ unit (left scale) vs. molar ratio R at a temperature 2 ° below the main transition temperature. Right scale: interchain distances.
335 almost twice as big as that o f pure D P P C liposomes (Fig. 7), in analogy with the behaviour observed at lower temperatures. Lastly it is interesting to note that, whereas isotropic diffraction is expected for tiposomes, X-ray pictures show that preferential orientations of the diffracting planes can exist for high R values. Moreover the microscopic textures are typical of planar lamellar phases [15] for R >1 0.2: thus the liposomes are destroyed at high propranolol molar ratios. R > 1. Liposomes destruction (as above reported) has been also detected by Eriksson in a NMR study [7] on the interaction o f propranoiol with egg yolk lecithin vesicles in NaCI solutions. In particular he found that at propranoloi molar ratio R = 2.2 the sample becomes transparent and the vescicles are destroyed. At such high concentration the high angle X-ray diffraction patterns at room temperature are characterized by a broad peak, indicating that the chains are melted (Fig. 8). In the low angle patterns peak positions in s -~ units are in the ratios I:V~:2 (Fig. 8). These facts indicate [hat the iamellar organization has been destroyed and a hexagonal phase appears: the obtained cell parameter is a = 5.85 nm. For still higher concentrations (R = 4) the mixtures are transparent, highly viscous, optically anisotropic and the X-ray diffraction patterns indicate lameilar organization with melted chains also at room temperature. l
R=22
I 10"
T=303K
x 20*
20
Fig. 8. X-ray diffraction pattern obtained at R = 2.2, T = 303 K. Lower scale: take off angle 20 (%
These results are in agreement with those obtained in Ref. 7 in the case o f egg yolk lecithin: also in that case a clear and very viscous system was obtained for molar ratio greater than 2.2. Discussion The calorimetric results show that for R > 0.08 the chain melting temperature decreases considerably, indicating that the drug induces a larger fluidity in the bilayer. The disappearance of the La, ~ Pa, transition, which can be related to the interaction between the choline moiety and the bound water [19], could indicate that the drug interacts with the polar region o f the phospholipids; the lowering of the chain melting transition without enthalpy decrease suggests that propranolol interacts with the head group and only partially penetrates between the chains
[201.
uJ zv . -lwo~
I 0
i
i
i
25
5
75 MOLAR RAI]O
R
Fig. 7. L a m e l l a r r e p e a t s p a c i n g vs. m o l a r r a t i o R at the t e m p e r a t u r e T = 321 K.
The X-ray diffraction-data indicate that at room temperature and for molar ratio 0.8 ~< R 1 the chains are perpendicular to the iamellar planes, the chain distance slightly decreases and the lamellar spacing greatly increases with drug concentration (Fig. 7). The strong increase of the lamellar thickness cannot be attributed only to the loss of the chain tilt, inferred from high angle data, as the effect is present also in the L° phase, where the chains are melted. Thus the
336 increase o f the repeat distance, induced by the drug, should be related to an increase of the water sublayer thickness: as a consequence the effects of the propranoiol" HCI charged molecule are similar to those induced by c h l o r p r o m a z i n e ' H C l in excess water [21]. In general the capability of increasing water layer thickness can he related to the charge of the molecule; in fact charged molecules are reported to increase it, while neutral molecules do not 1211. The charged amphiphilic drug chlorpromazine. HCI, presenting calorimetric [22] and X-ray diffraction results [21] similar to those of propranolol, interacts with polar head groups, slightly penetrates into the hydrocarbon region of the bilayer and at high molar ratio (R = 0.5) induces a chain interdigitation [21]. It could be interesting to calculate the electron density profile across the bilayer in order to determine if also the propranolol induces a chain interdigitation and to find the position of the propranolol molecule in the bilayer; we are trying now to perform a swelling experiment to determine the phases of the reflections for the electron density map calculation: unfortunately at high water contents the first order peak, although visible in the X-ray picture, is too close to the direct beam and a clear intensity determination is not possible. Preliminar measurements at molar ratio R = 0.3 and at low water content indicate that the lipid bilayer thickness at relative humidity of 10o70 and 20°70 has an almost constant value of 3.3 _ 0.2 nm and that the repeat distances are 4.5 and 4.8 nm, respectively (three peaks were considered and the only possible choice, physically significant, of the reflections phases was 0, rt, n). Therefore, water addition induces an increase of the water sublayer thickness but leaves unchanged the lipid sublayer thickness; moreover both profiles have two density maxima in correspondence of the head groups of phospholipids and a secondary maximum at the centre of the bilayer, thus suggesting a chain interdigitation; this conclusion i~ also supported by the lipid thickness value, which is too small as compared to the lipid thickness of pure
DPPC with the chains fully extended and perpendicular to the lamellae, which is 4.9 nm [21]. The chain interdigitation could be also related to a small decrease of the chain distance shown in Fig. 6: it seems that the chain packing is tighter, in apparent disagreement with the increased bilayer fluidity inferred from calorimetric data. Probably the lipid bilayer is partially disordered owing to the presence of defects in the chain packing a n d / o r to some fluctuations of the interlayer distance value, due to the non-homogeneous distribution of the drug: in fact for R = 0.6 the second and third order peaks (Fig. 3a) have the half height widths larger than those of the pure DPPC, probably due to small variations of the periodicity. Moreover a correlation can also exist between the increased water layer thickness and the lowering of the transition temperature; in fact it was shown [23] that the neutral amphiphilic molecule of dicarboxilic azelaic acid interacts with the polar head-group and induces chain interdigitation, as chlorpromazine" HCI and propranolol" HCI do, but does not induce an increase of the water thickness: chain melting temperature is not affected.
Conclusions The effects of propranolol are detectable only if sufficient amount of the drug is present in the system (drug/lipid molar ratio R >t 0.08). The presence of the drug retains lamellar arrangement but imparts fluidity to the lipids, almost doubles the lamellar repeat spacing, increasing the water thickness, and induces a /J conformation of the chains, which thus become perpendicular to the lamellae and seem to be interdigitated; moreover the liposomes are destroyed and the bilayers are organized in multilamellar planar stacks. At molar ratio R = 2.2 a hexagonal phase is obtained, which is optically transparent, and for still higher molar ratios a very viscous lamellar phase is obtained. The calorimetric and X-ray results suggest that the p r o p r a n o l o l ' H C l molecule interacts with the phospholipid head groups and slightly penetrates into the hydrocarbon chain region.
337
At high propranoiol content the observed behaviour has many similarities with that observed by adding propranolol to unilamellar vesicles of egg lecithin in NaCl solutions [7]. Acknowledgements It is a pleasure to thank Mr. S. Polenta and Mr. M. Pergolini for valuable technical assistance. This work was supported by Regione Marche. Part of the equipment was obtained on the basis of financial help from CNR and MPI. One of the authors (R.S. Phadke) would like to thank ICTP Trieste, Italy , Programme for Training and Research in Italian Laboratories, for the support towards her stay in Italy. References 1 2 3 4 5 6 7 8
D.T. Mason, A.N. De Maria, R. Asterdam, R. Zelis and R.A. Massumi 0973) Drugs 5, 261--349. B.N. Singh (1978) Drugs 15,218--225. D. Atlas, M.L. Steer and A. Levitzki 0974) Proc. Nat. Acad. Sci. U.S.A. 71, 4246--4248. M. Chabirani, E.G. Carbon, D. Mullikin, D. Leftkovitz and R.J. Leftkovitz (1976) Mol. Pharm. 12, 16--31. A. Barret and V.A. Cullum (1968) Br. J. Pharm. 34, 43--55. R.S. Phadke, N.V. Kumar, R.V. Hosur, A. Saran and G. Govil 0981) Int. J. Quantum Chem. 20, 55--65. L.E.G. Eriksson 0987) Biophys. Chem. 26, 9--18. G. AIbertini, C. Donati, F. Rustichelli and R.S. Phadke (1988) Nuovo Cimento D, 10, 1399--1408.
9 10
11 12 13 14
15
16
17 18 19 20 21 22 23
A.L. Lehninger (1977) in: Biochemistry, Worth Pub. inc., New York, pp. 807--813. F. Puisieux and J. Delattre (Eds.) (1985) Les Liposomes: Applications Therapeutiques, Technique et Documentation, Lavoisier, Paris. G. Johnson and C.G. Regardh (1976) Clin. Farmacokin. 1,233--310. J.A. Henry and S.L. Cassydy (1986) Lancet 1, 1414. R.N. McEIhaney (1982) Chem. Phys. Lipids 30, 229-259. A.D. Bangham (1986) in: J.A.F. Op Den Kamp, B. Roelofsen and K.W.A. Wirtz (Eds.), Lipids and Membranes: Past, Present and Future, Elsevier, New York, p. 95. M. Kleman, C. Colliex and M. Veyssie (1976) in: S. Friberg (Ed.), Lyotropic Liquid Crystals, Advances in Chemistry Series 152, American Chemical Society, Washington, pp. 71--123. V. Luzzati (1968) in: D. Chapman and F.H. Wallah (Eds.), Biological Membranes, Academic Press, Nev, York, London, pp. 21--123. A. Tardieu, V. Luzzati and F.C. Reman (1973) J. Mol. Biol. 75,711--723. J.L. Ranck, T. Keira and V. Luzzati (197"7) Biochim. Biophys. Acta 488, 432--441. M.J. Janiak, D.M. Small and G.G. Shypley (1976) Biochemistry 15, 4575--4580. J. Bermejo, A. Barbadillo, F. Taro and D. Chapman (1975) FEBS Lett. 52, 69--72. T.J. Mclntosh, R.V. McDaniel and S.A. Simon (1983) Biochim. Biophys. Acta 731, 109--114. J. Freznell, K. Arnold and P. Nuhn (1978) Biochim. Biophys. Acta 507, 185--197. G. Bossi, B. Dubini, P. Mariani, R.S. Phadke, M.G. Ponzi Bossi and F. Rustichelli 0990) Nuovo Cimento D, in press.
331
Thermodynamic and structural effects of propranolol on DPPC liposomes G. A l b e r t i n P , C. D o n a t i b, R . S . P h a d k e c, M . G . P o n z i Bossi b a n d F. Rustichelli b •Dipartimento di Scienze dei Materiali e della Terra, 60131 Ancona, °lstituto di Fisica Medica 60131 Ancona (Italy) and ~Chem. Phys., Tala Institute o f Fundamental Research, Homi Bhabha Road, Bombay 400005 (India) (Received January 15th, 1990; revision received July 2nd, 1990; accepted July 3rd, 1990)
Some thermodynamic and structural aspects of propranoloI-DPPC liposomes interaction were investigated by DSC and X-ray diffraction: the lamellar arrangement of the lipid matrix remains intact even at high concentrations of the drug (until I:l drug/ lipid molar ratio). However, the bilayer thickness increases significantly and the chains become perpendicular to the lamellar planes, for increasing drug content. At still higher propranolol concentrations a hexagonal phase occurs followed by a lamellar phase, in which the liposomes are destroyed. Moreover, the presence of propranolol has been found to impart fluidity to the lipid matrix. Keywords: DSC; X-ray diffraction; liposomes; propranolol; phase transitions.
Introduction
The pharmacological action of a drug is a complex phenomenon, sometimes involving specific binding to receptors as well as aspecific incorporation into lipid matrix. Moreover, the structure and function of membrane proteins and therefore of some receptors seems to depend on the physical properties such as structure, conformation and fluidity, of the surrounding lipidic medium, which in turn depend on the lipiddrug interaction. These facts justify the interest of a systematic investigation of the physical modifications induced by drugs in lipid aggregates. Propranolol, a prototype /3-adrenoreceptor blocking agent is known to regulate cardiac arrhythmias [1,2]. It is also known that 80070 of the administered drug binds aspecifically to membrane lipids leading to local anaesthesia [3,4]. In fact, the antiarrhythmic activity has been shown to be closely related to the anaesth-
Correspondence to: Professor Franco Rustichelli.
etic property rather than to its adrenergic /3receptor blocking activity [5]. It is therefore desirable to investigate in detail the effects of incorporation of the drug in the membrane matrix. Reports are available in the literature describing altered dynamics and local order of the matrix itself [6--8]. The present work reports some thermodynamic and structural aspects of the drug-liposome system based on DSC and Xray diffraction experiments. We have used L-a dipalmitoyl phosphatidyl choline (DPPC) iiposomes as membrane system, as 30070 of lipids in biological membranes are phosphocholines [9] and DPPC liposomes have been reported to be biocompatible drug carrier systems [10], so that the present investigation could possibly be interesting also for the technology of drug vehiculation. In fact it is particularly important in the case of propranolol to have a suitable carrier system as its bioavailibility is notoriously low on account of its high hepatic first-pass metabolism [1 1]. Lastly, the membrane activity of propranolol may be important to study its toxicity following
0009-3084/90/$03.50 © 1990 Elsevier Scientific Publishers Ireland Ltd. Published and Printed in Ireland
332 overdose [12], thus we have reported also the results obtained at high molar ratios, beyond physiological conditions. On the other hand, the high molar ratio results can help to understand where the drug molecule is localized in the bilayer. Experimental
procedures
Ni-filtered Cu-Ka radiation (i = 0.154 nm) was used in any case. Temperature was controlled by a BT300/301 control system supplied by SMC, (Grenoble, France).
Optical microscopy Optical observations were performed using a Leitz Ortolux 2 polarizing microscope equipped with a Mettler FP52 hot stage.
Materials and Methods Results
1, 2 - dipalmitoyl - 3 - s n - phosphatidylcholine (DPPC) was purchased from Sigma (St. Louis, MO) and was used without further purification. P r o p r a n o l o l . HCI was a gift from Cipla, India.
Sample preparations The appropriate quantities o f propranolol were added to weighted amounts o f D P P C in order to get the desired mixtures. The molar ratio R between propranoiol and D P P C used in the experiments ranged from R = 0 to R = 1; furthermore two samples at higher concentration were examined: R = 2.2 and R = 4. The mixtures were first dissolved in chloroform then dried in a slow nitrogen stream and lyophilized in order to evaporate residual solvents. Distilled water was added in a water to D P P C weight ratio x = 3. Liposomes were obtained by equilibrating the aqueous mixtures for at least 4 h at about 328 K with intermittent vortexing during this period.
DSC Calorimetric scans were obtained in the temperature range 290--330 K. D P P C liposomes without the drug (R = 0) showed characteristic peaks at 309 and 314.5 K (Fig. 1, curve a) corresponding to the phase transitions L~, -" P~, and Po, --- Lo (chain melting transition or main transition) [13]. Liposomes containing low concentrations of propranolol (R < 0.08) exhibited similar behaviour but the transitions occurred at lower temperatures (Fig. 1, curve b).
R=.6
E3 Z iii [
~
R=.I
Calorimetry Differential scanning calorimetry experiments were performed by using DSC 2C Perkin Elmer calorimeter with associated processor. Sealed containers having 20 ~! capacity were used as sample holders. The samples were heated or cooled at 2.5 K / m i n scan rate.
R:.0~
!
X-ray diffraction X-ray diffraction studies were performed by using powder diffractometers equipped either with a conventional X-ray source or with a Rigaku Denki RU300 rotating anode generator and also obtaining pictures using rotating anode source in conjunction with a low angle chamber.
R:0 a [
300
'
310 '
I
320
K
Fig. 1. Calorimetric heating scans for R = 0 (a), R = 0.01 (b), R = 0.1 (c) and R = 0.6 (d). The bar in every scan corresponds to 0.025 eal/(g's). Scan rate 2.5 K/rain.
333
Increasing propranolol content further (R > 0.08) resulted in the disappearance of the La, Pa. transition, whereas the main transition peak appears to be broadened and shifted to lower temperatures (Fig. 1, curves c and d ): X-ray diffraction data did confirm that this peak corresponds to the chain melting transition.The full width at half maximum of the main transition peak increases reaching a maximum near R = 0. I, then decreases. Figure 2 depicts the temperatures of the two phase transitions, versus the drug-to-DPPC molar ratio R. The enthalpy per DPPC mole does not diminish in the considered concentration range, increasing in case at molar ratios around R = 0.1, where phase coexistence was observed by using X-ray diffraction, as discussed below. For the two highest concentrations (R = 2.2 and R = 4) no calorimetric peaks were detected in the temperature range 290--330 K.
X-ray diffraction R < l. X-ray diffraction patterns show that mixtures having molar ratio R < 1 are iamellar both in gel and in melted chain phase. This fact is confirmed by the corresponding textures, seen in polarized light microscopy: in fact birefringence patterns characteristic of liposomes [14] or of planar lamellar arrangements [15] are seen. Room temperature. At room temperature, incorporation of low drug content (R < 0.08) does not alter significantly the diffraction pat-
3,5 Y
3,ol
terns with respect to that obtained from pure DPPC liposomes (Fig. 3a and 3b, lower curves); in fact in the low angle region the peak positions in s -~ units are in the ratio 1 : 2 : 3 : 4 . . . typical of a lameilar phase [16]; in the high angle region the well known composite peak appears, characteristic of the hydrocarbon chain two dimensional organization in a distorted hexagonal lattice with tilted chains (L#, phase). At higher drug content (R >1 0.08) the propranoloi induces important alterations (higher curves of Fig. 3a and 3b) and the layer thickness increases from 6.3 ± 0.3 nm (pure DPPC) to 11 ± 0.5 nm (molar ratio R >~ 0.6) (Fig. 4); these values were calculated from the low angle peak positions obtained by the diffractometric and photographic techniques. Coexistence of lameilar phases was observed for molar ratios around R = 0.I and the layers spacings reported in Fig. 4, correspond to the higher diffracted intensities, measured in the Xray pictures. 5
8
3
.... I /~Ii|
T'=303K
.~6 l
~3 •
,
i
.tO S I ~ ) ,
i
T=303K
\
!:
¢
II
'
~
2 S'(nm)
6
¢
305
o I i
i
i
MOLAR RATIO R Fig. 2. Calorimetric peak maximum temperatures vs. the propranolol DPPC molar ratio R. DPPC/water = 1/3 (w/ w). O, higher temperature peak; O, lower temperature peak.
1°
I
3*
I
I
I
5* 20
19" 20°
21°
I
22°
23°
2B Fig. 3. X-ray diffraction patterns from reference sample (R = 0, lower curves) and from R = 0.1 and R = 0.6: (a) low angle and (b) high angle patterns. Lower scale: take off angle 20 ( % Higher scale: s" units (nm). s = 2 * sin (O)/A.
334
.~
.1.2
.~ ~;~TTnl
I= 303 K
12
+
u'l LU Z
1
¥8 LU ~6
C
/.
8 MOLAR RATIO R
/i
Fig. 4. Lamellar repeat spacing vs. molar ratio R at the temperature T = 303 K .
In the high angle region, for molar ratio R >/ 0.08, the two peaks are replaced by one intense peak, which becomes sharper and shifts towards higher diffraction angles for increasing drug content (Fig. 3b). No other peaks were detected for s -t >/ 0.3 (diffraction angle 20 g 30). These facts indicate that the hydrocarbon chains become perpendicular to the lameUar planes and remain still elongated [17,18]. The high angle large peak obtained at R = 0.1 can be related to a coexistence of phases, which is confirmed by low angle data (see above) and by the large calorimetric peak (Fig. 1). Higher temperatures. Figure 5 reports the high angle diffraction patterns obtained at different concentrations at a temperature 2 K below the chain melting transition temperature; in all cases (R ~< l) the two dimensional lattice of the chains is hexagonal [17,18], the lattice parameter / being related to the peak positions by:
R=.I
/ I ~jo
2~
2#
22 = 23 ° 29
Fig. 5. High angle X-ray diffraction patterns from reference sample (P~. phase) and from R = 0.1 a n d R = 0 . 3 at a temperature 2 K below the main transition temperatures. Lower scale: take off angle 20 (o). Higher scale: s-' units
(nm).
angle peak is broad indicating that the chains are melted. The low angle peaks indicate that the phase is lamellar and that moreover the interlayer periodicity increases with propranolol content reaching at R = 0.6 a value which is t(~rn) c
~9
i.n ./.2 1.8
2
Is*V~
I..I /.7
Variations of peak positions in s "~ units (left scale) and of lattice parameter 1 (right scale) with drug content are reported in Fig. 6. At temperatures higher than chain melting temperatures, the diffraction patterns, for molar ratio R g 1 show a iamellar L, phase: the high
6 MOLAR
,9 RA'I'IO
R
Fig. 6. Position of the X-ray high angle peak in s -~ unit (left scale) vs. molar ratio R at a temperature 2 ° below the main transition temperature. Right scale: interchain distances.
335 almost twice as big as that o f pure D P P C liposomes (Fig. 7), in analogy with the behaviour observed at lower temperatures. Lastly it is interesting to note that, whereas isotropic diffraction is expected for tiposomes, X-ray pictures show that preferential orientations of the diffracting planes can exist for high R values. Moreover the microscopic textures are typical of planar lamellar phases [15] for R >1 0.2: thus the liposomes are destroyed at high propranolol molar ratios. R > 1. Liposomes destruction (as above reported) has been also detected by Eriksson in a NMR study [7] on the interaction o f propranoiol with egg yolk lecithin vesicles in NaCI solutions. In particular he found that at propranoloi molar ratio R = 2.2 the sample becomes transparent and the vescicles are destroyed. At such high concentration the high angle X-ray diffraction patterns at room temperature are characterized by a broad peak, indicating that the chains are melted (Fig. 8). In the low angle patterns peak positions in s -~ units are in the ratios I:V~:2 (Fig. 8). These facts indicate [hat the iamellar organization has been destroyed and a hexagonal phase appears: the obtained cell parameter is a = 5.85 nm. For still higher concentrations (R = 4) the mixtures are transparent, highly viscous, optically anisotropic and the X-ray diffraction patterns indicate lameilar organization with melted chains also at room temperature. l
R=22
I 10"
T=303K
x 20*
20
Fig. 8. X-ray diffraction pattern obtained at R = 2.2, T = 303 K. Lower scale: take off angle 20 (%
These results are in agreement with those obtained in Ref. 7 in the case o f egg yolk lecithin: also in that case a clear and very viscous system was obtained for molar ratio greater than 2.2. Discussion The calorimetric results show that for R > 0.08 the chain melting temperature decreases considerably, indicating that the drug induces a larger fluidity in the bilayer. The disappearance of the La, ~ Pa, transition, which can be related to the interaction between the choline moiety and the bound water [19], could indicate that the drug interacts with the polar region o f the phospholipids; the lowering of the chain melting transition without enthalpy decrease suggests that propranolol interacts with the head group and only partially penetrates between the chains
[201.
uJ zv . -lwo~
I 0
i
i
i
25
5
75 MOLAR RAI]O
R
Fig. 7. L a m e l l a r r e p e a t s p a c i n g vs. m o l a r r a t i o R at the t e m p e r a t u r e T = 321 K.
The X-ray diffraction-data indicate that at room temperature and for molar ratio 0.8 ~< R 1 the chains are perpendicular to the iamellar planes, the chain distance slightly decreases and the lamellar spacing greatly increases with drug concentration (Fig. 7). The strong increase of the lamellar thickness cannot be attributed only to the loss of the chain tilt, inferred from high angle data, as the effect is present also in the L° phase, where the chains are melted. Thus the
336 increase o f the repeat distance, induced by the drug, should be related to an increase of the water sublayer thickness: as a consequence the effects of the propranoiol" HCI charged molecule are similar to those induced by c h l o r p r o m a z i n e ' H C l in excess water [21]. In general the capability of increasing water layer thickness can he related to the charge of the molecule; in fact charged molecules are reported to increase it, while neutral molecules do not 1211. The charged amphiphilic drug chlorpromazine. HCI, presenting calorimetric [22] and X-ray diffraction results [21] similar to those of propranolol, interacts with polar head groups, slightly penetrates into the hydrocarbon region of the bilayer and at high molar ratio (R = 0.5) induces a chain interdigitation [21]. It could be interesting to calculate the electron density profile across the bilayer in order to determine if also the propranolol induces a chain interdigitation and to find the position of the propranolol molecule in the bilayer; we are trying now to perform a swelling experiment to determine the phases of the reflections for the electron density map calculation: unfortunately at high water contents the first order peak, although visible in the X-ray picture, is too close to the direct beam and a clear intensity determination is not possible. Preliminar measurements at molar ratio R = 0.3 and at low water content indicate that the lipid bilayer thickness at relative humidity of 10o70 and 20°70 has an almost constant value of 3.3 _ 0.2 nm and that the repeat distances are 4.5 and 4.8 nm, respectively (three peaks were considered and the only possible choice, physically significant, of the reflections phases was 0, rt, n). Therefore, water addition induces an increase of the water sublayer thickness but leaves unchanged the lipid sublayer thickness; moreover both profiles have two density maxima in correspondence of the head groups of phospholipids and a secondary maximum at the centre of the bilayer, thus suggesting a chain interdigitation; this conclusion i~ also supported by the lipid thickness value, which is too small as compared to the lipid thickness of pure
DPPC with the chains fully extended and perpendicular to the lamellae, which is 4.9 nm [21]. The chain interdigitation could be also related to a small decrease of the chain distance shown in Fig. 6: it seems that the chain packing is tighter, in apparent disagreement with the increased bilayer fluidity inferred from calorimetric data. Probably the lipid bilayer is partially disordered owing to the presence of defects in the chain packing a n d / o r to some fluctuations of the interlayer distance value, due to the non-homogeneous distribution of the drug: in fact for R = 0.6 the second and third order peaks (Fig. 3a) have the half height widths larger than those of the pure DPPC, probably due to small variations of the periodicity. Moreover a correlation can also exist between the increased water layer thickness and the lowering of the transition temperature; in fact it was shown [23] that the neutral amphiphilic molecule of dicarboxilic azelaic acid interacts with the polar head-group and induces chain interdigitation, as chlorpromazine" HCI and propranolol" HCI do, but does not induce an increase of the water thickness: chain melting temperature is not affected.
Conclusions The effects of propranolol are detectable only if sufficient amount of the drug is present in the system (drug/lipid molar ratio R >t 0.08). The presence of the drug retains lamellar arrangement but imparts fluidity to the lipids, almost doubles the lamellar repeat spacing, increasing the water thickness, and induces a /J conformation of the chains, which thus become perpendicular to the lamellae and seem to be interdigitated; moreover the liposomes are destroyed and the bilayers are organized in multilamellar planar stacks. At molar ratio R = 2.2 a hexagonal phase is obtained, which is optically transparent, and for still higher molar ratios a very viscous lamellar phase is obtained. The calorimetric and X-ray results suggest that the p r o p r a n o l o l ' H C l molecule interacts with the phospholipid head groups and slightly penetrates into the hydrocarbon chain region.
337
At high propranoiol content the observed behaviour has many similarities with that observed by adding propranolol to unilamellar vesicles of egg lecithin in NaCl solutions [7]. Acknowledgements It is a pleasure to thank Mr. S. Polenta and Mr. M. Pergolini for valuable technical assistance. This work was supported by Regione Marche. Part of the equipment was obtained on the basis of financial help from CNR and MPI. One of the authors (R.S. Phadke) would like to thank ICTP Trieste, Italy , Programme for Training and Research in Italian Laboratories, for the support towards her stay in Italy. References 1 2 3 4 5 6 7 8
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