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Lipid perturbation of liposomal membrane of dipalmitoyl phosphatidylcholine by chloroquine sulphate — a fluorescence anisotropic study
COLLOIDS AND SURFACES ELSEVIER
B
Colloids and Surfaces B: Biointerfaces 4 (1995) 1 4
Lipid perturbation of liposomal membrane of dipalmitoyl phosphatidylcholine by chloroquine sulphate - - a fluorescence anisotropic study A.K. Ghosh, R. Basu, P. Nandy * Physics Department, Jadavpur University, Calcutta 700 032, India
Received 16 August 1993; accepted 15 June 1994
Abstract
Chloroquine sulphate (CQS) is a water-soluble anti-malarial drug having blood schizontocidal activity against Plasmodium falciparum, P. ovale, P. vivax and P. malariae. It has been reported that the lipid-soluble chloroquine base, which has similar anti-malarial and other biochemical effects, rigidities (orders) the liposomal membrane in vivo. Here, in order to examine the lipid perturbation mechanism of CQS, we have studied the effect of incorporation of this drug into the liposomal membrane of dipalmitoyl phosphatidylcholine as measured by the fluorescence polarization of the membrane-embedded probe, 1,6-diphenyl-l,3,5-hexatriene. Our results show that the drug CQS also rigidities the liposomal membrane even though the mechanism of the drug metabolism in vivo plays an important role. Probably the anti-malarial and other biochemical effects of CQS are exercised through this membrane rigidification. Keywords: Chloroquine sulphate; Drug bound lipid; Fluorescence spectroscopy; Lipid ordering; Membrane fluidity
1. Introduction
Chloroquine sulphate (CQS) is used for the suppression and clinical cure of malaria caused by susceptible strains of Plasmodium falciparum, P. ovale, P. vivax, and P. malariae in spite of having many adverse side-effects like haemolytic anaemia, agranulocytosis, etc. [1]. Lipid perturbation by this drug may be the mechanism responsible for its different biochemical effects, as it is known that the membrane lipid ordering affects the structural and functional integrities of the membrane proteins and receptors [2,3]. Chandra et al. have shown that the lipid-soluble * Corresponding author. 0927-7765/95/$09.50 © 1995 Elsevier Science B.V. All rights reserved SSDI 0 9 2 7 - 7 7 6 5 ( 9 4 ) 0 1 1 5 5 - X
chloroquine base has a stabilizing effect on the microsomal membrane in vivo [4], but the effect of water-soluble CQS on membrane physical properties has not been reported although CQS is used as a more effective drug. We report here for the first time the effect of CQS on the lipid-ordering profile in liposomal membranes of dipalmitoyl phosphatidylcholine (DPPC) as measured by the fluorescence polarization of the membrane-embedded probe 1,6-diphenyl-l,3,5-hexatriene (DPH). To gain a better understanding of the drug-lipid interaction at the molecular level we have also calculated the fraction of the lipid molecules that are motionally restricted due to binding with the drug molecules.
2
A.K. Ghosh et al./Colloids Surfaces B: Biointerfaces 4 (1995) 1-4
2. Materials and methods
a thermostatted temperatures.
cell maintained
at constant
2.1. Reagents
Spectral grade solvents (chloroform, methanol, acetone, and N-N'-dimethylformamide) were supplied by Merck, India. Dipalmitoyl phosphatidylcholine (DPPC) and 1,6-diphenyl-l,3,5-hexatriene (DPH) from Sigma were used without further purification. Chloroquine sulphate was a gift of the Central Drug Laboratory, Calcutta, India. 2.2. Preparation of drug-incorporated liposome
DPPC in chloroform was taken in a round bottomed flask and dried to a thin film by rotary evaporation (the vacuum was maintained for several hours to remove the residual solvent). Finally, the film was suspended in double-distilled water. The drug was added to the lipid suspension in molar ratios of 0.04, 0.08 and 0.12 with respect to lipid. The final concentration of lipid was 0.1 mM. The aqueous suspension of lipid and drug was then sonicated using an Imeco bath sonicator until the solution became almost clear. For better formation of liposomes, the temperature of the bath was maintained at 50°C, which is approximately 5°C above the phase transition temperature of DPPC in water [5-8]. 2.3. Fluorescence anisotropy measurement
DPH solution (25pl; 4 x 1 0 - 4 M in N-N'dimethylformamide) was added to the sonicated aqueous dispersion of liposomes (with/without the drug). The DPH : lipid molar ratio was maintained at a level of 1:200. A Perkin-Elmer fluorescence spectrometer (MPF 44B) was used to measure the steady-state fluorescence anisotropy r [5,6,8] r = (I1 --
12)/(I1 4- 2 1 2 )
where 11 and I 2 a r e the vertical and horizontal components of the 428 nm emission band of DPH in the liposomes while the sample is excited by the vertical component of light at 360 nm. In all the experimental set-ups, fluorescence intensities were calculated after properly eliminating the light scattering effect [8]. All measurements were made in
2.4. Estimation of the number of motionally restricted lipid molecules
Following the method of Houbre et al. [9] we have estimated the fraction of motionally restricted lipids, x = LB/L where LB and L are the number of lipid molecules bound to drug molecules and total lipid molecules respectively. Referring to the fluorescence anisotropy, x can be calculated as follows r' - r = x(r'b - rb) + (1 -- x)(r'f-- rf)
where r, rb and rf are, respectively, the values of anisotropy due to the total lipid, to the motionally restricted lipids associated with the drug molecules (LB), and to the remaining bulk of the lipid molecules not associated with the drug molecules, and the prime refers to these values at a lower temperature.
3. Experimental results and discussion In Fig. 1, we have plotted the temperature profile of the fluorescence anisotropy of DPH-probed DPPC liposome and monitored its change due to CQS incorporation. From the figure it is evident that the drug does not change the phase transition temperature T~ (the temperature corresponding to the point of inflexion). However the drugincorporated liposomes show a higher anisotropic value throughout the experimental temperature range, which increases with the drug concentrations, indicating a more rigid (ordered) membrane. The rigidifying effect is greater below the T~. It is established that the interaction between most of the hydrophilic compounds and the lipid bilayer membrane is controlled by non-specific charge-charge and hydrophobic interactions [ 10]. Here, the charge-charge interaction between the drug and the head group of the lipid molecules may lead to the adsorption of CQS molecules on the liposomal surface, and subsequently hinders the motion of the fatty acyl chains of DPPC, i.e. the membrane becomes more rigid. From the fact
A.K. Ghosh et al./Colloids Surfaces B: Biointe(faces 4 (1995) 1-4
0.35
Lipid:Drug o-1:0 n1 : 0.04 a1 ; 0.08 --
*
3
0.4 0.3
•
0.2 ~.
x 0.2
O I_
0.1
o
0
o,15
i 0
0.05
0.10
0.15
Drug : Lipid
0.05 30
1 35
I 1 40 45 Temperature
I 50
Fig. 1. Fluorencence anisotropy as a function of temperature in DPH-probed DPPC liposomes (concentration, 10-4 M). Molar ratio of lipid to drug: (©) 1:0; ([3) 1:0.04; (AI 1:0.08; (T) 1:0.12. that the membrane fluidity accounts for the antihaemolytic effect of general anaesthetics [-11], we may infer that the CQS-induced membrane rigidity may account for its haemolytic effect. An interesting point of observation is that this rigidifying effect due to drug incorporation is in contrast with the effect observed in the presence of other small molecules like cholesterol, where the anisotropy value decreases below the phase transition temperature. However, a similar rigidifying effect has been reported by us in the case of glyceryl trinitrate and pentaerythritol tetranitrate incorporated in D P P C liposomal membranes [-8]. In Fig. 2 we have plotted along the y-axis the calculated values of x, the fraction of motionally restricted lipid molecules, and along the x-axis the molar ratio of drug to lipid. The figure shows that as the drug concentration is increased, x increases and tends to a saturation value. This indicates that the drug molecules, due to their interaction with the phospholipid head groups, increase the molecular packing of fatty acyl chains of the lipid molecules. The process continues as long as there are accessible phospholipid head groups. Thus it can be stated that hydrophilic CQS
Fig. 2. Variation of the fraction of motionally restricted lipids (x) with respect to the molar ratio of lipid to drug. rigidities the liposomal membrane in vitro, as does lipophilic chloroquine base in vivo [4], even though their modes of interaction are different due to the difference in the mechanism of drug metabolism as well as their solubilities in the lipid matrix. Our experimental results indicate that in spite of being an effective drug for the treatment of malaria, CQS should have a restricted use in the case of drug-resistant malaria where membrane impermeability is a major problem. This in vitro study has an important physical significance as it can provide support in favour of a lipid perturbation mechanism for different biochemical effects exerted by CQS. However, as there is a great deal of difference between the drug metabolism in vitro and in vivo, the results obtained here may be extrapolated to in vivo systems with some limitations.
Acknowledgements We are thankful to A. Ghoroi, Dr. A. Nandy and Dr. P.C. Sen for their help. We have obtained the instrumental facilities from RSIC of the Bose Institute, Calcutta. Financial support from CSIR, Government of India, is gratefully acknowledged.
References [1] LE.F. Reynolds (Ed.), MARTINDALE- The Extra Pharmacopoeia, 29th edn., The Pharmaceutical Press, London, 1989, p. 508.
4
A.K. Ghosh et aL/Colloids Surfaces B: Biointerfaces 4 (1995) 1-4
[2] D.K. Lawrence and E.W. Gill, Mol. Pharmacol., 11 (1975) 595. [3] H.H. Loh and P.Y. Law, Ann. Rev. Pharmacol. Toxicol., 20 (1980) 201. [4] S. Chandra, G. Adhikary, R. Sikdar and P.C. Sen, Mol. Cell. Biochem., 118 (1992) 15. [-5] M. Bhattacharya and P. Nandy, Arch. Biochem. Biophys., 275 (1989) 379. [6] M. Bhattacharya, B.B. Bhowmik and P. Nandy, Arch. Biochem. Biophys., 263 (1988) 117.
[7] M. Bhattacharya, M. Sarkar and P. Nandy, J. Surf. Sci. Technol., 7 (1991) 323. [8] A.K. Ghosh, J. Mukherjee, R. Basu, M. Bhattacharya and P. Nandy, Biochim. Biophys. Acta, 1153 (1993) 20. [9] D. Houbre, P. Schindler, E. Trifilieff, B. Luu and G. Duportail, Biochim. Biophys. Acta, 1029 (1990) 136. [10] A.G. Lee, Biochim. Biophys. Acta, 472 (1977) 285. [11] P. Seeman, T. Sauks, W. Argent and W.O. Kwant, Biochim. Biophys. Acta, 183 (1969) 476.
B
Colloids and Surfaces B: Biointerfaces 4 (1995) 1 4
Lipid perturbation of liposomal membrane of dipalmitoyl phosphatidylcholine by chloroquine sulphate - - a fluorescence anisotropic study A.K. Ghosh, R. Basu, P. Nandy * Physics Department, Jadavpur University, Calcutta 700 032, India
Received 16 August 1993; accepted 15 June 1994
Abstract
Chloroquine sulphate (CQS) is a water-soluble anti-malarial drug having blood schizontocidal activity against Plasmodium falciparum, P. ovale, P. vivax and P. malariae. It has been reported that the lipid-soluble chloroquine base, which has similar anti-malarial and other biochemical effects, rigidities (orders) the liposomal membrane in vivo. Here, in order to examine the lipid perturbation mechanism of CQS, we have studied the effect of incorporation of this drug into the liposomal membrane of dipalmitoyl phosphatidylcholine as measured by the fluorescence polarization of the membrane-embedded probe, 1,6-diphenyl-l,3,5-hexatriene. Our results show that the drug CQS also rigidities the liposomal membrane even though the mechanism of the drug metabolism in vivo plays an important role. Probably the anti-malarial and other biochemical effects of CQS are exercised through this membrane rigidification. Keywords: Chloroquine sulphate; Drug bound lipid; Fluorescence spectroscopy; Lipid ordering; Membrane fluidity
1. Introduction
Chloroquine sulphate (CQS) is used for the suppression and clinical cure of malaria caused by susceptible strains of Plasmodium falciparum, P. ovale, P. vivax, and P. malariae in spite of having many adverse side-effects like haemolytic anaemia, agranulocytosis, etc. [1]. Lipid perturbation by this drug may be the mechanism responsible for its different biochemical effects, as it is known that the membrane lipid ordering affects the structural and functional integrities of the membrane proteins and receptors [2,3]. Chandra et al. have shown that the lipid-soluble * Corresponding author. 0927-7765/95/$09.50 © 1995 Elsevier Science B.V. All rights reserved SSDI 0 9 2 7 - 7 7 6 5 ( 9 4 ) 0 1 1 5 5 - X
chloroquine base has a stabilizing effect on the microsomal membrane in vivo [4], but the effect of water-soluble CQS on membrane physical properties has not been reported although CQS is used as a more effective drug. We report here for the first time the effect of CQS on the lipid-ordering profile in liposomal membranes of dipalmitoyl phosphatidylcholine (DPPC) as measured by the fluorescence polarization of the membrane-embedded probe 1,6-diphenyl-l,3,5-hexatriene (DPH). To gain a better understanding of the drug-lipid interaction at the molecular level we have also calculated the fraction of the lipid molecules that are motionally restricted due to binding with the drug molecules.
2
A.K. Ghosh et al./Colloids Surfaces B: Biointerfaces 4 (1995) 1-4
2. Materials and methods
a thermostatted temperatures.
cell maintained
at constant
2.1. Reagents
Spectral grade solvents (chloroform, methanol, acetone, and N-N'-dimethylformamide) were supplied by Merck, India. Dipalmitoyl phosphatidylcholine (DPPC) and 1,6-diphenyl-l,3,5-hexatriene (DPH) from Sigma were used without further purification. Chloroquine sulphate was a gift of the Central Drug Laboratory, Calcutta, India. 2.2. Preparation of drug-incorporated liposome
DPPC in chloroform was taken in a round bottomed flask and dried to a thin film by rotary evaporation (the vacuum was maintained for several hours to remove the residual solvent). Finally, the film was suspended in double-distilled water. The drug was added to the lipid suspension in molar ratios of 0.04, 0.08 and 0.12 with respect to lipid. The final concentration of lipid was 0.1 mM. The aqueous suspension of lipid and drug was then sonicated using an Imeco bath sonicator until the solution became almost clear. For better formation of liposomes, the temperature of the bath was maintained at 50°C, which is approximately 5°C above the phase transition temperature of DPPC in water [5-8]. 2.3. Fluorescence anisotropy measurement
DPH solution (25pl; 4 x 1 0 - 4 M in N-N'dimethylformamide) was added to the sonicated aqueous dispersion of liposomes (with/without the drug). The DPH : lipid molar ratio was maintained at a level of 1:200. A Perkin-Elmer fluorescence spectrometer (MPF 44B) was used to measure the steady-state fluorescence anisotropy r [5,6,8] r = (I1 --
12)/(I1 4- 2 1 2 )
where 11 and I 2 a r e the vertical and horizontal components of the 428 nm emission band of DPH in the liposomes while the sample is excited by the vertical component of light at 360 nm. In all the experimental set-ups, fluorescence intensities were calculated after properly eliminating the light scattering effect [8]. All measurements were made in
2.4. Estimation of the number of motionally restricted lipid molecules
Following the method of Houbre et al. [9] we have estimated the fraction of motionally restricted lipids, x = LB/L where LB and L are the number of lipid molecules bound to drug molecules and total lipid molecules respectively. Referring to the fluorescence anisotropy, x can be calculated as follows r' - r = x(r'b - rb) + (1 -- x)(r'f-- rf)
where r, rb and rf are, respectively, the values of anisotropy due to the total lipid, to the motionally restricted lipids associated with the drug molecules (LB), and to the remaining bulk of the lipid molecules not associated with the drug molecules, and the prime refers to these values at a lower temperature.
3. Experimental results and discussion In Fig. 1, we have plotted the temperature profile of the fluorescence anisotropy of DPH-probed DPPC liposome and monitored its change due to CQS incorporation. From the figure it is evident that the drug does not change the phase transition temperature T~ (the temperature corresponding to the point of inflexion). However the drugincorporated liposomes show a higher anisotropic value throughout the experimental temperature range, which increases with the drug concentrations, indicating a more rigid (ordered) membrane. The rigidifying effect is greater below the T~. It is established that the interaction between most of the hydrophilic compounds and the lipid bilayer membrane is controlled by non-specific charge-charge and hydrophobic interactions [ 10]. Here, the charge-charge interaction between the drug and the head group of the lipid molecules may lead to the adsorption of CQS molecules on the liposomal surface, and subsequently hinders the motion of the fatty acyl chains of DPPC, i.e. the membrane becomes more rigid. From the fact
A.K. Ghosh et al./Colloids Surfaces B: Biointe(faces 4 (1995) 1-4
0.35
Lipid:Drug o-1:0 n1 : 0.04 a1 ; 0.08 --
*
3
0.4 0.3
•
0.2 ~.
x 0.2
O I_
0.1
o
0
o,15
i 0
0.05
0.10
0.15
Drug : Lipid
0.05 30
1 35
I 1 40 45 Temperature
I 50
Fig. 1. Fluorencence anisotropy as a function of temperature in DPH-probed DPPC liposomes (concentration, 10-4 M). Molar ratio of lipid to drug: (©) 1:0; ([3) 1:0.04; (AI 1:0.08; (T) 1:0.12. that the membrane fluidity accounts for the antihaemolytic effect of general anaesthetics [-11], we may infer that the CQS-induced membrane rigidity may account for its haemolytic effect. An interesting point of observation is that this rigidifying effect due to drug incorporation is in contrast with the effect observed in the presence of other small molecules like cholesterol, where the anisotropy value decreases below the phase transition temperature. However, a similar rigidifying effect has been reported by us in the case of glyceryl trinitrate and pentaerythritol tetranitrate incorporated in D P P C liposomal membranes [-8]. In Fig. 2 we have plotted along the y-axis the calculated values of x, the fraction of motionally restricted lipid molecules, and along the x-axis the molar ratio of drug to lipid. The figure shows that as the drug concentration is increased, x increases and tends to a saturation value. This indicates that the drug molecules, due to their interaction with the phospholipid head groups, increase the molecular packing of fatty acyl chains of the lipid molecules. The process continues as long as there are accessible phospholipid head groups. Thus it can be stated that hydrophilic CQS
Fig. 2. Variation of the fraction of motionally restricted lipids (x) with respect to the molar ratio of lipid to drug. rigidities the liposomal membrane in vitro, as does lipophilic chloroquine base in vivo [4], even though their modes of interaction are different due to the difference in the mechanism of drug metabolism as well as their solubilities in the lipid matrix. Our experimental results indicate that in spite of being an effective drug for the treatment of malaria, CQS should have a restricted use in the case of drug-resistant malaria where membrane impermeability is a major problem. This in vitro study has an important physical significance as it can provide support in favour of a lipid perturbation mechanism for different biochemical effects exerted by CQS. However, as there is a great deal of difference between the drug metabolism in vitro and in vivo, the results obtained here may be extrapolated to in vivo systems with some limitations.
Acknowledgements We are thankful to A. Ghoroi, Dr. A. Nandy and Dr. P.C. Sen for their help. We have obtained the instrumental facilities from RSIC of the Bose Institute, Calcutta. Financial support from CSIR, Government of India, is gratefully acknowledged.
References [1] LE.F. Reynolds (Ed.), MARTINDALE- The Extra Pharmacopoeia, 29th edn., The Pharmaceutical Press, London, 1989, p. 508.
4
A.K. Ghosh et aL/Colloids Surfaces B: Biointerfaces 4 (1995) 1-4
[2] D.K. Lawrence and E.W. Gill, Mol. Pharmacol., 11 (1975) 595. [3] H.H. Loh and P.Y. Law, Ann. Rev. Pharmacol. Toxicol., 20 (1980) 201. [4] S. Chandra, G. Adhikary, R. Sikdar and P.C. Sen, Mol. Cell. Biochem., 118 (1992) 15. [-5] M. Bhattacharya and P. Nandy, Arch. Biochem. Biophys., 275 (1989) 379. [6] M. Bhattacharya, B.B. Bhowmik and P. Nandy, Arch. Biochem. Biophys., 263 (1988) 117.
[7] M. Bhattacharya, M. Sarkar and P. Nandy, J. Surf. Sci. Technol., 7 (1991) 323. [8] A.K. Ghosh, J. Mukherjee, R. Basu, M. Bhattacharya and P. Nandy, Biochim. Biophys. Acta, 1153 (1993) 20. [9] D. Houbre, P. Schindler, E. Trifilieff, B. Luu and G. Duportail, Biochim. Biophys. Acta, 1029 (1990) 136. [10] A.G. Lee, Biochim. Biophys. Acta, 472 (1977) 285. [11] P. Seeman, T. Sauks, W. Argent and W.O. Kwant, Biochim. Biophys. Acta, 183 (1969) 476.