Effect of lanthanide ions on the phase behavior of dipalmitoylphosphatidylcholine multilamellar liposomes

Effect of Lanthanide Ions on the Phase Behavior of Dipalmitoylphosphatidylcholine Multilamellar Liposomes Xin-min Li, Ya-fei Zhang, Jia-zuan Ni, Jian-wen Chen, and Fen Hwang XL, YZ, JN . Laboratory of Rare Earth Chemistry and Physics, Changchun Institute of Applied Chemistry, Academia Sinica, Changchun, P . R. China .-JC, FH. Department of Biomembranes, Institute of Biophysics, Academia Sinica, Beijing, P . R China

ABSTRACT The effect of lanthanide ions (Ln 3+ ) and their coordination compounds of diethylenetriamine pentaacetic acid (DTPA) on the phase behavior of dipalmitoylphosphatidycholine (DPPC) multilamellar liposomes has been studied by differential scanning calorimetry (DSC), Raman spectroscopy, and freeze-fracture electron microscopic techniques . The displacement of Ca t+ binding on DPPC liposomes by lanthanide ions was also studied . The results show that the binding degree of four kinds of chloride salts with DPPC liposomes is : YbC13 > GdC1 3 > LaC1 3 > CaCl2 . Lanthanide ions increase the phase transition temperature of DPPC liposomes and decrease the membrane fluidity . Freeze-fracture electron microscopic results show that La 3+ enhances the DTPA on order of DPPC membrane . The effect of coordination compounds of lanthanides with , , the phase behavior of DPPC liposomes is smaller than that of their chlorides . La3+ Gd3+ and Yb3+ , can displace Ca t+ binding on DPPC liposomes, but there coordination compounds of DTPA can hardly displace Ca t+. Raman spectroscopic results show that a very slight effect in lateral packing order of DPPC liposomes was observed at various concentrations of lanthanides . Abbreviations DPPC, dipalmitoylphosphatidylcholine ; DSC ; differential scanning calorimetry; EDTA ; ethylene; diamine tetraacetic acid ; DTPA ; diethylenetriamine pentaacetic acid ; Ln3+ lanthanide ion.

Address reprint requests and correspondence to : Dr. X-m . Lin, Lab 5a, Laboratory of Rare Earth Chemistry and Physics, Changchun Institute of Applied Chemistry, Academia Sinica, Changchun 130022, Peoples Republic of China . Journal of Inorganic Biochemistry, 53, 139-149 (1994) © 1994 Elsevier Science Inc., 655 Avenue of the Americas, NY, NY 10010

139 0162-0134/94/$7.00

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INTRODUCTION The lanthanide series of elements comprises the 15 elements between lanthanum and lutetium in the periodic table . Much of the interest in lanthanide biochemistry flows from the ability of lanthanide ions to replace Ca 21 in a specific and often isomorphous manner [1]. It is well-known that Ca" plays a very important role in living cells, such as muscle contraction, gating of ionic channels, fertilization of oocytes, and as a secondary messenger following hormone action on the cell membrane [2, 3] . The functional changes in a molecule upon substituting Ca" by lanthanide ions help in ascribing roles to Ca 21 in the native structure [4] . It has been reported that the primary site of interaction of lanthanide ions with living cells is at the cytoplasma membranes [5]. Due to the complexity of biological membranes, artificial membranes have been widely used to study the interaction between phospholipids and metallic ions [6]. So far, the mechanism of action of lanthanide ions on biomembranes is an intriguing question [7-10]. It has been reported that lanthanide ions form 1 :1 complexes with the small molecules that make up the phosphotidylcholine polar group [11] ; they form 2 :1 complexes with vesicular phosphatidylcholine, this stoichiometry reflects the chelation of one lanthanide ion by two phosphate groups [12]. Binding of lanthanides is independent of pH between 3 and 7 but is enhanced by various anions in the order : Cl - < Br < NO - < SCN < I - < C' 0 4 [13] . In the present study, we have investigated the interaction of lanthanide ions and their coordinate compounds of DTPA with dipalmitoylphosphatidylcholine (DPPC) liposomes . Lanthanide ions can stabilize DPPC membranes and increase membrane rigidity . MATERIALS AND METHODS Chemicals Dipalmitoylphosphatidylcholine (DPPC) was purchased from Fluka (Switzerland) without further purification . A single spot was shown by thin-layer chromatography . LaCl 3 ; GdCl 3 and YbC1 3 were prepared by dissolving their oxides (99 .99%) in HCl with elimination of excess acid, and the concentration was determined by ethylenediamine tetraacetic acid (EDTA) ; the coordination compounds of Ln 3+ and Ca 21 with DTPA were prepared by the reaction of their oxides with DTPA ; the composition proved by elemental analysis was : CaC14 H 22 O 10 N3 , LaC 14 H 22O 111 N3 , GdC 14 H 22 O 10 N3 , and YbC t4 H 22 O 111 N3 . All other reagents were of analytical grade . Preparation of Multilamellar Liposomes DPPC was dissolved in CHC1 3 and vacuum desiccated overnight to remove traces of solvent . The lipid films were suspended in Tris-HCl buffer (pH 7 .0) and sonicated at about 50°C for 10 min and lanthanide was added, the lipid was sonicated for 10 min again and then concentrated to a final concentration of DPPC (120 mg/ml) for DSC and Raman spectroscopic determination . Pure lipid liposomes were used as control . Differential Scanning Calorimetry The samples were examined in sealed pans with empty pans as reference on a Perkin-Elmer DSC-2C differential scanning calorimeter supplemented with In-

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tracooler II . Temperature was calibrated with indium (P-E standard) . Scans were obtained with : range setting 1 mcal/sec, heating rate 5 K/min, nitrogen atmosphere . The transition temperature Tm was expressed by peak maximum . Raman Spectroscopy Raman spectra were recorded with a SPEX-1403 double monochromatic spectrometer . An argon ion laser was provided at the sample operating at 100 mw and 514 .5 nm ; slit width, 400 gm ; scanning rate, 0.5 cm - '/sec . All measurements were at 20°C . Freeze-fracture Electron Microscopy Free-fracture electron microscopy was performed according to an established procedure [14]. Prior to freeze-fracturing, lanthanide was added into the liposomes suspension with a final concentration and the suspension equilibrated for two h at the desired temperature . A small volume of sample was pipetted into a specimen block maintained at the same temperature, and quenched rapidly in the liquid nitrogen . Specimens were fractured with a cooled knife, and shadowed with platinum-carbon at a vacuum of 5 x 10 -5 torr. The replicas were cleaned in chloroform-methanol, picked up on copper grids, and examined in a JEM-100CX electron microscope . RESULTS Differential Scanning Calorimetry Interaction of Lanthanide and Calcium Ions with DPPC Liposomes . In agreement with that previously reported [15], pure DPPC multilamellar liposomes exhibited a sharp gel-liquid crystal phase transition temperature at 315 .2 K with pretransition temperature at 308 .8 K. The addition of LaC1 3 , GdC1 3 , YbCl 3 , and CaCl 2 leads to a drastic effect on the the DSC heating curves shown in Figure 1 . Lanthanide and calcium ions increase the phase transition temperature of DPPC, causing the pretransition peak of DPPC liposomes to disappear . LaC1 3 , GdCl 3 , and YbCl 3 all induce a phase separation of DPPC liposomes and form two peaks, the peaks in the lower temperature region of lanthanides/DPPC profile are the phase transition peaks of DPPC as a major component and the new peaks probably mainly represent the Ln 3+ -DPPC complexes . But the phase separation of DPPC liposomes only occurs at a higher concentration of CaCl 2 . With the increase in lanthanide and calcium concentration, the phase transition temperature (Tm) shifts to higher temperature, from 0 .5 K to 10 .5 K (Fig.1, Table 1). The addition of CaC1 2 , LaC1 3, GdCl 3 , and YbCl 3 causes the peak to broaden, and the peak shows an apparently poorer cooperativity than any one due to the addition of YbCl 3 . The effect of four kinds of chloride salts on membrane fluidity of DPPC liposomes are in the following order : YbCl 3 > GdC1 3 > LaCl 3 > CaCl 2 . As the concentration of lanthanide and calcium ions increases, the phase transition temperature Tm continues to increase, a sudden change appears (Fig . 2) and then increases only to a certain limit ; this suggests that the interaction of lanthanide and calcium ions with DPPC liposomes is near saturation, namely, they have nearly occupied the binding sites of DPPC liposomes .



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310 T K 320 310 T K 320 310 T K 320 310 T K320 330 FIGURE 1 . Differential scanning calorimetry curves showing the effect of different concentrations of lanthanides and calcium in molar ratios LnCl 3 /DPPC and CaC1 2 /DPPC on the gel to liquid-crystalline transition of DPPC liposomes . (A) CaC1 2 ; (B) LaC1 3 ; ( C) GdCl 3 ; (D) YbCl 3 . ( a) 0; (b) 0 .05; (c) 0 .1 ; (d) 0 .25 ; (e) 0 .5 .

Interaction of the Coordination Compounds of Ln 3+-DTPA and Ca z+-DTPA with DPPC Liposomes. Table 2 indicates that the effect of DTPA on the phase transition temperature of DPPC liposomes is not distinct . The effect of Ca z+ DTPA, La3+ -DTPA, Gd 3+ -DTPA, and Yb 3+-DTPA on the phase transition behavior of DPPC liposomes is smaller than their chlorides . Competition of Lanthanide Ions and Their Coordination Compounds of DTPA with Ca t+ Binding on DPPC Liposomes. When the interaction of Ca 21 with DPPC liposomes is near saturation and different amounts of LaC1 3 , GdCl 3 , and YbC1 3 are added, the resulting DSC curves (Fig . 3) are significantly different from that

TABLE 1 . Effect of CaC1 2 and LnCl 3 Concentrations of Phase Transition Temperature Tm of DPPC Liposomes Molar Ratios (sample/DPPC) 0 Sample CaC1 2 LaCI 3 GdC1 3 YbCI 3 n = 3 -4.

0.05

0.1

0 .25

0.5

Phase Transition Temperature Tm(K) 315 .2 ± 0 .2 315 .2±0 .2 315 .2±0 .2 315 .2±0 .2

315 .7±0.1 317 .6±0.2 317 .7±0.2 319 .5±0.1

316 .3±0.2 318 .6±0 .1 319 .1±0.1 322.0±0 .2

317 .1±0.1 319 .1 ±0 .1 319 .9±0.3 324 .6±0.1

317 .4±0 .2 319.6 ± 0.3 320 .2±0.1 325 .7±0.2

EFFECT OF LANTHANIDES ON DPPC

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FIGURE 2 . Effect of different molar ratios LnC1 3 /DPPC and CaC1 2/DPPC on the phase transition temperature of DPPC liposomes. (a) CaC1 2 ; (b) LaC1 3 ; (c) GdC13; (d) YbC1 3 .

0

of CaCl2 binding on DPPC liposomes, and they are very similar to the DSC curves of LaC1 3 , GdC1 3 , and YbC1 3 binding in DPPC liposomes (Fig. 1). Phase separation is obvious and the phase transition temperature increases, these indicate that La 3 +, Gd 3 +, and Yb 3 + have displaced Ca t+ binding on DPPC liposomes . The results are due to the stronger combination power of La 31 , Gd 3 +, and Yb 3 + than that of Cat + . La3 +-DTPA, Gd 3 +-DTPA, and Yb 3 +-DTPA have little effect on Ca t + binding on DPPC liposomes (Fig . 4), this further suggests that their effect on DPPC liposomes becomes smaller when lanthanide ions have formed stable coordination compounds with strong ligands . Raman Spectroscopy Interaction of LaCl3 and GdC13 with DPPC Liposomes. Raman spectra are studied in the C-H stretching vibration (2800-2950 cm - ') and the C-C stretch-

TABLE 2 . Effect of DTPA, Ca t+ -DTPA, and Ln 3+ -DTPA Concentrations on Phase Transition Temperature Tm of DPPC Liposomes Molar Ratios (sample/DPPC) 0 Sample DTPA Ca 2 +-DTPA La 3 +-DTPA Gd 3+-DTPA Yb 3 +-DTPA n = 3- 4.

0.5

1.0

Phase Transition Temperature Tm(K) 315.2±0.2 315 .2 t 0 .2 315.2 t 0 .2 315.2 ± 0 .2 315.2±0.2

315.6±0.1 316.1 t 0 .1 316.1 ± 0 .2 315.4±0.1 315 .3 ± 0 .1

315.3±0.3 316 .4 ± 0 .3 316.7 ± 0 .1 315.5 ± 0 .2 315.5±0.2



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Differential scanning calorimetry curves showing the effect of different concentrations Ln 3 +-DTPA and calcium in molar ratios Ln 3+ -DTPA/Ca 21 on the gel to liquid-crystalline transition of DPPC liposomes. (A) La 3+ -DTPA/ Ca 2+ . (B) Gd 3+ -DTPA/Ca 2 ; (C) FIGURE 4.

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In order to investigate the comformational change of DPPC liposomes with LaCl 3 and GdC1 3 , we have studied the Raman spectra of DPPC (solid), DPPC liposomes, La 3 +-DPPC, and Gd3+-DPPC at 20°C (Fig . 5). It can be seen that there is little direct effect of LaC1 3 and GdC1 3 on the C-C chain of DPPC liposomes from Table 3 . When the molar ratio of LaCl 3 or GdCl 3 with DPPC liposomes was 0 .1, there were no significant variation in 128822848 and S L . This indicates that low concentrations of LaC1 3 or GdC1 3 make the lateral coupling between inter-chains of DPPC liposomes change very little, the lateral packing disorders, and the membrane mobility changes slightly . But when the molar ratio is 0.5, 12882/2848 and S L slightly increases, the degree of order of the

TABLE 3 . Effect of Lanthanides on C-C and C-H Stretching Vibrations of DPPC Liposomes Parameter System DPPC(S) DPPC(L) LaC13/DPPC = 0 .1 GdCl 3 /DPPC = 0.1 LaCI 3/DPPC = 0.5 GdC1 3 /DPPC=0 .5

1 1095/ , 1124

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SL

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0.35 0.23 0.23 0.24 0.25 0.27

The data are averages of at least three measurements . The standard error of the mean of all intensity ratios was smaller than ±0 .01 .



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(a)

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FIGURE 6. Platinum-carbon replicas of liposome fracture faces quenched from 45° C . (A) DPPC liposomes x 30,000 ; (B) molar ratio : LaC1 3 /DPPC = 2 :1 x 30,000.

hydrocarbon chains increases a little, the membrane fluidity decreases slightly . This has the same effect as CaCI 2 on the hydrocarbon chains of DPPC liposomes [18]. Freeze-Fracture Electron Microscopy When DPPC liposomes are quenched from 45°C (above the phase transition temperature), a jumbled and irregular pattern (liquid crystal-line phase) can be observed on the fracture face (Fig . 6), and LaCl 3 is added into DPPC liposomes (molar ratio: LaC1 3/DPPC = 2 :1); after equilibrium at 45°C, the regular ripple repeat patterns on the fracture faces can be seen (Fig . 6) . These regular patterns are presumably related in some way to the crystal packing of the phospholipid molecules upon passing from fluid to gel phase [19] . This further indicates that LaC1 3 increases the order of the phospholipid molecules while the membrane fluidity decreases .

DISCUSSION It has been reported that changes in torsion angles of the phosphate segment and choline methylene groups induced by La" ions result in a conformational change of the choline head group of DPPC [20-22] . X-ray data indicate that a molar ratio of DPPC/La 3+ of 3 :1, there is a change in acyl chain from the chain title of DPPC relative to the normal, to the bilayer plan is lost [23]. High-sensitivity differential scanning calorimetry studies have demonstrated that the interaction of La" with DPPC causes an increase in transition temperature and alters the polar head group conformation [24]. Our results indicate that Ln 3+ ions increase the phase transition temperature of DPPC liposomes . The effect of three kinds of LnCl 3 on the membrane fluidity of DPPC liposomes are in the following order : YbC1 3 > GdC1 3 > LaC1 3 . Apparently, the ionic radii of heavy

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lanthanides is smaller, the ionic potential is greater, and the ionic binding strength is greater . All three kinds of Ln 3 + induce the phase separation of DPPC liposomes . This suggests the efficacy of interaction is dependent on the polarity of DPPC molecules through the polar-polar interaction . There is significant effect of Yb 3+ -DTPA, Gd 3+ -DTPA, and La3+ -DTPA on membrane fluidity of DPPC liposomes . The ionic radii of Ca t+ and Ln 3 + are similar while the electric charges and the ionic potential of Cat+ are smaller than that of Ln3 + . La3 +, Gd3 + and Yb 3 + can displace Ca 2+ binding on DPPC liposomes, but Ln3 +-DTPA cannot . Only Ln3 + ions, but not their coordination compounds of DTPA, can change the phase behavior of DPPC liposomes . Through monitoring the intensity alterations of vibrational modes in C-H stretching modes 2800-3000 cm - ' and C-C stretching modes 1000-1200 cm -1 regions of phospholipid acyl chains, it has been noticed that there is little direct effect of La3+ and Gd3+ on the hydrocarbon chains of DPPC liposomes . Since La3 + and Gd 3 + might interact with polar head P0 2 - of DPPC liposomes, it is plausible that the anomalously high values of S T for various ions are to be ascribed to the interaction between ions and the zwitter-ionic polar head of phosphatidylcholine ; such interaction certainly may affect the polarizability of the phosphoryl moiety, and are reflected in the Raman spectra of lipids as changes in the intensities of 1095 cm - ' band which include P0 2 - symmetric stretching modes and gauche rotation [25]. Hence 11095/11124 and ST cannot give the adequate information of the degree of order . In the C-H stretching mode region, La3+ and Gd3+ have only a very slight effect in the lateral packing order of DPPC liposomes ; it indicates that the order is probably due to secondary (perhaps head-group mediated) interaction, and ion head interaction may equally be the cause of the apparent changes observed in DSC studies . During the past twenty years, freeze-fracture electron microscopy is used to study the phase transition in phospholipid liposomes . Certain pure phosphatidylcholines which form lipid bilayers in the presence of water offer interesting possibilities for model membrane lateral distribution studies because they can display characteristic, highly regular surface patterns in freeze-fracture electron micrographs [26-29] . These regular patterns are presumably related in some way to the crystal packing of the phospholipid molecules upon passing from fluid to gel phase [30]. Regular patterns are not present in freeze-fracture electron micrographs when lipid samples are quenched from above their phase transition temperature [28, 29], instead, the lipid surface takes on a jumbled or even smooth appearance depending upon the quenching rate . LaCl 3 is added into DPPC liposomes (molar ratio : LaC1 3 /DPPC = 2:1), ripple repeat patterns appear at 45°C ; this indicates that the phase transition temperature of DPPC liposomes increases when the liposomes were prepared from the mixture of DPPC and LaC1 3 ; the order of the phospholipid increases while the membrane fluidity decreases . With extensive application of lanthanides in agriculture, clinical practice, etc . [31, 32], the study of the binding mechanism of lanthanides in biological study has become a field of intense interest, and the competition of lanthanide ions with Ca 2+ is also much concerned . A number of works on macrotoxicology of lanthanides had been made [33], but only a few are related to the effect of lanthanides on biomembranes [5, 13] . We have reported the effect of lanthanide ions on the phase behavior of DPPC liposomes in order to understand the

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molecule mechanism of the interaction of lanthanides with biomembranes ; further investigation is in progress . This project was supported by the National Natural Science Foundation of China . We wish to thank Li-xia Li and Dong-mei Li for measurement of DSC and Raman spectroscopic experiments .

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27. D . J. Fluck, A. F. Henson, and D. Chapman, J. Ultrastruct. Res. 29, 416 (1969). 28. P. Pinto da Silva, J. de Microsc . 12, 185 (1971). 29. P. H. J. Ververgaert, A . J . Verkleij, J. J. Verhoeven, and P . F. Elbers, Biochim . Biophys . Acta 311, 651 (1973) . 30. A. Tardieu, V . Luzzati, and F. C. Reman, J. Mol. Biol. 75, 711 (1973). 31 . C. H. Evans, in Biochemistry of the Lanthanides, Earl Frieden, Ed ., Plenum, New York and London, 1990, pp. 391-427 . 32 . R. B . Lauffer, Chem. Rev . 87, 901 (1987). 33. C. H. Evans, in Biochemistry of the Lanthanides, Earl Frieden, Ed ., Plenum, New York and London, 1990, pp. 339-390 . Received October 22, 1992; accepted March 23, 1993