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Effect of amino acids, polypeptides and proteins on electrophoretic mobilities of phospholipid liposomes
Colloids and Surfaces A: Physicochemical and Engineering Aspects 92 (1994) 87-93
!i#.iLorDs A SURFACES
Effect of amino acids, polypeptides and proteins on electrophoretic mobilities of phospholipid liposomes Hideo Matsumura a,*, Fumiko Mori b, Kiyoko Kawahara b, Chiharu Obata b, Kunio Furusawa b a Electrotechnical Laboratory, AIST, MITI, Tsukuba 305, Japan b The University of Tsukuba, Tsukuba 305, Japan Received 2 December
1993; accepted
28 January
1994
Abstract The electrophoretic mobilities of liposomes prepared from egg phosphatidylcholine (PC) immersed in solutions which contain amino acids, polypeptides or proteins have been studied. Amino acids (lysine - a basic amino acid,
or glutamic acid - an acidic amino acid) cause a pH variation which changes the acid-base dissociation equilibria of charged compounds in the PC membranes. Polypeptides (poly(L-lysine) - a cationic polymer and poly(L-glutamic acid) - an anionic polymer) can adsorb onto the membrane surface of the PC liposomes because of their high affinity for the membrane, which causes significant changes in the electrophoretic mobilities towards more positive values in the case of poly(L-lysine) and causes slight negative shifts in the case of poly(L-glutamic acid). Proteins (serum albumin: isoelectric point, pH 4.9; and cytochrome c: isoelectric point, pH 10.1) adsorb on the membrane surface, which decreases the negative value of the electrophoretic mobilities in both cases. Furthermore, both proteins shift the zero point of zeta potential of the lipid liposomes towards the isoelectric point of the proteins. Keywords: Electrophoresis;
Liposomes; Protein adsorption; Polypeptides
1. Introduction
The interaction of proteins with lipid membranes is an important subject from the point of view of fundamental principles and their applications. Many studies on the membrane-protein interaction concerning membrane fusion have been carried out. The study of protein-mediated fusion is one of the highlights of cell biology. However, the fusion mechanism is still obscure, and many more fundamental studies concerning the various aspects are required [l] (see also a review by Bentz and Ellens [2]). * Corresponding author. 0927-7757/94/$07.00 0 1994 Elsevier Science B.V. All rights reserved SSDI 0927-7757(94)02785-Q
Artificial lipid liposomes and natural vesicles in cells encounter various types of proteins and other biological substances in blood, extracellular fluid and cytoplasm. For example, liposomes acting as drug-carriers flowing in the blood stream encounter serum proteins, etc. until they reach the diseased part, and vesicles in the cytoplasm operating as transporters of nourishment must pass through the cytosol, which contains various biological substances, before their fusion with a cytomembrane. Therefore the surface properties of the liposomes/vesicles may be altered by the adsorption/binding of peptides or proteins. It is therefore fairly important to investigate the change in the surface properties of simple liposomes caused by
88
H. Matsumura et aL/Colloids Surfaces A Physicochem. Eng. Aspects 92 (1994) 87-93
the adsorption of well-defined biological substances such as peptides or proteins. In this work, the electrokinetic properties (electrophoretic mobilities) of phosphatidylcholine (PC) liposomes immersed in various biological materials (amino acids, peptides and proteins) have been studied, because electrostatic potentials on the membrane surface of liposomes are one of the significant factors which dominate membranemembrane interactions resulting in aggregation and fusion.
2. Materials and methods 2.1. Materials Liposomes were prepared from egg yolk phosphatidylcholine (PC; purchased from Sigma Chemical Co., Ltd.) by the sonication or vortexmixing methods in distilled and deionized water obtained from a Nanopure system (Barnstead). Amino acids (L-alanine, L-lysine and L-glutamic acid) were purchased from Sigma Chemical Co. As peptides, cationic (poly(L-lysine), PLL) and anionic (poly(L-glutamic acid), PLG) compounds are employed. PLL hydrobromide was purchased from Sigma Chemical Co. Ltd., and the sodium salt of PLG was obtained from Ajinomoto Co. Ltd., Japan. Two different types of protein, i.e. cytochrome c and serum albumin, were used. Horse-heart cytochrome c, which was purchased from Boehringer-Mannheim consists of small, almost spherical molecules (molecular weight M = 12 500; diameter, about 3 nm) and its isoelectric point is at approximately pH 10. Bovine serum albumin (BSA), which was purchased from Sigma, is composed of rather large (M = 69 000) and easily deformable molecules, and its isoelectric point is at approximately pH 5. These reagents were used without further purification. To demonstrate that the electrokinetic properties were substancedependent, no buffer systems and no extra salts were used.
mixtures were allowed to stand at room temperature for more than 1 h. Electrophoretic mobilities were measured with a microelectrophoretic apparatus (Rank Brothers MK-2) at 25°C. The sizes of the liposomes measured by microelectrophoresis were in the micrometre range (l-2 urn in diameter).
3. Results and discussion Initially, electrostatic properties of the egg PC liposomes used in this study were examined by measuring the electrophoretic mobilities as a function of calcium concentration (Fig. 1). In this experiment, the values of the zeta potentials were calculated from the mobility data using the Smoluchowski equation, because the value of rca ranges from about 20 ([Cal= lo-’ M) to 200 ([Cal = 10m3 M) and the mobility is not as large (see Ref. [3]), where l/~ is the Debye length and a is the radius of the particles (about 1 urn). It was found that the zeta potential of the egg PC liposomes had a negative value, which approached zero at a CaCl, concentration of around 1 x 10m3 M. This clearly indicates that egg PC liposomes carry negative charges on their surface, which may be due to a small amount of acidic impurities often found in natural lipids. From the pH dependence of the electrophoretic mobilities of the PC liposomes (the electrophoretic mobility has a value of zero between pH 4 and 5; see Fig. 8 10 F E.
$
0
-10
z 5
-20
: Li N
-30 -40
2.2. Electrophoretic
mobilities
The liposome dispersions were mixed with reagent solutions of various concentrations and the
WW
(Ml
Fig. 1. Zeta potentials of egg PC liposomes immersed aqueous CaCI, solutions at various concentrations.
in
H. Matsumura
et al. JColloids Surfaces A: Physicochem.
below), it can be seen that the most probable candidate for dissociation is the -COOH group, and the electrophoretic mobilities of the liposomes shift to more positive values when the pH decreases and to more negative values when the pH increases. Fig. 2 shows the electrophoretic mobilities of PC liposomes immersed in aqueous solutions having various concentrations of each amino acid: Lglutamic acid (isoelectric point: pl= 3.22; pK, = 2.10; pK, = 4.07, pK3 = 9.47), E-alanine (pl = 6.00; pK1 = 2.35; pK, = 9.87); L-lysine (pl= 9.7; pK, = 2.18; pK,=8.95; pK3 = 10.53). These amino acids have net negative, zero, and positive charges in the neutral pH region respectively. In this figure, pH changes in the aqueous solution on the addition of glutamic acid or lysine are also plotted. The concentration dependence of the electrophoretic
log [amino acid]
(M)
Eng. Aspects 92 (1994j 87-93
mobility obtained here is mainly attributable to the pH change in solution [4], i.e. the addition of L-glutamic acid to the solution causes the pH to shift to lower values and L-lysine addition causes a pH shift to higher values. These shifts make the electrophoretic mobility of the liposomes move to more positive values and more negative values respectively. No adsorption or slight adsorption of amino acids scarcely affects the electrophoretic mobility. Fig. 3 shows the electrophoretic mobilities of PC liposomes immersed in aqueous solutions having various concentrations of each polypeptide: poly(~glutamic acid) (PLG; A4= 100000) and poly(~lysine) (PLL; M = 100 000). So far, several researchers have reported theories of electrophoretic mobility for particles which are covered with a polymer layer (see for example, Refs. [ 51 and [ 61). However, structural information about the layer is required when applying these theories. Therefore, in this report, the electrophoretic mobility is used as a measure for qualitative discussions. The polycation PLL causes a significant positive change in the electrophoretic mobilities and the polyanion PLG causes a small negative change in the electrophoretie mobilities. These polypeptides can adsorb onto the PC liposomes and the surface charges are increased/decreased; PLL, especially reverses the sign of the electrophoretic mobility. Both PLG
W
-4
-3 log
Fig. 2. Electrophoretic mobilities of egg PC liposomes immersed in aqueous solutions of each amino acid at various concentrations: A, L-glutamic acid; Cl, L-alanine; 0, L-lysine. The pH change is also shown in the figure.
89
-2
[polypeptide]
-1
0
(mg/ml)
Fig. 3. Electrophoretic mobilities of egg PC liposomes immersed in aqueous solutions of each polypeptide at various concentrations: n , poly(L-glutamic acid); 0, poly(L-lysine).
H. Matsumura et al.~Colloi& Surfaces A: Physicochem. Eng. Aspects 92 (1994) 87-93
90
and the PC liposomes possess negative charges, so that the motivating force for the adsorption of PLG on the liposomes must originate from the hydrophobic interaction between them. In this case, the PLG must penetrate the liposome membranes and make contact with the hydrocarbon region of the phospholipid membranes, because the surfaces of egg PC liposomes are highly hydrophilic [7]. In contrast, PLL possesses positive charges, so that both electrostatic attraction and hydrophobic interactions contribute to the PLL-PC liposome interaction. Fig. 4 shows the electrophoretic mobilities of PC liposomes immersed in aqueous solutions having various concentrations of the two types of protein: cytochrome c and BSA. Cytochrome c carries net positive charges and BSA carries net negative charges in the neutral pH region. For the protein data, the electrophoretic mobilities were also not converted to zeta potentials because of the unknown surface structures of protein-adsorbed liposomes. Therefore the electrophoretic mobility was used as a measure for qualitative discussions. In this field, extensive theoretical work is needed. With increasing protein concentration, the negative values of electrophoretic mobilities of the liposomes decreases for both proteins. As can be seen from the fact that the change in the electrophoretic mobility caused by cytochrome c is larger than
3 W
-0.5
1 -4
I
I
I
I
I
-3
-2
-1
0
1
log [protein]
(mgiml)
Fig. 4. Electrophoretic mobilities of egg PC liposomes immersed in aqueous solutions of each protein at various concentrations: 0, bovine serum albumin; n , cytochrome c.
that caused by BSA, the surface charge compensation effect is dominant in the case of cytochrome c because of its net positive charge. However, in contrast to the situation for poly(L-lysine), the electrophoretic mobilities gradually decrease and approach zero, and do not reverse their sign over this experimental concentration range (the concentration range is above at which gives a plateau in the adsorption isotherm of cytochrome c on a glass surface at neutral pH). This suggests that the electrostatic attraction between cytochrome c and the liposome is a main motivating force for the adsorption, and is compatible with the fact that the adsorption of “hard” protein (i.e. one which upon adsorption, does not undergo a major structural change) on the hydrophilic surface is mainly attributable to the electrostatic interaction between them. However, the decrease in the electrophoretic mobility of the liposomes caused by BSA is not so clear. One of the interpretations is that the surface of the liposomes is partially/completely covered with adsorbed BSA and the surface then possesses the character of BSA partially/completely. The same behaviour has been observed for protein adsorption on latex particles [8-lo]. In this case, the adsorption of an acidic protein possessing negative charges on the negatively charged surface of the latex causes counterion incorporation into the interfacial region in order to reduce the electrostatic energy. The motivating force of the adsorption of the proteins in such a case is said to be the dehydrating effect of the hydrophobic surface of the latex and the hydrophobic domain of the BSA. However, the surface of egg PC liposomes is hydrophilic, large-sized liposomes especially being fairly hydrated because of an almost flat surface, so the mechanism of adsorption of BSA on the liposomes must be different from that described above. BSA is one of the “soft” proteins (easily changeable in their conformation upon adsorption onto surfaces); therefore it experiences a conformation change when it comes into contact with the liposomes. The invasion of BSA into the liposome membranes is one of the possible mechanisms, because this promotes hydrophobic-hydrophobic interactions between BSA and the membrane interior. Incidentally, the addition of proteins to the solution causes a pH shift which may affect the electro-
91
H. Matsumura et al. JColloids Surfaces A: Physicochem. Eng. Aspects 92 (1994) 87793
phoretic mobilities of the liposome, so the pH change due to BSA and cytochrome c was examined (Fig. 5). Cytochrome c causes the pH to shift to higher values but BSA has a minor effect on the pH, so the change in pH should cause a shift in the electrophoretic mobilities towards more negative values, but the data obtained here show an opposite trend i.e. a shift in the mobilities in the positive direction. Therefore the adsorption of proteins provides the main contribution to the change in the electrophoretic mobilities. The electrophoretic mobility of particles covered with proteins can be satisfactorily described by the linear combination of the electrophoretic mobility of bare particles and that of fully protein-covered particles. Matsumoto et al. [l l] have reported that l/d[ in the case of protein adsorption on hydroxyapatite has a linear relationship with l/C (where &’ is the change in the zeta potential [, and C is the equilibrium concentration of protein in the bulk solution). This means that the adsorbed protein molecules contribute to the total electrophoretic mobilities in the same manner throughout the whole concentration range. Generally, if the surface electrical properties of the liposomes are determined by a linear combination of those of the bare surface and those of the parts covered with adsorbate, and also Langmuir adsorption isotherms are assumed, the reciprocal of the change in electrical properties shows a linear relation with the reciprocal of the equilibrium concentration of
adsorbate in the solution: l/dZ = (l/AZ*) (1+ l/KC)
(1)
where dZ is the change in surface electrical properties, dZ* is the difference in the surface electrical properties between the bare and the covered parts, K is the equilibrium constant of adsorption, and C is the equilibrium concentration of adsorbate in the solution. The reciprocal of the change in electrophoretic mobility versus the reciprocal concentration of the polypeptides (from the data in Fig. 3) is plotted in Fig. 6. Only PLL shows a linear relationship between the two quantities. It can be said that PLG does not adsorb on the liposomes in a simple manner. Also, the relationship between l/(change in electrophoretic mobility) and l/C for the two proteins is plotted in Fig. 7. Only cytochrome c shows a linear relation between the two quantities. This means that BSA does not adsorb on the liposomes in a simple manner such as is found for cytochrome c on liposomes, or other proteins on solid particles. BSA has a high affinity for the liposomes, which may originate from the penetration of BSA into the liposome membranes. These considerations can be supported by the results of monolayer experiments. The latter, which were carried out to study the interaction of cytochrome c with phospholipid membranes, have revealed that cytochrome can be incorporated into the membrane only at low membrane pressures (cytochrome c, once incorporated into the mono-
9.5 n
,4
-3
-2 log
Fig. 5. The pH change bovine serum albumin;
[protein]
-1
0
0
500
1000
1500
l/C
(mg/ml)
of l/(electrophoretic
mobility)
(mgiml)
in solution caused by each protein: W, cytochrome c.
0,
Fig. 6. Plots
(0) and PLG (0).
2000
2500
vs. l/C
for PLL
H. Matsumura et al./Colloids Surfaces A: Physicochem. Eng. Aspects 92 (1994) 87-93
92
vu
-K
30
5 1
25
k
20
.g n
15
z
10
:
5 0'
0
I
I
50
100
1
150
I
200
I 250
1IC (ml/mg) Fig. 7. Plots of l/(electrophoretic (0) and cytochrome c (0).
mobility)
Lu
3
4
5
6
7
a
PH vs. l/C
for BSA
layer at a surface pressure of 2 mN m- ’ is expelled from the membrane at a pressure of 16 mN m-i) [ 121. This result supports the view that the cytochrome c does not easily penetrate into bilayer membranes because the lateral pressure of the bilayer is stated to be about 10 mN m-l [13]. Compared with cytochrome c, BSA penetrates rather easily into the PC monolayer at a surface pressure below about 20 mN m-i [ 141. These data therefore suggest that the liposomes can be almost covered with conformationally changed BSA but cannot be so well covered with cytochrome c. Fig. 8 shows the pH dependence of the electrophoretic mobilities of PC liposomes with adsorbed protein. The adsorption experiments were undertaken at a protein concentration of 0.05 mg mll’ at neutral pH (pure water) and the pH was then adjusted with NaOH/HCl aqueous solutions; the final concentration of the proteins was 0.025 mg ml - ‘. Liposomes with adsorbed BSA have an isoelectric point (IEP) close to that of the BSA molecules but liposomes with adsorbed cytochrome c have a far lower IEP compared with that of the cytochrome c molecules. These data can be explained by considering that the negative charges of the underlying PC surface are almost compensated by coadsorbed counterions from the bulk solution in the case of BSA adsorption, but they are partially compensated by the positive charges on the protein molecules in the case of cytochrome
Fig. 8. pH dependence of the electrophoretic mobilities of egg PC liposomes on which each protein has previously been adsorbed: 0, bovine serum albumin; 0, cytochrome c; 0, bare egg PC. In these experiments, each solution contains 0.061 M NaCI.
c; a similar situation has been observed in the adsorption of BSA on latex particles [lo].
4. Conclusions No adsorption or slight adsorption of amino acid scarcely affects the electrokinetic properties of the liposomes, unlike the polymers (the polypeptides) which show a large effect. Polypeptides are flexible linear polymers, so they can easily change their conformation upon adsorption, and the liposome surfaces are modified that they assume a polycation/polyanion like character. The “soft” protein, BSA, which carries a net negative charge at neutral pH, adsorbs onto the liposomes and causes their surfaces to become more BSA-like in nature. The “hard” protein, cytochrome c adsorbs on the liposomes via electrostatic attraction and compensates the surface charges, but the surfaces of the liposomes still partially retain their PC character.
References [l]
P.R. K. Hong, D. Papahajopoulos,
Meers, N. in J. Wilschut
and Duzgunes and D. Hoekstra
H. Matsumura et al./Colloids Surfaces A: Physicochetn. Eng. Aspects 92 (1994) 87-93
[Z] [3] [4] [5] [6] [7]
(Eds.), Membrane Fusion, Marcel Dekker, New York, 1991, p. 195. J. Bentz and H. Ellens, Colloids Surfaces, 30 (1988) 65. R. O’Brien and L. White, J. Chem. Sot., Faraday Trans., 2, 77 (1978) 1607. H. Matsumura, C. Obata, K. Kawahara and K. Furusawa, J. Colloid Interface Sci., 156 (1993) 269. S. Levine, M. Levine, K.A. Sharp and D.E. Brooks, Biophys. J., 42 (1983) 127. H. Ohshima and T. Kondo, J. Colloid Interface Sci., 116 (1987) 305. R.P. Rand and V.A. Parsegian, Biochim. Biophys. Acta, 988 (1989) 351.
[8)
93
W. Norde and J. Lyklema, J. Colloid Interface Sci., 66 (1978) 277. [9] T. Arai and W. Norde, Colloids Surfaces, 51 (1990) 1. [lo] A.V. Elgersma, R.J. Zsom, W. Norde and J. Lyklema, J. Colloid Interface Sci., 138 (1990) 145. [ll] M. Matsumoto, T. Miyake, H. Noshi, M. Kambara and K. Konishi, Colloids Surfaces, 40 (1989) 77. [ 123 M. Saint-Pierre-Chazalet, C. Fressigne, F. Billoudet and M.P. Pileni, Thin Solid Films, 210 (1992) 743. [ 131 0. Albrecht, H. Gruler and E. Sackmann, J. Phys. (Paris), 39 (1978) 301. [ 141 P. Quinn and R.M.C. Dawson, Biochem J., 119 (1970) 21.
!i#.iLorDs A SURFACES
Effect of amino acids, polypeptides and proteins on electrophoretic mobilities of phospholipid liposomes Hideo Matsumura a,*, Fumiko Mori b, Kiyoko Kawahara b, Chiharu Obata b, Kunio Furusawa b a Electrotechnical Laboratory, AIST, MITI, Tsukuba 305, Japan b The University of Tsukuba, Tsukuba 305, Japan Received 2 December
1993; accepted
28 January
1994
Abstract The electrophoretic mobilities of liposomes prepared from egg phosphatidylcholine (PC) immersed in solutions which contain amino acids, polypeptides or proteins have been studied. Amino acids (lysine - a basic amino acid,
or glutamic acid - an acidic amino acid) cause a pH variation which changes the acid-base dissociation equilibria of charged compounds in the PC membranes. Polypeptides (poly(L-lysine) - a cationic polymer and poly(L-glutamic acid) - an anionic polymer) can adsorb onto the membrane surface of the PC liposomes because of their high affinity for the membrane, which causes significant changes in the electrophoretic mobilities towards more positive values in the case of poly(L-lysine) and causes slight negative shifts in the case of poly(L-glutamic acid). Proteins (serum albumin: isoelectric point, pH 4.9; and cytochrome c: isoelectric point, pH 10.1) adsorb on the membrane surface, which decreases the negative value of the electrophoretic mobilities in both cases. Furthermore, both proteins shift the zero point of zeta potential of the lipid liposomes towards the isoelectric point of the proteins. Keywords: Electrophoresis;
Liposomes; Protein adsorption; Polypeptides
1. Introduction
The interaction of proteins with lipid membranes is an important subject from the point of view of fundamental principles and their applications. Many studies on the membrane-protein interaction concerning membrane fusion have been carried out. The study of protein-mediated fusion is one of the highlights of cell biology. However, the fusion mechanism is still obscure, and many more fundamental studies concerning the various aspects are required [l] (see also a review by Bentz and Ellens [2]). * Corresponding author. 0927-7757/94/$07.00 0 1994 Elsevier Science B.V. All rights reserved SSDI 0927-7757(94)02785-Q
Artificial lipid liposomes and natural vesicles in cells encounter various types of proteins and other biological substances in blood, extracellular fluid and cytoplasm. For example, liposomes acting as drug-carriers flowing in the blood stream encounter serum proteins, etc. until they reach the diseased part, and vesicles in the cytoplasm operating as transporters of nourishment must pass through the cytosol, which contains various biological substances, before their fusion with a cytomembrane. Therefore the surface properties of the liposomes/vesicles may be altered by the adsorption/binding of peptides or proteins. It is therefore fairly important to investigate the change in the surface properties of simple liposomes caused by
88
H. Matsumura et aL/Colloids Surfaces A Physicochem. Eng. Aspects 92 (1994) 87-93
the adsorption of well-defined biological substances such as peptides or proteins. In this work, the electrokinetic properties (electrophoretic mobilities) of phosphatidylcholine (PC) liposomes immersed in various biological materials (amino acids, peptides and proteins) have been studied, because electrostatic potentials on the membrane surface of liposomes are one of the significant factors which dominate membranemembrane interactions resulting in aggregation and fusion.
2. Materials and methods 2.1. Materials Liposomes were prepared from egg yolk phosphatidylcholine (PC; purchased from Sigma Chemical Co., Ltd.) by the sonication or vortexmixing methods in distilled and deionized water obtained from a Nanopure system (Barnstead). Amino acids (L-alanine, L-lysine and L-glutamic acid) were purchased from Sigma Chemical Co. As peptides, cationic (poly(L-lysine), PLL) and anionic (poly(L-glutamic acid), PLG) compounds are employed. PLL hydrobromide was purchased from Sigma Chemical Co. Ltd., and the sodium salt of PLG was obtained from Ajinomoto Co. Ltd., Japan. Two different types of protein, i.e. cytochrome c and serum albumin, were used. Horse-heart cytochrome c, which was purchased from Boehringer-Mannheim consists of small, almost spherical molecules (molecular weight M = 12 500; diameter, about 3 nm) and its isoelectric point is at approximately pH 10. Bovine serum albumin (BSA), which was purchased from Sigma, is composed of rather large (M = 69 000) and easily deformable molecules, and its isoelectric point is at approximately pH 5. These reagents were used without further purification. To demonstrate that the electrokinetic properties were substancedependent, no buffer systems and no extra salts were used.
mixtures were allowed to stand at room temperature for more than 1 h. Electrophoretic mobilities were measured with a microelectrophoretic apparatus (Rank Brothers MK-2) at 25°C. The sizes of the liposomes measured by microelectrophoresis were in the micrometre range (l-2 urn in diameter).
3. Results and discussion Initially, electrostatic properties of the egg PC liposomes used in this study were examined by measuring the electrophoretic mobilities as a function of calcium concentration (Fig. 1). In this experiment, the values of the zeta potentials were calculated from the mobility data using the Smoluchowski equation, because the value of rca ranges from about 20 ([Cal= lo-’ M) to 200 ([Cal = 10m3 M) and the mobility is not as large (see Ref. [3]), where l/~ is the Debye length and a is the radius of the particles (about 1 urn). It was found that the zeta potential of the egg PC liposomes had a negative value, which approached zero at a CaCl, concentration of around 1 x 10m3 M. This clearly indicates that egg PC liposomes carry negative charges on their surface, which may be due to a small amount of acidic impurities often found in natural lipids. From the pH dependence of the electrophoretic mobilities of the PC liposomes (the electrophoretic mobility has a value of zero between pH 4 and 5; see Fig. 8 10 F E.
$
0
-10
z 5
-20
: Li N
-30 -40
2.2. Electrophoretic
mobilities
The liposome dispersions were mixed with reagent solutions of various concentrations and the
WW
(Ml
Fig. 1. Zeta potentials of egg PC liposomes immersed aqueous CaCI, solutions at various concentrations.
in
H. Matsumura
et al. JColloids Surfaces A: Physicochem.
below), it can be seen that the most probable candidate for dissociation is the -COOH group, and the electrophoretic mobilities of the liposomes shift to more positive values when the pH decreases and to more negative values when the pH increases. Fig. 2 shows the electrophoretic mobilities of PC liposomes immersed in aqueous solutions having various concentrations of each amino acid: Lglutamic acid (isoelectric point: pl= 3.22; pK, = 2.10; pK, = 4.07, pK3 = 9.47), E-alanine (pl = 6.00; pK1 = 2.35; pK, = 9.87); L-lysine (pl= 9.7; pK, = 2.18; pK,=8.95; pK3 = 10.53). These amino acids have net negative, zero, and positive charges in the neutral pH region respectively. In this figure, pH changes in the aqueous solution on the addition of glutamic acid or lysine are also plotted. The concentration dependence of the electrophoretic
log [amino acid]
(M)
Eng. Aspects 92 (1994j 87-93
mobility obtained here is mainly attributable to the pH change in solution [4], i.e. the addition of L-glutamic acid to the solution causes the pH to shift to lower values and L-lysine addition causes a pH shift to higher values. These shifts make the electrophoretic mobility of the liposomes move to more positive values and more negative values respectively. No adsorption or slight adsorption of amino acids scarcely affects the electrophoretic mobility. Fig. 3 shows the electrophoretic mobilities of PC liposomes immersed in aqueous solutions having various concentrations of each polypeptide: poly(~glutamic acid) (PLG; A4= 100000) and poly(~lysine) (PLL; M = 100 000). So far, several researchers have reported theories of electrophoretic mobility for particles which are covered with a polymer layer (see for example, Refs. [ 51 and [ 61). However, structural information about the layer is required when applying these theories. Therefore, in this report, the electrophoretic mobility is used as a measure for qualitative discussions. The polycation PLL causes a significant positive change in the electrophoretic mobilities and the polyanion PLG causes a small negative change in the electrophoretie mobilities. These polypeptides can adsorb onto the PC liposomes and the surface charges are increased/decreased; PLL, especially reverses the sign of the electrophoretic mobility. Both PLG
W
-4
-3 log
Fig. 2. Electrophoretic mobilities of egg PC liposomes immersed in aqueous solutions of each amino acid at various concentrations: A, L-glutamic acid; Cl, L-alanine; 0, L-lysine. The pH change is also shown in the figure.
89
-2
[polypeptide]
-1
0
(mg/ml)
Fig. 3. Electrophoretic mobilities of egg PC liposomes immersed in aqueous solutions of each polypeptide at various concentrations: n , poly(L-glutamic acid); 0, poly(L-lysine).
H. Matsumura et al.~Colloi& Surfaces A: Physicochem. Eng. Aspects 92 (1994) 87-93
90
and the PC liposomes possess negative charges, so that the motivating force for the adsorption of PLG on the liposomes must originate from the hydrophobic interaction between them. In this case, the PLG must penetrate the liposome membranes and make contact with the hydrocarbon region of the phospholipid membranes, because the surfaces of egg PC liposomes are highly hydrophilic [7]. In contrast, PLL possesses positive charges, so that both electrostatic attraction and hydrophobic interactions contribute to the PLL-PC liposome interaction. Fig. 4 shows the electrophoretic mobilities of PC liposomes immersed in aqueous solutions having various concentrations of the two types of protein: cytochrome c and BSA. Cytochrome c carries net positive charges and BSA carries net negative charges in the neutral pH region. For the protein data, the electrophoretic mobilities were also not converted to zeta potentials because of the unknown surface structures of protein-adsorbed liposomes. Therefore the electrophoretic mobility was used as a measure for qualitative discussions. In this field, extensive theoretical work is needed. With increasing protein concentration, the negative values of electrophoretic mobilities of the liposomes decreases for both proteins. As can be seen from the fact that the change in the electrophoretic mobility caused by cytochrome c is larger than
3 W
-0.5
1 -4
I
I
I
I
I
-3
-2
-1
0
1
log [protein]
(mgiml)
Fig. 4. Electrophoretic mobilities of egg PC liposomes immersed in aqueous solutions of each protein at various concentrations: 0, bovine serum albumin; n , cytochrome c.
that caused by BSA, the surface charge compensation effect is dominant in the case of cytochrome c because of its net positive charge. However, in contrast to the situation for poly(L-lysine), the electrophoretic mobilities gradually decrease and approach zero, and do not reverse their sign over this experimental concentration range (the concentration range is above at which gives a plateau in the adsorption isotherm of cytochrome c on a glass surface at neutral pH). This suggests that the electrostatic attraction between cytochrome c and the liposome is a main motivating force for the adsorption, and is compatible with the fact that the adsorption of “hard” protein (i.e. one which upon adsorption, does not undergo a major structural change) on the hydrophilic surface is mainly attributable to the electrostatic interaction between them. However, the decrease in the electrophoretic mobility of the liposomes caused by BSA is not so clear. One of the interpretations is that the surface of the liposomes is partially/completely covered with adsorbed BSA and the surface then possesses the character of BSA partially/completely. The same behaviour has been observed for protein adsorption on latex particles [8-lo]. In this case, the adsorption of an acidic protein possessing negative charges on the negatively charged surface of the latex causes counterion incorporation into the interfacial region in order to reduce the electrostatic energy. The motivating force of the adsorption of the proteins in such a case is said to be the dehydrating effect of the hydrophobic surface of the latex and the hydrophobic domain of the BSA. However, the surface of egg PC liposomes is hydrophilic, large-sized liposomes especially being fairly hydrated because of an almost flat surface, so the mechanism of adsorption of BSA on the liposomes must be different from that described above. BSA is one of the “soft” proteins (easily changeable in their conformation upon adsorption onto surfaces); therefore it experiences a conformation change when it comes into contact with the liposomes. The invasion of BSA into the liposome membranes is one of the possible mechanisms, because this promotes hydrophobic-hydrophobic interactions between BSA and the membrane interior. Incidentally, the addition of proteins to the solution causes a pH shift which may affect the electro-
91
H. Matsumura et al. JColloids Surfaces A: Physicochem. Eng. Aspects 92 (1994) 87793
phoretic mobilities of the liposome, so the pH change due to BSA and cytochrome c was examined (Fig. 5). Cytochrome c causes the pH to shift to higher values but BSA has a minor effect on the pH, so the change in pH should cause a shift in the electrophoretic mobilities towards more negative values, but the data obtained here show an opposite trend i.e. a shift in the mobilities in the positive direction. Therefore the adsorption of proteins provides the main contribution to the change in the electrophoretic mobilities. The electrophoretic mobility of particles covered with proteins can be satisfactorily described by the linear combination of the electrophoretic mobility of bare particles and that of fully protein-covered particles. Matsumoto et al. [l l] have reported that l/d[ in the case of protein adsorption on hydroxyapatite has a linear relationship with l/C (where &’ is the change in the zeta potential [, and C is the equilibrium concentration of protein in the bulk solution). This means that the adsorbed protein molecules contribute to the total electrophoretic mobilities in the same manner throughout the whole concentration range. Generally, if the surface electrical properties of the liposomes are determined by a linear combination of those of the bare surface and those of the parts covered with adsorbate, and also Langmuir adsorption isotherms are assumed, the reciprocal of the change in electrical properties shows a linear relation with the reciprocal of the equilibrium concentration of
adsorbate in the solution: l/dZ = (l/AZ*) (1+ l/KC)
(1)
where dZ is the change in surface electrical properties, dZ* is the difference in the surface electrical properties between the bare and the covered parts, K is the equilibrium constant of adsorption, and C is the equilibrium concentration of adsorbate in the solution. The reciprocal of the change in electrophoretic mobility versus the reciprocal concentration of the polypeptides (from the data in Fig. 3) is plotted in Fig. 6. Only PLL shows a linear relationship between the two quantities. It can be said that PLG does not adsorb on the liposomes in a simple manner. Also, the relationship between l/(change in electrophoretic mobility) and l/C for the two proteins is plotted in Fig. 7. Only cytochrome c shows a linear relation between the two quantities. This means that BSA does not adsorb on the liposomes in a simple manner such as is found for cytochrome c on liposomes, or other proteins on solid particles. BSA has a high affinity for the liposomes, which may originate from the penetration of BSA into the liposome membranes. These considerations can be supported by the results of monolayer experiments. The latter, which were carried out to study the interaction of cytochrome c with phospholipid membranes, have revealed that cytochrome can be incorporated into the membrane only at low membrane pressures (cytochrome c, once incorporated into the mono-
9.5 n
,4
-3
-2 log
Fig. 5. The pH change bovine serum albumin;
[protein]
-1
0
0
500
1000
1500
l/C
(mg/ml)
of l/(electrophoretic
mobility)
(mgiml)
in solution caused by each protein: W, cytochrome c.
0,
Fig. 6. Plots
(0) and PLG (0).
2000
2500
vs. l/C
for PLL
H. Matsumura et al./Colloids Surfaces A: Physicochem. Eng. Aspects 92 (1994) 87-93
92
vu
-K
30
5 1
25
k
20
.g n
15
z
10
:
5 0'
0
I
I
50
100
1
150
I
200
I 250
1IC (ml/mg) Fig. 7. Plots of l/(electrophoretic (0) and cytochrome c (0).
mobility)
Lu
3
4
5
6
7
a
PH vs. l/C
for BSA
layer at a surface pressure of 2 mN m- ’ is expelled from the membrane at a pressure of 16 mN m-i) [ 121. This result supports the view that the cytochrome c does not easily penetrate into bilayer membranes because the lateral pressure of the bilayer is stated to be about 10 mN m-l [13]. Compared with cytochrome c, BSA penetrates rather easily into the PC monolayer at a surface pressure below about 20 mN m-i [ 141. These data therefore suggest that the liposomes can be almost covered with conformationally changed BSA but cannot be so well covered with cytochrome c. Fig. 8 shows the pH dependence of the electrophoretic mobilities of PC liposomes with adsorbed protein. The adsorption experiments were undertaken at a protein concentration of 0.05 mg mll’ at neutral pH (pure water) and the pH was then adjusted with NaOH/HCl aqueous solutions; the final concentration of the proteins was 0.025 mg ml - ‘. Liposomes with adsorbed BSA have an isoelectric point (IEP) close to that of the BSA molecules but liposomes with adsorbed cytochrome c have a far lower IEP compared with that of the cytochrome c molecules. These data can be explained by considering that the negative charges of the underlying PC surface are almost compensated by coadsorbed counterions from the bulk solution in the case of BSA adsorption, but they are partially compensated by the positive charges on the protein molecules in the case of cytochrome
Fig. 8. pH dependence of the electrophoretic mobilities of egg PC liposomes on which each protein has previously been adsorbed: 0, bovine serum albumin; 0, cytochrome c; 0, bare egg PC. In these experiments, each solution contains 0.061 M NaCI.
c; a similar situation has been observed in the adsorption of BSA on latex particles [lo].
4. Conclusions No adsorption or slight adsorption of amino acid scarcely affects the electrokinetic properties of the liposomes, unlike the polymers (the polypeptides) which show a large effect. Polypeptides are flexible linear polymers, so they can easily change their conformation upon adsorption, and the liposome surfaces are modified that they assume a polycation/polyanion like character. The “soft” protein, BSA, which carries a net negative charge at neutral pH, adsorbs onto the liposomes and causes their surfaces to become more BSA-like in nature. The “hard” protein, cytochrome c adsorbs on the liposomes via electrostatic attraction and compensates the surface charges, but the surfaces of the liposomes still partially retain their PC character.
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W. Norde and J. Lyklema, J. Colloid Interface Sci., 66 (1978) 277. [9] T. Arai and W. Norde, Colloids Surfaces, 51 (1990) 1. [lo] A.V. Elgersma, R.J. Zsom, W. Norde and J. Lyklema, J. Colloid Interface Sci., 138 (1990) 145. [ll] M. Matsumoto, T. Miyake, H. Noshi, M. Kambara and K. Konishi, Colloids Surfaces, 40 (1989) 77. [ 123 M. Saint-Pierre-Chazalet, C. Fressigne, F. Billoudet and M.P. Pileni, Thin Solid Films, 210 (1992) 743. [ 131 0. Albrecht, H. Gruler and E. Sackmann, J. Phys. (Paris), 39 (1978) 301. [ 141 P. Quinn and R.M.C. Dawson, Biochem J., 119 (1970) 21.