J. MICROENCAPSULATION,
1990, VOL. 7,
NO.
2, 179-184
Electrostatic interaction of microcapsules with guinea-pig polymorphonuclear leucocytes
Journal of Microencapsulation Downloaded from informahealthcare.com by UB der LMU Muenchen on 02/19/13 For personal use only.
SATOSHI YASUKAWA*, H I R O Y U K I OHSHIMAX, N O B U H I R O MURAMATSU*t and T A M O T S U KONDO*t$ Faculty of Pharmaceutical Sciences" and Research Institute for Biosciencest, Science University of Tokyo, Shinjuki-ku, Tokyo 162, Japan (Received 2 May 1989; accepted 2 June 1989)
Electrostatic interaction of microcapsules with guinea-pig polymorphonuclear leucocytes was investigated at different ionic strengths of the medium through measurements of the degree of phagocytosisby the cells of microcapsules. At any ionic strength, the interaction exhibited a maximum when the surface potential of microcapsules was equal to that of the leucocytes to make phagocytosis of microcapsules by the cells minimum. The experimental findings were analyzed by a novel model for the electrostatic interaction between ion-penetrable membranes. In this analysis, it was assumed that the leucocytes have an area (or areas) which permits cations to partition much more into the membrane than anions on their surface to reduce the electric potential of the area.
Introduction Although the phagocytic action of leucocytes has been extensively studied (Klebanott and Clark 1978, Gallin and Fauci 1982), its mechanism has not yet been fully elucidated. I n the process of phagocytosis, the attachment phase in which foreign particles attach to the phagocyte surface should be distinguished from the ingestion phase, during which leucocytes uptake the foreign particles to turn themselves into phagosomes. In order to start phagocytosis, foreign particles have to attach to the leucocyte surface (Silverstein et al. 1977). T h e attachment phase, the initial stage of phagocytosis, is considered to be nonspecific because the electrostatic interaction between the foreign particle surface and the phagocyte surface plays an important role in this stage. In our previous works (Watanabe et al. 1984, Sano et al. 1986), we reported that microcapsules having the same surface negative potential as that of leucocytes undergo less phagocytosis than those having surface negative potentials different from that of the phagocytes. However, the reason why microcapsules with surface negative potentials higher or lower than those of leucocytes are more easily phagocytosed is still unknown. This paper deals with the electrostatic interaction of microcapsules with guineapig polymorphonuclear leucocytes as a function of the ionic strength of the medium from the experimental as well as theoretical points of view, to find a plausible explanation for the fact that the phogocytosis is minimum when microcapsules and leucocytes possess the same surface negative potential. T h e use of microcapsules as foreign particles is beneficial since their surface potential can easily be controlled.
$ To whom correspondence should be addressed. 0265-2048/90 $3.00 0 1990 Taylor & Francis Ltd
Journal of Microencapsulation Downloaded from informahealthcare.com by UB der LMU Muenchen on 02/19/13 For personal use only.
180
S . Yasukawa et al.
Experimental Preparation of microcapsules Poly( 1,4-piperazinediylterephthaloyl)microcapsules were prepared by making use of the interfacial polycondensation reaction between piperazine dissolved in the aqueous phase and terephthaloyl dichloride dissolved in the oil phase, with aid of electrocapillary emulsification as described in the previous papers (Watanabe et al. 1984, Sano et al. 1986). T h e microcapsules thus prepared were found to have a mean diameter in the region of 0 5 pm and a surface potential of - 8.38 mV in a medium of p H 7.4 and ionic strength 0.1 54. Microcapsules with surface potential lower than that of poly( 1,4piperazinediylterephthaloyl)microcapsules were obtained by using mixed aqueous solutions of piperazine and 4,4’-diamino-stilbene-2,2’-disulfonicacid in several molar ratios as the aqueous phase for the interfacial polycondensation reaction in the microcapsule preparation. T h e surface potentials of the sulfonated polyamide microcapsules were -23.96 and -26.16mV in the same medium as above. Esterification with diazomethane of the carboxyl groups of poly( 1,4piperazinediylterephthaloyl) microcapsules resulted in an increase in the surface potential of the microcapsules to a value of - 3.95 mV in the same medium. Isolation of polymorphonuclear leucocytes from guinea-pig Preparation of guinea-pig polymorphonuclear leucocyte suspensions was made by the method reported previously (Sano et al. 1986). T h e leucocytes had a surface potential of - 8.94 mV at p H 7.4 and ionic strength 0.1 54. Measurement of surface potential The surface potentials of microcapsules and leucocytes were assumed to be approximately equal to their zeta potentials. T h e zeta potentials were measured with a Pen Kem System 3000 zeta potential measuring apparatus (Pen Kem, Inc.). Measurement of phagocytosis Phagocytosis of microcapsules by leucocytes was evaluated by oxygen consumption by the cells according to the method described previously (Sano et al. 1986).
Results and discussion Figure 1 shows the effect of the surface potential of microcapsules on phagocytosis by leucocytes at different ionic strengths. Increase in the surface potential of microcapsules did not cause a concomitant reduction in the degree of phagocytosis. Instead, there was an increase after an initial decrease at any ionic strength. Hence, a minimum was observed on the degree of phagocytosis versus surface potential curve and this minimum was found, as before, for those microcapsules which have the surface potential approximately equal to that of leucocytes. A rise in the ionic strength of the medium produced a shift of the point of minimum phagocytosis to the lower surface potential side. This means that phagocytosis is enhanced with increasing difference in the surface potential between the cells and microcapsules, irrespective of the ionic strength of the medium. In any case where phagocytosis occurs, microcapsules have to adhere to leucocytes. T h e adhesiveness of a microcapsule to the surface of a leucocyte is determined by the force acting between them, the resultant force of electrostatic
181
Journal of Microencapsulation Downloaded from informahealthcare.com by UB der LMU Muenchen on 02/19/13 For personal use only.
Electrostatic interaction of microcapsules
-15
-30
Zeta potential (mV) Figure 1. Effect of the surface potential of microcapsuleson their phagocytosisby leucocytes at different ionic strengths. Ionic strength: 0, 00154; 0 , 0100; 0 , 0,154.
repulsion and van der Waals attraction, which can, in principle, be calculated by the classical DLVO theory (Verwey and Overbeek 1948). Of these two forces, electrostatic repulsive force depends on the surface potentials of the interacting microcapsule and leucocyte. To a first approximation, this force is proportional to the product of the surface potentials, increasing as the surface negative potential of microcapsule decreases. Hence, the classical DLVO theory, which assumes that the fixed charges are located only at the particle surface of zero thickness and the surface is impermeable to electrolyte ions, can explain only the decreases in phagocytosis observed at surface potentials of microcapsules higher than and equal to that of leucocytes; it fails to give a plausible explanation for the increase in phagocytosis observed when the surface potential of microcapsules is lower than that of the cells. In this situation, certain modifications should be introduced into the classical DLVO theory because the above two assumptions are no longer valid for the membranes of biological cells and microcapsules.
Journal of Microencapsulation Downloaded from informahealthcare.com by UB der LMU Muenchen on 02/19/13 For personal use only.
182
S. Yasukawa et al.
Recently, we have proposed a novel model for the electrostatic interaction between ion-penetrable membranes, by taking into account the membrane structure in which the fixed charges are distributed through an ion-penetrable surface layer of non-zero thickness (Ohshima et al. 1987, Ohshima and Kondo 1987, Ohshima and Kondo 1988, Makino et al. 1987, Makino et al. 1988). I n these works, it is assumed that the electric potential far inside the membrane remains constant at the Donnan potential during interaction. We refer to this type of interaction as the double-layer interaction regulated by the Donnan potential. This model, therefore, is completely different from the conventional interaction models for ion-impenetrable solid surfaces, which assume that the surface potential or surface charge density remains constant during interaction. According to the classical DLVO theory, the electrostatic interaction energy, VR(H),is given by
VdH>=~wwJILJI,,
~ X (P- XH)
(1)
where H is the separation distance between a microcapsule and a leucocyte, E, is the relative permittivity of the medium, E,, is the permittivity of a vacuum, a is the microcapsule radius (which is assumed to be much smaller than the leucocyte radius), JIL and $ M C are, respectively, the surface potentials of the leucocyte and the microcapsule, and IC is the Debye-Huckel parameter. Now, we relate the surface potential of the leucocyte to its Donnan potential in the following form (Makino et al. 1987):
where k is the Boltzmann constant, T is the absolute temperature, e is the elementary electric charge, N is the volume density of the fixed charges in the leucocyte membrane, b, and b- are, respectively, the partition coefficients of cations and anions into the leucocyte membrane, and n is the electrolyte concentration in the medium. Here, let us assume that leucocytes have an area (or areas) that allows cations to partition much more than anions into their membrane, i.e., b+ >>!I-. Equations (2) and (3) can then be simplified to: kT
*DON
=p ln
(-.-> b+n
(4)
and
When a microcapsule with a highly negative surface potential approaches such area of the surface of a leucocyte, these equations are particularly suitable to apply since the microcapsule is accompanied by a high concentration of cations. This situation is
Electrostatic interaction of microcapsules
+@+ +
t;@+
183
+ + + +
Journal of Microencapsulation Downloaded from informahealthcare.com by UB der LMU Muenchen on 02/19/13 For personal use only.
L
I f
L b
a
C
Figure 2. Schematic representation of ion distributions near the leucocyte surface during microcapsule approach. Surface potential of microcapsule: a, low; b, medium; c, high.
illustrated in figure 2. The cation concentration around the leucocyte will be approximated by: n+ =n exp
(-F) e$MC
and hence, the surface potential of the leucocyte, * t = $ L - T$MC .
$t, becomes (7)
Substitution of this relation into Eq. (1) yields
It is noteworthy that the electrostatic interaction energy, VR(H),as a function of $Mc at a fixed value of H , is symmetrical around $ M C = $ L and exhibits a maximum at $MC = t,hL. Consequently, the electrostatic repulsion between microcapsules and leucocytes is reduced, if the surface potential of microcapsules, $Mc is higher or lower than that of leucocytes, $L, to raise the degree of phagocytosis by the cells of microcapsules.
References GALLIN, J. I., and FAUCI, A. S., 1982, Phugocytic Cells, Raven Press, New York. KLEBANOTT, S. J., and CLARK, R. A., 1978, The Neutrophil; Function and Clinical Disorders, North-Holland Publishing Co., Amsterdam. MAKINO, K., OHSHIMA, H., and KONDO, T., 1987, Electrostatic interaction of ion-penetrable membranes. Effects of ionic solubility. Colloid and Polymer Science, 265, 91 1-915.
Journal of Microencapsulation Downloaded from informahealthcare.com by UB der LMU Muenchen on 02/19/13 For personal use only.
184
Electrostatic interaction of microcapsules
MAKINO, K . , OHSHIMA, H., and KONDO,T., 1988, Electrostatic interaction of two dissimilar membranes: Effects of ionic solubility, permittivity and thickness of surface charge layers. Colloids and Surfaces, 33, 153-166. H., MAKINO,K., and KONDO,T., 1987, Electrostatic interaction of two parallel OHSHIMA, plates with surface charge layers. Journal of Colloid and Interface Science, 116,196-199. H., and KONDO,T., 1987, Electrostatic repulsion of ion penetrable charged OHSHIMA, membranes: Role of Donnan potential. Journal of Theoretical Biology, 128, 187-1 94. H., and KONDO,T., 1988, Double-layer interaction regulated by the Donnan OHSHIMA, potential. Journal of Colloid and Interface Science, 123, 136-142. N., and KONDO,T., 1986, Phagocytosis of microcapsules by guineaSANO,T., MURAMATSU, pig polymorphonuclear leucocytes. Journal of Microencapsulation, 3, 265-273. SILVERSTEIN, S. C., STEINMAN, R. M., and COHN,Z. A., 1977, Endocytosis. Annual Report of Biochemistry, 46, 669-722. E. J. W., and OVERBEEK, J. T. G., 1948, Theory of the Stability of Lyophobic Colloids, VERWEY, Elsevier, Amsterdam. WATANABE, Y., KONDO,T., MIYAMOTO, M., and SASAKAWA, S., 1984, Interaction of microcapsules with human polymorphonuclear leucocytes. Chemical and Pharmaceutical Bulletin, 32, 2788-2794.
1990, VOL. 7,
NO.
2, 179-184
Electrostatic interaction of microcapsules with guinea-pig polymorphonuclear leucocytes
Journal of Microencapsulation Downloaded from informahealthcare.com by UB der LMU Muenchen on 02/19/13 For personal use only.
SATOSHI YASUKAWA*, H I R O Y U K I OHSHIMAX, N O B U H I R O MURAMATSU*t and T A M O T S U KONDO*t$ Faculty of Pharmaceutical Sciences" and Research Institute for Biosciencest, Science University of Tokyo, Shinjuki-ku, Tokyo 162, Japan (Received 2 May 1989; accepted 2 June 1989)
Electrostatic interaction of microcapsules with guinea-pig polymorphonuclear leucocytes was investigated at different ionic strengths of the medium through measurements of the degree of phagocytosisby the cells of microcapsules. At any ionic strength, the interaction exhibited a maximum when the surface potential of microcapsules was equal to that of the leucocytes to make phagocytosis of microcapsules by the cells minimum. The experimental findings were analyzed by a novel model for the electrostatic interaction between ion-penetrable membranes. In this analysis, it was assumed that the leucocytes have an area (or areas) which permits cations to partition much more into the membrane than anions on their surface to reduce the electric potential of the area.
Introduction Although the phagocytic action of leucocytes has been extensively studied (Klebanott and Clark 1978, Gallin and Fauci 1982), its mechanism has not yet been fully elucidated. I n the process of phagocytosis, the attachment phase in which foreign particles attach to the phagocyte surface should be distinguished from the ingestion phase, during which leucocytes uptake the foreign particles to turn themselves into phagosomes. In order to start phagocytosis, foreign particles have to attach to the leucocyte surface (Silverstein et al. 1977). T h e attachment phase, the initial stage of phagocytosis, is considered to be nonspecific because the electrostatic interaction between the foreign particle surface and the phagocyte surface plays an important role in this stage. In our previous works (Watanabe et al. 1984, Sano et al. 1986), we reported that microcapsules having the same surface negative potential as that of leucocytes undergo less phagocytosis than those having surface negative potentials different from that of the phagocytes. However, the reason why microcapsules with surface negative potentials higher or lower than those of leucocytes are more easily phagocytosed is still unknown. This paper deals with the electrostatic interaction of microcapsules with guineapig polymorphonuclear leucocytes as a function of the ionic strength of the medium from the experimental as well as theoretical points of view, to find a plausible explanation for the fact that the phogocytosis is minimum when microcapsules and leucocytes possess the same surface negative potential. T h e use of microcapsules as foreign particles is beneficial since their surface potential can easily be controlled.
$ To whom correspondence should be addressed. 0265-2048/90 $3.00 0 1990 Taylor & Francis Ltd
Journal of Microencapsulation Downloaded from informahealthcare.com by UB der LMU Muenchen on 02/19/13 For personal use only.
180
S . Yasukawa et al.
Experimental Preparation of microcapsules Poly( 1,4-piperazinediylterephthaloyl)microcapsules were prepared by making use of the interfacial polycondensation reaction between piperazine dissolved in the aqueous phase and terephthaloyl dichloride dissolved in the oil phase, with aid of electrocapillary emulsification as described in the previous papers (Watanabe et al. 1984, Sano et al. 1986). T h e microcapsules thus prepared were found to have a mean diameter in the region of 0 5 pm and a surface potential of - 8.38 mV in a medium of p H 7.4 and ionic strength 0.1 54. Microcapsules with surface potential lower than that of poly( 1,4piperazinediylterephthaloyl)microcapsules were obtained by using mixed aqueous solutions of piperazine and 4,4’-diamino-stilbene-2,2’-disulfonicacid in several molar ratios as the aqueous phase for the interfacial polycondensation reaction in the microcapsule preparation. T h e surface potentials of the sulfonated polyamide microcapsules were -23.96 and -26.16mV in the same medium as above. Esterification with diazomethane of the carboxyl groups of poly( 1,4piperazinediylterephthaloyl) microcapsules resulted in an increase in the surface potential of the microcapsules to a value of - 3.95 mV in the same medium. Isolation of polymorphonuclear leucocytes from guinea-pig Preparation of guinea-pig polymorphonuclear leucocyte suspensions was made by the method reported previously (Sano et al. 1986). T h e leucocytes had a surface potential of - 8.94 mV at p H 7.4 and ionic strength 0.1 54. Measurement of surface potential The surface potentials of microcapsules and leucocytes were assumed to be approximately equal to their zeta potentials. T h e zeta potentials were measured with a Pen Kem System 3000 zeta potential measuring apparatus (Pen Kem, Inc.). Measurement of phagocytosis Phagocytosis of microcapsules by leucocytes was evaluated by oxygen consumption by the cells according to the method described previously (Sano et al. 1986).
Results and discussion Figure 1 shows the effect of the surface potential of microcapsules on phagocytosis by leucocytes at different ionic strengths. Increase in the surface potential of microcapsules did not cause a concomitant reduction in the degree of phagocytosis. Instead, there was an increase after an initial decrease at any ionic strength. Hence, a minimum was observed on the degree of phagocytosis versus surface potential curve and this minimum was found, as before, for those microcapsules which have the surface potential approximately equal to that of leucocytes. A rise in the ionic strength of the medium produced a shift of the point of minimum phagocytosis to the lower surface potential side. This means that phagocytosis is enhanced with increasing difference in the surface potential between the cells and microcapsules, irrespective of the ionic strength of the medium. In any case where phagocytosis occurs, microcapsules have to adhere to leucocytes. T h e adhesiveness of a microcapsule to the surface of a leucocyte is determined by the force acting between them, the resultant force of electrostatic
181
Journal of Microencapsulation Downloaded from informahealthcare.com by UB der LMU Muenchen on 02/19/13 For personal use only.
Electrostatic interaction of microcapsules
-15
-30
Zeta potential (mV) Figure 1. Effect of the surface potential of microcapsuleson their phagocytosisby leucocytes at different ionic strengths. Ionic strength: 0, 00154; 0 , 0100; 0 , 0,154.
repulsion and van der Waals attraction, which can, in principle, be calculated by the classical DLVO theory (Verwey and Overbeek 1948). Of these two forces, electrostatic repulsive force depends on the surface potentials of the interacting microcapsule and leucocyte. To a first approximation, this force is proportional to the product of the surface potentials, increasing as the surface negative potential of microcapsule decreases. Hence, the classical DLVO theory, which assumes that the fixed charges are located only at the particle surface of zero thickness and the surface is impermeable to electrolyte ions, can explain only the decreases in phagocytosis observed at surface potentials of microcapsules higher than and equal to that of leucocytes; it fails to give a plausible explanation for the increase in phagocytosis observed when the surface potential of microcapsules is lower than that of the cells. In this situation, certain modifications should be introduced into the classical DLVO theory because the above two assumptions are no longer valid for the membranes of biological cells and microcapsules.
Journal of Microencapsulation Downloaded from informahealthcare.com by UB der LMU Muenchen on 02/19/13 For personal use only.
182
S. Yasukawa et al.
Recently, we have proposed a novel model for the electrostatic interaction between ion-penetrable membranes, by taking into account the membrane structure in which the fixed charges are distributed through an ion-penetrable surface layer of non-zero thickness (Ohshima et al. 1987, Ohshima and Kondo 1987, Ohshima and Kondo 1988, Makino et al. 1987, Makino et al. 1988). I n these works, it is assumed that the electric potential far inside the membrane remains constant at the Donnan potential during interaction. We refer to this type of interaction as the double-layer interaction regulated by the Donnan potential. This model, therefore, is completely different from the conventional interaction models for ion-impenetrable solid surfaces, which assume that the surface potential or surface charge density remains constant during interaction. According to the classical DLVO theory, the electrostatic interaction energy, VR(H),is given by
VdH>=~wwJILJI,,
~ X (P- XH)
(1)
where H is the separation distance between a microcapsule and a leucocyte, E, is the relative permittivity of the medium, E,, is the permittivity of a vacuum, a is the microcapsule radius (which is assumed to be much smaller than the leucocyte radius), JIL and $ M C are, respectively, the surface potentials of the leucocyte and the microcapsule, and IC is the Debye-Huckel parameter. Now, we relate the surface potential of the leucocyte to its Donnan potential in the following form (Makino et al. 1987):
where k is the Boltzmann constant, T is the absolute temperature, e is the elementary electric charge, N is the volume density of the fixed charges in the leucocyte membrane, b, and b- are, respectively, the partition coefficients of cations and anions into the leucocyte membrane, and n is the electrolyte concentration in the medium. Here, let us assume that leucocytes have an area (or areas) that allows cations to partition much more than anions into their membrane, i.e., b+ >>!I-. Equations (2) and (3) can then be simplified to: kT
*DON
=p ln
(-.-> b+n
(4)
and
When a microcapsule with a highly negative surface potential approaches such area of the surface of a leucocyte, these equations are particularly suitable to apply since the microcapsule is accompanied by a high concentration of cations. This situation is
Electrostatic interaction of microcapsules
+@+ +
t;@+
183
+ + + +
Journal of Microencapsulation Downloaded from informahealthcare.com by UB der LMU Muenchen on 02/19/13 For personal use only.
L
I f
L b
a
C
Figure 2. Schematic representation of ion distributions near the leucocyte surface during microcapsule approach. Surface potential of microcapsule: a, low; b, medium; c, high.
illustrated in figure 2. The cation concentration around the leucocyte will be approximated by: n+ =n exp
(-F) e$MC
and hence, the surface potential of the leucocyte, * t = $ L - T$MC .
$t, becomes (7)
Substitution of this relation into Eq. (1) yields
It is noteworthy that the electrostatic interaction energy, VR(H),as a function of $Mc at a fixed value of H , is symmetrical around $ M C = $ L and exhibits a maximum at $MC = t,hL. Consequently, the electrostatic repulsion between microcapsules and leucocytes is reduced, if the surface potential of microcapsules, $Mc is higher or lower than that of leucocytes, $L, to raise the degree of phagocytosis by the cells of microcapsules.
References GALLIN, J. I., and FAUCI, A. S., 1982, Phugocytic Cells, Raven Press, New York. KLEBANOTT, S. J., and CLARK, R. A., 1978, The Neutrophil; Function and Clinical Disorders, North-Holland Publishing Co., Amsterdam. MAKINO, K., OHSHIMA, H., and KONDO, T., 1987, Electrostatic interaction of ion-penetrable membranes. Effects of ionic solubility. Colloid and Polymer Science, 265, 91 1-915.
Journal of Microencapsulation Downloaded from informahealthcare.com by UB der LMU Muenchen on 02/19/13 For personal use only.
184
Electrostatic interaction of microcapsules
MAKINO, K . , OHSHIMA, H., and KONDO,T., 1988, Electrostatic interaction of two dissimilar membranes: Effects of ionic solubility, permittivity and thickness of surface charge layers. Colloids and Surfaces, 33, 153-166. H., MAKINO,K., and KONDO,T., 1987, Electrostatic interaction of two parallel OHSHIMA, plates with surface charge layers. Journal of Colloid and Interface Science, 116,196-199. H., and KONDO,T., 1987, Electrostatic repulsion of ion penetrable charged OHSHIMA, membranes: Role of Donnan potential. Journal of Theoretical Biology, 128, 187-1 94. H., and KONDO,T., 1988, Double-layer interaction regulated by the Donnan OHSHIMA, potential. Journal of Colloid and Interface Science, 123, 136-142. N., and KONDO,T., 1986, Phagocytosis of microcapsules by guineaSANO,T., MURAMATSU, pig polymorphonuclear leucocytes. Journal of Microencapsulation, 3, 265-273. SILVERSTEIN, S. C., STEINMAN, R. M., and COHN,Z. A., 1977, Endocytosis. Annual Report of Biochemistry, 46, 669-722. E. J. W., and OVERBEEK, J. T. G., 1948, Theory of the Stability of Lyophobic Colloids, VERWEY, Elsevier, Amsterdam. WATANABE, Y., KONDO,T., MIYAMOTO, M., and SASAKAWA, S., 1984, Interaction of microcapsules with human polymorphonuclear leucocytes. Chemical and Pharmaceutical Bulletin, 32, 2788-2794.
