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The coalescence behavior of oil droplets stabilized by phospholipid emulsifiers
NOTES The Coalescence Behavior of Oil Droplets Stabilized by Phospholipid Emulsifiers INTRODUCTION
Coalescence Studies
Emulsions made from vegetable oils, stabilized by complex phospholipid mixtures in the form of egg or soy lecithins, are used in parenteral nutrition as a source of calories (1). The stability of these fat emulsions can be related to the physical nature of the layer of emulsifier at the oil-water interface and both mechanical and electrical barriers to droplet coalescence can be envisaged (1). Egg and soy lecithins are composed largely of phosphatidylcholine (PC) and phosphatidylethanolamine (PE) together with other minor lipid components such as phosphatidic acid, phosphatidylserine, cholesterol, etc., as well as hydrolysis products present in the original emulsifier or formed during the production of the emulsion (e.g., lysophosphatidylcholine). Rydhag (2) and Rydhag and Winton (3) have investigated the properties of phospholipid, water, and oil mixtures in terms of the relevant phase diagrams and have concluded that liquid crystalline gel systems can be formed when mixtures of phosphatidyleholine and phosphatidylethanolamine contain small proportions of ionizable lipids (either phospholipids or fatty acids). However, the optimal combination of phospholipids for maximum emulsion stability has not been defined. Therefore, we have studied the effect of phospholipid composition on the properties of soybean oil in water emulsion systems. In the present report we describe investigation of the effect of phospholipid composition on the coalescence behavior of soybean oil droplets, the phospholipid mixtures being made from pure phospholipid components.
The rest times of single droplets at the plane liquid interface were measured using a coalescence cell based on a design by Nielson et al. (6) as modified by Davis and Smith (7), The distribution of rest times for a minimum of 80 droplets was analyzed by plotting the log number n o t coalesced against time and were characterized by the first-order rate constant and half-life (Tu2). In some cases the plots demonstrated a lag time (t~) before the first order decrease in the number not coalesced. In such cases a time for 50% to coalesce (q/2) was obtained where (7)
EXPERIMENTAL
td = t m -
Tl/2.
RESULTS
Interfacial Tension The addition of phospholipid to the soybean/water system brought about a time-dependent reduction in interfacial tension. This fall was rapid over the first few minutes followed by a slower process of equilibration to 3 h. The addition of phosphatidylethanolamine (PE) at different ratios (4:1, 2:1, 1:1) to phosphatidylcholine (PC) solutions at a total phospholipid concentration of 10-4 mole dm -3 had no effect on the interracial tension. At a constant PC:PE ratio of 4:1 and a concentration of 10-4 mole dm -3 the effect of minor components was studied (Table I). Added material had little effect on the interracial tension of the PC:PE system, except for the case of lysophospbatidylcholine(LPC) where a significant reduction in interfacial tension was observed.
Materials and Methods Coalescence Soybean oil and the phospholipids were as described by Davis and Hansrani (4). Sphingomyelin (Type 1 from bovine brain) was obtained from Sigma. The purity of all phospholipids was checked using thin layer chromatography as before (4). Water was triple-distilled from an all glass still and the purity was monitored by surface tension and conductivity measurements. The interracial tension between the vegetable oil and aqueous phospholipid mixtures at 25 _+ 0.1°C was measured using the pendant drop method with an apparatus based on the design of Andreas et al. (5).
The effect of added PE on the stability of oil droplets in PC:PE mixed systems at a total concentration of 10-4 M is shown in Fig. 1. An increase in the proportion of PE to PC results in the T m changing from 2.7 s for pure PC to 5.2 for PC:PE at 4:1 (and 2:1) and 6.0 at PC:PE at 1:1. At constant PC:PE ratio (4:1) and concentration (10 -4 M) added minor phospholipids (concentration 5 to 30 #g/ml--approximately 5 × 10-6 to 6 × 10-5 M) had differing effects (Fig. 2). At low concentrations some additives increased the tendency to coalesce (e.g., SP,
285 0021-9797/85 $3.00 Journal of Colloid and Interface Science, Vol. 108, No. 1, November 1985
Copyright © 1985 by Academic Press, Inc. All rights of reproduction in any form reserved.
286
NOTES 100
TABLE I The Effect of Minor Components on Interfacial Tension between Soybean Oil and Phospholipid Sols (25°C, 300s Aging Period)
&o Phosphatide
None PC:PE (4:1, 10-4 M) with additives at 10/tg/ml Lysolecithin Phosphatidic acid Sphingomyelin Phosphatidylserine Phosphatidylinositol Cholesterol Cholesterol oleate
Interracial tension (raN m -~)
o~ u
45.3
0~
\
40.2 27.5 37.5 40.9 42.1 37.2 36.5 39.9
CH) but at concentrations of 10 /~g/ml and above, all additives except CH gave an enhanced stability. For a given concentration the phosphatides can be ranked as follows (with necessary interpolation): LPC > SP = PS =PA>CO>PI>CH. DISCUSSION It is known that fat emulsions prepared using pure phospholipid components (PC and PE) give rise to
100
4C ca
c
20
10 0
20
I S
I ~ 10 Time (secs)
I 15
I 20
FlG. 1. The effect of PC:PE ratio on droplet rest times at a constant phospholipid concentration 10-4 M. Legend: ©--PC; O--PC:PE (4:1); (A)--PC:PE (1:1). Journal of Colloid and Interface Science. Vol. 108, No. 1, November 1985
10
I 10
I I 20 30 Time (secs)
I 40
FIG. 2. The effect of added PS on droplet rest times (PC:PE, 4:1 at 10-4 M), Legend: A--5 ~g/ml PS; A - 10 ~g/ml PS; O--25 #g/ml PS; (3--75 #g/ml PS.
systems demonstrating poor stability (8) and Rydhag (2) has shown that it is necessary to incorporate so-called ionic lipids into lecithin (soy or egg) emulsifiers in order to produce stable emulsions. These materials, such as PA and PS as well as fatty acid soaps, not only increased the surface charge on the emulsion droplets but also formed complex liquid crystalline interfacial films at the o/w interface. These liquid crystalline gel structures would be expected to provide good mechanical stability and thereby retard coalescence. In this respect it is interesting to note that when simple phospholipid vesicles approach each other structural changes can occur that include separation (demixing) of component of a mixed system (9). Such a demixing process would explain the poor stability of emulsions stabilized by PC and PE alone. Added ionic lipids PS, PA, SP, LPC, and CHO all give an increased stability of droplets at the plane oilwater interface (Table II), probably through their ability to produce complex viscoelastic gel-like structures at the o/w interface with PC and PE (3). The complex film so created would also act to prevent demixing and early coalescence. The profound stabilizing effect of LPC is interesting. LPC, like the parent molecule PC, is zwitterionic and carries no net charge over a wide range of pH. It is a very effective surface active agent and is adsorbed strongly at the o/w interface (10). The incorporation of CH has little effect on the coalescence behavior of the droplets. This is rather surprising since CH is used to increase the stability of phospholipid vesicles by decreasing membrane fluidity (11). Clearly, the analogies between large emulsion droplets stabilized by phospholipids and small phospholipid vesicles cannot be extended too far. For example, it is known that LPC can intercalate into the vesicle bilayer
NOTES
287
TABLE I1
CONCLUSIONS
The Effect of Phosphatide Composition on the Stability of Single Droplets at the Plane Oil-Water Interfacea
The coalescence of oil droplets stabilized by phospholipids can be altered by the addition of small quantities of minor components that will form liquid crystalline phases at the oil/water interface. Lysophosphatidylcholine had a greater effect than did sphingomyelin, phosphatidylserine, and phosphatidic acid. Cholesterol was not particularly effective in changing interfacial characteristics.
Composition PC PC/PE 4:1 2:1 1:1
PC/PE 4:1 with added minor components Phosphatidylserine (PS) Phosphatidylinositol (PI) Phosphatidic acid (PA) Sphingomyelin (SP)
Lysolecithin (LPC)
Cholesterol (CH)
Cholesterol oleate (CHO)
Conc. 10-4 M
7"1/2s
Lagtime (s)
10-4 M 10-4 M
2.7 5.2 5.2 6.0
2.6 3.7 5.0 4.9
10-4 M
5.2
3.7
9.3 11.6 12.2 4.9 5.9 10.4 6.5 9.6 21.0 3.5 6.8 13.8 13,3 20.4 38.4 180.6 3.6 4.4 7.7 7.4 8.5 16.1 16.8
5.7 5.4 8.5 5.3 5.8 4.3 3.7 6.4 5.9 2. I 11.6 7.5 4.5 19.3 51.2 60.5 4.9 7.4 6.6 5.0 11.2 5.1 5.0
10 -4 M
~g]ml 5 10 25 10 20 30 2 7.5 15 1.3 2.5 10 5 10 20 30 10 20 40 5 l0 15 20
a Droplets aged for 30 s before detachment. Oil-water interface aged for 180 min.
and thereby increase the permeability of the vesicle to entrapped compounds (12), whereas in the present work LPC provides enhanced stability.
REFERENCES 1. Davis, S. S., in "Advances in Clinical Nutrition," I. D. A. Johnson, (Ed.), pp. 213-239. Lancaster, MTP Press, 1983. 2. Rydhag, L., Fette Seifen Anstriehm. 81, 168 (1979). 3. Rydhag, L., and Wilton, I., J. Amer. Oil Chem. Soc. 58, 830 (1981). 4. Davis, S. S., and Hansrani, P. K., Int. J. Pharmaceut., 23, 69 (1985). 5. Andreas, J. M., Hauser, E. A., and Tucker, W. B., J. Phys. Chem. 42, 1001 (1938). 6. Nielsen, L. E., Wall, R., and Adams, G., J. Colloid Sci. 13, 441 (1958). 7. Davis, S. S., and Smith, A., Kolloid-Z. Z. Polym. 251, 337 (1973). 8. Yeadon, D. A., Goldblatt, L. A., and Altschull, A. M., J. Amer. Oil Chem. Soc. 35, 458 (1958). 9. Rang, R. P., Ann. Rev. Biophys. Bioeng. 10, 277 (1981). 10. Saunders, L., and Thomas, I. L., J. Pharm. Pharmac. 10, 182T (1958). 11. Yatvin, M. B., and Lelkes, P. I., Med. Phys. 9, 149 (1982). 12. Attwood, D., and Florence, A. T., "Surfactant Systems, Their Chemistry, Pharmacy, and Biology." Chapman & Hall, London, 1983. S. S. DAVIS P. HANSRAN1
Department of Pharmacy University of Nottingham Nottingham, NG7 2RD, England Received September 18, 1984; accepted February 7, 1985
Journalof ColloMand InterfaceScience,Vol. 108,No. 1, November1985
Coalescence Studies
Emulsions made from vegetable oils, stabilized by complex phospholipid mixtures in the form of egg or soy lecithins, are used in parenteral nutrition as a source of calories (1). The stability of these fat emulsions can be related to the physical nature of the layer of emulsifier at the oil-water interface and both mechanical and electrical barriers to droplet coalescence can be envisaged (1). Egg and soy lecithins are composed largely of phosphatidylcholine (PC) and phosphatidylethanolamine (PE) together with other minor lipid components such as phosphatidic acid, phosphatidylserine, cholesterol, etc., as well as hydrolysis products present in the original emulsifier or formed during the production of the emulsion (e.g., lysophosphatidylcholine). Rydhag (2) and Rydhag and Winton (3) have investigated the properties of phospholipid, water, and oil mixtures in terms of the relevant phase diagrams and have concluded that liquid crystalline gel systems can be formed when mixtures of phosphatidyleholine and phosphatidylethanolamine contain small proportions of ionizable lipids (either phospholipids or fatty acids). However, the optimal combination of phospholipids for maximum emulsion stability has not been defined. Therefore, we have studied the effect of phospholipid composition on the properties of soybean oil in water emulsion systems. In the present report we describe investigation of the effect of phospholipid composition on the coalescence behavior of soybean oil droplets, the phospholipid mixtures being made from pure phospholipid components.
The rest times of single droplets at the plane liquid interface were measured using a coalescence cell based on a design by Nielson et al. (6) as modified by Davis and Smith (7), The distribution of rest times for a minimum of 80 droplets was analyzed by plotting the log number n o t coalesced against time and were characterized by the first-order rate constant and half-life (Tu2). In some cases the plots demonstrated a lag time (t~) before the first order decrease in the number not coalesced. In such cases a time for 50% to coalesce (q/2) was obtained where (7)
EXPERIMENTAL
td = t m -
Tl/2.
RESULTS
Interfacial Tension The addition of phospholipid to the soybean/water system brought about a time-dependent reduction in interfacial tension. This fall was rapid over the first few minutes followed by a slower process of equilibration to 3 h. The addition of phosphatidylethanolamine (PE) at different ratios (4:1, 2:1, 1:1) to phosphatidylcholine (PC) solutions at a total phospholipid concentration of 10-4 mole dm -3 had no effect on the interracial tension. At a constant PC:PE ratio of 4:1 and a concentration of 10-4 mole dm -3 the effect of minor components was studied (Table I). Added material had little effect on the interracial tension of the PC:PE system, except for the case of lysophospbatidylcholine(LPC) where a significant reduction in interfacial tension was observed.
Materials and Methods Coalescence Soybean oil and the phospholipids were as described by Davis and Hansrani (4). Sphingomyelin (Type 1 from bovine brain) was obtained from Sigma. The purity of all phospholipids was checked using thin layer chromatography as before (4). Water was triple-distilled from an all glass still and the purity was monitored by surface tension and conductivity measurements. The interracial tension between the vegetable oil and aqueous phospholipid mixtures at 25 _+ 0.1°C was measured using the pendant drop method with an apparatus based on the design of Andreas et al. (5).
The effect of added PE on the stability of oil droplets in PC:PE mixed systems at a total concentration of 10-4 M is shown in Fig. 1. An increase in the proportion of PE to PC results in the T m changing from 2.7 s for pure PC to 5.2 for PC:PE at 4:1 (and 2:1) and 6.0 at PC:PE at 1:1. At constant PC:PE ratio (4:1) and concentration (10 -4 M) added minor phospholipids (concentration 5 to 30 #g/ml--approximately 5 × 10-6 to 6 × 10-5 M) had differing effects (Fig. 2). At low concentrations some additives increased the tendency to coalesce (e.g., SP,
285 0021-9797/85 $3.00 Journal of Colloid and Interface Science, Vol. 108, No. 1, November 1985
Copyright © 1985 by Academic Press, Inc. All rights of reproduction in any form reserved.
286
NOTES 100
TABLE I The Effect of Minor Components on Interfacial Tension between Soybean Oil and Phospholipid Sols (25°C, 300s Aging Period)
&o Phosphatide
None PC:PE (4:1, 10-4 M) with additives at 10/tg/ml Lysolecithin Phosphatidic acid Sphingomyelin Phosphatidylserine Phosphatidylinositol Cholesterol Cholesterol oleate
Interracial tension (raN m -~)
o~ u
45.3
0~
\
40.2 27.5 37.5 40.9 42.1 37.2 36.5 39.9
CH) but at concentrations of 10 /~g/ml and above, all additives except CH gave an enhanced stability. For a given concentration the phosphatides can be ranked as follows (with necessary interpolation): LPC > SP = PS =PA>CO>PI>CH. DISCUSSION It is known that fat emulsions prepared using pure phospholipid components (PC and PE) give rise to
100
4C ca
c
20
10 0
20
I S
I ~ 10 Time (secs)
I 15
I 20
FlG. 1. The effect of PC:PE ratio on droplet rest times at a constant phospholipid concentration 10-4 M. Legend: ©--PC; O--PC:PE (4:1); (A)--PC:PE (1:1). Journal of Colloid and Interface Science. Vol. 108, No. 1, November 1985
10
I 10
I I 20 30 Time (secs)
I 40
FIG. 2. The effect of added PS on droplet rest times (PC:PE, 4:1 at 10-4 M), Legend: A--5 ~g/ml PS; A - 10 ~g/ml PS; O--25 #g/ml PS; (3--75 #g/ml PS.
systems demonstrating poor stability (8) and Rydhag (2) has shown that it is necessary to incorporate so-called ionic lipids into lecithin (soy or egg) emulsifiers in order to produce stable emulsions. These materials, such as PA and PS as well as fatty acid soaps, not only increased the surface charge on the emulsion droplets but also formed complex liquid crystalline interfacial films at the o/w interface. These liquid crystalline gel structures would be expected to provide good mechanical stability and thereby retard coalescence. In this respect it is interesting to note that when simple phospholipid vesicles approach each other structural changes can occur that include separation (demixing) of component of a mixed system (9). Such a demixing process would explain the poor stability of emulsions stabilized by PC and PE alone. Added ionic lipids PS, PA, SP, LPC, and CHO all give an increased stability of droplets at the plane oilwater interface (Table II), probably through their ability to produce complex viscoelastic gel-like structures at the o/w interface with PC and PE (3). The complex film so created would also act to prevent demixing and early coalescence. The profound stabilizing effect of LPC is interesting. LPC, like the parent molecule PC, is zwitterionic and carries no net charge over a wide range of pH. It is a very effective surface active agent and is adsorbed strongly at the o/w interface (10). The incorporation of CH has little effect on the coalescence behavior of the droplets. This is rather surprising since CH is used to increase the stability of phospholipid vesicles by decreasing membrane fluidity (11). Clearly, the analogies between large emulsion droplets stabilized by phospholipids and small phospholipid vesicles cannot be extended too far. For example, it is known that LPC can intercalate into the vesicle bilayer
NOTES
287
TABLE I1
CONCLUSIONS
The Effect of Phosphatide Composition on the Stability of Single Droplets at the Plane Oil-Water Interfacea
The coalescence of oil droplets stabilized by phospholipids can be altered by the addition of small quantities of minor components that will form liquid crystalline phases at the oil/water interface. Lysophosphatidylcholine had a greater effect than did sphingomyelin, phosphatidylserine, and phosphatidic acid. Cholesterol was not particularly effective in changing interfacial characteristics.
Composition PC PC/PE 4:1 2:1 1:1
PC/PE 4:1 with added minor components Phosphatidylserine (PS) Phosphatidylinositol (PI) Phosphatidic acid (PA) Sphingomyelin (SP)
Lysolecithin (LPC)
Cholesterol (CH)
Cholesterol oleate (CHO)
Conc. 10-4 M
7"1/2s
Lagtime (s)
10-4 M 10-4 M
2.7 5.2 5.2 6.0
2.6 3.7 5.0 4.9
10-4 M
5.2
3.7
9.3 11.6 12.2 4.9 5.9 10.4 6.5 9.6 21.0 3.5 6.8 13.8 13,3 20.4 38.4 180.6 3.6 4.4 7.7 7.4 8.5 16.1 16.8
5.7 5.4 8.5 5.3 5.8 4.3 3.7 6.4 5.9 2. I 11.6 7.5 4.5 19.3 51.2 60.5 4.9 7.4 6.6 5.0 11.2 5.1 5.0
10 -4 M
~g]ml 5 10 25 10 20 30 2 7.5 15 1.3 2.5 10 5 10 20 30 10 20 40 5 l0 15 20
a Droplets aged for 30 s before detachment. Oil-water interface aged for 180 min.
and thereby increase the permeability of the vesicle to entrapped compounds (12), whereas in the present work LPC provides enhanced stability.
REFERENCES 1. Davis, S. S., in "Advances in Clinical Nutrition," I. D. A. Johnson, (Ed.), pp. 213-239. Lancaster, MTP Press, 1983. 2. Rydhag, L., Fette Seifen Anstriehm. 81, 168 (1979). 3. Rydhag, L., and Wilton, I., J. Amer. Oil Chem. Soc. 58, 830 (1981). 4. Davis, S. S., and Hansrani, P. K., Int. J. Pharmaceut., 23, 69 (1985). 5. Andreas, J. M., Hauser, E. A., and Tucker, W. B., J. Phys. Chem. 42, 1001 (1938). 6. Nielsen, L. E., Wall, R., and Adams, G., J. Colloid Sci. 13, 441 (1958). 7. Davis, S. S., and Smith, A., Kolloid-Z. Z. Polym. 251, 337 (1973). 8. Yeadon, D. A., Goldblatt, L. A., and Altschull, A. M., J. Amer. Oil Chem. Soc. 35, 458 (1958). 9. Rang, R. P., Ann. Rev. Biophys. Bioeng. 10, 277 (1981). 10. Saunders, L., and Thomas, I. L., J. Pharm. Pharmac. 10, 182T (1958). 11. Yatvin, M. B., and Lelkes, P. I., Med. Phys. 9, 149 (1982). 12. Attwood, D., and Florence, A. T., "Surfactant Systems, Their Chemistry, Pharmacy, and Biology." Chapman & Hall, London, 1983. S. S. DAVIS P. HANSRAN1
Department of Pharmacy University of Nottingham Nottingham, NG7 2RD, England Received September 18, 1984; accepted February 7, 1985
Journalof ColloMand InterfaceScience,Vol. 108,No. 1, November1985