Colloids und Surfaces A: Physicochemical and Engineering Aspects, 84 (1994) 71-79 0927-7757/94/$07.00 0 1994 - Elsevier Science B.V. All rights reserved.
2 June 1993; accepted
S. Habif, Henri L. Rosano*
Department of Chemistry, The City College Street, New York, NY10031, USA
of the City University
of New York, Convent
Abstract Flocculation of oil-in-water emulsions composed of a perfluorochemical emulsified by phospholipids was studied using photon correlation spectroscopy and viscoelasticity measurements in unsteady oscillatory flow. Flocculation gives rise to emulsion instability but can be prevented by the addition of a negatively charged surfactant to phosphatidylcholine, the zwitterionic phospholipid used as a primary emulsifier, or by using a saccharide solution as a continuous phase. The study indicates that both electrostatic (Coulombic) repulsive forces and hydration (steric) forces play a role in preventing flocculation. Various minor components of the egg yolk phospholipids used in commercial emulsion preparation probably stabilize the emulsion by increasing both electrostatic and hydration repulsion. Key words: Emulsion;
Introduction Perfluorochemical (PFC) emulsions have already been developed for various biomedical applications including red blood cell substitutes and medical imaging [l-l 11. General criteria for such preparations include injectability (nontoxicity, sterility) and a stable shelf-life of several months, preferably at temperatures above 0°C. One practical formulation (developed by researchers at Alliance Pharmaceutical Corp.) consists of the PFC perflubron (perfluorooctylbromide; PFOB) dispersed in a saline solution with egg yolk phospholipids (EYP) as an emulsifier. This emulsion has been found to be stable over 4 years at 5°C [ 121 and has been characterized [ 131 as consisting of (a) PFOB droplets (about 250 nm in diameter) encapsulated in a phospholipid monolayer and (b) PFOB-free phospholipid vesicles (SO-100 nm in diameter). The present paper deals with the relationship between the stability of *Corresponding author. SSDI
concentrated PFC-in-aqueous emulsions and the interfacial properties of the phospholipid-based emulsifier used. Since EYP is a variable and complex mixture of phospholipids (essentially phosphatidylcholine (PC) and phosphatidylethanolamine (PE)) and minor components (cholesterol, acidic phospholipids, free fatty acids, sphingomyelin, pigments), we used phospholipon 90H (PL), a hydrogenated phospholipid with a simpler composition, as a standard and replacement. Materials and methods Phospholipon 90H (PL) was purchased from American Lecithin Company (Danbury, CT) and was used without further purification. Perflubron (PFOB) was generously supplied by Alliance Pharmaceutical Corp. (San Diego, CA) and was Ethyleneditwice before use. redistilled aminetetraacetic acid was purchased from Harleco (Philadelphia, PA). Dipalmitoyl phosphatidylcholine (DPPC), stearylamine (99%) and choles-
C.B. Oleksiak et al./Colloids
(tris salt) were purchased
Sigma Chemical Company. were purchased from Fisher
All other Scientific.
Surfaces A: Physicochem.
Several emulsions PFOB/saline concentration the
perature were prepared
ratios in order to find the optimum of the dispersed phase (PFOB) in 43352%
EYP/PFOB ratio was found to be 9.5% (w/v) corresponding to EYP concentrations of 4% and 5% (w/v) for 43% and 52% (v/v) PFOB emulsions respectively. For a given amount of the dispersed phase, the more surfactant in the emulsion, the smaller the particle size, until a limiting lower value is reached that corresponds to the maximum curvature of the film; this value depends on the structure and spatial configuration of the surfactant molecules at the interface. Ishii et al.  found a similar optimum EYP/oil ratio in their preparation of fat emulsions for parenteral nutrition. When more surfactant is used in the formulation (EYP/PFOB ratios larger than 9.5% (w/v)), the excess EYP will be present as PFOB-free phospholipid vesicles. The excess surfactant was deemed detrimental to the stability of the emulsion by other researchers [ 151. We found no evidence that the excess EYP was either detrimental or beneficial to emulsion stability, but smaller amounts of emulsifier are certainly desirable a priori from both biomedical and cost-efficiency standpoints
[ 161. In addition,
that the excess phospholipid
(as vesicles) has detri-
mental effects on cholesterol metabolism in the case of fat emulsions used in parenteral nutrition has been presented by Hajri et al. [ 161 and Untracht [ 171. In the experiments described in the present paper, the concentrations of dispersed phase and continuous phase are kept constant, at 43% (v/v) and 53% (v/v) respectively, as is the emulsifier concentration, at 50 mM (4% (w/v) for pure PL). (PL is a hydrogenated phospholipid with a phase transition temperature (PTT) of 52°C as deter-
mined by differential scanning calorimetry (DSC). To be water dispersible it should be in its liquid crystalline phase (above its PTT): the emulsion preparation
Eng. Aspects 84 (1994)
was accordingly greater
than the phospholipid
at a temPTT.)
The emulsifier is first dispersed in the aqueous phase by magnetic stirring at 65-70°C until a milky dispersion is obtained. The mixture is then submitted to mechanical work (3 min at 13 500 rev min at 65 ‘C) in an Ultra-Turrax
(IKA Works, Inc., Cincinnati, OH) fitted with a dispersing tool S25 KG-25 F. The oil is added and the system is mixed (5 min at 13 500 rev min-’ at 65°C) until it yields a homogeneous preemulsion which is then fed into a microfluidizer (MF) MllO-T (Microfluidics Corp., Newton, MA) (69 MPa (10 000 psi) with 5-7 passes) and the resulting emulsion, contained in glass vials with rubber stoppers and crimped aluminum seals, is sterilized in a static autoclave (AMSCO) at 121 “C and 140 kPa (20 psi) for 15 min. The total volume of each emulsion sample is 300 ml.
Particle size measurements Photon correlation spectroscopy (PCS) was used to evaluate the particle size of the emulsion. Measurements were carried out on a ZetaSizer 3 (ZS3) particle electrophoresis and multiangle particle size analyzer (Malvern) equipped with a 5 mW He-Ne laser of wavelength 633 nm. All the emulsion samples were diluted 500-2000 times prior to the measurements, which were carried out in the AZ4 cell (4 mm in diameter) at 25 “C at a fixed angle of 90’.
The zeta potential of the emulsion particles was measured by microelectrophoresis using the ZS3 operating in the cross-beam mode at 25°C with samples diluted 500-2000-fold and contained in the AZ4 cell.
C.B. Oleksiak et al.JColloids
Surfaces A: Physicochem.
Eng. Aspects 84 ( 1994 ) 71-79
‘surface isotherm measurements Shulman-Stenhagen [IS] [ 193 monolayer techniques
at 45” angle) scopy (TEM). and Shulman-Rideal were used to investi-
Results and discussion
gate the surface properties (surface pressure isotherms, collapse pressures, reversibility of the film compressions and surface potential isotherms) of
Pure phospholipids have been shown to reduce dramatically the PFOB-saline interfacial tension
of the free energy
strates. A complete description of the apparatus and experimental parameters has been given in a
for its stability is the presence of a structured interfacial film. Compression isotherms of monolayers of EYP, PL and DPPC (Fig. 1) show that in all cases the film has a high collapse pressure (45-65 mN m-r) and a high surface potential (+ 400 to + 550 mV for a molecular surface area of 50 A’) and that the compression/decompression curves have no, or very little, hysteresis. Emulsions are stabilized by the presence of a film at the oil water interface; the tighter the packing of the surfactant molecules in the interfacial monolayer, the more stable the emulsion . The collapse pressure corresponds to the pressure at which surfactant molecules are squeezed out of the monolayer. Since the energy of attraction between the surfactant acyl chains varies inversely as the sixth power of the distance, the tighter the packing, the higher the value of the collapse pressure. Therefore a high value of the collapse pressure proves that the phospholipid film is highly structured and regularly packed. Too many irregularities in the film structure would result in a weaker film unable to withstand high pressure (i.e. a film with low collapse pressure). A film of pure DPPC or PL (phospholipids with saturated lipid chains) has a collapse pressure of 65 mN m-l, evidence of a well-packed film. A lower value (45 mN m-‘) was found for EYP; the lesser degree of organization of the EYP film is due, first, to a kink in the unsaturated lipid chains resulting from double bonds in the cis position and, second, to the presence of impurities (minor components of EYP such as cholesterol or free fatty acids) capable of disturbing the packing. A high value of the surface potential is further
in oscillatory flow
Both the elastic and viscous moduli
of the emul-
sions (G’ and G” respectively) were determined using a Vilastic 3 apparatus (Vilastic Scientific, Inc.; Vilastic 3 viscoelasticity analyzer). Each system to be tested fills a precision cylindrical tube (radius, a; tube length, L) and is submitted to an oscillatory flow (frequency f= 2 Hz). The pressure gradient along, and the volume flow through, the tube (P and U respectively) are measured while the shear strain y is increased. The shear stress, shear strain and shear rate at the wall of the tube are calculated [20,21]. The part of the shear stress (TccP(a/2L)) that is in phase with the shear strain is called the viscous stress (r,). The part of the shear stress that is 90” out of phase with the shear strain is called the elastic stress (7s). The elastic and viscous moduli of the system are defined as follows: elastic modulus G’ = z,/y; viscous modulus G” = rvly. All systems were investigated at ature. Performance characteristics diameter of the measurement tube material under test. Data below the automatically discarded.
Sample preparation for transmission
room tempervary with the and with the noise level are
microscopy After rapid freezing in liquid propane, each sample was fractured and coated (platinum replica
in the emulsification process. has been prepared, a necessary
Once the condition
C.B. Oleksiak et al.,‘Colloids
Swfuces A: Phy.sicochem
EII~. Asprcts X4 ( 1994 ) 71- 79
of a highly organized potential
vector sum of the dipole moment of each phospholipid molecule under the surface of the air-ionizing electrode (about 1014 phospholipid molecules). A well-organized film results in aligned dipole moments,
which in turn
yield a maximum
for the surface potential . The compression/decompression
that the system does not lose its integrity while compressed. Hence, phospholipids yield emulsion droplets with a structured film that can take stress and return to its equilibrium state with no physical change. A series of emulsions was prepared using the process described above. The criteria used to assess the emulsion stability are (a) no phase separation, (b) no or very little coalescence, and (c) no flocculation. The emulsion particle size was measured by photon correlation spectroscopy (PCS) [2.5]. Emulsion particles are in constant Brownian motion, which causes the intensity of light scattered from the particles to vary with time. Large particles move more slowly than small particles, so the rate of fluctuation of the scattered light is also slower. PCS uses the rate of change of these light fluctuations to determine the size distribution of the particles scattering the light. The particle size was measured for samples prior to and after sterilization. To avoid multiple scattering, emulsions should be diluted. and we diluted each sample in two ways: in a solution with the same composition as the continuous phase, and in distilled water. If
surface area (Az/molecute)
Fig. 1. (Left) surface pressure and surface potential isotherms and (right) surface pressure isotherms (compression,‘decompression) of (a) commercial EYP, (b) DPPC and (c) PL monolayers on saline at pH 6.X and 25 C.
a sample yields a much smaller particle size value when diluted in water than when diluted in the continuous phase, the larger size is the size of a particle aggregate that breaks down into single droplets when diluted in water (due to the critical flocculation concentration of the emulsion ). If dilutions in the two solvents lead to the same observed particle size, no flocculation occurred and the “real” size of an individual droplet is measured in both solvents. An increase in “real”
C.B. Oleksiuk et ul./Colloids
coalescence. An emulsion pure
Surfaces A: Physicochem.
Eng. Aspects 84 (1994)
PL is unstable.
with of the
emulsions shows a settling of a gel-like phase with a high resistance to the flow (thixotropic behavior) at the bottom
of the vial (supernatant/infranatant
ratio is 50: 50 (v/v)). Such settling,
and the viscous
nature of the settled phase, are both characteristic of a flocculated system. Furthermore, the particle size increases from 650 nm before sterilization to about 1 ym after sterilization when the emulsion is diluted in saline solution, while with dilution in distilled water the particle size (about 300 nm) is similar before and after sterilization, indicating a flocculated system. The flocculation phenomenon was confirmed by viscoelastic measurements. With an increase in shear strain, the elastic modulus G’ (stored energy) decreases following a two-slope pattern (Fig. 2(a)). The initial decrease in elastic modulus (first slope) corresponds to a breakage of the floes: the smaller the floe, the less energy it can store [ 271. Once the shear strain is high enough to break all the floes, a further decrease in elastic modulus (second slope) results from the reduced ability of single droplets to store energy - itself a result of their stress-induced flattening. The droplets will not start to flatten until a threshold shear strain value is reached. If this value is higher than the minimum shear strain needed to break the floes, a plateau will be observed before the second slope. In the second part of the experiment, the shear strain is reduced to its original value. The curve of the viscous modulus G” versus shear strain follows the same pattern. First, the decrease in viscous modulus with increased shear strain is due to the breakage of the floes; then, once the floes are broken, the single droplets align themselves with the flow, resulting in a reduction of the viscous modulus. This system exhibits high viscous modulus values (100-2000 mPa) that make it unsuitable for intravenous injection. A transmission
for the same system after a 2 month at room
shows a multitude
of small clustered
around the surface of very large droplets (5-10 urn in diameter), evidence that PFOB-PL-saline systems flocculate and eventually coalesce. In contrast, PFOB-EYP-saline systems Corp., San (Imagent TM Alliance Pharmaceutical Diego, CA) are stable. After 28 months of storage at room temperature, the minimal settling displayed by the emulsion is readily reversible by slight hand shaking. Particle size measurements and a TEM study show that no coalescence or flocculation occurred [ 133. Viscoelastic measurements also show no evidence of flocculation (Fig. 2(b)): the viscous modulus is low (11 mPa) and decreases regularly (a single slope) with increasing shear strain due to deformation and alignment of the droplets with the flow, followed by a plateau when a maximum deformation is reached. The elastic modulus follows the same pattern when the shear strain is increased. Previous work on fat emulsions [26,28-311 showed that flocculation could be prevented by the addition of negatively charged surfactants to the pure phospholipids. An increase in the amplitude of the zeta potential was correlated with a lesser tendency to flocculate. The study of PFOB in saline emulsions using a binary mixture of PL and a positively or negatively charged surfactant, stearylamine (SA) and cholesteryl hemisuccinate (CHS) respectively, showed that electrostatic forces alone could not explain the flocculation phenomenon. That an emulsion (PL/SA 9 : 1 (mol/mol)) with a positive zeta potential (17 i 7 mV) flocculates is demonstrated in three ways. First, a viscous phase settles at the bottom of the vial (supernatant/ infranatant ratio is 27: 73 (v/v)). Second, the particle size is high (around 600 nm) both before and after sterilization when the emulsion is diluted in saline. Dilution in water yields a system with a 330 nm particle size both before and after sterilization. Finally, viscoelastic measurements (Fig. 2(c)) show a high viscous modulus (90-70 mPa) and a
C.B. Oleksiak et al./Colloids Surf'aces A: Physicochem. Eng. Aspects 84 ( 1994 10000 '
...... 100 1000
. . . . . . . ,] . . . . . . . . , .001 .01 .1
....... , 1
........ i ........ , ....... 10 100 1000
....... 100 1000 ,
1 O0 '
(e) Fig. 2. (Caption opposite.)
I G '
•0 0 1
, . '
C.B. Oleksiak et al./Colloids Surfaces A: Physicochem. Eng. Aspects 84 (1994) 71 79
high elastic modulus (100-10 mPa), both of which decrease with increasing shear strain according to a two-slope pattern. An emulsion (PL/CHS 9' 1 (mol/mol)) with a negative zeta potential (-14_+ 6 mV) is stable. After 4 months of storage at room temperature, the emulsion shows very little settling (the supernatant/infranatant ratio is 4:96 (v/v)), the particle size is around 300 nm before and after sterilization both in saline and water, and viscoelastic measurements (Fig. 2(d)) reveal a low viscous modulus (10 mPa), constant under increasing shear strain (hard-sphere behavior is further indicated by a very low elastic modulus (below noise level)). A negatively charged interface seems to prevent flocculation more effectively than either a positively charged interface or an uncharged one (a pure PL interface shows a zeta potential value of 3 _+ 7 mV). Two systems with zeta potentials of comparable magnitude but with opposite signs ( P L - S A and P L - C H S ) yield emulsions with different degrees of stability, so flocculation cannot be explained solely by electrostatic force considerations. The zeta potential is the potential at the surface of a sphere having a radius equal to the shear radius; its value is smaller than the value of the potential at the emulsifier head-group surface because of the distance and the screening effect of the counterions. A negatively charged interface will be screened by cations, a positively charged interface by anions. The difference in stability between the two systems studied (PL SA and P L - C H S ) probably arises from the fact that the counterions have different degrees of hydration. The cations, being more hydrated than the anions, are more effective in preventing flocculation because of the high repulsive hydration forces. P F O B emulsions prepared with pure PL in a saccharide solution show that hydration forces alone can significantly limit flocculation.
P F O B PL-9.5% sucrose solution emulsions show some settling (the supernatant/infranatant ratio is 25 : 75 (v/v)) after a 4 month storage period at room temperature. The particle size is somewhat larger when the emulsion is diluted in the continuous phase (sucrose solution) than when it is diluted in water (450 nm and 300 nm respectively) both before and after sterilization. The viscous modulus is low (15-10 mPa) and the change of slope due to the breakage of small flocs is hardly noticeable (Fig. 2(e)). The elastic modulus curve shows a two-slope pattern, but the amplitude of the stored energy is low compared with that observed for the P F O B - P L saline emulsion (3-0.1 mPa and 2000 8 mPa respectively), due probably to the smaller size of the flocs (450nm against 1 jam) and possibly to the strength of the interdroplet bonds. P F O B P L - 5 % dextrose solution emulsions show less flocculation: very little settling was observed after a 4 month storage period at room temperature (the supernatant/infranatant ratio is 10 : 90 (v/v)). The particle size is 360 nm when the emulsion is diluted in the 5% dextrose solution and 300 nm when it is diluted in water, both before and after sterilization. The viscous modulus is significantly smaller than that of the P F O B - P L - s a l i n e emulsion (40 11 mPa against 2000-90 mPa) and follows a one-slope pattern, as does (probably) the elasticity (Fig. 2(f)), provided we have a plateau for ~ <0.3. Previous work on the stability of fat emulsions for parenteral nutrition provides further evidence of the stabilizing effects of glucose and amino acids, showing the importance of hydration/steric forces in emulsion stability [-28,29,32,33].
Conclusions In the systems under study, emulsion instability takes the form of coalescence after prior floccula-
Fig. 2. Viscousmodulus G" (mPa) and elastic modulus G' (mPa) vs. shear strain of (a) PFOB PL saline, (b) PFOB EYP-saline, (c) PFOB-PL-SA (9 : 1 (mol/mol))-saline,(d) PFOB-PL CHS (9 : 1 (moI/mol))-saline,(e) PFOB PL-9.5% sucrose solution, and (f) PFOB-PL-5% dextrose solution at f= 2 Hz and 25°C.
tion; since flocculation
is a necessary
can be prevented,
in the short term, by preventing flocculation. It should be noted that long-term emulsion stability may be affected negatively by so-called Ostwald
“a process of gradual
at the expense
En,y. Aspects 84 ( 1994 ) 71-79
of the larger
ones by means
of the molecular diffusion; the process arises as a consequence of the Kelvin effect” [ 341. Our research has shown that the absence of flocculation in PFC emulsions cannot be accounted for by considerations of electrostatic repulsive forces alone: a negatively charged interface results in a stable system, while a positively charged interface results in flocculation. This difference in stability is probably due to interdroplet repulsive hydration forces. Positive counterions adsorbed at the negatively charged interface are more hydrated than the negative counterions. The role played by hydrationsteric forces was confirmed by the stabilizing role played by a saccharide solution when it was substituted for saline in systems with uncharged interfaces. The EYP used in the ImagentTM formulation provides a well-structured, tightly packed film at the PFOB-saline interface, as shown by surface isotherm measurements. It contains minor component - acidic phospholipids (phosphatidyl serine, phosphatidyl glycerol) and hydrated compounds (phosphatidyl inositol) that probably act by increasing both electrostatic and hydration repulsion, thus stabilizing the emulsion through a mechanism similar to that found with systems prepared with PL and a negatively charged additive (such as CHS) that forms a mixed film with PL but does not affect the film’s molecular packing. Acknowledgments We thank Dr. David Ikenberry and Dr. Jimbay Loh of General Foods-Kraft, Tarrytown, New York; Dr. Larry Boni of The Liposome Company, Princeton, NJ; and Stephane Fouris of CCNY for technical assistance. We thank Martha F. Browne for assistance in manuscript preparation. C.B.O.
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