Biomaterials, Artificial Cells and Immobilization Biotechnology
ISSN: 1055-7172 (Print) (Online) Journal homepage: http://www.tandfonline.com/loi/ianb18
Stabilization of Perflubron Emulsions with Egg Yolk Phospholipid T. J. Pelura, C. S. Johnson, T. E. Tarara & J. G. Weers To cite this article: T. J. Pelura, C. S. Johnson, T. E. Tarara & J. G. Weers (1992) Stabilization of Perflubron Emulsions with Egg Yolk Phospholipid, Biomaterials, Artificial Cells and Immobilization Biotechnology, 20:2-4, 845-848, DOI: 10.3109/10731199209119728 To link to this article: http://dx.doi.org/10.3109/10731199209119728
Published online: 11 Jul 2009.
Submit your article to this journal
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Citing articles: 2 View citing articles
Full Terms & Conditions of access and use can be found at http://www.tandfonline.com/action/journalInformation?journalCode=ianb18 Download by: [Universität Osnabrueck]
Date: 16 March 2016, At: 06:58
BIOMAT.,
ART. C E L L S & IPIMOB. BIOTECH.,
20(2-4), 845-848 (1992)
STABILIZATION OF PERFLUBRON EMULSIONS WITH EGG YOLK PHOSPHOLIPID
Downloaded by [Universität Osnabrueck] at 06:58 16 March 2016
T.J. Pelura, C.S. Johnson, T.E. Tarara and J.G.Weers Alliance Pharmaceutical Corp., San Diego, CA, USA
ABSTRACT Egg Yolk Phospholipid(EYP) has been used extensively as the primary surfactant in parented fat emulsions for many years. The simplicity, functionality and physiologic tolerance of EYP has contributed greatly to its success in the intravenous emulsion arena. The mechanism of stabilization in triglyceride emulsions is well understood; however, this is not the case with perthorocarbon emulsions. Interfacial models, as well as emulsion stability studies, have been conducted utilizing EYP of varied composition in order to derive a structurdfunction relationship. Our studies indicate that minor components, total unsaturation, acyl chain length and presence of charged species have significant impact on the functional properties of EYP and the subsequent stability of the emulsion product. These findings contribute to our ability to design and manipulate natural surfactants with superior properties for use in medical applications of prfluorocarbon emulsions. INTRODUCTION Egg Yolk Phospholipid(EYP) is the mixture of lipids obtained from hens’ egg yolks by solvent extraction followed by various purification steps including recrystallization and/or column chromatography. High purity and physiologically acceptable surfactants are readily obtained from yolk lipids due to their inherent simplicity. Modifications to functionality and stability can be made by catalytic hydrogenation. EYP is an excellent wetting and emulsifying agent due to the fatty acid containing phosphatides which are amphipathic in structure, having strongly lipophilic, fatty acid nuclei at one end of the molecule and a strongly hydrophilic amino or phosphoric acid nucleus at the opposite end. In heterogeneous systems such as oil and water, the phosphatide molecules arrange themselves in monomolecularlayers with the fatty acid portion facing the oil surface and the phosphoric acid or amine portion facing the water surface. This arrangement lowers the interfacial tension at the oil-water boundaries with a resultant benefit of emulsion stabilization. Commercial intravenous fat emulsions have long taken advantage of these properties for obvious reasons. The use of EYP, however, as a stabilizer of intravenous perfluorocarbon emulsions for contrast and therapy, is intuitive yet intriguing with regard to the nature of the
045 Copyright 0 1992 by Marcel Dekker, Inc.
846
PELURA ET AL.
Downloaded by [Universität Osnabrueck] at 06:58 16 March 2016
m
II
EhQspbolipid w o n. .o f EYP(wt%), I. V., iodine value; N.L.,neural lipids; PE, phosphatidylethanolamine; LPE, lysophosphatidylethanolamine; PC, phosphatidylcholine; SPH, sphingomyelin; LPC, lysophosphatidylcholine.
I
3
I
5.71
I
0.00
I
0.00
I
92.42
I
0.29
I
0.47
I
interface. This study attempts to characterize this interface and factors which affect the physicochemical characteristics of pertlubron emulsions such as phospholipid composition and acyl group unsaturation.
Emulsions containing 90% w/v perflubron (perfluorooctylbromide; PFOB), stabilized with 4% wlv egg yolk phospholipid (EYP), were made by high pressure homogenization. Natural and partially hydrogenated egg yolk phospholipids containing approximately 80 and 94 I phosphatidylcholine were obtained commercially. The emulsions are phosphate buffered to physiologic pH and contain an antioxidant and metal chelator to protect the oxidatively labile phospholipid surfactant. The emulsions are terminally sterilized in a rotating autoclave at 121°C. Particle size distributions were determined by photosedimentation on a Horiba CAPA700. Rheologic measurements were performed on a Brookfield DV-I1 Viscometer. Mechanical stress studies were accomplished by shaking the emulsion samples horizontally at a rate of 250 strokedminute. The stressed emulsions were subsequently centrifuged and the amount of unemulsified perflubron determined volumetrically. Determination of oxidative degradation of the phospholipid was accomplished with a RF-5O00 Shimadzu Spectrofluorophotometer. Interfacial tension studies were performed on a Kriss K12 Processor Tensiometer. Phospholipid class analyses were performed by HPLC separation and subsequent quantitation with a Cunow DDL 21 Light Scattering Detector. RESULTS A N D DISCUS= The lipid composition of natural and partially hydrogenated E-80 and E-100 phospholipid samples, as determined quantitatively by light scattering detection, is given in Table 1.
STABILIZATION O F PERFLUBRON EMULSIONS WITH EYP
Downloaded by [Universität Osnabrueck] at 06:58 16 March 2016
Table 2.
. .
Phvsical and Chemical Charac-ics of 90% wlv Pefflubron Emulsiom I.V., iodine value; Particle Size, median p; "S" Parameter, &/month; viscosity, CPS at shear rate I sec-'; Mechanical Stress, volume X wemuls@ed perjlubron after I hour shaking at 250 strokes/minute; Fluorescence. intemity after 7 days of oxidative stress(0, and Cu2' at 40°C).
-
Air/Saline Perf lubron/Saline
+
c 0 ' z 40 c W
I-
lE .-
30
0
L 20 L
W
= c
10
0 1 2 3 4 5 6 7 8 910111213 Egg Yolk Phospholipid Concentration, Fieure 1,
lo-'
.. . . . Critical h$k&zawn Concentriltl'on of Eee Yolk P m D l d-oS
M
.
.
.
Downloaded by [Universität Osnabrueck] at 06:58 16 March 2016
848
PELURA ET AL.
The phospholipidsused in this study differ mainly in the levels of phosphatidylethanolamine(PE) and phosphatidylcholine (PC), and iodine value. Iodine value is a measure of the total unsaturation of lipid materials. Natural EYP, as extracted from hens’ eggs, has an iodine value of 65 to 75. Physical and chemicai characteristics of 90% wlv perflubron emulsions made with the various E W s are shown in Table 2. The physical stability of 90% pertlubron emulsions, as a function of phospholipid composition and level of unsaturation, was assessed by mechanical stress and particle size growth studies. Particle size stability, expressed as parameter “S”(the linear increase of particle drop volume vs time, in units of pm’lmonth), is affected primarily by the level of phospholipid acyl group unsaturation and to a lesser extent by lipid class. Stability to mechanical stress, however, is affected by both phospholipid composition and unsaturation. This is most likely due to the dramatic increase in viscosity as a function of iodine value. Oxidative stability of the emulsions was determined spectrofluorophotometrically. Condensationof unsaturated aldehydes from hydroperoxide decomposition and primary amines, such as PE, yield conjugated Schiff bases. These compounds, when excited at 350nm, have an emission maxima of 435nm. Results indicate that oxidative decomposition is directly related to the level of unsaturation and the concentration of PE. Surface tension studies were performed to characterize the nature of the phospholipid membrane at the perflubronlsaline interface. A large increase in the critical micellization concentration (cmc) observed at the perflubronlsaline interface relative to the airlsaline interface is demonstrated in Figure 1. The cmc for EYP at the perflubronlsaline interface (7.1 x lO-’M) is approximately two times greater than that at the airlsaline interface (3.2 x lO-’M), which implies extensive solubilization of perflubron into EYP micelles. Perfluorocarbons have a profound effect on the packing of EYP alkyl chains in micelles, most likely by reducing the dispersive forces between those chains. This reduction in the hydrophobic contribution to the free energy of micellization raises the cmc.
CONCLUSIONS Increase in critical micellizationconcentration observed at the perflubronlsaline interface relative to the airlsaline interface indicates penetration of the lipid surfactant into the perfiubron core. Perflubronlsaline systems stabilized by phospholipid are conventional emulsions. Phospholipid composition and unsaturation (iodine value) of constituent acyl groups contribute to mechanical and oxidative stability of perflubron emulsions. Level of phospholipid fatty acid unsaturation as determined by iodine value affect the particle size stability of perflubron emulsions. Iodine value of the phospholipid inversely affects the viscosity of perflubron emulsions. EYP is an excellent surfactant candidate for perfluorocarbon emulsions. Subtle manipulatims in EYP composition and unsaturation can have profound effects on the functionality and subsequent bulk emulsion properties.
ISSN: 1055-7172 (Print) (Online) Journal homepage: http://www.tandfonline.com/loi/ianb18
Stabilization of Perflubron Emulsions with Egg Yolk Phospholipid T. J. Pelura, C. S. Johnson, T. E. Tarara & J. G. Weers To cite this article: T. J. Pelura, C. S. Johnson, T. E. Tarara & J. G. Weers (1992) Stabilization of Perflubron Emulsions with Egg Yolk Phospholipid, Biomaterials, Artificial Cells and Immobilization Biotechnology, 20:2-4, 845-848, DOI: 10.3109/10731199209119728 To link to this article: http://dx.doi.org/10.3109/10731199209119728
Published online: 11 Jul 2009.
Submit your article to this journal
Article views: 10
View related articles
Citing articles: 2 View citing articles
Full Terms & Conditions of access and use can be found at http://www.tandfonline.com/action/journalInformation?journalCode=ianb18 Download by: [Universität Osnabrueck]
Date: 16 March 2016, At: 06:58
BIOMAT.,
ART. C E L L S & IPIMOB. BIOTECH.,
20(2-4), 845-848 (1992)
STABILIZATION OF PERFLUBRON EMULSIONS WITH EGG YOLK PHOSPHOLIPID
Downloaded by [Universität Osnabrueck] at 06:58 16 March 2016
T.J. Pelura, C.S. Johnson, T.E. Tarara and J.G.Weers Alliance Pharmaceutical Corp., San Diego, CA, USA
ABSTRACT Egg Yolk Phospholipid(EYP) has been used extensively as the primary surfactant in parented fat emulsions for many years. The simplicity, functionality and physiologic tolerance of EYP has contributed greatly to its success in the intravenous emulsion arena. The mechanism of stabilization in triglyceride emulsions is well understood; however, this is not the case with perthorocarbon emulsions. Interfacial models, as well as emulsion stability studies, have been conducted utilizing EYP of varied composition in order to derive a structurdfunction relationship. Our studies indicate that minor components, total unsaturation, acyl chain length and presence of charged species have significant impact on the functional properties of EYP and the subsequent stability of the emulsion product. These findings contribute to our ability to design and manipulate natural surfactants with superior properties for use in medical applications of prfluorocarbon emulsions. INTRODUCTION Egg Yolk Phospholipid(EYP) is the mixture of lipids obtained from hens’ egg yolks by solvent extraction followed by various purification steps including recrystallization and/or column chromatography. High purity and physiologically acceptable surfactants are readily obtained from yolk lipids due to their inherent simplicity. Modifications to functionality and stability can be made by catalytic hydrogenation. EYP is an excellent wetting and emulsifying agent due to the fatty acid containing phosphatides which are amphipathic in structure, having strongly lipophilic, fatty acid nuclei at one end of the molecule and a strongly hydrophilic amino or phosphoric acid nucleus at the opposite end. In heterogeneous systems such as oil and water, the phosphatide molecules arrange themselves in monomolecularlayers with the fatty acid portion facing the oil surface and the phosphoric acid or amine portion facing the water surface. This arrangement lowers the interfacial tension at the oil-water boundaries with a resultant benefit of emulsion stabilization. Commercial intravenous fat emulsions have long taken advantage of these properties for obvious reasons. The use of EYP, however, as a stabilizer of intravenous perfluorocarbon emulsions for contrast and therapy, is intuitive yet intriguing with regard to the nature of the
045 Copyright 0 1992 by Marcel Dekker, Inc.
846
PELURA ET AL.
Downloaded by [Universität Osnabrueck] at 06:58 16 March 2016
m
II
EhQspbolipid w o n. .o f EYP(wt%), I. V., iodine value; N.L.,neural lipids; PE, phosphatidylethanolamine; LPE, lysophosphatidylethanolamine; PC, phosphatidylcholine; SPH, sphingomyelin; LPC, lysophosphatidylcholine.
I
3
I
5.71
I
0.00
I
0.00
I
92.42
I
0.29
I
0.47
I
interface. This study attempts to characterize this interface and factors which affect the physicochemical characteristics of pertlubron emulsions such as phospholipid composition and acyl group unsaturation.
Emulsions containing 90% w/v perflubron (perfluorooctylbromide; PFOB), stabilized with 4% wlv egg yolk phospholipid (EYP), were made by high pressure homogenization. Natural and partially hydrogenated egg yolk phospholipids containing approximately 80 and 94 I phosphatidylcholine were obtained commercially. The emulsions are phosphate buffered to physiologic pH and contain an antioxidant and metal chelator to protect the oxidatively labile phospholipid surfactant. The emulsions are terminally sterilized in a rotating autoclave at 121°C. Particle size distributions were determined by photosedimentation on a Horiba CAPA700. Rheologic measurements were performed on a Brookfield DV-I1 Viscometer. Mechanical stress studies were accomplished by shaking the emulsion samples horizontally at a rate of 250 strokedminute. The stressed emulsions were subsequently centrifuged and the amount of unemulsified perflubron determined volumetrically. Determination of oxidative degradation of the phospholipid was accomplished with a RF-5O00 Shimadzu Spectrofluorophotometer. Interfacial tension studies were performed on a Kriss K12 Processor Tensiometer. Phospholipid class analyses were performed by HPLC separation and subsequent quantitation with a Cunow DDL 21 Light Scattering Detector. RESULTS A N D DISCUS= The lipid composition of natural and partially hydrogenated E-80 and E-100 phospholipid samples, as determined quantitatively by light scattering detection, is given in Table 1.
STABILIZATION O F PERFLUBRON EMULSIONS WITH EYP
Downloaded by [Universität Osnabrueck] at 06:58 16 March 2016
Table 2.
. .
Phvsical and Chemical Charac-ics of 90% wlv Pefflubron Emulsiom I.V., iodine value; Particle Size, median p; "S" Parameter, &/month; viscosity, CPS at shear rate I sec-'; Mechanical Stress, volume X wemuls@ed perjlubron after I hour shaking at 250 strokes/minute; Fluorescence. intemity after 7 days of oxidative stress(0, and Cu2' at 40°C).
-
Air/Saline Perf lubron/Saline
+
c 0 ' z 40 c W
I-
lE .-
30
0
L 20 L
W
= c
10
0 1 2 3 4 5 6 7 8 910111213 Egg Yolk Phospholipid Concentration, Fieure 1,
lo-'
.. . . . Critical h$k&zawn Concentriltl'on of Eee Yolk P m D l d-oS
M
.
.
.
Downloaded by [Universität Osnabrueck] at 06:58 16 March 2016
848
PELURA ET AL.
The phospholipidsused in this study differ mainly in the levels of phosphatidylethanolamine(PE) and phosphatidylcholine (PC), and iodine value. Iodine value is a measure of the total unsaturation of lipid materials. Natural EYP, as extracted from hens’ eggs, has an iodine value of 65 to 75. Physical and chemicai characteristics of 90% wlv perflubron emulsions made with the various E W s are shown in Table 2. The physical stability of 90% pertlubron emulsions, as a function of phospholipid composition and level of unsaturation, was assessed by mechanical stress and particle size growth studies. Particle size stability, expressed as parameter “S”(the linear increase of particle drop volume vs time, in units of pm’lmonth), is affected primarily by the level of phospholipid acyl group unsaturation and to a lesser extent by lipid class. Stability to mechanical stress, however, is affected by both phospholipid composition and unsaturation. This is most likely due to the dramatic increase in viscosity as a function of iodine value. Oxidative stability of the emulsions was determined spectrofluorophotometrically. Condensationof unsaturated aldehydes from hydroperoxide decomposition and primary amines, such as PE, yield conjugated Schiff bases. These compounds, when excited at 350nm, have an emission maxima of 435nm. Results indicate that oxidative decomposition is directly related to the level of unsaturation and the concentration of PE. Surface tension studies were performed to characterize the nature of the phospholipid membrane at the perflubronlsaline interface. A large increase in the critical micellization concentration (cmc) observed at the perflubronlsaline interface relative to the airlsaline interface is demonstrated in Figure 1. The cmc for EYP at the perflubronlsaline interface (7.1 x lO-’M) is approximately two times greater than that at the airlsaline interface (3.2 x lO-’M), which implies extensive solubilization of perflubron into EYP micelles. Perfluorocarbons have a profound effect on the packing of EYP alkyl chains in micelles, most likely by reducing the dispersive forces between those chains. This reduction in the hydrophobic contribution to the free energy of micellization raises the cmc.
CONCLUSIONS Increase in critical micellizationconcentration observed at the perflubronlsaline interface relative to the airlsaline interface indicates penetration of the lipid surfactant into the perfiubron core. Perflubronlsaline systems stabilized by phospholipid are conventional emulsions. Phospholipid composition and unsaturation (iodine value) of constituent acyl groups contribute to mechanical and oxidative stability of perflubron emulsions. Level of phospholipid fatty acid unsaturation as determined by iodine value affect the particle size stability of perflubron emulsions. Iodine value of the phospholipid inversely affects the viscosity of perflubron emulsions. EYP is an excellent surfactant candidate for perfluorocarbon emulsions. Subtle manipulatims in EYP composition and unsaturation can have profound effects on the functionality and subsequent bulk emulsion properties.
