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UBC Theses and Dissertations
Role of blood proteins in liposome clearance Chonn, Arcadio
Abstract
The studies presented in this thesis were aimed at understanding the basis for the apparent recognition of liposomes as foreign particles by the reticuloendothelial system. This phenomenon represents a major problem to the development of optimal liposome targeted drug delivery systems. An understanding of the clearance process potentially will lead to the design of improved liposome carrier systems that display favorable and predictable pharmacokinetic properties. The studies fall into two main areas of investigation. The first area involved the study of the complement activating properties of liposomes composed of various lipid species. This is of interest because the activation of the complement system is known to provide the effector molecules that lead to opsonization and lysis of foreign particulates. It had not been previously determined whether liposomes composed of simple lipid species activate the complement system in the complex biological milieu. The studies presented here used a complement hemolytic assay to demonstrate that surface charge is a key determinant of complement-activating liposomes. The nature of the charge, whether negative or positive appeared to dictate which pathway of the complement system is activated. Negatively charged liposomes activated complement in a Ca2+ dependent manner suggesting that activation occurred via the classical pathway. Positively charged liposomes activated complement via the alternative pathway. Neutral liposomes failed to activate complement as measured by the hemolytic assays. Further, it was shown that unsaturated liposomes were more potent complement activators than saturated liposomes and that 45 mol% cholesterol promoted complement protein/liposome interactions. The relation between complement activation and clearance rate from the circulation was investigated. Although there appeared to be a direct relation between complement activation and clearance for liposomes composed of various anionic phospholipids, the relation appeared to break down for other liposomal systems. This suggests that other factors play a more important role in liposome clearance. The second area of investigation was directed at establishing an unambiguous role for proteins in mediating the clearance process in vivo. Liposomes were administered intravenously into CD1 mice and after various times the liposomes were recovered from the blood. The amount of total protein binding to the liposomes was quantitated using the bicinchoninic acid protein assay, and the proteins associated with the recovered liposomes were analyzed by SDS-polyacrylamide gel electrophoresis. In order to facilitate the isolation of liposomes from blood components, a simple and rapid procedure combining chromatographic and centrifugal methods was developed. This “spin column” procedure enabled the use of large unilamellar liposomes instead of multilamellar vesicles, thus making possible the measurement of parameters such as the amount of protein bound/vesicle. From these studies, it was established that the amount of protein bound to the liposomes was inversely related to the circulation half-life of the liposomes. The proteins associated predominantly with very rapidly cleared liposomes consisted of large amounts of opsonins including C3 fragments and IgG, as well as other proteins such as apolipoprotein H which as of yet has no recognized opsonic role. Furthermore, these studies demonstrated that the mechanism by which ganglioside GM1 prolongs the circulation half-life of liposomes was by reducing the total amount of blood protein bound to the liposomes in a relatively non-specific manner. These studies established unambiguously that the apparent differences in liposome clearance behavior observed in vivo is related to the amount and type of protein associated with the liposomes in vivo. This has important implications for the design of liposomes having stable properties in the circulation and for the design of biocompatible surfaces.
Item Metadata
Title |
Role of blood proteins in liposome clearance
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Creator | |
Publisher |
University of British Columbia
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Date Issued |
1992
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Description |
The studies presented in this thesis were aimed at understanding the basis for the
apparent recognition of liposomes as foreign particles by the reticuloendothelial system.
This phenomenon represents a major problem to the development of optimal liposome
targeted drug delivery systems. An understanding of the clearance process potentially
will lead to the design of improved liposome carrier systems that display favorable and
predictable pharmacokinetic properties.
The studies fall into two main areas of investigation. The first area involved the
study of the complement activating properties of liposomes composed of various lipid
species. This is of interest because the activation of the complement system is known to
provide the effector molecules that lead to opsonization and lysis of foreign particulates.
It had not been previously determined whether liposomes composed of simple lipid
species activate the complement system in the complex biological milieu. The studies
presented here used a complement hemolytic assay to demonstrate that surface charge is
a key determinant of complement-activating liposomes. The nature of the charge,
whether negative or positive appeared to dictate which pathway of the complement
system is activated. Negatively charged liposomes activated complement in a Ca2+
dependent manner suggesting that activation occurred via the classical pathway.
Positively charged liposomes activated complement via the alternative pathway. Neutral
liposomes failed to activate complement as measured by the hemolytic assays. Further, it
was shown that unsaturated liposomes were more potent complement activators than
saturated liposomes and that 45 mol% cholesterol promoted complement
protein/liposome interactions. The relation between complement activation and clearance rate from the circulation was investigated. Although there appeared to be a
direct relation between complement activation and clearance for liposomes composed of
various anionic phospholipids, the relation appeared to break down for other liposomal
systems. This suggests that other factors play a more important role in liposome
clearance.
The second area of investigation was directed at establishing an unambiguous role
for proteins in mediating the clearance process in vivo. Liposomes were administered
intravenously into CD1 mice and after various times the liposomes were recovered from
the blood. The amount of total protein binding to the liposomes was quantitated using
the bicinchoninic acid protein assay, and the proteins associated with the recovered
liposomes were analyzed by SDS-polyacrylamide gel electrophoresis. In order to
facilitate the isolation of liposomes from blood components, a simple and rapid
procedure combining chromatographic and centrifugal methods was developed. This
“spin column” procedure enabled the use of large unilamellar liposomes instead of
multilamellar vesicles, thus making possible the measurement of parameters such as the
amount of protein bound/vesicle. From these studies, it was established that the amount
of protein bound to the liposomes was inversely related to the circulation half-life of the
liposomes. The proteins associated predominantly with very rapidly cleared liposomes
consisted of large amounts of opsonins including C3 fragments and IgG, as well as other
proteins such as apolipoprotein H which as of yet has no recognized opsonic role.
Furthermore, these studies demonstrated that the mechanism by which ganglioside GM1
prolongs the circulation half-life of liposomes was by reducing the total amount of blood
protein bound to the liposomes in a relatively non-specific manner. These studies
established unambiguously that the apparent differences in liposome clearance behavior observed in vivo is related to the amount and type of protein associated with the
liposomes in vivo. This has important implications for the design of liposomes having
stable properties in the circulation and for the design of biocompatible surfaces.
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Extent |
2759086 bytes
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Genre | |
Type | |
File Format |
application/pdf
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Language |
eng
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Date Available |
2008-12-19
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Provider |
Vancouver : University of British Columbia Library
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Rights |
For non-commercial purposes only, such as research, private study and education. Additional conditions apply, see Terms of Use https://open.library.ubc.ca/terms_of_use.
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DOI |
10.14288/1.0086676
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URI | |
Degree | |
Program | |
Affiliation | |
Degree Grantor |
University of British Columbia
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Graduation Date |
1992-05
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Campus | |
Scholarly Level |
Graduate
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Aggregated Source Repository |
DSpace
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For non-commercial purposes only, such as research, private study and education. Additional conditions apply, see Terms of Use https://open.library.ubc.ca/terms_of_use.