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Lipid polymorphism and intracellular delivery Hafez, Ismail Mahmoud
Abstract
The role of lipid polymorphism and lipid-based systems used for intracellular delivery has been examined. First, the self-assembly properties of the anionic lipid cholesteryl hemisuccinate (CHEMS) were studied as a function of pH. CHEMS is an acidic cholesterol ester that self-assembles into bilayers in alkaline and neutral aqueous media and is commonly employed in mixtures with the nonbilayer lipid, dioleoylphosphatidylethanolamine (DOPE) to prepare pH-sensitive fusogenic liposomes. pH-sensitive liposomes can be used for the intracellular delivery of macromolecules through the endocytic pathway. It is shown that CHEMS itself adopts a nonbilayer phase at low pH. This is evident from the fusogenic properties of large unilamellar vesicles (LUVs) composed of CHEMS and direct visualization employing freeze-fracture electron microscopy. It is suggested that the pHdependent phase preferences of CHEMS contributes to the pH-sensitive fusion of LUVs composed of mixtures of CHEMS and DOPE. Next, the pH-dependent fusion properties of LUVs composed of binary mixtures of anionic and cationic lipids was investigated. It was found that stable LUVs can be prepared from the ionizable anionic lipid CHEMS and the permanently charged cationic lipid N,N-dioleoyl-N,N-dimethylammonium chloride (DODAC) at neutral pH values and that these LUVs undergo fusion as the pH is reduced. The critical pH at which fusion was observed (pHf) was dependent on the cationic lipid-to-anionic lipid ratio. LUVs prepared from DODAC/CHEMS mixtures at molar ratios of 0 to 0.85 resulted in vesicles with pHf values that ranged from pH 4.0 to 6.7, respectively. This behaviour is consistent with a model in which fusion occurs at pH values such that the DODAC/CHEMS LUV surface charge is zero. Related behaviour was observed for LUVs composed of the ionizable cationic lipid, 3α-[N-(N',N'- dimethylaminoethane)-carbamoyl] cholesterol hydrochloride (DC-Choi) and the acidic lipid dioleoylphosphatidic acid (DOPA). Freeze-fracture and ³¹P NMR evidence indicates that pH-dependent fusion results from a preference of mixtures of cationic and anionic lipid for "inverted" nonbilayer lipid phases under conditions where the surface charge is zero. It is concluded that tunable pH-sensitive LUVs composed of cationic and anionic lipids may be of utility for drug delivery applications. Finally, the mechanism of nucleic acid transfection mediated by cationic lipids (lipofection) is elucidated. Cationic lipids are widely used as non-viral gene transfer agents, but the mechanism by which cationic liposomes promote the intracellular delivery of membrane impermeable macromolecules such as plasmid DNA or antisense oligonucleotides is not well understood. In this work it is demonstrated that cationic lipids can destabilize cell membranes by promoting the formation of nonbilayer lipid structures. Using ³¹P NMR, it is shown that addition of cationic lipids to bilayer-adopting anionic phospholipids results in the formation of the nonbilayer inverted hexagonal (H[sub ii] phase. Further, the presence of "helper" lipids such as dioleoylphosphatidylethanolamine or cholesterol, lipids that enhance cationic lipidmediated transfection, also facilitates the formation of the H[sub ii] phase. It is suggested that the ability of cationic lipids to promote nonbilayer structure in combination with anionic phospholipids leads to disruption of the endosomal membrane following uptake of nucleic acid-cationic lipid complexes into cells, thus facilitating cytoplasmic release of the plasmid or oligonucleotide.
Item Metadata
Title |
Lipid polymorphism and intracellular delivery
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Creator | |
Publisher |
University of British Columbia
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Date Issued |
2000
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Description |
The role of lipid polymorphism and lipid-based systems used for intracellular
delivery has been examined. First, the self-assembly properties of the anionic lipid
cholesteryl hemisuccinate (CHEMS) were studied as a function of pH. CHEMS is an
acidic cholesterol ester that self-assembles into bilayers in alkaline and neutral
aqueous media and is commonly employed in mixtures with the nonbilayer lipid,
dioleoylphosphatidylethanolamine (DOPE) to prepare pH-sensitive fusogenic
liposomes. pH-sensitive liposomes can be used for the intracellular delivery of
macromolecules through the endocytic pathway. It is shown that CHEMS itself
adopts a nonbilayer phase at low pH. This is evident from the fusogenic properties
of large unilamellar vesicles (LUVs) composed of CHEMS and direct visualization
employing freeze-fracture electron microscopy. It is suggested that the pHdependent
phase preferences of CHEMS contributes to the pH-sensitive fusion of
LUVs composed of mixtures of CHEMS and DOPE.
Next, the pH-dependent fusion properties of LUVs composed of binary mixtures of
anionic and cationic lipids was investigated. It was found that stable LUVs can be
prepared from the ionizable anionic lipid CHEMS and the permanently charged
cationic lipid N,N-dioleoyl-N,N-dimethylammonium chloride (DODAC) at neutral pH
values and that these LUVs undergo fusion as the pH is reduced. The critical pH at
which fusion was observed (pHf) was dependent on the cationic lipid-to-anionic lipid
ratio. LUVs prepared from DODAC/CHEMS mixtures at molar ratios of 0 to 0.85
resulted in vesicles with pHf values that ranged from pH 4.0 to 6.7, respectively.
This behaviour is consistent with a model in which fusion occurs at pH values such
that the DODAC/CHEMS LUV surface charge is zero. Related behaviour was
observed for LUVs composed of the ionizable cationic lipid, 3α-[N-(N',N'-
dimethylaminoethane)-carbamoyl] cholesterol hydrochloride (DC-Choi) and the
acidic lipid dioleoylphosphatidic acid (DOPA). Freeze-fracture and ³¹P NMR
evidence indicates that pH-dependent fusion results from a preference of mixtures
of cationic and anionic lipid for "inverted" nonbilayer lipid phases under conditions
where the surface charge is zero. It is concluded that tunable pH-sensitive LUVs
composed of cationic and anionic lipids may be of utility for drug delivery
applications.
Finally, the mechanism of nucleic acid transfection mediated by cationic lipids
(lipofection) is elucidated. Cationic lipids are widely used as non-viral gene transfer
agents, but the mechanism by which cationic liposomes promote the intracellular
delivery of membrane impermeable macromolecules such as plasmid DNA or
antisense oligonucleotides is not well understood. In this work it is demonstrated
that cationic lipids can destabilize cell membranes by promoting the formation of
nonbilayer lipid structures. Using ³¹P NMR, it is shown that addition of cationic lipids
to bilayer-adopting anionic phospholipids results in the formation of the nonbilayer
inverted hexagonal (H[sub ii] phase. Further, the presence of "helper" lipids such as
dioleoylphosphatidylethanolamine or cholesterol, lipids that enhance cationic lipidmediated
transfection, also facilitates the formation of the H[sub ii] phase. It is suggested
that the ability of cationic lipids to promote nonbilayer structure in combination with
anionic phospholipids leads to disruption of the endosomal membrane following
uptake of nucleic acid-cationic lipid complexes into cells, thus facilitating
cytoplasmic release of the plasmid or oligonucleotide.
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Extent |
10426825 bytes
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Genre | |
Type | |
File Format |
application/pdf
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Language |
eng
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Date Available |
2009-07-23
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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 |
10.14288/1.0089758
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URI | |
Degree | |
Program | |
Affiliation | |
Degree Grantor |
University of British Columbia
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Graduation Date |
2000-11
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Campus | |
Scholarly Level |
Graduate
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Aggregated Source Repository |
DSpace
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Item Media
Item Citations and Data
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.