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Lipid-based vehicles for nucleic acid drugs Wong, Frances M. P.
Abstract
Lipid-based carriers are anticipated to be a viable option to deliver nucleic acid drugs (NAs) for gene therapy. This thesis describes a method of adding lipids to NAs in a manner that generates a hydrophobic lipid-NA complex and bypasses the aggregation events induced by use of conventional cationic liposomes. It is anticipated that careful control of the lipid components will facilitate generation of a carrier formulation that protects, directs and delivers NAs to a target and in a form capable of mediating a therapeutic response. The factors that govern the cationic lipid/DNA binding reaction were assessed using a hydrophobic lipid/DNA complex generated by Bligh and Dyer extraction. Efficient recovery of DNA (>95%) in an organic phase was achieved when monocationic lipids interacted with anionic phosphate groups to neutralise the DNA charge. Results indicate that the cationic lipid/DNA complex forms at the aqueous/organic interface and binding is dependent on cooperative, multivalent interactions. Based on the hydrophobic intermediate, self-assembling lipid-DNA particles (LDPs) were formed in detergent and were used to assess the role(s) of lipid components that govern the attributes of a novel lipid-based DNA transfer system. While plasmid DNA, formulated within LDPs containing phosphatidylethanolamine (PE)-lipids, was more sensitive to enzymatic cleavage than DNA in LDPs containing phosphatidylcholine (PC)-lipids, only LDPs with PE-lipids could mediate transgene expression. Another key factor effecting delivery of NAs is electrostatic mediated binding to the target cell. In an effort to model this interaction, a microelectrophoresis technique was used to provide information on LDP surface charge and binding to an anionic surface. It was confirmed that LDPs bound anionic latex beads through electrostatic interactions, where binding involved both the NA and associated lipids. While the previous studies assessed transfection activity of a reporter gene, a second goal of these studies was to use lipids with a therapeutically relevant NA. A particulate delivery system was formed through a hydrophobic intermediate containing cationic lipid, PE-lipid, poly(ethylene glycol)-phosphatidylethanolamine (PEG-PE) and an antisense oligonucleotide (ASO), shown to effect eradication of a B-cell lymphoma via downregulation of Bcl-2 protein. Although in vitro data did not demonstrate that lipid-ASO particles (LAPs) could mediate downregulation of Bcl-2 protein, in vivo data indicated that LAPs were more efficacious when compared to free ASOs. While the use of stabilised PEG-conjugated lipids may not be advantageous for delivery in vitro, the results suggest that LAPs can modulate the pharmacokinetic properties of an associated ASO leading to increased bioavailability.
Item Metadata
Title |
Lipid-based vehicles for nucleic acid drugs
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Creator | |
Publisher |
University of British Columbia
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Date Issued |
2000
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Description |
Lipid-based carriers are anticipated to be a viable option to deliver nucleic acid drugs
(NAs) for gene therapy. This thesis describes a method of adding lipids to NAs in a manner that generates a hydrophobic lipid-NA complex and bypasses the aggregation events induced by use of conventional cationic liposomes. It is anticipated that careful control of the lipid
components will facilitate generation of a carrier formulation that protects, directs and delivers NAs to a target and in a form capable of mediating a therapeutic response.
The factors that govern the cationic lipid/DNA binding reaction were assessed using a hydrophobic lipid/DNA complex generated by Bligh and Dyer extraction. Efficient recovery of DNA (>95%) in an organic phase was achieved when monocationic lipids interacted with
anionic phosphate groups to neutralise the DNA charge. Results indicate that the cationic lipid/DNA complex forms at the aqueous/organic interface and binding is dependent on cooperative, multivalent interactions. Based on the hydrophobic intermediate, self-assembling
lipid-DNA particles (LDPs) were formed in detergent and were used to assess the role(s) of lipid components that govern the attributes of a novel lipid-based DNA transfer system. While plasmid DNA, formulated within LDPs containing phosphatidylethanolamine (PE)-lipids, was more sensitive to enzymatic cleavage than DNA in LDPs containing phosphatidylcholine (PC)-lipids, only LDPs with PE-lipids could mediate transgene expression. Another key factor effecting delivery of NAs is electrostatic mediated binding to
the target cell. In an effort to model this interaction, a microelectrophoresis technique was used to provide information on LDP surface charge and binding to an anionic surface. It was confirmed that LDPs bound anionic latex beads through electrostatic interactions, where binding involved both the NA and associated lipids.
While the previous studies assessed transfection activity of a reporter gene, a second goal of these studies was to use lipids with a therapeutically relevant NA. A particulate delivery system was formed through a hydrophobic intermediate containing cationic lipid, PE-lipid, poly(ethylene glycol)-phosphatidylethanolamine (PEG-PE) and an antisense
oligonucleotide (ASO), shown to effect eradication of a B-cell lymphoma via downregulation
of Bcl-2 protein. Although in vitro data did not demonstrate that lipid-ASO particles (LAPs)
could mediate downregulation of Bcl-2 protein, in vivo data indicated that LAPs were more
efficacious when compared to free ASOs. While the use of stabilised PEG-conjugated lipids
may not be advantageous for delivery in vitro, the results suggest that LAPs can modulate the
pharmacokinetic properties of an associated ASO leading to increased bioavailability.
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Extent |
16732744 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 |
10.14288/1.0099524
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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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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.