Pharmaceutical formulations of bionanoparticles for siRNA delivery

Abdelkader Metwally

Research output: ThesisDoctoral Thesis

Abstract

The aims of this thesis are to design and synthesize non-viral cationic lipid vectors based on spermine, for the intracellular delivery of siRNA (short interfering RNA) and the subsequent siRNA mediated gene silencing. Two parameters were varied: the type of fatty acid and the cationic head-group. Among the symmetrical spermine conjugates, N4,N9-dierucoyl spermine (DES) resulted in higher siRNA delivery compared to N4,N9-dioleoyl spermine (DOS), while enhanced green fluorescent protein (EGFP) silencing in HeLa cells
showed that the unsaturated fatty acid conjugates are more efficient than the saturated fatty acid ones, and cell viability was 75%-85% for conjugates with chain length ≥ 18. Two cationic lipids with guanidine head-groups, N1,N12-diamidino-N4,N9-dioleoylspermine and N1,N12-diamidino-N4-linoleoyl-N9-oleoylspermine, were more efficient in EGFP gene silencing compared to cationic lipids with shorter C12 (lauroyl) and very long C22 (erucoyl) chains, with cell viability (64%-83% for chain length ≥ 18). Changing the cationic headgroup
to guanidine did not offer a significant advantage in gene silencing over the conjugates with terminal primary amine groups. The asymmetrical N4-linoleoyl-N9-oleoyl-1,12-diamino-4,9-diazadodecane (LinOS) resulted in the best gene silencing, while LigOS (with one lignoceroyl 24:0 chain) resulted in the best siRNA delivery. Conjugates with two unsaturated fatty chains generally resulted in better EGFP gene silencing, while conjugates with one saturated chain and one unsaturated chain resulted in better siRNA delivery. Increasing the chain length also resulted in increased siRNA delivery (cell viabilities of asymmetrical >
74%, LinOS 88%). siRNA lipoplexes prepared using mixtures of LinOS with either
cholesterol or DOPE (1,2-dioleoyl-sn-glycero-3-phosphoethanolamine) resulted in increased siRNA delivery, and enhanced EGFP silencing, with LinOS/Chol mixture (1:2 molar ratio) resulting in the highest siRNA delivery and the best gene silencing (EGFP reduced to 20%). Temperature studies of intracellular entry showed that the majority of lipoplexes are internalized by endocytosis, however the majority of gene-silencing occurs due to lipoplexes internalized via another mechanism.
LanguageEnglish
QualificationPh.D.
Awarding Institution
  • University of Bath
Supervisors/Advisors
  • Blagbrough, Ian, Supervisor
Award date31 Dec 2012
StatusUnpublished - 2012

Fingerprint

drug formulations
small interfering RNA
gene silencing
green fluorescent protein
spermine
cell viability
guanidines
lipids
primary amines
endocytosis
unsaturated fatty acids
saturated fatty acids
cholesterol
fatty acids

Keywords

  • siRNA
  • spermine
  • Nanoparticles

Cite this

Pharmaceutical formulations of bionanoparticles for siRNA delivery. / Metwally, Abdelkader.

2012. 202 p.

Research output: ThesisDoctoral Thesis

Metwally, A 2012, 'Pharmaceutical formulations of bionanoparticles for siRNA delivery', Ph.D., University of Bath.
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abstract = "The aims of this thesis are to design and synthesize non-viral cationic lipid vectors based on spermine, for the intracellular delivery of siRNA (short interfering RNA) and the subsequent siRNA mediated gene silencing. Two parameters were varied: the type of fatty acid and the cationic head-group. Among the symmetrical spermine conjugates, N4,N9-dierucoyl spermine (DES) resulted in higher siRNA delivery compared to N4,N9-dioleoyl spermine (DOS), while enhanced green fluorescent protein (EGFP) silencing in HeLa cells showed that the unsaturated fatty acid conjugates are more efficient than the saturated fatty acid ones, and cell viability was 75{\%}-85{\%} for conjugates with chain length ≥ 18. Two cationic lipids with guanidine head-groups, N1,N12-diamidino-N4,N9-dioleoylspermine and N1,N12-diamidino-N4-linoleoyl-N9-oleoylspermine, were more efficient in EGFP gene silencing compared to cationic lipids with shorter C12 (lauroyl) and very long C22 (erucoyl) chains, with cell viability (64{\%}-83{\%} for chain length ≥ 18). Changing the cationic headgroup to guanidine did not offer a significant advantage in gene silencing over the conjugates with terminal primary amine groups. The asymmetrical N4-linoleoyl-N9-oleoyl-1,12-diamino-4,9-diazadodecane (LinOS) resulted in the best gene silencing, while LigOS (with one lignoceroyl 24:0 chain) resulted in the best siRNA delivery. Conjugates with two unsaturated fatty chains generally resulted in better EGFP gene silencing, while conjugates with one saturated chain and one unsaturated chain resulted in better siRNA delivery. Increasing the chain length also resulted in increased siRNA delivery (cell viabilities of asymmetrical > 74{\%}, LinOS 88{\%}). siRNA lipoplexes prepared using mixtures of LinOS with either cholesterol or DOPE (1,2-dioleoyl-sn-glycero-3-phosphoethanolamine) resulted in increased siRNA delivery, and enhanced EGFP silencing, with LinOS/Chol mixture (1:2 molar ratio) resulting in the highest siRNA delivery and the best gene silencing (EGFP reduced to 20{\%}). Temperature studies of intracellular entry showed that the majority of lipoplexes are internalized by endocytosis, however the majority of gene-silencing occurs due to lipoplexes internalized via another mechanism.",
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N2 - The aims of this thesis are to design and synthesize non-viral cationic lipid vectors based on spermine, for the intracellular delivery of siRNA (short interfering RNA) and the subsequent siRNA mediated gene silencing. Two parameters were varied: the type of fatty acid and the cationic head-group. Among the symmetrical spermine conjugates, N4,N9-dierucoyl spermine (DES) resulted in higher siRNA delivery compared to N4,N9-dioleoyl spermine (DOS), while enhanced green fluorescent protein (EGFP) silencing in HeLa cells showed that the unsaturated fatty acid conjugates are more efficient than the saturated fatty acid ones, and cell viability was 75%-85% for conjugates with chain length ≥ 18. Two cationic lipids with guanidine head-groups, N1,N12-diamidino-N4,N9-dioleoylspermine and N1,N12-diamidino-N4-linoleoyl-N9-oleoylspermine, were more efficient in EGFP gene silencing compared to cationic lipids with shorter C12 (lauroyl) and very long C22 (erucoyl) chains, with cell viability (64%-83% for chain length ≥ 18). Changing the cationic headgroup to guanidine did not offer a significant advantage in gene silencing over the conjugates with terminal primary amine groups. The asymmetrical N4-linoleoyl-N9-oleoyl-1,12-diamino-4,9-diazadodecane (LinOS) resulted in the best gene silencing, while LigOS (with one lignoceroyl 24:0 chain) resulted in the best siRNA delivery. Conjugates with two unsaturated fatty chains generally resulted in better EGFP gene silencing, while conjugates with one saturated chain and one unsaturated chain resulted in better siRNA delivery. Increasing the chain length also resulted in increased siRNA delivery (cell viabilities of asymmetrical > 74%, LinOS 88%). siRNA lipoplexes prepared using mixtures of LinOS with either cholesterol or DOPE (1,2-dioleoyl-sn-glycero-3-phosphoethanolamine) resulted in increased siRNA delivery, and enhanced EGFP silencing, with LinOS/Chol mixture (1:2 molar ratio) resulting in the highest siRNA delivery and the best gene silencing (EGFP reduced to 20%). Temperature studies of intracellular entry showed that the majority of lipoplexes are internalized by endocytosis, however the majority of gene-silencing occurs due to lipoplexes internalized via another mechanism.

AB - The aims of this thesis are to design and synthesize non-viral cationic lipid vectors based on spermine, for the intracellular delivery of siRNA (short interfering RNA) and the subsequent siRNA mediated gene silencing. Two parameters were varied: the type of fatty acid and the cationic head-group. Among the symmetrical spermine conjugates, N4,N9-dierucoyl spermine (DES) resulted in higher siRNA delivery compared to N4,N9-dioleoyl spermine (DOS), while enhanced green fluorescent protein (EGFP) silencing in HeLa cells showed that the unsaturated fatty acid conjugates are more efficient than the saturated fatty acid ones, and cell viability was 75%-85% for conjugates with chain length ≥ 18. Two cationic lipids with guanidine head-groups, N1,N12-diamidino-N4,N9-dioleoylspermine and N1,N12-diamidino-N4-linoleoyl-N9-oleoylspermine, were more efficient in EGFP gene silencing compared to cationic lipids with shorter C12 (lauroyl) and very long C22 (erucoyl) chains, with cell viability (64%-83% for chain length ≥ 18). Changing the cationic headgroup to guanidine did not offer a significant advantage in gene silencing over the conjugates with terminal primary amine groups. The asymmetrical N4-linoleoyl-N9-oleoyl-1,12-diamino-4,9-diazadodecane (LinOS) resulted in the best gene silencing, while LigOS (with one lignoceroyl 24:0 chain) resulted in the best siRNA delivery. Conjugates with two unsaturated fatty chains generally resulted in better EGFP gene silencing, while conjugates with one saturated chain and one unsaturated chain resulted in better siRNA delivery. Increasing the chain length also resulted in increased siRNA delivery (cell viabilities of asymmetrical > 74%, LinOS 88%). siRNA lipoplexes prepared using mixtures of LinOS with either cholesterol or DOPE (1,2-dioleoyl-sn-glycero-3-phosphoethanolamine) resulted in increased siRNA delivery, and enhanced EGFP silencing, with LinOS/Chol mixture (1:2 molar ratio) resulting in the highest siRNA delivery and the best gene silencing (EGFP reduced to 20%). Temperature studies of intracellular entry showed that the majority of lipoplexes are internalized by endocytosis, however the majority of gene-silencing occurs due to lipoplexes internalized via another mechanism.

KW - siRNA

KW - spermine

KW - Nanoparticles

M3 - Doctoral Thesis

ER -