Profiling and exploiting lipid-based nanoparticles in vitro and in vivo
Author: Sork, Helena
Date: 2018-06-15
Location: Hörsalen, level 4, Hälsovägen 7-9, Novum, Karolinska Institutet, Flemingsberg
Time: 09.00
Department: Inst för laboratoriemedicin / Dept of Laboratory Medicine
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Thesis (925.2Kb)
Abstract
One of the major hurdles for therapeutic applications is the efficient delivery of bioactive molecules to the site of action. The high flexibility and biosafety of lipid-based nanoparticles has greatly enhanced their employment as delivery systems not only for synthetic but also for natural molecules such as proteins and nucleic acids. This thesis was brought about to investigate the nucleic acid delivery potential of synthetic lipid-based nanoparticles as well as to look into the composition and delivery patterns of their natural counterparts, extracellular vesicles (EVs), in order to set ground for future lipid-based therapeutic interventions.
Firstly, in Paper I we explored the potency of a lipid-based delivery agent, Lipofectamine 2000 which after being frozen and thawed showed orders of magnitude higher nucleic acid delivery efficiency in vitro and in vivo than the non-frozen counterpart. This effect was consistent across different cryo-manipulations, cell lines and also various types of nucleic acid. Further analysis with different methodologies revealed that the underlying potency plausibly relies on the elevated sedimentation and spreading of the complexes and/or relates to the specific structure or composition of the carrier. These findings illustrate that a simple freeze-thawing procedure allows to drastically reduce the amount of transfection reagent for cellular nucleic acid delivery, whilst not losing the desired activity.
Secondly, we shifted our focus to natural lipid-based carriers, EVs in order to shed light on the vesicular and non-vesicular (non-EV) small RNA patterns and their relation to the EV proteome (Paper II and III). Though the studies exploited different EV enrichment methods the relative depletion of vesicular small RNAs was confirmed in both instances. A detailed analysis of the secretory repertoire of small RNAs showed a significant depletion of microRNA (miRNA) sequences, matching well with the depletion of “miRNA related” proteins in EVs. The relative expression level of cellular, EV and non-EV miRNAs correlated well and though some differentially expressed (DE) miRNAs were detected, these had a relatively low expression in both the source cells as well as in the secretory fractions. We also quantified the total level of selected miRNAs in EVs and non-EV fraction investigating both the basal as well as overexpressed levels and could verify that the vast majority of mature miRNA is secreted to the non-EV portion of the secretome.
Paper IV was brought about to gain a comprehensive overview of the biodistribution of exogenous EVs. This study confirmed that fluorescent lipophilic dyes are suitable for membrane labelling and in vivo tracking of EVs. The general biodistribution pattern of EVs was seen to follow a common mononuclear phagocytic system (MPS) uptake pattern with the majority of EVs accumulating in the liver, spleen and lungs. Nevertheless, depending on the cell source, administration route, dose and the presence of targeting moieties this distribution could be altered.
The present findings are important to gain a thorough understanding of the nucleic acid delivery capacity of lipid-based nanoparticles, especially EVs and thereby progress their employment as therapeutic nucleic acid carriers.
Firstly, in Paper I we explored the potency of a lipid-based delivery agent, Lipofectamine 2000 which after being frozen and thawed showed orders of magnitude higher nucleic acid delivery efficiency in vitro and in vivo than the non-frozen counterpart. This effect was consistent across different cryo-manipulations, cell lines and also various types of nucleic acid. Further analysis with different methodologies revealed that the underlying potency plausibly relies on the elevated sedimentation and spreading of the complexes and/or relates to the specific structure or composition of the carrier. These findings illustrate that a simple freeze-thawing procedure allows to drastically reduce the amount of transfection reagent for cellular nucleic acid delivery, whilst not losing the desired activity.
Secondly, we shifted our focus to natural lipid-based carriers, EVs in order to shed light on the vesicular and non-vesicular (non-EV) small RNA patterns and their relation to the EV proteome (Paper II and III). Though the studies exploited different EV enrichment methods the relative depletion of vesicular small RNAs was confirmed in both instances. A detailed analysis of the secretory repertoire of small RNAs showed a significant depletion of microRNA (miRNA) sequences, matching well with the depletion of “miRNA related” proteins in EVs. The relative expression level of cellular, EV and non-EV miRNAs correlated well and though some differentially expressed (DE) miRNAs were detected, these had a relatively low expression in both the source cells as well as in the secretory fractions. We also quantified the total level of selected miRNAs in EVs and non-EV fraction investigating both the basal as well as overexpressed levels and could verify that the vast majority of mature miRNA is secreted to the non-EV portion of the secretome.
Paper IV was brought about to gain a comprehensive overview of the biodistribution of exogenous EVs. This study confirmed that fluorescent lipophilic dyes are suitable for membrane labelling and in vivo tracking of EVs. The general biodistribution pattern of EVs was seen to follow a common mononuclear phagocytic system (MPS) uptake pattern with the majority of EVs accumulating in the liver, spleen and lungs. Nevertheless, depending on the cell source, administration route, dose and the presence of targeting moieties this distribution could be altered.
The present findings are important to gain a thorough understanding of the nucleic acid delivery capacity of lipid-based nanoparticles, especially EVs and thereby progress their employment as therapeutic nucleic acid carriers.
List of papers:
I. Sork H, Nordin JZ, Turunen JJ, Wiklander OPB, Bestas B, Zaghloul EM, Margus H, Padari K, Duru AD, Corso G, Bost J, Vader P, Pooga M, Smith CIE, Wood MJA, Schiffelers RM, Hällbrink M, EL Andaloussi S. Lipid-based Transfection Reagents Exhibit Cryo-induced Increase in Transfection Efficiency. Molecular Therapy Nucleic Acids. 2016 Mar; 5(3): e290.
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II. Sork H, Corso G, Krjutskov K, Johansson HJ, Nordin JZ, Wiklander OPB, Lee YXF, Orzechowski Westholm J, Lehtiö J, Wood MJA, Mäger I, EL Andaloussi S. Heterogeneity and interplay of the extracellular vesicle small RNA transcriptome and proteome. [Manuscript]
III. Sork H, Conceicao M, Corso C, Nordin JZ, Lee YXF, Krjutskov K, Orzechowski Westholm J, Vader P, Wood MJA, EL Andaloussi S, Mäger I. Profiling the vesicular and non-vesicular miRNA secretome. [Manuscript]
IV. Wiklander OPB, Nordin JZ, O’Loughlin A, Gustafsson Y, Corso G, Mäger I, Vader P., Lee Y, Sork H, Seow Y, Heldring N, Alvarez-Erviti L, Smith CIE, Le Blanc K, Macchiarini P, Jungebluth P, Wood MJA, EL Andaloussi S. Extracellular vesicle in vivo biodistribution is determined by cell source, route of administration and targeting. Journal of Extracellular Vesicles. 2015 Apr 20;4:26316.
Fulltext (DOI)
Pubmed
View record in Web of Science®
I. Sork H, Nordin JZ, Turunen JJ, Wiklander OPB, Bestas B, Zaghloul EM, Margus H, Padari K, Duru AD, Corso G, Bost J, Vader P, Pooga M, Smith CIE, Wood MJA, Schiffelers RM, Hällbrink M, EL Andaloussi S. Lipid-based Transfection Reagents Exhibit Cryo-induced Increase in Transfection Efficiency. Molecular Therapy Nucleic Acids. 2016 Mar; 5(3): e290.
Fulltext (DOI)
Pubmed
View record in Web of Science®
II. Sork H, Corso G, Krjutskov K, Johansson HJ, Nordin JZ, Wiklander OPB, Lee YXF, Orzechowski Westholm J, Lehtiö J, Wood MJA, Mäger I, EL Andaloussi S. Heterogeneity and interplay of the extracellular vesicle small RNA transcriptome and proteome. [Manuscript]
III. Sork H, Conceicao M, Corso C, Nordin JZ, Lee YXF, Krjutskov K, Orzechowski Westholm J, Vader P, Wood MJA, EL Andaloussi S, Mäger I. Profiling the vesicular and non-vesicular miRNA secretome. [Manuscript]
IV. Wiklander OPB, Nordin JZ, O’Loughlin A, Gustafsson Y, Corso G, Mäger I, Vader P., Lee Y, Sork H, Seow Y, Heldring N, Alvarez-Erviti L, Smith CIE, Le Blanc K, Macchiarini P, Jungebluth P, Wood MJA, EL Andaloussi S. Extracellular vesicle in vivo biodistribution is determined by cell source, route of administration and targeting. Journal of Extracellular Vesicles. 2015 Apr 20;4:26316.
Fulltext (DOI)
Pubmed
View record in Web of Science®
Institution: Karolinska Institutet
Supervisor: EL Andaloussi, Samir
Co-supervisor: Smith, C.I. Edvard
Issue date: 2018-05-22
Rights:
Publication year: 2018
ISBN: 978-91-7831-083-8
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