Mechanistic studies of nanoparticle-mediated mRNA delivery

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dc.contributor.authorMasmanidou, Olga
dc.contributor.departmentChalmers tekniska högskola / Institutionen för biologi och biotekniksv
dc.contributor.departmentChalmers University of Technology / Department of Biology and Biological Engineeringen
dc.date.accessioned2019-07-05T11:52:43Z
dc.date.available2019-07-05T11:52:43Z
dc.date.issued2018
dc.description.abstractDiseases caused by gene dysfunctions and protein deficiencies are affecting an increasing number of people around the world. The current available medicines are not in many cases adequate for targeting these, often complex, diseases. Hence there is an emerging need of a new age of therapeutics. Scientists have focused their interest towards nucleic acid based drugs since these moieties are the precursors of proteins. mRNA therapeutics have gathered particular interest since they only need to reach the cytosol (not the nucleus) to be expressed. However, due to their high negative charge, mRNA cannot be delivered naked and carriers must be employed for efficiently transfer mRNA into target cells. In this thesis, two types of carriers were studied synthetically formulated lipid nanoparticles (LNPs) and naturally produced extracellular vesicles (EVs). Different batches of LNPs loaded with Cy5 labeled and eGFP expressing mRNA were tested on two hepatic cell lines (HepG2 and Huh-7). First we determined the eGFP:Cy5-eGFP mRNA ratio for optimal detection, then we investigated the physical and chemical properties of the formulation, with regards to cellular uptake and protein expression. It was found that eGFP:Cy5-eGFP mRNA ratio of 5:1 was optimal for simultaneous detection of mRNA uptake and translation. The comparison of LNP formulations differing in size and lipid surface composition indicates that lipid surface composition can directly affect the internalization of the LNPs and that size plays an essential role both for cellular uptake and protein expression. Larger in size LNPs with a constant surface composition were proven to be optimum for both cellular uptake and protein expression regardless of the cell type. However, eGFP positive EVs originating from bone marrow MSCs and Hek-293T cells were internalized by Huh-7 cells, but not by HepG2 pointing out the fine-tuning capabilities of these naturally derived vesicles. Cytotoxicity assessment revealed that EVs are non-toxic to cells, in contrast to LNPs, for which dose dependent cytotoxicity effects were observed. In general, this study showed that LNPs can successfully delivered mRNA intracellularly but size and lipid surface composition matter for cellular uptake as well as protein expression. EVs can potentially be promising alternative carriers for reducing cytotoxic effects though suitable pairs donor-recipient cells must be identified.
dc.identifier.urihttps://hdl.handle.net/20.500.12380/256819
dc.language.isoeng
dc.setspec.uppsokLifeEarthScience
dc.subjectLivsvetenskaper
dc.subjectCellbiologi
dc.subjectCell- och molekylärbiologi
dc.subjectMedicinsk bioteknologi (med inriktning mot cellbiologi)
dc.subjectLife Science
dc.subjectCell Biology
dc.subjectCell and Molecular Biology
dc.subjectMedical Biotechnology (with a focus on Cell Biology)
dc.titleMechanistic studies of nanoparticle-mediated mRNA delivery
dc.type.degreeExamensarbete för masterexamensv
dc.type.degreeMaster Thesisen
dc.type.uppsokH
local.programmeBiotechnology (MPBIO), MSc
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