Nanoemulsions For Gene Therapy
Author
Úbeda I Nicolau, Cristina
Term
4. term
Education
Publication year
2022
Pages
107
Abstract
This thesis investigates whether nanoemulsions can serve as non-viral vectors for gene therapy by encapsulating plasmid DNA and delivering it to mammalian cells. Nanoemulsions containing a plasmid encoding the fluorescent protein TagRFP were produced by electrospray and high-speed stirring and characterized by nanoparticle tracking analysis (NTA) and scanning electron microscopy; particle sizes ranged from about 200 to 1,300 nm. To control droplet formation, microfluidic chips were designed in AutoCAD/LayoutEditor, considered for fabrication by 3D printing and soft lithography, and evaluated in 2D COMSOL simulations using the level set method. Simulations indicated that flow-focusing geometries with 400 µm channels generated droplets around 60 µm, while 100 µm channels yielded droplets around 30 µm; no droplet formation was obtained for T-junction designs under the tested conditions. For biological evaluation, plasmid DNA isolated from Escherichia coli was used to transfect HeLa cells, and transfection efficiency was assessed by fluorescence microscopy. Fluorescence from TagRFP was detected after 24–48 hours when the plasmid was incubated with poly-L-lysine, whereas no transfection was observed when cells were incubated with the droplets. These results outline practical routes to produce and model nanoemulsions for gene delivery and highlight the need for further optimization to achieve effective cell transfection with droplet-based systems.
Denne afhandling undersøger, om nanoemulsioner kan fungere som ikke-virale vektorer til genterapi ved at indkapsle plasmid-DNA og levere det til pattedyrsceller. Nanoemulsioner med et plasmid, der koder for det fluorescerende protein TagRFP, blev fremstillet ved elektrospray og højhastighedsomrøring og karakteriseret med nanopartikelsporing (NTA) og scanningelektronmikroskopi; partikelstørrelserne lå omkring 200–1.300 nm. For at styre dråbedannelse blev mikrofluidiske chips designet i AutoCAD/LayoutEditor, tiltænkt fremstillet via 3D-print og blød litografi, og evalueret i 2D-COMSOL-simulationer med level-set-metoden. Simulationer viste, at flow-fokuserede geometrier med kanaler på 400 µm genererede dråber omkring 60 µm, mens 100 µm kanaler gav dråber omkring 30 µm; der sås ingen dråbedannelse i T-kryds-designs under de testede betingelser. Til biologisk evaluering blev plasmid-DNA isoleret fra Escherichia coli anvendt til transfektion af HeLa-celler, og transfektionseffektivitet blev vurderet med fluorescensmikroskopi. Fluorescens fra TagRFP blev påvist efter 24–48 timer, når plasmidet var inkuberet med poly-L-lysin, mens der ikke blev observeret transfektion, når cellerne blev inkuberet med dråberne. Resultaterne skitserer praktiske fremgangsmåder til at fremstille og modellere nanoemulsioner til genlevering og understreger behovet for videre optimering for at opnå effektiv celletransfektion med dråbebaserede systemer.
[This apstract has been generated with the help of AI directly from the project full text]
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