Characterization of fluorescein-labelled PVP-based polymeric nanocarriers & their uptake in mammalian cells.
Authors
Ameljan, Daniel ; Kumar, Tushar
Term
4. term
Education
Publication year
2021
Submitted on
2021-03-31
Pages
94
Abstract
Denne afhandling undersøger polymeriske miceller som nanobærere til lægemiddellevering med fokus på en amphifil poly(N-vinyl-2-pyrrolidon)-baseret blokcopolymer modificeret med akrylsyre og en thiooctadecyl hydrofob blok (PVP-OD-AA). Formålet er at klarlægge polymerens selvorganisering til stabile, fluorescensmærkede nanobærere og hvordan fremstillings- og mærkningsbetingelser påvirker deres egenskaber, så cellulær optagelse kan studeres uafhængigt af en hydrofob last. Polymerens fysikokemiske egenskaber og opløsningsmiddelkompatibilitet blev karakteriseret (FTIR-spektroskopi og opløsningsmiddel-screening), hvor ethanol blev identificeret som et velegnet opløsningsmiddel til opløsning og efterfølgende EDC/NHS-baseret carbodiimid-kobling. Fluorescein (5-aminofluorescein) blev koblet til akrylsyregrupper via EDC/NHS med forskellige aktiveringsstrategier, og miceller blev fremstillet ved direkte opløsning, cosolvent-fordampning og dialyse. Dynamisk lysspredning viste hydrodynamiske diametre og polydispersiteter omkring 97 nm (PDI 0,530) for direkte opløsning, 168,6 nm (PDI 0,114) for cosolvent-fordampning og 105,6 nm (PDI 0,172) for dialyse. Øvrig karakterisering omfattede UV-Vis- og fluorescensspektroskopi, zeta-potentialemålinger, fluorescensmikroskopi samt undersøgelser af kritisk micellekoncentration og koncentrationsregimer. Samlet set fastlægger arbejdet opløsningsmiddel- og konjugationsbetingelser og demonstrerer dannelsen af fluorescein-mærkede PVP-baserede miceller med justerbare størrelser, som kan bruges til at belyse optagelsesmekanismer i pattedyrsceller.
This thesis examines polymeric micelles as nanocarriers for drug delivery by focusing on an amphiphilic poly(N-vinyl-2-pyrrolidone) block copolymer modified with acrylic acid and a thiooctadecyl hydrophobic block (PVP-OD-AA). The aim is to determine whether this polymer self-assembles into stable, fluorescently trackable nanocarriers and how preparation and labeling conditions affect their properties, enabling studies of cellular uptake independently of a hydrophobic cargo. The polymer’s physicochemical features and solvent compatibility were characterized (FTIR spectroscopy and solvent screening), identifying ethanol as a suitable medium for dissolution and subsequent EDC/NHS carbodiimide coupling. Fluorescein (5-aminofluorescein) was conjugated to acrylic acid groups via EDC/NHS using different activation strategies, and micelles were prepared by direct dissolution, cosolvent evaporation, and dialysis. Dynamic light scattering showed hydrodynamic diameters and dispersities of about 97 nm (PDI 0.530) for direct dissolution, 168.6 nm (PDI 0.114) for cosolvent evaporation, and 105.6 nm (PDI 0.172) for dialysis. Additional characterization included UV-Vis and fluorescence spectroscopy, zeta potential measurements, fluorescence microscopy, and investigations of the critical micelle concentration and concentration regimes. Overall, the study establishes solvent and conjugation conditions and demonstrates the formation of fluorescein-labeled PVP-based micelles with tunable sizes, supporting their use as a platform to interrogate uptake mechanisms in mammalian cells.
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