Characterization of 12 kDa PVP-OD Nanocarriers and the Influence of Their Size on the Uptake in Mammalian Cells
Author
Nelemans, Levi Collin
Term
4. term
Education
Publication year
2019
Submitted on
2019-06-10
Pages
71
Abstract
Nanobærere er bittesmå partikler, der kan transportere lægemidler og forbedre biotilgængelighed, hvor længe et lægemiddel bliver i blodbanen, og hvor meget der når frem til celler. I denne kandidatopgave undersøges det 12 kDa amphifile blokcopolymer PVP-OD (med både vand- og fedtelskende dele) og hvordan nanobærerens størrelse påvirker optagelsen i fibroblaster (CRL 2429), som er bindevævsceller dyrket i laboratoriet. To størrelser blev fremstillet: cirka 220 nm (PDI 0,2) ved sonikering og cirka 21 nm (PDI 0,244) ved cosolvent-evaporation. PDI (polydispersitetsindeks) angiver, hvor ensartede partikelstørrelserne er. Begge typer blev optaget via en energikrævende proces, hvilket sås ved, at hæmning af celleenergi markant sænkede den mediane fluorescensintensitet (MFI) med henholdsvis 90 % og 97 %. MFI er et mål for, hvor meget af materialet der blev målt inde i cellerne ved hjælp af fluorescens. For de små nanobærere fremstillet ved cosolvent-evaporation overlappede signalet med LysoTracker, en farve der markerer cellens sure rum, hvilket peger på optagelse via makropinocytose eller clathrin-medieret endocytose. For at fastslå de præcise veje er der behov for yderligere forsøg med forskellige endocytosehæmmere.
Nanocarriers are tiny particles designed to transport drugs and can improve bioavailability, how long a drug circulates, and how much reaches target cells. This thesis examines the 12 kDa amphiphilic block copolymer PVP-OD (with both water- and fat-loving segments) and how nanocarrier size influences uptake in fibroblasts (CRL 2429), connective tissue cells grown in the lab. Two sizes were produced: about 220 nm (PDI 0.2) by sonication and about 21 nm (PDI 0.244) by cosolvent evaporation. PDI (polydispersity index) indicates how uniform the particle sizes are. Both types were taken up by an energy-dependent process, shown by a strong drop in median fluorescent intensity (MFI) when cellular energy was inhibited—by 90% and 97% for the sonicated and cosolvent-evaporated nanocarriers, respectively. MFI is a fluorescence-based measure of how much material was detected inside cells. For the smaller, cosolvent-evaporated nanocarriers, co-localization with LysoTracker, a dye that marks acidic cellular compartments, suggests uptake via macropinocytosis or clathrin-mediated endocytosis. Further experiments with different endocytic inhibitors are needed to confirm the exact pathways.
[This abstract was generated with the help of AI]
Keywords
Documents