Browsing by Subject "farmaseuttinen nanoteknologia, biofarmasia"

Sort by: Order: Results:

Now showing items 1-1 of 1
  • Kari, Otto (Helsingin yliopisto, 2020)
    Nanoparticles administered into the body are rapidly covered by biomolecules and assume a biological identity that mediates their biological interactions. Methodological constraints have limited our ability to study the formation of this “protein corona”. To increase our understanding of its formation and evolution in different biological environments, and hence improve the safety and efficacy of nanomedicines, there is a need for non-invasive and label-free methods that better mimic the conditions inside the human body. This thesis describes the development of a novel label-free workflow to determine the structure and composition of the hard and soft corona on liposomes. Multi-parametric surface plasmon resonance is adapted to probe corona structure without interference, including the soft corona of loosely bound proteins. Microfluidics is used to elute the corona subsections for proteomics analysis of their compositions by nanoliquid chromatography tandem mass spectrometry. The in vivo relevance of the method is improved by the use of undiluted biological fluids under dynamic flow conditions. The workflow was applied to the preclinical development of pH-responsive and light- activated liposomes intended for tumour targeting and ocular drug delivery. A novel hyaluronic acid (HA)-coated liposome was also synthesized. The results indicate that thin and sparse monolayer hard coronas form on the liposomes in plasma and vitreous. It is covered by a soft corona of monolayer thickness in plasma, but in vitreous, the soft corona is less defined. Formulation-dependent differences in corona structure and relative protein composition were observed in plasma, and the liposomes clustered based on their surface charge. In vitreous, corona composition was independent of formulation properties. In both environments, there is a high overlap in hard and soft corona compositions, and the most abundant proteins are predominantly hydrophilic and negatively charged irrespective of liposome properties. A common set of surfactant and immune system associated proteins adsorbs in both plasma and vitreous, possibly in response to the high surface free energy of lipid bilayers, labelling them “non-self” lipid particles. However, liposomal lipids induced the enrichment of stealth-mediating proteins in the plasma hard coronas regardless of pegylation. These also enriched in the soft coronas of most other formulations, suggesting that the soft corona is part of the biological identity and modulated by liposome surface properties. Protein-specific, rather than formulation- specific factors, are drivers of protein adsorption in the vitreous, possibly due to the molecular crowding activity of structural HA. Although the HA-coated liposome bound more proteins in vitreous, which may interact with its structural meshwork, corona formation did not significantly influence the vitreal mobility of HA or PEG-coated anionic liposomes. The HA-coating yielded improved plasma stability of the light-activated liposomes with otherwise comparable properties, making it a promising coating for both intravenous and ocular drug delivery applications. The workflow addresses significant methodological gaps in the research field by providing information on truly complementary hard and soft corona compositions together with their in situ structural properties. This is achieved with undiluted biofluids and under dynamic conditions without the use of interfering labels. The first description of vitreal corona formation on drug delivery systems contributes to our understanding of ocular pharmacokinetics. The method was also applied to determine the kinetics of opsonin binding directly on liposomes for the first time. The convenient and easily reproducible workflow is well-suited for the preclinical development of liposomes, and it can be combined with other omics methodologies and adapted to accelerate the preclinical development of different types of nanomedicines.