Explanation of inconsistencies in the determination of human serum albumin thermal stability.

Thermal denaturation of human serum albumin has been the subject of many studies in recent decades, but the results of these studies are often conflicting and inconclusive. To clarify this, we combined different spectroscopic and calorimetric techniques and performed an in-depth analysis of the structural changes that occur during the thermal unfolding of different conformational forms of human serum albumin. Our results showed that the inconsistency of the results in the literature is related to the different quality of samples in different batches, methodological approaches and experimental conditions used in the studies. We confirmed that the presence of fatty acids (FAs) causes a more complex process of the thermal denaturation of human serum albumin. While the unfolding pathway of human serum albumin without FAs can be described by a two-step model, consisting of subsequent reversible and irreversible transitions, the thermal denaturation of human serum albumin with FAs appears to be a three-step process, consisting of a reversible step followed by two consecutive irreversible transitions.

Hypericin can cross barriers in the chicken's chorioallantoic membrane model when delivered in low-density lipoproteins.

BACKGROUND: Low-density lipoproteins (LDL) were used as a natural drug delivery system for the transport of hypericin (Hyp) in the bloodstream of the chicken's chorioallantoic membrane model (CAM). Hyp was chosen as a model for hydrophobic drug used in photo-diagnosis and photo-treatments (PDT). The extravasation of the Hyp:LDL complexes for different concentration ratios and the redistribution of Hyp between different serum components were investigated with an innovative statistical treatment. METHODS: Hyp biodistribution was monitored in CAM by intravital fluorescence microscopy. The innovative statistical treatment of experimental data presented here enabled us to obtain highly detailed information from the weak Hyp fluorescence distribution in CAM blood vessels. Hyp redistribution between the serum components was studied by fluorescence spectroscopy in lipids/protein composed solutions. RESULTS: Extravasation of Hyp was dependent on Hyp:LDL concentration ratio. While Hyp:LDL = 50:1 resulted in a significant Hyp extravasation, the Hyp extravasation from Hyp:LDL = 100:1 was weak. The redistribution of Hyp was found to be faster for lipidic particles than for proteins. CONCLUSION: We have demonstrated that lipids composition has a significant control over Hyp delivery in CAM.

Negligible interaction of [Ru(Phen)3]2+ with human serum albumin makes it promising for a reliable invivo assessment of the tissue oxygenation.

The interaction between a ruthenium - based water soluble oxygen probe ([Ru(Phen)3]2+, phen - phenanthroline) and human serum albumin (HSA) was investigated with the aim of describing the influence of HSA on the [Ru(Phen)3]2+ luminescence properties. Nowadays, several oxygen sensitive luminescent probes are used to determine the oxygen level in different compartments of living organisms. However, they can interact, depending on their hydrophilic/hydrophobic characters, with various serum proteins, and/or lipids, during their utilization for invivo oxygen measurement. Since HSA is the most abundant serum protein in most biological organisms, its presence may affect the spectral properties of the employed probes and, consequently, the determination of the oxygen concentration. Having this in mind, we have applied several spectroscopic and calorimetric techniques to study [Ru(Phen)3]2+ - HSA mixtures. Only a negligible effect of HSA on the absorption and luminescence spectra of [Ru(Phen)3]2+ was observed. In addition, differential scanning calorimetric studies showed that [Ru(Phen)3]2+ does not significantly influence HSA thermal stability. Importantly, [Ru(Phen)3]2+ retained a reliable luminescence lifetime sensitivity to the oxygen concentration in solutions supplemented with HSA and in U87 MG cancer cells. Finally, the biodistribution of [Ru(Phen)3]2+ in the presence of serum proteins in the blood stream of chick embryo's chorioallantoic membrane (CAM) was investigated. Fast [Ru(Phen)3]2+ and similar extravasations were observed in the presence or absence of CAM-serum. We can conclude that HSA-[Ru(Phen)3]2+ complex interaction does not significantly influence the potential of [Ru(Phen)3]2+ to be a suitable candidate for a reliable oxygen probe in living organisms.

Estimation of PKCδ autophosphorylation in U87 MG glioma cells: combination of experimental, conceptual and numerical approaches.

Golgi apparatus (GA) is a center for lipid metabolism and the final target of ceramide pathway, which may result in apoptosis. In this work localization of highly hydrophobic hypericin is followed by time-resolved imaging of NBDC6 (fluorescent ceramide) in U87 MG glioma cells. Decrease of NBDC6 fluorescence lifetimes in cells indicates that hypericin can also follow this pathway. It is known that both, ceramide and hypericin can significantly influence protein kinase C (PKC) activity. Western blotting analysis shows increase of PKCδ autophosphorylation at Ser645 (p(S645)PKCδ) in glioma cells incubated with 500 nM hypericin and confocal-fluorescence microscopy distinguishes p(S645)PKCδ localization between GA related compartments and nucleus. Experimental and numerical methods are combined to study p(S645)PKCδ in U87 MG cell line. Image processing based on conceptual qualitative description is combined with numerical treatment via simple exponential saturation model which describes redistribution of p(S645)PKCδ between nucleus and GA related compartments after hypericin administration. These results suggest, that numerical methods can significantly improve quantification of biomacromolecules (p(S645)PKCδ) directly from the fluorescence images and such obtained outputs are complementary if not equal to typical used methods in biology.

Deeper insights into the drug defense of glioma cells against hydrophobic molecules.

By means of fluorescence microscopy the intracellular distribution of fluorescent drugs with different hydrophobicity (quinizarin, emodin and hypericin) was studied. Selective photoactivation of these drugs in precisely defined position (nuclear envelope) allowed moderately hydrophobic emodin enter the nucleus. Highly hydrophobic hypericin was predominantly kept in the membranes with no fluorescence observed in the nucleus. The redistribution of quinizarin, emodin and hypericin between lipids, proteins and DNA was studied in solutions and cells. Based on these results was proposed theoretical model of hydrophobic drugs' nuclear internalization after photo-activation. Molecular docking models showed that hypericin has the strongest affinity to P-glycoprotein involved in the cell detoxification. Presence of 10 µM quinizarin, emodin or hypericin increased P-glycoprotein function in U87 MG cells. Moreover, emodin pretreatment allowed quinizarin nuclear internalization without photo-activation, which was not the case for hypericin. The synergy of such pretreatment and photo-activation should lessen the drug doses with simultaneous increase of drug efficacy triggering cell apoptosis/necrosis.