Thioflavin T (ThT) is a fluorescent dye able to enhance significantly its fluorescence quantum yield upon binding to protein amyloids. ThT assay is widely used to detect and quantify amyloids in a variety of conditions, including solutions with different pH levels. In the present work, the effect of acidic and basic pH on the conformation of the ThT molecule and its absorption and fluorescence properties was studied. The results show that both acidic and basic pH decrease significantly the intensity of ThT absorption in the visible region and fluorescence emission intensity. Low pHs induce an immediate "all-or-nothing" decrease in the ThT signal, while in alkaline solutions the ThT signal decreases gradually over time. pH-induced signal quenching is less in the presence of glycerol or protein aggregates. Two different mechanisms are responsible for the ThT signal quenching-the ThT hydroxylation at basic pH and protonation of the nitrogen atom of the dimethylamino group at acidic pH. ThT assays should be carefully carried out at basic or acidic pH as strong pH dependence of ThT could be responsible for misinterpretation and false positive/negative experimental results. The potential unsuitability of ThT as a probe in solutions with high pH (>9) has been shown.

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L-arginine was introduced into protein-based freeze-dried formulations to study the ability of arginine to reduce/prevent from protein aggregation during manufacturing, storage and reconstitution of lyophilized protein-based pharmaceuticals. As L-arginine is known to be very hygroscopic, additional excipients which could provide a moisture buffering capacity need to be introduced into the formulation. In the first part of our study-excipient formulation development-the screening of a number of sugars/polyols has been done in order to select the best combination of excipients that, in a complex with L-arginine, can (i) produce freeze-dried cakes with elegant appearance, adequate mechanical properties and reconstitution times, and (ii) resist/minimise the moisture sorption. Various freeze-dried cakes containing L-arginine in combination with mannitol, trehalose, lactose and sucrose were produced and analysed by TGA, DSC, texture analysis, moisture sorption, cake shrinkage, TVIM and SEM. The non-linear dependencies of the physicochemical properties of the freeze-dried cakes on the sugar-to-mannitol ratios were found. The best combinations of excipients (L-arginine, mannitol and trehalose) were selected to be used in the second part of this work, in which the impact of each selected formulation will be studied in relation to the aggregation of a protein.

Hydrogels are polymeric materials used in many pharmaceutical and biomedical applications due to their ability to form the 3-D hydrophilic polymeric networks, which can absorb large amounts of water. In the present work polyethylene glycols (PEG) were introduced into the hydrogel liquid phase in order to improve the mechanical properties of hydrogels composed of 2-hydroxyethylacrylate and 2hydroxyethylmethacrylate (HEA-HEMA) synthesized with different co-monomer compositions and equilibrated in water or in 20% water-PEG 400 Da and 600 Da solutions. The thermoanalytical techniques (differential scanning calorimetry (DSC) and thermogravimetry (TG)) were used to evaluate the amount and properties of free and bound water in HEA-HEMA hydrogels. Internal structure and the mechanical properties of hydrogels were studied using scanning electron microscopy and friability assay. TG 'loss-on-drying' experiments were applied to study the water-retention properties of hydrogels whereas the combination of TG and DSC allowed estimating the total amount of freezable and non-freezing water in hydrogels. The results show that the addition of the viscous co-solvent (PEG) to the liquid medium results in significant improvement of the mechanical properties of HEA-HEMA hydrogels and also slightly retards the water loss from hydrogels. A redistribution of free and bound water in the hydrogels equilibrated in mixed solutions containing 20 vol% of PEGs takes place.