EPR studies of intermolecular interactions and competitive binding of drugs in a drug-BSA binding model

Understanding intermolecular interactions between drugs and proteins is very important in drug delivery studies. Here, we studied different binding interactions between salicylic acid and bovine serum albumin (BSA) using electron paramagnetic resonance (EPR) spectroscopy. Salicylic acid was labeled with a stable radical (spin label) in order to monitor its mobilized (free) or immobilized (bound to BSA) states. In addition to spin labeled salicylic acid (SL-salicylic acid), its derivatives including SL-benzoic acid, SL-phenol, SL-benzene, SL-cyclohexane and SL-hexane were synthesized to reveal the effects of various drug binding interactions. EPR results of these SL-molecules showed that hydrophobic interaction is the main driving force. Whereas each of the two functional groups (-COOH and -OH) on the benzene ring has a minute but detectable effect on the drug-protein complex formation. In order to investigate the effect of electrostatic interaction on drug binding, cationic BSA (cBSA) was synthesized, altering the negative net charge of BSA to positive. The salicylic acid loading capacity of cBSA is significantly higher compared to that of BSA, indicating the importance of electrostatic interaction in drug binding. Moreover, the competitive binding properties of salicylic acid, ibuprofen and aspirin to BSA were studied. The combined EPR results of SL-salicylic acid/ibuprofen and SL-ibuprofen/salicylic acid showed that ibuprofen is able to replace up to ∼83% of bound SL-salicylic acid, and salicylic acid can replace only ∼14% of the bound SL-ibuprofen. This indicates that ∼97% of all salicylic acid and ibuprofen binding sites are shared. On the other hand, aspirin replaces only ∼23% of bound SL-salicylic acid, and salicylic acid replaces ∼50% of bound SL-aspirin, indicating that ∼73% of all salicylic acid and aspirin binding sites are shared. These results show that EPR spectroscopy in combination with the spin labeling technique is a very powerful method to investigate drug binding dynamics in detail.Turkish Scientific and Technological Research Council (2232-114C082

Aronia melanocarpa (Michaux) Elliot Fruit Juice Attenuates Acetaminophen-induced Hepatotoxicity on Larval Zebrafish Model

Aronia melanocarpa (Michaux) Elliot (chokeberry) is a natural medicinal plant with a rich content of phenolic compounds such as procyanidins, anthocyanins, and phenolic acids. Chokeberry fruits are gaining worldwide popularity due to the strong bioactivities of their phenolic constituents, such as antioxidant, anti-inflammatory, anticancer, and liver-protective effects. In the present study, total phenolic, flavonoid, and anthocyanin contents of chokeberry juice were determined via the Folin-Ciocalteu method, a spectrophotometric method based on AlCl3 complexation, and pH differential method, respectively. Anthocyanin content was determined as 1.14% (equivalent to cyanidin-3-glucoside), while phenolic and flavonoid contents were measured as 5060.87 and 331.03 mg per 100 g of freeze-dried juice (equivalent to gallic acid and quercetin), respectively. The hepatoprotective effects of chokeberry fruit juice were evaluated using a zebrafish in vivo model for acetaminophen (APAP)-induced liver injury. Zebrafish is an emerging in vivo liver injury model that enables hepatoprotective bioactivity screening of samples on live organisms. The APAP-induced liver injury model was established by treating zebrafish larvae with 5 mM APAP from 2 days post fertilization (dpf) to 5 dpf. The hepatoprotective effect of chokeberry was evaluated via exposure to 1, 10, and 100 µg/mL of fruit juice. While chokeberry fruit juice did not cause any toxicity up to 100 µg/mL, it successfully reduced the injury induced by APAP when applied at 1 µg/mL concentration. To our knowledge, this is the first report evaluating the hepatoprotective effects of chokeberry using zebrafish in vivo liver injury model

Preparation of albumin nanoparticles in water-in-ionic liquid microemulsions

Ionic liquids (Its) with a variety of properties have been considered a unique class of solvents. Using ILs in microemulsions as oil substitutes provides environmentally benign media for various applications including nanoparticle synthesis. Here, bovine serum albumin nanoparticles (BSA NPs) widely used in drug delivery studies were prepared in nano-sized water droplets of water-in-IL (W/IL) microemulsion systems. A hydrophobic IL of 1-butyl-3-methylimidazolium hexafluorophosphate (BmimPF(6)) was used as oil component in place of oils (castor oil, olive oil, etc.) and/or conventional organic solvents (cyclohexane, dichloromethane, etc.) in an emulsification method. In order to obtain spherical BSA NPs, high speed homogenizer treatment was applied followed by glutaraldehyde addition. Effects of glutaraldehyde, speed of homogenizer, type of surfactants and compositional fractions of the microemulsion components on the formation of water droplets and/or preparation of BSA NPs were studied using FTIR, EPR, DLS, and SEM techniques. Optimization of these preparation parameters showed that 3 wt% of BSA in a water/Tween 20/BmimPF(6) microemulsion with 20:50:30 wt% yielded similar to 100 nm average sized BSA NPs based on the SEM analysis. Although, water droplet size strongly depends on the water content, BSA nanoparticle size did not show a significant dependency on the water content. On the other hand, surfactant/IL weight ratio is more crucial for obtaining more uniformly size distributed albumin nanoparticles. A significant cellular uptake of BSA NPs prepared in IL based microemulsions with high cell viability showed the potential of this technique in preparation of albumin nanoparticles that can be used also in drug delivery studies. (C) 2019 Elsevier B.V. All rights reserved