Bovine serum albumin (BSA) acts as a carrier for many endogenous and exogenous compounds, such as thyroid hormones or corresponding drugs. Binding of the hydrophilic levothyroxine drug (LT4) to BSA is of significant pharmacological importance. In this work, UV-vis measurements were used to determine the pH value at which LT4 interacts optimally with proteins. The binding mechanism and affinity of the interaction between LT4 and BSA were investigated using Fourier-transform infrared spectroscopy (FT-IR), fluorescence, fluorescence resonance energy transfer (FRET), Surface Plasmon Resonance (SPR), supplemented by molecular docking analysis. Fluorescence measurements revealed the quenching effect of LT4 on the BSA intrinsic fluorescence and LT4 binding with BSA is driven by a ground-state complex formation that may be accompanied by a nonradiative energy transfer process. The thermodynamic parameters correspond to an enthalpic process, driven mainly by hydrogen bonds and van der Waals forces. Using SPR, the adsorbed amount of biomolecules was calculated and the binding affinity of LT4 with confined-BSA was characterized, indicating that the BSA immobilization plays an important role in LT4 binding. Docking studies confirmed the formation of the LT4-BSA complex with LT4 bound to site I on the BSA structure mainly with amino acid residues Trp 213, Tyr 137, Tyr 147. The calculation of the apparent association constant confirms the result obtained in SPR. Communicated by Ramaswamy H. Sarma

In the last few years, a great amount of attention has been given to nanoparticles research due to their physicochemical properties that allow their use in analytical instruments or in promising imaging applications on biological systems. The use of ferrihydrite nanoparticles (Fh-NPs) in practical applications implies a particular control of their magnetic properties, stability, biocompatibility, interaction with the surface of the target, and low toxicity. In this study, the formation and organization of human serum albumin (HSA) molecules around the simple Fh-NPs and Fh-NPs doped with Co and Cu were examined by Scanning Electron Microscopy (SEM) and Atomic Force Microscopy (AFM) in terms of morphology and particle size. The topology of all Fh-NPs shows an organized area of HSA around each type of Fh-NP. Molecular docking studies were used in order to determine the probable location of the ferrihydrite in the HSA structure. The thermal stability of these nanohybrids was further investigated by fluorimetry, using 214-Trp residue from HSA as a spectral sensor. The denaturation temperature (T_m) was determined, and stabilization of the HSA structure in the presence of Fh-NPs was discussed. This study could be a starting point for the development of different applications targeting the structure and stability of Fh-NPs complexes with proteins.

Human serum transferrin (HST) is a glycoprotein involved in iron transport that may be a candidate for functionalized nanoparticles to bind and target cancer cells. In this study, the effects of the simple and doped with cobalt (Co) and copper (Cu) ferrihydrite nanoparticles (Fh-NPs, Cu-Fh-NPs, and Co-Fh-NPs) were studied by spectroscopic and molecular approaches. Fluorescence spectroscopy revealed a static quenching mechanism for all three types of Fh-NPs. All Fh-NPs interacted with HST with low affinity, and the binding was driven by hydrogen bonding and van der Waals forces for simple Fh-NPs and by hydrophobic interactions for Cu-Fh-NPs and Co-Fh-NPs binding, respectively. Of all samples, simple Fh-NPs bound the most to the HST binding site. Fluorescence resonance energy transfer (FRET) allowed the efficient determination of the energy transfer between HST and NPs and the distance at which the transfer takes place and confirmed the mechanism of quenching. The denaturation of the HST is an endothermic process, both in the case of apo HST and HST in the presence of the three types of Fh-NPs. Molecular docking studies revealed that Fh binds with a low affinity to HST (K_a = 9.17 × 10³ M⁻¹) in accord with the fluorescence results, where the interaction between simple Fh-NPs and HST was described by a binding constant of 9.54 × 10³ M⁻¹.

Protein-based particles are one of the most important research topics in nanomedicine, being used especially as drug delivery systems. From the wide variety of proteins, albumins offer several advantages in biomedical applications due to their special properties. Albumin nanoparticles play an important role as carriers in the drug delivery of chemical and biomolecular drugs, such as anticancer drugs; offer many advantages, such as biocompatibility and biodegradability; and are well-tolerated, without any side effects. In this work, various types of bovine serum albumin nanoparticles (BSA NPs), with or without ascorbic acid or glucose, were prepared via different nanoprecipitation methods. The obtained BSA NPs were characterized by UV–Vis absorption spectroscopy. Their size and morphology were studied by Scanning Electron Microscopy (SEM) and Atomic Force Microscopy (AFM). The stability in time of the developed BSA NPs was spectrally monitored. Three types of bio-entities containing BSA NPs and chlorophyll-labeled artificial cell membranes were “green” developed. The designed biohybrids were characterized by UV–Vis absorption and fluorescence emission spectroscopy, and their three-dimensional topography was investigated by AFM. Both the size and shape of the developed bio-entities were monitored through SEM analysis. These results could be exploited in the development of novel drug carrier systems or as bio-coatings to be used in the biomedical field.

Levothyroxine (LT4) is known for its use in various conditions including hypothyroidism. LT4 interaction with serum albumin may be influenced by the presence of vitamins. For this reason, we investigated the effect of vitamin C, vitamin B12, and folic acid on the complex of Bovine Serum Albumin with LT4 (BSA-LT4). UV-Vis spectroscopy was used to monitor the influence of vitamins on the BSA-LT4 complex. Fluorescence spectroscopy revealed a static quenching mechanism of the fluorescence of BSA-LT4 complex by the vitamin C and folic acid and a combined mechanism for vitamin B12. The interaction of vitamin C and folic acid with BSA-LT4 was moderate, while the binding of vitamin B12 was much stronger, extending the storage time of LT4 in blood plasma. Synchronous fluorescence found that the vitamins were closer to the vicinity of Trp than to Tyr and the effect was more pronounced for the binding of vitamin B12. The thermal stability of the BSA-LT4 complex was more evident, but no influence on the stability of BSA-LT4 complex was obtained for vitamin C. Molecular docking studies showed that vitamin C and folic acid bound the same site of the protein, while vitamin B12 bonded to a different site.