In this work we present a computational analysis together with experimental studies, focusing on the interaction between a benzothiazole (BTS) and lysozyme. Results obtained from isothermal titration calorimetry, UV-vis, and fluorescence were contrasted and complemented with molecular docking and machine learning techniques. The free energy values obtained both experimentally and theoretically showed excellent similarity. Calorimetry, UV-vis, and 3D/2D-lig-plot analysis revealed that the most relevant interactions between BTS and lysozyme are based on a predominance of aromatic, hydrophobic Van der Waals interactions, mainly aromatic edge-to-face (T-shaped) π-π stacking interactions between the benzene ring belonging to the 2-(methylthio)-benzothiazole moiety of BTS and the aromatic amino acid residue TRP108 of the lysozyme receptor. Next, conventional hydrogen bonding interactions contribute to the stability of the BTS-lysozyme coupling complex. In addition, mechanistic approaches performed using elastic network models revealed that the BTS ligand theoretically induces propagation of allosteric signals, suggesting non-physiological conformational flexing in large blocks of lysozyme affecting α-helices. Likewise, the BTS ligand interacts directly with allosteric residues, inducing perturbations in the conformational dynamics expressed as a moderate conformational softening in the α-helices H1, H2, and their corresponding β-loop in the lysozyme receptor, in contrast to the unbound state of lysozyme.

Among the several possible uses of nanoparticulated systems in biomedicine, their potential as theragnostic agents has received significant interest in recent times. In this work, we have taken advantage of the medical applications of Gadolinium as a contrast agent with the versatility and huge array of possibilities that microfluidics can help to create doped Hydroxyapatite nanoparticles with magnetic properties in an efficient and functional way. First, with the help of Computational Fluid Dynamics (CFD), we performed a complete and precise study of all the elements and phases of our device to guarantee that our microfluidic system worked in the laminar regime and was not affected by the presence of nanoparticles through the flow requisite that is essential to guarantee homogeneous diffusion between the elements or phases in play. Then the obtained biomaterials were physiochemically characterized by means of XRD, FE-SEM, EDX, confocal Raman microscopy, and FT-IR, confirming the successful incorporation of the lanthanide element Gadolinium in part of the Ca (II) binding sites. Finally, the magnetic characterization confirmed the paramagnetic behaviour of the nanoparticles, demonstrating that, with a simple and automatized system, it is possible to obtain advanced nanomaterials that can offer a promising and innovative solution in theragnostic applications.