The growing interest in high-energy emulsification is a result of its scalability, which is important from an industrial perspective and allows for a more reproducible and efficient production of pharmaceutical formulations. The aim of this study was to evaluate the effect of composition, mainly a fixed surfactant/cosurfactant (S_mix) ratio, their concentration, and the parameters of high-pressure homogenization (HPH) processing on the quality and stability of ophthalmic fluconazole-loaded nanoemulsions. After a physicochemical analysis of nanoemulsions containing 20% w/w of oil, as optimal conditions for the HPH process, three cycles at a pressure of 1000 bar were established, obtaining formulations with an average droplet diameter size in the range of 80.63–129.68 nm and PDI values below 0.25. While it was expected that an increasing cosurfactant concentration decreased the droplet size, in the case of formulations containing Tween 20 and 10% w/w of cosurfactants, “over-processing” was observed, identified by the droplet size and polydispersity index increase. Consecutively, the selected formulations were evaluated for in vitro drug release in Franz’s cell, antifungal activity, and 30-day stability using NMR spectroscopy. An antifungal activity test showed no significant difference in the antifungal activity between optimal fluconazole-loaded nanoemulsions and a 0.3% aqueous drug solution, but previously, research showed that prepared formulations were characterized by a higher viscosity and satisfactory prolonged release compared to a control. In a 30-day stability study, it was observed that higher HLB values of the used surfactants decreased the stability of the formulations in the following order: Kolliphor EL, Tween 80, Tween 20. The NMR spectra confirmed that Kolliphor EL-based formulations ensured the higher stability of the nanoemulsion composition in comparison to Tween 80 and a better stabilizing effect of propylene glycol as a cosurfactant in comparison to PEG 200. Therefore, the optimization of HPH technology should be focused on the selection of S_mix and the S_mix:oil ratio in order to prepare stable formulations of high quality.

Despite extensive use of micelles in materials and colloidal science, their supramolecular organization as well as host-guest interactions within these dynamic assemblies are poorly understood. Small guest molecules in the presence of micelles undergo constant exchange between a micellar aggregate and the surrounding solution, posing a considerable challenge for their molecular level characterisation. In this work we reveal the interaction maps between small guest molecules and surfactants forming micelles via novel applications of NMR techniques supported with state-of-the-art analytical methods used in colloidal science. Model micelles composed of structurally distinct surfactants (block non-ionic polymer Pluronic® F-127, non-ionic surfactant Tween 20 or Tween 80, and ionic surfactant sodium lauryl sulphate, SLS) were selected and loaded with model small molecules of biological relevance (i.e. the drugs fluconazole, FLU or indomethacin, IMC) known to have different partition coefficients. Molecular level organization of FLU or IMC within hydrophilic and hydrophobic domains of micellar aggregates was established using the combination of NMR methods (1D ¹H NMR, 1D ¹⁹F NMR, 2D ¹H-¹H NOESY and 2D ¹H-¹⁹F HOESY, and the multifrequency-STD NMR) and corroborated with molecular dynamics (MD) simulations. This is the first application of multifrequency-STD NMR to colloidal systems, enabling us to elucidate intricately detailed patterns of drug/micelle interactions in a single NMR experiment within minutes. Importantly, our results indicate that flexible surfactants, such as block copolymers and polysorbates, form micellar aggregates with a surface composed of both hydrophilic and hydrophobic domains and do not follow the classical core-shell model of the micelle. We propose that the magnitude of changes in ¹H chemical shifts corroborated with interaction maps obtained from DEEP-STD NMR and 2D NMR experiments can be used as an indicator of the strength of the guest-surfactant interactions. This NMR toolbox can be adopted for the analysis of broad range of colloidal host-guest systems from soft materials to biological systems.

The imidazole ring of histidine (His) and the methylcarboxylate function of aspartic acid (Asp) are crucial for Cu coordination in enzymes, artificial proteins and other bioinspired systems. In order to elucidate the effect of Asp at the C-terminus, a new branched peptide, H-Gly-Dap(H-Gly)-Asp-NH₂ (2GD, Dap = 2,3-diaminopropionic acid) has been synthesized to ligate Cu^II. While at pH < 8, 2GD favours dimeric complexes at 1 : 1 metal-to-ligand ratio, at increasingly alkaline pH the Asp at the C-terminus creates a {NH₂,N⁻,N⁻,O⁻}_eq set and the dominance of mononuclear CuLH₋₂ and CuLH₋₃ species, according to potentiometry, molecular spectroscopy (UV-visible, circular dichroism and X-band electron paramagnetic resonance) and ESI-MS analysis. The changes in speciation with pH are accompanied by shifts in the Cu^III/II redox current peaks that were analyzed by square wave voltammetry (SWV) and allowed the calculation of the pK_a values for the Cu^II and Cu^III forms. The 2GD complex exhibits electrocatalytic activity in water oxidation in phosphate buffer. However, when Asp residues are present at all three termini to produce 3D (H-Asp-Dap(H-Asp)-Asp-NH₂), the electrocatalysis of water oxidation decreases and, if present only at the N-termini in 2DG (H-Asp-Dap(H-Asp)-Gly-NH₂), it ceases. As for Cu^II ligated by 2GD, a TOF of ∼16 s⁻¹ was calculated at pH = 11 in 0.2 M phosphate electrolyte, which is crucial for catalysis, but also acts as inhibitor anion according to cyclic voltammetry observations. The system is highly tolerant to the presence of chloride, which is a feature of practical importance in efficient water oxidation catalysis.

Candida strains as the most frequent causes of infections, along with their increased drug resistance, pose significant clinical and financial challenges to the healthcare system. Some polymeric excipients were reported to interfere with the multidrug resistance mechanism. Bearing in mind that there are a limited number of marketed products with fluconazole (FLU) for the topical route of administration, Pluronic F-127 (PLX)/FLU formulations were investigated in this work. The aims of this study were to investigate (i) whether PLX-based formulations can increase the susceptibility of resistant Candida strains to FLU, (ii) whether there is a correlation between block polymer concentration and the antifungal efficacy of the FLU-loaded PLX formulations, and (iii) what the potential mode of action of PLX assisting FLU is. The yeast growth inhibition upon incubation with PLX formulations loaded with FLU was statistically significant. The highest efficacy of the azole agent was observed in the presence of 5.0 and 10.0% w/v of PLX. The upregulation of the CDR1/CDR2 genes was detected in the investigated Candida strains, indicating that the efflux of the drug from the fungal cell was the main mechanism of the resistance.