Preparation and Characterization of Diph@HsaFtH

Diph was dissolved in dimethyl sulfoxide (DMSO) at 5 mg/mL and diluted with Na₂HPO₄-NaH₂PO₄ (200 mM, pH 7) to 1 mg/mL. For encapsulation, HsaFtH was dissociated by adding 1.25 µL of 1 M HCl (pH 4) to 120 µL of 4.3 mg/mL HsaFtH, with subsequent incubation for 15 min at 20 °C. Then, 200 µL of Diph in pH 7.0 buffer was added, and the mixture was incubated for 15 min at 20 °C to induce reassembly of the HsaFtH quaternary structure and to physically entrap Diph in the HsaFtH cavity. To remove the residual unbound Diph, HsaFtH was diafiltrated using Amicon Ultra 50 K (Sigma-Aldrich) with 3× buffer exchange at 6000×g for 15 min. The concentration of encapsulated Diph was quantified via its absorption at 365 nm using a multimode Infinite 200 PRO microplate reader (Tecan, Männedorf, Switzerland). The hydrodynamic diameter (HDD) and polydispersity index (PdI) of Diph@HsaFtH were determined by DLS using a Zetasizer Nano ZS (Malvern Instruments). For this purpose, Diph@HsaFtH was diluted 100× with distilled water, placed in polystyrene cells, and measured at a detector angle of 173°, wavelength of 633 nm, and a temperature of 25 °C, with the refractive index of the dispersed phase 1.45 and 1.333 for a dispersive environment. The same approach was employed to analyze the stability of Diph@HsaFtH at 4 °C and 25 °C. The surface ζ-potential of Diph@HsaFtH diluted 50× with distilled water was analyzed using a Zetasizer Nano ZS (Malvern Instruments). The number of runs varied between 20 and 40, and the calculations considered the diminution of particle concentration based on the Smoluchowski model, with an F(ka) of 1.5 and an equilibration time of 120 s. Morphology was investigated by TEM with negative staining using Nano-W (Nanoprobes, NY, USA). 4 μL of each sample were deposited onto 400-mesh copper grids coated with a continuous carbon layer. The dried grids were imaged using a Tecnai F20 microscope (FEI). Successful reassociation of Diph@HsaFtH was also validated using 6% native polyacrylamide gel electrophoresis (PAGE) (200 V, 4 °C, 25 min) with NativeMark Unstained Protein Standard, subsequent Coomassie Brilliant Blue staining. Diph@HsaFtH was also analyzed using fast protein liquid chromatography - size-exclusion chromatography (FPLC-SEC) on the Superose 6 Increase 10/300 GL column using the BioLogic DuoFlow System (Bio-Rad, Hercules, CA, USA). The sample was injected using a 500 μL injection loop and eluted isocratically using phosphate-buffered saline (PBS, 0.5 mL/min, room temperature). Detection was performed at a wavelength of 280 nm.