Sephadex g 25 fine

To separate free ICG and NPs, a gel filtration procedure was developed by modifying literature methods [43 (link),44 (link)]. Here, 2 g Sephadex G-25 (Fine, Sigma Aldrich, Milan, Italy) was hydrated in distilled water, and a chromatographic column was packed by gravity. A flux of 0.4 mL/min was maintained by means of an HPLC pump (isocratic Kontron 420, Kontron Instruments, Milan, Italy). Then, 250 µL of ICG aqueous solution 0.4 M was loaded as reference. Subsequently, 1 mL fractions were collected and analyzed at 780 nm wavelength by means of a spectrophotometer (Perkin Elmer Lambda 25, Milan, Italy) versus a calibration curve. Different amounts of NPs were mixed with the same amount of ICG solution of the reference experiment and loaded in the column, obtaining molar ratios between ICG and CS (as a monomeric unit) ranging from 8:1 to 0.4:1. In these cases, as well, 1 mL volumes were collected and analyzed for ICG concentration. A similar procedure was followed when loading ads-ICG NPs and enc-ICG NPs, both at an ICG:CS molar ratio of 0.4. These were, however, eluted not only with distilled water but also with DMEM cell culture medium supplemented with 10% v/v inactivated fetal calf bovine serum (Euroclone, Milan, Italy).

Native αLA (6 mg) was dissolved in 600 µL of a 0.1 M tris(hydroxymethyl)aminomethane hydrochloride (Tris) buffer solution at pH 8.0 containing 5 M GdmCl and 0.2 mM DTT^red. After incubation for 2 h at 25 °C, the solution was passed through a size-exclusion column (Sephadex™ G-25 Fine, Sigma Aldrich, Tokyo, Japan), which was equilibrated at room temperature in a 0.1 M Tris buffer solution at pH 8.0, with a flow rate of 0.8 mL/min. The fraction containing R (4 mL) was collected and was diluted with the same buffer solution to adjust the final concentration of R to be 30 µM. The concentration was determined by the absorbance at 280 nm using a molar extinction coefficient of R (ε₂₈₀ = 27,330 cm⁻¹·M⁻¹) determined by amino acid analysis and UV spectrometry.