Ovalbumin (ova)

OVA-grafted NLC (NLC–OVA) were obtained as previously reported.^{20 (link)} Thiol-functionalized NLC (NLC–SH) were prepared by adding 5 mg of (2-pyridildithio) propionate-functionalized PEGylated surfactants to the aqueous phase and by reducing the disulfide bond using TCEP (tris(2-carboxyethyl) phosphine hydrochloride, Sigma Aldrich). The resulting NLC–SH were purified by dialysis and filtered on a 0.22 µm membrane. Functional maleimide groups were introduced on OVA (OVA–mal) by derivatization of 1 mg OVA (Bio Basic Inc.) with 200 µg sulfo-SMCC (sulfosuccinimidyl 4-(N-maleimidomethyl)cyclohexane-1-carboxylate, Thermo Scientific). Excess linker was removed by gel filtration on Sephadex G25 gel in a PD10 desalting column (GE Healthcare). OVA–mal (1 mg) was added dropwise to a solution of 17 mg NLC–SH, under gentle stirring at 4 °C. The reaction was allowed to proceed overnight at room temperature. Free thiols were capped using N-(2-hydroxyethyl)maleimide (Sigma Aldrich). NLC–OVA were then purified by size exclusion chromatography (SEC) on Superdex 200 gel (GE Healthcare). To determine the number of OVA per NLC, the same reaction was performed in parallel using fluorescently-labeled moieties: DiD-loaded NLC–SH and fluorescein-tagged OVA, and fluorescence titration was performed (both dyes from Invitrogen).

CHI (M_w = 190,000‒310,000 g/mol), ALG (M_w = 120,000‒190,000 g/mol), TA, DOX, and FITC were purchased from Sigma-Aldrich (St. Louis, MO, USA). Mg⁺ and Ca²⁺-free 10× PBS was purchased from Gibco (Waltham, MA, USA). The hyaluronic acid was purchased from SK Bioland (Cheonan, Chungnam, South Korea). The Ova was purchased from Bio Basic Inc. (Konrad Crescent, Markham, Ontario, Canada). The Ova fluorescein conjugate was purchased from Invitrogen (Carlsbad, CA, USA).