Conjugation of Peptide to tHBcAg VLNP

tHBcAg VLNP was produced and purified as described in Tan et al.^{32 (link)} and Yoon et al.^{67 (link)}. Peptide NRPDSAQFWLHHGGGSLLGRMKGA was conjugated at the spike of the tHBcAg VLNP by mixing tHBcAg: peptide (1:1) in phosphate buffer (25 mM NaH₂PO₄/Na₂HPO₄, pH 7) in the presence of EDC and Sulfo-NHS as described in Gan et al.^{13 (link)}. The conjugated product was dialyzed against phosphate buffer (pH 7) to remove the excessive cross-linkers, and concentrated with VIVASPIN 6 (30 kDa cut-off polyethersulfone membrane; VIVASCIENCE, Germany) at 4500×g, 4 °C. The CPP-tHBcAg VLNP (250 µg/mL) was applied to A431, HT29 and HeLa cells in order to study its rate of internalization into these cells. The cells were incubated at 37 °C for 16 h, washed, fixed, and permeabilized with ice cold methanol at − 20 °C for 6 min. Then, the cells were incubated with the mouse anti-HBcAg monoclonal antibody [1:100 dilutions in PBS containing BSA (0.2 mg/mL); Santa Cruz Biotechnology, Dallas, Texas, USA] for 1 h at RT, followed by incubation with FITC-conjugated goat anti-mouse antibody (1: 100 dilutions in PBS containing 0.2 mg/mL BSA; BD Biosciences, San Jose, CA, USA) for another 1 h at RT. After that, the cells were washed and stained with Hoechst 33342 prior to viewing under a fluorescence microscope.