2.5. Evaluation of Surface pKa by TNS Assay

For the 6-(p-Toluidino)-2-naphthalenesulfonyl chloride (TNS) assay, 20 mM citric acid/NaOH buffer (with 150 mM NaCl, pH 3.0, 3.5, 4.0, 4.5, 5.0, 5.5), 20 mM sodium dihydrogen phosphate/NaOH buffer (with 150 mM NaCl, pH 6.0, 6.4, 6.8, 7.2, 7.6, 8.0), and 20 mM Tris/HCl buffer (with 150 mM NaCl pH8.5, 9.0, 9.5, 10.0) were prepared. TNS was dissolved at 0.6 mM in water as a stock solution. In the wells of a 96-well black plate, 2 μL of the TNS solution, 12 μL of the LNP solution (0.5 mM total lipid) and 186 μL of the each of the buffers above were mixed. After shaking the incubation mixture (400 rpm, 10 min), the fluorescence of the TNS (Ex: 321/Em: 447) was measured. The apparent pKa of the surface was calculated as the pH at which the LNP showed 50% of the maximum fluorescence.