The liposome stock solution obtained after gel filtration was first diluted 100 times in PBS (1×, pH 7.4) to prepare 5 ml of solution A. Then, solution A was diluted 10 times in PBS (1×, pH 7.4), and the resulting solution (18 ml) was aliquoted into a 0.5-ml Protein LoBind Eppendorf tube (0.3 ml per tube). For each temperature (22°, 37°, 50°, 60°, and 70°C), 11 tubes corresponding to 11 time points were prepared and incubated at the corresponding temperatures. For every time point (t = 0, 0.5, 1, 1.5, 2, 3, 4, 5, 6, 7, and 8 hours), the fluorescence (λEm: 485 nm and λEx: 517 nm) of one tube/temperature was measured at room temperature in triplicate using a PerkinElmer EnSpire multimode plate reader and Costar 96-well half-area plates. Solution A was also diluted 10-fold in a solution of 0.5% Triton X-100 in PBS (1×, pH 7.4) to induce 100% leakage. After 15 min of incubation at room temperature, the fluorescence of the solution was measured as above. In addition, the values were corrected for the quenching of CF fluorescence by Triton X-100 using our plate reader (measured correction factor = 1.28). This leakage experiment was repeated at least three times per stock solution of liposome to ensure accuracy.

For each assay, the percent leakage (%leakage) was normalized using Eq. 1, where F0 represents fluorescence at t0, Ft represents fluorescence measurements at different times t, and Ftriton is the fluorescence measurement of the liposome solution including Triton X-100%leakage=((FtF0)(FtritonF0))×100(1)

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