*2.9. Temperature-Induced Release*

The release of calcein caused by heating was measured at 35, 37, 38, 39, 40, 41, 42, and 44 ◦C. Firstly, 490 µL of HEPES buffer was warmed to the desired temperature in an Eppendorf ThermoMixer C (Eppendorf AG, Hamburg, Germany). Once the target temperature was reached, 10 µL of the liposome solution was added into the pre-warmed buffer in the incubator, and the mixture was stirred for 10 min at 300 rpm. The sample was then rapidly cooled in a bucket of ice. The cold control that contained only 490 µL of buffer was kept in the fridge until the addition of 10 µL of the liposomes just before the measurements. Finally, the calcein fluorescence (excitation 493 nm, emission 518 nm) was measured with Varioskan LUX (Thermo Fisher Scientific). The calcein release (R) was calculated as percentages using Equation (3).

$$\text{IR} = \frac{\text{F} - \text{F}\_0}{\text{F}\_{100} - \text{F}\_0} \times 100\% \text{.} \tag{3}$$

where F is the fluorescence of the sample, F<sup>0</sup> denotes the background fluorescence measured from the control, and F<sup>100</sup> is the maximum fluorescence when the content is completely released from the liposomes (addition of 10 µL of 1% Triton X-100 solution for total decomposition).
