NP-induced vesicle leakage was tested by means of calcein release71. Calcein is a membrane-impermeable fluorescent probe, self-quenched at high concentration. It is commonly used for leakage assays involving Au NPs to probe the membrane integrity during the NP-membrane interaction25,57. Measurements were performed at 25 °C in a quartz cuvette (2.4 mL) at a lipid concentration of 0.035 mM, in a 100 mM NaCl, 2 mM histidine, 2 mM TES, 0.1 mM EDTA buffer, using a Fluorolog spectrofluorometer (Horiba Jobin-Ivon). Throughout the whole experiment, calcein fluorescence was monitored as a function of time (λex = 490 nm, λem = 520 nm). To ensure the homogeneity of the system before and after NP addition, the sample was continuously stirred with a magnetic bar. We observed that the introduction of the stirring bar in the cuvette caused a transient, reproducible fluorescence increase that reached a plateau after 30 min, as shown in Fig. 2b. This behavior was absent if fluorescence was recorded for the same length of time in the absence of the magnetic bar. We interpreted it as due to the adsorption of lipids by the hydrophobic Teflon surface of the magnetic bar and by the hydrophilic walls of the quartz cuvette, facilitated by stirring and resulting in the disruption of some vesicles with subsequent release of calcein. Similar behavior has already been observed for liposomes of different composition and the extent of release was shown to depend on the lipid mixture and the cuvette material72. Therefore, to avoid artifacts, NPs were always added to the POPC vesicle suspension 30 min after introducing the stirrer in the cuvette. In particular, filtered NP dispersions were added to POPC liposomes at a NP/lipid mass ratio, Rm, of 0.03 and 0.05. We chose to use such mass ratios (excluding higher values) as further precaution (in addition to NP Z-potential in buffer) to limit as much as possible NP aggregation before interaction with vesicles. These NP/lipid mass ratios are slightly higher than those used in the literature for similar experiments with Au NPs73. Besides, they correspond to a sufficiently high NP uptake measured by QCM as demonstrated in this investigation (Fig. 1b). Therefore, these Rm values represent a good compromise to study the effect of NP uptake on the bilayer integrity of calcein-loaded vesicles and to limit undesired NP aggregation in solution. The NP volumes added to POPC liposomes were in the range between 14.6 and 124 μL (Fig. 2d), depending on the concentration of NP aqueous dispersions after filtration. In a typical content release assay, fluorescence data are expressed as:

where F(t) is the time-dependent fluorescence, F0 the mean fluorescence level immediately before NP addition, and Fmax the maximum fluorescence (see Fig. 2b). Fmax is determined as the mean fluorescence level immediately after the addition of 0.5% (w/v) sodium cholate to the sample at the end of the experiment to cause the complete leakage of the dye. The data acquisition rate was 1 point per second.

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