Kalliope software

The zeta potential (ZP) and hydrodynamic diameter (HD) of the prepared particle dispersions were determined on a Litesizer500, Anton Paar (Graz, Austria) particle size analyzer at a temperature of 25 °C. The ZP was measured by electrophoretic light scattering (ELS), which measures the speed of the particles in the presence of an electric field. The HD was measured by dynamic light scattering (DLS). Particles suspended in a liquid are constantly undergoing random motion, and the speed of this motion depends on the size of the particles—smaller particles move faster than larger ones. Before analysis, the dispersion was stirred and adjusted to pH 4 with acetic acid, if necessary. For carrying out the measurements, a diluted sample was poured into an omega cuvette for ZP and size evaluation. The data were collected using Kalliope software (Anton Paar, Graz, Austria).

The LUV size distribution was determined using a Litesizer 500 dynamic light scattering instrument with the Kalliope software (Anton Paar). Using disposable cuvettes with 1.0 ml sample volume and a lipid concentration of 50 μM in Na buffer, the transmittance was ≈88%. The refractive index and viscosity of the Na buffer were set to 1.3318 and 0.9064 cP, respectively, the default settings in Kalliope. The default correlation function and fitting curve were used to calculate the diffusion coefficient, mean hydrodynamic diameter (d_LUV), and the polydispersity index (PDI), defined as ${\left(\sigma /d_{\text{LUV}}\right)}^2\text{,}$ where σ² denotes the variance of the size distribution (e.g., Clayton et al., 2016 (link)). Each sample was tested 1 d after extrusion with three independent measurements and at least seven repeats in each measurement. There was only one discernable peak (Fig. S2), with d_LUV = 130 ± 5 nm and a PDI of 0.09 ± 0.04 (n = 9). A PDI < 0.1 is considered to indicate a monodisperse sample (Clayton et al., 2016 (link)). For an LUV sample with d_LUV = 130 nm and a PDI = 0.06, 10% of the LUVs will have a diameter <87 nm and 10% of the LUVs will have a diameter >160 nm.

The average size of live, inactivated, and inactivated split viruses was assessed in a Litesizer 500 dynamic light scattering (DLS) instrument (Anton Paar) at an angle of 175° with 10 × 12 × 45 mm polystyrene cuvettes (Sarstedt) and 1 mL of sample. Triplicate measurements were performed per sample, each with 11 runs, and processed with the Kalliope software (Anton Paar). Live and inactivated virus samples were diluted 1:1,000 in 0.22 µm filtered PBS (pH = 7.4), while inactivated split virus samples were diluted 1:100.

Hydrodynamic diameter (D_h) and intensity-weighted size distribution of the droplets in the formulations were analyzed by DLS using Litesizer 500 device (Anton Paar GmbH, Graz, Austria). The formulations were taken from the previous experiment and poured carefully into a quartz cell (Hellma Analytics, Müllheim, Germany) to avoid air bubbles. The measurements were performed at a light wavelength of 658 nm, a detection angle of 175° (backscattering), and the temperature was set to 25 °C. D_h and the broadness of the intensity size distribution, called the polydispersity index (PDI), were derived from the intensity curve fitting by applying the autocorrelation function using Kalliope Software (Anton Paar GmbH).

The cell zeta potential was measured in 0.02 μm filtered saline with gold electrode Omega cuvettes (Anton Paar, Graz, Austria). For DLS measurements, a 1:50 dilution of the standardized cultures in filtered saline was read in polystyrene cuvettes. The zeta potential and DLS were measured using Anton Paar Litesizer 500 and Kalliope software (Anton Paar, Graz, Austria). Zeta potential measurements used standard aqueous method settings. For particle size analysis, samples were equilibrated at room temperature in aqueous 154 mM NaCl (refractive index 1.3323), and material refractive index set at 1.388 for bacteria. For DLS and zeta potential experiments, n ≥ 8 biological replicates per subpopulation.