Nta 3

A NanoSight LM10 (Malvern Instruments Ltd., UK) using nanoparticle tracking analysis (NTA) was used to determine the average size and size distribution of both liposomes and bacteriophages. NTA measurements were performed in a sample chamber equipped with a 640 nm laser to track the NPs. Typically, a 10 μl aliquot was taken from each sample and diluted 10²-10³ fold in order to achieve a particle concentration of 10⁷-10¹⁰ particles ml⁻¹. The sample was injected into the sample chamber using a sterile syringe and sample flow was maintained through the chamber until all air bubbles were removed. The temperature was registered with a thermometer (RTD Pt100, OMEGA, UK) and temperature correction was carried out. The software used for capturing and analyzing the data was NTA 3.0 (Malvern Instruments Ltd., UK). Data for each sample was captured over a period of 60 s and each measurement was repeated five times. The focus was set to achieve a uniform perfect spherical particle view. Before capturing the video, the camera had to set-up to ensure all the particles in the sample were clearly visible with no more than the 20% saturation. The single gain mode was used throughout the whole measurement process. Statistical analysis was carried out using the NTA software.

Nanoparticle tracking analysis (NTA) (Nanosight LM10, Malvern Panalytical, Malvern, UK) was used to determine the OMV concentration and size. The samples were diluted in PBS prior to injection. Videos of 60 s were captured in triplicate. The data were acquired and analyzed using NTA 3.0 (Malvern Panalytical, UK). The total number of OMVs was calculated based on the dilution factors of each condition. Particle size and concentration were analyzed using one-way analysis of variance (ANOVA) followed by Dunnett’s post hoc test using GraphPad Prism software version 9.4.1 (GraphPad Software, San Diego, CA, USA). Differences were considered significant when * p < 0.05, *** p < 0.001, and **** p < 0.0001.

The particle size distribution in the cell culture medium was determined by parallel particle tracking analyses (PTA) at the end of cell culture testing period. A NanoSight LM10 instrument equipped with a violet laser (405 nm), an Andor CCD camera, and particle tracking software (NTA3.0, Malvern Instruments GmbH, Herrenberg, Germany) was used. Cell-free approaches were carried out using 90 µg/mL of each SAS dispersed with either 18 or 270 J/mL. Suspensions were incubated in H₂O, KRPG buffer, and F-12K medium under cell culture conditions for 90 min and 16 h, respectively. Suspensions were retrieved from the wells and pipetted onto the laser-stage of the LM-10. Measurable concentrations (approximately 5 × 10⁸ particles/mL) were prepared by serial dilution. Results are presented together with the dilution factor (DF), which may be used to estimate the particle concentration.

Samples diluted in PBS were measured thrice for 60 s using the NanoSight LM-10 (Malvern Instruments Ltd.) and the NanoSight NTA 3.0 software on a stable table TS-140 at 25 °C. In the capture mode the camera level was set to 13 and the gain to 3.9, detection threshold was set to 5 for analysis. The particle number served for normalization in subsequent experiments.

The hydrodynamic diameter was determined by optical tracking analyses using a NanoSight LM10 instrument equipped with a violet laser (405 nm), an Andor CCD camera, and particle tracking software NTA3.0 (all from: Malvern Instruments GmbH, Herrenberg, Germany). Starting with the aqueous particle stock suspension, dilutions were prepared in KRPG and F-12K medium in the absence and presence of 10% FCS. Concentration was uniformly set to 90 µg/mL. Suspensions were incubated under cell culture conditions (37 °C, 5% CO₂, 100% humidity) for 90 min (KRPG) and 16 h (F-12K), respectively. Suspensions were further diluted to obtain measurable concentrations, approximately in the range of 5 × 10⁸ particle/mL.

In addition to the particle sizes provided in Table 1, we determined the particle size distribution in the cell culture medium, that is, under assay conditions by particle tracking analyses (PTA). A NanoSight LM10 instrument equipped with a violet laser (405 nm), an Andor CCD camera, and particle tracking software (NTA 3.0, Malvern Instruments GmbH, Herrenberg, Germany) was used. To measure particle and/or aggregate/agglomerate sizes of SAS suspensions under cell culture conditions, the aqueous particle suspensions were incubated under cell culture conditions (37°C, 5% CO₂) for 90 min in KRPG, or for 16 h in F-12K medium, respectively. The suspensions were serially diluted with the respective medium (H₂O, KRPG, or F-12K medium) to optimize the apparent particle concentration to PTA requirements (~5 × 10⁸ particles/ml). The results and respective dilution factors are presented in Table 2. Since the technique is limited by the light-scattering properties of particles, (colloidal) SAS particles smaller than 50 nm were not detected (Supplementary Figures S1, S2).