Nanosight software version 2

Exosomes were analysed by nanoparticle tracking analysis (NanoSight LM10, Malvern Panalytical, Malvern, UK) to determine the size range and distribution. Briefly, 1 mL of diluted exosome sample (1:50–1:100) was loaded on to the NanoSight machine, and particle concentration was determined and diluted in the range of 4 × 10⁸–12 × 10⁸ particles/mL. Parameters were set at a detection rate of 15,000 particles per minute for capture settings, and the smallest vesicle size was set at 30 nm, with analysis performed by NanoSight software version 2.3 (Malvern Panalytical, Malvern, UK). The rate at which exosomes were produced per cell per hour was calculated by dividing the total number of exosomes by the total number of cells after exosome harvesting and then dividing by the number of hours over which the sample was collected.

Dynamic light scattering (DLS) determines the size, size distribution, and polydispersity index (PDI) of nanocapsules. For these analyses, the samples were diluted with deionized water. The analyses were performed in triplicate, and the results are expressed as mean ± standard deviation. A ZetaSizer Nano ZS 90 analyzer (Malvern^®) was used to determine the zeta potential values with 1:100 dilutions of the samples in deionized water. The samples were diluted in deionized water (1:1000 v/v) and inserted into a cuvette for analysis. The results are expressed in mV, with the mean and standard deviation of three determinations. Nanoparticle tracking analyses (NTA) were performed using NanoSight LM14 equipment (green laser, 532 nm), where the images of the nanocapsules were collected by an sCMOS camera using NanoSight software version 2.3 (Malvern Instruments, UK). Initially, the formulations were diluted in deionized water (dilution factor: 1 × 10⁴). Subsequently, 1 mL of each formulation was injected into the volumetric cell to start the analysis of the nanocapsules. For each sample, approximately 143 particles/frame were obtained, resulting in a total of 1951 frames. Each study included five measurements.