*2.5. Nanoparticle Tracking Analysis (NTA)*

HIV-1 Gag-eGFP VLPs and total particle content were analyzed by NTA. A NanoSight® NS300 device (Malvern Panalytical, Malvern, United Kingdom) equipped with a blue filter module (488 nm) and a neutral filter was used to quantify GFP-fluorescent nanoparticles and total particle by light diffraction, respectively. The measurements were performed at Service of Preparation and Characterization of Soft Materials located at Institut de Ciència de Materials de Barcelona (ICMAB, CSIC, Campus UAB). The workflow used for HIV-1 Gag-eGFP VLP quantification is summarized in Figure 5A. Prior to injection into the device chamber, each sample was diluted to obtain 1 mL sample with a concentration of around 108 particles/mL. Sample injection into the chamber was continuously added using a pump to improve the robustness of the measurement and minimize the photobleaching effect due to fluorescence depletion over time (Figure 5B) [42]. The videos recorded were then analyzed with the NTA 3.2 software (Malvern Panalytical) by tracking the individual particle movement, where camera level and detection threshold were adjusted manually for each sample (Table 1). Three independent analyses were carried out and videos of 60 s were recorded at RT, with particles identified and tracked by their Brownian motion. HIV-1 Gag-eGFP VLP concentrations were calculated as the total fluorescent particles and the concentration of EVs was calculated as the difference between light scattering particles and fluorescent particles. PSD analyses were performed with NTA 3.2 software and SigmaPlot 12.0 software.


**Table 1.** NTA analysis settings.
