**3. Results**

#### *3.1. Transmission Electron Microscopy (TEM)-Negative Staining*

Virus-like particle (VLP) characterization by TEM with uranyl acetate as the negative staining solution is shown in Figure 1. VLPs were concentrated by ultracentrifugation according to previous studies [36]. VLPs produced in HEK 293 (Figure 1B,D) and Sf9 cells (Figure 1C,E) were examined in order to identify differences arising from the expression of these nanoparticles in each specific cell platform. Uranium strongly reacts with phosphate and amino groups and typically stains proteins, nucleic acids and lipid membranes [44]. Thus, VLPs were observed as spherical electrodense structures surrounded by a bright corona (white arrows) that might correspond to structured Gag-eGFP monomers surrounded by the lipid membrane [36,45]. As expected, the presence of baculoviruses (BV) (black arrows) was detected along with VLPs (white arrows) in infected Sf9 samples. EVs were observed as

less electrodense nanoparticles (dashed grey arrows) and that could also be identified in conditioned medium samples (Supplementary materials S1) [46]. Interestingly, differences between VLPs and EVs were less evident in Sf9 micrographs compared to HEK 293 ones. A mean population diameter of 165 ± 54 nm (*n* = 71) and 146 ± 33 nm (*n* = 69) was quantified in HEK 293 and Sf9 samples, respectively (Figure 1F,G). Moreover, TEM characterizations revealed that only the 52 ± 6% and 55 ± 14% of the nanoparticles analyzed were VLPs in HEK 293 and Sf9 concentrated samples. The use of negative staining for sample visualization hindered a more detailed characterization of the structure of the different specimens due to the presence of artifacts and background noise in all samples (Figure 1B,E) [1].

**Figure 1.** Transmission electron microscopy with negative staining analysis of HIV-1 Gag-eGFP VLPs produced in HEK 293 and Sf9 cells harvested at 72 hpt and 72 hpi, respectively. (**A**) Sample preparation

protocol; (**B**–**E**) samples corresponding to the 25–45% ultracentrifugation fraction from HEK 293 supernatants (**B**,**D**) and Sf9 supernatants (**C**,**E**). All samples were stained with uranyl acetate in Ca-Cu grids. (**F**,**G**) PSD analysis of HEK 293 and Sf9 concentrated samples, respectively. White and grey dashed arrows indicate the presence of VLPs and EVs in HEK 293 and Sf9 productions, respectively, and black arrows indicate the presence of BVs in Sf9 samples. Negative controls were assessed using the same conditions as in VLP samples (Supplementary materials S1).
