*4.3. Particle Size Distribution*

The PSD of VLPs has been studied with the six methods evaluated in this work. Typically, the VLP mean diameter is comprised between 100 and 200 nm, as observed by TEM, cryo-TEM, NTA and flow virometry [61]. Non-symmetric right-skewed distributions were observed for VLPs measured with the different techniques. Similar PSDs of HIV-1 Gag VLPs produced in CAP-T and CHO cells were shown by Gutierrez-Granados et al. and Steppert et al., respectively [37,62]. The presence of larger population of VLPs, especially when measuring Sf9 samples by NTA and flow virometry, could be related to the aggregation of VLPs or their interaction with other cellular compounds released to the medium. SEM and SRFM presented higher PSD in comparison to the rest of analytical technologies. SEM analysis

of HEK 293 samples resulted in a PSD of 296 ± 88 nm, which could indicate that several EVs were quantified instead of VLPs since microvesicles can reach up to 2000 nm [6]. HyVolution2 SRFM yielded the highest VLP PSD values in both cell platforms (>250 nm), which has also been observed by Xiao and co-workers when analyzing Cy5-labeled adeno-associated viruses [63], and could be attributed to the convolution effect of light. In this case, the individual fluorochrome intensity does not necessarily correlate with the real particle size and might result in particle size diameter overestimation [64]. The use of more powerful SRFM techniques, such as stimulated emission depletion (STED) or stochastic optical reconstruction microscopy (STORM) could reduce this effect [65]. However, STED and STORM require specific fluorophores that tolerate the high laser intensities applied and also a higher level of expertise and equipment infrastructure in comparison to HyVolution2 SRFM.

The PSD of EVs was assessed by NTA and flow virometry (Figures 5 and 6). In both cases, EVs presented an average diameter comprised between 100 and 200 nm, similar to that of VLPs. Nonetheless, a right-skewed distribution with vesicles diameters from 45 to 500 nm was observed, especially in Sf9 samples. These results correlate with the existence of several types of EVs, encompassing exosomes and microvesicles, and BVs as visualized in cryo-TEM micrographs (Figure 3). Of note, the PSD of rod-shaped baculoviruses could not be accurately characterized by any of these methods since they assume that all nanoparticles are spherical. Van der Pol and coworkers also observed the size heterogeneity of EVs, with diameters ranging between 70 and 800 nm. They report a minimum detection limit for EVs of 70–90 nm with NTA and 150–190 nm by flow virometry [5]. Lower EV detection limits of 45 nm with NTA and 100 nm by flow virometry were reported in this work. The sample composition and also the implementation of the V-SSC instead of the 488 nm SSC in flow virometry could contribute to explain these differences [34]. Still, a higher level of accuracy is possible by using National Institute of Standards and Technology (NIST)-traceable beads in the Mie correlation with flow virometry.
