(Figure 10.7). The VLPs’ liquid suspension is rapidly vitrified at −180°C in liquid

ethane, and then this frozen preparation is placed in the chamber of the electron

microscope, which is maintained at −173°C with liquid nitrogen. Cryo-TEM gives

more representative images of the particles as there is no interaction with the stain.

A further step is the use of electron cryotomography. Here, several cryo images

are taken at different angles collecting tilt series data sets so they can be later

processed to obtain a 3D model of the structure (Figure 10.7). Segmentation of the

different parts composing the VLP can be used to render a detailed 3D model

defining each of the Gag monomers that form the Gag shell [110].

Regarding quantification, there are some classical methods that have typically

been used for quantifying VLPs, such as the counting of particles in TEM with the

presence of a standard, ELISA to quantify the amount of an antigen, or HPLC

methods. There are also tag-based methods in which a fluorescence-based method is

used to facilitate the monitoring of these particles in a simple way [134].

In the field of nanotechnology and nanoscience, different techniques have

emerged, such as nanoparticle tracking analysis (NTA) and flow virometry methods

based in light scattering, that provide further insights in the quantification of VLPs.

They could be further combined with other fluoresce tags, not only characterizing

the total amount of nanoparticles that are present in the samples but if the protein

conforming the VLP is fused to a tag protein, the populations of VLPs and micro-

vesicles can be differentiated within the samples. Flow virometry has the same

concept as flow cytometry but is used to quantify particles that are 1,000-fold

smaller than cells achieving the limit of detection of this equipment.

The advances in all these different technologies to characterize VLPs pave the way

for further development of this platform, contributing to overcome the current chal-

lenges. In the last few years, the capacity to enhance VLP production, purification,

and characterization has significantly increased. However, as a complex cellular

product, this platform still faces the challenges of understanding the molecular

pathways governing the loading of internal components and custom modification of

the bounding cellular membrane. These next steps will certainly send forth this

promising platform beyond vaccine development towards the use of VLPs for specific

delivery strategies and specific protein-receptor interactions in future therapies.

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