Developing a new method for nanoparticle characterization and cell trafficking using expansion microscopy as high- resolution fluorescent microscopy technique

Abstract

Nucleic acid-based therapies constitute a promising tool for treatment of diseases like cancer.

However, the main challenge in this discipline consists in the development of safe and efficient

vectors to deliver the therapeutic genes to the target cells. In this way, poli(beta-aminoesters)

(pBAE)s were modified with cationic terminal oligopeptides (OM-pBAEs), to facilitate the

genetic material encapsulation and, in turn, help on achieving high transfection efficacy, while

maintaining the biocompatibility of the resulting nanoparticles (NPs).

Microscopy could be a good way to characterise these NPs and their cellular trafficking. Even

so, the diffraction limits of conventional microscopes hinder the resolution of particle smaller

than 200-300 nm. On the other hand, the acquisition of super-resolution microscopy involves a

large cost and time investment. Thus, a new super-resolution imaging technology, expansion

microscopy (ExM), has been developed. This technique consists on the use of common lab

equipment and inexpensive chemicals to physically magnify biological specimens, so that the

sample can be imaged with nanoscale resolution using a conventional microscope.

The main objective of this project has been to develop and optimise an ExM protocol, for the

study of OM-pBAE NPs trafficking in cells. To achieve the desired protocol it have been set: the appropriated cell density to seed (25·104 cells/mL), the concentration of nuclei staining (0.5 µg/mL), the ideal way of dyeing the cytoplasmic membrane (pre-gelation) and the incubation time with Proteinase (ProK) (3 hours)

to ensure an optimal and expansion of the sample. For the NPs, the protocol has been modified,

reducing the Acryloyl X treatment and polymerization to 30 minutes and removing the ProK

digestion, in order to keep the NPs formed and visualize them after the process. Lastly, the

characterization of the cellular trafficking of NPs showed differences between NPs with

different oligopeptide terminals, as a proof of concept of the utility of the technique.

Consequently, it can be stated that proExM protocols have been successfully optimized, which

helped to characterize OM-pBAEs NPs, which can be useful to understand the endocytic

pathways of the cell, once transfected with them