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

Castells Colldeforn, Pau
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 ​
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