Hyaluronic acid based nanoparticles are suitable carriers to muscle cells: the ultrastructural evidence

Myotonic dystrophies (DMs) are neuromuscular diseases of genetic origin that involve multiple organs and tissues, with particular emphasis on skeletal muscles. Although only palliative treatments are administered, some molecules (e.g. pentamidine) have been proven to be efficient in mitigating the pathological halmarks of DMs. Actually, the therapeutic application of these compounds is impaired by their low bioavailability and high toxicity. To overcome these limitations, polymeric hyaluronic-acid-based nanoparticles (HA-NPs) were developed as a therapeutic strategy to efficiently deliver pentamidine (PTM) to skeletal muscle cells [1]. Howerver, to obtain HA-NPs suitable for clinical applications, it is mandatory to elucidate their behaviors and interactions with the biological milieu. Electron microscopy represents a suitable tecnique able to characterize, localize and track the fate of NPs inside cells thanks to its high resolution [2]. Firstly, morphological analysis of HA-NPs and PTM-loaded NPs was carried out using trasmission electron microscopy (TEM) and Cryogenic transmission electron microscopy (CryoTEM). As shown in Fig. 1 (A,B), both HA-NPs and PTM-loaded NPs formed monodispersed populations of rounded shaped particles. Moreover, to precisely investigate NPs behaviour at the ultrastructural level, NPs must be unequivocally visualized. This is easily obtained for NPs containing electron dense components but it may be difficult for organic NPs having an intrinsic moderate electron density such as HA-NPs. A valid detection method to overcome this limitation is represented by the Alcian Blue (AB) staining, a long-estabilished histochemical technique [3]. In muscle cells treated with HA-NPs, the AB staining appeared as an irregular granular electron dense product found on weakly electron dense NPs (Fig.2) similar to those observed in cells processed for conventional ultrastructural morphology (Fig 3). Once HA-NPs were unequivocally identified, their internalization and trafficking were investigated by TEM. As shown in Fig. 3 (A,B), HA-NPs were internalized individually via endocytosis and localized throughout the cytoplasms, being never observed inside the cell nuclei. HA-NPs rapidly escaped endosomes occurring free in cytosol and, after 24 h, they formed large cluster. However, no organelle damage or alteration was ever observed. The results obtained underline the importance of transmission electron microscopy tecnique to investigate the behaviour of NPs inside cells and tissue, providing essential information on their actual spatial relationships with biological components.