Biocompatible nanocarriers for delivering drugs to skeletal muscle cells: a therapeutic option for myotonic dystrophy?

Myotonic dystrophy (DM) represents a genetic disorder characterized by progressive dysfunction of multiple organs and tissues (e.g. skeletal, cardiac, and smooth muscle; the central nervous systems) among which the most severely affected is the skeletal muscle; this condition leads patients to a progressive muscle weakness, wasting and myotonia. The molecular pathogenetic mechanism of DM type 1 (or Steinert’s) disease is the expansion of a (CTG)n triplet in 3’ UTR region of the Dystrophia Myotonica Protein Kinase (DMPK) gene, while the (CCTG)n quadruplet expansion in the first intron of the cellular Nucleic Acid Binding Protein/Zinc Finger 9 (CNBP/ZNF9) is responsible for DM type 2 (previously named “proximal Myotonic Myopathy” or “PROMM”). These expanded repeats are transcribed into toxic RNA that accumulates in nuclear RNA-protein aggregates (called foci), and lead to a general splicing alteration. The main problem of DM pathologies is that no therapies are currently available, and the commonly used treatments are administered to only manage symptoms. At present, the pharmaceutical research is screening small molecules such as pentamidine (PTM) able to repair the DM-associated splicing defects: PTM is an antimicrobial and antitumor compound that can mitigate the DM missplicing, but has limited applicability in humans due to its high systemic toxicity. To overcome these limitations, the administration via biocompatible nanoparticles (NPs) may represent a suitable approach, improving targeted delivery of the therapeutic drug and decreasing its systemic toxicity. Therefore, the main goal of the present experimental thesis was to set up an innovative experimental therapeutic strategy for DM based on biocompatible NPs loaded with PTM. To this aim, different types of NPs potentially suitable for drug delivery [liposomes, poly(lactic-co-glycolic acid) (PLGA) NPs, mesoporous silica NPs] were tested for biocompatibility in vitro on stabilized tumor cell lines and cultured primary human muscle cells. Conventional and confocal fluorescence microscopy and transmission electron microscopy allowed elucidating the mechanisms of NP internalization, intracellular distribution, fate and degradation. The tested NPs proved to be biocompatible for all the cell types investigated, although muscle-derived cells (especially the differentiated myotubes) showed lower internalization capability than cancer cells. In addition, novel hyaluronic acid-based nanocomplexes for hydrophilic drug encapsulation were synthesized in collaboration with the University of Lyon; these NPs proved to be biocompatible for both cancer and cultured muscle cells, and to efficiently deliver PTM to cancer cells; the effects of PTM-loaded NPs on muscle cells are currently under investigation. Finally, in the attempt to fill the gap between the conventional cell cultures and the organ complexity in vivo, an in vitro fluid dynamic system was set up to improve the preservation of explanted muscles and was then used for monitoring the biodistribution of NPs in this organ. Preliminary results revealed that PLGA NPs, which are easily internalized by cultured muscle cells, hardly enter the myofibers in the whole muscle since most of them accumulate in the connective tissue; consequently, modifications of the NP surface are in progress to improve targeting to and uptake by the muscle fibers.