Repurposing pentamidine using hyaluronic acid-based nanocarriers for skeletal muscle treatment in myotonic dystrophy Mathieu Repellin, PhD a , b , 1 , Flavia Carton, PhD a , 1 , 2 , Federico Boschi, PhD c , Mirco Galiè, PhD a , Massimiliano Perduca, PhD d , Laura Calderan, PhD a , Arnaud Jacquier, PhD e , f , Julien Carras, MSc e , f , Laurent Schaeffer, PhD e , f , Stéphanie Briançon, PhD b , Giovanna Lollo, PhD b , Manuela Malatesta, MSc a , ⁎ a Department of Neurosciences, Biomedicine and Movement Sciences, Anatomy and Histology Section, University of Verona, Strada Le Grazie 8, 37134 Verona, Italy b University of Lyon, Université Claude Bernard Lyon 1, CNRS, LAGEPP UMR 5007, 43 bd 11 Novembre 1918, 69622 Villeurbanne, France c Department of Computer Science, University of Verona, Strada Le Grazie 15, 37134 Verona, Italy d Department of Biotechnology, Biocrystallography and Nanostructure Laboratory, University of Verona, Strada Le Grazie 15, 37134 Verona, Italy e Institut NeuroMyogène, University of Lyon1, CNRS UMR 5310, INSERM U1217, 8 avenue Rockefeller, 69008 Lyon, France f Centre de Biotechnologie Cellulaire, CBC Biotec, CHU de Lyon – Hospices civils de Lyon groupement Est, Bron, France Revised 1 September 2022 Abstract In a context of drug repurposing, pentamidine (PTM), an FDA-approved antiparasitic drug, has been proposed to reverse the splicing defects associated in myotonic dystrophy type 1 (DM1). However, clinical use of PTM is hinder by substantial toxicity, leading to find alternative delivery strategies. In this work we proposed hyaluronic acid-based nanoparticles as a novel encapsulation strategy to efficiently deliver PTM to skeletal muscles cells. In vitro studies on C2C12 myoblasts and myotubes showed an efficient nanoparticles' internalization with minimal toxicity. More interestingly, our findings evidenced for the first time the endosomal escape of hyaluronic acid-based nano- carriers. Ex vivo studies showed an efficient nanoparticles' internalization within skeletal muscle fibers. Finally, the therapeutic efficacy of PTM-loaded nanosystems to reduce the number of nuclear foci has been demonstrated in a novel DM1 in vitro model. So far, current data demonstrated the potency of hyaluronic acid-based nanosystems as efficient nanocarrier for delivering PTM into skeletal muscle and mitigate DM1 pathology. © 2022 Elsevier Inc. All rights reserved. Keywords: Nanoparticles; Biomaterials; C2C12 cells; Muscular dystrophies; DM1 cell model Introduction Myotonic dystrophies (DM) are dominantly-inherited genetic disorders characterized by progressive myopathy, myotonia and multiorgan involvement, leading to possibly premature death. 1 Two distinct forms of DM have been identified, among which Myotonic Dystrophy type 1 (DM1) is the most common and severe form (OMIM:160900), with an estimated prevalence of 1/ 8000. 2 The most accredited molecular pathological mechanism relies on the abnormal CTG expansion located in the 3′ un- translated region of the DMPK (dystrophia myotonia protein kinase) gene. 3,4 This mutant (CTG) n DNA is transcribed into (CUG) n RNA, forming hairpin structures accumulating as nu- clear foci. It is well documented that these nuclear foci are as- sociated with a strong mis-regulation of splicing factors. 5–7 This deregulation is triggered by two leading biological events: on one hand the sequestration and loss-of-function of Muscleblind- like (MBNL) proteins, in particular Muscleblind-like 1 (MBNL1); on the other hand an up-regulation of CUG RNA- binding protein Elav-Like Family Member 1 (CELF1). 8–10 The Nanomedicine: Nanotechnology, Biology, and Medicine 47 (2023) 102623 nanomedjournal.com ⁎ Corresponding author. E-mail address: manuela.malatesta@univr.it (M. Malatesta). 1 These authors contributed equally to this work. 2 Present address: University of Eastern Piedmont, Department of Health Sciences, Via Solaroli 17, 28100 Novara, Italy. https://doi.org/10.1016/j.nano.2022.102623 1549-9634/© 2022 Elsevier Inc. All rights reserved. NANO-0000102623; No of Pages 16