title: Bolaamphiphile-based nanocomplex delivery of phosphorothioate gapmer antisense oligonucleotides as a treatment for Clostridium difficile.
abstract: Despite being a conceptually appealing alternative to conventional antibiotics, a major challenge toward the successful implementation of antisense treatments for bacterial infections is the development of efficient oligonucleotide delivery systems. Cationic vesicles (bolasomes) composed of dequalinium chloride ("DQAsomes") have been used to deliver plasmid DNA across the cardiolipin-rich inner membrane of mitochondria. As cardiolipin is also a component of many bacterial membranes, we investigated the application of cationic bolasomes to bacteria as an oligonucleotide delivery system. Antisense sequences designed in silico to target the expression of essential genes of the bacterial pathogen, Clostridium difficile, were synthesized as 2'-O-methyl phosphorothioate gapmer antisense oligonucleotides (ASO). These antisense gapmers were quantitatively assessed for their ability to block mRNA translation using luciferase reporter and C. difficile protein expression plasmid constructs in a coupled transcription-translation system. Cationic bolaamphiphile compounds (dequalinium derivatives) of varying alkyl chain length were synthesized and bolasomes were prepared via probe sonication of an aqueous suspension. Bolasomes were characterized by particle size distribution, zeta potential, and binding capacities for anionic oligonucleotide. Bolasomes and antisense gapmers were combined to form antisense nanocomplexes. Anaerobic C. difficile log phase cultures were treated with serial doses of gapmer nanocomplexes or equivalent amounts of empty bolasomes for 24 hours. Antisense gapmers for four gene targets achieved nanomolar minimum inhibitory concentrations for C. difficile, with the lowest values observed for oligonucleotides targeting polymerase genes rpoB and dnaE. No inhibition of bacterial growth was observed from treatments at matched dosages of scrambled gapmer nanocomplexes or plain, oligonucleotide-free bolasomes compared to untreated control cultures. We describe the novel application of cationic bolasomes to deliver ASOs into bacteria. We also report the first successful in vitro antisense treatment to inhibit the growth of C. difficile.
authors.affiliation: Deparment of Surgery, Pennsylvania State University College of Medicine Hershey, PA, USA.
authors.forename: John P
authors.initials: JP
authors.lastname: Hegarty
authors.affiliation: Department of Pharmacology, Penn State Hershey Cancer Institute, Hershey, PA, USA.
authors.forename: Jacek
authors.initials: J
authors.lastname: Krzeminski
authors.affiliation: Department of Pharmacology, Penn State Hershey Cancer Institute, Hershey, PA, USA.
authors.forename: Arun K
authors.initials: AK
authors.lastname: Sharma
authors.affiliation: Department of Pharmaceutical Sciences, College of Pharmacy, Nanomedicine Center of Excellence in Translational Research, Midwestern University, Glendale, AZ, USA.
authors.forename: Diana
authors.initials: D
authors.lastname: Guzman-Villanueva
authors.affiliation: Department of Pharmaceutical Sciences, College of Pharmacy, Nanomedicine Center of Excellence in Translational Research, Midwestern University, Glendale, AZ, USA.
authors.forename: Volkmar
authors.initials: V
authors.lastname: Weissig
authors.affiliation: Deparment of Surgery, Pennsylvania State University College of Medicine Hershey, PA, USA.
authors.forename: David B
authors.initials: DB
authors.lastname: Stewart
mesh_terms: D020121:5' Untranslated Regions / Q000235:genetics; D002412:Cations; D016360:Clostridioides difficile / Q000187:drug effects*; D003720:Densitometry; D003868:Dequalinium / Q000737:chemistry; D005663:Furans / Q000737:chemistry*; D017930:Genes, Reporter; D015151:Immunoblotting; D008156:Luciferases / Q000378:metabolism; D053758:Nanoparticles / Q000737:chemistry*; D009690:Nucleic Acid Conformation; D016376:Oligonucleotides, Antisense / Q000494:pharmacology*; D010316:Particle Size; D054735:Phosphorothioate Oligonucleotides / Q000494:pharmacology*; D010957:Plasmids / Q000378:metabolism; D014176:Protein Biosynthesis / Q000187:drug effects; D011728:Pyridones / Q000737:chemistry*; D055672:Static Electricity
chemical_list: D020121:5' Untranslated Regions; D002412:Cations; D005663:Furans; D016376:Oligonucleotides, Antisense; D054735:Phosphorothioate Oligonucleotides; D011728:Pyridones; C543575:bolaamphiphile; D003868:Dequalinium; D008156:Luciferases
keywords: Clostridium difficile; antisense; bacteria; cationic bolaamphiphiles; dequalinium derivatives; gapmers; nanocomplex