Bolaamphiphile-based nanocomplex delivery of phosphorothioate gapmer antisense oligonucleotides as a treatment for Clostridium difficile

John P Hegarty,1 Jacek Krzeminski,2 Arun K Sharma,2 Diana Guzman-Villanueva,3 Volkmar Weissig,3 David B Stewart Sr1 1Deparment of Surgery, Pennsylvania State University College of Medicine Hershey, PA, USA; 2Department of Pharmacology, Penn State Hershey Cancer Institute, Hershey, PA, USA; 3Departm...

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Autores principales:	Hegarty JP, Krzeminski J, Sharma AK, Guzman-Villanueva D, Weissig V, Stewart DB Sr
Formato:	article
Lenguaje:	EN
Publicado:	Dove Medical Press 2016
Materias:	Cationic bolaamphiphiles dequalinium derivatives nanocomplex antisense gapmers Clostridium difficile bacteria Medicine (General) R5-920
Acceso en línea:	https://doaj.org/article/c9d78932576c4ab2a1215177b95c157f
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spelling	oai:doaj.org-article:c9d78932576c4ab2a1215177b95c157f2021-12-02T07:13:45ZBolaamphiphile-based nanocomplex delivery of phosphorothioate gapmer antisense oligonucleotides as a treatment for Clostridium difficile1178-2013https://doaj.org/article/c9d78932576c4ab2a1215177b95c157f2016-08-01T00:00:00Zhttps://www.dovepress.com/bolaamphiphile-based-nanocomplex-delivery-of-phosphorothioate-gapmer-a-peer-reviewed-article-IJNhttps://doaj.org/toc/1178-2013John P Hegarty,1 Jacek Krzeminski,2 Arun K Sharma,2 Diana Guzman-Villanueva,3 Volkmar Weissig,3 David B Stewart Sr1 1Deparment of Surgery, Pennsylvania State University College of Medicine Hershey, PA, USA; 2Department of Pharmacology, Penn State Hershey Cancer Institute, Hershey, PA, USA; 3Department of Pharmaceutical Sciences, College of Pharmacy, Nanomedicine Center of Excellence in Translational Research, Midwestern University, Glendale, AZ, USA 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. Keywords: cationic bolaamphiphiles, dequalinium derivatives, nanocomplex, antisense, gapmers, Clostridium difficile, bacteriaHegarty JPKrzeminski JSharma AKGuzman-Villanueva DWeissig VStewart DB SrDove Medical PressarticleCationic bolaamphiphilesdequalinium derivativesnanocomplexantisensegapmersClostridium difficilebacteriaMedicine (General)R5-920ENInternational Journal of Nanomedicine, Vol 2016, Iss default, Pp 3607-3619 (2016)
institution	DOAJ
collection	DOAJ
language	EN
topic	Cationic bolaamphiphiles dequalinium derivatives nanocomplex antisense gapmers Clostridium difficile bacteria Medicine (General) R5-920
spellingShingle	Cationic bolaamphiphiles dequalinium derivatives nanocomplex antisense gapmers Clostridium difficile bacteria Medicine (General) R5-920 Hegarty JP Krzeminski J Sharma AK Guzman-Villanueva D Weissig V Stewart DB Sr Bolaamphiphile-based nanocomplex delivery of phosphorothioate gapmer antisense oligonucleotides as a treatment for Clostridium difficile
description	John P Hegarty,1 Jacek Krzeminski,2 Arun K Sharma,2 Diana Guzman-Villanueva,3 Volkmar Weissig,3 David B Stewart Sr1 1Deparment of Surgery, Pennsylvania State University College of Medicine Hershey, PA, USA; 2Department of Pharmacology, Penn State Hershey Cancer Institute, Hershey, PA, USA; 3Department of Pharmaceutical Sciences, College of Pharmacy, Nanomedicine Center of Excellence in Translational Research, Midwestern University, Glendale, AZ, USA 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. Keywords: cationic bolaamphiphiles, dequalinium derivatives, nanocomplex, antisense, gapmers, Clostridium difficile, bacteria
format	article
author	Hegarty JP Krzeminski J Sharma AK Guzman-Villanueva D Weissig V Stewart DB Sr
author_facet	Hegarty JP Krzeminski J Sharma AK Guzman-Villanueva D Weissig V Stewart DB Sr
author_sort	Hegarty JP
title	Bolaamphiphile-based nanocomplex delivery of phosphorothioate gapmer antisense oligonucleotides as a treatment for Clostridium difficile
title_short	Bolaamphiphile-based nanocomplex delivery of phosphorothioate gapmer antisense oligonucleotides as a treatment for Clostridium difficile
title_full	Bolaamphiphile-based nanocomplex delivery of phosphorothioate gapmer antisense oligonucleotides as a treatment for Clostridium difficile
title_fullStr	Bolaamphiphile-based nanocomplex delivery of phosphorothioate gapmer antisense oligonucleotides as a treatment for Clostridium difficile
title_full_unstemmed	Bolaamphiphile-based nanocomplex delivery of phosphorothioate gapmer antisense oligonucleotides as a treatment for Clostridium difficile
title_sort	bolaamphiphile-based nanocomplex delivery of phosphorothioate gapmer antisense oligonucleotides as a treatment for clostridium difficile
publisher	Dove Medical Press
publishDate	2016
url	https://doaj.org/article/c9d78932576c4ab2a1215177b95c157f
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Bolaamphiphile-based nanocomplex delivery of phosphorothioate gapmer antisense oligonucleotides as a treatment for Clostridium difficile

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