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Cationic amphiphilic bolaamphiphile-based delivery of antisense oligonucleotides provides a potentially microbiome sparing treatment for C. difficile

The Journal of Antibioticsvolume 71pages713721 (2018) | Download Citation


Conventional antibiotics for C. difficile infection (CDI) have mechanisms of action without organismal specificity, potentially perpetuating the dysbiosis contributing to CDI, making antisense approaches an attractive alternative. Here, three (APDE-8, CODE-9, and CYDE-21) novel cationic amphiphilic bolaamphiphiles (CABs) were synthesized and tested for their ability to form nano-sized vesicles or vesicle-like aggregates (CABVs), which were characterized based on their physiochemical properties, their antibacterial activities, and their toxicity toward colonocyte (Caco-2) cell cultures. The antibacterial activity of empty CABVs was tested against cultures of E. coli, B. fragilis, and E. faecalis, and against C. difficile by “loading” CABVs with 25-mer antisense oligonucleotides (ASO) targeting dnaE. Our results demonstrate that empty CABVs have minimal colonocyte toxicity until concentrations of 71 µM, with CODE-9 demonstrating the least toxicity. Empty CABVs had little effect on C. difficile growth in culture (MIC90 ≥ 160 µM). While APDE-8 and CODE-9 nanocomplexes demonstrated high MIC90 against C. difficile cultures (>300 µM), CYDE-21 nanocomplexes demonstrated MIC90 at CABV concentrations of 19 µM. Empty CABVs formed from APDE-8 and CODE-9 had virtually no effect on E. coli, B. fragilis, and E. faecalis across all tested concentrations, while empty CYDE-21 demonstrated MIC90 of >160 µM against E. coli and >40 µM against B. fragilisand E. faecalis. Empty CABVs have limited antibacterial activity and they can deliver an amount of ASO effective against C. difficile at CABV concentrations associated with limited colonocyte toxicity, while sparing other bacteria. With further refinement, antisense therapies for CDI may become a viable alternative to conventional antibiotic treatment.

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  1. 1.

    Leffler DA, Lamont JT. Clostridium difficile infection. N Engl J Med. 2015;372:1539–48.

  2. 2.

    Zhang S, Palazuelos-Munoz S, Balsells EM, et al. Cost of hospital management of Clostridium difficile infection in United States—a meta-analysis and modelling study. BMC Infect Dis. 2016;16:447.

  3. 3.

    Eyre DW, Walker AS, Wyllie D, et al. Predictors of first recurrence of Clostridium difficile infection: implications for initial management. Clin Infect Dis. 2012;55:S77–87.

  4. 4.

    Thomas C, Stevenson M, Riley TV. Antibiotics and hospital-acquired Clostridium difficile-associated diarrhoea: a systematic review. J Antimicrob Chemother. 2003;51:1339–50.

  5. 5.

    Schubert AM, Rogers MA, Ring C, et al. Microbiome data distinguish patients with Clostridium difficile infection and non-C. difficile-associated diarrhea from healthy controls. MBio. 2014;5:e01021–14.

  6. 6.

    Lamendella R, Wright JR, McLimans C, et al. Antibiotic treatments for Clostridium difficile infection are associated with distinct bacterial and fungal community structures. mSphere. 2018;3:e00572–17.

  7. 7.

    Daly SM, Sturge CR, Felder-Scott CF, et al. MCR-1 inhibition with peptide-conjugated phosphorodiamidate morpholino oligomers restores sensitivity to polymyxin in escherichia coli. MBio. 2017;8:e01315–17.

  8. 8.

    Otsuka T, Brauer AL, Kirkham C, et al. Antimicrobial activity of antisense peptide-peptide nucleic acid conjugates against non-typeable Haemophilus influenzae in planktonic and biofilm forms. J Antimicrob Chemother. 2017;72:137–44.

  9. 9.

    Hegarty JP, Krzeminski J, Sharma AK, et al. Bolaamphiphile-based nanocomplex delivery of phosphorothioate gapmer antisense oligonucleotides as a treatment for Clostridium difficile. Int J Nanomed. 2016;11:3607–19.

  10. 10.

    Stewart DB, Berg A, Hegarty J. Predicting recurrence of C. difficile colitis using bacterial virulence factors: binary toxin is the key. J Gastrointest Surg. 2013;17:118–24. discussion p.124-5

  11. 11.

    Ayhan DH, Tamer YT, Akbar M, et al. Sequence-specific targeting of bacterial resistance genes increases antibiotic efficacy. PLoS Biol. 2016;14:e1002552.

  12. 12.

    Howard JJ, Sturge CR, Moustafa DA, et al. Inhibition of pseudomonas aeruginosa by peptide-conjugated phosphorodiamidate morpholino oligomers. Antimicrob Agents Chemother. 2017;61:e01938–16.

  13. 13.

    Galanakis D, Davis CA, Del Rey Herrero B, et al. Synthesis and structure-activity relationships of dequalinium analogues as K+ channel blockers. Investigations on the role of the charged heterocycle. J Med Chem. 1995;38:595–606.

  14. 14.

    Weissig V, Lasch J, Erdos G, et al. DQAsomes: a novel potential drug and gene delivery system made from Dequalinium. Pharm Res. 1998;15:334–7.

  15. 15.

    Weissig V. From serendipity to mitochondria-targeted nanocarriers. Pharm Res. 2011;28:2657–68.

  16. 16.

    Mileykovskaya E, Dowhan W. Cardiolipin membrane domains in prokaryotes and eukaryotes. Biochim Biophys Acta. 2009;1788:2084–91.

  17. 17.

    Epand RM, Epand RF. Lipid domains in bacterial membranes and the action of antimicrobial agents. Biochim Biophys Acta. 2009;1788:289–94.

  18. 18.

    Marín-Menéndez A, Montis C, Díaz-Calvo T, et al. Antimicrobial nanoplexes meet model bacterial membranes: the key role of Cardiolipin. Sci Rep. 2017;7:41242.

  19. 19.

    Jenior ML, Leslie JL, Young VB, et al. Clostridium difficile colonizes alternative nutrient niches during infection across distinct murine gut microbiomes. mSystems. 2017;2:e00063–17.

  20. 20.

    Pepin J. Vancomycin for the treatment of Clostridium difficile infection: for whom is this expensive bullet really magic? Clin Infect Dis. 2008;46:1493–8.

  21. 21.

    McDonald LC, Gerding DN, Johnson S, Bakken JS, Carroll KC, Coffin SE, Dubberke ER, Garey KW, Gould CV, Kelly C, Loo V, Shaklee Sammons J, Sandora TJ, Wilcox MH. Clinical Practice Guidelines for Clostridium difficile infection in adults and children: 2017 update by the Infectious Diseases Society of America (IDSA) and Society for Healthcare Epidemiology of America (SHEA). Clin Infect Dis. 2018;66:987–94.

  22. 22.

    Ajami NJ, Cope JL, Wong MC, Petrosino JF, Chesnel L. Impact of oral fidaxomicin administration on the intestinal microbiota and susceptibility to Clostridium difficile colonization in mice. Antimicrob Agents Chemother. 2018. AAC.02112-17 [Epub ahead of print].

  23. 23.

    Weiss K, Allgren RL, Sellers S. Safety analysis of fidaxomicin in comparison with oral vancomycin for Clostridium difficile infections. Clin Infect Dis. 2012;55(Suppl 2):S110–5.

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The authors thank Dr. David Craft at Penn State Hershey Medical Center (Hershey, PA) for providing stocks of E. coli, B. fragilis, and E. faecalis used in this work. The authors also thank Ms. Maria Lozoya, M.S. at Midwestern University (Glendale, AZ) for her work with CAB preparations and size distribution measurements.


This project was funded by multi-principal investigator (Drs. Stewart and Sharma) award from the National Institutes of Health, National Institute of Allergy and Infectious Diseases (1R21AI132353-01).

Author information


  1. Department of Pharmacology, College of Medicine, The Pennsylvania State University, Hershey, PA, 17033, USA

    • Arun K. Sharma
    •  & Jacek Krzeminski
  2. Department of Pharmaceutical Sciences, Nanomedicine Center of Excellence, College of Pharmacy Midwestern University, Glendale, AZ, 85308, USA

    • Volkmar Weissig
  3. Department of Medicine, College of Medicine, The Pennsylvania State University, Hershey, PA, 17033, USA

    • John P. Hegarty
  4. Department of Surgery, University of Arizona – Banner University Medical Center, Tucson, AZ, 85724, USA

    • David B. Stewart


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The authors declare that they have no conflict of interest.

Corresponding author

Correspondence to David B. Stewart.

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