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.
C. difficile infection (CDI) is the most frequently reported nosocomial bacterial infection  in the United States, accounting for more than 450,000 new cases annually and for more than four billion dollars in CDI-attributable annual health care costs . CDI has a strong reliance on intestinal dysbiotic states, which, when combined with the presence of C. difficile in the human gut, represents the most common pathogenesis for CDI. The high prevalence of this infection is, in large part, due to formidable recurrence rates of 15–25% following first treatment  with conventional antibiotics (CAs). CAs have long been recognized as the most important risk factor for the development of CDI , due to their mechanisms of action lacking organismal specificity, leading to widespread changes in gut ecology , which can lead to CDI by disrupting the gut microbial community. Given the important role of intestinal dysbiosis in the development of CDI, there has also been recent interest in studying the effects of difficile-directed conventional antibiotics on the bacterial and fungal communities of human subjects being treated for CDI, as a way of potentially explaining the high persistence and recurrence rates of this disease. These more recent data  suggest that even difficile-directed conventional antibiotics could potentially contribute to the perpetuation of dysbiotic states, which in turn could perpetuate CDI, potentially leading to even primary treatment failures.
There has been previous [7, 8] interest in the development of antisense therapies to treat bacterial infections, in part due to concerns regarding antibiotic resistance to traditional drugs. Given the dependence of CDI on dysbiotic states, approaches using therapeutic antisense oligonucleotides (ASO) complimentary to specific C. difficile mRNAs could limit or prevent the expression of important bacterial genes leading to bacterial death, all while sparing other organisms. This approach would offer significant advantages over CAs, especially in terms of a more limited impact on gut microbial communities. Developing clinically effective antisense therapies targeting a Gram-positive organism requires several elements. Since antisense oligonucleotides will not be efficiently introduced into bacteria without assistance given the presence of both a cell membrane and a thick cell wall, a carrier molecule must be used to deliver the ASO. This carrier must complex with the ASO strongly enough to concentrate it, to protect it from degradation in the extracellular environment, and to focus its delivery on its target cell. In order to accomplish these activities, the carrier-ASO complex itself must be stable in the in vivo environment of the gut. Once at the cell, the carrier must be able to release its cargo. Simultaneously, the carrier must demonstrate both limited gut toxicity and limited antibacterial activity at the doses required to effectively treat the target bacteria.
Our group published the first  in vitro data for antisense therapies against CDI by complexing cyclohexyl dequalinium analogs to various ASO-targeting essential C. difficile genes. However, since dequalinium has both antibacterial activity as well as toxicity at higher doses, a better delivery compound for ASO is required if antisense approaches to CDI are to be further developed. Here, we report our data on vesicles formed from novel cationic amphiphilic bolaamphiphiles (CABs) as carriers for chimeric 25-mer 2′-O-methyl phosphorothioate ASO. CABs, characteristic of all bola-like compounds, have hydrophilic, positively charged end groups separated by a hydrophobic linker chain. This molecular structure enables CABs to form nano-sized vesicle-like aggregates (CABVs), which in turn allow them to complex with negatively charged oligonucleotides in addition to promoting electrostatic interactions with bacterial cell membranes for intracellular delivery of ASO. The synthesis, physiochemical properties, toxicity, and antibacterial properties of three novel CABs and their respective CABVs are described, and their specificity for C. difficile compared to several other organisms is also provided.
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