According to World Health Organization (WHO), antimicrobial resistance (AMR) is a leading cause of ∼ 700,000 deaths each year globally dictating the dire need for better therapeutic options (Larsson and Flach, 2022, Singh and Tandon, 2023, Mancuso et al., 2021) and WHO published a list of high priority pathogens designated by the acronym ESKAPE (Enterococcus faecium, Staphylococcus aureus, Klebsiella pneumoniae, Acinetobacter baumannii, Pseudomonas aeruginosa, and Enterobacter species) which are posing great threat (“WHO publishes list of bacteria for which new antibiotics are urgently needed,” 2017). Infections caused by AMR organisms which are challenging to treat are further constrained by limited number of existing antibiotics, and the limited supply of new antimicrobials in the pipeline. This is further exacerbated by the limited tissue penetration of systemic antimicrobials in the lung, spleen, brain and bone marrow the major organs of the reticulo-endothelial system (RES) (Devarajan et al., 2015, Welling et al., 2019). Furthermore, intracellular pathogens hijack and harbor within the mononuclear phagocytic cells (macrophages) of the RES thereby evading the drugs. Novel strategies that could ferry antibiotics inside the macrophages in augmented concentration, could provide great benefits (Butler et al., 2023, Gerace et al., 2022, Jindal et al., 2017, Liu et al., 2020, Patel et al., 2015).

Nanomedicine is one such promising approach. It is accepted that spherical particles 5 nm–15 μm in diameter are taken up by the mononuclear phagocytic system (MPS) of the liver, spleen, lung and bone marrow following IV administration (Lim et al., 2016, Zazo et al., 2016). Intravenous injection of carboxyl-coated polystyrene (PS) spheres of three different sizes namely 20, 100 and 1000 nm were found majorly in the RES, with a larger proportion of 1000 nm particles in the spleen (Sarlo et al., 2009). In another instance amino-modified PS of 1000-nm spherical particles showed a faster clearance (∼85 %, from the blood) and exhibited higher uptake by the liver compared to 100 nm particles, which exhibited reverse trend (Simon et al., 1995). In another study, PS particles of 2–10 μm particles in the size range of 6 and 10 μm exhibited optimal targeting to pulmonary capillaries (Kutscher et al., 2010). A study on size dependent splenic accumulation of blank nanoemulsions revealed splenic accumulation in the rank order 900 nm > 500 nm > 200 nm > 70 nm (Fan et al., 2020). In general, various studies demonstrate the importance of large particle size for RES targeting, while small particles < 150 nm are crucial for prolonged circulation. Furthermore, it is amply demonstrated that nanoparticles < 150 nm generally by pass the RES while sizes > 200 nm preferably accumulate in the RES (Devarajan et al., 2015, Jindal et al., 2017).

DNA gyrase and topoisomerase IV have been used as target for numerous antibiotics. However, the development of resistance through multiple mechanisms has limited their effectiveness. As an innovative alternative, researchers are exploring bacterial topoisomerase I as a drug target (Bansal et al., 2017). PPEF.3HCl (2′-(4-ethoxyphenyl)-5-(4-propylpiperazin-1-yl)-1H,1′H-2,5′bibenzo(d) imidazole -trihydrochloride), is a bis-benzimidazole, a broad-spectrum antibiotic, acting on a novel bacterial target (topoisomerase IA). It does not target human topoisomerase IB and II and Gyrase enzymes (Maurya et al., 2023, Singh et al., 2019) and is thus proposed to overcome AMR (Bansal et al., 2019, Seddek et al., 2021, Sinha et al., 2017). Protonation of tertiary nitrogen on its piperazine ring results in a positive charge at physiological pH, and facilitates interaction with the acidic triad of topo IA. PPEF functions as a poison inhibitor, preventing the DNA strands from rejoining and further hindering the enzymatic process (Bansal et al., 2019, Maurya et al., 2023, Nimesh et al., 2014, Singh et al., 2019). Previously, pharmacokinetic studies were performed on blood plasma sample of PPEF.3HCl treated BALB/c mice at a dose of 40 mg/kg bw i.v. The single i.v. dose of PPEF.3HCl showed maximum concentrations (Cmax) of 7813 ng/mL, plasma exposure (AUClast) 6435.28 h*ng/mL, half-life 5.09 h, mean residence time (MRTlast) 3.47 h and clearance 99.99 mL/min/kg (Maurya et al., 2023). In the present study, we harness the power of nanomedicine to design an aqueous nanosuspension of PPEF.3HCl to ensure targeted delivery to the reticuloendothelial system, a major site of infections. Conventional nanotechnology methodologies entail multiple steps, rely on high-end equipment and pose difficulties in isolation and stabilization, thereby hampering the translation of nanodrug delivery systems (Jahagirdar et al., 2019, Shevade et al., 2023). Our group has demonstrated In-situ nanotechnology as an elegant, simple and readily scalable solution that sidesteps these hurdles (John et al., 2021, Shevade et al., 2023). This simple approach merely involves introducing a monophasic preconcentrate containing the drug and optionally carriers (polymers/lipids) and stabilizers into an aqueous phase for instantaneous and on-site generation of drug nanosuspensions/drug loaded nanoparticles. The versatility of this approach has been demonstrated and In-situ nanotechnology has been successfully established for a range of drugs (John et al., 2021, Kapse et al., 2012, Shevade et al., 2023). Further, in one study an optimized In-situ SLN of nevirapine demonstrated size-based targeting in the rat model, to multiple reservoirs of HIV which included the major RES organs namely liver, spleen, as well as the brain, a remote HIV reservoir (Jindal et al., 2017).

Strategies to fabricate nanosuspensions of a water-soluble drug are complex. In one instance we achieved a nanodispersion of water soluble primaquine phosphate by complexation to form a nanocarboplex (Jahagirdar et al., 2019). However, nanosuspensions which comprise only drug particles preclude the use of complexing agents and other carriers. Herein we present yet another radical In-situ nanotechnology strategy which is practical, facile and involved simple pH modulation. The objective of the study was to reproducibly obtain a PPEF.3HCl nanosuspension of nano size and high precipitation efficiency by the facile In-situ nanotechnology. Yet another objective was to ensure high efficacy in the S. aureus induced murine sepsis model.