Fungal keratitis is one of the major causes of corneal blindness affecting 6 to 8 million people globally (WHO, 2019a, WHO, 2019b). Ocular trauma with contaminated vegetative matter and contact lenses is the leading cause of fungal keratitis. The common genera causing the infection are Aspergillus, Candida, and Fusarium (CDC, 2021). Topical natamycin suspension (5 %) is the first FDA-approved standard of care for the management of fungal keratitis (Alcon Laboratories Inc., 2008). Natamycin specifically blocks fungal ergosterol synthesis without interacting with human cellular membranes (Tania et al., 2014). Since it is poorly soluble in water, it is challenging to formulate into a solution and is therefore available as a suspension (5 % w/v). However, suspensions have many shortcomings, including poor bioavailability, rapid clearance, minor discomfort, blurred vision, and increased tear secretion (Deepta and Edelhauser, 2006). Further, poor water solubility and bioavailability necessitate frequent dosing schedules with consequent risk of poor patient compliance (Akash et al., 2017).

A variety of reformulation strategies like cyclodextrin (Thirumurthy et al., 2021), nanoparticles (Bhatta et al., 2012; Hardik et al., 2014), niosomes (Mansi and Parmar, 2017), and bilosomes (Yadav et al., 2018) based eye drops have been attempted by various research groups. Ex vivo and in vivo studies of previously developed formulations have shown improved precorneal residence time of natamycin; however, detailed safety and efficacy studies were not performed to support the frequency of dosage administration. Additionally, most of the attempted delivery systems have various other challenges including multiple preparation steps and increased production costs (Muthu Madaswamy and Wilson, 2012; Rishi et al., 2014). Therefore, an alternative approach is needed that could overcome the mentioned limitations and have the potential for clinical translation.

Nanomicelles entrap the poorly water soluble drug into its hydrophobic core, thereby improving its water solubility (Torchilin, 2007). Its size ranges from 20 to 100 nm, and selecting the appropriate polymer is crucial for its stability in the aqueous system (Rosenholm Jarl et al., 1980). Thermodynamics is one of the most important aspects responsible for the stability of nanomicelles (Fisicaro et al., 2008). A lack of information about the polymer's thermodynamic properties leads to product failure. Understanding these properties could help a formulator choose an appropriate polymer for the preparation of nanomicelles.

Nanomicelles reported earlier have improved the solubility of natamycin and superior permeability ex vivo, however, in vivo permeation and efficacy are not yet proven (Blanca et al., 2019). The in vivo studies could shed light on the impact of additional physiological barriers (pre-corneal residence, drainage, and non-corneal absorption) on permeation across the cornea (Maxime et al., 2019). Another study utilized crosslinked polymeric micelles to boost stability in water and performed in vitro as well as in vivo studies for efficacy, which showed a reduction of fungal growth. However, the synthesis of poly-ethylene glycol block poly-glycidyl methacrylate (PEG-b-PGMA) copolymer could be cumbersome and also will increase the production cost (Meyer, 2003). Therefore, a formulation that is simple to prepare, scalable, and superior to the current suspension is required. Moreover, marketed product such as Cequa® (Polyoxyl hydrogenated castor oil, Octoxynol-40 nanomicelles) have already demonstrated the clinical translation potential of nanomicelles (Abhirup et al., 2019; Tania et al., 2014).

In this study, we employed tocopheryl polyethylene glycol 1000 succinate (TPGS), Soluplus and Poloxamer 407 for the preparation of nanomicelles as these polymers are mucoadhesive, biocompatible and widely used as components for various ocular drug delivery systems. Among these polymers, we have selected TPGS for the preparation of nanomicelles, due to its ability to form nanomicelles at low concentrations (Zhiping et al., 2012) and also showed the highest entrapment of natamycin as compared to Soluplus and Poloxamer 407. Also, TPGS has a PEGylated end that enhances the contact with the tear film’s mucin, thereby improves the residence time in the pre-corneal region (Akash et al., 2018). Additionally, TPGS is one of the key components of the marketed eye drops (Navitae® Plus) that holds promising potential for ophthalmic use (Akash et al., 2017).

We hypothesize that 1 % Natcel (natamycin loaded nanomicelles) would improve the therapeutic outcome of fungal keratitis by; a. Improving natamycin solubility by encapsulating it in the hydrophobic center of nanomicelles, b. Facilitating corneal permeation due to the smaller size of nanomicelles, c. Enhancing contact with cornea due to the mucoadhesive nature of the polymeric nanomicelles. The developed nanomicelles could result in uniform drug dosing due to its solubilized form and also reduces the irritation due to its nano size as compared to micron-sized suspension. Hence, nanomicelles enhances the permeation of the drug across the cornea – reducing the dose and dosing frequency as compared to the 5 % suspension. Therefore, we developed a 1 % Natcel formulation that is simple to develop, stable, and biocompatible, and its detailed physicochemical characterization was carried out along with the screening of suitable polymer for nanomicelles preparation by calculating its thermodynamic properties. Further, the impact of improved natamycin solubility on transcorneal permeation in comparison with suspension was studied using ex vivo and in vivo models. The safety and therapeutic potential of 1 % Natcel was evaluated using in vitro, ex vivo and in vivo model.