(M1530-12-70) Reformulation of Niclosamide into Biofilm Penetrating Nanoparticles to Improve Its Repurposing for the Treatment of Candidiasis

Purpose: Niclosamide (NCL) is an FDA-approved antiparasitic drug that showed promise as an antifungal agent targeting NDU1, which is an essential protein for Candida mitochondrial respiration, and growth¹ and our studies showed that NCL interferes with C. albicans hyphenation and early and mature biofilms of drug-resistant and drug-sensitive Candida sp. While the repurposing of NCL to treat candidiasis holds great promise, poor water solubility, limited permeability, and intestinal metabolism limit its in vivo efficacy and translation as an antifungal agent. To overcome these limitations, we focused on the development of nanoparticles (NPs) of easily available, pharmaceutically acceptable, and low-cost polymethacrylate polymers (Eudragits) to improve local and oral delivery of NCL and to facilitate its preclinical development.

Methods: To select the suitable polymethacrylate for the development of NCL loaded NPs, we evaluated anionic and cationic Eudragits to stabilize NCL in their polymer matrix by preparing coevaporates followed by their characterization using Fourier-Transform Infrared (FT-IR) spectroscopy and nuclear magnetic resonance (NMR) spectroscopy. Furthermore, we also used the solvent-antisolvent precipitation method to study the ability of various Eudragits to stabilize NCL. We evaluated the ability of various pharmaceutically acceptable surfactants to obtain NCL loaded Eudragit EPO NPs with the lowest particle size and acceptable polydispersity index, high NCL loading, and long-term colloidal stability. The optimized poloxamer 407 stabilized NCL-EPO-NPs were evaluated for morphology using TEM and their physical (size, polydispersity index, surface charge, and pH) and chemical (NCL content) stability were evaluated for 4 weeks. In vitro antifungal activity of EPO-NCL-NPs against C. albicans and C. auris was evaluated and the ability of poloxamer 407 stabilized fluorescent EPO-NPs labeled with Coumarin 6 to penetrate the fungal biofilm was evaluated using confocal microscopy. Finally, we developed an in-situ gelling Poloxamer 407 formulation containing NCL-EPO-NPs and evaluated the intra-oral efficacy of NCL-EPO-NPs embedded in the in-situ gelling formulation using the mouse model of oropharyngeal candidiasis (OPC).

Results: Our FT-IR and NMR spectroscopic studies showed that cationic Eudragit EPO could easily stabilize the weakly acidic and hydrophobic NCL via electrostatic interactions (Fig. 1) in the solid-state as well as in the nanoparticulate form (Figure 2B). Among various nonionic surfactants that were screened, Poloxamer 407 (P407) showed optimal stabilization and colloidal stability in the initial screening (Fig.2A). Spectrofluorometric analysis of NCL and NCL-EPO-NPs (25 ppm) showed a significant red shift in the case of NCL-EPO-NPs indicating strong molecular interaction between NCL and EPO in the NPs (Fig. 2C). The encapsulation efficiency of NCL in P407 stabilized EPO-NPs was found to be >98%. NCL-EPO-NPs showed the highest NCL loading (40% w/w compared to EPO) with 70.2 nm, 0.23, and +4.5 mV particle size, polydispersity index, and zeta potential respectively. The TEM image showed the spherical morphology for NCL-EPO NPs (Fig. 2D). The in vitro studies demonstrated that NCL-EPO-NPs inhibited growth and filamentation of C. albicans as well as C. auris at a concentration as low as 0.5 μg/ml (1.5 μM) and confocal studies showed that Poloxamer 407 stabilized fluorescent EPO NPs completely penetrated mature biofilm of the C. albicans. In the OPC mouse model, the intra-oral NCL-EPO NPs thermosensitive gel treatment of mice intra-orally infected with drug-sensitive C. albicans showed significantly higher antifungal activity (~10-fold lower fungal burden) compared to untreated control and similar activity as fluconazole treatment whereas in mice infected with drug-resistant C. albicans, NCL-EPO NPs thermosensitive gel showed 1-1.5 log-fold higher antifungal effect compared to untreated control and fluconazole.

Conclusion: The reformulation of NCL into biofilm-penetrating nanoparticles of low-cost, pharmaceutically acceptable cationic bioenhancer Eudragit EPO showed several benefits in terms of pharmaceutical development and in vitro and in vivo antifungal activity and this pharmaceutically viable nanoformulation can be further developed to improve repurposing of NCL for the treatment of mucosal and systemic candidiasis.

References: 1) Mamouei, Z. et al. An evolutionarily diverged mitochondrial protein controls biofilm growth and virulence in 746 Candida albicans. PLoS Biol 19, e3000957, doi:10.1371/journal.pbio.3000957 (2021).


Figure 1: Niclosamide-Eudragit EPO molecular level interaction. (A) Fourier-transform infrared (FT-IR) spectroscopic study showed that -OH and/or NH stretching vibrations (3234 cm-1) of NCL disappeared in the NCL-EPO solid dispersion (SD) indicating strong interaction between NCL and EPO. Moreover, C=O stretching vibrations (1723 cm-1) and –CH stretching (2770 cm-1) of EPO showed red shift and reduction in the peak intensity indicates the possible interaction between NCL to EPO. (B) The nuclear magnetic resonance (NMR) spectroscopy studies showed that proton peak for -OH (#4) and -NH (#5) in NCL disappeared in NCL-EPO SD which indicates a strong electrostatic interaction. (C) The plausible interaction sites on Eudragit EPO and NCL; hydrogen bonding in green & orange line; while cation-π interaction in red line.


Figure 2: (A) FDA approved surfactant screening studies showed P407 (Poloxamer 407) stabilize EPO-NPs with acceptable particle size/PDI. Data expressed as mean ± S.D. (n =3); (B) Without EPO, NCL-P407 dispersion become turbid and precipitated. (C) Spectrofluorometric analysis of aqueous dispersion of NCL-EPO-NPs showed red shift compared to the hydroalcoholic solution of NCL at same concentration indicating strong molecular interaction between NCL and EPO in the NPs. (D) Transmission electron microscopy (TEM) image of P407-NCL-EPO-NPs (Scale bar = 200 nm).


Figure 3: Poloxamer 407 stabilized fluorescent (green) EPO NPs labeled with Coumarin 6 could penetrate the mature C. albicans biofilm.