Antileishmanial Activity of BNIPDaoct- and BNIPDanon-loaded Emulsomes on Leishmania infantum Parasites

Zeynep Islek,Mustafa Guzel,Elif Keskin,Ana G Gomes‐Alves,Fikrettin Sahin,Mehmet Hikmet Ucisik,Ana M Tomás,Bilgesu Onur Sucu

doi:10.3389/fnano.2021.773741

Abstract

Among bisnaphthalimidopropyl (BNIP) derivatives, BNIPDaoct and BNIPDanon recently came forward with antileishmanial activities beyond the standard, commercialized antileishmanial therapies. However, high-level toxicity on macrophages plus poor aqueous solubility and poor bioavailability of the compounds limit their application in therapies. Addressing these limitations, the present study introduces BNIPDaoct- and BNIPDanon-loaded emulsomes as lipid-based nanocarrier systems. Accordingly, emulsome formulations were prepared with the presence of BNIP compounds. The average diameters of BNIPDaoct- and BNIPDanon-loaded emulsomes were found as 363.1 and 337.4 nm, respectively; while empty emulsomes differed with a smaller average particle diameter, i.e., 239.1 nm. All formulations exhibited a negative zeta potential value. The formulations achieved the encapsulation of BNIPDaoct and BNIPDanon at approximately 0.31 mg/ml (501 µM) and 0.24 mg/ml (387 µM), respectively. The delivery of BNIP within the emulsomes improved the antileishmanial activity of the compounds. BNIPDaoct-loaded emulsome with 50% inhibitory concentration (IC50) value of 0.59 ± 0.08 µM was in particular effective againstLeishmania infantumpromastigotes compared to free BNIPDaoct (0.84 ± 0.09 µM), free BNIPDanon (1.85 ± 0.01 µM), and BNIPDanon-loaded emulsome (1.73 ± 0.02 µM). Indicated by at least ≥ 2-fold higher 50% cytotoxic concentration (CC50) values, the incorporation of BNIP into emulsomes significantly reduced the toxicity of BNIPs against macrophages, corresponding to up to 16-fold improvement in selectivity index (CC50/IC50) forL. infantumpromastigotes. The infection rates of macrophages were determined using dual-fluorescent flow cytometry as 68.6%. Both BNIP formulations at concentration of 1.87 µM reduced the parasitic load nearly to 40%, whereas BNIPDaoct-loaded emulosmes could further decrease the parasitic load below 20% at 7.5 µM and above. In conclusion, the incorporation of BNIPDaoct and BNIPDanon into emulsomes results in water-soluble dispersed emulsome formulations that do not only successfully facilitate the delivery of BNIP compounds into the parasites and the Leishmania-infected macrophagesin vitrobut also enhance antileishmanial efficacy as proven by the decline in IC50values. The selectivity of the formulation forL. infantumparasites further contributes to the challenging safety profile of the compounds. The promisingin vitroantileishmanial efficacy of BNIP-loaded emulsomes highlights the potential of the system for the futurein vivostudies.

Highlights

Leishmaniasis is a neglected tropical disease caused by the protozoan parasites of the genus Leishmania and affectsGRAPHICAL ABSTRACT | Graphical abstract illustrates the framework of the study
3.1.1 Size, Polydispersity and Zeta Potential At least five separate BNIPDaoct- and BNIPDanon-loaded emulsome formulations were analyzed for their size, polydispersity index (PDI), and zeta potential characteristics
half maximal cytotoxic concentration (CC50) values were calculated for BNIPDaoct, BNIPDaoct-loaded emulsomes, BNIPDanon, and BNIPDanon-loaded emulsomes as 1.52 ± 0.02 μM, 17.73 ± 0.03 μM, 3.07 ± 0.87 μM, and 5.96 ± 0.83 μM, respectively (Table 3). These results indicated that the incorporation of BNIP derivatives into emulsomes significantly reduced their toxicities to macrophages, as demonstrated by higher CC50 values, i.e., >11-fold for BNIPDaoct (p ≤ 0.0001) and nearly 2fold for BNIPDanon (p ≤ 0.001)

Summary

Introduction

Leishmaniasis is a neglected tropical disease caused by the protozoan parasites of the genus Leishmania and affectsGRAPHICAL ABSTRACT | Graphical abstract illustrates the framework of the study. The available treatment options of leishmaniasis rely on pentavalent antimonials—i.e., sodium stibogluconate (pentostan) and meglumine antimoniate (glucantim), pentamidine, conventional amphotericin B (AmpB) deoxycholate, miltefosine, paramomycin (aminosidine), micellar formulation of AmpB (fungizone), AmpB lipid complex (Abelcet), and liposomal AmpB (AmBisome) (Sundar et al, 2002a; Sundar et al, 2002b; Sundar et al, 2003; Köse et al, 2004; Sundar et al, 2004; Murray et al, 2005; Sundar and Chatterjee, 2006; Tiuman et al, 2011; Sundar and Chakravarty, 2013; Aronson, 2017) These therapies have certain drawbacks associated with 1) their limited efficacy on parasites (Gupta et al, 2013), 2) multiple adverse effects due to their low therapeutic index (Al-Natour, 2009), 3) requirement for careful and slow intravenous administration due to their toxicities (e.g., AmpB) (Freitas-Junior et al, 2012), and 4) drug resistance developed against the therapy (Sundar et al, 2002a, 2003; Ouellette et al, 2004; Murray et al, 2005; Croft et al, 2006; Sundar and Chatterjee, 2006; Gupta et al, 2007, 2013; Natera et al, 2007; Chakravarty and Sundar, 2010). Low water solubility and poor bioavailability are the other drawbacks limiting the use of current therapies and further increase the need for new medical therapies

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Conclusion