Nanoemulsions containing a synthetic chalcone as an alternative for treating cutaneous leshmaniasis: optimization using a full factorial design

Abstract

Nanoemulsions are drug delivery systems that may increase the penetration of lipophilic compounds through the skin, enhancing their topical effect. Chalcones are compounds of low water solubility that have been described as promising molecules for the treatment of cutaneous leishmaniasis (CL). In this context, the aim of this work was to optimize the development of a nanoemulsion containing a synthetic chalcone for CL treatment using a 2(2) full factorial design. The formulations were prepared by spontaneous emulsification and the experimental design studied the influence of two independent variables (type of surfactant - soybean lecithin or sorbitan monooleate and type of co-surfactants - polysorbate 20 or polysorbate 80) on the physicochemical characteristics of the nanoemulsions, as well as on the skin permeation/retention of the synthetic chalcone in porcine skin. In order to evaluate the stability of the systems, the antileishmanial assay was performed against Leishmania amazonensis 24 hours and 60 days after the preparation of the nanoemulsions. The formulation composed of soybean lecithin and polysorbate 20 presented suitable physicochemical characteristics (droplet size 171.9 nm; polydispersity index 0.14; zeta potential -39.43 mV; pH 5.16; and viscosity 2.00 cP), drug content (91.09%) and the highest retention in dermis (3.03 µg·g(-1)) - the main response of interest - confirmed by confocal microscopy. This formulation also presented better stability of leishmanicidal activity in vitro against L. amazonensis amastigote forms (half maximal inhibitory concentration value 0.32±0.05 µM), which confirmed the potential of the nanoemulsion soybean lecithin and polysorbate 20 for CL treatment.

Keywords: chalcone; full factorial; leishmaniasis; nanoemulsion; skin permeation.

Figure 1

Structure of (E)-3-(3-nitrophenyl)-1-(3,4,5-trimethoxyphenyl) prop-2-en-1-one.

Figure 2

Pareto chart (A) and interaction graphs (B) to physicochemical characteristics. Notes: A: lipophilic surfactant; B: hydrophilic surfactant. Continuous line: sorbitan monooleate (−1); dotted line: soybean lecithin (+1). Abbreviation: PDI, polydispersity index.

Figure 2

Pareto chart (A) and interaction graphs (B) to physicochemical characteristics. Notes: A: lipophilic surfactant; B: hydrophilic surfactant. Continuous line: sorbitan monooleate (−1); dotted line: soybean lecithin (+1). Abbreviation: PDI, polydispersity index.

Figure 3

Pareto chart to (A) content and (B) association efficiency. Notes: A: lipophilic surfactant; B: hydrophilic surfactant.

Figure 4

Transmission electronic microscopy images of the nanoemulsions ×100,000 magnification. Notes: (A) LP20, (B) LP80, (C) SP80, and (D) SP20 (scale bars 0.2 µm). Abbreviations: LP20, soybean lecithin and polysorbate 20; LP80, soybean lecithin and polysorbate 80; SP20, sorbitan monooleate and polysorbate 20; SP80, sorbitan monooleate and polysorbate 80.

Figure 5

Pareto chart (A) and interaction graphs (B) for SC skin retention. Notes: A: lipophilic surfactant; B: hydrophilic surfactant. Continuous line: sorbitan monooleate (−1); dotted line: soybean lecithin (+1). Abbreviation: SC, (E)-3-(3-nitrophenyl)-1-(3,4,5-trimethoxyphenyl) prop-2-en-1-one.

Figure 6

Phytomicrographs of skin after 8 hours of skin permeation (tenfold optical zoom). Notes: Untreated skin (A); skin treated with LP20 (B), LP80 (C), SP80 (D), and SP20 (E) nanoemulsions. Abbreviations: LP20, soybean lecithin and polysorbate 20; LP80, soybean lecithin and polysorbate 80; SP20, sorbitan monooleate and polysorbate 20; SP80, sorbitan monooleate and polysorbate 80.

Figure 7

Confocal micrograph images of porcine ear skin after 8 hours treatment with (A) LP20, (B) LP80, (C) SP80, and (D) SP20 with Nile red fluorescent dye. Fluorescence images recorded with 559 nm excitation and 636 nm emission wavelengths. Abbreviations: LP20, soybean lecithin and polysorbate 20; LP80, soybean lecithin and polysorbate 80; SP20, sorbitan monooleate and polysorbate 20; SP80, sorbitan monooleate and polysorbate 80.

Figure 8

Inhibitory effects of nanoemulsions on Leishmania amazonensis in vitro tested 24 hours after the preparation of formulations. Notes: Adherent THP-1 cells were infected at a ratio of 1:10 cell/parasite L. amazonensis and treated for 48 hours with free SC and SC loaded into nanoemulsions in a concentration range of 0.375–20 µg·mL⁻¹. Results are expressed as the mean ± SD of the percent inhibition of parasite growth (n=3). Abbreviations: LP20, soybean lecithin and polysorbate 20; LP80, soybean lecithin and polysorbate 80; SC, (E)-3-(3-nitrophenyl)-1-(3,4,5-trimethoxyphenyl) prop-2-en-1-one; SD, standard deviation; SP20, sorbitan monooleate and polysorbate 20; SP80, sorbitan monooleate and polysorbate 80.