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

Figures & data

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

Table 1 Nanoemulsion composition (%, w/w) and matrix factorial design

Compounds	LP20	SP80	LP80	SP20
Chalcone	0.1	0.1	0.1	0.1
MCT	10.0	10.0	10.0	10.0
Sorbitan monooleate	–	2.0^(−1)	–	2.0^(−1)
Soybean lecithin	2.0^(+1)	–	2.0^(+1)	–
Polysorbate 20	2.0^(+1)	–	–	2.0^(+1)
Polysorbate 80	–	2.0^(−1)	2.0^(−1)	–
Water qsp	100.0	100.0	100.0	100.0

Notes: Factor A: lipophilic surfactant. High level (+1): soybean lecithin. Low level (−1): sorbitan monooleate. Factor B: hydrophilic surfactant. High level (+1): polysorbate 20. Low level (−1): polysorbate 80. “–” Indicates absence of this compound.

Abbreviations: LP20, soybean lecithin and polysorbate 20; LP80, soybean lecithin and polysorbate 80; MCT, medium chain triglycerides; qsp, a sufficient quantity for; SP20, sorbitan monooleate and polysorbate 20; SP80, sorbitan monooleate and polysorbate 80.

Table 2 Physicochemical characteristics of nanoemulsions

Parameters analyzed	LP20	SP80	LP80	SP20
Content, mg·mL^−1 (SD)	0.91 (0.18)^a	0.89 (0.28)^a	0.92 (0.60)^a	0.88 (0.10)^a
Association efficiency (SD)	99.93 (0.005)^a	99.97 (0.04)^a	99.97 (0.02)^a	99.97 (0.03)^a
Particle size, nm (SD)	171.9 (18.73)^a	272.74 (8.85)^b	183.77 (1.94)^a	296.27 (2.81)^b
Polydispersity index (SD)	0.14 (0.01)^a	0.24 (0.04)^b	0.13 (0.02)^a	0.34 (0.07)^b
Zeta potential, mV (SD)	−39.43 (3.56)^a	−21.22 (4.59)^b	−36.61 (0.58)^a	−25.8 (2.68)^b
pH (SD)	5.16 (0.40)^a	5.81 (0.56)^a	5.47 (0.31)^a	5.68 (0.68)^a
Viscosity, cP (SD)	2.00 (0.13)^a	1.71 (0.06)^b	2.01 (0.07)^a	1.75 (0.19)^b

Note: Different letters indicate significant statistical differences (ANOVA, Tukey test, P<0.05).

Abbreviations: ANOVA, analysis of variance; LP20, soybean lecithin and polysorbate 20; LP80, soybean lecithin and polysorbate 80; SD, standard deviation; SP20, sorbitan monooleate and polysorbate 20; SP80, sorbitan monooleate and polysorbate 80.

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.

Table 3 In vitro chalcone retention (µg·g⁻¹) from different nanoemulsions in porcine ear skin after 8 hours (mean ± SD, n=6)

Samples	Receptor fluid	Stratum corneum	Epidermis	Dermis
Control	ND	<LLOQ	<LLOQ	<LLOQ
LP20	ND	<LLOQ	28.19±6.75^a	3.03±1.18^a
LP80	ND	<LLOQ	20.79±2.48^a	1.77±0.34^a
SP20	ND	<LLOQ	13.83±1.35^b	0.56±0.13^b
SP80	ND	<LLOQ	29.76±7.33^a	1.17±0.23^a

Notes: Different letters indicate significant statistical differences (ANOVA, Tukey test, P<0.05). <LLOQ: below LLOQ.

Abbreviations: ANOVA, analysis of variance; LLOQ, lower limit of quantification; LP20, soybean lecithin and polysorbate 20; LP80, soybean lecithin and polysorbate 80; ND, not detected; SD, standard deviation; 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.

Table 4 THP-1 CC₅₀, Leishmania amazonensis (amastigote) IC₅₀, and selectivity index values of free SC, nanoemulsion, and amphotericin B 24 hours after preparation of the formulations

	SC	LP20	LP80	SP80	SP20	ANFB
CC50	44.47±6.60	8.95±6.62	ND	17.53±6.19	ND	>10
IC50	0.42±0.13	0.32±0.05	0.94±0.17	0.26±0.06	0.53±0.1	0.07±0.06
SI	105.88	27.97	ND	67.42	ND	>142

Note: CC₅₀ and IC₅₀ (µg·mL⁻¹) are expressed as mean ± standard deviation (n=3).

Abbreviations: CC₅₀, 50% cytotoxic concentration; IC₅₀, half maximal inhibitory concentration; ANFB, amphotericin B; LP20, soybean lecithin and polysorbate 20; LP80, soybean lecithin and polysorbate 80; ND, not determined; SC, (E)-3-(3-nitrophenyl)-1-(3,4,5-trimethoxyphenyl) prop-2-en-1-one; SI, selectivity index (CC₅₀/IC₅₀); 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.