Nanoemulsions of sulfonamide carbonic anhydrase inhibitors strongly inhibit the growth of Trypanosoma cruzi

Figures & data

Figure 1. Preparation of nanoemulsion by high-energy method.

Figure 2. Sulfonamides 3F, 3G, 3W, 5B, 5C, and 5D used in the study and their TcCA inhibitory action.

Table 1. NEs size and polydispersity index

Formulation/drug	Drug (mg)	Oil Clove (ml)	AP (ml)	Size (nm)	PDI	Stability
NE	–	1	9	31.54 ± 0.413	0.105 ± 0.012	Stable
NE-3F	5	1	9	60.12 ± 2.36	0.274 ± 0.033	Stable
NE-3G	5	1	9	100.63 ± 2.05	0.262 ± 0.008	Stable
NE-3W	5	1	9	97.34 ± 2.82	0.264 ± 0.15	Stable
NE-5B	5	1	9	44.83 ± 0.753	0.123 ± 0.078	Stable
NE-5C	5	1	9	53.99 ± 1.12	0.233 ± 0.003	Stable
NE-5D	5	1	9	35.09 ± 0.575	0.165 ± 0.019	Stable

AP: aqueous phase containing tensioactive (Pluronic F127) and water, drug concentration 500 µg/mL, mean ± SD (from three different determinations).

Figure 3. Inhibition effects of different concentrations of nanoemulsions with sulfonamide derivatives (1–128 μM) 3F, 3G, 3W, 5B, 5C, 5D. (A and C) Epimastigotes T. cruzi Dm28c strain; (B and D) epimastigotes T. cruzi Y strain, after 5 days of incubation. CTL control nanoemulsions without sulfonamide derivatives, BZD: benznidazole reference drug (1–128 μM).

Table 2. IC₅₀ and IC₉₀ values derived from growth inhibition assays of Trypanosoma cruzi (DM28c, Y) and determination of cytotoxicity (CC₅₀), selectivity index (SI₅₀) of 3F, 3G, 3W, 5B, 5C, 5D NEs.

	Drug nanoemulsions
	3F	3G	3W	5B	5C	5D	BZN
Tc DM28c
IC50^1 µM	3.54^a ± 1.53	5.66^b ± 1.62	7.36^b ± 1.54	6.24^b ± 0.18	3.98^a ± 0.24	6.69^b ± 1.85	20.63^c ± 1.831
IC90^2 µM	49.56^a ± 9.61	84.87^b ± 5.16	68.64^c ± 23.47	84.46^b ± 6.80	64.34^c ± 6.47	120.54^d ± 7.89	>128
CC50^3 µM	8.13^a ± 1.19	6.77^b ± 1.07	3.21^c ± 0.55	6.51^b ± 1.11	8.04^a ± 1.33	6.75^b ± 0.98	127.54^d ± 12.04
SI50^4	2.25^a ± 0.17	1.20^b ± 0.21	0.44^c ± 0.12	1.08^b ± 0.11	2.02^a ± 0.22	1.05^b ± 0.09	5.54^d ± 1.82
TcY
IC50^1 µM	2.83^a ± 0.71	2.27^a ± 0.56	3.51^b ± 0.12	3.47^b ± 0.35	2.15^a ± 0.29	3.27^b ± 0.53	21.92^c ± 1.67
IC90^2 µM	>128	83.61^a ± 38.25	114.97^b ± 8.16	82.03^a ± 8.01	78.04^a ± 32.46	52.67^c ± 7.70	>128
CC50^3 µM	8.13^a ± 1.19	6.77^b ± 1.07	3.21^c ± 0.55	6.51^b ± 1.11	8.04^a ± 1.33	6.75^b ± 0.98	127.54^d ± 12.04
SI50^4	2.89^a ± 1.02	3.09^a ± 1.33	0.44^b ± 0.06	1.95^c ± 0.97	5.09^d ± 1.41	1.76^c ± 0.30	5.89^d ± 0.81

¹ Concentration in μM which reduced the proliferation of epimastigotes by 50%.

² Concentration in μM which reduced the proliferation of epimastigotes by 90%.

³ Concentration in cytotoxic μg ml⁻¹ to 50% of RAW 267.4 cells.

⁴ IS₅₀ Selectivity index of 50% = CC₅₀/IC₅₀.

^a,b,c,d in the rows, means followed by different letters differ statistically (p < .05).

Figure 4. Representative graphs of flow cytometric analysis for nanoemulsions of the sulfonamides 3F, 3G, 3W, 5B, 5C, 5D, and benznidazole (BZN) at 32 μΜ. (A, C) Necrosis using propidium iodide (PI) and (B, D) apoptosis using annexin V-FITC (ANV).

Figure 5. Histogram of epimastigotes representative of apoptosis analysis by flow cytometry using propidium iodide (PI) and annexin V-FITC. CTL: control without sulfonamides derivates (A) T. cruzi Dm28c and (B) T. cruzi Y Nanoemulsions of 3F, 3G, 3W, 5B, 5C, 5D sulfonamides derivatives at 32 μM. R1: Cells marked with PI only (necrosis); R2: cells labelled with annexin V and PI (late apoptosis); R3: unlabelled cells (viable cells); R4: cells marked only with annexin V (early apoptosis).