Chemical structure of the TPP+ derivatives.

<p>The structures of triphenyl(8-((3,4,5-trihydroxybenzoyl)oxy)-octyl) phosphonium bromide (TPP⁺-C₈), triphenyl(10-((3,4,5-trihydroxybenzoyl)oxy)-decyl) phosphonium bromide (TPP⁺-C₁₀), triphenyl(11-((3,4,5-trihydroxybenzoyl)oxy)-undecyl) phosphonium bromide (TPP⁺-C₁₁), and triphenyl(12-((3,4,5-trihydroxybenzoyl)oxy)-dodecyl) phosphonium bromide (TPP⁺-C₁₂) are shown. For details regarding their synthesis, see Jara <i>et al</i> (2014).</p

Effect of TPP⁺ derivatives on the parasite load in <i>T</i>. <i>cruzi</i>-infected cells.

<p>RAW 264.7 cells were infected with <i>T</i>. <i>cruzi</i> trypomastigotes (Y strain); the cells were treated for 48 hours with the different derivatives, and the parasite load was assessed by qPCR. The results are expressed as the relative quantification of the parasite DNA and mammalian DNA, with a ratio of 1 assigned to the control. Treated cells were compared with the control using the ^∆∆C_T method. *: <i>p</i> < 0.05; **: <i>p</i> < 0.01; and ***: <i>p</i> < 0.001, compared with the control, based on one-way ANOVA with Dunnett’s post-test.</p

Effect of TPP⁺ derivatives on the opening of mitochondrial permeability transition pore.

<p><i>T</i>. <i>cruzi</i> trypomastigotes were loaded with calcein AM dye and CoCl₂ and exposed to the different TPP⁺ derivatives for 1 hour. Transition pore opening was then evaluated using a flow cytometer based on the quenching of mitochondrial calcein fluorescence. <b>A.</b> Representative frequency histogram of the fluorescence emitted at 515 nm. Both panels show calcein fluorescence in intact cells (grey area). Cytosolic fluorescence was quenched by the addition of CoCl₂, allowing visualization of the fluorescence from <i>T</i>. <i>cruzi</i> mitochondria (black line). The median fluorescence intensity of cells incubated with calcein AM and CoCl₂ was used as a control. As a positive control, we incubated the cells with 0.5 μM ionomycin (black dashed line). Parasites were also incubated with TPP⁺-C₈ (red line), TPP⁺-C₁₀ (blue line), TPP⁺-C₁₁ (orange line), and TPP⁺-C₁₂ (green line). Upper panel: trypomastigotes exposed to 1 μM of the TPP⁺ derivatives. Lower panel: trypomastigotes exposed to 5 μM of the TPP+ derivatives. AU: Arbitrary Units of Fluorescence. <b>B.</b> Quantification of the median fluorescence at 515 nm. The results were calculated using the median fluorescence obtained from the histograms and normalized assuming 100% fluorescence of the controls. The results are expressed as the mean ± SD of three independent experiments, each performed in triplicate. *: <i>p</i> < 0.05 and ***: <i>p</i> < 0.001, compared with the control, based on one-way ANOVA with Dunnett’s post-test.</p

Effect of TPP⁺ derivatives on the amastigote number in <i>T</i>. <i>cruzi</i>-infected cells.

<p>VERO cells were infected with <i>T</i>. <i>cruzi</i> trypomastigotes (Y strain). The cells were treated for 48 hours with the different derivatives, and the parasite load was then assessed by DAPI staining and fluorescence microscopy visualization. <b>A.</b> Representative images showing the effect of the different TPP+ derivatives on the intracellular amastigote number. All of the compounds shown were used at 1 μM. The white arrows show amastigote nuclei stained with DAPI. <b>B.</b> Quantification of the effect of TPP⁺ derivatives on VERO cells infected with <i>T</i>. <i>cruzi</i>. For each condition, performed in triplicate, at least five images were taken. The left panel shows the percentage of infected cells in each microscopic field photographed. The right panel shows the number of amastigotes per infected cell in each microscopic field photographed. For all of the graphs in the figure, the results are expressed as the mean ± SD of three independent experiments, each performed in triplicate. ***: <i>p</i> < 0.001 compared with the control, based on one-way ANOVA with Dunnett’s post-test.</p

Effect of TPP⁺ derivatives on the mitochondrial transmembrane potential (ΔΨ_m) of <i>T</i>. <i>cruzi</i>.

<p><i>T</i>. <i>cruzi</i> trypomastigotes were exposed to the different TPP⁺ derivatives for 2 hours, and ΔΨ_m was evaluated through JC-1 fluorescence using a flow cytometer. <b>A.</b> Representative frequency histogram of the fluorescence emitted at 590 nm. Upper panel: trypomastigotes exposed to 0.1 μM of the TPP⁺ derivatives. Lower panel: trypomastigotes exposed to 1 μM of the TPP⁺ derivatives. In both panels, FCCP (100 μM, 15 minutes before measurement) is shown as a positive control. Control: grey line, TPP⁺-C₈: red line, TPP⁺-C₁₀: blue line, TPP⁺-C₁₁: orange line, TPP⁺-C₁₂: green line, FCCP: black dashed line. AU: Arbitrary Units of Fluorescence. <b>B.</b> Quantification of the ratio between the 590 and 490 nm fluorescence emissions in trypomastigotes exposed to the TPP⁺ derivatives. The ratios were calculated using the fluorescence medians obtained from the histograms. The results are expressed as the mean ± SD of three independent experiments, each performed in triplicate. *: <i>p</i> < 0.05 and ***: <i>p</i> < 0.001, compared with the control, based on one-way ANOVA with Dunnett’s post-test.</p

Effect of TPP⁺ derivatives on markers of cell death in <i>Trypanosoma cruzi</i>.

<p><i>T</i>. <i>cruzi</i> trypomastigotes (Y strain, 10⁷/mL) were exposed to TPP⁺-C₈, TPP⁺-C₁₀, TPP⁺- C₁₁, or TPP⁺-C₁₂ at 0.1, 0.5, or 1 μM for 24 hours. The measurement of cell death markers (Annexin-V linkage and propidium iodide incorporation) was performed by flow cytometry. <b>A.</b> Representative dot plot showing the effect of TPP⁺ derivatives at 0.5 μM. The numbers within the quadrants indicate the percentage of double-negative, Annexin-V+, propidium iodide+, or double-positive cells. <b>B.</b> Quantification of viable cells (double negative) after 24 hours of exposure to the different derivatives. The bars represent the mean ± SD of three independent experiments, each performed in triplicate. *: <i>p</i> < 0.05 and ***: <i>p</i> < 0.001, compared with the control, based on one-way ANOVA with Dunnett’s post-test.</p