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Effect of MSN-AVA-TPP@DOX on M. marinum uptake and intracellular replication. a Schematic representation of the protocol used for the internalization assay. b Percentage of infected monocyte-derivate macrophages (MDM) (left panel) and monocyte-derivate dendritic cells (MoDC) (right panel) measured by flow cytometry. Both cell types were infected with mScarlet-Mmar pre-incubated with control (gray), 50 or 200 µg/mL of MSN-AVA-TPP (orange), or MSN-AVA-TPP@DOX (purple). The experiment was performed with 3 donors, and 3 technical replicates for each donor. Each dot corresponds to the mean of 3 technical replicates for one donor, normalized to the control. Data are shown as median and interquartile range (n = 3). Comparisons were done using ordinary one-way ANOVA with Šídák’s multiple comparisons test. Asterisks above each bar correspond to comparison to non-treated condition (gray). Adjusted p values (from left to right): MDM: **p = 0.0026, ****p < 0.0001, ***p = 0.0007, ns p = 0.9995 (AVA-TPP 50 vs AVA-TPP@DOX 50), ns p = 0.9957 ((AVA-TPP 200 vs AVA-TPP@DOX 200); MoDC: ***p = 0.0005 (AVA-TPP 50), ****p < 0.0001, ***p = 0.0002 (AVA-TPP@DOX 50), ns p = 0.9844 (AVA-TPP 50 vs AVA-TPP@DOX 50), ns p = 0.9987 (AVA-TPP 200 vs AVA-TPP@DOX 200). c Schematic representation of the protocol used to quantify intracellular bacteria. d Quantity of bacteria per well of infected THP-1 cells, treated with different concentrations of MSN-AVA-TPP@DOX (125, 250, and 500 µg/mL) or free doxycycline (1.2, 3, 6, and 12 µg/mL). Three biological replicates were performed with at least 3 technical replicates each. Each dot corresponds to CFUs from one technical replicate. Data are represented as dot plots with median and interquartile range (n = 16 MSN-AVA-TPP@DOX; n = 12 DOX). Comparisons were done using ordinary one-way ANOVA with Šídák’s multiple comparisons test. Asterisks above each data set correspond to comparisons to non-treated condition (gray). Adjusted p values (from left to right): *p = 0.0416, ****p < 0.0001, ns p = 0.9433 (DOX 1.2), ns p = 0.8660 (DOX 3), ns p = 0.2435 (DOX 6), ns p = 0.3928 (AVA-TPP@DOX 125 vs DOX 1.2). e Representative confocal images of Mmar-infected THP-1 cells treated with 125, 250, and 500 µg/mL of MSN-AVA-TPP@DOX. Left panels correspond to xy orthogonal projections of the z-stack images. Right panels correspond to zoomed 3D representation of the white squares. Three independent experiments were performed. Created in BioRender. Kremer, L. (2025) https://BioRender.com/zsckfm1. Source data are provided as a SourceData file.
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