Synchrotron-based X-ray fluorescence microscopy reveals accumulation of polymyxins in single human alveolar epithelial cells

Abstract

Intravenous administration of the last-line polymyxins results in poor drug exposure in the lungs and potential nephrotoxicity; while inhalation therapy offers better pharmacokinetics/pharmacodynamics for pulmonary infections by delivering the antibiotic to the infection site directly. However, polymyxin inhalation therapy has not been optimized and adverse effects can occur. This study aimed to quantitatively determine the intracellular accumulation and distribution of polymyxins in single human alveolar epithelial A549 cells. Cells were treated with an iodine-labeled polymyxin probe FADDI-096 (5.0 and 10.0 μM) for 1, 4, and 24 h. Concentrations of FADDI-096 in single A549 cells were determined by synchrotron-based X-ray fluorescence microscopy. Concentration- and time-dependent accumulation of FADDI-096 within A549 cells was observed. The intracellular concentrations (mean ± SEM, n ≥ 189) of FADDI-096 were 1.58 ± 0.11, 2.25 ± 0.10, and 2.46 ± 0.07 mM following 1, 4 and 24 h of treatment at 10 μM, respectively. The corresponding intracellular concentrations following the treatment at 5 μM were 0.05 ± 0.01, 0.24 ± 0.04, and 0.25 ± 0.02 mM (n ≥ 189). FADDI-096 was mainly localized throughout the cytoplasm and nuclear region over 24 h. The intracellular zinc concentration increased in a concentration- and time-dependent manner. This is the first study to quantitatively map the accumulation of polymyxins in human alveolar epithelial cells and provides crucial insights for deciphering the mechanisms of their pulmonary toxicity. Importantly, our results may shed light on the optimization of inhaled polymyxins in patients and the development of new-generation safer polymyxins.

FIG 1

Intracellular distribution of FADDI-096 in single A549 cells determined with confocal fluorescence microscopy (signal from dansyl) and synchrotron XFM imaging (signal from iodine). (A and B) A549 cells without treatment (control) and treated with 5.0 or 10.0 μM FADDI-096 for 1, 4, and 24 h. Signal intensities are linearly scaled for iodine from the minimum to the maximum; the numbers at the top of the relevant panels show the maximum pixel value (μg/cm²) in each sample. Bar, 5 μm. (C) Representative signal intensities from low-resolution XFM images of A549 cells treated with 10.0 μM FADDI-096 for 24 h. Bar, 50 μm. (D) Intracellular accumulation of FADDI-096 in single A549 cells measured by XFM (mean ± standard error of the mean [SEM]; n = at least 189 cells; ****, P < 0.0001).

FIG 2

Fluorescence signals from the (A) dansyl group and (B) iodine of FADDI-096 loaded on to the silicon nitride windows by fluorescence microscopy and XFM imaging, respectively.

FIG 3

(A) Intracellular concentration of zinc in single A549 cells measured by XFM following treatments with 5.0 or 10.0 μM FADDI-096 for 1, 4, and 24 h (mean ± SEM; n = at least 189 cells; ****, P < 0.0001). (B) Correlation between the intracellular concentration of zinc and FADDI-096 in single human lung epithelial A549 cells treated with 5.0 or 10.0 μM FADDI-096 for 1, 4, and 24 h (P < 0.0001; Pearson’s r = 0.48).

FIG 4

Intracellular distribution of elements (P, K, Fe, Cl, and Ca) in single A549 cells determined with XFM. (A) Untreated A549 cells (control) and cells treated with 5.0 or 10.0 μM FADDI-096 for 1, 4, and 24 h. Signal intensities are linearly scaled separately for each element from the minimum to the maximum; the numbers at the top of the relevant panels show the maximum pixel value (μg/cm²) in each sample. Bar, 5 μm. (B) Intracellular accumulation of elements in single A549 cells measured by XFM (mean ± SEM; n = at least 189 cells; ****, P < 0.0001).