Dasatinib-loaded albumin nanoparticles possess diminished endothelial cell barrier disruption and retain potent anti-leukemia cell activity

Abstract

Dasatinib (DAS), a second-generation tyrosine kinase inhibitor, is highly effective in treating chronic myeloid leukemia and Philadelphia chromosome-positive acute lymphoblastic leukemia. However, its clinical use is limited due to serious adverse effects. DAS can disrupt endothelial barrier integrity and increase endothelial permeability which may cause peripheral edema and pleural effusion. Albumin nanoparticles (NPs) as a drug carrier may serve as a useful tool for cell-selective drug delivery to reduce DAS-induced endothelial hyperpermeability and maintain endothelial barrier integrity. In this study, we reported that DAS-loaded NPs exhibited potent anti-leukemia efficacy as DAS alone. Importantly, albumin NPs as a drug carrier markedly reduced DAS-induced endothelial hyperpermeability by restraining the inhibition of Lyn kinase signaling pathway in endothelial cells. Therefore, albumin NPs could be a potential tool to improve anti-leukemia efficacy of DAS through its cell-selective effects.

Keywords: drug carrier; endothelial barrier; leukemia; nanoparticles; tyrosine kinase.

Figure 1. Size (A) and Zeta potential (B) distribution of DAS-loaded albumin NPs

The size and Zeta potential distribution of DAS-loaded albumin NPs were measured. Data are representatives of three independent experiments.

Figure 2. Effects of DAS and DAS-loaded NPs on K562 cell viability and modulation of Lyn kinase

K562 cells were treated with DMSO, NPs, DAS (100 nM) or DAS-loaded NPs (DAS-NPs, 100 nM as DAS). (A) Cell viability was assessed by growth inhibition assay at 72 h after exposure. (B) Representative blots of phosphorylated Lyn (p-Lyn) and total Lyn (Lyn) kinases 2 h after the treatment. (C) Densitometry analysis is presented as relative ratios of p-Lyn to Lyn. Data were expressed as mean ± SEM. The experiments were repeated three times. *P < 0.05 versus DMSO group.

Figure 3. Effects of DAS and DAS-loaded NPs on FAK modulation in K562 cells

K562 cells were treated with DMSO, NPs, DAS (100 nM) or DAS-loaded NPs (DAS-NPs, 100 nM as DAS) for 2 h. (A) Representative blots of phosphorylated FAK (p-FAK-397, p-FAK-576/577 and p-FAK-925) and total FAK (FAK). (B–D) Densitometry analysis is presented as relative ratios of p-FAK-397, p-FAK-576/577 and p-FAK-925 to FAK. Data were expressed as mean ± SEM. The experiments were repeated three times. *P < 0.05 versus DMSO group.

Figure 4. Effects of DAS and DAS-loaded NPs on TER across HPAECs

HPAECs were grown to confluence on gold microelectrodes arrays, then treated with DMSO, NPs, DAS (100 nM) or DAS-loaded NPs (DAS-NPs, 100 nM as DAS). TER across HPAECs monolayers was measured. (A) TER dynamic changes in 2.5 h. (B) Representative TER values at 0.8 h after exposure. (C) Representative TER values at 2.5 h after exposure. Data were expressed as mean ± SEM of four independent experiments. *P < 0.05 versus DMSO group; ^#P < 0.05 versus DAS group.

Figure 5. Effects of DAS and DAS-loaded NPs on adherent junctions between HPAECs

HPAECs were treated with DMSO, NPs, DAS (100 nM) or DAS-loaded NPs (DAS-NPs, 100 nM as DAS) for 2 h. (A) Immunofluorescence staining for VE-cadherin subcellular distribution and formation of interendothelial junctional gaps (White arrow). Quantification of gaps (B) in 10 random images was expressed as mean ± SEM of three independent experiments. *P < 0.05 versus DMSO group; ^#P < 0.05 versus DAS group.

Figure 6. Effects of DAS and DAS-loaded NPs on Lyn signaling in HPAECs

HPAECs were treated with DMSO, NPs, DAS (100 nM) or DAS-loaded NPs (DAS-NPs, 100 nM as DAS) for 2 h. (A) Representative blots showing phosphorylated Lyn (p-Lyn) and total Lyn (Lyn) kinases. (B) Densitometry analysis is presented as relative ratios of p-Lyn to Lyn. Data were expressed as mean ± SEM. The experiments were repeated three times.*P < 0.05 versus DMSO group; ^#P < 0.05 versus DAS group.

Figure 7. Effects of DAS and DAS-loaded NPs on modulation of FAK in HPAECs

HPAECs were treated with DMSO, NPs, DAS (100 nM) or DAS-loaded NPs (DAS-NPs, 100 nM as DAS) for 2 h. (A) Representative blots showing phosphorylated (p-FAK-397, p-FAK-576/577 and p-FAK-925) and total FAK. (B–D) Densitometry analysis is presented as relative ratios of p-FAK-397, p-FAK-576/577 and p-FAK-925 to FAK. Data were expressed as mean ± SEM. The experiments were repeated three times. *P < 0.05 versus DMSO group; ^#P < 0.05 versus DAS group.

Figure 8. Uptake of albumin NPs by K562 and HPAECs

K562 cells and HPAECs were incubated with Alexa Fluor 488-conjugated albumin NPs for 2 h. Cells were then washed, fixed, and visualized by fluorescence microscopy. Images are representatives of three independent experiments.