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Cellular internalization of bystander nanomaterial induced by TAT-nanoparticles and regulated by extracellular cysteine

Cell penetrating ligands are often linked to nanoparticles (NPs) to stimulate cellular internalization by endocytic processes. On their own, these ligands are known to promote uptake of bystander cargo through a specialized endocytic pathway known as micropinocytosis. However, it is not clear if functionalized NPs linked to such ligands also influence the cellular uptake of bystander cargo.

In a recent publication of Nature Communications, Wei et al. investigated the bystander activity of NPs linked to TAT, a widely-used cell penetrating peptide. These functionalized NPs (T-NPs) and non-functionalized bystander NPs were conjugated to fluorescent CF® dyes to allow visualization of NP trafficking. The effect of T-NPs on uptake of bystander cargo was studied in CHO and H1975 cells by co-incubating with non-functionalized NPs and other labeled fluid-phase markers (ie. BSA and dextran). Internalization of T-NPs and bystander cargo was measured by flow cytometry and fluorescence microscopy. Results show T-NPs strongly promote the uptake of bystander NPs, but not fluid-phase markers. Meanwhile, monomeric TAT peptide enhanced bystander uptake of fluid-phase markers, but only certain NP types. This suggests a preferential NP bystander uptake in the presence of functionalized T-NPs. Moreover, the authors confirm the TAT interaction with cell surface receptors is necessary for T-NP associated bystander uptake. Lastly, the authors report T-NP internalization and bystander uptake is strongly stimulated by extracellular cysteine, suggesting a role for the cysteine transport machinery in this specialized endocytic pathway.

Demonstration of T-NP-induced bystander uptake under physiological conditions. a Bystander uptake of Ag-488 by live tumor slices. T-Ag and Ag-488 were incubated with live 4T1 tumor slices in AA-free, 20AA+, and Cys medium for 2 h. After incubation, slices were etched, stained with DAPI (blue) and imaged by confocal microscope (n = 3 mice for each group). Scale bars are 20 µm. b, c The bystander uptake of Ag-647 (b) or dextran (c) in 4T1 tumors. 4T1 tumors bearing mice were received Intratumoral (i.t.) injection of T-Ag (±Cys) prior to the intravenous (i.v.) injection of Ag-647 (±Cys) or FITC-dextran (±Cys). Two hours after circulation, mice were transcardial perfused with PBS, and then tumors were collected and further dissociated to single cells, etched and quantified by flow cytometry. The amount of Ag-647 or FITC-dextran positive cells in percentage was shown. Data are represented as the mean ± s.d. of at least three mice for each group (b n = 3 for control and n = 5 for experimental groups; n = 3 for c). Quantified data b, c were analyzed using one-way ANOVA with Tukey’s multiple comparisons test. One-way ANOVA, b, F = 60.141, P < 0.0001; c F = 33.373, P = 0.0100. Source data are provided as a Source Data file; credit Wei et. al, doi: 10.1038/s41467-019-11631-w.

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Full Citation

Wei, Y., Tang, T. & Pang, H.-B. Cellular internalization of bystander nanomaterial induced by TAT-nanoparticles and regulated by extracellular cysteine. Nat. Commun. 10, 3646 (2019).