Modulation of Signaling and Intracellular Trafficking Pathways by Surface-Engineered Hydrogel Nanoparticles in Tumor Cells.

[摘要] Surface engineering of a polyacrylamide (PAA) hydrogel nanoparticle (NP) with the tumor-targeting ligand, F3 peptide (KDEPQRRSARLSAKPAPPKPEPKPKKAPAKKC), confers binding specificity toward Nucleolin overexpressing tumor cells (9L rat gliosarcoma, and MDA-MB-435 human breast adenocarcinoma). In this study, the endocytic internalization, and intracellular trafficking of the non-targeted PAA-NPs (NTNPs), and F3-targeted PAA-NPs (F3NPs) in the above-mentioned cell lines, was investigated. Caveolae-mediated internalization of both types of PAA-NPs peaked at 2 hours post-delivery, although internalization of the NTNPs was ~2-fold greater than for the F3NPs.In contrast, clathrin-mediated internalization of both types of PAA-NPs was markedly faster; the NTNPs and F3NPs both reached similar peak colocalization levels with early endosome antigen-1 (EEA1, ~32%) at 30 minutes post-delivery. However, at 60 minutes post-delivery, the NTNPs exhibited faster egress from the early endosomes than the F3NPs, with a concomitant, sharp increase in trafficking to the lysosomes (acidic, degradative vesicles), whereas the F3NPs largely evaded trafficking to the lysosomes. Furthermore, the F3 peptides alone exhibited significantly higher accumulation within the lysosomes than both the NTNPs, and the F3NPs. The p38 Mitogen-Activated Protein Kinases (MAPKs), upon activation, promote (i) internalization of caveolae from the cell membrane, and (ii) rapid trafficking of early endosomes to the lysosomes by directly phosphorylating Caveolin1 and EEA1, respectively. Phospho-proteomic analyses, in MDA-MB-435 cells, revealed that the peak levels of activated p38β and p38δ MAPKs (at 2 hours post-delivery) elicited by the F3 peptides alone, and the NTNPs was ~2-fold greater than by the F3NPs. These data therefore provide compelling evidence that the intracellular trafficking behavior of the F3 peptides, NTNPs and F3NPs are attributable to their differential activation of the p38 MAPKs. Further analysis of the ERK MAPK, JNK MAPK, and Akt pathways revealed that the NTNPs elicit a pro-apoptotic signaling profile, whereas the F3 peptides, and F3NPs elicit proliferative profiles. The findings of this thesis suggest that the design of tumor-targeting nanoparticles also need to consider the MAPK signaling profiles that they elicit on the intended target cell type, due to the influence of the p38 MAPKs, in particular, on endocytic trafficking, and the survival status of the target tumor cell.

[发布机构] University of Michigan

[效力级别] endocytosis and intracellular trafficking

[关键词] hydrogel nanoparticles;endocytosis and intracellular trafficking;cell signaling pathways;nanoparticle surface engineering;Chemistry;Science;Chemical Biology