Confocal Microscopy Investigations of Biopolymeric PLGA Nanoparticle Uptake in <i>Arabidopsis thaliana</i> L. Cultured Cells and Plantlet Roots

Abstract

To date, most endocytosis studies in plant cells have focused on clathrin-dependent endocytosis, while limited evidence is available on clathrin-independent pathways. Since dynamin a is a key protein both in clathrin-mediated endocytosis and in clathrin-independent endocytic processes, this study investigated its role in the uptake of poly-(lactic-co-glycolic) acid (PLGA) nanoparticles (NPs). The experiments were performed on cultured cells and roots of Arabidopsis thaliana. Dynasore was used to inhibit the activity of dynamin-like proteins to investigate whether PLGA NPs enter plant cells through a dynamin-like-dependent or dynamin-like-independent endocytic pathway. Observations were performed by confocal microscopy using a fluorescent probe, coumarin 6, loaded in PLGA NPs. The results showed that both cells and roots of A. thaliana rapidly take up PLGA NPs. Dynasore was administered at different concentrations and exposure times in order to identify the effective ones for inhibitory activity. Treatments with dynasore did not prevent the NPs uptake, as revealed by the presence of fluorescence emission detected in the cytoplasm. At the highest concentration and the longest exposure time to dynasore, the fluorescence of NPs was not visible due to cell death. Thus, the results suggest that, because the NPs’ uptake is unaffected by dynasore exposure, NPs can enter cells and roots by following a dynamin-like-independent endocytic pathway.

Keywords

• endocytosis
• dynamin-independent endocytosis
• PLGA nanoparticles
• dynasore
• cell cultures
• root plantlets