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Endocytic pathways involved in PLGA nanoparticle uptake by grapevine cells and role of cell wall and membrane in size selection

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Abstract

Key message

PLGA NPs’ cell uptake involves different endocytic pathways. Clathrin-independent endocytosis is the main internalization route. The cell wall plays a more prominent role than the plasma membrane in NPs’ size selection.

Abstract

In the last years, many studies on absorption and cell uptake of nanoparticles by plants have been conducted, but the understanding of the internalization mechanisms is still largely unknown. In this study, polydispersed and monodispersed poly(lactic-co-glycolic) acid nanoparticles (PLGA NPs) were synthesized, and a strategy combining the use of transmission electron microscopy (TEM), confocal analysis, fluorescently labeled PLGA NPs, a probe for endocytic vesicles (FM4-64), and endocytosis inhibitors (i.e., wortmannin, ikarugamycin, and salicylic acid) was employed to shed light on PLGA NP cell uptake in grapevine cultured cells and to assess the role of the cell wall and plasma membrane in size selection of PLGA NPs. The ability of PLGA NPs to cross the cell wall and membrane was confirmed by TEM and fluorescence microscopy. A strong adhesion of PLGA NPs to the outer side of the cell wall was observed, presumably due to electrostatic interactions. Confocal microscopy and treatment with endocytosis inhibitors suggested the involvement of both clathrin-dependent and clathrin-independent endocytosis in cell uptake of PLGA NPs and the latter appeared to be the main internalization pathway. Experiments on grapevine protoplasts revealed that the cell wall plays a more prominent role than the plasma membrane in size selection of PLGA NPs. While the cell wall prevents the uptake of PLGA NPs with diameters over 50 nm, the plasma membrane can be crossed by PLGA NPs with a diameter of 500–600 nm.

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Abbreviations

PLGA NPs:

Poly(lactic-co-glycolic) acid nanoparticles

TEM:

Transmission electron microscope

FM4-64:

N-(3-triethylammoniumpropyl) -4-(6-(4-(diethylamino) phenyl)hexatrienyl)pyridinium dibromide

ACN:

Acetonitrile

HPLC:

High-performance liquid chromatography

Cu6:

Coumarin 6

CFM:

Continuous flow microfluidic

OBM:

Osmosis-based methodology

DLS:

Dynamic light scattering

PdI:

Polydispersity index

SEM:

Scanning electron microscopy

DMSO:

Dimethylsulfoxide

NAA:

α-Naphthaleneacetic acid

KIN:

Kinetin

PI3K:

Phosphatidylinositol 3-kinase

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Acknowledgements

This study was supported by the University of Rome La Sapienza, Grant No. C26A13L5HT. The authors wish to thank the Electron Microscopy Service (Department of Biology, University of Padova, Italy) for providing technical support in TEM observations.

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Authors and Affiliations

  1. Department of Chemistry, University of Rome La Sapienza, Piazzale A. Moro 5, 00185, Rome, Italy

    Cleofe Palocci, Laura Chronopoulou, Marco Bramosanti & Elisa Brasili

  2. Department of Environmental Biology, University of Rome La Sapienza, Piazzale A. Moro 5, 00185, Rome, Italy

    Alessio Valletta, Livia Donati & Gabriella Pasqua

  3. Department of Biology, University of Padua, Via Ugo Bassi 58/B, 35131, Padua, Italy

    Barbara Baldan

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  1. Cleofe Palocci
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  2. Alessio Valletta
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  5. Marco Bramosanti
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Correspondence to Alessio Valletta.

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Communicated by Kan Wang.

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299_2017_2206_MOESM1_ESM.jpg

Fig. S1 Size distribution measurements of PLGA NPs and Cu6-loaded PLGA NPs obtained with the microfluidic reactor (JPEG 3717 kb)

299_2017_2206_MOESM2_ESM.jpg

Fig. S2 Size distribution measurements of Cu6-loaded PLGA NPs obtained by OBM method before and after filtration (JPEG 3946 kb)

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Palocci, C., Valletta, A., Chronopoulou, L. et al. Endocytic pathways involved in PLGA nanoparticle uptake by grapevine cells and role of cell wall and membrane in size selection. Plant Cell Rep 36, 1917–1928 (2017). https://doi.org/10.1007/s00299-017-2206-0

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  • DOI: https://doi.org/10.1007/s00299-017-2206-0

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