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

Plant Cell Reports volume 36, pages 1917–1928 (2017)Cite this article

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

References

  • Aniento F, Robinson DG (2005) Testing for endocytosis in plants. Protoplasma 226:3–11

    CAS  Article  PubMed  Google Scholar 

  • Bandmann V, Homann U (2012) Clathrin-independent endocytosis contributes to uptake of glucose into BY-2 protoplasts. Plant J 70:578–584

    CAS  Article  PubMed  Google Scholar 

  • Bolte S, Talbot C, Boutte Y, Catrice O, Read ND, Satiat-Jeunemaitre B (2004) FM-dyes as experimental probes for dissecting vesicle trafficking in living plant cells. J Microsc 214:159–173

    CAS  Article  PubMed  Google Scholar 

  • Botha CE, Aoki N, Scofield GN, Liu L, Furbank TR, White RG (2008) A xylem sap retrieval pathway in ricer leaf blades: evidence of a role for endocytosis. J Exp Bot 59:2945–2954

    CAS  Article  PubMed  PubMed Central  Google Scholar 

  • Bramosanti M, Chronopoulou L, Grillo F, Valletta A, Palocci C (2017) Microfluidic-assisted nanoprecipitation of antiviral-loaded polymeric nanoparticles. Col Surf A. doi:10.1016/j.colsurfa.2017.04.062 (in press)

    Google Scholar 

  • Chronopoulou L, Fratoddi I, Palocci C, Venditti I, Russo MV (2009) Osmosis based method drives the self-assembly of polymeric chains into micro- and nanostructures. Langmuir 25:11940–11946

    CAS  Article  PubMed  Google Scholar 

  • Chronopoulou L, Nocca G, Amalfitano A, Callà C, Arcovito A, Palocci C (2015) Dexamethasone-loaded biopolymeric nanoparticles promote gingival fibroblasts differentiation. Biotechnol Progr 3:1381–1387

    Article  Google Scholar 

  • Couvreur P (2013) Nanoparticles in drug delivery: past, present and future. Adv Drug Deliv Rev 65:21–23

    CAS  Article  PubMed  Google Scholar 

  • daSilva LL, Taylor JP, Hadlington JL, Hanton SL, Snowden CJ, Fox SJ, Foresti O, Brandizzi F, Denecke J (2005) Receptor salvage from the prevacuolar compartment is essential for efficient vacuolar protein targeting. Plant Cell 17:132–148

    CAS  Article  PubMed  PubMed Central  Google Scholar 

  • Dhonukshe P, Aniento F, Hwang I, Robinson DG, Mravec J, Stierhof YD, Friml J (2007) Clathrin-mediated constitutive endocytosis of PIN auxin efflux carriers in Arabidopsis. Curr Biol 17:520–527

    CAS  Article  PubMed  Google Scholar 

  • Du Y, Tejos R, Beck M, Himschoot E, Li H, Robatzek S, Vanneste S, Friml J (2013) Salicylic acid interferes with clathrin-mediated endocytic protein trafficking. Proc Natl Acad Sci 110:7946–7951

    CAS  Article  PubMed  PubMed Central  Google Scholar 

  • Elkin SR, Oswald NW, Reed DK, Mettlen M, MacMillan JB, Schmid SL (2016) Ikarugamycin: a natural product inhibitor of clathrin-mediated endocytosis. Traffic 17:1139–1149

    CAS  Article  PubMed  PubMed Central  Google Scholar 

  • Etxeberria E, Baroja-Fernandez E, Muñoz FJ, Pozueta-Romero J (2005) Sucrose-inducible endocytosis as a mechanism for nutrient uptake in heterotrophic plant cells. Plant and Cell Physiol 46:474–481

    CAS  Article  Google Scholar 

  • Etxeberria E, Gonzalez P, Baroja-Fernandez E, Romero JP (2006) Fluid phase endocytic uptake of artificial nano-spheres and fluorescent quantum dots by sycamore cultured cells: evidence for the distribution of solutes to different intracellular compartments. Plant Signal Behav 1:196–200

    Article  PubMed  PubMed Central  Google Scholar 

  • Etxeberria E, Gonzalez P, Pozueta J (2009) Evidence for two endocytic transport pathways in plant cells. Plant Sci 177:341–348

    CAS  Article  Google Scholar 

  • Fan LM, Wang YF, Wang H, Wu WH (2001) In vitro Arabidopsis pollen germination and characterization of the inward potassium currents in Arabidopsis pollen grain protoplasts. J Exp Bot 52:1603–1614

    CAS  Article  PubMed  Google Scholar 

  • Fan L, Li R, Pan J, Ding Z, Lin J (2015) Endocytosis and its regulation in plants. Trends Plant Sci 20:388–397

    CAS  Article  PubMed  Google Scholar 

  • Fleischer A, O’Neill MA, Ehwald R (1999) The pore size of non-graminaceous plant cell walls is rapidly decreased by borate ester cross-linking of the pectic polysaccharide rhamnogalacturonan II. Plant Physiol 121:829–838

    CAS  Article  PubMed  PubMed Central  Google Scholar 

  • Geldner N, Jurgens G (2006) Endocytosis in signaling and transport. Curr Opin Plant Biol 9:589–594

    CAS  Article  PubMed  Google Scholar 

  • González-Melendi P, Fernández-Pacheco R, Coronado MJ, Corredor E, Testillano PS, Risueño MC, Ibarra MR, Rubiales B, Pérez-de-Luque A (2008) Nanoparticles as smart treatment-delivery systems in plants: assessment of different techniques of microscopy for their visualization in plant tissues. Ann Bot 101:187–195

    Article  PubMed  Google Scholar 

  • Holstein SE (2002) Clathrin and plant endocytosis. Traffic 3:614–620

    CAS  Article  PubMed  Google Scholar 

  • Ito E, Fujimoto M, Ebine K, Uemura T, Ueda T, Nakano A (2012) Dynamic behavior of clathrin in Arabidopsis thaliana unveiled by live imaging. Plant J 69:204–216

    CAS  Article  PubMed  Google Scholar 

  • Kankaanpää P, Paavolainen L, Tiitta S, Karjalainen M, Päivärinne J, Nieminen J, Marjomäki V, Heino J, White DJ (2012) BioImageXD: an open, general-purpose and high-throughput image-processing platform. Nat Methods 9:683–689

    Article  PubMed  Google Scholar 

  • Kettler K, Veltman K, van de Meent D, van Wezel A, Hendriks AJ (2014) Cellular uptake of nanoparticles as determined by particle properties, experimental conditions, and cell type. Environ Toxicol Chem 33:481–492

    CAS  Article  PubMed  Google Scholar 

  • Lam SK, Tse YC, Jiang L, Oliviusson P, Heinzerling O, Robinson DG (2005) Plant prevacuolar compartment and endocytosis. Plant Cell Monogr 1:37–61

    Article  Google Scholar 

  • Leborgne-Castel N, Lherminier J, Der C, Fromentin J, Houot V, Simon-Plas F (2008) The plant defense elicitor cryptogein stimulates clathrin-mediated endocytosis correlated with reactive oxygen species production in bright yellow-2 tobacco cells. Plant Physiol 146:1255–1266

    CAS  Article  PubMed  PubMed Central  Google Scholar 

  • Liu Q, Chen B, Wang Q, Shi X, Xiao Z, Lin J, Fang X (2009) Carbon nanotubes as molecular transporters for walled plant cells. Nano Lett 9:1007–1010

    CAS  Article  PubMed  Google Scholar 

  • Ma X, Geiser-Lee J, Deng Y, Kolmakov A (2010) Interactions between engineered nanoparticles (ENPs) and plants: phytotoxicity, uptake and accumulation. Sci Total Environ 408:3053–3061

    CAS  Article  PubMed  Google Scholar 

  • Meckel T, Hurst AC, Thiel G, Homann U (2004) Endocytosis against high turgor: intact guard cells of Viciafaba constitutively endocytose fluorescently labeled plasma membrane and GFP-tagged K-channel KAT1. Plant J 39:182–193

    CAS  Article  PubMed  Google Scholar 

  • Moscatelli A, Ciampolini F, Rodighiero S, Onelli E, Cresti M, Santo N, Idilli A (2007) Distinct endocytic pathways identified in tobacco pollen tubes using charged nanogold. J Cell Sci 120:3804–3819

    CAS  Article  PubMed  Google Scholar 

  • Mura S, Seddaiu G, Bacchini F, Roggero PP, Greppi GF (2013) Advances of nanotechnology in agro-environmental studies. Ital J Agron 8:18

    Article  Google Scholar 

  • Nair R, Varghese SH, Nair BG, Maekawa T, Yoshida Y, Kumar DS (2010) Nanoparticulate material delivery to plants. Plant Sci 179:154–163

    CAS  Article  Google Scholar 

  • Navarro E, Baun A, Behra R, Hartmann NB, Filser J, Miao AJ, Sigg L (2008) Environmental behavior and ecotoxicity of engineered nanoparticles to algae, plants, and fungi. Ecotoxicol 17:372–386

    CAS  Article  Google Scholar 

  • Onelli E, Prescianotto-Baschong C, Caccianiga M, Moscatelli A (2008) Clathrin-dependent and independent endocytic pathways in tobacco protoplasts revealed by labeling with charged nanogold. J Exp Bot 59:3051–3068

    CAS  Article  PubMed  PubMed Central  Google Scholar 

  • Pérez-Gómez J, Moore I (2007) Plant endocytosis: it is clathrin after all. Curr Biol 17:R217–R219

    Article  PubMed  Google Scholar 

  • Rico CM, Majumdar S, Duarte-Gardea M, Peralta-Videa JR, Gardea-Torresdey JL (2011) Interaction of nanoparticles with edible plants and their possible implications in the food chain. J Agric Food Chem 59:3485–3498

    CAS  Article  PubMed  PubMed Central  Google Scholar 

  • Robinson DG, Pimpl P (2014) Clathrin and post-golgi trafficking: a very complicated issue. Trends Plant Sci 19:134–139

    CAS  Article  PubMed  Google Scholar 

  • Robinson DG, Jiang L, Schumacher K (2008) The endosomal system of plants: charting new and familiar territories. Plant Physiol 147:1482–1492

    CAS  Article  PubMed  PubMed Central  Google Scholar 

  • Šamaj J, Read ND, Volkmann D, Menzel D, Baluška F (2005) The endocytic network in plants. Trends Cell Biol 15:425–433

    Article  PubMed  Google Scholar 

  • Sandvig K, Torgersen ML, Raa HA, Van Deurs B (2008) Clathrin-independent endocytosis: from nonexisting to an extreme degree of complexity. Histochem Cell Biol 129:267–276

    CAS  Article  PubMed  PubMed Central  Google Scholar 

  • Santamaria AR, Innocenti M, Mulinacci N, Melani F, Valletta A, Sciandra I, Pasqua G (2012) Enhancement of viniferin production in Vitis vinifera L. cv. Alphonse Lavallée Cell suspensions by low-energy ultrasound alone and in combination with methyl jasmonate. J Agric Food Chem 60:11135–11142

    CAS  Article  PubMed  Google Scholar 

  • Schwab F, Zhai G, Kern M, Turner A, Schnoor JL, Wiesner MR (2016) Barriers, pathways and processes for uptake, translocation and accumulation of nanomaterials in plants—critical review. Nanotoxicology 10:257–278

    CAS  PubMed  Google Scholar 

  • Serag MF, Kaji N, Gaillard C, Okamoto Y, Terasaka K, Jabasini M, Tokeshi M, Mizukami H, Bianco A, Baba Y (2010) Trafficking and subcellular localization of multiwalled carbon nanotubes in plant cells. ACS Nano 5:493–499

    Article  PubMed  Google Scholar 

  • Tripathi DK, Singh S, Singh S, Pandey R, Singh VP, Sharma NC, Prasad SM, Dubey NK, Chauhan DK (2017) An overview on manufactured nanoparticles in plants: uptake, translocation, accumulation and phytotoxicity. Plant Physiol Biochem 110:2–12

    CAS  Article  PubMed  Google Scholar 

  • Valletta A, Chronopoulou L, Palocci C, Baldan B, Donati L, Pasqua G (2014) Poly(lactic-co-glycolic) acid nanoparticles uptake by Vitis vinifera and grapevine-pathogenic fungi. J Nanopart Res 16:2744

    Article  Google Scholar 

  • Vlot AC, Dempsey DMA, Klessig DF (2009) Salicylic acid, a multifaceted hormone to combat disease. Ann Rev Phytopathol 47:177–206

    CAS  Article  Google Scholar 

  • Wang P, Lombi E, Zhao FJ, Kopittke PM (2016) Nanotechnology: a new opportunity in plant sciences. Trends Plant Sci 21:699–712

    CAS  Article  PubMed  Google Scholar 

  • Yamada K, Fuji K, Shimada T, Nishimura M, Hara-Nishimura I (2005) Endosomal proteases facilitate the fusion of endosomes with vacuoles at the final step of the endocytotic pathway. Plant J 41:888–898

    CAS  Article  PubMed  Google Scholar 

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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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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

Authors
  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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Additional information

Communicated by Kan Wang.

Electronic supplementary material

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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

Keywords

  • Microfluidics
  • Polymeric nanoparticles
  • Cell cultures
  • Endocytosis
  • Vitis vinifera