Optical ratiometric silica pH sensors unveil active transport and subcellular particle localization in protoplasts  

作　　者：Helena Iuele Lorenzo Maria Curci Alessio Bucciarelli Valentina Onesto Stefania Forciniti Giuseppe Gigli Loretta L.del Mercato Monica De Caroli Gabriella Piro 

机构地区：[1]Institute of Nanotechnology,National Research Council(CNR-NANOTEC),c/o Campus Ecotekne,via Monteroni,73100 Lecce,Italy [2]Department of Biological and Environmental Sciences and Technologies,University of Salento,c/o Campus Ecotekne,via Monteroni,73100 Lecce,Italy [3]BIOtech Research Center,Italy and European Institute of Excellence on Tissue Engineering and Regenerative Medicine,Department of Industrial Engineering,University of Trento,Via Delle Regole 101,38123 Trento,Italy [4]Department of Experimental Medicine,University of Salento,c/o Campus Ecotekne,via Monteroni,73100 Lecce,Italy [5]NBFC National Biodiversity Future Center,90133 Palermo,Italy

出　　处：《Nano Research》2025年第3期444-457,共14页纳米研究(英文版)

基　　金：supported by the European Research Council(ERC)under the European Union’s Horizon 2020 research and innovation program ERC Starting Grant“INTERCELLMED”(Nr.759959);the Associazione Italiana per la Ricerca contro il Cancro(AIRC)(MFAG-2019,Nr.22902);the PRIN 2022(Nr.2022CRFNCP_PE11_PRIN2022 and PRIN 2022-SAPPHIRE(CUP 2022MWK7Y7);funded by European Union-Next Generation EU,the“Tecnopolo per la medicina di precisione”(TecnoMed Puglia)-Regione Puglia:DGR n.2117 of 21/11/2018,CUP B84I18000540002);the Italian Ministry of Research(MUR)in the framework of the National Recovery and Resilience Plan(NRRP),“NFFA-DI”Grant(CUP B53C22004310006);“I-PHOQS”Grant(CUP B53C22001750006);under the complementary actions to the NRRP funded by NextGenerationEU,“Fit4MedRob”Grant(PNC0000007,CUP B53C22006960001).

摘　　要：Understanding the interaction mechanisms of engineered nanomaterials(ENMs)with plant membranes is crucial for their effective use in various applications.While passive transport of smaller ENMs is well-documented,the mechanisms underlying active transport of larger ENMs remain poorly understood.This study systematically investigates the active transport and subcellular distribution of ENMs(100-1000 nm)within protoplasts using optical ratiometric silica pH sensors for localization.Highly monodispersed ratiometric pH sensors,based on silica particles modified with fluorescein-5-isothiocyanate(FITC)and cyanine3 NHS ester(CY3)dyes,were employed to elucidate internalization mechanisms.Protoplasts from Nicotiana tabacum L.leaves successfully internalized the sensors.3D segmentation of protoplasts revealed distinct pH gradients,indicating vacuole accumulation.Colocalization analysis and cellular compartments staining further confirmed sensor distribution.High-throughput imaging flow cytometry showed efficient internalization rates,which decreased after cell wall regeneration.Notably,inhibition experiments with the salicylic acid(SA)and Tyrphostin A23(TyrA23)inhibitors confirmed clathrin-mediated endocytosis in particle uptake.This study establishes rational design principles for controlling active ENM uptake and subcellular localization via optical pH sensing in protoplasts.The findings enhance our understanding of plant cell trafficking mechanisms and hold promise for targeted delivery and applications in plant biology research.

关 键 词：optical ratiometric pH-sensors intracellular pH tracking PROTOPLAST endocytic pathway computational analysis 

分 类 号：Q94[生物学—植物学]