In Vivo Delivery of Nanoparticles into Plant Leaves

Honghong Wu1, Israel Santana1, Joshua Dansie1, Juan P. Giraldo1

1 Department of Botany and Plant Sciences, University of California, Riverside
Publication Name:  Current Protocols in Chemical Biology
Unit Number:   
DOI:  10.1002/cpch.29
Online Posting Date:  December, 2017
GO TO THE FULL TEXT: PDF or HTML at Wiley Online Library


Plant nanobiotechnology is an interdisciplinary field at the interface of nanotechnology and plant biology that aims to utilize nanomaterials as tools to study, augment or impart novel plant functions. The delivery of nanoparticles to plants in vivo is a key initial step to investigate plant nanoparticle interactions and the impact of nanoparticles on plant function. Quantum dots are smaller than plant cell wall pores, have versatile surface chemistry, bright fluorescence and do not photobleach, making them ideal for the study of nanoparticle uptake, transport, and distribution in plants by widely available confocal microscopy tools. Herein, we describe three different methods for quantum dot delivery into leaves of living plants: leaf lamina infiltration, whole shoot vacuum infiltration, and root to leaf translocation. These methods can be potentially extended to other nanoparticles, including nanosensors and drug delivery nanoparticles. © 2017 by John Wiley & Sons, Inc.

Keywords: confocal imaging; leaf infiltration; nanoparticle delivery; root to leaf translocation; vacuum infiltration; quantum dots

PDF or HTML at Wiley Online Library

Table of Contents

  • Introduction
  • Basic Protocol 1: Synthesis and Characterization of Thioglycolic Acid (TGA) Wrapped Quantum Dots
  • Basic Protocol 2: Infiltration of TGA‐Quantum Dots Through Leaf Lamina
  • Basic Protocol 3: TGA‐Quantum Dot Delivery to Leaves Via Vacuum Infiltration
  • Basic Protocol 4: Root‐To‐Leaf Delivery of TGA‐Quantum Dots
  • Basic Protocol 5: In Vivo Imaging of TGA‐Quantum Dots in Plant Leaves by Confocal Imaging
  • Reagents and Solutions
  • Commentary
  • Literature Cited
  • Figures
PDF or HTML at Wiley Online Library


Basic Protocol 1: Synthesis and Characterization of Thioglycolic Acid (TGA) Wrapped Quantum Dots

  • Sodium borohydride (NaBH 4; 96%, Sigma‐Aldrich)
  • Tellurium powder (Te; 99.8%, Sigma‐Aldrich)
  • Ethanol 100 proof (Sigma‐Aldrich)
  • Molecular biology water (Corning, cat. no. 46‐000‐CM)
  • Nitrogen gas
  • Cadmium chloride hydrate (CdCl 2·2.5 H 2O; Sigma‐Aldrich)
  • Thioglycolic acid (TGA; 98%, TCI)
  • Sodium hydroxide solution (NaOH; Sigma‐Aldrich)
  • Two‐necked flasks
  • Hot plate and magnetic stirrer
  • 1‐ml syringe equipped with a 21‐G needle

Basic Protocol 2: Infiltration of TGA‐Quantum Dots Through Leaf Lamina

  • Sunshine LC1 mix soil
  • Tap water
  • Wild‐type Arabidopsis (Col‐0) seeds
  • Deionized water
  • QD stock solution (see protocol 1 for the preparation)
  • Magnesium chloride (MgCl 2; Sigma‐Aldrich, cat. no. M8266)
  • 2‐[(2‐Hydroxy‐1,1‐bis(hydroxymethyl)ethyl)amino]ethanesulfonic acid (TES; Sigma‐Aldrich, cat. no. T1375)
  • 32 insert sheets disposable pots (2 × 2 in.)
  • 1020 greenhouse trays without holes (11 × 21.37 × 2.44 inch W/L/D)
  • Forceps
  • Adaptis 1000 growth chambers (Conviron)
  • 1‐ml sterile needleless syringe (NORM‐JECT®)
  • Kimwipes (Kimtech Science®)
  • Permanent marker (Sharpie, fine point)

Basic Protocol 3: TGA‐Quantum Dot Delivery to Leaves Via Vacuum Infiltration

  • Arabidopsis seedlings, aged four weeks (see protocol 2)
  • 5.18 nM Cd/Te Cd/S thioglycolic acid quantum dot (TGA‐QD) solution
  • 2‐[(2‐Hydroxy‐1,1‐bis(hydroxymethyl)ethyl)amino]ethanesulfonic acid (TES; Sigma, cat. no. T1375)
  • Industrial cling film
  • Tape
  • Sonicator (Elmasonic P)
  • 5‐ml syringes
  • 20‐nm pore‐size syringe filter (AnotopTM 25, WhatmanTM)
  • 50‐ml Falcon tube
  • 50‐ml beaker with ventilation hole
  • Vacuum desiccator (BelArt, cat. no. F42400‐2021)
  • Kimwipes
  • Growth chamber

Basic Protocol 4: Root‐To‐Leaf Delivery of TGA‐Quantum Dots

  • Arabidopsis thaliana seeds
  • 50% bleach solution
  • 70% ethanol
  • ddH 2O
  • Murashige and Skoog basal salt mixture (MS) medium (Sigma)
  • Potassium hydroxide (KOH)
  • Phytoagar (Bioworld)
  • TGA‐capped QD solution
  • KCl (Sigma‐Aldrich)
  • 1.5‐ml microcentrifuge tubes (Thermo Fisher)
  • Vortex mixer
  • Aluminum foil, optional
  • 85‐mm petri plates (Thermo Fisher)
  • 500‐ml bottles
  • Autoclave (Amsco® lab 110, STERIS Life Science)
  • Laminar flow hood (NuAire, cat. no. NU‐201‐530)
  • 200‐μl pipettes
  • Micropore tape (3M)
  • Growth chamber
  • 20‐nm pore‐size syringe filter (AnotopTM 25, WhatmanTM)
  • Forceps, sterilized

Basic Protocol 5: In Vivo Imaging of TGA‐Quantum Dots in Plant Leaves by Confocal Imaging

  • Plants incubated with TGA‐QD (see protocol 2)
  • Observation gel (Carolina)
  • Perfluorodecalin (PFD; Sigma)
  • Cork borers
  • Glass slides (Corning, cat. no. 2948‐75X25)
  • Glass Pasteur pipet and rubber bulb (Fisher, cat. nos. 13‐678‐20C and 03‐448‐22)
  • Coverslip (VWR, cat. no. 48366 045)
  • Argon 488‐nm laser
  • Zeiss confocal microscope imaging system (Zeiss 510)
PDF or HTML at Wiley Online Library



Literature Cited

Literature Cited
  Albersheim, P., Darvill, A., Roberts, K., Sederoff, R., & Staehelin, A. (2011). Cell walls and plant anatomy. Plant Cell Walls, 241–242.
  Al‐Jamal, W., Al‐Jamal, K. T., Tian, B., Lacerda, L., Bomans, P. H., Frederik, P. M., & Kostarelos, K. (2008). Lipid‐quantum dot bilayer vesicles enhance tumor cells uptake and retention in vitro and in vivo. ACS Nano, 2, 408–418. doi: 10.1021/nn700176a.
  Al‐Salim, N., Barraclough, E., Burgess, E., Clothier, B., Deurer, M., Green, S., … Weir, G. (2011). Quantum dot transport in soil, plants, and insects. The Science of the Total Environment, 409, 3237–3248. doi: 10.1016/j.scitotenv.2011.05.017.
  Brag, H. (1972). The influence of potassium on the transpiration rate and stomatal opening in Triticum aestivum and Pisum sativum. Physiologiae Plantarum, 26, 250–257. doi: 10.1111/j.1399‐3054.1972.tb03577.x.
  Buckley, T. N., & Mott, K. A. (2013). Modelling stomatal conductance in response to environmental factors. Plant, Cell & Environment, 36, 1691–1699. doi: 10.1111/pce.12140.
  Clough, S. J., & Bent, A. F. (1998). Floral dip: A simplified method for Agrobacterium‐mediated transformation of Arabidopsis thaliana. The Plant Journal, 16, 735–743. doi: 10.1046/j.1365‐313x.1998.00343.x.
  Da Costa, N. V. J., & Sharma, P. K. (2016). Effect of copper oxide nanoparticles on growth, morphology, photosynthesis, and antioxidant response on Oryza sativa. Photosynthetica, 54, 110–119. doi: 10.1007/s11099‐015‐0167‐5.
  Drake, P. L., Froend, R. H., & Franks, P. J. (2013). Smaller, faster stomata: Scaling of stomatal size, rate of response, and stomatal conductance. Journal of Experimental Botany, 64, 495–505. doi: 10.1093/jxb/ers347.
  Delehanty, J. B., Boeneman, K., Bradburne, C. E., Robertson, K., & Medintz, I. L. (2009). Quantum dots: A powerful tool for understanding the intricacies of nanoparticle‐mediated drug delivery. Expert Opinion on Drug Delivery, 6, 1091–1112. doi: 10.1517/17425240903167934.
  Fanourakis, D., Giday, H., Milla, R., Pieruschka, R., Kjaer, K. H., Bolger, M., … Ottosen, C. O. (2015). Pore size regulates operating stomatal conductance, while stomatal densities drive the partitioning of conductance between leaf discs. Annals of Botany, 15, 555–565. doi: 10.1093/aob/mcu247.
  Freitas, D. V., Dias, J. M. M., Passos, S. G. B., de Souza, G. C. S., Neto, E. T., & Navarro, M. (2014). Electrochemical synthesis of TGA‐capped CdTe and CdSe quantum dots. Green Chemistry, 16, 3247–3254. doi: 10.1039/c4gc00300d.
  Giraldo, J. P., Landry, M. P., Faltermeier, S. M., McNicholas, T. P., Iverson, N. M., Boghossian, A. A., … Strano, M. S. (2014). Plant nanobionics approach to augment photosynthesis and biochemical sensing. Nature Materials, 13, 400–408. doi: 10.1038/nmat3890.
  Giraldo, J. P., Landry, M. P., Kwak, S. Y., Jain, R. M., Wong, M. H., Ben‐Naim, M., & Strano, M. S. (2015). A ratiometric sensor from single chirality carbon nanotubes: Application to in vivo monitoring. Small, 11, 3973–3984. doi: 10.1002/smll.201403276.
  Gu, X. F., Yang, A. F., Meng, H., & Zhang, J. R. (2005). In vitro induction of tetraploid plants from Zizyphus jujube Mill. Cv. Zhanhua. Plant Cell Reports, 24, 671–676. doi: 10.1007/s00299‐005‐0017‐1.
  Hetherington, A. M., & Woodward, F. I. (2003). The role of stomata in sensing and driving environmental change. Nature, 424, 901–908. doi: 10.1038/nature01843.
  Hussain, H. I., Yi, Z., Rookes, J. E., Kong, L. X., & Cahill, D. M. (2013). Mesoporous silica nanoparticles as a biomolecule delivery vehicle in plants. Journal of Nanoparticle Research, 15, 1676. doi: 10.1007/s11051‐013‐1676‐4.
  Khodakovskaya, M. V., de Silva, K., Biris, A. S., Dervishi, E., & Villagarcia, H. (2012). Carbon nanotubes induce growth enhancement of tobacco cells. ACS Nano, 9, 115–123. doi: 10.1021/nn204643g.
  Khodakovskaya, M. V., Kim, B. S., Kim, J. N., Alimohammadi, M., Dervishi, E., Mustafa, T., & Cernigla, C. E. (2013). Carbon nanotubes as plant growth regulators: Effects on tomato growth, reproductive system, and soil microbial community. Small, 6, 2128–2135. doi: 10.1002/smll.201201225.
  Lichtenthaler, H. K., & Buschmann, C. (2001). Chlorophylls and carotenoids: Measurement and characterization by UV‐VIS spectroscopy. Current Protocols in Food Analytical Chemistry, F4.3.1–F4.3.8.
  Majumdar, S., Almeida, I. C., Arigi, E. A., Choi, H., VerBerkomes, N. C., Trujillo‐Reyes, J., … Gardea‐Torresdey, J. L. (2015). Environmental effects of nanoceria on seed production of common bean (Phaseolus vulgaris): A proteomic analysis. Environmental Science & Technology, 49, 13283–13293. doi: 10.1021/acs.est.5b03452.
  Marmiroli, M., Imperiale, D., Pagano, L., Villani, M., Zappettini, A., & Marmiroli, N. (2015). The proteomic response of Arabidopsis thaliana to cadmium sulfide quantum dots, and its correlation with the transcriptomic response. Frontiers in Plant Science, 6, 1104. doi: 10.3389/fpls.2015.01104.
  Mousavi, S. A. R., Chauvin, A., Pascaud, F., Kellenberger, S., & Farmer, E. E. (2013). Glutamate receptor‐like genes mediate leaf‐to‐leaf wound signaling. Nature, 500, 422–429. doi: 10.1038/nature12478.
  Muller, F., Houben, A., Barker, P. E., Xiao, Y., Kas, J. A., & Melzer, M. (2006). Quantum dots ‐ a versatile tool in plant science? Journal of Nanobiotechnology, 4, 5. doi: 10.1186/1477‐3155‐4‐5.
  Oddo, E., Inzerillo, S., Grisafi, F., Sajeva, M., Salleo, S., & Nardini, A. (2014). Does short‐term potassium fertilization improve recovery from drought stress in laurel? Tree Physiology, 34, 906–913. doi: 10.1093/treephys/tpt120.
  Peng, J., Liu, S., Wang, L., Liu, Z., & He, Y. (2009). Study on the interaction between CdSe quantum dots and chitosan by scattering spectra. Journal of Colloid and Interface Science, 338, 578–583. doi: 10.1016/j.jcis.2009.06.055.
  Probst, C. E., Zrazhevskiy, P., Bagalkot, V., & Gao, X. (2013). Quantum dots as a platform for nanoparticle drug delivery vehicle design. Advanced Drug Delivery Reviews, 65, 703–718. doi: 10.1016/j.addr.2012.09.036.
  Rico, C. M.; Lee, S.C., Rubenecia, R., Mukherjee, A., Hong, J., Peralta‐Videa, J. R., & Gardea‐ Torresdey, J. L. (2014). Cerium oxide nanoparticles impact yield and modify nutritional parameters in wheat (Triticum Aestivum L.). Journal of Agricultural and Food Chemistry, 62, 9669–9675. doi: 10.1021/jf503526r.
  Sabo‐Attwood, T., Unrine, J. M., Stone, J. W., Murphy, C. J., Ghoshroy, S., Blom, D., … Newman, L. A. (2011). Uptake, distribution and toxicity of gold nanoparticles in tobacco (Nicotiana xanthi) seedlings. Nanotoxicol, 6, 353–360. doi: 10.3109/17435390.2011.579631.
  Sparkes, I. A., Runions, J., Kearns, A., & Hawes, C. (2006). Rapid, transient expression of fluorescent fusion proteins in tobacco plants and generation of stably transformed plants. Nature Protocols, 1, 2019–2025. doi: 10.1038/nprot.2006.286.
  Sosan, A., Svitunenko, D., Straltsova, D., Tsiurkina, K., Smolich, I., Lawson, T., … Demidchik, V. (2016). Engineered silver nanoparticles are sensed at the plasma membrane and dramatically modify the physiology pf Arabidopsis thaliana plants. The Plant Journal, 85, 245–257. doi: 10.1111/tpj.13105.
  Sun, D., & Gang, O. (2012). DNA‐Functionalized quantum dots: Fabrication, structural, and physicochemical properties. Langmuir, 29, 7038–7046. doi: 10.1021/la4000186.
  Sun, D., Hussain, H. I., Yi, Z., Rookes, J. E., Kong, L., & Cahill, D. M. (2016). Mesoporus silica nanoparticles enhance seedling growth and photosynthesis in wheat and lupin. Chemosphere, 152, 81–91. doi: 10.1016/j.chemosphere.2016.02.096.
  Torney, F., Trewyn, B. G., Lin, V. S. Y., & Wang, K. (2007). Mesoporous silica nanoparticles deliver DNA and chemicals into plants. Nature Nanotechnology, 2, 95–300. doi: 10.1038/nnano.2007.108.
  Tu, V. A., Kaga, A., Gericke, K. H., Watanabe, N., Narumi, T., Toda, M., … Mase, N. (2014). Synthesis and characterization of quantum dot nanoparticles bound to the plant volatile precursor of hydroxyl‐apo‐10’‐carotenal. The Journal of Organic Chemistry, 79, 6808–6815. doi: 10.1021/jo500605c.
  Yuan, Z., Yang, P., Cao, Y., Yuan, Z., Yang, P., & Cao, Y. (2012). Time‐resolved photoluminescence spectroscopy evaluation of CdTe and CdTe/CdS quantum dots. ISRN Spectrosc, 2012, 1–8. doi: 10.5402/2012/894385.
  Wang, X., Yang, X., Chen, S., Li, Q., Wang, W., Hou, C., … Wang, S. (2016). Zinc oxide nanoparticles affect biomass accumulation and photosynthesis in Arabidopsis. Frontiers in Plant Science, 6, 1243. doi: 10.3389/fpls.2015.01243.
  Wang, Y., & Chen, L. (2011). Quantum dots, lighting up the research and development of nanomedicine. Nanomedicine, 7, 85–402. doi: 10.1016/j.nano.2010.12.006.
  Wong, M. H., Giraldo, J. P., Kwak, S. Y., Koman, V. B., Sinclair, R., Lew, T. T. S., … Strano, M. S. (2016). Nitroaromatic detection and infrared communication from wild‐type plants using plant nanobionics. Nature Materials, 16, 1161–1172. doi: 10.1038/nmat4771.
  Wroblewski, T., Tomczak, A., & Michelmore, R. (2005). Optimization of Agrobacterium‐mediated transietn assays of gene expression in lettuce, tomato and Arabidopsis. Plant Biotechnology Journal, 3, 259–273. doi: 10.1111/j.1467‐7652.2005.00123.x.
  Zhao, M. Z., & Zhu, B. J. (2016). The research and applications of quantum dots as nano‐carriers for targeted drug delivery and caner therapy. Nanoscale Research Letters, 11, 207. doi: 10.1186/s11671‐016‐1394‐9.
  Zuverza‐Mena, N., Armendariz, R., Peralta‐Videa, J. R., & Gardea‐Torresday, J. L. (2016). Effects of silver nanoparticles on radish sprouts: Root growth reduction and modifications in the nutritional value. Frontiers in Plant Science, 7, 90. doi: 10.3389/fpls.2016.00090.
PDF or HTML at Wiley Online Library