Zein Nanoparticles Uptake and Translocation in ... - ACS Publications

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Zein Nanoparticles Uptake and Translocation in Hydroponically Grown Sugarcane Plants Alisha Prasad, Carlos E Astete, Andreea Elena Bodoki, McKenzie Windham, Ede Bodoki, and Cristina M. Sabliov J. Agric. Food Chem., Just Accepted Manuscript • DOI: 10.1021/acs.jafc.7b02487 • Publication Date (Web): 02 Aug 2017 Downloaded from http://pubs.acs.org on August 3, 2017

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Journal of Agricultural and Food Chemistry is published by the American Chemical Society. 1155 Sixteenth Street N.W., Washington, DC 20036 Published by American Chemical Society. Copyright © American Chemical Society. However, no copyright claim is made to original U.S. Government works, or works produced by employees of any Commonwealth realm Crown government in the course of their duties.

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Journal of Agricultural and Food Chemistry

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Zein Nanoparticles Uptake and Translocation in Hydroponically

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Grown Sugarcane Plants

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Alisha Prasada, Carlos E. Astetea, Andreea E. Bodokib, McKenzie Windhama, Ede Bodokic*, and

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Cristina. M. Sabliova*

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a

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Agricultural and Mechanical College, and LSU AgCenter, Baton Rouge, LA 70803, USA

Department of Biological and Agricultural Engineering, Louisiana State University

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b

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University of Medicine and Pharmacy, 12, Ion Creanga St., 400010, Cluj-Napoca, Romania

General and Inorganic Chemistry Department, Faculty of Pharmacy, “Iuliu Hatieganu”

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c

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Medicine and Pharmacy, 4, Louis Pasteur St., 400349, Cluj-Napoca, Romania

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* Co-corresponding authors

Analytical Chemistry Department, Faculty of Pharmacy, “Iuliu Hatieganu” University of

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Key words: Zein nanoparticles, sugarcane, translocation, nanopesticides, zein-FITC

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Abstract

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The main objective of this study was to investigate the uptake and translocation of positively

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charged zein nanoparticles (ZNPs) in hydroponically grown sugarcane plants. Fluorescent ZNPs

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(spherical and measuring an average diameter 135±3 nm) were synthesized by emulsion-

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diffusion method from FITC-tagged zein. Fluorescent measurement following digestion of plant

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tissue indicated that sugarcane roots had a significant adhesion of ZNPs, 342.5±24.2 µg

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NPs/mg of dry matter, while sugarcane leaves contained a very limited amount, 12.9±1.2 µg

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NPs/mg dry matter for high dose after 12 hrs. Confocal microscopy studies confirmed

ACS Paragon Plus Environment

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presence of fluorescent ZNPs in the epidermis and endodermis of the root system. Given their

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ability to adhere to roots for extended periods of time, ZNPs are proposed as effective delivery

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systems for agrochemicals to sugarcane plants, but more studies are needed to identify effect of

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nanoparticle exposure to health of the plant.

32 33

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Until recently, nanomaterials were developed nearly exclusively for biomedical,

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electronics, and energy fields applications.1 As a result of significant achievements in

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these fields, research in the agricultural sector has begun to eagerly approach

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nanoparticles as pesticide vectors, while investigating their impact on the environment.2,3

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Current methods for administering pesticides such as manual backpack type spraying,

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self-propelled row-crop spraying, aerosol spraying etc. have several limitations most

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critically including the direct human and environment exposure, and uneven distribution

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of pesticides. Despite toxicity concerns, pesticides might undergo microbial degradation,

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degradation by sun exposure, and hydrolysis, such that a very low percentage of the

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sprayed chemical is delivered to the plant and insect.4,5 It has been reported that more

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than 90% of the conventional agrochemicals are lost and never reach the desired target

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due to application or environmental conditions. To maintain the agrochemical

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concentration over the minimum effective concentration, periodical applications are

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required. The process increases the cost, and excess of agrochemicals end up in the soil

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and water with negative environmental impacts.6,7

INTRODUCTION

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Nanomaterials offer a safer and more efficient method for pesticide delivery.8

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Pesticide concentration can be decreased by delivering it with engineered bio-adhesive


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nanodelivery systems and by decreasing degradation of the nano-entrapped pesticide

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during application.9,10,11 Some relevant examples of nanoparticles studied for

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agrochemical delivery are polymeric nanoparticles synthesized out of sodium alginate,10

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Eudragit S100,11 chitosan,12 poly(lactic acid),13 as well as solid lipid nanoparticles.14

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These nanoparticles showed promising results for agrochemical delivery relative to

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traditional formulations, but limited data about polymeric nanoparticle interaction with

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plants is available in the literature.

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Current research on nanoparticle-plant interaction has focused mainly on metallic

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nanoparticles such as TiO2, ZnO2, fullerenes, quantum dots, gold, silver, copper and iron

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oxide, showing evidence of nanomaterial uptake by plants through seeds, roots and

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leaves.15,16,17,18,19 Several factors affect uptake and translocation of metallic nanomaterials

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such as particle’s size, shape, dissolution rate, agglomeration state, surface chemistry,20

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but equally important, plant species has an enormous impact on whether or not the

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nanoparticle will translocate from the root system into the stems and leaves.21

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It has been established that metallic nanoparticles enter a plant through the roots.22 The

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soil is a place in which nanoparticles are likely to accumulate as a result of run-off,

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making the roots a potential primary organ of exposure, while some nanoparticles can be

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applied directly to the leaves by broadcasting.23 Through the roots, plants interact strongly

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with the environment, absorbing mainly water and nutrients, but also contaminants and

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other materials. Absorption of nanoparticles through the root is controlled based mainly

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on nanoparticle size, given the pore diameter of the root of 5 to 20 nm.1 The macro-

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structure of the root is responsible for the extent of nanoparticle absorption: plants with

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more lateral roots provide additional surface area for more absorption.24 Nanomaterials


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properties may also modulate interaction with the roots because of the possibility of

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mechanical adhesion, electrostatic adsorption, and hydrophobic affinity, which has been

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responsible for accumulation on the surface and in the epidermis of the root.25 Part of the

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reason nanoparticles are thought to adhere to the root surface is because of the release of

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exudates and mucilage, which is made up of amino acids and organic acids providing an

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overall negative surface charge.26, 27

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A subject of keen interest is nanoparticle translocation, which refers to the particle

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moving from the roots into the shoots. After the nanoparticles diffuse through the cell

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wall pores of the roots, there are two paths they can follow to move from the epidermis to

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the endodermis: the apoplastic and symplastic route, similar to ways in which water and

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nutrients are moved through the plant.28 To the authors’ knowledge, there is little-to-no

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literature available on the uptake and translocation of polymeric nanoparticles (NPs) in

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plants. The aim of this research was to study root nanoparticle uptake and translocation in

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sugarcane plants exposed to NPs hydroponically.

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The selected polymer for nanoparticle synthesis was zein, a protein derived from

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Zea mays L (corn). Zein is water insoluble, amphiphilic, renewable, and biodegradable29

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and hence preferred for agrochemical delivery. Zein nanoparticles (ZNPs) have shown

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potential applications as carrier vehicle for delivery of drugs,8,30 gene delivery,31 as well

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as surface coatings,32 etc. Various geometries of ZNPs have been explored such as

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spheres, hexagonal, sponge, lamellar phases, and nano-tubes etc. by tailoring the

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microphase behavior of zein based on ethanol: water ratio.33 This is possible since zein

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contains over 50% hydrophobic regions, and has the ability to form nanoparticles of

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specific surface chemistry due to its unique amino acid distribution.34 Various methods of


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ZNPs synthesis, their stability, and surface properties have already been well established.

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The main objective of this study was to synthesize ZNPs via emulsion-diffusion method

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using didodecyldimethylammonium bromide (ddMab) as a positively charged stabilizing

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surfactant and particles were fluorescently tagged to allow for qualitative and quantitative

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tracking in the plant. Fluorescence microscopy, fluorescence measurement, and

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transmission microscopy were employed to quantitatively and qualitatively assess

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nanoparticle association with sugarcane roots and their translocation to the leaves of the

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plant. The understanding of the ZNPs uptake, translocation, and distribution in plants

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would help in the development of zein nanoparticles as a potent carrier vehicle for

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delivery of agrochemicals with a broader impact both in food and agricultural

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applications. This work will advance the fields of controlled release materials, plant

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pathology, materials science/engineering, and agricultural chemistry.

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Materials. Zein powder, fluorescein isothiocyanate (FITC), dichloromethane (DCM), and

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didodecyldimethylammonium bromide (ddMab) were purchased from Sigma-Aldrich

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(Sigma

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dimethylformamide (DMF), triethylamine and acetone was purchased from Fisher

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Chemical (Fisher Scientific International, Fairlawn, NJ). Ultrapure (type 1, 18.2 MΩ)

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water was obtained using a NANOpure Diamond™ machine (Barnstead International, IA,

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USA). Hoagland medium was purchased from plant media bioWORLD (bioWORLD,

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Ohio, USA). Sugarcane plants (HoCP09-804 variety) were provided by Louisiana State

MATERIALS AND METHODS

Chemical

Co.

Ltd.,

St.

Louis,

MO).

Sodium


hydroxide

(NaOH),

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University’s Agricultural Center and collected from Sugar Research Station. All other

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chemicals used were of laboratory grade.

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Fluorescent tagging of zein via FITC. In order to track the ZNPs through the

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roots and leaves, prior to preparation of ZNPs, zein was conjugated with a fluorophore

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(FITC). Accurately weighed zein powder (3 g) was dissolved in 100 ml DMF, in a 250 ml

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round bottom flask completely covered with aluminum foil to avoid light exposure. Next,

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0.1 mL of triethylamine and 80 mg of FITC were slowly added under gentle stirring at

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room temperature. Stirring was stopped after 24 hours and the sample was concentrated

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(approximately 25 ml) under high vacuum conditions. Subsequently, the concentrated

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suspension was washed and precipitated with DCM (50 ml) and the cycle was performed

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for six times to ensure free dye removal. Finally, the precipitate was dried in an oven

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(40°C) under high vacuum for 24 hours and kept at 4oC in an amber container until

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further use. The absence of free FITC residue was further confirmed by HPLC analysis.

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ZNPs preparation. Emulsion-diffusion method was used to prepare fluorescent

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ZNPs. The organic phase was prepared by dissolving equal amounts of zein powder (200

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mg) and zein with covalently tagged FITC (zein-FITC, 200 mg) in 10 ml of an acetone:

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water solution (8:2, v/v) and kept under rigorous stirring in a closed container covered

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with aluminum foil until complete dissolution. The aqueous phase was prepared by

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mixing ddMab (57.8 mg) in 100 ml of water under stirring settings. Finally, the emulsion

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was prepared by adding the organic phase drop wise into the aqueous phase under

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rigorous stirring, and passing the resulting emulsion through a microfluidizer (M-110P,

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Microfluidics, MA, USA) at a pressure of 30,000 psi, thrice. Next, the acetone was

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evaporated with a rotavapor Buchi R-300 (Buchi Analytical Inc., DE, USA) under


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vacuum and the suspension was brought with additional water to a final volume of 110

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ml. For each experiment a freshly prepared fluorescent ZNPs suspension was used.

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Transmission electron microscopy. Transmission electron microscopy (TEM)

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was performed on a JEOL 1400, (Jeol USA Inc, Peabody, MA). A droplet of freshly

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prepared ZNPs suspension was placed over a carbon grid and allowed to dry. The dried

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patch was then stained with 2% uranyl acetate solution as a contrast agent and the excess

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droplet was removed with a filter paper. The dried film was then loaded into a TEM

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sample holder for visualization.

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Dynamic light scattering. Dynamic light scattering (DLS) was used to determine

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the mean particle size, zeta potential and polydispersity index (PDI) of the ZNPs using a

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ZetasizerNano (Malvern Instruments, Southborough, MA) at a pH of 6. The analysis was

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performed at 25 ºC on a freshly prepared ZNPs sample, and the values of size and

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polydispersity index were determined using mono-modal distribution.

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Exposure of sugarcane plants to ZNPs in a hydroponic system. To study the

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uptake and translocation of ZNPs in sugarcane plants of HoCP09-804 variety, the 2

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weeks old-plants were transferred in a hydroponic culture systems (1.6g/L) spiked with

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varying doses of freshly prepared fluorescent ZNPs. In this study, the plants were exposed

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to two doses containing 0.88 mg/mL low dose (LD), and 1.75mg/mL high dose (HD) of

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fluorescent ZNPs. The concentrations selected were correlated to relevant pesticide

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concentrations when entrapped in zein nanoparticles. Control plants were not exposed to

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ZNPs and contained only water and Hoagland hydroponic solution (1:1 ratio). All

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experiments were conducted in triplicates in beakers completely covered with aluminum

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foils to prevent evaporation and the photobleaching of the fluorescently labeled NPs. All


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plants were grown in a controlled environment chamber (Intellus Environmental

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Controller, Percival: Perry, IA) at 27°C under cool, white fluorescent light with 16/8 h

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light/dark cycle. Samples of roots and leaves were collected at 2, 4, 6, 8, 10, and 12 hours

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using separate batches of sugarcane plants for roots and leaves. The collected samples

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were freeze dried using a lyophilizer FreeZone 2.5 Plus (Labconco Corp., Kansas City,

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MO) for further study.

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Confocal laser-scanning microscopy. Confocal laser scanning microscopy

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(CLSM) performed on Leica TCS SP2 microscope (Leica Inc, Buffalo Grove, IL) was

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used to visualize the fluorescent ZNPs translocation in sugarcane root samples immersed

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into Hoagland solution spiked with two different doses of ZNPs. The accumulation of

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ZNPs in the sugarcane root system was detected based on the fluorescence imparted by

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FITC. CLSM images were obtained at Excitation/Emission: 480-495nm and 550/560nm.

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Images were taken under Leica 20x (NA 0·75) oil immersion objective lens. Argon laser

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were used for NR and GFP excitation. Pictures have brightness and contrast

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enhancements. Thin sections of the root samples were placed on a glass slide covered

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with a cover slip and few drops of distilled water was added to keep it hydrated. For

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imaging the internal root structure, a standard protocol was followed which involved four

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basic steps: tissue fixation, dehydration, rehydration and successive embedment of the

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root.35 Root samples from control, low dose and high dose were washed with PBS and

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subjected to 2.3 M sucrose gradient infiltration overnight at 4°C. The root samples where

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then placed on the cryostat and sectioned with optimum cutting temperature (OCT). The

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thinner cut sections were then placed on the glass slide with the sections facing upright.


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For each study three root tips were examined from one treatment group before

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representative images were selected.

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Uptake, biotransformation, and bioaccumulation of fluorescent ZNPs.

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Pulverized freeze dried root (3mg) and leaves (2mg) samples, originating from the low

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dose, high dose and control plant batches (5 samples/batch), were weighed in amber

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Eppendorf tubes. Next, 1 ml of 1N NaOH was added to each tube and placed into an

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agitated water bath at 70° C for 30 minutes. Then, the samples were centrifuged at

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50,000xg for 15 minutes at 16° C. After digestion, the obtained clear supernatant’s

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fluorescence was determined in triplicate on 100 µl sample aliquots in a dark 96-well

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plate using a Cell Imaging Multi-Mode reader Cytation 3 (BioTek Instruments, Inc.,

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Winooski, VT) equipped with Gen5 data analysis software. Three samples of 1N NaOH

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(100 µl) were used as blank. The nanoparticle concentration in each sample was

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calculated using a standard curve developed by measuring the fluorescence (λexc = 495

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nm; λem = 525 nm) of FITC-labeled ZNPs at known concentrations digested with 1N

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NaOH.

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Statistical analysis. One way analysis of means using all pairs Tukey Kramer

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HSD were performed using JMP Pro 13 software (SAS Institute, Cary NC) to

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demonstrate significant difference (p

Zein Nanoparticles Uptake and Translocation in ... - ACS Publications

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