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Novel brassinosteroid-modified PEG micelles for controlled release of agrochemicals Javier Pérez Quiñones, Oliver Brueggemann, Mohammad Shahavi, Jørgen Kjems, and Carlos Peniche Covas J. Agric. Food Chem., Just Accepted Manuscript • DOI: 10.1021/acs.jafc.7b05019 • Publication Date (Web): 29 Jan 2018 Downloaded from http://pubs.acs.org on January 31, 2018

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Journal of Agricultural and Food Chemistry

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“Novel brassinosteroid-modified PEG micelles for controlled release of agrochemicals”.

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Javier Pérez Quiñones†,*, Oliver Brüggemann†, Jørgen Kjems‡, Mohammad Shahavi‡, Carlos

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Peniche Covas§,

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†

7

Linz,

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[email protected]

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†,*

Oliver Brüggemann Affiliation: Institute of Polymer Chemistry, Johannes Kepler University Linz, Austria.

Address:

Altenberger

Strasse

69,

4040

Linz,

Austria.

Email:

Javier Pérez Quiñones. Corresponding Author. Email: [email protected] Telephone:

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+4368120399376

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‡

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Molecular Biology and Genetics, University of Aarhus, Aarhus, Denmark. Address: Gustav

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Wiedsvej 14, 8000 Aarhus C., Denmark. Email: [email protected] ; [email protected]

14

‡

15

§

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Ave. Universidad S/N entre G y Ronda, Vedado, 10400 La Habana, Cuba. Email:

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[email protected] ; [email protected]

Jørgen Kjems. Affiliation: Interdisciplinary Nanoscience Center (iNANO) and Department of

Mohammad Shahavi. Email: [email protected] Carlos Peniche Covas. Center of Biomaterials, University of Havana, Havana, Cuba. Address:

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1 ACS Paragon Plus Environment


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ABSTRACT: Two synthetic analogues of brassinosteroids (DI31 and S7) exhibit good plant

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growth enhancer activity. However, their hydrophobicity and quick metabolism in plants have

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limited their application and benefits in agriculture. Our objective was to prepare novel

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brassinosteroid-modified polyethylene glycol (PEG) micelles to achieve controlled release with

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extended stability, while retaining agrochemical activity. Spectroscopic studies confirmed

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quantitative di-substitution of studied PEGs with the brassinosteroids, while elemental analysis

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assessed purity of the synthesized conjugates. Conjugates were also characterized by X-ray

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diffraction and thermal analysis. Dynamic and static light scattering showed stable and

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homogeneous approximately spherical micelles with average hydrodynamic diameters of 22 to 120

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nm and almost neutral Ζ-potential. Spherical 30-140 nm micelles were observed by electron

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microscopy. Sustained in vitro releases at pH 5.5 were extended up to 96 hours. Prepared PEG

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micelles showed good agrochemical activity in radish seeds bioassay and no cytotoxicity to

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HMVEC cell line in MTS test.

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Keywords: agrochemicals, brassinosteroids, controlled release, PEG micelles

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INTRODUCTION

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Brassinosteroids are steroid plant hormones which affect the vegetal physiological processes at very

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low concentrations (in the order of 10-9 mol/L).1,2 In this sense, brassinosteroids together with other

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phytohormones are expressed in the vegetal proteome and regulate growth, xylem differentiation,

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senescence and disease resistance of the plants.1–3 24-epibrassinolide, 28-homobrassinolide and

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different synthetic analogues of brassinosteroids are able to stimulate the germination of seeds, the

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growth and efficiency of crops, and to promote the photosynthesis and senescence.4–6 Furthermore,

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different brassinosteroids and synthetic analogues of brassinosteroids are proposed as plaguicides

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due to their antiecdysteroid activity.7 Some brassinosteroids have been employed as potential

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agrochemicals because very small quantities (from 5 to 100 mg/ha) increase the yield and quality of

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several crops.4,8 This regulatory effect is observed at concentrations one hundred times smaller than

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those required for other vegetal hormones, and the interaction of the brassinosteroids with auxins

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and gibberellins in plants is well documented.9

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DI31 and S7 are two Cuban synthetic analogues of brassinosteroids widely employed as

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agrochemicals in different plantations, with increases in crop yields of 5% to 30%.6,10,11 Particularly,

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DI31 has been commercialized for agriculture as liquid extract at 100 to 1000 ppm in 50 vol.%

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ethanol/water and other additives to ensure stable dispersions (Biobras-16) for almost twenty years.6

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However the described potential benefits are not completely expressed in plants because these

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compounds are quickly metabolized and two or three foliar spray applications are often needed,

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increasing the economic cost of their use.6,10 Another problem with current commercial

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formulations of DI31 is the preparation, storage and transport as hydro-alcoholic suspensions,

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which also increases costs associated to the agrochemical application. It is envisaged that if the

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duration of their action may be prolonged their use as agrochemicals would be made more feasible.

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In addition, preparation of novel solid formulations of DI31 and S7 as potential agrochemical

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formulations is also envisioned.



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In this sense, the covalent linking of diosgenin (the synthetic substrate of DI31) to chitosan would

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allow to prepare pH dependent delivery systems for the controlled release of potential diosgenin-

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based agrochemicals.12 However, these diosgenin-chitosan conjugates were not appropriate for

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foliar spray applications in agriculture because they exhibited low aqueous solubility. Further

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research with the synthetic N,O6-acetyl chitosan allowed the synthesis of steroid-modified chitosan

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conjugates, which formed stable aqueous particle dispersions, suitable for agrochemical

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employment.13 But, the synthesis and purification of the water soluble N,O6-acetyl chitosan involve

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harsh chemicals and conditions, and must be carried out thoroughly (purification with dialysis for

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several days and lyophilization).13 Finally, in vitro agrochemical activity of brassinosteroid-chitosan

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conjugates was not observed when evaluated at the concentrations employed in agriculture (10-6 to

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10-7 mg mL-1).13 Therefore, this approach is not suitable for practical application in agriculture.

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On the other hand, brassinosteroid-modified PEG conjugates were synthesized, which self-assemble

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as micelles in water and are expected to allow sustained release of the brassinosteroids. These novel

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solid formulations should improve bioavailability of the parent brassinosteroids to the crops. The

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biosafety of the envisioned DI31-PEG micelles could be properly evaluated and compared with the

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traditional formulations of Biobras-16, thus also contributing to progress and assess the application

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of nanomaterials in agriculture.14–16

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In the present study, eight novel PEG conjugates of synthetic analogues of brassinosteroids (DI31

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and S7) were synthesized for their application as agrochemicals. Their chemical structure and

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particle properties were characterized. In addition, experiments were conducted to assess the

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stability of particles in aqueous dispersion and the release of the covalently linked brassinosteroids

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over an extended period. Agrochemical activity and safety of prepared PEG conjugates were also

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evaluated with the radish cotyledon test and MTS essay to HMVEC cell line. Thus, the results from

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this study on the PEGylation of agrochemicals to improve their bioavailability to plants might

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supply useful information to further research on nanomaterials in agriculture and preparation of

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novel commercial formulations of agrochemicals such as Biobras-16. 4 ACS Paragon Plus Environment

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MATERIALS AND METHODS

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Materials. Polyethylene glycols (PEG1000, PEG6000, PEG10000 and PEG20000 with Mw/Mn of

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1062/938, 7042/6288, 11038/9822 and 22814/19648 by gel permeation chromatography (GPC)

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according to Sigma Aldrich certificates of analysis), succinic anhydride, 1-Ethyl-3-(3-

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dimethylaminopropyl)carbodiimide hydrochloride (EDC) and 4-dimethylaminopyridine (DMAP)

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were purchased from Sigma-Aldrich (Germany). Brassinosteroid hemisuccinates were synthesized

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by base-catalyzed traditional esterification in pyridine of synthetic analogues of brassinosteroids

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DI31 and S7 (kindly provided by University of Havana, Cuba) with succinic anhydride.17 Cellulose

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dialysis membranes Spectra/Por1 MWCO 1 000 Da (Spectrum, USA) were used as supplied. Other

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chemicals and solvents employed were of the highest grade commercially available and were used

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as received.

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Synthesis of Brassinosteroid-modified PEG Conjugates. Synthetic analogues of brassinosteroids

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DI31 and S7 hemisuccinates were conjugated to PEG1000, PEG6000, PEG10000 and PEG20000

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by reaction with EDC and DMAP in methylene chloride, and the products were recrystallized from

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cold ethanol. A number of preliminary experiments were conducted to select the most appropriate

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synthesis conditions. Briefly, 100 to 2 000 mg (0.1 mmol) of PEG1000, PEG6000, PEG10000 or

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PEG20000 were dissolved in 10 mL of methylene chloride. Then, 55 mg (0.28 mmol) of EDC and

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10 mg (0.08 mmol) of DMAP were added with stirring. Finally, 100 mg (0.2 mmol) of DI31 or S7

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hemisuccinates were added and the solutions were stirred at room temperature in sealed vessels for

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24 hours. Once the synthesis was complete, methylene chloride was evaporated and conjugates

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were recrystallized from cold ethanol (0-5 oC) yielding colorless to white solids with yields from 68

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to 91%.

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Preparation of Brassinosteroid-modified PEG Micelles. The brassinosteroid-modified PEG

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conjugates were dispersed in bi-distilled water or phosphate buffer saline solution (PBS) at pH 7.4,



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vortexed for 2 min. The dispersions were sonicated (Branson Sonifier W-250, Heinemann,

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Germany) with an ultrasonic probe for 10 s at 15 W.

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Molecular Weight Determination of Brassinosteroid-modified PEG Conjugates. The number-

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average molecular weights and polydispersities of PEG and PEG conjugates were determined with

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gel permeation chromatography (GPC) using a Viscotek GPCmax (Malvern, Germany) with a PFG

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column from PSS, 300 x 8 mm2, 5 µm particle size. The samples (100 µL of injection volume, 2

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mg/mL) were eluted with 0.01 mol/L LiBr in (N,N)-dimethylformamide at a flow rate of 0.75

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mL/min at 60 oC. The PEG solutions were filtered through a 0.22 µm microporous nylon film

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syringe filter (Macherey-Nagel, Germany). The molecular weights were determined with a Viscotek

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TDA 305 Triple Detector Array (Malvern, Germany) with integrated refractive index, viscometer

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and light scattering detectors, using a multidetector calibration with polystyrene standard from PSS

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(Malvern, Germany).

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Physicochemical Characterization of Brassinosteroid-modified PEG Conjugates.

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Spectroscopic and Elemental Analyses. Fourier transform infrared (FTIR) spectra of conjugates

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were obtained by the potassium bromide pellet method using a Perkin-Elmer 1720 FTIR

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spectrophotometer (Perkin-Elmer Corporation, USA) with 32 scans and 4 cm-1 resolution. The 1H-

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NMR and

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UK) operating at 500.13 MHz for proton and 125.77 MHz for carbon at 25 oC with concentrations

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of ca. 25 mg/mL in d3-chloroform and analyzed with the VNMRJ software, version 2.2. Elemental

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analyses were performed in triplicate on a Vario MicroCube Analyzer (Elementar Analysensysteme

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GmbH, Germany) with burning temperature of 1150 oC.

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X-ray Diffraction and Thermal Analyses. Wide-angle X-ray diffraction of powered samples was

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performed using a Rigaku SmartLab X-Ray diffractometer (Rigaku, Japan) with Cu Kα radiation

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(40 kV, 180 mA, λ = 0.15418 nm). Data were collected at a scan rate of 5 o/min with a scan angle

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from 4 to 50o. Differential Scanning Calorimetry (DSC) was performed with a Perkin-Elmer

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Differential Scanning Calorimeter Pyris 1 (Perkin-Elmer Instrument Inc., USA) and analyzed with

13

C-NMR spectra were recorded with a Bruker Biospin GmbH spectrometer (Bruker,


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the Pyris 1 software (version 6.0.0.033). DSC studies were conducted using sample weights of

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approximately 5 mg, under nitrogen dynamic flow of 20.0 mL/min and a heating-cooling rate of 10

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ºC/min. Samples were heated and cooled from -30 to 300 ºC, with indium as reference.

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Thermogravimetric Analyses (TGA) were carried out with a Netzsch TG 209 C Iris system. All

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analyses were performed with a 10-15 mg sample in aluminum pans under dynamic nitrogen

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atmosphere between 25 and 601 oC. Experiments were run at a scanning rate of 10 oC/min.

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Physicochemical Characterization of Micelles. Dynamic Light Scattering (DLS) studies on the

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prepared micelles were performed using a Malvern Zetasizer Nano ZS (Malvern, UK) at 25 oC in

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bi-distilled water (ca. 0.5-1 mg/mL) to obtain the hydrodynamic particle size and zeta potential.

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Static Light Scattering (SLS) measurements were performed with a model 5000e compact

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goniometer system (ALV-Laser Vertriebsgesellschaft, Germany), which employed a 100mW

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Nd:YAG laser (Soliton, Germany) operating at a wavelength of 532 nm as the light source.

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Cylindrical quartz cuvettes with an outer diameter of 10 mm (Hellma, Germany) served as

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scattering cells. A C25 Haake thermostat (Haake, Germany) was used to set the temperature to 25

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o

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with angular increments of 5o. All samples were filtered with a 0.45 µm PET syringe filter

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(Macherey-Nagel, Germany) prior to experiment. Critical Micelle Concentration (CMC) of

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micelles was determined with the pyrene probe method by fluorescence spectroscopy (Photon

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Technology Int., Canada) with 337 nm excitation wavelength and emission scan from 320 to 420

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nm.18–20 The size and morphology of dried nanoparticles were examined by transmission electron

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microscopy (TEM) with a Philips CM20 (Philips, Netherlands) operating at 200 kV and scanning

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electron microscopy (SEM) with a Nova NanoSEM 600 (FEI, USA) electron microscope. Each

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sample was stirred for 48 hours in bi-distilled water (ca. 1 mg/mL), probe tip sonicated as

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previously described and a drop of the dispersion was deposited on the carbon plates. The excess

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solution was removed with filter paper and air-dried. SEM samples were sputter-coated with gold

C with a precision of 0.01 oC. Measurements were recorded in an angular range of 15o ˂ θ ˂ 150o



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(AJA Sputtering System, UK). TEM samples were negative stained with uranyl acetate solution

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(1%). All determinations were performed in triplicate.

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In Vitro Drug Release Studies. In vitro release of DI31 and S7 from brassinosteroid-modified

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PEG micelles was assessed by UV detection (Genesys 10 UV-Vis Spectrophotometer, Thermo

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Spectronic, USA) at 275 nm in PBS solution (pH 5.5). 10 mg of brassinosteroid-modified PEG

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micelles dispersed in PBS solution at pH 5.5 (5 mL) were placed in dialysis bags containing PBS

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solution at pH 5.5 (40 mL) and dialyzed against the release media at 30oC with constant agitation at

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100 rpm. The entire media were removed at determined time intervals, and replaced with the same

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volume of fresh media.13 The amount of brassinosteroids released was determined by UV

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spectrophotometry (Figure S1, supporting information) and calculated from a previously obtained

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calibration curve. These studies were conducted in triplicate for each sample.

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Biological Activity. Radish Cotyledon Test for Agrochemical Activity. The radish (Raphanus

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sativus) test was employed in order to detect plant growth activity. This bioassay is based on the

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increased weight of treated radish’s cotyledons (auxin type activity). Radish seeds previously

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sterilized by sodium hypochlorite treatment were germinated over wet filter paper in the dark at 25

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o

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DI31- or S7-modified PEGs micelles in water (10-1 to 10-7 mg/mL); DI31 or S7 solutions (10-1

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mg/mL in ethanol/water solution 50% (v/v) and diluted up to 10-2-10-7 mg/mL); PEGs aqueous

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solution (10-1 to 10-7 mg/mL) or pure water (control). After 72 hours, cotyledon weights were

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measured. These studies were conducted in triplicate for each sample and concentration (10

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cotyledons each run). Cell Culture and Cytotoxicity. Human microvascular endothelial cells

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(HMVEC) were cultured in Endothelial Cell Basal Medium-2 (Lonza) supplemented with EGM-2

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Single Quots (Lonza) containing h-EGF, VEGF, h-FGF-B, R3-IGF-1, hydrocortisone, and FBS (2%

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final concentration). The cytotoxicity of all the samples was tested on HMVEC cells by MTS assay

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(CellTiter 96® Aqueous One Solution Reagent). HMVEC cells in full growth media were seeded in

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96-well plate (1 × 104 cells/well). After cells were attached on the plate, the medium was changed to

C, for 72 hours.13 Cotyledons were separated from hypocotyls, weighted and treated with 5 mL of


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serum free media and DI31, S7 and brassinosteroid-modified PEG micelles dispersions in PBS at

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different concentrations were added. After incubation for 48 h, the medium was replaced with full

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growth medium, 20 µL MTS reagent was added to each well and incubated for additional 3 h before

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measuring the absorbance at 490 nm using a 96-well plate reader (µQuant, Bio-Tek Instruments

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Inc., USA). These studies were also conducted in triplicate.

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Statistical Analysis. Data are expressed as mean ± standard deviation (SD) for three replicates.

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One-way ANOVA was used to analyze the significant differences between the groups, followed by

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Tukey test for between-group comparisons, multiple comparisons procedure and Kruskall-Wallis

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test at 95% confidence by Statgraphics Plus 5.1, Professional edition, licensed to JKU University.

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Probabilities of p< 0.05 were considered statistically significant. No significant different means are

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represented with same letter (p> 0.05).

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RESULTS AND DISCUSSION

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Synthesis of brassinosteroid-modified PEG conjugates and preparation of micelles. PEGylation

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of proteins and hydrophilic drugs is widely used to improve stability and biocompatibility while

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increasing clearance times from the body, in order to achieve a better therapeutic effect.20

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Researchers have been working on the preparation of PEG-stabilized lipid nanoparticles, mostly

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formed by physical or emulsion methods, for application as pharmaceutical carriers. In the present

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study, four different PEGs (PEG1000, PEG6000, PEG10000 and PEG20000) were used to

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synthesize the brassinosteroid-modified PEG conjugates (Figure 1). Our results show that higher

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yields, purity and quantitative esterification (di-substitution) were obtained when using DMAP in

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enough quantity (three fold catalytic amounts) and EDC as carbodiimide instead of N,N´-

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dicyclohexylcarbodiimide. These hydrophobically-modified brassinosteroid-disubstituted PEG

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conjugates formed self-assembled micelles in aqueous solution upon probe tip sonication, due to

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their amphiphilic character.

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Figure 1 appears here. 9 ACS Paragon Plus Environment


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Table 1 shows the steroid weight content of brassinosteroid-disubstituted PEG conjugates and their

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molecular weights as estimated by GPC chromatography. Relative error of GPC molecular weights

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refers to estimated values from the analytical certificates as supplied by Sigma Aldrich.

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Table 1 appears here.

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GPC chromatography results showed that quantitative esterification (p< 0.05) of the studied PEG

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with the DI31 and S7 hemisuccinates was achieved. Besides, no significant changes in polymer

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chain sizes (Mw/Mn) were observed after the esterification reactions.

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Spectroscopic and Elemental Analysis Studies. FTIR and NMR spectroscopies are useful tools to

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elucidate chemical structures and to follow up chemical reactions through detection of new

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functional groups created from the reactants. In this research, FTIR spectra of the brassinosteroid-

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modified PEG conjugates showed a distinctive absorption peak around 1732-1734 cm-1, related to

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carbonyl stretching of ester bonds formed (Figure 2). Conversion of free carboxylic groups from

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brassinosteroid hemisuccinates (observed at 177 ppm) into PEG esters (observed at 171-172 ppm)

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were also confirmed by

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spectroscopy allowed quantification of the esterification degree in the synthesized conjugates, by

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comparing the intensity of the signals around 0.5-1.2 ppm, assigned to methyl groups of

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brassinosteroid moieties, to the intensity of the signal at 3.5 ppm, assigned to the -CH2-CH2-O-

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repeating unit of PEGs (Figure 3, Figure S3 of supporting information). Thus, the successful and

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quantitative di-esterification of PEGs with brassinosteroid hemisuccinates was confirmed. On the

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other hand, nitrogen was not found by elemental analysis in the brassinosteroid-modified PEG

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conjugates and good agreement between theoretical and experimental composition was achieved

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(data not shown). Therefore, recrystallization from cold ethanol was proven a simple and effective

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method to purify the PEG conjugates, by removal of formed EDC urea, DMAP and any unreacted

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EDC, PEG or brassinosteroid hemisuccinate.

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Figure 2 appears here.

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13

C-NMR spectroscopy (Figure S2, supporting information). 1H-NMR


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X-ray Diffraction and Thermal Studies. X-ray diffraction and DSC are useful methods to

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investigate the physicochemical properties and stability of obtained conjugates in solid state. X-ray

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diffraction patterns of pure PEGs and synthesized conjugates are almost identical, with almost same

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crystallinity (Figure S4, supporting information). It means that the crystal structure of PEGs was not

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affected upon esterification, probably due to the insignificant influence of the intermolecular

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hydrogen bond on very large PEG chains. It seems that large PEG chains are able to accommodate

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the hydrophobic brassinosteroid moieties without perturbation of the PEG crystalline structure.

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DSC and TGA studies of brassinosteroid-modified PEG conjugates and the pure PEGs showed that

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the thermal properties remained unaffected after esterification. Endothermic processes related with

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melting of PEG chains were observed at 38 to 64 oC, while exothermic processes related with

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thermal pyrolysis started at 225 to 300 oC (Figure S5, supporting information), with a weight loss of

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92 to 98 % (Figure S6, supporting information).

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Size, Zeta Potential, Critical Micelle Concentration Measurements and Morphological

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Analysis. DLS studies conducted in triplicate yielded average hydrodynamic diameters in bi-

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distilled water between 22 to 122 nm with polydispersity indices of 0.37 to 0.68 and very low zeta

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potentials (-4.9 to 0 mV) (Table 2). The very low zeta potential values observed in almost all

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conjugates might be associated with the presence of small quantities of negatively charged

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brassinosteroids adsorbed in PEG micelle surfaces that were not separated during purification of

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PEG conjugates by recrystallization in ethanol. Hydrodynamic diameters remained unaltered after

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storing PEG micelle dispersions in bi-distilled water at room temperature for 30 days (Table 2),

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showing stability in water. The ratios between the radius of gyration and hydrodynamic radius

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(Rg/Rh) extrapolated at zero concentrations from Zimm plots of recorded SLS data of

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brassinosteroid-modified PEG micelles in bi-distilled water were close to 0.775, this value

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corresponding to a homogeneous hard sphere (Table 2).20 SEM and TEM analysis were employed to

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obtain morphological information on the dried micelles. SEM micrographs showed almost spherical

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individual micelles and few aggregates with sizes lower than 200 nm (Figure 4). TEM images 11 ACS Paragon Plus Environment


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showed almost spherical micelles with mean diameters of 30-140 nm (Figure 5). The size measured

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by the DLS technique are the hydrodynamic diameters of hydrated micelles, while SEM and TEM

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micrographs depicted the size in the dried state; thus, particle size is slightly smaller in electron

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microscopy techniques. Critical micelle concentration values of prepared micelles in bi-distilled

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water determined by fluorescence spectroscopy with pyrene probe are also shown in Table 2. CMC

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is the minimal concentration needed to form micelles in water and CMC values increased with

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increasing the PEG chain length for each brassinosteroid. This is the result of the increase of the

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hydrophilic-lipophilic balance and the increase of the hydrophilic PEG shell in the flower-like

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micelle conformation (brassinosteroid moieties accommodated in the hydrophobic micelle core and

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the looped hydrophilic PEG chains pointing out and forming the micelle shell).20

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Table 2 appears here.

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295


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In Vitro Drug Release Studies. The release profiles of brassinosteroid-modified PEG micelles at

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30 oC in PBS (pH 5.5), expressed as percentage of cumulative release against time, are shown in

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Figure 6. These studies were performed at pH 5.5 because acidic conditions are needed to achieve

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the hydrolysis of the ester linkage and the release of brassinosteroids DI31 and S7.13 The PEG

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micelles presented sustained release with almost constant release rates during the first 8 h. Release

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was dependent on the particle sizes, ranging from 91% to 96% (PEG10000-DI31, PEG20000-DI31,

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PEG6000-S7, PEG10000-S7 and PEG20000-S7) after 96 h for the bigger aggregates; while smaller

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micelles released ca. 72% to 76% after 96 h (PEG1000-DI31, PEG6000-DI31 and PEG1000-S7).

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Thus, by selecting the proper PEG it is possible to regulate the release rate and the maximum

305

quantity released of agrochemicals DI31 and S7. Moreover, in all cases releases were extended for

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long time (4 days), showing that prepared PEG micelles are promising candidates for a controlled

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and extended delivery of the studied brassinosteroids to plants.

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Figure 6 appears here. 12 ACS Paragon Plus Environment

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Biological Activity. Figure 7 shows the plant growth biological activity of the synthetic

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brassinosteroid analogues DI31 and S7, and brassinosteroid-modified PEG micelles in the radish

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cotyledons bioassay.

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Plant growth stimulator activities of the synthetic brassinosteroids DI31 and S7 are very similar,

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showing best results at 10-3 and 10-4 mg/mL concentrations, but an almost doubled cotyledon weight

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was reached as compared to control with the lowest concentrations (10-6 and 10-7 mg/mL).

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PEG20000 exerts a very slight inhibitory effect at higher (10-1 to 10-2 mg/mL) concentrations; but

317

no activity is observed at lower concentration (10-3 to 10-7 mg/mL) (data not shown). The other

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PEGs did not show any agrochemical effect on the studied concentrations (10-1 to 10-7 mg/mL)

319

(data not shown). The prepared brassinosteroid-modified PEG micelles in aqueous solution

320

presented stimulatory activities at all concentrations (weight increased approximately one to four

321

times compared to control). Particularly, PEG1000-DI31 and PEG1000-S7 showed a similar

322

stimulatory activity at lowest concentrations (10-6 to 10-7 mg/mL) when compared with parent DI31

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and S7. The other PEG conjugates exhibited slightly less activity at lowest concentrations (10-6 to

324

10-7 mg/mL) when compared with pure DI31 and S7; but they are still suitable to exert a good

325

stimulatory effect on vegetal growth. These results are promising for a practical agrochemical

326

application of the synthesized brassinosteroid-modified PEG conjugates (parent DI31 and S7

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brassinosteroids are usually applied at concentrations of 10-5 to 10-7 mg mL-1 in agriculture).

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Cytotoxicity to the HMVEC cell line is an initial in vitro test for biocompatibility of different

329

potential biomaterials (drug delivery systems based on nanoparticles, micelles, nanocomposites,

330

hydrogels and implants). Synthetic brassinosteroids and brassinosteroid-modified PEG micelles

331

were not cytotoxic to the HMVEC cell line (HMVEC cell proliferation in treated samples higher

332

than 80% compared to control) (Figure 8). In summary, the results achieved suggest that the

333

synthesized brassinosteroid-modified PEG micelles might be good candidates to be applied as

334

agrochemicals. 13 ACS Paragon Plus Environment


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335


336

Abbreviations Used

337

Polyethylene glycol (PEG); Human microvascular endothelial cells (HMVEC); Gel permeation

338

chromatography (GPC); 1-Ethyl-3-(3-dimethylaminopropyl)carbodiimide hydrochloride (EDC);

339

4-dimethylaminopyridine (DMAP); Phosphate buffer saline (PBS); Fourier transform infrared

340

(FTIR); Nuclear magnetic resonance (NMR); Differential scanning calorimetry (DSC); Dynamic

341

light scattering (DLS); Static light scattering (SLS); Critical micelle concentration (CMC);

342

Transmission electron microscopy (TEM); Scanning electron microscopy (SEM); standard

343

deviation (SD); Thermogravimetric analyses (TGA).

344

Acknowledgements

345

Claudia Schmidt is acknowledged for hosting a five-months-stay of Javier Perez Quinones at the

346

Department of Chemistry at Paderborn University, and helpful discussion and comments on the

347

manuscript. Susanne Keuker-Baumman is acknowledged for elemental analysis and differential

348

scanning calorimetry measurements at Paderborn University. Authors wish to thank to Jacques

349

Chevallier and Karen E. Thomsen by electron microscopy and staining training at Aarhus

350

University. Jens-Erik Jørgensen and Niels N. Sandal are also acknowledged by X-ray diffraction

351

measurements and guidance on seed germination, respectively at Aarhus University.

352

Notes

353

The authors declare no competing financial interest.

354

Funding

355

This study was supported by the Deutscher Akademischer Austauschdienst (DAAD) (A/12/96452),

356

the Coimbra Group, and Erasmus Mundus, trough research grants to Javier Pérez-Quiñones.

357

Supporting Information description

358

Figure S1. UV spectra of DI31 (A) and S7 (B) at 1 mg/mL in PBS (pH 5.5).

359

Figure S2. Carbon nuclear magnetic resonance (13C-NMR) spectra of DI31 hemisuccinate (A), S7

360

hemisuccinate (B), PEG1000-DI31 conjugate (C) and PEG1000-S7 conjugate (D). 14 ACS Paragon Plus Environment

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361

Figure S3. Proton nuclear magnetic resonance (1H-NMR) spectra of DI31 hemisuccinate (A) and

362

S7 hemisuccinate (B).

363

Figure S4. X-ray diffraction patterns of PEG and PEG conjugates of DI31 and S7.

364

Figure S5. DSC curves of PEG and PEG conjugates of DI31 and S7.

365

Figure S6. TGA curves of PEG and PEG conjugates of DI31 and S7.

366 367 368 369

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(2) Choe, S. Brassinosteroid Biosynthesis and Metabolism. Plant Hormones 2010, B, 156–178.

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(3) Schmitz, G. J. H.; Andrade, J. M.; Valle, T. L.; Labate, C. A.; Nascimento, J. R. O.

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Comparative proteome analysis of the tuberous roots of six cassava (Manihot esculenta) varieties

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reveals proteins related to phenotypic traits. J. Agric. Food Chem. 2016, 10.1021/acs.jafc.5b05585

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(4) Hola, D.; Rothova, O.; Kocova, M.; Kohout, L.; Kvasnica, M. The effect of brassinosteroids

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on the morphology, development and yield of field-grown maize. Plant Growth Regulation 2010,

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61, 29–43.

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(5) Talaat, N. B.; Abdallah, A. M. Effect of 28-homobrassinolide and 24-epibrassinolide on the

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growth, productivity and nutritional value of two faba bean (Vicia faba L.) cultivars. Archives of

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Agronomy and Soil Science 2009, 1–21.

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(6) Nuñez, M. V.; Robaina, C.; Coll F. Synthesis and practical application of brassinosteroid

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analogs. In Brassinosteroids: Bioactivity and Crop Productivity; Hayat, S., Ahmad, A., Eds.;

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Springer Science+Media Business, B. V.: The Netherlands, 2003, 100–111.

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(7) Bhardwaj, R.; Khurma, U. R.; Ohri, P.; Sohal, S. K. Influence of 28-homobrassinolide on

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development of second-stage juveniles of Meloidogyne incognita (Kofoid & White) chitwood.

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Indian Journal of Nematology 2007, 37, 188–191.



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(8) Sirhindi, G.; Kumar, S.; Bhardwaj, R.; Kumar, M. Effects of 24-epibrassinolide and 28-

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homobrassinolide on the growth and antioxidant enzyme activities in the seedlings of Brassica

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juncea L. Physiology and Molecular Biology of Plants 2009, 15, 335–341.

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(9) Kuppusamy, K.; Walcher, C.; Nemhauser, J. Cross-regulatory mechanisms in hormone signaling. Plant Molecular Biology 2009, 69, 375–381.

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(10) Terry, E.; Diaz de Armas, M. M.; Ruiz, J.; Tejeda, T.; Zea, M. E.; Camacho-Ferre, F. Effects

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of different bioactive products used as growth stimulators in lettuce crops (Lactuca sativa L.).

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Journal of Food, Agriculture & Environment 2012, 10, 386–389.

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(11) Serrano, Y. C.; Fernandez, R. R.; Pineda, F. R.; Pelegrin, L. T. S.; Fernandez, D. G.; Cepero,

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M. C. G. Synergistic Effect of Low Doses of X-rays and Biobras-16 on Yield and Its Components

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in Tomato (Solanum lycopersicum L.) Plants. American Journal of Bioscience and Bioengineering

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2015, 3, 197–202.

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(12) Quiñones, J. P.; Szopko, R.; Schmidt, C.; Covas, C. P. Novel drug delivery systems: Chitosan

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conjugates covalently attached to steroids with potential anticancer and agrochemical activity.

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Carbohydr. Polym. 2011, 84, 858–864.

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(13) Quiñones, J. P.; Gothelf, K. V.; Kjems, J.; Caballero, A. M. H.; Schmidt, C.; Covas, C. P.

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N,O6-partially acetylated chitosan nanoparticles hydrophobically-modified for controlled release of

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steroids and vitamin E. Carbohydr. Polym. 2013, 91, 143–151.

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(14) Gogos, A.; Knauer, K.; Bucheli, T. D. Nanomaterials in Plant Protection and Fertilization:

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Current State, Foreseen Applications, and Research Priorities. J. Agric. Food Chem. 2012, 60,

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9781−9792

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(15) Kookana, R. S.; Boxall, A. B. A.; Reeves, P. T.; Ashauer, R.; Beulke, S.; Chaudhry, Q.;

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Cornelis, G.; Fernandes, T. F.; Gan, J.; Kah, M.; Lynch, I.; Ranville, J.; Sinclair, C.; Spurgeon, D.;

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Tiede, K.; Van den Brink, P. J. Nanopesticides: Guiding Principles for Regulatory Evaluation of

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Environmental Risks. J. Agric. Food Chem. 2014, 62, 4227−4240

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(16) Chen, H.; Seiber, J. N.; Hotze, M. ACS Select on Nanotechnology in Food and Agriculture: 16 ACS Paragon Plus Environment

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A Perspective on Implications and Applications. J. Agric. Food Chem. 2014, 62, 1209–1212. (17) Abe, T.; Hasunuma, K.; Kurokawa, M. Vitamin E orotate and a method of producing the same. US: 3,944,550, 1976.

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(18) Ashjari, M.; Khoee, S.; Mahdavian, A. R.; Rahmatolahzadeh, R. Self-assembled

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nanomicelles using PLGA–PEG amphiphilic block copolymer for insulin delivery: a

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physicochemical investigation and determination of CMC values. J. Mater. Sci.: Mater. Med. 2012,

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23, 943–53.

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(19) Sadoqi, M.; Lau-Cam, C. A.; Wu, S. H. Investigation of the micellar properties of the

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tocopheryl polyethylene glycol succinate surfactants TPGS 400 and TPGS 1000 by steady state

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fluorometry. J. Colloid Interface Sci. 2009, 333, 585–589.

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(20) de Graaf, A. J.; Boere, K. W. M.; Kemmink, J.; Fokkink, R. G.; van Nostrum, C. F.; Rijkers,

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D. T. S.; van der Gucht, J.; Wienk, H.; Baldus, M.; Mastrobattista, E.; Vermonden, T.; Hennink, W.

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E. Looped Structure of Flowerlike Micelles Revealed by 1H NMR Relaxometry and Light

425

Scattering. Langmuir 2011, 27, 9843–9848.

426 427

Figure Captions

428 429 430

Figure 1. Chemical reaction of brassinosteroid-modified PEG conjugates formation, their structures and schematic representation of obtained flower-like PEG micelles.

431

Figure 2. Fourier transform infrared (FTIR) spectra of PEG conjugates of DI31 (A) and S7 (B).

432

Figure 3. Proton nuclear magnetic resonance (1H-NMR) spectra of DI31 hemisuccinate and their

433

PEG1000 conjugate (A) and S7 hemisuccinate and their PEG1000 conjugate (B).

434

Figure 4. Scanning electron microscopy photographs of DI31-modified PEG micelles PEG1000-

435

DI31 (A), PEG6000-DI31 (B), PEG10000-DI31 (C), PEG20000-DI31 (D) and S7-

436

modified PEG micelles PEG1000-S7 (E), PEG6000-S7 (F), PEG10000-S7 (G),

437

PEG20000-S7 (H). 17 ACS Paragon Plus Environment


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438

Figure 5. Transmission electron microscopy photographs of DI31-modified PEG micelles

439

PEG1000-DI31 (A), PEG6000-DI31 (B), PEG10000-DI31 (C), PEG20000-DI31 (D) and

440

S7-modified PEG micelles PEG1000-S7 (E), PEG6000-S7 (F), PEG10000-S7 (G),

441

PEG20000-S7 (H).

442

Figure 6. In vitro cumulative release of DI31 from DI31-modified PEG micelles (A) and of S7

443

from S7-modified PEG micelles (B). The data represent the mean ± standard deviation of

444

three independent experiments (n = 3).

445

Figure 7. Biological activity as plant growth regulator of DI31 and S7 brassinosteroids (A),

446

PEG1000 conjugates of DI31 and S7 (B), other PEG conjugates of DI31 (C), and other

447

PEG conjugates of S7 (D) at 25 oC. (*) Not measured because cotyledons died as result of

448

high ethanol content. Data are the mean ± standard deviation of three independent

449

experiments (n = 3). Concentrations were measured for pure brassinosteroids (DI31 and

450

S7) or PEG conjugates (in the PEG conjugates), respectively. Concentrations of DI31 and

451

S7 in their PEG conjugates can be calculated from the steroid weight content showed in

452

Table 1.

453

Figure 8. Human microvascular endothelial cell (HMVEC) cytotoxic activity expressed as % cell

454

viability of DI31 and their PEG micelles (A), and S7 and their PEG micelles (B).

455

Concentrations are measured for pure brassinosteroid (DI31 and S7) or PEG conjugates

456

(in PEG conjugates). Concentrations of DI31 and S7 in their PEG conjugates can be

457

calculated from the steroid weight content shown in Table 1.

458 459

Table 1. Steroid weight contents (wt. %), molecular weights measured by GPC chromatography and

460

relative errors (error %) Mna Samples

wt. %

-

Mnc

error

(g/mol)

(g/mol)

(%)

938

-

-2.5

Mw/Mn (g/mol)

PEG1000

Mnb a

915

1.12


Page 19 of 36


PEG1000-DI31

45.0

1939

1.13

-

1997

-2.9

PEG1000-S7

47.0

1973

1.12

-

2022

-2.4

-

6240

1.10

6288

-

-0.8

PEG6000-DI31

13.3

7231

1.11

-

7347

-1.6

PEG6000-S7

13.7

7335

1.10

-

7372

-0.5

PEG10000

-

9801

1.14

9822

-

-0.2

PEG10000-DI31

8.8

10828

1.15

-

10881

-0.5

PEG10000-S7

9.0

10868

1.13

-

10906

-0.3

-

19339

1.16

19648

-

-1.6

PEG20000-DI31

4.4

20383

1.14

-

20707

-1.6

PEG20000-S7

4.5

20424

1.17

-

20732

-1.5

PEG6000

PEG20000

461

a

462

chromatography.

463

b

Number-average molecular weights determined from Sigma Aldrich analytical certificate.

464

c

Molecular weights calculated for di-substituted PEG conjugate.

Number-average molecular weights and polydispersities (Mw/Mn) measured by GPC



Page 20 of 36

Table 2. Hydrodynamic parameters and critical micelle concentration of brassinosteroid-PEG micellesa Samples PEG1000-DI31

a

Diameter1 (nm) 79 ± 1a

diameter2 (nm)

PDI1

PDI2

80 ± 2a

0.70 ± 0.05

0.68 ± 0.02

Rg/Rh1 ζ-potential1 CMC1 (mg/mL) (mV) 0.842

0i

0.07l

PEG6000-DI31

105.2 ± 0.3b 105.7 ± 0.8b 0.593±0.006 0.601± 0.002

0.901

-4.9 ± 0.9j

0.11

PEG10000-DI31

22.1 ± 0.4c

23.1 ± 0.6c

0.92 ± 0.02

0.96 ± 0.03

0.887

0i

0.23

PEG20000-DI31

120 ± 4d

125 ± 3d

0.65 ± 0.05

0.52 ± 0.05

0.906

-3.0 ± 0.7k

0.37

PEG1000-S7

56.0 ± 0.6e

55 ± 1e

0.37 ± 0.05

0.37 ± 0.04

0.875

-4.6 ± 0.8j

0.07l

PEG6000-S7

32 ± 1f

33.2 ± 0.7f

0.48 ± 0.08

0.54 ± 0.02

0.893

-2.3 ± 0.6k

0.15

PEG10000-S7

73 ± 3g

75 ± 2g

0.7 ± 0.1

0.67 ± 0.08

0.915

-4.3 ± 0.3j

0.30

PEG20000-S7

112 ± 3h

110 ± 1h

0.53 ± 0.05

0.56 ± 0.01

0.859

-2.1 ± 0.4k

0.48

Data represented as the mean ± standard deviation (except for Rg/Rh and CMC). Same letters

indicate no significant differences (p > 0.05). The numbers 1 and 2 in row of labels indicate samples measured freshly prepared or 30 days after dispersion in bi-distilled water, respectively.

Figure 1. Chemical reaction of brassinosteroid-modified PEG conjugates formation; their structures and schematic representation of obtained flower-like PEG micelles.


Page 21 of 36


Figure 2. Fourier transform infrared (FTIR) spectra of PEG conjugates of DI31 (A) and S7 (B).



Page 22 of 36

Figure 3. Proton nuclear magnetic resonance (1H-NMR) spectra of DI31 hemisuccinate and their PEG1000 conjugate (A) and S7 hemisuccinate and their PEG1000 conjugate (B).


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Figure 4. Scanning electron microscopy photographs of DI31-modified PEG micelles PEG1000DI31 (A), PEG6000-DI31 (B), PEG10000-DI31 (C), PEG20000-DI31 (D) and S7-modified PEG micelles PEG1000-S7 (E), PEG6000-S7 (F), PEG10000-S7 (G), PEG20000-S7 (H).

Figure 5. Transmission electron microscopy photographs of DI31-modified PEG micelles PEG1000-DI31 (A), PEG6000-DI31 (B), PEG10000-DI31 (C), PEG20000-DI31 (D) and S7modified PEG micelles PEG1000-S7 (E), PEG6000-S7 (F), PEG10000-S7 (G), PEG20000-S7 (H).



Page 24 of 36

Figure 6. In vitro cumulative release of DI31 from DI31-modified PEG micelles (A) and of S7 from S7-modified PEG micelles (B). The data represent the mean ± standard deviation of three experiments (n = 3).


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Figure 7. Biological activity as plant growth regulator of DI31 and S7 brassinosteroids (A), PEG1000 conjugates of DI31 and S7 (B), other PEG conjugates of DI31 (C), and other PEG conjugates of S7 (D) at 25 oC. (*) Not measured because cotyledons died as result of high ethanol content. Data are the mean ± standard deviation of three independent experiments (n = 3). Concentrations were measured for pure brassinosteroids (DI31 and S7) or PEG conjugates (in the PEG conjugates), respectively. Concentrations of DI31 and S7 in their PEG conjugates can be calculated from the steroid weight content shown in Table 1.



Page 26 of 36

Figure 8. Human microvascular endothelial cell (HMVEC) cytotoxic activity expressed as % cell viability of DI31 and their PEG micelles (A), and S7 and their PEG micelles (B). Data are the mean ± standard deviation of three independent experiments (n = 3). Concentrations are measured for pure brassinosteroid (DI31 and S7) or PEG conjugates (in PEG conjugates). Concentrations of DI31 and S7 in their PEG conjugates can be calculated from the steroid weight content shown in Table 1.


Page 27 of 36


Graphic for table of contents.



Figure 1. Chemical reaction of brassinosteroid-modified PEG conjugates formation, their structures and schematic representation of obtained flower-like PEG micelles. 261x109mm (96 x 96 DPI)

ACS Paragon Plus Environment

Page 28 of 36

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Figure 2. Fourier transform infrared (FTIR) spectra of PEG conjugates of DI31 (A) and S7 (B). 110x196mm (96 x 96 DPI)



Figure 3. Proton nuclear magnetic resonance (1H-NMR) spectra of DI31 hemisuccinate and their PEG1000 conjugate (A) and S7 hemisuccinate and their PEG1000 conjugate (B). 184x209mm (300 x 300 DPI)


Page 30 of 36

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Figure 4. Scanning electron microscopy photographs of DI31-modified PEG micelles PEG1000-DI31 (A), PEG6000-DI31 (B), PEG10000-DI31 (C), PEG20000-DI31 (D) and S7-modified PEG micelles PEG1000-S7 (E), PEG6000-S7 (F), PEG10000-S7 (G), PEG20000-S7 (H). 264x122mm (150 x 150 DPI)



Figure 5. Transmission electron microscopy photographs of DI31-modified PEG micelles PEG1000-DI31 (A), PEG6000-DI31 (B), PEG10000-DI31 (C), PEG20000-DI31 (D) and S7-modified PEG micelles PEG1000-S7 (E), PEG6000-S7 (F), PEG10000-S7 (G), PEG20000-S7 (H). 269x133mm (150 x 150 DPI)


Page 32 of 36

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Figure 6. In vitro cumulative release of DI31 from DI31-modified PEG micelles (A) and of S7 from S7modified PEG micelles (B). The data represent the mean ± standard deviation of three independent experiments (n = 3). 128x215mm (96 x 96 DPI)



Figure 7. Biological activity as plant growth regulator of DI31 and S7 brassinosteroids (A), PEG1000 conjugates of DI31 and S7 (B), other PEG conjugates of DI31 (C), and other PEG conjugates of S7 (D) at 25 oC. (*) Not measured because cotyledons died as result of high ethanol content. Data are the mean ± standard deviation of three independent experiments (n = 3). Concentrations were measured for pure brassinosteroids (DI31 and S7) or PEG conjugates (in the PEG conjugates), respectively. Concentrations of DI31 and S7 in their PEG conjugates can be calculated from the steroid weight content showed in Table 1. 161x135mm (600 x 600 DPI)


Page 34 of 36

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Figure 8. Human microvascular endothelial cell (HMVEC) cytotoxic activity expressed as % cell viability of DI31 and their PEG micelles (A), and S7 and their PEG micelles (B). Concentrations are measured for pure brassinosteroid (DI31 and S7) or PEG conjugates (in PEG conjugates). Concentrations of DI31 and S7 in their PEG conjugates can be calculated from the steroid weight content shown in Table 1. 127x214mm (96 x 96 DPI)



Graphic for table of contents. 204x109mm (96 x 96 DPI)


Page 36 of 36

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