Novel Brassinosteroid-Modified Polyethylene Glycol Micelles for

Article Cite This: J. Agric. Food Chem. 2018, 66, 1612−1619

pubs.acs.org/JAFC

Novel Brassinosteroid-Modified Polyethylene Glycol Micelles for Controlled Release of Agrochemicals Javier Pérez Quiñones,*,† Oliver Brüggemann,† Jørgen Kjems,‡ Mohammad Hassan Shahavi,‡,§ and Carlos Peniche Covas∥ †

Institute of Polymer Chemistry, Johannes Kepler University Linz, 4040 Linz, Austria Interdisciplinary Nanoscience Center (iNANO) and Department of Molecular Biology and Genetics, University of Aarhus, 8000 Aarhus, Denmark § Faculty of Engineering Modern Technologies, Amol University of Special Modern Technologies (AUSMT), Amol, Iran ∥ Center of Biomaterials, University of Havana, 10400 Havana, Cuba ‡

S Supporting Information *

ABSTRACT: Two synthetic analogues of brassinosteroids (DI31 and S7) exhibit good plant growth enhancer activity. However, their hydrophobicity and quick metabolism in plants have limited their application and benefits in agriculture. Our objective was to prepare novel brassinosteroid-modified polyethylene glycol (PEG) micelles to achieve controlled release with extended stability while retaining agrochemical activity. Spectroscopic studies confirmed quantitative disubstitution of studied PEGs with the brassinosteroids, while elemental analysis assessed purity of the synthesized conjugates. Conjugates were also characterized by X-ray diffraction and thermal analysis. Dynamic and static light scattering showed stable and homogeneous approximately spherical micelles with average hydrodynamic diameters of 22−120 nm and almost neutral ζ potential. Spherical 30−140 nm micelles were observed by electron microscopy. Sustained in vitro releases at pH 5.5 were extended up to 96 h. Prepared PEG micelles showed good agrochemical activity in the radish seed bioassay and no cytotoxicity to the human microvascular endothelial cell line in the MTS test. KEYWORDS: agrochemicals, brassinosteroids, controlled release, PEG micelles

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INTRODUCTION Brassinosteroids are steroid plant hormones that affect the vegetal physiological processes at very low concentrations (in the order of 10−9 mol/L).1,2 In this sense, brassinosteroids together with other phytohormones are expressed in the vegetal proteome and regulate growth, xylem differentiation, senescence, and disease resistance of the plants. 1−3 24epibrassinolide, 28-homobrassinolide, and different synthetic analogues of brassinosteroids are able to stimulate the germination of seeds and the growth and efficiency of crops and to promote photosynthesis and senescence.4−6 Furthermore, different brassinosteroids and synthetic analogues of brassinosteroids are proposed as plaguicides as a result of their antiecdysteroid activity.7 Some brassinosteroids have been employed as potential agrochemicals because very small quantities (from 5 to 100 mg/ha) increase the yield and quality of several crops.4,8 This regulatory effect is observed at concentrations 100 times smaller than those required for other vegetal hormones, and the interaction of the brassinosteroids with auxins and gibberellins in plants is well-documented.9 DI31 and S7 are two Cuban synthetic analogues of brassinosteroids widely employed as agrochemicals in different plantations, with increases in crop yields of 5−30%.6,10,11 Particularly, DI31 has been commercialized for agriculture as a liquid extract at 100−1000 ppm in 50 vol % ethanol/water and other additives to ensure stable dispersions (Biobras-16) for almost 20 years.6 However, the described potential benefits are not completely expressed in plants because these compounds © 2018 American Chemical Society

are quickly metabolized, and two or three foliar spray applications are often needed, increasing the economic cost of their use.6,10 Another problem with current commercial formulations of DI31 is the preparation, storage, and transport as hydro-alcoholic suspensions, which also increases costs associated with the agrochemical application. It is envisaged that, if the duration of their action may be prolonged, their use as agrochemicals would be made more feasible. In addition, the preparation of novel solid formulations of DI31 and S7 as potential agrochemical formulations is also envisioned. In this sense, the covalent linking of diosgenin (the synthetic substrate of DI31) to chitosan would allow for the preparation of pH-dependent delivery systems for the controlled release of potential diosgenin-based agrochemicals.12 However, these diosgenin−chitosan conjugates were not appropriate for foliar spray applications in agriculture because they exhibited low aqueous solubility. Further research with the synthetic N,O6acetyl chitosan allowed for the synthesis of steroid-modified chitosan conjugates, which formed stable aqueous particle dispersions, suitable for agrochemical employment.13 However, the synthesis and purification of water-soluble N,O6-acetyl chitosan involve harsh chemicals and conditions and must be carried out thoroughly (purification with dialysis for several Received: Revised: Accepted: Published: 1612

October 27, 2017 January 25, 2018 January 29, 2018 January 29, 2018 DOI: 10.1021/acs.jafc.7b05019 J. Agric. Food Chem. 2018, 66, 1612−1619

Article

Journal of Agricultural and Food Chemistry days and lyophilization).13 Finally, in vitro agrochemical activity of brassinosteroid−chitosan conjugates was not observed when evaluated at the concentrations employed in agriculture (from 10−6 to 10−7 mg/mL).13 Therefore, this approach is not suitable for practical application in agriculture. On the other hand, brassinosteroid-modified polyethylene glycol (PEG) conjugates were synthesized, which self-assemble as micelles in water and are expected to allow for sustained release of the brassinosteroids. These novel solid formulations should improve bioavailability of the parent brassinosteroids to the crops. The biosafety of the envisioned DI31−PEG micelles could be properly evaluated and compared to the traditional formulations of Biobras-16, thus also contributing to progress and assess the application of nanomaterials in agriculture.14−16 In the present study, eight novel PEG conjugates of synthetic analogues of brassinosteroids (DI31 and S7) were synthesized for their application as agrochemicals. Their chemical structure and particle properties were characterized. In addition, experiments were conducted to assess the stability of particles in aqueous dispersion and the release of the covalently linked brassinosteroids over an extended period. Agrochemical activity and safety of prepared PEG conjugates were also evaluated with the radish cotyledon test and MTS assay to the human microvascular endothelial cell (HMVEC) line. Thus, the results from this study on the PEGylation of agrochemicals to improve their bioavailability to plants might supply useful information to further research on nanomaterials in agriculture and preparation of novel commercial formulations of agrochemicals, such as Biobras-16.

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Molecular Weight Determination of Brassinosteroid-Modified PEG Conjugates. The number-average molecular weights and polydispersities of PEG and PEG conjugates were determined with GPC using a Viscotek GPCmax (Malvern, Germany) with a PFG column from PSS, 300 × 8 mm2, 5 μm particle size. The samples (100 μL of injection volume, 2 mg/mL) were eluted with 0.01 mol/L LiBr in N,N-dimethylformamide at a flow rate of 0.75 mL/min at 60 °C. The PEG solutions were filtered through a 0.22 μm microporous nylon film syringe filter (Macherey-Nagel, Germany). The molecular weights were determined with a Viscotek TDA 305 triple detector array (Malvern, Germany) with integrated refractive index, viscometer, and light scattering detectors, using a multidetector calibration with polystyrene standard from PSS (Malvern, Germany). Physicochemical Characterization of Brassinosteroid-Modified PEG Conjugates. Spectroscopic and Elemental Analyses. Fourier transform infrared (FTIR) spectra of conjugates were obtained by the potassium bromide pellet method using a PerkinElmer 1720 FTIR spectrophotometer (PerkinElmer, Inc., Waltham, MA, U.S.A.) with 32 scans and 4 cm−1 resolution. The 1H and 13C nuclear magnetic resonance (NMR) spectra were recorded with a Bruker Biospin GmbH spectrometer (Bruker, U.K.) operating at 500.13 MHz for proton and 125.77 MHz for carbon at 25 °C with concentrations of ca. 25 mg/mL in chloroform-d3 and analyzed with the VNMRJ software, version 2.2. Elemental analyses were performed in triplicate on a vario MICRO cube analyzer (Elementar Analysensysteme GmbH, Germany) with a burning temperature of 1150 °C. X-ray Diffraction (XRD) and Thermal Analyses. Wide-angle XRD of powered samples was performed using a Rigaku SmartLab X-ray diffractometer (Rigaku, Japan) with Cu Kα radiation (40 kV, 180 mA, and λ = 0.15418 nm). Data were collected at a scan rate of 5°/min with a scan angle from 4° to 70°. Differential scanning calorimetry (DSC) was performed with a PerkinElmer Differential Scanning Calorimeter Pyris 1 (PerkinElmer, Inc., Waltham, MA, U.S.A.) and analyzed with the Pyris 1 software (version 6.0.0.033). DSC studies were conducted using sample weights of approximately 5 mg, under nitrogen dynamic flow of 20.0 mL/min and a heating−cooling rate of 10 °C/min. Samples were heated and cooled from −30 to 300 °C, with indium as a reference. Thermogravimetric analyses (TGAs) were carried out with a Netzsch TG 209 C Iris system. All analyses were performed with a 10−15 mg sample in aluminum pans under a dynamic nitrogen atmosphere between 25 and 601 °C. Experiments were run at a scanning rate of 10 °C/min. Physicochemical Characterization of Micelles. Dynamic light scattering (DLS) studies on the prepared micelles were performed using a Malvern Zetasizer Nano ZS (Malvern, U.K.) at 25 °C in bidistilled water (ca. 0.5−1 mg/mL) to obtain the hydrodynamic particle size and ζ potential. Static light scattering (SLS) measurements were performed with a model 5000e compact goniometer system (ALV-Laser Vertriebsgesellschaft, Germany), which employed a 100 mW Nd:YAG laser (Soliton, Germany) operating at a wavelength of 532 nm as the light source. Cylindrical quartz cuvettes with an outer diameter of 10 mm (Hellma, Germany) served as scattering cells. A C25 Haake thermostat (Haake, Germany) was used to set the temperature to 25 °C with a precision of 0.01 °C. Measurements were recorded in an angular range of 15° < θ < 150°, with angular increments of 5°. All samples were filtered with a 0.45 μm PET syringe filter (Macherey-Nagel, Germany) prior to experiment. The critical micelle concentration (CMC) of micelles was determined with the pyrene probe method by fluorescence spectroscopy (Photon Technology International, Inc., London, Ontario, Canada) with a 337 nm excitation wavelength and emission scan from 320 to 420 nm.18−20 The size and morphology of dried nanoparticles were examined by transmission electron microscopy (TEM) with a Philips CM20 (Philips, Netherlands) operating at 200 kV and scanning electron microscopy (SEM) with a Nova NanoSEM 600 (FEI, Hillsboro, OR, U.S.A.) electron microscope. Each sample was stirred for 48 h in bidistilled water (ca. 1 mg/ mL), probe tip sonicated as previously described, and a drop of the dispersion was deposited on the carbon plates. The excess solution was removed with filter paper and air-dried. SEM samples were sputter-

MATERIALS AND METHODS

Materials. PEGs [PEG1000, PEG6000, PEG10000, and PEG20000 with Mw/Mn of 1062/938, 7042/6288, 11 038/9822, and 22 814/19 648 by gel permeation chromatography (GPC) according to Sigma-Aldrich certificates of analysis], succinic anhydride, 1-ethyl-3(3-(dimethylamino)propyl)carbodiimide hydrochloride (EDC), and 4dimethylaminopyridine (DMAP) were purchased from Sigma-Aldrich (Germany). Brassinosteroid hemisuccinates were synthesized by basecatalyzed traditional esterification in pyridine of synthetic analogues of brassinosteroids DI31 and S7 (kindly provided by the University of Havana, Havana, Cuba) with succinic anhydride.17 Cellulose dialysis membranes Spectra/Por 1 with a molecular weight cut-off (MWCO) of 1000 Da (Spectrum, Rancho Dominguez, CA, U.S.A.) were used as supplied. Other chemicals and solvents employed were of the highest grade commercially available and were used as received. Synthesis of Brassinosteroid-Modified PEG Conjugates. Synthetic analogues of brassinosteroid DI31 and S7 hemisuccinates were conjugated to PEG1000, PEG6000, PEG10000, and PEG20000 by reaction with EDC and DMAP in methylene chloride, and the products were recrystallized from cold ethanol. A number of preliminary experiments were conducted to select the most appropriate synthesis conditions. Briefly, 100−2000 mg (0.1 mmol) of PEG1000, PEG6000, PEG10000, or PEG20000 was dissolved in 10 mL of methylene chloride. Then, 55 mg (0.28 mmol) of EDC and 10 mg (0.08 mmol) of DMAP were added with stirring. Finally, 100 mg (0.2 mmol) of DI31 or S7 hemisuccinate was added, and the solutions were stirred at room temperature in sealed vessels for 24 h. Once the synthesis was complete, methylene chloride was evaporated and conjugates were recrystallized from cold ethanol (0−5 °C), yielding colorless to white solids with yields from 68 to 91%. Preparation of Brassinosteroid-Modified PEG Micelles. The brassinosteroid-modified PEG conjugates were dispersed in bidistilled water or phosphate buffer saline (PBS) solution at pH 7.4 and vortexed for 2 min. The dispersions were sonicated (Branson Sonifier W-250, Heinemann, Germany) with an ultrasonic probe for 10 s at 15 W. 1613

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Figure 1. Chemical reaction of brassinosteroid-modified PEG conjugate formation, their structures, and schematic representation of obtained flowerlike PEG micelles.

Table 1. Steroid Weight Contents (wt %), Molecular Weights Measured by GPC, and Relative Errors (Error, %) sample

wt %

Mna (g/mol)

Mw/Mna

PEG1000 PEG1000−DI31 PEG1000−S7 PEG6000 PEG6000−DI31 PEG6000−S7 PEG10000 PEG10000−DI31 PEG10000−S7 PEG20000 PEG20000−DI31 PEG20000−S7

45.0 47.0 13.3 13.7 8.8 9.0 4.4 4.5

915 1939 1973 6240 7231 7335 9801 10828 10868 19339 20383 20424

1.12 1.13 1.12 1.10 1.11 1.10 1.14 1.15 1.13 1.16 1.14 1.17

Mnb (g/mol)

Mnc (g/mol)

938 1997 2022 6288 7347 7372 9822 10881 10906 19648 20707 20732

error (%) −2.5 −2.9 −2.4 −0.8 −1.6 −0.5 −0.2 −0.5 −0.3 −1.6 −1.6 −1.5

a

Number-average molecular weights and polydispersities (Mw/Mn) measured by GPC. bNumber-average molecular weights determined from SigmaAldrich analytical certificate. cMolecular weights calculated for the disubstituted PEG conjugate.

coated with gold (AJA Sputtering System, U.K.). TEM samples were negative-stained with uranyl acetate solution (1%). All determinations were performed in triplicate. In Vitro Drug Release Studies. In vitro release of DI31 and S7 from brassinosteroid-modified PEG micelles was assessed by ultraviolet (UV) detection (Genesys 10 UV−vis spectrophotometer, Thermo Scientific Spectronic, Waltham, MA, U.S.A.) at 275 nm in PBS solution (pH 5.5). A total of 10 mg of brassinosteroid-modified PEG micelles dispersed in PBS solution at pH 5.5 (5 mL) were placed in dialysis bags containing PBS solution at pH 5.5 (40 mL) and dialyzed against the release media at 30 °C with constant agitation at 100 rpm. The entire media were removed at determined time intervals and replaced with the same volume of fresh media.13 The amount of brassinosteroids released was determined by UV spectrophotometry (Figure S1 of the Supporting Information) and calculated from a previously obtained calibration curve. These studies were conducted in triplicate for each sample. Biological Activity. Radish Cotyledon Test for Agrochemical Activity. The radish (Raphanus sativus) test was employed to detect plant growth activity. This bioassay is based on the increased weight of cotyledons of the treated radish (auxin-type activity). Radish seeds previously sterilized by sodium hypochlorite treatment were germinated over wet filter paper in the dark at 25 °C for 72 h.13 Cotyledons were separated from hypocotyls, weighted, and treated with 5 mL of DI31- or S7-modified PEG micelles in water (10−1−10−7 mg/mL), DI31 or S7 solution (10−1 mg/mL in 50% (v/v) ethanol/ water solution and diluted up to 10−2−10−7 mg/mL), PEG aqueous solution (10−1−10−7 mg/mL), or pure water (control). After 72 h, cotyledon weights were measured. These studies were conducted in triplicate for each sample and concentration (10 cotyledons each run). Cell Culture and Cytotoxicity. HMVECs were cultured in endothelial cell basal medium-2 (Lonza) supplemented with EGM-2

SingleQuots (Lonza) containing h-EGF, VEGF, h-FGF-B, R3-IGF-1, hydrocortisone, and FBS (2% final concentration). The cytotoxicity of all of the samples was tested on HMVECs by the MTS assay (CellTiter 96 Aqueous One Solution Reagent). HMVECs in full growth media were seeded in a 96-well plate (1 × 104 cells/well). After cells were attached on the plate, the medium was changed to serumfree media, and DI31, S7, and brassinosteroid-modified PEG micelle dispersions in PBS at different concentrations were added. After incubation for 48 h, the medium was replaced with full growth medium and 20 μL of MTS reagent was added to each well and incubated for an additional 3 h before measuring the absorbance at 490 nm using a 96-well plate reader (μQuant, Bio-Tek Instruments, Inc., Winooski, VT, U.S.A.). These studies were also conducted in triplicate. Statistical Analysis. Data are expressed as the mean ± standard deviation (SD) for three replicates. One-way analysis of variance (ANOVA) was used to analyze the significant differences between the groups, followed by the Tukey test for between group comparisons, multiple comparison procedure, and Kruskall−Wallis test at 95% confidence by Statgraphics Plus 5.1, Professional edition, licensed to Johannes Kepler University Linz. Probabilities of p < 0.05 were considered statistically significant. No significant different means are represented with the same letter (p > 0.05).

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RESULTS AND DISCUSSION

Synthesis of Brassinosteroid-Modified PEG Conjugates and Preparation of Micelles. PEGylation of proteins and hydrophilic drugs is widely used to improve stability and biocompatibility while increasing clearance times from the body, to achieve a better therapeutic effect.20

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PEG10000, and PEG20000) were used to synthesize the brassinosteroid-modified PEG conjugates (Figure 1). Our results show that higher yields, purity, and quantitative esterification (disubstitution) were obtained when using DMAP in enough quantity (3-fold catalytic amounts) and EDC as carbodiimide, instead of N,N′-dicyclohexylcarbodiimide. These hydrophobically modified brassinosteroid-disubstituted PEG conjugates formed self-assembled micelles in aqueous solution upon probe tip sonication as a result of their amphiphilic character. Table 1 shows the steroid weight content of brassinosteroiddisubstituted PEG conjugates and their molecular weights as estimated by GPC. The relative error of GPC molecular weights refers to estimated values from the analytical certificates as supplied by Sigma-Aldrich. GPC results showed that quantitative esterification (p < 0.05) of studied PEG with the DI31 and S7 hemisuccinates was achieved. Besides, no significant changes in polymer chain sizes (Mw/Mn) were observed after the esterification reactions. Spectroscopic and Elemental Analysis Studies. FTIR and NMR spectroscopies are useful tools to elucidate chemical structures and to follow up chemical reactions through detection of new functional groups created from the reactants. In this research, FTIR spectra of the brassinosteroid-modified PEG conjugates showed a distinctive absorption peak around 1732−1734 cm−1, related to carbonyl stretching of ester bonds formed (Figure 2). Conversion of free carboxylic groups from brassinosteroid hemisuccinates (observed at 177 ppm) into PEG esters (observed at 171−172 ppm) was also confirmed by 13 C NMR spectroscopy (Figure S2 of the Supporting Information). 1H NMR spectroscopy allowed for quantification of the esterification degree in the synthesized conjugates by comparing the intensity of the signals around 0.5−1.2 ppm, assigned to methyl groups of brassinosteroid moieties, to the intensity of the signal at 3.5 ppm, assigned to the −CH2− CH2−O− repeating unit of PEGs (Figure 3 and Figure S3 of the Supporting Information). Thus, the successful and quantitative diesterification of PEGs with brassinosteroid hemisuccinates was confirmed. On the other hand, nitrogen was not found by elemental analysis in the brassinosteroidmodified PEG conjugates, and good agreement between theoretical and experimental composition was achieved (data not shown). Therefore, recrystallization from cold ethanol was proven a simple and effective method to purify the PEG conjugates by removal of formed EDC urea, DMAP, and any unreacted EDC, PEG, or brassinosteroid hemisuccinate. XRD and Thermal Studies. XRD and DSC are useful methods to investigate the physicochemical properties and stability of obtained conjugates in the solid state. XRD patterns of pure PEGs and synthesized conjugates are almost identical, with almost the same crystallinity (Figure S4 of the Supporting Information). It means that the crystal structure of PEGs was not affected upon esterification, probably as a result of the insignificant influence of the intermolecular hydrogen bond on very large PEG chains. It seems that large PEG chains are able to accommodate the hydrophobic brassinosteroid moieties without perturbation of the PEG crystalline structure. DSC and TGA studies of brassinosteroid-modified PEG conjugates and the pure PEGs showed that the thermal properties remained unaffected after esterification. Endothermic processes related with melting of PEG chains were observed at 38−64 °C, while exothermic processes related with thermal pyrolysis started at 225−300 °C (Figure S5 of the Supporting Information), with a

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

Figure 3. 1H NMR spectra of (A) DI31 hemisuccinate and its PEG1000 conjugate and (B) S7 hemisuccinate and its PEG1000 conjugate.

Researchers have been working on the preparation of PEGstabilized lipid nanoparticles, mostly formed by physical or emulsion methods, for application as pharmaceutical carriers. In the present study, four different PEGs (PEG1000, PEG6000, 1615

DOI: 10.1021/acs.jafc.7b05019 J. Agric. Food Chem. 2018, 66, 1612−1619

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Journal of Agricultural and Food Chemistry Table 2. Hydrodynamic Parameters and CMC of Brassinosteroid−PEG Micellesa sample PEG1000−DI31 PEG6000−DI31 PEG10000−DI31 PEG20000−DI31 PEG1000−S7 PEG6000−S7 PEG10000−S7 PEG20000−S7

diameter1 (nm) 79 105.2 22.1 120 56.0 32 73 112

± ± ± ± ± ± ± ±

1a 0.3 b 0.4 c 4d 0.6 e 1f 3g 3h

diameter2 (nm) 80 105.7 23.1 125 55 33.2 75 110

± ± ± ± ± ± ± ±

2a 0.8 b 0.6 c 3d 1e 0.7 f 2g 1h

PDI1 0.70 0.593 0.92 0.65 0.37 0.48 0.7 0.53

± ± ± ± ± ± ± ±

0.05 0.006 0.02 0.05 0.05 0.08 0.1 0.05

PDI2

Rg/Rh1

± ± ± ± ± ± ± ±

0.842 0.901 0.887 0.906 0.875 0.893 0.915 0.859

0.68 0.601 0.96 0.52 0.37 0.54 0.67 0.56

0.02 0.002 0.03 0.05 0.04 0.02 0.08 0.01

ζ potential1 (mV) 0i −4.9 ± 0.9 0i −3.0 ± 0.7 −4.6 ± 0.8 −2.3 ± 0.6 −4.3 ± 0.3 −2.1 ± 0.4

j k j k j k

CMC1 (mg/mL) 0.07 l 0.11 0.23 0.37 0.07 l 0.15 0.30 0.48

Data represented as the mean ± SD (except for Rg/Rh and CMC). Same letters indicate no significant differences (p > 0.05). The numbers 1 and 2 indicate samples measured freshly prepared or 30 days after dispersion in bidistilled water, respectively. a

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

Figure 5. TEM photographs of DI31-modified PEG micelles (A) PEG1000−DI31, (B) PEG6000−DI31, (C) PEG10000−DI31, and (D) PEG20000−DI31 and S7-modified PEG micelles (E) PEG1000− S7, (F) PEG6000−S7, (G) PEG10000−S7, and (H) PEG20000−S7. Figure 6. In vitro cumulative release of (A) DI31 from DI31-modified PEG micelles and (B) S7 from S7-modified PEG micelles. The data represent the mean ± SD of three independent experiments (n = 3).

weight loss of 92−98% (Figure S6 of the Supporting Information). Size, ζ Potential, CMC Measurements, and Morphological Analysis. DLS studies conducted in triplicate yielded average hydrodynamic diameters in bidistilled water between 22 and 122 nm with polydispersity indices of 0.37−0.68 and very low ζ potentials (from −4.9 to 0 mV) (Table 2). The very low ζ potential values observed in almost all conjugates might be associated with the presence of small quantities of negatively charged brassinosteroids adsorbed in PEG micelle surfaces that were not separated during purification of PEG conjugates by recrystallization in ethanol. Hydrodynamic diameters remained unaltered after storing PEG micelle dispersions in bidistilled water at room temperature for 30 days (Table 2), showing stability in water. The ratios between the radius of gyration and hydrodynamic radius (Rg/Rh) extrapolated at zero concentrations from Zimm plots of recorded SLS data of

brassinosteroid-modified PEG micelles in bidistilled water were close to 0.775, with this value corresponding to a homogeneous hard sphere (Table 2).20 SEM and TEM analyses were employed to obtain morphological information on the dried micelles. SEM micrographs showed almost spherical individual micelles and few aggregates with sizes lower than 200 nm (Figure 4). TEM images showed almost spherical micelles with mean diameters of 30−140 nm (Figure 5). The sizes measured by the DLS technique are the hydrodynamic diameters of hydrated micelles, while SEM and TEM micrographs depicted the size in the dried state; thus, the particle size is slightly smaller in electron microscopy techniques. CMC values of prepared micelles in bidistilled 1616

DOI: 10.1021/acs.jafc.7b05019 J. Agric. Food Chem. 2018, 66, 1612−1619

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

water determined by fluorescence spectroscopy with the pyrene probe are also shown in Table 2. CMC is the minimal concentration needed to form micelles in water, and CMC values increased with increasing the PEG chain length for each brassinosteroid. This is the result of the increase of the hydrophilic−lipophilic balance and the increase of the hydrophilic PEG shell in the flower-like micelle conformation (brassinosteroid moieties accommodated in the hydrophobic micelle core and the looped hydrophilic PEG chains pointing out and forming the micelle shell).20 In Vitro Drug Release Studies. The release profiles of brassinosteroid-modified PEG micelles at 30 °C in PBS (pH 5.5), expressed as a percentage of the cumulative release against time, are shown in Figure 6. These studies were performed at pH 5.5 because acidic conditions are needed to achieve the hydrolysis of the ester linkage and the release of brassinosteroids DI31 and S7.13 The PEG micelles presented sustained release with almost constant release rates during the first 8 h. Release was dependent upon the particle sizes, ranging from 91 to 96% (PEG10000−DI31, PEG20000−DI31, PEG6000−S7, PEG10000−S7, and PEG20000−S7) after 96 h for the bigger aggregates, while smaller micelles released ca. 72−76% after 96 h (PEG1000−DI31, PEG6000−DI31, and PEG1000−S7). Thus, by selection of the proper PEG, it is possible to regulate

the release rate and the maximum quantity released of agrochemicals DI31 and S7. Moreover, in all cases, releases were extended for a long time (4 days), showing that prepared PEG micelles are promising candidates for a controlled and extended delivery of the studied brassinosteroids to plants. Biological Activity. Figure 7 shows the plant growth biological activity of the synthetic brassinosteroid analogues DI31 and S7 and brassinosteroid-modified PEG micelles in the radish cotyledon bioassay. Plant growth stimulator activities of the synthetic brassinosteroids DI31 and S7 are very similar, showing best results at 10−3 and 10−4 mg/mL concentrations, but an almost doubled cotyledon weight was reached in comparison to the control with the lowest concentrations (10−6 and 10−7 mg/mL). PEG20000 exerts a very slight inhibitory effect at higher (10−1− 10−2 mg/mL) concentrations, but no activity is observed at lower concentrations (10−3−10−7 mg/mL) (data not shown). The other PEGs did not show any agrochemical effect on the studied concentrations (10−1−10−7 mg/mL) (data not shown). The prepared brassinosteroid-modified PEG micelles in aqueous solution presented stimulatory activities at all concentrations (weight increased approximately 1−4 times compared to the control). Particularly, PEG1000−DI31 and PEG1000−S7 showed a similar stimulatory activity at lowest 1617

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ASSOCIATED CONTENT

S Supporting Information *

The Supporting Information is available free of charge on the ACS Publications website at DOI: 10.1021/acs.jafc.7b05019. UV spectra of (A) DI31 and (B) S7 at 1 mg/mL in PBS (pH 5.5) (Figure S1), 13C NMR spectra of (A) DI31 hemisuccinate, (B) S7 hemisuccinate, (C) PEG1000− DI31 conjugate, and (D) PEG1000−S7 conjugate (Figure S2), 1H NMR spectra of (A) DI31 hemisuccinate and (B) S7 hemisuccinate (Figure S3), XRD patterns of PEG and PEG conjugates of DI31 and S7 (Figure S4), DSC curves of PEG and PEG conjugates of DI31 and S7 (Figure S5), and TGA curves of PEG and PEG conjugates of DI31 and S7 (Figure S6) (PDF)

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AUTHOR INFORMATION

Corresponding Author

*Telephone: +4368120399376. E-mail: [email protected]. ORCID

Javier Pérez Quiñones: 0000-0002-4412-5652 Funding

This study was supported by the Deutscher Akademischer Austauschdienst (DAAD, A/12/96452), the Coimbra Group, and Erasmus Mundus, through research grants to Javier Pérez Quiñones. Notes

The authors declare no competing financial interest.

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ACKNOWLEDGMENTS Claudia Schmidt is acknowledged for hosting a 5 month stay of Javier Pérez Quiñones at the Department of Chemistry at Paderborn University and helpful discussion and comments on the manuscript. Susanne Keuker-Baumman is acknowledged for elemental analysis and DSC measurements at Paderborn University. The authors thank Jacques Chevallier and Karen E. Thomsen for electron microscopy and staining training at Aarhus University. Jens-Erik Jørgensen and Niels N. Sandal are also acknowledged for XRD measurements and guidance on seed germination, respectively, at Aarhus University.

Figure 8. HMVEC cytotoxic activity expressed as percent cell viability of (A) DI31 and its PEG micelles and (B) S7 and its PEG micelles. 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.

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concentrations (10−6−10−7 mg/mL) when compared to parent DI31 and S7. The other PEG conjugates exhibited slightly less activity at lowest concentrations (10−6−10−7 mg/mL) when compared to pure DI31 and S7, but they are still suitable to exert a good stimulatory effect on vegetal growth. These results are promising for a practical agrochemical application of the synthesized brassinosteroid-modified PEG conjugates (parent DI31 and S7 brassinosteroids are usually applied at concentrations of 10−5−10−7 mg/mL in agriculture). Cytotoxicity to the HMVEC line is an initial in vitro test for biocompatibility of different potential biomaterials (drug delivery systems based on nanoparticles, micelles, nanocomposites, hydrogels, and implants). Synthetic brassinosteroids and brassinosteroid-modified PEG micelles were not cytotoxic to the HMVEC line (HMVEC proliferation in treated samples higher than 80% compared to the control) (Figure 8). In summary, the results achieved suggest that the synthesized brassinosteroid-modified PEG micelles might be good candidates to be applied as agrochemicals.

ABBREVIATIONS USED PEG, polyethylene glycol; HMVEC, human microvascular endothelial cell; GPC, gel permeation chromatography; EDC, 1-ethyl-3-(3-(dimethylamino)propyl)carbodiimide hydrochloride; DMAP, 4-dimethylaminopyridine; PBS, phosphate buffer saline; FTIR, Fourier transform infrared; NMR, nuclear magnetic resonance; DSC, differential scanning calorimetry; DLS, dynamic light scattering; SLS, static light scattering; CMC, critical micelle concentration; TEM, transmission electron microscopy; SEM, scanning electron microscopy; SD, standard deviation; TGA, thermogravimetric analysis

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