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
See https://pubs.acs.org/sharingguidelines for options on how to legitimately share published articles.

∥
Center of Biomaterials, University of Havana, 10400 Havana, Cuba
*
S Supporting Information
Downloaded via UNIV DE EXTREMADURA on July 16, 2021 at 12:29:44 (UTC).

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

■ 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, sen-
escence, and disease resistance of the plants. 1−3 24-
epibrassinolide, 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 Further-
more, 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
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,O6-
acetyl 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: October 27, 2017
Revised: January 25, 2018
Accepted: January 29, 2018
Published: January 29, 2018

© 2018 American Chemical Society 1612 DOI: 10.1021/acs.jafc.7b05019

J. Agric. Food Chem. 2018, 66, 1612−1619
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 prepara-
tion of novel commercial formulations of agrochemicals, such
as Biobras-16.
■
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 4-
dimethylaminopyridine (DMAP) were purchased from Sigma-Aldrich
(Germany). Brassinosteroid hemisuccinates were synthesized by base-
catalyzed 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
Article
Molecular Weight Determination of Brassinosteroid-Modi-
fied 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-Modi-
fied 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, Ger-
many) 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 Vertriebsge-
sellschaft, 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 micro-
scope. 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-
DOI: 10.1021/acs.jafc.7b05019
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Journal of Agricultural and Food Chemistry Article

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

Table 1. Steroid Weight Contents (wt %), Molecular Weights Measured by GPC, and Relative Errors (Error, %)
sample wt % Mna (g/mol) Mw/Mna Mnb (g/mol) Mnc (g/mol) error (%)
PEG1000 - 915 1.12 938 −2.5
PEG1000−DI31 45.0 1939 1.13 1997 −2.9
PEG1000−S7 47.0 1973 1.12 2022 −2.4
PEG6000 - 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
PEG20000 - 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
a
Number-average molecular weights and polydispersities (Mw/Mn) measured by GPC. bNumber-average molecular weights determined from Sigma-
Aldrich 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 ultra-
violet (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
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 serum-
free 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).

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
1614
■
RESULTS AND DISCUSSION
Synthesis of Brassinosteroid-Modified PEG Conju-
gates 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
DOI: 10.1021/acs.jafc.7b05019
J. Agric. Food Chem. 2018, 66, 1612−1619
Journal of Agricultural and Food Chemistry
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 PEG-
stabilized lipid nanoparticles, mostly formed by physical or
emulsion methods, for application as pharmaceutical carriers. In
the present study, four different PEGs (PEG1000, PEG6000,
1615
Article
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′-dicyclohexylcarbodii-
mide. These hydrophobically modified brassinosteroid-disub-
stituted 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 brassinosteroid-
disubstituted 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 brassinosteroid-
modified 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
DOI: 10.1021/acs.jafc.7b05019
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Table 2. Hydrodynamic Parameters and CMC of Brassinosteroid−PEG Micellesa

sample diameter1 (nm) diameter2 (nm) PDI1 PDI2 Rg/Rh1 ζ potential1 (mV) CMC1 (mg/mL)
PEG1000−DI31 79 ± 1a 80 ± 2a 0.70 ± 0.05 0.68 ± 0.02 0.842 0i 0.07 l
PEG6000−DI31 105.2 ± 0.3 b 105.7 ± 0.8 b 0.593 ± 0.006 0.601 ± 0.002 0.901 −4.9 ± 0.9 j 0.11
PEG10000−DI31 22.1 ± 0.4 c 23.1 ± 0.6 c 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 ± 0.7 k 0.37
PEG1000−S7 56.0 ± 0.6 e 55 ± 1e 0.37 ± 0.05 0.37 ± 0.04 0.875 −4.6 ± 0.8 j 0.07 l
PEG6000−S7 32 ± 1f 33.2 ± 0.7 f 0.48 ± 0.08 0.54 ± 0.02 0.893 −2.3 ± 0.6 k 0.15
PEG10000−S7 73 ± 3g 75 ± 2g 0.7 ± 0.1 0.67 ± 0.08 0.915 −4.3 ± 0.3 j 0.30
PEG20000−S7 112 ± 3h 110 ± 1h 0.53 ± 0.05 0.56 ± 0.01 0.859 −2.1 ± 0.4 k 0.48
a
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.

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

weight loss of 92−98% (Figure S6 of the Supporting
Information).
Size, ζ Potential, CMC Measurements, and Morpho-
logical 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 concen-
trations from Zimm plots of recorded SLS data of
1616
PEG micelles and (B) S7 from S7-modified PEG micelles. The data
represent the mean ± SD of three independent experiments (n = 3).
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
DOI: 10.1021/acs.jafc.7b05019
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Journal of Agricultural and Food Chemistry Article

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 hydro-
philic 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 brassinoste-
roids 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
1617
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 brassinos-
teroids 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
DOI: 10.1021/acs.jafc.7b05019
J. Agric. Food Chem. 2018, 66, 1612−1619
Journal of Agricultural and Food Chemistry
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.
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, nano-
composites, hydrogels, and implants). Synthetic brassinoste-
roids 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.
1618
Article
■
*
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)
■ AUTHOR INFORMATION
Corresponding Author
*Telephone: +4368120399376. E-mail: javenator@gmail.com.
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.
■ 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.
■ ABBREVIATIONS USED
PEG, polyethylene glycol; HMVEC, human microvascular
endothelial cell; GPC, gel permeation chromatography; EDC,
1-ethyl-3-(3-(dimethylamino)propyl)carbodiimide hydrochlor-
ide; 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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