Novel brassinosteroid-modified PEG micelles for controlled release of

Subscriber access provided by UNIV OF NEW ENGLAND ARMIDALE

Article

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

Just Accepted “Just Accepted” manuscripts have been peer-reviewed and accepted for publication. They are posted online prior to technical editing, formatting for publication and author proofing. The American Chemical Society provides “Just Accepted” as a free service to the research community to expedite the dissemination of scientific material as soon as possible after acceptance. “Just Accepted” manuscripts appear in full in PDF format accompanied by an HTML abstract. “Just Accepted” manuscripts have been fully peer reviewed, but should not be considered the official version of record. They are accessible to all readers and citable by the Digital Object Identifier (DOI®). “Just Accepted” is an optional service offered to authors. Therefore, the “Just Accepted” Web site may not include all articles that will be published in the journal. After a manuscript is technically edited and formatted, it will be removed from the “Just Accepted” Web site and published as an ASAP article. Note that technical editing may introduce minor changes to the manuscript text and/or graphics which could affect content, and all legal disclaimers and ethical guidelines that apply to the journal pertain. ACS cannot be held responsible for errors or consequences arising from the use of information contained in these “Just Accepted” manuscripts.

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

Page 1 of 36

Journal of Agricultural and Food Chemistry

1

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

2 3

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

4

Peniche Covas§,

5 6

†

7

Linz,

8

[email protected]

9

†,*

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:

10

+4368120399376

11

‡

12

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

13

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

14

‡

15

§

16

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

17

[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:

18 19 20 21 22 23 24 25

1 ACS Paragon Plus Environment


Page 2 of 36

26

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

27

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

28

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

29

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

30

extended stability, while retaining agrochemical activity. Spectroscopic studies confirmed

31

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

32

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

33

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

34

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

35

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

36

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

37

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

38

HMVEC cell line in MTS test.

39 40

Keywords: agrochemicals, brassinosteroids, controlled release, PEG micelles

41 42 43 44 45 46 47 48 49 50 51 2 ACS Paragon Plus Environment

Page 3 of 36


52

INTRODUCTION

53

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

54

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

55

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

56

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

57

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

58

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

59

different brassinosteroids and synthetic analogues of brassinosteroids are proposed as plaguicides

60

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

61

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

62

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

63

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

64

and gibberellins in plants is well documented.9

65

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

66

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

67

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

68

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

69

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

70

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

71

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

72

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

73

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

74

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

75

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

76

formulations is also envisioned.



Page 4 of 36

77

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

78

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

79

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

80

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

81

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

82

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

83

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

84

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

85

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

86

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

87

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

88

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

89

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

90

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

91

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

92

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

93

of nanomaterials in agriculture.14–16

94

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

95

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

96

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

97

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

98

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

99

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

100

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

101

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

102

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

Page 5 of 36


103 104

MATERIALS AND METHODS

105

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

106

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

107

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

108

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

109

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

110

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

111

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

112

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

113

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

114

as received.

115

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

116

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

117

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

118

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

119

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

120

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

121

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

122

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

123

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

124

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

125

to 91%.

126

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

127

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



Page 6 of 36

128

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

129

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

130

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

131

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

132

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

133

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

134

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

135

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

136

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

137

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

138

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

139

(Malvern, Germany).

140

Physicochemical Characterization of Brassinosteroid-modified PEG Conjugates.

141

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

142

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

143

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

144

NMR and

145

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

146

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

147

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

148

GmbH, Germany) with burning temperature of 1150 oC.

149

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

150

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

151

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

152

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

153

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,


Page 7 of 36


154

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

155

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

156

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

157

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

158

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

159

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

160

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

161

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

162

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

163

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

164

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

165

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

166

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

167

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

168

o

169

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

170

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

171

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

172

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

173

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

174

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

175

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

176

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

177

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

178

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



Page 8 of 36

179

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

180

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

181

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

182

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

183

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

184

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

185

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

186

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

187

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

188

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

189

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

190

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

191

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

192

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

193

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

194

o

195

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

196

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

197

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

198

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

199

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

200

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

201

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

202

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

203

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

204

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


Page 9 of 36


205

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

206

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

207

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

208

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

209

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

210

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

211

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

212

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

213

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

214

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

215

represented with same letter (p> 0.05).

216 217

RESULTS AND DISCUSSION

218

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

219

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

220

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

221

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

222

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

223

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

224

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

225

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

226

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

227

dicyclohexylcarbodiimide. These hydrophobically-modified brassinosteroid-disubstituted PEG

228

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

229

their amphiphilic character.

230

Figure 1 appears here. 9 ACS Paragon Plus Environment


Page 10 of 36

231

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

232

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

233

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

234

Table 1 appears here.

235

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

236

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

237

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

238

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

239

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

240

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

241

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

242

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

243

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

244

were also confirmed by

245

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

246

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

247

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

248

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

249

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

250

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

251

conjugates and good agreement between theoretical and experimental composition was achieved

252

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

253

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

254

EDC, PEG or brassinosteroid hemisuccinate.

255

Figure 2 appears here.

256


13

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


Page 11 of 36


257

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

258

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

259

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

260

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

261

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

262

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

263

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

264

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

265

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

266

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

267

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

268

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

269

Size, Zeta Potential, Critical Micelle Concentration Measurements and Morphological

270

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

271

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

272

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

273

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

274

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

275

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

276

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

277

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

278

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

279

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

280

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

281

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

282

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


Page 12 of 36

283

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

284

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

285

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

286

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

287

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

288

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

289

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

290

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

291

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

292

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

293

Table 2 appears here.

294


295


296

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

297

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

298

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

299

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

300

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

301

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

302

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

303

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

304

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

306

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

307

and extended delivery of the studied brassinosteroids to plants.

308

Figure 6 appears here. 12 ACS Paragon Plus Environment

Page 13 of 36


309

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

310

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

311

cotyledons bioassay.

312


313

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

314

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

315

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

316

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

318

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

323

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

327

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

328

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


Page 14 of 36

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

Page 15 of 36


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

References (1) Clouse, S. D. Brassinosteroid Signal Transduction and Action. Plant Hormones 2010, D, 427– 450 (2010).

370

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

371

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

372

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

373

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

374

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

375

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

376

61, 29–43.

377

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

378

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

379

Agronomy and Soil Science 2009, 1–21.

380

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

381

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

382

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

383

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

384

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

385

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



Page 16 of 36

386

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

387

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

388

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

389 390

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

391

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

392

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

393

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

394

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

395

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

396

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

397

2015, 3, 197–202.

398

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

399

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

400

Carbohydr. Polym. 2011, 84, 858–864.

401

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

402

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

403

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

404

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

405

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

406

9781−9792

407

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

408

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

409

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

410

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

411

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

Page 17 of 36

412 413 414


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.

415

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

416

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

417

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

418

23, 943–53.

419

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

420

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

421

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

422

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

423

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

424

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


Page 18 of 36

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).


Page 23 of 36


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).


Page 25 of 36


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

Page 29 of 36


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

Page 31 of 36


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

Page 33 of 36


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

Page 35 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). 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

Novel brassinosteroid-modified PEG micelles for controlled release of

Recommend Documents