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European Polymer Journal

journal homepage: www.elsevier.com/locate/europolj

Macromolecular Nanotechnology

Self-assembled cellulose particles for agrochemical applications

Javier Pérez Quiñonesa,⁎, Cezarina Cela Mardareb, Achim Walter Hasselb,Oliver Brüggemanna

a Johannes Kepler University Linz, Institute of Polymer Chemistry, Altenberger Straβe 69, 4040 Linz, Austriab Johannes Kepler University Linz, Christian Doppler Laboratory for Combinatorial Oxide Chemistry (COMBOX) at Institute of Chemical Technologyof Inorganic Materials, Altenberger Straβe 69, 4040 Linz, Austria

A R T I C L E I N F O

Keywords:Self-assembled particlesCelluloseAgrochemicalSustained releaseSteroids

A B S T R A C T

The present work focuses on the hydrophobic functionalization of water soluble celluloses methylcellulose, hydroxyethyl cellulose and (hydroxypropyl)methyl cellulose with the anticancersteroid diosgenin, and two synthetic brassinosteroids (DI31 and S7) used as agrochemicals.Fourier transform infrared (FT-IR) and nuclear magnetic resonance (NMR) spectroscopiesconfirmed the cellulose modification. Prepared amphiphilic steroid-cellulose conjugates canself-assemble in water as stable and almost neutral particles with micelle-like structure, asdepicted using dynamic light scattering. Whereas scanning and transmission electron micro-scopies showed 50–300 nm almost spherical particles and aggregates in dried state, atomic forcemicroscopy assessed particles aggregates with mean sizes of 220–355 nm. These celluloseparticles showed sustained steroid release in acidic aqueous medium over 72 h, and goodstimulatory agrochemical activity in radish cotyledons assay. Thus, the outlined synthesis ofsteroid-cellulose conjugates, which would be capable to form self-assembled particles in water forcontrolled release of agrochemicals, is envisioned as a promising strategy.

1. Introduction

Cellulose, a natural linear polysaccharide based on repeating units of β(1 → 4) linked D-glucose, is the most abundant biopolymeras the key structural component of plants (33% of vegetal material). It combines biocompatibility, good biodegradability (glycosidehydrolases and cellulase enzymes in some ruminants, termites and fungi) and no toxicity, while exhibiting proper reactivity towardsesterification [1–3]. Even, when cellulose itself is not soluble in water, several cellulose esters show proper aqueous solubility, or areable to form stable aqueous nanoparticulate or micelle dispersions after further functionalization [4,5]. In this sense, methyl cellulose(MC), hydroxyethyl cellulose (HEC) and (hydroxypropyl)methyl cellulose (HPMC) are water-soluble cellulose esters widely used infood and pharmaceutical industry, and envisioned as promising materials for novel smart medicines [6–8]. Particularly, HPMC baseddrug delivery systems are well established in medical applications due to the polymer matrix biocompatibility and swelling propertiesupon contact with biological fluids [9–11]. Thus, cellulose based micro/nanoparticles, hydrogels, fibers, films and composites havebeen proposed for different medical applications (e.g. antibiotic and anticancer drug delivery) [12–16]. An added value of cellulose-based systems is their good biodegradability [17,18], antimicrobial behaviour observed after cationization or when prepared ascomposites or other formulations against L. monocytogenes and E. coli [14], and low cytotoxicity [15,19]. On the other hand, the self-assembly of stimuli-responsive amphiphilic celluloses as nanoparticulate systems for sustained release of different drugs is an active

http://dx.doi.org/10.1016/j.eurpolymj.2017.02.023Received 29 September 2016; Received in revised form 14 February 2017; Accepted 14 February 2017

⁎ Corresponding author.E-mail addresses: javenator@gmail.com (J. Pérez Quiñones), cezarina.mardare@jku.at (C. Cela Mardare), achimwalter.hassel@jku.at (A. Walter Hassel),

oliver.brueggemann@jku.at (O. Brüggemann).

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Available online 20 February 20170014-3057/ © 2017 Elsevier Ltd. All rights reserved.

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research area [5,20]. However, these self-assembled systems are mostly devoted to release of loaded hydrophobic drugs [5,20],instead of delivery of the covalently grafted compound. Furthermore, reports about preparation of agrochemical controlled deliverysystems are not as common as the ones devoted to medicine. Our research extends previous work on the field of synthesis and assay ofdifferent polymer-based systems for sustained release of brassinosteroids for agriculture [21].

Diosgenin ((25R)-spirost-5-en-3β-ol) is a steroidal sapogenin mostly obtained by basic hydrolysis of dioscin, the most availablesteroidal saponin. Both, dioscin and the derived diosgenin, exhibit antioxidant, anti-inflammatory, estrogenic activity, andcytotoxicity to some cancer cell lines [22–24]. Diosgenin is the main substrate in chemical synthesis of some steroids (i.e.progesterone, corticosteroids, and contraceptives), due to the fact that the required backbone and stereochemistry are already presentin diosgenin [24]. In this sense, diosgenin is the precursor of two Cuban synthetic analogues of brassinosteroids (DI31 and S7) [25]used as commercial agrochemicals over the last two decades (Biobras-16). Biobras-16 regulates plants growth and protects the cropsof biotic and abiotic stress once applied, with increases in harvest of 5–25% [26,27]. Nevertheless, the expected agrochemicalbenefits are not fully achieved in plants because the exogenous brassinosteroids are rapidly metabolized. Consequently, up to two orthree foliar spray applications are usually applied to crops, which increase economic cost of the Biobras-16 application [26].Moreover, the hydrophobicity of brassinosteroids DI31 and S7 limits their bioavailability to plants and commercial Biobras-16formulation includes plenty of ethanol, and some environmentally unfriendly additives (i.e. N,N-dimethylformamide, surfactants).Herein, it is proposed that synthesis of novel biodegradable conjugates of diosgenin, DI31 and S7, by conjugation to water solublecellulose esters via hydrolysable ester bonds, should improve bioavailability of the parent steroids and provide their sustained releaseover time. In the present research, we synthesised steroid-cellulose conjugates functionalized with three different steroids linked viaester bond, characterized them by attenuated total reflectance Fourier transform infrared (ATR-FTIR), proton nuclear magneticresonance (1H NMR) and bi-dimensional nuclear magnetic resonance (2D-NMR) spectroscopies, as well as assessed self-assembly ofthese conjugates by dynamic light scattering, atomic force microscopy, scanning and transmission electron microscopies. In vitro drugrelease of the steroids from conjugates was investigated in an acidic aqueous medium. In vitro agrochemical activity of the preparedcellulose nanoparticles towards radish (Raphanus sativus) was also studied. To the best of our knowledge, this is the first approach tothe preparation of cellulose self-assembled particulate-based system for the delivery of brassinosteroids as agrochemicals.

2. Experimental

2.1. Materials

Three water soluble celluloses named methyl cellulose (MC) (14 mPa s 2% in water at 20 °C, methoxyl content 30.2%, number-average molecular weight Mn ca. 14,000 g/mol), hydroxyethyl cellulose (HEC) (178.6 mPa s 1% in water at 20 °C, Mn ca. 220,000 g/mol) or (hydroxypropyl)methyl cellulose (HPMC) (22.1 mPa s 2% in water at 25 °C, methoxyl content 28.8% and hydroxypropylcontent 8.9%, Mn ca. 25,000 g/mol) (Sigma A.G.) were used to prepare the steroid-cellulose conjugates. Solvents and chemicals wereemployed as purchased from Sigma-Aldrich. The diosgenin and synthetic analogues of brassinosteroids (DI31 and S7) were suppliedby the Center of Natural Products at University of Havana, Cuba. Hemisuccinates of diosgenin and two synthetic analogues ofbrassinosteroids with agrochemical activity (DI-31 and S7) were synthesised by base-catalyzed traditional esterification in pyridinewith succinic anhydride [28].

2.2. Synthesis of steroid-cellulose conjugates

100 mg (0.4–0.6 mmol monosaccharide units) of methyl cellulose (MC), hydroxyethyl cellulose (HEC) or (hydroxypropyl)methylcellulose (HPMC) were stirred 48 h at room temperature with 20 mg (0.05 mmol) of diosgenin or two synthetic brassinosteroid DI31and S7 hemisuccinates (MSD, MSDI31 and MSS7), with 20 mg (0.1 mmol) of 1-ethyl-3-(3′-dimethylamino)carbodiimide hydro-chloride and 20 mg (0.16 mmol) of 4-(dimethylamino)pyridine in 10% LiCl in N,N-dimethylacetamide. Products were dialyzed(Spectra/Por 6, MWCO 1 kDa, Spectrum Lab., USA) against methanol (1 time, 600 mL, 12 h) and bi-distilled water (2 times, 1 L,24 h), and lyophilized affording white cotton wool like products. Dissolution of studied celluloses in 10% LiCl in N,N-dimethylacetamide prior to chemical reaction was conducted [29]. All studies were performed in triplicate for each sample.

2.3. Preparation of the self-assembled particles

The synthesised steroid-cellulose conjugates were able to form nanoparticles in aqueous solution after stirring overnight. To thisend, the cellulose conjugates (ca. 0.5–2.0 mg/mL) were stirred overnight at 100 rpm in bi-distilled water or phosphate buffered salinesolution (PBS, pH 7.4).

2.4. Characterization

The number-average molecular weight of celluloses and steroid-cellulose conjugates were determined with gel permeationchromatography (GPC) using a Viscotek GPCmax (Malvern, Germany) with a PFG column from PSS, 300 × 8 mm2, 5 μm particlesize. The samples (100 μL of injection volume, 2 mg/mL) were eluted with 0.01 mol/L LiBr in (N,N)-dimethylformamide at a flowrate of 0.75 mL/min at 60 °C. The cellulose 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,

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Germany) using a multidetector calibration with different polystyrene standards from PSS (Malvern, Germany). The steroid-celluloseconjugates were characterized by ATR-FT-IR spectroscopy using a Perkin-Elmer Spectrum 100 FT-IR systems spectrophotometer(Perkin-Elmer Corporation, Norwalk, Connecticut, USA) with 32 scans and 4 cm−1 resolution in the region of 4000–650 cm−1, andsolid samples were measured directly after 2 days in desiccator. 1H NMR and 2D-NMR (gradient heteronuclear single quantumcorrelation and gradient heteronuclear multiple bond correlation, g-HSQC and g-HMBC) spectra were recorded with a Varian UnityINOVA 600 MHz spectrometer, operating at 599.4 MHz for 1H at 70 °C with concentrations of approximately 25–30 mg/mL indeuterated water and d6-DMSO and analysed with the VNMRJ software, version 2.2. Steroid-cellulose particles were studied bydynamic light scattering (DLS) performed using a Zetasizer Nano ZS (Malvern Instruments, Malvern, UK) at 25 °C to obtain theparticle size and zeta potential. Size and morphology of dried steroid-cellulose particles were studied by transmission electronmicroscopy (TEM) with a Jeol JEM-2011 (Jeol Ltd., Tokyo, Japan) operating at 100 kV and images taken with a Gatan Slow-Scan CCD camera. Samples were stirred in bi-distilled water (ca. 1 mg/mL) for 12 h and a drop was deposited on a carbon coated coppergrid. The excess solution was removed, negatively stained with a drop of uranyl acetate solution (1 wt.%), and dried on air. Scanningelectron microscopy (SEM) of steroid-cellulose particles was performed in a field emission Zeiss Gemini 1540 XB SEM (Zeiss,Germany) with an acceleration voltage of 3 kV and secondary electrons detector. SEM samples were prepared by depositing a drop ofsteroid-cellulose particles suspension on a silicon wafer (0.5 × 0.2 cm2), room temperature evaporation of the water and gold coatingwith a HUMMER X (Anatech Ltd., Alexandria, VA, USA) sputter coater system.

2.5. In vitro drug release studies

In vitro drug release of diosgenin, DI31 or S7 from steroid-cellulose nanoparticles was studied by UV detection (SpectraMax M3spectrometer microplate reader, Molecular Devices, CA, USA) of the delivered steroids (from 200 nm to 500 nm) at pH 5.0. For thispurpose, ca. 5 mg of steroid-cellulose nanoparticles was dispersed in phosphate buffered saline (PBS) solutions (1.5 mL) at pH 5.0 andthe dispersions were placed in dialysis cups (Slide-A-Lyzer MINI Dialysis Devices, 3.5 KDa MWCO, 2 mL, ThermoSCIENTIFIC, IL,USA). The samples were dialyzed against the release media (PBS pH 5.0, 10 mL) at 25 °C with constant agitation at 100 rpm. Theentire media was removed at determined time intervals, and replaced with the same volume of fresh media. 200 μl aliquots wereanalyzed in a 96 well plate at 25 °C. The amount of released steroids was determined from a previously obtained calibration curve.These studies were conducted in triplicate for each sample. Linear fitting of release profiles of steroid-cellulose conjugates up to 8 hand growth-sigmoidal fitting to a SWeibull2 function (y = a − (a− b) ∗ exp(−(k ∗ x)^d) up to 72 h were conducted with the Origin2015 software (Microcal Origin, OriginLab, Northampton, MA, USA).

2.6. Agrochemical activity

The radish (Raphanus sativus) assay was employed in order to evaluate the plant growth activity. This bioassay consists ofdetermining the increased weight of the treated radish’s cotyledons (auxin type activity). To this end, radish seeds previouslysterilized by sodium hypochlorite treatment were germinated over wet filter paper in dark at 25 °C for 3 days [30]. Cotyledons wereseparated from hypocotyls, weighted and treated with 5 mL of brassinosteroid-cellulose nanoparticles in water (10−1–10−7 mg/mLof particles, brassinosteroid content 10−5.4–10−11.6 mol/L of DI31 or 10−4.8–10−11.2 mol/L of S7), DI31 or S7 solutions (10−1–10−7 mg/mL, 10−3.6–10−9.7 mol/L), or pure water (control). Parent brassinosteroid DI31 and S7 solutions were prepared at 1 mg/mL (10−2.6 mol/L) in ethanol, while required brassinosteroid concentrations were achieved upon dilution in water. After 72 h,cotyledons weights were measured. These studies were conducted in triplicate for each sample and concentration (10 cotyledons pereach run).

3. Results and discussion

Synthesis of cellulose conjugates involves mild and quantitative esterification (ca. 90% yields, referred to starting cellulosicmaterial) between the eOH group at C6 position of commercial water-soluble celluloses and the steroid hemisuccinates (Fig. 1).Preliminary synthesis optimization showed that steroid to cellulose feed ratios higher than 20 wt.% lead to conjugates thatprecipitated in aqueous solutions. Table 1 shows substitution degree (SD mol%), as measured by proton NMR in DMSO-d6 at 70 °C,steroid content (wt.%), nominal ratios of modifications defined by the steroid to saccharide unit feed molar ratios (R%), yield ofreaction based on starting cellulose materials (%), and number-average molecular weight (Mn).

3.1. Characterization

The ATR-FTIR spectra of the cellulose conjugates are dominated by the intense peaks of the saccharide moiety because the lowsubstitution degrees on steroid (< 5 mol%). Thus, the characteristic saccharide peaks observed in the methyl cellulose, hydroxyethylcellulose and (hydroxypropyl)methyl cellulose at 3460 or 3359 cm−1 (OeH stretching band), 2898 cm−1 and 2873 or 2835 cm−1

(aliphatic CeH stretching bands), 1454 cm−1 (methyl CeH vibrations), 1373 cm−1 (methyl CeH vibrations) and 1054 cm−1

(skeletal vibrations of CeO and CeC stretching, CeOeC bridge stretch) [31,32] (Fig. 2I) are dominant in the spectra of relatedsteroid-cellulose conjugates (Fig. 2II–IV). However, the C]O peak of the new ester linkage formed is still observed at 1735–1733 cm−1 (Fig. 2II–IV), while the C]O peak of ketone related to S7 steroid is visible at 1713–1717 cm−1 (Fig. 2IV). This confirmsthe functionalization of studied water-soluble celluloses with steroid hemisuccinates. The conjugates also exhibited an absorption

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peak at 1646 cm−1 (water vapour from atmosphere adsorbed onto cellulose surface) [33].1H NMR spectra of the cellulose conjugates in D2O and DMSO-d6 (Fig. 3I and Fig. 1 of supplementary material) supports the

formation of particles like-micelles in aqueous phase, with the grafted steroid moieties shielded in the hydrophobic core of theaggregates, and the hydrophilic cellulose chains forming the shell oriented to the continuous aqueous phase (Fig. 3II (a)). Thus,cellulose conjugates self-assembled as particles like-micelle or nanometric aggregates in D2O, making it not possible to observe the

Fig. 1. Structures and synthesis of steroid-cellulose conjugates.

Table 1Substitution degree (SD, mol%), steroid content (wt.%), nominal ratios of modifications of steroid to saccharide units (R%), yield of reaction (%), and number-averagemolecular weight (Mn, g/mol) of steroid-cellulose conjugates.

SD/mol% wt.% R% yields/% Mn/(g/mol)

MC-MSD 1.9 4.6 30 92 13,547HEC-MSD 1.5 3.0 15 87 221,458HPMC-MSD 1.8 4.4 27 90 25,052MC-MSDI31 0.6 1.6 8 93 14,249HEC-MSDDI31 0.5 1.1 5 88 220,891HPMC-MSDI31 0.7 1.9 9 94 26,105MC-MSS7 3.0 7.7 41 91 15,084HEC-MSS7 1.2 2.6 13 90 220,652HPMC-MSS7 1.9 5.1 25 93 24,616

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proton peaks of the steroid moieties (1H NMR of cellulose conjugates in D2O is too similar to related spectrum of MC, HEC and HPMC,Fig. 3I (a)–(b) and Fig. 1 of supplementary material). On the other hand the steroid peaks in cellulose conjugates are visible by 1HNMR in DMSO-d6 (Fig. 3I (c), Figs. 1 and 2 of supplementary material), then it was possible to determine the steroid content based onthe intense characteristic peaks related to them. Therefore, it is proposed that cellulose conjugates stand as extended chains in DMSO-d6 (Fig. 3II (b)). The 1H NMR peaks used to calculate substitution degrees were the H2 glucose proton at 2.83–2.84 ppm and theanomeric proton signal around 4.3–4.4 ppm of the celluloses [34], and the methyl group peaks of the steroids around 1 ppm (Fig. 3I(c), Figs. 1 and 2 of supplementary material). Particularly, the cellulose derivatives MC, HEC and HPMC showed the characteristicsaccharide peaks at 2.83–2.84 ppm (H2 of glucose repeating units), a broadband ca. 3.40 ppm (H3, H4, H5, H6 sugar protons ofglucose repeating units), and 4.34–4.38 ppm (Ha, anomeric sugar proton of glucose repeating units). In addition to these intensesugar peaks, the steroid-cellulose conjugates showed their typical steroid peaks at 0.61–1.09 ppm (methyl groups,H18 + H19 + H21 + H27 of diosgenin and DI31 moieties; H18 + H19 + H21 + H26 + H27 + H29 of S7 moiety), 5.34–5.35 ppm (methylene proton, H6 of diosgenin). Furthermore, covalent modification of celluloses is confirmed with shielding ofthe hemisuccinate proton chemical shifts from characteristic values in parent diosgenin and brassinosteroid hemisuccinates from ca.2.5 ppm to observed 2.09 ppm (singlet) or 2.14 and 2.28 ppm (doublets). 13C NMR spectra showed mostly the sugar peaks, while thesteroid signals were not observed, due to the low substitution degree and a lower sensitivity of this technique (data not shown). 2D-NMR experiments (g-HSQC and g-HMBC) confirmed the presence of steroid moieties in the cellulose conjugates, as the characteristicsteroid signals at 15–21 ppm (methyl groups, C18 + C19 + C21 + C27 of diosgenin and DI31;C18 + C19 + C21 + C26 + C27 + C29 of S7), 109.9–111.4 ppm (C22 of diosgenin and DI31), 124.6 and 142.7 ppm (C]C,C5 + C6 of diosgenin), 174.6–175.6 ppm (C]O, ester bond steroid succinates), 181.3 ppm (C]O, formed ester bond of steroid-cellulose conjugates), 207.7 ppm (C]O, C6 ketone of DI31 and S7 moieties) (Fig. 3 of supplementary material).

Dynamic light scattering studies of synthesised cellulose conjugates conducted in bi-distilled water, confirmed the formation ofparticles smaller than 850 nm (Table 2). HEC afforded the less substituted cellulose conjugates and significantly biggest particles(except for the HEC-MSD). It is due to the high molecular weight (ca. 220,000 g/mol) of studied HEC, which impedes the accessibilityof hydroxyl groups to esterification with steroid hemisuccinates. In spite of the almost neutral zeta potential (ca. 0 mV) of synthesisedsteroid-cellulose conjugates, particles remained stable in aqueous dispersion as observed by DLS after 1 month (similar hydrodynamicsizes and PDI values) (data not shown). The trends in the CMC values were related to the substitution degrees of different cellulose

Fig. 2. FT-IR spectra of cellulose derivatives and steroid-cellulose conjugates (I) (a) MC, (b) HEC, (c) HPMC; (II) (a) MC-MSD, (b) HEC-MSD, (c) HPMC-MSD; (III) MC-MSDI31, (b) HEC-MSDI31, (c) HPMC-MSDI31; (IV) (a) MC-MSS7, (b) HEC-MSS7, (c) HPMC-MSS7 (see Fig. 1 for structures).

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conjugates for each steroid. Thus, it was observed that the highest substitution degree in each serial of steroid-cellulose conjugates isrelated to the highest critical micelle concentration.

Atomic force microscopy, scanning electron microscopy and transmission electron microscopy showed some aggregates andindividual nanoparticles in dried state with almost spherical shapes and sizes from 50 to 300 nm. SEM showed some aggregates andalmost spherical particles of ca. 100–300 nm (Fig. 4). Additionally, the steroid-modified HEC particles appeared as the biggerparticles, in good agreement with the dynamic light scattering determinations. On the other hand, TEM micrographs showed almostcircular or disk shaped individual particles of ca. 55–160 nm, when dried (Fig. 5). Finally, AFM showed rounded aggregates ofparticles with mean sizes ca. 220–355 nm (Fig. 4 of supplementary material).

Fig. 3. (I) 1H NMR spectra of (a) MC, (b) MC-MSD in D2O at 25 °C, (c) MC-MSD in DMSO-d6 at 70 °C; (II) scheme of cellulose conjugates (a) self-assembly as particleslike-micelle or nanometric aggregates in D2O, (b) extended chains in DMSO-d6 (see Fig. 1 for structures).

Table 2Hydrodynamic diameters (d, nm), polydispersity index (PDI), zeta potential (ζ, mV) and critical micelle concentration (CMC, mg/mL) of cellulose conjugates in bi-distilled water at 25 °C.

d/nm PDI ζ/mV CMC/(mg/mL)

MC-MSD 152 ± 2 0.7 ± 0.1 −1.83 ± 0.09 0.02HEC-MSD 223 ± 4 0.31 ± 0.04 −0.74 ± 0.09 0.01HPMC-MSD 246 ± 5 0.67 ± 0.02 1.91 ± 0.07 0.01MC-MSDI31 139.8 ± 0.9 0.567 ± 0.003 −4.5 ± 0.2 0.04HEC-MSDI31 500 ± 8 0.798 ± 0.001 −3.3 ± 0.1 0.07HPMC-MSDI31 127 ± 2 0.71 ± 0.02 −3.3 ± 0.1 0.08MC-MSS7 170 ± 3 0.59 ± 0.03 −2.5 ± 0.1 0.09HEC-MSS7 842 ± 3 0.41 ± 0.02 −1.36 ± 0.01 0.04HPMC-MSS7 118 ± 2 0.681 ± 0.007 −2.06 ± 0.07 0.03

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Fig. 4. Scanning electron micrographs of steroid-cellulose conjugates (a) MC-MSD, (b) HEC-MSD, (c) HPMC-MSD, (d) MC-MSDI31, (e) HEC-MSDI31, (f) HPMC-MSDI31, (g) MC-MSS7, (h) HEC-MSS7 and (i) HPMC-MSS7 at 80,000× magnifications (see Fig. 1 for structures).

Fig. 5. Transmission electron micrographs of steroid-cellulose conjugates (a) MC-MSD, (b) HEC-MSD, (c) HPMC-MSD, (d) MC-MSDI31, (e) HEC-MSDI31, (f) HPMC-MSDI31, (g) MC-MSS7, (h) HEC-MSS7 and (i) HPMC-MSS7 at 100,000× magnifications (see Fig. 1 for structures).

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3.2. Drug delivery

In vitro drug delivery studies in PBS solution at pH 5.0 showed almost linear steroid release for the first 8 h (Fig. 5 and Table 1 ofsupplementary material), sustained and almost quantitative for 3 days (up to 75–89%) (Fig. 6I–III, fitting to a growth-sigmoidalfunction SWeibull2 with adjusted R-square from 0.9525 to 0.9984). In vitro drug releases were conducted at pH 5.0 because acidicconditions are needed to achieve the hydrolysis of the ester linkage and the steroid delivery. A control steroid delivery experimentwas also conducted in PBS at pH 7.0, when no release is expected, which resulted in less than 5% steroid delivered after 72 h (datashown only for diosgenin-celluloses, Fig. 6IV). Then, deviation of the release profiles from Fick’s law arises from the kinetics of esterhydrolysis. Thus, releases in PBS at pH 5.0 achieved 82% in MC-MSD, 88% in HEC-MSD, 77% in HPMC-MSD, 88% in MC-MSDI31,85% in HEC-MSDI31, 79% in HPMC-MSDI31, 75% in MC-MS7, 87% in HEC-MSS7 and 80% in HPMC-MSS7. It was observed thatreleases rates and extension were affected by the substitution degree and particles sizes. As expected, for each serial (same steroid,different cellulose substituent), the releases were generally faster and more quantitative with lower substitution degree and biggerparticles.

3.3. Agrochemical activity

Finally, the in vitro agrochemical activities based in the radish cotyledon assay [30] of the synthetic brassinosteroids DI31 and S7,and brassinosteroid-modified cellulose conjugates are shown in Fig. 7. Plant growth stimulator activities of the syntheticbrassinosteroids DI31 and S7 are quite similar, showing best results at 10−3 and 10−4 mg/mL concentrations, but almost a doubledcotyledons weight was reached as compared to control (no treatment, water) with the lowest concentrations (10−6 and 10−7 mg/mL)(Fig. 7I). On the other hand, the brassinosteroid-modified cellulose nanoparticles in aqueous solution showed very good stimulatoryactivities at studied concentration of particles (10−1–10−7 mg/mL, brassinosteroid concentrations of 10−5–10−11.5 mol/L), withcotyledons weight increased almost two-three times compared to control (Fig. 7II–III). The excellent stimulatory activity found atlowest concentration of particles (10−6 and 10−7 mg/mL, brassinosteroid concentrations of 10−9.8–10−11.6 mol/L) is particularly

Fig. 6. In vitro release profiles of steroid-cellulose conjugates (I) (a) MC-MSD, (b) HEC-MSD, (c) HPMC-MSD; (II) (a) MC-MSDI31, (b) HEC-MSDI31, (c) HPMC-MSDI31;(III) (a) MC-MSS7, (b) HEC-MSS7, (c) HPMC-MSS7 in PBS (pH 5.0) at 25 °C; (IV) (a) MC-MSD, (b) HEC-MSD, (c) HPMC-MSD in PBS (pH 7.0) at 25 °C (see Fig. 1 forstructures).

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promising for potential applications in agriculture. Thus, the brassinosteroid-modified cellulose conjugates exhibited 1.5–3 timesstimulatory effects compared to the parent brassinosteroids DI31 and S7, probably as a result of brassinosteroids controlled releaseand cellulose stimulatory effect in the radish cotyledons. Furthermore, in vivo plant growth stimulator effect of these brassinosteroid-modified cellulose conjugates is expected to be more important than the already observed results with the in vitro radish cotyledon’sbioassay, as well known for commercial formulations of DI31 in different crops [35,27].

4. Conclusions

Nine novel steroid-modified cellulose conjugates were synthesised in good yields, which exhibited sustained release of covalently

Fig. 7. Agrochemical activity of (I): (a) Control (C), (b) DI31, (c) S7; (II): (a) Control (C), (b) MC-MSDI31, (c) HEC-MSDI31, (d) HPMC-MSDI31; (III): (a) Control (C),(b) MC-MSS7, (c) HEC-MSS7, (d) HPMC-MSS7 at 25 °C. (*) Not measured because cotyledons died as result of high ethanol content. Data are the mean ± standarddeviation (n = 3) (see Fig. 1 for structures).

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linked steroids for 72 h in acidic conditions. Successful steroid functionalization of studied cellulose derivatives was assessed with ATR-FTIR, 1H NMR and 2D-NMR spectroscopies. Synthesised cellulose conjugates self-assembled as almost neutral particles in aqueousmedium and remained as stable dispersions over 30 days. These particles exhibited sustained release of studied steroids over 72 h.Brassinosteroid-modified cellulose particles showed to be more active as agrochemicals than parent DI31 and S7 in an in vitro radishcotyledons assay. Therefore, synthesised steroid-cellulose conjugates might find practical potential in agriculture, and they constitutea proof of concept about preparation of self-assembled particles in water of hydrophobically modified natural polymers and theirderivatives.

Acknowledgements

The authors wish to thank the Erasmus Mundus for a research grant to Javier Pérez Quinones. Günter Hesser is acknowledged forTEM training and help with TEM imaging of steroid-cellulose particles at ZONA facility of JKU Linz, Linz, Austria. Lisa MariaUiberlacker of JKU Linz, Linz, Austria and Dr. Pavlo Gordiichuk of Zernike Institute of Advanced Materials at University ofGroningen, The Netherlands are acknowledged for AFM measurements. The financial support by the Austrian Federal Ministry ofScience, Research and Economy and the National Foundation for Research, Technology and Development through the ChristianDoppler Laboratory for Combinatorial Oxide Chemistry (COMBOX) is gratefully acknowledged by CCM and AWH.

Appendix A. Supplementary material

Supplementary data associated with this article can be found, in the online version, at http://dx.doi.org/10.1016/j.eurpolymj.2017.02.023.

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