Solid Lipid Nanoparticles Co-loaded with Simazine ... - ACS Publications

Article pubs.acs.org/JAFC

Solid Lipid Nanoparticles Co-loaded with Simazine and Atrazine: Preparation, Characterization, and Evaluation of Herbicidal Activity Jhones Luiz de Oliveira,† Estefânia Vangelie Ramos Campos,†,‡ Camila Morais Gonçalves da Silva,‡ Tatiane Pasquoto,§ Renata Lima,§ and Leonardo Fernandes Fraceto*,†,‡ †

Department of Environmental Engineering, São Paulo State University, Sorocaba, Brazil Department of Biochemistry, Institute of Biology, State University of Campinas, Cidade Universitária Zeferino Vaz, Campinas, Brazil § Department of Biotechnology, University of Sorocaba, Sorocaba, Brazil ‡

ABSTRACT: Solid lipid nanoparticles (SLN) containing the herbicides atrazine and simazine were prepared and characterized, and in vitro evaluation was made of the release kinetics, herbicidal activity, and cytotoxicity. The stability of the nanoparticles was investigated over a period of 120 days, via analyses of particle size, ζ potential, polydispersion, pH, and encapsulation efficiency. SLN showed good physicochemical stability and high encapsulation efficiencies. Release kinetics tests showed that use of SLN modified the release profiles of the herbicides in water. Herbicidal activity assays performed with pre- and postemergence treatment of the target species Raphanus raphanistrum showed the effectiveness of the formulations of nanoparticles containing herbicides. Assays with nontarget organisms (Zea mays) showed that the formulations did not affect plant growth. The results of cytotoxicity assays indicated that the presence of SLN acted to reduce the toxicity of the herbicides. The new nanoparticle formulations enable the use of smaller quantities of herbicide and therefore offer a more environmentally friendly method of controlling weeds in agriculture. KEYWORDS: environmental nanotechnology, herbicides, nanoparticles, herbicidal activity

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INTRODUCTION

substances, reduce the quantities necessary, and diminish the risk of contamination of hydric resources due to leaching.9,10 A wide range of different particles can be used, obtained from a variety of matrices and preparation procedures.11 Solid lipid nanoparticles, whose matrices consist of lipids that are solid at ambient temperature, are particularly useful as carrier systems for nonpolar substances. They possess good physicochemical stability, promote sustained release of the active principle from the solid matrix, and can be metabolized by a variety of organisms.12,13 Many different types of carriers have been developed for the modified release of agricultural pesticides, including cyclodextrins,14,15 clays,16,17 silica,18 lignin,19 polymeric microparticles,20−24 and nanoparticles.25−27 Recent review articles have described advances in the use of modified release systems for active agents of interest in the agricultural sector.28,29 In a previous study,26 nanoparticles of poly(ε-caprolactone) were developed as a carrier system for atrazine, and the results obtained with target organisms (Brassica sp.) showed the effectiveness of these formulations of nanospheres and nanocapsules, compared to a commercial formulation. The objective of the present work was to prepare and characterize solid lipid nanoparticles used as a carrier system for a combination of the two herbicides atrazine and simazine. The use of a combination of two herbicides is a technique widely used in weed control, and in association with other procedures is an effective strategy for the control of vegetation. The

The growing demand for food to sustain the global population has resulted in increased efforts to maximize agricultural production.1 However, the presence of weeds that directly compete with crops for light, water, and nutrients can adversely affect productivity.2 Herbicides that are widely used to control weeds offer advantages such as rapid action, flexibility in terms of the timing of application, and the ability to treat extensive areas.3 Atrazine (2-chloro-4-ethylamine-6-isopropylamine-s-triazine) and simazine [2-chloro-4,6-bis(s-ethylaminotriazine)] are members of the triazine family of herbicides that have low solubility in water (30 and 5 mg/L, respectively) and a mechanism of action based on the inhibition of photosynthesis (due to their action on photosystem II); they are used in the pre- and postemergence control of weeds.4 A disadvantage associated with the use of herbicides lies in the fact that these substances have a wide range of different molecular structures and properties, which influences their behavior in the environment in terms of persistence, mobility, and toxicity.5 Repeated applications are often required in order to achieve the desired results, which can increase adverse effects on the environment and nontarget organisms.6 The use of nanotechnology in agriculture has received increasing attention, with the development of new formulations containing active compounds.6−8 The main objectives of these formulations are to (i) increase the solubility of active compounds, (ii) provide slow release, and (iii) provide protection against premature degradation. The use of carrier systems offers advantages, compared to conventional systems, because they can increase the biological activity of active © 2014 American Chemical Society

Received: August 18, 2014 Accepted: December 23, 2014 Published: December 23, 2014 422

DOI: 10.1021/jf5059045 J. Agric. Food Chem. 2015, 63, 422−432

Article

Journal of Agricultural and Food Chemistry

Determination of the efficiency of encapsulation of herbicides in nanoparticles was performed by the ultrafiltration/centrifugation technique, employing ultrafiltration devices containing a cellulose membrane with a molecular exclusion pore size of 10 kDa (Microcon, Millipore). The filtrate was collected, and the concentrations of the nonencapsulated herbicides were quantified by HPLC. The encapsulation efficiencies could then be determined by the difference between total amounts (100%) of the herbicides and the amounts found in the filtrate. The measurements were performed in triplicate after 0, 15, 30, 60, and 90 days.

presence of more than one herbicide can broaden the range of target weeds, reduce costs, and help to prevent the emergence of plants resistant to the herbicides. The advantage of using solid lipid nanoparticles loaded with two different herbicides lies in improved efficiency of the formulation, while at the same time helping to reduce toxicity to nontarget organisms and the wider environment. Concerning the use of polymeric nanoparticles,26 lipid nanoparticles were selected for use in the present work, because they offer high colloidal stability as well as competitive production costs and ease of scale-up. Evaluation of the characteristics and stability of the nanoparticles was performed by measurements of particle size (by means of dynamic light scattering), ζ potential, pH, polydispersion index, and encapsulation efficiency, as a function of time (up to 120 days). Particle morphology was investigated by transmission electron microscopy (TEM), and nanoparticle tracking analysis (NTA) was employed to measure the concentration and size distribution of the particles. The profiles of release of the herbicides from the SLN were investigated, and cytotoxicity of the formulations was evaluated via cellular viability assays with 3T3 cells. The effectiveness of the formulation was determined in herbicidal activity assays with pre- and postemergence treatment of the target species Raphanus raphanistrum. The same assays were repeated with Zea mays in order to determine the effects of the formulation on a nontarget organism. The goal was to develop a formulation suitable for future use in agriculture that provided increased effectiveness, while using smaller quantities of herbicides and reducing possible adverse impacts on the environment and human health.

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CHARACTERIZATION OF NANOPARTICLES Measurements of Size Distribution (by Dynamic Light Scattering) and ζ Potential. Hydrodynamic diameter and polydispersion of the particles were determined by the dynamic light scattering technique, on a Zetasizer ZS 90 instrument (Malvern Instruments). The ζ potential was determined by microelectrophoresis, on the Zetasizer ZS 90. Nanoparticles (with and without herbicides) were diluted in deionized water (1:1000 v/v) and analyzed at 25 °C, with the detector at a fixed angle of 90°. Results were expressed as the average of three determinations. In order to determine the stability of the system as a function of time, the samples were analyzed after 0, 15, 30, 60, and 90 days. Nanoparticle Morphology Determined by Transmission Electron Microscopy. SLN morphology was evaluated by use of a Zeiss-LEO microscope (Model 906). The samples were prepared from droplets of a suspension of nanoparticles (with or without herbicides), which were deposited onto 200-mesh copper grids and allowed to dry for 15 min. After this period, a droplet of uranyl acetate solution (2%) was added to each grid, as a contrast agent, and the excess solution was removed with tissue paper. After drying, the samples were analyzed by use of the microscope operated at a voltage of 80 kV.31 Determination of Particle Size Distribution and Concentration by Nanoparticle Tracking Analysis. Concentration and size distribution of the solid lipid nanoparticles was investigated by the nanoparticle tracking analysis technique. Equipment comprised a NanoSight LM 10 cell (green laser, 532 nm) and a sCMOS camera controlled via NanoSight v.2.3 software. The SLN suspensions were diluted 11 000 times, and each sample was analyzed in triplicate. In order to ensure that different particles were analyzed, each replicate used 1 mL of sample suspension injected into the volumetric cell in order to displace the contents that had been measured previously. Each repetition consisted of five measurements, with approximately 2000 particles counted in each analysis. The final result was the size-distributed number concentration of the particles.32 Evaluation of Interaction of Herbicides with Nanoparticles by Differential Scanning Calorimetry and Fourier Transform Infrared Spectroscopy. In differential scanning calorimetry (DSC) analyses, 5 mg portions of each sample (herbicides, nanoparticles, and lipids) were weighed on an analytical balance, placed in sealed aluminum pans, and analyzed on a model Q20 instrument (TA Instruments). The thermal program consisted of a heating ramp from 5 to 250 °C, at a rate of 10 °C/min, under a flow of nitrogen (50 mL/min). An empty pan was used as a reference. These measurements were performed for all the sample materials (atrazine, simazine, tripalmitin, a tripalmitin/atrazine/simazine physical mixture, SLN alone, and SLN containing atrazine and simazine).

MATERIALS AND METHODS

Materials. The reagents atrazine, simazine, poly(vinyl alcohol) (PVA; 30−70 kDa), and glycerol tripalmitate were obtained from Sigma−Aldrich. The solvents chloroform and acetone were acquired from Labsynth. Acetonitrile and methanol (HPLC-grade) were obtained from J. T. Baker. Seeds of Raphanus raphanistrum and Zea mays were purchased from ISLA Sementes. The substrate used to cultivate the plants was obtained from Hortaliça Mix. Production of Solid Lipid Nanoparticles Containing Herbicides. SLN were prepared according to the method of emulsification with solvent evaporation, described in a previous report.30 The lipid phase was prepared from 5 mL of chloroform, 250 mg of glycerol tripalmitate, and 5 mg each of atrazine (ATZ) and simazine (SMZ). After preparation, the lipid phase was inserted into an aqueous phase composed of 1.25% (m/v) PVA and the mixture was sonicated (Unique DES500) for 4 min at a power of 40 W. The pre-emulsion formed was then submitted to high-speed Ultraturrax homogenization (Tecnal TE-102) at 14 000 rpm for 7 min. After this step, the chloroform was removed by evaporation in a rotary evaporator and the sample was concentrated to obtain final concentrations of ATZ and SMZ of 0.5% (m/v). Quantification of Atrazine and Simazine and Determination of Encapsulation Efficiency. The herbicides were quantified by high-performance liquid chromatography (HPLC) employing a Varian ProStar instrument (Agilent Technologies) fitted with a model PS 210 pump, a UV−vis detector operated at a wavelength of 225 nm, and a Phenomenex Gemini C18 reverse-phase column (5 μm, 250 × 4.6 mm) kept at a temperature of 25 °C. The mobile phase was composed of acetonitrile and deionized water (50:50 v/v), at a flow rate of 0.8 mL/min. Both herbicides were quantified in the same chromatographic run. The limits of detection and quantification, respectively, were 0.21 μg/mL and 0.71 μg/mL (atrazine) and 0.18 μg/mL and 0.60 μg/mL (simazine). 423


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Figure 1. Stability of nanoparticles over a period of 120 days. SLN, solid lipid nanoparticles; SLN + ATZ + SMZ, solid lipid nanoparticles containing atrazine and simazine. (A) Hydrodynamic diameter of nanoparticles; (B) polydispersity index; (C) ζ potential; (D) pH; (E) encapsulation efficiency.

Fourier transform infrared (FTIR) analyses were performed on a Model 660 (Varian) spectrometer fitted with an attenuated total reflectance accessory (GladiATR, Pike Technologies) equipped with a diamond crystal (2.2 × 3.0 mm). The wavenumber range used was from 4000 to 400 cm−1, the incidence angle was 45°, and 32 scans were collected for each sample, at a resolution of 8 cm−1. The types of samples analyzed were the same as those used in the DSC analyses. Release Kinetics Assays and Mathematical Modeling of the Data. Kinetics of release of ATZ and SMZ, free or encapsulated in SLN, was investigated in a two-compartment system with donor and acceptor compartments. The donor compartment contained 2 mL of the formulation and was separated from the acceptor compartment by a Spectrapore cellulose membrane with a molecular exclusion pore size of 1 kDa. The system was constantly agitated under “sink” conditions. Aliquots of 1 mL were collected at intervals of 15, 30, and 60 min during the first 7 h, after which a further two aliquots were collected at 24 h intervals, up to a maximum time of 55 h. The analyte concentrations were determined by HPLC

and used to determine the percentage release of the herbicides. The experiments were performed in triplicate.26 The results of the release experiments were used to calculate the apparent flux values (Japp), according to Fick’s first law (eq 1), using the slope of the linear section of the plot of the values: M t = M 0 − SJt

(1)

where Mt is the cumulative amount of the active agent released through the membrane of area S, as a function of time t, and M0 is the total quantity of the active agent.33,34 The type of mechanism governing the release of herbicides from nanoparticles was elucidated by use of the Korsmeyer− Peppas model (also known as the power law).35,36 Evaluation of Cytotoxicity of Nanoparticles Containing Herbicides. Assessment of cytotoxicity was performed by the 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) assay. Balb-c 3T3 mouse fibroblast cells were kept in continuous culture at 37 °C, under a humid atmosphere containing 5% CO2. The culture medium used was Dulbecco’s modified Eagle’s medium (DMEM) supplemented with 10% fetal bovine serum, 100 units/mL penicillin, and 100 μL of 424


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Figure 2. Analysis of SLN morphology immediately after preparation (day 0) and following 120 days of storage: (A) SLN, day 0, 27800× magnification; (B) SLN + ATZ + SMZ, day 0, 21560× magnification; (C) SLN, 120 days, 27800× magnification; (D) SLN + ATZ + SMZ, 120 days, 21560× magnification (red arrows indicate particle deformations); (E) size distribution after 120 days, according to intensity (SLN + ATZ + SMZ); (F) size distribution after 120 days, according to volume (SLN + ATZ + SMZ).

postemergence treatment, the formulations were applied in the form of a spray 7 days after planting, and measurements of the aerial and root parts were made after a further 7 days of cultivation. The treatments were repeated for 1:10 (v/v) dilutions of the formulations (equivalent to application of herbicides at a rate of 0.3 kg/ha). Each test (pre- and postemergence) was performed with triplicates of five containers. The values obtained for the masses of the plants were calculated as means and standard deviations, and statistical evaluation employed analysis of variance (ANOVA) with the Tukey−Kramer test. These analyses were performed with GraphPad Prism v6 software.

streptomycin sulfate. The cells were plated out and, after a period of 48 h, were exposed for 24 h to a commercial formulation of herbicides (Primatop, Syngenta) and to nanoparticle suspensions (with and without herbicides). The herbicide concentrations tested were 0, 15.6, 31.25, and 62.5 μg/mL. Cellular viability was determined by the mitochondrial reductase activity of live cells, which causes conversion of the yellow tetrazolium salt to blue/purple crystals of formazan. After the 24 h period, the cells were incubated with MTT (0.5 mg/mL) for 3 h at 37 °C, followed by dissolution of the formazan crystals by agitation for 30 min in ethanol. The number of viable cells was then determined by colorimetric analysis (at a wavelength of 570 nm) employing a microplate reader (Multiskan MS, Labsystems).37,38 Herbicidal Activity Assays. Herbicidal activity was evaluated by means of pre- and postemergence treatments employing the target species Raphanus raphanistrum (wild radish) and nontarget species Zea mays (maize). Applications were made of commercial formulation (Primatop, Syngenta) and SLN containing herbicides, at concentrations equivalent to the recommended dosage (3 kg/ha). Control treatments consisted of nanoparticles without herbicides, applied at the same dilutions as the suspensions of nanoparticles containing herbicides, as well as water alone. The plants were cultivated in containers with a height of 17 cm and an upper diameter of 5.2 cm, which were completely filled with the substrate (Hortaliça Mix). One seed was planted in each container. For the preemergence treatment, the formulations were applied to the substrates by spraying soon after planting the seeds. After 13 days, the plants were collected and washed, and measurements were made of the fresh aerial parts and roots. For the

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RESULTS AND DISCUSSION The purpose of this work was to develop solid lipid nanoparticles as a carrier system for the two herbicides (simazine and atrazine), offering an alternative technique for weed control. In earlier work by our research group,26 polymeric carrier systems were developed for atrazine, and good results were obtained in terms of effectiveness of formulations. Meanwhile, in the present work, we use the strategy of associating two different herbicides in order to achieve more effective weed control, reducing costs, and helping to avoid the development of plant resistance.39,40 Combinations of the herbicides atrazine and simazine are available commercially under various brand names (Controller 500 SC, Extrazin SC, Herbimix SC, Primatop SC, and Triamex 500 SC), and all are specifically registered for use in maize cultivations. The products are indicated for use at the preemergence stage, or after emergence, with weeds at the stage of 2−4 leaves, at dosages varying from 1.75 to 3.5 kg/ha−1 425


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(equivalent to 3.5−7.0 L/ha of the commercial products). The dosage applied varies according to manufacturer and soil type, with smaller quantities used in the case of light soils, and greater quantities with clay soils or in the presence of high levels of organic matter.41 When the relative merits of solid lipid nanoparticles and polymeric nanoparticles of poly(ε-caprolactone) are considered, the great advantage of the former lies in their lower production costs, which makes them commercially attractive for use in agricultural applications. The following discussion describes the results obtained for this new formulation containing two herbicides. Stability of Solid Lipid Nanoparticles. The stability of the nanoparticles was investigated over a period of 120 days, by measurements of hydrodynamic diameter, ζ potential, polydispersion index, pH, and encapsulation efficiency (Figure 1). At the beginning of the period, the hydrodynamic diameter of SLN without herbicides was 272.2 ± 1.35 nm, while nanoparticles loaded with herbicides showed a hydrodynamic diameter of 255.43 ± 3.01 nm. Hydrodynamic diameters of all the formulations increased significantly after 15 days of storage (Figure 1A). After this period, the SLN without the herbicides then remained stable up to the end of the trial (120 days). The polydispersity index provides an indication of the homogeneity of size distribution of the particles. Values below 0.2 are considered to be indicative of good stability, with the particles present within a narrow size range.42 Soon after preparation, the formulations showed polydispersity index values below 0.15 (Figure 1B). The values then increased

Figure 3. Differential scanning calorimetry evaluation of interaction between herbicides and components of the SLN formulation: Thermograms for (A) ATZ, (B) SMZ, (C) tripalmitin, (D) physical mixture, (E) SLN, and (F) SLN + ATZ + SMZ.

Figure 4. Infrared spectroscopic evaluation of interaction between herbicides and components of the SLN formulation: FTIR spectra for (A) ATZ, (B) SMZ, (C) tripalmitin, (D) physical mixture, (E) SLN, and (F) SLN + ATZ + SMZ. Arrows indicate the main characteristic absorption bands in each spectrum. 426


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Figure 5. Nanoparticle number concentration as a function of particle size: (A) SLN; (B) SLN + ATZ + SMZ. Analyses were performed at 25 °C (n = 15). Red shading corresponds to the standard deviation of the analyses.

Figure 7. Evaluation of cytotoxicity of SLN (with and without herbicides) and commercial formulation, for herbicide concentrations of 0, 15.6, 31.25, and 62.5 μg/mL.

Figure 6. In vitro release kinetics profiles for atrazine and simazine, free or associated with SLN, determined in triplicate (n = 3) at 25 °C by use of the two-compartment system.

here the stability of the particles was related to steric hindrance caused by the presence of PVA molecules on the surfaces of nanoparticles and not by the surface charge, as reported previously.43 The SLN formulations showed negative ζ potential values that were not affected by encapsulation of the herbicides (Figure 1C). After 30 days of storage, an average value of −15 mV was obtained, which remained stable up to the end of the trial. Figure 1D shows the profile of variation of pH as a function of time, for all the formulations. The observed changes were the same for all the particles, probably due to the occurrence of hydrolytic processes in the medium (such as the hydrolysis of tripalmitin), as well as modification of the lipid matrix of the particles, including phenomena such as crystallization or recrystallization, which altered the ionic equilibrium of species present in the medium.44 Another possible contributing factor was the degradation of PVA by hydrolytic processes, which could cause changes in the medium leading to an increase in pH. However, further work will be needed in order to obtain a better understanding of these phenomena. Joint encapsulation of the herbicides resulted in encapsulation efficiencies of 89.7% ± 0.02% (atrazine) and 97.3% ± 0.05% (simazine), which remained stable over time. This indicated the effectiveness of SLN for encapsulation of herbicides, because despite the variations in particle size, pH, and ζ potential, the encapsulation efficiency remained

Table 1. Apparent Flux Values for Free and Encapsulated Herbicides Fapp (mg·m−2·h−1)

formulation ATZ SMZ SLN + ATZ + SMZ: ATZ SLN + ATZ + SMZ: SMZ

428.0 322.8 50.9 13.8

± ± ± ±

43.74 31.5 0.69 1.04

Table 2. Values of Release Constant k, Release Exponent n, and Correlation Coefficient r Obtained by Mathematical Modeling formulation

k (min−1)

n

r

SLN + ATZ + SMZ: ATZ SLN + ATZ + SMZ: SMZ

0.40 ± 0.03 0.31 ± 0.05

0.76 0.83

0.9829 0.9814

over the period of the trial, but remained below 0.2, indicating satisfactory stability of the formulations as a function of time. The ζ potential is a parameter that can be used to evaluate the stability of colloidal systems. However, in the present work, the PVA surfactant used during preparation of the formulations was adsorbed on the surface of the particles, creating a layer that provided steric stabilization. Hence, the measured ζ potential values reflected the charges on the particles, while 427


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Figure 8. Effects of solid lipid nanoparticles (SLN), commercial formulation (CF), and solid lipid nanoparticles loaded with herbicides (SLN + ATZ + SMZ) on plant growth (nontarget species), at dosages equivalent to 3 and 0.3 kg/ha: (A) fresh masses of aerial parts after pre-emergence treatment; (B) fresh masses of roots after pre-emergence treatment; (C) fresh masses of aerial parts after postemergence treatment; and (D) fresh masses of roots after postemergence treatment. (E, F) Images of Zea mays plants after pre-emergence treatment at dosages equivalent to 3 and 0.3 kg/ha, respectively.

while SLN containing herbicides showed deformations (indicated by arrows in Figure 2D). This was reflected in the appearance of a new particle population with low intensity (Figure 2E) but high volume (Figure 2F), which corroborated the results obtained for hydrodynamic diameter, where a significant increase in particle size was observed after 120 days. Interaction between Herbicides and Nanoparticles. Analyses employing differential scanning calorimetry (Figure 3) and infrared spectroscopy (Figure 4) were used to investigate the interactions of herbicides with SLN, as well as to determine whether the preparation process caused any changes in the components of the formulations. DSC results revealed the presence of two narrow endothermic peaks, at 179 and 232 °C (Figure 3A,B), corresponding to the fusion points of atrazine and simazine, respectively. Figure 3C shows a peak at 64 °C, corresponding

unaffected. The high values obtained can be explained by solubility of the herbicides in the lipid matrix, which is supported by their low solubility in water (30 mg/L for atrazine and 5 mg/L for simazine).45 Nanoparticle Morphology (Transmission Electron Microscopy). TEM micrographs of the SLN are shown in Figure 2. The particles obtained immediately after preparation were spherical, with average sizes of 111 ± 2.3 nm and 178 ± 3.1 nm in the presence and absence of herbicides, respectively (Figure 2A,B). These values were smaller than those obtained by photon correlation spectroscopy (PCS), which can be explained by the fact that the PCS technique measured the hydrodynamic diameter of the particles, while the TEM analyses employed dried samples.27 Microscopic analyses performed at the end of the storage period showed that in the absence of herbicides, SLN remained spherical (Figure 2C), 428


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Figure 9. Effects of solid lipid nanoparticles (SLN), commercial formulation (CF), and solid lipid nanoparticles loaded with herbicides (SLN + ATZ + SMZ) on plant growth (target species), at dosages equivalent to 3 and 0.3 kg/ha: (A) fresh masses of aerial parts after pre-emergence treatment; (B) fresh masses of roots after pre-emergence treatment; (C) fresh masses of aerial parts after postemergence treatment; and (D) fresh masses of roots after postemergence treatment. (E, F) Images of Raphanus raphanistrum plants after pre-emergence treatment at dosages equivalent to 3 and 0.3 kg/ha, respectively. A significance level of p < 0.05 was considered for the differences obtained between groups, at the same application level, where a* indicates significant difference relative to the control; b* indicates significant difference relative to the commercial formulation; and c* indicates significant difference relative to SLN loaded with herbicides.

to the fusion point of the β form of pure tripalmitin lipid.46 Results for the physical mixture of the components (Figure 3D) revealed two broad herbicide peaks, which were shifted due to interaction with tripalmitin. Analysis of SLN without herbicides (Figure 3E) showed a symmetrical peak at 64 °C, due to fusion of the β form of tripalmitin, and another peak at 48 °C, corresponding to recrystallization of the α form of tripalmitin.47 In colloidal suspensions, lipids can show changes in crystallization and polymorphism, which consequently affect the fusion point.47,48 In the case of SLN containing herbicides (Figure 3F), peaks characteristic of the fusion of forms α and β of tripalmitin were observed, but there were no symmetrical herbicide peaks at around 179 and 232 °C, indicating that the

herbicides were dispersed throughout the nanoparticle matrix and were not present in the form of crystals. The infrared spectra obtained for atrazine and simazine are shown in Figure 4 panels A and B, respectively. Similarities can be seen in the spectra, because both compounds belong to the triazine herbicide family, with specific bands at 3251 cm−1 corresponding to symmetrical and asymmetrical axial deformation of N−H, at 2972 cm−1 due to aliphatic C−H stretching and at 1616 and 1546 cm−1 due to deformation of CC and CN bonds, respectively. The spectrum of tripalmitin (Figure 4C) shows specific bands at 2914 cm−1 corresponding to stretching of alkyl group C−H, at 1733 cm−1 corresponding to stretching of ester CO, and at 1173 cm−1 due to angular 429


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lated, atrazine showed an initial release that was faster than that of simazine. This can be explained by the lower encapsulation efficiency obtained for atrazine, with initial fast release of the fraction of the herbicide that was not encapsulated. However, after the initial release, the release profiles for the two herbicides were very similar, indicating that the compounds had interacted in similar ways with the hydrophobic cores of the nanoparticles. Release profiles of the herbicides were analyzed by use of the Korsmeyer−Peppas mathematical model. Linear regression was used to calculate the values of release exponent n, release constant k, and correlation coefficient r (Table 2). Use of the Korsmeyer−Peppas model enables prediction of the mechanism of release of active compounds from nanostructured systems, which can essentially be by either Fickian diffusion and/or case II transport.36 Here, the values obtained for the release exponent were in the range 0.45 > n > 0.85, indicating that the kinetics could be described by anomalous transport, which involves a combination of the two different mechanisms. The values of the release constants k showed that atrazine was released faster than simazine, in agreement with the results obtained in kinetics and apparent flux experiments. Grillo et al.25 also obtained higher release constant values for atrazine when the Korsmeyer−Peppas model was used to investigate the mechanisms of release of the herbicides atrazine, simazine, and ametryn from nanocapsules composed of poly(εcaprolactone). In Vitro Cytotoxicity Assays. The MTT reduction test employing 3T3 fibroblast cells was used to determine whether encapsulation of the herbicides altered their cytotoxicity. The results (Figure 7) showed that in the absence of herbicides, SLN were not cytotoxic in the concentration range tested, since cellular viability remained at around 100%. When herbicides were incorporated into SLN, cellular viability of 80% was obtained for the highest concentration tested, while greater cytotoxicity was observed for the commercial formulation, with a value of 64% obtained at the same concentration. These findings confirmed that encapsulation of the herbicides in the solid lipid nanoparticles resulted in a decrease in cytotoxicity. Herbicidal Activity Assays. Effects of nanoparticle formulations, with and without herbicides, as well as the commercial formulation, were investigated for pre- and postemergence treatments applied to a target species (Raphanus raphanistrum) and a nontarget species (Zea mays), at concentrations equivalent to 0.3 and 3 kg/ha. Atrazine and simazine are used together to increase the spectrum of action in controlling weeds in maize plantations. Maize is resistant to these herbicides because of the presence in this species of a soluble enzyme, glutathione S-transferase, that detoxifies both atrazine and simazine.1,4 For the nontarget species (for pre- and postemergence treatments), none of the formulations showed any phytotoxic effects on the aerial and root parts of plants when compared with the control (Figure 8). For the target species, in the case of pre-emergence treatments, all the formulations showed effects on both the aerial and root parts of plants, compared to the control treatment (Figure 9). At the 3 kg/ha concentration level, effects of the commercial formulation and herbicides encapsulated in SLN were significantly greater, compared to SLN without herbicides, which could be attributed to the presence of the active agents. However, when the formulations were diluted 10 times (0.3 kg/ha), SLN without herbicides showed no

deformation of C−O. The spectrum for the physical mixture (Figure 4D) shows the characteristic bands of tripalmitin, together with deformation of the CC and CN bonds of the herbicides. The spectrum for SLN without herbicides (Figure 4E) shows most of the bands corresponding to tripalmitin, together with a band at around 3340 cm −1 indicative of the presence of the −O-H group in the structure, which probably resulted from the use of the water and poly(vinyl alcohol) as stabilizers during preparation of the nanoparticles. Another characteristic −O-H deformation band of water can be seen at 1640 cm−1 in this spectrum. Figure 4F shows the spectrum for SLN loaded with atrazine and simazine, with the presence of characteristic bands corresponding to poly(vinyl alcohol), tripalmitin, and (indicated by the arrows) shifted lower intensity bands at 1629 and 1561 cm−1 associated with deformation of CC and CN bonds of the herbicides. These features indicate that the herbicides were encapsulated and/or had interacted with the nanoparticles. In previous work, de Melo et al.49 used changes in FTIR spectra to show that the bioactive compound articaine interacted with nanospheres of alginate/chitosan, as well as with nanocapsules of PCL/PEG, confirming that both types of nanoparticle were suitable for loading with articaine. Nanoparticle Tracking Analysis. The nanoparticle tracking analysis technique was used to characterize SLN suspensions immediately after their preparation. Figure 5 shows the concentrations of nanoparticles as a function of particle size. For SLN without herbicides, the particle number concentration was (9.09 ± 0.70) × 1012 particles/mL and the average hydrodynamic diameter was 240.37 ± 95.54 nm. Encapsulation of the herbicides resulted in a concentration of (11.75 ± 0.64) × 1012 particles/mL and an average hydrodynamic diameter of 252.18 ± 91.56 nm. It can be seen that use of DLS and NTA techniques resulted in similar average hydrodynamic diameter values, as well as particle number concentrations that were in the same range, evidencing the satisfactory conditions used to prepare the formulations. An important point is that the formulations showed the monomodal size distributions associated with only one population of particles. This type of homogeneity is desirable in modified release formulations, because there are therefore likely to be fewer interferences in the process of release of the active agents.50 Release Kinetics Assays and Mathematical Modeling. Release kinetics assays were conducted in order to obtain further information concerning the interactions between herbicides and nanoparticles, as well as the mechanisms involved in herbicide release. Figure 6 shows the curves obtained for cumulative release of the herbicides as a function of time. In these experiments, the herbicide molecules were able to traverse the 1 kDa pores of the cellulose membrane separating the donor and acceptor compartments, enabling observation of the progressive release of herbicide into solution. The results (Figure 6) showed that the presence of SLN modified the release profiles, which were slower compared to those of the free herbicides. For the free herbicides, 50% release was obtained in 2.5 h (ATZ) and 5.3 h (SMZ). When the compounds were encapsulated together in SLN, 50% release of the same quantities of the herbicides was obtained in 52.9 h (ATZ) and 51.1 h (SMZ). The apparent fluxes (Fapp) for the free and encapsulated herbicides are listed in Table 1. The data showed that the mass flux of simazine was lower than that of atrazine, for both free and encapsulated forms. When the herbicides were encapsu430


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providing greater efficiency in weed control, reducing the quantities of active agents required, and at the same time decreasing toxicity toward nontarget organisms.

phytotoxicity, compared to the control, and the commercial formulation showed a significant effect only on the roots. On the other hand, SLN containing herbicides caused greater effects on both the aerial parts and roots of plants, compared to the commercial formulation. This provided evidence of the effectiveness and agricultural potential of SLN containing herbicides, since when applications were performed at the recommended concentrations, all the formulations caused inhibition of growth, while at 10 times lower concentrations, the effect of the commercial formulation decreased, while SLN containing herbicides remained effective (Figure 9E). In the case of postemergence treatment, SLN without herbicides showed no phytotoxic effects on aerial parts or roots, at either concentration, compared to the control. However, the commercial formulation and SLN loaded with herbicides showed phytotoxic effects on aerial parts and roots at both concentrations. No significant differences were observed between these phytotoxic effects. The phytotoxic effects of SLN in pre- and postemergence assays could have been due to interactions of SLN with roots of R. raphanistrum, resulting in changes in the processes of absorption of water and/or nutrients. It is also possible that absorption of nanoparticles by the roots caused toxic effects and reduced growth rates. There have been several reports of interactions between nanoparticles and plants.51−53 Meanwhile, it should be highlighted that the interaction of SLN with R. raphanistrum (Figure 9E,F) appeared to be species-specific, because no toxic effects of SLN were observed in assays with Z. mays, at the same concentration range (Figure 8E,F). The greater effectiveness of SLN containing herbicides could be explained by reduced soil sorption of the active agents, as well as physicochemical and microbiological protection provided by encapsulation and sustained release of herbicides provided by the nanoparticles. Similar findings were reported previously26 for encapsulation of atrazine in PCL nanoparticles (nanocapsules and nanopheres) and evaluation of the effects on mustard plants (Brassica sp.). In conclusion, the herbicides atrazine and simazine were successfully incorporated into solid lipid nanoparticles, with encapsulation efficiencies of around 90% for atrazine and 98% for simazine. SLN showed hydrodynamic diameters between 255 and 300 nm and remained stable over a period of 120 days. DSC and FTIR analyses confirmed that the herbicides were encapsulated and interacted with the lipid matrix. The use of TEM and NTA techniques demonstrated that the nanoparticles were spherical, with an average concentration of 12 × 1012 particles/mL. Encapsulation resulted in modification of the herbicide release profiles, with slower and more sustained release compared to the free forms of the herbicides. Evaluation of cytotoxicity by the MTT test showed that SLN provided a protective effect and acted to decrease the toxicity of the active agents. In herbicidal activity assays, postemergence treatment with SLN containing the two herbicides was as effective as use of the commercial formulation. In the case of pre-emergence treatment, SLN loaded with herbicides were more effective, compared to the commercial product, indicating the potential of these formulations for use in agriculture. An important finding was that the formulations of SLN containing two active agents were more effective, compared to the commercial formulation, and caused no toxicity in nontarget organisms (Z. mays plants and mouse fibroblast cells). The findings illustrate one of the ways in which nanotechnology can be used to develop effective systems for modified release of herbicides,

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AUTHOR INFORMATION

Corresponding Author

*Telephone 55 15 3238-3400, ext 3456; fax 55 15 3228-2842; e-mail [email protected]. Funding

We are grateful for the financial support provided by the São Paulo State Research Foundation (FAPESP, Grant 2012/ 20076-9), CNPq, and FUNDUNESP. Notes

The authors declare no competing financial interest.

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REFERENCES

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