1 Preparation and Characterization of Polymeric Microparticles Used for Controlled Release of Ametryn Herbicide

There is increasing pressure to improve agricultural productivity, due to rapid population growth, increased consumption and global demand for high quality products. As a result, agricultural chemicals have become essential for the control of weeds, pests and diseases in a wide range of crops. Ametryn (2-ethylamino-4-isopropylamino-6-methylthio-s-2,4,6triazine) is a selective herbicide belonging to the s-triazine family, whose activity is the result of inhibition of photosynthesis by blocking of electron transport. The ametryn molecule (Figure 1) contains a symmetrical hexameric aromatic ring in its chemical structure, consisting of three carbon atoms and three nitrogen atoms in alternate positions. The herbicide is classified as a methylthiotriazine, due to the presence of the SCH3 group (Tennant et al., 2001).


Introduction
There is increasing pressure to improve agricultural productivity, due to rapid population growth, increased consumption and global demand for high quality products.As a result, agricultural chemicals have become essential for the control of weeds, pests and diseases in a wide range of crops.Ametryn (2-ethylamino-4-isopropylamino-6-methylthio-s-2,4,6triazine) is a selective herbicide belonging to the s-triazine family, whose activity is the result of inhibition of photosynthesis by blocking of electron transport.The ametryn molecule (Figure 1) contains a symmetrical hexameric aromatic ring in its chemical structure, consisting of three carbon atoms and three nitrogen atoms in alternate positions.The herbicide is classified as a methylthiotriazine, due to the presence of the SCH 3 group (Tennant et al., 2001).Ametryn is used for the control of graminaceous and broad-leaved weeds in plantations of annual crops (Tennant et al., 2001).Once in the soil the herbicide may be taken up by plants, absorbed by the soil and plant residues, biodegraded, or undergo chemical transformations that increase its volatilization and photocatalytic decomposition.Studies have shown that prolonged human exposure to triazine herbicides can lead to serious health problems including contact dermatitis, intoxication, hormonal dysfunction and cancers (Friedmann et al., 2002).It is therefore desirable to develop techniques whereby the physico-chemical properties of these chemicals can be altered and their usage made safer.The goal is to enable the use of soil management strategies that can produce foods at the current high levels of demand, without significant human or environmental risk.
Micro-and nanostructured polymeric materials can be used as transport systems for active chemicals.Advantages of these materials include good physical, chemical and biological stability, simple and reproducible preparation procedures, and applicability to a wide range of chemicals.In use, the active principle is released slowly and continuously, enabling the use of smaller quantities with greater efficiency, which reduces the risk of adverse environmental impacts (Sinha et al., 2004;Sopena et al., 2009).
Controlled release systems have been extensively used in the food and pharmaceutical industries for active substances including nutrients, drugs and aromas (El Bahri & Taverdet, 2007;Grillo et al., 2008;Mello et al., 2008;Moraes et al., 2010), and there has been a recent increase in their application in medicine (Natarajan et al., 2011;Parajo et al., 2010;Vicente et al., 2010).
The objective of this work was to develop a novel release system for ametryn, employing microparticles prepared using two different polymers, PHB and PHBV (either individually or as mixtures).It was envisaged that the encapsulation of the herbicide in these microparticles would improve its chemical stability and enable the use of smaller quantities of the chemical, hence reducing the risk of environmental contamination.

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Preparation and Characterization of Polymeric Microparticles Used for Controlled Release of Ametryn Herbicide 5

Determination of ametryn
The HPLC analyses were performed using a Varian ProStar instrument fitted with a PS 210 pump, a UV-VIS detector (PS 325), a Metatherm oven and an automatic injector (PS 410).The chromatograms were acquired and processed using Galaxy Workstation software.The eluent used was acetonitrile/water (70:30, v/v), at a flow rate of 1.4 mL min -1 , and separation was achieved using a Phenomenex Gemini C 18 reversed phase column (5 μm, 110 Å, 150 mm x 4.60 mm i.d.).Ametryn was detected at a wavelength of 260 nm.The injection volume was 100 μL, and all samples were previously filtered through 0.22 µm nylon membranes.

Preparation of the polymeric microparticles containing ametryn
Microparticles were prepared with the PHB and PHBV polymers, used either individually or as a mixture, by formation of oil in water emulsions using the emulsification-solvent evaporation technique (Coimbra et al., 2008;Conti et al., 1995;Lionzo et al., 2007;Lobo et al., 2011).200 mg of polymer (PHB, PHBV or a mixture of the two polymers, as described in Table 1) and 10 mg of herbicide were dissolved in 10 mL of chloroform to form the organic phase.The aqueous phase (200 mL) was prepared using 0.5 % (w/v) polyvinyl alcohol, at 50 o C. The organic phase was transferred to the aqueous phase (at 50 o C) with magnetic stirring (1000 rpm for 15 min).The chloroform was then evaporated from the emulsion.The suspension of microparticles formed was stored in an amber flask (to avoid any photodegradation of the herbicide).The final concentration of ametryn was 50 mg L -1 .Table 1.Proportions of polymers used to prepare the different formulations.

Measurements of encapsulation efficiency
Portions (10 mg) of the different microparticles containing herbicide were dissolved in 50 mL of acetonitrile, and the association rate of the herbicide with the microparticles was determined by the technique described previously, which involves ultrafiltration/ centrifugation and analysis using HPLC (Kilic et al., 2005;Schaffazick et al., 2003).The samples were centrifuged in regenerated cellulose ultrafiltration filters that had a molecular size-exclusion pore size of 30 KDa (Microcon, Millipore), and the filtrate was analyzed using HPLC.The ametryn concentration was obtained from an analytical curve.The association rate of ametryn was calculated from the difference between the concentration measured in the filtrate and the total concentration (100 %) in the microparticle suspension.The total concentration was measured after diluting the suspension with acetonitrile, which dissolved the polymer and ensured complete release of the herbicide.The measurements were performed in triplicate for each formulation.The encapsulation efficiency (EE, %) was expressed as the ratio: Where, W s is the quantity of ametryn in the microparticles and W total is the amount of ametryn used in the formulation.

Scanning electron microscopy (SEM)
A scanning electron microscope (Model JSM-6700F, JEOL, Japan) was used to investigate the size distribution and surface morphology of the microparticles.Suspensions of microparticles containing the herbicide were filtered and the particles were then washed with 150 mL of distilled water.The solid residues were dried overnight over Na 2 SO 4 in a desiccator.The samples were then attached to metallic supports (stubs) with double-sided tape, and metalized by deposition of a gold layer at a current of 25 mA for 150 s.Images (electron micrographs) of the samples were then generated using the microscope.Particle sizes were measured using the ImageJ 1.42 program, and the size distributions of the different microparticles were obtained using OriginPro 7.0.At least 1000 individual particles of each sample were used for these measurements.

Release of ametryn from the microparticles
The release profiles of ametryn, either free or associated with the microparticles, were investigated using a two-compartment experimental system.A cellulose membrane (Spectrapore, with a molecular exclusion pore size of 1000 Da) separated the donor compartment, containing 4 mL of solution (or suspension) of the herbicide, from the acceptor compartment, which contained 50 mL of deionized water maintained under gentle agitation at ambient temperature (Paavola et al., 1995).The pore size of the membrane only allowed passage of the free herbicide, while the herbicide associated with the microparticles was retained in the donor compartment until the equilibrium was shifted so as to release the ametryn present within the particles.The size of the microparticles prevented their passage through the pores of the membrane.These experiments were conducted under dilution sink conditions, whereby the volume of the dissolution medium was sufficiently large that the herbicide concentration never exceeded 10 % of the value of its saturation concentration (Aulton et al., 2002).
Samples were retrieved from the acceptor compartment as a function of time, and analyzed by HPLC at a detector wavelength of 260 nm.During the first hour, samples were collected every 15 min, during the second hour every 30 min, and subsequently at hourly intervals until the peak area stabilized.The peak area values were then converted into the percentage of herbicide released as a function of time (De Araújo et al., 2004).

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Preparation and Characterization of Polymeric Microparticles Used for Controlled Release of Ametryn Herbicide 7

Mathematical modeling of ametryn release
Mathematical modeling is increasingly used to investigate the release profiles of bioactive compounds in polymeric systems, since it can provide important information concerning the release mechanism.Analysis of the mechanism of release of ametryn from the microparticles employed the zero order, first order, Higuchi and Korsmeyer-Peppas models (Colombo et al., 1995(Colombo et al., , 2005;;Costa & Lobo, 2001;Ferrero et al., 2000;Hariharam et al., 1994;Ritger & Peppas, 1987a, 1987b).

Results and discussion
The encapsulation efficiency values obtained for the different microparticles are listed in Table 2. Formulation A (100 % PHBV) showed the highest encapsulation efficiency (76.5 %).
The efficiency decreased as the proportion of PHBV decreased, and formulation E (100 % PHB) provided the lowest encapsulation efficiency (26.2 %).The values obtained for formulations A and B were fairly high, relative to values that have been reported in the literature for other active principles (Bazzo et al., 2009;Grillo et al., 2010;Lobo et al., 2011;Sendil et al., 1999).Grillo and colleagues (2010) showed that the encapsulation efficiency of the herbicide atrazine in PHBV microparticles was in excess of 30 %. Lobo et al. (2011), using an experimental design optimization procedure, obtained an encapsulation efficiency of 24 % for atrazine in PHBV microparticles.The relationship between the percentage of PHBV and the encapsulation efficiency is illustrated in Figure 2.There was a polynomial relationship between the encapsulation efficiency and the PHBV concentration, which was positive for PHBV and negative for PHB.This can probably be explained by the structural differences between the microparticles, due to the different polymer ratios used in their preparation (Table 1).

Formulation
The morphological characteristics of the microparticles, as well as the influence of the encapsulation of ametryn, were analyzed using the SEM procedure.Electron micrographs of the microparticles containing ametryn are illustrated in Figure 3.All types of microparticle were spherical, although the surface structures were different.Most of the PHB microparticles possessed smooth surfaces with few pores, while most of the PHBV microparticles were rough-surfaced with many cavities and pores, some of which were quite large, as can be clearly seen for formulation A (Figure 3, a1 and a2).Grillo et al. (2010) also found that PHBV microparticles, prepared using the same methodology as that described here, were rough-surfaced with pores, while PHB microparticles had smooth surfaces and fewer pores.A higher encapsulation efficiency of ametryn was therefore related to a greater number of pores in the microparticles, probably due to greater contact (and/or affinity) of the herbicide with the microparticles during the formulation preparation procedure.Ametryn is likely to have greater affinity for the PHBV polymer, since both of these molecules possess alkyl branches, with interaction being further enhanced by the porosity of the PHBV microparticles.
The size distribution profiles (Figure 4) differed between microparticle formulations (it was not possible to measure the size distribution of the formulation D microparticles due to focusing problems).The average size of the microparticles (Table 3) increased as the PHBV concentration decreased and the PHB concentration increased, and was greatest for the PHB microparticles (formulation E).These size differences could be related to the incorporation of the herbicide as well as to associations between the molecules (as discussed above).At higher encapsulation rates, the amount of ametryn present within the microparticle increased, and the potential for reactions and interactions with the polymer therefore also increased.Ametryn is likely to have a higher affinity for PHBV, and as a result of this affinity (and/or reaction) the polymer contracts due to the formation of linkages between the polymer chains.As the proportion of PHBV decreases, the affinity of ametryn for the polymer mixture also diminishes (due to the lower affinity of ametryn for PHB), so that there is less shrinkage.Table 3.Average sizes (± SD) of the different microparticles.

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The release profiles of free ametryn (as the reference) and ametryn encapsulated in the microparticles are illustrated in Figure 5, as a function of time (up to approximately 360 min).In these experiments the herbicide could traverse the pores of the membrane, while the microparticles were retained, so that it was possible to measure the influence of the association of ametryn with the polymeric matrix of the microparticles on its release rate.
The release kinetics of free ametryn was faster than that of the encapsulated herbicide, with www.intechopen.comThe release of other bioactive compounds from systems composed of microstructured polymers has been described in the literature, but usually for only one type of polymer (Grillo et al., 2010;Maqueda et al., 2009;Sendil et al., 1999;Singh et al., 2010;Wang et al., 2007).However, interpretation of release profiles relies to a large extent on knowledge of the composition and structural characteristics of the microparticles concerned, and in this respect studies that use more than one type of microparticle are advantageous.In the present work, the release of ametryn increased in line with the content of PHBV for formulations A-D, indicating that increased porosity aided the exit of ametryn molecules due to increased contact with the solvent.However formulation E was an exception to the rule, since it was composed of PHB alone and showed the fastest release of ametryn.There are two possible explanations for this observation.Firstly, the encapsulation efficiency of this formulation was lower than those achieved using the other formulations, which could have resulted in higher concentrations of ametryn crystals in the solution, and consequently higher release rates.Secondly, it is possible that lengthy refrigerated storage of this sample could have resulted in solubilization of the herbicide, due to increased contact time with the solvent.Analysis of release curves can provide important information concerning the mechanisms involved in the release of compounds from microparticles (Polakovic et al., 1999).Possible mechanisms include desorption from the surface of the polymeric matrix, diffusion through the pores or wall of the matrix, disintegration of the microparticle with subsequent release of the active principle, and dissolution and erosion of the matrix or the polymeric wall (Polakovic et al., 1999;Schaffazick et al., 2003).
A number of mathematical models have been extensively used to analyze the characteristics of the release of substances from polymeric systems (Costa & Lobo 2001).Here, the results of the release experiments (Figure 5) were analyzed using the zero order, first order, Higuchi and Korsmeyer-Peppas models (Table 4).For the formulations investigated, the Korsmeyer-Peppas model provided the best explanation of the ametryn release mechanism, according to the correlation coefficient obtained.The curves obtained for each formulation using this model are illustrated in Figure 6.

Zero order
First order Higuchi Korsmeyer-Peppas Formulation A n = 0,82641 Release constant (k) 4.59184 min -1 0.00667 min -1 4.49925 min -1/2 0.00628min The Korsmeyer-Peppas model is based on a semi-empirical equation (Korsmeyer & Peppas, 1991;Korsmeyer et al., 1983) that is widely used when the release mechanism is unknown.When the release exponent (n) is equal to 0.43 the mechanism involved is diffusion.When the value of the exponent is greater than 0.43 but smaller than 0.85, the release occurs due to anomalous transport that does not obey Fick's Law.Values less than 0.43 are indicative of porous systems in which transport occurs by a combination of diffusion through the polymeric matrix and diffusion through the pores.The values obtained ( according to formulation, as expected considering the different structural characteristics of the microparticles, so that the release mechanisms were not identical.Nonetheless, the values obtained for all formulations were in the range 0.43 < n < 0.85, indicating that in all cases the release occurred as a result of anomalous transport, involving diffusion and relaxation of the polymeric chains.This information concerning the release mechanism is of vital importance in order to be able to adjust and optimize the release of the active principle according to circumstances.

Conclusions
Ametryn herbicide was efficiently encapsulated in microparticles composed of PHB, PHBV and mixtures of the two polymers.The highest encapsulation efficiencies were achieved when higher proportions of PHBV were used.SEM analysis showed that the microparticles were spherical, although with different surface features (either smooth or rough with pores).The release profile of ametryn was modified when it was encapsulated, with slower and more sustained release compared to the free herbicide.This finding suggests that the use of encapsulated ametryn could help to mitigate adverse impacts on ecosystems and human health.This is particularly important given the increasingly widespread and intensive use of agents such as ametryn in modern agriculture.

Acknowledgments
The authors thank FAPESP, CNPq and Fundunesp for financial support.
Preparation and Characterization of Polymeric Microparticles Used for Controlled Release of Ametryn Herbicide 11 almost total release after 360 min.Association with the microparticles resulted in retarded release, with around 70 % (formulations A and B), 30 % (formulation C), 20 % (formulation D) and 40 % (formulation E) being released after 360 min.

Fig. 5 .
Fig. 5. Results of the release experiments, comparing the kinetic profiles of free ametryn and ametryn associated with the different microparticles (PHB, PHBV and PHBV+PHB), at ambient temperature (n = 3).

Fig. 6 .
Fig. 6. Results obtained using the Korsmeyer-Peppas model applied to formulations A-E.

Table 4 .
Results of the application of four mathematical models to the release curves of ametryn associated with different microparticles.