J. Agric. Food Chem. 2011, 59, 307–311
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DOI:10.1021/jf103640t
Slow-Release Formulation of a New Biological Pesticide, Pyoluteorin, with Mesoporous Silica JIE CHEN, WEI WANG, YUQUAN XU, AND XUEHONG ZHANG* Key Laboratory of Microbial Metabolism, Ministry of Education, and School of Life Sciences and Biotechnology, Shanghai Jiao Tong University, Shanghai 200240, People’s Republic of China
A slow-release formula of potential biological pesticide Pyoluteorin (Plt) was prepared by using nanophase material of silicon dioxide loading drugs. The final experimental formula was m(Plt: Brij56:TMOS:HCl(aq)) = 0.04:1.4:2:1, synthesized by a highly ordered monolith (HOM) method. This formula can continuously release 85.13 ( 2.03 % of Plt within 28 days. A characterization study showed the formula formed a well-ordered mesoporous structure, with a surface area of 822 m2 g-1, with a measured mesoporous volume of 0.41 cm3 g-1 and a narrow distribution for the pore size centered at 2.4 nm. A bioactivity experiment showed it authentically prolonged the antifungal effects. This study is the first to report mesoporous formulations for biological pesticides and indicates a potentially interesting drug carrier. The association of a nanostructured silica to the molecular state of the drug holds great interests for field applications as it overcomes the rapid loss of biological function during drug utilities. KEYWORDS: Slow release; biological pesticide; pyoluteorin; mesoporous silica
INTRODUCTION
Biological pesticides are considered to be less harmful to the environment and even more specific than chemical pesticides. People pay more and more attention to the development of new biological pesticides that display little or no nontarget toxicity (1). Pyoluteorin (Plt, 4,5-dichloropyrrol-2-yl 2,6-dihydroxyphenyl, structure shown in Figure 1) is a polyketide metabolite produced by fluorescent Pseudomonas. It controls a wide range of pests, including bacteria, epiphytes, and weeds. It has the potential to be widely used as a new biological pesticide. However, like most of the biological pesticides, ignorance of the stability of Plt is an obstacle to its utility. Several conditions including temperature, pH, and UV-vis light irradiation can rapidly decrease the biological activity of Plt to a low, ineffective level (2). Therefore, modification of Plt’s structure is essential for keeping its stability during storage and field applications. Recently, increased interests have been shown in novel formulations about pesticides to meet the need for prolonged and better control of drug. Slow-release formulations can significantly decrease drug leaching and surface migration, allow reduction of the applied amounts, and have been the focus of research about drug delivery systems (3). Among slow-release drug delivery systems, mesoporous silica materials have the potential to be widely used as drug carriers due to their nontoxic nature and drug-releasing properties, such as mesoporous structure stability, high surface area, large pore volume, regular nanopore sizes, and abundant pore surface Si-OH bonds for loading and releasing drug molecules (4-9). Recently, new applications of mesoporous silica as drug delivery
Figure 1. Structure of pyoluteorin (Plt).
systems have been explored (10-16). All of these systems are expected to enhance the bioavailability and lower the therapeutic doses of poorly absorbed drugs. The utilities of typical mesoporous materials in drug delivery systems, such as SBA-15 and MCM-41, have been studied well. Their mesoporous structure characteristics made it possible to load and release drugs under a controlled rate (13, 17). However, the drug storage capacities for those conventional mesoporous materials were relatively low (18, 19), and formulations for biological pesticides have not been well studied yet. Therefore, to overcome these disadvantages and make further studies to support the field application, here we report a novel slow-release drug system synthesized by a highly ordered monolith (HOM) method. In our experiments, we found that this system could directly load drugs with outstanding capacity for drug storage. By optimizing conditions including drug loading, surfactant, solvent, etc., we studied the final formulation in structure, drug-releasing rate, and bioactivity. MATERIALS AND METHODS
*Author to whom correspondence should be addressed (e-mail
[email protected]; phone 86-21-34206715; fax 86-21-34205081).
Materials. Plt (99.3% purity) was prepared in our laboratory (Laboratory of Microbial Resources and Metabolic Engineering) from
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Published on Web 12/08/2010
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J. Agric. Food Chem., Vol. 59, No. 1, 2011
Chen et al.
Table 1. Component of Plt-SiO2 by HOM Method formula
Plt (mg)
surfactant type
surfactant amount (g)
TMOS (g)
0.05 M HCl (g)
1 2 3 4 5 6
40 40 40 40 40 40
F127 F127 P123 P123 Brij56 Brij56
1.2 1.4 1.2 1.4 1.2 1.4
2 2 2 2 2 2
1 1 1 1 1 1
the culture of Pseudomonas sp. M18 (20, 21). Tetramethyl ο-silicate (TMOS) was used as silica source. Pluronic P123 (EO20PO70EO20, EO is ethylene oxide and PO is propylene oxide), Pluronic F127 (EO106PO70EO106), and Brij 56 (C16EO10) were used as surfactants. Dextrose and agar were purchased from Sigma-Aldrich. These reagents were of analytical grade and used directly. Synthesis of Drug-Loaded Nanoparticles. The typical synthetic procedure was as follows: 1.4 g of surfactant and 2 g of TMOS dissolved in a flask, followed by the addition of 40 mg of Plt drug with mild stirring at 80 °C in a water bath heater for 1-2 min. After yielding a clear solution (homogeneous), 1 g of HCl (aq, 0.05 M) was quickly added to this mixed solution and then stirred for another 10 min. After the reaction was complete, the transparent glassy monoliths were cured at 40 °C for an additional 48 h. After the curing material changed to a solid state, it was ground into powder for use. The components of all Plt-SiO2 (Plt-loaded mesoporous silica) are shown in Table 1. Characterization. X-ray diffraction (XRD) patterns were recorded on an MX Labo powder diffractometer equipped with Cu Ka radiation (40 kV, 20 mA) with a resolution of 0.02° and a scanning speed of 1° min-1 over the range of 0.7-4.0° (2θ). Samples were prepared as thin layers on glass slides. The nitrogen adsorption-desorption isotherms were collected at 77 K using an adsorption analyzer ASAP 2010 from Micromeritics. Samples were pretreated for 2 h at 120 °C and 1.33 10-4 Pa. The specific surface area was assessed according to the standard BET method. The primary mesopore volume, Vp, was determined by the high-resolution t-plot method. The pore size distribution was calculated from adsorption branches of isotherms by the BJH method. Transmission electron microscope (TEM) images of the samples deposited on carbon-copper grids were taken on a JEM 2010/INCA Oxford microscope. Analysis of Drug Release. The drug release analysis of Plt-SiO2 was carried out at room temperature in pure water (produced by a water purifier Aquapro DZG-303A). Dry Plt-SiO2 (100 mg) was dissolved into 15 mL of pure water. The solution was collected at certain time points and was filtered through a 0.45 μm Millipore filter. All experiments were performed in duplicate. The concentration of Plt was determined by HPLC with a UV-vis detector (Shimadzu, SPD-10A) and a C-18, 250 4.6 mm, column (Kromasil). The mobile phase was made up of methanol and water (7:3, v/v) and was delivered at a constant rate of 1.0 mL min-1. The absorbance detection was 310 nm. The concentrations of Plt in the tested samples were calculated on the basis of the standard curve generated with Plt standards. All statistical graphics and data analyses were carried out with Microsoft Excel 2007 or Origin 6.0. Antifungal Activity. Plt drug and Plt-SiO2 were dispersed into 15 mL glycine-sodium hydroxide (pH 8.6) to obtain fast degradation of the pesticide. The above samples were then tested for antifungal activity against Phytophthora capsici Leonian. Test fungi were cultured on potato dextrose agar (PDA) medium (peeled potato, 250 g; dextrose, 20 g; agar, 15 g; distilled water, 1 L). In culture plates, each Plt-containing sample was diluted to the theoretical concentration of 10 μg mL-1, at which the fungus was sensitive to Plt in preliminary experiments. We used the same amount of pure water, instead of Plt-containing sample for blank control, and dispersed Plt and Plt-SiO2 into natural water to compare the degradation under a pH 8.6 environment. Fungal plaques (L 5 mm) were picked up from 5-7-day-old culture with a sterilized inoculation needle and were put facedown upon plates. These plates were cultured at 28 ( 2 °C. RESULTS AND DISCUSSION
Drug Delivery Profiles. We used three commonly used surfactants, F127, P123, and Brij 56, and changed the amount of
Figure 2. Release rate of six formulations of Plt in pure water within 28 days. The formulations varied by type and concentration of surfactants: 1, F127, 1.2 g; 2, F127, 1.4 g; 3, P123, 1.2 g; 4, P123, 1.4 g; 5, Brij 56, 1.2 g; 6, Brij 56, 1.4 g.
Figure 3. XRD patterns of Plt-SiO2. A strong peak was observed at a 2θ angle in the range of 1.75-1.9.
surfactants to optimize the drug loading system. The storage amount of the drug loading was 47.5 mg g-1, which was calculated according to the formulation. In the samples after synthesis, only drug and SiO2 were retained; other components were volatilized. This drug loading amount was suitable for manufacturing and field application (4, 22-24). Higher drug loading can be achieved by adding more drugs. The release percentage of Plt in pure water is shown in Figure 2. The samples varied with different surfactant concentrations and surfactant types. The Plt release rate increased significantly as surfactant concentration increased using Brij 56 (formula 6 > 5), P123 (formula 4 > 3), and F127 (formula 2 > 1 after 20 days). Surfactant was used as structure template and structure stabilizer; the increasing amount perhaps indicated that the better structure was formed, but the instant synthesis of mesoporous monolithic materials did not easily form structure in good order. The amount of surfactant would lower the silica concentration and affect the stability, so the concentration was kept in a certain region (8, 15, 25). The best performance was with Brij56 as surfactant; the component was m(Plt:Brij56:TMOS:HCl(aq)) = 0.04:1.4:2:1. Plt-SiO2 using F127 and P123 as surfactant released
