Article pubs.acs.org/JAFC
Development of a Chlorantraniliprole Microcapsule Formulation with a High Loading Content and Controlled-Release Property Baoxia Liu,†,#,∥ Yan Wang,‡,∥ Fei Yang,†,# Haixin Cui,*,‡ and Decheng Wu*,†,# †
Beijing National Laboratory for Molecular Sciences, State Key Laboratory of Polymer Physics & Chemistry, Institute of Chemistry, Chinese Academy of Sciences, Beijing 100190, China ‡ Institute of Environment and Sustainable Development in Agriculture, Chinese Academic of Agriculture Sciences, Beijing 100081, China # University of Chinese Academy of Sciences, Beijing 100049, China ABSTRACT: Microcapsule formulations have been widely developed and used in agriculture to improve pesticide utilization and reduce environmental pollution. However, commercial formulations of chlorantraniliprole (CAP) are only traditional formulations due to poor solubility of CAP in organic solvents. Here, adopting a solid in oil in water (S/O/W) double-emulsion method combined with premix membrane emulsion, we successfully constructed CAP microcapsule formulations with a high loading content. The microcapsule formulations with good light and thermal stability showed a significantly sustained release for a long period, which could be optimally regulated by tuning the surface porosity and size of the porous microcapsules. Bioassay studies showed that control efficacy of the porous microcapsule formulations against Plutella xylostella was superior to that of the commercial formulation. These results demonstrated that such a porous microcapsule delivery system should have great potential for further exploration as a commercial CAP formulation. KEYWORDS: microcapsule formulations, chlorantraniliprole, controlled release, light stability, thermal stability
■
organic phase separation,20 in situ polymerization,21 interfacial polycondensation,22 nanoprecipitation,23,24 and suspension cross-linking.25 However, the size of particles is difficult to control, and the size distribution is very broad via mechanical stirring, homogenization, or ultrasonication in the above techniques, resulting in poor reproducibility in preparation, poor repeatability of the release behavior, and poor bioavailability in application. Therefore, it is necessary to obtain microcapsules with uniform and tailored size for construction of controlled-release drug carriers. Premix membrane emulsification (PME) combined with W/O/W double emulsion, a novel and mild improvement technique, has been widely used to obtain uniform microcapsule delivery systems.26−28 The resulting particle size is primarily controlled in a range from hundreds of nanometers to micrometers by the regulation of the process parameters including membrane pore size, applied membrane pressure, and viscosity of emulsion.29 In a word, the PME is a promising industrialized technique in the preparation of microcapsule formulations for controlled release of pesticide. The control of drug release behavior has been an important research subject in the field of drug delivery.30−33 For example, a broad range of carriers with diverse sizes have been designed to adjust release behaviors.20,34−36 Controlling the surface
INTRODUCTION The use of pesticides has greatly improved the quality and quantity of vegetables and crops in agriculture.1 However, the long-term extensive and inefficient use of traditional pesticide formulations such as emulsifiable concentrates (EC), suspension concentrate (SC), and wettable powders (WP) has posed a serious harm to both the environment and humans.2−5 In fact, >80% of applied pesticides are actually lost owing to biodegradation, chemical degradation, photolysis, and evaporation. Repeated applications of pesticides are thus required to replace this loss, which results in high cost and environmental pollution.6−11 Furthermore, the traditional pesticide formulations need considerable organic solvents and generate terrible odors in the spraying process. Currently, water-dispersed microcapsule formulations based on biodegradable materials have been developed and commercialized, which could not only reduce environmental pollution but also improve pesticide utilization by controlling its release behavior and decreasing its evaporation and degradation.12−15 Chlorantraniliprole (CAP) has been widely used to prevent and control pests as a broad-spectrum pesticide with long residual activity, low toxicity, and no cross-resistance with other pesticides. Currently, only traditional SC, EC, and WP formulations of CAP are available, and water-dispersed microcapsule formulations have not been industrialized yet and even rarely reported. The major challenge is that poor solubility of CAP restricts its loading content and subsequent release from the microcapsule. Thus, there is an urgent need to design efficient microcapsule formulations for CAP with a high loading content and controlled-release property. Various encapsulation techniques have been developed such as water in oil in water (W/O/W) double emulsion,16−19 © XXXX American Chemical Society
Special Issue: Nanotechnology Applications and Implications of Agrochemicals toward Sustainable Agriculture and Food Systems Received: March 22, 2017 Revised: May 2, 2017 Accepted: May 4, 2017
A
DOI: 10.1021/acs.jafc.7b01295 J. Agric. Food Chem. XXXX, XXX, XXX−XXX
Article
Journal of Agricultural and Food Chemistry
and four circulations through the 7.0 μm SPG membrane under 80 kPa pressure. The 0.65 μm microcapsules were prepared with 30 mg/ mL PLA solution as the oil phase, 0.1 wt % PVA aqueous solution as the external water phase, and three circulations through the 1.0 μm SPG membrane under 1000 kPa pressure. The microcapsules were marked PLAS1, PLAS2, and PLAS3 with sizes of 4.2, 2.2, and 0.65 μm, respectively. Characterization of CAP-Loaded PLA Microcapsules. The morphology of the microcapsules was observed via scanning electron microscopy (SEM; JSM-6700F, JEOL, Japan) with an accelerating voltage of 5 kV and a working distance of 8 mm. The specimens were mounted to metal stubs using double-sided tape and vacuum-coated with a thin layer of platinum using a sputter coater (EM SCD 500, Leica, GER). The sizes of the microcapsules were measured by laser scatter using a zetasizer (Zetasizer Nano ZS90, Malvern, UK). Fourier transform infrared spectra were recorded using a KBr method with a spectrometer (Tensor27, Bruker, GER). The pore characteristics of porous microcapsules were measured with a surface area and pore size analyzer (BK222, JingBo, China). The specific surface areas and pore volume were evaluated according to the Brunauer−Emmett−Teller (BET) and Barrett−Joyner−Halenda (BJH) methods from the adsorption branches of the isotherms. Drug Loading Content and Entrapment Efficiency. The CAP loading content and entrapment efficiency in microcapsules were measured by disruption of the microcapsules. CAP-loaded microcapsules were fully dissolved in methylene chloride. Then, the solution was dried via reduced-pressure distillation to obtain dry precipitation. The CAP in precipitate was dissolved in methanol. Finally, the methanol solution was filtered to form a clear solution for UV−vis spectrophotometer analysis at a wavelength of 272 nm. In Vitro Release of CAP. CAP release from the microcapsules was evaluated through a dynamic dialysis method. The CAP-loaded microcapsules were dispersed in 10 mL of a methanol/water mixture (70:30, v/v) in the dialysis membrane. The dialysis membrane was immersed in 90 mL of the mixed solvent of methanol and water in a 250 mL wide-mouth flask and then placed in a shaking incubator at 25 °C. At various time intervals, 5 mL of solution outside the dialysis membrane was withdrawn and was replaced with a fresh mixed solution. The concentration of CAP was measured using a UV−vis spectrophotometer to determine the released kinetic profile of CAP. Bioassay Studies. Bioassay studies were carried out to evaluate the control efficiency of various CAP-loaded microcapsule formulations. The commercial formulation was diluted to 100, 20, 5, 1, 0.5, or 0.1 mg/L CAP with 0.05% Triton. A series of aqueous microcapsule suspensions with the same CAP concentration were prepared. Cabbage leaves were immersed in the prepared solutions for 10 s and placed into Petri dishes after drying in the air. In the control treatments, the leaves were only immersed in 0.05% triton. Each treatment was carried with 10 second instars of P. xylostella and three replications. The mortalities of predators were evaluated after exposing the individuals to insecticide residues on cabbages leaves for 48 h. All of the P. xylostella were reared in a climatic chamber at 25 ± 1 °C temperature, 75 ± 10% relative humidity, and a 12:12 h (L/D) photoperiod at the biotechnology center. SPSS software was used to calculate toxicity regression equations, LC50, and LC90. Stability Studies. The microcapsules were packed in glass tubes and stored at 4 °C for 7 days and at 50 °C for 14 days, and the changes in CAP contents and apparent morphology of the microcapsules were studied. The UV-shielding properties of the microcapsules were tested as the following. A certain amount of CAP-loaded microcapsules was dispersed with a methanol/water mixture (70:30, v/v) and divided equally into culture dishes. In the center of the reactor, a 500 W (Emax = 365 nm) UV lamp was applied to the sample at a constant temperature of 25 °C during the experiments. At various time intervals, a culture dish was taken out of the reactor, and the CAP content in the culture dish was determined using the method described above.
porosity is also a possible approach to regulate release behaviors of the drug.37 The porous particles possess high surface area to volume ratio, increasing the diffusion and release of loading ingredients.38−41 To multiply and precisely control the drug release behaviors in a wide range, the porosity of polymeric microcapsules was regulated through an osmotic induction method, which is a simple and safe method based on the double-emulsion method by addition of osmotic pressure agents into the inner water phase. In this study, we prepared novel CAP microcapsule formulations with a high loading content by encapsulating highly insoluble CAP inside the microcapsules through PME combined with the solid in oil in water (S/O/W) doubleemulsion method. PLA microspheres with controlled surface morphology and size were obtained by changing osmotic agents (bovine serum albumin (BSA) and poly(vinyl alcohol) (PVA)) and process parameters of PME, respectively. The release behaviors of CAP could be precisely controlled by regulating the size and surface morphology of PLA microcapsules. Moreover, the CAP-loaded PLA microcapsules with good thermal and light stability showed control efficiency on Plutella xylostella (Linnaeus) superior to that of the commercial formulation.
■
MATERIALS AND METHODS
Chemicals. Polylactide (PLA) was kindly provided by Dongguan Zhuyou Plastic Co., Ltd. Technical and commercial CAP were purchased from DuPont Co., Ltd., and Shanghai Shengnong Pesticide Co., Ltd., respectively. BSA was obtained from Beijing Biodee Biotechnology Co., Ltd. PVA with a Mw of 30,000−70,000 and a hydrolysis of 87−89% was purchased from Aldrich. The dialysis membrane with a specification of 35,000 was purchased from Beijing Tianan Technology Co. Ltd. The fast membrane emulsification equipment (FMEM-500M) and Shirasu porous glass (SPG) membrane were purchased from the National Engineering Research Center for Biotechnology. The SPG membranes were annulus cylinders with pore sizes of 9.0, 7.0, and 1.0 μm. Other chemical reagents were of analytical grade and purchased from Beijing Chemical Works. Preparation of CAP-Loaded PLA Microcapsules. The CAPloaded PLA microcapsules were prepared via the S/O/W doubleemulsion method combined with PME. Briefly, CAP was dispersed in a BSA/PVA aqueous solution by sonication to produce CAP suspension and used as inner water phase (S). PLA was dissolved in methylene chloride and used as oil phase (O). The inner water phase was mixed with the oil phase and sonicated to form the S/O primary emulsion. PVA was dissolved in water and used as external water phase (W). The primary emulsion was immediately poured into the external water phase under mechanical stirring to prepare a coarse S/O/W double emulsion. The coarse double emulsion was passed through a SPG membrane under a certain nitrogen pressure several times to obtain a uniform double emulsion. The droplets were solidified under mechanical stirring overnight and then collected via centrifugation. The obtained microcapsules were freeze-dried using a lyophilizer, and the dried powder was stored at 4 °C prior to use. The porous microcapsules using BSA as an osmotic agent were marked PLAB1, PLAB2, and PLAB3, of which the mass percentages of BSA in internal water phase were 5, 15, and 30%, respectively. The porous microcapsules using 1.0 and 2.0 wt % aqueous PVA solution as internal water phase were denoted PLAB3P1 and PLAB3P2, respectively. The microcapsules with different sizes were prepared by various optimal process parameters. The 4.2 μm microcapsules were produced with 100 mg/mL PLA solution as the oil phase, 1 wt % PVA aqueous solution as the external water phase, and five circulations through a 9.0 μm SPG membrane under 100 kPa pressure. The 2.2 μm microcapsules were obtained with 50 mg/mL PLA solution as the oil phase, 1 wt % PVA aqueous solution as the external water phase, B
DOI: 10.1021/acs.jafc.7b01295 J. Agric. Food Chem. XXXX, XXX, XXX−XXX
Article
Journal of Agricultural and Food Chemistry
■
RESULTS AND DISCUSSION Preparation of CAP Microcapsule Formulation. CAP is a broad-spectrum and highly effective pesticide, but its current commercial formulations are only traditional EC, SC, and WP. For various encapsulation techniques, the hydrophobic pesticides are generally dissolved in the organic solvent and finally embedded in the carrier. However, CAP is excessively insoluble in organic solvent, and thus it is difficult to obtain its microcapsule formulation. Here, we directly dispersed CAP in the inner aqueous phase and encapsulated it inside microcapsules via the S/O/W method, which is an improved method of W/O/W. The innermost phase of W/O/W is aqueous solution or water, whereas the innermost of S/O/W is water suspension of solid and here denoted S phase. The SEM image showed that the obtained microcapsules had spherical shapes, smooth surface, and uniform size distribution with a polydispersity index of 0.064 (Figure 1a), demonstrating that PME combined with
pesticide formulations. It would not only save time, manpower, and resources during the preparation process but also avoid extensive use in the spraying process. To achieve efficient CAP microcapsule formulation, we investigated its loading content and entrapment efficiency in the microcapsule at various feed weight ratios of pesticides and PLA by fixing the PLA concentration in the oil phase. As shown in Figure 2, the loading content using a saturated solution of CAP as oil phase was only 0.52%, whereas the CAP
Figure 2. Loading content and entrapment efficiency of CAP at different feed ratios.
content in various microcapsules was >14.8% and could achieve 31% at the feed of 40 wt % CAP, suggesting that the loading content could be dramatically improved by encapsulating CAP in the inner core of the microcapsule. When the feed ratio was changed from 25 to 40 wt %, the loading content of CAP increased from 23.4 to 31.2%, but the entrapment efficiency decreased from 93.6 to 78.0%. The loading content and entrapment efficiency would both reduce when the feed ratio of CAP was >40 wt %. This is reasonable because CAP became difficult to disperse in the inner water phase and quickly settled at high feeding weight, which would be filtered during the membrane emulsification and subsequently lead to a decrease of the loading efficiency. Therefore, we chose 40 wt % feed content to produce CAP microcapsule formulation with high loading contents. Preparation of Microcapsules with Controlled Porous Morphology. The surface porosity of microcapsules would have a great effect on the release behavior of loaded pesticides. More and smaller pores on the microcapsule surface would accelerate penetration and release of loaded CAP due to a higher surface area. Here, we prepared porous microcapsules via osmosis induction method and regulated their surface porosity by changing the amount of BSA/PVA as osmotic agents. As shown in Figure 3, the addition of an osmotic agent in the inner water phase would induce an osmotic gradient between internal and external water phases during the double-emulsion process. Under the osmotic pressure, water molecules in the external water phase are inclined to transfer into the inner water phase and finally form a water zone in the oil phase. As the organic solvents evaporate and oil droplets solidify, the retained water would form pores in the microcapsules after drying. Furthermore, greater osmotic pressure generated at higher osmotic agent concentration would enhance the shift of water molecules and thus induce more pores in the microcapsules.
Figure 1. (a) SEM image of CAP-loaded PLA microcapsules; (b) IR spectra of CAP, PLA, and the CAP-loaded PLA microcapsules.
double-emulsion methods could successfully prepare uniform microcapsules. FTIR measurement was used to verify the encapsulation of CAP in the microcapsule. It was found that those microcapsules showed strong peaks at 2986, 1757, and 3386 cm−1 as well as 1635 cm−1, which were characteristic bands of PLA and CAP (Figure 1b). The results indicated that CAP was encapsulated into the microcapsule. As a result, we successfully produced uniform CAP-loaded microcapsules by encapsulating dispersed CAP in the inner aqueous phase based on PME combined with double-emulsion methods. Evaluation of CAP Content in PLA Microcapsules. High pesticide loading content and entrapment efficiency in carriers are extremely desirable for the design of efficient C
DOI: 10.1021/acs.jafc.7b01295 J. Agric. Food Chem. XXXX, XXX, XXX−XXX
Article
Journal of Agricultural and Food Chemistry
10.3 to 33.3 nm when the PVA concentration was enhanced from 0 to 1.0 to 2.0 wt % with 30% BSA in the inner water phase. However, the surface area of porous microspheres decreased from 12.384 to 8.497 to 0.017 cm2/g. When the PVA aqueous solution was adopted as inner aqueous phase, greater osmosis pressures generated were supposed to attract more water shift as dichloromethane evaporated and the oil phase solidified; the water zone was retained and formed the macropores after drying. This finding clearly indicates that we could tailor the porosity of microcapsules by regulating BSA and PVA concentrations in the internal water phase. Preparation of Porous Microcapsules with Different Sizes. The size of particles is another factor influencing their release kinetics and is recognized as a tunable parameter to obtain a controlled-release system. Size homogeneity of the microcapsules is a necessary prerequisite to obtain a controlledrelease formulation. Here, we prepared uniform microcapsules with sizes of 4.2, 2.2, and 0.65 μm by changing the size of the membrane pores, the viscosity of the emulsion, and the transmembrane pressure as previously reported. Figure 5 shows that the surfaces of these microcapsules were similar if the BSA concentration in the inner aqueous phase was fixed. Controlled Release of CAP from Porous Microspheres. In recent years, it was desirable to develop pesticide formulations with accurate and quantitative release instead of a slow and qualitative one. Controlling the size and the porosity of the microcapsules is a feasible approach to precisely control release of loading drug. The release behaviors of PLA microcapsules with different surface porosity and size were investigated, using technical CAP as a control. Compared with CAP technical and commercial formulations, all prepared microcapsules exhibited relatively slow release and maintained the sustained release for a longer period (Figure 6). After 24 h, the technical CAP was totally released, and the cumulative release of commercial formulation reached 90%. The effects of
Figure 3. Schematic description for the preparation of porous microcapsules via the osmosis induction method.
We prepared various porous PLA microcapsules at different amounts of BSA/PVA and studied the morphology change of microcapsules. Figure 4 showed surface morphology changes of PLA microcapsules depended on the amounts of osmotic agent. The surface of PLA microcapsules without osmotic agent was smooth as shown in Figure 1. Whereas a few micropores were observed on the surface of PLAB1 microspheres, the PLAB3 microcapsules were completely covered with micropores. This finding indicates that the addition of BSA in the internal water phases leads to pore formation on the surface of microcapsules and the pore increase with an increase of the BSA content. Next, fixing the BSA concentration, we tried to choose the PVA aqueous solution as internal water phase to further improve the osmotic pressures and studied the morphology of microcapsule. Figure 4 shows that the pore size on the PLA microcapsule would become bigger with the PVA solution as inner water phase. The BET analyses demonstrated that the average pore sizes of PLA microspheres increased from 9.5 to
Figure 4. SEM images of various microcapsules PLAB1 (a), PLAB2 (b), PLAB3 (c), PLAB1P1 (d), PLAB2P1 (e), PLA B3P1 (f), PLAB1P2 (g), PLAB2P1 (h), and PLA B2P2 (i) prepared at different amounts of osmotic agent (BSA/PVA) in the inner water phase. D
DOI: 10.1021/acs.jafc.7b01295 J. Agric. Food Chem. XXXX, XXX, XXX−XXX
Article
Journal of Agricultural and Food Chemistry
Figure 5. SEM images of PLA microcapsules of PLAS1 (a), PLAS2 (b), and PLAS3 (c) with sizes of 4.2, 2.2, and 0.65 μm, respectively.
Figure 6. Effects of the BSA (a, b), PVA (c), and size (d) on the release behavior of CAP from PLA microcapsules.
porosity and the size of the microcapsules on the release of CAP are described in more detail below. Effects of Surface Porosity on the Release of CAP. The release behaviors of pesticide from various porous microspheres by changing the osmotic agent content were investigated. Figure 6a shows that the release of CAP from various porous PLA microcapsules was much higher than that of microcapsules without the pore. The cumulative release of CAP after 24 h reached 40.5, 48.9, and 56.6% for PLAB1, PLAB2, and PLAB3, respectively. The cumulative release of CAP from PLAB3 achieved 92.2% after 290 h, whereas the amount released from PLA was only 65.1%. This result indicated that BSA as an osmotic agent could induce pore formation in the microcapsule and greatly improve the specific surface area of microcapsule, which enhanced the drug diffusion and release from the PLA microcapsule. The relationship of the microcapsule porosity and the BSA content was further quantitatively studied. Figure 6b shows that the specific surface area and the pore volume of the microcapsule exhibited a good linear relationship with BSA concentration. This result indicates that the porosity of the microcapsule actually rises with the increase of BSA concentration, and the specific surface area and pore volume of microcapsule could be readily predicted from the feed BSA concentration. That is to say, we could regulate the porosity of the microcapsule by changing the BSA feed amount, which is of great significance to explore controlled-release pesticide formulations.
Figure 6c shows that the PLAB3P1 microcapsules showed a cumulative release of 39.8 and 72.6% for 24 and 290 h, respectively. For PLAB3P2 with high PVA concentration in the inner aqueous phase, the cumulative releases of CAP at 24 and 290 h were 32.4 and 66.4%, respectively. Interestingly, upon increasing concentration of PVA in the inner aqueous phase, cumulative release and initial burst release were reduced. That was probably because the macropores on the microcapsule prepared at PVA concentration reduce the surface area and the penetration of CAP, which slow CAP release from the microcapsules. Effects of Size on the Release of CAP. Effects of microcapsule sizes on release behaviors of CAP are shown in Figure 6d. For PLAs1 in 4.2 μm, the cumulative release rates of CAP were about 40.0% at 24 h, whereas the cumulative release rates for PLAs3 in 0.65 μm reached 59.8%. Subsequently, the cumulative release rates for PLAs1 and PLAs3 increased to 69.8 and 96.5%, respectively, after 280 h. The results showed that the microcapsule delivery system with the smallest particle size had the fastest release of active ingredient due to the higher surface area being exposed to the surroundings, aiding the permeation and effusion of the pesticide located in the shell of the microcapsule. The results demonstrate the change of particle sizes is an effectively tunable way for the precise regulation of pesticide release. Bioassay Studies of CAP-Loading Microcapsules. To verify the feasibility of the porous microcapsule suspension as a novel pesticide formulation, the bioactivity of CAP-loaded E
DOI: 10.1021/acs.jafc.7b01295 J. Agric. Food Chem. XXXX, XXX, XXX−XXX
Article
Journal of Agricultural and Food Chemistry porous microcapsules against P. xylostella was tested. Figure 7 shows that compared with the commercial formulation, the
microcapsules was only 7%. Even after 72 h, only
