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Construction of a controlled-release delivery system for pesticides using biodegradable PLA-based microcapsules
Accepted Manuscript Title: Construction of a controlled-release delivery system for pesticides using biodegradable PLA-based microcapsules Author: Baoxia Liu Yan Wang Fei Yang Xing Wang Hong Shen Haixin Cui Decheng Wu PII: DOI: Reference:
S0927-7765(16)30255-7 http://dx.doi.org/doi:10.1016/j.colsurfb.2016.03.084 COLSUB 7791
To appear in:
Colloids and Surfaces B: Biointerfaces
Received date: Revised date: Accepted date:
18-11-2015 29-3-2016 31-3-2016
Please cite this article as: Baoxia Liu, Yan Wang, Fei Yang, Xing Wang, Hong Shen, Haixin Cui, Decheng Wu, Construction of a controlled-release delivery system for pesticides using biodegradable PLA-based microcapsules, Colloids and Surfaces B: Biointerfaces http://dx.doi.org/10.1016/j.colsurfb.2016.03.084 This is a PDF file of an unedited manuscript that has been accepted for publication. As a service to our customers we are providing this early version of the manuscript. The manuscript will undergo copyediting, typesetting, and review of the resulting proof before it is published in its final form. Please note that during the production process errors may be discovered which could affect the content, and all legal disclaimers that apply to the journal pertain.
Construction of a controlled-release delivery system for pesticides using biodegradable PLA-based microcapsules Baoxia Liu a, 1, Yan Wang b, 1, Fei Yang a, Xing Wang a, Hong Shen a, Haixin Cui b, *, Decheng Wu a, * a
Beijing National Laboratory for Molecular Sciences, State Key Laboratory of Polymer Physics & Chemistry, Institute of Chemistry, Chinese Academy of Sciences, Beijing 100190, China
b
Institute of Environment and Sustainable Development in Agriculture, Chinese Academic of Agriculture Sciences, Beijing 100081, China
* Corresponding authors at: No.2, Zhongguancun North First Street Haidian District, Beijing 100190, PR China Tel：010‐8261 1492. E-mail address: [email protected] (Decheng Wu), [email protected] (Haixin Cui) 1
These authors contributed equally to this work.
Graphical abstract
1
Highlights
An aqueous microcapsule formulation was yielded via premix membrane emulsification.
The loading content of Lambda－Cyhalothrin was higher than 40%.
The sizes of microcapsule systems were tunable from 0.68 to 4.6 µm.
The stable microcapsule formulation showed high efficacy on plutella xylostella.
Abstract Conventional pesticides usually need to be used in more than recommended dosages due to their loss and degradation, which results in a large waste of resources and serious environmental pollution. Encapsulation of pesticides in biodegradable carriers is a feasible approach to develop environment-friendly and efficient controlled-release delivery system. In this work, we fabricated three kinds of polylactic acid (PLA) carriers including microspheres, microcapsules, and porous microcapsules for controlled delivery of Lambda－ Cyhalothrin (LC) via premix membrane emulsification (PME). The microcapsule delivery system had better water dispersion than the other two systems. Various microcapsules with a high LC contents as much as 40% and tunable sizes from 0.68 to 4.6 µm were constructed by manipulating the process parameters. Compared with LC technical and commercial microcapsule formulation, the microcapsule systems showed a significantly sustained release of LC for a longer period. The LC release triggered by LC diffusion and matrix degradation could be optimally regulated by tuning LC contents and particle sizes of the microcapsules. This multi-regulated release capability is of great significance to achieve the precisely controlled release of pesticides. A preliminary bioassay against plutella xylostella revealed that 0.68 µm LC-loaded microcapsules with good UV and thermal stability exhibited an activity similar to a commercial microcapsule formulation. These results demonstrated such an aqueous microcapsule delivery system had a great potential to be further explored for developing an effective and environmentally friendly pesticide-release formulation. Keywords: Controlled UV-shielding properties
release;
Water
dispersion;
Biodegradable;
Microcapsule;
Thermal-stability;
1. Introduction Currently, about 2 million tons of pesticides are applied to control pathogens and pests every year all over the world. However, it is estimated that ~90% of the pesticides actually are lost due to their degradation, photolysis, evaporation and surface runoff, and only ~0.1% of the pesticides are finally deposited on the harmfully biological targets [1-2]. This long-term extensive and inefficient use of pesticides has caused serious social concerns on food safety and ecological environment [3-6]. The social concerns motivate the researchers to develop efficient, safe and green pesticide formulations [7-9]. Conventional formulations usually rapidly fall below the effective level after an initial burst release of pesticide. A controlled delivery system could achieve continuous and stable release of active ingredients, maintaining a predetermined minimum effective level of drugs for a specified period of time [10-12], so it is an effective way to improve the utilization of pesticide via prolonging the effective duration with reduced spraying times and pesticide dosages [13-14]. Controlled-release technology of pesticides is gradually developing from 2
the simple and qualitative release toward the precise and quantitative controlled release, ultimately achieving the more economic, safe and effective insect control and reducing environmental pollution. Encapsulation of pesticides into polymeric carriers has attracted emerging interests. The polymeric matrix prevents direct exposure of pesticides to environment, reducing loss of evaporation and degradation. Biodegradable polymers, including chitosan [15-16], alginate [8-9], polyacrylamide and starch [17-18] et al., has been widely investigated for constructing pesticide carriers [19]. Polylactic acid (PLA) is a FDA-approved material widely used as drug/cell carriers in the medical field [20-26]. However, PLA has been rarely reported and studied systemically as carrier materials in the field of pesticide. The final metabolized products of PLA in vivo are carbon dioxide and water, which have no harm to human and the environment. Industrial PLA is cheap (~US$3/kg) and affordable as pesticide carriers. The molecular weights, physical properties and degradation rates of PLA can be tuned to optimize release properties of pesticides [27-30]. It is preferred to construct PLA carriers for development of safe and green pesticide formulations. The conventional encapsulation techniques of pesticides include in situ polymerization [31], interfacial polycondensation [32-33], nanoprecipitation [34], suspension cross-linking [35], and solvent evaporation/extraction [36]. However, these techniques usually require complicated process and rigorous conditions. The size of particles is difficult to control and the size distribution is very broad via mechanical stirring, homogenization or ultrasonication. Poor dispersion and uniformity as well as residual organic solvents would hinder the applications of the resulting delivery systems. Good dispersion and uniform size of the carriers can improve their adhesion on the surface of foliage and permeability to harmful insects, increasing the utilization rate and biological activity of pesticides [37-39]. Premix membrane emulsification (PME) technology is a novel and simple method to prepare uniform particles [19,40-42]. The whole procedure of PME involves using an applied pressure to force coarse emulsion to pass through the membrance without intense ultrasound and heat, which avoids the degradation of pesticides under turbulent ultrasound and high temperature. The resulting particle size in a range from hundreds nanometers to micrometers is primarily controlled by membrane pore size as well as the process parameters including applied membrane, membrane pressure and viscosity of emulsion, which is suitable to obtain various reproduced carries [43-44]. In a word, the PME is a promising industrialized technique in construction of pesticide-delivery systems because of its distinguishing characters such as low energy consumption, easy operation and simple equipment, amendable for large scale production [45]. The reported microcapsules and commercial microcapsules for pesticides are generally in micron scale and inhomogeneous. Herein, we adopt the PME combining with the emulsion method to fabricate uniform PLA-based carriers with tunable sizes from 0.68 to 4.6 µm for controlled release of pesticides. Several delivery systems, employing microcapsules, microspheres, and porous microcapsules, were fabricated and investigated. The results showed that the microcapsule-based delivery system had better dispersion, high loading content and entrapment efficiency, an effective size-controlled property, and good ultraviolet (UV) shielding and stability.
2. Materials and methods 2.1. Materials Polylactide (PLA) and Lambda－Cyhalothrin (LC) were kindly provided by Dongguan Zhuyou Plastic Co., Ltd. (Dongguan, China), and Yangnong Chemical Co., Ltd. (Yangzhou, China), respectively. Bovine serum albumin (BSA) was obtained from Beijing Biodee Biotechnology Co., Ltd. (Beijing, China). Poly(vinyl alcohol) (PVA) with a molecular weight of 30,000-70,000 and a hydrolysis of 87-89% was purchased from Sigma-Aldrich (St. Louis, MO). The specification of the dialysis membrane purchased from Beijing Tianan 3
Technology Co., Ltd., (Beijing, China) was 50,000. Other chemical reagents were of analytical grade and purchased from Beijing Chemical Works (Beijing, China). 2.2. Preparation of the LC-loaded microcapsules The PLA microcapsules were prepared via a water-in-oil-in-water (W1/O/W2) double-emulsion method combined with PME. Briefly, PLA and LC were dissolved in methylene chloride as the oil phase. The oil phase was mixed with deionized water and sonicated to form the primary emulsion of W1/O. Then, the primary emulsion was immediately poured into a large volume of aqueous PVA solution under mechanical stirring to prepare coarse double emulsion. The coarse double emulsion was extruded through an SPG membrane under a certain nitrogen pressure several times. The uniform double emulsion was obtained and solidified under magnetic stirring overnight. The hardened microcapsules were collected via centrifugation and washed with deionized water three times. The obtained microcapsules were freeze-dried using a lyophilizer to yield a free-flowing powder. The dried powder was stored at 4 °C prior to use. The microcapsules with different sizes were prepared with various optimal process parameters. The 4.6 µm microcapsules were obtained with 100 mg/mL PLA in the oil phase, 1 wt.% PVA concentration in the external water phase, and 4 circulations through a 9.0 μm SPG membrane under 80 KPa pressure. The 2.4 µm microcapsules were produced with 50 mg/mL PLA in the oil phase, 1 wt.% PVA concentration in the external water phase, and 4 circulations through the 7.2 μm SPG membrane under 80 KPa pressure. The 0.68 µm microcapsules were yielded with 30 mg/mL PLA in the oil phase, 0.1 wt.% PVA concentration in the external water phase, and 3 circulations through the 1.0 μm SPG membrane under 1000 KPa pressure. The feed weight ratios of LC/PLA were 1/9, 1/4, 3/7, 2/3 and 1/1 to produce various microcapsules with different LC contents for further study. The preparation process of LC-loaded porous microcapsules (50 wt.% LC) was the same as that of producing 4.6 μm microcapsule except that 0.4 g BSA was added into 4 mL of the inner water phase to induce formation of micropores on surface of the microcapsules. 2.3. Preparation of the microspheres The PLA microspheres were prepared via an oil-in-water (O/W) emulsion method combined with PME. Briefly, PLA and LC were dissolved in methylene chloride. The solution was immediately poured into an aqueous PVA solution under mechanical stirring to prepare the coarse double emulsion. The coarse double emulsion was then extruded through an SPG membrane under a certain nitrogen pressure several times. The uniform emulsion was obtained and solidified under magnetic stirring overnight. The hardened microspheres were collected via centrifugation and washed with deionized water three times. The microspheres were further freeze-dried to yield a free-flowing powder. The dried powder was stored at 4 °C prior to use. The 4.6 µm LC-loaded microspheres (50 wt.% LC) were prepared by the following optimal process parameters: 200 mg/mL PLA in the oil phase, 1 wt.% PVA concentration in the water phase, and 4 circulations through a 9.0 μm SPG membrane under 100 KPa pressure. 2.4. Characterization of the LC-loaded microcapsules and microspheres The morphology of the microparticles was observed via scanning electron microscopy (SEM, JSM-6700F; JEOL Ltd., Tokyo, Japan) with an accelerating voltage of 5 kV and a working distance of 8 mm. The sample was 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 microparticles were measured with laser scatter using a zetasizer (Zetasizer Nano ZS90; Malvern Instruments LTD., Malvern, UK). 4
2.5. Drug content and entrapment efficiency The loading content and entrapment efficiency of LC in the formulations were determined by high-performance liquid chromatography (HPLC) (Agilent 1260, Agilent Technologies, Santa Clara, CA). An approximately 30 mg sample was fully dissolved in 30 mL of methylene chloride. The solution was added to ethyl alcohol and dried via reduced-pressure distillation. Then, 5 mL of HPLC-grade ethanol was added to soak the LC from the dried precipitation. Finally, the mixture was filtered to form a clear solution for HPLC analysis. The mobile phase consisting of acetonitrile/water (90:10) was applied. A reverse-phase Inertsil C-18 column (150 mm × 4.6 mm, pore size 5 mm, GL Science Inc., Tokyo, Japan) was used. The flow rate of the mobile phase was set at 1 mL/min. The sample was detected at 230 nm with a UV-vis detector. 2.6. In vitro release of LC To study in vitro release of the encapsulated LC from the microcapsules, a 10 mL of LC suspension in an ethanol/water mixture (30:70, v/v) was loaded in the dialysis membrane. Then the membrane was placed into a wild-mouth flask with 90 mL of the mixed solution of ethanol and water. The flask was incubated in the incubator shakers under shaking at 300 rpm. Next, 2 mL of the mixed solvent outside the dialysis membrane was withdrawn over a period of 250 h at various time intervals and was replaced with a fresh mixed solution. The concentration of LC was measured using a UV-vis spectrophotometer at a wavelength of 280 nm to determine the kinetic profile of the released LC. 2.7. Biological assay A preliminary bioassay of LC-loaded microcapsules with various sizes (from 0.68 to 4.6 µm) against plutella xylostella was evaluated using a commercial formulation as control. The LC-loaded microcapsules and commercial formulation with a LC concentration to be 25 g/L were diluted into different concentrations with 0.1 % triton to prepare the test suspensions according to statistical requirements. The 2rd instars of plutella xylostella were immersed in the prepared test suspensions for 10 seconds, and then were placed on cabbage leaves in petri dishes to investigate the mortalities after 48 h. Assessments were made on a dead/alive basis, and mortality rates were corrected using Abbott’s formula. SPSS software was used to calculate toxicity regression equations, LC50 and confidence limits. The toxicity regression equation was given as below: Y = bX + a Where Y is the mortality rate of treated group. X is the logarithm of pesticide dosage. LC50 means the dosage when the mortality rate of the treated group was 50%. 2.8. Stability test The microcapsules were packed in glass tubes and stored at 4 °C for 7 days and 50 °C for 14 days, and the changes in loading contents and surface of the microcapsules were studied. The UV-shielding properties of the microcapsules were tested as follows. A certain amount of LC-loaded microcapsules were mixed with an ethanol/water mixture (70:30, v/v), and the suspension was transferred into a culture dish. In the center of the reactor, a 500 W (Emax = 365 nm) UV lamp was applied to the sample for a desired period of time. The temperature was maintained at 25 °C during the experiments. At various time intervals, the culture dish was removed from the reactor, and the content of LC in the culture dish was determined using the method described above. The concentration of LC was measured with a UV-vis spectrophotometer at a wavelength of 280 nm to determine the kinetic profile of the released LC. 5
3. Results and discussion 3.1. Preparation of the LC delivery systems The structure of the delivery system is one of the most key factors affecting the dispersion of pesticide. Good uniformity and dispersion of the pesticide-delivery system is conducive to improving adhesion and permeability of the pesticide on target crops and to achieving effective utilization and high bioavailability of the pesticide. To obtain an optimal delivery system with stable and homogeneous dispersion, we constructed three kinds of LC-delivery systems based on microspheres, microcapsules and porous microcapsules, by combining the emulsion method (O/W for microspheres and W/O/W for microcapsules) with PME and the osmosis induction method for formation of the porous structures, as illustrated in Fig. 1. Fig. 2 depicts the morphologies and cross-sections of the various delivery systems. The microspheres (Fig. 2A) had an almost identical spherical shapes and smooth surfaces as the microcapsule systems (Fig. 2B), but a distinct solid inner structure instead of a hollow inner cavity for the microcapsule system from the cross-sectional images. The results demonstrated the O/W emulsion and W1/O/W2 double-emulsion methods combined with PME can successfully prepare solid-microsphere and hollow-microcapsule delivery systems, respectively. During the preparation of the microcapsule-based delivery systems, addition of porogen BSA in the inner water phase was favorable to form porous microcapsules. Fig. 2C indicated that some small pores appeared in the surfaces of the microcapsules. The formation of the pores should result from water shift under osmotic pressures between the internal and external water phases after introduction of BSA in the inner water phase. Some water molecules go into the inner, also resulting in formation of bigger hollow cavity than the microcapsules. It must note that the entire PME procedure used an applied pressure to force coarse emulsion to pass through a membrane without intense ultrasound or heat, avoiding the degradation of the pesticide under turbulent-ultrasound and high-temperature conditions. The dispersion capacity and stability of the pesticide delivery systems in water are vital for successful construction of their aqueous formulations. The aqueous microcapsule suspension could maintain stable and homogeneous even after standing for 4 h instead of obvious deposition observed from the microsphere and porous microcapsule suspensions, resulting from different gravity effects as shown in Fig. 2D. The microcapsule is light and has high buoyancy in water due to its hollow structure, which is favorable to slow down the settling of the microcapsule. The microsphere with a solid structure possesses high density, resulting in its rapid settlement. For the porous microcapsule suspension, the open pores allow water to go into the inner cores, causing a loss of buoyancy and subsequently accelerated precipitation. So the microcapsule is optimal to be further studied for developing the aqueous formulation of pesticides. 3.2. The loading content and entrapment efficiency of LC The loading content and entrapment efficiency of pesticides in the microcapsules have an important effect on overall performance of the pesticide delivery system. High entrapment efficiency and loading content are preferred to reduce the waste of pesticide during the preparation process as well as to avoid its extensive use in spraying and subsequent environmental pollution. To achieve high loading content as well as suitable entrapment efficiency, we constructed several specimens by feeding various weight ratios of PLA/LC. Table 1 indicated that all the specimens had loading efficiencies of higher than 82%. The results demonstrate the mild PME process can effectively avoid damage to the pesticide activity without a prolonged period of intense ultrasound and/or heating. As the feeding weight contents of LC increased from 10% to 50%, the loading 6
contents of LC increased from 9.5% to 41.0% with the loading efficiency decreased from 95% to 82%. It is reasonable as the LC feeding contents increased, the corresponding reduced concentrations of PLA lowered the LC encapsulation ability, subsequently leading to decrease of the loading efficiency. However, even for 50 wt% feeding contents of LC, the loading efficiency still can reach as high as 82%. So, we chose 50 wt% feeding contents of LC to produce the LC delivery systems with high loading contents. 3.3. Preparation of the LC-loaded microcapsules with different sizes The particle size is an important tunable parameter in controlled release system and definitely has a pronounced influence on the release kinetics due to its different surface-area-to-volume ratios. PME can easily adjust the size of the microcapsules in a large range by simply changing the size of membrane pores, the viscosity of the emulsion and the transmembrane pressure. Here, we adopted three SPG membranes with pore sizes of 9.0, 7.2 and 1.0 µm to produce various LC-loaded microcapsules with sizes to be 4.6, 2.4 and 0.68 µm and LC contents to be 40.8%，40.5% and 40.9%, designated as MC1, MC2, and MC3 as indicated in Fig. 3. All the microcapsules had smooth surfaces and spherical shapes, and narrow size distributions. 3.4. Controlled release of LC in vitro In recent years, development of pesticide-release systems has transited toward accurate and quantitative release from slow and qualitative release. To achieve controllable and precise release, we systematically investigated the release profiles of the LC-loaded microcapsules with various particle sizes and pesticide contents. Compared with LC technical and commercial microcapsule formulation, all our microcapsules released LC in relatively slow speeds and maintained its sustained release for longer periods (Fig. 4). The LC technical was totally released after 18 h, and the cumulative release of commercial microcapsule formulation reached 90% in 12 h. Our microcapsules had an initial burst release in the first 25 h and then maintained a sustained and stable release. In the initial stage, the loaded pesticide at the surface of the microcapsules would dissolve and leak into the surroundings quickly, resulting in the burst release. It must note that the release profiles in vitro obtained from an incubation medium of ethanol-water cannot precisely exhibit a practical release law in nature. Generally, when the microcapsules are exposed to air or aqueous environments in nature, the release rate of pesticides should turn slower, and initial rapid release may be reduced. However, the various release profiles of different drug delivery systems still can provide an insightful understanding to evaluate their discrepancies in pesticide release, especially for further release study in vivo. The effects of loading content and the size of the capsules on the release of LC are described in more detail below. 3.4.1. Effects of size on the LC release We investigated the effects of size on the release profile of LC. As shown in Fig. 4A, all the microcapsules had a slow release, lasting for 250 h. As the size decreased from 4.6 nm to 0.68 µm, the cumulative release increased from 67% to 82% after 48 h and from 80% to 95% after 250 h. The results showed that the LC-microcapsule delivery system with the smaller particle size had the faster release of the 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 change of particle sizes is an effectively tunable way for the precise regulation of pesticide release.
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3.4.2. Effects of the loading content on the LC release In order to achieve a multi-regulated release system, the effects of the LC loading content on the release behavior were further investigated. The microcapsules with different LC contents were obtained by changing the LC feeding content while maintaining the same PLA concentration. As shown in Fig. 4B, the cumulative release rates of LC increased from 78% to 95% after 250 h with increased loading content from 22% to 45%. Higher LC contents in MC6 occupy more spaces in the shells of the microcapsules. When the pesticide molecules migrated and dissolved to the surroundings, more voids in MC6 are formed, resulting in subsequent accelerated pesticide release. 3.4.3. The release mechanism of the LC-loaded micocapsules In order to investigate the release mechanism of the LC-loaded microcapsules, the release data were analyzed by applying the exponential relation proposed by Ritger and Peppas [46]: (1) Mt/M0=ktn Where Mt/M0 is the percentage of active ingredient released at time t, k is a constant, and n is the diffusion exponent. (The values of the diffusion exponent n and T50, the time taken for 50% of the active ingredient to be released into water, were calculated and presented in Table 2.) There was a good correlation between the release profiles of LC and the empirical equation with correlation coefficients greater than 0.98. The lowest values of T50 for MC3 and MC6 indicated the faster release of LC from suspensions of microcapsules with smaller capsule sizes and higher loading contents. The values of the diffusion exponent n were larger than 0.45, indicating that the LC release from the microcapsules was controlled by both a diffusion and matrix degradation. As depicted in Fig. 4(C, D), the surface of the microcapsules after 250 h in the medium became rough, and some pores appeared in the shells, indicating the degradation of the microcapsules. The degradation occurred because of the hydrolysis of the PLA on the surface. 3.5. Bioassay study of the LC-loaded microcapsules To verify feasibility of the microcapsule suspension as a novel pesticide formulation, bioactivity of LC in different microcapsules against plutella xylostella was tested. Fig. 5 indicated the MC3 microcapsule (0.68 µm) had a lower LC50 value and exhibited obviously higher activity than the other two microcapsules, suggesting that efficacy of pesticide gradually increased with the decrease of the microcapsule size. The MC3 microcapsule had the similar efficacy as a commercial formulation. The high efficacy should ascribe to that the nano-sized microcapsules can enhance the adhesivity and penetrability of pesticide on surface of crops, subsequently reducing leaking loss of pesticide during spraying operation. However, compared with the commercial formulation, our LC loaded microcapsules are more environmentally friendly because they could be directly dispersed in water, avoiding the usage of toxic organic solvents and a large number of pesticide adjuvants. 3.6. Stability of the LC-loaded microcapsules 3.6.1 UV-shielding properties of the microcapsules for LC The photodegradation of pesticides after spraying seriously destroys the activities of these pesticides and reduces their utilization rates. This encapsulation technology is an important mean to improve the optical stability of pesticides by incorporating pesticides into capsule shells, preventing photodegradation and slowing 8
down the release rate of pesticide. Fig. 6A showed that the decomposition rate of the encapsulated LC was significantly reduced compared to that of LC technical. The photolysis rate of LC technical was more than 11% after 12 h of UV irradiation, while that of LC in the microcapsules was only 3%. Even after 72 h, only less than 18% of LC was degraded for the microcapsules compared with up to 60% for the LC technical. The results clearly demonstrate that the microcapsules could effectively reduce LC photolysis, indicating the microcapsule formulation has the remarkable UV-shielding properties for LC. 3.6. 2Thermal-stability of the LC-loaded microcapsules The storage stability is another key factor that affects the quality of a pesticide formulation. During storage, delamination, caking, and degradation of the active pesticides may occur, reducing the utilization of the pesticides and thus wasting resources. To determine LC thermal stability of the microcapsules, the samples were stored at 4 °C for 7 days and 54 °C for 14 days. As shown in Fig. 6(C-E), there were no obvious morphologic changes observed, indicating that the microcapsules had good thermal stability. As shown in Fig. 6B, the LC content only had a negligible loss after storage at 4 °C for 7 days. A small loss of LC was observed after 14 days at 54 °C due to the degradation of LC at high temperatures. The results showed that the solid microcapsules had good storage stability.
4. Conclusion In this work, we developed an environment-friendly controlled-release delivery system for LC using biodegradable PLA as the microcapsule shells. The LC-loaded microcapsule system had good water dispersion and stability. Manipulating the process parameters constructed various systems with LC contents as higher than 40% as well as tunable sizes from 0.68 to 4.6 µm. Compared with both LC technical and commercial microcapsule formulations, the LC-loaded microcapsule systems yielded LC sustained release for a longer period. The LC release was controlled by LC diffusion and matrix degradation. The 0.68 µm microcapsule system with good UV and thermal stability had the similar efficacy as the commercial formulation. We envision that such an environmentally friendly novel pesticide formulation should be greatly potential for wide applications in the field of agriculture.
Acknowledgments The authors thank the Major National Scientific Research Program of China (Nos. 2014CB932200 and 2014BAI11B04), the National Young Thousand Talents Program and the National Natural Science Foundation of China (NSFC, Nos. 21174147, 21474115 and 51103165), and the Basic Scientific Research Fund of the National Nonprofit Institutes for financial support.
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[45] C. Charcosset, I. Limayem, H. Fessi, J. Chem. Technol. Biotechnol. 79 (2004) 209–218. [46] P.L. Ritger, N.A. Peppas, J. Control. Release 5 (1987) 23–36.
11
Fig. 1. Schematic description for preparation of microspheres, microcapsules and porous microcapsules (A) and PME process (B).
12
Fig. 2. SEM images of the (A) microspheres, (B) microcapsules, and (C) porous microspheres before and after cut by a super thin blade and optical photos of their suspensions (D) in water after setting for 4 h. The concentrations both are 2 mg/mL, respectively.
13
Fig. 3. SEM images of LC-loaded microcapsules (A) MC1, (B) MC2, and (C) MC3 with average sizes of 4.6, 2.4 and 0.68 µm and PDIs of 0.122, 0.090 and 0.066, respectively.
14
Fig. 4. Effects of the sizes (A; 4.6, 2.4 and 0.68 µm for MC1, MC2 and MC3 with 50% feeding contents of LC) and LC loading contents (B; 22%, 28% and 45% for MC4, MC5 and MC6 constructed using 1µm SPG membrane) on the release behavior of LC, and SEM images of surface morphology of the MC1 before (C) and after (D) soaking in an ethanol/water mixture (30:70, v/v) for 250 h.
15
Fig. 5. The LC50 values of the MC1, MC2, MC3 and commercial formulations in bioassay study.
16
Fig. 6. Comparison of the LC photolysis percentage of LC technical and MC3 formulations under UV irradiation (A) and LC contents (B) and its SEM images of the MC3 microcapsules before (C), and after 4 °C for 7 days (D) and 54 °C for 14 days (E).
17
Table 1 The LC loading content and loading efficiency of the microcapsules with different feed ratios of PLA/LC
Weight contents of LC (%) Loading content (%) Loading efficiency (%) 10
9.5
95.0
20
18.2
89.3
30
28.7
86.2
40
34.4
83.6
50
41.0
82.0
Table 2 Constants from fitting the generalized model of Eq. (1) to the release data of LC from the different capsule suspensions
Sample
r
n
T50
MC1
0.9891
0.73
16.0
MC2
0.9914
0.82
14.5
MC3
0.9942
0.83
14.1
MC4
0.9930
0.72
18.6
MC5
0.9934
0.75
16.2
MC6
0.9954
0.65
14.6
18
S0927-7765(16)30255-7 http://dx.doi.org/doi:10.1016/j.colsurfb.2016.03.084 COLSUB 7791
To appear in:
Colloids and Surfaces B: Biointerfaces
Received date: Revised date: Accepted date:
18-11-2015 29-3-2016 31-3-2016
Please cite this article as: Baoxia Liu, Yan Wang, Fei Yang, Xing Wang, Hong Shen, Haixin Cui, Decheng Wu, Construction of a controlled-release delivery system for pesticides using biodegradable PLA-based microcapsules, Colloids and Surfaces B: Biointerfaces http://dx.doi.org/10.1016/j.colsurfb.2016.03.084 This is a PDF file of an unedited manuscript that has been accepted for publication. As a service to our customers we are providing this early version of the manuscript. The manuscript will undergo copyediting, typesetting, and review of the resulting proof before it is published in its final form. Please note that during the production process errors may be discovered which could affect the content, and all legal disclaimers that apply to the journal pertain.
Construction of a controlled-release delivery system for pesticides using biodegradable PLA-based microcapsules Baoxia Liu a, 1, Yan Wang b, 1, Fei Yang a, Xing Wang a, Hong Shen a, Haixin Cui b, *, Decheng Wu a, * a
Beijing National Laboratory for Molecular Sciences, State Key Laboratory of Polymer Physics & Chemistry, Institute of Chemistry, Chinese Academy of Sciences, Beijing 100190, China
b
Institute of Environment and Sustainable Development in Agriculture, Chinese Academic of Agriculture Sciences, Beijing 100081, China
* Corresponding authors at: No.2, Zhongguancun North First Street Haidian District, Beijing 100190, PR China Tel：010‐8261 1492. E-mail address: [email protected] (Decheng Wu), [email protected] (Haixin Cui) 1
These authors contributed equally to this work.
Graphical abstract
1
Highlights
An aqueous microcapsule formulation was yielded via premix membrane emulsification.
The loading content of Lambda－Cyhalothrin was higher than 40%.
The sizes of microcapsule systems were tunable from 0.68 to 4.6 µm.
The stable microcapsule formulation showed high efficacy on plutella xylostella.
Abstract Conventional pesticides usually need to be used in more than recommended dosages due to their loss and degradation, which results in a large waste of resources and serious environmental pollution. Encapsulation of pesticides in biodegradable carriers is a feasible approach to develop environment-friendly and efficient controlled-release delivery system. In this work, we fabricated three kinds of polylactic acid (PLA) carriers including microspheres, microcapsules, and porous microcapsules for controlled delivery of Lambda－ Cyhalothrin (LC) via premix membrane emulsification (PME). The microcapsule delivery system had better water dispersion than the other two systems. Various microcapsules with a high LC contents as much as 40% and tunable sizes from 0.68 to 4.6 µm were constructed by manipulating the process parameters. Compared with LC technical and commercial microcapsule formulation, the microcapsule systems showed a significantly sustained release of LC for a longer period. The LC release triggered by LC diffusion and matrix degradation could be optimally regulated by tuning LC contents and particle sizes of the microcapsules. This multi-regulated release capability is of great significance to achieve the precisely controlled release of pesticides. A preliminary bioassay against plutella xylostella revealed that 0.68 µm LC-loaded microcapsules with good UV and thermal stability exhibited an activity similar to a commercial microcapsule formulation. These results demonstrated such an aqueous microcapsule delivery system had a great potential to be further explored for developing an effective and environmentally friendly pesticide-release formulation. Keywords: Controlled UV-shielding properties
release;
Water
dispersion;
Biodegradable;
Microcapsule;
Thermal-stability;
1. Introduction Currently, about 2 million tons of pesticides are applied to control pathogens and pests every year all over the world. However, it is estimated that ~90% of the pesticides actually are lost due to their degradation, photolysis, evaporation and surface runoff, and only ~0.1% of the pesticides are finally deposited on the harmfully biological targets [1-2]. This long-term extensive and inefficient use of pesticides has caused serious social concerns on food safety and ecological environment [3-6]. The social concerns motivate the researchers to develop efficient, safe and green pesticide formulations [7-9]. Conventional formulations usually rapidly fall below the effective level after an initial burst release of pesticide. A controlled delivery system could achieve continuous and stable release of active ingredients, maintaining a predetermined minimum effective level of drugs for a specified period of time [10-12], so it is an effective way to improve the utilization of pesticide via prolonging the effective duration with reduced spraying times and pesticide dosages [13-14]. Controlled-release technology of pesticides is gradually developing from 2
the simple and qualitative release toward the precise and quantitative controlled release, ultimately achieving the more economic, safe and effective insect control and reducing environmental pollution. Encapsulation of pesticides into polymeric carriers has attracted emerging interests. The polymeric matrix prevents direct exposure of pesticides to environment, reducing loss of evaporation and degradation. Biodegradable polymers, including chitosan [15-16], alginate [8-9], polyacrylamide and starch [17-18] et al., has been widely investigated for constructing pesticide carriers [19]. Polylactic acid (PLA) is a FDA-approved material widely used as drug/cell carriers in the medical field [20-26]. However, PLA has been rarely reported and studied systemically as carrier materials in the field of pesticide. The final metabolized products of PLA in vivo are carbon dioxide and water, which have no harm to human and the environment. Industrial PLA is cheap (~US$3/kg) and affordable as pesticide carriers. The molecular weights, physical properties and degradation rates of PLA can be tuned to optimize release properties of pesticides [27-30]. It is preferred to construct PLA carriers for development of safe and green pesticide formulations. The conventional encapsulation techniques of pesticides include in situ polymerization [31], interfacial polycondensation [32-33], nanoprecipitation [34], suspension cross-linking [35], and solvent evaporation/extraction [36]. However, these techniques usually require complicated process and rigorous conditions. The size of particles is difficult to control and the size distribution is very broad via mechanical stirring, homogenization or ultrasonication. Poor dispersion and uniformity as well as residual organic solvents would hinder the applications of the resulting delivery systems. Good dispersion and uniform size of the carriers can improve their adhesion on the surface of foliage and permeability to harmful insects, increasing the utilization rate and biological activity of pesticides [37-39]. Premix membrane emulsification (PME) technology is a novel and simple method to prepare uniform particles [19,40-42]. The whole procedure of PME involves using an applied pressure to force coarse emulsion to pass through the membrance without intense ultrasound and heat, which avoids the degradation of pesticides under turbulent ultrasound and high temperature. The resulting particle size in a range from hundreds nanometers to micrometers is primarily controlled by membrane pore size as well as the process parameters including applied membrane, membrane pressure and viscosity of emulsion, which is suitable to obtain various reproduced carries [43-44]. In a word, the PME is a promising industrialized technique in construction of pesticide-delivery systems because of its distinguishing characters such as low energy consumption, easy operation and simple equipment, amendable for large scale production [45]. The reported microcapsules and commercial microcapsules for pesticides are generally in micron scale and inhomogeneous. Herein, we adopt the PME combining with the emulsion method to fabricate uniform PLA-based carriers with tunable sizes from 0.68 to 4.6 µm for controlled release of pesticides. Several delivery systems, employing microcapsules, microspheres, and porous microcapsules, were fabricated and investigated. The results showed that the microcapsule-based delivery system had better dispersion, high loading content and entrapment efficiency, an effective size-controlled property, and good ultraviolet (UV) shielding and stability.
2. Materials and methods 2.1. Materials Polylactide (PLA) and Lambda－Cyhalothrin (LC) were kindly provided by Dongguan Zhuyou Plastic Co., Ltd. (Dongguan, China), and Yangnong Chemical Co., Ltd. (Yangzhou, China), respectively. Bovine serum albumin (BSA) was obtained from Beijing Biodee Biotechnology Co., Ltd. (Beijing, China). Poly(vinyl alcohol) (PVA) with a molecular weight of 30,000-70,000 and a hydrolysis of 87-89% was purchased from Sigma-Aldrich (St. Louis, MO). The specification of the dialysis membrane purchased from Beijing Tianan 3
Technology Co., Ltd., (Beijing, China) was 50,000. Other chemical reagents were of analytical grade and purchased from Beijing Chemical Works (Beijing, China). 2.2. Preparation of the LC-loaded microcapsules The PLA microcapsules were prepared via a water-in-oil-in-water (W1/O/W2) double-emulsion method combined with PME. Briefly, PLA and LC were dissolved in methylene chloride as the oil phase. The oil phase was mixed with deionized water and sonicated to form the primary emulsion of W1/O. Then, the primary emulsion was immediately poured into a large volume of aqueous PVA solution under mechanical stirring to prepare coarse double emulsion. The coarse double emulsion was extruded through an SPG membrane under a certain nitrogen pressure several times. The uniform double emulsion was obtained and solidified under magnetic stirring overnight. The hardened microcapsules were collected via centrifugation and washed with deionized water three times. The obtained microcapsules were freeze-dried using a lyophilizer to yield a free-flowing powder. The dried powder was stored at 4 °C prior to use. The microcapsules with different sizes were prepared with various optimal process parameters. The 4.6 µm microcapsules were obtained with 100 mg/mL PLA in the oil phase, 1 wt.% PVA concentration in the external water phase, and 4 circulations through a 9.0 μm SPG membrane under 80 KPa pressure. The 2.4 µm microcapsules were produced with 50 mg/mL PLA in the oil phase, 1 wt.% PVA concentration in the external water phase, and 4 circulations through the 7.2 μm SPG membrane under 80 KPa pressure. The 0.68 µm microcapsules were yielded with 30 mg/mL PLA in the oil phase, 0.1 wt.% PVA concentration in the external water phase, and 3 circulations through the 1.0 μm SPG membrane under 1000 KPa pressure. The feed weight ratios of LC/PLA were 1/9, 1/4, 3/7, 2/3 and 1/1 to produce various microcapsules with different LC contents for further study. The preparation process of LC-loaded porous microcapsules (50 wt.% LC) was the same as that of producing 4.6 μm microcapsule except that 0.4 g BSA was added into 4 mL of the inner water phase to induce formation of micropores on surface of the microcapsules. 2.3. Preparation of the microspheres The PLA microspheres were prepared via an oil-in-water (O/W) emulsion method combined with PME. Briefly, PLA and LC were dissolved in methylene chloride. The solution was immediately poured into an aqueous PVA solution under mechanical stirring to prepare the coarse double emulsion. The coarse double emulsion was then extruded through an SPG membrane under a certain nitrogen pressure several times. The uniform emulsion was obtained and solidified under magnetic stirring overnight. The hardened microspheres were collected via centrifugation and washed with deionized water three times. The microspheres were further freeze-dried to yield a free-flowing powder. The dried powder was stored at 4 °C prior to use. The 4.6 µm LC-loaded microspheres (50 wt.% LC) were prepared by the following optimal process parameters: 200 mg/mL PLA in the oil phase, 1 wt.% PVA concentration in the water phase, and 4 circulations through a 9.0 μm SPG membrane under 100 KPa pressure. 2.4. Characterization of the LC-loaded microcapsules and microspheres The morphology of the microparticles was observed via scanning electron microscopy (SEM, JSM-6700F; JEOL Ltd., Tokyo, Japan) with an accelerating voltage of 5 kV and a working distance of 8 mm. The sample was 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 microparticles were measured with laser scatter using a zetasizer (Zetasizer Nano ZS90; Malvern Instruments LTD., Malvern, UK). 4
2.5. Drug content and entrapment efficiency The loading content and entrapment efficiency of LC in the formulations were determined by high-performance liquid chromatography (HPLC) (Agilent 1260, Agilent Technologies, Santa Clara, CA). An approximately 30 mg sample was fully dissolved in 30 mL of methylene chloride. The solution was added to ethyl alcohol and dried via reduced-pressure distillation. Then, 5 mL of HPLC-grade ethanol was added to soak the LC from the dried precipitation. Finally, the mixture was filtered to form a clear solution for HPLC analysis. The mobile phase consisting of acetonitrile/water (90:10) was applied. A reverse-phase Inertsil C-18 column (150 mm × 4.6 mm, pore size 5 mm, GL Science Inc., Tokyo, Japan) was used. The flow rate of the mobile phase was set at 1 mL/min. The sample was detected at 230 nm with a UV-vis detector. 2.6. In vitro release of LC To study in vitro release of the encapsulated LC from the microcapsules, a 10 mL of LC suspension in an ethanol/water mixture (30:70, v/v) was loaded in the dialysis membrane. Then the membrane was placed into a wild-mouth flask with 90 mL of the mixed solution of ethanol and water. The flask was incubated in the incubator shakers under shaking at 300 rpm. Next, 2 mL of the mixed solvent outside the dialysis membrane was withdrawn over a period of 250 h at various time intervals and was replaced with a fresh mixed solution. The concentration of LC was measured using a UV-vis spectrophotometer at a wavelength of 280 nm to determine the kinetic profile of the released LC. 2.7. Biological assay A preliminary bioassay of LC-loaded microcapsules with various sizes (from 0.68 to 4.6 µm) against plutella xylostella was evaluated using a commercial formulation as control. The LC-loaded microcapsules and commercial formulation with a LC concentration to be 25 g/L were diluted into different concentrations with 0.1 % triton to prepare the test suspensions according to statistical requirements. The 2rd instars of plutella xylostella were immersed in the prepared test suspensions for 10 seconds, and then were placed on cabbage leaves in petri dishes to investigate the mortalities after 48 h. Assessments were made on a dead/alive basis, and mortality rates were corrected using Abbott’s formula. SPSS software was used to calculate toxicity regression equations, LC50 and confidence limits. The toxicity regression equation was given as below: Y = bX + a Where Y is the mortality rate of treated group. X is the logarithm of pesticide dosage. LC50 means the dosage when the mortality rate of the treated group was 50%. 2.8. Stability test The microcapsules were packed in glass tubes and stored at 4 °C for 7 days and 50 °C for 14 days, and the changes in loading contents and surface of the microcapsules were studied. The UV-shielding properties of the microcapsules were tested as follows. A certain amount of LC-loaded microcapsules were mixed with an ethanol/water mixture (70:30, v/v), and the suspension was transferred into a culture dish. In the center of the reactor, a 500 W (Emax = 365 nm) UV lamp was applied to the sample for a desired period of time. The temperature was maintained at 25 °C during the experiments. At various time intervals, the culture dish was removed from the reactor, and the content of LC in the culture dish was determined using the method described above. The concentration of LC was measured with a UV-vis spectrophotometer at a wavelength of 280 nm to determine the kinetic profile of the released LC. 5
3. Results and discussion 3.1. Preparation of the LC delivery systems The structure of the delivery system is one of the most key factors affecting the dispersion of pesticide. Good uniformity and dispersion of the pesticide-delivery system is conducive to improving adhesion and permeability of the pesticide on target crops and to achieving effective utilization and high bioavailability of the pesticide. To obtain an optimal delivery system with stable and homogeneous dispersion, we constructed three kinds of LC-delivery systems based on microspheres, microcapsules and porous microcapsules, by combining the emulsion method (O/W for microspheres and W/O/W for microcapsules) with PME and the osmosis induction method for formation of the porous structures, as illustrated in Fig. 1. Fig. 2 depicts the morphologies and cross-sections of the various delivery systems. The microspheres (Fig. 2A) had an almost identical spherical shapes and smooth surfaces as the microcapsule systems (Fig. 2B), but a distinct solid inner structure instead of a hollow inner cavity for the microcapsule system from the cross-sectional images. The results demonstrated the O/W emulsion and W1/O/W2 double-emulsion methods combined with PME can successfully prepare solid-microsphere and hollow-microcapsule delivery systems, respectively. During the preparation of the microcapsule-based delivery systems, addition of porogen BSA in the inner water phase was favorable to form porous microcapsules. Fig. 2C indicated that some small pores appeared in the surfaces of the microcapsules. The formation of the pores should result from water shift under osmotic pressures between the internal and external water phases after introduction of BSA in the inner water phase. Some water molecules go into the inner, also resulting in formation of bigger hollow cavity than the microcapsules. It must note that the entire PME procedure used an applied pressure to force coarse emulsion to pass through a membrane without intense ultrasound or heat, avoiding the degradation of the pesticide under turbulent-ultrasound and high-temperature conditions. The dispersion capacity and stability of the pesticide delivery systems in water are vital for successful construction of their aqueous formulations. The aqueous microcapsule suspension could maintain stable and homogeneous even after standing for 4 h instead of obvious deposition observed from the microsphere and porous microcapsule suspensions, resulting from different gravity effects as shown in Fig. 2D. The microcapsule is light and has high buoyancy in water due to its hollow structure, which is favorable to slow down the settling of the microcapsule. The microsphere with a solid structure possesses high density, resulting in its rapid settlement. For the porous microcapsule suspension, the open pores allow water to go into the inner cores, causing a loss of buoyancy and subsequently accelerated precipitation. So the microcapsule is optimal to be further studied for developing the aqueous formulation of pesticides. 3.2. The loading content and entrapment efficiency of LC The loading content and entrapment efficiency of pesticides in the microcapsules have an important effect on overall performance of the pesticide delivery system. High entrapment efficiency and loading content are preferred to reduce the waste of pesticide during the preparation process as well as to avoid its extensive use in spraying and subsequent environmental pollution. To achieve high loading content as well as suitable entrapment efficiency, we constructed several specimens by feeding various weight ratios of PLA/LC. Table 1 indicated that all the specimens had loading efficiencies of higher than 82%. The results demonstrate the mild PME process can effectively avoid damage to the pesticide activity without a prolonged period of intense ultrasound and/or heating. As the feeding weight contents of LC increased from 10% to 50%, the loading 6
contents of LC increased from 9.5% to 41.0% with the loading efficiency decreased from 95% to 82%. It is reasonable as the LC feeding contents increased, the corresponding reduced concentrations of PLA lowered the LC encapsulation ability, subsequently leading to decrease of the loading efficiency. However, even for 50 wt% feeding contents of LC, the loading efficiency still can reach as high as 82%. So, we chose 50 wt% feeding contents of LC to produce the LC delivery systems with high loading contents. 3.3. Preparation of the LC-loaded microcapsules with different sizes The particle size is an important tunable parameter in controlled release system and definitely has a pronounced influence on the release kinetics due to its different surface-area-to-volume ratios. PME can easily adjust the size of the microcapsules in a large range by simply changing the size of membrane pores, the viscosity of the emulsion and the transmembrane pressure. Here, we adopted three SPG membranes with pore sizes of 9.0, 7.2 and 1.0 µm to produce various LC-loaded microcapsules with sizes to be 4.6, 2.4 and 0.68 µm and LC contents to be 40.8%，40.5% and 40.9%, designated as MC1, MC2, and MC3 as indicated in Fig. 3. All the microcapsules had smooth surfaces and spherical shapes, and narrow size distributions. 3.4. Controlled release of LC in vitro In recent years, development of pesticide-release systems has transited toward accurate and quantitative release from slow and qualitative release. To achieve controllable and precise release, we systematically investigated the release profiles of the LC-loaded microcapsules with various particle sizes and pesticide contents. Compared with LC technical and commercial microcapsule formulation, all our microcapsules released LC in relatively slow speeds and maintained its sustained release for longer periods (Fig. 4). The LC technical was totally released after 18 h, and the cumulative release of commercial microcapsule formulation reached 90% in 12 h. Our microcapsules had an initial burst release in the first 25 h and then maintained a sustained and stable release. In the initial stage, the loaded pesticide at the surface of the microcapsules would dissolve and leak into the surroundings quickly, resulting in the burst release. It must note that the release profiles in vitro obtained from an incubation medium of ethanol-water cannot precisely exhibit a practical release law in nature. Generally, when the microcapsules are exposed to air or aqueous environments in nature, the release rate of pesticides should turn slower, and initial rapid release may be reduced. However, the various release profiles of different drug delivery systems still can provide an insightful understanding to evaluate their discrepancies in pesticide release, especially for further release study in vivo. The effects of loading content and the size of the capsules on the release of LC are described in more detail below. 3.4.1. Effects of size on the LC release We investigated the effects of size on the release profile of LC. As shown in Fig. 4A, all the microcapsules had a slow release, lasting for 250 h. As the size decreased from 4.6 nm to 0.68 µm, the cumulative release increased from 67% to 82% after 48 h and from 80% to 95% after 250 h. The results showed that the LC-microcapsule delivery system with the smaller particle size had the faster release of the 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 change of particle sizes is an effectively tunable way for the precise regulation of pesticide release.
7
3.4.2. Effects of the loading content on the LC release In order to achieve a multi-regulated release system, the effects of the LC loading content on the release behavior were further investigated. The microcapsules with different LC contents were obtained by changing the LC feeding content while maintaining the same PLA concentration. As shown in Fig. 4B, the cumulative release rates of LC increased from 78% to 95% after 250 h with increased loading content from 22% to 45%. Higher LC contents in MC6 occupy more spaces in the shells of the microcapsules. When the pesticide molecules migrated and dissolved to the surroundings, more voids in MC6 are formed, resulting in subsequent accelerated pesticide release. 3.4.3. The release mechanism of the LC-loaded micocapsules In order to investigate the release mechanism of the LC-loaded microcapsules, the release data were analyzed by applying the exponential relation proposed by Ritger and Peppas [46]: (1) Mt/M0=ktn Where Mt/M0 is the percentage of active ingredient released at time t, k is a constant, and n is the diffusion exponent. (The values of the diffusion exponent n and T50, the time taken for 50% of the active ingredient to be released into water, were calculated and presented in Table 2.) There was a good correlation between the release profiles of LC and the empirical equation with correlation coefficients greater than 0.98. The lowest values of T50 for MC3 and MC6 indicated the faster release of LC from suspensions of microcapsules with smaller capsule sizes and higher loading contents. The values of the diffusion exponent n were larger than 0.45, indicating that the LC release from the microcapsules was controlled by both a diffusion and matrix degradation. As depicted in Fig. 4(C, D), the surface of the microcapsules after 250 h in the medium became rough, and some pores appeared in the shells, indicating the degradation of the microcapsules. The degradation occurred because of the hydrolysis of the PLA on the surface. 3.5. Bioassay study of the LC-loaded microcapsules To verify feasibility of the microcapsule suspension as a novel pesticide formulation, bioactivity of LC in different microcapsules against plutella xylostella was tested. Fig. 5 indicated the MC3 microcapsule (0.68 µm) had a lower LC50 value and exhibited obviously higher activity than the other two microcapsules, suggesting that efficacy of pesticide gradually increased with the decrease of the microcapsule size. The MC3 microcapsule had the similar efficacy as a commercial formulation. The high efficacy should ascribe to that the nano-sized microcapsules can enhance the adhesivity and penetrability of pesticide on surface of crops, subsequently reducing leaking loss of pesticide during spraying operation. However, compared with the commercial formulation, our LC loaded microcapsules are more environmentally friendly because they could be directly dispersed in water, avoiding the usage of toxic organic solvents and a large number of pesticide adjuvants. 3.6. Stability of the LC-loaded microcapsules 3.6.1 UV-shielding properties of the microcapsules for LC The photodegradation of pesticides after spraying seriously destroys the activities of these pesticides and reduces their utilization rates. This encapsulation technology is an important mean to improve the optical stability of pesticides by incorporating pesticides into capsule shells, preventing photodegradation and slowing 8
down the release rate of pesticide. Fig. 6A showed that the decomposition rate of the encapsulated LC was significantly reduced compared to that of LC technical. The photolysis rate of LC technical was more than 11% after 12 h of UV irradiation, while that of LC in the microcapsules was only 3%. Even after 72 h, only less than 18% of LC was degraded for the microcapsules compared with up to 60% for the LC technical. The results clearly demonstrate that the microcapsules could effectively reduce LC photolysis, indicating the microcapsule formulation has the remarkable UV-shielding properties for LC. 3.6. 2Thermal-stability of the LC-loaded microcapsules The storage stability is another key factor that affects the quality of a pesticide formulation. During storage, delamination, caking, and degradation of the active pesticides may occur, reducing the utilization of the pesticides and thus wasting resources. To determine LC thermal stability of the microcapsules, the samples were stored at 4 °C for 7 days and 54 °C for 14 days. As shown in Fig. 6(C-E), there were no obvious morphologic changes observed, indicating that the microcapsules had good thermal stability. As shown in Fig. 6B, the LC content only had a negligible loss after storage at 4 °C for 7 days. A small loss of LC was observed after 14 days at 54 °C due to the degradation of LC at high temperatures. The results showed that the solid microcapsules had good storage stability.
4. Conclusion In this work, we developed an environment-friendly controlled-release delivery system for LC using biodegradable PLA as the microcapsule shells. The LC-loaded microcapsule system had good water dispersion and stability. Manipulating the process parameters constructed various systems with LC contents as higher than 40% as well as tunable sizes from 0.68 to 4.6 µm. Compared with both LC technical and commercial microcapsule formulations, the LC-loaded microcapsule systems yielded LC sustained release for a longer period. The LC release was controlled by LC diffusion and matrix degradation. The 0.68 µm microcapsule system with good UV and thermal stability had the similar efficacy as the commercial formulation. We envision that such an environmentally friendly novel pesticide formulation should be greatly potential for wide applications in the field of agriculture.
Acknowledgments The authors thank the Major National Scientific Research Program of China (Nos. 2014CB932200 and 2014BAI11B04), the National Young Thousand Talents Program and the National Natural Science Foundation of China (NSFC, Nos. 21174147, 21474115 and 51103165), and the Basic Scientific Research Fund of the National Nonprofit Institutes for financial support.
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Fig. 1. Schematic description for preparation of microspheres, microcapsules and porous microcapsules (A) and PME process (B).
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Fig. 2. SEM images of the (A) microspheres, (B) microcapsules, and (C) porous microspheres before and after cut by a super thin blade and optical photos of their suspensions (D) in water after setting for 4 h. The concentrations both are 2 mg/mL, respectively.
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Fig. 3. SEM images of LC-loaded microcapsules (A) MC1, (B) MC2, and (C) MC3 with average sizes of 4.6, 2.4 and 0.68 µm and PDIs of 0.122, 0.090 and 0.066, respectively.
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Fig. 4. Effects of the sizes (A; 4.6, 2.4 and 0.68 µm for MC1, MC2 and MC3 with 50% feeding contents of LC) and LC loading contents (B; 22%, 28% and 45% for MC4, MC5 and MC6 constructed using 1µm SPG membrane) on the release behavior of LC, and SEM images of surface morphology of the MC1 before (C) and after (D) soaking in an ethanol/water mixture (30:70, v/v) for 250 h.
15
Fig. 5. The LC50 values of the MC1, MC2, MC3 and commercial formulations in bioassay study.
16
Fig. 6. Comparison of the LC photolysis percentage of LC technical and MC3 formulations under UV irradiation (A) and LC contents (B) and its SEM images of the MC3 microcapsules before (C), and after 4 °C for 7 days (D) and 54 °C for 14 days (E).
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Table 1 The LC loading content and loading efficiency of the microcapsules with different feed ratios of PLA/LC
Weight contents of LC (%) Loading content (%) Loading efficiency (%) 10
9.5
95.0
20
18.2
89.3
30
28.7
86.2
40
34.4
83.6
50
41.0
82.0
Table 2 Constants from fitting the generalized model of Eq. (1) to the release data of LC from the different capsule suspensions
Sample
r
n
T50
MC1
0.9891
0.73
16.0
MC2
0.9914
0.82
14.5
MC3
0.9942
0.83
14.1
MC4
0.9930
0.72
18.6
MC5
0.9934
0.75
16.2
MC6
0.9954
0.65
14.6
18