Preparation, characterisation, and controlled release of sex pheromone-loaded MPEG-PCL diblock copolymer micelles for Spodoptera litura (Lepidoptera: Noctuidae).

Sex pheromones are important for agricultural pest control. The main sex pheromone components of Spodoptera litura are (Z,E)-9,11- and (Z,E)-9,12-tetradecadienyl acetate (Z9,E11-14:Ac; Z9,E12-14:Ac). In this study, we investigated the optimal conditions for encapsulation of S. litura sex pheromonesin micelles via the self-assembly method using monomethoxy poly (ethylene glycol)-poly (ε-caprolactone) (MPEG-PCL) as a biodegradable wall-forming material with low toxicity. In the L9(3(4) orthogonal experiment, 3 amphiphilic block copolymers, with different hydrophilicity to hydrophobicity ratios, were examined. Optimal encapsulation conditions included stirring of MPEG5000-PCL2000 at 1000 rpm at 30°C with 2.5:1 wall-forming: core material mass ratio. S. litura sex pheromone-loaded MPEG5000-PCL2000 micelles presented a homogeneous spherical morphology with apparent core-shell structure. The release kinetics of optimized MPEG5000-PCL2000 micelles was best explained by a first-order model. Encapsulated Z9,E11-14:Ac and Z9,E12-14:Ac were released slowly, not suddenly. Methyl oleate (MO) was used as an agent to control micellar release performance. When MO content equalled block content, micelle half-life could be prolonged, thereby controlling the release speed. Overall, our results showed MPEG-PCL as a promising controlled-release substrate for sex pheromones.

Introduction

Spodoptera litura Fabricius (Lepidoptera: Noctuidae), a type of polyphagous pest with an aggressive eating pattern, has a wide range of hosts, encompassing approximately 200 kinds of plants [1] including those of various vegetables, fruits, baccies, cotton, corn, tea, and other cash crops [2]. However, due to the overuse of chemical agents to prevent and control this pest, insecticide-resistance of S. litura has gradually increased [3-6]. The use of synthetic sex pheromone traps to monitor population densities of S. litura on crops is a simple and practical method applied in many cropping systems [7]. The main components of the sex pheromones of S. litura, namely, (Z9,E11)-tetradecadienyl acetate (Z9,E11-14:Ac) and (Z9,E12)-tetradecadienyl acetate (Z9,E12-14:Ac), were isolated and identified by Tamaki and colleagues in the early 1970s [8, 9]. In an attempt to replace traditional chemical insecticides, sex pheromones have been employed to control S. litura in various countries; sex pheromones have also been used to trap [10, 11] or disorient [12-14] pests. Sustained-release materials are an important part of the medium in which the pheromones are constituted and influence the effects and duration of release of the pheromones. At present, insect sex pheromone-releasing dispensers mainly consist of rubber septa heads, plastic tubes, hollow fibres, and paraffin oil [15-17].

Nanotechnology has enormous potential in various fields, used in the development of insecticides, pharmaceuticals [18,19], and electronics. In addition, in the field of agriculture, nanomaterials have been developed for the management of insect pests [20, 21]. Encapsulation of hydrophobic pheromones into nanoparticles is an advanced and effective application method. Recently, the use of microcapsules has also received increased attention [22, 23]. Microcapsulation can reduce the influence of the surrounding environment on encapsulated materials, prolong the duration of activity of the compounds, or reduce their toxicity, and avoid contamination of the effective ingredients by preventing mixture with other substances. The use of sex pheromone microcapsules in sustained-release dosage forms to disorient and control pests, such as Plutellaxy lostella (L.), Cydia pomonella (L.), Tuta absoluta (Meyrick), Grapholita molesta (Busck), Argyrotaenia velutinana (Walker), Choristoneura rosaceana (Harris), Pectinophora gossypiella (Saunders), and Keiferia lycopersicella (Walsingham) has been reported in various countries [13, 24–28].

Monomethoxy poly (ethyleneglycol)-poly (ε-caprolactone) (MPEG-PCL) has been widely studied due to its biocompatibility, biodegradability, easy production, non-toxicity, and thermo-sensitivity that promise its widespread applications [29-32]. Polymeric micelles are more stable than surfactant micelles and have lower critical micelle concentrations and degradation rates [33]. MPEG-PCL copolymers that self-assemble into nanoparticles with the core-shell structure include a hydrophobic PCL core and a hydrophilic MPEG shell. During the process of self-assembly, agents can be effectively loaded into the hydrophobic nucleus through physical packaging [34], which allows the agent to retain its activity, decreases its volatilisation, and permits sustained release [35, 36]. Thus, polymeric micelles can be a versatile system for the effective delivery of different classes of agents.

In this study, three kinds of MPEG-PCL, with different proportions of hydrophobicity and hydrophilicity, were selected as wall-forming materials for incorporation into S. litura sex pheromone containing micelles formed by amphiphilic block copolymer self-assembly, and the optimal experimental conditions and pheromone release performance were evaluated to examine the effect of controlled-release agent incorporation during micelle assembly.

Materials and methods

Materials

Z9,E11-14:Ac (97%) and Z9-E12-14:Ac (97%) were purchased from Sigma-Aldrich Inc. (St. Louis, MO, USA); MPEG5000-PCL2000, MPEG3000-PCL2300, and MPEG5000-PCL10000 were purchased from Jinan Daigang Bio-engineering Co., Ltd. (Jinan, China); N, N—dimethylformamide (DMF, analytical grade) and Tween-20 (analytical grade) were from Sinopharm Chemical Reagent Co., Ltd. (Shanghai, China); methyl oleate (MO, chemically pure) was from Macklin Reagent Co., Ltd. (Shanghai, China).

Critical micelle concentration

The critical micelle concentration (CMC) of MPEG-PCL was measured by a UV spectrophotometer (TU-1810, Beijing Purkinje General Instrument Co., Ltd.). Three kinds of MPEG-PCL, with different proportions of hydrophobic and hydrophilic components were dissolved in deionized water to obtain a stock solution of concentration 1.000 g/L. The absorption maxima of the different concentrations of MPEG-PCL (0.001, 0.005, 0.008, 0.01, 0.04, 0.05, 0.1, 0.2, 0.35, 0.5, 0.7, and 1 g/L) was recorded and mapped with the values of lgA-lgC (A: absorption, C: concentration); critical micelle concentration of block copolymer corresponded to the concentration at which the first derivative curve reached zero [37].

Preparation of micelles

MPEG-PCL micelles were prepared by the self-assembly method [38]. Briefly, desired amounts of MPEG-PCL and S. litura sex pheromones (Z9,E11-14:Ac: Z9,E12-14:Ac = 9:1) were individually dissolved in 1 mL DMF and mixed well by adding Tween-20 [0.01% (v/v)]. At a fixed temperature and stirring speed (RH D W S25 stirrer, IKA Company, Germany) (Table 1), 8 mL deionised water was added drop-wise into the solution. The micelle solution was stirred for 30 min, dialysed using dialysis tubing (MD44-3.5, Viskase Co., Lombard, IL, USA) for 24 h, accompanied by stirring. The resulting micellar solution was filtered using a syringe filter (pore size: 0.22 m) to remove aggregates.

Table 1

Experimental factors and their levels in orthogonal projects.

Level	W(A)	W/S ratio (w/w)(B)	T (°C)(C)	S (rpm)(D)
1	MPEG5000-PCL2000	2:1	30	800
2	MPEG3000-PCL2300	2.5:1	40	1000
3	MPEG5000-PCL10000	5:1	50	1200

W (wall-forming materials), W/S ratio (the mass ratio of sex pheromone to wall-forming materials), T (reaction temperature), S (stirring speed).

Orthogonal experimental design

An L9 (34) orthogonal table (Table 1) was adopted for this test. The investigated factors included wall-forming materials (W), mass ratio of sex pheromone to wall-forming materials (W/S ratios), reaction temperature (T), and stirring speed (S); encapsulation efficiency of the micelles (EE) was considered as the assessment index. The optimised formulation was prepared in triplicate.

Determination of entrapment efficiency

Briefly, 0.5 mL of the sex pheromone-loaded micelle solution was fully mixed with 0.5 mL ultrapure water. The solution was extracted with 1 mL n-hexane and completely disrupted using an ultrasound sonicator (Scientz-IID, Ningbo Scientz Biotechnology Co., China) on ice. The encapsulated sex pheromone was dissolved in the hexane after 30 min. The concentration of sex pheromone was determined by gas chromatography (GC, Agilent 7890B, Agilent Technologies, Santa Clara, CA, USA). For GC, a capillary column (HP-5, 30 m × 0.32 mm × 0.25 μm) with a flame ionisation detector and a splitless injector, with nitrogen as the carrier gas, was used. GC conditions were as follows: the column temperature set at 80°C (held for 5 min), raised to 210°C at 10°C/min, and held at 210°C for 15 min.

A standard curve was generated according to the concentration of sex pheromones and peak area; quantity of each component in the sex pheromone was determined from the standard curve. The standard curve regression equations of Z9,E11-14:Ac and Z9,E12-14:Ac were y = 16806x + 50.5 (R2 = 0.9997) and y = 18672x − 3.4706 (R2 = 0.9999). The sex pheromone entrapment efficiency was calculated using Eq 1:

Characterisation of micelles

Particle morphology

The micelle morphology was observed by transmission electron microscope (TEM, HT 7700, Hitachi, Tokyo, Japan), and the speeding voltage during the test was 80 kV. Samples were prepared by dropping the micelle solution on a carbon-coated copper net, followed by air drying, and dyeing with 0.2 wt% phosphotungstic acid.

Determination of particle size

The particle size and its distribution were analysed using a Malvern nanometre particle size analyser (MNPSA) (Zetasizer Nano S90, Malvern Instruments Ltd., Malvern, UK).

Stability of micelles

Micelles were stored at 2, 4, and 8°C in the dark. In order to evaluate the physical stability of nanoparticles during this storage period, particle size distribution was monitored at time intervals of 0, 15, and 30 days, using the method described in the section “Determination of particle size”.

Release performance

Sex pheromone release

To evaluate sex pheromone release, the micelles were transferred to a centrifuge tube and placed in an artificial climate chamber (MGC-450HP2, Shanghai Yiheng Co., China) with controlled temperature in the range of 35 ± 3°C, light: dark cycle of 12 h:12 h, and relative humidity of 75 ± 5% for a period of 28 days. The samples were taken out of the artificial climate chamber at regular time intervals for sex pheromone examination by GC. To evaluate the release of sex pheromones from micelles prepared under optimal conditions, the samples were examined every day during the first 14 days, and every 7 days during the subsequent 14 days. To evaluate the release from micelles containing the controlled-release agent, the samples were examined every 3 days over a period of 15 days. Three samples were used in each experiment.

Sex pheromone release was expressed as percentage of accumulated release, since this enabled the evaluation of performance of different micelles. Accumulated release was calculated using Eq 2: where W0 is the sex pheromone content at the initial time and Wt is the sex pheromone content at each recorded time.

Sex pheromone release kinetics for optimized micelles

For a better understanding of the efficacy of sex pheromones, their release kinetics were studied. Selection of a suitable kinetic model for fitting the sex pheromone release data helped determine the release characteristics. There are a number of kinetic models that describe the overall release of sex pheromone from the vehicle. The most common mathematical models used are: zero-order model (Eq 3), first-order model (Eq 4), Higuchi model (Eq 5), Korsmeyer-Peppasmodel (Eq 6), and Hixson-Crowell model (Eq 7) [39-45]: where

Ct—amount of drug released in time t,

C0—the initial amount of drug,

K0—zero-order kinetic constant,

K1—first-order kinetic constant,

KH—Higuchi kinetic constant,

KKP—Korsmeyer-Peppas release constant,

KHC—Hixson-Crowell release constant,

n—diffusional release exponent,

t—time.

Half-life calculations

Depletion of pheromone components from the micelle formulations was characterised by the first-order kinetic model: lnCt = lnC0 +K1·t. Half-lives (t1/2) for compounds were determined from the exponential equation, substituting calculated values of C0 and K1, and setting (Ct/C0) to 0.5 [46].

Statistical analysis

Statistical analysis was done with SPSS 17.0 software package (Chicago, IL, USA). One-way analysis of variance (ANOVA) for independent samples followed by Duncan’s multiple range tests were performed to evaluate the quantitative results. Data were obtained from triplicate samples and, expressed as mean ± standard error (SE); values of P ≤0.05 and P ≤ 0.01 were considered statistically significant and extremely significant, respectively.

Results and discussion

The CMC of MPEG5000-PCL2000, MPEG3000-PCL2300, and MPEG5000-PCL10000 were 0.011 g/L, 0.00199g/L, and0.00102g/L, respectively. The low CMC may better stabilise the micelles in suspension.

Optimisation of MPEG-PCL micelle formation

The results of the L9(34) orthogonal experiments using MPEG-PCL nanoparticles are shown in Tables 2 and 3. The range values (R) of the factors indicated their effects on the efficiency of encapsulation of Z9,E11-14:Ac in the order: S (D) > T (C) > W (A) > W/S ratios (B), and that of Z9,E12-14:Ac in the order: S (D) > W (A) > T (C) > W/S ratios (B). The K value showed the optimal encapsulation group to be W (MPEG5000-PCL2000)—W/S ratios (2.5:1)—T (30°C)—S (1000 rpm) for both Z9,E11-14:Ac and Z9,E12-14:Ac.

Table 2

Results of the L9(34) orthogonal experiment using Z9,E11-14:Ac MPEG-PCL nanoparticles.

Factor	W(A)	W/S ratio (w/w)(B)	T (°C)(C)	S (rpm)(D)	EE (%)(Z9:E11-14:Ac)%
1	1	1	1	1	76.76
2	1	2	2	2	76.51
3	1	3	3	3	51.10
4	2	1	2	3	53.95
5	2	2	3	1	65.45
6	2	3	1	2	78.04
7	3	1	3	2	61.26
8	3	2	1	3	55.77
9	3	3	2	1	60.74
K1	204.37	191.96	210.57	202.95
K2	197.44	197.73	191.20	215.81
K3	177.77	189.89	177.80	160.82
k1	68.12	63.99	70.19	67.65
k2	65.81	65.91	63.73	71.94
k3	59.26	63.30	59.27	53.61
R	8.87	2.61	10.92	18.33
Influence degree of factors	S> T> W> W/S ratios
Best group	A1B2C1D2W (MPEG5000-PCL2000)—W/S ratio (2.5:1)—T (30°C)—S (1000 rpm)

W (wall-forming materials), W/S ratio (the mass ratio of sex pheromone to wall-forming materials), T (reaction temperature), S (stirring speed), EE (encapsulation efficiency of micelle). The arrangements of A, B, C, D were decided by orthogonal design for 4 (factor) × 9 (run number).

Table 3

Results of the L9(34) orthogonal experiment using Z9,E12-14:Ac MPEG-PCL nanoparticles.

Factor	W(A)	W/S ratio (w/w)(B)	T (°C)(C)	S (rpm)(D)	EE (%)(Z9:E12-14:Ac)%
1	1	1	1	1	77.11
2	1	2	2	2	85.77
3	1	3	3	3	58.91
4	2	1	2	3	55.41
5	2	2	3	1	71.66
6	2	3	1	2	82.73
7	3	1	3	2	60.23
8	3	2	1	3	56.45
9	3	3	2	1	57.95
K1	221.79	192.74	216.29	206.72
K2	209.79	213.88	199.13	228.73
K3	174.63	199.59	190.80	170.77
k1	73.93	64.25	72.10	68.91
k2	69.93	71.29	66.38	76.24
k3	58.21	66.53	63.60	56.92
R	15.72	7.05	8.50	19.32
Influence degree of factors	S> W> T> W/S ratios
Best group	A1B2C1D2W (MPEG5000-PCL2000)—W/S ratio (2.5:1)—T (30°C)—S (1000 rpm)

W (wall-forming materials), W/S ratio (the mass ratio of sex pheromone to wall-forming materials), T (reaction temperature), S (stirring speed), and EE (encapsulation efficiency of micelle). The arrangements of A, B, C, D were decided by orthogonal design for 4 (factor) × 9 (run number).

Conversely, it was much easier to draw a more intuitive conclusion from the results by range analysis of the orthogonal experiment. However, the calculation processes were extensive and could not evaluate the errors; thus, it was necessary to carry out variance analysis of the orthogonal experiment results. It can be seen from the variance analysis tables (Tables 4 and 5) that except for the W/S ratios, all the other factors (including W, S, and T) had significant effects on the experimental results. The order of factors affecting the encapsulation efficiency of Z9,E11-14:Ac and Z9,E12-14:Ac, obtained from variance analysis, was the same as that from the range analysis.

Table 4

Analysis of the orthogonal experiment results of Z9,E11-14:Ac MPEG-PCL nanoparticles using ANOVA.

Source of variation	SS	Df	MS	F
A	380.931	2	190.466	7.554**
B	33.001	2	16.501	6.540*
C	542.744	2	271.372	10.763**
D	1655.042	2	827.521	32.821**
error	453.842	18	25.213

SS (Sum of square), df (degree of freedom), MS (mean square), F (critical value).

‘*’and ‘**’represent significant difference (P ≤ 0.05) and extremely significant difference (P ≤ 0.01), respectively.

Table 5

Analysis of the orthogonal experiment results of Z9,E12-14:Ac MPEG-PCL nanoparticles using ANOVA.

Source of variation	SS	Df	MS	F
A	121.833	2	600.917	20.087**
B	232.648	2	116.324	3.888*
C	337.751	2	168.875	5.645*
D	1711.861	2	855.930	28.611**
Consolidated error	538.493	18	29.916

SS (Sum of square), df (degree of freedom), MS (mean square), F (critical value).

‘*’and ‘**’represent significant difference (P ≤ 0.05) and extremely significant difference (P ≤ 0.01), respectively.

Encapsulation efficiency of the micelles was controlled by the length of hydrophobic or hydrophilic chain (wall-forming materials), W/S ratio, T, and S. Based on the two analyses, it was concluded that the order of effect of W and T on the encapsulation efficiency was different. Since the mass ratio of Z9,E11-14:Ac was much larger than that of Z9,E12-14:Ac, factor S was regarded as the most important factor affecting the encapsulation efficiency followed by T, W, and W/S ratios. S likely played an important role in the formation of micelles, since the sex pheromone should be well mixed in the process of micelle formation, and a certain speed would be required when water is added to the solution to conjugate the hydrophilic ends of the amphiphilic block copolymer. The influence of W was determined by the length of hydrophobic and hydrophilic chains, whereas T likely influenced micellar assembly and speed of sex pheromone volatility to lessen the encapsulation efficiency. However, the influence of W/S ratios on encapsulation efficiency was relatively small.

The optimal conditions for S. litura sex pheromone encapsulation with MPEG-PCL, determined from the above results, involved stirring MPEG5000-PCL2000 at a speed of 1000 rpm at 30°C with a 2.5:1 mass ratio of wall-forming to core materials. Based on these conditions, three parallel experiments with MPEG-PCL micelles were subsequently conducted (Table 6). The results consistently showed that entrapment efficiency was the highest among the combinations used in the orthogonal experiments, which verified the utility and feasibility of the conditions.

Table 6

Test of verification.

Best group	EE (%)
	Z9,E11-14:Ac	Z9,E12-14:Ac
A1B2C1D2	82.91	88.73
A1B2C1D2	84.54	87.84
A1B2C1D2	82.21	89.32
Mean ± SE	83.22 ± 0.35	88.63 ± 0.56

EE (encapsulation efficiency of micelle), A1B2C1D2 [best group: W (MPEG5000-PCL2000)—W/S ratio (2.5:1)—T (30°C)—S (1000 rpm)]

Characterisation of microcapsules

For fresh MPEG5000-PCL2000 nanoparticles, prepared according to the optimised formulation and preparation conditions, the particle size was 374 ± 5.13 nm by MNPSA (Fig 1). The formation of micellar nanostructures was confirmed by TEM. The MPEG5000-PCL2000 nanoparticles showed a homogeneous spherical morphology, with average diameter of 300 nm, presenting an apparent core-shell structure (Fig 2). The size of the MPEG5000-PCL2000 nanoparticles, measured by TEM, was smaller compared to that from MNPSA measurements, since the former was related to the collapsed nanoparticles after water evaporation, whereas the latter represented their hydrodynamic diameter [47].

Fig 1

Particle size distribution of sex pheromone-loaded MPEG5000-PCL2000 nanoparticles.

Fig 2

TEM micrographs of sex pheromone-loaded MPEG5000-PCL2000 nanoparticles.

Bars in panel A and panel B are 400 nm. Panel B is an enlarged view of the boxed area in Panel A.

After preparation, the micelles were dispersed in aqueous medium. Therefore, stability of their sizes was of great importance, both as a measure of particle structure integrity and as an indicator of the possible inter-particular associations (aggregation). At sub-zero temperatures, the solution solidified and the micellar structure lost its integrity. For this purpose, we chose 2, 4, and 8°C as the storage temperatures, at which the particle size was monitored in the dark over a period of 30 days. The variation of micellar size as a function of storage time is shown in Table 7. All the micelles increased slightly in size, throughout the measurement period, at different temperatures. This observation could not be an indicator of aggregation, which usually leads to a several-fold increase in size; instead, copolymer swelling and/or hydration may be responsible for this event [48]. Since the variation of micellar size was less when stored at 2°C, we chose to store the micelles at 2°C in the dark for the best storage conditions.

Table 7

Stability of micelles during storage period.

Store temperature (°C)	Mean size of micelles immediately after preparation (nm)	Mean size of micelles after 15 day (nm)	Mean size of micelles after 30 day (nm)
2	374	377	386
4	374	379	391
8	374	384	402

Sex pheromone release kinetics in optimized micelles

The sex pheromone release results of MPEG5000-PCL2000 micelles were used in various mathematical models to evaluate the kinetics and mechanism of release from the micelles. Based on the correlation coefficient (R) value in various models, the one that fit best with the release data was selected; the one with a high ‘R’ value was considered as the best fit. The release constant was calculated from the slopes of the appropriate models, and the regression coefficient (R2) was determined (Table 8).

Table 8

Results of release kinetics model fitting of MPEG5000-PCL2000 micelles.

Model	Z9,E11-14:Ac			Z9,E12-14:Ac
	Intercept	Slope	R2	Intercept	Slope	R2
Zero-order model	25.5268	3.2234	0.8599	22.2329	3.1157	0.8670
First-order model	0.2101	0.0853	0.9598	0.1886	0.0721	0.9608
Higuchi model	1.0032	-0.2014	0.9364	1.0237	-0.1932	0.9398
Korsmeyer-Peppas model	0.1221	0.69(n)	0.9298	0.0952	0.58(n)	0.9339
Hixson-Crowell model	0.4441	-0.0230	0.6861	0.4736	-0.0236	0.6987

According to the results shown in Table 8, the profile of Z9,E11-14:Ac and Z9,E12-14:Ac release from micelles fit best to the first-order kinetic model where the highest linearity was achieved (Z9,E11-14:Ac R2 = 0.9598; Z9,E12-14:Ac R2 = 0.9608). Therefore, the sex pheromone release mechanism was assumed to be diffusion-controlled. When analysed according to the Korsmeyer-Peppas model, the diffusion exponent was found between 0.5 and 1.0 (Z9,E11-14:Ac n = 0.69, Z9,E12-14:Ac n = 0.58), based on which the diffusional release was assumed to follow anomalous transport.

Release performance of optimized micelles

The plot of accumulated release from sex pheromone-loaded MPEG5000-PCL2000 micelles indicated that Z9,E11-14:Ac could be released from micelles faster than Z9,E12-14:Ac, in a sustained manner. The two components had a high release rate in the first 3 days, which was attributed to the fact that nanoparticles usually contain sex pheromone not only at the inner core but also on their surface. After this initial loss, sex pheromone release approximated first-order release rates more closely [49]. Accordingly, following the first burst release period, sex pheromone was released slowly, independent of the initial sex pheromone concentration in the micelles. As shown in Fig 3, from day 4 to 14, the release rate tended to slow down and remained constant. After 14 days, the release rate decreased further and tended to be stable, although the release rate of Z9,E11-14:Ac was less than that of Z9,E12-14:Ac. According to the first-order kinetic model, the half-life of Z9,E11-14:Ac and Z9,E12-14:Ac in the micelle was 5.6 and 7.0 days, respectively. The half-life difference of 1.4 days may have been due to the different proportions of sex pheromone components in the micelle. Based on the results of this study, we found that Z9,E11-14:Ac and Z9,E12-14:Ac were released slowly from MPEG5000-PCL2000 micelles, and that no sudden release occurred throughout the process, thereby indicating that diblock copolymer micelles were suitable for use as a controlled substrate. Our studies indicated that although MPEG-PCL diblock copolymer micelles did not maintain a constant release rate, they met the first-order kinetic model requirements, with adynamic rapid-to-slow release, lasting for almost a month. Other release carriers, such as PVC, have demonstrated equal or better release duration for that pheromone [46]. However, the micelles in this study were in aqueous solution and hence environment friendly; they were physically and chemically stable, non-toxic, and biodegradable.

Fig 3

Plot of accumulated release with time in case of sex pheromone-loaded MPEG5000-PCL2000 micelles.

The S. litura sex pheromone has two components, Z9,E11-14:Ac and Z9,E12-14:Ac. Data are expressed as means ± S.D. (n = 3).

Influence of a controlled-release agent on the controlled release performance of micelles

Table 9 shows that while the differences among the tested concentrations of MO were not significant, when the mass of wall-forming materials equalled that of MO (10 mg/mL), the half-life of Z9,E11-14:Ac and Z9,E12-14:Ac in the micelle increased by 3.7 and 4.2 days, respectively, compared to that of the control. With the increased content of controlled-release agent, the efficiency of controlled release declined, potentially due to the organic liquid which may have affected micelle formation and inhibited the encapsulation efficiency, thereby impacting the release rate. Fig 4 shows that MO, as a controlled-release agent, could retard the overall release rate of micelles, especially in the first 3 days without burst release. Compared to that of the control, release rate of the two components was slower over the first 6 days. From day 7 to 15, the release rates increased relative to that during the first 6 days. Thus, addition of appropriate quantities of MO into the micelle could prolong the half-life and control the release performance.

Table 9

Influence of a controlled-release agent on the controlled-release performance of sex pheromone-loaded MPEG5000-PCL2000 micelles.

Compound	Mass of MO (mg/mL)	Half-life (days)	First-order release parametersa
			Slope	Intercept	R2
Z9,E11-14:Ac	CK	5.4	-0.1395	0.0636	0.9866
	10	9.1	-0.1021	0.2326	0.9432
	20	8.7	-0.1064	0.2284	0.9485
	30	8.6	-0.1086	0.2377	0.9475
Z9,E12-14:Ac	CK	5.0	-0.1374	-0.0127	0.9919
	10	9.2	-0.0967	0.1935	0.9432
	20	8.9	-0.0998	0.1988	0.9435
	30	8.7	-0.1032	0.2064	0.9428

aThefirst-order kinetic model: lnCt = lnC0 +K1·t, R2 = regression coefficient.

Fig 4

Accumulated release of sex pheromone-loaded MPEG5000-PCL2000 micelles containing controlled-release agents (A) Z9,E11-14:Ac and (B) Z9,E12-14:Ac.

Data are expressed as means ± S.D. (n = 3). Asterisks (*) indicate significance (P≤ 0.05).

Conclusions

The optimal preparation conditions of S. litura sex pheromone-amphiphilic block copolymer micelles were shown to involve stirring MPEG5000-PCL2000 at a speed of 1000 rpm at 30°C with a 2.5:1 mass ratio of wall-forming to core materials. The nanoparticles presented a homogeneous spherical morphology with an apparent core-shell structure, and were free from the inter-micellar adhesion phenomena. The release kinetics of optimized MPEG5000-PCL2000 micelles was best explained by first-order model. Since the release from micelles was slow, without a sudden-release phenomenon, the amphiphilic copolymer was considered suitable for use as a controlled substrate. When the mass of added MO equalled that of wall-forming materials, the half-life could be prolonged, thereby allowing control of the release rate. These results indicated that the diblock copolymer could be a suitable controlled-release substrate, and the micelles could have potential use in the control applications of mass trapping and mating disruption in the field.

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(TIF)

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(DOC)

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(DOC)

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SS (Sum of square), df (degree of freedom), MS (mean square), F (critical value). ‘*’and ‘**’ represent significant difference (P ≤ 0.05) and extremely significant difference (P ≤ 0.01), respectively.

(DOC)

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SS (Sum of square), df (degree of freedom), MS (mean square), F (critical value). ‘*’and ‘**’ represent significant difference (P ≤ 0.05) and extremely significant difference (P ≤ 0.01), respectively.

(DOC)

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(DOC)

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(DOC)

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(DOC)

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The first-order kinetic model: lnCt = lnC0 +K1·t, R2 = regression coefficient.

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