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Optimization and characterization of biocompatible oil-in-water nano-emulsion for pesticide delivery Zhiping Du, Chuanxin Wang, Xiumei Tai, Guoyong Wang, and Xiaoying Liu ACS Sustainable Chem. Eng., Just Accepted Manuscript • DOI: 10.1021/ acssuschemeng.5b01058 • Publication Date (Web): 12 Feb 2016 Downloaded from http://pubs.acs.org on February 17, 2016

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ACS Sustainable Chemistry & Engineering

Optimization and characterization of biocompatible oil-in-water nano-emulsion for pesticide delivery Zhiping Dua,b,*, Chuanxin Wanga, Xiumei Taia, Guoyong Wanga, and Xiaoying Liua a

China Research Institute of Daily Chemical Industry, No. 34 Wenyuan Road, Taiyuan 030001, People’s Republic of China

b

Institute of Resources and Environment Engineering, Shanxi University, No. 92 Wucheng Road, Taiyuan 030006, People’s Republic of China

* Corresponding Author Zhiping Du Institute of Resources and Environment Engineering, Shanxi University, No. 92 Wucheng Road, Taiyuan 030006, People’s Republic of China Tel.: +86 3514084691; fax: +86 3514040802 E-mail: [email protected]

ACS Paragon Plus Environment


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ABSTRACT: The formation of promising oil-in-water nano-emulsion suitable for pesticide delivery, has been achieved by using methyl laurate as oil phase, alkyl polyglycoside (APG) and polyoxyethylene 3-lauryl ether (C12E3) as mixed surfactant. Effects of APG and C12E3 mixing ratios, oil weight fraction and total surfactant concentration on droplet size and distribution of the nano-emulsion were systematically investigated. Long-term stability of the nano-emulsions prepared with various surfactant mixing ratios were assessed by measuring droplet size at different time intervals, the results indicated that the main driving force for droplet size increase over time was Ostwald ripening. On this basis, a practical water-insoluble pesticide β-cypermethrin (β-CP) was incorporated into two optimized nano-emulsion systems to demonstrate potential applications. The results of dynamic light scattering (DLS) and transmission electron microscopy (TEM) measurements showed that the nano-emulsions had a nearly monodisperse droplet size distribution (PDI < 0.2) and incorporation of β-CP had no notable effect on the size and stability of the nano-emulsions. For consideration of practical application, dilution stability and spreading properties of the pesticide-loaded nano-emulsion were studied by DLS, contact angle and dynamic surface tension, respectively. The nano-emulsion was still homogeneous after dilution, although destabilization in droplet size was observed by DLS. The results of contact angle and dynamic surface tension demonstrated the excellent spreading performance of the optimized nano-emulsion. KEYWORDS: Green nano-emulsions; Droplet size; Stability; Alkyl polyglycoside; Wetting and spreading properties


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INTRODUCTION Nano-emulsions can be recognized and defined as emulsions consisting of nano-scale oil or water droplets (typically in the range of 20-200 nm) dispersed in the external phase of opposite polarity by the effect of surfactants arranging at the oil/water interface.1,

2

Recent years, nano-emulsions have drawn particular attentions in

different industrial fields, for their attractive characteristics such as monodisperses, high kinetic stability, low viscosity, etc. For example, nano-emulsions can serve as active ingredients delivery systems in the agrochemical and pharmaceutical fields,3, 4 as personal care formulations in cosmetics,5 and as polymerization reaction media for nanoparticle preparation.6 For pesticide delivery in agrochemicals, nano-emulsion have significant advantages in terms of cost and safety.7 First, o/w nano-emulsion formulation is water-based, construction of nano-emulsion uses much less organic solvent than conventional emulsifiable concentrate (EC).8 Also, unlike microemulsion which formulation requires a high surfactant concentration (about 20% or higher), nano-emulsion can be prepared with surfactant concentration typically between 3-10%.9 Besides, the small size of the droplets allows them to be deposited uniformly on plant leaves, wetting, spreading and permeating may also be enhanced as a result of the low surface tension of the whole system. The bioavailability of poorly water-soluble pesticides was reported to be strongly enhanced by solubilization in small oil droplets, indicating that using nano-emulsion as pesticide delivery system may probably improve the efficacy of pesticides.10 But up to now, there are remain some noticeable problems about



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nano-emulsions used as pesticide carriers. One is that the oil phase used in agrochemical emulsions may still be toxic, volatile and low flash organic solvents,11 such as toluene and xylene. Another one is the use of ungreen emulsifiers such as alkylphenol ethoxylates (APEO) series, which are commonly used in pesticide formulations.12 APEOs are highly toxic to aquatic organisms, and their primary degradation products (alkylphenols) are supposed to be more toxic.13 Furthermore, the wetting and spreading properties (which are often neglected) of nano-emulsion formulations on hydrophobic surface are also important parameters for their bioactivity.14 For the purpose of sustainable development and environmental protection, it is necessary to formulate nano-emulsions with nontoxic oils and eco-friendly surfactants. Fatty acid methyl ester (methyl laurate) was chosen as the oil phase in this study, for its advantages of high flash point, ready availability, low volatility and renewability.15 Nano-emulsions with fatty acid methyl esters as oil phase have already been produced and used as agrochemical formulations, and the oil adjuvant of methylated seed oil has been reported to increase the uptake of glyphosate.16 According to previous study,17 surfactant mixture generally perform better than single surfactant for nano-emulsion formation, this study adopted mixtures of alkyl polyglycoside (APG) and polyoxyethylene 3-lauryl ether (C12E3) to prepare methyl laurate o/w nano-emulsions. APGs18, 19 are produced from renewable raw materials such as corn, potatoes and wheat, due to their good dermatological compatibility and good biodegradability, they are also called “green surfactants” and widely used in washing,


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cleaning and cosmetic products. At the same time, APGs are superior surfactants with outstanding wetting properties and used as adjuvants for pesticide formation, for example, APGs were recognized by Syngenta (A leading agriculture company located at Basel, Switzerland) as potentiators for glyphosate.20 Polyoxyethylene 3-lauryl ether (C12E3) is an important kind of nonionic surfactant and be widely used as emulsifier, wetting agent and foaming agent.21 In this paper, a green o/w nano-emulsion system was prepared with methyl laurate as oil phase, the mixture of APG and C12E3 as surfactant. Nano-emulsion preparation method was high-shear stirring, which is extensively used for pesticide formulation preparation.22, 23 The mixing ratio of APG and C12E3, total surfactant concentration and oil weight fraction on the long-term stability of the nano-emulsions were investigated. To demonstrate potential application of these nano-emulsion systems, a water-insoluble pesticide β-CP, was incorporated into two optimized nano-emulsions, besides, dilution stability and spreading properties of the pesticide-loaded nano-emulsion were also characterized by dynamic light scattering, contact angle and dynamic surface tension measurements, respectively.

EXPERIMENTAL SECTION Materials. Methyl laurate (purity≥98%) was purchased from Nanjing Hand in Hand Chemical & Technology Co., Ltd (Nanjing, China). Dodecyl/tetradecyl polyglycoside with an average of 1.4 mol of glycoside per surfactant molecule (APG, ＞50%, HLB=13.0) and polyoxyethylene castor oil ether (EL-10, HLB=6.5) were supplied by China Research Institute of Daily Chemical Industry. Polyoxyethylene 3-lauryl ether



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(C12E3, HLB=6.8) was supplied by BASF Corporation. Nonylphenol ethoxylates ether (TX-10, HLB=13.2) and β-cypermethrin (β-CP, purity≥97%) were purchased from Zibo Nongbang Pesticides Co., Ltd (Zibo, China). A commercial 2.5% β-CP nano-emulsion with xylene as solvent was purchased from Zhongshan Kaizhong Agrochemical Corporation. All products were used without further purification. Deionized water (18.2 MΩ) was used for all experiments.

Methods and Techniques. Preparation of nano-emulsions. All nano-emulsions were prepared by the high-shear stirring emulsification method at room temperature (25 °C). It is well known that the hydrophilic and lipophilic surfactants often have synergistic effects on emulsion stability.24 In this study, a hydrophilic surfactant (APG, HLB=13) and a lipophilic surfactant (C12E3, HLB=6.8) were used in combination to stabilize the methyl laurate o/w nano-emulsions. First, C12E3 and methyl laurate were mixed together to form the oil phase, then APG was dissolved in deionized water completely to form the water phase by stirring in a beaker. Nano-emulsions were prepared by adding the water phase to oil phase using a high speed homogenizer (Model B25, Berthe Mechanical and Electrical Equipment Co., Ltd. Shanghai, China) at 13000 rpm for 10 min at room temperature. The influence of composition parameters, including the APG and C12E3 mixing ratio, total surfactant concentration and oil weight fraction on droplet size and stability of nano-emulsion was systematically investigated. The solubility of β-CP in methyl laurate (40.6 g/100g at 25 °C) is much higher than that in water (1.13×10-5 g/100g at 25 °C).10, 25 Pesticide-loaded nano-emulsions were


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prepared by dissolving β-CP completely in methyl laurate (25 g β-CP dissolved in 75 g methyl laurate to ensure that there will be no precipitation when the temperature changed) before nano-emulsion formation. An ordinary β-CP nano-emulsion (β-CP/methyl laurate/TX-10+EL-10/water, 2.5:7.5:5:85) with methyl laurate as solvent, TX-10 (APEO series) and EL-10 (6:4) as composite emulsifier was prepared in the same way. All the emulsions were kept at 25 °C by a constant temperature incubator for further study.

Droplet size and stability determination. The mean droplet size and PDI of all nano-emulsions were determined by dynamic light scattering (Zetasizer, Brookhaven, model BI-200SM, United States). Measurements were carried out at 25 °C with a scattering angle of 90 °, employing an argon laser (λ=633 nm). The sample was diluted about 100 times with deionized water just before the measurements.22, 26 PDI27 is a dimensionless measure of the width of the size distribution calculated by the instrument. Sample were considered polydisperse when PDI was higher than 0.2. Measurements were carried out 5 times for each nano-emulsion, then the average droplet size and the corresponding standard deviation were obtained. During the whole study period of three months, the nano-emulsion samples were enclosed in a screw-neck glass bottle, protected from light in a constant temperature incubator at 25 ± 1°C. Long-term stability of the nano-emulsions with different surfactant ratios was evaluated by measuring droplet size at different time intervals.

The cmc values of APG and C12E3 mixtures. Surface tension of the mixed surfactant



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aqueous solution was measured at 25 ± 0.1 °C with a Krüss K12 Processor Tensiometer (Krüss Company, Germany) by the Wilhelmy plate method. Before each set of measurements, the glass container was cleaned with chromic acid lotion and rinsed with deionized water. The Pt plate was also rinsed with deionized water and cleaned by flaming. The tension meter was then calibrated with ultra-pure water. For each mixing ratio, measurements were carried out three times at intervals of 60 s after stirring and with an average deviation of less than 0.2 mN m-1. The critical micelle concentration (cmc) and the surface tension at cmc (γcmc) were determined from the breakpoint of the surface tension and the logarithm of the concentration curve. The surface excess concentration (Гcmc) and the occupied area per surfactant molecule (Acmc) at the cmc can be calculated using the Gibbs adsorption isotherm equations as follows.28

Гcmc =

ቂ ቃ 2.303RT ∂ lg c

Acmc =

-1

1 N · Гcmc

∂γ

(1)

(2)

Where R is gas constant (8.314 J mol-1 K-1), c is the molar concentration of the surfactant (mol L-1), T is the absolute temperature, γ represents the surface tension (mN m-1) at the surfactant concentration of c, lg is base-10 logarithm, and N is Avogadro’s number.

Transmission electron microscopy (TEM). The microstructures of optimized nano-emulsions and pesticide-loaded nano-emulsions were observed using a


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JEM-1011 Transmission electron microscope (Joel Co., Japan). Samples were first diluted by 100 times with deionized water. A drop of the diluted nano-emulsion was added onto a copper grid and allowed to equilibrate for 15 min. The grid was then stained with 2% phosphotungstic acid solution. Excess liquid was removed by gently touching the edge of grid with filter paper, and the grid dried overnight at room temperature. After that, the grid was transferred to TEM operating at an acceleration voltage of 100 kV.

Dynamic Surface Tension. Dynamic surface tension for the diluted nano-emulsions was measured using a Krüss BP100 bubble-pressure tensiometer (Krüss Company, Germany) at 25 ± 0.1 °C. Before the measurements, the instrument was calibrated with deionized water (72.0 ± 0.2 mN/m).28 Measurements were always carried out with effective surface ages ranging from 10 to 100000 ms.

Contact Angle. Contact angle for the diluted nano-emulsions on hydrophobic substrate was measured with a DAS-100 drop shape analyzer (Krüss Company, Germany) at 25 ± 1 °C, the environmental humidity was kept constantly at 50 ± 5%. Paraffin film was chosen as solid substrate. According to previous research, contact angle of deionized water on paraffin film is around 106 °,29 so deionized water was used to check the instrument prior to the measurements.

RESULTS AND DISCUSSION Effects of the surfactant mixing ratios on mean droplet size and polydispersity index. One of the key factors for the formation of stable emulsion is the surfactant



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Hydrophilic-Lipophilic Balance (HLB) Value.30 Selection of surfactant is a critical step, in preliminary tests, several nonionic surfactants including APG, alkyl ethoxy polyglycosides (AEG), the Tween series, the Span series, and several polyoxyethylene lauryl ethers (C12En) were adopted to prepare nano-emulsions. The combination of APG and C12E3 achieved the best results. Different ratios of APG and C12E3 were used to satisfy the suitable HLB value (from 6.8 to 13) for nano-emulsion formation, the HLB value of the mixed surfactant (NHLB) was derived by multiplying the HLB values of APG and C12E3 with their weight fraction.31 Figure 1 shows the influence of the mixing ratios (weight ratio) of APG and C12E3, and surfactant HLB value on the mean droplet size and PDI. All the emulsions were prepared by keeping methyl laurate/APG+C12E3/water ratio constantly at 10:5:85. The mean droplet size and PDI both decreased with surfactant mixing ratio raised from 0:10 to 6:4 (APG:C12E3) and then increased gradually when the surfactant mixing ratio changed from 6:4 to 10:0. The smallest droplet size (113.5 nm) and PDI (0.129) were obtained when surfactant mixing ratio was 6:4 (NHLB=10.52). While only one surfactant was used (0:10 and 10:0), the mean droplet size and PDI were both too large to stabilize the emulsion, the addition of the second surfactant resulted in a remarkable decrease in mean droplet size and PDI. Similar trends have also been observed by other researchers.32 Hydrophilic emulsifier (APG, HLB=13) and lipophilic emulsifier (C12E3, HLB=6.8) are thought to align along side by side in order to provide greater rigidity and strength to the interfacial film.33


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0.4

500 size PDI 400

Mean droplet size (nm)

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0.3

300 0.2 200 0.1 100 0.0 7 0

8 20

9

10

11

NHLB 40

XAPG (%)

60

12 80

13 100

Figure 1. Effects of APG and C12E3 mixing ratios on mean droplet size and PDI in the methyl laurate/APG+C12E3/water (10:5:85) system. The XAPG was calculated as APG/(APG+C12E3) ×100%.

The nano-emulsions with the surfactant ratios at 1:9, 2:8, 3:7, 4:6 and 9:1 were considered polydisperse as the PDI was larger than 0.2,27, 34 so more attention was focused on the other four nano-emulsions (8:2, 7:3, 6:4, 5:5) with smaller droplet size (less than 150 nm) and PDI (less than 0.2), these four nano-emulsions had no apparent flocculation or creaming, and had uniform appearance over the entire study period of three months.

Stability of the nano-emulsions with different APG and C12E3 mixing ratios. Effect of surfactant mixing ratio on long-term stability of the nano-emulsion system (methyl laurate/APG+C12E3/water, 10:5:85) was also evaluated by measuring droplet size at different time intervals. As can be seen in Figure 2(a), just after preparation, the four nano-emulsions (8:2, 7:3, 6:4, 5:5) all displayed a relative small droplet size (D＜150 nm), which means the high-shear stirring method for nano-emulsion preparation was succeeded. While an increase in droplet size was observed over the 90 days study,



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especially in the first 25 days after nano-emulsion preparation. The oil/water interface provisionally created by the high energy input may not be fully covered with surfactant molecules, so small droplets tend to dissolve in larger ones to give reduce of oil/water interface area over time.23 However, once the systems reach equilibrium and oil droplets are tightly encircled by surfactant molecules, the increase in droplet size tends to slow, Asma Chebil et al. had reported the similar results.35 This process could also be explained by the classical disperse system instability theory: Ostwald Ripening. According to this theory, smaller drops have higher chemical potential, larger droplets will grow at the cost of the smaller ones to reduce the potential energy of the whole system. The droplet growth rate (ω) can be calculated using the following equation:35, 36

ω=

dr3 dt

=

8C∞ γVm D 9ρRT

(3)

where r is the mean droplet radius (nm), t is the abbreviation of time (s), C∞ is the bulk phase solubility of oil in aqueous phase (mol/m3), γ is the interfacial tension (N/m), Vm is the molar volume of oil phase (m3/mol), D is diffusion coefficient of the oil phase in the continuous phase (m2/s), ρ is the density of the oil (Kg/m3), R is the gas constant (8.314 J/mol K), T is the absolute temperature (K).


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(a)

240

8:2 7:3 6:4 5:5

220 200

(b) 10

8:2 7:3 6:4 5:5

8

r ×10 (nm )

3

180

6

6

160

3


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140 120

4

2

100 0

20

40

60

80

100

0

20

Time (d)

40

60

80

5

Time×10 (s)

Figure 2. Mean droplet size (a) and r3 (b) as a function of time in the methyl laurate/APG+C12E3/ water (10:5:85) nano-emulsion system for various surfactant mixing ratios of APG and C12E3 at storage temperature of 25 °C.

The cube of radius (r3) for the nano-emulsions with different surfactant mixing ratios (8:2, 7:3, 6:4, 5:5) were plotted as a function of time. Figure 2(b) indicated that in the first 25 days of storage, there were good correlations between r3 and time with regression coefficients (R) ＞0.85 for all nano-emulsions (Table 1), implying that the main driving force for instability was Ostwald ripening. Ostwald ripening rates for the nano-emulsions were obtained from the slopes of each straight line in Figure 2(b) and summarized in Table 1. The initial mean droplet size of nano-emulsions with different surfactant mixing ratios were very close, but they diverge a lot after storage, this might be attributed to the difference in polydispersity index.37 When the surfactant mixing ratio was 6:4, mean droplet size of nano-emulsion just increased from 113.5 nm to 127.6 nm after 90 days storage, and the minimum Ostwald ripening ratio was obtained, implying that this nano-emulsion was the most stable.



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Table 1 Initial mean droplet size, PDI and Ostwald ripening rate (ω) of the nano-emulsions prepared with different APG and C12E3 ratios. r0 ± SD

ω ± SD PDI ± SD

APG:C12E3 (nm)

Linear fitting

(×10-27 m3 s-1)

R

P-value

8:2

142.4 ± 6.58

0.192 ± 0.028

2.741 ± 0.344

0.9258

0.0014

7:3

122.2 ± 11.0

0.137 ± 0.035

1.924 ± 0.301

0.8957

0.0025

6:4

113.5 ± 7.81

0.128 ± 0.027

0.282 ± 0.043

0.8998

0.0027

5:5

129.1 ± 2.47

0.155 ± 0.031

0.595 ± 0.087

0.8883

0.0031

Taking into account the stability of nano-emulsions prepared with APG and C12E3 alone and the different stability properties of nano-emulsions with various mixing ratios, the observed effects on nano-emulsion stability may due to co-adsorption of APG and C12E3 at interface. Besides, different kind of surfactants often exhibit synergistic effects when they used together at many practical applications.37, 38 So in this section, surface properties including the critical micelle concentration (cmc), the surface tension at cmc (γcmc), the surface excess concentration (Гcmc), and the minimum surface area per surfactant molecule (Acmc) of the different ratio surfactant mixtures were all investigated to verify the co-adsorption, and the results are presented in Table 2. When APG and C12E3 mixing ratio was 6:4, the smallest Acmc and the largest Гcmc were obtained, these two values suggest that the surfactants packed more closely and adsorbed more strongly at the interface than the other ratios of APG and C12E3 mixture, therefore enhancing the strength of the interfacial film. The very low cmc and γcmc values make clear that it is favorable to form micelles in


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water, thus to enhance oil solubilization. So when the surfactant mixing ratio was 6:4, the mixture exhibited much better synergism and surface activity, and the corresponding nano-emulsion showed greater physical stability. Possible organizing mechanism between APG and C12E3 at the oil/water interface are as follows. First, APG and C12E3, as well as methyl laurate all have a similar hydrophobic alkyl chain length of about 12 carbon atoms, which means the hydrophobic tails can hold and intertwine each other steadily, and it has been reported that, for a nonionic surfactant system, the Ostwald ripening rate can be decreased by adding a second surfactant with the same alkyl chain length.1 Second, APG has a large hydrophilic head group (glycoside) but C12E3 has a relatively small one (EO), the small one may fill the gap between large ones when ranging at oil/water interface, resulting in a compact interfacial film, and the strong interfacial layer can effectively reduce the ageing rate caused by Ostwald ripening.35 Third, it has been reported that APGs and ethoxylated fatty alcohols (C12En) mixture show higher wettability on hydrophobic surfaces than pure surfactants, which mean they have synergistic effect in surface properties.39 The cmc, γcmc and Acmc of other mixing ratios slight increased compared to the 6:4 ratio, and the stability of the nano-emulsions decreased concomitantly.



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Table 2 Surface properties of the mixed surfactant with different mixing ratios. APG:C12E3

cmc (×10-5 mol L-1)

γcmc (mN m-1)

Гcmc (×10-6 mol m-2)

Acmc (nm2)

8:2

2.20

26.81

6.05

0.274

7:3

1.96

26.69

6.20

0.268

6:4

1.74

25.81

6.32

0.263

5:5

1.97

26.57

6.19

0.268

Effects of total surfactant concentration and oil weight fraction on mean droplet size and stability of nano-emulsion. Surfactant concentration and oil weight fraction are also very important factors for a stable o/w nano-emulsion system.40 Usually, people prefer to stabilize more oil phase in water but use relatively less surfactant, then more active ingredient can be dissolved in the nano-emulsion and the cost will reduce. In addition, the required amount of surfactant for a stable nano-emulsion system may have a close relationship with the weight fraction of oil phase. Therefore the influence of surfactant concentration and oil weight fraction on the mean droplet size and the stability of methyl laurate/APG+C12E3 (6:4)/water nano-emulsion system were evaluated in this section. The surfactant concentration and oil weight fraction are defined as the weight of mixed surfactant and oil in total weight of the system, respectively.


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800 3% surfactant 4% surfactant 5% surfactant 6% surfactant 7% surfactant

700


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600 500 400 300 200 100 5

10

15

20

25

Oil weight fraction (%)

Figure 3. Effect of oil weight fraction and surfactant concentration on mean droplet size in the methyl laurate/APG+C12E3 (6:4)/water nano-emulsion system.

The mean droplet size of the nano-emulsion system as a function of surfactant concentration and oil weight fraction was shown in Figure 3. The mean droplet size increased as more oil phase dispersed in water and decreased when more surfactant was used. Other authors have reported similar results showing that the droplet size of nano-emulsions decreases with increasing surfactant concentration due to the increase in interfacial area and decrease in interfacial tension,41 besides, increase of surfactant ratio means more emulsifier adsorption around the oil/water interface and result in a relatively thicker surfactant film that affords better steric stabilization against flocculation. Mean droplet size, however, shrank with higher surfactant concentration when the oil phase was kept constant. Taking the nano-emulsion with 10% dispersed phase (oil) as an example, the droplet size of the nano-emulsion decreased when surfactant concentration increased from 3% to 5%, indicating that low surfactant concentration can not produce droplets with large surface area. But there was no significant changes in mean droplet size when surfactant concentration rise from 5%



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to 7%, so the appropriate surfactant concentration for 10% oil phase was 5%, and 6% surfactant concentration seems sufficient to stabilize the nano-emulsion with 20% oil phase. Previously,42 Peng et al. had reported similar results showing that droplet size decreased when surfactant concentration rose, and leveled off with higher surfactant ratios due to the saturated adsorption of surfactant at the oil/water interface. The nano-emulsion system gradually lost its stability when oil weight fraction was greater than 25% even if more surfactant was used. Observable creaming was present above the emulsion surface after storage. It may be due to the close packing of droplets, encouraging the formation of tumbling clusters.26 Therefore, 5-6% surfactant composed of APG and C12E3 (6:4) was found to maintain the physical stability of methyl laurate in water nano-emulsions with an oil weight fraction below 25%.

β-CP solubilization. The advantages for nano-emulsion used as pesticide delivery system have been mentioned in the introduction section. In this work, a practical water-insoluble active ingredient, β-CP, was employed to investigate the potential application of the green nano-emulsions. Two optimized nano-emulsion systems were chosen for active-loading studies, the first one (A in Figure 4) is the nano-emulsion with mass composition of methyl laurate/APG+C12E3 (6:4)/water (10:5:85), the second one (C in Figure 4) is methyl laurate/APG+C12E3 (6:4)/water (20:6:74). Firstly, β-CP was completely dissolved in methyl laurate (25 g β-CP dissolved in 75 g methyl laurate to ensure that there would be no precipitation when the temperature changed) before nano-emulsion formation, then the solution of β-CP in methyl laurate was used as oil phase to prepare β-CP loaded nano-emulsions in the same way as for the


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active-free system, and two corresponding pesticide-loaded nano-emulsion systems were obtained (B and D). As can be seen from Figure 4, the pesticide-free (A and C) and pesticide-loaded (B and D) nano-emulsions are homogeneous liquid with slight blue tint, and within the experimental time of three months, these nano-emulsions had no apparent flocculation or creaming and remain homogeneous.

Figure 4: Photograph of β-CP free and β-CP loaded nano-emulsions three months after preparation.

A:

methyl

laurate/APG+C12E3

(6:4)/water

(10:5:85),

B:

β-CP/methyl

laurate/APG+C12E3 (6:4)/water (2.5:7.5:5:85), C: methyl laurate/APG+C12E3 (6:4)/water (20:6:74), D: β-CP /methyl laurate/APG+C12E3 (6:4)/water (5:15:6:74).

The droplet size distribution and microstructure of pesticide-free and pesticide-loaded nano-emulsions were also determined by DLS and TEM respectively. As can be seen in Figure 5(a), there were no significant changes in droplet size and distribution between the nano-emulsions in the presence and absence of the pesticide. From the TEM images in Figure 5(b), still no obvious difference in size was observed, which means incorporation of pesticide had no noticeable effect on droplet size and of the



final nano-emulsions, furthermore, the pesticide-loaded nano-emulsions also maintained the homogeneous state during the entire study period. There were two main possible reasons for the similar stability properties exhibited by the pesticide-free and pesticide-loaded nano-emulsions. One is that β-CP has relative high solubility in methyl laurate, indicating that these two components have similar polarity. The required HLB of them for emulsification may then be similar.31 The other one is that incorporation of β-CP makes the density of the mixed oil (0.997 g/cm3, 25 °C) close to water, almost the same density between the dispersion phase and continuous phase is beneficial for emulsion stability.4 The excellent solubilization capacity of these nano-emulsions indicated that this system is suitable for water-insoluble active delivery.

110

(a) 100

(b)

A B C D

90 80

Intensity

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

70 60 50 40 30 20 50

100

150

200

250

300

350

400

Droplet size (nm)

Figure 5. Droplet size distribution (a) and negative-stained TEM images (b) of the nano-emulsions described in Figure 4.

Dilution stability. The stability of the pesticide emulsions after dilution is very important for practical applications.43 The optimized pesticide-loaded nano-emulsion with a mass composition of 2.5% β-CP, 7.5% methyl laurate, 5% APG and C12E3 (6:4),


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85% water was selected to determine the dilution stability. The diluted nano-emulsions was obtained by adding the concentrated nano-emulsion to deionized water with slight stirring. Measurements were carried out just after dilution. Figure 6 shows the mean droplet size over time after 100-, 500- and 1000-fold dilution, visible increase in droplet size can be seen just after dilution, then the rate of growth slows after about 40 min. Addition of water may cause redistribution of surfactant throughout the system, part of surfactant diffuses from the interface to the aqueous phase, leading to a decline in the strength of the interfacial film and an increase in free energy.43 Therefore, small droplets tend to dissolve into larger ones to reduce the interfacial area and free energy. Once the newly formed interface film was strengthen enough, the droplet size tends to be relatively stable with time. After the dilution process, the samples remained homogeneous without any creaming or precipitation even if the droplet size increased after dilution, and the diluted samples had a translucent appearance with slight blue tint (Pictures in Figure 6). Therefore, a macroscopically stable dilute nano-emulsion was obtained. 270

100 500 1000

240


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210 180 150 Diluted with water

120 90 0

20

40

60

80

100

120

Time (min)

Figure 6. Mean droplet size obtained from dilution (100-, 500- and 1000-fold) of β-CP/methyl laurate/APG+C12E3 (6:4)/water (2.5:7.5:5:85) nano-emulsion as a function of time.



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Wetting and spreading properties. Leaves are typically covered with a layer of hydrophobic cuticular wax,44 therefore agrochemical sprays need to both wet and to spread on plant leaves and then penetrate to maximize biological activity. So rapid spreading of emulsion drops on plant leaves is very important for agrochemical biological

activity.

The

optimized

pesticide-loaded

nano-emulsion

system,

β-CP/methyl laurate/APG+C12E3 (6:4)/water (2.5:7.5:5:85) was used for these studies, and an ordinary 2.5% β-CP nano-emulsion with a mass composition of 2.5% β-CP, 7.5% methyl laurate, 5% TX-10 and EL-10 (6:4), 85% water, and a commercial 2.5% β-CP nano-emulsion with xylene as oil were also invited as comparison samples. Generally, contact angle can be considered as an important property and has been used to characterize the spreading ability of aqueous drops.45 In this work, a more hydrophobic substrate (compared to ordinary plant leaves), paraffin film, was used to simulate the leaf surface. Before measurement, the nano-emulsion was diluted 100-, 500- and 1000-fold with deionized water, respectively. The liquidity of these highly dilute nano-emulsions was excellent, so the influence of viscosity on spreading would appear negligible. A lower value for the contact angle represents better spreading. Figure 7(a) shows the dynamic contact angle (θ) against paraffin film of the

β-CP/methyl laurate/APG+C12E3 (6:4)/water (2.5:7.5:5:85) nano-emulsion system with different dilution times. θ declined rapidly from 0 to about 10 s, and then the rate of decline slowed until equilibrium was reached. Smaller dilution ratio resulted in a steeper slope of the curve and a lower equilibrium value of θ, which means that spreading velocity promoted with increasing of the nano-emulsion concentration.


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Starov46 had described the spreading mechanism of aqueous surfactant solutions over hydrophobic surfaces as a slow transfer of surfactant molecules onto the bare hydrophobic surfaces in front of the moving liquid. The transfer of surfactant molecule from bulk to the new liquid/solid interface is therefore the rate determining step. Higher nano-emulsion concentration imply higher surfactant content, leading to faster diffusion of surfactant molecules to the new surface, and therefore resulting in quicker reduction of contact angle. From Figure 8(a), it is easy to find that the contact angle of the optimized 2.5% β-CP nano-emulsion system dropped much faster than that of the corresponding ordinary and commercial ones under the same dilution conditions (500-flod), implying that a sprayed solution of the optimized nano-emulsion might spread on plant leaves more quickly. Dynamic surface tension is also an important property as it governs many industrial and biological processes including spreading of pesticides onto leaves.43,

47

Quick

decay of the dynamic surface tension over time represents fast wetting and spreading on hydrophobic surfaces. Dynamic surface tension measurements for the optimized

β-CP/methyl laurate/APG+C12E3 (6:4)/water (2.5:7.5:5:85) nano-emulsion system with different dilution times is shown in Figure 7(b), the curves of surface tension seem to begin with a quick decrease with surface age before reaching an equilibrium. The decay rate of surface tension increased with nano-emulsion concentration, the surface tension of the 100-fold diluted sample had nearly reached equilibrium at surface age of 5 s, whereas the 1000-fold diluted sample needed 10 s. After the same dilution time (500-flod), dynamic surface tension of the optimized, the ordinary and



the commercial 2.5% β-CP nano-emulsions can be seen in Figure 8(b), dynamic surface tension curve of the optimized nano-emulsion produces a steeper slope compared with the ordinary and commercial ones. This result is consistent with the dynamic contact angle, indicating that the optimized pesticide-loaded nano-emulsion possesses excellent wetting and spreading properties even after 1000-fold dilution in water, this may be attributed to the enhanced wetting power of alkyl polyglycosides and ethoxylated fatty alcohol mixtures.39 75

100 100 500 1000

Contact angle (°)

80 70 60 50 40

100 500 1000

70

65

Surface tension (mN/m)

90

80

(b) 70


(a)

60 55

60 50 40 30 20 0

2

4

6

8

10

12

14

Time (s)

50 45 40 35 30

30

25 0

10

20

30

40

50

60

0.01

0.1

Time (s)

1

10

100

Surface age, t (s)

Figure 7. Contact angles against paraffin film (a) and dynamic surface tension (b) of β-CP/methyl laurate/APG+C12E3 (6:4)/water (2.5:7.5:5:85) system with different dilution times.

(a)

110

75

Optimized Ordinary Commercial

(b) 70

90 80 70 60

Optimized Ordinary Commercial

65


100

Contact angle (°)

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60 55 50 45 40 35 30

50 0

10

20

30

40

50

60

25 0.01

0.1

Time (s)

1

10

100

Surface age, t (s)

Figure 8. Contact angles against paraffin film (a) and dynamic surface tension (b) of the optimized, the ordinary and the commercial 2.5% β-CP nano-emulsions after diluted 500-fold.


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CONCLUSIONS Biocompatible o/w nano-emulsions suitable for pesticide delivery were formed using methyl laurate as the oil phase, alkyl polyglycoside (APG) and polyoxyethylene 3-lauryl ether (C12E3) as mixed surfactant. Effects of APG and C12E3 mixing ratios, oil weight fraction and total surfactant concentration on droplet size distribution and stability of nano-emulsion was systematically investigated. The ideal mixing ratio of APG and C12E3 for methyl laurate oil-in-water nano-emulsion is 6:4, where the nano-emulsion was obtained with minimum droplet size and high kinetic stability. Analysis of mean droplet size over time showed that the main mechanism for instability was Ostwald ripening. Nano-emulsion droplet size decreased with increasing surfactant concentration, while 5% and 6% surfactant concentration seems to be sufficient to stabilize the nano-emulsions with 10% and 20% oil phase respectively. Incorporation of β-CP showed no notable effect on the droplet size and stability of two optimized nano-emulsion systems according to DLS and TEM results. Diluted samples of the pesticide-loaded nano-emulsion remained monophasic and homogeneous throughout the dilution process, although the droplet size increased after dilution. Compared to the ordinary and commercial β-CP nano-emulsions, the excellent wetting and spreading properties of the diluted nano-emulsion on hydrophobic surface were also clear from the results of contact angle and dynamic surface tension measurements. The nano-emulsion formulations may provide stable, biocompatible and fast spreading delivery systems for agrochemicals.



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Acknowledgements This research was supported by the Shanxi Science and Technology Innovation Project (#2012102008), the National Science & Technology Pillar Program during the Twelfth Five-year Plan Period (#2014BAE03B03) and Natural Science Found of Shanxi Province (#2014011014-1).

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Manuscript Title: Optimization and characterization of biocompatible oil-in-water nano-emulsion for pesticide delivery

Names of Authors: Zhiping Dua,b,*, Chuanxin Wanga, Xiumei Taia, Guoyong Wanga, and Xiaoying Liua

Notes The authors declare no competing financial interest.

E-mail address of all authors: Zhiping Du: [email protected] Chuanxin Wang: [email protected] Xiumei Tai: [email protected] Guoyong Wang: [email protected] Xiaoying Liu: [email protected]

Author Affiliations: a

China Research Institute of Daily Chemical Industry, No. 34 Wenyuan Road,

Taiyuan, People’s Republic of China. b

Institute of Resources and Environment Engineering Research Institute, Shanxi

University, No. 92 Wucheng Road, Taiyuan, People’s Republic of China.



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Table of contents graphic

Synopsis: Green oil-in-water nano-emulsion with fast spreading properties, suitable for pesticide delivery has been prepared with methyl laurate as oil phase, alkyl polyglycoside and polyoxyethylene 3-lauryl ether as mixed surfactant.


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Figure 1. Effects of APG and C12E3 mixing ratios on mean droplet size and PDI in the methyl laurate/APG+C12E3/water (10:5:85) system. The XAPG was calculated as APG/(APG+C12E3) ×100%. 296x209mm (300 x 300 DPI)



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Figure 2. Mean droplet size (a) and r3 (b) as a function of time in the methyl laurate/APG+C12E3/ water (10:5:85) nano-emulsion system for various surfactant mixing ratios of APG and C12E3 at storage temperature of 25 °C. 296x121mm (300 x 300 DPI)


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Figure 3. Effect of oil weight fraction and surfactant concentration on mean droplet size in the methyl laurate/APG+C12E3 (6:4)/water nano-emulsion system. 296x209mm (300 x 300 DPI)



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Figure 4: Photograph of β-CP free and β-CP loaded nano-emulsions three months after preparation. A: methyl laurate/APG+C12E3 (6:4)/water (10:5:85), B: β-CP/methyl laurate/APG+C12E3 (6:4)/water (2.5:7.5:5:85), C: methyl laurate/APG+C12E3 (6:4)/water (20:6:74), D: β-CP /methyl laurate/APG+C12E3 (6:4)/water (5:15:6:74). 38x23mm (600 x 600 DPI)


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Figure 5. Droplet size distribution (a) and negative-stained TEM images (b) of the nano-emulsions described in Figure 4. 296x121mm (300 x 300 DPI)



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Figure 6. Mean droplet size obtained from dilution (100-, 500- and 1000-fold) of β-CP/methyl laurate/APG+C12E3 (6:4)/water (2.5:7.5:5:85) nano-emulsion as a function of time. 296x209mm (300 x 300 DPI)


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Figure 7. Contact angles against paraffin film (a) and dynamic surface tension (b) of β-CP/methyl laurate/APG+C12E3 (6:4)/water (2.5:7.5:5:85) system with different dilution times. 296x121mm (300 x 300 DPI)



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Figure 8. Contact angles against paraffin film (a) and dynamic surface tension (b) of the optimized, the ordinary and the commercial 2.5% β-CP nano-emulsions after diluted 500-fold. 296x121mm (300 x 300 DPI)


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Table of contents graphic 84x47mm (300 x 300 DPI)


Optimization and Characterization of ... - ACS Publications

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