Biocompatible Colloidal Dispersions as Potential Formulations of

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Biocompatible colloidal dispersions as potential formulations of natural pyrethrins: A structural and efficacy study Argyro Kalaitzaki, Nikos E Papanikolaou, Filitsa Karamaouna, Vassilis Dourtoglou, Aristotelis Xenakis, and Vassiliki Papadimitriou Langmuir, Just Accepted Manuscript • DOI: 10.1021/acs.langmuir.5b00246 • Publication Date (Web): 06 May 2015 Downloaded from http://pubs.acs.org on May 12, 2015

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Biocompatible colloidal dispersions as potential formulations of natural pyrethrins: A structural and efficacy study Argyro Kalaitzaki,†,# Nikos E. Papanikolaou,‡ Filitsa Karamaouna,‡ Vassilis Dourtoglou,§ Aristotelis Xenakis, †,# and Vassiliki Papadimitriou,†,* †

Institute of Biology, Medicinal Chemistry & Biotechnology, National Hellenic Research Foundation, Athens, Greece

#

MTM Research Center, School of Science and Technology, Örebro University, Örebro, Sweden ‡

Benaki Phytopathological Institute, Kifissia, Attica, Greece §

VIORYL S.A, Afidnes, Greece

KEYWORDS: Microemulsions, Nanoemulsions, Pyrethrins, Insecticide, EPR, DLS, SAXS, Conductivity

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ABSTRACT

Biocompatible colloidal dispersions of the micro- and nanoemulsion type based on lemon oil terpenes, polysorbates, water and glycerol were used for the formulation of pyrethrins, botanical insecticides derived from the white pyrethrum daisy, Tanacetum cinerariifolium. The proposed formulation is based in pyrethrin containing water-in-oil (W/O) microemulsions that could be diluted in one step with aqueous phase to obtain kinetically stable oil-in-water (O/W) nanoemulsions. Structural characteristics of the micro- and nanoemulsions were evaluated by Electron Paramagnetic Resonance (EPR) spectroscopy, Dynamic Light Scattering (DLS), Small Angle X-Ray Scattering (SAXS) and electrical conductivity. Dynamic properties of the surfactants’ monolayer as evidenced by EPR measurements were affected by the water content, the surfactant and also the presence of the biocide. DLS and SAXS experiments of the nanoemulsions indicated the existence of two populations of oil droplets dispersed in the aqueous phase, globular droplets of 36-37 nm in diameter and also larger droplets with diameters >150 nm. All the applied techniques for structural determination revealed participation of the biocide in the nanostructure. The insecticidal effect of the encapsulated natural pyrethrin was evaluated in laboratory bioassays upon a target-insect pest, the cotton aphid Aphis gossypii Glover (Hemiptera: Aphididae) in eggplant and was found increased compared to the commercial pyrethrin formulation.

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INTRODUCTION Recent breakthroughs in the development and characterization of materials at the nanometer scale, exhibiting a wide range of novel physico-chemical properties and offering new technological possibilities, have made nanosized systems highly attractive in the field of basic and applied research. Liquid nanosized systems such as micro- and nanoemulsions could be of great fundamental interest offering new technological applications especially in the field of pharmaceutics, cosmetics and foods. Most of the work reported in the literature has been related to the development of micro- and nanoemulsions as encapsulation media of a variety of pharmaceuticals and nutraceuticals suitable for utilization in drugs, foods and beverages.1-7 However, relatively few articles have been published related to the use of micro- and nanoemulsions as carriers of pesticides with potential application in plant protection.8-12 Formulation of pesticides is a critical intermediate stage between production and application and determines the effectiveness and ease of implementation. There are several environmental factors such as ultraviolet radiation, rain, pH, temperature and physiology of foliage, hindering the effectiveness of the conventional formulations. The agrochemical industry has shown in recent years a strong interest in replacing conventional formulations of phytoprotective substances (wettable powders, emulsifiable concentrates, emulsions, aqueous suspension concentrates etc.) with new ones based on the modern technology of microencapsulation and nanodiaspersion. In this respect, the main aim of the present work was the development and characterization of colloidal dispersions such as micro- and nanoemulsions based on safe, biocompatible components, to be used as novel media for the formulation and effective delivery of natural insecticides. Between these two kinds of colloidal nanodispersions there are many similarities

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and important differences in terms of their composition, structure, stability and formulation. The most important common characteristics are stability, optical clarity, increased solubilisation capacity and the ability to tune particle size by an appropriate choice of materials and preparation method. Furthermore, the proposed formulations are specially designed to reduce or eliminate the generation of hazardous substances, being environmentally friendly and harmless to humans.13 In addition, the dispersions are formed spontaneously without high energy input.14,15 In recent years there is a renewed interest in the use of biopesticides for crop protection as environmentally friendly alternatives to the extensive use of synthetic pesticides. Pyrethrins are botanical insecticides derived from the extract of white pyrethrum daisy, Tanacetum cinerariifolium, and a member of the Asteraceae family. The active ingredient is concentrated in the flower head and is made up of a combination of six esters, pyrethrin I and II, cinerin I and II, jasmolin I and II. In these six esters, Pyrethin I and II are dominant (Figure 1). Because of the difficulty of separating these six components, the content of the pyrethrum extract is called as pyrethrins. Pyrethrins are highly effective insecticides against a broad range of insect pests but also safe against beneficial insects and other arthropods, used in domestic settings and for industrial and agricultural preparations. Pyrethrins are lipophilic (logP value 5.9) and exhibit high biodegradability by air and sunlight.16 In the present work the nanoformulation of natural pyrethrins in W/O microemulsions based exclusively on safe non-toxic materials is reported for the first time. Lemon oil terpenes and safe nonionic surfactants such as polysorbates were chosen for the construction of the systems. The aqueous phase was either tap water or a 2:1 mixture of water and glycerol. Notably the use of harmful co-surfactants was avoided. The proposed pyrethrin containing microemulsions could be

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diluted with aqueous phase to obtain kinetically stable O/W nanoemulsions ready to use in sprayers for plant protection. The concentration of the biocide in the micro- and nanoemulsions had to be at a level that guarantees its efficiency. Moreover, the encapsulated compound should not alter the structural characteristics of the hosting system in a way that could affect its stability. In addition the efficiency of the biocide upon encapsulation and aqueous dilution should be ensured. Furthermore, from the applied research perspective, structural characterization of the proposed pyrethrin formulations was of great importance. Interfacial properties of the surfactant monolayer in both microemulsions and nanoemulsions were studied by Electron Paramagnetic Resonance (EPR) spectroscopy using the spin probing technique.17 Dynamic Light Scattering (DLS) is a well-established and fast scattering technique for studying self-organizing amphiphilic systems like micro- and nanoemulsions in relatively simple and dilute systems.6 Finally, to fully elucidate the structure of the systems, Small Angle X-Ray Scattering (SAXS) was applied to free and loaded microemulsions and nanoemulsions.6 The insecticidal effect of the natural pyrethrin microemulsions was evaluated in laboratory bioassays upon a target-insect pest, the cotton aphid Aphis gossypii Glover (Hemiptera: Aphididae) in eggplant. Aphids are generally recognized as important pest insects in agriculture of the temperate climatic zones.18 In particular, A. gossypii is extremely polyphagous, attacking a variety of plants such as cotton, cucurbits, citrus, coffee, cocoa, eggplant, pepper, potato, okra, and many ornamental plants, where it is known to transmit over 50 plant viruses.19

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II

Figure 1. Chemical structure of Pyrethin I and II.

EXPERIMENTAL SECTION Materials. Lemon oil terpenes (97.5 %), polyoxyethylene sorbitan mono-oleate (Tween 80), natural pyrethrins (75%) and glycerol were donated from Vioryl S.A, Greece. Natural pyrethrins solution is a combination of six esters, pyrethrin I (38%), pyrethrin II (35%), cinerin I (7.3%), cinerin II (11.7%), jasmolin I (4%) and jasmolin II (4%). Polyoxyethylene sorbitan monolaurate (Tween 20) was obtained from Sigma, USA. 5-Doxyl stearic acid (5-DSA) was a product of Sigma-Aldrich, Germany. Ethanol absolute (EtOH) was purchased from Merck KGaA, Darmstadt, Germany. High-purity water was obtained from a Millipore Milli Q Plus water purification system. All the chemicals were of reagent grade and were used as received. Aphids used in the toxicity bioassays were reared in a colony of the aphid A. gossypii at the Laboratory of Agricultural Entomology, Benaki Phytopathological Institute, on eggplants, Solanum melongena cv. Bonika F1 (25 ± 1ºC, 16:8 (L:D) h). Preparation of the w/o microemulsions and solubilization of pyrethrins. The phase behavior of two different microemulsion systems consisting of lemon oil terpenes/tween 20/

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(water/glycerol) (2:1) (System 1) and lemon oil terpenes/tween 80/ (water/glycerol) (2:1) (System 2) was described on pseudo-ternary phase diagrams as reported recently.20 Microemulsions containing pyrethrins were prepared as follows: Initially pyrethrins were solubilized in the oily phase. Then mixtures of the oily phase with the appropriate surfactant were prepared and kept in a water bath at 25 °C. Water and glycerol were then added gradually to obtain single-phase W/O microemulsions with the desired composition. Microemulsions were formed spontaneously when the components were taken in appropriate proportion (Figure S1, Supporting Information) Formation of O/W nanoemulsions. Empty and loaded with pyrethrins O/W nanoemulsions

were prepared upon 1:100 aqueous dilution of the corresponding W/O microemulsions (Systems 1 and 2). The aqueous phase used for the dilution was either tap water or a solution of high purity water and glycerol (2:1). The tap water used was “hard water” with total hardness 150 mg/L CaCO3, 15 FH, 8.4 DH. The emulsification procedure followed was addition of the aqueous phase in the W/O microemulsion in one step followed by gentle mixing using a laboratory vortex mixer.21 Electrical conductivity measurements. The electrical conductivity measurements were

performed with a Metrohm 644 conductometer using a thermostated microcell (23 ± 1 °C). The cell constant, c, was equal to 0.1 cm−1. Empty and pyrethrin loaded microemulsions were prepared as follows: Mixtures of oily phase and surfactant at constant weight ratios were prepared. Certain increasing amounts of aqueous phase containing NaCl 1.7 mM were added to obtain different final aqueous phase concentrations. Electrical conductivity measurements were carried out along the dilution line D46 (40% surfactant-60% oil) for System 1 and along dilution line D37 (30% surfactant-70% oil) for System 2.

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Electron paramagnetic resonance (EPR) spectroscopy. Electron paramagnetic resonance

(EPR) measurements were performed at constant room temperature 25 °C, using a Bruker EMX EPR spectrometer operating at the X-Band. Samples were contained in a WG-813-Q Wilmad (Buena, NJ) Suprasil flat cell. Typical instrument setting were: center field, 0.348 T; scan range, 0.01 T; gain, 5.64x103; time constant, 163 ms; conversion time 5 ms; modulation amplitude, 0.4 mT; frequency, 9.77 GHz. Data collection and analysis were performed using the Bruker WinEPR acquisition and processing program. Experimental results were analyzed in terms of rotational correlation time (τR) and order parameter (S), of the spin probe 5-DSA as described elsewhere.

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To obtain the desired concentration of the spin probe in the empty and loaded

micro- and nanoemulsions, 1 g of each was added to a tube into which the appropriate amount of 5-DSA had been deposited previously. This was done by placing 10 µL of a stock 5-DSA solution in ethanol (10-2 M) in the tube and by further evaporating the ethanol. Dynamic light scattering (DLS). Mean droplet size and size distribution (polydispersity

Index) of the O/W nanoemulsions were measured by Dynamic Light Scattering using a Zetasizer NanoZS (ZEN3600) from Malvern Instruments (UK), equipped with aHe–Ne laser (632.8 nm) and using a non-invasive back scatter (NIBS) technology. Measurements were carried out at the scattering angle of 173 °. The droplet’s mean radius was computed by the Stokes–Einstein law: RH = kBT/(6πηD), where RH, kB, T, η and D, are the hydrodynamic radius of the droplet, Boltzmann’s constant, temperature in Kelvin, viscosity of the microemulsion, and diffusion constant, respectively. Measurement data were processed using the Malvern Zetasizer Nano software which fits a spherical model of diffusivities with polydispersity below a value of 0.1. The cummulants analysis only gives two values, a mean value for the size (Intensity mean), and a width parameter known as the Polydispersity Index (Pdi).

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All samples were filtered through 0.45 µm filters before light scattering measurements and contained in a quartz type cuvette. The temperature of the scattering cell was controlled at 25 °C. Small Angle X-ray Scattering (SAXS). Micro- and nanoemulsions prepared as described

above, were investigated by small angle X-ray scattering. SAXS spectra were recorded at 25 °C, using a SAXSess mc2 (Anton Paar) apparatus, using line-collimation system. It is attached to an ID 3003 laboratory X-Ray generator (General Electric), equipped with a sealed X-ray tube (PANalytical, λCu(Ka)=0.154 nm) operating at 40 kV and 50 mA. A multilayer mirror and a block collimator provide a monochromatic primary beam. A translucent beam stop allows the measurement of an attenuated primary beam at q=0. Samples of 40 µL were introduced in a quartz capillary and placed inside an evacuated chamber. Acquisition times were typically in the range of 5 to 20 minutes. Scattering of the X-ray beam was registered by a CCD detector. The scattered X-rays intensity, I(q), was measured as function of the scattering wave vector, q, defined by q = (4π/λ)sin(θ/2), where θ is the scattering angle of the X-ray beam. All data were corrected for the background scattering from the capillary and the solvent and for slit-smearing effects by a desmearing procedure. After correction, obtained intensities were scaled into absolute units using water as a reference material. The intensity is expressed as I(q) = P(q)S(q) where P(q) is the form factor and S(q) is the structure factor. Stability studies. The stability of selected O/W nanoemulsions was assessed by visual

inspection and droplet size analysis. Samples were stored in tightly closed tubes at room temperature (25±1 °C) in the dark for avoiding any physical changes. Droplet size variations were monitored by DLS for a time period of ten days. Insecticidal activity study. The insecticidal activity of the developed pyrethrin

nanoemulsions (System 1 and 2) were tested on 3-4 days old aphid nymphs in laboratory

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bioassays using the leaf-dip method. For each pyrethrin nanoemulsion, five concentrations were tested (450–7200 x 10-6 mg a.i./ml solution). Two commercial products, namely Pyrethro Vioryl 5 SC (pyrethrins 5%) at five concentrations (625–10,000 x 10-6 mg a.i./ml solution) and also Decis 2.5 EC (deltamethrin) at six concentrations (78.1-2,500 x 10-6 mg a.i./ml solution), were used as reference products whereas the control treatment consisted of water. Stalked leaves containing 15-50 individuals of A. gossypii were detached from eggplants and dipped for 10 s in each solution. After dipping, the leaves were transferred individually to transparent plastic cylinder-shaped containers (12 cm height x 7 cm diameter) bearing a ventilating hole at the top. The leaf moisture was kept by adjusting a wet cotton-wool bandage around the edge of the petiole) and were kept in a growth chamber at 25 ± 1 ºC, 65 ± 2% RH and photoperiod 16:8 (L:D) h. The mortality of the nymphs was assessed 24 hours after treatment. The trials were repeated 5 times (replications) for each treatment (test compound x concentration). Mortality data obtained from the dose-response trials were subjected to probit analysis and LC50 and LC90 values and 95 confidence intervals were estimated. LC50 or LC90 values were compared using the respective confidence intervals. Statistical analysis was conducted using Polo Plus.23

RESULTS AND DISCUSSION Phase diagrams. Phseudoternary phase diagrams of free and loaded with pyrethrins

microemulsions were constructed at constant temperature and are presented in Figure 2. In each case two different areas separated by the demixing line are obtained: An isotropic monophasic region that corresponds to microemulsions and a broad multiphase region of unexamined structure. It has been observed that in the presence of pyrethrins the solubilisation ability of the microemulsion systems was significantly decreased.

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Lemon oil terpenes/Pyrethrins

Single phase region

D46

o/w nanoemulsion area

Tween 20

Water/Glycerol (2:1)

Lemon oil terpenes/Pyrethrins

Single phase region D37

o/w nanoemulsion area

Water/Glycerol (2:1)

Tween 80

Figure 2. Phseudoternary phase diagrams of free and loaded microemulsions at 25 ˚C. Free: (□)

and (), loaded with pyrethrins: (■) and (▲). Structural studies. Complementary studies using a combination of advanced instrumental

techniques based mainly on spectroscopy and scattering were undertaken to elucidate the structural details of the proposed micro- and nanoemulsions systems in relation to their composition. Due to their complexity and the variety of structures involved, structural characterization of micro- and nanoemulsions is a rather difficult task and therefore very important when investigating their potential applications as carriers of bioactive compounds.

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Electrical conductivity. Electrical conductivity measurements of microemulsions are highly

useful to determine the nature of the continuous phase and to detect the phase inversion phenomenon. W/O microemulsions exhibit higher conductivity values compared to the bulk continuous medium due to the fact that microemulsions are able to transport charges along transient water channels formed upon aqueous dilution. Increased attractive interactions between aqueous droplets with water content increase lead to the formation of a network of conducting channels in the continuous oil phase.24 In the present study, electrical conductivity of the empty and loaded microemulsions, System 1 and System 2, was measured at constant temperature (23 ± 1 °C), along dilution line D46 for the System 1 (empty and loaded) and dilution line D37 for the System 2 (empty and loaded). Since nonionic surfactants were used in the preparation of the microemulsion systems, the aqueous phase was replaced with a NaCl 1.7 mM aqueous solution to better visualize conductivity changes. As shown in the literature, salt effects on nonionic microemulsions (salting out, absorption or depletion on the membrane) are not important at such low electrolyte concentration. 25,26 Figure 3 depicts the effect of the increased aqueous phase weight percentage on the electrical conductivity of the four systems. By increasing the water content, it can be observed that the electrical conductivity of the microemulsion systems was slightly increased. Although the observed increase of the conductivity indicates a small increase of interparticle interactions, this rise was not so important to signify structural transition from the water-in-oil to a percolating system and the bicontinuous state. Interestingly, microemulsions formulated with Tween 80 showed very low conductivity values possibly due to very weak interparticle interactions. It is worth noting that in the presence of pyrethrins the conductivity values of the W/O

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microemulsions were slightly decreased. That reveals that pyrethrins affect the nanostructure of the microemulsions possibly through participation on the surfactants monolayer. Since an effect of pyrethrins on the properties of the membrane was hypothesized, further structural investigation employing the method of EPR spectroscopy and also scattering techniques was considered necessary.

3.0

2.5 -

Conductivity [µS.cm 1]

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2.0

1.5

1.0

0.5

0.0 0

2

4

6

8

10

% Aqueous phase

Figure 3. Electrical conductivity of W/O microemulsions as a function of aqueous phase weight

content. System 1, empty: (□), System 1, loaded: (■), System 2, empty: () and System 2, loaded: (▲). EPR measurements. EPR spectroscopy using the spin-probing technique with 5-DSA as

probe, was undertaken to study the interfacial properties of the surfactants monolayer of both W/O microemulsions and O/W nanoemulsions in the presence and in the absence of pyrethrins. This interface-located fatty acid spin probe gives EPR spectra reflecting the rigidity/flexibility of its environment from the depth of the membrane where the doxyl-ring of the 5-DSA is located. EPR spectra of 5-DSA in free and loaded W/O microemulsions at aqueous phase content 10% w/w and the corresponding diluted O/W nanoemulsions are given in Supporting Information (Figures S2 and S3) . EPR spectra characteristic of nitroxides were obtained in both cases. In

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general, EPR spectra of unequal heights and widths are indicative of a restrictive motion of the spin probe in the membrane where it is located. In the present study, both rotational correlation time (τR) and order parameter (S) of the spin-probe were calculated from the spectral characteristics of all the microemulsions tested and the results are shown in Table 1. As it can be observed in Table 1, by increasing the percentage of the aqueous phase from 0 to 10 (System 1) and from 0 to 7 (System 2), rotational correlation times (τR) of 5-DSA were increased both in empty and loaded microemulsions indicating a decrease of spin probe’s mobility in the membrane with water content. As the percentage of the aqueous phase was increased the curvature of the surfactants monolayer was decreased due to the swelling of the micelles. The lower the curvature the more closely packed are the hydrocarbon chains of the surfactants thus hindering the motion of the spin probe located in the membrane.27 Moreover, as shown in Table 1, the values of order parameter of 5-DSA measured in the same systems remained almost constant with the aqueous content. This means that the rigidity of the membrane was not affected by the increased aqueous phase content. In addition, both τR and S values of 5DSA in microemulsions of System 1 were higher than those of System 2, indicating a more tight packing of Tween 20 in lemon oil terpenes compared to Tween 80 at comparable water content. Furthermore, Table 1 provides information regarding the interfacial properties of Systems 1 and 2 when pyrethrins were encapsulated. It is observed that in loaded microemulsion systems the values of τR and S are decreased as compared to the empty systems for aqueous phase weight percentages higher or equal 5% w/w. More specifically, the observed decrease of the rotational correlation time related to the empty and loaded microemulsion system for the same aqueous phase weight percentage is indicative of a faster movement of the probe in the membrane and pyrethrum encapsulation results in a more flexible membrane packing. It is therefore deduced

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that the encapsulation of pyrethrins in W/O microemulsions of aqueous phase higher or equal 5% w/w affects the interfacial properties of both systems and plays a role in the membrane, possibly acting as a “spacer”. Similar behavior has been also reported for other drug containing microemulsions regardless the hydrophobicity of the drug.6,7,28 The calculated rotational correlation time (τR) and order parameter (S) values of the spin-probe in free and loaded O/W nanoemulsions are shown in Table 2. From the τR and S values of Table 2 we can conclude that 1:100 aqueous dilution of the W/O microemulsions of Systems 1 and 2 gave O/W nanoemulsions with membranes that, in general, allow the spin probe 5-DSA to rotate slower in other words a more hindered motion was observed. This effect was also reflected in the order parameter values that were higher in almost all systems studied. Interestingly, this effect was more pronounced in the case of System 2, indicating a tighter packing of Tween 80 molecules after phase inversion and consequent change of their orientation in the membrane. Concerning the effect of the addition of pyrethrins in the diluted systems, it was again found that the values of τR and S of the loaded nanoemulsions were constantly decreased as compared to the empty ones. To summarize, EPR spectroscopy employing the spin-probing technique is a valuable tool to monitor interfacial structural changes of free and loaded colloidal dispersions upon dilution with aqueous phase. Both W/O microemulsions and O/W nanoemulsions were affected by the presence of natural pyrethrins thus indicating participation of the biocide in the nanostructure. Such information could be of importance in the perspective of using these systems as environmental friendly formulations of biocides since membrane rigidity is related to the permeability and bioavailability of the enclosed active compounds.29

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Dynamic light scattering (DLS) and stability. Dynamic light scattering measurements of

O/W nanoemulsions at constant aqueous phase weight content 99% w/w were carried out to evaluate the size and the polydispersity of the oil droplets in the presence and in the absence of natural pyrethrins. DLS measurements of the corresponding W/O microemulsions were not possible due to the high surfactant concentration of the samples placing severe method limitations. Table 3 shows the size and polydispersity index of the oil droplets in empty and loaded with pyrethrins O/W nanoemulsions obtained from the corresponding W/O microemulsions, System 1 and System 2, upon dilution with a mixture of water and glycerol (2:1). DLS results showed the existence of two populations of oil droplets dispersed in the continuous aqueous phase for both nanoemulsion systems examined. Differences in droplet sizes were observed making clear that sample preparation plays a considerable role in the size determination of the oil droplets of the nanoemulsions. In any case nanoemulsions obtained by diluting the microemulsions of System 1 had larger hydrodynamic diameters and were more polydispersed compared to those obtained from the microemulsions of System 2. Polydispersity is the ratio of standard deviation to mean droplet size, so it indicates the uniformity of droplet size within the formulation. Nevertheless, in both cases, addition of natural pyrethins in the oily phase resulted in the formation of smaller droplets indicating participation of the biocide in the nanostructure. The experimental results obtained from the DLS measurements of the O/W nanoemulsions are in accordance with the results obtained from the EPR measurements regarding participation of pyrethrins in the nanostructure of the dispersions.

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Stability study. Droplet size measurements are a good indicator of the stability of the O/W

nanoemulsions. To evaluate the effect of the emulsification procedure and also the presence of pyrethrins on the stability of selected nanoemulsions, droplet size and polydispersity of the preparations were monitored by DLS for a time period of 10 days (Figure 4). After production, the mean oil droplet diameter (1st peak) of both empty and loaded O/W nanoemulsions was in the range of 210-258 nm with the PI values of lower than 0.3 indicating a relatively narrow size of distribution for the formulations. In this study, the aqueous phase used for the dilution of the W/O microemulsions was a 2:1 solution of water and glycerol. As it can be observed, pyrethrins showed a stabilizing effect, allowing the system to remain monophasic for a considerably longer period of time. This effect could be also related to the formulation of smaller oil droplets in the presence of the lipophilic biocide. Taking into consideration that aqueous dilution of the pyrethins containing W/O microemulsions to produce O/W nanoemulsions ready to use in sprayers for plant protection will be most probably carried out using tap water, the effect of tap water on the stability and the size of the nanoemulsions was also investigated. Figure 4 presents the stability study of loaded with pyrethrins o/w nanoemulsions using tap water for the 1:100 aqueous dilution. As it can be observed tap water affected the size of the formulated oil droplets resulting in smaller sizes. The mean diameter of the dispersed oil droplets was in the range of 77 to 107 nm for the 1st peak and from 14 to 17 nm for the 2nd peak. The systems also remain monophasic for the time period under investigation.

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Table 1. Rotational Correlation time, τR, and Order Parameter, S, of 5-DSA in empty and loaded

W/O microemulsions (Systems 1 and 2) at different aqueous contents.

System System 1, empty

System 1, loaded

System 2, empty

System 2, loaded

Aqueous content (%/w/w)

τR (ns)

S

0

1.64

0.15

2

1.71

0.15

5

2.07

0.17

10

2.20

0.16

0

1.70

0.15

2

1.80

0.15

5

1.89

0.15

10

1.99

0.15

0

1.39

0.14

2

1.50

0.14

5

1.56

0.15

7

1.71

0.15

0

1.38

0.14

2

1.49

0.14

5

1.54

0.14

7

1.55

0.14

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Table 2. Rotational Correlation time, τR, and Order Parameter, S, of 5-DSA in empty and loaded

O/W nanoemulsions.

O/W nanoemulsion

Aqueous content of initial W/O microemulsion

System

% (w/w)

% (w/w)

τR (ns)

S

System 1, empty

99

0

1.82

0.13

99

2

1.88

0.15

99

5

2.22

0.16

99

10

1.76

0.16

99

0

1.63

0.15

99

2

1.92

0.15

99

5

1.85

0.16

99

10

1.77

0.16

99

0

1.92

0.15

99

2

1.99

0.19

99

5

1.91

0.16

99

7

1.91

0.16

99

0

1.52

0.15

99

2

1.70

0.14

99

5

1.70

0.15

99

7

1.70

0.14

Aqueous content of

System 1, loaded

System 2, empty

System 2, loaded

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Table 3. Mean droplet diameter (nm) and polydispersity index (PI) of O/W nanoemulsions at

constant aqueous content 99% w/w, in the absence and in the presence of pyrethrins. All measurements were performed in triplicate. System

System 1, empty

System 1, loaded

System 2, empty

System 2, loaded

Aqueous content of initial w/o microemulsions % (w/w) 0

Size, D [nm] 1

Size, D [nm] 2

PI

222 ± 2

53 ± 6

0.28 ± 0.02

2

248 ± 7

52 ± 7

0.26 ± 0.02

5

257 ± 5

47 ± 9

0.26 ± 0.01

10

252 ± 6

52 ± 4

0.26 ± 0.02

0

232 ± 8

50 ± 7

0.24 ± 0.02

2

179 ± 4

-

0.14 ± 0.01

5

230 ± 2

42 ± 3

0.28 ± 0.01

10

211 ± 7

40 ± 4

0.24 ± 0.03

0

156 ± 4

35 ± 1

0.20 ± 0.01

2

178 ± 3

39 ± 3

0.14 ± 0.01

5

176 ± 5

37 ± 5

0.17 ± 0.01

7

165 ± 3

37 ± 4

0.23 ± 0.01

0

190 ± 3

44 ± 4

0.17 ± 0.01

2

180 ± 3

-

0.17 ± 0.01

5

173 ± 3

-

0.17 ± 0.01

7

178 ± 0

33 ± 2

0.16 ± 0.01

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350

300

250

Size [d.nm]

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200

150

100

50

0 0

2

4

6

8

10

12

Time [Day]

Figure 4. Droplet size of the empty and loaded O/W nanoemulsions (System 1) at constant

aqueous content 99% w/w during a stability test. Empty system: (□), loaded system: (■), diluted with tap water: (▲). All measurements were performed in triplicate.

Small Angle X-ray Scattering (SAXS). In the present study, SAXS measurements of both

empty and pyrethrin loaded microemulsions and the corresponding inverted nanoemulsions were performed to evaluate the size of the dispersed droplets and also the participation of pyrethrin in the nanostructures. Microemulsions. Initially, W/O microemulsions of System 1 at 10% w/w aqueous phase were measured. Experimental spectra showed that the scattering intensity of the loaded microemulsions was higher as compared to the empty ones. Moreover the interaction peak was shifted towards higher q values, meaning that the average center-to-center distance between the droplets was smaller in the presence of pyrethrins (Figure S3, Supporting Information). This can be either attributed to the formulation of larger droplets or to an increase in the number of the droplets upon pyrethrins incorporation.

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To evaluate the effect of storage on the structural characteristics of the microemulsions, SAXS spectra of loaded systems were recorded for samples prepared a year earlier under identical experimental conditions. In that case the diffractogram was different compared to that obtained from the freshly prepared microemulsions. Although the level of intensity was the same meaning that the volume fraction of surfactant and aqueous phase was the same, the interaction peak was not observed. The observed change in the shape of the curve could be related to a change in the shape or the size of the aqueous droplets of the microemulsions (Figure S3, Supporting Information). In any case these changes did not affect the stability of the microemulsions. Nanoemulsions. Empty and pyrethrin containing O/W nanoemulsions derived from the corresponding W/O microemulsions upon 1:100 aqueous dilution were measured by SAXS. To evaluate the storage effect on the structural characteristics of the oily droplets both fresh and one year old microemulsions were used for the dilutions. All the derived nanoemulsions are considered highly diluted and for this reason the diffractograms of the measurements were very noisy. Presenting the results in log-log curves, a q-4 slope is observed at small angles (Figure S4, Supporting Information). This is characteristic of an interfacial scattering. More specifically, a q4 slope is consistent with an interfacial scattering in SAXS. That reflects that there are two media separated by a sharp interface. Furthermore, to evaluate the size and the shape of the nanoemulsions, scattering data were analysed with the Generalized Indirect Fourier Transformation (GIFT) analysis.30,31 The corresponding pair-distance distribution function (PDDF) P(r) is given in Figure 5. The curves exhibit pronounced maximum and provide quantitative information about the internal structure of the oil droplets. Moreover, the PDDF function, which represents a histogram of the distances inside the particle, provides the maximum dimension of the particle as well as its shape. A

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symmetric maximum indicates the presence of spherical aggregates. It is deduced that the oil droplets in these systems seem to be spherical with a total dimension of 36-37 nm. Interestingly storage of the initial microemulsions did not affect the profile of the pair-distance distribution function of the derived inverted nanoemulsions.

Figure 5. Corresponding pair-distance distribution function of O/W nanoemulsions derived from

W/O microemulsions (System 1) with 10 w/w aqueous phase. Empty (dashed line), loaded with pyrethrins (solid line) and loaded with pyrethrins prepared 1 year before (dotted line).

Taking into account the results from DLS and SAXS measurements, it was supposed that two populations of oil droplets might exist in the continuous aqueous phase, namely larger droplets with diameters >150 nm and also smaller globular droplets of 36-37 nm in diameter. It is essential to be noted that with the SAXS technique, large particles are difficult to be identified. At this point it should be noted that nanoemulsions are characterized by the possibility to contain more than one droplet populations whereas microemulsions tend to have one narrow peak in their particle size distribution.14 Concerning the structure of the dispersed oily phase the existence of spherical droplets seems quite

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Insecticidal activity study. The insecticidal activity of the developed pyrethrin O/W

nanoemulsions was tested in laboratory bioassays and compared to two commercial insecticide products, a suspension concentrate (SC) pyrethrum formulation and also an emulsifiable concentrate (EC) deltemethrin forlmulation.

Dose-response trials were carried out and the

mortality data assessed 24 hours after treatment were subjected to probit analysis estimating the LC50 and LC90 values and 95 confidence intervals. LC50 and LC90 are the minimal inhibition concentrations of a given product to cause 50% and 90% effect on the studied population, respectively. The results of the laboratory toxicity bioassays are shown in Table 4. As it can be observed lower LC50 and LC90 values of the natural pyrethrin nanoemulsions compared to the commercial pyrethrin product (Pyrethro Vioryl 5SC) were obtained meaning increased efficiency for A. gossypii control upon nanoencapsulation. Although the observed differences were not proved statistically significant at 95% confidence intervals (marginally in case of System 1), the increasing trend in the insecticidal effect of the natural pyrethrin in the nanoemulsions is promising and encourages further research. Nanoencapsulation of water insoluble pyrethrins in stable, non-toxic nanoemulsions probably enhances the pesticidal life and in parallel enables easier penetration due to the increased interfacial area of the system. In this task, more extended laboratory bioassays and field trials with the target-aphid on host plants will be needed in order to approximate the insecticidal activity of the nanoemulsions in agricultural conditions. Our results also showed that the toxicity of deltamethrin against A. gossypii was substantially higher than that of the natural pyrethrins, regardless the type of formulation. In general, pyrethroids are considered as more effective insecticides compared to natural pyrethrins, but they are also characterized by negative effects on aphid natural enemies, in contrast to harmless or moderate effects of pyrethrins.32,33 Differences in the relative susceptibility of target insect species and

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non-target beneficial insects to natural pyrethrins or synthetic pyrethroids have been attributed to differences in the production of the enzyme glutathione transferase, which is capable of metabolizing pyrethrins.32,34,35 Possible adverse effects of the developed nanodispersions on beneficial arthropods are yet to be investigated, however based on the general profile of natural pyrethrins and preliminary toxicity studies of the nanoemulsions on the aphid predator Coccinella septempuctata (Coleoptera: Coccinellidae), we expect the nanoemulsions to keep this advantage over pyrethroids, which makes them compatible plant protection products in organic farming and IPM systems. 36

Formatted: Font: Not Bold

Table 4. LC50 and LC90 values for the nanoemulsions of natural pyrethrins (System 1 and 2),

Pyrethro Vioryl 5SC (natural pyrethrins) and Decis 2.5 EC (deltamethrin) against Aphis gossypii nymphs on eggplant (laboratory bioassays using the leaf-dip method). Slope (±S.E.)

System 1

System 2

1.68 ± 0.15

1.78 ± 0.17

PyrethroVioryl 5SC (pyrethrins)

1.75 ± 0.14

Decis 2.5 EC (deltamethrin)

1.78 ± 0.14

LC50 (95% C.I.)

LC90 (95% C.I.)

(x 10-6 mg a.i./ml solution)

(x 10-6 mg a.i./ml solution)

609.1

3518.3

(443.2–773.3)

(2714.4-5031.7)

761.8

4011.2

(565.7–949.8)

(3007.4–6221.2)

965.5

5224.0

(742.0–1186.5)

(4050.0-7444.0)

134.5

704.8

(97.5–169.8)

(544.0–1002.5)

x2

Df

32.7

23

39.6

23

37.1

23

53.8

28

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CONCLUSION Biocompatible micro- and nanoemulsions based on lemon oil terpenes, polysorbates, water and glycerol were structurally characterized to be used for the formulation and effective delivery of natural pyrerthrins. Significant advantages of the proposed nanoformulations over other conventional formulations of phytoprotective substances are the use of safe nontoxic ingredients and the ability to prepare biocide loaded concentrates and then dilute them with water in one step with low energy input. Pyrethrins are botanical insecticides which upon encapsulation and subsequent release from nanoemulsions present a greener, environmental friendly and easy to apply alternative to chemical pesticides. As evidenced by the conductivity, EPR, DLS and SAXS measurements, structural characteristics of the hosting systems upon pyrethrins’ incorporation were not changed in a way that could affect their stability. In addition participation of the pyrethrum extract in the nanostucturation of both the concentrated and the diluted colloidal dispersions was shown. The nanoformulation seems to be able to enhance the insecticidal effect of the active substance for the control of the aphid A. gossypii, and should be further investigated as a new formulation for the improvement of the commercially available insecticide. The information generated in this study could potentially facilitate the design and development of novel biopesticide formulations based on safe biocompatible colloidal delivery systems, leading to reduced-risk plant protection products for the environment and humans.

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ACKNOWLEDGMENTS Dr. M.J. Stébé and M. Emo from Université de Lorraine are gratefully acknowledged for the SAXS measurements. G. Sotiroudis from the Institute of Biology, Medicinal Chemistry and Biotechnology, NHRF, is acknowledged for his involvement in the discussion concerning the SAXS results. Dr. D.P. Papachristos from Benaki Phytopathological Institute is acknowledged for his valuable comments and discussion on the efficacy study. This work was financed by the Greek Secretary of Research & Technology and VIORYL SA, Greece, within the frame of the common research project “Cooperation”, 09- ΣΥΝ -42-699 and the STSM, COST, Action CM 1101 scientific program on Colloidal Aspects of Nanoscience for Innovative Processes and Materials.

ASSOCIATED CONTENT EPR spectra of microemulsions and nanoemulsions, SAXS spectra of microemulsions and nanoemulsions. This material is available free of charge via the Internet at http://pubs.acs.org.

AUTHOR INFORMATION Corresponding Author Papadimitriou Vassiliki, E-mail: [email protected], Tel: +302107273763, Fax: +302107273758

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Biocompatible Colloidal Dispersions as Potential Formulations of

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