Synthesis and characterization of acetamiprid nanoemulsion by high-energy methods

Nanotechnology's application in agriculture has opened up new options for generating nanosized agrochemicals that have the potential to improve efficiency, improve stability, extend the effective duration, and reduce environmental impacts 1 . One of the most pressing difficulties in the agricultural industry is the need to handle pesticide-related issues such as environmental contamination, bioaccumulation, and increases in insect resistance, which necessitates reducing the amount of pesticide sprayed on crops and protecting stored products. Nano-technology is proving to be an appealing tool for achieving this goal since it provides new ways to synthesize and transport active ingredients known as na-nopesticides 1 . Nanoemulsions are particularly well suited to creating lipophilic functional agent delivery systems 2 . The current study uses a high-energy (ultrasound) emulsification approach to create oil-in-water (O/W) acetamiprid nanoemulsions with synthetic and natural additives. The acetamiprid nanoemulsions were spontaneously formed by adding a mixture of acetamiprid and solvent in an aqueous solution containing a surfactant (tween) with continuous stirring. The nanoemulsions were then formed by ultra-sonication. Various characterization techniques for acetamiprid nanoemulsions include particle size analysis (DLS), Fourier-transform infrared spectroscopy (FTIR), and transmission electron microscopy (TEM). Acetamiprid nanoemulsions are further evaluated by studying thermodynamic stability. This includes a Centrifugation assay, Freeze-thaw cycle nanoemulsions, Heating-cooling test, stability at room temperature of 25°C, pH measurement, and viscosity measurement. The droplet size and morphology of the acetamiprid nanoemulsions were measured by dynamic light scattering (DLS) and transmission electron microscopy (TEM). DLS and TEM measurements showed that acetamiprid nanoemulsions had an almost droplet size distribution (PDI < 200 nm). On this basis, an insecticide acetamiprid was incorporated into an optimized nanoemulsion system to demonstrate potential applications in pest control.


INTRODUCTION
However, forming stable nanoemulsions over a long period is a crucial but difficult feature to achieve. The specific methods by which nanoemulsions arise and how their characteristics can be regulated are still being studied at the fundamental level. Compared to traditional emulsions, nanoemulsions exhibit significantly superior gravitational separation and aggregation 17 . Ostwald ripening, flocculation, and coalescence are the additional Physico-chemical processes that break down nanoemulsions. To produce a stable nanoemulsion formulation, several parameters must be regulated, such as component composition, sequence of addition of components, and application of shear or surfactants in a way that efficiently ruptures droplets 14 . According to one study, increased surfactant concentrations resulted in more stable nanoemulsions. A combination of surfactants can sometimes give better stability than a single surfactant (Wang et al. 2007). The emulsifiers or surfactants act as a shear, which breaks down a dispersible liquid into small droplets (typically nanometers in size). This process is known as emulsification, where surfactants reduce the surface tension, and precipitation is no longer likely to occur. Nanoemulsions are an excellent choice for delivering poorly water-soluble active ingredients. However, nanoemulsion formulations that improve the bioavailability of active chemicals can distribute and encapsulate hydrophilic and hydrophobic pesticides. Nanoemulsions have considerable economic and safety advantages for pesticide delivery in agrochemicals. 21 .The main objective of this study was to prepare oil in water nanoemulsion containing an Acetamiprid insecticide. Moreover, a Nanoemulsions system was prepared with surfactant (Tween 80 or lecithin). The nanoemulsion preparation method was high shear agitation, which is widely used for pesticide preparation. The mixing ratio of the three levels of the active ingredient, solvent, co-solvent, surfactant, sonication time, and sonication cycle and power. The effect of these factors on the long-term stability of the nanoemulsions, droplet size distribution (PDI) by dynamic light scattering (DLS), (FTR)Fourier-transform infrared spectroscopy, (TEM)Transmission Electron Microscopy, viscosity, pH.

MATERIALS AND METHODS Chemicals and reagents
Acetamiprid grade (95%) was obtained from the Ministry of Agriculture National Center for Pesticides Control. (Abu Grabe, Baghdad, Iraq). (Polyethylene glycol 6000 PEG-6000), (Chitosan), (Sodium Tripolyphosphate Light Density STPP), (Linseed oil), (Acetic acid), surfactant (Tween 80 or lecithin), and Emulsifier SPAN 20 purchased from the chemical bureau(Bab Al Moatham, Baghdad, Iraq). Deionized water was used for all the experiments throughout the study. All other commercially available solvents and reagents were used without further purification.

Preparation of acetamiprid nanoemulsion
Preparation of active ingredient Acetamiprid (Nanoemulsion): For the preparation of nanoemulsion (active ingredient Acetamiprid), Many combinations with different proportions of materials were prepared; after passing all tests, this emulsion was chosen, which gives the greatest stability. The nanoemulsion was prepared according to the method described previously by Sugumar and co-authors with some modifications 22 .As well as the procedure with some modifications 23 . The nanoemulsion was prepared by taking 10 ml of Tween solution into a flask (surfactant) and adding 100 ml of thawed water. Added 30 ml of linseed oil, and the oil phase was slowly added to the aqueous phase with stirring at 4000 rpm for 30 minutes. Added 3 grams of the active ingredient, dissolved in 97 ml of solvent (ethanol), and added to the mixture. The formed emulsions were then sonicated with an ultrasonic probe (ultrasonic homogenizers). These combinations were tested to find out their stability.
Preparation of commercial insecticide ) nanoemulsion ( : The commercial insecticide nanoemulsion was prepared by taking 200 ml of the commercial pesticide and dissolved in 400 ml of deionized water to obtain a commercial emulsion with a high concentration. The formed emulsions were then sonicated with an ultrasonic probe (ultrasonic homogenizers). These combinations were tested to find out their stability 23 .
Thermodynamic stability studies to screen nanoemulsions. Centrifugation assay: The samples were centrifuged for 30 minutes at 5000 rpm, and phase separation, foaming, and cracking were observed. Nanoemulsions should have maximum stability not associated with phase separation (foaming and cracking). The successful formulations were subjected to other thermodynamic stability tests. The measurements were carried out in triplicate 24 .
Freeze-thaw cycle nanoemulsions: This test was performed to determine the increased stability of the nanoemulsion formulations. The formulations were exposed to two temperatures (-21 ° C and 21 ° C) for at least 24 hours for each temperature test. The measurements were carried out in triplicate 24 . Heating-cooling test: It is used to demonstrate the radiation effect of heating and cooling on the stability of prepared nanoemulsions, where the prepared nanoemulsions were held at 4 and 40 ° C with exposure for 48 hours for each temperature test. Formulations that are stable at this temperature are subject to further study. The measurements were carried out in triplicate 24 . Stability at room temperature of 25°C: A glass tube was filled with around 20 mL of freshly manufactured nanoemulsions. During an a4-week storage period, the shift from steady-state to creaming and coalescence was studied 24 . PH measurement: The pH value is one of the main parameters of nanoemulsions. Observation of the pH value follows from the determination of the stability of the nanoemulsion due to the change in pH in the presence of chemical reactions. In the present study, a digital pH meter was used to determine the pH values of the prepared nanoemulsions. The samples were repeated three times. Viscosity measurement: The nature and concentration of surfactant, oil phase components, droplet size, and the viscosity of the essence of oil production all influence the viscosity of nanoemulsions 25,26 . BROOKFIELD CAP 2000+viscometer at 25C was used to assess the nanoemulsion's dynamic (absolute) viscosity. The viscosity of the formulations was determined without the use of any additional dilution. Characterization of the prepared nanoemulsions Particle Size Analysis DLS: This technique is commonly known as dynamic light scattering (DLS) but is also called photon correlation spectroscopy (PCS) and quasi-elastic light scattering (QELS). The latter terms are more common in older literature. DLS is most commonly used to analyze nanoparticles. Examples include determining nanogold, protein, latex, and colloid sizes. The technique is generally best used for submicron particles and can be used to measure particles with sizes less than a nanometer. 27 . Nanoparticles may differ in physical properties, such as composition and concentration, as well as in size, shape, surface properties, crystallinity, or dispersion state. These properties are usually assessed by several methods, aiming for the complete characterization of the nanoparticles; dynamic light scattering (DLS) is one of the most commonly used 28,29 . The particles' size and polydispersity index (i.e., size distribution) were determined through Dynamic Light Scattering Technology (DLS) using a HORIBA model SZ-100 to determine particle size. All evaluations were performed in triplicate. Tests were carried out for samples in the Faculty of Biology, University of Tehran, Science Campus, Enghelab St., Tehran, Iran. Fourier-transform infrared spectroscopy FTIR Determination of the active groups contributing to the preparation of particles (Nanoemulsion): Infrared spectroscopy (FTIR) is an essential means of determining the effective groups that can be reducing agents, capping agents, and stabilizers for the prepared nanoparticles. In the current study, several peaks of energy units were obtained when analyzing the preparation of active ingredient acetamiprid nanoemulsion, preparation of commercial insecticide nanoemulsion, and standard commercial insecticide.
Transmission Electron Microscopy TEM: Transmission electron microscopy is, without a doubt, one of the most essential techniques for characterizing nanoparticles. TEM produces micrographs of nanoscale materials with a high lateral spatial resolution by focusing an electron beam on a thin (usually less than 200 nm) sample 25,26 . By eliminating picture distortion with aberration correctors, current electron microscopes may attain resolutions as low as 0.05-0.1 nm, resulting in high-resolution images with atomic resolution. 32,33 By spatially restricting and concentrating the impinging beam and measuring the resulting electron diffraction pattern, TEM may also analyze the crystalline structure of specific tiny sections of crystalline materials 34 TEM allows for the analysis of size, shape, and crystal structure at the single-particle level due to its great spatial resolution and selectivity 35 . The TEM technique is frequently used to characterize nanoemulsions as a supplemental tool for observing lipid particles directly and obtaining trustworthy data about the system's shape 36 . Tests were carried out for samples in the Faculty of Biology, University of Tehran, Science Campus, Enghelab St., Tehran, Iran. Table 1 summarizes the pH measurements of pesticide nanoemulsions, which indicated that the pH ranged from 5.00 to 5.60 for all formulations. Insecticides are more vulnerable to alkaline degradation than fungicides or plant growth regulators. Furthermore, insecticides are the most vulnerable to alkaline degradation or "high" pH solutions. The results in Table 1 show that all nanoemulsions were less than 25mPas.Each reading was collected after the sample had reached equilibrium for 2 minutes. Nanoemulsions of (Commercial Insecticide Acetamiprid Nanoemulsion), (Active ingredient acetamiprid Nanoemulsion), and (particles pesticide Acetamiprid) had low viscosity values (7.10, 6.10, and 24.23mPa.s, respectively).  In the current study of infrared spectroscopy (FTIR), the result has several peaks of energy units obtained when analyzing * Commercial insecticide acetamiprid nanoemulsion*Active ingredient acetamiprid nanoemulsion*pesticide acetamiprid. Also recorded values at 2179.01 1/cm, which represents the group aliphatic isonitrile s; CN stretching vibrations, well as recording peaks for the region of 1648.00 1/cm, which represents isolated group w-m; C=C stretching vibration, While the peak at 660.59 1/cm represents a Vinyl hydrocarbon compound w; C-H wagging vibration. Spectroscopy (FTIR) has shown *Active ingredient Acetamiprid Nanoemulsion, shape 2 having peaked at 3360.89 1/cm, which represents Imines group m; N-H stretching vibration (associated), Also recorded values at 2979.79 1/cm, which represents the group Ethers s-m; CH3 asym. Stretching vibration, As well as recording peaks for the region of 1644.26 1/cm, which represents isolated group w-m; C=C stretching vibration, While the peak at 686.72 1/cm represents a group of Vinyl hydrocarbon compound w; C-H wagging vibration. As shown by spectroscopy analysis, * particles pesticide acetamiprid in Figure 3 have peaks at 2962.51 1/cm, representing Ethers group s-m; CH3 asym. Stretching vibration, Also recorded values at 1662.66 1/cm, which represents the isolated group w-m; C=C stretching vibration, As well as recording peaks for the region of 1089.17 1/cm, which represents sat. Prim alcohol group s; C-O stretching vibration. Tests were carried out for samples in the Faculty of Biology, University of Tehran, Science Campus, Enghelab St., Tehran, Iran. http://www.science-and-fun.de/tools/ The micrograph of three nanoemulsions Figure 4,5,6 shows that the droplets have a spherical shape, typical of an oil-in-water nanoemulsion. According to TEM analysis, the compositions' droplet diameters are nanometric in size. The particle size of prepared nanoemulsions produced by Transmission Electron Microscopy (TEM), Figure 7, showed droplet size values of (Commercial insecticide acetamiprid nanoemulsion) with an average size of 61.5nm as well as recording droplet size values of Figure 8 (Active ingredient acetamiprid nanoemulsion) which has an average size of particles of 94.66nm. As shown by the microscope (particles pesticide acetamiprid) in Figure 9, the average size of particles is 84.3nm.

DISCUSSION
The optimal pH level should be between 5 and 7 37 . However, the (particle pesticide Acetamiprid) had the highest value (27.23 mPa.s). From these results, it can be concluded that viscosity increased slightly in the low water-loading nanoemulsion 38 . The size of emulsion droplets was estimated at an average of three measurements and was shown as an average diameter in nanometers. (DLS) values indicated a restricted size distribution providing good stability of nanoemulsions 39,40 . Spectroscopic analysis has shown *Commercial insecticide acetamiprid nanoemulsion, shape 1 Many peaks of energy units; it recorded peaks at 3366.53 1/cm, which represents the Imines group v; N-H stretching vibration (free) to the spectroscopic values table (FTIR table) 41 While the peak at 657.52 1/cm represents a group of Vinyl hydrocarbon compound w; C-H wagging vibration 41 , TEM analyses also confirmed that the droplet diameter of the formulations falls on a nanometric scale. The average size of a drop of oil-in-waterer type nanoemulsions is typically between 20 and 200 nm 42 . Several authors verified the nanodroplet size measurements, reporting that the microstructure and size distribution were produced with nanoemulsions containing specific pesticides 23,43,22 . Droplet size in nanoemulsions has been shown to improve water delivery of insoluble compounds and active compounds 43,44 . Understanding the physics of nanoemulsion generation is crucial for managing droplet volume 45 . It should be mentioned that previous research 46,23 surface concentration, type of oil, ultrasonic energy, time on drop diameter.  Oil-in-water (o/w) nanoemulsions containing different insecticides were prepared by ultrasonic emulsification. The active ingredient, solvent, co-solvent as oil phase, surfactant, and water as water phase were used. The conditions of ultrasonic emulsification were applied at a power of 10 kHz for 20 min and sonication pulses of 9 cycle/s. The Particle Size Analysis (DLS), determination of the active groups (FTIR), TEM, viscosity, and pH of the nanoemulsions were studied and confirmed that the products were found in the nanometric range (20-200 nm). Stable nanoemulsions are stable against degradation processes, such as interference, deposition, flocculation, and cohesion. Regarding cost, manufacturing safety, transport, and usage, nanoemulsions containing pesticides offer more substantial benefits than emulsifiable concentrations. These also aid in solubilizing lipophilic active substances by reducing organic solvents, widely used in agricultural applications. Nanoemulsions used at the nanoscale will have a high level of pest control effectiveness while posing no danger to the environment. Nanoemulsions containing pesticides, on the other hand, may hold promise for the creation of pesticide formulations, and further study is needed in this field. Future research will focus on assessing the toxicity of these pesticide nanoemulsions against several major agricultural pests, as well as comparing their efficacy to that of traditional and commercial formulations already in use.