Preparation and Release Performance of Carboxymethyl Chitosan Gel Microspheres for Spinetoram

Pesticide controlled release technology is an effective measure to improve the efficiency of pesticide utilization. In this study, the controlled releasing gel microspheres of spinetoram were prepared by suspension-crosslinking. Carboxymethyl chitosan was the carrier, and calcium chloride and glutaraldehyde were the cross-linking agent. The diameter and morphology of microspheres were observed. The thermal stabilities of spinetoram-loaded microspheres were evaluated. The release properties of spinetoram loaded gel microspheres were investigated. The results showed that pesticide loaded microspheres were elastic balls with high strength, more thermal stability than traditional pesticides, and exhibited good performance of pesticide sustained release. The release of spinetoram was governed by the combination of swelling and erosion of carboxymethyl chitosan. This study would provide reference for the application of carboxymethyl chitosan in pesticide reduction and efficiency.


Introduction
Spinetoram is a new type of spinosad insecticide produced by Dow Yinong Company.It has stomach toxicity and contact effect, and has special effect on lepidoptera, fly and thrips pests.At present, the commonly used formulation of spinetoram is suspension concentrater, with high application rates, which not only increases costs but also causes environmental pollution.Pesticide sustained and controlled release formulations can avoid these drawbacks, among which pesticide microspheres have the characteristics of targeted delivery, controlled release, and reduced dosage, and have been widely studied [1,2] .Pesticide microspheres mainly rely on carrier materials to regulate or modify the active components of pesticides to achieve sustained release function, so the selection of carrier materials is crucial [3] .Carboxymethyl chitosan has the advantages of non-toxicity, good water solubility, biodegradability, biocompatibility, and film-forming properties, as well as good compatibility with the vast majority of drugs/pesticides.It has received widespread attention as an ideal carrier material for controlled release and sustained release technology [4] .
We recently constructed the pesticide controlled releasing gel microspheres with carboxymethyl chitosan as the carrier.The pesticide-loaded gel microspheres prepared by suspension-crosslinking had high pesticide loading and encapsulation efficiency, and had a release time of over 6 days [5] .However, the stability and mechanism of pesticide release from microspheres is unclear.Here, the spinetoram loaded gel microspheres were prepared by the same method.The thermal stabilities and release properties of spinetoram loaded gel microspheres were investigated.These results would provide reference for the application of carboxymethyl chitosan in pesticide reduction and efficiency.

Materials and Instruments
Carboxymethyl chitosan was synthesized through the carboxylation reaction of chitosan [5] .Spinetoram (suspension concentrater, 60g/L) was obtained from pesticide companies.The other chemical reagents such as the glutaraldehyde and calcium chloride used in this study were bought from the market as high purity as possible.
Lambda 35 UV-Vis spectrophotometer (PerkinElmer, US) was used to record the UV-Vis absorption spectra.Milli-Q Element water purification system (Millipore Co., Billerica, MA, USA) was used to prepare the deionised water.Electron microscope (Nikon, Japan) was used to observe the morphology.

Preparation of Microspheres
Gel microspheres were prepared by suspension crosslinking according to reference 5. First, carboxymethyl chitosan in 30 mL deionised water and spinetoram in 5 mL dichloromethane were mixed and stirred at 40 °C for 2 h.Then, the suspension was slowly dropped into the calcium chloride and glutaraldehyde crosslinking solution through a 5 mL syringe.After crosslinking for 1 h, the solvent was removed by filtration, and the gel microspheres were obtained.

Observation of Diameter and Morphology of Microspheres
The diameter and morphology of gel microspheres were viewed by electron microscope.More than 200 particles were observed [5] .

Determination of Spinetoram Loading and Encapsulation Efficiency
The spinetoram loading and encapsulation efficiency were determinated by UV scanning according to reference 5.

Analysis of Thermal Stability of Spinetoram Loaded Microspheres
The spinetoram loaded microspheres in a glass tube were placed at 35 and 45 °C for 2h, 4h, 6h, 8h, 24h, 32h to determine the change of spinetoram content, respectively, with the traditional formulation as a control.

Swelling Release Properties
Weigh an appropriate amount of dry microspheres and disperse them in deionized water.Stir at 100 rpm at room temperature, take out the sample at regular intervals, and use filter paper to absorb the surface moisture before weighing.The swelling degree of the drug loading system is expressed by water absorption rate as shown in formula 1.
Where, W0 is the initial dry gel microspheres weight (g), Wt is the wet gel microspheres weight at different times during the swelling process (g).

Erosion Release Properties
Weigh an appropriate amount of dried microspheres and disperse them in deionized water.Stir at 100 rpm at room temperature, take out the sample at regular intervals, and replenish with fresh deionized water.Measure the absorbance of the pesticide at the maximum absorption wavelength and calculate the pesticide release amount based on the standard curve.After the in vitro release experiment, collect the residual microspheres, dry it at room temperature to a constant weight, and calculate the erosion rate using the following formula 2.
Where, W0 is the weight of dried gel microsphere before in vitro release experiment (g), Wf is the weight of dried gel microsphere after in vitro release experiment (g), Wt is the weight of pesticide released from pesticide loading system during in vitro release experiment (g).

Pesticide Release Model Analysis
The pesticide cumulative release data of chitosan gel microspheres were analyzed by Ritger's generalized model according to equation 3 [6] .
Where, Mt/M0 is the cumulative release percentage, t is the release time, k is a constant, and n is the diffusional exponent characteristic of the release mechanism.

Statistical Analysis
Determinations were carried out in triplicate.Data were collected and processed using Origin Pro 8.5 (Origin Lab Co., Northampton, MA, USA) and SPSS 18.0 (Chicago, IL, USA).Significant differences between means were determined by T-tests with p< 0.05 considered a statistically significant difference.

Preparation of Microspheres
Spinetoram loaded carboxymethyl chitosan gel microspheres were prepared using the optimized suspension crosslinking method [5] , and the morphology of gel microspheres was observated through electron microscope.As shown in figure 1, the microsphere was elastic with high strength, and the appearance was smooth with a particle size of 0.91~1.03mm.The interlaced network could be clearly seen in the interior of the gel microspheres, and the spinetoram was evenly embedded in the grid.

Spinetoram Loading and Encapsulation Efficiency
First, the standard curve of spinetoram was made by ultraviolet method and the blank microsphere was used to subtract the interference of the adjuvant in the microsphere.The standard curve equations of spinetoram was Y=0.0391X+0.0293,R 2 =0.9980, which had a good linear relationship between the absorbances and spinetoram mass concentrations within 0-30 μg/mL.The spinetoram loading and encapsulation efficiency were 43.51% and 87.00%, respectively, which were higher than imidacloprid and acetamiprid [4] .It had been found that the pesticide loading and encapsulation efficiency would be affected by the structural characteristics of carriers and the physicochemical properties of pesticides [7,8] .In this system, spinetoram might be interact with carboxymethyl chitosan more easily, and its volume might be more suitable to embed into the grid of net-like gel microspheres.

Thermal Stability of Spinetoram Loaded Microspheres
Figure 2 showed that the pesticide microspheres were more stable than the spinetoram suspension concentrate at high temperatures.At 35 °C, the decomposition of spinetoram suspension concentrate presented a rapid upward linear trend within the first 8 hours, and the decomposition rate reached 72.35%.After that, its decomposition tended to slow down, and at 32 hours, the decomposition rate reached 90.08%.The decomposition trend of spinetoram microspheres was the same as the spinetoram suspension concentrate, but slower than it, and at 32 hours, the decomposition rate of spinetoram microspheres was 81.83%.Compared to 35 °C, at 45 °C, the spinetoram suspension concentrate decomposed faster and exhibited sudden release within 1 hour, with a decomposition rate of 50.89%.At 32 hours, the spinetoram suspension concentrate was completely decomposed, and the decomposition rate of spinetoram microspheres was 85.50%.These findings indicated that spinetoram-loaded carboxymethyl chitosan microspheres had good thermal stability and could protect spinetoram from high-temperature decomposition.

Spinetoram Release Properties
Chitosan has swelling properties.At the same time, the cross-linked gel microspheres prepared by ionic cross-linking reaction are a reversible reaction, and their cross-linked structure will destroy the network due to ion dissolution, which means that they have erosion properties.Here, the swelling and erosion properties of spinetoram-loaded carboxymethyl chitosan microspheres were investigated.As shown in figure 3 (left), the water swelling rate of the gel microspheres increased with the increase of time.When the swelling equilibrium was reached, the water swelling rate of the gel microspheres tended to decrease.Blank chitosan microspheres reached the maximum swelling rate earlier than pesticide loaded chitosan microspheres.This might be due to the ion and covalent crosslinking between pesticides and carboxymethyl chitosan delayed the swelling of carboxymethyl chitosan, and this could slow down the release of pesticides.At the same time, at each time point except at 1h, the swelling rate of the blank chitosan microspheres was lower than that of the pesticide-loaded chitosan microspheres.This might be due to the interaction between the pesticide and carboxymethyl chitosan broken the dense molecular structure of carboxymethyl chitosan [9,10] , and thus the water swelling rate of the microspheres was increased.The cross-linking structure of gel microspheres prepared by ionic cross-linking reaction would be destroyed due to ion dissolution.The experiment measured the weight loss of carboxymethyl chitosan carrier at the end of pesticide release, and obtained the erosion rate of 33%.
The release of pesticides from the macromolecular network system could be divided into chemical reaction (chemical bond breaking between the pesticide and the matrix, matrix degradation and dissolution), diffusion, solvation (swelling) and mixed controlled release mechanism.As shown in figure 3 (right), for the spinetoram suspension concentrate, with the extension of release time, the release amount of the spinetoram showed a linear upward trend, and the spinetoram was completely released within 22 hours.For the spinetoram loaded chitosan gel microspheres, the spinetoram release showed a slow upward trend, and the spinetoram release time lasted to 72 hours.The cumulative release property of spinetoram loaded chitosan gel microspheres was analyzed by generalized model.According to the equation (3), when n > 0.85, the release belongs to the transfer mechanism of Super case II, that is, the release of drugs in gel microspheres is the result of swelling and erosion.Only when the matrix in the swelled gel microspheres breaks some ionic cross-linking bonds can erosion occur.The degree of erosion increases with the gradual increase of matrix swelling.As the erosion rate increases, the pores in the matrix increase, which accelerates the release rate of drug.Thus, a Super case II type transfer mechanism would be developed, where the release rate increases with time.When the n value decreases from more than 0.85 to less than 0.85, the transition from Super case II type transfer to irregular transfer mechanism might be related to the reduction of swelling and erosion of gel microspheres.Here, the release of spinetoram from calcium chloride glutaraldehyde crosslinked gel microspheres was jointly controlled by the swelling and erosion of the matrix materials in the microspheres.

Conclusion
In short, a smooth and elastic spinetoram-loaded microsphere was made by means of emulsion crosslinking.The spinetoram loading and encapsulation efficiency reached 43.51% and 87.00%, respectively.Compared to traditional spinetoram suspension concentrate, the spinetoram-loaded carboxymethyl chitosan microspheres had better thermal stability and longer release time.The release of spinetoram from calcium chloride glutaraldehyde crosslinked gel microspheres was jointly controlled by the swelling and erosion of the carboxymethyl chitosan.This work would further promote the application of carboxymethyl chitosan in pesticide controlled release agents.

Figure 2 .
Figure 2. Decomposition of different pesticide formulations at different temperatures.
This research was financially supported by the Science and Technology Key Project of Guizhou Province, China (JZ [2015]2006 and [2017]01), and the Science and Technology Project of Guizhou Institute of Biology ([2021]07).
Tao R P, You C Q, Qu Q L, Zhang X L, Deng Y K, Ma W J and Huang C B 2023 Environ.Sci.Nano 10 351 [2] Yang G T, ZhaoH M, Liu Ya L, Li Z L, Gao F, Zhang Q, Zou Peng, Liu Z G and Zhang M 2023 Arab.J. Chem.16 7.