Preparation of THI@BSA·NPs and Loading efficiency
To study the structure of the synthesized THI@BSA·NPs, SEM images were used to characterize the final products. As shown in Fig. 2A, the nanoparticles were uniformly spherical and evenly dispersed in the field of view. The surface of the nanoparticles was smooth, and no holes were formed. As shown in Fig. 2B, the average particle size was approximately 23 nm, and the particle size distribution showed a single peak, indicating that the particle size distribution of THI@BSA·NPs was concentrated and that no other by-product had been produced. Continuous sampling within 30 days showed that the particle size and zeta potential of the nanoparticle powder did not fluctuate greatly, indicating that the prepared nanoparticles had good stability (Figure S1).
The calibration curve of THI was linear in the concentration range of 2–25 µg/mL, the regression equation was y = 0.04594x, and the correlation coefficient was 0.9997 (Fig. 2C). The loading efficiency of THI@BSA·NPs was calculated by measuring the content of unencapsulated THI after the solvent was completely evaporated. As shown in Fig. 2D, with increasing concentrations of THI acetonitrile solution, the loading efficiency gradually increased. When the concentration exceeded 80 µg/mL, the loading efficiency was basically stable and stopped increasing. When the THI concentration reached 40 µg/mL, the optimal loading efficiency was approximately 64.8%. The above results showed that the THI@BSA·NPs prepared by encapsulating THI with BSA had a uniform nanostructure and an ideal THI loading efficiency.
Release performance
To study the controlled-release properties of THI@BSA·NPs, the release behaviors of THI@BSA·NPs were investigated in PBS at different temperatures and pH conditions (Fig. 3), and compared with those of the commercial THI microcapsule suspension (THI@M). The temperature and pH selected in this study represented the temperature and acid-base range under natural conditions, and the physiological conditions of MA. At a pH of 7.0, the cumulative release durations were over 15 d at 15 ℃, 25 ℃, and 35 ℃. The trends of the three release curves were basically the same, and the entire release process was stable. No sudden release occurred, and the cumulative release amounts under the three temperature conditions were approximately 93%. As the temperature increased, the release rate of THI@BSA·NPs increased to a certain extent, but the cumulative release amounts under the three temperature conditions were similar. The above results indicated that temperature conditions could affect only the release rate of THI@BSA·NPs, and did not affect the cumulative release of THI from THI@BSA·NPs. Next, at 25 ℃., the release behaviors of THI@BSA·NPs were investigated in PBS at pH 5.0, 7.0 and 9.0. Under these three pH conditions, the cumulative release durations of THI@BSA·NPs were over 10 d. The pH 5.0 treatment group had the highest cumulative release (97.9%), followed by the pH 9.0 treatment group (96.8%), and the lowest cumulative release (93.4%) was observed for the pH 7.0 treatment group. Under the three pH conditions, the trends of the three release curves were also the same, and the release process was relatively stable; no sudden release occurred. These results showed that the appropriate acid-base conditions would promote the release of THI and increase the cumulative release amount. This may be related to the destruction of the structure of the protein under acid-base conditions, as more THI was released from the incomplete shell structure.
Finally, the release behavior of THI@M was investigated under the same conditions (25 ℃, pH 7.0). After 3 d of treatment, THI@M reached a state of release equilibrium, with a cumulative release of approximately 90.0%. The above results show that THI@BSA·NPs had a better slow-release performance, a longer release duration, and a higher cumulative release amount, which is conducive for long-term control of pests. The development of THI@BSA·NPs might effectively reduce the frequency of application while ensuring a control effect.
Toxicity bioassay
By determining the contact toxicity and stomach toxicity of THI@BSA·NPs toward the 3rd instar larvae of MA, whether the BSA nanocarriers had an effect on the bioactivity of THI was evaluated. As shown in Fig. 4, for the contact toxicity of THI@BSA·NPs against larvae at 24 and 48 h, the LC50 was 61.8 and 35.6 µg/mL, respectively; for the stomach toxicity at 24 and 48 h, the LC50 was 55.1 and 25.9 µg/mL, respectively. The toxicity of THI was also determined for comparison. for the stomach toxicity against larvae at 24 h and 48 h, the LC50 was 74.5 and 39.0 µg/mL, respectively. The results suggested that the toxicity of THI@BSA·NPs at 24 and 48 h was significantly higher than that of THI in the two insecticidal modes. In the stomach toxicity experiment, the larvae were acclimated to drilling into artificial feed, and some active ingredients came in contact with the larval surface. This behavior explained that the stomach toxicity in this study included a certain contact toxicity, which was one of the reasons why the stomach toxicity was much higher than the contact toxicity. These results were consistent with previous research results. Kah summarized previous research results and found that in 21 related reports, the toxicity of nanopesticides was 2.0 times higher than that of pure active ingredients[42], and nanocarriers could improve the insecticidal activity of active ingredients on multiple levels. Next, the performance of THI@BSA·NPs in the two insecticidal mechanisms was studied separately.
Contact toxicity
There are three main steps by which contact pesticides exert their insecticidal effects: first, they come in contact with and remain on the surface; then, they penetrate the epidermis structure; and finally, they reach the target site. Due to the nanoscale particle size and large specific surface area of nanopesticide formulations, they could improve the coverage of, adhesion to and permeation into pests compared with conventional formulations[43]. Therefore, the distribution of the fluorescence of THI@BSA@FITC·NPs on the surface of the larvae and its coverage were examined to understand the performance of THI@BSA·NPs on the surface of the larvae (Fig. 5). As shown in Fig. 5B, there was some fluorescence distributed in the head, body and tail. Compared with the heads and tails of the larvae, the fluorescence distribution in the bodies was more uniform, and the fluorescence intensity was stronger. This might be related to the greater activity of the larval head and tail, compared with the body; the swing range of the tail and the head is large, causing the attached THI@BSA@FITC·NPs to fall off. In addition, the fluorescence was more concentrated on the internodes and small protrusions on the surface of the larvae, indicating that the nanoformulation had a stronger ability to adhere to these uneven structures than to the smooth surface, and more THI@BSA@FITC·NPs remained by the holes on the internodes. In addition, fluorescence distribution was observed on each of the small rosary warts that made up the step blisters (ambulatory ampullae) for crawling on the larval abdomen. As the larvae crawl mainly by using the step blisters (ambulatory ampullae) of the abdomen, this part had a long contact duration with the THI@BSA@FITC·NPs, indicating that the longer the contact duration is, the stronger the adhesion of THI@BSA@FITC·NPs on the larval surface. These phenomena proved that, as a nanopesticide delivery system, THI@BSA·NPs exhibited good coverage and adhesion to pests, which could allow the active ingredients to come in contact with and stay on the larval surface.
It is important to study how nanoparticles penetrate the epidermal structure of pests to better understand the mechanism underlying the increase in contact toxicity. Coomassie brilliant blue-stained THI@BSA·NPs were used to explore the main mechanism by which the nanoparticles penetrated the epidermal structure into the larvae. One phenomenon caught our attention (Fig. 5C) during analysis of the staining results. The longer the larvae were exposed to the stained THI@BSA·NPs, the more intensely the valve was stained on both sides of the larval body, indicating that an increasing number of THI@BSA·NPs remained at the valve. The valve is constantly opened or closed when the larvae breathe, and the nanoparticles could easily pass through the valve and enter the body. Perhaps the nanoparticles could penetrate the epidermal structure or interstitial membrane to directly enter the larvae to exert insecticidal activity; however, the valve channels were needed when a large number of nanoparticles entered the body.
Finally, whether THI@BSA·NPs could successfully enter the larvae still needed to be verified. THI@BSA@FITC·NPs were used to treat the larval surface, and then, the treated larvae were dissected to obtain transverse sections and observed under a fluorescence microscope (Fig. 5D). It could be seen that fluorescence was uniformly distributed on the entire cross-section. A mass of dispersed AVM@BSA@FITC·NPs was clearly observed, indicating that the nanoparticles could enter the larvae through the epidermal structure and move further into the center of the larval body to deliver the AVM to the target site. The entire epidermal structure emitted bright fluorescence, indicating that a large number of THI@BSA@FITC·NPs were enriched in the surface layer and had not yet entered the larvae. These results were also verified in our previous toxicity bioassay. Therefore, THI@BSA·NPs exhibited higher contact toxicity than THI.
Stomach toxicity
The efficiency of transport and conduction could be enhanced significantly, and the insecticidal toxicity could be accelerated, because the small particle size pf the nanoparticles improved the dispersal and permeability[44]. Therefore, the mechanism for the acceleration of stomach toxicity could be revealed (Fig. 6A) by comparing the transport efficiencies of THI@BSA·NPs and THI@M in the body. The total THI content detected in the THI@M treatment group was much smaller than that in the THI@BSA·NP treatment group. After 24 h of treatment, the THI levels in the head, body and tail of the THI@M treatment group were 0.11, 0.43, and 0.09 mg, respectively, and the total THI content was 0.63 mg. The THI levels in the head, body and tail of the THI@BSA·NP treatment group were 0.09, 0.62 and 0.15 mg, respectively, and the total THI content was 0.86 mg. After 48 h of treatment, the THI content in each treatment group increased, and the distribution trend was basically the same. It is known that a certain amount of odorous organic auxiliaries is added in the process of microcapsule pesticide production, which could cause antifeedant behavior, and the antifeedant behavior of larvae toward pesticides may lead to a decrease in the intake of active ingredients. In contrast, no other additives were used during the synthesis of THI@BSA·NPs, and the small amount of organic solvent used was removed by rotary evaporation. The active ingredient detected in the THI@BSA·NPs was higher than that in THI@M, indicating that the antifeedant behavior of THI@BSA·NPs was significantly lower than that of THI@M, which could lead to increased ingestion of active ingredients by pests. In the same treatment duration, in the THI@BSA·NP treatment group, more active ingredients were distributed in the body and tail; with prolongation of treatment duration, more THI was detected in the body and tail. This result suggests that THI@BSA·NPs could significantly increase the transport efficiency of active ingredients due to the size of the nanoparticles and the hydrophilicity of BSA itself. The midgut and hindgut, the major digestive and absorption systems of larvae, are concentrated in the middle and rear parts of the larvae. The higher the transport efficiency is, the greater the amount of active ingredients that could be absorbed, further improving the bioactivity of THI.
The larvae were fed artificial feed mixed with THI@BSA@FITC·NPs, and then, the treated larvae were collected and dissected to obtain intestinal tissue sections. Then, the fluorescence distribution of intestinal tissues was observed by fluorescence microscopy (Fig. 6B). Fluorescence distribution was also observed in the intestinal tissues, indicating that under oral feeding, THI@BSA@FITC·NPs had high transport efficiency inside larvae and could be smoothly transported to intestinal tissues. The villi inside the intestinal wall could clearly be seen, and the fluorescence intensity inside the intestinal wall was the strongest due to the adhesion of THI@BSA@FITC·NPs to these villi. Fluorescence was also observed on the fat particles outside the intestine, indicating that the intestine effectively absorbed THI@BSA@FITC·NPs and quickly converted them into nutrients. Shen's research showed that nanocarriers could efficiently penetrate insect midgut cells to reach the target site, thereby improving activity[28]. This phenomenon revealed that a small number of THI@BSA@FITC·NPs could escape directly through the intestinal tissue cells due to the nanoscale size of the particles.
Transport efficiency tree trunks
Since the larvae live in the trunk of the pine tree, which acts as a natural barrier against chemical control, THI@BSA·NPs were designed to be injected into the trunk during application; thus, the transport efficiency in the trunk of the pine tree would directly affect the application. Therefore, at the end of this study, the transport efficiencies of THI@M and THI@BSA·NPs in the trunk were compared (Fig. 7). As shown in the figure, the transport efficiency of THI@BSA·NPs was significantly higher than that of THI@M at the same concentration. The distribution of THI in the THI@BSA·NP treatment group was more even than that of THI@M; when the concentration was 50 mg/mL, the proportions of the four layers were 28.94%, 26.97%, 25.00% and 19.09% in the THI@BSA·NP treatment group and 45.41%, 26.97%, 14.50%, and 9.84% in the THI@M treatment group. On the other hand, as the concentration decreased, the transport efficiencies of the THI@BSA·NP treatment group and the THI@M treatment group improved. In the THI@M treatment group, the THI levels in layer 4 were 1.0, 1.6 and 3.0 mg/g when the concentrations were 200, 100, and 50 mg/mL, respectively, and in the THI@BSA·NP treatment group, the levels were 3.4, 4.6, and 5.8 mg/g, respectively. This showed that with decreasing concentration, greater amounts of active ingredients could be transported farther away, and the migration distance could be effectively increased. THI@BSA·NPs are nanosized particles with high dispersibility and a long migration distance in water, and all these features would allow the active ingredients reach the site where the target organism resides more efficiently, improving the application efficiency and reducing the pesticide application amount. In applications, the migration distance in the trunk could be increased by appropriately diluting the particles without affecting the control effect of THI to achieve the maximum performance.
THI@BSA·NPs were considered for treatment of diseased wood to reduce the cost of transporting the diseased wood in high-altitude areas to resettlement sites. The transport efficiencies of different concentrations and different formulations were also studied, and the distribution of nanoparticles in the trunks of dead trees was explored. The results, as shown in Fig. 8, were the same as those for live trees. When the concentration was 200 µg/mL, the THI content in the layers was 89.4, 96.5, 101.7, 62.6 and 29.7 µg/g, and the corresponding values in the THI@M group were 126.0, 112.9, 73.9, 33.6, 21.9 µg/g; the distribution of THI@BSA·NPs in the trunks of dead trees was more even. On the other hand, when the active ingredients are the same, the lower the concentration is and the higher the water consumption is, the more uniform the distribution