Sustainable nano-pesticide platform based on Pyrethrins II for prevention and control Monochamus alternatus | Journal of Nanobiotechnology


Materials and characterization

Pyrethrins II (PthII, 98%) was purchased from Shanghai Acmec Biochemical Co., Ltd (Shanghai, China); Trimesic acid (H3BTC, 98%), copper acetate (Cu(OAc)2H2O, 98%) and acetic acid (HAc, > 99.8%) were provided by Shanghai Macklin Biochemical Co., Ltd. (Shanghai, China); Fluorescein isothiocyanate (FITC, > 95%) and trypan blue were supplied by Coolaber Science & Technology Co., Ltd (Beijing, China); DAPI Staining Solution and 4%Paraformaldehyde Fixative Solution were supplied by Shanghai Beyotime Bio-Technology Co., Ltd (Shanghai, China); DMEM medium and Fetal bovine serum (FBS) were obtained from ThermoFisher scientific Co., Ltd (Shanghai, China); Insect cell culture medium sf9 was purchased from Thermo Fisher Scientific (Shanghai, China). Nematode growth medium (NGM) was obtained by Shijiazhuang cmore Technology Co., Ltd (Shijiazhuang, China); CCK-8 kit was got from New Cell & Molecular Bio-Technology Co., Ltd; The C. elegans, the Bristol strain N2 and escherichia coli OP50 (E. coli strain OP50) were obtained from Sunybiotech Science & Technology Co., Ltd (Fujian, China); DF-1 cells were C/E chicken embryo fibroblasts which were resistant to endogenous ALV in the E subgroup, and were a gift from the Institute of Poultry Diseases and Tumors of the United States Department of Agriculture that was preserved by our laboratory for generations. Other reagents were from Beijing Aitemon Co., Ltd (Beijing, China).

X-ray diffraction (XRD) patterns were recorded with a Bruker D8 Discover 25 Advance diffractometer using nickel-filtered Cu Kα radiation (λ = 1.5406 Å). UV-visible spectra were recorded on a DaoJin UV-2550 spectrophotometer. Thermogravimetric analysis (TGA) was performed on a TG-DTA6300. Transmission electron microscopy (TEM) images was taken by a JEOL JEM 2100F at an accelerating voltage of 200 kV. Scanning electron microscopy (SEM) images were obtained by a JEOL JSM-6700F field-emission SEM with an accelerating voltage of 10 kV. Nitrogen sorption were performed in a MicroActive ASAP 2460 (2.02 version) adsorption apparatus at 77 K up to 1 bar to obtain pore volume and pore size by analyzing nitrogen adsorption and desorption isotherms. Confocal laser scanning microscope (CLSM) images were performed on a Leica TCS SP8 microscope. Fluorescent inverted microscope (Nikon TE2000), flow cytometer (BD LSRFortessa 4), stereo light microscope (SLM, Nikon, SMZ25), biological microscope (Yoke, XSP-8CA), high performance liquid chromatography (HPLC) is configured with Waters 1525 column oven and Waters 2998 UV detector. Cell viability was measured on a ThermoFisher Varioskan Lux Multifunctional microplate reader.

Preparation of PthII@HKUST-1

HKUST-1 as a member of the MOFs is widely used in the fields of medicine, catalysis, membrane engineering, etc., which is chosen to load biopesticide into HKUST-1, hoping to avoid the application problems of small-molecule biopesticide on the basis of reducing environmental pollution, and realize the high-efficiency application of pesticides. However, the size of HKUST-1 is usually large, and although it is speculated that it can effectively kill the insect of MA, it may not be transmitted efficiently on the diseased pine trees. So, this will prompt us to consider this harsh problem. In this paper, nanoHKUST-1 was chosen for encapsulating PthII to research the toxicity mechanism, traceable biopesticide monitoring and environment assessment. Simply, 0.6 g Cu (OAc)2·H2O was dispersed in 10% aqueous acetic acid, and 0.42 g H3BTC/20 mL ethanol was added drop by drop under high speed stirring for 60 min. The blue precipitate was collected by centrifugation of 14,000 rpm for 30 min, and washed with ethanol several times, then the precipitation was replaced with deionized water twice, stored at  − 80 °C for 2 h before freeze-drying. The obtained lyophilized powder of nanoHKUST-1 was redispersed in 20 mg/mL PthII/ethanol and stirred for 48 h. The mixture was centrifuged to collect the precipitate and washed with ethanol several times to harvest PthII@HKUST-1.

Preparation of FITC@HKUST-1

1 mg FITC was ultrasonically dissolved in 40 mL deionized water, and 50 mg nanoHKUST-1 powder was added to the FITC solution to stir at 300 rpm for 4 h in the dark. The product was centrifuged at 14,000 rpm for 20 min, and the supernatant was removed. The obtained precipitate was washed with deionized water several times to harvest FITC@HKUST-1.

Detection of PthII@HKUST-1 biopesticide-loaded

5 mg nanoHKUST-1 powder was dispersed in different concentrations of PthII/ethanol solution, and stirred at 300 rpm for 48 h. After the reaction, the supernatant was collected at 14,000 rpm for 20 min to measure the content of free PthII using an UV spectrophotometer. The loading efficiency of PthII@HKUST-1 was calculated according to the following formula [18].

$$ {\text{Loading}}\,{\text{efficiency = }}\left( {\frac{{{\text{Weight}}\,{\text{of}}\,{\text{total}}\,{\text{PthII}}{ – }{\text{Weight}}\,{\text{of}}\,{\text{free}}\,{\text{PthII}}}}{{{\text{PthII@HKUST}}{ – }{1}}}} \right) \times 100\% $$

Release performance of PthII@HKUST-1

The release curve of PthII@HKUST-1 was determined according to the method described in literature [23]. PthII@HKUST-1 powder was dispersed in PBS, transferred it to a dialysis bag after being ultrasonically dispersed, and put the dialysis bag into the deionized water. The release system was placed in a shaking box (150 rpm) for incubation. At different time points, the precipitate in the dialysis bag was collected by centrifugation, eluted with ethanol ultrasonic for three times, and centrifuged to collect the supernatant, and then the release amount of PthII in PthII@HKUST-1 was detected by high performance liquid chromatography (HPLC, Waters 2998 UV detector) after filtration with 0.22 µm filter membrane. The liquid chromatography detection conditions are as follows: mobile phases were acetonitrile (A) and 0.1% formic acid in water (B), gradient elution conditions: 0–10 min was 60–80% A, 10–10.1 min was 80–100% A, 10.1–15 min was 100% A,15–15.1 min was 100–60% A, 15.1–20 min was 60% A, with a flow rate 0.3 mL/min, injection volume 1 µL, column temperature 40 °C. The maximum absorption wavelength of PthII is 228 nm.

Photodegradability of PthII@HKUST-1

1 mg PthII@HKUST-1 powder was ultrasonically dispersed in 10 mL of PBS with pH of 5.0, 7.0 and 9.0, and spread in a 9.0 cm petri dish, then natural dried in the dark. The petri dishes containing PthII@HKUST-1 were exposed to ultraviolet light for different time, Free PthII and PthII water emulsion (PthII/WE) were as a control. Free PthII was dissolved in ethanol and spread in 9.0 cm a petri dish, and PthII/WE was directly spread in 9.0 cm a petri dish. The anti-photolysis performance of PthII@HKUST-1 was measured by UV spectrophotometer at 228 nm, that is, the difference between the mass of PthII before irradiation and the remaining amount after irradiation.

Biodistribution and cellular uptake

Firstly, ten of 5th instar MA larvae were placed in 6 cm petri dishes, respectively. The dispersed FITC@HKUST-1 (10 μg/mL) was sprayed on the larvae by small pressure spray can (soLo-408). After incubating for 5 min, the larvae are repeatedly rinsed with deionized water by small pressure spray can to remove non-adhered FITC@HKUST-1. The treated larvae were paralyzed with ethyl acetate and placed under a stereo light microscope (Nikon, SMZ25) to observe the fluorescence distribution on body wall and stomata. The main respiratory system of most insect larvae is through larval stomata, and a small amount spread through the body wall. Therefore, larval stomata and body wall gully area were focused on detection during the shooting. Secondly, the evenly dispersed FITC@HKUST-1 (10 μg/mL) solution was mixed with the artificial diet to feed MA larvae after starving for 24 h. After the larvae had fully fed for 1 h, the larvae were stored in 4% paraformaldehyde fixative solution for 48 h in the dark, and observed the fluorescence distribution of gut through dissecting intestines and making paraffin section. The fluorescence distribution of the larvae’s intestines was observed with a fluorescence microscope. Finally, 50 of 5th instar MA larvae were placed in 6 cm petri dishes, respectively. PthII@HKUST-1 (10 g/mL) was sprayed on the larvae by small pressure spray can and extend the crawling time of the larvae (1, 2, 3, 4 and 5 h) according to the previous operation. The epidermis of the larvae was dissected to observe the distribution of PthII@HKUST-1 on the epidermis through SEM.

In order to further study the cellular uptake of PthII@HKUST-1, DF-1 cells (C/E chicken embryo fibroblasts) were obtained as a model cell line, FITC@HKUST-1 was used to indirectly characterize the distribution of PthII@HKUST-1 on the surface of DF-1 cells. Firstly, DF-1 cells were inoculated into a 24-well plate at a concentration of 5 × 104 cells/well, and cultured in DMEM medium containing 10% fetal bovine serum, 1% penicillin–streptomycin and 5% CO2 for 24 h at 37 °C. After incubating, the original medium of DF-1 cells were removed and added fresh medium containing FITC@HKUST-1 (10 μg/mL). After 4 h of incubation, DF-1 cells were washed with ice PBS for three times, and digest with 0.25% trypsin for 5 min, and then the reaction was terminated with ice PBS. DF-1 cells suspension was centrifuged at 2000 rpm for 5 min with the temperature of 4 °C to remove supernatant, and resuspend the cells in ice PBS. The cellular uptake of FITC@HKUST-1 was observed through CLSM.

Cell morphology

Primary cell culture medium including 80% insect cell culture medium, 10% fetal bovine serum FBS and 10% phenylthiourea saturated solution, and subculture medium including 80% insect cell culture medium and 20% fetal bovine serum FBS, were ready for the following experiment. Under sterile conditions, the well-developed MA larvae were soaked in 70% ethanol for 20 min and absorbed the ethanol through the sterile absorbent paper. Then the insect needle was pierced into larval pronotum, and the insect blood cells were taken with a dot capillary. The blood cells were put into the primary cell culture medium and cultured under aseptic conditions at 27 °C for 7 days. After cultivation, the cell culture medium was transferred to the subculture medium and continued to incubate, and half of the subculture medium was taken out and replaced with fresh medium every week. PthII@HKUST-1, PthII/WE, Free-PthII and nanoHKUST-1 were added to the subculture medium with same concentration of PthII under aseptic conditions at 27 °C for cultivation. After 3 days of cultivation, the cell suspension was diluted with PBS and observed the cell morphology with a biological microscope.

Cytotoxicity assessment of PthII@HKUST-1

DF-1 cells were inoculated into 96-well plates at a density of 5 × 103 cells/well, and cultured in DMEM medium containing 10% fetal bovine serum, 1% penicillin–streptomycin and 5% CO2 for 24 h at 37 °C. After incubating, the original medium of DF-1 cells were removed and added fresh medium containing PthII@HKUST-1, and then PBS, PthII/WE and Free PthII were as control groups. Except for the PBS group, PthII/WE and PthII@HKUST-1 had the same amount of Free PthII. After 4 h of incubation, the medium of DF-1 cells were changed to the original medium to continue cultivate for 96 h. The medium was removed and added 20 µL, 5 mg/mL of CCK-8 to incubate for 4 h in the dark. The supernatant was removed and added 100 µL DMSO to detect the absorbance at 450 nm by a microplate reader. The calculation formula of cell viability is as follows:

$$ \left[ {\left( {{\text{A}}_{{{\text{Sample}}}} – {\text{A}}_{{{\text{Blank}}}} } \right)/\left( {{\text{A}}_{{{\text{Control}}}} – {\text{A}}_{{{\text{Blank}}}} } \right)} \right] \times 100\% $$

Among them, ASample is the absorbance value of the sample group, ABlank is the absorbance value of the blank group, and AControl is the absorbance value of the PBS group.

Mechanism research of contact toxicity and stomachtoxicity

The contact toxicity and stomachtoxicity of PthII@HKUST-1 were determined by the biopesticide membrane contact method and the feed mixing biopesticide method [24, 25]. Firstly, the dispersed PthII@HKUST-1 solution was placed in a petri dish, and slowly spread the filter paper into it to make it completely soaked, then turned the filter paper sided down to the top, and placed the well-developed larva after starving for 24 h on the filter paper to observe the biopesticide intake of the larvae’s abdomen. The well-developed 3rd-instar larvae were picked and placed on the filter paper, allowing them to crawl so that the larval epidermis completely contact PthII@HKUST-1 solution. Then the treated MA larvae were put in an artificial breeding box of 6 cm3. There were 30 larvae under each concentration gradient, and 10 larvae are a set of repeated group, and the control group was treated with water.

Feed mixing biopesticide method: 3 g of artificial feed were put into the breeding box, and 1 mL PthII@HKUST-1 solution was added. The well-developed 3rd-instar larvae were put into the breeding box. There were 30 larvae under each concentration gradient, and 10 larvae are a set of repeated group, and the control group was treated with water. The breeding boxes were placed in a constant temperature incubator with a temperature of 26 ± 1 ℃ and a relative humidity (RH) of 65 ± 10%. The survival rate of MA larvae was counted at 24 h and 48 h after feeding. If the larva did not move when touched with a brush. HKUST-1, PthII/WE and Free PthII were as control.

Environmental assessment

Preparation of M9 buffer solution: Na2HPO4·12H2O (12.1 g), KH2PO4 (2.4 g), NaCl (4.0 g) and MgSO4·7H2O was dissolved in 1 L deionized water. Lysate solution: NaClO: 2 mol/L NaOH = 2:1 (v/v). The Caenorhabditis elegans, the Bristol strain N2 was cultured in inoculated Escherichia coli OP50 at 20 ℃. Adult nematodes in the egg-laying stage were washed with M9 buffer solution into the EP (Eppendorf) tube, then centrifuged to discard the supernatant, and M9 buffer solution was added to wash twice. The lysate solution was added and performed intermittent shaking fully lyse the nematodes. Then the M9 buffer solution was added and washed 3 times repeatedly to remove the lysate components. Finally, the M9 buffer solution was used to transfer the eggs that settle at the bottom of the centrifuge tube to an empty petri dish without medium, and incubated in an incubator at 20 °C for 12 h to obtain L1 instar larvae.

Before conducting the environmental safety assessment of nanomedicines, we firstly studied the stability of HKUST-1 in M9 buffer and also in nematode serum considering the stable delivery of HKUST-1 to nematodes, fetal bovine serum (FBS) was chosen for mimicing the in vivo serum environment of nematodes. Simply, HKUST-1 were dispersed in M9 buffer and 10% FBS for 7 days to observe their size changes by DLS detection. Then, HKUST-1, Free PthII and PthII@HKUST-1 were dispersed in M9 buffer solution, and mixed with E. coli OP50 solution at ratio 1:1 to obtain a culture solution with final concentrations of 0, 0.001, 0.01, 0.1, 1, 10, 100 and 1000 μg/mL. The prepared culture medium was spread evenly into the petri dish just poured with NGM medium and placed in a 37 °C incubator for 1 h. After the solution was dry, the petri dish was placed upside down and incubated for 11 h. The nematodes that reached L4 instar after being cultured for 2 days were transferred to a medium containing different concentrations of reagent. After 24 h of feeding, LC50, body length and width, and head swing frequency were measured.

Determination of LC50: different concentrations of reagent (200 μL) were added to the 96-well plate, and then 30 of L4 nematodes were picked into each well. Each concentration treatment was repeated three times. After contacting with the agent in the incubator at 20 °C for 24 h, counted dead nematodes under the microscope to calculate the LC50.

Determination of head swing frequency, body bending frequency and body length and width: the conventional application concentration 20 μg/mL of free PthII, HKUST-1 and PthII@HKUST-1 are configured to study the effect of three reagent on the body length, width and mobility of C. elegans. In one independent experiment, three L4 nematodes with the same activity were selected, one infected with Free PthII for 24 h, one infected with HKUST-1 for 24 h and one infected with PtII@HKUST-1 for 24 h, and then place them on three slides dripping with M9 buffer solution (60 μL). Each reagent was independently replicated 3 times, and total 90 nematodes were determined. After 1 min of stable adaptation, the head swing and body bending frequency of nematode were observed and recorded under a microscope within 1 min. The head of the nematode swings from one side of the body to the other, and the angle > 90° is defined as one head swing. A sine wave movement of the nematode relative to the long axis of the body is defined as a one body bending. In addition, the nematode to be tested was anesthetized, then observed and photographed with a microscope, and the body length and width was measured using Image J software.

In-tree delivery of PthII@HKUST-1

Although it has been clarified that PthII@HKUST-1 could effectively kill MA larvae in the direct application process, it is still a big problem that cannot be ignored whether it can effectively penetrate the bark of the diseased pine into the trunk and spread through the water transport. After all, realizing the practical application of PthII@HKUST-1 is our core value of preparing nano-pesticides. As an important evaluation standard, transmission of PthII@HKUST-1 in tree trunks were studied in this paper. Experiment base on studying transmission of PthII@HKUST-1 was is located at Taishan Forest Area in the city of Tai’an, Shandong Province, China (117.06 °E, 36.23 °N). An electric drill was used to drill a hole with a diameter of 5 mm and a depth of 4 cm in the trunk at a 45-degree angle downward from the ground of 30 cm. First, FITC@HKUST-1 was used to inject trees and the diseased pine was sawn off after different processing time. The diseased pine was cut into small wooden pieces along the direction of the pine pith to observe the transmission of FITC@HKUST-1 through the small animal image. Second, PthII@HKUST-1 was injected with the concentration 1.0 mg/L, 10 mg/L and 50 mg/L, respectively, and PthII/WE with the same concentration as a control. After 3 days, sawdust was taken 5 cm above the punch at different depths. The method of subcritical water was used to extract PthII from wood chips. Simply, the sawdust was mixed with deionized water and put into a small polytetrafluoroethylene tank, then tanked into the reaction kettle and placed in a high-temperature furnace at 120 ℃ for 30 min. The reaction kettle was taken out, and quickly cooled, centrifuged to remove the aqueous solution. Sawdust was added methanol and mechanically shaken for 2 min, then centrifuged to retain the supernatant, continued to repeat the above operation 2 times. The methanol solution was collected to measure the content of PthII by an ultraviolet spectrophotometer. Third, the dead wood was sprayed with the concentration 1.0 mg/L, 10 mg/L and 50 mg/L, respectively, and PthII/WE with the same concentration as a control. After 3 days, sawdust was taken 5 cm above the punch at different depths. The method of subcritical water was also used to extract PthII from wood chips with the same operation method.

Statistical analysis

All experiments in this work were repeated three times, and statistical analysis of the data was performed by analysis of variance (ANOVA), and all the data were subjected to normality and homogeneity tests using DPS v 7.05. All graphical data are reported as the mean ± standard deviation (SD). Significance levels were set at * p < 0.05.


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