An upconversion nanomaterial with both visual tracking and interface control capabilities and its applications

Abstract

This invention belongs to the field of nanomaterials and green pest control technology, specifically relating to an upconversion nanomaterial and its application, which combines visualization tracking capabilities with interface regulation capabilities. The upconversion nanomaterial, from the inside out, comprises: an upconversion nanoparticle core (UCNP) for emitting visible light under near-infrared excitation, enabling in vivo optical tracking; an aminated mesoporous silica coating layer (MSN) providing a high specific surface area for dsRNA loading and regulating surface charge and interfacial wettability; and a dsRNA loading layer, which is electrostatically adsorbed onto the aminated surface. This upconversion nanomaterial simultaneously achieves dsRNA protection, delivery, and visualization; improves the stability and utilization rate of dsRNA in insects; significantly improves compatibility with the insect epidermal interface; and exhibits stable RNAi effects, is applicable to a wide range of delivery methods, and is beneficial for large-scale green pest control applications.

Description

Technical Field

[0001] This invention belongs to the field of nanomaterials and green control technology for agricultural pests, specifically involving an upconversion nanomaterial with both visual tracking and interface control capabilities and its applications. Background Technology

[0002] The information disclosed in this background section is intended only to enhance understanding of the overall background of the invention and is not necessarily to be construed as an admission or in any way implying that such information constitutes prior art known to those skilled in the art.

[0003] RNA interference (RNAi) technology, with its advantages of strong targeting, environmental friendliness, and safety for non-target organisms, has become an important green control method for agricultural pests, replacing traditional chemical pesticides, and has shown broad application prospects in the field of pest control. Among them, lepidopteran pests such as the cotton bollworm are important pests causing damage in agricultural production, with a wide host range and high degree of damage. Moreover, long-term use of chemical pesticides has led to continuous enhancement of their resistance. Therefore, there is an urgent need for efficient and stable RNAi technology to control these pests.

[0004] However, existing RNAi technology faces numerous key technical challenges in its application to lepidopteran pests, resulting in low gene silencing efficiency and unstable effects of dsRNA, hindering its large-scale application. The main limiting factors include: dsRNA itself is easily degraded rapidly by nucleases within insects, leading to extremely low bioavailability; the insect epidermis and digestive tract possess natural hydrophobic barriers with poor wettability, impeding dsRNA adsorption and tissue penetration; existing delivery systems cannot achieve real-time tracking of dsRNA delivery pathways and distribution within insects, making it difficult to elucidate delivery mechanisms and hindering optimization and improvement; existing dsRNA delivery vectors, such as cationic polymers, liposomes, and single nanomaterials, suffer from limited loading efficiency, poor interface regulation capabilities, and a lack of optical tracer functionality; some vectors also pose toxicity risks, and RNAi efficacy is highly dependent on the delivery method, resulting in poor reproducibility. Summary of the Invention

[0005] This invention aims to address the problems of low dsRNA uptake efficiency, poor stability, lack of visualization of the delivery process, and insufficient interfacial penetration in existing RNAi delivery technologies for lepidopteran pests, and to provide an upconversion nanomaterial with both visual tracking and interfacial regulation capabilities and its applications.

[0006] To achieve the above-mentioned technical objectives, the present invention relates to the following technical solutions: In one aspect, the present invention provides an upconversion nanoparticle delivery material with both visual tracking and interface regulation capabilities, named UCNP@MSN-NH2@dsRNA; the upconversion nanoparticle delivery material comprises, from the inside out: an upconversion nanoparticle core (UCNP) for emitting visible light under near-infrared light excitation to achieve in vivo optical tracking; an aminated mesoporous silica coating layer (MSN) for providing a high specific surface area for dsRNA loading and regulating surface charge and interfacial wettability; and a dsRNA loading layer, which is bound to the aminated surface by electrostatic adsorption.

[0007] A second aspect of the present invention provides a method for preparing the above-mentioned upconversion nanodelivery material, the method comprising: synthesizing NaYF4:Yb,Tm@NaYF4 upconversion nanoparticles; coating their surface with mesoporous silica via a microemulsion method to obtain UCNP@MSN nanoparticles; performing amino modification with APTMS to obtain UCNP@MSN-NH2 nanoparticles; and mixing and incubating dsRNA with UCNP@MSN-NH2 nanoparticles to obtain UCNP@MSN-NH2@dsRNA.

[0008] A third aspect of the present invention provides the use of the above-described upconversion nanodelivery material in any one or more of the following: (a) Insect RNAi delivery or preparation of insect RNAi delivery products; (b) Controlling insects or preparing insect control products.

[0009] The delivery methods mentioned include, but are not limited to, epidermal contact delivery, feeding delivery, and insect cavity injection delivery, etc., and are not specifically limited here.

[0010] A fourth aspect of the present invention provides an insect RNAi delivery product or an insect control product, said product comprising at least the above-described upconversion nanodelivery material.

[0011] A fifth aspect of the present invention provides a method for controlling insects, the method comprising: applying the above-described upconversion nanodelivery material, insect RNAi delivery product, or insect control product to insects or an environment containing insects.

[0012] The beneficial technical effects of one or more of the above technical solutions are as follows: The nanodelivery platform provided by the above technical solution integrates functions such as stable protection of dsRNA, efficient delivery, optical visualization tracking, and interface wettability regulation. It effectively solves the core problems of low dsRNA uptake efficiency, poor in vivo stability, invisible delivery, and insufficient interface penetration in existing insect RNAi technologies, achieving efficient, controllable, and trackable delivery of dsRNA. The mesoporous silica coating provides physical protection for dsRNA, while amylation modification achieves efficient dsRNA loading (complete loading at a mass ratio of 1:10 to 1:20) through electrostatic interactions, effectively preventing dsRNA degradation by nucleases in the insect and significantly improving dsRNA bioavailability. The amylation surface modification layer regulates the positive charge density on the carrier surface, significantly improving the wettability of the superhydrophobic interface between the nanocarrier and the insect epidermis, enhancing the carrier's spreading ability on the insect surface, improving dsRNA's surface adsorption and tissue penetration capabilities, and solving the problem of hydrophobic barrier obstruction. The upconversion nanoparticle core emits visible light under near-infrared excitation, enabling real-time fluorescence imaging of the dsRNA delivery pathway and tissue distribution within insects using laser confocal microscopy. This provides intuitive experimental evidence for elucidating the delivery mechanism and optimizing delivery strategies. The nanodelivery platform can achieve stable dsRNA delivery through various methods, including epidermal contact, feeding, and intracavitary injection. By silencing the key detoxification metabolism gene UGT5, the sensitivity of cotton bollworm to lambda-cyhalothrin was significantly enhanced, greatly improving pest control efficacy. The nanodelivery system described above has been experimentally verified to have good biocompatibility, no significant toxicity to non-target organisms, and is environmentally friendly, meeting the requirements of green pest control. The preparation steps of the nanodelivery platform provided by the above technology are well-defined and controllable, the raw materials are readily available, and mass production is possible. Furthermore, the delivery method is simple to operate, suitable for large-scale field application, and has broad application prospects in the field of green pest control in agriculture. Attached Figure Description

[0013] The accompanying drawings, which form part of this invention, are used to provide a further understanding of the invention. The illustrative embodiments of the invention and their descriptions are used to explain the invention and do not constitute an improper limitation of the invention.

[0014] Figure 1 This is a schematic diagram of the nanoplatform structure in this invention.

[0015] Figure 2 The images show (A) transmission electron microscopy of UCNP nanoparticles, (B) UCNP@MSN, and (C) UCNP@MSN-NH2 successfully loaded with dsRNA to become UCNP@MSN-NH2@dsRNA.

[0016] Figure 3The contact angle between water, dsRNA, and UCNP@MSN-NH2@dsRNA droplets and the epidermis of the cotton bollworm in this invention is shown.

[0017] Figure 4 (A) Relative expression level of UGT5 under contact treatment in this invention, and (B) Mortality rate.

[0018] Figure 5 (A) Relative expression level of UGT5 under the feeding treatment in this invention, and (B) Mortality rate.

[0019] Figure 6 (A) Relative expression level of UGT5 under injection treatment in this invention, and (B) Mortality rate.

[0020] Figure 7 This invention uses co-focused imaging to detect UCNP@MSN-NH2@dsRNA attached to the larval epidermis.

[0021] Figure 8 The distribution of UCNP@MSN-NH2@dsRNA in the foregut, midgut, and hindgut of larvae was obtained by co-focusing imaging in this invention.

[0022] Figure 9 The distribution of dorsal blood vessels in larvae using co-focused imaging is UCNP@MSN-NH2@dsRNA, as described in this invention.

[0023] Figure 10 The images shown in this invention are: (A) fluorescence image of Sf9 cells treated with naked dsRNA, (B) fluorescence image of Sf9 cells treated with UCNP@MSN–NH2@dsRNA, (C) quantitative analysis of cell uptake and (D) RNAi efficiency.

[0024] Figure 11 For the safety assessment of (A) bees, (B) silkworms, and (C) zebrafish in this invention. Detailed Implementation

[0025] It should be noted that the following detailed descriptions are exemplary and intended to provide further illustration of the invention. Unless otherwise specified, all technical and scientific terms used in this invention have the same meaning as commonly understood by one of ordinary skill in the art to which this invention pertains.

[0026] It should be noted that the terminology used herein is for the purpose of describing particular embodiments only and is not intended to limit the exemplary embodiments according to the present invention. As used herein, the singular form is intended to include the plural form as well, unless the context clearly indicates otherwise. Furthermore, it should be understood that when the terms "comprising" and / or "including" are used in this specification, they indicate the presence of features, steps, operations, devices, components, and / or combinations thereof.

[0027] The following examples further illustrate the present invention, but do not constitute a limitation thereof. It should be understood that these examples are for illustrative purposes only and are not intended to limit the scope of the invention.

[0028] In a typical embodiment of the present invention, an upconversion nanoparticle delivery material with both visual tracking and interface regulation capabilities is provided, named UCNP@MSN-NH2@dsRNA. The structure of the upconversion nanoparticle delivery material, from the inside out, includes: an upconversion nanoparticle core (UCNP) for emitting visible light under near-infrared light excitation to achieve in vivo optical tracking; an aminated mesoporous silica coating layer (MSN) for providing a high specific surface area for dsRNA loading and regulating surface charge and interfacial wettability; and a dsRNA loading layer that is bound to the aminated surface by electrostatic adsorption.

[0029] The upconversion nanoparticle core is a core-shell structure NaYF4:Yb,Tm@NaYF4, which can emit visible light under near-infrared excitation, enabling optical visualization and tracking of the dsRNA delivery process within insects. The mesoporous silica coating layer has a high specific surface area and a hierarchical pore structure, providing ample loading sites for dsRNA and achieving efficient dsRNA loading. Simultaneously, its aminated surface modification can regulate the positive charge density on the carrier surface, enhancing the electrostatic adsorption between the carrier and the negatively charged dsRNA, thereby improving the dsRNA loading efficiency and stability. On the other hand, it improves the wettability of the superhydrophobic interface between the nanocarrier and the insect epidermis, enhancing the carrier's surface adsorption and tissue penetration capabilities. The dsRNA loading layer is adsorbed onto the aminated MSN surface through electrostatic interactions. The dsRNA can target the key insect detoxification metabolism gene UDP-glucuronyl transferase 5 (UGT5), including one or more of UGT5-19, UGT5-50, and UGT5-57, achieving efficient silencing of the target gene.

[0030] In another specific embodiment of the present invention, a method for preparing the above-mentioned upconversion nanodelivery material is provided, the method comprising: synthesizing NaYF4:Yb,Tm@NaYF4 upconversion nanoparticles; coating their surface with mesoporous silica via a microemulsion method to obtain UCNP@MSN nanoparticles; performing amination modification with APTMS to obtain UCNP@MSN-NH2 nanoparticles; and mixing and incubating dsRNA with UCNP@MSN-NH2 nanoparticles to obtain UCNP@MSN-NH2@dsRNA.

[0031] Furthermore, the specific method for synthesizing NaYF4:Yb,Tm@NaYF4 upconversion nanoparticles includes: Using Y(CH3CO2)3·xH2O, Yb(CH3CO2)3·4H2O, and Tm(CH3CO2)3·xH2O as precursors, 1-octadecene (ODE) and oleic acid (OA) were added, and the mixture was heated under an inert atmosphere to form a lanthanide oleic acid complex. Subsequently, a methanol solution containing NH4F and NaOH was added, and after stirring, the methanol was evaporated and the reaction was continued at a higher temperature. After precipitation with ethanol, centrifugation, and washing, the product was dispersed in cyclohexane. Then, a shell precursor solution was prepared using the same method (the only difference being that it did not contain Yb(CH3CO2)3·4H2O and Tm(CH3CO2)3·xH2O), and the above core particles were added. The reaction was carried out under an inert atmosphere to obtain the core-shell structure NaYF4:Yb,Tm@NaYF4.

[0032] The mass-volume ratio of Y(CH3CO2)3·xH2O, Yb(CH3CO2)3·4H2O, Tm(CH3CO2)3·xH2O, 1-octadecene (ODE) and oleic acid is 0.05-0.5:0.01-0.2:0.001-0.01:5-20:1-10 (g:g:g:mL:mL).

[0033] The inert atmosphere can be nitrogen; the formation of lanthanide oleic acid complex by heating under an inert atmosphere can be achieved by heating to 130-180℃ (preferably 150℃) under a nitrogen atmosphere for 0.5-5 h (preferably 1 h); the specific conditions for continuing the reaction by heating are 260-290℃ (preferably 285℃) for 60-120 min (preferably 90 min); the specific conditions for obtaining the core-shell structure NaYF4:Yb,Tm@NaYF4 by heating under an inert atmosphere are 260-290℃ (preferably 285℃) for 60-120 min (preferably 90 min) under nitrogen protection.

[0034] The specific method for obtaining UCNP@MSN nanoparticles by coating mesoporous silica on its surface using a microemulsion method includes: adding isopropanol and tetraethoxysilane (TEOS) to a cyclohexane solution containing UCNP and stirring, then mixing with an aqueous solution containing CTAB and urea to form an oil-in-water microemulsion system; after heating the reaction (e.g., reacting at 50-80℃ for 10-20 h), centrifuging and washing with ethanol to remove CTAB, thereby obtaining UCNP@MSN nanoparticles; The specific method for obtaining UCNP@MSN-NH2 nanoparticles by amination modification using APTMS includes: dispersing UCNP@MSN in anhydrous ethanol, adding 3-aminopropyltrimethoxysilane (APTMS) and heating under reflux (reflux at 80-90℃ for 10-20 h); the reaction product is centrifuged and washed with ethanol to obtain UCNP@MSN-NH2.

[0035] The specific method for obtaining UCNP@MSN-NH2@dsRNA by mixing and incubating dsRNA with UCNP@MSN-NH2 nanoparticles includes: dispersing UCNP@MSN-NH2 nanoparticles in RNase-free PBS and sonicating them. Then, slowly adding the dsRNA solution to the dispersion and stirring the mixture at room temperature allows the dsRNA to adsorb onto the nanoparticle surface via electrostatic attraction. After the reaction is complete, centrifugation and washing are performed to remove free dsRNA, yielding UCNP@MSN-NH2@dsRNA nanoparticles.

[0036] The mass ratio of dsRNA to UCNP@MSN-NH2 nanoparticles is 1:1 to 1:20; more specifically, it is 1:10 to 1:20.

[0037] In another specific embodiment of the present invention, the above-described upconversion nanodelivery material is provided for use in any one or more of the following: (a) Insect RNAi delivery or preparation of insect RNAi delivery products; (b) Controlling insects or preparing insect control products.

[0038] The delivery methods mentioned include, but are not limited to, epidermal contact delivery, feeding delivery, and insect cavity injection delivery, etc., and are not specifically limited here.

[0039] The product may be a pesticide, but no specific limitation is made here.

[0040] The insects mentioned can be pests, and more specifically, lepidopteran pests such as bollworms, etc., without being specifically limited here.

[0041] In another specific embodiment of the present invention, an insect RNAi delivery product or insect control product is provided, wherein the product comprises at least the above-mentioned upconversion nanodelivery material.

[0042] The product may be a pesticide, and further, the product may also contain at least one pesticide-acceptable excipient.

[0043] In another specific embodiment of the present invention, the excipients are selected from one or more of dispersants, wetting agents, disintegrants, binders, defoamers, antifreeze agents, thickeners, fillers, and solvents. The present invention does not impose any special restrictions on the sources of acceptable excipients for the bacterial agent; commercially available products are generally sufficient.

[0044] The dispersant is an anionic dispersant and / or a nonionic dispersant, and may be selected from one or more of sodium lignosulfonate, sodium naphthalenesulfonate formaldehyde condensate, sodium methylene bisnaphthalenesulfonate, formaldehyde condensate sulfate, polycarboxylate, alkylphenol polyoxyethylene phosphate, and fatty acid polyoxyethylene ester.

[0045] The wetting agent may be selected from one or more of sodium dodecyl sulfate, sodium dodecylbenzene sulfonate, soapberry powder, soapberry powder, tea seed cake powder, and splitting powder BX.

[0046] The disintegrant may be selected from one or more of bentonite, ammonium sulfate, aluminum chloride, urea, magnesium chloride, and glucose.

[0047] The binder may be selected from one or more of starch, diatomaceous earth, cyclodextrin, rosin, carboxymethyl cellulose, carboxyethyl cellulose, and carboxymethyl cellulose salts.

[0048] The defoamer may be selected from one or more of the following: C8-C20 fatty alcohols, C10-C20 saturated fatty acid compounds, epoxidized soybean oil, ethanol, silicone compounds, and organosilicon oil.

[0049] The antifreeze agent may be selected from one or more of sorbitol, ethylene glycol, polyethylene glycol, propylene glycol, glycerol, urea, and sodium chloride.

[0050] The thickener may be selected from one or more of gelatin, xanthan gum, polyethylene glycol, and polyvinyl alcohol.

[0051] The filler may be selected from one or more of the following: light calcium carbonate, diatomaceous earth, bentonite, attapulgite, and silica.

[0052] The solvent may be selected from water (preferably deionized water) or methyl oleate.

[0053] In another specific embodiment of the present invention, a method for controlling insects is provided, the method comprising: applying the above-mentioned upconversion nanodelivery material, insect RNAi delivery product, or insect control product to insects or an environment containing insects.

[0054] Example 1. Materials and Methods 1.1 Insect breeding and strain establishment cotton bollworm ( Helicoverpa armigera The strains were collected from Xinxiang City, Henan Province (35.31°N, 113.85°E) and purified using artificial feed for more than 10 generations. The sensitive strain (Group S) was taken from this long-term protected population; the highly resistant strain (Group R) was based on a laboratory population and purified using continuous LC-LC-MS with lambda-cyhalothrin. 50The strains were obtained through more than 10 generations of dose selection, exhibiting stable resistance levels. Both strains were reared at 27 ± 1 °C, relative humidity of 70-80%, and a light-dark cycle of 16:8 h. Larvae were fed artificial feed, while adults were provided with a 10% honey solution. Newly hatched larvae were reared separately to prevent cannibalism, and their feed was changed regularly. To avoid inbreeding depression, mating was performed periodically between different generations of the same strain. All larvae used in the experiment were derived from healthy, uniformly developed populations.

[0055] 1.2 Transcriptome Sequencing To compare the transcriptional expression differences between the R and S groups of cotton bollworms, third-instar larvae of both strains were starved for 12 hours before sampling, followed by feeding with a conventional artificial diet for 48 hours. Ten larvae from each treatment were collected as one biological replicate, for a total of three replicates. After flash freezing in liquid nitrogen and storage at -80 °C, total RNA was extracted using a commercially available kit. Genomic DNA was removed, and RNA quality (OD) was assessed. 260 / 280 = 1.8-2.2, RIN ≥ 7.0). Qualified samples were used for mRNA enrichment, cDNA synthesis, and library construction, and 150 bp paired-end sequencing was performed on a high-throughput platform. Data quality control was performed using FastQC and Trimmomatic, and alignment was performed using HISAT2 or STAR. Helicoverpa armigera Reference genome, featureCounts to count gene expression levels. Differential gene analysis was performed using DESeq2, with a significance threshold of FDR < 0.05; GO and KEGG enrichment analyses were performed using ClusterProfiler, and the results were visualized using R software.

[0056] 1.3 Validation of Transcriptome Data To validate the RNA-seq results, 10 differentially expressed genes (DEGs) were selected for qRT-PCR analysis. Three independent biological replicates were set up for each gene assay. Primers were designed based on NCBI sequences (Table 1). cDNA was synthesized using a Vazyme qRT-PCR kit (Vazyme, Nanjing, China). Each 20 μL reaction volume contained SYBR Green qPCR Mix, specific primers, and cDNA template. The amplification program was: 95 °C pre-denaturation for 2 min, followed by 35 cycles (95 °C 15 s, 64 °C 15 s). Relative gene expression levels were measured using a 22... ΔΔCt Methodological calculations. The correlation between qRT-PCR and RNA-seq data was analyzed using GraphPad Prism software.

[0057] Table 1. Primer Design Sequences for qRT-PCR

[0058] 1.4 Tissue-specific and developmental expression analysis To investigate tissue-specific expression of UGT5, the head, epidermis, fat body, foregut, midgut, hindgut, and Malpighian tubules of fifth-instar nymphs of the brown planthopper were collected and placed in grinding tubes containing DEPC-containing water. Three biological replicates were performed for each tissue, with 100 individuals per replicate. To analyze the developmental expression pattern of UGT5, samples were collected from eggs, 1st-5th instar nymphs, and both male and female adults. Samples at different developmental stages were obtained through single-pair reproduction. Three replicates were performed for each instar, with 50 individuals per replicate. After extracting total RNA, the relative expression level of UGT5 was detected by real-time quantitative PCR (qPCR).

[0059] 1.5 Preparation of dsRNA High-purity dsRNA was prepared using a bacterial induction expression system. Total RNA was extracted from *Bollworm* and cDNA was synthesized. Specific primers (Table 2) were designed for the 300-500 bp fragment located within the UGT5 ORF, and T7 promoter sequences were introduced at both ends. PCR amplification, gel purification, and ligation with the L4440 vector after restriction enzyme digestion (EcoRI-XhoI) were performed. The recombinant plasmid was transformed into HT115(DE3) (RNase III deletion) and incubated overnight at 37 °C. Single colonies were picked and inoculated with 5 mL of LB+Amp (100 mg / L) and shaken overnight. 1 mL of this inoculum was then transferred to 1 L of LB+Amp and cultured at 37 °C with shaking until OD (occurrence limit). 600 At a 0.4 mmol / L pH, the cells were induced with IPTG at a final concentration of 24 mg / L for 4 h. The bacterial culture was collected by centrifugation at 10,000 g for 2 min. The precipitate was washed with 75% ethanol-PBS and centrifuged again for 5-15 min. The bacterial culture was resuspended in 150 mM NaCl and incubated at room temperature for 1.5 h to extract total RNA. Then, RNase-free DNase (≥1 U) and RNase A (2 ng / µL) were added, and the cells were incubated at 37 °C for 10-30 min to remove DNA and ssRNA. An equal volume of isopropanol was added to precipitate the dsRNA. After centrifugation at 10,000-12,000 g for 10-15 min on ice for 10 min, the cells were washed twice with 75% ethanol, air-dried, and dissolved in DEPC water to obtain dsRNA. The concentration and purity (A260 / 280, A260 / 230) were determined using a spectrophotometer (NP80 / NanoDrop), and the integrity was confirmed by 1% agarose gel electrophoresis.

[0060] Table 2. Primer sequences for UGT5 gene dsRNA synthesis (5′→3′)

[0061] 1.6 Preparation and Characterization 1.6.1 Synthesis and Characterization of UCNP-NaYF4:Yb,Tm@NaF4 Using Y(CH3CO2)3·xH2O (0.2671 g), Yb(CH3CO2)3·4H2O (0.0700 g), and Tm(CH3CO2)3·xH2O (0.0035 g) as precursors, 15 mL of 1-octadecene (ODE) and 7 mL of oleic acid (OA) were added, and the mixture was heated to 150 °C for 1 h under a nitrogen atmosphere to form a lanthanide oleic acid complex. After cooling to 50 °C, 10 mL of methanol solution (containing 0.148 g of NH4F and 0.761 g of NaOH) was added, and the mixture was stirred for 30 min. The methanol was then evaporated to dryness, and the reaction was carried out at 285 °C for 90 min. After precipitation with ethanol, centrifugation at 8000 rpm, and washing, the product was dispersed in 5 mL of cyclohexane. Subsequently, a shell precursor solution was prepared using the same method (using only Y(CH3CO2)3·xH2O (0.2671 g) as the precursor), and the aforementioned core particles were added. The reaction was carried out at 285 °C for 1.5 h under nitrogen protection to obtain the core-shell structure NaYF4:Yb,Tm@NaYF4. The morphology and crystal form of the obtained product were characterized by transmission electron microscopy (TEM) and X-ray diffraction (XRD).

[0062] 1.6.2 Synthesis and Characterization of UCNP@MSN Nanoparticles 0.5 mL of isopropanol and 0.25 mL of tetraethoxysilane (TEOS) were added to a 20 mL cyclohexane solution containing 10 mg of UCNPs. After stirring for 10 min, the mixture was then mixed with a 10 mL aqueous solution containing 0.5 g of CTAB and 0.3 g of urea to form an oil-in-water microemulsion. The mixture was reacted at 70 °C for 16 h, centrifuged (12,000 rpm, 20 min), and washed repeatedly with ethanol. CTAB was removed by methanol-NaCl solution at 50 °C to obtain UCNP@MSN nanoparticles. The nanoparticles were characterized by TEM, XRD, and BET surface area analysis.

[0063] 1.6.3 Synthesis and Characterization of UCNP@MSN-NH2 Nanoparticles 100 mg of UCNP@MSN was dispersed in 50 mL of anhydrous ethanol, and 200 μL of 3-aminopropyltrimethoxysilane (APTMS) was added and refluxed at 85 °C for 12 h. The reaction product was centrifuged (12,000 rpm, 20 min) and washed with ethanol to obtain UCNP@MSN-NH2. The surface amino modification of the sample was confirmed by Fourier transform infrared spectroscopy (FTIR), and the particle size and zeta potential were measured by dynamic light scattering (DLS).

[0064] 1.6.4 Synthesis of UCNP@MSN-NH2@dsRNA 50-100 mg of UCNP@MSN-NH2 nanoparticles were added to 50 mL of RNase-free PBS buffer and ultrasonically dispersed using an ultrasonic homogenizer. The ultrasonic conditions were 100 W, 40 kHz, 15 min, and an ice bath. After ultrasonic treatment, the particle size was 50-150 nm, and the polydispersity index was less than 0.25. 50 mg of dsRNA was dissolved in 5 mL of RNase-free PBS to prepare a 10 mg / mL solution, and the concentration was verified by 260 nm UV absorption. Under magnetic stirring at 200-300 rpm, the dsRNA solution was slowly added to the nanoparticle dispersion over 60-120 seconds, in increments of 50-100 μL, with 10-15 s intervals. The liquid color gradually changed from blue to purplish-blue. After addition, the reaction was carried out at room temperature (25°C) with stirring for 6 h. dsRNA adsorbed onto the nanoparticle surface through Coulombic electrostatic interactions. The adsorption process consists of four stages: 30-40% adsorption is achieved from 0-30 min, 80-90% from 30 min to 2 h, 90-95% from 2-4 h, and approximately 95% equilibrium is reached from 4-6 h. After the reaction is complete, the mixture is separated by cold centrifugation at 8000 rpm, 4°C, for 10 min. The supernatant containing free dsRNA is discarded, and the precipitate is resuspended in 30 mL of RNase-free PBS. The mixture is then centrifuged repeatedly, and the washing cycle is repeated 3-5 times until the supernatant is colorless. Finally, the mixture is dried with 50 mL of anhydrous ethanol and centrifuged, followed by ultrasonic dispersion in 5 mL of anhydrous cyclohexane at 50 W for 15 min on ice, yielding UCNP@MSN-NH2@dsRNA at a concentration of 10-20 mg / mL.

[0065] 1.7 Water droplet impact on superhydrophobic surfaces A high-speed camera (FASTCAM mini UX100 Photron) was used to capture the impact of water droplets on a superhydrophobic surface from the side. The camera operated at 2000 frames per second with a shutter speed of 1 / 20000 second. The impact velocity of the water droplets remained at 1.98 m / s. -¹ This is achieved by allowing water droplets to fall freely from a stainless steel injection needle located 20 cm above the surface. The volume of the water droplets is adjusted by controlling the inner diameter of the needle using a LongerPump, ultimately obtaining uniform water droplets with a diameter of 2.0 mm.

[0066] 1.8 Delivery method and toxicity assessment To evaluate the silencing efficiency of different delivery routes for UCNP@MSN-NH2@dsRNA-mediated target genes, three treatment methods were set up: injection, feeding, and epidermal contact, with corresponding controls. In the injection group, 20 nL of UCNP@MSN-NH2@dsRNA solution at a concentration of 1000 mg / L was injected into the thorax of third instar larvae, with an equal volume of DEPC water as a control. In the feeding group, 200 μL of a 10% sucrose solution containing 1000 mg / L UCNP@MSN-NH2@dsRNA was encapsulated in a double-layered parafilm glass tube (2 × 15 cm) for the larvae to feed on, while the control group was fed only with the 10% sucrose solution. In the epidermal contact group, after anesthesia on ice, 0.5–1.0 μL of a solution containing 1000 mg / L UCNP@MSN-NH2@dsRNA (containing 0.05% Tween-80 as a wetting agent) was dripped onto the back of the larvae, while the control group was dripped with an equal volume of DEPC water. Samples were collected 24 h after each treatment, total RNA was extracted, treated with DNase I, and then reverse transcribed into cDNA. qPCR analysis was performed using gene-specific primers, with HaActin or HaRPL32 as internal control genes. ΔΔCt Relative expression levels were calculated. Furthermore, to assess the virulence effects of different delivery methods on UCNP@MSN-NH2@dsRNA-mediated virulence, indoor virulence assays were performed. After treatment with the above delivery methods, the survival rate, weight changes, and possible behavioral changes of cotton bollworms were observed and recorded to further evaluate the impact of gene silencing on growth and development.

[0067] 1.9 Enzyme Activity Analysis To assess the effect of gene silencing on metabolic enzyme activity, samples were taken from fifth-instar cotton bollworms after treatment for total UGT activity assay. Three individuals from each treatment were collected, and midgut tissue was obtained through dissection. The tissue was homogenized on ice with pre-chilled PBS, centrifuged at 12,000 rpm for 10 min at 4 °C, and the supernatant was used for enzyme activity analysis. UGT (UDP-glucuronyl transferase) activity was determined using a commercially available kit (Nanjing Jiancheng Biotechnology Institute, Nanjing, China), with UDP-glucuronic acid as the donor substrate. The reaction was terminated after 30 min at 37 °C, and the absorbance of the product was measured to calculate the protease activity per unit. Protein concentration was determined using the BCA method (Beyotime Biotechnology Institute, Shanghai, China) to standardize the results.

[0068] 1.10 Fluorescence localization analysis To investigate the penetration and distribution of UCNP@MSN-NH2@dsRNA within cotton bollworms, upconversion fluorescence imaging analysis was performed. Third-instar cotton bollworms, after treatment, were gently washed with PBS to remove residual particles from the surface, and then immediately subjected to whole-body imaging or observation via liquid nitrogen cryosectioning. Upconversion fluorescence detection (excitation wavelength 980 nm, emission wavelength 540-560 nm) was performed using a Leica SP8 laser confocal microscope to record the fluorescence signal distribution of UCNP@MSN-NH2@dsRNA on the body surface and inside the larvae. The fluorescence localization results were used to assess the penetration efficiency and spatial distribution characteristics of UCNP@MSN-NH2@dsRNA within the larvae.

[0069] 1.11 Cell transfection SF9 cells were spaced at 1.5 × 10⁶ cells per well. 5 The culture medium was inoculated into 24-well plates at the desired concentration and cultured for 1-2 days. After culture, 10% FBS medium was aspirated, and 300 µL of serum-free medium was added. The mixture was allowed to stand. 100 µL of serum-free medium was added to a 1.5 mL enzyme-free centrifuge tube, followed by 20 µL of dsRNA samples from different treatments. The mixture was thoroughly mixed and allowed to stand for 45 min, gently tapping to mix during this time. After standing, 200-300 µL of serum-free medium was added to the centrifuge tube and mixed well. The medium in the well plate was aspirated, and the medium containing the mixed dsRNA samples was added dropwise. The mixture was incubated for 5 h. After incubation, the medium in the well plate was aspirated, and 400 µL of serum-free medium was added to wash the cells. After washing, 800 µL of 10% FBS was added, and the mixture was allowed to stand for 24 h. The mixed solution was then observed under an inverted microscope. Cells showing fluorescence in the field of view were considered successfully transfected, and the transfection efficiency was calculated.

[0070] 1.12 Safety Evaluation To assess safety, bees ( Apis mellifera ),silkworm( Bombyx mori ) and zebrafish ( Danio rerio Exposure experiments were conducted on three representative non-target organisms. In the experiments, different dsRNA complexes (dsUGT5-19, dsUGT5-50, and dsUGT5-57) were loaded onto the UCNP@MSN-NH2 vector for treatment. Each non-target organism group had three biological replicates, with 20-30 individuals per group. Healthy adults were selected for exposure in bees and silkworms, while 2-3 day old embryos with well-developed gills were selected for zebrafish. Each group was treated using an immersion method to ensure that the UCNP@MSN-NH2@dsRNA complexes came into contact with the non-target organisms at an appropriate concentration. The experiment lasted 48 hours, with organism survival observed and recorded every 12 hours.

[0071] 1.13 Statistical Analysis All data analysis and plotting were performed using GraphPad Prism 9.5 (GraphPad Software, San Diego, USA) and Origin 2021 (OriginLab Corporation, Northampton, MA, USA). Differences between groups were assessed using one-way ANOVA and Tukey's post-hoc test, with significance levels set at P < 0.05 or P < 0.01. Results are expressed as mean ± standard error (SEM).

[0072] 2. Results This invention constructs a highly efficient RNAi nanodelivery system (UCNP@MSN-NH2@dsRNA), which combines upconversion nanoparticles (UCNPs) with mesoporous silica (MSN) and modifies the surface with amino groups to achieve efficient loading, stable protection, and trackable delivery of dsRNA. Figure 1 ).

[0073] Conjugating UCNPs with MSN and then amylating them provides an ideal platform for efficient, controllable, and traceable dsRNA delivery. Transmission electron microscopy (TEM) observations showed that the prepared upconversion nanoparticles (UCNPs) exhibited a highly uniform spherical distribution with an average particle size of approximately 20 nm. Figure 2 A) indicates that the preparation method can obtain nanoparticles with consistent morphology and controllable size. After coating the surface of UCNPs with dendritic mesoporous silica via a water-oil two-phase reaction, the particle diameter increased to approximately 110 nm. Figure 2 B). Agarose gel electrophoresis results ( Figure 2C) shows that when dsUGT is compounded with UCNP@MSN-NH2, the free dsRNA band disappears as the mass ratio increases from 1:1 to 1:10-1:20, indicating that dsRNA can be completely loaded onto the surface of the nanocarrier at higher ratios.

[0074] This study compared the static and dynamic behavior of dsRNA solution and UCNP@MSN-NH2@dsRNA composite droplets on the superhydrophobic surface of the cotton bollworm epidermis. The results showed that both systems maintained superhydrophobic characteristics but exhibited different wetting responses. Compared to pure water, dsRNA solution and UCNP@MSN-NH2@dsRNA droplets showed slight spreading and a flatter morphology, indicating that the biomolecules and nanocarriers in the solution slightly reduced the surface tension. Figure 3 Contact angle detection shows () Figure 3 The pure water droplet (121.2°) was significantly larger than that of dsRNA (111.9°) and UCNP@MSN-NH2@dsRNA (111.1°) (P=0.036), indicating that the wettability of the solution was slightly enhanced but the water repellency was still maintained.

[0075] In epidermal contact treatment, the silencing efficiency was significantly enhanced compared to naked dsRNA. Figure 4 A) suggests that nanocarriers can effectively protect dsRNA from degradation by surface or intestinal nucleases, while improving transmembrane absorption. In LCT LC 50 Under stress conditions, the corrected mortality rate of larvae increased from 52.39% in the control group to 69.74%-75.31%, an increase of 17.66%-23.48%. Figure 4 (B) indicates that gene silencing can effectively enhance toxicity sensitivity.

[0076] In the feeding experiment, UCNP@MSN-NH2@dsRNA showed a significantly enhanced silencing efficiency at 48 h compared to naked dsRNA. Figure 5 A). LCT LC 50 Under stress, the mortality rate of larvae increased significantly to 81.77%. Figure 5 B), which increased by 32.43% compared to the control group, shows that oral delivery can also achieve high silencing efficiency under the protection of nanocarriers.

[0077] In the injection treatment, UCNP@MSN-NH2@dsRNA decreased the expression of the target gene. Figure 6 A). LCT LC 50 Under these conditions, the mortality rate significantly increased to 87.52%-92.44%. Figure 6 B) is the most effective of the three delivery methods.

[0078] Fluorescence imaging showed that, under epidermal contact treatment, the distribution of UCNP@MSN-NH2@dsRNA in larvae was more concentrated on the larval body wall, indicating that a large number of nanodelivery systems entered the larval body. Figure 7 This provides direct evidence for the silencing effect. Tissue distribution analysis showed that after larvae fed, fluorescence was highly enriched in the larval digestive system. Figure 8 Fluorescence imaging showed that the fluorescence of the injection-delivered nano-delivery system was very uniformly distributed throughout the larvae, especially after injection as it circulated throughout the larvae's hemolymphatic system. Figure 9 This suggests that nanocarriers can be rapidly distributed in vivo and target important metabolic tissues.

[0079] Fluorescence microscopy revealed that UCNP@MSN-NH2@dsRNA significantly enhanced intracellular fluorescence signals compared to the naked dsRNA treatment group, suggesting that the nanocarrier can significantly improve the cellular uptake of dsRNA. Quantitative analysis further confirmed this: at 24 h of treatment, the uptake efficiency of UCNP@MSN-NH2@dsRNA was approximately 42%, while that of naked dsRNA was only 3%; after 48 h, the uptake rate of UCNP@MSN-NH2@dsRNA further increased to approximately 92%, far exceeding the less than 10% of naked dsRNA. Figure 10 C). The corresponding RNAi effect also showed a significant improvement, with silencing efficiencies reaching approximately 70% and 90% at 24 h and 48 h, respectively, while the efficiency of naked dsRNA under the same conditions was less than 15%. Figure 10 (D) This shows that the nanodelivery system can significantly improve the stability and effectiveness of dsRNA in cells, thereby enhancing the silencing effect of target genes.

[0080] Considering the environmental safety of RNAi biocontrol strategies, this study also conducted exposure experiments on three representative non-target organisms: bees, silkworms, and zebrafish. Figure 11 (AC). The results showed that, whether using the UCNP@MSN-NH2 vector itself or the composite system loaded with dsUGT5-19, dsUGT5-50, and dsUGT5-57, the survival rate of the three types of non-target organisms remained above 95%, with no significant difference compared to the control group. In particular, the survival of silkworms, which also belong to the Lepidoptera order, was not affected. This indicates that UCNP@MSN-NH2@dsRNA effectively improves RNAi efficiency while exhibiting good biocompatibility and environmental safety for typical non-target organisms, providing feasibility support for future large-scale applications.

[0081] The nucleotide sequence involved in this invention UGT5-19 (LOC110384519) UGT5-50 (LOC110378150) UGT5-57 (LOC110382757) Finally, it should be noted that the above description is merely a preferred embodiment of the present invention and is not intended to limit the present invention. Although the present invention has been described in detail with reference to the foregoing embodiments, those skilled in the art can still modify the technical solutions described in the foregoing embodiments or make equivalent substitutions for some of them. Any modifications, equivalent substitutions, improvements, etc., made within the spirit and principles of the present invention should be included within the protection scope of the present invention. Although the specific embodiments of the present invention have been described above, they are not intended to limit the protection scope of the present invention. Those skilled in the art should understand that various modifications or variations that can be made by those skilled in the art without creative effort based on the technical solutions of the present invention are still within the protection scope of the present invention. It should be noted that the above examples are only used to illustrate the technical solutions of the present invention and not to limit them. Although the present invention has been described in detail with reference to the given examples, those skilled in the art can modify or make equivalent substitutions for the technical solutions of the present invention as needed without departing from the spirit and scope of the technical solutions of the present invention.

Claims

1. An upconversion nanomaterial with both visual tracking and interface control capabilities, named UCNP@MSN-NH2@dsRNA, characterized in that... The upconversion nanomaterial comprises, from the inside out, an upconversion nanoparticle core (UCNP), an aminated mesoporous silica coating layer (MSN), and a dsRNA loading layer; wherein the upconversion nanoparticle core is a core-shell structure NaYF4:Yb, Tm@NaYF4.

2. The upconversion nanodelivery material as described in claim 1, characterized in that, The dsRNA targets UDP-glucuronyltransferase 5, a key gene in insect detoxification metabolism, including one or more of UGT5-19, UGT5-50, and UGT5-57.

3. The method for preparing the upconversion nanodelivery material according to any one of claims 1-2, characterized in that, The preparation method includes: synthesizing NaYF4:Yb,Tm@NaYF4 upconversion nanoparticles; coating their surface with mesoporous silica using a microemulsion method to obtain UCNP@MSN nanoparticles; performing amino modification with APTMS to obtain UCNP@MSN-NH2 nanoparticles; and mixing and incubating dsRNA with UCNP@MSN-NH2 nanoparticles to obtain UCNP@MSN-NH2@dsRNA.

4. The preparation method according to claim 3, characterized in that, The specific method for synthesizing NaYF4:Yb,Tm@NaYF4 upconversion nanoparticles includes: Using Y(CH3CO2)3·xH2O, Yb(CH3CO2)3·4H2O, and Tm(CH3CO2)3·xH2O as precursors, 1-octadecene and oleic acid were added, and the mixture was heated under an inert atmosphere to form a lanthanide oleic acid complex. Subsequently, a methanol solution containing NH4F and NaOH was added, and after stirring, the methanol was evaporated and the reaction was continued at a higher temperature. After precipitation with ethanol, centrifugation, and washing, the product was dispersed in cyclohexane. Then, a shell precursor solution was prepared using the same method, and the above core particles were added. The reaction was carried out under an inert atmosphere to obtain the core-shell structure NaYF4:Yb,Tm@NaYF4. Furthermore, the inert atmosphere is nitrogen; the formation of the lanthanide oleic acid complex by heating under an inert atmosphere involves heating to 130-180℃ under a nitrogen atmosphere for 0.5-5 h to form the lanthanide oleic acid complex; the specific conditions for continuing the reaction by heating are 260-290℃ for 60-120 min; the specific conditions for obtaining the core-shell structure NaYF4:Yb,Tm@NaYF4 by heating under an inert atmosphere are 260-290℃ for 60-120 min under nitrogen protection.

5. The preparation method according to claim 3, characterized in that, The specific method for obtaining UCNP@MSN nanoparticles by coating mesoporous silica on its surface using the microemulsion method includes: adding isopropanol and tetraethoxysilane to a cyclohexane solution containing UCNP and stirring, then mixing with an aqueous solution containing CTAB and urea to form an oil-in-water microemulsion system; after heating and reacting, centrifuging and washing with ethanol to remove CTAB, thereby obtaining UCNP@MSN nanoparticles.

6. The preparation method according to claim 3, characterized in that, The specific method for obtaining UCNP@MSN-NH2 nanoparticles by amination modification using APTMS includes: dispersing UCNP@MSN in anhydrous ethanol, adding 3-aminopropyltrimethoxysilane and heating under reflux; the reaction product is centrifuged and washed with ethanol to obtain UCNP@MSN-NH2.

7. The preparation method according to claim 3, characterized in that, The specific method for obtaining UCNP@MSN-NH2@dsRNA by mixing and incubating dsRNA with UCNP@MSN-NH2 nanoparticles includes: dispersing UCNP@MSN-NH2 nanoparticles in RNase-free PBS and ultrasonically dispersing them; then slowly adding the dsRNA solution to the above dispersion and stirring the reaction at room temperature, so that the dsRNA is adsorbed onto the surface of the nanoparticles through electrostatic interaction; after the reaction is completed, the free dsRNA is removed by centrifugation and washing to obtain UCNP@MSN-NH2@dsRNA nanoparticles; The mass ratio of dsRNA to UCNP@MSN-NH2 nanoparticles is 1:1 to 1:20; more specifically, it is 1:10 to 1:

20.

8. The use of the upconversion nanodelivery material according to claim 1 or 2 in any one or more of the following: (a) Insect RNAi delivery or preparation of insect RNAi delivery products; (b) Controlling insects or preparing insect control products; Furthermore, the delivery methods include epidermal contact delivery, feeding delivery, and insect coelomic injection delivery; Furthermore, the product is a pesticide; Furthermore, the insect is a pest, specifically a lepidopteran pest, and even more specifically, a cotton bollworm.

9. An insect RNAi delivery product or insect control product, characterized in that, The product comprises at least the upconversion nanodelivery material as described in claim 1 or 2.

10. A method for controlling insects, characterized in that, The method includes applying the upconversion nanodelivery material, insect RNAi delivery product, or insect control product as described in claim 1 or 2 to insects or an environment containing insects.

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