Spherical nanoparticles obtained from TMGMV to improve soil migration of small, hydrophobic agrochemicals

Abstract

Applicants described herein are TMGMV-derived spherical nanoparticles (SNPs) that use encapsulated small molecule loads to improve loading efficiency while minimizing upstream processing steps.

Description

CROSS-REFERENCE TO RELATED APPLICATIONS

[0001] This application claims priority under 35 U.S.C. §119(e) to U.S. Provisional Application No. 63 / 337,488, filed on May 2, 2022, the entire contents of which are incorporated herein by reference. Government funding statement

[0002] This invention was made with government support from the National Institute of Food and Agriculture (NIFA-2020-67021-31255) and the National Science Foundation (DMR2011924). The U.S. Government has certain rights in this invention. Reference Merge

[0003] All publications, patents, patent applications and appendices mentioned in this specification are incorporated into this specification by reference to the same extent as if each individual publication, patent, patent application or appendix was specifically and individually indicated to be incorporated by reference into this specification. background

[0004] Plant parasitic nematodes cause crop losses amounting to hundreds of billions of dollars, posing both economic risks and jeopardizing the global food supply. In order to improve the migration behavior and sustainable use of compounds used for nematode treatment, more advanced delivery vehicles are needed to help retain hydrophobic cargo in the soil and prevent leakage into groundwater. The present disclosure meets this need and has related advantages. Public content overview

[0005] One approach to address this unmet need is to use virus-like particles and viral nanoparticles as multifunctional platforms for drug delivery and small molecule loading. Due to their symmetry, surface reactive amino acids, and high stability compared to other protein delivery vehicles, they have demonstrated many successful applications as biocompatible and immunogenic platforms for cancer therapy, diagnostic imaging agents, hydrogels and polymer materials for sustained release, etc.

[0006] Recently, rod-shaped plant virus nanoparticles derived from tobacco mild green mosaic virus (TMGMV) delivered electrostatically and covalently bound small molecules in soil, showing higher mobility compared to icosahedral counterparts and other nanoparticle delivery systems. Since TMGMV is currently approved as a herbicide by the U.S. Environmental Protection Agency and is commercially available, this platform has the potential to be translated into a drug delivery platform for soil and agricultural applications. Electrostatically bound crystal violet (TMGMV-CV) effectively treated the nematode model organism Caenorhabditis elegans (C. elegans) in a nematode motility assay, indicating that the bound payload maintained its potency. However, improving the covalent loading efficiency of hydrophobic payloads on the exterior of the virus through bioconjugation is challenging due to solubility limitations and the molar equivalents required to drive the bioconjugation reaction. Therefore, there is a need to establish a low-cost method to load hydrophobic payloads onto TMGMV that circumvents the solubility and reaction efficiency issues.

[0007] Applicants describe here TMGMV-derived spherical nanoparticles (SNPs) that are used to encapsulate small molecule payloads, improving loading efficiency while minimizing upstream processing steps. First, TMGMV-SNPs were prepared by thermal reshaping at several concentrations to produce a range of nanoparticle diameters, and the resulting particles were analyzed using electron microscopy to determine the characteristics of the batches. In order to compare the release behavior of covalently linked molecules of encapsulated viruses, SNPs were prepared by two methods: 1) simple encapsulation of Cyanine 5 during thermal reshaping; 2) covalent linkage of TMGMV to Cyanine 5 before thermal reshaping. The release behavior was characterized by dialysis, and the soil mobility curve was characterized by SDS-PAGE and absorbance measurement. The absorption of these SNPs by the nematode model organism Caenorhabditis elegans was determined by fluorescence microscopy. Finally, the nematicide was loaded onto the SNP using method 1, and its migration and release in soil were determined by HPLC. These results suggest that SNPs can serve as a simple platform for encapsulating otherwise water-insoluble compounds for drug delivery in soil and display desirable drug release and soil mobility properties.

[0008] Therefore, the present disclosure provides a virus-like particle (VLP or "SNP") derived from tobacco light green mosaic virus (TMGMV) or a derivative thereof, which is optionally connected to an agent. In one aspect, the agent or its derivative is encapsulated in the VLP or covalently linked to the VLP. In another aspect, the diameter of the VLP nanoparticle may be between about 1 nanometer and about 2 microns. In one embodiment, the agent is selected from the list of agents or pesticides provided in Table 1, or avermectin or a derivative thereof. In another aspect, the avermectin is selected from ivermectin, abamectin, doramectin, eprinomectin, moxidectin or selamectin.

[0009] In one aspect, the agent is selected from the list of agents or pesticides provided in Table 1, or avermectin, or a derivative thereof. In another aspect, the avermectin is selected from ivermectin, abamectin, doramectin, eplerimectin, moxidectin, or selamectin. In another aspect, the agent is avermectin.

[0010] In another aspect, the agent or pesticide is present in an amount of about 5 mg mL -1 to about 10 mg mL -1 or wherein the agent is loaded at a concentration of about 0.5 mg mL -1 to about 0.2 mg mL -1 The concentration of loading.

[0011] The present disclosure also provides a plurality of VLPs that are the same as or different from each other.

[0012] Provided herein is a method for treating an agricultural environment (e.g., soil, fertilizer (feed) or plant (leaf, stem or root)), comprising, or consisting essentially of, or consisting of the following steps: delivering a VLP formulation or composition as described herein to or contacting the environment. They can also be used to deliver pesticides or other insoluble compounds to inhibit pathogenic infection of plants, roots and soil. In one aspect, the method is performed using a plurality of VLPs described herein, wherein the VLPs are the same or different from each other, and / or the medicaments are the same or different from each other, and / or the TMGMV or derivatives thereof are the same or different.

[0013] It will be apparent to one skilled in the art that the agents or pesticides are selected to treat the infestation or pest, see for example those listed in Table 2. BRIEF DESCRIPTION OF THE DRAWINGS

[0014] Figures 1A-1D : Schematic diagram of the heat-induced transformation of TMGMV from rod-shaped to SNP (Fig. 1A). Schematic phase space of the TMGMV to SNP transformation at 96 °C (Fig. 1B, left) and 30 s heating time (Fig. 1B, right), dark gray indicates no formation, light gray indicates partial transformation, and medium gray indicates reliable SNP formation. 5 mg mL -1 Phase space of TMGMV / SNP morphology after heating for 10-60 seconds at initial TMGMV concentration, between 60-96°C (Figure 1C). A zoomed-in view of the boxed nanoparticle features in phase space is shown (Figure 1D).

[0015] Figures 2A-2F : Soil mobility measurements of SNPs in 10 cm soil. Schematic diagram of SNP delivery and collection of fractions from a soil column (particles not drawn to scale) ( FIG2A ). Calculated relative band intensity of each 2 mL fraction collected from a soil column after addition of SNPs ( FIG2B ). Glue used for band intensity analysis and soil mobility of SNPs at a concentration of ( FIG2C ) 0.1 mg mL -1 , (Figure 2D) 1 mg mL -1 , (Figure 2E) 5 mg mL -1 and (Fig. 2F) 10 mg mL -1 The numbers on the gel images represent the elution volume of the soil column in mL. The marker on each gel is SeeBlue2 Plus (Invitrogen) in 1X MOPS, and each protein fraction is at approximately 17.5 kDa.

[0016] Figures 3A-3G : Encapsulation of small molecules using TMGMV SNPs. Schematic diagram of small molecule encapsulation in SNPs, where blue shapes represent small molecules (Figure 3A). SEM micrographs of SNPs containing Cy5 (blue spheres in the SNP subfigure) (Figure 3B) and ivermectin (Figure 3C). Encapsulation efficiency of Cy5 (1 mg mL-1) in TMGMV SNPs at several TMGMV concentrations (Figure 3D). Competitive ELISA results of ivermectin loaded in SNPs (sample preparation was 50 μM) (Figure 3E). [TMGMV] of Cy5 and ivermectin, SNP diameter, and calculated number of small molecules per unit TMGMV coat protein (2100 per viral particle) (Figure 3F). Soil mobility of superimposed Cy5 signal and SNP signal as a function of elution volume in a 10 cm soil column (Figure 3G).

[0017] Figures 4A-4F: Evaluation of the effect of ivermectin SNPs on paralysis in C. elegans. Schematic diagram of the burrowing assay of Pluronic F127 gel with E. coli attractants on the surface (Figure 4A). Image analysis workflow (10X magnification) identifying areas of the Pluronic F127 gel surface covered by C. elegans; bright field (Figure 4B), edge detection (Figure 4C), and merging (Figure 4D). Calculated surface velocity (Figure 4E) and number of nematodes (Figure 4F) when C. elegans were treated with SNPs of ivermectin at different concentrations in four formulations. For Figures 4E and 4F, from left to right, the standards are 100-200nm, 200-500nm, 500-1μm, and 1μm-2μm.

[0018] Figure 5A-5B : More SEM images of TMGMV transformation at different heating temperatures (columns) and heating times (rows). The images shown have 1.0 mg mL -1 (Figure 5A) and 0.1 mg mL -1 (Fig. 5B) Initial TMGMV concentration. Images are approximately 5000x magnification.

[0019] Figure 6: Effect of heating time on SNP formation at 10 mg mL -1 SEM images of the impacted TMGMV samples.

[0020] Figure 7A-7B : SNP formation for the analysis of sequence and integrity of TMGMV coat protein. SDS-PAGE shows that the expected molecular weight (band 1) is the main product under each condition, although SNP formation does lead to some degradation (band 2) and dimerization (top band) (Fig. 7A). Sequence coverage and proteomic analysis after LC / MS / MS (Fig. 7B).

[0021] Figures 8A-8B :SNP soil migration test in 10cm soil.Band intensity analysis for TMGMV and gel for soil migration analysis (Fig. 8A).The numbers on the gel image represent the elution volume (in mL) of the soil column.The mark on each gel is SeeBlue2 Plus (Invitrogen) in 1X MOPS, and each protein fraction is about 17.5kDa.The calculated relative band intensity (Fig. 8B) of each 2mL fraction collected from the soil column after loading TMGMV.

[0022] Figures 9A-9E: SEM images of ivermectin and SNP samples loaded with ivermectin. Images show ivermectin after heating (Fig. 9A), ivermectin after heating with TMGMV (Fig. 9B), ivermectin and TMGMV heated with 25% MeCN (Fig. 9C) and SNP yields under these conditions at 2 TMGMV concentrations (Fig. 9D and Fig. 9E). Magnification is 5000x (ac) and 1000x (Fig. 9D-9E).

[0023] Fig.10 : Competitive ELISA standard curve for ivermectin detection. The curve plotted is the absorbance at 450nm vs. ivermectin concentration in μM after 30 minutes of reaction. Each condition was repeated three times, and the average and standard deviation are shown (for some data points, the error is less than the data point).

[0024] Figures 11A-11B : CD spectra of TMGMV and SNP samples. The legend is located below the figure. Far-UV spectra (Figure 11A) and near-UV spectra (Figure 11B).

[0025] Fig.12 : Soil migration of IVN-SNP in 20 cm soil columns.

[0026] Figures 13A-13D : Example micrograph of C. elegans on a pluronic gel surface. Their movement paths are those wavy lines. The animals are indicated by red arrows. Each frame ( Figures 13A-13D ) indicate different regions of the same treatment group.

[0027] Fig.14 : Exemplary images of the Pluronic F127 gel surface before breakthrough of C. elegans.

[0028] Figures 15A-15E : Exemplary area scans of the C. elegans burrowing assay after animals were treated with ivermectin solutions (FIGs. 15A-15D) and TMGMV-only controls (FIG. 15E). 0 μM (FIG. 15A), 0.1 μM (FIG. 15B), 1.0 μM (FIG. 15C), and 10 μM (FIG. 15D). Each assay was repeated three times.

[0029] Figures 16A-16E : Example area scans of C. elegans burrowing assays after animals were treated with SNP 0.1 samples loaded with ivermectin. 0 μM ( FIG. 16A ), 0.1 μM ( FIG. 16B ), 1.0 μM ( FIG. 16C ), 10 μM ( FIG. 16D ) and 10 μM after soil column ( FIG. 16E ). Each assay was repeated three times.

[0030] Figures 17A-17E: Example area scans of C. elegans burrowing assays after animals were treated with SNP 1.0 samples loaded with ivermectin. 0 μM ( FIG. 17A ), 0.1 μM ( FIG. 17B ), 1.0 μM ( FIG. 17C ), 10 μM ( FIG. 17D ), and 10 μM after soil column ( FIG. 17E ). Each assay was repeated three times.

[0031] Figures 18A-18E : Example area scans of C. elegans burrowing assays after animals were treated with SNP 5.0 samples loaded with ivermectin. 0 μM ( FIG. 18A ), 0.1 μM ( FIG. 18B ), 1.0 μM ( FIG. 18C ), 10 μM ( FIG. 18D ) and 10 μM after soil column ( FIG. 18E ). Each assay was repeated three times.

[0032] Figures 19A-19E : Exemplary area scans of C. elegans burrowing assays after animals were treated with SNP 10 samples loaded with ivermectin. 0 μM ( FIG. 19A ), 0.1 μM ( FIG. 19B ), 1.0 μM ( FIG. 19C ), 10 μM ( FIG. 19D ), and 10 μM after soil column ( FIG. 19E ). Each assay was repeated three times. Detailed description of the public content

[0033] definition

[0034] Unless otherwise defined, all technical and scientific terms used herein have the same meaning as commonly understood by a person skilled in the art to which the claimed subject matter belongs. It should be understood that the foregoing general description and the following detailed description are exemplary and explanatory only and do not limit any subject matter claimed. In this application, unless otherwise specifically stated, the use of the singular includes the plural. It must be noted that the singular forms "one" and "the" used in the specification and the appended claims include the plural, unless the context clearly dictates otherwise. In this application, "or" is used to mean "and / or" unless otherwise stated. In addition, the use of the term "including" and other forms, such as grammatical variants, is not restrictive.

[0035] As used herein, ranges and quantities may be expressed as "about" a particular value or range. "About" also includes the exact amount. Thus, "about 5 μL" means both "about 5 μL" and "5 μL." In general, the term "about" includes amounts that are expected to be within the range of experimental error.

[0036] The section headings used herein are for organizational purposes only and are not to be construed as limiting the subject matter described.

[0037] The term "comprising" as used herein means that the method includes the steps or elements, but does not exclude other steps or elements. "Substantially consisting of..." means that the claim is open only to those steps or elements that do not substantially affect the basic and novel features of the claimed method. "Consisting of..." means excluding any elements or steps not specifically described in the claim. The embodiments defined by each of the above transition terms belong to the scope of the present disclosure.

[0038] As used herein, the terms "individual," "subject," and "patient" refer to any mammal. In certain embodiments, the mammal is a human. In certain embodiments, the mammal is a non-human. None of these terms require or are limited to situations characterized by monitoring (e.g., continuous or intermittent monitoring) by a medical professional (e.g., a physician, registered nurse, licensed practical nurse, physician's assistant, caregiver, or hospice staff).

[0039] “Agricultural products” means all or part of vegetation, including plants, trees, roots, flowers, branches, buds, stems, leaves or any other part thereof.

[0040] The term "treating" as used herein refers to obtaining a desired agricultural effect, such as an improvement. The effect may be preventive, i.e., completely or partially preventing pests or insects from infesting plants, vegetation, or other agricultural products. In one aspect, the term "treating" does not include prevention.

[0041] The term "improvement" refers to a detectable improvement in an agricultural product or vegetation (such as a plant, tree, flower, crop, root, stem or leaf). A detectable improvement includes a subjective or objective reduction, decrease, inhibition, suppression, restriction or control of the incidence, frequency, severity, progression or duration of an infection or presence of a pest or microorganism (such as C. elegans).

[0042] As used herein, the term "agent" refers to an active agent that treats or ameliorates the presence or infestation of agricultural products, such as a pesticide or insecticide, for example an avermectin, such as ivermectin.

[0043] Commonly used pesticides are shown in Table 1 below.

[0044] Table 1. Types of insecticides and their mechanisms of action

[0045] It will be apparent to one skilled in the art that the agent is selected to treat or ameliorate the presence of pests or agricultural infections. Non-limiting examples include avermectins. Fermectins are a class of drugs that occur naturally from fermentation products of the actinomycete Streptomyces avermitilis isolated from soil. Eight different structures are known, including ivermectin, abamectin, doramectin, eplerimectin, moxidectin, and selamectin, divided into four major components (A1a, A2a, B1a, and B2a) and four minor components (A1b, A2b, B1b, and B2b). Avermectins have anthelmintic and insecticidal properties and are commonly used as insecticides to treat pests and parasites. See El-Saber Batiha, et al. (2020), Pharmaceuticals, Aug 17; 13(8): 196. doi: 10.3390 / ph13080196. PMID: 32824399; PMCID: PMC7464486. Table 2 provides non-limiting examples of crops and commonly used pesticides for treating crops or vegetation. Table 2. Examples of crops and commonly used pesticides

[0046] Virus-like particles (VPL) and compositions

[0047] The terms "SNP" or "virus-like particle" or "VLP" as used herein refer to a non-replicative viral coat derived from one or more viruses, such as one or more plant viruses described herein. VLPs are typically composed of one or more viral proteins, such as but not limited to those proteins referred to as coat proteins, capsid proteins, coat proteins, surface proteins and / or envelope proteins, or particle-forming polypeptides derived from these proteins. VLPs can form spontaneously after recombinant expression of proteins in an appropriate expression system. VLPs can also be engineered, for example, comprising one or more viral proteins, or consisting essentially of them, or consisting of them, and these viral proteins include modifications, consist essentially of modifications, or consist of modifications. Methods for producing VLPs are known in the art. The presence of VLPs after recombinant expression of viral proteins can be detected by conventional techniques known in the art, such as electron microscopy, biophysical characterization, etc. In addition, VLPs can also be separated by known techniques, such as density gradient centrifugation, and identified by characteristic density bands. See, e.g., Baker et al. (1991) Biophys. J. 60: 1445-1456; and Hagensee et al. (1994) J. Viral. 68: 4503-4505; Vincente, J Invertebr Pathol., 2011; Schneider Ohrum and Ross, Curr. Top. Microbial. Immunol., 354: 53073, 2012).

[0048] As described herein, the VLP is a Tobacco Mild Green Mosaic Virus (TMGMV)-derived VLP, which comprises, consists essentially of, or consists of one or more viral particles, such as capsids, derived from Tobacco Mild Green Mosaic Virus (TMGMV) or a derivative thereof.

[0049] As used herein, the term "equivalent" when referring to a polynucleotide or protein (e.g., a coat protein or capsid protein) includes a polynucleotide or protein that contains at least 80%, 85%, 90%, 95%, 96%, 97%, 98% or 99% sequence identity (or consists essentially of or consists of these sequence identities) compared to the polynucleotide or protein being compared, while still retaining functional activity. In the case of referring to coat or capsid proteins, functional activity refers to the formation of VLPs, e.g., rod-shaped plant virus nanoparticles derived from tobacco light green mosaic virus (TMGMV) or a derivative thereof.

[0050] As used herein, the term "modification" includes replacement, addition, insertion and deletion of amino acid sequences, etc., which may be referred to as "variants". Exemplary sequence replacements, additions and insertions include full-length sequences or partial sequences in which one or more amino acids are replaced (or mutated), added or inserted, such as capsids derived from plant viruses. In some cases, the capsids described herein include, for example, modified capsids comprising at least 70%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99% or 100% sequence identity (or consisting essentially of or consisting of these sequence identities) compared to wild-type capsids. These or other modifications are within the scope of "or its derivatives".

[0051] The term "sequence identity" refers to the percentage of bases or amino acids that are identical and in the same relative position between two polynucleotide or polypeptide sequences. Therefore, a polynucleotide or polypeptide sequence has a certain percentage of sequence identity compared to another polynucleotide or polypeptide sequence. In sequence comparison, one sequence is usually used as a reference sequence to which a test sequence is compared. The term "reference sequence" refers to a molecule compared to a test sequence. A polynucleotide or polynucleotide region (or polypeptide or polypeptide region) having a certain percentage (e.g., 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 98% or 99%) of "sequence identity" with a reference sequence means that when aligned, the percentage base (or amino acid) at each position in the test sequence is the same as the base (or amino acid) at the same position in the reference sequence. This comparison and homology or sequence identity percentage can be determined using software programs known in the art, such as the software programs described in Ausubel et al. eds. (2007) Current Protocols in Molecular Biology. It is best to use the default parameters for the alignment. One alignment program is BLAST, using the default parameters. In particular, the BLASTN and BLASTP programs use the following default parameters: Genetic code = standard; filter = none; strand = both; cutoff = 60; expectation = 10; Matrix = BLOSUM62; Descriptions = 50 sequences; sort by = HIGH SCORE; Databases = non-redundant, GenBank + EMBL + DDBJ + PDB + GenBank CDS translations + SwissProtein + SPupdate + PIR. For more information on these programs, please visit the following website: ncbi.nlm.nih.gov / blast / Blast.cgi.

[0052] Modified capsid polypeptides include, for example, non-conservative and conservative substitutions of the capsid amino acid sequence. In one aspect, the modified capsid falls within the scope of the term "derivatives thereof".

[0053] As used herein, the term "conservative substitution" refers to replacing an amino acid residue with another chemically or biologically similar residue. Biologically similar means that the substitution does not destroy biological activity or function, such as viral capsid assembly.

[0054] Structural similarity refers to the similarity of the side chain lengths of the amino acids, such as alanine, glycine and serine, or similarity of size. Chemical similarity refers to the residues having the same charge or hydrophilicity or hydrophobicity. Specific examples of conservative substitutions include replacing another hydrophobic residue with a hydrophobic residue (such as isoleucine, valine, leucine or methionine), replacing another polar residue with a polar residue, such as replacing lysine with arginine, replacing aspartic acid with glutamic acid, or replacing asparagine with glutamine, etc. The term "conservative substitution" also includes replacing the unsubstituted parent amino acid with a substituted amino acid. This protein comprising amino acid substitutions can be encoded by a nucleic acid. Therefore, a nucleic acid sequence encoding a protein comprising amino acid substitutions is also provided.

[0055] Modified proteins also include substitution of one or more D-amino acids with L-amino acids (and mixtures thereof), structural and functional analogs (e.g., peptidomimetics and derivative forms with synthetic or non-natural amino acids or amino acid analogs). Modifications include cyclic structures, such as end-to-end amide bonds between the amino and carboxyl termini of the molecule, or intramolecular or intermolecular disulfide bonds.

[0056] Modified forms also include "chemical derivatives" in which the side chains of one or more amino acids are chemically changed or derivatized. For example, such derivatized polypeptides include amino acids such as free amino groups forming amine hydrochlorides, p-toluenesulfonyl groups, carboxyl groups; free carboxyl groups forming salts, methyl esters and ethyl esters; free hydroxyl groups forming O-acyl or O-alkyl derivatives and naturally occurring amino acid derivatives, such as 4-hydroxyproline (proline), 5-hydroxylysine (lysine), homoserine (serine), ornithine (lysine), etc. In addition, amino acid derivatives that can change covalent bonds are also included, for example, a disulfide bond formed between two cysteine ​​residues can produce a cyclized polypeptide.

[0057] Virus-like particles (VLP) provided herein are derived from tobacco light green mosaic virus (TMGMV) or its derivatives, optionally coupled with an agent. In one aspect, an agent or its derivatives are encapsulated in VLP or covalently attached to VLP. In another aspect, the diameter of VLP nanoparticles can be between about 1 nanometer to about 2 microns. In certain embodiments, the diameter of nanoparticles is less than about 2 μm, or less than about 1.5 μm, or less than about 1.25 μm, or less than about 1 μm, or less than about 0.9 μm, or less than about 0.8 μm, or less than about 0.7 μm, or less than about 0.5 μm. In other embodiments, the diameter of VLP nanoparticles is less than about 500nm, less than about 400nm, less than about 300nm, less than about 200nm, less than about 100nm or less than about 50nm. In one aspect, the diameter of VLP is between about 100 nanometers to about 200 nanometers, or the range value between them. In one aspect, the agent is selected from the list of agents or pesticides provided in Table 1, or avermectin or a derivative thereof. In another aspect, the avermectin is selected from ivermectin, abamectin, doramectin, eplerimectin, moxidectin or selamectin.

[0058] In one aspect, the agent is selected from the list of agents or pesticides provided in Table 1, or avermectin or a derivative thereof. In another aspect, the avermectin is selected from ivermectin, abamectin, doramectin, eplerimectin, moxidectin or selamectin. In another aspect, the agent is ivermectin. In another aspect, the VLP is TMGMV or a derivative thereof, the agent is ivermectin, and the diameter of the nanoparticles is about 100 nanometers to about 200 nanometers, or a range value therebetween. In another aspect, the nanoparticles are TMGMV or a derivative thereof, the agent is avermectin, such as ivermectin, and the diameter of the nanoparticles is about 100 nanometers to about 200 nanometers, or a range value therebetween.

[0059] In another aspect, the agent or pesticide is present in an amount of about 5 mg mL -1 to about 10 mg mL -1 Alternatively, the agent is loaded at a concentration of about 0.5 mg mL -1 to about 0.2 mg mL -1 In one aspect, the agent is selected from the list of agents or pesticides provided in Table 1, or avermectin or a derivative thereof, and the loading concentration is about 5 mg mL -1 to about 10 mg mL -1 , or, wherein the agent loading concentration is about 0.5 mg mL -1 to about 0.2 mg mL -1 In another aspect, the avermectin is selected from the group consisting of ivermectin, abamectin, doramectin, eplerimectin, moxidectin, or selamectin and is present in a concentration of about 5 mg mL -1to about 10 mg mL -1 , or, wherein the loading concentration of the agent is about 0.5 mg mL -1 to about 0.2 mg mL -1 In another aspect, the pharmaceutical agent is ivermectin and is loaded at a concentration of about 5 mg mL -1 to about 10 mg mL -1 , or, wherein the loading concentration of the agent is about 0.5 mg mL -1 to about 0.2 mg mL -1 In another aspect, the VLP is TMGMV or a derivative thereof, the agent is ivermectin, and the diameter of the nanoparticle is between about 100 nanometers and about 200 nanometers; the agent is an avermectin, such as ivermectin, and the diameter of the nanoparticle is between about 100 nanometers and about 200 nanometers.

[0060] In some cases, the VLPs described herein further include, a label or tag, e.g., a detectable label, or consist essentially of, or consist of. The detectable label can be attached to, e.g., the surface of the VLP. In one aspect, the rod-shaped plant virus nanoparticles derived from tobacco mild green mosaic virus (TMGMV) or a derivative thereof further include, or consist essentially of, or consist of a label or tag.

[0061] Non-limiting exemplary detectable labels also include radioactive materials, such as radioisotopes, metals, or metal oxides. Radioisotopes include radionuclides that emit alpha, beta, or gamma radiation. In certain embodiments, the radioisotope can be one or more of the following: 3 H, 10 B, 18 F, 11 C, 14 C, 13 N, 18 O, 15 O, 32 P,P 33 , 35 S, 35 Cl, 45 Ti, 46 Sc, 47 Sc, 51 Cr, 52 Fe, 59 Fe, . 57 Co, 60 Cu, 61 Cu, 62 Cu, 64 Cu, 67 Cu, 67 Ga, 68Ga, 72 As 76 Br, 77 Br, 81m Kr, 82 Rb, 85 Sr, 89 Sr, 86 Y, 90 Y, 95 Nb, 94m Tc, 99m Tc, 97 Ru, 103 Ru, 105 Rh, 109 Cd, 111 In, 113 Sn, 113m In, 114 In,I 125 ,I 131 , 140 La, 141 Ce, 149 Pm, 153 Gd, 157 Gd, 153 Sm, 161 Tb, 166 Dy, 166 Ho, 169 Er, 169 Y, 175 Yb, 177 Lu, 186 Re, 188 Re, 201 Tl, 203 Pb, 211 At, 212 Bior 225 Ac.

[0062] Other non-limiting exemplary detectable labels include metals or metal oxides. In particular embodiments, the metal or metal oxide is one or more of the following: gold, silver, copper, boron, manganese, gadolinium, iron, chromium, barium, europium, erbium, praseodymium, indium or technetium. In other embodiments, the metal oxide includes one or more of the following: Gd(III), Mn(II), Mn(III), Cr(II), Cr(III), Cu(II), Fe(III), Pr(III), Nd(III) Sm(III), Tb(III), Yb(III) Dy(III), Ho(III), Eu(II), Eu(III) or Er(III).

[0063] Further non-limiting exemplary detectable labels include contrast agents (e.g., gadolinium; manganese; barium sulfate; iodinated or non-iodinated agents; ionic or non-ionic agents); magnetic and paramagnetic agents (e.g., iron oxide chelates); nanoparticles; enzymes (horseradish peroxidase, alkaline phosphatase, β-galactosidase, or acetylcholinesterase); prosthetic groups (e.g., streptavidin / biotin and avidin / biotin); fluorescent materials (e.g., umbelliferone, fluorescein, fluorescein isothiocyanate, rhodamine, dichlorotriazinylamine fluorescein, dansyl chloride, or phycoerythrin); luminescent materials (e.g., luminol); or bioluminescent materials (e.g., luciferase, luciferin, aequorin).

[0064] Other non-limiting examples of labels and / or detectable tags include enzymes (horseradish peroxidase, urease, peroxidase, alkaline phosphatase, β-galactosidase, chloramphenicol transferase); enzyme substrates; ligands (such as biotin); receptors (avidin); GST, T7-, His-, myc-, HA- and Labels; electron-dense reagents; energy transfer molecules; paramagnetic tags; fluorophores (fluorescein, fluorescamine, rhodamine, phycoerythrin, phycocyanin, allophycocyanin); chromophores; chemiluminescence (imidazole, luciferase, acridinium, oxalate); and bioluminescent agents.

[0065] As described herein, detectable labels or markers can be connected or coupled (e.g., covalently) to VLPs. In various embodiments, detectable labels, such as radionuclides or metals or metal oxides, can be directly or indirectly combined or coupled to an agent. A connector or an intermediate functional group can be used to connect a molecule to a detectable label or marker. A connector includes an amino acid or peptidomimetic sequence inserted between a molecule and a label or marker so that the two entities at least partially maintain different functions or activities. A connector may have one or more properties, including a flexible conformation, an inability to form an ordered secondary structure, or hydrophobic or charged properties, which can promote or interact with any domain. Amino acids commonly present in flexible protein regions include Gly, Asn, and Ser. The length of the connector sequence can vary, but will not significantly affect function or activity.

[0066] Connectors also include chemical molecules, coupling agents and intermediate functional groups. Examples include molecules that react with free or semi-free amines, oxygen, sulfur, hydroxyl or carboxyl groups. Therefore, such functional groups include monofunctional and bifunctional cross-linking agents, such as sulfosuccinimidyl derivatives (sulfo-SMCC, sulfo-SMPB), particularly disuccinimidyl suberate (DSS), BS3 (Sulfo-DSS), disuccinimidyl glutarate (DSG) and disuccinimidyl tartrate (DST). Non-limiting examples include diethylenetriaminepentaacetic acid (DTPA) and ethylenediaminetetraacetic acid.

[0067] Also provided herein is VLP as herein described that further comprises additional medicament, or is basically made up of additional medicament, or is made up of additional medicament.The limiting examples of these additional medicaments include originally insoluble compounds and pesticides for drug delivery in soil, and they have desirable drug release and soil migration characteristics.These medicaments are covalently linked to VLP, or are encapsulated in VLP.Medicament also can be covalently linked to VLP by linker.

[0068] The size of the VLP nanoparticles can be between about 1 nanometer and about 2 microns. In certain embodiments, the diameter of the nanoparticles is less than about 2 μm, or less than about 1.5 μm, or less than about 1.25 μm, or less than about 1 μm, or less than about 0.9 μm, or less than about 0.8 μm, or less than about 0.7 μm, or less than about 0.5 μm. In other embodiments, the diameter of the VLP nanoparticles is less than about 500 nm, less than about 400 nm, less than about 300 nm, less than about 200 nm, less than about 100 nm or less than about 50 nm. In further embodiments, the diameter of the nanoparticles is about 75 nm to about 300 nm, or about 75 nm to about 275 nm, or about 75 nm to about 225 nm, or about 100 nm to about 300 nm, or about 100 nm to about 250 nm, or about 100 nm to about 200 nm, and ranges therebetween.

[0069] Also provided are a plurality of VLPs as described herein, wherein the VLPs are the same or different from each other, and / or the agents are the same or different from each other, and / or the TMGMV or derivatives thereof are the same or different from each other.

[0070] Composition

[0071] In another aspect, a composition is provided herein, comprising, or consisting essentially of, or consisting of VLPs and at least one carrier as provided herein, suitable for intended use, e.g., in soil or other agricultural environments. In one aspect, the VLPs are rod-shaped plant virus nanoparticles derived from tobacco mild green mosaic virus (TMGMV) or a derivative thereof.

[0072] Compositions comprising only VLP components (or consisting essentially of, or consisting of) or compositions comprising VLP components and other medicaments (or consisting essentially of, or consisting of) can be manufactured by conventional mixing, dissolving, granulating, grinding, emulsifying, encapsulating, encapsulating or lyophilizing processes. These preparations can be prepared in a conventional manner using one or more carriers, diluents, excipients or adjuvants, so as to facilitate the processing of the compound combinations provided herein into preparations that can be used for agriculture.

[0073] In some embodiments, agricultural formulations include, but are not limited to, lyophilized formulations, aqueous liquid dispersions, self-emulsifying dispersions, solid solutions, liposomal dispersions, aerosols, solid formulations, powders, immediate release formulations, controlled release formulations, fast melt formulations, tablets, capsules, pills, sustained release formulations, extended release formulations, pulsed release formulations, multi-particulate formulations (e.g., nanoparticle formulations), and immediate release and controlled release mixed formulations.

[0074] In one aspect, the nanoparticles are combined with carriers (e.g., organic solvents (e.g., water) or mineral clays), adjuvants (e.g., pastes or spreaders), stabilizers, safeners, or other chemicals that improve or enhance insecticidal activity. See, for example, http: / / npic.orst.edu / factsheets / formulations.html and references cited therein.

[0075] In certain embodiments, the composition includes one or more carrier materials selected based on compatibility with the composition disclosed herein and the release curve characteristics of the desired dosage form. Exemplary carrier materials include binders, suspending agents, disintegrants, fillers, surfactants, solubilizers, stabilizers, lubricants, wetting agents, diluents, etc. Carrier materials suitable for agriculture include, but are not limited to, gum arabic, gelatin, colloidal silicon dioxide, calcium glycerophosphate, calcium lactate, maltodextrin, glycerol, magnesium silicate, polyvinyl pyrrolidone (PVP), cholesterol, cholesterol ester, sodium caseinate, soy lecithin, taurocholic acid, phosphatidylcholine, sodium chloride, tricalcium phosphate, dipotassium hydrogen phosphate, cellulose and cellulose conjugates, sodium stearoyl lactylate, carrageenan, monoglycerides, diglycerides, pregelatinized starch, etc.

[0076] In some cases, the composition further includes a pH adjuster or buffer, including acids such as acetic acid, boric acid, citric acid, lactic acid, phosphoric acid and hydrochloric acid; bases such as sodium hydroxide, sodium phosphate, sodium borate, sodium citrate, sodium acetate, sodium lactate and tris; and buffers such as citrate / dextrose, sodium bicarbonate and ammonium chloride. The amount of these acids, bases and buffers should be such that the pH value of the composition is maintained within an acceptable range.

[0077] In some cases, the composition contains one or more salts in an amount necessary to bring the osmotic pressure of the composition to an acceptable level. Such salts include those having sodium, potassium or ammonium cations and chloride, citrate, ascorbate, borate, phosphate, bicarbonate, sulfate, thiosulfate or bisulfite anions, suitable salts including sodium chloride, potassium chloride, sodium thiosulfate, sodium bisulfite and ammonium sulfate.

[0078] In certain embodiments, the composition includes, but is not limited to, sugars such as trehalose, sucrose, mannitol, maltose, glucose, or salts such as potassium dihydrogen phosphate, sodium citrate, ammonium sulfate and / or other agents such as heparin to increase solubility and stability.

[0079] In some cases, the composition also includes a diluent for stabilizing the compound, as the diluent can provide a more stable environment. Salts dissolved in buffered solutions (which can also provide pH control or maintenance) are used as diluents in the art, including but not limited to phosphate buffered saline solutions. In some cases, the diluent can increase the volume of the composition to facilitate compression or to create sufficient volume to mix evenly for capsule filling. Such compounds can include, for example, lactose, starch, mannitol, sorbitol, glucose, microcrystalline cellulose (such as ), calcium hydrogen phosphate, dicalcium phosphate dihydrate, tricalcium phosphate, calcium phosphate, anhydrous lactose, spray-dried lactose, pregelatinized starch, compressible sugar (such as (Amstar), mannitol, hydroxypropyl methylcellulose, hydroxypropyl methylcellulose acetate stearate, sucrose-based diluent, confectioner's sugar, monobasic calcium sulfate monohydrate, calcium sulfate dihydrate, calcium lactate trihydrate, dextrin, hydrolyzed cereal solids, amylose, powdered cellulose, calcium carbonate, glycine, kaolin, mannitol, sodium chloride, inositol, bentonite, etc.

[0080] In some cases, the composition includes a disintegrant to promote the breakup or disintegration of the material. The term "disintegration" includes the dissolution and dispersion of the dosage form when it comes into contact with the gastrointestinal fluid. Examples of disintegrants include starch (e.g., natural starch, such as corn starch or potato starch), pregelatinized starch (e.g., National 1551 or ), or sodium starch glycolate (such as or ), cellulose (such as wood products), methyl crystalline cellulose (such as PH101, PH102, PH105, P100, MING and ), methylcellulose, cross-linked carboxymethylcellulose or cross-linked cellulose (such as cross-linked carboxymethylcellulose sodium ), cross-linked carboxymethyl cellulose or cross-linked carboxymethyl cellulose, cross-linked starch such as sodium glycolate, cross-linked polymers such as cross-linked povidone, cross-linked polyvinyl pyrrolidone, alginates (such as alginic acid or alginates such as sodium alginate), clays such as HV (magnesium aluminum silicate), colloids (such as agar, guar, locust bean, Karaya, pectin or tragacanth), sodium starch glycolate, bentonite, natural sponge, surfactants, resins (such as cation exchange resins), citrus pulp, sodium lauryl sulfate, sodium lauryl sulfate mixed starch, etc.

[0081] In some cases, the composition includes fillers such as lactose, calcium carbonate, calcium phosphate, calcium hydrogen phosphate, calcium sulfate, microcrystalline cellulose, cellulose powder, dextrose, dextran, dextran, starch, pregelatinized starch, sucrose, xylitol, lactitol, mannitol, sorbitol, sodium chloride, polyethylene glycol, etc.

[0082] Lubricants and glidants may also optionally be included in the compositions described herein to prevent, reduce or inhibit sticking or friction of the materials.

[0083] Exemplary lubricants include, for example, stearic acid, calcium hydroxide, talc, sodium stearate fumarate, hydrocarbons such as mineral oil or hydrogenated vegetable oils such as hydrogenated soybean oil. Higher fatty acids and their alkali metal and alkaline earth metal salts (such as aluminum, calcium, magnesium, zinc), stearic acid, sodium stearate, glycerin, talc, wax, Boric acid, sodium benzoate, sodium acetate, sodium chloride, leucine, polyethylene glycol (such as PEG-4000) or methoxy polyethylene glycol (such as CARBOWAX TM ), sodium oleate, sodium benzoate, glyceryl behenate, polyethylene glycol, sodium lauryl sulfate or magnesium dialkyl sulfate, colloidal silicon dioxide (such as SYLOID TM , ), starch (such as corn starch), silicone oil, surfactants, etc.

[0084] Plasticizers include compounds that are used to soften microcapsule materials or film coatings to make them less brittle. Suitable plasticizers include polyethylene glycols, such as PEG 300, PEG 400, PEG 600, PEG 1450, PEG 3350, and PEG 800, stearic acid, propylene glycol, oleic acid, triethylcellulose, and triacetin. Plasticizers can also be used as dispersants or wetting agents.

[0085] Solubilizers include, for example, triacetin, triethyl citrate, ethyl oleate, ethyl caprylate, sodium lauryl sulfate, sodium dimethyl acetate, vitamin E TPGS, dimethylacetamide, N-methylpyrrolidone, N-hydroxyethylpyrrolidone, polyvinylpyrrolidone, hydroxypropyl methylcellulose, hydroxypropyl cyclodextrin, ethanol, n-butanol, isopropanol, cholesterol, bile salts, polyethylene glycol 200-600, tetraethylene glycol ether, carbitol, propylene glycol and dimethyl isosorbide.

[0086] Stabilizers include compounds such as any antioxidant, buffer, acid, preservative, etc. Typical stabilizers include L-arginine hydrochloride, tromethamine, albumin (human), citric acid, benzyl alcohol, phenol, disodium diphosphate dehydrate, propylene glycol, m-cresol or m-cresol, zinc acetate, polysorbate-20 or 20 or tromethamine.

[0087] Suspending agents include compounds such as polyvinyl pyrrolidone, for example, polyvinyl pyrrolidone K12, polyvinyl pyrrolidone K17, polyvinyl pyrrolidone K25 or polyvinyl pyrrolidone K30, vinyl pyrrolidone / vinyl acetate copolymer (S630), polyethylene glycol (e.g., which may have a molecular weight of about 300 to about 6000, or about 3350 to about 4000, or about 7000 to about 5400), sodium carboxymethyl cellulose, methyl cellulose, hydroxypropyl methyl cellulose, hydroxymethyl Cellulose acetate stearate, polysorbate-80, hydroxyethylcellulose, sodium alginate, gums (such as gum tragacanth and gum arabic, guar gum, xanthan gum), sugars, cellulose products (such as sodium carboxymethylcellulose, methylcellulose, sodium carboxymethylcellulose, hydroxypropyl methylcellulose, hydroxyethylcellulose), polysorbate-80, sodium alginate, polyethoxylated sorbitan monolaurate, polyethoxylated sorbitan monolaurate, polyethoxylated sorbitan monolaurate, povidone, etc.

[0088] Surfactants include, for example, sodium lauryl sulfate, sodium docusate, Tween 60 or 80, triacetin, vitamin E TPGS, sorbitan monooleate, polyoxyethylene sorbitan monooleate, polysorbates, polaxomers, bile salts, glyceryl monostearate, copolymers of ethylene oxide and propylene oxide, e.g. (BASF) etc. Other surfactants include polyoxyethylene fatty acid glycerides and vegetable oils, such as polyoxyethylene (60) hydrogenated castor oil, and polyoxyethylene alkyl ethers and alkylphenyl ethers, such as octinoxate 10, octinoxate 40. Sometimes, surfactants are added to enhance physical stability or for other purposes.

[0089] Viscosifiers include, for example, methylcellulose, xanthan gum, carboxymethylcellulose, hydroxypropylcellulose, hydroxypropylmethylcellulose, hydroxypropylmethylcellulose stearate, hydroxypropylmethylcellulose phthalate, carbomer, polyvinyl alcohol, alginate, gum arabic, chitosan, and combinations thereof.

[0090] Wetting agents include compounds such as oleic acid, glyceryl monostearate, sorbitan monooleate, sorbitan monolaurate, triethanolamine oleate, polyoxyethylene sorbitan monooleate, sodium docusate, sodium oleate, sodium lauryl sulfate, sodium docusate, triacetin, Tween 80, vitamin E TPGS, ammonium salts, and the like.

[0091] In a further aspect, the composition is frozen or lyophilized and may contain agents to maintain stability and activity.

[0092] In some embodiments, one or more compositions disclosed herein are included in a kit. Thus, in some embodiments, the kits provided herein include, or are essentially composed of, or are composed of, one or more compositions disclosed herein and instructions for use thereof.

[0093] Dosage and dosage form

[0094] In some embodiments, the composition may be administered or delivered to the environment alone or as part of a formulation once a week, once a day, twice a day, three times a day, or four times a day, or even more frequently.

[0095] Administration of VLP formulations alone or in combination with compositions containing the same may be effected by any method capable of delivering to the site of action.

[0096] Therefore, based on the disclosure provided herein, those skilled in the art will understand to adjust dosages and administration regimens according to methods well known in the art.

[0097] Use of VLPs and compositions comprising VLPs

[0098] Provided herein is a method for processing an agricultural environment, such as soil, fertilizer or plant (leaf, stem or root), including delivering VLP preparations as herein described or compositions to an environment or contacting the environment, or consisting essentially of this step, or consisting of this step. They can also be used to deliver pesticides and other insoluble compounds to suppress the pathogenicity of plants, roots and soils. In one aspect, the method is performed with a plurality of VLPs described herein, wherein VLPs are identical or different from each other and / or medicaments are identical or different from each other and / or TMGMV or its derivatives are identical or different.

[0099] It will be apparent to one skilled in the art that the agent or pesticide is selected to treat the infection or pest, such as those listed in Table 2.

[0100] In one aspect, the method is implemented with a VLP derived from tobacco light green mosaic virus (TMGMV) or a derivative thereof, the VLP being optionally coupled to an agent. In another aspect, it is coupled to an agent. In one aspect, an agent or a derivative thereof is coupled by being encapsulated in a VLP or covalently attached to a VLP. In another aspect, the disclosed method is implemented using a VLP having a diameter of about 1 nm to about 2 μm. In certain embodiments, the diameter of the nanoparticle is less than about 2 μm, or less than about 1.5 μm, or less than about 1.25 μm or less than about 1 μm, or less than about 0.9 μm, or less than about 0.8 μm, or less than about 0.7 μm, or less than about 0.5 μm. In other embodiments, the diameter of the VLP nanoparticle in the method is less than about 500 nm, less than about 400 nm, less than about 300 nm, less than about 200 nm, less than about 100 nm, or less than about 50 nm. In one aspect, the diameter of the VLP in the method is from about 100 nm to about 200 nm and the range between the two. In one aspect, the agent in the method is selected from the list of agents or pesticides provided in Table 1, or is selected from avermectin or its derivatives. In another aspect, the avermectin is selected from ivermectin, abamectin, doramectin, eplerimectin, moxidectin or selamectin. In another aspect, the agent is avermectin, such as ivermectin for treating Caenorhabditis elegans.

[0101] In one aspect, the agent used in the method is selected from the list of agents or pesticides provided in Table 1, or avermectin or a derivative thereof. In another aspect, the avermectin is selected from ivermectin, abamectin, doramectin, eplerimectin, moxidectin or selamectin. In another aspect, the agent used in the method is ivermectin. In another aspect, the VLP used in the method is TMGMV or a derivative thereof, the agent is ivermectin, and the diameter of the nanoparticles is from about 100nm to about 200nm and a range of values ​​between the two. In another aspect, the nanoparticles used in the method are TMGMV or a derivative thereof, the agent is an avermectin such as ivermectin, and the diameter of the nanoparticles is from about 100nm to about 200nm and a range of values ​​between the two.

[0102] In another aspect, the agent or pesticide used in the method is present in an amount of about 5 mg mL -1 to about 10 mg mL -1 Alternatively, wherein the reagent is loaded at a concentration of about 0.5 mg mL -1 to about 0.2 mg mL -1 In one aspect, the agent used in the method is selected from the list of agents or pesticides provided in Table 1, or is avermectin or a derivative thereof, at a concentration of about 5 mg mL -1 to about 10 mg mL -1or at a concentration of approximately 0.5 mg mL -1 to about 0.2 mg mL -1 In another aspect, the avermectin used in the method is selected from about 5 mg mL -1 to about 10 mg mL -1 Ivermectin, abamectin, doramectin, eplerimectin, moxidectin or selamectin, or the loading concentration of the agent used in the method is about 0.5 mg mL -1 to about 0.2 mg mL -1 In another aspect, the agent used in the method is ivermectin, at about 5 mg mL -1 to about 10mgmL -1 Alternatively, the agent used in the method is loaded at a concentration of about 0.5 mg mL -1 to about 0.2 mg mL -1 In another aspect, the VLP used in the method is TMGMV or a derivative thereof, the agent is ivermectin, the diameter of the nanoparticles is from about 100 nm to about 200 nm and a range value therebetween, the agent is ivermectin or an avermectin, and the diameter of the nanoparticles is from about 100 nm to about 200 nm and a range value therebetween.

[0103] In some instances, the VLP used in the methods described herein further comprises a tag or label, or consists essentially of a tag or label, or consists of a tag or label, such as a detectable tag. For example, a detectable label can be attached to the surface of the VLP. In one aspect, the rod-shaped plant virus nanoparticle derived from tobacco mild green mosaic virus (TMGMV) or a derivative thereof further comprises a tag or label, or consists essentially of a tag or label, or consists of a tag or label.

[0104] Reagent test kit

[0105] As used herein, the kits or articles of manufacture described herein include carriers, packages or containers that are partitioned to receive one or more containers, such as vials, test tubes, etc., each of which contains, consists essentially of, or consists of one of the separate elements used in the methods described herein. Suitable containers include bottles, vials, syringes, and test tubes, etc. In one embodiment, the container is formed of a variety of materials, such as glass or plastic.

[0106] Articles of manufacture provided herein include packaging materials. Examples of agricultural packaging materials include, but are not limited to, blister packs, bottles, tubes, bags, containers, bottles, and any packaging material suitable for a selected formulation and intended mode of application and handling.

[0107] The kit typically includes a label listing the contents and / or instructions for use, and a package insert with instructions for use. A set of instructions is also typically included.

[0108] Although preferred embodiments of the present disclosure have been shown and described herein, it will be apparent to those skilled in the art that such embodiments are provided by way of example only. Many changes, variations, and substitutions will now occur to those skilled in the art without departing from the scope of the art. It should be understood that various alternatives to the disclosed embodiments described herein may be employed in implementing the disclosure. The following claims are intended to define the scope of the disclosure and to cover methods and structures within the scope of these claims and their equivalents.

[0109] experiment

[0110] These examples are for illustrative purposes only and are not intended to limit the scope of the claims provided herein.

[0111] The extensive use of pesticides in agriculture leads to the accumulation of these toxins on crops, in soil, and in drinking water and groundwater, which seriously endangers ecosystems and human health. The first step towards a healthier society is to enhance food security by improving quality and yield (i.e. more efficient crop treatment) while protecting the environment and agricultural ecosystems (i.e. preventing pesticides from leaching and accumulating in the environment). Most pesticides are hydrophobic, so soil mobility is poor. This can lead to overuse. As described herein, VLPs have enhanced pesticide delivery properties. In one aspect, a concise "plug and play" strategy is used, and a connector is added to load the pesticide in the VLP.

[0112] Alternatively, a non-covalent encapsulation technique is provided herein to encapsulate pesticides into nanoparticles from tobacco mild green mosaic virus (TMGMV). TMGMV is a good platform for precision agriculture because of its excellent soil mobility.

[0113] TMGMV baculovirus was thermally converted into spherical nanoparticles (called SNPs), during which the hydrophobic insecticide payload was encapsulated with high yield; the surprising finding was that thermal remodeling enabled the insecticide to be covalently or quasi-covalently bound to the carrier, thereby improving the soil mobility of the payload.

[0114] Biotin or avidin can be used as molecular linkers to load the insecticide onto TMGMV-VLP. Biomolecular linkers overcome the need for direct bioconjugation, which has proven challenging for insecticides (from both a chemistry and regulatory process perspective).

[0115] Thus, the disclosed VLPs are used as synthetic nanocarriers for pesticide delivery. VLPs are ideal choices because of their unique zwitterionic properties and modular shape, which enable these protein nanoparticles to have favorable soil transport.

[0116] Experimental Discussion

[0117] As the global population increases, strategies to protect crops are essential to meet the upcoming food demand. At current levels, 9.8% of the population is malnourished or hungry. Plant parasitic nematodes cause economic losses to the agricultural sector of up to $125B USD, and 14% of crops are lost worldwide each year. 1 The advent of pesticide use has overcome many challenges of crop loss, but has also created new challenges of human health, environmental toxicity, and public skepticism. Coupled with a strong regulatory environment, creating next-generation nanopesticides that can mitigate and address many of the concerns surrounding crop protection is an important step toward creating a sustainable agricultural system.

[0118] The present disclosure provides the use of nanoparticles for soil delivery of pesticides, many of which have poor soil mobility and rapid washoff, thus requiring delivery vehicles that remain effective and economically viable 2-5 Pesticide delivery systems derived from polymers and nanoparticles have been developed but are costly and may not have optimal soil transport properties. 2,4,6 One promising nanopesticide delivery platform is tobacco mild green mosaic virus (TMGMV) nanoparticles. TMGMV has many desirable properties as a nanocarrier for agrochemical delivery, is commercially available, and is currently approved by the U.S. EPA as a herbicide. 4,7-9 In previous studies, covalent and electrostatic strategies for cargo loading in TMGMV were investigated; model cargo molecules were explored and toxicity to nematodes was confirmed. 9 Importantly, the applicants here demonstrate that TMGMV exhibits good soil mobility, which is important because nematodes can infest the root systems of crops. 4 While successful, covalent and electrostatic loading efficiencies are fairly low; loading of agrochemicals has proven challenging even with a wide range of formulation chemistries. 10. Prior work has highlighted the need for improved economics of TMVGMV-based formulations to make them commercially viable candidates for agricultural nanotechnology. An important consideration for agrochemicals is that non-covalent strategies are preferred for nanoparticle formulations, as modifications to agrochemicals initiate new regulatory approval and registration processes. Without being bound by theory, applicants herein explore the thermal shape-switching properties that have been reported for some high-aspect ratio viruses, such as tobacco mosaic virus (TMV), Alternathera mosaic virus (AltMV), and potato virus X (PVX). 11-13 . To encapsulate agrochemical loads during this transition, the applicant developed a SNP formulation protocol for TMGMV. The structural properties of the SNPs were evaluated, and their loading capacity was evaluated by using a fluorophore (Cyanine 5) and a nematicide (ivermectin). The applicant demonstrated here the soil mobility and effect of ivermectin treatment on Caenorhabditis elegans using SNPs.

[0119] SNPs can be generated from high-aspect ratio plant viruses, but data show conditions need to be optimized for each system 13-16 To test whether SNPs from TMGMV can form, we investigated the phase space of the rod-to-SNP transition (Figure 1). We varied the concentration of TMGMV, heating time, and temperature, and confirmed the morphology by SEM. The conditions and transitions are summarized in Figure 1B. Figures 1C and 1D show the rod-to-SNP transition at 5 mg mL -1 Results of thermal remodeling of TMGMV at initial concentrations (see Figure 5 for other conditions). SNPs were formed at all tested TMGMV concentrations, but a minimum temperature and heating time were required for complete transformation. Below 80°C, SNPs were not formed within 1 minute after heating, but instead formed agglomerated rod bundles (Figure 6). Above 90°C, SNPs were formed after 30 seconds of heating. For temperatures and heating times below this threshold, aligned and agglomerated TMGMV bundles were observed (Figure 1C and Figure 6). At the threshold of 30 seconds at 96°C, SNPs were formed, and the SNP diameter was proportional to the initial TMGMV concentration: the lowest tested concentration of TMGMV (0.1 mgmL -1 ) resulted in particle sizes of approximately 100–200 nm, while the highest (5 and 10 mg mL -1 ) The concentrations tested resulted in particles in the 500nm-2μm range. The aggregation and clustering observed with the smaller SNPs is a result of the SEM drying process, but the rounded features observed in these samples were not present in any of the controls.

[0120] Since the TMGMV SNP had not been previously reported, the applicants characterized its protein content by SDS-PAGE and LC-MS-MS. SDS-PAGE showed differences between the protein content of rod-shaped TMGMV and its SNP (Figure 7): TMGMV consists of ~2100 copies of the same coat protein (CP) with a molecular weight of 17.6 kDa. Even under denaturing conditions, the dimeric CP of the SNP (35 kDa) was more prevalent, indicating that complete decomposition was hindered. In addition, smaller degradation byproducts (15 kDa) were observed at higher heating times. The sequence of the cleaved protein was unexpected in the absence of protease or bacterial contamination. LC-MS-MS and sequence analysis determined that the band originated from the TMGMV CP. Interestingly, this phenomenon has not yet been described for any rod-to-SNP transition. 11-13 The aspartate-proline (DP) bond in proteins is susceptible to cleavage at temperatures close to 100°C and under acidic conditions. 17-19 . Therefore, the applicants analyzed the sequences of TMGMV and other baculoviruses for which SNP conversion was reported. Both TMV and TMGMV have a DP at amino acid positions D20-P21, while PVX and AltMV do not have this motif. The N-terminal sequence preceding the putative cleavage site is 2.1 kDa in length and correlates with the mass shift on SDS-PAGE. This effect is reported to occur at longer heating times (on the order of several minutes). Since the conversion of TMGMV to SNP takes longer than TMV, this phenomenon may occur with TMV but has not been observed.

[0121] Applicants first determined the soil mobility of SNPs and TMGMV. TMGMV has previously been shown to have good soil mobility properties - superior to other synthetic and biological systems, which Applicants attribute to the zwitterionic nature of the protein-based macromolecule. 4 The applicants used SNP to completely replicate the experimental setup of the soil mobility assay previously performed by the applicants. 4. Briefly, a 0.3 cm diameter column was filled with Magic topsoil horticultural soil (Michigan Peat) to a depth of 10 cm and pre-wetted before bolus injection of TMGMV or SNP (0.5 mg viral nanoparticles in 1 mL DI H2O, injected at 5 mL min-1). Fractions were collected and TMGMV CP was analyzed by SDS-PAGE (Figures 2 and 8). Band analysis was performed to determine the elution curve, and the applicant determined that SNP had good soil mobility regardless of its size (Figure 2B). The independence from size may be because surface chemistry is the dominant factor in soil mobility. Interestingly, for a soil depth of 10 cm, the elution volume of SNP (maximum of approximately 8-10 mL elution volume) is longer than that of TMGMV rod-shaped particles (maximum of approximately 6 mL elution volume). This may be due to their larger length scale and the changes in surface chemistry of SNP compared to rod-shaped particles that promote more interactions between particles and soil. 20 At 1mgmL -1 and 5 mg mL -1 The overall signal intensity observed in the case of 0.1 mg mL -1 and 10 mg mL -1 In contrast, SNPs were more retained in the soil under these conditions.

[0122] Based on phase space, the applicant concluded that the most reliable rod-to-SNP transformation occurred at 96°C using a heating time of 60 seconds. Under these conditions, the SNPs were reproducibly formed as uniform spheres observed on the SEM. Therefore, the applicant selected the 60 second / 96°C condition as the active ingredient encapsulation condition and established a functional relationship with protein concentration (0.1, 1.0, 5.0 and 10 mg mL-1). Within this protein concentration range, the SNPs formed ranged from 100-200nm at the lowest protein concentration to 500nm-2μm at the highest protein concentration. Two small molecule loads were selected: Cy5 was used as the fluorescent model molecule and ivermectin was selected as the insecticide. Cyanine 5 (Cy5) was chosen because it is a photostable fluorophore that has previously been shown to remain fluorescent after heat treatment. 21 Small molecule loading into SNPs derived from TMV and other baculoviruses has been demonstrated previously. 11,12,14,16. However, in these systems, chemical conjugation strategies are used to achieve association before and after SNP formation. While these studies have demonstrated functional conjugation of fluorophores and other classes of payloads, these covalent modification strategies rely on modification of the active ingredient. This is highly undesirable in an agricultural setting because changing the chemical structure of the active ingredient would require a lengthy regulatory process and increase product development costs. In addition, many bioconjugation procedures are multi-step processes that include intermediate and final purification steps. These processes can be time-consuming, reduce yields, and increase costs. A one-step synthesis of SNPs loaded with active substances could alleviate these technical barriers (as shown in Figure 3A).

[0123] A fixed concentration of Cy5 (1 mg mL-1) was added during the thermal transition of TMGMV to SNP (60 s, 96 °C), with a mass ratio of 0.1 to 10 times compared to TMGMV. The resulting Cy5-SNP was then purified on a desalting column to remove excess Cy5. The purified sample significantly concentrated Cy5 (Figure 3B, inset). Cy5-SNP was imaged by SEM, and there was no obvious difference in the morphology of Cy5-SNP compared to unloaded SNP (Figure 3B). The synthesis produced fewer non-SNP aggregates, which may be attributed to the 5% volume of DMSO added to the synthesis by Cy5, which may contribute to the dissociation and thermal conversion of SNP. This indicates that this non-covalent encapsulation strategy does not interfere with the formation of SNP. To quantify Cy5 encapsulation, the applicant analyzed the absorbance of purified SNP at 647 nm. The applicant found that the removal of Cy5 (1 mg mL-1) from the solution to the particle phase was TMGMV concentration-dependent, with the removal efficiency increasing from 0.1 mg mL -1 25% of TMGMV to 10 mg mL -1 The encapsulation efficiency of TMGMV was close to 100% (Figure 3D). The calculated encapsulation efficiency is reported in Figure 3F, showing high encapsulation capacity at higher protein concentrations. Although there is still room for optimization of the loading efficiency of each TMGMV concentration, the applicants demonstrated that SNPs loaded with the load can be obtained in different sizes (from 100-200nm to 500nm-2μm) through a one-step synthesis without the need for chemical changes to the load. For Cy5, 77 molecules were loaded into 100-200nm Cy5-SNP (0.1mg mL -1 With increasing TMGMV concentration and particle size, the amount of Cy5 per CP tends to decrease for the largest particles (500nm-2μm), with less than 10 molecules per CP, but still exceeding the maximum achieved by bioconjugation and optimization.

[0124] Next, ivermectin is encapsulated. Ivermectin has limited water solubility and has previously proven to be an unwise choice for chemical conjugation. 22. Therefore, the applicant tested encapsulation during the transition from rod to SNP. When the applicant heated an aqueous solution containing ivermectin and TMGMV to encapsulate the active ingredients into SNP, the applicant observed the formation of a fluffy colloidal byproduct that interfered with the formation of SNP (Figure 9). The formation of this byproduct was unrelated to the presence of TMGMV and was identified as an aggregate of ivermectin. In order to increase the solubility of ivermectin and achieve SNP encapsulation, the applicant added 25% (by volume) of acetonitrile as a cosolvent. The use of a cosolvent does achieve encapsulation of hydrophobic loads, thus greatly increasing the types of loads that the applicant can encapsulate in SNP. The cosolvent is then removed using precipitation, dialysis or other buffer exchange techniques. The applicant observed correctly formed IVN-SNPs at all sizes (Figures 3C and 9).

[0125] Using competitive ELISA (Figure 3E) and standard curve ( Fig.10 ) was used to determine the amount of ivermectin per SNP formulation. When a lower concentration of TMGMV was used, the ivermectin loading per CP was relatively high (Figure 3F), matching the trend of Cy5. -1 TMGMV), up to ~50 ivermectin molecules can be detected per CP. For smaller SNPs (100-200 nm), the estimated loading per SNP is approximately 1x10 6 ivermectin, or 60% by mass ivermectin. In order to gain a deeper understanding of the structural changes that occur during the formation of SNPs using a cosolvent, the circular dichroism (CD) spectra of IVN-SNP and TMGMV were analyzed and compared (Figure 11). When comparing rod-shaped TMGMV with SNP, the data showed changes in signal intensity around 200-220nm, and the peak maximum shifted from 270nm to 260nm after thermal conversion. In the presence of acetonitrile as a cosolvent, the shift associated with SNP formation is exacerbated, but unheated acetonitrile has little effect on the CD spectra. In addition, the presence of ivermectin leaves a spectral fingerprint at 240-260nm. Since TMV (a close homolog of TGMMV) undergoes large changes in secondary structure during SNP formation, these changes in TMGMV are also expected.

[0126] Next, the effect of loading on soil mobility was evaluated. Applicants confirmed the soil mobility of the loaded SNPs, and the elution maximum matched that of the empty SNP, indicating that loading did not affect soil mobility (Figure 3G, Fig.12Importantly, we demonstrated in a 10 cm soil column that the Cy5 signal in the eluate overlapped well with the protein signal of the Cy5-SNP (this was true for all sizes tested, 500 nm-2 μm SNPs), indicating that non-covalent loading in the SNPs is robust and stable. This is in stark contrast to rod-shaped TMGMVs: here, we established a non-covalent loading strategy using electrostatic binding, and while stable under physiological conditions, we found that the loading stripped from the particles in soil. 7 .

[0127] As a final test, the applicant delivered IVN-SNPs to C. elegans (Figure 4). The nematodes were incubated in solution with SNPs, IVN-SNPs, and free IVN for 90 minutes. At the same time, the same (IVN-)SNPs were passed through a soil column and collected, and then the C. elegans in solution were processed. For all samples, the applicant performed a modified gel drilling assay, i.e., transferring the nematodes and throwing them into Pluronic F127 in a well plate (Figure 4A) 23 Surfaces were coated with E. coli OP-50 lysate to attract nematodes, and the number of C. elegans that penetrated the surface was counted. Nematode mobility was quantified based on total track coverage using edge detection (Figures 4B-4D, Figure 13, Fig.14 ). The mobility is then estimated using the following formula:

[0128]

[0129] where v is the approximate mobility, N is the total number of nematodes, A is the area covered by the tracks, A0 is the total area in the image, D is the diameter of the well plate, and Δt is the time interval of the experiment (see Figures 15-19). The effects were measured by reduced burrowing ability of C. elegans, soluble ivermectin, and IVN-SNP (Figures 4E and 4F); the SNP alone had no effect, as expected. Interestingly, a simple approximation of labeled surface area correlated well as an indicator of the number of nematodes on the surface, suggesting that this approach may be suitable for future high-throughput screening at lower magnifications. The highest tested concentration of ivermectin (10 μM) resulted in a 4.5-fold reduction in surface velocity and a 6-fold reduction in the number of nematodes on the surface.

[0130] Applicants also noted that the largest SNPs (5 and 10 mg mL -1 The smallest SNPs (100-200 nm, 0.1 mg mL -1) decreased the nematode surface velocity by 2.35-fold and reduced the number of nematodes on the surface by 2.9-fold. The ivermectin concentrations in these experiments were normalized, so Applicants speculate that smaller SNPs interact more efficiently with nematodes, either based on diffusion or a higher nanoparticle-to-nematode ratio, and / or that ivermectin may be more accessible and adsorbed to the surface of SNPs (thus potentially causing a burst release).

[0131] For 500nm-2μM IVN-SNP, the IVN-SNP formulation was about 35% less effective than 10μM soluble ivermectin. However, in an agricultural setting, nematicides need to reach root-feeding nematodes, so a soil environment needs to be included for a realistic comparison. Applicants added free ivermectin and IVN-SNP to a 10cm soil column and collected fractions before treating C. elegans and conducting burrowing assays. Only IVN-SNP caused a significant reduction in the number of nematodes and their surface mobility, while soluble ivermectin showed no reduction (Figure 4E, Figure 4F). At 0.1 and 1.0mg mL -1 IVN-SNPs formed under TMGMV resulted in 1.36- and 1.72-fold reductions in surface velocity and 1.74- and 1.81-fold reductions in nematode abundance. These data indicate that after 10 μM ivermectin was applied to soil, efficacy was reduced by 2.25-fold (100-200 nm SNPs) and 1.33-fold (500 nm-1 μm). This reduction in efficacy was not observed for larger SNPs: 5.0 and 10 mg mL -1 IVN-SNP showed similar efficacy before and after soil treatment. After passing through the soil column, the surface velocity of 5 mg mL-1 decreased by 2.14 times and the number of nematodes decreased by 2.25 times, while 10 mg mL -1 The surface velocity was reduced by 2.24 times and the number of nematodes was reduced by 2.30 times. From these data, the applicants concluded that the agrochemical loaded SNP encapsulation was able to achieve soil transport, thereby effectively treating nematodes.

[0132] In summary, SNP-based encapsulation is efficient for soil-based nematicide delivery. The applicant details the phase space of SNP synthesis, showing that heating times greater than 30 seconds and temperatures above 90°C lead to SNP formation. SNP size is related to protein concentration, and SNP size ranges from 100nm to 2μm in diameter were synthesized. The applicant demonstrated that Cy5 and ivermectin were effectively encapsulated into SNPs during the thermal TMGMV to SNP conversion. Finding a medium (such as acetonitrile) that can be heated to >90°C and maintain the solubility of the target molecule is very important for effective encapsulation. Future applications may require more sustainable cosolvents. Acetonitrile enhances the solubility of the active ingredient and improves SNP synthesis, possibly by helping to destabilize the TMGMV assembly. Importantly, the applicant demonstrated effective active ingredient loading and co-delivery in soil to target root-feeding nematodes. Although free ivermectin was ineffective after passing through the soil, IVN-SNP promoted soil mobility and effects on Caenorhabditis elegans. The applicants envision that these technologies could find applications beyond applying pesticides in the soil and extend to foliar delivery of active ingredients. TMGMV itself has been approved by the EPA as a bioherbicide, and this non-covalent encapsulation could simplify the regulatory process. The use of a one-step synthetic approach holds promise for production in a scalable industrial process.

[0133] experiment

[0134] TMGMV preparation

[0135] TMGMV from BioProdex (Gainesville, FL, USA) was prepared as previously described and stored at -20 ° C until use (Chariou, P et al., Nature Nanotechnology, 2019; Chariou, P et al., ACS Agricultural Science & Technology, 2021; Chariou, P et al., ChemBioChem, 2022; González-Gamboa, I et al., ChemBioChem, 2022). Briefly, thawed TMGMV was dialyzed with potassium phosphate buffer (KP; 10 mM, pH 7.2) at 4 ° C using 12-14 kDa dialysis tubing (Fisher Scientific S432700, Waltham, MA, USA) for 24 hours, and the process was repeated with fresh buffer within the next 48 hours. The virus solution was centrifuged at 10,000 x g for 20 minutes (Beckman Coulter Allegra centrifuge, Brea, CA, USA). The supernatant was collected and ultracentrifuged at 42,000 rpm for 2.5 hours at 4°C (Beckman Coulter Optima L-90k ultracentrifuge equipped with a 50.2Ti rotor, Brea, CA, USA). The pellet was resuspended in KP buffer at 4°C overnight. The concentration was determined using Nanodrop 2000 (ThermoScientific, Waltham, MA, USA).

[0136] SNP preparation

[0137] TMGMV solution was diluted to the desired concentration (0.1, 0.3, 0.5, 1.0, 5.0 or 10.0 mg mL-1) in DI H2O in a PCR tube in a volume of 100 μL. The run was programmed using a thermal cycler (MJ Research PTC200, Waltham, MA, USA) to heat to the desired temperature (60, 70, 80, 90, 96 and 98 °C) for a fixed duration (5, 10, 15, 20, 30, 60 and 120 seconds) and then decrease to 10 °C. Samples were stored at 4 °C between measurements.

[0138] SEM

[0139] The particle preparation was dried on a silica wafer, which was bonded to stainless steel mounting studs with carbon fiber tape. The sample was allowed to dry overnight and then iridium coated with a coating time of 12 seconds. The sample was imaged in a Zeiss 500 at 3-5 kV under vacuum.

[0140] SDS-PAGE

[0141] Denatured TMGMV SNP samples (10 μg) were loaded onto a 12% NuPAGE gel (Life Technologies, Inc., San Diego, CA). Technologies, Carlsbad, CA, USA) and in 1x MOPS running buffer (Life Technologies, Carlsbad, CA, USA) Technologies, Carlsbad, CA, USA). Cyanine 5 encapsulated in SNPs was observed under UV light. Encapsulated proteins were stained using Gel Code Blue stain (Life Technologies, Carlsbad, CA, USA) and observed under white light in a gel imager (ProteinSimple, Santa Clara, CA, USA).

[0142] Soil mobility test

[0143] Magic Topsoil (Michigan Peat) was weighed and filled into a cylindrical column with miracloth at the bottom. The tube had a conical outlet at the bottom for dropwise fraction collection. The soil was moistened with DI H2O and allowed to stand until dripping stopped. A 0.5 mg aliquot of TMGMV or SNP was added to the column and pumped at 5 mL min using a syringe pump containing DI H2O. -1 The eluate was collected in 2 mL fractions and run on SDS PAGE for analysis. The band intensity of the fractions on SDS PAGE was determined using ImageJ.

[0144] Encapsulation of small molecules in SNPs

[0145] Prepare 5 mg mL -1 Small molecule solutions (Cy5 in DI H2O, ivermectin in acetonitrile) were prepared. These solutions were diluted to 1 mg mL in the desired concentration of TMGMV in DI H2O (0.1, 0.3, 1.0, 5.0, 10 mg mL-1 final TMGMV concentration). -1Small molecules (ivermectin in 20% acetonitrile v / v). Samples were then prepared on a thermal cycler as previously described. Free / unencapsulated small molecules were removed by filtration by spin filtration (EMD Millipore UFC500324 3kDa, Burlington, MA, USA) at 5000x g for 5 min, and the process was repeated twice. The retentate was diluted in DI H2O. Sample absorbance of the Cy5 sample was collected using nanodrop. The concentration of Cy5 in the sample was determined using Beer-Lambert's law with an absorbance of 647nm and a molar extinction coefficient of 230,400cm-1M-1.

[0146] Competitive ELISA for quantification of ivermectin

[0147] According to the manufacturer's protocol, a sample containing ivermectin was diluted using a high-sensitivity ivermectin ELISA kit (Creative DiagnosticsDEIASL215, Shirley, NY, USA). Competitive ELISA of ivermectin was carried out in the 96-well plates provided. Then, the ivermectin-horseradish peroxidase conjugate in the sample well reacted with the provided substrate for 30 minutes, and then quenched with an equal volume of 1N HCl. The absorbance at 450 nm was measured on a microplate reader (Tecan Infinite M Plex, San Jose, CA, USA), and the concentration was fitted to the standard curve of ivermectin.

[0148] Circular dichroism

[0149] Aviv 215 CD spectrometer was used to collect CD spectra. Each sample was collected twice at 25°C using a quartz cuvette (Starna Cells, Atascadero, CA, USA) with a pathlength of 2 mm. Sample concentrations ranging from 0.025 mg / mL to 0.5 mg / mL were used, and a volume of 400 μL was obtained for each CD run. Near-UV and far-UV spectra were collected in two separate scans. For far-UV spectra, scans were made from 250 nm to 180 nm with a wavelength step of 1 nm and an average time of 1 second. For near-UV spectra, scans were made from 310 nm to 240 nm with a wavelength step of 0.5 nm and an average time of 1 second.

[0150] Cultivation of Caenorhabditis elegans

[0151] The N2 strain of C. elegans was obtained from the C. elegans Genetics Center (University of Minnesota, Minneapolis, MN, USA) and was grown on solid nematode growth medium (NGM) agar plates with OP-50 Escherichia coli as the main food source. Animals were maintained at 20°C.

[0152] Treatment of Caenorhabditis elegans with ivermectin and INV-SNP

[0153] The concentrated solution of C. elegans was aliquoted to ~100 animals per microcentrifuge tube. The animals were briefly centrifuged at 200 x g for 30 seconds to produce a pellet, and the supernatant was removed and replaced with a treatment group-specific solution (e.g., SNP or ivermectin). C. elegans was incubated in these solutions for 90 minutes and then transferred to the burrowing assay (Lesanpezeshki, L. et al., Scientific Reports 2019).

[0154] Pluronic-based drilling test

[0155] The Pluronic F127 solution was stored at 4°C to dissolve and maintain solution using the optimized Pluronic concentration of 26% w / w reported previously ((Lesanpezeshki, L. et al., Scientific Reports 2019). The Pluronic F127 solution was kept on ice while preparing the experiment. 30 μL of Pluronic F127 was added to a 6-well plate. The concentrated solution of treated C. elegans was deposited to introduce approximately 100 animals. After approximately 10 minutes, a layer of 2 mL of Pluronic was poured on top. OP-50 Escherichia coli lysate solution (20 μL OD600 = 0.5) was added to the surface. After 2 hours, an 11×11 area of ​​each well was scanned at 4x magnification (Keyence BZ-X800 fluorescence microscope). The number of nematodes on the surface was manually counted, and the area fraction of their tracks was determined by edge detection and binary image conversion in ImageJ.

[0156] Equivalent

[0157] Unless defined otherwise, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this technology belongs.

[0158] The present technology illustratively described herein may be suitably implemented in the absence of any one or more elements, limitations, not specifically disclosed herein. Thus, for example, the terms "including" and the like are to be understood broadly and without limitation. Furthermore, the terms and expressions used herein have been used as descriptive terms, rather than limiting terms, and there is no intention to use such terms and expressions to exclude any equivalents of the functions shown and described, or portions thereof, but it is recognized that various modifications may be made within the scope of the current technology claimed. Therefore, it should be understood that the materials, methods, and examples provided herein represent preferred aspects, are exemplary, and are not intended to limit the scope of the present technology.

[0159] The technology is described broadly and generically herein. Each narrower species and sub-generic grouping that falls within the generic disclosure also forms part of the technology. This includes a general description of the prior art, with a proviso or negative limitation deleting any subject matter from the genus, whether or not the deleted material is specifically cited herein.

[0160] In addition, where features or aspects of the technology are described in terms of Markush groups, those skilled in the art will recognize that the technology is also thereby described in terms of any individual member or subgroup of the Markush group.

[0161] All publications, patent applications, patents, and other references mentioned herein are expressly incorporated by reference in their entirety to the same extent as if each individual publication were individually incorporated by reference. In the event of a conflict, the present specification, including definitions, will control. References 1. Agriculture, UN FaHunger and food insecurity; United Nations: https: / / www.fao.org / hunger / en / , 2023. 2.Arias-Estévez,M.;López-Periago,E.;Martínez-Carballo,E.;Simal-Gándara,J.;Mejuto,J.-C.;García-Río,L.,The mobility and degradation ofpesticides in soils and the pollution of groundwater resources.Agriculture,Ecosystems&Environment 2008,123(4),247-260. 3. Brewer, A.; Dror, I.; Berkowitz, B., The Mobility of Plastic Nanoparticles in Aqueous and Soil Environments: A Critical Review. ACS ES&TWater 2021, 1(1), 48-57. 4.Chariou,P.L.;Dogan,A.B.;Welsh,A.G.;Saidel,G.M.;Baskaran,H.;Steinmetz,N.F.,Soil mobility of synthetic and virus-based modelnanopesticides.Nature Nanotechnology 2019,14(7),712-718. 5.Katagi,T.,Soil Column Leaching of Pesticides.In Reviews ofEnvironmental Contamination and Toxicology Volume 221,Whitacre,D.M.,Ed.Springer New York:New York,NY,2013;pp 1-105. 6.Beckers,S.J.;Staal,A.H.J.;Rosenauer,C.;Srinivas,M.;Landfester,K.;Wurm,F.R.,Targeted Drug Delivery for Sustainable Crop Protection:Transportand Stability of Polymeric Nanocarriers in Plants.Advanced Science 2021,8(11),2100067. 7.Chariou,P.L.;Steinmetz,N.F.,Delivery of Pesticides to PlantParasitic Nematodes Using Tobacco Mild Green Mosaic Virus as aNanocarrier.ACS Nano 2017,11(5),4719-4730. 8.Charudattan,R.;Hiebert,E.,A Plant Virus as a Bioherbicide forTropical Soda Apple,Solanum Viarum.Outlooks on Pest Management 2007,18(4),161-171. 9.Chariou,P.L.;Ma,Y.;Hensley,M.;Rosskopf,E.N.;Hong,J.C.;Charudattan,R.;Steinmetz,N.F.,Inactivated Plant Viruses as an Agrochemical DeliveryPlatform.ACS Agricultural Science&Technology 2021,1(3),124-130. 10.Shin,M.D.;Hochberg,J.D.;Pokorski,J.K.;Steinmetz,N.F.,Bioconjugation of Active Ingredients to Plant Viral Nanoparticles Is Enhancedby Preincubation with a Pluronic F127 Polymer Scaffold.ACS Applied Materials&Interfaces 2021,13(50),59618-59632. 11.Nikitin,N.;Ksenofontov,A.;Trifonova,E.;Arkhipenko,M.;Petrova,E.;Kondakova,O.;Kirpichnikov,M.;Atabekov,J.;Dobrov,E.;Karpova,O.,Thermalconversion of filamentous potato virus X into spherical particles withdifferent properties from virions.FEBS Letters 2016,590(10),1543-1551. 12.Manukhova,T.I.;Evtushenko,E.A.;Ksenofontov,A.L.;Arutyunyan,A.M.;Kovalenko,A.O.;Nikitin,N.A.;Karpova,O.V.,Thermal remodelling of Alternantheramosaic virus virions and virus-like particles into protein sphericalparticles.PLOS ONE 2021,16(7),e0255378. 13.Atabekov,J.;Nikitin,N.;Arkhipenko,M.;Chirkov,S.;Karpova,O.,Thermaltransition of native tobacco mosaic virus and RNA-free viral proteins intospherical nanoparticles.J Gen Virol 2011,92(Pt 2),453-6. 14.Bruckman,M.A.;Czapar,A.E.;VanMeter,A.;Randolph,L.N.;Steinmetz,N.F.,Tobacco mosaic virus-based protein nanoparticles and nanorods forchemotherapy delivery targeting breast cancer.Journal of Controlled Release2016,231,103-113. 15.Bruckman,M.A.;Hern,S.;Jiang,K.;Flask,C.A.;Yu,X.;Steinmetz,N.F.,Tobacco mosaic virus rods and spheres as supramolecular high-relaxivity MRIcontrast agents.Journal of Materials Chemistry B 2013,1(10),1482-1490. 16.Bruckman,M.A.;VanMeter,A.;Steinmetz,N.F.,Nanomanufacturing ofTobacco Mosaic Virus-Based Spherical Biomaterials Using a Continuous FlowMethod.ACS Biomaterials Science&Engineering 2015,1(1),13-18. 17.Deutsch,D.G.,Effect of prolonged 100℃ heat treatment in sodiumdodecyl sulfate upon peptide bond cleavage.Analytical Biochemistry 1976,71(1),300-303. 18.Kowit,J.D.;Maloney,J.,Protein cleavage by boiling in sodiumdodecyl sulfate prior to electrophoresis.Analytical Biochemistry 1982,123(1),86-93. 19.Kurien,B.T.;Scofield,R.H.,Common Artifacts and Mistakes Made inElectrophoresis.In Protein Electrophoresis:Methods and Protocols,Kurien,B.T.;Scofield,R.H.,Eds.Humana Press:Totowa,NJ,2012;pp 633-640. 20.Dobrov,EN / Nikitin,NA;Trifonova,EA;Parshina,EY;Makarov,VV;Maksimov,GV;Karpova,OV6Atabekov,JG,β-structure of the coat proteinsubunits in spherical particles generated by tobacco mosaic virus thermaldenaturation.Journal of Biomolecular Structure and Dynamics 2014,32(5),701-708. 21.Della Pelle,G.;Delgado Lopez,A.Salord Fiol,M. N.,Cyanine Dyes for Photo-Thermal Therapy:A Comparison of Synthetic Liposomes andNatural Erythrocyte-Based Carriers.International Journal of MolecularSciences 2021,22(13),6914. 22.Ahmadpour,E.Godrati-Azar,Z carriers of ivermectin as a novel drug delivery system in hydatidosis.Parasites&Vectors 2019,12(1),469. 23.Lesanpezeshki,L.Hewitt,JE;Laranjeiro,R.Antebi,A.Driscoll,M.Szewczyk,NJ.Blawzdziewicz,J.Lacerda,CMRVanapalli,SA,Pluronic gel-based burrowing assay for rapid assessment of neuromuscular health in general physiology.Scientific Reports 2019,9(1),15246.

Claims

1. Virus-like particles (VLPs) derived from Tobacco Mild Green Mosaic Virus (TMGMV) or a derivative thereof, optionally coupled to a pharmaceutical agent.

2. The VLP of claim 1, wherein the agent or derivative thereof is encapsulated in the VLP or covalently linked to the VLP.

3. The VLP of claim 1 or 2, wherein the VLP has a diameter of about 100 nm to about 2 μm.

4. The VLP of claim 1 or 2, wherein the VLP has a diameter of about 100 nm to about 200 nm.

5. The VLP according to any one of claims 1 to 4, wherein the agent is present in an amount of about 5 mg mL -1 to about 10 mg mL -1 The concentration of loading.

6. The VLP according to any one of claims 1 to 4, wherein the agent is present at about 0.5 mg mL -1 to about 0.2 mg mL -1 The concentration of loading.

7. The VLP of claim 3, wherein the agent is present at about 0.5 mg mL -1 to about 0.2 mg mL -1 The concentration of loading.

8. The VLP of claim 3, wherein the agent is present at about 0.5 mg mL -1 to about 0.2 mg mL -1 The concentration of loading.

9. The VLP of claim 4, wherein the agent is present at about 0.5 mg mL -1 to about 0.2 mg mL -1 The concentration of loading.

10. The VLP of claim 4, wherein the agent is present at about 0.5 mg mL -1 to about 0.2 mg mL -1 The concentration of loading.

11. The VLP according to any one of claims 1 to 10, wherein the agent is an insoluble compound or a pesticide.

12. The VLP according to any one of claims 1 to 10, wherein the insecticide is avermectin.

13. The VLP of claim 12, wherein the avermectin is selected from ivermectin, abamectin, doramectin, eplerimectin, moxidectin or selamectin.

14. The VLP according to any one of claims 1 to 12, wherein the pharmaceutical agent is ivermectin.

15. A plurality of VLPs according to any one of claims 1 to 14, wherein the VLPs are identical to or different from each other.

16. A plurality of VLPs according to claim 15, wherein the agents are the same or different from each other.

17. A plurality of VLPs according to any one of claims 1 to 15, wherein the TMGMVs ​​are the same as or different from each other.

18. A composition comprising the VLP of any one of claims 1 to 14 or a plurality of VLPs of any one of claims 15 to 17, and a carrier, optionally a carrier for agricultural application or delivery.

19. The composition according to claim 18, wherein the composition further comprises a preservative or a stabilizer.

20. The composition of claim 18 or 19, wherein the composition is lyophilized or frozen.

21. A method of treating an agricultural environment, the method comprising applying to the environment the VLP of any one of claims 1 to 14, the plurality of VLPs of any one of claims 15 to 17, or the composition of any one of claims 18 to 20.

22. The method of claim 21, wherein the application is to soil, fertilizer, or leaves, stems, flowers or roots of plants.

23. A kit comprising the VLP of any one of claims 1 to 14, the plurality of VLPs of any one of claims 15 to 17, or the composition of any one of claims 18 to 20; and instructions for use.

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