Spherical nanoparticles derived from TMGMV improve the soil transport of small hydrophobic pesticides

Abstract

Applicants herein describe spherical nanoparticles (SNPs) derived from TMGMV that are used to entrap small molecule cargo for improved loading efficiency while minimizing upstream processing steps. The TMGV-derived SNPs can be prepared by thermal reshaping at several concentrations to a range of functional nanoparticles. The small molecule cargo can be entrapped in the VLP or covalently attached to the VLP. The small molecule cargo can be selected from the list of drugs or insecticides provided in Table 1, or avermectins, or derivatives thereof.

Description

Technical Field

[0001] Cross - Reference to Related Applications This application claims priority under 35 U.S.C. § 119(e) to U.S. Provisional Application No. 63 / 337,488, filed May 2, 2022, the content of which is incorporated herein by reference in its entirety.

[0002] Statement Regarding Government Support This invention was made with government support under NIFA - 2020 - 67021 - 31255 awarded by the National Institute of Food and Agriculture and DMR2011924 awarded by the National Science Foundation. The government has certain rights in this invention.

[0003] Notice of Incorporation by Reference All publications, patents, and patent applications mentioned in this specification and the accompanying documents are incorporated herein by reference to the same extent as if each individual publication, patent, patent application, or document was specifically and individually indicated to be incorporated by reference.

Background Art

[0004] Background Damage from plant - parasitic nematodes amounts to hundreds of billions of dollars in crop losses, presenting both economic risk and risk to the global food supply. To improve the transport behavior and sustainable use of compounds for nematode treatment, more evolved delivery vehicles are needed that can hold hydrophobic cargo in the soil and help prevent leaching into groundwater. This disclosure meets this need and provides related advantages.

Summary of the Invention

Means for Solving the Problems

[0005] Summary of the Disclosure One means of addressing this yet unmet need is to use virus-like particles and virus nanoparticles as a versatile platform for drug delivery and small molecule loading. Due to their symmetry, the reactive amino acids on their surfaces, and their high stability compared to other proteinaceous delivery vehicles, they have shown many successful applications as a biocompatible and immunogenic platform for cancer therapy, diagnostic imaging agents, and hydrogels and polymeric materials for slow release.

[0006] In recent years, rod-shaped plant virus nanoparticles derived from tobacco mild green mosaic virus (TMGMV) have been delivering small molecules electrostatically and covalently in soil. This shows higher mobility than their icosahedral counterparts and other nanoparticle delivery systems. Since TMGMV is currently approved as a herbicide by the US 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 C. elegans (a model organism for nematodes) in a motility assay. This showed that the bound cargo maintained its efficacy. However, improving the covalent loading efficiency of hydrophobic cargo outside the virus by bioconjugation is challenging due to solubility limitations and the required molar equivalents needed to drive the bioconjugation reaction. Therefore, establishing a low-cost method for loading hydrophobic cargo onto TMGMV is necessary to cleverly circumvent solubility and reaction efficiency issues.

[0007] The applicant describes herein spherical nanoparticles (SNPs) derived from Tobacco mild green mosaic virus (TMGMV) for capturing low molecular weight cargo for improved loading efficiency while minimizing upstream processing steps. First, the TMGV-SNPs are prepared by thermal reshaping at several concentrations to produce a range of functional nanoparticles, and the resulting particles are analyzed using electron microscopy to characterize the batch. To compare the release behavior of molecules captured against covalently bound molecules, the SNPs are prepared using two methods: 1) simple capture of Cyanine 5 during thermal reshaping, and 2) covalently linking TMGMV to Cyanine 5 prior to thermal reshaping. The release behavior is characterized using dialysis, and the soil mobility profile is characterized using SDS-PAGE and absorbance measurements. Uptake of these SNPs into C. elegans (model nematode) is determined by measurement using fluorescence microscopy. As a final measure, nematicides are loaded onto the SNPs using Method 1, and the soil mobility and release profiles are determined by HPLC. These results indicate that SNPs can be developed as a simple platform for encapsulation of other insoluble compounds for drug delivery in soil, exhibiting desirable drug release and soil mobility characteristics.

[0008] Accordingly, the present disclosure provides virus-like particles (VLPs or "SNPs") derived from Tobacco mild green mosaic virus (TMGMV) or derivatives thereof conjugated to a drug as needed. In one aspect, the drug or derivative thereof is captured within the VLP or covalently bound to the VLP. In another aspect, the diameter of the VLP nanoparticles can range from about 1 nm to about 2 μm. In one embodiment, the drug is selected from the list of drugs or pesticides provided in Table 1, or abamectin, or a derivative thereof. In another aspect, the abamectin is selected from the group consisting of 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 abamectin, or a derivative thereof. In another aspect, the abamectin is selected from the group consisting of ivermectin, abamectin, doramectin, eprinomectin, moxidectin, or selamectin. In a further aspect, the agent is ivermectin.

[0010] In a further aspect, the agent or pesticide is loaded at a concentration of about 5 mg / mL -1 to about 10 mg / mL -1 or alternatively here the agent is loaded at a concentration of about 0.5 mg / mL -1 to about 0.2 mg / mL -1 of concentration.

[0011] A plurality of VLPs, which may be the same or different from each other, are also provided.

[0012] For example, a method of treating an agricultural environment such as soil, feed or a plant (leaf, stem or root), the method comprising delivering or contacting the VLP formulation or composition as described herein to the environment, consisting essentially of that step, or even consisting of that step, is provided herein. They are also useful for delivering pesticides and other insoluble compounds to inhibit pathogenic infestation of plants, roots, and soil. In one aspect, the method is performed with a plurality of VLPs as described herein, where 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 its derivatives are the same or different.

[0013] As will be apparent to those skilled in the art, the agent or pesticide is selected for the treatment of infections or pests. See, for example, those identified in Table 2. BRIEF DESCRIPTION OF THE DRAWINGS

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Mode for Carrying Out the Invention

[0033] Detailed Description of the Disclosure Definitions Unless otherwise defined, all technical and scientific terms used herein shall have the same meaning as commonly understood by one of ordinary skill in the art to which the claimed subject matter belongs. It is to be understood that the foregoing general description and the following detailed description are exemplary and explanatory only and are not restrictive of any claimed subject matter. In this application, the use of the singular includes the plural unless specifically stated otherwise. As used in this specification and the appended claims, the singular forms "a," "an," and "the" include plural references unless the context clearly dictates otherwise. In this application, the use of "or" means "and / or" unless stated otherwise. Further, the use of the terms "including," and other forms such as "include," "includes," and "included" is not limiting.

[0034] As used herein, ranges and amounts may be expressed as "about" a particular value or range. About 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 experimental error.

[0035] The section headings used herein are for organizational purposes only and are not to be construed as limiting the subject matter being described.

[0036] As used herein, the term "comprising" is intended to mean that the method includes the recited steps or elements but does not exclude others. "Consisting essentially of" is intended to mean that a claim is limited only as to the inclusion of steps or elements that do not materially affect the basic and novel characteristics of the claimed method. "Consisting of" is intended to mean that a claim excludes any element or step not specified therein. Embodiments defined by each of these transitional terms are within the scope of the present disclosure.

[0037] As used herein, the terms "individual(s)", "subject(s)", and "patient(s)" mean any mammal. In some embodiments, the mammal is a human. In some embodiments, the mammal is non-human. None of the terms require or are limited to a situation characterized by the supervision (e.g., constant or intermittent) of a healthcare provider (e.g., a physician, registered nurse, nurse practitioner, physician's assistant, attendant, or hospice staff).

[0038] "Agricultural product" is intended to mean vegetation, either as a whole or in part, and includes plants, trees, roots, flowers, branches, shoots, stems, leaves, or any other part thereof.

[0039] As used herein, the terms "treating," "treatment," etc. mean obtaining a desired agricultural effect such as an improvement. The above effects can be preventive in that they completely or partially prevent the infection of plants, vegetation, or other agricultural products by pests or insects. In one aspect, the terms "treatment," "treatment" exclude prevention.

[0040] The term "ameliorate" means a detectable improvement in an agricultural product or vegetation (e.g., a plant, tree, flower, crop, root, stem, or leaf). Detectable improvements include subjective or objective decreases, reductions, inhibitions, suppressions, limitations, or management in the occurrence, frequency, severity, progression, or duration of pest or microorganism (e.g., such as C. elegans) infection or presence.

[0041] As used herein, the term "agent" is intended to mean an active agent (e.g., an insecticide or insecticide, such as abamectin like ivermectin) that treats or ameliorates the presence or infection of an agricultural product.

[0042] Common insecticides are listed in Table 1 below.

Table 1-1

Table 1-2

[0043] As will be apparent to those skilled in the art, the above agents are selected to treat or ameliorate the presence of pests or agricultural infections. Non-limiting examples of such include abamectin. Fermectin is a group of drugs that occur naturally as products of the fermenting Streptomyces avermitilis (actinomycetes) isolated from soil. Eight different structures (including ivermectin, abamectin, doramectin, eprinomectin, moxidectin, and selamectin) are known and are divided into four main components (A1a, A2a, B1a, and B1b) and four minor components (A1b, A2b, B1b, and B2b). Abamectin is generally used as an insecticide for the treatment of pests and parasitic worms as a result of their anthelmintic and insecticidal properties. See El-Saber Batiha et al. (2020), Pharmaceuticals, Aug 17;13(8):196. doi: 10.3390 / ph13080196. PMID: 32824399; PMCID: PMC7464486. Non-limiting examples of common insecticides for treating crops, and crops or plants are provided in Table 2.

Table 2

[0044] Virus-like particles (VLPs) and compositions As used herein, the terms “SNP” or “virus-like particle” or “VLP” refer to a non-replicating viral shell derived from one or more viruses (e.g., one or more plant viruses described herein). VLPs generally consist of one or more viral proteins (e.g., but not limited to those proteins referred to as capsid, coat, shell, surface and / or envelope proteins, or particle-forming polypeptides derived from these proteins). VLPs can form naturally upon recombinant expression of the protein in a suitable expression system. VLPs can also be engineered (e.g., including, consisting essentially of, or even consisting of one or more viral proteins that are modified or derived from or even further derived from them). Methods for generating VLPs are known in the art. The presence of VLPs after recombinant expression of the viral protein can be detected using conventional techniques known in the art (e.g., by electron microscopy, biophysical characterization, etc.). Further, VLPs can be isolated by known techniques (e.g., density gradient centrifugation) and identified by characteristic density banding. See, for example, 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.

[0045] As used herein, a VLP is a VLP derived from Tobacco Mild Green Mosaic Virus (TMGMV), comprising, consisting essentially of, or even more consisting of one or more viral particles (e.g., capsids) derived from Tobacco Mild Green Mosaic Virus (TMGMV) or a derivative thereof.

[0046] As used herein, the term "equivalent thereof" in reference to a polynucleotide or protein (e.g., a capsid or coat protein) encompasses a polynucleotide or protein that has at least 80%, 85%, 90%, 95%, 96%, 97%, 98%, or 99% sequence identity with the respective polynucleotide or protein to which it is being compared, while still retaining functional activity. When referring to a capsid or coat protein, functional activity refers to the formation of rod-shaped plant virus nanoparticles derived from a VLP, e.g., Tobacco Mild Green Mosaic Virus (TMGMV) or a derivative thereof.

[0047] As used herein, the term "modification" includes, for example, substitutions, additions, insertions, and deletions to an amino acid sequence, which may be referred to as a "variant". Exemplary sequence substitutions, additions, and insertions include, for example, all or part of a sequence in which one or more amino acids of a capsid derived from a plant virus have been substituted (or mutated), added, or inserted. In some cases, the capsids described herein include modified capsids that have at least 70%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity with their representative wild-type versions, consisting essentially of, or even more consisting of. These or other modifications are intended to be within the scope of "or a derivative thereof".

[0048] The term "sequence identity" refers to the percentage of bases or amino acid sequences that are the same and in the same relative positions between two polynucleotide or polypeptide sequences. Thus, one polynucleotide or polypeptide sequence has a certain percentage of sequence identity when compared to another polynucleotide or polypeptide sequence. For sequence comparison, typically, one sequence acts as a reference sequence against which a test sequence is compared. The term "reference sequence" refers to the molecule to which the test sequence is compared. A polynucleotide or polynucleotide region (or polypeptide or polypeptide region) having a certain percentage of "sequence identity" to a reference sequence (e.g., 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 98% or 99%) means that, when aligned, the percentage of bases (or amino acids) at each position in the test sequence is identical to the bases (or amino acids) at the same position in the reference sequence. This alignment and percent homology or sequence identity can be determined using software programs known in the art (e.g., those described in Ausubel et al. eds. (2007) Current Protocols in Molecular Biology). Preferably, default parameters are used for the alignment. One alignment program is BLAST (using default parameters). In particular, the programs are BLASTN and BLASTP, and use the following default parameters: genetic code = standard; filter = none; strand = both; cutoff = 60; expect = 10; matrix = BLOSUM62; description = 50 sequences; sort = HIGH SCORE; database = non-redundant, GenBank + EMBL + DDBJ + PDB + GenBank CDS translations + SwissProtein + SPupdate + PIR. Details of these programs can be found at the following Internet address: ncbi.nlm.nih.gov / blast / Blast.cgi.

[0049] The modified capsid polypeptide includes, for example, non-conservative and conservative substitutions of the capsid amino acid sequence. In one aspect, the modified capsid is within the scope of the term "its derivatives".

[0050] As used herein, the term "conservative substitution" refers to the replacement of an amino acid residue with another chemically or biologically similar residue. Biologically similar means that the substitution does not disrupt biological activity or function, for example, the assembly of the viral capsid.

[0051] Structurally similar means that the amino acids have side chains of similar length (e.g., alanine, glycine, and serine) or similar size. Chemically similar means that the residues have the same charge or that both are hydrophilic or hydrophobic. Specific examples of conservative substitutions include the substitution of a hydrophobic residue (e.g., isoleucine, valine, leucine, or methionine) for another, the substitution of a polar residue for another (e.g., the substitution of arginine for lysine, glutamic acid for aspartic acid, or glutamine for asparagine, etc.). The term "conservative substitution" also includes the use of an amino acid that has been substituted in place of the unsubstituted parent amino acid. Such proteins containing amino acid substitutions can be encoded by nucleic acids. In conclusion, nucleic acid sequences encoding proteins containing amino acid substitutions are also provided.

[0052] The modified protein also includes one or more D-amino acids (and mixtures thereof) substituted for L-amino acids, structural and functional analogs (e.g., peptidomimetics and derivatized forms having synthetic or non-natural amino acids or amino acid analogs). The modifications include cyclic structures (e.g., end-to-end amide bonds between the amino and carboxy termini of the molecule) or intramolecular or intermolecular disulfide bonds.

[0053] The modified form further includes a "chemical derivative" having a side chain in which one or more amino acids are chemically altered or derivatized. Such derivatized polypeptides include, for example, amino acids in which the free amino group forms an amine hydrochloride, p-toluenesulfonyl group, carbobenzoxy group; amino acids in which the free carboxy group forms a salt, methyl and ethyl esters; amino acids in which the free hydroxyl group forms an O-acyl or O-alkyl derivative, as well as naturally occurring amino acid derivatives (e.g., 4-hydroxypropyl for proline, 5-hydroxylysine for lysine, homoserine for serine, ornithine for lysine, etc.). Amino acid derivatives capable of altering covalent bonds (e.g., disulfide linkages formed between two cysteine residues that result in a cyclic polypeptide) are also included.

[0054] Virus-like particles (VLPs) derived from tobacco mild green mosaic virus (TMGMV) or derivatives thereof conjugated to a drug as required are provided herein. In one aspect, the drug or its derivative is trapped within the VLP or covalently attached to the VLP. In another aspect, the diameter of the VLP nanoparticles can range from about 1 nm to about 2 μm. In certain embodiments, the nanoparticles are 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 diameter. 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 diameter. In one aspect, the diameter of the VLP is in the range of about 100 nm to about 200 nm. In one aspect, the drug is selected from the list of drugs or pesticides provided in Table 1, or abamectin, or a derivative thereof. In another aspect, the abamectin is selected from the group consisting of ivermectin, abamectin, doramectin, eprinomectin, moxidectin, or selamectin.

[0055] In one aspect, the drug is selected from the list of drugs or pesticides provided in Table 1, or abamectin, or a derivative thereof. In another aspect, the abamectin is selected from the group consisting of ivermectin, abamectin, doramectin, eprinomectin, moxidectin, or selamectin. In a further aspect, the drug is ivermectin. In a further aspect, the VLP is TMGMV or a derivative thereof, the drug is ivermectin, and the diameter of the nanoparticles is in the range of about 100 nm to about 200 nm. In another aspect, the nanoparticles are TMGMV or a derivative thereof, the drug is abamectin (e.g., ivermectin), and the diameter of the nanoparticles is in the range of about 100 nm to about 200 nm.

[0056] In a further aspect, the agent or insecticide is loaded at a concentration of about 5 mg / mL -1 to about 10 mg / mL -1 or alternatively, where the agent is at a concentration of about 0.5 mg / mL -1 to about 0.2 mg / mL -1 In one aspect, the agent is a list of agents or insecticides provided in Table 1, or abamectin, or a derivative thereof, loaded at a concentration of about 5 mg / mL -1 to about 10 mg / mL -1 or alternatively, where the agent is at a concentration of about 0.5 mg / mL -1 to about 0.2 mg / mL -1 In another aspect, the abamectin is selected from the group of ivermectin, abamectin, doramectin, eprinomectin, moxidectin, or selamectin at a concentration of about 5 mg / mL -1 to about 10 mg / mL -1 or alternatively, where the agent is at a concentration of about 0.5 mg / mL -1 to about 0.2 mg / mL -1 In a further aspect, the agent is ivermectin loaded at a concentration of about 5 mg / mL -1 to about 10 mg / mL -1 or alternatively, where the agent is at a concentration of about 0.5 mg / mL -1 to about 0.2 mg / mL -1 In a further aspect, the VLP is TMGMV or a derivative thereof, the agent is ivermectin, the diameter of the nanoparticles is about 100 nm to about 200 nm and in the range therebetween, the agent is abamectin (e.g., ivermectin), and the diameter of the nanoparticles is about 100 nm to about 200 nm and in the range therebetween.

[0057] In some cases, the VLPs described herein further comprise, consist essentially of, or even consist of a label or tag (such as a detectable label). The detectable label can be attached, for example, to the surface of the VLP. In one aspect, rod-shaped plant virus nanoparticles or derivatives thereof derived from tobacco mild green mosaic virus (TMGMV) further comprise, consist essentially of, or consist of the above label or tag.

[0058] Non-limiting and exemplary detectable labels also include radioactive substances (such as radioisotopes), metals or metal oxides. Radioisotopes include radionuclides that emit α, β or γ 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, 68 Ga, 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 Bi or 225 Ac.

[0059] Additional non - limiting and exemplary detectable labels include metals or metal oxides. In certain embodiments, the metal or metal oxide can be one or more of the following: gold, silver, copper, boron, manganese, gadolinium, iron, chromium, barium, europium, erbium, praseodymium, indium, or technetium. In further embodiments, the metal oxides include 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).

[0060] Further non-limiting and exemplary detectable labels include contrast agents (e.g., gadolinium; manganese; barium sulfate; iodinated or noniodinated agent; ionic agent or nonionic agent; magnetic and paramagnetic agents (e.g., iron oxide chelate compounds); nanoparticles; enzymes (horseradish peroxidase, alkaline phosphatase, β-galactosidase, or acetylcholinesterase); avidin-biotin families (e.g., streptavidin / biotin and avidin / biotin); fluorescent substances (e.g., umbelliferone, fluorescein, fluorescein isothiocyanate, rhodamine, dichlorotriazinylamine fluorescein, dansyl chloride or phycoerythrin); luminescent substances (e.g., luminol); or bioluminescent substances (e.g., luciferase, luciferin, aequorin).

[0061] Further non-limiting examples of tags and / or detectable labels include the following: enzymes (horseradish peroxidase, urease, catalase, alkaline phosphatase, β-galactosidase, chloramphenicol transferase); enzyme substrates; ligands (e.g., biotin); receptors (avidin); GST-, T7-, His-, myc-, HA- and FLAG®-tags; electron-dense reagents; energy transfer molecules; paramagnetic labels; fluorophores (fluorescein, fluorscamine, rhodamine, phycoerythrin, phycocyanin, allophycocyanin); chromophores; chemiluminescent (imidazole, luciferase, acridinium, oxalate); and bioluminescent agents.

[0062] As shown herein, detectable labels or tags can be linked or conjugated (e.g., covalently) to the above-mentioned VLPs. In various embodiments, detectable labels (e.g., radionuclides or metals or metal oxides) can be bound or conjugated to the above-mentioned agents either directly or indirectly. Linkers or intermediate functional groups can be used to link the molecule to a detectable label or tag. The linker includes an amino acid or peptidomimetic sequence and a label or tag inserted between the molecules, such that the two entities maintain at least partially distinct functions or activities. The linker can have one or more properties, including a flexible conformation, the inability to form a regular secondary structure, or hydrophobic or charged properties that can facilitate or interact with any domain. Amino acids typically found in flexible protein regions include Gly, Asn, and Ser. The length of the above linker sequence can vary without significantly affecting the function or activity.

[0063] The linker further includes a chemical entity, a conjugated agent, and an intermediate functional group. Examples include entities that react with free or semi-free amines, oxygen, sulfur, hydroxy, or carboxy groups. Such functional groups are thus monofunctional or bifunctional crosslinking agents (e.g., sulfo-succinimidyl derivatives (sulfo-SMCC, sulfo-SMPB), in particular, disuccinimidyl suberate (DSS), BS3 (sulfo-DSS), disuccinimidyl glutarate (DSG), and disuccinimidyl tartrate (DST). Non-limiting examples include diethylenetriaminepentaacetic acid (DTPA) and ethylenediaminetetraacetic acid.

[0064] Also provided are VLPs as described herein that further comprise, or consist essentially of, or still further consist of additional agents. Non-limiting examples of such include other insoluble compounds and insecticides for drug delivery in soil that exhibit desired drug release and soil mobility characteristics. These are covalently linked to and / or entrapped within the VLPs. The agents may also be covalently attached to the VLPs through the use of linkers.

[0065] The size of the VLP nanoparticles can range from about 1 nm to about 2 μm. In certain embodiments, the nanoparticles have a diameter of 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 VLP nanoparticles have a diameter of 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 nanoparticles are in the range of 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.

[0066] Also provided are a plurality of VLPs as described herein, wherein the VLPs are the same or different from one another, and / or the agents are the same or different from one another, and / or the TMGMV or its derivatives are the same or different.

[0067] Composition In another aspect, provided herein is a composition comprising, consisting essentially of, or consisting of a VLP as provided herein, and at least one carrier, suitable for its intended use, e.g., use in soil or other agricultural environments. In one aspect, the VLP is a rod-shaped plant virus nanoparticle or a derivative thereof derived from Tobacco mild green mosaic virus (TMGMV).

[0068] Compositions comprising, consisting essentially of, or consisting of the VLP compositions alone or in combination with other agents can be manufactured by conventional mixing, dissolving, granulating, tablet coating, wet milling, emulsifying, encapsulating, entrapping, or lyophilization processes. These can be formulated in a conventional manner using one or more carriers, diluents, excipients, or auxiliary substances that facilitate the processing of the provided compounds into preparations that can be used in agriculture.

[0069] In some embodiments, the agricultural formulations include, but are not limited to, lyophilized formulations, aqueous liquid dispersions, self-emulsifying dispersions, solid solutions, liposomal dispersions, aerosols, solid dosage forms, powders, immediate release agents, controlled release formulations, fast melt formulations, tablets, capsules, pills, sustained release formulations, long acting release agents, pulsatile release formulations, multiparticulate formulations (e.g., nanoparticle formulations), and mixed immediate and controlled release formulations.

[0070] In one aspect, the nanoparticles are combined with a carrier such as an organic solvent (e.g., water) or mineral clay, stickers or spreaders, stabilizers, safeners, or other adjuvants such as chemicals that improve or enhance pesticidal activity. See, for example, http: / / npic.orst.edu / factsheets / formulations.html, and the references cited therein.

[0071] In some embodiments, the composition includes a carrier or carrier substance selected based on its compatibility with the compositions disclosed herein and the release profile characteristics of the desired dosage form. Exemplary carrier substances include, for example, binders, suspending agents, disintegrants, fillers, surfactants, solubilizing agents, stabilizers, lubricants, wetting agents, diluents, and the like. Agriculturally compatible carrier substances include, but are not limited to, acacia, gelatin, colloidal silicon dioxide, calcium glycerophosphate, calcium lactate, maltodextrin, glycerin, magnesium silicate, polyvinylpyrrolidone (PVP), cholesterol, cholesterol esters, sodium caseinate, soybean lecithin, taurocholic acid, phosphatidylcholine, sodium chloride, tricalcium phosphate, dipotassium phosphate, cellulose and cellulose conjugates, sugars, sodium stearoyl lactate, carrageenan, monoglycerides, diglycerides, pregelatinized starch, and the like.

[0072] In some cases, the composition further comprises a pH regulator or buffer containing an acid (e.g., acetic acid, boric acid, citric acid, lactic acid, phosphoric acid, and hydrochloric acid), a base (e.g., sodium hydroxide, sodium phosphate, sodium borate, sodium citrate, sodium acetate, sodium lactate, and tris(hydroxymethyl)aminomethane), and a buffering substance (e.g., citrate / dextrose, sodium bicarbonate, and ammonium chloride). Such acids, bases, and buffering substances are included in amounts necessary to maintain the pH of the composition within an acceptable range.

[0073] In some cases, the composition contains one or more salts in amounts necessary to bring the weight osmolality of the composition within an acceptable range. Such salts include those having sodium, potassium, or ammonium cations, and chloride, citrate, ascorbate, borate, phosphate, bicarbonate, sulfate, thiosulfate, or bisulfite anions, and suitable salts include sodium chloride, potassium chloride, sodium thiosulfate, sodium bisulfite, and ammonium sulfate.

[0074] In some embodiments, the composition includes, but is not limited to, sugars such as trehalose, sucrose, mannitol, maltose, glucose, or salts such as potassium phosphate, sodium citrate, ammonium sulfate, and / or other agents such as heparin to increase solubility and stability.

[0075] In some cases, the above composition further includes a diluent used to stabilize the compound. This is because they can provide a more stable environment. Salts dissolved in a buffered solution (which can also provide pH control or maintenance) are utilized as diluents in the art, including but not limited to phosphate buffered saline aqueous solution. In certain cases, the diluent increases the bulk of the above composition to facilitate compression or create sufficient volume for a homogeneous blend for capsule filling. Such compounds include, for example, lactose, starch, mannitol, sorbitol, dextrose, microcrystalline cellulose (e.g., AVICEL (登録商標) ), dibasic calcium phosphate, dicalcium phosphate dihydrate, tricalcium phosphate, calcium phosphate, anhydrous lactose, spray-dried lactose, pregelatinized starch, compressible sugar (e.g., Di-PAC (登録商標) (Amstar)), mannitol, hydroxypropylmethylcellulose, hydroxypropylmethylcellulose acetate stearate, sucrose-based diluent, powdered sugar, monobasic calcium sulfate monohydrate, calcium sulfate dihydrate, calcium lactate trihydrate, dextrate, hydrolyzed cereal solid, amylose, powdered cellulose, calcium carbonate, glycine, kaolin, mannitol, sodium chloride, inositol, bentonite, etc.

[0076] In some cases, the above composition includes disintegration agents or disintegrants to promote the breakup or disintegration of the substance. The term "disintegrate" includes both dissolution and dispersion of the dosage form when in contact with digestive fluids. Examples of disintegrants include the following: starch, such as natural starch (e.g., corn starch or potato starch), pregelatinized starch (e.g., National 1551 or AMIJEL (登録商標) ), or sodium starch glycolate (e.g., PROMOGEL (登録商標)or EXPLOTAB (登録商標) ), cellulose such as wood products, crystalline methylcellulose (e.g., AVICEL (登録商標)、 AVICEL (登録商標) PH101, AVICEL (登録商標) PH102, AVICEL (登録商標) PH105, ELCEMA (登録商標) P100, EMCOCEL (登録商標) , VIVACEL (登録商標) , MING TIA (登録商標) , and SOLKA-FLOC (登録商標) ), methylcellulose, croscarmellose, or cross-linked cellulose (e.g., sodium cross-linked carboxymethylcellulose (AC-DI-SOL (登録商標) ), cross-linked carboxymethylcellulose, or cross-linked croscarmellose), cross-linked starch (e.g., sodium starch glycolate), cross-linked polymer (e.g., crospovidone), cross-linked polyvinylpyrrolidone, alginate (e.g., salts of alginic acid such as alginic acid or sodium alginate), clay (e.g., VEEGUM (登録商標) HV (magnesium aluminum silicate)), gum (e.g., agar, guar, locust bean, karaya, pectin, or tragacanth), sodium starch glycolate, bentonite, natural sponge, surfactant, resin (e.g., cation exchange resin), citrus pulp, sodium lauryl sulfate, sodium lauryl sulfate in combination starch, etc.

[0077] Optionally, the above compositions may contain fillers (e.g., lactose, calcium carbonate, calcium phosphate, dibasic calcium phosphate, calcium sulfate, microcrystalline cellulose, cellulose powder, dextrose, dextrate, dextran, starch, pregelatinized starch, sucrose, xylitol, lactitol, mannitol, sorbitol, sodium chloride, polyethylene glycol, etc.).

[0078] Lubricants and lubricating agents are also included in the compositions described herein, if desired, to prevent, reduce or inhibit the adhesion or friction of substances.

[0079] Exemplary lubricants include the following: for example, stearic acid, calcium hydroxide, talc, sodium stearyl fumarate, hydrocarbons (e.g., mineral oil), or hydrogenated vegetable oils (e.g., hydrogenated soybean oil (STEROTEX (登録商標) )), higher fatty acids and their alkali metal and alkaline earth metal salts (e.g., aluminum, calcium, magnesium, zinc), stearic acid, sodium stearate, glycerol, talc, wax, STEAROWET (登録商標) , boric acid, sodium benzoate, sodium acetate, sodium chloride, leucine, polyethylene glycol (e.g., PEG-4000) or methoxypolyethylene glycol (e.g., CARBOWAX TM ), sodium oleate, sodium benzoate, glyceryl behenate, polyethylene glycol, magnesium or sodium lauryl sulfate, colloidal silica (e.g., SYLOID TM , CAB-O-SIL (登録商標) ), starch (e.g., corn starch), silicone oil, surfactants, and the like.

[0080] Plasticizers include compounds used to soften microencapsulated substances or film coatings and reduce their handling difficulties. Suitable plasticizers include, for example, polyethylene glycol (e.g., PEG 300, PEG 400, PEG 600, PEG 1450, PEG 3350, and PEG 800), stearic acid, propylene glycol, oleic acid, triethyl cellulose, and triacetin. Plasticizers can also function as dispersants or wetting agents.

[0081] Examples of solubilizers include compounds such as triacetin, triethyl citrate, ethyl oleate, ethyl caprylate, sodium lauryl sulfate, sodium doxsate, vitamin E TPGS, dimethylacetamide, N-methylpyrrolidone, N-hydroxyethylpyrrolidone, polyvinylpyrrolidone, hydroxypropylmethylcellulose, hydroxypropyl cyclodextrin, ethanol, n-butanol, isopropyl alcohol, cholesterol, bile salts, polyethylene glycol 200 - 600, glycofurol, transcutol, propylene glycol, and dimethyl isosorbide.

[0082] Examples of stabilizers include compounds such as any antioxidant, buffer, acid, preservative, etc. Exemplary stabilizers include L-arginine hydrochloride, tromethamine, albumin (human), citric acid, benzyl alcohol, phenol, disodium biphosphate dehydrate, propylene glycol, metacresol or m-cresol, zinc acetate, polysorbate-20 or TWEEN (registered trademark) 20, or trometamol.

[0083] Examples of suspending agents include polyvinylpyrrolidone (e.g., polyvinylpyrrolidone K12, polyvinylpyrrolidone K17, polyvinylpyrrolidone K25, or polyvinylpyrrolidone K30, vinylpyrrolidone / vinyl acetate copolymer (S630)), polyethylene glycol (e.g., polyethylene glycol 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, hydroxyethyl methyl cellulose acetate stearate, polysorbate - 80, hydroxyethyl cellulose, sodium alginate, gums (e.g., such as tragacanth gum and gum arabic, guar gum, xanthan (including xanthan gum)), sugars, cellulosics (e.g., such as sodium carboxymethyl cellulose, methyl cellulose, sodium carboxymethyl cellulose, hydroxypropyl methyl cellulose, hydroxyethyl cellulose), polysorbate - 80, sodium alginate, polyethoxylated sorbitan monolaurate, polyethoxylated sorbitan monolaurate, povidone, and the like.

[0084] Examples of surfactants include sodium lauryl sulfate, sodium doxsate, Tween® 60 or 80, triacetin, vitamin E TPGS, sorbitan monolaurate, polyoxyethylene sorbitan monolaurate, polysorbate, poloxamer, bile salts, glyceryl monostearate, copolymers of ethylene oxide and propylene oxide (e.g., Pluronic® (BASF)), and the like. Further examples of surfactants include polyoxyethylene fatty acid glycerides and vegetable oils, such as polyoxyethylene (60) hydrogenated castor oil, and polyoxyethylene alkyl ethers and alkyl phenyl ethers, such as octoxynol 10, octoxynol 40. Occasionally, surfactants are included to enhance physical stability or for other purposes.

[0085] Examples of thickeners include, for example, methylcellulose, xanthan gum, carboxymethylcellulose, hydroxypropylcellulose, hydroxypropylmethylcellulose, hydroxypropylmethylcellulose acetate stearate, hydroxypropylmethylcellulose phthalate, carbomer, polyvinyl alcohol, alginate, gum arabic, chitosan, and combinations thereof.

[0086] Examples of wetting agents include compounds such as oleic acid, glyceryl monostearate, sorbitan monolaurate, sorbitan monolaurate, triethanolamine oleate, polyoxyethylene sorbitan monooleate, polyoxyethylene sorbitan monolaurate, sodium docusate, sodium oleate, sodium lauryl sulfate, sodium doccusate, triacetin, Tween® 80, vitamin E TPGS, ammonium salts, and the like.

[0087] In a further aspect, the composition may be frozen or lyophilized and contain agents for preserving stability and activity.

[0088] In some embodiments, one or more of the compositions disclosed herein are included in a kit. Thus, in some embodiments, a kit is provided herein that comprises, consists essentially of, or consists of one or more of the compositions disclosed herein and instructions for their use.

[0089] Dosage and dosage form In some embodiments, the composition may be administered or delivered to the environment once a week, once a day, twice a day, three times a day, or four times a day, or even more frequently, either alone or as part of a formulation.

[0090] Administration of the VLP formulations, alone or in combination with additional agents, and compositions containing them can be effected by any method that enables delivery to the site of action.

[0091] Accordingly, one of ordinary skill in the art will recognize that, based on the disclosure provided herein, the dosages and administration regimens are adjusted according to methods well known in the art.

[0092] Use of VLPs and Compositions Containing the Same A method of treating an agricultural environment (e.g., soil, feed, or a plant (leaf, stem, or root)), the method comprising, consisting essentially of, or further consisting of delivering or contacting the VLP formulation or composition as described herein to or with the environment. They are also useful for delivering pesticides and other insoluble compounds to inhibit pathogenic ectoparasitism of plants, roots, and soil. In one aspect, the method is performed with a plurality of VLPs as described herein, where 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 its derivatives are the same or different.

[0093] As will be apparent to one of ordinary skill in the art, the agents or pesticides are selected for treatment of infection or pests. See, for example, those identified in Table 2.

[0094] In one aspect, the method is performed with VLPs derived from tobacco mild green mosaic virus (TMGMV) or derivatives thereof conjugated to an agent as needed. In a further aspect, it is conjugated to the agent. In one aspect, the agent or derivative thereof is conjugated by being entrapped within or covalently attached to the VLPs. In another aspect, the method of the present disclosure is performed with VLPs having a diameter of about 1 nm to about 2 μm. In certain embodiments, the nanoparticles have a diameter of 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 in the method is less than about 500 nm, less than about 400 nm, less than about 300 nm, less than about 200 nm, about 100 nm, or less than about 50 nm. In one aspect, the diameter of the VLPs in the method is in the range of about 100 nm to about 200 nm. In one aspect, the agent in the method is selected from the list of agents or pesticides provided in Table 1, or abamectin or a derivative thereof. In another aspect, the abamectin is selected from the group consisting of ivermectin, abamectin, doramectin, eprinomectin, moxidectin, or selamectin. In a further aspect, the agent is abamectin (e.g., ivermectin) for treating C. elegans.

[0095] In one aspect, the agent used in the above method is selected from the list of agents or pesticides provided in Table 1, or abamectin, or a derivative thereof. In another aspect, the abamectin is selected from the group consisting of ivermectin, abamectin, doramectin, eprinomectin, moxidectin, or selamectin. In a further aspect, the agent used in the above method is ivermectin. In a further aspect, the VLP used in the above method is TMGMV or a derivative thereof, the agent is ivermectin, and the diameter of the nanoparticles is from about 100 nm to about 200 nm, and in the range therebetween. In another aspect, the nanoparticles used in the above method are TMGMV or a derivative thereof, the agent is abamectin (e.g., ivermectin), and the diameter of the nanoparticles is from about 100 nm to about 200 nm, and in the range therebetween.

[0096] In a further aspect, the agent or pesticide used in the above method is loaded at a concentration of about 5 mg mL -1 to about 10 mg mL -1 or, alternatively, where 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 used in the above method is loaded at a concentration of about 5 mg mL -1 to about 10 mg mL -1 and is selected from the list of agents or pesticides provided in Table 1, or abamectin, or a derivative thereof, or, alternatively, where the agent is loaded at a concentration of about 0.5 mg mL -1 to about 0.2 mg mL -1 In another aspect, the abamectin used in the above method is selected from ivermectin, abamectin, doramectin, eprinomectin, moxidectin, or selamectin at a concentration of about 5 mg mL -1 to about 10 mg mL -1 or, alternatively, where the agent used in the above method is loaded at a concentration of about 0.5 mg mL -1~about 0.2 mg / mL -1 is loaded at a concentration of. In a further aspect, the agent is ivermectin used in the above agent, about 5 mg / mL -1 ~about 10 mg / mL -1 is loaded at a concentration of, or alternatively, the agent used in the above method is about 0.5 mg / mL -1 ~about 0.2 mg / mL -1 is loaded at a concentration of. In a further aspect, the VLP used in the above method is TMGMV or a derivative thereof, the agent is ivermectin, the diameter of the above nanoparticles is about 100 nm to about 200 nm, and in the range therebetween, the agent is abamectin (e.g., ivermectin), and the diameter of the above nanoparticles is about 100 nm to about 200 nm, and in the range therebetween.

[0097] In some cases, the VLP used in the methods described herein further comprises, consists essentially of, or even consists of a label or tag (such as a detectable label). The detectable label can be, for example, bound to the surface of the VLP. In one aspect, a rod-shaped plant virus nanoparticle derived from Tobacco mild green mosaic virus (TMGMV) or a derivative thereof further comprises, consists essentially of, or consists of the above label or tag.

[0098] Kit As used herein, a kit or article of manufacture described herein is a container compartmentalized to receive a carrier, package, or one or more containers (such as vials, tubes, etc.) (each of the containers comprising, consisting essentially of, or even consisting of one of the distinct elements to be used in the methods described herein). Suitable containers include, for example, bottles, vials, syringes, and test tubes. In one embodiment, the containers are formed from various substances (such as glass or plastic).

[0099] The manufactured articles 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 the selected formulation and the intended mode of administration and handling.

[0100] 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.

[0101] Preferred embodiments of the present disclosure have been shown and described herein, but it will be apparent to those skilled in the art that such embodiments are provided by way of example only. Many variations, modifications, and substitutions will now occur to those skilled in the art without departing from the present disclosure. It should be understood that various alternatives to the embodiments of the disclosure described herein may be used in practicing the present disclosure. The following claims define the scope of the present disclosure, and it is intended that methods and structures within the scope of these claims and their equivalents be covered thereby.

Examples

[0102] Experiment These examples are provided for illustrative purposes only and are not provided to limit the scope of the claims provided herein.

[0103] The widespread use of pesticides in agriculture accumulates these toxins in crops, in the soil, and in drinking water and groundwater, posing a serious risk to ecosystems and human health. The first step towards a healthier society is to enhance food security by improving quality and yield (i.e., more effective crop treatment) while protecting the environment and agricultural ecosystems (i.e., preventing the leaching and accumulation of pesticides into the environment). Most pesticides are hydrophobic and thus do not have good soil mobility. This leads to overuse. VLPs with enhanced properties for pesticide delivery are provided herein. In one aspect, a linker is added to load the pesticide into the VLP using a proper "plug and play" strategy.

[0104] Alternatively, a non-covalent encapsulation technique is provided herein for encapsulating the pesticide into nanoparticles derived from Tobacco mild green mosaic virus (TMGMV). TMGMV is a good platform for precision agriculture because it has excellent soil mobility.

[0105] The TMGMV rod-shaped virus is thermally transitioned into spherical nanoparticles (referred to as SNPs) - during the encapsulation process, the hydrophobic pesticide cargo is encapsulated in high yield; the surprising discovery is also that the thermal reformation enables a covalent or covalent-like binding of the pesticide to the carrier, which enhances the soil mobility of the cargo.

[0106] Biotin or avidin can be used as a molecular linker to load the pesticide onto the TMGMV-VLP. The biomolecular linker overcomes the need for direct bioconjugation, which has been found to be a problem for pesticides (both from a chemical and a regulatory procedure perspective).

[0107] Accordingly, the VLPs of the present disclosure are used as synthetic nanocarriers for insecticide delivery. The VLPs are ideal due to their unique zwitterionic properties and modular shape that allows these protein nanoparticles to have favorable soil transport.

[0108] Experimental Considerations

[0109] As the world population increases, strategies to protect crops are essential to meet the coming food demands. At current levels, 9.8% of people are undernourished or starving. The economic losses in the agricultural sector due to plant-parasitic nematodes amount to $125 billion, and 14% of crops are lost worldwide every year. 1 Although the advent of insecticide use has overcome many of the challenges of crop loss, it has introduced new challenges in terms of human health, environmental toxicity, and skepticism among the general public. Creating next-generation nanoinsecticides that can mitigate and address many of the concerns around crop protection, in combination with a robust regulatory environment, is an essential step in creating a sustainable agricultural system.

[0110] The present disclosure provides the application of nanoparticles for the soil delivery of insecticides (many of which suffer from poor soil mobility and a rapid washout effect and require a delivery vehicle to remain effective and economically viable). 2-5 Insecticide delivery systems derived from polymers and nanoparticles have been developed but suffer from high costs and may not have optimal soil mobility characteristics. 2, 4, 6 One promising platform for nanoinsecticide delivery is tobacco mild green mosaic virus (TMGMV) nanoparticles. TMGMV has many desirable properties as a nanocarrier for pesticide delivery, is commercially available, and is currently approved as a herbicide by the US EPA. 4, 7-9。Previous studies have investigated covalent and electrostatic cargo loading strategies in TMGMV; model cargo molecules were explored and toxicity to nematodes was confirmed. 9 。Importantly, Applicants have shown here that TMGMV exhibits good soil mobility, which is important since nematodes parasitize the roots of crops. 4 Although successful, covalent and electrostatic loading were found to be rather inefficient; and pesticide loading was found to be a challenge even with extensive formulation chemistry. 10 。This previous research emphasizes that the economics of TMVGMV-based preparations need to be improved for them to be commercially viable candidates for agricultural nanotechnology. An important consideration for pesticides is that non-covalent strategies are preferred for nanoparticle formulation. This is because modifications to pesticides would initiate a new regulatory approval and registration process. Without being bound by theory, Applicants have explored here the thermal shape-switching properties 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 the pesticide cargo during this transition, Applicants developed a protocol for SNP formulations derived from TMGMV. The structural properties of the above SNPs were evaluated and their loading capabilities were evaluated by utilizing fluorophore (Cyanine 5) and nematicide (ivermectin). Applicants show here the soil mobility and efficacy against Caenorhabditis elegans (C. elegans) by treatment with ivermectin in the SNPs.

[0111] SNPs can be generated from high aspect ratio plant viruses, but the data indicate the conditions necessary for optimization for each system. 13-16To test whether SNPs derived from TMGMV can form, Applicants investigated the phase space for the rod-to-SNP transition (Figure 1). Applicants varied TMGMV concentration, 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 phase space for the rod-to-SNP transition at 5 mg mL -1 Thermal reshaping results for TMGMV at the initial concentration (remaining conditions in Figure 5) are shown. SNPs formed at all tested TMGMV concentrations, but required a minimum temperature and heating time to fully translocate. Below 80°C, SNPs did not form within 1 min of heating, instead forming bundles of aggregated rods (Figure 6). Above 90°C, SNPs formed after 30 s of heating. For temperatures and heating times below this threshold, bundles of aligned and aggregated TMGMV were observed (Figure 1C and Figure 6). At a threshold of 96°C for 30 s, SNPs formed and the diameter of the SNPs was proportional to the initial TMGMV concentration: at the lowest tested concentration of TMGMV (0.1 mg mL -1 ) resulted in particle sizes of approximately 100–200 nm in diameter, while the highest tested (5 and 10 mg mL -1 ) concentrations resulted in particles in the 500 nm to 2 μm range. The clumping and clustering observed in the cases with the smaller SNPs is a result of the SEM drying process, while the round features observed in these samples are not present in any of the controls.

[0112] Since the TMGMV SNPs have not been previously reported, the Applicant characterized their protein content by SDS-PAGE and LC-MS-MS. SDS-PAGE shows a distinction between the protein content of the TMGMV rods and their SNPs (Figure 7): TMGMV consists of approximately 2100 copies of the same coat protein (CP) with a molecular weight of 17.6 kDa. The SNPs are more dominant in the dimer CP (35 kDa), even under denaturing conditions, suggesting that complete disassembly was hindered. Additionally, smaller disassembly by-products (15 kDa) were observed with longer heating times. In the absence of protease or bacterial contamination, the cleaved protein sequences were unexpected. LC-MS-MS and sequence analysis identified this band as being derived from the TMGMV CP. Interestingly, this phenomenon has not been explained with respect to any transfer from rods to SNPs. 11-13 The aspartic acid-proline (DP) bond in proteins can be sensitive to cleavage at temperatures close to 100 °C and under acidic conditions. 17-19 Therefore, the Applicant analyzed the sequences of TMV and other rod-shaped viruses in which TMGMV and SNP transfer have been reported. Both TMV and TMGMV have DP at amino acid positions D20-P21, while PVX and AltMV do not have this motif. The length of the N-terminal sequence before the putative cleavage site is 2.1 kDa, correlating with the mass shift in SDS-PAGE. This effect has been reported to occur within a few minutes at longer heating times. Since the transfer from TMGMV to SNPs takes longer than in TMV, this phenomenon may occur in TMV but has not yet been observed.

[0113] The Applicant first assayed the soil mobility of the SNPs vs. TMGMV. TMGMV has previously been shown to have good soil mobility characteristics - superior performance compared to other synthetic and biological systems - which the Applicant attributed to the zwitterionic nature of the protein-based polymers. 4 The Applicant's soil mobility assay with SNPs 4The preceding experimental setup was reproduced. Briefly, a column with a 0.3 cm diameter was filled with Magic topsoil gardening soil (Michigan Peat) to a depth of 10 cm, pre-wetted, and then TMGMV or SNP was applied by bolus (0.5 mg virus nanoparticles in 1 mL DI H 2 2O, 5 mL aliquot -1 and perfused). Fractions were collected and analyzed for TMGMV CP by SDS-PAGE (Figures 2 and 8). Band analysis was performed to determine the elution profile, and the applicant determined that SNPs have good soil mobility independent of their size (Figure 2B). The size independence may be due to the fact that surface chemistry is the dominant factor with respect to soil mobility. It is interesting to note that for a soil depth of 10 cm, the above SNPs exhibit a longer elution volume (maximum at 8 - 10 mL elution volume) compared to TMGMV rod-shaped particles (maximum at 6 mL elution volume). This is likely due to their larger length scale and charge in the surface chemistry of SNPs compared to rods, promoting a greater interaction between the particles and the soil 20 . Approximately 90% lower overall signal intensity was observed for the cases of 1 mg mL -1 and 5 mg mL -1 (Figures 2D and 2E). This indicates that the above SNPs are retained more in the soil for these conditions compared to 0.1 mg mL -1 and 10 mg mL -1 .

[0114] Based on phase space, the applicant concluded that the most reliable transition from rods to SNPs occurs at 96 °C using a heating time of 60 seconds. Under these conditions, SNPs were formed reproducibly and uniform spherical shapes were observed by SEM. Thus, the applicant varied the protein concentration (0.1 mg mL -1 , 1.0 mg mL -1 , 5.0 mg mL -1 , and 10 mg mL -1) As a function of , the 60 s / 96 °C condition was selected for active ingredient encapsulation. In this protein concentration range, the SNPs formed ranged from 100 - 200 nm with respect to the lowest protein concentration to 500 nm - 2 μm with respect to the highest protein concentration. Two small molecule cargos were selected: Cy5 was used as a fluorescent model molecule and ivermectin was selected as an insecticide. Cyanine 5 (Cy5) was selected because it has been previously shown to retain fluorescence after heat treatment. 21 Selected as a photostable fluorophore. Loading of small molecules into SNPs derived from TMV and other rod-shaped viruses has been previously shown. 11, 12, 14, 16 . However, in these systems, association was achieved using chemical conjugation strategies both before and after SNP formation. These studies demonstrated the functional conjugation of fluorophores and other classes of cargos, but these covalent modification strategies rely on the modification of the active ingredient. In an agricultural environment, this is highly undesirable because changing the chemical structure of the active ingredient requires a long process by regulatory authorities and increases the cost in product development. Furthermore, many bioconjugation procedures are multi-step processes involving intermediate and final purification steps. The above processes can be time-consuming, reduce yields, and increase costs. A one-step synthesis process for active-laden SNPs can alleviate these technical barriers (outlined in Figure 3A).

[0115] Compared to TMGMV, in the range of mass ratios from 0.1-fold to 10-fold, at a fixed concentration of Cy5 (1 mg mL -1) was added during the thermal transfer from TMGMV to SNP (60 seconds, 96 °C). The resulting Cy5-SNP was then purified using a desalting column to remove excess Cy5. The purified sample was visibly concentrated with Cy5 (Figure 3B, inset). When Cy5-SNP was imaged by SEM, there were no apparent differences in the morphology of Cy5-SNP compared to unloaded SNP (Figure 3B). The synthesis produced fewer non-SNP aggregates. This may be due to the 5 volume % DMSO added to the synthesis via Cy5, which can assist in the dissociation and thermal conversion of SNP. This indicates that this non-covalent encapsulation strategy does not interfere with SNP formation. To quantify Cy5 encapsulation, the applicant analyzed the absorbance of the purified SNP at 647 nm. The applicant found a TMGMV concentration-dependent removal of Cy5 from the solution phase to the particle phase (1 mg mL -1 ). The removal efficiency ranged from 25% for 0.1 mg mL -1 TMGMV to nearly 100% for 10 mg mL -1 TMGMV (Figure 3D). The encapsulation efficiency was calculated and reported in Figure 3F. This indicates a high capacity with higher monovalent earth. There is still room to optimize the loading efficiency for each TMGMV concentration, but the applicant has shown that SNPs loaded with distinct sizes of cargo from 100 - 200 nm to 500 nm - 2 μm can be obtained in a one-step synthesis that does not require chemical modification of the cargo. For Cy5, 77 molecules per CP were loaded in 100 - 200 nm Cy5-SNP (0.1 mg mL -1 TMGMV). As the TMGMV concentration and particle size increased, the amount of Cy5 per CP tended to drop below 10 molecules per CP for the largest particles (500 nm - 2 μm), yet exceeded the maximum achieved via bioconjugation without optimization.

[0116] Next, ivermectin was encapsulated. Ivermectin has limited solubility in water and has previously been shown to be an inappropriate choice for chemical conjugation 22Therefore, the applicant tested the encapsulation during the transition from the rod to the SNP. When the applicant heated an aqueous solution containing ivermectin and TMGMV to trap its active ingredients into the SNP, the applicant observed the formation of a fluffy colloidal by-product that interfered with SNP formation (Figure 9). This by-product was formed regardless of the presence of TMGMV and was identified as an aggregate of ivermectin. To increase the solubility of ivermectin and enable SNP trapping, the applicant added 25% by volume of acetonitrile as a co-solvent. The use of the co-solvent actually enabled hydrophobic cargo encapsulation, greatly diversifying the types of cargo that the applicant could trap in the SNP. The co-solvent was then removed using precipitation, dialysis, or other buffer exchange techniques. The applicant observed properly formed IVN-SNPs of all sizes (Figure 3C and Figure 9).

[0117] The amount of ivermectin per SNP formulation was determined using competitive ELISA (Figure 3E) and a standard curve (Figure 10). The ivermectin loading per CP was relatively higher when using lower concentrations of TMGMV, consistent with the trend for Cy5 (Figure 3F). Up to about 50 ivermectin molecules per CP were detectable for SNPs with dimensions of 100 - 200 nm (0.1 mg mL -1 TMGMV). For smaller SNPs (100 - 200 nm), the predicted loading per SNP was 1 × 10 per SNP 6Ivermectin, or to the extent of 60% ivermectin by mass. To gain insight into the structural changes that occur during SNP formation using a co-solvent, circular dichroism (CD) spectra were analyzed for IVN-SNP compared to TMGMV (Figure 11). When comparing the TMGMV rod with the SNP, the data show a change in signal intensity of approximately 200 - 220 nm and a peak maximum shift from 270 nm to 260 nm after thermal transition. The shift associated with SNP formation deteriorates in the presence of acetonitrile as a co-solvent, while acetonitrile without heating shows little effect on the CD spectrum. Furthermore, the presence of ivermectin leaves a spectral fingerprint at 240 - 260 nm. TMV (a close homolog of TMGMV), which undergoes large changes in secondary structure during SNP formation, was similarly predicted to have these changes in TMGMV.

[0118] The effect of cargo loading on soil mobility was next evaluated. The applicant confirmed the soil mobility of the SNP loaded with the above cargo and found that the maximum elution was consistent with that of the empty SNP. This indicates that the above cargo does not affect soil mobility (Figure 3G, Figure 12). Importantly, the applicant confirmed that in a 10 cm soil column, the Cy5 signal in the eluent fully overlapped with the protein signal for Cy5-SNP (this was true for all sizes tested, 500 nm - 2 μm SNP). This indicates that non-covalent loading onto the SNP is robust and stable. This is in complete contrast to that regarding the TMGMV rod: here the applicant established a non-covalent loading strategy by utilizing electrostatic binding, which was stable under physiological conditions, but the applicant found that the cargo was detached from the particles in the soil. 7 。

[0119] As a final test, the Applicant delivered IVN-SNP to C. elegans (Figure 4). The nematodes were incubated in solution for 90 minutes with SNP, IVN-SNP, and free IVN. In parallel, the same (IVN-)SNP was passed through a soil column and collected before treating C. elegans in solution. For all samples, the Applicant performed a modified gel invasion assay (Figure 4A) by transferring the nematodes and placing them into Pluronic® F127 in a well plate. 23 . Its surface was covered with E. coli OP-50 lysate to attract the nematodes, and the number of C. elegans that advanced onto the surface was counted. Nematode motility was quantified based on their entire tracking application range using their edge detection (Figures 4B - 4D, Figure 13, Figure 14). Then, the motility was predicted using the following equation:

Number

[0120] The Applicant also noted that the largest SNPs (5 and 10 mg mL -1 at 500 - 2 μm) resulted in a >2-fold reduction in nematode surface velocity and a >2-fold reduction in the number of nematodes on the surface. The smallest SNP (0.1 mg mL-1 In (100 - 200 nm), a 2.35-fold reduction in nematode surface velocity and a 2.9-fold reduction in the number of nematodes on the surface were observed. Since the ivermectin concentrations in these experiments were normalized, the applicant hypothesizes that the smaller the SNP, the more efficiently it interacts with nematodes and / or the more accessible ivermectin is, and it may adsorb on the surface of the SNP (thus potentially causing burst release), either based on diffusion or a higher nanoparticle-to-nematode ratio.

[0121] The efficacy of the IVN-SNP formulation was approximately 35% lower than that of 10 μM soluble ivermectin for 500 nm - 2 μm IVN-SNP. However, in an agricultural environment, nematicides need to reach root-feeding nematodes, so a realistic comparison requires including the soil environment. The applicant added free ivermectin and IVN-SNP onto a 10 cm soil column, treated C. elegans, and collected fractions before performing a penetration assay. 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). 0.1 mg mL -1 and 1.0 mg mL -1 IVN-SNP formed in TMGMV caused a 1.36-fold and 1.72-fold reduction in surface velocity, and a 1.74-fold and 1.81-fold reduction in the number of nematodes. These data show a 2.25-fold (100 - 200 nm SNP) and 1.33-fold (500 nm - 1 μm) reduction in efficacy after soil application with 10 μM ivermectin. This reduction in efficacy was not observed for larger SNPs: 5.0 mg mL -1 and 10 mg mL -1 IVN-SNP at showed similar efficacy before and after soil treatment, and for 5 mg mL after passing through the soil column, there was a 2.14-fold reduction in surface velocity and a 2.25-fold reduction in the number of nematodes, and for 10 mg mL -1 there was a 2.14-fold reduction in surface velocity and a 2.25-fold reduction in the number of nematodes, and for 10 mg mL -1showed a 2.24-fold reduction in surface velocity and a 2.30-fold reduction in nematode numbers. From this data, the applicant concluded that SNP capture of the pesticide cargo enables soil mobility for effective treatment of nematodes.

[0122] In summary, SNP-based encapsulation was highly efficient for soil-based delivery of nematicides. The applicant detailed the phase space for SNP synthesis and showed that heating times longer than 30 seconds and temperatures higher than 90 °C result in SNP formation. The SNP size is protein concentration-dependent, and a size range of SNPs with diameters from 100 nm to 2 μm was synthesized. The applicant showed effective encapsulation of Cy5 and ivermectin into SNPs during the thermal transition from TMGMV to SNPs. Finding a medium that can be heated to >90 °C, such as acetonitrile, and maintain the solubility of the target molecule is important for effective encapsulation. Future applications may require more sustainable co-solvents. Acetonitrile likely enhanced the solubility of the active ingredient and improved SNP synthesis by perhaps assisting in the destabilization of TMGMV assembly. Importantly, the applicant showed efficient active ingredient loading and co-delivery in the soil to target root-feeding nematodes. Free ivermectin was not effective after soil passage, but IVN-SNPs promoted soil mobility and efficacy against C. elegans. The applicant anticipates that these technologies may find applications beyond pesticide delivery in the soil and may be extended to foliar delivery of active ingredients. TMGMV itself has already been approved as a bioherbicide by the EPA, and this non-covalent encapsulation may streamline the process for regulatory authorities. The use of a one-pot synthesis method is expected to result in an expandable industrial process.

[0123] Experiment

[0124] TMGMV Preparation

[0125] TMGMV from BioProdex (Gainesville, FL, USA) was prepared as previously described and stored at -20 °C until use (Chariou, P. L. et al., Nature Nanotechnology, 2019; Chariou, P. L. et al., ACS Agricultural Science & Technology, 2021; Chariou, P. L. et al., ChemBioChem, 2022; Gonzalez-Gamboa, I. et al., ChemBioChem, 2022). Briefly, the thawed TMGMV was dialyzed against potassium phosphate buffer (KP; 10 mM, pH 7.2) for 24 h at 4 °C using a 12–14 kDa dialysis tube (Fisher Scientific S432700, Waltham, MA, USA), and the process was repeated with fresh buffer over the next 48 h. The virus solution was centrifuged at 10,000×g for 20 min (Beckman Coulter Allegra centrifuge, Brea, CA, USA). The supernatant was collected and ultracentrifuged at 42,000 rpm for 2.5 h at 4 °C (Beckman Coulter Optima L-90k Ultracentrifuge with 50.2 Ti roter, Brea, CA, USA). The pellet was resuspended overnight in KP buffer at 4 °C. Its concentration was determined using a Nanodrop 2000 (Thermo Scientific, Waltham, MA, USA).

[0126] SNP preparation

[0127] The TMGMV solution was placed in a PCR tube in a volume of 100 μL of DI H2O at the desired concentration (0.1 mg mL -1 , 0.3 mg mL -1 , 0.5 mg mL -1 , 1.0 mg mL -1 , 5.0 mg mL -1 , or 10.0 mg mL -1) It was diluted. Using a thermocycler (MJ Research PTC200, Waltham, MA, USA), the run was programmed and heated for a duration (5 seconds, 10 seconds, 15 seconds, 20 seconds, 30 seconds, 60 seconds, and 120 seconds) fixed to the desired temperatures (60 °C, 70 °C, 80 °C, 90 °C, 96 °C, and 98 °C), and then cooled to 10 °C. The samples were stored at 4 °C during the measurement.

[0128] SEM

[0129] The particle preparation was dried on a silica wafer fixed to a stainless - steel mounting stud by carbon tape. The sample was dried overnight before iridium coating with a coating time of 12 seconds. The sample was imaged with a Zeiss 500 at 3 - 5 kV under vacuum.

[0130] SDS - PAGE

[0131] The denatured TMGMV SNP sample (10 μg) was loaded onto a 12% NuPAGE gel (Life Technologies, Carlsbad, CA, USA) and electrophoresed with 1×MOPS Running Buffer (Life Technologies, Carlsbad, CA, USA). The Cyanine 5 encapsulated in the SNP was visualized under UV light. Gel Code Blue dye (Life Technologies, Carlsbad, CA, USA) was used to stain the coat proteins, and they were visualized with a gel imager (ProteinSimple, Santa Clara, CA, USA) under white light.

[0132] Soil mobility assay

[0133] Magic Topsoil (Michigan Peat) was weighed and packed into a cylindrical column with a Miracloth attached to the bottom. There was a tapered outlet at the bottom of the cylinder for collecting the dripping fractions. The soil was wetted with DI H2O and left until the dripping stopped. An aliquot of 0.5 mg TMGMV or SNP was added to the column, and DI H2O was perfused using a syringe pump at a constant rate of 5 mL min -1 -1. The eluate was collected in 2 mL fractions and electrophoresed on SDS PAGE for analysis. The band intensity of the fractions on SDS PAGE was determined using ImageJ.

[0134] Encapsulation of low molecular weight in SNP

[0135] Solutions of low molecular weight were prepared at 5 mg mL -1 (Cy5 in DI H2O and ivermectin in acetonitrile). These solutions were diluted to the desired concentration of TMGMV in DI H2O (0.1 mg mL -1 , 0.3 mg mL -1 1.0 mg mL -1 5.0 mg mL -1 10 mg mL -1 final TMGMV concentration) in 1 mg mL -1 low molecular weight (20% acetonitrile v / v for ivermectin). The samples were then prepared in a thermocycler as described previously. Free / unencapsulated low molecular weight was removed by spin filtration at 5000 × g for 5 min (EMD Millipore UFC500324 3 kDa, Burlington, MA, USA), and the process was repeated twice. The retentate was diluted in DI H2O. The sample absorbance of the Cy5 samples was collected using a nanodrop. The concentration of Cy5 in the samples was determined using Lambert-Beer's law with the absorbance at 647 nm and a molar absorptivity of 230,400 cm-1 M-1.

[0136] Competitive ELISA for ivermectin quantification

[0137] Samples containing ivermectin were diluted according to the manufacturer's protocol using a High-Sensitivity Ivermectin ELISA kit (Creative Diagnostics DEIASL215, Shirley, NY, USA). Competitive ELISA for ivermectin was performed in the provided 96-well plates. The ivermectin-horseradish peroxidase conjugate in the sample wells was then reacted with the provided substrate for 30 minutes and then quenched with an equal volume of 1N HCl. Absorbance was measured at 450 nm with a plate reader (Tecan Infinite M Plex, San Jose, CA, USA), and the concentration was fitted to a standard curve of ivermectin.

[0138] Circular dichroism

[0139] CD spectra were collected using an Aviv model 215 CD spectrometer. Quartz cuvettes (Starna Cells, Atascadero, CA, USA) with a path length of 2 mm at 25 °C were used for each sample, and each measurement was collected twice. Sample concentrations between 0.025 mg / mL and 0.5 mg / mL were used to obtain a volume of 400 μL for each CD run. The near-ultraviolet and far-ultraviolet spectra were collected in two separate scans. For the far-ultraviolet spectrum, 250 nm to 180 nm was used with a wavelength step of 1 nm and an average time of 1 second. For the near-ultraviolet spectrum, 310 nm to 240 nm was scanned with a wavelength step size of 0.5 nm and an average time of 1 second.

[0140] C. elegans culture

[0141] The N2 strain of Caenorhabditis elegans was obtained from the Caenorhabditis Genetics Center (University of Minnesota, Minneapolis, MN, USA) and grown on solid Nematode Growth Medium (NGM) agar plates using OP-50 E. coli as the main food source. The animals were grown and maintained at 20 °C.

[0142] Treatment of C. elegans with ivermectin and IVN-SNP

[0143] A concentrated solution of C. elegans was aliquoted into microcentrifuge tubes at approximately 100 animals per tube. The animals were briefly centrifuged at 200×g for 30 seconds to form a pellet, the supernatant was removed, and replaced with a solution specific to the treatment group (e.g., with SNP or ivermectin). The C. elegans was incubated in these solutions for 90 minutes and then transferred to the submersion assay (Lesanpezeshki, L. et al., Scientific Reports 2019).

[0144] Pluronic®-based submersion assay

[0145] Using the previously reported optimized Pluronic® concentration of 26% w / w, the Pluronic® F127 solution was stored at 4 °C, and when the solute was dissolved, it remained soluble (Lesanpezeshki, L. et al., Scientific Reports 2019). The above Pluronic® F127 solution was maintained on ice while preparing the assay. 30 μL of Pluronic® F127 was added to a 6-well plate. A concentrated solution of treated C. elegans was placed to introduce approximately 100 animals. After approximately 10 minutes, a 2 mL layer of Pluronic® was poured on top. A solution of OP-50 E. coli lysate (20 μL with OD600 = 0.5) was added to the surface. After 2 hours, an 11×11 area scan at 4× magnification (Keyence BZ-X800 Fluorescence Microscope) was performed for each well. The number of nematodes on the surface was counted by hand, and the percentage of the area of their tracks was determined by edge detection and binary image conversion in ImageJ.

[0146] equivalent 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.

[0147] The technology described herein by way of example can be suitably practiced without any element that is not specifically disclosed herein, and without limitation. Thus, for example, terms such as "comprising", "including", "containing", etc. are to be read expansively and without limitation. Further, the terms and expressions employed herein are used as terms of description and not of limitation, and in using such terms and expressions there is no intention of excluding any equivalents of the features shown and described or portions thereof, but it is recognized that various modifications are possible within the scope of the technology claimed herein.

[0148] Accordingly, it should be understood that the materials, methods, and examples provided herein are representative of preferred aspects, are illustrative, and are not intended as limitations on the scope of the technology.

[0149] The technology is described herein broadly and generally. Narrower species and subgeneric inclusive groups that fall within the scope of the general disclosure also form part of the technology. This includes the general description of the technology with any provisos or negative limitations removing any subject matter from the genus, whether or not the excised material is specifically described herein.

[0150] Furthermore, when a feature or aspect of the technology is described in terms of a Markush group, one of ordinary skill in the art will recognize that the technology is also thereby described with respect to any individual member or sub-group of members of the Markush group.

[0151] All publications, patent applications, patents, and other references mentioned herein are hereby incorporated by reference in their entirety to the same extent as if each was individually incorporated by reference. In case of conflict, the present specification, including definitions, will control. References:

Table 3-1

Table 3-2

Table 3-3

Claims

1. Virus-like particles (VLPs) derived from tobacco mild green mosaic virus (TMGMV) or derivatives thereof conjugated to a drug if necessary.

2. The VLP according to claim 1, wherein the drug or its derivative is captured in the VLP or covalently bonded to the VLP.

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

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

5. The agent is loaded at a concentration of about 5 mg / mL －１ to about 10 mg / mL －１ of the VLP according to any one of claims 1 to 4.

6. The agent is loaded at a concentration of about 0.5 mg / mL －１ to about 0.2 mg / mL －１ The VLP according to any one of claims 1 to 4

7. The agent is loaded at a concentration of about 0.5 mg / mL －１ to about 0.2 mg / mL －１ of the VLP according to claim 3.

8. The agent is loaded at a concentration of about 0.5 mg / mL －１ to about 0.2 mg / mL －１ of the VLP according to claim 3.

9. The agent is loaded at a concentration of about 0.5 mg / mL －１ to about 0.2 mg / mL －１ The VLP according to claim 4, which is loaded at a concentration of

10. The agent is loaded at a concentration of about 0.5 mg / mL －１ to about 0.2 mg / mL －１ of the VLP according to claim 4.

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

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

13. The VLP according to claim 12, wherein the abamectin is selected from the group consisting of ivermectin, abamectin, doramectin, eprinomectin, moxidectin, or selamectin.

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

15. The VLP according to any one of claims 1 to 14, which is a plurality of VLPs that are the same or different from each other.

16. The drug according to claim 15, which is a plurality of VLPs that are the same or different from each other.

17. The TMGMV according to any one of claims 1 to 15, which is a plurality of VLPs that are the same or different from each other.

18. A composition comprising the VLP according to any one of claims 1 to 14, the plurality of VLPs according to 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, further comprising a preservative or a stabilizer.

20. The composition according to claim 18 or 19, which is lyophilized or frozen.

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

22. The method according to claim 21, wherein the administration is to soil, a supply, or the leaves, stems, flowers, or roots of a plant.

23. A kit comprising a VLP according to any one of claims 1 to 14, a plurality according to any one of claims 15 to 17, or a composition according to any one of claims 18 to 20, and an instruction manual.

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