Methods and compositions comprising tobacco mild green mosaic virus (TMGMV)

Abstract

This application relates in part to nanoparticles comprising tobamovirus, and to nanoparticles comprising tobamovirus and beta-cyclodextrin (β-CD or BCD). This application also relates in part to nanoparticles comprising tobamovirus and one or more active ingredients (AIs) non-covalently conjugated to the tobamovirus. This application also provides methods of making and using such nanoparticles, as well as compositions comprising the disclosed nanoparticles.

Description

Technical Field

[0001] Cross - Reference to Related Applications This application claims the benefit of U.S. Provisional Application No. 63 / 353,309, filed Jun. 17, 2022. The entire contents of the foregoing are hereby incorporated by reference in their entirety.

[0002] Federally Sponsored Research or Development This invention was made with government support under USDA awards 2020 - 67021 - 31255 and 2022 - 67012 - 36698, and NSF award DMR - 2011924. The government has certain rights in this invention.

Background Art

[0003] Pesticides are widely used in field food production. However, pesticides, and the methods used to apply them, are inefficient. Pesticides accumulate in the environment, on crops, and in drinking water. Pesticides are toxic to the environment and human health. It is important to develop better ways to apply pesticides. The widespread use of pesticides in agriculture causes these toxins to accumulate in crops, soil, as well as drinking water and groundwater, posing a great risk to ecosystems and human health. The first step towards a healthier society is to improve food security by improving quality and yield (i.e., more effective crop treatment) while protecting the environment and agro - ecosystems (i.e., preventing the leaching and accumulation of pesticides in the environment). Most pesticides are hydrophobic and thus do not have good soil mobility. This leads to over - use and, as a result, increases health and environmental problems. It is important to develop better ways to apply pesticides.

Summary of the Invention

[0004] This application is based in part on the surprising discovery that tobamovirus rods (e.g., Tobacco mild green mosaic virus (TMGMV)) can be used to load (also referred to throughout as encapsulate) target active ingredients (also called AIs, active substances), such as pesticides, drugs, and pharmaceuticals. Importantly, the compositions and methods described herein do not require any modification to any useful drug, pesticide, pharmaceutical, or compound. In part, this application is involved in non-covalent encapsulation or loading techniques for encapsulating pesticides and / or drugs within the nanoparticles described herein. Tobamovirus rods are an excellent platform for precision agriculture because they have excellent soil mobility, are described herein, and nanoparticles made using tobamovirus can have a soil distribution and / or soil mobility of up to over 30 cm. In some embodiments, the nanoparticles of the disclosure have a soil distribution and / or soil mobility of at least 5, 10, 15, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 21, 32, 33, 34, 35, 36, 37, 38, 39, or 40 cm.

[0005] Disclosed herein is, among other things, the use of a non-covalent encapsulation technique for encapsulating pesticides on nanoparticles including tobamovirus (tobacco mild green mosaic virus (TMGMV)). Tobamovirus nanoparticles have excellent soil mobility and thus are a good platform for precision agriculture. This application includes tobamovirus nanoparticles that use beta-cyclodextrin (also referred to throughout this disclosure as BCD, bCD, βCD, β-CD, etc.) as a cargo pocket, and tobamovirus nanoparticles capable of undergoing a structural transition that allows for the injection of molecules (e.g., AI) into the tobamovirus structure. In some embodiments, BCD is conjugated to the surface tobamovirus nanoparticles and, without being bound by theory, BCD functions as a pocket for loading cargos such as medical drugs and pesticides. In some embodiments, the structural transition that allows for the injection of molecules (e.g., AI) into the tobamovirus nanoparticles is caused by an external factor. In some embodiments, the external factor is to expose the tobamovirus nanoparticles to a pH change or a solvent (e.g., dimethyl sulfoxide or DMSO).

[0006] Although often shown as a rigid / solid structure, plant viruses including tobamovirus "breathe" in solution through carefully adjusting the pH, opening the structure to encapsulate at least one active ingredient or more than one active ingredient. Disclosed herein is, among other things, a method for causing a partial and reversible dissociation of one or more of its coat proteins in the tobamovirus nanoparticles by adjusting the pH or by contacting the tobamovirus nanoparticles with a solvent (e.g., DMSO). Such a method allows for the "breathing" or phase transition of the tobamovirus, which enables the injection of drug molecules or any AI into the tobamovirus structure. In the present disclosure, the AI can be one or more of pesticides and other drugs.

[0007] The disclosed nanoparticles have good soil mobility, and in some embodiments, the nanoparticles utilize two strategies for (1) β-CD as a cargo pocket for AI and (2) structural translocation for molecule and / or AI loading, using pesticides. In some embodiments, beta-cyclodextrin (β-CD) is conjugated to the surface of the tobamovirus nanoparticles, and without being bound by theory, β-CD functions as a pocket for loading cargo / AI such as medical drugs and pesticides.

[0008] Also, without being bound by theory, although often presented as a rigid / solid structure, plant viruses, including tobamovirus, "breathe" in solution by carefully adjusting the pH, opening the structure to encapsulate one or more AIs. As described herein, a method of breathing for tobamovirus has been developed that allows the tobamovirus to undergo a structural translocation and inject drug molecules into the structure. As described herein, the breathing method can be used to encapsulate or inject multiple AIs, including pesticides and other drugs, into the tobamovirus nanoparticles. In some embodiments, the compositions and methods described herein utilize the supramolecular interaction between β-cyclodextrin and the target AI to formulate multifunctional nanoparticles for delivery applications.

[0009] Β-cyclodextrin is a naturally occurring toroidal-shaped cyclic oligosaccharide. It has a hydrophilic outer surface and a hydrophobic internal cavity capable of accommodating a wide range of guest molecules. Furthermore, it is the most widely used host system in supramolecular chemistry, and is low-cost, with good water solubility and biocompatible properties. Without being bound by theory, the principle is to use a supramolecular strategy based on the interaction between β-CD and the target A.I. (e.g., pesticide). The Β-CD units are grafted onto the outer surface of the tobamovirus using an optimized bioconjugation reaction to capture one or more target AIs for efficient delivery to the soil.

[0010] In some embodiments, the nanoparticles described herein trap the pesticide in the tobamovirus by a pH change that traps the AI through the formation of "pockets" or "pores" between the CPs.

[0011] The principle is that by increasing the pH of the buffer or by the presence of a solvent (e.g., DMSO), the virus begins to dissociate and hydrophobic pockets or pores are created between the coat proteins of the virion. In an exemplary method, the AI is then added to interact with the virus particles, and then the pH is decreased to promote self - organization of the particles and entrapment of the AI on the hydrophobic pockets or pores. In yet another exemplary method, after adding a solvent (e.g., DMSO), the AI is added to interact with the virus particles to promote entrapment of the AI on the hydrophobic pockets or pores.

[0012] Certain aspects of the present disclosure are directed to nanoparticles comprising a tobamovirus and one or more active ingredients (AIs) non - covalently conjugated to the tobamovirus, wherein the tobamovirus comprises one or more coat proteins that reversibly and partially dissociate in response to an external factor.

[0013] In some embodiments, the one or more coat proteins reversibly and partially dissociate to form one or more pores. In some embodiments, one or more AIs are non - covalently conjugated to and entrapped within one or more pores of the tobamovirus. In some embodiments, one or more AIs are intercalated into one or more coat proteins of the tobamovirus. In some embodiments, one or more AIs are not chemically altered. In some embodiments, the external factor is a change in pH. In some embodiments, the external factor is the presence of a solvent. In some embodiments, the solvent is a polar aprotic solvent. In some embodiments, the solvent is a polar aprotic solvent that is miscible with water. In some embodiments, the polar aprotic solvent is dimethyl sulfoxide (DMSO). In some embodiments, the TM tobamovirus GMV is rod - shaped.

[0014] In some embodiments, described herein are nanoparticles comprising a tobamovirus and beta-cyclodextrin (βCD). In some embodiments, the nanoparticles further comprise an R group between the tobamovirus and the βCD. In some embodiments, the tobamovirus and the βCD are covalently linked. In some embodiments, the tobamovirus and the βCD are linked by an R group. In some embodiments, the R group is an alkyl, alkene, alkyne, ester, or other carbon-containing compound. In some embodiments, the R group is ethyne.

[0015] In some embodiments, the tobamovirus-AI nanoparticles have a width broader than that of the reference tobamovirus. In some embodiments, the reference tobamovirus molecules are processed under the same conditions as the tobamovirus-AI nanoparticles without adding AI. In some embodiments, the present application relates to nanoparticles comprising a tobamovirus and one or more active ingredients (AI), wherein the width of the tobamovirus-AI nanoparticles is broader than a reference value. In some embodiments, the reference value is the width of the tobamovirus molecules processed under the same conditions without adding AI. In some embodiments, the reference value is 15, 16, 17, or 18 nm. In some embodiments, the width of the tobamovirus-AI nanoparticles is 2% to 105% broader than the reference value. In some embodiments, the width of the tobamovirus-AI nanoparticles is about 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, 50, 51, 52, 53, 54, 55, 56, 57, 58, 59, 60, 61, 62, 63, 64, 65, 66, 67, 68, 69, 70, 71, 72, 73, 74, 75, 76, 77, 78, 79, 80, 81, 82, 83, 84, 85, 86, 87, 88, 89, 90, 91, 92, 93, 94, 95, 96, 97, 98, 99, 100, 101, 102, 103, 104, or 105% broader than the reference value. In some embodiments, the width of the tobamovirus-AI nanoparticles is 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, 50, 51. In some embodiments, the one or more AI comprise one or more of a drug, a pesticide, or a small molecule.In some embodiments, the agrochemical is a water-insoluble organic compound, an insecticide, a herbicide, a fungicide, a miticide, an algicide, an antibacterial agent, a biopesticide, a biocide, a disinfectant, a fumigant, an insect growth regulator, a plant growth regulator, an acaricide, a microbial pesticide, a molluscicide, a nematicide, an ovicide, a pheromone, a repellent, a rodenticide, a defoliant, a desiccant, a phytotoxicity reducer, or any combination thereof.In some embodiments, the pesticides are benzoyl ureas such as novaluron, lufenuron, chlorfluazuron, flufenoxuron, hexaflumuron, noviflumuron, teflubenzuron, triflumuron, and diflubenzuron; carbamates; pyrethroids such as cyhalothrin, and its isomers and isomer mixtures, lambda-cyhalothrin, deltamethrin, tau-fluvalinate, cyfluthrin, beta-cyfluthrin, tefluthrin, and bifenthrin; organophosphates such as azinfos-methyl, chlorpyrifos, diazinon, endosulfan, methidathion; neonicotinoids; phenylpyrazoles such as imidacloprid, acetamiprid, thiacloprid, dinotefuran, thiamethoxam, and fipronil; triazoles such as epoxiconazole, hexaconazole, propiconazole, prochloraz, imazalil, triadimenol, difenoconazole, microbutanil, prothioconazole, triticonazole, and tebuconazole; morpholines such as dimethomorph, fenpropidin, and fenpropimorph; strobilurins such as azoxystrobin, kresoxim-methyl, and their analogs; phthalonitriles such as chlorothalonil; mancozeb; fluazinam; pyrimidines such as bupirimate; aryloxyphenoxy derivatives; aryl ureas; aryl carboxylic acids; aryloxyalkanoic acid derivatives such as clodinafop-propargyl and its analogs, fenoxaprop-p-ethyl and its analogs, propaquizafop, quizalafop and its analogs; dintroanilines such as pendimethalin and trifluralin; diphenyl ethers such as oxyfluorfen; imidazolinones; sulfonyl ureas such as chlorosulfuron, nicosulfuron, rimsulfuron, tribenuron-methyl; sulfonamides; triazines; and triazinones such as metamitron.

[0016] In some embodiments, at least one AI comprises at least one of a drug, a pesticide, or a small molecule. In some embodiments, the drug can be a chemokine, an antibacterial agent, or any therapeutic compound. In some embodiments, the drug is a chemotherapeutic agent, an antiparasitic agent, an antibiotic, or an immunomodulatory agent. In some embodiments, the drug is a hydrophilic drug or a hydrophobic drug. In some embodiments, the tobamovirus is Tobacco mild green mosaic virus (TMGMV). In some embodiments, the tobamovirus is Tobacco mosaic virus (TMV).

[0017] In some embodiments, the nanoparticles comprise from about 1 to about 1500 AI molecules per tobamovirus.

[0018] Also described herein is a composition comprising any of the disclosed nanoparticles. In some embodiments, any of the nanoparticles or compositions described herein have a soil distribution and / or soil mobility of at least 5, 10, 15, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 21, 32, 33, 34, 35, 36, 37, 38, 39, or 40 cm. In some embodiments, the composition further comprises an excipient. In some embodiments, the excipient is a buffer or water.

[0019] Disclosed herein is, in certain embodiments, a method of making nanoparticles comprising a tobamovirus and βCD, the method comprising providing an isolated tobamovirus, coupling βCD to the tobamovirus thereby creating nanoparticles, and purifying the nanoparticles.

[0020] In some embodiments, the coupling step comprises forming a covalent bond between βCD, a linker, and tobacco mosaic virus. In some embodiments, the coupling step comprises a diazonium coupling reaction. In some embodiments of any of the compositions or methods described herein, the tobacco mosaic virus is modified or inactivated. In some embodiments, the linker is an R group. In some embodiments, the R group is an alkyl, alkene, alkyne, ester, or other carbon-containing compound. In some embodiments, the R group is ethyne.

[0021] Also disclosed herein is a method for producing nanoparticles comprising, in certain embodiments, tobacco mosaic virus and one or more active ingredients (AIs), the method comprising providing a buffer having a pH of about 7-9 to the isolated tobacco mosaic virus, adding the one or more AIs to the tobacco mosaic virus two or more times, thereby creating nanoparticles, and purifying the nanoparticles in a solution having a pH of about 5-9, wherein the one or more AIs are non-covalently conjugated to the tobacco mosaic virus and the tobacco mosaic virus comprises one or more coat proteins that dissociate reversibly and partially in response to a change in pH.

[0022] In some embodiments, one or more AIs are added for at least 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, or 21 days. In some embodiments, the buffer has a pH of about 7 - 7.5, 7.5 - 8, 7 - 8, 8 - 8.5, 8.5 - 9, or 8 - 9. In some embodiments, the pH of the buffer is such that the compound is 7.2 - 7.8, 7.3 - 7.8, 7.2 - 7.7, 7.3 - 7.7, 7.4 - 7.8, 7.4 - 7.7, 7.5 - 7.7, 7.5 - 7.8, 7.2 - 7.6, 7.3 - 7.6, 7.4 - 7.6, 7.5 - 7.6, 7.2 - 7.5, 7.3 - 7.5, 7.4 - 7.5, 7.2 - 7.9, 7.3 - 7.9, 7.4 - 7.9, 7.5 - 7.9, 7.3 - 7.99, 7.4 - 7.99, or 7.5 - 7.99, or the buffer has a pH of about 7.2, 7.3, 7.4, 7.5, 7., 7.7, 7.8, 7.9, or 7.99. In some embodiments, the solution has a pH of about 6.9, 7.0, 7.1, 7.2, or 7.3. 1. In some embodiments, the change in pH is about 0.5 - 1, about 0.5 - 2, 0.5 - 3, 1 - 2, or 1 - 3.

[0023] Disclosed herein is, in certain embodiments, a method of making nanoparticles comprising a tobamovirus and one or more active ingredients (AIs), the method comprising providing an isolated tobamovirus to a buffer having a pH of about 5 - 9 to create a tobamovirus - buffer, adding a solvent at a concentration of about 15% (v / v) - about 25% (v / v), adding one or more AIs to the tobamovirus - buffer, thereby creating nanoparticles, and purifying the nanoparticles in a solution having a pH of about 5 - 9, wherein the one or more AIs are non - covalently conjugated to the tobamovirus and the tobamovirus comprises one or more coat proteins that dissociate reversibly and partially in response to the presence of the solvent.

[0024] In some embodiments, the solvent is added dropwise. In some embodiments, one or more AIs are added dropwise. In some embodiments, one or more AIs are added stepwise over a period of time. In some embodiments, the period is from about 0.5 hours to about 10 days. In some embodiments, one or more AIs are added once a day. In some embodiments, the method further comprises incubating one or more AIs in a tobamovirus-buffer for about 4 hours to about 24 hours. In some embodiments, the solvent is a polar aprotic solvent. In some embodiments, the polar aprotic solvent is dimethyl sulfoxide (DMSO). In some embodiments, one or more coat proteins dissociate reversibly and partially to form one or more pores.

[0025] In some embodiments, one or more AIs are added repeatedly. In some embodiments, one or more AIs are added two or more times to the tobamovirus-buffer. In some embodiments, one or more AIs are added at least once a day. In some embodiments, one or more AIs are added until an equivalent ratio of about 10:1, 25:1, 50:1, 75:1, 100:1, 150:1, 200:1, 250:1, 300:1, 350:1, 400:1, 450:1, 500:1, 550:1, 600:1, 650:1, 700:1, 750:1, 800:1, 850:1, 900:1, 950:1, or 1000:1 is reached, or one or more AIs are added in a 1000, 1500, 2000, 2500, 3000, 3300, 4000, 4500, 5000, 5500, 6500, 7000, 7500, 8000, 8500, 9000, or 9500-fold molar excess relative to the tobamovirus, or 100, 150, 200, 250, 300, 350, 400, 450, or 500 nmol of one or more AIs per gram of the tobamovirus are added.

[0026] In some embodiments, one or more AIs are non-covalently conjugated to and confined within one or more pores of the tobamovirus. In some embodiments, one or more AIs are intercalated into one or more coat proteins of the tobamovirus. In some embodiments, one or more AIs are not chemically altered. In some embodiments, the tobamovirus is rod-shaped.

[0027] In some embodiments, the nanoparticles have a width greater than the width of the reference tobamovirus. In some embodiments, the reference tobamovirus molecules are treated under the same conditions as the tobamovirus-AI nanoparticles without the addition of AI. In some embodiments, the reference value is 15, 16, 17, or 18 nm. In some embodiments, the width of the nanoparticles is 2% to 105% greater than the reference value, or the width of the nanoparticles is about 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, 50, 51, 52, 53, 54, 55, 56, 57, 58, 59, 60, 61, 62, 63, 64, 65, 66, 67, 68, 69, 70, 71, 72, 73, 74, 75, 76, 77, 78, 79, 80, 81, 82, 83, 84, 85, 86, 87, 88, 89, 90, 91, 92, 93, 9, 95, 96, 97, 98, 99, 100, 101, 102, 103, 104, or 105% greater than the reference value. In some embodiments, the width of the nanoparticles is 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, 50, 51, 52, 53, 54, 55, 56, 57, 58, 59, 60, 61, 62, 63, 64, 65, 66, 67, 68, 69, 70, 71, 72, 73, 74, or 75 nm.

[0028] In some embodiments, one or more AIs include one or more of a drug, a pesticide, or a small molecule. In some embodiments, the pesticide is a water-insoluble organic compound, an insecticide, a herbicide, a fungicide, a miticide, an algicide, an antibacterial agent, a biopesticide, a biocide, a disinfectant, a fumigant, an insect growth regulator, a plant growth regulator, a miticide, a microbial pesticide, a molluscicide, a nematicide, an ovicide, a pheromone, a repellent, a rodenticide, a defoliant, a desiccant, a phytotoxicity reducer, or any combination thereof.In some embodiments, the pesticides are benzoyl ureas such as novaluron, lufenuron, chlorfluazuron, flufenoxuron, hexaflumuron, noviflumuron, teflubenzuron, triflumuron, and diflubenzuron; carbamates; pyrethroids such as cyhalothrin, and its isomers and isomer mixtures, lambda-cyhalothrin, deltamethrin, tau-fluvalinate, cyfluthrin, beta-cyfluthrin, tefluthrin, and bifenthrin; organophosphates such as azinphos-methyl, chlorpyrifos, diazinon, endosulfan, methidathion; neonicotinoids; phenylpyrazoles such as imidacloprid, acetamiprid, thiacloprid, dinotefuran, thiamethoxam, and fipronil; triazoles such as epoxiconazole, hexaconazole, propiconazole, prochloraz, imazalil, triadimenol, difenoconazole, microbutanil, prothioconazole, triticonazole, and tebuconazole; morpholines such as dimethomorph, fenpropidin, and fenpropimorph; strobilurins such as azoxystrobin, kresoxim-methyl, and their analogs; phthalonitriles such as chlorothalonil; mancozeb; fluazinam; pyrimidines such as bupirimate; aryloxyphenoxy derivatives; aryl ureas; aryl carboxylic acids; aryloxyalkanoic acid derivatives such as clodinafop-propargyl and its analogs, fenoxaprop-p-ethyl and its analogs, propaquizafop, quizalofop and its analogs; dinitroanilines such as pendimethalin and trifluralin; diphenyl ethers such as oxyfluorfen; imidazolinones; sulfonyl ureas such as chlorosulfuron, nicosulfuron, rimsulfuron, tribenuron-methyl; sulfonamides; triazines; and triazinones such as metamitron.

[0029] In some embodiments, the drug is a chemotherapeutic agent, an anti-parasitic agent, an antibiotic, or an immunomodulatory agent. In some embodiments, the drug is a hydrophilic drug or a hydrophobic drug. In some embodiments, the nanoparticles contain from about 1 to about 1500 AI molecules per tobacco mosaic virus. In some embodiments, the tobacco mosaic virus is the tobacco mild green mosaic virus (TMGMV). In some embodiments, the tobacco mosaic virus is the tobacco mosaic virus (TMV).

[0030] Also described herein is a method comprising administering any of the nanoparticles or compositions described herein to soil, a crop, or a plant, wherein the nanoparticles or composition are administered in an effective amount.

[0031] Also described herein is a pharmaceutical composition comprising any of the nanoparticles of the present disclosure.

[0032] In some embodiments, the pharmaceutical composition further comprises at least one pharmaceutically acceptable carrier, diluent, or excipient. In some embodiments, the pharmaceutical composition is formulated into a dosage form that is an injectable solution, a lyophilized powder, a suspension, or any combination thereof.

[0033] Also described herein is a method of doing so in a subject in need of treating cancer, the method comprising administering to the subject in need of treating cancer the nanoparticles of the present disclosure or the pharmaceutical composition of the present disclosure, wherein the nanoparticles or composition are administered in an effective amount.

[0034] In some embodiments, the cancer includes breast cancer, ovarian cancer, glioma, gastrointestinal cancer, prostate cancer, cancer, lung cancer, hepatocellular cancer, testicular cancer, cervical cancer, endometrial cancer, bladder cancer, head and neck cancer, lung cancer, gastro-esophageal cancer, gynecological cancer, or any combination thereof.

[0035] Also described herein is a method of doing so in a subject in need of treating an infection, the method comprising administering to the subject in need of treating an infection a nanoparticle of the present disclosure or a pharmaceutical composition of the present disclosure, wherein the nanoparticle or composition is administered in an effective amount.

[0036] In some embodiments, the infection is a bacterial infection, a viral infection, a fungal infection, a parasitic infection, or any combination thereof.

[0037] Furthermore, the present disclosure also relates to a method of combating harmful insects and / or phytopathogenic fungi, the method comprising contacting a plant, soil, or plant habitat in which harmful insects and / or phytopathogenic fungi are growing or can grow, or a plant or soil protected from attack or spread by said harmful insects and / or phytopathogenic fungi, with an effective amount of a formulation according to the present disclosure. Accordingly, the formulations according to the present disclosure can be used for the control of a number of phytopathogenic fungi or insects against various cultivated plants or weeds such as wheat, rye, barley, oats, rice, corn, grass, banana, cotton, soybean, coffee, sugarcane, vine, fruit, and ornamental plants, as well as vegetables such as cucumber, bean, tomato, potato, and cucumber.

[0038] The present disclosure also relates to a method of controlling unwanted vegetation, the method comprising enabling a herbicidally effective amount of a formulation according to the present disclosure to act on plants and their habitats. Control of unwanted vegetation is understood to mean the destruction of weeds. Weeds are understood to mean, in the broadest sense, all plants growing in an unwanted location.

[0039] Accordingly, the formulations and compositions according to the present disclosure can be used for the control of a number of phytopathogenic fungi or insects against various cultivated plants or weeds such as wheat, rye, barley, oats, rice, corn, grass, banana, cotton, soybean, coffee, sugarcane, vine, fruit, and ornamental plants, as well as vegetables such as cucumber, bean, tomato, potato, and cucumber, and the seeds of these plants.

[0040] Therefore, the formulations according to the present disclosure and the compositions according to the present disclosure are suitable for controlling common harmful plants in useful plants, especially crops such as oats, barley, millet, corn, rice, wheat, sugarcane, cotton, rapeseed, flax, lentils, sugar beets, tobacco, sunflowers, and soybeans, or perennial crops.

[0041] The embodiments disclosed below include nanoparticles comprising a tobamovirus and one or more active ingredients (AIs) non-covalently conjugated to the tobamovirus, compositions containing these nanoparticles, methods of using these nanoparticles, and methods of preparing these therapeutic nanoparticles. Some embodiments of the nanoparticles, compositions, and methods described herein can provide one or more of the following advantages.

[0042] First, certain embodiments of the present disclosure include nanoparticles and compositions that can be prepared in an efficient and cost-effective manner. Covalent conjugation is often used in the preparation of pesticides and nanoparticles. However, covalent conjugation is a complex and resource-intensive process, and pesticides are difficult to conjugate to proteins considering their high hydrophobicity. Furthermore, the costs and regulatory processes associated with using covalent conjugation strategies for pesticides linked to nanoparticles (including the classification, characterization, and approval of covalently modified chemicals) may exceed the benefits of agricultural use. Therefore, an efficient non-covalent method for loading nanoparticles is desired. The nanoparticles and compositions of the present disclosure address this need by including AIs that can be efficiently loaded or injected into tobamovirus nanoparticles without the need for chemical modification or covalent conjugation.

[0043] Second, certain embodiments of the present disclosure include nanoparticles and compositions that can be used in a variety of applications depending on which AI is selected and loaded onto the tobamovirus nanoparticles. For example, in some embodiments, the nanoparticles and compositions of the present disclosure can be used to treat diseases, to combat harmful insects and / or phytopathogenic fungi, and to control unwanted vegetation in a subject in need thereof.

[0044] Third, certain embodiments of the present disclosure include nanoparticles that can have a high AI loading efficiency. For example, in some embodiments, the nanoparticles and compositions of the present disclosure can load about 1100 or more AI molecules per virion.

[0045] Unless otherwise expressly stated, 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 invention belongs. Methods and materials for use in the present invention are described herein, and other suitable methods and materials known in the art may also be used. The materials, methods, and examples are illustrative only and not intended to be limiting. All publications, patent applications, patents, sequences, database entries, and other references mentioned herein are incorporated by reference in their entirety. In case of conflict, the present specification, including definitions, will control.

[0046] Other features and advantages of the present invention will be apparent from the following detailed description, drawings, and claims. BRIEF DESCRIPTION OF THE DRAWINGS

[0047]

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Mode for Carrying Out the Invention

[0048] All technical and scientific terms used in this specification are intended to have the same meaning as commonly understood by one of ordinary skill in the art, unless otherwise defined herein below. References to techniques used herein are intended to refer to techniques commonly understood in the art that include variations of those techniques and / or alternatives to equivalent techniques that would be apparent to one of ordinary skill in the art.

[0049] As used herein, the singular forms "a", "an", and "the" include plural referents unless the context clearly dictates otherwise.

[0050] As used herein, the terms "about" and "approximately" are used to modify a numerical value or an amount specified in a range, and when so used, a numerical value, as well as reasonable deviations from the known values by one of ordinary skill in the art, e.g., ±20%, ±15%, ±10%, ±5%, ±4%, ±3%, ±2%, or ±1% are within the intended meaning of the recited value.

[0051] The term "nanoparticle" means a substance having a length of about 2 nm to about 300 nm (e.g., about 2 nm to 100 nm, 2 nm to 200 nm, 2 nm to 250 nm, 2 nm to 300 nm, 100 nm to 200 nm, 100 nm to 250 nm, 100 nm to 300 nm, 150 nm to 250 nm, 200 nm to 300 nm, 200 nm to 250 nm). Non-limiting examples of nanoparticles include the nanoparticles described herein.

[0052] The term "subject" or "patient", as used herein, refers to any mammal (e.g., a human or veterinary subject, e.g., a dog, cat, horse, cow, goat, sheep, mouse, rat, or rabbit) to which the compositions or methods of the present disclosure can be administered, for example, for experimental, diagnostic, prophylactic, and / or therapeutic purposes. A subject may be seeking or in need of treatment, may be in need of treatment, may be receiving treatment, will receive treatment, or is under the care of a trained professional for a particular disease or condition.

[0053] The term "chemotherapeutic agent" means a molecule that can be used to reduce the growth rate of cancer cells or induce or mediate the death of cancer cells (e.g., necrosis or apoptosis) in a subject (e.g., a human). In non-limiting examples, a chemotherapeutic agent can be a small molecule, a protein (e.g., an antibody, an antigen-binding fragment of an antibody, or a derivative or conjugate thereof), a nucleic acid, or any combination thereof. Non-limiting examples of chemotherapeutic agents include cyclophosphamide, mechlorethamine, chlorambucil, melphalan, daunorubicin, doxorubicin, epirubicin, idarubicin, mitoxantrone, valrubicin, paclitaxel, docetaxel, etoposide, teniposide, talaporoside, azacitidine, azathioprine, capecitabine, cytarabine, doxifluridine, fluorouracil, gemcitabine, mercaptopurine, methotrexate, thioguanine, bleomycin, carboplatin, cisplatin, oxaliplatin, all-trans retinoic acid, vinblastine, vincristine, vindesine, vinorelbine, and bevacizumab (or an antigen-binding fragment thereof). Additional examples of chemotherapeutic agents are known in the art.

[0054] As described herein, the term "effective amount" means an amount that produces a desired result. Desired results in the context of this description can include the reduction of an undesirable characteristic (e.g., reduction of an undesirable organism, reduction of an undesirable plant, etc.).

[0055] This document provides nanoparticles, compositions, methods of making, and methods of applying tobamoviruses engineered for the purposes of drug and pesticide delivery. The nanoparticles, compositions, methods of making, and methods of use described herein can include any species of tobamovirus. For example, the nanoparticles, compositions, methods of making, and methods of use described herein can include Tobacco mild green mosaic virus (TMGMV). In another example, the nanoparticles, compositions, methods of making, and methods of use described herein can include Tobacco mosaic virus (TMV). In some embodiments, the nanoparticles, compositions, methods of making, and methods of use described herein include Pepper mild mottle virus (BPeMV), Brugmansia mild mottle virus, Cactus mild mottle virus (CMMoV), Crinodendron yellow vein virus, Cucumber fruit mottle mosaic virus, Cucumber green mottle mosaic virus (CGMMV), Cucumber mosaic virus, Frangipani mosaic virus (FrMV), Hibiscus latent Singapore virus (HLFPV), Hibiscus latent Singapore virus (HLSV), Cucumber green mottle mosaic virus, Maracuja mosaic virus (MarMV), Obuda pepper virus (ObPV), Odontoglossum ringspot virus (ORSV), Opuntia chlorotic ringspot virus, Pepper mild mottle virus, Passion fruit mosaic virus, Pepper mild mottle virus (PMMoV), Plumeria mosaic virus, Rattail cactus necrosis-associated virus (RCNaV), Acalypha mosaic virus, Ribgrass mosaic virus (HRV), Sammons’s Opuntia virus (SOV), Streptocarpus flower breakvirus), sunn-hemp mosaic virus (SHMV), tobacco latent virus, tomato brown rugose fruit virus (ToBRFV), tomato mosaic virus (ToMV), tomato mottle mosaic virus, tropical soda apple mosaic virus, turnip vein-clearing virus (TVCV), ullucus mild mosaic virus, wasabi mosaic virus (WMoV), yellowtail flower mild mottle virus, youcai mosaic virus (YoMV) also known as oilseed rape mosaic virus (ORMV), zucchini green mottle mosaic virus, or any combination thereof.

[0056] Tobamovirus is a genus of plus-strand RNA viruses in the family Virgaviridae. TMGMV and TMV are members of the Tobamovirus consisting of rod-shaped RNA viruses that are strictly plant pathogens.

[0057] TMGMV and TMV each have 2,130 identical coat proteins arranged helically around a single-stranded RNA genome, forming a hollow rigid rod measuring 300 × 18 nm with an internal channel of 4 nm. The outer surface of the coat protein is characterized by tyrosine side chains (Tyr2 and Tyr139) that are exposed to two solvents and can be functionalized using a diazonium coupling reaction. In some embodiments, the tobamovirus (e.g., TMGMV or TMV) is coupled to a carrier such as beta-cyclodextrin (BCD).

[0058] In some embodiments, the present disclosure is directed to a “breathing” method of tobamoviruses (e.g., TMGMV or TMV) based on careful pH adjustment (sometimes referred to herein as the “pH method”) or solvent concentration (sometimes referred to herein as the “solvent method”), and loaded with molecules such as, but not limited to, fluopyram, clothianidin, rifampicin, and ivermectin (see, e.g., FIGS. 17-24). As shown in FIGS. 26A-26D, doxorubicin and cyanine 5 (Cy5) are used as model active ingredients, and the fluorescence of doxorubicin and Cy5 provides a convenient means for characterization. Nanoparticle formulations and tobamovirus (e.g., TMGMV or TMV) structures prepared via the pH and solvent methods were characterized by a combination of techniques to determine particle integrity, AI injection, and secondary structure stability after injection, as described in Examples 5-10.

[0059] Nanoparticle In some embodiments, the nanoparticles of the present disclosure are viral nanoparticles. In some embodiments, the viral nanoparticles are tobamoviruses. In some embodiments, the viral nanoparticles are tobacco mosaic virus (TMV). In some embodiments, the viral nanoparticles are tobacco mild green mosaic virus (TMGMV). In some embodiments, the viral nanoparticles are one or more species of the genus Tobamovirus. In some embodiments, the viral nanoparticles are, for example, pepper mild mottle virus (BPeMV), Brugmansia mild mottle virus, cactus mild mottle virus (CMMoV), crinivirus yellow vein virus, cucumber fruit mottle mosaic virus, cucumber green mottle mosaic virus (CGMMV), cucumber mottle virus, frangipani mosaic virus (FrMV), hibiscus latent Singapore virus (HLSV), hibiscus latent Singapore virus, malvastrum mosaic virus (MarMV), odontoglossum ringspot virus (ORSV), opuntia chlorotic ringspot virus, paprika mild mottle virus, passion fruit mosaic virus, pepper mild mottle virus (PMMoV), plumeria mosaic virus, rat-tail cactus necrosis-associated virus (RCNaV), sida mosaic virus, ribgrass mosaic virus (HRV), solanum open leaf virus (SOV), streptocarpus spotted virus, sunn-hemp mosaic virus (SHMV), tobacco latent virus, tomato brown rugose fruit virus (ToBRFV), tomato mosaic virus (ToMV), tomato mottle mosaic virus, tropical soda apple mosaic virus, turnip vein clearing virus (TVCV), uruguay mild mottle virus, wasabi mottle virus (WMoV), yellowtail flower mild mottle virus, rape mosaic virus (YoMV) also known as turnip mosaic virus (ORMV), zucchini green mottle mosaic virus, or any combination thereof.

[0060] In some embodiments, the tobamovirus (e.g., TMGMV or TMV) is an engineered tobamovirus (e.g., TMGMV or TMV). In some embodiments, the engineered tobamovirus (e.g., TMGMV or TMV) is conjugated or linked to beta-cyclodextrin. In some embodiments, the beta-cyclodextrin is located on the outer surface of the tobamovirus (e.g., TMGMV or TMV). In some embodiments, the beta-cyclodextrin interacts with the active ingredient through supramolecular interactions. In some embodiments, the beta-cyclodextrin interacts with the active ingredient (AI) (e.g., pesticide) through hydrophobic and / or hydrophilic interactions.

[0061] In some embodiments, the engineered tobamovirus (e.g., TMGMV or TMV) is modified to be injected with, impregnated with, or contain an active ingredient (AI) (e.g., the tobamovirus (e.g., TMGMV or TMV) is modified to “breathe” in the AI). In some embodiments, the tobamovirus (e.g., TMGMV or TMV) dissociates partially and reversibly to allow incorporation of the AI into the tobamovirus (e.g., TMGMV or TMV) structure. In some embodiments, one or more coat proteins of the tobamovirus (e.g., TMGMV or TMV) dissociate reversibly and partially in response to an external factor. The external factor can be a change in pH or the presence of a solvent at a particular concentration. For example, a change in pH causes a change in the ionization of protein residues, which in turn affects the electrostatic interactions between protein residues. When the capsid protein begins to dissociate, the nanoparticle structure “breathes,” pores or pockets open, and access to the space between the coat proteins becomes possible. In addition to pH, the solvent can also cause assembly and disassembly. For example, in some embodiments, a solvent at a concentration of about 15% (v / v) to about 25% (v / v) destabilizes the protein and tends to cause dissociation and unfolding due to its effect on the charge state distribution and disruption of the structured water in the protein. In some embodiments, the solvent is a polar aprotic solvent. In some embodiments, the solvent is a polar aprotic solvent that is miscible with water. In some embodiments, the solvent is dimethyl sulfoxide (DMSO).

[0062] In some embodiments, one or more AIs are non-covalently conjugated to and confined within one or more pores or pockets of a tobamovirus (e.g., TMGMV or TMV). In some embodiments, one or more AIs are intercalated into one or more coat proteins of a tobamovirus (e.g., TMGMV or TMV). In some embodiments, one or more AIs are not chemically altered when loaded onto a tobamovirus (e.g., TMGMV or TMV). In some embodiments, an impregnated tobamovirus (e.g., TMGMV or TMV) contains an AI located within the tobamovirus (e.g., TMGMV or TMV). In some embodiments, an impregnated tobamovirus (e.g., TMGMV or TMV) contains an AI that is dispersed within and throughout the tobamovirus (e.g., TMGMV or TMV). In some embodiments, an impregnated tobamovirus (e.g., TMGMV or TMV) does not interact with an AI on the surface of the tobamovirus (e.g., TMGMV or TMV). In some embodiments, a TMG tobamovirus (e.g., TMGMV or TMV) MV does not have an AI shell located on the outer surface of the tobamovirus (e.g., TMGMV or TMV).

[0063] In some embodiments, the AI-loaded tobamovirus (e.g., TMGMV or TMV) and the non-loaded tobamovirus (e.g., TMGMV or TMV) are rod-shaped. In some embodiments, the engineered tobamovirus (e.g., TMGMV or TMV) has a different shape (e.g., different width) from the non-engineered tobamovirus (e.g., TMGMV or TMV). For example, as shown in FIG. 17A, when AI is loaded into a tobamovirus (e.g., TMGMV or TMV), the AI-loaded tobamovirus (e.g., TMGMV or TMV) may exhibit an increased width compared to the non-loaded tobamovirus (e.g., TMGMV or TMV) or the reference tobamovirus (e.g., TMGMV or TMV). In some embodiments, the AI-loaded tobamovirus (e.g., TMGMV or TMV) may appear to be inflated compared to the non-loaded tobamovirus (e.g., TMGMV or TMV) or the reference tobamovirus (e.g., TMGMV or TMV). In some embodiments, this change in the width of the AI-loaded tobamovirus (e.g., TMGMV or TMV) suggests the confinement of AI.

[0064] In some embodiments, the engineered tobamovirus (e.g., TMGMV or TMV)-AI nanoparticles are about 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, 50, 51, 52, 53, 54, 55, 56, 57, 58, 59, 60, 61, 62, 63, 64, 65, 66, 67, 68, 69, 70, 71, 72, 73, 74, 75, 76, 77, 78, 79, 80, 81, 82, 83, 84, 85, 86, 87, 88, 89, 90, 91, 92, 93, 94, 95, 96, 97, 98, 99, 100, 101, 102, 103, 104, or 105% wider than those of the non-engineered tobamovirus (e.g., TMGMV or TMV)-AI particles. In some embodiments, the width of the engineered tobamovirus (e.g., TMGMV or TMV)-AI nanoparticles is 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, 50, 51, 52, 53, 54, 55, 56, 57, 58, 59, 60, 61, 62, 63, 64, 65, 66, 67, 68, 69, 70, 71, 72, 73, 74, or 75 nm.

[0065] In some embodiments, the nanoparticles provided herein can be rod-shaped or amorphous-shaped. In some embodiments, the nanoparticles provided herein have a length in the range of about 2 nm to about 300 nm (e.g., about 2 nm to about 50 nm, about 2 nm to about 100 nm, about 2 nm to about 200 nm, about 2 nm to about 250 nm, about 2 nm to about 300 nm, about 50 nm to 75 nm, about 50 nm to about 100 nm, about 50 nm to about 125 nm, about 50 nm to about 150 nm, about 50 nm to about 175 nm, about 50 nm to about 200 nm, about 50 nm to about 225 nm, about 50 nm to about 250 nm, about 50 nm to about 275 nm, about 50 nm to about 300 nm, about 100 nm to about 125 nm, about 100 nm to about 150 nm, about 100 nm to about 175 nm, about 100 nm to about 200 nm, about 100 nm to about 225 nm, about 100 nm to about 250 nm, about 100 nm to about 275 nm, about 100 nm to about 300 nm, about 150 nm to about 175 nm, about 150 nm to about 200 nm, about 150 nm to about 225 nm, about 150 nm to about 250 nm, about 150 nm to about 275 nm, about 150 nm to about 300 nm, about 200 nm to about 225 nm, about 200 nm to about 250 nm, about 200 nm to about 275 nm, about 200 nm to about 300 nm, about 250 nm to about 275 nm, or about 250 nm to about 300 nm) that extends between a first end and a second end of the outer surface of the rod-shaped nanoparticles. In some embodiments, the nanoparticles provided herein have a length of about 50 nm to about 300 nm. In some embodiments, the nanoparticles provided herein have a length between about 50 nm. In some embodiments, the nanoparticles provided herein have a length between about 100 nm. In some embodiments, the nanoparticles provided herein have a length between about 150 nm. In some embodiments, the nanoparticles provided herein have a length between about 200 nm. In some embodiments, the nanoparticles provided herein have a length between about 250 nm. In some embodiments, the nanoparticles provided herein have a length between about 300 nm.

[0066] In some embodiments, the nanoparticles of the present disclosure comprise from about 1 to about 2000 or more AI molecules per TMGMV (e.g., from about 1 to 10, 1 to 15, 1 to 20, 1 to 50, 1 to 60, 1 to 75, 1 to 100, 1 to 150, 1 to 175, 1 to 185, 1 to 200, 1 to 300, 1 to 400, 1 to 500, 1 to 600, 1 to 700, 1 to 800, 1 to 900, 1 to 1000, 1 to 1100, 1 to 1500, 1 to 1999, 10 to 15, 10 to 20, 10 to 50, 10 to 60, 10 to 75, 10 to 100, 10 to 150, 10 to 175, 10 to 185, 10 to 200, 10 to 300, 10 to 400, 10 to 500, 10 to 600, 10 to 700, 10 to 800, 10 to 900, 10 to 1000, 10 to 1100, 10 to 1500, 10 to 2000, 50 to 60, 50 to 75, 50 to 100, 50 to 150, 50 to 175, 50 to 185, 50 to 200, 50 to 300, 50 to 400, 50 to 500, 50 to 600, 50 to 700, 50 to 800, 50 to 900, 50 to 1000, 50 to 1100, 50 to 1500, 50 to 2000, 150 to 175, 150 to 185, 150 to 200, 150 to 300, 150 to 400, 150 to 500, 150 to 600, 150 to 700, 150 to 800, 150 to 900, 150 to 1000, 150 to 1100, 150 to 1500, 150 to 2000, 500 to 600, 500 to 700, 500 to 800, 500 to 900, 500 to 1000, 500 to 1100, 500 to 1500, 500 to 2000, 750 to 800, 750 to 900, 750 to 1000, 750 to 1100, 750 to 1500, 750 to 2000, 1000 to 1100, 1000 to 1500, 1000 to 2000 or more AI molecules per tobacco mosaic virus (e.g., TMGMV or TMV)).

[0067] Composition In some embodiments, the compositions of the present disclosure contain a plurality of nanoparticles of the present disclosure. In some embodiments, the plurality of nanoparticles includes nanoparticles having the same engineered modification (e.g., a population of tobamoviruses conjugated to beta-cyclodextrin, an engineered population of tobamoviruses, or a population of tobamoviruses impregnated with a particular AI). In some embodiments, the plurality of nanoparticles includes nanoparticles carrying the same AI. In some embodiments, the composition includes a mixture of nanoparticles, with some of the nanoparticles in the mixture including tobamoviruses conjugated to beta-cyclodextrin (BCD), and some of the nanoparticles in the mixture including impregnated tobamoviruses. In some embodiments, the composition includes a mixture of nanoparticles, where the nanoparticles carry or are impregnated with different AIs (e.g., pesticides). For example, the compositions of the present disclosure may include a mixture of nanoparticles, where nanoparticle "A" composed of a plurality of BCD-conjugated tobamoviruses carrying pesticide "1" is mixed with nanoparticle "B" composed of a plurality of tobamoviruses impregnated with pesticide "1". Nanoparticle "A" composed of a plurality of BCD-conjugated tobamoviruses carrying pesticide "1" may be mixed with nanoparticle "B" composed of a plurality of BCD-conjugated tobamoviruses carrying pesticide "2", or nanoparticle "A" composed of a plurality of tobamoviruses impregnated with pesticide "1" may be mixed with nanoparticle "B" composed of a plurality of tobamoviruses impregnated with pesticide "2", among other non-limiting examples. In some embodiments, mixtures having three or more, four or more, or five or more of the plurality of nanoparticles of the present disclosure are also included.

[0068] In some embodiments, the disclosed nanoparticles can be formulated into an aqueous solution. In some embodiments, the nanoparticles can be formulated into a hydrogel. In some embodiments, the disclosed nanoparticles can be lyophilized. In some embodiments, the disclosed nanoparticles are formulated into a redispersible powder and an aqueous dispersion. In some embodiments, the nanoparticles contain a high weight percentage of a water-insoluble pesticide or other active ingredient. In some embodiments, the disclosed nanoparticles can be prepared from an oil-in-water microemulsion or nanoemulsion containing a water-insoluble non-halogenated volatile organic solvent, and the organic solvent and / or water are removed.

[0069] In some embodiments, a composition containing any of the disclosed nanoparticles can also contain a solvent. Examples of solvents include, but are not limited to, dimethyl sulfoxide (DMSO), ethanol, 1-propanol, 2-propanol, n-pentanol, n-butanol, ethyl acetate, tetrahydrofuran, propylene glycol, formamide, glycerol, polyethylene glycol, and mixtures thereof. In another embodiment, the co-solvent is present in an amount of about 5 to about 30% by weight, based on the total weight of the microemulsion.

[0070] In some embodiments, the disclosed nanoparticles are in a liquid formulation. In some embodiments, the liquid formulation contains an AI and the disclosed nanoparticles that are dissolved, emulsified as droplets, or suspended as matrix particles. In some embodiments, the liquid formulation contains the disclosed nanoparticles and an AI such as a pesticide that is dissolved or emulsified as droplets. In some embodiments, the pesticide is uniformly distributed throughout the particles.

[0071] In some embodiments, any nanoparticle formulation described herein can be used as such or, for example, in the form of a directly sprayable solution, powder, suspension, or dispersion, emulsion, oil dispersion, paste, dustable product, material for spraying, or granules, which can be prepared on-site, by means of spraying, atomizing, dusting, spreading, or injection. In some embodiments, the form of use depends entirely on the intended purpose. For example, the formulation is intended in each case to ensure the best possible distribution of the pesticide(s) and nanoparticles described herein.

[0072] In some embodiments, the aqueous form of use can also be prepared from an emulsion concentrate, paste, or wettable powder (sprayable powder, oil dispersion) by adding a suitable solvent, for example water. In some embodiments, the disclosed nanoparticles can be used individually or mixed with each other and / or with any AI already partially or fully disclosed herein for preparing the compositions according to the present disclosure.

[0073] In some embodiments, a composition comprising any of the disclosed nanoparticles may also include a surfactant or a mixture of surfactants. In one embodiment, the surfactant is any one or more of a cationic surfactant, an anionic surfactant, an amphoteric surfactant, a nonionic surfactant, and mixtures thereof. In some embodiments, the anionic surfactant is selected from the group consisting of alkylbenzene sulfonates (e.g., sodium alkyl naphthalene sulfonate), sodium dodecyl sulfate, sodium sulfosuccinate, sodium lauryl sulfate, concentrated sodium alkyl naphthalene sulfonate, sodium stearate, and mixtures thereof, the nonionic surfactant is selected from the group consisting of ethoxylated sorbitan esters, sorbitan esters, organosilicone surfactants, polyglycerol esters, sucrose esters, poloxamers, alkyl polyglucosides, polyalkylene oxide-modified heptamethyltrisiloxane, and allyloxy polyethylene glycol methyl ether, and mixtures thereof, the amphoteric surfactant is lecithin, and the cationic surfactant is selected from the group consisting of cetyltrimethylammonium bromide, cetyltrimethylammonium chloride, and mixtures thereof. In some embodiments, the surfactant is present in an amount of about 5 to about 35% by weight based on the total weight of the microemulsion. In some embodiments, the surfactant is Morwet® (sodium n-butylnaphthalene sulfonate). In some embodiments, the surfactant is Silwet® L-77 (an organosilicone surfactant comprising a blend of polyalkylene oxide-modified heptamethyltrisiloxane and allyloxy polyethylene glycol methyl ether).

[0074] In some embodiments, the nanoparticles of the present disclosure can be formulated within a matrix. As used herein, the term "matrix" has its ordinary meaning and refers to a mixture in which the nanoparticles are suspended throughout another substance. Thus, in some embodiments, the composition comprises dispersed or suspended nanoparticles. Within the scope of the present disclosure, the matrix fluid refers to a composition that is activated using any one or more of the activation means. In some embodiments, the matrix is a hydrogel.

[0075] In some embodiments, the compositions of the present disclosure comprise excipients. In some embodiments, the excipient is a buffer or water. In some embodiments, the buffer is a potassium phosphate buffer. In some embodiments, the water is deionized water.

[0076] Active ingredient Any of the nanoparticles described herein can comprise at least one active ingredient (AI) or one or more AIs. In some embodiments, at least one AI comprises at least one of a drug, a pesticide, or a small molecule. In some embodiments, the drug can be a chemokine, an antibacterial agent, or any therapeutic compound. In some embodiments, the drug is a chemotherapeutic agent, an antiparasitic agent, an antibiotic, an immunomodulator, an antifungal agent, an antiprotozoal agent, an antiviral agent, or any combination thereof. In some embodiments, the drug is a hydrophilic drug or a hydrophobic drug.

[0077] In some embodiments, the chemotherapeutic agent is a small molecule, a protein (e.g., an antibody, an antigen-binding fragment of an antibody, or a derivative or conjugate thereof), a nucleic acid, or any combination thereof. Non-limiting examples of chemotherapeutic agents include cyclophosphamide, mechlorethamine, chlorambucil, melphalan, daunorubicin, doxorubicin, epirubicin, idarubicin, mitoxantrone, valrubicin, paclitaxel, docetaxel, etoposide, teniposide, tafuriposide, azacitidine, azathioprine, capecitabine, cytarabine, doxifluridine, fluorouracil, gemcitabine, mercaptopurine, methotrexate, thioguanine, bleomycin, carboplatin, cisplatin, oxaliplatin, all-trans retinoic acid, vinblastine, vincristine, vindesine, vinorelbine, and bevacizumab (or an antigen-binding fragment thereof).

[0078] In some embodiments, the anti-parasitic drug is niclosamide, oxyclozanide, rafoxanide, closantel, dibromosalan, metabromosalan, tribromosalan, and nitazoxanide.

[0079] In some embodiments, the antibiotic is a beta-lactam antibiotic, an aminoglycoside, an ansamycin type antibiotic, an anthraquinone, an antibiotic azole, an antibiotic glycopeptide, a macrolide, an antibiotic nucleoside, an antibiotic peptide, an antibiotic polyene, an antibiotic polyether, a quinolone, an antibiotic steroid, a sulfonamide, a tetracycline, a dicarboxylic acid, an antibiotic metal, an oxidizing agent, a substance that releases free radicals and / or reactive oxygen species, a cationic antibacterial agent, a quaternary ammonium compound, a biguanide, a triguanide, a bisbiguanide and its analogs and polymers, a naturally occurring antibiotic compound, and any combination thereof. In some embodiments, the AI is rifampicin. In some embodiments, the AI is ivermectin. In some embodiments, the AI is fluopyram. In some embodiments, the AI is clothianidin.

[0080] In some embodiments, an immunomodulatory agent is a substance that can stimulate or suppress the immune system to help the body fight cancer, infection, or other diseases. In some embodiments, the immunomodulatory agent is, but not limited to, a checkpoint inhibitor, adoptive cell therapy (T cell transplantation therapy, monoclonal antibody therapy, cancer vaccine, immune system modulator (e.g., cytokine, and biological response modifiers such as thalidomide, lenalidomide, pomalidomide, and imiquimod), or any combination thereof, etc., cancer immunotherapy agents. In some embodiments, the immunomodulatory agent is a corticosteroid, disease-modifying antirheumatic drug (DMARD) (e.g., azathioprine, cyclosporine, hydroxychloroquine, leflunomide, methotrexate, and sulfasalazine), biological agent (e.g., tumor necrosis factor (TNF) inhibitor, interleukin-1 (IL-1) inhibitor, interleukin-6 (IL-6) inhibitor, T cell inhibitor, B cell inhibitor), Janus kinase inhibitor, or any combination thereof. In some embodiments, the immunomodulatory substance is GM-CSF (granulocyte-macrophage colony-stimulating factor).

[0081] Any nanoparticle described herein may contain at least one active ingredient (AI) or one or more AIs. In some embodiments, at least one AI includes at least one of a drug, a pesticide, or a small molecule. In some embodiments, the drug can be a chemokine, an antibacterial agent, or any therapeutic compound. In some embodiments, the drug is a chemotherapeutic agent, an antiparasitic agent, an antibiotic, or an immunomodulatory agent. In some embodiments, the drug is a hydrophilic drug or a hydrophobic drug. In some embodiments, the pesticide is a water-insoluble organic compound, an insecticide, a herbicide, a fungicide, a miticide, an algicide, an antibacterial agent, a biopesticide, a biocide, a disinfectant, a fumigant, an insect growth regulator, a plant growth regulator, a miticide, a microbial pesticide, a molluscicide, a nematicide, an ovicide, a pheromone, a repellent, a rodenticide, a defoliant, a desiccant, a phytotoxicity reducer, or any combination thereof.In some embodiments, the pesticides are benzoyl ureas such as novaluron, lufenuron, chlorfluazuron, flufenoxuron, hexaflumuron, noviflumuron, teflubenzuron, triflumuron, and diflubenzuron; carbamates; pyrethroids such as cyhalothrin, and its isomers and isomer mixtures, lambda-cyhalothrin, deltamethrin, tau-fluvalinate, cyfluthrin, beta-cyfluthrin, tefluthrin, and bifenthrin; organophosphates such as azinphos-methyl, chlorpyrifos, diazinon, endosulfan, methidathion; neonicotinoids; phenylpyrazoles such as imidacloprid, acetamiprid, thiacloprid, dinotefuran, thiamethoxam, and fipronil; triazoles such as epoxiconazole, hexaconazole, propiconazole, prochloraz, imazalil, triadimenol, difenoconazole, microbutanil, prothioconazole, triticonazole, and tebuconazole; morpholines such as dimethomorph, fenpropidin, and fenpropimorph; strobilurins such as azoxystrobin, kresoxim-methyl, and their analogs; phthalonitriles such as chlorothalonil; mancozeb; fluazinam; pyrimidines such as bupirimate; aryloxyphenoxy derivatives; aryl ureas; aryl carboxylic acids; aryloxyalkanoic acid derivatives such as clodinafop-propargyl and its analogs, fenoxaprop-p-ethyl and its analogs, propaquizafop, quizalofop and its analogs; dinitroanilines such as pendimethalin and trifluralin; diphenyl ethers such as oxyfluorfen; imidazolinones; sulfonyl ureas such as chlorosulfuron, nicosulfuron, rimsulfuron, tribenuron-methyl; sulfonamides; triazines; and triazinones such as metamitron; and any combination thereof. In some embodiments, any nanoparticles described herein may include one or more compounds selected from the group consisting of fungicides, insecticides, nematicides, herbicides, and / or phytotoxicity reducing agents or growth regulators. Any two or more of the aforementioned classes of pesticides may be used.One skilled in the art is familiar with useful drugs and pesticides, which can be found, for example, in the Pesticide Manual, 13th Ed. (2003), The British Crop Protection Council, London.

[0082] Any nanoparticle, composition, or method described herein may include any one or more of the following lists of pesticides, which is intended to illustrate possible combinations but not to impose any limitations: A) Strobilurin, azoxystrobin, dimoxystrobin, enestroburin, fluoxastrobin, kresoxim-methyl, metominostrobin, orysastrobin, picoxystrobin, pyraclostrobin, pyribencarb, trifloxystrobin, 2-(2-(6-(3-chloro-2-methyl-phenoxy)-5-fluoro-pyrimidin-4-yloxy)-phenyl)-2-methoxyimino-N-methyl-acetamide, 3-methoxy-2-(2-(N-(4-methoxy-phenyl)-cyclopropane-carboximidoyl-sulfanylmethyl)-phenyl)-acrylic acid methyl ester, methyl (2-chloro-5-[1-(3-methylbenzyloxyimino)ethyl]benzyl)carbamate, and 2-(2-(3-(2,6-dichlorophenyl)-1-methyl-allylideneaminooxymethyl)-phenyl)-2-methoxyimino-N-methyl-acetamide; B) Carboxamide, Carboxanilide: benalaxyl, benalaxyl-M, benodanil, bixafen, boscalid, carboxin, fenfuram, fenhexamid, flutolanil, flutriafol, isopyrazam, isothianil, kiralaxyl, mepronil, metalaxyl, metalaxyl-M (mefenoxam), ofurace, oxadixyl, oxycarboxin, penthiopyrad, tecloftalam, difenoconazole, thifluzamide, thiazinyl, 2-amino-4-methyl-thiazole-5-carboxanilide, 2-chloro-N-(1,1,3-trimethyl-indan-4-yl)-nicotinamide, N-(2′,4′-difluorobiphenyl-2-yl)-3-difluoromethyl-1-methyl-1H-pyrazole-4-carboxamide, N-(2′,4′-dichlorobiphenyl-2-yl)-3-difluoromethyl-1-methyl-1H-pyrazole-4-carboxamide, N-(2′,5′-difluorobiphenyl-2-yl)-3-difluoromethyl-1-methyl-1H-pyrazole-4-carboxamide, N-(2′,5′-dichlorobiphenyl-2-yl)-3-difluoromethyl-1-methyl-1H-pyrazole-4-carboxamide, N-(3′,5′-difluorobiphenyl-2-yl)-3-difluoromethyl-1-methyl-1H-pyrazole-4-carboxamide, N-(3′-fluorobiphenyl-2-yl)-3-difluoromethyl-1-methyl-1H-pyrazole-4-carboxamide, N-(3′-chlorobiphenyl-2-yl)-3-difluoromethyl-1-methyl-1H-pyrazole-4-carboxamide, N-(2′-fluorobiphenyl-2-yl)-3-difluoromethyl-1-methyl-1H-pyrazole-4-carboxamide, N-(2′-chlorobiphenyl-2-yl)-3-difluoromethyl-1-methyl-1H-pyrazole-4-carboxamide, N-(3′,5′-dichlorobiphenyl-2-yl)-3-difluoromethyl-1-methyl-1H-pyrazole-4-carboxamide, N-(3′,4′,5′-trifluorobiphenyl-2-yl)-3-difluoromethyl-1-methyl-1H-pyrazole-4-carboxamide, N-(2′,4′,5′-Trifluorobiphenyl-2-yl)-3-difluoromethyl-1-methyl-1H-pyrazole-4-carboxamide, N-[2-(1,1,2,3,3,3-hexafluoropropoxy)-phenyl]-3-difluoromethyl-1-methyl-1H-pyrazole-4-carboxamide, N-[2-(1,1,2,2-tetrafluoroethoxy)-phenyl]-3-difluoromethyl-1-methyl-1H-pyrazole-4-carboxamide, N-(4′-trifluoromethyl-thiobiphenyl-2-yl)-3-difluoromethyl-1-methyl-1H-pyrazole-4-carboxamide, N-(2-(1,3-dimethyl-butyl)-phenyl)-1,3-dimethyl-5-fluoro-1H-pyrazole-4-carboxamide, N-(2-(1,3,3-trimethylbutyl)-phenyl)-1,3-dimethyl-5-fluoro-1H-pyrazole-4-carboxamide, N-(4′-chloro-3′,5′-difluoro-biphenyl-2-yl)-3-difluoromethyl-1-methyl-1H-pyrazole-4-carboxamide, N-(4′-chloro-3′,5′-difluoro-biphenyl-2-yl)-3-trifluoromethyl-1-methyl-1H-pyrazole-4-carboxamide, N-(3′,4′-dichloro-5′-fluoro-biphenyl-2-yl)-3-trifluoromethyl-1-methyl-1H-pyrazole-4-carboxamide, N-(3′,5′-difluoro-4′-methyl-biphenyl-2-yl)-3-difluoromethyl-1-methyl-1H-pyrazole-4-carboxamide, N-(3′,5′-difluoro-4′-methyl-biphenyl-2-yl)-3-trifluoromethyl-1-methyl-1H-pyrazole-4-carboxamide, N-(2-bicyclopropyl-2-yl-phenyl)-3-difluoromethyl-1-methyl-1H-pyrazole-4-carboxamide, N-(cis-2-bicyclopropyl-2-yl-phenyl)-3-difluoromethyl-1-methyl-1H-pyrazole-4-carboxamide, N-(trans-2-bicyclopropyl-2-yl-phenyl)-3-difluoromethyl-1-methyl-1H-pyrazole-4-carboxamide, N-[1,2,3,4-tetrahydro-9-(1-methylethyl)-1,4-Methano-naphthalen-5-yl]-3-(difluoromethyl)-1-methyl-1H-pyrazole-4-carboxamide; Carboxylic acid morpholides: dimethomorph, flumorph; Benzoic acid amides: fluopicolide, flupyradifurone; Other carboxamides: carpropamid, dicyclomet, mandiproamid, oxytetracycline, silthiofarm, and N-(6-methoxy-pyridin-3-yl)cyclopropanecarboxamide; C) Azoles, Triazoles: azaconazole, bitertanol, bromoconazole, cyproconazole, difenoconazole, diniconazole, diniconazole-M, epoxiconazole, fenbuconazole, fluquinconazole, flusilazole, flutriafol, hexaconazole, imibenconazole, ipconazole, metconazole, myclobutanil, oxpoconazole, paclobutrazole, penconazole, propiconazole, prothioconazole,simeconazole, tebuconazole, tetraconazole, triadimefon, triadimenol, triticonazole, uniconazole, 1-(4-chloro-phenyl)-2-([1,2,4]triazol-1-yl)-cycloheptanol; Imidazoles: cyazofamid, imazalil, pefurazoate, prochloraz, triflumizole, Benzimidazoles: benomyl, carbendazim, fuberidazole, thiabendazole, Others: ethaboxam, etridiazole, hymexazol, and 2-(4-chloro-phenyl)-N-[4-(3,4-dimethoxy-phenyl)-isoxazol-5-yl]-2-prop-2-ynyloxy-acetamide; D) Heterocyclic compounds, Pyridine: fluazinam, pyrifenox, 3-[5-(4-chlorophenyl)-2,3-dimethylisoxazolidin-3-yl]pyridine, 3-[5-(4-methylphenyl)-2,3-dimethylisoxazolidin-3-yl]pyridine, 2,3,5,6-tetrachloro-4-methanesulfonylpyridine, 3,4,5-trichloropyridine-2,6-dicarbonitrile, N-(1-(5-bromo-3-chloropyridin-2-yl)ethyl)-2,4-dichloronicotinamide, N-[(5-bromo-3-chloropyridin-2-yl)methyl]-2,4-dichloronicotinamide; Pyrimidine: bupirimate, cyprodinil, diflumezopyrim, fenarimol, ferimzone, mepanipyrim, nitrapyrin, nuarimol, pyrimethanil; Piperazine: triforine; Pyrrole: fenpiclonil, fludioxonil; Morpholine: aldimorph, dodemorph, dodemorph acetate, fenpropimorph, tridemorph; Piperidine: fenpropidin; Dicarboximide: fluoroimide, iprodione, procymidone, vinclozolin; Non-aromatic 5-membered heterocycle: famoxadone, fenamidone, octhilinone, probenazole, 5-amino-2-isopropyl-3-oxo-4-o-tolyl-2,3-dihydro-1H-pyrazole-1-carbothioic acid S-allyl ester; And / or others: acibenzolar-S-methyl, amisulbrom, anilazine, blasticidin-S, captan, captophos, chinomethionat, dazomet, decarb, dichlormid, difenzoquat, difenzoquat-methyl-sulfate, fenoxanil, folpet, oxolinic acid, piperalin, proquinazid, pyroquilon, quinoxyfen, triazoxide, tricyclazole, 2-butoxy-6-iodo-3-propylchromen-4-one, 5-chloro-1-(4,6-dimethoxy-pyrimidin-2-yl)-2-methyl-1H-benzimidazole, 5-chloro-7-(4-methyl-piperidin-1-yl)-6-(2,4,6-trifluorophenyl)-[1,2,4]triazolo[1,5-a]pyrimidine, 6-(3,4-dichloro-phenyl)-5-methyl-[1,2,4]triazolo[1,5-a]pyrimidin-7-ylamine, 6-(4-tert-butyl-phenyl)-5-methyl-[1,2,4]triazolo[1,5-a]pyrimidin-7-ylamine, 5-methyl-6-(3,5,5-trimethyl-hexyl)-[1,2,4]triazolo[1,5-a]pyrimidin-7-ylamine, 5-methyl-6-octyl-[1,2,4]triazolo[1,5-a]pyrimidin-7-ylamine, 6-methyl-5-octyl-[1,2,4]triazolo[1,5-a]pyrimidin-7-ylamine, 6-ethyl-5-octyl-[1,2,4]triazolo[1,5-a]pyrimidin-7-ylamine, 5-ethyl-6-octyl-[1,2,4]triazolo[1,5-a]pyrimidin-7-ylamine, 5-ethyl-6-(3,5,5-trimethyl-hexyl)-[1,2,4]triazolo[1,5-a]pyrimidin-7-ylamine, 6-octyl-5-propyl-[1,2,4]triazolo[1,5-a]pyrimidin-7-ylamine, 5-methoxymethyl-6-octyl-[1,2,4]triazolo[1,5-a]pyrimidin-7-ylamine, 6-octyl-5-trifluoromethyl-[1,2,4]triazolo[1,5-a]pyrimidin-7-ylamine, and 5-trifluoromethyl-6-(3,5,5-trimethyl-hexyl)-[1,2,4]triazolo[1,5-a]pyrimidin-7-ylamine; E) Carbamates, Thio- and dithiocarbamates: ferbam, mancozeb, maneb, metam, methasulphocarb, methiram, propineb, thiram, zineb, ziram; Carbamates: benthiavalicarb, diethofencarb, flubenthiavalicarb, iprovalicarb, propamocarb, propamocarb hydrochloride, valiphenal, and N-(1-(1-(4-cyano-phenyl)-ethanesulphonyl)-butan-2-yl)carbamic acid (4-fluorophenyl) ester; F) Any other active substances, Guanidines: guanidine, dodine, dodine free base, guazatine, guazatine-acetate, iminoctadine, iminoctadine-triacetate, iminoctadine-tris(albesilate); Antibiotics: kasugamycin, kasugamycin hydrochloride hydrate, streptomycin, polyoxin, validamycin A; Nitrophenyl derivatives: binapacryl, dinobuton, dinocap, nitrthal-isopropyl, tecnazene, Organometallic compounds: fentin-acetate, fentin chloride, or fentin salts such as fentin hydroxide; Sulphur-containing heterocyclyl compounds: dithianon, isoprothiolane; Organophosphorus compounds: edifenphos, fosetyl, fosetyl-aluminium, iprobenfos, phosphoric acid and its salts, pyrazophos, tolclofos-methyl; Organochlorine compounds: chlorothalonil, dichlofluanid, dichlorophen, flusulphamide, hexachlorobenzene, pentachloronitrobenzene, pentachlorophenol and its salts, phthalide, quintozene, thiophanate-methyl, tolylfluanid, N-(4-chloro-2-nitro-phenyl)-N-ethyl-4-methyl-benzenesulphonamide; Inorganic active substances: Bordeaux mixture, copper acetate, copper hydroxide, copper oxychloride, basic copper sulphate, sulphur; Any one or more of the others: biphenyl, bronopol, cymoxanil, dimethomorph, diphenylamine, metrafenone, miltiradiene, oxine-copper, prohexadione-calcium, spiroxamine, tolylfluanid, N-(cyclopropylmethoxyimino-(6-difluoro-methoxy-2,3-difluoro-phenyl)-methyl)-2-phenylacetamide, N′-(4-(4-chloro-3-trifluoromethyl-phenoxy)-2,5-dimethyl-phenyl)-N-ethyl-N-methylformamidine, N′-(4-(4-fluoro-3-trifluoromethyl-phenoxy)-2,5-dimethyl-formamidine, N′-(2-methyl-5-trifluoromethyl-4-(3-trimethylsilanil-propoxy)-phenyl)-N-ethyl-N-methylformamidine, and N′-(5-difluoromethyl-2-methyl-4-(3-trimethylsilanil-propoxy)-phenyl)-N-ethyl-N-methylformamidine. G) Herbicides such as acetamide: acetochlor, alachlor, butachlor, dimethachlor, dimethenamid, flufenacet, mefenacet, metachlor, metazachlor, napropamide, naproanilide, pethoxamid, pretilachlor, propachlor, thenylchlor; Amino acid derivatives: bilanafos, glyphosate, glufosinate, sulfosate; Aryloxyphenoxypropionates: quizalofop, quizalofop-butyl, fenoxaprop, fluazifop, haloxyfop, metamifop, propaquizafop, quizalofop-P-tefuryl; Bipyridyl: diquat, paraquat; (Thio)carbamates: asulam, butylate, carbamido, desmedipham, dimepiperate, EPTC, esprocarb, molinate, oryzacarb, phenmedipham, prosulfocarb, pyributicarb, thiobencarb, triallate; Cyclohexanediones: butroxydim, clethodim, cycloxydim, profoxydim, sethoxydim, tepraloxydim, tralkoxydim; Dinitroaniline: Benefin, ethalfluralin, oryzalin, pendimethalin, pro-diamine, trifluralin; Diphenyl ether: Acifluorfen, acifluorfen-sodium, acifluorfen-methyl, acifluorfen-ethyl, acifluorfen-butyl, acifluorfen-isopropyl, acifluorfen-propyl, acifluorfen-isobutyl, acifluorfen-sec-butyl, acifluorfen-tert-butyl, acifluorfen-pentyl, acifluorfen-hexyl, acifluorfen-heptyl, acifluorfen-octyl, acifluorfen-nonyl, acifluorfen-decyl, acifluorfen-undecyl, acifluorfen-dodecyl, acifluorfen-tridecyl, acifluorfen-tetradecyl, acifluorfen-pentadecyl, acifluorfen-hexadecyl, acifluorfen-heptadecyl, acifluorfen-octadecyl, acifluorfen-nonadecyl, acifluorfen-eicosyl, acifluorfen-docosyl, acifluorfen-tetracosyl, acifluorfen-hexacosyl, acifluorfen-octacosyl, acifluorfen-triacontyl, acifluorfen-dotriacontyl, acifluorfen-tetracontyl, acifluorfen-pentacontyl, acifluorfen-hexacontyl, acifluorfen-heptacontyl, acifluorfen-octacontyl, acifluorfen-nonacontyl, acifluorfen-hectyl, acifluorfen-kilolyl, acifluorfen-megalyl, acifluorfen-gigalyl, acifluorfen-teragalyl, acifluorfen-petagalyl, acifluorfen-exagalyl, acifluorfen-zettalyl, acifluorfen-yottalyl, acifluorfen-xonnalyl, acifluorfen-wendyl, acifluorfen-vendyl, acifluorfen-uranyl, acifluorfen-thorium, acifluorfen-protactinium, acifluorfen-uranium, acifluorfen-neptunium, acifluorfen-plutonium, acifluorfen-americium, acifluorfen-curium, acifluorfen-berkelium, acifluorfen-californium, acifluorfen- einsteinium, acifluorfen-fermium, acifluorfen-mendelevium, acifluorfen-nobelium, acifluorfen-lawrencium, acifluorfen-rutherfordium, acifluorfen-dubnium, acifluorfen-seaborgium, acifluorfen-bohrium, acifluorfen-hassium, acifluorfen-meitnerium, acifluorfen-darmstadtium, acifluorfen-roentgenium, acifluorfen-copernicium, acifluorfen-nihonium, acifluorfen-flerovium, acifluorfen-moscovium, acifluorfen-livermorium, acifluorfen-tennessine, acifluorfen-oganesson; Hydroxybenzonitrile: Bromoxynil, dichlobenil, ioxynil; Imidazolinone: Imazamethabenz, imazamox, imazapic, imazapyr, imazakip, imazethapyr; Phenoxyacetic acid: Chloramben, 2,4-dichlorophenoxyacetic acid (2,4-D), 2,4-DB, dichlorprop, MCPA, MCPA-thioethyl, MCPB, mecoprop; Pyrazine: Chloridazon, flufenpyr-ethyl, fluthiacet, norflurazon, pyridate; Pyridine: Aminopyralid, clopyralid, diflufenican, dithiopyr, fluridone, fluroxypyr, picloram, picolinafen, thiazopyr; Sulfonylurea: Amidosulfuron, azimsulfuron, bensulfuron, chlorimuron-ethyl, chlorsulfuron, cinosulfuron, cyclosulfamuron, ethoxysulfuron, flazasulfuron, flucetosulfuron, flupyrsulfuron, foramsulfuron, halosulfuron, imazosulfuron, iodosulfuron, mesosulfuron, metsulfuron-methyl,nicosulfuron, oxasulfuron, primisulfuron, prosulfuron, pyrazosulfuron, rimsulfuron, sulfometuron, sulfosulfuron, thifensulfuron, triasulfuron, tribenuron, trifloxysulfuron, triflusulfuron, tritosulfuron, 1-((2-chloro-6-propyl-imidazo[1,2-b]pyridazin-3-yl)sulfonyl)-3-(4,6-dimethoxy-pyrimidin-2-yl)urea; Triazine: Ametryn, atrazine, cyanazine, dimethametryn, ethidimuron, hexazinone, metamitron, metribuzin, prometryn, simazine, terbuthylazine, terbutryn, triaziflam; Urea: Chlorotoluron, Dimethoate, Diuron, Fluometuron, Isoproturon, Linuron, Metabenzthiazuron, Tebuthiuron; Other acetolactate synthase inhibitors: Bispyribac-sodium, Chloransulam-methyl, Diclosulam, Florasulam, Flucarbazone, Flumetsulam, Metsulfuron, Orthosulfamuron, Penoxsulam, Propoxycarbazone, Pyribambenz-propyl, Pyribenzoxim, Pyriflufen, Pyriminobac-methyl, Pyrimisulfan, Pyrithiobac, Pyroxasulfone, Pyroxysulam; He: Amicarbazone, Aminotriazole, Anilophos, Beflubutamid, Benazolin, Bencarbazone, Benfluresate, Benzofenap, Bentazone, Benzobicyclon, Bromacil, Bromobutide, Butaphenacil, Butamiphos, Cafestrol, Carfentrazone, Cinidon-ethyl, Chlorotal, Cinmethylin, Chromazone, Cumyluron, Cyprosulfamide, Dicamba, Dienzoate, Diflufenican, Drechslera monoceras, Endothal, Ethofumesate, Ethobenzanid, Fentrazamide, Flumiclorac-pentyl, Flumioxazin, Flupoxam, Flurochloridone, Flutamon, Indanofan, Isoxaben, Isoxafutole, Lenacil, Propanil, Propyzamide, Kinklorac, Kimmelac, Mesotrione, Methylarsonous acid, Naptalam, Oxadiargyl, Oxadiazon, Oxadiquomefon, Pentoxazone, Pinoxaden, Pyraclonil, Pyraflufen-ethyl, Pyrazosulfotole, Pyrazoxyfen, Pyrazolineate, Quinoclamine, Saffufenacil, Sulcotrione, Sulfentrazone, Tebuthiuron, Tefluthrin, Tembotrione, Thiencarbazone, Topramezone, 4-hydroxy-3-[2-(2-methoxy-ethoxymethyl)-6-trifluoromethyl-pyridine-3-carbonyl]-bicyclo[3.2.1]oct-3-en-2-one, (3-[2-chloro-4-fluoro-5-(3-methyl-2,6-dioxo-4-trifluoromethyl-3,6-dihydro-2H-pyrimidin-1-yl)-phenoxy]-pyridin-2-yloxy)-acetic acid ethyl ester, 6-amino-5-chloro-2-cyclopropyl-pyrimidine-4-carboxylic acid methyl ester, 6-chloro-3-(2-cyclopropyl-6-methyl-phenoxy)-pyridazin-4-ol, 4-amino-3-chloro-6-(4-chloro-phenyl)-5-fluoro-pyridine-2-carboxylic acid, 4-amino-3-chloro-6-(4-chloro-2-fluoro-3-methoxy-phenyl)-pyridine-2-carboxylic acid methyl ester, and 4-amino-3-chloro-6-(4-chloro-3-dimethylamino-2-fluoro-phenyl)-pyridine-2-carboxylic acid methyl ester; (H) Any one or more of insecticides that can be selected from the group consisting of the following Organo(thio)phosphates: acephate, azamethiphos,azinphos-methyl, chlorpyrifos, chlorpyrifos-methyl, chlorfenvinphos, diazinon, dichlorvos, dichlorothophos, dimethoate, disulfoton, ethion, fenitrothion, fenthion, isoxathion, malathion, methamidophos, methidathion, methyl-parathion, mevinphos, monocrotophos, oxydemeton-methyl, paraoxon, parathion, phenthoate, hosalone, phosmet, phosphamidon, phorate, phoxim, pyrimiphos-methyl, profenofos, prothiofos, sulprofos, tetrachlorvinphos, terbufos, triazophos, trichlorfon; Carbamates: alanycarb, aldicarb, benfuracarb, benomyl, carbaryl, carbofuran, carbosulfan, fenoxycarb, furathiocarb, methiocarb, methomyl, oxamyl, pirimicarb, propoxur, thiodicarb, triazamate; Pyrethroids: allethrin, bifenthrin, cyfluthrin, cyhalothrin, cyphenothrin, permethrin, alpha-permethrin, beta-permethrin, zeta-permethrin, deltamethrin, esfenvalerate, etofenprox, fenpropathrin, fenvalerate, imiprothrin, lambda-cyhalothrin, permethrin, prallethrin, pyrethrin I and II, resmethrin, silafluofen, tau-fluvalinate, tefluthrin, tetramethrin, tralomethrin, transfluthrin, profluthrin, dimefluthrin; I) Any one or more of the insect growth regulators: a) Chitin synthesis inhibitors: benzoylureas: chlorfluazuron, cyramazin, diflubenzuron, flucycloxuron, flufenoxuron, hexaflumuron, lufenuron, novaluron, teflubenzuron, triflumuron; buprofezin, diofenolan, hexythiazox, etoxazole, chlorphentazine; b) Ecdysone antagonists: halofenozide, methoxyfenozide, tebufenozide, azadirachtin; c) Juvenoids: pyriproxyfen, methoprene, phenoxycarb; d) Lipid biosynthesis inhibitors: spirodiclofen, spirotetramat, spirodiclofen; J) Any one or more of any other compounds such as the following: Nicotinic receptor agonist / antagonist compounds: clothianidin, dinotefuran, imidacloprid, thiamethoxam, nitenpyram, acetamiprid, thiacloprid, 1-(2-chloro-thiazol-5-ylmethyl)-2-nitrimino-3,5-dimethyl-[1,3,5]triazinan; GABA antagonist compounds: endosulfan, ethiprole, fipronil, vaniliprole, pyrafluprole, pyrifluquinazon, 5-amino-1-(2,6-dichloro-4-methyl-phenyl)-4-sulfinamoyl-1H-pyrazole-3-carbothioic acid amide; Macrolide insecticides: abamectin, emamectin, milbemectin, lepimectin, spinosad, spinetoram; Mitochondrial electron transport inhibitor (METI) I acaricides: fenazaquin, pyridaben, tebufenpyrad, tolfenpyrad, flufenoxuron; METI II and III compounds: acequinocyl, fluacyprim, hydramethylnon; Uncoupling agent: chlorfenapyr; Oxidative phosphorylation inhibitors: hexythiazox, diafenthiuron, fenbutatin oxide, propargite; Molting inhibitor compounds: cryomazine; Mixed-function oxidase inhibitor: piperonyl butoxide; Sodium channel blocker: indoxacarb, metaflumizone; Others: benclothiaz, bifenazate, cartap, flonicamid, pyridalyl, pymetrozine, sulfur, thiosultap, flubendiamide, chlorantraniliprole, cyazypyr (HGW86), cyenopyrafen, flupyrazofos, siflumetofen, amidoflumet, imicyafos, bistrifluron, and pyriprole; K) The growth regulator may be selected from any one or more of abscisic acid, amidochlor, ancymidol, 6-benzylaminopurine, brassinolide, butralin, chlormequat (chlormequat chloride), choline chloride, cyclanilide, daminozide, dicamba, dimethipin, 2,6-dimethylpyridine, ethephon, flumetralin, flurprimidol, fluthiacet, forchlorfenuron, gibberellic acid, inabenfide, indole-3-acetic acid, maleic hydrazide, mefluidide, mepiquat (mepiquat chloride), naphthaleneacetic acid, N-6-benzyladenine, paclobutrazol, prohexadione (prohexadione-calcium), prohydrojasmon, thidiazuron, triapenthenol, tributyl phosphorotrithioate, 2,3,5-triiodobenzoic acid, trinexapac-ethyl, and uniconazole.

[0083] In some embodiments, the pesticide is sensitive to UV light. The sensitivity can be detected by a simple test in which the pesticide is exposed to UV light for a certain period of time. Thereafter, the residual pesticide that has not been decomposed can be quantified.

[0084] In some embodiments, the nanoparticles include herbicides such as napropamide, proparnil, bentazon, paraquat dichloride, cyclohexydim, setoxydim, ethalfluralin, oryzalin, pendimethalin, trifluralin, acifluren, acifluorfen, fomesafen, oxyfluoren, ioxtnil, imazetapyr, imazaquin, chloridazon, norflurazon, thiazopyr, triclopyr, dithiopyr, diflufenican, picolinafen, amidosulfuron, molinate, barmate, promethon, metribuzin, azafenidin, carfentrazone-ethyl, sulfentrazone, methoxuron, monolinuron, fluchloralin, and flurenol.

[0085] In some embodiments, the nanoparticles include fungicides such as cyprodinil, fuberidazole, dimethomorph, prochloraz, triflumizole, tridemorph, edifenphos, fenarimol, nuarimol, etirimol, quinoxylen, dithianon, metominostrobin, trifloxystrobin, dichlofluamid, bromoconazole, and microbutanil.

[0086] In some embodiments, the nanoparticles include insecticides such as acephate, azinphos-ethyl, azinphos-methyl, isofenphos, chlorpyrifos-methyl, dimethylvinphos, phorate, phoxim, prothiofos, hexythiazox, alanicarb, ethiofencarb, pyrimicarb, thiodicarb, fipronil, bioallethrin, bioresmethrin, deltamethrin, fenpropathin, flucythrinate, tau-fluvalinate, cypermethrin, zeta-cypermethrin, resmethin, tefluthrin, lambda-cyhalothrin, and hydramethylnon. In another preferred embodiment, the insecticide is metaflumizone or alpha-cypermethrin.

[0087] In some embodiments, the nanoparticles comprise metaflumizone or alpha-cypermethrin.

[0088] Method for producing nanoparticles In certain embodiments, the present disclosure is directed to a method (e.g., the "pH method") for producing nanoparticles comprising a tobamovirus (e.g., TMGMV and / or TMV) and one or more active ingredients (AIs), the method comprising adjusting the pH of a solution in which the nanoparticles are suspended. In some embodiments, the method comprises providing an isolated tobamovirus (e.g., TMGMV and / or TMV) to a buffer having a pH of about 7 to 9 to create a tobamovirus-buffer, adding one or more AIs to the tobamovirus-buffer two or more times, thereby creating nanoparticles, and purifying the nanoparticles in a solution having a pH of about 5 to 9. This method is further described in Example 5. In some embodiments, the one or more AIs are non-covalently conjugated to the tobamovirus (e.g., TMGMV and / or TMV). In some embodiments, the tobamovirus (e.g., TMGMV and / or TMV) comprises one or more coat proteins that dissociate reversibly and partially in response to a change in pH.

[0089] In some embodiments, one or more AIs are added at least once a day for at least 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, or 21 days. In some embodiments, the pH of the tobamovirus-buffer is about 7 - 7.5, 7.5 - 8, 7 - 8, 8 - 8.5, 8.5 - 9, or 8 - 9. In some embodiments, the pH of the tobamovirus-buffer is about 7.2 - 7.8, 7.3 - 7.8, 7.2 - 7.7, 7.3 - 7.7, 7.4 - 7.8, 7.4 - 7.7, 7.5 - 7.7, 7.5 - 7.8, 7.2 - 7.6, 7.3 - 7.6, 7.4 - 7.6, 7.5 - 7.6, 7.2 - 7.5, 7.3 - 7.5, 7.4 - 7.5, 7.2 - 7.9, 7.3 - 7.9, 7.4 - 7.9, 7.5 - 7.9, 7.3 - 7.99, 7.4 - 7.99, or 7.5 - 7.99. In some embodiments, the pH of the tobamovirus-buffer is about 7.2, 7.3, 7.4, 7.5, 7.6, 7.7, 7.8, 7.9, or 7.99. In some embodiments, the pH of the tobamovirus-buffer is 7.5.

[0090] In some embodiments, the solution in which the nanoparticles are purified has a pH of about 5 - about 9 (e.g., about 5 - 6, 5 - 7, 5 - 8, 5 - 8.9, 6 - 7, 6 - 8, 6 - 9, 7 - 8, 7 - 9, 8 - 9, 5.5 - 6.5, 5.5 - 7.5, 5.5 - 8.5, 5.5 - 8.9, 6.5 - 7.5, 6.5 - 8.5, 6.5 - 8.9, 7.5 - 8.5, 7.5 - 9, 8.5 - 9, 6.9 - 7.1, 6.9 - 7.2, 6.9 - 7.3, 5 - 6.5, 5 - 7.5, 5 - 8.5, 5 - 8.9, 6 - 7.5, 6 - 8.5, 6 - 9.5, 7 - 7.1, 7 - 7.2, 7 - 7.3, 7 - 7.4, 7 - 7.5, 7 - 7.6, 7 - 7.7, 7 - 7.8, 7 - 7.9, 7 - 9.5, 8 - 9.5, 5.5 - 6, 5.5 - 7, 5.5 - 8, 5.5 - 8.9, 6.5 - 7, 6.5 - 8, 6.5 - 9, or 7.5 - 8). In some embodiments, the solution in which the nanoparticles are purified has a pH of about 6.9, 7.0, 7.1, 7.2, or 7.3.

[0091] As described elsewhere in this specification, a change in pH causes a phase transition of the tobamovirus (e.g., TMGMV and / or TMV), thereby opening "pores" or "pockets" that can accept and non-covalently link one or more AI molecules therein. In some embodiments, the change in pH (e.g., the difference in pH between the tobamovirus-buffer and the solution in which the nanoparticles are purified) is from about 0.5 to 1, from about 0.5 to 2, 0.5 to 3, 1 to 2, or 1 to 3.

[0092] In certain embodiments, the present disclosure is directed to a method of making nanoparticles (e.g., which may be referred to throughout this disclosure as the "solvent method") that includes a tobamovirus (e.g., TMGMV and / or TMV) and one or more active ingredients (AI), the method including adjusting the concentration of a solvent (e.g., DMSO) present in the solution in which the nanoparticles are suspended. In some embodiments, the method includes providing an isolated tobamovirus (e.g., TMGMV and / or TMV) to a buffer having a pH of about 5 to 9 to create a tobamovirus-buffer, adding a solvent at a concentration of about 15% (v / v) to about 30% (v / v), adding one or more AI to the tobamovirus-buffer to thereby create nanoparticles, and purifying the nanoparticles in a solution having a pH of about 5 to 9.

[0093] In some embodiments, the step of providing an isolated tobamovirus comprises creating a tobamovirus-buffer using a buffer having a pH of about 5-6, 5-7, 5-8, 5-8.9, 6-7, 6-8, 6-9, 7-8, 7-9, 8-9, 5.5-6.5, 5.5-7.5, 5.5-8.5, 5.5-8.9, 6.5-7.5, 6.5-8.5, 6.5-8.9, 7.5-8.5, 7.5-9, 8.5-9, 6.9-7.1, 6.9-7.2, 6.9-7.3, 5-6.5, 5-7.5, 5-8.5, 5-8.9, 6-7.5, 6-8.5, 6-9.5, 7-7.1, 7-7.2, 7-7.3, 7-7.4, 7-7.5, 7-7.6, 7-7.7, 7-7.8, 7-7.9, 7-9.5, 8-9.5, 5.5-6, 5.5-7, 5.5-8, 5.5-8.9, 6.5-7, 6.5-8, 6.5-9, or 7.5-8. In some embodiments, the buffer has a pH of about 7, 7.1, 7.2, 7.3, 7.4, or 7.5.

[0094] In some embodiments, the solvent is a polar aprotic solvent. In some embodiments, the solvent is a polar aprotic solvent that is miscible with water. In some embodiments, the solvent is dimethyl sulfoxide (DMSO). In some embodiments, the solvent is acetone, acetonitrile, dichloromethane, dimethylformamide, dimethylpropyleneurea, dimethyl sulfoxide, ethyl acetate, hexamethylphosphoramide, pyridine, sulfolane, tetrahydrofuran, or any combination thereof.

[0095] In some embodiments, the solvent (e.g., DMSO) is added at a concentration of about 15% (v / v) to about 40% (v / v) (e.g., about 15%-20%, about 15%-25%, about 15%-30%, about 15%-35%, about 15%-40%, about 20%-25%, about 20%-30%, about 20%-35%, about 20%-40%, about 25%-30%, about 25%-35%, about 25%-40%, about 30%-35%, about 30%-40%). In some embodiments, the solution is added at a concentration of about 20% (v / v).

[0096] In some embodiments, one or more AIs are non-covalently conjugated to a tobamo virus (e.g., TMGMV and / or TMV). In some embodiments, the tobamo virus (e.g., TMGMV and / or TMV) comprises one or more coat proteins that reversibly and partially dissociate in response to the presence of a solvent (e.g., DMSO). This method is further described in Example 5.

[0097] In some embodiments, the solvent is added dropwise. In some embodiments, one or more AIs are added dropwise. In some embodiments, one or more AIs are added stepwise over a period of time. In some embodiments, one or more AIs are added dropwise over about 0.5 hours to about 10 days (e.g., about 0.5 - 1 hour, 0.5 - 2 hours, 0.5 - 3 hours, 0.5 - 4 hours, 0.5 - 5 hours, 0.5 - 6 hours, 0.5 - 7 hours, 0.5 - 8 hours, 0.5 - 9 hours, 0.5 - 10 hours, 0.5 - 11 hours, 0.5 - 12 hours, 0.5 - 13 hours, 0.5 - 14 hours, 0.5 - 15 hours, 0.5 - 16 hours, 0.5 - 17 hours, 0.5 - 18 hours, 0.5 - 19 hours, 0.5 - 20 hours, 0.5 - 21 hours, 0.5 - 22 hours, 0.5 - 23 hours, 0.5 - 24 hours, 0.5 hours - 2 days, 0.5 hours - 3 days, 0.5 hours - 4 days, 0.5 hours - 5 days, 0.5 hours - 6 days, 0.5 hours - 7 days, 0.5 hours - 8 days, 0.5 hours - 9 days, 0.5 hours - 9.9 days, 6 hours - 12 hours, 6 hours - 1 day, 6 hours - 2 days, 6 hours - 3 days, 6 hours - 4 days, 6 hours - 5 days, 6 hours - 6 days, 6 hours - 7 days, 6 hours - 8 days, 6 hours - 9 days, 6 hours - 10 days, 12 hours - 1 day, 12 hours - 2 days, 12 hours - 3 days, 12 hours - 12 days, 12 hours - 12 days, 12 hours - 6 days, 12 hours - 7 days, 12 hours - 8 days, 12 hours - 9 days, 12 hours - 10 days, 1 day - 2 days, 1 - 3 days, 1 - 4 days, 1 - 5 days, 1 - 6 days, 1 - 7 days, 1 - 8 days, 1 - 9 days, 1 - 10 days, 2 - 5 days, 2 - 10 days, or 5 - 10 days. In some embodiments, one or more AIs are added once a day.

[0098] In some embodiments, the method comprises adding a solvent (e.g., DMSO), adding one or more AIs to the tobamovirus-buffer, and then incubating the one or more AIs in the tobamovirus-buffer for about 0.5 hours to about 36 hours (e.g., about 0.5 to 1 hour, 0.5 to 2 hours, 0.5 to 3 hours, 0.5 to 4 hours, 0.5 to 5 hours, 0.5 to 6 hours, 0.5 to 7 hours, 0.5 to 8 hours, 0.5 to 9 hours, 0.5 to 10 hours, 0.5 to 11 hours, 0.5 to 12 hours, 0.5 to 13 hours, 0.5 to 14 hours, 0.5 to 15 hours, 0.5 to 16 hours, 0.5 to 17 hours, 0.5 to 18 hours, 0.5 to 19 hours, 0.5 to 20 hours, 0.5 to 21 hours, 0.5 to 22 hours, 0.5 to 23 hours, 0.5 to 24 hours, 0.5 to 30 hours, 0.5 to 35.Incubating for 9 hours, 1 - 2 hours, 1 - 3 hours, 1 - 4 hours, 1 - 5 hours, 1 - 6 hours, 1 - 7 hours, 1 - 8 hours, 1 - 9 hours, 1 - 10 hours, 1 - 11 hours, 1 - 12 hours, 1 - 13 hours, 1 - 14 hours, 1 - 15 hours, 1 - 16 hours, 1 - 17 hours, 1 - 18 hours, 1 - 19 hours, 1 - 20 hours, 1 - 21 hours, 1 - 22 hours, 1 - 23 hours, 1 - 24 hours, 1 - 30 hours, 1 - 36 hours, 2 - 3 hours, 2 - 4 hours, 2 - 5 hours, 2 - 6 hours, 2 - 7 hours, 2 - 8 hours, 2 - 9 hours, 2 - 10 hours, 2 - 11 hours, 2 - 12 hours, 2 - 13 hours, 2 - 14 hours, 2 - 15 hours, 2 - 16 hours, 2 - 17 hours, 2 - 18 hours, 2 - 19 hours, 2 - 20 hours, 2 - 22 hours, 2 - 22 hours, 2 - 23 hours, 2 - 24 hours, 2 - 30 hours, 2 - 36 hours, 3 - 4 hours, 3 - 5 hours, 3 - 6 hours, 3 - 7 hours, 3 - 8 hours, 3 - 9 hours, 3 - 10 hours, 3 - 11 hours, 3 - 12 hours, 3 - 13 hours, 3 - 14 hours, 3 - 15 hours, 3 - 16 hours, 3 - 17 hours, 3 - 18 hours, 3 - 19 hours, 3 - 20 hours, 3 - 21 hours, 3 - 22 hours, 3 - 23 hours, 3 - 24 hours, 3 - 30 hours, 3 - 36 hours, 4 - 5 hours, 4 - 6 hours, 4 - 7 hours, 4 - 8 hours, 4 - 9 hours, 4 - 10 hours, 4 - 11 hours, 4 - 12 hours, 4 - 13 hours, 4 - 14 hours, 4 - 15 hours, 4 - 16 hours, 4 - 17 hours, 4 - 18 hours, 4 - 19 hours, 4 - 20 hours, 4 - 21 hours, 4 - 22 hours, 4 - 23 hours, 4 - 24 hours, 4 - 30 hours, 4 - 36 hours, 8 - 9 hours, 8 - 10 hours, 8 - 11 hours, 8 - 12 hours, 8 - 13 hours, 8 - 18 hours, 8 - 15 hours, 8 - 16 hours, 8 - 17 hours, 8 - 18 hours, 8 - 19 hours, 8 - 20 hours, 8 - 21 hours, 8 - 22 hours, 8 - 23 hours, 8 - 24 hours, 8 - 30 hours, 8 - 36 hours, 12 - 13 hours, 12 - 18 hours, 12 - 15 hours, 12 - 16 hours, 12 - 17 hours, 12 - 18 hours, 12 - 19 hours, 12 - 20 hours, 12 - 21 hours, 12 - 22 hours, 12 - 23 hours, 12 - 24 hours, 12 - 30 hours, 12 - 36 hours, 24 - 30 hours, or 12 - 36 hours) further includes.

[0099] In some embodiments, after adding the solvent and one or more AIs, the solution in which the nanoparticles are purified has a pH of about 5 to about 9 (e.g., about 5 to 6, 5 to 7, 5 to 8, 5 to 8.9, 6 to 7, 6 to 8, 6 to 9, 7 to 8, 7 to 9, 8 to 9, 5.5 to 6.5, 5.5 to 7.5, 5.5 to 8.5, 5.5 to 8.9, 6.5 to 7.5, 6.5 to 8.5, 6.5 to 8.9, 7.5 to 8.5, 7.5 to 9, 8.5 to 9, 6.9 to 7.1, 6.9 to 7.2, 6.9 to 7.3, 5 to 6.5, 5 to 7.5, 5 to 8.5, 5 to 8.9, 6 to 7.5, 6 to 8.5, 6 to 9.5, 7 to 7.1, 7 to 7.2, 7 to 7.3, 7 to 7.4, 7 to 7.5, 7 to 7.6, 7 to 7.7, 7 to 7.8, 7 to 7.9, 7 to 9.5, 8 to 9.5, 5.5 to 6, 5.5 to 7, 5.5 to 8, 5.5 to 8.9, 6.5 to 7, 6.5 to 8, 6.5 to 9, or 7.5 to 8). In some embodiments, the solution in which the nanoparticles are purified has a pH of about 6.9, 7.0, 7.1, 7.2, or 7.3.

[0100] In some embodiments, when preparing the nanoparticles using the pH method or the solvent method, one or more AIs are added to the tobamovirus-buffer more than twice. In some embodiments, one or more AIs are added at least once a day. In some embodiments, when preparing the nanoparticles using the pH method or the solvent method, one or more AIs are added dropwise only once a day. In some embodiments, when preparing the nanoparticles using the pH method or the solvent method, one or more AIs are added until an equivalent ratio of about 10:1, 25:1, 50:1, 75:1, 100:1, 150:1, 200:1, 250:1, 300:1, 350:1, 400:1, 450:1, 500:1, 550:1, 600:1, 650:1, 700:1, 750:1, 800:1, 850:1, 900:1, 950:1, or 1000:1 is reached. In some embodiments, when preparing the nanoparticles using the pH method or the solvent method, one or more AIs are added in a 1000, 1500, 2000, 2500, 3000, 3300, 4000, 4500, 5000, 5500, 6500, 7000, 7500, 8000, 8500, 9000, or 9500-fold molar excess relative to the tobamovirus (e.g., TMGMV and / or TMV). In some embodiments, when preparing the nanoparticles using the pH method or the solvent method, one or more AIs of 100, 150, 200, 250, 300, 350, 400, 450, or 500 nmol per gram of the tobamovirus (e.g., TMGMV and / or TMV) are added.

[0101] Methods for formulating and delivering compositions to soil, crops, and plants are well known in the art (U.S. Pat. Nos. 5,091,188, 5,091,187, 5,250,236, 5,472,706, 5,750,142, 5,874,029, 5,879,715, 4,725,442, 6,835,396, 6,872,773, 8,404,263, 9,095,133, PCT Applications Nos. 2005 / 102507, 2005 / 020933, 2005 / 072680, 01 / 88046, 2007 / 014826, U.S. Application Nos. 2005 / 0170004, 2006 / 0063676, 2014 / 164418). The disclosed formulations can be mixed in any order in single or multi-stage mixing. One or more of the compounds / AIs can be added to a formulation comprising tobamovirus (e.g., TMGMV and / or TMV) nanoparticles, and examples of suitable agrochemical formulations are EC (emulsifiable concentrate) formulations; SL or LS (soluble concentrate) formulations; EW (emulsion, oil-in-water type) formulations; ME (microemulsion) formulations; MEC (microemulsifiable concentrate) formulations; CS (capsule suspension) formulations; TK (technical concentrate) formulations; OD (oil-based suspension concentrate) formulations; SC (suspension concentrate) formulations; SE (suspension emulsion) formulations; ULV (ultra-low volume liquid) formulations; SO (spray oil) formulations; AL (any other liquid) formulations; LA (lacquer) formulations; DC (dispersible concentrate) formulations, etc., liquid formulations; or WG (water-dispersible granule) formulations; TB (tablet) formulations; FG (fine granule) formulations; MG (microgranule) formulations; SG (soluble granule) formulations, etc., solid formulations. In some embodiments, the liquid formulations are EC, SL, LS, EW, ME, MEC, TK, OD, SC, SE, ULV, SO, AL, LA, and DC.

[0102] Pharmaceutical composition In certain embodiments, disclosed herein is a pharmaceutical composition comprising the nanoparticles described herein. Two or more (e.g., two, three, or four) of any of the types of therapeutic nanoparticles described herein may be present in the pharmaceutical composition in any combination. The pharmaceutical composition may be formulated in any manner known in the art.

[0103] In some embodiments, the pharmaceutical composition comprises at least one pharmaceutically acceptable carrier, diluent, or excipient. In some embodiments, the pharmaceutical composition is formulated into a dosage form that is an injectable solution, freeze-dried powder, suspension, or any combination thereof.

[0104] The pharmaceutical composition is formulated to be compatible with their intended route of administration (e.g., intravenous, intraarterial, intramuscular, intradermal, subcutaneous, or intraperitoneal). In some embodiments, the compositions provided herein may contain a pharmaceutically acceptable diluent (e.g., a sterile diluent). In some embodiments, the pharmaceutically acceptable diluent may be sterile water, sterile saline, an inert oil, polyethylene glycol, glycerin, propylene glycol, or other synthetic solvents, antibacterial or antifungal agents such as benzyl alcohol or methylparaben, chlorobutanol, phenol, ascorbic acid, or thimerosal, antioxidants such as ascorbic acid or sodium bisulfite, chelating agents such as ethylenediaminetetraacetic acid, buffers such as acetate, citrate, or phosphate, and isotonic agents such as sugars (e.g., dextrose), polyalcohols (e.g., mannitol or sorbitol), or salts (e.g., sodium chloride), or any combination thereof.

[0105] In some embodiments, the pharmaceutical compositions provided herein may include a pharmaceutically acceptable carrier. The preparations of the compositions may be formulated and enclosed within an ampoule, disposable syringe, or multiple-dose vial. If necessary (e.g., in injectable formulations), appropriate fluidity can be maintained by the use of coatings such as lecithin or surfactants. The absorption of nanoparticles can be extended by including agents that retard absorption (e.g., aluminum monostearate and gelatin). Alternatively, controlled release can be achieved by implant and microencapsulation delivery systems that may include biodegradable, biocompatible polymers (e.g., ethylene vinyl acetate, polyanhydrides, polyglycolic acid, collagen, polyorthoesters, and polylactic acid).

[0106] Compositions containing one or more of any of the nanoparticles described herein can be formulated in unit dosage form (i.e., physically discrete units containing a predetermined amount of the active compound for ease of administration and uniformity of dosage) for parenteral (e.g., intravenous, intraarterial, intramuscular, intradermal, subcutaneous, or intraperitoneal) administration. In some embodiments, compositions containing one or more of any of the nanoparticles described herein can be formulated into injectable dosage forms, lyophilized powders, suspensions, or any combination thereof.

[0107] The toxicity and therapeutic efficacy of the compositions can be determined by standard pharmaceutical procedures in cell cultures or experimental animals (e.g., monkeys). For example, the LD50 (the dose lethal to 50% of the population) and ED50 (the dose therapeutically effective in 50% of the population) can be determined, and the therapeutic index is the ratio of LD50:ED50. Agents with a high therapeutic index are preferred. If the agent exhibits undesirable side effects, care should be taken to minimize potential damage (i.e., reduce undesirable side effects). Toxicity and therapeutic efficacy can be determined by other standard pharmaceutical procedures.

[0108] In formulating an appropriate dosage of any given agent for use in a subject (e.g., a human), data obtained from cell culture assays and animal studies can be used. A therapeutically effective amount of one or more (e.g., one, two, three, or four) nanoparticles (e.g., any of the nanoparticles described herein) can be an amount that reduces cancer cell invasion or metastasis in a subject (e.g., a human) having cancer, or reduces and / or eliminates infection in a subject (e.g., a human).

[0109] The efficacy and dosage of any of the nanoparticles described herein can be determined by medical practitioners using methods known in the art, as well as by observation of one or more symptoms of a disease (e.g., cancer or infection) in a subject (e.g., a human). Certain factors (e.g., the severity of the disease or disorder, previous treatments, the general health status and / or age of the subject, and the presence of other diseases) can affect the dosage and timing required to effectively treat the subject.

[0110] One of ordinary skill in the art will understand that therapeutic agents containing the nanoparticles described herein vary in their potency and that an effective amount can be determined by methods known in the art. Typically, a relatively low dose is initially administered, and the attending physician (in the case of therapeutic use) or researcher (if still working in the development stage) may then gradually increase the dose until an appropriate response is obtained. In addition, the specific dosage level for any particular subject will depend on a variety of factors, including the activity of the specific compound used, the age, weight, general health status, sex, and diet of the subject, the time of administration, the route of administration, the rate of excretion, and the half-life of the nanoparticles in vivo.

[0111] The pharmaceutical composition can be included in a kit, container, pack, or dispenser, together with instructions for administration.

[0112] Method of treatment Also provided herein is a method of doing so in a subject in need of treating cancer. The method of treating cancer includes administering the nanoparticles of the present disclosure or the pharmaceutical composition of the present disclosure to a subject in need of cancer treatment. In some embodiments, the nanoparticles or pharmaceutical composition are administered in an effective amount. In some embodiments, the cancer includes breast cancer, ovarian cancer, glioma, gastrointestinal cancer, prostate cancer, cancer, lung cancer, hepatocellular cancer, testicular cancer, cervical cancer, endometrial cancer, bladder cancer, head and neck cancer, lung cancer, gastro-esophageal cancer, gynecological cancer, or any combination thereof.

[0113] Also provided herein is a method of doing so in a subject in need of treating an infection. The method of treating an infection includes administering the nanoparticles of the present disclosure or the pharmaceutical composition of the present disclosure to a subject in need of infection treatment. In some embodiments, the nanoparticles or pharmaceutical composition are administered in an effective amount. In some embodiments, the infection is a bacterial infection, viral infection, fungal infection, parasitic infection, or any combination thereof.

Examples

[0114] The present disclosure is further described in the following examples, which do not limit the scope of any of the embodiments recited in the claims.

[0115] Method Preparation of TMGMV TMGMV was obtained from BioProdex (Gainesville, FL, USA) and stored at -20 °C until use. The solution was thawed overnight at 4 °C and then dialyzed against potassium phosphate buffer (KP; 10 mM, pH 7.2) at 4 °C for 24 h using a 12–14 kDa dialysis tube (Fisher Scientific S432700; Waltham, MA, USA). The buffer solution was exchanged and dialysis was continued for an additional 48 h. The solution was then centrifuged at 10,000 × g for 20 min (Beckman Coulter Allegra or Avanti centrifuge). The supernatant was collected and ultracentrifuged at 42,000 rpm for 2.5 h 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 under rotary mixing. The sample concentration was then confirmed using a Nanodrop 2000 (Thermo Scientific; Waltham, MA, USA), and the concentration was adjusted to 10 mg mL - 1 in 10 mM KP and then stored at 4 °C (for TMGMV CP, ε 260 = 3 mL mg -1 cm -1 ).

[0116] Preparation of diazonium salts from 4-ethynylaniline In a 5 mL tube, 298 mg of 4-ethynylaniline was dissolved in 2 mL of methanol. In a 50 mL tube, 1.09 g of p-toluenesulfonic acid was dissolved in 20 mL of DIH2O. Both solutions were placed at -20 °C for 10 minutes to pre-cool. A 1.5 mL solution of 3 M sodium nitrite (258 mg in 1.5 mL of DIH2O) was prepared and placed at -20 °C for 5 minutes to pre-cool. A 50 mL beaker was submerged in an ice / water slurry on a stirring plate. The solutions were taken out of the freezer. A stir bar was added to the 20 mL of pre-cooled acid in the submerged beaker. After mixing, the methanol solution was added. The solution turned an opaque beige color. The nitrite solution was slowly added dropwise to the acid solution, and the mixture gradually turned yellow and finally red after a reaction time of 30 - 60 minutes. A 1 mL sample of the diazonium slurry was collected and centrifuged at 10,000×g for 2 minutes to isolate the diazonium salt. On ice, the supernatant was removed and the diazonium salt was resuspended in 1 mL of pre-cooled ethanol. The prepared diazonium salt was immediately used for tyrosine modification.

[0117] Coupling of Diazonium to TMGMV A 962 μL solution of 1 mg mL - of TMGMV in 100 mM borate buffer (pH 8.5) was prepared and pre-cooled on ice. The diazonium salt solution was added to the TMGMV solution in 80 μL volumes. The solution was mixed by inversion and reacted on ice for 30 minutes. The solution was centrifuged on a sucrose cushion (30% w / v) for 1 hour at 50,000 rpm in a benchtop ultracentrifuge (Beckman Optima MAX-XP equipped with a TLA-55 rotor). The virus pellet was resuspended in 10 mM KP at 4 °C overnight on a rotary shaker.

[0118] Copper-Catalyzed Alkyne Azide Cycloaddition Reaction To an ultracentrifugation tube (Beckman Coulter 357448, Indianapolis, IN, USA), 1 mg of TMGMV was added. The reaction medium consisted of 1 mM copper sulfate, 2 mM aminoguanidine, 2 mM L - ascorbic acid, and 3.7 mM tris(benzyltriazolylmethyl)amine. 50 equivalents of 6A - azido - 6A - deoxy - β - cyclodextrin (TCI Chemicals) per TMGMV coat protein were added, and the volume of 10 mM KP (pH 7) was adjusted to a final volume of 500 μL. The reaction was allowed to proceed on ice for 1 hour. To the bottom of the same tube, 200 μL of a sucrose cushion (30% w / v) was added, and then the sample was ultracentrifuged at 50,000 rpm for 1 hour at 4 °C (Beckman Optima MAX - XP equipped with a TLA - 55 rotor). The supernatant was removed, and the pellet was resuspended under rotary mixing overnight at 4 °C and then further characterized.

[0119] Characterization of Chemically Labeled β - CD - TMGMV SDS - PAGE: The denatured β - CD conjugate TMGMV sample (10 μg) was loaded onto a 12% NuPAGE gel (Life Technologies) and run on 1×MOPS Running Buffer (Life Technologies). The protein was stained using Gel Code Blue dye (Life Technologies) and visualized under white light.

[0120] FPLC (Size - Exclusion Chromatography): A Superose6 Increase 100GL column and an AKTA Pure25 chromatography system (GE Healthcare) using a flow rate of 0.5 mL / min in 10 mM KP (pH 7.4) were used to analyze the β - CD conjugate TMGMV sample (500 μL at 0.5 mg / mL). Absorbance at 260 and 280 nm was recorded.

[0121] TEM Imaging: 0.05 mg mL -1The sample was diluted to the concentration and absorbed onto a carbon-coated TEM grid (Electron Microscopy Sciences). Subsequently, the grid was washed three times with pure water. Then, for imaging, the grid was stained with 2% (w / v) uranyl acetate for 2 minutes. TEM was performed using an FEI Tecnai F30 transmission electron microscope operating at 300 kV.

[0122] Loading of pesticides onto β-CD-TMGMV To evaluate the loading of pesticides onto β-CD-TMGMV, a competition assay between doxorubicin (ApexBio) and the target molecule was completed. Samples with a volume of 100 μL were prepared in 96-well plates (Costar). In the sample wells, in addition to doxorubicin at 10 equivalents β-CD-TMGMV (molar equivalents relative to the β-CD conjugate TMGMV), 0.0825 mg of the β-CD conjugate TMGMV in KP buffer was added to the wells.

[0123] Three control conditions of 1 equivalent of TMGMV containing 1 equivalent of TMGMV, 575 equivalents of β-CD, and 575 equivalents of β-CD (non-conjugated) were utilized. These control wells also received doxorubicin at 10 equivalents. The plate was incubated overnight at 4 °C on a plate rocker (Fisher). Fluorescence top readings were taken on the plate via a UV-Vis Plate Reader (Tecan infinite200Pro) (excitation 470 nm, emission 595 nm, 25 flashes). At the completion of the fluorescence measurement, 0, 10, 100, or 1000 equivalents of β-CD-TMGMV either clothianidin (BASF), fluopyram (BASF), or tetracycline (Sigma-Aldrich) was added to the β-CD-TMGMV and control wells. After repeated incubation overnight at 4 °C on a plate rocker (Fisher), the fluorescence readings were repeated as before.

[0124] Samples were prepared in 96-well plates (Costar) in 100 μL volumes. In the sample wells, 0.0825 mg of β-CD-conjugated TMGMV in KP buffer was added to the wells in addition to doxorubicin at 10 equivalents (molar equivalents relative to β-CD-conjugated TMGMV). Three control conditions were utilized: 1 equivalent of TMGMV, 575 equivalents of β-CD, and 1 equivalent of TMGMV with 575 equivalents of β-CD (unconjugated). These control wells also received doxorubicin at 10 equivalents. Incubation was overnight at 4° C. on a plate rocker (Fisher) to allow doxorubicin to load onto the β-CD-TMGMV particles. A fluorescent top read was then taken on the plate via a UV-Vis Plate Reader (Tecan infinite200Pro) (excitation 470 nm, emission 595 nm, 25 flashes). Upon completion of fluorescence measurements, β-CD-TMGMV and control wells received either clothianidin (CTD; BASF), fluopyram (FLP; BASF), or tetracycline (TET; Sigma-Aldrich) at 10, 100, or 1000 equivalents. Incubation was repeated overnight at 4° C., and then these molecules competed against doxorubicin for entrapment on β-CD-TMGMV. Fluorescence was then read on a plate reader as previously described.

[0125] Partial dissociation (also called "breathing") 5 mg mL in KP buffer, pH 7.5 -1 TMGMV at a concentration of 0.01% was kept at 4°C for 5 days. The addition of the target AI to the solution was done every 24 hours until an equivalence ratio of 500:1 was reached and left to mix on a rotary shaker. The solution was then centrifuged on a sucrose cushion (30% w / v) for 1 hour in a tabletop ultracentrifuge (Beckman Optima MAX-XP with a TLA-55 rotor) at 50,000 rpm. The virus pellet was then resuspended in 10 mM KP (pH 7) overnight at 4°C on a rotary shaker. After complete resuspension, the solution was dialyzed for 48 hours to remove excess (unentrapped) AI.

[0126] TEM imaging 0.05 mg / mL -1 The sample was diluted to a concentration of and absorbed onto a carbon-coated TEM grid (Electron Microscopy Sciences). The grid was then washed three times with pure water. Then, for imaging, the grid was stained with 2% (w / v) uranyl acetate for 2 minutes. TEM was performed using an FEI Tecnai F30 transmission electron microscope operating at 300 kV.

[0127] Soil mobility assay 0.32 g / cm -3 A cylindrical column (diameter 28 mm, upper height 30 cm) was filled with Garden Magic Top Soil at a density of and saturated with deionized water to remove air pockets. Due to depth and time-dependent compression effects, the density of soil in the actual environment may be higher (0.6 - 1.6 g / cm -3 ). A bolus containing 1 mg of each formulation, with and without conjugated or injected dye molecules, was injected onto the top of the soil column, and the column was saturated with deionized water at a constant flow rate of 1.5 cm 3 / min - . The eluate was collected at the bottom of the column in fractions of 500 μL - 2 mL. A maximum of 200 fractions were collected in each test (two tests per depth for each formulation).

[0128] The eluate fractions were analyzed by SDS-PAGE to determine the mass and amount of the nanoparticles recovered in each eluate fraction. TMGMV nanoparticles were analyzed on a 4–12% NuPage polyacrylamide SDS gel cast according to the Surecast Handcast protocol (Invitrogen). 25 μl of each eluate fraction was mixed with 5 μl of 5× SDS loading buffer, and the samples were separated at 200 V and 120 mA for 1 h with a SeeBlue Plus2 ladder size. The gel was then stained with Gel Code Blue Stain (Life Technologies), microwave treated for 1 min, and then stirred for 5 min. The process was then repeated with deionized water for destaining. The gel was imaged using a FluorChem R system.

[0129] All the nanoparticles were imaged and analyzed using ImageJ.

[0130] Loading of Hydrophobic Cargo into TMGMV by pH Increase from pH 7 to pH 7.5 Loading of AI into TMGMV was carried out via the “pH method”. TMGMV at a concentration of 1 mg mL -1 in 10 mM KP buffer at pH 7.5 was maintained at 4 °C for 10 days. The following AIs were used: fluopyram and clothianidin (BASF, Berkeley, CA, USA), rifampicin and ivermectin (BioVision; Milpitas, CA, USA). Cy5 (Lumiprobe; Cockeysville, MD, USA) and doxorubicin (ApexBio; Houston, TX, USA) were also studied as proof-of-concept (fluorescent molecule and cancer chemotherapy). AI was added to TMGMV by daily addition of an excess 10:1 AI: coat protein (CP; each TMGMV rod is assembled from approximately 2,100 identical CPs) until a ratio of 100:1 was reached. During this process, the reactants were continuously mixed on a rotary shaker. 1 mL aliquots were obtained daily for further analysis.

[0131] After loading with AI, aliquots were spin-filtered using a 100K molecular weight cut-off 0.5 mL filter (MilliporeSigma, Burlington, MA, USA). 200 μL of the aliquot and 250 μL of KP solution were added, then centrifuged at 16,160×g for 5 minutes at 4 °C, the flow-through fraction was discarded, then 450 μL of KP was added and centrifuged again, and this step was repeated three times. After the third centrifugation, the filter was inverted in a new tube and centrifuged at 1000×g for 2 minutes to recover the supernatant, and subsequent characterization was performed.

[0132] Injection of Hydrophobic Cargo into TMGMV by Changing DMSO Concentration Loading of AI into TMGMV was carried out via the "DMSO method". TMGMV in 10 mM KP buffer (pH 7.2) was diluted to 5 mg mL-1 in 2 mL of buffer, transferred to a 25 mL beaker, and magnetically stirred at 300 rpm at room temperature. Solutions of DMSO and 10 mM KP were added dropwise to dilute the solution to 20% (v / v) DMSO and 2 mg mL-1 TMGMV. An aliquot of AI was added dropwise to the solution to prevent precipitation. The solution was stirred at room temperature for 24 hours. Samples were collected, spin-filtered as described above, and then stored at 4 °C.

[0133] TEM The TMGMV sample was diluted to a concentration of 0.05 mg mL-1 and absorbed onto a carbon-coated TEM grid (Electron Microscopy Sciences, Hatfield, PA, USA). The grid was then washed three times with pure water. The grid was then stained with 2% (w / v) uranyl acetate for 90 seconds. TEM was performed using an FEI Tecnai F30 transmission electron microscope operating at 300 kV. ImageJ software (https: / / imagej.nih.gov / ij / download.html) was used to perform image analysis. To determine the change in the width of the nanoparticles, the width of a 100 nm long section was measured for normalization purposes. Five different sections were measured for each micrograph, and a total of 30 sections were measured for each sample. Subsequently, for the complete particles, the length, perimeter, and area were measured, and the average width was calculated from the perimeter later.

[0134] Size exclusion chromatography The TMGMV sample (500 μL at 0.5 mg / mL) was analyzed using a Superose6 Increase 100GL column and an AKTA Pure 25 chromatography system (GE Healthcare, Chicago, Il, USA) using a flow rate of 0.5 mL / min in 10 mM KP (pH 7.4). Absorbance at 260 and 280 nm was recorded.

[0135] Circular dichroism spectroscopy The CD spectra were obtained using an Aviv Model 215 CD spectrometer (Lakewood, NJ, USA). All samples were run at 25 °C in a quartz cuvette with a 2 mm path length (Starna Cells, Atascadero, CA, USA). Samples were dissolved in 10 mM KP buffer at pH 7 to concentrations ranging from 0.025 mg / mL to 0.5 mg / mL to obtain a volume of 400 μL for each CD run. In separate scans, near-UV and far-UV spectra were obtained. For the far-UV spectra, the samples were scanned from 250 nm to 180 nm with a wavelength step of 1 nm and an averaging time of 1 s. For the near-UV spectra, the samples were scanned from 310 nm to 240 nm with a wavelength step size of 0.5 nm and an averaging time of 1 s. All spectra were scanned twice and averaged within each UV region.

[0136] Small molecule quantification by high performance liquid chromatography (HPLC) For HPLC, AI was extracted from TMGMV. Briefly, the concentration of the TMGMV sample was adjusted to 1.2 mg mL-1 in KP. The solution was diluted 4-fold in a 1:1 acetonitrile / methanol mixture and vortexed for 30 s. The solution was centrifuged at 10,000×g for 10 min at 4 °C, and the organic phase (bottom fraction) was collected and transferred to a 2 mL glass screw-top vial for HPLC (SureSTART, Thermo Scientific, Waltham, MA, USA).

[0137] After dilution 10-fold with acetonitrile, 500 μL of the extracted sample was injected and run on a 5 μm C18 column (20×100 mm) using a Shimadzu LC-40 HPLC system (Columbia, MD, USA). The method was run at 0.5 mL min-1 for 15 min per sample with a gradient of acetonitrile and 0.02% (v / v) phosphoric acid. Absorbance values were collected at 280 nm (fluopyram), 269 nm (clothianidin), 225 nm (ivermectin), and 330 nm (rifampicin) using a photodiode array. Absorbance values were fitted to a standard curve to identify the sample concentration with N = 3.

[0138] Example 1 β-CD-TMGMV formation Experiments were conducted to test the formation and integrity of the conjugated TMGMV particles before AI loading.

[0139] By SDS-PAGE, covalent attachment of β-CD to CP was confirmed as indicated by the additional higher molecular weight (MW) band in lane 3 of Figure 2, and the increase in MW corresponded to the size of β-CD. The conjugation efficiency was estimated to be approximately 35% based on ImageJ band density analysis of β-CD-TMGMV subunit protein versus TMGMV and TMGMV-alkyne. This suggests that using a 50:1 molar excess of β-CD to CP resulted in approximately 750 molecules per particle being conjugated.

[0140] To verify the structural integrity of the modified TMGMV particles, size exclusion chromatography (SEC) and transmission electron microscopy (TEM) were performed. As seen in Figure 3, SEC measurements showed no significant difference between native TMGMV and conjugated TMGMV (shown below), with an elution volume of approximately 9 mL and an A260:280 ratio of 1.2, similar to that of the native, indicating intact TMGMV. Furthermore, no obvious signs of aggregation or particle dissociation were observed. As seen in Figure 4, TEM imaging confirmed the structural integrity of β-CD-TMGMV after modification and purification.

[0141] Example 2 AI loading onto β-CD-TMGMV Experiments were conducted to test the loading (or "encapsulation") of AI into β-CD-TMGMV. By quantifying the loading ("encapsulation") of pesticides, many difficulties were presented due to the lack of fluorescence of the molecules. To quantify the encapsulation, a competitive assay was performed (see the schematic diagram in Figure 5). Doxorubicin ("DOX") with known excitation (470 nm) and emission (595 nm) wavelengths was added to the samples and control wells. Free doxorubicin can fluoresce at 595 nm, but encapsulated doxorubicin cannot, and this disparity enables the measurement of free doxorubicin in the solution (and the indirect quantification of the encapsulated molecules).

[0142] By measuring the fluorescence of unencapsulated doxorubicin, it was determined that 88.59% of what was loaded into the wells was encapsulated by β-CD-TMGMV. This encapsulation, measured as a decrease in the fluorescence of doxorubicin (as withdrawn from the solution), confirms the molecular loading capacity of β-CD-TMGMV. As expected, the addition of pesticides resulted in a dose-dependent replacement of doxorubicin from β-CD-TMGMV, with higher concentrations of pesticides replacing more doxorubicin. This increase in the amount of free doxorubicin in the wells resulted in an increase in relative fluorescence. As seen in the table in Figure 6, when challenged with the addition of clothianidin ("CTD"), fluopyram ("FLP"), or tetracycline ("TET"), 1000 equivalents of tetracycline replaced 47% of the doxorubicin that was initially bound to β-CD-TMGMV. 1000 equivalents of clothianidin and fluopyram replaced 17.60% and 18.34% of the doxorubicin, respectively. These data correlate with the degree of hydrophobicity of the molecules, and as expected, β-CD functioned as a bucket for most readily encapsulating hydrophobic molecules.

[0143] Example 3 AI Loading into TMGMV Experiments were conducted to load pesticides into TMGMV by strategically changing the pH. Without being constrained by theory, this entraps AI through the formation of "pockets" between coat proteins (CPs). Without being constrained by theory, the principle is by increasing the pH of the buffer, the virus begins to dissociate, creating hydrophobic pockets between the coat proteins of the virion. Then, AI is added to interact with the virus particles, and then the pH is decreased to promote self-assembly of the particles and entrapment of AI on the hydrophobic pockets (see the schematic diagram in Figure 7). As discussed above, TMGMV at a concentration of 5 mg mL-1 in KP buffer at pH 7.5 was held at 4 °C for 5 days, and the target AI was added to the solution every 24 hours until an equivalent ratio of 500:1 was reached. The solution was then centrifuged at 50,000 rpm for 1 hour on a sucrose cushion (30% w / v). The virus pellet was then resuspended in 10 mM KP at pH 7 at 4 °C overnight, and after complete resuspension, the solution was dialyzed for 48 hours to remove excess (unentrapped) AI. The samples were then observed by TEM and analyzed using ImageJ.

[0144] To determine the change in width (breathing / injection) of the nanoparticles, the width of sections of 100 nm long nanoparticles (as shown in the micrograph in Figure 8) was measured. This was done to normalize the measurements and determine the change in width. At least 5 different sections were measured for each micrograph, and a total of at least 30 sections were measured for each sample.

[0145] After measurement, the differences between native TMGMV (control) and TMGMV injected with doxorubicin, ATTO550, fluopyram, and clothianidin (Figures 9A - 9F) were determined. The largest increase was observed with doxorubicin, which had an average width of 35 nm compared to 18 nm for the control (DOX: 89% increase; Figure 9A compared to control Figure 9E). Clothianidin showed the second largest increase at 38% (Figure 9C; 26 nm versus 18 nm for the control). Fluopyram had a 21% increase in width (Figure 9B; 22 nm versus 18 nm), while the most hydrophilic ATTO550 of the AIs showed the smallest change, with a 7% increase in width (Figure 9D). This correlates with pockets that better confine hydrophobic compounds.

[0146] Example 4 Soil Mobility of TMGMV Nanoparticles Experiments were conducted to test the soil mobility of various TMGMV nanoparticles. The experimental setup is shown in the schematic of Figure 10A. The soil column setup was composed of cheesecloth to prevent the formation of depressions at the top of the soil. As described above (shown in Figure 10B), fractions were collected and analyzed via SDS - PAGE. The soil was treated with TMGMV and TMGMV injected and then returned to buffer after 5 days of treatment in a higher pH buffer. The gel was imaged (Figure 11A) and quantified, and the results showed that the injected TMGMV nanoparticles (Figure 11C) had the same penetration ability as those of TMGMV alone (Figure 11B). This finding confirmed that the TMGMV "breathing" technology works and that it does not affect the mobility of the nanoparticles.

[0147] TMGMV-DOX nanoparticles were prepared by loading doxorubicin onto TMGMV as described above and analyzed in soil columns via SDS-PAGE and a plate reader (shown in Figures 12A - 12B, respectively). The soil columns were 30 cm in length and divided into five fractions, each fraction being 6 cm (shown in Figure 12B). Thus, the first fraction represents the first 6 cm of soil closest to the top of the column, the third fraction (the middle fraction) is at a depth of 12 - 18 cm, and the fifth fraction is at a depth of 24 - 30 cm. TMGMV-DOX nanoparticles were found to be present in all five of the fractions (Figure 13A). The highest percentage of TMGMV-DOX nanoparticles was found in the third fraction, which represents the middle of the soil column, but more than 20% of the nanoparticles were present in the fifth fraction, which represents deep penetration at the bottom of the soil column, and more than 10% of the TMGMV-DOX nanoparticles were found in the first fraction, which is closest to the top of the soil. The results are supported by gel analysis of the fractions (Figure 13B).

[0148] TMGMV-Cy5 nanoparticles were prepared by loading Cy5 amine onto TMGMV as described above and analyzed in soil columns via SDS-PAGE and a plate reader (shown in Figures 12A - 12B, respectively). As described above, the column was divided into five fractions, and interestingly, TMGMV-Cy5 was evenly dispersed throughout all of the fractions (Figure 14A). Approximately 20% of the nanoparticles were present in all five of the fractions (Figure 14A). The results are supported by gel analysis of the soil fractions (Figure 14B).

[0149] When the soil mobility of Cy5-loaded TMGMV-βCD nanoparticles was also tested, the results showed that the loaded nanoparticles were also uniformly distributed throughout the soil. The results showed that there was no elution and the nanoparticles were mainly retained by the soil. As a reference, Cy5 was passed through the soil column. Cy5 could not penetrate well into the soil and was mainly located on top of the soil. This is supported by studies showing that Cy5 binds strongly to soil particles and cannot penetrate more than 4 cm into the soil (Chariou, et al. (2019) Nat. Nanotechnol. 14:712). These results are consistent with the data reported for abamectin (Chariou & Steinmetz (2017) ACS Nano 11,4719), fenamiphos and oxamyl (Hassan, et al. (2016) Plant Pathol. J 15,144), and other pesticides (Pestovsky & Martinez-Antonio (2017) J. Nanosci. Nanotechnol. 17,8699).

[0150] Regardless of the nanoparticle type used as the carrier, the mobility of Cy5 in the column was significantly improved compared to Cy5 alone and was better retained in the soil.

[0151] Example 5 AI Loading onto TMGMV via pH and DMSO Methods Experiments were conducted to investigate the assembly / disassembly phase diagram of TMGMV and determine conditions suitable for AI confinement. The main goal was to achieve "breathing" without complete disassembly. First, the experiments focused on pH-induced structural changes and AI injection. In this approach, the process required extensive optimization, carefully optimizing the parameters pH (7 - 8), incubation time (2 - 24 hours), protein concentration (100 - 500 equivalents of AI per CP), and AI addition interval (single supply vs. daily increments). The latter was an important parameter: bulk addition resulted in severe aggregation and insolubility - likely due to hydrophobic AI binding to the nanoparticle surface promoting interparticle association and aggregation (Figure 25). It was determined that the best results were obtained when AI was added in daily increments over 10 days, thereby ensuring particle stability and AI loading (see below). Briefly, 10 equivalents of AI per CP were supplied daily at pH 7.5, left stirring overnight, and this process was repeated for 10 days. Subsequently, the samples were spin-filtered to remove excess AI.

[0152] As a second approach, DMSO was used to disrupt coat protein - protein interactions. This was favorable for some AIs, especially those with high hydrophobicity (e.g., ivermectin and fluopyram). The advantages of this approach could be twofold: increased solubility of the AI could lead to a higher effective concentration to drive injection, and the co - solvent could prevent precipitation of the AI that would otherwise impede the injection process. To further improve this process, the TMGMV preparation was subjected to magnetic stirring and supplied from the top of the tube to prevent short - term sharp increases in AI concentration that could promote precipitation. In cases where immediate precipitation was previously shown under the pH approach, the DMSO approach showed no visible aggregates and was thus more likely to succeed by injection. Additionally, the increased stirring, the binding - disrupting effect of DMSO, and the increased concentration of AI in solution all suggested that injection should occur more rapidly under these conditions if the TMGMV nanoparticles maintained their structure.

[0153] The optimized procedures for each of the pH method and the DMSO method are shown in FIGS. 16A - 16C and are described under the "Methods" section of the above examples.

[0154] Example 6 TEM Characterization of AI-Loaded TMGMV Nanoparticles Prepared via the pH and DMSO Methods TEM imaging and quantitative TEM imaging analysis of AI-loaded TMGMV nanoparticles prepared by the pH method and the DMSO method were performed. As demonstrated by the TEM images in FIG. 17A, rod-shaped virions were observed in AI-loaded TMGMV nanoparticles prepared by both the pH and DMSO methods. Most interestingly, there were significant structural changes upon AI loading, and TMGMV loaded with AI appeared to be inflated, and the structural transition suggested AI encapsulation. To gain insights into the degree of the structural changes, quantitative TEM image analysis comparing native TMGMV to AI-loaded TMGMV was performed. Based on negative staining, the width of native virions was on average 15.7 nm (±1.9 nm), which is at least estimated for native TMGMV of 18 nm. This could be due to uranyl acetate negative staining bringing heavy shadows to the boundaries of the virions and significantly reducing their width. There was no statistical significance between the controls receiving both methodologies (FIG. 27), but for each compound, the increments (compared to native TMGMV) were different, and so were between the methodologies (FIG. 17A). Fluopyram-loaded TMGMV and ivermectin-loaded TMGMV showed maximum widths of 18 or 23 nm, respectively (FIG. 17B). This resulted in a 14% increase for fluopyram and a 46% increase in width for ivermectin compared to native TMGMV stained negatively. On the other hand, clothianidin and rifampicin loading resulted in rods of 22 - 27 nm, which were significantly thicker than native TMGMV (FIG. 17B). This resulted in a 65% increase for clothianidin and a 73% increase for rifampicin.

[0155] Example 7 Characterization of the Structure of AI-Loaded TMGMV Nanoparticles Prepared via the pH and DMSO Methods Circular dichroism (CD) was performed to observe any possible changes in the secondary structure of TMGMV after exposing the particles to breathing and injection (Figs. 18A - 18B). The effects of structural motifs on circular dichroism are additive and can be difficult to deconvolve. Rather, the differences between the spectra of the treatment groups can indicate whether a structural change has occurred. The most powerful signals in protein or viral CD are around 205 - 220 nm, which represents the sum of contributions from alpha helices, beta sheets, and aggregation. A shift in the minimum value from 208 nm to 220 nm suggests a greater contribution from aggregation behavior or alpha - helical content rather than beta sheets in both the pH and DMSO samples. Otherwise, CD did not show a change in structure as expected. Dissociating the coat protein (CP) and loading AI onto the interface - the structure did not change. In the range of 208 - 220 nm, all the AIs tested showed similar molar ellipticity profiles, suggesting no difference in secondary structure. In the near - UV range, a similar trend holds for the AIs, each sharing a signal profile of general shape. These results suggest that both the pH and DMSO approaches for breathing did not significantly change the secondary structure of TMGMV. In summary, the modest pH increase and relatively low volume fraction of DMSO used in these breathing experiments were not expected to change the tertiary rather than the secondary structure of the viral particles to enable inter - coat protein loading.

[0156] Size - exclusion chromatography (SEC) was performed to further verify the structural integrity of TMGMV particles loaded with AI during post - treatment and purification. The SEC measurements showed no significant difference between native TMGMV and TMGMV loaded with AI for any of the AIs, showing a typical elution profile from a Superose6 Increase column with elution at approximately 9 mL, indicating intact TMGMV, 1.2 A 260:280It has a ratio, where 260 nm shows RNA absorption and 280 nm shows protein absorption. In addition to the target pesticide Ai, due to the fluorescence characteristics of DOX and Cy5, they were used. DOX and Cy5 exhibit maximum absorbance at 480 nm and 647 nm, respectively. By SEC analysis, the co-localization of AI (480 nm and 647 nm) and TMGMV (260 / 280 nm) was confirmed. Using absorbance measurements and applying the Beer-Lambert law for TMGMV and AI (and their molar extinction coefficients), AI was detected, co-localized at approximately 9 mL, and the loading of approximately 615 molecules of DOX per TMGMV and approximately 80 Cy5 per TMGMV was detected. Aggregation was observed especially for Cy5, and significant particle dissociation (peaks at approximately 20 - 25 mL) was observed for Dox (Figures 26A - 26D).

[0157] Furthermore, the rods observed through TEM had non-uniform lengths, and some disks clearly showed partial degradation and damage. Also, in the ivermectin sample, globule-like structures were observed. These are thought to be precipitated ivermectin aggregates of highly hydrophobic ivermectin (Figures 17A - 17B).

[0158] The length of TMGMV nanoparticles prepared via the pH and DMSO methods was measured through image analysis. The pH treatment showed slightly less damage and had a higher distribution of lengths compared to the DMSO treatment (Figures 19A - 19J). The DMSO treatment showed that most particles were less than 100 nm. However, within AI, there was no significant difference between treatments or between compounds.

[0159] Example 8 Quantification of AI Loaded within TMGMV Nanoparticles Prepared via the pH and DMSO Methods The loaded AI was quantified using HPLC (Table 1). Using a pH-based method for injection, crothianidin and rifampicin showed successful loading 10 days after batch loading of AI in solution, achieving 1107.55 molecules per virion for crothianidin and 737.66 molecules per virion for rifampicin. In the case of fluopyram and ivermectin, a large amount of precipitate was observed, which likely removed the virus from the solution and made the AI inaccessible for diffusion into the virus. Fluopyram was calculated to have approximately 15.82 molecules per virion, and ivermectin was calculated to have 2.89 molecules per virion. Comparing these results with TEM micrographs and the change in the aspect ratio of the virus after treatment, the change in the amount and morphology of the loaded AI appears to be correlated, with crothianidin and rifampicin being the most enlarged and the change in particle width being more prominent for the virus particles than for fluopyram. Ivermectin loading appears to have a significant change in morphology, and although the amount of loaded AI was lower than that of fluopyram, it was calculated to have wider particles after treatment. This may be due to the extraction of ivermectin or issues in the molecular properties of ivermectin that permanently distort the structure of TMGMV without permanently loading the AI.

[0160] Using the DMSO method of injection over a 24-hour period, in most cases, the loading behavior of AI molecules was improved. For fluopyram, the loading increased 11.7-fold using DMSO, achieving 185.59 molecules per virion. Similar results were observed for ivermectin, with a 21.3-fold increase to 61.63 molecules per virion. Crothianidin had approximately 10% less loading using DMSO over a 24-hour period, reaching 995 molecules per virion. Higher crothianidin concentrations in solution or longer injection periods can continue to improve this number and approach the 10-day value of pH-based injection. Rifampicin loading was improved 1.5-fold using the DMSO method, reaching 1104 molecules per virion.

Table 1

[0161] These data indicate that DMSO dramatically improves the injection timeline compared to pH-based approaches. Morphologically, AI-injected TMGMV nanoparticles appear nearly identical using a 10-day pH approach compared to a 1-day DMSO approach, and using 20% DMSO indicates that particle integrity is not at risk. By reducing the time to achieve injection to 1 day, there are fewer particles degrading or precipitating out of solution, so the synthetic yield is also significantly improved. The DMSO co-solvent also reduces AI precipitation, so the effective concentration of AI for injection remains higher, driving the molecule into the TMGMV. The DMSO concentration, injection time, mixing rate, and AI-to-virus solution concentration can be further optimized.

[0162] When injecting molecules into rod-shaped TMGMV, several factors made the DMSO approach more productive. As previously mentioned, DMSO better retains the solubility of AI in aqueous buffers, thus preventing precipitation of AI and potential co-precipitation of the virus. This advantage is twofold since precipitated AI cannot diffuse into the virus particles and precipitated virus particles cannot be recovered from this process. Additionally, the dropwise addition of AI under magnetic stirring prevents pockets of insoluble concentrations of AI that drive precipitation from forming as the solution remains well mixed throughout the process. In using 20% v / v DMSO, a balance of structural strain of TMGMV is achieved, seemingly allowing penetration of AI between the coat proteins of TMGMV. In some embodiments, the loading of AI between the coat proteins in rod-shaped virus using DMSO results in a 10-fold reduction in synthesis time. This is composed of an improvement in synthetic yield by not losing particles in the precipitated state, as well as enabling the loading of fluopyram and ivermectin into TMGMV.

[0163] Example 9 Characterization of the Molecular Properties of the Active Ingredients and TMGMV Nanoparticles To gain a better understanding of the molecular properties that result in the loading between the coat proteins of the AIs, their aqueous and organic partition coefficients (logP), molecular weights, and surface charge distributions were compared. A summary of these properties can be found in Table 2 and Figures 20A - 20D. Among the AIs loaded within the TMGMV nanoparticles, ivermectin and fluopyram had the highest logP values of 4.4 and 3.33, respectively. Clothianidin and rifampicin had values of 1.3 and 2.4, respectively. The values of ivermectin and fluopyram indicate that the molecules are highly water-insoluble, which is in better agreement with what was observed in the loading experiments. This can explain why the same effective morphological changes were achieved using these AIs within one day using DMSO, compared to ten days using the pH approach, as the effective concentration of the AIs in solution was much higher. Another factor to consider regarding the injection efficiency is their size. Larger molecules may have the potential to have steric hindrance when entering the space between the coat proteins during these measurements. Analyzing the change in particle width using both approaches in comparison to their molecular weights, it was observed that clothianidin (249.68 Da) had the largest change in width, and fluopyram (396.71 Da) had the smallest change in width. Rifampicin (822.94 Da) and ivermectin (875.1 Da) had intermediate values in terms of the change in width. Since there is no clear trend in this set based on molecular weight, the AI size does not seem to be a limiting factor for loading using this approach. [Table 2]

[0164] Beyond the small molecule regime, steric hindrance is expected to become dominant. From the AI electron density plots, it was observed that ivermectin and rifampicin have large charge-free regions and small regions of small, mostly separated charges, creating mild amphiphilic molecules. In contrast, fluopyram and clothianidin are much smaller and have a higher charge surface area. TMGMV is essentially zwitterionic but also contains many hydrophobic interfaces, making it difficult to isolate the predicted morphological changes to a single physicochemical interaction. The amphiphilicity, charge, compactness, and flexible structure of clothianidin can all act together to change the morphology of TMGMV.

[0165] To gain some insights into how AI interacts with the coat protein surface, molecular docking experiments were performed on the TMGMV coat protein (CP) (PDB: 1VTM) and four AIs. In these analyses, the top 20 docking conformations were analyzed for their binding energies and residues involved in the stabilization of AI. These data do not suggest that AI is a suitable ligand for the TMGMV CP, but rather identify putative residues that may be involved in intercoat protein loading. 8 kcal mol -1 Actual ligand interactions for binding heats above 8 kcal mol have been reported, but most of these interactions are in the range of 3 - 8 kcal mol -1is within. Figures 29A - 29D summarize the binding regions, their functions against TMGMV, and the residues specifically identified to stabilize AI. Figures 21A - 21B, 22A - 22B, 23A - 23B, and 24A - 24B show examples of AI docked on TMGMV and related residues, and Figures 28A - 28D show the binding heats for each conformation calculated by Autodock4. From the simulated docking, it was observed that the 20 best binding sites on the TMGMV CP of all four AIs have many sites that are likely inaccessible. Depending on the separation mechanism of the TMGMV CP (inter - CP vs. inter - disk), ivermectin has up to 10 accessible sites, rifampicin has 8, fluopyram has 11, and clothianidin has 5 accessible sites. The binding energy distribution shows that rifampicin has the highest binding heat to the surface, followed by ivermectin, then fluopyram and clothianidin. Despite the numerous potential binding sites, ivermectin is a very large molecule and would require a high degree of separation of the CP to intercalate into the virion. Its relatively high affinity could appear as a transient surface binding that can disrupt the inter - CP binding, explaining the spread of TMGMV in the presence of ivermectin. Finally, it is suggested that ivermectin is not detectable during quantification and does not remain bound to TMGMV. Rifampicin has the highest binding heat to the TMGMV CP, is well - loaded onto TMGMV, and induces a morphological change in TMGMV. This shows improved loading in the presence of DMSO compared to the pH approach, suggesting that the structure change induced by DMSO allows this relatively large molecule to access the binding site. Despite having 11 potential binding sites, fluopyram also has somewhat the lowest binding heat and the highest affinity for the inner channel. Since this molecule is insoluble and relatively small, it can be preferentially allocated to the inner channel rather than loading between the CPs. Clothianidin had five accessible sites on the outside according to the docking model but also had somewhat the lowest binding energy.However, its relatively small size and surface charge distribution may assist in the binding and disruption of the structure between TMGMV CPs. Chlorothiazide shows somewhat the highest loading by HPLC and the largest difference in virion width, suggesting that the properties of this molecule are better suited for this approach. Using more robust docking analysis for loading between TMGMV CPs and a larger library of small molecules, it may be possible to identify the molecular properties of the individual residues of AI and TMGMV CP involved in these binding events.

[0166] Example 10 TMGMV nanoparticles prepared via the pH and DMSO methods encapsulate the target molecule and non-covalently load it therein The viral nanoparticle (VNP) AI loading methods described herein not only showed novel and interesting morphological changes in the virus, but also emphasized how these methods can be used to encapsulate target molecules within the virus and non-covalently load them therein and can be used as a delivery system. Careful adjustment of solution conditions such as pH and DMSO concentration allows TMGMV to "breathe", thereby creating structural changes and altering the interactions between structural motifs. These changes enable the loading and encapsulation of AI into the newly formed pockets and significantly improve the electrostatic loading capacity of TMGMV. The structural strain in the presence of AI results in an expansion of the short axis of TMGMV, which correlates with the degree of AI loading. These changes in particle size simplify the online measurement during VNP preparation and allow the degree of loading to be tracked in real time.

[0167] Both the pH and DMSO methodologies showed equal entrapment of rifampicin and clotianidin molecules during virus “breathing,” with up to 1000 AIs per TMGMV loaded. However, the DMSO methodology aided in the loading of ivermectin and fluopyram where, in the absence of DMSO, insoluble precipitates formed and the pH strategy did not show successful AI entrapment. Importantly, the DMSO strategy loaded AIs up to 10-fold faster than the pH strategy under the conditions tested, with no significant difference in particle integrity between the two conditions. Further refinement of the “breathing” conditions could help to precisely identify the phase transition of TMGMV, which could result in achieving higher loading, or loading of larger or several different molecules. Overall, the experiments led to further elucidated TMGMV as a highly versatile nanotechnology platform for cargo delivery.

[0168] Other embodiments The present invention has been described in conjunction with its detailed description, but it is understood that the foregoing description is intended to illustrate and not limit the scope of the invention as defined by the appended claims. Other aspects, advantages, and modifications are within the scope of the following claims.

Claims

**Claim 1** A nanoparticle comprising: a tobamovirus; and one or more active ingredients (AIs) non-covalently conjugated to the tobamovirus, wherein the tobamovirus comprises one or more coat proteins that reversibly and partially dissociate in response to an external factor. **Claim 2** The nanoparticle according to claim 1, wherein the one or more coat proteins reversibly and partially dissociate to form one or more pores. **Claim 3** The nanoparticle according to claim 2, wherein the one or more AIs are non-covalently conjugated to and confined within the one or more pores of the tobamovirus. **Claim 4** The nanoparticle according to claim 1, wherein the one or more AIs intercalate into the one or more coat proteins of the tobamovirus. **Claim 5** The nanoparticle according to any one of claims 1 to 4, wherein the one or more AIs are not chemically altered. **Claim 6** The nanoparticle according to any one of claims 1 to 5, wherein the external factor is a change in pH. **Claim 7** The nanoparticle according to any one of claims 1 to 5, wherein the external factor is the presence of a solvent. **Claim 8** The nanoparticle according to claim 9, wherein the solvent is a polar aprotic solvent. **Claim 9** The nanoparticle according to claim 10, wherein the polar aprotic solvent is dimethyl sulfoxide (DMSO). **Claim 10** The nanoparticle according to any one of claims 1 to 9, wherein the tobamovirus is rod-shaped. **Claim 11** The nanoparticle according to any one of claims 1 to 10, wherein the tobamovirus-AI nanoparticle has a width greater than the width of a reference tobamovirus. **Claim 12** The nanoparticle according to claim 11, wherein a reference tobamovirus molecule is treated under the same conditions as the tobamovirus-AI nanoparticle without the addition of AI. **Claim 13** The nanoparticle according to claim 11, wherein the reference tobamovirus has a width of 15, 16, 17, or 18 nm. **Claim 14** The nanoparticle according to any one of claims 11 to 13, wherein the width of the tobamovirus-AI nanoparticle is 2% to 105% greater than the width of the reference tobamovirus. **Claim 15** The width of the tobamovirus-AI nanoparticle is about 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, 50, 51, 52, 53, 54, 55, 56, 57, 58, 59, 60, 61, 62, 63, 64, 65, 66, 67, 68, 69, 70, 71, 72, 73, 74, 75, 76, 77, 78, 79, 80, 81, 82, 83, 84, 85, 86, 87, 88, 89, 90, 91, 92, 93, 9, 95, 96, 97, 98, 99, 100, 101, 102, 103, 104, or 105% wider than the width of the reference tobamovirus, the nanoparticle according to any one of claims 11 to 13.

16. The width of the tobamovirus-AI nanoparticle is 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, 50, 51, 52, 53, 54, 55, 56, 57, 58, 59, 60, 61, 62, 63, 64, 65, 66, 67, 68, 69, 70, 71, 72, 73, 74, or 75 nm, the nanoparticle according to claim 11.

17. The one or more AIs include one or more of a drug, a pesticide, or a small molecule, the nanoparticle according to any one of claims 1 to 16.

18. The pesticide includes a water-insoluble organic compound, a hydrophobic organic compound, an insecticide, a herbicide, a fungicide, a mite repellent, an algicide, an antibacterial agent, a biopesticide, a biocide, a disinfectant, a fumigant, an insect growth regulator, a plant growth regulator, a miticide, a microbial pesticide, a molluscicide, a nematicide, an ovicide, a pheromone, a repellent, a rodenticide, a defoliant, a desiccant, a phytotoxicity reducing agent, or any combination thereof, the nanoparticle according to claim 17.

19. The nanoparticle according to claim 17 or 18, wherein the pesticide is a benzoylurea such as novaluron, lufenuron, chlorfluazuron, flufenoxuron, hexaflumuron, noviflumuron, teflubenzuron, triflumuron, and diflubenzuron; a carbamate; a pyrethroid such as cyhalothrin, and its isomers and isomer mixtures, lambda-cyhalothrin, deltamethrin, tau-fluvalinate, cyfluthrin, beta-cyfluthrin, tefluthrin, and bifenthrin; an organophosphate such as azinphos-methyl, chlorpyrifos, diazinon, endosulfan, and methidathion; a neonicotinoid; a phenylpyrazole such as imidacloprid, acetamiprid, thiacloprid, dinotefuran, thiamethoxam, and fipronil; a triazole such as epoxiconazole, hexaconazole, propiconazole, prochloraz, imazalil, triadimenol, difenoconazole, microbutanil, prothioconazole, triticonazole, and tebuconazole; a morpholine such as dimethomorph, fenpropidin, and fenpropimorph; a strobilurin such as azoxystrobin, kresoxim-methyl, and their analogs; a phthalonitrile such as chlorothalonil; mancozeb; fluazinam; a pyrimidine such as bupirimate; an aryloxy phenoxy derivative; an arylurea; an aryl carboxylic acid; an aryloxy alkanoic acid derivative such as clodinafop-propargyl and its analogs, fenoxaprop-p-ethyl and its analogs, propaquizafop, quizalofop and its analogs; a dinitroaniline such as pendimethalin and trifluralin; a diphenyl ether such as oxyfluorfen; an imidazolinone; a sulfonylurea such as chlorosulfuron, nicosulfuron, rimsulfuron, tribenuron-methyl; a sulfonamide; a triazine; and a triazinone such as metamitron.

20. The nanoparticle according to claim 17, wherein the drug is a chemotherapeutic agent, an anti-parasitic agent, an antibiotic, or an immunomodulatory agent.

21. The nanoparticle according to claim 17, wherein the drug is a hydrophilic drug or a hydrophobic drug.

22. The nanoparticle according to any one of claims 1 to 21, wherein the nanoparticle contains about 1 to about 1500 AI molecules per tobacco mosaic virus.

23. The nanoparticle according to any one of claims 1 to 22, wherein the tobamovirus is tobacco mild green mosaic virus (TMGMV).

24. The nanoparticle according to any one of claims 1 to 23, wherein the tobamovirus is tobacco mosaic virus (TMV).

25. A composition comprising the nanoparticle according to any one of claims 1 to 24.

26. The composition according to claim 26, wherein the composition exhibits a soil distribution and / or soil mobility of at least 5, 10, 15, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 21, 32, 33, 34, 35, 36, 37, 38, 39, or 40 cm from the application point.

27. The composition according to claim 26, further comprising an excipient.

28. The composition according to claim 27, wherein the excipient is a buffer or water.

29. A method for producing a nanoparticle comprising a tobamovirus and one or more active ingredients (AIs), the method comprising: a) providing an isolated tobamovirus to a buffer having a pH of about 7 to 9 to create a tobamovirus-buffer; b) adding one or more AIs to the tobamovirus-buffer two or more times, thereby creating the nanoparticle; purifying the nanoparticle in a solution having a pH of about 5 to 9, and wherein the one or more AIs are non-covalently conjugated to the tobamovirus, and the tobamovirus comprises one or more coat proteins that dissociate reversibly and partially in response to a change in pH.

30. The method according to claim 29, wherein the one or more AIs are added at least once a day for at least 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, or 21 days.

31. The method according to claim 29 or 30, wherein the buffer has a pH of about 7 to 7.5, 7.5 to 8, 7 to 8, 8 to 8.5, 8.5 to 9, or 8 to 9.

32. The method according to claim 29 or 30, wherein the buffer has a pH of about 7.2 to 7.8, 7.3 to 7.8, 7.2 to 7.7, 7.3 to 7.7, 7.4 to 7.8, 7.4 to 7.7, 7.5 to 7.7, 7.5 to 7.8, 7.2 to 7.6, 7.3 to 7.6, 7.4 to 7.6, 7.5 to 7.6, 7.2 to 7.5, 7.3 to 7.5, 7.4 to 7.5, 7.2 to 7.9, 7.3 to 7.9, 7.4 to 7.9, 7.5 to 7.9, 7.3 to 7.99, 7.4 to 7.99, or 7.5 to 7.99, or the buffer has a pH of about 7.2, 7.3, 7.4, 7.5, 7.6, 7.7, 7.8, 7.9, or 7.

99.

33. The method according to any one of claims 29 to 32, wherein the solution has a pH of about 6.9, 7.0, 7.1, 7.2, or 7.

3.

34. The method according to any one of claims 29 to 33, wherein the change in pH is about 0.5 to 1, about 0.5 to 2, 0.5 to 3, 1 to 2, or 1 to 3.

35. A method for producing nanoparticles comprising a tobamovirus and one or more active ingredients (AIs), the method comprising: a) providing an isolated tobamovirus in a buffer having a pH of about 5 to 9 to create a tobamovirus-buffer; b) adding a solvent at a concentration of about 15% (v / v) to about 25% (v / v); c) adding one or more AIs to the tobamovirus-buffer, thereby creating the nanoparticles; purifying the nanoparticles in a solution having a pH of about 5 to 9, wherein the one or more AIs are non-covalently conjugated to the tobamovirus; and the tobamovirus comprises one or more coat proteins that are reversibly and partially dissociated in response to the presence of the solvent.

36. The method according to claim 35, wherein the solvent is added dropwise.

37. The method according to claim 35 or 36, wherein the one or more AIs are added dropwise.

38. The method according to claim 35 or 36, wherein the one or more AIs are added stepwise over a period of time.

39. The method according to claim 38, wherein the period is about 0.5 hours to about 10 days.

40. The method according to any one of claims 35 to 39, further comprising incubating the one or more AIs in the tobamovirus-buffer for about 4 hours to about 24 hours.

41. The method according to any one of claims 35 to 40, wherein the solvent is a polar aprotic solvent. **Claim 42** The method according to claim 41, wherein the polar aprotic solvent is dimethyl sulfoxide (DMSO). **Claim 43** The method according to any one of claims 29, 31 to 38, or 40 to 42, wherein the one or more AIs are added to the tobacco mosaic virus-buffer two or more times. **Claim 44** The method according to any one of claims 29, 31 to 38, or 40 to 43, wherein the one or more AIs are added at least once a day. **Claim 45** The one or more AIs are added until an equivalent ratio of about 10:1, 25:1, 50:1, 75:1, 100:1, 150:1, 200:1, 250:1, 300:1, 350:1, 400:1, 450:1, 500:1, 550:1, 600:1, 650:1, 700:1, 750:1, 800:1, 850:1, 900:1, 950:1, or 1000:1 is reached, or The one or more AIs are added in a 1000, 1500, 2000, 2500, 3000, 3500, 4000, 4500, 5000, 5500, 6500, 7000, 7500, 8000, 8500, 9000, or 9500-fold molar excess with respect to the tobacco mosaic virus, or The method according to any one of claims 29 to 44, wherein 100, 150, 200, 250, 300, 350, 400, 450, or 500 nmol of one or more AIs per gram of tobacco mosaic virus are added. **Claim 46** The method according to any one of claims 29 to 45, wherein the one or more coat proteins dissociate reversibly and partially to form one or more pores. **Claim 47** The method according to claim 46, wherein the one or more AIs are non-covalently conjugated to and confined within the one or more pores of the tobacco mosaic virus. **Claim 48** The method according to any one of claims 29 to 47, wherein the one or more AIs are intercalated into the one or more coat proteins of the tobacco mosaic virus. **Claim 49** The method according to any one of claims 29 to 48, wherein the one or more AIs are not chemically altered. **Claim 50** The method according to any one of claims 29 to 49, wherein the tobacco mosaic virus is rod-shaped. **Claim 51** The method according to any one of claims 29 to 50, wherein the nanoparticles have a width wider than the width of the reference tobamovirus.

52. The method according to any one of claims 29 to 51, wherein the reference tobamovirus molecule is treated under the same conditions as the tobamovirus-AI nanoparticles without adding AI.

53. The method according to claim 52, wherein the reference tobamovirus has a width of about 15, 16, 17, or 18 nm.

54. The width of the nanoparticles is 2% to 105% wider than the width of the reference tobamovirus, or The width of the nanoparticles is about 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, 50, 51, 52, 53, 54, 55, 56, 57, 58, 59, 60, 61, 62, 63, 64, 65, 66, 67, 68, 69, 70, 71, 72, 73, 74, 75, 76, 77, 78, 79, 80, 81, 82, 83, 84, 85, 86, 87, 88, 89, 90, 91, 92, 93, 9, 95, 96, 97, 98, 99, 100, 101, 102, 103, 104, or 105% wider than a reference value, the method according to claim 53.

55. The method according to any one of claims 29 to 54, wherein the width of the nanoparticles is 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, 50, 51, 52, 53, 54, 55, 56, 57, 58, 59, 60, 61, 62, 63, 64, 65, 66, 67, 68, 69, 70, 71, 72, 73, 74, or 75 nm.

56. The method according to any one of claims 29 to 55, wherein the one or more AIs include one or more of a drug, a pesticide, or a small molecule.

57. The method according to claim 56, wherein the pesticide comprises a water-insoluble organic compound, an insecticide, a herbicide, a fungicide, a mite control agent, an algicide, an antibacterial agent, a biopesticide, a biocide, a disinfectant, a fumigant, an insect growth regulator, a plant growth regulator, a miticide, a microbial pesticide, a molluscicide, a nematicide, an ovicide, a pheromone, a repellent, a rodenticide, a defoliant, a desiccant, a phytotoxicity reducing agent, or any combination thereof.

58. The method according to claim 56, wherein the pesticide comprises benzoyl ureas such as novaluron, lufenuron, chlorfluazuron, flufenoxuron, hexaflumuron, noviflumuron, teflubenzuron, triflumuron, and diflubenzuron; carbamates; pyrethroids such as cyhalothrin, and its isomers and isomer mixtures, lambda-cyhalothrin, deltamethrin, tau-fluvalinate, cyfluthrin, beta-cyfluthrin, tefluthrin, and bifenthrin; organophosphates such as azinphos-methyl, chlorpyrifos, diazinon, endosulfan, and methidathion; neonicotinoids; phenylpyrazoles such as imidacloprid, acetamiprid, thiacloprid, dinotefuran, thiamethoxam, and fipronil; triazoles such as epoxiconazole, hexaconazole, propiconazole, prochloraz, imazalil, triadimenol, difenoconazole, metconazole, prothioconazole, triticonazole, and tebuconazole; morpholines such as dimethomorph, fenpropizine, and fenpropimorph; strobilurins such as azoxystrobin, kresoxim-methyl, and their analogs; phthalonitriles such as chlorothalonil; mancozeb; fluazinam; pyrimidines such as bupirimate; aryloxyphenoxy derivatives; aryl ureas; aryl carboxylic acids; aryloxyalkanoic acid derivatives such as clodinafop-propargyl and its analogs, fenoxaprop-p-ethyl and its analogs, propaquizafop, quizalofop and its analogs; dinitroanilines such as pendimethalin and trifluralin; diphenyl ethers such as oxyfluorfen; imidazolinones; sulfonyl ureas such as chlorosulfuron, nicosulfuron, rimsulfuron, tribenuron-methyl; sulfonamides; triazines; and triazinones such as metiamidone.

59. The method according to claim 56, wherein the drug is a chemotherapeutic agent, an anti-parasitic drug, an antibiotic, or an immunomodulatory agent.

60. The method according to claim 56, wherein the drug is a hydrophilic drug or a hydrophobic drug.

61. The method according to any one of claims 29 to 60, wherein the nanoparticles contain about 1 to about 1500 AI molecules per tobacco mosaic virus.

62. The method according to any one of claims 29 to 61, wherein the tobacco mosaic virus is tobacco mild green mosaic virus (TMGMV).

63. The method according to any one of claims 29 to 61, wherein the tobacco mosaic virus is tobacco mosaic virus (TMV).

64. A method comprising administering the nanoparticles according to any one of claims 1 to 20 to the composition according to claims 21 to 24 to soil, crops, or plants, wherein the nanoparticles or the composition are administered in an effective amount.

65. A pharmaceutical composition comprising the nanoparticles according to any one of claims 1 to 24.

66. The pharmaceutical composition according to claim 65, further comprising at least one pharmaceutically acceptable carrier, diluent, or excipient.

67. The pharmaceutical composition according to claim 65 or 66, wherein the pharmaceutical composition is formulated into a dosage form that is an injectable solution, a lyophilized powder, a suspension, or any combination thereof.

68. A method of performing it in a subject in need of treating cancer, the method comprising administering the nanoparticles according to any one of claims 1 to 24 or the pharmaceutical composition according to any one of claims 65 to 67 to the subject in need of treating cancer, wherein the nanoparticles or the pharmaceutical composition are administered in an effective amount.

69. The method according to claim 68, wherein the cancer includes breast cancer, ovarian cancer, glioma, gastrointestinal cancer, prostate cancer, cancer, lung cancer, hepatocellular cancer, testicular cancer, cervical cancer, endometrial cancer, bladder cancer, head and neck cancer, lung cancer, gastric-esophageal cancer, gynecological cancer, or any combination thereof.

70. A method of performing it in a subject in need of treating an infection, the method being A method comprising administering to the subject in need of treatment of the infection a nanoparticle according to any one of claims 1 to 24 or a pharmaceutical composition according to any one of claims 65 to 67, wherein the nanoparticle or the pharmaceutical composition is administered in an effective amount.

71. The method according to claim 70, wherein the infection is a bacterial infection, a viral infection, a fungal infection, a parasitic infection, or any combination thereof.

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