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

Abstract

The present application relates, in part, to nanoparticles comprising the Tobacco Mosaic Virus and nanoparticles comprising the Tobacco Mosaic Virus and a beta-cyclodextrin (beta-CD or BCD). The present application also relates, in part, to nanoparticles comprising a tobacco mosaic virus and one or more active ingredients (AI) that are non-covalently conjugated to the tobacco mosaic virus. Also provided are methods of making such nanoparticles and methods of using such nanoparticles, as well as compositions comprising the disclosed nanoparticles.

Description

[0001] CROSS-REFERENCE TO RELATED APPLICATIONS

[0002] This application claims the benefit of U.S. Provisional Application No. 63 / 353,309, filed on June 17, 2022. The entire contents of the foregoing application are incorporated herein by reference in their entirety.

[0003] Federally funded research or development

[0004] This invention was made with government support from the United States Department of Agriculture under Grants 2020-67021-31255 and 2022-67012-36698 and from the National Science Foundation under Grant DMR-2011924. The government has certain rights in this invention. Background Art

[0005] Pesticides are widely used in food product production in the field. But pesticides and methods of using the pesticides are inefficient. Pesticides can accumulate in the environment, crops and drinking water. Pesticides are harmful to the environment and human health. It is important to develop better methods of applying pesticides. The widespread use of pesticides in agriculture causes these toxins to accumulate in crops, soil, drinking water and groundwater, seriously endangering ecosystems and human health. The first step towards a healthier society is to enhance food safety by improving quality and yield (i.e., more effective crop treatment) while protecting the environment and agricultural ecosystems (i.e., preventing leaching and accumulation of pesticides in the environment). Most pesticides are hydrophobic and therefore do not have good soil mobility. This leads to overuse, and therefore increases health and environmental problems. It is important to develop better methods of applying pesticides. Summary of the invention

[0006] The present application is based in part on the surprising discovery that Tobacco mosaic virus (e.g., Tobacco mild green mosaic virus (TMGMV)) rods can be used to load (also referred to as integrally encapsulate) target active ingredients (AIs, also referred to as active substances), such as pesticides, drugs, and pharmaceuticals. Importantly, the compositions and methods described herein do not require any modification of any useful drug, pesticide, pharmaceutical, or compound. In part, the present application relates to non-covalent encapsulation or loading techniques to encapsulate pesticides and / or drugs into nanoparticles described herein. Tobacco mosaic virus rods are a good platform for precision agriculture because the Tobacco mosaic virus rods have excellent soil fluidity, and as described herein, nanoparticles produced using Tobacco mosaic virus can have uniform soil distribution and / or soil fluidity, up to 30 cm and above. In some embodiments, the nanoparticles of the present disclosure have a soil distribution and / or soil mobility of at least 5 cm, 10 cm, 15 cm, 20 cm, 21 cm, 22 cm, 23 cm, 24 cm, 25 cm, 26 cm, 27 cm, 28 cm, 29 cm, 30 cm, 21 cm, 32 cm, 33 cm, 34 cm, 35 cm, 36 cm, 37 cm, 38 cm, 39 cm, or 40 cm.

[0007] In particular, the use of non-covalent encapsulation techniques to encapsulate pesticides onto nanoparticles containing tobacco mosaic virus (Tobacco mild green mosaic virus (TMGMV)) is described herein. Tobacco mosaic virus nanoparticles are a good platform for precision agriculture because the tobacco mosaic virus nanoparticles have excellent soil fluidity. The present application includes tobacco mosaic virus nanoparticles using β-cyclodextrin (also referred to as BCD, bCD, βCD, β-CD, etc. throughout this disclosure) as cargo pockets, and tobacco mosaic virus nanoparticles that can undergo structural transformations that enable molecules (e.g., AI) to be infused into the tobacco mosaic virus structure. In some embodiments, BCD is conjugated to the tobacco mosaic virus nanoparticles on the surface; without being bound by theory, BCD acts as a pocket for loading cargo (such as medical drugs and pesticides). In some embodiments, the structural transformation that enables molecules (e.g., AI) to be infused into tobacco mosaic virus nanoparticles is triggered by external factors. In some embodiments, the external factor is exposure of the tobamovirus nanoparticles to a pH change or a solvent (eg, dimethyl sulfoxide or DMSO).

[0008] Although usually described as rigid / solid structure, plant viruses including tobacco mosaic virus genus "breathe" in solution, and by carefully adjusting pH, the structure can be opened to encapsulate at least one active ingredient or one or more active ingredients. This paper especially describes the method for triggering tobacco mosaic virus genus nanoparticles to partially and reversibly dissociate one or more coat proteins in its coat protein by adjusting pH or by contacting tobacco mosaic virus genus nanoparticles with solvent (e.g., DMSO). Such methods allow tobacco mosaic virus genus "breathing" or allow drug molecules or any AI to be infused into the phase change in the tobacco mosaic virus genus structure. In the present disclosure, AI can be one or more of pesticides and other drugs.

[0009] The disclosed nanoparticles have good soil fluidity, and in some embodiments, the nanoparticles are loaded with pesticides using two strategies: (1) β-CD as a cargo pocket for AI, and (2) structural transformation of molecules and / or AI infusion. In some embodiments, β-cyclodextrin (β-CD) is conjugated to the surface of tobacco mosaic virus nanoparticles; without being bound by theory, β-CD acts as a pocket for loading cargo / AI (such as medical drugs and pesticides).

[0010] Also without being bound by theory, although generally described as rigid / solid structures, plant viruses including the genus Tobacco mosaic virus "breathe" in solution, and by carefully adjusting the pH, the structure can be opened to embed one or more AIs. As described herein, a breathing method for the genus Tobacco mosaic virus has been developed that allows the genus Tobacco mosaic virus to undergo structural transformation and infuse drug molecules into the structure. As described herein, the breathing method can be used to embed or infuse a variety of AIs, including pesticides and other drugs, in the genus Tobacco mosaic virus nanoparticles. In some embodiments, the compositions and methods described herein utilize supramolecular interactions between β-cyclodextrin and the target AI to formulate multifunctional nanoparticles for delivery applications.

[0011] B-cyclodextrin is a natural annular cyclic oligosaccharide. It has a hydrophilic outer surface and a hydrophobic inner cavity that can accommodate a variety of guest molecules. In addition, it is the most widely used host system in supramolecular chemistry, and has low cost, good water solubility and biocompatibility properties. Without being bound by theory, the basic principle is to use a supramolecular strategy based on the interaction between β-CD and a target AI (such as an insecticide). The B-CD unit is grafted onto the outer surface of the tobacco mosaic virus genus using an optimized bioconjugation reaction, which captures one or more target AIs for effective delivery to the soil.

[0012] In some embodiments, the nanoparticles described herein entrap insecticides in Tobaccomovirus through pH changes, which entrap the AI ​​by forming "pockets" or "holes" between the CPs.

[0013] The basic principle is that by increasing the pH of the buffer or by the presence of a solvent (e.g., DMSO), the virus will begin to dissociate and a hydrophobic pocket or pore will be created between the coat proteins of the virions. In an exemplary method, the AI ​​is then added to interact with the virions, and the pH is then lowered to promote self-assembly of the particles and embedding of the AI ​​on the hydrophobic pocket or pore. In yet another exemplary method, after adding a solvent (e.g., DMSO), the AI ​​is added to interact with the virions, thereby promoting embedding of the AI ​​on the hydrophobic pocket or pore.

[0014] Certain aspects of the present disclosure relate to a nanoparticle comprising: a tobacco mosaic virus; and one or more active ingredients (AIs) non-covalently conjugated to the tobacco mosaic virus, wherein the tobacco mosaic virus comprises one or more coat proteins that reversibly and partially dissociate in response to external factors.

[0015] In some embodiments, the one or more coat proteins are reversibly and partially dissociated to form one or more holes. In some embodiments, the one or more AIs are non-covalently conjugated to the one or more holes of the tobacco mosaic virus and embedded in the one or more holes. In some embodiments, the one or more AIs are embedded in the one or more coat proteins of the tobacco mosaic virus. In some embodiments, wherein the one or more AIs are not chemically changed. 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 miscible with water. In some embodiments, the polar aprotic solvent is dimethyl sulfoxide (DMSO). In some embodiments, TM tobacco mosaic virus GMV is rod-shaped.

[0016] In certain embodiments, the nanoparticle that comprises tobacco mosaic virus and beta-cyclodextrin (βCD) is described herein.In certain embodiments, the nanoparticle is further included in the R group between the tobacco mosaic virus and the βCD.In certain embodiments, the tobacco mosaic virus and the βCD are covalently attached.In certain embodiments, the tobacco mosaic virus and the βCD are connected with the R group.In certain embodiments, the R group is an alkyl, alkene, alkyne, ester or other carbon-containing compound.In certain embodiments, the R group is an acetylene.

[0017] In some embodiments, the width of the tobacco mosaic virus-AI nanoparticle is greater than the width of a reference tobacco mosaic virus. In some embodiments, a reference tobacco mosaic virus molecule is treated with the same conditions as the tobacco mosaic virus-AI nanoparticle without the addition of an AI. In some embodiments, the present application relates to nanoparticles comprising a tobacco mosaic virus and one or more active ingredients (AI), wherein the width of the tobacco mosaic virus-AI nanoparticle is greater than the width of the reference. In some embodiments, the reference is the width of a tobacco mosaic virus molecule treated under the same conditions without the addition of an AI. In some embodiments, the reference is 15nm, 16nm, 17nm or 18nm. In some embodiments, the width of the tobacco mosaic virus-AI nanoparticle is 2%-105% greater than the width of the reference. In some embodiments, 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%, 94%, 95%, 96%, 97%, 98%, 99%, 100%, 101%, 102%, 103%, 104%, 105%, 106%, 107%, 108%, 109%, 110%, 111 %, 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%, 95%, 96%, 97%, 98%, 99%, 100%, 101%, 102%, 103%, 104% or 105%. In some embodiments, the width of the tobacco mosaic virus-AI nanoparticle is 19nm, 20nm, 21nm, 22nm, 23nm, 24nm, 25nm, 26nm, 27nm, 28nm, 29nm, 30nm, 31nm, 32nm, 33nm, 34nm, 35nm, 36nm, 37nm, 38nm, 39nm, 40nm, 41nm, 42nm, 43nm, 44nm, 45nm, 46nm, 47nm, 48nm, 49nm, 50nm, 51nm. In some embodiments, the one or more AIs comprise 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, an herbicide, a fungicide, a miticide, an algaecide, an antimicrobial, 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, an insect repellent, a rodenticide, a defoliant, a desiccant, a safener, or any combination thereof. In some embodiments, the insecticide is a benzoylurea, such as fluazifop, lufenuron, chlorpyrifos, flufenoxuron, flufenoxuron, flubendiamide, fluthrin and flubendiamide; a carbamate; a pyrethroid, such as cyhalothrin and its isomers and isomer mixtures, high-efficiency cyhalothrin, deltamethrin, fluvalinate, cyfluthrin, high-efficiency fluthrin, tefluthrin and bifenthrin; an organophosphate, such as azinphos-methyl, chlorpyrifos, diazinon, endosulfan, methidathrin; a neonicotinoid; a phenylpyrazole, such as imidacloprid, acetamiprid, thiamethoxam, dimethomorph, thiamethoxam and fipronil; a conazole, such as epoxiconazole, hexaconazole, propiconazole, prochloraz, imazalil, triadimenol, difenoconazole, nitrile oxadiazole, prothioconazole, trichlorfon and tebuconazole; morpholines such as dimethomorph, fenpropidin and fenpropimorph; strobilurins such as azoxystrobin, kresoxim-methyl and their analogs; phthalonitriles such as thiocarb; mancozeb; fluazinam; pyrimidines such as ethoxystrobin; aryloxyphenoxy derivatives; aryl ureas; aryl carboxylic acids; aryloxyalkanoic acid derivatives such as clodinafop-butyl and its analogs, fenoxaprop-butyl and its analogs, chlorfenapyr, quizalofop-butyl and its analogs; dinitroanilines such as pendimethalin and trifluralin; diphenyl ethers such as oxyfluorfen; imidazolinones; sulfonylureas such as chlorsulfuron, nicosulfuron, sulfasulfuron-methyl, bensulfuron-methyl; sulfonamides; triazines; and triazinones such as metamitron-methyl.

[0018] In some embodiments, at least one AI comprises at least one of a drug, an insecticide or a small molecule. In some embodiments, the drug can be a chemokine, an antimicrobial or any therapeutic compound. In some embodiments, the drug is a chemotherapeutic, an antiparasitic, an antibiotic or an immunomodulator. In some embodiments, the drug is a hydrophilic or hydrophobic drug. In some embodiments, the tobacco mosaic virus genus is a tobacco mild green mosaic virus (TMGMV). In some embodiments, the tobacco mosaic virus genus is a tobacco mosaic virus (TMV).

[0019] In some embodiments, the nanoparticles comprise from about 1 to about 1500 AI molecules per tobacco mosaic virus genus.

[0020] Also described herein are compositions comprising any disclosed nanoparticles. In certain embodiments, the soil distribution and / or soil mobility of any nanoparticle or composition described herein is at least 5cm, 10cm, 15cm, 20cm, 21cm, 22cm, 23cm, 24cm, 25cm, 26cm, 27cm, 28cm, 29cm, 30cm, 21cm, 32cm, 33cm, 34cm, 35cm, 36cm, 37cm, 38cm, 39cm or 40cm. In certain embodiments, the composition further comprises an excipient. In certain embodiments, the excipient is a buffer or water.

[0021] In certain embodiments, disclosed herein are methods of preparing nanoparticles comprising a tobacco mosaic virus and βCD, the methods comprising: providing an isolated tobacco mosaic virus, conjugating the βCD to the tobacco mosaic virus, thereby producing the nanoparticle; and purifying the nanoparticle.

[0022] In certain embodiments, the coupling step is included in the formation of a covalent bond between the βCD, joint and the tobacco mosaic virus. In certain embodiments, the coupling step comprises a diazo coupling reaction. In some embodiments of any compositions or methods of description described herein, the tobacco mosaic virus is modified or inactivated. In certain embodiments, the joint is an R group. In certain embodiments, the R group is an alkyl, alkene, alkyne, ester or other carbon-containing compound. In certain embodiments, the R group is an acetylene.

[0023] In certain embodiments, the present invention also discloses a method for preparing nanoparticles comprising a tobacco mosaic virus and one or more active ingredients (AIs), the method comprising: providing a buffer having a pH of about 7 to 9 to separate tobacco mosaic virus; adding one or more AIs to the tobacco mosaic virus more than once, thereby producing 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 tobacco mosaic virus, and wherein the tobacco mosaic virus comprises one or more coat proteins that reversibly and partially dissociate in response to a change in pH.

[0024] In some embodiments, the one or more AIs are added for at least 1 day, 2 days, 3 days, 4 days, 5 days, 6 days, 7 days, 8 days, 9 days, 10 days, 11 days, 12 days, 13 days, 14 days, 15 days, 16 days, 17 days, 18 days, 19 days, 20 days, or 21 days. In some embodiments, the pH of the buffer is about 7 to 7.5, 7.5 to 8, 7 to 8, 8 to 8.5, 8.5 to 9, or 8 to 9. In some embodiments, the pH of the buffer 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 wherein the pH of the buffer is about 7.2, 7.3, 7.4, 7.5, 7., 7.7, 7.8, 7.9 or 7.99. In some embodiments, the pH of the solution is about 6.9, 7.0, 7.1, 7.2, or 7.3. 1. In some embodiments, the pH changes from about 0.5 to 1, about 0.5 to 2, 0.5 to 3, 1 to 2, or 1 to 3.

[0025] In certain embodiments, disclosed herein is a method for preparing nanoparticles comprising a tobacco mosaic virus and one or more active ingredients (AIs), the method comprising: providing an isolated tobacco mosaic virus to a buffer having a pH of about 5 to 9 to produce a tobacco mosaic virus-buffer; adding a solvent having a concentration of about 15% (v / v) to about 25% (v / v); adding one or more AIs to the tobacco mosaic virus-buffer, thereby producing the nanoparticles; and 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 tobacco mosaic virus, and wherein the tobacco mosaic virus comprises one or more coat proteins that reversibly and partially dissociate in response to the presence of the solvent.

[0026] In some embodiments, the solvent is added dropwise. In some embodiments, the one or more AIs are added dropwise. In some embodiments, the one or more AIs are added dropwise over a certain period of time. In some embodiments, the period of time is about 0.5 hours to about 10 days. In some embodiments, the one or more AIs are added once a day. In some embodiments, the method further comprises incubating the one or more AIs in the tobacco mosaic virus-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, the one or more coat proteins reversibly and partially dissociate to form one or more holes.

[0027] In some embodiments, the one or more AIs are added repeatedly. In some embodiments, the one or more AIs are added to the tobacco mosaic virus-buffer two or more times. In some embodiments, the one or more AIs are added at least once a day. In some embodiments, 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 wherein the one or more AIs are added 1,000 times, 1,500 times, 2,000 times, 2,500 times, fold, 3,000-fold, 3,300-fold, 4,000-fold, 4,500-fold, 5,000-fold, 5,500-fold, 6,500-fold, 7,000-fold, 7,500-fold, 8,000-fold, 8,500-fold, 9,000-fold or 9,500-fold molar excess to the tobacco mosaic virus genus; or wherein 100 nmol, 150 nmol, 200 nmol, 250 nmol, 300 nmol, 350 nmol, 400 nmol, 450 nmol or 500 nmol of one or more AI / gram of tobacco mosaic virus genus is added.

[0028] In some embodiments, the one or more AIs are non-covalently conjugated to the one or more holes of the tobacco mosaic virus and embedded in the one or more holes. In some embodiments, the one or more AIs are embedded in the one or more coat proteins of the tobacco mosaic virus. In some embodiments, wherein the one or more AIs are not chemically altered. In some embodiments, the tobacco mosaic virus is rod-shaped.

[0029] In some embodiments, the width of the nanoparticle is greater than the width of a reference Tobacco Mosaic Virus. In some embodiments, a reference Tobacco Mosaic Virus molecule is treated under the same conditions as the Tobacco Mosaic Virus-AI nanoparticle without the addition of AI. In some embodiments, the reference is 15 nm, 16 nm, 17 nm, or 18 nm. In some embodiments, the width of the nanoparticle is 2%-105% greater than the width of the reference; or wherein the width of the nanoparticle is approximately 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% greater than the width of the reference. %, 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%, 95%, 96%, 97%, 98%, 99%, 100%, 101%, 102%, 103%, 104%, or 105%. In some embodiments, the width of the nanoparticle is 19nm, 20nm, 21nm, 22nm, 23nm, 24nm, 25nm, 26nm, 27nm, 28nm, 29nm, 30nm, 31nm, 32nm, 33nm, 34nm, 35nm, 36nm, 37nm, 38nm, 39nm, 40nm, 41nm, 42nm, 43nm, 44nm, 45nm, 46nm, 47nm, 48nm, 49nm, 50nm, 51nm, 52nm, 53nm, 54nm, 55nm, 56nm, 57nm, 58nm, 59nm, 60nm, 61nm, 62nm, 63nm, 64nm, 65nm, 66nm, 67nm, 68nm, 69nm, 70nm, 71nm, 72nm, 73nm, 74nm or 75nm.

[0030] In some embodiments, the one or more AIs include one or more of a drug, an insecticide, or a small molecule. In some embodiments, the insecticide is a water-insoluble organic compound, an insecticidal drug, a herbicide, a fungicide, a miticide, an algicide, an antimicrobial agent, a biopesticide, a biocide, a disinfectant, a fumigant, an insect growth regulator, a plant growth regulator, a miticide, a microbial insecticide, a molluscicide, a nematicide, an ovicide, a pheromone, a repellent, a rodenticide, a defoliant, a desiccant, a safener, or any combination thereof. In some embodiments, the insecticide is a benzoylurea, such as diflubenzuron, lufenuron, chlorfluazuron, flufenoxuron, hexaflumuron, flucycloxuron, teflubenzuron, and diflubenzuron; a carbamate; a pyrethroid, such as cyhalothrin and its isomers and isomer mixtures, lambda-cyhalothrin, deltamethrin, fluvalinate, cyfluthrin, beta-cyfluthrin, tefluthrin, and bifenthrin; an organophosphate, such as azinphos-methyl, chlorpyrifos, diazinon, endosulfan, methidathion; a neonicotinoid; a phenylpyrazole, such as imidacloprid, acetamiprid, thiacloprid, dinotefuran, thiamethoxam, and fipronil; a triazole, such as tebuconazole, hexaconazole, propiconazole, prochloraz, imazalil, triadimenol, difenoconazole, myclobutanil, 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 ethirimol; an aryloxyphenoxy derivative; an arylurea; an arylcarboxylic acid; an aryloxyalkanoic acid derivative, such as clodinafop-propargyl and its analogs, fenoxaprop-p-ethyl and its analogs, oxaziclomefone, quizalofop-p-ethyl and its analogs; a dinitroaniline, such as pendimethalin and trifluralin; a diphenyl ether, such as oxyfluorfen; an imidazolinone; a sulfonylurea, such as chlorsulfuron, nicosulfuron, rimsulfuron, tribenuron-methyl; a sulfonamide; a triazine; and a triazinone, such as bentazone.

[0031] In some embodiments, the drug is a chemotherapeutic drug, an antiparasitic drug, an antibiotic drug, or an immunomodulator. In some embodiments, the drug is a hydrophilic drug or a hydrophobic drug. In some embodiments, the nanoparticle contains from about 1 to about 1500 AI molecules per tobamovirus. In some embodiments, the tobamovirus is Tobacco mild green mosaic virus (TMGMV). In some embodiments, the tobamovirus is Tobacco mosaic virus (TMV).

[0032] Also described herein are methods of applying any of the nanoparticles or compositions described herein to soil, crops, or plants, wherein the nanoparticles or compositions are applied in an effective amount.

[0033] Also described herein are pharmaceutical compositions comprising any of the nanoparticles of the present disclosure.

[0034] 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 as an injectable solution, a lyophilized powder, a suspension, or any combination thereof.

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

[0036] In some embodiments, the cancer wherein the cancer comprises breast cancer, ovarian cancer, glioma, gastrointestinal cancer, prostate cancer, carcinoma, lung cancer, hepatocellular carcinoma, testicular cancer, cervical cancer, endometrial cancer, bladder cancer, head and neck cancer, lung cancer, gastroesophageal cancer, gynecological cancer, or any combination thereof.

[0037] Also provided herein is a method of treating an infection in a subject in need thereof, the method comprising administering a nanoparticle of the present disclosure or a pharmaceutical composition of the present disclosure to the subject in need of treatment for the infection, wherein the nanoparticle or the composition is administered in an effective amount.

[0038] In some embodiments, the infection is a bacterial infection, a viral infection, a fungal infection, a parasitic infection, or any combination thereof.

[0039] Further, the present disclosure also relates to a method for combating harmful insects and / or plant pathogenic fungi, the method comprising contacting a plant, soil or plant habitat in which or on which the harmful insects and / or the plant pathogenic fungi are growing or may grow, a plant or soil to be protected from attack or infestation by the harmful insects and / or the plant pathogenic fungi with an effective amount of a formulation according to the present disclosure. Thus, the formulation according to the present disclosure can be used to control a variety of plant pathogenic fungi or insects on various cultivated plants or weeds (such as wheat, rye, barley, oats, rice, corn, grass, bananas, cotton, soybeans, coffee, sugarcane, vines, fruits and ornamental plants, and vegetables (such as cucumbers, beans, tomatoes, potatoes and gourds)).

[0040] The present disclosure also relates to a method for controlling undesirable vegetation, the method comprising allowing a herbicidally effective amount of a formulation according to the present disclosure to act on a plant, its habitat. Controlling undesirable vegetation is understood to mean eliminating weeds. In the broadest sense, weeds are understood to mean all those plants that grow in undesirable locations.

[0041] Thus, the formulations and compositions according to the present disclosure can be used to control a variety of plant pathogenic fungi or insects on various cultivated plants or weeds, such as wheat, rye, barley, oats, rice, corn, grass, bananas, cotton, soybeans, coffee, sugar cane, vines, fruits and ornamentals, and vegetables such as cucumbers, beans, tomatoes, potatoes and gourds, as well as on the seeds of these plants.

[0042] Thus, the formulations according to the disclosure and the compositions according to the disclosure are suitable for controlling common harmful plants in useful plants, in particular, common harmful plants in crops such as oats, barley, millet, corn, rice, wheat, sugarcane, cotton, rapeseed, flax, lentils, sugar beets, tobacco, sunflower and soybeans or perennial crops.

[0043] The embodiments disclosed below include nanoparticles, compositions containing these nanoparticles, methods of using these nanoparticles, and methods of preparing these therapeutic nanoparticles, wherein the nanoparticles include a tobacco mosaic virus and one or more active ingredients (AIs) non-covalently conjugated to the tobacco mosaic virus. Some embodiments of the nanoparticles, compositions, and methods described herein may provide one or more of the following advantages.

[0044] First, certain embodiments of the present disclosure include nanoparticles and compositions that can be prepared effectively and in a cost-effective manner. Covalent conjugation is commonly used in the preparation of pesticides and nanoparticles. However, covalent conjugation is a complex and resource-intensive process, and in view of the highly hydrophobic nature of proteins, it is challenging to conjugate pesticides to proteins. In addition, the costs and regulatory processes (including classification, characterization, and approval of covalently modified chemicals) associated with using covalent conjugation strategies to connect pesticides to nanoparticles may exceed the benefits of agricultural use. Therefore, an effective non-covalent method for loading nanoparticles is desired. Nanoparticles and compositions of the present disclosure meet this demand by including an AI that does not need to be chemically altered and does not require covalent conjugation to be effectively loaded or infused into tobacco mosaic virus nanoparticles.

[0045] 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 into the tobacco mosaic virus nanoparticles. For example, in some embodiments, the nanoparticles and compositions of the present disclosure can be used to treat diseases in subjects in need, to combat harmful insects and / or plant pathogenic fungi, and to control undesirable vegetation.

[0046] Third, certain embodiments of the present disclosure include nanoparticles that can have high AI loading efficiency. For example, in some embodiments, the nanoparticles and compositions of the present disclosure can load about 1100 AI molecules or more per viral particle.

[0047] Unless otherwise defined, all technical and scientific terms used herein have the same meaning as those generally understood by those of ordinary skill in the art to which the invention belongs. Methods and materials for use in the present invention are described herein; other suitable methods and materials known in the art may also be used. The materials, methods and examples are illustrative only and are 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 the event of a conflict, the present specification (including definitions) shall prevail.

[0048] Other features and advantages of the invention will be apparent from the following detailed description and drawings, and from the claims. BRIEF DESCRIPTION OF THE DRAWINGS

[0049] Figure 1 Schematic diagram of the formation of TMGMV-βCD (top row) and loading of AI onto TMGMV-βCD (bottom row).

[0050] Figure 2 Is a SDS-PAGE gel showing the size of TMGMV (lane 1), TMGMV-alkyne (lane 2), and TMGMV-βCD (lane 3).

[0051] Figure 3 is a size exclusion chromatography (SEC) profile showing the elution of TMGMV and TMGMV-βCD.

[0052] Figure 4 : is a transmission electron micrograph (TEM) showing purified β-CD-TMGMV. The bar indicates a size of 200 nm.

[0053] Figure 5 is a schematic diagram of the method used to detect and quantify the loading of AI onto β-CD-TMGMVs.

[0054] Figure 6 The table is a result of displacement of doxorubicin ("DOX") from β-CD-TMGMV by addition of Clothianidin ("CTD"), Fluopyram ("FLP"), or Tetracycline ("TET").

[0055] Figure 7 is a schematic diagram of a "breathing" phase transition diagram illustrating the effect of pH on the embedding of active ingredients (AI) into TMGMVs.

[0056] Figure 8is an Image J analysis depicting TEM images measured on TMGMV constructs. Images of TMGMV (control) and TMGMV infused with doxorubicin, ATTO550, fluopyram, and clothianidin were taken and analyzed.

[0057] Figures 9A-9F The increase in nanoparticle width of the TMGMV construct was revealed. TEM images show that the nanoparticles infused with doxorubicin ( Fig. 9A ; Bars indicate size of 50 nm), Fluopyram ( Fig. 9B ; Bars indicate size of 100 nm), clothianidin ( Fig. 9C ; Bars indicate size of 50 nm), ATTO550 ( Fig.9D ; bars indicate size of 100 nm) and TMGMV alone ( Fig.9E ; control; bar indicates size of 500 nm) and a bar graph showing the average width of each TMGMV construct in the TMGMV constructs ( Fig.9F ).

[0058] Figures 10A-10B is the soil column ( Fig. 10A ) and soil mobility analysis ( Fig. 10B ) schematic diagram of the experimental setup.

[0059] Figures 11A-11C The distribution of TMGMV and infused TMGMV is shown. Fig.11A Figure 2 is an SDS gel showing which soil fractions contained TMGMV (top gel) and infused TMGMV (bottom gel). The gels were quantified and the fractions containing TMGMV ( Fig. 11B ) and infused TMGMV ( Fig. 11C ) of the soil fractions.

[0060] Figures 12A-12B SDS-PAGE ( Fig. 12A ) and ELISA reader ( Fig. 12B ) Schematic diagram of the soil fluidity analysis.

[0061] Figures 13A-13B Soil mobility of doxorubicin-infused TMGMV is shown. Fig.13A is a plot of the dye in the soil fraction as a percentage of the total dye in the soil. Fig. 13B are the results of SDS-PAGE (top gel) and microplate reader (bottom gel) showing the presence of infused TMGMV in the soil fraction.

[0062] Figures 14A-14B Soil mobility of TMGMV infused with Cy5 amine is shown. Fig.14Ais a plot of the dye in the soil fraction as a percentage of the total dye in the soil. Fig. 14B are the results of SDS-PAGE (top gel) and microplate reader (bottom gel) showing the presence of infused TMGMV in the soil fraction.

[0063] Fig.15 is a graph of the absorbance at 646 nm of soil fractions treated with Cy5 amine alone or with TMGMV-βCD loaded with Cy5 amine.

[0064] Figures 16A-16C Schematic diagram of the method for infusing active ingredients (AI) into TMGMV nanoparticles. Fig.16A is a schematic diagram of the steps for infusing AI into TMGMV nanoparticles via the pH method. Fig. 16B is a schematic diagram of the steps for infusing AI into TMGMV nanoparticles via the dimethyl sulfoxide (DMSO) method. Fig. 16C is a schematic diagram of the characterization steps of AI-loaded TMGMV nanoparticles.

[0065] Figures 17A-17B Images and imaging quantification of TMGMV nanoparticles infused with (ie, non-covalently conjugated to) various active ingredients (AIs) are shown. Fig.17A TEM images of TMGMV nanoparticles loaded with fluopyram, clothianidin, ivermectin, and rifampicin. Fig. 17B Figure 2 shows the measurement of TMGMV nanoparticles infused with AI. Fig.17A Figure 1. TEM images of the samples analyzed by Image J; ****p value < 0.00001.

[0066] Figures 18A-18B is a graph showing the circular dichroism spectrum of the non-covalently loaded TMGMV sample. Fig.18A is a graph showing the circular dichroism spectra of TMGMV nanoparticles infused with AI by the pH method. Fig.18B is a graph showing the circular dichroism spectra of TMGMV nanoparticles infused with AI by the DMSO method.

[0067] Figures 19A-19J is a graph showing image analysis from transmission electron microscopy comparing the length of viral particles after AI infusion. Fig.19A is a graph outlining the quantification of viral particles and their lengths of AI-infused TMGMV nanoparticles prepared by the pH method. Figures 19B-19E The results show that the pH method can be used to evaluate the effect of clothianidin ( Fig.19B )、Ivermectin( Fig.19C )、Fluopyram( Fig.19D ) and rifampicin ( Fig.19E ) of the TMGMV nanoparticles and the quantification of their virus particles and length. Fig.19F is a graph outlining the quantification of viral particles and their lengths of AI-infused TMGMV nanoparticles prepared by the DMSO method. Figures 19G-19J The results show that the DMSO method can be used to treat the infusion of clothianidin ( Figure 19G )、Ivermectin( Fig.19H )、Fluopyram( Fig.19I ) and rifampicin ( Fig.19J ) of the TMGMV nanoparticles and the quantification of their virus particles and length.

[0068] Figures 20A-20D is an illustration showing the surface charge distribution of various AI molecules. Fig. 20A The surface charge distribution of clothianidin is shown. Fig. 20B The surface charge distribution of fluopyram is shown. Fig. 20C The surface charge distribution of ivermectin is shown. Fig.20D The surface charge distribution of rifampicin is shown.

[0069] Fig.21A and 21B is an image of a molecular modeling simulation of molecular docking of TMGMV coat protein and rifampicin.

[0070] Fig.22A and 22B is an image of a molecular modeling simulation of molecular docking of TMGMV coat protein and ivermectin.

[0071] Fig.23A and 23B is an image of a molecular modeling simulation of molecular docking of TMGMV coat protein and fluopyram.

[0072] Fig.24A and 24B This is an image of a molecular modeling simulation of molecular docking of TMGMV coat protein and clothianidin.

[0073] Fig.25 is a TEM image of TMGMV particle aggregation after AI infusion (eg, Cy5).

[0074] Figures 26A-26D Characterization of AI-infused TMGMV nanoparticles additionally loaded with cyanine 5 (Cy5) and doxorubicin is shown. Fig.26A is a transmission electron microscopy (TEM) image of AI-infused TMGMV nanoparticles loaded with Cy5. Fig.26B is a graph showing size exclusion chromatography of AI-infused TMGMV nanoparticles loaded with Cy5. Fig.26C is a TEM image of AI-infused TMGMV nanoparticles loaded with doxorubicin. Fig.26D is a graph showing size exclusion chromatography of AI-infused TMGMV nanoparticles loaded with doxorubicin.

[0075] Fig. 27 is a graph showing a comparison of the width of TMGMV nanoparticles prepared by the pH and DMSO methods. Wild-type TMGMV was exposed to pH 7.5 and 20% DMSO concentration as described in the "Methods" section of the Examples and Example 6. Transmission electron microscopy was performed and TMGMV nanoparticles were analyzed using ImageJ software to determine the length of the particles (n=150).

[0076] Figures 28A-28D Shown are the binding heats of each conformation of the docked AI on TMGMV and its related residues as calculated using molecular modeling simulation software. Fig.28A is a graph showing the binding heats of each conformation of docked ivermectin on TMGMV and its related residues. Fig.28B is a graph showing the binding heats of each conformation of docked fluopyram on TMGMV, its relevant residues. Fig.28C is a graph showing the binding heat of each conformation of docked clothianidin on TMGMV and its related residues. Fig.28D is a graph showing the binding heats of each conformation of docked rifampicin on TMGMV and its related residues.

[0077] Figures 29A-29D is to show that clothianidin ( Fig.29A )、Fluopyram( Fig.29B )、Ivermectin( Fig.29C ) and rifampicin ( Fig.29D ) binding regions, their functions towards TMGMV and a table of residues specifically identified to stabilize these AIs. DETAILED DESCRIPTION

[0078] Unless otherwise defined below, all technical and scientific terms used herein are intended to have the same meaning as those commonly understood by those of ordinary skill in the art. References to the techniques employed herein are intended to refer to techniques commonly understood in the art, including changes to those techniques and / or replacement of equivalent techniques that will be apparent to those skilled in the art.

[0079] As used herein, the singular forms "a," "an," and "the" include plural referents unless the content clearly dictates otherwise.

[0080] As used herein, the terms "about" and "approximately" when used to modify an amount specified as a value or range, indicate the value as well as reasonable deviations from the value known to those skilled in the art, e.g., ±20%, ±15%, ±10%, ±5%, ±4%, ±3%, ±2% or ±1% within the intended meaning of the value.

[0081] The term "nanoparticle" means an object having a length between about 2 nm and about 300 nm (e.g., between about 2 nm and 100 nm, between 2 nm and 200 nm, between 2 nm and 250 nm, between 2 nm and 300 nm, between 100 nm and 200 nm, between 100 nm and 250 nm, between 100 nm and 300 nm, between 150 nm and 250 nm, between 200 nm and 300 nm, between 200 nm and 250 nm). Non-limiting examples of nanoparticles include nanoparticles described herein.

[0082] As used herein, the term "subject" or "patient" 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 may be administered, e.g., for experimental, diagnostic, prophylactic, and / or therapeutic purposes. A subject may be seeking or in need of treatment, in need of treatment, currently receiving treatment, about to be treated, or being cared for by a trained professional for a particular disease or condition.

[0083] The term "chemotherapeutic agent" refers to a molecule that can be used to reduce the growth rate of cancer cells in a subject (e.g., a human) or to induce or mediate the death (e.g., necrosis or apoptosis) of cancer cells. 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, chlorabucil, melphalan, daunorubicin, doxorubicin, epirubicin, idarubicin, mitoxantrone, valrubicin, paclitaxel, docetaxel, etoposide, teniposide, tafluposide, The chemotherapeutic agents include, for example, cytarabine, azacitidine, axathioprine, capecitabine, cytarabine, doxifluridine, fluorouracil, gemcitabine, mercaptopurine, methotrexate, tioguanine, 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.

[0084] As described herein, the term "effective amount" is an amount that produces a desired result. In the context of this specification, a desired result may include a reduction in undesirable characteristics (e.g., a reduction in undesirable organisms, a reduction in undesirable plants, etc.).

[0085] This document provides nano-particles, compositions, preparation methods and methods for applying engineered tobacco mosaic virus for the purpose of drug and pesticide delivery.Nano-particles described herein, compositions, preparation methods and methods of use can include the tobacco mosaic virus of any species.For example, nano-particles described herein, compositions, preparation methods and methods of use can include tobacco mild green mosaic virus (TMGMV).In another example, nano-particles described herein, compositions, preparation methods and methods of use can include tobacco mosaic virus (TMV). In some embodiments, the nanoparticles, compositions, preparation methods, and methods of use described herein may include sweet pepper mottle virus (BPeMV), wood mandshurica mild mottle virus, cactus mild mottle virus (CMMoV), butterfly pea yellow mottle virus, cucumber mottle mosaic virus, cucumber green mottle mosaic virus (CGMMV), cucumber mottle virus, plumeria mosaic virus (FrMV), hibiscus latent Fort Pierce virus (HLFPV), hibiscus latent Singapore virus (HLSV), kyuri green mottle mosaic virus (kyuri green mottle mosaic virus), passion flower leaf virus (MarMV), obuda pepper virus (obuda pepper virus), and the like. virus (ObPV), Orchid ringspot virus (ORSV), Cactus chlorotic ringspot virus, Pepper mild mottle virus, Passion fruit mosaic virus, Pepper mild mottle virus (PMMoV), Plumeria mosaic virus, Raccoon necrosis-associated virus (RCNaV), Rehmannia mosaic virus, Longleaf plantain mosaic virus (HRV), Sammons's Opuntia virus (SOV), Dolphin flower break virus, Sun hemp mosaic virus (SHMV), Tobacco latent virus, Tomato brown wrinkled fruit virus (ToBRFV), Tomato mosaic virus (ToMV), Tomato mottle mosaic virus, Tropical soda apple mosaic virus, Turnip vein clear virus (TVCV), Chenopodium mild mottle virus, Wasabi mottle virus (WMoV), Yellowtail mild mottle virus, Rape mosaic virus (YoMV) (also known as rapeseed mosaic virus (ORMV)), Cucurbita pekinensis green mottle mosaic virus, or any combination thereof.

[0086] Tobacco mosaic virus is a genus of positive-stranded RNA viruses in the family Virgaviridae. TMGMV and TMV are members of the genus Tobacco mosaic virus, which consists of baculoviruses that are strictly plant pathogens.

[0087] Both TMGMV and TMV have 2,130 identical coat proteins that are helically arranged around a single-stranded RNA genome to form 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 two solvent-exposed tyrosine side chains (Tyr 2 and Tyr 139) that can be functionalized using a diazo coupling reaction. In some embodiments, a tobacco mosaic virus (e.g., TMGMV or TMV) is coupled to a carrier such as β-cyclodextrin (BCD).

[0088] In some embodiments, the present disclosure relates to a "breathing" method for tobacco mosaic virus (e.g., TMGMV or TMV) based on careful pH adjustment (sometimes referred to herein as "pH method") or concentration of solvent (sometimes referred to herein as "solvent method") and loaded molecules such as, but not limited to, fluopyram, clothianidin, rifampicin, and ivermectin (see, e.g., Figures 17-24). Figures 26A-26D As shown, doxorubicin and cyanine 5 (Cy5) are used as model active ingredients; the fluorescence of doxorubicin and Cy5 provides a convenient method for characterization. As explained in Examples 5-10, nanoparticle formulations prepared by pH and solvent methods and tobacco mosaic virus (e.g., TMGMV or TMV) structures are characterized by a combination of techniques to determine particle integrity, AI infusion, and secondary structure stability after infusion.

[0089] Nanoparticles

[0090] In some embodiments, the nanoparticles of the present disclosure are viral nanoparticles. In some embodiments, the viral nanoparticles are of the genus Tobacco Mosaic Virus. 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 Tobacco Mosaic Virus. In some embodiments, the viral nanoparticles are sweet pepper mottle virus (BPeMV), wood mandrake mild mottle virus), cactus mild mottle virus (CMMoV), butterfly pea yellow mottle virus, cucumber mottle mosaic virus, cucumber green mottle mosaic virus (CGMMV), cucumber mottle virus, plumeria mosaic virus (FrMV), hibiscus latent Fort Pierce virus (HLFPV), hibiscus latent Singapore virus (HLSV), kyuri green mottle mosaic virus, passion fruit leaf virus (MarMV), obuda pepper virus (ObPV), tooth orchid ring spot virus (ORSV), cactus chlorotic ring spot virus, pepper mild mottle virus, passion fruit mosaic virus, pepper mild mottle disease tomato mosaic virus (PMMoV), frangipani mosaic virus, rat tail cactus necrosis-associated virus (RCNaV), rehmannia mosaic virus, longleaf plantain mosaic virus (HRV), Samons cactus virus (SOV), dolphin flower break virus, sun hemp mosaic virus (SHMV), tobacco latent virus, tomato brown wrinkled fruit virus (ToBRFV), tomato mosaic virus (ToMV), tomato mottle mosaic virus, tropical soda apple mosaic virus, turnip vein clear virus (TVCV), tuberous quinoa mild mottle virus, wasabi mottle virus (WMoV), yellow tail flower mild mottle virus, rapeseed mosaic virus (YoMV) (also known as rapeseed mosaic virus (ORMV)), zucchini green mottle mosaic virus, or any combination thereof.

[0091] In some embodiments, the tobacco mosaic virus genus (e.g., TMGMV or TMV) is an engineered tobacco mosaic virus genus (e.g., TMGMV or TMV). In some embodiments, the engineered tobacco mosaic virus genus (e.g., TMGMV or TMV) is conjugated to or otherwise connected to beta-cyclodextrin. In some embodiments, beta-cyclodextrin is located on the outer surface of the tobacco mosaic virus genus (e.g., TMGMV or TMV). In some embodiments, beta-cyclodextrin interacts with active ingredients through supramolecular interactions. In some embodiments, beta-cyclodextrin interacts with active ingredients (AI) (e.g., pesticides) through hydrophobic and / or hydrophilic interactions.

[0092] In some embodiments, engineered tobacco mosaic virus (e.g., TMGMV or TMV) is modified to be infused, impregnated with, or otherwise contain an active ingredient (AI) (e.g., tobacco mosaic virus (e.g., TMGMV or TMV) is modified to "breathe" in the AI). In some embodiments, tobacco mosaic virus (e.g., TMGMV or TMV) is partially and reversibly dissociated to allow the AI ​​to be incorporated into the tobacco mosaic virus (e.g., TMGMV or TMV) structure. In some embodiments, in response to external factors, one or more coat proteins of tobacco mosaic virus (e.g., TMGMV or TMV) reversibly and partially dissociate. The external factor can be a change in pH or the presence of a specific concentration of solvent. For example, a change in pH results in a change in the ionization of protein residues, which in turn affects the electrostatic interactions therebetween. When the capsid proteins begin to dissociate, the nanoparticle structure "breathes", and the pores or pockets open, allowing access to the space between the coat proteins. In addition to pH, solvents can also trigger assembly and disassembly. For example, in some embodiments, a solvent having a concentration of about 15% (v / v) to about 25% (v / v) tends to destabilize the protein due to its effect on the charge state distribution and the destruction of structural water in the protein, thereby causing dissociation and unfolding. In some embodiments, the solvent is a polar aprotic solvent. In some embodiments, the solvent is a polar aprotic solvent that is miscible in water. In some embodiments, the solvent is dimethyl sulfoxide (DMSO).

[0093] In certain embodiments, one or more AIs are non-covalently conjugated to one or more holes or pockets of the tobacco mosaic virus (e.g., TMGMV or TMV) and embedded therein. In certain embodiments, one or more AIs are embedded in one or more coat proteins of the tobacco mosaic virus (e.g., TMGMV or TMV). In certain embodiments, when loaded into the tobacco mosaic virus (e.g., TMGMV or TMV), one or more AIs are not chemically altered. In certain embodiments, the impregnated tobacco mosaic virus (e.g., TMGMV or TMV) contains the AI ​​located in the tobacco mosaic virus (e.g., TMGMV or TMV). In certain embodiments, the impregnated tobacco mosaic virus (e.g., TMGMV or TMV) contains the AI ​​dispersed in the tobacco mosaic virus (e.g., TMGMV or TMV) and in the whole tobacco mosaic virus. In certain embodiments, the impregnated tobacco mosaic virus (e.g., TMGMV or TMV) does not interact with the AI ​​on the surface of the tobacco mosaic virus (e.g., TMGMV or TMV). In some embodiments, the TMG mosaic virus (eg, TMGMV or TMV) MV does not have an AI capsid on the outer surface of the mosaic virus (eg, TMGMV or TMV).

[0094] In some embodiments, the AI-loaded tobacco mosaic virus (e.g., TMGMV or TMV) and the unloaded tobacco mosaic virus (e.g., TMGMV or TMV) are rod-shaped. In some embodiments, the engineered tobacco mosaic virus (e.g., TMGMV or TMV) has a different shape (e.g., a different width) than the non-engineered tobacco mosaic virus (e.g., TMGMV or TMV). For example, when the tobacco mosaic virus (e.g., TMGMV or TMV) is loaded with AI, the AI-loaded tobacco mosaic virus (e.g., TMGMV or TMV) can exhibit a wider width than the unloaded tobacco mosaic virus (e.g., TMGMV or TMV) or the reference tobacco mosaic virus (e.g., TMGMV or TMV), such as Fig.17A In some embodiments, AI-loaded tobacco mosaic virus (e.g., TMGMV or TMV) may appear swollen compared to an unloaded tobacco mosaic virus (e.g., TMGMV or TMV) or a reference tobacco mosaic virus (e.g., TMGMV or TMV). In some embodiments, this change in width of AI-loaded tobacco mosaic virus (e.g., TMGMV or TMV) indicates AI encapsulation.

[0095] In some embodiments, the engineered tobacco mosaic virus (e.g., TMGMV or TMV)-AI nanoparticles are approximately 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%, %, 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%. In some embodiments, the width of the engineered tobacco mosaic virus (e.g., TMGMV or TMV)-AI nanoparticle is 19 nm, 20 nm, 21 nm, 22 nm, 23 nm, 24 nm, 25 nm, 26 nm, 27 nm, 28 nm, 29 nm, 30 nm, 31 nm, 32 nm, 33 nm, 34 nm, 35 nm, 36 nm, 37 nm, 38 nm, 39 nm, 40 nm, 41 nm, 42 nm, 43 nm, 44 nm, 45 nm, 46 nm, 47 nm, 48 nm, 49 nm, 50 nm, 51 nm, 52 nm, 53 nm, 54 nm, 55 nm, 56 nm, 57 nm, 58 nm, 59 nm, 60 nm, 61 nm, 62 nm, 63 nm, 64 nm, 65 nm, 66 nm, 67 nm, 68 nm, 69 nm, 70 nm, 3nm, 44nm, 45nm, 46nm, 47nm, 48nm, 49nm, 50nm, 51nm, 52nm, 53nm, 54nm, 55nm, 56nm, 57nm, 58nm, 59nm, 60nm, 61nm, 62nm, 63nm, 64nm, 65nm, 66nm, 67nm, 68nm, 69nm, 70nm, 71nm, 72nm, 73nm, 74nm or 75nm.

[0096] In some embodiments, the nanoparticles provided herein can be rod-shaped or can have an amorphous shape. In some embodiments, the nanoparticles provided herein have a diameter 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 1 In some embodiments, the nanoparticles provided herein have a length (extending between the first end and the second end of the outer surface of the rod-shaped nanoparticle) in the range of 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. In some embodiments, the nanoparticles provided herein have a length (extending between the first end and the second end of the outer surface of the rod-shaped nanoparticle). In certain embodiments, the length of the nanoparticle provided herein is between about 100nm. In certain embodiments, the length of the nanoparticle provided herein is between about 150nm. In certain embodiments, the length of the nanoparticle provided herein is between about 200nm. In certain embodiments, the length of the nanoparticle provided herein is between about 250nm. In certain embodiments, the length of the nanoparticle provided herein is between about 300nm.

[0097] In some embodiments, the nanoparticles of the present disclosure comprise about 1 to about 2000 AI molecules / TMGMV or more (e.g., 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 150, 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 1800, 10 to 1999, 10 to 15, 10 to 180 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 2 00, 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, 000-1500, 150-2000, 500-600, 500-700, 500-800, 500-900, 500-1000, 500-1100, 500-1500, 500-2000, 750-800, 750-900, 750-1000, 750-1100, 750-1500, 750-2000, 1000-1100, 1000-1500, 1000-2000 AI molecules per Tobacco Mosaic Virus (e.g., TMGMV or TMV), or more).

[0098] Composition

[0099] In certain embodiments, the composition of the present disclosure contains a plurality of nanoparticles of the present disclosure. In certain embodiments, the plurality of nanoparticles include nanoparticles with identical engineered modifications (e.g., engineered tobacco mosaic virus colony, wherein the tobacco mosaic virus colony is connected to beta-cyclodextrin or engineered tobacco mosaic virus colony, wherein the colony is impregnated with specific AI). In certain embodiments, a plurality of nanoparticles include nanoparticles carrying identical AI. In certain embodiments, the composition includes a mixture of nanoparticles, wherein some nanoparticles in the nanoparticles of the mixture include tobacco mosaic virus that beta-cyclodextrin (BCD) connects, and some nanoparticles in the nanoparticles of the mixture include impregnated tobacco mosaic virus. In certain embodiments, the composition includes a mixture of nanoparticles, wherein the nanoparticles carry or are impregnated with different AI (e.g., pesticides). For example, the composition of the present disclosure may include a mixture of nanoparticles, wherein the nanoparticles "A" of the tobacco mosaic virus that includes a plurality of BCDs carrying pesticides "1" are connected and the nanoparticles "B" of the tobacco mosaic virus that includes a plurality of pesticides "1" are mixed. Other non-limiting examples include a mixture of nanoparticles "A" containing multiple BCD-linked tobacco mosaic virus carrying insecticide "1" and nanoparticles "B" containing multiple BCD-linked tobacco mosaic virus carrying insecticide "2", or a mixture of nanoparticles "A" containing multiple tobacco mosaic virus impregnated with insecticide "1" and nanoparticles "B" containing multiple tobacco mosaic virus impregnated with insecticide "2". In some embodiments, mixtures of three or more, four or more, or five or more nanoparticles of the present disclosure are also included.

[0100] In some embodiments, the disclosed nanoparticles can be formulated into aqueous solutions. In some embodiments, the nanoparticles can be formulated into hydrogels. In some embodiments, the disclosed nanoparticles can be freeze-dried. In some embodiments, the disclosed nanoparticles are formulated into redispersible powders and aqueous dispersions. In some embodiments, the nanoparticles contain a high weight percentage of water-insoluble pesticides or other active ingredients. In some embodiments, the disclosed nanoparticles can be prepared from oil-in-water microemulsions or nanoemulsions containing water-insoluble, non-halogenated volatile organic solvents, from which the organic solvents and / or water have been removed.

[0101] In certain embodiments, the composition comprising any disclosed nanoparticles may also include 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, based on the gross weight of the microemulsion, a cosolvent is present in an amount of about 5 wt % to about 30 wt %.

[0102] In some embodiments, the disclosed nanoparticles are in a liquid formulation. In some embodiments, the liquid formulation comprises an AI and the disclosed nanoparticles, the nanoparticles being dissolved, emulsified as droplets, or suspended as matrix particles. In some embodiments, the liquid formulation comprises the disclosed nanoparticles and an AI (e.g., an insecticide), the AI ​​being dissolved or emulsified as droplets. In some embodiments, the insecticide is uniformly distributed throughout the particle.

[0103] In some embodiments, any nanoparticle formulation described herein can be used as is or in a form prepared therefrom by spraying, atomizing, dusting, spreading or pouring, for example, in the form of a solution, powder, suspension or dispersion, emulsion, oil dispersion, paste, dustable product, material for spreading or granules that can be sprayed directly. In some embodiments, the form of use depends entirely on the intended purpose; for example, the formulation is intended to ensure the finest possible distribution of the pesticides and nanoparticles described herein in each case.

[0104] In some embodiments, aqueous use forms can also be prepared from emulsion concentrates, pastes or wettable powders (sprayable powders, oil dispersions) by adding a suitable solvent (e.g., water). In some embodiments, the disclosed nanoparticles can be used alone, or have been partially or completely mixed with each other and / or with any AI disclosed herein to prepare compositions according to the present disclosure.

[0105] In some embodiments, the composition comprising any disclosed nanoparticles may further comprise a surfactant or a mixture of surfactants. In one embodiment, the surfactant is any one or more of the following: a cationic surfactant, an anionic surfactant, an amphoteric surfactant, a nonionic surfactant, and a mixture thereof. In some embodiments, the anionic surfactant is selected from the group consisting of: alkylbenzene sulfonate (e.g., sodium alkylnaphthalene sulfonate), sodium dodecyl sulfate, sodium sulfosuccinate, sodium lauryl sulfate, sodium salt of alkylnaphthalene sulfonate condensate, sodium stearate, and a mixture thereof; the nonionic surfactant is selected from the group consisting of: ethoxylated sorbitan esters, sorbitan esters, silicone surfactants, polyglycerol esters, sucrose esters, poloxamer, alkyl polyglucosides, polyoxyalkylene modified heptamethyl trisiloxane and allyloxy polyethylene glycol methyl ether, and a mixture thereof; the amphoteric surfactant is lecithin; and the cationic surfactant is selected from the group consisting of: hexadecyl trimethyl ammonium bromide, hexadecyl trimethyl ammonium chloride, and a mixture thereof. In some embodiments, the surfactant is present in an amount of about 5% by weight to about 35% by weight based on the total weight of the microemulsion. In some embodiments, the surfactant is (sodium n-butylnaphthalene sulfonate). In some embodiments, the surfactant is L-77 (a silicone surfactant comprising a blend of polyalkylene oxide-modified heptamethyltrisiloxane and allyloxy polyethylene glycol methyl ether.

[0106] In certain embodiments, nanoparticles of the present disclosure can be deployed in matrix. As used herein, term "matrix" has its common meaning and refers to the mixture that nanoparticles are suspended in another material. Therefore, in certain embodiments, compositions include dispersed or suspended nanoparticles. Within the scope of the present disclosure, matrix fluid refers to compositions activated by any one or more activation methods in the activation method. In certain embodiments, matrix is ​​a hydrogel.

[0107] In some embodiments, the compositions of the present disclosure include an excipient. 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.

[0108] Active ingredients

[0109] Any nanoparticle described herein may include 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 may be a chemokine, an antimicrobial, or any therapeutic compound. In some embodiments, the drug is a chemotherapeutic, an antiparasitic, an antibiotic, an immunomodulator, an antifungal, an antiprotozoal, an antiviral, or any combination thereof. In some embodiments, the drug is a hydrophilic drug or a hydrophobic drug.

[0110] 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, tafluposide, 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).

[0111] In some embodiments, the antiparasitic drug is niclosamide, oxyclozanide, rafoxanide, closantel, dibromsalan, metabromsalan, tribromsalan, and nitazoxanide.

[0112] In some embodiments, the antibiotic is a beta-lactam antibiotic, an aminoglycoside, a loop 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 oxidant, a substance that releases free radicals and / or active oxygen, a cationic antimicrobial, 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.

[0113] In some embodiments, immunomodulators are substances that stimulate or suppress the immune system and can help the body fight cancer, infection, or other diseases. In some embodiments, immunomodulators are cancer immunotherapeutics, such as, but not limited to, checkpoint inhibitors, adoptive cell therapy (T cell transfer therapy, monoclonal antibody therapy, cancer vaccines, immune system modulators (e.g., cytokines and biological response modifiers, such as thalidomide, lenalidomide, pomalidomide, and imiquimod), or any combination thereof. In some embodiments, immunomodulators are corticosteroids, disease-modifying antirheumatic drugs (DMARDs) (e.g., azathioprine), e, cyclosporine, hydroxychloroquine, leflunomide, methotrexate and sulfasalazine), biologics (e.g., tumor necrosis factor (TNF) inhibitors, interleukin-1 (IL-1) inhibitors, interleukin-6 (IL-6) inhibitors, T cell inhibitors, B cell inhibitors), Janus kinase inhibitors, or any combination thereof. In some embodiments, the immunomodulator is GM-CSF (granulocyte-macrophage colony stimulating factor).

[0114] Any nanoparticle described herein can be 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 antimicrobial, or any therapeutic compound. In some embodiments, the drug is a chemotherapeutic, an antiparasitic, an antibiotic, or an immunomodulator. 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 antimicrobial, 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, an anthelmintic, a rodenticide, a defoliant, a desiccant, a safener, or any combination thereof. In some embodiments, the insecticide is a benzoylurea, such as fluazifop, lufenuron, chlorpyrifos, flufenoxuron, noviflumuron, flubendiamide, fluthrin and flubendiamide; a carbamate; a pyrethroid, such as cyhalothrin and its isomers and isomer mixtures, lambda-cyhalothrin, deltamethrin, fluvalinate, cyfluthrin, lambda-cyhalothrin, tefluthrin and bifenthrin; an organophosphate, such as azinphos-methyl, chlorpyrifos, diazinon, endosulfan, methidathrin; a neonicotinoid; a phenylpyrazole, such as imidacloprid, acetamiprid, thiamethoxam, dimethomorph, dinotefuran, thiamethoxam and fipronil; a conazole, such as epoxiconazole, hexaconazole, propiconazole, prochloraz, imazalil, triadimenol, difenoconazole, myclobutanil, propylthiocyanate, chlorpyrifos ... oxadiazole, trichlorfon and tebuconazole; morpholines such as dimethomorph, fenpropidin and fenpropimorph; strobilurins such as azoxystrobin, kresoxim-methyl and their analogs; phthalonitriles such as thiocarb; mancozeb; fluazinam; pyrimidines such as ethoxystrobin; aryloxyphenoxy derivatives; aryl ureas; aryl carboxylic acids; aryloxyalkanoic acid derivatives such as clodinafop-butyl and its analogs, fenoxaprop-butyl and its analogs, chlorfenapyr, quizalofop-butyl and its analogs; dinitroanilines such as pendimethalin and trifluralin; diphenyl ethers such as oxyfluorfen; imidazolinones; sulfonylureas such as chlorsulfuron, nicosulfuron, sulfamethoxam, bensulfuron; sulfonamides; triazines; and triazinones such as metamitron; and any combination thereof. In some embodiments, any nanoparticle described herein can include one or more compounds selected from the group consisting of: fungicides, insecticides, nematicides, herbicides and / or safeners or growth regulators. Any pesticides of two or more of the above categories can be used. Those skilled in the art are familiar with useful drugs and pesticides, which can be found in, for example, Pesticide Manual, 13th edition (2003), The British Crop Protection Council, London.

[0115] Any of the nanoparticles, compositions, or methods described herein may include any one or more of the following list of pesticides, which list is intended to illustrate possible combinations but does not impose any limitation:

[0116] A) Strobilurins,

[0117] azoxystrobin, dimoxystrobin, enestroburin, fluoxastrobin, kystrobin, 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-carboximidothiolmethyl)-phenyl)-acrylate, methyl (2-chloro-5-[1-(3-methylbenzyloxyimino)ethyl]benzyl)carbamate and 2-(2-(3-(2,6-dichlorophenyl)-1-methyl-allyliminooxymethyl)-phenyl)-2-methoxyimino-N-methyl-acetamide;

[0118] B) formamide,

[0119] Formanilides: benalaxyl, benalaxyl-M, benodanil, bixafen, boscalid, carboxin, fenfuram, fenhexamid, flutolanil, furametpyr, isopyrazam, isotianil, kiralaxyl, mepronil, metalaxyl, metalaxyl-M (mefen oxam), ofurace, xadixyl, oxycarboxin, penthiopyrad, tecloftalam, thifluzamide, tiadinil, 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'-chlorobiphenyl-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'-chlorobiphenyl-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-(1,1,2,3,3,3-hexafluoropropoxy)-phenyl]-3-difluoromethyl-1-methyl-1H-pyrazole-4-carboxamide,2-tetrafluoroethyloxy)-phenyl]-3-difluoromethyl-1-methyl-1H-pyrazole-4-carboxamide, N-(4'-trifluoromethyl-thiophenyl-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-trimethyl-butyl)-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-dicyclopropyl-2-yl-phenyl)-3-difluoromethyl-1-methyl-1H-pyrazole-4-carboxamide, -(cis-2-dicyclopropyl-2-yl-phenyl)-3-difluoromethyl-1-methyl-1H-pyrazole-4-carboxamide, N-(trans-2-dicyclopropyl-2-yl-phenyl)-3-difluoromethyl-1-methyl-1H-pyrazole-4-carboxamide, N-[1,2,3,4-tetrahydro-9-(1-methylethyl)-1,4-methylene-naphthalen-5-yl]-3-(difluoromethyl)-1-methyl-1H-pyrazole-4-carboxamide;

[0120] Carboxymorpholines: dimethomorph, flumorph;

[0121] Benzoic acid amides: flumetover, fluopicolde, fluopyram;

[0122] Other carboxamides: carpropamid, dicyclomet, mandiproamid, oxytetracyclin, silthiofarm and N-(6-methoxy-pyridin-3-yl)cyclopropanecarboxylic acid amide;

[0123] C) azole,

[0124] Triazoles: azaconazole, bitertanol, bromuconazole, 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;

[0125] Imidazoles: cyazofamid, imazalil, pefurazoate, prochloraz, triflumizol;

[0126] Benzimidazoles: benomyl, carbendazim, fuberidazole, thiabendazole;

[0127] Others: ethaboxam, etridiazole, hymexazole and 2-(4-chloro-phenyl)-N-[4-(3,4-dimethoxy-phenyl)-isoxazol-5-yl]-2-prop-2-ynyloxy-acetamide;

[0128] D) heterocyclic compounds,

[0129] Pyridines: fluazinam, pyrifenox, 3-[5-(4-chloro-phenyl)-2,3-dimethyl-isoxazolidin-3-yl]-pyridine, 3-[5-(4-methyl-phenyl)-2,3-dimethyl-isoxazolidin-3-yl]-pyridine, 2,3,5,6-tetra-chloro-4-methanesulfonyl-pyridine, 3,4,5-trichloropyridine-2,6-di-carbonitrile, N-(1-(5-bromo-3-chloro-pyridin-2-yl)-ethyl)-2,4-dichloronicotinamide, N-[(5-bromo-3-chloro-pyridin-2-yl)-methyl]-2,4-dichloro-nicotinamide;

[0130] Pyrimidines: bupirimate, cyprodinil, diflumetorim, fenarimol, ferimzone, mepanipyrim, nitrapyrin, nuarimol, pyrimethanil;

[0131] Piperazines: triforine;

[0132] Apyrroles: fenpiclonil, fludioxonil;

[0133] Morpholines: aldimorph, dodemorph, dodemorph-acetate, fenpropimorph, tridemorph;

[0134] Piperidines: fenpropidin;

[0135] Dicarboximides: fluoroimid, iprodione, procymidone, vinclozolin;

[0136] Non-aromatic 5-membered heterocyclic ring: oxadone, fenamidone, octhilinone, probenazole, S-5-amino-2-isopropyl-3-oxo-4-o-tolyl-2,3-dihydro-pyrazole-1-carboxythioic acid allyl ester;

[0137] and / or any other: acibenzolar-S-methyl, amisulbrom, anilazin, blasticidin-S, captafol, captan, chinomethionat, dazomet, debacarb, diclomezine, difenzoquat, avenous sulphate-methyl, fenoxanil, folpet, oxolinic acid, 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 6-(2,4,6-trifluorophenyl)-[1,2,4]triazolo[1,5-a]pyrimidine, 6-(3,4-dichlorophenyl)-5-methyl-[1,2,4]triazolo[1,5-a]pyrimidine-7-ylamine, 6-(4-tert-butyl-phenyl)-5-methyl-[1,2,4]triazolo[1,5-a]pyrimidine-7-ylamine, 5-methyl-6-(3,5,5-trimethyl-hexyl)-[1,2,4]triazolo[1,5-a]pyrimidine-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, 5-(trifluoromethyl)-6-(3,5,5-trimethyl-hexyl)-[1,2,4]triazolo[1,5-a]pyrimidin-7-ylamine; 6-(3,5,5-trimethyl-hexyl)-[1,2,4]triazolo[1,5-a]pyrimidin-7-ylamine; 5-(trifluoromethyl)-6-(3,5,5-trimethyl-hexyl)-[1,2,4]triazolo[1,5-a]pyrimidin-7-ylamine;

[0138] E) carbamates,

[0139] Thiocarbamates and dithiocarbamates: ferbam, mancozeb, maneb, metam, methasulphocarb, metiram, propineb, thiram, zineb, ziram;

[0140] Carbamates: benthiavalicarb, diethofencarb, flubenthiavalicarb, iprovalicarb, propamocarb, propamocarb hydrochlorid, valiphenal and N-(1-(1-(4-cyano-phenyl)-ethanesulfonyl)-butan-2-yl)carbamic acid-(4-fluorophenyl) ester;

[0141] F) any other active substance,

[0142] Guanidines: guanidine, dodine, dodine free base, guazatine, guazatine acetate, iminoctadine, iminoctadine triacetate, iminoctadine tri(p-dodecylbenzenesulfonate);

[0143] Antibiotics: kasugamycin, kasugamycin hydrochloride-hydrate, streptomycin, polyoxine, validamycin A;

[0144] Nitrophenyl derivatives: binapacryl, dinobuton, dinocap, nitrthal-isopropyl, tecnazen; organometallic compounds: fentin salts, such as fentin acetate, fentin chloride or fentin hydroxide;

[0145] Sulfur-containing heterocyclic compounds: dithianon, isoprothiolane;

[0146] Organophosphorus compounds: edifenphos, fosetyl, fosetyl-aluminum, iprobenfos, phosphoric acid and its salts, pyrazophos, tolclofos-methyl;

[0147] Organochlorine compounds: chlorothalonil, dichlofluanid, dichlorophen, flusulfamide, hexachlorobenzene, pencycuron, pentachlorophenol and its salts, phthalide, quintozene, thiophanate-methyl, tolylfluanid, N-(4-chloro-2-nitro-phenyl)-N-ethyl-4-methyl-benzenesulfonamide;

[0148] Inorganic active substances: Bordeaux mixture, copper acetate, copper hydroxide, copper oxychloride, basic copper sulfate, sulfur;

[0149] Any one or more of the others: biphenyl, bronopol, cyflufenamid, cymoxanil, diphenylamine, metrafenone, mildiomycin, oxin-copper, prohexadione-calcium, spiroxamine, tolylfluanid, N-(cyclopropylmethoxyimino-(6-difluoro-methoxy)imino) 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-trimethylsilyl-propoxy)-phenyl)-N-ethyl-N-methylformamidine and N'-(5-difluoromethyl-2-methyl-4-(3-trimethylsilyl-propoxy)-phenyl)-N-ethyl-N-methylformamidine.

[0150] G) Herbicides, such as acetamides: acetochlor, alachlor, butachlor, dimethachlor, dimethenamid, flufenacet, mefenacet, metolachlor, metazachlor, naproxamide, naproanilide, pethoxamid, pretilachlor, propachlor, thenylchlor;

[0151] Amino acid derivatives: bialafos, glyphosate, glufosinate, sulfosate;

[0152] Aryloxyphenoxypropionate: clodinafop, cyhalofop-butyl, fenoxaprop, fluazifop, haloxyfop, metamifop, propaquizafop, quizalofop, quizalofop-P-tefuryl;

[0153] Bipyridines: diquat, paraquat;

[0154] (Sulfur) carbamates: asulam, butylate, carbetamide, desmedipham, dimepiperate, eptam (EPTC), esprocarb, molinate, orbencarb, phenmedipham, prosulfocarb, pyributicarb, thiobencarb, triallate;

[0155] Cyclohexanedione: butroxydim, clethodim, cycloxydim, profoxydim, sethoxydim, tepraloxydim, tralkoxydim;

[0156] Dinitroaniline: benfluralin, ethalfluralin, oryzalin, pendimethalin, prodiamine, trifluralin;

[0157] Diphenyl ethers: acifluorfen, aclonifen, bifenox, diclofop, ethoxyfen, fomesafen, lactofen, oxyfluorfen;

[0158] Hydroxybenzonitriles: bomoxynil, dichlobenil, ioxynil;

[0159] Imidazolinones: imazamethabenz, imazamox, imazapic, imazapyr, imazaquin, imazethapyr;

[0160] Phenoxyacetic acid: clomeprop, 2,4-dichlorophenoxyacetic acid (2,4-D), 2,4-DB, dichlorprop, MCPA, MCPA-thioethyl, MCPB, Mecoprop;

[0161] Pyrazines: chloridazon, flufenpyr-ethyl, fluthiacet, norflurazon, pyridate;

[0162] Pyridines: aminopyralid, clopyralid, diflufenican, dithiopyr, fluridone, fluroxypyr, picloram, picolinafen, thiazopyr;

[0163] Sulfonylureas: amidosulfuron, azimsulfuron, bensulfuron, chlorimuron-ethyl, chlorsulfuron, cinosulfuron, cyclosulfamuron, ethoxysulfuron, flazasulfuron, flucetosulfuron, flupyrsulfuron, foramsulfuron, halosulfuron, imazosulfuron, iodosulfuron, mesosulfuron, metsul furon-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;

[0164] Triazines: ametryn, atrazine, cyanazine, dimethametryn, ethiozin, hexazinone, metamitron, metribuzin, prometryn, simazine, terbuthylazine, terbutryn, triaziflam;

[0165] Ureas: chlorotoluron, daimuron, diuron, fluometuron, isoproturon, linuron, methabenzthiazuron, tebuthiuron;

[0166] Other acetolactate synthase inhibitors: bispyribac-sodium, cloransulam-methyl, diclosulam, florasulam, flucarbazone, flumetsulam, metosulam, ortho-sulfamuron, penoxsulam, propoxycarbazone, pyribambenz-propyl, pyribenzoxim, pyriftalid, pyriminobac-methyl, pyrimisulfan, pyrithiobac, pyroxasulfone, pyroxsulam;

[0167] Others: amicarbazone, aminotriazole, anilofos, beflubutamid, benazolin, bencarbazone, benfluresate, benzofenap, bentazone, benzobicyclon, bromacil, bromobutide, fluazifop-butyl butafenacil, butamifos, cafenstrole, carfentrazone, cinidon-ethlyl, chlorthal, cinmethylin, clomazone, cumyluron, cyprosulfamide, dicamba, avena cinerea, diflufenzopyr, Drechslera monoceras), endothal, ethofumesate, etobenzanid, fentrazamide, flumiclorac-pentyl, flumioxazin, flupoxam, flurochloridone, flurtamone, indanofan, isoxaben, isoxaflutole, lenacil, propanil, propyzamide, quinclorac, quinmerac, mesotrione, methylarsonicacid), naptalam, oxadiargyl, oxadiazon, oxaziclomefone, pentoxazone, pinoxaden, pyraclonil, pyraflufen-ethyl, pyrasulfotole, pyrazoxyfen, pyrazolynate, quinoclamine, saflufenacil, sulcotrione, sulfentrazone, terbacil, tefuryltrione, tembotrione, thiocarbamide (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)-ethyl acetate, 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;

[0168] H) any one or more insecticides, which may be selected from the group consisting of:

[0169] Organic (sulfur) phosphates: acephate, azamethiphos, azinphos-methyl, chlorpyrifos, chlorpyrifos-methyl, chlorfenvinphos, diazinon, dichlorvos, dicrotophos, dimethoate, disulfoton, ethion, fenitrothion, fenthion, isoxathion, malathion, methamidophos, methidathion, methyl parathion rathion), mevinphos, monocrotophos, oxydemeton-methyl, paraoxon, parathion, phenthoate, phosalone, phosmet, phosphamidon, phorate, phoxim, pirimiphos-methyl, profenofos, prothiofos, sulprophos, tetrachlorvinphos, terbufos, triazophos, trichlorfon;

[0170] Carbamates: alanycarb, aldicarb, bendiocarb, benfuracarb, carbaryl, carbofuran, carbosulfan, fenoxycarb, furathiocarb, methiocarb, methomyl, oxamyl, pirimicarb, propoxur, thiodicarb, triazamate;

[0171] Pyrethroids: allethrin, bifenthrin, cyfluthrin, cyhalothrin, cyphenothrin, cypermethrin, alpha-cypermethrin, beta-cypermethrin, zeta-cypermethrin, deltamethrin, esfenvalerate, etofenprox, fenpropathrin, fenvalerate, imiprothrin, lambda-cyhalothrin, permethrin, prallethrin, pyrethrin I and II II), resmethrin, silafluofen, tau-fluvalinate, tefluthrin, tetramethrin, tralomethrin, transfluthrin, profluthrin, dimefluthrin;

[0172] I) any one or more insect growth regulators: a) chitin synthesis inhibitors: benzyl urea: chlorfluazuron, cyramazin, diflubenzuron, flucycloxuron, flufenoxuron, hexaflumuron, lufenuron, novaluron, teflubenzuron, triflumuron; buprofezin, diofenolan, hexythiazox, etoxazole a) ecdysone antagonists: halofenozide, methoxyfenozide, tebufenozide, azadirachtin; c) juvenile hormone analogs: pyriproxyfen, methoprene, fenoxycarb; d) lipid biosynthesis inhibitors: spirodiclofen, spiromesifen, spirotetramat;

[0173] J) any one or more of any other compounds, such as:

[0174] Nicotinic receptor agonist / antagonist compounds: clothianidin, dinotefuran, imidacloprid, thiamethoxam, nitenpyram, acetamiprid, thiacloprid, 1-(2-chloro-thiazol-5-ylmethyl)-2-nitramino-3,5-dimethyl-[1,3,5]triazine;

[0175] GABA antagonist compounds: endosulfan, ethiprole, fipronil, vaniliprole, pyrafluprole, pyriprole, 5-amino-1-(2,6-dichloro-4-methyl-phenyl)-4-sulfinamido-1H-pyrazole-3-carbothioamide;

[0176] Macrolide insecticides: abamectin, emamectin, milbemectin, lepimectin, spinosad, spinetoram;

[0177] Mitochondrial electron transport inhibitors (METI) I acaricides: fenazaquin, pyridaben, tebufenpyrad, tolfenpyrad, flufenerim;

[0178] METI II and III compounds: acequinocyl, fluacyprim, hydramethylnon;

[0179] Uncoupler: chlorfenapyr;

[0180] Oxidative phosphorylation inhibitors: cyhexatin, diafenthiuron, fenbutatin oxide, propargite;

[0181] Molting disruptor compound: cryomazine;

[0182] Mixed function oxidase inhibitors: iperonyl butoxide;

[0183] Sodium channel blockers: indoxacarb, metaflumizone;

[0184] Others: benclothiaz, bifenazate, cartap, flonicamid, pyridalyl, pymetrozine, sulfur, thiocyclam, flubendiamide, chlorantraniliprole, cyazypyr (HGW86), cyenopyrafen, flupyrazofos, cyflumetofen, amidoflumet, imicyafos, bistrifluron and pyrifluquinazon;

[0185] K) Growth regulators may be selected from any one or more of the following: abscisic acid, amidochlor, ancymidol, 6-benzylaminopurine, brassinolide, butralin, chlormequat / chlormequat chloride, choline chloride, cyclanilide, daminozide, dikegulac, dimethipin, 2,6-dimethylpurine, ethephon, flumetralin, flurprimidol, fluthiacet, forchlorfenuron, gibberellic acids, inabenfide, indole-3-acetic acid, maleic hydrazide, mefluidide, mepiquat / mepiquat chloride, chloride), naphthylacetic acid, N-6-benzyladenine, paclobutrazol, prohexadione (prohexadione-calcium), prohydrojasmon, thidiazuron, triapenthenol, tributyl trithiophosphate, 2,3,5-triiodobenzoic acid, trinexapac-ethy, and uniconazole.

[0186] In some embodiments, the pesticide is sensitive to UV. The sensitivity can be detected by a simple test in which the pesticide is exposed to UV for a period of time. Subsequently, the undecomposed residual pesticide can be quantified.

[0187] In some embodiments, the nanoparticles comprise herbicides such as naproamid, proparnil, bentazone, paraquat dichlorid, cycloxydim, sethoxydim, ethalfluralin, oryzalin, pendimethalin, trifluralin, acifluren, aclonifen, fomesafen, oxyfluoren, ioxybenzonitrile, imazetapyr, imazaquin, cypermethrin, chlorpyrifos, chloranil ... The compounds include chloridazon, norfloxacin, thiamethoxam, triclopyr, dithiopyr, fluazifop, flupyralid, acylosulfuron, vernolate, promethon, metribuzin, azafenidin, carfentrazone-ethyl, sulfentrazone, metoxuron, monolinuron, fluchloralin and flurenol.

[0188] In some embodiments, the nanoparticles comprise a fungicide such as cyprodinil, fenpropimorph, dimethomorph, procloraz, trifloxystrobin, chlorpyrifos, edifenfos, chloranil, flufenoxanol, ethirimol, quinoxylen, dithianon, oxazolidinone, trifloxystrobin, dichlofluamid, bromuconazole, and myclobutanil.

[0189] In some embodiments, the nanoparticles contain insecticides such as acephate, azinphos-ethyl, methyl azinphos, isofenphos, chlorpyriphos-methyl, dimethylvinphos, phorate, phoxim, propylthiophos, tricyclamate, ethiofencarb, pirimicarb, thiodicarb, fipronil, bioallethrin, bioresmethin, deltamethrin, fenpropathin, flucythrinate, taufluvalinate, cypermethrin, zeta-cypermethrin, resmethin, tefluthrin, lambda-cyhalothrin and hydrazone. In another preferred embodiment, the insecticide is metaflumizone or cis-cypermethrin.

[0190] In some embodiments, the nanoparticles comprise metaflumizone or cypermethrin.

[0191] Method for preparing nanoparticles

[0192] In certain embodiments, the present disclosure relates to a method of preparing nanoparticles comprising a tobacco mosaic virus (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 (e.g., a "pH method"). In some embodiments, the method comprises the steps of providing an isolated tobacco mosaic virus (e.g., TMGMV and / or TMV) to a buffer having a pH of about 7 to 9 to produce a tobacco mosaic virus buffer, adding one or more AIs to the tobacco mosaic virus buffer more than once, thereby producing the 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, one or more AIs are non-covalently conjugated to a tobacco mosaic virus (e.g., TMGMV and / or TMV). In some embodiments, a tobacco mosaic virus (e.g., TMGMV and / or TMV) comprises one or more coat proteins that reversibly and partially dissociate in response to a change in pH.

[0193] 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 tobacco mosaic virus buffer is about 7 to 7.5, 7.5 to 8, 7 to 8, 8 to 8.5, 8.5 to 9, or 8 to 9. In some embodiments, the pH of the tobacco mosaic virus-buffer is 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. In some embodiments, the pH of the tobacco mosaic virus-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.

[0194] In some embodiments, the pH of the solution in which the nanoparticles are purified is 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 , 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 pH of the solution in which the nanoparticles are purified is about 6.9, 7.0, 7.1, 7.2, or 7.3.

[0195] As described elsewhere herein, the change in pH results in a phase transition of the tobacco mosaic virus (e.g., TMGMV and / or TMV), thereby opening a "pore" or "pocket" that can receive and non-covalently bind one or more AI molecules therein. In some embodiments, the change in pH (e.g., the difference in pH of the tobacco mosaic virus-buffer relative to the solution in which the nanoparticles are purified) is about 0.5 to 1, about 0.5 to 2, 0.5 to 3, 1 to 2, or 1 to 3.

[0196] In certain embodiments, the present disclosure relates to a method for preparing nanoparticles comprising a tobacco mosaic virus (e.g., TMGMV and / or TMV) and one or more active ingredients (AIs), the method comprising adjusting the concentration of a solvent (e.g., DMSO) present in a solution in which the nanoparticles are suspended (e.g., this method is sometimes referred to as a "solvent method" throughout this disclosure). In some embodiments, the method comprises the steps of providing an isolated tobacco mosaic virus (e.g., TMGMV and / or TMV) to a buffer having a pH of about 5 to 9 to produce a tobacco mosaic virus-buffer, adding a solvent having a concentration of about 15% (v / v) to about 30% (v / v), adding one or more AIs to the tobacco mosaic virus-buffer, thereby producing nanoparticles, and purifying the nanoparticles in a solution having a pH of about 5 to 9.

[0197] In some embodiments, the step of providing an isolated tobamovirus comprises using a buffer having a pH of 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 7.5. .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 pH of the buffer is about 7, 7.1, 7.2, 7.3, 7.4, or 7.5.

[0198] In some embodiments, the solvent is a polar aprotic solvent. In some embodiments, the solvent is a polar aprotic solvent miscible with water. In some embodiments, the solvent is dimethyl sulfoxide (DMSO). In some embodiments, the solvent is acetone, acetonitrile, dichloromethane, dimethylformamide, dimethylpropylene urea, dimethyl sulfoxide, ethyl acetate, hexamethylphosphoramide, pyridine, sulfolane, tetrahydrofuran or any combination thereof.

[0199] In some embodiments, the concentration of the solvent (e.g., DMSO) added is about 15% (v / v) to about 40% (v / v) (e.g., about 15% to 20%, about 15% to 25%, about 15% to 30%, about 15% to 35%, about 15% to 40%, about 20% to 25%, about 20% to 30%, about 20% to 35%, about 20% to 40%, about 25% to 30%, about 25% to 35%, about 25% to 40%, about 30% to 35%, about 30% to 40%). In some embodiments, the solvent is added at a concentration of about 20% (v / v).

[0200] In some embodiments, the one or more AIs are non-covalently conjugated to a tobacco mosaic virus (e.g., TMGMV and / or TMV). In some embodiments, the tobacco mosaic 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.

[0201] In some embodiments, the solvent is added dropwise. In some embodiments, the one or more AIs are added dropwise. In some embodiments, the one or more AIs are added dropwise over a period of time. In some embodiments, the one or more AIs are added dropwise over a period of time of about 0.5 hours to about 10 days (e.g., about 0.5 hours to 1 hour, 0.5 hours to 2 hours, 0.5 hours to 3 hours, 0.5 hours to 4 hours, 0.5 hours to 5 hours, 0.5 hours to 6 hours, 0.5 hours to 7 hours, 0.5 hours to 8 hours, 0.5 hours to 9 hours, 0.5 hours to 10 hours, 0.5 hours to 11 hours, 0.5 hours to 12 hours, 0.5 hours to 14 hours, 0.5 hours to 15 hours, 0.5 hours to 16 hours, 0.5 hours to 17 hours, 0.5 hours to 18 hours, 0.5 hours to 19 hours, 0.5 hours to 20 hours, 0.5 hours to 21 hours, 0.5 hours to 22 hours, 0.5 hours to 24 hours, 0.5 hours to 25 hours, 0.5 hours to 26 hours, 0.5 hours to 27 hours, 0.5 hours to 28 hours, 0.5 hours to 29 hours, 0.5 hours to 30 hours, 0.5 hours to 31 hours, 0.5 hours to 32 hours, 0.5 hours to 34 hours, 0.5 hours to 36 hours, 0.5 hours to 37 hours, 0.5 hours to 38 hours, 0.5 hours to 39 hours, 0.5 hours to 40 hours, 0.5 hours to 41 hours, 0.5 hours to 42 hours, 0.5 hours to 43 hours, 0.5 hours to 0.5 hour to 12 hours, 0.5 hour to 13 hours, 0.5 hour to 14 hours, 0.5 hour to 15 hours, 0.5 hour to 16 hours, 0.5 hour to 17 hours, 0.5 hour to 18 hours, 0.5 hour to 19 hours, 0.5 hour to 20 hours, 0.5 hour to 21 hours, 0.5 hour to 22 hours, 0.5 hour to 23 hours, 0.5 hour to 24 hours, 0.5 hour to 2 days, 0.5 hour to 3 days, 0.5 hour Hours to 4 days, 0.5 hours to 5 days, 0.5 hours to 6 days, 0.5 hours to 7 days, 0.5 hours to 8 days, 0.5 hours to 9 days, 0.5 hours to 9.9 days, 6 hours to 12 hours, 6 hours to 1 day, 6 hours to 2 days, 6 hours to 3 days, 6 hours to 4 days, 6 hours to 5 days, 6 hours to 6 days, 6 hours to 7 days, 6 hours to 8 days, 6 hours to 9 days, 6 hours to 10 days, 12 hours to 1 day, 12 hours to 2 days, 12 hours From 1 day to 3 days, 12 hours to 12 days, 12 hours to 12 days, 12 hours to 6 days, 12 hours to 7 days, 12 hours to 8 days, 12 hours to 9 days, 12 hours to 10 days, 1 day to 2 days, 1 day to 3 days, 1 day to 4 days, 1 day to 5 days, 1 day to 6 days, 1 day to 7 days, 1 day to 8 days, 1 day to 9 days, 1 day to 10 days, 2 days to 5 days, 2 days to 10 days or 5 days to 10 days. In some embodiments, the one or more AIs are added once a day.

[0202] In some embodiments, the method further comprises, after adding the solvent (e.g., DMSO) and adding the one or more AIs to the tobacco mosaic virus-buffer, incubating the one or more AIs in the tobacco mosaic virus-buffer for about 0.5 hours to about 36 hours (e.g., about 0.5 hours to 1 hour, 0.5 hours to 2 hours, 0.5 hours to 3 hours, 0.5 hours to 4 hours, 0.5 hours to 5 hours, 0.5 hours to 6 hours, 0.5 hours to 7 hours, 0.5 hours to 8 hours, 0.5 hours to 9 hours, 0.5 hours to 10 hours, 0.5 hours to 11 hours, 0.5 hours to 12 hours, 0.5 hours to 13 hours, 0.5 hours to 14 hours). , 0.5 hour to 15 hours, 0.5 hour to 16 hours, 0.5 hour to 17 hours, 0.5 hour to 18 hours, 0.5 hour to 19 hours, 0.5 hour to 20 hours, 0.5 hour to 21 hours, 0.5 hour to 22 hours, 0.5 hour to 23 hours, 0.5 hour to 24 hours, 0.5 hour to 30 hours, 0.5 hour to 35.9 hours, 1 hour to 2 hours, 1 hour to 3 hours, 1 hour to 4 hours, 1 hour to 5 hours, 1 hour to 6 hours, 1 hour to 7 hours, 1 hour to 8 hours, 1 hour to 9 hours, 1 hour to 10 hours, 1 hour to 11 hours, 1 hour to 12 hours, 1 hour to 13 hours, 1 hour to 14 hours, 1 hour to 15 hours, 1 hour to 16 hours, 1 hour to 17 hours, 1 hour to 18 hours, 1 hour to 19 hours, 1 hour to 20 hours, 1 hour to 21 hours, 1 hour to 22 hours, 1 hour to 23 hours, 1 hour to 24 hours, 1 hour to 30 hours, 1 hour to 36 hours, 2 hours to 3 hours, 2 hours to 4 hours, 2 hours to 5 hours, 2 hours to 6 hours, 2 hours to 7 hours, 2 hours to 8 hours, 2 hours to 9 hours, 2 hours to 10 hours, 2 hours to 11 hours, 2 hours to 12 hours, 2 hours to 13 hours, 2 hours to 14 hours, 2 hours to 15 hours, 2 hours to 16 hours, 2 hours to 17 hours, 2 hours to 18 hours, 2 hours to 19 Hours, 2 hours to 20 hours, 2 hours to 22 hours, 2 hours to 22 hours, 2 hours to 23 hours, 2 hours to 24 hours, 2 hours to 30 hours, 2 hours to 36 hours, 3 hours to 4 hours, 3 hours to 5 hours, 3 hours to 6 hours, 3 hours to 7 hours, 3 hours to 8 hours, 3 hours to 9 hours, 3 hours to 10 hours, 3 hours to 11 hours, 3 hours to 12 hours, 3 hours to 13 hours, 3 hours to 14 hours, 3 hours to 15 hours, 3 hours to 16 hours, 3 hours to 17 hours, 3 hours to 18 hours, 3 hours to 19 hours, 3 hours to 20 hours, 3 hours to 21 hours, 3 hours to 22 hours, 3 hours to 23 hours, 3 hours to 24 hours,3 hours to 30 hours, 3 hours to 36 hours, 4 hours to 5 hours, 4 hours to 6 hours, 4 hours to 7 hours, 4 hours to 8 hours, 4 hours to 9 hours, 4 hours to 10 hours, 4 hours to 11 hours, 4 hours to 12 hours, 4 hours to 13 hours, 4 hours to 14 hours, 4 hours to 15 hours, 4 hours to 16 hours, 4 hours to 17 hours, 4 hours to 18 hours, 4 hours to 19 hours, 4 hours to 20 hours, 4 hours to 21 hours, 4 hours to 22 hours, 4 hours to 23 hours, 4 hours to 24 hours, 4 hours to 30 hours, 4 hours to 36 hours, 8 hours to 9 hours, 8 hours to 10 hours, 8 hours to 11 hours, 8 hours to 12 hours, 8 hours to 13 hours, 8 hours to 18 hours, 8 hours 12 hours to 36 hours, 12 hours to 13 hours, 12 hours to 18 hours, 12 hours to 15 hours, 12 hours to 16 hours, 12 hours to 17 hours, 12 hours to 18 hours, 12 hours to 19 hours, 12 hours to 20 hours, 12 hours to 21 hours, 12 hours to 22 hours, 12 hours to 23 hours, 12 hours to 24 hours, 12 hours to 30 hours, 12 hours to 36 hours, 24 hours to 30 hours or 12 hours to 36 hours).

[0203] In some embodiments, after adding the solvent and the one or more AIs, the pH of the solution in which the nanoparticles are purified is 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 7.1, 7.2, or 7.3). In some embodiments, the pH of the solution in which the nanoparticles are purified is about 6.9, 7.0, 7.1, 7.2, or 7.3.

[0204] In certain embodiments, when using pH method or solvent method to prepare nanoparticle, one or more AIs are added to tobacco mosaic virus-buffer twice or more. In certain embodiments, one or more AIs are added at least once a day. In certain embodiments, when using pH method or solvent method to prepare nanoparticle, one or more AIs are added dropwise only once a day. In certain embodiments, when using pH method or solvent method to prepare nanoparticle, one or more AIs are added until reaching 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. In some embodiments, when the pH method or the solvent method is used to prepare nanoparticles, the one or more AIs are added to the tobacco mosaic virus (e.g., TMGMV and / or TMV) at a 1,000-fold, 1,500-fold, 2,000-fold, 2,500-fold, 3,000-fold, 3,300-fold, 4,000-fold, 4,500-fold, 5,000-fold, 5,500-fold, 6,500-fold, 7,000-fold, 7,500-fold, 8,000-fold, 8,500-fold, 9,000-fold, or 9,500-fold molar excess. In some embodiments, when the pH method or the solvent method is used to prepare nanoparticles, 100 nmol, 150 nmol, 200 nmol, 250 nmol, 300 nmol, 350 nmol, 400 nmol, 450 nmol or 500 nmol of the one or more AIs per gram of tobacco mosaic virus (e.g., TMGMV and / or TMV) is added.

[0205] Methods for formulating compounds 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 Application Nos. WO 2005 / 102507; WO 2005 / 020933; WO 2005 / 072680; WO 01 / 88046; WO 2007 / 014826; US Application Nos. 2005 / 0170004; 2006 / 0063676; 2014 / 164418). The disclosed formulations can be mixed in any order in a single step or multiple step mixing. One or more compounds / AIs can be added to a formulation comprising nanoparticles of the tobacco mosaic virus genus (e.g., TMGMV and / or TMV), and examples of suitable agrochemical formulations are liquid formulations such as EC (emulsifiable concentrate) formulations; SL or LS (soluble concentrate) formulations; EW (emulsion, oil in water) formulations; ME (microemulsion) formulations; MEC (microemulsifiable concentrate) formulations; CS (capsule suspension) formulations; TK (technical concentrate) formulations; OD (oil-based suspension concentrate) formulation; SC (suspension concentrate) formulation; SE (suspoemulsion) formulation; ULV (ultra-low volume liquid) formulation; SO (film spreading oil) formulation; AL (any other liquid) formulation; LA (paint) formulation; DC (dispersible concentrate) formulation; or solid formulations such as WG (water dispersible granules) formulation; TB (tablet) formulation; FG (fine granules) formulation; MG (microgranules) formulation; SG (soluble granules). In some embodiments, the liquid formulation is EC, SL, LS, EW, ME, MEC, TK, OD, SC, SE, ULV, SO, AL, LA and DC.

[0206] Pharmaceutical composition

[0207] In certain embodiments, disclosed herein are pharmaceutical compositions comprising nanoparticles as described herein. Two or more (e.g., two, three, or four) therapeutic nanoparticles of any type of therapeutic nanoparticles described herein can be present in a pharmaceutical composition in any combination. The pharmaceutical composition can be formulated in any manner known in the art.

[0208] In some embodiments, the pharmaceutical composition comprises at least one pharmaceutically acceptable carrier, diluent or excipient. In some embodiments, the pharmaceutical composition is formulated as an injectable solution, a lyophilized powder, a suspension, or any combination thereof.

[0209] The pharmaceutical composition is formulated to be compatible with its intended route of administration (e.g., intravenous, intra-arterial, intramuscular, intradermal, subcutaneous or intraperitoneal). In some embodiments, the composition provided herein may include a pharmaceutically acceptable diluent (e.g., a sterile diluent). In some embodiments, a pharmaceutically acceptable diluent may be sterile water or sterile saline, fixed oil, polyethylene glycol, glycerol, propylene glycol or other synthetic solvent, an antibacterial or antifungal agent, such as benzyl alcohol or methyl paraben, chlorobutanol, phenol, ascorbic acid, thimerosal, etc.; an antioxidant, such as ascorbic acid or sodium bisulfite; a chelating agent, such as ethylenediaminetetraacetic acid; a buffer, such as acetate, citrate or phosphate; and an isotonic agent, such as a sugar (e.g., dextrose), a polyol (e.g., mannitol or sorbitol) or a salt (e.g., sodium chloride) or any combination thereof.

[0210] In certain embodiments, the pharmaceutical composition provided herein may include a pharmaceutically acceptable carrier. The preparation of the composition may be formulated and packaged in an ampoule, a disposable syringe or a multiple dose vial. Where necessary (e.g., in an injectable formulation), suitable fluidity may be maintained, for example, by using a coating (e.g., lecithin) or a surfactant. The absorption of nanoparticles may be extended by including a medicament (e.g., aluminum monostearate and gelatin) that delays absorption. Alternatively, controlled release may be achieved by an implant and a microencapsulated delivery system that may include a biodegradable biocompatible polymer (e.g., ethylene vinyl acetate, polyanhydrides, polyglycolic acid, collagen, polyorthoesters and polylactic acid).

[0211] Compositions containing one or more of the nanoparticles described herein can be formulated for parenteral (e.g., intravenous, intraarterial, intramuscular, intradermal, subcutaneous or intraperitoneal) administration in dosage unit form (i.e., physically discrete units containing a predetermined amount of active compound for ease of administration and uniform dosage). In some embodiments, compositions containing one or more of any nanoparticles described herein can be formulated into injectable, lyophilized powder, suspension, or any combination thereof.

[0212] The toxicity and therapeutic efficacy of the composition can be determined by standard pharmaceutical procedures in cell culture or experimental animals (e.g., monkeys). For example, the LD50 (the dose lethal to 50% of the population) and the ED50 (the dose therapeutically effective in 50% of the population) can be determined: the therapeutic index is the ratio of LD50:ED50. Agents that exhibit high therapeutic indices are preferred. In the case where 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.

[0213] The data obtained from cell culture assays and animal studies can be used to formulate the appropriate dosage of any given medicament for a subject (e.g., a person). The therapeutically effective amount of one or more (e.g., one, two, three or four) nanoparticles (e.g., any nanoparticle described herein) can be to reduce cancer cell invasion or metastasis in a subject with cancer in a subject (e.g., a person), or to reduce and / or eliminate the amount of the infection of a subject (e.g., a person).

[0214] The effectiveness and dosage of any nanoparticle described herein can be determined by health care professionals using methods known in the art, as well as by observing one or more symptoms of a disease (e.g., cancer or infection) in a subject (e.g., a human). Certain factors may affect the dosage and time course (e.g., the severity of the disease or condition, previous treatment, the overall health status and / or age of the subject, and the presence of other diseases) required for effective treatment of the subject.

[0215] Those of ordinary skill in the art will appreciate that the efficacy of therapeutic agents (including nanoparticles described herein) varies, and effective amounts can be determined by methods known in the art. Typically, a relatively low dose is first administered, and the attending health care professional (in the case of therapeutic applications) or researcher (when still working in the research and development phase) can subsequently and gradually increase the dose until an appropriate response is obtained. In addition, it should be understood that the specific dosage level of any particular subject will depend on various factors, including the activity of the specific compound used, the subject's age, weight, overall health, sex and diet, administration time, administration route, excretion rate and nanoparticle half-life in vivo.

[0216] The pharmaceutical compositions can be included in a kit, container, pack, or dispenser together with instructions for administration.

[0217] Treatment

[0218] Also provided herein are methods for treating cancer in a subject in need thereof. The method for treating cancer comprises administering a nanoparticle of the present disclosure or a pharmaceutical composition of the present disclosure to a subject in need of treating cancer. In some embodiments, the nanoparticle or the pharmaceutical composition is administered in an effective amount. In some embodiments, the cancer comprises breast cancer, ovarian cancer, glioma, gastrointestinal cancer, prostate cancer, carcinoma, lung cancer, hepatocellular carcinoma, testicular cancer, cervical cancer, endometrial cancer, bladder cancer, head and neck cancer, lung cancer, gastroesophageal cancer, gynecological cancer, or any combination thereof.

[0219] Also provided herein is a method for treating an infection in a subject in need thereof. The method for treating an infection comprises administering a nanoparticle of the present disclosure or a pharmaceutical composition of the present disclosure to a subject in need of treating the infection. In some embodiments, the nanoparticle or the pharmaceutical composition is administered in an effective amount. In some embodiments, the infection is a bacterial infection, a viral infection, a fungal infection, a parasitic infection, or any combination thereof.

[0220] Examples

[0221] The present disclosure is further described in the following examples, which do not limit the scope of any embodiments described in the claims.

[0222] method

[0223] Preparation of TMGMVs

[0224] 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 for 24 hours at 4°C using 12-14kDa dialysis tubing (Fisher Scientific S432700; Waltham, MA, USA) for potassium phosphate buffer (KP; 10mM, pH 7.2). The buffer solution was replaced and the dialysis was continued for another 48 hours. The solution was then centrifuged at 10,000xg for 20 minutes (Beckman Coulter Allegra or Avanti centrifuge). The supernatant was collected and ultracentrifuged at 42,000 rpm for 2.5 hours at 4°C (Beckman Coulter Optima L-90k ultracentrifuge with 50.2Ti rotor; Brea, CA, USA). The pellet was mixed overnight by rotation in KP buffer at 4°C. The concentration in 10 mM KP was then adjusted to 10 mg mL using a Nanodrop 2000 (Thermo Scientific; Waltham, MA, USA) before storage at 4°C. -1 (For TMGMV CP, ε 260 =3mL mg -1 cm -1 ).

[0225] Preparation of diazonium salt from 4-ethynylaniline

[0226] In a 5 mL tube, dissolve 298 mg of 4-ethynylaniline in 2 mL of methanol. In a 50 mL tube, dissolve 1.09 g of p-toluenesulfonic acid in 20 mL of DIH 2O. Both solutions were placed at -20 ° C for 10 minutes to precool. Prepare 1.5 mL of 3M sodium nitrite solution (258 mg in 1.5 mL DIH2O) and place at -20 ° C for 5 minutes to precool. A 50 mL beaker was immersed in an ice / water slurry on a stirring plate. The solution was taken out of the refrigerator. A stirring bar was added to the 20 mL precooled acid in the immersed beaker. Once mixed, a methanol solution was added. The solution became opaque and the color was beige. The nitrite solution was gradually dripped into the acid solution, and the mixture gradually turned yellow and finally turned red after a reaction time of 30-60 minutes. A sample of 1 mL of diazo slurry was collected and centrifuged at 10,000 x g for 2 minutes to separate the diazonium salt. On ice, the supernatant was removed, and the diazonium salt was resuspended in 1 mL of precooled ethanol. The prepared diazonium salt was used immediately for tyrosine modification.

[0227] Coupling of Diazo with TMGMV

[0228] Prepare 962 μL of 2 mg mL -1 TMGMV solution in 100mM borate buffer (pH 8.5), and precooled on ice. The diazonium salt solution is added to the TMGMV solution with a volume of 80 μL. The solution is mixed by inversion, and reacted on ice for 30 minutes. The solution is centrifuged at 50,000rpm for 1 hour on a sucrose cushion (30% w / v) in a desktop ultracentrifuge (Beckman Optima MAX-XP, with TLA-55 rotor). The virus pellet is resuspended overnight in 10mM KP at 4 ° C on a rotary shaker.

[0229] Copper-catalyzed azide-alkyne cycloaddition

[0230] 1 mg of TMGMV was added to an ultracentrifuge tube (Beckman Coulter 357448, Indianapolis, IN, USA). The reaction medium consisted of 1 mM copper sulfate, 2 mM aminoguanidine, 2 mM L-ascorbic acid, and 3.7 mM tris(benzyltriazolylmethyl)amine. Fifty equivalents of 6A-azido-6A-deoxy-b-cyclodextrin (TCI Chemicals) / TMGMV coat protein were added and the volume was adjusted to a final volume of 500 μL with 10 mM KP pH 7. The reaction was allowed to proceed on ice for 1 hour. A 200 μL sucrose cushion (30% w / v) was added to the bottom of the same tube, and the sample was then ultracentrifuged at 50,000 rpm for 1 hour at 4°C (Beckman Optima MAX-XP with TLA-55 rotor). The supernatant was removed and the pellet was resuspended with rotation mixing at 4°C overnight before further characterization.

[0231] Characterization of chemically labeled β-CD-TMGMV

[0232] SDS-PAGE: Denatured β-CD-conjugated TMGMV samples (10 μg) were loaded onto 12% NuPAGE gels (Life Technologies) and run on 1× MOPS running buffer (Life Technologies). Proteins were stained using gel-coded blue stain (Life Technologies) and visualized under white light.

[0233] FPLC (size exclusion chromatography): using a Superose6 Increase 100GL column and β-CD conjugated TMGMV samples (500 μL, 0.5 mg / mL) were analyzed using a Pure25 chromatography system (GE Healthcare) in 10 mM KP (pH 7.4) using a flow rate of 0.5 mL / min. The absorbance at 260 nm and 280 nm was recorded.

[0234] TEM imaging: dilute the sample to 0.05 mg mL -1 The concentration of 50 μg / ml was 100 μg / ml and adsorbed onto carbon-coated TEM grids (Electron Microscopy Sciences). The grids were then washed three times with pure water. The grids were then stained with 2% (w / v) uranyl acetate for 2 minutes for imaging. TEM was performed using a FEI Tecnai F30 transmission electron microscope operating at 300 kV.

[0235] Loading of insecticides into β-CD-TMGMV

[0236] To evaluate the loading of pesticides into β-CD-TMGMV, a competition assay was completed between doxorubicin (ApexBio) and the target molecule. Samples were prepared in 100 μL volumes in 96-well plates (Costar). For the sample wells, 0.0825 mg of β-CD-conjugated TMGMV in KP buffer was added to the wells, and 10 equiv. β-CD-TMGMV (molar equivalent of TMGMV-conjugated to β-CD) of doxorubicin.

[0237] Three control conditions were used: 1 equivalent of TMGMV, 575 equivalents of β-CD and 1 equivalent of TMGMV and 575 equivalents of β-CD (unconjugated). These control wells also received 10 equivalents of doxorubicin. The plate was incubated overnight at 4 ° C on a plate shaker (Fisher). Fluorescence top readings (excitation 470nm, emission 595nm, 25 flashes) were performed on the plate by a UV-Vis microplate reader (Tecan infinite 200Pro). After completing the fluorescence measurement, β-CD-TMGMV and control wells received 0 equivalents, 10 equivalents, 100 equivalents or 1000 equivalents. β-CD-TMGMV After repeated overnight incubations at 4°C on a plate shaker (Schirmjer), fluorescence readings were repeated as before.

[0238] Samples of 100 μL volume were prepared in 96-well plates (Cosda). For sample wells, 0.0825 mg of KP buffer containing β-CD-conjugated TMGMV was added to the wells, and 10 equivalents (molar equivalents of TMGMV conjugated with β-CD-) of doxorubicin were added in addition. 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 10 equivalents of doxorubicin. Incubate overnight at 4 ° C on a plate shaker (Shi Er Company), thereby allowing doxorubicin to be loaded onto β-CD-TMGMV particles. Fluorescence top readings (excitation 470nm, emission 595nm, 25 flashes) were then performed on the plate by a UV-Vis microplate reader (Tecan infinite 200Pro). After the fluorescence measurement was completed, β-CD-TMGMV and control wells received 10 equivalents, 100 equivalents, or 1000 equivalents of clothianidin (CTD; BASF), fluopyram (FLP; BASF), or tetracycline (TET; Sigma-Aldrich). Overnight incubations were repeated at 4°C, and these molecules were then allowed to compete with doxorubicin for entrapment on β-CD-TMGMV. Fluorescence was then read on a microplate reader as described previously.

[0239] Partial dissociation (also called "breathing")

[0240] The concentration was 5 mg mL in KP buffer (pH 7.5). -1TMGMV was kept at 4 ° C for 5 days. The target AI was added to the solution every 24 hours until an equivalent ratio of 500: 1 was reached and mixed on a rotary shaker. Afterwards, the solution was centrifuged at 50,000 rpm on a sucrose cushion (30% w / v) in a benchtop ultracentrifuge (Beckman Optima MAX-XP with TLA-55 rotor) for 1 hour. The viral pellet was then resuspended overnight in 10mM KP pH 7 at 4 ° C on a rotary shaker. After complete resuspension, the solution was dialyzed for 48 hours to remove excess (unembedded) AI.

[0241] TEM imaging

[0242] Dilute the sample to 0.05 mg mL -1 The concentration of 50 μg / cm2 was 0.177 μg / cm2 and adsorbed onto carbon-coated TEM grids (Electron Microscopy Sciences, Inc.). The grids were then washed three times with pure water. The grids were then stained with 2% (w / v) uranyl acetate for 2 minutes for imaging. TEM was performed using a FEI Tecnai F30 transmission electron microscope operating at 300 kV.

[0243] Soil fluidity test

[0244] The density is 0.32 g cm -3 Garden Magic topsoil was loaded into cylindrical columns (28 mm diameter, 30 cm top height) and saturated with deionized water to remove air pockets. The density of soil in real environments may be higher (0.6–1.6 g cm) due to compaction effects with depth and over time. -3 A pellet containing 1 mg of each formulation (with or without conjugated or infused dye molecules) was injected at the top of the soil column and 3 min -1 The column was saturated with deionized water at a constant flow rate of 100 ng / min. The eluate was collected at the bottom of the column in fractions of 500 μl-2 mL. Up to 200 fractions were collected in each experiment (two experiments / depth for each formulation).

[0245] The elution fractions were analyzed by SDS-PAGE to determine the quality and amount of nanoparticles recovered in each elution fraction. TMGMV nanoparticles were analyzed on 4-12% NuPage polyacrylamide SDS gel casting according to the Surecast Handcast protocol (Invitrogen). 25 μl of each elution fraction was mixed with 5 μl 5× SDS loading buffer and the samples were separated by SeeBlue Plus2 ladder size at 200V and 120mA for 1 hour. The gel was then stained with gel encoding blue stain (Life Technologies) and microwaved for 1 minute and then stirred for 5 minutes. This process was then repeated with deionized water for destaining. The gel was imaged using the FluorChem R system.

[0246] All nanoparticles were imaged and analyzed using ImageJ.

[0247] Infusion of hydrophobic cargo in TMGMVs ​​by increasing pH from pH 7 to pH 7.5

[0248] AI was loaded into TMGMVs ​​by the “pH method”. The concentration of AI was 1 mg mL in 10 mM KP buffer (pH 7.5). -1 TMGMV was kept 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 molecules and cancer chemotherapy). AI was added to TMGMV by adding an excess of 10:1 AI: coat protein (CP; each TMGMV rod was assembled from approximately 2,100 identical CPs) every day until a ratio of 100:1 was reached. During this process, the reaction was kept mixed on a rotary shaker. 1 mL aliquots were obtained every day for further analysis.

[0249] After AI loading, aliquots were spin filtered using a 100K molecular weight cutoff 0.5 mL filter (MilliporeSigma, Burlington, MA, USA). 200 μL of aliquots and 250 μL of KP solution were added, and then centrifuged at 16,160 x g for 5 minutes at 4 ° C, the flow-through was discarded, and then 450 μL of KP was added and centrifuged again, and this step was repeated 3 times. After the third centrifugation, the filter was inverted in a new tube and centrifuged at 1000 x g for 2 minutes to recover the supernatant and perform subsequent characterization.

[0250] Infusion of hydrophobic cargo in TMGMVs ​​by varying DMSO concentration

[0251] AI was loaded into TMGMV by the "DMSO method". 10mM KP buffer (pH 7.2) containing TMGMV was diluted to 5mg mL-1 in 2mL of buffer and transferred to a 25mL beaker and magnetically stirred at 300rpm at room temperature. A solution of DMSO and 10mM KP was added dropwise to dilute the solution to 20% (v / v) concentration of DMSO and 2mg mL-1 of TMGMV. Aliquots of AI were added dropwise to the solution to prevent precipitation. The solution was stirred at room temperature for 24 hours. Before storage at 4°C, samples were collected and spin filtered as described above.

[0252] TEM

[0253] The TMGMV samples were diluted to a concentration of 0.05 mg mL-1 and adsorbed onto carbon-coated TEM grids (Electron Microscopy Sciences, Hatfield, PA, USA). The grids were then washed three times with pure water. The grids were then stained with 2% (w / v) uranyl acetate for 90 seconds. TEM was performed using a FEI Tecnai F30 transmission electron microscope operating at 300 kV. Image analysis was performed using ImageJ software (https: / / imagej.nih.gov / ij / download.html). In order to determine the variation in the width of the nanoparticles, the width of slices with a length of 100 nm was measured for standardization purposes. Five different slices were measured for each micrograph, and a total of 30 slices were measured for each sample. Subsequently, for intact particles, the length, perimeter, and area were measured to subsequently calculate the average width from the perimeter.

[0254] Size Exclusion Chromatography

[0255] Using Superose6 Increase 100GL column and TMGMV samples (500 μL, 0.5 mg / mL) were analyzed using a Pure25 chromatography system (GE Healthcare, Chicago, IL, USA) in 10 mM KP, pH 7.4, using a flow rate of 0.5 mL / min. The absorbance at 260 nm and 280 nm was recorded.

[0256] Circular dichroism spectroscopy

[0257] CD spectra were obtained using an Aviv type 215CD spectrometer (Lakewood, NJ, USA). All samples were run at 25°C in a quartz cuvette with an optical path length of 2 mm (Starna Cells, Atascadero, CA, USA). The samples were dissolved in 10 mM KP buffer (pH 7) in a range of 0.025 mg / mL to 0.5 mg / mL to obtain a volume of 400 μL for each CD run. Near and far UV spectra were obtained in separate scans. For far UV spectra, samples were scanned from 250 nm to 180 nm, with a wavelength step size of 1 nm, and an average time of 1 second. For near UV spectra, samples were scanned from 310 nm to 240 nm, with a wavelength step size of 0.5 nm, and an average time of 1 second. All spectra were scanned twice, and averaged in each UV region.

[0258] Small molecule quantification by high performance liquid chromatography (HPLC)

[0259] For HPLC, AI was extracted from TMGMV. In short, in KP, the concentration of TMGMV sample was adjusted to 1.2 mg mL-1. The solution was diluted 4 times in a 1:1 acetonitrile / methanol mixture and vortexed for 30 seconds. The solution was centrifuged at 10,000 x g for 10 minutes at 4 ° C, and the organic phase (bottom fraction) was collected and transferred to a HPLC 2 mL glass screw cap vial (SureSTART, Thermo Fisher Scientific, Waltham, MA, USA).

[0260] After diluting 10 times in acetonitrile, the extracted samples were injected with 500 μL and run on a 5 μm C18 column (20×100 mm) using a Shimadzu LC-40HPLC system (Columbia, MD, USA). The method was run for 15 minutes at a flow rate of 0.5 mL min-1 in a gradient of acetonitrile and 0.02% (v / v) phosphoric acid. A photodiode array was used to collect absorbance values ​​at 280 nm (fluopyram), 269 nm (thiamethoxam), 225 nm (ivermectin) and 330 nm (rifampicin). The absorbance values ​​were fitted to a standard curve to identify the sample concentration of N=3.

[0261] Example 1: β-CD-TMGMV formation

[0262] Experiments were performed to test the formation and integrity of AI-loaded prefixed TMGMV particles.

[0263] SDS-PAGE confirmed the covalent attachment of β-CD to CP, as Figure 2 The increase in MW corresponds to the size of β-cyclodextrin. Based on the band density analysis of β-CD-TMGMV subunit protein relative to TMGMV and TMGMV-alkyne by ImageJ, the conjugation efficiency is roughly estimated to be about 35%. This shows that when a molar excess of β-CD to CP is 50:1, the conjugation produces approximately 750 molecules / particle.

[0264] To verify the structural integrity of the modified TMGMV particles, size exclusion chromatography (SEC) and transmission electron microscopy (TEM) were performed. Figure 3 As shown, SEC measurements showed no significant difference between native TMGMV and conjugated TMGMV (as shown below), showing that the elution volume of native TMGMV was about 9 mL, and the A260:280 ratio was 1.2, indicating intact TMGMV. In addition, no obvious signs of aggregation or particle decomposition were observed. Figure 4 As shown, TEM imaging confirmed the structural integrity of β-CD-TMGMV after modification and purification.

[0265] Example 2: Loading AI into β-CD-TMGMV

[0266] Experiments were performed to test the loading (or "entrapment") of AI into β-CD-TMGMV. Quantification of the loading (or "entrapment") of the insecticide presents difficulties due to the molecule's lack of fluorescence. To quantify the entrapment, a competition assay was performed (see Figure 5Schematic diagram of the method). Doxorubicin ("DOX") with known excitation (470 nm) and emission (595 nm) wavelengths is added to sample and control wells. While free doxorubicin is able to fluoresce at 595 nm, entrapped doxorubicin is not; this difference allows measurement of free doxorubicin in solution (and indirectly quantification of entrapped molecules).

[0267] By measuring the fluorescence of unembedded doxorubicin, it was determined that 88.59% of the doxorubicin loaded into the hole was embedded by β-CD-TMGMV. This embedding measurement is a decrease in the fluorescence of doxorubicin (when the doxorubicin is pulled out of solution), thereby confirming the molecular carrying capacity of β-CD-TMGMV. As expected, the addition of pesticides leads to a dose-dependent displacement of doxorubicin from β-CD-TMGMV, with higher concentrations of pesticides displacing more doxorubicin. The increase in the amount of free doxorubicin in the hole leads to an increase in relative fluorescence. When challenged by the addition of clothianidin ("CTD"), fluopyram ("FLP") or tetracycline ("TET"), as Figure 6 As shown in the table, 1000 equivalents of tetracycline displaced 47% of the doxorubicin initially bound to β-CD-TMGMV. 1000 equivalents of clothianidin and fluopyram displaced 17.60% and 18.34% of doxorubicin, respectively. These data correlate with the degree of hydrophobicity of the molecules, and as expected, β-CD acts as a barrel to most easily entrap hydrophobic molecules.

[0268] Example 3: Loading AI into TMGMV

[0269] Experiments were conducted to load pesticides into TMGMV by strategically varying the pH. Without being bound by theory, this entraps the AI ​​through the formation of a "pocket" between the coat proteins (CP). Without being bound by theory, the basic principle is that by increasing the pH of the buffer, the virus will begin to dissociate and a hydrophobic pocket will be created between the coat proteins of the virion. The AI ​​is then added to interact with the virion, and the pH is then lowered to promote self-assembly of the particles and entrapment of the AI ​​on the hydrophobic pocket (see Figure 7 Schematic diagram of ). As discussed above, TMGMV at a concentration of 5 mg mL-1 in KP buffer (pH 7.5) was maintained 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. Afterwards, the solution was 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 pH 7 at 4°C overnight, and after complete resuspension, the solution was dialyzed for 48 hours to remove excess (unembedded) AI. The samples were then observed under TEM and analyzed using ImageJ.

[0270] To determine the change in nanoparticle width (breathing / infusion), the width of the slices with a nanoparticle length of 100 nm was measured ( Figure 8 This was done to normalize the measurements and determine the variation in width. At least 5 different sections were measured per micrograph and at least 30 sections were measured per sample in total.

[0271] After measurement, the differences between native TMGMV (control) and TMGMV infused with doxorubicin, ATTO550, fluopyram, and clothianidin were determined ( Figures 9A-9F The greatest increase was observed with doxorubicin, where the average width was 35 nm (DOX: 89% increase; Fig. 9A Compared with Fig.9E Clothianidin showed the second largest increase of 38% ( Fig. 9C ; 26 nm vs. 18 nm for the control). The width of Fluopyram increased by 21% ( Fig. 9B ; 22nm vs. 18nm), while ATTO550, the most hydrophilic AI, showed the smallest change, with a 7% increase in width ( Fig.9D ). This would be associated with a pocket that better entraps hydrophobic compounds.

[0272] Example 4: Soil mobility of TMGMV nanoparticles

[0273] Experiments were conducted to test the soil mobility of various TMGMV nanoparticles. Fig. 10A The experimental setup is depicted in the schematic diagram of . The soil column apparatus contained cheesecloth that prevented depressions from forming in the soil surface layer. Fractions were collected and analyzed by SDS-PAGE as described above ( Fig. 10B ). Soil was treated with TMGMV and infused with TMGMV that had been treated for 5 days in a buffer with a higher pH and returned to the buffer. The gel was imaged ( Fig.11A ) and quantified, and the results showed that the infused TMGMV nanoparticles ( Fig. 11C ) has the same characteristics as the individual TMGMV ( Fig. 11B ) with the same penetration ability. This finding confirms that the TMGMV “breathing” technology is effective and does not affect the mobility of the nanoparticles.

[0274] TMGMV-DOX nanoparticles were prepared by loading doxorubicin onto TMGMV as described above and analyzed by SDS-PAGE and microplate reader on soil columns (see Table 2, respectively). Figures 12A-12B The length of the soil column was 30 cm, and the soil column was divided into 5 fractions, each fraction was 6 cm ( Fig. 12Bas depicted). Thus, the first fraction represents the top 6 cm of soil closest to the column, and the 3rd fraction (middle fraction) will be 12 - 18 cm deep, and the 5th fraction will be 24 - 30 cm deep. TMGMV-DOX nanoparticles were found in all five fractions ( Fig.13A ). The highest percentage of TMGMV-DOX nanoparticles was found in the 3rd fraction, which represents the middle of the soil column, but over 20% of the nanoparticles were present in the 5th fraction, which represents deep penetration at the bottom of the soil column, and over 10% of the TMGMV-DOX nanoparticles were found in the 1st fraction, which is closest to the surface of the soil. These results were supported by fraction gel analysis ( Fig. 13B ).

[0275] TMGMV-Cy5 nanoparticles were prepared by loading Cy5 amine onto TMGMV as described above and analyzed on the soil column by SDS-PAGE and a microplate reader (as depicted respectively in Figures 12A-12B ). As described above, the column was divided into five fractions, and interestingly, TMGMV-Cy5 was evenly dispersed in all fractions ( Fig.14A ). Approximately 20% of the nanoparticles were present in all five fractions ( Fig.14A ). These results were supported by gel analysis of the soil fractions ( Fig. 14B ).

[0276] The soil mobility of TMGMV-βCD nanoparticles loaded with Cy5 was also tested, and the results showed that the loaded nanoparticles were also evenly distributed throughout the soil. The results showed no elution, and the nanoparticles were mainly retained by the soil. For reference, Cy5 passed through the soil column. Cy5 could not penetrate into the soil and was mainly located on the surface layer of the soil. This was supported by studies showing that Cy5 could not penetrate more than 4 cm into the soil because it binds strongly to soil particles (Chariou et al., (2019) Nat. Nanotechnol. 14:712). These results are comparable to the data reported for abamectin (Chariou and Steinmetz (2017) ACS Nano 11, 4719), fenamiphos, and carbofuran (Hassan et al., (2016) Plant Pathol. J. 15, 144), and other pesticides (Pestovsky and Martínez-Antonio (2017) J. Nanosci. Nanotechnol. 17, 8699).

[0277] Regardless of which nanoparticle type was used as the support, the mobility of Cy5 within the column was significantly enhanced and it was better retained in the soil than Cy5 alone.

[0278] Example 5: Loading of AI into TMGMVs ​​by pH and DMSO methods

[0279] Experiments were performed to study the assembly / disassembly phase diagram of TMGMV and to determine conditions suitable for AI encapsulation. The main goal was to achieve "breathing" without complete disassembly. First, the experiments focused on pH-induced structural changes and AI infusion. In this approach, the process requires extensive optimization, and the parameters pH (7 to 8), incubation time (2 hours to 24 hours), protein concentration (100 to 500 AI equivalents / CP), and AI addition interval (one-time feed versus daily increments) are carefully optimized. The latter is a critical parameter: large additions lead to severe aggregation and insolubility - this is probably due to the binding of hydrophobic AIs to the nanoparticle surface, thereby promoting inter-particle association and aggregation ( Fig.25 ). It was determined that the best results were obtained when the AI ​​was added in daily increments over 10 days; this ensured particle stability and AI loading (see below). Briefly, 10 equivalents of AI / CP were fed daily at pH 7.5, stirred overnight, and this process was repeated for 10 days. Afterwards, the samples were spin filtered to remove any excess AI.

[0280] The second method is that DMSO is used to destroy the interaction between the coat proteins. For some AIs, specifically highly hydrophobic AIs (e.g., ivermectin and fluopyram), this is advantageous. The benefits of this method are twofold: increasing the solubility of the AI ​​can lead to a higher effective concentration to drive the infusion, and the cosolvent can prevent the AI ​​precipitation that interferes with the infusion process. In order to further improve this process, the TMGMV preparation is subjected to magnetic stirring and fed from the top of the tube, thereby preventing any short-term spike-in of the AI ​​concentration that may promote precipitation. For the situation that the pH method previously showed immediate precipitation, the DMSO method shows no visible aggregates, and is therefore more likely to be successfully infused. Additionally, the increase in stirring, the bond breaking effect of DMSO, and the higher concentration of AI in the solution all indicate that if the TMGMV nanoparticles maintain their structure, infusion should occur faster under these conditions.

[0281] The optimization procedures for the pH method and the DMSO method are as follows Figures 16A-16C and described in the “Methods” section of the Examples above.

[0282] Example 6: TEM Characterization of AI-Loaded TMGMV Nanoparticles Prepared by pH and DMSO Methods

[0283] TEM imaging and quantitative TEM imaging analysis were performed on AI-loaded TMGMV nanoparticles prepared by the pH method and the DMSO method. Fig.17A As demonstrated by TEM images of , rod-shaped virus particles were observed in AI-loaded TMGMV nanoparticles prepared by both the pH and DMSO methods. Most interesting are the significant structural changes after AI loading: AI-loaded TMGMV appear swollen, and structural shifts indicate AI entrapment. To gain insight into the extent of the structural changes, quantitative TEM image analysis was performed comparing native TMGMV relative to AI-loaded TMGMV. Based on negative staining, the average width of native virus particles was 15.7nm (±1.9nm), which is an underestimate of the 18nm native TMGMV. This is likely due to the fact that uranyl acetate negative staining produces heavy shadows at the edges of the virus particles, significantly reducing their width. Although there was no statistically significant difference between the controls subjected to both methods ( Fig. 27 ), but the increment (compared to native TMGMV) was different for each compound and also varied between methods ( Fig.17A ). Fluopyram-loaded and ivermectin-loaded TMGMVs ​​showed a maximum width of 18 nm or 23 nm, respectively ( Fig. 17B This resulted in a 14% increase in the width of fluopyram and a 46% increase in the width of ivermectin compared to negatively stained native TMGMV. Meanwhile, clothianidin and rifampicin loading produced rods of 22-27 nm, significantly thicker than native TMGMV ( Fig. 17B ). This resulted in a 65% increase in clothianidin and a 73% increase in rifampicin.

[0284] Example 7: Characterization of the structure of AI-loaded TMGMV nanoparticles prepared by pH and DMSO methods

[0285] Circular dichroism (CD) was performed to observe any possible changes in the secondary structure of TMGMV after particle exposure to respiration and infusion ( Figures 18A-18B). The effect of structural motifs on circular dichroism is additive and difficult to deconvolute. On the contrary, the difference between the spectra of the treatment groups can indicate whether structural changes have occurred. The strongest signal of protein or virus CD is about 205nm-220nm, which represents the sum of contributions from α helix, β sheet and aggregation. The global minimum changes from 208nm to 220nm, indicating that in both pH and DMSO samples, the contribution of aggregation behavior or α helix content is greater than β sheet. In addition, CD shows that the structure does not change-as expected. The coat protein (CP) is dissociated to load AI at the interface-the structure does not change. In the range of 208nm-220nm, the AIs tested all show similar molar ellipticity curves, indicating that there is no difference in the secondary structure. In the near UV range, AI also has a similar trend, in which each AI shares the general shape of the signal curve. These results show that both the pH and DMSO methods used for breathing do not significantly change the secondary structure of TMGMV. In summary, the modest pH increase and relatively low volume fraction of DMSO used in these respiration experiments are not expected to alter the secondary structure of the viral particles, but rather to alter the tertiary structure to allow for inter-coat protein loading.

[0286] Size exclusion chromatography (SEC) was performed to further verify the structural integrity of the AI-loaded TMGMV particles during post-processing and purification. SEC measurements showed no significant difference between native TMGMV and AI-loaded TMGMV for any AI, showing a typical elution profile from a Superose 6 Increase column, eluting at about 9 mL, and A 260:280 The ratio is 1.2, thereby indicating a complete TMGMV, wherein 260nm indicates RNA absorption and 280nm protein absorption. In addition to the target insecticide Ai, DOX and Cy5 were also used because they have fluorescent properties, and DOX and Cy5 show maximum absorbance at 480nm and 647nm, respectively. SEC analysis confirmed the co-localization of AI (480nm and 647nm) and TMGMV (260 / 280nm). By using absorbance measurement and according to the Beer-Lambert law (Beer-Lambert law) (and its molar extinction coefficient) of TMGMV and AI, at about 9mL, AI was detected and co-localized, and the loading of about 615 DOX molecules / TMGMV and about 80 kinds of Cy5 / TMGMV was determined. Aggregation was observed, especially for Cy5, and for the significant particle dissociation of Dox (peak around about 20-25mL) ( Figures 26A-26D ).

[0287] Furthermore, by TEM, the observed rods were not uniform in length; several discs were evident indicating partial disassembly and rupture. In addition, droplet-like structures were observed in the ivermectin samples. These are believed to be ivermectin aggregates ( Figures 17A-17B ).

[0288] The length of TMGMV nanoparticles prepared by pH and DMSO methods was measured by image analysis. Compared with DMSO treatment, pH treatment showed slightly less fragmentation with a higher length distribution ( Figures 19A-19J ). DMSO treatment showed that most particles were smaller than 100 nm. However, there were no significant differences within AI either between treatments or between compounds.

[0289] Example 8: Quantification of AI loaded in TMGMV nanoparticles prepared by pH and DMSO method

[0290] HPLC was used to quantify the AI ​​loaded (Table 1). Infusion was performed using a pH-based method, and after loading AI in batches in the solution for 10 days, clothianidin and rifampicin showed successful loading, thereby achieving 1107.55 molecules / virion of clothianidin and 737.66 molecules / virion of rifampicin. In the case of fluopyram and ivermectin, a large amount of precipitation was observed, which may strip the virus from the solution and prevent the AI ​​from diffusing into the virus. It is calculated that fluopyram has about 15.82 molecules / virion, and ivermectin has 2.89 molecules / virion. When these results are compared with TEM micrographs and the change in the aspect ratio of the virus after treatment, the amount of the AI ​​loaded and the change in morphology seem to be related, wherein clothianidin and rifampicin have the largest virus particles and the change in particle width that is more obvious than fluopyram. Ivermectin loading does change significantly in morphology, and is calculated to have wider particles after treatment, although the amount of the AI ​​loaded is lower than that of fluopyram. This may be attributed to the challenges of extracting ivermectin or the molecular properties of ivermectin that permanently distort the structure of TMGMV without permanent loading of the AI.

[0291] Infusions were performed using the DMSO method over a 24-hour period, and in most cases the loading behavior of the AI ​​molecules was improved. Fluopyram loading increased 11.7-fold when using DMSO, reaching 185.59 molecules / virion. Similar results were observed for ivermectin, where an increase of 21.3-fold was observed to reach 61.63 molecules / virion. Clothianidin loading decreased by approximately 10% over the 24-hour period using DMSO, reaching 995 molecules / virion. Higher clothianidin concentrations in the solution or longer infusion time periods may continue to increase this value and approach the 10-day value of pH-based infusion. Rifampicin loading was improved 1.5-fold using the DMSO method, reaching 1104 molecules / virion.

[0292] Table 1. Quantification of AI in samples by HPLC.

[0293]

[0294] These data show that DMSO significantly improves the timeline of infusion compared to pH-based methods. Morphologically, TMGMV nanoparticles infused with AI look almost the same using the 10-day pH method versus the one-day DMSO method, indicating that the use of 20% DMSO does not compromise particle integrity. Reducing the time to achieve infusion to one day also greatly improves the synthesis yield because fewer particles degrade or precipitate from the solution. Because the DMSO cosolvent also mitigates AI precipitation, the effective concentration of the AI ​​used for infusion remains high and drives the molecule into TMGMV. The DMSO concentration, infusion time, mixing rate, and solution concentration of the AI ​​relative to the virus can be further optimized.

[0295] When the molecule is infused into rod-shaped TMGMV, several factors lead to the DMSO method being more efficient. As previously mentioned, DMSO keeps the solubility of AI in aqueous buffer higher, thereby preventing the potential co-precipitation of AI precipitation and virus. This benefit is twofold, because the precipitated AI cannot diffuse into the viral particles, and the precipitated viral particles cannot be recovered from this process. Additionally, since the solution remains well mixed throughout the process, the dropwise addition of AI under magnetic stirring prevents the pocket of AI driving precipitation at an insoluble concentration. When 20% v / v DMSO is used, it seems that a balance of TMGMV structural distortion has been achieved, allowing AI to penetrate between the coat proteins of TMGMV. In some embodiments, the inter-coat protein loading of AI in rod-shaped viruses using DMSO results in a 10-fold reduction in synthesis time. This is exacerbated by the fact that the synthesis yield is increased by not losing particles in the precipitate and by being able to load fluopyram and ivermectin into TMGMV.

[0296] Example 9: Characterization of the molecular properties of active ingredients and TMGMV nanoparticles

[0297] To better understand the molecular properties that lead to inter-coat protein loading of the AIs, the aqueous and organic partition coefficients (logP), molecular weights, and surface charge distributions of the AIs were compared. A summary of these properties can be found in Tables 2 and Figures 20A-20D Found in . Among the AIs loaded into TMGMV nanoparticles, the highest logP values ​​for ivermectin and fluopyram were 4.4 and 3.33, respectively. The values ​​for clothianidin and rifampicin were 1.3 and 2.4, respectively. The values ​​for ivermectin and fluopyram indicate that the molecules are highly water-insoluble, which matches well with what was observed in the loading experiments. This may explain why the same effective morphological changes using these AIs were achieved within 1 day using DMSO versus 10 days using the pH method, as the effective concentration of the AI ​​in solution is much higher. Another factor to consider regarding infusion efficiency is its size. During these measurements, larger molecules may have steric hindrance when they enter the space between the coat proteins. When comparing the changes in particle width analyzed using both methods with their molecular weight, the largest width change was observed for clothianidin (249.68 Da), and the smallest width change was observed for fluopyram (396.71 Da). The width change values ​​for rifampicin (822.94 Da) and ivermectin (875.1 Da) were intermediate. There was no clear trend in this group based on molecular weight, so AI size did not appear to be a limiting factor for loading using this method.

[0298] Table 2. Comparison of aqueous and organic partition coefficients (logP) and molecular weights of AIs.

[0299]

[0300] Outside the range of small molecules, steric hindrance is expected to dominate. Based on the electron density map of AI, ivermectin and rifampicin were observed to have large uncharged regions and small low-charged regions that are largely separated, resulting in mildly amphiphilic molecules. In contrast, fluopyram and clothianidin are much smaller and have larger charged surface areas. Because TMGMV is zwitterionic in nature but also contains many hydrophobic interfaces, it is challenging to separate the predicted changes in morphology into a single physicochemical interaction. The amphiphilicity, charged, compacted, and flexible structure of clothianidin may work together to change the morphology of TMGMV.

[0301] To gain insight into how the AIs interact with the coat protein surface, molecular docking experiments with the TMGMV coat protein (CP) (PDB: 1VTM) and four AIs were performed. In these analyses, the binding energies of the top 20 docked conformations and the residues involved in stabilizing the AIs were analyzed. These data do not indicate that the AIs are suitable ligands for the TMGMV CP, but rather identify putative residues that may be involved in inter-coat protein loading. Binding heats of bona fide ligand interactions were reported to be greater than 8 kcal mol -1 , and most of these interactions fall within the range of 3–8 kcal mol -1 Inside. Figures 29A-29D The binding region, its function towards TMGMV and the residues specifically identified to stabilize AI are summarized. Figures 21A-21B , 22A-22B, 23A-23B, and 24A-24B show examples of AI and related residues docked on TMGMV, and Figures 28A-28DThe binding heat of each conformation calculated by Autodock 4 is shown. According to the docking of simulation, it is observed that among the 20 best binding sites on TMGMV CP, all 4 AIs have many possible inaccessible sites. According to the separation mechanism of TMGMV CP (between CP relative to between discs), ivermectin has at most 10 accessible sites, rifampicin has 8 accessible sites, fluopyram has 11 accessible sites, and clothianidin has 5 accessible sites. The binding energy distribution shows that the binding heat of rifampicin with the surface is the highest, followed by ivermectin, then fluopyram and clothianidin. Although there are a large number of potential binding sites, ivermectin is a very large molecule, and needs to be highly separated from CP to be embedded in virions. Its relatively high affinity may show as instantaneous surface binding, and the instantaneous surface binding can destroy the key between CP, thereby explaining the widening of TMGMV in the presence of ivermectin. Finally, ivermectin was not detected during the quantitative period, indicating that the ivermectin was not combined with TMGMV. The binding heat of rifampicin and TMGMV CP was the highest, well loaded on TMGMV, and induced the morphological changes of TMGMV. Compared with the pH method, it shows the loading improved in the presence of DMSO, indicating that the structural changes induced by DMSO allow this relatively large molecule to approach the binding site. Although there are 11 potential binding sites, fluopyram also has some of the lowest binding heats, and has the highest affinity to the internal channel. Because this molecule is insoluble and relatively small, the molecule may be preferentially assigned to the internal channel, rather than loading between CP. According to the docking model, clothianidin has 5 accessible sites outside, but also has some of the lowest binding energies. However, its relatively small size and surface charge distribution may contribute to its combination and destruction of the structure between TMGMV CP. The maximum difference in some of the highest loadings and virion widths of clothianidin was demonstrated by HPLC, indicating that the characteristic of this molecule makes it very suitable for this method. Through more robust docking analysis and loading of larger libraries of small molecules between TMGMV CP, it may be possible to pinpoint the molecular identities of the AI ​​and the individual residues of TMGMV CP involved in these binding events.

[0302] Example 10: TMGMV nanoparticles prepared by pH and DMSO method encapsulate target molecules instead of covalently loading them

[0303] The virus nanoparticle (VNP) AI loading method described herein not only shows novel and interesting morphological changes in the virus, but also highlights how these methods can be used to embed and non-covalently load target molecules into the virus, and how to be used as a delivery system. Carefully adjust the solution conditions (such as pH and DMSO concentration) to allow TMGMV to "breathe", thereby producing structural changes and changing the interactions between structural motifs. These changes enable AI to be loaded and embedded in the newly formed pocket, thereby greatly improving the electrostatic loading capacity of TMGMV. Structural distortion in the presence of AI causes the short axis of TMGMV to widen, which is related to the degree of AI loading. These changes in particle size can simplify online measurements during VNP preparation to track the degree of loading in real time.

[0304] Both pH and DMSO methods show equal embedding of rifampicin and clothianidin molecules during virus "breathing", where 1000 AI / TMGMV are loaded. However, the DMSO method helps to load ivermectin and fluopyram, which form insoluble precipitates in the absence of DMSO, and does not show successful AI embedding using the pH strategy. Importantly, under the conditions tested, the speed at which the DMSO strategy loads AI is 10 times that of the pH strategy, although there is no significant difference in particle integrity between the two conditions. Additional refinement of the "breathing" conditions helps to pinpoint the phase transition of TMGMV, which may result in achieving higher loading, or loading larger or several different molecules. In general, the experiments performed further inspired TMGMV as a multifunctional nanotechnology platform for cargo delivery.

[0305] Other embodiments

[0306] It should be understood that although the invention has been described in conjunction with the specific description of the invention, the foregoing description is intended to illustrate rather than limit the scope of the invention, which is defined by the scope of the appended claims. Other aspects, advantages and modifications are within the scope of the following claims.

Claims

1. A nanoparticle comprising: Tobacco mosaic virus; and one or more active ingredients (AI), said one or more AI being non-covalently conjugated to said tobacco mosaic virus, Wherein the tobacco mosaic virus genus comprises one or more coat proteins that reversibly and partially dissociate in response to external factors.

2. The nanoparticle of claim 1, wherein the one or more coat proteins reversibly and partially dissociate to form one or more pores.

3. The nanoparticle of claim 2, wherein the one or more AIs are non-covalently conjugated to and embedded within the one or more pores of the tobacco mosaic virus.

4. The nanoparticle of claim 1, wherein the one or more AIs are embedded in the one or more coat proteins of the tobacco mosaic virus genus.

5. The nanoparticle of any one of claims 1 to 4, wherein the one or more AIs are not chemically altered.

6. The nanoparticle according to any one of claims 1 to 5, wherein the external factor is a change in pH.

7. The nanoparticle according to any one of claims 1 to 5, wherein the external factor is the presence of a solvent.

8. The nanoparticle according to any one of claims 9, wherein the solvent is a polar aprotic solvent.

9. The nanoparticle according to any one of claims 10, wherein the polar aprotic solvent is dimethyl sulfoxide (DMSO).

10. The nanoparticle of any one of claims 1 to 9, wherein the tobacco mosaic virus is rod-shaped.

11. The nanoparticle of any one of claims 1 to 10, wherein the width of the Tobaccomovirus-AI nanoparticle is greater than the width of a reference Tobaccomovirus.

12. The nanoparticle of claim 11, wherein the reference tobamovirus molecule is treated with the same conditions as the tobamovirus-AI nanoparticle without the addition of AI.

13. The nanoparticle of claim 11, wherein the width of the reference tobacco mosaic virus is 15 nm, 16 nm, 17 nm, or 18 nm.

14. The nanoparticle of any one of claims 11 to 13, wherein the width of the Tobaccomovirus-AI nanoparticle is 2%-105% greater than the width of the reference Tobaccomovirus width.

15. The nanoparticle of any one of claims 11 to 13, wherein the width of the tobamovirus-AI nanoparticle is approximately 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% greater than the width of the reference tobamovirus width. %, 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%, 95%, 96%, 97%, 98%, 99%, 100%, 101%, 102%, 103%, 104%, or 105%.

16. The nanoparticle of claim 11, wherein the width of the tobacco mosaic virus-AI nanoparticle is 19 nm, 20 nm, 21 nm, 22 nm, 23 nm, 24 nm, 25 nm, 26 nm, 27 nm, 28 nm, 29 nm, 30 nm, 31 nm, 32 nm, 33 nm, 34 nm, 35 nm, 36 nm, 37 nm, 38 nm, 39 nm, 40 nm, 41 nm, 42 nm, 43 nm, 44 nm, 45 nm, 46 nm, 47 nm, 48 nm, 49 nm, 50 nm, 51 nm, 52 nm, 53 nm, 54 nm, 55 nm, 56 nm, 57 nm, 58 nm, 59 nm, 60 nm, 61 nm, 62 nm, 63 nm, 64 nm, 65 nm, 66 nm, 67 nm, 68 nm, 69 nm, 70 nm, 3nm, 44nm, 45nm, 46nm, 47nm, 48nm, 49nm, 50nm, 51nm, 52nm, 53nm, 54nm, 55nm, 56nm, 57nm, 58nm, 59nm, 60nm, 61nm, 62nm, 63nm, 64nm, 65nm, 66nm, 67nm, 68nm, 69nm, 70nm, 71nm, 72nm, 73nm, 74nm or 75nm.

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

18. The nanoparticle of claim 17, wherein the pesticide comprises a water-insoluble organic compound, a hydrophilic organic compound, an insecticide, a herbicide, a fungicide, a miticide, an algaecide, an antimicrobial, a biopesticide, a biocide, a disinfectant, a fumigant, an insect growth regulator, a plant growth regulator, a miticide, a microbial insecticide, a molluscide, a nematicide, an ovicide, a pheromone, an insect repellent, a rodenticide, a defoliant, a desiccant, a safener, or any combination thereof.

19. The nanoparticle of claim 17 or 18, wherein the insecticide comprises a benzoyl urea such as novaluron, lufenuron, chlorfluazuron, flufenoxuron, hexaflumuron, noviflumuron, teflubenzuron, triflumuron and diflubenzuron; a carbamate; a pyrethroid such as cyhalothrin and its isomers and isomers; mixtures of isomers, lambda-cyhalothrin, deltamethrin, tau-fluvalinate, cyfluthrin, beta-cyfluthrin, tefluthrin and bifenthrin; organophosphates, such as azinfos-methyl, chlorpyrifos, diazinon, endosulfan and methidathion; neonicotinoids; phenylpyrazoles, such as imidacloprid, acetamiprid, thiacloprid, dinotefuran, thiamethoxam, and fipronil; conazoles, such as epoxiconazole, hexaconazole, propiconazole, prochloraz, imazalil, triadimenol, and difenocarb; difenoconazole, myclobutanil, prothioconazole, triticonazole and tebuconazole; morpholines such as dimethomorph, fenpropidine 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; dinitroaniline; itroaniline, such as pendimethalin and trifluralin; diphenyl ethers, such as oxyfluorfen; imidazolinones; sulfonylureas, such as chlorsulfuron, nicosulfuron, rimsulfuron, tribenuron-methyl; sulfonamides; triazines; and triazinones, such as metamitron. ; 20. The nanoparticle of claim 17, wherein the drug is a chemotherapeutic drug, an antiparasitic drug, an antibiotic drug, or an immunomodulator.

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

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

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

24. The nanoparticle of any one of claims 1 to 23, wherein the Tobacco Mosaic Virus is Tobacco Mosaic Virus (TMV).

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

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

27. The composition of claim 26, further comprising an excipient.

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

29. A method of preparing nanoparticles comprising a tobacco mosaic virus and one or more active ingredients (AI), the method comprising: a) providing the isolated tobamovirus to a buffer having a pH of about 7 to 9 to produce a tobamovirus-buffer; b) adding one or more AIs to the tobamovirus-buffer more than once, thereby producing the nanoparticles; and The nanoparticles are purified in a solution having a pH of about 5 to 9. in, The one or more AIs are non-covalently conjugated to the tobacco mosaic virus, and Wherein the tobacco mosaic virus comprises one or more coat proteins that reversibly and partially dissociate in response to a change in pH.

30. The method of 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 of claim 29 or claim 30, wherein the pH of the buffer is 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 of claim 29 or claim 30, wherein the pH of the buffer is 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 wherein the pH of the buffer is about 7.2, 7.3, 7.4, 7.5, 7.6, 7.7, 7.8, 7.9, or 7.

99.

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

3.

34. The method of 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 of preparing nanoparticles comprising a tobacco mosaic virus and one or more active ingredients (AI), the method comprising: a) providing the isolated tobamovirus to a buffer having a pH of about 5 to 9 to produce 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 producing the nanoparticles; and Purifying the nanoparticles in a solution having a pH of about 5 to 9, in, The one or more AIs are non-covalently conjugated to the tobacco mosaic virus, and wherein the tobacco mosaic virus comprises one or more coat proteins that reversibly and partially dissociate in response to the presence of the solvent.

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

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

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

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

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

41. The method of any one of claims 35 to 40, wherein the solvent is a polar aprotic solvent.

42. The method of claim 41, wherein the polar aprotic solvent is dimethyl sulfoxide (DMSO).

43. The method of any one of claims 29, 31 to 38, or 40 to 42, wherein the one or more AIs are added to the tobamovirus-buffer two or more times.

44. The method of any one of claims 29, 31-38, or 40-43, wherein the one or more AIs are added at least once a day.

45. The method of any one of claims 29 to 44, wherein the one or more AIs are added to 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; or wherein the one or more AIs are added to the tobacco mosaic virus at a 1,000-fold, 1,500-fold, 2,000-fold, 2,500-fold, 3,000-fold, 3,300-fold, 4,000-fold, 4,500-fold, 5,000-fold, 5,500-fold, 6,500-fold, 7,000-fold, 7,500-fold, 8,000-fold, 8,500-fold, 9,000-fold, or 9,500-fold molar excess; or 100 nmol, 150 nmol, 200 nmol, 250 nmol, 300 nmol, 350 nmol, 400 nmol, 450 nmol or 500 nmol of one or more AI / gram of tobacco mosaic virus is added.

46. ​​The method of any one of claims 29 to 45, wherein the one or more coat proteins reversibly and partially dissociate to form one or more pores.

47. The method of claim 46, wherein the one or more AIs are non-covalently conjugated to and embedded within the one or more pores of the Tobacco Mosaic Virus.

48. The method of any one of claims 29 to 47, wherein the one or more AIs are embedded in the one or more coat proteins of the Tobacco Mosaic Virus genus.

49. The method of any one of claims 29 to 48, wherein the one or more AIs are not chemically altered.

50. The method of any one of claims 29 to 49, wherein the tobacco mosaic virus is baculovirus.

51. The method of any one of claims 29 to 50, wherein the width of the nanoparticle is greater than the width of a reference tobacco mosaic virus.

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

53. The method of claim 52, wherein the width of the reference Tobacco Mosaic Virus is about 15 nm, 16 nm, 17 nm, or 18 nm.

54. The method of claim 53, wherein the width of the nanoparticle is 2%-105% greater than the width of the reference tobacco mosaic virus; or wherein the width of the 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%, %, 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%, 95%, 96%, 97%, 98%, 99%, 100%, 101%, 102%, 103%, 104% or 105%.

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

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

57. The method of claim 56, wherein the pesticide comprises a water-insoluble organic compound, an insecticide, an herbicide, a fungicide, a miticide, an algaecide, an antimicrobial, 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, an insect repellent, a rodenticide, a defoliant, a desiccant, a safener, or any combination thereof.

58. The method of claim 56, wherein the insecticide comprises benzoylurea, such as fluazifop, lufenuron, chlorfluazuron, flufenoxuron, hexaflumuron, noviflumuron, fenfluramide, triflumuron and diflubenzuron; carbamates; pyrethroids, such as cyhalothrin and its isomers and isomer mixtures, lambda-cyhalothrin, deltamethrin, fluvalinate, cyfluthrin, lambda-cyhalothrin, tefluthrin and bifenthrin; organophosphates, such as azinphos-methyl, chlorpyrifos, diazinon, endosulfan, methidathion; neonicotinoids; phenylpyrazoles, such as imidacloprid, acetamiprid, thiacloprid, dinotefuran, thiamethoxam and fipronil; conazoles, such as epoxiconazole, hexaconazole, propiconazole, prochloraz, imazalil, triadimenol , fenpropimorph, myclobutanil, prothioconazole, trichlorfon and tebuconazole; morpholines such as dimethomorph, fenpropidin and fenpropimorph; strobilurins such as azoxystrobin, kresoxim-methyl and their analogs; phthalonitriles such as thiocarb; mancozeb; fluazinam; pyrimidines such as ethimol sulfonate; aryloxyphenoxy derivatives; aryl ureas; aryl carboxylic acids; aryloxyalkanoic acid derivatives such as clodinafop-butyl and its analogs, fenoxaprop-butyl and its analogs, fenthiocarb-butyl, quizalofop-butyl and its analogs; dinitroanilines such as pendimethalin and trifluralin; diphenyl ethers such as oxyfluorfen; imidazolinones; sulfonylureas such as chlorsulfuron, nicosulfuron, sulfasulfuron-methyl, bensulfuron-methyl; sulfonamides; triazines; and triazinones such as metamitron-methyl.

59. The method of claim 56, wherein the drug is a chemotherapeutic drug, an antiparasitic drug, an antibiotic drug, or an immunomodulator.

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

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

62. The method of any one of claims 29 to 61, wherein the Tobacco Mosaic Virus is Tobacco Mild Green Mosaic Virus (TMGMV).

63. The method of any one of claims 29 to 61, wherein the Tobacco Mosaic Virus is Tobacco Mosaic Virus (TMV).

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

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

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

67. The pharmaceutical composition of claim 65 or claim 66, wherein the pharmaceutical composition is formulated as an injectable solution, a lyophilized powder, a suspension, or any combination thereof.

68. A method for treating cancer in a subject in need thereof, the method comprising administering to the subject in need of cancer treatment 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.

69. The method of claim 68, wherein the cancer wherein the cancer comprises breast cancer, ovarian cancer, glioma, gastrointestinal cancer, prostate cancer, carcinoma, lung carcinoma, hepatocellular carcinoma, testicular cancer, cervical cancer, endometrial cancer, bladder cancer, head and neck cancer, lung cancer, gastroesophageal cancer, gynecological cancer, or any combination thereof.

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

71. The method of 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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