WO2022204714A1

WO2022204714A1 - Zinc-based physionanocomposites and methods of use thereof

Info

Publication number: WO2022204714A1
Application number: PCT/US2022/071334
Authority: WO
Inventors: Robert Delong; Megan NIEDERWERDER
Original assignee: Kansas State University Research Foundation
Priority date: 2021-03-24
Filing date: 2022-03-24
Publication date: 2022-09-29

Abstract

The present disclosure provides ZnS-based compositions that treat cancer and viral infections. The ZnS compositions can further include manganese or iron and/or be doped with manganese (Mn), iron (Fe), nickel (Ni), cobalt Co), cobalt ferrite (CoFe), and any combination thereof.

Description

ZINC -BASED PHYSIONANOCOMPOSITES AND METHODS OF USE

THEREOF

CROSS REFERENCE TO RELATED APPLICATIONS

[0001] This application relates to and claims priority to U.S. Provisional Patent Application No. 63/200,720, which was filed on March 24, 2021. The teachings and contents of this reference is incorporated herein in its entirety.

STATEMENT REGARDING FEDERALLY SPONSORED RESEARCH & DEVELOPMENT

[0002] The invention was made with government support under National Science Foundation Grant number #2029579. The government has certain rights in the invention.

BACKGROUND OF THE DISCLOSURE

[0003] The field of the invention relates generally to the antiviral and anticancer mechanisms of zinc-based physiometacomposite complexes.

BRIEF DESCRIPTION OF THE DISCLOSURE

[0004] With the COVID-19 pandemic and deaths from metastatic cancer at an all-time high, the search for a biocompatible chemotherapeutic targeted nanomedicine has never been more important. Antimicrobial, antiviral and anticancer activity of zinc oxide nanoparticles (ZnO NPs) have been linked to ROS, zinc ion intracellular gradients and enzyme inhibition. Over the years a variety of different methods to synthesize nanoparticles have been developed. One early focus was on the use of gold, but gold is non-physiological and as a precious metal somewhat expensive. This focus has moved on to copper and more recently zinc-based materials knowing that in cells and tissues, zinc is often used to stabilize protein and nucleic acids and cells have evolved ways to re-use and recycle Zn, and other bio-metals like magnesium and iron. Cells also use them for a variety of reasons and other metals for example in the electron transport chain proteins or other metabolic enzymes.

[0005] In another aspect, a little cobalt or nickel will be incorporated onto the surface of ZnS, MnZnS, or FeZnS and will increase its biocompatibility and importantly could be used to attach nucleic acids forming an amino conjugate.

[0006] In one aspect, the distribution, tolerance, and anticancer/antiviral activity of Zn-based physiometacomposites (PMCs) was determined. Manganese, iron, nickel and cobalt doped ZnO, ZnS, or ZnSe were synthesized. “Doped” refers to a combination of one material with another, wherein one of the two materials is smaller and/or in a lesser amount than the other material. For example, in the present disclosure, nanoparticles can be doped with a separate component, such as an element, wherein the element is both smaller in size and in a lesser amount (by weight) than the nanoparticle.” Cell uptake and distribution into 3- D culture and mice, as well as biochemical and chemotherapeutic activity were studied by fluorescence/bioluminescence, confocal microscopy, flow cytometry, viability, antitumor and virus titer assays. Luminescence and inductively coupled plasma mass spectrometry analysis showed that nanoparticle distribution was liver>spleen>kidney>lung>brain, without tissue or blood pathology. Photophysical characterization as ex vivo tissue probes and LL37 peptide (SEQ ID NO. 2), antisense oligomer (ASO) or aptamer delivery targeting RAS/RBD. 25 pg/ml 48-hour treatment showed >98-99% cell viability, and 3-D organoid uptake. Such data support the preclinical development of PMCs for imaging and delivery targeting cancer and infectious disease.

[0007] In another aspect, the present disclosure provides a nanoparticle. In some forms, the nanoparticle is ZnS, MnZnS, or FeZnS. In some forms, the nanoparticle is doped with or combined with an element. In some forms, the element is selected from the group consisting of manganese, iron, nickel, cobalt and any combination thereof. In some forms, the nanoparticle is combined with or complexed with a protein or peptide. In some forms, the nanoparticle is combined with LL37 peptide (SEQ ID NO. 2), preferably having the sequence of SEQ ID NO. 2, an antisense oligomer (ASO), aptamer, or any combination thereof. “Complexed” refers to a combination of components that can be in direct contact or indirect contact with one another. In some forms, the nanoparticle delivery targets a specific domain or organ. In some forms, the organ is selected from the group consisting of liver, spleen, kidney, lung, brain, or any combination thereof. In some forms, the domain is a particular protein segment. In some forms, the segment is RAS/RBD or a spike protein. In some forms the sequence is selected from the group consisting of SEQ ID NO. 3 or SEQ ID NO. 4.

[0008] In another aspect, the pharmacokinetics of ZnO NPs versus silica coated ZnO NPs was investigated. This disclosure demonstrates that bioavailability is better for the naked uncoated material. Previously, it was observed that zinc-based physiometacomposite (PMC) nanoparticles doped with cobalt (5%CoZnO) were biocompatible and capable of forming amino or ami do-conjugates with antisense oligomer (ASO) for delivery into cells. We observed that by similarly doping magnesium into ZnO (5%MgZnO) this could red-shift its fluorescence excitation/emission. Thus, here we expanded on this chemistry to include manganese (Mn), iron (Fe), nickel (Ni) or cobalt ferrite (CoFe) doped oxide (ZnO) or the corresponding doped ZnS or ZnSe. The Zn-based physiologically-based metal composites, physiometacomposites (PMC) were comparatively characterized, their fluorescence, biocompatibility and delivery were investigated. Biocompatibility and photophysical properties of the PMC series was investigated with the MnZnS and MnZnSe nanoparticles yielding promising results. Indeed, MnZnS showed dose- dependent inhibition of beta-galactosidase (b-Gal) activity and significant antiviral activity against porcine reproductive respiratory virus (PRRSV). Anticancer activity of these and the nickel-doped zinc oxide (Ni/ZnO) was then compared against drug- resistant melanoma in conjunction with Ras binding domain (RBD) or RAS -targeted antisense or aptamer oligonucleotides. Nanoscale physiometacomposite (PMC) materials containing zinc oxide, sulfide, or selenide doped with manganese, iron, nickel or cobalt were synthesized.

[0009] In another aspect, this disclosure provides compositions comprising ZnO-based physiometacomposite (PMC) nanoparticles. In some forms, these PMC nanoparticles are combined or doped with cobalt, magnesium, manganese, iron, nickel, cobalt ferrite, oxide, or any combination thereof. In some forms, the material combined or doped with is present in an amount of 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, or more weight percent. In some forms, the doped zinc-based PMC nanoparticles are further formed into amino or amido-conjugates. In some forms, the amino or amido- conjugates are with ASO. In some forms, any of the above PMCs can be delivered into cells. In some forms, the above PMCs are administered to a subject in need thereof. In some forms, the administration is as described herein. In some forms, the administration is systemic. In some forms, the administration is via injection or infusion. In some forms, a PMC composition described herein is used to treat or prevent cancer or infection with or clinical signs or symptoms caused by a virus.

[0010] In another aspect, the disclosure provides a method for administering a nanoparticle as described herein to a subject in need thereof. In some forms, the administration targets a desired body part or organ. In some forms, the organ is selected from the group consisting of liver, spleen, kidney and lung with heart and brain. In some forms, the administration is systemic. In some forms, the administration is via any conventional route including injection and/or intravenously. In some forms, the administration occurs 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, ormore times including on a routine basis hourly, daily, bi-daily, weekly, monthly, yearly, or the like. In some forms, the composition administered includes nanoscale physiometacomposite (PMC) materials containing zinc oxide, sulfide, or selenide doped with manganese, iron, nickel or cobalt.

[0011] The MnZnS and especially MnZnSe achieved two-three log order improvement in fluorescence approaching the red range for emission.

[0012] MnZnS and MnZnSe were also quite biocompatible, cells tolerating dosages of up to 25 microgram/ml for up to 48 hours of continuous exposure with almost 100% viability with very little fluorescence quenching in serum, tumor or liver extracts.

[0013] Importantly, the MnZnS gives a two-three log inhibition of B- Galactosidase an enzyme previously associated with antimicrobial activity and in this concentration range significantly inhibited the model coronavirus, porcine reproductive respiratory virus (PRRSV).

[0014] In another aspect, the present disclosure provides compositions for treating viral infections or cancer. Due to the antiviral activity of the compositions, any virus can be treated. In some forms, the composition comprises a PMC nanoparticle disclosed herein. In some forms, the PMC NP is selected from the group consisting of ZnS, MnZns, FeZnS, ZnS doped with manganese (Mn), iron (Fe), nickel (Ni) or cobalt ferrite (CoFe), MnZnS doped with Mn, Fe, Ni, or CoFe. In some forms, the virus is one that infects an animal. In some forms, the animal is a mammal or a bird. In some forms, the animal is a human, dog, cat, bird, cow, pig, sheep, goat, or horse. In some forms, the virus is one that infects humans and is selected from the group consisting of Adeno-associated virus; Aichi virus; Australian bat lyssavirus; BK polyomavirus; Banna virus; Barmah forest Virus; Bunyamwera virus; Bunyavirus La Crosse; Bunyavirus snowshoe hare; Cercopithecine herpesvirus; Chandipura virus; Chikungunya virus; Cosavirus A; Cowpox virus; Coxsackievirus; Crimean-Congo hemorrhagic fever virus; Dengue virus; Dhori virus; Dugbe virus; Duvenhage virus; Eastern equine encephalitis virus; Ebolavirus; Echovirus; Encephalomyocarditis virus; Epstein-Barr virus; European bat lyssavirus; GB virus C/Hepatitis G virus; Hantaan virus; Hendra virus; Hepatitis A virus; Hepatitis B virus; Hepatitis C virus; Hepatitis E virus; Hepatitis delta virus; Horsepox virus; Human adenovirus; Human astrovirus; Human coronavirus; Human cytomegalovirus; Human enterovirus; Human herpesvirus 1; Human herpesvirus 2; Human herpesvirus 6; Human herpesvirus 7; Human herpesvirus 8; Human immunodeficiency virus; Human papillomavirus 1; Human papillomavirus 2; Human papillomavirus; Human parainfluenza; Human parvovirus B19; Human respiratory syncytial virus; Human rhinovirus; Human SARS coronavirus; Human spumaretrovirus; Human T-lymphotropic virus; Human torovirus; Influenza A virus; Influenza B virus; Influenza C virus; Isfahan virus; JC polyomavirus; Japanese encephalitis virus; Junin arenavirus; KI Polyomavirus; Kunjin virus; Lagos bat virus; Lake Victoria Marburgvirus; Langat virus; Lassa virus; Lordsdale virus; Louping ill virus; Lymphocytic choriomeningitis virus; Machupo virus; Mayaro virus; MERS coronavirus; Measles virus; Mengo encephalomyocarditis virus; Merkel cell polyomavirus; Mokola virus; Molluscum contagiosum virus; Monkeypox virus; Mumps virus; Murray valley encephalitis virus; New York virus; Nipah virus; Norwalk virus; O'nyong-nyong virus; Orf virus; .Oropouche virus; Pichinde virus; Poliovirus; Punta toro phlebovirus; Puumala virus; Rabies virus; Rift valley fever virus; Rosavirus A; Ross river virus; Rotavirus A; Rotavirus B; Rotavirus C; Rubella virus; Sagiyama virus; Salivirus A; Sandfly fever Sicilian virus; Sapporo virus; SARS coronavirus 2; Semliki forest virus; Seoul virus; Simian foamy virus; Simian virus 5; Sindbis virus; Southampton virus; St. louis encephalitis virus; Tick-borne powassan virus; Torque teno virus; Toscana virus; Uukuniemi virus; Vaccinia virus; Varicella-zoster virus; Variola virus; Venezuelan equine encephalitis virus; Vesicular stomatitis virus; Western equine encephalitis virus; WU polyomavirus; West Nile virus; Yaba monkey tumor virus; Yaba-like disease virus; Yellow fever virus; Zika virus; and any combination thereof. In some forms, virus is one that infects swine or pigs and is selected from the group consisting of Adenovirus; African Swine Fever Virus, Alphavirus such as Eastern equine encephalomyelitis viruses; Classical swine fever virus; Coronavirus, Porcine Respiratory Corona virus; Hemagglutinating encephalomyelitis virus; Japanese Encephalitis Virus; Porcine Circovirus; Porcine cytomegalovirus; Porcine Parvovirus; Porcine Reproductive and Respiratory Syndrome (PRRS) Virus; Pseudorabies virus; Rotavirus; Swine herpes virus; Swine Influenza Virus; Swine pox virus; Vesicular stomatitis virus; Virus of vesicular exanthema of swine; porcine epidemic diarrhea virus (PEDV); foot and mouth disease virus (FMDV); porcine enteroviruses; porcine toroviruses (PToV); porcine sapelovirus (PSV); porcine bocavirus (PBoV); porcine kobuvirus (PKBV); porcine Torque teno sus virus (TTSuV); Atypical Porcine Pestivirus; Linda virus; Mapuera Virus; Turisops Trucatus Parainfluenzavirus 1 (PIV1); Severe Acute Diarrhea Syndrome Coronavirus; Swine Enteric Alphacoronavirus; Seneca Valley Virus; Influenza virus D; Parainfluenzavirus; Parainfluenzavirus 5; Nipah virus; Swine vesicular disease virus; Transmissible gastroenteritis virus; and any combination thereof. In some forms, the virus is one that infects cows or cattle and is selected from the group consisting of Infectious Bovine Rhinotracheitis (IBR) virus; Bovine Virus Diarrhea (BVD) Types 1 and 2; Parainfluenza 3 (PI3) virus; Bovine Respiratory Syncytial Virus (BRSV); Bovine Herpesvirus; Bovine Leukemia Virus; Lumpky Skin Disease Virus; Allerton Virus; Bovine Mammilitis Virus; Infectious Bovine Keratoconjunctivitis Virus; Maligbnant Catarrhal Fever Virus; Pseudorabies Virus; Bovine Papilloma Virus; Bovine Papular Stomatitis Virus; Cowpox Virus; Paravaccinia Virus; Rift Valley Fever Virus; Rinderpest Virus; Enterovirus; Rhinovirus; Encephalomyocarditis Virus; Reovirus; Pseudorabies virus; Bluetongue virus; Japanese encephalitis virus; Rabies virus; Vesicular stomatitis virus; West Nile fever virus; and any combination thereof. In some forms, the virus is one that infects canines or dogs and is selected from the group consisting of Canine Influenza; Morbillivirus; Canine Parvovirus; Norovirus; Astrovirus; Adenovirus; Parainfluenza Virus; Reovirus; Rotavirus, Flavivirus; Wesselsbron Virus; Poxvirus; Herpesvirus; Orbivirus; Calicivirus; Coronavirus; Pseudorabies; Phlebovirus; and any combination thereof. In some forms, the virus is one that infects cats or felines and is selected from the group consisting of Feline Immunodeficiency Virus; Feline Coronavirus; Feline Leukemia Virus; Feline Panleukopenia Virus; Feline Calicivirus; Feline Herpesvirus; Rabies; Feline Infectious Peritonitis; and any combination thereof. In some forms, the virus is one that infects sheep and/or goats and is selected from the group consisting of Caprine arthritis and encephalitis virus; Sheeppox virus; Goatpox virus; and any combination thereof. In some forms, the virus is one that infects horses or equine and is selected from the group consisting of African horse sickness virus; Eastern equine encephalomyelitis virus; Western equine encephalomyelitis virus; Equine infectious anemia virus; Equine influenza virus; Equine herpesvirus 4; Equine arteritis virus; Venezuelan equine encephalomyelitis virus; West Nile Virus; Rabies; and any combination thereof. In some forms, the virus is one that infects birds or avian species and is selected from the group consisting of Avian infectious bronchitis virus; Infectious laryngotracheitis virus; Duck hepatitis virus; High and low pathogenic avian influenza viruses; Marek’s disease virus; Newcastle disease virus; Avian metapneumo virus; Avian Polyomavirus; Avian Bomavirus; West Nile Virus; Herpesvirus; Psittacine circovirus; Poxvirus; Paramyxovirus; and any combination thereof. It is understood that many of these viruses can infect multiple different types of animals, so inclusion in one list does not exclude it from another.

[0015] In some forms, the composition is administered to an animal in need thereof in an amount effective for inhibiting viral infection or cancer. In some forms, the amount of PMC NP is at least 10, 20, 30, 40, 50, 60, 70, 80, 90, 100, 150, 200, 250, 300, 350, 400, 450, 500, 550, 600, 650, 700, 750, 800, 850, 900, 950, 1000, 1250, 1500, 1750, 2000, 2500, 3000, or pg/kg of the animal.

[0016] In one aspect, the composition of the present disclosure further comprises at least one additional element. The at least one additional element is preferably selected from, but not limited to, pharmaceutical-acceptable-carrier(s) and/or veterinary-acceptable carrier(s), diluent(s), solvent(s), dispersion media, coating(s), adjuvant(s), preservatives, isotonic agent(s), adsorption delaying agent(s), protectant(s), antibacterial and/or antifungal agent(s), stabilizers, colors, flavors, and any combination(s) thereof.

[0017] “Adjuvants” as used herein, can include aluminum hydroxide and aluminum phosphate, saponins e.g., Quil A, QS-21 (Cambridge Biotech Inc., Cambridge MA), GPI-0100 (Galenica Pharmaceuticals, Inc., Birmingham, AL), water- in-oil emulsion, oil-in-water emulsion, water-in-oil-in-water emulsion. The emulsion can be based in particular on light liquid paraffin oil (European Pharmacopea type); isoprenoid oil such as squalane or squalene oil resulting from theoligomerization of alkenes, in particular of isobutene or decene; esters of acids or of alcohols containing a linear alkyl group, more particularly plant oils, ethyl oleate, propylene glycol di- (caprylate/caprate), glyceryl tri-(caprylate/caprate) or propylene glycol dioleate; esters of branched fatty acids or alcohols, in particular isostearic acid esters. The oil is used in combination with emulsifiers to form the emulsion. The emulsifiers are preferably nonionic surfactants, in particular esters of sorbitan, of mannide (e.g. anhydromannitol oleate), of glycol, of poly glycerol, of propylene glycol and of oleic, isostearic, ricinoleic or hydroxystearic acid, which are optionally ethoxylated, and polyoxypropylene- polyoxyethylene copolymer blocks, in particular the Pluronic products, especially L121. See Hunter et al., The Theory and Practical Application of Adjuvants (Ed.Stewart-Tull, D. E. S.)· JohnWiley and Sons, NY, pp51-94 (1995) and Todd et al., Vaccine 15:564-570 (1997).

[0018] For example, it is possible to use the SPT emulsion described on page 147 of “Vaccine Design, The Subunit and Adjuvant Approach” edited by M. Powell and M. Newman, Plenum Press, 1995, and the emulsion MF59 described on page 183 of this same book.

[0019] A further instance of an adjuvant is a compound chosen from the polymers of acrylic or methacrylic acid and the copolymers of maleic anhydride and alkenyl derivative. Advantageous adjuvant compounds are the polymers of acrylic or methacrylic acid which are cross-linked, especially with poly alkenyl ethers of sugars or polyalcohols. These compounds are known by the term carbomer (Phameuropa Vol. 8, No. 2, June 1996). Persons skilled in the art can also refer to U. S. Patent No. 2,909,462 which describes such acrylic polymers cross-linked with a polyhydroxylated compound having at least 3 hydroxyl groups, preferably not more than 8, the hydrogen atoms of at least three hydroxyls being replaced by unsaturated aliphatic radicals having at least 2 carbon atoms. The preferred radicals are those containing from 2 to 4 carbon atoms, e.g. vinyls, allyls and other ethylenically unsaturated groups. The unsaturated radicals may themselves contain other substituents, such as methyl. The products sold under the name Carbopol ; (BF Goodrich, Ohio, USA) are particularly appropriate. They are cross-linked with an allyl sucrose or with allyl pentaerythritol. Among then, there may be mentioned Carbopol 974P, 934P and 97 IP. Among the copolymers of maleic anhydride and alkenyl derivative, the copolymers EMA (Monsanto) which are copolymers of maleic anhydride and ethylene. The dissolution of these polymers in water leads to an acid solution that will be neutralized, preferably to physiological pH, in order to give the adjuvant solution into which the immunogenic, immunological or vaccine composition itself will be incorporated. [0020] Further suitable adjuvants include, but are not limited to, the RIBI adjuvant system (Ribi Inc.), Block co-polymer (CytRx, Atlanta GA), SAF-M (Chiron, Emeryville CA), monophosphoryl lipid A, Avridine lipid-amine adjuvant, heat-labile enterotoxin from E. coli (recombinant or otherwise), cholera toxin, IMS 1314 or muramyl dipeptide among many others.

[0021] Preferably, the adjuvant is added in an amount of about 100 pg to about 10 mg per dose. Even more preferably, the adjuvant is added in an amount of about 100 pg to about 10 mg per dose. Even more preferably, the adjuvant is added in an amount of about 500 pg to about 5 mg per dose. Even more preferably, the adjuvant is added in an amount of about 750 pg to about 2.5 mg per dose. Most preferably, the adjuvant is added in an amount of about 1 mg per dose.

[0022] A “protectant” as used herein, refers to an anti-microbiological active agent, such as for example Gentamycin, Merthiolate, and the like. In particular, adding a protectant is most preferred for the preparation of a multi-dose composition. Those anti-microbiological active agents are added in concentrations effective to prevent the composition of interest from any microbiological contamination or for inhibition of any microbiological growth within the composition of interest.

[0023] In some preferred forms, the present disclosure contemplates immunogenic or vaccine compositions comprising from about lug/ml to about 60 pg/ml of protectant, and more preferably less than about 30 pg/ml of protectant.

[0024] A “stabilizing agent”, as used herein, refers to an ingredient, such as for example saccharides, trehalose, mannitol, saccharose, albumin and alkali salts of ethylendiamintetracetic acid, and the like, to increase and/or maintain product shelf-life and/or to enhance stability.

[0025] Those of skill in the art will understand that the composition herein may incorporate known injectable, physiologically acceptable, sterile solutions. For preparing a ready-to-use solution for parenteral injection or infusion, aqueous isotonic solutions, such as e.g. saline or corresponding plasma protein solutions are readily available. In addition, as noted above, the compositions of the present disclosure can include diluents, isotonic agents, stabilizers, or adjuvants. Diluents can include water, saline, dextrose, ethanol, glycerol, and the like. Isotonic agents can include sodium chloride, dextrose, mannitol, sorbitol, and lactose, among others. Suitable adjuvants and stabilizers, are those described above.

[0026] According to a further aspect, the composition of the present disclosure further comprises a pharmaceutical acceptable salt, preferably a phosphate salt in physiologically acceptable concentrations. Preferably, the pH of said composition is adjusted to a physiological pH, meaning between about 6.5 and 7.5.

[0027] According to a further aspect, the compositions described herein can further include one or more other immunomodulatory agents such as, e. g., interleukins, interferons, or other cytokines.

[0028] According to a further aspect, the compositions described herein can further include an immune stimulant. It is understood that any immune stimulant known to a person skilled in the art can also be used. “Immune stimulant” as used herein, means any agent or composition that can trigger a general immune response, preferably without initiating or increasing a specific immune response, for example the immune response against a specific pathogen.

[0029] In another aspect, the present disclosure provides a method for treating, reducing the duration, incidence, or severity of clinical symptoms or signs associated with a viral infection or cancer. The method preferably includes the steps of administration of the composition of the present disclosure to an animal or human in need thereof. The dosage is preferably provided in an effective amount.

[0030] The clinical signs or symptoms are preferably reduced in duration, incidence, or severity by about 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, 95%, or even by 100% when compared to those animals or humans not provided the composition of the present disclosure. Such reduction can be applied to individual animals as well as groups or herds of animals. [0031] The method preferably includes the steps of administration of the composition of the present disclosure to an animal or human in need thereof. The composition can be administered once as a single dose composition or several times. When administered more than once, the second or subsequent doses will be administered at least 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 days, or more after the initial or previous administration. In preferred forms, the administration will lessen the severity, frequency, and/or duration of at least one clinical sign of the viral infection or cancer by at least 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, 95%, 96%, 97%, 98%, 99%, or even 100% in comparison to a group of animals or humans that did not receive an administration of the composition. Protection can include the complete prevention of clinical signs of infection, or a lessening of the severity, duration, or likelihood of the manifestation of one or more clinical signs of infection. Methods are known in the art for determining or titrating suitable dosages of active agent to find minimal effective dosages based on the weight of the subject, concentration of the agent and other typical factors.

[0032] In some preferred forms, said method also includes the administration of an immune stimulant. Preferably, said immune stimulant shall be given at least twice. Preferably, at least 3, more preferably at least 5, and even more preferably at least 7 days are between the first and the second or any further administration of the immune stimulant. Preferably, the immune stimulant is given at least 10 days, preferably 15, even more preferably 20, and still even more preferably at least 22 days beyond the initial administration of the composition. It is understood that any immune stimulant known to a person skilled in the art can also be used. “Immune stimulant” as used herein, means any agent or composition that can trigger a general immune response, preferably without initiating or increasing a specific immune response, for example the immune response against a specific pathogen. It is further instructed to administer the immune stimulant in a suitable dose.

[0033] The composition of the disclosure can conveniently be administered intranasally, transdermally (i.e., applied on or at the skin surface for systemic absorption), orally, parenterally, etc. The parenteral route of administration includes, but is not limited to, intramuscular, intravenous, intraperitoneal, intradermal (i.e., injected or otherwise placed under the skin) routes and the like.

[0034] When administered as a liquid, the present composition may be prepared in the form of an aqueous solution, syrup, an elixir, a tincture and the like. Such formulations are known in the art and are typically prepared by dissolution of the active agent (active agent for this disclosure is the PMC NP) and other typical additives in the appropriate carrier or solvent systems. Suitable carriers or solvents include, but are not limited to, water, saline, ethanol, ethylene glycol, glycerol, etc. Typical additives are, for example, certified dyes, flavors, sweeteners and antimicrobial preservatives such as thimerosal (sodium ethylmercurithiosalicylate). Such solutions may be stabilized, for example, by addition of partially hydrolyzed gelatin, sorbitol or cell culture medium, and may be buffered by conventional methods using reagents known in the art, such as sodium hydrogen phosphate, sodium dihydrogen phosphate, potassium hydrogen phosphate, potassium dihydrogen phosphate, a mixture thereof, and the like.

[0035] Liquid formulations also may include suspensions and emulsions that contain suspending or emulsifying agents in combination with other standard co-formulants. These types of liquid formulations may be prepared by conventional methods. Suspensions, for example, may be prepared using a colloid mill. Emulsions, for example, may be prepared using a homogenizer.

[0036] Parenteral formulations, designed for injection into body fluid systems, require proper isotonicity and pH buffering to the corresponding levels of body fluids. Isotonicity can be appropriately adjusted with sodium chloride and other salts as needed. Suitable solvents, such as ethanol or propylene glycol, can be used to increase the solubility of the ingredients in the formulation and the stability of the liquid preparation. Further additives that can be employed in the present vaccine include, but are not limited to, dextrose, conventional antioxidants and conventional chelating agents such as ethylenediamine tetraacetic acid (EDTA). Parenteral dosage forms must also be sterilized prior to use.

[0037] A method for eliciting an immune response against a viral infection and/or clinical signs or symptoms of viral infection is also provided. Such a method follows the same methodology as set forth above. In preferred forms, the virus is disclosed in the list above.

[0038] In some forms, the composition further includes an antimicrobial or anti-cancer peptide.

[0039] Interestingly, while the anticancer activity of ZnO had been reported, Ni/ZnO PMC inhibits melanoma cell invasion and ERK and AKT expression, two markers often associated with drug-resistant cancers.

[0040] The Ni/ZnO anticancer activity is enhanced with LL37 peptide (SEQ ID NO. 2), and the ZnO or MnZnS in conjunction with RAS/RBD targeted antisense oligomer or aptamer.

[0041] In another aspect, the compositions of the disclosure are used to treat cancer. In some forms, the composition is combined with a peptide. In some forms, the peptide is known to have anticancer activity. In some forms, the cancer is melanoma or brain cancer. In some forms, the peptide is LL337 (SEQ ID NO. 2). In some forms, the cancer is drug-resistant. In some forms, the composition is combined with an ASO or aptamer. In some forms, the ASO or aptamer targets RAS or RBD.

[0042] The following examples demonstrate certain aspects of the present disclosure. However, it is to be understood that these examples are for illustration only and do not purport to be wholly definitive as to conditions and scope of this disclosure. It should be appreciated that when typical reaction conditions (e.g., temperature, reaction times, etc.) have been given, the conditions both above and below the specified ranges can also be used, though generally less conveniently. The examples are conducted at room temperature (about 23^°C to about 28^°C) and at atmospheric pressure unless otherwise noted. All parts and percents referred to herein are on a weight basis and all temperatures are expressed in degrees centigrade unless otherwise specified. Further unless noted otherwise, all components of the disclosure are understood to be disclosed to cover “comprising”, “consisting essentially of’, and “consisting of’ claim language as those terms are commonly used in patent claims.

BRIEF DESCRIPTION OF THE DRAWINGS

[0043] Figure 1 is a series of graphs comparing the DSC analysis for the melting temperature increase for poly I:C upon interaction to ZnO shown by DSC;

[0044] Fig. 2 is a graph illustrating the structural stability imparted to poly I:C upon binding to ZnO NP as shown by circular dichroism wherein the peak of the lines occurring between 270 and 290 nm represents Poly(PC) for the lowest line, Poly(PC)-ZnO at both 1:1 and 10:1 for the middle line, and Poly(PC) at 20:1 for the top line;

[0045] Fig. 3 is a 2D graph illustrating the fluorescence shift of NP- proteimRNA tripartite complexes formed with Zn-based nanomaterials;

[0046] Fig. 4 is a photograph of a RNA agarose electrophoresis (RAGE) illustrating that protamine coating MSN or ZnO NP imparts RNA stability to TY-RNA when incubated at 4° as a PBS suspension;

[0047] Fig. 5 is a photograph of a RAGE illustrating the stability of dry powders incubated for 1 or 2 days at 30, 40, or 50°C in comparison with PBS suspensions stored in the refrigerator for 1 day or 1 week and left out on the bench overnight prior to RAGE analysis;

[0048] Fig. 6 upper left panel is a schematic representation of Cy5.5- ZnO click chemistry synthesis, the upper middle panel is a graph representing the hydrodynamic size wherein the ZnO has a higher peak signal intensity, the upper right panel is a graph illustrating the zeta potential characterization wherein the ZnO-PEG has a higher total counts, and the bottom panel is a graph illustrating the stability data when incubated in serum-containing Media (10% FBS/DMEM);

[0049] Fig. 7A is a set of photographs illustrating bioimaging wherein two mice were administered a 2 mg/kg single intravenous dose in 100 microliters PBS of either ZnO-NP or cy5.5-ZnO-PEG NP into the tail vein and imaged directly in the bio-imager in the near infra-red (700 nm) or sacrificed at 5 hours the brain, heart, lungs, liver, spleen and kidneys were removed and imaged in the bio-imager;

[0050] Fig. 7B is a set of photographs illustrating histopathological analysis of the corresponding mice when sacrificed after 3 days;

[0051] Fig. 7C is a graph illustrating the relative fluorescence and zinc content per milligram tissue determined by fluorescence spectroscopy and ICP/MS analysis after 5 hours wherein the tissues were removed and weighed, and homogenized in PBS buffer;

[0052] Fig. 7D is a dot blot of the free cy5.5 dye or cy5.5-ZnO showing fluorescence quenching of the conjugate (D);

[0053] Fig. 8A is a graph illustrating the nanoscale confirmation of the pysiometacomposite materials, NiZnO, MnZnS, FeZnS, MnZnSe and others described in the manuscript by NTA;

[0054] Fig. 8B is a photograph illustrating the nanoscale confirmation of the pysiometacomposite materials, NiZnO, MnZnS, FeZnS, MnZnSe and others described in the manuscript by TEM analysis

[0055] Fig. 8C is a graph illustrating the biocompatibility of the different compositions after 48 hour treatment of continuous exposure in serum containing media to NIH3T3 cells as shown by MTT assay wherein in each set of 3 bars, the respective amounts of the compositions is 10, 20, and 25 pg/ml, respectively and error bars shown are standard deviation of 4 independent wells; [0056] Fig. 9A is a set of illustrations of the photophysical properties of PMC nanoparticles that were spiked into PBS, serum, tumor or liver homogenates and their fluorescence versus concentration curves obtained;

[0057] Fig. 9B is a set of illustrations of bioluminescence assays that were conducted in the presence of FeZnS or MnZnS with/without Luciferase enzyme and substrate;

[0058] Fig. 9C is a set of photographs illustrating Caco-2 spheroids incubated with cy5-5-ZnO that were imaged by confocal microscopy as described in experimental methods;

[0059] Fig. 9D is a set of photographs illustrating HeLa cell tumor spheroids that were established and treated with cy5.5-NP complexes, rinsed with PBS and imaged in the bioimager;

[0060] Fig. 9E is a set of photographs illustrating ex vivo slices of mouse brain, liver and lung (shown) that were injected with MnZnS or MnZnSe and imaged directly in the bio-imager;

[0061] Fig. 10A is a set of graph illustrating the biochemical and antiviral activity of the PMC nanoparticles wherein B-Gal enzyme inhibition using the fluorescence-based assay with silver or ZnP NP controls relative to the other PMC nanoparticles (inset is a parallel dose-response experiment with Luciferase enzyme) is depicted and wherein NiO is the bottom line, NiZnO is the 2^nd to the bottom line, FeZnS is the 3^rd to the bottom line, MnZnS is the 2^nd line from the top, and the blank is the top line;

[0062] Fig. 10B is a graph illustrating the dose-response inactivation curve of PRRSV-GFP after exposure to MnZnS NP in cell culture wherein data is shown as the logio TCIDso/ml PRRSV-GFP titer post-exposure to different concentrations of MnZnS NP; TCIDso/ml calculations were performed for each sample in triplicate; positive controls are represented by the 0 pg/ml NP concentration; and results are based on †three or {four independent titration experiments with mean quantity calculated and shown;

[0063] Fig. 11 A is a schematic representation of the scratch assay and 3-D tumor spheroid studies;

[0064] Fig. 1 IB is a set of photographs illustrating a scratch assay showing B16F 10 cells re-invade the scratch unless 20 microgr/ml ZnO or Ni/ZnO PMC is present;

[0065] Fig. llC is a set of photographs illustrating a tumor spheroid assay showing inhibition of NiZnO nanoparticle composite;

[0066] Fig. 1 ID is a graph illustrating ERK/AKT expression as a function of NiZnO treatment;

[0067] Fig. 11E is a graph illustrating the percent cytotoxicity of NiZnO treatment to M5 canine mucosal melanoma cells;

[0068] Fig. 12A is a Circos plot of B16F10-B ALB/C tumor protein analysis associating RAS/ERK/AKT and BCL pathways;

[0069] Fig. 12B is a graph illustrating ZnO, NiO, C03O4 NP delivery of cy5.5-ASO into B16F10 cells by flow cytometry;

[0070] Fig. 12C is a graph and a photograph of a gel illustrating RT- PCR of exon3/intron4 correction by ASO in A375 cells;

[0071] Fig. 12D is a set of photographs illustrating ZnO or C03O4 NP delivery of cy5.5-ASO into A375 cells shown by confocal microscopy;

[0072] Fig. 12 E is a set of graphs illustrating the activity of RAS/RBD ASO or aptamer compared to nanoparticle-LL37 (SEQ ID NO. 2) against B16F10;

[0073] Fig. 13 is a graph illustrating the validation of RBD target in B16F10 cells by delivery of RBD protein via Co/ZnO or CoFe/ZnO PMC nanoparticles increasing anticancer activity against B16F 10 cells with %viability determined relative to untreated controls by MTT assay;

[0074] Fig. 14 is a graph illustrating the binding of LL37 peptide (SEQ ID NO. 2) to various PMC NP compositions as a function of zeta potential surface charge shift wherein for each NP composition, the PMC alone is on the left and the PMC+LL37 (SEQ ID NO. 2) is on the right; and

[0001] Fig. 15 is a photograph illustrating the binding of LL37 (SEQ ID NO. 2) peptide to FeZnS shown by gel shift and interaction to poly I:C RNA.

DETAILED DESCRIPTION OF THE DISCLOSURE

[0002] This writen description uses examples to disclose the invention, including the best mode, and also to enable any person skilled in the art to practice the invention, including making and using any devices or systems and performing any incorporated methods. The patentable scope of the invention is defined by the claims, and may include other examples that occur to those skilled in the art. Such other examples are intended to be within the scope of the claims if they have structural elements that do not differ from the literal language of the claims, or if they include equivalent structural elements with insubstantial differences from the literal languages of the claims.

[0003] EXAMPLE 1

[0004] Materials and methods

[0005] Differential scanning calorimetry (DSC) was conducted by Dr. Akshay Jain at the University of Kansas Center for Vaccine Stabilization and Characterization according to a protocol developed by the group for protein vaccine subunits here applied to poly I:C exposed to 20 ug/ml ZnO NP in phosphate buffered saline. Circular dichroism experiments were conducted as we have previously described. Poly inosinic:poly cytidylic acid [poly(I:C)] and Torula yeast RNA (TY- RNA) were obtained from Sigma- Aldrich. 100 nm and 14 nm ZnO NP were obtained from Sigma- Aldrich and PlasmaChem respectively. MgO NP was obtained from Sigma-Aldrich. Cobalt oxide and nickel oxide nanoparticles were synthesized by Dr. Kartik Ghosh as previously reported. ASO sequence: (3-

CCUCUUACCUCAGUUACA-5) (SEQ ID NO. 1) was obtained from Trilink Biotechnologies. Zeta potential measurements and UV payload experiments were conducted as previously described in Comparative functional dynamics studies on the enzyme nano-bio interface. Thomas SE, Comer J, Kim MJ, Marroquin S, Murthy V, Ramani M, Hopke TG, McCall J, Choi SO, DeLong RK.Thomas SE, et al. Int J Nanomedicine. 2018 Aug 8;13:4523-4536; Comparing the effects of physiologically- based metal oxide nanoparticles on ribonucleic acid and RAS/RBD-targeted triplex and aptamer interactions, Huslig G, Marchell N, Hoffman A, Park S, Choi SO, DeLong RK.Huslig G, et al. Biochem Biophys Res Commun. 2019 Sep 10;517(l):43-48; and Amino/Amido Conjugates Form to Nanoscale Cobalt Physiometacomposite (PMC) Materials Functionally Delivering Nucleic Acid Therapeutic to Nucleus Enhancing Anticancer Activity via RAS-targeted Protein Interference. RK DeLong, J Dean, G Glaspell, M Jaberi-Douraki, K Ghosh, D Davis, Nancy Moneteiro-Riviere, P. Chandran, T. Ngyuen, S. Aryal, C. Russel Middaugh, S. Chan Park, S-0 Choi and Meghana Ramani. ACS Applied Bio Materials 3 (1), 175-1792020.

[0006] Results and discussion

[0007] Zeta potential and payload analysis:

[0008] In cells and tissues, zinc is used to stabilize RNA and protein structure and interactions. Thus using zeta potential analysis the change in apparent charge at the surface of the nanoparticle was assessed, comparing RNA interaction to zinc oxide nanoparticle (ZnO NP) versus the other common physiologically-based metal oxide nanoparticles with a known active nanobio interface. These data are summarized in Table 1 below:

[0009] Table 1 shows RNA interaction to the physiologically-based metal oxide nanoparticles on the basis of apparent charge at the nanoparticle surface indicated by zeta potential (ZP) analysis, where notably all nanoparticles undergo an anionic shift to the negative in the presence of either antisense oligomer (ASO) or poly I:C. Interestingly the change in ZP for the larger RNA was greatest for ZnO NP consistent with our prior work, whereas ASO interaction favored Co304, consistent with its bioconjugation. The RNA payload in units of micrograms/milligram nanoparticle was then obtained by microcentrifugation of the RNA and nanoparticle sample, the loss of UV absorbance in the supernatant when the RNA and nanoparticle controls were background subtracted was used to estimate the payload of RNA per nanoparticle mass. This parameter was significant with the payload increasing dramatically.

[0010] Zinc oxide nanoparticle increases RNA melting temperature:

[0011] We had previously examined the effect of the physiological metal oxides on duplex and triplex formation and on RNA secondary structure analysis for a microbial RNA obtainable in bulk, torula yeast RNA (TY-RNA) by UV or circular dichroism analysis. Poly I:C is an immunologically active double-stranded RNA viral mimetic and in the presence of ZnO NP its melting temperature shifts from 63.9-64.7 degrees Celsius (°C) to 70.1-71.6 indicating gain in temperature-stability (Fig. 1): [0012] Zinc oxide nanoparticle binding imparts RNA structural stability:

[0013] Previously we had examined the effect of physiological nanoparticle interaction to TY-RNA by circular dichroism. Given the temperature- stabilization shown above, we examined the effect of increasing the stoichiometric ratio of ZnO NP to poly I:C by CD similarly. Surprisingly, the presence of ZnO NP at lower stoichiometries, 1 : 10 or 1 :20 actually stabilized the RNA causing the pattern to amplify suggesting structural stability was imparted to the RNA by its interaction to ZnO NP (Fig. 2):

[0014] As shown in Fig. 2, the CD pattern is actually accentuated in the presence of ZnO NP suggesting structural stability is imparted to the RNA upon ZnO NP interaction. These data are consistent with the data above, that ZnO NP interaction to poly I:C is RNA-stabilizing.

[0015] NP-protein:RNA tri-complexes

[0016] We had previously reported that 2-dimensional fluorescence difference spectroscopy (2-D FDS) could be used to detect ZnO NP surface modification and interaction to RNA. Mg/ZnO or FeZnS nanomaterials were incubated with antiviral LL-37 peptide (SEQ ID NO. 2) with/without poly I:C (pIC) and the fluorescence shift and gel electrophoresis mobility shift as a function of the NP: protein: RNA tripartite species is shown (Fig. 3).

[0017] The 2-D FDS shift that occurs when Mg/ZnO interacts with LL-37 (SEQ ID NO. 2) and subsequent RNA interaction to form the tripartite species is shown in Fig. 3. These data suggest that the surface of zinc-based nanoparticles can be coated with cationic protein enabling the subsequent attachment of the RNA.

[0018] Protamine coated nanoparticles protect RNA from temperature degradation: [0019] In our previous work we reported protamine could condense DNA or RNA into nanoparticles which could be loaded onto an inorganic surface such as gold and this could impart accelerated stability to DNA vaccine allowing the plasmid DNA vector to retain gel staining intensity. Previously it was reported surface- functionalized mesoporous silica nanoparticle (MSN) could temperature stabilize RNA, and here it could be shown that coating the surface of either MSN or ZnO NP with protamine enhanced the stability of the RNA as shown by RNA agarose gel electrophoresis (RAGE) analysis when incubated at 4 degrees Celsius (4 °C) for up to 4 days when stored as a suspension in PBS buffer (Fig. 4).

[0020] As can be seen in Fig. 4, the RNA band staining intensity is retained when the samples, either MSN or ZnO NP are coated with protamine and can be stored in the refrigerator for up to 4 days without losing band intensity. By contrast formulations what were dried to a powder and stored near 60°C for the same amount of time, very little intact RNA could be detected. It should be noted that in the previous paper, MSN was surface-functionalized prior to RNA loading which protected the RNA and protamine was not used in these experiments. However in that case, RT-PCR amplification was used as a read-out for stability enhancement, and the RAGE method is expected to be a truer reflection of the degree to which RNA structure is maintained over the time course.

[0021] Accelerated stability protocol:

[0022] TY-RNA was formulated onto ZnO NP (14 nm) by coating first with protamine, alcohol precipitated, air dried and incubated for 1 or 2 days at 30, 40 and 50 °C, the RNA eluted from the particles and analyzed by RAGE as shown (Fig. 5).

[0023] As can be seen in Fig. 5, when stored as a dry powder, the ZnO- protamine-RNA formulations are stable at 30 or 40 degrees Celsius for several days, the RNA band retaining considerable staining intensity. The formulations stored at 4 deg C for 1 day or 1 week could also be stored at room temperature as a dry powder and considerable intact RNA could still be detected. These data indicate that the protamine coated ZnO NP can temperature stabilize RNA, both as a suspension in PBS and as a dry powder.

[0024] EXAMPLE 2

[0025] Materials and Methods

[0026] Nanomaterials and Reagents: Zinc oxide (ZnO) nanoparticles (NPs) of 100 nm (cat-544906-108 and lot-MKBV5880V), Dichloromethane (DCM; Cat-439223) and 3-Mercaptopropionic acid (cat-M5801) were purchased from Sigma- Aldrich, MO, USA. Diethyl ether (cat-AC364330025; 99.5%) and ethanol (cat - 9111) were from Thermo Fisher. Briefly, Cy5.5-ZnO NPs were synthesized similar to Shi, J. Hao Hong, Yong Ding et al. Evolution of zinc oxide nanostructures through kinetics control. J. Mater. Chem. 21, 9000-9008 (2011) and Hong, H. Jian Shi, Yunan Yang, et al. Cancer-Targeted Optical Imaging with Fluorescent Zinc Oxide Nanowires. Nano Lett. 11, 3744-3750 (2011) with some modifications. A total of 3 mg of 3- mercaptopropionic acid was added to the ZnO NP suspension (3 mg/mL) in DCM and the mixture stirred at 300 rpm for 3 hr at room temperature. The DCM solvent was removed by evaporation in a hood. The carboxylic functional group from the 3- mercaptopropionic acid stabilized the ZnO NPs by forming a bidentate coordination bonds with zinc atoms. These acid functionalized ZnO NPs (ZnO-SH) were washed with 100% ethanol by centrifugation at 1000 rpm for 5 min and the supernatant was discarded. After repeated washing with ethanol, particles were washed with dry diethyl ether to obtain a dry powder of ZnO-SH. For PEG passivation stability, similar to Nikam, D. S. Swati V. Jadhav, Vishwajeet M. Khot et al. Colloidal stability of polyethylene glycol functionalized Co0.5Zn0.5Fe204 nanoparticles: effect of pH, sample and salt concentration for hyperthermia application. RSCAdv. 4, 12662-12671 (2014) and Marasini, R., Pitchaimani, A., Thanh Nguyen, T. D., Comer, J. & Aryal, S. The influence of polyethylene glycol passivation on the surface plasmon resonance induced photothermal properties of gold nanorods. Nanoscale 10, 13684-13693 (2018). 5 mg of ZnO-SH was incubated with 153 pg of maleimide-polyethylene glycol- succinimidylcarboxy methyl ester (Mal-PEG-NH2; Creative PEGWorks; MW, 5 kDa) in water for 1 h at room temperature, followed by the addition of 5 pg of Cy5.5 dye in waterDMSO (v/v) solution. The final volume of the reaction mixture was made to 2 mL with lx phosphate buffer saline, gently vortexed for 1 min, and the reaction mixture was stirred overnight at 40 rpm in a rotating shaker. Finally, the product was washed three times with ethanol followed by washing with water using centrifugation at 1000 rpm for 5 min and the supernatant was discarded. The product was lyophilized to get the dry powder and stored at -20C until further use. Cy5.5-ZnO size, shape, zeta potential and fluorescence was characterized as previously reported (Robert K. DeLong, John Dean, Garry Glaspell et al, Amino/ Amido conjugates form to nanoscale cobalt physiometacomposite (PMC) materials functionally delivering nucleic acid therapeutic to nucleus enhancing anticancer activity via Ras-targeted protein interference. ACS Applied Bio Materials. 3, 1, 175-179 (2020) and Amanda Hoffman, Xiaotong Wu, Jianjie Wang el al, Two-Dimensional Fluorescence Difference Spectroscopy ofZnO and Mg Composites in the Detection of Physiological Protein and RNA Interactions. Materials (Basel). 10(12), 1430 (2017).

[0027] 5%MgZnO and MnZnSe were provided by Dr Wanekeya (Missouri State University, MO, USA) and Dr McLaurin (Kansas State University, KS, USA) as described previously. Nanorod shaped < 200 nm cobalt zinc oxide (CoZnO) and nickel zinc oxide (NiZnO) nanoparticles were synthesized as previously described (Robert K. DeLong, John Dean, Garry Glaspell et al, Amino/Amido conjugates form to nanoscale cobalt physiometacomposite (PMC) materials functionally delivering nucleic acid therapeutic to nucleus enhancing anticancer activity via Ras-targeted protein interference. ACS Applied Bio Materials. 3, 1, 175-179 (2020)). MnZnS and FeZnS were synthesized by the same procedure, where briefly pure powders of MnS, FeS or ZnS were physically mixed at the 5% ratio, heated to a flux, allowed to cool in an oxygen purged atmosphere and jet ball milled to nanoscale confirmed by transmission electron microscopy and nanoparticle tracking analysis. Cobalt ferrite PMCs were synthesized by Dr. KC Ghosh’s laboratory (Missouri State University) by a similar method. Pure cobalt and nickel oxide used as controls were obtained from Sigma-Aldrich or PlasmaChem GmbH (Berlin, Germany). Cy5.5-labelled SSO (sequence: 3-CCUCUUACCUCAGUUACA-5) (SEQ ID NO. 1) was obtained from Trilink Biotechnologies linked through standard automated solid support chemistry. Clinical-grade LL-37 peptide (SEQ ID NO. 2) was obtained from Dr. Cheng Kao (Indiana University). NIH3T3, B16F10 and A375 cells for cytotoxicity studies were obtained from the American Tissue Culture Collection (ATCC). Canine mucosal melanoma cells (M5) were obtained from Dr. Raelene Wouda (Kansas State University). All NPs and RNA were precipitated from 70% alcohol/H₂0 washed once with 100% alcohol, air dried in the biosafety cabinet prior to RNA and protein complexation, cell or animal administration. The NPs were washed with double- distilled water, 70% ethanol/water, ethanol, and were stored dry prior to use. Costar (Coming, NY, USA) 96-well black, clear bottom assay plates were used for the assays. Luciferase enzyme (Photinus pyralis, >10xl0¹⁰ (units/mg protein) was obtained from Sigma Aldrich and diluted it to a 0.2% solution [1:500 dilution with PBS buffer] PBS buffer at 10X concentration was diluted to a 10% solution with de-ionized water [ddH20] Luciferase enzyme substrate buffer (ATP, Mg) was diluted to a 1:1 vol/vol ratio with PBS buffer. b-Galactosidase (b-Gal) from Aspergillus oryzae was obtained from Sigma Aldrich (>8.0 units/mg solid, Louis, MI, USA) and was diluted to a 1 mg/kg solution in spectral grade ¾0. The B-Gal substrate resorufm b-D-galactopyranoside was purchased in a 10 g vial from Marker Gene Technologies (Eugene, OR, USA) and was diluted down into ten 10 mg/kg aliquots in spectral grade ¾0 and re-suspended into a 1 mg/kg solution for experimentation.

[0028] Animals

[0029] Animal procedures were approved by Kansas State University IACUC 4064.1. Female 6-week-old BALB/C Nu/Nu mice were obtained from Charles River and allowed to acclimate for several weeks prior to the experiment. Mice were anesthetized using oxygen/isoflurane prior to administration with treatment and bioimaging. Mice were intravenously injected into the tail vein with 100 pi of PBS or ZnO NP or ZnO NP-Cy5.5 at the dose rate of 2 mg/kg body weight. Two mice were used for the PBS sham injection as a control, one for the blood and tissue samples at 5 hours and another at 3 days. Similarly, two mice were used for ZnO and cy5.5-ZnO, one for the 5 hour time-point, and one for the 3 day time-point. A total of 6 mice were used for the in vivo study.

[0030] ICP-MS for zinc determination in tissue: Samples were prepared from; (1) PBS alone (n=l), (2) ZnO NP (n=2), and (3) ZnO NP-Cy5.5 (n=2). The concentration of zinc (Zn) in the mouse tissues was determined using ICP-MS analysis following the standard protocol. In brief, tissues were digested using 2 ml of 70% nitric acid (HNO3) for liver and 1 ml for brain, heart, lungs, spleen and kidneys. The digestion was performed in SC154 HotBlock^® (Environmental Express, USA) at 90°C overnight. Following overnight digestion, the tissue digests were diluted by an addition of 9 ml deionized water. The diluted digests were further diluted by combining 1 ml of the digest with 4 ml of 2% HNO3 and filtered using 0.2 pm filter. Zinc concentration was measured on a PerkinElmer NexION® 350D ICP-MS. Zinc present in tissues without nanoparticle treatment was used as control for background subtraction. This work was conducted in the accredited Kansas State University Veterinary Diagnostic Laboratory.

[0031] Relative fluorescence in tissues: Tissues were collected from mice treated with, (1) PBS alone (n=l), (2) ZnO NP (n=2), and (3) ZnO-Cy5.5 NP (n=2). Fluorescence was measured in the following tissues:

(1) PBS treated: Liver and Kidney

(2) ZnO NP treated: a. Mouse 1: Liver and kidneys b. Mouse 2: brain, heart, lungs, spleen and kidneys

(3) ZnO NP-Cy5.5 treated: brain, heart, lungs, spleen and kidneys from both mice

[0032] From the tissues collected, a portion was cut off, weighed and homogenized using SONICS VCX Vibra 130 Tissue Sonicator (PRO Scientific Inc.) at an amplitude of 50 at a pulse rate of 10s(on) and 5s (off) for 20 minutes. From the homogenate, 200 pi was transferred to a 96-well plate and fluorescence was measured using SpectraMax^® i3x multimode microplate reader (Molecular Devices, California, USA). Excitation and emission wavelengths used were 660 nm and 695 nm respectively. PBS was used as the blank. The experiment was done in triplicate.

[0033] Histopathology and hematology analyses: were performed at the Veterinary Medical Diagnostic Laboratory, Kansas State University. The remainder of the collected tissues were fixed in 10% neutral buffered formalin. Sections of fixed tissue were routinely processed on a Sakura Tissue-TEK VIP 6 Processor prior to paraffin embedding. Slides were cut at 4 pm and routinely stained with hematoxylin and eosin on a Leica Autostainer XL ST5010. Representative images at lOx magnification were captured on an Olympus LC20 camera mounted on an Olympus BX53F2 light microscope with CellSens (Olympus Corporation).

[0034] Animal Model Imaging: Imaging experiments were conducted on 5 mice using a Pearl® Trilogy Small Animal Imaging System (LI-COR Biosciences, USA) immediately before and after administration, and 5 hours after administration of either ZnO NP or cy5.5-ZnO NP. At 5 hours or 3 days after administration animals were euthanized under anesthesia and the blood or tissues analyzed as above with assistance from the Nanotechnology Innovation Center Kansas State and Comparative Laboratory Animal Medicine group. Images were collected using the near infrared filter (700 nm).

[0035] MTT Assay: For the cytotoxicity (MTT) assay, NIH3T3 fibroblast cells were seeded on a 96-well plate with 5,000 cells/well and allowed to grow for 24 h in DMEM with 10% fetal bovine serum (FBS) and 1% penicillin/streptomycin. After 24 h, the medium was replaced with PMCs (5% NiZnO, MnZnS, 5% MgZnO, FeZnS and MnZnSe) dissolved in DMEM at 10, 20 and 25 pg/ml. Each treatment was tested on four wells. Four wells containing DMEM alone served as the blank and four wells with untreated cells served as the control. The cells were monitored daily for visible cytotoxicity using a light microscope. After 24/48/72/96 h with the treatment, the treatment was removed. All the wells were rinsed once with PBS, followed by the addition of 110 pL of 1:20 mix of MTT: indicator-free DMEM. After incubating the plate for 5 h at 37°C, 85 pL of the mixture was removed from each well and 75 pL of dimethyl sulfoxide was added to solubilize the crystals. The plate was then kept at 37°C in an orbital shaker with 175 revolutions/minute. After 15 minutes on the orbital shaker, the plate was then read on a Synergy HI Hybrid Multi- Mode Microplate Reader for absorbance at a wavelength of 562 nm.

[0036] Photophysical characterization: To obtain excitation, emission, and intensity data, a Molecular Devices Spectramax i3x spectrophotometer was used. The microplate was scanned without the lid utilizing the Spectral Optimization Wizard that is included in the Softmax Pro 6.4.2 accompaniment software (Sunnyvale, CA, USA). For the cy5.5-ZnO NP Images were acquired at the indicated excitation wavelength (Ex 678 nm) and emission range (Em 694 nm). The device was set to read the fluorescence endpoints of unknown wavelengths. The photomultiplier (PMT) gain was set to high, flashes per read was six, and wavelength increment was 5 nm. Before the first read, the microplate is shaken at medium intensity in a linear mode. The microplate was read from the top at a height of 1 mm. The ranges of excitation and emission wavelengths were set to 250-830 nm and 270-850 nm, respectively. PMCs (1 mg/ml) were spiked into tissue slurry /homogenate and then placed into BRAND® 96- well black bottomed plates (CAT# 781668). 200 pi of PMC/tissue slurry /homogenate was assayed via SpectraMax i3x by Molecular Devices (San Jose, CA, USA) for 2- Dimensional Fluorescence Difference Spectroscopy (2D-FDS). For NP-Luc 2-D FDS bioluminescent readings, an all-black, FB brand 96-well titer plate was loaded with 200 mΐ of NP (FeZnS and MnZnS) in a 1: 1 vol/vol solution. For the enzyme:NP conjugate, Luc was added (10 mΐ) with 200 mΐ of NP and spun down at 140 RPM for one minute and re-suspended and added to the microtiter plate. The wavelength settings were set to unknown, spin before read, and optimization settings were set at excitation (250-830 nm) and emission (270-850 nm). [0037] 3D Spheroid Culture of Caco2 cells: Human Caco2 cells (ATCC^®, passage 30) were seeded onto a 35 mm sterile glass-bottomed cell culture dish (FluoroDish™-World Precision Instruments) to form 3D spheroids in a thin layer of 10% Matrigel (Coming® Matrigel® Basement Membrane Matrix, LDEV-free). Culture medium was comprised of IX Minimum Essential Media (MEM, L-glutamine free), 10% Fetal bovine serum, 1% L-glutamine, and 1% Pen/Strep. Caco2 spheroids were in culture for approximately 24-hours prior to imaging. Delivery of ZnO-PEG- Cy5.5 (ZnOCy5.5) nanoparticles (NPs) to human Caco2 spheroids: Immediately prior to delivery, ZnOCy5.5 NPs were diluted to a stock concentration in Ham’s F12 medium (160 pg/mL) sonicated for 60 seconds at room temperature (Fisher Scientific 60 Sonic Dismembrator Model F60 Cell Disrupter). Caco2 Matrigel-embedded spheroids were exposed to 20 pg/mL of ZnOCy5.5 NPs in culture media overnight in a 5% C0₂ humidified incubator at 37 °C. Post-treatment (16-hours), the Caco2 cells were evaluated for NP uptake by confocal microscopy using a FluoView FV1000 Inverted Confocal Microscope. Images were acquired with a 60X oil objective and the following laser settings (CHS1: Cy5.5, 795v, lx Gain, 7% offset, laser 635 (1%), TDl:235v, lx Gain, 0% offset, laser 488 (11%). Images were imported into SlideBook Version 5.0 (SB 5.0.0.14 5/13/2010) for presentation. 3-D tumor spheroids were formed onto Insphero plates using HeLa cells per manufacturer’s recommendation and exposed to nanoparticle bound cy 5.5 -nucleic acid as previously described and imaged directly in the plate on the Licor Pearl Trilogy imaging system.

[0038] Ex-Vivo imaging: Mouse specimens were provided by Comparative Medicine Group. Lung sections were removed and were evenly divided into 2 sections. Ex-vivo imaging was performed in the Pearl® Trilogy Bioimaging system. 1 mg/ml of MnZnSe was diluted to a 1:3 with HPLC water and injected into individual sample and then imaged under white light, 700, and 800 nm filter, with 85 pm resolution and “0” focus. Increasing volumes (pi) were injected (1-20) with increasing fluorescent output. Tissue slurry /homogenate preparation consisted of heart, liver, kidney, brain, spleen, and lung from three different mice. Tissues were weighed on the XS204 Mettler Toledo (Columbus, OH, USA) analytical balance. Sectioned samples 100 mg per ml were then placed in sterile 10% PBS buffer, and homogenized via Vibra-Cell Processor VCX 130 (Newton, CT, USA) for 2 minutes, with 10 second pulses and 5 seconds rest. Slurry composition contained stromal tissue homogenized in with the sample (liver and kidney); homogenate composition had stroma removed via centrifugation and removal of supernatant to a separate tube (lung, heart, small intestine, liver, kidney, and spleen).

[0039] B-Galactosidase (b-Gal ) inhibition assays: b-Galactosidase (//- Gal ) was diluted to a 1 mg/kg solution in spectral grade ¾0. b -Gal substrate was diluted down into ten 10 mg/kg aliquots in spectral grade ¾0 and re-suspended into a 1 mg/kg solution for experimentation. Fluorometric readings were taken on the Synergy HI (Winooski, VT, USA) in BRAND 96-well black plates, clear flat bottom. Settings for Synergy HI testing set to Fluorescence Spectrum, fixed excitation 360 nm and emission 400 nm to 430 nm in 5 nm steps. Read height 7 mm at 37°C with a 5 second linear shake. Spectral readings were taken at 0, 5, 15, 30, 45, and 60 minutes. An optimization assay was performed in advance to establish optimal wavelength criteria. Bioluminescence and fluorescent readings were taken on PerkinElmer (Caliper) LifeSciences IVIS Lumina II imager. Fluorescent readings were set to 60 second exposure, medium binning, 1 F/stop, dsRed emission filter, and intensity viewed through Rainbow setting. 200 pi of NPs (FeZnS and 3% MnZnS) ± luciferase were added to an all-black FB brand 9-well microtiter plate in PBS solution (1:1 vol/vol). Nanomaterials were incubated with b-Gal in a 2: 1 enzyme: substrate ratio (200 and 100 pg/ml respectively at a NP concentration of 1, 2, 5, 10, 50, 100, 200 and 400 pg/ml dose. Time course measurements looking at enzymemanoparticle interaction were taken at 0, 10, 30, and 60 minutes. The dose-dependent assay utilized 1 mg of each NP was weighed out on aXS204 Mettler Toledo (Columbus, OH, USA) analytical balance, placed into Eppendorf tubes, and made into a 1 mg/ml suspension with PBS buffer stock solution. Each well had a total volume of 200 mΐ. Bio-fluorescence detection was performed by the SpectraMax i3x by Molecular Devices (San Jose, CA, USA) at 1, 10, 30, and 60 minutes. [0040] Virus mitigation assay: A North American genotype-2 porcine reproductive and respiratory syndrome virus infectious clone containing green fluorescence protein (PRRSV-GFP) was utilized for in vitro anti-viral activity experiments. PRRSV-GFP was propagated and titrated on MARC- 145 cells derived from African green monkey kidney cells. Assays to determine the antiviral activity of MnZnS nanoparticles (NP) were similar to previous work investigating the in vitro efficacy of fatty acid and formaldehyde-based additives on reducing the titer of African swine fever virus. Dilutions of MnZnS NP (100 pg/ml, 50 pg/ml, 20 pg/ml, 10 pg/ml) were prepared in Minimum Essential Medium (Coming^® Eagle's MEM; Fisher Scientific) supplemented with fetal bovine serum, antibiotics, and anti-mycotics. Each dilution of MnZnS NPs was mixed with an equal volume of PRRSV-GFP (titer 10⁶ 50% tissue culture infectious dose per ml, TCIDso/ml) for testing anti-viral activity. Positive controls included PRRSV-GFP mixed with an equal volume of MEM. Cytotoxicity controls included MnZnS NPs mixed with an equal volume of MEM. Ten fold serial dilutions of each NP/virus mixture or control were prepared in triplicate and added to confluent monolayers of MARC-145 cells in a 96-well tissue culture plate. Cells were incubated at 37°C in 5% CO2 for 3 days prior to examination of cells for fluorescence under an inverted microscope. PRRSV titers (TCIDso/ml) were calculated using the method of Spearman and Karber. Mean titers were determined using results from a minimum of 3 independent replicates performed on different days.

[0041] Anticancer activity: Scratch assays were conducted by standard procedures, briefly, B16F10 were plated and scratched with glass Pasteur pipettes treated with poly I:C, nanoparticles or complexes as previously described (DeLong RK, Mitchell JA, Morris RT, el al, Enzyme and Cancer Cell Selectivity of Nanoparticles: Inhibition of 3D Metastatic Phenotype and Experimental Melanoma by Zinc Oxide. J Biomed Nanotechnol . 13(2), 221-31 (2017)) and light microscope images were obtained on an Olympus CKX41 inverted microscope. Spheroids were cultured and exposed to nanoparticles as above (Ni/ZnO was used here) intravital stained using live/dead green/red stain (Invitrogen Corp/Thermofisher) per manufacturers recommendations and imaged on the confocal microscopy in the K-State CVM confocal microscopy core facility (https://www.k-state.edu/cobre/confocal_core/). Briefly, the canine mucosal melanoma cells (M5) were assayed after Ni/ZnO treatment by ERK/AKT RT-PCR or by MTT assay using procedures similar to those described above.

[0042] Targeted delivery: Mouse tumors isolated from two previous studies (Meghana Ramani, Miranda C Mudge, R Tyler Morris el al, Zinc Oxide Nanoparticle-Poly I:C RNA Complexes: Implication as Therapeutics against Experimental Melanoma. Mol Pharm, 14(3), 614-625 (2017) and DeLong RK, Mitchell JA, Morris RT, et al, Enzyme and Cancer Cell Selectivity of Nanoparticles: Inhibition of 3D Metastatic Phenotype and Experimental Melanoma by Zinc Oxide. J Biomed Nanotechnol. 13(2), 221-31 (2017)) were stored frozen at -79-80 °C. Samples were thawed on ice, 20 mg samples were lysed and the proteins extracted (2-3 tumors were pooled) and standardized to A280 (Molecular Devices Spectramax i3x, Sunnyvale, CA, USA). Slides were incubated with Cy3-Streptavidin (Sigma Aldrich, St. Louis, MO, USA), dried by centrifugation and stored under dark conditions and imaged using Molecular Devices Genepix 4000B (Sunnyvale, CA, USA). For the circos high throughput tumor proteomics analysis, in total we conducted experiments for approximately 150 unique proteins with 12 independent readings/protein. The data arrays represent feature pixel intensities wavelength (532 nm) based on mean and standard deviation analysis reflecting the relative abundance of all proteins. The high throughput tumor proteomics data were analyzed to identify the most over-expressed targets in the tumor at-the-time-of-metastasis. The network plot shows the associations found using model selection by cross validation for control cancer data against experimental data. Cohen’s d analysis was used for the cross-validation procedure. Instead of testing the relationships for each pair of proteins that requires numerous hypothesis testings such as Bonferroni correction multiple comparisons methods, we employed Markov random fields defined here: Given n = 12 independent and identically distributed samples (readings)

where X^(L> is a p- dimensional vector of the expression changes (p = 150) of proteins in ith sample; we reconstruct the underlying network G defined by the set of proteins V corresponding to p random variables, and weighted edges E representing the conditional dependencies between the expression changes of proteins associated these linkages. This network is known as Markov random field and represents non-zero joint dependencies between expression changes of all protein pairs given changes of others. Markov networks follow pairwise Markov property: if there is no edge between random variables A, B e V, they are conditionally independent, i.e., X_A 1 X_B\X\_A,B . associated with melanoma tumor using the Markov network. Delivery procedures were also conducted as previously described using the same instrumentation with B16F10 cells or A375 cells. Fluorescent microscopy was also conducted on an Olympus 1X73 inverted microscope within a poly-D-lysine coated 8 chamber slide, cells inoculum density (5x10⁴) after o/n adherence exposed to 20 ug/ml NP with/without cy5.5-ASO versus cy5.5-ASO control (200 nM) control and imaged in the Texas Red/Rhodamine filter/ channel 48 hours after treatment. Similarly an equivalent concentration of LL37 (SEQ ID NO. 2) (20 ug/ml) was complexed to nanoparticle and treated as above. Into a 6 well culture plate, 50,000 cells were inoculated per well and allowed to adhere overnight, in some cases each well contained a cover slide for microscopy analysis. Alternatively, after rinsing with PBS, the cells are trypsinized to remove any surface bound material and analyzed for cellular fluorescence by flow cytometry (K-State VDL core lab).

[0043] Results

[0044] I. IN VIVO DISTRIBUTION AND TOLERANCE.

[0045] To stabilize and track the ZnO NP in vivo, PEG and Cy5.5 dye functionalization was carried out. The surface charge of the NPs was negative as shown by the zeta potential graph before and after PEG functionalization. The hydrodynamic size of ZnO NPs in water was 300 ±6 nm with 0.49 polydispersity index (PDI) before PEG functionalization while the size of the PEG-coated ZnO NPs decreases significantly to 200±30 nm with PDI 0.1 and fluorescence intensity was stable after incubation in serum for one week (Fig. 6). Tissue distribution of ZnO or cy5.5-ZnO by fluorescence, ICP-MS and in vivo whole animal luminescence imaging was compared. Although the cy5.5-ZnO was quenched, distribution into the liver and kidneys could clearly be seen by whole body imaging immediately after intravenous administration. 5 hours after tail vein injection, animals were humanely sacrificed, the tissues obtained and subjected to ex vivo imaging in comparison to sham animals injected with PBS as controls (Fig. 7A). After background subtraction of untreated animal tissue, for fluorescence or ICP-MS zinc analysis, both techniques were quite comparable, enabling the direct quantification of distribution into these tissues which was in the order; liver > spleen > > kidney > lung > heart ~ brain, in units of relative fluorescence (RFU) or nanogram (ng) zinc on a per mass tissue basis (Fig. 7).

[0046] Significantly, Fig. 7 shows ZnO NPs or their cy5.5 fluorophore conjugate distributed in the liver, kidney, spleen, lungs and brain tissue. Imaging the whole animal immediately after cy5.5-ZnO injection, distribution into the liver and kidney could clearly be seen. However, 5 hours later when these tissues were removed and imaged ex vivo, the fluorescence intensity of tissues from the cy5.5-ZnO treated was only marginally higher than for ZnO NPs, likely because of the quenching of the cy5.5-ZnO (shown in the dot blot inset panel d). Signal in all tissues including the liver was above that of the background from the sham PBS injected animals. With a single 2 mg/kg dose, the 1700-1800 ng of zinc seen in the liver (as quantified by ICP-MS after background subtraction) represents approximately 9-10% of the injected dose (ID) which is in excellent agreement with a previous study determined by radiotracer analysis. The relative fluorescence determined by fluorescence spectroscopy per mass tissue, after subtracting background fluorescence, also compares quite well with the zinc amount by ICP-MS. Interestingly, 5 hours after administration, about one third to one half of either the zinc or fluorescence signal in liver was present in the spleen, kidney and lung. These data suggest that as much as 4 to 6% of the injected dose (ID) is available in these key organs. 5 hours after administration, blood cell count or serum protein was unchanged, as shown in Table 2. Similarly, 3 days after administration no tissue damage was observed relative to control PBS injected mice or reference standards as can be seen in Fig. 7B, in the histopathology analysis of these tissues. Fluorescence and zinc measured in the brain was above background as shown in Fig. 7 A and 7C.

Table 2

[0047] II. Zn-BASED PHYSIOMETACOMPOSITES (PMC).

[0048] Fig. 8 summarizes the physiometacomposite (PMC) nanoparticles compared in this study, namely ZnO, ZnS or ZnSe doped with manganese, cobalt, nickel, or iron either as binary (2-component), ternary (3- component) or quaternary (4-component) systems (Fig. 8).

[0049] As shown in Fig. 8, PMC nanoparticles synthesized were less than 200 nm and nanorod shaped the typical size and morphology for this synthetic method. The example represents high-level composite material containing ZnO, Fe and Co (Table 3) as shown by transmission electron microscopy (Fig. 8C) and Nanosight analysis (Fig. 8C). TEM images of other PMCs are not shown, a limitation of the study. As expected, the magnesium-doped ZnO (5%MgZnO) were quite toxic, but the iron, nickel or manganese doped nanoparticles were surprisingly biocompatible with as much as 80-90% viability after treating NIH3T3 cells at 25 micrgr/ml concentration for 48 hours. It should be noted here, 5%MgZnO and MnZnSe were made by microwave- based syntheses described by our collaborators and for all others a solid-phase physico chemical scalable method was used.

Table 3

[0050] III. PHOTOPHYSICAL PROPERTIES.

[0051] Next, to evaluate imaging potential, zinc sulfide (ZnS) or zinc selenide (ZnSe) nanoparticles doped with iron (Fe) or manganese (Mn) at a similar percentage (3%) as they were for Co and Ni were characterized. The fluorescence excitation, emission and intensity of the PMC nanoparticles and their bioluminescence as complexes with Luciferase were characterized in PBS or in serum containing media, liver and tumor homogenates and slurries of the other key organs (kidney, spleen, lung and brain) (Fig. 9).

[0052] Importantly Fig. 9 shows the optimal fluorescence excitation, emission and intensity for each of the PMC nanoparticles before and after spiking into tissue homogenates and slurries. Depending on the tissue, the fluorescent yield ex vivo ranged from 9.9 to 57-fold above background (Table 4). Unquenchable fluorescent yield of the PMC nanoparticles in serum, liver and tumor homogenate is shown in Fig. 9A. MnZnS and FeZnS-Luciferase complexes greatly red-shifted the bioluminescence (675-695 nm) amplifying the signal to 10⁴-10⁵ relative light units above background (Fig. 9B). In the absence of these nanoparticles enzyme only bioluminescence would be expected at 545-562 nm (data not shown). Whereas control untreated 3-D organoids showed no background fluorescence in the red channel, tissues exposed to cy5.5-ZnO NPs clearly showed uptake by confocal microscopy overlaid with brightfield (Fig. 9C). Similarly, ZnO NPs complexed to cy5.5-fluorophore tagged poly I:C RNA clearly labeled 3-D tumor spheroids (Fig. 9D). PMC nanoparticles showed some tissue- specificity. For example, MnZnSe fluorescence shifts in the kidney slurry and the MnZnS fluorescence was enhanced in the liver homogenate (Table 4). Dose-dependent signal intensity was also enhanced in the ex vivo lung tissue, especially for MnZnSe (Fig. 9E). A dose-dependent increase in relative fluorescence intensity after injecting MnZnS or MnZnSe was also seen in the ex vivo slices of the brain and kidney (data not shown).

[0053] IV. BIOCHEMICAL AND ANTIVIRAL ACTIVITY

[0054] The antimicrobial activity of ZnO NPs was previously correlated with its biomimetic inhibition of beta-galactosidase enzyme (b-Gal). We screened the activity of the PMC nanoparticles against b-Gal by developing a fluorescence-based enzyme assay to measure its activity. Our results show, Ni/ZnO and MnZnS gave a 2-log or 3-log order level of inhibition of the enzyme respectively. Although ZnO NP has shown some ability to inhibit flu virus infectivity in cell culture the activity of the MnZnS against porcine reproductive respiratory virus (PRRSV), considered a safer surrogate model coronavirus to SARS-CoV-2, has never been explored. Therefore, we evaluated its activity against PRRSV which does not require BSL-III/IV conditions to test its antiviral activity (Fig. 10).

[0055] Fig. 10 shows significant b-Gal enzyme inhibition, with Ni/ZnO and MnZnS PMC nanoparticles giving a 2-log or 3 -log order inhibition respectively in comparison to ZnO NP or silver (Ag) nanoparticle controls. Dose- response (Fig. 10A) inset) was similar for both Luciferase and b-Gal enzymes with an LD50 generally between 20 to 50 mg/ml. In the virus mitigation assay, no cytotoxicity of MARC-145 cells was observed for any of the MnZnS NP concentrations tested (100 pg/ml, 50 pg/ml, 20 pg/ml, 10 pg/ml). Anti-viral activity assays revealed a dose- dependent reduction in PRRSV-GFP titer post-exposure to MnZnS NP (Fig. 10B). Exposure of PRRSV-GFP to concentrations of MnZnS NP from 10 pg/ml to 50 pg/ml resulted in similar reductions to virus titer (approximately 0.5 logio TCIDso/ml) compared to the untreated positive control samples. However, exposure of PRRSV- GFP to 100 pg/ml MnZnS NP resulted in an approximate 88.6% or 1.0 logio TCIDso/ml reduction in virus concentration relative to the positive control; 4.9 logio TCIDso/ml after 100 pg/ml NP exposure compared to 5.9 logio TCIDso/ml for the untreated control. No NP concentration tested in the current experiment reduced the virus titer below the level of detection on cell culture.

[0056] V. ANTICANCER ACTIVITY

[0057] ZnO NP has been shown to inhibit both mouse and human melanoma cells in culture. Next ZnO NP inhibition in the scratch cell invasion assay and antitumor activity in 3-D spheroid assay was compared to Ni/ZnO PMC nanoparticle. The nanoparticles effect on ERK and AKT expression associated with drug resistant cancer and on canine mucosal melanoma a comparative oncology model cell line was tested (Fig. 11). [0058] As shown in Fig. 11, ZnO NP or PMC NP (Ni/ZnO) were able to inhibit melanoma cell invasion in the scratch assay. Untreated cells or those treated with the positive control poly I:C RNA caused the cells to migrate into the space left by the scratch. In stark contrast, cells which had been exposed to ZnO NP or NiZnO PMC (Fig. 11B) did not migrate to fill the space. Intravital staining of tumor spheroids showed a small population of dead cells in the interior of the spheroid. When treated with NiZnO PMC the spheroids broke apart as shown by light microscopy and the dead cells which stained red in the confocal image were clearly increased (Fig. 11C) consistent with previous data which showed uptake of the ZnO NP into the 3-D tissues. Given the potent anti-migration and anti-tumor activity of the Ni/ZnO we also investigated its effect on ERK and AKT expression, markers most often associated with melanoma, brain, and other drug-resistant cancers. The RT-PCR assay suggested > 90% inhibition of both ERK and AKT. Ni/ZnO nanoparticles also exhibited significant anticancer activity against drug-resistant canine mucosal melanoma (M5) considered an excellent comparative oncology model (Fig. 11E).

[0059] VI. TARGETED DELIVERY

[0060] High throughput proteomics analysis of syngeneic immuno competent B16F10-BALB/C tumor model was conducted. Associations between the RAS pathway and its downstream targets ERK and AKT often associated with cancer drug-resistance and the BCL-apoptosis related targets was seen. To address this, antisense oligomer (ASO) sequences targeting BCL-xL [21] or Ras binding domain (RBD) were compared and nanoparticle-mediated uptake of the cy5.5-conjugated ASO into B16F10, human melanoma (A375) or another RAS-dependent cell (132N1) a brain cancer line was studied by flow cytometry, confocal and inhibition assays (Fig. 12).

[0061] As shown in Fig. 12, the proteomics pattern of the B16F10/BALB-C tumor implicated RAS/ERK/AKT and BCL-related pathways (Fig. 12A). Initially, functional ASO delivery of first generation physiological metal oxide NPs was investigated (Table 5). [0062] Table 5

[0063] ASO targeting either RBD or BCL-xL significantly inhibited (14-19% viability) the growth of mouse and human melanoma and brain cancer cells in culture (Table 6).

Table 6

NAT (5’-3’) Target % viability

Doxorubicin (positive control) Non-specific 14 +- 3.1 (B16F10)

GAGGACAGTG/ gtgagtcagt RBD 19% +/- 1.2 (A375)

(exon 3/Intron 4) 15.2 +/- 0.8 (132N1)

TGGTTCTTACCCAGCCGCCG BCL-xL 14.9 +- 0.9 (B16F10)

[0064] NPs were able to increase ASO uptake and intracellular delivery using a cy5.5-labeled ASO as previously described shown by flow cytometry (Fig. 12C) and confocal fluorescence microscopy. ZnO NPs delivered ASO to the cytosol whereas Co-based nanoparticles delivered it to the nucleus (Fig. 12D). The data clearly show the effect of the NP-conjugation on the uptake of the fluorescently- labelled ASO. As a plausible mechanism for anticancer activity, unlabeled ASO complementary to the RBD exon3/intron 4 alternative splice junction was shown to correct splicing in the targeted site with intron4 excluded by RT-PCR (Fig. 12F). The RBD target was also validated by delivery of the protein decoy as shown in (Fig. 13). LL37 peptide (SEQ ID NO. 2) has been used in clinical trials against drug-resistant melanoma, and the data show its complexation by zeta potential (Fig. 14) and gel shift (Fig. 15) to Ni/ZnO increases anticancer activity with > 60% cytotoxicity (Fig. 12E). Similarly, complexation to RAS-targeted ASO improves anticancer activity of the ZnO NP. Interestingly no anticancer activity is observed for MnZnS nanoparticle unless complexed to the RBD-targeted aptamer (Fig. 12E).

[0065] Discussion

[0066] In this study, a zinc-based composite nanoparticle series was expanded from oxide to sulfide and selenide to include manganese-doped materials. Their fluorescence characteristics, biocompatibility, delivery, enzyme inhibition, antiviral and anticancer activity was studied. Previous pharmacokinetic studies with radiolabeled zinc oxide have showed that silica coating increased protein corona, causing greater uptake into the liver and reducing the half-life compared to naked ZnO NP. PEG-modified red fluorescent ZnO-NP radiolabeled with copper (⁶⁴Cu) show similar blood kinetics. Importantly in that study, a distinguishing feature of Zn-based nanoparticles is that significant localization occurs in extrahepatic tissues, for example distribution into spleen, lung and kidney (5-10% ID) similar to our results, albeit in tumor-bearing nude mice. Unfortunately, however, the cy5.5-ZnO exhibited fluorescence quenching, limiting its utility as an in vivo fluorescence probe, yet the relative fluorescence levels in these tissues was comparable to the levels of zinc measured by ICP-MS analysis after background subtraction, further supporting our biodistribution data. 1-2% ID into the brain seen at 5 hours appears to be somewhat unique, and merits investigation at longer time points.

[0067] Nanoparticle composites or metamaterials have unique photo physical properties. The Mirkin group was the first to synthesize the precious metal composite series doped with cobalt and nickel. However, the biocompatibility, fluorescence and delivery characteristics of these nanoparticles had not yet been reported. Early highly fluorescent quantum dot materials were zinc sulfide (ZnS)-based, but doped with toxic non-physiological metals such as lead or cadmium, limiting their biological utility. Importantly here, manganese zinc sulfide (MnZnS) and selenide (MnZnSe) show both excellent biocompatibility and fluorescence characteristics. Despite quenching of the cy5.5-ZnO NP, its uptake into 3-D tissue organoids could be readily observed by confocal microscopy. By contrast when incubated in ex vivo serum, liver, kidney, lung and tumor homogenates, Co/ZnO, Ni/ZnO and especially MnZnSe PMC nanoparticles were less prone to fluorescence quenching.

[0068] Nanoparticles inhibit enzyme activity, and in the case of ZnO NPs of similar size and shape to the PMCs used here, its inhibition of Beta- Galactosidase (b-Gal) had been previously correlated with antimicrobial activity. Our results show that ZnO NP does give 2-log b-Gal inhibition, with Ni/ZnO and Co/ZnO showing comparable activity. Yet surprisingly the MnZnS nanoparticles gave >3-log enzyme inhibition. Recently some antiviral activity of zinc oxide nanoparticle or surface-coated materials has been reported. Whereas antimicrobial and anticancer activity of ZnO NP had been previously reported, its antiviral mechanism is unknown. In addition to exploiting the nickel and manganese sulfides or selenide compositions as luminescent cell and tissue probes, it is intriguing to speculate that antiviral activity of Zn-based nanoparticles may be due to key protein and/or enzyme inhibition. Interestingly, whereas ZnO NPs did not inhibit PRSSV infection, the MnZnS did give one log inhibition. The mechanism for this is currently under investigation.

[0069] In the scratch assay, sub-IC50 dose was used (20 microgr/ml), but we cannot rule out an anti-proliferative effect rather than invasion inhibition. Indeed, the increased zone of killing in the intravital stained tumor spheroid assays as a function of Ni/ZnO NP treatment also at 20 mg/ml suggests growth inhibition effects may predominate. Antisense oligomer (ASO) and aptamer have been clinically approved and a number of oligonucleotide conjugates are under clinical evaluation. ASO targeting alternatively spliced Bcl-x(L) has shown antitumor activity in a mouse B16F10 model. To add mechanistic insight, we performed high throughput proteomics analysis of this tumor, associating the RAS pathway and its downstream effectors ERK and AKT often activated in cancer drug resistance, and BCL-apoptosis related targets. Our results show activity of BCL-xL or Ras binding domain (RBD) targeted ASO were comparable against all three RAS-dependent lines, B16F10, human melanoma (A375) and 132N1 brain cancer (Fig. 12). Mechanism of melanoma drug resistance has been associated with alternatively splicing of RBD exon 3/intron4, and evidence of splicing correction in this target site was observed by RT-PCR (Fig. 12C). Finally, activity of ASO and aptamer delivery by ZnO, Ni/ZnO or MnZnS was compared against the standard B16F 10 melanoma line. Unlike ZnO or Ni/ZnO, the MnZnS nanoparticle itself showed no anticancer activity in the absence of the RBD-targeted aptamer. However, the activity of both the clinically tested control peptide, LL37 (SEQ ID NO. 2), and the RAS-targeted ASO could be enhanced by complexation to Ni/ZnO or ZnO respectively.

[0070] Conclusion

[0071] To summarize the key results of the disclosure, Zn-based conjugates, either ZnO NP or the cy5.5 derivative are shown to distribute into liver, kidney, lung, spleen and brain, based on comparative fluorescence and ICP-MS analysis. A 2 mg/kg dosage after a single intravenous administration of ZnO NP or cy5.5-ZnO NP was well-tolerated based on blood cell counts and tissue histopathology after 5 hours or 3 days. Fluorescence-enhancement could be achieved by synthesis of manganese or iron doped zinc sulfide or selenide (MnZnS, FeZnS, MnZnSe) and these physiometacomposite (PMC) nanoparticles could be applied as fluorescent probes in these tissues ex vivo including kidney, lung and brain, and were not limited by fluorescence quenching in serum, liver or tumor homogenate. One biocompatible composition, MnZnS inhibited b-Gal enzyme activity by more than three log orders and also inhibited PRSSV infection in cell culture. The chemical composition of Ni/ZnO, ZnO, or MnZnS being quite distinct, these three materials exhibited marked differences in anti cancer activity which could be improved by complexation to anti cancer peptide, antisense or aptamer oligomers. Taken together the data suggest the clinical potential of the PMC nanoparticles, as conjugates with nucleic acid therapeutics, anticancer and antiviral peptides. Future work will explore the role of PMCs/MnZnS against SARS- CoV-2.

Claims

WHAT IS CLAIMED IS:

1. A composition for treating cancer or a viral infection comprising a zinc-based physiometacomposite (PMC).

2. The composition of claim 1, wherein the PMC includes ZnS.

3. The composition of claim 2, wherein the PMC further comprises manganese (Mn), iron (Fe), nickel (Ni), cobalt ferrite (CoFe), or any combination thereof.

4. The composition of claim 1, wherein the PMC includes or is combined or complexed with a peptide.

5. The composition of claim 4, wherein the peptide has anti-cancer or anti-microbial properties.

6. The composition of claim 4, wherein the peptide includes SEQ ID NO. 2.

7. The composition of claim 4, wherein the peptide is an antisense oligomer (ASO) or aptamer.

8. The composition of claim 4, wherein the ASO or aptamer is targeted to a specific domain or organ.

9. The composition of claim 8, wherein the organ is selected from the group consisting of liver, spleen, kidney, lung, brain, or any combination thereof.

10. The composition of claim 8, wherein the domain is a particular protein segment.

11. The composition of claim 10, wherein the particular protein segment is RAS/RBD or a spike protein.

12. The composition of claim 11, wherein the segment includes a sequence selected from the group consisting of SEQ ID NO. 3 or SEQ ID NO. 4.

13. A method of treating a viral infection comprising the step of administering the composition of claim 1 to a subject in need thereof.

14. The method of claim 13, wherein the administration of the composition reduces the severity, duration, or incidence of at least one clinical sign of the viral infection.

15. The method of claim 14, wherein the severity, duration, or incidence of the at least one clinical sign of the viral infection is reduced by at least 10%.

16. The method of claim 13, wherein the administration is oral, parenteral, topical, or via infusion.

17. The method of claim 13, wherein the subject in need thereof is selected from the group consisting of a human, dog, cat, bird, cow, pig, sheep, goat, or horse.

18. The method of claim 17, wherein when the subject in need thereof is a human, the viral infection is caused by a virus selected from the group consisting of Adeno- associated virus; Aichi virus; Australian bat lyssavirus; BK polyomavirus; Banna virus; Barmah forest Virus; Bunyamwera virus; Bunyavirus La Crosse; Bunyavirus snowshoe hare; Cercopithecine herpesvirus; Chandipura virus; Chikungunya virus; Cosavirus A; Cowpox virus; Coxsackievirus; Crimean-Congo hemorrhagic fever virus; Dengue virus; Dhori virus; Dugbe virus; Duvenhage virus; Eastern equine encephalitis virus; Ebolavirus; Echovirus; Encephalomyocarditis virus; Epstein-Barr virus; European bat lyssavirus; GB virus C/Hepatitis G virus; Hantaan virus; Hendra virus; Hepatitis A virus; Hepatitis B virus; Hepatitis C virus; Hepatitis E virus; Hepatitis delta virus; Horsepox virus; Human adenovirus; Human astrovirus; Human coronavirus; Human cytomegalovirus; Human enterovirus; Human herpesvirus 1; Human herpesvirus 2; Human herpesvirus 6; Human herpesvirus 7; Human herpesvirus 8; Human immunodeficiency virus; Human papillomavirus 1; Human papillomavirus 2; Human papillomavirus; Human parainfluenza; Human parvovirus B19; Human respiratory syncytial virus; Human rhinovirus; Human SARS coronavirus; Human spumaretrovirus; Human T-lymphotropic virus; Human torovirus; Influenza A virus; Influenza B virus; Influenza C virus; Isfahan virus; JC polyomavirus; Japanese encephalitis virus; Junin arenavirus; KI Polyomavirus;

Kunjin virus; Lagos bat virus; Lake Victoria Marburgvirus; Langat virus; Lassa virus; Lordsdale virus; Louping ill virus; Lymphocytic choriomeningitis virus; Machupo virus; Mayaro virus; MERS coronavirus; Measles virus; Mengo encephalomyocarditis virus; Merkel cell polyomavirus; Mokola virus; Molluscum contagiosum virus; Monkeypox virus; Mumps virus; Murray valley encephalitis virus; New York virus; Nipah virus; Norwalk virus; O'nyong-nyong virus; Orf virus; .Oropouche virus; Pichinde virus; Poliovirus; Punta toro phlebovirus; Puumala virus; Rabies virus; Rift valley fever virus; Rosavirus A; Ross river virus; Rotavirus A; Rotavirus B; Rotavirus C; Rubella virus; Sagiyama virus; Salivirus A; Sandfly fever Sicilian virus; Sapporo virus; SARS coronavirus 2; Semliki forest virus; Seoul virus; Simian foamy virus; Simian virus 5; Sindbis virus; Southampton virus; St. louis encephalitis virus; Tick- borne powassan virus; Torque teno virus; Toscana virus; Uukuniemi virus; Vaccinia virus; Varicella-zoster virus; Variola virus; Venezuelan equine encephalitis virus; Vesicular stomatitis virus; Western equine encephalitis virus; WU polyomavirus; West Nile virus; Yaba monkey tumor virus; Yaba-like disease virus; Yellow fever virus; Zika virus; and any combination thereof.

19. The method of claim 17, wherein when the subject in need thereof is a swine or pig, the viral infection is caused by a virus selected from the group consisting of Adenovirus; African Swine Fever Virus, Alphavirus such as Eastern equine encephalomyelitis viruses; Classical swine fever virus; Coronavirus, Porcine Respiratory Corona virus; Hemagglutinating encephalomyelitis virus; Japanese Encephalitis Virus; Porcine Circovirus; Porcine cytomegalovirus; Porcine Parvovirus; Porcine Reproductive and Respiratory Syndrome (PRRS) Virus; Pseudorabies virus; Rotavirus; Swine herpes virus; Swine Influenza Virus; Swine pox virus; Vesicular stomatitis virus; Virus of vesicular exanthema of swine; porcine epidemic diarrhea virus (PEDV); foot and mouth disease virus (FMDV); porcine enteroviruses; porcine toroviruses (PToV); porcine sapelovirus (PSV); porcine bocavirus (PBoV); porcine kobuvirus (PKBV); porcine Torque teno sus virus (TTSuV); Atypical Porcine Pestivirus; Linda virus; Mapuera Virus; Turisops Trucatus Parainfluenzavirus 1 (PIV1); Severe Acute Diarrhea Syndrome Coronavirus; Swine Enteric Alphacoronavirus; Seneca Valley Virus; Influenza virus D;

Parainfluenzavirus; Parainfluenzavirus 5; Nipah virus; Swine vesicular disease virus; Transmissible gastroenteritis virus; and any combination thereof.

20. The method of claim 17, wherein when the subject in need thereof is a cow or cattle, the viral infection is caused by a virus selected from the group consisting of Infectious Bovine Rhinotracheitis (IBR) virus; Bovine Virus Diarrhea (BVD) Types 1 and 2; Parainfluenza 3 (PI3) virus; Bovine Respiratory Syncytial Virus (BRSV); Bovine Herpesvirus; Bovine Leukemia Virus; Lumpky Skin Disease Virus; Allerton Virus; Bovine Mammilitis Virus; Infectious Bovine Keratoconjunctivitis Virus; Maligbnant Catarrhal Fever Virus; Pseudorabies Virus; Bovine Papilloma Virus; Bovine Papular Stomatitis Virus; Cowpox Virus; Paravaccinia Virus; Rift Valley Fever Virus; Rinderpest Virus; Enterovirus; Rhinovirus; Encephalomyocarditis Virus; Reovirus; Pseudorabies virus; Bluetongue virus; Japanese encephalitis virus; Rabies virus; Vesicular stomatitis virus; West Nile fever virus; and any combination thereof.

21. The method of claim 17, wherein when the subject in need thereof is a dog or canine, the viral infection is caused by a virus selected from the group consisting of Canine Influenza; Morbillivirus; Canine Parvovirus; Norovirus; Astrovirus; Adenovirus; Parainfluenzavirus; Reovirus; Rotavirus, Flavivirus; Wesselsbron Virus; Poxvirus; Herpesvirus; Orbivirus; Calicivirus; Coronavirus; Pseudorabies; Phlebovirus; and any combination thereof.

22. The method of claim 17, wherein when the subject in need thereof is a cat or feline, the viral infection is caused by a virus selected from the group consisting of Feline Immunodeficiency Virus; Feline Coronavirus; Feline Leukemia Virus; Feline Panleukopenia Virus; Feline Calicivirus; Feline Herpesvirus; Rabies; Feline Infectious Peritonitis; and any combination thereof.

23. The method of claim 17, wherein when the subject in need thereof is a sheep or goat, the viral infection is caused by a virus selected from the group consisting of Caprine arthritis and encephalitis virus; Sheeppox virus; Goatpox virus; and any combination thereof.

24. The method of claim 17, wherein when the subject in need thereof is a horse or equine, the viral infection is caused by a virus selected from the group consisting of African horse sickness virus; Eastern equine encephalomyelitis virus; Western equine encephalomyelitis virus; Equine infectious anemia virus; Equine influenza virus; Equine herpesvirus 4; Equine arteritis virus; Venezuelan equine encephalomyelitis virus; West Nile Virus; Rabies; and any combination thereof.

25. The method of claim 17, wherein when the subject in need thereof is a bird or avian species, the viral infection is caused by a virus selected from the group consisting of Avian infectious bronchitis virus; Infectious laryngotracheitis virus; Duck hepatitis virus; High and low pathogenic avian influenza viruses; Marek’s disease virus; Newcastle disease virus; Avian metapneumo virus; Avian Polyomavirus; Avian Bomavirus; West Nile Virus; Herpesvirus; Psittacine circovirus; Poxvirus; Paramyxovirus; and any combination thereof.

26. A method of treating cancer comprising the step of administering the composition of claim 1 to a subject in need thereof.

27. The method of claim 26, wherein the cancer is drug-resistant.

28. The method of claim 26, wherein the cancer is a melanoma or brain cancer.

29. The method of claim 26, wherein the administration of the composition reduces the severity, duration, or incidence of at least one clinical sign of the cancer.

30. The method of claim 29, wherein the severity, duration, or incidence of the at least one clinical sign of the cancer is reduced by at least 10%.

31. The method of claim 26, wherein the administration is oral, parenteral, topical, or via infusion.

32. The method of claim 26, wherein the subject in need thereof is selected from the group consisting of a human, dog, cat, bird, cow, pig, sheep, goat, or horse.