WO2022204714A1 - Composites de physio-nanocomposites à base de zinc et leurs méthodes d'utilisation - Google Patents

Composites de physio-nanocomposites à base de zinc et leurs méthodes d'utilisation Download PDF

Info

Publication number
WO2022204714A1
WO2022204714A1 PCT/US2022/071334 US2022071334W WO2022204714A1 WO 2022204714 A1 WO2022204714 A1 WO 2022204714A1 US 2022071334 W US2022071334 W US 2022071334W WO 2022204714 A1 WO2022204714 A1 WO 2022204714A1
Authority
WO
WIPO (PCT)
Prior art keywords
virus
human
zno
composition
porcine
Prior art date
Application number
PCT/US2022/071334
Other languages
English (en)
Inventor
Robert Delong
Megan NIEDERWERDER
Original Assignee
Kansas State University Research Foundation
Priority date (The priority date is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the date listed.)
Filing date
Publication date
Application filed by Kansas State University Research Foundation filed Critical Kansas State University Research Foundation
Publication of WO2022204714A1 publication Critical patent/WO2022204714A1/fr

Links

Classifications

    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P29/00—Non-central analgesic, antipyretic or antiinflammatory agents, e.g. antirheumatic agents; Non-steroidal antiinflammatory drugs [NSAID]
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K33/00—Medicinal preparations containing inorganic active ingredients
    • A61K33/24—Heavy metals; Compounds thereof
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K33/00—Medicinal preparations containing inorganic active ingredients
    • A61K33/24—Heavy metals; Compounds thereof
    • A61K33/26—Iron; Compounds thereof
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K33/00—Medicinal preparations containing inorganic active ingredients
    • A61K33/24—Heavy metals; Compounds thereof
    • A61K33/30—Zinc; Compounds thereof
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K33/00—Medicinal preparations containing inorganic active ingredients
    • A61K33/24—Heavy metals; Compounds thereof
    • A61K33/32—Manganese; Compounds thereof
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K38/00—Medicinal preparations containing peptides
    • A61K38/16—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
    • A61K38/17—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans
    • A61K38/1703—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans from vertebrates
    • A61K38/1709—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans from vertebrates from mammals
    • A61K38/1729—Cationic antimicrobial peptides, e.g. defensins
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P31/00—Antiinfectives, i.e. antibiotics, antiseptics, chemotherapeutics
    • A61P31/12—Antivirals
    • A61P31/14—Antivirals for RNA viruses
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P35/00—Antineoplastic agents
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N15/00—Mutation or genetic engineering; DNA or RNA concerning genetic engineering, vectors, e.g. plasmids, or their isolation, preparation or purification; Use of hosts therefor
    • C12N15/09—Recombinant DNA-technology
    • C12N15/11—DNA or RNA fragments; Modified forms thereof; Non-coding nucleic acids having a biological activity
    • C12N15/111—General methods applicable to biologically active non-coding nucleic acids
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N15/00—Mutation or genetic engineering; DNA or RNA concerning genetic engineering, vectors, e.g. plasmids, or their isolation, preparation or purification; Use of hosts therefor
    • C12N15/09—Recombinant DNA-technology
    • C12N15/11—DNA or RNA fragments; Modified forms thereof; Non-coding nucleic acids having a biological activity
    • C12N15/113—Non-coding nucleic acids modulating the expression of genes, e.g. antisense oligonucleotides; Antisense DNA or RNA; Triplex- forming oligonucleotides; Catalytic nucleic acids, e.g. ribozymes; Nucleic acids used in co-suppression or gene silencing
    • C12N15/1135—Non-coding nucleic acids modulating the expression of genes, e.g. antisense oligonucleotides; Antisense DNA or RNA; Triplex- forming oligonucleotides; Catalytic nucleic acids, e.g. ribozymes; Nucleic acids used in co-suppression or gene silencing against oncogenes or tumor suppressor genes
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N2310/00—Structure or type of the nucleic acid
    • C12N2310/10—Type of nucleic acid
    • C12N2310/11—Antisense
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N2310/00—Structure or type of the nucleic acid
    • C12N2310/10—Type of nucleic acid
    • C12N2310/17—Immunomodulatory nucleic acids
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N2310/00—Structure or type of the nucleic acid
    • C12N2310/30—Chemical structure
    • C12N2310/35—Nature of the modification
    • C12N2310/351—Conjugate
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N2320/00—Applications; Uses
    • C12N2320/30—Special therapeutic applications
    • C12N2320/32—Special delivery means, e.g. tissue-specific

Definitions

  • the field of the invention relates generally to the antiviral and anticancer mechanisms of zinc-based physiometacomposite complexes.
  • ZnO NPs zinc oxide nanoparticles
  • the distribution, tolerance, and anticancer/antiviral activity of Zn-based physiometacomposites 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.
  • 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.”
  • a separate component such as an element
  • 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.
  • the present disclosure provides a nanoparticle.
  • the nanoparticle is ZnS, MnZnS, or FeZnS.
  • the nanoparticle is doped with or combined with an element.
  • the element is selected from the group consisting of manganese, iron, nickel, cobalt and any combination thereof.
  • the nanoparticle is combined with or complexed with a protein or peptide.
  • 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.
  • the nanoparticle delivery targets a specific domain or organ.
  • the organ is selected from the group consisting of liver, spleen, kidney, lung, brain, or any combination thereof.
  • the domain is a particular protein segment.
  • the segment is RAS/RBD or a spike protein.
  • the sequence is selected from the group consisting of SEQ ID NO. 3 or SEQ ID NO. 4.
  • MnZnS manganese
  • Fe iron
  • Ni nickel
  • CoFe cobalt ferrite
  • ZnO doped oxide
  • PMC physiometacomposites
  • Biocompatibility and photophysical properties of the PMC series was investigated with the MnZnS and MnZnSe nanoparticles yielding promising results.
  • MnZnS showed dose-dependent inhibition of beta-galactosidase (b-Gal) activity and significant antiviral activity against porcine reproductive respiratory virus (PRRSV).
  • Ni/ZnO nickel-doped zinc oxide
  • RBD Ras binding domain
  • RAS 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.
  • compositions comprising ZnO-based physiometacomposite (PMC) nanoparticles.
  • PMC nanoparticles are combined or doped with cobalt, magnesium, manganese, iron, nickel, cobalt ferrite, oxide, or any combination thereof.
  • 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.
  • the doped zinc-based PMC nanoparticles are further formed into amino or amido-conjugates.
  • the amino or amido- conjugates are with ASO.
  • any of the above PMCs can be delivered into cells.
  • the above PMCs are administered to a subject in need thereof.
  • the administration is as described herein.
  • the administration is systemic.
  • the administration is via injection or infusion.
  • a PMC composition described herein is used to treat or prevent cancer or infection with or clinical signs or symptoms caused by a virus.
  • the disclosure provides a method for administering a nanoparticle as described herein to a subject in need thereof.
  • the administration targets a desired body part or organ.
  • the organ is selected from the group consisting of liver, spleen, kidney and lung with heart and brain.
  • the administration is systemic.
  • the administration is via any conventional route including injection and/or intravenously.
  • 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.
  • the composition administered includes nanoscale physiometacomposite (PMC) materials containing zinc oxide, sulfide, or selenide doped with manganese, iron, nickel or cobalt.
  • PMC nanoscale physiometacomposite
  • the MnZnS and especially MnZnSe achieved two-three log order improvement in fluorescence approaching the red range for emission.
  • 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.
  • 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).
  • PRRSV porcine reproductive respiratory virus
  • the present disclosure provides compositions for treating viral infections or cancer. Due to the antiviral activity of the compositions, any virus can be treated.
  • the composition comprises a PMC nanoparticle disclosed herein.
  • 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.
  • the virus is one that infects an animal. In some forms, the animal is a mammal or a bird.
  • the animal is a human, dog, cat, bird, cow, pig, sheep, goat, or horse.
  • 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
  • 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.
  • 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);
  • 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
  • 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.
  • 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.
  • 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.
  • 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.
  • 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.
  • the composition is administered to an animal in need thereof in an amount effective for inhibiting viral infection or cancer.
  • 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.
  • 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.
  • adjuvants 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.
  • mannide e.g. anhydromannitol oleate
  • glycol of poly glycerol
  • propylene glycol and of oleic isostearic, ricinoleic or hydroxystearic acid, which are optionally ethoxylated
  • polyoxypropylene- polyoxyethylene copolymer blocks in particular the Pluronic products, especially L121.
  • 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.
  • Carbopol 974P, 934P and 97 IP there may be mentioned Carbopol 974P, 934P and 97 IP.
  • 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.
  • 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.
  • 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.
  • 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.
  • 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.
  • 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.
  • compositions herein may incorporate known injectable, physiologically acceptable, sterile solutions.
  • aqueous isotonic solutions such as e.g. saline or corresponding plasma protein solutions are readily available.
  • 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.
  • the composition of the present disclosure further comprises a pharmaceutical acceptable salt, preferably a phosphate salt in physiologically acceptable concentrations.
  • a pharmaceutical acceptable salt preferably a phosphate salt in physiologically acceptable concentrations.
  • the pH of said composition is adjusted to a physiological pH, meaning between about 6.5 and 7.5.
  • compositions described herein can further include one or more other immunomodulatory agents such as, e. g., interleukins, interferons, or other cytokines.
  • immunomodulatory agents such as, e. g., interleukins, interferons, or other cytokines.
  • compositions described herein can further include an immune stimulant.
  • immune stimulant any immune stimulant known to a person skilled in the art can also be used.
  • Immuno stimulant 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.
  • 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.
  • 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.
  • 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.
  • 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.
  • said method also includes the administration of an immune stimulant.
  • said immune stimulant shall be given at least twice.
  • 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.
  • 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.
  • Immunune 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.
  • 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.
  • 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.
  • the present composition 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.
  • 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.
  • 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.
  • EDTA ethylenediamine tetraacetic acid
  • 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.
  • the composition further includes an antimicrobial or anti-cancer peptide.
  • Ni/ZnO PMC inhibits melanoma cell invasion and ERK and AKT expression, two markers often associated with drug-resistant cancers.
  • 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.
  • compositions of the disclosure are used to treat cancer.
  • the composition is combined with a peptide.
  • the peptide is known to have anticancer activity.
  • the cancer is melanoma or brain cancer.
  • the peptide is LL337 (SEQ ID NO. 2).
  • the cancer is drug-resistant.
  • the composition is combined with an ASO or aptamer.
  • the ASO or aptamer targets RAS or RBD.
  • 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;
  • 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;
  • Fig. 3 is a 2D graph illustrating the fluorescence shift of NP- proteimRNA tripartite complexes formed with Zn-based nanomaterials
  • 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;
  • 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;
  • 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
  • the bottom panel is a graph illustrating the stability data when incubated in serum-containing Media (10% FBS/DMEM);
  • 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;
  • Fig. 7B is a set of photographs illustrating histopathological analysis of the corresponding mice when sacrificed after 3 days;
  • 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;
  • Fig. 7D is a dot blot of the free cy5.5 dye or cy5.5-ZnO showing fluorescence quenching of the conjugate (D);
  • 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;
  • 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
  • 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;
  • 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;
  • 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;
  • 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;
  • 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;
  • 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;
  • 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;
  • 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;
  • Fig. 11 A is a schematic representation of the scratch assay and 3-D tumor spheroid studies
  • 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;
  • Fig. llC is a set of photographs illustrating a tumor spheroid assay showing inhibition of NiZnO nanoparticle composite
  • Fig. 1 ID is a graph illustrating ERK/AKT expression as a function of NiZnO treatment
  • Fig. 11E is a graph illustrating the percent cytotoxicity of NiZnO treatment to M5 canine mucosal melanoma cells
  • Fig. 12A is a Circos plot of B16F10-B ALB/C tumor protein analysis associating RAS/ERK/AKT and BCL pathways;
  • Fig. 12B is a graph illustrating ZnO, NiO, C03O4 NP delivery of cy5.5-ASO into B16F10 cells by flow cytometry;
  • Fig. 12C is a graph and a photograph of a gel illustrating RT- PCR of exon3/intron4 correction by ASO in A375 cells;
  • Fig. 12D is a set of photographs illustrating ZnO or C03O4 NP delivery of cy5.5-ASO into A375 cells shown by confocal microscopy;
  • 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;
  • 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;
  • 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
  • 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.
  • DSC Differential scanning calorimetry
  • 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.
  • 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.
  • ZP zeta potential
  • 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.
  • Zinc oxide nanoparticle increases RNA melting temperature:
  • 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.
  • 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).
  • 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.
  • 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.
  • MSN was surface-functionalized prior to RNA loading which protected the RNA and protamine was not used in these experiments.
  • 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.
  • 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).
  • the ZnO- protamine-RNA formulations when stored as a dry powder, 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.
  • 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.
  • 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.
  • 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.
  • 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).
  • NPs and RNA were precipitated from 70% alcohol/H 2 0 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, >10xl0 10 (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 3 ⁇ 40.
  • 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 3 ⁇ 40 and re-suspended into a 1 mg/kg solution for experimentation.
  • mice 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.
  • the concentration of zinc (Zn) in the mouse tissues was determined using ICP-MS analysis following the standard protocol.
  • 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.
  • Mouse 1 Liver and kidneys
  • Mouse 2 brain, heart, lungs, spleen and kidneys
  • 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).
  • 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.
  • MTT cytotoxicity
  • 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.
  • 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.
  • PMT photomultiplier
  • the microplate 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).
  • NP-Luc 2-D FDS bioluminescent readings an all-black, FB brand 96-well titer plate was loaded with 200 m ⁇ of NP (FeZnS and MnZnS) in a 1: 1 vol/vol solution.
  • NP conjugate Luc was added (10 m ⁇ ) with 200 m ⁇ 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).
  • 3D Spheroid Culture of Caco2 cells Human Caco2 cells (ATCC ® , passage 30) were seeded onto a 35 mm sterile glass-bottomed cell culture dish (FluoroDishTM-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.
  • MEM Minimum Essential Media
  • 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% C0 2 humidified incubator at 37 °C.
  • 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.
  • 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).
  • B-Galactosidase (b-Gal ) inhibition assays b-Galactosidase (//- Gal ) was diluted to a 1 mg/kg solution in spectral grade 3 ⁇ 40.
  • b -Gal substrate was diluted down into ten 10 mg/kg aliquots in spectral grade 3 ⁇ 40 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.
  • 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.
  • 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.
  • MnZnS NPs Each dilution of MnZnS NPs was mixed with an equal volume of PRRSV-GFP (titer 10 6 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.
  • 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.
  • 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.
  • 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.
  • 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 (5x10 4 ) 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.
  • an equivalent concentration of LL37 (SEQ ID NO. 2) (20 ug/ml) was complexed to nanoparticle and treated as above.
  • 50,000 cells were inoculated per well and allowed to adhere overnight, in some cases each well contained a cover slide for microscopy analysis.
  • the cells are trypsinized to remove any surface bound material and analyzed for cellular fluorescence by flow cytometry (K-State VDL core lab).
  • 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.
  • the 1700-1800 ng of zinc seen in the liver 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.
  • 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.
  • 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).
  • 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.
  • 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.
  • 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.
  • ZnS zinc sulfide
  • ZnSe zinc selenide
  • Fe iron
  • Mn manganese
  • 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.
  • 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 10 4 -10 5 relative light units above background (Fig. 9B).
  • ZnO NPs The antimicrobial activity of ZnO NPs was previously correlated with its biomimetic inhibition of beta-galactosidase enzyme (b-Gal).
  • b-Gal beta-galactosidase enzyme
  • Ni/ZnO and MnZnS gave a 2-log or 3-log order level of inhibition of the enzyme respectively.
  • PRRSV porcine reproductive respiratory virus
  • 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.
  • 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.
  • ZnO NP has been shown to inhibit both mouse and human melanoma cells in culture.
  • 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).
  • 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.
  • cells which had been exposed to ZnO NP or NiZnO PMC 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.
  • 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.
  • Ni/ZnO Ni/ZnO nanoparticles also exhibited significant anticancer activity against drug-resistant canine mucosal melanoma (M5) considered an excellent comparative oncology model (Fig. 11E).
  • 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.
  • 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).
  • complexation to RAS-targeted ASO improves anticancer activity of the ZnO NP.
  • no anticancer activity is observed for MnZnS nanoparticle unless complexed to the RBD-targeted aptamer (Fig. 12E).
  • 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.
  • 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.
  • ZnS zinc sulfide
  • MnZnS manganese zinc sulfide
  • MnZnSe selenide
  • 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.
  • b-Gal Beta- Galactosidase
  • Our results show that ZnO NP does give 2-log b-Gal inhibition, with Ni/ZnO and Co/ZnO showing comparable activity.
  • 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.
  • 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.
  • MnZnS inhibited b-Gal enzyme activity by more than three log orders and also inhibited PRSSV infection in cell culture.
  • 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.
  • 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.

Landscapes

  • Health & Medical Sciences (AREA)
  • Life Sciences & Earth Sciences (AREA)
  • Chemical & Material Sciences (AREA)
  • General Health & Medical Sciences (AREA)
  • Veterinary Medicine (AREA)
  • Public Health (AREA)
  • Medicinal Chemistry (AREA)
  • Pharmacology & Pharmacy (AREA)
  • Animal Behavior & Ethology (AREA)
  • Engineering & Computer Science (AREA)
  • Epidemiology (AREA)
  • Organic Chemistry (AREA)
  • Genetics & Genomics (AREA)
  • Biomedical Technology (AREA)
  • Inorganic Chemistry (AREA)
  • Chemical Kinetics & Catalysis (AREA)
  • General Chemical & Material Sciences (AREA)
  • Bioinformatics & Cheminformatics (AREA)
  • Zoology (AREA)
  • Molecular Biology (AREA)
  • Nuclear Medicine, Radiotherapy & Molecular Imaging (AREA)
  • Wood Science & Technology (AREA)
  • Virology (AREA)
  • Biotechnology (AREA)
  • General Engineering & Computer Science (AREA)
  • Communicable Diseases (AREA)
  • Pharmaceuticals Containing Other Organic And Inorganic Compounds (AREA)
  • Pain & Pain Management (AREA)
  • Marine Sciences & Fisheries (AREA)
  • Biochemistry (AREA)
  • Rheumatology (AREA)
  • Proteomics, Peptides & Aminoacids (AREA)
  • Gastroenterology & Hepatology (AREA)
  • Plant Pathology (AREA)
  • Immunology (AREA)
  • Biophysics (AREA)
  • Physics & Mathematics (AREA)
  • Oncology (AREA)
  • Microbiology (AREA)

Abstract

La présente divulgation concerne des compositions à base de ZnS de traitement du cancer et d'infections virales. Les compositions de ZnS peuvent, en outre, comprendre du manganèse ou du fer et/ou être dopées avec du manganèse (Mn), du Fer (Fe), du nickel (Ni), du cobalt (Co), de la ferrite de cobalt (CoFe) et toute combinaison de ceux-ci.
PCT/US2022/071334 2021-03-24 2022-03-24 Composites de physio-nanocomposites à base de zinc et leurs méthodes d'utilisation WO2022204714A1 (fr)

Applications Claiming Priority (2)

Application Number Priority Date Filing Date Title
US202163200720P 2021-03-24 2021-03-24
US63/200,720 2021-03-24

Publications (1)

Publication Number Publication Date
WO2022204714A1 true WO2022204714A1 (fr) 2022-09-29

Family

ID=83396108

Family Applications (1)

Application Number Title Priority Date Filing Date
PCT/US2022/071334 WO2022204714A1 (fr) 2021-03-24 2022-03-24 Composites de physio-nanocomposites à base de zinc et leurs méthodes d'utilisation

Country Status (1)

Country Link
WO (1) WO2022204714A1 (fr)

Citations (3)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO1999050403A2 (fr) * 1998-03-27 1999-10-07 Ribozyme Pharmaceuticals, Inc. Procede et reactifs pour le traitement de maladies ou d'affections associees a des molecules impliquees dans les reactions angiogeniques
WO2011022350A1 (fr) * 2009-08-17 2011-02-24 Saint Louis Unversity Nanoparticules de zinc pour le traitement d’infections et du cancer
WO2019169219A1 (fr) * 2018-03-02 2019-09-06 Ionis Pharmaceuticals, Inc. Modulateurs de l'expression d'irf4

Patent Citations (3)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO1999050403A2 (fr) * 1998-03-27 1999-10-07 Ribozyme Pharmaceuticals, Inc. Procede et reactifs pour le traitement de maladies ou d'affections associees a des molecules impliquees dans les reactions angiogeniques
WO2011022350A1 (fr) * 2009-08-17 2011-02-24 Saint Louis Unversity Nanoparticules de zinc pour le traitement d’infections et du cancer
WO2019169219A1 (fr) * 2018-03-02 2019-09-06 Ionis Pharmaceuticals, Inc. Modulateurs de l'expression d'irf4

Non-Patent Citations (2)

* Cited by examiner, † Cited by third party
Title
MATHEW ELZA NEELIMA: "Investigating the biophysical, biochemical, and biological activity of anti-cancer zinc oxide nanoparticle and its physiometacomposite (PMC) nanoparticles.", DOCTORAL DISSERTATION, 1 January 2020 (2020-01-01), XP055976739, Retrieved from the Internet <URL:https://krex.k-state.edu/dspace/bitstream/handle/2097/40927/ElzaMathew2020.pdf?sequence=5&isAllowed=y> [retrieved on 20221101] *
TAVAKOLI, A ET AL.: "Polyethylene glycol-coated zinc oxide nanoparticie: an efficient nanoweapon to fight against herpes simplex virus type 1.", NANOMEDICINE, vol. 13, no. 21, 22 October 2018 (2018-10-22), pages 2675 - 2690, DOI: 10.2217/nnm-2018-0089 *

Similar Documents

Publication Publication Date Title
Kerry et al. Nano-based approach to combat emerging viral (NIPAH virus) infection
Huang et al. Magnetic virus-like nanoparticles in N. benthamiana plants: a new paradigm for environmental and agronomic biotechnological research
Gaspar et al. Bioreducible poly (2-ethyl-2-oxazoline)–PLA–PEI-SS triblock copolymer micelles for co-delivery of DNA minicircles and Doxorubicin
Son et al. Self-crosslinked human serum albumin nanocarriers for systemic delivery of polymerized siRNA to tumors
Tahara et al. Chitosan-modified poly (D, L-lactide-co-glycolide) nanospheres for improving siRNA delivery and gene-silencing effects
Boylan et al. Enhancement of airway gene transfer by DNA nanoparticles using a pH-responsive block copolymer of polyethylene glycol and poly-L-lysine
DK2591792T3 (en) COMPOSITION FOR NUCLEIC ACID RELEASE, CARRIER COMPOSITION, PHARMACEUTICAL COMPOSITION WITH COMPOSITION FOR NUCLEIC ACID RELEASE OR CARRIER COMPOSITION AND PROCEDURE FOR NUCLEAR PROCESS
Lee et al. Tumoral gene silencing by receptor-targeted combinatorial siRNA polyplexes
CN105939708A (zh) 包覆TiO2的上转换纳米颗粒均匀核壳结构及其应用
KR20100123674A (ko) 나노치료제 콜로이드성 금속 조성물 및 방법
Yang et al. The effect of quantum dot size and poly (ethylenimine) coating on the efficiency of gene delivery into human mesenchymal stem cells
CN113197880B (zh) 一种巨噬细胞外泌体膜包被仿生纳米颗粒及其制备方法和应用
Duan et al. Nanoparticle approaches against SARS-CoV-2 infection
Zhai et al. Highly fluorescent, photostable, and biocompatible silicon theranostic nanoprobes against Staphylococcus aureus infections
US20160312222A1 (en) Manganese ion coated nanoparticles for delivery of compositions into the central nervous system by nasal insufflation
Kopp et al. Delivery of the autofluorescent protein R-phycoerythrin by calcium phosphate nanoparticles into four different eukaryotic cell lines (HeLa, HEK293T, MG-63, MC3T3): Highly efficient, but leading to endolysosomal proteolysis in HeLa and MC3T3 cells
Goldshtein et al. Co-assembled Ca2+ alginate-sulfate nanoparticles for intracellular plasmid DNA delivery
Chen et al. General and mild modification of food-derived extracellular vesicles for enhanced cell targeting
Bhattarai et al. N-hexanoyl chitosan-stabilized magnetic nanoparticles: enhancement of adenoviral-mediated gene expression both in vitro and in vivo
Deng et al. A plug-and-play ratiometric pH-sensing nanoprobe for high-throughput investigation of endosomal escape
Bakrania et al. DEAE-Dextran coated paclitaxel nanoparticles act as multifunctional nano system for intranuclear delivery to triple negative breast cancer through VEGF and NOTCH1 inhibition
JP2013517297A (ja) ナノ粒子を介するがん細胞標的化送達のための方法および組成物
CN110996923B (zh) 磁性病毒粒子及其制备方法与用途
WO2022204714A1 (fr) Composites de physio-nanocomposites à base de zinc et leurs méthodes d&#39;utilisation
Yao et al. Efficient gene delivery system mediated by cis-aconitate-modified chitosan-g-stearic acid micelles

Legal Events

Date Code Title Description
121 Ep: the epo has been informed by wipo that ep was designated in this application

Ref document number: 22776840

Country of ref document: EP

Kind code of ref document: A1

NENP Non-entry into the national phase

Ref country code: DE

122 Ep: pct application non-entry in european phase

Ref document number: 22776840

Country of ref document: EP

Kind code of ref document: A1