WO2018164883A1 - Dna-templated micelle and uses thereof - Google Patents

Dna-templated micelle and uses thereof Download PDF

Info

Publication number
WO2018164883A1
WO2018164883A1 PCT/US2018/019926 US2018019926W WO2018164883A1 WO 2018164883 A1 WO2018164883 A1 WO 2018164883A1 US 2018019926 W US2018019926 W US 2018019926W WO 2018164883 A1 WO2018164883 A1 WO 2018164883A1
Authority
WO
WIPO (PCT)
Prior art keywords
micelle
dna
payload
micelle system
templated
Prior art date
Application number
PCT/US2018/019926
Other languages
French (fr)
Inventor
Kam W. Leong
Yeh-Hsing Lao
Tzu-Chieh HO
Smruthi SURYAPRAKASH
Original Assignee
The Trustees Of Columbia University In The City Of New York
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 The Trustees Of Columbia University In The City Of New York filed Critical The Trustees Of Columbia University In The City Of New York
Priority to US16/492,320 priority Critical patent/US11311629B2/en
Publication of WO2018164883A1 publication Critical patent/WO2018164883A1/en
Priority to US17/727,549 priority patent/US20220313839A1/en

Links

Classifications

    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K47/00—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient
    • A61K47/50—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates
    • A61K47/69—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates the conjugate being characterised by physical or galenical forms, e.g. emulsion, particle, inclusion complex, stent or kit
    • A61K47/6905—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates the conjugate being characterised by physical or galenical forms, e.g. emulsion, particle, inclusion complex, stent or kit the form being a colloid or an emulsion
    • A61K47/6907—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates the conjugate being characterised by physical or galenical forms, e.g. emulsion, particle, inclusion complex, stent or kit the form being a colloid or an emulsion the form being a microemulsion, nanoemulsion or micelle
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K31/00—Medicinal preparations containing organic active ingredients
    • A61K31/13—Amines
    • A61K31/135—Amines having aromatic rings, e.g. ketamine, nortriptyline
    • A61K31/136—Amines having aromatic rings, e.g. ketamine, nortriptyline having the amino group directly attached to the aromatic ring, e.g. benzeneamine
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K31/00—Medicinal preparations containing organic active ingredients
    • A61K31/33—Heterocyclic compounds
    • A61K31/395—Heterocyclic compounds having nitrogen as a ring hetero atom, e.g. guanethidine or rifamycins
    • A61K31/41—Heterocyclic compounds having nitrogen as a ring hetero atom, e.g. guanethidine or rifamycins having five-membered rings with two or more ring hetero atoms, at least one of which being nitrogen, e.g. tetrazole
    • A61K31/4164—1,3-Diazoles
    • A61K31/4178—1,3-Diazoles not condensed 1,3-diazoles and containing further heterocyclic rings, e.g. pilocarpine, nitrofurantoin
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K31/00—Medicinal preparations containing organic active ingredients
    • A61K31/70—Carbohydrates; Sugars; Derivatives thereof
    • A61K31/7088—Compounds having three or more nucleosides or nucleotides
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K31/00—Medicinal preparations containing organic active ingredients
    • A61K31/70—Carbohydrates; Sugars; Derivatives thereof
    • A61K31/7088—Compounds having three or more nucleosides or nucleotides
    • A61K31/7105—Natural ribonucleic acids, i.e. containing only riboses attached to adenine, guanine, cytosine or uracil and having 3'-5' phosphodiester links
    • 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
    • A61K38/43—Enzymes; Proenzymes; Derivatives thereof
    • A61K38/46—Hydrolases (3)
    • A61K38/465—Hydrolases (3) acting on ester bonds (3.1), e.g. lipases, ribonucleases
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K47/00—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient
    • A61K47/50—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates
    • A61K47/51—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates the non-active ingredient being a modifying agent
    • A61K47/54—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates the non-active ingredient being a modifying agent the modifying agent being an organic compound
    • A61K47/549—Sugars, nucleosides, nucleotides or nucleic acids
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K47/00—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient
    • A61K47/50—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates
    • A61K47/51—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates the non-active ingredient being a modifying agent
    • A61K47/54—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates the non-active ingredient being a modifying agent the modifying agent being an organic compound
    • A61K47/55—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates the non-active ingredient being a modifying agent the modifying agent being an organic compound the modifying agent being also a pharmacologically or therapeutically active agent, i.e. the entire conjugate being a codrug, i.e. a dimer, oligomer or polymer of pharmacologically or therapeutically active compounds
    • A61K47/551—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates the non-active ingredient being a modifying agent the modifying agent being an organic compound the modifying agent being also a pharmacologically or therapeutically active agent, i.e. the entire conjugate being a codrug, i.e. a dimer, oligomer or polymer of pharmacologically or therapeutically active compounds one of the codrug's components being a vitamin, e.g. niacinamide, vitamin B3, cobalamin, vitamin B12, folate, vitamin A or retinoic acid
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K47/00—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient
    • A61K47/50—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates
    • A61K47/51—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates the non-active ingredient being a modifying agent
    • A61K47/56—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates the non-active ingredient being a modifying agent the modifying agent being an organic macromolecular compound, e.g. an oligomeric, polymeric or dendrimeric molecule
    • A61K47/59—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates the non-active ingredient being a modifying agent the modifying agent being an organic macromolecular compound, e.g. an oligomeric, polymeric or dendrimeric molecule obtained otherwise than by reactions only involving carbon-to-carbon unsaturated bonds, e.g. polyureas or polyurethanes
    • A61K47/60—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates the non-active ingredient being a modifying agent the modifying agent being an organic macromolecular compound, e.g. an oligomeric, polymeric or dendrimeric molecule obtained otherwise than by reactions only involving carbon-to-carbon unsaturated bonds, e.g. polyureas or polyurethanes the organic macromolecular compound being a polyoxyalkylene oligomer, polymer or dendrimer, e.g. PEG, PPG, PEO or polyglycerol
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K47/00—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient
    • A61K47/50—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates
    • A61K47/51—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates the non-active ingredient being a modifying agent
    • A61K47/62—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates the non-active ingredient being a modifying agent the modifying agent being a protein, peptide or polyamino acid
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K47/00—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient
    • A61K47/50—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates
    • A61K47/51—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates the non-active ingredient being a modifying agent
    • A61K47/62—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates the non-active ingredient being a modifying agent the modifying agent being a protein, peptide or polyamino acid
    • A61K47/64—Drug-peptide, drug-protein or drug-polyamino acid conjugates, i.e. the modifying agent being a peptide, protein or polyamino acid which is covalently bonded or complexed to a therapeutically active agent
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K47/00—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient
    • A61K47/50—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates
    • A61K47/51—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates the non-active ingredient being a modifying agent
    • A61K47/68—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates the non-active ingredient being a modifying agent the modifying agent being an antibody, an immunoglobulin or a fragment thereof, e.g. an Fc-fragment
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K47/00—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient
    • A61K47/50—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates
    • A61K47/51—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates the non-active ingredient being a modifying agent
    • A61K47/68—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates the non-active ingredient being a modifying agent the modifying agent being an antibody, an immunoglobulin or a fragment thereof, e.g. an Fc-fragment
    • A61K47/6835—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates the non-active ingredient being a modifying agent the modifying agent being an antibody, an immunoglobulin or a fragment thereof, e.g. an Fc-fragment the modifying agent being an antibody or an immunoglobulin bearing at least one antigen-binding site
    • A61K47/6845—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates the non-active ingredient being a modifying agent the modifying agent being an antibody, an immunoglobulin or a fragment thereof, e.g. an Fc-fragment the modifying agent being an antibody or an immunoglobulin bearing at least one antigen-binding site the antibody targeting a cytokine, e.g. growth factors, VEGF, TNF, a lymphokine or an interferon
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K47/00—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient
    • A61K47/50—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates
    • A61K47/51—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates the non-active ingredient being a modifying agent
    • A61K47/68—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates the non-active ingredient being a modifying agent the modifying agent being an antibody, an immunoglobulin or a fragment thereof, e.g. an Fc-fragment
    • A61K47/6835—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates the non-active ingredient being a modifying agent the modifying agent being an antibody, an immunoglobulin or a fragment thereof, e.g. an Fc-fragment the modifying agent being an antibody or an immunoglobulin bearing at least one antigen-binding site
    • A61K47/6849—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates the non-active ingredient being a modifying agent the modifying agent being an antibody, an immunoglobulin or a fragment thereof, e.g. an Fc-fragment the modifying agent being an antibody or an immunoglobulin bearing at least one antigen-binding site the antibody targeting a receptor, a cell surface antigen or a cell surface determinant
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K47/00—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient
    • A61K47/50—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates
    • A61K47/51—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates the non-active ingredient being a modifying agent
    • A61K47/68—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates the non-active ingredient being a modifying agent the modifying agent being an antibody, an immunoglobulin or a fragment thereof, e.g. an Fc-fragment
    • A61K47/6835—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates the non-active ingredient being a modifying agent the modifying agent being an antibody, an immunoglobulin or a fragment thereof, e.g. an Fc-fragment the modifying agent being an antibody or an immunoglobulin bearing at least one antigen-binding site
    • A61K47/6851—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates the non-active ingredient being a modifying agent the modifying agent being an antibody, an immunoglobulin or a fragment thereof, e.g. an Fc-fragment the modifying agent being an antibody or an immunoglobulin bearing at least one antigen-binding site the antibody targeting a determinant of a tumour cell
    • A61K47/6867—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates the non-active ingredient being a modifying agent the modifying agent being an antibody, an immunoglobulin or a fragment thereof, e.g. an Fc-fragment the modifying agent being an antibody or an immunoglobulin bearing at least one antigen-binding site the antibody targeting a determinant of a tumour cell the tumour determinant being from a cell of a blood cancer
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K47/00—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient
    • A61K47/50—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates
    • A61K47/69—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates the conjugate being characterised by physical or galenical forms, e.g. emulsion, particle, inclusion complex, stent or kit
    • A61K47/6901—Conjugates being cells, cell fragments, viruses, ghosts, red blood cells or viral vectors
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K48/00—Medicinal preparations containing genetic material which is inserted into cells of the living body to treat genetic diseases; Gene therapy
    • A61K48/0008—Medicinal preparations containing genetic material which is inserted into cells of the living body to treat genetic diseases; Gene therapy characterised by an aspect of the 'non-active' part of the composition delivered, e.g. wherein such 'non-active' part is not delivered simultaneously with the 'active' part of the composition
    • A61K48/0025—Medicinal preparations containing genetic material which is inserted into cells of the living body to treat genetic diseases; Gene therapy characterised by an aspect of the 'non-active' part of the composition delivered, e.g. wherein such 'non-active' part is not delivered simultaneously with the 'active' part of the composition wherein the non-active part clearly interacts with the delivered nucleic acid
    • A61K48/0041—Medicinal preparations containing genetic material which is inserted into cells of the living body to treat genetic diseases; Gene therapy characterised by an aspect of the 'non-active' part of the composition delivered, e.g. wherein such 'non-active' part is not delivered simultaneously with the 'active' part of the composition wherein the non-active part clearly interacts with the delivered nucleic acid the non-active part being polymeric
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K9/00—Medicinal preparations characterised by special physical form
    • A61K9/10—Dispersions; Emulsions
    • A61K9/107—Emulsions ; Emulsion preconcentrates; Micelles
    • A61K9/1075—Microemulsions or submicron emulsions; Preconcentrates or solids thereof; Micelles, e.g. made of phospholipids or block copolymers
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P35/00—Antineoplastic agents
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P35/00—Antineoplastic agents
    • A61P35/02—Antineoplastic agents specific for leukemia
    • 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
    • C12N15/87—Introduction of foreign genetic material using processes not otherwise provided for, e.g. co-transformation
    • C12N15/88—Introduction of foreign genetic material using processes not otherwise provided for, e.g. co-transformation using microencapsulation, e.g. using amphiphile liposome vesicle

Definitions

  • the present invention discloses a multifunctional DNA-templated micelle system which comprises a payload carrier comprising at least a DNA bridge and a functionalized polyethylene glycol (PEG) segment.
  • the DNA-templated micelle system can be used to deliver various molecules, such as, compounds, drugs, or polynucleotides, for pharmaceutical uses.
  • Nanoparticles have been utilized for clinical applications as drug carriers.
  • the development of nano-medicines for the treatment of cancer has been done to enhance drug efficacy or to reduce adverse drug effects by targeted delivery locally, such as the delivery of chemotherapeutic drugs to the tumor site.
  • Nano-medicines provide the advantages of increasing the therapeutic efficacy of anti-cancer drugs with less delivery barriers.
  • a wide range of organic and inorganic nanoparticles with dimensions of less than several hundred nanometers have been used for cancer therapy for tumor targeting.
  • Nano-medicines utilizing nano-carriers loaded with drugs are promising in cancer treatment due to their unique physico- chemical characteristics, such as increasing solubility and stability for targeted delivery, and improving distribution of anti-cancer drugs while decreasing drug resistance of tumors.
  • nanoparticles can provide enhanced permeability and retention effect that allows for accumulation of therapeutics in tumors at a much higher concentration than that in healthy cells, which reduces the systemic toxicity of anti-cancer drugs.
  • Some nano-medicines for cancer therapy are effective in terms of improved bioavailability and tolerability, but there is a need to increase drug performance, such as to improve the capacity of drug carriers to deliver high doses of anti-cancer drugs to specific locations in the body.
  • Some stimuli responsive carriers have been developed to release drugs through internal or external stimuli, such as liposomes, polymeric micelles, nanogels and mesoporous silica nanoparticles.
  • van Elk et al. Nanomedicines for advanced cancer treatments: Transitioning towards responsive system, Int J Pharm. 2016 Oct; 515(1-2): pp. 132-164
  • Several multifunctional nano-carrier systems have been published. Kataoka et al.
  • nucleic acid encapsulating polymer micelle complex comprising a block polymer containing an uncharged hydrophilic polymer chain block, a cationic polymer chain block, and double-stranded or single-stranded DNAs.
  • a micelle composition comprising a micelle containing polyethylene glycol-phosphatidyl ethanolamine (PEG-PE), a DC-cholesterol and a dioleoylphosphatidyl-ethanolamine (DOPE) coated with either or both of a pharmaceutical compound core and a polynucleotide coating.
  • PEG-PE polyethylene glycol-phosphatidyl ethanolamine
  • DOPE dioleoylphosphatidyl-ethanolamine
  • the present invention now provides a multifunctional DNA-templated micelle system comprising a payload carrier which comprises at least a DNA bridge and a functionalized polyethylene glycol (PEG) segment.
  • the micelle system can be used to deliver molecules, such as drugs and polynucleotides, to targeted cells for pharmaceutical uses and benefits.
  • the DNA- templated micelle system can also be used as a stabilizer of nanoparticle formation.
  • the present invention provides a micelle system for delivering at least one payload to cells, the micelle system comprising at least one payload carrier, wherein the payload carrier comprises at least one DNA bridge and at least one functionalized PEG segment, wherein the functionalized PEG segment is covalently bound to the DNA bridge, wherein the DNA bridge can carry at least one payload through intercalation or nucleotide base pairing.
  • the functionalized PEG segment of the micelle system of the present invention can further conjugated to at least one ligand with a controllable ligand density in the range of from 0% to 100%.
  • the payload carrier of the micelle system can be preloaded with at least one payload prior to the formation of micelle and can be further conjugated with at least one ligand prior to the formation of micelle.
  • the functionalized PEG segment of the payload carrier can be an amine-functionalized PEG segment with the molecular weight in the range of from 1 kD to 30 kD.
  • the DNA bridge of the payload carrier can be a single-stranded DNA in the range of from 8 nucleotides to 100 nucleotides.
  • the payload of the micelle system of the present invention may be selected from the group that includes small molecules, compounds, drugs, chemodrugs, polynucleotides, DNA, genes, messenger RNA (mRNA), aptamers, and combinations thereof.
  • the ligand of micelle system of the present invention is different from the payload and may be selected from the group that includes of small molecules, peptide, antibodies, aptamers, polynucleotides, and combinations thereof.
  • the size of the micelle of the present invention is from about 5 nm to about 250 mm with the polydispersity of less than 0.1.
  • the present invention provides a method for delivering a payload to cells of a subject for disease treatment, comprising administering to the subject a pharmaceutical composition comprising the micelle system of the present invention, including the disease treatments of cell transfections, gene therapy, chemotherapy, immunotherapy, cancer treatment, disease monitoring, brain tumor chemotherapy, glioblastoma chemotherapy, or leukemia immunotherapy.
  • the present invention provides a kit for performing a cell transfection reaction, which comprises the micelle system of the present invention.
  • FIG. 1 shows a payload carrier comprising at least a DNA bridge and a functionalized- polyethylene glycol (PEG).
  • PEG polyethylene glycol
  • FIG. 2 shows the formation of a DNA-templated micelle through nano-precipitation.
  • the micelles include a payload carrier comprising at least a DNA bridge and at least a functionalized-PEG.
  • FIG. 3 shows the tunable ranges of micelle size, PEG length, and ligand density.
  • FIG. 4 shows the influence of the lengths of PEG and single-stranded DNA motif.
  • FIG. 5 shows the influence of the lengths of mRNA payloads.
  • FIG. 6 shows the particle sizes of DNA-templated micelles.
  • FIG. 7 shows the influence of ligand density using large protein ligand.
  • FIG. 8 shows controllable ligand density in the DNA-templated micelle for maximizing cellular uptake using a small chemical ligand.
  • FIG. 9 shows the results of micelle stability study.
  • FIG. 10 shows the results of cytotoxicity study of the DNA template micelles.
  • FIG. 1 1 shows the application of DNA-templated micelles in transfecting adherent cells.
  • FIG. 12 shows size tunable, highly stable DNA-templated micelles with high drug loading efficiency.
  • FIG. 13 shows specific glioblastoma (GBM) cell killing using DNA-templated micelle/MSC hybrid cluster.
  • the present invention discloses a multifunctional DNA-templated micelle system which can be used to deliver various components, such as small molecules, compounds, drugs, chemodrugs, polynucleotides, DNA, genes, messenger RNA (mRNA), aptamers or combinations thereof, to targeted cells or tumor sites for pharmaceutical uses, such as cell transfections, gene therapy, chemotherapy, immunotherapy, cancer treatment, disease monitoring, brain tumor chemotherapy, glioblastoma chemotherapy, or leukemia
  • the present invention provides a micelle system for delivering at least one payload to cells, the micelle system comprising at least one payload carrier, wherein the payload carrier comprises at least one DNA bridge and at least one functionalized polyethylene glycol (PEG) segment, wherein the functionalized PEG segment is covalently bound to the DNA bridge, wherein the DNA bridge can carry at least one payload through intercalation or nucleotide base pairing.
  • the payload carrier comprises at least one DNA bridge and at least one functionalized polyethylene glycol (PEG) segment, wherein the functionalized PEG segment is covalently bound to the DNA bridge, wherein the DNA bridge can carry at least one payload through intercalation or nucleotide base pairing.
  • PEG polyethylene glycol
  • the DNA-templated micelle system of the present invention includes a payload carrier which comprises at least a DNA bridge and a functionalized PEG segment (FIG. 1), such as an amine-functionalized PEG.
  • the payload carrier of the present invention can be used to form DNA-templated micelles through nano-precipitation (FIG. 2).
  • the formed DNA- templated micelle has low toxicity and are narrowly dispersed and colloidally stable.
  • the DNA bridge of the payload carrier contains a single-stranded DNA and serves as a delivery vehicle for various payloads (such as small molecules, compounds, drugs, chemodrugs, polynucleotides, DNA, mRNA, aptamers, or combinations thereof), including delivering drugs through intercalation, or delivering polynucleotides through Watson-Crick nucleotide base pairing.
  • payloads such as small molecules, compounds, drugs, chemodrugs, polynucleotides, DNA, mRNA, aptamers, or combinations thereof
  • payloads such as small molecules, compounds, drugs, chemodrugs, polynucleotides, DNA, mRNA, aptamers, or combinations thereof
  • payloads such as small molecules, compounds, drugs, chemodrugs, polynucleotides, DNA, mRNA, aptamers, or combinations thereof
  • delivering drugs through intercalation or delivering polynucleotides through Watson-Crick nucleotide base pairing.
  • the functionalized PEG segment can be conjugated to at least one ligand, e.g. various ligands (such as small molecule, peptide, antibody, aptamer, polynucleotide, or combinations thereof) for tumor targeting.
  • the DNA bridge of the payload carrier is a single-stranded DNA with the size of in the range of from 8 to 100 nucleotides, preferred in the range of from 20 to 40 nucleotides, and the molecular weight of the PEG segment is in the range of from 1 kD to 30 kD, preferred in the range of from 2 kD to 20 kD.
  • the DNA-templated micelle of the present invention is tunable with regards to size, ligand type, ligand density, and stability (FIG. 3), which is precisely controllable and provides the advantages of stability, specificity, and versatility.
  • the payload carrier of the micelle may or may not be conjugated to a ligand.
  • the ligand density of this particular micelle is defined as 0% ligand density.
  • the ligand density of this particular micelle is defined as 100% ligand density.
  • the versatility of the DNA-templated micelle system of the present invention can be achieved by mixing payload carriers with or without conjugated ligands in a desired ratio, from 0% to 100% of ligand density, to form micelles with various ligand densities. Therefore, the ligand density of the DNA-templated micelle of the present invention is precisely controllable for the needs of designing various cancer therapy strategies.
  • Payloads and ligands can be preloaded separately before the formation of the DNA- templated micelles.
  • the DNA-templated micelle is capable of carrying various payloads and ligands simultaneously to provide versatile strategies for cancer treatment for enhanced therapeutic index and outcome.
  • the properties of the DNA bridge can be tuned precisely to fit the requirement of various applications, such as a partial complementary to a specific polynucleotide with a desirable melting temperature. Loading efficiency and ligand density of the multifunctional DNA-templated micelles are therefore controllable.
  • the DNA-templated micelle of the present invention provides the advantages of simultaneously delivering multiple therapeutic payloads and ligands with high efficiency and low cytotoxicity to enhance the efficacy of cancer therapy.
  • the typical size of the DNA-templated micelles of the present invention ranges from 5 nm to 250 nm, preferred in the range of from 15 nm to 200 nm, depending on the sizes of the components in the micelles, such as the sizes of the DNA bridge, PEG, ligands, or loaded polynucleotides.
  • the size of PEG-DNA and the presence of PEG are critical to control the formation of micelle in nano-sized.
  • the polydispersity or heterogeneity of the DNA-templated micelles is typically lower than 0.1.
  • the present invention provides a system to transfect cells with polynucleotides, including mRNAs and small nucleic acids.
  • Polynucleotides are loaded to the DNA bridge through Watson-Crick nucleotide base pairing.
  • the loaded micelles can deliver polynucleotides to a wide spectrum of cell lines, including adherent cells and suspension cells, with 50-70% transfection efficiency and ⁇ 10% cytotoxicity.
  • the design of the payload carrier can contain a long PEG segment to reduce undesirable uptake by the normal cells, and the long PEG segment can be conjugated with a specific ligand targeting a unique type of tumor. Therefore, micelles containing such drug-loaded payload carriers can deliver drugs to and specifically kill tumors expressing the specific target of the ligand.
  • the present invention also provides a DNA-templated micelle for leukemia immunotherapy.
  • two types of the payload carriers are designed. One type is conjugated with the antibody that recognizes a leukemic stem cell biomarker, and the other type is conjugated with human Fc protein to recruit immune cells.
  • the payload carriers are loaded with drugs. Micelles containing such modified and loaded payload carrier can specifically kill cancer cells and also induce phagocytosis and antibody-dependent cell- mediated cytotoxicity of immune cells.
  • the DNA-templated micelle of the present invention can be used as a superior delivery vehicle for both drug and gene delivery. It can be a potential modality for cancer therapy as its properties are programmable and can be further customized to resolve the heterogeneous issues on cancer patients.
  • the present invention provides a method for delivering a payload to cells of a subject for disease treatment.
  • the present invention provides a kit for performing a cell transfection reaction, which comprises the micelle system of the present invention.
  • the payload carrier comprised at least an amine-functionalized PEG segment and a DNA bridge (FIG. 1) was synthesized using Click chemistry and purified by reversed-phase high performance liquid chromatography (HPLC).
  • Amine-PEG-azides were purchased from Nanocs Inc. (New York, NY) or Laysan Bio, Inc. (Arab, AL).
  • the single-stranded 5'-hexynyl- DNA was synthesized by IDT (Coralville, IA), and its molecular weight was confirmed by ESI-MS (electrospray ionization mass spectrometry).
  • reaction was carried out in 200 mM trithylamiimum buffer (pH 7) containing 50% DMSO (dimethyl sulfoxide) with single- stranded DNA (50 ⁇ ), amine-PEG-azide (250 ⁇ ), L-ascorbic acid (10 mM) and copper(II)-TBTA complex (500 ⁇ ) (TBTA is
  • the DNA-templated micelle was formed through nano- precipitation (FIG. 2).
  • the micelle was formed by adding equal volume of the Tris buffer containing 250 mM calcium chloride and 0.1 mM EDTA (pH 7.6) to the loaded/modified payload carriers in a solution (50 mM HEPES buffer with 1.3 mM sodium phosphate and 140 mM sodium chloride, pH 7.1), then mixing for 1 min at room temperature.
  • the sizes of resulted DNA-templated micelles ranged from 15 nm to 200 nm.
  • the resulted micelles had the polydispersity at lower than 0.1 and was stable in serum-containing environment, such as in complete cell culture medium.
  • a DNA library was prepared for constructing the DNA bridge of the payload carrier.
  • the payload carriers include various single-stranded DNA motifs (such as polyT, polyGC) with different melting temperatures (such as 37°C or 52°C).
  • the payload carriers also comprise various PEG segments in the range of from 2 kD to 20 kD, such as in the size of 2 kD, 5 kD, 10 kD or 20 kD.
  • the linkage between DNA and PEG was non-biodegradable or biodegradable with various termini, such as methylated, free amine, dye-conjugated or ligand- conjugated.
  • GFP green fluorescent protein
  • mRNA (1 ⁇ g) was preloaded on a payload carrier, such as PEG5-T20 (PEG length at 5 kD and single-stranded DNA length at 20 nucleotides).
  • PEG5-T20 PEG length at 5 kD and single-stranded DNA length at 20 nucleotides.
  • payload carriers with the length of PEG in the range of 5-20 kD and the length of DNA at 20 or 40 nucleotides were used in this study, including PEG5-T20, PEG10-T20, PEG20-T20, PEG5- T40, PEG10-T40 and PEG20-T40.
  • the micelle was formed through nano-precipitation.
  • TFIP1 cell was seeded in a 24- well plate in complete media without antibiotics at 4 hr prior to the transfection.
  • Cells were treated with the micelles for 48 hr, and transfection efficiency with GFP expression in the live cell population was determined by FACS (fluorescence-activated cell sorting) with DAPI (4',6-diamidino-2-phenylindole) staining.
  • FACS fluorescence-activated cell sorting
  • DAPI 4,6-diamidino-2-phenylindole
  • THP1 cells were transfected with different mRNA payloads to study the influence of the lengths of mRNA payloads.
  • GFP mRNA (1 ⁇ g, mRNA length at approximately 996 nucleotides) or Cas9-GFP mRNA (1 ⁇ g, mRNA length at approximately 5,000 nucleotides) was preloaded on the payload carrier, PEG10-T20 (PEG at 10 kD, DNA at 20 nucleotides).
  • the micelle was formed through nano-precipitation.
  • THP1 cell was seeded in a 24-well plate in complete media without antibiotics at 4 hr prior to the transfection. Cells were treated with the micelles for 48 hr, and transfection efficiency was determined by FACS with DAPI staining as shown in FIG. 5.
  • Lipofectamine Messenger Max was used as a comparison (denoted as Lipofectamine in FIG. 5).
  • the DNA-templated micelle containing PEG10-T20 payload carrier showed comparable transfection rate compared to Lipofectamine.
  • the DNA-templated micelle containing PEG10-T20 payload carrier showed exceptional higher transfection rate compared to
  • DNA templates at the length of 20 or 40 nucleotides with or without PEG were used to form micelles, such as T20 (DNA at 20 nucleotides without PEG), or PEG10-T40
  • the DNA templates used in this study including T20, PEG10-T20, PEG20-T20, T40, PEG10-T40, and PEG20-T40.
  • the micelles were formed through nano-precipitation. The sizes of the micelles were measured using Malvern ZetaSizer NS90. In the presence of PEG, the micelles have the polydispersity (PDI) of less than 0.1 with the particle sizes in the range of 100-200 nm as shown in FIG. 6.
  • the size of PEG-DNA and the presence of PEG were critical to control the formation of micelle in nano-sized with the polydispersity (PDI) of less than 0.1.
  • FIG. 7 shows the influence of ligand density, when large protein ligand, e.g. an antibody, was included in the DNA-templated micelle.
  • Anti- IL1RAP antibody (denoted as mAb in FIG. 7) was used as a large protein ligand to be conjugated to PEG5-pGC20 payload carrier.
  • AML Acute Myeloid Leukemia
  • THP1 high surface ILIRAP expression
  • K562 low surface ILIRAP expression
  • Bisantrene (BIS) a chemodrug to treat AML, was preloaded to the payload carriers prior to the formation of the micelles. Micelle were formed through nano-precipitation.
  • FIG. 8 shows controllable ligand density in the DNA-templated micelle for maximizing cellular uptake using a small chemical ligand.
  • a small chemical ligand, folate was used to optimize the delivery of a chemodrug, mitoxantrone (MTX, Sigma- Aldrich M6545), through folate-conjugated DNA-templated micelle.
  • Folate was used as a small chemical ligand to be conjugated to PEG20-T40 payload carrier.
  • CHO-K1 wild-type, folate receptor expressed
  • CHO/FR folate receptor deficient
  • PEG20-T40 payload carriers with or without folate, were mixed in a desired ratio to achieve various ligand densities in the range of 0 to 50% of folate.
  • 0% of folate indicated the absence of folate in the micelle.
  • Mitoxantrone (MTX) was preloaded to the payload carriers prior to the formation of the micelles. Micelle were formed through nano-precipitation.
  • FIG. 9 shows the results of micelle stability study.
  • PEG20-T40 payload carrier was conjugated with folate and preloaded with GFP mRNA (1 ⁇ g).
  • the micelles were formed through nano-precipitation and subsequently incubated with 450 ⁇ , of complete media (RPMI1640 with 10% FBS).
  • Example 9 Cytotoxicity study
  • FIG. 10 shows the results of cytotoxicity study of the DNA template micelles.
  • panel A indicates GFP mRNA transfection
  • panel B indicates Cas9-GFP mRNA transfection.
  • GFP mRNA or Cas9-GFP mRNA (1 ⁇ g each) was preloaded on the payload carrier.
  • the micelles were formed through nano-precipitation.
  • THPl cell was seeded in a 24-well plate in complete media without antibiotics at 4 hr prior to the transfection. Cells were treated with the micelles for 48 hr, and the cell viability was determined by FACS with DAPI staining.
  • NC negative control
  • LiMM Lipofectamine Messenger Max.
  • FIG. 1 1 shows the application of DNA-templated micelles in transfecting adherent cells, HeLa and U87MG cells.
  • GFP mRNA (1 or 2 ⁇ g) was preloaded on payload carrier (PEG5-T20). Micelles were formed through nano-precipitation.
  • HeLa and U87MG cells were seeded in a 24-well plate in complete media one day prior to the transfection. Cells were treated with the micelles for 24 hr, and transfection efficiency of GFP in the live cell population was determined as shown in FIG. 11.
  • Example 11 Application of the multifunctional DNA-templated micelles in specifically delivering drugs to brain tumors
  • the DNA bridge of the payload carrier was conjugated with a long amine-functionalized PEG segment with a molecular weight of 20 kD, and the amine-functionalized PEG segment was conjugated with a targeting peptide, Pepl, which recognizes interleukin 13 receptor alpha 2 (TL13RA2). 25% of the amino-groups on the surfaces of the DNA-templated micelles were replaced by Pepl .
  • mitoxantrone was preloaded onto the payload carrier through intercalation through a gradient-annealing process from 94 °C to 4 °C with a cooling rate at -1 °C /sec.
  • the Pepl -modified and mitoxantrone-loaded DNA-templated micelle was tested on different types of brain tumor cells with low, medium and high expression of IL13RA2, respectively. The results showed that this Pepl-modified and mitoxantrone-loaded DNA-templated micelle specifically killed the IL13RA2-expressed tumor cells.
  • Example 12 DNA-templated micelle/MSC cluster for glioblastoma therapeutics
  • Payload carriers were purified by reversed-phased HPLC using an acetonitrile/PBS (phosphate-buffered saline) mixture as an eluent.
  • the therapeutic payloads (such as chemodrugs and polynucleotides) were loaded onto the payload carrier through a gradient annealing process with a cooling rate of -l°C/min.
  • Payload-carrying DNA-templated micelles were formed via nano-precipitation. The formulation of forming this DNA-templated micelle was optimized for simultaneously loading of drug and nucleic acid.
  • the size of the loaded DNA-templated micelles was tunable and ranged from 15 nm to 150 nm with a narrow polydispersity (PDI ⁇ 0.1).
  • the micelles were colloidally stable in serum-containing environment.
  • FIG. 12 shows size tunable, highly stable DNA-templated micelles with high drug loading capacity. As shown in FIG. 12, the loading efficiency reached nearly 100 %.
  • DNA-templated micelles were mixed with an alginate-containing MSC suspension at the ratio of 1 : 1, and the micelle-MSC clusters were subsequently formed via a microfluidic technology.
  • a micelle-MSC hybrid cluster was developed for co-delivering multiple anti-cancer drugs.
  • MSCs hold tumor homing property as they express tumor-associated cytokine receptors. MSCs in the cluster form showed higher migration toward GBM cells.
  • MSCs In micelle-MSC clusters, more than 50% of the DNA-templated micelles were loaded on the extracellular matrix of the micelle-MSC cluster and were not internalized by the MSCs.
  • MSC migration was not affected by the decoration of DNA-templated micelles.
  • These DNA-templated micelles were size- tunable, colloidally stable, relatively monodispersed and low toxic, which can be integrated synergistically with other anti-cancer approaches to improve the efficacy in cancer therapy.
  • FIG. 13 shows specific glioblastoma (GBM) cell killing using DNA-templated micelle/MSC hybrid cluster.
  • the targeted, drug-preloaded micelle was formed with Pepl- conjugated DNA template (25% coverage) and mitoxantrone (MTX, Sigma-Aldrich M6545). Subsequently the micelle MSC hybrid cluster, free MTX/MSC cluster or MSC cluster was formed.
  • MSC mesenchymal stem cell
  • MTX mitoxantrone
  • NP nano- particle
  • GBM glioblastoma.
  • Example 13 Application of DNA-templated micelles in transfecting cells with mRNAs and small nucleic acids
  • the DNA bridge of the payload carrier was designed as a partial complement to a polynucleotide (as a payload of the DNA bridge of the payload carrier) with a 37 °C melting temperature.
  • Micelles containing this payload carrier was tested in a wide spectrum of cell lines, including adherent cells, such as HeLa and U87MG cells, and suspension cells, such as THP-1 cell.
  • This payload carrier was used to deliver green fluorescent protein (GFP) mRNA to these cells with 50-70% transfection efficiency which was comparable to the commercially- available transfection reagent, Lipofectamine Messenger Max (available from Thermo Fisher Scientific).
  • GFP green fluorescent protein
  • the micelle containing this payload carrier resulted in less cytotoxicity to the cells at ⁇ 10% compared to Lipofectamine at 50-70%.
  • This payload carrier also was used to transfect the hard-to-transfect cell line, THP1, with a long mRNA, such as Cas9 mRNA for CRISPR/Cas9 genome editing system, at 40% efficiency, whereas Lipofectamine reached only 5-10% efficiency with the same long mRNA.
  • Co-delivery of Cas9 mRNA and its guide RNA (gRNA) of the CRISPR/Cas9 genome editing system was also validated in a cellular uptake assay, which showed that 98-99% of the Cas9 mRNA and gRNA co-localized inside the transfected cells.
  • Example 14 Application of the multifunctional DNA-templated micelle for leukemia immunotherapy
  • Two types of payload carriers were designed. One type was conjugated with an antibody that recognizes a leukemic stem cell biomarker, and the other type was conjugated with human Fc protein that can recruit immune cells, including natural killer (NK) cells and macrophage.
  • the payload carrier was loaded with a chemodrug, bisantrene.
  • the modified and loaded DNA-templated micelle was found to not only specifically kill cancer cells, but also was able to induce phagocytosis of immune cells.

Abstract

The present invention discloses a multifunctional DNA-templated micelle system that has a payload carrier of at least a DNA bridge and a functionalized polyethylene glycol (PEG) segment. The micelle can be used to deliver molecules, such as drugs and polynucleotides, to targeted cells for pharmaceutical uses. The PEG segment provides a functional group, such as amine, for ligand conjugation. The DNA-templated micelle of the present invention is highly controllable in size, loading efficiency and tissue targeting, and can carry multiple payloads for targeted combination strategies in cancer therapy, such as gene delivery, gene therapy, and immunotherapy.

Description

DNA-TEMPLATED MICELLE AND USES THEREOF
FIELD OF THE INVENTION
The present invention discloses a multifunctional DNA-templated micelle system which comprises a payload carrier comprising at least a DNA bridge and a functionalized polyethylene glycol (PEG) segment. The DNA-templated micelle system can be used to deliver various molecules, such as, compounds, drugs, or polynucleotides, for pharmaceutical uses.
BACKGROUND OF THE INVENTION
Nanoparticles have been utilized for clinical applications as drug carriers. The development of nano-medicines for the treatment of cancer has been done to enhance drug efficacy or to reduce adverse drug effects by targeted delivery locally, such as the delivery of chemotherapeutic drugs to the tumor site. Nano-medicines provide the advantages of increasing the therapeutic efficacy of anti-cancer drugs with less delivery barriers. A wide range of organic and inorganic nanoparticles with dimensions of less than several hundred nanometers have been used for cancer therapy for tumor targeting.
The limitation of effectiveness of cancer treatment is the high toxicity of anti-cancer agents and the lack of selective delivery of drugs to the tumor site. Nano-medicines utilizing nano-carriers loaded with drugs are promising in cancer treatment due to their unique physico- chemical characteristics, such as increasing solubility and stability for targeted delivery, and improving distribution of anti-cancer drugs while decreasing drug resistance of tumors. In addition, nanoparticles can provide enhanced permeability and retention effect that allows for accumulation of therapeutics in tumors at a much higher concentration than that in healthy cells, which reduces the systemic toxicity of anti-cancer drugs.
Some nano-medicines for cancer therapy are effective in terms of improved bioavailability and tolerability, but there is a need to increase drug performance, such as to improve the capacity of drug carriers to deliver high doses of anti-cancer drugs to specific locations in the body. Some stimuli responsive carriers have been developed to release drugs through internal or external stimuli, such as liposomes, polymeric micelles, nanogels and mesoporous silica nanoparticles. (van Elk et al., Nanomedicines for advanced cancer treatments: Transitioning towards responsive system, Int J Pharm. 2016 Oct; 515(1-2): pp. 132-164) Several multifunctional nano-carrier systems have been published. Kataoka et al. (US 2016/0184457 Al, Nucleic acid-encapsulating polymer micelle complex and method for producing the same) discloses a nucleic acid encapsulating polymer micelle complex comprising a block polymer containing an uncharged hydrophilic polymer chain block, a cationic polymer chain block, and double-stranded or single-stranded DNAs. Haag et al. (US Patent No. 9, 102,595 B2, Compounds suited as nanocarriers for active agents and their use) discloses a compound suited as a nano-carrier for active agents comprising a polyglycerol core with an amine-containing residue, and further recites a nano-carrier system comprising a nucleic acid or pharmacologically active small molecules to be carried by and bound to said nano-carrier in a covalent, ionic or complexed manner. Mohapatra et al. (US 2013/0243867 Al, Micelle compositions and methods for their use) discloses a micelle composition comprising a micelle containing polyethylene glycol-phosphatidyl ethanolamine (PEG-PE), a DC-cholesterol and a dioleoylphosphatidyl-ethanolamine (DOPE) coated with either or both of a pharmaceutical compound core and a polynucleotide coating.
For cancer therapy, single drug treatment may not be sufficient to completely eliminate cancer cells at least partially due to their heterogeneity and insensitivity against the induced inhibitory signals. There are needs to develop nano-medicines to improve the therapeutic strategies against cancer to obtain sophisticated targeting strategies with multi-functionalities for synergistic cancer therapies, such as co-delivering multiple anti-cancer drugs and DNA in a controlled manner with improved pharmacokinetic profile, pharmacodynamics profile, chemical stability, solubility, controlled release, and bio-distribution of anti-cancer drugs. In addition, there remain concerns over potential toxicity of nano-particles, high heterogeneity of nanoparticles (high polydispersity) in different batches, loss of drug efficacy during encapsulation and delivery, and limitations of the scalability of nano-medicines. The present invention now addresses these needs and provides viable improvements that have not been previously disclosed in the art.
SUMMARY OF THE INVENTION
The present invention now provides a multifunctional DNA-templated micelle system comprising a payload carrier which comprises at least a DNA bridge and a functionalized polyethylene glycol (PEG) segment. The micelle system can be used to deliver molecules, such as drugs and polynucleotides, to targeted cells for pharmaceutical uses and benefits. The DNA- templated micelle system can also be used as a stabilizer of nanoparticle formation. The present invention provides a micelle system for delivering at least one payload to cells, the micelle system comprising at least one payload carrier, wherein the payload carrier comprises at least one DNA bridge and at least one functionalized PEG segment, wherein the functionalized PEG segment is covalently bound to the DNA bridge, wherein the DNA bridge can carry at least one payload through intercalation or nucleotide base pairing.
The functionalized PEG segment of the micelle system of the present invention can further conjugated to at least one ligand with a controllable ligand density in the range of from 0% to 100%. The payload carrier of the micelle system can be preloaded with at least one payload prior to the formation of micelle and can be further conjugated with at least one ligand prior to the formation of micelle. The functionalized PEG segment of the payload carrier can be an amine-functionalized PEG segment with the molecular weight in the range of from 1 kD to 30 kD. The DNA bridge of the payload carrier can be a single-stranded DNA in the range of from 8 nucleotides to 100 nucleotides.
The payload of the micelle system of the present invention may be selected from the group that includes small molecules, compounds, drugs, chemodrugs, polynucleotides, DNA, genes, messenger RNA (mRNA), aptamers, and combinations thereof. The ligand of micelle system of the present invention is different from the payload and may be selected from the group that includes of small molecules, peptide, antibodies, aptamers, polynucleotides, and combinations thereof. The size of the micelle of the present invention is from about 5 nm to about 250 mm with the polydispersity of less than 0.1.
In one aspect, the present invention provides a method for delivering a payload to cells of a subject for disease treatment, comprising administering to the subject a pharmaceutical composition comprising the micelle system of the present invention, including the disease treatments of cell transfections, gene therapy, chemotherapy, immunotherapy, cancer treatment, disease monitoring, brain tumor chemotherapy, glioblastoma chemotherapy, or leukemia immunotherapy.
In yet another aspect, the present invention provides a kit for performing a cell transfection reaction, which comprises the micelle system of the present invention.
The details of the preferred embodiments of the present invention are set forth in the accompanying figures and detailed description herein. Once these details of the invention are known, numerous additional innovations and changes will become obvious and
implementable to one of ordinary skill in the art. BRIEF DESCRIPTION OF THE FIGURES
Further features of the inventive concept, its nature and various advantages will be more apparent from the following detailed description, taken in conjunction with the accompanying figures:
FIG. 1 shows a payload carrier comprising at least a DNA bridge and a functionalized- polyethylene glycol (PEG).
FIG. 2 shows the formation of a DNA-templated micelle through nano-precipitation. The micelles include a payload carrier comprising at least a DNA bridge and at least a functionalized-PEG.
FIG. 3 shows the tunable ranges of micelle size, PEG length, and ligand density.
FIG. 4 shows the influence of the lengths of PEG and single-stranded DNA motif.
FIG. 5 shows the influence of the lengths of mRNA payloads.
FIG. 6 shows the particle sizes of DNA-templated micelles.
FIG. 7 shows the influence of ligand density using large protein ligand.
FIG. 8 shows controllable ligand density in the DNA-templated micelle for maximizing cellular uptake using a small chemical ligand.
FIG. 9 shows the results of micelle stability study.
FIG. 10 shows the results of cytotoxicity study of the DNA template micelles.
FIG. 1 1 shows the application of DNA-templated micelles in transfecting adherent cells.
FIG. 12 shows size tunable, highly stable DNA-templated micelles with high drug loading efficiency.
FIG. 13 shows specific glioblastoma (GBM) cell killing using DNA-templated micelle/MSC hybrid cluster.
DETAILED DESCRIPTION OF THE INVENTION
Throughout this description, the preferred embodiments and examples provided herein should be considered as exemplar, rather than as limitations of the present invention.
The present invention discloses a multifunctional DNA-templated micelle system which can be used to deliver various components, such as small molecules, compounds, drugs, chemodrugs, polynucleotides, DNA, genes, messenger RNA (mRNA), aptamers or combinations thereof, to targeted cells or tumor sites for pharmaceutical uses, such as cell transfections, gene therapy, chemotherapy, immunotherapy, cancer treatment, disease monitoring, brain tumor chemotherapy, glioblastoma chemotherapy, or leukemia
immunotherapy.
The present invention provides a micelle system for delivering at least one payload to cells, the micelle system comprising at least one payload carrier, wherein the payload carrier comprises at least one DNA bridge and at least one functionalized polyethylene glycol (PEG) segment, wherein the functionalized PEG segment is covalently bound to the DNA bridge, wherein the DNA bridge can carry at least one payload through intercalation or nucleotide base pairing.
The DNA-templated micelle system of the present invention includes a payload carrier which comprises at least a DNA bridge and a functionalized PEG segment (FIG. 1), such as an amine-functionalized PEG. The payload carrier of the present invention can be used to form DNA-templated micelles through nano-precipitation (FIG. 2). The formed DNA- templated micelle has low toxicity and are narrowly dispersed and colloidally stable. The DNA bridge of the payload carrier contains a single-stranded DNA and serves as a delivery vehicle for various payloads (such as small molecules, compounds, drugs, chemodrugs, polynucleotides, DNA, mRNA, aptamers, or combinations thereof), including delivering drugs through intercalation, or delivering polynucleotides through Watson-Crick nucleotide base pairing. For example, for loading a chemodrug through the DNA bridge of the payload carrier, a complementary single-stranded DNA fragment without further PEG is added to form a DNA-duplex with the DNA bridge, then the chemodrug can be loaded to the payload carrier through intercalation by inserting the chemodrug to the DNA duplex.
The functionalized PEG segment can be conjugated to at least one ligand, e.g. various ligands (such as small molecule, peptide, antibody, aptamer, polynucleotide, or combinations thereof) for tumor targeting. In one embodiment, the DNA bridge of the payload carrier is a single-stranded DNA with the size of in the range of from 8 to 100 nucleotides, preferred in the range of from 20 to 40 nucleotides, and the molecular weight of the PEG segment is in the range of from 1 kD to 30 kD, preferred in the range of from 2 kD to 20 kD.
The DNA-templated micelle of the present invention is tunable with regards to size, ligand type, ligand density, and stability (FIG. 3), which is precisely controllable and provides the advantages of stability, specificity, and versatility. The payload carrier of the micelle may or may not be conjugated to a ligand. When the micelle is formed by mixing the payload carriers without ligand, the ligand density of this particular micelle is defined as 0% ligand density. When the micelle is formed by mixing the payload carriers which are all conjugated with ligands, the ligand density of this particular micelle is defined as 100% ligand density. The versatility of the DNA-templated micelle system of the present invention can be achieved by mixing payload carriers with or without conjugated ligands in a desired ratio, from 0% to 100% of ligand density, to form micelles with various ligand densities. Therefore, the ligand density of the DNA-templated micelle of the present invention is precisely controllable for the needs of designing various cancer therapy strategies.
Payloads and ligands can be preloaded separately before the formation of the DNA- templated micelles. The DNA-templated micelle is capable of carrying various payloads and ligands simultaneously to provide versatile strategies for cancer treatment for enhanced therapeutic index and outcome. The properties of the DNA bridge can be tuned precisely to fit the requirement of various applications, such as a partial complementary to a specific polynucleotide with a desirable melting temperature. Loading efficiency and ligand density of the multifunctional DNA-templated micelles are therefore controllable. The DNA-templated micelle of the present invention provides the advantages of simultaneously delivering multiple therapeutic payloads and ligands with high efficiency and low cytotoxicity to enhance the efficacy of cancer therapy.
The typical size of the DNA-templated micelles of the present invention ranges from 5 nm to 250 nm, preferred in the range of from 15 nm to 200 nm, depending on the sizes of the components in the micelles, such as the sizes of the DNA bridge, PEG, ligands, or loaded polynucleotides. In particular, the size of PEG-DNA and the presence of PEG are critical to control the formation of micelle in nano-sized. The polydispersity or heterogeneity of the DNA-templated micelles is typically lower than 0.1.
In one aspect, the present invention provides a system to transfect cells with polynucleotides, including mRNAs and small nucleic acids. Polynucleotides are loaded to the DNA bridge through Watson-Crick nucleotide base pairing. The loaded micelles can deliver polynucleotides to a wide spectrum of cell lines, including adherent cells and suspension cells, with 50-70% transfection efficiency and <10% cytotoxicity.
In one embodiment, the design of the payload carrier can contain a long PEG segment to reduce undesirable uptake by the normal cells, and the long PEG segment can be conjugated with a specific ligand targeting a unique type of tumor. Therefore, micelles containing such drug-loaded payload carriers can deliver drugs to and specifically kill tumors expressing the specific target of the ligand.
The present invention also provides a DNA-templated micelle for leukemia immunotherapy. As an example, two types of the payload carriers are designed. One type is conjugated with the antibody that recognizes a leukemic stem cell biomarker, and the other type is conjugated with human Fc protein to recruit immune cells. In addition, the payload carriers are loaded with drugs. Micelles containing such modified and loaded payload carrier can specifically kill cancer cells and also induce phagocytosis and antibody-dependent cell- mediated cytotoxicity of immune cells.
The DNA-templated micelle of the present invention can be used as a superior delivery vehicle for both drug and gene delivery. It can be a potential modality for cancer therapy as its properties are programmable and can be further customized to resolve the heterogeneous issues on cancer patients. In addition, the present invention provides a method for delivering a payload to cells of a subject for disease treatment. In yet another aspect, the present invention provides a kit for performing a cell transfection reaction, which comprises the micelle system of the present invention.
EXAMPLE
The following examples illustrate the benefits and advantages of the present invention. The applications of the multifunctional DNA-templated micelle are shown in the examples.
Example 1. Synthesis of payload carriers and the formation of DNA-templated micelles
The payload carrier comprised at least an amine-functionalized PEG segment and a DNA bridge (FIG. 1) was synthesized using Click chemistry and purified by reversed-phase high performance liquid chromatography (HPLC). Amine-PEG-azides were purchased from Nanocs Inc. (New York, NY) or Laysan Bio, Inc. (Arab, AL). The single-stranded 5'-hexynyl- DNA was synthesized by IDT (Coralville, IA), and its molecular weight was confirmed by ESI-MS (electrospray ionization mass spectrometry). For Click chemistry, the reaction was carried out in 200 mM trithylamiimum buffer (pH 7) containing 50% DMSO (dimethyl sulfoxide) with single- stranded DNA (50 μΜ), amine-PEG-azide (250 μΜ), L-ascorbic acid (10 mM) and copper(II)-TBTA complex (500 μΜ) (TBTA is
tris(benzyltriazolylmethyl)amine; copper(II)-TBTA is a catalyst used for Click Chemistry conjugation reaction) in an argon atmosphere for overnight at room temperature. The product was purified by reversed-phase HPLC and then lyophilized.
After the payload carrier was loaded with payloads (such as chemodrugs or polynucleotides) or was conjugated with ligands (such as small molecules, peptides, antibodies, aptamers, or nucleotides), the DNA-templated micelle was formed through nano- precipitation (FIG. 2). The micelle was formed by adding equal volume of the Tris buffer containing 250 mM calcium chloride and 0.1 mM EDTA (pH 7.6) to the loaded/modified payload carriers in a solution (50 mM HEPES buffer with 1.3 mM sodium phosphate and 140 mM sodium chloride, pH 7.1), then mixing for 1 min at room temperature.
The sizes of resulted DNA-templated micelles ranged from 15 nm to 200 nm. The resulted micelles had the polydispersity at lower than 0.1 and was stable in serum-containing environment, such as in complete cell culture medium.
Example 2. Preparation of a DNA template library
A DNA library was prepared for constructing the DNA bridge of the payload carrier. The payload carriers include various single-stranded DNA motifs (such as polyT, polyGC) with different melting temperatures (such as 37°C or 52°C). The payload carriers also comprise various PEG segments in the range of from 2 kD to 20 kD, such as in the size of 2 kD, 5 kD, 10 kD or 20 kD. The linkage between DNA and PEG was non-biodegradable or biodegradable with various termini, such as methylated, free amine, dye-conjugated or ligand- conjugated.
Example 3. The influence of the lengths of PEG and single-stranded DNA motif
The influence of the lengths of PEG and single-stranded DNA motif in the performance of the DNA-templated micelle was studied as shown in FIG. 4. GFP (green fluorescent protein) mRNA (1 μg) was preloaded on a payload carrier, such as PEG5-T20 (PEG length at 5 kD and single-stranded DNA length at 20 nucleotides). Several payload carriers with the length of PEG in the range of 5-20 kD and the length of DNA at 20 or 40 nucleotides were used in this study, including PEG5-T20, PEG10-T20, PEG20-T20, PEG5- T40, PEG10-T40 and PEG20-T40.
The micelle was formed through nano-precipitation. TFIP1 cell was seeded in a 24- well plate in complete media without antibiotics at 4 hr prior to the transfection. Cells were treated with the micelles for 48 hr, and transfection efficiency with GFP expression in the live cell population was determined by FACS (fluorescence-activated cell sorting) with DAPI (4',6-diamidino-2-phenylindole) staining. Abbreviations in FIG 4: NC, negative control; LiMM, Lipofectamine Messenger Max (available from Thermo Fisher Scientific).
Example 4. The influence of the lengths of mRNA payloads
THP1 cells were transfected with different mRNA payloads to study the influence of the lengths of mRNA payloads. GFP mRNA (1 μg, mRNA length at approximately 996 nucleotides) or Cas9-GFP mRNA (1 μg, mRNA length at approximately 5,000 nucleotides) was preloaded on the payload carrier, PEG10-T20 (PEG at 10 kD, DNA at 20 nucleotides). The micelle was formed through nano-precipitation. THP1 cell was seeded in a 24-well plate in complete media without antibiotics at 4 hr prior to the transfection. Cells were treated with the micelles for 48 hr, and transfection efficiency was determined by FACS with DAPI staining as shown in FIG. 5. Lipofectamine Messenger Max was used as a comparison (denoted as Lipofectamine in FIG. 5).
As shown in FIG. 5, for transfecting mRNA at approximately 996 nucleotides (GFP mRNA), the DNA-templated micelle containing PEG10-T20 payload carrier showed comparable transfection rate compared to Lipofectamine. For transfecting large mRNA at approximately 5,000 nucleotides (Cas9-GFP mRNA), the DNA-templated micelle containing PEG10-T20 payload carrier showed exceptional higher transfection rate compared to
Lipofectamine (PEG10-T20 vs. Lipofectamine = 40% vs. 10%).
Example 5. Particle sizes of DNA-templated micelles
Several DNA templates at the length of 20 or 40 nucleotides with or without PEG were used to form micelles, such as T20 (DNA at 20 nucleotides without PEG), or PEG10-T40
(DNA at 40 nucleotides with PEG at 10 kD). The DNA templates used in this study including T20, PEG10-T20, PEG20-T20, T40, PEG10-T40, and PEG20-T40. The micelles were formed through nano-precipitation. The sizes of the micelles were measured using Malvern ZetaSizer NS90. In the presence of PEG, the micelles have the polydispersity (PDI) of less than 0.1 with the particle sizes in the range of 100-200 nm as shown in FIG. 6. The size of PEG-DNA and the presence of PEG were critical to control the formation of micelle in nano-sized with the polydispersity (PDI) of less than 0.1.
Example 6. The influence of ligand density using large protein ligand
The ligand density is controllable in the formation of the DNA-templated micelles to achieve desired chemotherapy strategies. FIG. 7 shows the influence of ligand density, when large protein ligand, e.g. an antibody, was included in the DNA-templated micelle. Anti- IL1RAP antibody (denoted as mAb in FIG. 7) was used as a large protein ligand to be conjugated to PEG5-pGC20 payload carrier.
Two types of AML (Acute Myeloid Leukemia) cells, THP1 (high surface ILIRAP expression) and K562 (low surface ILIRAP expression), were seeded in a 96 well plate 4 hr prior to the treatment. Two types of PEG5-pGC20 payload carriers, with or without anti- ILIRAP, were mixed in a desired ratio to achieve various ligand densities in the range of 0 to 100% of anti-ILlRAP. 0% of anti-ILlRAP indicated the absence of anti-ILlRAP in the micelle. Bisantrene (BIS), a chemodrug to treat AML, was preloaded to the payload carriers prior to the formation of the micelles. Micelle were formed through nano-precipitation.
THP1 and K562 cells were treated with BIS (denoted as free BIS in FIG. 7, BIS without DNA-templated micelle) or micelle formulated BIS (denoted as PEG5-pGC20MC in FIG. 7) for 48 hr (equivalent BIS concentration = 1 μΜ). Cell viability was determined by FACS with DAPI staining.
Example 7. Optimization of the ligand density using a small chemical ligand
FIG. 8 shows controllable ligand density in the DNA-templated micelle for maximizing cellular uptake using a small chemical ligand. A small chemical ligand, folate, was used to optimize the delivery of a chemodrug, mitoxantrone (MTX, Sigma- Aldrich M6545), through folate-conjugated DNA-templated micelle. Folate was used as a small chemical ligand to be conjugated to PEG20-T40 payload carrier.
Two types of CHO cells, CHO-K1 (wild-type, folate receptor expressed) and CHO/FR (folate receptor deficient), were seeded in a 96 well plate one day prior to the treatment. Two types of PEG20-T40 payload carriers, with or without folate, were mixed in a desired ratio to achieve various ligand densities in the range of 0 to 50% of folate. 0% of folate indicated the absence of folate in the micelle. Mitoxantrone (MTX), was preloaded to the payload carriers prior to the formation of the micelles. Micelle were formed through nano-precipitation.
The CHO-K1 and CHO/FR cells were treated with free MTX (MTX without micelle) or micelle-formulated MTX for 24 hr (equivalent MTX concentration = 1.5 μΜ). Cell viability was determined by MTS assay. The efficacy of delivering MTX was enhanced by optimizing the ligand (folate) density as shown in FIG. 8. The controllable ligand density in the DNA-templated micelle demonstrated the feasibility of maximizing cellular uptake.
Example 8. Micelle stability study
FIG. 9 shows the results of micelle stability study. PEG20-T40 payload carrier was conjugated with folate and preloaded with GFP mRNA (1 μg). The micelles were formed through nano-precipitation and subsequently incubated with 450 μΐ, of complete media (RPMI1640 with 10% FBS). The size of the micelle was measured using Malvern ZetaSizer NS90 in a time course. The result at each time point was normalized to that at time = 0. The micelle held a z-average diameter of 141.8 ± 10.18 nm at time = 0. Example 9. Cytotoxicity study
FIG. 10 shows the results of cytotoxicity study of the DNA template micelles. As shown in FIG. 10, panel A indicates GFP mRNA transfection, panel B indicates Cas9-GFP mRNA transfection. GFP mRNA or Cas9-GFP mRNA (1 μg each) was preloaded on the payload carrier. The micelles were formed through nano-precipitation. THPl cell was seeded in a 24-well plate in complete media without antibiotics at 4 hr prior to the transfection. Cells were treated with the micelles for 48 hr, and the cell viability was determined by FACS with DAPI staining. Abbreviations in FIG. 10: NC, negative control; LiMM, Lipofectamine Messenger Max.
Example 10. Transfection on adherent cells
FIG. 1 1 shows the application of DNA-templated micelles in transfecting adherent cells, HeLa and U87MG cells. GFP mRNA (1 or 2 μg) was preloaded on payload carrier (PEG5-T20). Micelles were formed through nano-precipitation. HeLa and U87MG cells were seeded in a 24-well plate in complete media one day prior to the transfection. Cells were treated with the micelles for 24 hr, and transfection efficiency of GFP in the live cell population was determined as shown in FIG. 11.
Example 11. Application of the multifunctional DNA-templated micelles in specifically delivering drugs to brain tumors
In order to reduce undesirable uptake of the DNA-templated micelles by normal cells, the DNA bridge of the payload carrier was conjugated with a long amine-functionalized PEG segment with a molecular weight of 20 kD, and the amine-functionalized PEG segment was conjugated with a targeting peptide, Pepl, which recognizes interleukin 13 receptor alpha 2 (TL13RA2). 25% of the amino-groups on the surfaces of the DNA-templated micelles were replaced by Pepl . For drug loading, mitoxantrone was preloaded onto the payload carrier through intercalation through a gradient-annealing process from 94 °C to 4 °C with a cooling rate at -1 °C /sec. The Pepl -modified and mitoxantrone-loaded DNA-templated micelle was tested on different types of brain tumor cells with low, medium and high expression of IL13RA2, respectively. The results showed that this Pepl-modified and mitoxantrone-loaded DNA-templated micelle specifically killed the IL13RA2-expressed tumor cells.
Example 12. DNA-templated micelle/MSC cluster for glioblastoma therapeutics
Payload carriers were purified by reversed-phased HPLC using an acetonitrile/PBS (phosphate-buffered saline) mixture as an eluent. The therapeutic payloads (such as chemodrugs and polynucleotides) were loaded onto the payload carrier through a gradient annealing process with a cooling rate of -l°C/min. Payload-carrying DNA-templated micelles were formed via nano-precipitation. The formulation of forming this DNA-templated micelle was optimized for simultaneously loading of drug and nucleic acid. The size of the loaded DNA-templated micelles was tunable and ranged from 15 nm to 150 nm with a narrow polydispersity (PDI < 0.1). The micelles were colloidally stable in serum-containing environment. FIG. 12 shows size tunable, highly stable DNA-templated micelles with high drug loading capacity. As shown in FIG. 12, the loading efficiency reached nearly 100 %.
To decorate a mesenchymal stem cell (MSC) cluster with DNA-templated micelles, DNA-templated micelles were mixed with an alginate-containing MSC suspension at the ratio of 1 : 1, and the micelle-MSC clusters were subsequently formed via a microfluidic technology.
To enhance the therapeutic efficacy in glioblastoma (GBM) treatment, a micelle-MSC hybrid cluster was developed for co-delivering multiple anti-cancer drugs. MSCs hold tumor homing property as they express tumor-associated cytokine receptors. MSCs in the cluster form showed higher migration toward GBM cells. In micelle-MSC clusters, more than 50% of the DNA-templated micelles were loaded on the extracellular matrix of the micelle-MSC cluster and were not internalized by the MSCs. In addition, MSC migration was not affected by the decoration of DNA-templated micelles. These DNA-templated micelles were size- tunable, colloidally stable, relatively monodispersed and low toxic, which can be integrated synergistically with other anti-cancer approaches to improve the efficacy in cancer therapy.
FIG. 13 shows specific glioblastoma (GBM) cell killing using DNA-templated micelle/MSC hybrid cluster. The targeted, drug-preloaded micelle was formed with Pepl- conjugated DNA template (25% coverage) and mitoxantrone (MTX, Sigma-Aldrich M6545). Subsequently the micelle MSC hybrid cluster, free MTX/MSC cluster or MSC cluster was formed. Three GBM cells, LN18 (Pep 1 -receptor deficient), U87MG (high Pepl-receptor expression) and primary GBM cells, were co-cultured with the clusters. Cell viability of the GBM cell was determined using the Promoga CellTiter-Glo Luminescent Cell Viability Assay kit. Abbreviations in FIG. 13 : MSC, mesenchymal stem cell; MTX, mitoxantrone; NP, nano- particle, e.g. DNA-templated micelle; GBM, glioblastoma.
Example 13. Application of DNA-templated micelles in transfecting cells with mRNAs and small nucleic acids
The DNA bridge of the payload carrier was designed as a partial complement to a polynucleotide (as a payload of the DNA bridge of the payload carrier) with a 37 °C melting temperature. Micelles containing this payload carrier was tested in a wide spectrum of cell lines, including adherent cells, such as HeLa and U87MG cells, and suspension cells, such as THP-1 cell. This payload carrier was used to deliver green fluorescent protein (GFP) mRNA to these cells with 50-70% transfection efficiency which was comparable to the commercially- available transfection reagent, Lipofectamine Messenger Max (available from Thermo Fisher Scientific). However, the micelle containing this payload carrier resulted in less cytotoxicity to the cells at <10% compared to Lipofectamine at 50-70%. This payload carrier also was used to transfect the hard-to-transfect cell line, THP1, with a long mRNA, such as Cas9 mRNA for CRISPR/Cas9 genome editing system, at 40% efficiency, whereas Lipofectamine reached only 5-10% efficiency with the same long mRNA. Co-delivery of Cas9 mRNA and its guide RNA (gRNA) of the CRISPR/Cas9 genome editing system was also validated in a cellular uptake assay, which showed that 98-99% of the Cas9 mRNA and gRNA co-localized inside the transfected cells. Example 14. Application of the multifunctional DNA-templated micelle for leukemia immunotherapy
Two types of payload carriers were designed. One type was conjugated with an antibody that recognizes a leukemic stem cell biomarker, and the other type was conjugated with human Fc protein that can recruit immune cells, including natural killer (NK) cells and macrophage. The payload carrier was loaded with a chemodrug, bisantrene. The modified and loaded DNA-templated micelle was found to not only specifically kill cancer cells, but also was able to induce phagocytosis of immune cells.
The term "about" as used herein means that the value is not necessarily precise and could vary by + 10% or even + 5%.
It is to be understood that the present invention is not to be limited to the exact description and embodiments as illustrated and described herein. To those of ordinary skill in the art, one or more variations and modifications will be understood to be contemplated from the present disclosure. Accordingly, all expedient modifications readily attainable by one of ordinary skill in the art from the disclosure set forth herein, or by routine experimentation therefrom, are deemed to be within the true spirit and scope of the invention as defined by the appended claims.

Claims

THE CLAIMS What is claimed is:
1. A micelle system for delivering at least one payload to cells, the micelle system comprising at least one payload carrier, wherein the payload carrier comprises at least one DNA bridge and at least one functionalized polyethylene glycol (PEG) segment, wherein the functionalized PEG segment is covalently bound to the DNA bridge, wherein the DNA bridge can carry at least one payload through intercalation or nucleotide base pairing.
2. The micelle system of claim 1, wherein the functionalized PEG segment is further conjugated to at least one ligand.
3. The micelle system of claim 2, wherein a density of the ligand of the micelle system is controllable in the range of from 0% to 100%.
4. The micelle system of claim 1, wherein the payload carrier is preloaded with at least one payload prior to the formation of micelle.
5. The micelle system of claim 1, wherein the payload carrier is further conjugated with at least one ligand prior to the formation of micelle.
6. The micelle system of claim 1, wherein the functionalized PEG segment is an amine-functionalized PEG segment.
7. The micelle system of claim 1, wherein the DNA bridge is a single-stranded DNA in the range of from about 8 nucleotides to about 100 nucleotides.
8. The micelle system of claim 1 , wherein the payload is a small molecule, compound, drug, chemodrug, polynucleotide, DNA, gene, messenger RNA (mRNA), aptamer, or a combination thereof.
9. The micelle system of claim 2, wherein the ligand is a small molecule, peptide, antibody, aptamer, polynucleotide, or a combination thereof.
10. The micelle system of claim 1, wherein the size of the micelle is from about 5 nm to about 250 mm.
11. The micelle system of claim 1, wherein the molecular weight of the PEG segment is in the range of from about 1 kD to about 30 kD.
12. The micelle system of claim 1, wherein the polydispersity of the micelle is less than about 0.1.
13. A method for delivering a payload to cells of a subject for disease treatment, comprising administering to the subject a pharmaceutical composition comprising the micelle system of claim 1.
14. The method of claim 13, wherein the disease treatment is cell transfections, gene therapy, chemotherapy, immunotherapy, cancer treatment, disease monitoring, brain tumor chemotherapy, glioblastoma chemotherapy, or leukemia immunotherapy.
15. A kit for performing a cell transfection reaction, comprising the micelle system of claim 1.
PCT/US2018/019926 2017-03-09 2018-02-27 Dna-templated micelle and uses thereof WO2018164883A1 (en)

Priority Applications (2)

Application Number Priority Date Filing Date Title
US16/492,320 US11311629B2 (en) 2017-03-09 2018-02-27 DNA-templated micelle and uses thereof
US17/727,549 US20220313839A1 (en) 2017-03-09 2022-04-22 Dna-templated micelle and uses thereof

Applications Claiming Priority (2)

Application Number Priority Date Filing Date Title
US201762469494P 2017-03-09 2017-03-09
US62/469,494 2017-03-09

Related Child Applications (2)

Application Number Title Priority Date Filing Date
US16/492,320 A-371-Of-International US11311629B2 (en) 2017-03-09 2018-02-27 DNA-templated micelle and uses thereof
US17/727,549 Continuation US20220313839A1 (en) 2017-03-09 2022-04-22 Dna-templated micelle and uses thereof

Publications (1)

Publication Number Publication Date
WO2018164883A1 true WO2018164883A1 (en) 2018-09-13

Family

ID=63447934

Family Applications (1)

Application Number Title Priority Date Filing Date
PCT/US2018/019926 WO2018164883A1 (en) 2017-03-09 2018-02-27 Dna-templated micelle and uses thereof

Country Status (2)

Country Link
US (2) US11311629B2 (en)
WO (1) WO2018164883A1 (en)

Non-Patent Citations (4)

* Cited by examiner, † Cited by third party
Title
ELIAS, DR ET AL.: "Effect of ligand density, receptor density, and nanoparticle size on cell targeting", NANOMEDICINE, vol. 9, no. 2, February 2013 (2013-02-01), pages 194 - 201, XP055606285 *
MAKINO, A ET AL.: "Size control of core-shell-type polymeric micelle with a nanometer precision", LANGMUIR, vol. 30, no. 2, 21 January 2014 (2014-01-21), pages 669 - 674, XP055237861, DOI: doi:10.1021/la404488n *
MAY, M: "Gene Transfer: Taming Diversity", SCIENCE, vol. 324, no. 5934, 18 June 2009 (2009-06-18), pages 1587 *
ZHOU, J ET AL.: "Cell-type-specific, Aptamer-functionalized Agents for Targeted Disease Therapy", MOLECULAR THERAPY NUCLEIC ACIDS, vol. 3, no. e169, 17 June 2014 (2014-06-17), pages 1 - 17, XP055430156 *

Also Published As

Publication number Publication date
US20220313839A1 (en) 2022-10-06
US20210128746A1 (en) 2021-05-06
US11311629B2 (en) 2022-04-26

Similar Documents

Publication Publication Date Title
Mahmoodi Chalbatani et al. Small interfering RNAs (siRNAs) in cancer therapy: a nano-based approach
Yi Xue et al. Lipid-based nanocarriers for RNA delivery
Lee et al. Delivery strategies and potential targets for siRNA in major cancer types
Gandhi et al. Nanocarrier mediated delivery of siRNA/miRNA in combination with chemotherapeutic agents for cancer therapy: current progress and advances
US10087442B2 (en) Polycation-functionalized nanoporous silicon carrier for systemic delivery of gene silencing agents
Sun et al. Simultaneous delivery of siRNA and paclitaxel via a “two-in-one” micelleplex promotes synergistic tumor suppression
Darvishi et al. Stimuli-responsive mesoporous silica NPs as non-viral dual siRNA/chemotherapy carriers for triple negative breast cancer
Sheikh et al. Recent development of aptamer conjugated chitosan nanoparticles as cancer therapeutics
Kabilova et al. Targeted delivery of nucleic acids into xenograft tumors mediated by novel folate-equipped liposomes
Kozlowska et al. Functionalized bioengineered spider silk spheres improve nuclease resistance and activity of oligonucleotide therapeutics providing a strategy for cancer treatment
Sharma et al. Nanoparticulate RNA delivery systems in cancer
Padayachee et al. HER-2/neu and MYC gene silencing in breast cancer: therapeutic potential and advancement in nonviral nanocarrier systems
Huang et al. Nonviral delivery systems for antisense oligonucleotide therapeutics
Yuan et al. A polyethylenimine-containing and transferrin-conjugated lipid nanoparticle system for antisense oligonucleotide delivery to AML
Rajeev et al. Knocking down barriers: advances in siRNA delivery
Xiong et al. Engineered Aptamer‐Organic Amphiphile Self‐Assemblies for Biomedical Applications: Progress and Challenges
Chen et al. Nanoparticle‐mediated siRNA delivery and multifunctional modification strategies for effective cancer therapy
Lin et al. Development and clinical applications of siRNA-encapsulated lipid nanoparticles in cancer
Chen et al. The Hydrophobicity of AIE Dye Facilitates DNA Condensation for Carrier‐Free Gene Therapy
Charbgoo et al. Aptamer‐incorporated nanoparticle systems for drug delivery
US20220313839A1 (en) Dna-templated micelle and uses thereof
Farooqi et al. TRAIL and microRNAs in the treatment of prostate cancer: therapeutic potential and role of nanotechnology
Patel et al. Targeting nanocarriers containing antisense oligonucleotides to cancer cell
Jung et al. Linear polyethyleneimine-doxorubicin conjugate for pH-responsive synchronous delivery of drug and microRNA-34a
Yan et al. Design, strategies, and therapeutics in nanoparticle-based siRNA delivery systems for breast cancer

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: 18763756

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: 18763756

Country of ref document: EP

Kind code of ref document: A1