WO2020093748A1 - 一种靶向线粒体的自组装蛋白质纳米颗粒的制备与应用 - Google Patents

一种靶向线粒体的自组装蛋白质纳米颗粒的制备与应用 Download PDF

Info

Publication number
WO2020093748A1
WO2020093748A1 PCT/CN2019/100706 CN2019100706W WO2020093748A1 WO 2020093748 A1 WO2020093748 A1 WO 2020093748A1 CN 2019100706 W CN2019100706 W CN 2019100706W WO 2020093748 A1 WO2020093748 A1 WO 2020093748A1
Authority
WO
WIPO (PCT)
Prior art keywords
protein
gstp1
self
nucleic acid
protein nanoparticle
Prior art date
Application number
PCT/CN2019/100706
Other languages
English (en)
French (fr)
Inventor
林坚
朱新杰
徐良
许诺
陈龙
Original Assignee
北京大学
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 北京大学 filed Critical 北京大学
Priority to US17/050,188 priority Critical patent/US20210100916A1/en
Publication of WO2020093748A1 publication Critical patent/WO2020093748A1/zh

Links

Images

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
    • A61K47/6921—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 particulate, a powder, an adsorbate, a bead or a sphere
    • A61K47/6927—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 particulate, a powder, an adsorbate, a bead or a sphere the form being a solid microparticle having no hollow or gas-filled cores
    • A61K47/6929—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 particulate, a powder, an adsorbate, a bead or a sphere the form being a solid microparticle having no hollow or gas-filled cores the form being a nanoparticle, e.g. an immuno-nanoparticle
    • 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
    • A61K31/335—Heterocyclic compounds having oxygen as the only ring hetero atom, e.g. fungichromin
    • A61K31/337—Heterocyclic compounds having oxygen as the only ring hetero atom, e.g. fungichromin having four-membered rings, e.g. taxol
    • 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
    • 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
    • A61K47/62—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
    • A61K47/65—Peptidic linkers, binders or spacers, e.g. peptidic enzyme-labile linkers
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K49/00—Preparations for testing in vivo
    • A61K49/001—Preparation for luminescence or biological staining
    • A61K49/0013—Luminescence
    • A61K49/0017—Fluorescence in vivo
    • A61K49/0019—Fluorescence in vivo characterised by the fluorescent group, e.g. oligomeric, polymeric or dendritic molecules
    • A61K49/0021—Fluorescence in vivo characterised by the fluorescent group, e.g. oligomeric, polymeric or dendritic molecules the fluorescent group being a small organic molecule
    • A61K49/0032—Methine dyes, e.g. cyanine dyes
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K49/00—Preparations for testing in vivo
    • A61K49/001—Preparation for luminescence or biological staining
    • A61K49/0013—Luminescence
    • A61K49/0017—Fluorescence in vivo
    • A61K49/005—Fluorescence in vivo characterised by the carrier molecule carrying the fluorescent agent
    • A61K49/0056—Peptides, proteins, polyamino acids
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K49/00—Preparations for testing in vivo
    • A61K49/001—Preparation for luminescence or biological staining
    • A61K49/0063—Preparation for luminescence or biological staining characterised by a special physical or galenical form, e.g. emulsions, microspheres
    • A61K49/0069—Preparation for luminescence or biological staining characterised by a special physical or galenical form, e.g. emulsions, microspheres the agent being in a particular physical galenical form
    • A61K49/0089—Particulate, powder, adsorbate, bead, sphere
    • A61K49/0091—Microparticle, microcapsule, microbubble, microsphere, microbead, i.e. having a size or diameter higher or equal to 1 micrometer
    • A61K49/0093—Nanoparticle, nanocapsule, nanobubble, nanosphere, nanobead, i.e. having a size or diameter smaller than 1 micrometer, e.g. polymeric nanoparticle
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K49/00—Preparations for testing in vivo
    • A61K49/06—Nuclear magnetic resonance [NMR] contrast preparations; Magnetic resonance imaging [MRI] contrast preparations
    • A61K49/08—Nuclear magnetic resonance [NMR] contrast preparations; Magnetic resonance imaging [MRI] contrast preparations characterised by the carrier
    • A61K49/10—Organic compounds
    • A61K49/14—Peptides, e.g. proteins
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K49/00—Preparations for testing in vivo
    • A61K49/06—Nuclear magnetic resonance [NMR] contrast preparations; Magnetic resonance imaging [MRI] contrast preparations
    • A61K49/18—Nuclear magnetic resonance [NMR] contrast preparations; Magnetic resonance imaging [MRI] contrast preparations characterised by a special physical form, e.g. emulsions, microcapsules, liposomes
    • A61K49/1818—Nuclear magnetic resonance [NMR] contrast preparations; Magnetic resonance imaging [MRI] contrast preparations characterised by a special physical form, e.g. emulsions, microcapsules, liposomes particles, e.g. uncoated or non-functionalised microparticles or nanoparticles
    • A61K49/1821—Nuclear magnetic resonance [NMR] contrast preparations; Magnetic resonance imaging [MRI] contrast preparations characterised by a special physical form, e.g. emulsions, microcapsules, liposomes particles, e.g. uncoated or non-functionalised microparticles or nanoparticles coated or functionalised microparticles or nanoparticles
    • A61K49/1824—Nuclear magnetic resonance [NMR] contrast preparations; Magnetic resonance imaging [MRI] contrast preparations characterised by a special physical form, e.g. emulsions, microcapsules, liposomes particles, e.g. uncoated or non-functionalised microparticles or nanoparticles coated or functionalised microparticles or nanoparticles coated or functionalised nanoparticles
    • A61K49/1827—Nuclear magnetic resonance [NMR] contrast preparations; Magnetic resonance imaging [MRI] contrast preparations characterised by a special physical form, e.g. emulsions, microcapsules, liposomes particles, e.g. uncoated or non-functionalised microparticles or nanoparticles coated or functionalised microparticles or nanoparticles coated or functionalised nanoparticles having a (super)(para)magnetic core, being a solid MRI-active material, e.g. magnetite, or composed of a plurality of MRI-active, organic agents, e.g. Gd-chelates, or nuclei, e.g. Eu3+, encapsulated or entrapped in the core of the coated or functionalised nanoparticle
    • A61K49/1866—Nuclear magnetic resonance [NMR] contrast preparations; Magnetic resonance imaging [MRI] contrast preparations characterised by a special physical form, e.g. emulsions, microcapsules, liposomes particles, e.g. uncoated or non-functionalised microparticles or nanoparticles coated or functionalised microparticles or nanoparticles coated or functionalised nanoparticles having a (super)(para)magnetic core, being a solid MRI-active material, e.g. magnetite, or composed of a plurality of MRI-active, organic agents, e.g. Gd-chelates, or nuclei, e.g. Eu3+, encapsulated or entrapped in the core of the coated or functionalised nanoparticle the nanoparticle having a (super)(para)magnetic core coated or functionalised with a peptide, e.g. protein, polyamino acid
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K51/00—Preparations containing radioactive substances for use in therapy or testing in vivo
    • A61K51/02—Preparations containing radioactive substances for use in therapy or testing in vivo characterised by the carrier, i.e. characterised by the agent or material covalently linked or complexing the radioactive nucleus
    • A61K51/04—Organic compounds
    • A61K51/08—Peptides, e.g. proteins, carriers being peptides, polyamino acids, proteins
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K51/00—Preparations containing radioactive substances for use in therapy or testing in vivo
    • A61K51/12—Preparations containing radioactive substances for use in therapy or testing in vivo characterised by a special physical form, e.g. emulsion, microcapsules, liposomes, characterized by a special physical form, e.g. emulsions, dispersions, microcapsules
    • A61K51/1241—Preparations containing radioactive substances for use in therapy or testing in vivo characterised by a special physical form, e.g. emulsion, microcapsules, liposomes, characterized by a special physical form, e.g. emulsions, dispersions, microcapsules particles, powders, lyophilizates, adsorbates, e.g. polymers or resins for adsorption or ion-exchange resins
    • A61K51/1244—Preparations containing radioactive substances for use in therapy or testing in vivo characterised by a special physical form, e.g. emulsion, microcapsules, liposomes, characterized by a special physical form, e.g. emulsions, dispersions, microcapsules particles, powders, lyophilizates, adsorbates, e.g. polymers or resins for adsorption or ion-exchange resins microparticles or nanoparticles, e.g. polymeric nanoparticles
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P35/00—Antineoplastic agents
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K14/00—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K14/00—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
    • C07K14/825—Metallothioneins
    • 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
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N15/00—Mutation or genetic engineering; DNA or RNA concerning genetic engineering, vectors, e.g. plasmids, or their isolation, preparation or purification; Use of hosts therefor
    • C12N15/09—Recombinant DNA-technology
    • C12N15/11—DNA or RNA fragments; Modified forms thereof; Non-coding nucleic acids having a biological activity
    • C12N15/113—Non-coding nucleic acids modulating the expression of genes, e.g. antisense oligonucleotides; Antisense DNA or RNA; Triplex- forming oligonucleotides; Catalytic nucleic acids, e.g. ribozymes; Nucleic acids used in co-suppression or gene silencing
    • 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
    • C12N5/00—Undifferentiated human, animal or plant cells, e.g. cell lines; Tissues; Cultivation or maintenance thereof; Culture media therefor
    • C12N5/0012—Cell encapsulation
    • 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
    • C12N9/00—Enzymes; Proenzymes; Compositions thereof; Processes for preparing, activating, inhibiting, separating or purifying enzymes
    • C12N9/0004—Oxidoreductases (1.)
    • C12N9/0051—Oxidoreductases (1.) acting on a sulfur group of donors (1.8)
    • 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
    • C12N9/00—Enzymes; Proenzymes; Compositions thereof; Processes for preparing, activating, inhibiting, separating or purifying enzymes
    • C12N9/10—Transferases (2.)
    • C12N9/1085—Transferases (2.) transferring alkyl or aryl groups other than methyl groups (2.5)
    • C12N9/1088—Glutathione transferase (2.5.1.18)

Definitions

  • the present invention relates to the field of bioengineering and medicine, in particular to the fields of tumor imaging and therapy, and drug delivery technology, and in particular to a novel protein nanoparticle targeting mitochondria (GSTP1-MT3), which has mitochondrial targeting and causes mitochondria Relevant properties of functional disorders, enrichment around tumors, and inhibition of tumor growth.
  • GSTP1-MT3 novel protein nanoparticle targeting mitochondria
  • Cancer is a worldwide problem that people urgently need to solve. According to the statistics of the World Health Organization, nearly 14 million people die of cancer each year. Among them, breast cancer is the second largest cancer among women after dermatosis. According to statistics from the US Department of Health, nearly 260,000 women die of breast cancer each year. Therefore, the treatment of breast cancer has also received increasing attention.
  • the present invention provides a novel mitochondrial-targeted protein nanoparticle (GSTP1-MT3).
  • the protein nanoparticle can be fused and expressed by using E. coli as a host bacterium, and then obtained by metal ion induction.
  • 4T1 breast cancer cells were transplanted into Balb / c mice to construct a breast cancer model of mice. It was found that GSTP1-MT3 protein nanoparticles can be enriched at the tumor site and quickly enter the mitochondria of tumor cells, resulting in a decrease in mitochondrial membrane potential and cells The increase of internal ROS content can ultimately effectively inhibit the growth of tumors.
  • the present invention provides a new type of self-assembled protein nanoparticles targeted by mitochondria, characterized in that the protein nanoparticles are formed by self-assembly of protein monomers, the protein monomers include metallothionein, connecting peptides and Glutathione thiotransferase is connected in sequence from the amino terminal to the carboxy terminal of the fusion protein, and the amino acid sequence of the connecting peptide is shown in SEQ ID NO: 3.
  • the protein nanoparticle of the present invention is characterized in that the glutathione sulfur transferase is GSTP1, and the amino acid sequence of the GSTP1 is shown in SEQ ID NO: 1.
  • the protein nanoparticle of the present invention is characterized in that the metallothionein is preferably MT3, and the amino acid sequence of the MT3 is shown in SEQ ID NO: 2.
  • the self-assembled protein nanoparticle of the present invention is characterized in that the protein nanoparticle is a fusion protein induced by metal ions, and its amino acid sequence is shown in SEQ ID NO: 4.
  • the metal ions include metal ions such as Cd 2+ , Gd 3+ , Cr 3+ , Ni 2+ , Fe 2+ , Mn 2+ , Co 2+ and the like.
  • the protein nanoparticle of the present invention is characterized in that glutathione sulfur transferase is located at the C-terminus of the fusion protein.
  • the present invention also provides a nucleic acid encoding the fusion protein in the protein nanoparticle, and the nucleotide sequence of the nucleic acid is shown in SEQ ID NO: 5.
  • the present invention also provides a construct comprising the encoding nucleic acid, the construct including an expression cassette and a vector.
  • the vector of the present invention includes a prokaryotic expression vector, and the expression vector includes a promoter, a terminator and other regulatory elements.
  • the present invention also provides a host expression bacterium containing the encoding nucleic acid and nucleic acid construct.
  • the host cell of the present invention includes a prokaryotic host expression bacterium, the prokaryotic host BL21 (DE3).
  • the present invention also provides the application of the mitochondrial-targeted protein nanoparticles in cancer diagnosis.
  • the protein nanoparticles can also be connected with an active substance, and the active substance is a diagnostic reagent.
  • the protein nanoparticles can be enriched around the tumor.
  • the diagnostic reagents include fluorophores, isotopes, MRI contrast agents, radioisotopes and other substances.
  • the present invention also provides the application of the mitochondrial-targeted protein nanoparticles in tumor therapy.
  • the protein nanoparticles targeted by the mitochondria can inhibit tumor growth.
  • the protein nanoparticles are also connected with an active substance, the active substance is a tumor therapeutic drug, the tumor therapeutic drug is paclitaxel, and the tumor is breast cancer.
  • the present invention also provides a novel method for preparing self-assembled protein nanoparticles targeted by mitochondria.
  • the method for preparing novel protein nanoparticles targeted by mitochondria is characterized in that protein monomers may be induced by 0.3 mM metal ions, and may utilize the coordination of amino groups and sulfhydryl groups on metals and proteins to form proteins by self-assembly Nanoparticles.
  • novel protein nanoparticles targeted by mitochondria wherein the metal ions include Cd 2+ , Gd 3+ , Cr 3+ , Ni 2+ , Fe 2+ , Mn 2+ , Co 2+, etc. Metal ion.
  • the method for preparing novel protein nanoparticles targeted by mitochondria according to the present invention also includes a step of purifying protein nanoparticles formed by self-assembly using a GST purification column.
  • the present invention provides a protein nanoparticle that can significantly increase the water solubility of paclitaxel.
  • the combination of the protein nanoparticles and paclitaxel significantly prolongs the survival time of 4T1 breast cancer mice.
  • the first report of metal-mediated self-assembly protein nanoparticles which can rapidly target mitochondria and monitor mitochondrial changes in real time.
  • the protein nanoparticles have better biocompatibility and water solubility, and the targeting speed is faster, which can reflect the real situation of mitochondria in real time.
  • the protein nanoparticles can cause a decrease in mitochondrial membrane potential and an increase in intracellular ROS content, which in turn can cause cell apoptosis.
  • the multi-drug resistance of cancer to chemical drugs is the main factor that reduces the effectiveness of chemical drugs.
  • Mitochondria as an intracellular ATP supply station, play an important role in cell growth and division. Because the damage of mitochondria is irreversible, the dysfunction of mitochondria can effectively overcome the problems of multi-drug tolerance.
  • the protein nanoparticles play an important role in overcoming multi-drug resistance.
  • the protein can be effectively enriched around the tumor, which can reduce the damage of drugs to other organs, thus reducing the damage to normal tissues.
  • FIG. 1 Amino acid sequence composition of protein nanoparticles targeted by GSTP1-MT3 mitochondria
  • Figure 3 The expression level of Co 2+ at different concentrations.
  • FIG. 4 GSTP1-MT3 (Co 2+ ) targets mitochondria.
  • FIG. 6 GSTP1-MT3 protein nanoparticles can cause an increase in intracellular ROS content.
  • FIG. 8 GSTP1-MT3 (Co 2+ ) is enriched around the tumor.
  • FIG. 10 GSTP1-MT3 (Co 2+ ) coupled with PTX (paclitaxel) prolongs the survival of 4T1 breast cancer mice.
  • Figure 11 Schematic diagram of the anti-tumor mechanism of GSTP1-MT3 protein nanoparticles in vivo.
  • Example 1 Construction of GSTP1-MT3 mitochondrial targeted protein nanoparticle amino acid sequence:
  • GSTP1-MT3 is mainly composed of GSTP1 and MT3, where MT3 is at the N-terminus of the amino acid sequence and GSTP1 is at the amino acid sequence At the C-terminus, MT3 and GSTP1 are coupled through the GGGGS sequence.
  • the complete amino acid sequence of GSTP1-MT3 is shown in SEQ ID NO: 4, its molecular weight is 30.566 kDa, and its isoelectric point PI is 5.14; the coding nucleotide sequence of GSTP1-MT3 is shown in SEQ ID NO: 5.
  • a prokaryotic expression vector in which pET-28a (+) was selected as the prokaryotic expression vector, and the enzyme cleavage sites HindIII and NdeI on it were used as The nucleotide sequence encoding GSTP1-MT3 shown in SEQ ID NO: 5 was connected to pET-28a (+), and the recombinant expression vector pET-28a (+)-GSTP1 was successfully obtained after digestion, electrophoresis and monoclonal sequencing detection -MT3.
  • AKTA purification system For purification using the AKTA purification system, first equilibrate with 5 times (5V) column volume of PBS, then use AKTA to bind the supernatant from step 4 to the GST column, and continue to use 5V column volume of PBS for washing.
  • the collected protein was removed using a 10 kDa ultrafiltration tube to remove GSH from the protein, and finally filtered using a 0.22 ⁇ m filter.
  • Short-term storage at 4 ° C (such as long-term storage at -80 ° C, it needs to be stored in 10% glycerol).
  • GSTP1-MT3 with a molecular weight of about 30kDa can be obtained through Co 2+ or Ni 2+ induction (Figure 2A); and 1mmol / L of different metal ions (metal ions can be Cd 2+ , Gd 3+ , Cr 3+ , Ni 2+ , Zn 2+ , Fe 2+ , Mn 2+ , Co 2+, etc.) can be used to induce GSTP1-MT3 protein nanoparticles, of which CO 2+ is the most inducible (Figure 2B ), Therefore, the level of expression of Co 2+ at different concentrations was also explored in the follow-up.
  • metal ions can be Cd 2+ , Gd 3+ , Cr 3+ , Ni 2+ , Zn 2+ , Fe 2+ , Mn 2+ , Co 2+, etc.
  • U87MG cell line 5 ⁇ 10 4 U87MG cells were added to the confocal dish, incubated at 37 ° C., 5% CO 2 overnight, and GSTP1-MT3 (Co 2+ ) -Cy5 prepared in Example 4 was added thereto. 5 (final concentration 6 ⁇ mo / L), then wash three times with PBS, and finally add Green FM (final concentration 2 ⁇ mol / L), continue to incubate at 37 ° C., 5% CO 2 for 30 minutes, finally wash with PBS 3 times, use confocal imaging, and use Image J to calculate Pearson's co-localization efficiency.
  • Example 6 GSTP1-MT3 (Co 2+ ) is enriched in tumor
  • Balb / c mice were inoculated with 3 * 10 6 4T1 (Luciferase stably transformed) breast cancer cells, when the tumor grew to 150 mm 3 .
  • 100 ⁇ L of 30 ⁇ mol / L GSTP1-MT3 (Co 2+ ) -Cy5.5 was injected through the tail vein, the intensity of fluorescence was observed at different time points, and then Luciferase substrate was injected through the abdominal cavity to observe the co-localization of the tumor.
  • Example 7 Treatment of breast cancer with GSTP1-MT3 (Co 2+ )
  • Balb / c mice were inoculated with 3 ⁇ 10 6 4T1 (Luciferase stably transformed) breast cancer cells, when the tumor grew to 50-70 mm 3 .
  • 100 ⁇ L 30 ⁇ mol / L GSTP1-MT3 (Co 2+ ) was injected through the tail vein as the experimental group; 100 ⁇ L PBS was injected through the tail vein as the control group.
  • inject once every two days, that is, on the 3rd, 5th, 7th, 9th, and 11th days of the start of the experiment tail vein injections were given, a total of 5 injections, and the weight of the mice was recorded at different time points And the size of the tumor.
  • the body weights of the mice in the experimental group and the control group are not much different, indicating that GSTP1-MT3 (Co 2+ ) has no obvious toxic and side effects on the mice; from the perspective of tumor volume and weight, the experimental group
  • the tumor volume of mice and control mice was significantly different (P ⁇ 0.05), and the tumor weight of experimental mice and control mice was significantly different (p ⁇ 0.05), indicating that GSTP1-MT3 protein nanoparticles can significantly inhibit tumor growth .

Landscapes

  • Health & Medical Sciences (AREA)
  • Life Sciences & Earth Sciences (AREA)
  • Chemical & Material Sciences (AREA)
  • Engineering & Computer Science (AREA)
  • General Health & Medical Sciences (AREA)
  • Bioinformatics & Cheminformatics (AREA)
  • Medicinal Chemistry (AREA)
  • Public Health (AREA)
  • Animal Behavior & Ethology (AREA)
  • Veterinary Medicine (AREA)
  • Genetics & Genomics (AREA)
  • Organic Chemistry (AREA)
  • Epidemiology (AREA)
  • Proteomics, Peptides & Aminoacids (AREA)
  • Biomedical Technology (AREA)
  • Pharmacology & Pharmacy (AREA)
  • Zoology (AREA)
  • Wood Science & Technology (AREA)
  • Molecular Biology (AREA)
  • Nanotechnology (AREA)
  • Biotechnology (AREA)
  • General Engineering & Computer Science (AREA)
  • Biochemistry (AREA)
  • Microbiology (AREA)
  • Immunology (AREA)
  • Physics & Mathematics (AREA)
  • Nuclear Medicine, Radiotherapy & Molecular Imaging (AREA)
  • Biophysics (AREA)
  • Gastroenterology & Hepatology (AREA)
  • Optics & Photonics (AREA)
  • Plant Pathology (AREA)
  • Radiology & Medical Imaging (AREA)
  • Cell Biology (AREA)
  • Chemical Kinetics & Catalysis (AREA)
  • General Chemical & Material Sciences (AREA)
  • Dispersion Chemistry (AREA)
  • Peptides Or Proteins (AREA)
  • Medicinal Preparation (AREA)
  • Medicines That Contain Protein Lipid Enzymes And Other Medicines (AREA)
  • Pharmaceuticals Containing Other Organic And Inorganic Compounds (AREA)

Abstract

一种线粒体靶向的蛋白质纳米颗粒，包含其氨基酸序列，编码核酸序列，以及所述编码核酸的载体、宿主表达菌等相关信息。在金属离子的诱导下，利用大肠杆菌表达系统可获得自组装的上述蛋白质纳米颗粒。所述蛋白质纳米颗粒可应用于癌症诊断和治疗中，相比于现有的线粒体靶向小分子(TPP、MPP等)，该蛋白质纳米颗粒能够实现肿瘤富集、线粒体靶向、造成细胞内ROS含量增加、引发细胞发生凋亡以及抑制肿瘤生长等性能。

Description

一种靶向线粒体的自组装蛋白质纳米颗粒的制备与应用 技术领域
本发明涉及生物工程及医药领域，特别涉及肿瘤成像和治疗以及药物递送技术领域，具体涉及一种新型靶向线粒体的蛋白质纳米颗粒(GSTP1-MT3)，该蛋白质纳米颗粒具有线粒体靶向、引起线粒体功能性紊乱、在肿瘤周围富集以及抑制肿瘤生长等相关性质。
背景技术
癌症是当前人们亟待解决的世界性难题，根据世界卫生组织统计，每年将近有1400万人死于癌症，其中乳腺癌是在女性中发病率仅次于皮肤病的第二大癌症。根据美国卫生局统计，每年将近有26万的女性死于乳腺癌。因此，对于乳腺癌的治疗也越来越受到人们的关注。
然而，病人一般在癌症晚期才会发现自己的相关症状，同时很多的癌症病人会对很多的化疗药物产生一定的多重耐药性等问题，这给癌症的治疗带来了很大的挑战。线粒体作为细胞内主要的ATP供应站，对于维持细胞的生长与代谢起着非常重要的作用。由于线粒体功能性损伤，会细胞造成不可逆的损伤，因此越来越多的人在探索基于线粒体功能性紊乱的癌症治疗方案。最近人们发现对线粒体功能性紊乱，能够从一定程度上克服癌症的多重耐药性这一难题。然而现在能够靶向线粒体的药物主要是一些三苯基膦类似物以及线粒体穿膜肽等小分子，但是这些小分子存在半衰期短、在肿瘤周围不能有效富集等问题，这些不足极大限制了其在癌症治疗中的应用。
发明内容
为了有效解决上述问题，本发明提供一种新型线粒体靶向的蛋白质纳米颗粒(GSTP1-MT3)，该蛋白质纳米颗粒能够以大肠杆菌作为宿主菌进行融合表达，再经金属离子诱导而获得。在Balb/c小鼠中移植4T1乳腺癌细胞构建小鼠的乳腺癌模型，发现GSTP1-MT3蛋白质纳米颗粒能够在肿瘤部位富集，快速进入到肿瘤细胞的线粒体中，造成线粒体膜电位下降以及细胞内ROS含量的增加，最终能够有效地抑制肿瘤的生长。同时我们还发现GSTP1-MT3(Co 2+)通过化学偶联的方式和紫杉醇联用，不仅能够提高紫杉醇的水溶性，而且还极大地延长 了小鼠的生存期，为临床上乳腺癌治疗提供了广阔的前景。
一方面，本发明提供一种新型线粒体靶向的自组装蛋白质纳米颗粒，其特征在于：所述蛋白质纳米颗粒由蛋白质单体自组装形成，所述蛋白质单体包括由金属硫蛋白、连接肽以及谷胱甘肽硫转移酶以从氨基端到羧基端的顺序依次连接组成的融合蛋白，所述连接肽的氨基酸序列如SEQ ID NO：3所示。
本发明所述蛋白质纳米颗粒，其特征在于：所述谷胱甘肽硫转移酶为GSTP1，所述GSTP1的氨基酸序列如SEQ ID NO：1所示。
本发明所述蛋白质纳米颗粒，其特征在于：所述金属硫蛋白优选为MT3，所述MT3的氨基酸序列如SEQ ID NO：2所示。
本发明所述自组装蛋白质纳米颗粒，其特征在于：所述蛋白质纳米颗粒是由金属离子诱导而形成的融合蛋白，其氨基酸序列如SEQ ID NO：4所示。所述金属离子包含Cd 2+、Gd 3+、Cr 3+、Ni 2+、Fe 2+、Mn 2+、Co 2+等金属离子。
本发明所述蛋白质纳米颗粒，其特征在于：谷胱甘肽硫转移酶位于所述融合蛋白的C端。
第二方面，本发明还提供编码所述的蛋白质纳米颗粒中的融合蛋白的核酸，所述核酸的核苷酸序列如SEQ ID NO：5所示。
第三方面，本发明还提供包含所述编码核酸的构建体，所述构建体包括表达盒、载体。
本发明所述载体包括原核表达载体，所述表达载体中包括启动子、终止子和其他调控元件。
第四方面，本发明还提供一种含有所述编码核酸、核酸构建体的宿主表达菌。
本发明所述宿主细胞包括原核宿主表达菌，所述原核宿主BL21(DE3)。
第五方面，本发明还提供所述线粒体靶向的蛋白质纳米颗粒在癌症诊断中的应用。
其中，所述蛋白质纳米颗粒还能够连接有活性物质，所述活性物质为诊断试剂。
其中，所述蛋白质纳米颗粒能够在肿瘤周围富集。所述诊断试剂包括荧光基团、同位素、MRI造影剂、放射性同位素等物质。
第六方面，本发明还提供所述线粒体靶向的蛋白质纳米颗粒在肿瘤治疗中的应用。
其中，所述线粒体靶向的蛋白质纳米颗粒能够抑制肿瘤生长。
其中，所述蛋白质纳米颗粒还连接有活性物质，所述活性物质为肿瘤治疗药物，所述肿瘤治疗药物为紫杉醇、所述肿瘤为乳腺癌。
第七方面，本发明还提供一种新型的线粒体靶向的自组装蛋白质纳米颗粒制备方法。
本发明所述制备线粒体靶向的新型蛋白质纳米颗粒的方法，其特征在于：蛋白质单体在0.3mM的金属离子的诱导下，可能利用金属和蛋白质上的氨基和巯基配位，自组装形成蛋白质纳米颗粒。
本发明所述制备线粒体靶向的新型蛋白质纳米颗粒的方法，其中所述金属离子包含Cd 2+、Gd 3+、Cr 3+、Ni 2+、Fe 2+、Mn 2+、Co 2+等金属离子。
本发明所述制备线粒体靶向的新型蛋白质纳米颗粒的方法，其还包括利用GST纯化柱对自组装形成的蛋白质纳米颗粒进行纯化的步骤。
第八方面，本发明提供了一种能显著提高紫杉醇水溶性的蛋白质纳米颗粒。
其中，所述蛋白质纳米颗粒和紫杉醇联用后，显著延长4T1乳腺癌小鼠的生存期。
与现有技术相比，本发明的技术方案具有以下优点：
第一、首次报道了通过金属介导的自组装蛋白质纳米颗粒，该蛋白质纳米颗粒能够快速靶向到线粒体，实时监测线粒体的变化。相比于其它的靶向线粒体的小分子而言，该蛋白质纳米颗粒的生物相容性和水溶性较好，而且靶向速度快，更能实时反映线粒体真实情况。
第二、该蛋白纳米颗粒能够造成线粒体膜电位下降、细胞内ROS含量的增加，进而引起细胞发生凋亡。
第三、癌症对化学药物的多重耐药性是降低化学药物治疗效果的主要因素。线粒体作为细胞内ATP供应站，在细胞的生长与分裂中扮演着重要的角色。由于线粒体的损伤是不可逆性的，因此对线粒体功能性紊乱能够有效地克服多重药物耐受等问题，该蛋白质纳米颗粒在克服多重耐药性方面起着重要的作用。
第四、相比于其它的线粒体靶向小分子而言，该蛋白质能够在肿瘤周围进 行有效地富集，能够减少药物对其它器官的损伤，因此减少对正常组织的损伤。
附图说明
通过阅读下文优选实施方式的详细描述，各种其他的优点和益处对于本领域普通技术人员将变得清楚明了。附图仅用于示出优选实施方式的目的，而并不认为是对本发明的限制。
图1：GSTP1-MT3线粒体靶向的蛋白质纳米颗粒的氨基酸序列构成
图2：GSTP1-MT3蛋白质纳米颗粒的相关表征
A：分别在金属Co 2+、Ni 2+诱导下的蛋白表达情况；
B：在1mmol/L不同金属诱导下，蛋白质的表达量高低；
C：GSTP1-MT3(Co 2+)粒径大小测定；
D：GSTP1-MT3(Co 2+)蛋白质纳米颗粒在不同pH条件下二级结构的变化；
E-F：GSTP1-MT3(Co 2+)蛋白质纳米颗粒形貌测定(E)扫描电镜、(F)透射电镜。
图3：不同浓度下Co 2+的表达量水平高低。
图4：GSTP1-MT3(Co 2+)靶向线粒体。
图:5：线粒体的Pearson’s co-localization efficiency测定
图6：GSTP1-MT3蛋白质纳米颗粒能够引起细胞内ROS含量的增加。
图7：细胞内ROS含量的测定。
图8：GSTP1-MT3(Co 2+)在肿瘤周围富集。
图9：GSTP1-MT3(Co 2+)抑制肿瘤生长
A：小鼠体重的变化，其中箭头代表给药的时间点；
B：小鼠肿瘤大小的测定，其中箭头代表给药的时间点；
C：第21天时，小鼠的实体图(上)以及小鼠的肿瘤实体图片(下)；
D：第21天时，小鼠肿瘤的体重。
图10：GSTP1-MT3(Co 2+)偶联PTX(紫杉醇)延长4T1乳腺癌小鼠的生存期。
图11：GSTP1-MT3蛋白纳米颗粒体内抗肿瘤作用机理示意图。
具体实施方式
下面将参照附图更详细地描述本公开的示例性实施方式。虽然附图中显示了本公开的示例性实施方式，然而应当理解，可以以各种形式实现本公开而不应被这里阐述的实施方式所限制。相反，提供这些实施方式是为了能够更透彻 地理解本公开，并且能够将本公开的范围完整的传达给本领域的技术人员。
根据本发明的实施方式，提出以下实施案例
实施例一、构建GSTP1-MT3线粒体靶向的蛋白质纳米颗粒氨基酸序列:
基于前期实验研究，构建了如下的线粒体靶向的蛋白质纳米颗粒表达载体，如图1所示，GSTP1-MT3主要由GSTP1和MT3两部分组成，其中MT3在氨基酸序列的N端，GSTP1在氨基酸序列的C端，MT3和GSTP1中间通过GGGGS序列偶联。
GSTP1氨基酸序列：
Figure PCTCN2019100706-appb-000001
MT3氨基酸序列：
Figure PCTCN2019100706-appb-000002
GSTP1和MT3之间的linker：
Figure PCTCN2019100706-appb-000003
完整的GSTP1-MT3氨基酸序列如SEQ ID NO：4所示，其分子量为30.566kDa，等电点PI为5.14；GSTP1-MT3的编码核苷酸序列如SEQ ID NO：5所示。
实施例二、GSTP1-MT3蛋白质纳米颗粒载体构建
为了利于后续的GSTP1-MT3蛋白质纳米颗粒在重组细胞中的表达，构建了其原核表达载体，其中选择pET-28a(+)作为原核表达载体，利用其上的酶切位点HindIII、NdeI将如SEQ ID NO：5所示编码GSTP1-MT3的核苷酸序列连接到pET-28a(+)中，经酶切、电泳及单克隆测序检测后成功获得重组表达载体pET-28a(+)-GSTP1-MT3。
实施例三、GSTP1-MT3蛋白质纳米颗粒的重组表达
采用大肠杆菌作为宿主菌进行重组表达，具体表达方法如下：
1、质粒转化
取2μL 42ng/μL pET-28a(+)-GSTP1-MT3质粒，加入到20μL BL21(DE3)感受态细胞，在冰上预混15-30分钟，然后放在42℃水浴锅中加热90秒，随后冰上再放置10分钟。加入800μL的无抗性的LB培养基，在37℃220rpm培养1小时，然后在3500rpm离心10分钟，移去600μL的上清，剩余的200μL混匀后，待用。
2、抗性筛选
将步骤1中剩余的200μL菌液加入到含有卡那霉素的琼脂糖平板中，37℃培养箱中培养2小时后，将平板倒置培养过夜。
3、单克隆挑选
挑选单个克隆，加入到10mL含有卡那霉素的LB培养基中，37℃220rpm培养10小时，溶液逐渐变浑浊。
4、蛋白质诱导表达
将步骤3中的10mL菌液加入到1L含有卡那霉素的LB培养基中，37℃220rpm培养4小时后，再分别加入1mL 0.1mol/L IPTG(培养基中终浓度0.1mmol/L)和1mmol/L不同金属离子(金属离子可为Cd 2+、Gd 3+、Cr 3+、Ni 2+、Zn 2+、Fe 2+、Mn 2+、Co 2+等)继续诱导表达过夜，4℃4000rpm离心20分钟，弃去上清，加入20mL GST重悬液(pH＝8.0、50mM Tris/HCl、100mM NaCl、60mMβ-Mercaptoethanol)进行超声波碎(30％的功率,SCIENTZ，JY 92-IIN)，4℃12000rpm超速离心，收集上清，使用0.22μm的滤膜进行过滤，有待进一步纯化。
5、蛋白纯化
利用AKTA纯化系统进行纯化，首先使用5倍(5V)柱体积的PBS平衡，然后利用AKTA将步骤4中上清结合到GST柱子上，继续使用5V柱体积的PBS进行冲洗待基线平稳后，使用GST洗脱液(pH＝8.0、10mmol/L GSH、50mM Tris/HCl、100mM NaCl、60mMβ-Mercaptoethanol)洗脱并收集。收集的蛋白使用10kDa超滤管除去蛋白中的GSH，最后使用0.22μm的滤膜过滤。4℃短 期保存(如长期-80℃保存，需要保存在10％的甘油中)。
如图2所示，经由Co 2+或Ni 2+诱导均能获得分子量约30kDa的GSTP1-MT3(图2A)；而1mmol/L的不同金属离子(金属离子可为Cd 2+、Gd 3+、Cr 3+、Ni 2+、Zn 2+、Fe 2+、Mn 2+、Co 2+等)均能够用于诱导GSTP1-MT3蛋白质纳米颗粒，其中CO 2+的诱导能力最强(图2B)，因此，后续还探索了Co 2+在不同浓度下表达量的高低，结果表明Co 2+最适宜的诱导浓度为0.3mmol/L(图3)(无特殊说明所有的金属诱导浓度均为0.3mmol/L)。采用Co 2+诱导的蛋白质纳米颗粒进行功能分析；图2D给出了GSTP1-MT3蛋白质纳米颗粒在不同的pH条件下二级结构情况；由图2C、图2E-2F可知，GSTP1-MT3经由金属离子诱导后形成了大小分布均匀的纳米颗粒。
实施例四、GSTP1-MT3标记Cy5.5荧光
以实施例3获得的GST-MT3(Co 2+)为例，将500μL 100μM GST-MT3(Co 2+)加入到pH＝9.0的Tris/HCl缓冲液中，充分混匀后，向其中加入6.7μL 15mM Cy5.5-NHS(Cy5.5-NHS溶解在DMSO中)，快速混匀，放到25℃1000rpm混匀器中反应过夜(全程避光)，利用脱盐柱除去没有反应完的Cy5.5-NHS，最后用0.22μm的滤膜过滤，反应后的产物记为GSTP1-MT3(Co 2+)-Cy5.5，4℃避光保存。
实施例五、GSTP1-MT3靶向线粒体
以U87MG细胞株为例，向共聚焦皿中加入5×10 4个U87MG细胞，37℃、5％CO 2培养过夜，向其中加入实施例4制备的GSTP1-MT3(Co 2+)-Cy5.5(终浓度6μmo/L)，然后使用PBS清洗三次，最后加入
Figure PCTCN2019100706-appb-000004
Green FM(终浓度2μmol/L)，继续在37℃、5％CO 2培养30分钟，最终使用PBS清洗3次，使用共聚焦成像，并使用Image J计算Pearson’s co-localization efficiency。
结果如图4和图5可知，该蛋白质纳米颗粒能够很好地定位到线粒体，其中GSTP1在线粒体靶向中起着关键性作用。
以U87MG细胞株为例，向共聚焦皿中加入5×10 4个U87MG细胞，37℃、 5％CO2培养过夜，向其中加入实施例3制备的GSTP1-MT3(Fe 2+)和GSTP1-MT3(Co 2+)(终浓度6μmol/L)继续培养24小时，然后使用PBS清洗三次，随后加入10μmol/L DCFH-DA(ROS探针)，37℃孵育30分钟后，使用PBS清洗三次，最后进行共聚焦成像，并统计ROS的含量高低。如图6-7结果表明GSTP1-MT3(Co 2+)能够显著引起细胞内ROS含量的增加。
实施例六、GSTP1-MT3(Co 2+)在肿瘤富集
以Balb/c小鼠的乳腺癌为例，向Balb/c小鼠接种3*10 6 4T1(稳转Luciferase)乳腺癌细胞，当肿瘤生长到150mm 3。通过尾静脉注射100μL 30μmol/L GSTP1-MT3(Co 2+)-Cy5.5，在不同时间点观察荧光的强度，然后通过腹腔注射Luciferase底物，观察肿瘤的共定位情况。
结果如图8所示，Cy5.5荧光能和Luciferase生物发光很好地重合在一起，表明GSTP1-MT3(Co 2+)-Cy5.5能够很好地在肿瘤部位富集。
实施例七、GSTP1-MT3(Co 2+)对乳腺癌的治疗
以Balb/c小鼠的乳腺癌为例，向Balb/c小鼠接种3×10 6 4T1(稳转Luciferase)乳腺癌细胞，当肿瘤生长到50-70mm 3。通过尾静脉注射100μL 30μmol/L GSTP1-MT3(Co 2+)作为实验组；通过尾静脉注射100μL PBS作为对照组。每两天注射一次，即在实验开始的第3天、第5天、第7天、第9天和第11天分别进行尾静脉注射，一共注射5次，在不同时间点记录小鼠的体重以及肿瘤大小。
如图6所示，实验组小鼠和对照组小鼠的体重相差不大，表明GSTP1-MT3(Co 2+)对小鼠无明显的毒副作用；从肿瘤体积和重量来看，实验组小鼠和对照组小鼠的肿瘤体积差异显著(P<0.05)，而且实验组小鼠和对照组小鼠的肿瘤重量差异显著(p<0.05)，表明GSTP1-MT3蛋白质纳米颗粒能够显著抑制肿瘤生长。
同时，结果发现GSTP1-MT3(Co 2+)偶联紫杉醇以后，能够显著延长4T1乳腺癌小鼠的生存期(如图10)。GSTP1-MT3体内抗肿瘤作用机理示意图如图11所示。
以上所述，仅为本发明较佳的具体实施方式，但本发明的保护范围并不局限于此，任何熟悉本技术领域的技术人员在本发明揭露的技术范围内，可轻易想到的变化或替换，都应涵盖在本发明的保护范围之内。因此，本发明的保护范围应以所述权利要求的保护范围为准。
Figure PCTCN2019100706-appb-000005
Figure PCTCN2019100706-appb-000006
Figure PCTCN2019100706-appb-000007
Figure PCTCN2019100706-appb-000008

Claims (10)

  1. 一种线粒体靶向的自组装蛋白质纳米颗粒，其特征在于：所述蛋白质纳米颗粒由蛋白质单体自组装形成，所述蛋白质单体包括由金属硫蛋白、连接肽、谷胱甘肽硫转移酶以从氨基端到羧基端的顺序依次连接组成的融合蛋白，所述连接肽的氨基酸序列如SEQ ID NO：3所示。
  2. 如权利要求1所述的自组装蛋白质纳米颗粒，其特征在于：所述谷胱甘肽硫转移酶为GSTP1，所述GSTP1的氨基酸序列如SEQ ID NO：1所示；所述金属硫蛋白为MT3，所述MT3的氨基酸序列如SEQ ID NO：2所示。
  3. 如权利要求1所述的自组装蛋白质纳米颗粒，其特征在于：所述蛋白质纳米颗粒是由金属离子诱导而形成的融合蛋白，其氨基酸序列如SEQ ID NO：4所示；所述金属离子包含Cd 2+、Gd 3+、Cr 3+、Ni 2+、Fe 2+、Mn 2+、Co 2+等。
  4. 一种线粒体靶向的自组装蛋白质纳米颗粒制备方法，其特征在于：蛋白质单体在金属离子诱导下，利用金属离子和蛋白质上的氨基和巯基配位自组装成蛋白质纳米颗粒。
  5. 编码权利要求1-3所述的蛋白质纳米颗粒中的融合蛋白的核酸，其特征在于：所述核酸的核苷酸序列如SEQ ID NO：5所示。
  6. 包含权利要求5所述核酸的核酸构建体，所述构建体包括表达盒、载体。
  7. 含有权利要求5所述核酸或权利要求6所述核酸构建体的原核宿主细胞或真核宿主细胞。
  8. 权利要求1-3所述线粒体靶向的蛋白质纳米颗粒在癌症诊断药物或治疗药物中的用途。
  9. 权利要求1-3所述蛋白质纳米颗粒在制备肿瘤治疗药物中的用途。
  10. 如权利要求8或9所述的用途，其特征在于：所述蛋白质纳米颗粒能够连接活性物质，所述活性物质为诊断标记或肿瘤治疗药物，所述活性物质主要包括荧光分子、同位素、MRI造影剂、放射物质、抗氧化以及抗癌药物等。
PCT/CN2019/100706 2018-11-09 2019-08-15 一种靶向线粒体的自组装蛋白质纳米颗粒的制备与应用 WO2020093748A1 (zh)

Priority Applications (1)

Application Number Priority Date Filing Date Title
US17/050,188 US20210100916A1 (en) 2018-11-09 2019-08-15 Preparation and use of mitochondrion-targeting self-assembled protein nanoparticle

Applications Claiming Priority (2)

Application Number Priority Date Filing Date Title
CN201811336263.4A CN109529046B (zh) 2018-11-09 2018-11-09 一种靶向线粒体的自组装蛋白质纳米颗粒的制备与应用
CN201811336263.4 2018-11-09

Publications (1)

Publication Number Publication Date
WO2020093748A1 true WO2020093748A1 (zh) 2020-05-14

Family

ID=65846696

Family Applications (1)

Application Number Title Priority Date Filing Date
PCT/CN2019/100706 WO2020093748A1 (zh) 2018-11-09 2019-08-15 一种靶向线粒体的自组装蛋白质纳米颗粒的制备与应用

Country Status (3)

Country Link
US (1) US20210100916A1 (zh)
CN (1) CN109529046B (zh)
WO (1) WO2020093748A1 (zh)

Cited By (1)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
CN114316057A (zh) * 2022-03-14 2022-04-12 深圳市人民医院 一种靶向线粒体外膜转位因子复合体tom20亚基的纳米抗体及其应用

Families Citing this family (7)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
CN109529046B (zh) * 2018-11-09 2020-10-27 北京大学 一种靶向线粒体的自组装蛋白质纳米颗粒的制备与应用
CN112274650A (zh) * 2019-07-24 2021-01-29 江苏集萃分子工程研究院有限公司 基于蛋白质纳米颗粒的免疫激动剂及其应用
CN112546239B (zh) * 2020-11-19 2022-09-02 北京大学 一种蛋白质纳米颗粒用于制备抗病毒产品的用途
CN112472821B (zh) * 2020-11-19 2022-11-18 北京大学 一种蛋白质纳米颗粒用于制备抗vsv病毒产品的用途
CN112574318B (zh) * 2020-12-23 2023-04-18 非零和(北京)投资管理有限公司 非洲猪瘟病毒p22蛋白纳米颗粒及其制备方法与应用
CN112574319B (zh) * 2020-12-23 2023-04-18 非零和(北京)投资管理有限公司 非洲猪瘟病毒p12蛋白纳米颗粒及其制备方法与应用
CN114533894A (zh) * 2022-02-28 2022-05-27 郑州大学 一种具有线粒体靶向功能的自组装多肽药物及其制备方法和应用

Citations (8)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO2014124425A1 (en) * 2013-02-11 2014-08-14 University Of Georgia Research Foundation, Inc. Generation of functional dendritic cells
WO2015157409A1 (en) * 2014-04-08 2015-10-15 University Of Georgia Research Foundation, Inc. Mitochondria-targeting platinum(iv) prodrug
CN105726481A (zh) * 2014-12-12 2016-07-06 中国科学院大连化学物理研究所 一种基于复合瓷质体的靶向线粒体纳米粒的制备及应用
CN106317018A (zh) * 2016-07-21 2017-01-11 湖南大学 一种肿瘤靶向性亲脂性阳离子‑苯丁酸氮芥化合物及制备方法和在白蛋白纳米药物中的应用
CN106957436A (zh) * 2015-03-10 2017-07-18 中国药科大学 共递送药物和基因的纳米载体及其制备方法和用途
CN107158405A (zh) * 2017-05-24 2017-09-15 电子科技大学 一种线粒体靶向的纳米光敏免疫复合药物及其制备方法和应用
CN107501245A (zh) * 2017-08-02 2017-12-22 浙江工业大学 一种线粒体靶向的双信号turn‑on的甲醛荧光纳米探针及其制备与应用
CN109529046A (zh) * 2018-11-09 2019-03-29 北京大学 一种靶向线粒体的自组装蛋白质纳米颗粒的制备与应用

Family Cites Families (5)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
CN1141395C (zh) * 2001-10-18 2004-03-10 复旦大学 金属硫蛋白的制备方法
US8758745B2 (en) * 2009-10-30 2014-06-24 Medizinische Universitaet Wien Use of GSTP1
WO2016146042A1 (en) * 2015-03-13 2016-09-22 Acro Biotech Co., Ltd. Zinc binding fusion protein of glutathione s-transferase and metallothionein
CN105543262B (zh) * 2015-12-22 2018-11-23 盘古基因生物工程(南京)股份有限公司 一种人类金属硫蛋白-3融合蛋白表达载体
CN106309375B (zh) * 2016-10-11 2019-03-26 中国科学院过程工程研究所 一种纳米粒制剂、其制备方法及应用

Patent Citations (8)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO2014124425A1 (en) * 2013-02-11 2014-08-14 University Of Georgia Research Foundation, Inc. Generation of functional dendritic cells
WO2015157409A1 (en) * 2014-04-08 2015-10-15 University Of Georgia Research Foundation, Inc. Mitochondria-targeting platinum(iv) prodrug
CN105726481A (zh) * 2014-12-12 2016-07-06 中国科学院大连化学物理研究所 一种基于复合瓷质体的靶向线粒体纳米粒的制备及应用
CN106957436A (zh) * 2015-03-10 2017-07-18 中国药科大学 共递送药物和基因的纳米载体及其制备方法和用途
CN106317018A (zh) * 2016-07-21 2017-01-11 湖南大学 一种肿瘤靶向性亲脂性阳离子‑苯丁酸氮芥化合物及制备方法和在白蛋白纳米药物中的应用
CN107158405A (zh) * 2017-05-24 2017-09-15 电子科技大学 一种线粒体靶向的纳米光敏免疫复合药物及其制备方法和应用
CN107501245A (zh) * 2017-08-02 2017-12-22 浙江工业大学 一种线粒体靶向的双信号turn‑on的甲醛荧光纳米探针及其制备与应用
CN109529046A (zh) * 2018-11-09 2019-03-29 北京大学 一种靶向线粒体的自组装蛋白质纳米颗粒的制备与应用

Non-Patent Citations (2)

* Cited by examiner, † Cited by third party
Title
YUE, C. X ET AL: "ROS-Responsive Mitochondria-Targeting Blended Nanoparticles: Chemo- and Photodynamic Synergistic Therapy for Lung Cancer with On-Demand Drug Release upon Irradiation with a Single Light Source", THERANOSTICS, vol. 6, no. 13, 1 October 2016 (2016-10-01), XP055705480, ISSN: 1838-7640, DOI: 10.7150/thno.15433 *
ZHU, X. J.: "A Novel Self-Assembled Mitochondria-Targeting Protein Nano- particle Acting as Theranostic Platform for Cancer", SMALL, vol. 15, no. 2, 19 November 2018 (2018-11-19), pages 1 - 7, XP055705474, ISSN: 1613-6810, DOI: 10.1002/smll.201803428 *

Cited By (2)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
CN114316057A (zh) * 2022-03-14 2022-04-12 深圳市人民医院 一种靶向线粒体外膜转位因子复合体tom20亚基的纳米抗体及其应用
CN114316057B (zh) * 2022-03-14 2022-05-10 深圳市人民医院 一种靶向线粒体外膜转位因子复合体tom20亚基的纳米抗体及其应用

Also Published As

Publication number Publication date
CN109529046B (zh) 2020-10-27
US20210100916A1 (en) 2021-04-08
CN109529046A (zh) 2019-03-29

Similar Documents

Publication Publication Date Title
WO2020093748A1 (zh) 一种靶向线粒体的自组装蛋白质纳米颗粒的制备与应用
US11466085B2 (en) Anti-PD-L1 nanobody, coding sequence and use thereof
Vannucci et al. Selective targeting of melanoma by PEG-masked protein-based multifunctional nanoparticles
US11707527B2 (en) Nanoscale drug carrier capable of passing through blood-brain barrier
CN102153653B (zh) 肿瘤血管靶向多肽与组织因子的融合蛋白及其制备方法
WO2021008454A1 (zh) 基于铁蛋白重链亚基的药物载体
CN102863537A (zh) 一种肿瘤靶向性肿瘤坏死因子相关凋亡配体变体及其应用
US20240189443A1 (en) Pd-l1 and tlr7 double-targeting nanobody coupling drug and use thereof in anti-tumor
CN103805621B (zh) 靶向性抗肿瘤融合蛋白质lpo的新型制备工艺
WO2010049375A1 (en) Use of bacteria for the sensing and killing of cancer cells
CN102993314B (zh) 一种抗肿瘤融合蛋白及其制备方法和用途
CN107236046A (zh) 一种重组人内皮抑素融合蛋白及其制备方法和应用
CN101987873A (zh) P53融合蛋白及应用
US8852576B2 (en) Modified sodium iodide symporter proteins and uses thereof
CN101117635B (zh) Ptd、hif的odd与肿瘤抑制基因的融合表达及其应用
CN103755813B (zh) 一种靶向性抗肿瘤融合蛋白及其编码基因与表达质粒
WO2017152873A1 (zh) 微小rna31前体编码多肽在制备免疫调节药物中的应用
CN105985447B (zh) 一种白蛋白结合型肿瘤坏死因子相关凋亡诱导配体变异体及其制备方法和用途
CN103865899B (zh) 具有vegfr2/kdr受体特异性的融合毒素及其编码基因与应用
CN114716566A (zh) 一种融合蛋白及其在制备肿瘤药物中的应用
JP5529609B2 (ja) 標的結合部を有する薬剤運搬体
WO2024067295A1 (zh) 修饰的红细胞及其用于递送药物的用途
WO2022179536A1 (zh) 铁蛋白重链亚基突变体及其应用
KR20230055678A (ko) 암세포 투과성 단백질 전달용 조성물 및 그 용도
CN107141354A (zh) 一种融合蛋白及光敏剂复合物及其制备方法和应用

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: 19883265

Country of ref document: EP

Kind code of ref document: A1

NENP Non-entry into the national phase

Ref country code: DE

32PN Ep: public notification in the ep bulletin as address of the adressee cannot be established

Free format text: NOTING OF LOSS OF RIGHTS PURSUANT TO RULE 112(1) EPC (EPO FORM 1205 DATED 24-08-2021)

122 Ep: pct application non-entry in european phase

Ref document number: 19883265

Country of ref document: EP

Kind code of ref document: A1