WO2023165467A1 - Vecteur de nanocage de ferritine chargé d'un médicament à petite molécule à base d'acide nucléique dans une cavité interne et utilisation - Google Patents

Vecteur de nanocage de ferritine chargé d'un médicament à petite molécule à base d'acide nucléique dans une cavité interne et utilisation Download PDF

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WO2023165467A1
WO2023165467A1 PCT/CN2023/078704 CN2023078704W WO2023165467A1 WO 2023165467 A1 WO2023165467 A1 WO 2023165467A1 CN 2023078704 W CN2023078704 W CN 2023078704W WO 2023165467 A1 WO2023165467 A1 WO 2023165467A1
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nucleic acid
ferritin
small nucleic
nanocarrier
cage
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PCT/CN2023/078704
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Chinese (zh)
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孙国明
陈向茹
尹雨芳
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南京纳么美科技有限公司
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    • 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
    • A61K47/6949—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 inclusion complexes, e.g. clathrates, cavitates or fullerenes
    • 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
    • A61K38/00—Medicinal preparations containing peptides
    • A61K38/16—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
    • A61K38/17—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans
    • 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
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P31/00—Antiinfectives, i.e. antibiotics, antiseptics, chemotherapeutics
    • A61P31/12—Antivirals
    • 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
    • A61P43/00—Drugs for specific purposes, not provided for in groups A61P1/00-A61P41/00
    • B—PERFORMING OPERATIONS; TRANSPORTING
    • B82—NANOTECHNOLOGY
    • B82Y—SPECIFIC USES OR APPLICATIONS OF NANOSTRUCTURES; MEASUREMENT OR ANALYSIS OF NANOSTRUCTURES; MANUFACTURE OR TREATMENT OF NANOSTRUCTURES
    • B82Y5/00—Nanobiotechnology or nanomedicine, e.g. protein engineering or drug delivery
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K14/00—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
    • C07K14/435—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans
    • C07K14/46—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans from vertebrates
    • C07K14/47—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans from vertebrates from mammals
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K14/00—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
    • C07K14/435—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans
    • C07K14/79—Transferrins, e.g. lactoferrins, ovotransferrins

Definitions

  • the invention belongs to the field of ferritin biological nanomaterials, and relates to a preparation method and application of a ferritin nanocage carrier loaded with small nucleic acid drugs in an inner cavity.
  • Nucleic acid drugs can specifically target disease-causing genes and precisely target and regulate diseases at the gene level. In recent years, they have received extensive attention in the field of precision and personalized treatment. However, free nucleic acid drugs have problems of poor stability and low bioavailability in application. After entering the blood, naked nucleic acid is easily degraded by nucleases, and is easily eliminated through the kidneys, with a short half-life. At the same time, exogenous nucleic acid molecules are immunogenic and can easily cause immune responses in the human body. In addition, most nucleic acid drugs are negatively charged hydrophilic molecules, which are difficult to be taken up by cells through the plasma membrane. Small nucleic acid drugs will not be effective if they cannot enter cells for endocytosis. Therefore, it is urgent to develop a safe and efficient nucleic acid drug delivery vehicle in vivo.
  • Clathrin widely exists in nature, such as viral capsid, ferritin, small heat shock protein and Dps protein, etc. Its unique water solubility, high dispersion, symmetry and uniformity make it widely used in the field of drug carrier materials s concern.
  • ferritin is a representative class of natural cage-like proteins, and its 24 subunits can form a hollow spherical nanoprotein cage with an inner diameter of 8 nm and an outer diameter of 12 nm through a reversible self-assembly process.
  • Natural protein cages have many advantages as drug carriers, including: 1. Uniform nanometer size and hollow cage structure; 2. High stability, low immunogenicity and high biocompatibility; 3.
  • the drug can be loaded into the inner cavity simply by adjusting the acid-base of the buffer or adding a denaturant to mediate the disassembly and self-assembly of the protein cage; 4. It is easy to pass genetic engineering 5.
  • Certain cage proteins (such as ferritin) also have receptor-mediated endocytosis and inherent tumor targeting; cage proteins have been widely used Applied to loading small molecule drugs.
  • ferritin due to the limitations of the nature of the natural protein cage cavity, its nucleic acid loading performance is not satisfactory. For example, the charge distribution in the cavity of ferritin is dominated by negative charges, which makes it difficult to effectively load nucleic acid drugs that are also negatively charged.
  • the technical problem to be solved by the present invention is how to efficiently load small nucleic acid drugs into the inner cavity of the cage protein. Aiming at the defects of the existing cage protein variants, the present invention provides a safer and more efficient ferritin cage nanocarrier with internal positive mutation for loading small nucleic acid drugs in the inner cavity, and further provides its design method and application.
  • the first aspect of the present invention provides a method for preparing a ferritin cage nanocarrier for small nucleic acid drug delivery, the method comprising changing the charge of the ferritin cage cavity from negative to positive.
  • the ferritin "HFn" in the present invention refers to any ferritin that can form a cage structure, which can be ferritin from natural sources, or recombinantly expressed ferritin, or its mutants, which can be derived from prokaryotes , protists, fungi, plants or animals, e.g. from bacteria, fungi, insects, reptiles, avians, amphibians, fish, mammals, e.g. from rodents, ruminants, non-human primates or Humans, eg mice, rats, guinea pigs, dogs, cats, cows, horses, sheep, monkeys, gorillas, humans. from From bacteria to humans, although the ferritin amino acid sequences of different organisms have great differences, their structures are similar, and they can all form protein shell structures.
  • the ferritin is human heavy chain ferritin HFn, and its amino acid sequence is SEQ ID NO.1.
  • said altering comprises the steps of,
  • the mutation sites in the step (A) are Glu61, Glu64, Glu140 and Glu147.
  • the positively charged amino acid in the step (A) is selected from any one of arginine, lysine and histidine.
  • the replacement in step (B) is any one or more of the following,
  • the functional motif sequence with electropositive peptide is shown in SEQ ID NO.7 (GRKKRRQRRR).
  • the functional motif sequence with nucleic acid binding peptide is shown in SEQ ID NO.8 (QSTEKGAADKARRKSA).
  • the functional motif sequence with the cell penetrating peptide is shown in SEQ ID NO. 9 (YWHHHHH) or SEQ ID NO. 10 (KHHHKHHHKHHHKHHH).
  • the second aspect of the present invention provides a ferritin cage nanocarrier for small nucleic acid drug delivery prepared by the preparation method of the first aspect of the present invention, and the lumen of the ferritin cage nanocarrier is positively charged.
  • the ferritin cage nanocarrier contains mutation sites Glu61, Glu64, Glu140 and Glu147.
  • the ferritin cage nanocarrier contains any one of the amino acid sequences of SEQ ID NO.7-10.
  • the small nucleic acid drug includes but is not limited to ssDNA, ASO, siRNA, shRNA, miRNA, mRNA, IncRNA, nucleic acid aptamer and the like.
  • the amino acid sequence of the ferritin cage nanocarrier is any one or more of SEQ ID NO.2-6.
  • the small nucleic acid drug loading capacity of the ferritin cage nanocarrier is 2-8 nucleic acid molecules per protein cage.
  • the small nucleic acid drug loading capacity of the ferritin cage nanocarrier is 5-6 nucleic acid molecules per protein cage.
  • the third aspect of the present invention provides a small nucleic acid drug delivery system, which includes the small nucleic acid delivery ferritin cage nanocarrier provided by the second aspect of the present invention and the small nucleic acid drug.
  • the molar mass ratio of the ferritin cage nanocarrier to the small nucleic acid drug is 1:2-1:10, preferably, the molar mass ratio is 1:5.
  • the fourth aspect of the present invention provides a method for encapsulating small nucleic acid drugs using the small nucleic acid drug delivery system provided by the third aspect of the present invention, comprising the following steps,
  • Step S1 preparing and purifying the ferritin cage nanocarrier in the small nucleic acid drug delivery system according to claim 15;
  • Step S2 dissolving the small nucleic acid drug in DEPC water and diluting it to a certain concentration
  • Step S3 adding the ferritin cage nanocarrier obtained in step S1 into an acidic buffer solution with a pH of 1-3, and incubating at 4°C for 30-45 minutes to obtain an acid depolymerization system;
  • Step S4 add the small nucleic acid drug solution prepared in step S2 into the alkaline buffer solution with pH 9-11, mix well, add the acid depolymerization system obtained in step S3, and incubate at 4°C for 1-3 hours to reassemble A ferritin nanocage with the small nucleic acid drug loaded in the inner cavity is obtained.
  • the acid buffer solution described in step S3 is an HCl solution, preferably an HCl solution with a pH of 1.5-1.6.
  • the alkaline buffer described in step S4 includes but not limited to Na 2 CO 3 /NaHCO 3 , Na 2 CO 3 , NaHCO 3 , Tris, NaOH solution, etc., preferably, the alkaline buffer Na 2 CO 3 /NaHCO 3 solution at pH 9-10.
  • the molar mass ratio of protein cage nanocarriers and small nucleic acid drugs prepared in step S4 is 1:2-1:10, preferably, the molar mass ratio is 1:5.
  • the fifth aspect of the present invention provides the application of the small nucleic acid drug delivery ferritin cage nanocarrier described in the second aspect of the present invention and the small nucleic acid drug delivery system described in the third aspect of the present invention in drug delivery.
  • the drug is a small nucleic acid drug.
  • the small nucleic acid drug includes but not limited to ssDNA, ASO, siRNA, shRNA, miRNA, mRNA, IncRNA, nucleic acid aptamer and the like.
  • the present invention also provides the application of the ferritin cage nanocarrier for small nucleic acid drug delivery and the small nucleic acid drug delivery system in the preparation of drugs for the treatment of anti-tumor, anti-viral and related genetic diseases.
  • the present invention transforms the negatively charged inner cavity of ferritin into positively charged by means of genetic engineering (including based on amino acid mutations on its inner surface or fusion of functional peptides at the C-terminus) to construct a new nucleic acid-loaded protein nanocage carrier.
  • genetic engineering including based on amino acid mutations on its inner surface or fusion of functional peptides at the C-terminus
  • negatively charged small nucleic acid drugs can be efficiently loaded into ferritin nanocages, significantly improving the efficiency of small nucleic acid drugs.
  • the ferritin cage nanocarrier constructed by the invention can be used as a universal small nucleic acid drug loading platform and widely used in the treatment of anti-tumor, anti-virus and related gene diseases.
  • Figure 1 The inner cavity of the protein cage is electropositively mutated to realize the internal loading of small nucleic acid drugs.
  • FIG. 2 Construction and characterization of lumen-positive mutein cage nanocarriers.
  • A Schematic diagram of designing and constructing lumen positively charged protein cage nanocarriers by performing point mutations on the amino acids on the inner surface of the cage protein, that is, replacing negatively charged amino acids with positively charged amino acids;
  • B Purified wild-type HFn and mutant HFn( +) 15% SDS-PAGE analysis of protein;
  • C Native-PAGE analysis of purified wild-type HFn and mutant HFn(+) proteins;
  • D TEM characterization of mutant HFn(+) proteins;
  • E The particle size of mutant HFn(+) protein was characterized by DLS.
  • Figure 3 Characterization of protein cage nanocarriers with functional motifs modified at the C-terminus.
  • A TEM characterization and particle size analysis of the HFn(+) protein mutant modified at the C-terminus with a positive electropeptide
  • B TEM characterization and particle size analysis of the HFn(+) protein mutant with a nucleic acid-binding peptide modified at the C-terminus
  • C TEM characterization and particle size analysis of HFn(+) protein mutants with C-terminal modified cell-penetrating peptides.
  • FIG. 4 Preparation and characterization of CpG@HFn(+).
  • A Schematic diagram of loading CpG ODN into the lumen of protein cage nanocarriers based on the pH-mediated protein cage disassembly/reassembly process;
  • B TEM characterization of CpG@HFn(+);
  • C CpG@HFn (+) Native-PAGE analysis;
  • D particle size detection of CpG@HFn(+);
  • E comparison of CpG loading capacity between protein cage nanocarrier HFn(+) and wild-type HFn;
  • F after loading CpG , Comparison of protein recovery between protein cage nanocarrier HFn(+) and wild-type HFn.
  • FIG. 5 Preparation and characterization of miRNA@HFn(+).
  • A Protein cage nanocarrier HF Comparison of miRNA loading capacity between n(+) and wild-type HFn;
  • B Comparison of protein recovery between protein cage nanocarrier HFn(+) and wild-type HFn after miRNA loading.
  • Figure 6 Preparation and characterization of siRNA@HFn(+).
  • A Comparison of siRNA loading capacity between protein cage nanocarrier HFn(+) and wild-type HFn;
  • B Comparison of protein recovery between protein cage nanocarrier HFn(+) and wild-type HFn after loading siRNA.
  • FIG. 7 Evaluation of the cellular uptake efficiency and immune activation function of CpG@HFn(+).
  • A CLSM analysis of CpG uptake in DC cells;
  • B-C CLSM analysis and quantification of CpG uptake in DC cells;
  • D expression of surface activation markers CD80 and CD86 in DC cells after CpG@HFn(+) treatment
  • E-F The secretion of cytokines TNF ⁇ and IL-6 by DC cells after CpG@HFn(+) treatment.
  • Fig. 8 Antitumor efficacy and in vivo immune activation of CpG@HFn(+) on 4T1 breast cancer.
  • A-B Tumor volumes of in situ and distant tumors;
  • C body weights of mice;
  • D-E levels of cytokines IL-6 and TNF ⁇ in serum.
  • the negatively charged amino acids located on the inner surface of HFn are point-mutated by genetic engineering means, that is, the 61st, 64th, and 64th subunits of HFn subunits are specifically replaced with positively charged lysine and arginine. Glutamate at positions 140 and 147, specifically Mutation to E61K/E64R/E140K/E147K.
  • amino acid sequence (SEQ ID No.2) of HFn (+) its cDNA sequence was designed, and it was cloned into the Escherichia coli (E.coli) expression vector pET30a plasmid with NdeI and BamHI restriction enzyme sites. Sequence identification sequence is correct.
  • HFn(+) protein transform the plasmid obtained above into the expression strain BL21(DE3), and grow and amplify in LB medium containing 100mg/L kanamycin or ampicillin, and add the final concentration of 0.5mM IPTG was cultured at 30°C and 200rpm for 9.5h to induce protein expression.
  • HFn(+) protein collect the bacterial liquid, centrifuge at 4000g to collect the bacterial cells, and use 20 Resuspend in mM Tris-HCl (pH 8.0) buffer. After high-pressure homogenization, centrifuge at 12,000g to remove Escherichia coli fragments, collect the supernatant and heat it in a 72°C water bath for 15 minutes to denature and precipitate most of the impurity proteins. The supernatant was collected by centrifugation at 12000g. The supernatant was initially purified by an anion exchange column Q-Sepharose Fast Flow, and then further purified by superdex 200 molecular sieves.
  • CpG ODN is an oligonucleotide with a single-stranded DNA structure, and it is a Toll-like receptor 9 agonist.
  • the method of CpG ODN loading based on protein cage nanocarriers is described in detail below: add in HCl solution (pH 1-3) Equal volume of 10mg/mL protein cage nanocarrier HFn(+) solution, mix thoroughly, and incubate at 4°C for 30-45 minutes to mediate protein cage depolymerization in a strong acid environment.
  • the CpG ODN solution with Na 2 CO 3 /NaHCO 3 solution (pH 8-10), then add it to the acid depolymerization system of the protein cage and mix well, neutralize the system to neutral (pH 6.5-7.5), Incubate at 4°C for 1-2 hours. After taking out the sample solution Ultrafiltration was performed to remove free nucleic acids. Finally, the ssDNA and Protein kits of Qubit 4 Fluorometer were used to quantify the concentrations of CpG and ferritin, respectively, and the CpG loading rate and ferritin recovery rate of CpG@HFn(+) were calculated.
  • CpG@HFn(+) The morphology of CpG@HFn(+) was characterized by TEM; the 24-mer assembly of CpG@HFn(+) was identified by Native-PAGE electrophoresis; the particle size of CpG@HFn(+) nanoparticles was characterized by DLS.
  • the average CpG loading rate of HFn(+) with positively charged lumen was 3.4 ⁇ 0.4 CpG molecules per ferritin molecule, about 12 times that of wild-type HFn (HFn loading rate was 0.3 ⁇ 0.1 CpG molecule/per ferritin molecule), the nucleic acid loading capacity of ferritin has been greatly improved.
  • the lumen modification did not affect the stability of the ferritin cage itself, and the protein recovery rate of HFn(+) was basically the same as that of wild-type HFn.
  • Example 4 Method for loading ssRNA small nucleic acid drugs (taking miRNA as an example) on protein cage nanocarriers
  • miRNA is an oligonucleotide with a single-stranded RNA structure.
  • the method of miRNA loading based on protein cage nanocarriers is described in detail below: add an equal volume of protein cage nanocarrier HFn(+) solution to HCl solution (pH 1-3) , after mixing thoroughly, incubate at 4°C for 30-45 minutes to mediate protein cage depolymerization in a strong acid environment. Take the miRNA solution and Na 2 CO 3 /NaHCO 3 solution (pH 8-10) pre-mixed, then add it to the acid depolymerization system of the protein cage and mix well, neutralize the system to neutral (pH 6.5-7.5), 4 Incubate for 2 hours at °C.
  • the miRNA loading rate of the protein cage nanocarrier HFn(+) was 2.43 ⁇ 0.19 miRNA molecules per ferritin molecule on average, about 7 times that of wild-type HFn (HFn loading The ratio is 0.34 ⁇ 0.07 miRNA molecule/per ferritin molecule), and the nucleic acid loading capacity of ferritin has been greatly improved.
  • the lumen modification did not affect the stability of the ferritin cage itself, and the protein recovery rate of HFn(+) was basically the same as that of wild-type HFn.
  • siRNA is an oligonucleotide with a double-stranded RNA structure.
  • the method of siRNA loading based on protein cage nanocarriers is described in detail below: add an equal volume of protein cage nanocarrier HFn(+) solution to HCl solution (pH 1-2) , after mixing thoroughly, incubate at 4°C for 30-45 minutes to mediate protein cage depolymerization in a strong acid environment. Take the siRNA solution and NaOH solution (pH 8.5-9.5) pre-mixed, then add it to the acid depolymerization system of the protein cage and mix well, neutralize the system to neutral (pH 6.5-7.5), and incubate at 4°C for 1.5 hours.
  • siRNA and ferritin were quantified with BR RNA and Protein kits of Qubit 4 Fluorometer, respectively, and the siRNA loading rate and protein recovery rate of HFn(+) were calculated.
  • the experimental results were analyzed, and the results are shown in Figure 6.
  • the siRNA loading rate of the protein cage nanocarrier HFn(+) was 2.01 ⁇ 0.28 siRNA molecules/per ferritin molecule on average, which was about 7 times that of wild-type HFn (HFn loading The average rate is 0.40 ⁇ 0.08 siRNA molecule/per ferritin molecule), and the nucleic acid loading capacity of the ferritin cage has been greatly improved.
  • the lumen modification did not affect the stability of the ferritin cage itself, and the protein recovery rate of HFn(+) was basically the same as that of wild-type HFn.
  • Example 6 HFn (+) nanocarriers promote the cell uptake efficiency and immune activation function of CpG (in vitro cell verification)
  • DC cell uptake (1) Fluorescence confocal microscopy (CLSM) evaluation: first, FAM-CpG ODN molecules labeled with FAM green fluorescence were synthesized. Spread DC2.4 on a confocal dish, add Free FAM-CpG and FAM-CpG@HFn(+) to the control group and the experimental group, respectively, and the CpG concentration of both is 1 ⁇ M. After co-incubation for 1 h and 2 h, the cells were taken out, the nuclei were stained with DAPI, and the FAM CpG fluorescence inside the DC cells was observed with CLSM to characterize the cellular uptake.
  • CLSM Fluorescence confocal microscopy
  • Activation of DC cells (1) Detection of surface activation markers by flow cytometry: Plate DC2.4 in 12-well plates and co-incubate with PBS, HFn(+), CpG and CpG@HFn(+) respectively at 37°C. After 24 hours, the cells were collected, stained with FITC-CD80 and APC-CD86 fluorescent dyes, and CD80+CD86+ cells were detected by flow cytometry. (2) ELISA detection of cytokines related to cellular immune activation. Mouse bone marrow-derived DC cells (BMDC) were plated in a 24-well plate and co-acted with PBS, HFn(+), CpG and CpG@HFn(+) respectively.
  • BMDC Mouse bone marrow-derived DC cells
  • Example 7 Protein cage nanocarriers enhance the anti-tumor and immune activation functions of CpG (in vivo efficacy verification)
  • Serum cytokine detection 4T1 tumor-bearing mice were randomly divided into groups, and PBS, HFn(+), Free CpG and CpG@HFn(+) were administered intravenously to each group, and the dose of CpG was 0.5 mg/Kg. After 7 days, the mice were taken from the eyes to collect blood, and the levels of TNF- ⁇ and IL-6 in the serum were detected with ELISA kits.
  • ferritin nanocarriers were pre-labeled with fluorescent molecule Cy5.5.
  • a 4T1 subcutaneous tumor mouse model was established, and equal amounts of Cy5.5@HFn and Cy5.5@HFn(+) were injected through the tail vein. 1, 2, and 4 hours after injection, the mice were subjected to in vivo near-infrared imaging by the IVIS spectral imaging system.

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Abstract

L'invention concerne un procédé de préparation d'un vecteur de nanocage de ferritine chargé d'un médicament à petite molécule à base d'acide nucléique dans la cavité interne et une utilisation. La cavité interne chargée négativement de ferritine est modifiée pour être chargée positivement par génie génétique. Les moyens de modification comprennent la construction d'un nouveau vecteur de nanocage protéique chargé d'acide nucléique sur la base de mutations d'acides aminés dans sa surface interne ou la fusion d'un peptide fonctionnel à l'extrémité C-terminale. Un médicament à petite molécule à base d'acide nucléique chargé négativement peut être efficacement chargé dans la nanocage protéique au moyen d'une adsorption électrostatique, de telle sorte que la stabilité de l'administration in vivo et in vitro du médicament à petite molécule à base d'acide nucléique, l'efficacité de l'absorption cellulaire et l'efficacité de la thérapie ciblée sont améliorées de manière significative.
PCT/CN2023/078704 2022-03-04 2023-02-28 Vecteur de nanocage de ferritine chargé d'un médicament à petite molécule à base d'acide nucléique dans une cavité interne et utilisation WO2023165467A1 (fr)

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CN202210209855.X 2022-03-04
CN202210209855.XA CN117547618A (zh) 2022-03-04 2022-03-04 内腔装载小核酸药物的铁蛋白纳米笼载体及应用

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