WO2023165467A1 - Ferritin nanocage vector loaded with small nucleic acid drug in inner cavity and use - Google Patents
Ferritin nanocage vector loaded with small nucleic acid drug in inner cavity and use Download PDFInfo
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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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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
- A61K47/6949—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
Provided are a method for preparing a ferritin nanocage vector loaded with a small nucleic acid drug in the inner cavity and use. The negatively charged inner cavity of ferritin is modified to be positively charged by means of genetic engineering. The means of modification include construction of a novel, nucleic-acid-loaded protein nanocage vector based on amino acid mutations in the inner surface thereof or fusion of a functional peptide at the C-terminus. A negatively charged small nucleic acid drug can be efficiently loaded into the protein nanocage by means of electrostatic adsorption, so that the stability of in-vivo and in-vitro delivery of the small nucleic acid drug, the efficiency of cellular uptake and the efficacy of targeted therapy are significantly improved.
Description
本发明属于铁蛋白生物纳米材料领域，涉及内腔装载小核酸药物的铁蛋白纳米笼载体的制备方法及应用。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.
核酸药物(nucleic acid drug)可特异性针对致病基因，在基因水平上精准靶向调控治疗疾病，近年来在精准和个性化治疗领域受到了广泛关注。然而，游离的核酸药物在应用中存在着稳定差和生物利用率低的问题，进入血液之后裸核酸极易被核酸酶降解，且容易通过肾脏清除，半衰期短。同时外源核酸分子具有免疫原性，容易引起人体的免疫反应。此外，核酸药物大多为带负电的亲水性分子，难以透过细胞质膜被细胞摄取。如果不能进入细胞实现胞吞，小核酸药物将无法发挥作用。因此，亟需开发一个安全高效的核酸药物体内递送载体。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.
笼形蛋白广泛存在于自然界中，如病毒衣壳，铁蛋白、小热激蛋白和Dps蛋白等，其独特的水溶性、高分散性、对称性、均一性使其在药物载体材料领域受到广泛的关注。例如，铁蛋白是一类具有代表性的天然笼状蛋白，其24个亚基可以通过可逆自组装过程形成内径为8nm，外径为12nm的空心球形纳米蛋白笼。天然的蛋白笼作为药物载体具有众多优点，包括：1、具有均一的纳米尺寸和中空的笼状结构；2、高稳定性、低免疫原性和高生物相容性；3、可逆的自组装性质，可简单地通过调节缓冲液的酸碱或添加变性剂介导蛋白笼的解组装和自组装，进而实现药物的内腔装载；4、易于通过基因工程
手段和化学修饰方法对其进行性质及功能改造；5、某些笼状蛋白(如铁蛋白)还具有受体介导的细胞内吞作用和固有肿瘤靶向性；目前笼状蛋白已经被广泛应用于装载小分子药物。但是由于天然蛋白笼内腔性质的限制，其核酸装载表现不尽人意，如铁蛋白内腔电荷分布以负电为主导，难以有效装载同样带负电的核酸药物。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. For example, 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. Reversible self-assembly properties, 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. However, 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.
虽然目前存在少数研究对笼状蛋白进行改造以实现核酸装载的目的，包括在蛋白笼外表面融合正电肽或阳离子聚合物以借助静电作用来吸附核酸药物。但是在复杂的血液循环环境，存在血浆剪切力会提前剥离外部装载的核酸及血浆蛋白干扰的问题。因此，如何将带负电的核酸药物装载至蛋白笼内腔以进一步提高其稳定性和安全性是领域目前面临的一大瓶颈难题。Although there are currently a few studies on the modification of cage proteins to achieve the purpose of nucleic acid loading, including fusing positively charged peptides or cationic polymers on the outer surface of protein cages to adsorb nucleic acid drugs by electrostatic interaction. However, in the complex blood circulation environment, there is a problem that the plasma shear force will strip the externally loaded nucleic acid and plasma protein interference in advance. Therefore, how to load negatively charged nucleic acid drugs into the inner cavity of protein cages to further improve their stability and safety is a major bottleneck problem currently facing the field.
发明内容Contents of the invention
本发明要解决的技术问题在于如何高效地将小核酸药物装载至笼状蛋白内腔。针对现有笼状蛋白变体的缺陷，本发明提供了更加安全高效的内部正电突变的铁蛋白笼纳米载体用于内腔装载小核酸药物，并进一步提供其设计方法和应用。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.
本发明第一方面，提供了一种小核酸药物递送铁蛋白笼纳米载体的制备方法，所述方法包括将所述铁蛋白笼内腔电荷性进行由负转正的改变。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.
本发明所述铁蛋白“HFn”是指可以形成笼状结构的任何铁蛋白，其可以是天然来源的铁蛋白，也可以是重组表达的铁蛋白，或其突变体，其可以来源于原核生物、原生生物、真菌、植物或动物，例如来源于细菌、真菌、昆虫、爬行动物、禽类、两栖动物、鱼类、哺乳动物，例如来源于啮齿类动物、反刍动物、非人灵长类动物或人类，例如小鼠、大鼠、豚鼠、犬类、猫、牛、马、羊、猴、大猩猩、人。从
细菌到人类，尽管不同生物的铁蛋白氨基酸序列具有极大的差别，但其结构相似，均可以形成蛋白壳结构。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.
在某些实施例中，所述铁蛋白为人重链铁蛋白HFn，其氨基酸序列为SEQ ID NO.1。In some embodiments, the ferritin is human heavy chain ferritin HFn, and its amino acid sequence is SEQ ID NO.1.
在某些实施例中，所述改变包括以下步骤，In some embodiments, said altering comprises the steps of,
(A)将空间上分布于所述铁蛋白内表面的带负电或不带电氨基酸突变为带正电氨基酸，和/或，(A) mutating negatively charged or uncharged amino acids spatially distributed on the inner surface of said ferritin to positively charged amino acids, and/or,
(B)将铁蛋白C端的E螺旋替换为具有核酸亲和力的功能肽。(B) The E-helix at the C-terminus of ferritin was replaced with a functional peptide with nucleic acid affinity.
在某些实施例中，所述步骤(A)中突变位点为Glu61，Glu64，Glu140和Glu147。In some embodiments, the mutation sites in the step (A) are Glu61, Glu64, Glu140 and Glu147.
在某些实施例中，所述步骤(A)中带正电氨基酸选自精氨酸、赖氨酸和组氨酸的任一种。In some embodiments, the positively charged amino acid in the step (A) is selected from any one of arginine, lysine and histidine.
在某些实施例中，所述步骤(B)中的替换为以下任意一种或多种，In some embodiments, the replacement in step (B) is any one or more of the following,
(a)将所述铁蛋白C端的E螺旋替换为带有正电肽的功能性基序，(a) replacing the E-helix at the C-terminus of the ferritin with a functional motif with a positive electropeptide,
(b)将所述铁蛋白C端的E螺旋替换为带有核酸结合肽的功能性基序，(b) replacing the E-helix at the C-terminus of the ferritin with a functional motif with a nucleic acid binding peptide,
(c)将所述铁蛋白C端的E螺旋替换为带有细胞穿透肽的功能性基序。(c) Replacement of the E-helix at the C-terminus of the ferritin with a functional motif bearing a cell-penetrating peptide.
在某些实施例中，所述带有正电肽的功能性基序序列如SEQ ID NO.7(GRKKRRQRRR)所示。In some embodiments, the functional motif sequence with electropositive peptide is shown in SEQ ID NO.7 (GRKKRRQRRR).
在某些实施例中，所述带有核酸结合肽的功能性基序序列如SEQ ID NO.8(QSTEKGAADKARRKSA)所示。In some embodiments, the functional motif sequence with nucleic acid binding peptide is shown in SEQ ID NO.8 (QSTEKGAADKARRKSA).
在某些实施例中，带有细胞穿透肽的功能性基序序列如SEQ ID NO.9(YWHHHHH)或SEQ ID NO.10(KHHHKHHHKHHHKHHH)所示。
In certain embodiments, the functional motif sequence with the cell penetrating peptide is shown in SEQ ID NO. 9 (YWHHHHH) or SEQ ID NO. 10 (KHHHKHHHKHHHKHHH).
本发明第二方面，提供了一种由本发明第一方面的制备方法制备获得的小核酸药物递送铁蛋白笼纳米载体，所述铁蛋白笼纳米载体内腔带正电。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.
在某些实施例中，所述铁蛋白笼纳米载体含有突变位点为Glu61，Glu64，Glu140和Glu147。In some embodiments, the ferritin cage nanocarrier contains mutation sites Glu61, Glu64, Glu140 and Glu147.
在某些实施例中，所述铁蛋白笼纳米载体含有氨基酸序列SEQ ID NO.7-10中任意一种。In some embodiments, the ferritin cage nanocarrier contains any one of the amino acid sequences of SEQ ID NO.7-10.
在某些实施例中，所述小核酸药物为包括但不限于ssDNA、ASO、siRNA、shRNA、miRNA、mRNA、IncRNA、核酸适配体等。In some embodiments, the small nucleic acid drug includes but is not limited to ssDNA, ASO, siRNA, shRNA, miRNA, mRNA, IncRNA, nucleic acid aptamer and the like.
在某些实施例中，所述铁蛋白笼纳米载体的氨基酸序列为SEQ ID NO.2-6中任意一种或多种。In some embodiments, the amino acid sequence of the ferritin cage nanocarrier is any one or more of SEQ ID NO.2-6.
在某些实施例中，所述铁蛋白笼纳米载体的小核酸药物装载量为2-8个核酸分子/每个蛋白笼。In some embodiments, the small nucleic acid drug loading capacity of the ferritin cage nanocarrier is 2-8 nucleic acid molecules per protein cage.
在某些实施例中，所述铁蛋白笼纳米载体的小核酸药物装载量为5-6个核酸分子/每个蛋白笼。In some embodiments, the small nucleic acid drug loading capacity of the ferritin cage nanocarrier is 5-6 nucleic acid molecules per protein cage.
本发明第三方面，提供了一种小核酸药物递送系统，所述小核酸药物递送系统包括本发明第二方面提供的小核酸递送铁蛋白笼纳米载体和小核酸药物。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.
在某些实施例中，所述所述铁蛋白笼纳米载体与小核酸药物的摩尔质量比为1:2～1:10，优选的，摩尔质量比为1:5。In some embodiments, 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.
本发明第四方面，提供了一种采用本发明第三方面提供的小核酸药物递送系统包载小核酸药物的方法，包括以下步骤，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,
步骤S1、制备并纯化权利要求15所述的小核酸药物递送系统中的铁蛋白笼纳米载体；Step S1, preparing and purifying the ferritin cage nanocarrier in the small nucleic acid drug delivery system according to claim 15;
步骤S2、将所述小核酸药物溶解于DEPC水，稀释成一定浓度；
Step S2, dissolving the small nucleic acid drug in DEPC water and diluting it to a certain concentration;
步骤S3、将步骤S1获得的铁蛋白笼纳米载体加入pH 1-3的酸性缓冲液中，4℃共孵育30-45分钟，获得酸解聚体系；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;
步骤S4、将步骤S2配置的小核酸药物溶液加入pH 9-11的碱性缓冲液中，混合均匀后，加入步骤S3所得的酸解聚体系，4℃共孵育1-3小时，以重组装内腔包载有小核酸药物的铁蛋白纳米笼，即得。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.
在某些实施例中，步骤S3中所述的酸性缓冲液为HCl溶液，优选为pH 1.5-1.6的HCl溶液。In some embodiments, the acid buffer solution described in step S3 is an HCl solution, preferably an HCl solution with a pH of 1.5-1.6.
在某些实施例中，步骤S4中所述的碱性缓冲液包括但不限于Na2CO3/NaHCO3、Na2CO3、NaHCO3、Tris、NaOH溶液等，优选的，碱性缓冲液为pH 9-10的Na2CO3/NaHCO3溶液。In some embodiments, 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.
在某些实施例中，步骤S4中的蛋白笼纳米载体与小核酸药物制备的投料摩尔质量比为1:2～1:10，优选的，摩尔质量比为1:5。In some embodiments, 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.
本发明第五方面，提供了本发明第二方面所述的小核酸药物递送铁蛋白笼纳米载体、本发明第三方面所述的小核酸药物递送系统在药物递送中的应用。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.
在某些实施例中，所述药物为小核酸药物。In certain embodiments, the drug is a small nucleic acid drug.
在某些实施例中，所述小核酸药物包括但不限于ssDNA、ASO、siRNA、shRNA、miRNA、mRNA、IncRNA、核酸适配体等。In some embodiments, the small nucleic acid drug includes but not limited to ssDNA, ASO, siRNA, shRNA, miRNA, mRNA, IncRNA, nucleic acid aptamer and the like.
本发明第六方面，本发明还提供了所述的小核酸药物递送铁蛋白笼纳米载体、所述的小核酸药物递送系统在制备抗肿瘤、抗病毒及相关基因疾病治疗的药物中的应用。In the sixth aspect of the present invention, 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.
本发明对于现有技术而言，通过基因工程手段，将铁蛋白的负电内腔改造为正电(包括基于其内表面氨基酸突变或在C端融合功能肽)构建新型载核酸蛋白纳米笼载体。通过静电吸附作用，呈负电性的小核酸药物可被高效装载至铁蛋白纳米笼内部，显著提升小核酸药物的
体内外递送稳定性、细胞摄取效率和靶向治疗疗效。本发明构建的铁蛋白笼纳米载体可作为一种普适性的小核酸药物装载平台广泛应用于抗肿瘤、抗病毒及相关基因疾病治疗。Compared with the prior art, 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. Through electrostatic adsorption, negatively charged small nucleic acid drugs can be efficiently loaded into ferritin nanocages, significantly improving the efficiency of small nucleic acid drugs. In vivo and in vitro delivery stability, cellular uptake efficiency and targeted therapy efficacy. 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.
为了更清楚地说明本发明具体实施方式或现有技术中的技术方案，下面将对具体实施方式描述中所需要使用的附图作简单地介绍。In order to more clearly illustrate specific embodiments of the present invention or technical solutions in the prior art, the following briefly introduces the accompanying drawings that are used in the description of specific embodiments.
图1将蛋白笼内腔进行正电突变以实现小核酸药物的内部装载。Figure 1. The inner cavity of the protein cage is electropositively mutated to realize the internal loading of small nucleic acid drugs.
图2内腔正电突变蛋白笼纳米载体的构建及表征。(A)通过对笼状蛋白内表面氨基酸进行点突变，即用正电氨基酸替代负电氨基酸，设计构建内腔正电蛋白笼纳米载体的示意图；(B)纯化后野生型HFn及突变体HFn(+)蛋白的15％SDS-PAGE分析；(C)纯化后野生型HFn及突变体HFn(+)蛋白的Native-PAGE分析；(D)突变体HFn(+)蛋白的TEM表征；(E)通过DLS表征突变体HFn(+)蛋白粒径。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.
图3C端修饰功能基序的蛋白笼纳米载体的表征。(A)C端修饰正电肽的HFn(+)蛋白突变体的TEM表征和粒径分析；(B)C端修饰核酸结合肽的HFn(+)蛋白突变体的TEM表征和粒径分析；(C)C端修饰细胞穿透肽的HFn(+)蛋白突变体的TEM表征和粒径分析。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.
图4CpG@HFn(+)的制备及表征。(A)基于pH介导的蛋白笼解组装/再组装过程，将CpG ODN装载蛋白笼纳米载体内腔的示意图；(B)CpG@HFn(+)的透射电镜表征；(C)CpG@HFn(+)的Native-PAGE分析；(D)CpG@HFn(+)的粒径检测；(E)蛋白笼纳米载体HFn(+)与野生型HFn的CpG装载能力对比；(F)装载CpG后，蛋白笼纳米载体HFn(+)与野生型HFn的蛋白回收率对比。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.
图5miRNA@HFn(+)的制备及表征。(A)蛋白笼纳米载体HF
n(+)与野生型HFn的miRNA装载能力对比；(B)装载miRNA后，蛋白笼纳米载体HFn(+)与野生型HFn的蛋白回收率对比。Figure 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.
图6siRNA@HFn(+)的制备及表征。(A)蛋白笼纳米载体HFn(+)与野生型HFn的siRNA装载能力对比；(B)装载siRNA后，蛋白笼纳米载体HFn(+)与野生型HFn的蛋白回收率对比。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.
图7CpG@HFn(+)的细胞摄取效率及免疫激活功能评价。(A)CLSM分析DC细胞中CpG摄取情况；(B-C)CLSM分析及定量DC细胞中CpG摄取情况；(D)CpG@HFn(+)处理后DC细胞的表面活化标志分子CD80和CD86的表达情况；(E-F)CpG@HFn(+)处理后DC细胞分泌细胞因子TNFα和IL-6情况。Figure 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 TNFα and IL-6 by DC cells after CpG@HFn(+) treatment.
图8CpG@HFn(+)对4T1乳腺癌的抗肿瘤功效及体内免疫激活作用。(A-B)原位瘤和远位瘤的肿瘤体积；(C)小鼠体重；(D-E)血清中细胞因子IL-6和TNFα水平。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 TNFα in serum.
图9HFn(+)的体内肿瘤靶向蓄积情况。Figure 9 In vivo tumor-targeted accumulation of HFn(+).
下面将对本发明技术方案的实施例进行详细的描述。以下实施例仅用于更加清楚地说明本发明的技术方案，因此只是作为示例，而不能以此来限制本发明的保护范围。需要注意的是，除非另有说明，本申请使用的技术术语或者科学术语应当为本发明所属领域技术人员所理解的通常意义。Embodiments of the technical solution of the present invention will be described in detail below. The following examples are only used to illustrate the technical solution of the present invention more clearly, so they are only examples, and should not be used to limit the protection scope of the present invention. It should be noted that, unless otherwise specified, the technical terms or scientific terms used in this application shall have the usual meanings understood by those skilled in the art to which the present invention belongs.
实施例1内腔正点突变铁蛋白笼纳米载体HFn(+)重组质粒的构建Example 1 Construction of a positive point mutation ferritin cage nanocarrier HFn (+) recombinant plasmid in the lumen
基于蛋白笼内表面氨基酸突变进行设计：通过基因工程手段对位于HFn内表面的负电氨基酸进行点突变，即用带正电的赖氨酸和精氨酸特异性替换HFn亚基第61、64、140和147位的谷氨酸，具体
突变为E61K/E64R/E140K/E147K。根据HFn(+)的氨基酸序列(SEQ ID No.2)设计出其cDNA序列，将其克隆到具有NdeI和BamHI限制酶切位点的大肠杆菌(E.coli)表达载体pET30a质粒上，经DNA测序鉴定序列正确。Design based on amino acid mutations on the inner surface of the protein cage: 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. According to the 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.
基于铁蛋白C端修饰正电肽功能性基序进行设计：通过基因工程手段将铁蛋白C端的E螺旋替换为带有正电肽的功能性基序。根据HFn(+)的氨基酸序列(SEQ ID No.3)设计出其cDNA序列，将其克隆到具有NdeI和BamHI限制酶切位点的E.coli表达载体pET22b质粒上，经DNA测序鉴定序列正确。Design based on the functional motif of the positive electropeptide modified at the C-terminus of ferritin: the E-helix at the C-terminus of ferritin was replaced by a functional motif with the electropositive peptide by genetic engineering. According to the amino acid sequence (SEQ ID No.3) of HFn (+), its cDNA sequence was designed, and it was cloned into the E.coli expression vector pET22b plasmid with NdeI and BamHI restriction enzyme sites, and the sequence was confirmed to be correct by DNA sequencing .
基于铁蛋白C端修饰核酸结合肽功能性基序进行设计：通过基因工程手段将铁蛋白C端的E螺旋替换为带有核酸结合肽的功能性基序。根据HFn(+)的氨基酸序列(SEQ ID No.4)设计出其cDNA序列，将其克隆到具有NdeI和BamHI限制酶切位点的E.coli表达载体pET22b质粒上，经DNA测序鉴定序列正确。Design based on the functional motif of the nucleic acid-binding peptide modified at the C-terminus of ferritin: the E-helix at the C-terminus of ferritin was replaced by a functional motif with a nucleic acid-binding peptide by means of genetic engineering. According to the amino acid sequence (SEQ ID No.4) of HFn (+), its cDNA sequence was designed, and it was cloned into the E.coli expression vector pET22b plasmid with NdeI and BamHI restriction sites, and the sequence was confirmed to be correct by DNA sequencing .
基于铁蛋白C端修饰细胞穿透肽功能性基序进行设计：通过基因工程手段将铁蛋白C端的E螺旋替换为带有细胞穿透肽的功能性基序。根据HFn(+)的氨基酸序列(SEQ ID No.5)设计出其cDNA序列，将其克隆到具有NdeI和BamHI限制酶切位点的E.coli表达载体pET22b质粒上，经DNA测序鉴定序列正确。Design based on the functional motif of cell-penetrating peptide modified at the C-terminus of ferritin: the E-helix at the C-terminus of ferritin was replaced by a functional motif with a cell-penetrating peptide through genetic engineering. According to the amino acid sequence (SEQ ID No.5) of HFn (+), its cDNA sequence was designed, and it was cloned into the E.coli expression vector pET22b plasmid with NdeI and BamHI restriction sites, and the sequence was confirmed to be correct by DNA sequencing .
实施例2内腔正点突变铁蛋白笼纳米载体的构建Example 2 Construction of Lumen Positive Point Mutation Ferritin Cage Nanocarrier
HFn(+)蛋白表达：将上述所得质粒转化入表达菌株BL21(DE3)中，并在含100mg/L卡那霉素或氨苄霉素的LB培养基中生长扩增，添加终浓度为0.5mM IPTG，在30℃，200rpm环境下培养9.5h进行蛋白诱导表达。Expression of 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(+)蛋白纯化：收集菌液，4000g离心收集菌体，菌体用20
mM Tris-HCl(pH 8.0)缓冲液重悬。高压均质破菌后，12000g离心去除大肠杆菌残片，收集上清液置于72℃水浴中加热15分钟，变性沉淀绝大部分杂蛋白。12000g离心收集上清。上清液先经过阴离子交换柱Q-Sepharose Fast Flow进行初步纯化，再用superdex 200分子筛进行进一步纯化。用BCA法测定野生型HFn及突变体HFn(+)的蛋白浓度，用15％SDS-PAGE电泳鉴定HFn及HFn(+)纯度，用Native-PAGE电泳鉴定突变体HFn(+)的24聚体组装情况，用透射电子显微镜(TEM)和动态光散射(DLS)进一步表征HFn(+)的形貌及粒径大小。Purification of 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. Determine the protein concentration of wild-type HFn and mutant HFn(+) by BCA method, identify the purity of HFn and HFn(+) by 15% SDS-PAGE electrophoresis, and identify the 24-mer of mutant HFn(+) by Native-PAGE electrophoresis Assembled, the morphology and particle size of HFn(+) were further characterized by transmission electron microscopy (TEM) and dynamic light scattering (DLS).
对实验结果进行分析，如图2和图3显示，SDS-PAGE结果显示表明，突变体HFn(+)的蛋白条带出现在21kDa分子量处，与理论值保持一致。在Native-PAGE胶上，突变体HFn(+)和野生型HFn的蛋白条带处于相似位置，说明内腔改造并不会影响铁蛋白的组装能力，HFn(+)亚基可顺利自组装形成24聚体。TEM和DLS分析显示内腔正电氨基酸突变以及C端修饰的突变体HFn(+)具有直径13nm左右的纳米笼状结构。The experimental results were analyzed, as shown in Figure 2 and Figure 3, the SDS-PAGE results showed that the protein band of the mutant HFn(+) appeared at the molecular weight of 21kDa, which was consistent with the theoretical value. On the Native-PAGE gel, the protein bands of mutant HFn(+) and wild-type HFn are in similar positions, indicating that the lumen modification will not affect the assembly ability of ferritin, and the HFn(+) subunit can self-assemble smoothly 24-mer. TEM and DLS analysis showed that the mutant HFn(+) with lumen positively charged amino acid mutation and C-terminal modification had a nanocage structure with a diameter of about 13nm.
实施例3蛋白笼纳米载体装载ssDNA小核酸药物(以CpG为例)的方法Example 3 Method for Loading ssDNA Small Nucleic Acid Drugs (Taking CpG as an Example) on Protein Cage Nanocarriers
CpG ODN是具有单链DNA结构的寡聚核苷酸，是Toll样受体9激动剂，以下具体描述基于蛋白笼纳米载体进行CpG ODN包载的方法：在HCl溶液(pH 1-3)加入等体积10mg/mL蛋白笼纳米载体HFn(+)溶液，充分混匀后，4℃共孵育30-45分钟，在强酸环境下介导蛋白笼解聚。取CpG ODN溶液与Na2CO3/NaHCO3溶液(pH 8-10)预混，随后加入到蛋白笼的酸解聚体系中充分混匀，中和体系至中性(pH 6.5-7.5)，4℃共孵育1-2小时。取出后对样品溶液
进行超滤以除去游离核酸。最后，用Qubit 4 Fluorometer的ssDNA和Protein试剂盒分别定量CpG及铁蛋白的浓度，并计算CpG@HFn(+)的CpG装载率及铁蛋白回收率。用TEM表征CpG@HFn(+)的形貌；用Native-PAGE电泳鉴定CpG@HFn(+)的24聚体组装情况；用DLS表征CpG@HFn(+)纳米粒的粒径大小。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. Pre-mix 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. 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.
对实验结果进行分析，如图4所示，TEM表征和Native-PAGE鉴定结果均表明，在基于pH法内腔装载CpG ODN之后，CpG@HFn(+)依旧可以成功再组装成24聚体的纳米笼状结构。DLS分析表明CpG@HFn(+)的纳米尺寸相较于未装载CpG的HFn(+)有小幅度增大，平均粒径在14nm左右。在核酸能力方面，内腔正电改造的HFn(+)的CpG装载率平均为3.4±0.4个CpG分子/每铁蛋白分子，是野生型HFn的12倍左右(HFn装载率是0.3±0.1个CpG分子/每铁蛋白分子)，铁蛋白的核酸装载能力得到了大幅度提升。与此同时，内腔改造不影响铁蛋白笼自身的稳定性，HFn(+)的蛋白回收率基本和野生型HFn保持一致。Analysis of the experimental results, as shown in Figure 4, both TEM characterization and Native-PAGE identification results show that CpG@HFn(+) can still be successfully reassembled into a 24-mer after loading CpG ODN in the cavity based on the pH method. nanocage structure. DLS analysis showed that the nanometer size of CpG@HFn(+) was slightly larger than that of HFn(+) without CpG, and the average particle size was around 14nm. In terms of nucleic acid ability, 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. At the same time, 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.
实施例4蛋白笼纳米载体装载ssRNA小核酸药物(以miRNA为例)的方法Example 4 Method for loading ssRNA small nucleic acid drugs (taking miRNA as an example) on protein cage nanocarriers
miRNA是具有单链RNA结构的寡聚核苷酸，以下具体描述基于蛋白笼纳米载体进行miRNA包载的方法：在HCl溶液(pH 1-3)加入等体积蛋白笼纳米载体HFn(+)溶液，充分混匀后，4℃共孵育30-45分钟，在强酸环境下介导蛋白笼解聚。取miRNA溶液与Na2CO3/NaHCO3溶液(pH 8-10)预混，随后加入到蛋白笼的酸解聚体系中充分混匀，中和体系至中性(pH 6.5-7.5)，4℃共孵育2小时。取出后对样品溶液进行超滤以除去游离miRNA药物。最后，用Qubit 4 Fluorometer的micro RNA和Protein试剂盒分别定量miRNA及
铁蛋白的浓度，并计算miRNA@HFn(+)的miRNA装载率及铁蛋白回收率。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. After removal, the sample solution was subjected to ultrafiltration to remove free miRNA drugs. Finally, the micro RNA and Protein kits of Qubit 4 Fluorometer were used to quantify miRNA and The concentration of ferritin was calculated, and the miRNA loading rate and ferritin recovery rate of miRNA@HFn(+) were calculated.
对实验结果进行分析，结果如图5所示，蛋白笼纳米载体HFn(+)的miRNA装载率平均为2.43±0.19个miRNA分子/每铁蛋白分子，是野生型HFn的7倍左右(HFn装载率是0.34±0.07个miRNA分子/每铁蛋白分子)，铁蛋白的核酸装载能力得到了大幅度提升。与此同时，内腔改造不影响铁蛋白笼自身的稳定性，HFn(+)的蛋白回收率基本和野生型HFn保持一致。The experimental results were analyzed, as shown in Figure 5, 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. At the same time, 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.
实施例5蛋白笼纳米载体装载dsRNA小核酸药物(以siRNA为例)的方法Example 5 Method for Loading dsRNA Small Nucleic Acid Drugs (taking siRNA as an Example) on Protein Cage Nanocarriers
siRNA是具有双链RNA结构的寡聚核苷酸，以下具体描述基于蛋白笼纳米载体进行siRNA包载的方法：在HCl溶液(pH 1-2)加入等体积蛋白笼纳米载体HFn(+)溶液，充分混匀后，4℃共孵育30-45分钟，在强酸环境下介导蛋白笼解聚。取siRNA溶液与NaOH溶液(pH 8.5-9.5)预混，随后加入到蛋白笼的酸解聚体系中充分混匀，中和体系至中性(pH 6.5-7.5)，4℃共孵育1.5小时。取出后对样品溶液进行超滤以除去游离siRNA药物。最后，用Qubit 4 Fluorometer的BR RNA和Protein试剂盒分别定量siRNA及铁蛋白的浓度，并计算HFn(+)的siRNA装载率及蛋白回收率。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. After removal, the sample solution was subjected to ultrafiltration to remove free siRNA drugs. Finally, the concentrations of 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.
对实验结果进行分析，结果如图6所示，蛋白笼纳米载体HFn(+)的siRNA装载率平均为2.01±0.28个siRNA分子/每铁蛋白分子，是野生型HFn的7倍左右(HFn装载率平均是0.40±0.08个siRNA分子/每铁蛋白分子)，铁蛋白笼的核酸装载能力得到了大幅度提升。与此同时，内腔改造不影响铁蛋白笼自身的稳定性，HFn(+)的蛋白回收率基本和野生型HFn保持一致。
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. At the same time, 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.
实施例6 HFn(+)纳米载体对CpG的细胞摄取效率及免疫激活功能的促进(体外细胞验证)Example 6 HFn (+) nanocarriers promote the cell uptake efficiency and immune activation function of CpG (in vitro cell verification)
DC细胞摄取：(1)荧光共聚焦显微镜(CLSM)法评价：首先合成FAM绿色荧光标记的FAM-CpG ODN分子。将DC2.4铺于共聚焦小皿，对照组和实验组分别加入Free FAM-CpG和FAM-CpG@HFn(+)，其中二者CpG浓度均为1μM。在共孵育1h和2h后分别取出，用DAPI对细胞核进行染色，用CLSM观察DC细胞内部的FAM CpG荧光以表征细胞摄取情况。(2)流式细胞法评价：将DC2.4铺板12孔板，与Free FAM-CpG和CpG@HFn(+)置于37℃共孵育4h后收集各组细胞，用流式细胞仪检测FAM-CpG荧光并进行定量分析。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 μM. 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. (2) Evaluation by flow cytometry: Plate DC2.4 on a 12-well plate, incubate with Free FAM-CpG and CpG@HFn(+) at 37°C for 4 hours, collect cells in each group, and detect FAM by flow cytometry -CpG fluorescence and quantitative analysis.
DC细胞激活：(1)流式细胞法检测表面活化Marker：将DC2.4铺板12孔板，分别与PBS、HFn(+)、CpG以及CpG@HFn(+)在37℃共孵育。24h后收集细胞，用FITC-CD80和APC-CD86荧光染料进行细胞染色，用流式细胞仪检测CD80+CD86+细胞。(2)ELISA检测细胞免疫激活相关的细胞因子。将小鼠骨髓来源DC细胞(BMDC)铺于24孔板，分别与PBS、HFn(+)、CpG以及CpG@HFn(+)共作用。37℃孵育8h(用于TNF-α)或24h(用于IL-6)后，吸出各孔上清，用ELISA试剂盒检测细胞培养上清液中TNFα及IL-6浓度。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. After incubation at 37°C for 8h (for TNF-α) or 24h (for IL-6), the supernatant of each well was aspirated, and the concentrations of TNFα and IL-6 in the cell culture supernatant were detected with an ELISA kit.
对实验结果进行分析，结果如图7所示，从CLSM和流式分析结果可见，由于亲水性和负电性，游离核酸的难以透过细胞质膜被细胞摄取，因此在游离CpG组的DC细胞中几乎观察不到FAM CpG荧光。CpG@HFn(+)实验组中，CpG借助于蛋白笼纳米载体的细胞摄取作用，更有效透过细胞质膜，可在DC细胞内部观察到明显FAM
CpG荧光。流式定量分析显示DC细胞中CpG@HFn(+)的CpG摄取量是游离CpG ODN的4倍。同时，相较于游离CpG，CpG@HFn(+)处理后DC细胞表面活化标志分子CD80和CD86表达明显上调，免疫激活细胞因子TNFα和IL-6分泌量也显著增多，说明蛋白笼纳米载体HFn(+)可以有效促进CpG发挥免疫激活的生物学作用。The results of the experiment were analyzed, as shown in Figure 7. From the results of CLSM and flow cytometry analysis, it can be seen that due to hydrophilicity and negative charge, it is difficult for free nucleic acids to permeate the plasma membrane and be taken up by cells. Therefore, DC cells in the free CpG group FAM CpG fluorescence was hardly observed in . In the CpG@HFn(+) experimental group, CpG penetrates the plasma membrane more effectively through the cell uptake of protein cage nanocarriers, and obvious FAM can be observed inside DC cells CpG fluorescence. Flow cytometry analysis showed that the CpG uptake of CpG@HFn(+) in DC cells was 4 times that of free CpG ODN. At the same time, compared with free CpG, after CpG@HFn(+) treatment, the expression of CD80 and CD86 on the surface of DC cells was significantly up-regulated, and the secretion of immune activation cytokines TNFα and IL-6 was also significantly increased, indicating that the protein cage nanocarrier HFn (+) can effectively promote CpG to play the biological role of immune activation.
实施例7蛋白笼纳米载体增强CpG的抗肿瘤及免疫激活功能(体内药效验证)Example 7 Protein cage nanocarriers enhance the anti-tumor and immune activation functions of CpG (in vivo efficacy verification)
抗肿瘤药效评价(双侧瘤模型)：在Balb/C雌鼠的左侧背部皮下注射1×106个4T1细胞/只，构建4T1皮下瘤模型。将4T1荷瘤小鼠随机分组，并分别静脉给药PBS、HFn(+)、Free CpG和CpG@HFn(+)，其中CpG给药剂量为0.5mg/Kg。给药后24h，再在右侧背部皮下注射1×105个4T1细胞/只，构建4T1远位瘤。每隔两天对小鼠的双侧肿瘤体积及体重进行检测记录。Evaluation of antitumor efficacy (bilateral tumor model): 1×10 6 4T1 cells/mouse were subcutaneously injected into the left back of Balb/C female mice to construct a 4T1 subcutaneous tumor model. The 4T1 tumor-bearing mice were randomly divided into groups, and PBS, HFn(+), Free CpG and CpG@HFn(+) were administered intravenously, and the dose of CpG was 0.5 mg/Kg. 24 hours after administration, 1×10 5 4T1 cells/mouse were subcutaneously injected on the right back to construct 4T1 distant tumors. The bilateral tumor volume and body weight of the mice were detected and recorded every two days.
血清细胞因子检测：取4T1荷瘤小鼠，随机分组，各组分别静脉给药PBS、HFn(+)、Free CpG和CpG@HFn(+)，其中CpG给药剂量为0.5mg/Kg。7天后对小鼠进行摘眼球取血，用ELISA试剂盒对血清中TNF-α和IL-6水平进行检测。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-α and IL-6 in the serum were detected with ELISA kits.
对实验结果进行分析，如图8所示，体内抗肿瘤药效结果表明，游离CpG由于血浆稳定性差和生物利用率低，在体内未显示出肿瘤抑制作用。虽然HFn(+)载体本身不具有抗肿瘤作用，但是以HFn(+)作为核酸载体的CpG@HFn(+)对原位肿瘤表现出抗肿瘤效果，同时具有远端肿瘤抑制效应，可以观察到对远位瘤的生长抑制作用。血清中细胞因子检测表明，相较于游离CpG，CpG@HFn(+)可以更有效引发全身性免疫激活。此外，铁蛋白笼纳米载体HFn(+)和CpG@HFn(+)具有良好的体内安全性，均未引起体重减轻等副作用。
Analyzing the experimental results, as shown in Figure 8, the in vivo anti-tumor drug efficacy results showed that free CpG did not show tumor-inhibitory effect in vivo due to poor plasma stability and low bioavailability. Although the HFn(+) carrier itself does not have an anti-tumor effect, CpG@HFn(+) using HFn(+) as a nucleic acid carrier exhibits an anti-tumor effect on orthotopic tumors, and at the same time has a distant tumor inhibitory effect, which can be observed Growth inhibitory effect on distant tumors. The detection of cytokines in serum showed that, compared with free CpG, CpG@HFn(+) could trigger systemic immune activation more effectively. In addition, the ferritin cage nanocarriers HFn(+) and CpG@HFn(+) had good in vivo safety, and neither caused side effects such as weight loss.
实施例8蛋白笼纳米载体HFn(+)的体内肿瘤靶向性Example 8 In vivo tumor targeting of protein cage nanocarrier HFn(+)
为了对HFn(+)进行体内生物分布研究，用荧光分子Cy5.5预先对铁蛋白纳米载体进行标记。建立4T1皮下瘤小鼠模型，通过尾静脉注射等量的Cy5.5@HFn和Cy5.5@HFn(+)。注射1、2和4小时后，通过IVIS光谱成像系统对小鼠进行在体近红外成像。For in vivo biodistribution studies of HFn(+), 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.
体内分析结果表明，如图9所示，仅对内腔进行改造并不会影响铁蛋白外表面对肿瘤组织的特异性识别，HFn(+)保持有和野生型HFn一样的肿瘤靶向蓄积能力。The in vivo analysis results show that, as shown in Figure 9, only the modification of the lumen does not affect the specific recognition of the ferritin surface to tumor tissue, and HFn(+) maintains the same tumor-targeted accumulation ability as wild-type HFn .
最后应说明的是：以上各实施例仅用以说明本发明的技术方案，而非对其限制；尽管参照前述各实施例对本发明进行了详细的说明，本领域的普通技术人员应当理解：其依然可以对前述各实施例所记载的技术方案进行修改，或者对其中部分或者全部技术特征进行等同替换；而这些修改或者替换，并不使相应技术方案的本质脱离本发明各实施例技术方案的范围，其均应涵盖在本发明的权利要求和说明书的范围当中。
Finally, it should be noted that: the above embodiments are only used to illustrate the technical solutions of the present invention, rather than limiting them; although the present invention has been described in detail with reference to the foregoing embodiments, those of ordinary skill in the art should understand that: It is still possible to modify the technical solutions described in the foregoing embodiments, or perform equivalent replacements for some or all of the technical features; and these modifications or replacements do not make the essence of the corresponding technical solutions deviate from the technical solutions of the various embodiments of the present invention. All of them should be covered by the scope of the claims and description of the present invention.
Claims (24)
- 一种小核酸药物递送铁蛋白笼纳米载体的制备方法，其特征在于，所述方法包括将所述铁蛋白笼内腔电荷性进行由负转正的改变。A method for preparing ferritin cage nanocarriers for delivery of small nucleic acid drugs, characterized in that the method includes changing the charge of the inner cavity of the ferritin cage from negative to positive.
- 根据权利要求1所述的制备方法，其特征在于，所述改变包括以下步骤，The preparation method according to claim 1, wherein said change comprises the following steps,(A)将空间上分布于所述铁蛋白内表面的带负电或不带电氨基酸突变为带正电氨基酸，和/或，(A) mutating negatively charged or uncharged amino acids spatially distributed on the inner surface of said ferritin to positively charged amino acids, and/or,(B)将铁蛋白C端的E螺旋替换为具有核酸亲和力的功能肽。(B) The E-helix at the C-terminus of ferritin was replaced with a functional peptide with nucleic acid affinity.
- 根据权利要求2所述的制备方法，其特征在于，所述步骤(A)中突变位点为Glu61，Glu64，Glu140和Glu147。The preparation method according to claim 2, characterized in that the mutation sites in the step (A) are Glu61, Glu64, Glu140 and Glu147.
- 根据权利要求2所述的制备方法，其特征在于，所述步骤(A)中带正电氨基酸选自精氨酸、赖氨酸和组氨酸的任一种。The preparation method according to claim 2, characterized in that, in the step (A), the positively charged amino acid is selected from any one of arginine, lysine and histidine.
- 根据权利要求2所述的制备方法，其特征在于，所述步骤(B)中的替换为以下任意一种或多种，The preparation method according to claim 2, characterized in that, the replacement in the step (B) is any one or more of the following,(a)将所述铁蛋白C端的E螺旋替换为带有正电肽的功能性基序，(a) replacing the E-helix at the C-terminus of the ferritin with a functional motif with a positive electropeptide,(b)将所述铁蛋白C端的E螺旋替换为带有核酸结合肽的功能性基序，(b) replacing the E-helix at the C-terminus of the ferritin with a functional motif with a nucleic acid binding peptide,(c)将所述铁蛋白C端的E螺旋替换为带有细胞穿透肽的功能性基序。(c) Replacement of the E-helix at the C-terminus of the ferritin with a functional motif bearing a cell-penetrating peptide.
- 根据权利5所述的制备方法，其特征在于，所述带有正电肽的功能性基序序列如SEQ ID NO.7(GRKKRRQRRR)所示。According to the preparation method described in right 5, it is characterized in that, the functional motif sequence with electropositive peptide is as shown in SEQ ID NO.7 (GRKKRRQRRR).
- 根据权利5所述的制备方法，其特征在于，所述带有核酸结合肽的功能性基序序列如SEQ ID NO.8(QSTEKGAADKARRKSA)所示。The preparation method according to claim 5, wherein the functional motif sequence with nucleic acid binding peptide is shown in SEQ ID NO.8 (QSTEKGAADKARRKSA).
- 根据权利5所述的制备方法，其特征在于，带有细胞穿透肽的功能性基序序列如SEQ ID NO.9(YWHHHHH)或SEQ ID NO.10 (KHHHKHHHKHHHKHHH)所示。The preparation method according to claim 5, characterized in that the functional motif sequence with cell penetrating peptides such as SEQ ID NO.9 (YWHHHHH) or SEQ ID NO.10 (KHHHKHHHKHHHKHHH).
- 一种权利要求1-8任一所述的制备方法制备获得的小核酸药物递送铁蛋白笼纳米载体，其特征在于，所述铁蛋白笼纳米载体内腔带正电。A ferritin cage nanocarrier for small nucleic acid drug delivery prepared by the preparation method according to any one of claims 1-8, characterized in that the lumen of the ferritin cage nanocarrier is positively charged.
- 根据权利要求9所述的小核酸药物递送铁蛋白笼纳米载体，其特征在于，所述铁蛋白笼纳米载体含有突变位点为Glu61，Glu64，Glu140和Glu147。The ferritin cage nanocarrier for small nucleic acid drug delivery according to claim 9, wherein the ferritin cage nanocarrier contains mutation sites Glu61, Glu64, Glu140 and Glu147.
- 根据权利要求9所述的小核酸药物递送铁蛋白笼纳米载体，其特征在于，所述铁蛋白笼纳米载体含有氨基酸序列SEQ ID NO.7-10中任意一种或多种。The ferritin cage nanocarrier for small nucleic acid drug delivery according to claim 9, wherein the ferritin cage nanocarrier contains any one or more of the amino acid sequences of SEQ ID NO.7-10.
- 根据权利要求9所述的小核酸药物递送铁蛋白笼纳米载体，其特征在于，所述小核酸药物为包括但不限于ssDNA、ASO、siRNA、shRNA、miRNA、mRNA、IncRNA、核酸适配体等。The ferritin cage nanocarrier for delivery of small nucleic acid drugs according to claim 9, wherein the small nucleic acid drugs include but are not limited to ssDNA, ASO, siRNA, shRNA, miRNA, mRNA, IncRNA, nucleic acid aptamers, etc. .
- 根据权利要求9所述的小核酸药物递送铁蛋白笼纳米载体，其特征在于，所述铁蛋白笼纳米载体的氨基酸序列为SEQ ID NO.2-6中任意一种。The ferritin cage nanocarrier for small nucleic acid drug delivery according to claim 9, wherein the amino acid sequence of the ferritin cage nanocarrier is any one of SEQ ID NO.2-6.
- 根据权利要求9所述的小核酸药物递送铁蛋白笼纳米载体，其特征在于，所述铁蛋白笼纳米载体的小核酸药物装载量为2-8个核酸分子/每个蛋白笼，优选为5-6个。The ferritin cage nanocarrier for small nucleic acid drug delivery according to claim 9, characterized in that, the small nucleic acid drug loading capacity of the ferritin cage nanocarrier is 2-8 nucleic acid molecules/each protein cage, preferably 5 -6 pcs.
- 一种小核酸药物递送系统，其特征在于，所述小核酸药物递送系统包括权利要求9-14任一所述的小核酸递送铁蛋白笼纳米载体和小核酸药物。A small nucleic acid drug delivery system, characterized in that the small nucleic acid drug delivery system comprises the small nucleic acid delivery ferritin cage nanocarrier and the small nucleic acid drug according to any one of claims 9-14.
- 根据权利要求15所述的递送系统，其特征在于，所述铁蛋白笼纳米载体与小核酸药物的摩尔质量比为1:2～1:10，优选的，摩尔质量比为1:5。 The delivery system according to claim 15, wherein 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.
- 一种采用权利要求15-16任一所述的小核酸药物递送系统包载小核酸药物的方法，其特征在于，包括以下步骤，A method for encapsulating small nucleic acid drugs using the small nucleic acid drug delivery system according to any one of claims 15-16, characterized in that it comprises the following steps,步骤S1、制备并纯化权利要求15所述的小核酸药物递送系统中的铁蛋白笼纳米载体；Step S1, preparing and purifying the ferritin cage nanocarrier in the small nucleic acid drug delivery system according to claim 15;步骤S2、将所述小核酸药物溶解于DEPC水，稀释成一定浓度；Step S2, dissolving the small nucleic acid drug in DEPC water and diluting it to a certain concentration;步骤S3、将步骤S1获得的铁蛋白笼纳米载体加入pH 1-3的酸性缓冲液中，4℃共孵育30-45分钟，获得酸解聚体系；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;步骤S4、将步骤S2配置的小核酸药物溶液加入pH 9-11的碱性缓冲液中，混合均匀后，加入步骤S3所得的酸解聚体系，4℃共孵育1-3小时，以重组装内腔包载有小核酸药物的铁蛋白纳米笼，即得。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.
- 根据权利要求17所述的方法，其特征在于，步骤S3中所述的酸性缓冲液为HCl溶液，优选为pH 1.5-1.6的HCl溶液。The method according to claim 17, characterized in that, the acidic buffer solution described in step S3 is an HCl solution, preferably an HCl solution with a pH of 1.5-1.6.
- 根据权利要求17所述的方法，其特征在于，步骤S4中所述的碱性缓冲液包括但不限于Na2CO3/NaHCO3、Na2CO3、NaHCO3、Tris、NaOH溶液等，优选的，碱性缓冲液为pH 9-10的Na2CO3/NaHCO3溶液。The method according to claim 17, characterized in that the alkaline buffer 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 Yes, the alkaline buffer is a Na 2 CO 3 /NaHCO 3 solution with a pH of 9-10.
- 根据权利要求17所述的方法，其特征在于，步骤S4中的铁蛋白笼纳米载体与小核酸药物制备的投料摩尔质量比为1:2～1:10，优选的，摩尔质量比为1:5。The method according to claim 17, characterized in that, the molar mass ratio of ferritin cage nanocarrier and small nucleic acid drug preparation in step S4 is 1:2～1:10, preferably, the molar mass ratio is 1: 5.
- 权利要求9-14任一所述的小核酸药物递送铁蛋白笼纳米载体、权利要求15-16任一所述的小核酸药物递送系统在药物递送中的应用。Application of the small nucleic acid drug delivery ferritin cage nanocarrier according to any one of claims 9-14 and the small nucleic acid drug delivery system described in any one of claims 15-16 in drug delivery.
- 根据权利要求21所述的应用，其特征在于，所述药物为小核酸药物。The use according to claim 21, characterized in that the drug is a small nucleic acid drug.
- 根据权利要求21所述的应用，其特征在于，所述小核酸药物包括 但不限于ssDNA、ASO、siRNA、shRNA、miRNA、mRNA、IncRNA、核酸适配体等。The application according to claim 21, wherein the small nucleic acid drug comprises But not limited to ssDNA, ASO, siRNA, shRNA, miRNA, mRNA, IncRNA, nucleic acid aptamer, etc.
- 权利要求9-14任一所述的小核酸药物递送铁蛋白笼纳米载体、权利要求15-16任一所述的小核酸药物递送系统在制备抗肿瘤、抗病毒及相关基因疾病治疗的药物中的应用。 The small nucleic acid drug delivery ferritin cage nanocarrier according to any one of claims 9-14, and the small nucleic acid drug delivery system according to any one of claims 15-16 are used in the preparation of drugs for anti-tumor, anti-viral and related genetic disease treatments Applications.
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