WO2023164991A1 - 一种重组靶向铁蛋白-纳米硒杂化复合物及其应用 - Google Patents

一种重组靶向铁蛋白-纳米硒杂化复合物及其应用 Download PDF

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WO2023164991A1
WO2023164991A1 PCT/CN2022/086284 CN2022086284W WO2023164991A1 WO 2023164991 A1 WO2023164991 A1 WO 2023164991A1 CN 2022086284 W CN2022086284 W CN 2022086284W WO 2023164991 A1 WO2023164991 A1 WO 2023164991A1
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ferritin
targeting
selenium
nano
recombinant
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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
    • A61K33/00—Medicinal preparations containing inorganic active ingredients
    • A61K33/04—Sulfur, selenium or tellurium; Compounds thereof
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K39/00—Medicinal preparations containing antigens or antibodies
    • A61K39/0005—Vertebrate antigens
    • A61K39/0011—Cancer antigens
    • 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
    • A61P35/00—Antineoplastic agents
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P35/00—Antineoplastic agents
    • A61P35/02—Antineoplastic agents specific for leukemia
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P37/00—Drugs for immunological or allergic disorders
    • A61P37/02—Immunomodulators
    • A61P37/04—Immunostimulants
    • 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
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K39/00—Medicinal preparations containing antigens or antibodies
    • A61K2039/60—Medicinal preparations containing antigens or antibodies characteristics by the carrier linked to the antigen
    • A61K2039/6031—Proteins
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K2319/00—Fusion polypeptide
    • C07K2319/33—Fusion polypeptide fusions for targeting to specific cell types, e.g. tissue specific targeting, targeting of a bacterial subspecies

Definitions

  • the technical field of ferritin modification of the present invention relates to a recombinant targeting ferritin-nano selenium hybrid complex and its application.
  • Ferritin a natural iron storage protein, consists of a 24-mer composed of heavy and light chains. Utilizing ferritin's 8-12nm inner cavity, uniform size and dispersed nanoparticles can be synthesized through biomineralization. Targeting can be conferred by chemically or genetically displaying targeting sequences on its surface. In 2007, Fe3O4 nanozyme was reported for the first time. In 2012, by loading Fe3O4 into the lumen of human heavy chain ferritin, based on the characteristic of human heavy chain ferritin that can target and bind to tfR1, the visual diagnosis of tumors was realized. Since then, researches have used ferritin nanozymes loaded with different metal elements for the treatment of various diseases. Ferritin has important application potential in the diagnosis and treatment of diseases.
  • Selenium is an essential element in the human body and has potential anti-tumor effects. Studies have also shown that selenium has the function of immune activation. In 2001, there was research on the synthesis of red nano-selenium, which has good anti-tumor properties and low toxicity. Selenium is used in the treatment of leukemia, and studies have found that it can induce apoptosis of leukemia cells. Nano-selenium is used in the treatment of acute myeloid leukemia, which can cause apoptosis of acute myeloid leukemia cells.
  • the present invention synthesizes nano-selenium biomimetically in the inner cavity of ferritin, and uses genetic engineering to display tumor-targeting peptides on the surface of ferritin, which can effectively realize targeted and precise treatment of tumors.
  • the present invention provides a recombinant targeting ferritin, the recombinant targeting ferritin contains a targeting peptide displayed at the amino terminal of ferritin.
  • Ferritin “HFn” 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, birds, amphibians, fish, mammals, e.g. from rodents, ruminants, non-human primates or humans, e.g. small Rats, rats, guinea pigs, dogs, cats, cows, horses, sheep, monkeys, gorillas, humans. 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 has a nucleotide sequence of SEQ ID NO.1.
  • the targeting peptide consists of 5-200 amino acids, preferably 5-100 amino acids, preferably 5-50 amino acids, preferably 5-25 amino acids, preferably 5 - Composition of 15 amino acids.
  • the targeting peptide is a leukemia targeting peptide.
  • the nucleotide sequence of recombinant targeting ferritin is SEQ ID NO.2.
  • the amino acid sequence of the targeting peptide is CLL1 target targeting peptide C DLRSAAVC (SEQ ID NO.3) or VLA-4 target targeting peptide CPLDIDFYC (SEQ ID NO.4).
  • the second aspect of the present invention provides a ferritin-nano-selenium hybrid complex, which is loaded with nano-selenium particles in the lumen of the recombinant targeting ferritin provided in the first aspect of the present invention.
  • the loading ratio of the recombinant targeting ferritin to selenium atoms is 1:10-1:1000, preferably 1:500-1:700.
  • the third aspect of the present invention provides the application of the recombinant targeting ferritin-nanometer selenium hybrid complex of the second aspect of the present invention in the preparation of drugs for targeted treatment of tumors.
  • the tumors include solid tumors and hematological tumors.
  • the hematological tumor is leukemia.
  • the leukemia is acute myeloid leukemia (AML) or acute promyelocytic leukemia (APL).
  • AML acute myeloid leukemia
  • APL acute promyelocytic leukemia
  • the fourth aspect of the present invention provides the application of the recombinant targeting ferritin-nano selenium hybrid complex of the second aspect of the present invention in the preparation for inducing differentiation and/or apoptosis of leukemia cells.
  • the fifth aspect of the present invention provides an immune activator, which contains the recombinant targeting ferritin-nano-selenium hybrid complex described in the second aspect of the present invention as an active ingredient.
  • the present invention displays tumor targeting peptide at the amino terminal of human heavy chain ferritin (HFn) through genetic engineering method for the first time, and synthesizes nano-selenium in ferritin lumen through biomimetic synthesis.
  • Ferritin nano-selenium has a good anti-tumor effect (solid tumors, blood tumors), especially for the treatment of leukemia, ferritin nano-selenium can obviously induce leukemia cell differentiation and apoptosis.
  • Ferritin nanoselenium (AE-HFn-Se) can be used as a histone deacetylase (HDAC) inhibitor for the treatment of tumors (solid tumors, blood tumors).
  • HDAC histone deacetylase
  • ferritin nano-selenium can activate the tumor immune system and act as a tumor vaccine.
  • the ferritin nano selenium prepared by the invention has important biomedical application potential of resisting solid tumors, inducing leukemia cell differentiation and apoptosis, and activating tumor immune system.
  • FIG. 1 Purification and characterization results of AE-HFn recombinant ferritin.
  • Figure A is the molecular weight of AE-HFn single subunit verified by SDS-PAGE
  • Figure B is the size of ferritin shown by SEC showing AE-HFn assembled into ferritin
  • Figure C is DLS showing that the average radius of AE-HFn is about 7.4nm
  • Figure D is Transmission electron microscopy showed that AE-HFn was spherical and had good monodispersity.
  • Figure 2AE-HFn-Se characterization results are Among them, Figure A is the negative staining result of TEM AE-HFn-Se, Figure B is the result of TEM AE-HFn-Se non-staining, Figure C is the high-resolution transmission electron microscope image of AE-HFn-Se, Figure D is the XPS detection of AE- HFn-Se binding energy absorption peak. Figure D shows the selenium content in AE-HFn-Se measured by ICP-MS.
  • Figure 3 Sensitivity detection of tumor cells (leukemia and solid tumors) to AE-HFn-Se
  • Figure A is the sensitivity test results of leukemia cell lines Kasumi-1, K562, U937
  • Figure B is the solid tumor cell line A549 , HepG2, U87 sensitivity detection results to AE-HFn-Se.
  • FIG. 4 Targeting detection results of ferritin nano-selenium on leukemia cells.
  • Figure A is siRNA knockdown of ITGA
  • Figure B is the targeting detection of AE-HFn-FITC on ITGA knockdown cell Kasumi-1
  • Figure C is the detection of AE-HFn-FITC targeting CD71.
  • Figure 5 Detection of ferritin nano-selenium induced leukemia cell differentiation.
  • panel A is Swiss staining
  • panel B is the effect of AE-HFn-Se on the expression of CD11b in Kasumi-1 cells
  • panel C is the effect of AE-HFn-Se on the expression of CD14 in Kasumi-1 cells.
  • FIG. 6 Detection results of apoptosis of leukemia cells induced by ferritin nano-selenium.
  • Figure A shows that AE-HFn-Se induces Kasumi-1 cell apoptosis in a time- and dose-dependent manner detected by flow cytometry
  • Figure B shows that AE-HFn-Se induces an increase in the expression of Cleaved caspase 3 protein by WB detection .
  • FIG. 7 The detection results of the effect of ferritin nano-selenium on H3K9 acetylation in leukemia.
  • Figure A is the effect of AE-HFn-Se on the acetylation of H3K9 in Kasumi-1 cells
  • Figure B is the effect of AE-HFn-Se on the acetylation of H3K9 in NB4 cells.
  • Fig. 8 Changes of CD80 expression in dendritic cells induced by HFn-Se.
  • the invention relates to a protein-nano-selenium hybrid complex, which mainly contains targeting protein and nano-selenium.
  • the protein-nanometer selenium hybrid complex can be used for treating tumors (solid tumors, blood tumors) and as an immune vaccine.
  • the present invention uses a genetic engineering method to display a tumor targeting peptide at the amino terminal of human heavy chain ferritin (HFn).
  • HFn human heavy chain ferritin
  • Nano-selenium was synthesized in the inner cavity of ferritin by biomimetic synthesis.
  • Ferritin nano-selenium has a good anti-tumor effect (solid tumors, blood tumors), especially for the treatment of leukemia, ferritin nano-selenium can obviously induce leukemia cell differentiation and apoptosis.
  • Ferritin nanoselenium (AE-HFn-Se) can be used as a histone deacetylase (HDAC) inhibitor for the treatment of tumors (solid tumors, blood tumors). At the same time, ferritin nano-selenium can activate the tumor immune system and act as a tumor vaccine.
  • the ferritin nano selenium prepared by the invention has important biomedical application potential of resisting solid tumors, inducing leukemia cell differentiation and apoptosis, and activating tumor immune system.
  • 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 , such as mice, rats, guinea pigs, dogs, cats, cows, horses, sheep, monkeys, gorillas, humans. 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.
  • AE is the abbreviation of AML1-ETO, displaying nine amino acid gene sequences at the amino-terminus of the HFn full gene sequence (SEQ ID NO.1) (which mainly targets AML1-ETO type leukemia, referred to as AE), to obtain targeted recombination HFn sequence (AE-HFn), the sequence is as SEQ ID No.2 (among them, the base sequence in bold italics is the targeting peptide sequence, and the base sequence underlined is the whole gene sequence of HFn), select NdeI/BamHI restriction endonuclease As a restriction site, it was constructed on the pET22b plasmid, which was constructed by Shanghai Jierui.
  • Transform AE-HFn into E.coli BL21 (Trans Gen) expression strain smear ampicillin LB plate, pick a single clone, and shake the bacteria overnight. Then add ampicillin-resistant LB medium, after expanding the culture, centrifuge at 6000rpm, collect the bacterial liquid, and resuspend in 50mM Tris, pH 8.0. After resuspension, after high-pressure homogenization and crushing, centrifuge at 12000rpm for 20min, then heat-treat at 72°C for 15min, centrifuge at 12000rpm for 40min, collect the supernatant, filter with a 0.22um filter head, and add sodium chloride to 0.75mM.
  • AE-HFn was purified by a hydrophobic column, concentrated after purification, and further separated and purified by superdex200 molecular sieves.
  • concentration of AE-HFn protein was determined using BCA protein detection kit with bovine serum albumin as the standard.
  • FIG. 1 The results are shown in FIG. 1 .
  • the purified AE-HFn was verified by SDS-PAGE, and the single subunit molecular weight of AE-HFn was larger than that of HFn (A in FIG. 1 ).
  • SEC showed that AE-HFn peaked between 50-75ml, indicating that AE-HFn was assembled into the size of ferritin (B in Figure 1).
  • DLS showed that the average radius of AE-HFn was about 7.4 nm (C in Figure 1).
  • Transmission electron microscopy showed that AE-HFn was spherical and had good monodispersity (D in Figure 1).
  • XPS shows that the binding energy is 55.3eV, and it has an obvious absorption peak, indicating that it is zero-valent nano-selenium.
  • the selenium content in AE-HFn-Se was measured by ICP-MS, and each AE-HFn contained about 530 selenium atoms.
  • CCK8 Cell Proliferation-Toxicity Detection Kit
  • tumor cells leukemia and solid tumors
  • leukemia cells show good sensitivity to AE-HFn-Se
  • leukemia cells Kasumi-1 to AE-HFn-Se HFn-Se is more sensitive.
  • ITGA is a subunit of the leukemia target VLA-4, and the targeting peptide displayed at the amino terminal of ferritin mainly binds to ITGA.
  • siRNA electroporation Low ITGA testing the targeting of AE-HFn-FITC to Kasumi-1 cells.
  • CD71 targeting detection HFn has good targeting specificity to CD71, we detected CD71 on the surface of Kasumi-1 by HFn-FITC.
  • Kasumi-1 cells were induced with 30ug/ml, 90ug/ml, and 180ug/ml AE-HFn-Se, respectively, and the expression changes of cell surface molecules CD11b and CD14 were detected at 24h, 48h, and 72h.
  • Flow cytometry Induce Kasumi-1 cells with 30ug/ml, 90ug/ml, and 180ug/ml AE-HFn-Se, respectively, and perform Annexin V-FITC and PI double staining at 24h, 48h, and 72h to detect Kasumi-1 Cell apoptosis.
  • Example 7 Ferritin nano-selenium inhibits the activity of histone deacetylase, causing acetylation of histone H3
  • Kasumi-1 and NB4 cells were stimulated with different concentrations of AE-HFn-Se (60ug/ml, 180ug/ml, 270ug/ml), and after 48h, the effects of AE-HFn-Se on Kasumi-1 and NB4 were detected by Western Blot experiment. Effect of histone H3K9 acetylation level in NB4 cells. The results are shown in Figure 7, AE-HFn-Se significantly induced a dose-dependent up-regulation of histone H3K9 acetylation levels in Kasumi-1 and NB4 cells.
  • HFn-Se Stimulate DC2.4 with HFn-Se (20ug/ml), and after 24h, detect the expression changes of DC2.4 CD80 in mouse dendritic cells induced by HFn-Se by flow cytometry, and judge whether dendritic cells can be induced Mature.
  • the results are shown in Figure 8, HFn-Se induces up-regulation of DC2.4 CD80 expression in mouse dendritic cells, which can induce the differentiation and maturation of mouse dendritic cells.

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Abstract

涉及通过基因工程方法，在笼状蛋白氨基端展示肿瘤靶向肽。通过仿生合成方式在笼状蛋白内腔合成纳米硒。笼状蛋白纳米硒具有良好的抗肿瘤(实体瘤、血液肿瘤)效果，尤其对于白血病治疗，笼状蛋白纳米硒明显的诱导白血病细胞分化和凋亡。同时，笼状蛋白纳米硒可以激活肿瘤免疫系统，作为一种肿瘤疫苗。涉及制备的笼状蛋白纳米硒，具有抗实体瘤和诱导白血病细胞分化和凋亡、激活肿瘤免疫系统的重要生物医学应用潜力。

Description

一种重组靶向铁蛋白-纳米硒杂化复合物及其应用 技术领域
本发明铁蛋白修饰技术领域，涉及一种重组靶向铁蛋白-纳米硒杂化复合物及其应用。
背景技术
铁蛋白，一种天然的储铁蛋白，由重链和轻链组成的24聚体构成。利用铁蛋白8～12nm内腔，通过生物矿化的方式可以合成尺寸均一、分散的纳米颗粒。通过化学或者遗传学方式在其表面展示靶向序列，可以赋予其靶向性。2007年，四氧化三铁纳米酶被首次报道，2012年通过在人重链铁蛋白内腔装载四氧化三铁，基于人重链铁蛋白可以靶向结合tfR1特点，实现肿瘤可视化诊断。此后，有研究将装载不同金属元素的铁蛋白纳米酶，用于多种疾病的治疗。铁蛋白在疾病诊疗过程中具有重要的应用潜力。
硒是人体中的一种必须元素，具有潜在的抗肿瘤效应。也有研究显示，硒具有免疫激活的功能。2001年，有研究合成红色纳米单质硒，纳米硒具有良好的抗肿瘤特性和低毒性特点。将硒用于白血病治疗，研究发现其可以引起白血病细胞发生凋亡。纳米硒被用于急性髓系白血病治疗，可以引起急性髓系白血病细胞发生凋亡。
但是有关铁蛋白-纳米硒的合成及应用并未见报道。
发明内容
为了克服上述现有技术的不足，本发明通过在铁蛋白内腔仿生合成纳米硒，运用基因工程的方法在铁蛋白表面展示肿瘤靶向肽，可以有效的实现对肿瘤的靶向精准治疗。
第一方面，本发明提供了一种重组靶向铁蛋白，所述重组靶向铁蛋白含有在铁蛋白氨基端展示的靶向肽。
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。
在某些实施例中，所述铁蛋白为核苷酸序列为SEQ ID NO.1。
在某些实施例中，所述靶向肽由5-200个氨基酸组成，优选为5-100个氨基酸组成，优选为5-50个氨基酸组成，优选为5-25个氨基酸组成，优选为5-15个氨基酸组成。
在某些实施例中，所述靶向肽为白血病靶向肽。
在某些实施例中，重组靶向铁蛋白的核苷酸序列为SEQ ID NO.2。
在某些实施例中，所述靶向肽的氨基酸序列为CLL1靶点靶向肽C DLRSAAVC(SEQ ID NO.3)或VLA-4靶点靶向肽CPLDIDFYC(SEQ ID NO.4)。
本发明第二方面，提供了一种铁蛋白-纳米硒杂化复合物，其为在本发明第一方面提供的重组靶向铁蛋白内腔中装载纳米硒颗粒。
在某些实施例中，所述重组靶向铁蛋白与硒原子的装载比为1:10～1:1000,优选的1:500～1:700。
本发明第三方面，提供了本发明第二方面的重组靶向铁蛋白-纳米硒杂化复合物在制备靶向治疗肿瘤的药物中的应用。
在某些实施例中，所述肿瘤包括实体瘤和血液肿瘤。
在某些实施例中，所述血液肿瘤为白血病。
在某些实施例中，所述白血病为急性髓系白血病(AML)或急性早幼粒细胞白血病(APL)。
本发明第四方面，提供了本发明第二方面的重组靶向铁蛋白-纳米硒杂化复合物在制备用于诱导白血病细胞分化和/或凋亡中的应用。
本发明第五方面，提供了一种免疫激活剂，所述免疫激活剂中作为有效成分含有本发明第二方面所述的重组靶向铁蛋白-纳米硒杂化复合物。
本发明相对于现有技术，首次通过基因工程方法，在人重链铁蛋白(HFn)氨基端展示肿瘤靶向肽，并通过仿生合成方式在铁蛋白内腔合成纳米硒。铁蛋白纳米硒具有良好的抗肿瘤(实体瘤、血液肿瘤)效果，尤其对于白血病治疗，铁蛋白纳米硒明显的诱导白血病细胞分化和凋亡。铁蛋白纳米硒(AE-HFn-Se)可以作为一种组蛋白去乙酰化酶(HDAC)抑制剂，用于肿瘤(实体瘤、血液肿瘤)治疗。同时，铁蛋白纳米硒可以激活肿瘤免疫系统，作为一种肿瘤疫苗。利用本发明制备的铁蛋白纳米硒，具有抗实体瘤和诱导白血病细胞分化和凋亡、激活肿瘤免疫系统的重要生物医学应用潜力。
附图说明
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。
图1AE-HFn重组铁蛋白的纯化及表征结果。其中，图A为SDS-PAGE验证AE-HFn单亚基分子量；图B为SEC显示AE-HFn组装成铁蛋白的大小；图C为DLS显示AE-HFn平均半径大小约7.4nm；图D为透射电镜显示AE-HFn呈球状且单分散性较好。
图2AE-HFn-Se表征结果。其中，图A为TEM AE-HFn-Se负 染色结果，图B为TEM AE-HFn-Se不染色结果，图C为AE-HFn-Se高分辨透射电镜图，图D为XPS检测的AE-HFn-Se结合能吸收峰。图D为ICP-MS测AE-HFn-Se中的硒含量。
图3肿瘤细胞(白血病和实体瘤)对AE-HFn-Se的敏感性检测，其中，图A为白血病细胞系Kasumi-1、K562、U937的敏感性检测结果，图B为实体瘤细胞系A549、HepG2、U87对AE-HFn-Se的敏感性检测结果。
图4铁蛋白纳米硒对白血病细胞靶向性检测结果。其中，图A为siRNA敲低ITGA，图B为AE-HFn-FITC对ITGA敲低细胞Kasumi-1的靶向性检测，图C为AE-HFn-FITC对CD71靶向性检测。
图5铁蛋白纳米硒诱导白血病细胞分化检测。其中，图A为瑞士染色，图B为AE-HFn-Se对Kasumi-1细胞CD11b表达的影响，图C为AE-HFn-Se对Kasumi-1细胞CD14表达的影响。
图6铁蛋白纳米硒诱导白血病细胞凋亡检测结果。其中，图A为流式细胞术检测AE-HFn-Se诱导Kasumi-1细胞呈时间和剂量依赖性发生凋亡，图B为WB检测发现AE-HFn-Se诱导Cleaved caspase 3蛋白表达水平升高。
图7铁蛋白纳米硒对白血病H3K9乙酰化影响检测结果。其中，图A为AE-HFn-Se对Kasumi-1细胞H3K9乙酰化影响，图B为AE-HFn-Se对NB4细胞H3K9乙酰化影响。
图8 HFn-Se诱导树突状细胞CD80表达变化图。
发明详述
总体概述
本发明涉及一种蛋白-纳米硒杂化复合物，其主要含具有靶向性 的蛋白和纳米硒。该蛋白-纳米硒杂化复合物可用作治疗肿瘤(实体瘤、血液肿瘤)及作为一种免疫疫苗。具体而言，本发明通过基因工程方法，在人重链铁蛋白(HFn)氨基端展示肿瘤靶向肽。通过仿生合成方式在铁蛋白内腔合成纳米硒。铁蛋白纳米硒具有良好的抗肿瘤(实体瘤、血液肿瘤)效果，尤其对于白血病治疗，铁蛋白纳米硒明显的诱导白血病细胞分化和凋亡。铁蛋白纳米硒(AE-HFn-Se)可以作为一种组蛋白去乙酰化酶(HDAC)抑制剂，用于肿瘤(实体瘤、血液肿瘤)治疗。同时，铁蛋白纳米硒可以激活肿瘤免疫系统，作为一种肿瘤疫苗。利用本发明制备的铁蛋白纳米硒，具有抗实体瘤和诱导白血病细胞分化和凋亡、激活肿瘤免疫系统的重要生物医学应用潜力。
本发明中铁蛋白“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。
具体实施方式
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。
实施例1 重组改造人重链铁蛋白纯化及表征
1.AE(AML1-ETO简称)-HFn质粒的构建
AE是AML1-ETO简称，在HFn全基因序列(SEQ ID NO.1)氨基端展示九个氨基酸基因序列(其主要靶向结合AML1-ETO类型白血病，简称AE)，获得具有靶向性的重组HFn序列(AE-HFn)，序列如SEQ ID No.2(其中，加粗斜体碱基序列为靶向肽序列，下划线碱基序列为HFn全基因序列)，选择NdeI/BamHI限制性内切酶为酶切位点，将其构建到pET22b质粒上，由上海捷瑞构建。
Figure PCTCN2022086284-appb-000001
2.AE-HFn蛋白纯化
将AE-HFn转化E.coli BL21(Trans Gen)表达菌株，涂氨苄LB平板，挑取单克隆，摇菌过夜。然后加入氨苄抗性LB培养基，扩 大培养后，6000rpm离心，收集菌液，用50mM Tris,pH 8.0中重悬。重悬后，进行高压匀浆破碎后，12000rpm，离心20min，然后在72℃热处理15min，12000rpm，离心40min，收集上清，用0.22um滤头滤过，补氯化钠至0.75mM。然后用疏水柱纯化AE-HFn，纯化后浓缩，用superdex200分子筛进一步分离纯化。以牛血清白蛋白为标准，采用BCA蛋白检测试剂盒测定AE-HFn蛋白浓度。
3.AE-HFn蛋白表征
通过SDS-PAGE验证重组表达后AE-HFn单亚基分子量大小。superdex200分子筛和透射电镜检测AE-HFn组装情况。
结果如图1显示，纯化后的AE-HFn，通过SDS-PAGE验证发现，AE-HFn单亚基分子量较HFn大(图1中A)。SEC显示AE-HFn在50-75ml之间出峰，说明AE-HFn组装成铁蛋白大小(图1中B)。DLS显示AE-HFn平均半径大小约7.4nm(图1中C)。透射电镜显示AE-HFn呈球状且单分散性较好(图1中D)。
实施例2 铁蛋白纳米硒合成及表征
1.铁蛋白纳米硒的合成
将2mM亚硒酸钠加入含有1μM AE-HFn的50Mm tris-HCL 0.15M氯化钠溶液中，室温赋予3h，加入硼氢化钠还原1h，10000rpm离心15min，用Hiprep TM 26/10Desalting脱盐，然后用superdex200(10/300GL，GE Healthcare)进行纯化分离。
2.铁蛋白纳米硒表征
准备0.1mg/ml AE-HFn-Se，在PBS溶液中，PH＝7.4，通过TEM负染色和不负染色分别检测AE-HFn-Se组装和硒纳米颗粒。准备0.1mg/ml AE-HFn-Se，在PBS溶液中，送中科百测，通过ICP-MS和高分别透射电镜，分别检测其含量和纳米硒晶格间距。将50mg/ml  AE-HFn-Se，在PBS溶液中，将其缓冲液置换成水，冻干成粉末，送中科百测，测XPS。
结果如图2显示，将合成的AE-HFn-Se经Hiprep TM 26/10 Desalting脱盐，然后用superdex200(10/300GL,GE Healthcare)纯化分离后呈红色。TEM负染色，显示AE-HFn-Se具有球壳结构，且单分散性好。TEM不负染色显示AE-HFn-Se含有约4.5nm大小的纳米颗粒。高分辨透射电镜显示AE-HFn-Se中纳米颗粒具有约0.216nm晶格间距。XPS显示在结合能55.3eV，具有明显的吸收峰，说明其为零价态纳米硒。ICP-MS测AE-HFn-Se中的硒含量，每个AE-HFn约含有530个硒原子。
实施例3 肿瘤细胞(白血病和实体瘤)对铁蛋白纳米硒的敏感性
使用细胞增殖-毒性检测试剂盒(CCK8)，分别检测白血病细胞系(Kasumi-1、K562、U937)和实体瘤细胞系(A549、HepG2、U87)对AE-HFn-Se的敏感性，计算半数致死剂量(IC50)。
结果如图3显示，肿瘤细胞(白血病和实体瘤)对AE-HFn-Se具有一定的敏感性，白血病细胞对AE-HFn-Se显示出较好的敏感性，白血病细胞Kasumi-1对AE-HFn-Se较敏感。
实施例4 铁蛋白纳米硒对白血病细胞靶向性检测
VLA-4靶向性检测：ITGA作为白血病靶点VLA-4的一个亚基，铁蛋白氨基端展示的靶向肽，主要结合ITGA，为了验证AE-HFn的靶向性，我们利用siRNA电转敲低ITGA，检测AE-HFn-FITC对Kasumi-1细胞的靶向性。
CD71靶向性检测：HFn对CD71具有较好的靶向特异性，我们通过HFn-FITC对Kasumi-1表面CD71进行检测。
结果如图4所示，与对照组相比，AE-HFn-FITC对ITGA敲低组靶向结合性减弱。证明AE-HFn对VLA-4具有靶向性。同时，HFn可以抑制AE-HFn对Kasumi-1的结合特异性，证明AE-HFn对CD71也具有靶向性。
实施例5 铁蛋白纳米硒诱导白血病细胞分化
瑞士染色：使用90ug/ml AE-HFn-Se诱导Kasumi-1细胞，每两天换液一次，连续诱导4天，观察细胞形态和细胞核变化。
流式细胞术：分别用30ug/ml、90ug/ml、180ug/ml AE-HFn-Se诱导Kasumi-1细胞，在24h、48h、72h检测细胞表面分子CD11b、CD14表达变化。
结果如图5显示，瑞士染色显示，AE-HFn-Se诱导Kasumi-1细胞发生分化。AE-HFn-Se诱导Kasumi-1细胞表面分子CD11b、CD14呈剂量和时间依赖性上调。
实施例6 铁蛋白纳米硒诱导白血病细胞凋亡
流式细胞术：分别用30ug/ml、90ug/ml、180ug/ml AE-HFn-Se诱导Kasumi-1细胞，在24h、48h、72h，进行Annexin V-FITC和PI双染色，检测Kasumi-1细胞凋亡情况。
Western Blot：分别用90ug/ml、180ug/ml AE-HFn-Se诱导Kasumi-1细胞，在24h、48h，检测Cleaved caspase3蛋白表达水平。
结果如图6显示，流式细胞术显示，AE-HFn-Se诱导Kasumi-1细胞呈时间和剂量依赖性发生凋亡。WB检测发现AE-HFn-Se诱导Cleaved caspase3蛋白表达水平升高。
实施例7 铁蛋白纳米硒抑制组蛋白去乙酰化酶的活性，引起组蛋白H3乙酰化
分别用不同浓度AE-HFn-Se(60ug/ml、180ug/ml、270ug/ml)刺 激Kasumi-1和NB4细胞，48h后，通过Western Blot实验，分别检测AE-HFn-Se对Kasumi-1和NB4细胞组蛋白H3K9乙酰化水平的影响。结果如图7显示，AE-HFn-Se明显引起Kasumi-1和NB4细胞组蛋白H3K9乙酰化水平呈剂量依赖性上调。
实施例8 铁蛋白纳米硒诱导树突状细胞成熟
使用HFn-Se(20ug/ml)刺激DC2.4，24h后，通过流式细胞术，检测HFn-Se诱导小鼠树突状细胞DC2.4 CD80表达变化情况，判断是否可以诱导树突状细胞成熟。结果如图8显示，HFn-Se诱导小鼠树突状细胞DC2.4 CD80表达上调，其可以诱导小鼠树突状细胞分化成熟。
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。

Claims (14)

  1. 一种重组靶向铁蛋白，其特征在于，所述重组靶向铁蛋白含有在铁蛋白氨基端展示的靶向肽。
  2. 根据权利要求1所述的重组靶向铁蛋白，其特征在于，所述铁蛋白核苷酸序列为SEQ ID NO.1。
  3. 根据权利要求1所述的重组靶向铁蛋白，其特征在于，所述靶向肽由5-200个氨基酸组成，优选为5-15个氨基酸组成。
  4. 根据权利要求1所述的重组靶向铁蛋白，其特征在于，所述靶向肽为白血病靶向肽。
  5. 根据权利要求1所述的重组靶向铁蛋白，其特征在于，所述靶向肽的氨基酸序列为CLL1靶点靶向肽CDLRSAAVC或VLA-4靶点靶向肽CPLDIDFYC。
  6. 根据权利要求5所述的重组靶向铁蛋白，其特征在于，重组靶向铁蛋白的核苷酸序列为SEQ ID NO.2。
  7. 一种重组靶向铁蛋白-纳米硒杂化复合物，其特征在于，在权利要求1-6任一所述的重组靶向铁蛋白内腔中装载纳米硒颗粒。
  8. 根据权利要求7所述的重组靶向铁蛋白-纳米硒杂化复合物，其特征在于，所述重组靶向铁蛋白与硒原子的装载比为1:10～1:1000,优选的1:500～1:700。
  9. 权利要求7-8任一所述的重组靶向铁蛋白-纳米硒杂化复合物在制备靶向治疗肿瘤的药物中的应用。
  10. 根据权利要求9所述的应用，其特征在于，所述肿瘤包括实体瘤和血液肿瘤。
  11. 根据权利要求10所述的应用，其特征在于，所述血液肿瘤为白血病。
  12. 根据权利要求11所述的应用，其特征在于，所述白血病为急性髓 系白血病(AML)或急性早幼粒细胞白血病(APL)。
  13. 权利要求7-8任一所述的重组靶向铁蛋白-纳米硒杂化复合物在制备用于诱导白血病细胞分化和/或凋亡中的应用。
  14. 一种免疫激活剂，其特征在于，所述免疫激活剂中作为有效成分含有权利要求7-8任一所述的重组靶向铁蛋白-纳米硒杂化复合物。
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