WO2024239165A1 - 一种正交反应调控的相分离凝集体及制备其的方法和应用 - Google Patents

一种正交反应调控的相分离凝集体及制备其的方法和应用 Download PDF

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WO2024239165A1
WO2024239165A1 PCT/CN2023/095383 CN2023095383W WO2024239165A1 WO 2024239165 A1 WO2024239165 A1 WO 2024239165A1 CN 2023095383 W CN2023095383 W CN 2023095383W WO 2024239165 A1 WO2024239165 A1 WO 2024239165A1
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protein
nucleic acid
drug
double
tco
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French (fr)
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顾臻
李洪军
梁亭西子
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Jinhua Institute Of Zhejiang University
Zhejiang University
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Jinhua Institute Of Zhejiang University
Zhejiang University
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    • 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
    • A61K31/7028—Compounds having saccharide radicals attached to non-saccharide compounds by glycosidic linkages
    • A61K31/7034—Compounds having saccharide radicals attached to non-saccharide compounds by glycosidic linkages attached to a carbocyclic compound, e.g. phloridzin
    • A61K31/704—Compounds having saccharide radicals attached to non-saccharide compounds by glycosidic linkages attached to a carbocyclic compound, e.g. phloridzin attached to a condensed carbocyclic ring system, e.g. sennosides, thiocolchicosides, escin, daunorubicin
    • 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
    • A61K47/30—Macromolecular organic or inorganic compounds, e.g. inorganic polyphosphates
    • A61K47/42—Proteins; Polypeptides; Degradation products thereof; Derivatives thereof, e.g. albumin, gelatin or zein
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K47/00—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient
    • A61K47/50—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates
    • A61K47/51—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates the non-active ingredient being a modifying agent
    • A61K47/56—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates the non-active ingredient being a modifying agent the modifying agent being an organic macromolecular compound, e.g. an oligomeric, polymeric or dendrimeric molecule
    • 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
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P35/00—Antineoplastic agents

Definitions

  • the present invention relates to the field of drug delivery, and in particular to a phase separation aggregate regulated by an orthogonal reaction and a method for preparing the same and an application thereof.
  • Liquid-Liquid phase separation has attracted widespread attention in recent years because it forms membraneless organelles such as stress granules, nucleoli or Cajal bodies through multiple interactions between biomacromolecules such as proteins, DNA and RNA.
  • Current research on liquid-liquid phase separation tends to explore the mechanism, focusing on revealing the intrinsic driving force of liquid-liquid phase separation, the specific process of intracellular compartmentalization and its association with a series of physiological activities, such as intracellular communication, transcriptional regulation, stress response and disease occurrence, but there are relatively few explorations on the practical application of liquid-liquid phase separation systems.
  • Bioorthogonal reactions refer to reactions that can be carried out in living cells or tissues without interfering with the biochemical reactions of the organism itself. They have good biochemical reaction control capabilities and biosafety, and can provide a solution to the instability of the above-mentioned biomolecular aggregates. Therefore, we proposed a synthetic method for in situ assembly of liquid-liquid phase separation aggregates, and used the bioorthogonal click-release strategy to achieve in vitro sealing of the phase separation system and intracellular recovery, thereby forming controllable long-term drug retention aggregates and enhancing the cancer treatment effect.
  • the purpose of the present invention is to provide a phase separation aggregate regulated by an orthogonal reaction and a method for preparing the same and an application thereof, so as to solve the problem of drug resistance in the treatment of cancer or drug-resistant bacterial infection.
  • the modified protein is a (2E)TCO-modified protein, ie, protein-TCO
  • the modified nucleic acid is a tetrazine-modified nucleic acid, ie, nucleic acid-Tz.
  • the concentration of the protein is greater than 1 mg/mL, preferably less than 5 mg/mL, and the concentration of the DNA is greater than 20 ⁇ M, preferably less than 100 uM.
  • the concentration of the protein is greater than 50 ⁇ g/mL, preferably less than 200 ⁇ g/mL, and the concentration of the DNA is greater than 1 ⁇ M, preferably less than 10 ⁇ M.
  • the concentration of the protein is greater than 100 ⁇ g/mL and less than 200 ⁇ g/mL, and the concentration of the DNA is greater than 5 ⁇ M and less than 10 ⁇ M.
  • the protein is selected from proteins with phase separation ability, preferably amino-rich proteins;
  • the nucleic acid is selected from nucleic acids rich in repetitive base sequences and capable of stably existing, such as double-stranded DNA or double-stranded RNA containing repeated C-G base pairs.
  • the amino-rich protein comprises a histone or a prion-like protein, such as FUS (fused us sarcoma).
  • the protein has a molecular weight range of 10-20 kDa.
  • the nucleic acid is 25 to 30 bp in length.
  • the histone is selected from one or more of histones H1, H2A, H2B, H3 and H4; the two single-stranded nucleotide sequences of the DNA are respectively shown as SEQ ID NO: 1 (sequence-1) and SEQ ID NO: 2 (sequence-2).
  • the second aspect of the present invention provides a drug combination, which comprises the nucleic acid protein combination described in the first aspect of the present invention and a drug.
  • the drug is intercalated into a C-G base pair of a nucleic acid.
  • the drug is selected from one or more of doxorubicin, mitoxantrone, cisplatin and tetracycline.
  • the third aspect of the present invention provides a kit, the kit comprising the nucleic acid protein described in the first aspect of the present invention. White combination.
  • the kit further comprises one or more of (2E)TCO, (4E)TCO, azide, and tetrazine.
  • the kit further comprises a drug selected from one or more of doxorubicin, mitoxantrone, cisplatin, and tetracycline.
  • a fourth aspect of the present invention provides a method for preparing an agglomerate, the method comprising the following steps:
  • the protein-TCO and nucleic acid-Tz in the nucleic acid-protein combination described in the first aspect of the present invention are reacted to produce the aggregate.
  • the reaction is performed in vitro in a cell-free solution or intracellularly.
  • the method further comprises the following steps:
  • a fifth aspect of the present invention provides a method for preparing a drug-containing aggregate, the method comprising the following steps:
  • nucleic acid-Tz such as double-stranded DNA-Tz or double-stranded RNA-Tz in the nucleic acid-protein combination described in the first aspect of the present invention
  • the reaction is performed in vitro in a cell-free solution or intracellularly.
  • the method comprises the following steps:
  • the protein-TCO is reacted with double-stranded DNA-Tz or double-stranded RNA-Tz loaded with drugs to generate drug-containing aggregates.
  • the reaction is performed in vitro in a cell-free solution or intracellularly.
  • the sixth aspect of the present invention provides the use of the nucleic acid protein combination described in the first aspect of the present invention or the drug combination described in the second aspect of the present invention in the preparation of a drug or kit for treating tumors or drug-resistant bacterial infections.
  • the reagents and raw materials used in the present invention are commercially available.
  • the present invention innovatively proposes a nucleic acid protein combination and a method for preparing a phase-separated aggregate using the nucleic acid protein combination; controllable preparation of the aggregate and long-term retention of the active ingredients are achieved.
  • the nucleic acid protein combination includes two separated components outside the cell, and can form a phase-separated aggregate through a click chemistry reaction inside the cell, which can significantly improve the anti-tumor effect of the drug and significantly prolong the survival time of mice with tumors.
  • the drug retention strategy based on the aggregate can significantly improve the efficacy of the drug.
  • Figure 1A shows the morphology of droplets formed by histones and DNA with fluorescein FITC under a confocal microscope.
  • the scale bar is 20 ⁇ m.
  • FIG1B is a photograph of histone, DNA and aggregate solutions.
  • FIG1C shows the turbidity test of histones, DNA and aggregates.
  • Figure 1D shows the fluorescence recovery of the droplet after photobleaching.
  • FIG. 2A shows the double-stranded DNA sequence structure bound to the drug doxorubicin.
  • Figure 3A shows the type of click-release reaction.
  • FIG3D shows the release curve of the loaded molecule Rhodamine B (RB) after the (2E)TCO and (4E)TCO groups reacted with the Tz groups, respectively.
  • FIG4B is a mass spectrometric representation of the molecular weight change of azide-modified DNA before and after the reaction with DBCO-Tz.
  • FIG5 is a diagram showing the formation process of liquid-liquid phase separation aggregates triggered by a bioorthogonal reaction in an embodiment of the present invention.
  • the upper part of the diagram shows the formation process of aggregates triggered by a click-release reaction, and the lower part shows the control group of a click-ligation reaction.
  • Figure 6 shows the formation of aggregates in cells in an embodiment of the present invention.
  • the left figure shows a confocal image of 2E-conjugate forming aggregates by click-release reaction in cells
  • the right figure shows a control group in which 4E-conjugate fails to trigger the formation of aggregates by click-ligation reaction in cells.
  • the scale bar is 20 microns.
  • FIG. 7A shows the effect of 2E-conjugate on the activity of 4T1 cancer cells.
  • FIG. 7B is a toxicity test of 2E-conjugate on drug-resistant cancer cells 4T1/ADR.
  • FIG8A is a verification of the killing effect of a single drug and drug-loaded aggregates formed within cells on 4T1 cells.
  • FIG8B is a verification of the killing effect of single drug and drug-loaded aggregates formed in cells on 4T1/ADR cells.
  • FIG9A shows the retention rates of drug alone, aggregates/drug, and non-aggregates/drug in 4T1 cells after 12 hours of action.
  • FIG9B shows the retention rates of drug alone, aggregates/drug and non-aggregates/drug in 4T1/ADR cells after 12 hours of action.
  • FIG9C is a time-dependent drug retention assay of different groups in 4T1/ADR cells.
  • FIG. 10A shows the content of doxorubicin, aggregate/drug and non-aggregate/drug groups in cells changing with time.
  • FIG. 10B is a statistical analysis of FIG. 10A , showing the average intracellular drug fluorescence intensity at different time points.
  • FIG. 11B is a statistical analysis of FIG. 11A , showing the fluorescence co-localization coefficients of drugs and aggregates in different groups at different time points.
  • FIG11C is a statistical analysis of FIG11A , showing the average drug adsorption intensity of different groups at different time points.
  • FIG. 12A is a graph showing bioluminescent imaging of tumors in each group of mice at different days after treatment in the 4T1 mouse tumor model.
  • FIG. 12B shows the tumor growth of mice in each group after treatment in the 4T1 mouse tumor model.
  • FIG. 13A is the average tumor growth curve of each group of mice after treatment in the 4T1 mouse tumor model.
  • FIG. 13B is a survival curve of mice in each group after treatment in the 4T1 mouse tumor model.
  • FIG. 14B shows the tumor growth of mice in each group after treatment in the 4T1/ADR mouse tumor model.
  • FIG. 14C is a photo of the tumors in each group of mice in the 4T1/ADR mouse tumor model after 12 days of treatment.
  • FIG. 15A shows drug retention after intratumoral injection of drugs and formulations.
  • FIG15B shows the adsorption of drugs injected via the tail vein by the drug reservoir formed in the tumor.
  • Dox doxorubicin
  • Example 3 Formation of intracellular aggregates, biocompatibility testing and anticancer effects of aggregates at the cellular level
  • 4T1/ADR cells were incubated with equal concentrations of protein-nucleic acid conjugates 2E-conjugate or 4E-conjugate, with histone and DNA double-strand concentrations of 50 ⁇ g/mL and 1 ⁇ M, respectively, and the formation of aggregates at different time points was observed under a confocal microscope. The results showed that the formation of phase-separated aggregates triggered by the click-release reaction could also be observed in the cells (Figure 6).
  • the drug-loaded 2E-conjugate i.e., 2E-conjugate/Dox
  • doxorubicin group the same below
  • agglutination/drug group the same below
  • the drug-resistant cells were inoculated in a 96-well plate, and Dox, 2E-conjugate/Dox (i.e., agglutination/drug group, the same below) and 4E-conjugate/Dox (i.e., non-agglutination/drug group, the same below) were incubated for 4 hours after 24 hours of adhesion (drug concentration was 10 ⁇ M). Subsequently, the culture medium was removed, washed twice with PBS, and fresh medium without drugs was incubated. At a certain time interval, the culture medium was removed and replaced with fresh culture medium, and the intracellular drug fluorescence intensity was measured.
  • Dox, 2E-conjugate/Dox i.e., agglutination/drug group, the same below
  • 4E-conjugate/Dox i.e., non-agglutination/drug group, the same below
  • the cells were seeded on the confocal dish and adhered to the wall for 24 hours.
  • the single drug Dox, 2E-conjugate/Dox and 4E-conjugate/Dox were incubated for different time periods.
  • the drug concentration was 10 ⁇ M
  • the concentrations of histone and DNA double strand were 200 ⁇ g/mL and 10 ⁇ M, respectively.
  • the culture medium was then removed, the cells were washed twice with PBS, the cell nuclei were stained with Hoechst 33342, and the cells were fixed to observe the fluorescence intensity of the drug in the cells.
  • the results showed that through the confocal qualitative experiment, the fluorescence intensity increased with time (Figure 10A), confirming that the red fluorescent drug can be retained in the cell for a long time by the aggregate ( Figure 10B).
  • 4T1/ADR cells were pretreated with drug-free 2E-conjugate-Cy5 (when preparing DNA-Tz, DNA was simultaneously modified with Cy5) and 4E-conjugate-Cy5 for 4 h to form intracellular aggregates.
  • the culture medium was then removed and replaced with a medium containing the drug (drug concentration was 10 ⁇ M) for incubation for different times.
  • drug concentration was 10 ⁇ M
  • the fluorescence colocalization of the drug and Cy5-labeled DNA was observed under a confocal microscope.
  • the fluorescence colocalization coefficient and drug adsorption amount were analyzed using Image J software.
  • mice 4T1 or 4T1/ADR cells were inoculated on the back of Balb/c nude mice, and when the tumor volume grew to about 100 mm 3 , the mice were randomly divided into four groups, 6 in each group, and were administered drugs respectively, G1 group: PBS; G2 group: 2E-conjugate; G3 group: Dox; G4 group: 2E-conjugate/Dox, for 4T1 and 4T1/ADR tumor-bearing mice, the drug concentrations were 0.25 mg/kg and 2.5 mg/kg, respectively, and the drug was administered once every three days, and the tumor volume and mouse body weight were measured every two days. When the tumor volume of the mouse exceeded 1500 mm 3 , the mouse was euthanized.
  • mice were injected with Dox, 2E-conjugate/Dox, and 4E-conjugate/Dox. 24 hours later, the mice were euthanized, the tumor tissues were removed, frozen sections were made, and the cells were stained with DAPI, and the retention of the drugs in the tumor tissues was observed under a confocal microscope.
  • Tumor-bearing mice were injected with PBS, 2E-conjugate, and 4E-conjugate intratumorally, and doxorubicin was injected into the tail vein at the same time. After 24 hours, the mice were euthanized, the tumor tissues were removed, frozen sections were made, and the cells were stained with DAPI. The adsorption of drugs in the tumor tissue was observed under a confocal microscope.
  • tumor tissue sections confirmed that the bioorthogonal click-release reaction can not only be used as a drug delivery system to improve the retention of drugs in cells by using the larger size blocking effect (Figure 15A), but also can adsorb the drugs injected into the tail vein through the drug reservoir formed in the tumor, thereby achieving drug enhancement in the tumor tissue (Figure 15B) and enhancing the anti-tumor efficacy.

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Abstract

一种正交反应调控的相分离凝集体及其制备方法和应用。可形成相分离凝集体的核酸蛋白组合包含：修饰的蛋白质和修饰的核酸，所述核酸包括DNA和RNA，所述修饰的蛋白质与修饰的核酸可发生点击-释放反应，使蛋白质与核酸发生相分离，形成所述相分离凝集体。所述核酸蛋白组合实现了凝集体可控的制备和活性成分的长效保留。所述核酸蛋白组合在胞外包括分离的两种组分，在胞内可以通过点击化学反应形成相分离凝集体，提高药物的抗肿瘤效果，延长患肿瘤小鼠的生存时间。

Description

一种正交反应调控的相分离凝集体及制备其的方法和应用 技术领域
本发明涉及药物递送领域，具体涉及一种正交反应调控的相分离凝集体及制备其的方法和应用。
背景技术
在利用化疗药物治疗癌症的过程中，会逐渐产生多药耐药性，进入肿瘤细胞的药物会被耐药细胞过表达的蛋白(ABC转运蛋白)主动泵出，从而造成治疗失败，并引起肿瘤复发和转移。通过研究新的策略增强药物在耐药细胞中的保留可维持有效治疗剂量，获得最佳治疗效果。
液-液相分离(Liquid-Liquid phase separation,LLPS)通过蛋白质、DNA和RNA等生物大分子之间发生多重相互作用，形成无膜细胞器，如应激颗粒(stress granules)、核仁(nucleoli)或卡哈尔体(Cajal bodies)等，近年来引起了广泛的关注。当下关于液-液相分离的研究偏向于机制探究，集中于揭示液-液相分离的内在驱动力、细胞内区室化的具体过程以及其与一系列生理活动的关联，如细胞内通讯、转录调节、压力响应和疾病发生等，但关于液-液相分离体系的实际应用探索相对较少。在细胞内拥挤环境中，充满着蛋白质、核酸等生物大分子，在体积排斥的情况下，这些生物大分子倾向于形成浓缩的凝集体，使得其中的分子浓度比细胞质高几百倍，从而改变分子的存在状态和生化反应平衡。受此启发，申请人设想相分离凝集体可被用于浓缩癌症治疗制剂，从而提高有效药物剂量，避免耐药肿瘤对药物的清除，从而提高癌症治疗效果。但是由于凝集体的动态性质及其易受环境参数影响，对凝集体进行可控制备具有较大挑战。
生物正交反应是指能在活体细胞或组织中，在不干扰生物自身生化反应的条件下可以进行的反应，具有较好的生化反应控制能力和生物安全性，可为上述生物分子凝集体的不稳性提供解决方案。因此，我们提出了一种原位组装液-液相分离凝集体的合成手段，利用生物正交的点击-释放策略实现了相分离体系的体外封存及细胞内的恢复，从而形成可控的长效保留药物的凝集体，增强了癌症治疗效果。
发明内容
本发明的目的在于提供一种正交反应调控的相分离凝集体及制备其的方法和应用，用于解决癌症或耐药菌感染治疗过程中的耐药性问题。
为解决上述技术问题，本发明第一方面提供了一种可形成相分离凝集体的核酸蛋白组合，其包含：修饰的蛋白质和修饰的核酸，所述核酸包括DNA和RNA，所述修饰的蛋白质与修饰的核酸可发生点击-释放反应，使蛋白质与核酸发生相分离，形成所述相分离凝集体。
在一些实施方案中，所述点击-释放反应在体外无细胞的溶液中或胞内进行。
在一些实施方案中，所述修饰的蛋白质为(2E)TCO修饰的蛋白质即蛋白质-TCO，所述修饰的核酸为四嗪修饰的核酸即核酸-Tz。
在一些实施方案中，当所述点击-释放反应在体外无细胞的溶液中进行时，所述蛋白质的浓度大于1mg/mL，优选小于5mg/mL，所述DNA的浓度大于20μM，优选小于100uM。
在一些实施方案中，所述蛋白质的浓度大于2mg/mL且小于5mg/mL，所述DNA的浓度大于30μM且小于100uM。
在一些实施方案中，当所述点击-释放反应在胞内进行时，所述蛋白质的浓度大于50μg/mL，优选小于200μg/mL，所述DNA的浓度大于1μM，优选小于10μM。
在一些实施方案中，所述蛋白质的浓度大于100μg/mL且小于200μg/mL，所述DNA的浓度大于5μM且小于10μM。
在一些实施方案中，所述蛋白质选自具相分离能力的蛋白质，优选富含氨基的蛋白质；所述核酸选自富含重复碱基序列且能稳定存在的核酸，例如包含重复C-G碱基对的双链DNA或双链RNA。
在一些实施方案中，所述富含氨基的蛋白质包括组蛋白或朊病毒样蛋白，例如FUS(融合肉瘤)。
在一些实施方案中，所述蛋白质的分子量范围为10-20kDa。
在一些实施方案中，所述核酸的长度为25～30bp。
在一些实施方案中，所述组蛋白选自组蛋白H1、H2A、H2B、H3和H4中的一种或多种；所述DNA的两条单链核苷酸序列分别如SEQ ID NO:1(序列-1)和SEQ ID NO:2(序列-2)所示。
本发明第二方面提供了一种药物组合，其包含本发明第一方面所述的核酸蛋白组合和药物。
在一些实施方案中，所述药物嵌入核酸的C-G碱基对中。
在一些实施方案中，所述药物选自阿霉素、米托蒽醌(Mitoxantrone)、顺铂(Cisplatin)和四环素中的一种或多种。
本发明第三方面提供了一种试剂盒，所述试剂盒包含本发明第一方面所述的核酸蛋 白组合。
在一些实施方案中，所述试剂盒还包含(2E)TCO、(4E)TCO、叠氮和四嗪中的一种或多种。
在一些实施方案中，所述试剂盒还包含选自阿霉素、米托蒽醌、顺铂和四环素中的一种或多种的药物。
本发明第四方面提供了一种制备凝集体的方法，所述方法包含如下步骤：
将本发明第一方面所述的核酸蛋白组合中的蛋白质-TCO和核酸-Tz反应，产生所述凝集体。
在一些实施方案中，所述反应在体外无细胞的溶液中或胞内进行。
在一些实施方案中，所述方法还包括以下步骤：
(1)将蛋白质与(2E)TCO-PEG4-NHS反应，制备蛋白质-TCO；
(2)将叠氮修饰的双链DNA或双链RNA与DBCO-Tz反应，制备双链DNA-Tz或双链RNA-Tz。
本发明第五方面提供了一种制备含药物的凝集体的方法，所述方法包含如下步骤：
(1)将药物载入本发明第一方面所述的核酸蛋白组合中的核酸-Tz例如双链DNA-Tz或双链RNA-Tz；
(2)将蛋白质-TCO和包载药物的双链DNA-Tz或双链RNA-Tz反应，产生所述含药物的凝集体。
在一些实施方案中，所述反应在体外无细胞的溶液中或胞内进行。
在一些实施方案中，所述方法包括以下步骤：
(1)将蛋白质与(2E)TCO-PEG4-NHS反应，制备蛋白质-TCO；
(2)将叠氮修饰的双链DNA或双链RNA与DBCO-Tz反应，制备双链DNA-Tz或双链RNA-Tz；
(3)将药物载入双链DNA-Tz或双链RNA-Tz；
(4)将所述蛋白质-TCO和包载药物的双链DNA-Tz或双链RNA-Tz反应，产生含药物的凝集体。
在一些实施方案中，所述反应在体外无细胞的溶液中或胞内进行。
本发明第六方面提供了本发明第一方面所述的核酸蛋白组合或本发明第二方面所述的药物组合在制备治疗肿瘤或耐药菌感染的药物或试剂盒中的应用。
在符合本领域常识的基础上，上述各优选条件，可任意组合，即得本发明各较佳实例。
本发明所用试剂和原料均市售可得。
本发明的积极进步效果在于：
本发明创新性地提出了一种核酸蛋白组合和利用该核酸蛋白组合制备相分离凝集体的方法；实现了凝集体可控制备和活性成分的长效保留。所述核酸蛋白组合在胞外包括分离的两种组分，在胞内可以通过点击化学反应形成相分离凝集体，可以显著提高药物的抗肿瘤效果，使患肿瘤的小鼠生存时间显著延长，基于所述凝集体的药物保留策略可显著提高药物疗效。
附图说明
图1A为组蛋白和带荧光素FITC的DNA形成的液滴在共聚焦显微镜下的形貌。比例尺为20微米。
图1B为组蛋白、DNA和凝集体溶液的照片。
图1C为组蛋白、DNA和凝集体的浊度测试。
图1D为液滴的荧光漂白后恢复情况。
图2A为结合药物阿霉素的DNA双链序列构成。
图2B为所构建的凝集体对药物的吸附情况。
图2C为药物吸附至凝集体中的时间曲线。
图3A为点击-释放反应的类型。
图3B为基于(2E)TCO基团的点击-释放反应和基于(4E)TCO基团的点击-连接反应的示意图。
图3C为和TCO基团反应后，Tz基团在530nm处吸光度的衰减。
图3D为(2E)TCO和(4E)TCO基团分别和Tz基团反应后，负载分子罗丹明B(RB)的释放曲线。
图4A为组蛋白与(2E)TCO-PEG4-NHS反应前后的分子量变化的质谱表征。
图4B为叠氮修饰的DNA与DBCO-Tz反应前后的分子量变化的质谱表征。
图5为本发明实施例中生物正交反应触发的液液相分离凝集体形成过程，图上方为点击-释放反应触发的凝集体形成过程，下方为点击-连接反应的对照组。
图6为本发明实施例中细胞内凝集体的形成，图左为2E-conjugate在细胞内发生点击-释放反应形成凝集体的共聚焦图，图右为4E-conjugate在细胞内发生点击-连接反应未能触发凝集体形成的对照组。比例尺为20微米。
图7A为2E-conjugate对4T1癌细胞活性的影响。
图7B为2E-conjugate对耐药癌细胞4T1/ADR的毒性测试。
图8A为单独药物和细胞内形成的载药凝集体对4T1细胞的杀伤效果验证。
图8B为单独药物和细胞内形成的载药凝集体对4T1/ADR细胞杀伤效果验证。
图9A为单独药物、凝集体/药物及非凝集体/药物作用12小时后，在4T1细胞内的保留率。
图9B为单独药物、凝集体/药物及非凝集体/药物作用12小时后，在4T1/ADR细胞内的保留率。
图9C为不同组别在4T1/ADR细胞的随时间变化的药物保留检测。
图10A为阿霉素、凝集体/药物及非凝集体/药物组在细胞内随时间变化的含量。
图10B为图10A的统计学分析，表明了不同时间点细胞内的平均药物荧光强度。
图11A为Cy5标记的凝集体+药物组和Cy5标记的非凝集体+药物组作用于细胞后，药物和凝集体的荧光共定位情况。比例尺为20微米。
图11B为图11A的统计学分析，表明了不同时间点不同组别的药物与凝集体的荧光共定位系数。
图11C为图11A的统计学分析，表明了不同时间点不同组别的平均药物吸附强度。
图12A为在4T1小鼠肿瘤模型中，各组小鼠治疗后不同天数的肿瘤生物发光成像图。
图12B为在4T1小鼠肿瘤模型中，各组小鼠治疗后肿瘤生长情况。
图13A为在4T1小鼠肿瘤模型中，各组小鼠治疗后平均肿瘤生长曲线。
图13B为在4T1小鼠肿瘤模型中，各组小鼠治疗后的生存曲线。
图14A为在4T1/ADR小鼠肿瘤模型中，各组小鼠治疗后平均肿瘤生长曲线。
图14B为在4T1/ADR小鼠肿瘤模型中，各组小鼠治疗后肿瘤生长情况。
图14C为在4T1/ADR小鼠肿瘤模型中，治疗12天后各组小鼠肿瘤实物图。
图15A为瘤内注射药物和制剂后的药物保留情况。
图15B为瘤内形成的药物库对经尾静脉注射药物的吸附情况。
具体实施方式
下面通过实施例的方式进一步说明本发明，但并不因此将本发明限制在所述的实施例范围之中。下列实施例中未注明具体条件的实验方法，按照常规方法和条件，或按照商品说明书选择。
实施例1：凝集体的制备和药物存储库的构建
将4μL组蛋白(5mg/mL，生工生物工程(上海)股份有限公司)，4μL DNA(100μM，生工生物工程(上海)股份有限公司)和2μL PEG 3350(250mg/mL，上海麦克林生化科技股份有限公司)充分混合均匀，转移到载玻片上，盖上盖玻片，置于显微镜下观察形貌。用酶标仪测定600nm处的吸光度值以反映溶液浊度，在激光共聚焦显微镜下观察，利用激光漂白选定区域，监测漂白区域荧光强度恢复情况。结果表明，组蛋白和DNA之间会发生液-液相分离，形成液滴(图1A)，伴随溶液浊度增加(图1B、图1C)，且液滴具有动态性质，在激光漂白其中部分区域后，可在50s内恢复DNA的荧光强度(图1D)。
将等量的Seq-1(序列-1)和Seq-2(序列-2)溶于PBS缓冲液(pH＝7.4)，40℃孵育15min，形成DNA双链。将50μL PBS，组蛋白(2mg/mL)，DNA双链(50μM)和凝集体(C histone＝2mg/mL,C DNA duplex＝50μM)分别和50μL阿霉素(以下简称Dox)(5μM，上海麦克林生化科技股份有限公司)混合，在30min内监测阿霉素的荧光强度变化。结果表明，通过设计DNA双链序列，使得抗癌药物阿霉素可以嵌入DNA双链的C-G碱基对中(图2A)，药物的荧光强度淬灭证实成功负载(图2B)。DNA在与组蛋白形成相分离凝结体后，也可用于吸附药物，并在30分钟内达到吸附平衡(图2C)。
实施例2：生物正交反应
1、点击反应：
申请人对不同类型点击-释放反应(图3A)进行了实验摸索，发现仅一种反应类型适用于胞内形成组蛋白和核酸的凝集体，即基于Tz-TCO反应对的点击-释放反应，其他基于Tz-vinyl ethers和Tz-vinyl boronic acid的点击-释放正交反应无法触发胞内相分离凝集体形成。在进行此类型反应时，选择(2E)TCO基团和(4E)TCO基团分别修饰组蛋白，发现仅(2E)TCO基团修饰的情况下能够形成凝集体。具体操作如下：
将5μL梯度浓度的(2E)TCO-PEG4-NHS或(4E)TCO-PEG4-NHS(0.02，0.05，0.1，0.2，0.5，1mM)(西安瑞禧生物科技有限公司)分别和5μL Methyltetrazine-DBCO(1mM，美国Click Chemistry Tools公司)在室温下反应5min后测溶液吸光度，Tz基团在530nm处的衰减用于反映TCO和Tz基团之间的点击反应。
2、释放反应：
首先将RB-PEG-NH2与(2E)TCO-PEG4-NHS Ester或(4E)TCO-PEG4-NHS Ester反应得到(2E)TCO-RB或(4E)TCO-RB。然后，将75μL(2E)TCO-RB或(4E)TCO-RB(120μg/mL)与75μL Duplex-Tz((100μM))混合后，置于微量透析管内管(Slide-A-LyzerTM MINI Dialysis Device，10K MWCO)，外管透析液为13.5mL PBS。在特定时间检测，100μL外管透析液 被取出用于检测RB-PEG-NH2(上海阿拉丁生化科技股份有限公司)释放量(测定透析液在530nm波长激发下，在590nm处的发射荧光强度)。结果表明，基于(2E)TCO基团和Tz基团的点击-释放反应可以释放出带氨基的负载物，基于(4E)TCO基团和Tz基团的点击-连接反应无法释放负载物(图3B)，因此被用于后续实验的对照组。点击-释放和点击-连接反应均可使得Tz基团的结构发生改变，在530nm处的吸光度值降低(图3C)。相比于点击-连接反应，点击-释放反应可在约4小时内释放出80％以上的负载物(图3D)。
3、组蛋白修饰：
将2.5mg histone溶于10mLNaHCO3水溶液(0.1M，pH＝8.4)，加入50μL(2E)TCO-PEG4-NHS或(4E)TCO-PEG4-NHS(10mg/mL in DMSO)。室温反应4h后，超纯水透析1天后冷冻干燥。
4、DNA修饰：
将1mL DNA-N3(20μM in PBS buffer)与100μL DBCO-Tz(1mM in PBS buffer)混合，置于振荡器上，600rpm，室温反应72h。透析后冻干。结果表明，将组蛋白与(2E)TCO-PEG4-NHS反应，将叠氮修饰的DNA与DBCO-Tz反应，使得组蛋白和DNA上分别连接TCO基团和Tz基团，记为Histone-TCO和DNA-Tz，质谱表征修饰前后分子量的变化，证实了组蛋白与TCO基团、DNA与Tz基团连接的成功(图4A、图4B)。
将4μL(2E)Histone-TCO(5mg/mL)或(4E)Histone-TCO(5mg/mL)，4μL DNA-FITC(100μM)和2μL PEG 3350(250mg/mL)充分混合后，转移到载玻片上，盖上盖玻片，置于共聚焦显微镜下观察。结果表明，将DNA-Tz与(2E)Histone-TCO或(4E)Histone-TCO反应，(2E)Histone-TCO与DNA-Tz发生点击-释放反应，释放出带氨基的组蛋白与DNA发生相分离，形成大量凝集体；而(4E)Histone-TCO与DNA-Tz发生点击-连接反应，无法释放出带氨基的组蛋白，凝集体形成量有限(图5)。说明本发明中所提出的生物正交反应控制的液液相分离是可行的。
实施例3：细胞内凝集体的形成、生物相容性测试和凝集体在细胞层面的抗癌效果
用4T1/ADR细胞孵育等浓度的蛋白核酸连接物2E-conjugate或4E-conjugate，组蛋白和DNA双链浓度分别为50μg/mL和1μM，共聚焦显微镜下观察不同时间点凝集体的形成。结果表明，细胞内也可观察到点击-释放反应触发的相分离凝集体形成(图6)。
将4T1和4T1/ADR细胞分别以5×103/well的密度接种于九十六孔板，贴壁24h后，将梯度浓度的2E-conjugate加入到培养基中作用48h，随后，细胞孵育MTT溶液(0.5mg/mL)4h。随后100μL DMSO用于溶解形成的蓝紫色结晶甲臜。细胞活性通过酶标 仪测定490nm吸光度反映。计算公式：Cell viability＝OD490nm(Treatment group)/OD490nm(Control  group)×100％。结果表明，细胞内形成的凝集体对4T1癌细胞和耐药4T1细胞(4T1/ADR)活性均无显著影响(图7A、图7B)，说明了制剂具有较好的生物相容性。
将负载药物的2E-conjugate(即2E-conjugate/Dox)，以梯度浓度单独药物Dox作为对照组(即阿霉素组，下同)，梯度浓度2E-conjugate/Dox作为实验组(即凝集体/药物组)，其余操作不变，观察4T1和4T1/ADR细胞的细胞活性。结果表明，细胞内形成的凝集体显著提高了药物对4T1细胞细胞和4T1/ADR细胞的杀伤效果，且随着阿霉素浓度增加，杀伤能力越强(图8A、图8B)。
实施例4：细胞内药物保留情况
将耐药细胞接种于九十六孔板，贴壁24h后孵育Dox、2E-conjugate/Dox(即凝集体/药物组，下同)和4E-conjugate/Dox(即非凝集体/药物组，下同)4h(药物浓度均为10μM)。随后，除去培养基，PBS洗涤两遍，孵育不含药物的新鲜培养基，在一定时间间隔内，撤去培养基换上新鲜培养基，并测定细胞内药物荧光强度。结果表明，通过定量细胞内药物荧光强度，证实了凝集体在4T1细胞和4T1/ADR细胞中均提高了药物的保留率(图9A、图9B)，且随着时间变化，在作用24h后依旧可以检测到药物保留(图9C)。
细胞接种于共聚焦皿，贴壁24h后，持续孵育单独药物Dox、2E-conjugate/Dox和4E-conjugate/Dox不同时间，这些制剂中，药物浓度均为10μM，组蛋白和DNA双链浓度分别为200μg/mL和10μM。随后除去培养基，PBS洗涤细胞两遍，Hoechst 33342染色细胞核后固定细胞，观察细胞内药物荧光强度。结果表明，通过共聚焦定性实验，随着时间变化，荧光强度增加(图10A)，证实了带红色荧光的药物可以被凝集体长效保留在细胞内(图10B)。
实施例5：凝集体对药物的吸附
用4T1/ADR细胞预处理不含药物的2E-conjugate-Cy5(在制备DNA-Tz时，用Cy5对DNA同时进行修饰)和4E-conjugate-Cy5 4h以形成细胞内凝集体，随后除去培养基，换为包含药物的培养基(药物浓度为10μM)孵育不同时间，共聚焦显微镜下观察药物和Cy5标记的DNA的荧光共定位情况。荧光共定位系数和药物吸附量用Image J软件分析。结果表明，相比于非凝集体组，带红色荧光的药物可以被吸附至Cy5标记的凝集体中(图11A)，且表明了不同时间点凝集体+药物组的荧光共定位系数高于非凝集体+药物组(图11B)，平均药物吸附强度也是凝集体+药物组高于凝集体+药物组(图11C)，证实了凝集 体对药物的吸附能力强。
实施例6：体内药效实验
将4T1或4T1/ADR细胞分别接种于Balb/c裸鼠后背部，待肿瘤体积增长到约100mm3，将小鼠随机分为四组，每组6只，并分别给药，G1组：PBS；G2组：2E-conjugate；G3组：Dox；G4组：2E-conjugate/Dox，对于4T1和4T1/ADR荷瘤小鼠，药物浓度分别为0.25mg/kg和2.5mg/kg，每三天给一次药，每隔两天测量肿瘤体积和小鼠体重，当小鼠肿瘤体积超过1500mm3，安乐死小鼠。结果表明，在4T1小鼠肿瘤模型中肿瘤发光成像图显示凝集体/药物组的肿瘤最小(图12A)，凝集体/药物组的肿瘤尺寸最小(图12B)，随着治疗天数增加，凝集体/药物组肿尺寸增加缓慢(图13A)，且存活比率显著增加(图13B)。在4T1及4T1/ADR小鼠肿瘤模型中，给药后凝集体/药物组肿尺寸增加缓慢(图14A)、凝集体/药物组的肿瘤尺寸最小(图14B)且小鼠肿瘤体积有了显著程度的抑制(图14C)。体内药效实验证实了凝集体可以显著提高药物的抗肿瘤效果，小鼠生存时间显著延长，基于凝集体的药物保留策略可显著提高药物疗效。
实施例7：药物在肿瘤组织的积累情况
1、药物保留：
荷瘤小鼠瘤内注射Dox，2E-conjugate/Dox，4E-conjugate/Dox，24h后，安乐死小鼠，取出肿瘤组织，制作冰冻切片，DAPI染色细胞后，共聚焦显微镜下观察药物在肿瘤组织内的保留情况。
2、药物吸附：
荷瘤小鼠瘤内注射PBS、2E-conjugate,4E-conjugate，同时尾静脉注射阿霉素，24h后，安乐死小鼠，取出肿瘤组织，制作冰冻切片，DAPI染色细胞后，共聚焦显微镜下观察药物在肿瘤组织内的吸附情况。结果表明，肿瘤组织切片证实了生物正交的点击-释放反应不仅可作为药物递送体系，利用较大的尺寸阻滞效果提高药物在细胞内的保留(图15A)，还可通过瘤内形成的药物库吸附尾静脉注射的药物，从而实现肿瘤组织内的药物提升(图15B)，增强抗肿瘤疗效。
虽然以上描述了本发明的具体实施方式，但是本领域的技术人员应当理解，这些仅是举例说明，在不背离本发明的原理和实质的前提下，可以对这些实施方式做出多种变更或修改。因此，本发明的保护范围由所附权利要求书限定。

Claims (10)

  1. 一种可形成相分离凝集体的核酸蛋白组合，其特征在于，其包含：修饰的蛋白质和修饰的核酸，所述核酸包括DNA和RNA，所述修饰的蛋白质与修饰的核酸可发生点击-释放反应，使蛋白质与核酸发生相分离，形成所述相分离凝集体。
  2. 如权利要求1所述的核酸蛋白组合，其特征在于，所述点击-释放反应在体外无细胞的溶液中或胞内进行；和/或，所述修饰的蛋白质为(2E)TCO修饰的蛋白质即蛋白质-TCO，所述修饰的核酸为四嗪修饰的核酸即核酸-Tz；
    较佳地，当所述点击-释放反应在体外无细胞的溶液中进行时，所述蛋白质的浓度大于1mg/mL，优选小于5mg/mL，所述DNA的浓度大于20μM，优选小于100μM；更佳地，所述蛋白质的浓度大于2mg/mL且小于5mg/mL，所述DNA的浓度大于30μM且小于100μM；
    或，当所述点击-释放反应在胞内进行时，所述蛋白质的浓度大于50μg/mL，优选小于200μg/mL，所述DNA的浓度大于1μM，优选小于10μM；更佳地，所述蛋白质的浓度大于100μg/mL且小于200μg/mL，所述DNA的浓度大于5μM且小于10μM。
  3. 如权利要求1或2所述的核酸蛋白组合，其特征在于，所述蛋白质选自具相分离能力的蛋白质，优选富含氨基的蛋白质；所述核酸选自富含重复碱基序列且能稳定存在的核酸，例如包含重复C-G碱基对的双链DNA或双链RNA；
    优选地，所述富含氨基的蛋白质包括组蛋白或朊病毒样蛋白，例如FUS；和/或，所述蛋白质的分子量范围为10-20kDa；和/或，所述核酸的长度为25～30bp；
    更优选地，所述组蛋白选自组蛋白H1、H2A、H2B、H3和H4中的一种或多种；所述DNA的两条单链核苷酸序列分别如SEQ ID NO:1和SEQ ID NO:2所示。
  4. 一种药物组合，其特征在于，其包含如权利要求1～3任一项所述的核酸蛋白组合和药物；
    优选地，所述药物嵌入核酸的C-G碱基对中；和/或，所述药物选自阿霉素、米托蒽醌、顺铂和四环素中的一种或多种。
  5. 一种试剂盒，其特征在于，所述试剂盒包含如权利要求1～3任一项所述的核酸蛋白组合；
    优选地，还包含(2E)TCO、(4E)TCO、叠氮和四嗪中的一种或多种；和/或，还包含选自阿霉素、米托蒽醌、顺铂和四环素中的一种或多种的药物。
  6. 一种制备凝集体的方法，其特征在于，所述方法包含如下步骤：
    将如权利要求1～3任一项所述的核酸蛋白组合中的蛋白质-TCO和核酸-Tz反应，产 生所述凝集体；
    优选地，所述反应在体外无细胞的溶液中或胞内进行。
  7. 如权利要求6所述的方法，其特征在于，所述方法还包括以下步骤：
    (1)将蛋白质与(2E)TCO-PEG4-NHS反应，制备蛋白质-TCO；
    (2)将叠氮修饰的双链DNA或双链RNA与DBCO-Tz反应，制备双链DNA-Tz或双链RNA-Tz。
  8. 一种制备含药物的凝集体的方法，其特征在于，所述方法包含如下步骤：
    (1)将药物载入如权利要求1～3任一项所述的核酸蛋白组合中的核酸-Tz例如双链DNA-Tz或双链RNA-Tz；
    (2)将蛋白质-TCO和包载药物的双链DNA-Tz或双链RNA-Tz反应，产生所述含药物的凝集体；优选地，所述反应在体外无细胞的溶液中或胞内进行。
  9. 如权利要求8所述的方法，其特征在于，所述方法包括以下步骤：
    (1)将蛋白质与(2E)TCO-PEG4-NHS反应，制备蛋白质-TCO；
    (2)将叠氮修饰的双链DNA或双链RNA与DBCO-Tz反应，制备双链DNA-Tz或双链RNA-Tz；
    (3)将药物载入双链DNA-Tz或双链RNA-Tz；
    (4)将所述蛋白质-TCO和包载药物的双链DNA-Tz或双链RNA-Tz反应，产生含药物的凝集体；优选地，所述反应在体外无细胞的溶液中或胞内进行。
  10. 如权利要求1～3任一项所述的核酸蛋白组合或如权利要求4所述的药物组合在制备治疗肿瘤或耐药菌感染的药物或试剂盒中的应用。
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