WO2024227301A1 - 一种递送生物制剂的蛋白质-核酸凝集体及其制备方法 - Google Patents

一种递送生物制剂的蛋白质-核酸凝集体及其制备方法 Download PDF

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WO2024227301A1
WO2024227301A1 PCT/CN2023/092187 CN2023092187W WO2024227301A1 WO 2024227301 A1 WO2024227301 A1 WO 2024227301A1 CN 2023092187 W CN2023092187 W CN 2023092187W WO 2024227301 A1 WO2024227301 A1 WO 2024227301A1
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nucleic acid
stranded nucleic
hollow
vector
histone
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顾臻
李洪军
文苹
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Zhejiang University
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    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K47/00—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient
    • A61K47/50—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates
    • A61K47/69—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates the conjugate being characterised by physical or galenical forms, e.g. emulsion, particle, inclusion complex, stent or kit
    • A61K47/6949—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates the conjugate being characterised by physical or galenical forms, e.g. emulsion, particle, inclusion complex, stent or kit inclusion complexes, e.g. clathrates, cavitates or fullerenes
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • 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
    • A61K47/06—Organic compounds, e.g. natural or synthetic hydrocarbons, polyolefins, mineral oil, petrolatum or ozokerite
    • A61K47/26—Carbohydrates, e.g. sugar alcohols, amino sugars, nucleic acids, mono-, di- or oligo-saccharides; Derivatives thereof, e.g. polysorbates, sorbitan fatty acid esters or glycyrrhizin
    • 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
    • A61K47/54—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 compound
    • 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
    • A61K47/54—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 compound
    • A61K47/543—Lipids, e.g. triglycerides; Polyamines, e.g. spermine or spermidine
    • A61K47/544—Phospholipids
    • 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
    • A61K47/54—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 compound
    • A61K47/554—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 compound the modifying agent being a steroid plant sterol, glycyrrhetic acid, enoxolone or bile acid
    • 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
    • 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

Definitions

  • the present invention relates to the medical field, and in particular to a protein-nucleic acid aggregate for delivering biological agents and a preparation method thereof.
  • Biomacromolecules such as proteins and nucleic acids form membrane-less organelles or aggregates through weak multivalent interactions and liquid-liquid phase separation (LLPS) mediated by conformational entropy, which participate in the regulation of many key cellular life activities and are closely related to a variety of human diseases. Aggregates formed by LLPS allow the enrichment of biomolecules with different physical and chemical properties, while isolating other biomolecules outside the aggregates. Aggregates, as separate compartments within cells, create an isolated environment for the enriched molecules, thereby separating and regulating biochemical reactions within cells.
  • the LLPS phenomenon is not limited to playing a role in biology or pathology, but can also provide inspiring clues for biomimetic applications.
  • the aggregates can absorb and recruit biomacromolecules (peptides, proteins, etc.) into the compartments, and have the advantages of efficiently enriching guest molecules and effectively retaining their biological activity, showing the potential as a delivery carrier for biological agents.
  • biomacromolecules peptides, proteins, etc.
  • the aggregates reversibly and dynamically formed by LLPS are not restricted by the membrane structure, are easy to aggregate, and are affected by environmental factors such as temperature, pH, ionic strength, etc., which limits their application in the field of drug delivery.
  • the present invention designs and generates a new delivery carrier, which is composed of protein and nucleic acid agglomerates, wherein the agglomerates are hollow.
  • the structure has the following advantages: excellent physiological environment stability; biocompatibility, the ability to efficiently recruit biological agents and improve the efficiency of cell uptake of biological agents, and ultimately achieve effective delivery of biological agents, thereby improving the treatment effect of diseases.
  • the object of the present invention is to provide a delivery vector, wherein the vector is formed by the aggregation of protein and nucleic acid, wherein the vector is a hollow structure or contains space inside the vector.
  • the vector is used to load biological agents, which may be polypeptides, proteins, antibody drugs, nucleic acids, bacteria and viruses, etc., for treating or preventing diseases, such as for preparing vaccines, infectious disease vaccines, tumor vaccines, etc.
  • the protein is a histone.
  • the histone is a mixture of multiple proteins.
  • the histone is derived from an extract of calf thymus.
  • the histone includes any one or more of H1, H2A, H2B, H3 and H4.
  • non-histone sequence-specific DNA binding proteins may also be included.
  • the histones described in the present invention have the following characteristics: they are positively charged, contain Arg and Lys and are alkaline proteins, and are synthesized only in the S phase of the cell cycle. There are five types in total, which are core histones, H2A, H2B, H3, H4 and linker histones (H1). The structure of histones is highly conserved, especially H4.
  • the core histones are composed of a globular part and a tail.
  • the globular part uses the electrostatic interaction between Arg and the phosphodiester backbone to wrap the DNA molecule around the histone core to form a nucleosome.
  • the tail contains a large amount of Arg and Lys, which is the site of post-translational modification.
  • H1 is diverse and has genus and tissue specificity.
  • the non-histone sequence-specific DNA binding protein of the present invention has the following characteristics: it is negatively charged, an acidic protein, is synthesized throughout the cell cycle, can recognize specific DNA sequences, and binds with hydrogen bonds and ionic bonds.
  • the function of the non-histone sequence-specific DNA binding protein is to help DNA folding and replication; and regulate gene expression.
  • the hollow aggregate formed by the present invention has a surface with positive charge or close to neutral charge, and interacts with the cell membrane with negative charge; the hollow aggregate can maintain a stable structure under physiological conditions.
  • the nucleic acid is a modified single-stranded nucleic acid, such as deoxyribonucleic acid (DNA) or ribonucleic acid (RNA), or a mixture of DNA and RNA.
  • DNA deoxyribonucleic acid
  • RNA ribonucleic acid
  • the modification group includes substances similar to mammalian cell membranes.
  • the modified group can be any one or more of cholesterol, phospholipids, glycolipids, and glycoproteins.
  • the protein here can be a protein that constitutes a cell membrane. It is understood that all molecules that constitute a cell membrane can be used to modify the single-stranded nucleic acid defined in the present invention, such as lipids, proteins, and a small amount of sugars, such as phospholipids, or substances similar to the components that constitute the cell membrane, such as amphiphilic substances, block copolymers, modified drugs, etc., can be used for modification. Mechanistically, any substance similar to cell membrane components can be used to modify single-stranded nucleic acids. For example, cholesterol in one embodiment of the present invention is similar to cell membrane components and can assist in the transmembrane transport of other components.
  • the modification group is modified at the 3' end or 5' end of the single-stranded nucleic acid, or at both the 3' and 5' ends. Of course, it can also be modified at a position close to the end (modification at the 3' end or 5' end). Optionally, it can also be modified in the middle of the single-stranded nucleic acid.
  • the modification ratio is 0.1%-99%, which can be 1-95%, 1-85%, 1-75%, 1-55%, 1-45%.
  • the so-called ratio here refers to the substitution reaction of the modified purine base or pyrimidine base, ribose or deoxyribose and phosphate functional groups in all the single-stranded nucleotides to carry out the modification of the present invention.
  • the length of the cholesterol-modified single-stranded deoxyribonucleic acid is any DNA fragment, and the length is about 5-1000bp, preferably, 10-500bp, 15-300bp, 15-100bp.
  • the nucleotides can be composed of A, U, C, T, G in any order (5′-adenylic acid, 5′-thymidylic acid, 5′-guanylic acid, 5′-inosinic acid, 3′-xanthylic acid, 5′-cytidylic acid, 5′-uridylic acid), or a certain length composed of single nucleotide molecules A, U, C, T, G.
  • the so-called single-stranded nucleic acid here refers to a nucleic acid that is mainly a single strand, and also includes a single-stranded nucleic acid with a hairpin structure.
  • the single-stranded nucleic acid here is a concept opposite to the double-stranded nucleic acid.
  • the single-stranded nucleic acid is artificially synthesized or extracted from plants, animals, humans, or lower organisms.
  • the molar ratio of the histone to the modified single-stranded nucleic acid is 1:500 to 1:20.
  • the histone and the modified single-stranded nucleic acid are mixed at a concentration ratio of 1 ⁇ M-50 ⁇ M ssDNA and 0.25 mM-2 mM histone.
  • the final concentration of the histone is 0.5 mM
  • the final concentration of the modified single-stranded nucleic acid ssDNA is 10 ⁇ M.
  • the vector includes biological agents that can be delivered by the vector, and the biological agents include one or more of polypeptides, proteins, antibody drugs, nucleic acids, bacteria, and viruses.
  • the vector is formed by condensation of histones and modified single-stranded nucleic acids.
  • the present invention provides a method for preparing a hollow protein-nucleic acid condensate carrier: the carrier is prepared by mixing histones with modified single-stranded nucleic acids and a solvent, wherein the carrier is a hollow structure.
  • the molar ratio of the histones to the modified single-stranded nucleic acids is 1:500 to 1:20.
  • the solvent used in the preparation step is any one or more of PBS solution, citric acid-sodium citrate buffer solution, disodium hydrogen phosphate-sodium dihydrogen phosphate buffer solution.
  • the solvent is 1X PBS, with a pH of 7.4; or 0.1-0.01 M citric acid-sodium citrate buffer, with a pH of 5-6.5; or 0.1-0.01 M disodium hydrogen phosphate-sodium dihydrogen phosphate buffer, with a pH of 6.5-8.
  • the histone is mixed with a biological agent to be delivered, and then mixed with the modified single-stranded nucleic acid.
  • the biological agent includes one or more of: polypeptides, proteins, antibody drugs, nucleic acids, bacteria, and viruses.
  • the final concentration of the modified single-stranded nucleic acid is 0.1 ⁇ M to 50 ⁇ M, and the final concentration of histone is 0.025 mM to 2 mM.
  • ssDNA with 5'-end modified cholesterol is mixed with 2.25*10 7 pfu of ad11 adenovirus at a final concentration of 10 ⁇ M and a final histone concentration of 0.5 mM, and the virus molecules are selectively enriched in the phase separation region of the hollow agglutinate through molecular enrichment of phase separation to form agglutinates containing adenovirus.
  • the agglutinate preparation containing ad11 adenovirus is subcutaneously injected to treat mouse tumors.
  • the present invention provides use of a hollow protein-nucleic acid aggregate carrier for preparing a delivery carrier for delivering a biological agent to an organism.
  • the present invention provides the use of a hollow protein-nucleic acid aggregate vector encapsulating adenovirus for preparing a preparation for improving the therapeutic effect of tumors.
  • the present invention provides the use of modified single-stranded nucleic acids for preparing hollow protein-nucleic acid condensate carriers.
  • the modified groups are one or more of cholesterol, phospholipids, glycolipids, and glycoproteins.
  • the modification may be cholesterol modification or modification of substances similar to cell membranes, including phospholipids, proteins, and the like.
  • the modifying group is cholesterol.
  • modified single-stranded nucleic acid which is modified into a molecule similar to the components of biological cell membranes.
  • the modified single-stranded nucleic acid is not restricted, and there are many choices of modifiable molecules and optional modification methods, which has a wide range of applications.
  • the hollow aggregate carrier formed has a stable structure in the physiological environment, high biocompatibility, and can efficiently recruit biological agents and improve the efficiency of cell uptake of biological agents, thereby achieving effective delivery and assisting disease treatment.
  • the formation of hollow aggregates avoids the problems of low delivery efficiency caused by the large volume, easy aggregation, poor structural stability, irregular structure, and excessive size of non-hollow aggregates.
  • the biological agents that can be delivered include peptides, proteins, antibody drugs, nucleic acids, bacteria, viruses, etc., and have a wide range of applications.
  • Figure 1 (a)-(c) are phase diagrams and phase forms based on liquid-liquid phase separation between protein and nucleic acid in Example 1 of the present invention.
  • FIG1( a ) shows the morphology of aggregates and hollow aggregates formed by LLPS of rhodamine B-labeled histones and 6-FAM-labeled modified single-stranded nucleotide ssDNA under a confocal microscope; the scale bar is 10 ⁇ m.
  • Figure 1(b) is a partial phase diagram between histones and ssDNA. It can be seen from Figure 1(b) that the ratio of the two is critical, and a hollow structure can only be obtained within a certain range.
  • FIG1( c ) is a potential diagram obtained by mixing histones and ssDNA at different ratios.
  • Figure 1(d) is the structural formula of cholesterol modified at the 3’ end of single-stranded deoxyribonucleic acid.
  • Figure 1(e) is the structural formula of cholesterol modified on the 5’ end of single-stranded deoxyribonucleic acid.
  • Figure 2 is a liquid-liquid phase separation phase diagram and phase form between histone and single-stranded deoxyribonucleic acid (5’-AAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAA-3’) of unmodified cholesterol in Example 2 of the present invention.
  • Figure 2 is a partial phase diagram between histones and single-stranded deoxyribonucleic acid without cholesterol modification.
  • (b) is a mixed solution formed by histones and nucleic acids labeled with Cy5.
  • (c) is an aggregate formed by LLPS of histones and nucleic acids labeled with cyanine dye Cy5 (red).
  • (d) is an aggregate formed by LLPS of histones and nucleic acids labeled with Cy5.
  • the scale bar is 50 microns. Single-stranded deoxyribonucleic acid without cholesterol modification and histones cannot form a hollow aggregate structure under the conditions of Figure a.
  • Figure 3 is a structural and morphological characterization of the aggregate and hollow aggregate in the embodiment of the present invention.
  • (a, d) are three-dimensional confocal microscope images of the aggregate and hollow aggregate, respectively.
  • (b, e) are scanning electron microscope images of the aggregate droplets and hollow aggregate, respectively.
  • (c, f) are cryo-transmission electron microscope images of the aggregate droplets and hollow aggregate, respectively.
  • FIG4 is a performance characterization of the assembly formed by the LLPS process in an embodiment of the present invention.
  • (a) is the fusion phenomenon of the aggregate droplets.
  • (b) is a confocal microscopy group of fluorescence recovery after bleaching of the aggregate droplets and fluorescence intensity analysis.
  • (c) is a confocal microscopy group of fluorescence recovery after bleaching of the hollow aggregate and fluorescence intensity analysis.
  • the scale bar is 5 ⁇ m.
  • FIG5 is a phase diagram and phase form of virus loading based on liquid-liquid phase separation between proteins and nucleic acids in an embodiment of the present invention.
  • (a) is the morphology of aggregates and hollow aggregates formed by LLPS of histones labeled with rhodamine B, ssDNA labeled with 6-FAM, and adenovirus ad11 (Cy5-ad11) labeled with Cy5 (blue) under a confocal microscope.
  • the scale bar is 10 microns.
  • (b) is a partial phase diagram between the three.
  • Figure 6 shows the enrichment of biological agents by agglomerate droplets and hollow agglomerates in an embodiment of the present invention.
  • Figure 6(a) is a fluorescence intensity analysis diagram of histones, ssDNA and ad11 virus, with a scale of 5 microns.
  • Figure 6(b) is a comparison diagram of the loading rate of ad11 virus by agglomerates and hollow agglomerates.
  • FIG7 shows the distribution of viruses in a phase separation system in an embodiment of the present invention.
  • (a) is a transmission electron microscopy image of a single virus.
  • (b) is a transmission electron microscopy image of a virus in a protein-nucleic acid solution.
  • (c) is a cryo-transmission electron microscopy image of a virus in a protein-nucleic acid aggregate droplet.
  • (d) is a cryo-transmission electron microscopy image of a virus in a protein-nucleic acid hollow aggregate.
  • the scale bar is 100 nanometers.
  • FIG8 is a study on the structural stability of agglomerate droplets and hollow agglomerates in an embodiment of the present invention.
  • (a) shows the turbidity and morphology changes of agglomerate droplets and hollow agglomerates at the initial moment and after 48 hours.
  • (b) shows the particle size changes of agglomerate droplets and hollow agglomerates at the initial moment and after 48 hours.
  • the scale bar is 10 microns.
  • the particle size of the hollow agglomerates changes little, and there is basically no change after 48 hours (the two bar graphs on the left of FIG8(b)); while the particle size of the agglomerate droplets changes significantly, from about 2321 nm to 8619 nm (the two bar graphs on the right of FIG8(b)).
  • FIG9A shows the viral uptake of agglomerate droplets and hollow agglomerates as viral vectors in mouse lung epithelial cells (TC-1) and TC-1 cell lines that highly express the receptor CD46 in an embodiment of the present invention.
  • (a) shows the uptake of ad11 or ad11 solution, ad11/agglomerates, and ad11/hollow agglomerates in TC-1 cell lines and the statistics of viral content.
  • (b) shows the uptake of ad11 or ad11 solution, ad11/agglomerates, and ad11/hollow agglomerates in TC-1 cell lines that highly express CD46 and the statistics of viral content.
  • the scale bar is 20 ⁇ m.
  • 1 represents a single virus (ad11)
  • 2 represents a single virus solution (ad11 solution)
  • 3 represents an aggregate/virus (ad11/aggregate) (non-hollow);
  • 4 represents a hollow aggregate/virus (ad11/hollow aggregate), indicating that the hollow one loaded with virus is more stable, and the number of viruses taken up by cells is the highest, which is significantly different from the non-hollow one (normalized).
  • the number of pure virus group is 1, the loading capacity of ad11/solution is about 3, ad11/aggregate is about 5, and ad11/hollow aggregate is about 7).
  • Figure 10 shows the accumulation of ad11 in tumor tissues in an embodiment of the present invention.
  • the scale bar is 100 micrometers.
  • FIG. 11 is the in vivo efficacy data of the present invention in the TC-1 mouse tumor model.
  • (a) is a real picture of the tumor of each group of mice after 10 days of treatment.
  • (b) is the weight of the tumor of each group of mice after 10 days of treatment.
  • (c) is the average tumor growth curve of each group of mice after treatment.
  • tumor size in terms of tumor size, the size of the small tumor treated with ad11/hollow aggregates remains basically unchanged, while the size of the tumor under other treatments (PBS, simple hollow aggregates, simple virus treatment, and non-hollow aggregates) increases rapidly within 12 days, showing a very significant difference with the size of the small tumor treated with ad11/hollow aggregates). It can also be seen from FIG.
  • the weight under ad11/hollow aggregate treatment is about 0.5g, while the weight under PBS, simple hollow aggregates, simple virus treatment, and non-hollow aggregates is about 2.4g, 2.3g, 1.9g, and 1.8g, respectively., which is significantly or very significantly different from these treatments.
  • Example 1 Histones and modified single-stranded nucleic acids form hollow aggregates.
  • Figure 1(b) shows the phase diagram under the corresponding conditions by observing the morphology of the phase in the system under a laser confocal microscope. As the concentration of the two and the potential in the system change, three phase forms are shown: uniform solution, droplet-shaped aggregate (non-hollow) and hollow aggregate.
  • the conditions under which hollow aggregates can be formed are that 1 ⁇ M-50 ⁇ M ssDNA is mixed with 0.25 mM-2 mM histone. Among them, the proportion of hollow aggregates was the highest when the final histone concentration was 0.5 mM and the final ssDNA concentration was 10 ⁇ M.
  • nucleic acids it can be any single-stranded nucleic acid material, such as DNA, RNA, etc. These nucleic acid materials can be artificially synthesized, or they can be single-stranded nucleic acids extracted from plants, animals, humans, and lower organisms.
  • the length of these single-stranded nucleic acid materials can be any length, generally less than 1000bp, or less than 500bp, less than 100bp, or between 10-80bp.
  • these single-stranded nucleotides can be one or more different sequences, mixtures of different lengths, but these single-stranded nucleic acids need to be modified.
  • the modified position it can be the two ends of the nucleic acid, such as the 5' or 3' end, or the 5' and 3' ends, or it can be close to the end of the nucleic acid, such as the position of 10-1 nucleotides away from the 5' or 3' end. This can be arbitrarily selected through the disclosure of the present invention, such as modifying the middle position of the nucleic acid. Through our experiments, the above selections and combinations can also obtain hollow aggregates.
  • solutions of different concentrations of Histone (the same as in Example 1) and ssDNA labeled with cyanine dye Cy5 (sequence 5'-AAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAA-Cy5-3') were prepared respectively, and the two solutions were mixed to a final concentration of Histone of 2mM, 1mM, 0.5mM, 0.25mM, 0.1mM, 0.05mM, 0.025mM, and a final concentration of ssDNA of 50 ⁇ M, 25 ⁇ M, 10 ⁇ M, 5 ⁇ M, 1 ⁇ M, 0.5 ⁇ M, 0.1 ⁇ M.
  • the morphology of the phase in the system was observed under a fluorescence microscope to draw a phase diagram under the corresponding conditions. As the concentration and ratio of the two changed, three forms of phases were shown: uniform solution, droplet-shaped aggregates, and aggregates.
  • the ssDNA and histones without modified cholesterol failed to obtain hollow aggregates under this condition.
  • Example 3 Characterization of the structure and morphology of the aggregates and hollow aggregates in the examples of the present invention
  • the aggregates were prepared under the conditions of a final histone concentration of 0.25mM and a final ssDNA concentration of 50 ⁇ M, and the hollow aggregates were prepared under the conditions of a final histone concentration of 0.5mM and a final ssDNA concentration of 10 ⁇ M.
  • the aggregate droplets were solid spheres, the hollow aggregates were aqueous phase cavities, and the hollow sphere structure obtained by phase separation (as described in the text of Figure 3d) was specifically
  • the histone and the modified single-stranded nucleic acid are the same as those in Example 1.
  • Example 4 Characterization of the performance of the assembly formed by the LLPS process in the present invention
  • the condensate was prepared under the conditions of a final histone concentration of 0.25mM and a final ssDNA concentration of 50 ⁇ M, and the hollow condensate was prepared under the conditions of a final histone concentration of 0.5mM and a final ssDNA concentration of 10 ⁇ M (the specific histone and the modified single-stranded nucleic acid are the same as those in Example 1).
  • the structure assembled by phase separation has the characteristics of a liquid system, such as molecular mobility and deformability. After adjacent droplets contact each other (0s), fusion will occur.
  • the photobleaching fluorescence recovery experiment can also be used to prove that the assembly is formed by the LLPS process.
  • the specific areas of the condensate droplets and the hollow condensate are photobleached (50% light intensity, photobleaching time is 10s), and then the fluorescence recovery is detected.
  • Example 5 Phase diagram and phase form of virus loading based on liquid-liquid phase separation between protein and nucleic acid in the example of the present invention.
  • solutions of different concentrations of rhodamine B-labeled histone (Histone) and 6-carboxyfluorescein (6-FAM)-labeled cholesterol-modified ssDNA were prepared respectively, and the two solutions were mixed to a final concentration of 2mM, 1mM, 0.5mM, 0.25mM, 0.1mM, 0.05mM, 0.025mM for Histone, and a final concentration of 50 ⁇ M, 25 ⁇ M, 10 ⁇ M, 5 ⁇ M, 1 ⁇ M, 0.5 ⁇ M, 0.1 ⁇ M for ssDNA.
  • Cy5-ad11 was obtained from Beijing Bio-Targeting Therapeutics Technology Co., Ltd (Beijing, China) (plaque forming unit, abbreviated as pfu), histones and modified single-stranded nucleic acids recruit Cy5-ad11 into the assembled compartments during the phase separation process.
  • the system with Cy5-ad11 addition also contains three forms: uniform solution, droplet-shaped aggregates, and hollow aggregates.
  • Example 6 Enrichment of biological agents by agglomerate droplets and hollow agglomerates in the present invention
  • the aggregates were prepared under the conditions of a final histone concentration of 0.25mM and a final ssDNA concentration of 50 ⁇ M, and the hollow aggregates were prepared under the conditions of a final histone concentration of 0.5mM and a final ssDNA concentration of 10 ⁇ M.
  • the total volume of each system was 40 ⁇ L, and the number of Cy5-ad11 was 9*10 7 pfu.
  • the phase separation system has excellent molecular enrichment ability and promotes the efficient loading of biological agents.
  • the analysis of the fluorescence intensity and distance distribution of histones, ssDNA and viruses confirmed the effective enrichment of viruses by the phase separation domain (scale bar is 5 microns).
  • the analysis software is Image J.
  • Two virus-enriched assemblies were prepared under conditions of a final histone concentration of 0.25 mM and a final ssDNA concentration of 50 ⁇ M, and under conditions of a final histone concentration of 0.5 mM and a final ssDNA concentration of 10 ⁇ M.
  • the virus loading rates of the two assemblies were calculated using real-time fluorescence quantitative technology.
  • the loading rates of the aggregates were 92.8%, and those of the hollow aggregates were 81.4%, as shown in Figure 6(b).
  • Example 7 Distribution of viruses in a phase separation system in an example of the present invention.
  • a solution was obtained under the conditions of a final histone concentration of 0.5mM and a final ssDNA concentration of 0.1 ⁇ M; an aggregate was prepared under the conditions of a final histone concentration of 0.25mM and a final ssDNA concentration of 50 ⁇ M; a hollow aggregate was prepared under the conditions of a final histone concentration of 0.5mM and a final ssDNA concentration of 10 ⁇ M; the total volume of each system was 40 ⁇ L, and the number of Cy5-ad11 was 9*10 7 pfu.
  • the virus in the solution was in a state of aggregation (Figure 7b). The virus was separated into the aggregate droplets and the hollow aggregate by the molecular enrichment of phase separation, and was isolated from the external environment.
  • the virus in the aggregate, was evenly dispersed in the aggregate droplets.
  • the virus in the hollow aggregate, was selectively enriched in the phase separation area of the hollow aggregate, distributed in the annular area, and co-localized with histones and single-stranded nucleic acids.
  • Example 8 Study on the structural stability of aggregate droplets and hollow aggregates in the embodiments of the present invention
  • the condensate was prepared under the conditions of a final histone concentration of 0.25mM and a final ssDNA concentration of 50 ⁇ M
  • the hollow condensate was prepared under the conditions of a final histone concentration of 0.5mM and a final ssDNA concentration of 10 ⁇ M.
  • the condensate (non-hollow, B in Figure 8(b)) droplets have stronger liquid fluidity and deformability, so the condensate droplets tend to aggregate over time. This characteristic is manifested in the gradual decrease in turbidity of the condensate system, the aggregation of the condensate, and the significant increase in particle size.
  • the efficiency of the subsequent condensate delivery of the virus to the tumor cells is reduced, thereby affecting the tumor treatment effect.
  • the liquid fluidity and deformability of the hollow condensate or carrier droplet are weaker than those of the non-hollow one, so the hollow condensate will not condense over time. In this way, the structure is more stable, more regular, and of suitable size, and the required substance can be delivered to the cell.
  • Example 9 Viral uptake of aggregate droplets and hollow aggregates as viral vectors in mouse lung epithelial cells (TC-1) and TC-1 cell lines that highly express the receptor CD46 in the present invention
  • the aggregates were prepared under the conditions of a final histone concentration of 0.25mM and a final ssDNA concentration of 50 ⁇ M, and the hollow aggregates were prepared under the conditions of a final histone concentration of 0.5mM and a final ssDNA concentration of 10 ⁇ M.
  • the number of Cy5-ad11 (adenovirus) was 1*10 7 pfu.
  • the above solution was added to mouse lung epithelial cells (TC-1) and TC-1 cell lines that highly expressed the receptor CD46, with a cell number of 500,000 and a virus number of 20pfu per cell.
  • the above system was incubated in a cell culture incubator at 37°C and 5% CO2 for 4 hours to remove free viruses/assemblies.
  • Example 10 Accumulation of ad11 in tumor tissue in the present invention (scale bar is 100 micrometers)
  • Aggregates were prepared under the conditions of a final histone concentration of 0.25 mM and a final ssDNA concentration of 50 ⁇ M, and hollow aggregates were prepared under the conditions of a final histone concentration of 0.5 mM and a final ssDNA concentration of 10 ⁇ M.
  • untreated TC-1 cells were cultured, and 100 ⁇ L of subcutaneous tumor-bearing cells of 2*10 6 were injected to construct an animal model.
  • C57/BL-6J, 8-week-old female mice were selected as experimental mice. The experimental mice were raised until the tumor size was 100 mm 3 , and the preparations were injected intratumorally.
  • the PBS group was the control group, and 50 ⁇ L 1XPBS (pH 7.4) was injected intratumorally;
  • the ad11 group was the virus-only group, and the intratumoral injection dose was 50 ⁇ L, and the number of viruses was 1*10 8 pfu;
  • the composition of the ad11/aggregate group the final concentration of histones was 0.25 mM, the final concentration of ssDNA was 50 ⁇ M, the number of viruses was 1*10 8 pfu, and the intratumoral injection dose was 50 ⁇ L;
  • the composition of the ad11/hollow aggregate group the final concentration of histones was 0.5 mM, the final concentration of ssDNA was 10 ⁇ M, the number of viruses was 1*10 8 pfu, and the intratumoral injection dose was 50 ⁇ L.
  • the virus was label
  • Example 11 In vivo efficacy data of the present invention in the TC-1 mouse tumor model
  • Aggregates were prepared under the conditions of a final histone concentration of 0.25 mM and a final ssDNA concentration of 50 ⁇ M, and hollow aggregates were prepared under the conditions of a final histone concentration of 0.5 mM and a final ssDNA concentration of 10 ⁇ M.
  • untreated TC-1 cells were cultured, and 100 ⁇ L of subcutaneous tumor-bearing cells of 2*10 6 were injected to construct an animal model.
  • C57/BL-6J, 8-week-old female mice were selected as experimental mice. The experimental mice were raised until the tumor size reached 100 mm 3 , and the preparations were injected intratumorally.
  • the PBS group was the control group, and 50 ⁇ L was injected intratumorally.
  • 1XPBS pH 7.4
  • ad11 group is a simple virus group, the intratumoral injection dose is 50 ⁇ L, the virus number is 1*10 8 pfu
  • ad11/aggregate group composition the final histone concentration is 0.25mM, the final ssDNA concentration is 50 ⁇ M, the virus number is 1*10 8 pfu, and the intratumoral injection dose is 50 ⁇ L
  • ad11/hollow aggregate group composition the final histone concentration is 0.5mM, the final ssDNA concentration is 10 ⁇ M, the virus number is 1*10 8 pfu, and the intratumoral injection dose is 50 ⁇ L.
  • the preparation was injected once every two days, and the tumor size was recorded.
  • In vivo efficacy experiments confirmed that hollow aggregates can significantly improve the anti-tumor effect of the virus.
  • the tumor volume of the mouse was significantly inhibited after administration.
  • Hollow aggregates can significantly improve the therapeutic efficacy as a virus delivery carrier.
  • the use of hollow agglomerates to deliver anti-tumor drugs can significantly inhibit the increase in tumor volume or weight, which is significantly different from the use of agglomerates carrying the same anti-tumor drugs.

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Abstract

本发明提供了一种递送生物制剂的蛋白质-核酸凝集体载体，该载体包括组蛋白和被基团修饰的单链核酸；其中所述的载体为中空的结构，利用这样的载体稳定地递送生物制剂。将组蛋白与被修饰的单链核酸在溶剂中按照特定的终浓度混合后可获得中空的蛋白质-核酸凝集体载体，可包载各种类的生物制剂用于辅助治疗。此载体在生物体内结构稳定，递送效果好，具有良好的应用前景。

Description

一种递送生物制剂的蛋白质-核酸凝集体及其制备方法
本申请主张中国在先申请，申请号:202310490378.3，申请日2023年5月4日的优先权；其所有的内容作为本发明的一部分。
技术领域
本发明涉及医学领域，具体涉及一种用于递送生物制剂的蛋白质-核酸凝集体及其制备方法。
背景技术
蛋白质和核酸等生物大分子通过弱多价相互作用及构象熵介导的液-液相分离(Liquid-liquid phase separation,LLPS)形成无膜的细胞器或凝集体，参与众多细胞关键生命活动的调控并与多种人类疾病密切相关。经LLPS形成的凝集体允许富集不同理化性质的生物分子，同时将其他生物分子隔离在凝集体之外。凝集体作为细胞内单独的区室，为富集的分子创造一个隔离的环境，从而实现分隔和调节细胞内的生化反应。LLPS现象不仅局限于在生物学或病理学上发挥作用，也可以为仿生应用提供启发性线索。
随着多肽类、蛋白质、抗体药物、核酸、细菌和病毒等新型生物制剂的出现，对递送载体的要求也在不断的变化。多肽类和蛋白质的稳定性、核酸药物易被降解以及递送效率低、病毒存在安全性和易被机体免疫清除等局限限制了新型生物制剂在疾病治疗中的应用。针对这些挑战，传统的药物载体，如脂质体、纳米颗粒等，存在其载药量有限、易渗漏及生产工艺复杂等问题，因此药物递送领域亟需新型药物载体来解决生物制剂在疾病治疗方面新出现的挑战。
凝集体在形成过程中可以将生物大分子(多肽，蛋白质等)吸收招募到区室内，具备高效富集客体分子及有效保留其生物活性的优点，展现出了作为生物制剂递送载体的潜力。然而由LLPS可逆、动态形成的凝集体不受膜结构的限制，易聚结及受环境因素，如温度、pH、离子强度等影响，从而限制了其在药物递送领域中的应用。
这就需要进行对现有的递送系统进行改良，提供新的递送生物制剂的载体。
发明内容
本发明设计并生成了新的递送载体，该载体由蛋白与核酸凝集而成，其中，所述的凝集体为中空的。该结构的具有如下的优点：具备优异的生理环境稳定性；生物相容性，能够高效地招募生物制剂并提升细胞对生物制剂的摄取效率，最终实现生物制剂的有效递送，从而改善疾病治疗效果。
本发明的目的在于提供一种递送载体，所述的载体是由蛋白质与核酸凝集而成，其中所述的载体是中空的结构或者载体内含有空间。利用该载体装载生物制剂，生物制剂可以是多肽类、蛋白质、抗体药物、核酸、细菌和病毒等，用于治疗疾病或者预防疾病，例如用于制备疫苗，传染病疫苗，肿瘤疫苗等等。
进一步的，所述的蛋白为组蛋白。在一些方式中，所述的组蛋白为多种蛋白的混合体。在一些方式中，所述的组蛋白来源于小牛胸腺的提取物。在一些方式中，所述的组蛋白包括H1、H2A、H2B、H3和H4中的任意一种或多种。
在一些方式中，除了包含组蛋白，还可以包含非组蛋白序列特异性DNA结合蛋白。
本发明所述的组蛋白，具有如下特征：带正电荷，含Arg、Lys属碱性蛋白，只在细胞周期中的S期合成。共五种，分为核心组蛋白，H2A、H2B、H3、H4和连接组蛋白(H1)。组蛋白结构高度保守，尤其是H4。核心组蛋白由球形部和尾部构成，球形部借Arg与磷酸二脂骨架间的静电作用使DNA分子缠绕在组蛋白核心上，形成核小体，尾部含有大量Arg和Lys，为转译后修饰的部位。H1具有多样性，具有属和组织特异性。
本发明所述的非组蛋白序列特异性DNA结合蛋白具有如下特征：带负电，属酸性蛋白，在整个细胞周期都合成，能识别特异DNA序列，以氢键和离子键结合。非组蛋白序列特异性DNA结合蛋白的功能是帮助DNA折叠、复制；调节基因表达。
本发明所形成的中空凝集体表面带正电荷或接近电中性，与带负电荷的细胞膜产生相互作用；中空凝集体在生理条件下能保持结构稳定。
进一步的，所述的核酸为被修饰的单链核酸，例如脱氧核糖核酸(DNA)或者核糖核酸(RNA)，或者DNA与RNA的混合体。
进一步的，所述的修饰基团包括与哺乳动物细胞膜类似的物质。
在一些方式中，修饰的基团可以是胆固醇、磷脂、糖脂、糖蛋白中的任意一种或多种。这里的蛋白可以是构成细胞膜的蛋白。可以理解，凡是构成细胞膜的分子都可以被用来修饰本发明所定义的单链核酸，如脂质、蛋白质以及少量糖类，比如磷脂，或者与构成细胞膜成分类似的物质，如两亲型物质，嵌段共聚物，经修饰的药物等，都可以用来修饰。从 机理上，凡是那些与细胞膜成分相似的物质都可以被用来对单链核酸进行修饰。比如本发明的一个具体实施方式中的胆固醇与细胞膜成分相似，可辅助其他组分跨膜运输。
在一些方式中，所述的修饰基团在单链核酸的3’端或者5’饰，或者在3’和5’端同时修饰，当然也可以在靠近端部(3’端或者5’端修饰)的位置修饰，可选的，也可以在单链核酸的中间位置进行修饰。修饰的比例为0.1％-99％，可以是1-95％，1-85％，1-75％，1-55％，1-45％。这里所谓的比例是指在所有组成单链核苷酸中被修饰的嘌呤碱或嘧啶碱基、核糖或脱氧核糖以及磷酸的官能团进行取代反应从而进行本发明的修饰。
进一步的，所述的胆固醇修饰的单链脱氧核糖核酸的长度是任何DNA片段，长度大概在5-1000bp，优选的，10-500bp，15-300bp，15-100bp。所述的核苷酸可以是A，U，C，T，G任意顺序组成的(5′-腺苷酸(5′-adenylic acid)，5′-胸苷酸(5′-thymidylic acid)5′-鸟苷酸(5′-guanylic acid)5′-肌苷酸(5′-inosinic acid)3′-黄苷酸(3′-xanthylic acid)5′-胞苷酸(5′-cytidylic acid)5′-尿苷酸(5′-uridylic acid))，也可以是全部由单个的核苷酸分子A，U，C，T，G组成一定长度的。例如5-AAAAAAAAAAAAAA-3，也可以是5-UUUUUUUUUUUUUUU-3，或者5-TTTTTTTTTTTTTTTTTTTTTT-3，或者5-CCCCCCCCCCCCCCCCCCCCCCCCCCCCC-3，或者5-GGGGGGGGGGGGGGGGGGGGGGGG-3。当然，也可以是以上5种碱基组任意组成的单链核苷酸，例如5-AAGGCCTTTTGTACTGGG-3等等。这里所谓的单链，是主要为单链条的核酸，也包括具有发夹结构的单链核酸。这里的单链是与双链相对立的概念。
进一步的，所述单链核酸为人工合成或从植物，动物，人，低等生物中提取。
进一步的，所述的组蛋白与被修饰的单链核酸摩尔比为1:500～1:20。
进一步的，所述的组蛋白与修饰的单链核酸与浓度比例为：1μM-50μM的ssDNA与0.25mM-2mM的组蛋白混合。在一些方式中，所述的组蛋白终浓度为0.5mM，修饰的单链核酸ssDNA终浓度为10μM。
进一步的，所述的载体中包括能够被载体递送的生物制剂，所述的生物制剂包括多肽类、蛋白质、抗体药物、核酸、细菌、病毒中的一种或多种。
进一步的，所述的载体是由组蛋白与修饰的单链核酸凝集而成。
另一方面，本发明提供一种中空的蛋白质-核酸凝集体载体的制备方法：将组蛋白与被修饰的单链核酸、溶剂混合制得，所述的载体为中空的结构。在一些方式中，所述组蛋白与被修饰的单链核酸摩尔比为1：500～1：20。
进一步的，制备步骤所采用的溶剂为PBS溶液、柠檬酸-柠檬酸钠缓冲液、磷酸氢二钠-磷酸二氢钠缓冲液中的任意一种或多种。
在一些方式中，溶剂为1XPBS，其pH为7.4；或者0.1～0.01M柠檬酸-柠檬酸钠缓冲液，其pH为5～6.5；或者0.1～0.01M磷酸氢二钠-磷酸二氢钠缓冲液，其pH为6.5～8。
进一步的，所述的组蛋白与需要递送的生物试剂混合，然后再与修饰的单链核酸混合。
进一步的，所述生物制剂包括：多肽类、蛋白质、抗体药物、核酸、细菌、病毒中的一种或多种。
进一步的，所述被修饰的单链核酸的终浓度为0.1μM～50μM，组蛋白的终浓度为0.025mM～2mM。
在一些方式中，采用5’端修饰胆固醇的ssDNA在终浓度为10μM，与组蛋白终浓度为0.5mM的条件下，混合2.25*107pfu的ad11腺病毒，病毒分子通过相分离的分子富集作用，选择性地富集在中空凝集体的相分离区域，形成包载腺病毒的凝聚体。将包载了ad11腺病毒的凝集体制剂皮下注射用于治疗小鼠肿瘤。
再一方面，本发明提供中空的蛋白质-核酸凝集体载体用于制备递送载体的用途，所述递送载体用于向生物体递送生物制剂。
再一方面，本发明提供中空的蛋白质-核酸凝集体载体包载腺病毒用于制备提高肿瘤治疗效果的制剂的用途。
另一方面，本发明提供修饰的单链核酸用于制备中空的蛋白质-核酸凝集体载体的用途，在一些方式中，所述修饰的基团为胆固醇、磷脂、糖脂、糖蛋白中的一种或多种。
在一些方式中，所述的修饰可以是胆固醇修饰或者与细胞膜近似的物质的修饰，包括磷脂、蛋白质等修饰。
进一步的，所述的修饰基团为胆固醇。
本发明所提供的中空的蛋白质-核酸凝集体具有以下有益效果：
(1)明确了产生中空凝集体所必须的条件为被修饰的单链核酸，其修饰为与生物细胞膜成分相似的分子，被修饰的单链核酸不受限制，可修饰的分子选择多、可选修饰方式多，适用范围广。
(2)明确了产生中空凝集体所需要的组蛋白与被修饰单链核酸的浓度配比，在本发明所提供的浓度范围内可稳定地形成中空凝集体，在最优的两者浓度配比下可以获得的中空凝集体比例最高。
(3)形成的中空凝集体载体在生理环境内结构稳定，生物相容性高，高效招募生物制剂并提升细胞对生物制剂的摄取效率，实现有效递送，辅助疾病治疗。形成中空凝集体避免了非中空凝集体体积太大、容易集团、结构稳定性差、结构不规则、尺寸过大，导致其递送效率低的问题。
(4)可递送的生物制剂包括多肽类、蛋白质、抗体药物、核酸、细菌、病毒等，应用范围广泛。
附图说明
图1(a)-(c)为本发明实施例1中的基于蛋白质和核酸之间的液-液相分离相图及相的形式。
图1(a)为罗丹明B标记的组蛋白和6-FAM标记的经过修饰的单链核苷酸ssDNA经LLPS形成的凝集体及中空凝集体在共聚焦显微镜下的形貌；比例尺为10微米。
图1(b)为组蛋白和ssDNA之间的部分相图，从图1(b)可以看出，两者的比例比较关键的，在一个范围内，才能得到中空的结构。
图1(c)为通过将组蛋白和ssDNA以不同比例混合的电位图。
图1(d)为胆固醇修饰在单链脱氧核糖核酸的3’端的结构式。
图1(e)为胆固醇修饰在单链脱氧核糖核酸的5’端的结构式。
图2为本发明实施例2中的组蛋白及未修饰胆固醇的单链脱氧核糖核酸(5’-AAAAAAAAAAAAAAAAAAAAAAAAAAAAAA-3’)之间的液-液相分离相图及相的形式。
图2(a)为组蛋白和未修饰胆固醇的单链脱氧核糖核酸之间的部分相图。(b)为组蛋白和Cy5标记的核酸形成的混合溶液。(c)为组蛋白和花氰染料Cy5(红色)标记的核酸经LLPS形成的凝集体。(d)为组蛋白和Cy5标记的核酸经LLPS形成的团聚物。比例尺为50微米。未经胆固醇修饰的单链脱氧核糖核酸与组蛋白在图a的条件下不能形成中空凝集体结构。
图3为本发明实施例中凝集体与中空凝集体的结构与形貌表征。(a，d)分别为凝集体与中空凝集体的三维共聚焦显微镜图像。(b，e)分别为凝集体液滴与中空凝集体的扫描电镜图。(c，f)分别为凝集体液滴与中空凝集体的冷冻透射电镜图。
图4为本发明实施例中对LLPS过程形成的组装体的性能表征。(a)为凝集体液滴的融合现象。(b)凝集体液滴的荧光漂白后恢复共聚焦显微镜组图及荧光强度分析。(c)为中空凝集体的荧光漂白后恢复共聚焦显微镜组图及荧光强度分析。比例尺为5微米。
图5为本发明实施例中的基于蛋白质和核酸之间的液-液相分离装载病毒的相图及相的形式。(a)为罗丹明B标记的组蛋白、6-FAM标记的ssDNA及Cy5(蓝色)标记的腺病毒ad11(Cy5-ad11)经LLPS形成的凝集体及中空凝集体在共聚焦显微镜下的形貌。比例尺为10微米。(b)为三者之间的部分相图。
图6为本发明实施例中凝集体液滴及中空凝集体对生物制剂的富集。图6(a)为对组蛋白、ssDNA以及ad11病毒的荧光强度分析图，比例尺为5微米。图6(b)为凝集体和中空凝集体对ad11病毒的装载率对比图。
图7为本发明实施例中病毒在相分离体系中的分布。(a)为单独病毒的透射电镜图片。(b)为病毒在蛋白质-核酸溶液中的透射电镜图片。(c)为病毒在蛋白质-核酸凝集体液滴内的冷冻透射电镜图片。(d)为病毒在蛋白质-核酸中空凝集体内的冷冻透射电镜图片。比例尺为100纳米。
图8为本发明实施例中凝集体液滴及中空凝集体的结构稳定性研究。(a)为凝集体液滴及中空凝集体在初始时刻以及48小时后的浊度以及形貌变化。(b)为凝集体液滴及中空凝集体在初始时刻以及48小时后的粒径变化。比例尺为10微米。从图8(b)可以看出,中空的凝集的粒径变化小,在48小时后基本没有变化(图8(b)的左边两个柱状图)；而凝集体液滴的粒径显著变化,从大约2321nm变化到8619nm(图8(b)的右边的两个柱状图)。
图9A为本发明实施例中凝集体液滴及中空凝集体作为病毒载体在小鼠肺上皮细胞(TC-1)及高表达受体CD46的TC-1细胞系中的病毒摄取情况。(a)为ad11或者ad11溶液、ad11/凝集体及ad11/中空凝集体在TC-1细胞系中的摄取情况及病毒含量统计。(b)为ad11或者ad11溶液、ad11/凝集体及ad11/中空凝集体在高表达CD46的TC-1细胞系中的摄取情况及病毒含量统计。比例尺为20微米。
图9B中的1表示单独的病毒(ad11)，2表示单独的病毒溶液(ad11溶液)，3表示凝集体/病毒(ad11/凝集体)(非中空)；4表示中空凝集体/病毒(ad11/中空凝集体)，说明中空的装载有病毒更加稳定，被细胞摄取的病毒数量最高，与非中空的相比，显著差异(是归一化处理 后，以单纯病毒组的数量为1的情况下，ad11/溶液大概为3，而ad11/凝集体大概为5，而ad11/中空凝集体的载量大概为7)。
图10为本发明实施例中ad11在肿瘤组织的积累情况。比例尺为100微米。
图11为本发明实施例中在TC-1小鼠肿瘤模型中的体内药效数据。(a)为治疗10天后各组小鼠肿瘤实物图。(b)为各组小鼠治疗10天后肿瘤重量。(c)为各组小鼠治疗后平均肿瘤生长曲线。从图可以看出,对于肿瘤尺寸来讲,ad11/中空凝集体处理小肿瘤的尺寸基本保持不变，而其他处理(PBS，单纯的中空凝集体，单纯的病毒处理，非中空的凝集体下的肿瘤尺寸在12天内快速的增大，与ad11/中空凝集体处理小肿瘤的尺寸呈现极显著差异)，从图11B中也可以看出，10天后重量也出现显著变化，ad11/中空凝集体处理下的重量大约是0.5g,而PBS，单纯的中空凝集体，单纯的病毒处理，非中空的凝集体下的重量分别大约为2.4g,2.3g,1.9g,1.8g.，与这些处理呈显著或者极显著差异。
具体实施方式
下面结合附图和实施例对本发明作进一步详细描述，需要指出的是，以下所述实施例旨在便于对本发明的理解，而对其不起任何限定作用。本实施例中使用的试剂均为已知产品，通过购买市售产品获得。
实施例子1：组蛋白与经过修饰的单链核酸形成中空的凝集体。
分别配置罗丹明B标记的组蛋白(Histone)(购买于生工生物工程(上海)股份有限公司，产品编号A002544)和6-羧基荧光素(6-FAM)标记的胆固醇修饰的ssDNA(5’-Cholestery-AAAAAAAAAAAAAAAAAAAAAAAAAAAAAA-6-FAM-3’(SEQ NO：1))不同浓度的溶液，两种溶液混合至Histone终浓度为2mM、1mM、0.5mM、0.25mM、0.1mM、0.05mM、0.025mM，ssDNA终浓度为50μM、25μM、10μM、5μM、1μM、0.5μM、0.1μM。溶剂为1XPBS(pH 7.4)。图1(b)为在激光共聚焦显微镜下观察体系内相的形态来绘制对应条件下的相图，随着二者浓度以及体系内电势变化，分别展现出相的三种形式：均一的溶液、液滴状凝集体(非中空)以及中空凝集体。实验发现，胆固醇修饰在单链脱氧核糖核酸的5’或者3’端，图1(d)为胆固醇修饰在单链脱氧核糖核酸的3’端的结构式，图1(e)为胆固醇修饰在单链脱氧核糖核酸的5’端的结构式，都可以形成上述相分离组装体。
能够形成中空凝集体的条件为1μM-50μM的ssDNA与0.25mM-2mM的组蛋白混合。 其中，在组蛋白终浓度为0.5mM，ssDNA终浓度为10μM的条件下，产生中空的凝集的比例最高。
可以理解，除了本实施例子1的具体方式外，能够修饰的基团除了胆固醇之外，其它的修饰也是可行的，例如磷脂、蛋白质等，与细胞膜的组成相似的物质都可以用来对核酸进行修饰。通过我们的实验，都是可以获得中空的凝集体。另外，对于核酸，可以是任意的单链核酸物质，例如DNA，RNA等等，这些核酸物质可以是人工合成的，也可以是从植物，动物，人，低等生物中提取出来的单链核酸，这些单链核酸物质的长度可以是任意的长度，一般是小于1000bp,或者小于500bp,小于100bp，或者10-80bp之间。在另外的一些方式中，这些单链核苷酸可以是一条或者多条不同的序列，不同长度的混合体，不过这些单链核酸都需要进过修饰。对于修饰的位置，可以是核酸的两端，例如5’或者3’端，或者5’和3’端，也可以是靠近核酸的端部，例如距离5’或者3’端的10-1个核苷酸的位置进行修饰也是可以的。这个通过本发明的公开，是可以任意进行选择，比如在核酸的中间位置进行修饰。通过我们的实验，以上的选择以及组合在一起，我们也能够获得中空的凝集体。
实施例子2：组蛋白与未经修饰的单链核苷酸不能形成中空的凝集体
如图2，分别配置Histone(与实施例子1相同的)和花氰染料Cy5标记的ssDNA(序列为5’-AAAAAAAAAAAAAAAAAAAAAAAAAAAAAA-Cy5-3’)不同浓度的溶液，两种溶液混合至Histone终浓度为2mM、1mM、0.5mM、0.25mM、0.1mM、0.05mM、0.025mM，ssDNA终浓度为50μM、25μM、10μM、5μM、1μM、0.5μM、0.1μM。在荧光显微镜下观察体系内相的形态来绘制对应条件下的相图。随着二者浓度以及比例变化，展现出相的三种形式：均一的溶液、液滴状凝集体以及团聚物。未修饰胆固醇的ssDNA与组蛋白在该条件下未能得到中空凝集体。
本实验说明，单链核酸必须经过修饰，才能形成中空的结构，而不经过修饰，是不能形成中空的结构的，至于修饰的比例、与组蛋白的浓度比，都是可以通过实验调整出来的。
实施例子3：本发明实施例中凝集体与中空凝集体的结构与形貌表征
如图3，在组蛋白终浓度为0.25mM，ssDNA终浓度为50μM的条件下制备得到凝集体，在组蛋白终浓度为0.5mM，ssDNA终浓度为10μM的条件下制备得到中空凝集体。根据共聚焦显微镜、扫描电镜及冷冻透射电镜的表征方法，证明了凝集体液滴为实心的球体、中空凝集体为含水相内腔，相分离得到的中空球体结构(如图3d文字描述)，具体 组蛋白与修饰的单链核酸与实施例子1相同。
实施例子4：本发明实施例中对LLPS过程形成的组装体的性能表征
如图4，在组蛋白终浓度为0.25mM，ssDNA终浓度为50μM的条件下制备得到凝集体，在组蛋白终浓度为0.5mM，ssDNA终浓度为10μM的条件下制备得到中空凝集体(具体组蛋白与修饰的单链核酸与实施例子1相同)。相分离组装出来的结构具有液态体系的特征，如分子运动性和可变形性。相邻液滴相互接触后(0s)会发生融合现象，融合后(180秒)在液滴表面张力的作用下趋于形成球形的结构，以此验证蛋白质-核酸发生相分离。此外，还可以通过光漂白荧光恢复实验来证明组装体是由LLPS过程形成。通过对凝集体液滴及中空凝集体的特定区域(白色箭头)进行光漂白(50％光强，光漂白时间为10s)，然后检测荧光恢复情况。共聚焦显微镜图像显示两种组装体的漂白区域的荧光逐渐恢复，其中凝集体在60s后荧光几乎完全恢复，而中空凝集体1200s后荧光恢复到60％，这证明两种相分离组装体具有液体的分子运动性，而凝集体分子流动性更强。这在后面的应用中其实是缺点，流动性强，导致结构稳定性变差，限制后续应用，相反，中空凝集体流动性并不强，结构稳定，方便后续的实际生物制剂的递送。
实施例子5：本发明实施例中的基于蛋白质和核酸之间的液-液相分离装载病毒的相图及相的形式。
如图5，分别配置罗丹明B标记的组蛋白(Histone)和6-羧基荧光素(6-FAM)标记的胆固醇修饰的ssDNA(5’-Cholestery-AAAAAAAAAAAAAAAAAAAAAAAAAAAAAA-6-FAM-3’)不同浓度的溶液，两种溶液混合至Histone终浓度为2mM、1mM、0.5mM、0.25mM、0.1mM、0.05mM、0.025mM，ssDNA终浓度为50μM、25μM、10μM、5μM、1μM、0.5μM、0.1μM，在组蛋白与ssDNA混合前先与病毒数量为2.25*107pfu的Cy5标记的腺病毒(Cy5-ad11)混合(Cy5-ad11由Beijing Bio-Targeting Therapeutics Technology Co.,Ltd(Beijing,China)赠送)(空斑形成单位(plaque forming unit)，缩写pfu)，组蛋白与被修饰的单链核酸在相分离的过程中将Cy5-ad11招募到组装的区室内。与单纯的蛋白质和核酸的体系相比，Cy5-ad11加入的体系同样含均一的溶液、液滴状凝集体以及中空凝集体三种形式。
通过我们的实验，如果把同样的腺病毒先与修饰的核酸混合，然后再与组蛋白混合，也可以获得三种不同的状体的物质：均一的溶液、液滴状凝集体以及中空凝集体三种形式。相对而言，让腺病毒与组蛋白混合，获得的中空的凝集体的效率低一些。
实施例子6：本发明实施例中凝集体液滴及中空凝集体对生物制剂的富集
如图6(a)，在组蛋白终浓度为0.25mM，ssDNA终浓度为50μM的条件下制备得到凝集体，在组蛋白终浓度为0.5mM，ssDNA终浓度为10μM的条件下制备得到中空凝集体，各体系总体积为40μL，Cy5-ad11数量为9*107pfu。相分离体系具备优异的分子富集能力，促进对生物制剂的高效负载。通过对组蛋白、ssDNA和病毒的荧光强度和距离分布的分析，证实了相分离结构域对病毒的有效富集(比例尺为5微米)。分析软件为Image J。在组蛋白终浓度为0.25mM，ssDNA终浓度为50μM的条件下以及在组蛋白终浓度为0.5mM，ssDNA终浓度为10μM的条件下制备富集病毒的两种组装体，利用实时荧光定量技术计算两种组装体对病毒的装载率，凝集体装载率为92.8％，中空凝集体为81.4％，如图6(b)。
实施例子7：本发明实施例中病毒在相分离体系中的分布。
如图7，在组蛋白终浓度为0.5mM，ssDNA终浓度为0.1μM的条件下得到溶液；在组蛋白终浓度为0.25mM，ssDNA终浓度为50μM的条件下制备得到凝集体；在组蛋白终浓度为0.5mM，ssDNA终浓度为10μM的条件下制备得到中空凝集体；各体系总体积为40μL，Cy5-ad11数量为9*107pfu。溶液中病毒是团聚在一起的状态(图7b)。病毒通过相分离的分子富集作用分隔在凝集体液滴以及中空凝集体内，与外界环境隔绝。其中，如图7(c)，在凝集体中，病毒均匀分散在凝集体液滴内。如图7(d)在中空凝集体中，病毒选择性的富集到中空凝集体相分离区域，分布在环形区域内，与组蛋白以及单链核酸共定位。
实施例子8：本发明实施例中凝集体液滴及中空凝集体的结构稳定性研究
如图8，在组蛋白终浓度为0.25mM，ssDNA终浓度为50μM的条件下制备得到凝集体，在组蛋白终浓度为0.5mM，ssDNA终浓度为10μM的条件下制备得到中空凝集体。与中空凝集体(图8(b)中的A)相比，凝集体(非中空，图8(b)中的B)液滴具备更强的液体流动性和可变形性，因此随着时间变化凝集体液滴之间趋于聚结。该特性表现在凝集体体系浊度逐渐降低、凝集体聚结成团以及粒径显著增加。由于凝集体结构稳定性差、结构不规则、尺寸过大，导致后续凝集体递送病毒至肿瘤细胞内的效率降低，进而影响肿瘤治疗效果。相反，中空的凝集体或者载体滴液体流动性和可变形性比非中空的要弱一些，因此随着时间变化中空的凝集体之间不会凝结，这样，结构更加稳定，更加规则，尺寸合适，能够把所需要递送的物质递送到细胞内。
实施例子9：本发明实施例中凝集体液滴及中空凝集体作为病毒载体在小鼠肺上皮细胞(TC-1)及高表达受体CD46的TC-1细胞系中的病毒摄取情况
如图9，在组蛋白终浓度为0.25mM，ssDNA终浓度为50μM的条件下制备得到凝集体，在组蛋白终浓度为0.5mM，ssDNA终浓度为10μM的条件下制备得到中空凝集体，Cy5-ad11(腺病毒)数量为1*107pfu。将上述溶液加入到小鼠肺上皮细胞(TC-1)及高表达受体CD46的TC-1细胞系中，细胞数量为500000，病毒数量每个细胞20pfu。上述体系在37℃，5％CO2的细胞培养箱中孵育4个小时，去除游离的病毒/组装体。通过鬼笔环肽染细胞骨架，以及Hoechst 33342染细胞核的过程及激光共聚焦显微镜的观察，证实了以中空凝集体作为载体，标记Cy5的腺病毒ad11在细胞内的摄取效率显著提升。利用实时荧光定量技术测定细胞内病毒的含量，同样验证了上述结论。
实施例子10：本发明实施例中ad11在肿瘤组织的积累情况(比例尺为100微米)
在组蛋白终浓度为0.25mM，ssDNA终浓度为50μM的条件下制备得到凝集体，在组蛋白终浓度为0.5mM，ssDNA终浓度为10μM的条件下制备得到中空凝集体。如图10，培养未处理的TC-1细胞，皮下荷瘤细胞数量2*106注射100μL构建动物模型。实验鼠选择C57/BL-6J，8周龄雌鼠。饲养实验鼠至肿瘤尺寸为100mm3，瘤内注射制剂。制剂包括：PBS组、ad11组、ad11/凝集体组和ad11/中空凝集体组。其中PBS组为对照组，瘤内注射50μL1XPBS(pH 7.4)；ad11组为单纯病毒组，瘤内注射剂量为50μL，病毒数量为1*108pfu；ad11/凝集体组组成：组蛋白终浓度为0.25mM，ssDNA终浓度为50μM，病毒数量为1*108pfu，瘤内注射剂量为50μL；ad11/中空凝集体组组成：在组蛋白终浓度为0.5mM，ssDNA终浓度为10μM，病毒数量为1*108pfu，瘤内注射剂量为50μL。其中病毒标记了Cy5荧光，对肿瘤组织的冰冻切片进行DAPI染色处理。
利用荧光显微镜拍摄肿瘤组织切片图片，证实了中空凝集体作为生物制剂递送载体，利用优异的递送能力，提高细胞对病毒摄取效率。具体表现在ad11/中空凝集体处理的肿瘤组织中Cy5荧光强度高于其他组别，实现肿瘤组织内的病毒含量提升，溶瘤病毒通过自我复制在肿瘤细胞中大量增殖，最终使肿瘤细胞裂解，进而释放更多的溶瘤病毒，引起肿瘤溶解来增强抗肿瘤疗效。
实施例子11：本发明实施例中在TC-1小鼠肿瘤模型中的体内药效数据
在组蛋白终浓度为0.25mM，ssDNA终浓度为50μM的条件下制备得到凝集体，在组蛋白终浓度为0.5mM，ssDNA终浓度为10μM的条件下制备得到中空凝集体。如图10，培养未处理的TC-1细胞，皮下荷瘤细胞数量2*106注射100μL构建动物模型。实验鼠选择C57/BL-6J，8周龄雌鼠。饲养实验鼠至肿瘤尺寸为100mm3，瘤内注射制剂。制剂包括：PBS组、ad11组、ad11/凝集体组和ad11/中空凝集体组。其中PBS组为对照组，瘤内注射50μL 1XPBS(pH 7.4)；ad11组为单纯病毒组，瘤内注射剂量为50μL，病毒数量为1*108pfu；ad11/凝集体组组成：组蛋白终浓度为0.25mM，ssDNA终浓度为50μM，病毒数量为1*108pfu，瘤内注射剂量为50μL；ad11/中空凝集体组组成：在组蛋白终浓度为0.5mM，ssDNA终浓度为10μM，病毒数量为1*108pfu，瘤内注射剂量为50μL。两天注射一次制剂，记录肿瘤尺寸。体内药效实验证实了中空凝集体可以显著提高病毒的抗肿瘤效果，在TC-1小鼠肿瘤模型中，给药后小鼠肿瘤体积有了显著程度的抑制，中空凝集体作为病毒递送载体可显著改善治疗疗效。从图11可以看出，采用中空的凝集体递送抗肿瘤药物，可以显著抑制肿瘤的体积增大或者重量的增加，与凝集体带有相同的抗肿瘤药物相比，具有极显著差异。
本发明说明书中提到的所有专利和出版物都表示这些是本领域的公开技术，本发明可以使用。这里所引用的所有专利和出版物都被同样列在参考文献中，跟每一个出版物具体的单独被参考引用一样。这里所述的本发明可以在缺乏任何一种元素或多种元素，一种限制或多种限制的情况下实现，这里这种限制没有特别说明。例如这里每一个实例中术语“包含”，“实质由……组成”和“由……组成”可以用两者之一的其余2个术语代替。这里的所谓的“一个”仅仅表示“一”的意思，而不排除仅仅只是包括一个，也可以表示包括2个以上。这里采用的术语和表达方式所为描述方式，而不受其限制，这里也没有任何意图来指明此书描述的这些术语和解释排除了任何等同的特征，但是可以知道，可以在本发明和权利要求的范围内做任何合适的改变或修改。可以理解，本发明所描述的实施例子都是一些优选的实施例子和特点，任何本领域的一般技术人员都可以根据本发明描述的精髓下做一些更改和变化，这些更改和变化也被认为属于本发明的范围和独立权利要求以及附属权利要求所限制的范围内。

Claims (28)

  1. 一种载体，其特征在于，该载体包括组蛋白和被基团修饰的单链核酸，其中所述的载体为中空的结构。
  2. 如权利要求1所述的载体，其特征在于，所述的修饰基团包括与哺乳动物细胞膜类似的物质。
  3. 如权利要求2所述的载体，其特征在于，修饰基团为脂质、蛋白质、糖类、两亲性物质、嵌段共聚物、药物中的任意一种或多种。
  4. 如权利要求3所述的载体，其特征在于，所述的修饰的基团为胆固醇、磷脂、糖脂、糖蛋白中的任意一种或多种。
  5. 如权利要求4所述的载体，其特征在于，所述的修饰基团为胆固醇。
  6. 如权利要求1所述的载体，其特征在于，所述的基团修饰单链核酸的比例为0.1％-99％。
  7. 如权利要求1所述的载体，所述组蛋白与被修饰的单链核酸摩尔比为1:500～1:20。
  8. 如权利要求1所述的载体，其特征在于，所述基团修饰单链核酸的位置是在单链核酸的3’端或者5’端，或者在靠近3’端或者5’端的位置，或者在核酸中间的位置。
  9. 如权利要求1所述的载体，其特征在于，所述单链核酸为DNA和/或RNA。
  10. 如权利要求1所述的载体，其特征在于，单链核酸为A、U、C、T、G任意顺序组成。
  11. 如权利要求10所述的载体，其特征在于，所述单链核酸为人工合成或从植物，动物，人，低等生物中提取。
  12. 如权利要求1所述的载体，其特征在于，所述单链核酸的长度为小于1000bp。
  13. 如权利要求12所述的载体，其特征在于，所述单链核酸的长度为小于500bp，或小于100bp，或10-80bp。
  14. 如权利要求1所述的载体，其特征在于，所述组蛋白包括H1、H2A、H2B、H3和H4中的任意一种或多种。
  15. 如权利要求14所述的载体，其特征在于，除了包含组蛋白，还可以包含非组蛋白序列特异性DNA结合蛋白。
  16. 如权利要求1所述的载体，其特征在于，所述被修饰的单链核酸的终浓度为0.1μM～50μM，组蛋白的终浓度为0.025mM～2mM。
  17. 如权利要求16所述的载体，其特征在于，所述被修饰的单链核酸的终浓度为10μM，组蛋白的终浓度为0.5mM。
  18. 根据权利要求1所述的载体，所述的载体中包括能够被载体递送的生物制剂，所述的生物制剂包括多肽类、蛋白质、抗体药物、核酸、细菌、病毒中的一种或多种。
  19. 根据权利要求1所述的载体，所述的载体是由组蛋白与修饰的单链核酸凝集而成。
  20. 一种中空载体的制备方法：其特征在于，将组蛋白与被修饰的单链核酸、溶剂混合制得，所述的载体为中空的结构。
  21. 根据权利要求20所述的方法，其特征在于，所述溶剂为PBS溶液、柠檬酸-柠檬酸钠缓冲液、磷酸氢二钠-磷酸二氢钠缓冲液中的任意一种或多种。
  22. 根据权利要求20所述的方法，其特征在于，所述的组蛋白与需要递送的生物试剂混合，然后再与修饰的单链核酸混合。
  23. 根据权利要求22所述的方法，其特征在于，所述生物制剂包括：多肽类、蛋白质、抗体药物、核酸、细菌、病毒中的一种或多种。
  24. 根据权利要求20所述的方法，其特征在于，所述被修饰的单链核酸的终浓度为0.1μM～50μM，组蛋白的终浓度为0.025mM～2mM。
  25. 如权利要求1～15任一项所述的中空的蛋白质-核酸凝集体载体用于制备递送载体的用途，其特征在于，所述递送载体用于向生物体递送生物制剂。
  26. 如权利要求1～15任一项所述的中空的蛋白质-核酸凝集体载体包载腺病毒用于制备提高肿瘤治疗效果的制剂的用途。
  27. 修饰的单链核酸用于制备中空的蛋白质-核酸凝集体载体的用途，其特征在于，所述修饰的基团为胆固醇、磷脂、糖脂、糖蛋白中的一种或多种。
  28. 根据权利要求27所述的用途，所述的修饰基团为胆固醇。
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