WO2018228178A1 - 一种核酸载体及其用途 - Google Patents

一种核酸载体及其用途 Download PDF

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WO2018228178A1
WO2018228178A1 PCT/CN2018/088859 CN2018088859W WO2018228178A1 WO 2018228178 A1 WO2018228178 A1 WO 2018228178A1 CN 2018088859 W CN2018088859 W CN 2018088859W WO 2018228178 A1 WO2018228178 A1 WO 2018228178A1
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nucleic acid
compound
formula
pharmaceutically acceptable
tat
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PCT/CN2018/088859
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English (en)
French (fr)
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孟庆斌
康子瑶
孟昭
孙超
杨森
刘克良
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中国人民解放军军事科学院军事医学研究院
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Publication of WO2018228178A1 publication Critical patent/WO2018228178A1/zh

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    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K7/00—Peptides having 5 to 20 amino acids in a fully defined sequence; Derivatives thereof
    • C07K7/04—Linear peptides containing only normal peptide links
    • C07K7/06—Linear peptides containing only normal peptide links having 5 to 11 amino acids
    • 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
    • A61K48/00—Medicinal preparations containing genetic material which is inserted into cells of the living body to treat genetic diseases; Gene therapy
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K48/00—Medicinal preparations containing genetic material which is inserted into cells of the living body to treat genetic diseases; Gene therapy
    • A61K48/005—Medicinal preparations containing genetic material which is inserted into cells of the living body to treat genetic diseases; Gene therapy characterised by an aspect of the 'active' part of the composition delivered, i.e. the nucleic acid delivered
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P25/00—Drugs for disorders of the nervous system
    • 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
    • A61P9/00—Drugs for disorders of the cardiovascular system
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K19/00—Hybrid peptides, i.e. peptides covalently bound to nucleic acids, or non-covalently bound protein-protein complexes
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N15/00—Mutation or genetic engineering; DNA or RNA concerning genetic engineering, vectors, e.g. plasmids, or their isolation, preparation or purification; Use of hosts therefor
    • C12N15/09—Recombinant DNA-technology
    • C12N15/63—Introduction of foreign genetic material using vectors; Vectors; Use of hosts therefor; Regulation of expression
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N15/00—Mutation or genetic engineering; DNA or RNA concerning genetic engineering, vectors, e.g. plasmids, or their isolation, preparation or purification; Use of hosts therefor
    • C12N15/09—Recombinant DNA-technology
    • C12N15/87—Introduction of foreign genetic material using processes not otherwise provided for, e.g. co-transformation
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K2319/00—Fusion polypeptide
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K2319/00—Fusion polypeptide
    • C07K2319/01—Fusion polypeptide containing a localisation/targetting motif
    • C07K2319/10—Fusion polypeptide containing a localisation/targetting motif containing a tag for extracellular membrane crossing, e.g. TAT or VP22

Definitions

  • the invention belongs to the field of biomedicine, and relates to a nucleic acid carrier and a use thereof.
  • the invention also relates to a complex comprising the nucleic acid vector and a nucleic acid molecule, and a method of preparation and use of the nucleic acid vector/nucleic acid molecule complex.
  • the US Food and Drug Administration defines gene therapy as a treatment for treating diseases by exogenous genes that are transcribed or translated, transported, and/or integrated into the host genome.
  • Gene therapy is the most potentially effective treatment for cancer, monogenic diseases, cardiovascular diseases and neurological diseases. Since 1990, gene therapy has entered clinical trials for the first time, and there have been more than 1,800 clinical trials of gene therapy in the world. In 2004, China became the first country to introduce genetic drugs (Gendicine) on the market. Because the nucleic acid drug molecule used in gene therapy has the characteristics of large molecular weight, rich in negative charge and high degradation, the biggest problem facing gene therapy at present is the lack of ideal transport vector. Therefore, it is necessary to find efficient and safe gene transfer carrier. the key of.
  • Non-viral vector transport gene drugs mostly carry DNA or RNA through the ionic interaction between their own cations and the anion of the gene drug to form smaller-scale nanoparticles, transport DNA or RNA into the host cell, and then integrate into the host genome. expression.
  • Polypeptides are a good choice for gene therapy vectors because of their good biocompatibility, functional diversity and ease of synthesis.
  • Cell-Penetrating Peptides are a class of short peptides that efficiently mediate the entry of biological macromolecules such as nucleic acids and proteins into cells through cell membranes.
  • the length of peptide sequences is generally less than 30 amino acids, most of which are transparent.
  • Membrane peptides are rich in basic amino acids such as lysine and arginine.
  • TAT 49-57
  • HAT (49-57) is the smallest fragment of cell membrane-permeable protein peptide fragment that can fully function through cell membrane and is not cytotoxic.
  • the TAT (49-57) peptide sequence is Arg-Lys-Lys.
  • the transmembrane peptide can bind to the negative charge of the nucleic acid under physiological conditions, and the nucleic acid is loaded to thereby mediate the entry of the nucleic acid into the cell by endocytosis, and the TAT peptide fragment has a certain nuclear targeting ability.
  • the TAT/DNA or RNA complex that enters the cell by endocytosis first enters the endosome, an internal lipid membrane structure that is acidic, and is ultimately transported to lysosomal degradation.
  • Laminin (LN) is a macromolecular collagen glycoprotein. With the in-depth study of LN, LN has been found to have a variety of biological effects: promoting cell adhesion, spreading, mitosis, accelerating axon growth and cell migration, and inducing cell differentiation, as well as morphogenesis and tumor metastasis.
  • Laminin receptor (LN-R) is widely distributed on the surface of epithelial cells, endothelial cells, peripheral nerve cells, macrophages and most tumor cells, and exhibits high affinity, competition and concentration in combination with LN in vitro. Time dependent.
  • LN-R When malignant tumors metastasize, the adhesion of LN-R to LN on the cell surface is a key step in the interaction between tumor cells and the basement membrane, providing a molecular basis for tumor adhesion and invasion to break through the basement membrane; while LN-R can not only promote cells Adhesion on the LN matrix, and can induce chemotactic migration of cells and secretion of collagen type 1V; it is also found that LN-R and LN in the early stage are critical for tumor neovascularization.
  • nucleic acid carrier that is, a compound of the formula I.
  • nucleic acid vector has a high transfection efficiency and is low in cytotoxicity.
  • nucleic acid vector is also capable of better achieving endosomal escape.
  • One aspect of the invention relates to a compound of formula I, or a pharmaceutically acceptable salt thereof,
  • A represents a cell-permeable peptide
  • C represents one or more Lys, and/or one or more Arg, or C is missing
  • D represents a laminin receptor targeting fragment
  • connection joint Between A and B, between B and C, and / or between C and D, for direct connection or by connection joint;
  • the compound of formula I is further linked to one or more hydrophobic molecules E, and said hydrophobic molecule E is attached to the amino group of the side chain of Lys and/or Arg in said compound of formula I.
  • the direct linkage may be such that an amide bond is formed between a carboxyl group of one of A and B and an amino group of the other.
  • a similar understanding can be made between B and C, and between C and D.
  • connection joint includes, but is not limited to, one or more Gly, one or more Lys, and/or one or more Cys.
  • the plurality may be, for example, 2-20, 2-15, 2-10, 2-8, 2-6, 2, 3, 4, 5 or 6.
  • connection joints between A and B, between B and C, and between C and D may be the same or different.
  • the attachment of the linker to A, B, C or D is also via the formation of an amide bond between the carboxyl group and the amino group.
  • the compound of Formula I is characterized by any one or more of the following items (1) to (5):
  • the endosomal escape segment is a proton pump endosomal escape segment, preferably one or more Hiss, such as 4-10, 4-8, 4-6, 4, 5, 6, 7, or 8 His; preferably 6 His;
  • the laminin receptor targeting fragment is YIGSR (SEQ ID NO: 17) or YIGSK (SEQ ID NO: 18);
  • the hydrophobic molecule is selected from the group consisting of fatty acids having a carbon number greater than or equal to 12 (for example, stearic acid, palmitic acid, oleic acid, linoleic acid, palmitic acid), cholesterol, and phospholipids;
  • hydrophobic molecule is attached to the amino group of the side chain of Lys and/or Arg in C.
  • the compound of Formula I or a pharmaceutically acceptable salt thereof, wherein the compound is represented by SEQ ID NO: 1, SEQ ID NO: 2 or SEQ ID NO: 4.
  • the compound of Formula I or a pharmaceutically acceptable salt thereof, wherein the N-terminus of the amino acid sequence is acetylated and/or C-terminally amidated.
  • the imidazole group in histidine has a pKa of about 6.0, which can absorb a large amount of protons in the acidic endosome, forming a "proton sponge” effect, causing the rupture of the endosomes.
  • the positively charged hydrophilic amino acid to fully contact with the negatively charged nucleic acid drug to form a stable and dense nanocomposite, which is beneficial to cells.
  • the local concentration of aggregates can be increased by hydrophobic interaction, thereby increasing the local charge density, increasing the load capacity of DNA or RNA, improving the stability of the nucleic acid vector/DNA or RNA complex, and at the same time, through its hydrophobic alkyl chain. The fusion with the cell membrane, thereby effectively improving the efficiency of the membrane and the ability to destroy the endosomes.
  • sequence of the nucleic acid vector is Gly-Arg-Lys-Lys-Arg-Arg-Gln-Arg-Arg-Arg-His-His-His-His-His-His-Lys ( C18)-Tyr-Ile-Gly-Ser-Arg (SEQ ID NO: 1).
  • sequence of the nucleic acid vector is Gly-Arg-Lys-Lys-Arg-Arg-Gln-Arg-Arg-Arg-His-His-His-His-His-His-His--Lys (C18)-Tyr-Ile-Gly-Ser-Lys (SEQ ID NO: 2).
  • sequence of the nucleic acid vector is Gly-Arg-Lys-Lys-Arg-Arg-Gln-Arg-Arg-Arg-His-His-His-His-His-His-His-His-His-Lys (C18) (SEQ ID NO: 3).
  • sequence of the nucleic acid vector is Gly-Arg-Lys-Lys-Arg-Arg-Gln-Arg-Arg-Arg-His-His-His-His-His-His-His-His-Lys- Tyr-Ile-Gly-Ser-Arg (SEQ ID NO: 4).
  • the nucleic acid vector has the sequence C18-Gly-Arg-Lys-Lys-Arg-Arg-Gln-Arg-Arg-Arg-Arg-His-His-His-His-His-His-His-His- Lys-Tyr-Ile-Gly-Ser-Arg (SEQ ID NO: 5).
  • the nucleic acid vector has the sequence His-His-His-His-His-His-His-His-Gly-Arg-Lys-Lys-Arg-Arg-Gln-Arg-Arg-Arg-Lys ( C18)-Tyr-Ile-Gly-Ser-Arg (SEQ ID NO: 6).
  • the C18 means stearic acid (attached to the amino group of the side chain of Arg or Lys by the carboxyl group of stearic acid).
  • the cell membrane peptide is TAT.
  • the sequence of the TAT is Arg-Lys-Lys-Arg-Arg-Gln-Arg-Arg-Arg (RKKRRQRRR, SEQ ID NO: 7) or Gly-Arg-Lys-Lys- Arg-Arg-Gln-Arg-Arg-Arg (GRKKRRQRRR, SEQ ID NO: 8).
  • the phospholipid mainly comprises two major classes of glycerophospholipids and sphingomyelins, wherein the glycerophospholipids can be further divided into phosphatidylcholine, phosphatidylethanolamine, phosphatidylserine, phosphatidylglycerol, diphosphatidylglycerol and There are several types of phosphatidylinositol, and each type can be several depending on the fatty acid composition.
  • Another aspect of the invention relates to a complex comprising a compound of formula I as claimed in any one of the invention, or a pharmaceutically acceptable salt thereof, and a nucleic acid molecule such as DNA or RNA.
  • the complex wherein the charge ratio of the compound of Formula I or a pharmaceutically acceptable salt thereof to the nucleic acid molecule is (1-10):1, for example, (2- 8): 1, 1:1, 1.5:1, 2:1, 2.5:1, 3:1, 3.5:1, 4:1, 4.5:1, 5:1, 6:1, 7:1 or 8 : 1.
  • the nucleic acid carrier has a positive charge
  • the nucleic acid molecule has a negative charge
  • the nucleic acid carrier and the nucleic acid molecule form a complex by attraction between positive and negative charges.
  • a further aspect of the invention relates to a host cell comprising a compound of formula I according to any one of the invention, or a pharmaceutically acceptable salt thereof, or a complex of the invention.
  • the host cell may be a prokaryotic cell or a eukaryotic cell, such as an E. coli cell, such as a yeast cell or a mammalian cell, such as a source. Cells of humans, mice, rats, monkeys, and the like.
  • the mammalian cell is a 293T cell or a B16F10 cell.
  • a further aspect of the invention relates to a method of transfecting a eukaryotic cell comprising the step of loading a target nucleic acid molecule (eg DNA or RNA) to a compound of formula I according to any one of the invention or a pharmaceutically acceptable salt thereof .
  • a target nucleic acid molecule eg DNA or RNA
  • a method of introducing the nucleic acid vector or complex into a cell to obtain a host cell is known in the art, for example, by introducing the nucleic acid vector or complex into the cell by culturing it with the cell.
  • a further aspect of the invention relates to a pharmaceutical composition
  • a pharmaceutical composition comprising a compound of formula I according to any one of the invention, or a pharmaceutically acceptable salt thereof, or a complex of the invention, and at least one pharmaceutically acceptable excipient .
  • a further aspect of the invention relates to a kit comprising a compound of formula I according to any one of the invention, or a pharmaceutically acceptable salt thereof, or a complex of the invention, and a nucleic acid of interest.
  • the composition or kit is used to load or transport a nucleic acid (e.g., DNA or RNA) molecule into a cell, and thus it may contain a corresponding buffer, a detection reagent, or a pharmaceutically acceptable adjuvant or the like.
  • a nucleic acid e.g., DNA or RNA
  • the composition or kit may contain a corresponding buffer, a detection reagent, or a pharmaceutically acceptable adjuvant or the like.
  • a further aspect of the invention relates to the use of YIGSR (SEQ ID NO: 17) or YIGSK (SEQ ID NO: 18) for the preparation of a medicament or agent for loading and/or transporting a nucleic acid, such as DNA or RNA.
  • a further aspect of the invention relates to a compound of formula I according to any one of the invention, or a pharmaceutically acceptable salt thereof, for use in the manufacture of a medicament or agent or gene therapy for loading and/or transporting a nucleic acid, such as DNA or RNA the use of.
  • a further aspect of the invention relates to a polypeptide of SEQ ID NO: 17 or SEQ ID NO: 18 for use in the preparation of a medicament or agent for loading and/or transporting a nucleic acid.
  • a further aspect of the invention relates to a compound of formula I, or a pharmaceutically acceptable salt thereof, according to any of the invention, for use in loading and/or transporting nucleic acids, or for gene therapy.
  • a further aspect of the invention relates to a method of loading and/or transporting a nucleic acid, such as DNA or RNA, or a method of preparing a complex of the invention, comprising a compound of formula I according to any of the invention a step of contacting a pharmaceutically acceptable salt thereof with a nucleic acid molecule (eg, DNA or RNA);
  • a nucleic acid molecule eg, DNA or RNA
  • the charge ratio of the compound or a pharmaceutically acceptable salt thereof to the nucleic acid molecule is (1-10):1, for example, (2-8): 1, 1:1, 1.5:1, 2:1 , 2.5:1, 3:1, 3.5:1, 4:1, 4.5:1, 5:1, 6:1, 7:1 or 8:1;
  • the contact is carried out under conditions of 36 ° C - 38 ° C (eg 37 ° C) for more than 20 min (eg 30 min);
  • the compound of formula I or a pharmaceutically acceptable salt thereof is mixed with the nucleic acid molecule prior to incubation; preferably, vortexing or stirring is carried out.
  • a further aspect of the invention relates to a method of gene therapy comprising administering to a subject in need thereof an effective amount of a compound of formula I according to any one of the invention, or a pharmaceutically acceptable salt thereof, or a compound of the invention The steps of the object.
  • the nucleic acid molecule refers to binding a nucleic acid vector to a nucleic acid (DNA or RNA) molecule.
  • the transport nucleic acid (DNA or RNA) molecule refers to the transfer or introduction of a nucleic acid molecule into a cell.
  • the subject is a mammal, such as a bovine, an equine, a sheep, a porcine, a canine, a feline, a rodent, a primate;
  • the subjects are human.
  • the nucleic acid vector of the present invention or the complex of the present invention when used for gene therapy of a mammal, can be introduced into a mammal or a tissue or an organ to be treated by various routes, for example, by intravenous injection, local administration. Injection and other methods.
  • the gene therapy refers to a method of introducing an exogenous nucleic acid molecule (such as a normal gene, a functional nucleic acid molecule, etc.) into a body or a cell of a subject in need thereof to achieve a disease treatment.
  • the nucleic acid molecule can be a DNA molecule or an RNA molecule.
  • the DNA molecule may be, for example, a gene encoding an enzyme (for example, adenosine deaminase), a gene capable of killing a tumor cell (for example, a tumor necrosis factor), a gene of a functional protein, or the like.
  • the RNA fraction may be, for example, an antisense oligonucleotide molecule or the like.
  • the cell-permeable membrane peptide refers to a polypeptide which enters cells directly through the cell membrane in a non-receptor-dependent manner, and which is not more than 30 amino acids in length and is alkaline. Amino acids, amino acid sequences are usually positively charged, such as the examples listed above.
  • the nucleic acid includes a DNA or RNA molecule.
  • the nucleic acid molecule can be a vector, a reporter gene, an effector gene (eg, an anti-cancer gene), or a DNA or RNA molecule having other functions.
  • the length of the nucleic acid is not particularly required, and may be, for example, in the range of several bases to several thousand bases.
  • One skilled in the art can adjust the amount of nucleic acid vector according to the size of the nucleic acid length, that is, when the length of the nucleic acid is small, a smaller number of nucleic acid carriers are required, and when the length of the nucleic acid is large, a larger number of nucleic acid carriers are required.
  • the charge ratio of the nucleic acid carrier and the nucleic acid molecule is calculated by calculating the number of charges carried by each of the nucleic acid carrier and the nucleic acid molecule.
  • the number of charges carried by the nucleic acid carrier refers to the number of positive charges carried by the polypeptide, and refers to the number of positively charged Lys and Arg in the polypeptide sequence, defined as N, ie, a Lys or an Arg with a positive charge;
  • the number of charges carried refers to the number of phosphate groups of a nucleic acid base, defined as P, that is, a phosphate group carries a negative charge.
  • the invention designs and synthesizes a series of polypeptide nucleic acid carriers containing a cell membrane-permeable peptide, a histidine-rich endosomal escape structure, a receptor targeting structure and an optional hydrophobic molecule, which effectively solves the process of gene transfer. Problems such as loading, transport, and intracellular endosomal escape have achieved higher transfection efficiency than commercial Lipofectamine 2000, and have lower toxic side effects.
  • Figure 1 Schematic diagram of the structure of a polypeptide gene vector.
  • Figure 2 A carrier/DNA complex agarose gel electrophoresis pattern in which numbers above the electropherogram, such as 0, 1.0, 1.5, 2.0, 2.5, 3.0, etc., represent the charge ratio of the vector to the DNA, respectively.
  • Figure 2A is a TAT-H6-K(C18)-YIGSR/DNA complex
  • Figure 2B is a TAT-H6-K(C18)-YIGSK/DNA complex
  • Figure 2C is a TAT-H6-K(C18)/DNA complex
  • Figure 2D is a TAT-H6-K-YIGSR/DNA complex
  • Figure 2E is a C18-TAT-H6-K-YIGSR/DNA complex
  • Figure 2F is a H6-TAT-K(C18)-YIGSR/DNA complex Things.
  • Figure 3 Vector/DNA complex fluid particle size and zeta potential map, where the abscissa is the charge ratio of the polypeptide to the DNA, and the ordinate is the particle size (A) and the zeta potential (B), respectively.
  • Figure 4 Projection electron micrograph of a complex with a peptide/DNA charge ratio of 6 (the ratio in the figure indicates 100 nm).
  • the samples in Figures 4A to 4F are as follows:
  • 4A is a TAT-H6-K(C18)-YIGSR/DNA complex
  • 4B is a TAT-H6-K(C18)-YIGSK/DNA complex
  • 4C is a TAT-H6-K (C18)/DNA complex
  • 4D is a TAT-H6-K-YIGSR/DNA complex
  • 4E is a C18-TAT-H6-K-YIGSR/DNA complex
  • 4F is a H6-TAT-K(C18)-YIGSR/DNA complex.
  • Figure 5 Outer transfection efficiency map of vector/DNA complexes, where the abscissa is the charge ratio of the polypeptide to the DNA and the ordinate is the luciferase activity (i.e., RLU). among them,
  • Figure 5A shows the transfection efficiency of the vector/DNA complex in 293T cells
  • Figure 5B shows the transfection efficiency of the vector/DNA complex in B16F10 cells.
  • Figure 6 Evaluation of 293T cytotoxicity of vector/DNA complexes, where the abscissa is the charge-to-DNA ratio and the ordinate is the cell viability.
  • the solid phase synthesis carrier Rink-amide resin used in the preparation example is the product of Tianjin Nankai Synthetic Co., Ltd. HOBT, HBTU, DIEA and Fmoc-protected amino acids provided by Shanghai Jill Biochemical Co., Ltd.
  • Arg stands for arginine
  • Gln stands for glutamine
  • Gly represents glycine
  • Tyr represents tyrosine
  • Lys represents lysine
  • Ile represents isoleucine
  • Fmoc represents fluorenylmethoxycarbonyl
  • HBTU stands for 2-(1H-1-hydroxybenzotriazole)-1,1,3,3-tetramethylurea hexafluorophosphate
  • DIEA stands for N,N-diisopropylethylamine
  • TFA stands for trifluoroacetic acid
  • TAE-buffer represents tris(hydroxymethyl)aminomethane-acetic acid-ethylenediaminetetraacetic acid buffer
  • ninhydrin reaction 5 ml of 2% hydrazine hydrate was used as a deprotecting reagent, and reacted at room temperature for three minutes and repeated three times. Whether the deprotection was successful was detected by the ninhydrin reaction. After confirming that the deprotection was successful, DIC was used as a condensing agent, and a DMF solution of stearic acid was introduced, and the reaction was carried out at room temperature for 4 hours or more. Whether the stearic acid linkage was successful was detected by the ninhydrin reaction.
  • ninhydrin reaction 5 ml of 2% hydrazine hydrate was used as a deprotecting reagent, and reacted at room temperature for three minutes and repeated three times. Whether the deprotection was successful was detected by the ninhydrin reaction. After confirming that the deprotection was successful, DIC was used as a condensing agent, and a DMF solution of stearic acid was introduced, and the reaction was carried out at room temperature for 4 hours or more. Whether the stearic acid linkage was successful was detected by the ninhydrin reaction.
  • ninhydrin reaction 5 ml of 2% hydrazine hydrate was used as a deprotecting reagent, and reacted at room temperature for three minutes and repeated three times. Whether the deprotection was successful was detected by the ninhydrin reaction. After confirming that the deprotection was successful, DIC was used as a condensing agent, and a DMF solution of stearic acid was introduced, and the reaction was carried out at room temperature for 4 hours or more. Whether the stearic acid linkage was successful was detected by the ninhydrin reaction.
  • a solution of stearic acid DMF was added and reacted at room temperature for 4 hours or more. Whether the stearic acid linkage was successful by the ninhydrin reaction (stearic acid linked to the exposed N-terminal amino group of the peptide chain).
  • ninhydrin reaction stearic acid linked to the exposed N-terminal amino group of the peptide chain.
  • the peptide is removed from the protecting group and cleaved from the resin.
  • the solution was purified by RP-HPLC, RP-HPLC conditions, phase A: 0.05% TFA / water; phase B: 0.05% TFA / 70% ACN / water; column: C8.
  • MALDI-Tof-MS 3189.81.
  • ninhydrin reaction 5 ml of 2% hydrazine hydrate was used as a deprotecting reagent, and reacted at room temperature for three minutes and repeated three times. Whether the deprotection was successful was detected by the ninhydrin reaction. After confirming that the deprotection was successful, DIC was used as a condensing agent, and a DMF solution of stearic acid was introduced, and the reaction was carried out at room temperature for 4 hours or more. Whether the stearic acid linkage was successful was detected by the ninhydrin reaction.
  • the amino acid sequence is from the N-terminus to the C-terminus from left to right.
  • the amino group at the N-terminus of the amino acid sequence was acetylated, and the carboxyl group at the C-terminal was amidated.
  • chemically synthesized peptides often carry free amino groups and free carboxyl groups, and the sequence of the peptide often represents the sequence of the parent protein.
  • the peptide ends are often blocked, ie, N-terminal acetyl.
  • the nucleic acid vector prepared above was applied to the following Preparation Examples 7-12.
  • TAT-H6-K(C18)-YIGSR Take 1 ⁇ g of PGL-3 plasmid DNA (purchased from Promega), dilute to 25 ⁇ l, and mix TAT-H6-K(C18)-YIGSR into a 1 mg/ml solution according to the peptide/DNA charge ratio (N/P)0. 1, 2, 2.5, 3, 3.5, 4, 6, 8 absorb different volumes of peptide solution, dilute to 25 ⁇ l, mix the peptide and DNA solution, vortex for 10s, incubate at 37 ° C for 30min, form TAT- with different charge ratio H6-K (C18)-YIGSR/DNA complex.
  • N/P peptide/DNA charge ratio
  • TAT-H6-K (C18) Take 1 ⁇ g of PGL-3 plasmid DNA, dilute to 25 ⁇ l, and mix TAT-H6-K (C18) into a 1 mg/ml solution according to peptide/DNA charge ratio (N/P) 0, 1, 2, 2.5, 3, 3.5, 4,6,8 draw different volume of peptide solution, dilute to 25 ⁇ l, mix the peptide and DNA solution, vortex for 10s, incubate at 37 °C for 30min, form TAT-H6-K(C18)/DNA complex with different charge ratio .
  • N/P peptide/DNA charge ratio
  • TAT-H6-K-YIGSR Take 1 ⁇ g of PGL-3 plasmid DNA, dilute to 25 ⁇ l, and mix TAT-H6-K-YIGSR into a 1 mg/ml solution according to peptide/DNA charge ratio (N/P) 0, 1, 2, 2.5, 3, 3.5, 4, 6, and 8 were pipetted into different volume peptide solutions, diluted to 25 ⁇ l, and the peptide and DNA solutions were mixed, vortexed for 10 s, and incubated at 37 ° C for 30 min to form TAT-H6-K-YIGSR/DNA complexes with different charge ratios.
  • N/P peptide/DNA charge ratio
  • nucleic acid vector/DNA complex used in the following Experimental Examples 1-5 was prepared by referring to Preparation Examples 7 to 12; wherein the specific charge ratio can also be appropriately adjusted.
  • the present invention investigated agarose gel electrophoresis of a nucleic acid vector/DNA complex.
  • Agar/TAE buffer solution with a mass/volume ratio of 1% was prepared, dissolved in a microwave oven, poured into an electrophoresis tank mold for 30 minutes, placed in an electrophoresis tank, and added with 10 ⁇ TAE buffer.
  • a nucleic acid vector/DNA complex prepared according to the above method was used, wherein the DNA was 0.1 ⁇ g, the volume was 10 ⁇ l, and the voltage was 100 V for 60 min.
  • the cells were stained for 15 min in EB staining solution, and then photographed under a gel imager to observe the DNA migration bands in the electrophoresis.
  • FIG. 2A shows that the TAT-H6-K(C18)-YIGSR/DNA complex charge ratio can be completely loaded with DNA at 3 o'clock (the polypeptide is completely loaded with DNA to form a nanocomplex that cannot enter the agarose gel, so there is no migration band; Unloaded DNA enters the agarose gel and EB staining reveals a bright DNA band under UV light).
  • Figure 2B shows that the TAT-H6-K(C18)-YIGSK/DNA complex charge ratio can completely load the DNA at 3.5, indicating that the YIGSR to YIGSK in the sequence has little effect on the DNA carrier-loaded DNA.
  • FIG. 2C shows that the TAT-H6-K(C18)/DNA complex charge ratio can be fully loaded with DNA at 3.0. It is indicated that the presence of the targeting sequence YIGSR has little effect on the ability of the complex to load DNA.
  • FIG. 2D shows that the charge ratio of the TAT-H6-K-YIGSR/DNA complex can be fully loaded at 2.5.
  • Figure 2E shows that the C18-TAT-H6-K-YIGSR/DNA complex charge ratio can completely load DNA at 7.
  • the placement of the transmembrane peptide in the middle of the peptide chain affects the positive charge of the basic amino acid and the negative charge binding of the nucleic acid, thereby affecting the ability of the vector to carry DNA.
  • Figure 2F shows that the H6-TAT-K(C18)-YIGSR/DNA complex charge ratio can completely load DNA at 6 o'clock.
  • the placement of the transmembrane peptide in the middle of the peptide chain affects the positive charge of the basic amino acid and the negative charge binding of the nucleic acid, thereby affecting the ability of the vector to carry DNA.
  • the particle size and zeta potential were detected by Zetasizer Nano ZS90, Malvern laser particle size analyzer, and the detection temperature was 25 ° C. Each sample was repeated three times, and the test results are shown in Fig. 3.
  • the particle size of the TAT-H6-K(C18)-YIGSR/DNA complex increased from 114.51 nm to 211 nm and then decreased to 126.53 nm as the charge ratio increased.
  • the zeta potential increased from -8.08 mv to 15.57 mv as the charge ratio increased.
  • the particle size of the TAT-H6-K(C18)-YIGSK/DNA complex increased from 144.97 nm to 161.63 nm and then decreased to 105.94 nm. From the results, it can be seen that the target sequence was changed by YIGSR. The effect of YIGSK on the particle size of the composite is not large. At the same time, the zeta potential did not change significantly, and the zeta potential increased from -11.68 mv to 14.63 mv as the positive-negative charge ratio increased.
  • the particle size of the TAT-H6-K(C18)/DNA complex increased from 133.33 nm to 441.7 nm and then decreased to 76.24 nm as the charge ratio increased.
  • the zeta potential increased from -8.24 mv to 14.9 mv as the positive and negative charge ratio increased.
  • the particle size of the TAT-H6-K-YIGSR/DNA complex increased from 589.86 nm to 657.63 nm and then decreased to 140.43 nm as the charge ratio increased.
  • the zeta potential increased from 5.74 mv to 14 mv as the positive and negative charge ratio increased.
  • the particle size of the C18-TAT-H6-K-YIGSR/DNA complex increased from 439.4 to 540.33 nm and then decreased to 107 nm as the charge ratio increased.
  • the zeta potential increased from 5.56 mv to 15.86 mv as the positive and negative charge ratio increased.
  • the particle size of the H6-TAT-K(C18)-YIGSR/DNA complex increased from 454.9 to 622 nm and then slightly increased to 86.48 nm as the charge ratio increased.
  • the zeta potential increased from 3.71 mv to 15.3 mv as the positive and negative charge ratio increased.
  • the sample tested was a complex with a peptide/DNA charge ratio of 6.
  • TAT-H6-K(C18)-YIGSR/DNA 5 ⁇ l of the prepared TAT-H6-K(C18)-YIGSR/DNA complex solution was added dropwise to a copper mesh, and after 30 min, it was blotted with filter paper at Hitachi. Photographed under the H-7650 microscope. Similarly, microscopic photographs were taken of the remaining five complexes.
  • the TAT-H6-K(C18)-YIGSR/DNA complex is a nanoparticle of about 55 nm, which is smaller than that measured by DLS (Dynamic Light Scattering) because of the test of transmission electron microscopy.
  • the composite is a sample after drying, which is smaller than the particle size of the fluid tested in the aqueous solution.
  • the TAT-H6-K(C18)-YIGSK/DNA complex is a nanoparticle of about 55 nm, which is comparable to the nanoparticle size of the TAT-H6-K(C18)-YIGSR/DNA complex.
  • this scale is smaller than that measured by DLS because the composites tested for TEM are air-dried samples that are smaller than the particle size of the fluids tested in aqueous solutions.
  • the TAT-H6-K(C18)/DNA complex is a nanoparticle of about 65 nm, which is larger than the TAT-H6-K(C18)/DNA complex nanoparticle, and it can be seen that a target is introduced into the polypeptide.
  • the group YIGSR promotes the formation of amphiphilic ⁇ -helical conformation to facilitate the formation of denser nanoparticles with DNA. Again, this scale is smaller than that measured by DLS because the composites tested for TEM are air-dried samples that are smaller than the particle size of the fluids tested in aqueous solutions.
  • the TAT-H6-K-YIGSR/DNA complex is a nanoparticle of about 78 nm, which is larger than the TAT-H6-K(C18)-YIGSR/DNA complex nanoparticle, and it can be seen that hydrophobicity is introduced into the polypeptide.
  • the group is more favorable for promoting the formation of amphipathic ⁇ -helical conformation and forming denser nanoparticles with DNA.
  • This scale is smaller than that measured by DLS because the composite when tested for transmission electron microscopy is a sample that has been dried, which is smaller than the particle size of the fluid tested in aqueous solution.
  • the C18-TAT-H6-K-YIGSR/DNA complex is a nanoparticle of about 45 nm, which is smaller than that measured by DLS (Dynamic Light Scattering) because of the measurement of TEM.
  • the composite is a sample after drying, which is smaller than the particle size of the fluid tested in the aqueous solution.
  • the H6-TAT-K(C18)-YIGSR/DNA complex is a nanoparticle of about 35 nm, which is smaller than that measured by DLS (Dynamic Light Scattering) because of the test of transmission electron microscopy.
  • the composite is a sample after drying, which is smaller than the particle size of the fluid tested in the aqueous solution.
  • the cell transfection efficiency of the peptide/DNA complex of the present invention was evaluated in two cell lines, 293T and B16F10 cells (purchased from the Concord Cell Bank, the Cell Resource Center of the Institute of Basic Medical Sciences, Chinese Academy of Medical Sciences).
  • TAT-H6-K(C18)-YIGSR/DNA complex (0.6 ⁇ g of PGL-3 plasmid DNA per well, charge ratio (N/P) of 4, 6, and 8 respectively) was diluted to 300 ⁇ l with DMEM medium, respectively Add to the cell culture plate and incubate for 4 hours in a 37 ° C, 5% CO 2 cell incubator.
  • TAT-H6-K(C18)-YIGSR rationally combines the transmembrane functional fragment, the concealed escape function fragment, the hydrophobic functional fragment and the receptor targeting functional fragment, and the transfection efficiency of the formed nucleic acid vector reaches the positive control Lipo2000 5 -10 times.
  • TAT-H6-K(C18)-YIGSK/DNA complex (0.6 ⁇ g of PGL-3 plasmid DNA per well, charge ratio (N/P) 4-8) was diluted to 300 ⁇ l with DMEM medium and added to cell culture. Plates were incubated for 4 hours in a 37 ° C, 5% CO 2 cell incubator.
  • luciferase expression levels of both cells reached a maximum at a charge ratio of 6:1, and luciferase activity (RLU) was 4.19 ⁇ 10 6 (293T cells) and 1.80 ⁇ 10 5 (B16F10 cells), respectively.
  • RLU luciferase activity
  • the expression efficiency of luciferase was slightly lower than that in the experimental example (1), and YIGSR was the key to improve transfection efficiency.
  • TAT-H6-K(C18)-YIGSK is replaced with TAT-H6-K(C18)-YIGSR, and the arginine in the receptor targeting sequence is replaced with a lysine of structural similarity.
  • TAT-H6-K(C18)-YIGSK rationally combines the transmembrane functional fragment, the concealed escape function fragment, the hydrophobic functional fragment and the receptor-targeted functional fragment-derived fragment, and the resulting nucleic acid vector transfection efficiency also reaches the positive control. 5-10 times that of Lipo2000.
  • TAT-H6-K(C18)/DNA complex (0.6 ⁇ g of PGL-3 plasmid DNA per well, charge ratio (N/P) 4-8) was diluted to 300 ⁇ l with DMEM medium and added to the cell culture plate, respectively. Incubate for 4 hours in a 37 ° C, 5% CO 2 cell incubator.
  • TAT-H6-K (C18) rationally combines the transmembrane functional fragment, the concealed escape function fragment, and the hydrophobic functional fragment to form a transfection efficiency of the nucleic acid vector relative to the TAT-H6-K(C18)-YIGSR transfection efficiency. Significantly reduced, indicating that the presence of receptor-targeted fragments can greatly improve transfection efficiency.
  • TAT-H6-K-YIGSR/DNA complex (0.6 ⁇ g of PGL-3 plasmid DNA per well, charge ratio (N/P) 4-8) was diluted to 300 ⁇ l with DMEM medium and added to the cell culture plate, respectively. Incubate for 4 hours in a 37 ° C, 5% CO 2 cell culture incubator.
  • TAT-H6-K-YIGSR rationally combines the transmembrane functional fragment, the concealed escape function fragment, and the receptor targeting functional fragment, and the resulting nucleic acid vector has low transfection efficiency. Irrespective of theoretical limitations, hydrophobic groups can enhance the ability of water-soluble peptides to fuse with phospholipid bilayer membranes, promote cell uptake and endosomal escape, and therefore play an important role in gene transfection.
  • the cell transfection efficiency of the peptide/DNA complex of the present invention was evaluated in two cell line 293T and B16F10 cells (purchased from the Concord Cell Bank, the Cell Resource Center of the Institute of Basic Medical Sciences, Chinese Academy of Medical Sciences).
  • 293T and B16F10 cells were seeded in 48-well plates, and each well was inoculated with 3 ⁇ 10 4 (500 ⁇ l DMEM cell suspension) cells. After 24 hours, the cell confluence reached 80%, and the medium was aspirated.
  • C18-TAT-H6-K-YIGSR/DNA complex (0.6 ⁇ g of PGL-3 plasmid DNA per well, charge ratio (N/P) 4-8) was diluted to 300 ⁇ l with DMEM medium and added to the cell culture plate. Incubate for 4 hours in a 37 ° C, 5% CO 2 cell incubator.
  • C18-TAT-H6-K-YIGSR combines a transmembrane functional fragment, an escape function escape fragment, a hydrophobic functional fragment, and a receptor-targeting functional fragment, but the transmembrane peptide is located in the middle of the peptide chain to affect its ability to load DNA, forming
  • the nucleic acid vector has a low transfection efficiency.
  • the cell transfection efficiency of the peptide/DNA complex of the present invention was evaluated in two cell lines, 293T and B16F10 cells (purchased from the Concord Cell Bank, the Cell Resource Center of the Institute of Basic Medical Sciences, Chinese Academy of Medical Sciences).
  • H6-TAT-K(C18)-YIGSR/DNA complex (0.6 ⁇ g of PGL-3 plasmid DNA per well, charge ratio (N/P) 4-8) was diluted to 300 ⁇ l with DMEM medium and added to cell culture. Plates were incubated for 4 hours in a 37 ° C, 5% CO 2 cell incubator.
  • C18-TAT-H6-K-YIGSR combines a transmembrane functional fragment, an escape function escape fragment, a hydrophobic functional fragment, and a receptor-targeting functional fragment, but the transmembrane peptide is located in the middle of the peptide chain to affect its ability to load DNA, forming
  • the nucleic acid vector has a low transfection efficiency.
  • the polypeptides other than TAT-H 6 -K(C 18 )-YIGSR and TAT-H 6 -K(C 18 )-YIGSK synthesized are transfected.
  • the efficiency of DNA is also relatively low, such as TAT-H 6 -K-YIGSR and H 6 -TAT-K(C 18 )-YIGSR.
  • the cytotoxicity evaluation experiment of the present invention was evaluated in 293T cells using the CellTiter of Promega.
  • 293T cells were seeded in 96-well plates at 1 ⁇ 10 4 cells per well, and after 24 hours of culture at 37° C. under 5% CO 2 , the cell confluence reached 70%-80%.
  • the medium was aspirated, and 100 ⁇ l of the TAT-H6-K(C18)-YIGSR/DNA complex obtained in Preparation Example 7-12 was added and incubated for 4 hours. After 4 hours, the medium was aspirated and replaced with fresh DMEM containing 10% FBS, and cultured for further 20 hours, and then the cell survival rate was measured by the CCK method.
  • TAT-H6-K(C18)-YIGSR/DNA complex The cell survival rate reached more than 90% under different conditions, and the TAT-H6-K(C18)-YIGSR/DNA complex of the present invention can be seen. Has low cytotoxicity.
  • the nucleic acid vector of the present invention has significant advantages over the 80% cytotoxicity of the positive control Lipo2000.
  • TAT-H6-K(C18)-YIGSK/DNA complex Under different conditions, the cell survival rate is over 90%, and the TAT-H6-K(C18)-YIGSK/DNA complex of the present invention can be seen. Has low cytotoxicity.
  • the nucleic acid vector of the present invention has significant advantages over the 80% cytotoxicity of the positive control Lipo2000.
  • TAT-H6-K(C18)/DNA complex Under different conditions, the cell survival rate reached more than 85%, and it can be seen that the TAT-H6-K(C18)/DNA complex of the present invention has a lower Cytotoxicity.
  • TAT-H6-K-YIGSR/DNA complex Under different conditions, the cell survival rate is above 90%, and the TAT-H6-K-YIGSR/DNA complex of the present invention has low cytotoxicity. .
  • C18-TAT-H6-K-YIGSR/DNA complex Under different conditions, the cell survival rate reached more than 75%, and the C18-TAT-H6-K-YIGSR/DNA of the present invention is relative to the complex. TAT-H6-K(C18)-YIGSR has high cytotoxicity.
  • H6-TAT-K(C18)-YIGSR/DNA complex Under different conditions, the cell survival rate reached more than 70%, and the H6-TAT-K(C18)-YIGSR/DNA complex of the present invention can be seen. It has high cytotoxicity relative to the complex TAT-H6-K(C18)-YIGSR.

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Abstract

公开了一种核酸载体及其用途。还公开了含有所述核酸载体和核酸分子的复合物，以及所述核酸载体/核酸分子复合物的制备方法及用途。所述核酸载体为A-B-C-D所示的化合物或其可药用盐，A为细胞透膜肽，B为内涵体逃逸片段，C为一个或多个Lys和/或一个或多个Arg，或者C缺失，D为层粘连蛋白受体靶向片段。

Description

一种核酸载体及其用途 技术领域
本发明属于生物医药领域，涉及一种核酸载体及其用途。本发明还涉及含有所述核酸载体和核酸分子的复合物，以及所述核酸载体/核酸分子复合物的制备方法及用途。
背景技术
美国食品药品管理局(FDA)将基因治疗定义为通过转录或翻译、转运和/或整合到宿主基因组中的外源基因进行治疗疾病的一种治疗方法。基因治疗是治疗癌症、单基因疾病、心血管疾病和神经性疾病等的潜在的最有效的治疗方法。自1990年，基因治疗第一次进入临床试验开始，全球已经有超过1800例基因治疗临床试验开展。我国更是在2004年，成为第一个在市场上引进基因药物(Gendicine)的国家。由于用于基因治疗的核酸药物分子具有分子量大、富含大量负电荷以及极易降解等特点，目前基因治疗面临的最大难题是缺乏理想的转运载体，因此寻找高效、安全的基因转运载体成为研究的重点。
目前开展的临床试验大多采用腺病毒和逆转录病毒作为基因药物的载体，但病毒载体的缺点在于转运能力低、使用成本高昂和潜在的安全性问题等。在1994年，因为在临床试验中使用病毒基因载体造成病人死亡，致使基因治疗研究一度陷入停滞。因此，近些年非病毒类载体研究发展迅速，包括脂质体、阳离子聚合物和多肽等。非病毒载体转运基因药物多数通过自身的阳离子与基因药物的阴离子之间的离子相互作用负载DNA或RNA，形成较小尺度的纳米颗粒，转运DNA或RNA进入宿主细胞，进而整合到宿主基因组中进行表达。多肽以其较好的生物相容性、功能多样性和易于合成等特点成为基因治疗载体的较好选择。
细胞透膜肽(Cell-Penetrating Peptides，CPPs)是一类能够高效介导核酸、蛋白等生物大分子透过细胞膜进入细胞的一类短肽，一般肽序列长度不超过30个氨基酸，大部分透膜肽富含赖氨酸和精氨酸等碱性氨基酸。其中，TAT(49-57)(HIV tat protein)是细胞透膜蛋白肽片段中可完全行使细胞透膜功能且无细胞毒性的最小片段，TAT(49-57)肽序列为Arg-Lys-Lys-Arg-Arg-Gln-Arg-Arg-Arg(RKKRRQRRR，SEQ ID NO：7)。该段透膜肽可以通过在生理条件下其碱性氨基酸的正电荷与核酸的负电荷结合，负载核酸进而通过内吞作用介导核酸进入细胞，并且TAT肽片段有一定的细胞核靶向能力。然而，通过内 吞作用进入细胞的TAT/DNA或RNA复合体首先进入内涵体(endosome，一种内部呈酸性环境的脂膜结构)，最终运送至溶酶体降解。富含精氨酸的透膜肽肽序列R n(n＝6-10)也具有和TAT相似的特性。由于TAT和Rn缺乏逃逸内涵体的能力，因此很难单独作为递送DNA的载体达到理想的转染效率。
实现内涵体逃逸的方法主要有两种，一种是利用酸性环境(pH＜6.0)下具有离子缓冲作用的材料，通过质子泵作用破坏内涵体，使DNA或RNA复合体逃逸；另一种是利用带有α-螺旋双亲性结构的多肽与脂膜作用，形成空洞，释放出来DNA或RNA复合体。组氨酸能够在酸性的内涵体中不断吸收质子，通过“质子海绵”效应涨破内涵体，进而起到破坏内涵体脂质囊泡的作用。
层粘连蛋白(laminin，LN)是一种大分子胶原糖蛋白。随着对LN的深入研究，发现LN具有多种生物学作用：促进细胞的粘着、铺展、有丝分裂、加速神经轴突生长及细胞移动，并诱导细胞分化，还与形态发生和肿瘤转移有关。层粘连蛋白受体(LN-R)广泛存在于上皮细胞、内皮细胞、周围神经细胞、巨噬细胞以及大部分肿瘤细胞表面，在体外与LN的结合表现出高亲和性、竞争性、浓度与时间依赖性。当恶性肿瘤发生转移时，细胞表面的LN-R与LN粘附是肿瘤细胞与基底膜相互作用关键的一步，为肿瘤黏附、侵袭突破基底膜提供了分子基础；同时LN-R不仅能促进细胞在LN基质上黏附，而且能诱导细胞的趋化性迁移和lV型胶原酶的分泌；也发现早期阶段的LN-R和LN对肿瘤的新生血管形成至关重要。
目前，尚需要开发转染效率高、细胞毒性低的新的核酸载体。
发明内容
本发明人经过深入的研究和创造性的劳动，得到了一种核酸载体即式I化合物。本发明人惊奇地发现，该核酸载体具有较高转染效率并且细胞毒性较低。此外，该核酸载体还能够较好地实现内涵体逃逸。由此提供了下述发明：
本发明的一个方面涉及式I所示的化合物，或其药学上可接受的盐，
A-B-C-D
式I
其中，
A表示细胞透膜肽，
B表示内涵体逃逸片段，
C表示一个或多个Lys，和/或一个或多个Arg，或者C缺失，
D表示层粘连蛋白受体靶向片段，并且
A和B之间，B和C之间，和/或C和D之间，为直接连接或者通过连接接头连接；
优选地，式I化合物还连接有一个或多个疏水分子E，并且所述疏水分子E连接在所述式I化合物中的Lys和/或Arg的侧链的氨基上。
所述直接连接可以是，例如A和B之中的一个的羧基和另一个的氨基之间形成酰胺键。B和C之间、C和D之间也可做类似理解。
所述连接接头，包括但不限于：一个或多个Gly、一个或多个Lys、和/或者一个或多个Cys。所述多个可以是例如2-20个、2-15个、2-10个、2-8个、2-6个、2个、3个、4个、5个或6个。所述A和B之间，B和C之间，以及C和D之间的连接接头可以相同或不同。连接接头与A、B、C或D的连接也是通过羧基和氨基形成酰胺键连接。
在本发明的一个实施方案中，所述的式I化合物或其药学上可接受的盐，其特征在于如下的(1)-(5)项中的任意一项或者多项：
(1)所述细胞透膜肽选自TAT、R n(n＝6、7、8、9或10)、penetratin、MAP、pVEC、MPG、MPGΔNLS、Stearyl-R8、EB1和Tat-DRBD中的任意一种或者多种；
(2)所述内涵体逃逸片段为质子泵内涵体逃逸片段，优选地，为一个或多个His，例如4-10、4-8、4-6、4、5、6、7或8个His；优选为6个His；
(3)所述层粘连蛋白受体靶向片段为YIGSR(SEQ ID NO：17)或YIGSK(SEQ ID NO：18)；
(4)所述疏水分子选自碳原子数大于或等于12的脂肪酸(例如硬脂酸、棕榈酸、油酸、亚油酸、软脂酸)、胆固醇和磷脂；
(5)所述疏水分子连接在C中的Lys和/或Arg的侧链的氨基上。
在本发明的一个实施方案中，所述的式I化合物或其药学上可接受的盐，其中，所述化合物如SEQ ID NO：1、SEQ ID NO：2或SEQ ID NO：4所示。
在本发明的一个实施方案中，所述的式I化合物或其药学上可接受的盐，其中，氨基酸序列的N末端乙酰化和/或C末端酰胺化。
根据本发明中任一项所述的式I化合物或其可药用盐或者本发明的复合物，其用于基因治疗。
在本发明中，组氨酸中咪唑基的pKa约为6.0，能够在酸性内涵体中大量吸收质子，形成“质子海绵”效应，造成内涵体的破裂。
不拘于理论的限制，通过连接疏水性分子，提供疏水性环境，有利于带正电荷的亲水性氨基酸与带负电荷的核酸药物充分接触，进而形成稳定致密的纳米复合物，有利于细胞的摄取，可以通过疏水相互作用提高聚集体的局部浓度，进而提高局部电荷密度，提高DNA或RNA的负载能力、提高核酸载体/DNA或RNA复合体的稳定性，同时，通过其疏水性烷基链与细胞膜的融合作用，进而有效提高透膜效率和破坏内涵体的能力。
在本发明的一个实施方案中，所述核酸载体的序列为Gly-Arg-Lys-Lys-Arg-Arg-Gln-Arg-Arg-Arg-His-His-His-His-His-His-Lys(C18)-Tyr-Ile-Gly-Ser-Arg(SEQ ID NO：1)。
在本发明的一个实施方案中，所述核酸载体的序列为Gly-Arg-Lys-Lys-Arg-Arg-Gln-Arg-Arg-Arg-His-His-His-His-His-His--Lys(C18)-Tyr-Ile-Gly-Ser-Lys(SEQ ID NO：2)。
在本发明的一个实施方案中，所述核酸载体的序列为Gly-Arg-Lys-Lys-Arg-Arg-Gln-Arg-Arg-Arg-His-His-His-His-His-His--Lys(C18)(SEQ ID NO：3)。
在本发明的一个实施方案中，所述核酸载体的序列为Gly-Arg-Lys-Lys-Arg-Arg-Gln-Arg-Arg-Arg-His-His-His-His-His-His-Lys-Tyr-Ile-Gly-Ser-Arg(SEQ ID NO：4)。
在本发明的一个实施方案中，所述核酸载体的序列为C18-Gly-Arg-Lys-Lys-Arg-Arg-Gln-Arg-Arg-Arg-His-His-His-His-His-His-Lys-Tyr-Ile-Gly-Ser-Arg(SEQ ID NO：5)。
在本发明的一个实施方案中，所述核酸载体的序列为His-His-His-His-His-His-Gly-Arg-Lys-Lys-Arg-Arg-Gln-Arg-Arg-Arg-Lys(C18)-Tyr-Ile-Gly-Ser-Arg(SEQ ID NO：6)。
在本发明中，如果没有特别说明，所述C18是指硬脂酸(通过硬脂酸的羧基连接到Arg或Lys的侧链的氨基上)。
在本发明的一个实施方案中，所述细胞透膜肽为TAT。在本发明的具体实施方案中，所述TAT的序列为Arg-Lys-Lys-Arg-Arg-Gln-Arg-Arg-Arg(RKKRRQRRR，SEQ ID NO：7)或Gly-Arg-Lys-Lys-Arg-Arg-Gln-Arg-Arg-Arg(GRKKRRQRRR，SEQ ID NO： 8)。
在本发明的一个实施方案中，其中所述细胞透膜肽选自TAT、R n(n＝6-10)(表示6-10个精氨酸组成的肽)、penetratin(RQIKIWFQNRRMKWKK，SEQ ID NO：9)、MAP(KLALKLALKALKAALKLA，SEQ ID NO：10)、pVEC(LLIILRRRIRKQAHAHSK，SEQ ID NO：11)、MPG(Ac-GALFLGFLGAAGSTMGAWSQPKKKRKV-NH 2，SEQ ID NO：12)、MPGΔNLS(Ac-GALFLGFLGAAGSTMGAWSQPKSKRKV-NH 2，SEQ ID NO：13)、Stearyl-R8(st-RRRRRRRR-NH 2，SEQ ID NO：14)、EB1(LIKLWSHLIHIWFQNRRLKWKKK-NH 2，SEQ ID NO：15)和Tat-DRBD(GRKKRRQRRRPQ-DRBD，SEQ ID NO：16)。
在本发明中，所述磷脂主要包括甘油磷脂和鞘磷脂两大类，其中所述甘油磷脂又可分为磷脂酰胆碱、磷脂酰乙醇胺、磷脂酰丝氨酸、磷脂酰甘油、二磷脂酰甘油及磷脂酰肌醇等几类，每一类中又可因组成的脂肪酸不同而有若干种。
本发明的另一方面涉及一种复合物，其包含本发明中任一项所述的式I化合物或其药学上可接受的盐，以及核酸分子例如DNA或RNA。
在本发明的一个实施方案中，所述的复合物，其中，所述式I化合物或其药学上可接受的盐与核酸分子的电荷比为(1-10)∶1，例如为(2-8)∶1、1∶1、1.5∶1、2∶1、2.5∶1、3∶1、3.5∶1、4∶1、4.5∶1、5∶1、6∶1、7∶1或8∶1。
在本发明中，所述的核酸载体带有正电荷，核酸分子带有负电荷，核酸载体与核酸分子之间通过正负电荷间的吸引作用形成复合物。
本发明的再一方面涉及一种宿主细胞，其含有本发明中任一项所述的式I化合物或其可药用盐或者本发明的复合物。
在本发明中，所述宿主细胞可以为原核细胞或真核细胞，所述原核细胞例如为大肠杆菌细胞，所述真核细胞例如为酵母细胞或哺乳动物细胞，所述哺乳动物细胞例如为来源于人、小鼠、大鼠、猴等的细胞。在本发明的实施方案中，所述哺乳动物细胞为293T细胞或B16F10细胞。
本发明的再一方面涉及一种转染真核细胞的方法，包括将目标核酸分子(例如DNA或RNA)负载至本发明中任一项所述的式I化合物或其可药用盐的步骤。
在本发明中，将所述核酸载体或复合物导入细胞得到宿主细胞的方法为本领域所 公知，例如，可通过将核酸载体或复合物与细胞孵育的方式将其引入细胞中。
本发明的再一方面涉及一种药物组合物，其含有本发明中任一项所述的式I化合物或其可药用盐或者本发明的复合物，以及至少一种药学上可接受的辅料。
本发明的再一方面涉及一种试剂盒，其含有本发明中任一项所述的式I化合物或其可药用盐或者本发明的复合物，以及目标核酸。
在本发明中，所述组合物或试剂盒用于负载或转运核酸(例如DNA或RNA)分子进入细胞，因此其可以含有相应的缓冲液、检测试剂、或药学上可接受的辅料等。
本发明的再一方面涉及YIGSR(SEQ ID NO：17)或YIGSK(SEQ ID NO：18)在制备负载和/或转运核酸(例如DNA或RNA)的药物或试剂中的用途。
本发明的再一方面涉及本发明中任一项所述的式I化合物或其可药用盐在制备用于负载和/或转运核酸(例如DNA或RNA)的药物或试剂或者基因治疗药物中的用途。
本发明的再一方面涉及SEQ ID NO：17或SEQ ID NO：18所示的多肽，其用于制备负载和/或转运核酸的药物或试剂。
本发明的再一方面涉及本发明中任一项所述的式I化合物或其可药用盐，其用于负载和/或转运核酸，或者用于基因治疗。
本发明的再一方面涉及一种负载和/或转运核酸(例如DNA或RNA)的方法，或者一种制备本发明的复合物的方法，包括使本发明中任一项所述的式I化合物或其可药用盐与核酸分子(例如DNA或RNA)接触的步骤；
优选地，所述化合物或其药学上可接受的盐与核酸分子的电荷比为(1-10)∶1，例如为(2-8)∶1、1∶1、1.5∶1、2∶1、2.5∶1、3∶1、3.5∶1、4∶1、4.5∶1、5∶1、6∶1、7∶1或8∶1；
优选地，所述接触的条件为，36℃-38℃(例如37℃)条件下孵育20min以上(例如30min)；
优选地，在孵育之前，将式I化合物或其可药用盐与核酸分子进行混合；优选地，进行涡旋或搅拌。
本发明的再一方面涉及一种基因治疗方法，所述方法包括给予有需要的受试者有效量的本发明中任一项所述的式I化合物或其可药用盐或者本发明的复合物的步骤。
在本发明中，所述负载核酸分子(例如DNA或RNA)是指使核酸载体与核酸(DNA或RNA)分子结合。
在本发明中，所述转运核酸(DNA或RNA)分子是指将核酸分子转入或导入细胞。
在本发明中，所述受试者为哺乳动物，例如牛科动物、马科动物、羊科动物、猪科动物、犬科动物、猫科动物、啮齿类动物、灵长类动物；其中优选的受试者为人。
在本发明中，当用于哺乳动物的基因治疗时，可以通过多种途径将本发明的核酸载体或者本发明的复合物导入哺乳动物体内或者需要治疗的组织或器官，例如通过静脉注射、局部注射等方式。
在本发明中，所述基因治疗是指将外源核酸分子(如正常基因、功能核酸分子等)导入有需要的受试者的体内或细胞中以达到治疗疾病的方法。所述核酸分子可以为DNA分子或RNA分子。所述DNA分子例如可以为编码酶(例如腺苷脱氨酶)的基因、能够杀死肿瘤细胞的分子(例如肿瘤坏死因子)的基因、功能蛋白的基因等。所述RNA分例如可以为反义寡核苷酸分子等。在本发明中，所述细胞透膜肽是指一类以非受体依赖方式，非经典内吞方式直接穿过细胞膜进入细胞的多肽，它们的长度一般不超过30个氨基酸且富含碱性氨基酸，氨基酸序列通常带正电荷，例如前面所列举的例子。
在本发明中，所述核酸包括DNA或RNA分子。所述核酸分子可以为载体、报告基因、效应基因(例如抗癌基因)或者具有其它功能的DNA或RNA分子。在本发明中，对核酸的长度没有特别要求，例如可以在几个碱基-几千个碱基的范围内。本领域技术人员可以根据核酸长度的大小调节核酸载体的用量，即当核酸的长度较小时，需要较少摩尔数的核酸载体，当核酸的长度较大时，需要较多摩尔数的核酸载体。
在本发明中，核酸载体和核酸分子的电荷比是通过计算核酸载体和核酸分子各自所带的电荷数计算出来的。其中，核酸载体所带的电荷数是指多肽携带的正电荷数，是指多肽序列中带正电荷的Lys和Arg的数量，定义为N，即一个Lys或一个Arg带一个正电荷；核酸分子所带的电荷数是指核酸碱基的磷酸基团数，定义为P，即一个磷酸基团带一个负电荷。
有益效果
本发明设计合成了一系列含有细胞透膜肽、富含组氨酸的内涵体逃逸结构、受体靶向结构以及任选的疏水性分子的多肽类核酸载体，有效解决了基因转运过程中的负 载、转运、细胞内内涵体逃逸等问题，实现了比商业化的Lipofectamine 2000更高的转染效率，同时具有更低的毒副作用。
附图说明
图1：多肽基因载体结构示意图。
图2：载体/DNA复合物琼脂糖凝胶电泳图，其中电泳图上方的数字例如0、1.0、1.5、2.0、2.5、3.0等分别表示载体与DNA的电荷比。
图2A为TAT-H6-K(C18)-YIGSR/DNA复合物；图2B为TAT-H6-K(C18)-YIGSK/DNA复合物；图2C为TAT-H6-K(C18)/DNA复合物；图2D为TAT-H6-K-YIGSR/DNA复合物；图2E为C18-TAT-H6-K-YIGSR/DNA复合物；图2F为H6-TAT-K(C18)-YIGSR/DNA复合物。
图3：载体/DNA复合物流体粒径和zeta电位图，其中横坐标为多肽与DNA的电荷比，纵坐标分别为颗粒粒径(A)和zeta电位(B)。
图4：多肽/DNA电荷比为6的复合物投射电镜图(图中的比例表示100nm)。图4A至图4F中的样品依次如下：
4A为TAT-H6-K(C18)-YIGSR/DNA复合物；
4B为TAT-H6-K(C18)-YIGSK/DNA复合物；
4C为TAT-H6-K(C18)/DNA复合物；
4D为TAT-H6-K-YIGSR/DNA复合物；
4E为C18-TAT-H6-K-YIGSR/DNA复合物；
4F为H6-TAT-K(C18)-YIGSR/DNA复合物。
图5：载体/DNA复合物体外转染效率图，其中横坐标为多肽与DNA的电荷比，纵坐标为荧光素酶活性(即RLU)。其中，
图5A为载体/DNA复合物在293T细胞的转染效率；图5B为载体/DNA复合物在B16F10细胞的转染效率。
图6：载体/DNA复合物的293T细胞毒性评价，其中横坐标为载体与DNA的电荷比，纵坐标为细胞存活率。
具体实施方式
下面将结合实施例对本发明的实施方案进行详细描述，但是本领域技术人员将会 理解，下列实施例仅用于说明本发明，而不应视为限定本发明的范围。实施例中未注明具体条件者，按照常规条件或制造商建议的条件进行。所用试剂或仪器未注明生产厂商者，均为可以通过市购获得的常规产品。
制备例所用固相合成载体Rink-酰胺树脂为天津南开合成责任有限公司产品HOBT、HBTU、DIEA以及Fmoc-保护的氨基酸由上海吉尔生化公司提供。
缩写词的含义
Arg       表示精氨酸
Ser       表示丝氨酸
Gln       表示谷氨酰胺
Gly       表示甘氨酸
Tyr       表示酪氨酸
Lys       表示赖氨酸
Ile       表示异亮氨酸
His       表示组氨酸
Fmoc      表示芴甲氧羰基
DMF      表示二甲基甲酰胺
HBTU   表示2-(1H-1-羟基苯并三唑)-1，1，3，3-四甲基脲六氟磷酸盐
HOBT     表示 1-羟基苯并三唑
DIEA     表示 N，N-二异丙基乙胺
TFA      表示三氟乙酸
RP-HPLC  表示反相高效液相色谱
TAE-buffer表示三羟甲基氨基甲烷-乙酸-乙二胺四乙酸缓冲液
其它未标示的缩写具有本领域公知的含义。
制备例1-6：不同核酸载体的制备和表征
(1)TAT-H6-K(C18)-YIGSR的合成
根据TAT-H6-K(C18)-YIGSR的氨基酸序列，合成Ac-Gly-Arg-Lys-Lys-Arg-Arg-Gln-Arg-Arg-Arg-His-His-His-His-His-His-Lys(C18)-Tyr- Ile-Gly-Ser-Arg-NH 2，以Rink-酰胺树脂为固相载体，以HBTU-HOBt为缩合剂，采取标准Fmoc策略(其中特殊氨基酸为Dde保护的赖氨酸)，利用微波多肽合成仪(CEM，USA)合成目标肽序列。以5ml 2％水合肼作为脱保护试剂，室温反应三分钟，重复三次。通过茚三酮反应检测脱保护是否成功。确认脱保护成功后，以DIC为缩合剂，投入硬脂酸的DMF溶液，室温反应4h以上。通过茚三酮反应检测硬脂酸连接是否成功。确认成功后，以20ml三氟乙酸∶苯甲硫醚∶间甲酚∶乙二硫醇∶水(8.25∶0.5∶0.5∶0.25∶0.5，体积比)作裂解液，0℃反应30分钟，室温反应150分钟，将肽脱除保护基并从树脂上裂解下来。溶液经RP-HPLC纯化，RP-HPLC条件，A相：0.05％TFA/水；B相：0.05％TFA/70％ACN/水；色谱柱：C8。MALDI-Tof-MS：3232.835。
(2)TAT-H6-K(C18)-YIGSK的合成
根据TAT-H6-K(C18)-YIGSK的氨基酸序列，合成Ac-Gly-Arg-Lys-Lys-Arg-Arg-Gln-Arg-Arg-Arg-His-His-His-His-His-His-Lys(C18)-Tyr-Ile-Gly-Ser-Lys-NH 2，以Rink-酰胺树脂为固相载体，以HBTU-HOBt为缩合剂，采取标准Fmoc策略(其中特殊氨基酸为Dde保护的赖氨酸)，利用微波多肽合成仪(CEM，USA)合成目标肽序列。以5ml 2％水合肼作为脱保护试剂，室温反应三分钟，重复三次。通过茚三酮反应检测脱保护是否成功。确认脱保护成功后，以DIC为缩合剂，投入硬脂酸的DMF溶液，室温反应4h以上。通过茚三酮反应检测硬脂酸连接是否成功。确认成功后，以20ml三氟乙酸∶苯甲硫醚∶间甲酚∶乙二硫醇∶水(8.25∶0.5∶0.5∶0.25∶0.5，体积比)作裂解液，0℃反应30分钟，室温反应150分钟，将肽脱除保护基并从树脂上裂解下来。溶液经RP-HPLC纯化，RP-HPLC条件，A相：0.05％TFA/水；B相：0.05％TFA/70％ACN/水；色谱柱：C8。MALDI-Tof-MS：3203.83。
(3)TAT-H6-K(C18)的合成
根据TAT-H6-K(C18)的氨基酸序列，合成Ac-Gly-Arg-Lys-Lys-Arg-Arg-Gln-Arg-Arg-Arg-His-His-His-His-His-His-Lys(C18)-NH 2(其中C18为硬脂酸)，以Rink-酰胺树脂为固相载体，以HBTU-HOBt为缩合剂，采取标准Fmoc策略(其中特殊氨基酸为Dde保护的赖氨酸)，利用微波多肽合成仪(CEM，USA)合成目标肽序列。以5ml 2％水合肼作为脱保护试剂，室温反应三分钟，重复三次。通过茚三酮反应检测脱保护是否成功。确认脱保护成功后，以DIC为缩合剂，投入硬脂酸的DMF溶液，室温反应4h以上。通过茚三酮反应检测硬脂酸连接是否成功。确认成功后，以20ml三氟乙酸∶苯甲硫醚∶间甲酚∶乙二硫醇∶水(8.25∶0.5∶0.5∶0.25∶0.5，体积比) 作裂解液，0℃反应30分钟，室温反应150分钟，将肽脱除保护基并从树脂上裂解下来。溶液经RP-HPLC纯化，RP-HPLC条件，A相：0.05％TFA/水；B相：0.05％TFA/70％ACN/水；色谱柱：C8。MALDI-Tof-MS：2655.2。
(4)TAT-H6-K-YIGSR的合成
根据TAT-H6-K-YIGSR的氨基酸序列，合成Ac-Gly-Arg-Lys-Lys-Arg-Arg-Gln-Arg-Arg-Arg-His-His-His-His-His-His-Lys-Tyr-Ile-Gly-Ser-Arg-NH 2，以Rink-酰胺树脂为固相载体，以HBTU-HOBt为缩合剂，采取标准Fmoc策略，利用微波多肽合成仪(CEM，USA)合成目标肽序列。以20ml三氟乙酸∶苯甲硫醚∶间甲酚∶乙二硫醇∶水(8.25∶0.5∶0.5∶0.25∶0.5，体积比)作裂解液，0℃反应30分钟，室温反应150分钟，将肽脱除保护基并从树脂上裂解下来。溶液经RP-HPLC纯化，RP-HPLC条件，A相：0.05％TFA/水；B相：0.05％TFA/70％ACN/水；色谱柱：C8。MALDI-Tof-MS：2965.36。
(5)C18-TAT-H6-K-YIGSR的合成
根据C18-TAT-H6-K-YIGSR的氨基酸序列，合成C18-Gly-Arg-Lys-Lys-Arg-Arg-Gln-Arg-Arg-Arg-His-His-His-His-His-His-Lys-Tyr-Ile-Arg-Ser-Arg-NH 2，以Rink-酰胺树脂为固相载体，以HBTU-HOBt为缩合剂，采取标准Fmoc策略，利用微波多肽合成仪(CEM，USA)合成目标肽序列。以DIC为缩合剂，投入硬脂酸DMF溶液，室温反应4h以上。通过茚三酮反应检测硬脂酸连接是否成功(硬脂酸连载肽链的N端裸露的氨基上)。以20ml三氟乙酸∶苯甲硫醚∶间甲酚∶乙二硫醇∶水(8.25∶0.5∶0.5∶0.25∶0.5，体积比)作裂解液，0℃反应30分钟，室温反应150分钟，将肽脱除保护基并从树脂上裂解下来。溶液经RP-HPLC纯化，RP-HPLC条件，A相：0.05％TFA/水；B相：0.05％TFA/70％ACN/水；色谱柱：C8。MALDI-Tof-MS：3189.81。
(6)H6-TAT-K(C18)-YIGSR的合成
根据H6-TAT-K(C18)-YIGSR的氨基酸序列，合成Ac-His-His-His-His-His-His-Gly-Arg-Lys-Lys-Arg-Arg-Gln-Arg-Arg-Arg-Lys(C18)-Tyr-Ile-Gly-Ser-Arg-NH 2，以Rink-酰胺树脂为固相载体，以HBTU-HOBt为缩合剂，采取标准Fmoc策略(其中特殊氨基酸为Dde保护的赖氨酸)，利用微波多肽合成仪(CEM，USA)合成目标肽序列。以5ml 2％水合肼作为脱保护试剂，室温反应三分钟，重复三次。通过茚三酮反应检测脱保护是否成功。确认脱保护成功后，以DIC为缩合剂，投入硬脂酸的DMF溶液，室温反应4h以上。通过茚三酮反应检测硬脂酸连接是否成功。 确认成功后，以20ml三氟乙酸∶苯甲硫醚∶间甲酚∶乙二硫醇∶水(8.25∶0.5∶0.5∶0.25∶0.5，体积比)作裂解液，0℃反应30分钟，室温反应150分钟，将肽脱除保护基并从树脂上裂解下来。溶液经RP-HPLC纯化，RP-HPLC条件，A相：0.05％TFA/水；B相：0.05％TFA/70％ACN/水；色谱柱：C8。MALDI-Tof-MS：3232.835。
备注：上面的制备例1-6中，氨基酸序列自左至右为从N末端至C末端。其中，对氨基酸序列N端的氨基进行了乙酰化修饰，对C端的羧基进行了酰胺化修饰。这是因为，化学合成的肽往往携带游离的氨基和游离的羧基，而肽的序列往往代表了母本蛋白的序列，为了与母本蛋白更为接近，肽末端往往进行封闭，即N端乙酰化和C端酰胺化，这些修饰会减少多肽的总电荷，降低多肽的溶解度，也可以使肽模拟它在母本蛋白中α氨基和羧基的原始状态。因此氨基酸序列依然是左端为N末端，右端为C末端。这些修饰不会对多肽的生物学活性产生显著的影响。
以上制备的核酸载体应用于下面的制备例7-12。
制备例7-12：不同核酸载体/DNA复合物的制备
(1)TAT-H6-K(C18)-YIGSR/DNA复合物的制备
取PGL-3质粒DNA(购自Promega公司)1μg，稀释到25μl，TAT-H6-K(C18)-YIGSR配成1mg/ml的溶液，分别按照peptide/DNA电荷比(N/P)0、1、2、2.5、3、3.5、4、6、8吸取不同体积的肽溶液，稀释到25μl，将肽和DNA溶液混合，涡旋10s，在37℃孵育30min，形成不同电荷比的TAT-H6-K(C18)-YIGSR/DNA复合物。
(2)TAT-H6-K(C18)-YIGSK/DNA复合物的制备
取PGL-3质粒DNA，1μg，稀释到25μl，TAT-H6-K(C18)-YIGSK配成1mg/ml的溶液，分别按照peptide/DNA电荷比(N/P)0、1、2、2.5、3、3.5、4、6、8吸取不同体积的肽溶液，稀释到25μl，将肽和DNA溶液混合，涡旋10s，在37℃孵育30min，形成不同电荷比的TAT-H6-K(C18)-YIGSK复合物。
(3)TAT-H6-K(C18)/DNA复合物的制备
取PGL-3质粒DNA1μg，稀释到25μl，TAT-H6-K(C18)配成1mg/ml的溶液，按照peptide/DNA电荷比(N/P)0、1、2、2.5、3、3.5、4、6、8吸取不同体积的肽溶液，稀释到25μl，将肽和DNA溶液混合，涡旋10s，在37℃孵育30min，形成不同电荷比的TAT-H6-K(C18)/DNA复合物。
(4)TAT-H6-K-YIGSR/DNA复合物的制备
取PGL-3质粒DNA1μg，稀释到25μl，TAT-H6-K-YIGSR配成1mg/ml的溶液，分别按照peptide/DNA电荷比(N/P)0、1、2、2.5、3、3.5、4、6、8吸取不同体积的肽溶液，稀释到25μl，将肽和DNA溶液混合，涡旋10s，在37℃孵育30min，形成不同电荷比的TAT-H6-K-YIGSR/DNA复合物。
(5)C18-TAT-H6-K-YIGSR/DNA复合物的制备
取PGL-3质粒DNA(购自Promega公司)1μg，稀释到25μl，C18-TAT-H6-K-YIGSR配成1mg/ml的溶液，分别按照peptide/DNA电荷比(N/P)0、2、3、4、5、6、7、8吸取不同体积的肽溶液，稀释到25μl，将肽和DNA溶液混合，涡旋10s，在37℃孵育30min，形成不同电荷比的C18-TAT-H6-K-YIGSR/DNA复合物。
(6)H6-TAT-K(C18)-YIGSR/DNA复合物的制备
取PGL-3质粒DNA(购自Promega公司)1μg，稀释到25μl，TAT-H6-K(C18)-YIGSR配成1mg/ml的溶液，分别按照peptide/DNA电荷比(N/P)0、2、3、4、5、6、7、8吸取不同体积的肽溶液，稀释到25μl，将肽和DNA溶液混合，涡旋10s，在37℃孵育30min，形成不同电荷比的H6-TAT-K(C18)-YIGSR/DNA复合物。
如果没有特别说明，下面的实验例1-5所用的核酸载体/DNA复合物为参照制备例7-12所制备；其中，具体的电荷比亦可适当调整。
实验例1：核酸载体负载能力的测定
为了评价核酸载体1-6载体负载DNA的能力，本发明研究了核酸载体/DNA复合物的琼脂糖凝胶电泳。
配制质量/体积比为1％的琼脂糖/TAE缓冲溶液，在微波炉中加热溶解，倒入电泳槽模具中冷却30min，放入电泳槽中，加入10×TAE缓冲液。取按照上述方法制备的核酸载体/DNA复合物，其中DNA为0.1μg，体积10μl上样，电压100v，60min。在EB染色液中染色15min，然后在凝胶成像仪下拍照，观察电泳中DNA迁移条带。
结果如图2所示。
图2A显示，TAT-H6-K(C18)-YIGSR/DNA复合物电荷比在3时可以完全负载DNA(多肽完全负载DNA形成纳米复合物无法进入琼脂糖凝胶，因此没有迁移条带；而未负载的DNA进入琼脂糖凝胶，经过EB染色在紫外灯下呈现明亮的DNA条带)。
图2B显示，TAT-H6-K(C18)-YIGSK/DNA复合物电荷比在3.5时可以完全负载DNA，说明序列中YIGSR变为YIGSK对核酸载体负载DNA的影响很小。
图2C显示，TAT-H6-K(C18)/DNA复合物电荷比在3.0时可以完全负载DNA。说明靶向序列YIGSR的存在对复合物负载DNA能力影响很小。
图2D显示，TAT-H6-K-YIGSR/DNA复合物电荷比在2.5时可以完全负载DNA。
图2E显示，C18-TAT-H6-K-YIGSR/DNA复合物电荷比在7时可以完全负载DNA。透膜肽放置于肽链中间会影响碱性氨基酸的正电荷与核酸的负电荷结合，从而影响载体负载DNA的能力。
图2F显示，H6-TAT-K(C18)-YIGSR/DNA复合物电荷比在6时可以完全负载DNA。透膜肽放置于肽链中间会影响碱性氨基酸的正电荷与核酸的负电荷结合，从而影响载体负载DNA的能力。
实验例2：核酸载体DNA复合物的粒径和zeta电位分析
粒径和zeta电位检测采用Zetasizer Nano ZS90，Malvern激光粒度仪，检测温度25℃，每个样品重复三次，检测结果如图3所示。
结果显示：
TAT-H6-K(C18)-YIGSR/DNA复合物的粒径随着电荷比增大，先由114.51nm升至211nm，然后降至126.53nm。zeta电位随着电荷比增加由-8.08mv升至15.57mv。
TAT-H6-K(C18)-YIGSK/DNA复合物的粒径随着电荷比增大，先由144.97nm升至161.63nm，然后降至105.94nm，从结果可以看出靶向序列由YIGSR变为YIGSK对复合物粒径的影响并不大。同时，zeta电位也无明显变化，zeta电位随着正负电荷比增加由-11.68mv升至14.63mv。
TAT-H6-K(C18)/DNA复合物的粒径随着电荷比增大，先由133.23nm升至441.7nm，然后降至76.24nm。zeta电位随着正负电荷比增加由-8.24mv升至14.9mv。
TAT-H6-K-YIGSR/DNA复合物的粒径随着电荷比增大，先由589.86nm升至657.63nm，然后降至140.43nm。zeta电位随着正负电荷比增加由5.74mv升至14mv。
C18-TAT-H6-K-YIGSR/DNA复合物的粒径随着电荷比增大，先由439.4升至540.33nm，然后降至107nm。zeta电位随着正负电荷比增加由5.56mv升至15.86mv。
H6-TAT-K(C18)-YIGSR/DNA复合物的粒径随着电荷比增大，先由454.9降至622nm，然后稍微升至86.48nm。zeta电位随着正负电荷比增加由3.71mv升至15.3mv。
实验例3：核酸载体/DNA复合物的透射电镜表征
测试的样品为多肽/DNA电荷比为6的复合物。TAT-H6-K(C18)-YIGSR/DNA为例，将制备好的TAT-H6-K(C18)-YIGSR/DNA复合物溶液5μl滴加在铜网上，30min后用滤纸吸干，在Hitachi H-7650显微镜下拍照。类似地，对其余5种复合物进行显微拍照。
结果如图4所示。
如图4A所示，TAT-H6-K(C18)-YIGSR/DNA复合物为约55nm的纳米颗粒，这一尺度比DLS(动态光散射)所测的结果较小，是因为测试透射电镜时的复合物为晾干后的样品，比其在水溶液中测试的流体粒径尺度偏小。
如图4B所示，TAT-H6-K(C18)-YIGSK/DNA复合物为约55nm的纳米颗粒，与TAT-H6-K(C18)-YIGSR/DNA复合物的纳米颗粒尺度相当。同样，这一尺度比DLS所测的结果较小，是因为测试透射电镜时的复合物为晾干后的样品，比其在水溶液中测试的流体粒径尺度偏小。
如图4C所示，TAT-H6-K(C18)/DNA复合物为约65nm的纳米颗粒，比TAT-H6-K(C18)/DNA复合物纳米颗粒更大，可见在多肽中引入靶向基团YIGSR，促进双亲性α-螺旋构象形成有利于和DNA形成更加致密的纳米颗粒。同样，这一尺度比DLS所测的结果较小，是因为测试透射电镜时的复合物为晾干后的样品，比其在水溶液中测试的流体粒径尺度偏小。
如图4D所示，TAT-H6-K-YIGSR/DNA复合物为约78nm的纳米颗粒，比TAT-H6-K(C18)-YIGSR/DNA复合物纳米颗粒更大，可见在多肽中引入疏水性基团，更有利于促进双亲性α-螺旋构象形成，并且和DNA形成更加致密的纳米颗粒。这一尺度比DLS所测的结果较小，是因为测试透射电镜时的复合物为晾干后的样品，比其在水溶液中测试的流体粒径尺度偏小。
如图4E所示，C18-TAT-H6-K-YIGSR/DNA复合物为约45nm的纳米颗粒，这一尺度比DLS(动态光散射)所测的结果较小，是因为测试透射电镜时的复合物为晾干后的样品，比其在水溶液中测试的流体粒径尺度偏小。
如图4F所示，H6-TAT-K(C18)-YIGSR/DNA复合物为约35nm的纳米颗粒，这一尺度比DLS(动态光散射)所测的结果较小，是因为测试透射电镜时的复合物为晾干后的样品，比其在水溶液中测试的流体粒径尺度偏小。
实验例4：转染效率测定
(1)TAT-H6-K(C18)-YIGSR/DNA复合物的转染效率
本发明的peptide/DNA复合物的细胞转染效率在两种细胞系293T和B16F10细胞(购自协和细胞库，即中国医学科学院基础医学研究所细胞资源中心)中评价。
将293T和B16F10细胞分别接种于48孔板中，每孔接种3×10 4(500μl DMEM细胞悬液)个细胞，24小时后细胞汇合度达到80％，吸出培养基。将TAT-H6-K(C18)-YIGSR/DNA复合物(每孔PGL-3质粒DNA 0.6μg，电荷比(N/P)分别为4、6、8)用DMEM培养基稀释到300μl，分别加入到细胞培养板，在37℃，5％CO 2的细胞培养箱中孵育4小时。4小时后吸出培养基，加入300μl含10％ FBS的DMEM培养基再培养44小时，然后检测细胞内荧光素酶表达。测试采用Promega公司的荧光素酶检测试剂盒，利用
Figure PCTCN2018088859-appb-000001
M5(Molecular Devices，WI，USA)酶标仪采集荧光信号值采集时间为500ms。统计结果绘图，数据如图5A、图5B中所示，实验数据每组重复三次，以商用脂质体Lipofectamine 2000作为阳性对照。两种细胞的荧光素酶表达量在电荷比为6∶1时达到最大，荧光素酶活性(RLU)分别为4.30×10 6(293T细胞)和2.76×10 5(B16F10细胞)。TAT-H6-K(C18)-YIGSR将透膜功能片段、内涵题逃逸功能片段、疏水功能片段和受体靶向功能片段合理组合在一起，形成的核酸载体转染效率达到阳性对照Lipo2000的5-10倍。
(2)TAT-H6-K(C18)-YIGSK/DNA复合物的转染效率
将293T和B16F10细胞分别接种于48孔板中，每孔接种3×10 4(500μl DMEM细胞悬液)个细胞，24小时后细胞汇合度达到80％，吸出培养基。将TAT-H6-K(C18)-YIGSK/DNA复合物(每孔PGL-3质粒DNA 0.6μg，电荷比(N/P)4-8)用DMEM培养基稀释到300μl，分别加入到细胞培养板，在37℃，5％CO 2的细胞培养箱中孵育4小时。4小时后吸出培养基，加入300μl含10％FBS的DMEM培养基再培养44小时，然后检测细胞内荧光素酶表达。测试采用Promega公司的荧光素酶检测试剂盒，利用
Figure PCTCN2018088859-appb-000002
M5(Molecular Devices，WI，USA)酶标仪采集荧光信号值采集时间为500ms。统计结果绘图，数据如图5A、图5B中所示，实验数据每组重复三次，以商用脂质体Lipofectamine 2000作为阳性对照。两种细胞的荧光素酶表达量在电荷比为6∶1时达到最大，荧光素酶活性(RLU)分别为4.19×10 6(293T细胞)和1.80×10 5(B16F10细胞)。荧光素酶的表达效率比本实验例(1)中有了少许降低，YIGSR是提高转染效率的关键。TAT-H6-K(C18)-YIGSK相对于TAT-H6-K(C18)-YIGSR，受体靶向序列中的精氨酸替换成结构极为相似的赖氨酸。TAT-H6-K(C18)-YIGSK将透膜功能片段、内涵题逃逸功能片段、疏水功能片段和受体靶向功能片段衍生片段合理组合在一起，形成的核酸载体转染效率同样达到阳性对照Lipo2000的5-10倍。
(3)TAT-H6-K(C18)/DNA复合物的转染效率
将293T和B16F10细胞分别接种于48孔板中，每孔接种3×10 4(500μl DMEM细胞悬液)个细胞，24小时后细胞汇合度达到80％，吸出培养基。将TAT-H6-K(C18)/DNA复合物(每孔PGL-3质粒DNA 0.6μg，电荷比(N/P)4-8)用DMEM培养基稀释到300μl，分别加入到细胞培养板，在37℃，5％CO 2的细胞培养箱中孵育4小时。4小时后吸出培养基，加入300μl含10％FBS的DMEM培养基再培养44小时，然后检测细胞内荧光素酶表达。测试采用Promega公司的荧光素酶检测试剂盒，利用
Figure PCTCN2018088859-appb-000003
M5(Molecular Devices，WI，USA)酶标仪采集荧光信号值采集时间为500ms。统计结果绘图，数据如图5A、图5B中所示，实验数据每组重复三次，以商用脂质体Lipofectamine 2000作为阳性对照。两种细胞的荧光素酶表达量在电荷比为6∶1时达到最大，荧光素酶活性(RLU)分别为6.70×10 4(293T细胞)和2.33×10 4(B16F10细胞)。TAT-H6-K(C18)将透膜功能片段、内涵题逃逸功能片段、疏水功能片段合理组合在一起，形成的核酸载体转染效率相对于TAT-H6-K(C18)-YIGSR转染效率大大降低，说明受体靶向片段的存在能够极大提高转染效率。
(4)TAT-H6-K-YIGSR/DNA复合物的转染效率
将293T和B16F10细胞分别接种于48孔板中，每孔接种3×10 4(500μl DMEM细胞悬液)个细胞，24小时后细胞汇合度达到80％，吸出培养基。将TAT-H6-K-YIGSR/DNA复合物(每孔PGL-3质粒DNA 0.6μg，电荷比(N/P)4-8)用DMEM培养基稀释到300μl，分别加入到细胞培养板，在37℃，5％CO 2的细胞培养箱中孵育4小时。4小时后吸出培养基，加入300μl含10％ FBS的DMEM培养基再培养44小时，然后检测细胞内荧光素酶表达。测试采用Promega公司的荧光素酶检测试剂盒，利用
Figure PCTCN2018088859-appb-000004
M5(Molecular Devices，WI，USA)酶标仪采集荧光信号值采集时间为500ms。统计结果绘图，数据如图5A、图5B中所示，实验数据每组重复三次，以商用脂质体Lipofectamine 2000作为阳性对照。两种细胞的荧光素酶表达量在电荷比为6∶1时达到最大，荧光素酶活性(RLU)分别为7649(293T细胞)和19503(B16F10细胞)。TAT-H6-K-YIGSR将透膜功能片段、内涵题逃逸功能片段、受体靶向功能片段合理组合在一起，形成的核酸载体转染效率很低。不拘于理论的限制，疏水性基团能够提高水溶性多肽与磷脂双层膜融合的能力，促进细胞的摄取以及内涵体逃逸，因此在基因转染过程中发挥着重要作用。
(5)C18-TAT-H6-K-YIGSR/DNA复合物的转染效率
本发明的peptide/DNA复合物的细胞转染效率在两种细胞系293T和B16F10细胞(购 自协和细胞库，即中国医学科学院基础医学研究所细胞资源中心)中评价。
将293T和B16F10细胞分别接种于48孔板中，每孔接种3×10 4(500μl DMEM细胞悬液)个细胞，24小时后细胞汇合度达到80％，吸出培养基。将C18-TAT-H6-K-YIGSR/DNA复合物(每孔PGL-3质粒DNA 0.6μg，电荷比(N/P)4-8)用DMEM培养基稀释到300μl，分别加入到细胞培养板，在37℃，5％CO 2的细胞培养箱中孵育4小时。4小时后吸出培养基，加入300μl含10％FBS的DMEM培养基再培养44小时，然后检测细胞内荧光素酶表达。测试采用Promega公司的荧光素酶检测试剂盒，利用
Figure PCTCN2018088859-appb-000005
M5(Molecular Devices，WI，USA)酶标仪采集荧光信号值采集时间为500ms。统计结果绘图，数据如图5A、图5B中所示，实验数据每组重复三次，以商用脂质体Lipofectamine 2000作为阳性对照。两种细胞的荧光素酶表达量在电荷比为6∶1时达到最大，荧光素酶活性(RLU)分别为5047(293T细胞)和387(B16F10细胞)。C18-TAT-H6-K-YIGSR将透膜功能片段、内涵题逃逸功能片段、疏水功能片段和受体靶向功能片段组合在一起，但是透膜肽位于肽链中间影响其负载DNA能力，形成的核酸载体转染效率很低。
(6)H6-TAT-K(C18)-YIGSR/DNA复合物的转染效率
本发明的peptide/DNA复合物的细胞转染效率在两种细胞系293T和B16F10细胞(购自协和细胞库，即中国医学科学院基础医学研究所细胞资源中心)中评价。
将293T和B16F10细胞分别接种于48孔板中，每孔接种3×10 4(500μl DMEM细胞悬液)个细胞，24小时后细胞汇合度达到80％，吸出培养基。将H6-TAT-K(C18)-YIGSR/DNA复合物(每孔PGL-3质粒DNA 0.6μg，电荷比(N/P)4-8)用DMEM培养基稀释到300μl，分别加入到细胞培养板，在37℃，5％CO 2的细胞培养箱中孵育4小时。4小时后吸出培养基，加入300μl含10％FBS的DMEM培养基再培养44小时，然后检测细胞内荧光素酶表达。测试采用Promega公司的荧光素酶检测试剂盒，利用
Figure PCTCN2018088859-appb-000006
M5(Molecular Devices，WI，USA)酶标仪采集荧光信号值采集时间为500ms。统计结果绘图，数据如图5A、图5B中所示，实验数据每组重复三次，以商用脂质体Lipofectamine 2000作为阳性对照。两种细胞的荧光素酶表达量在电荷比为6∶1时达到最大，荧光素酶活性(RLU)分别为27720(293T细胞)和117(B16F10细胞)。C18-TAT-H6-K-YIGSR将透膜功能片段、内涵题逃逸功能片段、疏水功能片段和受体靶向功能片段组合在一起，但是透膜肽位于肽链中间影响其负载DNA能力，形成的核酸载体转染效率很低。
综合上述实验结果可见，在本发明，所合成的除了TAT-H 6-K(C 18)-YIGSR和TAT-H 6-K(C 18)-YIGSK以外的多肽，即使含有YIGSR片段，转染DNA的效率也相对 很低，例如TAT-H 6-K-YIGSR和H 6-TAT-K(C 18)-YIGSR等。这些结果说明，即使含有可以和受体结合的YIGSR片段，如果缺乏其他功能片段或者改变功能片段序列，基因递送效率仍旧不够理想。因此，这些载体的高转染效率不仅仅是一个功能片段的作用，更主要的是各个功能片段有机结合，发挥协同作用的结果。
实验例5：核酸载体DNA复合物的细胞毒性评价
本发明的细胞毒性评价实验在293T细胞中评价，实验采用Promega公司的CellTiter
Figure PCTCN2018088859-appb-000007
AQueous One Solution reagent试剂盒分别评价了核酸载体/DNA复合物在固定DNA用量(2μg/μl)，不同电荷比(N/P＝4、6、8)的细胞毒性。
将293T细胞接种于96孔板中，每孔1×10 4个细胞，在37℃，5％CO 2条件下培养24小时后，细胞汇合度达到70％-80％。吸出培养基，加入按制备例7-12中所得TAT-H6-K(C18)-YIGSR/DNA复合物100μl，孵育4个小时。4小时后吸出培养基，换成新鲜的含10％FBS的DMEM，再培养20小时，然后按照CCK方法检测细胞存活率。
结果如图6所示。
TAT-H6-K(C18)-YIGSR/DNA复合物：在不同的条件下，细胞的存活率都达到了90％以上，可见本发明的TAT-H6-K(C18)-YIGSR/DNA复合物具有较低的细胞毒性。与阳性对照Lipo2000 80％的细胞毒性相比，本发明的核酸载体具有明显的优势。
TAT-H6-K(C18)-YIGSK/DNA复合物：在不同的条件下，细胞的存活率都达到了90％以上，可见本发明的TAT-H6-K(C18)-YIGSK/DNA复合物具有较低的细胞毒性。与阳性对照Lipo2000 80％的细胞毒性相比，本发明的核酸载体具有明显的优势。
TAT-H6-K(C18)/DNA复合物：在不同的条件下，细胞的存活率都达到了85％以上，可见本发明的TAT-H6-K(C18)/DNA复合物具有较低的细胞毒性。
TAT-H6-K-YIGSR/DNA复合物：在不同的条件下，细胞的存活率都达到了90％以上，可见本发明的TAT-H6-K-YIGSR/DNA复合物具有较低的细胞毒性。
C18-TAT-H6-K-YIGSR/DNA复合物：在不同的条件下，细胞的存活率都达到了75％以上，可见本发明的C18-TAT-H6-K-YIGSR/DNA相对于复合物TAT-H6-K(C18)-YIGSR具有较高的细胞毒性。
H6-TAT-K(C18)-YIGSR/DNA复合物：在不同的条件下，细胞的存活率都达到了70％以上，可见本发明的H6-TAT-K(C18)-YIGSR/DNA复合物相对于复合物TAT-H6-K(C18)-YIGSR具有较高的细胞毒性。
尽管本发明的具体实施方式已经得到详细的描述，本领域技术人员将会理解。根据已经公开的所有教导，可以对那些细节进行各种修改和替换，这些改变均在本发明的保护范围之内。本发明的全部范围由所附权利要求及其任何等同物给出。

Claims (15)

  1. 式I所示的化合物，或其药学上可接受的盐，
    A-B-C-D
    式I
    其中，
    A表示细胞透膜肽，
    B表示内涵体逃逸片段，
    C表示一个或多个Lys，和/或一个或多个Arg，或者C缺失，
    D表示层粘连蛋白受体靶向片段，并且
    A和B之间，B和C之间，和/或C和D之间，为直接连接或者通过连接接头连接；
    优选地，式I化合物还连接有一个或多个疏水分子E，并且所述疏水分子E连接在所述式I化合物中的Lys和/或Arg的侧链的氨基上。
  2. 根据权利要求1所述的式I化合物或其药学上可接受的盐，其特征在于如下的(1)-(7)项中的任意一项或者多项：
    (1)所述细胞透膜肽选自TAT、R n(n＝6、7、8、9或10)、penetratin、MAP、pVEC、MPG、MPGANLS、Stearyl-R8、EB1和Tat-DRBD中的任意一种或者多种；
    (2)所述内涵体逃逸片段为质子泵内涵体逃逸片段，优选地，为一个或多个His，例如4-8个His，优选为6个His；
    (3)所述层粘连蛋白受体靶向片段为如SEQ ID NO：17或SEQ ID NO：18所示；
    (4)所述疏水分子选自碳原子数大于或等于12的脂肪酸(例如硬脂酸、棕榈酸、油酸、亚油酸或软脂酸)、胆固醇和磷脂；
    (5)所述疏水分子连接在C中的Lys和/或Arg的侧链的氨基上；
    (6)所述连接接头为一个或多个Gly、一个或多个Lys或者一个或多个Cys；
    (7)式I化合物的氨基酸序列的N末端乙酰化和/或C末端酰胺化。
  3. 根据权利要求1或2所述的式I化合物或其药学上可接受的盐，其中，所述化合物如SEQ ID NO：1、SEQ ID NO：2或SEQ ID NO：4所示。
  4. 一种复合物，其包含权利要求1至3中任一权利要求所述的式I化合物或其药学上可接受的盐，以及核酸分子。
  5. 根据权利要求4所述的复合物，其中，所述式I化合物或其药学上可接受的盐与核酸分子的电荷比为(1-10)∶1，例如为(2-8)∶1、1∶1、1.5∶1、2∶1、2.5∶1、3∶1、3.5∶1、4∶1、4.5∶1、5∶1、6∶1、7∶1或8∶1。
  6. 一种宿主细胞，其含有权利要求1至3中任一权利要求所述的式I化合物或其可药用盐或者权利要求4或5所述的复合物。
  7. 一种药物组合物，其含有权利要求1至3中任一权利要求所述的式I化合物或其可药用盐或者权利要求4或5所述的复合物，以及至少一种药学上可接受的辅料。
  8. 一种试剂盒，其含有权利要求1至3中任一权利要求所述的式I化合物或其可药用盐或者权利要求4或5所述的复合物，以及目标核酸。
  9. SEQ ID NO：17或SEQ ID NO：18所示的多肽在制备负载和/或转运核酸的药物或试剂中的用途。
  10. 权利要求1至3中任一权利要求所述的式I化合物或其可药用盐在制备用于负载和/或转运核酸的药物或试剂或者基因治疗药物中的用途。
  11. 一种负载和/或转运核酸的方法，或者一种制备权利要求4或5所述的复合物的方法，包括使权利要求1至3中任一权利要求所述的式I化合物或其可药用盐与核酸分子(例如DNA或RNA)接触的步骤；
    优选地，所述化合物或其药学上可接受的盐与核酸分子的电荷比为(1-10)∶1，例如为(2-8)∶1、1∶1、1.5∶1、2∶1、2.5∶1、3∶1、3.5∶1、4∶1、4.5∶1、5∶1、6∶1、7∶1或8∶1；
    优选地，所述接触的条件为，36℃-38℃(例如37℃)条件下孵育20min以上(例如30min)；
    优选地，在孵育之前，将式I化合物或其可药用盐与核酸分子进行混合；优选地，进行涡旋或搅拌。
  12. 一种转染真核细胞的方法，包括将目标核酸分子负载至权利要求1至3中任一权利要求所述的式I化合物或其可药用盐的步骤。
  13. SEQ ID NO：17或SEQ ID NO：18所示的多肽，其用于制备负载和/或转运核酸的药物或试剂。
  14. 根据权利要求1至3中任一权利要求所述的式I化合物或其可药用盐，其用于负载和/或转运核酸，或者用于基因治疗。
  15. 一种基因治疗方法，包括给予有需要的受试者以有效量的权利要求1至3中任一权利要求所述的式I化合物或其可药用盐或者权利要求4或5所述的复合物的步骤。
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Publication number Priority date Publication date Assignee Title
CN101899091A (zh) * 2009-05-31 2010-12-01 首都医科大学 脂肪烷基五肽缀合物及其制备方法和在医学中的应用
CN101906140A (zh) * 2009-06-02 2010-12-08 首都医科大学 脂肪链和yigsr五肽缀合物及其合成方法和应用
CN105463002A (zh) * 2014-08-08 2016-04-06 中国人民解放军军事医学科学院毒物药物研究所 多肽类核酸载体、其制备方法及用途

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* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
CN101899091A (zh) * 2009-05-31 2010-12-01 首都医科大学 脂肪烷基五肽缀合物及其制备方法和在医学中的应用
CN101906140A (zh) * 2009-06-02 2010-12-08 首都医科大学 脂肪链和yigsr五肽缀合物及其合成方法和应用
CN105463002A (zh) * 2014-08-08 2016-04-06 中国人民解放军军事医学科学院毒物药物研究所 多肽类核酸载体、其制备方法及用途

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