CN109134659B

CN109134659B - Nucleic acid vector and application thereof

Info

Publication number: CN109134659B
Application number: CN201710452216.5A
Authority: CN
Inventors: 孟庆斌; 康子瑶; 孟昭; 孙超; 杨森; 刘克良
Original assignee: Institute of Pharmacology and Toxicology of AMMS
Current assignee: Institute of Pharmacology and Toxicology of AMMS
Priority date: 2017-06-15
Filing date: 2017-06-15
Publication date: 2022-04-26
Anticipated expiration: 2037-06-15
Also published as: CN109134659A; WO2018228178A1

Abstract

The invention belongs to the field of biomedicine, and relates to a nucleic acid vector and application thereof. The invention also relates to a compound containing the nucleic acid carrier and the nucleic acid molecule, and a preparation method and application of the nucleic acid carrier/nucleic acid molecule compound. Specifically, the invention relates to a nucleic acid vector which is a compound shown as a formula I or a pharmaceutically acceptable salt thereof. The nucleic acid vector has high transfection efficiency, has the advantages of simple preparation method, low cytotoxicity and the like, and provides an effective technical means for gene therapy. A-B-C-D is of formula I.

Description

Nucleic acid vector and application thereof

Technical Field

The invention belongs to the field of biomedicine, and relates to a nucleic acid vector and application thereof. The invention also relates to a compound containing the nucleic acid carrier and the nucleic acid molecule, and a preparation method and application of the nucleic acid carrier/nucleic acid molecule compound.

Background

The U.S. Food and Drug Administration (FDA) defines gene therapy as a therapeutic approach for treating diseases by transcription or translation, transport and/or integration of foreign genes into the host genome. Gene therapy is potentially the most effective therapeutic approach for the treatment of cancer, monogenic diseases, cardiovascular diseases and neurological diseases, among others. Since the first entry of gene therapy into clinical trials in 1990, over 1800 clinical trials of gene therapy have been conducted worldwide. China is the first country to introduce gene medicine (Gendicine) in the market in 2004. Because nucleic acid drug molecules for gene therapy have the characteristics of large molecular weight, abundant negative charges, high degradability and the like, the biggest problem of the current gene therapy is lack of an ideal transfer vector, and therefore, the search for an efficient and safe gene transfer vector becomes a key point of research.

The clinical tests carried out at present mostly adopt adenovirus and retrovirus as carriers of gene drugs, but the virus carriers have the defects of low transport capacity, high use cost, potential safety problems and the like. In 1994, gene therapy studies were once stuck because of death of patients caused by the use of viral gene vectors in clinical trials. Thus, non-viral vectors have been rapidly studied in recent years, including liposomes, cationic polymers, polypeptides, and the like. Most of non-viral vector transported gene drugs load DNA or RNA through ionic interaction between cations of the non-viral vector and anions of the gene drugs to form nanoparticles with smaller scale, and the transported DNA or RNA enters host cells and is further integrated into host genomes for expression. The polypeptide becomes a better choice of gene therapy vectors due to the characteristics of better biocompatibility, functional diversity, easy synthesis and the like.

Cell-Penetrating Peptides (CPPs) are short Peptides capable of efficiently mediating biomacromolecules such as nucleic acids and proteins to penetrate Cell membranes to enter cells, and generally, the length of a peptide sequence does not exceed 30 amino acids, and most of the Cell-Penetrating Peptides are rich in basic amino acids such as lysine and arginine. Wherein TAT (49-57) (HIV TAT protein) is the smallest fragment of a peptide fragment of a cell membrane-penetrating protein that can fully exert a cell membrane-penetrating function without cytotoxicity, and the peptide sequence of TAT (49-57) is Arg-Lys-Lys-Arg-Arg-Gln-Arg-Arg (RKKRRQRRR, SEQ ID NO: 7). The membrane-penetrating peptide can be combined with negative charges of nucleic acid through positive charges of basic amino acids of the membrane-penetrating peptide under physiological conditions, the loaded nucleic acid further mediates the nucleic acid to enter cells through endocytosis, and the TAT peptide fragment has certain cell nucleus targeting capacity. However, TAT/DNA or RNA complexes that enter cells by endocytosis first enter the endosome (a lipid membrane structure with an acidic environment inside) and are eventually transported to lysosomes for degradation. Arginine-rich membrane-penetrating peptide sequence R_n(n-6-10) also has similar properties to TAT. ByTAT and Rn lack the ability to escape endosomes, and thus it is difficult to achieve the desired transfection efficiency as a vector for delivering DNA alone.

The method for realizing endosome escape mainly comprises two methods, one method is to utilize a material with an ion buffering effect in an acidic environment (pH is less than 6.0) to destroy the endosome through the action of a proton pump so as to enable a DNA or RNA complex to escape; the other is to use polypeptide with alpha-spiral amphipathic structure to react with lipid membrane to form cavity and release DNA or RNA complex. Histidine can continuously absorb protons in acidic endosomes, and the endosomes are broken through the proton sponge effect, so that the effect of destroying endosome lipid vesicles is achieved.

Laminin (LN) is a macromolecular collagen glycoprotein. With the intensive research on LN, it was found that LN has various biological effects: promote cell adhesion, spreading, mitosis, accelerate neurite growth and cell migration, and induce cell differentiation, and are also associated with morphogenesis and tumor metastasis. Laminin receptors (LN-R) are widely present on the surface of epithelial cells, endothelial cells, peripheral nerve cells, macrophages, and most tumor cells, and exhibit high affinity, competitive, concentration-and time-dependent in vitro binding to LN. When malignant tumor is transferred, LN-R and LN on the cell surface are adhered as a key step of interaction between tumor cells and a basement membrane, and a molecular basis is provided for tumor adhesion and invasion to break through the basement membrane; meanwhile, LN-R can not only promote the adhesion of cells on the LN matrix, but also induce the chemotactic migration of cells and the secretion of collagenase IV; LN-R and LN in the early stages were also found to be critical for tumor neovascularization.

At present, there is a need for the development of novel nucleic acid vectors having high transfection efficiency and low cytotoxicity.

Disclosure of Invention

The present inventors have made intensive studies and creative efforts to obtain a nucleic acid vector, i.e., a compound of formula I. The present inventors have surprisingly found that the nucleic acid vector has a higher transfection efficiency and is less cytotoxic. In addition, the nucleic acid vector can well realize endosome escape. The following invention is thus provided:

one aspect of the present invention relates to compounds of formula I, or a pharmaceutically acceptable salt thereof,

A–B–C–D

formula I

Wherein the content of the first and second substances,

a represents a cell-permeable membrane peptide,

b represents an endosome escape fragment,

c represents one or more Lys, and/or one or more Arg, or a C deletion,

d represents a laminin receptor targeting fragment, and

a and B, B and C, and/or C and D are directly connected or connected through a connecting joint;

preferably, the compound of formula I is further attached to one or more hydrophobic molecules E, and said hydrophobic molecules E are attached to the amino groups of the side chains of Lys and/or Arg in said compound of formula I.

The direct linkage may be, for example, formation of an amide bond 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.

The connecting 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. The connecting joints between A and B, between B and C, and between C and D may be the same or different. The linking linker to A, B, C or D is also attached through a carboxyl and amino group to form an amide linkage.

In one embodiment of the invention, the compound of formula I, or a pharmaceutically acceptable salt thereof, is characterized by any one or more of the following items (1) - (5):

(1) the cell permeable peptide is selected from TAT and R_n(n ═ 6, 7, 8, 9 or 10), pendatin, MAP, pVEC, MPG Δ NLS, Stearyl-R8, EB1 and Tat-DRBD;

(2) the endosome escape fragment is a proton pump endosome escape fragment, preferably, one or more His, e.g., 4-10, 4-8, 4-6, 4, 5, 6, 7, or 8 His; preferably 6 His;

(3) the laminin receptor targeting fragment is YIGSR (SEQ ID NO: 17) or YIGSK (SEQ ID NO: 18);

(4) the hydrophobic molecule is selected from fatty acids having a number of carbon atoms greater than or equal to 12 (e.g., stearic acid, palmitic acid, oleic acid, linoleic acid, palmitic acid), cholesterol, and phospholipids;

(5) the hydrophobic molecule is attached to the amino group of the side chain of Lys and/or Arg in C.

In one embodiment of the invention, the compound of formula I or a pharmaceutically acceptable salt thereof is shown as SEQ ID NO 1, SEQ ID NO 2 or SEQ ID NO 4.

In one embodiment of the invention, the compound of formula I or a pharmaceutically acceptable salt thereof, wherein the N-terminal of the amino acid sequence is acetylated and/or C-terminal amidated.

A compound of formula I according to any one of the invention or a pharmaceutically acceptable salt thereof or a complex of the invention for use in gene therapy.

In the invention, the pKa of the imidazolyl group in histidine is about 6.0, and the imidazolyl group can absorb a large amount of protons in acidic endosomes to form a 'proton sponge' effect, so that the endosomes are cracked.

Without being limited by theory, by connecting hydrophobic molecules, a hydrophobic environment is provided, the full contact between hydrophilic amino acid with positive charges and nucleic acid medicine with negative charges is facilitated, and then a stable and compact nano compound is formed, the uptake of cells is facilitated, the local concentration of aggregates can be improved through hydrophobic interaction, and then the local charge density is improved, the loading capacity of DNA or RNA is improved, the stability of a nucleic acid carrier/DNA or RNA complex is improved, and meanwhile, the membrane penetration efficiency and the capacity of damaging endosomes are effectively improved through the fusion effect of hydrophobic alkyl chains and cell membranes.

In one embodiment of the present invention, the sequence of the nucleic acid vector is Gly-Arg-Lys-Arg-Arg-Gln-Arg-Arg-Arg-His-His-His-His-His-Lys (C18) -Tyr-Ile-Gly-Ser-Arg (SEQ ID NO: 1).

In one embodiment of the present invention, the sequence of the nucleic acid vector is Gly-Arg-Lys-Lys-Arg-Arg-Gln-Arg-Arg-Arg-His-His-His-His-His-Lys (C18) -Tyr-Ile-Gly-Ser-Lys (SEQ ID NO: 2).

In one embodiment of the present invention, the sequence of the nucleic acid vector is Gly-Arg-Lys-Arg-Arg-Gln-Arg-Arg-Arg-His-His-His-His-His-Lys (C18) (SEQ ID NO: 3).

In one embodiment of the present invention, the sequence of the nucleic acid vector is Gly-Arg-Lys-Arg-Arg-Gln-Arg-Arg-Arg-His-His-His-His-His-Lys-Tyr-Ile-Gly-Ser-Arg (SEQ ID NO: 4).

In one embodiment of the present invention, the sequence of the nucleic acid vector is C18-Gly-Arg-Lys-Lys-Arg-Arg-Gln-Arg-Arg-His-His-His-His-His-Lys-Tyr-Ile-Gly-Ser-Arg (SEQ ID NO: 5).

In one embodiment of the present invention, the sequence of the nucleic acid vector is 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).

In the present invention, the C18 means stearic acid (bonded to the amino group of the side chain of Arg or Lys through the carboxyl group of stearic acid) if not specifically mentioned.

In one embodiment of the invention, the cell-penetrating peptide is TAT. In a specific embodiment of the invention, 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 (GRKKRRQRRR, SEQ ID NO: 8).

In one embodiment of the invention, wherein said cell-penetrating peptide is selected from TAT, R_n(n-6-10) (indicating a peptide consisting of 6-10 arginines), pentatin (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-NH₂，SEQ ID NO：13)、Stearyl-R8(st-RRRRRRRR-NH₂，SEQ ID NO：14)、EB1(LIKLWSHLIHIWFQNRRLKWKKK-NH₂SEQ ID NO: 15) and Tat-DRBD (GRKKRRQRRRPQ-DRBD, SEQ ID NO: 16).

In the present invention, the phospholipids mainly include two main classes of glycerophospholipids and sphingomyelin, wherein the glycerophospholipids can be further classified into several classes, such as phosphatidylcholine, phosphatidylethanolamine, phosphatidylserine, phosphatidylglycerol, diphosphatidylglycerol, phosphatidylinositol, and the like, and each of the classes can be further classified into several classes according to the constituent fatty acids.

Another aspect of the invention relates to a complex comprising a compound of formula I as described in any one of the invention, or a pharmaceutically acceptable salt thereof, and a nucleic acid molecule, such as DNA or RNA.

In one embodiment of the invention, 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.

in the invention, the nucleic acid carrier has positive charges, the nucleic acid molecule has negative charges, and the nucleic acid carrier and the nucleic acid molecule form a complex through the attraction between the positive charges and the negative charges.

A further aspect of the invention relates to a host cell comprising a compound of formula I as described in any one of the invention or a pharmaceutically acceptable salt thereof or a complex of the invention.

In the present invention, the host cell may be a prokaryotic cell such as an escherichia coli cell or a eukaryotic cell such as a yeast cell or a mammalian cell such as a cell derived from human, mouse, rat, monkey, etc. In an embodiment of the invention, the mammalian cell is a 293T cell or a B16F10 cell.

Yet another aspect of the present invention relates to a method of transfecting a eukaryotic cell comprising the step of loading a target nucleic acid molecule (e.g. DNA or RNA) to a compound of formula I as described in any one of the present invention or a pharmaceutically acceptable salt thereof.

In the present invention, a method of introducing the nucleic acid vector or complex into a cell to obtain a host cell is well known in the art, and for example, the nucleic acid vector or complex can be introduced into a cell by incubating the cell with it.

A further aspect of the present invention relates to a pharmaceutical composition comprising a compound of formula I as described in any of the present invention or a pharmaceutically acceptable salt thereof or a complex of the present invention, together with 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.

In the present invention, the composition or kit is used for loading or transporting nucleic acid (e.g., DNA or RNA) molecules into cells, and thus it may contain a corresponding buffer, a detection reagent, or a pharmaceutically acceptable adjuvant, etc.

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 reagent for loading and/or transporting a nucleic acid, such as DNA or RNA.

A further aspect of the invention relates to the use of a compound of formula I according to any one of the invention or a pharmaceutically acceptable salt thereof for the preparation of a medicament or agent for loading and/or transporting nucleic acids (e.g. DNA or RNA) or a medicament for gene therapy.

Yet another aspect of the invention relates to a method of loading and/or transporting a nucleic acid (e.g., DNA or RNA), or a method of making a complex of the invention, comprising the step of contacting a compound of formula I, or a pharmaceutically acceptable salt thereof, as described in any one of the invention with a nucleic acid molecule (e.g., DNA or RNA);

preferably, the charge ratio of the compound or pharmaceutically acceptable salt thereof to the nucleic acid molecule is (1-10): 1, e.g. (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;

preferably, the contacting is carried out under conditions of 36-38 ℃ (e.g., 37 ℃) for 20min or more (e.g., 30 min);

preferably, 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 performed.

Yet another aspect of the present invention relates to a method of gene therapy comprising the step of administering to a subject in need thereof an effective amount of a compound of formula I as described in any one of the present invention or a pharmaceutically acceptable salt thereof or a complex of the present invention.

In the present invention, the loading of a nucleic acid molecule (e.g., DNA or RNA) refers to the binding of a nucleic acid vector to a nucleic acid (DNA or RNA) molecule.

In the present invention, the transfer of a nucleic acid (DNA or RNA) molecule refers to the transfer or introduction of a nucleic acid molecule into a cell.

In the present invention, the subject is a mammal, e.g., bovine, equine, ovine, porcine, canine, feline, rodent, primate; wherein the preferred subject is a human.

In the present invention, when used for gene therapy of mammals, the nucleic acid vector of the present invention or the complex of the present invention can be introduced into the body of the mammal or a tissue or organ to be treated by various routes, for example, by intravenous injection, local injection, etc.

In the present invention, the gene therapy refers to a method of introducing an exogenous nucleic acid molecule (e.g., a normal gene, a functional nucleic acid molecule, etc.) into the body or cells of a subject in need thereof to achieve a treatment of a disease. The nucleic acid molecule may be a DNA molecule or an RNA molecule. The DNA molecule may be, for example, a gene encoding an enzyme (e.g., adenosine deaminase), a gene encoding a molecule capable of killing tumor cells (e.g., tumor necrosis factor), a gene encoding a functional protein, or the like. The RNA component may be, for example, an antisense oligonucleotide molecule or the like. In the present invention, the cell membrane penetrating peptide refers to a kind of polypeptide which directly penetrates cell membrane into cell in a receptor independent mode, not a classical endocytosis mode, and the length of the polypeptide generally does not exceed 30 amino acids and is rich in basic amino acids, and the amino acid sequence is generally positively charged, such as the examples listed above.

In the present invention, the nucleic acid includes a DNA or RNA molecule. The nucleic acid molecule may be a vector, a reporter gene, an effector gene (e.g., an anti-cancer gene), or a DNA or RNA molecule with other functions. In the present invention, the length of the nucleic acid is not particularly limited, and may be, for example, in the range of several bases to several kilobases. The amount of nucleic acid vector used can be adjusted by one skilled in the art according to the length of the nucleic acid, i.e., when the length of the nucleic acid is small, a smaller number of moles of nucleic acid vector is required, and when the length of the nucleic acid is large, a larger number of moles of nucleic acid vector is required.

In the present invention, the charge ratio of the nucleic acid vector and the nucleic acid molecule is calculated by counting the charges of the nucleic acid vector and the nucleic acid molecule, respectively. Wherein, the number of charges carried by the nucleic acid carrier refers to the number of positive charges carried by the polypeptide, refers to the number of Lys and Arg with positive charges in the polypeptide sequence, and is defined as N, namely one Lys or one Arg carries one positive charge; the number of charges carried by a nucleic acid molecule refers to the number of phosphate groups of a nucleic acid base, and is defined as P, i.e., one phosphate group carries one negative charge.

Advantageous effects

The invention designs and synthesizes a series of polypeptide nucleic acid vectors containing cell membrane penetrating peptide, an endosome escape structure rich in histidine, a receptor targeting structure and optional hydrophobic molecules, effectively solves the problems of loading, transferring, endosome escape in cells and the like in the gene transfer process, realizes higher transfection efficiency than commercial Lipofectamine 2000, and has lower toxic and side effects.

Drawings

FIG. 1: schematic structure of polypeptide gene vector.

FIG. 2: the agarose gel electrophoresis pattern of the vector/DNA complex, wherein the numbers above the electrophoresis pattern, such as 0, 1.0, 1.5, 2.0, 2.5, 3.0, etc., respectively, indicate the charge ratio of the vector to the DNA.

FIG. 2A is TAT-H₆-K(C₁₈) -YIGSR/DNA complex; FIG. 2B is TAT-H₆-K(C₁₈) -YIGSK/DNA complex; FIG. 2C is TAT-H₆-K(C₁₈)/DA NA complex; FIG. 2D is TAT-H₆-K-YIGSR/DNA complex; FIG. 2E is C₁₈-TAT-H₆-K-YIGSR/DNA complex; FIG. 2F is H₆-TAT-K(C₁₈) YIGSR/DNA complex.

FIG. 3: particle size of the carrier/DNA complex fluid and zeta potential diagram, where the abscissa is the charge ratio of polypeptide to DNA and the ordinate is the particle size (A) and zeta potential (B), respectively.

FIG. 4: a projection electron micrograph of the complexes with a polypeptide/DNA charge ratio of 6 (scale in the figure indicates 100 nm). The samples in fig. 4A to 4F are in the following order:

4A is TAT-H₆-K(C₁₈) -YIGSR/DNA complex;

4B is TAT-H₆-K(C₁₈) -YIGSK/DNA complex;

4C is TAT-H₆-K(C₁₈) a/DNA complex;

4D is TAT-H₆-K-YIGSR/DNA complex;

4E is C₁₈-TAT-H₆-K-YIGSR/DNA complex;

4F is H₆-TAT-K(C₁₈) YIGSR/DNA complex.

FIG. 5: vector/DNA complex in vitro transfection efficiency plots, with polypeptide to DNA charge ratio on the abscissa and luciferase activity (i.e., RLU) on the ordinate. Wherein the content of the first and second substances,

FIG. 5A shows the transfection efficiency of vector/DNA complexes in 293T cells; FIG. 5B shows the transfection efficiency of vector/DNA complexes in B16F10 cells.

FIG. 6: 293T cytotoxicity assessment of vector/DNA complexes, with carrier to DNA charge ratio on the abscissa and cell viability on the ordinate.

Detailed Description

Embodiments of the present invention will be described in detail below with reference to examples, but those skilled in the art will appreciate that the following examples are only illustrative of the present invention and should not be construed as limiting the scope of the present invention. The examples, in which specific conditions are not specified, were conducted under conventional conditions or conditions recommended by the manufacturer. The reagents or instruments used are not indicated by the manufacturer, and are all conventional products commercially available.

Preparation examples the solid-phase synthesis carrier Rink-amide resin used was HOBT, HBTU, DIEA, and Fmoc-protected amino acids, products of Tianjin Nankai Synthesis responsibility Ltd, supplied by Shanghai Jier Biochemical company.

Meaning of abbreviations

Arg represents arginine

Ser represents serine

Gln represents glutamine

Gly represents glycine

Tyr represents tyrosine

Lys represents lysine

Ile represents isoleucine

His represents histidine

Fmoc represents fluorenylmethyloxycarbonyl

DMF means dimethylformamide

HBTU represents 2- (1H-1-hydroxybenzotriazole) -1,1,3, 3-tetramethyluronium hexafluorophosphate

HOBT represents 1-hydroxybenzotriazole

DIEA means N, N-diisopropylethylamine

TFA represents trifluoroacetic acid

RP-HPLC stands for reversed phase high performance liquid chromatography

TAE-buffer means Tris-acetate-EDTA buffer

Other abbreviations not indicated have the meaning known in the art.

Preparation examples 1 to 6: preparation and characterization of different nucleic acid vectors

(1)TAT-H₆-K(C₁₈) Synthesis of-YIGSR

According to TAT-H₆-K(C₁₈) -amino acid sequence of YIGSR, synthesizing Ac-Gly-Arg-Lys-Lys-Arg-Arg-Gln-Arg-Arg-His-His-His-His-His-Lys (C18) -Tyr-Ile-Gly-Ser-Arg-NH₂Rink-amide resin is used as solid phase carrierThe target peptide sequence was synthesized using HBTU-HOBt as a condensing agent, using a standard Fmoc strategy (in which the specific amino acid was Dde-protected lysine), and using a microwave peptide synthesizer (CEM, USA). 5ml of 2% hydrazine hydrate was used as a deprotection reagent, and the reaction was repeated three times at room temperature for three minutes. The success of deprotection was checked by ninhydrin reaction. After confirming the success of deprotection, DIC was used as a condensing agent, and a solution of stearic acid in DMF was added thereto, followed by reaction at room temperature for 4 hours or more. The success of stearic acid attachment was checked by ninhydrin reaction. After confirming success, the reaction mixture was mixed with 20ml of trifluoroacetic acid: thioanisole: m-cresol: ethanedithiol: water (8.25: 0.5: 0.5: 0.25: 0.5, vol.) as a lysis buffer was reacted at 0 ℃ for 30 minutes and at room temperature for 150 minutes to deprotect the peptide and cleave it 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; a chromatographic column: C8. MALDI-Tof-MS: 3232.835.

(2)TAT-H₆-K(C₁₈) Synthesis of YIGSK

According to TAT-H₆-K(C₁₈) -amino acid sequence of YIGSK, synthesizing Ac-Gly-Arg-Lys-Lys-Arg-Arg-Gln-Arg-Arg-His-His-His-His-His-Lys (C18) -Tyr-Ile-Gly-Ser-Lys-NH₂The method comprises the steps of taking Rink-amide resin as a solid phase carrier, taking HBTU-HOBt as a condensing agent, adopting a standard Fmoc strategy (wherein special amino acid is Dde protected lysine), and synthesizing a target peptide sequence by using a microwave polypeptide synthesizer (CEM, USA). 5ml of 2% hydrazine hydrate was used as a deprotection reagent, and the reaction was repeated three times at room temperature for three minutes. The success of deprotection was checked by ninhydrin reaction. After confirming the success of deprotection, DIC was used as a condensing agent, and a solution of stearic acid in DMF was added thereto, followed by reaction at room temperature for 4 hours or more. The success of stearic acid attachment was checked by ninhydrin reaction. After confirming success, the reaction mixture was mixed with 20ml of trifluoroacetic acid: thioanisole: m-cresol: ethanedithiol: water (8.25: 0.5: 0.5: 0.25: 0.5, vol.) as a lysis buffer was reacted at 0 ℃ for 30 minutes and at room temperature for 150 minutes to deprotect the peptide and cleave it 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; a chromatographic column: C8. MALDI-Tof-MS: 3203.83.

(3)TAT-H₆-K(C₁₈) Synthesis of (2)

According to TAT-H₆-K(C₁₈) Synthesizing Ac-Gly-Arg-Lys-Lys-Arg-Arg-Gln-Arg-Arg-Arg-His-His-His-His-Lys (C18) -NH₂(wherein C18 is stearic acid), Rink-amide resin is used as a solid phase carrier, HBTU-HOBt is used as a condensing agent, a standard Fmoc strategy (wherein special amino acid is Dde protected lysine) is adopted, and a microwave polypeptide synthesizer (CEM, USA) is utilized to synthesize a target peptide sequence. 5ml of 2% hydrazine hydrate was used as a deprotection reagent, and the reaction was repeated three times at room temperature for three minutes. The success of deprotection was checked by ninhydrin reaction. After confirming the success of deprotection, DIC was used as a condensing agent, and a solution of stearic acid in DMF was added thereto, followed by reaction at room temperature for 4 hours or more. The success of stearic acid attachment was checked by ninhydrin reaction. After confirming success, the reaction mixture was mixed with 20ml of trifluoroacetic acid: thioanisole: m-cresol: ethanedithiol: water (8.25: 0.5: 0.5: 0.25: 0.5, vol.) as a lysis buffer was reacted at 0 ℃ for 30 minutes and at room temperature for 150 minutes to deprotect the peptide and cleave it 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; a chromatographic column: C8. MALDI-Tof-MS: 2655.2.

(4)TAT-H₆synthesis of (E) -K-YIGSR

According to TAT-H₆-K-YIGSR amino acid sequence, synthesizing Ac-Gly-Arg-Lys-Lys-Arg-Arg-Gln-Arg-Arg-Arg-His-His-His-His-His-Lys-Tyr-Ile-Gly-Ser-Arg-NH₂The target peptide sequence is synthesized by taking Rink-amide resin as a solid phase carrier, taking HBTU-HOBt as a condensing agent and adopting a standard Fmoc strategy and utilizing a microwave polypeptide synthesizer (CEM, USA). With 20ml of trifluoroacetic acid: thioanisole: m-cresol: ethanedithiol: water (8.25: 0.5: 0.5: 0.25: 0.5, vol.) as a lysis buffer was reacted at 0 ℃ for 30 minutes and at room temperature for 150 minutes to deprotect the peptide and cleave it 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; a chromatographic column: C8. MALDI-Tof-MS: 2965.36.

(5)C₁₈-TAT-H₆synthesis of (E) -K-YIGSR

According to C₁₈-TAT-H₆Amino acid sequence of-K-YIGSR, C18-Gly-Arg-Lys-Lys-Arg-Arg-Gln-Arg-Arg-Arg-His-His-His-His-His-Lys-Tyr-Ile-Arg-Ser-Arg-NH₂The target peptide sequence is synthesized by taking Rink-amide resin as a solid phase carrier, taking HBTU-HOBt as a condensing agent and adopting a standard Fmoc strategy and utilizing a microwave polypeptide synthesizer (CEM, USA). DIC is used as a condensing agent, stearic acid DMF solution is added, and reaction is carried out for more than 4 hours at room temperature. Whether the stearic acid connection is successful or not is detected by ninhydrin reaction (on the naked amino group at the N-terminal of the stearic acid peptide-linked chain). With 20ml of trifluoroacetic acid: thioanisole: m-cresol: ethanedithiol: water (8.25: 0.5: 0.5: 0.25: 0.5, vol.) as a lysis buffer was reacted at 0 ℃ for 30 minutes and at room temperature for 150 minutes to deprotect the peptide and cleave it 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; a chromatographic column: C8. MALDI-Tof-MS: 3189.81.

(6)H₆-TAT-K(C₁₈) Synthesis of-YIGSR

Synthesizing Ac-His-His-His-His-His-Gly-Arg-Lys-Lys-Arg-Arg-Gln-Arg-Arg-Arg-Lys (C18) -Tyr-Ile-Gly-Ser-Arg-NH according to the amino acid sequence of H6-TAT-K (C18) -YIGSR₂The method comprises the steps of taking Rink-amide resin as a solid phase carrier, taking HBTU-HOBt as a condensing agent, adopting a standard Fmoc strategy (wherein special amino acid is Dde protected lysine), and synthesizing a target peptide sequence by using a microwave polypeptide synthesizer (CEM, USA). 5ml of 2% hydrazine hydrate was used as a deprotection reagent, and the reaction was repeated three times at room temperature for three minutes. The success of deprotection was checked by ninhydrin reaction. After confirming the success of deprotection, DIC was used as a condensing agent, and a solution of stearic acid in DMF was added thereto, followed by reaction at room temperature for 4 hours or more. The success of stearic acid attachment was checked by ninhydrin reaction. After confirming success, the reaction mixture was mixed with 20ml of trifluoroacetic acid: thioanisole: m-cresol: ethanedithiol: water (8.25: 0.5: 0.5: 0.25: 0.5, vol.) as a lysis buffer was reacted at 0 ℃ for 30 minutes and at room temperature for 150 minutes to deprotect the peptide and cleave it 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; a chromatographic column: C8. MALDI-Tof-MS: 3232.835.

remarking: in the above preparation examples 1 to 6, the amino acid sequence was from the N-terminus to the C-terminus from left to right. Wherein, the amino at the N terminal of the amino acid sequence is acetylated and modified, and the carboxyl at the C terminal is amidated and modified. This is because chemically synthesized peptides tend to carry free amino and carboxyl groups, while the sequence of the peptide often represents the sequence of the parent protein, and in order to be closer to the parent protein, the peptide ends are often blocked, i.e., N-terminal acetylated and C-terminal amidated, which modifications reduce the overall charge of the polypeptide, decrease the solubility of the polypeptide, and also allow the peptide to mimic its original state of alpha amino and carboxyl groups in the parent protein. Thus, the amino acid sequence is still N-terminal at the left end and C-terminal at the right end. These modifications do not have a significant effect on the biological activity of the polypeptide.

The nucleic acid vectors prepared above were applied to the following preparation examples 7 to 12.

Preparation examples 7 to 12: preparation of different nucleic acid vector/DNA complexes

(1)TAT-H₆-K(C₁₈) Preparation of the-YIGSR/DNA Complex

Mu.g of PGL-3 plasmid DNA (purchased from Promega) was diluted to 25. mu.l of TAT-H₆-K(C₁₈) Preparing 1mg/ml solution from YIGSR, sucking peptide solutions with different volumes according to peptide/DNA charge ratio (N/P)0, 1, 2, 2.5, 3, 3.5, 4, 6 and 8, diluting to 25 μ l, mixing peptide and DNA solutions, vortexing for 10s, and incubating at 37 deg.C for 30min to form TAT-H with different charge ratios₆-K(C₁₈) YIGSR/DNA complex.

(2)TAT-H₆-K(C₁₈) Preparation of the YIGSK/DNA Complex

The PGL-3 plasmid DNA was diluted to 25. mu.l, TAT-H, at 1. mu.g₆-K(C₁₈) Preparing 1mg/ml solution from YIGSK, sucking peptide solutions with different volumes according to peptide/DNA charge ratio (N/P)0, 1, 2, 2.5, 3, 3.5, 4, 6 and 8, diluting to 25 μ l, mixing peptide and DNA solutions, vortexing for 10s, and incubating at 37 deg.C for 30min to form TAT-H with different charge ratios₆-K(C₁₈) -YIGSK complex.

(3)TAT-H₆-K(C₁₈) Preparation of DNA Complex

The PGL-3 plasmid DNA 1. mu.g was diluted to 25. mu.l, TAT-H₆-K(C₁₈) Preparing 1mg/ml solution, sucking peptide solution with different volumes according to peptide/DNA charge ratio (N/P)0, 1, 2, 2.5, 3, 3.5, 4, 6, 8, diluting to 25 μ l, mixing peptide and DNA solution, vortexing for 10s, and incubating at 37 deg.C for 30min to form TAT-H with different charge ratios₆-K(C₁₈) A DNA complex.

(4)TAT-H₆Preparation of the-K-YIGSR/DNA Complex

The PGL-3 plasmid DNA 1. mu.g was diluted to 25. mu.l, TAT-H₆Preparing 1mg/ml solution from-K-YIGSR, sucking peptide solutions with different volumes according to peptide/DNA charge ratio (N/P)0, 1, 2, 2.5, 3, 3.5, 4, 6, 8, diluting to 25 μ l, mixing peptide and DNA solution, vortexing for 10s, and incubating at 37 deg.C for 30min to form TAT-H with different charge ratios₆-K-YIGSR/DNA complex.

(5)C₁₈-TAT-H₆Preparation of the-K-YIGSR/DNA Complex

Mu.g of PGL-3 plasmid DNA (purchased from Promega corporation) was diluted to 25. mu.l, C₁₈-TAT-H₆Preparing 1mg/ml solution of-K-YIGSR, sucking peptide solutions with different volumes according to peptide/DNA charge ratio (N/P)0, 2, 3, 4, 5, 6, 7, 8, respectively, diluting to 25 μ l, mixing peptide and DNA solution, vortexing for 10s, incubating at 37 deg.C for 30min to form C with different charge ratio₁₈-TAT-H₆-K-YIGSR/DNA complex.

(6)H₆-TAT-K(C₁₈) Preparation of the-YIGSR/DNA Complex

Mu.g of PGL-3 plasmid DNA (purchased from Promega) was diluted to 25. mu.l of TAT-H₆-K(C₁₈) Preparing 1mg/ml solution from YIGSR, sucking peptide solutions with different volumes according to peptide/DNA charge ratio (N/P)0, 2, 3, 4, 5, 6, 7, 8, diluting to 25 μ l, mixing peptide and DNA solutions, vortexing for 10s, and incubating at 37 deg.C for 30min to form H solutions with different charge ratios₆-TAT-K(C₁₈) YIGSR/DNA complex.

If not otherwise specified, the nucleic acid vector/DNA complexes used in the following Experimental examples 1-5 were prepared for reference preparations 7-12; wherein the specific charge ratio can be adjusted appropriately.

Experimental example 1: determination of the Loading Capacity of nucleic acid vectors

In order to evaluate the DNA-carrying ability of the nucleic acid vectors 1 to 6, agarose gel electrophoresis of the nucleic acid vector/DNA complex was investigated.

Preparing agarose/TAE buffer solution with the mass/volume ratio of 1%, heating and dissolving in a microwave oven, pouring into an electrophoresis tank mould, cooling for 30min, placing into the electrophoresis tank, and adding 10 XTAE buffer solution. The nucleic acid vector/DNA complex prepared as described above was applied at a volume of 10. mu.l and a DNA content of 0.1. mu.g for 60min at a voltage of 100 v. Staining is carried out for 15min in EB staining solution, then photographing is carried out under a gel imager, and DNA migration bands in electrophoresis are observed.

The results are shown in FIG. 2.

FIG. 2A shows TAT-H₆-K(C₁₈) The YIGSR/DNA complex charge ratio can be fully loaded with DNA at 3 (the polypeptide is fully loaded with DNA to form a nano-complex which cannot enter the agarose gel and thus has no migration band; while the unloaded DNA entered an agarose gel, which was EB stained to present a bright DNA band under uv light).

FIG. 2B shows TAT-H₆-K(C₁₈) The YIGSK/DNA complex charge ratio can completely load DNA at 3.5, which shows that the change of YIGSR into YIGSK in the sequence has little influence on the loading of the DNA by the nucleic acid carrier.

FIG. 2C shows TAT-H₆-K(C₁₈) the/DNA complex charge ratio was 3.0 to fully load the DNA. The existence of the targeting sequence YIGSR has little influence on the capability of the complex to load DNA.

FIG. 2D shows TAT-H₆The charge ratio of the-K-YIGSR/DNA complex is 2.5, and the DNA can be completely loaded.

FIG. 2E shows that₁₈-TAT-H₆The charge ratio of the-K-YIGSR/DNA complex is 7, so that the DNA can be completely loaded. The membrane-penetrating peptide is placed in the middle of a peptide chain, so that the positive charge of basic amino acid is combined with the negative charge of nucleic acid, and the capability of a carrier for loading DNA is influenced.

FIG. 2F shows that H₆-TAT-K(C₁₈) The YIGSR/DNA complex charge ratio can be completely loaded with DNA at 6. The membrane-penetrating peptide is placed in the middle of a peptide chain, so that the positive charge of basic amino acid is combined with the negative charge of nucleic acid, and the capability of a carrier for loading DNA is influenced.

Experimental example 2: particle size and zeta potential analysis of nucleic acid Carrier DNA complexes

Particle size and zeta potential measurements were carried out using a Zetasizer Nano ZS90, Malvern laser particle sizer, at a detection temperature of 25 ℃ in triplicate for each sample, and the results are shown in FIG. 3.

The results show that:

TAT-H₆-K(C₁₈) The particle size of the YIGSR/DNA complex increases from 114.51nm to 211nm and then decreases to 126.53nm as the charge ratio increases. The zeta potential increases with increasing charge ratio from-8.08 mv to 15.57 mv.

TAT-H₆-K(C₁₈) The particle size of the YIGSK/DNA complex increases with the charge ratio, and increases from 144.97nm to 161.63nm and then decreases to 105.94nm, and the effect of changing the targeting sequence from YIGSR to YIGSK on the particle size of the complex is not great. Meanwhile, the zeta potential has no obvious change, and the zeta potential is increased from-11.68 mv to 14.63mv along with the increase of the positive-negative charge ratio.

TAT-H₆-K(C₁₈) The particle size of the/DNA complex increases with increasing charge ratio, from 133.23nm to 441.7nm and then decreases to 76.24 nm. The zeta potential increases from-8.24 mv to 14.9mv with increasing positive to negative charge ratio.

TAT-H₆The particle size of the-K-YIGSR/DNA complex increases from 589.86nm to 657.63nm and then decreases to 140.43nm as the charge ratio increases. The zeta potential increases from 5.74mv to 14mv with increasing positive-negative charge ratio.

C₁₈-TAT-H₆The particle size of the-K-YIGSR/DNA complex increases from 439.4 to 540.33nm and then decreases to 107nm as the charge ratio increases. The zeta potential increases from 5.56mv to 15.86mv with increasing positive-negative charge ratio.

H₆-TAT-K(C₁₈) The particle size of the YIGSR/DNA complex increases with charge ratio and starts at 454.9 dropped to 622nm and then slightly microlitres to 86.48 nm. The zeta potential increases from 3.71mv to 15.3mv with increasing positive to negative charge ratio.

Experimental example 3: transmission electron microscopy characterization of nucleic acid vector/DNA complexes

The sample tested was a complex with a polypeptide/DNA charge ratio of 6. TAT-H₆-K(C₁₈) For example, TAT-H is prepared by₆-K(C₁₈) Mu.l of the YIGSR/DNA complex solution was dropped on a copper mesh, and after 30min, it was blotted with filter paper and photographed under a Hitachi H-7650 microscope. Similarly, the remaining 5 complexes were photographed microscopically.

The results are shown in FIG. 4.

TAT-H as shown in FIG. 4A₆-K(C₁₈) The YIGSR/DNA complex is a nanoparticle of about 55nm, which is smaller than that measured by DLS (dynamic light scattering), because the complex is an air-dried sample when tested under a transmission electron microscope, which is smaller than the fluid particle size scale when tested in aqueous solution.

TAT-H as shown in FIG. 4B₆-K(C₁₈) -nanoparticles of YIGSK/DNA complex of about 55nm, with TAT-H₆-K(C₁₈) The nanoparticle size of the YIGSR/DNA complex is comparable. Again, this scale is smaller than that measured by DLS because the composites when tested under transmission electron microscopy are air-dried samples that are smaller than the fluid particle size scale they were tested in aqueous solution.

TAT-H as shown in FIG. 4C₆-K(C₁₈) the/DNA complex is about 65nm nanoparticle to TAT-H₆-K(C₁₈) The DNA compound nano-particle is larger, and a targeting group YIGSR is introduced into the polypeptide to promote the formation of amphiphilic alpha-helix conformation, so that the formation of the denser nano-particle with DNA is facilitated. Again, this scale is smaller than that measured by DLS because the composites when tested under transmission electron microscopy are air-dried samples that are smaller than the fluid particle size scale they were tested in aqueous solution.

TAT-H as shown in FIG. 4D₆Nanoparticles of the-K-YIGSR/DNA complex of about 78nm, larger than TAT-H6-K (C18) -YIGSR/DNA complex nanoparticles, and possibly larger than TAT-H6-K (C18) -YIGSR/DNA complex nanoparticlesThe hydrophobic groups are introduced into the polypeptide, so that the amphiphilic alpha-helix conformation is promoted to be formed, and the polypeptide and DNA form more compact nano-particles. This scale is smaller than that measured by DLS because the composites when tested by transmission electron microscopy are air-dried samples, smaller than the fluid particle size scale they were tested in aqueous solution.

As shown in FIG. 4E, C₁₈-TAT-H₆the-K-YIGSR/DNA complex is about 45nm nanoparticles, which is smaller than the result measured by DLS (dynamic light scattering), because the complex is an air-dried sample when tested by transmission electron microscopy, which is smaller than the particle size of the fluid when tested in aqueous solution.

As shown in FIG. 4F, H₆-TAT-K(C₁₈) The YIGSR/DNA complex is a nanoparticle of about 35nm, which is smaller than that measured by DLS (dynamic light scattering), because the complex is an air-dried sample when tested under a transmission electron microscope, which is smaller than the fluid particle size scale when tested in aqueous solution.

Experimental example 4: transfection efficiency assay

(1)TAT-H₆-K(C₁₈) Transfection efficiency of the YIGSR/DNA Complex

The cell transfection efficiency of the peptide/DNA complexes of the invention was evaluated in two cell lines 293T and B16F10 cells (purchased from cooperative cell banks, the cell resource center of the institute of basic medicine of the Chinese academy of medicine).

293T and B16F10 cells were seeded in 48-well plates, 3X 10 cells per well, respectively⁴(500. mu.l DMEM cell suspension) cells, after 24 hours the cell confluence reached 80%, and the medium was aspirated. Reacting TAT-H₆-K(C₁₈) YIGSR/DNA complexes (0.6. mu.g of PGL-3 plasmid DNA per well, charge ratio (N/P)4, 6, 8, respectively) diluted to 300. mu.l in DMEM medium, added to cell culture plates at 37 ℃ with 5% CO₂Was incubated for 4 hours in the cell incubator of (1). After 4 hours, the medium was aspirated, and 300. mu.l of DMEM medium containing 10% FBS was added thereto for another 44 hours, and then intracellular luciferase expression was measured. The test was carried out using luciferase assay kit available from Promega corporation

The fluorescence signal was collected with a microplate reader M5(Molecular Devices, Wis., USA) for 500 ms. Statistical results were plotted and the data are shown in fig. 5A, 5B, with three replicates per group of experimental data, with commercial liposome Lipofectamine 2000 as a positive control. The luciferase expression amounts of both cells reached maximum at a charge ratio of 6:1, and the luciferase activities (RLU) were 4.30X 10, respectively⁶(293T cells) and 2.76X 10⁵(B16F10 cells). TAT-H₆-K(C₁₈) The YIGSR reasonably combines the membrane-penetrating functional fragment, the endosymis escape functional fragment, the hydrophobic functional fragment and the receptor targeting functional fragment together, and the transfection efficiency of the formed nucleic acid vector reaches 5-10 times of that of positive control Lipo 2000.

(2)TAT-H₆-K(C₁₈) Transfection efficiency of YIGSK/DNA Complex

293T and B16F10 cells were seeded in 48-well plates, 3X 10 cells per well, respectively⁴(500. mu.l DMEM cell suspension) cells, after 24 hours the cell confluence reached 80%, and the medium was aspirated. Reacting TAT-H₆-K(C₁₈) YIGSK/DNA complex (PGL-3 plasmid DNA 0.6. mu.g per well, charge ratio (N/P)4-8) diluted to 300. mu.l with DMEM medium, added to the cell culture plate separately, at 37 ℃ with 5% CO₂Was incubated for 4 hours in the cell incubator of (1). After 4 hours, the medium was aspirated, and 300. mu.l of DMEM medium containing 10% FBS was added thereto for another 44 hours, and then intracellular luciferase expression was measured. The test was carried out using luciferase assay kit available from Promega corporation

The fluorescence signal was collected with a microplate reader M5(Molecular Devices, Wis., USA) for 500 ms. Statistical results were plotted and the data are shown in fig. 5A, 5B, with three replicates per group of experimental data, with commercial liposome Lipofectamine 2000 as a positive control. The luciferase expression amounts of both cells reached maximum at a charge ratio of 6:1, and the luciferase activities (RLU) were 4.19X 10, respectively⁶(293T cells) and 1.80X 10⁵(B16F10 cells). List of luciferasesThe efficiency of transfection was slightly lower than that in the experimental example (1), and YIGSR is the key to improve the transfection efficiency. TAT-H₆-K(C₁₈) YIGSK arginine in the receptor targeting sequence was replaced by a structurally very similar lysine relative to TAT-H6-K (C18) -YIGSR. TAT-H₆-K(C₁₈) The YIGSK reasonably combines the membrane-permeable functional fragment, the endosymis escape functional fragment, the hydrophobic functional fragment and the receptor targeting functional fragment derivative fragment together, and the transfection efficiency of the formed nucleic acid vector also reaches 5-10 times of that of positive control Lipo 2000.

(3)TAT-H₆-K(C₁₈) Transfection efficiency of DNA Complex

293T and B16F10 cells were seeded in 48-well plates, 3X 10 cells per well, respectively⁴(500. mu.l DMEM cell suspension) cells, after 24 hours the cell confluence reached 80%, and the medium was aspirated. Reacting TAT-H₆-K(C₁₈) the/DNA complexes (0.6. mu.g of plasmid DNA for PGL-3 per well, charge ratio (N/P)4-8) were diluted to 300. mu.l with DMEM medium, added to the cell culture plates, respectively, at 37 ℃ with 5% CO₂Was incubated for 4 hours in the cell incubator of (1). After 4 hours, the medium was aspirated, and 300. mu.l of DMEM medium containing 10% FBS was added thereto for another 44 hours, and then intracellular luciferase expression was measured. The test was carried out using luciferase assay kit available from Promega corporation

The fluorescence signal was collected with a microplate reader M5(Molecular Devices, Wis., USA) for 500 ms. Statistical results were plotted and the data are shown in fig. 5A, 5B, with three replicates per group of experimental data, with commercial liposome Lipofectamine 2000 as a positive control. The luciferase expression amounts of the two cells reached maximum at a charge ratio of 6:1, and the luciferase activities (RLU) were 6.70X 10, respectively⁴(293T cells) and 2.33X 10⁴(B16F10 cells). TAT-H₆-K(C₁₈) The membrane-permeable functional fragment, the endoscopical escape functional fragment and the hydrophobic functional fragment are reasonably combined together, so that the transfection efficiency of the formed nucleic acid vector is greatly reduced compared with the transfection efficiency of TAT-H6-K (C18) -YIGSR, and the transfection efficiency can be greatly improved due to the existence of the receptor targeting fragment.

(4)TAT-H₆Transfection efficiency of the-K-YIGSR/DNA Complex

293T and B16F10 cells were seeded in 48-well plates, 3X 10 cells per well, respectively⁴(500. mu.l DMEM cell suspension) cells, after 24 hours the cell confluence reached 80%, and the medium was aspirated. Reacting TAT-H₆the-K-YIGSR/DNA complex (0.6. mu.g of plasmid DNA PGL-3 per well, charge ratio (N/P)4-8) was diluted to 300. mu.l with DMEM medium, added to each cell culture plate, and incubated at 37 ℃ with 5% CO₂Was incubated for 4 hours in the cell incubator of (1). After 4 hours, the medium was aspirated, and 300. mu.l of DMEM medium containing 10% FBS was added thereto for another 44 hours, and then intracellular luciferase expression was measured. The test was carried out using luciferase assay kit available from Promega corporation

The fluorescence signal was collected with a microplate reader M5(Molecular Devices, Wis., USA) for 500 ms. Statistical results were plotted and the data are shown in fig. 5A, 5B, with three replicates per group of experimental data, with commercial liposome Lipofectamine 2000 as a positive control. The luciferase expression levels of both cells reached their maximum at a charge ratio of 6:1, with luciferase activities (RLU) of 7649(293T cells) and 19503(B16F10 cells), respectively. TAT-H₆The membrane-permeable functional fragment, the endosymis escape functional fragment and the receptor targeting functional fragment are reasonably combined together by the K-YIGSR, and the transfection efficiency of the formed nucleic acid vector is very low. Without being limited by theory, the hydrophobic group can improve the fusion capacity of the water-soluble polypeptide and the phospholipid bilayer membrane and promote the uptake of cells and the escape of endosomes, thereby playing an important role in the gene transfection process.

(5)C₁₈-TAT-H₆Transfection efficiency of the-K-YIGSR/DNA Complex

293T and B16F10 cells were seeded in 48-well plates, 3X 10 cells per well, respectively⁴(500 μ l DMEM cell suspension) cells, the cell confluence reached 80% after 24 hours, and the medium was aspirated. C is to be₁₈-TAT-H₆the-K-YIGSR/DNA complex (0.6. mu.g of plasmid DNA PGL-3 per well, charge ratio (N/P)4-8) was diluted to 300. mu.l with DMEM medium, added to each cell culture plate, and incubated at 37 ℃ with 5% CO₂Was incubated for 4 hours in the cell incubator of (1). After 4 hours, the medium was aspirated, and 300. mu.l of DMEM medium containing 10% FBS was added thereto for another 44 hours, and then intracellular luciferase expression was measured. The test was carried out using luciferase assay kit available from Promega corporation

The fluorescence signal was collected with a microplate reader M5(Molecular Devices, Wis., USA) for 500 ms. Statistical results were plotted and the data are shown in fig. 5A, 5B, with three replicates per group of experimental data, with commercial liposome Lipofectamine 2000 as a positive control. Luciferase expression was maximized at a charge ratio of 6:1 for both cells, with luciferase activities (RLU) of 5047(293T cells) and 387(B16F10 cells), respectively. C18-TAT-H6-K-YIGSR combines a membrane penetrating functional fragment, an endosymis escaping functional fragment, a hydrophobic functional fragment and a receptor targeting functional fragment together, but the membrane penetrating peptide is positioned in the middle of a peptide chain to influence the DNA loading capacity of the peptide chain, and the transfection efficiency of the formed nucleic acid vector is low.

(6)H₆-TAT-K(C₁₈) Transfection efficiency of the YIGSR/DNA Complex

293T and B16F10 cells were seeded in 48-well plates, 3X 10 cells per well, respectively⁴(500. mu.l DMEM cell suspension) cells, after 24 hours the cell confluence reached 80%, and the medium was aspirated. H is to be₆-TAT-K(C₁₈) YIGSR/DNA complex (PGL-3 plasmid DNA 0.6. mu.g per well, charge ratio (N/P)4-8) diluted to 300. mu.l with DMEM medium, added to the cell culture plate separately, at 37 ℃ with 5% CO₂Was incubated for 4 hours in the cell incubator of (1). 4 smallThe medium was aspirated at intervals, and 300. mu.l of DMEM medium containing 10% FBS was added thereto for further culture for 44 hours, followed by detection of luciferase expression in the cells. The test was carried out using luciferase assay kit available from Promega corporation

The fluorescence signal was collected with a microplate reader M5(Molecular Devices, Wis., USA) for 500 ms. Statistical results were plotted and the data are shown in fig. 5A, 5B, with three replicates per group of experimental data, with commercial liposome Lipofectamine 2000 as a positive control. Luciferase expression levels were maximized at a charge ratio of 6:1 for both cells, with luciferase activities (RLU) of 27720(293T cells) and 117(B16F10 cells), respectively. C18-TAT-H6-K-YIGSR combines a membrane penetrating functional fragment, an endosymis escaping functional fragment, a hydrophobic functional fragment and a receptor targeting functional fragment together, but the membrane penetrating peptide is positioned in the middle of a peptide chain to influence the DNA loading capacity of the peptide chain, and the transfection efficiency of the formed nucleic acid vector is low.

As can be seen from the above experimental results, in the present invention, compounds other than TAT-H are synthesized₆-K(C₁₈) -YIGSR and TAT-H₆-K(C₁₈) Polypeptides other than YIGSK, which transfect DNA with relatively low efficiency even when containing YIGSR fragments, e.g. TAT-H₆-K-YIGSR and H₆-TAT-K(C₁₈) YIGSR and the like. These results indicate that even if the YIGSR fragment capable of binding to the receptor is contained, the gene delivery efficiency is still not ideal if other functional fragments are absent or the sequence of the functional fragment is changed. Therefore, the high transfection efficiency of these vectors is not only a function of one functional fragment, but more importantly, a result of the synergistic effect of the organic combination of the functional fragments.

Experimental example 5: evaluation of cytotoxicity of nucleic acid vector DNA complexes

The cytotoxicity evaluation test of the present invention was evaluated in 293T cells using CellTiter from Promega corporation

AQueous One Solution reagent kit respectively evaluatesCytotoxicity of nucleic acid vector/DNA complex at fixed DNA doses (2 μ g/μ l) with different charge ratios (N/P ═ 4, 6, 8).

293T cells were seeded in 96-well plates at 1X 10 per well⁴Individual cells, 5% CO at 37 ℃₂After culturing for 24 hours under the condition, the cell confluency reaches 70-80%. The medium was aspirated, and 100. mu.l of TAT-H6-K (C18) -YIGSR/DNA complex obtained in preparation examples 7 to 12 was added thereto, followed by incubation for 4 hours. After 4 hours, the medium was aspirated, replaced with fresh DMEM containing 10% FBS, and cultured for another 20 hours, and then the cell viability was measured according to the CCK method.

The results are shown in FIG. 6.

TAT-H₆-K(C₁₈) -YIGSR/DNA complex: under different conditions, the survival rate of the cells reaches more than 90 percent, and the TAT-H of the invention₆-K(C₁₈) The YIGSR/DNA complex has low cytotoxicity. Compared with the cytotoxicity of positive control Lipo 200080%, the nucleic acid vector has obvious advantages.

TAT-H₆-K(C₁₈) -YIGSK/DNA complex: under different conditions, the survival rate of the cells reaches more than 90 percent, and the TAT-H of the invention₆-K(C₁₈) The YIGSK/DNA complex has low cytotoxicity. Compared with the cytotoxicity of positive control Lipo 200080%, the nucleic acid vector has obvious advantages.

TAT-H₆-K(C₁₈) The DNA complex: under different conditions, the survival rate of the cells reaches more than 85 percent, so that the TAT-H of the invention₆-K(C₁₈) the/DNA complex has low cytotoxicity.

TAT-H₆-K-YIGSR/DNA complex: under different conditions, the survival rate of the cells reaches more than 90 percent, and the TAT-H of the invention₆the-K-YIGSR/DNA complex has low cytotoxicity.

C₁₈-TAT-H₆-K-YIGSR/DNA complex: under different conditions, the survival rate of the cells reaches over 75 percent, and the C of the invention can be seen₁₈-TAT-H₆-K-YIGSR/DNA vs. complexCompound TAT-H₆-K(C₁₈) YIGSR has high cytotoxicity.

H₆-TAT-K(C₁₈) -YIGSR/DNA complex: under different conditions, the survival rate of the cells reaches more than 70 percent, and the H of the invention is shown₆-TAT-K(C₁₈) YIGSR/DNA Complex vs. Complex TAT-H₆-K(C₁₈) YIGSR has high cytotoxicity.

Although specific embodiments of the invention have been described in detail, those skilled in the art will appreciate. Various modifications and substitutions of those details may be made in light of the overall teachings of the disclosure, and such changes are intended to be within the scope of the present invention. The full scope of the invention is given by the appended claims and any equivalents thereof.

SEQUENCE LISTING

<110> institute of poison drug of military medical science institute of liberty military of China

<120> a nucleic acid vector and use thereof

<130> IDC170077

<160> 18

<170> PatentIn version 3.2

<210> 1

<211> 22

<212> PRT

<213> Artificial

<220>

<223> nucleic acid vector 1

<220>

<221> MOD_RES

<222> (17)..(17)

<223> formation of amide bond between side chain amino group of lysine at position 17 and carboxyl group of stearic acid

<400> 1

Gly Arg Lys Lys Arg Arg Gln Arg Arg Arg His His His His His His

1 5 10 15

Lys Tyr Ile Gly Ser Arg

20

<210> 2

<211> 22

<212> PRT

<213> Artificial

<220>

<223> nucleic acid vector 2

<220>

<221> MOD_RES

<222> (17)..(17)

<400> 2

Gly Arg Lys Lys Arg Arg Gln Arg Arg Arg His His His His His His

1 5 10 15

Lys Tyr Ile Gly Ser Lys

20

<210> 3

<211> 17

<212> PRT

<213> Artificial

<220>

<223> nucleic acid vector 3

<220>

<221> MOD_RES

<222> (17)..(17)

<400> 3

Gly Arg Lys Lys Arg Arg Gln Arg Arg Arg His His His His His His

1 5 10 15

Lys

<210> 4

<211> 22

<212> PRT

<213> Artificial

<220>

<223> nucleic acid vector 4

<400> 4

Gly Arg Lys Lys Arg Arg Gln Arg Arg Arg His His His His His His

1 5 10 15

Lys Tyr Ile Gly Ser Arg

20

<210> 5

<211> 22

<212> PRT

<213> Artificial

<220>

<223> nucleic acid vector 5

<220>

<221> MOD_RES

<222> (1)..(1)

<223> amide bond formation between free amino group of first glycine and stearic acid

<400> 5

Gly Arg Lys Lys Arg Arg Gln Arg Arg Arg His His His His His His

1 5 10 15

Lys Tyr Ile Gly Ser Arg

20

<210> 6

<211> 22

<212> PRT

<213> Artificial

<220>

<223> nucleic acid vector 6

<220>

<221> MOD_RES

<222> (17)..(17)

<223> formation of an amide bond between the amino group of the side chain of lysine at position 17 and the carboxyl group of stearic acid

<400> 6

His His His His His His Gly Arg Lys Lys Arg Arg Gln Arg Arg Arg

1 5 10 15

Lys Tyr Ile Gly Ser Arg

20

<210> 7

<211> 9

<212> PRT

<213> Artificial

<220>

<223> cell membrane penetrating peptide TAT

<400> 7

Arg Lys Lys Arg Arg Gln Arg Arg Arg

1 5

<210> 8

<211> 10

<212> PRT

<213> Artificial

<220>

<223> Another sequence of cell membrane penetrating peptide TAT

<400> 8

Gly Arg Lys Lys Arg Arg Gln Arg Arg Arg

1 5 10

<210> 9

<211> 16

<212> PRT

<213> Artificial

<220>

<223> cell membrane-permeable peptide penetratin

<400> 9

Arg Gln Ile Lys Ile Trp Phe Gln Asn Arg Arg Met Lys Trp Lys Lys

1 5 10 15

<210> 10

<211> 18

<212> PRT

<213> Artificial

<220>

<223> cell membrane penetrating peptide MAP

<400> 10

Lys Leu Ala Leu Lys Leu Ala Leu Lys Ala Leu Lys Ala Ala Leu Lys

1 5 10 15

Leu Ala

<210> 11

<211> 18

<212> PRT

<213> Artificial

<220>

<223> cell membrane penetrating peptide pVEC

<400> 11

Leu Leu Ile Ile Leu Arg Arg Arg Ile Arg Lys Gln Ala His Ala His

1 5 10 15

Ser Lys

<210> 12

<211> 27

<212> PRT

<213> Artificial

<220>

<223> cell membrane penetrating peptide MPG

<220>

<221> MOD_RES

<222> (1)..(1)

<223> blocking of the Nitrogen terminus by a carboxyl group

<220>

<221> MOD_RES

<222> (27)..(27)

<223> carboxyl group at carbon terminal is blocked

<400> 12

Gly Ala Leu Phe Leu Gly Phe Leu Gly Ala Ala Gly Ser Thr Met Gly

1 5 10 15

Ala Trp Ser Gln Pro Lys Lys Lys Arg Lys Val

20 25

<210> 13

<211> 27

<212> PRT

<213> Artificial

<220>

<223> transmembrane peptide MPG Delta NLS

<220>

<221> MOD_RES

<222> (1)..(1)

<223> blocking of the Nitrogen terminus by a carboxyl group

<220>

<221> MOD_RES

<222> (27)..(27)

<223> carbon terminal blocked by amino group

<400> 13

Gly Ala Leu Phe Leu Gly Phe Leu Gly Ala Ala Gly Ser Thr Met Gly

1 5 10 15

Ala Trp Ser Gln Pro Lys Ser Lys Arg Lys Val

20 25

<210> 14

<211> 8

<212> PRT

<213> Artificial

<220>

<223> Stearyl-R8

<220>

<221> MOD_RES

<222> (1)..(1)

<223> Nitrogen terminal blocked by stearoyl group

<220>

<221> MOD_RES

<222> (8)..(8)

<223> carbon terminal blocked by amino group

<400> 14

Arg Arg Arg Arg Arg Arg Arg Arg

1 5

<210> 15

<211> 23

<212> PRT

<213> Artificial

<220>

<223> cell membrane penetrating peptide EB1

<220>

<221> MOD_RES

<222> (23)..(23)

<223> carbon terminal blocked by amino group

<400> 15

Leu Ile Lys Leu Trp Ser His Leu Ile His Ile Trp Phe Gln Asn Arg

1 5 10 15

Arg Leu Lys Trp Lys Lys Lys

20

<210> 16

<211> 16

<212> PRT

<213> Artificial

<220>

<223> cell membrane penetrating peptide Tat-DRBD

<400> 16

Gly Arg Lys Lys Arg Arg Gln Arg Arg Arg Pro Gln Asp Arg Asx Asp

1 5 10 15

<210> 17

<211> 5

<212> PRT

<213> Artificial

<220>

<223> laminin receptor targeting fragment 1

<400> 17

Tyr Ile Gly Ser Arg

1 5

<210> 18

<211> 5

<212> PRT

<213> Artificial

<220>

<223> laminin receptor targeting fragment 2

<400> 18

Tyr Ile Gly Ser Lys

1 5

Claims

1. A compound of formula I, or a pharmaceutically acceptable salt thereof,

A–B–C–D

formula I

Wherein the content of the first and second substances,

a represents a cell-permeable peptide which is TAT,

b represents an endosome escape fragment, the endosome escape fragment is 4-8 His,

c represents a Lys, and/or an Arg,

d represents a laminin receptor targeting segment, the laminin receptor targeting segment is shown as SEQ ID NO. 17 or SEQ ID NO. 18,

the compounds of formula I are also linked to one or more hydrophobic molecules E, which are stearic, palmitic, oleic, linoleic or palmitic acids and which are linked to the amino group of the side chain of Lys and/or Arg in C.

2. A compound of formula I according to claim 1, or a pharmaceutically acceptable salt thereof, characterized by any one or more of the following items (1) - (3):

(1) the endosome escape fragment is 6 His;

(2) the linker is 1-6 Gly, 1-6 Lys or 1-6 Cys;

(3) n-terminal acetylation and/or C-terminal amidation of the amino acid sequence of the compounds of formula I.

3. A compound of formula I according to claim 1 or 2, or a pharmaceutically acceptable salt thereof, wherein the compound is represented by SEQ ID No. 1 or SEQ ID No. 2.

4. A complex comprising a compound of formula I, or a pharmaceutically acceptable salt thereof, as claimed in any one of claims 1 to 3, and a nucleic acid molecule.

5. The complex of claim 4, 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.

6. the complex of claim 4, wherein the charge ratio of the compound of formula I, or a pharmaceutically acceptable salt thereof, to the nucleic acid molecule is (2-8): 1.

7. the complex of claim 4, wherein the charge ratio of the compound of formula I, or a pharmaceutically acceptable salt thereof, to the nucleic acid molecule is 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.

8. a host cell containing a compound of formula I according to any one of claims 1 to 3 or a pharmaceutically acceptable salt thereof or a complex according to any one of claims 4 to 7.

9. A pharmaceutical composition comprising a compound of formula I according to any one of claims 1 to 3 or a pharmaceutically acceptable salt thereof or a complex according to any one of claims 4 to 7, together with at least one pharmaceutically acceptable excipient.

10. A kit comprising a compound of formula I according to any one of claims 1 to 3 or a pharmaceutically acceptable salt thereof or a complex according to any one of claims 4 to 7, and a nucleic acid of interest.

11. Use of a compound of formula I according to any one of claims 1 to 3 or a pharmaceutically acceptable salt thereof for the preparation of a medicament or agent for loading and/or transporting nucleic acids or a gene therapy medicament.

12. A method for loading and/or transporting nucleic acids or a method for preparing a complex according to any of claims 4 to 7, comprising the step of contacting a compound of formula I according to any of claims 1 to 3 or a pharmaceutically acceptable salt thereof with a nucleic acid molecule.

13. The method of claim 12, wherein the nucleic acid molecule is DNA.

14. The method of claim 12, wherein the nucleic acid molecule is RNA.

15. The method of claim 12, wherein the charge ratio of the compound or pharmaceutically acceptable salt thereof to the nucleic acid molecule is (1-10): 1.

16. The method of claim 12, wherein the charge ratio of the compound or pharmaceutically acceptable salt thereof to the nucleic acid molecule is (2-8).

17. The method of claim 12, wherein the charge ratio of the compound or pharmaceutically acceptable salt thereof to the nucleic acid molecule is 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.

18. the method of claim 12, wherein the contacting is performed at a temperature of 36-38 ℃ for 20min or more.

19. The method of claim 18, wherein the contacting is performed under conditions of incubation at 37 ℃ for 20min or more.

20. The method of claim 18, wherein the contacting is at a temperature of 36-38 ℃ for 30 min.

21. The method of claim 18, wherein the contacting is at conditions of 37 ℃ for 30 min.

22. The method according to claim 12, wherein the compound of formula I or a pharmaceutically acceptable salt thereof is mixed with the nucleic acid molecule prior to the incubation.

23. The method of claim 22, wherein the mixing of the compound of formula I or a pharmaceutically acceptable salt thereof with the nucleic acid molecule is performed by vortexing or stirring prior to the incubation.

24. A method of transfecting a eukaryotic cell comprising the step of loading a nucleic acid molecule of interest into a compound of formula I as claimed in any one of claims 1 to 3 or a pharmaceutically acceptable salt thereof.