WO2019080504A1 - 一种细胞穿透肽和细胞穿透肽复合物及二者的应用 - Google Patents

一种细胞穿透肽和细胞穿透肽复合物及二者的应用

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WO2019080504A1
WO2019080504A1 PCT/CN2018/089742 CN2018089742W WO2019080504A1 WO 2019080504 A1 WO2019080504 A1 WO 2019080504A1 CN 2018089742 W CN2018089742 W CN 2018089742W WO 2019080504 A1 WO2019080504 A1 WO 2019080504A1
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preparation
drug
cell
penetrating peptide
cell penetrating
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PCT/CN2018/089742
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English (en)
French (fr)
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汪华
李小龙
赵龙
张雁
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中山大学附属口腔医院
广州一代医药科技有限公司
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Publication of WO2019080504A1 publication Critical patent/WO2019080504A1/zh

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    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K49/00—Preparations for testing in vivo
    • A61K49/001—Preparation for luminescence or biological staining
    • A61K49/0013—Luminescence
    • A61K49/0017—Fluorescence in vivo
    • A61K49/005—Fluorescence in vivo characterised by the carrier molecule carrying the fluorescent agent
    • A61K49/0056—Peptides, proteins, polyamino acids
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K51/00—Preparations containing radioactive substances for use in therapy or testing in vivo
    • A61K51/02—Preparations containing radioactive substances for use in therapy or testing in vivo characterised by the carrier, i.e. characterised by the agent or material covalently linked or complexing the radioactive nucleus
    • A61K51/04—Organic compounds
    • A61K51/06—Macromolecular compounds, carriers being organic macromolecular compounds, i.e. organic oligomeric, polymeric, dendrimeric molecules
    • A61K51/065—Macromolecular compounds, carriers being organic macromolecular compounds, i.e. organic oligomeric, polymeric, dendrimeric molecules conjugates with carriers being macromolecules
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K51/00—Preparations containing radioactive substances for use in therapy or testing in vivo
    • A61K51/02—Preparations containing radioactive substances for use in therapy or testing in vivo characterised by the carrier, i.e. characterised by the agent or material covalently linked or complexing the radioactive nucleus
    • A61K51/04—Organic compounds
    • A61K51/08—Peptides, e.g. proteins, carriers being peptides, polyamino acids, proteins
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K14/00—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
    • 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
    • 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 present invention relates to a cell membrane penetrating peptide and a cell penetrating peptide complex, and to the use of the cell penetrating peptide and cell penetrating peptide complex.
  • the cell membrane is a phospholipid bilayer, which is a semi-permeable barrier between the inner and outer environment of the cell. It has selective permeability and ensures the relative stability of the intracellular environment.
  • the cell membrane barrier prevents certain substances, especially harmful substances, from entering the cells from the blood, helping to maintain the normal functional activities of the cells.
  • Penicillin acts on the cell wall of prokaryotic cells.
  • the structure of penicillin is similar to that of D-alanyl-D-alanine in the component of the cell wall. It can compete with the latter for transpeptidase, hinder the formation of peptides and cause cell wall. The defect causes the bacteria to lose the permeation barrier of the cell wall and kill the bacteria.
  • Eukaryotic cells lack cell walls, so penicillin has almost no toxic effects on eukaryotes.
  • an important cellular immune mechanism of eukaryotes has been found to attack the cell membrane integrity of target cells.
  • the cytoplasm of cytotoxic T lymphocytes (CTL) and natural killer cells (NK) stores a membrane-forming protein, Pore-forming protein, also known as perforin.
  • CTL and NK cells When in close contact with target cells such as virus-infected cells or tumor cells, CTL and NK cells release perforin, forming a multi-perforin tubular channel on the cell membrane of the target cell, resulting in rapid dissolution and destruction of the target cells.
  • the perforin has a molecular weight of about 67 kDa and is composed of 534 amino acids.
  • perforin molecules are inserted into the target cell membrane, and multimerization forms a plurality of abnormal tubular channels with an inner diameter of about 16 nm on average.
  • cell membrane-destroying drug molecules like perforin have been rarely reported, and the invention of perforin drugs will undoubtedly open up new avenues for the treatment of diseases such as tumors and viruses.
  • CPPs Cell penetrating peptides
  • TAT human immunodeficiency virus-1 transcription activator
  • TAT has an activation domain (sequence 37-48, region II), the nuclear localization domain (sequences 49-57, region III), and an activation-enhancing domain (sequences 58-72, region IV), which play an important role in the transport of the virus to the nucleus.
  • CPPs can carry a variety of biologically active substances of different sizes and properties into cells, including proteins, peptides, oligo-nucleotides, small molecule compounds, dyes, peptide nucleoproteins (PNA), and plasmid DNA that are difficult to penetrate cell membranes. , siRNA, 200nm liposomes, phage particles and superparamagnetic particles, etc., this property provides a good carrier for targeting drugs.
  • CPPs can be divided into cationic CPPs, amphiphilic CPPs and hydrophobic CPPs.
  • the main functional sequence of TAT is GRKKRRQRRR, which is critical for TAT cell penetration and vector transport.
  • the penetration characteristics of TAT on cell membranes are energy independent, but can also enter cells by endocytosis of cell membranes.
  • Penetratin is found in the natural Drosophila tentacles homologous protein, which mediates the endogenousization of extracellular proteins.
  • the cell membrane permeability of Penetratin is mediated through the domain formed by liposomes or ceramides on the cell membrane.
  • the cationic CPPs sequence mainly contains cationic amino acids such as arginine, lysine and histidine.
  • the thiol group of arginine can form a hydrogen bond with a negatively charged phosphate, sulfate or the like on the cell membrane. Therefore, a short peptide containing arginine can penetrate the cell membrane under physiological conditions and enter the cell.
  • the positive charge of lysine is similar to that of arginine, but it has no sulfhydryl group and therefore has a lower penetration ability than arginine.
  • cationic CPP requires at least 8 positive charges to penetrate the cell membrane.
  • Amphiphilic CPPs have a lipophilic group and a hydrophilic group that penetrate the cell membrane by binding to the mucopolysaccharide on the cell membrane.
  • Transportan GWTLNSAGYLLGKINLKALAALAKKIL
  • Hydrophobic CPPs are mainly composed of hydrophobic amino acids. The charge is less and the cell membrane permeability is weak. However, hydrophobic CPPs can enter the cells through non-covalent bonds, which have less effect on cell membrane and can preserve cell viability to the utmost.
  • CPPs are carriers to carry molecules that are difficult to enter cells, such as nanoparticles, oligo-nucleotides, anticancer drugs, etc. into cells. Improve the ability of cells to absorb these molecules, reduce drug resistance, and reduce side effects.
  • Existing studies have shown that there are a variety of CPPs for drug molecule transport.
  • CPPs increase the permeability of tumor cell membranes, and thus can resist the MDR of tumor cells and increase the local drug concentration of tumor drugs in tumor cells.
  • CPP is used as a carrier to enter cells and exert biological activities, including proteins and nucleic acids.
  • Proteins include functional proteins such as enzymes, hormones, growth factors, neurotrophic factors, apoptotic factors, and intracellular signal transduction molecules;
  • nucleic acids include plasmids, double-stranded DNA, antisense oligonucleotides, and interfering small RNAs ( Small interfering RNA, siRNA), etc.
  • siRNA is usually used to regulate the activity of a target gene, but siRNA has a strong negative charge and is difficult to pass through the cell membrane.
  • CPP has a positive charge, and can form a strong covalent bond or a loose non-covalent bond when mixed with siRNA according to a certain ratio.
  • Some researchers have connected CPP and siRNA with a disulfide bond, and the transfection effect of CPP into cells is excellent.
  • Lipofectamin-siRNA the blood-brain barrier strictly limits the entry of macromolecules such as protein drugs.
  • Some researchers have demonstrated that CPP can guide macromolecular neurotrophic substances through the blood-brain barrier, such as anti-apoptosis through CPP.
  • the protein Bcl-xL can increase the absorption of the protein in the brain tissue, inhibit the apoptosis caused by ischemia, and significantly reduce the necrotic area and nerve damage in the brain region.
  • CPPs in combination with other targeting polypeptides, are linked to a drug molecule polymer to achieve targeted delivery and targeted release of the drug, including MT1-7F7p polypeptide, pH-sensitive polypeptide, and the like.
  • MT1-7F7p is a polypeptide that specifically binds to the peri-tumor neovascular and MT1-MMP molecules with high expression on the surface of glioma.
  • the binding of MT1-7F7p and CCPs to the Taxol-PEG-PLA antitumor drug polymer can enhance the targeted release of paclitaxel drugs into gliomas and achieve targeted drug release.
  • H7K(R2)2 is a pH-sensitive cell penetrating peptide which is stable in a normal physiological environment of pH 7.4, but is activated in an acidic environment of pH 6.0 and has membrane permeability. Since the tumor microenvironment is acidic and the pH value is lowered, H7K(R2)2 is activated in the acidic microenvironment of the tumor, and the antitumor drug is delivered to the tumor cells to increase the local drug concentration in the tumor microenvironment. More and more CPPs have been applied to the targeted transport of targeted anti-tumor drugs, directed release studies.
  • Photosensitive drugs such as porphyrin-type photosensitizer (Photofrin), Aminolevulinic acid (ALA), Hemoporfin, Verteporfin, Duperoporphyrin, mTHPC, porphyrin Tin (SnEtz), Methoxsalen, Methylene Blue, Methylene Blue, Benzoporphyrin Derivatives, Lutelium texaphyrins (Lu-Tex) and Sefium Sodium
  • the photosensitizing drugs have limited therapeutic effects due to the low tumor selective uptake rate. According to the cell penetration and carrier ability of CPPs, the attachment of photosensitizing drugs to CPPs may also increase the entry of photosensitizing drugs into cells, which is beneficial to improve the photodynamic therapy effect of tumors.
  • Rhodamine is a fluorescent dye belonging to the catechol polycyclic compound, mainly including Rhodamine B, Rhodamine 6G, Rhodamine 123, and cell fluorescence.
  • Rhodamine B molecular formula C28H31ClN2O3; molecular weight 479.0175, long-term use is carcinogenic and mutagenic
  • Rhodamine 6G is heated by equal weight of Rhodamine B and aniline hydrochloride to 185 ⁇ 190 ° C for 1.5 ⁇ 2h, then esterified with ethanol and mineral acid
  • Rhodamine 6G alone has the selective cytotoxicity of tumor cells, which can chemically react with proteins in the cytoplasm to deactivate cells; Rhodamine 123 can penetrate the cell membrane and accumulate in the mitochondria of living cells, and It emits yellow-green fluorescence.
  • Rhodamine 123 is used to stain a wide variety of cells, including plant cells and bacteria. Since there is a correlation between the amount of intracellular ATP and the fluorescence intensity of rhodamine 123, this fluorescent dye is applied to detect ATP in cells. There are no reports on the use of Rhodamine B and Rhodamine 123 for anti-tumor drug development.
  • Monoclonal antibodies include monoclonal antibodies, nanomaterials, liposomes, adenoviruses, and cell penetrating peptides (CPPs).
  • CPPs cell penetrating peptides
  • monoclonal antibody vectors have the advantages of high specificity, strong affinity, and good stability.
  • the shortcoming is that the monoclonal mouse-derived antibody will inevitably cause human anti-mouse antibody immune response in the human body, leaving a medical safety hazard.
  • the antibody has a larger molecular weight, less drug loading, and tissue cell permeability.
  • the cell membrane surface of the tumor overexpresses negatively charged proteins such as O-glycosylated mucin, phosphatidylserine and sialic ganglioside, resulting in a network-like negative charge on the surface of tumor cells.
  • Some drug molecules have a role in targeting cell membrane destruction, such as ophiobolin, polybia-MPI wasp venom polypeptide, cyclotide, cinnamycin, cinnamamide geldanamycin And duramycin.
  • Polybia-MPI is a cationic anti-tumor polypeptide extracted from the venom of the venomous wasp.
  • IDWKKLLDAAKQIL Its sequence is IDWKKLLDAAKQIL, which can form an alpha-helical conformation, targeting non-polar lipid cell membranes, increasing cell membrane permeability and lysing cells. .
  • Bladder cancer, prostate cancer, and tumor vascular endothelial cells are sensitive to Polybia-MPI.
  • Kahalalide F is a cyclic peptide extracted from Haitian cattle, which selectively inhibits prostate cancer and breast cancer cells at a concentration of 80 mg/kg, resulting in mitochondrial membrane lysis and increased lysosomes. Kahalalide F has entered Phase I/II clinical studies in the treatment of melanoma and non-small cell lung cancer.
  • Didemnin B Another cyclic polypeptide, Didemnin B, is derived from the extract of sea squirt, which prevents cells from entering the S phase from the G1 phase and promotes apoptosis of tumor cells.
  • Didemnin B has entered phase II clinical studies in the treatment of colorectal cancer, metastatic breast cancer, and glioblastoma.
  • phage display technology screened the naked mouse CD133-bound 7-peptide: LQNAPRS (LS-7), FGF3 receptor-bound 7 peptide: VLWLKNR (FP16), APRIL-bound peptides AAAPLAQPHMWA, SSTTTSDKYLSA and SNLHDNNTEKNV showed better Tumor suppressive effect.
  • linking cell membrane disrupting drugs to CPPs may also increase the entry of drugs into cells, which is beneficial to reducing toxicity and improving tumor treatment.
  • tumor cell membranes overexpress phosphatidylserine and hyperglycosylated mucin (Peetla et al. 2013 ; Hoskin and Ramamoorthy 2008 ) with a large proportion of anions; whereas cells in normal state are mainly composed of phosphatidylcholine, It is composed of phosphatidylethanolamine and sphingomyelin and is neutral (Spector and Yorek 1985 ).
  • the present invention provides a novel cell penetrating peptide and cell penetrating peptide complex and their use in order to compensate for the deficiencies of the prior art.
  • a first aspect of the invention provides a cell penetrating peptide selected from the group consisting of the following amino acid sequences: amino acid sequence of sequence 1, sequence 2, sequence 3, sequence 4 or sequence 5, or with any of sequences 1-5
  • the sequence has an amino acid sequence of at least 50% identity, for example, the identity can be 50% or more, 60% or more, 70% or more, 80% or more, 90% or more, 95% or more, and the like.
  • the cell penetrating peptide is preferably an amino acid sequence as shown in SEQ ID NO: 1 or SEQ ID NO: 5, or an amino acid sequence having at least 50% identity to SEQ ID NO: 1.
  • a second aspect of the present invention provides a cell penetrating peptide complex comprising one or two or more cell penetrating peptides, the cell penetrating peptide being at least one selected from the group consisting of peptides of the following amino acid sequence: Sequence 1, The amino acid sequence shown in SEQ ID NO: 2, SEQ ID NO: 3, SEQ ID NO: 4, SEQ ID NO: 5, or an amino acid sequence having at least 50% identity with any of the sequences of 1-5, for example, the identity may be 50% or more and 60%. Above, 70% or more, 80% or more, 90% or more, 95% or more, and the like.
  • the cell-penetrating peptide complex comprises at least one or more of the following amino acid sequences: the amino acid sequence shown in SEQ ID NO: 1 or 5, or at least 50% from the sequence 1 or 5. The amino acid sequence of identity.
  • a third aspect of the invention provides a nucleic acid molecule encoding the amino acid sequence described above.
  • a fourth aspect of the invention provides a vector comprising the nucleic acid molecule described above.
  • a fifth aspect of the invention provides a host cell comprising the vector described above.
  • a sixth aspect of the present invention provides a complex comprising a compound in which a cell penetrating peptide and a cargo molecule are coupled, the cell penetrating peptide being the cell penetrating peptide described above, and further comprising a cell-penetrating peptide
  • the cargo molecule on the peptide preferably, the complex comprises at least one of sequences 1 and 5.
  • the coupling may be by way of covalent or non-covalent bonds.
  • the cargo molecule is preferably, but not limited to, at least one of a pharmaceutically active molecule, a labeling molecule, and a targeting molecule.
  • the biofilm disrupting drug is coupled to the amino acid sequence terminus of the cell penetrating peptide, preferably to the amino terminus.
  • a seventh aspect of the invention provides a pharmaceutical composition comprising the complex described above; preferably, further comprising a pharmaceutically acceptable carrier or adjuvant; preferably, the pharmaceutical composition comprises, but is not limited to, a capsule, Soft capsules, tablets, oral liquids, dispersible tablets, freeze-dried powder, injections or dropping pills.
  • the carrier or excipient may be a diluent, an excipient, a binder, a wetting agent, a disintegrant, an absorption enhancer, a surfactant, an adsorption carrier, a lubricant, and the like which are commonly used in the pharmaceutical field, and are specifically required according to the dosage form. select.
  • An eighth aspect of the present invention provides an application, wherein the cell penetrating peptide or cell penetrating peptide complex described above is used in the preparation of at least one of the following a) to j):
  • a preparation for treating or preventing a tumor preferably, the tumor comprises leukemia, sarcoma, squamous cell carcinoma or adenocarcinoma;
  • a fluorescent nuclides or radiolabeling reagent preferably, said fluorescent nuclides or radiolabeling reagents for disease diagnosis;
  • the cell penetrating peptide is coupled to at least one of an antitumor drug or a rhodamine B compound, wherein the antitumor drug is preferably doxorubicin, At least one of docetaxel, methotrexate, cytarabine, 5-fluorouracil, cisplatin, cyclophosphamide or vincristine, said rhodamine B compound is preferably rhodamine B;
  • the cell penetrating peptide When used in the preparation of the preparation of the b), the cell penetrating peptide is coupled to at least one of the antiviral infectious drugs;
  • the cell penetrating peptide is coupled to at least one of a biofilm disrupting drug, preferably a rhodamine B compound, more preferably a rhodamine.
  • a biofilm disrupting drug preferably a rhodamine B compound, more preferably a rhodamine.
  • the cell penetrating peptide When used in the preparation of the preparation of the d), the cell penetrating peptide is coupled to at least one of a labeling fluorescent reagent, a nuclides or a radioactive reagent;
  • the cell penetrating peptide is coupled to at least one of a cytotoxic drug, preferably selected from the group consisting of doxorubicin, docetaxel, At least one of methotrexate, cytarabine, 5-fluorouracil, cisplatin, cyclophosphamide, and vincristine;
  • a cytotoxic drug preferably selected from the group consisting of doxorubicin, docetaxel, At least one of methotrexate, cytarabine, 5-fluorouracil, cisplatin, cyclophosphamide, and vincristine;
  • the cell penetrating peptide is coupled to at least one of a photosensitizing drug, preferably selected from the group consisting of methoxacil, aminolevulinic acid hydrochloride, and Heim.
  • a photosensitizing drug preferably selected from the group consisting of methoxacil, aminolevulinic acid hydrochloride, and Heim.
  • a photosensitizing drug preferably selected from the group consisting of methoxacil, aminolevulinic acid hydrochloride, and Heim.
  • a photosensitizing drug preferably selected from the group consisting of methoxacil, aminolevulinic acid hydrochloride, and Heim.
  • the cell penetrating peptide is coupled to at least one of the gene drugs
  • the cell penetrating peptide is coupled to at least one of the neurotrophic molecules.
  • the cell penetrating peptide is coupled to at least one of the molecules having a targeting effect
  • the cell penetrating peptide When used in the preparation of the preparation of said j), the cell penetrating peptide is coupled to at least one of stem cell regulatory factors.
  • a ninth aspect of the present invention provides an application, such as the cell penetrating peptide or cell penetrating peptide complex described above, as a carrier for carrying a drug molecule, which delivers the drug molecule to the cytoplasm and/or nucleus of the cell of interest. It can be in vitro or in vivo.
  • the drug molecule may include at least one of an antitumor drug, an antiviral drug, a biofilm destroying drug, a cytotoxic drug, a photosensitizing drug, a gene drug, a neurotrophic molecule, and a stem cell regulating factor. .
  • the pharmaceutically active molecule includes an antitumor drug, an antiviral drug, a biofilm destroying drug, a photosensitizing drug, a cytotoxic drug, a gene drug, a neurotrophic molecule, and a stem cell regulating factor. At least one.
  • the biofilm-destroying drug is preferably a rhodamine B compound.
  • the gene drug may be: double-stranded DNA, antisense oligonucleotide, and small interfering RNA (siRNA).
  • siRNA small interfering RNA
  • CPP links anti-apoptotic protein Bcl-xL, vascular endothelial growth factor VEGF siRNA and epidermal growth factor HER siRNA.
  • the biofilm-destroying drug is preferably a Rhodamine B compound, Rhodamine 6G, Rhodamine 123 or the like; more preferably Rhodamine B.
  • the photosensitizing drug may be selected from the group consisting of Methoxsalen, Aminolevulinic acid (ALA), Hemoporfin, and Verteporfin. , Duperoporphyrin, mTHPC, bismuth tin (SnEtz), methylene blue and xylene, benzophenone derivatives, lutelium texaphyrins (Lu-Tex), dysprosium And at least one of a hematoporphyrin derivative (HpD).
  • the cytotoxic drug may be selected from the group consisting of doxorubicin, docetaxel, methotrexate, cytarabine, 5-fluorouracil, cisplatin, cyclophosphamide, and At least one of vincristine.
  • the neurotrophic molecule may be, for example, nerve growth factor NGF, brain derived neurotrophic factor (BDNF), neurotrophic factor 3 (NT-3), nerve Nutritional factors 4/5 (NT-4/5), neurotrophic factor 6 (NT-6), ciliary neurotrophic factor (CNTF), glial cell line-derived neurotrophic Factor, GDNF) and insulin like-growth factor-I (IGF-I) and the like.
  • NGF nerve growth factor
  • BDNF brain derived neurotrophic factor
  • NT-3 neurotrophic factor 3
  • nerve Nutritional factors 4/5 NT-4/5
  • neurotrophic factor 6 NT-6
  • CNTF ciliary neurotrophic factor
  • GDNF insulin like-growth factor-I
  • IGF-I insulin like-growth factor-I
  • the stem cell regulatory factor may be, for example, Cyclin and Cyclin-Dependent Kinase which regulate the cell cycle, Gene transcription factor Oct4, and affected cells.
  • Cyclin and Cyclin-Dependent Kinase which regulate the cell cycle
  • Gene transcription factor Oct4 Gene transcription factor Oct4
  • affected cells Asymmetric division of cytokines and stem cell growth factors (SCF) and the like.
  • the labeling molecule comprises at least one of a fluorescent reagent, a nuclides or a radioactive reagent.
  • the fluorescent reagent is specifically a rhodamine compound or an isothiocyanate.
  • the targeting molecule is, for example, a polypeptide or antibody that specifically binds to CD34, CD56, CD3, epidermal growth factor receptor HER, and vascular endothelial growth factor receptor VEGFR.
  • the cell penetrating peptide of the invention can be used as a carrier for carrying drugs to guide cytotoxic drugs into the cytoplasm and nucleus, increase drug utilization efficiency and reduce complications; and can also serve as a carrier for carrying drugs to guide photosensitizing drugs into cytoplasm and nucleus, and increase photodynamic efficacy.
  • siRNA double-stranded DNA
  • antisense oligonucleotides and other gene drugs into the cytoplasm and nucleus, avoiding the use of some traditional gene therapy methods such as viral vectors
  • It can also be used as a carrier for drug delivery to guide neurotrophic molecules to penetrate the blood-brain barrier into neurons; it can also serve as a carrier for carrier drugs to guide stem cell regulatory factors into the cytoplasm and nucleus, regulating stem cell growth, differentiation and wound healing.
  • the present invention provides a penetrating polypeptide (or cell penetrating peptide) which is easy to prepare, has a small molecular weight, is not easy to produce an immunological rejection reaction, has a strong penetrating ability, and has a long intracellular half-life.
  • the cell penetrating peptide provided by the invention is rich in basic amino acids, has a positive charge, can quickly penetrate the cell membrane and the nuclear membrane, can be artificially synthesized, has the characteristics of no cytotoxicity and high safety.
  • the cell penetrating peptide provided by the invention can be made into a diagnostic reagent by combining fluorescent or radioactive element identification, and the cell peptide is rich in cations, can selectively bind to tumor cells, is enriched in tumor cells and tissues thereof, and can be used for tumors, etc. Identification of the disease.
  • the cell penetrating peptide provided by the invention binds to the penetrating peptide cell membrane targeted destruction drug formed by rhodamine B, and can bind to the tumor cell membrane, penetrate the tumor cell membrane into the cytoplasm and the nucleus, and cause cell membrane destruction and cell lysis death, so that the present invention is not significant.
  • the anticancer effect of Rhodamine B has a significant anticancer effect upon binding to the penetrating polypeptide of the present invention.
  • the present invention is based on the discovery of a penetrating peptide cell membrane targeted disrupting drug, and also provides a method for preparing a penetrating peptide cell membrane targeted disrupting drug by coupling a compound having a membrane protein or phospholipid destruction to a cell penetrating peptide end.
  • the cation of the cell penetrating peptide is combined with the anion in the cell biofilm to expose the targeting molecule of the compound, and synergistically cooperate to damage the biofilm structure, dissolve the cell, and treat the disease.
  • the invention also provides a photosensitizing drug which is combined with a penetrating polypeptide, which improves the cell membrane and nuclear nucleus penetration of the photosensitizing drug, and has better anticancer effect.
  • the invention also provides an anticancer drug formed by penetrating polypeptide combined with doxorubicin, which increases the cell membrane and nuclear penetration of doxorubicin, and has remarkable anticancer effect.
  • a large proportion of selectively bound tumor cells, enriched in tumor tissue, can be used for tumor tissue marker diagnosis; 2) can penetrate cell membrane and nuclear membrane, used for drug development of targeted cell membrane; 3) can be used for Positioning the nucleus; 4) can be used as a carrier to carry molecules into tumor cells; 5) can act as a carrier to carry anti-tumor drugs into the nucleus; 6) can bind rhodamine B and other cell membrane destruction compounds, selectively destroy tumor cell membrane, leading to tumor cell lysis death.
  • the peptide has strong penetrating ability, can penetrate the tumor cell membrane and enter the nucleus in 10 minutes; 2) the polypeptide has strong carrying capacity and can carry a variety of molecules into the nucleus; 3) the polypeptide has small molecular weight and good permeability. It is not easy to be denatured and has low immunogenicity. 4) The peptide is easy to prepare and low in cost, and can be directly synthesized by artificial synthesis or by genetic engineering; 5) The polypeptide is non-cytotoxic, trans, liver, kidney and stomach. Intestinal metabolism.
  • the penetrating peptide rhodamine B cell membrane destroys the drug, selectively destroys the tumor cell membrane, causes tumor cell lysis to die, and has low toxicity to normal cells.
  • the penetrating peptide rhodamine B cell membrane destroying drug has better killing effect on leukemia and sarcoma cells; has no significant killing effect on squamous epithelial cancer cells and normal cells; 3) the penetrating peptide rhodamine B cell membrane destroys drug height Gathering in tumor tissue is beneficial to the anti-tumor effect of the drug.
  • FIG. 1 Schematic diagram of the three-dimensional structure predicted by the cell-penetrating peptide TCPP (TCPP) in the embodiment of the present invention
  • FIG. 1 Cell-penetrating peptide FITC-TCPP binds to tumor cell membrane and cell permeation process
  • Figure 3 in Figure 3 shows the concentration dependence of cell-penetrating peptide FITC-TCPP on tumor cell binding and osmosis;
  • Figure B in Figure 3 shows the effect of cell-penetrating peptide FITC-TCPP on tumor cell proliferation inhibition;
  • Panels C and D in Figure 3 are the results of in vivo distribution and labeling studies of FITC-TCPP polypeptides.
  • Figure 4 is a diagram showing the design and preparation of the cell-penetrating peptide cytotoxic drug Doxorubicin (Tumour Cell Penetrating Peotides-Doxycycline, TCPP-Dox) and the anti-tumor effect of the present invention, wherein the AC in Figure 4 is TCPP- Dox penetrating peptide binding to tumor cell membrane and cell penetration ability; 4D: tumor cell killing efficiency study of TCPP-Dox penetrating peptide;
  • TCPP-DM Tumour Cell Penetrating Peotides Damage Membrane
  • 6 is a flow cytometric analysis result of the binding efficiency of the tumor cell penetrating peptide targeting cell membrane disrupting drug TCPP-DM and the tumor cell line K562;
  • Figure 7 is a graph showing the results of an anti-tumor application study of the tumor cell-penetrating peptide targeting cell membrane disrupting drug TCPP-DM of the present invention; wherein, Figure A, B of Figure 7 is a tumor of a TCPP-DM penetrating peptide drug. Inhibition of cell proliferation; Figure C, Figure D, Figure D: Animal experiment of TCPP-DM penetrating peptide drug for pre-stage tumor; Figure E, Figure F, Figure: Animal experiment of TCPP-DM penetrating peptide drug in late stage tumor Figure G in Figure 7, H: Distribution analysis of in vivo animals in the treatment of tumors with TCPP-DM penetrating peptide drugs.
  • Figure 8 Results of flow cytometry studies of TCPP-DM penetrating peptide drugs to induce apoptosis
  • Figure 9 Fitting analysis of TCPP-DM penetrating peptide drugs on tumor cell proliferation inhibition experiments
  • Figure A-B in Figure 10 is an optimized experimental result of the tumor cell penetrating peptide and its complex of the present invention
  • 11-12 are the ultrastructural dynamic changes of the anti-tumor treatment of the tumor cell-penetrating peptide cell membrane targeted destruction drug TCPP-DM according to the present invention.
  • CD14A recombinant protein was purchased in PeproTech, USA; T7Select human liver cancer cDNA library (Novagen, USA); peptide purchased in Qiang Yao Biotechnology Co., Ltd., China; Cell Counting Kit-8 (CCK-8) toxicity test kit, Annexin
  • V-FITC/PI apoptosis detection kit was purchased and placed in DOJINDO (Dongren Chemical Technology Co., Ltd., Japan); Importzole and Ivermectin were purchased in MCE, USA.
  • Importzole and Ivermectin were purchased from MCE, USA; K562, Jurkat, MNNG, MG63 cell lines were all from the laboratory of Professor Zhang Yan of the School of Life Sciences of Sun Yat-sen University; cell culture media were purchased in Sigma-Aldrich, USA.
  • Experimental animals were purchased at the East Campus of Experimental Animal Center of Sun Yat-sen University, SCXK (Guangdong) 2016-0029, and the animal experiment ethics was approved by the Ethics Committee of Sun Yat-Sen University.
  • the leukemia cell line K562 and Jurkat cells were cultured in RPMI 1640+10% FBS complete medium, and the osteosarcoma cell lines MG63 and MNNG cells were cultured in DF+5% FBS complete medium, and HFF cells were cultured in DF+10%.
  • FBS complete medium HUVEC cells were cultured in SFM + 5 ng / mL EGF + 10% FBS complete medium, and all cells were cultured in a 37 ° C, 5% CO 2 incubator, and passaged when the cell density reached 80%. Or cell experiments.
  • the suspension cells are directly centrifuged to discard the culture solution, washed once with PBS buffer, and the cells are counted. A suspension of 5 ⁇ 10 ⁇ 5/ml cell concentration is added, and the TCPP polypeptide is added to make the final concentration 3 ⁇ mol. Light, incubate for 15 minutes at room temperature. The adherent cells were washed once with PBS, digested with 0.25% TE, centrifuged, counted, taken 2 ⁇ 10 ⁇ 5/ml cells, planted in 6-well plates containing coverslips, cultured at 37 ° C, 5% CO 2 Incubate overnight in the box.
  • the cell penetrating peptide TCPPs obtained by the present invention were added to a final concentration of 3 ⁇ Mol, protected from light, and incubated at room temperature for 5 min, 10 min, 15 min, 20 min, 25 min, 30 min, 1 h. Wash 3 times in PBS buffer for 4 minutes each time. Fix with 15% PFA for 15 minutes. Wash 3 times in PBS buffer for 4 minutes each time. The photographs were taken under a laser confocal microscope using a DAPI anti-fluorescence quencher.
  • the Importin ⁇ / ⁇ blockade experiment was performed by adding Importzole 25 ⁇ Mol or Ivermectin 40 ⁇ Mol before incubating the peptide, incubating for 1 hour at 37°C, and then incubating with 3 ⁇ Mol polypeptide for 30min.
  • the suspension cells are directly centrifuged to discard the culture solution, and the PBS buffer is washed once.
  • the adherent cells were washed once with PBS buffer, and the cells were digested with 0.25% TE, centrifuged, and counted.
  • Take 5 ⁇ l of 5 ⁇ 10 ⁇ 5/ml cell concentration suspension add the fluorescent penetrating peptide obtained in the subsequent examples of the present invention to 1.5 ⁇ Mol or 3 ⁇ Mol, incubate at room temperature for 15 minutes, wash 3 times with PBS buffer, and filter with 40 ⁇ m.
  • Membrane filtration, flow analyzer analysis are used to analyze the fluorescent penetrating peptide obtained in the subsequent examples of the present invention.
  • the suspension cells are directly centrifuged to discard the culture solution, washed once with PBS buffer, and the cells are counted.
  • the adherent cells were washed once with PBS buffer, and the cells were digested with 0.25% TE, centrifuged, and counted.
  • K562 and Jurkat cells were taken 5 ⁇ 10 ⁇ 5/ml, MG63, MNNG cells were 1 ⁇ 10 ⁇ 5/ml, and planted in 96-well plates at 100 ⁇ l per well.
  • the cell killing experiments were divided into a penetrating peptide drug NC group (control group), a 1.5 ⁇ Mol group, a 3 ⁇ Mol group, a 7.5 ⁇ Mol group, and a 15 ⁇ Mol group. After 24 hours and 48 hours of drug treatment, 10 ⁇ l of CCK8 reagent was added, and the cells were incubated for 1 hour at 37 ° C in a 5% CO 2 incubator, and the absorbance was measured at a wavelength of 450 nm using a multi-plate reader, and each group was repeated 3 times. Calculate the percentage of cell activity.
  • K562 cells were directly centrifuged to discard the culture solution, washed once with PBS buffer, and counted. K562 cells were taken 1 ⁇ 10 ⁇ 5/ml and planted in 24-well plates. Each cell was divided into control group and peptide drug treatment group. There are 3 auxiliary holes in each group. Penetrating peptide drugs were added daily to a final concentration of 3 [mu]Mol. From day 2, cell counts were taken daily.
  • mice were anesthetized with 50 mg/kg sodium pentobarbital by intraperitoneal injection at 50 mg/kg; 100 ⁇ l of a penetrating peptide drug at a concentration of 15 ⁇ mol was injected into the MNNG subcutaneous subcutaneous tumor-forming nude mice.
  • the nude mice were imaged using a PerkinElmer Lumina III small animal in vivo imaging system, and a picture was taken every 10 minutes using a spectral separation method for 2 hours; spectral separation was performed on the fluorescence of the mice using spectral separation, and fluorescence was analyzed in nude mice. Distribution. The mice were sacrificed by cervical dislocation, and the tissues of the heart, liver, spleen, lung, kidney, tongue, brain, tumor, etc.
  • Tissue frozen sections fixed in acetone, washed 3 times with PBS for 5 min, DAPI stained for 5 min, washed with PBS 3 times for 5 min each time, anti-quenching fluorescent seals were mounted, and observed under laser confocal.
  • MNNG osteosarcoma cells were seeded in a laser confocal dish at 2 x 10 ⁇ 5 cells, overnight at 37 ° C in a 5% CO 2 incubator. MNNG was cultured in Axio Observer Z1 under the conditions of 37 ° C, 5% CO 2 . Add 4.5 ⁇ mol of penetrating peptide drug, take a photo every 1 min at 1 h, and take a photo every 2 min at 2 h for a total of 2 h. Use the software to synthesize photos into videos.
  • the MNNG osteosarcoma cells were cultured in a 10 cm culture dish, and when grown to 80% to 90%, 7.5 ⁇ mol of the penetrating peptide drug was added, and the cells were incubated at 37 ° C in a 5% CO 2 incubator for 24 hours. Wash 3 times with PBS, add 2.5% glutaraldehyde at 4 ° C overnight, and wash 3 times with PBS for 10 min each time. Fix with 1% hungry acid, let stand for 1.5h, wash 3 times with PBS for 10min each time. 2% uranyl acetate fast dyed, 4 ° C, 1 h. Dehydration, infiltration, embedding, ultrathin sectioning, double staining of uranium lead, transmission electron microscopy.
  • the tumor cell membrane protein was used as a target molecule, and the phage binding to the cell membrane protein was enriched by the "T7Select human lung cancer cDNA library (Novagen)", and the phage binding, elution and enrichment were repeated several times until the phage library recovery rate no longer increased.
  • the binding efficiency of each monoclonal phage to the tumor cell membrane protein was measured by a spectrophotometer.
  • Monoclonal phage with high binding rate to tumor cell membrane molecules were selected, and PCR and sequence analysis confirmed that amino acid sequence 1 might be a cell-penetrating peptide, totaling 54 amino acids, including 29 basic amino acids, accounting for 53.7%.
  • Arginine Histidine is 18:10:1, rich in lysine and arginine, named TCPP (Tumour Cell Penetrating Peotide), and its three-dimensional structure prediction is shown in Figure 1.
  • TCPP1, TCPP2, TCPP3 and TCPP4 were designed based on TCPP, and their amino acid sequences are shown in sequence 1-5 in the sequence listing.
  • Example 2 Design and preparation of penetrating peptide markers, in vitro and in vivo cell labeling and permeability studies
  • the solid phase synthesis method was used to synthesize FITC-TCPP according to the above-mentioned TCPPs amino acid sequence (synthesized by commercial synthesis company Shanghai Qiang Yao Biotechnology Co., Ltd.).
  • the direction of synthesis is from C to N.
  • the carboxyl group of the carboxy terminal amino acid of the peptide chain to be synthesized is first linked to an insoluble polymer resin by a covalent bond structure, and then the amino acid bonded to the solid phase carrier is used as an amino group.
  • the component is subjected to deamination of the protecting group and reacts with an excess of the activated carboxyl component to bind the long peptide chain.
  • This step is repeated a number of times, namely condensation-washing-deprotection-neutralization and washing--the next round of condensation, and finally the length of the peptide chain required for synthesis.
  • the specific synthesis consists of the following cycles: 1 Deprotection: Fmoc protected columns and monomers must be protected with a piperidine to remove the protecting group of the amino group. 2 Activation and cross-linking: The carboxyl group of the next amino acid is activated by an activator. The activated monomer reacts with the free amino group to form a peptide bond. 3 cycles: The two steps of the reaction are repeated until the synthesis is complete. The synthesized peptide is then cleaved and deprotected from the resin. Finally, it was precipitated, eluted, and lyophilized with a purity of >95%. The N-terminal coupled FITC (green fluorescent) tag FITC-TCPP was used for cytological experiments.
  • the tumor cells were cultured in a 37 ° C, 5% CO 2 incubator, and the cell density reached 80%.
  • the suspension cells were directly centrifuged to discard the culture solution, washed once with PBS buffer, and counted, and 5 ⁇ 10 5 /ml cells were taken.
  • the suspension was added to the FITC-TCPP polypeptide to give a final concentration of 3 ⁇ mol, protected from light, and incubated at room temperature for 15 minutes.
  • the adherent cells were washed once with PBS, digested with 0.25% TE, centrifuged, counted, taken 2 ⁇ 10 ⁇ 5/ml cells, planted in 6-well plates containing coverslips, cultured at 37 ° C, 5% CO 2 Incubate overnight in the box.
  • the FITC-TCPP polypeptide was added to a final concentration of 20 ⁇ g/ml, protected from light, and incubated at room temperature for 15 minutes. Wash 3 times in PBS buffer for 4 minutes each time. Fix with 15% PFA for 15 minutes. Wash 3 times in PBS buffer for 4 minutes each time.
  • the photographs were taken under a laser confocal microscope using a DAPI anti-fluorescence quencher. The results are shown in Fig. 2.
  • the gray color in the figure indicates the position of the FITC-TCPP polypeptide binding, and it was confirmed that the polypeptide first binds to the tumor cell membrane, then penetrates into the cytoplasm, and then penetrates into the nucleus through the nuclear membrane.
  • Figure 3A shows the binding efficiency of different concentrations of FITC-TCPP and K562 cells for 30 min. With the increase of FITC-TCPP concentration, the number of positive cells increased, and the binding of peptide to K562 was correlated.
  • Figure 3B shows the cytotoxicity test of FITC-TCPP against K562 and MG63. It was found that FITC-TCPP had no killing effect on K562 and MNG63.
  • mice were anesthetized with conventional intraperitoneal injection of 2% pentobarbital sodium.
  • the mice were injected with FITC-TCPP polypeptide 100 ⁇ l (1 mg/ml) in the tail vein.
  • In vivo imaging was started 10 minutes later.
  • the nude mice were imaged using the PerkinElmer Lumina III small animal in vivo imaging system. The picture was taken every five minutes.
  • Spectral separation method was used to separate the FITC fluorescence in mice. The distribution of fluorescence in nude mice was analyzed for 4 hours. The mice were sacrificed by cervical dislocation and the heart, liver and spleen were dissected.
  • Figure 3D shows fluorescence imaging of mouse heart, liver, spleen, lung, kidney, brain, tongue and submandibular tissue in vitro; in the experiment, by observing the tissue sections of each organ, it was found that FITC-TCPP was mainly enriched in the liver. , kidney, lung, tongue and submandibular tissue.
  • cell penetrating peptide marker of the present invention can be used for labeling of tissues and organs such as liver, kidney, lung, tongue and submandibular tissue.
  • Example 3 Design, preparation and anti-tumor effect of penetrating peptide cytotoxic drugs
  • doxorubicin purchased from Italian fascia
  • FITC-TCPP-Dox ie, TCPP-Dox
  • FITC-Dox ie, TCPP-Dox
  • FITC-Dox ie, TCPP-Dox
  • FITC-Dox FITC-Dox
  • the polypeptide was purified by HPLC, and the purified solution was lyophilized to protect it from light.
  • a small amount of the finished polypeptide was taken, and the molecular weight identification of MS and the purity of HPLC analysis were performed. The identification results showed that the FITC-TCPP-Dox drug and the FITC-Dox drug were successfully synthesized.
  • the K562 and MNNG cell concentrations were 1 ⁇ 10 5 /ml and 5 ⁇ 10 4 /ml, respectively, and RPMI1640 and DF medium were used respectively, and implanted in a 96-well plate at 100 ⁇ l per well; 3.5 ⁇ M, 7 ⁇ M, 13.5 ⁇ M, 35 ⁇ M, 3 sub-wells were set for each concentration; the drug was added at 0h and 24h after culture, and 10 ⁇ l of CCK8 reagent was added at 24h and 48h after culture. After 1h incubation, the absorbance of CCK8 was determined to calculate the cell killing effect. The results of the experiment are shown in Figure 4A.
  • Figure 4C shows the results of flow cytometry analysis of K562 cells combined with FITC-TCPP-Dox and FITC-Dox.
  • Figure 4D compares the killing effect of FITC-TCPP-Dox and FITC-Dox on K562 and MNNG cells. The results show that FITC-TCPP-Dox has a killing effect on K562 and MNNG at 24h, with fast onset and cytotoxicity compared with FITC. -Dox is strong.
  • test results of the third embodiment show that the tumor cell penetrating peptide-conjugated anti-tumor drug of the present invention is beneficial to increase drug absorption and increase anti-tumor therapeutic effect.
  • Example 4 Design and preparation of Tumour Cell Penetrating Peotides Damage Membrane (TCPP-DM) and anti-tumor application
  • the cell membrane compound Rhodamine B red fluorescence
  • TCPP-DM tumor-penetrating peptide cell membrane targeted destruction drug
  • TCPP-DM tumor cell binding and permeation ability of the tumor-penetrating peptide cell membrane targeting drug TCPP-DM was analyzed. 3 ⁇ Mol of TCPP-DM drug was added to K526 cell culture medium, respectively, 5 min, 10 min, 15 min, 20 min, 25 min after the addition. At 30 min, 1 h, the binding and permeation characteristics of the drug were observed by confocal microscopy. The results are shown in Fig. 5. At 5 min, TCPP-DM binds to the cell membrane, penetrates into the cytoplasm at 10 min, and penetrates into the cytoplasm and nucleus at 15 min, 20 min. The cell nucleus was infiltrated with 30 min of TCPP-DM, and there was no DAPI staining in the 1 hour cell nucleus, indicating apoptosis necrosis.
  • Figure 6 shows the results of flow cytometry analysis of the binding efficiency of TCPP-DM to tumor cell line K562.
  • the binding efficiency of different concentrations of TCPP-DM and K562 cells was 30 min. As the concentration of TCPP-DM increased, the number of positive cells increased. The binding of TCPP-DM to K562 is concentration dependent.
  • FIGS. 7A and 7B show that TCPP-DM inhibits CCK8 cell proliferation inhibition in different types of tumor cells, and found that TCPP-DM has significant proliferation inhibition effect on K562, Jurkat, MG63, MNNG and other tumor cells from 3 ⁇ Mol concentration, and When the concentration was increased, the inhibitory effect was enhanced, but the proliferation of the normal vascular endothelial cell line HUVEC was not significantly inhibited.
  • Figure 7B further investigates the long-term inhibitory effect of TCPP-DM on proliferation of K562 cells. It was found that K562 proliferation was inhibited from the third day, and the cell survival rate was counted on days 5 and 6. It was found that the number of cells in the TCPP-DM treatment group was significantly lower than that. Blank control group. By studying the effect of TCPP-DM on the proliferation of normal vascular endothelial cell line HUVEC, it was found that TCPP-DM had no significant inhibitory effect on HUVEC cell proliferation.
  • FIG. 8 Using Annexin V/PI staining to analyze the apoptosis of K562 and foreskin fibroblast cell line HFF by TCPP-DM treatment. The results showed that 22.7% of K562 cells were in late apoptosis after treatment with TCPP-DM drug at 3 ⁇ Mol for 24h. The HFF was 3.76% in the late stage of apoptosis.
  • Osteosarcoma cells were cultured in DF + 5% FBS, and cells were harvested when MNNG was grown to 80%-90%. MMNG was injected into the submandibular skin of 25 4-8 week old BALB/c nod mice at a number of 1 ⁇ 10 ⁇ 6 cells. Nude mice were randomly divided into early treatment group and late treatment group, with 5 rats in each group. In the early treatment group, the tail vein was administered 24 hours after subcutaneous injection of MNNG osteosarcoma cells 1 ⁇ 10 ⁇ 6 cells. The late treatment group was injected with MNNG 7 days later and the tail vein was administered every other day.
  • the early treatment component was control ( The NC group, the TCPP-DM drug 5 mg/kg group; the late treatment component was the control (NC) group, the TCPP-DM drug 5 mg/kg group, and the doxorubicin (Dox) 4 mg/kg group, which were administered 5 times.
  • the tumor volume was monitored daily from the time of tumor emergence.
  • the tumor volume was measured every other day after the tumor appeared, and the mice were sacrificed 5 times after administration.
  • Figures 7C and 7D show the results of animal experiments with TCPP-DM anti-tumor therapy in the early treatment group, as two nude mice in the early treatment group and the control group were not tumorigenic and were excluded.
  • Figures 7E and 7F show the results of animal experiments with the anti-tumor treatment of TCPP-DM in the late treatment group.
  • the volume of subcutaneous tumor formation of MNNG in the anti-tumor treatment of TCPP-DM was smaller than that in the untreated control group, and was similar to the treatment effect of 4 mg/kg doxorubicin. This animal experiment shows that TCPP-DM has obvious anti-tumor therapeutic effects.
  • TCPP-DM is mainly enriched in tumor areas except for the metabolic organs such as liver, kidney and lung distributed in mice, which is conducive to drug diagnosis and treatment of tumors.
  • This tumor enrichment effect may be related to the electrostatic binding of cationic TCPP to tumor cell membrane, which is not only beneficial for tumor selective treatment of TCPP-DM, but also for in vivo labeling and diagnosis of tumor.
  • TCPP1, TCPP2, TCPP3 and TCPP4 based on TCPP, and coupled with Rhodamine B to prepare TCPP1-DM, TCPP2-DM, TCPP3-DM and TCPP4-DM.
  • 10A and 10B show flow cytometric analysis of tumor cell binding and permeation experiments of the above polypeptide drugs, and the ability of TCPP1-DM penetrating peptide drugs to penetrate K562 cells was found after changing two KKRK sequences in the TCPP sequence into AAAA sequences.
  • TCPP2-DM and TCPP3-DM which changed the KKRK sequence alone to AAAA, had no significant effect on the ability of the penetrating peptide to penetrate K562 cells; it is worth noting that TCPP4-DM after removing the amino acid after the EGGR sequence in TCPP Significantly improved the penetration of TCPP.
  • Example 6 Ultrastructural dynamic changes of tumor cell penetrating peptide cell membrane targeting disruptive drug TCPP-DM antitumor application
  • FIG. 11 Observed the dynamic observation of TCPP incubated with MNNG for 2 h under Axio Observer Z1. The results showed that the penetration of the penetrating peptide caused the cell membrane to form gradually increasing vacuoles, and the cells gradually shrunk. When the vacuole ruptured, the cells completely coagulated and died. The contrast between the intracellular vacuoles and cell shrinkage deaths indicated by the same morphological arrows.
  • Figure 12 shows the transmission electron microscopic observation of MNNG cells treated with TCPP-DM 7.5 ⁇ Mol for 24 h. The results showed that vacuoles were formed in MNNG cells, partial bilayer nuclear membrane separation, deep nucleolar staining, mitochondrial and ribosome decomposition.

Abstract

提供了一种细胞穿透肽和细胞穿透肽复合物及二者的应用。本发明提供的细胞穿透肽，其选自如下氨基酸序列：序列1、序列2、序列3、序列4或序列5所示的氨基酸序列，或与序列1-5中的任一序列具有至少50％同一性的氨基酸序列。

Description

一种细胞穿透肽和细胞穿透肽复合物及二者的应用 技术领域
本发明涉及一种细胞膜穿透肽和细胞穿透肽复合物，还涉及该细胞穿透肽和细胞穿透肽复合物的应用。
背景技术
细胞膜是一种磷脂双分子层，它是细胞内、外环境间的半透性屏障，具有选择通透性，保证了细胞内环境的相对稳定性。细胞膜屏障能够阻止某些物质，特别是有害物质由血液进入细胞内，有助于维持细胞的正常功能活动。青霉素作用于原核细胞的细胞壁，青霉素的结构与细胞壁的成分粘肽结构中的D-丙氨酰-D-丙氨酸近似，可与后者竞争转肽酶，阻碍粘肽的形成，造成细胞壁的缺损，使细菌失去细胞壁的渗透屏障，对细菌起到杀灭作用。真核细胞缺乏细胞壁，因此青霉素对真核生物几乎无任何毒性作用。近年发现真核生物一项重要的细胞免疫机制是攻击靶细胞的细胞膜完整性。细胞毒性T淋巴细胞(CTL)和自然杀伤细胞(NK)的细胞质中储存有细胞膜成孔蛋白(Pore-forming protein)，又称穿孔素(Perforin)。当与病毒感染的细胞或肿瘤细胞等靶细胞密切接触相互作用后，CTL和NK细胞释放穿孔素，在靶细胞的细胞膜上形成多聚穿孔素管状通道，导致靶细胞迅速溶解破坏。穿孔素分子量为67kDa左右，由534个氨基酸组成。12-16个穿孔素分子插入靶细胞膜，多聚化形成多个异常的管状通道，其内径平均约16nm。细胞外的Na+、水分经该管道流入细胞内，而细胞内的K+及大分子物质经该管道流出，引起细胞渗透压改变，迅速导致细胞溶解死亡。然而，类似穿孔素的细胞膜破坏药物分子罕见报道，发明穿孔素类的药物无疑将开辟肿瘤和病毒等疾病治疗的新途径。
细胞穿透肽(cell penetrating peptides，CPPs)首先发现于1型人免疫缺陷病毒转录激活因子TAT(human immunodeficiency virus-1 transcription activator，HIV-1TAT)，TAT具有一个活化域(序列37-48，region Ⅱ)，核定位域(序列49-57，regionⅢ)，还有一个活化增强域(序列58-72，regionⅣ)，它对病毒转运到细胞核发挥着重要作用。随着细胞穿透肽(cell penetrating peptides，CPPs)的发现和研究深入，CPPs药物开发逐渐显露出诱人的应用前景。近年的研究确定CPPs是一类以非受体依赖方式穿过细胞膜进入细胞的多肽，由50个左右氨基酸组成，可以通过人工合成，具有细胞毒性低，穿透能力强，免疫反应小，细胞代谢慢等优点。CPPs可以携带多种不同大小和性质的生物活性物质进入细胞，包括难以穿透细胞膜的蛋白质、多肽、寡链核苷酸、小分子化合物、染料、多肽核酸(peptide nucleo acid,PNA)、质粒DNA、siRNA、200nm的脂质体、噬菌体颗粒和超顺磁性粒子等，这一性质为其成为靶向药物的良好载体提供了可能。
CPPs可分为阳离子CPPs、两亲性CPPs和疏水性CPPs三类，至今发现的阳离子CPPs主要有TAT49–57，Penetratin，Polyarginine，P22N，DPV3，DPV6等。TAT主要的功能序列是GRKKRRQRRR， 该序列对于TAT的细胞穿透作用及载体运输作用至关重要。TAT对细胞膜的穿透特性是能量非依赖性，但是也可通过细胞膜的内吞作用进入细胞内。Penetratin是从自然界果蝇触角同源异构蛋白中发现的，可介导细胞外蛋白的内源化，Penetratin的细胞膜穿透性通过细胞膜上脂质体或者神经酰胺形成的结构域介导。阳离子CPPs序列主要含有精氨酸，赖氨酸，组氨酸等阳离子氨基酸。其中精氨酸的胍基基团可以和细胞膜上的带负电荷的磷酸盐、硫酸盐等形成氢键。因此，含有精氨酸的短肽可以在生理情况下穿透细胞膜，进入细胞内。赖氨酸正电荷和精氨酸类似，但是它没有胍基基团，因此穿透能力较精氨酸弱。有研究表明，阳离子CPP穿透细胞膜至少需要带有8个正电荷。两亲性CPPs具有亲脂基团及亲水基团，通过与细胞膜上粘多糖结合而穿透细胞膜。Transportan(GWTLNSAGYLLGKINLKALAALAKKIL)是一种将甘丙肽和黄蜂毒素结合而形成的CPP，以能量依赖性和能量非依赖性两种形式进入细胞，它的细胞膜穿透作用强于TAT及Penetratin。疏水性CPPs主要由疏水性氨基酸组成，荷电量较少，细胞膜穿透性较弱，但疏水性CPPs可通过非共价键进入细胞内，对细胞膜影响较小，能最大限度保存细胞活力。
目前，越来越多的研究应用CPPs作为载体，将难以进入细胞内的分子，如纳米微粒，寡链核苷酸，抗癌药物等运载进细胞内。提高细胞对这些分子的吸收能力，降低耐药性，降低副作用。现有研究表明，有多种CPPs可进行药物分子的转运。在研究中将紫杉醇，甲氨蝶呤和阿霉素连接上CPPs后，可提高抗癌药物的抗癌作用，降低用药量，同时降低了抗癌药物的毒副作用。CPPs增加了肿瘤细胞膜的穿透性，因此可以抵抗肿瘤细胞的MDR，提高肿瘤药物在肿瘤细胞内的局部药物浓度。
CPPs连接抗肿瘤药物有多种不同的方法，其中有CPPs直接连接抗肿瘤药物。这主要应用于阿霉素，紫杉醇等较稳定抗癌药物的连接。在Jue-Yeon Lee的研究中发现，阿霉素连接TAT细胞穿透肽可提高阿霉素77.5％抗肿瘤活性，同时降低毒副作用。有学者将TAT同时连接阿霉素，紫杉醇两种抗癌药物，达到联合用药的效果，在阿霉素-紫杉醇聚合体中连接少量的TAT即可大幅度提高抗肿瘤药物的活性。
除了细胞毒性肿瘤化疗药物以外，以CPP为载体，进入细胞并发挥生物活性的还有蛋白质和核酸两类外源性物质分子。蛋白质类包括酶类、激素、生长因子、神经营养因子、细胞凋亡因子、细胞内信号转导分子等功能蛋白；核酸类包括质粒、双链DNA、反义寡核苷酸和干扰小RNA(small interfering RNA，siRNA)等。通常应用siRNA调节目标基因的活性，但是siRNA带有强烈的负电荷，很难通过细胞膜。而CPP带正电荷，按照一定比例与siRNA混合后可形成牢固的共价键或松散的非共价键连结，有学者将CPP与siRNA用双硫键进行连接，CPP导入细胞的转染效果优于脂质体siRNA复合物(Lipofectamin-siRNA)；血脑屏障严格限制蛋白质药物等大分子的进入，有学者论证了CPP可引导大分子神经营养物质通过血脑屏障，如通过CPP连结抗凋亡蛋白Bcl—xL，可增加该蛋白在脑组织的吸收，能抑制缺血引起的细胞凋亡，并显著降低了脑区坏死面积和神经损伤。
CPPs联合其它靶向性多肽，连接于药物分子聚合物上，实现药物对肿瘤的靶向运输及定向释放，其 中有MT1-7F7p多肽，pH敏感型多肽等。MT1-7F7p是一种能特异性结合于肿瘤周新生血管及神经胶质瘤表面高表达的MT1-MMP分子上的多肽。将MT1-7F7p和CCPs结合于Taxol-PEG-PLA抗肿瘤药物聚合物上，可提高紫杉醇药物靶向释放于神经胶质瘤中，实现药物定向释放。H7K(R2)2是一种pH敏感性细胞穿透肽，它在pH值7.4的正常生理环境中处于稳定状态，但在pH值6.0的酸性环境中被活化，具有胞膜穿透性。由于肿瘤微环境呈酸性，pH值降低，因此H7K(R2)2在到达肿瘤酸性微环境中被活化，将抗肿瘤药物输送到肿瘤细胞中，提高肿瘤微环境局部药物浓度。CPPs越来越多的被应用于抗肿瘤药物靶向运输，定向释放的研究。
光敏药物如卟啉型光敏药物(Photofrin)、盐酸氨基酮戊酸(Aminolevulinic acid,ALA)、海姆泊芬(Hemoporfin)、维替泊芬、多替泊芬(Duteroporphyrin)、mTHPC、初卟啉锡(SnEtz)、甲氧沙林(Methoxsalen)、亚甲基兰、亚甲苯兰、苯卟啉衍生物，lutelium texaphyrins(Lu-Tex)和卟非姆钠
Figure PCTCN2018089742-appb-000001
等光敏药物由于肿瘤选择性摄取率不高，治疗效果有限。依据CPPs细胞渗透和载体能力，将光敏药物连接于CPPs上同样可能增加光敏药物进入细胞内，有利于提高肿瘤光动力治疗效果。
罗丹明(Rhodamine)是一种荧光染料，属于邻苯二酚类多环化合物，主要包括罗丹明B(Rhodamine B)、罗丹明6G(Rhodamine 6G)、罗丹明123(Rhodamine 123)，是细胞荧光标记的常用试剂。罗丹明B分子式C28H31ClN2O3；分子量479.0175，长期服用具有致癌和致突变性；罗丹明6G由等重量的罗丹明B和盐酸苯胺加热至185～190℃保持1.5～2h，再用乙醇和无机酸加以酯化制得，罗丹明6G单独应用具有肿瘤细胞选择性细胞毒性，可与细胞浆中蛋白质发生化学反应，而使细胞失去活力；罗丹明123可透过细胞膜且在活细胞的线粒体内聚集，并发出黄绿色荧光。罗丹明123用于对许多种细胞进行染色，包括植物细胞和细菌。由于细胞内ATP的量与罗丹明123的荧光强度之间有相关性，此荧光染料被应用于检测细胞内的ATP。目前未见罗丹明B和罗丹明123用于抗肿瘤药物开发的研究报道。
目前的药物载体，主要有单克隆抗体、纳米材料、脂质体、腺病毒和细胞穿透肽(cell penetrating peptides，CPPs)等。其中，只有单克隆抗体作为载体可能达到靶向特异性。单克隆抗体载体具有靶向特异性高、亲和力强、稳定性好等优点。缺点是单克隆鼠源性抗体，在应用过程中将不可避免的引起人体内的人抗鼠抗体免疫应答，遗留医疗安全隐患；其次，抗体分子量较大，载药量少，组织细胞渗透性较弱，网状内皮系统和肝脏对抗体的非特异性吸收，均影响实体肿瘤的治疗效果；第三，抗体易受酸碱破坏，药物与抗体连接比例和连接效率不确定；第四，单抗的生产成本高，成功率低，批量生产复杂，生产周期较长，第五，单抗本身属于生物大分子，细胞膜的穿透能力较弱，不能满足携带药物进入细胞内的要求。纳米材料的优势在于对小分子或多肽药物的吸附力和载药量都很强，能运输足够的药物到组织细胞内。然而，纳米材料的组织细胞特异性较差，很容易被正常组织细胞吸附带来不必要的药物副作用。同时，纳米材料的人体毒性和稳定性尚未确定，有待长期临床研究论证。
肿瘤的细胞膜表面过度表达着O-糖基化粘蛋白、磷脂酰丝氨酸和唾液酸神经节苷脂等带负电荷的蛋白，导致肿瘤细胞表面携带网状负电荷。一些药物分子具有靶向细胞膜破坏的作用，如蛇孢假壳素(ophiobolin)、polybia-MPI黄蜂毒液多肽、大环寡肽(cyclotide)、肉桂霉素(cinnamycin)、肉桂酰胺格尔德霉素和耐久霉素(duramycin)。Polybia-MPI是一种从群居黄蜂毒液中提取出来的阳离子抗肿瘤多肽，其序列为IDWKKLLDAAKQIL，可形成α螺旋构象，以非极性脂质细胞膜为靶点，增加细胞膜通透性，使细胞溶解。膀胱癌、前列腺癌以及肿瘤血管内皮细胞对Polybia-MPI敏感。Kahalalide F是一种从海天牛鼠中提取出来的环缩肽，在80mg/kg浓度下对前列腺癌及乳腺癌细胞等具有选择性抑制作用，导致线粒体膜的溶解及溶酶体的增多。Kahalalide F治疗黑色素瘤和非小细胞肺癌已经进入Ⅰ/Ⅱ期临床研究。另一种环状多肽Didemnin B来源于海鞘提取物，阻碍细胞从G1期进入S期，促进肿瘤细胞的凋亡。Didemnin B治疗直结肠癌、转移性乳腺癌和胶质细胞瘤等已经进入Ⅱ期临床研究。此外，噬菌体展示技术筛选得到裸鼠CD133结合的7肽：LQNAPRS(LS-7)、FGF3受体结合的7肽：VLWLKNR(FP16)，APRIL结合的多肽AAAPLAQPHMWA,SSTTTSDKYLSA和SNLHDNNTEKNV均显示了较好的肿瘤抑制效果。依据CPPs细胞渗透和载体能力，将细胞膜破坏药物连接于CPPs上同样可能增加药物进入细胞内，有利于降低毒性，提高肿瘤治疗效果。
几十年来国内外已经投入了相当大的努力发展新的靶向性药物，但是这些抗肿瘤药物依然局限于肿瘤增殖的禁止药物(Parenti et al.,2014)、肿瘤细胞周期调节药物(Gabrielli et al.,2012)和肿瘤细胞凋亡和自噬引导药物等(Amaravadi et al.,2011)。这些药物对肿瘤靶向性不足常常使得肿瘤治疗过程中对正常细胞损伤过大，产生严重的并发症；进一步，传统化学治疗过程中容易发生肿瘤细胞耐药性，究其原因是肿瘤细胞分泌ATP转运蛋白等将化疗药物通过胞吐作用外排到细胞外，影响肿瘤治疗效果。因此有必要发现新的靶向性药物，提高抗肿瘤效率。
已经注意到肿瘤细胞膜过量表达磷脂酰丝氨酸和高糖基化粘蛋白(Peetla et al. 2013；Hoskin and Ramamoorthy  2008)，带有较大比例的阴离子；而正常状态的细胞主要由磷脂酰胆碱、磷脂酰乙醇胺和鞘磷脂构成，呈现中性(Spector and Yorek  1985)。Polybia-MPI抗肿瘤阳离子多肽的研究显示，这样的结构有利于阳离子CPPs与肿瘤细胞膜静电结合，不仅为开发靶向肿瘤细胞的穿透肽药物载体提供了分子基础，也为开发破坏肿瘤细胞膜的穿透肽药物平台提供了可能，但是至今鲜见这种极具前景的破坏肿瘤细胞膜复合药物的报道。
发明内容
本发明为弥补现有技术的不足，提供一种新颖的细胞穿透肽和细胞穿透肽复合物及它们的应用。
本发明第一方面提供一种细胞穿透肽，其选自如下氨基酸序列：序列1、序列2、序列3、序列4或序列5所示的氨基酸序列，或与序列1-5中的任一序列具有至少50％同一性的氨基酸序列，例如，同一性 可以达到50％以上、60％以上、70％以上、80％以上、90％以上、95％以上等等。
在更为优选的方案中，所述细胞穿透肽优选为序列1或序列5所示的氨基酸序列，或与序列1或5具有至少50％同一性的氨基酸序列。
本发明第二方面提供一种细胞穿透肽复合物，其包含一种或两种以上细胞穿透肽，所述细胞穿透肽选自如下氨基酸序列的肽中的至少一种：序列1、序列2、序列3、序列4、序列5所示的氨基酸序列，或与序列1-5中的任一序列具有至少50％同一性的氨基酸序列，例如，同一性可以达到50％以上、60％以上、70％以上、80％以上、90％以上、95％以上等等。在更为优选的方案中，所述细胞穿透肽复合物至少包括如下氨基酸序列中的一种或几种：序列1或序列5所示的氨基酸序列，或与序列1或5具有至少50％同一性的氨基酸序列。
本发明第三方面提供一种核酸分子，其编码上文所述的氨基酸序列。
本发明第四方面提供一种载体，包含上文所述的核酸分子。
本发明第五方面提供一种宿主细胞，其包含上文所述的载体。
本发明第六方面提供一种复合物，包含细胞穿透肽和货物分子偶联而成的化合物，所述细胞穿透肽为上文所述的细胞穿透肽，还包含偶联于细胞穿透肽上的货物分子；优选的，所述复合物包含序列1和5中的至少一种。所述偶联可以是通过共价键或非共价键的方式连接。
本发明的优选实施方式中，所述货物分子优选为但不局限于具有药物活性的分子、具有标记作用的分子、具有靶向作用的分子中的至少一种。
本发明较为优选的实施方式中，所述生物膜破坏药物偶联于细胞穿透肽的氨基酸序列末端，优选为偶联于氨基末端。
本发明第七方面提供一种药物组合物，包括上文所述的复合物；优选的，还包括药学上允许的载体或辅料；优选的，所述药物组合物的剂型包括但不限于胶囊、软胶囊、片剂、口服液、分散片、冻干粉针、注射液或滴丸。所述载体或辅料可以是药学领域常用的稀释剂、赋形剂、粘合剂、湿润剂、崩解剂、吸收促进剂、表面活性剂、吸附载体和润滑剂等等，根据剂型需要而具体选择。
本发明第八方面提供一种应用，上文所述的细胞穿透肽或细胞穿透肽复合物在制备如下a)-j)中的至少一种制剂中的应用：
a)用于治疗或预防肿瘤的制剂，优选的，所述肿瘤包括白血病、肉瘤、鳞状细胞癌或腺癌；
b)用于治疗或预防病毒感染的制剂；
c)生物膜破坏制剂；
d)荧光核素或放射标识试剂，优选的，所述荧光核素或放射标识试剂用于疾病诊断；
e)细胞毒性制剂；
f)光敏制剂；
g)基因治疗制剂；
h)神经营养制剂；
i)靶向制剂；
j)干细胞调节因子制剂。
在制备所述a)的制剂中应用时，所述细胞穿透肽与抗肿瘤药物或罗丹明B类化合物中的至少一种相偶联，其中，所述抗肿瘤药物优选为阿霉素、多西他赛、甲氨蝶呤、阿糖胞苷、5-氟尿嘧啶、顺铂、环磷酰胺或长春新碱中的至少一种，所述罗丹明B类化合物优选罗丹明B；
在制备所述b)的制剂中应用时，所述细胞穿透肽与抗病毒感染药物中的至少一种相偶联；
在制备所述c)的制剂中应用时，所述细胞穿透肽与生物膜破坏药物中的至少一种偶联，所述生物膜破坏药物优选为罗丹明B类化合物，更优选为罗丹明B；
在制备所述d)的制剂中应用时，所述细胞穿透肽与具有标记作用荧光试剂、核素或放射试剂中的至少一种偶联；
在制备所述e)的制剂中应用时，所述细胞穿透肽与细胞毒类药物中的至少一种相偶联，所述细胞毒类药物优选选自阿霉素、多西他赛、甲氨蝶呤、阿糖胞苷、5-氟尿嘧啶、顺铂、环磷酰胺和长春新碱中的至少一种；
在制备所述f)的制剂中应用时，所述细胞穿透肽与光敏药物中的至少一种相偶联，所述光敏药物优选选自甲氧沙林、盐酸氨基酮戊酸、海姆泊芬、维替泊芬、多替泊芬、mTHPC、初卟啉锡、亚甲基兰和亚甲苯兰、苯卟啉衍生物，lutelium texaphyrins(Lu-Tex)、卟非姆钠和血卟啉衍生物中的至少一种；
在制备所述g)的制剂中应用时，所述细胞穿透肽与基因药物中的至少一种相偶联；
在制备所述h)的制剂中应用时，所述细胞穿透肽与神经营养分子中的至少一种相偶联。
在制备所述i)的制剂中应用时，所述细胞穿透肽与具有靶向作用的分子中的至少一种相偶联；
在制备所述j)的制剂中应用时，所述细胞穿透肽与干细胞调节因子中的至少一种相偶联。
本发明第九方面提供一种应用，如上文所述的细胞穿透肽或细胞穿透肽复合物作为运载药物分子的载体，将运载的药物分子递送至目的细胞的细胞质和/或细胞核。可以是体外应用或体内应用。
本发明的优选实施方式中，所述药物分子可以包括抗肿瘤药物、抗病毒感染药物、生物膜破坏药物、细胞毒性药物、光敏药物、基因药物、神经营养分子、干细胞调节因子中的至少一种。
本发明的优选实施方式中，所述具有药物活性的分子包括抗肿瘤药物、抗病毒感染药物、生物膜破坏药物、光敏药物、细胞毒类药物、基因药物、神经营养分子、干细胞调节因子中的至少一种。生物膜破坏药物优选为罗丹明B类化合物。
本发明中，所述基因药物可以是：双链DNA、反义寡核苷酸和干扰小RNA(small interfering RNA，siRNA)等。如CPP连结抗凋亡蛋白Bcl—xL，血管内皮生长因子VEGF siRNA和表皮生长因子HER siRNA等。
本发明的一种优选方案中，所述生物膜破坏药物优选为罗丹明B(Rhodamine B)类化合物，罗丹明6G和罗丹明123等；更优选为罗丹明B。
本发明中，在部分优选实施方式中，所述光敏药物可以选自甲氧沙林(Methoxsalen)、盐酸氨基酮戊酸(Aminolevulinic acid,ALA)、海姆泊芬(Hemoporfin)、维替泊芬、多替泊芬(Duteroporphyrin)、mTHPC、初卟啉锡(SnEtz)、亚甲基兰和亚甲苯兰、苯卟啉衍生物，lutelium texaphyrins(Lu-Tex)、卟非姆钠
Figure PCTCN2018089742-appb-000002
和血卟啉衍生物(HpD)中的至少一种。
本发明中，在部分优选实施方式中，所述细胞毒类药物可以选自阿霉素、多西他赛、甲氨蝶呤、阿糖胞苷、5-氟尿嘧啶、顺铂、环磷酰胺和长春新碱中的至少一种。
本发明中，在部分优选实施方式中，所述神经营养分子例如可以是：神经生长因子NGF、脑源神经营养因子(brain derived neurotrophic factor,BDNF)，神经营养因子3(NT-3)、神经营养因子4/5(NT-4/5)、神经营养因子6(NT-6)、睫状神经营养因子(ciliary neurotrophic factor,CNTF)、胶质细胞源神经营养因子(glial cell line-derived neurotrophic factor,GDNF)和胰岛素样生长因子Ⅰ(insulin like-growth factor-Ⅰ,IGF-Ⅰ)等等。
本发明中，在部分优选实施方式中，所述干细胞调节因子例如可以是：调节细胞周期的各种周期素(Cyclin)和周期素依赖激酶(Cyclin－Dependent Kinase)、基因转录因子Oct4、影响细胞不对称分裂的细胞质因子和干细胞生长因子(Stem cell growth factors,SCF)等等。
本发明中，在部分优选实施方式中，所述具有标记作用的分子包括荧光试剂、核素或放射试剂中的至少一种，作为一种示例，荧光试剂具体如罗丹明类化合物、异硫氰酸荧光素FITC、四氯荧光素(TET)、噻唑橙(TO)、吖啶橙(AO)和荧光素酶等等；核素或放射试剂如碳14、氢3(氚)、碘125、碘131、硫35、磷32等放射性同位素等等。
本发明中，在部分优选实施方式中，所述具有靶向作用的分子例如为CD34、CD56、CD3、表皮生长因子受体HER和血管内皮生长因子受体VEGFR等特异性结合的多肽或抗体。
本发明的细胞穿透肽作为运载药物载体可以引导细胞毒性药物进入细胞质和细胞核，增加药物利用效率，减少并发症的应用；也可以作为运载药物载体引导光敏药物进入细胞质和细胞核，增加光动力疗效，减少并发症的应用；也可以作为运载药物载体在引导siRNA、双链DNA、反义寡核苷酸等基因药物进入细胞质和细胞核，避免使用病毒载体等一些传统基因治疗方法时发生的毒副作用；还可以作为运载药物载体在引导神经营养分子通透血脑屏障进入神经元；还可以作为运载药物载体在引导干细胞调节因 子进入细胞质和细胞核，调节干细胞生长、分化和创面愈合。
本发明提供的技术方案具有如下有益效果：
本发明提供一种易于制备，分子量小，不易产生免疫排斥反应，穿透能力强，细胞内半衰期长的穿透性多肽(或称为细胞穿透肽)。
本发明提供的细胞穿透肽富含碱性氨基酸，带正电荷，可迅速穿透细胞膜和核膜，可人工合成，具有无细胞毒性，安全性高等特点。
本发明提供的细胞穿透肽通过结合荧光或放射元素标识可制成诊断试剂，该细胞肽富含阳离子，可以选择性地与肿瘤细胞结合，富集于肿瘤细胞及其组织，可用于肿瘤等疾病的标识诊断。
本发明提供的细胞穿透肽结合罗丹明B形成的穿透肽细胞膜靶向破坏药物，可以与肿瘤细胞膜结合，渗透肿瘤细胞膜进入细胞质和细胞核，导致细胞膜破坏和细胞溶解死亡，使得本不具有显著抗癌效果的罗丹明B在与本发明的穿透性多肽结合后出现了显著的抗癌效果。
本发明基于穿透肽细胞膜靶向破坏药物的发现，也提供一种穿透肽细胞膜靶向破坏药物的制备方法，该方法是将具有细胞膜蛋白或磷脂破坏作用的化合物耦合于细胞穿透肽末端，利用细胞穿透肽的阳离子与细胞生物膜内的阴离子结合，暴露化合物作用的靶向分子，达到协同合作靶向破坏生物膜结构，溶解细胞，治疗疾病的作用。
本发明还提供一种穿透性多肽结合的光敏药物，增进了光敏药物的细胞膜和细胞核渗透，具有较好的抗癌效果。
本发明还提供一种穿透性多肽结合阿霉素形成的抗癌药物，增加了阿霉素的细胞膜和细胞核渗透，具有显著的抗癌效果。
采用本发明的细胞穿透肽，能够满足以下医学应用和医学研究的要求：
1)较大比例地选择性结合肿瘤细胞，富集于肿瘤组织，可以用于肿瘤组织标记诊断；2)可以穿透细胞膜和核膜，用于靶向细胞膜的药物开发；3)可以用于定位细胞核；4)可以作为载体，运载分子进入肿瘤细胞；5)可以作为载体，运载抗肿瘤药物进入细胞核；6)可以耦合罗丹明B等细胞膜破坏化合物，选择性破坏肿瘤细胞膜，导致肿瘤细胞溶解死亡。
本发明的细胞穿透肽具有以下优点：
1)该多肽穿透能力强，十分钟即可穿透肿瘤细胞膜，进入细胞核；2)该多肽运载能力强，能携带多种分子进入细胞核；3)该多肽分子量小，具有通透性好，不易变性，免疫原性低的特点；4)该多肽制备方便，成本低，可用人工合成方法直接合成，也可以通过基因工程的方法制备；5)该多肽无细胞毒性，经肝脏、肾脏和胃肠代谢。
本发明在优选方案中所提供的穿透肽罗丹明B细胞膜靶向破坏药物具有以下优点：
1)该穿透肽罗丹明B细胞膜破坏药物，选择性破坏肿瘤细胞膜，导致肿瘤细胞溶解死亡，对正常细胞毒性低。2)该穿透肽罗丹明B细胞膜破坏药物对白血病和肉瘤细胞杀伤效果较好；对鳞状上皮癌细胞和正常细胞无显著杀伤效果；3)该穿透肽罗丹明B细胞膜破坏药物能高度聚集于肿瘤组织，有利于该药的抗肿瘤效果。
附图说明
图1：本发明实施例中细胞穿透肽TCPP(Tumor Cell Penetrating Peotides,TCPP)预测的三维结构示意图；
图2：细胞穿透肽FITC-TCPP与肿瘤细胞膜结合和细胞渗透过程；
图3中的图A为细胞穿透肽FITC-TCPP与肿瘤细胞结合和渗透作用的浓度相关性；图3中的图B为细胞穿透肽FITC-TCPP对肿瘤细胞增殖抑制作用的影响；图3中的图C、D为FITC-TCPP多肽的动物体内分布和标记研究结果。
图4为本发明细胞穿透肽细胞毒性药物阿霉素(Tumour Cell Penetrating Peotides-Doxycycline，TCPP-Dox)的设计制备及其抗肿瘤作用的相关结果，其中，图4中的图A-C为TCPP-Dox穿透肽与肿瘤细胞膜结合和细胞渗透能力研究；4D：TCPP-Dox穿透肽的肿瘤细胞杀伤效率研究；
图5为本发明细胞穿透肽靶向细胞膜破坏药物(Tumour Cell Penetrating Peotides Damage Membrane,TCPP-DM)的设计制备和抗肿瘤应用研究的相关结果，共聚焦显微镜观察TCPP-DM穿透肽药物与肿瘤细胞膜结合和细胞质、细胞核渗透的观察结果；
图6为本发明肿瘤细胞穿透肽靶向细胞膜破坏药物TCPP-DM与肿瘤细胞株K562结合效率的流式细胞分析结果；
图7中各图为本发明肿瘤细胞穿透肽靶向细胞膜破坏药物TCPP-DM的抗肿瘤应用研究的相关结果；其中，图7中的图A，B：TCPP-DM穿透肽药物的肿瘤细胞增殖抑制作用；图7中的图C，D：TCPP-DM穿透肽药物治疗前期肿瘤的动物实验；图7中的图E，F：TCPP-DM穿透肽药物治疗后期肿瘤的动物实验；图7中的图G，H：TCPP-DM穿透肽药物治疗肿瘤的动物体内分布分析。
图8：TCPP-DM穿透肽药物诱导细胞凋亡的流式细胞研究结果；
图9：TCPP-DM穿透肽药物对肿瘤细胞增殖抑制实验的拟合分析；
图10中的图A-B为本发明肿瘤细胞穿透肽及其复合物的优化实验结果；
图11-12为本发明肿瘤细胞穿透肽细胞膜靶向破坏药物TCPP-DM抗肿瘤治疗的超微结构动态变化研究。
具体实施方式
为了更好的理解本发明的技术方案，下面结合具体实施例和附图进一步阐述本发明的技术方案。
1.实验方法介绍
1.1试剂及仪器
试剂：CD14A重组蛋白购置于PeproTech,USA；T7Select人肝癌cDNA文库(Novagen,USA)；多肽购置于强耀生物科技有限公司，China；Cell Counting Kit-8(CCK-8)毒性检测试剂盒、Annexin V-FITC/PI凋亡检测试剂盒购置于DOJINDO(东仁化学科技有限公司，Japan)；Importzole、Ivermectin购置于MCE，USA。
Importzole、Ivermectin购置于MCE，USA；K562，Jurkat，MNNG，MG63细胞株均来自于中山大学生命科学院张雁教授实验室；细胞培养基均购置于Sigma-Aldrich，USA。
使用仪器：流式细胞仪(分析)(FACSCalibur)，BD，USA；激光扫描共聚焦显微镜(TCS-SP5)，LEICA，Germany；全自动倒置荧光成像系统(Axio Observer Z1)，ZEISS，Germany；透射电镜，JEOL，Japan；小动物活体成像系统(IVIS Lumina XP SeriesⅢ)，PerkinElmer，USA。
实验动物：实验动物购置于中山大学实验动物中心东校区，SCXK(粤)2016-0029，动物实验伦理由中山大学伦理委员会审批通过。
1.2.细胞培养实验
将白血病细胞株K562、Jurkat细胞培养于RPMI 1640+10％FBS完全培养基中，将骨肉瘤细胞株MG63、MNNG细胞培养于DF+5％FBS完全培养基中，HFF细胞培养于DF+10％FBS完全培养基中，HUVEC细胞培养于SFM+5ng/mL EGF+10％FBS完全培养基中，将所有细胞培养于37℃，5％CO 2培养箱中，当细胞密度达到80％时进行传代或者细胞实验。
1.3.共聚焦显微镜荧光观察实验
待细胞密度达到80％时，悬浮细胞直接离心弃去培养液，PBS缓冲液清洗一次，细胞计数，取5×10^5/ml细胞浓度悬液，加入TCPP多肽，使终浓度为3μmol，避光，常温下孵育15分钟。贴壁细胞则用PBS清洗1次，0.25％TE消化，离心，细胞计数，取2×10^5/ml细胞，种植于含有盖玻片的6孔板中，37℃，5％CO 2培养箱中培养过夜。加入本发明得到的细胞穿透肽TCPPs，使终浓度为3μMol，避光，常温下孵育5min，10min，15min，20min，25min，30min，1h。PBS缓冲液清洗3次，每次4分钟。用4％PFA固定15分钟。PBS缓冲液清洗3次，每次4分钟。使用含有DAPI防荧光淬灭剂封片，激光共聚焦显微镜下观察拍照。Importinα/β阻断实验则在孵育多肽前加入Importzole 25μMol或者Ivermectin 40μMol，37℃，孵育1h，后加入3μMol多肽孵育30min。
1.4.流式细胞检测
待细胞密度达到80％时，悬浮细胞直接离心弃去培养液，PBS缓冲液清洗一次。贴壁细胞使用PBS缓冲液清洗一次，0.25％TE消化细胞，离心，细胞计数。取5×10^5/ml细胞浓度悬液100μl，加入本发明 后续实施例中所获得的的荧光穿透肽至1.5μMol或3μMol,常温孵育15分钟，PBS缓冲液洗3次，使用40μm滤膜过滤，流式分析仪分析。
1.5.细胞杀伤实验
在细胞培养过程中，当细胞密度达到80％时，悬浮细胞直接离心弃去培养液，PBS缓冲液清洗一次，细胞计数。贴壁细胞使用PBS缓冲液清洗一次，0.25％TE消化细胞，离心，细胞计数。取K562及Jurkat细胞5×10^5/ml，MG63，MNNG细胞1×10^5/ml，种植于96孔板中，每孔100μl。细胞杀伤实验分为穿透肽药物NC组(对照组)，1.5μMol组，3μMol组，7.5μMol组，15μMol组。在药物处理24小时及48小时后加入10μl CCK8试剂，37℃，5％CO2培养箱中培养1小时后使用多功能酶标仪在450nm波长下检测吸光值，每组重复3次。计算细胞活性百分比。
1.6.细胞增殖实验
将K562细胞直接离心弃去培养液，PBS缓冲液清洗一次，细胞计数。取K562细胞1×10^5/ml，种植于24孔板中，各细胞分为对照组及多肽药物处理组。每组设3个副孔。每天加入穿透肽药物，使终浓度为3μMol。从第2天开始，每天细胞计数。
1.7.多肽在小鼠体内分布实验
使用20mg/kg戊巴比妥钠以50mg/kg腹腔注射麻醉小鼠；将浓度为15μmol的穿透肽药物100μl鼠尾静脉注射到MNNG颌下皮下成瘤后裸鼠体内。使用PerkinElmer LuminaⅢ小动物活体成像系统对裸鼠成像，使用光谱分离方法，每10分钟拍摄图片一张，共拍摄2小时；使用光谱分离方法对小鼠体内荧光进行光谱分离，分析荧光在裸鼠体内分布情况。颈椎脱臼处死小鼠，解剖心，肝，脾，肺，肾，舌，脑，肿瘤等组织，再次曝光，确认多肽在组织分布情况。组织冰冻切片，丙酮固定，PBS洗3次，每次5min，DAPI染色5min，PBS洗3次，每次5min，防淬灭荧光封片剂封片，激光共聚焦下观察。
1.8.Axio Observer Z1观察
将MNNG骨肉瘤细胞以2×10^5细胞数种植于激光共聚焦皿中，37℃，5％CO 2培养箱中过夜。将MNNG培养于Axio Observer Z1中，培养条件37℃，5％CO 2。加入4.5μmol穿透肽药物，第1h每隔1min拍摄一张照片，第2h每隔2min拍摄一张照片，共拍摄2h。使用软件将照片合成视频。
1.9.透射电镜
将MNNG骨肉瘤细胞培养于10cm培养皿中，当生长至80％～90％时加入7.5μmol穿透肽药物，放置于37℃，5％CO 2培养箱中孵育24h。PBS洗3次，加入2.5％戊二醛4℃固定过夜，PBS清洗3次每次10min。用1％饿酸固定，静置1.5h，PBS清洗3次每次10min。2％醋酸铀快染，4℃，1h。脱水，渗透，包埋，超薄切片，铀铅双染色，透射电镜观察。
下面结合附图对本发明的技术方案做进一步说明：
实施例一、细胞渗透肽的筛选、序列设计和合成
1.噬菌体展示技术筛选肿瘤细胞渗透肽
以肿瘤细胞膜蛋白为靶分子，利用“T7Select人肺癌cDNA文库(Novagen)”富集与细胞膜蛋白结合的噬菌体，经噬菌体结合、洗脱和富集的多次循环，直至噬菌体文库回收率不再增加，随机挑选48个单克隆噬菌体扩增后，通过分光光度计检测每个单克隆噬菌体与肿瘤细胞膜蛋白的结合效率。挑选与肿瘤细胞膜分子结合率高的单克隆噬菌体，经PCR及序列测定分析，确定氨基酸序列1可能为细胞穿透肽，共计54个氨基酸，其中碱性氨基酸29个，占比53.7％，赖氨酸：精氨酸：组氨酸是18：10：1，富含赖氨酸和精氨酸，命名为TCPP(Tumour Cell Penetrating Peotide)，其三维结构预测如图1。在TCPP基础上设计了TCPP1，TCPP2，TCPP3和TCPP4，其氨基酸序列依次如序列表中序列1-5所示。
实施例二、穿透肽标记物的设计制备、体内外细胞标记及渗透性研究
1、穿透肽标记物FITC-TCPP的合成
应用固相合成法，按照上述TCPPs氨基酸序列合成FITC-TCPP(通过商业合成公司上海强耀生物科技有限公司合成)。合成的方向是从C到N端，先将所要合成肽链的羧末端氨基酸的羧基以共价键的结构同一个不溶性的高分子树脂相连，然后以此结合在固相载体上的氨基酸作为氨基组分经过脱去氨基保护基并同过量的活化羧基组分反应接长肽链。这样的步骤重复多次进行下去，即缩合——洗涤——去保护——中和和洗涤——下一轮缩合，最后达到所需要合成的肽链长度。具体合成由下列几个循环组成：①去保护：Fmoc保护的柱子和单体必须用piperidine去除氨基的保护基团。②激活和交联：下一个氨基酸的羧基被一种活化剂所活化。活化的单体与游离的氨基反应交联，形成肽键。③循环：这两步反应反复循环直到合成完成。接着合成的肽从树脂切割和去保护。最后被沉淀、洗脱、冷冻干燥，纯度＞95％，N末端耦合FITC(绿色荧光)标签FITC-TCPP，用于细胞学实验。
2、穿透肽标记物FITC-TCPP在肿瘤细胞标记中的应用
将肿瘤细胞培养于37℃，5％CO 2培养箱中，细胞密度达到80％时，悬浮细胞直接离心弃去培养液，PBS缓冲液清洗一次，细胞计数，取5×10^5/ml细胞浓度悬液，加入FITC-TCPP多肽，使终浓度为3μmol，避光，常温下孵育15分钟。贴壁细胞则用PBS清洗1次，0.25％TE消化，离心，细胞计数，取2×10^5/ml细胞，种植于含有盖玻片的6孔板中，37℃，5％CO 2培养箱中培养过夜。加入FITC-TCPP多肽，使终浓度为20μg/ml，避光，常温下孵育15分钟。PBS缓冲液清洗3次，每次4分钟。用4％PFA固定15分钟。PBS缓冲液清洗3次，每次4分钟。使用含有DAPI防荧光淬灭剂封片，激光共聚焦显微镜下观察拍照。结果如图2所示，图中灰色为FITC-TCPP多肽结合的位置，证明了该多肽首先与肿瘤细胞膜结合，然后渗透到细胞质，再通过核膜渗透到细胞核内。图3A是不同浓度FITC-TCPP与K562细胞孵育30min结合效率，随着FITC-TCPP浓度升高，结合阳性细胞数量也升高，多肽与K562结合呈浓度相关。图3B 显示FITC-TCPP对K562及MG63细胞毒性实验，结果发现FITC-TCPP对K562及MNG63并没有杀伤作用。
3、穿透肽标记物FITC-TCPP在体内组织器官标记中的应用
常规腹腔注射2％戊巴比妥钠麻醉小鼠，鼠尾静脉注射FITC-TCPP多肽100μl(1mg/ml),十分钟后开始小鼠体内成像；使用PerkinElmer LuminaⅢ小动物活体成像系统对裸鼠成像，每五分钟拍摄图片一张，使用光谱分离方法对小鼠体内FITC荧光进行光谱分离，分析荧光在裸鼠体内分布情况,共拍摄4小时；颈椎脱臼处死小鼠，解剖心，肝，脾，肺，肾等组织，再次曝光，确认多肽在组织分布情况；并进行组织冰冻切片，用含DAPI防淬灭封片剂封片，激光共聚焦显微镜下观察，结果如图3C所示，随着时间变化，穿透肽标记物FITC-TCPP经血液循环开始分布于口腔、头颈部、胸腹部。随着时间增加，头部、胸腹部荧光逐步消退，但是4小时以后依然在口腔、肝脏、肾脏、膀胱及肠道依然聚集，尿液中可见荧光排泄。图3D显示小鼠心、肝、脾、肺、肾、脑、舌、颌下组织在体外的荧光成像；在实验中通过对各脏器的组织切片观察，发现FITC-TCPP主要富集在肝、肾、肺、舌及颌下组织。
这些实验结果表明本发明的细胞穿透肽标记物可用于肝、肾、肺、舌及颌下组织等组织器官的标记。
实施例三、穿透肽细胞毒性药物的设计制备及抗肿瘤作用
1、肿瘤细胞穿透肽阿霉素(TCPP-DOX)细胞毒性药物的设计制备
在上述实施例二穿透肽TCPP和FITC-TCPP制备过程中，分别偶联阿霉素(购自意法玛西亚)，制备成FITC-TCPP-Dox(即TCPP-Dox)和FITC-Dox。用HPLC纯化多肽，将纯化后的溶液冻干，避光，既得到纯品。在密封包装，-20度保存以前，分别取少量的成品多肽，做MS的分子量鉴定和HPLC分析的纯度鉴定，鉴定结果显示成FITC-TCPP-Dox药物及FITC-Dox药物合成成功。
2、肿瘤细胞穿透肽阿霉素FITC-TCPP-Dox及FITC-Dox药物的肿瘤细胞杀伤实验
按照常规细胞培养方法，K562和MNNG细胞浓度分别为1×10 5/ml和5×10 4/ml，各自应用RPMI1640和DF培养液，种植于96孔板中，每孔100μl；药物浓度分组为3.5μM，7μM，13.5μM，35μM，每浓度各设置3个副孔；于培养0h及24h添加药物，培养后24h及48h加入CCK8试剂10μl，孵育1h后测定CCK8吸光值，计算细胞杀伤效果。实验结果参见图4A，K562及MNNG这两种肿瘤细胞系对FITC-TCPP-Dox肿瘤药物与FITC-Dox抗肿瘤药物吸收率对比分析，结果显示K562及MNNG对FITC-TCPP-Dox的细胞结合效率较FITC-Dox抗肿瘤药物高。图4B两组荧光密度分析显示FITC-TCPP-Dox与K562及MNNG两种肿瘤细胞结合荧光强度(平均IDO：0.077±0.022及0.055±0.015)显著高于FITC-Dox组(平均IDO：0.028±0.033及0.019±0.006)。图4C显示K562细胞与FITC-TCPP-Dox及FITC-Dox结合的流式细胞分析结果，FITC-TCPP-Dox与K562细胞结合率(93.7％)显著高于FITC-Dox组(6.3％)。图4D比较FITC-TCPP-Dox及FITC-Dox对K562及MNNG细胞杀伤效果分析，结果显示 FITC-TCPP-Dox对K562及MNNG在24h时即产生杀伤作用，起效快，且细胞毒性作用较FITC-Dox强。
实施例三的试验结果表明本发明的肿瘤细胞穿透肽偶联抗肿瘤药物有利于增加药物的吸收，增加抗肿瘤治疗效果。
实施例四、肿瘤细胞穿透肽靶向细胞膜破坏药物(Tumour Cell Penetrating Peotides Damage Membrane,TCPP-DM)的设计制备和抗肿瘤应用研究
1、肿瘤穿透肽细胞膜靶向破坏药物TCPP-DM的设计制备和筛选
在上述实施例二穿透肽TCPP制备过程中，偶联细胞膜化合物罗丹明B(Rhodamine B，红色荧光)，制备成肿瘤穿透肽细胞膜靶向破坏药物TCPP-DM，用HPLC纯化多肽，将纯化后的溶液冻干，避光，既得到纯品。在密封包装，-20度保存以前，分别取少量的成品多肽，做MS的分子量鉴定和HPLC分析的纯度鉴定，鉴定结果显示成TCPP-DM药物合成成功。
2、肿瘤穿透肽细胞膜靶向破坏药物TCPP-DM的肿瘤细胞增殖抑制实验
首先分析肿瘤穿透肽细胞膜靶向破坏药物TCPP-DM的肿瘤细胞结合和渗透能力，在K526细胞培养液中添加3μMol的TCPP-DM药物，分别于添加后5min,10min,15min,20min,25min,30min,1h用共聚焦显微镜观察药物与细胞结合和渗透特性，结果如图5所示：在5min时TCPP-DM结合于细胞膜，10min时渗透到细胞浆，15min时渗透到细胞浆和细胞核，20min和30minTCPP-DM渗入的细胞核固缩，1hour细胞核无DAPI染色，表明细胞凋亡坏死。
图6显示TCPP-DM与肿瘤细胞株K562结合效率的流式细胞分析结果，不同浓度TCPP-DM与K562细胞孵育30min结合效率，随着TCPP-DM浓度升高，结合阳性细胞数量也升高，TCPP-DM与K562结合呈浓度相关。
图7A和7B显示TCPP-DM对不同类型肿瘤细胞进行CCK8细胞增殖抑制实验，结果发现从3μMol浓度开始TCPP-DM对K562、Jurkat、MG63、MNNG等肿瘤细胞即有显著的增殖抑制作用，且随浓度升高，抑制作用增强，但是对正常血管内皮细胞株HUVEC的增殖无显著抑制作用。图7B进一步研究TCPP-DM对K562细胞增殖的长期抑制效果，结果发现从第三天开始K562增殖被抑制，统计第5、6天细胞存活率，发现TCPP-DM处理组细胞生存数量显著低于空白对照组。通过研究TCPP-DM对正常血管内皮细胞株HUVEC的增殖影响，结果发现TCPP-DM对HUVEC细胞增殖无显著抑制作用。
图8应用Annexin Ⅴ/PI染色，分析TCPP-DM处理引导K562及包皮成纤维细胞株HFF的细胞凋亡情况，结果显示TCPP-DM药物3μMol浓度处理24h后K562有22.7％细胞处在凋亡晚期，而HFF处在凋亡晚期细胞为3.76％。
图9，对K562及MG63细胞增殖抑制实验进行拟合分别为y K562＝-9.9233x K562+119.05,R K562 2＝0.9281，y MG63＝-9.0082x MG63+118.81,R MG63 2＝0.901，拟合程度较好，经计算，TCPP-DM对K562及MG63的半数致死量分别为10.35μMol及11.4μMol。
以上实验结果表明：将一些本身细胞毒性较小的化合物与肿瘤细胞穿透肽耦合以后，可以选择性诱导肿瘤细胞凋亡，显著抑制肿瘤细胞增殖，这些肿瘤包括但不限于白血病细胞K562、Jurkat和骨肉瘤细胞MG63、MNNG。
3、肿瘤穿透肽细胞膜靶向破坏药物TCPP-DM抗肿瘤治疗的动物实验研究
培养骨肉瘤细胞MNNG于DF+5％FBS中，当MNNG生长至80％-90％时收集细胞。将MMNG以1×10^6细胞数注射于25只4～5周龄BALB/c nod小鼠颌下皮下。裸鼠随机分组，分为早期治疗组及后期治疗组，每组5只。早期治疗组于皮下注射MNNG骨肉瘤细胞1×10^6细胞数后24小时开始鼠尾静脉给药，后期治疗组注射MNNG 7天后开始隔天鼠尾静脉给药，早期治疗组分为对照(NC)组，TCPP-DM药物5mg/kg组；后期治疗组分为对照(NC)组，TCPP-DM药物5mg/kg组，阿霉素(Dox)4mg/kg组，共给药5次。早期治疗组从出现肿瘤后开始每天监测肿瘤体积，后期治疗组从出现肿瘤后开始隔天检测肿瘤体积，给药5次后处死小鼠。
图7C和7D显示早期治疗组TCPP-DM抗肿瘤治疗的动物实验结果，因早期治疗组及其对照组中各有两只裸鼠未成瘤，予以排除。图7E和7F显示后期治疗组TCPP-DM抗肿瘤治疗的动物实验结果。TCPP-DM抗肿瘤治疗的动物MNNG皮下成瘤的体积均小于未治疗对照组，且与4mg/kg阿霉素治疗效果相类似。该动物实验表明TCPP-DM具有明显的抗肿瘤治疗效果。
进一步的研究，如图7G和7H显示，TCPP-DM除了分布于小鼠体内的肝、肾、肺等代谢器官以外，主要富集肿瘤区域，有利于药物对肿瘤治疗和利用药物进行肿瘤诊断及随访。这种肿瘤富集效应可能与阳离子TCPP与肿瘤细胞膜静电结合有关，不仅有利于TCPP-DM的肿瘤选择性治疗，也可用于肿瘤的体内标记和诊断。
实施例五、肿瘤细胞穿透肽及其复合物的优化实验
为了进一步明确TCPP的功能区域和优化其细胞穿透能力，发明人在TCPP基础上设计和合成了TCPP1，TCPP2，TCPP3和TCPP4，并分别偶联罗丹明B制备成TCPP1-DM，TCPP2-DM，TCPP3-DM和TCPP4-DM。图10A和10B显示上述多肽药物的肿瘤细胞结合和渗透实验的流式细胞分析，将TCPP序列中的两个KKRK序列改变成AAAA序列后发现TCPP1-DM穿透肽药物对K562细胞的穿透能力显著下降；将单独KKRK序列改变成AAAA的TCPP2-DM和TCPP3-DM对穿透肽渗透K562细胞的能力 并没有明显影响；值得注意的是将TCPP中EGGR序列后的氨基酸去掉后的TCPP4-DM显著提升了TCPP的渗透能力。
实施例六、肿瘤细胞穿透肽细胞膜靶向破坏药物TCPP-DM抗肿瘤应用的超微结构动态变化
图11，在Axio Observer Z1下观察TCPP与MNNG孵育2h的动态观察，结果显示穿透肽渗透使细胞膜形成逐渐增大的空泡，同时细胞逐步皱缩，当空泡破裂后，细胞完全凝固死亡。相同形态箭头所示细胞内空泡及细胞皱缩死亡情况的前后对比。
图12显示是MNNG细胞在TCPP-DM 7.5μMol处理24h后的透射电镜观察，结果显示MNNG细胞内形成空泡，部分双层核膜分离，核仁深染，线粒体及核糖体分解。
以上所述，仅是本发明的较佳实施例而已，并非对本发明做任何形式上的限制，故凡未脱离本发明技术方案的内容，依据本发明的技术实质对以上实施例所做的任何简单修改、等同变化与修饰，均仍属于本发明技术方案的范围内。

Claims (18)

  1. 一种细胞穿透肽，其特征在于，其选自如下氨基酸序列：序列1、序列2、序列3、序列4或序列5所示的氨基酸序列，或与序列1-5中的任一序列具有至少50％同一性的氨基酸序列。
  2. 一种细胞穿透肽复合物，其特征在于，其包含一种或两种以上的细胞穿透肽，所述细胞穿透肽选自具有如下氨基酸序列的肽：序列1、序列2、序列3、序列4或序列5所示的氨基酸序列，或与序列1-5中的任一序列具有至少50％同一性的氨基酸序列。
  3. 一种核酸分子，其特征在于，其编码权利要求1或2所述的氨基酸序列。
  4. 一种载体，其特征在于，包含权利要求3所述的核酸分子。
  5. 一种宿主细胞，其特征在于，其包含权利要求4所述的载体。
  6. 一种复合物，其特征在于，包含细胞穿透肽和货物分子偶联而成的化合物，所述细胞穿透肽为权利要求1所述的细胞穿透肽；优选的，所述细胞穿透肽的氨基酸序列如序列1或序列5所示。
  7. 根据权利要求6所述的复合物，其特征在于，所述货物分子为罗丹明B(Rhodamine B)，所述罗丹明B偶联于细胞穿透肽的氨基酸序列末端，优选为偶联于氨基末端。
  8. 根据权利要求6所述的复合物，其特征在于，所述货物分子选自具有药物活性的分子、具有标记作用的分子、具有靶向作用的分子中的至少一种。
  9. 根据权利要求8所述的复合物，其特征在于，所述具有药物活性的分子包括抗肿瘤药物、抗病毒感染药物、生物膜破坏药物、光敏药物、细胞毒类药物、基因药物、神经营养分子、干细胞调节因子中的至少一种。
  10. 根据权利要求9所述的复合物，其特征在于，所述生物膜破坏药物偶联于细胞穿透肽的氨基酸序列末端，优选为偶联于氨基末端。
  11. 根据权利要求9或10所述的复合物，其特征在于，所述生物膜破坏药物为蛇孢假壳素(ophiobolin)、大环寡肽(cyclotide)、肉桂霉素(cinnamycin)和耐久霉素(duramycin)类化合物；优选为蛇孢假壳素。
  12. 根据权利要求9所述的复合物，其特征在于，所述光敏药物选自甲氧沙林、盐酸氨基酮戊酸、海姆泊芬、维替泊芬、多替泊芬、mTHPC、初卟啉锡、亚甲基兰和亚甲苯兰、苯卟啉衍生物，lutelium texaphyrins(Lu-Tex)、卟非姆钠和血卟啉衍生物中的至少一种；
    所述细胞毒类药物选自阿霉素、多西他赛、甲氨蝶呤、阿糖胞苷、5-氟尿嘧啶、顺铂、环磷酰胺和长春新碱中的至少一种。
  13. 根据权利要求8所述的复合物，其特征在于，所述具有标记作用的分子包括荧光试剂、核素或放射试剂中的至少一种。
  14. 一种药物组合物，其特征在于，包括权利要求6-13任一项所述的复合物；优选的，还包括药学上允许的载体或辅料；优选的，所述药物组合物的剂型为胶囊、软胶囊、片剂、口服液、分散片、冻干粉针、注射液或滴丸。
  15. 一种应用，其特征在于，权利要求1所述的细胞穿透肽或权利要求2所述的细胞穿透肽复合物在制备如下a)-j)中的至少一种制剂中的应用：
    a)用于治疗或预防肿瘤的制剂，优选的，所述肿瘤包括白血病、肉瘤、鳞状细胞癌或腺癌；
    b)用于治疗或预防病毒感染的制剂；
    c)生物膜破坏制剂；
    d)荧光核素或放射标识试剂，优选的，所述荧光核素或放射标识试剂用于疾病诊断；
    e)细胞毒性制剂；
    f)光敏制剂；
    g)基因治疗制剂；
    h)神经营养制剂；
    i)靶向制剂；
    j)干细胞调节因子制剂。
  16. 根据权利要求15所述的应用，其特征在于，在制备所述a)的制剂中应用时，所述细胞穿透肽与抗肿瘤药物或罗丹明B类化合物中的至少一种相偶联，其中，所述抗肿瘤药物优选为阿霉素、多西他赛、甲氨蝶呤、阿糖胞苷、5-氟尿嘧啶、顺铂、环磷酰胺或长春新碱中的至少一种，所述罗丹明B类化合物优选罗丹明B；
    在制备所述b)的制剂中应用时，所述细胞穿透肽与抗病毒感染药物中的至少一种相偶联；
    在制备所述c)的制剂中应用时，所述细胞穿透肽与生物膜破坏药物中的至少一种偶联，所述生物膜破坏药物优选为罗丹明B类化合物，更优选为罗丹明B；
    在制备所述d)的制剂中应用时，所述细胞穿透肽与具有标记作用荧光试剂、核素或放射试剂中的至少一种偶联；
    在制备所述e)的制剂中应用时，所述细胞穿透肽与细胞毒类药物中的至少一种相偶联，所述细胞毒类药物优选选自阿霉素、多西他赛、甲氨蝶呤、阿糖胞苷、5-氟尿嘧啶、顺铂、环磷酰胺和长春新碱中的至少一种；
    在制备所述f)的制剂中应用时，所述细胞穿透肽与光敏药物中的至少一种相偶联，所述光敏药物优选选自甲氧沙林、盐酸氨基酮戊酸、海姆泊芬、维替泊芬、多替泊芬、mTHPC、初卟啉锡、亚甲基兰和亚甲苯兰、苯卟啉衍生物，lutelium texaphyrins(Lu-Tex)、卟非姆钠和血卟啉衍生物中的至少一种；
    在制备所述g)的制剂中应用时，所述细胞穿透肽与基因药物中的至少一种相偶联；
    在制备所述h)的制剂中应用时，所述细胞穿透肽与神经营养分子中的至少一种相偶联。
    在制备所述i)的制剂中应用时，所述细胞穿透肽与具有靶向作用的分子中的至少一种相偶联；
    在制备所述j)的制剂中应用时，所述细胞穿透肽与干细胞调节因子中的至少一种相偶联。
  17. 一种应用，其特征在于，如权利要求1所述的细胞穿透肽或权利要求2所述的细胞穿透肽复合物作为运载药物分子的载体，将运载的药物分子递送至目的细胞的细胞质和/或细胞核。
  18. 根据权利要求17所述的应用，其特征在于，所述药物分子包括抗肿瘤药物、抗病毒感染药物、生物膜破坏药物、细胞毒性药物、光敏药物、基因药物、神经营养分子、干细胞调节因子中的至少一种。
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