WO2019047299A1 - 具有核定位能力的透皮短肽及其应用 - Google Patents

具有核定位能力的透皮短肽及其应用 Download PDF

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WO2019047299A1
WO2019047299A1 PCT/CN2017/105051 CN2017105051W WO2019047299A1 WO 2019047299 A1 WO2019047299 A1 WO 2019047299A1 CN 2017105051 W CN2017105051 W CN 2017105051W WO 2019047299 A1 WO2019047299 A1 WO 2019047299A1
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short peptide
transdermal
cells
protein
nuclear localization
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PCT/CN2017/105051
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French (fr)
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张舒羽
曹建平
余道江
朱巍
王文洁
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苏州大学
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Priority to US16/484,153 priority Critical patent/US10858408B2/en
Publication of WO2019047299A1 publication Critical patent/WO2019047299A1/zh
Priority to US17/090,125 priority patent/US11261222B2/en

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    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K14/00—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
    • C07K14/435—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans
    • C07K14/46—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans from vertebrates
    • C07K14/47—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans from vertebrates from mammals
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K14/00—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K47/00—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient
    • A61K47/30—Macromolecular organic or inorganic compounds, e.g. inorganic polyphosphates
    • A61K47/42—Proteins; Polypeptides; Degradation products thereof; Derivatives thereof, e.g. albumin, gelatin or zein
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K47/00—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient
    • A61K47/50—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates
    • A61K47/51—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates the non-active ingredient being a modifying agent
    • A61K47/62—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates the non-active ingredient being a modifying agent the modifying agent being a protein, peptide or polyamino acid
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K49/00—Preparations for testing in vivo
    • A61K49/001—Preparation for luminescence or biological staining
    • A61K49/0013—Luminescence
    • A61K49/0017—Fluorescence in vivo
    • A61K49/0019—Fluorescence in vivo characterised by the fluorescent group, e.g. oligomeric, polymeric or dendritic molecules
    • A61K49/0021—Fluorescence in vivo characterised by the fluorescent group, e.g. oligomeric, polymeric or dendritic molecules the fluorescent group being a small organic molecule
    • A61K49/0041—Xanthene dyes, used in vivo, e.g. administered to a mice, e.g. rhodamines, rose Bengal
    • A61K49/0043—Fluorescein, used in vivo
    • 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
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P17/00—Drugs for dermatological disorders
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N9/00—Enzymes; Proenzymes; Compositions thereof; Processes for preparing, activating, inhibiting, separating or purifying enzymes
    • C12N9/0004—Oxidoreductases (1.)
    • C12N9/0089—Oxidoreductases (1.) acting on superoxide as acceptor (1.15)
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12Y—ENZYMES
    • C12Y115/00—Oxidoreductases acting on superoxide as acceptor (1.15)
    • C12Y115/01—Oxidoreductases acting on superoxide as acceptor (1.15) with NAD or NADP as acceptor (1.15.1)
    • C12Y115/01001—Superoxide dismutase (1.15.1.1)
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K38/00—Medicinal preparations containing peptides
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K2319/00—Fusion polypeptide
    • C07K2319/01—Fusion polypeptide containing a localisation/targetting motif
    • C07K2319/09—Fusion polypeptide containing a localisation/targetting motif containing a nuclear localisation signal
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K2319/00—Fusion polypeptide
    • C07K2319/60—Fusion polypeptide containing spectroscopic/fluorescent detection, e.g. green fluorescent protein [GFP]
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N9/00—Enzymes; Proenzymes; Compositions thereof; Processes for preparing, activating, inhibiting, separating or purifying enzymes
    • C12N9/14—Hydrolases (3)
    • C12N9/16—Hydrolases (3) acting on ester bonds (3.1)
    • C12N9/18—Carboxylic ester hydrolases (3.1.1)

Definitions

  • the invention relates to the field of biotechnology, in particular to a transdermal short peptide having nuclear localization ability and application thereof.
  • the skin is the largest organ in the human body. It covers the surface of the whole body, accounting for about 16% of the body weight, and the total area is 1.2 to 2 square meters.
  • the skin consists of the epidermis layer, the dermis layer and the subcutaneous tissue. It is rich in blood vessels, nerves and various skin appendages, including hair, hair follicles, sweat glands, and sebaceous glands. It is the body's first line of defense, preventing tissue and internal organs from being attacked by external physical, chemical, and biological stimuli. In addition, the skin also has immune, stimulating, regulating body temperature, absorption and excretion and excretion.
  • the stratum corneum is the outermost part of the epidermis and consists mainly of 10-20 flat, dead cells without nuclei. When these cells fall off, the cells below the basal layer are pushed up to form a new stratum corneum.
  • the function of the stratum corneum is to protect the skin physically, mechanically, chemically and biologically, to block the entry of most macromolecular substances from harmful substances and to maintain the integrity of their functions. But the barrier of the stratum corneum also prevents the absorption of exogenous nutrients and drugs that are supplied by the epidermis and dermal tissue through the epidermis.
  • Transdermal penetration enhancers are one type of compounds which can accelerate the penetration of topical drugs into the skin, such as pyrrolidone, azone, anthraquinones, amino acid lipids and the like.
  • topical drugs such as pyrrolidone, azone, anthraquinones, amino acid lipids and the like.
  • the existing transdermal penetration-inducing compounds have a short duration, are easily metabolized, and have a poor transdermal effect, and have skin toxicity at a high concentration, and have little effect on transdermal penetration of biological macromolecules such as proteins.
  • macromolecular proteins and peptide drugs are hydrophilic macromolecules that hardly penetrate the lipophilic barrier of the stratum corneum of the skin. Even with the help of transdermal penetration enhancers, protein molecules with molecular weights greater than 500 Da are difficult to penetrate through the stratum corneum. Therefore, transdermal administration of biologically active macromolecular proteins has not been solved for a long time. How to make biological macromolecules with important value can be administered through the epidermal route has become a difficult and hot topic of research. Physical methods are used to mediate a wide range of protein transdermal studies, including ultrasound, electric shock, microneedle, etc., but because of the dependence of the above methods on equipment, it is difficult to obtain large-scale applications.
  • transdermal short peptide having nuclear localization ability has attracted widespread attention in recent years. After finding a new short peptide that can mediate macroporation, it is expected that the relevant diseases will be treated by transdermal administration by linking functional proteins, polypeptides and compounds to transdermal short peptides. Therefore, it is of great practical significance to provide a transdermal short peptide having nuclear localization ability.
  • the nucleus is the largest and most important organelle in eukaryotic cells, the regulatory center of cytogenetics and metabolism, and one of the most prominent markers of eukaryotic cells distinguished from prokaryotic cells.
  • Both the cell membrane and the nuclear membrane are selectively permeable membranes, which are a kind of protection for cells and nuclei, but also organize the entry of functional cells and nuclear protective molecules.
  • the screening of short peptides that can mediate key molecules into the cell membrane and nuclear membrane and localize to the nucleus is important for carrying important molecules to protect the nucleus.
  • an object of the present invention is to provide a transdermal short peptide having nuclear localization ability and use thereof, and the transdermal short peptide of the present invention can enter cells spontaneously and localize to the nucleus, and can penetrate the stratum corneum of the skin. Entering the cells in the dermis; the short peptide is conveniently synthesized and can be administered transdermally.
  • the present invention provides a transdermal short peptide having nuclear localization ability, the amino acid sequence of which is shown in SEQ ID No. 1, and the transdermal short peptide can enter the eukaryotic cell autonomously.
  • transdermal short peptide is also linked to fluorescein.
  • fluorescein is fluorescein isothiocyanate (FITC), rhodamine or carboxyfluorescein (FAM).
  • the eukaryotic cells are HaCaT cells, WS1 cells, TE-1 cells, Eca-109 cells, HeLa cells or primary skin cells.
  • the transdermal short peptide with nuclear localization ability has an isoelectric point of 10.92 and a molecular weight of 2465 Da.
  • the transdermal short peptide enters eukaryotic cells in a dose- and time-dependent manner at a concentration of 0.1-10 ⁇ mol/L, and can successfully enter the cells after 0.1-240 min.
  • the present invention also provides a fusion protein having nuclear localization ability, comprising a macromolecular protein, one end of which is linked to the above-mentioned transdermal short peptide having a nuclear localization ability (amino acid sequence is shown in SEQ ID No. 1).
  • transdermal short peptide is also linked to fluorescein.
  • the other end of the macromolecular protein is linked to a fluorescent protein.
  • the fluorescent protein is green fluorescent protein (EGFP).
  • the molecular weight of the macromolecular protein is 10-50 KD.
  • amino acid sequence of the macromolecular protein is as shown in SEQ ID No. 2; or the macromolecular protein is human superoxide dismutase 1 (SOD1).
  • the invention also discloses the use of the above-mentioned transdermal short peptide having nuclear localization ability (amino acid sequence as shown in SEQ ID No. 1) for preparing a medicament for treating skin diseases or a transdermal preparation.
  • the skin disease is skin damage, and more preferably, the skin disease is radiation skin damage.
  • transdermal short peptide is also linked to fluorescein.
  • the present invention also provides a medicament for treating a skin disease, comprising the above-mentioned transdermal short peptide having nuclear localization ability and a pharmaceutically acceptable adjuvant.
  • the dosage form of the medicament for treating a skin disease is an infusion, a powder, a lotion, an elixir, an oil, an emulsion, an ointment, a plaster or an aerosol.
  • the present invention has at least the following advantages:
  • the transdermal short peptide disclosed in the present invention has the ability to localize to the nucleus after entering the cell autonomously; on the other hand, it has transdermal ability to penetrate the stratum corneum of the skin and enter the cells in the dermis layer.
  • the short peptide has a small molecular weight, is convenient for artificial synthesis, and can be administered through the epidermis, and has the potential to carry transdermal therapeutic drugs for treating skin diseases.
  • the transdermal short peptide disclosed in the present invention can be fused with a macromolecular protein, and the obtained fusion protein can enter the cell and perform nuclear localization, indicating that the transdermal short peptide of the present invention has a macromolecular protein linked thereto and enters the cell and enters The function of nuclear positioning.
  • Figure 3 is a test result of the transdermal short peptide of the present invention and the control short peptide after FITC labeling into TE-1 cells and nuclear localization ability;
  • Figure 4 is a graph showing the dose- and time-dependent detection of the percentage of HaCaT cells transdermally labeled with FITC in the transdermal short peptide of the present invention
  • Figure 5 is a test result of the fusion protein of the present invention into HaCaT cells and localized to the nucleus;
  • Fig. 6 is a view showing the results of transdermal action of the transdermal short peptide of the present invention.
  • the raw materials and reagents used in the transdermal short peptide having nuclear localization ability provided by the present invention are commercially available.
  • the N-terminal short peptide labeled with FITC was synthesized by the fmoc method, and the amino acid sequence thereof is shown in SEQ ID NO.
  • PBS phosphate buffer
  • In vitro culture of HaCaT The cells were grown to a density of 50%, and a polypeptide solution having a final concentration of 10 ⁇ mol/L was added to the medium, and after 4 hours, DAPI was added for 30 min. The culture solution was discarded, rinsed three times with PBS, and the free fluorescent polypeptide was removed and photographed under a confocal fluorescence microscope.
  • a control polypeptide set forth in SEQ ID NO. 3 was synthesized using the above method, wherein the N-terminus of the control polypeptide was also labeled with FITC.
  • the fluorescent peptide was tested by culturing the cells according to the above method using a control polypeptide. The results were compared with the untreated control cells, and the results are shown in Fig. 1.
  • Fig. 1 shows that a green fluorescent signal can be observed in the nucleus of the cells (the polypeptide-treating group of interest) to which the short peptide of the present invention is added, and is not involved in the addition. There was no green fluorescence in the cells of the control polypeptide (unrelated polypeptide-treated group), indicating that the short peptide of the present invention has a function of entering HaCaT cells.
  • the transdermal short peptides of the present invention and the control polypeptides were synthesized according to the method of Example 1, and the N-termini of both polypeptides were labeled with FITC.
  • the human fibroblast WS1 was cultured according to the method of Example 1. When it was grown to a density of 50%, a polypeptide solution or a control polypeptide solution having a final concentration of 10 ⁇ mol/L was added to the medium, and DAPI was added for 30 min after 4 hours. . The culture solution was discarded, rinsed three times with PBS, and the free fluorescent polypeptide was removed and photographed under a confocal fluorescence microscope. The results were compared with the untreated control cells, and the results are shown in Fig. 2. Fig.
  • transdermal short peptides of the present invention and the control polypeptides were synthesized according to the method of Example 1, and the N-termini of both polypeptides were labeled with FITC.
  • TE-1 cells were cultured according to the method of Example 1. When the growth was carried out to a density of 50%, a polypeptide solution or a control polypeptide solution having a final concentration of 10 ⁇ mol/L was added to the medium, and 4 hours later, DAPI was added for 16 min. The culture solution was discarded, rinsed three times with PBS, and the free fluorescent polypeptide was removed and photographed under a confocal fluorescence microscope. The results were compared with the untreated control cells, and the results are shown in Fig. 3. Fig.
  • the transdermal short peptide of the present invention was synthesized according to the method of Example 1, and the N-terminus of the polypeptide was labeled with FITC.
  • HaCaT cells were cultured in a plurality of culture dishes according to the method of Example 1, and when they were grown to a density of 50%, final concentrations of 0 ⁇ mol/L, 3 ⁇ mol/L, 6 ⁇ mol/L, and 10 ⁇ mol/ were respectively added to each medium.
  • the peptide solution of L was added to DAPI for 4 min after 4 hours.
  • the culture solution was discarded, rinsed three times with PBS, and the free fluorescent polypeptide was removed and photographed under a confocal fluorescence microscope.
  • a polypeptide solution having a final concentration of 10 ⁇ mol/L is added to the medium.
  • DAPI was added for 30 min.
  • the culture solution was discarded, rinsed three times with PBS, and the free fluorescent polypeptide was removed and photographed under a confocal fluorescence microscope.
  • the percentage of cells in the nucleus of the cells in the cells to which the short peptide of the present invention was added was dose-dependent (shown in Fig. 4A) and time-dependent (Fig. 4B). As the dose of the polypeptide increases and as time passes, the more polypeptides that enter the cell.
  • This embodiment provides a fusion protein and detects its ability to enter cells and nuclear localization.
  • the specific methods are as follows:
  • a short peptide having a nuclear localization and transdermal ability (amino acid sequence as shown in SEQ ID NO. 1) was constructed, and a protein represented by SEQ ID No. 2 and a protein fused to green fluorescent protein (EGFP) were constructed.
  • the gene encoding the above protein is in the middle, EGFP is located at its N-terminus, and the nuclear localization and transdermal ability of the short peptide gene is located at its C-terminus.
  • the nucleic acid was designed and synthesized based on the sequence of the polypeptide and the protein, and then inserted into the pET-28a vector (Novagen, USA) to construct pET-28a-EGFP-pep, and the inserted sequence was sequenced and identified, and the sequence was correct.
  • E. coli BL21 (DE3) was transformed with the constructed vector pET-28a-EGFP-pep.
  • IPTG final concentration: 1 mmol/L
  • the lysozyme was added to the above-mentioned induced bacterial solution for one hour, and after ultrasonication, it was centrifuged at 12,000 rpm for 30 minutes at 4 ° C, and the expression product was dissolved in the supernatant. After centrifugation, the supernatant was transferred to a 10 mL Eppendorf tube, and Ni-NTA (Novagen, USA) was added, and shaken at 50 ° C for 2 h at 4 ° C. The above mixture was transferred to a chromatography column, and 4 ml of Wash Buffer (containing PBS and 0.1 M imidazole) was added while the liquid was running out, and the column was washed.
  • Wash Buffer containing PBS and 0.1 M imidazole
  • the HaCaT cells cultured in vitro were grown to a density of 50%, and the above purified fusion protein was added to the culture medium at a final concentration of 10 ⁇ mol/L. After 4 hours, DAPI was added to the nucleus for 30 min. After rinsing three times with PBS, the free fusion protein was removed and photographed under a confocal fluorescence microscope. The results showed that after the addition of the short peptide of the present invention to the fusion protein of the above polypeptide and EGFP, a green fluorescent signal was observed in the nucleus of HaCaT cells (Fig. 5), indicating that the short peptide has a macromolecular protein molecule attached thereto and enters the cell. And the function of nuclear positioning.
  • transdermal short peptide of the present invention (amino acid sequence shown in SEQ ID NO. 1) is used for the preparation of a medicament for treating skin diseases, and after adding a conventional excipient, an aqueous infusion preparation is prepared.
  • the transdermal short peptide of the present invention (amino acid sequence shown in SEQ ID NO. 1) was fused with human superoxide dismutase 1 (SOD1) and smeared on the surface of the mouse whose back epidermis was lost once a day.
  • the control group was applied with SOD1 not linked to the polypeptide of the present invention. The results showed that after three days, the wounds of the experimental group were reduced by 38% compared with the control group, indicating that the short peptide of the invention mediated the entry of SOD1 into cells can effectively treat skin wounds.
  • transdermal short peptide of the present invention (amino acid sequence as shown in SEQ ID NO. 1) is used for the preparation of a medicament for treating skin diseases, and after adding a conventional excipient, a powder is prepared.
  • transdermal short peptide of the present invention (amino acid sequence as shown in SEQ ID NO. 1) is used for the preparation of a medicament for treating skin diseases, and after adding a conventional excipient, an oil preparation is prepared.
  • transdermal short peptide of the present invention (amino acid sequence as shown in SEQ ID NO. 1) is used for the preparation of a medicament for treating skin diseases, and after adding a conventional excipient, an emulsion is prepared.
  • transdermal short peptide of the present invention (amino acid sequence as shown in SEQ ID NO. 1) is used for the preparation of a medicament for treating skin diseases After adding conventional excipients, an ointment is prepared.
  • transdermal short peptide of the present invention (amino acid sequence as shown in SEQ ID NO. 1) is used for the preparation of a medicament for treating skin diseases, and after adding a conventional excipient, a plaster is prepared.
  • transdermal short peptide of the present invention (amino acid sequence as shown in SEQ ID NO. 1) is used for the preparation of a medicament for treating skin diseases, and after adding a conventional excipient, an aerosol is prepared.

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Abstract

本发明提供了一种具有核定位能力的透皮短肽，其氨基酸序列如SEQ ID No.1所示；还提供了一种具有核定位能力的融合蛋白，包括大分子蛋白，大分子蛋白的一端连接上述透皮短肽。还公开了上述透皮短肽在制备治疗皮肤病的药物或透皮制剂中的应用。本发明进一步提供了一种治疗皮肤病的药物，包括上述透皮短肽以及药学上可接受的辅料。

Description

具有核定位能力的透皮短肽及其应用 技术领域
本发明涉及生物技术领域，尤其涉及一种具有核定位能力的透皮短肽及其应用。
背景技术
皮肤是人体最大的器官，它覆盖于全身的表面，约占体重的16％，总面积达1.2～2平方米。皮肤由表皮层、真皮层和皮下组织组成，含有丰富的血管、神经以及多种皮肤附属器，包括毛发、毛囊、汗腺、皮脂腺等。它是机体的第一道防线，防止组织和内脏器官受到外来的物理、化学、生物刺激因子对机体的侵袭。除此之外，皮肤还具有免疫、感受刺激、调节体温、吸收分泌和排泄等作用。角质层是表皮最外层的部分，主要由10-20层扁平、没有细胞核的死亡细胞组成。当这些细胞脱落时，其下方位于基底层的细胞会被推上来，形成新的角质层。角质层的功能是对皮肤进行物理性、机械性、化学性以及生物性的保护，能够阻挡绝大多数大分子物质的进入，使其免受有害物质的损伤，保持其功能的完整性。但是角质层这一屏障也阻止了表皮和真皮组织对通过表皮补给的外源营养物质和药物的吸收。
透皮促渗剂是一类是指可以加快外用药物穿透皮肤的化合物，如吡咯酮类、氮酮、萜类、氨基酸脂类等化合物。然而现有的透皮促渗化合物持续时间短，易被代谢掉，且透皮效果不理想，高浓度时具有皮肤毒性，尤其对于蛋白质等生物大分子的透皮几乎没有作用。
正是由于大分子蛋白质和多肽类药物均系亲水性大分子，几乎不能透过皮肤角质层的亲脂性屏障。即使在透皮促渗剂的帮助下，分子量大于500Da的蛋白质分子也难以透过角质层。因此，长期以来，具有生物活性的大分子蛋白质的透皮给药一直没有解决。如何使得具有重要价值的生物大分子能够通过表皮途径给药成为研究的难点与热点。采用物理方法介导蛋白质透皮研究广泛，包括超声、电击、微针等方法，但是由于上述方法对设备具有依赖性，难以得到大规模应用。
近年来采用短肽介导的蛋白质透皮引起了广泛的关注。寻找到新的能介导大分子透皮的短肽后，有望通过将功能性蛋白质、多肽及化合物与透皮短肽相连后，通过透皮给药的方式治疗相关疾病。因此，提供一种具有核定位能力的透皮短肽具有重要的现实意义。
细胞核(nucleus)是真核细胞内最大、最重要的细胞器，是细胞遗传与代谢的调控中心，是真核细胞区别于原核细胞最显著的标志之一。细胞膜及细胞核膜均是选择性通透的膜，这一方面对于细胞及细胞核是一种保护，但也组织了功能性细胞及细胞核保护分子的进入。因 而筛选能介导关键分子进入细胞膜及核膜并定位于细胞核的短肽对于携带重要分子保护细胞核具有重要意义。但目前未出现关于同时具有核定位及透皮能力的短肽的报道。
发明内容
为解决上述技术问题，本发明的目的是提供一种具有核定位能力的透皮短肽及其应用，本发明的透皮短肽可自主进入细胞并定位于细胞核，且能透过皮肤角质层，进入真皮层中的细胞中；该短肽人工合成方便、能够通过透皮给药。
本发明提供了一种具有核定位能力的透皮短肽，其氨基酸序列如SEQ ID No.1所示，透皮短肽可自主进入真核细胞。
进一步地，透皮短肽还连接有荧光素。
进一步地，荧光素为异硫氰酸荧光素(FITC)、罗丹明或羧基荧光素(FAM)。
进一步地，真核细胞为HaCaT细胞、WS1细胞、TE-1细胞、Eca-109细胞、Hela细胞或原代皮肤细胞。
具有核定位能力的透皮短肽的等电点为10.92，分子量为2465Da。
透皮短肽以剂量依赖性及时间依赖性进入真核细胞，浓度为0.1-10μmol/L，在0.1-240min后可成功进入细胞。
本发明还提供了一种具有核定位能力的融合蛋白，包括大分子蛋白，大分子蛋白的一端连接上述具有核定位能力的透皮短肽(氨基酸序列如SEQ ID No.1所示)。
进一步地，透皮短肽还连接有荧光素。
进一步地，大分子蛋白的另一端连接有荧光蛋白。
进一步地，荧光蛋白为绿色荧光蛋白(EGFP)。
进一步地，大分子蛋白的分子量为10-50KD。
进一步地，大分子蛋白的氨基酸序列如SEQ ID No.2所示；或者大分子蛋白为人超氧化物歧化酶1(SOD1)。
本发明还公开了上述具有核定位能力的透皮短肽(氨基酸序列如SEQ ID No.1所示)在制备治疗皮肤病的药物或透皮制剂中的应用。
优选地，皮肤病为皮肤损伤，更优选地，皮肤病为放射性皮肤损伤。
进一步地，透皮短肽还连接有荧光素。
本发明还提供了一种治疗皮肤病的药物，包括上述具有核定位能力的透皮短肽以及药学上可接受的辅料。
进一步地，治疗皮肤病的药物的剂型为水浸剂、粉剂、洗剂、酊剂、油剂、乳剂、软膏、硬膏或气雾剂。
借由上述方案，本发明至少具有以下优点：
本发明公开的透皮短肽一方面具有自主进入细胞后定位于细胞核的能力；另一方面具有透皮能力，能透过皮肤角质层，进入真皮层中的细胞中。该短肽分子量小，具有人工合成方便、能够通过表皮给药等特点，具有携带治疗皮肤疾病治疗药物透皮的潜力。
本发明公开的透皮短肽可以与大分子蛋白融合后，所得到的融合蛋白能够进入细胞中并进行核定位，说明本发明的透皮短肽具有携带与其相连的大分子蛋白进入细胞并进入核定位的功能。
上述说明仅是本发明技术方案的概述，为了能够更清楚了解本发明的技术手段，并可依照说明书的内容予以实施，以下以本发明的较佳实施例并配合附图详细说明如后。
附图说明
图1是本发明的透皮短肽以及对照短肽经FITC标记后进HaCaT细胞及核定位能力检测结果；
图2是本发明的透皮短肽以及对照短肽经FITC标记后进WS1细胞及核定位能力检测结果；
图3是本发明的透皮短肽以及对照短肽经FITC标记后进TE-1细胞及核定位能力检测结果；
图4是本发明的透皮短肽经FITC标记后进HaCaT细胞百分比的剂量依赖性和时间依赖性检测结果；
图5是本发明的融合蛋白进HaCaT细胞并定位于细胞核的检测结果；
图6是本发明的透皮短肽的透皮作用结果示意图。
具体实施方式
下面结合附图和实施例，对本发明的具体实施方式作进一步详细描述。以下实施例用于说明本发明，但不用来限制本发明的范围。
实施例1
本发明提供的一种具有核定位能力的透皮短肽中所用原料及试剂均可由市场购得。
采用fmoc法合成N端为FITC标记的短肽，其氨基酸序列如SEQ ID NO.1所示。合成后的短肽溶于磷酸缓冲液(PBS，pH＝7.4)，中，配成浓度为500μmol/L的溶液。体外培养HaCaT 细胞，待其生长到密度为50％时，在培养基中加入终浓度为10μmol/L的多肽溶液，4小时后加入DAPI染核30min。弃掉培养液，用PBS漂洗三次，去除游离的荧光多肽后置于共聚焦荧光显微镜下拍照。为了作为对照，使用上述方法合成SEQ ID NO.3所示的对照多肽，其中对照多肽的N端同样用FITC标记。并使用对照多肽，按照上述方法培养细胞后测试荧光结果。与未处理的对照组细胞进行对比，结果如图1所示，图1表明，加入本发明短肽的细胞(目的多肽处理组)内的细胞核中能观察到绿色荧光信号，而在加入了无关对照多肽的细胞(无关多肽处理组)中没有绿色荧光，这表明本发明的短肽具有进入HaCaT细胞的功能。
实施例2
按照实施例1的方法合成本发明的透皮短肽以及对照多肽，且两种多肽的N端均采用FITC进行标记。按照实施例1的方法培养人成纤维细胞WS1，待其生长到密度为50％时，在培养基中加入终浓度为10μmol/L的多肽溶液或对照多肽溶液，4小时后加入DAPI染核30min。弃掉培养液，用PBS漂洗三次，去除游离的荧光多肽后置于共聚焦荧光显微镜下拍照。与未处理的对照组细胞进行对比，结果如图2所示，图2表明，加入本发明短肽的细胞(目的多肽处理组)内的细胞核中能观察到绿色荧光信号，而在加入了无关对照多肽的细胞(无关多肽处理组)中没有绿色荧光，这表明本发明的短肽具有进入WS1细胞的功能。
实施例3
按照实施例1的方法合成本发明的透皮短肽以及对照多肽，且两种多肽的N端均采用FITC进行标记。按照实施例1的方法培养TE-1细胞，待其生长到密度为50％时，在培养基中加入终浓度为10μmol/L的多肽溶液或对照多肽溶液，4小时后加入DAPI染核30min。弃掉培养液，用PBS漂洗三次，去除游离的荧光多肽后置于共聚焦荧光显微镜下拍照。与未处理的对照组细胞进行对比，结果如图3所示，图3表明，加入本发明短肽的细胞(目的多肽处理组)内的细胞核中能观察到绿色荧光信号，而在加入了无关对照多肽的细胞(无关多肽处理组)中没有绿色荧光，这表明本发明的短肽具有进入TE-1细胞的功能。
实施例4
按照实施例1的方法合成本发明的透皮短肽，且多肽的N端采用FITC进行标记。按照实施例1的方法在多个培养皿中培养HaCaT细胞，待其生长到密度为50％时，在各培养基中分别加入终浓度为0μmol/L、3μmol/L、6μmol/L和10μmol/L的多肽溶液，4小时后加入DAPI染核30min。弃掉培养液，用PBS漂洗三次，去除游离的荧光多肽后置于共聚焦荧光显微镜下拍照。
或待HaCaT细胞生长到密度为50％时，在培养基中加入终浓度为10μmol/L的多肽溶液， 于0、60、120和240分钟后，加入DAPI染核30min。弃掉培养液，用PBS漂洗三次，去除游离的荧光多肽后置于共聚焦荧光显微镜下拍照。结果如图4所示，加入本发明短肽的细胞内的细胞核中荧光信号的细胞百分比呈剂量依赖性(图4A所示)和加入时间依赖性(图4B所示)。随多肽剂量的增大以及时间的延长，进入细胞中的多肽越多。
实施例5
本实施例提供了一种融合蛋白，并对其进入细胞及核定位能力进行了检测，具体方法如下：
1、构建偶联蛋白与具有核定位和透皮能力短肽融合的基因
构建具有核定位和透皮能力短肽(氨基酸序列如SEQ ID NO.1所示)，并构建SEQ ID No.2所示的蛋白和和绿色荧光蛋白(EGFP)融合的蛋白质。其中上述蛋白的编码基因位于中间，EGFP位于其N端，具有核定位和透皮能力短肽基因位于其C端。依据多肽及蛋白质的序列设计并合成核酸，然后插入pET-28a载体(美国Novagen公司)中，构建成为pET-28a-EGFP-pep，插入序列经测序鉴定，序列正确。
2、融合基因在大肠杆菌中的表达
用构建的载体pET-28a-EGFP-pep转化E.coli BL21(DE3)。菌液生长至OD600＝0.6时，加入IPTG(终浓度为1mmol/L)，30℃下诱导10小时。
3、融合蛋白纯化
向上述诱导表达的菌液中加入溶菌酶处理一小时，经过超声破碎后，4℃12000rpm离心30分钟，表达产物溶于上清液中。取离心后上清至10mL Eppendorf管中，加入Ni-NTA(美国Novagen公司)，4℃，50rpm震荡2h。将上述混合物转移至层析柱中，待液体快流尽时加入4ml Wash Buffer(含有PBS及0.1M的咪唑)，洗涤层析柱。待液体快流尽时加入300μL洗脱液，(含有PBS及0.4M的咪唑)，在核酸蛋白质检测仪作用下收集流出的蛋白峰。纯化后液体经SDS-PAGE检验，分子量大小约为50KD，与计算值相符，上述方法可获得纯度大于90％的融合蛋白。
4、融合蛋白的进细胞及核定位能力检测
体外培养的HaCaT细胞，生长到密度为50％时，在培养基中加入终浓度为10μmol/L的上述纯化的融合蛋白，4小时后加入DAPI染核30min。用PBS漂洗三次，去除游离的融合蛋白后置于共聚焦荧光显微镜下拍照。结果显示：加入本发明短肽融合上述多肽及EGFP的融合蛋白后，在HaCaT细胞核中能观察到绿色荧光信号(图5)，这表明该短肽具有携带与之相连的大分子蛋白质分子进入细胞并进行核定位的功能。
实施例6
本实施例对短肽的透皮作用进行检测，具体方法如下：
采用8周龄SD大鼠(体重250g)进行实验。将SD大鼠背部毛剃掉，将实施例1制备的FITC标记的短肽和对照短肽分别溶于磷酸缓冲液(PBS，pH＝7.4)中，配成浓度为500μmol/L的溶液。将SD大鼠分为三组：1)对照组，背部不涂抹多肽溶液；2)大鼠背部表皮上涂抹SEQ ID NO.3所示的对照短肽；3)大鼠背部表皮上涂抹SEQ ID NO.1所示的短肽。组2)和组3)均涂抹50μL多肽溶液。三小时后洗去涂抹区域的多肽；处死上述SD大鼠，取涂抹多肽处的皮肤，冰冻切片后在分别置于荧光显微镜下观察。试验结果表明：对照组(未处理大鼠皮肤)和对照短肽组(涂抹无关对照组)大鼠皮肤的真皮组织中未见绿色荧光(图6中箭头指部位为具有自发荧光的角质层)；而涂抹本发明的短肽(涂抹目的多肽)的大鼠皮肤切片中，真皮区域的皮肤附属器中能明显观察到荧光信号(图6中椭圆圈中所示的荧光信号)，表明该短肽可透皮进入真皮组织中。
实施例7
将本发明的透皮短肽(氨基酸序列如SEQ ID NO.1所示)用于制备治疗皮肤疾病的的药物，添加常规辅料后，制备水浸剂。
将本发明的透皮短肽(氨基酸序列如SEQ ID NO.1所示)与人超氧化物歧化酶1(SOD1)融合表达后，涂抹在背部表皮丢失的小鼠表面，每日一次。对照组涂抹未与本发明多肽相连接的SOD1。结果表明，三天后，实验组小鼠创面较对照组减小38％，表明该发明短肽介导SOD1进入细胞可有效治疗皮肤创伤。
实施例8
将本发明的透皮短肽(氨基酸序列如SEQ ID NO.1所示)用于制备治疗皮肤疾病的的药物，添加常规辅料后，制备粉剂。
实施例9
将本发明的透皮短肽(氨基酸序列如SEQ ID NO.1所示)用于制备治疗皮肤疾病的的药物，添加常规辅料后，制备油剂。
实施例10
将本发明的透皮短肽(氨基酸序列如SEQ ID NO.1所示)用于制备治疗皮肤疾病的的药物，添加常规辅料后，制备乳剂。
实施例11
将本发明的透皮短肽(氨基酸序列如SEQ ID NO.1所示)用于制备治疗皮肤疾病的的药 物，添加常规辅料后，制备软膏。
实施例12
将本发明的透皮短肽(氨基酸序列如SEQ ID NO.1所示)用于制备治疗皮肤疾病的的药物，添加常规辅料后，制备硬膏。
实施例13
将本发明的透皮短肽(氨基酸序列如SEQ ID NO.1所示)用于制备治疗皮肤疾病的的药物，添加常规辅料后，制备气雾剂。
以上所述仅是本发明的优选实施方式，并不用于限制本发明，应当指出，对于本技术领域的普通技术人员来说，在不脱离本发明技术原理的前提下，还可以做出若干改进和变型，这些改进和变型也应视为本发明的保护范围。
Figure PCTCN2017105051-appb-000001
Figure PCTCN2017105051-appb-000002

Claims (10)

  1. 一种具有核定位能力的透皮短肽，其特征在于：其氨基酸序列如SEQ ID No.1所示，所述透皮短肽可自主进入真核细胞。
  2. 根据权利要求1所述的透皮短肽，其特征在于：所述透皮短肽还连接有荧光素。
  3. 根据权利要求2所述的透皮短肽，其特征在于：所述荧光素为异硫氰酸荧光素、罗丹明或羧基荧光素。
  4. 根据权利要求1所述的透皮短肽，其特征在于：所述真核细胞为HaCaT细胞、WS1细胞、TE-1细胞、Eca-109细胞或Hela细胞。
  5. 一种具有核定位能力的融合蛋白，其特征在于：包括大分子蛋白，所述大分子蛋白的一端连接权利要求1-4中任一项所述的透皮短肽。
  6. 根据权利要求5所述的具有核定位能力的融合蛋白，其特征在于：所述大分子蛋白的另一端连接有荧光蛋白。
  7. 根据权利要求5或6所述的具有核定位能力的融合蛋白，其特征在于：所述大分子蛋白的分子量为10-50KD。
  8. 根据权利要求5或6所述的具有核定位能力的融合蛋白，其特征在于：所述大分子蛋白的氨基酸序列如SEQ ID No.2所示；或者所述大分子蛋白为人超氧化物歧化酶。
  9. 根据权利要求1-4中任一项所述的透皮短肽在制备治疗皮肤病药物或透皮制剂中的应用。
  10. 一种治疗皮肤病的药物，其特征在于：包括权利要求1-4中任一项所述的透皮短肽以及药学上可接受的辅料。
PCT/CN2017/105051 2017-09-05 2017-09-30 具有核定位能力的透皮短肽及其应用 WO2019047299A1 (zh)

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