WO2019169697A1 - Hepatoma cell-targeting antimicrobial peptide chimera m27-39-htpp and use therof - Google Patents

Hepatoma cell-targeting antimicrobial peptide chimera m27-39-htpp and use therof Download PDF

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WO2019169697A1
WO2019169697A1 PCT/CN2018/083403 CN2018083403W WO2019169697A1 WO 2019169697 A1 WO2019169697 A1 WO 2019169697A1 CN 2018083403 W CN2018083403 W CN 2018083403W WO 2019169697 A1 WO2019169697 A1 WO 2019169697A1
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htpp
targeting
antimicrobial peptide
peptide chimera
liver cancer
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卢雪梅
陈朝霞
桂水清
金小宝
朱家勇
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广东药科大学
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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
    • C07K14/43504—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans from invertebrates
    • C07K14/43563—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans from invertebrates from insects
    • C07K14/43577—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans from invertebrates from insects from flies
    • 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
    • C07K14/43504—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans from invertebrates
    • C07K14/43563—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans from invertebrates from insects
    • C07K14/43577—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans from invertebrates from insects from flies
    • C07K14/43581—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans from invertebrates from insects from flies from Drosophila
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P35/00—Antineoplastic agents
    • 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
    • C07K14/44—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans from protozoa
    • C07K14/445—Plasmodium
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K38/00—Medicinal preparations containing peptides
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    • 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
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K2319/00—Fusion polypeptide
    • C07K2319/33—Fusion polypeptide fusions for targeting to specific cell types, e.g. tissue specific targeting, targeting of a bacterial subspecies

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  • the invention relates to the field of biotechnology, in particular to a liver cancer cell-targeting antimicrobial peptide chimera M27-39-HTPP and application thereof.
  • Liver cancer is the second most common disease in the world. It is a highly malignant tumor. It is a frequently-occurring disease and a major disease in China. Despite the combination of surgical resection and radiotherapy/chemotherapy, the mortality rate remains high. In 2013, the World Health Organization announced that primary liver cancer caused 745,517 deaths worldwide. About 383,000 people die of liver cancer each year in China, accounting for 51% of the total number of liver cancer deaths worldwide, which has brought serious social and economic burdens to the country. At present, there is still no effective drug for the prevention and treatment of liver cancer. Targeted molecules are used as carriers, and anti-cancer drugs are used as biological targets for warheads. It is a hot topic in liver cancer research in the past 20 years and has great social and economic significance. .
  • Hepatocyte-targeting and penetrating peptide is derived from the upstream of the conserved I region of Plasmodium circumsporozoite protein (CSP). Studies have shown that HTPP not only contains heparan sulfate binding sequence, but also specifically binds to liver.
  • the cell surface receptor, heparan sulfate proteoglycans (HSPG) also contains a PELEX/VTS motif, which can effectively penetrate the cell membrane and mediate CSP entry into the cell to achieve subcellular localization.
  • HSPG is a complex macromolecule composed of nucleoprotein molecules and glycosaminoglycan (GAG) covalently bonded by glycosidic bonds.
  • HSPG of the liver has a unique GAG chain, and its degree of sulfation is significantly higher than other tissues.
  • the expression of HSPG in liver cancer tissues is significantly different from that in normal liver tissues, and the expression level is directly proportional to the metastatic potential of cancer cells. Therefore, HTPP can not only act as a liver targeting molecule, but also enrich the drug with the liver, and can also interfere with HSPG receptor function and inhibit tumor metastasis.
  • Antimicrobial peptides are small molecular peptides produced by the biological immune system against external pathogen infections. They have a series of attractive biological activities, including antibacterial, anti-inflammatory, anti-viral, anti-parasitic, and inhibition. Tumor cells and immunomodulatory activities.
  • the antibacterial peptide can destroy the bacterial cell membrane or pass through the cell membrane to act on the intracellular target site, and the mechanism of action is unique, and it is not easy to produce drug resistance, and is toxic to normal human cells and has few side effects. Therefore, in the current research and development and clinical effects of traditional antibacterial, antiviral and antitumor drugs are not satisfactory, the above characteristics of antimicrobial peptides have shown good application development prospects.
  • Musca domestica cecropin is an insect antibacterial peptide cloned from the fatty acid cDNA library of Musca domestica larvae from Guangdong Pharmaceutical University/Guangdong Provincial Key Laboratory of Bioactive Drug Research.
  • the ORF region of this gene is 192 bp in length and can encode 63 amino acids.
  • the precursor protein, amino acid positions 1 to 23 are signal peptides ending in a conserved 4 peptide, and the mature peptide contains 40 amino acids, which has strong antibacterial and antitumor activities, and we have further studied the derived peptides on a preliminary basis.
  • M27-39 Compared with natural Musca domestica cecropin, M27-39 not only has more remarkable physiological activity in anti-tumor, but also has smaller molecules and easier entry into cells than natural Musca domestica cecropin.
  • liver cancer cell-targeting antimicrobial peptide chimera M27-39-HTPP it is an object of the present invention to provide a liver cancer cell-targeting antimicrobial peptide chimera M27-39-HTPP.
  • Another object of the present invention is to provide the use of the liver cancer cell-targeting antimicrobial peptide chimera M27-39-HTPP for the preparation of a targeted anti-hepatocarcinoma drug.
  • liver cancer cell of the present invention targets the antimicrobial peptide chimera M27-39-HTPP, and the amino acid sequence thereof is shown in SEQ ID NO: 1.
  • the SEQ ID NO: 1 is VAQQAANVAATLKNSRSLGENDDGNNEDNEKLR.
  • the liver cancer cell of the invention targets the antibacterial peptide chimera M27-39-HTPP, and adopts a solid phase chemical synthesis method to synthesize a crude polypeptide by a polypeptide synthesizer; and synthesizes the polypeptide by solid phase synthesis.
  • the synthesized polypeptide was purified by reverse-phase high performance liquid chromatography, and the synthesized polypeptide was identified by electrospray mass spectrometry to complete the preparation of the polypeptide.
  • the liver cancer cell targeted antibacterial peptide chimera M27-39-HTPP designed and developed by the invention has remarkable liver-targeting transmembrane action and anti-hepatocarcinoma activity, and can be used for preparing targeted anti-liver cancer drugs.
  • Figure 1 is a high performance liquid chromatogram of a liver cancer cell-targeted antimicrobial peptide chimera M27-39-HTPP.
  • Figure 2 is a mass spectrum of a liver cancer cell-targeted antimicrobial peptide chimera M27-39-HTPP.
  • Figure 3 shows the results of tissue binding experiments showing that the liver cancer cell-targeting antimicrobial peptide chimera M27-39-HTPP has liver targeting properties.
  • Figure 4 shows the results of laser confocal observation.
  • the hepatocellular carcinoma cell-targeting antimicrobial peptide chimera M27-39-HTPP can enter the liver cancer cells through the cell membrane.
  • Figure 5 shows that the MTT assay showed that the hepatocellular carcinoma cell-targeting antimicrobial peptide chimera M27-39-HTPP has more significant anti-tumor cell proliferation activity.
  • Figure 6 shows the results of flow cytometry showing that the hepatocellular carcinoma cell-targeting antimicrobial peptide chimera M27-39-HTPP has a more pronounced pro-apoptotic effect.
  • test methods used in the following examples are all conventional methods unless otherwise specified; the materials, reagents and the like used are, if not specified, commercially available reagents and materials.
  • Example 1 Solid phase chemical synthesis synthesis of liver cancer cells targeting antimicrobial peptide chimera M27-39-HTPP
  • the amino acid sequence of the liver cancer cell-targeting antimicrobial peptide chimera M27-39-HTPP is: VAQQAANVAATLKNSRSLGENDDGNNEDNEKLR, as shown in SEQ ID NO: 1, containing 33 amino acids.
  • the preparation of the liver cancer cell-targeting antibacterial peptide chimera M27-39-HTPP was carried out one by one from the C-terminus to the N-terminus, and was completed by a polypeptide synthesizer.
  • Fmoc-X (X is the first amino acid of the C-terminus of the antibacterial peptide MDC) is added to the Wang resin, and then the Fmoc group is removed to obtain the X-Wang resin; then Fmoc-Y-Trt-OH (9- ⁇ ) Methoxycarboxy-trimethyl-Y, Y is the second amino acid of the C-terminal of the antibacterial peptide chimera M27-39-HTPP of liver cancer cells; according to this procedure, it is synthesized from the C-terminus to the N-terminus until the synthesis is completed.
  • the peptide was dissolved in 90% acetonitrile aqueous solution for 30 min, filtered, and subjected to C18 reverse pressure atmospheric column using gradient elution ( The de-agent is methanol and sodium sulfate aqueous solution in a volume ratio of 30:70 to 70:30, the flow rate is 1 mL/min, the detection wave is 220 nm, the main peak is collected, and lyophilized; further purified by reverse phase C18 column, eluting
  • the liquid A was 0.1% TFA/water solution; the eluent B was 0.1% TFA/acetonitrile solution, the elution concentration was
  • mice were deeply anesthetized with sodium pentobarbital (100 mg/kg, IP), normal saline (10 ml, >2 min) and 25 ml of 4% paraformaldehyde (0.1 M sodium phosphate buffer, pH 7.4) (perfused)
  • Pump speed 5ml/min) heart 25G cannulated left ventricle perfusion, collect heart, liver, spleen, lung, kidney and other organs to cut 2-3 mm thick tissue block, fixed with 4% paraformaldehyde for 2h, transfer To 30% sucrose solution (0.1 mmol/L sodium phosphate buffer, pH 7.4) overnight at 4 °C.
  • Tissue blocks were embedded in Tissue Freezing Medium and sliced using a frozen tissue microtome with a section thickness of 10-12 ⁇ m. Tissue frozen sections were washed three times with PBS, 10% BSA blocking solution was added, and the non-specific sites were blocked by incubation at room temperature for 30 min; the blocking solution was discarded, FITC-labeled M27-39-HTPP was added, overnight at 4 ° C, washed with PBS, and mounted. Observed by fluorescence microscope.
  • HepG 2 cells were subcultured in 5% CO 2 in an incubator at 37 ° C with saturated humidity.
  • the medium was DMEM containing 10% fetal bovine serum, 100 U/mL ampicillin and 100 U/mL streptomycin, when the cells grew close to 80%.
  • a single cell suspension was prepared by digesting with 0.25% trypsin and accurately counted.
  • the medium was discarded, and after washing the plate with PBS, 10 ⁇ L of 5 mg/ml MTT solution and 100 ⁇ l of the medium were added to each well and placed in a constant temperature incubator for further 4 hours. The plate was taken out, the supernatant was discarded, 100 ⁇ L of DMSO was added to each well, and the plate was shaken for 30 min. After the crystals produced by the oxidation of MTT were completely dissolved, the OD value was measured using a microplate reader, the measurement wavelength was 570/630 nm, and the experiment was repeated 3 times, and the average value was taken.
  • the HepG 2 cells growing in log phase were adjusted to a concentration of 3.0-5.0 ⁇ 10 5 /mL and inoculated in 6-well tissue culture plates. After incubation at 37 ° C, 5.0% CO 2 , saturated humidity for 24 h, the control wells were added. 2.0 mL of sugar DMEM medium, 2.0 mL of medium containing different concentrations of the drug was added to the test well; after 72 hours of continuous culture, the cells of each well were collected by 0.25% trypsin digestion, washed 2-3 times with PBS (pH 7.4), and 500 ⁇ L Binding was added.
  • Buffer was mixed to make a single cell suspension; add 5 ⁇ L of FITC-labeled Annexin V (50 ⁇ g/mL) and 5 ⁇ L of PI (50 ⁇ g/mL), and react at room temperature for 30 min in the dark; shake and mix, and filter through 500 mesh copper mesh.
  • Flow cytometry was used to quantitatively detect the apoptosis rate of each group (Apoptosis%), and a tube without Annexin V-FITC and PI was used as a negative control.
  • the results showed that the antimicrobial peptide chimera M27-39-HTPP had a more pronounced pro-apoptotic effect (Fig. 6).

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Abstract

Disclosed is a hepatoma cell-targeting antimicrobial peptide chimera M27-39-HTPP and the use thereof, wherein same is obtained by the fusion of positions 27 to 39 of Musca domestica cecropin with a hepatocyte-targeting and penetrating peptide (HTPP), the amino acid sequence of which is as shown in SEQ ID NO: 1. The present invention uses a solid phase chemical synthesis method to obtain the crude M27-39-HTPP targeting antimicrobial peptide chimera, and uses reverse-phase high performance liquid chromatography and electrospray mass spectrometry to purify and identify the synthesized polypeptide. The hepatocyte-targeting M27-39-HTPP has a targeted anti-liver cancer effect, and has great practical significance and broad application prospects in the fields of medicine and bio-pharmacy, especially in the field of the preparation of anti-hepatoma drugs.

Description

一种肝癌细胞靶向抗菌肽嵌合体M27-39-HTPP及其应用Target of antibacterial peptide chimera M27-39-HTPP for liver cancer cells and application thereof 技术领域Technical field
本发明涉及生物技术领域，具体涉及一种肝癌细胞靶向抗菌肽嵌合体M27-39-HTPP及其应用。The invention relates to the field of biotechnology, in particular to a liver cancer cell-targeting antimicrobial peptide chimera M27-39-HTPP and application thereof.
背景技术Background technique
肝癌是世界上发病率第二高的疾病，属于高度恶性肿瘤，是中国的多发病和重大疾病，尽管采取手术切除、放/化疗综合疗法，死亡率仍高居不下。2013年，世界卫生组织宣布原发性肝癌引发全球745517人死亡，我国每年约有38.3万人死于肝癌，占全球肝癌死亡病例数的51％，给国家带来了严重的社会和经济负担。目前对肝癌的预防和治疗仍缺乏有效药物，以靶向分子为载体，携带抗癌药物为弹头的生物靶向治疗，是近20年以来肝癌研究的热点课题，具有较大的社会和经济意义。Liver cancer is the second most common disease in the world. It is a highly malignant tumor. It is a frequently-occurring disease and a major disease in China. Despite the combination of surgical resection and radiotherapy/chemotherapy, the mortality rate remains high. In 2013, the World Health Organization announced that primary liver cancer caused 745,517 deaths worldwide. About 383,000 people die of liver cancer each year in China, accounting for 51% of the total number of liver cancer deaths worldwide, which has brought serious social and economic burdens to the country. At present, there is still no effective drug for the prevention and treatment of liver cancer. Targeted molecules are used as carriers, and anti-cancer drugs are used as biological targets for warheads. It is a hot topic in liver cancer research in the past 20 years and has great social and economic significance. .
肝靶向穿膜肽(hepatocyte-targeting and penetrating peptide，HTPP)来源于疟原虫环子孢子蛋白(circumsporozoite protein,CSP)保守I区上游，研究显示HTPP不仅含有硫酸肝素结合序列，可特异性结合肝细胞表面的受体—硫酸乙酰肝素蛋白聚糖(heparan sulfateproteoglycans，HSPG)，还含有一个PELEX/VTS基序，能够有效穿透细胞膜，介导CSP进入细胞内部，实现细胞亚定位。HSPG是由核蛋白分子和糖胺聚糖(gly-cosaminoglycan，GAG)通过糖苷键共价结合组成的复合大分子，研究显示肝脏的HSPG具有独特的GAG链，其硫酸化程度显著高于其它组织，此外，肝癌组织中HSPG表达与正常肝组织有显著差异，且表达水平与癌细胞转移潜能成正比。因此，HTPP不仅可作为肝靶向分子，将药物富集肝脏，还可以干扰HSPG受体功能，抑制肿瘤转移。Hepatocyte-targeting and penetrating peptide (HTPP) is derived from the upstream of the conserved I region of Plasmodium circumsporozoite protein (CSP). Studies have shown that HTPP not only contains heparan sulfate binding sequence, but also specifically binds to liver. The cell surface receptor, heparan sulfate proteoglycans (HSPG), also contains a PELEX/VTS motif, which can effectively penetrate the cell membrane and mediate CSP entry into the cell to achieve subcellular localization. HSPG is a complex macromolecule composed of nucleoprotein molecules and glycosaminoglycan (GAG) covalently bonded by glycosidic bonds. Studies have shown that HSPG of the liver has a unique GAG chain, and its degree of sulfation is significantly higher than other tissues. In addition, the expression of HSPG in liver cancer tissues is significantly different from that in normal liver tissues, and the expression level is directly proportional to the metastatic potential of cancer cells. Therefore, HTPP can not only act as a liver targeting molecule, but also enrich the drug with the liver, and can also interfere with HSPG receptor function and inhibit tumor metastasis.
抗菌肽(Antimicrobial peptides,AMP)是生物免疫***产生的一类抵抗外界病原体感染的小分子多肽，具有一系列引人注目的生物学活性，包括抗菌、抗炎、抗病毒、抗寄生虫、抑制肿瘤细胞及免疫调节活性等。抗菌肽能够破坏细菌细胞膜或穿过细胞膜作用于胞内靶位点，作用机制独特，不易产生耐药性，对正常人体细胞毒、副作用小。因此，在传统抗菌、抗病毒和抗肿瘤药物研发和临床效果不尽如人意的现今，抗菌肽的上述特点使其显示出了良好的应用开发前景。Musca domestica cecropin是广东药科大学/广东省生物活性药物研究重点实验室从家蝇幼虫脂肪体cDNA文库中克隆的一种昆虫抗菌肽，该基因的ORF区全长为192bp，可编码63个氨基酸的前体蛋白，1～23位氨基酸是以保守4肽结尾的信号肽，其成熟肽含有40个氨基酸，具有较强的抗菌和抗肿瘤活性，并且我们在前期基础上，进一步研 究发现衍生肽M27-39与天然Musca domestica cecropin相比，不仅在抗肿瘤方面具有更显著优异的生理活性，且相较天然Musca domestica cecropin相比分子更小，更易进入胞内。Antimicrobial peptides (AMP) are small molecular peptides produced by the biological immune system against external pathogen infections. They have a series of attractive biological activities, including antibacterial, anti-inflammatory, anti-viral, anti-parasitic, and inhibition. Tumor cells and immunomodulatory activities. The antibacterial peptide can destroy the bacterial cell membrane or pass through the cell membrane to act on the intracellular target site, and the mechanism of action is unique, and it is not easy to produce drug resistance, and is toxic to normal human cells and has few side effects. Therefore, in the current research and development and clinical effects of traditional antibacterial, antiviral and antitumor drugs are not satisfactory, the above characteristics of antimicrobial peptides have shown good application development prospects. Musca domestica cecropin is an insect antibacterial peptide cloned from the fatty acid cDNA library of Musca domestica larvae from Guangdong Pharmaceutical University/Guangdong Provincial Key Laboratory of Bioactive Drug Research. The ORF region of this gene is 192 bp in length and can encode 63 amino acids. The precursor protein, amino acid positions 1 to 23 are signal peptides ending in a conserved 4 peptide, and the mature peptide contains 40 amino acids, which has strong antibacterial and antitumor activities, and we have further studied the derived peptides on a preliminary basis. Compared with natural Musca domestica cecropin, M27-39 not only has more remarkable physiological activity in anti-tumor, but also has smaller molecules and easier entry into cells than natural Musca domestica cecropin.
目前没有相关研究报道将Musca domestica cecropin衍生肽M27-39与HTPP进行融合改造，构建具有肝癌细胞靶向作用的嵌合体，用于制备靶向抗肝癌药物中的应用。At present, there is no related research report that Musca domestica cecropin-derived peptide M27-39 is fused with HTPP to construct a chimera with targeting effect of liver cancer cells for the preparation of targeted anti-hepatocarcinoma drugs.
发明内容Summary of the invention
为解决现有技术存在的问题，本发明的目的在于提供一种肝癌细胞靶向抗菌肽嵌合体M27-39-HTPP。In order to solve the problems existing in the prior art, it is an object of the present invention to provide a liver cancer cell-targeting antimicrobial peptide chimera M27-39-HTPP.
本发明的另一个目的在于提供所述的肝癌细胞靶向抗菌肽嵌合体M27-39-HTPP在制备靶向抗肝癌药物中的应用。Another object of the present invention is to provide the use of the liver cancer cell-targeting antimicrobial peptide chimera M27-39-HTPP for the preparation of a targeted anti-hepatocarcinoma drug.
为了实现上述目的，本发明是通过以下技术方案予以实现的：In order to achieve the above object, the present invention is achieved by the following technical solutions:
本发明肝癌细胞靶向抗菌肽嵌合体M27-39-HTPP，其氨基酸序列如SEQ ID NO:1所示。The liver cancer cell of the present invention targets the antimicrobial peptide chimera M27-39-HTPP, and the amino acid sequence thereof is shown in SEQ ID NO: 1.
具体地，所述的SEQ ID NO:1为VAQQAANVAATLKNSRSLGENDDGNNEDNEKLR。Specifically, the SEQ ID NO: 1 is VAQQAANVAATLKNSRSLGENDDGNNEDNEKLR.
本发明肝癌细胞靶向抗菌肽嵌合体M27-39-HTPP，采用固相化学合成法，通过多肽合成仪合成多肽粗品；用固相合成法合成多肽。将合成的多肽使用反相高效液相色谱进行纯化，并利用电喷射质谱法对合成的多肽进行鉴定，完成多肽的制备。The liver cancer cell of the invention targets the antibacterial peptide chimera M27-39-HTPP, and adopts a solid phase chemical synthesis method to synthesize a crude polypeptide by a polypeptide synthesizer; and synthesizes the polypeptide by solid phase synthesis. The synthesized polypeptide was purified by reverse-phase high performance liquid chromatography, and the synthesized polypeptide was identified by electrospray mass spectrometry to complete the preparation of the polypeptide.
本发明研发设计的肝癌细胞靶向抗菌肽嵌合体M27-39-HTPP，具有显著的肝靶向穿膜作用和抗肝癌活性，可用于制备靶向抗肝癌药物。The liver cancer cell targeted antibacterial peptide chimera M27-39-HTPP designed and developed by the invention has remarkable liver-targeting transmembrane action and anti-hepatocarcinoma activity, and can be used for preparing targeted anti-liver cancer drugs.
附图说明DRAWINGS
图1为肝癌细胞靶向抗菌肽嵌合体M27-39-HTPP的高效液相色谱图。Figure 1 is a high performance liquid chromatogram of a liver cancer cell-targeted antimicrobial peptide chimera M27-39-HTPP.
图2为肝癌细胞靶向抗菌肽嵌合体M27-39-HTPP的质谱图。Figure 2 is a mass spectrum of a liver cancer cell-targeted antimicrobial peptide chimera M27-39-HTPP.
图3为组织结合实验结果显示肝癌细胞靶向抗菌肽嵌合体M27-39-HTPP具有肝靶向性。Figure 3 shows the results of tissue binding experiments showing that the liver cancer cell-targeting antimicrobial peptide chimera M27-39-HTPP has liver targeting properties.
图4为激光共聚焦观察结果显示肝癌细胞靶向抗菌肽嵌合体M27-39-HTPP可透过细胞膜进入肝癌细胞内部。Figure 4 shows the results of laser confocal observation. The hepatocellular carcinoma cell-targeting antimicrobial peptide chimera M27-39-HTPP can enter the liver cancer cells through the cell membrane.
图5为MTT检测结果显示肝癌细胞靶向抗菌肽嵌合体M27-39-HTPP具有更显著抗肿瘤细胞增殖活性。Figure 5 shows that the MTT assay showed that the hepatocellular carcinoma cell-targeting antimicrobial peptide chimera M27-39-HTPP has more significant anti-tumor cell proliferation activity.
图6为流式检测结果显示肝癌细胞靶向抗菌肽嵌合体M27-39-HTPP具有更显著促凋亡作用。Figure 6 shows the results of flow cytometry showing that the hepatocellular carcinoma cell-targeting antimicrobial peptide chimera M27-39-HTPP has a more pronounced pro-apoptotic effect.
具体实施方式Detailed ways
下面结合说明书附图和具体实施例对本发明作出进一步地详细阐述，所述实施例只用于解释本发明，并非用于限定本发明的范围。下述实施例中所使用的试验方法如无特殊说明， 均为常规方法；所使用的材料、试剂等，如无特殊说明，为可从商业途径得到的试剂和材料。The invention is further described in detail with reference to the accompanying drawings and specific embodiments, which are intended to illustrate the invention and not to limit the scope of the invention. The test methods used in the following examples are all conventional methods unless otherwise specified; the materials, reagents and the like used are, if not specified, commercially available reagents and materials.
实施例1固相化学合成法合成肝癌细胞靶向抗菌肽嵌合体M27-39-HTPPExample 1 Solid phase chemical synthesis synthesis of liver cancer cells targeting antimicrobial peptide chimera M27-39-HTPP
肝癌细胞靶向抗菌肽嵌合体M27-39-HTPP的氨基酸序列为：VAQQAANVAATLKNSRSLGENDDGNNEDNEKLR，如SEQ ID NO:1所示，含有33个氨基酸。肝癌细胞靶向抗菌肽嵌合体M27-39-HTPP的制备从C端到N端逐一进行，通过多肽合成仪来完成。首先将Fmoc-X(X是抗菌肽MDC C端的第一个氨基酸)接入到Wang树脂，然后脱去Fmoc基团后得到X-Wang树脂；再将Fmoc-Y-Trt-OH(9-芴甲氧羧基-三甲基-Y，Y为肝癌细胞靶向抗菌肽嵌合体M27-39-HTPP的C端第二个氨基酸)；按照这个程序依次从C端合成到N端，直至合成完毕，得到脱去Fmoc基团的侧链保护的树脂；在上述得到的肽树脂中，加入切割试剂，20℃避光下反应2h，过滤；沉淀TFA(三氟乙酸)洗涤，将洗液与上述滤液混合，旋转蒸发仪浓缩，再加入10倍左右体积的预冷无水***，-20℃沉淀3h，析出白色粉末物，以2500g离心10min，收集沉淀，再用无水***洗涤沉淀，真空干燥，得到多肽，其中切割试剂由TFA、水和TIS(三异丙基氯硅烷)按照质量比95:2.5:2.5混合而成；使用0.2mol/L硫酸钠(磷酸调节至pH7.5)进行柱平衡30min，用90％乙腈水溶液溶解多肽，过滤，C18反相常压柱，采用梯度洗脱(洗脱剂为甲醇和硫酸钠水溶液按照体积比为30:70～70:30混合)，流速为1mL/min，检测波为220nm，收集主峰，冻干；再利用反相C18柱进一步纯化，洗脱液A为0.1％TFA/水溶液；洗脱液B为0.1％TFA/乙腈溶液，洗脱浓度为25％B～40％B，洗脱时间为12min，流速为1mL/min，再同上收集主峰，冻干；将上述得到的M27-39-HTPP经过反相高效液相色谱和电喷雾质谱法分析验证。反相高效液相色谱图如图1所示，质谱图如图2所示，结果显示肝癌细胞靶向抗菌肽嵌合体M27-39-HTPP的纯度为97.46％，分子量为3556.19，与理论分子量基本一致。The amino acid sequence of the liver cancer cell-targeting antimicrobial peptide chimera M27-39-HTPP is: VAQQAANVAATLKNSRSLGENDDGNNEDNEKLR, as shown in SEQ ID NO: 1, containing 33 amino acids. The preparation of the liver cancer cell-targeting antibacterial peptide chimera M27-39-HTPP was carried out one by one from the C-terminus to the N-terminus, and was completed by a polypeptide synthesizer. First, Fmoc-X (X is the first amino acid of the C-terminus of the antibacterial peptide MDC) is added to the Wang resin, and then the Fmoc group is removed to obtain the X-Wang resin; then Fmoc-Y-Trt-OH (9-芴) Methoxycarboxy-trimethyl-Y, Y is the second amino acid of the C-terminal of the antibacterial peptide chimera M27-39-HTPP of liver cancer cells; according to this procedure, it is synthesized from the C-terminus to the N-terminus until the synthesis is completed. Obtaining a resin for protecting the side chain of the Fmoc group; adding the cleavage reagent to the peptide resin obtained above, reacting at 20 ° C for 2 h in the dark, filtering; washing the precipitate with TFA (trifluoroacetic acid), and washing the filtrate with the filtrate The mixture was concentrated by a rotary evaporator, and then added with about 10 times of pre-cooled anhydrous diethyl ether. The precipitate was precipitated at -20 ° C for 3 hours to precipitate a white powder, which was centrifuged at 2500 g for 10 min, and the precipitate was collected, and the precipitate was washed with anhydrous diethyl ether and dried in vacuo. Obtaining a polypeptide in which a cleavage reagent is prepared by mixing TFA, water and TIS (triisopropylchlorosilane) at a mass ratio of 95:2.5:2.5; column equilibration is carried out using 0.2 mol/L sodium sulfate (phosphoric acid adjusted to pH 7.5) The peptide was dissolved in 90% acetonitrile aqueous solution for 30 min, filtered, and subjected to C18 reverse pressure atmospheric column using gradient elution ( The de-agent is methanol and sodium sulfate aqueous solution in a volume ratio of 30:70 to 70:30, the flow rate is 1 mL/min, the detection wave is 220 nm, the main peak is collected, and lyophilized; further purified by reverse phase C18 column, eluting The liquid A was 0.1% TFA/water solution; the eluent B was 0.1% TFA/acetonitrile solution, the elution concentration was 25% B-40% B, the elution time was 12 min, the flow rate was 1 mL/min, and the main peak was collected as above. Lyophilization; M27-39-HTPP obtained above was verified by reversed-phase high performance liquid chromatography and electrospray ionization mass spectrometry. The reversed-phase high performance liquid chromatogram is shown in Figure 1. The mass spectrum is shown in Figure 2. The results show that the purity of the liver cancer cell-targeting antimicrobial peptide chimera M27-39-HTPP is 97.46%, the molecular weight is 3556.19, and the theoretical molecular weight is basically Consistent.
实施例2组织结合实验观察肝靶向作用Example 2 Tissue Binding Experiment to Observe Liver Targeting
Balb/C小鼠用戊巴比妥钠深度麻醉(100mg/kg，IP)，生理盐水(10ml，>2min)和25毫升4％多聚甲醛(0.1M sodium phosphate buffer，pH7.4)(灌注泵速5ml/min)心脏(25G套管的心脏左心室)灌注，收集心、肝、脾、肺、肾等器官切取2-3毫米厚组织块，用4％多聚甲醛后固定2h，转移至30％的蔗糖溶液中(0.1mmol/L sodium phosphate buffer，PH7.4)4℃过夜。利用Tissue Freezing Medium包埋组织块，使用冰冻组织切片机切片，切片厚度为10-12μm。组织冰冻切片用PBS洗三次，加入10％BSA封闭液，室温孵育30min封闭非特异性位点；吸弃封闭液，加入带FITC标记的M27-39-HTPP，4℃过夜，PBS洗涤，封片，荧光显微镜观察。荧光显微镜观察M27-39-HTPP作用后肝组织切片可见明显荧光结合，且主要 分布在肝窦状隙边缘，而心、脾、肺和肾组织均未见特异荧光结合(图3)。Balb/C mice were deeply anesthetized with sodium pentobarbital (100 mg/kg, IP), normal saline (10 ml, >2 min) and 25 ml of 4% paraformaldehyde (0.1 M sodium phosphate buffer, pH 7.4) (perfused) Pump speed 5ml/min) heart (25G cannulated left ventricle) perfusion, collect heart, liver, spleen, lung, kidney and other organs to cut 2-3 mm thick tissue block, fixed with 4% paraformaldehyde for 2h, transfer To 30% sucrose solution (0.1 mmol/L sodium phosphate buffer, pH 7.4) overnight at 4 °C. Tissue blocks were embedded in Tissue Freezing Medium and sliced using a frozen tissue microtome with a section thickness of 10-12 μm. Tissue frozen sections were washed three times with PBS, 10% BSA blocking solution was added, and the non-specific sites were blocked by incubation at room temperature for 30 min; the blocking solution was discarded, FITC-labeled M27-39-HTPP was added, overnight at 4 ° C, washed with PBS, and mounted. Observed by fluorescence microscope. Fluorescence microscopy showed that the liver tissue sections showed obvious fluorescence binding after M27-39-HTPP treatment, and mainly distributed in the edge of the hepatic sinusoid, but no specific fluorescence binding was observed in the heart, spleen, lung and kidney tissues (Fig. 3).
实施例3肝癌细胞靶向抗菌肽嵌合体M27-39-HTPP的肝细胞穿膜作用研究Example 3 Study on Hepatocyte Transmembrane Effect of Hepatoma Cell Targeting Antibacterial Peptide Chimera M27-39-HTPP
制备HepG 2细胞爬片，加入FITC标记的肝癌细胞靶向抗菌肽嵌合体M27-39-HTPP，室温孵育10min后，用PBS洗涤4次，封片后用激光共聚焦显微镜观察肝癌细胞靶向抗菌肽嵌合体M27-39-HTPP的细胞定位情况。结果显示M27-39-HTPP作用后细胞内充满绿色荧光，提示M27-39-HTPP已经透过细胞膜进入HepG 2细胞内部(图4)。Prepare HepG 2 cell slides, add FITC-labeled liver cancer cells to target antibacterial peptide chimera M27-39-HTPP, incubate for 10 min at room temperature, wash 4 times with PBS, and observe the targeted antibacterial activity of liver cancer cells by laser confocal microscopy. Cellular localization of the peptide chimera M27-39-HTPP. The results showed that the cells were filled with green fluorescence after M27-39-HTPP treatment, suggesting that M27-39-HTPP has penetrated into the HepG 2 cells through the cell membrane (Fig. 4).
实施例4肝癌细胞靶向抗菌肽嵌合体M27-39-HTPP对肝癌细胞增殖的影响Example 4 Effect of Hepatoma Cell Targeting Antibacterial Peptide Chimera M27-39-HTPP on Proliferation of Hepatoma Cells
采用MTT法评价药物对HepG 2细胞增殖的影响，具体方法如下：The effect of drugs on the proliferation of HepG 2 cells was evaluated by MTT assay. The specific methods are as follows:
HepG 2细胞于37℃饱和湿度的恒温箱中5％CO 2传代培养，培养基为含10％胎牛血清，100U/mL氨苄青霉素和100U/mL链霉素的DMEM，当细胞生长接近80％融合度时，用0.25％胰蛋白酶消化制成单细胞悬液，准确计数。 HepG 2 cells were subcultured in 5% CO 2 in an incubator at 37 ° C with saturated humidity. The medium was DMEM containing 10% fetal bovine serum, 100 U/mL ampicillin and 100 U/mL streptomycin, when the cells grew close to 80%. At the time of fusion, a single cell suspension was prepared by digesting with 0.25% trypsin and accurately counted.
调整细胞浓度为1×10 5cell/mL，接种于96孔细胞培养板(分3组，每组3个复孔)，培养24小时待细胞贴壁后，弃培养液，分别加入含Musca domestica cecropin和Musca domestica cecropin衍生肽M27-39及抗菌肽嵌合体M27-39-HTPP的培养液，阴性对照组加不含药物的培养液。 Adjust the cell concentration to 1×10 5 cell/mL, inoculate 96-well cell culture plates (3 groups, 3 replicate wells per group), culture for 24 hours, after the cells are attached, discard the culture solution and add Musca domestica. A culture medium of cecropin and Musca domestica cecropin-derived peptide M27-39 and an antimicrobial peptide chimera M27-39-HTPP, and a negative control group plus a drug-free culture solution.
第3天弃培养基，用PBS洗板后，每孔加入10μL的5mg/ml MTT溶液和100μl培养基置于恒温培养箱内继续培养4h。取出培养板，弃去上清，每孔加入100μL DMSO，将培养板振摇30min。待MTT氧化产生的结晶完全溶解之后使用酶标仪测定OD值，测定波长为570/630nm，实验重复3次，取平均值。MTT结果显示上述药物均具有抗肿瘤细胞增殖活性，但Musca domestica cecropin衍生肽M27-39活性明显强于Musca domestica cecropin，而抗菌肽嵌合体M27-39-HTPP抗肿瘤细胞增殖活性略强于M27-39(图5)。On the third day, the medium was discarded, and after washing the plate with PBS, 10 μL of 5 mg/ml MTT solution and 100 μl of the medium were added to each well and placed in a constant temperature incubator for further 4 hours. The plate was taken out, the supernatant was discarded, 100 μL of DMSO was added to each well, and the plate was shaken for 30 min. After the crystals produced by the oxidation of MTT were completely dissolved, the OD value was measured using a microplate reader, the measurement wavelength was 570/630 nm, and the experiment was repeated 3 times, and the average value was taken. MTT results showed that the above drugs all had anti-tumor cell proliferation activity, but Musca domestica cecropin-derived peptide M27-39 activity was significantly stronger than Musca domestica cecropin, while antibacterial peptide chimera M27-39-HTPP anti-tumor cell proliferation activity was slightly stronger than M27- 39 (Figure 5).
实施例5肝癌细胞靶向抗菌肽嵌合体M27-39-HTPP对肝癌细胞凋亡影响研究Example 5 Effect of Hepatoma Cell Targeting Antibacterial Peptide Chimera M27-39-HTPP on Apoptosis of Hepatoma Cells
选取对数期生长的HepG 2细胞，调整浓度为3.0-5.0×10 5/mL，分别接种于6孔组织培养板中；37℃，5.0％CO 2，饱和湿度培养24h后，对照孔加入高糖DMEM培养基2.0mL，实验孔加入含不同浓度药物的培养基2.0mL；继续培养72h后0.25％胰蛋白酶消化收集各孔细胞，用PBS(pH7.4)洗2-3次，加入500μL Binding Buffer混匀制成单细胞悬液；再加入FITC标记的Annexin V(50μg/mL)5μL、PI(50μg/mL)5μL，室温避光反应30min后；振荡混匀，经500目铜网过滤后立即进行流式细胞术定量检测各组细胞凋亡率(Apoptosis％)，同时以不加Annexin V-FITC及PI的一管作为阴性对照。结果显示抗菌肽嵌合体M27-39-HTPP具有更显著促凋亡作用(图6)。 The HepG 2 cells growing in log phase were adjusted to a concentration of 3.0-5.0×10 5 /mL and inoculated in 6-well tissue culture plates. After incubation at 37 ° C, 5.0% CO 2 , saturated humidity for 24 h, the control wells were added. 2.0 mL of sugar DMEM medium, 2.0 mL of medium containing different concentrations of the drug was added to the test well; after 72 hours of continuous culture, the cells of each well were collected by 0.25% trypsin digestion, washed 2-3 times with PBS (pH 7.4), and 500 μL Binding was added. Buffer was mixed to make a single cell suspension; add 5 μL of FITC-labeled Annexin V (50 μg/mL) and 5 μL of PI (50 μg/mL), and react at room temperature for 30 min in the dark; shake and mix, and filter through 500 mesh copper mesh. Flow cytometry was used to quantitatively detect the apoptosis rate of each group (Apoptosis%), and a tube without Annexin V-FITC and PI was used as a negative control. The results showed that the antimicrobial peptide chimera M27-39-HTPP had a more pronounced pro-apoptotic effect (Fig. 6).
以上仅是本发明的优选实施方式，应当指出的是，上述优选实施方式不应视为对本发明的限制，本发明的保护范围应当以权利要求所限定的范围为准。对于本技术领域的普通技术人员来说，在不脱离本发明的精神和范围内，还可以做出若干改进和润饰，这些改进和润饰也应视为本发明的保护范围。The above is only a preferred embodiment of the present invention, and it should be noted that the above-described preferred embodiments are not to be construed as limiting the scope of the invention, and the scope of the invention should be determined by the scope defined by the claims. It will be apparent to those skilled in the art that various modifications and improvements can be made without departing from the spirit and scope of the invention.

Claims (2)

  1. 一种肝癌细胞靶向抗菌肽嵌合体M27-39-HTPP，其特征在于，其氨基酸序列如SEQ ID NO:1所示。A liver cancer cell-targeting antimicrobial peptide chimera M27-39-HTPP, characterized in that the amino acid sequence thereof is shown in SEQ ID NO: 1.
  2. 如权利要求1所述的肝癌细胞靶向抗菌肽嵌合体M27-39-HTPP在制备靶向抗肝癌药物中的应用。The use of the liver cancer cell-targeting antimicrobial peptide chimera M27-39-HTPP according to claim 1 for the preparation of a medicament for targeting anti-hepatocarcinogenesis.
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