技术领域technical field
本发明涉及纳米药物领域，涉及一种抗肿瘤多肽纳米药物及其制备方法和应用。The invention relates to the field of nano-medicine, and relates to an anti-tumor polypeptide nano-medicine and a preparation method and application thereof.
背景技术Background technique
近年来抗肿瘤多肽类药物的研发备受关注。与抗体和小分子药物相比，多肽药物免疫原性低、受体结合率高、制备成本低且易于改造和联合应用，但它最大的缺点是体内半衰期短，易被蛋白酶降解和肝肾等脏器代谢。因此，改善多肽的药代动力学而不降低其疗效是多肽药物研发的重要方向。In recent years, the research and development of anti-tumor peptide drugs has attracted much attention. Compared with antibodies and small-molecule drugs, peptide drugs have low immunogenicity, high receptor binding rate, low preparation cost, and are easy to modify and combined application. Organ metabolism. Therefore, improving the pharmacokinetics of polypeptides without reducing their efficacy is an important direction for the development of polypeptide drugs.
纳米药物是新兴的药物剂型，通过设计和调控有机或无机材料的纳米特性，制备结构稳定、功能多样和生物相容性好的纳米载体，可显著延长药物半衰期、提高靶向性、降低用药剂量并实现联合用药。通过合理调控多肽的分子结构和改变外界环境，某些多肽分子间或多肽分子中某一片段和另一片段之间可利用非共价的弱相互作用力，如氢键、范德华力、静电力、疏水作用和π-π堆积作用等，自发或触发地自组装成具有特定排列顺序的分子聚集体。多肽自身具有良好的生物相容性和可控的降解性能，但是其在体内的稳定性不够，分子结构易于被破坏。如果将抗肿瘤多肽通过改造使其能直接组装成纳米结构，可开发出分散性好、纯度高、毒副作用低和稳定性高的抗肿瘤纳米药物。Nano-drugs are emerging drug dosage forms. By designing and regulating the nano-characteristics of organic or inorganic materials, nano-carriers with stable structures, diverse functions and good biocompatibility can be prepared, which can significantly prolong the half-life of drugs, improve targeting, and reduce drug dosage. And realize the combination medicine. By rationally regulating the molecular structure of polypeptides and changing the external environment, non-covalent weak interaction forces, such as hydrogen bonds, van der Waals forces, electrostatic forces, Hydrophobic interaction and π-π stacking interaction, etc., spontaneously or triggered self-assembly into molecular aggregates with a specific arrangement order. Peptide itself has good biocompatibility and controllable degradation performance, but its stability in vivo is not enough, and its molecular structure is easily destroyed. If anti-tumor polypeptides can be directly assembled into nanostructures through modification, anti-tumor nano-drugs with good dispersion, high purity, low side effects and high stability can be developed.
此外，研究表明，恶性肿瘤组织的pH环境为微酸性，肿瘤细胞部位pH值在6.7-7.2之间。因此，为了减少药物对于正常组织细胞的毒副作用，期望能够得到一种在体内运输过程中可以稳定存在，而当到达肿瘤部位时可以释放药物以达到治疗肿瘤目的的pH响应性纳米药物。In addition, studies have shown that the pH environment of malignant tumor tissue is slightly acidic, and the pH value of tumor cell sites is between 6.7-7.2. Therefore, in order to reduce the toxic and side effects of drugs on normal tissue cells, it is expected to obtain a pH-responsive nanomedicine that can exist stably during in vivo transportation and release drugs when it reaches the tumor site to achieve the purpose of treating tumors.
发明内容Contents of the invention
针对现有技术的不足，本发明的目的在于提供一种抗肿瘤多肽纳米药物及其制备方法和应用。本发明所述的抗肿瘤多肽纳米药物克服了抗肿瘤多肽药物半衰期短的瓶颈，提供一种具有肿瘤部位弱酸环境响应性、生物相容性好、稳定性强、安全性高和生物利用度高的抗肿瘤多肽纳米药物。Aiming at the deficiencies of the prior art, the purpose of the present invention is to provide an anti-tumor polypeptide nano drug and its preparation method and application. The anti-tumor polypeptide nano-medicine of the present invention overcomes the bottleneck of short half-life of anti-tumor polypeptide drugs, and provides a kind of anti-tumor polypeptide drug with responsiveness to weak acid environment of tumor site, good biocompatibility, strong stability, high safety and high bioavailability anti-tumor peptide nanomedicine.
为达到此发明目的，本发明采用以下技术方案：To achieve this purpose of the invention, the present invention adopts the following technical solutions:
一方面，本发明提供一种抗肿瘤多肽纳米药物，所述抗肿瘤多肽纳米药物包含两亲性抗肿瘤多肽以及与两亲性抗肿瘤多肽偶联的酸响应性功能分子。In one aspect, the present invention provides an anti-tumor polypeptide nano-medicine, which comprises an amphiphilic anti-tumor polypeptide and an acid-responsive functional molecule coupled to the amphiphilic anti-tumor polypeptide.
目前存在的具有抗肿瘤性的多肽分子大多数是亲水性的，但是其体内半衰期短，易被蛋白酶降解和肝肾等脏器代谢，并且一些少数的两亲性多肽分子由于疏水与亲水片段之间的比例不协调造成很难形成纳米颗粒，因此，本发明对亲水性抗肿瘤多肽进行改造，将亲水性抗肿瘤多肽片段和疏水性多肽片段相结合，改造成两亲性抗肿瘤多肽，而后与酸响应性功能分子偶联得到的抗肿瘤多肽纳米药物，相容性好，稳定，使得其在体内的半衰期延长，也提高了对肿瘤部位的靶向性以及抗肿瘤多肽分子的生物利用度，另外，由于该药物中含有酸响应性功能分子使得所述纳米药物具有酸响应性，以在肿瘤细胞外基质pH值为酸性的环境下，达到较好的治疗效果。Most of the currently existing anti-tumor polypeptide molecules are hydrophilic, but their half-life in vivo is short, and they are easily degraded by proteases and metabolized by organs such as liver and kidney. The uncoordinated ratio between fragments makes it difficult to form nanoparticles. Therefore, the present invention transforms the hydrophilic anti-tumor polypeptide fragments and combines the hydrophilic anti-tumor polypeptide fragments and hydrophobic polypeptide fragments to transform them into amphiphilic anti-tumor polypeptide fragments. Tumor polypeptides, and then coupled with acid-responsive functional molecules to obtain anti-tumor polypeptide nano-drugs, have good compatibility and stability, which prolongs their half-life in vivo, and also improves the targeting of tumor sites and anti-tumor polypeptide molecules. In addition, because the drug contains acid-responsive functional molecules, the nano-medicine is acid-responsive, so as to achieve a better therapeutic effect in an environment where the pH value of the tumor extracellular matrix is acidic.
本发明将所述抗肿瘤多肽药物制备成纳米颗粒，是因为纳米颗粒体系稳定，肿瘤富集作用强，具有靶向性，此外，将其制备纳米颗粒还可以使得亲水性抗肿瘤多肽上的受体结合位点不暴露在外，提高了亲水性抗肿瘤多肽分子在体内的稳定性。The present invention prepares the anti-tumor polypeptide drug into nanoparticles because the nano-particle system is stable, has a strong tumor enrichment effect, and has targeting properties. In addition, the preparation of nanoparticles can also make the hydrophilic anti-tumor polypeptide The receptor binding site is not exposed, which improves the stability of the hydrophilic anti-tumor polypeptide molecule in vivo.
由于多肽分子侧链可带不同电荷，通过设计和修饰使多肽分子具有微酸性pH响应性，其中最简易的方法就是通过两亲性多肽自组装方式构建。两亲性多肽在水溶液中倾向于将其亲水部分暴露在外层与水分子形成交界面，疏水部分则聚集于内。在中性环境中，多肽分子主要通过疏水作用和氢键的物理相互作用力，自组装形成纳米颗粒；在微酸性环境中，多肽分子疏水端被质子化，疏水端之间的正电荷排斥力使自组装体解聚，释放出多肽分子。该方法可使抗肿瘤多肽药物在生理环境中形成纳米结构，延长其在生物体内的半衰期，纳米结构在微酸性肿瘤微环境中特异响应性解聚，释放出多肽分子，发挥抗肿瘤作用。Since the side chains of polypeptide molecules can have different charges, the polypeptide molecules can be designed and modified to have slightly acidic pH responsiveness, and the simplest method is to construct them through self-assembly of amphiphilic polypeptides. In aqueous solution, amphiphilic polypeptide tends to expose its hydrophilic part on the outer layer to form an interface with water molecules, while the hydrophobic part gathers inside. In a neutral environment, polypeptide molecules self-assemble into nanoparticles mainly through the physical interaction of hydrophobic interactions and hydrogen bonds; in a slightly acidic environment, the hydrophobic ends of polypeptide molecules are protonated, and the positive charge repulsion between hydrophobic ends Depolymerize the self-assembly and release the polypeptide molecule. The method enables the anti-tumor polypeptide drug to form a nanostructure in a physiological environment, prolonging its half-life in vivo, and the nanostructure depolymerizes specifically and responsively in a slightly acidic tumor microenvironment, releasing polypeptide molecules to exert an anti-tumor effect.
本发明所述抗肿瘤多肽纳米药物中，所述两亲性抗肿瘤多肽包含通过酰胺键偶联在一起的亲水性抗肿瘤多肽和疏水性多肽。In the anti-tumor polypeptide nanomedicine of the present invention, the amphipathic anti-tumor polypeptide comprises a hydrophilic anti-tumor polypeptide and a hydrophobic polypeptide coupled together through an amide bond.
优选地，所述酸响应性功能分子带有与氨基反应的官能团；Preferably, the acid-responsive functional molecules have functional groups reactive with amino groups;
优选地，所述酸响应性功能分子为异氰酸酯类有机分子，进一步优选为3-(二乙基氨基)丙基硫代异氰酸酯；Preferably, the acid-responsive functional molecule is an isocyanate organic molecule, more preferably 3-(diethylamino)propylthioisocyanate;
优选地，所述述酸响应性功能分子进行酸响应的pH值为6.7-7.2，例如6.7、6.8、6.9、7.0、7.1或7.2。Preferably, the acid-responsive functional molecule has a pH value of 6.7-7.2, such as 6.7, 6.8, 6.9, 7.0, 7.1 or 7.2.
本发明所述的酸响应的弱酸性pH值是相对于人体内正常组织内的pH值而言的，人体正常组织的pH值为7.4，而肿瘤部位的pH值为6.7-7.2，因此相对于人体正常组织来说，肿瘤组织部位的pH环境为弱酸性。The acid-responsive weakly acidic pH value of the present invention is relative to the pH value in the normal tissue in the human body. The pH value of the normal tissue in the human body is 7.4, while the pH value of the tumor site is 6.7-7.2. For normal tissues of the human body, the pH environment of the tumor tissue site is weakly acidic.
酸响应性功能分子在pH值为6.7-7.2时，可以被质子化，导致多肽疏水端之间产生正电荷排斥力，使自组装纳米颗粒形态被破坏。The acid-responsive functional molecules can be protonated when the pH value is 6.7-7.2, resulting in positive charge repulsion between the hydrophobic ends of the polypeptide, and destroying the morphology of self-assembled nanoparticles.
由于肿瘤部位的pH值为弱酸性环境即(6.7-7.2)，该药物在pH值为6.7-7.2，会使得药物纳米颗粒被破坏，抗肿瘤多肽上的结合位点裸露出来，达到治疗的目的。Since the pH value of the tumor site is a weakly acidic environment (6.7-7.2), the pH value of the drug is 6.7-7.2, which will destroy the drug nanoparticles and expose the binding site on the anti-tumor polypeptide to achieve the purpose of treatment .
优选地，所述酸响应性功能分子通过疏水性多肽末端氨基偶联至疏水性多肽上。Preferably, the acid-responsive functional molecule is coupled to the hydrophobic polypeptide through the terminal amino group of the hydrophobic polypeptide.
优选地，所述抗肿瘤多肽纳米药物还包含与疏水性多肽连接的赖氨酸。Preferably, the anti-tumor polypeptide nanomedicine further comprises lysine linked to a hydrophobic polypeptide.
优选地，所述酸响应性功能分子通过赖氨酸连接至疏水性多肽上。Preferably, the acid-responsive functional molecule is linked to the hydrophobic polypeptide through lysine.
本发明中所述酸响应性功能分子是指在一定酸性环境下的刺激下具有发生某种反应的能力的分子，例如，3-(二乙基氨基)丙基硫代异氰酸酯分子在酸性环境下可以被质子化。The acid-responsive functional molecules described in the present invention refer to molecules that have the ability to undergo a certain reaction under the stimulation of a certain acidic environment, for example, 3-(diethylamino)propyl thioisocyanate molecules can react in an acidic environment. can be protonated.
本发明所述抗肿瘤多肽纳米药物中，所述与疏水性多肽连接的赖氨酸为1个赖氨酸或2-5个赖氨酸形成的肽链，例如疏水性多肽可以连接1个赖氨酸，或者疏水性多肽可以连接有2、3、4或5个赖氨酸形成的肽链。在疏水性多肽上引入赖氨酸，而后再通过赖氨酸引入酸响应性功能分子，这样可以在抗肿瘤多肽上引入多个酸响应性功能分子，例如，当疏水性多肽不连接赖氨酸时，通过疏水性氨基酸末端氨基连接一个酸响应性功能分子，而当疏水性多肽连接一个赖氨酸时，可以通过赖氨酸的末端氨基和侧链氨基共连接2个酸响应性功能分子，如果疏水性多肽连接的是4个赖氨酸形成的肽链，则可以通过赖氨酸肽链的末端氨基和侧链氨基共连接5个酸响应性功能分子。连接多个酸响应性功能分子能够保证两亲性多肽疏水端的疏水性，并使得药物在肿瘤部位发生酸响应的动力更强。In the anti-tumor polypeptide nanomedicine of the present invention, the lysine connected to the hydrophobic polypeptide is a peptide chain formed by 1 lysine or 2-5 lysines, for example, the hydrophobic polypeptide can be connected to 1 lysine Lysine, or a hydrophobic polypeptide can be connected with a peptide chain formed by 2, 3, 4 or 5 lysines. Introduce lysine on the hydrophobic polypeptide, and then introduce acid-responsive functional molecules through lysine, so that multiple acid-responsive functional molecules can be introduced on the anti-tumor polypeptide, for example, when the hydrophobic polypeptide is not connected to lysine When connecting an acid-responsive functional molecule through the terminal amino group of a hydrophobic amino acid, and when a hydrophobic polypeptide is connected to a lysine, two acid-responsive functional molecules can be co-connected through the terminal amino group and the side chain amino group of the lysine, If the hydrophobic polypeptide is connected to a peptide chain formed by 4 lysines, then 5 acid-responsive functional molecules can be connected through the terminal amino group and side chain amino group of the lysine peptide chain. Linking multiple acid-responsive functional molecules can ensure the hydrophobicity of the hydrophobic end of the amphiphilic polypeptide, and make the drug more acid-responsive at the tumor site.
优选地，所述赖氨酸的末端氨基和侧链氨基均与所述酸响应性功能分子连接。Preferably, both the terminal amino group and the side chain amino group of the lysine are connected to the acid-responsive functional molecule.
本发明所述抗肿瘤多肽纳米药物中，所述亲水性抗肿瘤多肽为具有抑制肿瘤细胞、肿瘤血管、肿瘤淋巴管或肿瘤间质细胞功能的多肽分子中的任意一种或至少两种的组合。In the anti-tumor polypeptide nanomedicine of the present invention, the hydrophilic anti-tumor polypeptide is any one or at least two of the polypeptide molecules that can inhibit tumor cells, tumor blood vessels, tumor lymphatic vessels or tumor stromal cells. combination.
优选地，所述亲水性抗肿瘤多肽为含有5-30个氨基酸的可溶性多肽，例如可以是含有5、6、7、8、9、10、13、15、18、20、24、26、28或30个氨基酸的可溶性多肽。Preferably, the hydrophilic anti-tumor polypeptide is a soluble polypeptide containing 5-30 amino acids, for example, it may contain 5, 6, 7, 8, 9, 10, 13, 15, 18, 20, 24, 26, Soluble peptides of 28 or 30 amino acids.
本发明所述抗肿瘤多肽纳米药物中，所述疏水性多肽为含有5-40个疏水性氨基酸的多肽，例如在具体的实施方案中，疏水性多肽可以含有5、6、7、8、9、10、13、15、18、20、24、26、28、30、32、34、36、38或40个疏水性氨基酸。In the anti-tumor polypeptide nanomedicine of the present invention, the hydrophobic polypeptide is a polypeptide containing 5-40 hydrophobic amino acids. For example, in a specific embodiment, the hydrophobic polypeptide may contain 5, 6, 7, 8, 9 , 10, 13, 15, 18, 20, 24, 26, 28, 30, 32, 34, 36, 38 or 40 hydrophobic amino acids.
优选地，所述疏水性氨基酸为亮氨酸、丙氨酸、甘氨酸、异亮氨酸、亮氨酸、蛋氨酸、缬氨酸或酪氨酸中的任意一种或至少两种的组合。Preferably, the hydrophobic amino acid is any one or a combination of at least two of leucine, alanine, glycine, isoleucine, leucine, methionine, valine or tyrosine.
本发明所述抗肿瘤多肽纳米药物的粒径为10-200nm。该范围内的纳米颗粒稳定，具有肿瘤富集作用，可以提高药物的靶向性以及药物的生物利用度。The particle size of the anti-tumor polypeptide nano-medicine of the present invention is 10-200nm. Nanoparticles within this range are stable, have a tumor-enriching effect, and can improve drug targeting and bioavailability of drugs.
另一方面，本发明提供了如本发明第一方面所述的抗肿瘤多肽纳米药物的制备方法，所述方法为：以氨基酸为原料，合成两亲性抗肿瘤多肽分子，而后在两亲性抗肿瘤多肽分子上引入酸响应性功能分子，然后进行自组装形成所述抗肿瘤多肽纳米药物。On the other hand, the present invention provides a preparation method of the anti-tumor polypeptide nano-drug according to the first aspect of the present invention, the method is: using amino acids as raw materials, synthesizing amphiphilic anti-tumor polypeptide molecules, and then An acid-responsive functional molecule is introduced into the anti-tumor polypeptide molecule, and then self-assembled to form the anti-tumor polypeptide nano-medicine.
优选地，所述两亲性抗肿瘤多肽包含通过酰胺键偶联在一起的亲水性抗肿瘤多肽和疏水性多肽。Preferably, the amphiphilic anti-tumor polypeptide comprises a hydrophilic anti-tumor polypeptide and a hydrophobic polypeptide coupled together by an amide bond.
优选地，亲水性抗肿瘤多肽为具有抑制肿瘤细胞、肿瘤血管、肿瘤淋巴管或肿瘤间质细胞功能的多肽分子中的任意一种或至少两种的组合。Preferably, the hydrophilic anti-tumor polypeptide is any one or a combination of at least two polypeptide molecules that have the function of inhibiting tumor cells, tumor blood vessels, tumor lymphatic vessels or tumor stromal cells.
优选地，所述亲水性抗肿瘤多肽为含有5-30个氨基酸的可溶性多肽。Preferably, the hydrophilic anti-tumor polypeptide is a soluble polypeptide containing 5-30 amino acids.
优选地，所述疏水性多肽为含有5-40个疏水性氨基酸的多肽。Preferably, the hydrophobic polypeptide is a polypeptide containing 5-40 hydrophobic amino acids.
优选地，所述疏水性氨基酸为亮氨酸、丙氨酸、甘氨酸、异亮氨酸、蛋氨酸、缬氨酸或酪氨酸中的任意一种或至少两种的组合。Preferably, the hydrophobic amino acid is any one or a combination of at least two of leucine, alanine, glycine, isoleucine, methionine, valine or tyrosine.
优选地，所述两亲性抗肿瘤多肽分子的疏水性多肽末端连接有赖氨酸。Preferably, the hydrophobic polypeptide end of the amphipathic anti-tumor polypeptide molecule is linked with lysine.
优选地，所述两亲性抗肿瘤多肽分子的合成通过固相合成法实现。Preferably, the synthesis of the amphiphilic anti-tumor polypeptide molecule is achieved by solid-phase synthesis.
优选地，所述酸响应性功能分子带有与氨基反应的官能团。Preferably, the acid-responsive functional molecule has a functional group reactive with an amino group.
优选地，所述酸响应性功能分子为异氰酸酯类有机分子，进一步优选为3-(二乙基氨基)丙基硫代异氰酸酯。Preferably, the acid-responsive functional molecule is an isocyanate-based organic molecule, more preferably 3-(diethylamino)propylthioisocyanate.
优选地，所述酸响应性功能分子与两亲性抗肿瘤多肽分子的疏水性多肽末端的赖氨酸偶联。Preferably, the acid-responsive functional molecule is coupled to the lysine at the end of the hydrophobic polypeptide of the amphipathic anti-tumor polypeptide molecule.
作为本发明的优选技术方案，本发明的抗肿瘤多肽纳米药物的制备方法包括以下步骤：As a preferred technical solution of the present invention, the preparation method of the anti-tumor polypeptide nano drug of the present invention comprises the following steps:
(1)以氨基酸为原料，利用固相合成法合成疏水性多肽末端连接有赖氨酸的两亲性抗肿瘤多肽分子；(1) using amino acids as raw materials, using solid-phase synthesis to synthesize amphiphilic anti-tumor polypeptide molecules with lysine connected to the end of the hydrophobic polypeptide;
(2)使步骤(1)合成的两亲性抗肿瘤多肽分子的疏水性多肽末端的赖氨酸与酸响应性功能分子偶联；(2) coupling the lysine at the end of the hydrophobic polypeptide of the amphiphilic anti-tumor polypeptide molecule synthesized in step (1) to an acid-responsive functional molecule;
(3)将步骤(2)的产物在中性水环境中自组装得到所述抗肿瘤多肽纳米药物。(3) The product of step (2) is self-assembled in a neutral water environment to obtain the anti-tumor polypeptide nano drug.
本发明所述的抗肿瘤多肽纳米药物的制备方法中，步骤(1)以利用氨基酸之间发生缩合反应得到两亲性抗肿瘤多肽分子，可以在两亲性抗肿瘤多肽分子的疏水性多肽上偶联有赖氨酸。在步骤(2)中两亲性抗肿瘤多肽分子末端连接的赖氨酸上的氨基与酸响应性功能分子中可与氨基反应的官能团进行反应，从而在两亲性抗肿瘤多肽分子上偶联上酸响应性功能分子。In the preparation method of the anti-tumor polypeptide nano-medicine of the present invention, in step (1), the amphiphilic anti-tumor polypeptide molecule can be obtained by using the condensation reaction between amino acids, which can be used on the hydrophobic polypeptide of the amphiphilic anti-tumor polypeptide molecule. Conjugated with lysine. In step (2), the amino group on the lysine connected to the end of the amphiphilic anti-tumor polypeptide molecule reacts with the functional group that can react with the amino group in the acid-responsive functional molecule, thereby coupling the amphiphilic anti-tumor polypeptide molecule Acid-responsive functional molecules.
优选地，步骤(3)所述中性水环境为pH值为7.4的磷酸盐缓冲溶液。Preferably, the neutral water environment in step (3) is a phosphate buffer solution with a pH value of 7.4.
作为进一步的优选技术方案，本发明的抗肿瘤多肽纳米药物的制备方法具体包括以下步骤：As a further preferred technical solution, the preparation method of the anti-tumor polypeptide nano drug of the present invention specifically includes the following steps:
(1)以氨基酸为原料，利用固相合成法合成疏水性多肽末端连接有赖氨酸的两亲性抗肿瘤多肽分子；(1) using amino acids as raw materials, using solid-phase synthesis to synthesize amphiphilic anti-tumor polypeptide molecules with lysine connected to the end of the hydrophobic polypeptide;
(2)使步骤(1)合成的两亲性抗肿瘤多肽分子的疏水性多肽末端的赖氨酸与酸响应性功能分子3-(二乙基氨基)丙基硫代异氰酸酯偶联；(2) coupling the lysine at the end of the hydrophobic polypeptide of the amphiphilic anti-tumor polypeptide molecule synthesized in step (1) to the acid-responsive functional molecule 3-(diethylamino)propylthioisocyanate;
(3)将步骤(2)的产物在pH值为7.4的磷酸盐缓冲溶液中自组装得到所述抗肿瘤多肽纳米药物。(3) The product of step (2) is self-assembled in a phosphate buffer solution with a pH value of 7.4 to obtain the anti-tumor polypeptide nano drug.
其中，在上述步骤(2)中3-(二乙基氨基)丙基硫代异氰酸酯分子中的异氰酸酯基可以与赖氨酸的氨基发生反应而偶联在一起。Wherein, in the above step (2), the isocyanate group in the 3-(diethylamino)propyl thioisocyanate molecule can react with the amino group of lysine to couple together.
本发明所述将亲水性抗肿瘤多肽和和疏水性氨基酸以及任选的赖氨酸偶联获得两亲性抗肿瘤多肽分子的过程，可采用现有技术中已知的固相合成方法(例如，Lihong Liu等，Nature Nanotechnology,2009,4:457-463；以及Ying Zhao等，J Control Release,2014,177:11-19中提供的方法)来实现，为了实现亲水性抗肿瘤多肽与疏水性氨基酸以及任选的赖氨酸之间以特定方式连接，在合成两亲性多肽分子之前将原料中的不同种氨基利用化学修饰方法进行保护(采用现有技术中氨基的保护方法进行)。由于氨基保护的氨基酸产品可以商业购买到，因此，本发明利用从自吉尔生化(上海)有限公司购买的氨基酸完成本发明。本发明所述抗肿瘤多肽纳米药物的制备过程可以采用如下详细步骤完成：The process of coupling the hydrophilic anti-tumor polypeptide with the hydrophobic amino acid and optional lysine to obtain the amphipathic anti-tumor polypeptide molecule described in the present invention can adopt the solid-phase synthesis method known in the prior art ( For example, Lihong Liu et al., Nature Nanotechnology, 2009, 4:457-463; and Ying Zhao et al., the method provided in J Control Release, 2014, 177:11-19) to achieve, in order to realize the hydrophilic anti-tumor polypeptide and Hydrophobic amino acids and optional lysine are connected in a specific way, and different amino groups in the raw materials are protected by chemical modification methods before synthesizing amphiphilic polypeptide molecules (using the protection method of amino groups in the prior art) . Since amino acid products protected by amino groups can be purchased commercially, the present invention utilizes amino acids purchased from Jill Biochemical (Shanghai) Co., Ltd. to complete the present invention. The preparation process of the anti-tumor polypeptide nano-medicine of the present invention can be completed by the following detailed steps:
(1)本发明用于多肽合成的氨基酸均购自吉尔生化(上海)有限公司，所购氨基酸的末端氨基均由Fmoc(笏甲氧羰基)保护，用于合成亲水性抗肿瘤多肽的赖氨酸侧链氨基由Boc(叔丁氧羰基)保护，用于偶联功能分子的赖氨酸侧链的氨基由CBZ(苄氧羰基)保护。(1) The amino acids used in the synthesis of polypeptides in the present invention are all purchased from Jill Biochemical (Shanghai) Co., Ltd., and the terminal amino groups of the purchased amino acids are all protected by Fmoc (Wat methoxycarbonyl), which is used to synthesize hydrophilic anti-tumor polypeptides. The amino group of the amino acid side chain is protected by Boc (tert-butoxycarbonyl), and the amino group of the lysine side chain used for coupling functional molecules is protected by CBZ (benzyloxycarbonyl).
(2)将所要合成的两亲性多肽分子的第一个氨基酸(末端氨基由Fmoc保护)的末端羧基与CLEAR-酰胺树脂(引入CLEAR-酰胺树脂的目的是将氨基酸的羧基末端固定，以便使其氨基端发生反应)的氨基末端连接，通过20％哌啶/N,N-二甲基甲酰胺脱去Fmoc保护基，然后以此结合在树脂上的氨基酸作为氨基组分，同过量的含活化羧基的下一个氨基酸反应接长肽链，重复上述操作直至所有的氨基酸缩合完毕，形成肽链上氨基被保护的两亲性抗肿瘤多肽。(2) The terminal carboxyl group of the first amino acid (terminal amino group is protected by Fmoc) of the amphiphilic polypeptide molecule to be synthesized and CLEAR-amide resin (the purpose of introducing CLEAR-amide resin is to fix the carboxyl terminal of amino acid, so that make The amino terminal of its amino terminal reacts) is connected to the amino terminal, and the Fmoc protecting group is removed by 20% piperidine/N,N-dimethylformamide, and then the amino acid combined on the resin is used as the amino component, and the excess containing The next amino acid of the activated carboxyl group is reacted to extend the peptide chain, and the above operation is repeated until all the amino acids are condensed to form an amphipathic anti-tumor polypeptide with protected amino groups on the peptide chain.
(3)将两亲性抗肿瘤多肽用20％哌啶/N,N-二甲基甲酰胺脱去Fmoc保护基，用催化氢解法脱去CBZ保护基，使酸响应性功能分子与脱去保护的氨基反应，从而将酸响应性功能分子连接至两亲性抗肿瘤多肽上。(3) Use 20% piperidine/N,N-dimethylformamide to remove the Fmoc protecting group of the amphiphilic anti-tumor polypeptide, remove the CBZ protecting group by catalytic hydrogenolysis, and remove the acid-responsive functional molecule and The protected amino group is reacted to connect the acid-responsive functional molecule to the amphipathic anti-tumor polypeptide.
(4)用高浓度三氟乙酸的二氯甲烷溶液将肽链从树脂上裂解下来，C16Y片段中的Boc保护基团也将同时除去，经过纯化等处理，即得到连接有酸响应性功能分子的两亲性抗肿瘤多肽。(4) Cleavage the peptide chain from the resin with a dichloromethane solution of high-concentration trifluoroacetic acid, and the Boc protecting group in the C16Y fragment will also be removed at the same time. After purification and other treatments, the acid-responsive functional molecule is obtained. amphipathic antitumor polypeptide.
(5)将所得到的连接有酸响应性功能分子的两亲性抗肿瘤多肽在中性水环境中进行自组装性能纳米颗粒体系，即得到所述抗肿瘤多肽纳米药物。(5) The obtained amphiphilic anti-tumor polypeptide linked with acid-responsive functional molecules is subjected to self-assembly nanoparticle system in a neutral water environment to obtain the anti-tumor polypeptide nano drug.
本发明所述两亲性抗肿瘤多肽的自组装采用本领域技术人员熟知的现有技术进行。The self-assembly of the amphiphilic anti-tumor polypeptide of the present invention is carried out using the prior art well known to those skilled in the art.
本发明制备的抗肿瘤多肽纳米药物的粒径在10-200nm，粒径较均一，有利于在肿瘤部位的富集，提高了药物的靶向性，减少了对正常细胞的毒副作用。The particle size of the anti-tumor polypeptide nano-medicine prepared by the invention is 10-200nm, and the particle size is relatively uniform, which is beneficial to the enrichment at the tumor site, improves the targeting of the drug, and reduces the toxic and side effects on normal cells.
另一方面，本发明提供了如本发明第一方面所述的抗肿瘤多肽纳米药物在制备抗肿瘤药物中的应用。In another aspect, the present invention provides the application of the anti-tumor polypeptide nanomedicine according to the first aspect of the present invention in the preparation of anti-tumor drugs.
相对于现有技术，本发明具有以下有益效果：Compared with the prior art, the present invention has the following beneficial effects:
本发明的抗肿瘤多肽纳米药物生物相容性好，毒副作用低，具有酸性pH响应性，纳米材料本身即为药物，生物利用度高。本发明的抗肿瘤多肽纳米药物以多肽和氨基酸为原料制备而成，具有高度的生物安全性。与裸肽比较，在中性环境下，抗肿瘤多肽纳米药物形成高度有序的纳米结构，隐藏了多肽上的受体结合而位点，从而延长了在体内循环的半衰期，稳定性高，并可富集于肿瘤部位，在肿瘤部位的弱酸性环境中，纳米药物的纳米粒子形态解聚，暴露出抗肿瘤多肽上的受体结合位点，以达到抗肿瘤治疗的作用，提高了抗肿瘤多肽药物的生物利用度。本发明的制备方法简单，自组装过程中不生成共价健，没有逆反应，制备得到的抗肿瘤多肽纳米药物具有广阔的应用前景。The anti-tumor polypeptide nano-medicine of the invention has good biocompatibility, low toxicity and side effects, has acidic pH responsiveness, and the nano-material itself is a drug with high bioavailability. The anti-tumor polypeptide nano-medicine of the present invention is prepared from polypeptide and amino acid as raw materials, and has high biological safety. Compared with naked peptides, in a neutral environment, anti-tumor peptide nano-drugs form a highly ordered nano-structure, which hides the receptor binding sites on the peptide, thereby prolonging the half-life of circulation in the body, high stability, and It can be enriched in the tumor site. In the weakly acidic environment of the tumor site, the nanoparticles of the nanomedicine depolymerize to expose the receptor binding site on the anti-tumor polypeptide, so as to achieve the effect of anti-tumor therapy and improve the anti-tumor effect. Bioavailability of peptide drugs. The preparation method of the invention is simple, no covalent bond is generated in the self-assembly process, and there is no reverse reaction, and the prepared anti-tumor polypeptide nano-medicine has broad application prospects.
附图说明Description of drawings
图1为实施例1中偶联有DEAP的两亲性抗肿瘤多肽的高效液相色谱图；Fig. 1 is the high-performance liquid chromatogram of the amphipathic anti-tumor polypeptide coupled with DEAP in Example 1;
图2为实施例1中偶联有DEAP的两亲性抗肿瘤多肽的质谱图；Fig. 2 is the mass spectrogram of the amphiphilic anti-tumor polypeptide coupled with DEAP in Example 1;
图3A为实施例1中抗肿瘤多肽纳米药物在中性磷酸盐缓冲液中的电镜形貌图；Fig. 3A is the electron microscope morphology of the anti-tumor polypeptide nano drug in neutral phosphate buffer in Example 1;
图3B为实施例1中抗肿瘤多肽纳米药物在中性磷酸盐缓冲液中的粒径分布图；Figure 3B is a particle size distribution diagram of the anti-tumor polypeptide nano drug in neutral phosphate buffer in Example 1;
图4A为实施例1中制备的抗肿瘤多肽纳米药物在pH值为6.8的弱酸性磷酸盐缓冲液中的电镜形貌图；Fig. 4A is the electron micrograph of the anti-tumor polypeptide nano-medicine prepared in Example 1 in a weakly acidic phosphate buffer with a pH value of 6.8;
图4B为实施例1中制备的抗肿瘤多肽纳米药物在pH值为6.8的弱酸性磷酸盐缓冲液中的粒径分布图；Figure 4B is a particle size distribution diagram of the anti-tumor polypeptide nanomedicine prepared in Example 1 in a weakly acidic phosphate buffer with a pH value of 6.8;
图5为实施例8中对实施例1制备的抗肿瘤多肽纳米药物进行肿瘤部位响应性测定的结果图；FIG. 5 is a result diagram of the tumor site responsiveness determination of the anti-tumor polypeptide nanomedicine prepared in Example 1 in Example 8;
图6为实施例9中测定的实施例1制备的抗肿瘤多肽纳米药物在小鼠体内血液循环中的稳定性结果图；Figure 6 is a graph showing the stability results of the anti-tumor polypeptide nanomedicine prepared in Example 1 in the blood circulation of mice measured in Example 9;
图7为实施例10中对实施例1制备的的抗肿瘤多肽纳米药物测定的抑制肿瘤生长的效果图。FIG. 7 is a graph showing the effect of inhibiting tumor growth measured by the anti-tumor polypeptide nanomedicine prepared in Example 1 in Example 10.
具体实施方式detailed description
下面通过具体实施方式来进一步说明本发明的技术方案。本领域技术人员应该明了，所述实施例仅仅是帮助理解本发明，不应视为对本发明的具体限制。The technical solutions of the present invention will be further described below through specific embodiments. It should be clear to those skilled in the art that the embodiments are only for helping to understand the present invention, and should not be regarded as specific limitations on the present invention.
实施例1Example 1
在本实施例中，通过以下方法制备抗肿瘤多肽纳米药物，所述方法为：In this example, the anti-tumor polypeptide nano drug is prepared by the following method, which is:
选用M.Lourdes Ponce等，Cancer Research,2003,63:5060-5064提供的抑制肿瘤新生血管生成和肿瘤生长的C16Y多肽(从氨基端开始的序列为：天冬氨酸-苯丙氨酸-赖氨酸-亮氨酸-苯丙氨酸-丙氨酸-缬氨酸-酪氨酸-异亮氨酸-赖氨酸-酪氨酸-精氨酸)作为亲水性抗肿瘤多肽，按照文献(Lihong Liu等，Nature Nanotechnology,2009,4:457-463和Ying Zhao等，J Control Release,2014,177:11-19)提供的固相合成法以及多肽纯化方法，将8个亮氨酸和2个甘氨酸组成的疏水性多肽连接至抗肿瘤多肽C16Y上，并在疏水性多肽的氨基末端连接由3个赖氨酸形成的三肽，而后通过赖氨酸上的氨基(包括末端氨基和侧链氨基)连接4个3-(二乙基氨基)丙基硫代异氰酸酯功能分子(DEAP)，获得偶联有酸响应性功能分子的两亲性抗肿瘤多肽。所述合成过程如下：M. Lourdes Ponce et al., Cancer Research, 2003, 63: 5060-5064 provide the C16Y polypeptide that inhibits tumor angiogenesis and tumor growth (the sequence starting from the amino terminal is: aspartic acid-phenylalanine-lysine amino acid-leucine-phenylalanine-alanine-valine-tyrosine-isoleucine-lysine-tyrosine-arginine) as a hydrophilic anti-tumor polypeptide, according to Literature (Lihong Liu et al., Nature Nanotechnology, 2009, 4:457-463 and Ying Zhao et al., J Control Release, 2014, 177:11-19) provide solid-phase synthesis and peptide purification methods, the eight leucine The hydrophobic polypeptide composed of 2 glycines is connected to the anti-tumor polypeptide C16Y, and the tripeptide formed by 3 lysines is connected at the amino terminal of the hydrophobic polypeptide, and then the amino group on the lysine (including the terminal amino group and side chain amino) to link four 3-(diethylamino)propylthioisocyanate functional molecules (DEAP) to obtain an amphiphilic anti-tumor polypeptide coupled with an acid-responsive functional molecule. The synthesis process is as follows:
(1)用于合成C16Y的氨基酸以及亮氨酸、甘氨酸和赖氨酸的末端氨基均由Fmoc(笏甲氧羰基)保护，用于合成C16Y的赖氨酸侧链氨基由Boc(叔丁氧羰基)保护，用于偶联功能分子的赖氨酸侧链的氨基由CBZ(苄氧羰基)保护，以上氨基酸均购自吉尔生化(上海)有限公司。(1) The amino acid used to synthesize C16Y and the terminal amino groups of leucine, glycine and lysine are all protected by Fmoc (watt methoxycarbonyl), and the amino acid side chain of lysine used to synthesize C16Y is protected by Boc (tert-butoxy Carbonyl) protection, the amino group of the lysine side chain used for coupling functional molecules is protected by CBZ (benzyloxycarbonyl), and the above amino acids were purchased from Jill Biochemical (Shanghai) Co., Ltd.
(2)使C16Y羧基末端氨基酸的羧基与CLEAR-酰胺树脂(引入CLEAR-酰胺树脂的目的是将氨基酸的羧基末端固定，以便使其氨基端发生反应)的氨基末端连接，通过20％哌啶/N,N-二甲基甲酰胺脱去该氨基酸上的Fmoc保护基以暴露出氨基，然后以此结合在树脂上的氨基酸作为氨基组分，同过量的含活化羧基的下一个氨基酸反应接长肽链，重复上述操作直至所有的氨基酸缩合完毕，形成肽链上氨基被保护的两亲性抗肿瘤多肽。(2) The carboxyl of the C16Y carboxyl-terminal amino acid is connected to the amino-terminal of the CLEAR-amide resin (the purpose of introducing the CLEAR-amide resin is to fix the carboxyl-terminus of the amino acid so that the amino-terminus reacts), through 20% piperidine/ N,N-Dimethylformamide removes the Fmoc protecting group on the amino acid to expose the amino group, and then uses the amino acid bound to the resin as the amino component to react with an excess of the next amino acid containing an activated carboxyl group. Peptide chain, repeat the above operation until all the amino acids are condensed to form an amphipathic anti-tumor polypeptide with amino groups protected on the peptide chain.
(3)将两亲性抗肿瘤多肽用20％哌啶/N,N-二甲基甲酰胺脱去Fmoc保护基，用催化氢解法脱去CBZ保护基，使酸响应性功能分子DEAP与脱去保护的氨基反应，从而将酸响应性功能分子DEAP连接至两亲性抗肿瘤多肽上。(3) Use 20% piperidine/N,N-dimethylformamide to remove the Fmoc protecting group of the amphiphilic anti-tumor polypeptide, and remove the CBZ protecting group by catalytic hydrogenolysis, so that the acid-responsive functional molecule DEAP and the The deprotected amino group is reacted to link the acid-responsive functional molecule DEAP to the amphipathic anti-tumor polypeptide.
(4)用高浓度三氟乙酸的二氯甲烷溶液将肽链从树脂上裂解下来，C16Y片段中的Boc保护基团也将同时除去，经过纯化等处理，即得到偶联有DEAP的两亲性抗肿瘤多肽。(4) Cleavage the peptide chain from the resin with a dichloromethane solution of high-concentration trifluoroacetic acid, and the Boc protecting group in the C16Y fragment will also be removed at the same time. After purification and other treatments, the amphiphile coupled with DEAP can be obtained. antitumor polypeptides.
(5)取0.5mg偶联有DEAP的两亲性抗肿瘤多肽溶解于10μL二甲基亚砜中，接着加入到1mL的pH值为7.4的磷酸盐缓冲液中，将混合液于功率为600W的超声波清洗仪中超声处理2min。超声完毕后，样品于室温静置2h后即得到抗肿瘤多肽纳米药物体系。体系中的二甲基亚砜通过在pH 7.4的磷酸盐缓冲液中透析除去。(5) Dissolve 0.5 mg of the amphiphilic anti-tumor polypeptide coupled with DEAP in 10 μL of dimethyl sulfoxide, then add it to 1 mL of phosphate buffer with a pH value of 7.4, and put the mixture at a power of 600W Ultrasonic treatment for 2 min in an ultrasonic cleaner. After the sonication, the sample was allowed to stand at room temperature for 2 hours to obtain the anti-tumor polypeptide nano drug system. Dimethyl sulfoxide in the system was removed by dialysis against pH 7.4 phosphate buffer.
通过高效液相色谱和电喷雾离子化质谱等手段证实了本实施例得到的偶联有DEAP的两亲性抗肿瘤多肽的结构为：C16Y-(甘氨酸形成的二肽)-(亮氨酸形成的八肽)-(赖氨酸形成的三肽)-(DEAP)4，图1为抗肿瘤多肽的高效液相色谱图，图2为合成的抗肿瘤多肽的质谱图，表1中总结了图1的高效液相色谱图中各峰的出峰时间、峰面积、高度以及含量数据。The structure of the amphiphilic anti-tumor polypeptide coupled with DEAP obtained in this example was confirmed by means of high performance liquid chromatography and electrospray ionization mass spectrometry: C16Y-(dipeptide formed by glycine)-(formed by leucine) Octapeptide)-(tripeptide formed by lysine)-(DEAP)4 , Fig. 1 is a high performance liquid chromatogram of an anti-tumor polypeptide, Fig. 2 is a mass spectrogram of a synthetic anti-tumor polypeptide, summarized in Table 1 The peak time, peak area, height and content data of each peak in the high performance liquid chromatogram of Fig. 1.
表1Table 1
由图1和图2的结果分析得出主峰3为合成的抗肿瘤多肽的峰，由表1的结果可以看出，合成的抗肿瘤多肽纯度在90％以上。From the analysis of the results in Figure 1 and Figure 2, it can be concluded that the main peak 3 is the peak of the synthesized anti-tumor polypeptide, and it can be seen from the results in Table 1 that the purity of the synthesized anti-tumor polypeptide is above 90%.
利用透射电镜(美国FEI，Tecnai G2 20 S-TWIN，200kV)和激光粒度仪(英国Malvern,Zetasizer Nano ZS90)对得到的抗肿瘤多肽纳米药物体系进行形态以及粒径表征，如图3所示，其中图3A为抗肿瘤多肽纳米药物体系的透射电镜图，从图中可以看出，制备得到的抗肿瘤多肽纳米药物呈球形，颗粒大小较均一；图3B为粒径分布图，所得抗肿瘤多肽纳米药物的粒径分布为15-60nm，平均粒径约为30nm，分散指数(PDI)为0.373，与电镜图所测得的结果相符合。The shape and particle size of the obtained anti-tumor polypeptide nano drug system were characterized by transmission electron microscopy (US FEI, Tecnai G2 20 S-TWIN, 200kV) and laser particle size analyzer (UK Malvern, Zetasizer Nano ZS90), as shown in Figure 3. Among them, Figure 3A is a transmission electron microscope image of the anti-tumor polypeptide nano-drug system. It can be seen from the figure that the prepared anti-tumor polypeptide nano-drug is spherical, and the particle size is relatively uniform; Figure 3B is a particle size distribution diagram, and the obtained anti-tumor polypeptide The particle size distribution of the nanomedicine is 15-60nm, the average particle size is about 30nm, and the dispersion index (PDI) is 0.373, which is consistent with the results measured by the electron microscope.
实施例2Example 2
在本实施例中，通过与实施例1相同的合成方法以及步骤，实现了在抗肿瘤多肽C16Y上偶联3个异亮氨酸和2个蛋氨酸以及1个赖氨酸，并在赖氨酸的末端氨基和侧链氨基上连接了DEAP分子，得到了偶联有DEAP的两亲性抗肿瘤多肽。经与实施例1相同的自组装过程得到抗肿瘤多肽纳米药物体系。In this example, through the same synthesis method and steps as in Example 1, 3 isoleucine, 2 methionine and 1 lysine were coupled to the anti-tumor polypeptide C16Y, and the lysine DEAP molecules are connected to the terminal amino group and the side chain amino group, and the amphipathic anti-tumor polypeptide coupled with DEAP is obtained. The anti-tumor polypeptide nano drug system was obtained through the same self-assembly process as in Example 1.
经过高效液相色谱和质谱等手段证实了本实施例得到的偶联有DEAP的两亲性抗肿瘤多肽的结构为：C16Y-(异亮氨酸形成的三肽)-(蛋氨酸形成的二肽)-赖氨酸-(DEAP)2。The structure of the amphiphilic anti-tumor polypeptide coupled with DEAP obtained in this example is confirmed by means of high performance liquid chromatography and mass spectrometry: C16Y-(tripeptide formed by isoleucine)-(dipeptide formed by methionine) )-lysine-(DEAP)2 .
利用透射电镜和激光粒度仪对得到的抗肿瘤多肽纳米药物体系进行形态以及粒径表征，结果表明制备得到的抗肿瘤多肽纳米药物呈球形，颗粒大小较均一，抗肿瘤多肽纳米药物的粒径分布为40-200nm，平均粒径约为150nm，分散指数(PDI)为0.368。The morphology and particle size of the obtained anti-tumor polypeptide nano-drug system were characterized by transmission electron microscope and laser particle size analyzer. It is 40-200nm, the average particle size is about 150nm, and the dispersion index (PDI) is 0.368.
实施例3Example 3
在本实施例中，通过与实施例1相同的合成方法以及步骤，实现了在抗肿瘤多肽C16Y上偶联20个丙氨酸以及5个赖氨酸，并在赖氨酸的末端氨基和侧链氨基上连接了DEAP分子，得到了偶联有DEAP的两亲性抗肿瘤多肽。经与实施例1相同的自组装过程得到抗肿瘤多肽纳米药物体系。In this example, through the same synthesis method and steps as in Example 1, 20 alanines and 5 lysines were coupled to the anti-tumor polypeptide C16Y, and the terminal amino group and side of lysine A DEAP molecule is connected to the chain amino group, and an amphipathic anti-tumor polypeptide coupled with DEAP is obtained. The anti-tumor polypeptide nano drug system was obtained through the same self-assembly process as in Example 1.
经过高效液相色谱和质谱等手段证实了本实施例得到的偶联有DEAP的两亲性抗肿瘤多肽的结构为：C16Y-(20个丙氨酸形成的多肽)-(5个赖氨酸形成的多肽)-(DEAP)6。The structure of the amphiphilic anti-tumor polypeptide coupled with DEAP obtained in this example was confirmed by means of high performance liquid chromatography and mass spectrometry: C16Y-(polypeptide formed by 20 alanines)-(5 lysines Formed polypeptide)-(DEAP)6 .
利用透射电镜和激光粒度仪对得到的抗肿瘤多肽纳米药物体系进行形态以及粒径表征，结果表明制备得到的抗肿瘤多肽纳米药物呈球形，颗粒大小较均一，抗肿瘤多肽纳米药物的粒径分布为30-160nm，平均粒径约为100.5nm，分散指数(PDI)为0.324。The morphology and particle size of the obtained anti-tumor polypeptide nano-drug system were characterized by transmission electron microscope and laser particle size analyzer. It is 30-160nm, the average particle size is about 100.5nm, and the dispersion index (PDI) is 0.324.
实施例4Example 4
在本实施例中，通过与实施例1相同的合成方法以及步骤，实现了在抗肿瘤多肽C16Y上偶联20个缬氨酸、10个酪氨酸以及5个赖氨酸，并在赖氨酸的末端氨基和侧链氨基上连接了DEAP分子，得到了偶联有DEAP的两亲性抗肿瘤多肽。经与实施例1相同的自组装过程得到抗肿瘤多肽纳米药物体系。In this example, through the same synthesis method and steps as in Example 1, 20 valines, 10 tyrosines, and 5 lysines were coupled to the anti-tumor polypeptide C16Y, and lysine The terminal amino group and the side chain amino group of the acid are connected with DEAP molecule, and the amphipathic anti-tumor polypeptide coupled with DEAP is obtained. The anti-tumor polypeptide nano drug system was obtained through the same self-assembly process as in Example 1.
经过质谱以及核磁等手段证实了本实施例得到的偶联有DEAP的两亲性抗肿瘤多肽的结构为：C16Y-(20个缬氨酸形成的多肽)-(10个酪氨酸形成的多肽)-(5个赖氨酸形成的多肽)-(DEAP)6。The structure of the amphiphilic anti-tumor polypeptide coupled with DEAP obtained in this example was confirmed by means of mass spectrometry and NMR as follows: C16Y-(polypeptide formed by 20 valines)-(polypeptide formed by 10 tyrosines )-(polypeptide formed by 5 lysines)-(DEAP)6 .
利用透射电镜和激光粒度仪对得到的抗肿瘤多肽纳米药物体系进行形态以及粒径表征，结果表明制备得到的抗肿瘤多肽纳米药物呈球形，颗粒大小较均一，抗肿瘤多肽纳米药物的粒径分布为10-180nm，平均粒径约为123.6nm，分散指数(PDI)为0.348。The morphology and particle size of the obtained anti-tumor polypeptide nano-drug system were characterized by transmission electron microscope and laser particle size analyzer. It is 10-180nm, the average particle size is about 123.6nm, and the dispersion index (PDI) is 0.348.
实施例5Example 5
在本实施例中，通过与实施例1相同的合成方法以及步骤，实现了在抗肿瘤多肽C16Y上偶联40个亮氨酸以及4个赖氨酸，并在赖氨酸的末端氨基和侧链氨基上连接了DEAP分子，得到了偶联有DEAP的两亲性抗肿瘤多肽。经与实施例1相同的自组装过程得到抗肿瘤多肽纳米药物体系。In this example, through the same synthesis method and steps as in Example 1, 40 leucines and 4 lysines were coupled to the anti-tumor polypeptide C16Y, and the terminal amino group and the side of lysine A DEAP molecule is connected to the chain amino group, and an amphipathic anti-tumor polypeptide coupled with DEAP is obtained. The anti-tumor polypeptide nano drug system was obtained through the same self-assembly process as in Example 1.
经过质谱以及核磁等手段证实了本实施例得到的偶联有DEAP的两亲性抗肿瘤多肽的结构为：C16Y-(40个亮氨酸形成的多肽)-(4个赖氨酸形成多肽)-(DEAP)5。The structure of the amphiphilic anti-tumor polypeptide coupled with DEAP obtained in this example is confirmed by means of mass spectrometry and NMR: C16Y-(polypeptide formed by 40 leucines)-(polypeptide formed by 4 lysines) -(DEAP)5 .
利用透射电镜和激光粒度仪对得到的抗肿瘤多肽纳米药物体系进行形态以及粒径表征，结果表明制备得到的抗肿瘤多肽纳米药物呈球形，颗粒大小较均一，抗肿瘤多肽纳米药物的粒径分布为10-120nm，平均粒径约为78.6nm，分散指数(PDI)为0.311。The morphology and particle size of the obtained anti-tumor polypeptide nano-drug system were characterized by transmission electron microscope and laser particle size analyzer. It is 10-120nm, the average particle size is about 78.6nm, and the dispersion index (PDI) is 0.311.
实施例6Example 6
在本实施例中，通过与实施例1相同的合成方法以及步骤，实现了在文献(Stephanie Filleur等，Cancer Research,2005,65:5144-5152)提供的含有11个氨基酸的来源于色素上皮细胞衍生因子的多肽TGA(该多肽能抑制肿瘤血管生成和肿瘤生长)上偶联10个亮氨酸和3个甘氨酸以及2个赖氨酸，并在赖氨酸的末端氨基和侧链氨基上连接了DEAP分子，得到了偶联有DEAP的两亲性抗肿瘤多肽。经与实施例1相同的自组装过程得到抗肿瘤多肽纳米药物体系。In this example, through the same synthesis method and steps as in Example 1, the pigment epithelial cell containing 11 amino acids provided in the literature (Stephanie Filleur et al., Cancer Research, 2005, 65:5144-5152) was realized. The peptide TGA of the derivative factor (the polypeptide can inhibit tumor angiogenesis and tumor growth) is coupled with 10 leucines, 3 glycines and 2 lysines, and is connected to the terminal amino group and the side chain amino group of the lysine The DEAP molecule was obtained, and the amphipathic anti-tumor polypeptide coupled with DEAP was obtained. The anti-tumor polypeptide nano drug system was obtained through the same self-assembly process as in Example 1.
利用透射电镜和激光粒度仪对得到的抗肿瘤多肽纳米药物体系进行形态以及粒径表征，结果表明制备得到的抗肿瘤多肽纳米药物呈球形，颗粒大小较均一，抗肿瘤多肽纳米药物的粒径分布为10-60nm，平均粒径约为21.04，分散指数(PDI)为0.292。The morphology and particle size of the obtained anti-tumor polypeptide nano-drug system were characterized by transmission electron microscope and laser particle size analyzer. It is 10-60nm, the average particle size is about 21.04, and the dispersion index (PDI) is 0.292.
实施例7Example 7
本实施例目的在于测定抗肿瘤多肽纳米药物自组装纳米颗粒在微酸性溶液中的形貌和粒径。The purpose of this example is to determine the morphology and particle size of self-assembled nanoparticles of anti-tumor polypeptide nano-drugs in a slightly acidic solution.
将实施例1中得到的抗肿瘤多肽纳米药物体系样品的pH值调整至6.8，室温静置2h后，通过透射电镜观察形貌并用激光粒度仪测定粒径分布。如图4A所示，在酸性溶液中，多肽已无明显的纳米球形结构，如图4B所示，粒径分布为4-18nm，平均粒径为7nm。另外将抗肿瘤多肽纳米药物体系样品的pH值分别调整至6.7、7.0、7.1和7.2时均得到了与将pH值调整至6.8相似的结果，这说明在酸性溶液中，抗肿瘤多肽纳米药物不再是纳米球状，而是由于酸响应性分子带有正电荷之后，使得纳米球结构产生排斥力，使得药物不再聚集为纳米球。The pH value of the anti-tumor polypeptide nano drug system sample obtained in Example 1 was adjusted to 6.8, and after standing at room temperature for 2 hours, the morphology was observed with a transmission electron microscope and the particle size distribution was measured with a laser particle size analyzer. As shown in Figure 4A, in the acidic solution, the polypeptide has no obvious nano-spherical structure, as shown in Figure 4B, the particle size distribution is 4-18 nm, and the average particle size is 7 nm. In addition, when the pH value of the anti-tumor polypeptide nano-drug system sample was adjusted to 6.7, 7.0, 7.1 and 7.2, the results similar to those of adjusting the pH value to 6.8 were obtained, which shows that in acidic solution, the anti-tumor polypeptide nano-drug is not It is nano-spherical again, but because the acid-responsive molecules are positively charged, the nano-spherical structure generates repulsive force, so that the drug no longer aggregates into nano-spherical.
实施例8Example 8
本实施例目的在于测定抗肿瘤多肽纳米药物自组装纳米颗粒在体内对肿瘤酸性环境的响应性。The purpose of this example is to determine the responsiveness of self-assembled nanoparticles of anti-tumor polypeptide nano-drugs to the acidic environment of tumors in vivo.
取0.5mg实施例1步骤(4)得到的偶联有DEAP的两亲性抗肿瘤多肽(DEAP-两亲性抗肿瘤多肽)，与0.1mg四甲基罗丹明-5-异硫氰酸酯荧光分子和0.1mg淬灭分子共同溶解于10μL二甲基亚砜中，接着加入到1mL的pH值为7.4的磷酸盐缓冲液中，将混合液于功率为600W的超声波清洗仪中超声处理2min。超声完毕后，样品于室温静置2h后，于10000g离心5min，取上清即得到同时包载有荧光分子和淬灭分子的DEAP-两亲性抗肿瘤多肽自组装纳米颗粒，经测定该纳米颗粒为大小较均一、稳定的球形结构。Take 0.5 mg of the amphiphilic anti-tumor polypeptide (DEAP-amphipathic anti-tumor polypeptide) coupled with DEAP obtained in step (4) of Example 1, and 0.1 mg of tetramethylrhodamine-5-isothiocyanate Fluorescent molecules and 0.1 mg of quencher molecules were dissolved in 10 μL of dimethyl sulfoxide, then added to 1 mL of phosphate buffer with a pH value of 7.4, and the mixture was ultrasonically treated in an ultrasonic cleaner with a power of 600W for 2 min . After the ultrasound was completed, the sample was left to stand at room temperature for 2 hours, then centrifuged at 10,000 g for 5 minutes, and the supernatant was taken to obtain DEAP-amphiphilic anti-tumor polypeptide self-assembled nanoparticles loaded with fluorescent molecules and quencher molecules at the same time. The particles are relatively uniform in size and stable spherical structure.
取100μL制备的纳米颗粒，从尾静脉处注射至荷瘤小鼠体内，于1h、3h和5h用小动物活体成像仪(美国的Cambridge Research&Instrumentation，MaestroTM)检测体内荧光分布，如图5所示，荧光信号主要分布于肿瘤部位，这说明本发明制备的抗肿瘤多肽纳米药物能响应弱酸性的肿瘤环境而解聚释放出荧光分子。Take 100 μL of the prepared nanoparticles and inject them into the tumor-bearing mice from the tail vein, and detect the fluorescence distribution in vivo with a small animal in vivo imager (Cambridge Research & Instrumentation, MaestroTM , USA) at 1 h, 3 h and 5 h, as shown in Figure 5 , the fluorescent signal is mainly distributed in the tumor site, which indicates that the anti-tumor polypeptide nano drug prepared in the present invention can depolymerize and release fluorescent molecules in response to the weakly acidic tumor environment.
实施例9Example 9
本实施例目的在于测定抗肿瘤多肽纳米药物自组装纳米颗粒在血液循环中的稳定性。The purpose of this example is to determine the stability of self-assembled nanoparticles of anti-tumor polypeptide nanomedicine in blood circulation.
利用实施例1中制备的抗肿瘤多肽纳米药物(简记为DEAP-C16Y)进行测定，以C16Y多肽为对照样品，各取1μmol的C16Y和DEAP-C16Y，将3μmol的荧光分子Cy5.5分别偶联至C16Y和DEAP-C16Y多肽分子的氨基上，获得偶联荧光分子的多肽。将偶联Cy5.5的C16Y多肽直接溶解在1mL的pH值为7.4的磷酸盐缓冲液中；将偶联Cy5.5的DEAP-C16Y溶解于10μL二甲基亚砜中，接着加入到1mL的pH值为7.4的磷酸盐缓冲液中，将混合液于功率为600W的超声波清洗仪中超声处理2min，室温静置2h后获得偶联荧光分子的DEAP-C16Y多肽自组装纳米颗粒。体系中的二甲基亚砜以及多余的荧光分子通过在pH 7.4的磷酸盐缓冲液中透析除去。Utilize the anti-tumor polypeptide nano-medicine prepared in Example 1 (abbreviated as DEAP-C16Y) to measure, take the C16Y polypeptide as a control sample, take 1 μmol of C16Y and DEAP-C16Y, and couple 3 μmol of fluorescent molecule Cy5.5 to each Linked to the amino groups of C16Y and DEAP-C16Y polypeptide molecules to obtain polypeptides coupled with fluorescent molecules. Dissolve the Cy5.5-coupled C16Y polypeptide directly in 1 mL of phosphate buffer with a pH value of 7.4; dissolve the Cy5.5-coupled DEAP-C16Y in 10 μL of dimethyl sulfoxide, and then add it to 1 mL of In a phosphate buffer solution with a pH value of 7.4, the mixture was ultrasonically treated in an ultrasonic cleaner with a power of 600W for 2 minutes, and after standing at room temperature for 2 hours, DEAP-C16Y polypeptide self-assembled nanoparticles coupled with fluorescent molecules were obtained. Dimethyl sulfoxide and excess fluorescent molecules in the system were removed by dialysis in pH 7.4 phosphate buffer.
将偶联荧光分子的C16Y和DEAP-C16Y自组装纳米颗粒，于尾静脉处注射入BALB/c小鼠体内，于1h、3h、5h、8h、12h和24h等时间点分别从尾静脉处取小鼠血液30μL，于4000g离心10min后，取上清获得血浆。利用小动物活体成像仪检测血浆中的荧光信号，与注射荧光标记的C16Y组比较，如图6所示，注射荧光标记的DEAP-C16Y自组装纳米颗粒组的血浆中的荧光信号存在的时间显著延长，这说明本发明制备的抗肿瘤多肽纳米药物(DEAP-C16Y)比C16Y在体内血液循环中更稳定。The C16Y and DEAP-C16Y self-assembled nanoparticles coupled with fluorescent molecules were injected into the BALB/c mice at the tail vein, and were taken from the tail vein at 1h, 3h, 5h, 8h, 12h and 24h respectively. 30 μL of mouse blood was centrifuged at 4000 g for 10 min, and the supernatant was collected to obtain plasma. Utilize the small animal in vivo imager to detect the fluorescent signal in the plasma, compared with the group injected with fluorescently labeled C16Y, as shown in Figure 6, the fluorescent signal in the plasma of the group injected with fluorescently labeled DEAP-C16Y self-assembled nanoparticles exists for a significant time This shows that the anti-tumor polypeptide nano drug (DEAP-C16Y) prepared by the present invention is more stable in the blood circulation in vivo than C16Y.
实施例10Example 10
本实施例的目的在于测试多肽自组装纳米颗粒抑制肿瘤生长的效果。The purpose of this example is to test the effect of polypeptide self-assembled nanoparticles on inhibiting tumor growth.
利用实施例1中制备的抗肿瘤多肽纳米药物(简记为DEAP-C16Y)进行测定，以C16Y多肽为对照样品。将BALB/c裸鼠于乳腺脂肪垫处接种MDA-MB-231乳腺癌细胞，待肿瘤生长至体积为100mm3时，尾静脉注射中性磷酸盐(PBS)缓冲液、C16Y或DEAP-C16Y，其中C16Y为每天注射一次，DEAP-C16Y每两天注射一次和每三天注射一次各设一组，其中C16Y多肽剂量均为6.5μmol/kg，PBS为每三天注射一次，每组5只小鼠。处理14天并继续观察一周后，将小鼠处死，取出肿瘤组织，称取瘤重。如图7所示，DEAP-C16Y每两天注射一次时抑制肿瘤生长的效果最显著，明显优于DEAP-C16Y每三天注射一次以及C16Y每天注射一次的抑瘤率，而DEAP-C16Y每三天注射一次与C16Y每天注射一次的抑瘤率相当，这说明应用DEAP-C16Y和C16Y对肿瘤进行治疗时，要达到相同的治疗效果，在每次注射中所含C16Y剂量相同的情况下，要多次注射单独的C16Y，而DEAP-C16Y每三天注射一次即可，减少了用药次数，这也侧面反映了注射单独的C16Y时，由于C16Y在体内循环时不稳定而造成药物利用率低，本发明的抗肿瘤纳米药物很好地克服了此缺陷，具有很好的应用前景。The anti-tumor polypeptide nanomedicine prepared in Example 1 (abbreviated as DEAP-C16Y) was used for determination, and the C16Y polypeptide was used as a control sample. BALB/c nude mice were inoculated with MDA-MB-231 breast cancer cells in the mammary fat pad, and when the tumor grew to a volume of 100 mm, neutral phosphate (PBS) buffer, C16Y or DEAP-C16Y was injected into the tail vein, Among them, C16Y was injected once a day, DEAP-C16Y was injected once every two days and once every three days, and each group was set up. The dose of C16Y polypeptide was 6.5 μmol/kg, and PBS was injected once every three days. There were 5 small animals in each group. mouse. After 14 days of treatment and continued observation for one week, the mice were sacrificed, the tumor tissues were taken out, and the tumor weight was weighed. As shown in Figure 7, DEAP-C16Y had the most significant tumor growth inhibitory effect when injected once every two days, which was significantly better than the tumor inhibition rate of DEAP-C16Y injected once every three days and C16Y injected once a day, while DEAP-C16Y was injected every three days. The tumor inhibition rate of once-a-day injection is equivalent to that of C16Y injection once a day, which shows that when DEAP-C16Y and C16Y are used to treat tumors, to achieve the same therapeutic effect, when the dose of C16Y contained in each injection is the same, the Multiple injections of C16Y alone, while DEAP-C16Y can be injected once every three days, which reduces the number of medications, which also reflects that when C16Y is injected alone, the drug utilization rate is low due to the instability of C16Y in the circulation in the body. The anti-tumor nano drug of the invention well overcomes this defect and has a good application prospect.
申请人声明，本发明通过上述实施例来说明本发明的抗肿瘤多肽纳米药物及其制备方法和应用，但本发明并不局限于上述实施例，即不意味着本发明必须依赖上述实施例才能实施。所属技术领域的技术人员应该明了，对本发明的任何改进，对本发明所选用原料的等效替换及辅助成分的添加、具体方式的选择等，均落在本发明的保护范围和公开范围之内。The applicant declares that the present invention illustrates the anti-tumor polypeptide nanomedicine of the present invention and its preparation method and application through the above examples, but the present invention is not limited to the above examples, that is, it does not mean that the present invention must rely on the above examples to achieve implement. Those skilled in the art should understand that any improvement of the present invention, the equivalent replacement of the selected raw materials in the present invention, the addition of auxiliary components, the selection of specific methods, etc., all fall within the scope of protection and disclosure of the present invention.
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