WO2010051667A1

WO2010051667A1 - Pharmaceutical compositions comprising cyclic peptide nanotubes and the uses thereof

Info

Publication number: WO2010051667A1
Application number: PCT/CN2008/073015
Authority: WO
Inventors: 吴伟; 陈健; 邱怡婷; 张蓓; 卢懿
Original assignee: 复旦大学
Priority date: 2008-11-10
Filing date: 2008-11-11
Publication date: 2010-05-14

Abstract

The pharmaceutical compositions composed of cyclic peptide nanotubes and therapeutic agents and their uses are disclosed. The nanotubes are assembled by cyclic peptides and their Van Der Waals interior diameter is 0.75-1.3 nm, while the length of the nanotubes is 10-100 nm. The cyclic peptides are obtained by cyclization of 8, 10 or 12 amino acids. The diameter of the therapeutic agents in the compositions is less than the Van Der Waals interior diameter of the nanotubes. Nanometer artificial transmembrane channels can be formed by the nanotubes and the therapeutic agents can be transported into cells through the channels by passive diffusion. The compositions can be used for treating cancer, microbial infection or viral infection.

Description

一种环肽纳米管药物组合物及其应用 Cyclic peptide nanotube pharmaceutical composition and application thereof

【技术领域】本发明涉及一种药物组合物，具体地说，是关于一种环肽纳米管药物组合物及其应用。 TECHNICAL FIELD The present invention relates to a pharmaceutical composition, and more particularly to a cyclic peptide nanotube pharmaceutical composition and use thereof.

【背景技术】【Background technique】

偶数个 D，L -氨基酸交替连接可形成闭合环肽。为了使分子内氨基酸侧链与侧链及侧与主链间的相互作用最小，环肽采取平面构型。（X -碳上的取代基平行于环面并沿着环的半径指向环外，主链骨架上的羰基与氨基均垂直与环面。中空的环肽分子通过主链骨架酰氨基中的 C=0和 N - H以 β - 片层的方式形成分子间的氢键网络而堆积成性质稳定、末端开放、尺寸在分子范围的管状结构一自组装环肽纳米管（J Am Chem Soc, 1998 ， 120 (35) : 8949. ) 。这种 β -片层即可以是顺势平行也可以是反势平行，但是计算机分析和实验均表明，基于此原理设计的环肽纳米管的 β -片层氢键在热力学上优先选择反势平行结构（Ma t Sc i Eng C, 1997, 4 (4) : 207. ) 。以这种方式形成纳米管具有 2个尤为突出的优点： ①可以通过控制环肽氨基酸的个数来调节纳米管的孔径； ②可以通过控制环肽的氨基酸种类来改变纳米管的外壁性质。 An even number of D,L-amino acids are alternately linked to form a closed loop peptide. In order to minimize the interaction between the side chain of the amino acid in the molecule and the side chain and the side and the main chain, the cyclic peptide adopts a planar configuration. (The substituent on the X-carbon is parallel to the annulus and is directed outside the ring along the radius of the ring. The carbonyl group and the amino group on the backbone of the main chain are perpendicular to the torus. The hollow cyclic peptide molecule passes through the C in the main chain skeleton acylamino group. =0 and N-H form an intermolecular hydrogen bond network in a β-sheet fashion and accumulate into a tubular structure with a stable nature, open ends, and a molecular size ranging from a self-assembled cyclic peptide nanotube (J Am Chem Soc, 1998). , 120 (35) : 8949. ) This β-sheet can be parallel or parallel, but computer analysis and experiments show that the β-sheet of cyclic peptide nanotubes designed based on this principle Hydrogen bonding is thermodynamically preferred to the counter-parallel structure (Ma t Sc i Eng C, 1997, 4 (4): 207.). The formation of nanotubes in this way has two outstanding advantages: 1 can pass the control loop The number of peptide amino acids is used to modulate the pore size of the nanotube; 2 the outer wall properties of the nanotube can be altered by controlling the amino acid species of the cyclic peptide.

通过改变环肽的氨基酸数目可调节环肽纳米管的孔径，但是环肽的尺寸太小时，环内产生巨大张力不利于亚单元间形成氢键网络；太大时，由于环骨架的松散性也无法自组装成稳定的管状结构（J Am Chem The pore size of the cyclic peptide nanotube can be adjusted by changing the number of amino acids of the cyclic peptide, but the size of the cyclic peptide is too small, and a large tension in the ring is disadvantageous for forming a hydrogen bond network between the subunits; when it is too large, due to the looseness of the ring skeleton Unable to self-assemble into a stable tubular structure (J Am Chem

Soc ， 1996， 118 (1) : 43. ) 。 6、 8、 10、 12个氨基酸环肽均可以自组装成孔径依次增大的环肽纳米管，其中 8、 10、 12氨基酸组成的环肽纳米管的孔径约在 0· 75、 1. 0、 1. 3nm ( J Pept Res， 2001, 57 (4)： 301 ; Curr Op in Biotech, 1999, 10 (1) : 94 ; Angew Chem Int Ed, 2001 , 40 (6) : 988. ) 。 Soc, 1996, 118 (1) : 43. ). 6, 8, 10, 12 amino acid cyclic peptides can self-assemble into cyclic peptide nanotubes with increasing pore diameter, wherein the pore size of cyclic peptide nanotubes composed of 8, 10, 12 amino acids About 0·75, 1. 0, 1. 3nm (J Pept Res, 2001, 57 (4): 301; Curr Op in Biotech, 1999, 10 (1): 94; Angew Chem Int Ed, 2001, 40 ( 6) : 988. ).

与天然蛋白质、多肽一样， D，L -环肽的氨基酸残基顺序应该利于它处于稳定的空间结构，并且环肽中氨基酸的性质对不同条件下的各种自组装均产生重要的影响。环肽纳米管的优越性在于可以通过选择合适的氨基酸来调节纳米管的表面性质，这使得我们可以根据不同的用途来设计不同氨基酸组成的环肽，它可以在不同的化学环境中自组装成性质各异的纳米管。 Like natural proteins and peptides, the amino acid residue sequence of the D,L-cyclic peptide should be favorable for its stable spatial structure, and the nature of the amino acid in the cyclic peptide has an important influence on various self-assembly under different conditions. The advantage of cyclic peptide nanotubes is that the surface properties of the nanotubes can be adjusted by selecting suitable amino acids, which allows us to design cyclic peptides of different amino acids according to different uses, which can self-assemble in different chemical environments. Nanotubes of different nature.

蛋白质或多肽构成的天然离子通道控制着离子的转运、细胞内外物质的交换及信号转导，因此发挥着极其重要的生物学功能。尽管我们对离子通道的结构与功能有了深入的理解，但人工离子通道仍然是当前重要的研究对象之一。理论而言，低电导常数的脂质双分子膜利于环肽在其中的自组装。 1994 年 Ghadi r l 第 1 次通过脂质体模型及一系列光谱学手段验证了这种可能性。将足够浓度 cyc lo [- (Trp-D- Leu) ₃_G ln_D_Leu_]加到脂质体的混悬液中，环肽的疏水性侧链使它们迅速***脂质双分子层中，并依靠分子间的氢键在脂膜中堆积成内径约为 0. 75 謹的纳米管。通过用微膜片钳记录离子单通道电流，证明环肽纳米管对 K+、 Na+转运的活性分别为 2. 2 X 10\ 1. 8 X 10⁷ions - s"¹ , 是相同实验条件下自然界的类似物短杆菌肽 A的 3倍。而 eye lo [- (Gln-D-Leu) -]和 cyc lo [- (^MeN-D-Ala-Phe) -]在相同的实验条件下却无法形成纳米管（J Pept Res , 2001 ，57 (4) ： 301. ) 。这说明环肽分子要在脂质双分子膜中自组装成纳米管，环肽侧链必须具备恰当的疏水性，以使环肽可以进入质膜，并且氨基酸侧链的空间位阻要小，使得环肽分子间能够形成广泛且稳定的氢键。 The natural ion channels formed by proteins or peptides control the transport of ions, the exchange of substances inside and outside the cells, and signal transduction, thus playing an extremely important biological function. Although we have a deep understanding of the structure and function of ion channels, artificial ion channels are still one of the most important research objects. Theoretically, a lipid bimolecular membrane with a low conductance constant facilitates self-assembly of the cyclic peptide therein. In 1994, Ghadi rl verified this possibility for the first time through a liposome model and a series of spectroscopic methods. A sufficient concentration of cyc lo [- (Trp-D- Leu) ₃ _G ln_D_Leu_] is added to the suspension of liposomes, and the hydrophobic side chains of the cyclic peptides allow them to rapidly insert into the lipid bilayer and rely on the molecule The inter-hydrogen bond is deposited in the lipid film to an inner diameter of about 0.75. The activity of the cyclic peptide nanotubes for K+ and Na+ transport was 2. 2 X 10\ 1. 8 X 10 ⁷ ions - s" ¹ , which is the natural state under the same experimental conditions, by recording the ion single-channel current with a micro-film clamp. The analog is 3 times higher than the gramicidin A. However, eye lo [- (Gln-D-Leu) -] and cyc lo [- ( ^Me ND-Ala-Phe) -] cannot form nanometer under the same experimental conditions. Tube (J Pept Res, 2001, 57 (4): 301.) This indicates that the cyclic peptide molecule self-assembles into a nanotube in the lipid bilayer membrane, and the cyclic peptide side chain must have proper hydrophobicity to make the ring Peptides can enter the plasma membrane, and the steric hindrance of the amino acid side chains is small, enabling the formation of broad and stable hydrogen bonds between the cyclic peptide molecules.

人工合成的 D，L _ α -环肽可以嵌入细菌细胞膜，并在脂膜中堆积形成中空的管状通道。体内外试验均表明这些环肽纳米管可以有效杀死耐曱氧西林的金黄色葡萄球菌、耐万古霉素的屎肠球菌及其他致病菌 ( Na ture, 2001， 412 (26) : 452. ) 。自组装环肽纳米管作为新型抗生素的优势在于环肽设计灵活、易于合成及对蛋白酶稳定。通过选择不同的氨基酸组成可以提高它们对细菌细胞膜的选择性和渗透性，降低对人工红细胞的溶血作用。与其他抗生素的作用机制不同，环肽纳米管并没有产生化学作用，而仅仅利用在细菌细胞膜上形成纳米尺寸的孔道而发挥抗菌活性，这种独特的作用机制缩短了杀菌时间，并可以降低耐药性的产生。 Synthetic D,L_α-cyclic peptides can be inserted into the bacterial cell membrane and deposited in the lipid membrane to form a hollow tubular channel. Both in vitro and in vivo tests have shown that these cyclic peptide nanotubes can effectively kill methicillin-resistant Staphylococcus aureus, vancomycin-resistant Enterococcus faecium and other pathogenic bacteria. (Na ture, 2001, 412 (26) : 452. ). The advantage of self-assembling cyclic peptide nanotubes as novel antibiotics is that the cyclic peptides are flexible in design, easy to synthesize, and stable to proteases. By selecting different amino acid compositions, they can increase their selectivity and permeability to bacterial cell membranes and reduce the hemolysis of artificial red blood cells. Unlike other antibiotics, cyclic peptide nanotubes do not produce chemical effects, but only use nano-sized pores on bacterial cell membranes to exert antibacterial activity. This unique mechanism of action shortens sterilization time and reduces resistance. The production of medicinal properties.

鉴于环肽在细胞膜上自组装形成纳米管，允许直径小于其内径的小分子药物通过被动扩散进入细胞内，因此可利用环肽纳米管的这种性质，构建小分子药物输送***，使小分子药物以高浓度快速进入细胞内，提高其细胞毒作用，与环肽纳米管协同作用，在肿瘤治疗以及抗细胞、病毒等病原体感染方面具有应用潜力。 In view of the self-assembly of cyclic peptides on the cell membrane to form nanotubes, small molecules with a diameter smaller than their inner diameter are allowed to diffuse into the cells by passive diffusion. Therefore, this property of cyclic peptide nanotubes can be utilized to construct a small molecule drug delivery system to make small molecules. The drug rapidly enters the cell at a high concentration, enhances its cytotoxic effect, and synergizes with the cyclic peptide nanotubes, and has potential application in tumor treatment and infection against pathogens such as cells and viruses.

【发明内容】 [Summary of the Invention]

本发明的目的是，提供一种环肽纳米管药物组合物。 It is an object of the present invention to provide a cyclic peptide nanotube pharmaceutical composition.

本发明的再一的目的是，提供一种环肽纳米管药物组合物的用途。为实现上述目的，本发明采取的技术方案是：一种环肽纳米管药物组合物，由环肽纳米管和药物组成，所述的环肽纳米管范德华内径为 0. 75-1. 3nm, 管长为 10-100nm, 环肽纳米管基本组成单元为环肽，环肽由 8、 10、 12个氨基酸环合而成，所述的药物分子的直径小于环肽纳米管范德华内径。 It is yet another object of the present invention to provide a use of a cyclic peptide nanotube pharmaceutical composition. _________________________________________________________________________________________________________________________________________________________________________________________________________________________________________________________________________________________________________________________________________________________________________________________________ The tube length is 10-100 nm, the basic constituent unit of the cyclic peptide nanotube is a cyclic peptide, and the cyclic peptide is formed by cyclizing 8, 10, 12 amino acids, and the diameter of the drug molecule is smaller than the inner diameter of the cyclic peptide nanotube van der Waals.

所述的环肽纳米管范德华内径为 0. 75-1. 3nm, 管长为 10-100nm, 环肽纳米管基本组成单元为环肽，环肽由 8、 10、 12 个氨基酸环合而成，所述的药物分子的直径小于环肽纳米管范德华内径。所述的环肽纳米管中的环肽由相同数量的 D型和 L型氨基酸交替共价结合而成，所述的药物分子是亲水性药物。 The cyclic peptide nanotube van der Waals inner diameter is 0. 75-1. 3nm, the tube length is 10-100nm, the basic constituent unit of the cyclic peptide nanotube is a cyclic peptide, and the cyclic peptide is composed of 8, 10, 12 amino acids. The diameter of the drug molecule is smaller than the inner diameter of the cyclic peptide nanotube van der Waals. The cyclic peptide in the cyclic peptide nanotube is formed by alternately covalently bonding the same amount of D-form and L-form amino acid, and the drug molecule is a hydrophilic drug.

所述的环肽纳米管中环肽的氨基酸序列如 SEQ ID NO: 1、 SEQ ID NO: 2、 SEQ ID NO: 3、 SEQ ID NO: 4、 SEQ ID NO: 5、 SEQ ID NO: 6、 SEQ ID NO: 7、 SEQ ID NO: 8、 SEQ ID NO: 9、 SEQ ID NO: 10、 SEQ ID NO: 11、 SEQ ID NO: 12、 SEQ ID NO: 13、 SEQ ID NO: 14、 SEQ ID NO: 15、 SEQ ID NO: 16 , SEQ ID NO: 17 , SEQ ID NO: 18、 SEQ ID NO: 19 , SEQ ID NO: 20、 SEQ ID NO: 2 SEQ ID NO: 22、 SEQ ID NO: 23或 SEQ ID NO: 24所示，所述的药物选自 5 -氟尿嘧啶、氟胞嘧啶、替加氟、水杨酸、对氨基水杨酸、顺铂、卡铂、氮芥、环磷酰胺、异环磷酰胺、氮曱、甘磷酰芥、美法仑、卡莫司汀、洛莫司汀、司莫司汀、尼莫司汀、多巴胺、异烟肼、异硫异烟胺、乙胺丁醇、利巴韦林、齐多夫定或曱硝唑。 The amino acid sequence of the cyclic peptide in the cyclic peptide nanotube is SEQ ID NO: 1, SEQ ID NO: 2, SEQ ID NO: 3, SEQ ID NO: 4, SEQ ID NO: 5, SEQ ID NO: 6, SEQ ID NO: 7. SEQ ID NO: 8, SEQ ID NO: 9, SEQ ID NO: 10, SEQ ID NO: 11, SEQ ID NO: 12, SEQ ID NO: 13, SEQ ID NO: 14, SEQ ID NO: : SEQ ID NO: 16 , SEQ ID NO: 17, SEQ ID NO: 18, SEQ ID NO: 19, SEQ ID NO: 20, SEQ ID NO: 2 SEQ ID NO: 22, SEQ ID NO: 23 or SEQ ID NO: 24, wherein the drug is selected from the group consisting of 5-fluorouracil, flucytosine, tegafur, salicylic acid, p-aminosalicylic acid, cisplatin, carboplatin, nitrogen mustard, cyclophosphamide, and different Cyclophosphamide, guanidine, glyphosate, melphalan, carmustine, lomustine, semustine, nimustine, dopamine, isoniazid, isothiosulphonamide, ethylamine Butanol, ribavirin, zidovudine or metronidazole.

所述的环肽纳米管中环肽的氨基酸序列如 SEQ ID N0: 23或 SEQ ID NO: 24 所示，所述的药物选自 5 -氟尿嘧啶、替加氟、顺铂或卡铂。 The amino acid sequence of the cyclic peptide in the cyclic peptide nanotube is as shown in SEQ ID NO: 23 or SEQ ID NO: 24, and the drug is selected from the group consisting of 5-fluorouracil, tegafur, cisplatin or carboplatin.

为实现上述第二个目的，本发明采取的技术方案是：一种环肽纳米管药物组合物在制备***疾病、细菌感染疾病或病毒感染疾病药中的应用，所述的环肽纳米管药物组合物由环肽纳米管和药物组成。 In order to achieve the above second object, the technical solution adopted by the present invention is: a cyclic peptide nanotube pharmaceutical composition for preparing a medicament for treating a tumor disease, a bacterial infection disease or a virus infection disease, the cyclic peptide nanotube The pharmaceutical composition consists of a cyclic peptide nanotube and a drug.

所述的环肽纳米管范德华内径为 0. 75-1. 3nm, 管长为 10-100nm, 环肽纳米管基本组成单元为环肽，环肽由 8、 10、 12 个氨基酸环合而成，所述的药物分子的直径小于环肽纳米管范德华内径。 The cyclic peptide nanotube van der Waals inner diameter is 0. 75-1. 3nm, the tube length is 10-100nm, the basic constituent unit of the cyclic peptide nanotube is a cyclic peptide, and the cyclic peptide is composed of 8, 10, 12 amino acids. The diameter of the drug molecule is smaller than the inner diameter of the cyclic peptide nanotube van der Waals.

所述的环肽纳米管中的环肽由相同数量的 D型和 L型氨基酸交替共价结合而成，所述的药物分子是亲水性药物。所述的环肽纳米管中环肽的氨基酸序列如 SEQ ID NO: 1、 SEQ ID NO: 2、 SEQ ID NO: 3、 SEQ ID NO: 4、 SEQ ID NO: 5、 SEQ ID NO: 6、 SEQ ID NO: 7、 SEQ ID NO: 8、 SEQ ID NO: 9、 SEQ ID NO: 10、 SEQ ID NO: 11、 SEQ ID NO: 12、 SEQ ID NO: 13、 SEQ ID NO: 14、 SEQ ID NO: 15、 SEQ ID NO: 16 , SEQ ID NO: 17 , SEQ ID NO: 18、 SEQ ID NO: 19 , SEQ ID NO: 20、 SEQ ID NO: 2 SEQ ID NO: 22、 SEQ ID NO: 23或 SEQ ID NO: 24所示，所述的药物选自 5 -氟尿嘧啶、氟胞嘧啶、替加氟、水杨酸、对氨基水杨酸、顺铂、卡铂、氮芥、环磷酰胺、异环磷酰胺、氮曱、甘磷酰芥、美法仑、卡莫司汀、洛莫司汀、司莫司汀、尼莫司汀、多巴胺、异烟肼、异硫异烟胺、乙胺丁醇、利巴韦林、齐多夫定或曱硝唑。 The cyclic peptide in the cyclic peptide nanotube is formed by alternately covalently bonding the same amount of D-form and L-form amino acid, and the drug molecule is a hydrophilic drug. The amino acid sequence of the cyclic peptide in the cyclic peptide nanotube is SEQ ID NO: 1, SEQ ID NO: 2, SEQ ID NO: 3, SEQ ID NO: 4, SEQ ID NO: 5, SEQ ID NO: 6, SEQ ID NO: 7. SEQ ID NO: 8, SEQ ID NO: 9, SEQ ID NO: 10, SEQ ID NO: 11, SEQ ID NO: 12, SEQ ID NO: 13, SEQ ID NO: 14, SEQ ID NO: : SEQ ID NO: 16 , SEQ ID NO: 17, SEQ ID NO: 18, SEQ ID NO: 19, SEQ ID NO: 20, SEQ ID NO: 2 SEQ ID NO: 22, SEQ ID NO: 23 or SEQ ID NO: 24, wherein the drug is selected from the group consisting of 5-fluorouracil, flucytosine, tegafur, salicylic acid, p-aminosalicylic acid, cisplatin, carboplatin, nitrogen mustard, cyclophosphamide, and different Cyclophosphamide, guanidine, glyphosate, melphalan, carmustine, lomustine, semustine, nimustine, dopamine, isoniazid, isothiosulphonamide, ethylamine Butanol, ribavirin, zidovudine or metronidazole.

所述的环肽纳米管中环肽的氨基酸序列如 SEQ ID NO: 23或 SEQ ID NO: 24 所示，所述的药物选自 5 -氟尿嘧啶、替加氟、顺铂或卡铂。 The amino acid sequence of the cyclic peptide in the cyclic peptide nanotube is as shown in SEQ ID NO: 23 or SEQ ID NO: 24, and the drug is selected from the group consisting of 5-fluorouracil, tegafur, cisplatin or carboplatin.

上述技术方案通过以下技术方法来实现的： The above technical solutions are implemented by the following technical methods:

环肽纳米管在细胞膜上自组装，可建立贯穿细胞内外的通道，介导离子如 H ⁺、 ¾+、 K+等跨膜转运，因此打破细胞内外离子平衡，导致细胞死亡，从而具有抗菌、抗病毒作用。尚发现环肽纳米管可输送生命重要分子葡萄糖和谷氨酸跨膜转运。 The self-assembly of cyclic peptide nanotubes on the cell membrane can establish channels through the inside and outside of the cell, mediating the transmembrane transport of ions such as H ⁺ , 3⁄4+, K+, etc., thus breaking the intracellular and extracellular ion balance, leading to cell death, thus having antibacterial and antibiotic resistance. The role of the virus. It has also been found that cyclic peptide nanotubes can transport transmembrane transport of vital molecules, glucose and glutamate.

本发明的发明人通过研究发现，自组装环肽纳米管可介导小分子药物通过细胞膜转运，其速率大于小分子药物不经纳米管向细胞内的扩散速率。在本发明实施例中提供的对肿瘤细胞的活性研究中发现，环肽纳米管和药物联用具有协同抑瘤作用。 The inventors of the present invention have found through research that self-assembled cyclic peptide nanotubes can mediate transport of small molecule drugs through cell membranes at a rate greater than that of small molecule drugs without diffusion into the cells via nanotubes. In the study of the activity of tumor cells provided in the examples of the present invention, it was found that the combination of cyclic peptide nanotubes and drugs has a synergistic antitumor effect.

本发明中环肽自组装形成的纳米管药物输送***，由环肽纳米管和药物共同组成。 A nanotube drug delivery system formed by self-assembly of a cyclic peptide in the present invention, comprising a cyclic peptide nanotube and a drug The things are made up together.

基于环肽纳米管外周疏水的性质，环肽纳米管在细胞膜的磷脂双分子层中自组装，其管长视细胞膜或病原体外壳的厚度，介于 10-100nm, 优选介于 20- 50nm。 Based on the hydrophobic nature of the cyclic peptide nanotubes, the cyclic peptide nanotubes self-assemble in the phospholipid bilayer of the cell membrane, and the thickness of the tube long-term cell membrane or pathogen shell is between 10-100 nm, preferably between 20-50 nm.

环肽纳米管的基本组成单元为环肽，环肽取平观结构，分子间通过氢键层层自组装形成纳米管，为了有利于形成分子间氢键并形成稳定的纳米管，环肽的氨基酸残基数为 8 - 12个， D型和 L型氨基酸各半交替共价结合而成。受环肽氨基酸残基数的限制，环肽纳米管的内径为 0.75-1.3nm。 The basic constituent unit of the cyclic peptide nanotube is a cyclic peptide, and the cyclic peptide adopts a mesostructure, and the intermolecular self-assembly through the hydrogen bonding layer forms a nanotube, in order to facilitate the formation of intermolecular hydrogen bonds and form a stable nanotube, the cyclic peptide The number of amino acid residues is 8 - 12, and the D and L amino acids are alternately covalently combined. The inner diameter of the cyclic peptide nanotubes is 0.75-1.3 nm, limited by the number of amino acid residues of the cyclic peptide.

形成环肽纳米管的氨基酸序列可选自 S P S -、 SWFKTKSK-、 The amino acid sequence forming the cyclic peptide nanotube may be selected from the group consisting of S P S -, SWFKTKSK-,

SWFKHKSK-, SWBYKNKSK-, KKHKWLWK-, SKSWLWLW-, THSWLWLW-, RGDWLWLW-, KQRWLWLW-, K^RWLWLW-, RQRWLWLW-, KQKWLWLW-, KSKWLWLW-, SHKWLWLW-, SKHWLWLW -、 EKHWLWLW-, KKKWLWLW-, RRKWLWLW-, KAKWLWLW-, RRRWLWLW-, HKHWLWLW-, KHKWLWLW-, WLWLWLQL-, LWLWLWLWLQ-₀ 根据公认的氨基酸单字母代号， A、 D、 E、 F、 G、 H、 L、 K、 Q、 R、 S、 T、 W、 Y、 B分别代表丙氨酸、天门冬氨酸、谷氨酸、苯丙氨酸、甘氨酸、组氨酸、亮氨酸、赖氨酸、谷氨酰胺、精氨酸、丝氨酸、苏氨酸、色氨酸、酪氨酸、天门冬酰胺 /天门冬氨酸；字母下划线代表 D构型氨基酸，其余均为 L型氨基酸；氨基酸序列后的 "-" 代表该氨基酸序列首尾相连，构成环肽。环肽的氨基酸序列尤其选自 wy^w _、 ^wyvyvy^Q -。 SWFKHKSK-, SWBYKNKSK-, KKHKWLWK-, SKSWLWLW-, THSWLWLW-, RGDWLWLW-, KQRWLWLW-, K^RWLWLW-, RQRWLWLW-, KQKWLWLW-, KSKWLWLW-, SHKWLWLW-, SKHWLWLW-, EKHWLWLW-, KKKWLWLW-, RRKWLWLW-, KAKWLWLW-, RRRWLWLW-, HKHWLWLW-, KHKWLWLW-, WLWLWLQL-, LWLWLWLWLQ- ₀ according to the recognized amino acid single letter code, A, D, E, F, G, H, L, K, Q, R, S, T, W, Y, B represent alanine, aspartic acid, glutamic acid, phenylalanine, glycine, histidine, leucine, lysine, glutamine, arginine, serine, sul Acid, tryptophan, tyrosine, asparagine/aspartate; underlined to represent D-configuration amino acids, the rest are L-form amino acids; "-" after amino acid sequence means that the amino acid sequence is connected end to end, constitute Cyclic peptide. The amino acid sequence of the cyclic peptide is especially selected from the group consisting of wy^w _, ^wyvyvy^Q -.

本发明环肽的合成可采用通用的固相或液相合成方法，在通用反应器或固相合成仪中进行。合成的环肽通过制备液相分离，纯度大于 95%。本发明实施例中给出了 yvyvyvy^Q-的液相合成路线。药物通过环肽纳米管跨膜转运模式为被动扩散，受环肽内径大小的限制，药物的分子直径应小于环肽纳米管的内径。本发明实施例中提供的环十肽 yvyvyvy^Q-形成的纳米管的内径为 1. Onm, 可允许分子直径小于 1. 0謹的小分子通过。环十二肽形成的纳米管内径为 1. 3nm, 可允许分子直径小于 1. 3nm的药物通过。考虑到分子直径越大在穿透时遇到的阻力越大，分子直径应明显小于纳米管内径。本发明所述的环肽纳米管药物输送***，其中药物分子的直径小于 1. 3nm, 优选为小于 1. Onm。 The synthesis of the cyclic peptide of the present invention can be carried out by a general-purpose solid phase or liquid phase synthesis method in a general-purpose reactor or a solid phase synthesizer. The synthesized cyclic peptide is separated by preparative liquid phase and has a purity of more than 95%. A liquid phase synthesis route of yvyvyvy^Q- is given in the embodiment of the present invention. The transmembrane transport mode of the drug through the cyclic peptide nanotubes is passive diffusion, which is limited by the inner diameter of the cyclic peptide, and the molecular diameter of the drug should be smaller than the inner diameter of the cyclic peptide nanotube. The inner diameter of the nanotubes formed by the cyclic decapeptide yvyvyvy^Q- provided by the present invention is 1. Onm, which allows the molecular diameter to be less than 1.0. The drug having a molecular diameter of less than 1.3 nm is allowed to pass through. Considering that the larger the molecular diameter, the greater the resistance encountered during penetration, the molecular diameter should be significantly smaller than the inner diameter of the nanotube. The on-line peptide nanotube drug delivery system, wherein the diameter of the drug molecule is less than 1. 3 nm, preferably less than 1. Onm.

本发明的环肽纳米管药物输送***适用于多种情况下小分子药物经纳米管介导跨膜转运，药物的选择并不受限于其治疗目的。从抑杀细胞、病原体的角度分析，本发明适用于抗肿瘤药物、抗菌剂、抗病毒剂。其应用范围可根据本发明的技术原理延伸。 The cyclic peptide nanotube drug delivery system of the present invention is suitable for use in a plurality of cases in which small molecule drugs are transmembrane-transported via a nanotube, and the selection of the drug is not limited to its therapeutic purpose. From the perspective of killing cells and pathogens, the present invention is applicable to antitumor drugs, antibacterial agents, and antiviral agents. The scope of application can be extended in accordance with the technical principles of the present invention.

本发明的环肽纳米管药物输送***适用的药物选自 5 -氟尿嘧啶、氟胞嘧啶、替加氟、水杨酸、对氨基水杨酸、顺铂、卡铂、氮芥、环磷酰胺、异环磷酰胺、氮曱、甘磷酰芥、美法仑、卡莫司汀、洛莫司汀、司莫司汀、尼莫司汀、多巴胺、异烟肼、异硫异烟胺、乙胺丁醇、利巴韦林、齐多夫定、曱硝唑。尤其选自 5 -氟尿嘧啶、替加氟、顺铂、卡铂。 The drug for the cyclic peptide nanotube drug delivery system of the present invention is selected from the group consisting of 5-fluorouracil, flucytosine, tegafur, salicylic acid, p-aminosalicylic acid, cisplatin, carboplatin, nitrogen mustard, cyclophosphamide, Isophosphoramide, guanidine, glycolysate, melphalan, carmustine, lomustine, semustine, nimustine, dopamine, isoniazid, isothiazepine, B Aminobutanol, ribavirin, zidovudine, and metronidazole. It is especially selected from the group consisting of 5-fluorouracil, tegafur, cisplatin and carboplatin.

本发明的实施例中提供了环肽纳米管介导小分子药物跨脂质体人工磷脂双分子层膜转运的效果。脂质体人工磷脂双分子层膜被公认为最佳的模拟细胞膜的模型，适用于评价本发明的药物输送***。本发明实施例中依次评价了不同环肽用量情况下，氢离子、 5 -氟尿嘧啶、替加氟、顺铂的跨膜转运效果，结果表明随着环肽用量的增加，氢离子或小分子药物的转运速率加快。阿糖胞苷的分子直径为 1. l lnm, 不能透过本发明实施例中的环十肽纳米管。结果证实阿糖胞苷难以透过脂质体双分子层膜，增加环肽用量亦无法提高其跨膜转运率。 Loop effector nanotubes mediate the effect of small molecule drugs across liposome artificial phospholipid bilayer membrane transport in embodiments of the invention. The liposome artificial phospholipid bilayer membrane is recognized as the best model for simulating cell membranes and is suitable for use in evaluating the drug delivery system of the present invention. In the examples of the present invention, the transmembrane transport effects of hydrogen ion, 5-fluorouracil, tegafur, and cisplatin were evaluated in the order of different cyclic peptides, and the results showed that hydrogen ions or small molecule drugs increased with the amount of cyclic peptide. The transfer rate is accelerated. The molecular diameter of cytarabine is 1. l lnm, which cannot pass through the ring in the embodiment of the present invention. Decimal nanotubes. The results confirmed that cytarabine was difficult to penetrate the liposome bilayer membrane, and increasing the amount of cyclic peptide could not increase its transmembrane transport rate.

本发明实施例中选择人肝癌 BEL-7402 细胞株，考察环肽纳米管作用于肿瘤细胞膜后，对 5-FU的敏感性的改变情况。实验操作时将肿瘤细胞株接种于 96孔板上， 3000个 /孔， 24h后加入含一定浓度 CP和 5-FU的培养液作用 48h, MTT法检测药物的细胞毒性。结果表明，环肽（64 g/ml)作用于人肝癌 BEL-7402细胞株后， 5-FU对该细胞的 IC50从 47. 93 μ g/ml降低至 25. 02 g/ml。这说明环肽能够在肿瘤细胞膜上自组装形成环肽纳米管，并介导外部的小分子药物 5-FU通过该通道快速进入肿瘤细胞内而产生细胞毒性。本发明优点在于： In the embodiment of the present invention, a human liver cancer BEL-7402 cell line was selected, and the sensitivity of the 5-FU to the tumor cell membrane was examined. In the experimental operation, the tumor cell line was seeded in a 96-well plate at 3000 cells/well. After 24 hours, a medium containing a certain concentration of CP and 5-FU was added for 48 hours, and the cytotoxicity of the drug was detected by MTT assay. The results showed that after the cyclic peptide (64 g/ml) was applied to human hepatoma BEL-7402 cell line, the IC50 of 5-FU decreased from 47.93 μg/ml to 25.02 g/ml. This indicates that the cyclic peptide can self-assemble on the tumor cell membrane to form a cyclic peptide nanotube, and mediate the external small molecule drug 5-FU to rapidly enter the tumor cell through the channel to produce cytotoxicity. The advantages of the invention are:

1、本发明药物组合物以环肽纳米管作为跨膜人工纳米通道，使小分子药物通过被动扩散方式进入细胞内。 1. The pharmaceutical composition of the present invention uses cyclic peptide nanotubes as transmembrane artificial nanochannels to allow small molecule drugs to enter cells by passive diffusion.

2、本发明发掘了环肽纳米管药物组合物新的医疗用途。 2. The present invention explores new medical uses of cyclic peptide nanotube pharmaceutical compositions.

3、药物以高浓度快速进入细胞内，提高其细胞毒作用，与环肽纳米管协同作用，在肿瘤治疗以及抗细菌、病毒等病原体感染方面具有应用潜力。 3. The drug rapidly enters the cell at a high concentration, improves its cytotoxicity, and synergizes with the cyclic peptide nanotubes, and has potential application in tumor treatment and infection against pathogens such as bacteria and viruses.

【附图说明】 [Description of the Drawings]

附图 1 环十肽 eye 10 [LWLWLWLWLQ ]的结构式。 Figure 1 Structural formula of cyclic decapeptide eye 10 [LWLWLWLWLQ ].

附图 2 环十肽 cyclo [LWLWLWLWLQ]的合成路线图。附图 3A 束状环肽纳米管的光镜（ 600倍）图。 Figure 2 is a synthetic route diagram of cyclodecapeptide cyclo [LWLWLWLWLQ]. Figure 3A Light microscopy (600x) of a bundle of cyclic peptide nanotubes.

附图 3B 束状环肽纳米管的透射电镜（ 2000倍）图。 Figure 3B Transmission electron microscopy (2000 times) of bundled cyclic peptide nanotubes.

附图 4 环肽纳米管介导氢离子（H+)跨人工脂质膜转运图。附图 5 环肽纳米管介导 5-FU跨人工脂质膜转运图。附图 6 环肽纳米管介导顺铂跨人工脂质膜转运图。附图 7 环肽纳米管介导替加氟跨人工脂质膜转运图。附图 8 肽纳米管介导阿糖孢苷跨人工脂质膜转运图。 Figure 4 Loop peptide nanotubes mediate hydrogen ion (H+) transport across the artificial lipid membrane. Figure 5 Loop peptide nanotubes mediate the transport of 5-FU across artificial lipid membranes. Figure 6 Loop peptide nanotubes mediate transport of cisplatin across artificial lipid membranes. Figure 7 Cyclic peptide nanotubes mediate the transport of tegafur across artificial lipid membranes. Figure 8 Peptide nanotubes mediate transport of glucosinolates across artificial lipid membranes.

【具体实施方式】下面结合附图对本发明提供的一种环肽纳米管药物组合物的具体实施方式做详细说明。实施例 1 DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS A specific embodiment of a cyclic peptide nanotube pharmaceutical composition provided by the present invention will be described in detail below with reference to the accompanying drawings. Example 1

环肽的合成 Synthesis of cyclic peptides

采用固相 -液相结合的方法合成 c c o wyvyvy^Q] ,该环肽的范德化内径为 1. Onm, 其结构式如附图 1所示。 The c c o wyvyvy^Q] was synthesized by a solid-liquid phase binding method, and the van der Waals inner diameter of the cyclic peptide was 1. Onm, and its structural formula is shown in Fig. 1.

合成方法：采用 Boc_Trp (For) _PAM res in (PAM树脂）先固相法合成多肽，再液相法进行环合。合成路线图如附图 2所示。 Synthetic method: The peptide was synthesized by Boc_Trp (For) _PAM res in (PAM resin) first solid phase method, and then cyclized by liquid phase method. The synthetic route map is shown in Figure 2.

取树脂 l l Omg (取代度： 0. 6mmol/g, 0. 066mmol )置于 10ml多肽反应管中，加入 50%三氟醋酸（TFA) /CH₂C1₂溶液，搅拌反应脱除色氨酸 Boc保护基 ( 3ml , 15min, 2次）, 依次用 CH₂C1₂和 DMF搅拌洗涤树脂（ CH₂C1₂: 3ml , lmin, 5 次； DMF: 3ml , lmin, 4 次）。茚三酮溶液检查反应完全后，取树脂 4倍摩尔量的 Boc_D_Leu_0H和 HOBt , 8倍摩尔量的 DIPEA溶解于 4ml DMF 中，活化 30min后，加入反应管中，搅拌反应 30min, 依次用 DMF和 CH₂C1₂ 搅拌洗涤和旨（DMF: 3ml , lmin, 4 次； CH₂C1₂: 3ml , lmin, 5 次）。再加入 50%三氟醋酸（TFA) /CH₂C1₂溶液脱除 Boc保护基后，按照序列依次接入后续氨基酸。氨基酸全部接上后，加入 20%哌啶 /DMF溶液，搅拌反应 ( 3ml , l Omin, 2次）， CH₂C1₂搅拌洗涤树脂（ 3ml , lmin, 6次）。将树脂转入 HF反应管中，加入 10ml液体 HF和适量的对曱苯酚，冰浴下搅拌反应 lh, 负压除去 HF, 加入冰***沉淀并反复洗涤多肽，加入 50%乙腈 /水（0. 1%TFA ) 溶解环肽。半制备纯化，冷冻干燥，既得白色多肽粉末。 Take the resin ll Omg (degree of substitution: 0.6 mmol / g, 0. 066 mmol) in a 10 ml peptide reaction tube, add 50% trifluoroacetic acid (TFA) / CH ₂ C1 ₂ solution, stir the reaction to remove tryptophan Boc The protecting group (3 ml, 15 min, 2 times) was washed with CH ₂ C1 ₂ and DMF (CH ₂ C1 ₂ : 3 ml, lmin, 5 times; DMF: 3 ml, lmin, 4 times). After checking the reaction of ninhydrin solution, take 4 times the molar amount of Boc_D_Leu_0H and HOBt, 8 times the molar amount of DIPEA dissolved in 4ml DMF, activate for 30min, add to the reaction tube, stir the reaction for 30min, and then use DMF and CH. ₂ C1 ₂ Stirring and washing (DMF: 3 ml, lmin, 4 times; CH ₂ C1 ₂ : 3 ml, lmin, 5 times). After removing the Boc protecting group by adding 50% trifluoroacetic acid (TFA) / CH ₂ C1 ₂ solution, the subsequent amino acids were sequentially inserted according to the sequence. After all the amino acids connected, was added 20% piperidine / DMF solution and the reaction stirred (3ml, l Omin, 2 times), CH _₂ C1 ₂ was washed resin (3ml, lmin, 6 times) with stirring. Transfer the resin into the HF reaction tube, add 10 ml of liquid HF and an appropriate amount of p-nonylphenol, stir the reaction for 1 h under ice bath, negative pressure The HF was removed, precipitated with ice diethyl ether and the peptide was washed repeatedly, and 50% acetonitrile/water (0.1% TFA) was added to dissolve the cyclic peptide. Semi-preparative purification, freeze-drying, obtained white peptide powder.

取经半制备纯化后的多肽 20mg 置于圓底烧瓶中，加入 5 倍摩尔量的 PyBOP和 10倍摩尔量的 DIPEA, 20ml DMF溶解，室温搅拌反应约 20h。反应液加 20ml水稀释后，半制备纯化，冷冻干燥，既得白色环肽粉末。产品收率 30~40% , 纯度高 > 95 %。实施例 2 20 mg of the semi-prepared and purified polypeptide was placed in a round bottom flask, 5 times the molar amount of PyBOP and 10 times the molar amount of DIPEA, 20 ml of DMF were dissolved, and the reaction was stirred at room temperature for about 20 hours. After the reaction solution was diluted with 20 ml of water, it was semi-prepared and purified, and lyophilized to obtain a white cyclic peptide powder. Product yield 30~40%, high purity > 95%. Example 2

多肽和环肽的半制备纯化 Semi-preparative purification of peptides and cyclic peptides

色语条件： Ec i lpse XDB-C18 柱， 9. 4 m x 250mm; 流动相 A: 0. 1% TFA/H₂0, 流动相 B: 0. 1% TFA/乙腈；梯度条件（多肽）： 0- 3min 30%B, 3- 4min 30%B→50°/oB, 4- 6min 50%B, 6- 26min 50%B→ 60%B, 26- 30min 60%B→ 30%B; 梯度条件（环肽）： 0-5min 30%B, 5- 7min 30%B→ 50%B, 7-10min 50%B, 10- 30min 50%B→60°/oB , 30- 35min 60%B, 35- 40min 60%B→ 30%B; 检测波长： 280nm; 流速： 4ml/min; 柱温： 25 °C ; 进样量： 1ml。 Chromatic conditions: Ec i lpse XDB-C18 column, 9. 4 mx 250 mm; mobile phase A: 0.1% TFA/H ₂ 0, mobile phase B: 0. 1% TFA/acetonitrile; Gradient conditions (polypeptide): 0- 3min 30%B, 3- 4min 30%B→50°/oB, 4- 6min 50%B, 6- 26min 50%B→ 60%B, 26- 30min 60%B→ 30%B; Gradient conditions (cyclic peptide): 0-5min 30%B, 5- 7min 30%B→ 50%B, 7-10min 50%B, 10- 30min 50%B→60°/oB, 30-35min 60%B, 35 - 40 min 60% B → 30% B; Detection wavelength: 280 nm; Flow rate: 4 ml/min; Column temperature: 25 ° C; Injection volume: 1 ml.

合成的环十肽 c c o ^Wyvyvy^Q]经 HPLC、 LC/MS、 FT- IR、 UV、 'HNMR 表征，验证了其结构。实施例 3 The synthesized cyclic decapeptide c c o ^Wyvyvy^Q] was characterized by HPLC, LC/MS, FT-IR, UV, 'HNMR. Example 3

环肽纳米管的制备与表征 Preparation and characterization of cyclic peptide nanotubes

在溶剂中对环肽纳米管自组装行为进行观察。环十肽 lmg置于 1. 5ml离心管中，用 0. 5ml 1%TFA/CHC 1₃溶解，静置观察结晶析出情况。放置数小时或十多小时后，逐渐观察到溶液中有短针状结晶析出。当结晶析出程度达到最大时，离心（l OOOOrpm, l Omin) , 除去有机溶剂，所得沉淀物加适量纯净水混悬，作进一步的分析。形成的环肽纳米管进行光学显镜和透射电镜观察，如附图 3A和附图 3B所示。所观察到的环肽纳米管呈束状聚集，为长棒状。实施例 4 The self-assembly behavior of cyclic peptide nanotubes was observed in a solvent. The l-decyl peptide 1 mg was placed in a 1.5 ml centrifuge tube, and dissolved in 0.5 ml of 1% TFA/CHC 1 ₃ , and the crystal precipitation was observed by standing. After standing for several hours or more than ten hours, it was gradually observed that short needle crystals were precipitated in the solution. When the degree of crystallization was maximized, centrifugation (l OOOOrpm, l Omin), the organic solvent was removed, and the resulting precipitate was suspended with an appropriate amount of purified water for further analysis. The formed cyclic peptide nanotubes were observed by optical eigenoscopy and transmission electron microscopy as shown in Fig. 3A and Fig. 3B. The observed cyclic peptide nanotubes were bundled and formed into a long rod shape. Example 4

脂质体騎脂双分子层模型上环肽纳米管介导药物跨膜转运考察 Cyclic peptide nanotube-mediated drug transmembrane transport in liposome riding bilayer model

脂质体采用逆相蒸发法制备，药物为 5 -氟尿嘧啶。制备过程如下：取磷脂（纯度 > 80%) 60mg、胆固醇 15mg, 溶解于 6ml CHC1₃, 加入 2ml 5mg/ml 药物 /PBS溶液；采用超声探头进行超声乳化，超声参数：超 3s、停 ls、 40 次、功率 150W; 将制得的均一稳定的 W/0乳剂转入圓底烧瓶中，旋转蒸发除去（¾(1₃制得凝胶，参数：水浴温度 30士 2°C、真空度 0.07-0.08MPa; 加入 1.5-2ml PBS 溶液进行水化，制得药物脂质体混悬液，参数：水浴温度 30 ± 2°C、真空度 0.09-0. lOMPa, 时间 40- 60min; 过 1.2 μηι微孔滤膜；透析或过葡聚糖凝胶柱除去未包封的游离药物，既得含药脂质体。以上述方法制备的含药脂质体，其中 5 -氟尿嘧啶、顺铂、替加氟、阿糖胞苷的包封率分别为： 14.43%、 8.38%, 12.13%、 16.25%; 粒径分别为 794、 603、 470、 454、 371nm。 Liposomes were prepared by reverse phase evaporation and the drug was 5-fluorouracil. The preparation process is as follows: Take phospholipid (purity > 80%) 60mg, cholesterol 15mg, dissolve in 6ml CHC1 ₃ , add 2ml 5mg / ml drug / PBS solution; use ultrasonic probe for phacoemulsification, ultrasonic parameters: super 3s, stop ls, 40 Times, power 150W; The uniformly stable W/0 emulsion prepared was transferred to a round bottom flask and removed by rotary evaporation ( _3⁄4 (1 ₃ gel), parameters: water bath temperature 30 ± 2 ° C, vacuum 0.07 - 0.08MPa; adding 1.5-2ml PBS solution for hydration, to obtain a drug liposome suspension, parameters: water bath temperature 30 ± 2 ° C, vacuum degree 0.09-0. lOMPa, time 40-60min; over 1.2 μηι micro Porous membrane; dialysis or perdextran gel column to remove unencapsulated free drug, obtained drug-containing liposome. Drug-containing liposome prepared by the above method, wherein 5-fluorouracil, cisplatin, tegafur The encapsulation efficiency of cytarabine was 14.43%, 8.38%, 12.13%, 16.25%, and the particle sizes were 794, 603, 470, 454, and 371 nm, respectively.

氢离子 (H+)转运考察： Hydrogen ion (H+) transport inspection:

利用 5 (6) -羧曱基荧光素对 pH的敏感性，环肽纳米管介导 H+跨膜转运，改变脂质体内水相的 pH值，从而引起体系荧光强度的变化。取 5 (6)-羧曱基荧光素脂质体 100μ 1 (内水相 pH7.3), 混悬于 2.5ml pH4.0 PBS中，加入不同浓度的环肽 /DMF溶液 25 μ 1 (环肽浓度： Omg/ml, lmg/ml, 2mg/ml ), 置于荧光分光光度计上测定荧光强度的变化，每 3min测定一次，每个环肽浓度作 6个复管。相对荧光强度（％ ) / =ϋ X 100% 其中， /。： 0时刻荧光强度， i_t'. t时刻荧光强度， /_∞: 破乳后的荧光强度。 /„对时间 t作图，即得到环肽纳米管介导氢离子（H+)跨人工脂质膜转运情况，如附图 4所示。随着环肽浓度的增加，氢离子的转运速度加快。 Using the sensitivity of 5(6)-carboxymethyl fluorescein to pH, cyclic peptide nanotubes mediate H+ transmembrane transport and alter the pH of the aqueous phase in the liposome, resulting in changes in the fluorescence intensity of the system. Take 5 (6)-carboxymethyl fluorescein liposome 100μ 1 (internal aqueous phase pH7.3), suspend in 2.5ml pH4.0 PBS, add different concentrations of cyclic peptide / DMF solution 25 μ 1 (ring Peptide concentration: Omg/ml, lmg/ml, 2mg/ml), the fluorescence intensity was measured on a fluorescence spectrophotometer, measured every 3 minutes, and each loop peptide concentration was made up of 6 complex tubes. Relative fluorescence intensity (%) / = ϋ X 100% where /. : fluorescence intensity at time 0, fluorescence intensity at time i _t '. t, / _∞: fluorescence intensity after demulsification. / „Draw to time t, that is, the cyclic peptide nanotubes mediate the transport of hydrogen ions (H+) across the artificial lipid membrane, as shown in Figure 4. As the concentration of cyclic peptide increases, the transport speed of hydrogen ions accelerates. .

5-氟尿嘧啶（5- FU)转运考察：取 5-FU脂质体 1ml, 装入透析袋（3.5kD)中，加入不同浓度的环肽 /DMF 溶液 25 μ ΐ (环肽浓度： Omg/ml, lmg/ml, 2mg/ml, 4mg/ml ), 置于装有 30ml PBS (pH7.3)緩沖液的锥形瓶中，振荡器 lOOrpm, 37°C水浴，释放 90min, 每 3min取样一次，每次 40 μ 1,每批重复考察 6个样品。色谱条件： Agilent C18柱， 4.6 mx l50mm; 流动相： 10%CH₃OH/H₂0; 检测波长： 265nm; 流速： 0.8ml/min; 柱温： 30°C; 进样量： 20ul。释放百分率（％ ) = ^C' ~ ^C° X 100% 5-fluorouracil (5-FU) transport investigation: Take 1 ml of 5-FU liposome, put into dialysis bag (3.5kD), add 25 μΐ of different concentrations of cyclic peptide/DMF solution (cyclopeptide concentration: Omg/ml, lmg/ml, 2mg/ml, 4mg/ Ml), placed in an Erlenmeyer flask containing 30 ml of PBS (pH 7.3) buffer, shaken at 100 rpm, 37 ° C water bath, release for 90 min, sample every 3 min, 40 μl each time, repeat each sample 6 Samples. Chromatographic conditions: Agilent C18 column, 4.6 mx l50 mm; mobile phase: 10% CH ₃ OH/H ₂ 0; detection wavelength: 265 nm; flow rate: 0.8 ml/min; column temperature: 30 ° C; injection volume: 20 ul. Percent release (%) = ^C ' ~ ^C ° X 100%

c_∞-c₀ 其中， C。： 0时刻药物浓度， C_t: t时刻药物浓度， c_∞: 破乳后的药物浓度。释放百分率对时间 t作图，即得到环肽纳米管介导 5-FU跨人工脂质膜转运情况，如附图 5所示。在未添加环肽的情况下，氟尿嘧啶的跨膜转运速率极低，随着环肽量的增加，转运速度加快。 c _∞ -c ₀ where C. : drug concentration at time 0, C _t : drug concentration at time t, c _∞ : drug concentration after demulsification. The percent release is plotted against time t, which gives the cyclic peptide nanotubes mediating the transport of 5-FU across the artificial lipid membrane, as shown in Figure 5. In the absence of a cyclic peptide, the transmembrane transport rate of fluorouracil is extremely low, and as the amount of cyclic peptide increases, the transport rate increases.

顺铂（DDP)转运考察： Cisplatin (DDP) translocation inspection:

取顺铂脂质体 1ml, 装入透析袋（3.5kD)中，加入不同浓度的环肽 /DMF 溶液 25 μ ΐ (环肽浓度： Omg/ml, lmg/ml, 2mg/ml, 4mg/ml ), 置于装有 50ml 0.2%NaCl 的溶出杯中，小桨法搅拌， lOOrpm, 37°C水浴，释放 90min, 每 5min取样一次，每次 0.5ml, 每批重复考察 5个样品。样品采用石墨炉原子吸收光谱法进行测定。释放百分率对时间 t作图，即得到环肽纳米管介导顺铂跨人工脂质膜转运情况，如附图 6 所示。在未添加环肽的情况下，顺铂的跨膜转运速率较低，随着环肽量的增加，转运速度加快。 Take 1 ml of cisplatin liposome, put it into dialysis bag (3.5kD), add 25 μΐ of different concentrations of cyclic peptide/DMF solution (cyclopeptide concentration: Omg/ml, lmg/ml, 2mg/ml, 4mg/ml ), placed in a dissolution cup containing 50 ml of 0.2% NaCl, stirred by a small paddle, lOO rpm, 37 ° C water bath, released for 90 min, sampled every 5 min, 0.5 ml each time, 5 samples were repeated for each batch. The samples were measured by graphite furnace atomic absorption spectrometry. The percent release is plotted against time t, which gives the cyclic peptide nanotubes mediated transport of cisplatin across the artificial lipid membrane, as shown in Figure 6. In the absence of a cyclic peptide, the transmembrane transport rate of cisplatin is low, and as the amount of cyclic peptide increases, the transport rate increases.

替加氟转运考察： Tigafur transfer:

取替加氟脂质体 lml, 装入透析袋（3.5kD)中，加入不同浓度的环肽 /DMF 溶液 25 μ ΐ (环肽浓度： Omg/ml, lmg/ml, 2mg/ml, 4mg/ml ), 置于装有 50ml PBS (pH7.3)緩沖液的溶出杯中，小桨法搅拌 , lOOrpm, 37°C水浴，释放 90min, 每 5min取样一次，每次 0.5ml,每批重复考察 5个样品。色谱条件： Agilent C18柱， 4.6 mx l50mm; 流动相： 15%CH₃0H/H₂0; 检测波长： 282nm; 流速： 1. Oml/min; 柱温： 30°C; 进样量： 20ul。释放百分率对时间 t作图，即得到环肽纳米管介导替加氟跨人工脂质膜转运情况，如附图 7所示。 Replace 1 ml of fluorolipid liposome into dialysis bag (3.5kD) and add 25 μΐ of different concentrations of cyclic peptide/DMF solution (cyclopeptide concentration: Omg/ml, lmg/ml, 2mg/ml, 4mg/ Ml), placed in a dissolution cup containing 50 ml of PBS (pH 7.3) buffer, stirred by small paddle, lOOrpm, 37 ° C water bath, released for 90 min, sampled every 5 min, 0.5 ml each time, repeated inspections per batch 5 samples. Chromatographic conditions: Agilent C18 column, 4.6 mx l50 mm; mobile phase: 15% CH ₃ 0H/H ₂ 0; detection wavelength: 282 nm; flow rate: 1. Oml/min; Column temperature: 30 ° C; Injection volume: 20 ul. The percent release is plotted against time t, which gives the cyclic peptide nanotubes mediating the transport of tegafur across the artificial lipid membrane, as shown in Figure 7.

阿糖胞苷（Ara- C)转运考察： Cytarabine (Ara-C) transport investigation:

取 Ara-C脂质体 1ml,装入透析袋（3.5kD)中，加入不同浓度的环肽 /DMF 溶液 25ul (环肽浓度： Omg/ml, lmg/ml, 2mg/ml, 4mg/ml ), 置于装有 30ml PBS (pH7.3)緩沖液的锥形瓶中，振荡器 lOOrpm, 37°C水浴，释放 90min, 每 3m in取样一次，每次 40 μ 1 ,每批重复考察 6个样品。色谱条件： D i amons i 1 C18柱， 4· 6 μηιχ 250匪；流动相： CH₃OH/0. Olmol/1 PBS (pH4.0, 0. Olmol/1 SDS) ( 50: 50 ); 检测波长： 270nm; 流速： lml/min; 柱温： 30°C; 进样量： 20ul。 Take 1ml of Ara-C liposome, put it into dialysis bag (3.5kD), add 25ul of different concentration of cyclic peptide/DMF solution (cyclopeptide concentration: Omg/ml, lmg/ml, 2mg/ml, 4mg/ml) Place in a conical flask containing 30 ml of PBS (pH 7.3) buffer, shaker at 100 rpm, 37 ° C water bath, release for 90 min, sample once every 3 m in 40 μl each time, and repeat 6 samples per batch. sample. Chromatographic conditions: D i amons i 1 C18 column, 4· 6 μηιχ 250匪; mobile phase: CH ₃ OH/0. Olmol/1 PBS (pH 4.0, 0. Olmol/1 SDS) (50: 50 ); Wavelength: 270 nm; Flow rate: lml/min; Column temperature: 30 ° C; Injection volume: 20 ul.

释放百分率对时间 t作图，即得到环肽纳米管介导 Ara-C跨人工脂质膜转运情况，如附图 8 所示。 Ara-C 的分子大小为 l. llnm, 大于环十肽 c o Wyvyvy^Q]的范德化内径（lnm), 理论上该药物不能通过环肽纳米管介导跨膜转运。结果表明顺铂在未添加和添加环肽的条件下，均不能较好的穿透双分子层膜。实施例 5 The percent release is plotted against time t, which results in cyclic peptide nanotube-mediated translocation of Ara-C across the artificial lipid membrane, as shown in Figure 8. The molecular size of Ara-C is l. llnm, which is larger than the van der Waals inner diameter (lnm) of the cyclic decapeptide c o Wyvyvy^Q]. Theoretically, the drug cannot mediate transmembrane transport via cyclic peptide nanotubes. The results showed that cisplatin could not penetrate the bilayer membrane better without adding or adding a cyclic peptide. Example 5

环肽纳米管对肿瘤细胞作用的考察 Study on the effect of cyclic peptide nanotubes on tumor cells

肿瘤细胞株：人肝癌 BEL-74Q2细胞株。 Tumor cell line: Human liver cancer BEL-74Q2 cell line.

考察环肽纳米管作用于肿瘤细胞膜后，对 5-FU的敏感性的改变情况。实验操作：将肿瘤细胞株接种于 96孔板上， 3000个 /孔， 24h后加入含一定浓度 CP和 5-FU的培养液作用 48h, MTT法检测药物的细胞毒性。 The change in sensitivity to 5-FU after cyclic peptide nanotubes were applied to tumor cell membranes. Experimental operation: The tumor cell line was inoculated into a 96-well plate at 3000 cells/well. After 24 hours, a medium containing a certain concentration of CP and 5-FU was added for 48 hours, and the cytotoxicity of the drug was examined by MTT assay.

结果表明，环肽（64 g/ml)作用于人肝癌 BEL-7402细胞株后， 5-FU对该细胞的 IC50从 47.93 g/ml降低至 25.02 μ g/ml。这说明环肽能够在肿瘤细胞膜上自组装形成环肽纳米管，并介导外部的小分子药物 5-FU通过该通道快速进入肿瘤细胞内而产生细胞毒性。因为 5-FU通过该通道的转运速率比跨膜扩散途径要快，因而能够更快速的杀灭肿瘤细胞。 The results showed that after the cyclic peptide (64 g/ml) was applied to the human liver cancer BEL-7402 cell line, the IC50 of 5-FU decreased from 47.93 g/ml to 25.02 μg/ml. This indicates that the cyclic peptide can self-assemble on the tumor cell membrane to form a cyclic peptide nanotube, and mediate the external small molecule drug 5-FU to rapidly enter the tumor cell through the channel to produce cytotoxicity. Because of the speed of 5-FU transfer through the channel The rate is faster than the transmembrane diffusion pathway, thus enabling faster killing of tumor cells.

以上所述仅是本发明的优选实施方式，应当指出，对于本技术领域的普通技术人员，在不脱离本发明方法的前提下，还可以做出若干改进和补充，这些改进和补充也应视为本发明的保护范围。 The above description is only a preferred embodiment of the present invention, and it should be noted that those skilled in the art can make several improvements and additions without departing from the method of the present invention. These improvements and additions should also be considered. It is the scope of protection of the present invention.

Claims

权利要求 Rights request

1. 一种环肽纳米管药物组合物在制备***疾病、细菌感染疾病或病毒感染疾病药中的应用，所述的环肽纳米管药物组合物由环肽纳米管和药物组成。 A use of a cyclic peptide nanotube pharmaceutical composition for the preparation of a medicament for treating a neoplastic disease, a bacterial infection disease or a viral infection, wherein the cyclic peptide nanotube pharmaceutical composition comprises a cyclic peptide nanotube and a drug.

2. 根据权利要求 1所述的应用，其特征在于：所述的环肽纳米管范德华内径为 0. 75-1. 3nm, 管长为 10-100nm, 环肽纳米管基本组成单元为环肽，环肽由 8、 10、 12 个氨基酸环合而成，所述的药物分子的直径小于环肽纳米管范德华内径。 The application of the cyclic peptide nanotube van der Waals is 0. 75-1. 3nm, the tube length is 10-100nm, and the basic constituent unit of the cyclic peptide nanotube is a cyclic peptide. The cyclic peptide is formed by cyclizing 8, 10, 12 amino acids, and the diameter of the drug molecule is smaller than the inner diameter of the cyclic peptide nanotube van der Waals.

3. 根据权利要求 2所述的应用，其特征在于：所述的环肽纳米管中的环肽由相同数量的 D型和 L型氨基酸交替共价结合而成，所述的药物分子是亲水性药物。 3. The use according to claim 2, wherein: the cyclic peptide in the cyclic peptide nanotube is formed by alternately covalently bonding the same amount of D-form and L-form amino acid, and the drug molecule is a pro Water-based drugs.

4. 根据权利要求 3所述的应用，其特征在于：所述的环肽纳米管中环肽的氨基酸序列如 SEQ ID NO: SEQ ID NO: 2、 SEQ ID NO: 3、 SEQ ID NO: 4、 SEQ ID NO: 5、 SEQ ID NO: 6 , SEQ ID NO: 7 , SEQ ID NO: 8、 SEQ ID NO: 9、 SEQ ID NO: 10 , SEQ ID NO: 11、 SEQ ID NO: 12、 SEQ ID NO: 13、 SEQ ID NO: 14、 SEQ ID NO: 15、 SEQ ID NO: 16、 SEQ ID NO: 17、 SEQ ID NO: 18、 SEQ ID NO: 19、 SEQ ID NO: 20、 SEQ ID NO: 21、 SEQ ID NO: 22、 SEQ ID NO: 23或 SEQ ID NO: 24所示，所述的药物选自 5 -氟尿嘧啶、氟胞嘧啶、替加氟、水杨酸、对氨基水杨酸、顺铂、卡铂、氮芥、环磷酰胺、异环磷酰胺、氮曱、甘磷酰芥、美法仑、卡莫司汀、洛莫司汀、司莫司汀、尼莫司汀、多巴胺、异烟肼、异硫异烟胺、乙胺丁醇、利巴韦林、齐多夫定或曱硝唑。 4. The use according to claim 3, wherein: the amino acid sequence of the cyclic peptide in the cyclic peptide nanotube is SEQ ID NO: SEQ ID NO: 2, SEQ ID NO: 3, SEQ ID NO: 4. SEQ ID NO: 5, SEQ ID NO: 6, SEQ ID NO: 7, SEQ ID NO: 8, SEQ ID NO: 9, SEQ ID NO: 10, SEQ ID NO: 11, SEQ ID NO: 12, SEQ ID NO: 13, SEQ ID NO: 14, SEQ ID NO: 15, SEQ ID NO: 16, SEQ ID NO: 17, SEQ ID NO: 18, SEQ ID NO: 19, SEQ ID NO: 20, SEQ ID NO: 21. SEQ ID NO: 22, SEQ ID NO: 23 or SEQ ID NO: 24, wherein the drug is selected from the group consisting of 5-fluorouracil, flucytosine, tegafur, salicylic acid, p-aminosalicylic acid, Cisplatin, carboplatin, nitrogen mustard, cyclophosphamide, ifosfamide, guanidine, glyphosate, melphalan, carmustine, lomustine, semustine, nimustine, Dopamine, isoniazid, isothiazolidine, ethambutol, ribavirin, zidovudine or metronidazole.

5. 根据权利要求 4所述的应用，其特征在于：所述的环肽纳米管中环肽的氨基酸序列如 SEQ ID NO: 23或 SEQ ID NO: 24所示，所述的药物选自 5 -氟尿嘧啶、替加氟、顺铂或卡铂。 5. The use according to claim 4, wherein: the amino acid sequence of the cyclic peptide in the cyclic peptide nanotube is as shown in SEQ ID NO: 23 or SEQ ID NO: 24, and the drug is selected from the group consisting of 5- Fluoride Pyrimidine, tegafur, cisplatin or carboplatin.

6. 一种环肽纳米管药物组合物，其特征在于：由环肽纳米管和药物组成，所述的环肽纳米管范德华内径为 0. 75-1. 3nm, 管长为 10-100nm, 环肽纳米管基本组成单元为环肽，环肽由 8、 10、 12 个氨基酸环合而成，所述的药物分子的直径小于环肽纳米管范德华内径。 The inner diameter of the cyclic peptide nanotube van der Waals is 0. 75-1. 3nm, the tube length is 10-100nm, and the composition of the cyclic peptide nanotube is composed of a cyclic peptide nanotube and a drug. The basic constituent unit of the cyclic peptide nanotube is a cyclic peptide, and the cyclic peptide is formed by cyclizing 8, 10, 12 amino acids, and the diameter of the drug molecule is smaller than the inner diameter of the cyclic peptide nanotube van der Waals.

7. 根据权利要求 6所述的药物组合物，其特征在于：所述的环肽纳米管中的环肽由相同数量的 D型和 L型氨基酸交替共价结合而成，所述的药物分子是亲水性药物。 The pharmaceutical composition according to claim 6, wherein the cyclic peptide in the cyclic peptide nanotube is formed by alternately covalently bonding the same amount of D-type and L-type amino acids, and the drug molecule It is a hydrophilic drug.

8. 根据权利要求 6所述的药物组合物，其特征在于：所述的环肽纳米管中环肽的氨基酸序列如 SEQ ID NO: SEQ ID NO: 2、 SEQ ID NO: 3、 SEQ ID NO: 4、 SEQ ID NO: 5、 SEQ ID NO: 6、 SEQ ID NO: 7、 SEQ ID NO: 8、 SEQ ID NO: 9、 SEQ ID NO: 10、 SEQ ID NO: 11、 SEQ ID NO: 12、 SEQ ID NO: 13、 SEQ ID NO: 14、 SEQ ID NO: 15、 SEQ ID NO: 16、 SEQ ID NO: 17、 SEQ ID NO: 18、 SEQ ID NO: 19、 SEQ ID NO: 20、 SEQ ID NO: 21、 SEQ ID NO: 22、 SEQ ID NO: 23或 SEQ ID NO: 24所示。 The pharmaceutical composition according to claim 6, wherein the amino acid sequence of the cyclic peptide in the cyclic peptide nanotube is SEQ ID NO: SEQ ID NO: 2, SEQ ID NO: 3, SEQ ID NO: 4. SEQ ID NO: 5, SEQ ID NO: 6, SEQ ID NO: 7, SEQ ID NO: 8, SEQ ID NO: 9, SEQ ID NO: 10, SEQ ID NO: 11, SEQ ID NO: SEQ ID NO: 13, SEQ ID NO: 14, SEQ ID NO: 15, SEQ ID NO: 16, SEQ ID NO: 17, SEQ ID NO: 18, SEQ ID NO: 19, SEQ ID NO: 20, SEQ ID NO: 21, SEQ ID NO: 22, SEQ ID NO: 23 or SEQ ID NO: 24.

9. 根据权利要求 6所述的药物组合物，其特征在于：所述的药物选自 5 -氟尿嘧啶、氟胞嘧啶、替加氟、水杨酸、对氨基水杨酸、顺铂、卡铂、氮芥、环磷酰胺、异环磷酰胺、氮曱、甘磷酰芥、美法仑、卡莫司汀、洛莫司汀、司莫司汀、尼莫司汀、多巴胺、异烟肼、异硫异烟胺、乙胺丁醇、利巴韦林、齐多夫定或曱硝唑。 The pharmaceutical composition according to claim 6, wherein the drug is selected from the group consisting of 5-fluorouracil, flucytosine, tegafur, salicylic acid, p-aminosalicylic acid, cisplatin, carboplatin , nitrogen mustard, cyclophosphamide, ifosfamide, guanidine, glyphosate, melphalan, carmustine, lomustine, semustine, nimustine, dopamine, isoniazid , isothiazolidine, ethambutol, ribavirin, zidovudine or metronidazole.

10.根据权利要求 6 所述的药物组合物，其特征在于：所述的环肽纳米管中环肽的氨基酸序列如 SEQ ID NO: 23或 SEQ ID NO: 24所示，所述的药物选自 5 -氟尿嘧啶、替加氟、顺铂或卡铂，环肽纳米管管长为 20-50nm, 所述的环肽纳米管范德华内径为 1. Onm, 所述的药物分子的直径小于 1. Onm。 The pharmaceutical composition according to claim 6, wherein the amino acid sequence of the cyclic peptide in the cyclic peptide nanotube is as shown in SEQ ID NO: 23 or SEQ ID NO: 24, and the drug is selected from the group consisting of 5 The fluorouracil, tegafur, cisplatin or carboplatin, the cyclic peptide nanotube has a length of 20-50 nm, the cyclic peptide nanotube van der Waals inner diameter is 1. Onm, the diameter of the drug molecule is less than 1. Onm .