Ph-sensitive membrane rupture sequence polypeptide and use thereof

Abstract

A pH-sensitive membrane rupture sequence polypeptide having a structure as shown in formula (I) or a stereoisomer or a pharmaceutically acceptable salt thereof, and a use thereof. Under a normal physiological pH, the macromolecular material is hydrophobic and electrically neutral, can be self-assembled into nanoparticles, is tightly assembled, and has weak interaction with cell membranes. Under slightly acidic pH conditions, the macromolecular material can be protonated to form an amphiphilic structure consisting of a hydrophobic domain and a cationic domain, has strong interaction with cell membranes, and has strong membrane rupture activity, so that tumor cells can be efficiently and highly selectively killed.

Description

pH-sensitive membrane-permeabilizing peptides and their applications Technical Field

[0001] The present invention relates to the technical field of polymer materials and pharmaceuticals, and in particular to a pH-sensitive membrane-permeabilizing sequence polypeptide and applications thereof. Background Art

[0002] Some amphiphilic polymers with cationic groups can kill tumor cells and pathogens such as bacteria by disrupting cell membranes. This metabolic-independent killing method offers broad-spectrum efficacy and is less susceptible to drug resistance, holding great promise for the treatment of tumors and infectious diseases. Membrane-disrupting polymers interact with cell membranes primarily through electrostatic and hydrophobic interactions. First, the cationic domain of the polymer binds to the cell membrane surface through electrostatic interactions with the negatively charged cell membrane. Then, the hydrophobic domain of the polymer inserts into the lipid layer of the cell membrane, causing irreparable damage and ultimately killing the cell. The amphiphilic conformation, composed of cationic and hydrophobic groups, is crucial for macromolecular interactions with cell membranes and is also a key factor in their cytotoxicity to normal tissue cells. The ratio of these two structures is crucial for their interaction with cell membranes. Generally speaking, purely cationic polymers readily bind to cell membranes but poorly insert into them, while hydrophobic polymers have difficulty binding to the cell membrane surface and are unable to effectively disrupt them. Therefore, it is of great significance to design polymer materials so that they present cationic or hydrophobic structures in normal tissues and transform into amphiphilic conformations composed of cationic and hydrophobic domains in lesion sites to solve the problem of high toxicity of membrane-rupturing polymer materials.

[0003] The research team of the present invention previously prepared a polymethacrylate polymer material containing tertiary amine side chains with membrane-breaking activity. The activation of the membrane-breaking activity can be controlled at different pH levels by protonating the tertiary amine in the side chains. By introducing hydrophobic links through copolymerization, low hemolysis and killing can be achieved at pH = 7.4. In a slightly acidic environment, precise activation of the membrane-breaking activity can be achieved, causing irreversible damage to the tumor cell membrane and achieving highly selective killing of tumor cells. However, the polymethacrylate main chain is difficult to degrade, and long-term use can easily lead to toxicity due to accumulation in the body.

[0004] Summary of the Invention

[0005] Based on this, the present invention provides a pH-sensitive membrane-permeabilizing polypeptide material with side groups containing tertiary amines and hydrophobic groups. At normal physiological pH, this macromolecular material is hydrophobic and electrically neutral, with weak interactions with cell membranes. Under slightly acidic pH conditions, it can be protonated to form an amphiphilic structure composed of a hydrophobic domain and a cationic domain, which has strong interactions with cell membranes and possesses strong membrane-permeabilizing activity, thereby effectively and selectively killing tumor cells. In addition, the polypeptide can be synthesized using solid-phase synthesis methods, and the sequence and number of amino acids in the resulting polypeptide material can be kept fixed. The present invention includes the following technical solutions.

[0006] In one aspect, the present invention provides a membrane permeabilization sequence polypeptide having a structure represented by formula (I), or a stereoisomer thereof, or a pharmaceutically acceptable salt thereof:

[0007] Wherein, Q is a polypeptide group composed of n A and m B arranged in any sequence;

[0008] B is the residue after removing one amino hydrogen and one hydroxyl group from an amino acid with a hydrophobic side chain;

[0009] A is the residue of lysine with a modified group, after removing one amino hydrogen and one hydroxyl group, and its structure is:

[0010] R is selected from: -R3-N(R4R5), -R3-R',

[0011] R' is selected from:

[0012] L is selected from: -NH-C(=O)O-, -NH-C(=O)-,, -C(=O)-NH-, -C(=O)-O-;

[0013] R1 is selected from: alkylene;

[0014] R3 is selected from: alkylene, C6-C 14 Aryl-substituted alkylene;

[0015] R4 and R5 are independently selected from: alkyl, C6-C 14 an alkyl group substituted with an aryl group, or R3, R4 and the nitrogen atom to which they are attached together form a heterocycloalkyl group;

[0016] R6 is selected from: C1-C 15 Alkyl, C6-C 14 Aryl, C6-C 14 Aryl-substituted C1-C 15 alkyl;

[0017] y is selected from: 2-150;

[0018] n+m is greater than 0, and n is not 0;

[0019] q is selected from: 0, 1, 2, 3, 4.

[0020] Preferably, the structural formula of the membrane-permeabilizing sequence polypeptide is as shown in formula (II):

[0021] Wherein, Q1 is a polypeptide group composed of n1 A and m1 B arranged in any sequence;

[0022] n1+m1 is greater than 0, and n1 is not 0;

[0023] x1 is greater than 0;

[0024] The product of n1 and x1 is n; the product of m1 and x1 is m.

[0025] Preferably, the structural formula of the membrane-permeabilizing sequence polypeptide is as shown in formula (III):

[0026] Wherein, Q2 is a polypeptide group composed of n2 A and m2 B arranged in any sequence;

[0027] Q3 is a polypeptide group composed of n3 As and m3 Bs arranged in any sequence;

[0028] n2+n3+m2+m3 is greater than 0, and n2+n3 is not 0;

[0029] The sum of the product of n2 and x2 plus n3 is n, and the sum of the product of m2 and x2 plus m3 is m.

[0030] On the other hand, the present invention also provides a membrane-permeabilizing sequence polypeptide nanoparticle, including the following technical solution.

[0031] A membrane-breaking sequence polypeptide nanoparticle is formed by self-assembly of the membrane-breaking sequence polypeptide in an aqueous medium.

[0032] On the other hand, the present invention also provides a method for preparing membrane-permeabilizing sequence polypeptide nanoparticles, which includes the following technical solutions.

[0033] A method for preparing membrane-permeabilizing sequence polypeptide nanoparticles comprises the following steps: dissolving the membrane-permeabilizing sequence polypeptide in an organic solvent or a hydrochloric acid solution with a pH of 1.5-2.5, then adding the resulting solution dropwise into water under stirring, continuing stirring, and dialyzing to remove the solvent, thereby obtaining the membrane-permeabilizing sequence polypeptide nanoparticles.

[0034] On the other hand, the present invention also provides the use of the above-mentioned membrane-perforating sequence polypeptide or membrane-perforating sequence polypeptide nanoparticles, including the following technical solutions.

[0035] Use of the membrane-permeabilizing sequence polypeptide or its stereoisomer or its pharmaceutically acceptable salt in the preparation of drugs for preventing and / or treating tumors.

[0036] Application of the membrane-breaking sequence polypeptide nanoparticles in the preparation of drugs for preventing and / or treating tumors.

[0037] On the other hand, the present invention also provides a drug for preventing and / or treating tumors, including the following technical solutions.

[0038] A drug for preventing and / or treating tumors is prepared from active ingredients and pharmaceutically acceptable excipients, wherein the active ingredients include the membrane-breaking sequence polypeptide or its stereoisomer or its pharmaceutically acceptable salt, and / or the membrane-breaking sequence polypeptide nanoparticles.

[0039] On the other hand, the present invention also provides a method for preventing and / or treating tumors, including the following technical solutions.

[0040] A method for preventing and / or treating tumors, comprising: administering a safe and effective amount of the membrane-permeable sequence polypeptide of the present invention, or a stereoisomer thereof, or a pharmaceutically acceptable salt thereof; and / or,

[0041] Administering a safe and effective amount of the membrane-permeabilizing sequence polypeptide nanoparticles of the present invention; and / or,

[0042] Administer a safe and effective amount of the drug of the present invention.

[0043] The present invention provides a tertiary amine-modified membrane-permeable sequence polypeptide material. The macromolecular material is hydrophobic and electrically neutral at normal physiological pH, and the polypeptide fragment is hydrophobic and has a weak interaction with the cell membrane, thereby having the advantage of low toxicity to normal tissues when circulating in the body; under the slightly acidic pH conditions of tumor tissue, the tertiary amine portion of the macromolecular material will be protonated, so that the sequence polypeptide fragment forms an amphiphilic structure composed of a hydrophobic domain and a cationic domain, thereby having extremely strong interaction with the cell membrane and strong membrane-permeable activity, thereby being able to kill tumor cells and bacteria efficiently and highly selectively. The membrane-permeable sequence polypeptide macromolecular material of the present invention can be used to prepare anti-tumor drugs, and has the advantages of good anti-tumor effect, high selectivity, and low toxicity. And because the polymeric sequence polypeptide has the advantages of being degradable and the degradation products are non-biotoxic, it will have a wider range of biomedical applications. BRIEF DESCRIPTION OF THE DRAWINGS

[0044] Figure 1 shows the mass spectrum of (KW)5K.

[0045] Figure 2 shows the mass spectrum of (KW)6K.

[0046] Figure 3 shows the mass spectrum of (KW)7K.

[0047] Figure 4 shows the mass spectrum of (KW)8K.

[0048] Figure 5 shows the mass spectrum of (KWWK)2KKW.

[0049] FIG6 is the mass spectrum of (KWWK)3K.

[0050] FIG7 is the mass spectrum of (KWWK)3KKW.

[0051] FIG8 is the mass spectrum of (KWWK)4K.

[0052] Figure 9 shows the mass spectrum of (KWK)3.

[0053] Figure 10 is the mass spectrum of (KWK)4.

[0054] FIG11 is the mass spectrum of (KWK)5.

[0055] FIG12 is the mass spectrum of (KWK)6.

[0056] FIG13 is the mass spectrum of (KWK)7.

[0057] FIG14 is a mass spectrum of (KWKK)3.

[0058] FIG15 is the mass spectrum of (KWKK)4.

[0059] FIG16 is a mass spectrum of (KWKK)5.

[0060] FIG17 is the mass spectrum of (KWKK)7.

[0061] FIG18 is a mass spectrum of (KKWKK)3.

[0062] FIG19 is a mass spectrum of (KKWKK)5.

[0063] Figure 20 shows mPEG 45 -(K1W) x H NMR spectrum of K1.

[0064] Figure 21 shows mPEG 45 -(K1WWK1) x H NMR spectrum of K1.

[0065] Figure 22 shows mPEG 45 -(K1WWK1) x H NMR spectrum of K1K1W.

[0066] Figure 23 shows mPEG 45-(K1WK1) x 's H NMR spectrum.

[0067] Figure 24 shows mPEG 45 -(K1WK1K1) x 's H NMR spectrum.

[0068] Figure 25 shows mPEG 45 -(K1K1WK1K1) x 's H NMR spectrum.

[0069] Figure 26 shows mPEG 45 -(K1) n (W) m The protonation rate of α is changed with pH.

[0070] Figure 27 shows mPEG 45 -(K1) n (W) m Cytotoxicity against MC38 cells at pH 7.4.

[0071] Figure 28 shows mPEG 45 -(K1) n (W) m Cytotoxicity against MC38 cells at pH 6.8.

[0072] Figure 29 shows mPEG 45 -(K1) n (W) m Maximum tolerated dose (MTD) in ICR mice.

[0073] Figure 30 shows the mPEG imaged by a high-content imaging system. 45 -(K1) n (W) m Images of cells incubated with Panc02mCherry / GFP at different pH levels.

[0074] FIG31 is a tumor growth curve of the MC38 subcutaneous tumor model after treatment with fixed sequence peptides.

[0075] FIG32 shows the changes in body weight of mice after treatment with fixed sequence peptides in the MC38 subcutaneous tumor model.

[0076] FIG33 is a photograph of the tumor in the MC38 subcutaneous tumor model after treatment with fixed sequence peptides.

[0077] Figure 34 shows mPEG 45 -(K1WK1)5 images of Panc02mCherry / GFP cells incubated at pH 6.8 for different times.

[0078] Figure 35 shows the expression of mPEG in the presence of Annexin V-FITC and pyridinium iodide (PI). 45 - Images of MC38 cells incubated with (K1WK1)5 at pH 6.8 for different times.

[0079] Figure 36 is mPEG 45 -(K2K2WK2K2)5 H NMR spectrum.

[0080] Figure 37 is mPEG 45 -H NMR spectrum of (K3K3WK3K3)5.

[0081] Figure 38 is mPEG 45 -H NMR spectrum of (K4K4WK4K4)5.

[0082] Figure 39 is mPEG 45 -H NMR spectrum of (K5K5WK5K5)5.

[0083] Figure 40 shows mPEG 45 -H NMR spectrum of (K6K6WK6K2)5.

[0084] Figure 41 is mPEG 45 -(K7K7WK7K7)5 H NMR spectrum.

[0085] Figure 42 shows mPEG 45 -(K8K8WK8K8)5 H NMR spectrum.

[0086] Figure 43 shows mPEG modified with different tertiary amines 45 -(KKWKK)5 after the protonation rate of the peptide changes with pH.

[0087] Figure 44 shows mPEG modified with different tertiary amines 45 -(KKWKK)5-followed peptides showed cytotoxicity to MC38 cells at pH 7.4 and 6.8.

[0088] Figure 45 shows mPEG with different lengths of PEG y -(K1K1WK1K1)5 H NMR spectrum.

[0089] Figure 46 shows mPEG with different lengths of PEG y -The protonation rate of (K1K1WK1K1)5 changes with pH.

[0090] Figure 47 shows mPEG with different lengths of PEG y -(K1K1WK1K1)5 cytotoxicity to MC38 cells at pH 7.4 and 6.8.

[0091] Figure 48 is the mass spectrum of (KKKKW)5.

[0092] Figure 49 shows (K)5(W)2(K) 10 Mass spectrum of (W)3(K)5.

[0093] Figure 50 is the mass spectrum of (K)5(W)2(K)5W(K)5(W)2(K)5.

[0094] Figure 51 shows mPEG 45 -(K1K1WK1K1)5 constructed into peptides with different arrangements.

[0095] Figure 52 shows the mPEG 45 -(K1K1WK1K1)5 constructs a graph showing the protonation rate of peptides with different arrangements as a function of pH.

[0096] Figure 53 shows mPEG 45 -(K1K1WK1K1)5 constructed with different arrangements of peptides showed cytotoxicity against MC38 cells at pH 7.4 and 6.8.

[0097] Figure 54 is the mass spectrum of (KKKFKKK)2K.

[0098] Figure 55 is the mass spectrum of (KKFKK)3.

[0099] Figure 56 is the mass spectrum of (KFK)4KKK.

[0100] Figure 57 is the mass spectrum of (KFK)5.

[0101] Figure 58 is mPEG 45 -(K1) n (F) m 's H NMR spectrum.

[0102] Figure 59 is mPEG 45 -(K9) n (F) m 's H NMR spectrum.

[0103] Figure 60 shows mPEG 45 -(K1) n (F) m The protonation rate of α is changed with pH.

[0104] Figure 61 is mPEG 45 -(K9) n (F) m The protonation rate of α is changed with pH.

[0105] Figure 62 shows mPEG 45 -(K1)n (F) m Cytotoxicity against MC38 cells at pH 7.4 and pH 6.8.

[0106] Figure 63 is mPEG 45 -(K9) n (F) m Cytotoxicity against MC38 cells at pH 7.4 and pH 6.8. DETAILED DESCRIPTION

[0107] The experimental methods in the following examples of the present invention, where specific conditions are not specified, are generally carried out under conventional conditions or conditions recommended by the manufacturers. The various commonly used chemical reagents used in the examples are all commercially available products.

[0108] Unless otherwise defined, all technical and scientific terms used in the present invention have the same meaning as those commonly understood by those skilled in the art. The terms used in the specification of the present invention are only for the purpose of describing specific embodiments and are not intended to limit the present invention.

[0109] The terms "comprise," "comprising," and "having," and any variations thereof, are intended to cover non-exclusive inclusions. For example, a process, method, apparatus, product, or device comprising a series of steps is not limited to the listed steps or modules but may optionally include steps not listed, or other steps inherent to the process, method, product, or device.

[0110] In this application, "plurality" refers to two or more. "And / or" describes the relationship between related objects, indicating that three possible relationships exist. For example, "A and / or B" can mean: A exists alone, A and B exist simultaneously, or B exists alone. The character " / " generally indicates that the related objects are in an "or" relationship.

[0111] In the compounds of the present invention, when any variable (e.g., R1, R2, etc.) occurs more than once in any component, its definition at each occurrence is independent of its definition at every other occurrence. Likewise, combinations of substituents and variables are permitted so long as such combinations render the compound stable. A line drawn from a substituent into the ring system indicates that the indicated bond may be attached to any substitutable ring atom. If the ring system is polycyclic, this means that such a bond is attached only to any appropriate carbon atom of an adjacent ring. It will be understood that one of ordinary skill in the art can select the substituents and substitution patterns of the compounds of the present invention to provide compounds that are chemically stable and that can be readily synthesized from readily available raw materials by techniques in the art and the methods set forth below. If a substituent is itself substituted with more than one group, it will be understood that these groups may be on the same carbon atom or on different carbon atoms so long as the structure is stable. In the present invention, the "*" and "*" in a chemical group or chemical structure are Both represent key connection locations.

[0112] The term "alkyl" is intended to include both branched and straight-chain saturated aliphatic hydrocarbon groups having a specified number of carbon atoms. For example, the definition of "C1-C6 alkyl" includes groups having 1, 2, 3, 4, 5, or 6 carbon atoms in a straight or branched arrangement. For example, "C1-C6 alkyl" specifically includes methyl, ethyl, n-propyl, isopropyl, n-butyl, tert-butyl, isobutyl, pentyl, and hexyl.

[0113] The term "alkylene" refers to an "alkyl" radical with one hydrogen less, for example, -CH2-, -CH2CH2-, -CH2CH2CH2-, -CH2CH2CH2CH2-, etc.

[0114] The term "heterocycloalkyl" is a saturated monocyclic ring substituent, wherein one or more ring atoms are selected from N, O or S(O)m (wherein m is an integer from 0 to 2) and the remaining ring atoms are carbon, for example: piperidinyl, pyrrolidinyl, etc.

[0115] The term "fixed sequence" or "sequence fixed" means that the order of amino acids in a polypeptide chain is fixed.

[0116] In one embodiment of the present invention, a membrane permeabilization sequence polypeptide having a structure represented by formula (I) or a stereoisomer thereof or a pharmaceutically acceptable salt thereof is provided:

[0117] Wherein, Q is a polypeptide group composed of n A and m B arranged in any sequence;

[0118] B is the residue after removing one amino hydrogen and one hydroxyl group from an amino acid with a hydrophobic side chain;

[0119] A is the residue of lysine with a modified group, after removing one amino hydrogen and one hydroxyl group, and its structure is:

[0120] R is selected from: -R3-N(R4R5), -R3-R',

[0121] R' is selected from:

[0122] L is selected from: -NH-C(=O)O-, -NH-C(=O)-, -C(=O)-NH-, -C(=O)-O-;

[0123] R1 is selected from: alkylene;

[0124] R3 is selected from: alkylene, C6-C 14 Aryl-substituted alkylene;

[0125] R4 and R5 are independently selected from: alkyl, C6-C 14 an alkyl group substituted with an aryl group, or R3, R4 and the nitrogen atom to which they are attached together form a heterocycloalkyl group;

[0126] R6 is selected from: C1-C 15 Alkyl, C6-C 14 Aryl, C6-C 14 Aryl-substituted C1-C 15 alkyl;

[0127] y is selected from: 2-150;

[0128] n+m is greater than 0, and n is not 0;

[0129] q is selected from: 0, 1, 2, 3, 4.

[0130] In some embodiments, the structural formula of the membrane permeabilization sequence polypeptide is shown in Formula (II):

[0131] Wherein, Q1 is a polypeptide group composed of n1 A and m1 B arranged in any sequence;

[0132] n1+m1 is greater than 0, and n1 is not 0;

[0133] x1 is greater than 0;

[0134] The product of n1 and x1 is n; the product of m1 and x1 is m.

[0135] In some embodiments, the structural formula of the membrane permeabilization sequence polypeptide is shown in Formula (III):

[0136] Wherein, Q2 is a polypeptide group composed of n2 A and m2 B arranged in any sequence;

[0137] Q3 is a polypeptide group composed of n3 As and m3 Bs arranged in any sequence;

[0138] n2+n3+m2+m3 is greater than 0, and n2+n3 is not 0;

[0139] The sum of the product of n2 and x2 plus n3 is n, and the sum of the product of m2 and x2 plus m3 is m.

[0140] In the present invention, Q, Q1, Q2, and Q3 refer to polypeptide groups, wherein the amino acids can be arranged in any defined sequence. For example, the n As and m Bs in Q can be arranged in any sequence. For example, when n is 2 and m is 2, Q can be arranged in the following sequence: ABAB, ABBA, AABB, BBAA, BAAB, BABA, etc. The polypeptide can be synthesized using solid-phase synthesis. Therefore, the sequence and number of Q, Q1, Q2, and Q3 can be kept fixed and controllable as desired.

[0141] Among them, the amino acids with hydrophobic side chains can be: L-amino acids, tryptophan, phenylalanine, alanine, valine, leucine, isoleucine, etc.

[0142] In some embodiments, B is

[0143] R2 is selected from: C1-C 12 Alkyl, C6-C 14 Aryl, C6-C 14 Aryl-substituted C1-C 12 Alkyl, benzyloxycarbonyl substituted C1-C 12 Alkyl, 5-10 membered heteroaryl substituted C1-C 12 alkyl.

[0144] In some embodiments, R2 is selected from: C1-C8 alkyl, phenyl, naphthyl, C1-C6 alkyl substituted with phenyl, C1-C6 alkyl substituted with benzyloxycarbonyl, and C1-C6 alkyl substituted with 5-10 membered heteroaryl.

[0145] In some embodiments, R2 is selected from the group consisting of: methyl, ethyl, n-propyl, isopropyl, n-butyl, isobutyl, pentyl, hexyl, heptyl, octyl, nonyl, decyl, undecyl, dodecyl, phenyl, naphthyl, benzyl, ethyl substituted with benzyloxycarbonyl, ethyl substituted with benzopyrrole, or their chiral isomers.

[0146] In some embodiments, R1 is selected from: C1-C6 alkylene.

[0147] In some embodiments, R1 is selected from: -(CH2) p -, wherein p is selected from: 1, 2, 3, 4, 5, 6.

[0148] In some embodiments, A is: or their chiral isomers;

[0149] Wherein, X is: -O- or none.

[0150] In some embodiments, R3 is selected from: C1-C6 alkylene, phenyl-substituted C1-C6 alkylene.

[0151] In some embodiments, R3 is selected from: -(CH2) p -, phenyl-substituted-(CH2) p -; wherein p is selected from: 1, 2, 3, 4, 5, 6.

[0152] In some embodiments, R is selected from: -R3-N(R4R5),

[0153] In some embodiments, R4 and R5 are independently selected from: C1-C6 alkyl, phenyl-substituted C1-C6 alkyl, naphthyl-substituted C1-C6 alkyl, or R4, R5 and the nitrogen atom to which they are attached form a 5-10 membered heterocycloalkyl.

[0154] In some embodiments, R4 and R5 are independently selected from: C1-C4 alkyl, phenyl-substituted C1-C3 alkyl, naphthyl-substituted C1-C3 alkyl, or R4, R5 and the nitrogen atom to which they are attached form a 5-8 membered heterocycloalkyl.

[0155] In some embodiments, R4 and R5 are independently selected from: C3 alkyl, C4 alkyl, C5 alkyl, or R4, R5 and the nitrogen atom to which they are connected form a 7-membered heterocycloalkyl.

[0156] In some embodiments, R4, R5 and the nitrogen atom to which they are attached together form the following group:

[0157] In some embodiments, R6 is selected from the group consisting of: C1-C6 alkyl, phenyl, naphthyl, and phenyl-substituted C1-C6 alkyl.

[0158] In some embodiments, R6 is selected from the group consisting of: C1-C3 alkyl, phenyl, naphthyl, and benzyl.

[0159] In some embodiments, R is selected from: -R3-N(R4R5),

[0160] Wherein, R3 is selected from: -CH2-CH2-, -CH2-CH2-CH2-, -CH2-(CH2)3-CH2-, R4, R5 and the nitrogen atom to which they are connected together form the following group:

[0161] R6 is benzyl.

[0162] In some embodiments, R is selected from: -R3-N(R4R5);

[0163] Wherein, R3 is selected from: -CH2-CH2-; R4, R5 and the nitrogen atom to which they are connected together form the following group:

[0164] R2 is selected from: or a chiral isomer thereof.

[0165] In some embodiments, y is selected from: 10-120, preferably 20-120, preferably 20-50, 20-25, 40-48 or 108-116, more preferably 20, 21, 22, 23, 24, 43, 44, 45, 46, 111, 112, 113, 114.

[0166] In some embodiments, n+m is not less than 5, preferably not less than 9, preferably not less than 12, preferably not less than 15, preferably 6-200, more preferably 6-150, more preferably 6-110; more preferably 6-20 or 5-40 or 10-30; more preferably 13-30, 13-28, 13-21, or 15-25, and can be 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29 or 30.

[0167] In some embodiments, n is 5-30, and m is 0-15 or 1-15.

[0168] In some embodiments, n is 10-20, and m is 2-10.

[0169] In some embodiments, n:m = 1-10:1, preferably 1-5:1, more preferably 1.1-1.2:1, 2:1, 3:1 or 4:1.

[0170] In some embodiments, x1 is 1-30, preferably 3, 4, 5, 6, 7, 8, 9, or 10.

[0171] In some embodiments, the product of (n1+m1) and x1 is not less than 5, preferably not less than 9, preferably not less than 15, preferably 6-200, more preferably 6-150, more preferably 6-110; more preferably 6-20, 5-40, 9-30, or 15-28, more preferably 15-25, more preferably 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29 or 30.

[0172] In some embodiments, the product of n1 and x1 is 5-30, and the product of m1 and x1 is 0-15 or 1-15.

[0173] In some embodiments, the product of n1 and x1 is 10-20, and the product of m1 and x1 is 3-7.

[0174] In some embodiments, n1:m1=1-10:1, preferably 2-4:1, more preferably 2:1, 3:1 or 4:1.

[0175] In some embodiments, n1+m1=2-10, preferably 3, 4, 5, or 6.

[0176] In some embodiments, x2 is 1-30, preferably 3, 4, 5, 6, 7, 8, 9, or 10.

[0177] In some embodiments, the product of (n2+m2) and x2 plus the sum of n3 and m3 is not less than 5, preferably not less than 9, preferably not less than 12, preferably not less than 15, preferably 6-200, more preferably 6-150, more preferably 6-110; more preferably 6-20 or 5-40 or 10-30, more preferably 13-21, more preferably 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25.

[0178] In some embodiments, (the product of n2 and x2 plus the sum of n3): (the product of m2 and x2 plus the sum of m3) = 1-10:1, preferably 1-4:1, and more preferably 1.1-1.2:1.

[0179] In some embodiments, m3 is 0, n3 is 1, the product of n2 and x2 is 6-12, and the product of m2 and x2 is 6-12.

[0180] In some embodiments, the product of n2 and x2 is 6, 8, or 10, and the product of m2 and x2 is 6, 8, or 10.

[0181] In another embodiment of the present invention, a membrane-permeabilizing sequence polypeptide nanoparticle is provided, which is formed by self-assembly of the membrane-permeabilizing sequence polypeptide in an aqueous medium.

[0182] In another embodiment of the present invention, a method for preparing the membrane-permeable sequence polypeptide nanoparticles is also provided, comprising the following steps: dissolving the membrane-permeable sequence polypeptide in an organic solvent or a hydrochloric acid solution with a pH of 1.5-2.5, then adding the resulting solution dropwise into water under stirring, continuing stirring, and dialyzing to remove the solvent to obtain the membrane-permeable sequence polypeptide nanoparticles.

[0183] In some embodiments, the organic solvent is N,N-dimethylformamide.

[0184] In some embodiments, the ratio of the membrane-permeabilizing sequence polypeptide, the organic solvent or hydrochloric acid solution, and the water is 20 mg to 40 mg: 1 mL: 2 mL to 5 mL.

[0185] In some embodiments, the preparation method of the membrane-permeable sequence polypeptide nanoparticles includes the following steps: dissolving the membrane-permeable sequence polypeptide in N,N-dimethylformamide at a ratio of 10 mg to 30 mg: 1 mL, then adding the resulting solution dropwise to 2 mL to 5 mL of water under stirring at a speed of 400 to 800 rpm, continuing to stir at a speed of 200 to 600 rpm for 8 to 20 minutes, and dialyzing in water using a dialysis bag with a molecular weight cutoff of 10,000 to 20,000 to remove the solvent, thereby obtaining the membrane-permeable sequence polypeptide nanoparticles.

[0186] In another embodiment of the present invention, there is also provided the use of the membrane-permeabilizing sequence polypeptide or its stereoisomer or its pharmaceutically acceptable salt in the preparation of a drug for preventing and / or treating tumors.

[0187] In another embodiment of the present invention, there is also provided the use of membrane-perforating sequence polypeptide nanoparticles in the preparation of drugs for preventing and / or treating tumors.

[0188] In some embodiments, the tumor is pancreatic cancer, melanoma, colorectal cancer, colon cancer, lung cancer, tongue squamous cell carcinoma, cervical cancer, ovarian cancer, osteosarcoma, liver cancer, breast cancer, bladder cancer, or ovarian epithelial cancer.

[0189] In another embodiment of the present invention, a drug for preventing and / or treating tumors is provided, which is prepared from active ingredients and pharmaceutically acceptable excipients. The active ingredients include the membrane-breaking sequence polypeptide or its stereoisomer or its pharmaceutically acceptable salt, and / or the membrane-breaking sequence polypeptide nanoparticles.

[0190] In another embodiment of the present invention, a method for preventing and / or treating tumors is provided, comprising: administering a safe and effective amount of the membrane-permeable sequence polypeptide of the present invention or its stereoisomer or its pharmaceutically acceptable salt; and / or,

[0191] Administering a safe and effective amount of the membrane-permeabilizing sequence polypeptide nanoparticles of the present invention; and / or,

[0192] Administer a safe and effective amount of the drug of the present invention.

[0193] The compounds of formula (I)-(III) of the present invention can be used in combination with other known anti-tumor drugs. When administered in combination, the compounds of formula (I)-(III) and the known drug can each be an independent administration unit, or together form a combined administration unit; the compounds of formula (I)-(III) can be administered simultaneously with other known anti-tumor drugs or administered separately. When the compounds of formula (I)-(III) are taken simultaneously with one or more other drugs, it is preferred to use a pharmaceutical composition containing one or more known drugs and the compounds of formula (I)-(III). Drug combination also includes taking the compounds of formula (I)-(III) and one or more other known drugs at overlapping time periods. When the compounds of formula (I)-(III) are used in combination with one or more other known drugs, the dosage of the compounds of formula (I)-(III) or the known drug can be the same as the dosage when taken alone, or it can be lower than the dosage when taken alone.

[0194] The drugs or active ingredients that can be used in combination with the compounds of formula (I) to formula (III) include but are not limited to: immune checkpoint inhibitors, estrogen receptor modulators, androgen receptor modulators, retinoid receptor modulators, cytotoxins / cytostatics, antiproliferative agents, protein transferase inhibitors, HMG-CoA reductase inhibitors, HIV protein kinase inhibitors, reverse transcriptase inhibitors, angiogenesis inhibitors, cell proliferation and survival signal inhibitors, drugs that interfere with cell cycle checkpoints and apoptosis inducers, cytotoxic drugs, tyrosine protein inhibitors, EGFR inhibitors, VEGFR inhibitors, serine / Threonine protein inhibitors, Bcr-Abl inhibitors, c-Kit inhibitors, Met inhibitors, Raf inhibitors, MEK inhibitors, MMP inhibitors, topoisomerase inhibitors, histidine deacetylase inhibitors, proteasome inhibitors, CDK inhibitors, Bcl-2 family protein inhibitors, MDM2 family protein inhibitors, IAP family protein inhibitors, STAT family protein inhibitors, PI3K inhibitors, AKT inhibitors, integrin blockers, interferon-α, interleukin-12, COX-2 inhibitors, p53, p53 activators, VEGF antibodies, EGF antibodies, etc.

[0195] In some embodiments, the drugs or active ingredients that can be used in combination with the compounds of formula (I) to formula (III) include but are not limited to: aldesleukin, alendronic acid, interferon, atrenoin, allopurinol, allopurinol sodium, palonosetron hydrochloride, hexamethylmelamine, aminoglutethimide, amifostine, amrubicin, anacrine, anastrozole, dolasetron, aranesp, arglabin, arsenic trioxide, arnosine, 5-azacytidine, azathioprine, BCG or tice BCG, betadine, betamethasone acetate, betamethasone sodium phosphate preparation, bexarotene, bleomycin sulfate, bromourea, bortezomib, busulfan, calcitonin, alezotocin injection, capecitabine, carboplatin, castel , cefesone, cymoleukin, daunorubicin, chlorambucil, cisplatin, cladribine, cladribine, clodronate, cyclophosphamide, cytarabine, dacarbazine, actinomycin D, liposomal daunorubicin, dexamethasone, dexamethasone phosphate, estradiol valerate, denileukin-2, depomet, deslorelin, delazoxane, diethylstilbestrol, diflucan, docetaxel, doxorubicin, doxorubicin-166-chitosan complex, eligard, rasburicase, epirubicin hydrochloride, aprepitant, epirubicin, epoetin alfa, erythropoietin, epoetin, levamisole tablets, estradiol preparations, 17-beta-estradiol, estramustine sodium phosphate, ethinyl estradiol, amifostine, hydroxyphosphodiesterase, fenbifostine, etoposide Glycosides, fadrozole, tamoxifen preparations, filgrastim, finasteride, firaprestin, floxuridine, fluconazole, fludarabine, 5-fluorodeoxyuridine monophosphate, 5-fluorouracil, fluoxymesterone, flutamide, formestane, 1-β-D-arabinofuranosylcytosine-5'-stearoyl phosphate, fotemustine, fulvestrant, immunoglobulin, gemcitabine, gemtuzumab tuzumab, imatinib mesylate, carmustine rice paper capsules, goserelin, granisiron hydrochloride, histrelin, holmesin, hydrocortisone, erythro-hydroxynonyl adenine, hydroxyurea, tentanamib, idarubicin, ifosfamide, interferon α, interferon-α2, interferon α-2A, interferon α-2B, interferon α-nl, interferon α- n3, interferon beta, interferon gamma-la, interleukin-2, intron A, Iressa, irinotecan, Kateri, lentinan sulfate, letrozole, leucovorin, leuprorelin acetate, levamisole, levofolinate calcium salt, levothyroxine sodium, levothyroxine sodium preparations, lomustine, lonidamine, dronabinol, nitrogen mustard, methylcobalamin, medroxyprogesterone acetate, megestrol acetate, melphalan, esterified estrogen, 6-mercaptopurine, mesna, methotrexate, methyl aminolevulinate, miltefosine, minocycline, mitomycin C, mitotane, mitoxantrone, trilostane, liposomal doxorubicin citrate, nedaplatin, pegfilgrastim, oprelleukin, neupogen, nilutamide,Tamoxifen, NSC-631570, recombinant human interleukin-1-beta, octreotide, ondansetron hydrochloride, prednisone oral solution, oxaliplatin, paclitaxel, prednisone sodium phosphate, pegaspargase, PEGASYS, pentostatin, streptozotocin, pilocarpine hydrochloride, pirarubicin, plicamycin, porfimer sodium, prednimustine, stirprednisolone, prednisone, Premarin, procarbazine, recombinant human erythropoietin, raltitrexed, rebifurcation, etidronate rhenium-1 86. Rituximab, Dextromethorphan-A, Romotide, Pilocarpine Hydrochloride Tablets, Octreotide, Sarmustine, Semustine, Sizoran, Sobuzosine, Methylprednisolone Sodium, Pafosic Acid, Stem Cell Therapy, Streptozocin, Strontium-89 Chloride, Levothyroxine Sodium, Tamoxifen, Tamsulosin, Tasoflavin, Tastolactone, Taxotere, Tesithium, Temozolomide, Teniposide, Testosterone Propionate, Methyltestosterone, Thioguanine, Thiotepa, Thyroid-Stimulating Hormone, Tiludronic Acid, Tuoxifen Protecan, toremifene, tositumomab, trastuzumab, treosulfan, tretinoin, methotrexate tablets, trimethylmelamine, trimetrexate, triptorelin acetate, triptorelin pamoate, eufotaxime, uridine, valrubicin, veslanone, vinblastine, vincristine, vinsamide, vinorelbine, virulizine, dextromethorphan, statins, zofran, paclitaxel protein stabilizer, acolbifene, interferon r-lb, affinitak, amino Pterin, arzoxifene, asoprisnil, atamestane, atrasentan, BAY43-9006, Avastin, CCI-779, CDC-501, Celebrex, cetuximab, clenatol, cyproterone acetate, decitabine, DN-101, doxorubicin-MTC, dSLIM, dutasteride, edotecarin, eflornithine, exitecan, fenretinide, histamine dihydrochloride, histrelin hydrogel implant, holmium-166 DOTMP, ibandronic acid, interferon gamma, intron-PEG, ixabepilone, keyhole limpet hemocyanin, L-651582, lanreotide, lasofoxifene, libra, lonafamib, miproxifene, minocycline, MS-209, liposomal MTP-PE, MX-6, nafarelin, nemorubicin, nevastatin, noratriptide, oblimersen, onco-TCS, osidem, paclitaxel polyglutamate, sodium brasilate, PN- 401, QS-21, quasitan, R-154, raloxifene, ranpirnase, 13-cis retinoic acid, satraplatin, ciocalcitol, T-138067, tarceva, paclitaxel docosahexaenoic acid, thymosin α1, gazofurin, tipifarnib, tirapazamine, TLK-286, toremifene, trans-MID-lo7R, valspodar, vapreotide, vatalanib, verteporfin, vinflunine, Z-100, and zoledronic acid, or a combination thereof.

[0196] The drug for preventing and / or treating tumors of the present invention can be used in non-human mammals or humans.

[0197] The present invention provides a drug or method for preventing and / or treating tumors, comprising (administering) a safe and effective amount of an active ingredient (i.e., the tertiary amine-modified membrane-permeable peptide or nanoparticle thereof) and a pharmaceutically acceptable excipient. Administration of the drug involves administering a safe and effective amount of the active ingredient to a mammal (e.g., a human) in need of treatment, wherein the dosage is a pharmaceutically effective dosage. Of course, the specific dosage should also take into account factors such as the route of administration and the patient's health status, all of which are within the skill of a skilled physician.

[0198] Here, "safe and effective amount" means: the amount of active ingredient sufficient to significantly improve the condition without causing serious side effects. "Pharmaceutically acceptable excipients" refers to: one or more compatible solid or liquid fillers or gel substances that are suitable for human use and must be of sufficient purity and sufficiently low toxicity. "Compatibility" here refers to the ability of the various components of the composition to blend with the active ingredient of the present invention (the tertiary amine-modified sequence polypeptide membrane-breaking material of Formula I), and with each other, without significantly reducing the efficacy of the active ingredient.

[0199] The pharmaceutically acceptable excipients used in the drug for preventing and / or treating tumors of the present invention include, but are not limited to, one or more of the following materials: at least one of solvents, excipients, fillers, extenders, binders, humectants, disintegrants, dissolution buffers, absorption accelerators, adsorbents, diluents, solubilizers, emulsifiers, lubricants, wetting agents, suspending agents, flavoring agents, and fragrances.

[0200] Examples of pharmaceutically acceptable excipients include cellulose and its derivatives (such as sodium carboxymethyl cellulose, sodium ethyl cellulose, cellulose acetate, etc.), gelatin, talc, solid lubricants (such as stearic acid, magnesium stearate), calcium sulfate, vegetable oils (such as soybean oil, sesame oil, peanut oil, olive oil, etc.), polyols (such as propylene glycol, glycerol, mannitol, sorbitol, etc.), emulsifiers (such as ), wetting agents (such as sodium lauryl sulfate), colorants, flavorings, stabilizers, antioxidants, preservatives, pyrogen-free water, etc.

[0201] There is no particular limitation on the administration of the active ingredient or pharmaceutical composition of the present invention. Representative administration routes include (but are not limited to): oral, rectal, parenteral (intravenous, intramuscular or subcutaneous) and the like.

[0202] Solid dosage forms for oral administration include capsules, tablets, pills, powders, and granules.

[0203] In these solid dosage forms, the active ingredient is mixed with at least one conventional inert excipient (or carrier), such as sodium citrate or dicalcium phosphate, or with:

[0204] (a) fillers or extenders, for example, starch, lactose, sucrose, glucose, mannitol and silicic acid;

[0205] (b) binders, for example, hydroxymethylcellulose, alginate, gelatin, polyvinylpyrrolidone, sucrose and gum arabic;

[0206] (c) humectants, for example, glycerin;

[0207] (d) disintegrants, for example, agar-agar, calcium carbonate, potato or tapioca starch, alginic acid, certain complex silicates, and sodium carbonate;

[0208] (e) a buffering solvent, for example, paraffin;

[0209] (f) absorption accelerators, for example, quaternary ammonium compounds;

[0210] (g) wetting agents, for example, cetyl alcohol and glyceryl monostearate;

[0211] (h) adsorbents, for example, kaolin; and

[0212] (i) Lubricants, for example, talc, calcium stearate, magnesium stearate, solid polyethylene glycol, sodium lauryl sulfate, or mixtures thereof. In capsules, tablets and pills, the dosage form may also contain a buffering agent.

[0213] The solid dosage forms can also be prepared using coatings and shells, such as enteric coatings and other materials known in the art. They can contain opacifying agents, and the release of the active ingredient in such compositions can be delayed in a certain portion of the digestive tract. Examples of embedding components that can be used are polymeric substances and waxes.

[0214] Liquid dosage forms for oral administration include pharmaceutically acceptable emulsions, solutions, suspensions, syrups, or tinctures. In addition to the active ingredient, the liquid dosage form may contain inert diluents commonly used in the art, such as water or other solvents, solubilizers and emulsifiers, for example, ethanol, isopropyl alcohol, ethyl carbonate, ethyl acetate, propylene glycol, 1,3-butylene glycol, dimethylformamide, and oils, particularly cottonseed oil, peanut oil, corn germ oil, olive oil, castor oil, and sesame oil, or mixtures thereof. In addition to these inert diluents, the composition may also contain adjuvants, such as wetting agents, emulsifiers and suspending agents, sweeteners, flavoring agents, and fragrances.

[0215] In addition to the active ingredients, suspensions may contain suspending agents such as, for example, ethoxylated isostearyl alcohol, polyoxyethylene sorbitol and sorbitan esters, microcrystalline cellulose, aluminum methoxide and agar, or mixtures of these substances.

[0216] Compositions for parenteral injection may comprise physiologically acceptable sterile aqueous or anhydrous solutions, dispersions, suspensions or emulsions, and sterile powders for reconstitution into sterile injectable solutions or dispersions. Suitable aqueous and non-aqueous carriers, diluents, solvents or excipients include water, ethanol, polyols and suitable mixtures thereof.

[0217] The following are specific examples.

[0218] In the following examples, mPEG y -COOH reacts with the primary amine at the N-terminus of the peptide to synthesize a sequence peptide with an mPEG group. Then, a tertiary amine is introduced through the side chain primary amine of lysine to obtain a series of membrane-breaking sequence peptide polymer materials. The reaction formula and corresponding abbreviations are as follows:

[0219] In the general reaction formula of the present invention, n lysines or tertiary amine-modified lysines, and m hydrophobic amino acids can be arranged in any predetermined sequence. For example, in the present invention and the following examples, referred to as (K) n (W) m It refers to a polypeptide composed of n lysine (or tertiary amine-modified lysine) and m tryptophan arranged in a certain sequence through peptide bonds, abbreviated as (K) n (F) m It represents a polypeptide composed of n lysines (or lysines modified with tertiary amines) and m phenylalanines arranged in a defined sequence via peptide bonds. The specific sequence is described in detail in the following examples.

[0220] mPEG y The structural formula of -COOH is as follows:

[0221] y is 22, 45, or 113.

[0222] R-CDI is the product of the reaction between an alcohol (R-OH) with a tertiary amine structure and N,N'-carbonyldiimidazole (CDI). R contains a tertiary amine structure that can be protonated as the pH changes.

[0223] The alcohols (R-OH) containing tertiary amine structures used for side chain modification are all commercial products available on the market. R-OH is first reacted with N,N'-carbonyldiimidazole (CDI) to prepare R-CDI, which is then used for side chain modification.

[0224] R-CDI is prepared by reacting R-OH with CDI in dichloromethane. After the reaction, deionized water is added to remove unreacted CDI, and the solution is separated by dichloromethane extraction and dried over anhydrous magnesium sulfate to obtain a dichloromethane solution of R-CDI, which is then drained.

[0225] The side chain modification method is as follows: dissolve the sequence polypeptide in N,N-dimethylformamide (DMF), add R-CDI (5-fold excess), and add triethylamine and stir to react for 12 hours, drain, dissolve with DMSO, dialyze in deionized water with a dialysis bag with a molecular weight cutoff of 3500 for 24 hours, change the water every 2 hours, and freeze-dry to obtain the membrane-permeable sequence polypeptide.

[0226] Example 1: 2-(Dibutylamino)ethanol-modified methoxypolyethylene glycol-lysine and tryptophan fixed sequence polypeptide

[0227] R-OH is 2-(dibutylamino)ethanol (DB), R2 is a tryptophan residue, and y is 45. 45 -COOH and (K) n (W) m mPEG is obtained by reacting amino groups in fixed sequence peptides 45 -(K) n (W) m The tertiary amine was modified onto the side chain of lysine by CDI to obtain a fixed sequence peptide modified with a tertiary amine. The effects of different sequences and molecular weights on the pKa and pH selective killing of the fixed sequence peptide were studied.

[0228] (A) Preparation of fixed-sequence peptides modified with 2-(dibutylamino)ethanol.

[0229] The reaction equation is as follows:

[0230] The specific steps are as follows:

[0231] (1) Synthesize different sequences of H-[Lys(Boc)] n [Trp(Boc)] m -Resin, each time an amino acid was connected, ninhydrin reagent was used to detect whether the amino group had reacted completely. A portion of the resin was cleaved with a cleavage solution composed of 95% TFA, 2.5% H2O, and 2.5% TIS. The products were characterized by mass spectrometry. The results are shown in Figures 1 to 19, indicating that a series of polypeptide materials were successfully obtained.

[0232] (2) Synthesize mPEG with different sequences by solid phase synthesis (SPPS) 45 -[Lys(Boc)] n [Trp(Boc)] m-Resin, after the reaction is completed, ninhydrin reagent is used to detect whether the amino group has reacted completely.

[0233] (3) Cleavage of mPEG using a lysis solution consisting of 95% TFA, 2.5% H2O, and 2.5% TIS 45 -[Lys(Boc)] n [Trp(Boc)] m The sequence peptide was fixed for 3 hours, the lysate was concentrated with nitrogen and precipitated in ice ether, the precipitate was centrifuged and dissolved in deionized water, and dialyzed in deionized water with a 3500 dialysis bag for 24 hours, with the water changed every 2 hours, and white powder mPEG was obtained after freeze-drying. 45 -(K) n (W) m Fixed sequence polypeptide. The specific sequence is: mPEG 45 -KWKWKWKWKWK, mPEG 45 -KWKWKWKWKWKWK, mPEG 45 -KWKWKWKWKWKWKWK, mPEG 45 -KWKWKWKWKWKWKWKWK, mPEG 45 -KWKWKWKWKWKWKWKWKWK, mPEG 45 -KWWKKWWKKKW, mPEG 45 -KWWKKWWKKWWKK, mPEG 45 -KWWKKWWKKWWKKKW, mPEG 45 -KWWKKWWKKWWKKWWKK, mPEG 45 -KWWKKWWKKWWKKWWKKWWKK, mPEG 45 -KWKKWKKWK, mPEG 45 -KWKKWKKWKKWK, mPEG 45 -KWKKWKKWKKWKKWK, mPEG 45 -KWKKWKKWKKWKKWKKWK, mPEG 45 -KWKKWKKWKKWKKWKKWK, mPEG 45 -KWKKKWKKKWKK, mPEG 45 -KWKKKWKKKKWKKWKK, mPEG 45 -KWKKKWKKKKWKKKWKKWKK, mPEG 45 -KWKKKWKKKWKKKWKKKWKKKWKK,mPEG 45-KWKKKWKKKWKKKWKKKWKKKWKKKWKK, mPEG 45 -KKWKKKKWKKKKWKK, mPEG 45 -KKWKKKKWKKKKWKKKKWKK, mPEG 45 -KKWKKKKWKKKKWKKKKWKKKKWKK and mPEG 45 -KKWKKKKWKKKKWKKKKWKKKKWKKKKWKK are respectively abbreviated as mPEG 45 -(KW)5K, mPEG 45 -(KW)6K, mPEG 45 -(KW)7K, mPEG 45 -(KW)8K, mPEG 45 -(KW) 10 K, mPEG 45 -(KWWK)2KKW, mPEG 45 -(KWWK)3K, mPEG 45 -(KWWK)3KKW, mPEG 45 -(KWWK)4K, mPEG 45 -(KWWK)5K, mPEG 45 -(KWK)3, mPEG 45 -(KWK)4, mPEG 45 -(KWK)5, mPEG 45 -(KWK)6, mPEG 45 -(KWK)7, mPEG 45 -(KWKK)3, mPEG 45 -(KWKK)4, mPEG 45 -(KWKK)5, mPEG 45 -(KWKK)6, mPEG 45 -(KWKK)7, mPEG 45 -(KKWKK)3, mPEG 45 -(KKWKK)4, mPEG[[ID=SS]] 45 -(KKWKK)5 and mPEG 45 -(KKWKK)6.

[0234] (4) CDI was placed in a round-bottom flask, and anhydrous dichloromethane was added and stirred to disperse (1.0 g CDI plus 5 mL dichloromethane). The flask was sealed with a rubber stopper. 2-(dibutylamino)ethanol (3 times excess CDI) was slowly added with a syringe. The solution gradually became clear. After reacting for 12 h, deionized water equal to the amount of dichloromethane was added and stirred until no bubbles emerged. The lower dichloromethane phase was removed with a separatory funnel, and anhydrous magnesium sulfate was added to dry for 2 h. The solid was removed by filtration to obtain a solution and dried to obtain 2-(dibutylamino)ethanol-CDI (DB-CDI).

[0235] (5) mPEG 45 -(K) n (W) m The fixed sequence peptide was dissolved in DMF, DB-CDI (5-fold excess) was added, triethylamine (equimolar to the lysine side chain amino group) was added, and the reaction was continued for 12 hours. The solvent was pumped out with an oil pump, and the peptide was dissolved in DMSO. The peptide was loaded into a 3.5k dialysis bag and dialyzed with deionized water for 24 hours, with the water changed every 2 hours. The peptide was lyophilized to obtain a fixed sequence peptide modified with DB. The DB-modified lysine was designated as K1, and the resulting DB-modified fixed sequence peptide was referred to as mPEG. 45 -(K1W)5K1,mPEG 45 -(K1W)6K1,mPEG 45 -(K1W)7K1,mPEG 45 -(K1W)8K1,mPEG 45 -(K1W) 10 K1, mPEG 45 -(K1WWK1)2K1K1W, mPEG 45 -(K1WWK1)3K1,mPEG 45 -(K1WWK1)3K1K1W, mPEG 45 -(K1WWK1)4K1, mPEG 45 -(K1WWK1)5K1,mPEG 45 -(K1WK1)3,mPEG 45 -(K1WK1)4,mPEG 45 -(K1WK1)5,mPEG 45 -(K1WK1)6,mPEG 45 -(K1WK1)7,mPEG 45 -(K1WK1K1)3,mPEG 45 -(K1WK1K1)4,mPEG 45 -(K1WK1K1)5,mPEG 45 -(K1WK1K1)6,mPEG 45 -(K1WK1K1)7,mPEG45 -(K1K1WK1K1)3,mPEG 45 -(K1K1WK1K1)4,mPEG 45 -(K1K1WK1K1)5 and mPEG 45 -(K1K1WK1K1)6. Its structure was characterized by H NMR spectroscopy (Figures 20-25). The peak of the methylene group connected to the lysine side chain and the primary amino group disappeared, indicating that it was completely bonded.

[0236] The structural formula of the DB-modified fixed-sequence polypeptide prepared in this example is as follows:

[0237] (ii) pKa of the test sequence polypeptide.

[0238] Take 100 μL of concentrated hydrochloric acid (37%, 12 mol) and add it to 100 mL of deionized water to fully dissolve to obtain a clear and transparent solution. Dissolve 10 mg of the fixed sequence polypeptide material prepared in step (5) in 10 mL of hydrochloric acid solution, and insert the pH probe into the liquid surface. Under stirring (stirring speed Mot = 3), titrate with 0.5 M sodium hydroxide titrant. After the pH meter stabilizes, record the reading until the titration result reaches pH = 11. By taking the derivative of the titration curve, the extreme points are obtained and defined as the protonation rate of 1 and 0 points, and a pH and protonation rate curve is established. According to the definition of the dissociation equilibrium constant (pKa), the pH corresponding to the protonation rate of the polypeptide equal to 0.5 is defined as the pKa of the material. The results are shown in Figure 26. The protonation degree of this type of polypeptide undergoes a significant jump with pH, ​​and the pKa of the polypeptide shows a decreasing trend with increasing molecular weight.

[0239] (III) Cytotoxicity of fixed sequence peptides at different pH values.

[0240] Take 10 mg of the polypeptide prepared in step (5) in a 5 mL sample bottle and add 0.3 mL of DMF to fully dissolve it for later use. Take another 0.9 mL of sterile water in a pre-sterilized 25 mL round-bottom flask, place it on a magnetic stirring table, stir at RPM = 600 r / min, and add the polypeptide DMF solution dropwise to the round-bottom flask using a pipette. Continue stirring at RPM = 400 r / min for 15 minutes, then use a dialysis bag with a molecular weight cutoff of 14,000 to dialyze in 4 L of ultrapure water at 4 ° C for 24 hours. Change the water every 1 hour for the first 6 hours and every 6 hours for the next 18 hours to obtain polypeptide nanoparticles. After dialysis, remove and quantify with a pipette and store in a 4 ° C refrigerator.

[0241] The CCK-8 assay was used to evaluate the tumor cell killing effects of peptide nanoparticles at pH 7.4 and pH 6.8. DMEM culture medium was adjusted to pH 6.8 using 6 mol / L HCl solution and set aside. Mouse colorectal cancer cells MC38 (purchased from ATCC) were cultured in DMEM culture medium containing 10% (v / v) fetal bovine serum. At pH 7.4 and pH 6.8, different concentrations of the peptide nanoparticles prepared in this example were added to the cells (concentration of 1×10 5 The cells were incubated with 100 μL of CCK-8 solution at 37°C in a CO2 incubator for 24 hours. The absorbance at OD 450 nm was measured using a microplate reader.

[0242] The test results are shown in Figures 27 and 28. The DB-modified tryptophan and lysine sequence-fixed peptides are basically non-toxic to tumor cells at pH 7.4, except for mPEG 45 -(K1K1WK1K1)6 has a certain degree of toxicity and has strong killing toxicity at pH 6.8. This series of peptide materials shows good pH-selective killing ability.

[0243] (IV) In vivo toxicity of sequence-fixed peptides.

[0244] The toxicity of the polypeptide was tested in mice by testing the maximum lethal dose (MTD). Six-week-old ICR female mice (purchased from Hunan Shrek Jingda Experimental Animal Co., Ltd.) were divided into 10 groups with similar average weights, with 5 mice in each group. Each polypeptide material was administered starting at 200 mg / kg body weight, and the same dose was administered to one ICR female mouse. If no experimental animal died after 24 hours, the administration continued. If a dead experimental mouse appeared, the dose was reduced by 10 mg / kg and administered again until all mice did not die after the dose was administered. The maximum dose in which no mouse died was recorded as the maximum tolerated dose of the ICR mouse, and a graph was drawn, as shown in Figure 29: The sequence-fixed polypeptide materials in this embodiment all have low in vivo toxicity, and the MTD of most polypeptides is ≥200 mg / kg.

[0245] (V) High-content observation of pH-sensitive membrane disruption of sequence-immobilized peptides.

[0246] 1) Panc02 cells expressing GFP on the cell membrane and mCherry in the cytoplasm were cultured at a rate of 1.5-1.8×10 5 Cells at 1000 μg / mL were seeded in 96-well plates and cultured overnight in a constant temperature cell incubator before use;

[0247] 2) preparing DMEM culture media of different pH values, and using these media to prepare drug solutions with a concentration of 400 μg / mL at different pH values;

[0248] 3) Replace the culture medium with the prepared solution and perform imaging 4 hours later. The results are shown in Figure 30: These peptide materials exhibit pH-sensitive membrane permeabilization, allowing complete leakage of mCherry fluorescence within a small pH range.

[0249] This example shows that DB-modified lysine and tryptophan sequence polypeptides have excellent pH-selective killing ability and low toxicity in vivo, showing a pH-supersensitive membrane-breaking effect and can achieve leakage of intracellular mCherry fluorescence within a small pH range.

[0250] (VI) Therapeutic Experiment of Sequence-Fixed Peptides on MC38 Subcutaneous Colorectal Cancer Model

[0251] 1) Construction of mouse subcutaneous MC38 colorectal cancer tumor model: MC38 cells were resuspended in serum-free culture medium and the concentration was adjusted to 1.0×10 7 cells / mL, after depilation of the right back of female C57BL / 6 mice, 100 μL of cell suspension was subcutaneously injected at the depilated site;

[0252] 2) When the tumor volume is about 50 mm 3 The tumor-bearing mice were randomly divided into 2 groups, 3 mice in each group. The following groups were injected with PBS and mPEG. 45 -(K1WK1)5 (60 mg / kg), administered every other day for six consecutive treatments;

[0253] 3) Measure and record the long and short diameters of the tumor, calculate the tumor volume, and detect and record the weight of the mice.

[0254] The results are shown in Figures 31 to 33. 45 -(K1WK1)5 showed excellent anti-tumor effects, with the tumors of all three mice disappearing, and the mice did not lose weight during administration.

[0255] (7) mPEG 45 -(K1WK1)5 membrane rupture and killing mechanism

[0256] 1) mPEG 45 -(K1WK1)5 kills Panc02 cells expressing GFP on the cell membrane and mCherry in the cytoplasm: The cells were plated at a density of 100,000 / well in a confocal dish and cultured overnight in a 37°C, CO2 incubator. After adjusting the parameters on the instrument, a pH 6.8 solution containing 400 μg / mL mPEG was added. 45-(K1WK1)5 solution was added to the confocal dish and photographed after incubation for a certain period of time.

[0257] The results are shown in Figure 34, where red is mCherry protein, green is GFP protein, and purple is Cy5-labeled mPEG. 45 -(K1WK1)5. At pH 6.8, mPEG 45 -(K1WK1)5 causes leakage of cytoplasmic mCherry protein.

[0258] 2) mPEG 45 Killing of MC38 cells by -(K1WK1)5: MC38 cells were plated at a density of 100,000 / well in a confocal dish and cultured in a CO2 incubator at 37°C for 24 h. 1 mL of 400 μg / mL mPEG at pH 6.8 was prepared. 45 To the -(K1WK1)5 solution, add 5μL of Annexin V-FITC and 1μL of iodide and mix thoroughly. After adjusting the instrument parameters, replace the supernatant in the confocal dish with the above solution and incubate for a specified period before imaging.

[0259] The results are shown in Figure 35, where the green is Annexin V-FITC, which binds to the negatively charged phospholipid PS on the cell membrane to mark the cell membrane; the red is Cy5-labeled mPEG 45 -(K1WK1)5; blue is a membrane-impermeable dye iodide, which is used to mark the nuclei of dead cells. As shown in the figure, mPEG can be seen 45 -(K1WK1)5 material binds to the cell membrane, and the cell quickly dies by bubbling. At this time, PI enters the cell, indicating cell death.

[0260] These data suggest that mPEG 45 -(K1WK1)5 causes cell death by destroying the cell membrane.

[0261] Example 2: Methoxypolyethylene glycol-lysine and tryptophan fixed sequence peptides modified with different tertiary amines

[0262] R-OH is diethylaminoethanol (DE), N-hydroxyethylpiperidine (C6), 2-(hexamethyleneimine)ethanol (C7), N-benzyl-4-hydroxypiperidine (C6P), N-methyl-N-hydroxyethylbenzylamine (MP), N-benzyl-L-prolinol (C5P), (S)-3-hydroxy-1-benzylpyrrolidine (C5P2), R2 is a tryptophan residue, and y is 45. mPEG is used 45 -COOH and (K) n (W) m mPEG is obtained by reacting amino groups in fixed sequence peptides45 -(K) n (W) m The tertiary amine was modified onto the side chain of lysine by CDI to obtain a fixed sequence peptide modified with a tertiary amine. The effects of different tertiary amine modifications on the pKa and pH selective killing of the sequence peptide were tested.

[0263] (A) Preparation of R-OH modified fixed sequence peptides.

[0264] The reaction equation is as follows:

[0265] The specific steps are as follows:

[0266] (1) Synthesis of H-[Lys(Boc)] by solid phase synthesis (SPPS) n [Trp(Boc)] m -Resin, each time an amino acid was connected, ninhydrin reagent was used to detect whether the amino group had reacted completely. A portion of the resin was cleaved with a cleavage solution composed of 95% TFA, 2.5% H2O, and 2.5% TIS. The product was characterized by mass spectrometry for related structure. The results are shown in Figure 19, indicating that a polypeptide with the sequence (KKWKK)5 was obtained.

[0267] (2) Synthesize mPEG by solid phase synthesis (SPPS) 45 -[Lys(Boc)] n [Trp(Boc)] m -Resin, after the reaction is completed, use ninhydrin reagent to detect whether the amino group has reacted completely.

[0268] (3) Cleavage of mPEG using a lysis solution consisting of 95% TFA, 2.5% H2O, and 2.5% TIS 45 -[Lys(Boc)] n [Trp(Boc)] m The sequence peptide was fixed for 3 hours, the lysate was concentrated with nitrogen and precipitated in ice ether, the precipitate was centrifuged and dissolved in deionized water, and dialyzed in deionized water with a 3500 dialysis bag for 24 hours, with the water changed every 2 hours, and white powder mPEG was obtained after freeze-drying. 45 -(K) n (W) m Fixed sequence polypeptide. The specific sequence is mPEG 45 -KKWKKKKKWKKKKKKKKKWKK, referred to as mPEG 45 -(KKWKK)5.

[0269] (4) CDI was placed in a round-bottom flask, and anhydrous dichloromethane was added and stirred to disperse (1.0 g CDI plus 5 mL dichloromethane). The flask was sealed with a rubber stopper, and R-OH (3 times excess CDI) was slowly added with a syringe. The solution gradually became clear. After reacting for 12 h, deionized water equal to the amount of dichloromethane was added and stirred until no bubbles emerged. The lower dichloromethane phase was removed with a separatory funnel, and anhydrous magnesium sulfate was added to dry for 2 h. The solid was removed by filtration to obtain a solution and dried. R-CDI was obtained.

[0270] (5) mPEG 45 -(KKWKK)5 fixed sequence polypeptide was dissolved in DMF, R-CDI (5 times excess) was added, triethylamine (equimolar to the lysine side chain amino group) was added, and the reaction was carried out for 12 hours. The solvent was drained with an oil pump, and then dissolved with DMSO, loaded with a 3.5k dialysis bag, dialyzed with deionized water for 24 hours, and the water was changed every 2 hours. The fixed sequence polypeptide modified with different tertiary amines was obtained. The DB-modified lysine was denoted as K1, the DE-modified lysine was denoted as K2, the C6-modified lysine was denoted as K3, the C7-modified lysine was denoted as K4, the C6P-modified lysine was denoted as K5, the MP-modified lysine was denoted as K6, the C5P-modified lysine was denoted as K7, and the C5P2-modified lysine was denoted as K8. The tertiary amine-modified fixed sequence polypeptide obtained in this example is referred to as mPEG. 45 -(K1K1WK1K1)5, mPEG 45 -(K2K2WK2K2)5, mPEG 45 -(K3K3WK3K3)5, mPEG 45 -(K4K4WK4K4)5, mPEG 45 -(K5K5WK5K5)5, mPEG 45 -(K6K6WK6K6)5, mPEG 45 -(K7K7WK7K7)5 and mPEG 45 -(K8K8WK8K8)5. Its structure was characterized by H NMR spectroscopy (Figures 25, 36-42). The peak of the methylene group connected to the lysine side chain and the primary amino group disappeared, proving that it was completely bonded. For MP-modified polypeptides, i.e., mPEG 45 -(K6K6WK6K6)5, the ratio of the signal peak integral of the methylene hydrogen atom at the h position of the lysine side chain to the signal peak integral of the methyl hydrogen atom at the o position of the tertiary amine MP is about 2:3, indicating that mPEG 45 -(KKWKK)5 is fully modified with tertiary amine MP, i.e. mPEG 45 -(K6K6WK6K6)5 was successfully synthesized.

[0271] The structural formula of the fixed sequence polypeptide prepared in this example is as follows:

[0272] (2) The effect of different tertiary amine-modified lysine side chains on the pKa of sequence peptides.

[0273] Take 100 μL of concentrated hydrochloric acid (37%, 12 mol) and add it to 100 mL of deionized water to fully dissolve it to obtain a clear and transparent solution. Dissolve 10 mg of the fixed sequence polypeptide material prepared in step (5) in 10 mL of hydrochloric acid solution, and insert the pH probe into the liquid surface. Under stirring (stirring speed Mot = 3), titrate with 0.5 M sodium hydroxide titrant. After the pH meter stabilizes, record the reading until the titration result reaches pH = 11. By taking the derivative of the titration curve, the extreme points are obtained and defined as the protonation rate of 1 and 0 points, and a pH and protonation rate curve is established. According to the definition of the dissociation equilibrium constant (pKa), the pH corresponding to the protonation rate of the polypeptide equal to 0.5 is defined as the pKa of the material. The results are shown in Figure 43. The protonation degree of this type of polypeptide undergoes a significant jump with pH, ​​and the pKa of the polypeptide shows an increasing trend as the hydrophobicity of the tertiary amine decreases.

[0274] (III) Cytotoxicity of fixed sequence peptides at different pH values.

[0275] Take 10 mg of the polypeptide prepared in step (5) in a 5 mL sample bottle and add 0.3 mL of DMF to fully dissolve it for later use. Take another 0.9 mL of sterile water in a pre-sterilized 25 mL round-bottom flask, place it on a magnetic stirring table, stir at RPM = 600 r / min, and add the polypeptide DMF solution dropwise to the round-bottom flask using a pipette. Continue stirring at RPM = 400 r / min for 15 minutes, then use a dialysis bag with a molecular weight cutoff of 14,000 to dialyze in 4 L of ultrapure water at 4 ° C for 24 hours. Change the water every 1 hour for the first 6 hours and every 6 hours for the next 18 hours to obtain polypeptide nanoparticles. After dialysis, remove and quantify with a pipette and store in a 4 ° C refrigerator.

[0276] The CCK-8 assay was used to evaluate the tumor cell killing effects of peptide nanoparticles at pH 7.4 and pH 6.8. DMEM culture medium was adjusted to pH 6.8 using a 6 mol / L HCl solution. Mouse colorectal cancer cells MC38 (purchased from ATCC) were cultured in DMEM culture medium containing 10% (v / v) fetal bovine serum. At pH 7.4 and pH 6.8, different concentrations of the peptide nanoparticles prepared in this example were incubated with the cells (concentration of 1×10 5The cells were incubated with 100 μL of CCK-8 solution at 37°C in a CO2 incubator for 24 hours. The absorbance at OD 450 nm was measured using a microplate reader.

[0277] The test results are shown in Figure 44. The sequence of DE modified tryptophan and lysine fixed polypeptide, namely mPEG 45 -(K2K2WK2K2)5, has low toxicity to tumor cells at pH 7.4, and its killing toxicity at pH 6.8 is not strong, and it does not have good pH selectivity; C6 modified tryptophan and lysine sequence fixed polypeptide, namely mPEG 45 -(K3K3WK3K3)5, has strong cytotoxicity to tumor cells at both pH 7.4 and pH 6.8, and does not have good pH selectivity; C6P-modified tryptophan and lysine sequence fixed polypeptide, namely mPEG 45 -(K5K5WK5K5)5, does not cause tumor cell death at pH 7.4, but its cytotoxicity at pH 6.8 is also low, and it does not have good pH selectivity; MP-modified tryptophan and lysine sequence-fixed polypeptide, i.e. mPEG 45 -(K6K6WK6K6)5, does not cause tumor cell death at pH 7.4, but its cytotoxicity at pH 6.8 is also weak and does not have good pH selectivity; C5P-modified tryptophan and lysine sequence-fixed peptides, namely mPEG 45 -(K7K7WK7K7)5, does not cause tumor cell death at pH 7.4 and 6.8, and has no pH selectivity; C5P2 modified tryptophan and lysine sequence fixed polypeptide, namely mPEG 45 -(K8K8WK8K8)5, does not cause tumor cell death at pH 7.4, has certain killing toxicity at pH 6.8, and has certain pH selective killing toxicity; C7 modified tryptophan and lysine sequence fixed polypeptide, namely mPEG 2k -(K4K4WK4K4)5, the tumor cell toxicity at pH 6.8 is better than that at pH 7.4, and it has a certain pH selective killing toxicity; but mPEG 45 -(K8K8WK8K8)5 and mPEG 2k -(K4K4WK4K4)5 has no selectivity compared to DB-modified tryptophan and lysine sequence-fixed peptides (mPEG 45 -(K1K1WK1K1)5) Good. It can be seen that for the same fixed sequence peptide, different tertiary amine modifications show different tumor cell killing effects.

[0278] Example 3: PEG-modified methoxypolyethylene glycol-lysine and tryptophan fixed sequence peptides of different lengths

[0279] R-OH is 2-(dibutylamino)ethanol (DB), R2 is a tryptophan residue, and y is 22, 45, and 113. 22 -COOH, mPEG 45 -COOH and mPEG 113 -COOH and (K) n (W) m mPEG is obtained by reacting amino groups in fixed sequence peptides 22 -(K) n (W) m , mPEG 45 -(K) n (W) m and mPEG 113 -(K) n (W) m The tertiary amine was modified onto the side chain of lysine by CDI to obtain a fixed sequence peptide modified with a tertiary amine. The effects of different PEG lengths on the pKa and pH selectivity of the fixed sequence peptide were tested.

[0280] (A) Preparation of fixed-sequence peptides modified with 2-(dibutylamino)ethanol.

[0281] The reaction equation is as follows:

[0282] The specific steps are as follows:

[0283] (1) Synthesis of H-[Lys(Boc)] by solid phase synthesis (SPPS) n [Trp(Boc)] m -Resin, each time an amino acid was connected, ninhydrin reagent was used to detect whether the amino group had reacted completely. A portion of the resin was cleaved with a cleavage solution composed of 95% TFA, 2.5% H2O, and 2.5% TIS. The product was characterized by mass spectrometry for related structure. The results are shown in Figure 19, indicating that a polypeptide with the sequence (KKWKK)5 was obtained.

[0284] (2) Synthesize mPEG by solid phase synthesis (SPPS) 22 -[Lys(Boc)] n [Trp(Boc)] m -Resin, mPEG 45 -[Lys(Boc)] n [Trp(Boc)] m -Resin and mPEG 113 -[Lys(Boc)]n [Trp(Boc)] m -Resin, after the reaction is completed, use ninhydrin reagent to detect whether the amino group has reacted completely.

[0285] (3) Cleavage of mPEG using a lysis solution consisting of 95% TFA, 2.5% H2O, and 2.5% TIS 22 -[Lys(Boc)] n [Trp(Boc)] m -Resin, mPEG 45 -[Lys(Boc)] n [Trp(Boc)] m -Resin and mPEG 113 -[Lys(Boc)] n [Trp(Boc)] m -Resin fixed sequence peptide for 3 hours, the lysate was concentrated with nitrogen and precipitated into ice ether, the precipitate was centrifuged and dissolved in deionized water, and dialyzed in deionized water with a 3500 dialysis bag for 24 hours, with the water changed every 2 hours, and freeze-dried to obtain white powder mPEG. 22 -(K) n (W) m , mPEG 45 -(K) n (W) m and mPEG 113 -(K) n (W) m Fixed sequence polypeptide. The specific sequence is mPEG 22 -KKWKKKKWKKKKWKKKKWKKKKWKK,mPEG 45 -KKWKKKKWKKKKWKKKKWKKKKWKK and mPEG 113 -KKWKKKKKWKKKKKKKKKWKK, referred to as mPEG 22 -(KKWKK)5,mPEG 45 -(KKWKK)5 and mPEG 113 -(KKWKK)5.

[0286] (4) CDI was placed in a round-bottom flask, and anhydrous dichloromethane was added and stirred to disperse (1.0 g CDI plus 5 mL dichloromethane). The flask was sealed with a rubber stopper, and DB-OH (3 times excess CDI) was slowly added with a syringe. The solution gradually became clear. After reacting for 12 h, deionized water equal to the amount of dichloromethane was added and stirred until no bubbles emerged. The lower dichloromethane phase was removed with a separatory funnel, and anhydrous magnesium sulfate was added to dry for 2 h. The solid was removed by filtration to obtain a solution and dried. DB-CDI was obtained.

[0287] (5) mPEG 22 -(KKWKK)5,mPEG 45 -(KKWKK)5 and mPEG 113 -(KKWKK)5 fixed sequence peptide was dissolved in DMF, R-CDI (5-fold excess) was added, triethylamine (equimolar to the lysine side chain amino group) was added, and the reaction was carried out for 12 hours. The solvent was pumped out with an oil pump, and then dissolved in DMSO, loaded into a 3.5k dialysis bag, dialyzed with deionized water for 24 hours, changing the water every 2 hours, and lyophilized to obtain fixed sequence peptides modified with PEG of different lengths. The DB-modified lysine is denoted as K1, that is, the fixed sequence peptides modified with PEG of different lengths obtained in this example are referred to as mPEG. 22 -(K1K1WK1K1)5, mPEG 45 -(K1K1WK1K1)5 and mPEG 113 -(K1K1WK1K1)5. Its structure was characterized by H NMR spectroscopy (Figure 45), and the peak of the methylene group connected to the lysine side chain and the primary amino group disappeared, indicating that they were completely bonded.

[0288] The structural formula of the fixed sequence polypeptide prepared in this example is as follows:

[0289] (2) The effect of different lengths of PEG on the pKa of sequence peptides.

[0290] Take 100 μL of concentrated hydrochloric acid (37%, 12 mol) and add it to 100 mL of deionized water to fully dissolve to obtain a clear and transparent solution. Dissolve 10 mg of the fixed sequence polypeptide material prepared in step (5) in 10 mL of hydrochloric acid solution, and insert the pH probe into the liquid surface. Under stirring (stirring speed Mot = 3), titrate with 0.5 M sodium hydroxide solution. After the pH meter stabilizes, record the reading until the titration result reaches pH = 11. By taking the derivative of the titration curve, the extreme points are obtained and defined as the protonation rate of 1 and 0 points, and a pH and protonation rate curve is established. According to the definition of the dissociation equilibrium constant (pKa), the pH corresponding to the protonation rate of the polypeptide equal to 0.5 is defined as the pKa of the material. The results are shown in Figure 46. The protonation degree of this type of polypeptide undergoes a significant jump with pH, ​​and the PEG length has little effect on the pKa of the material.

[0291] (III) Cytotoxicity of fixed sequence peptides at different pH values.

[0292] Dissolve 10 mg of peptide in a 5 mL sample vial in 0.3 mL of DMF for further use. Dissolve 0.9 mL of sterile water in a pre-sterilized 25 mL round-bottom flask. Place the vial on a magnetic stirrer at 600 rpm. Add the peptide solution in DMF dropwise to the round-bottom flask using a pipette. Continue stirring at 400 rpm for 15 minutes. Dialyze the vial in 4 L of ultrapure water at 4°C for 24 hours using a 14,000 molecular weight cutoff (MWCO) dialysis bag. Change the water every hour for the first 6 hours and every 6 hours for the next 18 hours to obtain peptide nanoparticles. After dialysis, remove the vial using a pipette, quantify the amount, and store in a refrigerator at 4°C.

[0293] The CCK-8 assay was used to test the tumor cell killing effects of the peptide nanoparticles at pH 7.4 and pH 6.8. DMEM culture medium was adjusted to pH 6.8 using a 6 mol / L HCl solution. Mouse colorectal cancer cells MC38 (purchased from ATCC) were cultured in DMEM culture medium containing 10% (v / v) fetal bovine serum. At pH 7.4 and pH 6.8, different concentrations of the peptide nanoparticles prepared in this example were added to the cells (concentration of 1×10 5 The cells were incubated with 100 μL of CCK-8 solution at 37°C in a CO2 incubator for 24 hours. The absorbance at OD 450 nm was measured using a microplate reader.

[0294] The test results are shown in Figure 47. The DB-modified sequence-fixed polypeptides with different PEG lengths have very low toxicity to tumor cells at pH 7.4 and strong tumor cell toxicity at pH 6.8, that is, they all have good selective killing effects on tumor cells. When the PEG molecular weight is about 5000, that is, y is 113, the tumor cell toxicity of the polypeptide at pH 6.8 is weakened.

[0295] Example 4: 2-(Dibutylamino)ethanol Modified Methoxypolyethylene glycol-Lysine and Tryptophan Fixed Sequence Peptide

[0296] R-OH is 2-(dibutylamino)ethanol (DB), R2 is a tryptophan residue, and y is 45. 45 -COOH and (K) n (W) m mPEG is obtained by reacting amino groups in fixed sequence peptides ,45 -(K) n ( W) mThe tertiary amine was modified onto the side chain of lysine by CDI to obtain a fixed sequence peptide modified with a tertiary amine. The effects of different amino acid arrangements on the pKa and pH selectivity of the fixed sequence peptide were tested.

[0297] (A) Preparation of fixed-sequence peptides modified with 2-(dibutylamino)ethanol.

[0298] The reaction equation is as follows:

[0299] The specific steps are as follows:

[0300] (1) Synthesis of H-[Lys(Boc)] by solid phase synthesis (SPPS) n [Trp(Boc)] m -Resin, each time an amino acid was connected, ninhydrin reagent was used to detect whether the reaction of the amino group was complete. Part of the resin was cleaved with a lysis buffer prepared with 95% TFA, 2.5% H2O, and 2.5% TIS. The relevant structures of the products were characterized by mass spectrometry. The results are shown in Figures 19 and 48-50, indicating that polypeptides with sequences KKWKKKKWKKKKKWKKKKKWKKKWKK, KKKKWKKKKWKKKKWKKKKWKKK, KKKKKWWKKKKKKKKKKKWKKKW, KKKKKWWKKKKKKKKKKWWKKKKK and KKKKKWWKKKKKKKWKKKKKWWKKKKK were obtained.

[0301] (2) Synthesize mPEG by solid phase synthesis (SPPS) 45 -[Lys(Boc)] n [Trp(Boc)] m -Resin, after the reaction is completed, use ninhydrin reagent to detect whether the amino group has reacted completely.

[0302] (3) Cleavage of mPEG using a lysis solution consisting of 95% TFA, 2.5% H2O, and 2.5% TIS 45 -[Lys(Boc)] n [Trp(Boc)] m The sequence peptide was fixed for 3 hours, the lysate was concentrated with nitrogen and precipitated in ice ether, the precipitate was centrifuged and dissolved in deionized water, and dialyzed in deionized water with a 3500 dialysis bag for 24 hours, with the water changed every 2 hours, and white powder mPEG was obtained after freeze-drying. 45 -(K) n (W) m Fixed sequence polypeptide. The specific sequence is mPEG 45 -KKWKKKKWKKKKWKKKKWKKKKWKK,mPEG 45-KKKKWKKKKWKKKKWKKKKWKKKKW,mPEG 45 -KKKKKWWKKKKKKKKKKWWWKKKKK and mPEG 45 -KKKKKWWKKKKKWKKKKKKWWKKKKK, respectively referred to as mPEG 45 -(KKWKK)5,mPEG 45 -(KKKKW)5,mPEG 45 -(K)5(W)2(K) 10 (W)3(K)5 and mPEG 45 -(K)5(W)2(K)5W(K)5(W)2(K)5.

[0303] (4) CDI was placed in a round-bottom flask, and anhydrous dichloromethane was added and stirred to disperse (1.0 g CDI plus 5 mL dichloromethane). The flask was sealed with a rubber stopper. 2-(dibutylamino)ethanol (3 times excess CDI) was slowly added with a syringe. The solution gradually became clear. After reacting for 12 h, deionized water equal to the amount of dichloromethane was added and stirred until no bubbles emerged. The lower dichloromethane phase was removed with a separatory funnel, and anhydrous magnesium sulfate was added to dry for 2 h. The solid was removed by filtration to obtain a solution and dried to obtain 2-(dibutylamino)ethanol-CDI (DB-CDI).

[0304] (5) mPEG 45 -(K) n (W) m The fixed sequence peptide was dissolved in DMF, DB-CDI (5-fold excess) was added, triethylamine (equimolar to the lysine side chain amino group) was added, and the reaction was carried out for 12 hours. The solvent was pumped out with an oil pump, and then dissolved in DMSO. The peptide was loaded into a 3.5k dialysis bag and dialyzed with deionized water for 24 hours, with the water changed every 2 hours. The peptide was lyophilized to obtain a fixed sequence peptide modified with DB. The DB-modified lysine was designated as K1, and the DB-modified fixed sequence peptide obtained in this example was abbreviated as mPEG. 45 -(K1K1WK1K1)5,mPEG 45 -(K1K1K1K1W)5,mPEG 45 -(K1)5(W)2(K1) 10 (W)3(K1)5 and mPEG 45 -(K1)5(W)2(K1)5W(K1)5(W)2(K1)5. Its structure was characterized by H NMR spectroscopy (Figure 51). The peak of the methylene group connected to the lysine side chain and the primary amino group disappeared, indicating that they were completely bonded.

[0305] The structural formula of the fixed sequence polypeptide prepared in this example is as follows:

[0306] (2) The effect of different arrangements of amino acids on the pKa of sequence polypeptides.

[0307] Take 100 μL of concentrated hydrochloric acid (37%, 12 mol) and add it to 100 mL of deionized water, and dissolve it thoroughly to obtain a clear and transparent solution. Use 10 mL of hydrochloric acid solution to dissolve 10 mg of the fixed sequence polypeptide material prepared in step (5), and insert the pH probe into the liquid surface. Under stirring (stirring speed Mot = 3), titrate with 0.5 M sodium hydroxide titrant. After the pH meter stabilizes, record the reading until the titration result reaches pH = 11. By deriving the titration curve, the extreme points are obtained, which are defined as the protonation rate of 1 and 0 points, and a pH and protonation rate curve is established. According to the definition of the dissociation equilibrium constant (pKa), the pH corresponding to the protonation rate of the polypeptide equal to 0.5 is defined as the pKa of the material. The results are shown in Figure 52. The different arrangements of the sequences have little effect on their protonation degree.

[0308] (III) Cytotoxicity of fixed sequence peptides at different pH values.

[0309] Dissolve 10 mg of peptide in a 5 mL sample vial in 0.3 mL of DMF for further use. Dissolve 0.9 mL of sterile water in a pre-sterilized 25 mL round-bottom flask. Place the vial on a magnetic stirrer at 600 rpm. Add the peptide solution in DMF dropwise to the round-bottom flask using a pipette. Continue stirring at 400 rpm for 15 minutes. Dialyze the vial in 4 L of ultrapure water at 4°C for 24 hours using a 14,000 molecular weight cutoff (MWCO) dialysis bag. Change the water every hour for the first 6 hours and every 6 hours for the next 18 hours to obtain peptide nanoparticles. After dialysis, remove the vial using a pipette, quantify the amount, and store in a refrigerator at 4°C.

[0310] The CCK-8 assay was used to test the tumor cell killing effects of the peptide nanoparticles at pH 7.4 and pH 6.8. DMEM culture medium was adjusted to pH 6.8 using a 6 mol / L HCl solution. Mouse colorectal cancer cells MC38 (purchased from ATCC) were cultured in DMEM culture medium containing 10% (v / v) fetal bovine serum. At pH 7.4 and pH 6.8, different concentrations of the peptide nanoparticles prepared in this example were added to the cells (concentration of 1×10 5 The cells were incubated with 100 μL of CCK-8 solution at 37°C in a CO2 incubator for 24 hours. The absorbance at OD 450 nm was measured using a microplate reader.

[0311] The test results are shown in FIG53 , which indicate that different arrangements (sequences) of lysine and tryptophan have little effect on the cytotoxicity and selectivity of the polypeptide.

[0312] Example 5: Tertiary amine-modified methoxypolyethylene glycol-lysine and phenylalanine fixed sequence polypeptide

[0313] R-OH is 2-(dibutylamino)ethanol (DB) or (S)-3-dimethylamino-3-phenylpropanol (DMP2), R2 is a phenylalanine residue, and y is 45. 45 -COOH and (K) n (F) m mPEG is obtained by reacting amino groups in fixed sequence peptides 45 -(K) n (F) m The tertiary amine was modified onto the side chain of lysine by CDI to obtain a fixed sequence peptide modified with a tertiary amine. The effect of replacing the hydrophobic amino acid with phenylalanine on the pKa and pH selectivity of the fixed sequence peptide was tested.

[0314] (A) Preparation of tertiary amine-modified fixed-sequence peptides.

[0315] The reaction equation is as follows:

[0316] The specific steps are as follows:

[0317] (1) Synthesis of H-[Lys(Boc)] by solid phase synthesis (SPPS) n F m -Resin, each time an amino acid was connected, ninhydrin reagent was used to detect whether the reaction of the amino group was complete. Part of the resin was cleaved with a cleavage solution prepared with 95% TFA, 2.5% H2O, and 2.5% TIS. After cleavage, mass spectrometry was performed. The results are shown in Figures 54 to 57, indicating that polypeptides with sequences of KKKFKKKKKKFKKK, KKFKKKKKFKKKKFK, KFKKFKKFKKKFKKK, KFKKFKKFKKFKKKFK, KFKFKFKKFKKFKKFK, KFKFKFKFKFKFKFKFK and KFFKKFFKKFFKKKF were obtained.

[0318] (2) Synthesize mPEG by solid phase synthesis (SPPS) 45 -[Lys(Boc)] n F m -Resin, after the reaction is completed, use ninhydrin reagent to detect whether the amino group has reacted completely.

[0319] (3) Cleavage of mPEG using a lysis solution consisting of 95% TFA, 2.5% H2O, and 2.5% TIS 45-[Lys(Boc)] n F m The sequence peptide was fixed for 3 hours, the lysate was concentrated with nitrogen and precipitated in ice ether, the precipitate was centrifuged and dissolved in deionized water, and dialyzed in deionized water with a 3500 dialysis bag for 24 hours, with the water changed every 2 hours, and white powder mPEG was obtained after freeze-drying. 45 -(K) n (F) m Fixed sequence polypeptide. The specific sequence is mPEG 45 -KKKFKKKKKKFKKKK, mPEG 45 -KKFKKKKFKKKKFKK, mPEG 45 -KFKKFKKFKKFKKKK, mPEG 45 -KFKKFKKFKKFKKFK, mPEG 45 -KFKFKFKFKFKFKFK, mPEG 45 -KFFKKFFKKFFKKKF and mPEG 45 -(KKKFKKK)2K,mPEG 45 -(KKFKK)3,mPEG 45 -(KFK)4KKK, mPEG 45 -(KFK)5,mPEG 45 -(KF)7K and mPEG 45 -(KFFK)3KKF.

[0320] (4) CDI was placed in a round-bottom flask, and anhydrous dichloromethane was added and stirred to disperse (1.0 g CDI plus 5 mL dichloromethane). The flask was sealed with a rubber stopper, and R-OH (3 times excess CDI) was slowly added with a syringe. The solution gradually became clear. After reacting for 12 h, deionized water equal to the amount of dichloromethane was added and stirred until no bubbles emerged. The lower dichloromethane phase was removed with a separatory funnel, and anhydrous magnesium sulfate was added to dry for 2 h. The solid was removed by filtration to obtain a solution and dried. R-CDI was obtained.

[0321] (5) mPEG 2k -(K) n (F) m The fixed sequence polypeptide was dissolved in DMF, R-CDI (5-fold excess) was added, triethylamine (equimolar to the lysine side chain amino group) was added, and the reaction was carried out for 12 hours. The solvent was drained with an oil pump, and then dissolved in DMSO. The solution was loaded into a 3.5k dialysis bag and dialyzed with deionized water for 24 hours, with the water changed every 2 hours. The solution was lyophilized to obtain a fixed sequence polypeptide modified with DB or DMP2. The DB-modified lysine was designated as K1, and the DMP2-modified lysine was designated as K9. The fixed sequence polypeptide obtained in this example is referred to as mPEG.45 -(K1K1K1FK1K1K1)2K1,mPEG 45 -(K1K1FK1K1)3,mPEG 45 -(K1FK1)4K1K1K1,mPEG 45 -(K1FK1)5,mPEG 45 -(K9FK9)5,mPEG 45 -(K9F)7K9 and mPEG 45 The structure of the lysine (K9FFK9)3K9K9F was characterized by H NMR spectroscopy (Figures 58-59). For DB-modified lysine, the peaks for the methylene groups connecting the lysine side chain to the primary amino group disappeared, indicating complete bonding. For DMP2-modified lysine, l+m+n:q=K:F, indicating complete bonding.

[0322] The structural formula of the fixed sequence polypeptide prepared in this example is as follows:

[0323] (2) The effects of tertiary amine modification of lysine side chains and different hydrophobic amino acids on the pKa of sequence peptides.

[0324] Take 100 μL of concentrated hydrochloric acid (37%, 12 mol) and add it to 100 mL of deionized water to fully dissolve to obtain a clear and transparent solution. Use 10 mL of hydrochloric acid solution to dissolve 10 mg of the fixed sequence polypeptide material prepared in step (5), and insert the pH probe into the liquid surface. Under stirring (stirring speed Mot = 3), titrate with 0.5 M sodium hydroxide titrant. After the pH meter stabilizes, record the reading until the titration result reaches pH = 11. By deriving the titration curve, the extreme points are obtained and defined as the protonation rate of 1 and 0 points, and a pH and protonation rate curve is established. According to the definition of the dissociation equilibrium constant (pKa), the pH corresponding to the protonation rate of the polypeptide equal to 0.5 is defined as the pKa of the material. The results are shown in Figures 60 and 61. The protonation degree of this type of polypeptide undergoes a significant jump with pH.

[0325] (III) Cytotoxicity of fixed sequence peptides at different pH values.

[0326] Dissolve 10 mg of peptide in a 5 mL sample vial in 0.3 mL of DMF for further use. Dissolve 0.9 mL of sterile water in a pre-sterilized 25 mL round-bottom flask. Place the vial on a magnetic stirrer at 600 rpm. Add the peptide solution in DMF dropwise to the round-bottom flask using a pipette. Continue stirring at 400 rpm for 15 minutes. Dialyze the vial in 4 L of ultrapure water at 4°C for 24 hours using a 14,000 molecular weight cutoff (MWCO) dialysis bag. Change the water every hour for the first 6 hours and every 6 hours for the next 18 hours to obtain peptide nanoparticles. After dialysis, remove the vial using a pipette, quantify the amount, and store in a refrigerator at 4°C.

[0327] The CCK-8 assay was used to test the tumor cell killing effects of the peptide nanoparticles at pH 7.4 and pH 6.8. DMEM culture medium was adjusted to pH 6.8 using a 6 mol / L HCl solution. Mouse colorectal cancer cells MC38 (purchased from ATCC) were cultured in DMEM culture medium containing 10% (v / v) fetal bovine serum. At pH 7.4 and pH 6.8, different concentrations of the peptide nanoparticles prepared in this example were added to the cells (concentration of 1×10 5 The cells were incubated with 100 μL of CCK-8 solution at 37°C in a CO2 incubator for 24 hours. The absorbance at OD 450 nm was measured using a microplate reader.

[0328] The test results are shown in Figures 62 and 63. For peptides with DB-modified lysine and phenylalanine sequences, the toxicity of the peptides to tumor cells decreased with increasing phenylalanine ratios, but none of the peptides exhibited pH-selective killing. For peptides with DMP2-modified lysine and phenylalanine sequences, the toxicity at pH 7.4 decreased with increasing phenylalanine ratios, but all three DMP2-modified peptides showed relatively weak toxicity at pH 6.8.

[0329] The above-described embodiments merely illustrate several implementations of the present invention, and while their descriptions are relatively specific and detailed, they should not be construed as limiting the scope of the present invention. It should be noted that a person skilled in the art would be able to make numerous variations and improvements without departing from the spirit of the present invention, all of which fall within the scope of protection of the present invention. Therefore, the scope of protection of the present invention shall be determined by the appended claims.

Claims

1. A membrane-permeable polypeptide having a structure represented by formula (I) or a stereoisomer thereof or a pharmaceutically acceptable salt thereof: in, Q is a polypeptide group composed of n A's and m B's arranged in any sequence; B is the residue after removing one amino hydrogen and one hydroxyl group from an amino acid with a hydrophobic side chain; A is the residue after removing one amino hydrogen and one hydroxyl group from lysine with a modified group, and its structure is: R is selected from: -R3-N(R4R5), -R3-R', R' is selected from: L is selected from: -NH-C(=O)O-, -NH-C(=O)-, -C(=O)-NH-, -C(=O)-O-; R1 is selected from: alkylene; R3 is selected from: alkylene, C6-C 14 Aryl-substituted alkylene; R4 and R5 are independently selected from: alkyl, C6-C 14 an alkyl group substituted with an aryl group, or R3, R4 and the nitrogen atom to which they are attached together form a heterocycloalkyl group; R6 is selected from: C1-C 15 Alkyl, C6-C 14 Aryl, C6-C 14 Aryl substituted C1-C 15 alkyl; y is selected from: 2-150; n+m is greater than 0, and n is not 0; q is selected from: 0, 1, 2, 3, 4.

2. The membrane permeabilization sequence polypeptide or its stereoisomer or its pharmaceutically acceptable salt according to claim 1, characterized in that: The structural formula of the membrane-permeabilizing sequence polypeptide is shown in formula (II): Among them, Q1 is a polypeptide group composed of n1 A and m1 B arranged in any sequence; n1+m1 is greater than 0, and n1 is not 0; x1 is greater than 0; The product of n1 and x1 is n; the product of m1 and x1 is m.

3. The membrane permeabilization sequence polypeptide or its stereoisomer or its pharmaceutically acceptable salt according to claim 1, characterized in that: The structural formula of the membrane-permeabilizing sequence polypeptide is shown in formula (III): Wherein, Q2 is a polypeptide group composed of n2 A's and m2 B's arranged in any sequence; Q3 is a polypeptide group composed of n3 A's and m3 B's arranged in any sequence; n2+n3+m2+m3 is greater than 0, and n2+n3 is not 0; The sum of the product of n2 and x2 plus n3 is n, and the sum of the product of m2 and x2 plus m3 is m.

4. The membrane permeabilization sequence polypeptide or its stereoisomer or its pharmaceutically acceptable salt according to any one of claims 1 to 3, characterized in that: B is R2 is selected from: C1-C 12 Alkyl, C6-C 14 Aryl, C6-C 14 Aryl substituted C1-C 12 Alkyl, benzyloxycarbonyl substituted C1-C 12 Alkyl, 5-10 membered heteroaryl substituted C1-C 12 alkyl.

5. The membrane permeabilization sequence polypeptide or its stereoisomer or its pharmaceutically acceptable salt according to claim 4, characterized in that: R2 is selected from the group consisting of: C1-C8 alkyl, phenyl, naphthyl, C1-C6 alkyl substituted by phenyl, C1-C6 alkyl substituted by benzyloxycarbonyl, and C1-C6 alkyl substituted by 5-10 membered heteroaryl.

6. The membrane permeabilization sequence polypeptide or its stereoisomer or its pharmaceutically acceptable salt according to claim 5, characterized in that: R2 is selected from the group consisting of methyl, ethyl, n-propyl, isopropyl, n-butyl, isobutyl, pentyl, hexyl, heptyl, octyl, nonyl, decyl, undecyl, dodecyl, phenyl, naphthyl, benzyl, ethyl substituted with benzyloxycarbonyl, ethyl substituted with benzopyrrole, or their chiral isomers.

7. The membrane permeabilization sequence polypeptide or its stereoisomer or its pharmaceutically acceptable salt according to any one of claims 1 to 3, characterized in that: R1 is selected from: C1-C6 alkylene.

8. The membrane permeabilization sequence polypeptide or its stereoisomer or its pharmaceutically acceptable salt according to claim 7, characterized in that: R1 is selected from: -(CH2) p -, wherein p is selected from: 1, 2, 3, 4, 5, 6.

9. The membrane permeabilization sequence polypeptide or its stereoisomer or its pharmaceutically acceptable salt according to any one of claims 1 to 3, characterized in that: A is: or or their chiral isomers; Wherein, X is: -O- or none.

10. The membrane permeabilization sequence polypeptide or its stereoisomer or its pharmaceutically acceptable salt according to any one of claims 1 to 3, characterized in that: R3 is selected from: C1-C6 alkylene, phenyl-substituted C1-C6 alkylene.

11. The membrane permeabilization sequence polypeptide or its stereoisomer or its pharmaceutically acceptable salt according to claim 10, characterized in that: R3 is selected from: -(CH2) p -, phenyl-substituted-(CH2) p -; wherein p is selected from: 1, 2, 3, 4, 5, 6.

12. The membrane permeabilization sequence polypeptide or its stereoisomer or its pharmaceutically acceptable salt according to any one of claims 1 to 11, characterized in that: R is selected from: -R3-N(R4R5), 13. The membrane permeabilization sequence polypeptide or its stereoisomer or its pharmaceutically acceptable salt according to any one of claims 1 to 11, characterized in that: R4 and R5 are independently selected from: C1-C6 alkyl, phenyl-substituted C1-C6 alkyl, naphthyl-substituted C1-C6 alkyl, or R4, R5 and the nitrogen atom to which they are connected form a 5-10 membered heterocycloalkyl.

14. The membrane permeabilization sequence polypeptide or its stereoisomer or its pharmaceutically acceptable salt according to claim 13, characterized in that: R4 and R5 are independently selected from: C1-C4 alkyl, C1-C3 alkyl substituted by phenyl, C1-C3 alkyl substituted by naphthyl, or R4, R5 and the nitrogen atom to which they are connected form a 5-8 membered heterocycloalkyl.

15. The membrane permeabilization sequence polypeptide or its stereoisomer or its pharmaceutically acceptable salt according to claim 14, characterized in that: R4 and R5 are independently selected from: C3 alkyl, C4 alkyl, C5 alkyl, or R4, R5 and the nitrogen atom to which they are connected form a 7-membered heterocycloalkyl.

16. The membrane permeabilization sequence polypeptide or its stereoisomer or its pharmaceutically acceptable salt according to claim 14, characterized in that: R4, R5 and the nitrogen atom to which they are connected together form the following group:

17. The membrane permeabilization sequence polypeptide or its stereoisomer or its pharmaceutically acceptable salt according to any one of claims 1 to 11, characterized in that: R6 is selected from: C1-C6 alkyl, phenyl, naphthyl, phenyl-substituted C1-C6 alkyl; Preferably, R6 is selected from: C1-C3 alkyl, phenyl, naphthyl, benzyl.

18. The membrane permeabilization sequence polypeptide or its stereoisomer or its pharmaceutically acceptable salt according to any one of claims 1 to 11, characterized in that: R is selected from: -R3-N(R4R5), Wherein, R3 is selected from: -CH2-CH2-, -CH2-CH2-CH2-, -CH2-(CH2)3-CH2-, R4, R5 and the nitrogen atom to which they are connected together form the following group: R6 is benzyl.

19. The membrane permeabilization sequence polypeptide or its stereoisomer or its pharmaceutically acceptable salt according to any one of claims 4 to 11, characterized in that: R is selected from: -R3-N(R4R5); Wherein, R3 is selected from: -CH2-CH2-; R4, R5 and the nitrogen atom connected thereto together form the following group: R2 is selected from: or a chiral isomer thereof.

20. The membrane permeabilization sequence polypeptide or its stereoisomer or its pharmaceutically acceptable salt according to any one of claims 1 to 11, characterized in that: y is selected from: 10-120, preferably 20-120, preferably 20-50, 20-25, 40-48 or 108-116, more preferably 20, 21, 22, 23, 24, 43, 44, 45, 46, 111, 112, 113, 114.

21. The membrane permeabilization sequence polypeptide according to any one of claims 1 and 4 to 11, or a stereoisomer thereof, or a pharmaceutically acceptable salt thereof, characterized in that: n+m is not less than 5, preferably not less than 9, preferably not less than 12, preferably not less than 15, preferably 6-200, more preferably 6-150, more preferably 6-110; more preferably 6-20 or 5-40 or 10-30; more preferably 13-30, 13-28, 13-21, or 15-25, and can be 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29 or 30.

22. The membrane permeabilization sequence polypeptide according to any one of claims 1 and 4 to 11, or a stereoisomer thereof, or a pharmaceutically acceptable salt thereof, characterized in that: n is 5-30, m is 0-15 or 1-15; Preferably, n is 10-20 and m is 2-10.

23. The membrane permeabilization sequence polypeptide according to any one of claims 1 and 4 to 11, or a stereoisomer thereof, or a pharmaceutically acceptable salt thereof, characterized in that: n:m=1-10:1, preferably 1-5:1, more preferably 1.1-1.2:1, 2:1, 3:1 or 4:

1.

24. The membrane permeabilization sequence polypeptide according to any one of claims 2 and 4 to 11, or a stereoisomer thereof, or a pharmaceutically acceptable salt thereof, characterized in that: x1 is 1-30, preferably 3, 4, 5, 6, 7, 8, 9, or 10.

25. The membrane permeabilization sequence polypeptide according to any one of claims 2 and 4 to 11, or a stereoisomer thereof, or a pharmaceutically acceptable salt thereof, characterized in that: The product of (n1+m1) and x1 is not less than 5, preferably not less than 9, preferably not less than 15, preferably 6-200, more preferably 6-150, more preferably 6-110; more preferably 6-20, 5-40, 9-30, or 15-28, more preferably 15-25, more preferably 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29 or 30.

26. The membrane permeabilization sequence polypeptide according to any one of claims 2 and 4 to 11, or a stereoisomer thereof, or a pharmaceutically acceptable salt thereof, characterized in that: The product of n1 and x1 is 5-30, and the product of m1 and x1 is 0-15 or 1-15; Preferably, the product of n1 and x1 is 10-20, and the product of m1 and x1 is 3-7.

27. The membrane permeabilization sequence polypeptide according to any one of claims 2 and 4 to 11, or a stereoisomer thereof, or a pharmaceutically acceptable salt thereof, characterized in that: n1:m1=1-10:1, preferably 2-4:1, more preferably 2:1, 3:1 or 4:

1.

28. The membrane permeabilization sequence polypeptide according to any one of claims 2 and 4 to 11, or a stereoisomer thereof, or a pharmaceutically acceptable salt thereof, characterized in that: n1+m1=2-10, preferably 3, 4, 5, 6.

29. The membrane permeabilization sequence polypeptide or its stereoisomer or its pharmaceutically acceptable salt according to any one of claims 3 to 11, characterized in that: x2 is 1-30, preferably 3, 4, 5, 6, 7, 8, 9, 10.

30. The membrane permeabilization sequence polypeptide or its stereoisomer or its pharmaceutically acceptable salt according to any one of claims 3 to 11, characterized in that: The product of (n2+m2) and x2 plus the sum of n3 and m3 is not less than 5, preferably not less than 9, preferably not less than 12, preferably not less than 15, preferably 6-200, more preferably 6-150, more preferably 6-110; more preferably 6-20 or 5-40 or 10-30, more preferably 13-21, more preferably 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25.

31. The membrane permeabilization sequence polypeptide or its stereoisomer or its pharmaceutically acceptable salt according to any one of claims 3 to 11, characterized in that: (The product of n2 and x2 plus the sum of n3): (the product of m2 and x2 plus the sum of m3) = 1-10:1, preferably 1-4:1, and more preferably 1.1-1.2:

1.

32. The membrane permeabilization sequence polypeptide or its stereoisomer or its pharmaceutically acceptable salt according to any one of claims 3 to 11, characterized in that: m3 is 0, n3 is 1, the product of n2 and x2 is 6-12, and the product of m2 and x2 is 6-12; Preferably, the product of n2 and x2 is 6, 8, or 10, and the product of m2 and x2 is 6, 8, or 10.

33. The membrane permeabilization sequence polypeptide or its stereoisomer or its pharmaceutically acceptable salt according to claim 1, characterized in that: The membrane permeabilization sequence polypeptide is selected from:

34. A membrane-permeabilizing sequence polypeptide nanoparticle, characterized in that: It is formed by self-assembly of the membrane-permeable sequence polypeptide according to any one of claims 1 to 33 in an aqueous medium.

35. A method for preparing membrane-permeabilizing sequence polypeptide nanoparticles according to claim 34, characterized in that: The method comprises the following steps: dissolving the membrane-permeable sequence polypeptide in an organic solvent or a hydrochloric acid solution with a pH of 1.5-2.5, then adding the obtained solution dropwise into water under stirring, continuing stirring, and dialyzing to remove the solvent, thereby obtaining the membrane-permeable sequence polypeptide nanoparticles; Preferably, the organic solvent is N,N-dimethylformamide; Preferably, the ratio of the membrane permeabilization sequence polypeptide, the organic solvent or hydrochloric acid solution, and the water is 20 mg to 40 mg: 1 mL: 2 mL to 5 mL; Preferably, the preparation method of the membrane-breaking sequence polypeptide nanoparticles comprises the following steps: dissolving the membrane-breaking sequence polypeptide in N,N-dimethylformamide at a ratio of 10 mg to 30 mg: 1 mL, then dropwise adding the resulting solution into 2 mL to 5 mL of water under stirring at a speed of 400 to 800 rpm, continuing to stir at a speed of 200 to 600 rpm for 8 to 20 minutes, dialyzing in water using a dialysis bag with a molecular weight cutoff of 10,000 to 20,000 to remove the solvent, and obtaining the membrane-breaking sequence polypeptide nanoparticles. Column polypeptide nanoparticles.

36. Use of the membrane permeabilization sequence polypeptide according to any one of claims 1 to 33, or a stereoisomer thereof, or a pharmaceutically acceptable salt thereof in the preparation of a drug for preventing and / or treating tumors.

37. Use of the membrane-permeabilizing sequence polypeptide nanoparticles according to claim 34 in the preparation of drugs for preventing and / or treating tumors.

38. The use according to any one of claims 36-37, characterized in that: The tumor is pancreatic cancer, melanoma, colorectal cancer, colon cancer, lung cancer, tongue squamous cell carcinoma, cervical cancer, ovarian cancer, osteosarcoma, liver cancer, breast cancer, bladder cancer, and ovarian epithelial cancer.

39. A drug for preventing and / or treating tumors, characterized in that: It is prepared from active ingredients and pharmaceutically acceptable excipients, wherein the active ingredient includes the membrane-destroying sequence polypeptide or its stereoisomer or its pharmaceutically acceptable salt according to any one of claims 1 to 33, and / or the membrane-destroying sequence polypeptide nanoparticles according to claim 34.

40. A method for preventing and / or treating tumors, characterized in that: The method comprises: administering a safe and effective amount of any one of 1-33 membrane permeabilization sequence polypeptides or stereoisomers thereof or pharmaceutically acceptable salts thereof; and / or, Administering a safe and effective amount of the membrane permeabilization sequence polypeptide nanoparticles according to claim 34; and / or, Administer a safe and effective amount of the drug of claim 39.

41. The method according to claim 40, characterized in that The tumors are: pancreatic cancer, melanoma, colorectal cancer, colon cancer, lung cancer, tongue squamous cell carcinoma, cervical cancer, ovarian cancer, osteosarcoma, liver cancer, breast cancer, bladder cancer, and ovarian epithelial cancer.

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