PH-sensitive membrane cracking material based on sequence immobilized polylysine and application of pH-sensitive membrane cracking material

Abstract

The invention provides a pH-sensitive membrane cracking material with a structure as shown in a formula (I), or a stereoisomer or a pharmaceutically acceptable salt thereof, and an application of the pH-sensitive membrane cracking material or the stereoisomer or the pharmaceutically acceptable salt thereof. The macromolecular material is hydrophobic and electrically neutral under normal physiological pH, can be self-assembled into nano-particles, is compact in assembly, and has weak interaction with cell membranes; under the condition of slightly acidic pH, the amphipathic polymer can be protonated to form an amphipathic structure consisting of a hydrophobic structural domain and a cationic structural domain, has strong interaction with cell membranes and has strong membrane rupture and membrane lysis activity, so that the amphipathic polymer can efficiently and selectively kill tumor cells. # imgabs0 #

Description

pH-sensitive membrane lysis materials based on sequence-immobilized polylysine 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 lysis material based on sequence-fixed polylysine 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 lysis material based on sequence-fixed polylysine with a side group containing a tertiary amine. This macromolecular material is hydrophobic and electrically neutral at normal physiological pH, and has a weak interaction with the cell membrane; 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 a strong interaction with the cell membrane and has membrane lysis activity, thereby being able to kill tumor cells efficiently and selectively. In addition, the polypeptide in the pH-sensitive membrane lysis material can be synthesized using a solid-phase synthesis method, and the sequence and number of polypeptide amino acids in the resulting material can be kept fixed. The present invention includes the following technical solutions.

[0006] A pH-sensitive membrane lysis material having a structure represented by formula (I) or a stereoisomer thereof or a pharmaceutically acceptable salt thereof:

[0007] Wherein, R is selected from: -R3-N(R4R5), -R3-R',

[0008] R' is selected from:

[0009] L is selected from: -NH-C(=O)O-, -NH-C(=O)-;

[0010] R3 is selected from: alkylene, C6-C 14 Aryl-substituted alkylene;

[0011] 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;

[0012] R6 is selected from: C1-C 15 Alkyl, C6-C 14 Aryl, C6-C 14 Aryl-substituted C1-C 15 alkyl;

[0013] y is selected from: an integer between 2 and 150;

[0014] n is selected from: an integer between 6 and 30;

[0015] q is selected from: 0, 1, 2, 3, 4.

[0016] On the other hand, the present invention also provides a pH-sensitive membrane lysis nanoparticle, including the following technical solution.

[0017] The invention discloses a pH-sensitive membrane-lysis nanoparticle, which is formed by self-assembly of the pH-sensitive membrane-lysis material in an aqueous medium.

[0018] On the other hand, the present invention also provides a method for preparing pH-sensitive membrane lysis nanoparticles, which includes the following technical solutions.

[0019] A method for preparing pH-sensitive membrane lysis nanoparticles is characterized in that it includes the following steps: dissolving the pH-sensitive membrane lysis material 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 pH-sensitive membrane lysis nanoparticles.

[0020] On the other hand, the present invention also provides applications of the above-mentioned pH-sensitive membrane lysis material or pH-sensitive membrane lysis nanoparticles, including the following technical solutions.

[0021] Use of the pH-sensitive membrane lysis material or its stereoisomer or its pharmaceutically acceptable salt in the preparation of drugs for preventing and / or treating tumors.

[0022] Application of the pH-sensitive membrane-lysis nanoparticles in the preparation of drugs for preventing and / or treating tumors.

[0023] On the other hand, the present invention also provides a drug for preventing and / or treating tumors, including the following technical solutions.

[0024] A drug for preventing and / or treating tumors is prepared from active ingredients and pharmaceutically acceptable excipients, wherein the active ingredients include the pH-sensitive membrane-lytic material or its stereoisomers or pharmaceutically acceptable salts described in the present invention, and / or the pH-sensitive membrane-lytic nanoparticles described in the present invention.

[0025] On the other hand, the present invention also provides a method for preventing and / or treating tumors, including the following technical solutions.

[0026] A method for preventing and / or treating tumors, comprising: administering a safe and effective amount of the pH-sensitive membrane lysis material of the present invention, or a stereoisomer thereof, or a pharmaceutically acceptable salt thereof; and / or,

[0027] administering a safe and effective amount of the pH-sensitive membrane-lytic nanoparticles of the present invention; and / or,

[0028] Administer a safe and effective amount of the drug of the present invention.

[0029] The present invention provides a tertiary amine-modified pH-sensitive membrane lysis material based on sequence-fixed polylysine. The macromolecular material is hydrophobic and electrically neutral at normal physiological pH, and has a weak interaction with cell membranes, 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 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-breaking and membrane-lysis activity, thereby being able to kill tumor cells efficiently and highly selectively. The pH-sensitive membrane lysis 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 sequence polypeptide has the advantages of degradability and the degradation products are non-biotoxic, it will have a wider range of biomedical applications. BRIEF DESCRIPTION OF THE DRAWINGS

[0030] Figure 1 shows mPEG 45 -K n -C6P H NMR spectrum.

[0031] Figure 2 shows mPEG 45 -K 12 -C6P mass spectrum.

[0032] Figure 3 shows mPEG 45 -K 13 -C6P mass spectrum.

[0033] Figure 4 shows mPEG 45 -K 14 -C6P mass spectrum.

[0034] Figure 5 shows mPEG 45 -K 15 -C6P mass spectrum.

[0035] Figure 6 shows mPEG 45 -K 16 -C6P mass spectrum.

[0036] Figure 7 shows mPEG 45 -K 18 -C6P mass spectrum.

[0037] Figure 8 shows mPEG 45 -K 20 -C6P mass spectrum.

[0038] Figure 9 shows mPEG 45 -K n -C6P protonation rate as a function of pH.

[0039] Figure 10 shows mPEG 45 -K12 -Secondary structure curves of C6P at different pH values.

[0040] Figure 11 shows mPEG 45 -K 13 -Secondary structure curves of C6P at different pH values.

[0041] Figure 12 shows mPEG 45 -K 14 -Secondary structure curves of C6P at different pH values.

[0042] Figure 13 shows mPEG 45 -K 15 -Secondary structure curves of C6P at different pH values.

[0043] Figure 14 shows mPEG 45 -K 16 -Secondary structure curves of C6P at different pH values.

[0044] Figure 15 shows mPEG 45 -K 18 -Secondary structure curves of C6P at different pH values.

[0045] Figure 16 shows mPEG 45 -K 20 -Secondary structure curves of C6P at different pH values.

[0046] Figure 17 shows mPEG 45 -K 14 -Transmission electron microscopy image of C6P at pH = 6.8.

[0047] Figure 18 shows mPEG 45 -K 14 -Transmission electron microscopy image of C6P at pH = 7.4.

[0048] Figure 19 shows mPEG 45 -K 14 -C5P2 H NMR spectrum.

[0049] Figure 20 shows mPEG 45 -K 14 -DMP H NMR spectrum.

[0050] Figure 21 shows mPEG 45 -K 14 -DB H NMR spectrum.

[0051] Figure 22 shows mPEG 45 -K 14 -C5P H NMR spectrum.

[0052] Figure 22 shows mPEG45 -K 14 -C5P and mPEG 45 -K 14 -DB protonation rate as a function of pH.

[0053] Figure 24 shows mPEG 45 -K 14 -Secondary structure curves of C5P2 at different pH values.

[0054] Figure 25 shows mPEG 45 -K 14 -Secondary structure curves of DMP at different pH values.

[0055] Figure 26 shows mPEG 45 -K 14 -DB secondary structure curves at different pH.

[0056] Figure 27 shows mPEG 45 -K 14 -Secondary structure curves of C5P at different pH values.

[0057] Figure 28 shows mPEG 22 -K 14 -C6P H NMR spectrum.

[0058] Figure 29 shows mPEG 113 -K 14 -C6P H NMR spectrum.

[0059] Figure 30 shows mPEG 22 -K 14 -C6P and mPEG 113 -K 14 -C6P protonation rate as a function of pH.

[0060] Figure 31 is mPEG 22 -K 14 -Secondary structure curves of C6P at different pH values.

[0061] Figure 32 shows mPEG 113 -K 14 -Secondary structure curves of C6P at different pH values.

[0062] Figure 33 is mPEG 45 -K n -C6P cytotoxicity to MC38 cells at pH 7.4.

[0063] Figure 34 shows mPEG 45 -K n -C6P cytotoxicity to MC38 cells at pH 6.8.

[0064] Figure 35 is mPEG 45 -K 14 -C5P cytotoxicity to MC38 cells at pH 7.4 and 6.8.

[0065] Figure 36 is mPEG 45 -K 14 -DB cytotoxicity to MC38 cells at pH 7.4 and 6.8.

[0066] Figure 37 is mPEG 22 -K 14 -C6P cytotoxicity to MC38 cells at pH 7.4 and 6.8.

[0067] Figure 38 is mPEG 113 -K 14 -C6P cytotoxicity to MC38 cells at pH 7.4 and 6.8.

[0068] Figure 39 shows the effect of endocytic pathway inhibitor wortmannin on mPEG 45 -K 14 -C6P cytotoxicity.

[0069] Figure 40 shows the effect of methyl-β-cyclodextrin on mPEG endocytosis pathway inhibitor 45 -K 14 -C6P cytotoxicity.

[0070] Figure 41 shows the effect of chlorpromazine hydrochloride, an inhibitor of the endocytic pathway, on mPEG 45 -K 14 -C6P cytotoxicity.

[0071] Figure 42 shows the effect of chloroquine, a lysosomal inhibitor, on mPEG 45 -K 14 -C6P cytotoxicity.

[0072] Figure 43 shows the effect of lysosomal inhibitor bafilomycin A1 on mPEG 45 -K 14 -C6P cytotoxicity.

[0073] Figure 44 shows the effect of lysosomal inhibitor Concanavalin A on mPEG 45 -K 14 -C6P cytotoxicity.

[0074] Figure 45 is mPEG 45 -K 14 -C6P destruction of liposomes loaded with fluorescent dye ANTS and quencher DPX at different pH.

[0075] Figure 46 shows mPEG 45 -K 14 -Maximum tolerated dose of C6P in ICR mice.

[0076] Figure 47 shows the effect of MC38 cells on mPEG at 37°C or 4°C. 45 -K 14 -Uptake of C6P.

[0077] Figure 48 is mPEG 45 -K 14 - Colocalization of C6P with lysosomes at pH 7.4 or pH 6.8.

[0078] Figure 49 is mPEG 45 -K 14 - Colocalization of Gal3-GFP puncta with LAMP1-mCherry at pH 7.4 or pH 6.8 in C6P.

[0079] Figure 50 shows mPEG under pH 6.8 conditions 45 -K 14 -C6P treatment of MC38 cells and the localization of CTSB-mCherry in the cells.

[0080] Figure 51 is mPEG 45 -K 14 -C6P treatment of MC38 cells at pH 6.8 resulted in the extracellular release of LDH.

[0081] Figure 52 shows mPEG 45 -K 14 -C6P treatment of MC38 cells at pH 6.8 resulted in the release of ATP into the extracellular space.

[0082] Figure 53 is mPEG 45 -K 14 -C6P treatment of MC38 cells at pH 6.8 resulted in the extracellular release of HMGB1.

[0083] Figure 54 shows the observation of mPEG using a laser confocal microscope. 45 -K 14 -C6P treatment and calreticulin expression in tumor cells.

[0084] Figure 55 shows Panc02 mice injected with mPEG 45 -K 14 -Subcutaneous tumor growth curve after intravenous C6P treatment.

[0085] Figure 56 shows Panc02 mice injected with mPEG 45 -K14 -Statistical results of tumor mass after C6P treatment.

[0086] Figure 57 shows Panc02 mice injected with mPEG 45 -K 14 -Monitoring endpoint after C6P treatment: photograph the tumor in vitro.

[0087] Figure 58 shows Panc02 mice injected with mPEG 45 -K 14 -Weight change curve after C6P treatment.

[0088] Figure 59 shows MC38 mice injected with mPEG 45 -K 14 -Subcutaneous tumor growth curve after intravenous C6P treatment.

[0089] Figure 60 shows MC38 mice injected with mPEG 45 -K 14 -Statistical results of tumor mass after C6P treatment.

[0090] Figure 61 shows MC38 mice injected with mPEG 45 -K 14 -Monitoring endpoint after C6P treatment: photograph the tumor in vitro.

[0091] Figure 62 shows MC38 mice injected with mPEG 45 -K 14 -Weight change curve after C6P treatment. DETAILED DESCRIPTION

[0092] 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.

[0093] 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.

[0094] 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.

[0095] 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.

[0096] In the compounds of the present invention, when any variable (such as R, 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 bond referred to 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.

[0097] 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.

[0098] The term "alkylene" refers to an "alkyl" radical with one hydrogen less, for example, -CH2-, -CH2CH2-, -CH2CH2CH2-, -CH2CH2CH2CH2-, etc.

[0099] 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.

[0100] The term "fixed sequence" or "sequence fixed" means that the order of amino acids in a polypeptide chain is fixed.

[0101] In one embodiment of the present invention, a pH-sensitive membrane lysis material having a structure represented by formula (I) or a stereoisomer thereof or a pharmaceutically acceptable salt thereof is provided:

[0102] Wherein, R is selected from: -R3-N(R4R5), -R3-R',

[0103] R' is selected from:

[0104] L is selected from: -NH-C(=O)O-, -NH-C(=O)-;

[0105] R3 is selected from: alkylene, C6-C 14 Aryl-substituted alkylene;

[0106] 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;

[0107] R6 is selected from: C1-C 15 Alkyl, C6-C 14 Aryl, C6-C 14 Aryl-substituted C1-C 15 alkyl;

[0108] y is selected from: an integer between 2 and 150;

[0109] n is selected from: an integer between 6 and 30;

[0110] q is selected from: 0, 1, 2, 3, 4.

[0111] In some embodiments, R3 is selected from: C1-C6 alkylene, phenyl-substituted C1-C6 alkylene.

[0112] In some embodiments, R3 is selected from: -(CH2) p -, phenyl-substituted-(CH2) p -; wherein p is selected from: 1, 2, 3, 4, 5, 6.

[0113] In some embodiments, R is selected from: -R3-N(R4R5),

[0114] Wherein, R3 is selected from: -(CH2) p -, phenyl-substituted-(CH2) p -; wherein p is selected from: 1, 2, 3, 4.

[0115] 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.

[0116] 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.

[0117] In some embodiments, R4, R5 and the nitrogen atom to which they are attached together form the following group:

[0118] In some embodiments, R6 is selected from the group consisting of: C1-C6 alkyl, phenyl, naphthyl, and phenyl-substituted C1-C6 alkyl.

[0119] In some embodiments, R6 is selected from the group consisting of: C1-C3 alkyl, phenyl, naphthyl, and benzyl.

[0120] In some embodiments, 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:

[0121] R6 is benzyl.

[0122] In some embodiments, the pH sensitive membrane lysis material has a structure shown in the following formula (II):

[0123] In some embodiments, y is selected from: 30-120, preferably 40-116, more preferably 40-48 or 108-116, more preferably 44-46, more preferably 42, 43, 44, 45, 46.

[0124] In some embodiments, n is 10-30, or 6-20, preferably 10-25, more preferably 12-22; more preferably 13-20, more preferably 14-20, more preferably 14-18, more preferably 14-16, preferably 12, 13, 14, 15, 16, 17, 18, 19, 20.

[0125] In some embodiments, the pH sensitive membrane lysis material is selected from the following polymers:

[0126] wherein n is 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, or 20.

[0127] In another embodiment of the present invention, a pH-sensitive membrane-lysis nanoparticle is provided, which is formed by self-assembly of the pH-sensitive membrane-lysis material of the present invention in an aqueous medium.

[0128] In another embodiment of the present invention, a method for preparing the pH-sensitive membrane lysis nanoparticles is also provided, comprising the following steps: dissolving the pH-sensitive membrane lysis material of the present invention in an organic solvent or a hydrochloric acid solution with a pH of 1.5-2.5, and then adding the resulting solution dropwise into water under stirring, continuing stirring, and dialyzing to remove the solvent to obtain the pH-sensitive membrane lysis nanoparticles.

[0129] In some embodiments, the organic solvent is dimethyl sulfoxide.

[0130] In some embodiments, the ratio of the pH-sensitive membrane lysis material, the organic solvent or hydrochloric acid solution, and the water is 10 mg to 30 mg: 1 mL: 2 to 5 mL.

[0131] In another embodiment of the present invention, there is also provided use of the pH-sensitive membrane lysis material of the present invention, or a stereoisomer thereof, or a pharmaceutically acceptable salt thereof, in the preparation of a drug for preventing and / or treating tumors.

[0132] In another embodiment of the present invention, there is provided the use of pH-sensitive membrane-lytic nanoparticles in the preparation of drugs for preventing and / or treating tumors.

[0133] 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 pH-sensitive membrane lysis material or its stereoisomer or its pharmaceutically acceptable salt described in the present invention, and / or the pH-sensitive membrane lysis nanoparticles.

[0134] In another embodiment of the present invention, a method for preventing and / or treating tumors is also provided, which comprises: administering a safe and effective amount of the pH-sensitive membrane lysis material of the present invention or its stereoisomer or its pharmaceutically acceptable salt; and / or administering a safe and effective amount of the pH-sensitive membrane lysis nanoparticles of the present invention; and / or administering a safe and effective amount of the drug of the present invention.

[0135] 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.

[0136] The compound of formula (I) of the present invention can be used in combination with other known anti-tumor drugs. When administered in combination, the compound of formula (I) and the known drug can each become an independent administration unit, or together form a combined administration unit; the compound of formula (I) can be administered simultaneously with other known anti-tumor drugs or administered separately. When the compound of formula (I) is taken simultaneously with one or more other drugs, it is preferred to use a pharmaceutical composition containing one or more known drugs and the compound of formula (I). Drug combination also includes taking the compound of formula (I) and one or more other known drugs in overlapping time periods. When the compound of formula (I) is used in combination with one or more other known drugs, the dosage of the compound of formula (I) or the known drug can be the same as the dosage when used alone, or it can be lower than the dosage when they are used alone.

[0137] The drugs or active ingredients that can be used in combination with the compound of formula (I) include but are not limited to: immune checkpoint inhibitors, estrogen receptor modulators, androgen receptor modulators, retinal 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.

[0138] 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, stiprenisolone, prednisone, premarin, procarbazine, recombinant human erythropoietin, raltitrexed, rebifurcation, rhenium-186 etidronate, rituximab, dapoxetine-A, romotide, pilocarpine hydrochloride tablets, octreotide, sarmustine, semustine, sizolan, sobuzosine, methylprednisolone sodium, paphos acid, stem cell therapy, streptozocin, strontium-89 chloride, levothyroxine sodium , tamoxifen, tamsulosin, tasofenamin, tastolactone, taxotere, tesifizine, temozolomide, teniposide, testosterone propionate, methyltestosterone, thioguanine, thiotepa, thyroid-stimulating hormone, tiludronic acid, topotecan, 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-stabilized formulation, acolbifene, interferon r-lb, affinitak, aminopterin, 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-166DOTMP, ibandronic acid, interferon gamma, intron-PEG, ixabepilone, keyhole limpet hemocyanin, L-651582, lanreotide, lasofoxifene, libra, lonafamib, miproxifene, minodecan, MS -209, liposomal MTP-PE, MX-6, nafarelin, nemorubicin, nevastatin, noratript, oblimersen, onco-TCS, osidem, paclitaxel polyglutamate, sodium pyrimidine, PN-401, QS-21, quasitan, R-154, raloxifene, ranpirnase, 13-cis retinoic acid, satraplatin, ciocalcitol, T-138067, tarceva, paclitaxel docosahexaenoic acid, thymosin alpha 1, gazofurin, tipifarnib, tirapazamine, TLK-286, toremifene, trans-MID-lo7R, valspodar, vapreotide, vatalanib, verteporfin, vinflunine, Z-100, and zoledronic acid, or a combination thereof.

[0139] The drug for preventing and / or treating tumors of the present invention can be used in non-human mammals or humans.

[0140] 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 pH-sensitive membrane lysis material of the present invention, or a pharmaceutically acceptable salt or stereoisomer thereof, or nanoparticles thereof), and a pharmaceutically acceptable excipient. When administering the drug, a safe and effective amount of the active ingredient is administered 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.

[0141] Here, "safe and effective amount" means an amount of the active ingredient sufficient to significantly improve the condition without causing serious side effects. "Pharmaceutically acceptable excipients" refer to one or more compatible solid or liquid fillers or gels suitable for human use, of sufficient purity, and with sufficiently low toxicity. "Compatibility" here refers to the ability of the components of the composition to blend with the active ingredient of the present invention, and with each other, without significantly reducing the efficacy of the active ingredient.

[0142] 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.

[0143] 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 Tween ), wetting agents (such as sodium lauryl sulfate), colorants, flavorings, stabilizers, antioxidants, preservatives, pyrogen-free water, etc.

[0144] 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.

[0145] Solid dosage forms for oral administration include capsules, tablets, pills, powders, and granules.

[0146] 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:

[0147] (a) fillers or extenders, for example, starch, lactose, sucrose, glucose, mannitol and silicic acid;

[0148] (b) binders, for example, hydroxymethylcellulose, alginate, gelatin, polyvinylpyrrolidone, sucrose and gum arabic;

[0149] (c) humectants, for example, glycerin;

[0150] (d) disintegrants, for example, agar-agar, calcium carbonate, potato or tapioca starch, alginic acid, certain complex silicates, and sodium carbonate;

[0151] (e) a buffering solvent, for example, paraffin;

[0152] (f) absorption accelerators, for example, quaternary ammonium compounds;

[0153] (g) wetting agents, for example, cetyl alcohol and glyceryl monostearate;

[0154] (h) adsorbents, for example, kaolin; and

[0155] (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.

[0156] 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.

[0157] 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.

[0158] 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.

[0159] 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.

[0160] The following are specific examples.

[0161] In the following examples, mPEG y -NHS reacts with the primary amine at the N-terminus of the peptide to synthesize mPEG y The sequence-fixed polypeptide is then fixed by introducing a tertiary amine through the side chain primary amine of lysine in the sequence-fixed polypeptide to obtain a series of pH-sensitive membrane lysis materials based on sequence-fixed polylysine. The reaction formula and corresponding abbreviations are as follows:

[0162] Among them, mPEG y -NHS structural formula is as follows:

[0163] 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.

[0164] 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.

[0165] 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.

[0166] The side chain modification method is as follows: the sequence polypeptide is dissolved in N,N-dimethylformamide (DMF), R-CDI (5-fold excess) is added, and triethylamine is added and stirred for 12 hours, then the mixture is drained, dissolved in DMSO, and dialyzed in deionized water for 24 hours using a dialysis bag with a molecular weight cutoff of 3500, with the water changed every 2 hours, and freeze-dried to obtain a pH-sensitive membrane lysis material based on sequence-fixed polylysine.

[0167] Example 1 N-benzyl-piperidine modified methoxypolyethylene glycol-lysine fixed sequence polypeptide

[0168] n=12, 13, 14, 15, 16, 18, 20, R-OH is N-benzyl-4-hydroxypiperidin. 45 -NHS and K n mPEG is obtained by reacting amino groups in fixed sequence peptides 45 -K n , N-benzyl-4-hydroxypiperidine was modified onto the side chain of lysine via CDI to obtain an N-benzyl-piperidine-modified fixed-sequence peptide. The effects of different lysine side chain lengths on the pKa and helical structure of the fixed-sequence peptide were tested.

[0169] The reaction equation is as follows:

[0170] The specific steps are as follows:

[0171] (1) Synthesis of H-[K(Boc)] by solid phase synthesis (SPPS) n -Resin.

[0172] (2) Weigh 480 mg of mPEG 45 -NHS, 380 mg HBTU and 0.5 mL 4-methylmorpholine were placed in a 15 mL centrifuge tube, dissolved in 5 mL DMF, and then added to a 0.2 mmol H-[K(Boc)] n -Resin syringe, react on a shaker for 24 hours, use ninhydrin reagent to detect the complete reaction of amino groups, and obtain mPEG 45 -[K(Boc)] n Fixed sequence peptide, ready for use.

[0173] (3) Cleavage of mPEG using a lysis solution consisting of 95% TFA, 2.5% H2O, and 2.5% TIS 45 -[K(Boc)] n The sequence peptide was fixed 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 1000 dialysis bag for 24 hours, with the water changed every 2 hours, and freeze-dried to obtain white powder mPEG. 45 -K n Fixed sequence peptides.

[0174] (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. N-benzyl-4-hydroxypiperidine (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 N-benzyl-4-hydroxypiperidine-CDI (C6P-CDI).

[0175] (5) mPEG 45 -K n The fixed sequence peptide was dissolved in DMF, C6P-CDI (5 equivalents in excess) was added, triethylamine (equivalent 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 mPEG modified with N-benzyl-piperidine. 45 -K n -C6P, standby. Its structure was characterized by H NMR spectroscopy (Figure 1) and mass spectrometry (Figures 2-8), proving that it was fully bonded.

[0176] (6) 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 mPEG prepared in step (5) in 10 mL of hydrochloric acid solution. 45 -K n -C6P, and insert the pH probe under the liquid surface, under stirring (stirring speed Mot = 3), use 0.5M sodium hydroxide titrant to titrate. 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 determined as the protonation rate of 1 and 0, and the pH and protonation rate curve is established, as shown in Figure 9. The fixed sequence polypeptide material mPEG synthesized by the above steps 45 -K n -C6P all have sharp pH transitions, and by comparing the pKa results between different degrees of polymerization, it can be found that with the increase of the number of lysine, the pKa of the polypeptide decreases.

[0177] (7) Accurately weigh 20 mg of the fixed sequence polypeptide material mPEG prepared in step (5) 45 -K n-C6P was dissolved in 1 mL DMSO and added dropwise to 3 mL deionized water while stirring. After the addition was complete, stirring was continued for 20 min. The product was loaded into a 14000 dialysis bag and dialyzed with deionized water for 24 hours, with the water changed every 2 hours to obtain mPEG with a concentration of 5 mg / mL. 45 -K n -C6P particle stock solution. Dilute with buffer of different pH to obtain mPEG with a concentration of 1 mg / mL at the corresponding pH 45 -K n -C6P particle solution. Circular dichroism spectrometer was used to measure the fixed sequence peptide mPEG at different pH 45 -K n -C6P secondary structure, the results are shown in Figures 10-16. By calculating the proportion of various secondary structures of polypeptide materials at different pH, it can be found that mPEG 45 -K n -C6P exhibits a high proportion of α-helical structure at pH = 6.0-6.8, and the helicity decreases at pH = 7.0-7.4.

[0178] (8) mPEG was diluted with dilute hydrochloric acid 45 -K n -C6P nanoparticles (0.5 mg / mL) were adjusted to pH = 7.4 and 6.8. Two copper meshes were taken from the clean bench and placed in a 10 cm culture dish and marked. 10 μL of nanoparticles with different pH values ​​were taken with a pipette and dropped onto the corresponding marked copper mesh. The culture dish was covered to prevent dust from falling. After standing for 10 minutes, the excess liquid was removed from the edge of the copper mesh with filter paper. After drying naturally, the fixed sequence peptide mPEG was observed using a transmission electron microscope. 45 -K 14 -C6P morphology (Figure 17-18), from the results of transmission electron microscopy, we can see that mPEG 45 -K 14 -C6P particles showed a vesicle structure with a particle size of ∼200 nm at pH = 7.4, and the particle size was slightly reduced at pH = 6.8.

[0179] Example 2 Different Tertiary Amine Modified Methoxypolyethylene glycol-Lysine Fixed Sequence Peptides

[0180] Select mPEG 45 -NHS-modified H-[K(Boc)] 14 -Resin, tertiary amines were modified onto the side chains of polylysine by CDI to obtain a series of pH-sensitive membrane lysis materials based on sequence-fixed polylysine with different tertiary amine modifications. The effects of different tertiary amine structures on the pKa and secondary structure of sequence-fixed polylysine were tested. The reaction equation is as follows:

[0181] The specific operation steps are as shown in Example 1. The structures of the obtained sequence-immobilized polylysine modified with different tertiary amines were characterized by nuclear magnetic hydrogen spectroscopy, as shown in Figures 19 to 22, which prove that the pH-sensitive membrane lysis material based on sequence-immobilized polylysine has the following structure:

[0182] The obtained sequence-fixed polylysine modified with different tertiary amines was titrated using the method in step (6) of Example 1 to obtain a protonation rate curve (Figure 23). According to the pKa results, it can be found that when the tertiary amine group C5P with a hydrophobicity similar to that in Example 1 is used to modify lysine, the pKa of the obtained material is similar to that of mPEG. 45 -K 14 -C6P; when the more hydrophobic DB modification was selected, the pKa of the obtained material increased significantly, which shows that a series of fixed sequence polypeptide materials with different pKa can be obtained by changing the hydrophilicity of the side chain groups.

[0183] The secondary structure curves of sequence-fixed polylysine modified with different tertiary amines at different pH values ​​were obtained using the method of step (7) of Example 1 (Figures 24-27). By calculating the proportion of the secondary structure of the material, it was found that the proportion of the secondary structure of the fixed sequence polypeptide materials modified with DB, DMP, C5P, and C5P2 did not change significantly with the change of pH.

[0184] Example 3 Lysine fixed sequence polypeptides modified with methoxypolyethylene glycol of different lengths

[0185] Select mPEG 22 -NHS and mPEG 113 -NHS-modified H-[K(Boc)] 14 -Resin, N-benzyl-4-hydroxypiperidine was modified onto the side chain of lysine via CDI to obtain an N-benzyl-piperidine-modified fixed-sequence peptide. The effects of different mPEG lengths on the pKa and helical structure of the fixed-sequence peptide were tested. The reaction equation is as follows:

[0186] The specific operation steps were similar to those in Example 1, and fixed-sequence peptides with different lengths of methoxypolyethylene glycol and N-benzyl-piperidine modifications were obtained. The structure of the resulting fixed-sequence polylysine was characterized by H-NMR spectroscopy, as shown in Figures 28 and 29 , demonstrating the following structure of a pH-sensitive membrane lysis material based on fixed-sequence polylysine:

[0187] The obtained sequence-fixed polylysine was titrated using the method of step (6) of Example 1 to obtain a protonation rate curve (Figure 30). According to the pKa results, it can be found that mPEG 22 Modified and mPEG113 The pKa of the modified fixed sequence peptides did not change much.

[0188] The secondary structure curves of the obtained sequence-fixed polylysine at different pH values ​​were measured using the method of step (7) of Example 1 (Figures 31-32). The ratio of the secondary structure of the material was calculated to obtain mPEG. 22 -K 14 -C6P and mPEG 113 -K 14 -C6P exhibits a high proportion of α-helical structure at pH = 6.0-6.8, and the helicity decreases at pH = 7.0-7.4.

[0189] Example 4 pH-responsive anticancer activity of tertiary amine-modified methoxypolyethylene glycol-lysine fixed sequence polypeptides

[0190] (1) The tumor cell killing activity of the polypeptide particles prepared according to the method of step (7) of Example 1 was determined by MTT assay at pH = 6.8, while the control group was at pH = 7.4. The specific steps were as follows:

[0191] 1) MC38 tumor cells were plated at 10,000 cells per well in a 96-well plate and cultured overnight before use;

[0192] 2) Prepare a series of peptide concentrations using a pH 6.8 culture medium, and use a drug-free group as a control group;

[0193] 3) Remove the well plate, remove the culture supernatant, and add 100 μL / well of the corresponding peptide-containing culture medium. Place the cells in a 37°C incubator for culture.

[0194] 4) At a specific time point, remove the cells, discard the supernatant, add culture medium containing 0.5 mg / mL MTT, and continue culturing for 2-4 hours;

[0195] 5) Discard the supernatant and add 100 μL of DMSO to each well. Incubate on a shaker in the dark for 10 minutes to fully dissolve the crystals.

[0196] 6) Use a microplate reader to measure the absorbance at 490 nm, and define the absorbance of the experimental group incubated with the polypeptide as I 实验组 The absorbance value of the culture medium group without polypeptide and cells was defined as I negative control, and the cells without polypeptide were used as positive control group, and the absorbance value was defined as I 阳性对照 ; Then according to the formula [(I 实验组 -I 阴性对照 ) / (I 阳性对照 -I 阴性对照 )]×100%, and the cell survival rate was calculated.

[0197] Cell viability results at pH 6.8 and pH 7.4 are shown in Figures 33-38. When the mPEG molecular weight was 2000 and the tertiary amine group was C6P, fixed-sequence peptides with 10-20 lysine residues exhibited no significant cytotoxicity against MC38 cells at pH 7.4 when treated at concentrations of 400 μg / mL or less for 24 hours. At pH 6.8, peptides with 12-20 lysine residues exhibited strong cytotoxicity against MC38 cells, indicating that C6P-modified lysine-fixed sequence peptides exhibit selective cytotoxicity against tumor cells, with peptides with 14-16 lysine residues showing the strongest cytotoxicity against MC38 cells. By modifying the tertiary amine group, fixed-sequence peptides modified with C5P exhibited no significant tumor cell cytotoxicity at both pH 7.4 and pH 6.8. Fixed-sequence peptides modified with DB exhibited slightly stronger tumor cell cytotoxicity than those modified with C5P, but weaker than those modified with C6P, and showed no significant selectivity. Changing the molecular weight of mPEG 22 The modified lysine fixed sequence peptide showed no significant tumor cell toxicity at pH 7.4 and 6.8. 113 The modified lysine fixed sequence polypeptide showed strong tumor cell toxicity at pH = 7.4 and 6.8 at a certain dose, and its selectivity was worse than that of the C6P modified fixed sequence polypeptide.

[0198] Example 5 mPEG 45 -K 14 Determination of the maximum tolerated dose of -C6P in ICR mice

[0199] 1) Prepare peptide mPEG using glucose solution with a final concentration of 5% 45 -K 14 -C6P isotonic solution was first injected into the tail vein of healthy 6-7 week old female ICR mice at a dose of 200 mg / kg, and the survival status of the mice was observed;

[0200] 2) After more than 24 hours of intravenous injection of the peptide, if the mouse shows no obvious adverse symptoms and its vitality is not affected, continue intravenous injection of the peptide into another healthy mouse at the current dose and observe;

[0201] 3) When no obvious adverse effects occur after more than 5 consecutive mice are given the drug at this dose, it is determined as the MTD value;

[0202] 4) If mice die at this dose, the dose should be reduced to 100 mg / kg and the above-mentioned measurements and observations should be continued until at least 5 mice survive at a certain dose, which is recorded as the MTD value.

[0203] The results are shown in Figure 46. 45 -K 14 -C6P, mPEG 45 -K 15 -C6P and mPEG 44 -K 16 -The MTD values ​​of C6P are relatively high, especially mPEG 45 -K 14 -C6P and mPEG 45 -K 15 The MTD value of -C6P is greater than 200 mg / kg, indicating that it has high safety in vivo.

[0204] Example 6 Study on the Cell Killing Mode of Methoxypolyethylene glycol-Lysine Fixed Sequence Peptide

[0205] (1) MTT method to explore mPEG 45 -K 14 -C6P-dependent pathways and methods for killing tumor cells

[0206] 1) After digestion, MC38 cells were collected and counted, seeded at 15,000 cells / well in a 96-well plate, and cultured overnight for adherence before use;

[0207] 2) Inhibitor Preparation: Various inhibitor working solutions were prepared using complete DMEM medium (pH 6.8). Endocytic inhibitors included wortmannin (5, 10, 25, and 50 nM), methyl-β-cyclodextrin (5, 10, 25, and 50 μM), and chlorpromazine hydrochloride (5, 10, 25, and 50 μM). Lysosomal inhibitors included chloroquine (10, 50, and 200 μM), bafilomycin A1 (10, 50, and 100 nM), and concanamycin A (10, 50, 100, and 200 nM).

[0208] 3) Inhibitor pretreatment: Add the prepared inhibitor working solution to the well plate and incubate in a 37°C incubator for 30 minutes;

[0209] 4) Material processing: Use the above inhibitor working solution to prepare mPEG 45 -K 14-C6P working solution to obtain a mixture of inhibitors and peptides with a final concentration of 200 μg / mL; after the pretreatment, the original culture medium was removed, and the mixture of inhibitors and peptides was added, and the cells were incubated in a 37°C incubator for another 24 hours;

[0210] 5) Perform MTT assay after 24 hours.

[0211] The results are shown in Figures 39 to 44. For endocytosis inhibitors, the macropinocytosis inhibitor Wortmannin (Wor), the caveolin-dependent endocytosis inhibitor Methyl-beta-cyclodextrin (m-β-CD) and the clathrin-mediated endocytosis inhibitor Chlorpromazine hydrochloride (CPZ) were used in combination with mPEG. 45 -K 14 -C6P co-treatment of MC38 cells did not inhibit the killing of tumor cells by peptides, suggesting that mPEG 45 -K 14 -C6P does not only rely on a specific endocytosis method to enter tumor cells, but may enter cells through multiple endocytosis methods. For lysosomal inhibitors, chloroquine (CQ), bafilomycin A1 and concanamycin A, which are inhibitors of lysosomal acidity, are used to inhibit free H + After the level, mPEG 45 -K 14 -C6P weakened the killing ability of tumor cells, indicating that mPEG 45 -K 14 -C6P kills tumor cells by further protonation in lysosomes, leading to the destruction of lysosomal membranes.

[0212] (2) Liposome fluorescence leakage experiment testing peptide mPEG 45 -K 14 -C6P membrane-permeabilizing activity

[0213] The phospholipids used in this experiment are DOPC and POPS, with a molar ratio of 9:1, to simulate the tumor cell membrane structure; and the fluorescent dye ANTS and the quencher DPX are encapsulated inside the liposomes. When the liposome membrane structure is normal, ANTS and DPX are co-encapsulated in the liposomes, and the ANTS fluorescence is quenched. When the liposome membrane structure is destroyed, ANTS and DPX are released into the buffer solution, and the ANTS fluorescence is enhanced. Therefore, the peptide mPEG can be characterized by detecting the ANTS fluorescence intensity. 45 -K 14 -C6P destroys the liposome membrane structure. The specific operation is as follows:

[0214] 1) Prepare 25 mg / mL DOPC and POPS solutions in chloroform, then take 16 μL DOPC and 144 μL POPS, respectively, and mix them in 800 μL chloroform.

[0215] 2) Using a rotary evaporator, heat the mixed solution in a 37°C water bath. First, rotate at 150 rpm for 3 minutes at normal pressure, then slowly reduce the pressure to 350 mbar, hold for 10 minutes, and then reduce the pressure to 100 mbar, hold for 30 minutes.

[0216] 3) Vacuum for 2 hours to remove all chloroform;

[0217] 4) Add 2 mL of a solution containing 45 mM DPX and 12.5 mM MANTS (prepared using 10 mM sodium dihydrogen phosphate solution, pH 7.0) and hydrate at 37°C with stirring at 200 rpm for 1 hour. Repeat freeze-thaw cycles 10 times.

[0218] 5) Use a Sephadex column with 10 mM sodium hydrogen phosphate and 90 mM NaCl (pH 7.0) as the mobile phase to collect the liposome solution and store at 4°C until use.

[0219] 6) Dilute the liposome solution 10-fold with 1× PBS at pH 7.4, pH 6.8, and pH 5.5;

[0220] 7) mPEG prepared with 1× PBS solution of corresponding pH 45 -K 14 -C6P solution (final concentration 200 μg / mL) was incubated with the liposome solution;

[0221] 8) The negative control was a 1×PBS solution of the corresponding pH, and the positive control was a 0.2% (w / v) Triton X-100 solution; 9) The fluorescence intensity at 360 nm / 536 nm was measured using a microplate reader.

[0222] The results are shown in Figure 45. At pH 7.4, mPEG 45 -K 14 No obvious ANTS fluorescence signal was detected after liposomes were treated with -C6P, but obvious ANTS fluorescence leakage was detected under pH 6.8 and pH 5.5 conditions, indicating that mPEG 45 -K 14 -C6P's membrane-breaking activity is significantly enhanced under acidic conditions, especially lysosomal acidity.

[0223] (III) Flow cytometry detection of tumor cells in slightly acidic environment to mPEG 45 -K 14 -C6P intake

[0224] 1) Pancreatic cancer Panc02 cells were plated at 100,000 cells / well in a 24-well plate and cultured overnight before use;

[0225] 2) Prepare FITC-PEG in a series of concentrations ranging from 12.5 to 200 μg / mL using culture medium at pH 7.4 and pH 6.8, respectively. 2k -K 14 -C6P fixed sequence peptide, the drug was added to the cells and cultured in a 37°C incubator;

[0226] 3) After 2 hours, remove the cells, remove the drug-containing supernatant, and wash the cells 2-3 times with room temperature PBS;

[0227] 4) Add trypsin to digest the cells, collect the cells by centrifugation at 4°C / 1000 rpm for 5 minutes, and resuspend the cells in PBS containing 1% BSA;

[0228] 5) Pass the cells through a 200-mesh sieve and add PI (10 μg / mL) and 0.04% trypan blue to a final concentration of 10 μg / mL before loading.

[0229] 6) The detection channels used are 633 / 660nm and 495 / 519nm.

[0230] The results are shown in Figure 47. At pH 7.4, tumor cells are sensitive to mPEG. 45 -K 14 -C6P uptake was low, while at pH 6.8, tumor cells were more sensitive to mPEG. 45 -K 14 -C6P uptake increased significantly with increasing concentration; and after the cell metabolism was inhibited at low temperature (4 ° C), the cell's ability to uptake the peptide was weakened at pH 6.8, indicating that mPEG 45 -K 14 -C6P also enters tumor cells via an energy-dependent endocytosis pathway.

[0231] (IV) Observation of Cy5-PEG by confocal laser scanning microscopy (CLSM) 45 -K 14 - Co-localization of C6P with lysosomes

[0232] 1) Cell seeding: 100,000 MC38 tumor cells were seeded into a glass-bottomed confocal dish and cultured overnight before use.

[0233] 2) Material treatment: Cy5-PEG was prepared using DMEM medium at pH 7.4 and pH 6.8. 45 -K 14 -C6P material solution (50 μg / mL), aspirate the original culture medium, add the material and continue culturing;

[0234] 3) Cell staining: Prepare Lyso-Tracker Red working solution in complete culture medium at a working concentration of 50-75 nM. Dilute the nuclear dye Hoechst 33342 at a dilution of 1:1000. Discard the original culture medium, add the staining solution, and incubate in a cell culture incubator for 30 minutes.

[0235] 4) Discard the staining solution, wash three times with 1× PBS, and then add complete culture medium to prepare for imaging;

[0236] 5) The images were taken using an Olympus spinning disk confocal microscope (60x oil objective lens, 405 nm / 561 nm / 640 nm channels).

[0237] The results are shown in Figure 48. No Cy5-PEG was obviously observed inside the cell at pH 7.4. 45 -K 14 -C6P fluorescence signal, while at pH 6.8, Cy5-PEG in tumor cells 45 -K 14 The uptake of -C6P was significantly increased and colocalized with the lysosomal dye Lyso-Tracker Red, indicating that tumor cells were sensitive to mPEG at pH 6.8. 45 -K 14 -C6P uptake was significantly enhanced and localized to lysosomes.

[0238] (V) Observation of mPEG by confocal laser scanning microscopy (CLSM) 45 -K 14 -C6P causes lysosomal damage

[0239] 1) Cell plating: MC38Gal3-GFP / LAMP1-mCherry cells (stable expression of Gal3-GFP and LAMP1-mCherry proteins) were seeded at 150,000 cells / well in a glass-bottomed confocal dish and allowed to adhere overnight before use.

[0240] 2) Material processing: mPEG was prepared using pH 7.4 and pH 6.8 DMEM complete medium. 45 -K n -C6P material solution (200 μg / mL), discard the original culture medium, add the material to treat the cells, and place in an incubator away from light and continue to culture for 8-12 hours;

[0241] 3) Remove the culture medium and wash twice with room temperature 1× PBS solution. Then, stain with Hoechst 33342 nuclear staining solution (prepared at a 1:1000 dilution) for 15 minutes before preparing for imaging.

[0242] 4) After staining, wash twice with room temperature 1× PBS solution, then add 300 μL complete culture medium and prepare for imaging;

[0243] 5) The images were taken using an Olympus spinning disk confocal microscope (60x oil objective lens, 405 nm / 488 nm / 561 nm channels).

[0244] The results are shown in Figure 49. At pH 6.8, mPEG 45 -K 14 -Gal3-GFP spots aggregated in MC38Gal3-GFP / LAMP1-mCherry cells treated with C6P, formed spots, and colocalized with the lysosomal membrane protein LAMP1-mCherry, indicating that mPEG 45 -K 14 -C6P can cause lysosomal damage.

[0245] (VI) Observation of mPEG by confocal laser scanning microscopy (CLSM) 45 -K 14 Effect of -C6P on cathepsin B leakage

[0246] 1) Cell plate: MC38CTSB-mCherry cells (stable expression of cathepsin B protein with mCherry fluorescent labeling) were seeded at 150,000 cells per well in a glass-bottomed confocal dish and allowed to adhere overnight before use.

[0247] 2) Material treatment: mPEG was prepared in pH 6.8 DMEM complete medium 45 -K 14 -C6P material solution (200 μg / mL), discard the original culture medium, add the material to treat the cells, and place them in an incubator away from light to continue culturing;

[0248] 3) Use confocal microscopy to capture images at 2, 8, and 12 hours;

[0249] 4) The images were taken using an Olympus spinning disk confocal microscope (60x oil objective lens, 561 nm channel).

[0250] The results are shown in Figure 50. In the Control group, the cells were not treated with mPEG. 45 -K 14 -C6P peptide material treated tumor cell tissue proteases are located in the lysosomal compartment, so the mCherry fluorescence appears as a dot; 45 -K 14After -C6P treatment, the punctate fluorescence of cells gradually diffused with the extension of incubation time, showing sheet-like aggregation, and could be clearly observed to be diffused in the cytoplasmic space at 12 hours, indicating that the polypeptide destroyed the lysosomal membrane, leading to the cytoplasmic leakage of CSTB.

[0251] (VII) Lactate dehydrogenase toxicity assay to investigate the release of tumor cell contents

[0252] 1) Cell seeding: After digestion, MC38 cells were harvested and counted, seeded at 15,000 cells / well in a 96-well plate, and cultured overnight in a 37°C cell culture incubator until adherent.

[0253] 2) Material processing: mPEG was prepared using pH 7.4 and pH 6.8 DMEM culture media 45 -K 14 -C6P material solution with concentrations of 100, 200, and 400 μg / mL was then added to the 96-well plate and incubated in the incubator for 24 hours;

[0254] 3) Control group treatment: negative control wells were treated without adding mPEG 45 -K 14 -C6P material in pH 7.4 or pH 6.8 DMEM medium, incubation time is 24 hours; positive control wells use the LDH release reagent in the kit, added to the wells without material treatment at a 10% volume ratio (10 μL) 1 hour before the end of the scheduled time;

[0255] 4) After 24 hours, centrifuge the plate at 400 g for 5 minutes at 4°C, and transfer 60 μL of the supernatant to a new 96-well plate for testing.

[0256] 5) Prepare LDH detection working solution according to the kit instructions, add 30 μL of LDH detection working solution to the above sample wells, incubate at room temperature for 30 minutes, and then detect;

[0257] 6) LDH detection: Use a multifunctional microplate reader to measure the absorbance at 490 nm;

[0258] 7) Data processing: mPEG 45 -K 14 -C6P material treatment group absorbance value is called I 实验组 The absorbance value of the positive control group is called I positive control The absorbance value of the negative control group was I negative control Then according to the formula LDH release (%) = [(I 实验组 -I negative control ) / (I positive control -I negative control)] × 100% to calculate the LDH release rate of each group, and finally GraphPad Prism 9.5.1 software was used to draw the curve and perform data analysis.

[0259] The results are shown in Figure 51. At pH 6.8, mPEG 45 -K 14 -C6P leads to the release of cytoplasmic component LDH into the extracellular space, indicating that mPEG 45 -K 14 -C6P ultimately leads to the destruction of the cell membrane, allowing the cell contents to leak out.

[0260] (8) ATP release detection

[0261] 1) Cell seeding: After digestion, MC38 cells were harvested and counted, seeded at 15,000 cells / well in a 96-well plate, and cultured overnight in a 37°C cell culture incubator until adherent.

[0262] 2) Material processing: mPEG was prepared using pH 6.8 DMEM medium 45 -K 14 -C6P material solution with concentrations of 0, 25, 50, 100, 200, and 400 μg / mL was added to a 96-well plate, and the cells were placed in an incubator and cultured for 12 hours;

[0263] 3) After 12 hours, the plate was centrifuged at 2000 rpm at 4°C for 5 minutes, and the supernatant was taken as the test sample and stored at 4°C for testing.

[0264] 4) Preparation of ATP standard: dilute the ATP standard solution with ATP detection lysis buffer to a concentration gradient of 0.01, 0.03, 0.1, 0.3, 1, 3, and 10 μM;

[0265] 5) Prepare ATP assay working solution according to the kit instructions, with a volume ratio of 1:9 between assay reagent and assay diluent. Add 100 μL of assay working solution to a 96-well plate and incubate at room temperature for 3 minutes to reduce background.

[0266] 6) Sample detection: Take 40 μL of ATP standard solution and the sample to be tested and add them to the above-mentioned detection solution measurement wells. After thorough mixing, immediately place them in a multifunctional microplate reader and select the Luminometer module to detect the RLU value;

[0267] 7) Data processing: Draw a standard curve using the RLU values ​​measured with the ATP standard solution, and then calculate the ATP concentration in the sample according to the standard curve equation.

[0268] The results are shown in Figure 52. At pH 6.8, mPEG 45 -K 14-C6P causes the release of ATP from the cytoplasm to the extracellular space, indicating that mPEG 45 -K 14 -C6P ultimately leads to the destruction of the cell membrane, allowing the cell contents to leak out.

[0269] (IX) Western Blot Detection of HMGB1 Protein Leakage

[0270] 1) Cell seeding: After digestion, MC38 cells were harvested and counted, seeded at 200,000 cells / well in a 24-well plate, and cultured overnight in an incubator until ready for use.

[0271] 2) Material processing: mPEG was prepared using pH 7.4 and pH 6.8 DMEM culture media 45 -K 14 -C6P material solution with a concentration of 200 and 400 μg / mL was then added to a 24-well plate and incubated in an incubator for 24 h;

[0272] 3) After 24 hours, the supernatant was collected, and then centrifuged at 2000 rpm for 5 minutes, and then the supernatant was collected by centrifugation at 12000 rpm for 5 minutes;

[0273] 4) Prepare the supernatant into 1× electrophoresis sample using 4× Western blot loading buffer (containing DTT), mix thoroughly, and heat at 95°C for 10 minutes.

[0274] 5) Western blot: The samples were subjected to SDS-PAGE electrophoresis, transferred to a membrane, and subsequently incubated with antibodies and developed. The primary antibody was Recombinant Anti-HMGB1 antibody (ab79823) at a dilution of 1:10,000, and the secondary antibody was Goat Anti-Rabbit IgG, Peroxidase Conjugated, H+L (BL003A) at a dilution of 1:2,000.

[0275] The results are shown in Figure 53. At pH 6.8, mPEG 45 -K 14 -C6P leads to the release of cytoplasmic component HMGB1 protein into the extracellular space, indicating that mPEG 45 -K 14 -C6P ultimately leads to the destruction of the cell membrane, allowing the cell contents to leak out.

[0276] (10) Detection of calreticulin on the cell membrane by flow cytometry

[0277] 1) Cell seeding: After digestion, MC38 cells were harvested and counted, seeded at 150,000 cells / well in a 24-well plate, and cultured overnight in an incubator until ready for use.

[0278] 2) Material processing: mPEG was prepared using pH 7.4 and pH 6.8 DMEM culture media 45 -K 14 -C6P material solutions with concentrations of 50 and 100 μg / mL were added to 24-well plates and treated for 12 hours.

[0279] 3) After 12 hours, cells were digested and collected, and the cell pellet was collected by centrifugation at 2000 rpm for 5 minutes;

[0280] 4) Resuspend the cells in 0.5% PBA and centrifuge at 2000 rpm for 5 minutes to wash the cells;

[0281] 5) Antibody incubation: Dilute Recombinant Alexa Fluor 500 at 1:500 647 Anti-Calreticulin antibody was added to the cell pellet, and the cells were resuspended and incubated on ice for 1 hour;

[0282] 6) Wash the cells with 1 mL of 0.5% PBA and resuspend them in 200 μL of 0.5% PBA. Filter the cells through a 0.22 μm nylon mesh.

[0283] 7) Flow cytometric detection: DAPI dye was added at a final concentration of 1 μg / mL during detection, and an 18-color flow cytometer was used to detect the Ex 405nm / Em 488nm and Ex 640 / Em 670nm channels.

[0284] 8) FlowJo V10 software was used for data analysis and graphing, and GraphPad Prism 9.5.1 software was used to analyze the mean fluorescence intensity (MFI).

[0285] The results are shown in Figure 54. Under pH 6.8 conditions, the CRT level on the cell membrane surface increased significantly, indicating that mPEG 45 -K 14 -C6P kills tumor cells by inducing lysosomal damage and promoting the release of tumor cell immunogenic markers.

[0286] Example 7 In vivo anti-tumor effect of methoxypolyethylene glycol-lysine fixed sequence polypeptide

[0287] (1) mPEG 45 -K 14 -C6P therapeutic experiment on Panc02 subcutaneous pancreatic cancer model

[0288] 1) Construction of subcutaneous Panc02 pancreatic cancer tumor model in mice: Resuspend Panc02 cells in serum-free culture medium and adjust the concentration 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;

[0289] 2) When the tumor volume is about 50 mm 3 The tumor-bearing mice were randomly divided into 3 groups, with 6 mice in each group. The following groups were injected with 5% glucose solution (control) and mPEG 45 -K 14 -C6P (30 / 60 mg / kg), administered every other day for three consecutive treatments;

[0290] 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.

[0291] 4) Results are shown in Figures 55-58. mPEG was injected intravenously. 45 -K 14 -C6P polypeptide particles can effectively inhibit tumor growth, using high doses (60mg / kg) of mPEG 45 -K 14 -C6P significantly inhibited the growth of Panc02 tumors, and there was no significant weight loss in mice after three injections.

[0292] (2)mPEG 45 -K 14 -C6P therapeutic experiment on MC38 subcutaneous colorectal cancer model

[0293] 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;

[0294] 2) When the tumor volume is about 50 mm 3 The tumor-bearing mice were randomly divided into two groups, with 6 mice in each group. The following groups were injected with 5% glucose solution (control) and mPEG 45 -K 14 -C6P (30 mg / kg), administered every other day for three consecutive treatments;

[0295] 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.

[0296] The results are shown in Figures 59-62. mPEG was injected intravenously 45 -K 14 -C6P polypeptide particles can effectively inhibit tumor growth. A dose of 30 mg / kg can significantly inhibit the growth of MC38 tumors, and there is no significant weight loss in mice after three injections.

[0297] 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 pH-sensitive membrane lysis material having a structure represented by formula (I) or a stereoisomer thereof or a pharmaceutically acceptable salt thereof: in, R is selected from: -R3-N(R4R5), -R3-R', R' is selected from: L is selected from: -NH-C(=O)O-, -NH-C(=O)-; 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: an integer between 2 and 150; n is selected from: an integer between 6 and 30; q is selected from: 0, 1, 2, 3, 4.

2. The pH-sensitive membrane lysis material or its stereoisomer or its pharmaceutically acceptable salt according to claim 1, characterized in that: R3 is selected from: C1-C6 alkylene, phenyl-substituted C1-C6 alkylene.

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

4. The pH-sensitive membrane lysis material or its stereoisomer or its pharmaceutically acceptable salt according to claim 1, characterized in that: R is selected from: -R3-N(R4R5), Wherein, R3 is selected from: -(CH2) p -, phenyl-substituted-(CH2) p -; wherein p is selected from: 1, 2, 3, 4.

5. The pH-sensitive membrane lysis material according to any one of claims 1 to 4, or a stereoisomer thereof, or a pharmaceutically acceptable salt thereof, 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.

6. The pH-sensitive membrane lysis material or its stereoisomer or its pharmaceutically acceptable salt according to claim 5, 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.

7. The pH-sensitive membrane lysis material or its stereoisomer or its pharmaceutically acceptable salt according to claim 6, characterized in that: R4, R5 and the nitrogen atom to which they are connected together form the following group:

8. The pH-sensitive membrane lysis material according to any one of claims 1 to 4, or a stereoisomer thereof, or a pharmaceutically acceptable salt thereof, characterized in that: R6 is selected from the group consisting of: C1-C6 alkyl, phenyl, naphthyl, and C1-C6 alkyl substituted with phenyl.

9. The pH-sensitive membrane lysis material or its stereoisomer or its pharmaceutically acceptable salt according to claim 8, characterized in that: R6 is selected from: C1-C3 alkyl, phenyl, naphthyl, benzyl.

10. The pH-sensitive membrane lysis material according to any one of claims 1 to 4, or a stereoisomer thereof, or a pharmaceutically acceptable salt thereof, 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.

11. The pH-sensitive membrane lysis material or its stereoisomer or its pharmaceutically acceptable salt according to claim 1, characterized in that: The pH sensitive membrane lysis material has a structure shown in the following formula (II):

12. The pH-sensitive membrane lysis material according to any one of claims 1 to 4 or claim 11, or a stereoisomer thereof, or a pharmaceutically acceptable salt thereof, characterized in that: y is selected from: 30-120, preferably 40-116, more preferably 40-48 or 108-116, more preferably 44-46, more preferably 42, 43, 44, 45, 46.

13. The pH-sensitive membrane lysis material according to any one of claims 1 to 4 or claim 11, or a stereoisomer thereof, or a pharmaceutically acceptable salt thereof, characterized in that: n is 10-30, or 6-20, preferably 10-25, more preferably 12-22; more preferably 13-20, more preferably 14-20, more preferably 14-18, more preferably 14-16, preferably 12, 13, 14, 15, 16, 17, 18, 19, 20.

14. The pH-sensitive membrane lysis material according to claim 1, or its stereoisomer or its pharmaceutically acceptable salt, characterized in that: The pH sensitive membrane lysis material is selected from the following polymers: wherein n is 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, or 20.

15. A pH-sensitive membrane lytic nanoparticle, characterized in that The pH-sensitive membrane lysis material according to any one of claims 1 to 14 is self-assembled in an aqueous medium.

16. A method for preparing pH-sensitive membrane-lytic nanoparticles according to claim 15, characterized in that: The method comprises the following steps: dissolving the pH-sensitive membrane lysis material 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 pH-sensitive membrane lysis nanoparticles; Preferably, the organic solvent is dimethyl sulfoxide; preferably, the ratio of the pH-sensitive membrane lysis material, the organic solvent or hydrochloric acid solution, and the water is 10 mg to 30 mg: 1 mL: 2 to 5 mL.

17. Use of the pH-sensitive membrane lysis material according to any one of claims 1 to 14, or a stereoisomer thereof, or a pharmaceutically acceptable salt thereof in the preparation of a drug for preventing and / or treating tumors.

18. Use of the pH-sensitive membrane-lytic nanoparticles according to claim 15 in the preparation of drugs for preventing and / or treating tumors.

19. The use according to any one of claims 17-18, 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.

20. A drug for preventing and / or treating tumors, characterized in that: The composition is prepared from active ingredients and pharmaceutically acceptable excipients, wherein the active ingredients include the pH-sensitive membrane lysis material or its stereoisomer or its pharmaceutically acceptable salt according to any one of claims 1 to 14, and / or the pH-sensitive membrane lysis nanoparticles according to claim 15.

21. A method for preventing and / or treating tumors, characterized in that: The method comprises: administering a safe and effective amount of the pH-sensitive membrane lysis material or a stereoisomer thereof or a pharmaceutically acceptable salt thereof according to any one of items 1 to 14; and / or, Administering a safe and effective amount of the pH-sensitive membrane-lytic nanoparticles of claim 15; and / or, Administer a safe and effective amount of the drug according to claim 20.

22. The method according to claim 21, 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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