CN116635080A - PH-sensitive membrane-rupture polypeptide and application thereof - Google Patents

PH-sensitive membrane-rupture polypeptide and application thereof Download PDF

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Publication number
CN116635080A
CN116635080A CN202280005645.7A CN202280005645A CN116635080A CN 116635080 A CN116635080 A CN 116635080A CN 202280005645 A CN202280005645 A CN 202280005645A CN 116635080 A CN116635080 A CN 116635080A
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polypeptide
membrane
pharmaceutically acceptable
stereoisomer
acceptable salt
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熊梦华
李�杰
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South China University of Technology SCUT
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South China University of Technology SCUT
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    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K47/00—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient
    • A61K47/50—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates
    • A61K47/51—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates the non-active ingredient being a modifying agent
    • A61K47/56—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates the non-active ingredient being a modifying agent the modifying agent being an organic macromolecular compound, e.g. an oligomeric, polymeric or dendrimeric molecule
    • A61K47/59—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates the non-active ingredient being a modifying agent the modifying agent being an organic macromolecular compound, e.g. an oligomeric, polymeric or dendrimeric molecule obtained otherwise than by reactions only involving carbon-to-carbon unsaturated bonds, e.g. polyureas or polyurethanes
    • A61K47/60—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates the non-active ingredient being a modifying agent the modifying agent being an organic macromolecular compound, e.g. an oligomeric, polymeric or dendrimeric molecule obtained otherwise than by reactions only involving carbon-to-carbon unsaturated bonds, e.g. polyureas or polyurethanes the organic macromolecular compound being a polyoxyalkylene oligomer, polymer or dendrimer, e.g. PEG, PPG, PEO or polyglycerol
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P31/00—Antiinfectives, i.e. antibiotics, antiseptics, chemotherapeutics
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P35/00—Antineoplastic agents
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K14/00—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof

Abstract

The invention provides a pH-sensitive membrane-rupture polypeptide with a structure shown in a formula (I) or a stereoisomer or a pharmaceutically acceptable salt thereof, and application thereof. The macromolecular material is hydrophobic and neutral at normal physiological pH, can self-assemble into nano particles, is tightly assembled and has weak interaction with cell membranes; under the pH condition of the micro-acid, the cell membrane can be protonated to form an amphipathic structure consisting of a hydrophobic structural domain and a cationic structural domain, has strong interaction with the cell membrane and strong membrane rupture activity, and can kill tumor cells or bacteria efficiently and selectively.

Description

PH-sensitive membrane-rupture polypeptide and application thereof Technical Field
The invention relates to the technical field of high polymer materials and medicines, in particular to a pH-sensitive membrane-rupture polypeptide and application thereof.
Background
Some amphiphilic polymers with cationic groups can kill pathogens such as tumor cells and bacteria by destroying cell membranes, and the amphiphilic polymers are independent of killing forms of metabolic pathways, have the advantages of broad-spectrum killing effect, difficult drug resistance generation and the like, and have wide application prospects in treatment of tumors and infectious diseases. The membrane-breaking polymer material and the cell membrane are acted through electrostatic and hydrophobic interaction, firstly, the cationic structural domain of the polymer and the negative charge cell membrane are combined to the surface of the cell membrane through electrostatic interaction, then the hydrophobic structural domain of the polymer is inserted into the lipid layer of the cell membrane, and irreparable membrane damage is formed on the cell membrane, so that the cell is killed. The amphiphilic structure consisting of the cationic group and the hydrophobic group is a key structure of the action of a macromolecular material and a cell membrane, and is also an important cause of cytotoxicity on normal tissue cells. The structural proportion of the two is important for the interaction of the two with the cell membrane. In general, a simple cationic polymer is easy to bind to a cell membrane but is not easy to insert into the cell membrane, and a hydrophobic polymer is difficult to bind to the surface of the cell membrane, so that the cell membrane cannot be effectively destroyed. Therefore, the macromolecule material is designed to be in a cationic or hydrophobic structure in normal tissues, and is converted into an amphipathic conformation consisting of a cationic structural domain and a hydrophobic structural domain in a lesion part, so that the problem of high toxicity of the membrane-breaking macromolecule material is solved, and the membrane-breaking macromolecule material has important significance.
The polymethacrylate high polymer material with membrane rupture activity and tertiary amine side chain is prepared in the early stage, the activation of the membrane rupture activity can be realized under different pH values through the tertiary amine protonation of the side chain, and the low hemolysis and killing under the pH value of 7.4 can be realized through copolymerization and introduction of hydrophobic links, and the precise activation of the membrane rupture activity can be realized under the slightly acidic environment, so that the tumor cell membrane is irreversibly damaged, and the tumor cell is killed with high selectivity. However, the polymethacrylate main chain is difficult to degrade, and is easy to cause toxicity after long-term use and accumulation in vivo.
Disclosure of Invention
Based on the above, the invention provides a pH sensitive membrane-breaking polypeptide material with a lateral group containing tertiary amine and hydrophobic groups, wherein the macromolecular material is hydrophobic and neutral in electricity and weak in interaction with cell membranes under normal physiological pH; under the pH condition of the micro-acid, the cell membrane can be protonated to form an amphipathic structure consisting of a hydrophobic structural domain and a cationic structural domain, has strong interaction with the cell membrane and strong membrane rupture activity, and can kill tumor cells or bacteria efficiently and selectively. The invention comprises the following technical scheme.
A membrane-disrupting polypeptide having a structure represented by formula (I):
Wherein R is selected from: -R 3 -N(R 4 R 5 )、-R 3 -R’、
R' is selected from:
l is selected from: -NH-C (=o) O-, -NH-C (=o) -, -C (=o) -NH-, -C (=o) -O-;
R 1 selected from: an alkylene group;
R 2 selected from: c (C) 1 -C 12 Alkyl, C 6 -C 14 Aryl, C 6 -C 14 Aryl substituted C 1 -C 12 Alkyl-and benzyloxycarbonyl-substituted C 1 -C 12 Alkyl, 5-10 membered heteroaryl substituted C 1 -C 12 An alkyl group;
R 3 selected from: alkylene, C 6 -C 14 Aryl-substituted alkylene;
R 4 、R 5 each independently selected from: alkyl, C 6 -C 14 Aryl substituted alkyl, or R 3 、R 4 And the nitrogen atom to which it is attached form a heterocycloalkyl group;
y is selected from: 2-150;
R 6 selected from: c (C) 1 -C 15 Alkyl, C 6 -C 14 Aryl, C 6 -C 14 Aryl substituted C 1 -C 15 An alkyl group;
n+m is greater than 0, and n is not 0;
q is selected from: 0. 1, 2, 3 and 4.
On the other hand, the invention also provides a membrane-rupture polypeptide nanoparticle, which comprises the following technical scheme.
A membrane-rupture polypeptide nanoparticle is formed by self-assembling the membrane-rupture polypeptide in an aqueous medium.
On the other hand, the invention also provides a preparation method of the membrane-rupture polypeptide nano-particles, which comprises the following technical scheme.
A preparation method of membrane-breaking polypeptide nano-particles comprises the following steps: dissolving the membrane-broken polypeptide in an organic solvent or hydrochloric acid solution with the pH value of 1.5-2.5, then dropwise adding the obtained solution into water in a stirring state, continuously stirring, and removing the solvent by low-temperature dialysis to obtain the membrane-broken polypeptide nano particles.
On the other hand, the invention also provides application of the membrane-rupture polypeptide or the membrane-rupture polypeptide nano-particles, which comprises the following technical scheme.
The application of the membrane-rupture polypeptide or the stereoisomer or the pharmaceutically acceptable salt thereof in preparing medicaments for preventing and/or treating tumors.
The application of the membrane-rupture polypeptide nano-particles in preparing medicaments for preventing and/or treating tumors.
The application of the membrane-rupture polypeptide or the stereoisomer or the pharmaceutically acceptable salt thereof in combination with an immune checkpoint inhibitor in preparing medicines for preventing and/or treating tumors.
The application of the membrane-rupture polypeptide nanoparticle combined immune checkpoint inhibitor in preparing medicaments for preventing and/or treating tumors.
The application of the membrane-breaking polypeptide or the stereoisomer or the pharmaceutically acceptable salt thereof in preparing the medicines for resisting bacterial infection.
The application of the membrane-rupture polypeptide nano-particles in preparing medicaments for resisting bacterial infection.
On the other hand, the invention also provides a medicine for preventing and/or treating tumors, which comprises the following technical scheme.
The medicine for preventing and/or treating tumor is prepared with active component and pharmaceutically acceptable supplementary material and/or carrier, and the active component includes the membrane breaking polypeptide, its stereoisomer or pharmaceutically acceptable salt and/or the membrane breaking polypeptide nanometer particle.
On the other hand, the invention also provides a combined medicament for preventing and/or treating tumors, which comprises the following technical scheme.
A combination for the prevention and/or treatment of tumors, comprising, as active ingredients:
component 1: the membrane-rupture polypeptide or the stereoisomer or the pharmaceutically acceptable salt thereof, and/or the membrane-rupture polypeptide nano-particles; and
component 2: antitumor drugs other than component 1;
the component 1 and the component 2 are each independent administration units, or the component 1 and the component 2 together form a combined administration unit.
On the other hand, the invention also provides a medicine for resisting bacterial infection, which comprises the following technical scheme.
The antibacterial infection resisting medicine is prepared from active ingredients and pharmaceutically acceptable auxiliary materials and/or carriers, wherein the active ingredients comprise the membrane-breaking polypeptide or stereoisomers or pharmaceutically acceptable salts thereof and/or membrane-breaking polypeptide nano particles.
The invention provides a tertiary amine modified membrane-rupture polypeptide material, which has hydrophobic and neutral electricity under normal physiological pH, and polypeptide fragments have hydrophobic property and weak interaction with cell membranes, so that the material has the advantage of low toxicity to normal tissues during in vivo circulation; under the pH condition of tumor tissue or bacterial infection micro acid, the tertiary amine part of the macromolecular material can be protonated, so that the polypeptide fragment forms an amphipathic structure consisting of a hydrophobic structural domain and a cationic structural domain, and the polypeptide fragment has extremely strong interaction with a cell membrane and extremely strong membrane rupture activity, thereby killing tumor cells and bacteria with high efficiency and high selectivity. The invention also discovers that the polypeptide containing the benzene ring structure can improve the selectivity. The membrane-rupture polypeptide macromolecular material can be used for preparing antitumor or antibacterial drugs, and has the advantages of good antitumor and antibacterial effects, high selectivity and low toxicity. And the polymerized polypeptide has the advantages of degradability and no biological toxicity of degradation products, so that the polymerized polypeptide has wider biomedical application.
Drawings
FIG. 1 is mPEG 44 -PLys(Z) 33 Is a gel permeation chromatogram of (2).
FIG. 2 is mPEG 44 -PLys(Z) 33 Nuclear magnetic hydrogen spectrogram of (2).
FIG. 3 is mPEG 44 -PLys 33 Nuclear magnetic hydrogen spectrogram of (2).
FIG. 4 shows the nuclear magnetic hydrogen spectrum of DE-CDI.
FIG. 5 is mPEG 44 -PLys-DE 33 Nuclear magnetic hydrogen spectrogram of (2).
FIG. 6 shows the protonation curve (A) and the helicity change curve (B) before and after the tertiary amine modified polypeptide of example 1.
FIG. 7 is R 3 Nuclear magnetic hydrogen spectrograms of polypeptides modified for different alkylene groups.
FIG. 8 is R 3 A protonation curve (A) and a helicity change curve (B) for different alkylene modified polypeptides.
FIG. 9 is a gel permeation chromatogram of DE tertiary amine modified different polyethylene glycol initiated polypeptides.
FIG. 10 is a nuclear magnetic resonance spectrum of DE tertiary amine modified different polyethylene glycol initiated polypeptide.
FIG. 11 shows the PEGylation curves of PEG-initiated polypeptides of varying degrees of polymerization.
FIG. 12 is a different tertiary amine modified mPEG 44 -PLys 86 Nuclear magnetic hydrogen spectrogram of (2).
FIG. 13 is a different tertiary amine modified mPEG 44 -PLys 86 A protonation curve (A) and a helicity change curve (B).
FIG. 14 is mPEG 44 -NH 2 Gel permeation chromatograms of polypeptides of different degrees of polymerization obtained by triggering Lys (Z) -NCA.
FIG. 15 is a nuclear magnetic resonance hydrogen spectrum of polylysine with different degrees of polymerization.
FIG. 16 is a graph of the different degrees of polymerization of the polypeptide mPEG 44 -PLys n -protonation curve (a) and helicity change curve (B) of DB.
FIG. 17 is a nuclear magnetic resonance hydrogen spectrum of a C5P2 tertiary amine modified polypeptide of different degrees of polymerization.
FIG. 18 is a nuclear magnetic resonance hydrogen spectrum of a C5P tertiary amine modified polypeptide of varying degrees of polymerization.
FIG. 19 is a nuclear magnetic resonance hydrogen spectrum of a C6P tertiary amine modified polypeptide of varying degrees of polymerization.
FIG. 20 is a nuclear magnetic resonance hydrogen spectrum of a DB tertiary amine modified polypeptide of varying degrees of polymerization.
FIG. 21 is a nuclear magnetic resonance hydrogen spectrum of a DMP2 tertiary amine modified polypeptide of varying degrees of polymerization.
FIG. 22 is a nuclear magnetic resonance hydrogen spectrum of a DMP tertiary amine modified polypeptide of varying degrees of polymerization.
FIG. 23 is mPEG 44 -PLys-CC6 86 Nuclear magnetic hydrogen spectrogram of (2).
FIG. 24 is mPEG 44 -PLys-CC6 86 Is a proton rate curve of (2).
FIG. 25 is mPEG 44 -PBLG 30 Is a gel permeation chromatogram of (2).
FIG. 26 is mPEG 44 -PBLG 30 Nuclear magnetic hydrogen spectrogram of (2).
FIG. 27 is mPEG 44 -PLG-DB 30 Nuclear magnetic hydrogen spectrogram of (2).
FIG. 28 is mPEG 44 -PLG-DB 30 Is a proton rate curve of (2).
FIG. 29 shows the hemolytic activity of different tertiary amine modified polypeptides of different degrees of polymerization.
Fig. 30 is a graph showing cell killing curves of different tertiary amine modified polypeptides of different degrees of polymerization at ph=6.8 for 24h in a Panc02 organelle.
Fig. 31 is a graph showing cell killing curves of different tertiary amine modified polypeptides of different degrees of polymerization at ph=6.8 for 24h in MC38 organelles.
FIG. 32 is mPEG 44 -PLys-C6P 10 24h cell killing curves for MC38 (A) and Panc02 (B) at different pH values.
FIG. 33 is mPEG 44 -PLys-DB 86 4h cell killing curves for MC38 (A) and Panc02 (B) at different pH values.
FIG. 34 is mPEG 44 -PLys-DB 86 Membrane rupture activity at ph=6.8 in Panc02 organelle.
FIG. 35 is mPEG 44 -PLys-DB 86 Is effective in treating tumor in vivo and in treating and combining with alpha PD 1; wherein A is a schematic of a dosing regimen; b is mPEG at different doses 44 -PLys-DB 86 Is effective in treating tumor in vivo; c is mPEG 44 -PLys-DB 86 Therapeutic effects in combination with alpha PD 1.
FIG. 36 shows the antimicrobial activity of polypeptides modified with different tertiary amines to different degrees of polymerization.
FIG. 37 is mPEG 44 -PLys-C5P 33 At different pH valuesAntibacterial activity.
FIG. 38 is a gel permeation chromatogram of a polypeptide obtained by copolymerizing leucine and lysine (benzyloxycarbonyl protected) in different ratios.
FIG. 39 is a nuclear magnetic resonance spectrum of a polypeptide obtained by copolymerizing leucine and lysine in different proportions.
FIG. 40 shows nuclear magnetic resonance spectra of polypeptide obtained by copolymerization of N-hydroxyethylpiperidine modified leucine and lysine in different proportions.
FIG. 41 is a nuclear magnetic resonance spectrum of a polypeptide obtained by copolymerizing 2- (hexamethyleneimine) ethanol modified leucine and lysine in different proportions.
FIG. 42 shows the protonation rate curve (A) and the helicity curve (B) of the polypeptide obtained by copolymerization of N-hydroxyethylpiperidine modified leucine and lysine in different proportions.
FIG. 43 shows the protonation rate curve (A) and the helicity curve (B) of the polypeptide obtained by copolymerization of 2- (hexamethyleneimine) ethanol modified leucine and lysine in different proportions.
FIG. 44 is mPEG 44 -NH 2 Gel permeation chromatograms of the resulting polypeptide were initiated by copolymerization of phenylalanine and lysine (benzyloxycarbonyl protected) in different ratios.
FIG. 45 is mPEG 44 -NH 2 And (3) inducing different proportions of phenylalanine and lysine (benzyloxycarbonyl protection) to copolymerize to obtain the nuclear magnetic hydrogen spectrogram of the polypeptide.
FIG. 46 is a nuclear magnetic resonance spectrum of a polypeptide obtained by copolymerizing N-hydroxyethylpiperidine modified phenylalanine and lysine in different proportions.
FIG. 47 is a nuclear magnetic resonance spectrum of a polypeptide obtained by copolymerizing 2- (hexamethyleneimine) ethanol-modified phenylalanine and lysine in different proportions.
FIG. 48 shows the protonation rate curves of the polypeptides obtained by copolymerization of N-hydroxyethylpiperidine (A) and 2- (hexamethyleneimine) ethanol (B) modified phenylalanine and lysine in different proportions.
FIG. 49 is mPEG 112 -NH 2 Gel permeation chromatograms of the resulting polypeptide were initiated by copolymerization of phenylalanine and lysine (benzyloxycarbonyl protected) in different ratios.
FIG. 50 is mPEG 112 -NH 2 And (3) initiating the nuclear magnetic resonance hydrogen spectrogram of the polypeptide obtained by copolymerization of phenylalanine and lysine in different proportions.
FIG. 51 is N-hydroxyethyl piperidine modified mPEG 112 -NH 2 Nuclear magnetic hydrogen spectrogram of polypeptide obtained by copolymerization of initiated phenylalanine and lysine in different proportions.
FIG. 52 is N-hydroxyethyl piperidine modified mPEG 112 -NH 2 And (3) a protonation rate curve of the polypeptide obtained by copolymerization of the initiated phenylalanine and lysine in different proportions.
FIG. 53 is a gel permeation chromatogram of a polypeptide obtained by copolymerizing norleucine and lysine (benzyloxycarbonyl protected) in different ratios.
FIG. 54 is a nuclear magnetic resonance spectrum of a polypeptide obtained by copolymerizing norleucine and lysine (benzyloxycarbonyl protection) in different ratios.
FIG. 55 shows nuclear magnetic resonance spectra of the polypeptide obtained by copolymerizing norleucine and lysine in different proportions.
FIG. 56 is a nuclear magnetic resonance spectrum of a polypeptide obtained by copolymerizing N-hydroxyethyl piperidine modified norleucine and lysine in different proportions.
FIG. 57 is a nuclear magnetic resonance spectrum of a polypeptide obtained by copolymerizing N-hydroxyethyl piperidine modified L-aminocaprylic acid and lysine in different proportions.
FIG. 58 is a nuclear magnetic resonance spectrum of a polypeptide obtained by 1:1 copolymerization of N-hydroxyethyl piperidine modified tryptophan and lysine.
FIG. 59 shows the haemolytic activity of C6 tertiary amine modified co-lysines of different hydrophobic amino acids.
FIG. 60 is a graph of tumor selective killing of C6 tertiary amine modified co-lysines of different hydrophobic amino acids.
FIG. 61 is a graph showing tumor selective killing of C6 tertiary amine modified tryptophan and lysine co-polypeptide at different pH.
FIG. 62 is mPEG 44 -P(Lys-C6 50 -co-Trp 50 ) Inducing cell membrane breakage to release LDH.
FIG. 63 is mPEG 44 -P(Lys-C6 50 -co-Trp 50 ) A kind of electronic deviceAn in vivo tumor treatment effect; wherein A is a schematic of a dosing regimen, and B is mPEG 44 -P(Lys-C6 50 -co-Trp 50 ) Is effective in treating tumor.
Detailed Description
The experimental methods of the present invention, in which specific conditions are not specified in the following examples, are generally conducted under conventional conditions or under conditions recommended by the manufacturer. The various chemicals commonly used in the examples are commercially available.
Unless defined otherwise, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this invention belongs. The terminology used in the description of the invention herein is for the purpose of describing particular embodiments only and is not intended to be limiting of the invention.
The terms "comprising" and "having" and any variations thereof, are intended to cover a non-exclusive inclusion. For example, a process, method, apparatus, article, or device that comprises a list of steps is not limited to the elements or modules listed but may alternatively include additional steps not listed or inherent to such process, method, article, or device.
In the present invention, the term "plurality" means two or more. "and/or", describes an association relationship of an association object, and indicates that there may be three relationships, for example, a and/or B, and may indicate: a exists alone, A and B exist together, and B exists alone. The character "/" generally indicates that the context-dependent object is an "or" relationship.
In the compounds of the invention, when any variable (e.g., R 1 、R 2 Etc.) occur more than once in any component, the definition of each occurrence is independent of the definition of each other occurrence. Also, combinations of substituents and variables are permissible provided that such combinations stabilize the compounds. The lines drawn from the substituents into the ring system indicate that the bond referred to may be attached to any substitutable ring atom. If the ring system is polycyclic, it means that such bonds are only attached to any of the adjacent ringsOn a suitable carbon atom. It is to be understood that substituents and substitution patterns of the compounds of this invention may be selected by one of ordinary skill in the art to provide compounds that are chemically stable and that may be readily synthesized from readily available starting materials by techniques in the art and methods set forth below. If the substituent itself is substituted with more than one group, it is understood that these groups may be on the same carbon atom or on different carbon atoms, as long as the structure is stabilized.
The term "alkyl" is meant to include both branched and straight chain saturated aliphatic hydrocarbon groups having a specified number of carbon atoms. For example, "C 1 -C 6 Alkyl "medium" C 1 -C 6 The definition of "includes groups having 1, 2, 3, 4, 5 or 6 carbon atoms arranged in a straight or branched chain. For example, "C 1 -C 6 The alkyl group includes, in particular, methyl, ethyl, n-propyl, isopropyl, n-butyl, t-butyl, isobutyl, pentyl, hexyl.
The term "alkylene" refers to groups having one less hydrogen on an "alkyl" basis, e.g., -CH 2 -、-CH 2 CH 2 -、-CH 2 CH 2 CH 2 -、-CH 2 CH 2 CH 2 CH 2 -and the like.
The term "heterocycloalkyl" is a saturated monocyclic ring substituent in which one or more ring atoms are selected from heteroatoms of N, O or S (O) m (where m is an integer from 0 to 2) and the remaining ring atoms are carbon, for example: piperidinyl, pyrrolidinyl, and the like.
In one embodiment of the present invention, there is provided a membrane-disrupting polypeptide having a structure represented by formula (I):
wherein R is selected from: -R 3 -N(R 4 R 5 )、-R 3 -R’、
R' is selected from:
l is selected from: -NH-C (=o) O-, -NH-C (=o) -, -C (=o) -NH-, -C (=o) -O-;
R 1 selected from: an alkylene group;
R 2 selected from: c (C) 1 -C 12 Alkyl, C 6 -C 14 Aryl, C 6 -C 14 Aryl substituted C 1 -C 12 Alkyl-and benzyloxycarbonyl-substituted C 1 -C 12 Alkyl, 5-10 membered heteroaryl substituted C 1 -C 12 An alkyl group;
R 3 selected from: alkylene, C 6 -C 14 Aryl-substituted alkylene;
R 4 、R 5 each independently selected from: alkyl, C 6 -C 14 Aryl substituted alkyl, or R 3 、R 4 And the nitrogen atom to which it is attached form a heterocycloalkyl group;
y is selected from: 2-150;
R 6 selected from: c (C) 1 -C 15 Alkyl, C 6 -C 14 Aryl, C 6 -C 14 Aryl substituted C 1 -C 15 An alkyl group;
n+m is greater than 0, and n is not 0;
q is selected from: 0. 1, 2, 3 and 4.
In some of these embodiments, R 1 Selected from: c (C) 1 -C 6 An alkylene group.
In some of these embodiments, R 1 Selected from: - (CH) 2 ) x -wherein x is selected from: 1. 2, 3, 4, 5, 6.
In some embodiments, the membrane-disrupting polypeptide has a structure according to formula (II):
wherein X is: -O-or none.
In some embodiments, the membrane-disrupting polypeptide has a structure according to formula (III):
in some embodiments, R is selected from: -R 3 -N(R 4 R 5 )、
In some of these embodiments, R 3 Selected from: c (C) 1 -C 6 Alkylene-phenyl-substituted C 1 -C 6 An alkylene group.
In some of these embodiments, R 3 Selected from: - (CH) 2 ) x -, phenyl-substituted- (CH) 2 ) x The method comprises the steps of carrying out a first treatment on the surface of the Wherein x is selected from: 1. 2, 3, 4, 5, 6.
In some of these embodiments, R 4 、R 5 Each independently selected from: c (C) 1 -C 12 Alkyl-substituted C 1 -C 6 Alkyl, naphthyl substituted C 1 -C 6 Alkyl, or R 4 、R 5 And the nitrogen atom to which it is attached form a 5-to 10-membered heterocycloalkyl.
In some of these embodiments, R 4 、R 5 Each independently selected from: c (C) 1 -C 6 Alkyl-substituted C 1 -C 3 Alkyl, naphthyl substituted C 1 -C 3 Alkyl, or R 4 、R 5 And the nitrogen atom to which it is attached form a 5-8 membered heterocycloalkyl.
In some of these embodiments, R 4 、R 5 And the nitrogen atom to which it is attached form the following group:
in some of these embodiments, R 6 Selected from: c (C) 1 -C 6 Alkyl, phenyl, naphthyl, phenyl-substituted C 1 -C 6 An alkyl group.
In some embodiments, R is selected from: -R 3 -N(R 4 R 5 )、
Wherein R is 3 Selected from: -CH 2 -CH 2 -、-CH 2 -CH 2 -CH 2 -、-CH 2 -(CH 2 ) 3 -CH 2 -、 R 4 、R 5 And the nitrogen atom to which it is attached form the following group:
R 6 is benzyl.
In some of these embodiments, R 2 Selected from: c (C) 1 -C 8 Alkyl, phenyl, naphthyl, phenyl-substituted C 1 -C 6 Alkyl-and benzyloxycarbonyl-substituted C 1 -C 6 Alkyl, 5-10 membered heteroaryl substituted C 1 -C 6 An alkyl group.
In some of these embodiments, R 2 Selected from: methyl, ethyl, n-propyl, isopropyl, n-butyl, isobutyl, pentyl, hexyl, heptyl, octyl, nonanyl, decyl, undecyl, dodecyl, phenyl, naphthyl, benzyl, benzyloxycarbonyl-substituted ethyl, benzopyrrole-substituted ethyl.
In some embodiments, y is selected from: 5-120.
In some embodiments, y is selected from: 30-120.
In some embodiments, y is selected from: 40-48 or 108-116.
In some embodiments, y is selected from: 9. 44, 112.
In some of these embodiments, n+m is not less than 5, more preferably not less than 10.
In some of these embodiments, n+m is 10 to 200, more preferably 10 to 150, and even more preferably 10 to 110.
In some embodiments, m is 0; n is 5 to 200, preferably 10 to 150, preferably 10 to 110.
In some embodiments, m is 0; n is 10-15, 30-35, 60-65, 80-90, or 120-130.
In some embodiments, m is 0; n is 10, 30, 33, 61, 86, 128.
In some of these embodiments, m is 0-60%, more preferably 0-50%, of n+m.
In some embodiments, m is 0-30% of n+m.
In some of these embodiments, m is 0-25% of n+m.
In some of these embodiments, m is 10-23% of n+m.
In some embodiments, m is 15-25% of n+m.
In some of these embodiments, m is 25-35% of n+m.
In some of these embodiments, m is 35-45% of n+m.
In some embodiments, m is 40-50% of n+m.
In some of these embodiments, m is 45-50% of n+m.
In some embodiments, R is selected from: -R 3 -N(R 4 R 5 )、
Wherein R is 3 is-CH 2 -CH 2 -；R 4 、R 5 And the nitrogen atom to which it is attached form the following group:
R 6 is benzyl; y is 40-48.
In some embodiments, R is selected from: -R 3 -N(R 4 R 5 )；
Wherein R is 3 Selected from: -CH 2 -CH 2 -；R 4 、R 5 And the nitrogen atom to which it is attached form the following group:
R 2 selected from: methyl, ethyl, n-propyl, isopropyl, n-butyl, isobutyl, pentyl, hexyl, heptyl, octyl, nonanyl, decyl, undecyl, dodecyl, phenyl, naphthyl, benzyl, benzyloxycarbonyl-substituted ethyl, benzopyrrole-substituted ethyl.
In some embodiments, the membrane-disrupting polypeptide is selected from the group consisting of:
in another embodiment of the invention, a membrane-rupture polypeptide nanoparticle is provided, which is formed by self-assembling the membrane-rupture polypeptide in an aqueous medium.
In another embodiment of the present invention, a method for preparing the membrane-broken polypeptide nanoparticle is provided, which comprises the following steps: dissolving the membrane-broken polypeptide in an organic solvent or hydrochloric acid solution with the pH value of 1.5-2.5, then dropwise adding the obtained solution into water in a stirring state, continuously stirring, and removing the solvent by low-temperature dialysis to obtain the membrane-broken polypeptide nano particles.
In some of these embodiments, the organic solvent is N, N-dimethylformamide.
In some embodiments, the ratio of the membrane-breaking polypeptide, the organic solvent or the hydrochloric acid solution and the water is 10 mg-30 mg:1mL:4-6mL.
In some embodiments, the method for preparing the membrane-broken polypeptide nanoparticle comprises the following steps: the membrane-breaking polypeptide is prepared from 10mg to 30mg:1mL is dissolved in N, N-dimethylformamide, then the obtained solution is dropwise added into water under the stirring state of 400-800 rpm, the stirring is continued for 8-20 min at the speed of 200-600 rpm, and a dialysis bag with the molecular weight cutoff of 10000-20000 is used for dialysis in water to remove the solvent, thus obtaining the membrane-breaking polypeptide nano-particles.
In another embodiment of the invention, the application of the membrane-breaking polypeptide or the stereoisomer or the pharmaceutically acceptable salt thereof in preparing a medicament for preventing and/or treating tumors is also provided.
In another embodiment of the invention, the application of the membrane-rupture polypeptide nano-particles in preparing a medicament for preventing and/or treating tumors is also provided.
In another embodiment of the invention, the use of a membrane-breaking polypeptide or a stereoisomer or a pharmaceutically acceptable salt thereof in combination with an immune checkpoint inhibitor for the preparation of a medicament for the prevention and/or treatment of a tumor is also provided.
In another embodiment of the invention, the application of the membrane-rupture polypeptide nanoparticle combined immune checkpoint inhibitor in preparing medicaments for preventing and/or treating tumors is also provided.
In some of these embodiments, the immune checkpoint inhibitor is a PD-1 inhibitor.
In some embodiments, the tumor is pancreatic cancer, melanoma, colorectal cancer, colon cancer, lung cancer, tongue squamous carcinoma, cervical cancer, ovarian cancer, osteosarcoma, liver cancer, breast cancer, bladder cancer, ovarian epithelial cancer.
In another embodiment of the invention, the application of the membrane-breaking polypeptide or the stereoisomer or the pharmaceutically acceptable salt thereof in preparing the antibacterial infection resisting medicine is also provided.
In another embodiment of the invention, the application of the membrane-rupture polypeptide nanoparticle in preparing a medicament for resisting bacterial infection is also provided.
In some embodiments, the bacterium is a gram-negative bacillus, a gram-negative pseudomonas, a gram-positive staphylococcus, a gram-positive coccus, a gram-positive bacillus, a streptococcus.
In some embodiments, the bacterium is escherichia coli, salmonella, staphylococcus aureus, klebsiella pneumoniae, pseudomonas aeruginosa, enterococcus faecalis, streptococcus pyogenes, streptococcus pneumoniae, acinetobacter baumannii, diplococcus pneumoniae, pseudomonas aeruginosa.
In another embodiment of the invention, a medicine for preventing and/or treating tumor is provided, which is prepared from an active ingredient and pharmaceutically acceptable auxiliary materials and/or carriers, wherein the active ingredient comprises the membrane-breaking polypeptide or stereoisomer or pharmaceutically acceptable salt thereof, and/or the membrane-breaking polypeptide nano-particles.
In another embodiment of the present invention, there is also provided a combination for preventing and/or treating tumors, the active ingredients of which include:
component 1: the membrane-rupture polypeptide or the stereoisomer or the pharmaceutically acceptable salt thereof, and/or the membrane-rupture polypeptide nano-particles; and
component 2: antitumor drugs other than component 1;
the component 1 and the component 2 are each independent administration units, or the component 1 and the component 2 together form a combined administration unit.
In some of these embodiments, the component 2 is an immune checkpoint inhibitor.
In some of these embodiments, the immune checkpoint inhibitor is a PD-1 inhibitor.
The compounds of formula (I) -formula (III) of the present invention may be used in combination with other known antitumor agents. When administered in combination, the compounds of formula (I) -formula (III) and the known agent may each be separate administration units or together form a combined administration unit; the compounds of formula (I) -formula (III) may be administered simultaneously with or separately from other known antitumor agents. When the compounds of formula (I) -formula (III) are administered simultaneously with one or more other drugs, it is preferred to use a pharmaceutical composition containing one or more known drugs together with the compounds of formula (I) -formula (III). Drug combinations also include administration of the compounds of formulas (I) - (III) with one or more other known drugs over overlapping time periods. When the compounds of formula (I) -formula (III) are administered in combination with one or more other known drugs, the dosage of the compounds of formula (I) -formula (III) or the known drugs may be the same as the dosage administered alone or may be lower than the dosage at which they are administered alone.
Drugs or active ingredients that may be used in combination with the compounds of formulas (I) - (III) include, but are not limited to: immune checkpoint inhibitors, estrogen receptor modulators, androgen receptor modulators, retinoid receptor modulators, cytotoxin/cytostatics, antiproliferatives, protein transferase inhibitors, HMG-CoA reductase inhibitors, HIV protein kinase inhibitors, reverse transcriptase inhibitors, angiogenesis inhibitors, cell proliferation and survival signaling inhibitors, agents 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 activators, VEGF antibodies, EGF antibodies, and the like.
In some of these embodiments, the drugs or active ingredients that may be used in combination with the compounds of formulas (I) - (III) include, but are not limited to: albumin, alendronic acid, interferon, al Qu Nuoying, allopurinol sodium, palonosetron hydrochloride, altretamine, aminoglutethimide, amifostine, amrubicin, an Ya pyridine, anastrozole, dolasetron, aranesp, arglabin, arsenic trioxide, minoxin, 5-azacytidine, azathioprine, BCG or tice BCG, betadine, betamethasone acetate, betamethasone sodium phosphate formulation, bexarotene, bleomycin sulfate, british, bortezomib, busulfan, calcitonin, alezomib injection, capecitabine, carboplatin, kang Shide, cefesone, cet Mo Baijie, daunorubicin, chlorambucil, cisplatin, cladribine, clofaxine, cyclophosphamide, cytarabine, dacarbazine, dactinomycin, daunorubicin, dexamethasone dexamethasone phosphate, estradiol valerate, deniinterleukin 2, dibaume, dulorelin, delazocine, diethylstilbestrol, dafukang, docetaxel, deoxyfluorouridine, doxorubicin, dronabinol, jejunum-166-chitosan complex, eligard, labyrinase, epirubicin hydrochloride, aprepitant, epirubicin, alfuzoxetine, erythropoietin, eplatin, levamisole, estradiol formulations, 17-beta-estradiol, estramustine sodium phosphate, ethinyl estradiol, amifostine, hydroxy phosphate, petrolatum, etoposide, fadrozole, tamoxifen formulations, febuxostat, finasteride, feveride, fluorouridine, fluconazole, fludarabine, 5-fluorodeoxyuridine monophosphate, 5-fluorouracil, fluoxytestosterone, flusteramine, fotemustine, fludarabine, 1-beta-D-arabinofuranosyl-cytothiadine-5' -stearoyl phosphate, fotemustine, fulvestrant, progastrin, gemcitabine, gemtuzumab, imatinib mesylate, carmustine wafer capsule, goserelin, glatiramer hydrochloride, histrelin, and meflozin, hydrocortisone, erythro-hydroxynonyladenine, hydroxyurea, tetan iso Bei Moshan antibody, idarubicin, ifosfamide, interferon alpha 2A, interferon alpha 2B, interferon alpha nl, interferon alpha n3, interferon beta, interferon gamma la, interleukin 2, intron A, iressa, irinotecan, ketjel, lentinan sulfate, letrozole, leucovorin, leuprorelin acetate, levamisole calcium levofolinate, sodium levothyroxine formulations, lomustine, lonidamine, dronabinol, nitrogen mustard, mecobalamin, medroxyprogesterone acetate, megestrol acetate, melphalan, esterified estrogens, 6-borazine, mesna, methotrexate, methyl aminolevulinate, miltefosine, melomycin, mitomycin C, mitotane, mitoquinone, trospine, doxorubicin citrate liposomes, nedaplatin, pegylated febuxostat, olpreninterleukin, neunogen, nilutamide, tamoxifen, NSC-631570, recombinant human interleukin 1-beta, octreotide, ondansetron hydrochloride, dehydrohydrocortisone oral solution, oxaliplatin, paclitaxel, prednisone sodium phosphate formulations, pegine, roxyprogesterone, euphorbia, pernicid, and the like, pennisetum, streptozotocin, pilocarpine hydrochloride, bicubicin, plicamycin, porphin sodium, prednimustine, setprednisolone, prednisone, beclomethamine, procarbazine, recombinant human erythropoietin, raltitrexed, liratio, etidronate rhenium-186, mevalhua, dynamics stretch-A, romidepide, pilocarpine hydrochloride tablet, octreotide, sarustine, semustine, sirolimus, sibutramine, sibutrazol, sodium methylprednisolone, palustric acid, stem cell therapy, streptozocin, strontium chloride-89, levothyroxine sodium, tamoxifen, tamsulosin, testolazine, taxotere, temozolomide, teniposide, testosterone, thioguanine, thiotepa, somatostatin, temozolomide, toldronic acid, topotecan, tolnaftate, tolizumab, toxidan trastuzumab, trocounter, treoshu, tretinoin, methotrexate tablet, trimethamine, trimetraxazole, triptorelin acetate, trastuline pamoate, ulipraline, uridine, valrubicin, valdecolonil, vinblastine, vincristine, vinlamide, vinorelbine, vitamin Lu Liqin, dexpropimine, neat-Ding Sizhi, pivalonine, paclitaxel protein stabilized formulation, acolbifene, interferon r-lb, affinitak, aminopterin, alzoxifene, asorisnil, atomestane, atrasentan, BAY43-9006, avastin, CCI-779, CDC-501, celebantam, cetuximab, crizotrope, cyproterone acetate, decitabine, DN-101, doxorubicin-MTC, dIM, dutasteride, edoxin, irinotecan, flunine, valirbestrol, bivalirudin, amiloride, daphne hydrochloride, daphne, holmium-166 DOTMP, ibandronic acid, interferon gamma, intron-PEG, ixabepilone, keyhole limpet hemocyanin, L-651582, lanreotide, lasofoxifene, libra, lonafamib, milbexifene, mi Nuoqu acid ester, MS-209, liposomal MTP-PE, MX-6, nafarelin, nemorubicin, neovalproate, norlabratex, olimarson, onco-TCS, oside, paclitaxel polyglutamate, sodium silk-miate, PN-401, QS-21, quaternary, R-154, raloxifene, ranpirnase, 13-cis-retinoic acid, satraplatin, orcalcitol, T-138067, tarceva, docosahexaenoic acid paclitaxel, thymol, prostaglandin furin, tipifarnib, tiramimine, TLK-286, toremio, 7R, valproan, prandin, ibvanadn, R-154, raloxifene, MID-100, and combinations thereof.
In another embodiment of the invention, an antibacterial agent is provided, which is prepared from an active ingredient and pharmaceutically acceptable auxiliary materials and/or carriers, wherein the active ingredient comprises the membrane-breaking polypeptide or a stereoisomer or a pharmaceutically acceptable salt thereof, and/or the membrane-breaking polypeptide nano-particles.
The medicament for preventing and/or treating tumors or the medicament for resisting bacterial infection of the present invention may be used for non-human mammals or humans.
The pharmaceutically acceptable auxiliary materials used in the medicine for preventing and/or treating tumor or the medicine for resisting bacterial infection of the invention are as follows: one or more compatible solid or liquid filler or gel materials which are suitable for human use and must be of sufficient purity and sufficiently low toxicity.
"compatibility" as used herein means that the components of the composition are capable of blending with and between the active ingredients of the present invention (tertiary amine modified polypeptide rupture materials of formulas I-III) without significantly reducing the efficacy of the active ingredient.
Pharmaceutically acceptable excipients used in the medicament 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 a solvent, excipient, filler, compatibilizer, binder, humectant, disintegrant, slow solvent, absorption accelerator, adsorbent, diluent, solubilizer, emulsifier, lubricant, wetting agent, suspending agent, flavoring agent, and perfume.
Examples of pharmaceutically acceptable adjuvant parts include cellulose and its derivatives (such as sodium carboxymethylcellulose, sodium ethylcellulose, 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.), polyalcohols (such as propylene glycol, glycerol, mannitol, sorbitol, etc.), milkChemokines (e.g. Tween) Wetting agents (such as sodium lauryl sulfate), coloring agents, flavoring agents, stabilizing agents, antioxidants, preservatives, pyrogen-free water and the like.
The mode of administration of the active ingredient or pharmaceutical composition of the present invention is not particularly limited, and representative modes of administration include (but are not limited to): oral, rectal, parenteral (intravenous, intramuscular, or subcutaneous), and the like.
Solid dosage forms for oral administration include capsules, tablets, pills, powders and granules.
In these solid dosage forms, the active ingredient is admixed with at least one conventional inert excipient (or carrier), such as sodium citrate or dicalcium phosphate, or with the following ingredients:
(a) Fillers or compatibilizers, for example, starch, lactose, sucrose, glucose, mannitol and silicic acid;
(b) Binders, for example, hydroxymethyl cellulose, alginate, gelatin, polyvinylpyrrolidone, sucrose and acacia;
(c) Humectants, for example, glycerin;
(d) Disintegrants, for example, agar-agar, calcium carbonate, potato or tapioca starch, alginic acid, certain complex silicates, and sodium carbonate;
(e) Slow solvents, such as paraffin;
(f) Absorption accelerators, for example quaternary amine compounds;
(g) Wetting agents, for example cetyl alcohol and glycerol monostearate;
(h) Adsorbents, such as kaolin; and
(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 forms may also comprise buffering agents.
The solid dosage forms may also be prepared using coatings and shells, such as enteric coatings and other materials known in the art. They may contain opacifying agents and the release of the active ingredient in such a composition may be released in a delayed manner in a certain part of the digestive tract. Examples of embedding components that can be used are polymeric substances and waxes.
Liquid dosage forms for oral administration include pharmaceutically acceptable emulsions, solutions, suspensions, syrups or tinctures. In addition to the active ingredient, the liquid dosage forms may contain inert diluents commonly used in the art such as, for example, water or other solvents, solubilizing agents and emulsifiers such as ethyl alcohol, isopropyl alcohol, ethyl carbonate, ethyl acetate, propylene glycol, 1, 3-butylene glycol, dimethylformamide and oils, in particular, cottonseed, groundnut, corn germ, olive, castor and sesame oils or mixtures of these substances and the like. In addition to these inert diluents, the compositions can also include adjuvants such as wetting agents, emulsifying and suspending agents, sweetening, flavoring, and perfuming agents.
Suspensions, in addition to the active ingredient, may contain suspending agents as, for example, ethoxylated isostearyl alcohols, polyoxyethylene sorbitol and sorbitan esters, microcrystalline cellulose, aluminum methoxide and agar or mixtures of these substances, and the like.
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 nonaqueous carriers, diluents, solvents or excipients include water, ethanol, polyols and suitable mixtures thereof.
The following are specific examples.
In the examples below by mPEG-NH 2 Initiating R 1a -NCA、R 2 Polymerization of NCA copolymer A polypeptide was synthesized by R 1a And introducing tertiary amine to obtain a series of membrane-breaking polypeptide high polymer materials. The reaction formula and corresponding abbreviation are as follows:
wherein mPEG-NH 2 Is an initiator, R 1a -NCA is an N-carboxylic anhydride of an amino acid monomer with a modifiable side chain, R 2 -NCA is the N-carboxylic anhydride of a hydrophobic amino acid monomer, -N (R 4 R 5 ) Is a tertiary amine structure that can be protonated with changes in pH.
When R is 1a when-NCA is Lys (Z) -NCA, TFA/HBr/CH is required before modification of the side chain 3 COOH deprotection. Alcohols containing tertiary amine structures (R-OH) for side chain modification are commercially available products, and R-OH is reacted with carbonyl imidazole (CDI) to prepare R-CDI, which is then used for side chain modification.
The R-CDI is prepared by reacting R-OH and CDI in dichloromethane, adding deionized water to remove unreacted CDI after the reaction is finished, extracting and separating by dichloromethane, drying by anhydrous magnesium sulfate to obtain dichloromethane solution of R-CDI, and pumping to dryness.
The side chain modification method is as follows: dissolving the deprotected polypeptide in N, N-Dimethylformamide (DMF), adding R-CDI (2 times of excess) by a syringe, adding triethylamine, stirring for reaction for 24 hours, precipitating in diethyl ether, pumping to dryness, dissolving with deionized water, dialyzing in deionized water for 24 hours by a dialysis bag with molecular weight cutoff of 3500, changing water every 2 hours, and lyophilizing to obtain the membrane-broken polypeptide.
When R is 1a When NCA is BLG-NCA, TFA/HBr/CH is required before modification of the side chain 3 COOH deprotection. Primary amines (R-NH) containing tertiary amine structures for side chain modification 2 ) All are commercial products available in the market, and carboxyl groups of polyglutamic acid side chains react with BOP-Cl/DMAP and then react with R-NH 2 And (3) performing reaction, namely dialyzing in deionized water for 24 hours by using a dialysis bag with the molecular weight cut-off of 3500, changing water every 2 hours, and freeze-drying to obtain the membrane-breaking polypeptide.
Example 1: tertiary amine modified polyethylene glycol-polylysine copolymer (polypeptide)
(one) N, N-diethyl ethanol modified Polypeptides
y＝44, n=33, m=0, r-OH is N, N-diethyl ethanol. When using mPEG 44 -NH 2 Deprotection upon initiation of Lys (Z) -NCA gives mPEG 44 -PLys 33 N, N-diethyl ethanol is modified to a side chain of polylysine through CDI, and the influence of tertiary amine modified polylysine side chain on the pKa and the helix structure of polylysine is studied. The reaction equation is as follows:
the method comprises the following specific steps:
(1) 10g of Lys (Z) is weighed, pumped overnight by an oil pump, transferred into a glove box, 250mL of Tetrahydrofuran (THF) is added, the mixture is transferred out, placed on an ice bath for stirring, 11.5g of triphosgene is added, a condenser tube is connected, the mixture is stirred for about 10min, transferred into the oil bath for reaction at 50 ℃ for about 2.5h, transferred into the glove box after being pumped out, dissolved by ethyl acetate, recrystallized three times in normal hexane and pumped out, and Lys (N) -NCA is obtained for standby.
(2) Azeotropic removal of mPEG from toluene 44 -NH 2 The water vapor in the water vapor is pumped and then transferred into a glove box for standby.
(3) 500mg of mPEG was weighed out 44 -NH 2 Dissolving in 5mL of dichloromethane to obtain mPEG 44 -NH 2 A solution. 10.0g of Lys (N) -NCA was weighed into 30mL of N, N-dimethylformamide and mPEG was taken up in one portion with a syringe 44 -NH 2 Adding the solution into DMF solution, reacting for 24h, pumping off the reaction liquid, adding 10mL of dichloromethane for dissolving, dripping into n-hexane for precipitation, removing the supernatant, and pumping off to obtain polymer mPEG 44 -PLys(Z) 33 And (5) standby. The polymer was a unimodal distribution of pdi=1.12 as characterized by gel permeation chromatography, and the structure was characterized by nuclear magnetic hydrogen spectroscopy as shown in fig. 1, and the degree of polymerization was calculated as 33 from the integrated area of the nuclear magnetism as shown in fig. 2.
(4) Weighing the polymer mPEG prepared in the step (3) 44 -PLys(Z) 33 5.0g, dissolved in 5mL of CF 3 COOH, 6.0mLHBr/CH was added 3 COOH, allowing reaction for 4h, pumping solution with oil pump, adding DMF for dissolving, precipitating into diethyl ether, removing supernatant, pumping, dissolving with deionized water, dialyzing with dialysis bag with molecular weight cut-off of 3500 in deionized water for 24h, changing water every 2h, and lyophilizing to obtain mPEG 44 -PLys 33 And (5) standby. The structure was characterized by nuclear magnetic resonance hydrogen spectrum, as shown in FIG. 3, the benzyloxycarbonyl leaving peak disappeared at 5.0ppm and 7.25ppm, and the g peak was significantly changed, demonstrating successful deprotection.
(5) CDI was placed in a round bottom flask, anhydrous dichloromethane was added to disperse (1.0 g CDI plus 5mL dichloromethane), the plug was sealed, N-diethyl ethanol (2 fold excess CDI) was slowly added with a syringe, the solution was gradually clarified, deionized water equivalent to dichloromethane was added after 4h of reaction, the reaction was about 5min, the lower dichloromethane phase was removed by a separating funnel, anhydrous magnesium sulfate was added to dry for 1h, the solids were removed by filtration to give a solution and the solution was drained, i.e., N-diethyl ethanol-CDI (DE-CDI). The success of the DE-CDI bonding is proved by the characterization of the nuclear magnetic hydrogen spectrum, as shown in figure 4, with the integration area of each peak corresponding.
(6) mPEG (methyl polyethylene glycol) 44 -PLys 33 Dissolving in DMF, adding DE-CDI (2 times excess), adding triethylamine (equivalent to lysine side chain amino group), reacting for 24 hr, dripping into anhydrous diethyl ether for precipitation, removing diethyl ether, draining, dissolving in DMSO, loading in 3.5k dialysis bag, dialyzing in deionized water for 24 hr, changing water for 2 hr, and lyophilizing to obtain mPEG modified with N, N-diethyl ethanol 44 -PLys 33 Standby (mPEG) 44 -PLys-DE 33 ). The structure is characterized by nuclear magnetic hydrogen spectrum, as shown in figure 5, g peak is obviously shifted to low field after modification, and the complete bonding is proved by calculation.
(7) 100. Mu.L of concentrated hydrochloric acid (37%, 12 mol) was added to 100mL of deionized water and dissolved well to give a clear and transparent solution. 10mg of the polymer material (mPEG) prepared in step (6) was dissolved in 10mL of hydrochloric acid solution 44 -PLys-DE 33 ) Extending pH probe under the liquid surface, stirringIn the stirred state (stirring speed mot=3), titration with 0.5M sodium hydroxide titration solution was performed. After the pH meter is stable, the reading is recorded, meanwhile, liquid round-off chromatography is adopted at some pH values, and the measurement is put back and titration is continued. Until the titration result is ph=11. By deriving the titration curve to obtain extreme points, defining the protonation rate as 1 and 0 points, and establishing a pH and protonation rate curve, as shown as A in FIG. 6, the pKa of the polypeptide is obviously reduced after tertiary amine modification of the amino group of the polylysine side chain. The relationship between polymer helicity and pH was obtained by computational analysis of a circular dichroism spectrum, as shown in FIG. 6B, which shows that the critical pH for the helical transition of the polypeptide was significantly reduced when the tertiary amine modified the amino group of the polylysine side chain. The results are shown in the following table:
Polypeptide pKa Critical pH for spiral transition
mPEG 44 -PLys 33 9.47 9.98
mPEG 44 -PLys-DE 33 7.89 7.4
From the results, the tertiary amine modification can obviously reduce the pKa of the polylysine, and is expected to realize low protonation in the environment of ph=7.4, so that the membrane rupture activity of the polylysine under physiological conditions is reduced.
(II) R 3 Polypeptides of different alkylene groups
y=44, n=33, m=0, i.e. the initiator is mPEG 44 -NH 2 (molecular weight 2000), lysine polymerization degree of 33, R 4 And R is 5 The structure of the ethyl group and the nitrogen atom is as follows:study R 3 Is the effect of different alkylene groups on polylysine pKa and helix structure. The specific reaction equation is as follows:
wherein the structure of R-OH is as follows:
i.e. R 3 The method comprises the following steps of: ethylene, propylene, pentylene.
The method comprises the following specific steps:
mPEG 44 -Lys 33 and mPEG in example 1 44 -Lys 33 Is the same batch of polypeptide.
The CDI is placed in a round bottom flask, anhydrous dichloromethane is added for stirring and dispersing (1 g of CDI is added with 5mL of dichloromethane), a rubber plug is sealed, R-OH (2 times of CDI excess) is slowly added by a syringe, the solution is gradually clarified, deionized water with the same amount as the dichloromethane is added after the reaction for 4 hours, the reaction is carried out for about 5 minutes, a separating funnel takes down the dichloromethane phase of the lower layer, anhydrous magnesium sulfate is added for drying for 1 hour, solids are removed by filtration, and the solution is obtained and pumped to dryness, namely the R-CDI. mPEG (methyl polyethylene glycol) 44 -PLys 33 Dissolving in DMF, adding R-CDI (2 times excess), adding triethylamine (equivalent to lysine side chain amino group), reacting for 24 hr, dripping into anhydrous diethyl ether for precipitation, removing diethyl ether, draining, dissolving in DMSO, loading in 3500 dialysis bag, dialyzing in deionized water for 24 hr, changing water for 2 hr, and lyophilizing to obtain R 3 Tertiary amine modified mPEG of ethylene, propylene and pentylene respectively 44 -PLys 33 (designated as mPEG respectively) 44 -PLys-DE 33 、mPEG 44 -PLys-C 3 -DE 33 、mPEG 44 -PLys-C 5 -DE 33 ) The nuclear magnetic hydrogen spectrum is shown in FIG. 7.
Titration of the pKa and circular dichroism of the three polypeptides in the same way as in (A) and (B) results are shown in FIG. 8 when R 3 As one increases from ethylene to pentylene, the central nitrogen atom of the tertiary amine is further from the backbone, the pKa is greater and the critical pH for helix transition is greater. The detailed results are shown in the following table:
R 3 polypeptide pKa Critical pH for spiral transition
C 2 mPEG 44 -PLys-DE 33 7.89 7.40
C 3 mPEG 44 -PLys-C 3 -DE 33 8.20 7.83
C 5 mPEG 44 -PLys-C 5 -DE 33 8.69 8.20
Note that: c (C) 2 /C 3 /C 5 Expressed as ethylene, propylene, pentylene.
(III) Effect of PEG molecular weight on pH responsiveness of polypeptide
Selection of PEG-NH of different molecular weights 2 As an initiator, polylysine with similar polymerization degree is obtained, tertiary amine modification with a structure of N, N-diethyl ethanol is selected, and the influence of PEG with different molecular weights on modified polylysine pKa and a spiral structure is researched. The reaction equation is as follows:
Wherein mPEG-NH 2 The method comprises the following steps of: mPEG (polyethylene glycol) 9 -NH 2 、mPEG 44 -NH 2 、mPEG 112 -NH 2 。
The method comprises the following specific steps:
(1) Azeotropic removal of mPEG from toluene 9 -NH 2 、mPEG 44 -NH 2 、mPEG 112 -NH 2 The water vapor in the mixture is pumped overnight and transferred into a glove box.
(2) Lys (Z) -NCA was weighed in a glove box in a round bottom flask according to 35 times the initiator (mPEG) 9 -NH 2 、mPEG 44 -NH 2 、mPEG 112 -NH 2 ) Weighing, adding DMF for dissolution, weighing an initiator for dissolution in 3mL of dichloromethane, adding the solution of Lys (Z) -NCA for stirring reaction for 24 hours, carrying out infrared tracing, pumping out the solvent after the reaction is finished, dissolving the solvent by 3mL of dichloromethane, dripping the solvent into diethyl ether and n-hexane (V: V=1:1) for precipitation, removing the supernatant, precipitating for 2 times, pumping out, and all the polymers are unimodal distribution as shown in figure 9.
(3) Weighing 2.0g of the polymer prepared in the step (2), and dissolving in 3mL of CF 3 COOH, 3.0mLHBr/CH was added 3 COOH, carrying out reaction for 4h, pumping the solution by an oil pump, adding DMF for dissolving, precipitating into diethyl ether, removing supernatant, dissolving by deionized water after pumping, dialyzing in deionized water for 24h by a dialysis bag with molecular weight cutoff of 3500, changing water every 2h, and freeze-drying for later use.
(4) Placing CDI into a round bottom flask, adding anhydrous dichloromethane, stirring for dispersion (1 g of CDI and 5mL of dichloromethane), sealing a rubber plug, slowly adding N, N-diethyl ethanol with 2 times of CDI excess by using a syringe, gradually clarifying the solution, adding deionized water equivalent to the dichloromethane after reacting for 4 hours, reacting for about 5 minutes, taking the lower dichloromethane phase by using a separating funnel, adding anhydrous magnesium sulfate for drying for 1 hour, filtering to remove solids, obtaining solution, and pumping the solution, namely N, N-diethyl ethanol-CDI. Dissolving the polymer prepared in the step (3) in DMF, adding N, N-diethyl ethanol-CDI (2 times of excess) by a syringe, adding triethylamine (the same amount as the amino group of a lysine side chain), reacting for 24 hours, dripping the mixture into anhydrous diethyl ether for precipitation, removing diethyl ether, pumping to dryness, dissolving the mixture in DMSO, loading the mixture by a 3500 dialysis bag, dialyzing the mixture in deionized water for 24 hours, changing water for 2 hours, and freeze-drying the mixture to obtain the polylysine modified by the N, N-diethyl ethanol for later use, wherein the nuclear magnetic hydrogen spectrum is shown in figure 10.
Meanwhile, 1) The method of (2) titrates to obtain the protonation rate curve and the helicity change curve of the three polypeptides. The results are shown in FIG. 11 and the following table, the longer the PEG chain length, the greater the pKa, but the PEG 9 Is more hydrophilic than PEG 44 Poor stability of the nanoparticle formed by polymer assembly compared to PEG 44 And (3) difference.
y pKa
9 7.45
44 7.89
112 7.94
Note that: y is the degree of polymerization of PEG.
(IV) Effect of modification of different tertiary amines on pH responsiveness of the polypeptide
Selection of mPEG 44 -NH 2 Initiation of Lys (Z) -NCA polymerization and polymerization in HBr/CH 3 Deprotection in COOH gives mPEG 44 -PLys 86 And (3) modifying tertiary amine to a polylysine side chain through CDI to obtain a series of polylysine membrane-breaking high polymer materials modified by different tertiary amines, and researching the influence of different tertiary amine structures on the pKa and the spiral structure of the polylysine. The reaction equation is as follows:
wherein R-OH is one of the following structures:
the method comprises the following specific steps:
synthesis of mPEG in large quantity by the same method as that of step (one) 44 -PLys(Z) 86 By reacting polylysine in HBr/CH 3 Deprotection in COOH gives mPEG 44 -PLys 86 The alcohols R-OH each containing a tertiary amine structure are bonded to the polypeptide mPEG via CDI 44 -PLys 86 And its structure is characterized by nuclear magnetic hydrogen spectroscopy, as shown in figure 12. Titration is carried out by the same method as the first step to obtain a protonation rate curve and a helicity change curve.
Comparing the effect of 6 tertiary amine modifications on the pKa and helix structure of the polypeptide, the results are shown in fig. 13, where with increasing hydrophobicity, pKa gradually decreases and the critical pH for helix transition gradually decreases, with the N, N-dibutyl glycol modified polypeptide having the lowest pKa. The specific table is as follows:
R-OH pKa Critical pH for spiral transition
DE 7.34 7.18
DiP 7.32 7.18
DB 6.09 5.94
C5 7.76 7.40
C6 7.12 7.00
C7 7.33 6.81
(V) Effect of polylysine molecular weight on pH responsiveness
y=44, m=0, r-OH is N, N-dibutyl ethanol. Selection of mPEG 44 -NH 2 Initiation of Lys (Z) -NCA to obtain polypeptide with different polymerization degree and in HBr/CH 3 Deprotection in COOH to obtain mPEG-PLys, modifying N, N-dibutyl glycol to polypeptide side chains with different polymerization degrees by CDI, and researching the influence of different polymerization degrees on modified polypeptide pKa and a spiral structure.
Where n= 10/33/61/86/128.
Regulating initiator mPEG 44 -NH 2 And the ratio of monomers Lys (Z) -NCA, polymerization gives mPEG-PLys (Z) of different degrees of polymerization, which are characterized by GPC, as shown in FIG. 14, each polymer having monodispersity.
Further deprotection to obtain mPEG-PLys (nuclear magnetic hydrogen spectrum is shown in figure 15), modifying N, N-dibutyl glycol to polypeptide side chains with different polymerization degrees by CDI, and titrating the modified polymer to obtain a protonation curve and a helicity curve (the method is the same as (one)). As a result, as shown in FIG. 16, the pKa of the tertiary amine modified polypeptide decreased significantly with increasing degree of polymerization, and the critical pH for helix transition decreased first and then increased. The specific table is as follows:
n pKa critical pH for spiral transition
10 7.25 6.80
33 6.85 6.20
61 6.33 5.55
86 6.09 5.94
128 5.85 5.81
(VI) polypeptide libraries modified by tertiary amines with different molecular weights (nuclear magnetic resonance hydrogen spectra are shown in figures 17-22) were synthesized by the same method as in the example, wherein R-OH is DB or one of the following structures:
The pKa of the polypeptide was titrated in a solution containing 150mM NaCl, and the specific pKa and polypeptide molecular weight relationship is shown in the following table:
backbone structure C5P2 DMP C6P DMP2 C5P DB
mPEG 112 -PLys 30 6.21 6.56 6.52 7.20 6.22 6.61
mPEG 44 -PLys 10 6.50 6.77 6.64 7.31 6.48 6.76
mPEG 44 -PLys 33 6.0 6.02 6.35 7.05 6.30 6.59
mPEG 44 -PLys 86 5.97 6.08 6.03 7.05 6.25 6.48
From the pKa results, it can be seen that overall, pKa increases with increasing mPEG molecular weight; as the molecular weight of polylysine increases, the pKa decreases; as the hydrophobicity of the tertiary amine increases, the pKa decreases. mPEG (polyethylene glycol) 44 -PLys-DB 86 In a solution containing 150mM NaCl, the pKa of the polypeptide was higher than in deionized water, indicating that the pKa of the polypeptide was also affected by the salt.
(seventh) introduction of tertiary amine groups in different ways
In addition to introducing tertiary amine groups into the polypeptide side chains by the reaction of tertiary amine containing hydroxyl groups with lysine side chain amino groups, tertiary amine can also be introduced by the reaction of tertiary amine containing carboxyl groups with polylysine side chain amino groups, exemplified by the reaction of 1-piperidineacetic acid with polylysine.
0.7g of 1-piperidineacetic acid, 1.5g of DCC and 0.7g of NHS are dissolved in 10mL of anhydrous dichloromethane to react for 4h, the floating impurity DCC is removed by filtration, and the dichloromethane solution is pumped to dryness to obtain an activated intermediate. 300mg of mPEG was taken 44 -PLys 86 Dissolving in 5mL of DMF, adding the solution to the activated intermediate, adding 100mg of triethylamine, continuing the reaction overnight, dialyzing with dialysis bag with molecular weight cutoff of 3500, and lyophilizing to obtain mPEG 44 -PLys-CC6 86 . The nuclear magnetic spectrum is shown in figure 23, which proves that the structure is correct; by titration, a pKa of 7.15 was obtained as shown in fig. 24.
EXAMPLE 2 tertiary amine modified polyethylene glycol-polyglutamic acid copolymer (polypeptide)
In addition to introducing tertiary amine groups into the polypeptide side chains by amino reaction of tertiary amine containing hydroxyl groups or tertiary amine containing carboxyl groups and lysine side chains, tertiary amine groups can also be introduced into the polypeptide side chains by carboxyl reaction of tertiary amine containing amino groups and glutamic acid side chains, etc., the present example prepares tertiary amine modified polyethylene glycol-polyglutamic acid copolymer by reaction of N, N-dibutylethylamine and polyglutamic acid.
The method comprises the following specific steps:
(1) Weighing 4.0g of BLG in a 250mL round bottom flask, pumping overnight with an oil pump, transferring into a glove box, adding 150mL of Tetrahydrofuran (THF), transferring out, placing on an ice bath for stirring, adding 6.0g of triphosgene, connecting a condenser, stirring for about 10min, transferring into the oil bath, reacting for about 2.5h at 50 ℃, transferring into the glove box after pumping, dissolving with ethyl acetate, recrystallizing three times in normal hexane, pumping to obtain BLG-NCA for later use.
(2) Azeotropic removal of mPEG from toluene 44 -NH 2 The water vapor in the water vapor is pumped and then transferred into a glove box for standby.
(3) 500mg of mPEG was weighed out 44 -NH 2 Dissolving in 5mL of dichloromethane to obtain mPEG 44 -NH 2 A solution. 2.0g of BLG-NCA was weighed into 20mL of N, N-dimethylformamide and mPEG was taken up in one portion with a syringe 44 -NH 2 Adding the solution into DMF solution, reacting for 24h, pumping off the reaction liquid, adding 10mL of dichloromethane for dissolving, dripping into n-hexane for precipitation, removing the supernatant, and pumping off to obtain polymer mPEG 44 -PBLG 30 And (5) standby. The polymer was unimodal in distribution by gel permeation chromatography as in fig. 25, and its structure by nuclear magnetic hydrogen spectrometry as in fig. 26, and the degree of polymerization was calculated as 30 from the integrated area of the nuclear magnetism.
(4) Weighing the polymer mPEG prepared in the step (3) 44 -PBLG 30 2.0g, dissolved in 5mL of CF 3 COOH, 6.0mLHBr/CH was added 3 COOH, allowing reaction for 4h, pumping solution with oil pump, adding DMF for dissolving, precipitating into diethyl ether, removing supernatant, pumping, dissolving with deionized water, dialyzing with dialysis bag with molecular weight cut-off of 3500 in deionized water for 24h, changing water every 2h, and lyophilizing to obtain mPEG 44 -PLG 30 And (5) standby.
(5) mPEG (methyl polyethylene glycol) 44 -PLG 30 (repeating unit eq 1), BOP-Cl (eq 7) and DMAP (eq 0.7) were added to N-methylpyrrolidone, stirred in an ice bath and purged with nitrogen for 15min, then with a 2-fold excess of N, N-dibutylethylamine and nitrogen for 15min, then with TEA (eq 7) and reacted at constant temperature of 50℃for 72h. After the reaction was completed, the product was dialyzed in ultrapure water for 48 hours, with water being changed every hour. Lyophilization of post-dialysis product in ultra-pure water Processing to obtain mPEG 44 -PLG-DB 30 The nuclear magnetic hydrogen spectrum is shown in fig. 27, and the structure is correct.
(6) 100. Mu.L of concentrated hydrochloric acid (37%, 12 mol) was added to 100mL of deionized water (containing 150mM NaCl) and dissolved well to give a clear and transparent solution. 10mg of the polymer material (mPEG) prepared in step (5) was dissolved in 10mL of hydrochloric acid solution 44 -PLG-DB 30 ) And the pH probe was extended below the liquid surface and titrated with 0.5M sodium hydroxide titration solution under stirring (stirring speed mot=3). After the pH meter is stable, the reading is recorded, meanwhile, liquid round-off chromatography is adopted at some pH values, and the measurement is put back and titration is continued. Until the titration result is ph=11, its pKa is 7.86 (as shown in fig. 28), compared to mPEG 44 -PLys-DB 33 (pKa 6.59), the pKa increased significantly.
EXAMPLE 3 cytotoxicity of tertiary amine modified polyethylene glycol-polypeptide (polylysine or polyglutamic acid) at pH 7.4
The cytotoxicity of the polypeptide at pH 7.4 was determined by erythrocyte hemolysis experiments. The specific detection method comprises the following steps:
1) After the sheep whole blood is gently shaken up, 1mL of whole blood is taken into a 50mL centrifuge tube, diluted to 25mL (namely 4% sheep blood is prepared) by 1X PBS, and temporarily stored at 4 ℃ for standby;
2) Preparation of polypeptide stock solution: dissolving the polypeptide into a stock solution of 10mg/ml with deionized water, and adjusting the pH to pH 7.4;
3) Preparing 2 x drug solution: diluting the polypeptide stock solution to 1600 mug/mL by PBS, and then carrying out gradient dilution by PBS to obtain a series of 2X medicine stock solutions of each polypeptide;
4) Sample adding: taking the prepared 2X medicine solution in an EP tube, adding 4% sheep blood with equal volume, and finally, lightly blowing and mixing the solution by a liquid transfer device, wherein the concentration of the working solution of each polypeptide is 50-800 mug/mL;
5) Meanwhile, the final concentration of 0.1% Triton-X100 is used as a positive control, and PBS solution is used as a negative control;
6) All samples were placed in a 37 ℃ incubator for 1 hour;
7) Taking out the sample, placing the sample in a centrifuge, and centrifuging at 1000rpm for 5 minutes at room temperature;
8) After centrifugation, 100 μl of each sample supernatant was aspirated into a 96-well plate; the absorbance at 576nm was measured using an enzyme-labeled instrument;
9) The absorbance of the experimental group incubated with the drug was defined as I Experimental group The absorbance of the control incubated with PBS and erythrocytes was defined as I Negative control The absorbance of the control incubated with red blood cells at a final concentration of 0.1% Triton-X100 was defined as I Positive control The method comprises the steps of carrying out a first treatment on the surface of the And then according to the formula [ (I) Experimental group -I Negative control )/(I Positive control -I Negative control )]The erythrocyte hemolysis rate was calculated by 100%.
The results are shown in FIG. 29: most of the polypeptide had no apparent hemolytic activity at a high concentration of 800. Mu.g/mL; when the tertiary amine is DMP2, the polypeptide has stronger hemolytic activity; when the tertiary amine is DB and the polymerization degree of lysine is about 10 or 30, the polypeptide shows certain hemolytic activity, mPEG 44 -PLG-DB 30 Also has a higher haemolytic activity, probably due to its higher pKa. The DMP2 tertiary amine modified polypeptide has higher hemolytic activity, probably due to the higher pKa of the series of polypeptides; while other tertiary amine modified polypeptides containing benzene rings have lower hemolytic activity, possibly the benzene rings make the nanoparticles more stable; the N, N-Dibutyl (DB) modified polypeptide has hemolytic activity at low polymerization degree and low hemolytic activity at high polymerization degree, because the polypeptide has lower pKa at higher polymerization degree, can be assembled into relatively compact particles and has lower hemolysis.
EXAMPLE 4 pH-responsive anticancer Activity of tertiary amine modified polyethylene glycol-polypeptide (polylysine or polyglutamic acid)
(one) determination of the killing activity of the polypeptide on tumor cells by MTT method at ph=6.8:
1) Tumor cells are spread in 96-well plates according to 1 ten thousand cells per well, and are used after being cultured overnight;
2) Preparing polypeptide with serial concentration by using culture medium with pH of 6.8, and taking the group without medicine as control group;
3) Taking out the pore plate, removing the supernatant of the culture medium, adding the corresponding culture medium containing the medicine according to 100 mu L/hole, and placing the cells in a 37 ℃ incubator for culture;
4) At a specific time point, taking out the cells, removing the supernatant, adding a culture medium containing 0.5mg/mLMTT, and continuing to culture for 2-4 hours;
5) Removing the supernatant, adding 100 mu LDMSO into each hole, and shaking for 10 minutes in a shaking table in a dark place to fully dissolve the crystals;
6) Absorbance at 490nm was measured using an enzyme-labeled instrument, and absorbance for the experimental group incubated with drug was defined as I Experimental group The absorbance of the medium without drug and cells was defined as I negative control, and the cells without drug as positive control, absorbance was defined as I Positive control The method comprises the steps of carrying out a first treatment on the surface of the And then according to the formula [ (I) Experimental group -I Negative control )/(I Positive control -I Negative control )]Cell viability was calculated by 100% and plotted.
Results show mPEG 44 -PLys-DB 86 And mPEG 44 -PLys-C6P 10 mPEG (polyethylene glycol) 44 -PLys-CC6 86 The polypeptide had high killing activity against both Panc02 cells (fig. 30) and MC38 cells (fig. 31).
(II) test mPEG 44 -PLys-DB 86 And mPEG 44 -PLys-C6P 10 Killing selectivity of two Polypeptides against tumor cells at different pH
The same procedure as in example (one) was used to test the killing selectivity of two polypeptides against tumor cells at different pH's, and the results showed that: polypeptide mPEG 44 -PLys-C6P 10 Has better pH selectivity to tumor cells at 4h (FIG. 32), and polypeptide mPEG 44 -PLys-DB 86 Has better pH selectivity for tumor cells at 24 hours (FIG. 33).
EXAMPLE 5 investigation of the cell killing pattern of the polypeptide
In this example, the cell killing mode of the polypeptide was studied by high content, and the specific steps were as follows:
1) Cell seed plates: the cells were grown in high content 96-well plates at 15000cells/well density and incubated overnight in a 5% carbon dioxide incubator at 37 ℃.
2) Preparing the medicine: the polypeptide was prepared using each pH medium for a total of 12 pH (pH 7.4-6.3). For convenient dosing, the materials were placed in 96-well plates for bacteria, 120. Mu.L of medium of the corresponding pH was added to each well, and then 4.8. Mu.L of material (5 mg/ml) was added and mixed well using a lance.
3) And (3) drug treatment: the original medium in the high content 96-well plate was aspirated, and 100. Mu.L of the medium of each pH of the pre-prepared polypeptide was aspirated by a lance and gently added to the high content 96-well plate.
4) Cell imaging: two hours and eight hours after material treatment, images were taken of each well center 3*3 (EGFP, mCherry two channels and bright field), respectively.
5) A suitable picture (four types: bright field, GFP, mCherry and merge plots).
As a result, as shown in FIG. 34, a large amount of green fluorescence (cell membrane) appears in the visual field with a decrease in pH and an increase in time, and the red mCherry fluorescence quenches, indicating that the polypeptide breaks the cell membrane structure, resulting in leakage of the content mCherry.
EXAMPLE 6 in vivo anti-tumor Effect
In this example, the antitumor effect was evaluated by in vivo tumor suppression experiments, and the specific steps are as follows:
1) EMT6 tumor cells were cultured by amplification using DMEM medium (Gibco) containing 10% fetal bovine serum;
2) Cells were washed with 1 XPBS, added with 0.25% pancreatin (Biyun) containing EDTA, and digested at 37℃for several minutes; centrifuging at 1000rpm for 5 min, discarding supernatant, and resuspending cell pellet with serum-free medium;
3) Model construction:
in situ EMT6 breast cancer tumor model in mice: serum-free medium was resuspended and the concentration of EMT6 cells was adjusted to 6.0X10 6 cells/mL; injecting 50 μl of cell suspension into a second mammary fat pad on the right side of female BABL/C mice;
4) The calculation method of the tumor volume of the EMT6 tumor model is shown in the formula: v=length x width 2 /2。
5) To treat tumor volume about 50mm 3 At this time, tumor-bearing mice were randomly divided into 3 groups. Tail vein injections were grouped as follows: PBS,30mg/kg, 60mg/kg of the drug to be evaluated.
6) And measuring and recording the long diameter and the short diameter of the tumor, and calculating the tumor volume according to a formula.
The results are shown in FIG. 35, mPEG 44 -PLys-DB 86 Shows obvious dose dependency, and the 60mg/kg administration dose can well inhibit tumor growth without obvious toxicity, and has combined treatment effect with the antibody (alpha PD 1) of the anti-apoptosis ligand 1.
EXAMPLE 7 pH responsive antibacterial Activity of tertiary amine modified polyethylene glycol-polypeptide
Many of the polypeptides in the polypeptide pool prepared in example 1 have low hemolytic activity and antibacterial activity in addition to antitumor activity. Bacterial strains used in the antibacterial experiments of this example include gram-negative bacteria (E.coli, ATCC35218, P.aeruginosa ATCC 27853).
Bactericidal activity of tertiary amine modified polyethylene glycol-polypeptide on escherichia coli
The preparation of the reagents used in the antimicrobial experiments is briefly described as follows:
1) Preparation of LB (Luria-Bertani) medium: 10g of peptone, 5g of yeast powder and 10g of sodium chloride were weighed and dissolved in 1L of ultrapure water, sterilized at high temperature and high pressure (sterilization at 121 ℃ C. For 20 min), cooled to room temperature and stored at 4 ℃ for later use.
2) Preparation of LB agar: 36g of LB agar powder was weighed, dissolved in 1L of ultrapure water, and sterilized at high temperature under high pressure (sterilization at 121 ℃ C. For 20 min). When the LB agar is reduced to a proper temperature (50 ℃), pouring the LB agar into a 60mm sterile culture dish, and preserving the LB agar for later use at 4 ℃ after solidification.
3) Preparation of M9 medium: weigh 17.1g Na 2 HPO 4 ·12H 2 O，3.0g KH 2 PO 4 ，0.5gNaCl，1.0g NH 4 Cl and 4g D- (+) -glucose were dissolved in 1L of sterile ultra-pure water, and 0.1mL of 1mol/L CaCl was added sequentially 2 Solution and 2mL of 1mol/L MgSO 4 After complete dissolution, the pH values are adjusted to 6.0, 6.2, 6.4, 6.5, 6.6, 6.8, 7.0, 7.2 and 7.4,0.22 μm sterile filter membranes for sterilization and are preserved at 4 ℃ for standby.
The method for culturing and treating bacteria used in the antibacterial test is briefly described as follows:
1) ATCC35218 (E.coli) was cultured in a bench-type thermostatic shaker at 37℃and 220rpm based on LB medium. The platform bacteria were used for passage 12-16h with the passage ratio of LB medium: bacterial liquid=200:1 (v: v);
2) Unless otherwise indicated, all the bacteria used in the experiments were used after the following treatments: the bacteria were resuspended using sterile 1 XPBS after the last centrifugation, 100. Mu.L of the above-mentioned bacteria were added to 900. Mu.L of sterile PBS (10-fold dilution), 10-fold dilution was taken into quartz cuvettes, and the background was subtracted with 1 XPBS, the absorbance of the bacteria was measured at 600nm, and the bacterial concentration of the original bacteria was calculated from the bacterial absorbance.
All bacteria related experiments were run in biosafety cabinet and all reagents and consumables used were autoclaved (121 ℃ C. For 20 min) or treated with 0.22 μm sterile filters.
After bacterial cultivation and pretreatment, the platform bacterial solution was diluted to 1X 10 with M9 medium pH 6.5 6 CFU/mL; preparing polypeptide into 32 mug/mL with M9 culture medium with pH of 6.5, uniformly mixing serial polypeptide with bacteria in equal volume, uniformly mixing control group with blank M9 culture medium with equal volume with bacteria, and incubating all the systems at 37 ℃ for 2h; after vortexing well, plates were smeared and the agar plates were placed in the incubator overnight for incubation, the number of colonies between each material group was compared, and the smaller the number of colonies, the better the antibacterial effect was shown compared to the blank group.
As shown in FIG. 36, the polypeptide of the present invention has a bactericidal effect, wherein mPEG 44 -PLys-DB 33 And mPEG 44 -PLys-C5P 33 The sterilizing effect of the two polypeptides is better.
(II) mPEG 44 -PLys-C5P 33 Bactericidal activity against pseudomonas aeruginosa.
After the bacteria were cultured and pretreated (method I), the platform phase bacterial solutions were diluted to 1X 10 with M9 medium at pH 7.4, 7.2, 7.0, 6.8, 6.6, 6.4, 6.2, 6.0, respectively 6 CFU/mL, prepare 32 mug/mL polypeptide solution with M9 culture medium of pH 7.4, 7.2, 7.0, 6.8, 6.6, 6.4, 6.2, 6.0, incubate polypeptide solution with bacterium in equal volume, blank control is equal volume of M9 culture medium with different pH, incubate for 4h at 37 ℃, dilute 10 times, 100 times with cold LB, take dilution plate after sufficient vortex, after agar plate is placed in incubator for culture overnight, count colony number. The bacterial count on the surface of the blank M9 medium group was used as a blank control group for the corresponding pH. Bacterial viability was calculated as the mean ± s.d. of 2 parallel agar plates (n=2). The results are shown in FIG. 37, which illustrates that the polypeptide has a good bactericidal activity around pKa.
Example 8: hydrophobic group doped polypeptide
This example synthesizes a series of polypeptides containing amino acids with different hydrophobic groups copolymerized with lysine.
Leucine copolymerized polypeptide
This example selects mPEG 44 -NH 2 The Lys (Z) -NCA and the Leu-NCA are initiated to be copolymerized in different proportions to obtain a polymer mPEG-P (Lys (Z) -co-Leu) with approximate total polymerization degree (n+m) and the polymer mPEG-P is prepared in HBr/CH 3 Deprotection in COOH to obtain mPEG-P (Lys-co-Leu), respectively modifying polylysine side chain with N-hydroxyethyl piperidine and 2- (hexamethyleneimine) ethanol, and researching influence of hydrophobic leucine doping proportion on polypeptide pKa and helix structure. The reaction equation is as follows:
wherein R-OH is: i.e. R 4 、R 5 And the nitrogen atom to which it is attached form the structure:
the proportions of the synthetic mPEG-P (Lys (Z) -co-Leu) are given in the following table, which is numbered for convenience:
numbering device 0 1 2 3 4 5
Leu theoretical degree of polymerization 0 10 20 30 40 50
Lys (Z) theory polymerization degree 100 90 80 70 60 50
mPEG 44-NH 2 feeding mass/g 0.1 0.1 0.1 0.1 0.1 0.1
Leu-NCA feed quality/g 0 0.0756 0.1512 0.2268 0.3023 0.3779
Lys (Z) -NCA charge mass/g 1.5316 1.3784 1.2252 1.0721 0.9619 0.7658
The method comprises the following specific steps:
(1) mPEG (methyl polyethylene glycol) 44 -NH 2 The toluene is used for azeotropically removing a small amount of water vapor, an oil pump is used for pumping, and the glove box is transferred for standby.
(2) Leu is weighed according to the above table-NCA and Lys (Z) -NCA in each sample bottle, 5mL DMF was added for dissolution, and 0.1g mPEG was weighed separately 44 -NH 2 Dissolving in dichloromethane, adding the solution into monomer, reacting for 24h, infrared tracing, pumping out the solvent after the reaction is finished, dissolving with 2.0mL of dichloromethane, precipitating into diethyl ether and n-hexane, removing the supernatant, repeating the precipitation twice, and pumping out for later use. By gel chromatography analysis, it was confirmed that the polymers all had monodispersity, as shown in FIG. 38.
(3) Dissolving the polymer prepared in the step (2) in 2mL of CF respectively 3 COOH, 2.0mLHBr/CH was added 3 COOH, carrying out reaction for 4h, pumping the solution by an oil pump, adding DMF for dissolving, precipitating into diethyl ether, removing supernatant, dissolving by deionized water after pumping, dialyzing in deionized water for 24h by a dialysis bag with molecular weight cutoff of 3500, changing water every 2h, and freeze-drying for later use. The correct structure was confirmed by nuclear magnetic characterization, and the deprotection was complete as shown in fig. 39.
(4) Placing CDI into a round bottom flask, adding anhydrous dichloromethane, stirring and dispersing (1 g of CDI and 5mL of dichloromethane), sealing a rubber plug, slowly adding N-hydroxyethyl piperidine or 2- (hexamethyleneimine) ethanol (2 times of CDI excess) by using a syringe, gradually clarifying the solution, adding deionized water equivalent to dichloromethane after 4 hours of reaction, reacting for about 5 minutes, taking the lower dichloromethane phase by a separating funnel, adding anhydrous magnesium sulfate, drying for 1 hour, filtering to remove solids, obtaining solution, and pumping to dryness, namely R-CDI.
(5) Dissolving the series of polymers in DMF respectively, adding R-CDI (2 times excess) by a syringe, adding triethylamine (the same amount as the amino group of a lysine side chain), reacting for 24 hours, dripping into anhydrous diethyl ether for precipitation, removing diethyl ether, pumping to dryness, dissolving in DMSO, loading by a 3500 dialysis bag, dialyzing in deionized water for 24 hours, changing water for 2 hours, and freeze-drying for later use. The correct structure and complete modification are proved by nuclear magnetic characterization, as shown in fig. 40 and 41.
The resulting modified polymer was titrated to give a protonation profile and a helicity profile (the procedure is as in example 1). Wherein,as shown in fig. 42, the modified polypeptide had a gradual decrease in pKa with increasing leucine doping ratio, and the tertiary amine of the polypeptide side chain was fully protonated at ph=2, at which time the polypeptide had a charge interaction from non-helical to stable helical structure with increasing leucine doping ratio, probably with dispersing the side chain of leucine in the copolymer. The protonation rate and helicity at ph=6.8 and ph=7.4 were calculated and analyzed using the proportion of leucine to the polypeptide block as a reference index, PD indicated the protonation rate, and specific data are shown in the following table:
when (when)After modification of the polypeptide, similar variation rules can be obtained as shown in FIG. 43, and since the seven-membered ring is more hydrophobic, the pKa variation is also more pronounced. The specific table is shown below:
(di) phenylalanine copolypeptides
This example selects mPEG 44 -NH 2 (mPEG 2k -NH 2 ) The Lys (Z) -NCA and Phe-NCA are initiated to copolymerize in different proportions to obtain a polymer with a total polymerization degree (n+m) close to that of the polymer, and the polymer is polymerized in HBr/CH 3 Deprotection in COOH to obtain mPEG-P (Lys-co-Phe), respectively modifying a polylysine side chain by N-hydroxyethyl piperidine and 2- (hexamethyleneimine) ethanol, and researching influence of a hydrophobic phenylalanine doping proportion on pKa and a spiral structure of polypeptide. The reaction equation is as follows:
Wherein R-OH is:i.e. R 4 、R 5 And the nitrogen atom to which it is attached form the structure:
the specific procedure is the same as in (one) of example 2, and the specific feeding is shown in the following table:
numbering device 2 3 4 5
Phe theory polymerization degree 20 30 40 50
Lys (Z) theory polymerization degree 80 70 60 50
mPEG 44-NH 2 feeding mass/g 0.1 0.1 0.1 0.1
Phe-NCA charge mass/g 0.19 0.29 0.38 0.48
Lys (Z) -NCA charge mass/g 1.22 1.07 0.92 0.76
The resulting unmodified polymer mPEG-P (Lys (Z) -co-Phe) was characterized by gel permeation chromatography, as shown in fig. 44, as a unimodal distribution; the nuclear magnetic spectrum is shown in FIG. 45, and the degree of polymerization is calculated. Deprotection under acidic conditions, the polypeptide nuclear magnetism modified by N-hydroxyethyl piperidine is shown in FIG. 46, and has been modified completely. The polypeptide nuclear magnetism modified by 2- (hexamethyleneimine) ethanol is shown in FIG. 47, and has been completely modified. By titration, as shown in fig. 48, the pKa of the polypeptide gradually decreased as the proportion of phenylalanine increased.
Titration results after modification of N-hydroxyethyl piperidine are shown in the following table:
sequence number n m pKa
2 85 22 7.28
3 76 32 7.2
4 62 43 7.06
5 54 53 6.92
Titration results after 2- (hexamethyleneimine) ethanol modification are shown in the following table:
sequence number n m pKa
2 85 22 7.22
3 76 32 6.9
4 62 43 6.98
5 54 53 6.83
mPEG in the present example (two) 44 -NH 2 Replacement with mPEG 112 -NH 2 The procedure for the synthesis of the polypeptide comprising Lys (Z) -NCA and Phe-NCA copolymerized in different proportions was the same as in (one) of this example.
The specific feeding ratio is shown in the following table:
numbering device 0 1 2 3 4 5
Phe theory polymerization degree 0 10 20 30 40 50
Lys (Z) theory polymerization degree 100 90 80 70 60 50
mPEG 112-NH 2 feeding mass/g 0.2 0.2 0.2 0.2 0.2 0.2
Phe-NCA charge mass/g 0 0.076 0.152 0.229 0.306 0.382
Lys (Z) -NCA charge mass/g 1.225 1.103 0.980 0.857 0.735 0.612
The resulting polymer was characterized by gel permeation chromatography, as shown in fig. 49, as a unimodal distribution. The characteristics of the nuclear magnetic resonance spectrum after deprotection are shown in a figure 50, the complete deprotection is realized, the polypeptide product is obtained through modification of N-hydroxyethyl piperidine, and the characteristics of the nuclear magnetic resonance spectrum are shown in a figure 51. As shown in FIG. 52, it can be seen that the pKa of the polypeptide gradually decreases and is higher than mPEG as the proportion of phenylalanine increases, and the protonation rate curve is obtained by titration (the method is the same as in example 1) 44 -NH 2 The pKa of the initiated polymer is greater.
Sequence number n m pKa
0 90 0 7.33
1 80 10 7.29
2 70 20 7.25
3 65 35 7.16
4 55 40 7.05
5 50 50 6.95
Polypeptide of NorLeu, L-aminocapryline or tryptophan
By adopting the same method as in the first embodiment, a series of polymers are obtained by copolymerizing norleucine in different proportions, the gel permeation chromatography characterization of which is shown in fig. 53, and the polymers are all unimodal; calculating the polymerization degree of the polymer from the nuclear magnetism as shown in FIG. 54; the nuclear magnetic resonance spectrum after deprotection is shown in FIG. 55, which shows that the deprotection is complete; finally, the polypeptide product with pH response is obtained by tertiary amine modification of N-hydroxyethyl piperidine, and the nuclear magnetic hydrogen spectrum of the polypeptide product is shown in figure 56, thus proving complete modification.
By adopting the same method as in the first embodiment, the piperidine tertiary amine modified L-amino octanoic acid copolymerized polypeptide is prepared, and the nuclear magnetism is shown in fig. 57, so that the structure is correct.
The same method as in (one) of this example was used to obtain a polypeptide mPEG with tryptophan as the hydrophobic amino acid and 50% doping 44 -P(Lys-C6 50 -co-Trp 50 ) The nuclear magnetic resonance results are shown in FIG. 58, which demonstrates the correct structure.
The pKa of the polypeptide was titrated in a system containing 150mM sodium chloride and the results are shown in the table below: the pKa of the polypeptide is lower with higher doping ratio of the hydrophobic monomer.
And (IV) antitumor activity and selectivity of the polypeptide doped with the hydrophobic group.
The serial copolymerized polypeptide macromolecular materials prepared in this example were tested for cytotoxicity at pH characteristic of normal and tumor tissues, and specific experimental methods were the same as those of example 3 and example 4.
The result of hemolytic toxicity of the polypeptide is shown in FIG. 59, and the hemolytic activity of the polypeptide is related not only to the pKa but also to the hydrophilicity and hydrophobicity of the hydrophobic monomer, and the result shows that the polypeptide containing phenylalanine copolymerization has lower hemolytic activity, which may be related to the beta-sheet structure.
The tumor cell killing results at different pH values are shown in the graph 60 and the graph 61, the polypeptide doped with the hydrophobic group has better tumor killing selectivity, wherein the polypeptide containing the benzene ring structure has better tumor killing selectivity and lower hemolytic toxicity, which is probably because the benzene ring structure is more beneficial to the stability of the nano particles.
(V) killing mechanism of hydrophobic group doped polypeptide
Disruption of cell membrane structure by apoptosis or necrosis results in release of enzymes in the cytoplasm into the culture broth, including lactate dehydrogenase (lactate dehydrogenase, LDH) which has relatively stable enzymatic activity. The polypeptide of the present invention has an amphiphilic structure and kills cells by acting on the cytoplasmic membrane. With mPEG 44 -P(Lys-C6 50 -co-Trp 50 ) For example, LDH release during cell death was measured as follows:
1) Preparation of 5.0mg/mL mPEG 44 -P(Lys-C6 50 -co-Trp 50 ) An aqueous solution.
2) And (3) drug treatment: gradient dilution for preparing mPEG 44 -P(Lys-C6 50 -co-Trp 50 ) Polypeptide solution, note: in order to avoid the influence of lactate dehydrogenase in serum, serum is not needed to be added during the drug treatment. 100 μl of mPEG at the corresponding concentration was added to each well 44 -P(Lys-C6 50 -co-Trp 50 ) The polypeptide solution was incubated in a 37℃incubator for 4 hours.
3) And adding an LDH release reagent into the control hole with the maximum enzyme activity one hour before the treatment time is finished, wherein the adding amount is 10 percent of the volume of the original culture solution, repeatedly blowing and beating for a plurality of times, uniformly mixing, and then continuously incubating in a cell culture box.
4) After a predetermined time, the cell culture plates were centrifuged for 5min with 400g in a multi-well plate centrifuge. 80. Mu.L of the supernatant from each well was added to a new 96-well plate, and the sample was assayed.
5) 40. Mu.L of LDH detection working solution was added to each well.
6) Mixing, and incubating at room temperature (about 25deg.C) in dark place for 30min (optionally wrapping with aluminum foil, and slowly shaking on horizontal shaking table or side shaking table). The absorbance was then measured at 490 nm. The dual wavelength measurement is performed using either 600nm or more than 600nm as a reference wavelength.
7) Calculation (measured absorbance for each group should be subtracted from background blank well absorbance): cytotoxicity or mortality (%) = (absorbance of treated sample-absorbance of sample control wells)/(absorbance of maximum enzyme activity of cells-absorbance of sample control wells) ×100.
As shown in fig. 62, mPEG 44 -P(Lys-C6 50 -co-Trp 50 ) No LDH release was induced at ph=7.4, cell death with simultaneous LDH release at ph=6.8, indicating mPEG 44 -P(Lys-C6 50 -co-Trp 50 ) It is possible to kill the cells by rupture of the membranes.
(six) in vivo therapeutic Effect of hydrophobic group-doped polypeptide
The procedure is as in example 6, mPEG 44 -P(Lys-C6 50 -co-Trp 50 ) For example, as shown in FIG. 63, mPEG 44 -P(Lys-C6 50 -co-Trp 50 ) Can well inhibit the growth of tumor.
The technical features of the above-described embodiments may be arbitrarily combined, and all possible combinations of the technical features in the following embodiments are not described for brevity of description, however, as long as there is no contradiction between the combinations of the technical features, they should be considered as the scope of the description.
The foregoing examples illustrate only a few embodiments of the invention and are described in detail herein without thereby limiting the scope of the invention. It should be noted that it will be apparent to those skilled in the art that several variations and modifications can be made without departing from the spirit of the invention, which are all within the scope of the invention. Accordingly, the scope of protection of the present invention is to be determined by the appended claims.

Claims (40)

  1. A membrane-disrupting polypeptide having a structure represented by formula (I):
    wherein R is selected from: -R 3 -N(R 4 R 5 )、-R 3 -R’、
    R' is selected from:
    l is selected from: -NH-C (=o) O-, -NH-C (=o) -, -C (=o) -NH-, -C (=o) -O-;
    R 1 selected from: an alkylene group;
    R 2 selected from: c (C) 1 -C 12 Alkyl, C 6 -C 14 Aryl, C 6 -C 14 Aryl substituted C 1 -C 12 Alkyl-and benzyloxycarbonyl-substituted C 1 -C 12 Alkyl, 5-10 membered heteroaryl substituted C 1 -C 12 An alkyl group;
    R 3 selected from: alkylene, C 6 -C 14 Aryl-substituted alkylene;
    R 4 、R 5 each independently selected from: alkyl, C 6 -C 14 Aryl substituted alkyl, or R 3 、R 4 And the nitrogen atom to which it is attached form a heterocycloalkyl group;
    y is selected from: 2-150;
    R 6 selected from: c (C) 1 -C 15 Alkyl, C 6 -C 14 Aryl, C 6 -C 14 Aryl substituted C 1 -C 15 An alkyl group;
    n+m is greater than 0, and n is not 0;
    q is selected from: 0. 1, 2, 3 and 4.
  2. The membrane-disrupting polypeptide or stereoisomer or pharmaceutically acceptable salt thereof of claim 1, wherein R 1 Selected from: c (C) 1 -C 6 An alkylene group.
  3. The membrane-disrupting polypeptide or stereoisomer or pharmaceutically acceptable salt thereof of claim 2, wherein R 1 Selected from: - (CH) 2 ) x -wherein x is selected from: 1. 2, 3, 4, 5, 6.
  4. The membrane-disrupting polypeptide or a stereoisomer or a pharmaceutically acceptable salt thereof according to claim 1, which has a structure represented by the following formula (II):
    wherein X is: -O-or none.
  5. The membrane-disrupting polypeptide or a stereoisomer or a pharmaceutically acceptable salt thereof according to claim 1, which has a structure represented by the following formula (III):
  6. the membrane-disrupting polypeptide or stereoisomer thereof or a pharmaceutically acceptable salt thereof according to any of claims 1-5, wherein R is selected from: -R 3 -N(R 4 R 5 )、
  7. The membrane-disrupting polypeptide or stereoisomer or pharmaceutically acceptable salt thereof of claim 6, wherein R 3 Selected from: c (C) 1 -C 6 Alkylene-phenyl-substituted C 1 -C 6 An alkylene group.
  8. The membrane-disrupting polypeptide or stereoisomer or pharmaceutically acceptable salt thereof of claim 7, wherein R 3 Selected from: - (CH) 2 ) x -, phenyl-substituted- (CH) 2 ) x -; wherein x is selected from: 1. 2, 3, 4, 5, 6.
  9. The membrane-disrupting polypeptide or stereoisomer or pharmaceutically acceptable salt thereof according to any of claims 1-5, wherein R 4 、R 5 Each independently selected from: c (C) 1 -C 6 Alkyl-substituted C 1 -C 6 Alkyl, naphthyl substituted C 1 -C 6 Alkyl, or R 4 、R 5 And the nitrogen atom to which it is attached form a 5-to 10-membered heterocycloalkyl.
  10. The membrane-disrupting polypeptide or stereoisomer or pharmaceutically acceptable salt thereof of claim 9, wherein R 4 、R 5 Each independently selected from: c (C) 1 -C 4 Alkyl-substituted C 1 -C 3 Alkyl, naphthyl substituted C 1 -C 3 Alkyl, or R 4 、R 5 And the nitrogen atom to which it is attached form a 5-8 membered heterocycloalkyl.
  11. The membrane-disrupting polypeptide or stereoisomer or pharmaceutically acceptable salt thereof of claim 10, wherein R 4 、R 5 And the nitrogen atom to which it is attached form the following group:
  12. the membrane-disrupting polypeptide or stereoisomer or pharmaceutically acceptable salt thereof according to any of claims 1-5, wherein R 6 Selected from: c (C) 1 -C 6 Alkyl, phenyl, naphthyl, phenyl-substituted C 1 -C 6 An alkyl group.
  13. The membrane-disrupting polypeptide or stereoisomer thereof or a pharmaceutically acceptable salt thereof according to any of claims 1-5, wherein R is selected from: -R 3 -N(R 4 R 5 )、
    Wherein R is 3 Selected from: -CH 2 -CH 2 -、-CH 2 -CH 2 -CH 2 -、-CH 2 -(CH 2 ) 3 -CH 2 -、 R 4 、R 5 And the nitrogen atom to which it is attached form the following group:
    R 6 is benzyl.
  14. The membrane-disrupting polypeptide or stereoisomer or pharmaceutically acceptable salt thereof according to any of claims 1-5, wherein R 2 Selected from: c (C) 1 -C 8 Alkyl, phenyl, naphthyl, phenyl-substituted C 1 -C 6 Alkyl-and benzyloxycarbonyl-substituted C 1 -C 6 Alkyl, 5-10 membered heteroaryl substituted C 1 -C 6 An alkyl group.
  15. The membrane-disrupting polypeptide or stereoisomer or pharmaceutically acceptable salt thereof according to any of claims 1-5, wherein R 2 Selected from: methyl, ethyl, n-propyl, isopropyl, n-butyl, isobutyl, pentyl, hexyl, heptyl, octyl, nonanyl, decyl, undecyl, dodecyl, phenyl, naphthyl, benzyl, benzyloxycarbonyl-substituted ethyl, benzopyrrole-substituted ethyl.
  16. The membrane-disrupting polypeptide or stereoisomer thereof or a pharmaceutically acceptable salt thereof according to any of claims 1-5, wherein y is selected from the group consisting of: 30-120, preferably 40-48 or 108-116.
  17. The membrane-disrupting polypeptide or stereoisomer thereof or the pharmaceutically acceptable salt thereof according to any of claims 1-5, wherein n+m is not less than 5, more preferably not less than 10.
  18. The membrane-disrupting polypeptide of claim 17, or a stereoisomer or a pharmaceutically acceptable salt thereof, wherein n+m is from 10 to 200, more preferably from 10 to 150, more preferably from 10 to 110.
  19. The membrane-disrupting polypeptide of any of claims 1-5, or a stereoisomer or pharmaceutically acceptable salt thereof, wherein m is 0; n is 5 to 200, preferably 10 to 150, preferably 10 to 110, further preferably 10 to 15, 30 to 35, 60 to 65, 80 to 90, or 120 to 130.
  20. The membrane-disrupting polypeptide or stereoisomer thereof or the pharmaceutically acceptable salt thereof according to any of claims 1-5, wherein m is from 0 to 60%, more preferably from 0 to 50% of n+m.
  21. The membrane-disrupting polypeptide or stereoisomer thereof or a pharmaceutically acceptable salt thereof according to any of claims 1-5, wherein R is selected from: -R 3 -N(R 4 R 5 )、
    Wherein R is 3 is-CH 2 -CH 2 -；R 4 、R 5 And the nitrogen atom to which it is attached form the following group:
    R 6 is benzyl; y is 40-48.
  22. The membrane rupture of any of claims 1-5 A polypeptide or a stereoisomer thereof or a pharmaceutically acceptable salt thereof, wherein R is selected from: -R 3 -N(R 4 R 5 )；
    Wherein R is 3 Selected from: -CH 2 -CH 2 -；R 4 、R 5 And the nitrogen atom to which it is attached form the following group:
    R 2 selected from: methyl, ethyl, n-propyl, isopropyl, n-butyl, isobutyl, pentyl, hexyl, heptyl, octyl, nonanyl, decyl, undecyl, dodecyl, phenyl, naphthyl, benzyl, benzyloxycarbonyl-substituted ethyl, benzopyrrole-substituted ethyl.
  23. The membrane-disrupting polypeptide of claim 1, or a stereoisomer or a pharmaceutically acceptable salt thereof, wherein the polymer is selected from the group consisting of:
  24. a membrane-disrupting polypeptide nanoparticle formed by self-assembly of the membrane-disrupting polypeptide of any of claims 1-23 in an aqueous medium.
  25. A method of preparing the membrane-broken polypeptide nanoparticle of claim 24, comprising the steps of: dissolving the membrane-broken polypeptide in an organic solvent or hydrochloric acid solution with the pH value of 1.5-2.5, then dropwise adding the obtained solution into water in a stirring state, continuously stirring, and removing the solvent through low-temperature dialysis to obtain the membrane-broken polypeptide nano particles;
    Preferably, the organic solvent is N, N-dimethylformamide;
    preferably, the proportion of the membrane-breaking polypeptide, the organic solvent or the hydrochloric acid solution and the water is 10 mg-30 mg:1mL:4-6mL;
    preferably, the preparation method of the membrane-breaking polypeptide nanoparticle comprises the following steps: the membrane-breaking polypeptide is prepared from 10mg to 30mg:1mL is dissolved in N, N-dimethylformamide, then the obtained solution is dropwise added into water under the stirring state of 400-800 rpm, the stirring is continued for 8-20 min at the speed of 200-600 rpm, and a dialysis bag with the molecular weight cutoff of 10000-20000 is used for dialysis in water to remove the solvent, thus obtaining the membrane-breaking polypeptide nano-particles.
  26. Use of a membrane-breaking polypeptide according to any one of claims 1-23, or a stereoisomer or a pharmaceutically acceptable salt thereof, for the manufacture of a medicament for the prevention and/or treatment of a tumor.
  27. Use of the membrane-broken polypeptide nanoparticle of claim 24 in the preparation of a medicament for the prevention and/or treatment of a tumor.
  28. Use of a membrane-breaking polypeptide according to any one of claims 1-23, or a stereoisomer thereof, or a pharmaceutically acceptable salt thereof, in combination with an immune checkpoint inhibitor for the preparation of a medicament for the prevention and/or treatment of a tumor.
  29. Use of a membrane-rupture polypeptide nanoparticle in combination with an immune checkpoint inhibitor as defined in claim 24 for the manufacture of a medicament for the prevention and/or treatment of a tumour.
  30. The use according to claim 28 or 29, wherein the immune checkpoint inhibitor is a PD-1 inhibitor.
  31. The use according to any one of claims 26 to 29, wherein the tumour is pancreatic cancer, melanoma, colorectal cancer, colon cancer, lung cancer, squamous carcinoma of the tongue, cervical cancer, ovarian cancer, osteosarcoma, liver cancer, breast cancer, bladder cancer, ovarian epithelial cancer.
  32. Use of a membrane-breaking polypeptide according to any one of claims 1-23, or a stereoisomer or a pharmaceutically acceptable salt thereof, for the preparation of a medicament for combating bacterial infections.
  33. Use of the membrane-broken polypeptide nanoparticle of claim 24 in the preparation of a medicament against bacterial infection.
  34. The use according to claim 32 or 33, wherein the bacteria are gram-negative bacilli, gram-negative pseudomonas, gram-positive staphylococci, gram-positive cocci, streptococci.
  35. The use according to claim 34, wherein the bacteria are escherichia coli, salmonella, staphylococcus aureus, klebsiella pneumoniae, pseudomonas aeruginosa, enterococcus faecalis, streptococcus pyogenes, streptococcus pneumoniae, acinetobacter baumannii, diplococcus pneumoniae, pseudomonas aeruginosa.
  36. A medicament for preventing and/or treating tumors, which is prepared from an active ingredient and pharmaceutically acceptable auxiliary materials and/or carriers, wherein the active ingredient comprises the membrane-rupture polypeptide or a stereoisomer or a pharmaceutically acceptable salt thereof according to any one of claims 1 to 23 and/or the membrane-rupture polypeptide nano-particles according to claim 24.
  37. A combination for the prophylaxis and/or treatment of tumors, characterized in that the active ingredients thereof comprise:
    component 1: the membrane-disrupting polypeptide of any of claims 1-23, or a stereoisomer or a pharmaceutically acceptable salt thereof, and/or the membrane-disrupting polypeptide nanoparticle of claim 24; and
    component 2: antitumor drugs other than component 1;
    the component 1 and the component 2 are each independent administration units, or the component 1 and the component 2 together form a combined administration unit.
  38. The combination for preventing and/or treating tumors of claim 37, characterized in that said component 2 is an immune checkpoint inhibitor.
  39. The combination for preventing and/or treating tumors of claim 38, wherein said immune checkpoint inhibitor is a PD-1 inhibitor.
  40. A medicament for resisting bacterial infection, which is prepared from an active ingredient and pharmaceutically acceptable auxiliary materials and/or carriers, wherein the active ingredient comprises the membrane-breaking polypeptide or a stereoisomer or a pharmaceutically acceptable salt thereof according to any one of claims 1 to 23, and/or the membrane-breaking polypeptide nano-particles according to claim 24.
CN202280005645.7A 2021-12-21 2022-12-20 PH-sensitive membrane-rupture polypeptide and application thereof Pending CN116635080A (en)

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