Use of polymers in preparation of compositions for binding membrane structures

Abstract

The present invention discloses the use of a polymer in the preparation of a composition for binding membrane structures. The polymer disclosed by the invention is prepared by a specific method, and has the capability of combining cell membranes and virus membranes and splitting, so that the effects of resisting tumors (splitting tumor cells) and resisting viruses (splitting virus membranes or virus infection cell membranes) are realized.

Description

[0001] This application is a divisional application with an application date of February 16, 2022, application number CN202210143238.4, and invention name “Polymers capable of binding to membranes, preparation methods and uses thereof”, which is incorporated herein by reference in its entirety. Technical Field

[0002] The present invention relates to a (meth)acrylate polymer, in particular to a (meth)acrylate polymer having membrane binding ability or membrane cleavage ability and its use, especially its medical use. Background Art

[0003] The information in the background technology is only for illustrating the general background of the present invention and should not be regarded as admitting or suggesting in any form that such information constitutes the prior art known to a person skilled in the art. Summary of the invention

[0004] In order to solve at least some of the technical problems in the prior art, the present invention provides the use of a polymer in preparing a composition for bonding a membrane structure. The present invention is completed at least partially based on this. Specifically, the present invention includes the following contents.

[0005] In a first aspect of the present invention, there is provided use of a polymer in preparing a composition for bonding a membrane structure, wherein the polymer is obtained by reacting a monomer and an initiator in an organic solvent in the presence of a catalyst.

[0006] In certain embodiments, the use of the polymer according to the present invention in preparing a composition for bonding a membrane structure, wherein the catalyst comprises a metal catalyst and a ligand bonded thereto.

[0007] In certain embodiments, according to the use of the polymer of the present invention in preparing a composition for bonding a membrane structure, the initiator is selected from bromoisobutyrate and / or chloroisobutyrate.

[0008] In certain embodiments, the polymer according to the present invention is used in the preparation of a composition for bonding a membrane structure, wherein the monomer is selected from at least one of the group consisting of AMA, DMA, DEA, C7A, EPA, iDPA, nDPA, PBA, DBA and D5A.

[0009] In certain embodiments, according to the use of the polymer of the present invention in preparing a composition for bonding a membrane structure, the initiator is obtained by a substitution reaction of a hydroxy compound with a 2-halogenated isobutyryl halide, and the metal catalyst is a metal halide.

[0010] In certain embodiments, the use of the polymer according to the present invention in preparing a composition for bonding a membrane structure, wherein the polymer further comprises a hydrophilic segment.

[0011] In certain embodiments, in the use of the polymer according to the present invention in preparing a composition for binding a membrane structure, the hydrophilic segment is PEG with a molecular weight of 1000-10000 kDa.

[0012] In certain embodiments, the use of the polymer according to the present invention in preparing a composition for bonding a membrane structure, wherein the polymer has the NMR spectrum shown in Table 1.

[0013] In certain embodiments, the use of the polymer according to the present invention in preparing a composition for binding a membrane structure, wherein the composition is a nanoparticle suspension or a pharmaceutical composition.

[0014] In certain embodiments, the polymer according to the present invention is used in preparing a composition for bonding a membrane structure, wherein the composition is obtained by dispersing the polymer in an aqueous solution.

[0015] In a second aspect, the present invention provides a method for binding and lysing a membrane structure, which comprises the step of contacting a polymer or a nanoparticle suspension obtained therefrom with the membrane structure.

[0016] In certain embodiments, according to the method for binding and cracking a membrane structure according to the present invention, the polymer is obtained by reacting a monomer and an initiator in an organic solvent in the presence of a catalyst, the catalyst comprising a metal catalyst and a ligand bound thereto; the initiator is selected from bromoisobutyrate and / or chloroisobutyrate; and the monomer is selected from at least one of the group consisting of AMA, DMA, DEA, C7A, EPA, iDPA, nDPA, PBA, DBA and D5A.

[0017] In certain embodiments, according to the method for binding to and lysing a membrane structure of the present invention, the membrane structure is selected from the group consisting of a cell membrane, a virus membrane, a bacterial membrane, and an artificial lipid membrane.

[0018] According to the third aspect of the present invention, a method for predicting or adjusting the membrane binding ability or membrane lysis ability of a polymer is provided, wherein the polymerization monomer of the polymer is selected from at least one of the group consisting of AMA, DMA, DEA, C7A, EPA, iDPA, nDPA, PBA, DBA and D5A, and the method includes the step of predicting or adjusting the polymer according to the content of each polymerization monomer and the contribution to the membrane binding ability or membrane lysis ability, wherein the contribution decreases in the order of AMA, DMA, DEA, C7A, EPA, iDPA, nDPA, PBA, DBA to D5A.

[0019] The polymer of the present invention has the ability to bind to cell membranes and viral membranes and lyse them. According to the exemplary polymer monomers, they can be divided into the following categories: AMA, DMA, DEA, C7A, EPA, iDPA, nDPA, PBA, DBA, D5A. It is further found that its membrane binding ability gradually weakens, and the ability to lyse membranes also gradually weakens. Different monomers are matched to produce different membrane binding abilities. On this basis, a series of polymer materials can be synthesized, which can bind to cell membranes, cell membranes after virus infection, and viral membranes. The stronger the membrane binding ability of the material, the easier it is to bind to the cell membrane, and the stronger its corresponding ability to lyse membranes is; as the membrane binding ability of the material weakens, it gradually tends to bind to the viral membrane, and the ability to lyse membranes also gradually weakens. The polymer of the present invention has a broad-spectrum effect on a variety of cells and RNA viruses, thereby achieving anti-tumor (lyse tumor cells) and anti-viral (lyse viral membranes or virally infected cell membranes) effects. BRIEF DESCRIPTION OF THE DRAWINGS

[0020] Figure 1 is the NMR spectrum of exemplary compound P1.

[0021] Figure 2 is the NMR spectrum of exemplary compound P2.

[0022] Figure 3 is the NMR spectrum of exemplary compound P3.

[0023] Figure 4 is the NMR spectrum of exemplary compound P4.

[0024] Figure 5 is the NMR spectrum of exemplary compound P5.

[0025] Figure 6 is the NMR spectrum of exemplary compound P6.

[0026] Figure 7 is the NMR spectrum of exemplary compound P7.

[0027] Figure 8This is a Confocal co-localization image after incubation of the polymer prepared in Example 1 of the present invention with cells (P1 is the fluorescence of polymer P1, DiI represents the fluorescence staining of dye DiI, and Merge represents the spectrum obtained by superimposing P1 and DiI dye).

[0028] Fig. 9 This is a Confocal co-localization image of the cell membrane destroyed after incubation of the polymer prepared in Example 1 of the present invention with cells (P1 is the fluorescence of polymer P1, DiI represents the fluorescence staining of dye DiI, Merge represents the spectrum obtained by superimposing P1 and DiI dye).

[0029] Fig.10 This is a transmission electron micrograph of the polymer prepared in Example 2 of the present invention after incubation with VSV virus.

[0030] Fig.11 This is a Confocal co-localization image after incubation of the polymer prepared in Example 2 with virus-infected cells (NP is the fluorescence of polymer P2, DiI represents the fluorescence staining of dye DiI, GFP is the fluorescence of virus, Merge represents the spectrum obtained by superimposing the fluorescence of P2, DiI dye and virus GFP).

[0031] Fig.12 These are transmission electron micrographs of the VSV virus, model liposomes, copolymer nanoparticle suspension P4, and P4 after incubation with the former two in Example 3.

[0032] Fig.13 The figure shows the effect of different drug treatments on viral infectivity in Example 10.

[0033] Fig.14 This is a Confocal co-localization image after incubation of the polymer prepared in Example 5 with virus-infected cells (PDEA is the fluorescence of polymer P5, DiI represents the fluorescence staining presented by dye DiI, GFP is the fluorescence of virus, Merge represents the spectrum obtained by superimposing the fluorescence of P5, DiI dye and virus GFP).

[0034] Fig.15 This is a Confocal co-localization image after incubation of the polymer prepared in Example 5 with Hela cells overexpressing various viral ion channel proteins (PDEA is the fluorescence of polymer P5, DiI represents the fluorescence staining of dye DiI, GFP is the fluorescence of overexpressed viral ion channel protein, Merge represents the spectrum obtained by superimposing the fluorescence of P5, DiI dye and GFP).

[0035] Fig.16This is the Confocal co-localization map after incubation of P1, P2, P3 and P4 with normal cells or virus-infected cells (UPS is the fluorescence of polymers P1-P4, DiI represents the fluorescence staining of dye DiI, GFP is the fluorescence of virus, Merge represents the map obtained by superimposing the fluorescence of polymer, DiI dye and virus GFP).

[0036] Fig.17 The figure is a statistical graph showing the ability of exemplary nanoparticles to bind to cell membranes.

[0037] Fig.18 Schematic diagram of polymer binding to virus. DETAILED DESCRIPTION

[0038] Various exemplary embodiments of the present invention will now be described in detail. This detailed description should not be considered as limiting the present invention, but should be understood as a more detailed description of certain aspects, features, and embodiments of the present invention.

[0039] It should be understood that the terms described in the present invention are only for describing special embodiments and are not intended to limit the present invention. In addition, for the numerical range in the present invention, it should be understood that the upper and lower limits of the scope and each intermediate value therebetween are specifically disclosed. Each smaller range between the intermediate value in any stated value or stated range and any other stated value or intermediate value in the described range is also included in the present invention. The upper and lower limits of these smaller ranges can be independently included or excluded in the scope.

[0040] Unless otherwise specified, all technical and scientific terms used herein have the same meanings as commonly understood by those skilled in the art. Although only preferred methods and materials are described herein, any methods and materials similar or equivalent to those described herein may also be used in the practice or testing of the present invention. All documents mentioned in this specification are incorporated by reference to disclose and describe the methods and / or materials related to the documents. In the event of a conflict with any incorporated document, the content of this specification shall prevail. Unless otherwise specified, "%" is a percentage based on weight.

[0041] As used herein, the term "aminoalkyl (meth)acrylate" refers to an ester formed by acrylic acid and / or methacrylic acid and an aminoalkyl group. The aminoalkyl group refers to an alkyl compound containing an amino group and can be represented by the general formula -CH2N(R1)R2, wherein R1 and R2 each independently represent the same or different groups and each independently represent H, C 1-10 Straight or branched chain alkyl or C 1-10 Cycloalkyl.

[0042] Herein, the term "alkyl" includes straight and branched chain alkyl groups having 1 to about 10 carbon atoms, and typically 1 to 8 carbon atoms, or in some embodiments 1 to 5 carbon atoms. Herein, "alkyl" includes cycloalkyl groups defined as follows. Alkyl groups may be substituted or unsubstituted. Examples of straight chain alkyl groups include, but are not limited to methyl, ethyl, n-propyl, n-butyl, n-pentyl, n-hexyl, n-heptyl and n-octyl. Examples of branched chain alkyl groups include, but are not limited to isopropyl, isobutyl, sec-butyl, tert-butyl, neopentyl and isopentyl. Exemplary substituted alkyl groups may be substituted one or more times by, for example, amino, sulfenyl, hydroxyl, cyano, alkoxy and / or halogen atoms such as F, Cl, Br and I groups. The term "haloalkyl" is an alkyl group with one or more halogen groups. In some embodiments, haloalkyl refers to a perhaloalkyl group. Generally, in addition to those listed above, the alkyl group may include, but is not limited to, 2-pentyl, 2-methylbutyl, 3-methylbutyl, 1,2-dimethylpropyl, 1,1-dimethylpropyl, 2,2-dimethylpropyl, 1-ethylpropyl, 2-hexyl, 2-methylpentyl, 3-methylpentyl, 4-methylpentyl, 1,2-dimethylbutyl, 1,3-dimethylbutyl, 2,3-dimethylbutyl, 1,1-dimethylbutyl, 2,2-dimethylbutyl, 3,3- dimethylbutyl, 1,1,2-trimethylpropyl, 1,2,2-trimethylpropyl, 1-ethylbutyl, 2-ethylbutyl, 1-ethyl-2-methylpropyl, 2-heptyl, 3-heptyl, 2-ethylpentyl, 1-propylbutyl, 2-ethylhexyl, 2-propylheptyl, 1,1,3,3-tetramethylbutyl, nonyl, decyl, n-undecyl, n-dodecyl, n-tridecyl, isotridecyl, n-tetradecyl, n-hexadecyl, n-octadecyl, n-eicosyl, and the like.

[0043] Herein, "cycloalkyl" is a cyclic alkyl group such as, but not limited to, cyclopropyl, cyclobutyl, cyclopentyl, cyclohexyl, cycloheptyl, and cyclooctyl. In some embodiments, the cycloalkyl group has 3 to 8 ring members, while in other embodiments, the number of ring carbon atoms ranges from 3 to 5, 6, or 7. The cycloalkyl group may be substituted or unsubstituted.

[0044] Herein, "degree of polymerization" refers to the number of monomers polymerized, which can be measured by methods known in the art. An exemplary measurement method is nuclear magnetic resonance spectroscopy. Specifically, PEG 5k For example, according to the polymer 1 In the H-NMR spectrum, the signal at each chemical shift was integrated, and the integral value at the chemical shift of 3.79-3.50 (i.e., the H number of PEG, which is a determined value) was set to 450. The integral values ​​at other chemical shifts were divided by the H number at the corresponding structural attribution position to obtain the degree of polymerization of each monomer.

[0045] Herein, the term "membrane" or "membrane structure" has the same meaning, and both refer to a membrane formed by phospholipids, or a phospholipid membrane, including a biological membrane or an artificial membrane. A biological membrane refers to a naturally occurring membrane structure derived from an organism or part thereof, examples of which include but are not limited to cell membranes, virus membranes, bacterial membranes, exosome membranes, organelle membranes, etc. An artificial membrane refers to a phospholipid membrane obtained by artificial manipulation using phospholipid materials such as soybean lecithin, DOTAP, DOPE, etc., such as a phospholipid bilayer, examples of which include but are not limited to liposomes.

[0046] In this article, "membrane binding capacity" and "membrane binding efficiency" have the same meaning. The measurement of membrane binding capacity is measured by the percentage of cells bound to the material in a random field of view under a laser confocal microscope to the number of cells in the current field of view, or the percentage of cells bound to the material in the total number of cells detected by flow cytometry. The membrane rupture capacity is measured by the percentage of the number of membrane-ruptured cells in a random field of view under a laser confocal microscope to the number of cells in the current field of view, or the number of viable cells detected by flow cytometry.

[0047] Herein, the measurement conditions of the nuclear magnetic resonance spectrum are as follows: after the polymer is fully dried, more than 50 mg is weighed, dissolved in deuterated chloroform, and measured using a 300 / 400 / 500 MHz nuclear magnetic resonance spectrometer.

[0048] [polymer]

[0049] According to a first aspect of the present invention, there is provided a polymer capable of being combined with a membrane, which is obtained by reacting a monomer and an initiator in an organic solvent in the presence of a catalyst.

[0050] In the present invention, the preferred examples of monomers are aminoalkyl (meth)acrylates, examples of which include but are not limited to AMA, DMA, DEA, C7A, EPA, iDPA, nDPA, PBA, DBA and D5A. The structural formulas of these monomers are as follows:

[0051]

[0052] In the present invention, the type of monomer in each polymer may be one or more, such as two, three, four or more. In the case where the monomer of the polymer is one, the degree of polymerization of the polymer is not particularly limited, for example, it may be in the range of 10-500, preferably in the range of 20-300. In the case where the monomer of the polymer is multiple, the matching ratio between different monomers is not particularly limited, and may be equal proportion matching, or any other ratio. For example, in the case of two monomers, the molar ratio of the two may be in the range of 1:100-100:1. The total degree of polymerization of multiple monomers is not particularly limited, for example, it may be in the range of 10-500, preferably in the range of 20-300.

[0053] The catalyst of the present invention comprises a metal catalyst and a ligand combined therewith. The metal catalyst is preferably a metal halide, such as a metal chloride, a metal bromide, or a metal iodide. Examples of metals include divalent or monovalent metals, such as copper, magnesium, iron, nickel, ruthenium, rhodium, and cuprous. Examples of ligands include, but are not limited to, PMDETA, TPMA, bpy, and the like.

[0054] Examples of organic solvents of the present invention include, but are not limited to, isopropanol, dimethylformamide, tetrahydrofuran, 1,4-dioxane, and the like. The present invention may use one or two of these solvents. In the case of using two solvents, the ratio of the two is not particularly limited, and those skilled in the art may freely set the ratio as required. The exemplary ratio is generally in the range of 1:10-10:1.

[0055] In an exemplary embodiment, the polymer of the present invention is a polymer of a single monomer, such as PAMA x 、PDMA x , PDEA x 、PC7A x 、PEPA x 、iPDPA x 、nPDPA x PPBA x , PDBA x and PD5A x , wherein x represents the number of monomers, which is generally an integer of 10-500, preferably an integer of 20-400, and more preferably an integer of 20-300.

[0056] In another exemplary embodiment, the polymer of the present invention is a polymer of two monomers, such as PDEA x -nPDPA y 、PAMA x -PDMA y , PDEA x -PC7A y 、iPDPA x -nPDPA y PPBA x -PDBA y etc., wherein x and y each independently represent the number of monomers, and are generally integers of 10-500, preferably integers of 20-400, and more preferably integers of 20-300.

[0057] In other exemplary embodiments, the polymer of the present invention is a polymer of three monomers, for example, PAMA x -PDMA y -PDEA z 、PC7A x -PEPAy -iPDPA z 、nPDPA x -PPBA y -PDBA z and PPBA x -PDBA y -PD5A z Etc. wherein x, y and z each independently represent the number of monomers, and are generally integers of 10-500, preferably integers of 20-400, and more preferably integers of 20-300.

[0058] In the polymer of the present invention, in addition to the above-mentioned segments obtained by polymerization of monomers (sometimes referred to herein as "hydrophobic segments"), a hydrophilic segment may be further included. The hydrophobic segment of the polymer is generally used to bind to the membrane surface, preferably the inside of the membrane. The hydrophilic segment of the polymer is generally exposed to the outer surface of the membrane. Examples of hydrophilic segments include polyethylene glycol, etc. For example, PEG (20-20000) .

[0059] The present invention has found that different monomers in the polymer have different abilities to bind and cleave the membrane structure. The different degrees of membrane binding and cleavage abilities of each monomer to the overall polymer can be called contribution. It is further found that the contribution decreases in the order of AMA, DMA, DEA, C7A, EPA, iDPA, nDPA, PBA, DBA to D5A. For example, PDMA 100 The homopolymer has greater membrane binding and cleavage capabilities than PDEA 100 Homopolymer. In addition, the content of each monomer in the polymer also affects the contribution. For example, PDMA 70 -PDEA 30 The polymer has a greater ability to bind and cleave membranes than PDMA 50 -PDEA 50 polymer.

[0060] [Suspension]

[0061] The suspension of the present invention is preferably a nanoparticle suspension, which can be obtained by dispersing a polymer in an aqueous solution.

[0062] In an exemplary embodiment, the polymer can be dissolved in THF, and then; distilled water is added while being ultrasonically dispersed to obtain a dispersion; the dispersion is transferred to an ultrafiltration tube with a molecular weight of 100 kD, ultrafiltered at 5000 rpm for 20 min, and then the resulting material is washed five times with distilled water; centrifuged at 10000 rpm for 5 min to remove the precipitate; the supernatant is fixed to 1 mL to obtain a diblock copolymer nanoparticle suspension.

[0063] Example 1

[0064] PEG-PDEA 100 Synthesis

[0065] First, 2 mmol PEG 5k Dissolve it in 100 mL toluene, reflux at 110°C for 1 hour, and spin-dry the toluene to remove water. Then dissolve it in dichloromethane, add 4 mmol TEA and 2.4 mmol BiBB, and stir at room temperature for 12 hours. After the reaction is completed, filter out the residue, spin-dry the dichloromethane, redissolve it in tetrahydrofuran, dialyze it with pure water for 2 days, and freeze-dry it to obtain the product PEG-Br.

[0066] 20μmol of product PEG-Br, 2mmol of monomer DEA, and 20μmol of PMDETA were dissolved in 10mL of anhydrous DMF:IPA (1:1) solution, mixed and added to the polymerization tube, and the reaction system was frozen with liquid nitrogen, then the valve was unscrewed, and the vacuum pump was used to evacuate for about 5 minutes, the valve was screwed on, and the reaction system was placed in water to slowly dissolve. After the reaction system was completely dissolved, it was frozen and evacuated again with liquid nitrogen. After four cycles (i.e., freezing and evacuation cycles), 20μmol of CuBr was added to the reaction tube under a nitrogen atmosphere. After the reaction, the valve was tightened and placed in a 40℃ oil bath for 12h; after the reaction, the system was frozen with liquid nitrogen, the valve was unscrewed to ventilate and quench the reaction, and it was dialyzed with pure water for 2d, and freeze-dried to obtain the product PEG-PDEA. 100 , denoted as P1.

[0067]

[0068] Example 2

[0069] PEG-PDEA 39 -nPDPA 61 Synthesis

[0070] First, 2 mmol PEG 5k Dissolve it in 100 mL toluene, reflux at 110°C for 1 hour, and spin-dry the toluene to remove water. Then dissolve it in dichloromethane, add 4 mmol TEA and 2.4 mmol BiBB, and stir at room temperature for 12 hours. After the reaction is completed, filter out the residue, spin-dry the dichloromethane, redissolve it in tetrahydrofuran, dialyze it with pure water for 2 days, and freeze-dry it to obtain the product PEG-Br.

[0071] 20 μmol of product PEG-Br, 800 μmol of monomer DEA, 1.2 mmol of monomer DPA, 100 nmol of TPMA, 100 nmol of CuBr2 and 4 μmol of AIBN were dissolved in 10 mL of anhydrous DMF:IPA (1:1) solution, mixed and added to the polymerization tube, purged with high-purity argon for 0.5 h, stirred while purging, and after the end, the valve was tightened and placed in a 70°C oil bath for 48 h. After the reaction was completed, it was dialyzed with pure water for 2 days and freeze-dried to obtain the product PEG-nPDPA. 100 , denoted as P2.

[0072]

[0073] Example 3

[0074] PEG-iPDPA 102 Synthesis

[0075] First, 2 mmol PEG 5k Dissolve it in 100 mL toluene, reflux at 110°C for 1 hour, and spin-dry the toluene to remove water. Then dissolve it in dichloromethane, add 4 mmol TEA and 2.4 mmol BiBB, and stir at room temperature for 12 hours. After the reaction is completed, filter out the residue, spin-dry the dichloromethane, redissolve it in tetrahydrofuran, dialyze it with pure water for 2 days, and freeze-dry it to obtain the product PEG-Br.

[0076] 20μmol of product PEG-Br, 2mmol of monomer DPA, and 20μmol of PMDETA were dissolved in a 10mL anhydrous DMF:IPA (1:1) solution, mixed and added to the polymerization tube, and the reaction system was frozen with liquid nitrogen, then the valve was unscrewed, and the vacuum pump was used to evacuate for about 5 minutes, the valve was screwed on, and the reaction system was placed in water to slowly dissolve. After the reaction system was completely dissolved, it was frozen and evacuated again with liquid nitrogen. After four cycles (i.e., freezing and evacuation cycles), 20μmol of CuBr was added to the reaction tube under a nitrogen atmosphere. After the reaction, the valve was tightened and placed in a 40℃ oil bath for 12h; after the reaction, the system was frozen with liquid nitrogen, the valve was unscrewed to ventilate and quench the reaction, and it was dialyzed with pure water for 2d, and freeze-dried to obtain the product PEG-iPDPA. 102 , denoted as P3.

[0077]

[0078] Example 4

[0079] PEG-nPDPA 100 Synthesis

[0080] First, 2 mmol PEG 5kDissolve it in 100 mL toluene, reflux at 110°C for 1 hour, and spin-dry the toluene to remove water. Then dissolve it in dichloromethane, add 4 mmol TEA and 2.4 mmol BiBB, and stir at room temperature for 12 hours. After the reaction is completed, filter out the residue, spin-dry the dichloromethane, redissolve it in tetrahydrofuran, dialyze it with pure water for 2 days, and freeze-dry it to obtain the product PEG-Br.

[0081] 20 μmol of product PEG-Br, 2 mmol of monomer nDPA, 100 nmol of TPMA, 100 nmol of CuBr2 and 4 μmol of AIBN were dissolved in 10 mL of anhydrous DMF:IPA (1:1) solution, mixed and added to the polymerization tube, purged with high-purity argon for 0.5 h, stirred while purging, and after the end, the valve was tightened and placed in a 70°C oil bath for 48 h. After the reaction was completed, it was dialyzed with pure water for 2 days and freeze-dried to obtain the product PEG-nPDPA. 100 , denoted as P4.

[0082]

[0083] Example 5

[0084] MeO-PDEA 100 Synthesis

[0085] First, 4 mmol TEA and 2.4 mmol BiBB were added dropwise into 4 mmol methanol and stirred at room temperature for 12 h. After the reaction was completed, the residue was removed by suction filtration, dialyzed with pure water for 2 days, and freeze-dried to obtain the product MeO-Br.

[0086] 20 μmol of product MeO-Br, 2 mmol of monomer DEA, 100 nmol of TPMA, 100 nmol of CuBr2 and 4 μmol of AIBN were dissolved in 10 mL of anhydrous DMF:IPA (1:1) solution, mixed and added to the polymerization tube, purged with high-purity argon for 0.5 h, stirred while purging, and after the end, the valve was tightened and placed in a 70°C oil bath for 48 h. After the reaction was completed, it was dialyzed with pure water for 2 days and freeze-dried to obtain the product MeO-PDEA. 100 , denoted as P5.

[0087]

[0088] Example 6

[0089] PEG-PEPA 100 Synthesis

[0090] First, 2 mmol PEG 5kDissolve it in 100 mL toluene, reflux at 110°C for 1 hour, and spin-dry the toluene to remove water. Then dissolve it in dichloromethane, add 4 mmol TEA and 2.4 mmol BiBB, and stir at room temperature for 12 hours. After the reaction is completed, filter out the residue, spin-dry the dichloromethane, redissolve it in tetrahydrofuran, dialyze it with pure water for 2 days, and freeze-dry it to obtain the product PEG-Br.

[0091] 20 μmol of product PEG-Br, 2 mmol of monomer EPA, 100 nmol of TPMA, 100 nmol of CuBr2 and 4 μmol of AIBN were dissolved in 10 mL of anhydrous DMF:IPA (1:1) solution, mixed and added to the polymerization tube, purged with high-purity argon for 0.5 h, stirred while purging, and after the end, the valve was tightened and placed in a 70°C oil bath for 48 h. After the reaction was completed, it was dialyzed with pure water for 2 days and freeze-dried to obtain the product PEG-PEPA 100 , denoted as P6.

[0092]

[0093] Example 7

[0094] PEG-PC7A 100 Synthesis

[0095] First, 2 mmol PEG 5k Dissolve it in 100 mL toluene, reflux at 110°C for 1 hour, and spin-dry the toluene to remove water. Then dissolve it in dichloromethane, add 4 mmol TEA and 2.4 mmol BiBB, and stir at room temperature for 12 hours. After the reaction is completed, filter out the residue, spin-dry the dichloromethane, redissolve it in tetrahydrofuran, dialyze it with pure water for 2 days, and freeze-dry it to obtain the product PEG-Br.

[0096] 20 μmol of product PEG-Br, 2 mmol of monomer C7A, 100 nmol of TPMA, 100 nmol of CuBr2 and 4 μmol of AIBN were dissolved in 10 mL of anhydrous DMF:IPA (1:1) solution, mixed and added to the polymerization tube, purged with high-purity argon for 0.5 h, stirred while purging, and after the end, the valve was tightened and placed in a 70°C oil bath for 48 h. After the reaction was completed, it was dialyzed with pure water for 2 days and freeze-dried to obtain the product PEG-PC7A. 100 , denoted as P7.

[0097]

[0098] Example 8

[0099] Weigh 11 mg of P1-P7 copolymer and dissolve it in 1.5 mL of THF; add 9 mL of distilled water while ultrasonicating to disperse it to obtain a dispersion; transfer the dispersion to an ultrafiltration tube with a molecular weight of 100 kD, perform ultrafiltration at 5000 rpm for 20 min, and then wash the obtained material with distilled water five times; centrifuge at 10000 rpm for 5 min to remove the precipitate; and adjust the supernatant to 1 mL to obtain a nanoparticle suspension of the corresponding copolymer.

[0100] Example 9

[0101] 1) PEG-PDEA of Example 1 100 Binding to cell membrane studies

[0102] Hela cells were cultured in DMEM medium (supplemented with 10% FBS), and when the cells were confluent to 80%, PBS buffer (control) or aqueous solution of P1 copolymer nanoparticles (NP-1) was added to the cells to a concentration of 80 μM. The cells were incubated at 37°C for 1-2 h, and the cells were stained with fluorescent dye DiI and then observed by fluorescence confocal microscopy. The results are shown in Figure 8 , the results clearly showed that the P1 copolymer was bound to the cell membrane surface.

[0103] 2) PEG-PDEA of Example 1 100 Study on inducing cell membrane rupture

[0104] Hela cells were cultured in DMEM medium (supplemented with 10% FBS), and when the cells were confluent to 80%, PBS buffer (control) or aqueous solution of P1 copolymer nanoparticles (NP-1) was added to the cells to a concentration of 80 μM. The cells were incubated at 37°C for an appropriate time, and the cells were stained with the fluorescent dye DiI, and then observed by fluorescence confocal microscopy. The results are shown in Fig. 9 The results clearly showed that the P1 copolymer bound to the cell membrane surface and caused the cell bleb rupture.

[0105] Example 10

[0106] 1) PEG-PDEA of Example 2 39 -nPDPA 61 Direct binding to viral envelope

[0107] Take 3×10 9 PFU / mL of vesicular stomatitis virus (VSV) was mixed with an equal volume of 80 μM NP-1 aqueous solution or PBS buffer and incubated at 4°C. After incubation for 1 hour, the mixture was observed using a transmission electron microscope. The results are shown in Fig.10(Scale bar: 50 nm) The figure shows the binding of P2 copolymer to the VSV viral envelope.

[0108] 2) PEG-PDEA of Example 2 39 -nPDPA 61 Study on direct binding to virus-infected cell membrane

[0109] Hela cells were cultured in DMEM medium (supplemented with 10% FBS), and when the cells were 80% confluent, 3×10 9 PFU / mL of VSV, and PBS buffer (control) or aqueous solution of copolymer nanoparticles was added to a concentration of 80 μM. The cells were incubated at 37°C for a suitable time, and the cells were stained with fluorescent dye DiI and then observed by fluorescence confocal microscopy. The results are shown in Fig.11 , the results clearly showed that the P2 copolymer was bound to the surface of virus-infected cell membranes.

[0110] 3) PEG-PDEA of Example 2 39 -nPDPA 57 Research on direct destruction of viral envelope

[0111] Experiment (a)

[0112] Take 3×10 9 PFU / mL of VSV or liposomes prepared by hydrogenated soybean lecithin, cholesterol, and phosphatidylserine were mixed with an equal volume of 80 μM NP-2 aqueous solution or PBS buffer and incubated at 4°C; after incubation for 1 hour, the mixture was observed using a transmission electron microscope. The results are shown in Fig.12 (Scale bar: 50 nm) The figure shows the disruption of the VSV viral envelope by the P2 copolymer.

[0113] Experiment (b)

[0114] To 1×10 5 An equal volume of 80 μM P4 aqueous solution or DMEM medium (control) was added to 100 PFU / mL of vesicular stomatitis virus expressing green fluorescent protein (VSV-GFP), and PEG 550 or PEG 2000 Solution was treated at 4°C for 1 hour. One hour later, HeLa cells were infected with the treated virus at an MOI of 0.01. After 16 hours of infection, the infected cells were detected using laser confocal microscopy. The results are shown in Fig.13 In the figure, UT means no treatment, and as a control, Vehicle means only the medium was added without the addition of nanoparticles. The results show that the destruction of the viral envelope by the copolymer of the present invention can be inhibited by the known cell pyroptosis inhibitor PEG 2000Suppressed.

[0115] Embodiment 11

[0116] 1) MeO-PDEA of Example 5 100 Study on direct binding to virus-infected cell membrane

[0117] Hela cells were cultured in DMEM medium (supplemented with 10% FBS), and when the cells were 80% confluent, 3×10 9 PFU / mL of VSV, and PBS buffer (control) or aqueous solution of copolymer nanoparticles P5 was added to a concentration of 80 μM. The cells were incubated at 37°C for a suitable time, stained with fluorescent dye DiI, and then observed by fluorescence confocal microscopy. The results are shown in Fig.14 , the results clearly showed that no PEG polymer was bound to the surface of virus-infected cell membranes.

[0118] 2) Direct binding of Hela cells overexpressing viral ion channel-related proteins with MeO-PDEA100 from Example 5

[0119] Hela cells overexpressing various viral ion channel proteins were cultured in DMEM medium (supplemented with 10% FBS). When the cells were confluent to 80%, an aqueous solution of copolymer nanoparticles P5 was added to the cells to a concentration of 80 μM. The cells were incubated at 37°C for an appropriate time, stained with fluorescent dye DiI, and then observed by fluorescence confocal microscopy. The results are shown in Fig.15 , the results clearly showed that the polymers were bound to the surface of cell membranes overexpressing various viral ion channel proteins.

[0120] Example 12

[0121] Binding of P1-P4 of the present invention to normal cells or cell membranes after virus infection

[0122] Hela cells were cultured in DMEM medium (supplemented with 10% FBS), and when the cells were 80% confluent, 3×10 9 PFU / mL of VSV, and an aqueous solution of copolymer nanoparticles P1-P4 was added to a concentration of 80 μM. The cells were incubated at 37°C for a suitable time, and the cells were stained with the fluorescent dye DiI, and then observed by fluorescence confocal microscopy. The results are shown in Fig.16 , the results clearly showed the binding of the polymer to normal cells as well as virus-infected cell membranes.

[0123] Example 13

[0124] Hela cells were cultured in DMEM medium (supplemented with 10% FBS), and when the cells were confluent to 80%, PBS buffer (control) or aqueous solution of copolymer nanoparticles was added to the cells to a concentration of 80 μM. The cells were incubated at 37°C for 1-2 h, and the cells were stained with fluorescent dye DiI, and then observed by fluorescence confocal microscopy. The results are shown in Table 1. Some exemplary membrane binding efficiencies are shown in Table Fig.17 , the results clearly showed the ability of different copolymers to bind to the cell membrane surface.

[0125] Table 1

[0126]

[0127]

[0128]

[0129]

[0130]

[0131] Note:

[0132] 1. Unless otherwise specified, the molecular weight of PEG in the table is 5000.

[0133] 2. In the NMR spectrum, except for material 11, the total NMR part is 3.79-3.50 (450), 3.38 (3). The numbers outside the brackets are chemical shifts, and the numbers inside the brackets are the H numbers at the chemical shift.

[0134] 3. Under the same conditions, the degree of polymerization of the monomer has little effect on the membrane binding efficiency of the polymer. When the degree of polymerization differs by less than 50, there is no substantial effect on the membrane binding efficiency.

[0135] It can be seen from the above embodiments that the present invention provides copolymers that can bind to and lyse cell membranes, virus membranes, cell membranes after virus infection, liposome membranes, etc. The copolymers provided by the present invention can lyse or destroy the outer membrane of cells or viruses, thereby exhibiting corresponding biological properties.

[0136] It can also be seen from the above embodiments that the membrane binding ability and membrane splitting ability of the polymer of the present invention gradually weaken in the order of polymer monomers AMA, DMA, DEA, C7A, EPA, iDPA, nDPA, PBA, DBA, and D5A. Different ratios of monomers can produce different membrane binding abilities.

[0137] The polymer provided by the present invention has a broad-spectrum effect on cells and RNA viruses, and can achieve a broad-spectrum membrane binding and membrane lysis effect.

[0138] Although the present invention has been described with reference to exemplary embodiments, it should be understood that the present invention is not limited to the disclosed exemplary embodiments. Various adjustments or changes may be made to the exemplary embodiments of the present specification without departing from the scope or spirit of the present invention. The scope of the claims should be based on the broadest interpretation to cover all modifications and equivalent structures and functions.

Claims

1. Use of a polymer in the preparation of a composition for bonding a membrane structure, characterized in that The polymer is obtained by reacting a monomer and an initiator in an organic solvent in the presence of a catalyst, wherein: The catalyst includes a metal catalyst and a ligand bound thereto; The initiator is selected from bromoisobutyrate and / or chloroisobutyrate; The monomer is at least one selected from the group consisting of AMA, DMA, DEA, C7A, EPA, iDPA, nDPA, PBA, DBA and D5A.

2. The use according to claim 1, characterized in that The initiator is obtained by reacting a hydroxy compound with 2-halogenated isobutyryl halide via substitution reaction, and the metal catalyst is a metal halide.

3. The use according to claim 1, characterized in that The polymer further comprises a hydrophilic segment.

4. The use according to claim 3, characterized in that The hydrophilic segment is PEG with a molecular weight of 1000-10000 kDa.

5. The use according to claim 1, characterized in that: The polymer has the NMR spectrum shown in Table 1.

6. The use according to claim 1, characterized in that: The composition is a nanoparticle suspension or a pharmaceutical composition.

7. The use according to claim 6, characterized in that The composition is obtained by dispersing the polymer in an aqueous solution.

8. A method for binding and lysing a membrane structure, characterized in that: The step of contacting a polymer or a nanoparticle suspension obtained therefrom with a membrane structure; wherein: The polymer is obtained by reacting a monomer and an initiator in an organic solvent in the presence of a catalyst, wherein the catalyst comprises a metal catalyst and a ligand bound thereto; the initiator is selected from bromoisobutyrate and / or chloroisobutyrate; and the monomer is selected from at least one of the group consisting of AMA, DMA, DEA, C7A, EPA, iDPA, nDPA, PBA, DBA and D5A.

9. The method according to claim 8, characterized in that The membrane structure is selected from the group consisting of cell membranes, virus membranes, bacterial membranes and artificial lipid membranes.

10. A method for predicting or regulating the membrane binding ability or membrane lysis ability of a polymer, characterized in that: The polymerization monomer of the polymer is selected from at least one of the group consisting of AMA, DMA, DEA, C7A, EPA, iDPA, nDPA, PBA, DBA and D5A, and the method includes the step of predicting or adjusting the polymer according to the content of each polymerization monomer and the contribution to membrane binding ability or membrane lysis ability, wherein the contribution decreases in the order of AMA, DMA, DEA, C7A, EPA, iDPA, nDPA, PBA, DBA to D5A.

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