CN114456331A

CN114456331A - Polymer capable of being combined with membrane and preparation method and application thereof

Info

Publication number: CN114456331A
Application number: CN202210143238.4A
Authority: CN
Inventors: 杨圆圆
Original assignee: Peking University
Current assignee: Peking University
Priority date: 2022-02-16
Filing date: 2022-02-16
Publication date: 2022-05-10

Abstract

The invention discloses a polymer capable of being combined with a membrane, a preparation method and application thereof. The polymer of the invention is prepared by a specific method, and has the capacity of combining and splitting cell membranes and virus membranes, thereby realizing the functions of resisting tumors (splitting tumor cells) and resisting viruses (splitting virus membranes or virus infection cell membranes).

Description

Polymer capable of being combined with membrane and preparation method and application thereof

Technical Field

The present invention relates to (meth) acrylate polymers, in particular to (meth) acrylate polymers having membrane-binding or membrane-cleaving ability, to a process for their preparation and to their use, in particular their pharmaceutical use.

Background

The information in this background is only for the purpose of illustrating the general background of the invention and should not be taken as an acknowledgement or any form of suggestion that this information forms the prior art that is known to a person skilled in the art.

Disclosure of Invention

To solve at least part of the technical problems of the prior art, the present invention provides a polymer capable of bonding to a membrane. The present invention has been accomplished, at least in part, based on this. Specifically, the present invention includes the following.

In a first aspect of the present invention, there is provided a polymer capable of bonding to a membrane, the polymer being 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 monomer comprises aminoalkyl (meth) acrylate having the formula;

wherein R is₁、R₂Represent the same or different groups and each independently represent H, C_1-10Linear, branched or cyclic alkyl; the initiator is selected from bromoisobutyrate and/or chloroisobutyrate.

The polymer according to the present invention, illustratively, the monomer is at least one selected from the group consisting of AMA, DMA, DEA, C7A, EPA, iDPA, nDPA, PBA, DBA, and D5A.

According to the polymer of the invention, the initiator is obtained by substitution reaction of a hydroxyl compound and 2-halogenated isobutyl acyl halide, and the metal catalyst is metal halide.

The polymer according to the present invention, exemplarily, the polymer further comprises a hydrophilic segment.

According to the polymer of the present invention, the hydrophilic segment is illustratively PEG with a molecular weight of 1000-10000 kDa.

In a second aspect of the invention, there is provided a polymer having a nuclear magnetic spectrum as shown in table 1.

In a third aspect of the invention, there is provided a nanoparticle suspension or a pharmaceutical composition obtained by dispersing the polymer of the first or second aspect in an aqueous solution.

In a fourth aspect of the invention, there is provided the use of a polymer according to the first or second aspect or a nanoparticle suspension or pharmaceutical composition according to the third aspect in the preparation of a composition for binding a membrane structure.

In a fifth aspect of the invention, there is provided a method for binding and lysing a membrane structure, comprising the step of contacting a polymer according to the first or second aspect or a nanoparticle suspension according to the third aspect with the membrane structure.

According to the method of the present invention, the membrane structure is illustratively a phospholipid membrane.

According to the method of the present invention, the membrane structure is illustratively selected from the group consisting of a cell membrane, a viral membrane, a bacterial membrane, and an artificial lipid membrane.

In a sixth aspect of the present invention, there is provided a method for predicting or adjusting a membrane-binding capacity or a membrane-cleavage capacity of a polymer, of which polymerized monomer is at least one selected from the group consisting of AMA, DMA, DEA, C7A, EPA, iDPA, nDPA, PBA, DBA, and D5A, the method comprising the step of predicting or adjusting the polymer according to the content of each polymerized monomer and a degree of contribution to the membrane-binding capacity or the membrane-cleavage capacity, wherein the degree of contribution becomes smaller in order from AMA, DMA, DEA, C7A, EPA, iDPA, nDPA, PBA, DBA to D5A.

The polymers of the present invention have the ability to bind to and cleave cell membranes, viral membranes. According to the polymer monomer, the following groups can be exemplarily classified: AMA, DMA, DEA, C7A, EPA, iDPA, nDPA, PBA, DBA and D5A, and further finds that the membrane binding capacity is gradually weakened, the membrane splitting capacity is also gradually weakened, and different membrane binding capacities can be generated by matching different monomers. On the basis, a series of polymer materials can be synthesized, and can be combined with cell membranes, cell membranes after virus infection and virus membranes respectively. The stronger the membrane binding capacity of the material, the easier the material is to bind with a cell membrane, and the stronger the corresponding membrane splitting capacity is; as the binding capacity of the material membrane decreases, there is a gradual bias towards binding to the viral membrane, and the ability to split the membrane also decreases. The polymer of the invention has broad spectrum effect on various cells and RNA viruses, thereby realizing the effect of resisting tumor (cracking tumor cells) and virus (cracking virus membranes or virus infected cell membranes).

Drawings

Fig. 1 is an NMR spectrum of exemplary compound P1.

Fig. 2 is an NMR spectrum of exemplary compound P2.

Fig. 3 is an NMR spectrum of exemplary compound P3.

Fig. 4 is an NMR spectrum of exemplary compound P4.

Fig. 5 is an NMR spectrum of exemplary compound P5.

Fig. 6 is an NMR spectrum of exemplary compound P6.

Fig. 7 is an NMR spectrum of exemplary compound P7.

FIG. 8 is a plot of the Confocal co-localization of the polymer prepared in example 1 of the present invention after incubation with cells (P1 is the fluorescence of polymer P1, DiI represents the fluorescent staining exhibited by dye DiI, and Merge represents the overlay of P1 and DiI dye).

FIG. 9 shows the Confocal mapping of the disruption of the cell membrane after incubation of the polymer prepared in example 1 with cells (P1 is the fluorescence of the polymer P1, DiI shows the fluorescence staining with the dye DiI, and Merge shows the mapping obtained by superimposing the P1 with the DiI dye).

FIG. 10 is a transmission electron micrograph of a polymer prepared in example 2 of the present invention after incubation with VSV virus.

FIG. 11 is a Confocal map of the polymer prepared in example 2 incubated with virus-infected cells (NP is the fluorescence of polymer P2, DiI is the fluorescence of dye DiI, GFP is the fluorescence of the virus, Merge is the overlay of P2 with the DiI dye and the fluorescence of the virus GFP).

FIG. 12 is a transmission electron micrograph of VSV virus, model liposomes, copolymer nanoparticle suspensions P4 and P4 from example 3 after incubation with both.

FIG. 13 is a graph showing the effect of different drug treatments on the ability to infect viruses in example 10.

FIG. 14 is a Confocal map of the incubation of the polymer prepared in example 5 with virus-infected cells (PDEA is the fluorescence of polymer P5, DiI indicates the fluorescence staining with dye DiI, GFP is the fluorescence of the virus, Merge is the overlay of P5 with the DiI dye and the fluorescence of the virus GFP).

FIG. 15 is a Confocal colocalization map of the polymer prepared in example 5 incubated with Hela cells overexpressing various viral ion channel proteins (PDEA is the fluorescence of polymer P5, DiI is the fluorescence staining exhibited by dye DiI, GFP is the fluorescence of overexpressed viral ion channel proteins, Merge is the map obtained by superimposing the fluorescence of P5 with DiI dye and GFP).

FIG. 16 is a plot of the Confocal positions of P1, P2, P3 and P4 after incubation with normal cells or with cells infected with viruses (UPS indicates the fluorescence of polymers P1-P4, DiI indicates the fluorescence staining with dye DiI, GFP indicates the fluorescence of viruses, and Merge indicates the superposition of polymer with DiI dye and virus GFP fluorescence).

Fig. 17 is a statistical plot of the binding capacity of exemplary nanoparticles to cell membranes.

FIG. 18 is a schematic representation of polymer binding to a virus.

Detailed Description

Reference will now be made in detail to various exemplary embodiments of the invention, the detailed description should not be construed as limiting the invention but as a more detailed description of certain aspects, features and embodiments of the invention.

It is to be understood that the terminology used herein is for the purpose of describing particular embodiments only and is not intended to be limiting of the invention. Further, for numerical ranges in this disclosure, it is understood that the upper and lower limits of the range, and each intervening value therebetween, is specifically disclosed. Every smaller range between any stated value or intervening value in a stated range and any other stated or intervening value in a stated range is encompassed within the invention. The upper and lower limits of these smaller ranges may independently be included or excluded in the range.

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. Although only preferred methods and materials are described herein, any methods and materials similar or equivalent to those described herein can be used in the practice or testing of the present invention. All documents mentioned in this specification are incorporated by reference herein for the purpose of disclosing and describing the methods and/or materials associated with the documents. In case of conflict with any incorporated document, the present specification will control. Unless otherwise indicated, "%" is percent by weight.

As used herein, the term "aminoalkyl" (meth) acrylates "refers to esters of acrylic acid and/or methacrylic acid with aminoalkyl groups. Wherein aminoalkyl refers to an amino-containing alkyl compound represented by the general formula-CH₂N(R₁)R₂Is represented by the formula (I), wherein R₁And R₂Each independently represents the same or different group, and each independently represents H, C_1-10Straight or branched alkyl or C_1-10A cycloalkyl group.

As used herein, the term "alkyl" includes both straight and branched chain alkyl groups having from 1 to about 10 carbon atoms, and typically from 1 to 8 carbon atoms, or in some embodiments from 1 to 5 carbon atoms. Herein, "alkyl" includes cycloalkyl as defined below. 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 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 with, for example, amino, thio, hydroxy, cyano, alkoxy, and/or halogen atoms such as F, Cl, Br, and I groups. The term "haloalkyl" is an alkyl group having one or more halo groups. In some embodiments, haloalkyl refers to perhaloalkyl. Typically, alkyl groups may include, but are not limited to, 2-pentyl, 2-methylbutyl, 3-methylbutyl, 1, 2-dimethylpropyl, 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-dimethylbutyl, 2, 2-dimethylbutyl, 3-dimethylbutyl, 1, 2-trimethylpropyl, 1,2, 2-trimethylpropyl, 1-ethylbutyl, 2-ethylbutyl, 1-ethyl-2-methylpropyl, methyl-propyl, ethyl-2-methylpropyl, methyl-pentyl, hexyl, octyl, decyl, octyl, decyl, and the like, 2-heptyl, 3-heptyl, 2-ethylpentyl, 1-propylbutyl, 2-ethylhexyl, 2-propylheptyl, 1,3, 3-tetramethylbutyl, nonyl, decyl, n-undecyl, n-dodecyl, n-tridecyl, isotridecyl, n-tetradecyl, n-hexadecyl, n-octadecyl, n-eicosyl and the like.

As used herein, "cycloalkyl" is a cyclic alkyl group such as, but not limited to, cyclopropyl, cyclobutyl, cyclopentyl, cyclohexyl, cycloheptyl, and cyclooctyl. In some embodiments, cycloalkyl groups have 3 to 8 ring members, while in other embodiments the number of ring carbon atoms ranges from 3 to 5, 6, or 7. Cycloalkyl groups may be substituted or unsubstituted.

Herein, "degree of polymerization" means the number of polymerization of monomers, and can be measured by a method known in the art. An exemplary measurement method is nuclear magnetic spectroscopy. In particular, with PEG_5kObtained, for example, from polymers¹And (3) performing H-NMR spectrum, integrating signals at each chemical shift, setting the integral value of 3.79-3.50 chemical shifts (namely the H number of PEG, which is a determined value) as 450, and dividing the integral values at other chemical shifts by the H number of the corresponding structure home position respectively to obtain the polymerization degree of each monomer.

Herein, the term "membrane" or "membrane structure" has the same meaning and both refer to a membrane formed of phospholipids, or phospholipid membrane, including biological or artificial membranes. Biological membranes refer to naturally occurring membrane structures derived from an organism or portion thereof, examples of which include, but are not limited to, cell membranes, viral membranes, bacterial membranes, exosome membranes, organelle membranes, and the like. The artificial membrane refers to a phospholipid membrane obtained by artificial manipulation using a phospholipid material such as soybean lecithin, DOTAP, DOPE, etc., such as a phospholipid bilayer, and examples thereof include, but are not limited to, liposomes.

Herein, "membrane-binding capacity" has the same meaning as "membrane-binding efficiency". The magnitude of the membrane binding capacity is measured by using the percentage of cells bound to the material in a random field of view under confocal laser microscopy 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-splitting capacity is measured by the percentage of the number of ruptured cells in the random field of view in the current field of view under a laser confocal microscope or the survival number of the cells detected by flow cytometry.

Herein, the measurement conditions of the nuclear magnetic resonance spectrum are as follows: and (3) fully drying the polymer, weighing more than 50mg, dissolving by using deuterated chloroform, and measuring by using an 300/400/500M nuclear magnetic resonance spectrometer.

[ Polymer ]

In a first aspect of the present invention, there is provided a polymer capable of bonding to a membrane, which is obtained by reacting a monomer and an initiator in an organic solvent in the presence of a catalyst.

In the present invention, preferable examples of the monomer are aminoalkyl (meth) acrylates, and examples thereof include, but are not limited to, AMA, DMA, DEA, C7A, EPA, iDPA, nDPA, PBA, DBA, and D5A, and the like. The structural formulae of these monomers are shown below:

in the present invention, the kind of the monomer in each polymer may be one kind or plural kinds, for example, two kinds, three kinds, four kinds or more kinds. In the case where the monomer of the polymer is one, the polymerization degree of the polymer is not particularly limited, and may be, for example, in the range of 10 to 500, preferably in the range of 20 to 300. In the case where the monomers of the polymer are plural, the blending ratio between different monomers is not particularly limited, and may be an equal ratio 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 polymerization degree of the plurality of monomers is not particularly limited, and may be, for example, in the range of 10 to 500, preferably in the range of 20 to 300.

The catalyst of the present invention comprises a metal catalyst and a ligand bound thereto. 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, cuprous and the like. Examples of ligands include, but are not limited to, PMDETA, TPMA, bpy, and the like.

Examples of organic solvents of the present invention include, but are not limited to, isopropanol, dimethylformamide, tetrahydrofuran, 1, 4-dioxane, and the like. One or both of them may be used in the present invention. In the case of using two solvents, the ratio of the two solvents is not particularly limited, and can be freely set by those skilled in the art as needed. Exemplary ratios are generally in the range of 1:10 to 10: 1.

In exemplary embodiments, the polymer of the present invention is a single monomer polymer, such as PAMA_x、PDMA_x、PDEA_x、PC7A_x、PEPA_x、iPDPA_x、nPDPA_x、PPBA_x、PDBA_xAnd PD5A_xWherein x represents the number of monomers and is generally an integer from 10 to 500, preferably an integer from 20 to 400, more preferably an integer from 20 to 300.

In further exemplary embodiments, the polymer of the present invention is a polymer of two monomers, e.g., PDEA_x-nPDPA_y、PAMA_x-PDMA_y、PDEA_x-PC7A_y、iPDPA_x-nPDPA_y、PPBA_x-PDBA_yEtc., wherein x, y each independently represent the number of monomers, and are generally an integer of 10 to 500, preferably an integer of 20 to 400, and more preferably an integer of 20 to 300, respectively.

In other exemplary embodiments, the polymer of the present invention is a polymer of three monomers, e.g., PAMA_x-PDMA_y-PDEA_z、PC7A_x-PEPA_y-iPDPA_z、nPDPA_x-PPBA_y-PDBA_zAnd PPBA_x-PDBA_y-PD5A_zAnd the like. Wherein x, y and z each independently represent the number of monomers, and are generally an integer of 10 to 500, preferably an integer of 20 to 400, and more preferably an integer of 20 to 300, respectively.

The polymer of the present invention may further contain a hydrophilic segment in addition to the above-mentioned segment obtained by polymerization of the monomer (sometimes referred to herein simply as "hydrophobic segment"). The hydrophobic segment of the polymer is generally used to interact with the membrane surfaceAnd selecting membrane internal combination. The hydrophilic segments of the polymer are typically exposed at the outer surface of the membrane. Examples of hydrophilic segments include polyethylene glycol and the like. For example PEG_(20-20000)。

The present inventors have found that different monomers in a polymer have different binding and cleaving abilities to a membrane structure, and that the magnitude of each monomer differs in the membrane binding and cleaving abilities to the whole polymer, and further, have found that the degree of contribution becomes smaller in the order from AMA, DMA, DEA, C7A, EPA, iDPA, nDPA, PBA, DBA to D5A. For example, PDMA₁₀₀The homopolymer has a binding and splitting capacity for the film greater than PDEA₁₀₀A homopolymer. In addition, the content of each monomer in the polymer also affects the degree of contribution. For example, PDMA₇₀-PDEA₃₀The binding and splitting capacity of the polymer to the membrane is greater than that of PDMA₅₀-PDEA₅₀A polymer.

[ suspension ]

The suspension of the invention is preferably a nanoparticle suspension, obtainable by dispersing a polymer in an aqueous solution.

In exemplary embodiments, the polymer may be dissolved in THF, then; adding distilled water while performing ultrasonic treatment for dispersion to obtain a dispersion liquid; transferring the dispersion liquid into an ultrafiltration tube with the molecular weight of 100kD, carrying out ultrafiltration for 20min at the rotating speed of 5000rpm, and then washing the obtained material with distilled water for five times; centrifuging at 10000rpm for 5min to remove precipitate; and (4) fixing the volume of the supernatant to 1mL to obtain diblock copolymer nanoparticle suspension.

Example 1

PEG-PDEA₁₀₀Synthesis of (2)

Firstly, 2mmol of PEG_5kDissolved in 100mL of toluene, refluxed at 110 ℃ for 1h, and the toluene was spin-dried to remove water. And then dissolving in dichloromethane, adding 4mmol of TEA and 2.4mmol of BiBB, stirring at room temperature for 12h, after the reaction is finished, performing suction filtration to remove residues, spin-drying dichloromethane, dissolving again with tetrahydrofuran, dialyzing for 2d with pure water, and freeze-drying to obtain the product PEG-Br.

Mu. mol of the product PEG-Br, 2mmol of the monomeric DEA, 20. mu. mol of PMDETA, dissolved in 10mL of anhydrous DMF: IPA (1:1) solution, mixing, addingAnd (2) putting the reaction system into a polymerization tube, freezing the reaction system by using liquid nitrogen, then unscrewing a valve, pumping air for about 5min by using a vacuum pump, screwing the valve, putting the reaction system into water to slowly dissolve the reaction system, freezing and pumping air by using the liquid nitrogen again after the reaction system is completely dissolved, circulating for four times (namely freezing and pumping air circulation), and then adding 20 mu mol of CuBr into the reaction tube under the nitrogen atmosphere. Screwing the valve after finishing, and placing in an oil bath at 40 ℃ for 12 h; after the reaction is finished, freezing the system by using liquid nitrogen, unscrewing a valve, ventilating and quenching the reaction, dialyzing for 2d by using pure water, and freeze-drying to obtain a product PEG-PDEA₁₀₀And is denoted as P1.

Example 2

PEG-PDEA₃₉-nPDPA₆₁Synthesis of (2)

20 mu mol of the product PEG-Br, 800 mu mol of monomer DEA, 1.2mmol of monomer DPA, 100nmol of TPMA and 100nmol of CuBr₂And 4. mu. mol AIBN dissolved in 10mL anhydrous DMF: IPA (1:1) solution is added into a polymerization tube after being mixed evenly, purging is carried out for 0.5h by using high-purity argon gas, stirring is carried out while purging is carried out, a valve is screwed after the completion, the mixture is placed in an oil bath at 70 ℃ for 48h, after the reaction is finished, dialysis is carried out for 2d by using pure water, and freeze-drying is carried out to obtain a product PEG-nPDPA₁₀₀And is denoted as P2.

Example 3

PEG-iPDPA₁₀₂Synthesis of (2)

Mu. mol of product PEG-Br, 2mmol of monomeric DPA, 20. mu. mol of PMDETA, dissolved in 10mL of anhydrous DMF: IPA (1:1) solution is added into a polymerization tube after being mixed evenly, a reaction system is frozen by liquid nitrogen, then a valve is unscrewed, air is pumped by a vacuum pump for about 5min, the valve is screwed on, the reaction system is placed into water to be slowly dissolved, after the reaction system is completely dissolved, the reaction system is frozen by the liquid nitrogen again and pumped, the circulation is carried out for four times (namely, freezing and air pumping circulation), and 20 mu mol of CuBr is added into the reaction tube under the nitrogen atmosphere. Screwing the valve after finishing, and placing in an oil bath at 40 ℃ for 12 h; after the reaction is finished, freezing the system by using liquid nitrogen, unscrewing a valve, ventilating and quenching the reaction, dialyzing for 2d by using pure water, and freeze-drying to obtain a product PEG-iDPA₁₀₂And is denoted as P3.

Example 4

PEG-nPDPA₁₀₀Synthesis of (2)

20. mu. mol of product PEG-Br, 2mmol of monomer nDPA, 100nmol of TPMA, 100nmol of CuBr₂And 4. mu. mol AIBN dissolved in 10mL anhydrous DMF: IPA (1:1) solution is added into a polymerization tube after being mixed evenly, high-purity argon is used for blowing for 0.5h, stirring is carried out while blowing, a valve is screwed after the blowing, the mixture is placed in an oil bath at 70 ℃ for 48h, after the reaction is finished, pure water is used for dialysis for 2d, and freeze-drying is carried out to obtain a product PEG-nPDPA₁₀₀And is denoted as P4.

Example 5

MeO-PDEA₁₀₀Synthesis of (2)

Firstly, dropwise adding 4mmol of TEA and 2.4mmol of BiBB into 4mmol of methanol, stirring at room temperature for 12 hours, after the reaction is finished, carrying out suction filtration to remove residues, dialyzing for 2d with pure water, and freeze-drying to obtain a product MeO-Br.

20 mu mol of product MeO-Br, 2mmol of monomer DEA, 100nmol of TPMA and 100nmol of CuBr₂And 4. mu. mol AIBN dissolved in 10mL anhydrous DMF: IPA (1:1) solution is added into a polymerization tube after being mixed evenly, purging is carried out for 0.5h by using high-purity argon gas, stirring is carried out while purging, a valve is screwed after the reaction is finished, the mixture is placed in an oil bath at 70 ℃ for 48h, after the reaction is finished, dialysis is carried out for 2d by using pure water, and freeze-drying is carried out to obtain a product MeO-PDEA₁₀₀And is denoted as P5.

Example 6

PEG-PEPA₁₀₀Synthesis of (2)

Mu. mol of the product PEG-Br, 2mmol of monomer EPA, 100nmol of TPMA, 100nmol of CuBr₂And 4. mu. mol AIBN dissolved in 10mL anhydrous DMF: IPA (1:1) solution is added into a polymerization tube after being mixed evenly, purging is carried out for 0.5h by using high-purity argon gas while stirring is carried out, a valve is screwed after the purging is finished, the mixture is placed in an oil bath at 70 ℃ for 48h, after the reaction is finished, the mixture is dialyzed for 2d by using pure water and is freeze-dried to obtain a product PEG-PEPA₁₀₀And is denoted as P6.

Example 7

PEG-PC7A₁₀₀Synthesis of (2)

Mu. mol of product PEG-Br, 2mmol of monomer C7A, 100nmol of TPMA, 100nmol of CuBr₂And 4. mu. mol AIBN dissolved in 10mL anhydrous DMF: IPA (1:1) solution is added into a polymerization tube after being mixed evenly, purging is carried out for 0.5h by using high-purity argon gas, stirring is carried out while purging is carried out, a valve is screwed after the completion, the mixture is placed in oil bath at 70 ℃ for 48h, after the reaction is finished, dialysis is carried out for 2d by using pure water, and freeze-drying is carried out to obtain a product PEG-PC7A₁₀₀And is denoted as P7.

Example 8

Weighing 11mg of P1-P7 copolymer, and dissolving in 1.5mL of THF; adding 9mL of distilled water while performing ultrasonic treatment to disperse to obtain a dispersion liquid; transferring the dispersion liquid into an ultrafiltration tube with the molecular weight of 100kD, carrying out ultrafiltration for 20min at the rotating speed of 5000rpm, and then washing the obtained material with distilled water for five times; centrifuging at 10000rpm for 5min to remove precipitate; and (4) fixing the volume of the supernatant to 1mL to obtain the nanoparticle suspension of the corresponding copolymer.

Example 9

1) PEG-PDEA of example 1₁₀₀Cell membrane binding study

HeLa cells were cultured in DMEM medium (with 10% FBS), and when the cells were confluent to 80%, PBS buffer (control) or an aqueous solution of P1 copolymer nanoparticles (NP-1) was added to the cells to a concentration of 80. mu.M. After incubation for 1-2h at 37 ℃, cells were stained with the fluorescent dye DiI and then observed by a fluorescence confocal microscope, and the result is shown in fig. 8, which clearly shows that the P1 copolymer is bound on the surface of cell membrane.

2) PEG-PDEA of example 1₁₀₀Study of induced cell Membrane rupture

HeLa cells were cultured in DMEM medium (with 10% FBS), and when the cells were confluent to 80%, PBS buffer (control) or an aqueous solution of P1 copolymer nanoparticles (NP-1) was added to the cells to a concentration of 80. mu.M. After incubation at 37 ℃ for a suitable period of time, cells were stained with the fluorescent dye DiI and subsequently visualized by fluorescence confocal microscopy, the results are shown in fig. 9, which clearly shows that P1 copolymer binds to the cell membrane surface resulting in cell blister rupture.

Example 10

1) PEG-PDEA of example 2₃₉-nPDPA₆₁Study of direct binding to viral outer Membrane

Take 3X 10⁹PFU/mL Vesicular Stomatitis Virus (VSV) was mixed with an equal volume of 80. mu.M NP-1 in water or PBS buffer and incubated at 4 ℃; after 1 hour of incubation, the mixture was observed using a transmission electron microscope, and the result is shown in FIG. 10 (scale: 50 nm). The binding of the P2 copolymer to the VSV viral envelope is shown.

2) PEG-PDEA of example 2₃₉-nPDPA₆₁Study of direct binding to viral infected cell membranes

Hela cells were cultured in DMEM (10% FBS) medium, and 3X 10 cells were added to the cells after the cells were 80% confluent⁹PFU/mL of VSV, while adding PBS buffer (control) or an aqueous solution of copolymer nanoparticles to a concentration of 80. mu.M. After incubation at 37 ℃ for a suitable period of time, cells were stained with the fluorescent dye DiI and subsequently visualized by fluorescence confocal microscopy, the results are shown in fig. 11, which clearly shows that the P2 copolymer binds to the surface of the cell membrane of the virus infection.

3) PEG-PDEA of example 2₃₉-nPDPA₅₇Investigation of direct disruption of the outer viral Membrane

Experiment (a)

Take 3X 10⁹PFU/mL VSV or liposome prepared by hydrogenated soybean lecithin, cholesterol and phosphatidylserine is mixed with 80 mu M NP-2 aqueous solution or PBS buffer solution with the same volume, and incubation is carried out at 4 ℃; after 1 hour of incubation, the mixture was observed using a transmission electron microscope, and the result is shown in FIG. 12 (scale: 50 nm). The disruption of the VSV viral envelope by the P2 copolymer is shown.

Experiment (b)

To 1X 10⁵PFU/mL vesicular stomatitis virus expressing Green fluorescent protein (VSV-GFP) was added with an equal volume of 80. mu.MP 4 aqueous solution or DMEM medium (control) along with PEG₅₅₀Or PEG₂₀₀₀The solution was treated at 4 ℃ for 1 h. After 1 hour, HeLa cells were infected with the treated virus to give an MOI of 0.01. 16 hours after infection, infected cells were detected using confocal laser, and the results are shown in FIG. 13. In the figure, UT means that no treatment was performed, and as a control, Vehicle means that only medium was added and no nanoparticles were added. The results show that the destruction of the viral envelope by the copolymers of the invention can be inhibited by the known inhibitors of cellular apoptosis PEG₂₀₀₀Thereby inhibiting the growth of the cell.

Example 11

1) MeO-PDEA of example 5₁₀₀Study of direct binding to viral infected cell membranes

Hela cells were cultured in DMEM (10% FBS) medium, and 3X 10 cells were added to the cells after the cells were 80% confluent⁹PFU/mL of VSV, while adding PBS buffer (control) or an aqueous solution of copolymer nanoparticles P5 to a concentration of 80. mu.M. After incubation at 37 ℃ for a suitable period of time, cells were stained with the fluorescent dye DiI and subsequently observed by fluorescence confocal microscopy, the results are shown in fig. 14, which clearly shows that no PEG polymer was bound to the surface of the cell membrane infected by the virus.

2) Direct binding of Hela cells to the protein associated with the viral ion channel after overexpression of the protein in example 5MeO-PDEA100

HeLa cells overexpressing various viral ion channel proteins were cultured in DMEM medium (with 10% FBS added) and, after the cells were confluent to 80%, an aqueous solution of copolymer nanoparticles P5 was added to the cells to a concentration of 80. mu.M. After incubation at 37 ℃ for a suitable period of time, cells were stained with the fluorescent dye DiI and subsequently visualized by fluorescence confocal microscopy, the results are shown in fig. 15, which clearly shows that the polymer binds to the cell membrane surface overexpressing various viral ion channel proteins.

Example 12

The binding condition of the P1-P4 and the cell membrane of normal cells or after virus infection

Hela cells were cultured in DMEM (10% FBS) medium until the cells were 80% confluent, and 3X 10 cells were added to the cells⁹PFU/mL VSV, while adding an aqueous solution of copolymer nanoparticles P1-P4 to a concentration of 80. mu.M. After incubation at 37 ℃ for a suitable period of time, cells were stained with the fluorescent dye DiI and subsequently visualized by fluorescence confocal microscopy, as shown in fig. 16, which clearly shows the binding of the polymer to normal cells and to virus-infected cell membranes.

Example 13

HeLa cells were cultured in DMEM medium (with 10% FBS), and when the cells were confluent to 80%, PBS buffer (control) or an aqueous solution of copolymer nanoparticles was added to the cells to a concentration of 80. mu.M. Incubation was carried out at 37 ℃ for 1-2h, cells were stained with the fluorescent dye DiI, followed by observation by fluorescence confocal microscopy, and the results are shown in table 1. A portion of the exemplary membrane binding efficiencies are shown in fig. 17, which clearly demonstrates the ability of the different copolymers to bind to the cell membrane surface.

TABLE 1

Note:

1. the PEG molecular weight in the table is 5000 unless otherwise specified.

2. The nuclear magnetic spectrum of the material except the material 11 has nuclear magnetic parts of 3.79-3.50(450) and 3.38 (3). The number outside the brackets is the chemical shift and the number inside the brackets is the number of H at that chemical shift.

3. Under the same conditions, the influence of the polymerization degree of the monomer on the membrane binding efficiency of the polymer is small, and when the difference in polymerization degree is 50 or less, the membrane binding efficiency is not substantially affected.

As can be seen from the above examples, the present invention provides a copolymer capable of binding to and cleaving cell membranes, viral membranes, cell membranes after viral infection, liposome membranes, etc., which can cleave or destroy the outer membrane of cells or viruses, thereby exhibiting corresponding biological properties.

From the above examples, it can be seen that the polymer of the present invention has gradually reduced membrane binding capacity and reduced membrane splitting capacity according to the sequence of the monomers AMA, DMA, DEA, C7A, EPA, iDPA, nDPA, PBA, DBA, and D5A, and different membrane binding capacities can be generated by mixing different monomers.

The polymer provided by the invention has broad spectrum effect on cells and RNA viruses, and can realize broad spectrum membrane combination and membrane splitting effect.

While the present invention has been described with reference to exemplary embodiments, it is to be understood that the invention is not limited to the disclosed exemplary embodiments. Many modifications and variations may be made to the exemplary embodiments of the present description without departing from the scope or spirit of the present invention. The scope of the claims is to be accorded the broadest interpretation so as to encompass all modifications and equivalent structures and functions.

Claims

1. A polymer capable of bonding to a membrane, 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 monomer comprises aminoalkyl (meth) acrylate having the formula;

wherein R is₁、R₂Represent the same or different groups and each independently represent H, C_1-10Linear, branched or cyclic alkyl;

the initiator is selected from bromoisobutyrate and/or chloroisobutyrate.

2. The polymer of claim 1, wherein the monomer is at least one selected from the group consisting of AMA, DMA, DEA, C7A, EPA, iDPA, nDPA, PBA, DBA, and D5A.

3. The polymer of claim 1, wherein the initiator is obtained by substitution reaction of a hydroxyl compound with a 2-haloisobutyryl halide and the metal catalyst is a metal halide.

4. The polymer of claim 1, wherein the polymer further comprises a hydrophilic segment.

5. The polymer of claim 4, wherein the hydrophilic segment is PEG with a molecular weight of 1000-10000 kDa.

6. A polymer characterized by the nmr spectrum shown in table 1.

7. A nanoparticle suspension or pharmaceutical composition obtained by dispersing the polymer of any one of claims 1-6 in an aqueous solution.

8. Use of a polymer according to claims 1-6 or a nanoparticle suspension or pharmaceutical composition according to claim 7 for the preparation of a composition for binding membrane structures.

9. A method for binding and lysing a membrane structure, characterized by the step of contacting a polymer according to any of claims 1 to 6 or a nanoparticle suspension according to claim 7 with the membrane structure.

10. The method of claim 9, wherein the membrane structure is a phospholipid membrane.

11. The method of claim 9, wherein the membrane structure is selected from the group consisting of a cell membrane, a viral membrane, a bacterial membrane, and an artificial lipid membrane.

12. A method for predicting or adjusting a membrane-binding ability or a membrane-cleavage ability of a polymer, wherein a polymerized monomer of the polymer is at least one selected from the group consisting of AMA, DMA, DEA, C7A, EPA, iDPA, nDPA, PBA, DBA, and D5A, the method comprising the step of predicting or adjusting the polymer according to a content of each polymerized monomer and a degree of contribution to the membrane-binding ability or the membrane-cleavage ability, wherein the degree of contribution becomes smaller in order from AMA, DMA, DEA, C7A, EPA, iDPA, nDPA, PBA, DBA to D5A.