CN118005913A

CN118005913A - Block copolymer with cell membrane pore-forming property and preparation method and application thereof

Info

Publication number: CN118005913A
Application number: CN202410424674.8A
Authority: CN
Inventors: 丁明明; 刘聪聪; 严悦; 郑毅; 王一唯; 彭川; 刘洋
Original assignee: Sichuan University
Current assignee: Sichuan University
Priority date: 2024-04-10
Filing date: 2024-04-10
Publication date: 2024-05-10
Anticipated expiration: 2044-04-10
Also published as: CN118005913B

Abstract

The application discloses a block copolymer with cell membrane pore-forming property, a preparation method and application thereof, and relates to the technical field of biomedical materials. The block copolymer with cell membrane pore-forming property is composed of a cationic hydrophilic segment and a spiral hydrophobic segment; wherein the cationic hydrophilic segment is a cationic polymer having a random coil structure; the helical hydrophobic segment is polyamino acid and derivatives thereof capable of forming stable alpha-helical structure. Thus, the block copolymer with cell membrane pore-forming property provided by the application can balance the interaction with cell membranes through the effective separation and combination of the non-helical cationic hydrophilic segment and the helical hydrophobic segment, so that the block copolymer not only has excellent cell membrane pore-forming capability, but also has reversibility of formed pores, thereby improving the drug delivery efficiency and the safety of a carrier material.

Description

Block copolymer with cell membrane pore-forming property and preparation method and application thereof

Technical Field

The application relates to the field of biomedical materials, in particular to a block copolymer with cell membrane pore-forming property, a preparation method and application thereof.

Background

Efficient delivery of drugs to specific target cells or organs is a key challenge in the field of biological therapy. To achieve this goal, drug carriers must overcome the natural barrier of the cell membrane to efficiently transport biologically active molecules with therapeutic potential into the cell interior. In recent years, researchers have been exploring the use of various nanomaterials and polymeric complexes to achieve efficient drug delivery, gene transfection, protein delivery, and intracellular molecular imaging applications. However, one major obstacle often faced by drug carriers is endosomal entrapment by endocytosis, which can cause the drug to be degraded in the acid lysosomes, thereby reducing delivery efficiency and therapeutic efficacy. Therefore, the development of novel materials that safely and effectively penetrate cell membranes directly into cells has become an important research direction to solve this problem.

In nature, there is a special class of proteins such as perforins and melittins that are capable of free cellular internalization through the mechanism of cell membrane pore formation. These proteins with cell membrane pore-forming properties typically have amphiphilic character, presenting cationic and hydrophobic residues on opposite sides of their structure, respectively, with high affinity for negatively charged biological membranes. Furthermore, such proteins often exhibit a helical secondary structure, either in solution or upon interaction with the cell membrane. Cationic polypeptides having a helical structure are capable of interacting with anionic functional groups on the surface of a cell membrane, and interaction of such molecules with the membrane can result in translocation, instability, or pore formation of the membrane, thereby facilitating efficient uptake by the cell. In light of this, scientists have begun to explore the use of physical or chemical methods to construct cationic polypeptides having a helical structure. However, strong intramolecular charge repulsion between cationic groups can hinder the formation and stabilization of backbone helix structures, and thus artificial design of cationic polypeptides with stabilized helix structures presents a great challenge. Studies have shown that the helical structure of cationic polypeptides can be stabilized by extending the distance between the side chain charge groups and the backbone, thereby reducing the helical surface charge density to minimize the repulsive interactions between the side chain cationic charges. Based on this strategy, CN111187340a published a cationic α -helical polypeptide; CN106589355A discloses a class of non-viral gene transfection vector materials based on cationic helical polypeptides; US2023094088A1 discloses a three-dimensional spherical alpha-helical cationic polypeptide with high-efficiency gene delivery capability, and a preparation method and application thereof. However, this strategy requires the introduction of specific chemical structures in the amino acid side chains, thereby introducing additional synthetic steps and subsequent processing. In addition, these cationic helical polypeptides often have too high membrane activity, which inevitably results in irreversible pore formation or destruction of the cell membrane while increasing the efficiency of cell entry delivery, thereby leading to cell death. Accordingly, there is a strong need in the art to develop a novel polymer that overcomes the above-mentioned problems, and that allows for efficient and safe intracellular delivery and biological applications by means of cell membrane pore-forming.

Disclosure of Invention

The application aims to provide a block copolymer with cell membrane pore-forming property, a preparation method and application thereof, and the block copolymer balances interaction with cell membranes in a mode of separating and effectively combining a cationic hydrophilic segment and a spiral hydrophobic segment, so that the reversible pore-forming property of the cell membranes is realized, and meanwhile, the efficiency and the safety of drug delivery are improved, and a new thought is provided for solving the problems of complex and complicated preparation and synthesis processes and safety of the traditional cationic spiral polypeptides.

In order to achieve the above object, the technical solution of the embodiment of the present application is:

the first aspect of the present application provides a block copolymer having cell membrane pore-forming properties, the block copolymer being composed of a cationic hydrophilic segment and a helical hydrophobic segment;

Wherein the cationic hydrophilic segment is a cationic polymer having a random coil structure;

The helical hydrophobic segment is polyamino acid and derivatives thereof capable of forming stable alpha-helical structure.

Preferably in combination with the first aspect, the block copolymer is one of a linear block copolymer, a star-shaped block copolymer, a ring-shaped block copolymer, a comb-shaped block copolymer and a (super) branched block copolymer, and the block number m ₁ of the cationic hydrophilic segment is more than or equal to 1; and/or the block number m ₂ of the spiral hydrophobic block is more than or equal to 1.

With reference to the first aspect, preferably, when the block copolymer is a linear block copolymer, the block number m ₁ is less than or equal to 2, and/or m ₂ is less than or equal to 2, and the sum of m ₁ and m ₂ is 2-3, and the cationic hydrophilic segment is at least one of polymers containing primary amine, secondary amine, tertiary amine, quaternary amine, pyridine, imidazolium, guanidinium, sulfonium ion and phosphine ion; the spiral hydrophobic segment is at least one of phenylalanine, methionine, poly-2-amino isobutyric acid, polylysine derivative, polyaspartic acid derivative, polyglutamic acid derivative and polycysteine derivative.

With reference to the first aspect, preferably, when the block copolymer is a linear block copolymer, the block number m ₁ is not less than 2 and m ₂ is not less than 2, and the cationic hydrophilic segment is at least one of polymers containing primary amine, secondary amine, tertiary amine, quaternary amine, pyridine, imidazolium, guanidinium, sulfonium ion and phosphine ion; the spiral hydrophobic segment is at least one of polyalanine, polyleucine, polyphenylalanine, polymethine, poly-2-amino isobutyric acid, polylysine derivatives, polyaspartic acid derivatives, polyglutamic acid derivatives and polycysteine derivatives.

With reference to the first aspect, preferably, the block copolymer is a star block copolymer, a ring block copolymer, a comb block copolymer, a (hyper) branched block copolymer, and the cationic hydrophilic segment is at least one of a polymer containing primary amine, secondary amine, tertiary amine, quaternary amine, pyridine, imidazolium, guanidinium, sulfonium ion, and phosphine ion; the spiral hydrophobic segment is at least one of polyalanine, polyleucine, polyphenylalanine, polymethine, poly-2-amino isobutyric acid, polylysine derivatives, polyaspartic acid derivatives, polyglutamic acid derivatives and polycysteine derivatives.

Preferably in combination with the first aspect, the cationic hydrophilic segment is preferably at least one of a polymer containing secondary, tertiary, quaternary, pyridine, imidazolium, sulfonium ions.

Preferably in combination with the first aspect, the helical hydrophobic segment is preferably at least one of a polylysine derivative, a polyaspartic acid derivative, a polyglutamic acid derivative, and a polycysteine derivative.

Preferably in combination with the first aspect, the cationic hydrophilic segment is at least one of a polymer containing secondary amine, tertiary amine, quaternary amine, guanidine salt, sulfonium ion, and the helical hydrophobic segment is at least one of polyalanine, polyleucine, polyalanine, polylysine derivative, polyaspartic acid derivative, polyglutamic acid derivative, and polycysteine derivative.

In a second aspect, the present application provides a method for preparing a block copolymer having cell membrane pore-forming properties according to the first aspect, comprising:

The cationic polymer is prepared by taking a cationic compound monomer as a raw material through polymerization reaction; wherein the molecular weight Mn of the cationic polymer is 1000-25000;

The method comprises the steps of taking a cationic polymer as a raw material, and preparing a block copolymer by initiating small molecule ring-opening polymerization of amino acid and derivatives thereof;

and/or coupling and grafting reaction with the spiral hydrophobic segment to prepare a segmented copolymer; wherein the polymerization degree n of the polyamino acid and the derivative thereof is 10-100.

A third aspect of the present application provides a use of the block copolymer having cell membrane pore-forming properties of the first aspect for self-assembly, biosensing, drug delivery or disease diagnosis.

Compared with the prior art, the embodiment of the application has the advantages or beneficial effects that at least the advantages or beneficial effects comprise:

According to the block copolymer provided by the application, on one hand, through the effective separation and combination of the non-helical cationic hydrophilic segment and the hydrophobic segment with a helical structure, the interaction with a cell membrane can be balanced, so that the block copolymer not only has excellent cell membrane pore-forming capability, but also has reversibility of formed pores, thereby improving the drug delivery efficiency, and simultaneously improving the safety of a carrier material; on the other hand, compared with the traditional cationic helical polypeptides which rely on fine structural design and complex chemical means to stabilize the main chain helical structure and realize the pore-forming performance of the cell membrane, the application selects the existing general cationic polymer, and can realize efficient pore-forming of the cell membrane by combining and synergistically enhancing the existing general cationic polymer with the designed hydrophobic helical segment, thereby avoiding complex synthetic steps and subsequent treatment processes and bringing different possibilities for the pore-forming field of the cell membrane; in the third aspect, unlike the existing carrier materials, which comprise some common cationic polymers, most of the cationic polymers enter cells through entrapment of therapeutic drugs and endocytosis, the block copolymer provided by the application mediates the action of bioactive molecules directly entering cells through permeation by virtue of the unique cell membrane reversible pore-forming action mechanism, so that the capture and degradation of bioactive molecules by lysosomes are effectively avoided, and the drug delivery efficiency and bioavailability are remarkably improved; in addition, the variety of bioactive molecules delivered in this manner is not limited, providing a broader potential for their use; in the fourth aspect, compared with the complex, tedious and high-cost design and synthesis technology required by the existing cation helix and cell membrane pore-forming materials, the segmented copolymer provided by the application has the advantages of clear structure, simple and rapid preparation process, obvious reduction of synthesis and production cost and improvement of clinical transformation potential. In addition, the cationic hydrophilic segment and the spiral hydrophobic segment in the structure have more abundant selection diversity and modifier property, so that the copolymer can accurately regulate and control the performance and function under different biomedical application scenes; in a fifth aspect, the block copolymer provided by the application has great application potential in self-assembly, biosensing, drug delivery, disease diagnosis, treatment and the like.

Drawings

FIG. 1 is a graph showing the particle size and distribution of the block copolymer self-assemblies prepared in example 3;

FIG. 2 is a transmission electron microscope image of the block copolymer self-assembly prepared in example 3;

FIG. 3 is a circular dichroism spectrum of the block copolymer self-assembly prepared in example 3;

FIG. 4 is a confocal laser photograph of the integrity of the cell membrane after the self-assembly of the block copolymer prepared in examples 1 and 5 has been reacted with the cells;

FIG. 5 is a confocal laser photograph of the delivery properties of the block copolymer self-assemblies prepared in example 4;

FIG. 6 is a quantitative plot of the delivery performance of a block copolymer self-assembly prepared in part of the examples;

FIG. 7 is a confocal laser photograph of the cell entry mechanism of the block copolymer self-assembly prepared in example 9;

FIG. 8 is a graph of the quantification of the biocompatibility of the block copolymer self-assemblies prepared in example 12;

FIG. 9 is a graph showing the in vitro antitumor results of the block copolymer self-assembly prepared in example 7.

Detailed Description

The present application will be further described in detail with reference to the accompanying drawings, for the purpose of making the objects, technical solutions and advantages of the present application more apparent, and the described embodiments should not be construed as limiting the present application, and all other embodiments obtained by those skilled in the art without making any inventive effort are within the scope of the present application.

In the following description, reference is made to "some embodiments" which describe a subset of all possible embodiments, but it is to be understood that "some embodiments" can be the same subset or different subsets of all possible embodiments and can be combined with one another without conflict. 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 embodiments of this application belong. The terminology used in the embodiments of the application is for the purpose of describing embodiments of the application only and is not intended to be limiting of the application.

In the following description of the present embodiment, the terms "include," "comprise," "have," "contain," and the like are open-ended terms, meaning including, but not limited to.

It should be noted that all the raw materials/reagents in the examples of the present application can be purchased on the market or prepared according to conventional methods well known to those skilled in the art; the term "and/or" in the embodiment of the present application is only used to describe the association relationship of the associated objects, and indicates that three relationships may exist, for example, a and/or B indicates that there are three cases of a alone, B alone, and a and B simultaneously, where A, B may be singular or plural, and the character "/" generally indicates that the associated objects are an "or" relationship.

In the following description of the present embodiments, the term "at least one" means one or more, and "a plurality" means two or more. "at least one of" or the like means any combination of these items, including any combination of single item(s) or plural items(s). For example, "at least one (individual) of a, b, or c," or "at least one (individual) of a, b, and c" may each represent: a, b, c, a-b (i.e., a and b), a-c, b-c, or a-b-c, wherein a, b, c may be single or multiple, respectively.

It should be understood by those skilled in the art that, in the following description of the present embodiment, the sequence number does not mean that the execution sequence is sequential, and some or all of the steps may be executed in parallel or sequentially, and the execution sequence of each process should be determined by its functions and inherent logic, and should not constitute any limitation on the implementation process of the embodiment of the present application.

The terminology used in the embodiments of the application is for the purpose of describing particular embodiments only and is not intended to be limiting of the application. As used in this application and the appended claims, the singular forms "a," "an," and "the" are intended to include the plural forms as well, unless the context clearly indicates otherwise.

It will be appreciated by those skilled in the art that the numerical ranges in the embodiments of the present application are to be understood as also specifically disclosing each intermediate value between the upper and lower limits of the range. Every smaller range between any Chen Shuzhi and any stated range, and any other stated or intervening values in that stated range, is encompassed within the application. The upper and lower limits of these smaller ranges may independently be included or excluded in the range.

Unless otherwise defined, technical/scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this application 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 embodiments or testing examples of the present application. All documents referred to in this specification are generally incorporated by reference herein to disclose and describe the methods and/or materials in connection with which the documents are referred to. In case of conflict with any incorporated document, the present specification will control.

It should be noted that all the raw materials and/or reagents in the examples of the present application are commercially available or prepared according to conventional methods well known to those skilled in the art.

In a first aspect, embodiments of the present application provide a block copolymer having cell membrane pore-forming properties, the block copolymer being comprised of a cationic hydrophilic segment and a helical hydrophobic segment;

According to the block copolymer provided by the application, on one hand, through the effective separation and combination of the non-helical cationic hydrophilic segment and the hydrophobic segment with a helical structure, the interaction with a cell membrane can be balanced, so that the block copolymer not only has excellent cell membrane pore-forming capability, but also has reversibility of formed pores, thereby improving the drug delivery efficiency, and simultaneously improving the safety of a carrier material; on the other hand, compared with the traditional cationic helical polypeptides which rely on fine structural design and complex chemical means to stabilize the main chain helical structure and realize the pore-forming performance of the cell membrane, the application selects the existing general cationic polymer, and can realize efficient pore-forming of the cell membrane by combining and synergistically enhancing the existing general cationic polymer with the designed hydrophobic helical segment, thereby avoiding complex synthetic steps and subsequent treatment processes and bringing different possibilities for the pore-forming field of the cell membrane; in the third aspect, unlike the existing carrier materials, which comprise some common cationic polymers, most of the cationic polymers enter cells through entrapment of therapeutic drugs and endocytosis, the block copolymer provided by the application mediates the action of bioactive molecules directly entering cells through permeation by virtue of the unique cell membrane reversible pore-forming action mechanism, so that the capture and degradation of bioactive molecules by lysosomes are effectively avoided, and the drug delivery efficiency and bioavailability are remarkably improved. In addition, the variety of bioactive molecules delivered in this manner is not limited, providing a broader potential for their use; in the fourth aspect, compared with the complex, tedious and high-cost design and synthesis technology required by the existing cation helix and cell membrane pore-forming materials, the segmented copolymer provided by the application has the advantages that the structure is clear, the preparation process is simple and quick, the synthesis and production cost is obviously reduced, and the clinical transformation potential is improved; in addition, the cationic hydrophilic segment and the spiral hydrophobic segment in the structure have more abundant selection diversity and modifier property, so that the copolymer can accurately regulate and control the performance and function under different biomedical application scenes; in a fifth aspect, the block copolymer provided by the application has great application potential in self-assembly, biosensing, drug delivery, disease diagnosis, treatment and the like.

In a specific embodiment, the block copolymer of the embodiment of the application is preferably one of a linear block copolymer, a star-shaped block copolymer, a ring-shaped block copolymer, a comb-shaped block copolymer and a (super) branched block copolymer, and the block number of the cationic hydrophilic segment is preferably m ₁ or more than 1; and/or the number of blocks of the spiral hydrophobic segment is preferably m ₂ to be more than or equal to 1.

In a specific embodiment, when the block copolymer of the embodiment of the application is preferably a linear block copolymer, the block number m ₁ is less than or equal to 2 and/or m ₂ is less than or equal to 2, and the sum of m ₁ and m ₂ is preferably 2-3, and the cationic hydrophilic segment is one of polymers containing primary amine, secondary amine, tertiary amine, quaternary amine, pyridine, imidazolium, guanidine salt, sulfonium ion and phosphine ion; further, one of the polymers containing secondary amine, tertiary amine, quaternary amine, pyridine, imidazolium, sulfonium ion is preferable.

Wherein the cationic polymers are capable of rapidly binding to negatively charged lipid molecules on cell membranes by electrostatic interactions.

The spiral hydrophobic segment is one of phenylalanine, methionine, poly-2-amino isobutyric acid, polylysine derivative, polyaspartic acid derivative, polyglutamic acid derivative and polycysteine derivative; further, one of polylysine derivatives, polyaspartic acid derivatives, polyglutamic acid derivatives, and polycysteine derivatives is preferable.

Wherein, the polyamino acid and the derivative thereof can spontaneously form a stable hydrophobic alpha-helical structure, further disturb the cell membrane by hydrophobic action and form reversible transient pores on the cell membrane by synergistic action with the cationic hydrophilic segment.

In a specific embodiment, when the block copolymer of the embodiment of the application is preferably a linear block copolymer, the block number m ₁ is more than or equal to 2, and m ₂ is more than or equal to 2, and the cationic hydrophilic segment is one of polymers containing primary amine, secondary amine, tertiary amine, quaternary amine, pyridine, imidazolium, guanidine salt, sulfonium ion and phosphine ion; further, one of the polymers containing secondary amine, tertiary amine, quaternary amine, guanidine salt, sulfonium ion is preferable;

it should be noted that these cationic polymers are capable of binding to the cell membrane by efficient electrostatic interactions, and that their random coil structure does not produce strong biotoxicity.

The spiral hydrophobic segment is one of polyalanine, polyleucine, polyphenylalanine, polymethine, poly-2-amino isobutyric acid, polylysine derivatives, polyaspartic acid derivatives, polyglutamic acid derivatives and polycysteine derivatives; further, one of polyalanine, polyleucine, polyphenylalanine, polylysine derivatives, polyaspartic acid derivatives, polyglutamic acid derivatives, and polycysteine derivatives is preferable.

It should be noted that these polyamino acids and their derivatives can form stable hydrophobic alpha-helix structure, and this rigid hydrophobic structure can produce synergy with cationic hydrophilic segment, promote efficient uptake and translocation of cell, and avoid dissolution and rupture of cell membrane due to too strong hydrophobic effect.

In a specific embodiment, the block copolymer of the embodiment of the present application is preferably a star-shaped block copolymer, a ring-shaped block copolymer, a comb-shaped block copolymer, or a (hyper) -branched block copolymer, and the cationic hydrophilic segment is one of polymers containing primary amine, secondary amine, tertiary amine, quaternary amine, pyridine, imidazolium, guanidinium, sulfonium ion, and phosphine ion; further, one of the polymers containing secondary amine, tertiary amine, quaternary amine, guanidine salt, sulfonium ion is preferable.

It should be noted that, the cationic hydrophilic segments with stronger positive charges are combined with the cell membrane through electrostatic interaction, and are widely existing in some compounds or polymers with simple structures, and are not required to be obtained by complex physical and chemical means, so that modification and modification are facilitated, the multifunctional cell membrane has multifunctional performance, and multifunctional application is realized.

It should be noted that the polyamino acids and derivatives thereof have stronger tendency to form an α -helix structure and better structural stability, and the amino acid or derivative monomers thereof are simple and easy to obtain, have wide sources, and can form polymers through various chemical reaction modes.

In a second aspect, an embodiment of the present application provides a method for preparing a block copolymer having cell membrane pore-forming properties according to the first aspect, including:

In a third aspect, embodiments of the present application provide a use of the block copolymer with cell membrane pore-forming properties of the first aspect for self-assembly, biosensing, drug delivery or disease diagnosis.

The technical method of the present application will be further described with reference to specific examples.

Example 1

The present example 1 provides a preparation method of polyarginine-phenylalanine linear diblock copolymer, which comprises the following specific steps:

(1) Preparation of sulfonyl-L-arginine cyclic anhydride (Pbf-L-Arg-NCA)

10G sulfonyl-L-arginine (Pbf-Arg), 200 mL Tetrahydrofuran (THF) and 15 mL Propylene Oxide (PO) are sequentially added into a 350 mL reaction bottle, after magnetic stirring is uniform, 6.3 g triphosgene is quickly added and the container is immediately sealed, the reaction is carried out at 25 ℃ until the solution is clear, and the solid is completely disappeared. The reaction mixture was cooled in an ice bath, excess triphosgene was added by quenching with 70 mL% cold water, the mixture was extracted with 50mL ×2 ethyl acetate at room temperature, the combined organic phases were washed with saturated brine and dried over anhydrous MgSO ₄, after removal of the solvent by rotary evaporation, the crude product was purified by crystallization 3 times from n-hexane/tetrahydrofuran and dried in vacuo to give the product Pbf-L-Arg-NCA in 75% yield.

(2) Preparation of phenylalanine endocarboxylic acid anhydride (L-Phe-NCA)

Sequentially adding 5g phenylalanine (Phe), 75 mL THF and 10 mL PO into a 120 mL reaction bottle, magnetically stirring uniformly, rapidly adding 4.5 g triphosgene, immediately sealing the container, and reacting at 25 ℃ until the solution is clear, and completely removing solids. The reaction mixture was cooled in an ice bath, excess triphosgene was quenched with 70 mL added cold water with stirring, the mixture was extracted with 50 mL ×2 ethyl acetate at room temperature, the combined organic phases were washed with saturated brine and dried over anhydrous MgSO ₄, after removal of the solvent by rotary evaporation, the crude product was purified by crystallization 3 times from n-hexane/tetrahydrofuran and dried in vacuo to give the product L-Phe-NCA in 72% yield.

(3) Preparation of polysulfonyl arginine (PEG-P (Pbf) Arg-NH ₂)

1.84 G of polyethyleneglycol amine (PEG-NH ₂) serving as an initiator is dissolved in NaHCO ₃ aqueous solution, 2g Pbf-L-Arg-NCA is added, 12 h is reacted under ice water bath, the aqueous solution obtained by the reaction is transferred into a dialysis bag with 3500 molecular weight cutoff (MWCO 3500), deionized water is dialyzed for 2 days, and the product PEG-P (Pbf) Arg-NH ₂ is obtained by freeze drying, wherein the yield is 75%.

(4) Preparation of polysulfonyl lysine-phenylalanine (PEG-P (Pbf) Arg-PPhe)

PEG-P (Pbf) Arg-NH ₂ (2 g) is dissolved in dichloromethane (DCM, 27.55 mL), and BCP buffer (0.1 mol/L boric acid, 0.025 mol/L citric acid, 0.05 mol/L trisodium phosphate) with pH=7 is added, and the mixture is subjected to ultrasonic emulsification for 30min for standby; after that, L-Phe-NCA (1.93 g) was dissolved in DCM (27.55 mL), the two were mixed, magnetically stirred for reaction 2h, after the reaction was completed, the crude product was purified by crystallization 3 times with anhydrous diethyl ether/n-hexane (v/v=1:1), and dried in vacuo to give the product PEG-P (Pbf) Arg-PPhe in 74% yield.

(5) Preparation of polyarginine-phenylalanine (PEG-PArg-PPhe)

PEG-P (Pbf) Arg-PPhe (2 g) is dissolved in 20mL trifluoroacetic acid (TFA), stirring is carried out at room temperature under the protection of argon, reaction is carried out for 1h, the TFA is removed by rotary evaporation of a mixture obtained by the reaction, the crude product is purified by precipitation with glacial ethyl ether for 3 times, the obtained product is dissolved in anhydrous N, N-Dimethylformamide (DMF), then the obtained product is transferred into a dialysis bag of MWCO 3500, deionized water is dialyzed for 2 days, and the product PEG-PArg-PPhe (the molecular weight Mn=4700 of polyarginine, the polymerization degree of phenylalanine is n=30, the number of cationic blocks is m ₁ =1, and the number of hydrophobic helical blocks is m ₂ =1) is obtained after freeze drying, and the yield is 80%.

Example 2

The present example 2 provides a method for preparing polylysine-dibenzylcysteine linear multiblock copolymer, comprising the following specific steps:

(1) Preparation of poly (Boc) lysine-polybenzyl cysteine (P (Boc) Lys-P (Bn) Cys) multiblock copolymer

Boc-L-Lys-NCA (2.0 g) is dissolved in 20mL anhydrous DMF, then n-hexylamine (97 mu L) is added as an initiator, the reaction is carried out for 3 days at 37 ℃ under the protection of argon, when the reaction is carried out for 3 days, anhydrous DMF solution (1.94 g,20 mL) in which benzyl cysteine endocyclic carboxylic anhydride (Bn-L-Cys-NCA) is dissolved in advance is quickly injected into the system through a syringe for continuous reaction for 3 days, when the reaction is carried out for 3 days, the anhydrous DMF solution (2.0 g,20 mL) in which Boc-L-Lys-NCA is dissolved is injected through a syringe for continuous reaction, P (Boc) Lys-P (Bn) Cys multiblock copolymers with different block numbers are obtained by circulation, after the final reaction is finished, the crude product is precipitated in glacial diethyl ether for 3 times, and the yield of the product P (Boc) Lys-P (Bn) Cys multiblock copolymer is 65%.

(2) Preparation of polylysine-Poly (benzyl) cysteine (PLys-P (Bn) Cys) multiblock copolymers

Dissolving the prepared P (Boc) Lys-P (Bn) Cys multiblock copolymer (2 g) in 20mL TFA under the protection of argon, stirring at room temperature for reaction 1h, rotationally evaporating the mixture obtained by the reaction to remove TFA, precipitating the crude product with glacial ethyl ether for 3 times, dissolving the obtained crude product in DMF again, transferring to a dialysis bag of MWCO 3500, dialyzing with deionized water for 2 days, and freeze-drying to obtain PLys-P (Bn) Cys multiblock copolymer (polylysine molecule Mn=1300; benzyl cysteine polymerization degree n=10; cation block number m ₁ =3; hydrophobic helix block number m ₂ =3), wherein the yield is 80%.

Example 3

This example 3 provides a method for preparing PLys-P (Bn) Cys multiblock copolymer self-assemblies, comprising the following steps:

Taking PLys-P (Bn) Cys multiblock copolymer 10mg obtained in example 2, dissolving in 1mL DMF, slowly dripping into rapidly stirred deionized water (9 mL) at the speed of 30 s/d, and continuing stirring for 30 min after dripping; then transferring the liquid into a dialysis bag of MWCO 3500, dialyzing with deionized water for 2 days, centrifuging (3500 r/min), filtering (0.45 μm), and fixing the volume to obtain PLys-P (Bn) Cys multiblock copolymer self-assembly body solution.

Example 4

The embodiment 4 provides a preparation method of a four-arm polyethylene glycol-quaternary ammonium salt modified polycysteine-polyleucine star-shaped block copolymer, which comprises the following specific steps:

(1) Preparation of four-arm polyethylene glycol-cholesterol modified polycysteine (G4-PEG-P (Chol) Cys-NH ₂)

Cholesterol-modified cysteine cyclic anhydride (Chol-L-Cys-NCA, 3.5G) was dissolved with 30 mL anhydrous THF, then, anhydrous THF (10 mL) of quadrifilar polyethylene glycol (G4-PEG-HN ₂, 1G) was added to the reaction system, reacted at 37 ℃ for 3 days under argon, the mixed solution was concentrated and then purified 3 times with glacial diethyl ether to give a pale yellow solid product G4-PEG-P (Chol) Cys-NH ₂ in 75% yield.

(2) Preparation of four-arm polyethylene glycol-cholesterol modified Polycysteine-Polyleucine (G4-MPEG-P (Chol) Cys-PLeu)

Taking leucine ring dicarboxylic anhydride (L-Leu-NCA, 2.0G), dissolving in 20mL anhydrous DMF, adding anhydrous DMF solution (1.5G, 15 mL) in which G4-MPEG-P (Chol) Cys-NH ₂ is dissolved in advance through a syringe, reacting for 3 days at 37 ℃ under the protection of argon, precipitating and purifying in glacial ethyl ether for 3 times after the reaction is finished, and vacuum drying to obtain the product G4-PEG-P (Chol) Cys-PLeu, wherein the yield is 69%.

(3) Preparation of four-arm polyethylene glycol-Quaternary ammonium salt modified Polycysteine-Polyleucine (G4-PEG-P (Q-Chol) Cys-PLeu)

50Mg of G4-MPEG-P (Chol) Cys-PLeu is dissolved by 10mL anhydrous THF, then 2, 3-epoxypropyl trimethyl ammonium chloride (25 mg) is dissolved in 5mL of glacial acetic acid, the solution is slowly added into a reaction system through a syringe to react at 37 ℃ for 24 h, then 2, 3-epoxypropyl trimethyl ammonium chloride of 25 mg is added, the reaction is continued for 24 h, after the reaction is finished, the mixture is transferred into an MWCO 3500 dialysis bag, deionized water is used for dialysis for 2 days, and freeze drying is carried out to obtain the product G4-MPEG-P (Q-Chol) Cys-PLeu (quaternary ammonium salt modified polycysteine molecular weight Mn=4600, leucine polymerization degree n=40, cation block number m ₁ =2, hydrophobic helix block number m ₂ =1), and the yield is 70%.

Example 5

This example 5 provides a process for the preparation of a polyamidoamine-dibenzyl aspartate-polyaniline (hyper) branched block copolymer comprising the following steps:

(1) Preparation of polyamidoamine-Polybenzyl ester aspartic acid (PAMAM-P (Bn) Asp-NH ₂)

The initiator polyamidoamine (PAMAM, mn=3000, 2 g) is dissolved in 20mL anhydrous DMF by ultrasonic heating, then an anhydrous DMF solution in which aspartic acid-4-benzyl ester endocyclic carboxylic anhydride (Bn-L-Asp-NCA, 2.0 g) is dissolved in advance is added into a reaction system by a syringe, the reaction is carried out for 3 days at 37 ℃ under the protection of argon, after the reaction is finished, the mixed solution is precipitated and purified in glacial ethyl ether for 3 times, and the product PAMAM-P (Bn) Asp-NH ₂ is obtained by vacuum drying, wherein the yield is 78%.

(2) Preparation of polyamidoamine-Polybenzyl aspartate-polyarginine (PAMAM-P (Bn) Asp-PArg)

The PAMAM-P (Bn) Asp-NH ₂ is ultrasonically dissolved in 20mL anhydrous DMF, then arginine cyclic anhydride (L-Arg-NCA, 1 g) which is pre-dissolved in the anhydrous DMF is added into a reaction system through a syringe to react for 3 days at 37 ℃ under the protection of argon, after the reaction is finished, the mixed solution is precipitated and purified in glacial ethyl ether for 3 times, and the product PAMAM-P (Bn) Asp-PArg (benzyl ester aspartic acid polymerization degree n=25; polyarginine molecular weight Mn=6500) is obtained after vacuum drying, and the yield is 74 percent.

Example 6

The embodiment 6 provides a preparation method of poly (Boc) lysine grafted chitosan, which comprises the following specific steps:

(1) Preparation of N-phthaloyl chitosan

1G chitosan (Chi-6 k) and 2.76 g phthalic anhydride were dispersed in 200 mL anhydrous DMF and reacted at 90℃with stirring for 8h. After the reaction, the solvent was evaporated, 50mL absolute ethanol was precipitated, then the precipitate was filtered, washed successively with water, ethanol and diethyl ether, and finally dried in vacuo to give the product in 53% yield.

(2) Preparation of N-phthaloyl-6-O-trityl chitosan

Triphenylchloromethane (3.06 g) and N-phthaloyl chitosan (1 g) were dissolved in 20 mL anhydrous pyridine and reacted at 90℃with stirring for 24h, and after evaporation of the solvent, the product was washed with ethanol and diethyl ether in 60% yield.

(3) Preparation of 6-O-trityl chitosan (Tr-Chi)

N-phthaloyl-6-O-trityl chitosan (1 g) and water hydrazine (50 wt%) of 18 mL are stirred and dissolved in 35 mL deionized water under the protection of argon gas at 100 ℃, the reaction is carried out for 15 h, after the reaction is finished, the suspension is filtered, and the product is washed with water, ethanol and diethyl ether, wherein the yield is 85%.

(4) Preparation of Poly (Boc) lysine grafted 6-O-trityl Chitosan copolymer (Tr-Chi-P (Boc) Lys)

Tr-Chi (1 g) and Boc-L-Lys-NCA (1 g) were dissolved in 30 mL anhydrous N, N-dimethylacetamide (DMAc) and reacted at room temperature for 3 days. Precipitating and purifying the mixed solution in glacial diethyl ether for 3 times, and vacuum drying to obtain a product Tr-Chi-P (Boc) Lys (6-O-trityl chitosan molecular weight Mn=6000 and Boc lysine polymerization degree n=100), wherein the yield is 72%.

Example 7

The embodiment 7 provides a preparation method of polylysine grafted dextran modified by phenylalanine-quaternary ammonium salt, which comprises the following specific steps:

(1) Preparation of oxidized dextran (O-Dex)

Oxidized dextran (O-Dex) was obtained by reacting dextran with equimolar potassium periodate in water, purifying with DOWEX-1 anion exchange resin (acetate form), dialyzing with deionized water, and lyophilizing.

(2) Preparation of polyphenylalanine-polylysine grafted oxidized dextran (PPhe-PLys-O-Dex)

100ML deionized water solution of O-Dex (1 g) is slowly added into alkaline buffer solution (50 mL, 0.1M borate, pH=11) containing equimolar amount of phenylalanine-polylysine (PPhe-PLys) in 5 h, the mixture is stirred at room temperature for reaction 24h, excessive sodium borohydride (1 g) is added, stirring is continued at room temperature for 48 h, the obtained light yellow solution is transferred into MWCO 3500 dialysis bag, deionized water is dialyzed for 2 days, and freeze drying is carried out, thus obtaining the product PPhe-PLys-O-Dex, and the yield is 50%.

(3) Preparation of Polyphenylalanine-Quaternary ammonium salt modified polylysine grafted dextran (PPhe-P (Q) Lys-Dex)

PPhe-PLys-O-Dex (2 g) is dissolved in 100 mL deionized water, 400 mg sodium hydroxide (NaOH) is added, the solution is magnetically stirred and heated to 60 ℃, 24 mL glycidyl trimethyl ammonium chloride (GTMAC) is added, 4 h is reacted at 60 ℃, after the reaction is finished, the mixture is cooled and transferred into an MWCO 12000 dialysis bag, deionized water is dialyzed for 2 days, and after freeze drying, the product PPhe-P (Q) Lys-Dex (molecular weight Mn=10000 of dextran; molecular weight Mn=2000 of quaternary ammonium salt modified polylysine; phenylalanine polymerization degree n=20; cation block number m ₁ =3; hydrophobic helical block number m ₂ =2) is obtained, and the yield is 85%.

Example 8

The example 8 provides a preparation method of a polyethylene glycol-linear polyethylenimine-poly (tert-butyl) dimethyl glutamic acid linear block copolymer, which comprises the following specific steps:

(1) Preparation of polyethylene glycol-Linear polyethylenimine (PEG-LPEI)

100. Mu.L of ethanolamine was dissolved in 5 mL deionized water, then 5 min was irradiated with synchrotron X-rays (4-30 keV,10 ⁵ Gy/s) at room temperature, and the resulting mixed solution was transferred to an MWCO3500 dialysis bag, dialyzed against deionized water for 2 days, and lyophilized to obtain the product PEG-LPEI in a yield of 70%.

(2) Preparation of polyethylene glycol-Polylinear ethyleneimine-Poly (t-butyldimethyl glutamic acid) (PEG-LPEI-P (TBS) Glu)

The product PEG-LPEI (2.0 g) is dissolved in 20 mL anhydrous DMF, anhydrous DMF solution in which tert-butyl dimethyl glutamate endocyclic carboxylic anhydride (TBS-L-Glu-NCA, 1 g) is dissolved in advance is quickly added into a reaction system through a syringe, the reaction is carried out for 3 days at 37 ℃ under the protection of argon, after the reaction is finished, the mixed solution is precipitated and purified in glacial diethyl ether for 3 times, and the product PEG-LPEI-P (TBS) Glu (linear polyethylenimine molecular weight Mn=25000; tert-butyl dimethyl glutamate polymerization degree n=10) is obtained after vacuum drying, and the yield is 74%.

Example 9

This example 9 provides a method for preparing poly-2-aminoisobutyric acid-polyacrylamide, comprising the following steps:

(1) Preparation of N- (2- (2- (2-aminoethoxy) prop-2-yloxy) ethyl) -trifluoroacetamide

Triethylamine (TEA, 2.06 g) and 1,1'- (2, 2' - (propane-2, 2-diylbis (oxy)) bis (ethane-2, 1-diyl)) diurea (2.2 g) were dissolved in 10 mL absolute methanol followed by ethyl trifluoroacetate (1.93 g) likewise in 10 mL absolute methanol, and the resulting mixture was slowly added dropwise to an ice solution of the aforementioned starting materials, the reaction mixture was stirred under ice bath for reaction 6 h, the mixture was then extracted with 3 x 30 mL dichloromethane, the organic layers were combined and evaporated, and finally the product was purified by silica gel chromatography to give a white solid product with a yield of 45%.

(2) Preparation of N- (2- (2- (2- (2- (trifluoroacetamido) ethoxy) prop-2-yloxy) ethyl) acrylamide

TEA (2.19 g) and the product from step 1 above (1.40 g) were dissolved in 10 mL dioxane and the mixed solution was kept in an ice bath, an acryloyl chloride solution (0.98 g) was prepared in 10 mL dioxane and slowly added to the solution of the above starting materials, stirred for reaction 10min, extracted with 3X 100mL ethyl acetate and then purified by gradient chromatography on silica gel to give the product as a yellow oil in 69% yield.

(3) Preparation of Poly (2-aminoisobutyric acid) -Polyacrylamide

Poly (2-aminoisobutyric acid) (0.1 g) was dissolved in 2mL anhydrous methanol, methanol 30 min was evaporated under high vacuum, the resulting solution was dissolved in 10mL anhydrous DMF, then 0.73 g of the product obtained in the above step 2 and 0.23 g TEA were added, the resulting mixture was stirred at 45℃for 5 days, anhydrous diethyl ether was precipitated and purified 3 times, and after vacuum drying, the product poly (2-aminoisobutyric acid-polyacrylamide) (molecular weight Mn=5000; polymerization degree of 2-aminoisobutyric acid n=60; cation block number m ₁ =1; hydrophobic helical block number m ₂ =2) was obtained in a yield of 78%.

Example 10

The present example 10 provides a preparation method of a dendritic polyethyleneimine coupled polyethylene glycol-poly benzyloxycarbonyl lysine (hyper) branched block copolymer, comprising the following specific steps:

(1) Preparation of polyethylene glycol-Polybenzyloxycarbonyl lysine conjugate (P (Cbz) Lys-PEG-VS)

Polybenzyloxycarbonyl polylysine (P (Cbz) Lys) was dissolved in anhydrous DMF containing excess TEA. N-hydroxysuccinimide-vinyl sulfone polyethylene glycol (NHS-PEG-VS) was then likewise dissolved in anhydrous DMF, immediately followed by mixing, reaction 2h at room temperature, adding glacial ethyl ether to the reaction solution to precipitate the conjugate as a white precipitate, and drying the precipitate in vacuo to give the product P (Cbz) Lys-PEG-VS conjugate in 50% yield.

(2) Preparation of dendritic polyethyleneimine coupled polyethylene glycol-Polybenzyloxycarbonyl lysine (BPEI-PEG-P (Cbz) Lys)

Excess P (Cbz) Lys-PEG-VS conjugate was mixed with a solution of dendritic polyethylenimine (BPEI) in ph=9.0 sodium carbonate buffer and reacted overnight at room temperature. After lyophilization by dialysis separation, the final product BPEI-PEG-P (Cbz) Lys (dendritic polyethyleneimine molecular weight mn=1200; degree of polymerization of benzyloxycarbonyl lysine n=40) was obtained in a yield of 65%.

Example 11

This example 11 provides a method for preparing a six-arm polyamidoamine-phenylalanine radial block copolymer, comprising the following steps:

(1) Preparation of six-arm vinyl-terminated polyamidoamine

N, N' -methylenebisacrylamide (MBA, 3.083 g) was dissolved in 20 mL methanol/water (2/1, v/v) mixture, then 1- (2-aminoethyl) piperazine (AEPZ, 1.291 g) dissolved in another 10 mL identical mixed solvent was added and polymerization was carried out with vigorous stirring at 50℃until 25% of total vinyl groups remained.

(2) Preparation of six-arm polyamidoamine-phenylalanine (G6-PAMAM-PPhe)

Polyphenyl alanine (2.265 g) was added to the reaction solution of step 1, and the reaction was stirred at 45℃for 150: 150 h. After concentration, precipitation in acetone and vacuum drying at 40 ℃ gave a pale yellow powder G6-PAMAM-PPhe (six-arm polyamidoamine molecular weight mn=5000, phenylalanine polymerization degree n=80; cation block number m ₁ =1; hydrophobic helical block number m ₂ =1) in 91.4% yield.

Example 12

This example 12 provides a method for preparing polylysine-poly-dimethyl ester aspartate-shaped diblock copolymer, comprising the following steps:

(1) Preparation of polylysine-4-azidoaniline (PDL)

Polylysine (PLys), N- (3-dimethylaminopropyl) -N' -ethylcarbodiimide hydrochloride (EDAC) and 4-azidoaniline were mixed and dissolved in 20: 20 mL deionized water with a molar ratio of EDAC to PLL=40:1, 4-azidoaniline to EDAC=4:1, and reacted in a dark environment at 4℃for 4 h. The resulting mixture was dialyzed through MWCO 500 cellulose ester membrane (spectra) to remove unreacted materials, yielding the product PDL.

(2) Preparation of polylysine-Poly (dimethyl ester) aspartic acid (PLys-P (OMe) Asp)

Polylysine-4-azidoaniline (PDL) was dissolved in 6 mL phosphate buffer and the dispersion was added to 0.12 g poly (dimethyl ester) aspartic acid (P (OMe) Asp) powder, followed by placing the P (OMe) Asp/PDL solution in a polystyrene dish (Falcon) and irradiating 2 min at a distance of about 5 cm from an ultraviolet light source (BlakRay lamp, model B100 AP, wavelength 360nm, 100W) to thoroughly wash the polylysine-poly (dimethyl ester) aspartic acid 5 times to remove unbound reactive P (OMe) Asp, freeze-drying to give product PLys-P (OMe) Asp (polylysine molecular weight mn=6400; degree of polymerization of dimethyl ester aspartic acid n=50; number of cationic blocks m ₁ =1; number of hydrophobic helical blocks m ₂ =1).

Example 13

The present example 13 provides a preparation method of a linear multiblock copolymer of four-arm polyamidoamine-polyalanine-polyethylene glycol-polylysine-polyleucine, which comprises the following specific steps:

(1) Preparation of acetylated four-arm polyamidoamine dendrimers (G4-PAMAM-NHAC)

TEA (0.11 mL) and four-arm polyamidoamine (G4-PAMAM-NH ₂, 172 mg) were dissolved in anhydrous methanol (10 mL), excess acetic anhydride (0.08 mL) was added, the resulting mixture was stirred at room temperature for reaction 24h, methanol was evaporated under reduced pressure, and the resulting residue was dissolved in 2 mL deionized water, then transferred to a MWCO 2000 dialysis bag, dialyzed against deionized water for 2 days, and freeze-dried to give acetylated G4-PAMAM-NHAC dendrimer in a yield of 72%.

(2) Preparation of a four-arm polyamidoamine-polyalanine (G4-PAMAM-PAla)

The G4-PAMAM-NHAC (2G) is dissolved in 20mL anhydrous DMF by ultrasonic heating, then the anhydrous DMF solution which is dissolved with alanine endocyclic carboxylic anhydride monomer (Ala-NCA, 2.0G) in advance is quickly added into a reaction system by a syringe, the reaction is carried out for 3 days at 37 ℃ under the protection of argon, after the reaction is finished, the mixed solution is precipitated and purified in glacial ethyl ether for 3 times, and the product G4-PAMAM-PAla is obtained by vacuum drying, wherein the yield is 75%.

(3) Preparation of a four-arm polyamidoamine-polyalanine-polyethylene glycol (G4-PAMAM-PAla-PEG-COOH)

Bis (2-carboxyethyl) polyethylene glycol (15 mg) and G4-PAMAM-PAla (83 mg) were dissolved in a mixed solvent of DCM (5 mL) and DMSO (5 mL), after stirring reaction at room temperature for 10 min, N- (3-dimethylaminopropyl) -N-ethylcarbodiimide hydrochloride (EDC HCl,1 mg) and 4- (methylamino) pyridine (DMAP, 0.5 mg) were added to the reaction mixture, stirred at room temperature for 36 h again, the solvent was removed under reduced pressure, the residue was dissolved in deionized water, and dialyzed against deionized water using a Spectra/Por dialysis membrane (MWCO 6000) for 2 days, followed by lyophilization after passing through a Sephadex G10 column using water as eluent, to obtain the white solid product G4-PAMAM-PAla-PEG-COOH after further purification in 69% yield.

(4) Preparation of four-arm polyamidoamine-polyalanine-polyethylene glycol-polylysine (G4-PAMAM-PAla-PEG-PLys)

TEA (0.2 mL) and poly-L-lysine hydrobromide (22 mg) were dissolved in anhydrous DMSO (3 mL), the reaction mixture was further diluted with anhydrous DCM (5 mL), then G4-PAMAM-PAla-PEG-COOH (22 mg) was added, stirred at room temperature for reaction 15 min, HCl (1.5 mg) and PLys (0.5 mg) were added to the reaction mixture, the resulting solution was stirred again at room temperature for reaction 36 h, the by-product carbodiimide urea was filtered off, the solvent was removed under reduced pressure, the residue was dissolved in water and purified by dialysis using a Spectra/Por dialysis membrane (MWCO 10000) in deionized water for 2 days, lyophilized after passing through a Sephadex G10 column using water as eluent, and further purified to give G4-PAMAM-PAla-PEG-PLys as a white solid with a yield of 72%.

(5) Preparation of four-arm polyamidoamine-polyalanine-polyethylene glycol-polylysine-Polyleucine (G4-PAMAM-PAla-PEG-PLys-PLeu)

The G4-PAMAM-PAla-PEG-PLys (2G) is heated and dissolved in 20 mL anhydrous DMF, then an anhydrous DMF solution in which Ala-NCA monomer (2.0G) is dissolved in advance is quickly added into a reaction system through a syringe to react for 3 days at the temperature of 37 ℃ under the protection of argon, after the reaction is finished, the mixed solution is precipitated in glacial diethyl ether to purify the crude product for 3 times, and the crude product is obtained after vacuum drying, the product G4-PAMAM-PAla-PEG-PLys-PLeu (the molecular weight Mn=2000 of the four-arm polyamide amine, the polymerization degree n of alanine=60, the molecular weight Mn=12800 of polylysine, the polymerization degree n=40 of leucine, the cationic block number m ₁ =2, the hydrophobic helix block number m ₂ =2) is obtained, and the yield is 73%.

Example 14

The present example 14 provides a method for preparing polylysine-polyalanine linear multiblock copolymer, which comprises the following specific steps:

(1) Preparation of amino acid vinyl monomers

Taking Boc-L-Ala-OH as an example, boc-L-Ala-OH (6.0 g) was dissolved in anhydrous DCM (50 mL), followed by dicyclohexylcarbodiimide (DCC, 5.13 g) and 4-dimethylaminopyridine (DMAP, 0.28 g) were added to the system. 2-hydroxyethyl methacrylate (HEMA, 3.24 g) was then slowly added dropwise to the reaction mixture with stirring under ice-water bath, reaction 30 min followed by transfer to room temperature followed by reaction 24 h, removal of insoluble N, N' -Dicyclohexylurea (DCU) by suction filtration, further addition of 80 mL deionized water to the filtrate followed by extraction 4 times with 120 mL anhydrous DCM, further washing of the organic layer with NaHCO ₃ 3 times and drying with anhydrous NaSO ₄, removal of the solvent by rotary evaporation and further purification by silica gel column chromatography with hexane/ethyl acetate as mobile phase (v/v) to give the colorless liquid amino acid vinyl monomer Boc-Ala-HEMA in 93% yield. The process for preparing Boc-Lys-HEMA from Boc-lysine is similar.

(2) Preparation of Poly (Boc) lysine (P (Boc-Lys-HEMA) -macroCTA)

Amino acid vinyl monomer (0.50 g), 4-cyano-4- (dodecylthiocarbonyl) thiolpentanoic acid (CDP, 10.7 mg), 2' -azobisisobutyronitrile (AIBN, 8.7 mg) were dissolved in 11 mL anhydrous DMF, reacted under argon protection at 70 ℃, finally quenched for polymerization by cooling in ice water bath and exposing the solution to air, followed by dilution of the solution with acetone, precipitation purification of the crude product 3 times in acetone/hexane, vacuum drying to give product P (Boc-Lys-HEMA) -macroCTA in 78% yield.

(3) Preparation of poly (Boc) lysine-poly (Boc) phenylalanine (P (Boc) Lys-P (Boc) Ala) multiblock copolymers

Boc-Ala-HEMA (0.143 g), P (Boc-Lys-HEMA) -macroCTA (0.11 g), AIBN (5.9 mg) were dissolved in 10 mL anhydrous DMF and reacted at 70℃under argon for a predetermined time, after which the reaction flask was cooled in an ice-water bath and the reaction mixture was diluted with acetone as required, finally, the crude product was precipitated and purified 3 times in acetone/hexane and dried under vacuum at room temperature to give a pale yellow solid in 75% yield. This cycle gives a P (Boc) Lys-P (Boc) Ala multiblock copolymer.

(4) Preparation of polylysine-phenylalanine (PLys-PAla) multiblock copolymers

Removing Boc protecting group of block copolymer side chain with TFA in DCM to obtain polymer with free primary amine group, adding P (Boc) Lys-P (Boc) Phe multiblock copolymer (50 mg) to anhydrous DCM of 1mL, stirring 10min to ensure uniformity of mixture, then dropping 0.5 mL TFA under ice water bath condition, keeping stirring reaction 2h, precipitating and purifying 3 times in glacial diethyl ether, vacuum drying to obtain PLys-PAla multiblock copolymer (polylysine molecular weight mn=1940; n=15 of alanine polymerization; cation block number m ₁ =5; hydrophobic helical block number m ₂ =5) with yield of 80%.

Comparative example 1

The comparative example provides a preparation method of a polyethylene glycol-poly (D, L) lactide-poly (arginine) triblock copolymer, which comprises the following specific steps:

(1) Activated polyethylene glycol-poly (D, L) lactide (PEG-PLA-PNP)

4-Nitrobenzoate (PNP, 118 mg), pyrimidine (64 μL) was dissolved in 6mL anhydrous DCM at 0deg.C; then, rapidly injecting an anhydrous DCM solution (2 mL) dissolved with polyethylene glycol-poly (D, L) lactide (1 g) into the reaction system through a syringe, stirring for 30 minutes at 0 ℃, then transferring to room temperature, and stirring and reacting for 2 days under the protection of argon; after the reaction, the crude product was precipitated and purified 3 times in glacial ethyl ether and dried under reduced pressure to obtain the product PEG-PLA-PNP, the yield of which was 66%.

(2) Preparation of polyethylene glycol-Poly (D, L) lactide-polyarginine (PEG-PLA-R15)

PEG-PLA-PNP (660 mg), poly-arginine (R15-NH 2, 140, mg), TEA (26. Mu.L) were dissolved in 10 mL anhydrous DMF and reacted at room temperature under the protection of argon for 2 days. And (3) passing the solution obtained by the reaction through an MWCO 3500 dialysis bag, dialyzing with deionized water for 2 days, and freeze-drying to obtain the product PEG-PLA-R15 (the molecular weight Mn=2360 of the poly-arginine), wherein the yield is 70%.

Comparative example 2

The comparative example provides a preparation method of a deferoxamine grafted polyethylene glycol-poly benzyl ester glutamic acid diblock copolymer, which comprises the following specific steps:

(1) Preparation of polyethylene glycol-Poly benzyl ester glutamic acid (PEG-PBLG)

The macroinitiator PEG-NH ₂ is adopted to initiate Bn-L-Glu-NCA ring-opening polymerization method to prepare polyethylene glycol-poly benzyl ester glutamic acid. Bn-L-Glu-NCA (8.32 g) is dissolved in 40 mL anhydrous DMF, then, a PEG-NH ₂ dissolved anhydrous DMF solution is added into the system through a syringe, the mixture is stirred and reacted for 3 days at 37 ℃ under the protection of argon, the crude product is precipitated and purified in glacial ethyl ether for 3 times, and the product (PEG-PBLG) is obtained after vacuum drying, wherein the yield is 70%.

(2) Preparation of deferoxamine grafted polyethylene glycol-Poly benzyl ester glutamic acid (PEG-PBLG-DFO)

PEG-PBLG and Desferrioxamine (DFO) are subjected to ammonolysis reaction to prepare polyethylene glycol-poly benzyl ester glutamic acid grafted with DFO. PEG-PBLG (0.5 g) and DFO (0.48-0.90 g) are dissolved in a mixed solvent of anhydrous DMF/methanol (v: v=1:1), TEA (0.15-0.27 g) is dripped under the protection of argon, then the reaction is continued for 48 h at 45 ℃, the obtained solution is subjected to dialysis by a dialysis bag (MWCO 3500) and deionized water for 2 days, and the product PEG-PBLG-DFO (poly benzyl ester glutamic acid polymerization degree n=20; DFO grafting number is 6) is obtained after freeze drying, and the yield is 65%.

Comparative example 3

The comparative example provides a preparation method of a cationic alpha-helical polypeptide, which comprises the following specific steps:

(1) Preparation of 1, 2-dipalmitoyloxy-3-aminopropane (DPAP)

Tert-butyl N- (2, 3-dihydroxypropyl) carbamate (1.6 g) and palmitic acid (4.6 g) were dissolved in 100mL anhydrous DCM; 1-ethyl-3- (3-dimethylaminopropyl) carbodiimide hydrochloride (EDC HCl,10 g) and 4-dimethylaminopyridine (DMAP, 0.5 g) were then added to the reaction system; after stirring 48 h at room temperature, the reaction was stopped and the organic phase was washed with 30 mL x 2 dilute hydrochloric acid, 30 mL x 2 brine and 30 mL deionized water, respectively; then the obtained organic phase is dried by anhydrous Na2SO4, filtered, and DCM is removed by rotary evaporation to obtain a white crude product; purifying the crude product by basic alumina column chromatography to obtain N-Boc-1-imino-2, 3-dipalmitoyloxy propane; finally, TFA was used in an ice-water bath: deprotection of Boc with DCM (1:6) followed by precipitation in glacial diethyl ether gave the product DPAP in 36% yield.

(2) Preparation of gamma-allyl-L-glutamic acid cyclic anhydride (ALG-NCA)

Glutamic acid (L-Glu, 20 g) and allyl alcohol (50 mL) were placed in a flask immersed in ice water, and after stirring for 10 minutes, 8 mL sulfuric acid (98%) was added dropwise to the flask. After continuing to stir 24 h, the mixture was slowly poured into 100 mL water (27 g sodium bicarbonate in advance) to neutralize the sulfuric acid; then, standing overnight at-20deg.C, filtering to obtain crude product, and recrystallizing with methanol; finally, gamma-allyl-L-glutamate cyclic anhydride is obtained after vacuum drying, and the yield is 45 percent.

Gamma-allyl-L-glutamate cyclic anhydride (10 g) and triphosgene (5.4 g) were placed in a dry three-neck flask, then 200 mL anhydrous THF was added under argon protection, after stirring reaction at 50 ℃ for 10 min, the solution was clarified, after 20 min, the solution was poured into 1000 mL glacial hexane and stored overnight at-20 ℃, after discarding the supernatant, the crude product was dissolved in 200 mL cold ethyl acetate, then washed with 50mL x 2 ice water, the organic phase was dried over anhydrous sodium sulfate, filtered, and the ethyl acetate was removed in vacuo. Finally, ALG-NCA was obtained as a yellow viscous oil in a yield of 64%.

(3) Preparation of 1, 2-dipalmitoyloxy-3-aminopropyl-poly (gamma-allyl-L-glutamate) (DPAP-PALG)

DPAP (1.0 g) is dissolved in 250 mL anhydrous THF, then gamma-allyl-L-glutamic acid N-carboxylic anhydride (ALG-NCA, 9.4 g) dissolved in 150mL anhydrous THF is added rapidly, after stirring reaction at room temperature under the protection of argon for 72 h, solvent is removed by rotary evaporation, the crude product is redissolved in DMF, and then dialysis is carried out in deionized water for 2 days by a dialysis bag (MWCO 2000) in the absence of light, and freeze drying is carried out, thus obtaining DPAP-PALG white powder with the yield of 69%.

(4) Preparation of cationic spiral Polypeptides (ACPP)

DPAP-PALG (1.1 g), cysteamine hydrochloride (2.4 g) and Irgacure (0.24 g) were dissolved in DMF/H ₂ O (80 mL/5 mL) mixed solvent. The solution was bubbled with argon gas 20 min to remove oxygen in the system, then the flask was sealed, uv-irradiated (ʎ =365 nm,15mw/cm ²) for 30min, then stirred at room temperature for 48 h, and the mixture was dialyzed against deionized water for 2 days using a dialysis bag (MWCO 3500); finally, freeze-drying gave the product ACPP in 86% yield.

The particle size of the self-assembled body obtained was measured by Dynamic Light Scattering (DLS), as shown in fig. 1, and the result was 143 nm; the morphology of the self-assembly was characterized by Transmission Electron Microscopy (TEM), and the result is shown in fig. 2, and the self-assembly prepared was a vesicle structure. The secondary conformation of the self-assembly was tested using a circular dichroscope and the results are shown in figure 3, the self-assembly produced having a mixed structure of random coil and alpha-helix.

To verify the integrity of the cell membrane after a period of co-culture of the block copolymer self-assemblies prepared in examples 1 and 5 above with the cells. The self-assembly of the block copolymer and 4T1 cells are continuously cultured for 4 h, then the cell membrane is dyed by fluorescent dye Dio, and then the cell membrane is observed by means of a laser confocal microscope, the result is shown in figure 4, and figure 4 is a laser confocal photograph of the integrity of the cell membrane after the self-assembly of the block copolymer prepared in example 1 and 5 acts; wherein a represents the result of the self-assembly obtained in example 1, and b represents the result of the self-assembly obtained in example 5.

As can be seen from fig. 4, after the block copolymers prepared in the selected examples 1 and 5 were co-cultured with cells 4h, the cell membranes were complete in shape and clear in outline, which suggests that the cell membranes are not significantly damaged during the process of forming transient pores by the interaction of the block copolymers with the cell membranes, thereby ensuring the integrity of the cell membranes and providing a guarantee for biosafety during the drug delivery process.

To verify the delivery performance of the four-arm polyethylene glycol-quaternary ammonium salt modified polycysteine-polyleucine star block copolymer prepared in example 4, the self-assembly prepared in example 4 was co-cultured with 4T1 cells for 2 h after being labeled with FITC, and then the delivery performance was characterized, and the results are shown in fig. 5, and fig. 5 is a confocal laser photograph of the delivery performance of the star block copolymer self-assembly prepared in example 4; wherein a represents the result of continuous incubation of the self-assembled body obtained in example 4 with cells for 2 h; b shows the result of the culture after washing the self-assembly body and further culturing 2 h.

In the examples of the present application, one group of block copolymer self-assemblies prepared in example 4 was selected and continuously cultured with 4T1 cells using fluorescent substance FITC-labeled and fluorescent small molecule Pyridine Iodide (PI) impermeable to cell membranes for 2 h. Another group was to culture the block copolymer self-assemblies with 4T1 cells alone for 2h, then to wash the self-assemblies, to add PI for further culture for 2h after 30min, and then to observe the self-assemblies and the entry of PI by laser confocal.

As can be seen from fig. 5, when the self-assembled body of the block copolymer prepared in example 4 is co-cultured with PI, PI can enter cells with high efficiency, which means that the block copolymer has cell membrane pore-forming effect and can mediate fluorescent substances which are impermeable to cell membranes to enter cells in a molecular permeation manner; in contrast, after the block copolymer self-assembly was washed for 30 min, PI was added to the mixture to co-culture with the cells, and PI was not allowed to enter the cells. This demonstrates that the holes formed by the block copolymer on the cell membrane are reversible and can recover by themselves, thereby avoiding irreversible damage to the cell membrane and improving the safety of drug delivery.

To verify the pore-forming properties of the cell membrane of the block copolymer self-assemblies prepared in the above examples, some examples and comparative examples were selected for performance testing. First, fluorescence quantitative analysis is carried out on the selected self-assemblies of each example and comparative example by using a fluorescent substance FITC to label and co-culture with PI and 4T1 cells for 4 h, and a fluorescence quantitative chart is shown in FIG. 6; fig. 6 a: examples 2, b: example 8,c: examples 6, d: examples 14, e: examples 13, f: example 11, g, example 7,h: examples 10, i: comparative example 1, j: comparative example 2.

As can be seen from FIG. 6, the block copolymers prepared in the above examples all have cell membrane pore-forming ability and mediate PI entry into cells through the transient pores formed. In contrast, PEG-PLA-R15 with only cationic blocks (comparative example 1) and PEG-PBLG-DFO with only helical structures (comparative example 2) failed to mediate PI entry into cells through transient pores because they did not possess cell membrane pore-forming properties.

To verify the cell entry mechanism of the block copolymer self-assembly prepared in the above example, performance test was performed on the block copolymer self-assembly prepared in example 9, and the results are shown in fig. 7, and fig. 7 is a confocal laser photograph of the cell entry mechanism of the block copolymer self-assembly prepared in example 9; wherein a is a graph of the result of the block copolymer obtained in example 9; b. c is a graph of the results of comparative examples 1 and 2, respectively.

In the examples of the present application, the self-assembly of the block copolymer obtained in example 9 was selected, and the degree of capture of the block copolymer by lysosomes was monitored by labeling the lysosomes in the cells and evaluating their fluorescent co-localization with the block copolymer.

As can be seen from FIG. 7, the block copolymer self-assembly obtained in example 9 showed little co-localization by capturing only a few lysosomes after incubation with cells of 2 h. However, the fluorescence of both PEG-PLA-R15 having only cationic blocks (comparative example 1) and PEG-PBLG-DFO having only a helical structure (comparative example 2) overlapped greatly with that of lysosome, indicating that both the cationic polymer prepared in comparative example 1 and the helical polymer prepared in comparative example 2 were trapped in lysosomes and could not enter cytoplasm. Furthermore, the fluorescence intensities of comparative examples 1 and 2 were also considerably weaker than that of the block copolymer prepared in example 9. The above results show that the block copolymer prepared in example 9 has excellent cell entry performance, and enters cells through cell membrane pore forming, and can not be captured and degraded by lysosomes, thus greatly improving delivery efficiency.

To verify the biocompatibility of the block copolymer self-assembly prepared in example 12 above, the survival rate of cells was evaluated using CCK-8 cytotoxicity assay kit, and the results are shown in fig. 8, and fig. 8 is a quantitative graph of the biocompatibility of the block copolymer self-assembly prepared in example 12; wherein a represents the result graph of the block copolymer self-assembly obtained in example 12, and b represents the result graph of the cationic helical polypeptide ACPP (comparative example 3). The concentration of the polymer is 10, 50, 100, 200 and 300 mug/mL from left to right.

In the embodiment of the application, the block copolymer self-assembly body obtained in the embodiment 12 is selected to be co-cultured with 3T3 cells for 24h, the survival rate of the cells is estimated by using a CCK-8 cytotoxicity detection kit, and a survival rate quantitative graph of the 3T3 cells along with the change of the concentration of the block copolymer assembly body is drawn.

As can be seen from FIG. 8, the block copolymer prepared in example 12 had less effect on cell viability, and the cell viability after treatment with different concentrations of the block copolymer was above 85%. However, the cationic helical polypeptide ACPP prepared in comparative example 3 showed a high cytotoxicity in dependence on the concentration, and the cell viability was lower than 50% at a concentration of 300. Mu.g/mL. This demonstrates that the block copolymer prepared in example 12 has better biosafety compared to cationic helical polypeptides.

To verify the in vivo drug delivery and anti-tumor properties of the block copolymer self-assembly prepared in example 7 above, the block copolymer self-assembly prepared in example 7 was subjected to a performance test, the results of which are shown in fig. 9, and fig. 9 is a graph of the in vivo anti-tumor results of the block copolymer self-assembly prepared in example 7; wherein a is a result graph of a normal saline group, and b is a result graph of a free RNase A group; c is a graph of the results of the self-assembly obtained in example 7.

In embodiments of the application, ribonuclease A (RNase A) was selected as a model of therapeutic drug delivered to evaluate anti-tumor effects in vivo. The block copolymer +RNase A, free RNase A and physiological salt (Saline) prepared in example 7 were injected into tumor-bearing mice inoculated with 4T1 cells, and tumor volumes at different times were measured. On day 15 after the start of dosing, mice were humane sacrificed and tumors were exfoliated and weighed. As shown in FIG. 9, free RNaseA was very poor in tumor inhibition due to its difficulty in entering tumor cells. The block copolymer prepared in example 7 can mediate RNase A to enter tumor cells through a cell membrane pore-forming mode, and has a remarkable tumor inhibition effect in vivo.

The foregoing is merely illustrative of the present application, and the present application is not limited thereto, and any person skilled in the art will readily recognize that variations or substitutions are within the scope of the present application. Therefore, the protection scope of the present application shall be subject to the protection scope of the claims.

Claims

1. A block copolymer having cell membrane pore-forming properties, wherein the block copolymer is composed of a cationic hydrophilic segment and a helical hydrophobic segment;

2. The block copolymer according to claim 1, wherein the block copolymer is one of a linear block copolymer, a radial block copolymer, a cyclic block copolymer, a comb block copolymer, and a (hyperbranched) block copolymer, and the cationic hydrophilic segment has a block number m ₁ not less than 1;

and/or the block number m ₂ of the spiral hydrophobic block is more than or equal to 1.

3. The block copolymer according to claim 2, wherein when the block copolymer is a linear block copolymer, the block number m ₁ is not more than 2, and/or m ₂ is not more than 2, and the sum of m ₁ and m ₂ is 2 to 3, and the cationic hydrophilic segment is at least one of a polymer containing a primary amine, a secondary amine, a tertiary amine, a quaternary amine, pyridine, imidazolium, a guanidine salt, sulfonium ion and phosphine ion; the spiral hydrophobic segment is at least one of phenylalanine, methionine, poly-2-amino isobutyric acid, polylysine derivative, polyaspartic acid derivative, polyglutamic acid derivative and polycysteine derivative.

4. The block copolymer according to claim 2, wherein when the block copolymer is a linear block copolymer, the block number m ₁ is not less than 2 and m ₂ is not less than 2, and the cationic hydrophilic segment is at least one of a polymer containing a primary amine, a secondary amine, a tertiary amine, a quaternary amine, a pyridine, an imidazolium, a guanidine salt, a sulfonium ion, and a phosphine ion; the spiral hydrophobic segment is at least one of polyalanine, polyleucine, polyphenylalanine, polymethine, poly-2-amino isobutyric acid, polylysine derivatives, polyaspartic acid derivatives, polyglutamic acid derivatives and polycysteine derivatives.

5. The block copolymer of claim 2, wherein when the block copolymer is a radial block copolymer, a cyclic block copolymer, a comb block copolymer, a (hyperbranched block copolymer), the cationic hydrophilic segment is at least one of a polymer comprising primary, secondary, tertiary, quaternary, pyridine, imidazolium, guanidinium, sulfonium, and phosphonium ions; the spiral hydrophobic segment is at least one of polyalanine, polyleucine, polyphenylalanine, polymethine, poly-2-amino isobutyric acid, polylysine derivatives, polyaspartic acid derivatives, polyglutamic acid derivatives and polycysteine derivatives.

6. A block copolymer according to claim 3, characterized in that the cationic hydrophilic segment is preferably at least one of polymers containing secondary, tertiary, quaternary, pyridine, imidazolium, sulfonium ions.

7. A block copolymer according to claim 3, characterized in that the helical hydrophobic segment is preferably at least one of polylysine derivatives, polyaspartic acid derivatives, polyglutamic acid derivatives, polycysteine derivatives.

8. The block copolymer according to any one of claims 4 to 5, wherein the cationic hydrophilic segment is preferably at least one of a polymer containing secondary amine, tertiary amine, quaternary amine, guanidine salt, sulfonium ion, and the helical hydrophobic segment is preferably at least one of polyalanine, polylysine, polyalanine, polylysine derivative, polyaspartic acid derivative, polyglutamic acid derivative, and polycysteine derivative.

9. A method for producing a block copolymer having cell membrane pore-forming properties according to any one of claims 1 to 8, comprising:

10. Use of the block copolymer with cell membrane pore-forming properties of claim 9 for self-assembly, biosensing, drug delivery or disease treatment and diagnosis.