Polymer-cell-penetrating peptide conjugate, preparation method thereof, nano adjuvant and nano vaccine

Abstract

The invention discloses a polymer-cell-penetrating peptide conjugate, a preparation method of the polymer-cell-penetrating peptide conjugate, a nano adjuvant and a nano vaccine, and relates to the field of biomedicine. A series of polypeptides are grafted into a polymer structure through an exchange or coupling reaction to obtain the polymer-cell-penetrating peptide conjugate, when the pH value of the polymer-cell-penetrating peptide conjugate is neutral, the polymer-cell-penetrating peptide conjugate can be self-assembled to serve as hidden polypeptides of nanoparticles, and protonation can occur in an acid environment, so that the polymer-cell-penetrating peptide conjugate is formed. Therefore, the polypeptide is exposed to selectively destroy a lipid membrane of an endosome. Experimental results show that the nano vaccine taking the polymer-cell-penetrating peptide conjugate provided by the invention as an adjuvant can cause Th1 type cellular immune response and increase the proportion of antigen-specific T cells.

Description

Technical Field

[0001] The present invention relates to the field of biomedicine, and in particular to a polymer-membrane-penetrating peptide conjugate and a preparation method thereof, a nano adjuvant and a nano vaccine. Background Art

[0002] Vaccination is one of the most effective strategies for dealing with infectious diseases. Its principle is to produce a protective immune response in the body. The premise for the body to produce an immune response is that the immunogenicity of the vaccine is strong. Vaccines composed solely of antigens have weak immunogenicity and often cannot prevent infectious diseases. Therefore, adjuvants are needed to work together with antigens to enhance the immune response.

[0003] Vaccine adjuvants are a class of substances that can enhance or improve the immunogenicity of vaccines. They enhance the adaptive immunity of vaccines by activating innate immune cells. However, most current vaccine adjuvants mainly act to induce humoral immune responses and cannot induce cellular immune responses very well. Therefore, there is an urgent need to provide a vaccine adjuvant that can effectively induce cellular immune responses and improve antigen-specific immune responses. Summary of the invention

[0004] In view of this, the present invention provides a polymer-membrane-penetrating peptide conjugate and a preparation method thereof, a nanoadjuvant and a nanovaccine. The polymer-membrane-penetrating peptide conjugate provided by the present invention can induce the occurrence of cellular immune response in the body when used as a nanoadjuvant, enhance the ability of antigen cross-presentation, and significantly increase the proportion of specific T cells.

[0005] The present invention provides a polymer-membrane-penetrating peptide conjugate having a structure of formula (I):

[0006] (I);

[0007] wherein n is selected from an integer of 2 to 500, preferably an integer of 20 to 300, more preferably an integer of 50 to 200; x is selected from an integer of 2 to 300, preferably an integer of 20 to 250, more preferably an integer of 40 to 200; y is selected from an integer of 1 to 10, preferably an integer of 1 to 8, more preferably an integer of 1 to 5; z is selected from an integer of 1 to 20, preferably an integer of 1 to 10, more preferably an integer of 1 to 5;

[0008] Peptide is selected from the residue after removing the sulfhydryl group of cysteine ​​from a polypeptide containing cysteine;

[0009] X is selected from C1~C 30 Alkyl, at least one substituent substituted C1~C 30 Alkyl, C2~C 30 ether, at least one substituent substituted C2~C30 Ether, C2~C 30 sulfide, C2~C 30 Sulfide, C1~C 30 Alkoxy, at least one substituent substituted C1~C 30 Alkoxy, C1~C 30 alkylthio, C1~C 30 Alkylthio, C6~C 30 aromatic group or at least one substituent substituted C6~C 30 The aromatic group, the substituent is selected from halogen, C1~C 30 Alkyl, C6~C 30 Aromatic groups, C6~C 30 Heteroaryl, C1~C 30 Alkoxy, C1~C 30 Alkylthio, C2~C 30 Ether, C1~C 30 Esters, C2~C 30 Sulfide, C2~C 30 At least one of amine, hydroxyl, carboxyl, aldehyde, carbonyl, amino, cyano, sulfhydryl and thiocarbonyl; and the X is connected to the peptide through an S atom;

[0010] R1, R4 and R6 are independently selected from hydrogen, C1~C 30 Alkyl, at least one substituent substituted C1~C 30 Alkyl, C3~C 30 cycloalkyl, C3~C 30 Cycloalkyl, C2~C 30 heterocycloalkyl, C2~C 30 Heterocycloalkyl, C6~C 30 aromatic group, at least one substituent substituted with C6~C 30 Aromatic groups, C6~C 30 heteroaryl, C6~C 30 Heteroaryl, C1~C 30 Alkoxy, at least one substituent substituted C1~C 30 Alkoxy, C1~C 30 alkylthio, C1~C 30 Alkylthio, C2~C 30 ether, at least one substituent substituted C2~C 30 Ether, C1~C 30 esters, C1~C 30Esters, C2~C 30 sulfide, C2~C 30 Sulfide, C2~C 30 amine or at least one substituent substituted C2~C 30 The amine, the substituent is selected from C1~C 30 Alkyl, C6~C 30 Aromatic groups, C6~C 30 Heteroaryl, C1~C 30 Alkoxy, C1~C 30 Alkylthio, C2~C 30 Ether, C1~C 30 Esters, C2~C 30 Sulfide, C2~C 30 At least one of an amine, hydroxyl, carboxyl, aldehyde, carbonyl, amino, cyano, thiol and thiocarbonyl group;

[0011] R2 and R3 are independently selected from hydrogen, C1~C 30 Alkyl, at least one substituent substituted C1~C 30 Alkyl, C3~C 30 cycloalkyl, C3~C 30 Cycloalkyl, C2~C 30 heterocycloalkyl, C2~C 30 Heterocycloalkyl, C6~C 30 aromatic group, at least one substituent substituted with C6~C 30 Aromatic groups, C6~C 30 heteroaryl, C6~C 30 Heteroaryl, C1~C 30 Alkoxy, at least one substituent substituted C1~C 30 Alkoxy, C1~C 30 alkylthio, C1~C 30 Alkylthio, C2~C 30 ether, at least one substituent substituted C2~C 30 Ether, C1~C 30 esters, C1~C 30 Esters, C2~C 30 sulfide, C2~C 30 Sulfide, C2~C 30 amine or at least one substituent substituted C2~C 30 The amine, the substituent is selected from C1~C 30 Alkyl, C6~C 30Aromatic groups, C6~C 30 Heteroaryl, C1~C 30 Alkoxy, C1~C 30 Alkylthio, C2~C 30 Ether, C1~C 30 Esters, C2~C 30 Sulfide, C2~C 30 at least one of amine, hydroxyl, carboxyl, aldehyde, carbonyl, amino, cyano, thiol and thiocarbonyl; or R2 and R3 and the N atom to which they are connected together form a 3-30 membered heterocyclic ring.

[0012] The applicant of the present invention was inspired by the effective endosomal escape mechanism adopted by adenovirus, and introduced a polypeptide structure into the polymer by means of functional modification to obtain a polymer-membrane-penetrating peptide conjugate. When the pH is neutral, the polymer-membrane-penetrating peptide conjugate can self-assemble as nanoparticles to hide the contained polypeptide; in an acidic environment, a protonation reaction will occur, exposing the polypeptide to selectively destroy the endosomal membrane, thereby enhancing the cytoplasmic delivery of vaccine antigens, antigen cross-presentation and immune cell activation, solving the problem of low endosomal escape efficiency of antigen substances in current vaccines, and enhancing the specific immune response and immune memory of the antigen.

[0013] In some specific implementations, the polypeptide is selected from commercially available or customizable polypeptides. The present invention has no particular limitation on the source of the polypeptide.

[0014] In some specific implementations, the polypeptide can be Melittin (GIGAVLKVLTTGLPALISWIKRKRQQC), FL-20 (GIGAILKVLATGLPTLISWIC), CMA-2 (GIGAVLKVLTTGLPALISWIHHHHEEC), TAT (CYGRKKRRQRRR), Arg8 (RRRRRRRRC), Penetrain (RQIKIWFQNRRMKWKKC) or MPG (GALFLGFLGAAGSTMGAWSQPKKKRKVC), wherein C in the polypeptide sequence represents cysteine ​​(Cys), and the thiol group in the Cys can be connected to the polymer main chain through exchange or coupling reaction.

[0015] In some specific implementations, the X is selected from C1 to C 25 Alkyl, at least one substituent substituted C1~C 25 Alkyl, C2~C 25 ether, at least one substituent substituted C2~C 25 Ether, C2~C 25 sulfide, C2~C 25Sulfide, C1~C 25 Alkoxy, at least one substituent substituted C1~C 25 Alkoxy, C1~C 25 or at least one substituent substituted with C1~C 25 The alkylthio group, wherein the substituent is selected from halogen, C1~C 25 Alkyl, C1~C 25 Halogenated alkyl, C6~C 25 Aromatic groups, C6~C 25 Halogenated aromatic groups, C2~C 25 Ether, C1~C 25 Esters and C2~C 25 At least one of the sulfides of C1~C 20 Alkyl, at least one substituent substituted C1~C 20 Alkyl, C1~C 20 alkylthio, C1~C 20 Alkylthio, C2~C 20 ether or at least one substituent substituted C2~C 20 The ether, the substituent is selected from halogen and C6~C 20 At least one of the halogenated aromatic groups; more preferably the following structure:

[0016] , , ;

[0017] Wherein, f, g, h and i are independently selected from integers of 1 to 5;

[0018] Z is selected from S or O;

[0019] R e , R f , R g and R h independently selected from halogen;

[0020] In some specific implementations, R1 and R4 are independently selected from hydrogen, C1~C 25 Alkyl, C3~C 25 Cycloalkyl, C6~C 25 Aromatic or C6~C 25 The heteroaryl group is preferably C1~C 20 Alkyl, C3~C 20 Cycloalkyl or C6~C 20 The aromatic group is more preferably a methyl group.

[0021] In some specific implementations, the R6 is selected from the following structures:

[0022] ;

[0023] R a Selected from C1~C 25 Alkyl, C3~C 25 Cycloalkyl, C1~C 25 Alkoxy, C1~C 25 Alkylthio, C6~C 25 Aromatic or C6~C 25 The heteroaryl group.

[0024] In some specific implementations, the R6 is preferably the following structure:

[0025] ;

[0026] R a Selected from C1~C 20 of alkyl.

[0027] In some specific implementations, R2 and R3 are independently selected from C1-C5 alkyl groups or amino-substituted C1-C5 alkyl groups; or R2, R3 and the N atom to which they are connected together form a ring having the following structure:

[0028] , , ;

[0029] Wherein, a, b, c, d and e are independently selected from integers of 1 to 5;

[0030] R b and R c Independently selected from hydrogen or C1~C5 alkyl;

[0031] —Y— is selected from —O—, or ,

[0032] R d Selected from hydrogen or C1~C5 alkyl.

[0033] In some specific implementations, the R2 and R3 and the N atom to which they are commonly connected preferably form a ring having the following structure:

[0034] ;

[0035] Wherein, a is selected from an integer of 1 to 5;

[0036] More preferably, it is a ring having the following structure:

[0037] .

[0038] In some specific implementations, the polymer-membrane-penetrating peptide conjugate of the present invention has the following structure:

[0039] ;

[0040] Wherein, x is selected from an integer of 20 to 250, and y is selected from an integer of 1 to 8.

[0041] In some specific implementations, the number average molecular weight of the polymer-membrane-penetrating peptide conjugate having the structure of formula (I) is 1,000-200,000, preferably 2,000-150,000, and more preferably 3,500-100,000.

[0042] The present invention also provides a method for preparing a polymer-membrane-penetrating peptide conjugate, comprising:

[0043] a) a chain transfer agent of formula (II), a monomer of formula (III), a monomer of formula (IV) and an initiator undergo a polymerization reaction to obtain a polymer having a structure of formula (V);

[0044] b) the polymer having the structure of formula (V) obtained in step a), the polypeptide having the structure of formula (VI) and a buffer are subjected to a functionalization reaction to obtain a polymer-membrane-penetrating peptide conjugate having the structure of formula (I);

[0045] (II); (III); (IV);

[0046] (V); (VI);

[0047] (I);

[0048] wherein n is selected from an integer of 2 to 500; x is selected from an integer of 2 to 300; y is selected from an integer of 1 to 10; and z is selected from an integer of 1 to 20;

[0049] Peptide is selected from the residue after removing the sulfhydryl group of cysteine ​​from a polypeptide containing cysteine;

[0050] X is selected from C1~C 30 Alkyl, at least one substituent substituted C1~C 30 Alkyl, C2~C 30 ether, at least one substituent substituted C2~C 30 Ether, C2~C 30 sulfide, C2~C 30 Sulfide, C1~C30 Alkoxy, at least one substituent substituted C1~C 30 Alkoxy, C1~C 30 alkylthio, C3~C 10 Alkylthio, C6~C 30 aromatic group or at least one substituent substituted C6~C 30 The aromatic group, the substituent is selected from halogen, C1~C 30 Alkyl, C6~C 30 Aromatic groups, C6~C 30 Heteroaryl, C1~C 30 Alkoxy, C1~C 30 Alkylthio, C2~C 30 Ether, C1~C 30 Esters, C2~C 30 Sulfide, C2~C 30 At least one of amine, hydroxyl, carboxyl, aldehyde, carbonyl, amino, cyano, sulfhydryl and thiocarbonyl; and the X is connected to the peptide through an S atom;

[0051] R1, R4 and R6 are independently selected from hydrogen, C1~C 30 Alkyl, at least one substituent substituted C1~C 30 Alkyl, C3~C 30 cycloalkyl, C3~C 30 Cycloalkyl, C2~C 30 heterocycloalkyl, C2~C 30 Heterocycloalkyl, C6~C 30 aromatic group, at least one substituent substituted with C6~C 30 Aromatic groups, C6~C 30 heteroaryl, C6~C 30 Heteroaryl, C1~C 30 Alkoxy, at least one substituent substituted C1~C 30 Alkoxy, C1~C 30 alkylthio, C1~C 30 Alkylthio, C2~C 30 ether, at least one substituent substituted C2~C 30 Ether, C1~C 30 esters, C1~C 30 Esters, C2~C 30 sulfide, C2~C 30 Sulfide, C2~C 30amine or at least one substituent substituted C2~C 30 The amine, the substituent is selected from C1~C 30 Alkyl, C6~C 30 Aromatic groups, C6~C 30 Heteroaryl, C1~C 30 Alkoxy, C1~C 30 Alkylthio, C2~C 30 Ether, C1~C 30 Esters, C2~C 30 Sulfide, C2~C 30 At least one of an amine, hydroxyl, carboxyl, aldehyde, carbonyl, amino, cyano, thiol and thiocarbonyl group;

[0052] R5 is selected from C1~C 30 Alkyl, C2~C 30 Alkenyl, C2~C 30 Alkenyl, C2~C 30 Alkynyl, C2~C 30 Alkynyl, C2~C 30 ether, at least one substituent substituted C2~C 30 sulfide, at least one substituent substituted with C1~C 30 Alkoxy, at least one substituent substituted C1~C 30 or at least one substituent substituted with C6~C 30 The aromatic group, the substituent is selected from halogen, C1~C 30 Alkyl, C1~C 30 haloalkyl, C2~C 30 Alkenyl, C2~C 30 Halogenated alkenyl, C2~C 30 Alkynyl, C2~C 30 Haloalkynyl, C6~C 30 Aromatic groups, C6~C 30 Halogenated aromatic groups, C6~C 30 Heteroaryl, C1~C 30 Alkoxy, C1~C 30 Alkylthio, C2~C 30 Ether, C1~C 30 Esters, C2~C 30 Sulfide, C2~C 30 At least one of an amine, hydroxyl, carboxyl, aldehyde, carbonyl, amino, cyano, thiol and thiocarbonyl group;

[0053] R2 and R3 are independently selected from hydrogen, C1~C30 Alkyl, at least one substituent substituted C1~C 30 Alkyl, C3~C 30 cycloalkyl, C3~C 30 Cycloalkyl, C2~C 30 heterocycloalkyl, C2~C 30 Heterocycloalkyl, C6~C 30 aromatic group, at least one substituent substituted with C6~C 30 Aromatic groups, C6~C 30 heteroaryl, C6~C 30 Heteroaryl, C1~C 30 Alkoxy, at least one substituent substituted C1~C 30 Alkoxy, C1~C 30 alkylthio, C1~C 30 Alkylthio, C2~C 30 ether, at least one substituent substituted C2~C 30 Ether, C1~C 30 esters, C1~C 30 Esters, C2~C 30 sulfide, C2~C 30 Sulfide, C2~C 30 amine or at least one substituent substituted C2~C 30 The amine, the substituent is selected from C1~C 30 Alkyl, C6~C 30 Aromatic groups, C6~C 30 Heteroaryl, C1~C 30 Alkoxy, C1~C 30 Alkylthio, C2~C 30 Ether, C1~C 30 Esters, C2~C 30 Sulfide, C2~C 30 at least one of amine, hydroxyl, carboxyl, aldehyde, carbonyl, amino, cyano, thiol and thiocarbonyl; or R2 and R3 and the N atom to which they are connected together form a 3-30 membered heterocyclic ring.

[0054] The present invention first uses a hydrophilic macromolecular chain transfer agent having a structure of formula (II), a (meth)acrylate monomer containing a tertiary amine having a structure of formula (III), and a (meth)acrylate monomer having a structure of formula (IV) as raw materials, and obtains a polymer having a structure of formula (V) by a reversible addition-fragmentation chain transfer polymerization (RAFT) reaction under the action of an initiator; and then the obtained polymer is subjected to a functionalization reaction with a polypeptide to obtain a polymer-membrane-penetrating peptide conjugate having a structure of formula (I).

[0055] In the present invention, the hydrophilic macromolecular chain transfer agent having the structure of formula (II) provides easily breakable chemical bonds, so that an exchange reaction occurs between the polymerized monomer and the chain transfer agent during the reaction, thereby interrupting or extending the polymer chain, thereby achieving regulation of the polymer molecular chain and molecular weight distribution.

[0056] In some specific implementations, the hydrophilic macromolecular chain transfer agent having the structure of formula (II) is selected from the following structures:

[0057] , ;

[0058] Wherein, k is selected from an integer of 0 to 20, preferably an integer of 1 to 15, and more preferably an integer of 1 to 11; n is selected from an integer of 2 to 500, preferably an integer of 30 to 300, and more preferably an integer of 50 to 200.

[0059] In some specific implementations, the hydrophilic macromolecular chain transfer agent having the structure of formula (II) is synthesized by the following method:

[0060] A carboxylic acid chain transfer agent, monomethyl ether polyethylene glycol, 4-dimethylaminopyridine (DMAP) and N,N'-diisopropylcarbodiimide (DIC) react in a solvent to obtain a hydrophilic macromolecular chain transfer agent having a structure of formula (II).

[0061] In some specific implementations, the solvent is selected from dichloromethane (DCM), preferably anhydrous DCM. In some specific implementations, the carboxylic acid chain transfer agent is selected from 4-cyano-4-(dodecylsulfanylthiocarbonyl)sulfanylpentanoic acid (CDTPA), 4-cyano-4-(n-hexylsulfanylthiocarbonyl)sulfanylpentanoic acid (CHTPA), 4-cyano-4-(ethylsulfanylthiocarbonyl)sulfanylpentanoic acid (CETPA) or 4-cyano-4-(phenylcarbonylthiothiothio)pentanoic acid (CPTPA). In some specific implementations, the reaction temperature is 0-50°C, preferably 10-40°C, and more preferably 20-30°C. In some specific implementations, the reaction time is 6-72 h, preferably 15-60 h, and more preferably 24-48 h. In some specific implementations, the molar ratio of the monomethyl ether polyethylene glycol to the carboxylic acid chain transfer agent is 1:2-5, preferably 1:3-4. In some specific implementations, the molar ratio of the monomethyl ether polyethylene glycol to DMAP is 1:0.1-0.5, preferably 1:0.2-0.4, more preferably 1:0.2-0.3. In some specific implementations, the molar ratio of the monomethyl ether polyethylene glycol to DIC is 1:3-8, preferably 1:4-7, more preferably 1:4-6.

[0062] After obtaining the compound having the structure of formula (II), it is preferably purified to obtain a pure compound. In some specific implementations, the purification includes precipitating the reaction product through ice anhydrous ether, and obtaining a crude product by suction filtration, and then dissolving the crude product with deionized water and transferring it to a dialysis bag (3500 MWCO) for dialysis for 72 hours, and obtaining a pure compound having the structure of formula (II) after freeze-drying. The present invention has no particular limitation on the purification method.

[0063] In the present invention, the monomer having the structure of formula (III) provides a hydrophobic group containing a tertiary amine structure for the polymer main chain, which promotes the polymer to pass through the lipid membrane through the hydrophobic effect, thereby enhancing the transmembrane delivery ability.

[0064] In some specific implementations, the monomer having the structure of formula (III) is synthesized by the following method:

[0065] The tertiary amine compound, triethylamine (TEA) and the inhibitor are dissolved in DCM, cooled to 0°C in an ice bath, and the DCM solution of the acyl chloride compound is slowly added dropwise, and then the mixed solution is transferred to room temperature and stirred for 6 to 12 hours. After the reaction is completed, the insoluble impurities are removed to obtain a filtrate, which is purified to obtain a monomer having a structure of formula (III). Specifically, the monomer having a structure of formula (III) can be synthesized according to the route shown in the following scheme:

[0066] (III);

[0067] The selection ranges of R1, R2, R3 and z are the same as those described above, and the present invention will not be repeated here.

[0068] In some specific implementations, the DCM is preferably dry DCM. In some specific implementations, the tertiary amine compound is selected from N-(2-hydroxyethyl) hexamethylenediamine. In some specific implementations, the inhibitor is selected from hydroquinone. In some specific implementations, the acyl chloride compound is selected from acryloyl chloride or methacryloyl chloride. In some specific implementations, the purification includes concentrating the filtrate and purifying it by column chromatography or vacuum distillation, preferably vacuum distillation purification. The present invention has no particular restrictions on the purification method.

[0069] In some specific implementations, the molar ratio of the tertiary amine compound to the acyl chloride compound is 1:1-2, preferably 1:1-1.5, and more preferably 1:1-1.1. In some specific implementations, the molar ratio of the tertiary amine compound to TEA is 1:2-5, preferably 1:2-4, and more preferably 1:2-3. In some specific implementations, the molar ratio of the tertiary amine compound to the inhibitor is 1:0.005-0.02, preferably 1:0.008-0.02, and more preferably 1:0.01-0.02.

[0070] In the present invention, the monomer having the structure of formula (IV) provides a reactive group for the functionalization reaction between the polymer and the polypeptide, and the R5 substituent contained therein can undergo an exchange or coupling reaction with the thiol in the polypeptide, thereby introducing the polypeptide structure into the polymer main chain to obtain a polymer-membrane-penetrating peptide conjugate.

[0071] In some specific implementations, the monomer having the structure of formula (IV) is synthesized by the following method:

[0072] The alcohol compound, TEA and inhibitor are dissolved in DCM, cooled to 0°C in an ice bath, and the DCM solution of the acyl chloride compound is slowly added dropwise, and then the mixed solution is transferred to room temperature and stirred for 6 to 12 hours. After the reaction is completed, the insoluble impurities are removed and purified to obtain a monomer having a structure of formula (IV). Specifically, the monomer having a structure of formula (IV) can be synthesized according to the route shown in the following scheme:

[0073] (IV),

[0074] The selection ranges of R4 and R5 are the same as those described above, and the present invention will not be repeated here.

[0075] In some specific implementations, the DCM is preferably dry DCM. In some specific implementations, the alcohol compound is selected from 2-(2-pyridyldithio)ethanol. In some specific implementations, the inhibitor is selected from hydroquinone. In some specific implementations, the acyl chloride compound is selected from acryloyl chloride or methacryloyl chloride. In some specific implementations, the purification includes concentrating the filtrate and purifying it by column chromatography or vacuum distillation, preferably column chromatography purification. The present invention has no particular restrictions on the purification method.

[0076] In some specific implementations, the molar ratio of the alcohol compound to the acyl chloride compound is 1:1-2, preferably 1:1-1.5, and more preferably 1:1-1.1. In some specific implementations, the molar ratio of the alcohol compound to TEA is 1:2-5, preferably 1:2-4, and more preferably 1:2-3. In some specific implementations, the molar ratio of the alcohol compound to the inhibitor is 1:0.005-0.02, preferably 1:0.008-0.02, and more preferably 1:0.01-0.02.

[0077] The present invention firstly uses a hydrophilic macromolecular chain transfer agent having a structure of formula (II), a (meth)acrylate monomer containing a tertiary amine having a structure of formula (III), and a (meth)acrylate monomer having a structure of formula (IV) as raw materials, and dissolves them in a solvent with an initiator, deoxygenates the reaction system, and causes a RAFT polymerization reaction under sealed conditions. After the reaction is completed, the reaction is quenched to obtain a polymer having a structure of formula (V).

[0078] In some specific implementations, the solvent of the RAFT polymerization reaction is one or more of dioxane, N,N-dimethylacetamide and N,N-dimethylformamide, preferably N,N-dimethylacetamide or N,N-dimethylformamide, more preferably N,N-dimethylacetamide. In some specific implementations, the initiator is selected from azobisisobutyronitrile (AIBN) or azobiscyanovaleric acid (V-501), preferably AIBN, more preferably AIBN after recrystallization. In some specific implementations, the deoxygenation operation includes deoxygenation using a high-purity inert gas, preferably nitrogen or argon, more preferably argon. The present invention has no special restrictions on the deoxygenation method. In some specific implementations, the temperature of the RAFT polymerization reaction is 35~70°C, preferably 40~70°C, more preferably 45~70°C. In some specific implementations, the time of the RAFT polymerization reaction is 12~48h, preferably 15~40h, more preferably 20~30h. In some specific implementations, the quenching reaction is to place the reaction system directly under low temperature after the reaction is completed. In some specific implementations, the low temperature is -10 to -50°C, preferably -10 to -40°C, and more preferably -10 to -30°C.

[0079] The present invention preferably performs a purification operation on the polymer having the structure of formula (V) to obtain a pure polymer. In some specific implementations, the purification includes placing the reaction solution in a dialysis bag, dialyzing with methanol and deionized water, and freeze-drying after the dialysis to obtain a pure polymer. In some specific implementations, the molecular weight cutoff of the dialysis bag is 2500~4000MWCO, preferably 2800~3800MWCO, and more preferably 3200~3600MWCO. In some specific implementations, the dialysis time is 48~96h, preferably 60~84h, and more preferably 65~80h. The present invention has no special restrictions on the specific method of purification.

[0080] In some specific implementations, the polymer having the structure of formula (V) can also be produced by atom transfer radical polymerization (ATRP), which will not be elaborated herein.

[0081] After obtaining the polymer having the structure of formula (V), it is subjected to a functionalization reaction with a polypeptide having the structure of formula (VI) and a buffer to obtain a polymer-membrane-penetrating peptide conjugate having the structure of formula (I).

[0082] In some specific implementations, the functionalization reaction refers to an exchange or coupling reaction between the R5 substituent in the polymer having the structure of formula (V) and the thiol group in the polypeptide having the structure of formula (VI).

[0083] In some specific implementations, the buffer is sodium dihydrogen phosphate, and the solvent is at least one of methanol and water, preferably a mixed solution of methanol and water.

[0084] In some specific implementations, the molar ratio of the polymer having the structure of formula (V) to the polypeptide having the structure of formula (VI) is 1:0.8-2, preferably 1:1-1.8, and more preferably 1:1.1-1.7.

[0085] After obtaining the polymer-membrane-penetrating peptide conjugate having the structure of formula (I), the present invention preferably purifies it to obtain a pure polymer-membrane-penetrating peptide conjugate. In some specific implementations, the purification includes placing the reaction solution in a dialysis bag, dialyzing with deionized water, and freeze-drying after the dialysis to obtain a pure polymer-membrane-penetrating peptide conjugate. In some specific implementations, the molecular weight cutoff of the dialysis bag is 2500~4000MWCO, preferably 2800~3800MWCO, and more preferably 3200~3600MWCO. In some specific implementations, the dialysis time is 48~96h, preferably 60~84h, and more preferably 65~80h. The present invention has no particular restrictions on the specific method of purification.

[0086] In some specific implementations, the polymer-membrane-penetrating peptide conjugate having the structure of formula (I) can also be obtained by a click reaction of a polymer having the structure of formula (V) and a polypeptide having the structure of formula (VI), which will not be described in detail in the present invention.

[0087] The present invention provides a nanoparticle, comprising the polymer-membrane-penetrating peptide conjugate having the structure of formula (I). In some specific implementations, the particle size of the nanoparticle is 20-100 nm.

[0088] The present invention provides a nano adjuvant, comprising the polymer-membrane-penetrating peptide conjugate or nanoparticle having the structure of formula (I).

[0089] The present invention provides a method for preparing a nano adjuvant, comprising:

[0090] The polymer-membrane-penetrating peptide conjugate is dissolved in a sodium dihydrogen phosphate solution to adjust the pH value of the solution to 5.4, and then the sodium dihydrogen phosphate solution is added to adjust the pH value to 7.4 to obtain a nano adjuvant.

[0091] In some specific implementations, the concentration of the sodium dihydrogen phosphate solution is 0.1-0.5M, preferably 0.15-0.3M, and more preferably 0.15-0.25M.

[0092] In some specific implementations, the nanoadjuvant can also be prepared by a nanoprecipitation method, as follows:

[0093] The polymer-membrane-penetrating peptide conjugate is dissolved in N,N-dimethylformamide to obtain a solution with a concentration of 10-150 mg / mL, preferably 20-130 mg / mL, and more preferably 20-80 mg / mL. Deionized water is added under stirring, and the N,N-dimethylformamide is dialyzed in deionized water to obtain a nano adjuvant.

[0094] The present invention provides a nano vaccine, comprising the above-mentioned nano adjuvant, antigen and an adjuvant acceptable in the vaccine. In some specific implementations, the antigen includes a model antigen, a tumor-associated antigen, a viral antigen, a bacterial antigen, a malarial antigen, and the genetic material of the antigen such as RNA and DNA. In some specific implementations, the nano vaccine provided by the present invention can prevent or treat different disease models according to the different co-mixed antigens.

[0095] The present invention provides a polymer-membrane-penetrating peptide conjugate having a structure of formula (I), which is protonated in the acidic environment of the endosome of an antigen-presenting cell to expose the polypeptide structure, and destroys the endosomal membrane by the polypeptide, thereby achieving efficient endosomal escape and cytoplasmic delivery of antigens, activating antigen-presenting cells, inducing Th1-type immune responses, and enhancing antigen cross-presentation and antigen-specific immune responses. BRIEF DESCRIPTION OF THE DRAWINGS

[0096] Figure 1 The structure and hydrogen nuclear magnetic resonance spectrum of the polymer P (N7-PDSEMA) provided by the present invention;

[0097] Figure 2 The reaction between the polymer and the polypeptide provided by the present invention;

[0098] Figure 3 The average particle size of nanoparticles formed by the polymer-membrane-penetrating peptide conjugate provided by the present invention;

[0099] Figure 4 The experimental results of specific immune response in mice after injection of the nano vaccine provided by the present invention;

[0100] Figure 5 It is the proportion of antigen-specific T cells in mice after injection of the nano vaccine provided by the present invention. DETAILED DESCRIPTION

[0101] It should be understood that the expression "one or more of..." includes each of the items recited after the expression individually and in various different combinations of two or more of the recited items, unless otherwise understood from the context and usage. The expression "and / or" in combination with three or more recited items should be understood to have the same meaning, unless otherwise understood from the context.

[0102] The use of the terms "comprising", "having" or "containing", including their grammatical synonyms, should generally be understood as open and non-restrictive, for example not excluding other unrecited elements or steps, unless otherwise specifically stated or otherwise understood from the context.

[0103] It should be understood that the order of steps or the order in which certain actions are performed is not important as long as the present invention remains operable. In addition, two or more steps or actions may be performed simultaneously.

[0104] The use of any and all examples or exemplary language, such as "for example" or "including", herein is intended only to better illustrate the invention and does not limit the scope of the invention unless otherwise claimed. No language in this specification should be construed as indicating that any non-claimed element is essential to the practice of the invention.

[0105] In addition, the numerical ranges and parameters used to define the present invention are approximate values, and the relevant values ​​in the specific embodiments have been presented as accurately as possible. However, any numerical value inherently inevitably contains standard deviations due to individual test methods. Therefore, unless otherwise expressly stated, it should be understood that all ranges, quantities, values ​​and percentages used in this disclosure are modified by "about". Here, "about" generally means that the actual value is within plus or minus 10%, 5%, 1% or 0.5% of a specific value or range.

[0106] The present invention is further described below in conjunction with the following embodiments. The protection scope of the present invention is not limited by the following embodiments.

[0107] Example 1 Preparation of polymer P(N7-PDSEMA)

[0108] Weigh poly(ethylene glycol) monomethyl ether (4-cyano-4-pentanoic acid dodecyl trithiocarbonate) (0.459 g, 85.0 μmol), monomer 2-(azepan-1-yl) ethyl methacrylate (1.883 g, 8924.2 μmol), monomer pyridyl disulfide ethyl methacrylate (0.1402 g, 549.8 μmol), initiator AIBN (6.25 mg, 38.1 μmol) and add them to a single-necked flask with a volume of 5 mL, and add 5 mL of N,N-dimethylacetamide. Seal the single-necked flask with a soft rubber stopper, then insert two needles, one end of which is below the liquid surface and the other end is above the liquid surface. Blow argon gas below the liquid surface for 30 minutes, and then seal the bottle mouth with a sealing film. Place the flask in an oil bath at 70 °C for reaction for 24 h. After the reaction is completed, place the flask directly in a -20 °C refrigerator to quench the reaction. The reaction solution was then directly transferred to a dialysis bag (3500 MWCO) and dialyzed with deionized water for 72 h. After the dialysis, the product was freeze-dried to obtain the polymer P (N7-PDSEMA), a pure light yellow powder (2.001 g, 80.6%). Figure 1 The spectrum shown is the hydrogen nuclear magnetic resonance spectrum of the polymer P (N7-PDSEMA); the peaks labeled k and l in the structure shown are the hydrogen area peaks corresponding to the polymerization units that can react with thiols, and the peaks labeled a~j correspond to the hydrogen areas of the molecular chains and other polymerization units.

[0109]

[0110] P(N7-PDSEMA)

[0111] Example 2

[0112] The polymer P (N7-PDSEMA) (20.00 mg, 0.889 μmol) of Example 1 was weighed into a 4 mL small glass bottle, and 1 mL of 0.2M sodium dihydrogen phosphate solution was added to dissolve. The polypeptide FL-20 (GIGAILKVLATGLPTLISWIC) (2.85 mg, 1.333 μmol) was weighed and added to the above glass bottle, and then 1 mL of 0.2M sodium dihydrogen phosphate solution and 0.5 mL of methanol were added, and the reaction was carried out at room temperature for one day. The reaction solution was then directly transferred to a dialysis bag (3500 MWCO) and dialyzed with deionized water for 72 hours. After the dialysis, the product was lyophilized to obtain a polymer-membrane-penetrating peptide conjugate P (N7-FL-20), a pure light yellow powder (20.25 mg, 88.6%).

[0113] Example 3

[0114] The polymer P (N7-PDSEMA) (20.00 mg, 0.889 μmol) of Example 1 was weighed into a 4 mL small glass bottle, and 1 mL of 0.2 M sodium dihydrogen phosphate solution was added to dissolve. The polypeptide MPG (GALFLGFLGAAGSTMGAWSQPKKKRKVC) (3.88 mg, 1.333 μmol) was weighed and added to the above glass bottle, and then 1 mL of 0.2 M sodium dihydrogen phosphate solution and 0.5 mL of methanol were added, and the reaction was carried out at room temperature for one day. The reaction solution was then directly transferred to a dialysis bag (3500 MWCO) and dialyzed with deionized water for 72 hours. After the dialysis, the product was lyophilized to obtain a polymer-membrane-penetrating peptide conjugate P (N7-MPG), a pure light yellow powder (19.98 mg, 83.7%).

[0115] Example 4

[0116] The polymer P (N7-PDSEMA) (20.00 mg, 0.889 μmol) of Example 1 was weighed into a 4 mL small glass bottle, and 1 mL of 0.2M sodium dihydrogen phosphate solution was added to dissolve. The polypeptide CMA-2 (GIGAVLKVLTTGLPALISWIHHHHEEC) (3.91 mg, 1.333 μmol) was weighed and added to the above glass bottle, and 1 mL of 0.2M sodium dihydrogen phosphate solution was added to react at room temperature for one day. The reaction solution was then directly transferred to a dialysis bag (3500 MWCO) and dialyzed with deionized water for 72 hours. After the dialysis, the product was lyophilized to obtain a polymer-membrane-penetrating peptide conjugate P (N7-CMA-2), a pure light yellow powder (20.36 mg, 85.2%).

[0117] Example 5

[0118] The polymer P (N7-PDSEMA) (20.00 mg, 0.889 μmol) of Example 1 was weighed into a 4 mL small glass bottle, and 1 mL of 0.2M sodium dihydrogen phosphate solution was added to dissolve. The polypeptide TAT (CYGRKKRRQRRR) (2.22 mg, 1.333 μmol) was weighed and added to the above glass bottle, and then 1 mL of 0.2M sodium dihydrogen phosphate solution was added to react at room temperature for one day. The reaction solution was then directly transferred to a dialysis bag (3500 MWCO) and dialyzed with deionized water for 72 hours. After the dialysis, the product was lyophilized to obtain a polymer-membrane-penetrating peptide conjugate P (N7-TAT), a pure light yellow powder (20.08 mg, 90.4%).

[0119] Example 6

[0120] The polymer P (N7-PDSEMA) (20.00 mg, 0.889 μmol) of Example 1 was weighed into a 4 mL small glass bottle and dissolved by adding 1 mL of 0.2M sodium dihydrogen phosphate solution. The polypeptide Arg8 (RRRRRRRRC) (1.83 mg, 1.333 μmol) was weighed and added to the above glass bottle, and then 1 mL of 0.2M sodium dihydrogen phosphate solution was added to react at room temperature for one day. The reaction solution was then directly transferred to a dialysis bag (3500 MWCO) and dialyzed with deionized water for 72 hours. After the dialysis, the product was lyophilized to obtain a polymer-membrane-penetrating peptide conjugate P (N7-Arg8), a pure light yellow powder (19.88 mg, 91.1%).

[0121] Example 7

[0122] The polymer P (N7-PDSEMA) (20.00 mg, 0.889 μmol) of Example 1 was weighed into a 4 mL small glass bottle, and 1 mL of 0.2M sodium dihydrogen phosphate solution was added to dissolve. The polypeptide Penetrain (RQIKIWFQNRRMKWKKC) (3.13 mg, 1.333 μmol) was weighed and added to the above glass bottle, and then 1 mL of 0.2M sodium dihydrogen phosphate solution was added to react at room temperature for one day. The reaction solution was then directly transferred to a dialysis bag (3500 MWCO) and dialyzed with deionized water for 72 hours. After the dialysis, the product was lyophilized to obtain a polymer-penetrating peptide conjugate P (N7-Penetrain), a pure light yellow powder (20.02 mg, 86.6%).

[0123] Example 8

[0124] The polymer P (N7-PDSEMA) (20.00 mg, 0.889 μmol) of Example 1 was weighed into a 4 mL small glass bottle, and 1 mL of 0.2M sodium dihydrogen phosphate solution was added to dissolve. The polypeptide Melittin (GIGAVLKVLTTGLPALISWIKRKRQQC) (3.93 mg, 1.333 μmol) was weighed and added to the above glass bottle, and 1 mL of 0.2M sodium dihydrogen phosphate solution was added to react at room temperature for one day. The reaction solution was then directly transferred to a dialysis bag (3500 MWCO) and dialyzed with deionized water for 72 hours. After the dialysis, the product was lyophilized to obtain a polymer-membrane-penetrating peptide conjugate P (N7-Melittin), a pure light yellow powder (19.88 mg, 83.1%).

[0125] Example 9

[0126] Take 1 mg of the polymer or polymer-penetrating peptide conjugate of Examples 1 to 8, add 200 μL of 0.2 M sodium dihydrogen phosphate solution to dissolve, then add 200 μL of 20 mM dithiothreitol (DTT) aqueous solution to reduce, and react at 37°C for 2 h. Each unreacted PDSEMA group will release a pyridine-2-thiol molecule, and then use a UV-visible spectrophotometer to detect the absorption value at 343 nm before and after the reduction reaction. Figure 2 (c) The formula is used to calculate the percentage of PDSEMA reaction. The efficiency of PDSEMA reaction is the grafting efficiency of peptide. Figure 2 As shown, Figure 2 (a) shows the absorbance of the polymer solution at 343 nm before the reduction reaction. Figure 2 (b) shows the absorbance of the polymer solution at 343 nm after the reduction reaction. Figure 2 (c) shows the formula for calculating the percentage of reactive PDSEMA, Figure 2 (d) Shown is the percentage of reacted PDSEMA in each polymer.

[0127] Depend on Figure 2 It can be seen that except for the grafting rate of P (N7-FL-20) polypeptide of 76.8%, the grafting rates of other polymer-membrane-penetrating peptide conjugates were all 100%.

[0128] Comparative Example 1

[0129] Weigh poly(ethylene glycol) monomethyl ether (4-cyano-4-pentanoic acid dodecyl trithiocarbonate) (0.100 g, 18.5 μmol), monomer 2-(azepan-1-yl) ethyl methacrylate (0.410 g, 1943.1 μmol), initiator AIBN (1.35 mg, 8.23 ​​μmol) into a 5 mL single-necked flask, and add 2 mL of N,N-dimethylacetamide. Seal the single-necked flask with a soft rubber stopper, and then insert two needles, one end of which is below the liquid surface and the other end is above the liquid surface. Blow argon gas below the liquid surface for 30 minutes, and then seal the bottle mouth with a sealing film. Place the flask in an oil bath at 70 °C for 24 h. After the reaction is completed, place the flask directly in a -20 °C refrigerator to quench the reaction. The reaction solution was then directly transferred to a dialysis bag (3500 MWCO) and dialyzed with deionized water for 72 h. After the dialysis, the product was freeze-dried to obtain polymer P (N7), a pure light yellow powder (0.432 g, 84.8%).

[0130] Example 10

[0131] Weigh 10 mg of each of the polymer-penetrating peptide conjugates described in Examples 2 to 8 and the polymer described in Comparative Example 1, add them to a clean glass vial, add 4 mL of 0.2 M sodium dihydrogen phosphate solution to dissolve, and prepare a 2.5 mg / mL solution. Take 40 μL of the solution, add 160 μL of 0.2 M sodium dihydrogen phosphate under ultrasonic conditions, and adjust the pH value to around 7.4 to form nanoparticles. The average particle size of the nanoparticles is as follows: Figure 3 shown.

[0132] Embodiment 11

[0133] Weigh the polymer-penetrating peptide conjugates described in Examples 2 to 8 and the polymer described in Comparative Example 1 in equal amounts and add them to a clean glass vial. Taking Comparative Example 1 as an example, 10 mg of polymer was dissolved in 4 mL of 0.2 M sodium dihydrogen phosphate solution to prepare a 2.5 mg / mL polymer solution. Then take 400 μL of the polymer solution, add 20 μL of an aqueous solution of the antigen OVA (10 mg / mL), and then add 160 μL of 0.2 M sodium dihydrogen phosphate under ultrasonic conditions to make the pH value reach around 7.4 to obtain a nanovaccine. Take 400 μL of 0.2 M sodium dihydrogen phosphate solution, add 20 μL of an aqueous solution of the antigen OVA (10 mg / mL), and then add 1600 μL of 0.2 M sodium dihydrogen phosphate under ultrasonic conditions to obtain an OVA group nanovaccine; take 400 μL of 0.2 M sodium dihydrogen phosphate solution, add 1600 μL of 0.2 M sodium dihydrogen phosphate under ultrasonic conditions, and record it as the Control group.

[0134] On day 0, mice were subcutaneously injected with 200 μL of the above-mentioned polymer nanovaccine, which was labeled as P (N7-MPG) ~ P (N7-Melittin) and PN7 groups according to different formulations; the OVA group was injected with nanovaccine, labeled as OVA group; and the Control group was injected with solution, labeled as Control group. On day 2, the inguinal lymph nodes of mice were dissected. After mechanical disruption at 37°C, the samples were filtered through a 200-mesh nylon mesh filter to obtain a single-cell suspension, and the cells were counted using a cell counting plate. Then, they were incubated with anti-CD11c, anti-CD86, anti-CD40, anti-MHC-Ⅱ and anti-SIINFKEL-H2Kb antibodies at a concentration of 1:100 for 30 minutes at 4°C to analyze the antigen cross-presentation and activation effects of DCs in the lymph nodes. The samples acquired flow data on a flow cytometer (BD FACSCelesta) and analyzed using FlowJo software. The results are shown in Figure 2. Figure 4 shown.

[0135] On day 0 and day 14, mice were subcutaneously injected with PN7, P(N7-FL-20), P(N7-MPG), P(N7-CMA-2), P(N7-TAT), P(N7-Arg8), P(N7-Penetrain), P(N7-Melittin) group nanovaccine, OVA group nanovaccine and Control group solution. On day 21, mice were dissected to remove spleen. The cells were mechanically crushed at 37°C, and then the samples were filtered through a 200-mesh nylon mesh filter to obtain a single cell suspension, and the cells were counted by a cell counting plate. 2×10 cells were taken from each sample. 6 The cells were plated in a 48-well plate, and then the SIINFEKL peptide was added to the experimental system at a final concentration of 10 μg / mL. After 1 hour, the protein transport inhibitor Brefeldin A was added to a final concentration of 10 μg / mL. After 5 hours, the spleen cells were incubated with the dead and live dye Aqua, anti-CD3, and anti-CD8 for 30 minutes, and then the cells were stained with anti-IFN-γ for 30 minutes according to the intracellular factor staining method to analyze the antigen-specific immune response. In addition, 2×10 6 Cells were plated into 48-well plates, and then 2 mg / mL OVA peptide was added. After 1 hour, 10 μg / mL Brefeldin A, a protein transport inhibitor, was added. After 5 hours, the spleen cells were incubated with the dead and live dye Aqua, anti-CD3, and anti-CD4 for 30 minutes, and then the cells were stained with anti-IFN-γ for 30 minutes according to the intracellular factor staining method to analyze the antigen-specific immune response. The samples were acquired on a flow cytometer (BD FACS Celesta) and analyzed by FlowJo software. The results are shown in Figure 5 shown.

[0136] Figure 4 (a) is the activation ratio of DC cells in the draining lymph nodes of mice after incubation with anti-CD86. Figure 4 (b) is the activation ratio of DC cells in the draining lymph nodes of mice after incubation with anti-CD40. Figure 4 (c) is the activation ratio of DC cells in the draining lymph nodes of mice after incubation with anti-MHC-Ⅱ. Figure 4 (d) The ratio of antigen cross-presentation in DC cells in the draining lymph nodes of mice after incubation with anti-SIINFKEL-H2Kb. Figure 4 (a) ~ Figure 4 (c) shows that the DC cell activation ratio and SIINFKEL-H-2Kb after mice were injected with the nanovaccines described in Examples 2 to 8 and Comparative Example 1 +The antigen cross-presentation ratio was significantly increased compared with the Control group and the OVA group, and compared with the P (N7) group, the antigen cross-presentation ratios of the nanovaccines prepared with the polymer-penetrating peptide conjugate adjuvants in Examples 2 to 8 were increased to varying degrees.

[0137] Figure 5 (a) CD8 in mice after injection of different nanovaccines + Cellular heatmap of antigen-specific T cells, Figure 5 (b) CD8 in mice after injection of different nanovaccines + Antigen-specific T cell ratio chart, Figure 5 (c) CD4 in mice after injection of different nanovaccines + Graph showing the proportion of antigen-specific T cells. Figure 5 It can be seen that compared with the Control group and the OVA group, the proportion of antigen-specific T cells in mice injected with nanovaccines increased significantly, and compared with the P (N7) group, the proportion of antigen-specific T cells in the nanovaccines prepared with polymer-penetrating peptide conjugate adjuvants in Examples 2 to 8 increased significantly. However, nanovaccines prepared with polymer-penetrating peptide conjugate adjuvants such as P (N7-MPG) and P (N7-FL-20) did not significantly increase the proportion of antigen-specific T cells, which may be because some penetrating peptides can cause toxicity in the body, resulting in no increase in the final immune efficacy.

[0138] In summary, the polymer-membrane-penetrating peptide conjugate provided by the present invention can achieve the regulation of the type, quantity and ratio of hydrophilic and hydrophobic groups of the polypeptide in the structure. When used as a nanoadjuvant, it can improve the endosomal escape efficiency of antigen substances in vaccines, effectively enhance the cross-presentation of antigens, induce Th1 type cellular immune response, and increase the proportion of antigen-specific T cells.

Claims

1. A polymer-membrane-penetrating peptide conjugate, characterized in that: Having the structure of formula (I): （I）； wherein n is selected from an integer of 2 to 500; x is selected from an integer of 2 to 300; y is selected from an integer of 1 to 10; and z is selected from an integer of 1 to 20; Peptide is selected from the residue after removing the sulfhydryl group of cysteine ​​from a polypeptide containing cysteine; X is selected from C1~C 30 Alkyl, at least one substituent substituted C1~C 30 Alkyl, C2~C 30 ether, at least one substituent substituted C2~C 30 Ether, C2~C 30 sulfide, C2~C 30 Sulfide, C1~C 30 Alkoxy, at least one substituent substituted C1~C 30 Alkoxy, C1~C 30 alkylthio, C1~C 30 Alkylthio, C6~C 30 aromatic group or at least one substituent substituted C6~C 30 The aromatic group, the substituent is selected from halogen, C1~C 30 Alkyl, C1~C 30 Halogenated alkyl, C6~C 30 Aromatic groups, C6~C 30 Halogenated aromatic groups, C6~C 30 Heteroaryl, C1~C 30 Alkoxy, C1~C 30 Alkylthio, C2~C 30 Ether, C1~C 30 Esters, C2~C 30 Sulfide, C2~C 30 At least one of amine, hydroxyl, carboxyl, aldehyde, carbonyl, amino, cyano, sulfhydryl and thiocarbonyl; and the X is connected to the peptide through an S atom; R1, R4 and R6 are independently selected from hydrogen, C1~C 30 Alkyl, at least one substituent substituted C1~C 30 Alkyl, C3~C 30 cycloalkyl, C3~C 30 Cycloalkyl, C2~C 30 heterocycloalkyl, C2~C 30 Heterocycloalkyl, C6~C 30 aromatic group, at least one substituent substituted with C6~C 30 Aromatic groups, C6~C 30 heteroaryl, C6~C 30 Heteroaryl, C1~C 30 Alkoxy, at least one substituent substituted C1~C 30 Alkoxy, C1~C 30 alkylthio, C1~C 30 Alkylthio, C2~C 30 ether, at least one substituent substituted C2~C 30 Ether, C1~C 30 esters, C1~C 30 Esters, C2~C 30 sulfide, C2~C 30 Sulfide, C2~C 30 amine or at least one substituent substituted C2~C 30 The amine, the substituent is selected from C1~C 30 Alkyl, C6~C 30 Aromatic groups, C6~C 30 Heteroaryl, C1~C 30 Alkoxy, C1~C 30 Alkylthio, C2~C 30 Ether, C1~C 30 Esters, C2~C 30 Sulfide, C2~C 30 At least one of an amine, hydroxyl, carboxyl, aldehyde, carbonyl, amino, cyano, thiol and thiocarbonyl group; R2 and R3 are independently selected from hydrogen, C1~C 30 Alkyl, at least one substituent substituted C1~C 30 Alkyl, C3~C 30 cycloalkyl, C3~C 30 Cycloalkyl, C2~C 30 heterocycloalkyl, C2~C 30 Heterocycloalkyl, C6~C 30 aromatic group, at least one substituent substituted with C6~C 30 Aromatic groups, C6~C 30 heteroaryl, C6~C 30 Heteroaryl, C1~C 30 Alkoxy, at least one substituent substituted C1~C 30 Alkoxy, C1~C 30 alkylthio, C1~C 30 Alkylthio, C2~C 30 ether, at least one substituent substituted C2~C 30 Ether, C1~C 30 esters, C1~C 30 Esters, C2~C 30 sulfide, C2~C 30 Sulfide, C2~C 30 amine or at least one substituent substituted C2~C 30 The amine, the substituent is selected from C1~C 30 Alkyl, C6~C 30 Aromatic groups, C6~C 30 Heteroaryl, C1~C 30 Alkoxy, C1~C 30 Alkylthio, C2~C 30 Ether, C1~C 30 Esters, C2~C 30 Sulfide, C2~C 30 at least one of amine, hydroxyl, carboxyl, aldehyde, carbonyl, amino, cyano, thiol and thiocarbonyl; or R2 and R3 and the N atom to which they are connected together form a 3-30 membered heterocyclic ring.

2. The polymer-membrane-penetrating peptide conjugate according to claim 1, characterized in that The X is selected from C1~C 25 Alkyl, at least one substituent substituted C1~C 25 Alkyl, C2~C 25 ether, at least one substituent substituted C2~C 25 Ether, C2~C 25 sulfide, C2~C 25 Sulfide, C1~C 25 Alkoxy, at least one substituent substituted C1~C 25 Alkoxy, C1~C 25 or at least one substituent substituted with C1~C 25 The alkylthio group, wherein the substituent is selected from halogen, C1~C 25 Alkyl, C1~C 25 Halogenated alkyl, C6~C 25 Aromatic groups, C6~C 25 Halogenated aromatic groups, C2~C 25 Ether, C1~C 25 Esters and C2~C 25 At least one of the sulfides of The R1 and R4 are independently selected from hydrogen, C1~C 25 Alkyl, C3~C 25 Cycloalkyl, C6~C 25 Aromatic or C6~C 25 heteroaryl; The R6 is selected from the following structures: ； R a Selected from C1~C 25 Alkyl, C3~C 25 Cycloalkyl, C1~C 25 Alkoxy, C1~C 25 Alkylthio, C6~C 25 Aromatic or C6~C 25 heteroaryl; The R2 and R3 are independently selected from C1-C5 alkyl or amino-substituted C1-C5 alkyl; or R2, R3 and the N atom to which they are connected together form a ring having the following structure: 、 、 ； Wherein, a, b, c, d and e are independently selected from integers of 1 to 5; R b and R c Independently selected from hydrogen or C1~C5 alkyl; —Y— is selected from —O—, or , R d Selected from hydrogen or C1~C5 alkyl.

3. The polymer-membrane-penetrating peptide conjugate according to claim 2, characterized in that: The X is selected from C1~C 20 Alkyl, at least one substituent substituted C1~C 20 Alkyl, C1~C 20 alkylthio, C1~C 20 Alkylthio, C2~C 20 ether or at least one substituent substituted C2~C 20 The ether, the substituent is selected from halogen and C6~C 20 At least one of the halogenated aromatic groups; The R1 and R4 are independently selected from C1 to C 20 Alkyl, C3~C 20 Cycloalkyl or C6~C 20 Aromatic groups; The R6 is selected from the following structures: ； R a Selected from C1~C 20 The alkyl group; The R2 and R3 and the N atom to which they are commonly connected form a ring having the following structure: ； Wherein, a is selected from an integer of 1 to 5.

4. The polymer-membrane-penetrating peptide conjugate according to claim 3, characterized in that The —X— is selected from the following structures: 、 、 ； Wherein, f, g, h and i are independently selected from integers of 1 to 5; Z is selected from S or O; R e , R f , R g and R h are independently selected from halogen.

5. The polymer-membrane-penetrating peptide conjugate according to claim 1, characterized in that: The polymer-membrane-penetrating peptide conjugate has the following structure: ； Wherein, x is selected from an integer of 20 to 250, and y is selected from an integer of 1 to 8.

6. A method for preparing a polymer-membrane-penetrating peptide conjugate, characterized in that: include: a) a chain transfer agent of formula (II), a monomer of formula (III), a monomer of formula (IV) and an initiator undergo a polymerization reaction to obtain a polymer having a structure of formula (V); b) the polymer having the structure of formula (V) obtained in step a), the polypeptide having the structure of formula (VI) and a buffer are subjected to a functionalization reaction to obtain a polymer-membrane-penetrating peptide conjugate having the structure of formula (I); (II)) (III)) (IV)) (V); (VI); （I）； wherein n is selected from an integer of 2 to 500; x is selected from an integer of 2 to 300; y is selected from an integer of 1 to 10; and z is selected from an integer of 1 to 20; Peptide is selected from the residue after removing the sulfhydryl group of cysteine ​​from a polypeptide containing cysteine; X is selected from C1~C 30 Alkyl, at least one substituent substituted C1~C 30 Alkyl, C2~C 30 ether, at least one substituent substituted C2~C 30 Ether, C2~C 30 sulfide, C2~C 30 Sulfide, C1~C 30 Alkoxy, at least one substituent substituted C1~C 30 Alkoxy, C1~C 30 alkylthio, C1~C 30 Alkylthio, C6~C 30 aromatic group or at least one substituent substituted C6~C 30 The aromatic group, the substituent is selected from halogen, C1~C 30 Alkyl, C6~C 30 Aromatic groups, C6~C 30 Heteroaryl, C1~C 30 Alkoxy, C1~C 30 Alkylthio, C2~C 30 Ether, C1~C 30 Esters, C2~C 30 Sulfide, C2~C 30 At least one of amine, hydroxyl, carboxyl, aldehyde, carbonyl, amino, cyano, sulfhydryl and thiocarbonyl; and the X is connected to the peptide through an S atom; R1, R4 and R6 are independently selected from hydrogen, C1~C 30 Alkyl, at least one substituent substituted C1~C 30 Alkyl, C3~C 30 cycloalkyl, C3~C 30 Cycloalkyl, C2~C 30 heterocycloalkyl, C2~C 30 Heterocycloalkyl, C6~C 30 aromatic group, at least one substituent substituted with C6~C 30 Aromatic groups, C6~C 30 heteroaryl, C6~C 30 Heteroaryl, C1~C 30 Alkoxy, at least one substituent substituted C1~C 30 Alkoxy, C1~C 30 alkylthio, C1~C 30 Alkylthio, C2~C 30 ether, at least one substituent substituted C2~C 30 Ether, C1~C 30 esters, C1~C 30 Esters, C2~C 30 sulfide, C2~C 30 Sulfide, C2~C 30 amine or at least one substituent substituted C2~C 30 The amine, the substituent is selected from C1~C 30 Alkyl, C6~C 30 Aromatic groups, C6~C 30 Heteroaryl, C1~C 30 Alkoxy, C1~C 30 Alkylthio, C2~C 30 Ether, C1~C 30 Esters, C2~C 30 Sulfide, C2~C 30 At least one of an amine, hydroxyl, carboxyl, aldehyde, carbonyl, amino, cyano, thiol and thiocarbonyl group; R5 is selected from C1~C 30 Alkyl, C2~C 30 Alkenyl, C2~C 30 Alkenyl, C2~C 30 Alkynyl, C2~C 30 Alkynyl, C2~C 30 ether, at least one substituent substituted C2~C 30 sulfide, C1~C 30 Alkoxy, at least one substituent substituted C1~C 30 or at least one substituent substituted with C6~C 30 The aromatic group, the substituent is selected from halogen, C1~C 30 Alkyl, C1~C 30 haloalkyl, C2~C 30 Alkenyl, C2~C 30 Halogenated alkenyl, C2~C 30 Alkynyl, C2~C 30 Haloalkynyl, C6~C 30 Aromatic groups, C6~C 30 Halogenated aromatic groups, C6~C 30 Heteroaryl, C1~C 30 Alkoxy, C1~C 30 Alkylthio, C2~C 30 Ether, C1~C 30 Esters, C2~C 30 Sulfide, C2~C 30 At least one of an amine, hydroxyl, carboxyl, aldehyde, carbonyl, amino, cyano, thiol and thiocarbonyl group; R2 and R3 are independently selected from hydrogen, C1~C 30 Alkyl, at least one substituent substituted C1~C 30 Alkyl, C3~C 30 cycloalkyl, C3~C 30 Cycloalkyl, C2~C 30 heterocycloalkyl, C2~C 30 Heterocycloalkyl, C6~C 30 aromatic group, at least one substituent substituted with C6~C 30 Aromatic groups, C6~C 30 heteroaryl, at least one substituent substituted with C6~C 30 Heteroaryl, C1~C 30 Alkoxy, at least one substituent substituted C1~C 30 Alkoxy, C1~C 30 alkylthio, C1~C 30 Alkylthio, C2~C 30 ether, at least one substituent substituted C2~C 30 Ether, C1~C 30 esters, C1~C 30 Esters, C2~C 30 sulfide, C2~C 30 Sulfide, C2~C 30 amine or at least one substituent substituted C2~C 30 The amine, the substituent is selected from C1~C 30 Alkyl, C6~C 30 Aromatic groups, C6~C 30 Heteroaryl, C1~C 30 Alkoxy, C1~C 30 Alkylthio, C2~C 30 Ether, C1~C 30 Esters, C2~C 30 Sulfide, C2~C 30 at least one of amine, hydroxyl, carboxyl, aldehyde, carbonyl, amino, cyano, thiol and thiocarbonyl; or R2 and R3 and the N atom to which they are connected together form a 3-30 membered heterocyclic ring.

7. The preparation method according to claim 6, characterized in that: The polymerization reaction in step a) is carried out under anaerobic conditions at a temperature of 35-70° C. for 12-48 hours; The solvent of the polymerization reaction is selected from one or more of dioxane, N,N-dimethylacetamide and N,N-dimethylformamide, and the mass ratio of the solvent to the chain transfer agent is 10-30:1; The initiator is selected from azobisisobutyronitrile or azobiscyanovaleric acid, and the molar ratio of the initiator to the chain transfer agent is 1:2-5.

8. The preparation method according to claim 6, characterized in that: The buffer in step b) is sodium dihydrogen phosphate; The molar ratio of the polymer having the structure of formula (V) to the polypeptide having the structure of formula (VI) is 1:0.8-2; The functionalization reaction is carried out at a temperature of 10-40° C., for a time of 12-48 hours, and with a solvent of at least one of methanol and water.

9. A nanoparticle, characterized in that: It is formed by the polymer-membrane-penetrating peptide conjugate according to any one of claims 1 to 5 or the polymer-membrane-penetrating peptide conjugate prepared by the preparation method of any one of claims 6 to 8.

10. A nano adjuvant, characterized in that: It includes the polymer-membrane-penetrating peptide conjugate according to any one of claims 1 to 5, the polymer-membrane-penetrating peptide conjugate prepared by the preparation method of any one of claims 6 to 8, or the nanoparticle according to claim 9.

11. A vaccine drug, characterized in that: The invention comprises the nano adjuvant according to claim 11, an antigen and an adjuvant acceptable to the vaccine.

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