Fluorine-containing polymer, nano particle, nano adjuvant, nano vaccine and preparation method thereof

Abstract

The invention discloses a series of fluorine-containing polymers, nano particles, nano adjuvants, nano vaccines and preparation methods thereof, and relates to the field of biomedicine. A fluorine-containing monomer is copolymerized to a polymer main chain to obtain a series of fluorine-containing polymers, and when the fluorine-containing polymers are used as a nano adjuvant, the fluorine-containing polymers can quickly penetrate through a lipid membrane of an endosome by utilizing the hydrophobicity and lipophobicity of the fluorine, so that efficient endosome escape and antigen cytoplasm delivery are realized; and antigen cross presentation and antigen-specific immune response are enhanced. Experimental results show that the nano vaccine with the fluorine-containing polymer as the adjuvant can effectively improve the antigen cross presentation capacity of antigen presenting cells and the proportion of antigen-specific T cells, and enhance the immune efficacy of the vaccine. # imgabs0 # (I)

Description

Technical Field

[0001] The present invention relates to the field of biomedicine, and in particular to fluorine-containing polymers, nanoparticles, nanoadjuvants, nanovaccines and preparation methods thereof. Background Art

[0002] Vaccination is considered to be the most effective and economical public health intervention measure to prevent, resist and control infectious diseases. With the continuous development of modern biotechnology, various types of vaccines are constantly being developed. The strength of the vaccine in the body can be directly reflected by the level of antigen-specific T cells. However, most vaccines have weak immunogenicity and cannot induce a strong immune response. The level of antigen-specific T cells is not ideal.

[0003] Immune adjuvants are various components that enhance the immunogenicity of vaccines when used in combination with antigen vaccines. They can enhance the intensity, breadth and durability of the immune response and improve the efficacy of vaccines. The research demand is particularly urgent for antigen vaccines with weak immunogenicity. Summary of the invention

[0004] In view of this, the present invention provides fluorine-containing polymers, nanoparticles, nanoadjuvants, nanovaccines and preparation methods thereof. The fluorine-containing polymers provided by the present invention can be used as immune adjuvants of antigen vaccines to enhance the immune response in vivo and increase the proportion of antigen-specific T cells.

[0005] Specifically, the present invention provides a fluorine-containing polymer having a structure of formula (I):

[0006] (I);

[0007] wherein n is selected from an integer of 2 to 500, preferably an integer of 30 to 300, more preferably an integer of 50 to 200; x and y are independently selected from integers of 2 to 300, x is preferably an integer of 20 to 250, more preferably an integer of 40 to 200, y is preferably an integer of 4 to 100, more preferably an integer of 5 to 50; 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] 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~C30 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, C1~C 30 ether, at least one substituent substituted C1~C 30 Ether, C1~C 30 esters, C1~C 30 Esters, C1~C 30 sulfide, at least one substituent substituted with C1~C 30 Sulfide, C1~C 30 amine or at least one substituent substituted C1~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, C1~C 30 Ether, C1~C 30 Esters, C1~C 30 Sulfide, C1~C 30 At least one of an amine, hydroxyl, carboxyl, aldehyde, carbonyl, amino, cyano, thiol and thiocarbonyl group;

[0009] 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 30alkylthio, C1~C 30 Alkylthio, C1~C 30 ether, at least one substituent substituted C1~C 30 Ether, C1~C 30 esters, C1~C 30 Esters, C1~C 30 sulfide, at least one substituent substituted with C1~C 30 Sulfide, C1~C 30 amine or at least one substituent substituted C1~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, C1~C 30 Ether, C1~C 30 Esters, C1~C 30 Sulfide, C1~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;

[0010] R5 is selected from at least one fluorine-substituted C1~C 30 Alkyl, at least one fluorine-substituted C2~C 30 alkenyl, at least one fluorine-substituted C2~C 30 alkynyl, at least one fluorine-substituted C3~C 30 cycloalkyl, at least one fluorine-substituted C3~C 30 cycloalkenyl, at least one fluorine-substituted C3~C 30 cycloalkynyl, at least one fluorine-substituted C6~C 30 aromatic group, or at least one fluorine-substituted C6~C 30 The heteroaryl group.

[0011] The present invention uses poly (meth) acrylate as the skeleton, and copolymerizes the monomer containing fluorine element to the polymer body. Such fluorine-containing monomer has both hydrophobicity and lipophobicity. The hydrophobicity can make the polymer quickly fuse with the cell lipid membrane, and the lipophobicity can make the polymer diffuse through the lipid membrane, thereby improving the ability of the polymer to mediate the transmembrane delivery of cargo to the cytoplasm. When such fluorine-containing polymer is used as a nanoadjuvant, it will be protonated in the acidic environment of the antigen-presenting cell endosome, exposing the cationic structure and fluorine-containing structure, and using the hydrophobicity and lipophobicity of the fluorine element to destroy the endosomal membrane, achieve efficient endosomal escape and cytoplasmic delivery of antigens, and enhance antigen cross-presentation and antigen-specific immune response.

[0012] In some specific implementations, R1 and R4 are independently selected from hydrogen, C1~C 30 Alkyl, C3~C 30 Cycloalkyl, C6~C 30 Aromatic or C6~C 30 The heteroaryl group is preferably C1~C 30 The alkyl group is more preferably a methyl group.

[0013] 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 nitrogen atom where they are located together form a ring having the following structure:

[0014] , , ;

[0015] Wherein, a, b, c, d and e are independently selected from integers of 1 to 5;

[0016] R a and R b Independently selected from hydrogen or C1~C5 alkyl;

[0017] —Y— is selected from —O—, or ,

[0018] R c Selected from hydrogen or C1~C5 alkyl.

[0019] In some specific implementations, the R2 and R3 preferably form a ring with the nitrogen atom where they are located together with the nitrogen atom having the following structure:

[0020] ;

[0021] Wherein, a is selected from an integer of 1 to 5.

[0022] In some specific implementations, the R2 and R3 are more preferably formed together with the nitrogen atom where they are located to form a ring having the following structure:

[0023] .

[0024] In some specific implementations, the R5 is selected from at least one fluorine-substituted C1~C 30 Alkyl, at least one fluorine-substituted C3~C 30 cycloalkyl, or at least one fluorine-substituted C6~C 30 An aromatic group; preferably the following structure:

[0025] , , ;

[0026] wherein X is selected from H or F;

[0027] f, h and j are independently selected from integers of 0 to 20;

[0028] g and i are independently selected from integers of 1 to 20;

[0029] More preferably ;

[0030] X is selected from H or F;

[0031] f is an integer selected from 0 to 11;

[0032] g is selected from an integer of 1 to 11.

[0033] In some specific implementations, the R6 is selected from the following structures:

[0034] ;

[0035] R d Selected from C1~C 30 Alkyl, C3~C 30 Cycloalkyl, C1~C 30 Alkoxy, C1~C 30 Alkylthio, C6~C 30 Aromatic or C6~C 30 The heteroaryl group is preferably C1~C 30 Alkylthio or C6~C 30 The alkoxy group is preferably C1~C 30 of alkylthio.

[0036] In some specific implementations, the number average molecular weight of the fluorine-containing polymer having the structure of formula (I) is 1,000 to 200,000, preferably 2,000 to 150,000, and more preferably 3,500 to 100,000.

[0037] The present invention also provides a method for preparing a fluorine-containing polymer, comprising:

[0038] A chain transfer agent having a structure of formula (II), a monomer having a structure of formula (III), a monomer having a structure of formula (IV) and an initiator undergo a polymerization reaction to obtain a fluorine-containing polymer having a structure of formula (I);

[0039] (II); (III); (IV);

[0040] (I);

[0041] wherein n is selected from an integer of 2 to 500; x and y are independently selected from an integer of 2 to 300; z is selected from an integer of 1 to 20;

[0042] 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, C1~C 30 ether, at least one substituent substituted C1~C 30 Ether, C1~C 30 esters, C1~C 30 Esters, C1~C 30 sulfide, at least one substituent substituted with C1~C 30 Sulfide, C1~C 30amine or at least one substituent substituted C1~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, C1~C 30 Ether, C1~C 30 Esters, C1~C 30 Sulfide, C1~C 30 At least one of an amine, hydroxyl, carboxyl, aldehyde, carbonyl, amino, cyano, thiol and thiocarbonyl group;

[0043] 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, C1~C 30 ether, at least one substituent substituted C1~C 30 Ether, C1~C 30 esters, C1~C 30 Esters, C1~C 30 sulfide, at least one substituent substituted with C1~C 30 Sulfide, C1~C 30 amine or at least one substituent substituted C1~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, C1~C30 Ether, C1~C 30 Esters, C1~C 30 Sulfide, C1~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;

[0044] R5 is selected from at least one fluorine-substituted C1~C 30 Alkyl, at least one fluorine-substituted C2~C 30 alkenyl, at least one fluorine-substituted C2~C 30 alkynyl, at least one fluorine-substituted C3~C 30 cycloalkyl, at least one fluorine-substituted C3~C 30 cycloalkenyl, at least one fluorine-substituted C3~C 30 cycloalkynyl, at least one fluorine-substituted C6~C 30 aromatic group, or at least one fluorine-substituted C6~C 30 The heteroaryl group.

[0045] The present invention 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 fluorine-containing (meth)acrylate monomer having a structure of formula (IV) as raw materials, and under the action of an initiator, a reversible addition-fragmentation chain transfer polymerization (RAFT) reaction is performed to obtain a fluorine-containing polymer having a structure of formula (I). Specifically, the fluorine-containing polymer having a structure of formula (I) can be synthesized according to the route shown in the following scheme:

[0046] .

[0047] 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.

[0048] In some specific implementations, the hydrophilic macromolecular chain transfer agent having the structure of formula (II) is selected from the following structures:

[0049] , ;

[0050] 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.

[0051] In some specific implementations, the hydrophilic macromolecular chain transfer agent having the structure of formula (II) is synthesized by the following method:

[0052] 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).

[0053] 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.

[0054] 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.

[0055] In the present invention, the monomer having the structure of formula (III) provides the polymer with a hydrophobic group containing a tertiary amine structure, which enables the polymer to pass through the lipid membrane through the hydrophobic effect, thereby enhancing the transmembrane delivery ability.

[0056] In some specific implementations, the monomer having the structure of formula (III) is synthesized by the following method:

[0057] 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:

[0058] (III);

[0059] The selection ranges of R1, R2 and R3 are the same as those described above, and the present invention will not be repeated here.

[0060] 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.

[0061] 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.

[0062] In the present invention, the monomer having the structure of formula (IV) provides the polymer with a hydrophobic group of a fluorine-containing structure, and such a fluorine-containing structure can easily fuse with membrane lipids through hydrophobic interactions, and its lipophobicity can ensure that the fluorine-containing structure quickly diffuses through the lipid membrane, thereby enhancing its ability to mediate transmembrane delivery of cargo to the cell cytoplasm, achieving efficient endosomal escape and antigen cytoplasmic delivery, and enhancing antigen cross-presentation ability.

[0063] In some specific implementations, the monomer having the structure of formula (IV) is synthesized by the following method:

[0064] The fluorinated 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:

[0065] (IV),

[0066] The selection ranges of R4 and R5 are the same as those described above, and the present invention will not be repeated here.

[0067] In some specific implementations, the DCM is preferably dry DCM. In some specific implementations, the fluorine-containing alcohol compound is selected from 3,3,3-trifluoro-1-propanol. 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.

[0068] In some specific implementations, the molar ratio of the fluorinated 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 fluorinated 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 fluorinated 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.

[0069] The present invention firstly dissolves a hydrophilic macromolecular chain transfer agent having a structure of formula (II), a (meth)acrylate monomer containing a tertiary amine structure having a structure of formula (III), a fluorine-containing (meth)acrylate monomer having a structure of formula (IV), and an initiator in a solvent, 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 fluorine-containing polymer having a structure of formula (I).

[0070] 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.

[0071] After obtaining the fluorinated polymer having the structure of formula (I), the present invention preferably purifies it to obtain a pure fluorinated polymer. In some specific implementations, the purification includes placing the reaction solution in a dialysis bag, dialyzing it with methanol and deionized water, and freeze-drying after the dialysis to obtain a pure fluorinated 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.

[0072] In some specific implementations, the fluorine-containing polymer having the structure of formula (I) can also be obtained by atom transfer radical polymerization (ATRP), which will not be described in detail in the present invention.

[0073] The present invention provides a nanoparticle, comprising the fluorine-containing polymer having the structure of formula (I) above. In some specific implementations, the particle size of the nanoparticle is 20-100 nm.

[0074] The present invention provides a nano adjuvant, comprising the above-mentioned fluorine-containing polymer or nanoparticle having the structure of formula (I).

[0075] The present invention provides a method for preparing a nano adjuvant, comprising:

[0076] The fluorine-containing polymer is dissolved in a sodium dihydrogen phosphate solution to make the pH value of the solution reach 5.4, and then the sodium dihydrogen phosphate solution is added to make the pH value reach 7.4 to obtain a fluorine-containing polymer adjuvant.

[0077] 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.

[0078] In some specific implementations, the nanoadjuvant can also be prepared by a nanoprecipitation method, as follows:

[0079] The fluorine-containing polymer is dissolved in N,N-dimethylformamide to obtain a solution with a concentration of 50-150 mg / mL, preferably 70-130 mg / mL, and more preferably 80-120 mg / mL. Deionized water is added under stirring, and the N,N-dimethylformamide is dialyzed in deionized water to obtain a fluorine-containing polymer adjuvant.

[0080] 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.

[0081] The present invention provides a fluorine-containing polymer having a structure of formula (I), which utilizes the hydrophobicity and lipophobicity of the fluorine-containing group to enhance its ability to deliver substances across biological membranes. The hydrophobic effect can accelerate the contact time between the fluorine-containing polymer and the lipid membrane, promoting its fusion with the lipid membrane, while the lipophobic effect can enable it to quickly pass through the lipid membrane to reach the cell cytoplasm. Experimental results show that the nanovaccine obtained by blending the fluorine-containing polymer provided by the present invention and the antigen can achieve efficient endosomal escape and antigen cytoplasmic delivery in the body, enhance the ability of antigen cross-presentation, and increase the proportion of antigen-specific T cells. BRIEF DESCRIPTION OF THE DRAWINGS

[0082] Figure 1 The structure and hydrogen nuclear magnetic resonance spectrum of the polymers P(N7-3F10)~P(N7-3F40) provided by the present invention;

[0083] Figure 2 The structure and hydrogen nuclear magnetic resonance spectrum of the polymers P(N7-9F10)~P(N7-9F40) provided by the present invention;

[0084] Figure 3 The structure and hydrogen nuclear magnetic resonance spectrum of the polymers P (N7-13F10) ~ P (N7-13F40) provided by the present invention;

[0085] Figure 4 The structure and hydrogen nuclear magnetic resonance spectrum of the polymers P (N7-17F10) ~ P (N7-17F40) provided by the present invention;

[0086] Figure 5 The average particle size of the nanoparticles formed by the polymer provided by the present invention;

[0087] Figure 6 The specific immune response experimental results of mice after injection of the nano vaccine provided by the present invention;

[0088] Figure 7 It is the proportion of antigen-specific T cells in mice after injection of the nano vaccine provided by the present invention. DETAILED DESCRIPTION

[0089] 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.

[0090] 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.

[0091] 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.

[0092] 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.

[0093] 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.

[0094] The present invention is further described below in conjunction with the following examples. The protection scope of the present invention is not limited by the following examples.

[0095] Example 1

[0096] 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), monomer 3,3,3-trifluoropropyl methacrylate (0.0367 g, 201.7 μmol), initiator azobisisobutyronitrile (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. The flask was placed in an oil bath at 70 °C for 24 h. After the reaction, the single-necked flask was directly placed 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 hours. After the dialysis, the product was freeze-dried to obtain polymer P (N7-3F10), a pure light yellow powder (0.481 g, 88.0%), with the structure shown below, where the relationship between the degree of polymerization x and the degree of polymerization y is 70:10. Figure 1 In the H NMR spectrum shown, the spectrum line corresponding to number 1 on the right is the H NMR spectrum of polymer P (N7-3F10); the peaks numbered 1 and 2 in the structure shown are the hydrogen area peaks corresponding to the fluorine-containing polymerization units, and the peaks numbered a~j correspond to the hydrogen areas of the molecular chains and other polymerization units.

[0097]

[0098] P(N7-3F10)

[0099] Example 2

[0100] 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), monomer 3,3,3-trifluoropropyl methacrylate (0.0731 g, 401.7 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 reaction for 24 h. After the reaction is completed, place the single-necked 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 hours. After the dialysis, the product was freeze-dried to obtain polymer P (N7-3F20), a pure light yellow powder (0.504 g, 86.5%), with the structure shown below, where the relationship between the degree of polymerization x and the degree of polymerization y is 70:20. Figure 1 In the H NMR spectrum shown, the spectrum corresponding to number 2 on the right is the H NMR spectrum of polymer P (N7-3F20); the peaks labeled 1 and 2 in the structure shown are the hydrogen area peaks corresponding to the fluorine-containing polymerization units, and the peaks labeled a~j correspond to the hydrogen areas of the molecular chains and other polymerization units.

[0101]

[0102] P(N7-3F20)

[0103] Example 3

[0104] 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), monomer 3,3,3-trifluoropropyl methacrylate (0.1101 g, 605.0 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 single-necked 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 hours. After the dialysis, the product was freeze-dried to obtain polymer P (N7-3F30), a pure light yellow powder (0.531 g, 85.6%), with the structure shown below, where the relationship between the degree of polymerization x and the degree of polymerization y is 70:30. Figure 1 In the H NMR spectrum shown, the spectrum corresponding to number 3 on the right is the H NMR spectrum of polymer P (N7-3F30); the peaks numbered 1 and 2 in the structure shown are the hydrogen area peaks corresponding to the fluorine-containing polymerization units, and the peaks numbered a~j correspond to the hydrogen areas of the molecular chains and other polymerization units.

[0105]

[0106] P(N7-3F30)

[0107] Example 4

[0108] 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), monomer 3,3,3-trifluoropropyl methacrylate (0.1468 g, 806.6 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 reaction for 24 h. After the reaction is completed, place the single-necked 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 hours. After the dialysis, the product was freeze-dried to obtain polymer P (N7-3F40), a pure light yellow powder (0.549 g, 83.6%), with the structure shown below, where the relationship between the degree of polymerization x and the degree of polymerization y is 70:40. Figure 1 In the H NMR spectrum shown, the spectrum corresponding to line number 4 on the right is the H NMR spectrum of polymer P (N7-3F40); the peaks numbered 1 and 2 in the structure shown are the hydrogen area peaks corresponding to the fluorine-containing polymerization units, and the peaks numbered a~j correspond to the hydrogen areas of the molecular chains and other polymerization units.

[0109]

[0110] P(N7-3F40)

[0111] Example 5

[0112] 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), monomer perfluorobutyl ethyl methacrylate (0.0624 g, 187.8 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 reaction for 24 h. After the reaction is completed, place the single-necked 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 hours. After the dialysis, the product was freeze-dried to obtain polymer P (N7-9F10), a pure light yellow powder (0.490 g, 85.6%), with the structure shown below, where the relationship between the degree of polymerization x and the degree of polymerization y is 70:10. Figure 2 In the H NMR spectrum shown, the spectrum line corresponding to number 1 on the right is the H NMR spectrum of polymer P (N7-9F10); the peaks numbered 1 and 2 in the structure shown are the hydrogen area peaks corresponding to the fluorine-containing polymerization units, and the peaks numbered a~j correspond to the hydrogen areas of the molecular chains and other polymerization units.

[0113]

[0114] P(N7-9F10)

[0115] Example 6

[0116] 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), monomer perfluorobutyl ethyl methacrylate (0.1248 g, 375.7 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 single-necked 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 hours. After the dialysis, the product was freeze-dried to obtain polymer P (N7-9F20), a pure light yellow powder (0.519 g, 81.8%), with the structure shown below, where the relationship between the degree of polymerization x and the degree of polymerization y is 70:20. Figure 2 In the H NMR spectrum shown, the spectrum line corresponding to number 2 on the right is the H NMR spectrum of polymer P (N7-9F20); the peaks numbered 1 and 2 in the structure shown are the hydrogen area peaks corresponding to the fluorine-containing polymerization units, and the peaks numbered a~j correspond to the hydrogen areas of the molecular chains and other polymerization units.

[0117]

[0118] P(N7-9F20)

[0119] Example 7

[0120] 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), monomer perfluorobutyl ethyl methacrylate (0.1871 g, 563.2 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 reaction for 24 h. After the reaction is completed, place the single-necked 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 hours. After the dialysis, the product was freeze-dried to obtain polymer P (N7-9F30), a pure light yellow powder (0.583 g, 83.6%), with the structure shown below, where the relationship between the degree of polymerization x and the degree of polymerization y is 70:30. Figure 2 In the H NMR spectrum shown, the spectrum corresponding to number 3 on the right is the H NMR spectrum of polymer P (N7-9F30); the peaks labeled 1 and 2 in the structure shown are the hydrogen area peaks corresponding to the fluorine-containing polymerization units, and the peaks labeled a~j correspond to the hydrogen areas of the molecular chains and other polymerization units.

[0121]

[0122] P(N7-9F30)

[0123] Example 8

[0124] 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), monomer perfluorobutyl ethyl methacrylate (0.2495 g, 751.1 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 single-necked 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 hours. After the dialysis, the product was freeze-dried to obtain polymer P (N7-9F40), a pure light yellow powder (0.629 g, 82.8%), with the structure shown below, where the relationship between the degree of polymerization x and the degree of polymerization y is 70:40. Figure 2 In the H NMR spectrum shown, the spectrum corresponding to number 4 on the right is the H NMR spectrum of polymer P (N7-9F40); the peaks labeled 1 and 2 in the structure shown are the hydrogen area peaks corresponding to the fluorine-containing polymerization units, and the peaks labeled a~j correspond to the hydrogen areas of the molecular chains and other polymerization units.

[0125]

[0126] P(N7-9F40)

[0127] Example 9

[0128] 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), monomer perfluorohexyl ethyl methacrylate (0.0809 g, 187.2 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 single-necked 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 hours. After the dialysis, the product was freeze-dried to obtain polymer P (N7-13F10), a pure light yellow powder (0.510 g, 86.3%), with the structure shown below, where the relationship between the degree of polymerization x and the degree of polymerization y is 70:10. Figure 3 In the H NMR spectrum shown, the spectrum line corresponding to number 1 on the right is the H NMR spectrum of polymer P (N7-13F10); the peaks numbered 1 and 2 in the structure shown are the hydrogen area peaks corresponding to the fluorine-containing polymerization units, and the peaks numbered a~j correspond to the hydrogen areas of the molecular chains and other polymerization units.

[0129]

[0130] P(N7-13F10)

[0131] Example 10

[0132] 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), monomer perfluorohexyl ethyl methacrylate (0.1618 g, 374.4 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 reaction for 24 h. After the reaction is completed, place the single-necked 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 hours. After the dialysis, the product was freeze-dried to obtain polymer P (N7-13F20), a pure light yellow powder (0.555 g, 82.6%), with the structure shown below, where the relationship between the degree of polymerization x and the degree of polymerization y is 70:20. Figure 3 In the H NMR spectrum shown, the spectrum corresponding to number 2 on the right is the H NMR spectrum of polymer P (N7-13F20); the peaks numbered 1 and 2 in the structure shown are the hydrogen area peaks corresponding to the fluorine-containing polymerization units, and the peaks numbered a~j correspond to the hydrogen areas of the molecular chains and other polymerization units.

[0133]

[0134] P(N7-13F20)

[0135] Embodiment 11

[0136] 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), monomer perfluorohexyl ethyl methacrylate (0.2428 g, 561.6 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 single-necked 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 hours. After the dialysis, the product was freeze-dried to obtain polymer P (N7-13F30), a pure light yellow powder (0.629 g, 83.6%), with the structure shown below, where the relationship between the degree of polymerization x and the degree of polymerization y is 70:30. Figure 3 In the H NMR spectrum shown, the spectrum corresponding to number 3 on the right is the H NMR spectrum of polymer P (N7-13F30); the peaks numbered 1 and 2 in the structure shown are the hydrogen area peaks corresponding to the fluorine-containing polymerization units, and the peaks numbered a~j correspond to the hydrogen areas of the molecular chains and other polymerization units.

[0137]

[0138] P(N7-13F30)

[0139] Example 12

[0140] 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), monomer perfluorohexyl ethyl methacrylate (0.3237 g, 749.0 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 single-necked 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 hours. After the dialysis, the product was freeze-dried to obtain polymer P (N7-13F40), a pure light yellow powder (0.674 g, 80.8%), with the structure shown below, where the relationship between the degree of polymerization x and the degree of polymerization y is 70:40. Figure 3 In the H NMR spectrum shown, the spectrum corresponding to line number 4 on the right is the H NMR spectrum of polymer P (N7-13F40); the peaks numbered 1 and 2 in the structure shown are the hydrogen area peaks corresponding to the fluorine-containing polymerization units, and the peaks numbered a~j correspond to the hydrogen areas of the molecular chains and other polymerization units.

[0141]

[0142] P(N7-13F40)

[0143] Embodiment 13

[0144] 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), monomer perfluorooctyl ethyl methacrylate (0.0986 g, 185.3 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 reaction for 24 h. After the reaction is completed, place the single-necked 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 hours. After the dialysis, the product was freeze-dried to obtain polymer P (N7-17F10), a pure light yellow powder (0.527 g, 86.6%), with the structure shown below, where the relationship between the degree of polymerization x and the degree of polymerization y is 70:10. Figure 4 In the H NMR spectrum shown, the spectrum line corresponding to number 1 on the right is the H NMR spectrum of polymer P (N7-17F10); the peaks numbered 1 and 2 in the structure shown are the hydrogen area peaks corresponding to the fluorine-containing polymerization units, and the peaks numbered a~j correspond to the hydrogen areas of the molecular chains and other polymerization units.

[0145]

[0146] P(N7-17F10)

[0147] Embodiment 14

[0148] 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), monomer perfluorooctyl ethyl methacrylate (0.1971 g, 370.4 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 single-necked 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 hours. After the dialysis, the product was freeze-dried to obtain polymer P (N7-17F20), a pure light yellow powder (0.570 g, 80.6%), with the structure shown below, where the relationship between the degree of polymerization x and the degree of polymerization y is 70:20. Figure 4 In the H NMR spectrum shown, the spectrum corresponding to number 2 on the right is the H NMR spectrum of polymer P (N7-17F20); the peaks numbered 1 and 2 in the structure shown are the hydrogen area peaks corresponding to the fluorine-containing polymerization units, and the peaks numbered a~j correspond to the hydrogen areas of the molecular chains and other polymerization units.

[0149]

[0150] P(N7-17F20)

[0151] Embodiment 15

[0152] 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), monomer perfluorooctyl ethyl methacrylate (0.2957 g, 555.6 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 single-necked 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 hours. After the dialysis, the product was freeze-dried to obtain polymer P (N7-17F30), a pure light yellow powder (0.659 g, 81.8%), with the structure shown below, where the relationship between the degree of polymerization x and the degree of polymerization y is 70:30. Figure 4 In the H NMR spectrum shown, the spectrum corresponding to number 3 on the right is the H NMR spectrum of polymer P (N7-17F30); the peaks labeled 1 and 2 in the structure shown are the hydrogen area peaks corresponding to the fluorine-containing polymerization units, and the peaks labeled a~j correspond to the hydrogen areas of the molecular chains and other polymerization units.

[0153]

[0154] P(N7-17F30)

[0155] Example 16

[0156] 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), monomer perfluorooctyl ethyl methacrylate (0.3942 g, 740.7 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 single-necked 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 hours. After the dialysis, the product was freeze-dried to obtain polymer P (N7-17F40), a pure light yellow powder (0.785 g, 86.8%), with the structure shown below, where the relationship between the degree of polymerization x and the degree of polymerization y is 70:40. Figure 4 In the H NMR spectrum shown, the spectrum corresponding to line number 4 on the right is the H NMR spectrum of polymer P (N7-17F40); the peaks numbered 1 and 2 in the structure shown are the hydrogen area peaks corresponding to the fluorine-containing polymerization units, and the peaks numbered a~j correspond to the hydrogen areas of the molecular chains and other polymerization units.

[0157]

[0158] P(N7-17F40)

[0159] Comparative Example 1

[0160] 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 reaction for 24 h. After the reaction is completed, place the single-necked 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 hours. After the dialysis, the product was freeze-dried to obtain polymer PN7, a pure light yellow powder (0.432 g, 84.8%).

[0161] Embodiment 17

[0162] Weigh 10 mg of polymers P(N7-3F10)~P(N7-17F40) and PN7 respectively, 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 polymer solution. Take 40 μL of the polymer solution, add 160 μL of 0.2 M sodium dihydrogen phosphate under ultrasonic conditions, and make the pH value reach about 7.4 to form nanoparticles. The average particle size of the nanoparticles formed by each polymer is as follows: Figure 5 shown.

[0163] Embodiment 18

[0164] Weigh equal amounts of polymers P(N7-3F10)~P(N7-17F40) and PN7 respectively and add them to a clean glass vial. Taking the polymer PN7 described in Comparative Example 1 as an example, 10 mg of the 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 1600 μL of 0.2 M sodium dihydrogen phosphate under ultrasonic conditions to make the pH value reach around 7.4 to obtain a polymer nanovaccine. Take 400 μL of 0.2 M sodium dihydrogen phosphate solution, add 20 μL of antigen OVA aqueous solution (10 mg / mL), and then add 1600 μL of 0.2 M sodium dihydrogen phosphate under ultrasonic conditions to obtain the 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.

[0165] On day 0, mice were subcutaneously injected with 200 μL of the above-mentioned polymer nanovaccine, which was marked as P (N7-3F10) ~ P (N7-17F40) and PN7 groups according to different formulations; the OVA group was injected with nanovaccine, marked as OVA group; and the Control group was injected with solution, marked as Control group. On day 2, the inguinal lymph nodes of mice were dissected and the mass of the lymph nodes was weighed. 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, 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 by FlowJo software. The results are shown in Figure 2. Figure 6 shown.

[0166] On day 0 and day 14, mice in the second group were subcutaneously injected with polymer PN7, P(N7-3F30), P(N7-3F40), P(N7-9F20)~P(N7-9F40), P(N7-13F10), P(N7-13F20), P(N7-17F10) and P(N7-17F20) nanovaccines, OVA group nanovaccines and control group solutions. On day 21, the mice were dissected to remove the spleen, which was 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 using a cell counting plate. 2×10 cells were taken from each sample. 6The cells were plated into 48-well plates, and SIINFEKL peptide was added at a final concentration of 10 μg mL -1 One hour later, protein transport inhibitor Brefeldin A was added to reach 10 μg mL -1 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. The samples were acquired on a flow cytometer (BD FACSCelesta) and analyzed by FlowJo software. The results are shown in Figure 7 shown.

[0167] Figure 6 middle, Figure 6 (a) The mass of draining lymph nodes after mice were injected with different nanovaccines. Figure 6 (b) Antigen cross-presentation ratio of DC cells in draining lymph nodes after mice were injected with different nanovaccines. Figure 6 (c) The activation ratio of DC cells in the draining lymph nodes of mice after injection of different nanovaccines. Figure 6 (a) It can be seen that after mice were injected with nanovaccines formed by polymers P (N7-3F10) ~ P (N7-17F40) and PN7, the mass of draining lymph nodes increased by more than double. The mass of lymph nodes in the control group and OVA group mice was 2.4 mg and 2.7 mg, respectively. After injection of nanovaccines, the lymph nodes of mice increased to between 6.3 and 7.5 mg.

[0168] Depend on Figure 6(b) It can be seen that compared with the Control group and the OVA group, the antigen cross-presentation ratio of mice injected with the nanovaccine formed by the polymer P (N7-3F10) ~ P (N7-17F40) and PN7 increased significantly. And compared with the PN7 group, the antigen cross-presentation ratio of the polymer P (N7-3F10) ~ P (N7-17F40) nanovaccine prepared in Examples 1 to 16 increased to varying degrees, showing an obvious structure-activity relationship. For example, in the polymer P (N7-3F10) ~ P (N7-3F40) nanovaccine, the hydrophobic group is trifluoropropyl, and as the proportion of trifluoropropyl units increases, the antigen cross-presentation ratio increases. In the polymer P (N7-9F10) ~ P (N7-9F40) nanovaccine, the hydrophobic group is perfluorobutylethyl, and as the proportion of perfluorobutylethyl units increases, the antigen cross-presentation ratio first increases and then decreases. When the polymerization ratio is 30, the antigen cross-presentation ratio is the largest. In the polymer P (N7-17F10) ~ P (N7-17F40) nanovaccines, the hydrophobic group is perfluorooctylethyl. As the proportion of perfluorooctylethyl units increases, the antigen cross-presentation ratio gradually decreases.

[0169] Depend on Figure 6 (c) It can be seen that compared with the Control group and the OVA group, the activation ratio of DC cells in mice injected with the nanovaccine formed by the polymer P (N7-3F10) ~ P (N7-17F40) and PN7 increased significantly. After the injection of the P (N7-3F10) ~ P (N7-17F40) nanovaccines prepared in Examples 1 to 16, the activation ratio of DC cells showed an obvious structure-activity relationship. For example, in the polymer P (N7-3F10) ~ P (N7-3F40) nanovaccine, the hydrophobic group is trifluoropropyl, and as the proportion of trifluoropropyl units increases, the activation ratio of DC cells increases. In the polymer P (N7-9F10) ~ P (N7-9F40) nanovaccine, the hydrophobic group is perfluorobutylethyl, and as the proportion of perfluorobutylethyl units increases, the activation of DC cells first increases and then decreases. When the polymerization ratio is 30, the activation ratio of DC cells is the largest. In the polymer P (N7-17F10) ~ P (N7-17F40) nanovaccine, the hydrophobic group is perfluorooctylethyl. As the proportion of perfluorooctylethyl units increases, the activation ratio of DC cells gradually decreases. Compared with the PN7 group, the modification of polymers with fluorinated groups will affect the activation of DC cells. The appropriate fluorinated hydrophobic group and proportion can improve the DC activation effect of nanovaccines.

[0170] Figure 7 (a) is the proportion of antigen-specific T cells in mice after injection of the above nanovaccine. Figure 7(b) is the corresponding cell heat map. It can be seen that compared with the Control group and the OVA group, the proportion of antigen-specific T cells increased significantly after the injection of the nanovaccines formed by the polymers of different embodiments and comparative example 1. Compared with the PN7 group, the proportion of antigen-specific T cells increased significantly by adding different fluorinated hydrophobic units and different proportions of nanovaccines, and showed a structure-activity relationship similar to that of the antigen cross-presentation experiment. Among the nanovaccines formed by polymers P (N7-3F30) ~ P (N7-17F20), the proportion of antigen-specific T cells of P (N7-9F30) was the highest. When the polymerization ratio of the hydrophobic unit increased from 30 of P (N7-9F30) to 40 of P (N7-9F40), the proportion of antigen-specific T cells decreased. For P (N7-17F10) and P (N7-17F20) whose hydrophobic groups are perfluorooctylethyl, the proportion of antigen-specific T cells decreased as the polymerization ratio of the hydrophobic group increased.

[0171] In summary, the modification of polymer vaccine adjuvants with fluorinated hydrophobic groups has a significant structure-activity relationship with DC cell activation, antigen cross-presentation ratio and antigen-specific T cell ratio of nanovaccines. By increasing the level of antigen cross-presentation and improving the proportion of antigen-specific T cells, the level of antigen-specific immune response can be further improved, thereby enhancing the immune efficacy of the vaccine.

Claims

1. A fluorine-containing polymer, characterized in that Having the structure of formula (I): （I）； wherein n is selected from an integer of 2 to 500; x and y are independently selected from an integer of 2 to 300; z is selected from an integer of 1 to 20; 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, C1~C 30 ether, at least one substituent substituted C1~C 30 Ether, C1~C 30 esters, C1~C 30 Esters, C1~C 30 sulfide, C1~C 30 Sulfide, C1~C 30 amine or at least one substituent substituted C1~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, C1~C 30 Ether, C1~C 30 Esters, C1~C 30 Sulfide, C1~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, C1~C 30 ether, at least one substituent substituted C1~C 30 Ether, C1~C 30 esters, C1~C 30 Esters, C1~C 30 sulfide, C1~C 30 Sulfide, C1~C 30 amine or at least one substituent substituted C1~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, C1~C 30 Ether, C1~C 30 Esters, C1~C 30 Sulfide, C1~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; R5 is selected from at least one fluorine-substituted C1~C 30 Alkyl, at least one fluorine-substituted C2~C 30 alkenyl, at least one fluorine-substituted C2~C 30 alkynyl, at least one fluorine-substituted C3~C 30 cycloalkyl, at least one fluorine-substituted C3~C 30 cycloalkenyl, at least one fluorine-substituted C3~C 30 cycloalkynyl, at least one fluorine-substituted C6~C 30 aromatic group, or at least one fluorine-substituted C6~C 30 The heteroaryl group.

2. The fluorinated polymer according to claim 1, characterized in that The R1 and R4 are independently selected from hydrogen, C1~C 30 Alkyl, C3~C 30 Cycloalkyl, C6~C 30 Aromatic or C6~C 30 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 a and R b Independently selected from hydrogen or C1~C5 alkyl; —Y— is selected from —O—, or , R c Selected from hydrogen or C1~C5 alkyl; The R5 is selected from at least one fluorine-substituted C1~C 30 Alkyl, at least one fluorine-substituted C3~C 30 or at least one fluorine-substituted C6~C 30 Aromatic groups; The R6 is selected from the following structures: ； R d Selected from C1~C 30 Alkyl, C3~C 30 Cycloalkyl, C1~C 30 Alkoxy, C1~C 30 Alkylthio, C6~C 30 Aromatic or C6~C 30 The heteroaryl group.

3. The fluorine-containing polymer according to claim 2, characterized in that The R2, R3 and the N atom to which they are connected together form a ring having the following structure: ； Wherein, a is selected from an integer of 1 to 5; R5 is selected from the following structures: 、 、 ； wherein X is selected from H or F; f, h and j are independently selected from integers of 0 to 20; g and i are independently selected from integers of 1-20.

4. The fluorine-containing polymer according to claim 3, characterized in that The R5 is selected from the following structures: ； wherein X is selected from H or F; f is an integer selected from 0 to 11; g is selected from an integer of 1 to 11.

5. A method for preparing a fluorine-containing polymer, characterized in that: include: A chain transfer agent having a structure of formula (II), a monomer having a structure of formula (III), a monomer having a structure of formula (IV) and an initiator undergo a polymerization reaction to obtain a fluorine-containing polymer having a structure of formula (I); (II)) (III)) (IV)) （I）； wherein n is selected from an integer of 2 to 500; x and y are independently selected from an integer of 2 to 300; z is selected from an integer of 1 to 20; 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, C1~C 30 ether, at least one substituent substituted C1~C 30 Ether, C1~C 30 esters, C1~C 30 Esters, C1~C 30 sulfide, C1~C 30 Sulfide, C1~C 30 amine or at least one substituent substituted C1~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, C1~C 30 Ether, C1~C 30 Esters, C1~C 30 Sulfide, C1~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, C1~C 30 ether, at least one substituent substituted C1~C 30 Ether, C1~C 30 esters, C1~C 30 Esters, C1~C 30 sulfide, C1~C 30 Sulfide, C1~C 30 amine or at least one substituent substituted C1~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, C1~C 30 Ether, C1~C 30 Esters, C1~C 30 Sulfide, C1~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; R5 is selected from at least one fluorine-substituted C1~C 30 Alkyl, at least one fluorine-substituted C2~C 30 alkenyl, at least one fluorine-substituted C2~C 30 alkynyl, at least one fluorine-substituted C3~C 30 cycloalkyl, at least one fluorine-substituted C3~C 30 cycloalkenyl, at least one fluorine-substituted C3~C 30 cycloalkynyl, at least one fluorine-substituted C6~C 30 aromatic group, or at least one fluorine-substituted C6~C 30 The heteroaryl group.

6. The preparation method according to claim 5, characterized in that: The solvent for 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.

7. The preparation method according to claim 5, characterized in that: 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 5, characterized in that: The polymerization reaction is carried out under anaerobic conditions; The polymerization reaction temperature is 35-70° C. and the reaction time is 12-48 hours.

9. A nanoparticle, characterized in that: The fluorine-containing polymer is formed by the fluorine-containing polymer according to any one of claims 1 to 4 or the fluorine-containing polymer prepared by the preparation method according to any one of claims 5 to 8.

10. A nanoadjuvant, characterized in that: The invention comprises the fluorine-containing polymer according to any one of claims 1 to 4, the fluorine-containing polymer prepared by the preparation method according to any one of claims 5 to 8, or the nanoparticles according to claim 9.

11. A nano vaccine, characterized in that: The invention comprises the nano adjuvant according to claim 10, an antigen and an adjuvant acceptable to the vaccine.

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