A polycarbonate polymer, a method for preparing the same, a nano-adjuvant and a vaccine

Abstract

The application discloses a polycarbonate polymer, a preparation method and application, and relates to the biomedical field. The polycarbonate polymer synthesized by the application has simple synthesis and definite structure, can induce higher neutralizing antibody level when used as a rabies vaccine nano adjuvant, and can carry and transport antigens to corresponding immune cells to fully exert the immune effect. The experimental results show that the polycarbonate polymer provided by the application can significantly improve the antibody level in the body of a mouse, enhance the antigen cross-presentation ability, effectively enhance the immune response intensity, and has a synergistic effect after the polycarbonate polymer, LTB protein and antigen are combined.

Description

TECHNICAL FIELD

[0001] The present application relates to the biomedical field, and in particular to a polycarbonate polymer and a preparation method thereof, a nano-adjuvant and a vaccine. BACKGROUND

[0002] Human or animal exposure to rabies virus can cause rabies, which is harmful to the nervous system and even life-threatening. At present, there is still no effective treatment for rabies, and vaccination against rabies is the only feasible method after exposure to the rabies virus. Currently, commercially available rabies vaccines are all inactivated vaccines with good safety, but they have the problems of low immunogenicity and long immune cycle, and it is difficult to induce effective immune response. Therefore, it is necessary to introduce an immune adjuvant into the rabies vaccine to enhance the immunogenicity of the antigen and improve the overall efficacy of the vaccine. However, the currently used immune adjuvants mainly stimulate the body to produce Th2 response, and the immune effect is not ideal.

[0003] Polymer immune adjuvants are a new type of adjuvant material, which have the advantages of simplicity, high efficiency and multifunctionality. Polymer adjuvant materials generally have good biocompatibility, can sustainably release antigens, and can carry and transport antigens to corresponding immune cells to fully exert immune effects, can enhance humoral immunity, cellular immunity and mucosal immunity, and have greater flexibility in the administration route. SUMMARY

[0004] Therefore, the present application provides a polycarbonate polymer and a preparation method thereof, a nano-adjuvant and a vaccine. The polycarbonate polymer provided by the present application can be used as an immune adjuvant for vaccines to enhance the immune response in vivo and improve the immune efficacy of the vaccine.

[0005] The polycarbonate polymer provided by the present application has the structure of formula (I):

[0006]

[0007] wherein n is selected from an integer from 2 to 10, preferably an integer from 2 to 8, and more preferably an integer from 2 to 6; m is selected from an integer from 10 to 1000, preferably an integer from 50 to 1000, and more preferably an integer from 100 to 1000;

[0008] R1 is selected from C1-C6 alkyl, C1-C6 alkyl substituted with at least one substituent, C2-C6 alkenyl, C2-C6 alkenyl substituted with at least one substituent, C2-C6 alkynyl, C2-C6 alkynyl substituted with at least one substituent, C6-C10 aryl, C6-C10 aryl substituted with at least one substituent, C4-C10 cycloalkyl, C4-C10 cycloalkyl substituted with at least one substituent, C2-C10 heterocycloalkyl, C2-C10 heterocycloalkyl substituted with at least one substituent, C6-C10 heteroaryl, and C6-C10 heteroaryl substituted with at least one substituent. 20 20 20 20 20 20 20 ​​​​​​The aromatic group, or at least one substituent, of the C6-C6 group. 20 Aromatic group, C6~C 20 C6-C substituted with heteroaryl or at least one substituent 20 The heteroaryl group, wherein the substituent is selected from C1 to C2. 20 Alkyl groups, C2-C 20 alkenyl, C2~C 20 alkynyl group, C6-C 20 Aromatic groups and C6-C 20 heteroaryl groups;

[0009] R2 is selected from C1 to C2. 20 Alkyl groups, C1-C 20 alkoxy groups, C1-C 20 alkylthio groups or C2-C 20 ether.

[0010] This invention introduces a carbonate structure into a polymer, resulting in a polycarbonate polymer with immunostimulatory activity. The polymer exhibits a well-defined structure, is easy to modify, and has high biocompatibility. It possesses advantages such as immunomodulatory properties, biocompatibility, biodegradability, low toxicity, good biosafety, sustainable antigen release, and the ability to induce higher levels of neutralizing antibodies. Furthermore, it can be used as a nano-adjuvant to carry and transport antigens to corresponding immune cells, thereby maximizing the immune effect.

[0011] In the polycarbonate polymer provided by the present invention, R1 is preferably C1 to C2. 10 Alkyl group, or at least one substituent substituted C1-C2 10 Alkyl groups, C2-C 10 The alkenyl group, or at least one substituent, of the C2-C group 10 alkenyl, C2~C 10 The C2-C group substituted with an alkynyl group or at least one substituent 10 The alkynyl group, wherein the substituent is selected from C1 to C2. 10 Alkyl groups, C2-C 10 alkenyl, C2~C 10 acetylinyl group and C6~C 10 The aromatic group; more preferably, the C2-C group is substituted with at least one substituent. 10 The alkenyl group, wherein the substituent is selected from C1 to C2. 10 Alkyl groups and C6-C 10 Aromatic groups.

[0012] In some specific implementations, R2 is preferably C1 to C2. 10 Alkyl groups, C1-C 10 alkoxy or C1-C 10 The alkylthio group, more preferably C1 to C2, is a thioalkyl group.10 alkoxy groups.

[0013] Specifically, the polycarbonate provided by this invention can have the following structure:

[0014]

[0015] The selection ranges of n and m are the same as those described above, and will not be repeated here.

[0016] The present invention also provides a method for preparing a polycarbonate polymer, comprising: polymerizing an aromatic compound of formula (II), a monomer of formula (III) and a catalyst in a CO2 system to obtain a polycarbonate polymer;

[0017]

[0018] Where n is selected from integers from 2 to 10;

[0019] R1 is selected from C1 to C1. 20 Alkyl group, or at least one substituent substituted C1-C2 20 Alkyl groups, C2-C 20 The alkenyl group, or at least one substituent, of the C2-C group 20 alkenyl, C2~C 20 The alkynyl group, or at least one substituent, substituted C2-C 20 alkynyl group, C6-C 20 The aromatic group, or at least one substituent, of the C6-C6 group. 20 Aromatic group, C6~C 20 C6-C substituted with heteroaryl or at least one substituent 20 The heteroaryl group, wherein the substituent is selected from C1 to C2. 20 Alkyl groups, C2-C 20 alkenyl, C2~C 20 alkynyl group, C6-C 20 Aromatic groups and C6-C 20 heteroaryl groups;

[0020] R2 is selected from C1 to C2. 20 Alkyl groups, C1-C 20 alkoxy groups, C1-C 20 alkylthio groups or C2-C 20 ether.

[0021] The aromatic compound of formula (II), the monomer of formula (III) and the catalyst are mixed first, then the gas in the reaction system is converted into CO2, the epoxy group of the monomer of formula (III) is opened under high pressure, and the polymerization reaction occurs between the hydroxyl group in the aromatic compound of formula (II) and the carbon dioxide, so as to obtain the polycarbonate polymer. Specifically, the polycarbonate polymer can be synthesized according to the following reaction route:

[0022]

[0023] The polymerization reaction is carried out under high pressure, and the pressure is 1-5 MPa, preferably 2-5 MPa, and more preferably 2-4 MPa. The temperature of the polymerization reaction is 15-40℃, preferably 15-35℃, and more preferably 20-35℃; the time is 8-25h, preferably 8-20h, and more preferably 8-15h.

[0024] In some specific embodiments, the molar ratio of the aromatic compound of formula (II), the monomer of formula (III) and the catalyst is (1-20):(300-500):(1-5), preferably (1-15):(300-450):(1-4), and more preferably (1-12):(350-450):(1-3).

[0025] In some specific embodiments, the catalyst is selected from at least one of salenCoCl and PPNCL, and preferably salenCoCl and PPNCL jointly catalyze the reaction. In some specific embodiments, the molar ratio of salenCoCl and PPNCL is 1:1-8, preferably 1:1-5, and more preferably 1:1-3. The structures of salenCoCl and PPNCL are as follows:

[0026]

[0027] In some specific embodiments, the aromatic compound of formula (II) is preferably of formula (IV),

[0028]

[0029] R3, R4 and R5 are independently selected from hydrogen, C1-C 20 alkyl substituted with at least one substituent, C2-C 20 alkenyl, C2-C 20 alkenyl substituted with at least one substituent, C2-C 20 alkynyl, C2-C 20 alkynyl substituted with at least one substituent, C6-C 20 aryl, C1-C20 The aromatic group, or at least one substituent, of the C6-C6 group. 20 Aromatic group, C6~C 20 C6-C substituted with heteroaryl or at least one substituent 20 The heteroaryl group, wherein the substituent is selected from C1 to C2. 20 Alkyl groups, C2-C 20 alkenyl, C2~C 20 alkynyl group, C6-C 20 Aromatic groups and C6-C 20 Mixed aromatic compounds.

[0030] Aromatic compounds with the structure of formula (IV) can be synthesized by dissolving benzophenone compounds, hydroxybenzophenone compounds and catalysts at low temperature, and then heating to carry out the reaction to obtain aromatic compounds with the structure of formula (IV).

[0031] In some specific implementations, the temperature at which the reactants and catalyst dissolve at low temperatures is -20 to -60°C, preferably -25 to -55°C, and more preferably -30 to -50°C. In some specific implementations, the temperature at which the reaction is heated is 50 to 90°C, preferably 55 to 85°C, and more preferably 60 to 80°C. In some specific implementations, the reaction time is 8 to 16 hours, preferably 9 to 15 hours, and more preferably 10 to 13 hours.

[0032] In some specific implementations, the molar ratio of the benzophenone compound, the hydroxybenzophenone compound and the catalyst is (1-2):(1-2):(4-10), preferably (1-2):(1-2):(4-8), and more preferably (1-2):(1-2):(6-8).

[0033] In some specific implementations, the catalyst is zinc powder and titanium tetrachloride, and the molar ratio of zinc powder to titanium tetrachloride is 2 to 6:1, preferably 2 to 5:1, and more preferably 2 to 4:1.

[0034] After obtaining an aromatic compound with the structure of formula (IV), it is preferable to purify it to obtain a pure compound. The purification method can be recrystallization, column chromatography, etc. The present invention does not have any special restrictions on the purification method.

[0035] In this invention, the monomer having the structure of formula (III) can be synthesized by the following method:

[0036] Epoxides, alcohols, and catalysts undergo substitution reactions to yield monomers with the structure of formula (III).

[0037] In some specific embodiments, the epoxy compound is selected from the group consisting of epichlorohydrin, epichlorobutane and the like. In some specific embodiments, the alcohol compound is selected from the group consisting of 2-(2-methoxyethoxy)ethan-1-ol or 2-[2-(2-methoxyethoxy)ethoxy]ethanol. In some specific embodiments, the catalyst is a basic compound, preferably NaOH.

[0038] In some specific embodiments, the molar ratio of the epoxy compound, the alcohol compound and the catalyst is (1-4):(2-6):(1-3), preferably (2-4):(3-6):(1-2), more preferably (3-4):(4-6):(1-2).

[0039] In some specific embodiments, the temperature of the substitution reaction is 10-40℃, preferably 15-35℃, more preferably 20-30℃. In some specific embodiments, the time of the substitution reaction is 2-5h, preferably 2.5-4.5h, more preferably 3-4h.

[0040] Meanwhile, the inventors of the present application found that the polycarbonate polymer provided by the present application has a synergistic effect when used together with LTB protein as a nano-adjuvant, and the immunization intensity is greater than when the polycarbonate polymer or LTB protein is used as an adjuvant alone. Therefore, the present application provides a nano-adjuvant comprising the above-mentioned polycarbonate polymer and LTB protein.

[0041] The present application also provides a vaccine composition comprising the above-mentioned polycarbonate polymer or nano-adjuvant, antigen and acceptable adjuvant in vaccines. The polycarbonate polymer provided by the present application has immunostimulatory activity, high biocompatibility, can sustainably release antigens, can induce higher neutralizing antibody levels, can carry and transport antigens to corresponding immune cells to fully exert immune effects, and the polycarbonate has a synergistic effect after being combined with LTB protein (Escherichia coli heat-labile enterotoxin B subunit protein) and antigen, and can be applied to the preparation of a rabies virus vaccine. Therefore, the antigen in the above-mentioned vaccine composition can be a rabies virus antigen.

[0042] This invention provides a polycarbonate polymer that forms an antigen-nanoparticle complex by encapsulating viral antigens within nanoparticles or by displaying conjugated viral antigens on the surface of nanoparticles. This complex promotes uptake by dendritic cells, slow release of antigens, targeted and cross-presentation, and enhanced antigen stability. Simultaneously, this nanoadjuvant can induce spleen cell proliferation and cytokine secretion, promote the proliferation and differentiation of antigen-specific T cells, and enhance the in vivo immune response. Experimental results show that the polycarbonate polymer provided by this invention has a well-defined, easily controllable structure and is biosafety-free. It can induce spleen cell proliferation and cytokine secretion, protecting mice attacked by rabies virus. Furthermore, it can bind to LTB protein, exhibiting superior immunomodulatory effects compared to either the polycarbonate polymer or LTB protein alone as an adjuvant, achieving a synergistic effect. Attached Figure Description

[0043] Figure 1 For TPE-OH 1 H NMR spectrum;

[0044] Figure 2 For ME2GE and ME3GE 1 H NMR spectrum;

[0045] Figure 3 For polycarbonate polymers P-ME2GE and P-ME3GE 1 H NMR spectrum;

[0046] Figure 4 GPC trajectory diagrams of polycarbonate polymers P-ME2GE and P-ME3GE with different molecular weights;

[0047] Figure 5 Linear relationship between Mn and M / I for polycarbonate polymers P-ME2GE and P-ME3GE with different molecular weights;

[0048] Figure 6 MALDI-TOF-MS spectra of polycarbonate polymers P-ME2GE and P-ME3GE;

[0049] Figure 7 Characterization diagram of polycarbonate polymer;

[0050] Figure 8 A schematic diagram of the immunoreaction experiment with polycarbonate nano-adjuvants and the results of neutralizing antibody titers;

[0051] Figure 9 Evaluation of the protective effect of polycarbonate nano-adjuvants against viral challenge after immunization;

[0052] Figure 10 Evaluation of cellular immune activation after immunization with polycarbonate nano-adjuvants;

[0053] Figure 11 Safety evaluation of polycarbonate nano-adjuvants. DETAILED DESCRIPTION

[0054] It should be understood that the expression "one or more of the following" includes each of the objects recited after the expression individually as well as various different combinations of two or more of the recited objects, unless otherwise understood from the context and usage. The expression "and / or" in connection with three or more recited objects should be understood to have the same meaning, unless otherwise understood from the context.

[0055] The use of the terms "including," "has," or "has" including their grammatical variants, should generally be understood as open-ended and non-limiting, for example, not excluding additional unrecited elements or steps, unless otherwise specifically stated or understood from the context.

[0056] It should be understood that the order of steps or order for performing certain actions is immaterial so long as the application remains operable. Moreover, two or more steps or actions can be conducted simultaneously.

[0057] The use of any and all examples or exemplary language herein is intended merely to better illuminate the application and does not pose a limitation on the scope of the application unless otherwise claimed. No language in the specification should be construed as indicating any non-claimed element as essential to the practice of the application.

[0058] Further, the numerical ranges and parameters setting forth the broadest scope of the application are approximations, and are merely intended to convey general information as to the scope of the application. Consequently, any numerical values inherent in the application are inherently approximations. Accordingly, it is contemplated that any numerical value, including numerical values explicitly recited, should inherently be interpreted as modified by the term "about" the same number of significant figures as recited. It is also understood that the numerical ranges and parameters set forth are approximate, and that the application is intended to encompass all reasonable ranges and numbers within the range.

[0059] The application introduces CO2 into a polymer reaction system to obtain a series of polycarbonate polymers, which have immune stimulating activity, are easy to modify, have high biocompatibility, have immune regulation, biocompatibility, biodegradability, low toxicity, good biosafety, can sustainably release antigens, can induce higher levels of neutralizing antibodies, and have the advantages of being able to induce higher levels of neutralizing antibodies. When used as a nano-adjuvant, the virus antigen can be packaged inside the nanoparticles or displayed on the surface of the nanoparticles by coupling the virus antigen, forming an antigen-nanoparticle complex, promoting the absorption of dendritic cells, slowly releasing the antigen, targeting and cross-presentation, and enhancing the stability of the antigen. The nanoparticles enhance the interaction between the virus antigen and the immune system of the body by protecting the antigen from protease degradation, improving biocompatibility, and prolonging the release time of the antigen, and induce higher levels and longer periods of immune response. Experimental results show that the polycarbonate polymer provided by the application can effectively protect mice attacked by RABV, and can also be combined with LTB protein to enhance the immune efficacy.

[0060] The application will be further described below in conjunction with examples. The protection scope of the application is not limited by the following examples.

[0061] Example 1

[0062] Take 3.2 g of benzophenone, 3.48 g of 4-hydroxybenzophenone and 4.59 g of zinc powder into a two-necked flask, add 50 ml of anhydrous tetrahydrofuran, slowly drop 6.66 g of titanium tetrachloride at-40℃ under nitrogen atmosphere, the molar ratio of the benzophenone, 4-hydroxybenzophenone, zinc powder and titanium tetrachloride is 1:1:4:2, then reflux overnight in a 70℃ oil bath, remove the solid by filtration, dry the solution by rotary evaporation, and separate by column chromatography to obtain yellow solid TPE-OH with a yield of 76%.

[0063] Figure 1 The TPE-OH synthesized in this example is shown in the following figure 1 The H NMR spectrum and structure are shown in the following figure, in which the peaks numbered a-d correspond to the hydrogen areas numbered in the structure.

[0064] Example 2

[0065] Take 14.4 g of sodium hydroxide and 44.43 g of epichlorohydrin into a round-bottom flask, slowly drop 50 g of diethylene glycol monomethyl ether into it at room temperature, the molar ratio of the sodium hydroxide, epichlorohydrin and diethylene glycol monomethyl ether is 1.5:2:1, continue stirring at room temperature for 3 h, remove the solid by filtration, and distill to obtain colorless transparent liquid ME2GE with a yield of 67%.

[0066] Figure 2 The monomer ME2GE synthesized in this example is shown in the following figure 1H NMR spectrum and structure, the peaks numbered a~f respectively correspond to the hydrogen area of the numbered hydrogen in the structure.

[0067] Example 3

[0068] Synthesis of ME3GE: 11.89 g of sodium hydroxide and 36.67 g of epichlorohydrin were placed in a round-bottom flask, and 50 g of diethylene glycol monomethyl ether was slowly added dropwise at room temperature, the molar ratio of sodium hydroxide, epichlorohydrin and diethylene glycol monomethyl ether was 1.5:2:1, and stirring was continued at room temperature for 3 h, and the solid was removed by filtration, and a colorless transparent liquid ME3GE was obtained by distillation, with a yield of 65%.

[0069] Figure 2 (b) shows the monomer ME3GE synthesized in this example 1 H NMR spectrum and structure, the peaks numbered a~f respectively correspond to the hydrogen area of the numbered hydrogen in the structure, it can be seen that the hydrogen numbered f in the structure of ME3GE increases compared with ME2GE, and the peak area f in the spectrum also increases.

[0070] Example 4

[0071] 3 g of ME2GE monomer, 27.2 mg of salenCoCl, 24.4 mg of PPNCL and 14.8 mg of chain transfer agent TPE-OH were respectively placed in a 10 mL autoclave, the molar ratio of ME2GE monomer, salenCoCl, PPNCL and chain transfer agent TPE-OH was 400:1:1:1, the CO2 concentration was adjusted to 3.0 MPa, and polymerization was carried out at 25°C for 12 h, to obtain a polycarbonate polymer P-ME2GE with a number average molecular weight of 17100.

[0072] Example 5

[0073] 3 g of ME2GE monomer, 27.2 mg of salenCoCl, 24.4 mg of PPNCL and 29.7 mg of chain transfer agent TPE-OH were respectively placed in a 10 mL autoclave, the molar ratio of ME2GE monomer, salenCoCl, PPNCL and chain transfer agent TPE-OH was 400:1:1:2, the CO2 concentration was adjusted to 3.0 MPa, and polymerization was carried out at 25°C for 12 h, to obtain a polycarbonate polymer P-ME2GE with a number average molecular weight of 11300.

[0074] Example 6

[0075] 3g of ME2GE monomer, 27.2mg of salenCoCl, 24.4mg of PPNCL and 74.2mg of chain transfer agent TPE-OH were respectively placed in a 10mL autoclave, the molar ratio of the ME2GE monomer, salenCoCl, PPNCL and chain transfer agent TPE-OH was 400:1:1:5, the CO2 concentration was adjusted to 3.0MPa, and polymerization was carried out at 25℃ for 12h to obtain a polycarbonate polymer P-ME2GE with a number average molecular weight of 8100.

[0076] Example 7

[0077] 3g of ME2GE monomer, 27.2mg of salenCoCl, 24.4mg of PPNCL and 74.2mg of chain transfer agent TPE-OH were respectively placed in a 10mL autoclave, the molar ratio of the ME2GE monomer, salenCoCl, PPNCL and chain transfer agent TPE-OH was 400:1:1:5, the CO2 concentration was adjusted to 3.0MPa, and polymerization was carried out at 25℃ for 12h to obtain a polycarbonate polymer P-ME2GE with a number average molecular weight of 8100.

[0078] Figure 3 (a) shows the 1H NMR spectrum of the polycarbonate polymer P-ME2GE with a molecular weight of 6400. 1 H NMR spectrum and structure, where the peaks labeled a-e correspond to the hydrogen area of the structural polymer unit, and the enlarged curve on the left side of the spectrum corresponds to the hydrogen area of the TPE unit in the structure.

[0079] Example 8

[0080] 3g of ME3GE monomer, 21.8mg of salenCoCl, 19.6mg of PPNCL and 11.9mg of chain transfer agent TPE-OH were respectively placed in a 10mL autoclave, the molar ratio of the ME3GE monomer, salenCoCl, PPNCL and chain transfer agent TPE-OH was 400:1:1:1, the CO2 concentration was adjusted to 3.0MPa, and polymerization was carried out at 25℃ for 12h to obtain a polycarbonate polymer P-ME3GE with a number average molecular weight of 17900.

[0081] Example 9

[0082] 3g of ME3GE monomer, 21.8mg of salenCoCl, 19.6mg of PPNCL and 59.3mg of chain transfer agent TPE-OH were placed in a 10mL autoclave, the molar ratio of the ME3GE monomer, salenCoCl, PPNCL and chain transfer agent TPE-OH was 400:1:1:5, the CO2 concentration was adjusted to 3.0MPa, and polymerization was carried out at 25°C for 12h to obtain a polycarbonate polymer P-ME3GE with a number average molecular weight of 9700.

[0083] Example 10

[0084] 3g of ME3GE monomer, 21.8mg of salenCoCl, 19.6mg of PPNCL and 59.3mg of chain transfer agent TPE-OH were placed in a 10mL autoclave, the molar ratio of the ME3GE monomer, salenCoCl, PPNCL and chain transfer agent TPE-OH was 400:1:1:5, the CO2 concentration was adjusted to 3.0MPa, and polymerization was carried out at 25°C for 12h to obtain a polycarbonate polymer P-ME3GE with a number average molecular weight of 9700.

[0085] Example 11

[0086] 3g of ME3GE monomer, 21.8mg of salenCoCl, 19.6mg of PPNCL and 59.3mg of chain transfer agent TPE-OH were placed in a 10mL autoclave, the molar ratio of the ME3GE monomer, salenCoCl, PPNCL and chain transfer agent TPE-OH was 400:1:1:5, the CO2 concentration was adjusted to 3.0MPa, and polymerization was carried out at 25°C for 12h to obtain a polycarbonate polymer P-ME3GE with a number average molecular weight of 9700.

[0087] Figure 3 (b) shows the H NMR spectrum of the polycarbonate polymer P-ME3GE with a number average molecular weight of 7500 1 H NMR spectrum and structure, where the peaks labeled a-e correspond to the hydrogen area of the structural polymer unit, and the enlarged curve on the left side of the spectrum corresponds to the hydrogen area of the TPE unit in the structure.

[0088] Test Example 1

[0089] Polycarbonate polymer selectivity (polymer) test method:

[0090] Each polymer was dissolved in CDCl3to obtain a 5mg / mL solution, and the 1HNMR spectra give the chemical shift of hydrogen in polymer solution, and the content of carbonate unit (CU) in polycarbonate polymer is determined by comparing the integral of product peak and byproduct peak.

[0091] The number average molecular weight of polycarbonate polymer is determined by gel permeation chromatography (GPC) test instrument calibrated by polystyrene standard at 35°C, CH2Cl2 as eluent, column pressure 447 Pa, flow rate 1 mL / min, and then each polymer is dissolved in CH2Cl2 to obtain a 3 mg / mL solution, and the GPC trace of the polymer is obtained in the same way, and the number average molecular weight (Mn) and polydispersity of the polymer are determined by integration.

[0092] The test data obtained above are summarized in Table 1.

[0093] Table 1 Experimental results of polycarbonate polymers

[0094]

[0095] As can be seen from Table 1, the selectivity levels of polycarbonate polymers P-ME2GE and P-ME3GE for cyclic carbonate are high, which indicates that the active centers of the catalyst effectively inhibit the backbiting reaction of the active chain; at the same time, the content of carbonate unit in the polycarbonate polymer is more than 99%, which indicates that no continuous homopolymerization side reaction of epoxide occurs.

[0096] Test Example 2

[0097] The GPC traces of polycarbonate polymers P-ME2GE and P-ME3GE with different molecular weights are tested by gel permeation chromatography. The GPC test instrument is calibrated by polystyrene standard at 35°C, CH2Cl2 as eluent, column pressure 447 Pa, flow rate 1 mL / min, and then each polymer is dissolved in CH2Cl2 to obtain a 3 mg / mL solution, and the GPC trace of the polymer is obtained in the same way. The results are shown in Figure 4 . Figure 4 (a) shows the GPC curve of polycarbonate polymer P-ME2GE with different molecular weights, Figure 4 (b) shows the GPC curve of polycarbonate polymer P-ME3GE with different molecular weights, from which it can be seen that the trace curve of polycarbonate polymers P-ME2GE and P-ME3GE gradually changes from bimodal distribution to unimodal distribution as the polymerization monomer ratio decreases.

[0098] The linear relationship diagram is drawn with the Mn value of polycarbonate polymers P-ME2GE and P-ME3GE with different molecular weights as the ordinate and the M / I value as the abscissa, and the correlation coefficient (R 2 ) and linear equation are calculated, and the results are shown in Figure 5 .Figure 5 (a) is a linear plot of polycarbonate polymer P-ME2GE, Figure 5 (b) is a linear plot of polycarbonate polymer P-ME3GE, from which it can be seen that the Mn of the polycarbonate polymer is highly linearly correlated with M / I, which indicates that TPE-OH can well quantitatively control the molecular weight of the polymer, so as to obtain a polycarbonate polymer immunoadjuvant suitable for a specific antigen or immune response.

[0099] Test Example 3

[0100] The polycarbonate polymer P-ME2GE prepared in Example 7 and the polycarbonate polymer P-ME3GE prepared in Example 11 were respectively dissolved in tetrahydrofuran to obtain a solution of 1 mg / mL. With 10 mg / mL DCTB tetrahydrofuran solution as a matrix and 1 mg / mL sodium trifluoroacetate aqueous solution as a cationic agent, the polymer, the matrix and the cationic agent were mixed in a ratio of 1:1:1 (v / v / v), and the mixed solution was repeatedly spotted on a stainless steel MALDI plate for 5 times and left to dry in a fume hood overnight. The MALDI plate was moved into a matrix-assisted laser desorption mass spectrometer, and the matrix-assisted laser desorption ionization time-of-flight mass spectrum (MALDI-TOF-MS) was recorded in reflection mode, and the results are shown in Figure 6 Figure 6 (a) is a spectrum of the polycarbonate polymer P-ME2GE prepared in Example 7, Figure 6 (b) is a spectrum of the polycarbonate polymer P-ME3GE prepared in Example 11, from which it can be seen that the structures of the two polymers are clear and the molecular weight is unimodal.

[0101] Test Example 4

[0102] The polycarbonate polymer P-ME2GE prepared in Example 7 was dissolved in water to obtain a solution of 5 mg / mL, and after ultrasonic stirring, the P-ME2GE nano-adjuvant was obtained. CVS11 was incubated with 10 nm gold-labeled antibody, and LTB protein was incubated with 18 nm gold-labeled antibody, and the incubated CVS11 and LTB protein were mixed with the above P-ME2GE nano-adjuvant (mass ratio 1:1:4). Figure 7 (a) is P-ME2GE observed under an electron microscope; Figure 7 (b) is CVS11 virus particles and LTB observed by immunoelectron microscopy, from which it can be seen that the CVS11 virus particles and LTB are attached to the surface of P-ME2GE; Figure 7 (c) is a Zeta potential plot of the combination of P-ME2GE, CVS11 and LTB, from which it can be seen that the Zeta potential continuously decreases during the combination of the three. The above experimental data show that P-ME2GE, CVS11 and LTB are successfully combined. ​

[0103] Test Example 5

[0104] The polycarbonate polymer P-ME2GE prepared in Example 7 was dissolved in water to obtain a 5 mg / mL solution, and P-ME2GE nano-adjuvant was obtained after ultrasonic stirring. Inactivated CVS11 antigen was combined with aluminum hydroxide adjuvant, LTB, P-ME2GE, LTB and P-ME2GE (mass ratio 1:1) at an immunization adjuvant to vaccine antigen mass ratio of 1:4 to obtain vaccines, which are denoted as experimental groups. PBS buffer solution was mixed with P-ME2GE to obtain a 5 mg / mL solution, and inactivated CVS11 antigen was combined with the P-ME2GE solution to obtain a control group.

[0105] Equal volumes of the above-mentioned experimental group and control group vaccines were used to immunize mice at 0, 2, 4 weeks, and after three immunizations, the mice were taken blood from the orbital vein at 0-8 weeks, the serum samples were collected by centrifugation, the neutralizing antibody titers in the serum were determined by rabies neutralization test, and the data of each group per week were recorded and summarized in Table 2. Figure 8 (a) is a time axis for injecting vaccines into mice, Figure 8 (b) is a schematic diagram of blending P-ME2GE nano-adjuvant, antigen and LTB, Figure 8 (c) is the neutralizing antibody titer in the serum of mice.

[0106] Table 2. Neutralizing antibody titer data of serum of mice in each group

[0107]

[0108] As can be seen from Table 2 and Figure 8 , P-ME2GE as an adjuvant can produce antibody titers much higher than the standard 0.5 IU / mL level, indicating that P-ME2GE induces a large amount of protective antibodies against RABV in mice. In addition, the effect of P-ME2GE and LTB as adjuvants is higher than that of P-ME2GE or LTB alone as adjuvants, indicating that P-ME2GE can function to deliver LTB.

[0109] Test Example 6

[0110] BALB / c mice (4-6 weeks old) were randomly divided into 6 groups, 10 mice in each group, and the experimental group and control group vaccines in Test Example 5 were used to immunize mice at 0, 2, 4 weeks, and at the 5th week, the mice were challenged with RABV CVS11 strain at a dose of 10 5.75 TCID 50 per mouse by intramuscular injection, Figure 9 (a) is a time diagram of injecting vaccines and RABV antigens into mice. The clinical symptoms and survival rate of mice were observed every day, and the survival rate and survival time data of mice were summarized in Table 3. Figure 9(b) The survival rate of each group of mice, from which it can be seen that 90% of the mice in the control PBS immunization group died of rabies within 10 days, while 80% of the mice in the P-ME2GE immunization group survived. Figure 9 (c) The clinical score of mice injected with vaccines in the experimental and control groups, from which it can be seen that the experimental group injected with P-ME2GE and LTB as adjuvants scored the highest, indicating that the mixture of P-ME2GE and LTB as adjuvants provided complete protection to the mice.

[0111] Table 3 Number of surviving mice in each group

[0112]

[0113] As can be seen from Table 3, the mice in the experimental group injected with P-ME2GE as adjuvants survived for a longer time, and the mice in the experimental group injected with P-ME2GE and LTB as adjuvants survived for the longest time, indicating that P-ME2GE as an adjuvant provided effective protection against RABV infection, and the effect of P-ME2GE and LTB as adjuvants was higher than that of P-ME2GE or LTB as adjuvants alone.

[0114] Test Example 7

[0115] The experimental and control groups of vaccines in Test Example 4 were used to immunize mice at 0, 2, 4 weeks, and one week after the third immunization, the spleens of the mice were collected, 3 mice from each group were taken, and the spleen cells were re-stimulated with inactivated CVS11 to perform an ex vivo spleen cell proliferation test to evaluate the effect of adjuvants on the proliferation response of spleen cells, and the results are summarized in Table 4. Figure 10 (a) is a schematic diagram of the test time, and the numbers in the arrows represent the days. Figure 10 (b) is a schematic diagram of the mouse spleen cell proliferation index, from which it can be found that the spleen cell proliferation efficiency of mice in the P-ME2GE and LTB mixed group is higher than that of mice in other groups.

[0116] The ability of spleen cells from mice in the experimental and control groups to secrete IL-4 and IFN-γ was compared using enzyme-linked immunosorbent spot (ELISPOT) assay, and the results are summarized in Table 5. Figure 10 (c) and Figure 10 (d) are diagrams of the number of mouse IL-4 spots and IFN-γ spots, respectively, Figure 10 (e) is a diagram of the distribution of mouse IL-4 spots and IFN-γ spots. From which it can be seen that the levels of the two cytokines in the P-ME2GE and LTB mixed group are significantly increased.

[0117] Table 4 Spleen cell proliferation index of mice in each group

[0118]

[0119] Table 5: ELISPOT IL-4 spot and IFN-γ spot number of each group of mice

[0120]

[0121] As can be seen from Table 4 and Table 5, P-ME2GE can induce the proliferation of splenocytes and the secretion of cytokines. The increase of IFN-γ in the spleen can promote the proliferation and differentiation of CD8 + T and IL-4 promotes the differentiation of Th0 into Th2 cells, thereby triggering the humoral immune response.

[0122] Test Example 8

[0123] The 5 mg / mL P-ME2GE solution prepared in Test Example 4 was injected into mice by intramuscular injection, and PBS solution was used as a control. The blood of the mice was collected for blood biochemical detection and blood routine detection, and the data of each index were summarized in Table 6. Figure 11 (a) is the result of blood biochemical detection, Figure 11 (b) is the result of blood routine detection, from which it can be seen that the blood of the mice injected with P-ME2GE solution has no abnormal index compared with the control group injected with PBS solution. At the same time, the physiological sections of the main organs (including brain, heart, liver, spleen, lung, kidney, muscle and lymph node) of the mice were collected and stained with hematoxylin-eosin (H&E), Figure 11 (c) is the physiological section diagram after staining, and it can be seen that the mice do not have any obvious histological abnormalities.

[0124] Table 6: Mouse blood index data

[0125]

[0126] The above results show that P-ME2GE is a safe biological material and is suitable for use as an in vivo vaccine adjuvant.

[0127] The above describes only the preferred specific embodiments of the present application, but the protection scope of the present application is not limited thereto, and any person skilled in the art can make equivalent replacements or changes to the technical solutions and inventive concepts of the present application within the technical scope disclosed by the present application, which should be covered within the protection scope of the present application.

Claims

1. A polycarbonate polymer, characterized in that, It has the following structure: ； Where n is selected from integers from 2 to 10, and m is selected from integers from 10 to 1000.

2. The polycarbonate polymer according to claim 1, characterized in that, The number-average molecular weight is 5~20 kg / mol.

3. A method for preparing a polycarbonate polymer, characterized in that, include: Aromatic compounds of formula (1), monomers of formula (III) and catalysts are polymerized in a CO2 system to obtain polycarbonate polymers; （1）； (III); Where n is selected from integers from 2 to 10; R2 is selected from methoxy groups.

4. The preparation method according to claim 3, characterized in that, The pressure of the CO2 system is 1~5 MPa.

5. A nano-adjuvant, characterized in that, Including LTB protein, the polycarbonate polymer according to any one of claims 1 to 2, or the polycarbonate polymer prepared by the preparation method according to any one of claims 3 to 4.

6. A vaccine composition, characterized in that, It includes the polycarbonate polymer according to any one of claims 1 to 2, the polycarbonate polymer prepared by the preparation method according to any one of claims 3 to 4, or the nano-adjuvant according to claim 5, as well as antigens and other excipients; The antigen is a rabies virus antigen.

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