Polycarbonate polymer and preparation method thereof, nano adjuvant and vaccine

Abstract

The invention discloses a polycarbonate polymer as well as a preparation method and application thereof, and relates to the field of biomedicine. The polycarbonate polymer synthesized by the invention is simple to synthesize and clear in structure, can induce a higher neutralizing antibody level when being used as a rabies vaccine nano adjuvant, and can carry and transport antigens into corresponding immune cells to fully play an immune effect. Experimental results show that when the polycarbonate polymer provided by the invention is blended with the RABV CVS11 antigen, the antibody level in a mouse body can be remarkably improved, the antigen cross presentation capability is enhanced, the immunoreaction intensity is effectively enhanced, and the polycarbonate polymer, LTB protein and the antigen have a synergistic effect after being combined.

Description

Technical Field

[0001] The invention relates to the field of biomedicine, and in particular to a polycarbonate polymer and a preparation method thereof, a nano adjuvant and a vaccine. Background Art

[0002] After people or animals are exposed to rabies virus, they will develop rabies, which will cause damage to the nervous system and even endanger life. There is currently a lack of effective treatment for rabies, and vaccination with rabies vaccine is the only feasible way after exposure to rabies virus. Currently, all commercially available rabies vaccines are inactivated vaccines with good safety, but they have the problems of low immunogenicity and long immune cycle, and it is difficult to induce an 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 a Th2 response, and the immune effect is not ideal.

[0003] Polymer immune adjuvant is a new type of adjuvant material with the advantages of simplicity, high efficiency and versatility. Polymer adjuvant materials generally have good biocompatibility, can release antigens continuously, and can carry and transport antigens to the corresponding immune cells to fully exert immune effects, can enhance humoral immunity, cellular immunity and mucosal immunity, and have greater flexibility in the route of administration. Summary of the invention

[0004] In view of this, the present invention provides a polycarbonate polymer and a preparation method thereof, a nano adjuvant and a vaccine. The polycarbonate polymer provided by the present invention can be used as an immune adjuvant of a vaccine to enhance the immune response in the body and improve the immune efficacy of the vaccine.

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

[0006]

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

[0008] R1 is selected from C1~C 20 alkyl, C1~C 20 Alkyl, C2~C 20 Alkenyl, C2-C 20 Alkenyl, C2~C 20 Alkynyl, C2-C 20 Alkynyl, C6~C 20aromatic group, C6~C 20 Aromatic groups, C6~C 20 or a C6-C 20 The substituent is selected from C1 to C 20 Alkyl, C2~C 20 Alkenyl, C2~C 20 Alkynyl, C6~C 20 Aromatic groups and C6~C 20 heteroaryl;

[0009] R2 is selected from C1~C 20 Alkyl, C1~C 20 Alkoxy, C1~C 20 Alkylthio or C2~C 20 of ether.

[0010] The present invention introduces a carbonate structure into the polymer, so that the synthesized polycarbonate polymer has immunostimulatory activity, a clear structure, is easy to modify, and has high biocompatibility. It has the advantages of immunoregulatory, biocompatibility, biodegradability, low toxicity, good biosafety, sustainable antigen release, and the ability to induce higher neutralizing antibody levels. At the same time, it can be used as a nanoadjuvant to carry and transport antigens to corresponding immune cells to fully exert the immune effect.

[0011] In the polycarbonate polymer provided by the present invention, the R1 is preferably C1 to C 10 alkyl, C1~C 10 Alkyl, C2~C 10 Alkenyl, C2-C 10 Alkenyl, C2~C 10 Alkynyl or at least one substituent substituted C2-C 10 The alkynyl group, the substituent is selected from C1 to C 10 Alkyl, C2~C 10 Alkenyl, C2~C 10 Alkynyl and C6~C 10 More preferably, at least one substituent is substituted with C2 to C 10 The substituent is selected from C1 to C 10 Alkyl and C6~C 10 Aromatic base.

[0012] In some specific implementations, the R2 is preferably C1 to C 10 Alkyl, C1~C 10 Alkoxy or C1~C 10 The alkylthio group is more preferably C1 to C10 of alkoxy.

[0013] Specifically, the polycarbonate provided by the present invention can be the following structure:

[0014]

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

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

[0017]

[0018] Wherein, n is selected from an integer of 2 to 10;

[0019] R1 is selected from C1~C 20 alkyl, C1~C 20 Alkyl, C2~C 20 Alkenyl, C2-C 20 Alkenyl, C2~C 20 Alkynyl, C2-C 20 Alkynyl, C6~C 20 aromatic group, C6~C 20 Aromatic groups, C6~C 20 or a C6-C 20 The substituent is selected from C1 to C 20 Alkyl, C2~C 20 Alkenyl, C2~C 20 Alkynyl, C6~C 20 Aromatic groups and C6~C 20 heteroaryl;

[0020] R2 is selected from C1~C 20 Alkyl, C1~C 20 Alkoxy, C1~C 20 Alkylthio or C2~C 20 of ether.

[0021] The present invention first mixes an aromatic compound of formula (II), a monomer of formula (III) and a catalyst, then converts the gas in the reaction system into CO2, and opens the epoxy group of the monomer of formula (III) under high pressure, and polymerizes with the hydroxyl group in the aromatic compound of formula (II) and carbon dioxide to obtain a polycarbonate polymer. Specifically, the polycarbonate polymer of the present invention can be synthesized according to the following reaction route:

[0022]

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

[0024] In some specific implementations, the molar ratio of the aromatic compound having the structure of formula (II), the monomer having the structure 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 implementations, the catalyst is selected from at least one of salenCoCl and PPNCL, preferably salenCoCl and PPNCL jointly catalyze the reaction. In some specific implementations, the molar ratio of salenCoCl to PPNCL is 1:1 to 8, preferably 1:1 to 5, and more preferably 1:1 to 3. The structures of salenCoCl and PPNCL are as follows:

[0026]

[0027] In some specific implementations, 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, C1~C 20 Alkyl, C2~C 20 Alkenyl, C2-C 20 Alkenyl, C2~C 20 Alkynyl, C2-C 20 Alkynyl, C6~C20 aromatic group, C6~C 20 Aromatic groups, C6~C 20 or a C6-C 20 The heteroaryl group is selected from C1 to C 20 Alkyl, C2~C 20 Alkenyl, C2~C 20 Alkynyl, C6~C 20 Aromatic groups and C6~C 20 The heteroaryl group.

[0030] The aromatic compound of formula (IV) can be synthesized by the following method: dissolving a benzophenone compound, a hydroxybenzophenone compound and a catalyst at low temperature, and then heating the mixture to react to obtain an aromatic compound of formula (IV).

[0031] In some specific implementations, the temperature at which the reactants and catalysts are dissolved 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 of the temperature-raising reaction 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 the 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 the aromatic compound having the structure of formula (IV), it is preferably purified to obtain a pure compound. The purification method may be recrystallization, column chromatography, etc. The present invention has no particular limitation on the purification method.

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

[0036] The epoxy compound, the alcohol compound and the catalyst undergo a substitution reaction to obtain a monomer having a structure of formula (III).

[0037] In some specific implementations, the epoxy compound is selected from epoxy compounds such as epichlorohydrin and epichlorohydrin. In some specific implementations, the alcohol compound is selected from 2-(2-methoxyethoxy)ethane-1-ol or 2-[2-(2-methoxyethoxy)ethoxy]ethanol. In some specific implementations, the catalyst is an alkaline compound, preferably NaOH.

[0038] In some specific implementations, 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), and more preferably (3-4):(4-6):(1-2).

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

[0040] At the same time, the inventors of the present invention have found that the polycarbonate polymer provided by the present invention and the LTB protein have a synergistic effect when used together as a nanoadjuvant, and the immune strength is greater than when the polycarbonate polymer or the LTB protein is used alone as an adjuvant. Therefore, the present invention provides a nanoadjuvant, including the above-mentioned polycarbonate polymer and the LTB protein.

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

[0042] The present invention provides a polycarbonate polymer, which forms an antigen-nanoparticle complex by packaging viral antigens inside nanoparticles or by displaying coupled viral antigens on the surface of nanoparticles, promotes the absorption of dendritic cells, slowly releases antigens, and presents them in a targeted and cross-directed manner, thereby enhancing the stability of the antigen. At the same time, such nanoadjuvants can induce splenocyte proliferation and cytokine secretion, promote the proliferation and differentiation of antigen-specific T cells, and enhance the immune response in vivo. Experimental results show that the polycarbonate polymer provided by the present invention has a clear structure, is easy to control, and has biosafety. It can induce splenocyte proliferation and cytokine secretion, and protect individual mice attacked by rabies virus; at the same time, it can be combined with LTB protein, and its immune effect is better than that of polycarbonate polymer or LTB protein alone as an adjuvant, achieving synergistic enhancement. BRIEF DESCRIPTION OF THE DRAWINGS

[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 traces of polycarbonate polymers P-ME2GE and P-ME3GE with different molecular weights;

[0047] Figure 5 The linear relationship diagram of Mn and M / I of 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 is a characterization diagram of a polycarbonate polymer;

[0050] Figure 8 Schematic diagram of the polycarbonate nanoadjuvant immune response experiment and the neutralizing antibody titer results;

[0051] Fig. 9 Evaluation of polycarbonate nanoadjuvants for protection against virus attack after immunization;

[0052] Fig.10 To evaluate the cellular immune activation after immunization with polycarbonate nanoadjuvants;

[0053] Fig.11 Safety evaluation of polycarbonate nanoadjuvants. DETAILED DESCRIPTION

[0054] 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.

[0055] 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.

[0056] 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.

[0057] 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.

[0058] 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.

[0059] The present invention obtains a series of polycarbonate polymers by introducing CO2 into a polymer reaction system. Such polymers have immunostimulatory activity, and have clear structures, are easy to modify, have high biocompatibility, have immunoregulatory properties, biocompatibility, biodegradability and low toxicity, good biosafety, can continuously release antigens, and can induce higher neutralizing antibody levels. When used as a nano adjuvant, viral antigens can be packaged inside nanoparticles or by displaying coupled viral antigens on the surface of nanoparticles to form antigen-nanoparticle complexes, promote the absorption of dendritic cells, slowly release antigens, and present them in a targeted and cross-characteristic manner, thereby enhancing the stability of antigens. Nanoparticles enhance the interaction between viral antigens and the body's immune system by protecting antigens from protease degradation, improving biocompatibility and prolonging the antigen release time, thereby inducing higher levels and longer immune responses. Experimental results show that the polycarbonate polymer provided by the present invention can effectively protect mice attacked by RABV, and can also enhance immune efficacy by combining with LTB protein.

[0060] 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.

[0061] Example 1

[0062] 3.2 g of benzophenone, 3.48 g of 4-hydroxybenzophenone and 4.59 g of zinc powder were placed in a two-necked flask, 50 ml of anhydrous tetrahydrofuran was added thereto, and 6.66 g of titanium tetrachloride was slowly added dropwise at -40 ° C. under a nitrogen atmosphere, wherein the molar ratio of benzophenone, 4-hydroxybenzophenone, zinc powder and titanium tetrachloride was 1:1:4:2, and then refluxed in an oil bath at 70 ° C. overnight, the solid was filtered out, the solution was spin-dried and separated 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 FIG. 1 H NMR spectrum and structure, where the peaks numbered a to d correspond to the numbered hydrogen areas in the structure.

[0064] Example 2

[0065] 14.4 g of sodium hydroxide and 44.43 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. Stirring was continued at room temperature for 3 h, the solid was removed by filtration, and colorless transparent liquid ME2GE was obtained by distillation. The yield was 67%.

[0066] Figure 2 (a) shows the monomer ME2GE synthesized in this example. 1H NMR spectrum and structure, where the peaks numbered a to f correspond to the numbered hydrogen areas 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 thereto at room temperature. The molar ratio of the sodium hydroxide, epichlorohydrin and diethylene glycol monomethyl ether was 1.5:2:1. Stirring was continued at room temperature for 3 h, the solid was removed by filtration, and colorless transparent liquid ME3GE was obtained by distillation. The yield was 65%.

[0069] Figure 2 (b) shows the monomer ME3GE synthesized in this example. 1 H NMR spectrum and structure. The peaks numbered a to f correspond to the numbered hydrogen areas in the structure. It can be seen that the number of hydrogens numbered f in the ME3GE structure is more than that in ME2GE, and the f peak area in the spectrum also increases accordingly.

[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, where the molar ratio of the 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 the ME2GE monomer, salenCoCl, PPNCL and chain transfer agent TPE-OH being 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] 3 g of ME2GE monomer, 27.2 mg of salenCoCl, 24.4 mg of PPNCL and 74.2 mg of chain transfer agent TPE-OH were respectively placed in a 10 mL autoclave, the molar ratio of the ME2GE monomer, salenCoCl, PPNCL and chain transfer agent TPE-OH being 400:1:1:5, 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 8100.

[0076] Example 7

[0077] 3 g of ME2GE monomer, 27.2 mg of salenCoCl, 24.4 mg of PPNCL and 148.3 mg of chain transfer agent TPE-OH were respectively placed in a 10 mL autoclave, the molar ratio of the ME2GE monomer, salenCoCl, PPNCL and chain transfer agent TPE-OH being 400:1:1:10, 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 6400.

[0078] Figure 3 (a) shows the molecular weight of polycarbonate polymer P-ME2GE with a molecular weight of 6400. 1 H NMR spectrum and structure, where the peaks labeled a to e correspond to the hydrogen areas of the polymer units in the structure, 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] 3 g of ME3GE monomer, 21.8 mg of salenCoCl, 19.6 mg of PPNCL and 11.9 mg of chain transfer agent TPE-OH were respectively placed in a 10 mL autoclave, the molar ratio of the ME3GE monomer, salenCoCl, PPNCL and chain transfer agent TPE-OH being 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-ME3GE with a number average molecular weight of 17900.

[0081] Example 9

[0082] 3 g of ME3GE monomer, 21.8 mg of salenCoCl, 19.6 mg of PPNCL and 23.7 mg of chain transfer agent TPE-OH were respectively placed in a 10 mL autoclave, wherein the molar ratio of the ME3GE 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-ME3GE with a number average molecular weight of 15400.

[0083] Example 10

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

[0085] Embodiment 11

[0086] 3 g of ME3GE monomer, 21.8 mg of salenCoCl, 19.6 mg of PPNCL and 118.7 mg of chain transfer agent TPE-OH were respectively placed in a 10 mL autoclave, the molar ratio of the ME3GE monomer, salenCoCl, PPNCL and chain transfer agent TPE-OH being 400:1:1:10, 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-ME3GE with a number average molecular weight of 7500.

[0087] Figure 3 (b) shows the molecular weight of polycarbonate polymer P-ME3GE with a molecular weight of 7500. 1 H NMR spectrum and structure, where the peaks labeled a to e correspond to the hydrogen areas of the polymer units in the structure, 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 for cyclic carbonate (polymer) test method:

[0090] Each polymer was dissolved in CDCl3 to obtain a 5 mg / mL solution and then heated at 273 K. 1The HNMR spectrum obtains the chemical shift peak of hydrogen in the polymer solution, and the content of carbonate units (CU) in the polycarbonate polymer is determined by comparing the integral of the product peak and the by-product peak.

[0091] The number average molecular weight of polycarbonate polymers was determined using gel permeation chromatography: the GPC tester was calibrated with polystyrene standards at 35°C, the eluent was CH2Cl2, the column pressure was 447 Pa, and the flow rate was 1 mL / min. Each polymer was then dissolved in CH2Cl2 to obtain a 3 mg / mL solution. The GPC trace of the polymer was obtained in the same way, and the number average molecular weight (Mn) and polydispersity of the polymer were determined by integration.

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

[0093] Table 1 Experimental results of polycarbonate polymer

[0094]

[0095] As can be seen from Table 1, the polycarbonate polymers P-ME2GE and P-ME3GE have a high selectivity level for cyclic carbonates, which indicates that the catalyst active center effectively inhibits the backbiting reaction of the active chain; at the same time, the content of carbonate units in the polycarbonate polymers is above 99%, which indicates that no continuous homopolymerization side reaction of epoxide occurs.

[0096] Test Example 2

[0097] GPC traces of polycarbonate polymers P-ME2GE and P-ME3GE with different molecular weights were tested using gel permeation chromatography. The GPC tester was calibrated with polystyrene standards at 35°C, the eluent was CH2Cl2, the column pressure was 447Pa, and the flow rate was 1mL / min. After that, each polymer was dissolved in CH2Cl2 to obtain a 3mg / mL solution, and the GPC traces of the polymers were obtained. The results are shown in Figure 4 shown. Figure 4 (a) shows the GPC curves of polycarbonate polymer P-ME2GE with different molecular weights. Figure 4 (b) shows the GPC curves of polycarbonate polymers P-ME3GE with different molecular weights. It can be seen that the trajectory curves of polycarbonate polymers P-ME2GE and P-ME3GE gradually change from bimodal distribution to unimodal distribution as the proportion of polymerization monomer decreases.

[0098] A linear relationship diagram was drawn with the Mn value of polycarbonate polymers P-ME2GE and P-ME3GE of different molecular weights as the ordinate and the M / I value as the abscissa, and the correlation coefficient (R 2 ) and linear equations, the results are as follows Figure 5 shown. Figure 5 (a) is the linear relationship diagram of polycarbonate polymer P-ME2GE, Figure 5 (b) is the linear relationship diagram of the 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, thereby obtaining a polycarbonate polymer immune adjuvant that adapts to specific antigens or immune responses.

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

[0101] Test Example 4

[0102] The polycarbonate polymer P-ME2GE prepared in Example 7 was dissolved in water to obtain a 5 mg / mL solution, and the P-ME2GE nanoadjuvant was obtained after ultrasonic stirring. CVS11 was co-incubated with 10 nm gold-labeled antibody, and LTB protein was co-incubated with 18 nm gold-labeled antibody. After incubation, CVS11 and LTB protein were blended with the above P-ME2GE nanoadjuvant (mass ratio 1:1:4). Figure 7 (a) shows P-ME2GE observed under electron microscopy; Figure 7 (b) CVS11 virus particles and LTB observed by immunoelectron microscopy, from which it can be seen that CVS11 virus particles and LTB are attached to the surface of P-ME2GE; Figure 7 (c) is the Zeta potential diagram of the combination of P-ME2GE, CVS11 and LTB. It can be seen that the Zeta potential continues to decrease during the combination process. The above experimental data show that P-ME2GE, CVS11 and LTB 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 the P-ME2GE nanoadjuvant was obtained after ultrasonic stirring. The inactivated CVS11 antigen was combined with aluminum gel adjuvant, LTB, P-ME2GE, LTB and P-ME2GE (mass ratio 1:1) in a ratio of 1:4 of the mass of the immune adjuvant to the vaccine antigen to obtain a vaccine, which was recorded as the experimental group. The PBS buffer solution was mixed with P-ME2GE to obtain a 5 mg / mL solution, which was recorded together with the inactivated CVS11 antigen as the control group.

[0105] Mice were immunized with equal volumes of the above experimental group and control group vaccines at 0, 2, and 4 weeks. After three immunizations, blood was collected from the orbital vein of the mice at 0 to 8 weeks, and serum samples were collected by centrifugation. The neutralizing antibody titer in the serum was determined by rabies neutralization test, and the data of each group per week were recorded and summarized in Table 2. Figure 8 (a) Timeline of vaccination of mice. Figure 8 (b) is a schematic diagram of the mixing of P-ME2GE nanoadjuvant, antigen and LTB. Figure 8 (c) is the neutralizing antibody titer in mouse serum.

[0106] Table 2. Serum neutralizing antibody titer data of mice in each group

[0107]

[0108] From Table 2 and Figure 8 As can be seen, P-ME2GE as an adjuvant can produce antibody titers far higher than the standard 0.5 IU / mL level, indicating that P-ME2GE induced a large number of protective antibodies against RABV in mice. In addition, the effects of P-ME2GE and LTB as adjuvants were both higher than P-ME2GE or LTB alone, indicating that P-ME2GE can play a role in delivering 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 Experimental Example 5 were used to immunize the mice at 0, 2, and 4 weeks. In the 5th week, 10 mice were injected intramuscularly. 5.75 TCID 50 The mice were challenged with RABV CVS11 at a dose of Fig. 9 (a) is a schematic diagram of the time of injection of vaccine and RABV antigen into mice. The clinical symptoms and survival rate of mice were observed every day, and the data of survival rate and survival time of mice are summarized in Table 3. Fig. 9(b) is the survival rate of mice in each group, 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. Fig. 9 (c) is the clinical scores of mice injected with experimental and control group vaccines. It can be seen that the experimental group injected with P-ME2GE and LTB as adjuvants had the highest score, which indicates that the mixture of P-ME2GE and LTB as adjuvants provides complete protection for mice.

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

[0112]

[0113] As can be seen from Table 3, the mice in the experimental group injected with P-ME2GE as an adjuvant survived longer, and the mice in the experimental group injected with P-ME2GE and LTB as adjuvants survived the longest, which indicates that P-ME2GE as an adjuvant provides effective protection against RABV infection, and the effects of P-ME2GE and LTB as adjuvants are both higher than those of P-ME2GE or LTB alone as an adjuvant.

[0114] Test Example 7

[0115] The experimental group and control group vaccines in Experimental Example 4 were used to immunize mice at 0, 2, and 4 weeks. The spleens of mice were collected one week after the third immunization. Three mice were taken from each group. Splenocytes were restimulated with inactivated CVS11, and an in vitro splenocyte proliferation test was performed to evaluate the effect of the adjuvant on the splenocyte proliferation response. The results are summarized in Table 4. Fig.10 (a) is a schematic diagram of the test time, and the numbers in the arrows represent days. Fig.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 the P-ME2GE and LTB mixed group of mice is higher than that of the other groups of mice.

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

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

[0118]

[0119] Table 5 Number of ELISPOT IL-4 spots and IFN-γ spots in each group of mice

[0120]

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

[0122] Test Example 8

[0123] The 5 mg / mL P-ME2GE solution prepared in Experimental Example 4 was injected intramuscularly into mice, and the PBS solution was used as a control. The blood of the mice was collected for blood biochemical tests and blood routine tests, and the data of various indicators are summarized in Table 6. Fig.11 (a) is the result of blood biochemical test, Fig.11 (b) is the result of routine blood test, from which it can be seen that compared with the control group injected with PBS solution, the blood of mice injected with P-ME2GE solution had no abnormal indicators. At the same time, physiological sections of the main organs of mice (including brain, heart, liver, spleen, lung, kidney, muscle and lymph node) were collected and stained with hematoxylin-eosin (H&E). Fig.11 (c) is a physiological section after staining, from which it can be seen that there are no obvious histological abnormalities in the mice.

[0124] Table 6 Mouse blood index data

[0125]

[0126] The above results indicate that P-ME2GE is a safe biomaterial suitable for in vivo vaccine adjuvant.

[0127] The above description is only a preferred specific implementation manner of the present invention, but the protection scope of the present invention is not limited thereto. Any technician familiar with the technical field can make equivalent replacements or changes according to the technical scheme and inventive concept of the present invention within the technical scope disclosed by the present invention, which should be covered by the protection scope of the present invention.

Claims

1. A polycarbonate polymer, characterized in that Having the structure of formula (I): wherein n is selected from an integer of 2 to 10, and m is selected from an integer of 10 to 1000; R1 is selected from C1~C 20 Alkyl, C1~C 20 Alkyl, C2~C 20 Alkenyl, C2-C 20 Alkenyl, C2~C 20 Alkynyl, C2-C 20 Alkynyl, C6~C 20 aromatic group, C6~C 20 Aromatic groups, C6~C 20 or a C6-C 20 The heteroaryl group is selected from C1 to C 20 Alkyl, C2~C 20 Alkenyl, C2~C 20 Alkynyl, C6~C 20 Aromatic groups and C6~C 20 heteroaryl; R2 is selected from C1~C 20 Alkyl, C1~C 20 Alkoxy, C1~C 20 Alkylthio or C2~C 20 of ether.

2. The polycarbonate polymer according to claim 1, characterized in that The R1 is selected from C1~C 10 Alkyl, C1~C 10 Alkyl, C2~C 10 Alkenyl, C2-C 10 Alkenyl, C2~C 10 Alkynyl or at least one substituent substituted C2-C 10 The alkynyl group, the substituent is selected from C1 to C 10 Alkyl, C2~C 10 Alkenyl, C2~C 10 Alkynyl and C6~C 10 Aromatic groups; The R2 is selected from C1~C 10 Alkyl, C1~C 10 Alkoxy or C1~C 10 of alkylthio.

3. The polycarbonate polymer according to claim 2, characterized in that The R1 is selected from C2 to C 10 The substituent is selected from C1 to C 10 Alkyl and C6~C 10 Aromatic groups; The R2 is selected from C1~C 10 of alkoxy.

4. The polycarbonate polymer according to claim 1, characterized in that It has the following structure: Herein, n is selected from an integer of 2 to 10, and m is selected from an integer of 10 to 1000.

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

6. A method for preparing a polycarbonate polymer, characterized in that: include: The aromatic compound of formula (II), the monomer of formula (III) and a catalyst are polymerized in a CO2 system to obtain a polycarbonate polymer; Wherein, n is selected from an integer of 2 to 10; R1 is selected from C1~C 20 Alkyl, C1~C 20 Alkyl, C2~C 20 Alkenyl, C2-C 20 Alkenyl, C2~C 20 Alkynyl, C2-C 20 Alkynyl, C6~C 20 aromatic group, C6~C 20 Aromatic groups, C6~C 20 or a C6-C 20 The heteroaryl group is selected from C1 to C 20 Alkyl, C2~C 20 Alkenyl, C2~C 20 Alkynyl, C6~C 20 Aromatic groups and C6~C 20 heteroaryl; R2 is selected from C1~C 20 Alkyl, C1~C 20 Alkoxy, C1~C 20 Alkylthio or C2~C 20 of ether.

7. The polycarbonate polymer according to claim 6, characterized in that The pressure of the CO2 system is 1-5 MPa.

8. A nanoadjuvant, characterized in that: The invention comprises LTB protein, the polycarbonate polymer according to any one of claims 1 to 5 or the polycarbonate polymer prepared by the preparation method according to any one of claims 6 and 7.

9. A vaccine composition, characterized in that The invention comprises the polycarbonate polymer according to any one of claims 1 to 5, the polycarbonate polymer prepared by the preparation method according to any one of claims 6 and 7, or the nano adjuvant according to claim 8, as well as antigens and other auxiliary materials.

10. The vaccine composition according to claim 9, characterized in that The antigen is a rabies virus antigen.

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