Fluorinated or alkylated polyaspartic acid as well as preparation method and application thereof in vaccine delivery

Abstract

The invention relates to the technical field of nano vaccines, in particular to fluorinated or alkylated polyaspartic acid as well as a preparation method and application thereof in vaccine delivery. The fluorinated or alkylated polyaspartic acid has a structure as shown in a formula I in the specification. The fluorinated or alkylated N-substituted polyaspartic acid provided by the invention has the advantages of excellent self-assembly capability, good biocompatibility and relatively low cytotoxicity. When the nano vaccine is prepared, the loaded antigen and fluorinated or alkylated polyaspartic acid can be co-assembled into nano particles through hydrophobic interaction. In addition, the fluorinated or alkylated N-substituted polyaspartic acid retains the relatively high cell intake and endosome escape levels of the N-substituted polyaspartic acid per se. Thus, the fluorinated or alkylated N-substituted polyaspartic acid may enhance antigen uptake and facilitate cross presentation. # imgabs0 #

Description

Technical Field

[0001] The present invention relates to the technical field of nano vaccines, and in particular to a fluorinated or alkylated polyaspartic acid and a preparation method thereof and application thereof in vaccine delivery. Background Art

[0002] Cancer is a major intractable disease that threatens people's life and health, and it imposes a heavy burden on my country's medical and health system. At present, traditional cancer treatments such as chemotherapy and radiotherapy have problems such as insufficient efficacy, large side effects, drug resistance, and tumor metastasis and recurrence after treatment. Tumor immunotherapy is a rapidly developing cancer treatment strategy after surgical therapy, chemotherapy, and radiotherapy, which aims to restore or enhance immune cell recognition and elimination of tumors. Among them, tumor vaccines are an important component and cutting-edge hotspot in current cancer immunotherapy. Tumor vaccines are composed of tumor antigens, adjuvants, and carrier materials. Vaccine carriers produce specific CD8 by delivering appropriate tumor antigens or immunostimulatory adjuvants to antigen presenting cells (APCs). + T cells and CD4 + T cells, thereby clearing primary tumor cells and micrometastatic lesions, and stimulating the body to produce tumor-specific immune memory cells, establishing a long-lasting anti-tumor immune memory response, and preventing tumor recurrence.

[0003] In order to achieve efficient anti-tumor effects, vaccine carriers, as an important component of tumor vaccines, affect the efficacy, quality and stability of vaccines. Vaccine carriers need to meet the following requirements: 1. High safety and storage stability, easy to prepare on a large scale; 2. Effectively protect antigens and immunostimulants, and prevent antigen and immunostimulant degradation; 3. Enhance the enrichment of antigens and immunostimulants in lymphoid organs and the uptake of antigen-presenting cells, so as to achieve the simultaneous delivery of antigen molecules and immunostimulants to APCs; 4. Enhance the cross-presentation ability of antigens, and promote antigens to enter the cytoplasm across endosomes / lysosomes for processing and presentation; 5. Have multi-mode APCs activation characteristics to enhance the immune activation effect of vaccines. Currently, commercial aluminum adjuvants, Poly (I: C) and viral vectors have weak antigen protection, strong side effects and gene integration risks. In particular, the preparation cost and technical threshold of viral vectors are high, and the antigen load is low, which makes it difficult to meet the current requirements for therapeutic vaccine carriers. The development of safe vaccine carrier materials with strong cellular immune response ability is still a bottleneck problem that needs to be solved in the field of therapeutic vaccines. Summary of the invention

[0004] In view of this, the technical problem to be solved by the present invention is to provide a fluorinated or alkylated polyaspartic acid and a preparation method thereof and an application in vaccine delivery, wherein the prepared nano vaccine has efficient cytoplasmic delivery and self-adjuvant properties.

[0005] To achieve the above object, the present invention provides a fluorinated or alkylated polyaspartic acid having a structure shown in Formula I:

[0006]

[0007] Wherein, m is any integer from 1 to 9;

[0008] n is any integer from 1 to 250;

[0009] x is any integer from 1 to 250, and n is greater than x;

[0010] R is selected from the following structure shown in Formula II or Formula III:

[0011]

[0012] In formula II, a is any integer from 1 to 9; b is any integer from 0 to 9; c is any integer from 1 to 200;

[0013] In formula III, d is any integer from 1 to 18;

[0014] * indicates the binding site of the group.

[0015] Preferably, m is any integer from 2 to 4. In some specific embodiments of the present invention, m is 2, 3 or 4.

[0016] Preferably, n is any integer from 70 to 140.

[0017] Preferably, x is any integer from 25 to 75.

[0018] Preferably, in Formula II, a is any integer from 1 to 2; b is any integer from 2 to 6; and c is any integer from 1 to 60.

[0019] Preferably, in Formula III, d is any integer from 2 to 9.

[0020] The molecular weight of the fluorinated or alkylated polyaspartic acid is preferably 4,000 to 60,000 Daltons, more preferably 10,000 to 30,000 Daltons.

[0021] The present invention combines N-substituted polyaspartic acid with corresponding amine compounds to obtain a series of polycationic polymers with the same skeleton but with a variety of cationic side chain structures. Fine-tuning / arrangement of the aminoethylene structure in the polymer chain can significantly reduce the cytotoxicity of the polycation, produce superior pH sensitivity, possess stronger buffering capacity and unique changes in the main protonated structure. The N-substituted polyaspartic acid material with multi-chain has excellent cell transfection ability and can achieve efficient cytoplasmic delivery. Modification of N-substituted polyaspartic acid with fluorine-containing or alkyl chains can better regulate the local cationic charge, achieve efficient antigen loading, and realize efficient antigen cross-presentation, thereby enhancing the anti-tumor immune effect.

[0022] The test results show that the N-substituted polyaspartic acid prepared by the present invention can significantly improve the bioavailability and precise controllability of synthesis when used as the main chain, produce excellent pH sensitivity, have stronger buffering capacity, and reduce the cytotoxicity of polycations.

[0023] The present invention provides a method for preparing the above-mentioned fluorinated or alkylated polyaspartic acid, comprising the following steps:

[0024] S1) using an initiator having an amine terminal as shown in formula IV to initiate the ring-opening polymerization of L-aspartic acid-4-benzyl ester anhydride as shown in formula V to obtain polyaspartic acid as shown in formula VI;

[0025] S2) subjecting the polyaspartic acid represented by formula VI to an aminolysis reaction with diethylenetriamine to obtain an N-substituted polyaspartic acid represented by formula VII;

[0026] S3) reacting the N-substituted polyaspartic acid represented by formula VII with the alkylene oxide represented by formula VIII-a or the fluorinated alkylene oxide represented by formula VIII-b to obtain the fluorinated or alkylated polyaspartic acid represented by formula I;

[0027]

[0028]

[0029] R is selected from the following structure shown in Formula II or Formula III:

[0030]

[0031] Wherein, m is any integer from 1 to 9;

[0032] n is any integer from 1 to 250;

[0033] x is any integer from 1 to 250, and n is greater than x.

[0034] In formula III and formula VIII-a, d is any integer from 1 to 18;

[0035] In Formula II and Formula VIII-b, a is an arbitrary integer from 1 to 9; b is an arbitrary integer from 0 to 9; and c is an arbitrary integer from 1 to 200.

[0036] The ranges of m, n, x, a, b, c, and d are the same as above and will not be repeated here.

[0037] In the step S1), the solvent for the ring-opening polymerization is preferably dimethylformamide and dichloromethane. The volume ratio of dimethylformamide to dichloromethane is preferably 1:5-20, more preferably 1:8-12.

[0038] The temperature of the ring-opening polymerization is preferably 0 to 50° C., more preferably 30 to 40° C.; the time of the ring-opening polymerization is preferably 48 to 96 h, more preferably 48 to 72 h.

[0039] Preferably, in the present invention, after the above ring-opening polymerization reaction is completed, the product is precipitated by glacial acetic acid, filtered and dried to obtain polyaspartic acid represented by formula VI, denoted as PBLA.

[0040] In some specific embodiments of the present invention, the reaction equation of step S1) is as follows:

[0041]

[0042] Preferably, the step S1) is specifically:

[0043] The initiator with an amine terminal as shown in Formula IV is dissolved in dimethylformamide and then diluted with dichloromethane. After it is completely dissolved, the initiator diluted with dichloromethane is added to the L-aspartic acid-4-benzyl ester internal anhydride solution as shown in Formula V, and reacted under dry oxygen-free conditions.

[0044] Then the aminolysis reaction is carried out.

[0045] The temperature of the ammonolysis reaction is preferably 0-10°C, more preferably 0°C; the time of the ammonolysis reaction is preferably 1-4h, more preferably 1-2h.

[0046] Preferably, after reacting under the above conditions, the reaction is further carried out at room temperature for 1 to 4 hours, more preferably at room temperature for 1 to 2 hours.

[0047] Preferably, the molar ratio of the polyaspartic acid represented by formula VI to diethylenetriamine is 1:10-40, more preferably 1:20-30.

[0048] Preferably, after the aminolysis reaction is completed, the product is precipitated by glacial acetic acid, filtered and dried to obtain N-substituted polyaspartic acid.

[0049] In some specific embodiments of the present invention, the reaction equation of step S2) is as follows:

[0050]

[0051] Preferably, the step S2) is specifically:

[0052] The polyaspartic acid (PBLA) obtained in step S1) is dissolved in N-methylpyrrolidone (NMP), and then diethylenetriamine is diluted with NMP. Under ice bath conditions, the PBLA solution is added dropwise into the diethylenetriamine solution to react.

[0053] Then, alkyl modification or fluorine-containing modification is performed.

[0054] The reaction temperature of the N-substituted polyaspartic acid and the alkylene oxide represented by formula VIII-a is preferably 15-35°C, more preferably 20-30°C; the reaction time is preferably 24-72h, more preferably 36-48h.

[0055] The molar ratio of the N-substituted polyaspartic acid to the alkylene oxide represented by formula VIII-a is preferably 1:5-50, more preferably 1:5-30.

[0056] Preferably, an acid binding agent is added during the reaction. The present invention has no particular limitation on the type of the acid binding agent, and the acid binding agent may be an acid binding agent well known to those skilled in the art. In some specific embodiments of the present invention, the acid binding agent is preferably triethylamine.

[0057] The molar ratio of the N-substituted polyaspartic acid to the acid-binding agent is preferably 1:1-10, more preferably 1:2-4.

[0058] Preferably, in the present invention, after the N-substituted polyaspartic acid and the alkylene oxide represented by formula VIII-a react completely, the product is placed in a dialysis bag and dialyzed in methanol, then dialyzed against deionized water, and finally freeze-dried to obtain the alkylated N-substituted polyaspartic acid.

[0059] Preferably, the reaction is specifically:

[0060] The N-substituted polyaspartic acid obtained above was dissolved in methanol, and the alkylene oxide molecules dissolved in methanol were added dropwise to the methanol, and triethylamine (C6H 15 N) stirring the reaction, dialyzing and freeze-drying to obtain the alkylated N-substituted polyaspartic acid.

[0061] The alkylene oxide is preferably a C3-C10 alkylene oxide, more preferably a C3-C10 ethylene oxide. In some specific embodiments of the present invention, the alkylene oxide is heptyl ethylene oxide.

[0062] The reaction temperature of the N-substituted polyaspartic acid and the fluorinated alkylene oxide represented by formula VIII-b is preferably 15 to 35°C, more preferably 20 to 30°C; the reaction time is preferably 24 to 72 hours, more preferably 36 to 48 hours.

[0063] The molar ratio of the N-substituted polyaspartic acid to the fluorinated alkylene oxide represented by formula VIII-b is preferably 1:5-50, more preferably 1:5-30.

[0064] Preferably, an acid binding agent is added during the reaction. The present invention has no particular limitation on the type of the acid binding agent, and the acid binding agent may be an acid binding agent well known to those skilled in the art. In some specific embodiments of the present invention, the acid binding agent is preferably triethylamine.

[0065] The molar ratio of the N-substituted polyaspartic acid to the acid-binding agent is preferably 1:1-10, more preferably 1:2-4.

[0066] Preferably, in the present invention, after the N-substituted polyaspartic acid and the fluorinated alkylene oxide shown in formula VIII-b are completely reacted, the product is placed in a dialysis bag and dialyzed in methanol, then dialyzed against deionized water, and finally freeze-dried to obtain the fluorinated N-substituted polyaspartic acid.

[0067] Preferably, the reaction is specifically:

[0068] The N-substituted polyaspartic acid obtained above was dissolved in methanol, and the fluorinated alkylene oxide molecules dissolved in methanol were added dropwise to the methanol, and triethylamine (C6H 15 N) stirring the reaction, dialyzing and freeze-drying to obtain fluorinated N-substituted polyaspartic acid.

[0069] The fluorinated alkylene oxide is preferably a C3-C10 fluorinated alkylene oxide, more preferably a C3-C10 fluorinated ethylene oxide. In some specific embodiments of the present invention, the fluorinated alkylene oxide is tridecafluoroheptylethylene oxide.

[0070] The present invention provides the use of the fluorinated or alkylated polyaspartic acid or the fluorinated or alkylated polyaspartic acid prepared by the above preparation method as an antigen carrier and / or immune adjuvant of a nano vaccine.

[0071] Based on this, the present invention provides a nano vaccine comprising an antigen and the above-mentioned fluorinated or alkylated polyaspartic acid.

[0072] The fluorinated or alkylated polyaspartic acid is used as an antigen carrier and / or immune adjuvant.

[0073] Preferably, the nanovaccine is an anti-tumor vaccine.

[0074] The antigen includes, but is not limited to, one or more of protein, polypeptide, tumor lysate, DNA and RNA.

[0075] Furthermore, the antigen includes one or more of tumor-specific proteins, tumor neoantigen polypeptides, tumor neoantigen mRNA and tumor lysates.

[0076] The above-mentioned fluorinated or alkylated N-substituted polyaspartic acid can be effectively recruited to lymph nodes, enhance the cross-presentation ability of antigen-presenting cells to antigens, and activate antigen-specific CD8 immune responses.

[0077] The present invention has no particular limitation on the preparation method of the above-mentioned nano-vaccine, and the nano-vaccine can be prepared according to methods well known to those skilled in the art, including but not limited to self-assembly methods.

[0078] In some specific embodiments of the present invention, the fluorinated or alkylated N-substituted polyaspartic acid is mixed with an antigen in water to obtain a nanovaccine.

[0079] The antigen includes but is not limited to chicken ovalbumin (OVA) and the like.

[0080] The mixing time is preferably 10 to 50 minutes, more preferably 30 minutes.

[0081] In the above-mentioned nano-vaccine, the antigen loading rate can reach more than 90%.

[0082] The present invention provides the use of the nano vaccine in preparing a drug for preventing, treating and / or alleviating tumors.

[0083] The tumor nanovaccine provided by the present invention can not only have a certain preventive effect on tumors, but also can effectively inhibit the growth of tumors as a therapeutic vaccine.

[0084] The tumor includes but is not limited to B16-OVA melanoma tumor.

[0085] The fluorinated or alkylated N-substituted polyaspartic acid provided by the present invention can not only be used as a carrier of various types of antigens, directly delivering antibodies to the cytoplasm of antigen presenting cells (APCs), which is beneficial to the cross-presentation of antigens; it can also induce the activation of antigen presenting cells APCs, produce the function of immune adjuvant, and induce antigen-specific cellular immune response. This dual function is not possessed by general immune adjuvants or carriers.

[0086] Compared with the prior art, the present invention provides a fluorinated or alkylated polyaspartic acid having a structure shown in formula I.

[0087] The fluorinated or alkylated polyaspartic acid provided by the present invention has excellent self-assembly ability, good biocompatibility and low cytotoxicity. When preparing nano vaccines, the loaded antigen can be co-assembled into nanoparticles with the fluorinated or alkylated N-substituted polyaspartic acid through hydrophobic interaction. In addition, the fluorinated or alkylated N-substituted polyaspartic acid retains the higher cellular uptake and endosomal escape levels of the N-substituted polyaspartic acid itself. Therefore, the fluorinated or alkylated N-substituted polyaspartic acid can enhance antigen uptake and promote cross presentation. BRIEF DESCRIPTION OF THE DRAWINGS

[0088] Figure 1 This is the H-NMR spectrum of the N-substituted polyaspartic acid prepared in Example 1;

[0089] Figure 2 This is the H NMR spectrum of the alkylated N-substituted polyaspartic acid prepared in Example 2;

[0090] Figure 3 This is the H NMR spectrum of the fluorinated N-substituted polyaspartic acid prepared in Example 3;

[0091] Figure 4 is the particle size distribution diagram of different fluorinated or alkylated N-substituted polyaspartic acid;

[0092] Figure 5 Confocal fluorescence images of DC2.4 cells after incubation with different nanovaccines for 24 h;

[0093] Figure 6 The levels of SIINFEKL-H2Kb expressed by BMDC cells after 24 h of stimulation with different nanovaccines;

[0094] Figure 7 is the level of IFN-β secreted in the supernatant after 24 h of stimulation of BMDC cells with different nanovaccines;

[0095] Figure 8 is the accumulation of different nanovaccines in lymph nodes;

[0096] Fig. 9 The percentage and statistical graph of IFN-γ positive T cells in spleen cells stimulated by different nanovaccines;

[0097] Fig.10 The figure shows the therapeutic effects of different nanovaccines on the B16-OVA tumor model. DETAILED DESCRIPTION

[0098] In order to further illustrate the present invention, the following is a detailed description in conjunction with the embodiments. However, it should be understood that these descriptions are only for further illustrating the features and advantages of the present invention, rather than limiting the claims of the invention.

[0099] All raw materials of the present invention have no particular limitation on their sources, and can be purchased from the market or prepared according to conventional methods known to those skilled in the art.

[0100] Example 1

[0101] Preparation of N-substituted polyaspartic acid

[0102] Synthesis of poly(β-benzyl-L-aspartic acid) (PBLA). PBLA was synthesized by ring-opening polymerization of L-aspartic acid-4-benzyl ester-N-carboxyl cyclic anhydride (BLA-NCA) initiated by n-hexylamine. 5 g, 10 g and 15 g of BLA-NCA were weighed and dissolved in DMF (10 mL), and then diluted with CH2Cl2 (100 mL). n-Hexylamine (41.22 mg) diluted with CH2Cl2 was added to the BLA-NCA solution. The reaction solution was stirred at 35°C for 3 days. All processes were carried out under a dry argon environment. It was precipitated in an excess of ice ether, filtered and dried to obtain PBLA with a degree of polymerization of 50 (4.02 g, yield 95.2%), PBLA with a degree of polymerization of 100 (8.10 g, yield 97.9%) and PBLA with a degree of polymerization of 150 (12.11 g, yield 96.0%).

[0103] Aminolysis of PBLA. The above-mentioned dried PBLA (1.1 g) with a degree of polymerization of 50, 100, and 150 were dissolved in 8 mL of N-methylpyrrolidone (NMP), and then diethylenetriamine (13.8 g) was diluted with NMP. Under ice bath conditions, the PBLA solution was added dropwise to the diethylenetriamine solution. The reaction was stirred at 0°C for 1 hour, and then stirred at room temperature for another 2 hours. It was precipitated in excess ice ether, filtered and dried to obtain poly [N- (2-aminoethyl)] asparagine (hereinafter abbreviated as DET) with three degrees of polymerization, among which DET with a degree of polymerization of 50 was obtained. 50 (0.85 g, yield 79.4%), DET with a polymerization degree of 100 100 (0.87 g, yield 81.3%), DET with a degree of polymerization of 150 150 (0.96 g, yield 89.7%).

[0104] Figure 1 The three degrees of polymerization DET described in this example are 1 H NMR.

[0105] Example 2

[0106] Preparation of alkylated N-substituted polyaspartic acid

[0107] 1,2-Epoxydecane was added dropwise to a DET methanol solution with a polymerization degree of 100 at a ratio of 10%, 20%, and 30%, and stirred for 24 hours. Specific steps: 3 portions of DET (200 mg each) were weighed and dissolved in 2 mL of methanol (CH3OH). In addition, 15.6 mg, 31.2 mg, and 46.8 mg of heptyl oxirane were weighed and dissolved in 2 mL of CH3OH, and 500 μL of triethylamine (C6H 15 N), stirred at room temperature for 48 h, then dialyzed against CH3OH overnight and deionized water for two more days, and freeze-dried to obtain three different alkyl-modified DET white powders DET-C10 68 mg, DET-C20 120 mg, and DET-C30 171 mg.

[0108] Figure 2 The three different alkyl-modified N-substituted polyaspartic acids prepared in this example are 1 H NMR. As the alkyl modification rate of the side chains of DET-C10 to DET-C30 increases, the grafting ratio also increases.

[0109] Example 3

[0110] Preparation of Fluorinated N-Substituted Polyaspartic Acid

[0111] 10%, 20% and 30% were added dropwise to a DET methanol solution with a polymerization degree of 100 and stirred for 24 hours. Specific steps: 3 portions of DET (200 mg each) were weighed and dissolved in 2 mL of CH3OH. In addition, 37.8 mg, 75.2 mg and 112.8 mg of tridecafluoroheptyl oxirane were weighed and dissolved in 2 mL of CH3OH, and 500 μL of triethylamine (C6H 15 N), stirred at room temperature for 48 h. Then dialyzed against CH3OH overnight and deionized water for two more days, and freeze-dried to obtain three fluorine-modified white powders DET-F10 84 mg, DET-F20 165 mg, and DET-F30 201 mg. 19 The synthesized materials were characterized by F NHR.

[0112] Figure 3 The fluorinated N-substituted polyaspartic acid DET-F30 prepared in this example 19 F NHR. It was calculated that each DET-F30 molecule was actually grafted with 26.6 tridecafluoro molecules.

[0113] Example 4

[0114] Preparation of Nano-Vaccines Loaded with Mimic Antigens

[0115] Loading with simulated antigen hen egg white albumin (OVA): The above polymer carrier was loaded with OVA by self-assembly method to investigate the assembly properties of different modified materials with OVA. They were physically mixed with OVA simulated protein in deionized water for 30 minutes according to the weight ratio, and the size of the formed DET-C and DET-F nanoparticles with different modification degrees was measured by a particle size analyzer.

[0116] Figure 4 The particle size distribution diagram of different fluorinated DET prepared in this example. Different fluorinated DET simply mixes polycations with OVA protein in an aqueous environment through electrostatic interaction to produce multiple ion pairs. In the absence of mixing with OVA, as the fluorinated modification ratio increases, it is easier to form nanoparticles with uniform particle size. In addition, as the amount of OVA loading increases, its particle size also increases.

[0117] Example 5

[0118] Determination of loading efficiency of OVA-loaded nanoparticle vaccine

[0119] The DET obtained in Example 3 was modified with 10%, 20%, and 30% of tridecafluoroheptyl oxirane (DET-F10, DET-F20, and DET-F30), and the DET obtained in Example 2 was modified with 10%, 20%, and 30% of heptyl oxirane (DET-C10, DET-C20, and DET-C30), respectively, and was physically mixed with OVA-FITC at a mass ratio of 1:0.2, 1:0.5, 1:1, and 1:2 for 30 minutes. Subsequently, 400 μL of each of the above mixtures was added to an ultrafiltration tube, and the OVA-FITC not loaded with the compound was separated by two low-speed centrifugations at 3000 rpm, and the content of OVA-FITC at the bottom of the ultrafiltration tube was measured, and the loading of different materials and OVA-FITC was calculated.

[0120] Table 1 Loading of different materials and OVA-FITC

[0121]

[0122] Table 1 is a summary table of the loading of different materials and OVA-FITC in this embodiment. As the ratio of OVA loaded increases, the loading efficiency of the polymer carrier material gradually decreases. For example, after DET-C10 and OVA are assembled at a ratio of 1:0.2, the loading ratio is 80.66%, while after DET-C10 and OVA are assembled at a ratio of 1:2, the loading ratio drops to 51.09%. When the polymer carrier and OVA are assembled at a weight ratio of 1:0.5, the loading efficiency of the fluorine-modified DET polymer carrier reaches more than 90%.

[0123] Example 6

[0124] Endosomal escape effect of nanovaccines

[0125] Nanovaccines not only require a suitable delivery vehicle, but also need to be transported into cells and release the loaded antigen into the cytoplasm through endosomal escape. In order to evaluate the endosomal escape effect of nanovaccines, OVA labeled with FITC (OVA-FITC) was used, and DET, DET-C30, DET-F30 and OVA-FITC were physically mixed for 30 minutes at a mass ratio of 1:0.5 screened in Example 5 to prepare nanovaccines. Endosomal escape of nanovaccines was investigated by DC2.4 cells, and 2×10 5 Cells were cultured in complete medium (1% penicillin / streptomycin, 10% fetal bovine serum and 89% RPMI-1640) for 12 h. Subsequently, free OVA-FTTC and 10 μg / mL nanovaccine were co-incubated with DC2.4 cells for 24 h. The cells were washed three times with PBS and confocal fluorescence imaging was performed.

[0126] Figure 5 Confocal fluorescence images of DC2.4 cells incubated with different nanovaccines described in this example for 24 hours. The intracellular green fluorescence of DC2.4 cells incubated with DET-F30 was stronger than that of the control group, indicating that it can enhance the intracellular delivery of OVA through cationic and hydrophobic interactions. In addition, in DC2.4 cells incubated with DET-F30, most of the intracellular OVA-FITC was not co-localized with lysosomes, indicating that the DET-F30 polymer has endosomal escape properties.

[0127] Example 7

[0128] Effects of Nanovaccines on Antigen Cross-Presentation

[0129] The importance of antigen cross-presentation in nano vaccines is that it can significantly enhance the immune effect of vaccines, especially in activating cellular immune responses. According to the 1:0.5 weight ratio screened in Example 5, the above-mentioned differently modified nanomaterials were physically mixed with OVA for 30 minutes, and the formed nano vaccines were co-cultured with BMDC cells at a concentration of 10 μg / mL for 24 hours. The expression of SIINFEKL-H2Kb in CD11c positive cells was detected by flow cytometry (FCM) to investigate the antigen cross-presentation effect of nano vaccines in BMDC cells. The supernatant solution after nano vaccine stimulation of BMDC cells was collected, and the IFN-β level secreted in the supernatant was determined by IFN-β ELISA.

[0130] Figure 6The levels of antigen cross-presentation of different nanovaccines in BMDC cells described in this example. After 24 hours of stimulation of BMDC cells, the levels of SIINFEKL-H2Kb expressed in CD11c-positive cells by fluorinated or alkylated DET nanovaccines were significantly increased, indicating that they can improve the ability of antigen cross-presentation, and with the increase of the proportion of alkyl or fluorinated modification, the ability of antigen cross-presentation is enhanced.

[0131] Example 8

[0132] Accumulation of nanovaccines by draining lymph nodes

[0133] In order to evaluate the accumulation of nanovaccines in lymph nodes after injection, OVA (OVA-Cy5) was labeled with CY5, and DET, DET-C30, and DET-F30 nanovaccines loaded with fluorescently labeled OVA-Cy5 (wherein OVA-Cy5 was 15 μg) were prepared by the preferred 1:0.5 weight ratio of Example 5. After subcutaneous injection of free OVA-Cy5 and nanovaccines into the right inguinal groove of male C57BL / 6N mice for 24 hours, the draining lymph nodes were collected and ex vivo fluorescence imaging was performed using an IVIS imaging system at low magnification.

[0134] Figure 8 This example describes the statistical analysis of in vitro drainage lymph node imaging and fluorescence of different nanovaccine treatments. It can be clearly observed that after injection of free OVA-Cy5, the mouse draining lymph nodes cannot accumulate it well; while fluorinated and alkylated DET-F and DET-C can enhance the accumulation of OVA-Cy5 in the draining lymph nodes compared with DET.

[0135] Example 9

[0136] Cellular immune response of nanovaccines in vivo

[0137] The DET, DET-C30, and DET-F30 nanovaccines (75 μg) loaded with model protein antigen (OVA, 15 μg) were injected subcutaneously into the back of C57BL / 6N mice on days 0 and 7, respectively. 6 Mouse spleen cells were incubated with antigen SIINFEKL for 12 h under the stimulation of brefeldin A and monensin. IFN-γ-secreting T cells (CD3 + CD8 + IFN-γ).

[0138] Fig. 9 This example describes the use of flow cytometry to analyze the CD3 T cell subsets in the spleen of mice treated with different nanovaccines. + CD8 +The results showed that as the ratio of fluorination or alkylation increased, the nanovaccine induced IFN-γ + The proportion of T lymphocytes increased. It is worth noting that under the same modification ratio, the fluorinated DET nanovaccine induced higher levels of IFN-γ than the alkylated DET nanovaccine. Among them, DET-F30 was able to induce CD8 + T cells produced the highest level of IFN-γ, indicating that it can significantly enhance the immune response of specific T cells.

[0139] Example 10

[0140] The therapeutic effect of nanovaccines in vivo

[0141] The B16-OVA tumor model was established to investigate the therapeutic effect of the nanovaccine in vivo. 2×10 5 B16-OVA tumor cells were placed on the left back and counted as day 0. On days 3, 7, 11, and 15, 150 μL of PBS, OVA, DET-C30, and DET-F30 nanovaccines were injected (75 μg each, n=5). After vaccination, tumor growth was measured every two days using a digital caliper, and the calculation formula was tumor volume = 0.5 × length × width 2 When the tumor volume reaches 1500mm 3 The mice were euthanized at 4 pm and their body weights were recorded every two days.

[0142] Fig.10 The different nanovaccines described in this example were used to treat the B16-OVA tumor model. It can be seen that the DET-F30 treatment group achieved a better tumor inhibition rate than the other treatment groups, and the effect was better than the OVA and DET-C30 vaccine treatment groups alone. In addition, the survival rate of mice in the DET-F30 treatment group was the highest.

[0143] The above embodiments are only used to help understand the method and core idea of ​​the present invention. It should be noted that, for those skilled in the art, several improvements and modifications can be made to the present invention without departing from the principles of the present invention, and these improvements and modifications also fall within the scope of protection of the claims of the present invention.

Claims

1. A fluorinated or alkylated polyaspartic acid having the structure shown in Formula I: in, m is any integer from 1 to 9; n is any integer from 1 to 250; x is any integer from 1 to 250, and n is greater than x; R is selected from the following structure shown in Formula II or Formula III: In formula II, a is any integer from 1 to 9; b is any integer from 0 to 9; c is any integer from 1 to 200; In formula III, d is any integer from 1 to 18; * indicates the binding site of the group.

2. The fluorinated or alkylated polyaspartic acid according to claim 1, characterized in that The m is any integer from 2 to 4; The n is any integer from 70 to 140; The x is any integer from 25 to 75, and n is greater than x; In formula II, a is any integer from 1 to 2; b is any integer from 2 to 6; c is any integer from 1 to 60; In formula III, d is any integer from 2 to 9.

3. The method for preparing the fluorinated or alkylated polyaspartic acid according to any one of claims 1 to 2, comprising the following steps: S1) using an initiator having an amine terminal as shown in formula IV to initiate the ring-opening polymerization of L-aspartic acid-4-benzyl ester anhydride as shown in formula V to obtain polyaspartic acid as shown in formula VI; S2) subjecting the polyaspartic acid represented by formula VI to an aminolysis reaction with diethylenetriamine to obtain an N-substituted polyaspartic acid represented by formula VII; S3) reacting the N-substituted polyaspartic acid represented by formula VII with the alkylene oxide represented by formula VIII-a or the fluorinated alkylene oxide represented by formula VIII-b to obtain the fluorinated or alkylated N-substituted polyaspartic acid represented by formula I; R is selected from the following structure shown in Formula II or Formula III: Wherein, m is any integer from 1 to 9; n is any integer from 1 to 250; x is any integer from 1 to 250, and n is greater than x. In formula III and formula VIII-a, d is any integer from 1 to 18; In Formula II and Formula VIII-b, a is an arbitrary integer from 1 to 9; b is an arbitrary integer from 0 to 9; and c is an arbitrary integer from 1 to 200.

4. The preparation method according to claim 3, characterized in that: In the step S1), the solvent for the ring-opening polymerization is dimethylformamide and dichloromethane; The volume ratio of dimethylformamide to dichloromethane is 1:5-20; The temperature of the ring-opening polymerization is 0 to 50° C.; the time of the ring-opening polymerization is 48 to 96 hours; After the above ring-opening polymerization reaction is completed, the product is precipitated by glacial acetic acid, filtered and dried to obtain the polyaspartic acid represented by formula VI.

5. The preparation method according to claim 3, characterized in that: The temperature of the ammonolysis reaction is 0 to 10° C.; the time of the ammonolysis reaction is 1 to 4 hours; The molar ratio of the polyaspartic acid represented by formula VI to diethylenetriamine is 1:10-40; After the above aminolysis reaction is completed, the product is precipitated by glacial acetic acid, filtered and dried to obtain N-substituted polyaspartic acid.

6. The preparation method according to claim 3, characterized in that: The reaction temperature of the N-substituted polyaspartic acid and the alkylene oxide represented by formula VIII-a is 15 to 35° C.; the reaction time is 24 to 72 hours; The molar ratio of the N-substituted polyaspartic acid to the alkylene oxide represented by formula VIII-a is 1:5 to 50; The reaction temperature of the N-substituted polyaspartic acid and the alkylene oxide represented by formula VIII-b is 15 to 35° C.; the reaction time is 24 to 72 hours; The molar ratio of the N-substituted polyaspartic acid to the alkylene oxide represented by formula VIII-b is 1:5 to 50; Adding an acid binding agent during the reaction; The molar ratio of the N-substituted polyaspartic acid to the acid binding agent is 1:1-10.

7. Use of the fluorinated or alkylated polyaspartic acid according to any one of claims 1 to 2 or the fluorinated or alkylated polyaspartic acid prepared by the preparation method according to any one of claims 3 to 6 as an antigen carrier and / or immune adjuvant for nanovaccines.

8. A nano vaccine comprising an antigen and the fluorinated or alkylated polyaspartic acid according to any one of claims 1 to 2 or the fluorinated or alkylated polyaspartic acid prepared by the preparation method according to any one of claims 3 to 6.

9. The nanovaccine according to claim 8, characterized in that The antigen includes one or more of protein, polypeptide, tumor lysate, DNA and RNA.

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