A fluorinated or alkylated polyaspartic acid, its method of preparation and use in vaccine delivery

Abstract

The present application relates to the technical field of nano-vaccine, and particularly relates to fluorinated or alkylated polyaspartic acid, a preparation method thereof and application thereof in vaccine delivery. The fluorinated or alkylated polyaspartic acid has a structure shown in formula I. The fluorinated or alkylated N-substituted polyaspartic acid provided by the present application has excellent self-assembly capacity, and has good biocompatibility and low cytotoxicity. In the preparation of a nano-vaccine, the loaded antigen can be co-assembled into nanoparticles with the fluorinated or alkylated polyaspartic acid through hydrophobic interaction. In addition, the fluorinated or alkylated N-substituted polyaspartic acid retains the high cell uptake and endosome escape level of the N-substituted polyaspartic acid itself. Therefore, the fluorinated or alkylated N-substituted polyaspartic acid can enhance antigen uptake and promote cross-presentation.

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, a preparation method thereof, and an application thereof in vaccine delivery. Background Art

[0002] Tumors are a serious and intractable disease that threatens people's lives and health, and it also places a heavy burden on my country's medical and health system. At present, traditional tumor 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] To achieve effective anti-tumor effects, vaccine vectors, as a crucial component of cancer vaccines, influence the vaccine's efficacy, quality, and stability. Vaccine vectors must meet the following requirements: 1. High safety and storage stability for easy scalable production; 2. Effectively protect antigens and immunostimulants, preventing degradation; 3. Enhance their accumulation in lymphoid organs and uptake by antigen-presenting cells, enabling simultaneous delivery of antigens and immunostimulants to APCs; 4. Enhance antigen cross-presentation, promoting antigen transport across endosomes / lysosomes for cytoplasmic processing and presentation; and 5. Demonstrate multimodal APC activation, enhancing the vaccine's immune-stimulating effect. Currently commercially available aluminum adjuvants, poly(I:C), and viral vectors suffer from weak antigen protection, significant side effects, and the risk of gene integration. In particular, viral vectors are characterized by high production costs and technical barriers, coupled with low antigen payloads, making them difficult to meet current requirements for therapeutic vaccine vectors. The development of safe vaccine vector materials that demonstrate robust cellular immune responses remains a pressing bottleneck 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 its preparation method and application in vaccine delivery, and the prepared nanovaccine 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 the 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 structure shown in the following 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, resulting in superior pH sensitivity, 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 experimental 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 amino terminal as shown in Formula IV to initiate a 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 aminolysis with diethylenetriamine to obtain an N-substituted polyaspartic acid represented by formula VII;

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

[0027]

[0028]

[0029] R is selected from the structure shown in the following 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 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 hours, more preferably 48 to 72 hours.

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

[0043] The amino-terminated initiator of Formula IV is dissolved in dimethylformamide and then diluted with dichloromethane. After complete dissolution, the initiator diluted with dichloromethane is added to the L-aspartic acid-4-benzyl ester anhydride solution of Formula V, and the reaction is carried out 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, in the present invention, after the aminolysis reaction is completed, the product is precipitated with 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 as follows:

[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 to 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 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 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 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 15-35°C, more preferably 20-30°C; the reaction time is preferably 24-72h, more preferably 36-48h.

[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 represented by 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 proteins, polypeptides, tumor lysates, 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 nanovaccine, and the nanovaccine 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 serve as a carrier for various types of antigens, directly delivering antibodies to the cytoplasm of antigen-presenting cells (APCs), facilitating cross-presentation of antigens, but can also induce activation of APCs, functioning as an immune adjuvant and inducing antigen-specific cellular immune responses. This dual functionality is not possessed by conventional 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 interactions. In addition, the fluorinated or alkylated N-substituted polyaspartic acid retains the high 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 expression levels of SIINFEKL-H2Kb in BMDC cells after 24 h of stimulation with different nanovaccines;

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

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

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

[0097] Figure 10 The results of different nanovaccines in the treatment of B16-OVA tumor model. DETAILED DESCRIPTION

[0098] To further illustrate the present invention, the following is a detailed description of the embodiments. However, it should be understood that these descriptions are only for the purpose of further illustrating the features and advantages of the present invention, rather than for limiting the scope of the invention.

[0099] All raw materials of the present invention are not particularly limited in their sources and can be purchased from the market or prepared according to conventional methods well 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 4-benzyl L-aspartate-N-carboxylic anhydride (BLA-NCA) initiated by n-hexylamine. 5 g, 10 g, and 15 g of BLA-NCA were 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 procedures were performed under a dry argon atmosphere. The product was precipitated in an excess of glacial ether and filtered to dryness to obtain PBLA with a degree of polymerization of 50 (4.02 g, 95.2% yield), PBLA with a degree of polymerization of 100 (8.10 g, 97.9% yield), and PBLA with a degree of polymerization of 150 (12.11 g, 96.0% yield).

[0103] Aminolysis of PBLA. The 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, then stirred at room temperature for another 2 hours, 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 degree of polymerization 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 embodiment are 1 H NMR.

[0105] Example 2

[0106] Preparation of alkylated N-substituted polyaspartic acid

[0107] 1,2-Epoxydecane was added dropwise at a ratio of 10%, 20%, and 30% 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 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) and stirred at room temperature for 48 h. The product was then dialyzed against CH 3 OH overnight and then deionized water for two more days, followed by lyophilization 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 1 H NMR. As the number of alkyl modifications on 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] The mixture 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 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 days, and lyophilized 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. Calculations show that each DET-F30 molecule is actually grafted with 26.6 tridecafluoro molecules.

[0113] Example 4

[0114] Preparation of nanoparticle vaccines loaded with simulated antigens

[0115] Loading with the mimetic antigen hen ovalbumin (OVA): The above-mentioned polymer carriers were loaded with OVA via a self-assembly method to examine the assembly properties of the various modified materials with OVA. The materials were physically mixed with the OVA mimetic protein in deionized water at a specific weight ratio for 30 minutes. The resulting DET-C and DET-F nanoparticles with varying degrees of modification were then measured using a particle size analyzer.

[0116] Figure 4 This figure shows the particle size distribution of various fluorinated DETs prepared in this example. The various fluorinated DETs were simply mixed with polycations and OVA protein in an aqueous environment through electrostatic interactions to generate multiple ion pairs. In the absence of OVA, increasing the proportion of fluorinated DET modification resulted in a more uniform nanoparticle size. Furthermore, the particle size increased with increasing OVA loading.

[0117] Example 5

[0118] Determination of loading efficiency of OVA-loaded nanovaccine

[0119] The DET obtained in Example 3 modified with 10%, 20%, and 30% tridecafluoroheptyloxirane (DET-F10, DET-F20, and DET-F30), and the DET obtained in Example 2 modified with 10%, 20%, and 30% heptyloxirane (DET-C10, DET-C20, and DET-C30), were physically mixed with OVA-FITC at mass ratios of 1:0.2, 1:0.5, 1:1, and 1:2 for 30 minutes. Subsequently, 400 μL of each mixture 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. The content of OVA-FITC at the bottom of the ultrafiltration tube was measured, and the loading of different materials with OVA-FITC was calculated.

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

[0121]

[0122] Table 1 summarizes the loading efficiency of different materials with OVA-FITC in this example. The loading efficiency of the polymer carrier material decreases as the OVA loading ratio increases. For example, when DET-C10 and OVA are assembled at a weight ratio of 1:0.2, the loading ratio is 80.66%, while when DET-C10 and OVA are assembled at a weight ratio of 1:2, the loading ratio drops to 51.09%. When the weight ratio of the polymer carrier to OVA is 1:0.5, the loading efficiency of the fluorinated DET polymer carrier exceeds 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, FITC-labeled OVA (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 cells were seeded per well in a 24-well plate. 5 DC2.4 cells were cultured in complete culture medium (1% penicillin / streptomycin, 10% fetal bovine serum, and 89% RPMI-1640) for 12 hours. Free OVA-FTTC and 10 μg / mL of the nanovaccine were then incubated with the cells for 24 hours. The cells were washed three times with PBS and then subjected to confocal fluorescence imaging.

[0126] Figure 5 Confocal fluorescence images of DC2.4 cells incubated with the various nanovaccines described in this example for 24 hours. DC2.4 cells incubated with DET-F30 exhibited stronger intracellular green fluorescence than the control group, demonstrating its ability to enhance intracellular delivery of OVA through cationic and hydrophobic interactions. Furthermore, in DC2.4 cells incubated with DET-F30, the majority of intracellular OVA-FITC was not colocalized with lysosomes, suggesting that the DET-F30 polymer possesses 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 the vaccine, 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 min, and the formed nano vaccines were co-cultured with BMDC cells at a concentration of 10 μg / mL for 24 h. 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 in BMDCs by the different nanovaccines described in this example are shown. Fluorinated or alkylated DET nanovaccines significantly increased SIINFEKL-H2Kb expression in CD11c-positive BMDCs 24 hours after stimulation, demonstrating enhanced antigen cross-presentation, with increasing alkyl or fluorinated modification ratios leading to enhanced antigen cross-presentation.

[0131] Example 8

[0132] Accumulation of nanovaccines in draining lymph nodes

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

[0134] Figure 8 This example describes in vitro imaging and statistical analysis of fluorescence in draining lymph nodes treated with different nanovaccines. Clearly, free OVA-Cy5 does not accumulate well in the draining lymph nodes of mice following injection; however, fluorinated and alkylated DET-F and DET-C enhance OVA-Cy5 accumulation in draining lymph nodes compared to 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 the antigen SIINFEKL for 12 h under the stimulation of brefeldin A and monensin. IFN-γ secreting T cells (CD3 + CD8 + IFN-γ).

[0138] Figure 9 This example describes the application 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 can 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 implanted on the left side of the back, and day 0 was designated. On days 3, 7, 11, and 15, 150 μL of PBS, OVA, DET-C30, and DET-F30 nanovaccines (75 μg each, n=5) were injected, respectively. After vaccination, tumor growth was measured every two days using a digital caliper. The calculation formula was tumor volume = 0.5 × length × width. 2 When the tumor volume reaches 1500mm 3 The mice were euthanized at 4 hr and their body weights were recorded every two days.

[0142] Figure 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 intended to help understand the method and core concept of the present invention. It should be noted that, without departing from the principles of the present invention, a number of improvements and modifications may be made to the present invention by those skilled in the art, and such 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 structure shown in the following 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; Said 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 amino terminal as shown in Formula IV to initiate a 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 aminolysis 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 structure shown in the following 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 ring-opening polymerization reaction is completed, the product is precipitated with 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 to 40; After the aminolysis reaction is completed, the product is precipitated with 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 in the preparation of antigen carriers and / or immune adjuvants 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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