A Polylysine-Based Polymer Adjuvant Material, Its Preparation Method and Application

Abstract

The application discloses a polylysine-based polymer adjuvant material and a preparation method and application thereof, and belongs to the technical field of biological medicines. The polylysine-based polymer adjuvant material has any one of structures shown in formula (I)-(III), has the dual functions of a carrier and an immune stimulant, has inherent immune stimulation activity, and has a clear structure, is easy to modify, has high biocompatibility, is degradable, and has good biological safety. The branched side chains of poly-L-lysine are modified first to obtain PLL-2V and PLL-4V, then are bonded with a five-membered, six-membered or seven-membered nitrogen-containing heterocyclic group or a heterocyclic small molecule with a benzene structure which is substituted by a carboxyl group or a hydroxyl group, to obtain the polylysine-based polymer adjuvant material. The polylysine-based polymer adjuvant material can be applied to the preparation of a vaccine preparation, and is of great significance to improving the preparation efficiency of the vaccine.

Description

Technical Field

[0001] This invention relates to the field of biomedical technology, and in particular to a polymer adjuvant material based on polylysine, its preparation method, and its application. Background Technology

[0002] Polymer adjuvant materials, namely polymer materials with immunostimulatory activity, are still in the early stages of vaccine research, but their advantages such as simplicity, efficiency, and versatility give them enormous application potential. Unlike small molecule adjuvants, polymer materials not only exhibit immunostimulatory activity but can also carry protein, peptide, or nucleic acid antigens, making them important for the design of protein vaccines or nucleic acid vaccine carriers and accelerating the clinical translation of novel adjuvant vaccines.

[0003] The basic components of a common vaccine are a carrier, an immunostimulant, and an antigen. Adjuvants are substances that enhance the strength and persistence of antigen-specific immune responses during vaccination. They include carriers and immunostimulants. However, due to their complex composition, vaccines prepared with existing adjuvant materials have low efficiency and are difficult to convert. Therefore, the need for novel vaccine adjuvant materials is urgent. Previously, Adv Mater. 2022, 34(10):e2109254. reported on a derivative material based on branched polyethyleneimine (bPEI-M), which was found to have a self-adjuvant function that stimulates the activation of interferon gene stimulating factor (STING). Using this PEI-M in tumor vaccine design can achieve high vaccine efficacy without the presence of additional adjuvant molecules, simplifying vaccine design.

[0004] However, the non-degradability, physiological toxicity, and structural uncertainty of bPEI-M pose certain obstacles to its application and transformation. Therefore, it is crucial to research and develop new polymer adjuvant materials to achieve highly efficient vaccine immunization. Summary of the Invention

[0005] In view of this, the technical problem to be solved by the present invention is to provide a polylysine-based polymer adjuvant material, its preparation method, and its application. The polylysine-based polymer adjuvant material not only possesses immunostimulatory activity, but also has a well-defined structure, is easy to modify, has high biocompatibility, is biodegradable, and has good biosafety.

[0006] To achieve the above objectives, the technical solution adopted by the present invention is as follows:

[0007] This invention provides a polymeric adjuvant material based on polylysine, having the structure shown in any of formulas (I) to (III):

[0008]

[0009]

[0010] Wherein, n is the degree of polymerization, preferably 10≤n≤200; more preferably 20≤n≤80; and even more preferably 40 or 75.

[0011] Preferably, x1 = 0.2 to 0.5, y1 = 1 - x1; more preferably, x1 = 0.3 to 0.4, y1 = 1 - x1; even more preferably, x1 = 0.35, y1 = 0.65.

[0012] Preferably, x2 = 0.1 to 0.4, y2 = 1 - x2; more preferably, x2 = 0.2 to 0.3, y2 = 1 - x2; even more preferably, x2 = 0.23, y2 = 0.77.

[0013] Preferably, x3 = 0.05 to 0.35, y3 = 1 - x3; more preferably, x3 = 0.1 to 0.2, y3 = 1 - x3; even more preferably, x3 = 0.15, y3 = 0.85.

[0014] Preferably, R1 is selected from -NHC(O)R2 or -NHC(O)O(CH2). m R2;

[0015] Preferably, R2 is selected from five-membered, six-membered, or seven-membered nitrogen-containing heterocyclic groups or their benzo[a] structures;

[0016] Preferably, m is selected from an integer between 1 and 4; more preferably, m is selected from 2.

[0017] Preferably, the R2 is selected from one or more of the following: oxazolyl, isoxazolyl, benzoxazolyl, thiazolyl, benzothiazolyl, pyrazolyl, indazole, imidazolyl, benzimidazole, piperidinyl, tetrahydroisoquinolinyl, pyridinyl, quinolinyl, isoquinolinyl, pyridazinyl, pyrimidinyl, quinazolinyl, piperazinyl, pyrazinyl, quinoxalinyl, and cyclohexylimino.

[0018] The present invention also provides a method for preparing the above-mentioned polylysine-based polymer adjuvant material, wherein PLL-1V, PLL-2V, or PLL-4V is subjected to an amidation reaction with a carboxyl-substituted five-, six-, or seven-membered nitrogen-containing heterocyclic group or its benzo[a] structure to obtain a polylysine-based polymer adjuvant material.

[0019] Alternatively, PLL-1V, PLL-2V, or PLL-4V can be hydroxylated with a hydroxyl-substituted five-, six-, or seven-membered nitrogen-containing heterocyclic group or its benzo[a] structure, and then subjected to hydroxylation reaction in the presence of N'N-carbonyldiimidazole to obtain a polymer adjuvant material based on polylysine.

[0020] After the above amidation or hydroxylation reaction is completed, acidification post-dialysis treatment is also included.

[0021] The acidification dialysis preferably uses Milli Q water with a pH gradient of 3-7, with a pH adjustment range of 1. Finally, the mixture is dialyzed three times with pure Milli Q water at pH 7, then freeze-dried, and the product is collected to obtain the polymer adjuvant material based on polylysine.

[0022] In the above preparation method, the carbonyl group in N'N-carbonyldiimidazole is activated by imidazole (simultaneously losing two imidazole groups to generate carbonyl derivatives), thus exhibiting very high formylation reactivity. This allows the substituent hydroxyl groups of heterocyclic small molecules (five-membered, six-membered, or seven-membered nitrogen-containing heterocyclic groups or their benzo[a] structures) to react with the branched side chain amino groups of polylysine derivatives to generate aminocarboxylic acid ester bonds.

[0023] In some specific embodiments of the present invention, N-(2-hydroxyethyl)hexamethylenediamine (HPip) and PLL-4V undergo a hydroxylation reaction in the presence of N'N-carbonyldiimidazole to obtain a polylysine-based polymer adjuvant material PLL-4V-Hpip, the structure of which is shown below:

[0024]

[0025] The following example of the preparation route for benzimidazole-4-carboxylic acid (4BImi) modified polylysine derivatives illustrates the synthetic route for the polylysine-based polymer adjuvant material described in this invention. The specific route is as follows:

[0026]

[0027] The above-mentioned synthetic route for polylysine-based polymer adjuvant materials first involves modifying the branched side chains of the PLL-1V (also known as PLL) structure to prepare PLL-2V and PLL-4V. Then, heterocyclic small molecules are introduced into the ends of the branched side chains of PLL-1V, PLL-2V, or PLL-4V to finally prepare the polylysine-based polymer adjuvant materials.

[0028] The modification specifically involves: methyl acrylate undergoing an addition reaction with the branched side chain amino group of PLL-1V to obtain an intermediate (PLL-MA), and then ammonolyzing the methyl ester structure at the end of the branched side chain with ethylenediamine or tris(2-aminoethyl)amine to prepare PLL-2V and PLL-4V respectively.

[0029] The preferred molar number of methyl acrylate in the addition reaction is twice the number of PLL-branched side-chain amino groups.

[0030] The molar number of ethylenediamine or tri(2-aminoethyl)amine is preferably 20 times that of the methyl ester structure at the end of the branched side chain, so that the ammonolysis reaction can proceed fully.

[0031] The PLL-1V, PLL-2V, or PLL-4V described in this invention are bonded together with heterocyclic small molecules via amide bonds or aminocarboxylic acid ester bonds to obtain the polylysine-based polymer adjuvant material.

[0032] The present invention refers to the polylysine-based polymer adjuvant materials as PLL-1V-M, PLL-2V-M, and PLL-4V-M.

[0033] M is a carboxyl-substituted five-, six-, or seven-membered nitrogen-containing heterocyclic group or its benzo[a] structure, or a hydroxyl-substituted five-, six-, or seven-membered nitrogen-containing heterocyclic group or its benzo[a] structure.

[0034] Preferably, the carboxyl-substituted five-, six-, or seven-membered nitrogen-containing heterocyclic group or its benzo[a] structure is selected from oxazole-5-carboxylic acid (5Oxa), isoxazole-5-carboxylic acid (5Iso), benzo[oxazole-5-carboxylic acid (5BOxa), benzo[oxazole-6-carboxylic acid (6BOxa), thiazole-5-carboxylic acid (5Thi), benzo[thiazole-5-carboxylic acid (5BThi), benzo[thiazole-6-carboxylic acid (6BThi), pyrazole-4-carboxylic acid (4Pyr), indazole-4-carboxylic acid (4Ind), indazole-5-carboxylic acid (5Ind), indazole-6-carboxylic acid (6Ind), indazole-7-carboxylic acid (7Ind), imidazole-4-carboxylic acid (4Imi), benzimidazole-4- One or more of the following: carboxylic acid (4BImi), benzimidazole-5-carboxylic acid (5BImi), 1-piperidinylacetic acid (1Ppd), (2-ethylpiperidin-1-yl)acetic acid (PipE), 1,2,3,4-tetrahydroisoquinoline-3-carboxylic acid (3TIql), pyridine-4-carboxylic acid (4Prd), quinoline-3-carboxylic acid (3Qnl), isoquinoline-1-carboxylic acid (1Iql), pyridazine-4-carboxylic acid (4Pdz), cyclophosphine-4-carboxylic acid (4Cnl), pyrimidine-5-carboxylic acid (5Pmd), quinazolin-6-carboxylic acid (6Qzl), piperazine-2-carboxylic acid (2Ppz), pyrazine-2-carboxylic acid (2Prz), and quinoxaline-2-carboxylic acid (2Qol);

[0035] More preferably, the carboxyl-substituted five-, six-, or seven-membered nitrogen-containing heterocyclic group or its benzo[a] structure is selected from one or more of thiazol-5-carboxylic acid, indazole-4-carboxylic acid, indazole-5-carboxylic acid, imidazole-4-carboxylic acid, benzimidazole-4-carboxylic acid, benzimidazole-5-carboxylic acid, (2-ethylpiperidin-1-yl)acetic acid, 1,2,3,4-tetrahydroisoquinoline-3-carboxylic acid, isoquinoline-1-carboxylic acid, and pyrazine-2-carboxylic acid;

[0036] Further preferably, the carboxyl-substituted five-, six-, or seven-membered nitrogen-containing heterocyclic group or its benzo[a] structure is selected from one or more of thiazol-5-carboxylic acid, imidazole-4-carboxylic acid, benzimidazole-4-carboxylic acid, (2-ethylpiperidin-1-yl)acetic acid, 1,2,3,4-tetrahydroisoquinoline-3-carboxylic acid, and pyrazine-2-carboxylic acid. In some specific embodiments of the present invention, benzimidazole-4-carboxylic acid (4BImi) is preferred.

[0037] Preferably, the hydroxyl-substituted five-, six-, or seven-membered nitrogen-containing heterocyclic group or its benzo[a] group is selected from N-(2-hydroxyethyl)hexamethylenediamine (HPip).

[0038] In some specific embodiments of the present invention, preferably, performance tests were conducted on PLL-1V-4BImi, PLL-2V-4BImi, PLL-4V-4Bimi, PLL-4V-PipE, PLL-4V-3Tlql, PLL-4V-1lql, PLL-4V-2Prz, and PLL-4V-Hpip, demonstrating that the polylysine-based polymer adjuvant materials of the present invention have innate immunostimulatory activity and higher biocompatibility.

[0039] Preferably, the molar ratio of the carboxyl-substituted five-, six-, or seven-membered nitrogen-containing heterocyclic group or its benzo[a] structure, or the hydroxyl-substituted five-, six-, or seven-membered nitrogen-containing heterocyclic group or its benzo[a] structure, to PLL-1V is (12–37.5):1; more preferably (22.5–30):1; and even more preferably 26.3:1.

[0040] Preferably, the molar ratio of the carboxyl-substituted five-, six-, or seven-membered nitrogen-containing heterocyclic group or its benzo[a] structure, or the hydroxyl-substituted five-, six-, or seven-membered nitrogen-containing heterocyclic group or its benzo[a] structure, to PLL-2V is (7.5–30):1; more preferably (15–22.5):1; and even more preferably 17.3:1.

[0041] Preferably, the molar ratio of the carboxyl-substituted five-, six-, or seven-membered nitrogen-containing heterocyclic group or its benzo[a] structure, or the hydroxyl-substituted five-, six-, or seven-membered nitrogen-containing heterocyclic group or its benzo[a] structure, to PLL-4V is (3.8–27.4):1. More preferably, it is (7.5–15):1; even more preferably, it is 11.3:1.

[0042] Preferably, the molar ratio of the hydroxyl-substituted five-, six-, or seven-membered nitrogen-containing heterocyclic group or its benzo[a] group to the N'N-carbonyldiimidazole is (0.7–1):1. In some specific embodiments of the present invention, a ratio of 0.8:1 is preferred.

[0043] Preferably, when PLL-1V, PLL-2V, or PLL-4V undergoes an amidation reaction with a carboxyl-substituted five-, six-, or seven-membered nitrogen-containing heterocyclic group or its benzo[a] structure, a condensing agent is also added in the preparation method.

[0044] Preferably, the condensing agent is selected from 1-ethyl-(3-dimethylaminopropyl)carbodiimide hydrochloride (EDC·HCl) and / or N-hydroxysuccinimide (NHS).

[0045] The present invention also provides the application of the above-mentioned polylysine-based polymer adjuvant material or the polylysine-based polymer adjuvant material prepared by the above-mentioned preparation method in the preparation of antitumor vaccine formulations.

[0046] Preferably, the polylysine-based polymer adjuvant material can be used in the preparation of protein carriers or gene carriers in vaccine formulations.

[0047] This invention obtains polylysine-based polymer adjuvant materials with different branched side chains through heterocyclic small molecule modification, which enables them to activate the interferon-stimulating factor (STING) pathway and can regulate this immunostimulatory function by different heterocyclic small molecules bonded to the branched side chains.

[0048] The present invention also provides a vaccine formulation comprising the above-described polylysine-based polymer adjuvant material or the polylysine-based polymer adjuvant material prepared by the above-described preparation method.

[0049] Compared with existing technologies, the polylysine-based polymer adjuvant material provided by this invention has the structure shown in any of formulas (I) to (III). This polylysine-based polymer adjuvant material combines the dual functions of a carrier and an immunostimulant, possessing inherent immunostimulatory activity, and exhibits a well-defined structure, ease of modification, high biocompatibility, degradability, and good biosafety. This invention first modifies the branched side chains of poly-L-lysine (PLL) to obtain PLL-2V and PLL-4V, and then bonds them respectively to five-, six-, or seven-membered nitrogen-containing heterocyclic groups substituted with carboxyl or hydroxyl groups or their benzo[a]-structured heterocyclic small molecules to obtain the aforementioned polylysine-based polymer adjuvant material. This polylysine-based polymer adjuvant material can be applied to the preparation of vaccine formulations, which is of great significance for improving vaccine preparation efficiency. Attached Figure Description

[0050] Figure 1 The 1H NMR characterization spectrum of PLL-MA obtained in Example 1;

[0051] Figure 2 The 1H NMR characterization spectrum of PLL-2V obtained in Example 2;

[0052] Figure 3 The 1H NMR characterization spectrum of PLL-4V obtained in Example 2;

[0053] Figure 4 The 1H NMR characterization spectrum of PLL-1V-4BImi obtained in Example 16;

[0054] Figure 5 The 1H NMR characterization spectrum of PLL-2V-4BImi obtained in Example 44;

[0055] Figure 6 The 1H NMR characterization spectrum of PLL-4V-4BImi obtained in Example 72;

[0056] Figure 7 For PEI-4BImi obtained in Comparative Example 1 1 H NMR characterization spectrum;

[0057] Figure 8 The graph shows the concentration of IFN-β in the cell supernatant 24 hours after treating DC2.4 cells with PLL-1V-4Bimi, PLL-2V-4Bimi, PLL-4V-4BImi and PEI-4BImi in Example 1.

[0058] Figure 9 The graph shows the concentration of IFN-β in the cell supernatant after treating DC2.4 cells with PLL-4V-PipE, PLL-4V-3Tlql, PLL-4V-1lql, PLL-4V-2Prz, and PLL-4V-HPip at a concentration of 80 μg / mL for 24 hours, as described in Example 1.

[0059] Figure 10 The cytotoxicity of PLL-1V-4Bimi, PLL-2V-4Bimi, PLL-4V-4Bimi and PEI-4Bimi materials to DC2.4 cells at 24 h was measured in Application Example 2.

[0060] Figure 11 The graph shows the phosphorylation expression of STING pathway-related proteins in THP-1 cells after 4 hours of treatment with PLL-1V-4Bimi, PLL-2V-4Bimi, and PLL-4V-4BImi in Example 3. In Figure a, p-TBK1 phosphorylation is shown; in Figure b, p-STING phosphorylation is shown; and in Figure c, p-IRF3 phosphorylation is shown.

[0061] Figure 12Laser confocal imaging of THP-1 cells after 4 hours of treatment with 40 μg / mL PLL-1V-4Bimi-Cy5, PLL-2V-4Bimi-Cy5, and PLL-4V-4BImi-Cy5 as described in Example 4, showing the aggregation of STING protein in the cells and its co-localization with the material.

[0062] Figure 13 Figure showing the concentration of IFN-β in the cell supernatant after 24 hours of treatment with 20 μg / mL PLL-4V-4BImi and bPEI-4BImi from Example 5.

[0063] Figure 14 The images show the DLS characterization of the nanovaccine prepared in Example 6, where Figure a is the DLS characterization of PLL-1V-4Bimi / OVA nanoparticles, Figure b is the DLS characterization of PLL-2V-4Bimi / OVA nanoparticles, and Figure c is the DLS characterization of PLL-4V-4BImi / OVA nanoparticles. Detailed Implementation

[0064] To further illustrate the present invention, the following detailed description of the polylysine-based polymer adjuvant materials, their preparation methods, and applications, in conjunction with embodiments, is provided by the present invention.

[0065] Table 1 shows the structural formulas of the heterocyclic small molecules involved in the specific embodiments.

[0066]

[0067] Example 1

[0068] Preparation of PLL-1V intermediate (PLL-MA).

[0069] Taking a PLL with 75 repeating units as an example, 450 equivalents of methyl acrylate and 75 equivalents of triethylamine were dissolved in 10 volumes of methanol and placed in an ice-water bath for 15 minutes to cool to 0°C. The methanol suspension of the PLL was then slowly added dropwise to the methyl acrylate solution, and the mixture was stirred in an ice-water bath for 10 minutes. The reaction system was then slowly heated to 60°C and stirred for 8 hours until the reaction was complete. The reaction system was precipitated with 10 volumes of diethyl ether, then reconstituted with methanol. This precipitation and reconstitution process was repeated twice to remove excess methyl acrylate and triethylamine. Finally, the mixture was vacuum dried overnight, and the product was collected as a white, viscous solid.

[0070] Figure 1 The PLL-MA obtained in Example 1 1 H NMR characterization spectrum. 1H NMR (300MHz, TFA-d1): δ = 4.37 (s, 75H; COCHNH), 3.62 (s, 458.2H; COOCH3), 3.35 (s, 300.7H; N (CH2)2), 3.13 (s, 147.6H; CH2N), 2. 81 (t, 301.3H; (CH2COO)2), 1.68 (m, 294.1H; CHCH2CH2CH2CH2), 1.37 (s, 147.7H; CHCH2CH2CH2CH2), 0.65ppm (t, 3.08H; CH3CH2).

[0071] Example 2

[0072] Preparation of PLL-2V and PLL-4V materials.

[0073] One equivalent of PLL-MA with 75 repeating units obtained in Example 1 was dissolved in 10 times its mass volume of methanol and placed in an ice-water bath for 15 minutes to cool to 0°C. Then, 3000 times its mass equivalent of ethylenediamine or tris(2-aminoethyl)amine was slowly added dropwise, and the mixture was stirred in the ice-water bath for another 10 minutes. The reaction system was then slowly heated to 60°C and stirred for 8 hours until the reaction was complete. The reaction system was precipitated with 10 times its volume of diethyl ether, dried under vacuum overnight, dissolved in Milli-Q water, and dialyzed for 48 hours, changing the water at least 8 times. The product was then freeze-dried and collected. PLL-2V and PLL-4V were obtained, both as white, fluffy solids.

[0074] The structural formulas for PLL-2V and PLL-4V are as follows:

[0075]

[0076] Figure 2 The PLL-2V obtained in Example 2 1 H NMR characterization spectrum. 1 H NMR (300MHz, D2O): δ = 4.27 (s, 75H; COCHNH), 3.44 (t, 304.6H; NHCH2CH2NH2), 3.08 (t,296.4H; NHCH2CH2NH2),2.97(s,303.1H;N(CH2)2),2.68(s,147.7H;CH2N),2. 54(s,305.1H;(CH2CO)2),1.70(s,155.6H;CHCH2CH2CH2CH2),1.54(s,154.5H;CH CH2CH2CH2CH2), 1.31 (s, 154.1H; CHCH2CH2CH2CH2), 0.78ppm (t, 3.08H; CH3CH2).

[0077] Figure 3 The PLL-4V obtained in Example 2 1 H NMR characterization spectrum. 1 H NMR (300MHz, D2O): δ = 4.19 (s, 75H; COCHNH), 3.18 (m, 449.6H; CONHCH2, CH2N), 2.99 (m, 594.8H; N (CH2CH2) 2), 2 .59-2.28(m,1383.9H; CH2NCH2CH2NH2), 1.63-1.44(m,452.1H; CHCH2CH2CH2), 0.73ppmNN(t,3.08H; CH3CH2).

[0078] Examples 3-86

[0079] Preparation of PLL-1V, PLL-2V, or PLL-4V materials modified with heterocyclic molecules containing carboxyl substituents. One equivalent of PLL-1V, PLL-2V, or PLL-4V material was placed in a reaction flask, dried, and then protected with nitrogen. Subsequently, 28 heterocyclic small molecules (molecules 1-28 as shown in Table 2, corresponding to Examples 3-87, respectively) were added at equivalents of 35%, 23%, and 15% of the amino terminus of PLL, PLL-2V, and PLL-4V, respectively. Examples 3-30 were adjuvant materials prepared by bonding the amino terminus of PLL-1V with molecules 1-28, and Examples 31-58 were PLL-1V adjuvant materials. The adjuvant materials prepared by bonding the amino terminus of LL-2V to molecules 1-28 respectively (Examples 59-86 show adjuvant materials prepared by bonding the amino terminus of PLL-4V to molecules 1-28 respectively) were then added to the above examples with 53%, 35%, and 23% equivalents of EDC·HCl of the amino terminus of PLL-1V, PLL-2V, and PLL-4V respectively, and 70%, 46%, and 30% equivalents of NHS of the amino terminus of PLL-1V, PLL-2V, and PLL-4V respectively. Finally, anhydrous DMSO was injected into the system at ten times the total mass of the reactants for dissolution, and the reaction was stirred at 35°C for 72 hours. Subsequently, the reaction system was precipitated with 10 times the volume of diethyl ether, the supernatant was discarded, and the residual diethyl ether was dried under vacuum. The reaction product was then dissolved in sterile water, and the reaction system was dialyzed using a dialysis bag with a molecular weight cutoff of 7000 Da. The pH gradient was increased from 3 to 7, and the water was changed every 6-8 hours. The pH adjustment range was 1. Finally, the system was dialyzed three times with pure MilliQ water at pH 7, and then freeze-dried. The collected product was a loose solid.

[0080] Polylysine-based polymer adjuvant materials (PLL-1V-4Bimi, PLL-2V-4Bimi, PLL-4V-4BImi) obtained by grafting representative heterocyclic small molecule 4BImi onto polymers were characterized by NMR.

[0081] The structural formulas of PLL-1V-4BImi, PLL-2V-4BImi, and PLL-4V-4BImi are shown below:

[0082]

[0083]

[0084] Figure 4 The PLL-1V-4BImi obtained in Example 16 1 H NMR characterization spectrum. 1 H NMR (300MHz, D2O+DCl): δ=9.04 (s, 23.4H; CH), 7.56 (m, 84.0H; CHCHCH), 4.20 (m, 75 H; COCHNH), 3.24-2.90 (m, 158.0H; CHCH2), 1.68-1.33ppm (m, 432.7H; CH2CH2CH2).

[0085] Figure 5 The PLL-2V-4BImi obtained in Example 44 1 H NMR characterization spectrum. 1 H NMR (300MHz, D2O+DCl): δ=9.1 (s, 35.4H; CH), 7.70-7.45 (m, 107.3H; CHCHCH), 4.02 (m, 75H; COCH NH), 3.31-2.18 (m, 1820.4H; CHN (CH2CH2CONHCH2CH2)2), 1.59-1.28ppm (m, 438.2H; CH2CH2CH2).

[0086] Figure 6 The PLL-4V-4BImi obtained in Example 72 1 H NMR characterization spectrum. 1 H NMR (300MHz, D2O+DCl): δ=9.07 (s, 53.6H; CH), 7.70-7.39 (m, 162.9H; CHCHCH), 4.02-2.15 ( m, 2547.3H; CHN(CH2CH2CONH(CH2CH2N(CH2CH2)2)2), 1.69-1.34ppm(m, 450H; CH2CH2CH2).

[0087] Table 2 Heterocyclic small molecules in the examples

[0088]

[0089]

[0090] Examples 87-89

[0091] The preparation of PLL-1V, PLL-2V, or PLL-4V materials modified with heterocyclic molecules containing hydroxyl substituents involved adding heterocyclic small molecules (such as molecule 29 shown in Table 2, where the adjuvant material obtained by bonding the amino terminus of PLL-1V with molecule 29 is Example 87, the adjuvant material obtained by bonding the amino terminus of PLL-2V with molecule 29 is Example 88, and the adjuvant material obtained by bonding the amino terminus of PLL-4V with molecule 29 is Example 89) according to 35%, 23%, and 15% of the amino terminus of PLL-1V, PLL-2V, and PLL-4V materials respectively. Then, N'N-carbonyldiimidazole (CDI) was added according to 42%, 28%, and 18% of the amino terminus of PLL-1V, PLL-2V, and PLL-4V materials respectively. The mixture was dissolved in anhydrous DMSO at 10 times the total mass volume of the reactants, and then stirred at 35°C for 12 hours to carry out the reaction of hydroxyl groups and CDI. One equivalent of heterocyclic small molecule modified PLL-1V, PLL-2V, or PLL-4V material was placed in a reaction flask, dried, and protected with nitrogen. DMSO was added to dissolve the material, and the solution was injected into the heterocyclic small molecule reaction system. The reaction was continued at 35°C with stirring for 36 hours. Subsequently, the reaction system was settled with 10 volumes of diethyl ether, the supernatant was discarded, and the residual diethyl ether was dried under vacuum. The reaction product was then dissolved in sterile water and dialyzed in MilliQ water using a dialysis bag with a molecular weight cutoff of 7000 Da. The pH gradient was increased from 3 to 7, with the water changed every 6-8 hours and the pH adjustment span being 1. Finally, the mixture was dialyzed three times with pure MilliQ water at pH 7, and then freeze-dried. The product was collected as a white solid.

[0092] Comparative Example 1

[0093] The preparation of hyperbranched PEI material (PEI-4BImi) modified with heterocyclic small molecules was completed according to the literature reported in Adv Mater. 2022, 34(10):e2109254, as Comparative Example 1 of this invention. 40 equivalents of nitrogen-containing heterocyclic small molecules, 60 equivalents of EDC·HCl, and 80 equivalents of NHS were weighed into a reaction flask equipped with a stir bar. Anhydrous DMSO was added, and the mixture was stirred at 35°C for 30 minutes to activate the carboxyl groups on the small molecules. Subsequently, 1 equivalent of hyperbranched PEI-10kDa, pre-dissolved in DMSO, was added, and the mixture was stirred at 35°C for 72 hours. The reaction system was then settled with 10 times its volume of diethyl ether, the supernatant was discarded, and the residual diethyl ether was dried. The reaction product was then dissolved in sterile water and dialyzed in MilliQ water using a dialysis bag with a molecular weight cutoff of 7000 Da. The pH gradient was increased from 3 to 7, and the water was changed every 6-8 hours with a pH adjustment range of 1. Finally, the product was dialyzed three times with pure MilliQ water at pH 7, then freeze-dried and collected as a loose solid.

[0094] Figure 7 For PEI-4BImi obtained in Comparative Example 1 1 H NMR characterization spectrum: 1 H NMR (300MHz, D2O+DCl): δ = 8.46 (s, 36H; CH), 7.33 (s, 108H; CH, CH, CH), 3.25ppm (m, 930H; - (CH2CH2NH)n-)

[0095] Application Example 1

[0096] The heterocyclic small molecule modified polylysine derivatives (PLL-4V-M) PLL-4V-PipE, PLL-4V-3Tlql, PLL-4V-1lql, PLL-4V-2Prz, PLL-4V-HPip in Examples 3-86 and Examples 87-89, as well as the heterocyclic small molecule modified hyperbranched PEI material (PEI-4BImi) in Comparative Example 1, induced the release of IFN-β from DC2.4 cells.

[0097] PLL-1V-4Bimi, PLL-2V-4Bimi, PLL-4V-4Bimi, and PEI-4Bimi were thoroughly dissolved in sterile water and then added to 96-well plates containing 15,000 DC2.4 cells per well. The treatment concentrations of PLL-1V-4Bimi, PLL-2V-4Bimi, and PLL-4V-4Bimi were 40 μg / mL or 80 μg / mL, and the treatment concentration of PEI-4Bimi was 50 μg / mL. After 24 hours, the culture medium in the wells was collected and centrifuged at 3000 rpm for 5 minutes. The supernatant was collected, and the concentration of IFN-β was detected using an ELISA kit. The ELISA assay procedure is as follows (all procedures are performed at room temperature): Incubate the capture antibody overnight in a high-adhesion 96-well plate, wash three times with PBST (containing 0.05% PBS solution), block with PBS containing 1% BSA for 1 hour, wash three times with PBST, add the sample to be tested (3-4 replicates) and standard curve sample and incubate for 2 hours, wash three times with PBST, add the detection antibody and incubate for 2 hours, wash three times with PBST, add streptavidin-HRP and incubate for 20 minutes, wash three times with PBST, add TMB chromogenic solution and develop color in the dark for 20 minutes, add 2N H2SO4 to stop the assay, and use a microplate reader to detect the absorbance at 450nm and subtract the background value at 540nm.

[0098] Figure 8 The concentration of IFN-β in the cell supernatant after 24 hours of treatment with PLL-1V-4Bimi, PLL-2V-4Bimi, PLL-4V-4BImi, PLL-1V, PLL-2V, PLL-4V, and PEI-4BImi at concentrations of 40 μg / mL or 80 μg / mL, respectively. This indicates that PLL grafted with the heterocyclic small molecule 4BImi possesses immunostimulatory activity, and the stimulatory activity gradually increases with increasing branching valence, exhibiting a concentration-dependent relationship. (Appendix) Figure 8 In this context, PLL-4Bimi is the same as PLL-1V-4Bimi.

[0099] Figure 9The concentration of IFN-β in the cell supernatant after treating DC2.4 cells with PLL-4V-PipE, PLL-4V-3Tlql, PLL-4V-1lql, PLL-4V-2Prz, and PLL-4V-HPip at a concentration of 80 μg / mL for 24 hours was measured. This indicates that PLL-4V grafted with heterocyclic molecules exhibits innate immunostimulatory activity. When six- or seven-membered nitrogen heterocyclic molecules are grafted onto PLL-4V, similar concentrations of IFN-β secreted by DC2.4 cells can be observed.

[0100] Application Example 2

[0101] The cytotoxicity of PEI-M material (PEI-4BImi) prepared in Comparative Example 1 and heterocyclic small molecule modified polylysine derivatives (PLL-1V-4Bimi, PLL-2V-4Bimi, PLL-4V-4BImi) from Examples 16, 44, and 72 against DC2.4 cells were incubated overnight in 96-well plates at a density of 5000 cells / well. The next day, materials were added at six concentration gradients: 10 μg / mL, 20 μg / mL, 40 μg / mL, 80 μg / mL, 160 μg / mL, and 320 μg / mL. After incubation for 24 h, 20 μL of CCK8 solution was added to each well. After incubation for 1-2 h, the absorbance (OD value) at 560 nm was measured using a microplate reader. Cell viability was calculated as [OD value of experimental wells / OD value of PBS wells] × 100%.

[0102] Figure 10 To determine the 24-hour cytotoxicity of the obtained PEI-M material (PEI-4BImi) and the heterocyclic small molecule modified polylysine derivatives (PLL-1V-4Bimi, PLL-2V-4Bimi, PLL-4V-4BImi) to DC2.4 cells in Examples 16, 44, and 72, the following analysis was conducted. Figure 10 It can be seen that the polylysine derivatives modified with heterocyclic small molecules (PLL-1V-4Bimi, PLL-2V-4Bimi, PLL-4V-4BImi) have higher biocompatibility.

[0103] Application Example 3

[0104] Examples 16, 44, and 72 validated the phosphorylation expression of STING pathway-related proteins induced in THP-1 cells using PLL-1V-4Bimi, PLL-2V-4Bimi, and PLL-4V-4BImi materials. THP-1 cells were seeded at 500,000 cells per well in six-well plates and incubated for 4 hours in medium containing 40 μg / mL PLL-1V, 2V, and 4V-4BImi. Cells were collected, washed with PBS, and centrifuged (1200 rpm, 5 min) once. Immobilization with paraformaldehyde and permeabilization with 90% methanol was performed according to the antibody staining instructions, with sufficient PBS used to wash away excess paraformaldehyde or methanol at each step. Then, diluted primary antibodies (PE-labeled PTBK-1, AF488-labeled PSTING, and AF647-labeled PIRF3) were added for staining for 1 hour. Excess antibody was washed away, followed by a final wash and fixation. Characterization was performed by flow cytometry.

[0105] Figure 11 It is PLL-1V-4Bimi (with a concentration of 40 μg / mL) Figure 11 After treating THP-1 cells with PLL-4Bimi (PLL-1V-4Bimi), PLL-2V-4Bimi, or PLL-4V-4BImi for 4 hours, the phosphorylation expression of STING pathway-related proteins in the cells was observed, indicating the stimulatory activity of STING. In particular, after incubation with PLL-2V-4BImi and PLL-4V-4BImi, increased phosphorylation of p-TBK1 (Figure a), p-STING (Figure b), and p-IRF3 (Figure c) was observed.

[0106] Application Example 4

[0107] Examples 16, 44, and 72 verify the induction of STING protein aggregates in THP-1 cells by PLL-1V-4Bimi, PLL-2V-4Bimi, and PLL-4V-4BImi materials, and verify the co-localization of PLL-1V, 2V, and 4V-4BImi materials with STING protein. PLL-1V-4Bimi, PLL-2V-4Bimi, and PLL-4V-4BImi materials were labeled with Cy5 fluorescence. Cy5-NHS was added at a 4BImi molar ratio of 1 / 15, and the mixture was stirred overnight in water at 25°C. Subsequently, the mixture was dialyzed in Milli-Q water for 24 hours using a dialysis bag with a molecular weight cutoff of 7000 Da. The resulting solid product, PLL-1V, 2V, and 4V-4BImi-Cy5, was obtained by freeze-drying. Three × 10⁶ THP-1 cells were seeded in each well of a 6-well plate. Then, 2 mL of medium containing 40 μg / mL Cy5-labeled PLL-1V, 2V, 4V-4BImi was added and incubated for 4 hours. After incubation, THP-1 cells were collected, fixed with 4% PFA for 15 minutes, permeabilized with 0.1% Triton X-100 for 15 minutes, and stained with rabbit anti-human STING primary antibody (1:50 dilution in PBS) for 1 hour. Then, FITC-labeled goat anti-rabbit IgG (1:50 dilution in PBS) was added, and the cells were incubated for another 20 minutes. Hoechst 33342 (1:100 dilution in PBS) was added and incubated for 3–5 minutes for nuclear staining. After each step, the cells were washed twice with PBS or PBST. Finally, THP-1 cells were collected and resuspended in PBS for confocal laser imaging (CLSM).

[0108] Figure 12 Laser confocal imaging of THP-1 cells treated with 40 μg / mL PLL-1V-4Bimi-Cy5, PLL-2V-4Bimi-Cy5, and PLL-4V-4BImi-Cy5 for 4 hours shows the aggregation of STING protein in the cells and its co-localization with the materials. The images show that significant STING protein clusters were observed in THP-1 cells treated with PLL-2V-4BImi and PLL-4V-4BImi, further confirming the activation of the intracellular STING pathway.

[0109] Application Example 5

[0110] The IFN-β release induced by the PLL-4V-4BImi material prepared in Example 72 and the bPEI-4BImi obtained in Comparative Example 1 in THP-1 cells and STING- / -THP-1-Cas9 cells was demonstrated, proving the STING pathway dependence of the material. PLL-4V-4BImi and bPEI-4BImi were thoroughly dissolved in sterile water and added to 96-well plates containing 16,000 THP-1 cells and STING- / -THP-1-Cas9 cells per well, with a treatment concentration of 20 μg / mL. After 24 hours, the culture medium in the wells was collected and centrifuged at 3000 rpm for 5 minutes. The supernatant was collected, and the IFN-β concentration was detected using an ELISA kit. The ELISA assay procedure is as follows (all procedures are performed at room temperature): Incubate the capture antibody overnight in a high-adhesion 96-well plate, wash three times with PBST (containing 0.05% PBS solution), block with PBS containing 1% BSA for 1 hour, wash three times with PBST, add the sample to be tested (3-4 replicates) and standard curve sample and incubate for 2 hours, wash three times with PBST, add the detection antibody and incubate for 2 hours, wash three times with PBST, add streptavidin-HRP and incubate for 20 minutes, wash three times with PBST, add TMB chromogenic solution and develop color in the dark for 20 minutes, add 2N H2SO4 to stop the assay, and use a microplate reader to detect the absorbance at 450nm and subtract the background value at 540nm.

[0111] Figure 13 The graph shows the IFN-β concentration in the supernatant of THP-1 and STING- / -THP-1-Cas9 cells after treatment with 20 μg / mL PLL-4V-4BImi and bPEI-4BImi for 24 hours. The results, as measured by ELISA, indicate that IFN-β secretion in STING- / -THP-1-Cas9 cells was significantly reduced compared to THP-1-Cas9 cells. This suggests that the innate stimulatory activity of this type of heterocyclic branched polymer is dependent on STING.

[0112] Application Example 6

[0113] Examples 16, 44, and 72 describe the preparation of nanovaccines using PLL-1V-4Bimi, PLL-2V-4Bimi, and PLL-4V-4BImi materials as ovalbumin (OVA) antigen proteins. PEI-M and OVA were dissolved in sterile water to a concentration of 1 mg / mL. The OVA solution was then slowly added dropwise to the solutions of PLL-1V-4Bimi, PLL-2V-4Bimi, and PLL-4V-4BImi under vortex conditions. After vortexing for 10 minutes, OVA antigen proteins PLL-1V-4Bimi / OVA, PLL-2V-4Bimi / OVA, and PLL-4V-4BImi / OVA supported by polylysine-based polymer adjuvant materials were obtained.

[0114] Figure 14 DLS characterization of the PLL-1V-4Bimi / OVA, PLL-2V-4Bimi / OVA, and PLL-4V-4BImi / OVA nanoparticles prepared in Example 6 showed that they could form nanovaccines with stable assembly.

[0115] The above description of the embodiments is only for the purpose of helping to understand the method and core ideas of the present invention. It should be noted that those skilled in the art can make several improvements and modifications to the present invention without departing from the principles of the present invention, and these improvements and modifications also fall within the protection scope of the claims of the present invention.

Claims

1. A polylysine-based polymeric adjuvant material, characterized in that, Any one of formula (I)~(III): Formula (I); Formula (II); Formula (III); Wherein, n is the degree of polymerization, 10≤n≤200; x1=0.2~0.5, y1=1-x1; x2=0.1~0.4, y2=1-x2; x3=0.05~0.35, y3=1-x3; R1is selected from -NHC(O)R2or -NHC(O)O(CH2) m R2; The R2 is selected from one or more of oxazolyl, isoxazolyl, benzoxazolyl, thiazolyl, benzothiazolyl, pyrazolyl, indazolyl, imidazolyl, benzimidazolyl, piperidinyl, tetrahydroisoquinolinyl, pyridyl, quinolinyl, isoquinolinyl, pyridazinyl, cinnolinyl, pyrimidinyl, quinazolinyl, piperazinyl, pyrazinyl, quinoxalinyl, cycloheximide group. The R2 is selected from one or more of oxazolyl, isoxazolyl, benzoxazolyl, thiazolyl, benzothiazolyl, pyrazolyl, indazolyl, imidazolyl, benzimidazolyl, piperidinyl, tetrahydroisoquinolinyl, pyridyl, quinolinyl, isoquinolinyl, pyridazinyl, cinnolinyl, pyrimidinyl, quinazolinyl, piperazinyl, pyrazinyl, quinoxalinyl, cycloheximide group.

2. The polylysine-based polymeric adjuvant material of claim 1, wherein, The R2 is selected from one or more of oxazolyl, isoxazolyl, benzoxazolyl, thiazolyl, benzothiazolyl, pyrazolyl, indazolyl, imidazolyl, benzimidazolyl, piperidinyl, tetrahydroisoquinolinyl, pyridyl, quinolinyl, isoquinolinyl, pyridazinyl, cinnolinyl, pyrimidinyl, quinazolinyl, piperazinyl, pyrazinyl, quinoxalinyl, cycloheximide group.

3. The method of producing a polylysine-based polymeric adjuvant material according to claim 1, characterized in that, The R2 is selected from one or more of oxazolyl, isoxazolyl, benzoxazolyl, thiazolyl, benzothiazolyl, pyrazolyl, indazolyl, imidazolyl, benzimidazolyl, piperidinyl, tetrahydroisoquinolinyl, pyridyl, quinolinyl, isoquinolinyl, pyridazinyl, cinnolinyl, pyrimidinyl, quinazolinyl, piperazinyl, pyrazinyl, quinoxalinyl, cycloheximide group. The R2 is selected from one or more of oxazolyl, isoxazolyl, benzoxazolyl, thiazolyl, benzothiazolyl, pyrazolyl, indazolyl, imidazolyl, benzimidazolyl, piperidinyl, tetrahydroisoquinolinyl, pyridyl, quinolinyl, isoquinolinyl, pyridazinyl, cinnolinyl, pyrimidinyl, quinazolinyl, piperazinyl, pyrazinyl, quinoxalinyl, cycloheximide group. 。 4. The production method according to claim 3, characterized by, The R2 is selected from one or more of oxazolyl, isoxazolyl, benzoxazolyl, thiazolyl, benzothiazolyl, pyrazolyl, indazolyl, imidazolyl, benzimidazolyl, piperidinyl, tetrahydroisoquinolinyl, pyridyl, quinolinyl, isoquinolinyl, pyridazinyl, cinnolinyl, pyrimidinyl, quinazolinyl, piperazinyl, pyrazinyl, quinoxalinyl, cycloheximide group. The R2 is selected from one or more of oxazolyl, isoxazolyl, benzoxazolyl, thiazolyl, benzothiazolyl, pyrazolyl, indazolyl, imidazolyl, benzimidazolyl, piperidinyl, tetrahydroisoquinolinyl, pyridyl, quinolinyl, isoquinolinyl, pyridazinyl, cinnolinyl, pyrimidinyl, quinazolinyl, piperazinyl, pyrazinyl, quinoxalinyl, cycloheximide group.

5. The preparation method according to claim 3, characterized in that, The R2 is selected from one or more of oxazolyl, isoxazolyl, benzoxazolyl, thiazolyl, benzothiazolyl, pyrazolyl, indazolyl, imidazolyl, benzimidazolyl, piperidinyl, tetrahydroisoquinolinyl, pyridyl, quinolinyl, isoquinolinyl, pyridazinyl, cinnolinyl, pyrimidinyl, quinazolinyl, piperazinyl, pyrazinyl, quinoxalinyl, cycloheximide group. ​ The molar ratio of the carboxyl-substituted five-, six- or seven-membered nitrogen-containing heterocyclic group or its benzo structure or the hydroxyl-substituted five-, six- or seven-membered nitrogen-containing heterocyclic group or its benzo structure to PLL-4V is (3.8-27.4):

1.

6. The preparation method according to claim 3, characterized in that, The molar ratio of the hydroxyl-substituted five-, six- or seven-membered nitrogen-containing heterocyclic group or its benzo structure to N’N-carbonyldiimidazole is (0.7-1):

1.

7. The preparation method according to claim 3, characterized in that, When PLL-1V or PLL-2V or PLL-4V is subjected to amidation reaction with the carboxyl-substituted five-, six- or seven-membered nitrogen-containing heterocyclic group or its benzo structure, a condensing agent is further added in the preparation method.

8. The preparation method according to claim 7, characterized in that, The condensing agent is selected from 1-ethyl-(3-dimethylaminopropyl)carbodiimide hydrochloride and / or N-hydroxysuccinimide.

9. Use of the polylysine-based polymeric adjuvant material according to any one of claims 1-2 or the polylysine-based polymeric adjuvant material prepared by the preparation method according to any one of claims 3-8 in the preparation of an antitumor vaccine preparation.

10. A vaccine formulation, characterized in that, The polylysine-based polymeric adjuvant material according to any one of claims 1-2 or the polylysine-based polymeric adjuvant material prepared by the preparation method according to any one of claims 3-8.

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