Modified alumina composite adjuvant and method for preparing the same

Abstract

The application discloses a modified alumina composite adjuvant and a preparation method thereof, and belongs to the technical field of biological medicines. The modified alumina composite adjuvant takes nano-alumina as a core, introduces a metal organic framework structure, is functionally modified by using a nano-drug carrier material, and forms the adjuvant with a multi-level core-shell structure. The structure enables the adjuvant to have a high encapsulation rate and good dispersion stability; meanwhile, the adjuvant can slowly release antigens in a physiological environment, prolongs the immune stimulation time, realizes intelligent release of the antigens in an acid or a microenvironment with specific enzymes, and thus better simulates a natural infection process and synergistically enhances an immune response. The application has a simple preparation process, mild conditions, good repeatability, a wide clinical application prospect and industrialization value.

Description

Technical Field

[0001] This invention relates to the field of biomedical technology, and in particular to a modified alumina composite adjuvant and its preparation method. Background Technology

[0002] Adjuvants are key components for enhancing antigen-specific immune responses. Currently, aluminum adjuvants are mainly aluminum hydroxide and aluminum phosphate. Due to their long-term safety record and low production cost, they are the only adjuvants widely approved and marketed in human vaccines. Aluminum hydroxide typically exhibits a positively charged boehmite structure with a high specific surface area, enabling it to adsorb antigens through ligand exchange and other mechanisms. Aluminum phosphate, on the other hand, is amorphous, and its surface charge and isoelectric point vary with the phosphate ratio.

[0003] The existing technology CN120643686A, which prepares highly adsorbent aluminum hydroxide adjuvant, possesses good adsorption potential, but its encapsulation efficiency for weakly immunogenic antigens such as peptides and recombinant proteins is low, leading to easy loss of free antigens, limited immunization effects, and difficulty in direct application to human vaccines, with generally poor final potency. Furthermore, this adjuvant is prone to aggregation in electrolyte-containing solutions, affecting its stability. CN112569350A discloses an aluminum hydroxide adjuvant with a boehmite-like structure, which improves protein adsorption capacity, but neglects key process issues in actual injection and storage. In recent years, metal-organic frameworks (MOFs) have been explored as vaccine carriers due to their high specific surface area and biodegradability; however, the mixing of MOFs with aluminum adjuvants still faces problems such as particle aggregation, insufficient biocompatibility, and limited functionality. Therefore, the vaccine development field urgently needs to develop a novel modified alumina composite adjuvant that functionalizes alumina through a metal-organic framework, possessing high encapsulation efficiency, anti-aggregation, long-term storage stability, microenvironment-responsive release, and further surface functionalization capabilities. This has become an urgent need in the field of vaccine delivery technology. Summary of the Invention

[0004] Existing compound adjuvants struggle to simultaneously achieve the safety of aluminum adjuvants and the high loading and intelligent release characteristics of carrier materials. This invention functionalizes alumina using a metal-organic framework, resulting in properties such as high encapsulation efficiency, anti-aggregation, long-term storage stability, microenvironment-responsive release, and surface functionalizability, thus meeting the urgent needs of the vaccine delivery field.

[0005] A method for preparing a modified alumina composite adjuvant is as follows:

[0006] S1, Preprocessing Stage

[0007] Take 10-30 mg of nano-alumina and disperse it in 10-15 mL of water using ultrasonication at 200-500 W to prepare a nano-alumina dispersion for later use; take 0.1-0.5 g of zinc nitrate hexahydrate and dissolve it in 10-15 mL of water, mix well to prepare a zinc salt aqueous solution for later use.

[0008] S2, Reaction Stage

[0009] Dissolve 0.1-0.8g of 2-methylimidazole in 20-30mL of water, mix to obtain an aqueous solution of 2-methylimidazole, add the prepared nano-alumina dispersion and zinc salt aqueous solution under stirring at 500-1000rpm, and stir for 30-60min.

[0010] S3, Purification Stage

[0011] Centrifuge at 10000-15000 rpm for 10-20 min at 2-6℃; collect the precipitate and wash with water;

[0012] S4, Modification Stage

[0013] The washed precipitate was resuspended in 1-10 mL of water or buffer solution to obtain a resuspension; 1-10 mg of nano-drug carrier material was dispersed in 0.1-2 mL of acetonitrile, and then mixed with the resuspension under light-protected conditions, and stirred at 100-300 rpm for 1-5 h; then centrifuged at 12000 rpm for 15 min at 2-6 °C; the precipitate was collected and washed with water to obtain the modified alumina composite adjuvant;

[0014] S5, Storage Stage

[0015] Resuspend the resulting final product in 1-5 mL of water and store at 2-6 °C.

[0016] The nanomedicine carrier material in step S4 is one or more of polylactic acid-glycolic acid copolymer, polyethyleneimine, galactose-polyethyleneimine copolymer, polyethylene glycol-polylactic acid-glycolic acid copolymer-polyethyleneimine triblock copolymer, or enzyme-responsive peptide chain modified polymer.

[0017] The preparation method of the nanomedicine carrier material involved in step S4 is as follows: 50-200 mg of branched polyethyleneimine and 30-100 mg of lactobionic acid are dissolved in 5-20 mL of phosphate buffer, and then 10-50 mg of 1-ethyl-3-(3-dimethylaminopropyl)carbodiimide and 10-50 mg of N-hydroxysuccinimide are added. The mixture is stirred continuously at room temperature (10℃-35℃) and protected from light for 18-24 h. After dialysis and freeze-drying, galactose-polyethyleneimine copolymer is obtained.

[0018] The preparation method of the nanomedicine carrier material involved in step S4 is as follows: 20-100 mg of polylactic acid-glycolic acid copolymer-polyethylene glycol-carboxyl group, 10-50 mg of N-hydroxysuccinimide, and 10-50 mg of 1-ethyl-3-(3-dimethylaminopropyl)carbodiimide are added to 5-20 mL of anhydrous dimethyl sulfoxide and stirred at 300 rpm for 2 h at room temperature (10℃-35℃) in the dark. Separately, 10-50 mg of polyethyleneimine and 1-5 mL of anhydrous dimethyl sulfoxide are mixed and stirred for 18-24 h at room temperature (10℃-35℃) in the dark. After dialysis and freeze-drying, polyethylene glycol-polylactic acid-glycolic acid copolymer-polyethyleneimine triblock copolymer is obtained.

[0019] The copolymer dialysis treatment involves transferring the above-obtained reaction solution into a 3500Da dialysis bag, dialyzing with a 20-50 vol% dimethyl sulfoxide aqueous solution for 3-6 hours, and then dialyzing with water for 12-24 hours, changing the water every 3-6 hours.

[0020] The preparation method of the nanomedicine carrier material involved in step S4 is as follows: 10-50 mg of polyethylene glycol-succinimide ester is dissolved in 1-5 mL of phosphate buffer, 1-5 mg of matrix metalloproteinase substrate peptide is added, and the mixture is stirred and reacted at room temperature (10℃-35℃) for 1-4 h; the reaction solution is first dialyzed and freeze-dried to obtain grafted polypeptide-polyethylene glycol; then 20-100 mg of polyethyleneimine is dissolved in 5-10 mL of phosphate buffer, 20-100 mg of polylactic acid-glycolic acid copolymer-carboxyl group is dispersed in the above buffer, and 10-50 mg of N-hydroxysuccinimide and 10-50 mg of... 1-Ethyl-3-(3-dimethylaminopropyl)carbodiimide was stirred at room temperature (10℃-35℃) for 1-2 hours; then the obtained grafted polypeptide-polyethylene glycol was added, and the reaction was continued for 8-12 hours; the reaction solution was then dialyzed twice and freeze-dried to obtain the enzyme-responsive triblock copolymer.

[0021] The matrix metalloproteinase substrate peptide is at least one of matrix metalloproteinase 2 / 9 non-responsive mutant peptide chains and matrix metalloproteinase 2 / 9 responsive peptides.

[0022] The initial dialysis of the copolymer is as follows: the reaction solution is dialyzed with water with a molecular weight cutoff of 1000 Da for 24 hours and then freeze-dried to obtain the grafted polypeptide-polyethylene glycol; the second dialysis is as follows: the reaction solution is transferred into a 3500 Da dialysis bag and dialyzed with water for 12-24 hours, with the water changed every 3-6 hours.

[0023] The buffer solution involved in step S4 of the copolymer preparation method includes phosphate buffer or borate buffer with a pH range of 7.0-9.0.

[0024] The freeze-drying process of the copolymer is as follows: the cold trap temperature is set to -50°C to -60°C, the vacuum degree is 5-20Pa, and the drying time is 4-8h.

[0025] The beneficial effects of this invention are:

[0026] 1. This invention significantly enhances antigen loading capacity by in-situ growing metal-organic frameworks on the surface of nano-alumina. Metal-organic frameworks, with their high specific surface area and porous structure, can efficiently capture and immobilize antigen molecules during synthesis; they maintain structural stability at physiological pH, slowing antigen release and preventing loss through systemic circulation; when the adjuvant is phagocytosed by antigen-presenting cells and enters the acidic lysosomal environment, 2-methylimidazole undergoes protonation, leading to rapid dissociation of the entire framework structure, thereby achieving on-demand release.

[0027] 2. This invention constructs a multi-layered composite structure of alumina core-intermediate layer-functional polymer shell. Through the synergistic effect between the components, the metal shell provides an electrostatic repulsion barrier, while the outer triblock copolymer integrates electrostatic, hydrophobic and steric stabilization mechanisms, which together enhance the physical stability and biocompatibility of the adjuvant, making it suitable for various scenarios such as preventive and therapeutic vaccines, and has broad clinical application prospects. Detailed Implementation

[0028] The parameters and sources of some substances in the examples are as follows:

[0029] Nano-alumina: CAS No.: 1344-28-1; Model: NanoArc® Alumina Powder (γ-phase); Particle size ≤20nm; Source: Alfa Aesar.

[0030] Polyethylene glycol-succinimide ester: CAS No.: 1008402-79-6; Molecular weight: 2000 Da.

[0031] 2-Methylimidazole: CAS No.: 693-98-1.

[0032] Polylactic acid-glycolic acid copolymer: CAS No.: 26780-50-7; Model: RESERMER® RG503H; Source: Evonik Industries.

[0033] Polyethyleneimine: CAS No.: 9002-98-6; Molecular weight < 25000 Da; Density: 1.07-1.08 g / cm³ 3 .

[0034] Branched polyethyleneimine: CAS No.: 9002-98-6; Molecular weight < 15000 Da; Density: 0.997-1.08 g / cm³3 .

[0035] Lactobionic acid: CAS No.: 96-82-2.

[0036] Methoxylated polyethylene glycol-succinimide ester: CAS No.: 756525-94-7; where polyethylene glycol has a molecular weight of 2000 Da; Source: Yu Si Pharmaceutical.

[0037] Polylactic acid-glycolic acid copolymer-polyethylene glycol-carboxyl group: molecular weight: 15000 Da; of which polyethylene glycol has a molecular weight of 2000 Da; the ratio of lactic acid / glycolic acid monomers is 75 / 25; source: Weihua Bio.

[0038] Polylactic acid-glycolic acid copolymer - carboxyl group: molecular weight: 5000-15000 Da, polylactic acid / glycolic acid monomer ratio is 50 / 50; catalog number: L-212043; source: Aolong Biotechnology.

[0039] Matrix metalloproteinase 2 / 9 responsive peptide: Sequence: GGGPQGIWGQGK; Source: Shanghai Meiluo Technology Co., Ltd.

[0040] Non-responsive mutant peptide chains of matrix metalloproteinases 2 / 9: Sequence: GGGPQGIAGQGK; Source: Shanghai Meiluo Technology Co., Ltd.

[0041] 4-(N-maleimidemethyl)cyclohexane-1-carboxylic acid sulfonate succinimide ester: CAS No.: 92921-24-9.

[0042] Ovalbumin: CAS No.: 9006-59-1.

[0043] Example 1

[0044] A method for preparing a modified alumina composite adjuvant is as follows:

[0045] S1, Preprocessing Stage

[0046] 20 mg of nano-alumina was ultrasonically dispersed in 12.5 mL of water at 300 W to obtain a nano-alumina dispersion for later use; 0.293 g of zinc nitrate hexahydrate was dissolved in 12.5 mL of water, and the mixture was used to obtain a zinc salt aqueous solution for later use.

[0047] S2, Reaction Stage

[0048] Dissolve 0.328 g of 2-methylimidazole in 25 mL of water, mix to obtain an aqueous solution of 2-methylimidazole, add the prepared nano alumina dispersion and zinc salt aqueous solution under stirring at 800 rpm, and stir for 60 min.

[0049] S3, Purification Stage

[0050] Centrifuge at 12000 rpm for 15 min at 4℃; collect the precipitate and wash it three times with 5 mL of water to obtain the nano-alumina composite adjuvant.

[0051] S4, Storage Stage

[0052] The resulting final product was resuspended in 5 mL of water and stored at 4 °C.

[0053] Example 2

[0054] A method for preparing a modified alumina composite adjuvant is as follows:

[0055] S1, Preprocessing Stage

[0056] 20 mg of nano-alumina was ultrasonically dispersed in 12.5 mL of water at 300 W to obtain a nano-alumina dispersion for later use; 0.293 g of zinc nitrate hexahydrate was dissolved in 12.5 mL of water, and the mixture was used to obtain a zinc salt aqueous solution for later use.

[0057] S2, Reaction Stage

[0058] Dissolve 0.328 g of 2-methylimidazole in 25 mL of water, mix to obtain an aqueous solution of 2-methylimidazole, add the prepared nano alumina dispersion and zinc salt aqueous solution under stirring at 800 rpm, and stir for 60 min.

[0059] S3, Purification Stage

[0060] Centrifuge at 12000 rpm for 15 min at 4℃; collect the precipitate and wash it three times with 5 mL of water;

[0061] S4, Modification Stage

[0062] The washed precipitate was resuspended in 5 mL of pH 8.5 borate buffer to obtain a resuspension; 2 mg of methoxy polyethylene glycol-succinimide ester was mixed with 0.5 mL of acetonitrile, and then mixed with the above resuspension under light-protected conditions, and stirred at 200 rpm for 4 h; then centrifuged at 12000 rpm for 15 min at 4 °C, the precipitate was collected, and washed 3 times with 5 mL of water to obtain the modified alumina composite adjuvant;

[0063] S5, Storage Stage

[0064] The resulting final product was resuspended in 5 mL of water and stored at 4 °C.

[0065] Example 3

[0066] A method for preparing a modified alumina composite adjuvant is as follows:

[0067] S1, Preprocessing Stage

[0068] 20 mg of nano-alumina was ultrasonically dispersed in 12.5 mL of water at 300 W to obtain a nano-alumina dispersion for later use; 0.293 g of zinc nitrate hexahydrate was dissolved in 12.5 mL of water, and the mixture was used to obtain a zinc salt aqueous solution for later use.

[0069] S2, Reaction Stage

[0070] Dissolve 0.328 g of 2-methylimidazole in 25 mL of water, mix to obtain an aqueous solution of 2-methylimidazole, add the prepared nano alumina dispersion and zinc salt aqueous solution under stirring at 800 rpm, and stir for 60 min.

[0071] S3, Purification Stage

[0072] Centrifuge at 12000 rpm for 15 min at 4℃; collect the precipitate and wash it three times with 5 mL of water;

[0073] S4, Modification Stage

[0074] The washed precipitate was resuspended in 5 mL of water to obtain a resuspension; 2 mg of polylactic acid-glycolic acid copolymer was dispersed in 0.5 mL of acetonitrile, and then mixed with the above resuspension under light-protected conditions. The mixture was stirred at 200 rpm for 4 h; then centrifuged at 12000 rpm for 15 min at 4 °C; the precipitate was collected and washed three times with 5 mL of water to obtain the modified alumina composite adjuvant.

[0075] S5, Storage Stage

[0076] The resulting final product was resuspended in 5 mL of water and stored at 4 °C.

[0077] Example 4

[0078] A method for preparing a modified alumina composite adjuvant is as follows:

[0079] S1, Preprocessing Stage

[0080] 20 mg of nano-alumina was ultrasonically dispersed in 12.5 mL of water at 300 W to obtain a nano-alumina dispersion for later use; 0.293 g of zinc nitrate hexahydrate was dissolved in 12.5 mL of water, and the mixture was used to obtain a zinc salt aqueous solution for later use; 0.1 g of polyethyleneimine was dissolved in 10 mL of water to obtain a polyethyleneimine aqueous solution for later use.

[0081] S2, Reaction Stage

[0082] Dissolve 0.328 g of 2-methylimidazole in 25 mL of water, mix to obtain an aqueous solution of 2-methylimidazole, add the prepared nano alumina dispersion and zinc salt aqueous solution under stirring at 800 rpm, and stir for 60 min.

[0083] S3, Purification Stage

[0084] Centrifuge at 12000 rpm for 15 min at 4℃; collect the precipitate and wash it three times with 5 mL of water;

[0085] S4, Modification Stage

[0086] The precipitate obtained after washing was resuspended in 5 mL of water to obtain a resuspension; 5 mL of spare polyethyleneimine aqueous solution was added and mixed with the above resuspension under the dark, and stirred at 200 rpm for 4 h; then centrifuged at 12000 rpm for 15 min at 4 °C; the precipitate was collected and washed 3 times with 5 mL of water to obtain the modified alumina composite adjuvant.

[0087] S5, Storage Stage

[0088] The resulting final product was resuspended in 5 mL of water and stored at 4 °C.

[0089] Example 5

[0090] A method for preparing a modified alumina composite adjuvant is as follows:

[0091] S1, Preprocessing Stage

[0092] 20 mg of nano-alumina was ultrasonically dispersed in 12.5 mL of water at 300 W to obtain a nano-alumina dispersion for later use; 0.293 g of zinc nitrate hexahydrate was dissolved in 12.5 mL of water, and the mixture was used to obtain a zinc salt aqueous solution for later use.

[0093] 100 mg of branched polyethyleneimine and 60 mg of lactobionic acid were dissolved in 10 mL of phosphate buffer at pH 7.4. 25 mg of 1-ethyl-3-(3-dimethylaminopropyl)carbodiimide and 25 mg of N-hydroxysuccinimide were added. The mixture was stirred continuously for 24 h at room temperature (10℃-35℃) in the dark. The reaction solution was then transferred to a 3500 Da dialysis bag and dialyzed against 50 vol% dimethyl sulfoxide aqueous solution for 6 h, followed by dialyzed against water for 24 h, with the water changed every 6 h. The liquid in the dialysis bag was dried at -55℃ and a vacuum of 10 Pa for 8 h to obtain the galactose-polyethyleneimine copolymer, which was sealed and stored at -20℃ for later use.

[0094] S2, Reaction Stage

[0095] Dissolve 0.328 g of 2-methylimidazole in 25 mL of water, mix to obtain an aqueous solution of 2-methylimidazole, add the prepared nano alumina dispersion and zinc salt aqueous solution under stirring at 800 rpm, and stir for 60 min.

[0096] S3, Purification Stage

[0097] Centrifuge at 12000 rpm for 15 min at 4℃; collect the precipitate and wash it three times with 5 mL of water;

[0098] S4, Modification Stage

[0099] The precipitate obtained after washing was resuspended in 5 mL of water to obtain a resuspension; 5 mg of the galactose-polyethyleneimine copolymer prepared above and 0.5 mL of acetonitrile were mixed with the above resuspension under light-protected conditions and stirred at 200 rpm for 4 h; then centrifuged at 12000 rpm for 15 min at 4 °C; the precipitate was collected and washed three times with 5 mL of water to obtain the modified alumina composite adjuvant.

[0100] S5, Storage Stage

[0101] The resulting final product was resuspended in 5 mL of water and stored at 4 °C.

[0102] Example 6

[0103] A method for preparing a modified alumina composite adjuvant is as follows:

[0104] S1, Preprocessing Stage

[0105] Take 20 mg of nano-alumina and ultrasonically disperse it in 12.5 mL of water at 300 W to prepare a nano-alumina dispersion for later use; take 0.293 g of zinc nitrate hexahydrate and dissolve it in 12.5 mL of water, mix well to prepare a zinc salt aqueous solution for later use.

[0106] Take 50 mg of polylactic acid-glycolic acid copolymer-polyethylene glycol-carboxyl group, 25 mg of N-hydroxysuccinimide, and 25 mg of 1-ethyl-3-(3-dimethylaminopropyl)carbodiimide and add them to 5 mL of anhydrous dimethyl sulfoxide. Stir at 300 rpm for 2 h at room temperature (10℃-35℃) in the dark. Separately, take 20 mg of polyethyleneimine and 2 mL of anhydrous dimethyl sulfoxide and stir at room temperature (10℃-35℃) in the dark for 24 h. Transfer the two reaction solutions into a 3500 Da dialysis bag. Dialyze with 50 vol% dimethyl sulfoxide aqueous solution for 6 h, then dialyze with water for 24 h, changing the water every 6 h. Dry the liquid in the dialysis bag at -55℃ and a vacuum of 10 Pa for 8 h to obtain the polyethylene glycol-polylactic acid-glycolic acid copolymer-polyethyleneimine triblock copolymer. Seal and store at -20℃ for later use.

[0107] S2, Reaction Stage

[0108] Dissolve 0.328 g of 2-methylimidazole in 25 mL of water, and mix to obtain an aqueous solution of 2-methylimidazole. Add the prepared nano-alumina dispersion and zinc salt aqueous solution under stirring at 800 rpm, and stir for 60 min.

[0109] S3, Purification Stage

[0110] Centrifuge at 12000 rpm for 15 min at 4℃; collect the precipitate and wash it three times with 5 mL of water;

[0111] S4, Modification Stage

[0112] The precipitate obtained after washing was resuspended in 5 mL of water to obtain a resuspension; 5 mg of the prepared polyethylene glycol-polylactic acid-glycolic acid copolymer-polyethyleneimine triblock copolymer was dispersed in 0.5 mL of acetonitrile, and then mixed with the above resuspension under light-protected conditions, and stirred at 200 rpm for 4 h; then centrifuged at 12000 rpm for 15 min at 4 °C; the precipitate was collected and washed three times with 5 mL of water to obtain the modified alumina composite adjuvant;

[0113] S5, Storage Stage

[0114] The resulting final product was resuspended in 5 mL of water and stored at 4 °C.

[0115] Example 7

[0116] A method for preparing a modified alumina composite adjuvant is as follows:

[0117] S1, Preprocessing Stage

[0118] Take 20 mg of nano-alumina and ultrasonically disperse it in 12.5 mL of water at 300 W to prepare a nano-alumina dispersion for later use; take 0.293 g of zinc nitrate hexahydrate and dissolve it in 12.5 mL of water, mix well to prepare a zinc salt aqueous solution for later use.

[0119] 10 mg of polyethylene glycol-succinimide ester was dissolved in 2 mL of pH 7.4 phosphate buffer, and 5 mg of matrix metalloproteinase 2 / 9 responsive peptide was added. The mixture was stirred at 25 °C for 4 h. The reaction solution was dialyzed against 1000 Da molecular weight cutoff water for 24 h, and dried at -55 °C and 10 Pa for 6 h to obtain grafted polypeptide-polyethylene glycol. 100 mg of polyethyleneimine was then dissolved in 10 mL of pH 7.4 phosphate buffer, and 50 mg of polylactic acid-glycolic acid copolymer-carboxyl group was dispersed in the buffer. 25 mg of N-hydroxysuccinimide and 25 mg of... 1-Ethyl-3-(3-dimethylaminopropyl)carbodiimide was stirred at 25°C for 2 hours; then the obtained grafted polypeptide-polyethylene glycol was added, and the reaction was continued for 12 hours; the reaction solution was then dialyzed at a molecular weight cutoff of 3500 Da for 24 hours, and the liquid in the dialysis bag was dried at -55°C and a vacuum of 10 Pa for 8 hours to obtain the enzyme-responsive triblock copolymer; it was sealed and stored at -20°C for later use.

[0120] S2, Reaction Stage

[0121] Dissolve 0.328 g of 2-methylimidazole in 25 mL of water, and mix to obtain an aqueous solution of 2-methylimidazole. Add the prepared nano-alumina dispersion and zinc salt aqueous solution under stirring at 800 rpm, and stir for 60 min.

[0122] S3, Purification Stage

[0123] Centrifuge at 12000 rpm for 15 min at 4℃; collect the precipitate and wash it three times with 5 mL of water;

[0124] S4, Modification Stage

[0125] The washed precipitate was resuspended in 5 mL of water to obtain a resuspension; 5 mg of the prepared enzyme-response triblock copolymer was dispersed in 0.5 mL of acetonitrile, and then mixed with the above resuspension under light-protected conditions and stirred at 200 rpm for 4 h; then centrifuged at 12000 rpm for 15 min at 4 °C; the precipitate was collected and washed three times with 5 mL of water to obtain the modified alumina composite adjuvant.

[0126] S5, Storage Stage

[0127] The resulting final product was resuspended in 5 mL of water and stored at 4 °C.

[0128] Comparative Example 1

[0129] A method for preparing an alumina composite adjuvant is as follows:

[0130] 20 mg of nano-alumina was ultrasonically dispersed in 12.5 mL of water at 300 W to obtain a nano-alumina dispersion for later use; 0.293 g of zinc nitrate hexahydrate was dissolved in 12.5 mL of water, and the mixture was used to obtain a zinc salt aqueous solution for later use.

[0131] Dissolve 0.328 g of 2-methylimidazole in 25 mL of water, add zinc salt aqueous solution while stirring at 800 rpm, and stir for 60 min; then centrifuge at 12000 rpm for 15 min at 4 °C, wash the precipitate three times with 5 mL of water, resuspend it in 5 mL of water; then mix with 5 mL of the above nano alumina dispersion, vortex for 3 min to obtain the final product.

[0132] The resulting final product was resuspended in 5 mL of water and stored at 4 °C.

[0133] Comparative Example 2

[0134] A method for preparing a modified alumina composite adjuvant is as follows:

[0135] S1, Preprocessing Stage

[0136] Take 20 mg of nano-alumina and ultrasonically disperse it in 12.5 mL of water at 300 W to prepare a nano-alumina dispersion for later use; take 0.293 g of zinc nitrate hexahydrate and dissolve it in 12.5 mL of water, mix well to prepare a zinc salt aqueous solution for later use.

[0137] S2, Reaction Stage

[0138] Dissolve 0.328 g of 2-methylimidazole in 25 mL of water, and mix to obtain an aqueous solution of 2-methylimidazole. Add the prepared nano-alumina dispersion and zinc salt aqueous solution under stirring at 800 rpm, and stir for 60 min.

[0139] S3, Purification Stage

[0140] Centrifuge at 12000 rpm for 15 min at 4℃; collect the precipitate and wash it three times with 5 mL of water;

[0141] S4, Antigen loading stage by electrostatic adsorption

[0142] The washed precipitate was resuspended in 4 mL of 10 mmol / L, pH 7.4 phosphate buffer for later use; 2 mg of ovalbumin was added to 1 mL of 10 mmol / L, pH 7.4 phosphate buffer, filtered through 0.22 μm, and the prepared buffer was added dropwise; the mixture was incubated at 4 °C and 200 rpm in the dark for 2 h; the precipitate was collected by centrifugation at 12000 rpm for 15 min and washed twice with 5 mL of water to obtain the modified alumina composite adjuvant;

[0143] S5, Storage Stage

[0144] The resulting final product was resuspended in 5 mL of water and stored at 4 °C.

[0145] Comparative Example 3

[0146] A method for preparing a modified alumina composite adjuvant is as follows:

[0147] S1, Preprocessing Stage

[0148] Take 20 mg of nano-alumina and ultrasonically disperse it in 12.5 mL of water at 300 W to prepare a nano-alumina dispersion for later use; take 0.293 g of zinc nitrate hexahydrate and dissolve it in 12.5 mL of water, mix well to prepare a zinc salt aqueous solution for later use.

[0149] 10 mg of polyethylene glycol-succinimide ester was dissolved in 2 mL of pH 7.4 phosphate buffer, and 5 mg of matrix metalloproteinase 2 / 9 non-responsive mutant peptide chain was added. The reaction was stirred at 25 °C for 4 h. The reaction solution was dialyzed against 1000 Da molecular weight cutoff water for 24 h, and dried at -55 °C and 10 Pa vacuum for 6 h to obtain grafted non-responsive polypeptide-polyethylene glycol. Then, 100 mg of polyethyleneimine was dissolved in 10 mL of pH 7.4 phosphate buffer, and 50 mg of polylactic acid-glycolic acid copolymer-carboxyl group was dispersed in the buffer. 25 mg of N-hydroxysuccinimide and 25 mg of [unclear text - possibly a typo, should be "added"]. 1-Ethyl-3-(3-dimethylaminopropyl)carbodiimide was stirred at 25°C for 2 hours; then the obtained grafted non-responsive peptide-polyethylene glycol was added, and the reaction was continued for 12 hours; the reaction solution was then dialyzed at a molecular weight cutoff of 3500 Da for 24 hours, and the liquid in the dialysis bag was dried at -55°C and a vacuum of 10 Pa for 8 hours to obtain the non-responsive mutant peptide chain-triblock copolymer; it was sealed and stored at -20°C for later use.

[0150] S2, Reaction Stage

[0151] Dissolve 0.328 g of 2-methylimidazole in 25 mL of water, and mix to obtain an aqueous solution of 2-methylimidazole. Add the prepared nano-alumina dispersion and zinc salt aqueous solution under stirring at 800 rpm, and stir for 60 min.

[0152] S3, Purification Stage

[0153] Centrifuge at 12000 rpm for 15 min at 4℃; collect the precipitate and wash it three times with 5 mL of water;

[0154] S4, Modification Stage

[0155] The washed precipitate was resuspended in 5 mL of water to obtain a resuspension; 5 mg of the prepared non-responsive mutant peptide-triblock copolymer was dispersed in 0.5 mL of acetonitrile, and then mixed with the above resuspension under light-protected conditions and stirred at 200 rpm for 4 h; then centrifuged at 12000 rpm for 15 min at 4 °C; the precipitate was collected and washed three times with 5 mL of water to obtain the modified alumina composite adjuvant;

[0156] S5, Storage Stage

[0157] The resulting final product was resuspended in 5 mL of water and stored at 4 °C.

[0158] Test Example 1

[0159] Particle size distribution test

[0160] Pretreatment: The modified alumina composite adjuvants prepared in Examples 1-7 and Comparative Examples 1-3 were appropriately diluted 100 times with ultrapure water until they were translucent; then 1 mL of the diluted sample was placed in the sample cell of the Zetasizer Nano ZS90 Malvern particle size analyzer.

[0161] Particle size and PDI: After equilibration at 25℃ for 60s, measurements were taken using dynamic and static laser light scattering mode with a scattering angle set to 90°; each sample was measured 3 times and the average value was taken.

[0162] Zeta potential: After equilibration at 25℃ for 60 seconds, it was measured using a folded capillary electrophoresis pool combined with potential detection technology. Each sample was measured three times, and the average value was taken. The results are shown in Table 1.

[0163] Table 1. Results of particle size distribution test

[0164]

[0165] Note: PDI is the polydispersity index, and a value < 0.2 ensures the uniformity of particles.

[0166] In Example 1, a continuous lattice coating layer was formed on the surface of nano-alumina by in-situ growth of a metal-organic framework, generating an electrostatic repulsion barrier, which effectively avoided the multi-peak aggregation phenomenon that occurred in the basic physical mixing in Comparative Example 1; while in Comparative Example 2, without the steric hindrance provided by the polymer, the attractive forces such as van der Waals forces between particles dominated, resulting in decreased stability.

[0167] Building upon Example 1, Example 2 introduces polyethylene glycol modification. Its long chains create steric hindrance, shielding surface charges. In a physiological environment, this effectively resists non-specific protein adsorption and rapid clearance by the immune system, thereby prolonging in vivo circulation time and imparting better storage and usage stability to the adjuvant system. Example 3 introduces polylactic acid-glycolic acid copolymer modification. The carboxyl groups at the molecular ends provide strong electrostatic repulsion, helping to maintain dispersion stability. Example 4 uses polyethyleneimine modification to optimize its adsorption capacity for negatively charged protein antigens and facilitates endocytosis by negatively charged cell membranes via electrostatic interactions.

[0168] Furthermore, Examples 5-7 underwent functionalization modifications: In Example 5, the introduction of galactose-polyethyleneimine not only endowed the particles with targeting function, but its galactose portion also partially neutralized the strong positive charge of the polyethyleneimine chain segment, achieving a better balance between functionality and biocompatibility. Example 6 used a polyethyleneimine-polylactic acid-glycolic acid copolymer-polyethylene glycol triblock copolymer, taking into account positive charge, biodegradability, and long cycling. Example 7 further introduced matrix metalloproteinase 2 / 9 responsive peptides to form cleavage sites, endowing subsequent vaccine preparation with structural relaxation and drug release functions triggered by the lesion microenvironment, achieving precise immune activation.

[0169] In contrast, Comparative Example 3 utilizes a matrix metalloproteinase 2 / 9 non-responsive mutant peptide chain to covalently anchor the antigen to the triblock-modified alumina surface. Stable thioether bonds are formed through Michael addition of maleimide groups to the terminal thiol groups of the peptides, providing an outer hydration barrier that masks the positive charge and maintains particle integrity. Example 7, building upon the triblock copolymer modification of Example 6, utilizes an enzyme-responsive peptide chain and polyethylene glycol to form a more dense and regular higher-order structure on the particle surface, providing the strongest steric stabilization.

[0170] Test Example 2

[0171] Encapsulation test

[0172] Preparation of antigen stock solution: Accurately weigh 10 mg of ovalbumin and dissolve it in 1 mL of pH 7.4 phosphate buffer; prepare fresh before use.

[0173] Standard curve plotting: Antigen standard solutions were diluted with phosphate buffer at pH 7.4 to obtain concentrations of 2000, 1500, 1000, 750, 500, 250, 125, and 0 μg / mL. 25 μL of each concentration of standard solution was added to a 96-well plate, and 200 μL of diquinoline formic acid working solution was added to each well. The plate was sealed and incubated at 37°C in the dark for 30 min. The absorbance of each well was then measured at 562 nm using a SpectraMax iD5 microplate reader. A standard curve was plotted with the 0 μg / mL standard as a blank control, and the absorbance as the Y-axis and the antigen concentration as the X-axis.

[0174] Total antigen content: Take an equal volume of pure antigen solution (without adjuvant) as used in the antigen loading process, dilute it to 500 μg / mL with pH 7.4 phosphate buffer, and measure it at a wavelength of 562 nm. This value is the total antigen added, which is W. 总 ;

[0175] Free antigen concentration: Take 2 mL of adjuvant dispersion, centrifuge at 12000 rpm for 15 minutes at 4℃, carefully aspirate the supernatant, dilute to 500 μg / mL with pH 7.4 phosphate buffer, and measure at 562 nm. This value is the free antigen concentration, expressed as W. 游离 The results are shown in Table 2.

[0176] Encapsulation rate / % = (W 总 -W 游离 ) / W 总 ×100%.

[0177] Table 2 Encapsulation efficiency test results

[0178]

[0179] Compared to the physical dispersion methods of Comparative Examples 1-2, Example 1 employs in-situ synthesis of a metal-organic framework on the surface of nano-alumina. This allows the ovalbumin antigen in the solution to be directly captured and confined within the pores or cavities of the metal-organic material during its growth process, resulting in a slightly higher encapsulation efficiency. In contrast, the physical mixing in Comparative Example 1 relies solely on weak van der Waals forces between particles, leading to unstable loading. Although Comparative Example 2 uses the same core material as Example 1, its antigen loading is achieved through static electrostatic adsorption after particle synthesis. This method only utilizes the outermost surface area and charge of the particles, failing to leverage the vast specific surface area and pores within the metal-organic framework. Consequently, the adsorbed antigen may easily detach during subsequent washing and storage.

[0180] The long chains of polyethylene glycol form a dense hydrophilic layer on the particle surface, which can reduce non-specific protein adsorption, prolong in vivo circulation time, and hinder antigen diffusion, thus delaying immune clearance. Example 3 utilizes the interaction between the hydrophobic segments of the polylactic-co-glycolic acid copolymer and the hydrophobic regions of the antigen, making it suitable for hydrophobic or amphiphilic antigens. Example 4 introduces polyethyleneimine, imparting strong positive charge to the particles, enhancing electrostatic attraction with negatively charged ovalbumin, and improving encapsulation efficiency. In Example 5, galactose and polyethyleneimine form a targeting copolymer, which can enhance binding to the antigen through various intermolecular forces, such as the recognition effect of galactose and the electrostatic interaction of polyethyleneimine.

[0181] In Example 6, the copolymer was modified into a triblock copolymer, integrating electrostatic attraction, hydrophobic interaction, and hydrophilic regulation to achieve multifunctional synergy. In contrast, Comparative Example 3 immobilized the antigen via covalent bonds, rather than encapsulating it in pores or adsorbing it through reversible forces, thus failing to reflect its true immune response effect. In Example 7, the matrix metalloproteinase-responsive peptide chain itself acts as a linker; its specific amino acid sequence and conformation may have additional, specific molecular recognition or interaction with the antigen molecule, thereby providing precise supramolecular chemical anchoring during encapsulation.

[0182] Test Example 3

[0183] Simulated release test

[0184] Sample preparation: Take 500 μg of the adjuvants obtained in Examples 1-7 and Comparative Examples 1-3 and put them into dialysis bags made of regenerated cellulose material with a retention capacity of 100 kDa.

[0185] Release media: Place the dialysis bag into a 50mL centrifuge tube and add 20mL of each of the two release media respectively.

[0186] Medium A: To simulate the physiological environment, 138 mmol / L sodium chloride and 2.7 mmol / L potassium chloride were used, and the pH was adjusted to 7.4 with phosphoric acid. The medium was then filtered through a 0.22 μm filter membrane for sterilization.

[0187] Medium B: to simulate an acidic enzyme response environment; 0.05 mol / L pH 5.0 acetate buffer solution containing 100 ng / mL matrix metalloproteinase 2 / 9, 5 mmol / L calcium chloride and 0.02 wt% sodium azide;

[0188] Matrix metalloproteinases 2 / 9: Activity ≥100 U / mg, R&D Systems;

[0189] Release process: Place the centrifuge tube in a ZYC-200 constant temperature shaker at 37±0.5℃ and shake slowly at 100rpm to avoid continuous friction between the dialysis bag and the tube wall;

[0190] Sampling and determination: At predetermined time points of 0.5, 8, 24 and 48 h, 1 mL of solution was taken from the release medium and 1 mL of fresh corresponding release medium preheated to 37±0.5℃ was added at the same time to maintain the system volume constant; the taken samples were filtered through a 0.22 μm filter membrane and then stored in a refrigerator at 4℃ for testing.

[0191] Preparation of standard curve: Using the BCA Protein Quantification Thermo Kit (catalog number 23225), antigen standard solutions with concentrations of 0-100 μg / mL were prepared, and the absorbance was measured at a wavelength of 562 nm. A standard curve was plotted with antigen concentration on the x-axis and absorbance on the y-axis.

[0192] After processing the test samples according to the BCA kit instructions, the antigen concentration in each sample taken out was measured at a wavelength of 562 nm. After the experiment, the residual sample in the dialysis bag was demulsified with PBS containing 0.5% Triton X-100, and the amount of residual antigen was measured. The release rate was then calculated. The results are shown in Table 3.

[0193] Calculation: Cumulative release rate = (total amount of antigen released at each time point / total antigen load) × 100%.

[0194] Table 3 Results of in vitro antigen release and intelligent responsiveness tests

[0195]

[0196] In an acidic enzyme environment, Example 1 exhibited accelerated release in response to environmental changes, while Comparative Example 1 showed no significant difference in release. This indicates that Example 1 achieved controlled release in response to environmental changes, while Comparative Example 1 only experienced passive and rapid leakage. Under physiological conditions, the metal-organic framework can firmly confine the antigen within its pores, allowing antigens in the adjacent surface areas to be released slowly through diffusion, effectively preventing antigen loss before reaching the target site. When in an acidic enzyme environment, 2-methylimidazole can bind to protons, leading to dissociation of the framework structure and thus achieving a burst release of the antigen. In contrast, the physical mixture of Comparative Example 1 lacked this structural protection and control; the antigen was adsorbed only by weak forces and easily dissociated rapidly under any conditions.

[0197] In Example 2, the polyethylene glycol layer forms a diffusion barrier, further delaying antigen release and helping to prolong the immune stimulation time. In Example 3, the hydrophobicity and degradability of the polylactic-co-glycolic acid copolymer (PLCA) effectively block water molecule penetration and antigen diffusion through its tightly packed hydrophobic segments. In Example 4, the release was slightly faster in an acidic environment, possibly due to the increased protonation of polyethyleneimine under acidic conditions, leading to increased electrostatic repulsion between chains and a looser structure, thus promoting antigen release. Example 5 shows that the introduction of galactose did not significantly alter the acid-responsive release mechanism based on the metal-organic framework and polyethyleneimine. The slow release behavior in Example 6 at physiological pH is a result of the combined effect of the hydrophobic barrier effect of the PLA and the steric hindrance effect of polyethylene glycol. Compared to Comparative Example 3, the antigen release rate was extremely low in any release medium because it was directly related to non-responsive mutant peptide chains that could not be recognized by matrix metalloproteinases, which conversely confirms that controlled release depends on the specific cleavage of enzyme-responsive peptide chains, rather than simple material degradation or physical diffusion. Furthermore, Example 7 introduces an enzyme-responsive peptide chain. Under physiological pH, this composite adjuvant can maintain the densest and most stable surface structure, effectively delaying antigen release. When in an acidic environment, the metal-organic framework will dissociate, and the matrix metalloproteinases present in the environment can specifically cleave the peptide chain, causing the outer structure to disintegrate, thereby achieving rapid and intelligent release of the antigen.

Claims

1. A method for preparing a modified alumina composite adjuvant, characterized in that, Includes the following steps: S1, Preprocessing Stage Take 10-30 mg of nano-alumina and disperse it in 10-15 mL of water using ultrasonication at 200-500 W to prepare a nano-alumina dispersion for later use; take 0.1-0.5 g of zinc nitrate hexahydrate and dissolve it in 10-15 mL of water, mix well to prepare a zinc salt aqueous solution for later use. S2, Reaction Stage Dissolve 0.1-0.8g of 2-methylimidazole in 20-30mL of water, mix to obtain an aqueous solution of 2-methylimidazole, add the prepared nano-alumina dispersion and zinc salt aqueous solution under stirring at 500-1000rpm, and stir for 30-60min. S3, Purification Stage Centrifuge at 10,000-15,000 rpm for 10-20 min at 2-6℃; After collecting the sediment, wash with water; S4, Modification Stage The washed precipitate was resuspended in 1-10 mL of water or buffer solution to obtain a resuspension; 1-10 mg of nano-drug carrier material was dispersed in 0.1-2 mL of acetonitrile, and then mixed with the resuspension under light-protected conditions, and stirred at 100-300 rpm for 1-5 h; then centrifuged at 12000 rpm for 15 min at 2-6 °C; the precipitate was collected and washed with water to obtain the modified alumina composite adjuvant; S5, Storage Stage Resuspend the resulting final product in 1-5 mL of water and store at 2-6 °C. The preparation method of the nanomedicine carrier material involved in step S4 is as follows: 10-50 mg of polyethylene glycol-succinimide ester is dissolved in 1-5 mL of buffer solution, 1-5 mg of matrix metalloproteinase 2 / 9 responsive peptide is added, and the mixture is stirred and reacted at room temperature of 10℃-35℃ for 1-4 h; the reaction solution is first dialyzed and freeze-dried to obtain grafted polypeptide-polyethylene glycol; then 20-100 mg of polyethyleneimine is dissolved in 5-10 mL of buffer solution, 20-100 mg of polylactic acid-glycolic acid copolymer-carboxyl group is dispersed in the above buffer solution, 10-50 mg of N-hydroxysuccinimide and 10-50 mg of 1-ethyl-3-(3-dimethylaminopropyl)carbodiimide are added, and the mixture is stirred and reacted at room temperature of 10℃-35℃ for 1-2 h; then the obtained grafted polypeptide-polyethylene glycol is added, and the reaction is continued for 8-12 h; the reaction solution is then dialyzed a second time and freeze-dried to obtain enzyme-responsive triblock copolymer.

2. The preparation method of the modified alumina composite adjuvant according to claim 1, characterized in that, The initial dialysis was performed by dialyzing the reaction solution with water at a molecular weight cutoff of 1000 Da for 24 hours and then freeze-drying it to obtain the grafted polypeptide-polyethylene glycol. The second dialysis was performed by transferring the reaction solution into a 3500 Da dialysis bag and dialyzing it with water for 12-24 hours, changing the water every 3-6 hours.

3. The preparation method of the modified alumina composite adjuvant according to claim 1, characterized in that, The buffer solution involved in step S4 is a phosphate buffer or a borate buffer with a pH range of 7.0-9.

0.

4. The preparation method of the modified alumina composite adjuvant according to claim 1, characterized in that, The freeze-drying process is as follows: the cold trap temperature is set to -50℃ to -60℃, the vacuum degree is 5-20Pa, and the drying time is 4-8h.

5. A modified alumina composite adjuvant, characterized in that, Prepared by the method described in any one of claims 1-4.

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