Dendritic polyamino acid, preparation method therefor, and nanoadjuvant and nanovaccine thereof

Abstract

Provided in the present invention is a dendritic polyamino acid. Compared with the prior art, with regard to the dendritic polyamino acid provided in the present invention, different particle sizes can be precisely controlled by means of adjusting the degree of polymerization of polymer units and polyamino acid units, and the structure is clear. The dendritic polyamino acid is monodisperse as a nanoadjuvant and also has abundant positive charges on the surface. Therefore, the dendritic polyamino acid can be efficiently loaded with various antigens by means of electrostatic adsorption so as to be used as a nanovaccine. The obtained nanovaccine has the function of promoting endocytosis and activating antigen-presenting cells; can stimulate DC cells to release Th1-type cytokines, such as IL-6, IL-12, IFN-γ and TNF-α; and promote antigen cross-presentation of the DC cells in vivo, thereby further triggering a strong antigen-specific immune response. In summary, the dendritic polyamino acid provided in the present invention, as a nanoadjuvant, has the advantages of monodispersity, precisely controllable structure and particle size, a stable effect and biodegradability, and has the possibility of clinical applications in the future.

Description

Dendritic polyamino acid and preparation method thereof, nanoadjuvant and nanovaccine

[0001] This application claims priority to the Chinese patent application filed with the China Patent Office on March 22, 2024, with application number 2024103338812 and invention name “A dendritic polyamino acid and its preparation method, nanoadjuvant and nanovaccine”, the entire contents of which are incorporated herein by reference. Technical Field

[0002] The present invention belongs to the field of biomedicine technology, and in particular relates to a dendritic polyamino acid and a preparation method thereof, a nano adjuvant and a nano vaccine. Background Art

[0003] In recent years, tumor vaccines have garnered widespread attention as a key branch of tumor immunotherapy. The primary goal of tumor vaccines is to activate / stimulate antigen-presenting cells (APCs), particularly dendritic cells (DCs). Following subcutaneous or intramuscular injection, these vaccines can generate a reservoir of antigens known as the depot effect, which recruits and activates APCs. These activated APCs then enter the lymph nodes. Furthermore, vaccines can directly reach the lymph nodes via the lymphatic circulation. Activated APCs in the lymph nodes then present antigens via major histocompatibility complex (MHC) I or MHC II molecules, stimulating CD8 T cells or CD4 T cells to effectively expand into cytotoxic T lymphocytes (CTLs) or helper T (Th) cells (including Th1 and Th2 cells), respectively. Th2 cells can promote B cell differentiation into plasma cells, which produce antibodies that specifically bind to tumor cells and eliminate them through associated cytotoxicity. Th2-mediated humoral immune responses play a positive role in suppressing tumorigenesis, thereby contributing to a preventive effect. On the other hand, Th1 cells can secrete cytokines such as interferon γ (IFNγ) to promote the generation of CTLs. The amplified CTLs then migrate and invade tumor tissues, identifying and dissolving tumor cells.

[0004] The principle of tumor vaccines is generally to express specific, immunogenic tumor antigens, and with the help of immune adjuvants, activate or enhance the body's own anti-tumor immune response, thereby achieving the goal of killing and eliminating tumor cells. Among them, immune adjuvants in tumor vaccines can prolong the antigen's retention time in the body by changing its physical shape. Immune adjuvants can also stimulate lymphocyte differentiation, thereby increasing and expanding the immune response. Therefore, the development of an effective immune adjuvant to stimulate the body to produce an enhanced immune response is crucial.

[0005] Adjuvants are vaccine components that enhance the strength, breadth, and durability of immune responses. Since aluminum adjuvants became the first human adjuvant approved by the US FDA in the 1920s, vaccine adjuvant research and development has been slow. In the nearly 100 years since aluminum adjuvants were first introduced, only six FDA-approved vaccine adjuvants have been marketed for human use: MF59, AS04, AS03, AS01, CpG1018, and Matrix-M, approved for emergency use against COVID-19. However, aluminum adjuvants remain a key adjuvant component in all vaccines, acting as an antigen reservoir, slowly releasing antigens and effectively inducing a Th2-type humoral immune response. However, aluminum adjuvants do not mediate a Th1-type cellular immune response, making them a poor choice for vaccines requiring a highly potent cellular immune response, such as cancer and HIV vaccines. Furthermore, after injection, aluminum adjuvants are captured by immune cells and transported to distant organs and the brain, leading to selective, long-term neurotoxicity at low doses. Therefore, developing effective adjuvants to stimulate an enhanced immune response is crucial.

[0006] Nanoadjuvants are immune adjuvants prepared using nanotechnology or in combination with nanomaterials. Nanoparticles have the characteristics of large specific surface area, multiple active centers, and multiple reaction types. Therefore, nanotechnology is applied to immune adjuvants. The nanoadjuvants developed have good biocompatibility and biotargeting, controllable release characteristics, safety and high efficiency, showing unique advantages that other types of adjuvants cannot match. Nanoadjuvants can be used as vaccine delivery systems, carrying tumor antigens, and enriching them in lymph nodes through passive targeting. They also act as immune adjuvants to enhance the endocytosis of antigen-presenting cells and cause the activation of antigen-presenting cells. Among them, existing liposome nanovaccines have been widely used using liposomes as adjuvants and carriers for protein and polypeptide antigens, but their composition is complex and their stability is poor, making it difficult to achieve stable and uniform batches.

[0007] Summary of the Invention

[0008] In view of this, the technical problem to be solved by the present invention is to provide a dendritic polyamino acid and a preparation method thereof, a nanoadjuvant and a nanovaccine, wherein the nanoadjuvant has monodispersity, controllable structure and particle size and stable effect.

[0009] The present invention provides a dendritic polyamino acid, comprising a polymer unit and a polyamino acid unit; the polymer unit is connected to the polyamino acid unit via an amide bond;

[0010] The polymer unit is formed by losing a hydrogen atom from the terminal amino group of the polymer; the polymer is selected from dendritic polymers and / or branched polymers;

[0011] The polyamino acid unit is shown in formula (I):

[0012] Wherein, n is an integer from 2 to 500;

[0013] R is selected from one of the structures shown in formula (1) to formula (3):

[0014] m1 to m3 are each independently selected from integers of 0-5.

[0015] Preferably, the number of generations of the dendritic polymer is 0 to 9;

[0016] The number average molecular weight of the branched polymer is 300 to 100,000;

[0017] The carbon atom connected to R in the amino acid unit is a chiral carbon atom;

[0018] The configuration of the chiral carbon atom is L-type or D-type, and preferably the configuration of the chiral carbon atom is D-type.

[0019] Preferably, the polymer is selected from one or more of polyamide-amine, polypropylene imine and branched polyethylene imine.

[0020] Preferably, m1 is 3, m2 is 2, and m3 is 1.

[0021] The present invention also provides a method for preparing a dendritic polyamino acid, comprising the following steps:

[0022] S1) performing a ring-opening polymerization reaction on a polymer having terminal amino groups and a carboxylic anhydride represented by formula (II) to obtain an intermediate; the polymer is a dendritic polymer and / or a branched polymer;

[0023] S2) deprotecting the intermediate to obtain a dendritic polyamino acid;

[0024] Wherein, R' is selected from one of the structures represented by formula (1) to formula (3) in which the amino group is protected:

[0025] m1 to m3 are each independently selected from integers of 0-5.

[0026] Preferably, the molar ratio of the polymer having terminal amino groups to the carboxylic anhydride represented by formula (II) is (0.00005-0.1):1;

[0027] The number average molecular weight of the branched polymer is 300 to 100,000;

[0028] The ring-opening polymerization reaction is carried out in a solvent; the solvent is selected from one or more of dichloromethane, chloroform and N, N-dimethylformamide;

[0029] The temperature of the ring-opening polymerization reaction is 10°C to 65°C;

[0030] The ring-opening polymerization reaction time is 1 to 72 hours.

[0031] The present invention also provides a nanoparticle, which is obtained by self-assembly of the above-mentioned dendritic polyamino acid in water or a water-containing solvent.

[0032] The present invention also provides a nano adjuvant comprising the above-mentioned dendritic polyamino acid or the above-mentioned nanoparticles.

[0033] The present invention also provides a nano vaccine, comprising the above-mentioned nano adjuvant, antigen and an adjuvant acceptable to the vaccine.

[0034] Preferably, the mass ratio of the antigen to the nanoadjuvant is 1:0.01-100.

[0035] The present invention provides a dendritic polyamino acid, comprising a polymer unit and a polyamino acid unit; the polymer unit is connected to the polyamino acid unit by an amide bond; the polymer unit is a dendritic polymer unit and / or a branched polymer unit; the polyamino acid unit is as shown in formula (I). Compared with the prior art, the dendritic polyamino acid provided by the present invention can accurately control different particle sizes by adjusting the degree of polymerization of the polymer unit and the polyamino acid unit and has a clear structure. It has monodispersity as a nanoadjuvant and has rich positive charges on the surface. It can be efficiently loaded with different antigens by electrostatic adsorption for use as a nanovaccine. The obtained nanovaccine has the function of promoting endocytosis and activation of antigen-presenting cells, can stimulate DC cells to release Th1 type cytokines, such as IL-6, IL-12, IFN-γ and TNF-α, and promote antigen cross-presentation of DC cells in vivo, further triggering a strong antigen-specific immune response. In summary, the dendritic polyamino acid provided by the present invention has the advantages of monodispersity, precise controllable structure and particle size, stable effect, and biodegradability as a nanoadjuvant, and has the possibility of clinical application in the future. BRIEF DESCRIPTION OF THE DRAWINGS

[0036] FIG1 is a schematic diagram of the structure of a dendritic polyamino acid provided by the present invention;

[0037] FIG2 is a hydrogen spectrum of the dendritic D-polylysine prepared in Examples 1 to 5 of the present invention;

[0038] FIG3 is a hydrogen spectrum of the dendritic D-polylysine prepared in Examples 6 to 9 of the present invention;

[0039] FIG4 is a hydrogen spectrum of the dendritic D-polylysine prepared in Examples 11 to 15 of the present invention;

[0040] FIG5 is a hydrogen spectrum of the dendritic D-polylysine prepared in Examples 16 to 20 of the present invention;

[0041] FIG6 is a hydrogen spectrum of the dendritic D-polylysine prepared in Examples 21 to 23 and Example 25 of the present invention;

[0042] FIG7 is a graph showing the gel permeation chromatography results of the dendritic D-polylysine prepared in Examples 1 to 25 of the present invention;

[0043] FIG8 is a graph showing the average particle size of the nanoparticles and nanovaccines prepared in Example 27 of the present invention;

[0044] FIG9 is a graph showing the average particle size of nanoparticles and nanovaccines prepared in Example 27 of the present invention;

[0045] FIG10 is a graph showing the average particle size of the nanoparticles and nanovaccines prepared in Example 27 of the present invention;

[0046] FIG11 is a graph showing the average particle size of the nanoparticles and nanovaccines prepared in Example 27 of the present invention;

[0047] FIG12 is a graph showing the results of a dendritic cell (BMDC) activation experiment in Example 28 of the present invention;

[0048] FIG13 is a diagram showing the results of an in vivo lymph node (LN) analysis test in Example 29 of the present invention;

[0049] FIG14 is a graph showing the results of a dendritic cell (BMDC) activation experiment in Example 30 of the present invention;

[0050] FIG15 is a graph showing the experimental results of in vivo tumor treatment in Example 31 of the present invention. DETAILED DESCRIPTION

[0051] The following will be combined with the embodiments of the present invention to clearly and completely describe the technical solutions in the embodiments of the present invention. Obviously, the embodiments described are only part of the embodiments of the present invention, not all of the embodiments. Based on the embodiments of the present invention, all other embodiments obtained by ordinary technicians in this field without making creative efforts are within the scope of protection of the present invention.

[0052] The present invention provides a dendritic polyamino acid, comprising a polymer unit and a polyamino acid unit; the polymer unit is connected to the polyamino acid unit via an amide bond; the polymer unit is formed by losing a hydrogen atom from the terminal amino group of the polymer; the polymer is selected from a dendritic polymer and / or a branched polymer.

[0053] The structure of the dendritic polyamino acid provided by the present invention is shown in FIG1 . In FIG1 , the spherical initiator represents a polymer unit, and the polyamino acid unit is only one of them. The number of polyamino acid units can be adjusted by controlling the feed ratio of amino acid carboxylic acid anhydride to the primary amino group contained in the polymer of the polymer unit.

[0054] In the present invention, the polymer unit is obtained by losing a hydrogen atom from the terminal amino group of the polymer; the polymer is a dendrimer and / or a branched polymer; more specifically, the polymer is preferably one or more of polyamide-amine, polypropyleneimine and branched polyethyleneimine; the number of generations of the dendrimer is preferably 0 to 9 generations, more preferably 0 to 6 generations, more preferably 0 to 4 generations, and most preferably 0 to 3 generations; in some embodiments provided by the present invention, the number of generations of the dendrimer can specifically be 0 generation (PAMAM G0), 1 generation (PAMAM G1), 2 generation (PAMAM G2), 3 generation or 4 generation; the number average molecular weight of the branched polymer is preferably 300 to 500,000, more preferably 300 to 200,000, and more preferably 300 to 100,000.

[0055] The polyamino acid unit is shown in formula (I):

[0056] Wherein, n is an integer of 2 to 500, preferably an integer of 5 to 400, more preferably an integer of 5 to 300, further preferably an integer of 5 to 200, and most preferably an integer of 5 to 100; in some embodiments provided by the present invention, n is specifically 10, 25, 50, 100 or 200.

[0057] R is one of the structures shown in formula (1) to formula (3):

[0058] m1 to m3 are each independently an integer of 0 to 5; more specifically, m1 is preferably an integer of 1 to 5, more preferably an integer of 2 to 4, and more preferably 3; m2 is preferably an integer of 0 to 4, more preferably an integer of 1 to 3, and more preferably 2; m3 is preferably an integer of 0 to 4, more preferably an integer of 0 to 3, and more preferably 1 or 3.

[0059] In the present invention, most preferably, R is one of the structures shown in formula (4) to formula (6):

[0060] In the present invention, the carbon atom connected to R in the amino acid unit is a chiral carbon atom; the configuration of the chiral carbon atom can be L-type or D-type. Preferably, the configuration of the chiral carbon atom is D-type, which has a stronger immunostimulatory effect.

[0061] The polymer unit is connected to the polyamino acid unit via an amide bond, that is, the amino group of the polymer initiates the ring-opening polymerization of the amino acid carboxylic anhydride.

[0062] According to the present invention, the number average molecular weight of the dendritic polyamino acid is preferably 1,000 to 10,000,000, more preferably 1,000 to 5,000,000, further preferably 1,000 to 1,000,000, further preferably 2,000 to 800,000, and most preferably 3,500 to 600,000.

[0063] The present invention also provides a method for preparing the above-mentioned dendritic polyamino acid, comprising the following steps: S1) subjecting a polymer having terminal amino groups to a ring-opening polymerization reaction with a carboxylic anhydride represented by formula (II) to obtain an intermediate; the polymer is a dendritic polymer and / or a branched polymer; S2) deprotecting the amino groups of the intermediate to obtain a dendritic polyamino acid;

[0064] Wherein, R′ is selected from one of the structures represented by formula (1) to formula (3) in which the amino group is protected; in the present invention, the amino-protecting group is a group well known to those skilled in the art and is not particularly limited, and specifically can be benzyloxycarbonyl or trityl.

[0065] m1 to m3 are each independently selected from integers of 0 to 5; more specifically, m1 is preferably an integer of 1 to 5, more preferably an integer of 2 to 4, and more preferably 3; m2 is preferably an integer of 0 to 4, more preferably an integer of 1 to 3, and more preferably 2; m3 is preferably an integer of 0 to 4, more preferably an integer of 0 to 3, and more preferably 1 or 3.

[0066] According to the present invention, the polymer having terminal amino groups is preferably a dendritic polymer and / or a branched polymer, more preferably one or more of polyamide-amine, polypropyleneimine and branched polyethyleneimine; the number of generations of the dendritic polymer is preferably 0 to 9 generations, more preferably 0 to 6 generations, more preferably 0 to 4 generations, and most preferably 0 to 3 generations; in some embodiments provided by the present invention, the number of generations of the dendritic polymer unit can specifically be 0 generations, 1 generations, 2 generations, 3 generations or 4 generations; the number average molecular weight of the branched polymer is preferably 300 to 500,000, more preferably 300 to 200,000, and more preferably 300 to 100,000.

[0067] According to the present invention, the carboxylic anhydride is specifically one or more of formula (II-1) to formula (II-3):

[0068] In the present invention, the carboxylic anhydride represented by formula (II) can be prepared according to methods well known to those skilled in the art without any special limitation.

[0069] In a specific embodiment provided by the present invention, the carboxylic acid anhydride represented by formula (II-1) is prepared according to a method well known to those skilled in the art, specifically, by reacting N-benzyloxycarbonyl-lysine and triphosgene in tetrahydrofuran solvent to obtain the carboxylic acid anhydride represented by formula (II-1). The temperature for the reaction of N-benzyloxycarbonyl-lysine and triphosgene is preferably 40°C to 70°C, more preferably 50°C to 65°C; the reaction time is preferably 0.5 to 6 hours, more preferably 1 to 5 hours, and even more preferably 3 to 5 hours. In some embodiments provided by the present invention, N-benzyloxycarbonyl-lysine and triphosgene can be reacted under nitrogen protection. The mass ratio of N-benzyloxycarbonyl-lysine to triphosgene is 1:0.5 to 2, more preferably 1:0.5 to 1. After the reaction is complete, the product is preferably precipitated with icy petroleum ether and filtered to obtain a crude product. The crude product is further dissolved in cold ethyl acetate and transferred to a separatory funnel. Insoluble impurities and by-products are then washed with a cold saturated sodium chloride solution to purify the carboxylic anhydride solution represented by formula (II-1). Anhydrous magnesium sulfate is then added to dry the compound solution for 6 to 24 hours. Finally, the anhydrous magnesium sulfate is removed by filtration, and the solution is dried to obtain the pure carboxylic anhydride represented by formula (II-1).

[0070] In a specific embodiment provided by the present invention, the carboxylic acid anhydride represented by formula (II-2) is prepared according to a method well known to those skilled in the art, specifically: in a protective atmosphere, tri-N-benzyloxycarbonyl-D-arginine is mixed with dichloromethane, and then α,α'-dichloromethyl ether is added and heated for reaction. After the reaction, volatiles are removed in vacuo, and the residue is crystallized from a mixture of tetrahydrofuran and n-hexane (1:3) in a drying oven to obtain the carboxylic acid anhydride represented by formula (II-2); the temperature of the heating reaction is preferably 40°C to 70°C, more preferably 45°C to 60°C, and more preferably 48°C to 50°C; the heating reaction time is preferably 10 to 50 hours, more preferably 20 to 45 hours, more preferably 30 to 40 hours, and most preferably 36 hours; in some embodiments provided by the present invention, the steps are specifically followed: tri-N-benzyloxycarbonyl-D-arginine (2.0 g, 3.5 mmol) that has been dried in vacuum for 1 hour is placed in a 250 ml dry flask with a magnet. Under nitrogen, dry dichloromethane (100 mL) was added to the flask via cannula. After the amino acid was dissolved, α,α'-dichloromethyl ether (0.5 mL 5.5 mmol) was added via cannula under nitrogen. The flask was then equipped with a reflux condenser under nitrogen and heated at 48°C for 36 hours. The volatiles were then removed under vacuum and the reaction flask was transferred to a glove box under nitrogen. The residue was crystallized five times in a drying oven from a mixture of tetrahydrofuran and n-hexane (1:3) to obtain the carboxylic anhydride represented by formula (II-2) (0.85 g, 73%) as a white solid.

[0071] In a specific embodiment provided by the present invention, the carboxylic acid anhydride represented by formula (II-3) is prepared according to a method well known to those skilled in the art. Specifically, 30 mL of anhydrous tetrahydrofuran is quickly added to a single-necked flask containing 6 g of dry Boc-His(trt)-OH powder while stirring in an ice bath. 1.5 mL of thionyl chloride diluted with 10 mL of anhydrous tetrahydrofuran is then slowly added dropwise to the above solution. The reaction is carried out in an ice bath for 3 hours, followed by precipitation with 300 mL of anhydrous ether and filtration. The crude product obtained by filtration is dissolved in cold ethyl acetate and transferred to a separatory funnel. Insoluble impurities and by-products are then washed with a cold saturated sodium chloride solution to purify the carboxylic acid anhydride solution represented by formula (II-3). Anhydrous magnesium sulfate is then added to dry the above compound solution for 6 to 24 hours. Finally, the anhydrous magnesium sulfate is removed by filtration, and the solution is dried to obtain the pure carboxylic acid anhydride represented by formula (II-3).

[0072] A polymer having a terminal amino group is subjected to a ring-opening polymerization reaction with a carboxylic anhydride represented by formula (II); the molar ratio of the polymer having a terminal amino group to the carboxylic anhydride represented by formula (II) is preferably (0.00005-0.1):1, more preferably (0.00005-0.05):1, and even more preferably (0.00005-0.01:1, which can be specifically selected according to the number of terminal amino groups in the polymer; in some embodiments provided by the present invention, the molar ratio of the polymer having a terminal amino group to the carboxylic anhydride represented by formula (II) is preferably 0.025:1, 0.01:1, 0.005:1, 0.0025:1, 0.00125:1, 0.0125:1, 0.000625:1, 0.00625:1, 0.000313:1, 0.00313:1, 0.0013:1, 0.000156:1, 0.00 1562:1 or 0.000078:1; the ring-opening polymerization reaction is preferably carried out in a solvent; the solvent is preferably one or more of dichloromethane, chloroform and N,N-dimethylformamide; in the present invention, it is preferred to first mix and dissolve the carboxylic anhydride represented by formula (II) with the solvent and then add the polymer having terminal amino groups to carry out the ring-opening polymerization reaction; the concentration of the carboxylic anhydride represented by formula (II) in the system after mixed dissolution is preferably 0.01 to 0.1 mol / L, more preferably 0.05 mol / L; the temperature of the ring-opening polymerization reaction is preferably 10°C to 65°C, more preferably 15°C to 50°C, more preferably 20°C to 40°C, and most preferably 25°C to 35°C; the time of the ring-opening polymerization reaction is preferably 1 to 72 h, more preferably 1 to 50 h, more preferably 1 to 40 h, more preferably 1 to 24 h, more preferably 1 to 12 h, and most preferably 2 to 6 h.

[0073] After the ring-opening polymerization reaction is completed, the intermediate is obtained by precipitation with ice anhydrous ether and centrifugal drying.

[0074] The intermediate is subjected to amino deprotection; the amino deprotection method can be selected according to the type of amino protecting group and can be performed according to methods well known to those skilled in the art without any special limitation. In a specific embodiment provided by the present invention, the amino-amino protecting group is a benzyloxycarbonyl group, and trimethylsilyl iodide is used for amino deprotection, specifically: the intermediate is mixed with trimethylsilyl iodide in an organic solvent for amino deprotection; the organic solvent is an organic solvent well known to those skilled in the art and is not particularly limited. In the present invention, it is preferably one or more of dichloromethane, chloroform and N,N-dimethylformamide; the temperature for amino deprotection is preferably 10°C to 40°C, more preferably 20°C to 30°C, and more preferably 25°C; the time for amino deprotection is preferably 5 to 40 hours, more preferably 10 to 30 hours, more preferably 20 to 30 hours, and most preferably 24 hours; after amino deprotection, the solvent is preferably removed by vacuum, saturated sodium bicarbonate solution and deionized water are added to dissolve the resulting residue, and then NaS2O3 is added to form a colorless solution, and anhydrous ether is added to wash the aqueous phase to remove iodobenzyl to obtain an aqueous solution containing a dendritic polyamino acid.

[0075] The aqueous solution containing the dendritic polyamino acid is dialyzed and dried to obtain the dendritic polyamino acid; the specifications of the dialysis bag used for the dialysis are preferably 3500-10000 MWCO; the dialysis is preferably performed in deionized water; the dialysis time is preferably 20-100 h, more preferably 40-90 h, and even more preferably 60-72 h; the drying is preferably freeze-drying.

[0076] The present invention uses different dendritic polymers and / or branched polymers as initiators to initiate the polymerization of D-amino acid-N-carboxylic anhydride (NCA) units and controls different polymerization degrees, thereby precisely controlling the molecular weight of the obtained dendritic polyamino acid, and then obtaining nanoparticles of different particle sizes through self-assembly.

[0077] The present invention also provides a nanoparticle, which is obtained by self-assembly of the above-mentioned dendritic polyamino acid in water or a water-containing solvent.

[0078] The aqueous solvent is any aqueous solvent well known to those skilled in the art without any particular limitation. In the present invention, PBS buffer or PB buffer is preferred.

[0079] In a specific embodiment provided by the present invention, the dendritic polyamino acid is obtained by ultrasonic self-assembly in water or an aqueous solvent. The mass concentration of the dendritic polyamino acid in the solution formed in water or an aqueous solvent is preferably 0.1 to 10 mg / mL, more preferably 0.1 to 6 mg / mL, more preferably 0.1 to 4 mg / mL, and even more preferably 0.1 to 2 mg / mL; the ultrasonic temperature is preferably 5°C to 30°C, more preferably 5°C to 25°C; the ultrasonic time is preferably 5 to 60 minutes; the ultrasonic power is preferably 0.1 to 1 kW, more preferably 0.3 to 0.6 kW, even more preferably 0.4 to 0.5 kW, and most preferably 0.45 kW.

[0080] According to the present invention, the particle size of the nanoparticles is preferably 4 to 100 nm, more preferably 4 to 80 nm, and even more preferably 4 to 60 nm.

[0081] The present invention also provides a nano adjuvant comprising the above-mentioned dendritic polyamino acid or the above-mentioned nanoparticles.

[0082] The present invention also provides a use of the nanoparticles as a nanoadjuvant.

[0083] The nanoadjuvant provided by the present invention has the ability to significantly stimulate dendritic cell activation, improve antigen cross-presentation, and enhance adaptive immune response.

[0084] The present invention also provides a nano vaccine, comprising the above-mentioned nano adjuvant, antigen and an adjuvant acceptable to the vaccine.

[0085] In a specific embodiment provided by the present invention, the antigen is preferably a tumor antigen and / or a viral antigen; in some examples provided by the present invention, the antigen is specifically OVA.

[0086] In a specific embodiment provided by the present invention, the mass ratio of the antigen to the nanoadjuvant is preferably 1:0.001-100, more preferably 1:0.001-50, further preferably 1:0.005-30, further preferably 1:0.01-20, further preferably 1:0.1-10, further preferably 1:1-10, and most preferably 1:1-8.

[0087] The dendritic polyamino acids provided by the present invention are monodisperse and can be used as nanoadjuvants to efficiently load antigens and prepare nanovaccines. Depending on the added antigen, the nanovaccines can be used in different disease models for the prevention and treatment of different diseases, including tumor models, bacterial and viral infection models. The resulting nanovaccines all have the function of promoting the endocytosis and activation of antigen-presenting cells, stimulating DCs to release Th1 cytokines such as IL-6, IL-12, IFN-γ, and TNF-α. They also promote antigen cross-presentation by DCs in vivo, further triggering a strong antigen-specific immune response.

[0088] To further illustrate the present invention, a dendritic polyamino acid, a preparation method thereof, a nanoadjuvant and a nanovaccine provided by the present invention are described in detail below with reference to the examples.

[0089] All reagents used in the following examples were commercially available; yield of each product = actual mass of product obtained / theoretical mass of product obtained × 100%; the dendrimers used in the examples were purchased from Sigma-Aldrich, wherein PAMAM G0 had a molecular weight of 517, PAMAM G1 had a molecular weight of 1430, PAMAM G2 had a molecular weight of 3256, PAMAM G3 had a molecular weight of 6909, and PAMAM G4 had a molecular weight of 14214; the OVA protein used in the examples was commercially available chicken ovalbumin, purchased from Sigma-Aldrich, catalog number A2512; all Elisa kits in the examples were purchased from Thermo Fisher Scientific (Waltham, MA, USA); and all flow cytometry antibodies in the examples were purchased from BioLegend.

[0090] Example 1: Dendritic D-polylysine PAMAM-GO-Dlys 10 Preparation

[0091] Weigh N-benzyloxycarbonyl-D-lysine-N-carboxylic anhydride (0.306 g, 1.0 mmol) into an oven-dried round-bottom flask and add 20 mL of anhydrous dichloromethane for stirring and dissolution. PAMAM-G0 has four primary amine groups. PAMAM G0 initiator (12.9 mg 0.025 mmol) was added at the required monomer / initiator ratio and the reaction was carried out at 25 ° C for 2 h. After the reaction was completed, the product was precipitated with ice-cold anhydrous ether and centrifuged to obtain undeprotected PAMAM-G0-Dlys(Z) 10 (0.262 g, 95.3%).

[0092] Weigh the above undeprotected dendritic D-polylysine PAMAM-GO-Dlys(Z) 10(50.0 mg) was added to a clean flask, 4 mL of dichloromethane was added and stirred to dissolve. After complete dissolution, trimethylsilyl iodide (163 μL, 1.14 mmol, 6 equivalents per benzyl group) was added to form a slightly yellow solution. The reaction was carried out at 25°C for 24 hours. After the reaction was completed, the solvent was removed in vacuo, saturated NaHCO3 solution (4 mL) and deionized water (4 mL) were added to dissolve the resulting residue, and a small amount of NaS2O3 was added to form a colorless solution. After the residue was completely dissolved, the aqueous phase was washed with 3×10 mL of anhydrous ether to remove iodobenzyl. The resulting colorless aqueous solution was transferred to a dialysis bag (3500 MWCO) and dialyzed against deionized water for 72 hours. The deprotected dendritic D-polylysine was freeze-dried to a white powder, PAMAM-G0-Dlys 10 (21.6 mg, 88.6%). The NMR results are shown in Figure 2.

[0093] Example 2: Dendritic D-polylysine PAMAM-GO-Dlys 25 Preparation

[0094] Weigh N-benzyloxycarbonyl-D-lysine-N-carboxylic anhydride (0.306 g, 1.0 mmol) into an oven-dried round-bottom flask and add 20 mL of anhydrous dichloromethane for stirring and dissolution. PAMAM-G0 has four primary amine groups. PAMAM G0 initiator (5.2 mg 0.010 mmol) was added at the required monomer / initiator ratio and the reaction was carried out at 25°C for 2 h. After the reaction was completed, the product was precipitated with ice-cold anhydrous ether and centrifuged to obtain undeprotected PAMAM-G0-Dlys(Z). 25 (0.254 g, 95.1%).

[0095] Weigh the above undeprotected dendritic D-polylysine PAMAM-GO-Dlys(Z) 25 (50.0 mg) was added to a clean flask, 4 mL of dichloromethane was added and stirred to dissolve. After complete dissolution, trimethylsilyl iodide (163 μL, 1.14 mmol, 6 equivalents per benzyl group) was added to form a slightly yellow solution. The reaction was carried out at 25°C for 24 hours. After the reaction was completed, the solvent was removed in vacuo, and saturated NaHCO3 solution (4 mL) and deionized water (4 mL) were added to dissolve the resulting residue. A small amount of NaS2O3 was added to form a colorless solution. After the residue was completely dissolved, the aqueous phase was washed with 3×10 mL of anhydrous ether to remove the iodobenzyl. The resulting colorless aqueous solution was transferred to a dialysis bag (3500 MWCO) and dialyzed against deionized water for 72 hours. The deprotected dendritic D-polylysine was freeze-dried to a white powder, PAMAM-G0-Dlys 25 (21.3 mg, 85.5%). The NMR results are shown in Figure 2.

[0096] Example 3: Dendritic D-polylysine PAMAM-GO-Dlys 50 Preparation

[0097] Weigh N-benzyloxycarbonyl-D-lysine-N-carboxylic anhydride (0.306 g, 1.0 mmol) into an oven-dried round-bottom flask and add 20 mL of anhydrous dichloromethane for stirring and dissolution. PAMAM-G0 has four primary amine groups. PAMAM G0 initiator (2.58 mg 0.005 mmol) was added at the required monomer / initiator ratio and the reaction was carried out at 25 ° C for 2 h. After the reaction was completed, the product was precipitated with ice-cold anhydrous ether and centrifuged to obtain undeprotected PAMAM-G0-Dlys(Z) 50 (0.252 g, 95.1%).

[0098] Weigh the above undeprotected dendritic D-polylysine PAMAM-GO-Dlys(Z) 50 (50.0 mg) was added to a clean flask, 4 mL of dichloromethane was added and stirred to dissolve. After complete dissolution, trimethylsilyl iodide (163 μL, 1.14 mmol, 6 equivalents per benzyl group) was added to form a slightly yellow solution. The reaction was carried out at 25°C for 24 hours. After the reaction was completed, the solvent was removed in vacuo, and saturated NaHCO3 solution (4 mL) and deionized water (4 mL) were added to dissolve the resulting residue. A small amount of NaS2O3 was added to form a colorless solution. After the residue was completely dissolved, the aqueous phase was washed with 3×10 mL of anhydrous ether to remove the iodobenzyl. The resulting colorless aqueous solution was transferred to a dialysis bag (10000 MWCO) and dialyzed against deionized water for 72 hours. The deprotected dendritic D-polylysine was freeze-dried to a white powder, PAMAM-G0-Dlys 50 (21.4 mg, 86.6%). The NMR results are shown in Figure 2.

[0099] Example 4: Dendritic D-polylysine PAMAM-GO-Dlys 100 Preparation

[0100] N-Benzyloxycarbonyl-D-lysine-N-carboxylic anhydride (0.306 g, 1.0 mmol) was weighed into an oven-dried round-bottom flask and 20 mL of anhydrous dichloromethane was added for stirring and dissolution. PAMAM-G0 has four primary amine groups. PAMAM G0 initiator (1.29 mg 2.5 μmol) was added at the required monomer / initiator ratio and the reaction was carried out at 25 ° C for 2 h. After the reaction was completed, the product was precipitated with ice-cold anhydrous ether and centrifuged to obtain undeprotected PAMAM-G0-Dlys(Z) 100 (0.246 g, 93.3%).

[0101] Weigh the above undeprotected dendritic D-polylysine PAMAM-GO-Dlys(Z) 100 (50.0 mg) was added to a clean flask, 4 mL of dichloromethane was added and stirred to dissolve. After complete dissolution, trimethylsilyl iodide (163 μL, 1.14 mmol, 6 equivalents per benzyl group) was added to form a slightly yellow solution. The reaction was carried out at 25°C for 24 hours. After the reaction was completed, the solvent was removed in vacuo, and saturated NaHCO3 solution (4 mL) and deionized water (4 mL) were added to dissolve the resulting residue. A small amount of NaS2O3 was added to form a colorless solution. After the residue was completely dissolved, the aqueous phase was washed with 3×10 mL of anhydrous ether to remove the iodobenzyl. The resulting colorless aqueous solution was transferred to a dialysis bag (10000 MWCO) and dialyzed against deionized water for 72 hours. The deprotected dendritic D-polylysine was freeze-dried to a white powder, PAMAM-G0-Dlys 100 (20.3 mg, 82.5%). The NMR results are shown in Figure 2.

[0102] Example 5: Dendritic D-polylysine PAMAM-GO-Dlys 200 Preparation

[0103] N-Benzyloxycarbonyl-D-lysine-N-carboxylic anhydride (0.306 g, 1.0 mmol) was weighed into an oven-dried round-bottom flask and 20 mL of anhydrous dichloromethane was added for stirring and dissolution. PAMAM-G0 has four primary amine groups. PAMAM G0 initiator (0.64 mg 1.25 μmol) was added at the required monomer / initiator ratio and the reaction was carried out at 25 ° C for 2 h. After the reaction was completed, the product was precipitated with ice-cold anhydrous ether and centrifuged to obtain undeprotected PAMAM-G0-Dlys(Z) 200 (0.245 g, 93.2%).

[0104] Weigh the above undeprotected dendritic D-polylysine PAMAM-GO-Dlys(Z) 200 (50.0 mg) was added to a clean flask, 4 mL of dichloromethane was added and stirred to dissolve. After complete dissolution, trimethylsilyl iodide (163 μL, 1.14 mmol, 6 equivalents per benzyl group) was added to form a slightly yellow solution. The reaction was carried out at 25°C for 24 hours. After the reaction was completed, the solvent was removed in vacuo, and saturated NaHCO3 solution (4 mL) and deionized water (4 mL) were added to dissolve the resulting residue. A small amount of NaS2O3 was added to form a colorless solution. After the residue was completely dissolved, the aqueous phase was washed with 3×10 mL of anhydrous ether to remove the iodobenzyl. The resulting colorless aqueous solution was transferred to a dialysis bag (10000 MWCO) and dialyzed against deionized water for 72 hours. The deprotected dendritic D-polylysine was freeze-dried to a white powder, PAMAM-G0-Dlys 200(20.8 mg, 84.9%). The NMR results are shown in Figure 2.

[0105] Example 6: Dendritic D-polylysine PAMAM-G1-Dlys 10 Preparation

[0106] Weigh N-benzyloxycarbonyl-D-lysine-N-carboxylic anhydride (0.306 g, 1.0 mmol) into an oven-dried round-bottom flask and add 20 mL of anhydrous dichloromethane for stirring and dissolution. PAMAM-G1 has 8 primary amine groups. PAMAM G1 initiator (17.875 mg 0.0125 mmol) was added at the required monomer / initiator ratio and the reaction was carried out at 25 ° C for 2 h. After the reaction was completed, the mixture was precipitated with ice-cold anhydrous ether and centrifuged to dry to obtain undeprotected PAMAM-G1-Dlys(Z) 10 (0.265 g, 94.6%).

[0107] Weigh the undeprotected dendritic D-polylysine PAMAM-G1-Dlys(Z) 10 (50.0 mg) was added to a clean flask, 4 mL of dichloromethane was added and stirred to dissolve. After complete dissolution, trimethylsilyl iodide (163 μL, 1.14 mmol, 6 equivalents per benzyl group) was added to form a slightly yellow solution. The reaction was carried out at 25°C for 24 hours. After the reaction was completed, the solvent was removed in vacuo, and saturated NaHCO3 solution (4 mL) and deionized water (4 mL) were added to dissolve the resulting residue. A small amount of NaS2O3 was added to form a colorless solution. After the residue was completely dissolved, the aqueous phase was washed with 3×10 mL of anhydrous ether to remove the iodobenzyl. The resulting colorless aqueous solution was transferred to a dialysis bag (3500 MWCO) and dialyzed against deionized water for 72 hours. The deprotected dendritic D-polylysine was freeze-dried to a white powder, PAMAM-G1-Dlys 10 (23.3 mg, 89.3%). The NMR results are shown in Figure 3.

[0108] Example 7: Dendritic D-polylysine PAMAM-G1-Dlys 25 Preparation

[0109] N-benzyloxycarbonyl-D-lysine-N-carboxylic anhydride (0.306 g, 1.0 mmol) was weighed into an oven-dried round-bottom flask and 20 mL of anhydrous dichloromethane was added for stirring and dissolution. PAMAM-G1 has 8 primary amine groups. PAMAM G1 initiator (7.15 mg 0.005 mmol) was added at the required monomer / initiator ratio and the reaction was carried out at 25 ° C for 2 h. After the reaction was completed, the mixture was precipitated with ice-cold anhydrous ether and centrifuged to obtain undeprotected PAMAM-G1-Dlys(Z) 25(0.245 g, 90.9%).

[0110] Weigh the undeprotected dendritic D-polylysine PAMAM-G1-Dlys(Z) 25 (50.0 mg) was added to a clean flask, 4 mL of dichloromethane was added and stirred to dissolve. After complete dissolution, trimethylsilyl iodide (163 μL, 1.14 mmol, 6 equivalents per benzyl group) was added to form a slightly yellow solution. The reaction was carried out at 25°C for 24 hours. After the reaction was completed, the solvent was removed in vacuo, and saturated NaHCO3 solution (4 mL) and deionized water (4 mL) were added to dissolve the resulting residue. A small amount of NaS2O3 was added to form a colorless solution. After the residue was completely dissolved, the aqueous phase was washed with 3×10 mL of anhydrous ether to remove the iodobenzyl. The resulting colorless aqueous solution was transferred to a dialysis bag (3500 MWCO) and dialyzed against deionized water for 72 hours. The deprotected dendritic D-polylysine was freeze-dried to a white powder, PAMAM-G1-Dlys 25 (21.8 mg, 86.9%). The NMR results are shown in Figure 3.

[0111] Example 8: Dendritic D-polylysine PAMAM-G1-Dlys 50 Preparation

[0112] N-Benzyloxycarbonyl-D-lysine-N-carboxylic anhydride (0.306 g, 1.0 mmol) was weighed into an oven-dried round-bottom flask and 20 mL of anhydrous dichloromethane was added for stirring and dissolution. PAMAM-G1 has 8 primary amine groups. PAMAM G1 initiator (3.58 mg 2.5 μmol) was added at the required monomer / initiator ratio and the reaction was carried out at 25°C for 2 h. After the reaction was completed, the product was precipitated with ice-cold anhydrous ether and centrifuged to dryness to obtain undeprotected PAMAM-G1-Dlys(Z) 50 (0.255 g, 95.9%).

[0113] Weigh the undeprotected dendritic D-polylysine PAMAM-G1-Dlys(Z) 50(50.0 mg) was added to a clean flask, 4 mL of dichloromethane was added and stirred to dissolve. After complete dissolution, trimethylsilyl iodide (163 μL, 1.14 mmol, 6 equivalents per benzyl group) was added to form a slightly yellow solution. The reaction was carried out at 25°C for 24 hours. After the reaction was completed, the solvent was removed in vacuo, and saturated NaHCO3 solution (4 mL) and deionized water (4 mL) were added to dissolve the resulting residue. A small amount of NaS2O3 was added to form a colorless solution. After the residue was completely dissolved, the aqueous phase was washed with 3×10 mL of anhydrous ether to remove the iodobenzyl. The resulting colorless aqueous solution was transferred to a dialysis bag (10000 MWCO) and dialyzed against deionized water for 72 hours. The deprotected dendritic D-polylysine was freeze-dried to a white powder, PAMAM-G1-Dlys 50 (21.6 mg, 87.2%). The NMR results are shown in Figure 3.

[0114] Example 9: Dendritic D-polylysine PAMAM-G1-Dlys 100 Preparation

[0115] N-Benzyloxycarbonyl-D-lysine-N-carboxylic anhydride (0.306 g, 1.0 mmol) was weighed into an oven-dried round-bottom flask and 20 mL of anhydrous dichloromethane was added for stirring and dissolution. PAMAM-G1 has 8 primary amine groups. PAMAM G1 initiator (1.79 mg 1.25 μmol) was added at the required monomer / initiator ratio and the reaction was carried out at 25 ° C for 2 h. After the reaction was completed, the product was precipitated with ice-cold anhydrous ether and centrifuged to dry to obtain undeprotected PAMAM-G1-Dlys(Z) 100 (0.241 g, 91.3%).

[0116] Weigh the undeprotected dendritic D-polylysine PAMAM-G1-Dlys(Z) 100 (50.0 mg) was added to a clean flask, 4 mL of dichloromethane was added and stirred to dissolve. After complete dissolution, trimethylsilyl iodide (163 μL, 1.14 mmol, 6 equivalents per benzyl group) was added to form a slightly yellow solution. The reaction was carried out at 25°C for 24 hours. After the reaction was completed, the solvent was removed in vacuo, and saturated NaHCO3 solution (4 mL) and deionized water (4 mL) were added to dissolve the resulting residue. A small amount of NaS2O3 was added to form a colorless solution. After the residue was completely dissolved, the aqueous phase was washed with 3×10 mL of anhydrous ether to remove the iodobenzyl. The resulting colorless aqueous solution was transferred to a dialysis bag (10000 MWCO) and dialyzed against deionized water for 72 hours. The deprotected dendritic D-polylysine was freeze-dried to a white powder, PAMAM-G1-Dlys 100 (21.3 mg, 86.6%). The NMR results are shown in Figure 3.

[0117] Example 10: Dendritic D-polylysine PAMAM-G1-Dlys 200 Preparation

[0118] Weigh N-benzyloxycarbonyl-D-lysine-N-carboxylic anhydride (0.306 g, 1.0 mmol) into an oven-dried round-bottom flask and add 20 mL of anhydrous dichloromethane for stirring and dissolution. PAMAM-G1 has 8 primary amine groups. PAMAM G1 initiator (0.89 mg 0.625 μmol) is added at the required monomer / initiator ratio and the reaction is carried out at 25 ° C for 2 h. After the reaction is completed, the product is precipitated with ice-cold anhydrous ether and centrifuged to obtain undeprotected PAMAM-G1-Dlys(Z). 200 (0.245 g, 93.1%).

[0119] Weigh the undeprotected dendritic D-polylysine PAMAM-G1-Dlys(Z) 200 (50.0 mg) was added to a clean flask, 4 mL of dichloromethane was added and stirred to dissolve. After complete dissolution, trimethylsilyl iodide (163 μL, 1.14 mmol, 6 equivalents per benzyl group) was added to form a slightly yellow solution. The reaction was carried out at 25°C for 24 hours. After the reaction was completed, the solvent was removed in vacuo, and saturated NaHCO3 solution (4 mL) and deionized water (4 mL) were added to dissolve the resulting residue. A small amount of NaS2O3 was added to form a colorless solution. After the residue was completely dissolved, the aqueous phase was washed with 3×10 mL of anhydrous ether to remove the iodobenzyl. The resulting colorless aqueous solution was transferred to a dialysis bag (10000 MWCO) and dialyzed against deionized water for 72 hours. The deprotected dendritic D-polylysine was freeze-dried to a white powder, PAMAM-G1-Dlys 200 (21.2 mg, 86.5%).

[0120] Example 11: Dendritic D-polylysine PAMAM-G2-Dlys 10 Preparation

[0121] N-benzyloxycarbonyl-D-lysine-N-carboxylic anhydride (0.306 g, 1.0 mmol) was weighed into an oven-dried round-bottom flask and 20 mL of anhydrous dichloromethane was added for stirring and dissolution. PAMAM-G2 has 16 primary amine groups. PAMAM G2 initiator (20.35 mg 0.00625 mmol) was added at the required monomer / initiator ratio and the reaction was carried out at 25 ° C for 2 h. After the reaction was completed, the mixture was precipitated with ice-cold anhydrous ether and centrifuged to obtain undeprotected PAMAM-G2-Dlys(Z) 10 (0.269 g, 95.3%).

[0122] Weigh the undeprotected dendritic D-polylysine PAMAM-G2-Dlys(Z) 10 (50.0 mg) was added to a clean flask, 4 mL of dichloromethane was added and stirred to dissolve. After complete dissolution, trimethylsilyl iodide (163 μL, 1.14 mmol, 6 equivalents per benzyl group) was added to form a slightly yellow solution. The reaction was carried out at 25°C for 24 hours. After the reaction was completed, the solvent was removed in vacuo, saturated NaHCO3 solution (4 mL) and deionized water (4 mL) were added to dissolve the resulting residue, and a small amount of NaS2O3 was added to form a colorless solution. After the residue was completely dissolved, the aqueous phase was washed with 3×10 mL of anhydrous ether to remove the iodobenzyl. The resulting colorless aqueous solution was transferred to a dialysis bag (3500 MWCO) and dialyzed against deionized water for 72 hours. The deprotected dendritic D-polylysine was freeze-dried to a white powder, PAMAM-G2-Dlys 10 (23.5 mg, 89.4%). The NMR results are shown in Figure 4.

[0123] Example 12: Dendritic D-polylysine PAMAM-G2-Dlys 25 Preparation

[0124] N-benzyloxycarbonyl-D-lysine-N-carboxylic anhydride (0.306 g, 1.0 mmol) was weighed into an oven-dried round-bottom flask and 20 mL of anhydrous dichloromethane was added for stirring and dissolution. PAMAM-G2 has 16 primary amine groups. PAMAM G2 initiator (8.14 mg 0.0025 mmol) was added at the required monomer / initiator ratio and the reaction was carried out at 25 ° C for 2 h. After the reaction was completed, the product was precipitated with ice-cold anhydrous ether and centrifuged to obtain undeprotected PAMAM-G2-Dlys(Z) 25 (0.245 g, 90.5%).

[0125] Weigh the undeprotected dendritic D-polylysine PAMAM-G2-Dlys(Z) 25 (50.0 mg) was added to a clean flask, 4 mL of dichloromethane was added and stirred to dissolve. After complete dissolution, trimethylsilyl iodide (163 μL, 1.14 mmol, 6 equivalents per benzyl group) was added to form a slightly yellow solution. The reaction was carried out at 25°C for 24 hours. After the reaction was completed, the solvent was removed in vacuo, and saturated NaHCO3 solution (4 mL) and deionized water (4 mL) were added to dissolve the resulting residue. A small amount of NaS2O3 was added to form a colorless solution. After the residue was completely dissolved, the aqueous phase was washed with 3×10 mL of anhydrous ether to remove the iodobenzyl. The resulting colorless aqueous solution was transferred to a dialysis bag (10000 MWCO) and dialyzed against deionized water for 72 hours. The deprotected dendritic D-polylysine was freeze-dried to a white powder, PAMAM-G2-Dlys 25(22.1 mg, 87.8%). The NMR results are shown in Figure 4.

[0126] Example 13: Dendritic D-polylysine PAMAM-G2-Dlys 50 Preparation

[0127] N-benzyloxycarbonyl-D-lysine-N-carboxylic anhydride (0.306 g, 1.0 mmol) was weighed into an oven-dried round-bottom flask and 20 mL of anhydrous dichloromethane was added for stirring and dissolution. PAMAM-G2 has 16 primary amine groups. PAMAM G2 initiator (4.07 mg 1.25 μmol) was added at the required monomer / initiator ratio and the reaction was carried out at 25 ° C for 2 h. After the reaction was completed, the product was precipitated with ice-cold anhydrous ether and centrifuged to obtain undeprotected PAMAM-G2-Dlys(Z) 50 (0.255 g, 95.9%).

[0128] Weigh the undeprotected dendritic D-polylysine PAMAM-G2-Dlys(Z) 50 (50.0 mg) was added to a clean flask, 4 mL of dichloromethane was added and stirred to dissolve. After complete dissolution, trimethylsilyl iodide (163 μL, 1.14 mmol, 6 equivalents per benzyl group) was added to form a slightly yellow solution. The reaction was carried out at 25°C for 24 hours. After the reaction was completed, the solvent was removed in vacuo, and saturated NaHCO3 solution (4 mL) and deionized water (4 mL) were added to dissolve the resulting residue. A small amount of NaS2O3 was added to form a colorless solution. After the residue was completely dissolved, the aqueous phase was washed with 3×10 mL of anhydrous ether to remove the iodobenzyl. The resulting colorless aqueous solution was transferred to a dialysis bag (10000 MWCO) and dialyzed against deionized water for 72 hours. The deprotected dendritic D-polylysine was freeze-dried to a white powder, PAMAM-G2-Dlys 50 (21.8 mg, 87.8%). The NMR results are shown in Figure 4.

[0129] Example 14: Dendritic D-polylysine PAMAM-G2-Dlys 100 Preparation

[0130] Weigh N-benzyloxycarbonyl-D-lysine-N-carboxylic anhydride (0.306 g, 1.0 mmol) into an oven-dried round-bottom flask and add 20 mL of anhydrous dichloromethane for stirring and dissolution. PAMAM-G2 has 16 primary amine groups. PAMAM G2 initiator (2.04 mg 0.625 μmol) is added at the required monomer / initiator ratio and the reaction is carried out at 25 ° C for 2 h. After the reaction is completed, the product is precipitated with ice-cold anhydrous ether and centrifuged to dry to obtain undeprotected PAMAM-G2-Dlys(Z) 100(0.247 g, 93.3%).

[0131] Weigh the undeprotected dendritic D-polylysine PAMAM-G2-Dlys(Z) 100 (50.0 mg) was added to a clean flask, 4 mL of dichloromethane was added and stirred to dissolve. After complete dissolution, trimethylsilyl iodide (163 μL, 1.14 mmol, 6 equivalents per benzyl group) was added to form a slightly yellow solution. The reaction was carried out at 25°C for 24 hours. After the reaction was completed, the solvent was removed in vacuo, and saturated NaHCO3 solution (4 mL) and deionized water (4 mL) were added to dissolve the resulting residue. A small amount of NaS2O3 was added to form a colorless solution. After the residue was completely dissolved, the aqueous phase was washed with 3×10 mL of anhydrous ether to remove the iodobenzyl. The resulting colorless aqueous solution was transferred to a dialysis bag (10000 MWCO) and dialyzed against deionized water for 72 hours. The deprotected dendritic D-polylysine was freeze-dried to a white powder, PAMAM-G2-Dlys 100 (21.2 mg, 86.1%). The NMR results are shown in Figure 4.

[0132] Example 15: Dendritic D-polylysine PAMAM-G2-Dlys 200 Preparation

[0133] Weigh N-benzyloxycarbonyl-D-lysine-N-carboxylic anhydride (0.306 g, 1.0 mmol) into an oven-dried round-bottom flask and add 20 mL of anhydrous dichloromethane for stirring and dissolution. PAMAM-G2 has 16 primary amine groups. PAMAM G2 initiator (1.02 mg 0.313 μmol) is added at the required monomer / initiator ratio and the reaction is carried out at 25 ° C for 2 h. After the reaction is completed, the product is precipitated with ice-cold anhydrous ether and centrifuged to obtain undeprotected PAMAM-G2-Dlys(Z). 200 (0.249 g, 94.5%) Weigh the undeprotected dendritic D-polylysine PAMAM-G2-Dlys(Z) 200(50.0 mg) was added to a clean flask, 4 mL of dichloromethane was added and stirred to dissolve. After complete dissolution, trimethylsilyl iodide (163 μL, 1.14 mmol, 6 equivalents per benzyl group) was added to form a slightly yellow solution. The reaction was carried out at 25°C for 24 hours. After the reaction was completed, the solvent was removed in vacuo, and saturated NaHCO3 solution (4 mL) and deionized water (4 mL) were added to dissolve the resulting residue. A small amount of NaS2O3 was added to form a colorless solution. After the residue was completely dissolved, the aqueous phase was washed with 3×10 mL of anhydrous ether to remove the iodobenzyl. The resulting colorless aqueous solution was transferred to a dialysis bag (10000 MWCO) and dialyzed against deionized water for 72 hours. The deprotected dendritic D-polylysine was freeze-dried to a white powder, PAMAM-G2-Dlys 200 (21.8 mg, 88.8%). The NMR results are shown in Figure 4.

[0134] Example 16: Dendritic D-polylysine PAMAM-G3-Dlys 10 Preparation

[0135] Weigh N-benzyloxycarbonyl-D-lysine-N-carboxylic anhydride (0.306 g, 1.0 mmol) into an oven-dried round-bottom flask and add 20 mL of anhydrous dichloromethane for stirring and dissolution. PAMAM-G3 has 32 primary amine groups. PAMAM G3 initiator (21.59 mg 0.00313 mmol) is added at the required monomer / initiator ratio and the reaction is carried out at 25 ° C for 2 h. After the reaction is completed, the product is precipitated with ice-cold anhydrous ether and centrifuged to obtain undeprotected PAMAM-G3-Dlys(Z). 10 (0.272 g, 95.9%).

[0136] Weigh the undeprotected dendritic D-polylysine PAMAM-G3-Dlys(Z) 10 (50.0 mg) was added to a clean flask, 4 mL of dichloromethane was added and stirred to dissolve. After complete dissolution, trimethylsilyl iodide (163 μL, 1.14 mmol, 6 equivalents per benzyl group) was added to form a slightly yellow solution. The reaction was carried out at 25°C for 24 hours. After the reaction was completed, the solvent was removed in vacuo, saturated NaHCO3 solution (4 mL) and deionized water (4 mL) were added to dissolve the resulting residue, and a small amount of NaS2O3 was added to form a colorless solution. After the residue was completely dissolved, the aqueous phase was washed with 3×10 mL of anhydrous ether to remove the iodobenzyl. The resulting colorless aqueous solution was transferred to a dialysis bag (3500 MWCO) and dialyzed against deionized water for 72 hours. The deprotected dendritic D-polylysine was freeze-dried to a white powder, PAMAM-G3-Dlys 10 (23.0 mg, 87.1%). The NMR results are shown in Figure 5.

[0137] Example 17: Dendritic D-polylysine PAMAM-G3-Dlys 25 Preparation

[0138] N-benzyloxycarbonyl-D-lysine-N-carboxylic anhydride (0.306 g, 1.0 mmol) was weighed into an oven-dried round-bottom flask and 20 mL of anhydrous dichloromethane was added for stirring and dissolution. PAMAM-G3 has 32 primary amine groups. PAMAM G3 initiator (8.64 mg 0.0013 mmol) was added at the required monomer / initiator ratio and the reaction was carried out at 25 ° C for 2 h. After the reaction was completed, the mixture was precipitated with ice-cold anhydrous ether and centrifuged to obtain undeprotected PAMAM-G3-Dlys(Z) 25 (0.244 g, 90.1%).

[0139] Weigh the undeprotected dendritic D-polylysine PAMAM-G3-Dlys(Z) 25 (50.0 mg) was added to a clean flask, 4 mL of dichloromethane was added and stirred to dissolve. After complete dissolution, trimethylsilyl iodide (163 μL, 1.14 mmol, 6 equivalents per benzyl group) was added to form a slightly yellow solution. The reaction was carried out at 25°C for 24 hours. After the reaction was completed, the solvent was removed in vacuo, and saturated NaHCO3 solution (4 mL) and deionized water (4 mL) were added to dissolve the resulting residue. A small amount of NaS2O3 was added to form a colorless solution. After the residue was completely dissolved, the aqueous phase was washed with 3×10 mL of anhydrous ether to remove the iodobenzyl. The resulting colorless aqueous solution was transferred to a dialysis bag (10000 MWCO) and dialyzed against deionized water for 72 hours. The deprotected dendritic D-polylysine was freeze-dried to a white powder, PAMAM-G3-Dlys 25 (21.9 mg, 86.8%). The NMR results are shown in Figure 5.

[0140] Example 18: Dendritic D-polylysine PAMAM-G3-Dlys 50 Preparation

[0141] N-benzyloxycarbonyl-D-lysine-N-carboxylic anhydride (0.306 g, 1.0 mmol) was weighed into an oven-dried round-bottom flask and 20 mL of anhydrous dichloromethane was added for stirring and dissolution. PAMAM-G3 has 32 primary amine groups. PAMAM G3 initiator (4.32 mg 0.625 μmol) was added at the required monomer / initiator ratio and the reaction was carried out at 25 ° C for 2 h. After the reaction was completed, the product was precipitated with ice-cold anhydrous ether and centrifuged to obtain undeprotected PAMAM-G3-Dlys(Z) 50 (0.258 g, 96.9%).

[0142] Weigh the undeprotected dendritic D-polylysine PAMAM-G3-Dlys(Z) 50 (50.0 mg) was added to a clean flask, 4 mL of dichloromethane was added and stirred to dissolve. After complete dissolution, trimethylsilyl iodide (163 μL, 1.14 mmol, 6 equivalents per benzyl group) was added to form a slightly yellow solution. The reaction was carried out at 25°C for 24 hours. After the reaction was completed, the solvent was removed in vacuo, and saturated NaHCO3 solution (4 mL) and deionized water (4 mL) were added to dissolve the resulting residue. A small amount of NaS2O3 was added to form a colorless solution. After the residue was completely dissolved, the aqueous phase was washed with 3×10 mL of anhydrous ether to remove the iodobenzyl. The resulting colorless aqueous solution was transferred to a dialysis bag (10000 MWCO) and dialyzed against deionized water for 72 hours. The deprotected dendritic D-polylysine was freeze-dried to a white powder, PAMAM-G3-Dlys 50 (20.9 mg, 84.2%). The NMR results are shown in Figure 5.

[0143] Example 19: Dendritic D-polylysine PAMAM-G3-Dlys 100 Preparation

[0144] N-benzyloxycarbonyl-D-lysine-N-carboxylic anhydride (0.306 g, 1.0 mmol) was weighed into an oven-dried round-bottom flask and 20 mL of anhydrous dichloromethane was added for stirring and dissolution. PAMAM-G3 has 32 primary amine groups. PAMAM G3 initiator (2.16 mg 0.313 μmol) was added at the required monomer / initiator ratio and the reaction was carried out at 25 ° C for 2 h. After the reaction was completed, the product was precipitated with ice-cold anhydrous ether and centrifuged to obtain undeprotected PAMAM-G3-Dlys(Z) 100 (0.253 g, 95.6%).

[0145] Weigh the undeprotected dendritic D-polylysine PAMAM-G3-Dlys(Z) 100 (50.0 mg) was added to a clean flask, 4 mL of dichloromethane was added and stirred to dissolve. After complete dissolution, trimethylsilyl iodide (163 μL, 1.14 mmol, 6 equivalents per benzyl group) was added to form a slightly yellow solution. The reaction was carried out at 25°C for 24 hours. After the reaction was completed, the solvent was removed in vacuo, and saturated NaHCO3 solution (4 mL) and deionized water (4 mL) were added to dissolve the resulting residue. A small amount of NaS2O3 was added to form a colorless solution. After the residue was completely dissolved, the aqueous phase was washed with 3×10 mL of anhydrous ether to remove the iodobenzyl. The resulting colorless aqueous solution was transferred to a dialysis bag (10000 MWCO) and dialyzed against deionized water for 72 hours. The deprotected dendritic D-polylysine was freeze-dried to a white powder, PAMAM-G3-Dlys 100(21.4 mg, 86.8%). The NMR results are shown in Figure 5.

[0146] Example 20: Dendritic D-polylysine PAMAM-G3-Dlys 200 Preparation

[0147] Weigh N-benzyloxycarbonyl-D-lysine-N-carboxylic anhydride (0.306 g, 1.0 mmol) into an oven-dried round-bottom flask and add 20 mL of anhydrous dichloromethane for stirring and dissolution. PAMAM-G3 has 32 primary amine groups. PAMAM G3 initiator (1.08 mg 0.156 μmol) is added at the required monomer / initiator ratio and the reaction is carried out at 25 ° C for 2 h. After the reaction is completed, the product is precipitated with ice-cold anhydrous ether and centrifuged to obtain undeprotected PAMAM-G3-Dlys(Z). 200 (0.251 g, 95.5%).

[0148] Weigh the undeprotected dendritic D-polylysine PAMAM-G3-Dlys(Z) 200 (50.0 mg) was added to a clean flask, 4 mL of dichloromethane was added and stirred to dissolve. After complete dissolution, trimethylsilyl iodide (163 μL, 1.14 mmol, 6 equivalents per benzyl group) was added to form a slightly yellow solution. The reaction was carried out at 25°C for 24 hours. After the reaction was completed, the solvent was removed in vacuo, and saturated NaHCO3 solution (4 mL) and deionized water (4 mL) were added to dissolve the resulting residue. A small amount of NaS2O3 was added to form a colorless solution. After the residue was completely dissolved, the aqueous phase was washed with 3×10 mL of anhydrous ether to remove the iodobenzyl. The resulting colorless aqueous solution was transferred to a dialysis bag (10000 MWCO) and dialyzed against deionized water for 72 hours. The deprotected dendritic D-polylysine was freeze-dried to a white powder, PAMAM-G3-Dlys 200 (20.8 mg, 84.8%). The NMR results are shown in Figure 5.

[0149] Example 21: Dendritic D-polylysine PAMAM-G4-Dlys 10 Preparation

[0150] N-benzyloxycarbonyl-D-lysine-N-carboxylic anhydride (0.306 g, 1.0 mmol) was weighed into an oven-dried round-bottom flask and 20 mL of anhydrous dichloromethane was added for stirring and dissolution. PAMAM-G4 has 64 primary amine groups. PAMAM G4 initiator (22.2 mg 1.562 μmol) was added at the required monomer / initiator ratio and the reaction was carried out at 25 ° C for 2 h. After the reaction was completed, the product was precipitated with ice-cold anhydrous ether and centrifuged to obtain undeprotected PAMAM-G4-Dlys(Z) 10(0.269 g, 94.6%).

[0151] Weigh the undeprotected dendritic D-polylysine PAMAM-G4-Dlys(Z) 10 (50.0 mg) was added to a clean flask, 4 mL of dichloromethane was added and stirred to dissolve. After complete dissolution, trimethylsilyl iodide (163 μL, 1.14 mmol, 6 equivalents per benzyl group) was added to form a slightly yellow solution. The reaction was carried out at 25°C for 24 hours. After the reaction was completed, the solvent was removed in vacuo, and saturated NaHCO3 solution (4 mL) and deionized water (4 mL) were added to dissolve the resulting residue. A small amount of NaS2O3 was added to form a colorless solution. After the residue was completely dissolved, the aqueous phase was washed with 3×10 mL of anhydrous ether to remove the iodobenzyl. The resulting colorless aqueous solution was transferred to a dialysis bag (3500 MWCO) and dialyzed against deionized water for 72 hours. The deprotected dendritic D-polylysine was freeze-dried to a white powder, PAMAM-G4-Dlys 10 (23.2 mg, 87.8%). The NMR results are shown in Figure 6.

[0152] Example 22: Dendritic D-polylysine PAMAM-G4-Dlys 25 Preparation

[0153] Weigh N-benzyloxycarbonyl-D-lysine-N-carboxylic anhydride (0.306 g, 1.0 mmol) into an oven-dried round-bottom flask and add 20 mL of anhydrous dichloromethane for stirring and dissolution. PAMAM-G4 has 64 primary amine groups. PAMAM G4 initiator (8.88 mg 0.625 μmol) is added at the required monomer / initiator ratio and the reaction is carried out at 25 ° C for 2 h. After the reaction is completed, the product is precipitated with ice-cold anhydrous ether and centrifuged to dry to obtain undeprotected PAMAM-G4-Dlys(Z) 25 (0.253 g, 93.2%).

[0154] Weigh the undeprotected dendritic D-polylysine PAMAM-G4-Dlys(Z) 25(50.0 mg) was added to a clean flask, 4 mL of dichloromethane was added and stirred to dissolve. After complete dissolution, trimethylsilyl iodide (163 μL, 1.14 mmol, 6 equivalents per benzyl group) was added to form a slightly yellow solution. The reaction was carried out at 25°C for 24 hours. After the reaction was completed, the solvent was removed in vacuo, and saturated NaHCO3 solution (4 mL) and deionized water (4 mL) were added to dissolve the resulting residue. A small amount of NaS2O3 was added to form a colorless solution. After the residue was completely dissolved, the aqueous phase was washed with 3×10 mL of anhydrous ether to remove the iodobenzyl. The resulting colorless aqueous solution was transferred to a dialysis bag (10000 MWCO) and dialyzed against deionized water for 72 hours. The deprotected dendritic D-polylysine was freeze-dried to a white powder, PAMAM-G4-Dlys 25 (21.6 mg, 85.6%). The NMR results are shown in Figure 6.

[0155] Example 23: Dendritic D-polylysine PAMAM-G4-Dlys 50 Preparation

[0156] Weigh N-benzyloxycarbonyl-D-lysine-N-carboxylic anhydride (0.306 g, 1.0 mmol) into an oven-dried round-bottom flask and add 20 mL of anhydrous dichloromethane for stirring and dissolution. PAMAM-G4 has 64 primary amine groups. PAMAM G4 initiator (4.44 mg 0.313 μmol) is added at the required monomer / initiator ratio and the reaction is carried out at 25 ° C for 2 h. After the reaction is completed, the product is precipitated with ice-cold anhydrous ether and centrifuged to obtain undeprotected PAMAM-G4-Dlys(Z). 50 (0.256 g, 95.8%).

[0157] Weigh the undeprotected dendritic D-polylysine PAMAM-G4-Dlys(Z) 50 (50.0 mg) was added to a clean flask, 4 mL of dichloromethane was added and stirred to dissolve. After complete dissolution, trimethylsilyl iodide (163 μL, 1.14 mmol, 6 equivalents per benzyl group) was added to form a slightly yellow solution. The reaction was carried out at 25°C for 24 hours. After the reaction was completed, the solvent was removed in vacuo, and saturated NaHCO3 solution (4 mL) and deionized water (4 mL) were added to dissolve the resulting residue. A small amount of NaS2O3 was added to form a colorless solution. After the residue was completely dissolved, the aqueous phase was washed with 3×10 mL of anhydrous ether to remove the iodobenzyl. The resulting colorless aqueous solution was transferred to a dialysis bag (10000 MWCO) and dialyzed against deionized water for 72 hours. The deprotected dendritic D-polylysine was freeze-dried to a white powder, PAMAM-G4-Dlys 50 (21.3 mg, 85.8%). The NMR results are shown in Figure 6.

[0158] Example 24: Dendritic D-polylysine PAMAM-G4-Dlys 100 Preparation

[0159] Weigh N-benzyloxycarbonyl-D-lysine-N-carboxylic anhydride (0.306 g, 1.0 mmol) into an oven-dried round-bottom flask and add 20 mL of anhydrous dichloromethane for stirring and dissolution. PAMAM-G4 has 64 primary amine groups. PAMAM G4 initiator (2.22 mg 0.156 μmol) is added at the required monomer / initiator ratio and the reaction is carried out at 25 ° C for 2 h. After the reaction is completed, the product is precipitated with ice-cold anhydrous ether and centrifuged to obtain undeprotected PAMAM-G4-Dlys(Z). 100 (0.250 g, 94.6%).

[0160] Weigh the undeprotected dendritic D-polylysine PAMAM-G4-Dlys(Z) 100 (50.0 mg) was added to a clean flask, 4 mL of dichloromethane was added and stirred to dissolve. After complete dissolution, trimethylsilyl iodide (163 μL, 1.14 mmol, 6 equivalents per benzyl group) was added to form a slightly yellow solution. The reaction was carried out at 25°C for 24 hours. After the reaction was completed, the solvent was removed in vacuo, and saturated NaHCO3 solution (4 mL) and deionized water (4 mL) were added to dissolve the resulting residue. A small amount of NaS2O3 was added to form a colorless solution. After the residue was completely dissolved, the aqueous phase was washed with 3×10 mL of anhydrous ether to remove the iodobenzyl. The resulting colorless aqueous solution was transferred to a dialysis bag (10000 MWCO) and dialyzed against deionized water for 72 hours. The deprotected dendritic D-polylysine was freeze-dried to a white powder, PAMAM-G4-Dlys 100 (21.9 mg, 88.8%).

[0161] Example 25: Dendritic D-polylysine PAMAM-G4-Dlys 200 Preparation

[0162] Weigh N-benzyloxycarbonyl-D-lysine-N-carboxylic anhydride (0.306 g, 1.0 mmol) into an oven-dried round-bottom flask and add 20 mL of anhydrous dichloromethane for stirring and dissolution. PAMAM-G4 has 64 primary amine groups. PAMAM G4 initiator (1.11 mg 0.078 μmol) is added at the required monomer / initiator ratio and the reaction is carried out at 25 ° C for 2 h. After the reaction is completed, the product is precipitated with ice-cold anhydrous ether and centrifuged to dry to obtain undeprotected PAMAM-G4-Dlys(Z) 200 (0.252 g, 95.8%).

[0163] Weigh the undeprotected dendritic D-polylysine PAMAM-G4-Dlys(Z) 200 (50.0 mg) was added to a clean flask, 4 mL of dichloromethane was added and stirred to dissolve. After complete dissolution, trimethylsilyl iodide (163 μL, 1.14 mmol, 6 equivalents per benzyl group) was added to form a slightly yellow solution. The reaction was carried out at 25°C for 24 hours. After the reaction was completed, the solvent was removed in vacuo, and saturated NaHCO3 solution (4 mL) and deionized water (4 mL) were added to dissolve the resulting residue. A small amount of NaS2O3 was added to form a colorless solution. After the residue was completely dissolved, the aqueous phase was washed with 3×10 mL of anhydrous ether to remove the iodobenzyl. The resulting colorless aqueous solution was transferred to a dialysis bag (10000 MWCO) and dialyzed against deionized water for 72 hours. The deprotected dendritic D-polylysine was freeze-dried to a white powder, PAMAM-G4-Dlys 200 (21.0 mg, 85.6%). The NMR results are shown in Figure 6.

[0164] Example 26: Dendritic L-polylysine PAMAM-GO-Llys 50 Preparation

[0165] Weigh N-benzyloxycarbonyl-L-lysine-N-indolecarboxylic anhydride (0.306 g, 1.0 mmol) into an oven-dried round-bottom flask and add 20 mL of anhydrous dichloromethane for stirring and dissolution. PAMAM-G0 has four primary amine groups. PAMAM G0 initiator (2.58 mg 0.005 mmol) is added at the required monomer / initiator ratio and the reaction is carried out at 25 ° C for 2 h. After the reaction is completed, the product is precipitated with ice-cold anhydrous ether and centrifuged to dry to obtain undeprotected PAMAM-G0-Llys(Z) 50 (0.252 g, 95.1%).

[0166] Weigh the above undeprotected dendritic L-polylysine PAMAM-G0-Llys(Z) 50 (50.0 mg) was added to a clean flask, 4 mL of dichloromethane was added and stirred to dissolve. After complete dissolution, trimethylsilyl iodide (163 μL, 1.14 mmol, 6 equivalents per benzyl group) was added to form a slightly yellow solution. The reaction was carried out at 25°C for 24 hours. After the reaction was completed, the solvent was removed in vacuo, saturated NaHCO3 solution (4 mL) and deionized water (4 mL) were added to dissolve the resulting residue, and a small amount of NaS2O3 was added to form a colorless solution. After the residue was completely dissolved, the aqueous phase was washed with 3×10 mL of anhydrous ether to remove the iodobenzyl. The resulting colorless aqueous solution was transferred to a dialysis bag (10000 MWCO) and dialyzed against deionized water for 72 hours. The deprotected dendritic L-polylysine was freeze-dried to a white powder, PAMAM-G0-Llys 50(21.4 mg, 86.6%).

[0167] Example 27: Average Particle Size of Dendritic Polyamino Acid Nanoparticles

[0168] Preparation of Nanoparticles: 4 mg of each of the dendritic polyamino acids prepared in Examples 1-25 was weighed and added to a clean glass vial. 4 mL of PBS was then added to prepare a 1 mg / mL PBS solution. The vial was then sonicated (at 25°C, 60 minutes, and 0.45 kW power) to obtain dendritic amino acid nanoparticles.

[0169] Preparation of nanovaccine: At 4 degrees Celsius, 1 mg / mL of nanoparticles was compounded with 1 mg / mL of antigen (OVA) PBS solution in a volume ratio of 1:1 and stirred for 15 minutes.

[0170] The relative molecular weight of the dendritic polyamino acid was detected by gel permeation chromatography, and the GPC result was obtained, as shown in FIG7 .

[0171] The average particle sizes of nanoparticles and nanovaccines are shown in Figures 8 to 11 .

[0172] Example 28: Bone marrow-derived dendritic cell (BMDC) activation experiment

[0173] BMDC preparation: Dendritic cells were isolated from the bone marrow of C57BL / 6N mice. Bone marrow was collected from the femur and tibia, and single-cell suspensions were cultured in RPMI 1640 supplemented with 10% heat-inactivated fetal bovine serum, 1% penicillin-streptomycin, 20 ng / mL GM-CSF, and 10 ng / mL IL-4. Non-adherent and loosely adherent cells were collected at day 7-10 of differentiation for use in this study.

[0174] Test steps

[0175] 1. Add 250,000 BMDCs to each well of a 24-well plate.

[0176] 2. Add the prepared partial dendritic polyamino acid nanoparticles of Example 27 to each well to make the final concentration of the nanoparticles 4 μg / mL.

[0177] 3. Place the 24-well plate in a 37°C incubator for 12 hours.

[0178] 4. The cell supernatant was collected and the cytokine IFN-γ level was measured by enzyme-linked immunosorbent assay (ELISA). The results are shown in FIG11 . In FIG11 , the horizontal axis G0-10 represents the dendritic D-polylysine PAMAM-G0-Dlys 10Prepared nanoparticles, G0-25 is a dendritic D-polylysine PAMAM-G0-Dlys 25 Prepared nanoparticles, G0-50 is a dendritic D-polylysine PAMAM-G0-Dlys 50 The prepared nanoparticles are deduced by analogy, where G0 represents the generation of the initiator and the following numbers represent the degree of polymerization of the polyamino acid units.

[0179] The results in FIG12 show that the dendritic polyamino acid of the present invention significantly promotes the activation of BMDCs, significantly increases the secretion of IFN-γ, enhances the immune response, and improves the body's protective ability.

[0180] Example 29: In vivo lymph node (LN) analysis

[0181] Weigh the dendritic D-polylysine PAMAM-GO-Dlysine of Example 3 50 4 mg of the amino acid was added to a clean glass vial, and 4 mL of PBS was added to prepare a 1 mg / mL PBS solution. The vial was then ultrasonicated (the ultrasonic temperature was 25°C, the time was 60 minutes, and the power was 0.45 kW) to obtain dendritic amino acid nanoparticles.

[0182] Preparation of nanovaccine: At 4 degrees Celsius, nanovaccine was prepared by combining OVA solution with nanoparticles in different ratios. The mass ratios of OVA to nanoparticles were 1:1, 1:2, 1:4, 1:6, and 1:8, respectively.

[0183] On day 0, mice were subcutaneously injected with the nanovaccines at different compound ratios (the nanovaccine injection dose was 20 μg of OVA per mouse). On day 2, the inguinal lymph nodes of the mice were dissected and mechanically disrupted at 37°C. The samples were then filtered through a 200-mesh nylon filter to obtain a single-cell suspension, and the cells were counted using a cell counter. The cells were then incubated with anti-CD11c, anti-CD80, anti-MHC-II, and anti-SIINFKEL-H2Kb for 30 minutes at 4°C to analyze DC activation and antigen cross-presentation within the lymph nodes. Flow cytometer (BD FACSCelesta) was used to acquire flow data and analyze them using FlowJo software. The results are shown in Figure 13.

[0184] In vivo experiments were conducted to evaluate the dendritic D-polylysine PAMAM-GO-Dlys 50The results in Figure 13 show that the dendritic D-polylysine nanovaccine caused an increase in the mass of draining lymph nodes and an increase in the number of lymphocytes in the lymph nodes. In addition, the DC cell activation markers CD80 and MHC-Ⅱ in the lymph nodes increased, indicating that the nanovaccine caused the activation of DC cells. In addition, the level of SIINFKEL-H2Kb on the surface of DC cells increased, indicating that the nanovaccine caused antigen cross-presentation of DC cells in the lymph nodes. 50 When the composite mass ratio was 1:6, the DC cell activation and antigen cross-presentation effects were the strongest.

[0185] Example 30: Bone marrow-derived dendritic cell (BMDC) activation experiment

[0186] Weigh the dendritic D-polylysine PAMAM-GO-Dlys prepared in Example 3 respectively 50 and the dendritic L-polylysine PAMAM-GO-Llys prepared in Example 26 50 4 mg of the amino acid was added to a clean glass vial, and 4 mL of PBS was added to prepare a 1 mg / mL PBS solution. The vial was then ultrasonicated (the ultrasonic temperature was 25°C, the time was 60 minutes, and the power was 0.45 kW) to obtain dendritic amino acid nanoparticles.

[0187] Preparation of mouse Lewis lung cancer cell antigen:

[0188] (1) Lewis lung cancer cells were injected subcutaneously into the axilla of C57BL / 6N mice at a density of 2 million cells per mouse to establish a Lewis lung cancer subcutaneous xenograft tumor model.

[0189] (2) When the tumor volume of the animal model grows to about 300 mm 3 At 3 , the mice were euthanized and the tumors were removed under sterile conditions. The tumors were washed with serum-free PRMI-1640 medium containing 3 times the concentration of double antibodies and the necrotic part of the tumor tissue was removed. Finally, the tumor tissue was washed with serum-free PRMI-1640 to remove the double antibodies in the system.

[0190] (3) The tumor tissue was disrupted by a micro-electric tissue homogenizer (IKA, Germany), and then the tumor cells were fully disrupted by three freeze-thaw cycles at -80°C → 37°C → -80°C;

[0191] (4) Extract tumor cell membrane proteins according to the standard method provided by the Biyuntian Cell Membrane Protein and Cytoplasmic Protein Extraction Kit;

[0192] (5) Obtain LEWIS tumor cell membrane antigen protein (CM) according to the above steps.

[0193] Preparation of nanovaccine: At 4 degrees Celsius, 1 mg / mL nanoparticles were compounded with 0.25 mg / mL antigen (CM) PBS solution in a volume ratio of 1:1 and stirred for 15 minutes.

[0194] Test steps

[0195] 1. Add 250,000 BMDCs to each well of a 24-well plate.

[0196] 2. Add the above-mentioned nanovaccine to each well to make the final concentration of the nanovaccine material 4 μg / mL.

[0197] 3. Place the 24-well plate in a 37°C incubator for 24 hours.

[0198] 4. BMDCs were harvested and stained with fluorescently labeled antibodies against CD11c, CD86, CD80, and MHC-II for 30 minutes before flow cytometry analysis. Cell supernatants were collected and analyzed for TNF-α and IL-6 levels using enzyme-linked immunosorbent assay (ELISA). The results are shown in Figure 14.

[0199] The results in FIG14 show that the dendritic polyamino acid of the present invention significantly promotes the activation of BMDCs, enhances the immune response, and improves the body's protective ability.

[0200] Example 31: In vivo tumor treatment experiment

[0201] On day 0, B16-OVA cells (2.0×10 5 Cells) were subcutaneously inoculated on the left dorsal surface of 6-8 week old female C57BL / 6N mice. On days 4, 7, 11, and 14, different concentrations of PBS, OVA, and composite OVA (OVA to nanoparticles mass ratio of 1:6) were added to the dendritic D-polylysine PAMAM-G0-Dlys of Example 3. 50 (OVA+D) nanovaccine and Example 26 dendritic L-polylysine PAMAM-GO-Llys 50 The (OVA+L) nanovaccine was subcutaneously injected into the right dorsal surface of the mice (injection dose: 20 μg OVA + 120 μg nanoparticles per mouse). The OVA+D+αPD-L1 group received an intraperitoneal injection of anti-PD-L1 antibody (αPD-1) at a dose of 50.0 μg / mouse on the first day after nanovaccine injection. The weight of the mice and the volume of the tumor were then checked every other day. When the tumor volume reached 2000 mm 3When the skin was severely ulcerated, the mice were killed. The results are shown in Figure 15. As shown in Figure 15, the OVA+D nanovaccine of Example 3 injected with compound OVA can effectively inhibit tumor growth, with obvious advantages. By combining it with immune checkpoint inhibitor therapy, its tumor inhibition effect is more significant, and it has potential application value.

[0202] The above is only a preferred embodiment of the present invention. It should be pointed out that for ordinary technicians in this technical field, several improvements and modifications can be made without departing from the principles of the present invention. These improvements and modifications should also be regarded as within the scope of protection of the present invention.

Claims

1. A dendritic polyamino acid, characterized in that Comprising a polymer unit and a polyamino acid unit; the polymer unit is connected to the polyamino acid unit via an amide bond; The polymer unit is formed by losing a hydrogen atom from the terminal amino group of the polymer; the polymer is selected from dendritic polymers and / or branched polymers; The polyamino acid unit is shown in formula (I): Wherein, n is an integer from 2 to 500; R is selected from one of the structures shown in formula (1) to formula (3): m1 to m3 are each independently selected from integers of 0-5.

2. The dendritic polyamino acid according to claim 1, characterized in that The number of generations of the dendritic polymer is 0 to 9; The number average molecular weight of the branched polymer is 300 to 100,000; The carbon atom connected to R in the amino acid unit is a chiral carbon atom; The configuration of the chiral carbon atom is L-type or D-type, and preferably the configuration of the chiral carbon atom is D-type.

3. The dendritic polyamino acid according to claim 1, characterized in that The polymer is selected from one or more of polyamide-amine, polypropylene imine and branched polyethylene imine.

4. The dendritic polyamino acid according to claim 1, characterized in that The m1 is 3, m2 is 2, and m3 is 1.

5. A method for preparing a dendritic polyamino acid, characterized in that: The following steps are involved: S1) performing a ring-opening polymerization reaction on a polymer having terminal amino groups and a carboxylic anhydride represented by formula (II) to obtain an intermediate; the polymer is a dendritic polymer and / or a branched polymer; S2) deprotecting the intermediate to obtain a dendritic polyamino acid; Wherein, R' is selected from one of the structures represented by formula (1) to formula (3) in which the amino group is protected: m1 to m3 are each independently selected from integers of 0-5.

6. The preparation method according to claim 5, characterized in that The molar ratio of the polymer having terminal amino groups to the carboxylic anhydride represented by formula (II) is (0.00005-0.1):1; The number average molecular weight of the branched polymer is 300 to 100,000; The ring-opening polymerization reaction is carried out in a solvent; the solvent is selected from one or more of dichloromethane, chloroform and N,N-dimethylformamide; The temperature of the ring-opening polymerization reaction is 10°C to 65°C; The ring-opening polymerization reaction time is 1 to 72 hours.

7. A nanoparticle, characterized in that: The dendritic polyamino acid is obtained by self-assembly in water or a water-containing solvent by the dendritic polyamino acid according to any one of claims 1 to 4 or the dendritic polyamino acid prepared by the preparation method according to any one of claims 5 to 7.

8. A nanoadjuvant, characterized in that The invention comprises the dendritic polyamino acid according to any one of claims 1 to 4, the dendritic polyamino acid prepared by the preparation method according to any one of claims 5 to 7, or the nanoparticles according to claim 7.

9. A nano vaccine, characterized in that The invention comprises the nano-adjuvant according to claim 8, an antigen and an adjuvant acceptable to the vaccine.

10. The nanovaccine according to claim 9, characterized in that The mass ratio of the antigen to the nano adjuvant is 1:0.01-100.

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