Polyethylene glycol-modified polylysine, preparation method thereof, nano vaccine and application thereof

Abstract

The present invention provides polyethylene glycol-modified polylysine, its preparation method, a nanovaccine, and its application. The polyethylene glycol-modified polylysine comprises a dendritic polymer initiator and chiral polylysine linked by amide bonds. By grafting polyethylene glycol onto the side chains of polylysine, the present invention modulates charge density and enhances nanoparticle migration to lymph nodes. Finally, the nanovaccine is prepared by binding to antigens through electrostatic adsorption. Research has shown that the resulting antigen-complexed nanovaccine promotes endocytosis by antigen-presenting cells (APCs) and their migration to lymph nodes, activating immune cells there. It can stimulate dendritic cells (DCs) to mature and release inflammatory cytokines. It also promotes antigen cross-presentation by DCs in vivo, triggering a strong antigen-specific immune response.

Description

Technical Field

[0001] The present invention belongs to the field of biomedicine technology, and specifically relates to polyethylene glycol-modified polylysine and a preparation method thereof, a nano vaccine and applications thereof. Background Art

[0002] In recent years, using tumor vaccines to trigger the body's own immunity has become an effective method for treating cancer. Generally, after subcutaneous or intramuscular injection of a tumor vaccine, the vaccine passively migrates through intercellular spaces and lymphatic vessels to reach the lymph nodes, where it is taken up by antigen-presenting cells and delivered to the lymph nodes, thereby activating immune cells in the lymph nodes. After maturation, the T lymphocytes in these lymph nodes migrate and infiltrate into the tumor site, attacking tumor cells and inducing tumor cell death. During this period, substances released by tumor cells will act as antigens to continue to trigger an immune response in the body. Therefore, in tumor vaccine treatment, lymph nodes, as the center of immune cell differentiation and maturation, are crucial to the therapeutic effect.

[0003] Currently, in order for tumor vaccines to effectively reach lymph nodes, they must overcome four barriers. Barrier 1: The surrounding interstitial matrix blocks the passive transport of tumor vaccines to the lymph nodes; Barrier 2: The afferent lymphatic vessels have a small internal diameter, typically only allowing passage of substances around 10-100 nm; Barrier 3: Macrophages in the subcutaneous sinus of the lymph nodes clear larger particles from the lymph fluid; Barrier 4: The very small internal diameter of the vessels poses a challenge to reaching the deeper regions of the lymph nodes. Clearly, only by overcoming these four major physiological barriers to the lymph nodes can tumor vaccines effectively activate the body's own immune system.

[0004] Currently, studies have shown that by changing the size of the material during migration, the material can be better transported through the lymphatic vessels of the lymph nodes, or by modifying the material with a prodrug to allow it to accumulate specifically in the lymph node sinus region, or by modifying the material's surface potential, hydrophilicity, and other properties to improve its migration efficiency to the lymph nodes. However, while these methods have effectively improved the material's migration efficiency to the lymph nodes, the following problems still exist: the material's poor mobility in the intercellular space and low passive transport efficiency to the lymph nodes. Summary of the Invention

[0005] In view of this, the present invention aims to provide a polyethylene glycol-modified polylysine and its preparation method, a nano vaccine and its application. The nano vaccine has a better efficiency in migrating to lymph nodes.

[0006] To achieve this object, the present invention adopts the following technical solutions:

[0007] In a first aspect, the present invention provides a polyethylene glycol-modified polylysine, comprising a dendrimer initiator and polylysine connected via an amide bond, wherein polyethylene glycol is grafted onto the side chain of the polylysine.

[0008] Preferably, the dendrimer initiator is formed by losing one hydrogen atom from the terminal amino group of a dendrimer, and the dendrimer comprises polyamide-amine; the generation number of the polyamide-amine is 0 to 5.

[0009] Preferably, the lysine in the polylysine is chiral lysine.

[0010] Preferably, the molecular weight of the polyethylene glycol is 750-2000 Da.

[0011] Preferably, the grafting rate of the polyethylene glycol is 5% to 15%.

[0012] Preferably, the polyethylene glycol-modified polylysine has the structural formula shown in Formula I below:

[0013] Formula I;

[0014] Where x = any number from 2 to 8, y = any number from 42 to 48, and x + y = n = 50.

[0015] Preferably, the polyethylene glycol-modified polylysine is spherical and has a particle size of 10 to 100 nm.

[0016] In a second aspect, the present invention provides a method for preparing the polyethylene glycol-modified polylysine, characterized in that it comprises the following steps:

[0017] S1: amino protected lysine- N - Internal carboxylic acid anhydride is mixed with the dendrimer and undergoes ring-opening polymerization, and after deprotection, an intermediate is obtained;

[0018] S2: reacting the intermediate with mPEG-COOH to obtain polyethylene glycol-modified polylysine.

[0019] Preferably, the ring-opening polymerization reaction in step S1 is carried out at a temperature of 10-30° C. and for a time of 0.5-6 h.

[0020] Preferably, the reaction temperature in step S2 is 10-30° C., and the reaction time is 1-7 days.

[0021] Preferably, the amino group is protected lysine- N The molar ratio of the internal carboxylic acid anhydride to the dendritic polymer is (10~200):1.

[0022] Preferably, the molar ratio of the intermediate to mPEG-COOH is (6-20):1.

[0023] In a third aspect, the present invention provides a nanoadjuvant comprising the polyethylene glycol-modified polylysine and a solvent.

[0024] Preferably, the solvent is selected from water or PBS buffer solution.

[0025] Preferably, the concentration of the nanoadjuvant is 0.1-5 mg / mL.

[0026] In a third aspect, the present invention provides a nano vaccine obtained by complexing the above-mentioned polyethylene glycol-modified polylysine or nano adjuvant with an antigen.

[0027] Preferably, the nanoadjuvant comprises the polyethylene glycol-modified polylysine mentioned above, and a solvent.

[0028] Preferably, the solvent is selected from water or PBS buffer solution.

[0029] Preferably, the concentration of the nanoadjuvant is 0.1-5 mg / mL.

[0030] Preferably, the mass ratio of the antigen to polyethylene glycol-modified polylysine is 1:(1-10).

[0031] Preferably, the antigen comprises chicken ovalbumin, tumor extract or tumor cell membrane extract. The tumor in the tumor extract or tumor cell membrane extract includes but is not limited to melanoma (B16) or Lewis lung carcinoma (LLC).

[0032] In a fifth aspect, the present invention provides a use of the above-mentioned polyethylene glycol-modified polylysine, the above-mentioned nanoadjuvant or the above-mentioned nanovaccine in the preparation of a vaccine for preventing tumors.

[0033] Preferably, the tumor includes but is not limited to melanoma (B16) and Lewis lung carcinoma (LLC).

[0034] Compared with the prior art, the present invention has the following beneficial effects:

[0035] The present invention provides a polyethylene glycol-modified polylysine, and uses it as a substrate to prepare a nanoadjuvant and a nanovaccine with an immune activation function. The nanovaccine has an immunomodulatory function and the function of activating the body's own immunity, and can be used to prepare a vaccine for tumor prevention. The polyethylene glycol-modified polylysine in the present invention is a nanoparticle, comprising a dendrimer initiator and polylysine connected by an amide bond. The presence of the dendrimer initiator can regulate the topological structure of the nanoadjuvant, and the presence of the chiral lysine structural unit can regulate the ability of the nanoadjuvant to activate immune cells. The present invention further grafts polyethylene glycol on the side chain of polylysine to adjust the charge density, thereby enhancing the effect and transport efficiency of the passive migration of nanoparticles to lymph nodes. Finally, the antigen is modified by electrostatic adsorption, further enhancing the antigen cross-presentation of the nanoparticles in the lymph nodes.

[0036] Research has shown that the nanovaccine obtained after complexing with antigens has the function of promoting the endocytosis of antigen-presenting cells and their migration to lymph nodes to activate immune cells in the lymph nodes. It can stimulate the maturation of dendritic cells (DC cells) and release inflammatory-related cytokines, such as IL-6, IL-12, IFN-γ and TNF-α, and promote antigen cross-presentation of DC cells in the body, further triggering a strong antigen-specific immune response. BRIEF DESCRIPTION OF THE DRAWINGS

[0037] Figure 1 Schematic diagram of the synthesis route of the products at each stage in Example 1;

[0038] in, Figure 1 A is mPEG2k-COOH and N(ε)-benzyloxycarbonyl-D-lysine- N - internal carboxylic anhydride (Cbz- D -LysNCA) synthesis route diagram, Figure 1 B is G0-P D -Lys 50 Schematic diagram of the synthetic route, Figure 1 C is G0-P D -Lys 50 -Schematic diagram of the synthesis route of mPEG2k;

[0039] Figure 2 G0-P with different polyethylene glycol grafting ratios D -Lys 50 -mPEG2k 1 H NMR and FT-IR spectra;

[0040] in, Figure 2 A is G0-P with different polyethylene glycol grafting ratios D -Lys 50 -mPEG2k1 H NMR spectrum, Figure 2 B is G0-P D -Lys 50 And G0-P with different polyethylene glycol grafting ratios D -Lys 50 -mPEG2k and G0-P D -Lys 50 FT-IR spectrum of

[0041] Figure 3 G0-P with different polyethylene glycol grafting ratios D -Lys 50 -Particle size distribution and surface potential of mPEG2k nanoadjuvant before and after OVA antigen complexing;

[0042] in, Figure 3 A is G0-P D -Lys 50 -Particle size distribution of mPEG2k (4.9%), Figure 3 B is G0-P D -Lys 50 -Particle size distribution of mPEG2k (4.9%) / OVA, Figure 3 C is G0-P D -Lys 50 -Particle size distribution of mPEG2k (9.8%), Figure 3 D is G0-P D -Lys 50 -Particle size distribution of mPEG2k (9.8%) / OVA, Figure 3 E is G0-P D -Lys 50 -Particle size distribution of mPEG2k (14.8%), Figure 3 F is G0-P D -Lys 50 -mPEG2k(14.8%) / OVA particle size distribution, Figure 3 G is G0-P with different polyethylene glycol grafting ratios D -Lys 50 -Surface potential diagram of mPEG2k nanoadjuvant before and after complexing with antigen OVA;

[0043] Figure 4 G0-P D -Lys 50 -mPEG2k(4.9%), G0-P D -Lys 50 -mPEG2k(9.8%), G0-P D -Lys 50-mPEG2k(14.8%), G0-P D -Lys 50 -mPEG1000(5%)、G0-P D -Lys 50 -mPEG750(5%), G0-P D -Lys 50 Figure 2 shows the results of cytotoxicity experiments on DC cells;

[0044] in, Figure 4 A~C are G0-P D -Lys 50 -mPEG2k(4.9%), G0-P D -Lys 50 -mPEG2k(9.8%), G0-P D -Lys 50 -mPEG2k (14.8%) cytotoxicity test results on DC cells; Figure 4 D is G0-P D -Lys 50 -The results of the cytotoxicity experiment of mPEG1000 (5%) on DC cells; Figure 4 E is G0-P D -Lys 50 -The results of the cytotoxicity experiment of mPEG750 (5%) on DC cells; Figure 4 F is G0-P D -Lys 50 Figure 2 shows the results of cytotoxicity experiments on DC cells;

[0045] Figure 5 G0-P D -Lys 50 -mPEG2k(4.9%), G0-P D -Lys 50 -mPEG2k(9.8%), G0-P D -Lys 50 -Drug loading efficiency diagram of nanovaccine obtained at different mass ratios of mPEG2k (14.8%) and OVA;

[0046] Figure 6 G0-P D -Lys 50 -mPEG2k(4.9%) / OVA group, G0-P D -Lys 50 -mPEG2k(9.8%) / OVA group, G0-P D -Lys 50-The stimulation effect of mPEG2k (14.8%) / OVA group, PBS group, and OVA group on BMDC;

[0047] in, Figure 6 A to D correspond to the proportion of cells expressing CD80, CD40, MHCⅡ, and TNF-α, respectively;

[0048] Figure 7 G0-P D -Lys 50 -mPEG2k(4.9%) / OVA group, G0-P D -Lys 50 -mPEG2k(9.8%) / OVA group, G0-P D -Lys 50 -mPEG2k (14.8%) / OVA group, PBS group, OVA group and G0-P D -Lys 50 In vivo imaging of animals in the / OVA group and data of relative fluorescence intensity in mouse lymph nodes;

[0049] in, Figure 7 A In vivo imaging of animals in different groups; Figure 7 B is the relative fluorescence intensity data of different groups in mouse lymph nodes;

[0050] Figure 8 G0-P D -Lys 50 -mPEG2k (4.9%) / OVA group, PBS group, OVA group and G0-P D -Lys 50 The effect of the / OVA group on B16 tumor treatment. DETAILED DESCRIPTION

[0051] The following will clearly and completely describe the technical solutions of the present invention in conjunction with the embodiments of the present invention. Obviously, the embodiments described are only some embodiments of the present invention, not all 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] To address the problems in the prior art of poor tumor vaccine mobility in the extracellular matrix and low passive transport efficiency to lymph nodes, the present invention provides a polyethylene glycol-modified polylysine comprising a dendrimer initiator and polylysine connected by amide bonds.

[0053] In the present invention, the presence of a dendrimer initiator can adjust the dispersibility of the nanoparticles in solution. This initiator is generally formed by the loss of a hydrogen atom from the terminal amino group of the dendrimer. Preferably, the dendrimer comprises polyamidoamine (PAMAM), which offers advantages over other dendrimers, such as polyethyleneimine, due to its lower cost and higher number of surface reactive groups. In the present invention, the polyamidoamine is preferably of generation 0 to 5, such as 0, 1, 2, 3, 4, and 5, with generation 0 being more preferred. This enhances the stability of the nanoadjuvant in PBS or water. In other words, in some embodiments of the present invention, the dendrimer is preferably PAMAM-GO.

[0054] In the present invention, the presence of polylysine can regulate and activate relevant immune cells.

[0055] In the present invention, the lysine in the polylysine structural unit can be chiral ("D", "L") lysine, or achiral lysine, preferably chiral ("D") lysine. Compared with achiral and chiral ("L") lysine, the prepared polyethylene glycol-modified polylysine can better promote the antigen presentation function of immune cells.

[0056] In the present invention, polyethylene glycol is grafted onto the side chain of the polylysine. The introduction of the polyethylene glycol can adjust the charge density, thereby promoting the migration of the material to the lymph nodes in the body.

[0057] In the present invention, the molecular weight of the polyethylene glycol affects the size of the nanoparticles. Therefore, the molecular weight of the polyethylene glycol is preferably 750 to 2000 Da, such as 750, 1000, 1200, 1500, 1800 or 2000 Da, and more preferably 2000 Da.

[0058] In the present invention, the grafting rate of the polyethylene glycol cannot be too high, otherwise the material will be metabolized too quickly in the organism and lose its effect. At the same time, it cannot be too low, otherwise the effect of regulating the charge density cannot be achieved. Therefore, the grafting rate of the polyethylene glycol in the present invention is preferably 5-15%, such as 5%, 8%, 10%, 12%, 15%, etc., more preferably 5%.

[0059] In some specific preferred embodiments of the present invention, the polyethylene glycol-modified polylysine has the structural formula shown in the following formula I:

[0060] Formula I;

[0061] Here, x = any number from 2 to 8, y = any number from 42 to 48, and x + y = n = 50, which is the number of lysine structural units in polylysine. For example, if x = 2, then y = 48; or if x = 5, then y = 45; or if x = 8, then y = 42. The same applies to other values ​​of x and y.

[0062] It should be noted that the number of lysine in the structural unit of polylysine affects the effect of activating immune cells. Therefore, after screening, the present invention preferably selects x+y=n=50.

[0063] The present invention performs SEM characterization on the polyethylene glycol-modified polylysine, and the results show that the polyethylene glycol-modified polylysine has a spherical appearance and a particle size between 10 and 100 nm, such as 10, 20, 30, 40, 50, 60, 70, 80, 90 or 100 nm.

[0064] The point values ​​listed above in the present invention are only for illustration purposes, and other point values ​​within the corresponding numerical range are applicable and will not be repeated here.

[0065] The present invention also provides a method for preparing the polyethylene glycol-modified polylysine, comprising the following steps:

[0066] S1: amino protected lysine- N - Internal carboxylic acid anhydride is mixed with the dendrimer and undergoes ring-opening polymerization, and after deprotection, an intermediate is obtained;

[0067] S2: reacting the intermediate with mPEG-COOH to obtain polyethylene glycol-modified polylysine.

[0068] According to the present invention, first, the amino-protected lysine- N - carboxylic acid anhydride is mixed with the dendrimer and undergoes ring-opening polymerization, and after deprotection, an intermediate is obtained. N -internal carboxylic acid anhydride (abbreviated as: Lys NCA), preferably D-lysine with protected amino group- N -internal carboxylic acid anhydride (abbreviated as: D-Lys NCA), more preferably D-lysine protected by benzyloxycarbonyl- N - internal carboxylic anhydride (abbreviated as: Cbz- D -Lys NCA), the Cbz- D The molar ratio of -Lys NCA to dendrimer is (10-200):1, preferably (30-100):1, more preferably 50:1. D -Lys NCA and dendritic polymer are mixed in the above ratio and undergo ring-opening polymerization. The ring-opening polymerization reaction is carried out in the presence of a solvent, which can be anhydrous dichloromethane, anhydrousN , N -dimethylformamide, etc. In the present invention, the ring-opening polymerization reaction can be carried out at room temperature, 10-30°C, preferably 15-25°C, and for 0.5-6 hours, preferably 1-5 hours, and more preferably 2-4 hours. After the ring-opening reaction is completed, deprotection is performed to obtain an intermediate. The present invention does not particularly limit the deprotection method and can be carried out according to methods well known to those skilled in the art.

[0069] In the present invention, the above-mentioned Cbz- D -Lys NCA can be prepared according to methods well known to those skilled in the art, such as:

[0070] In tetrahydrofuran solvent, Cbz- D -Lys and triphosgene react at 55-70°C for 2-5 hours. After the reaction is complete, the product is precipitated with ice petroleum ether and filtered to obtain a crude product. Further, the crude product is dissolved in cold ethyl acetate and transferred to a separatory funnel. Then, the insoluble impurities and by-products are washed with a cold saturated sodium chloride aqueous solution to purify the obtained carboxylic anhydride solution. Anhydrous magnesium sulfate is then added to dry the above compound solution for 12-24 hours. Finally, the anhydrous magnesium sulfate is removed by filtration and the solution is dried to obtain pure Cbz- D -Lys NCA product.

[0071] Illustratively, in a specific embodiment of the present invention, the intermediate is prepared according to the following method:

[0072] Cbz- D -Lys NCA is placed in a dry round-bottom flask and a solvent, preferably anhydrous dichloromethane, is added and stirred to dissolve. PAMAM-G0 initiator has four primary amine groups. PAMAM-G0 initiator is added at the desired monomer / initiator ratio and the reaction is carried out at 10-30°C for 0.5-6 hours. After the reaction is completed, the product is precipitated with ice-cold anhydrous ether and centrifuged to dry to obtain the undeprotected G0-P(Cbz- D -Lys 50 ) (ie, in Cbz- D -Lys NCA in the presence of PAMAM-GO initiator, the product obtained by ring-opening polymerization). Weigh the above undeprotected dendrimer G0-P D -Lys 50Pour the mixture into a clean flask, add dichloromethane and stir to dissolve. After complete dissolution, add trimethylsilyl iodide to form a slightly yellow solution. The reaction is carried out at 10-30°C for 12-36 hours. After the reaction is completed, remove the solvent in vacuo, add saturated NaHCO3 solution and deionized water to dissolve the residue, and add a small amount of NaS2O3 to form a colorless solution. After the residue is completely dissolved, wash the aqueous phase with 3×50 mL (wash 3 times, 50 mL each time) of anhydrous ether to remove iodobenzyl. Transfer the resulting colorless aqueous solution to a dialysis bag with a molecular weight cutoff of 3500-5000 Da and dialyze against deionized water for 3-5 days. D -Lys 50 After freeze-drying, the product becomes a white powder, i.e., an intermediate.

[0073] After obtaining the intermediate, the intermediate is reacted with mPEG-COOH according to the present invention to obtain polyethylene glycol-modified polylysine. In this step, the amino group of the intermediate undergoes a condensation reaction with the carboxyl group of mPEG-COOH, wherein the molar ratio of the intermediate to mPEG-COOH is (6~20):1, preferably (10~20):1, and more preferably 20:1. The reaction can be carried out at room temperature, the temperature is 10~30°C, preferably 15~25°C; the time is 1~7 days, preferably 1~5 days, and more preferably 3 days. In the present invention, the reaction is carried out in the presence of a catalyst, and the catalyst preferably includes 1-ethyl-(3-dimethylaminopropyl)carbodiimide (abbreviated as: EDC) and N -Hydroxysuccinimide (abbreviation: NHS).

[0074] For example, in a specific embodiment of the present invention, polyethylene glycol-modified polylysine is prepared according to the following method:

[0075] G0-P was washed with deionized water D -Lys 50 Dissolve mPEG-COOH (preferably mPEG2k-COOH) in a round-bottom flask. Then, dissolve EDC and NHS in the solution and allow to react at room temperature for 1-5 days, preferably 3 days. The resulting product is dialyzed for 3-5 days using a dialysis bag with a molecular weight cutoff of 3500-5000 Da. Change the dialysis water 3-5 times per day. Lyophilize the solution using a freeze dryer to obtain a white solid, namely, polyethylene glycol-modified polylysine.

[0076] In the above scheme, the present invention can obtain polyethylene glycol-modified polylysine with different polyethylene glycol grafting rates by controlling the amount of mPEG-COOH.

[0077] It should be noted that the mPEG-COOH can be prepared according to methods well known to those skilled in the art, such as:

[0078] Add mPEG to a round-bottom flask. Then, add dichloromethane and pyridine and stir until the mPEG2k is fully dissolved. Once dissolved, add succinic anhydride to the flask and allow to react for 1-5 days, preferably 3 days. After the reaction, precipitate the product with anhydrous ether. Dissolve the resulting crude product in water and dialyze it in a dialysis bag with a molecular weight cutoff of 1000-2000 Da for 3-5 days. Lyophilize the solution using a freeze dryer to obtain the pure product, mPEG-COOH.

[0079] The preparation method of the polyethylene glycol-modified polylysine provided by the present invention is simple and easy to implement, and is conducive to large-scale production or industrial production.

[0080] The present invention also provides a nano adjuvant, which comprises the polyethylene glycol-modified polylysine and a solvent.

[0081] In the present invention, the solvent is selected from water or PBS buffer solution. Considering the subsequent application in the preparation of tumor prevention vaccines for human body, PBS buffer solution is preferred.

[0082] In the present invention, the concentration of the nanoadjuvant is 0.1-5 mg / mL, preferably 0.5-3 mg / mL, and more preferably 1-1.5 mg / mL.

[0083] The present invention also provides a nano vaccine, which is obtained by combining the polyethylene glycol-modified polylysine or nano adjuvant with an antigen.

[0084] In the present invention, the antigen can be selected from chicken ovalbumin (abbreviated as: OVA), tumor extract or tumor cell membrane extract.

[0085] In the present invention, the mass ratio of the antigen to polyethylene glycol-modified polylysine is 1:(1-10), such as 1:1, 1:2, 1:3, 1:4, 1:5, 1:6, 1:7, 1:8, 1:9 or 1:10.

[0086] Taking chicken ovalbumin as an example, the present invention explores the effect of different mass ratios of chicken ovalbumin to polyethylene glycol-modified polylysine on the chicken ovalbumin loading rate in polyethylene glycol-modified polylysine with polyethylene glycol grafting rates of 5%, 10%, and 15%. It was found that when the mass ratio of the two was 1:5, the chicken ovalbumin loading rates of the polyethylene glycol-modified polylysine obtained at the above three grafting rates were not much different. Therefore, the preferred mass ratio of chicken ovalbumin to polyethylene glycol-modified polylysine is 1:5.

[0087] The present invention regulates the size of nanoparticles by changing the density of modified polyethylene glycol. Taking chicken egg white albumin as an example, the nano vaccine obtained after compounding chicken egg white albumin has the function of promoting antigen presenting cell endocytosis and migration to lymph nodes to activate immune cells in lymph nodes. It can stimulate dendritic cells (DC cells) to mature and release inflammation-related 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. The nano vaccine provided by the present invention has a better migration effect in vivo than traditional tumor vaccines, allowing the vaccine to better aggregate in the lymph nodes, thereby promoting immune cell activation, maturation and differentiation, solving the problem that other vaccines in the research field are easily retained at the injection site and have poor immune regulation ability.

[0088] Based on this, the present invention also provides a use of the above-mentioned polyethylene glycol-modified polylysine, nanoadjuvant or nanovaccine in the preparation of a vaccine for preventing tumors.

[0089] The tumor includes but is not limited to melanoma (B16) and Lewis lung carcinoma (LLC).

[0090] To further illustrate the present invention, the following examples are provided for detailed description. The PAMAM-GO initiator used in the following examples of the present invention was purchased from Aladdin, with a molecular weight of 715.6; OVA was purchased from Sigma, model C0112-1g; CCK8 kit was purchased from Beyotime; BSA protein loading kit was purchased from Biolegend; DC2.4 cell cryopreservation solution was purchased from Cellorlab; C57BL / 6N female mice were provided by Beijing Weitong Lihua; the following P D -Lys 50 Refers to polylysine.

[0091] Example 1-G0-P D Synthesis of -Lys50-mPEG2k

[0092] (1) Preparation of modified monomethoxy polyethylene glycol with a molecular weight of 2000. The synthetic route is as follows: Figure 1 As shown in Figure A, the process is as follows: 4 g of mPEG2k was added to a 200 mL round-bottom flask. Then, 40 mL of dichloromethane and 20 mL of pyridine were added and stirred until the mPEG2k was fully dissolved. Once dissolved, 16 g of succinic anhydride was added to the flask and allowed to react for 3 days. After the reaction, the product was precipitated with 200 mL of anhydrous ether. The resulting crude product was dissolved in water and dialyzed in a dialysis bag with a molecular weight cutoff of 1000 Da for 5 days. The solution was lyophilized to obtain the pure product, mPEG2k-COOH.

[0093] Meanwhile, 18 g Cbz- D -Lys and 9 g of triphosgene were reacted at 55 ° C for 2 h. After the reaction was complete, the product was precipitated with ice petroleum ether and filtered to obtain a crude product. Further, the crude product was dissolved in cold ethyl acetate and transferred to a separatory funnel. Then, the insoluble impurities and by-products were washed with a cold saturated sodium chloride aqueous solution to purify the obtained carboxylic anhydride solution. Then, anhydrous magnesium sulfate was added to dry the above compound solution for 12 h. Finally, the anhydrous magnesium sulfate was removed by filtration, and the solution was dried to obtain pure Cbz- D -LysNCA product, see route Figure 1 A.

[0094] (2) Preparation of G0-P D -Lys 50 , the synthetic route is as follows Figure 1 As shown in B, the process is as follows: Cbz- D -Lys NCA (0.46 g, 2.0 mmol) was weighed into an oven-dried round-bottom flask and dissolved in 10 mL of anhydrous dichloromethane. PAMAM-G0 initiator (1.68 mg, 8 μmol) was added at the desired monomer / initiator ratio, and the reaction was carried out at 25°C for 2 h. After the reaction, the product was precipitated with ice-cold anhydrous ether and centrifuged to dryness, yielding the undeprotected G0-P(Cbz- D -Lys) 50 Weigh the undeprotected dendrimer G0-P(Cbz- D -Lys) 50 To a clean flask, add 10 mL of dichloromethane and stir to dissolve. After complete dissolution, add trimethylsilyl iodide (180 μL, 1.25 mmol), 6 equivalents for each benzyloxycarbonyl group, to form a slightly yellow solution. The reaction was carried out at 25 ° C for 24 h. After the reaction was completed, the solvent was removed in vacuo, and saturated sodium bicarbonate (NaHCO3) solution (4 mL) and deionized water (4 mL) were added to dissolve the residue. A small amount of sodium thiosulfate (NaS2O3) was added to form a colorless solution. After the residue was completely dissolved, the aqueous phase was washed with 3×50 mL of anhydrous ether to remove iodobenzyl. The resulting colorless aqueous solution was transferred to a dialysis bag with a molecular weight cutoff of 3500 Da and dialyzed against deionized water for 3 days. The deprotected G0-P D -Lys 50 After freeze-drying, it becomes white powder.

[0095] (3) Preparation of the final product G0-P by EDC / NHS reaction D -Lys 50-mPEG2k, synthetic route as Figure 1 C, the process is as follows: 1.0 g G0-P was added to 50 mL of deionized water. D -Lys 50 0.2, 0.3, and 0.4 g of mPEG2k-COOH were dissolved in a 100 mL round-bottom flask, and then 3.2 g of EDC and 0.8 g of NHS were dissolved in it and allowed to react at room temperature for 3 days. The resulting product was dialyzed for 5 days using a dialysis bag with a molecular weight cutoff of 3500 Da. The dialysis water was changed five times a day. The solution was freeze-dried using a freeze dryer to obtain white solids with a graft ratio of 5%, 10%, and 15% of G0-P00303. D -Lys 50 -mPEG2k (abbreviated as G0-P D -Lys 50 -mPEG2k(4.9%), G0-P D -Lys 50 -mPEG2k(9.8%), G0-P D -Lys 50 -mPEG2k (14.8%)).

[0096] In the present invention, the above-mentioned grafted mPEG2k-COOH ratio is calculated by dividing the molar mass of the input mPEG2k-COOH by the input intermediate product (i.e., G0-P D -Lys 50 ) is calculated from its molar mass.

[0097] The G0-P D -Lys 50 -mPEG2k(4.9%), G0-P D -Lys 50 -mPEG2k(9.8%), G0-P D -Lys 50 The structural formulas of -mPEG2k (14.8%) are shown in Formula I, wherein G0-P D -Lys 50 -mPEG2k (4.9%), x = 2, y = 48, n = 50; G0-P D -Lys 50 -mPEG2k (9.8%), x = 5, y = 45, n = 50; G0-P D -Lys50-mPEG2k (14.8%), x=7, y=43, n=50.

[0098] Example 2 - Preparation of Nanoadjuvants and Nanovaccines

[0099] Preparation of nanoadjuvant: Weigh G0-P D -Lys 50 -mPEG2k(4.9%), G0-P D -Lys 50 -mPEG2k (9.8%) or G0-P D -Lys 50 -mPEG2k (14.8%) was added to a clean glass vial and PBS was added to prepare 1, 1.2, and 1.4 mg / mL PBS solutions, namely G0-P D -Lys 50 -mPEG2k (4.9%) corresponds to 1 mg / mL PBS solution, G0-P D -Lys 50 -mPEG2k (9.8%) corresponds to 1.2 mg / mL PBS solution, G0-P D -Lys 50 -mPEG2k (14.8%) corresponds to 1.4 mg / mL PBS solution. Then the vial was ultrasonicated (the ultrasonic temperature was 25 ° C, the time was 30 min, and the power was 0.45 KW) to obtain the nano adjuvant G0-P D -Lys 50 -mPEG2k(4.9%), G0-P D -Lys 50 -mPEG2k (9.8%) or G0-P D -Lys 50 -mPEG2k (14.8%).

[0100] Preparation of nano vaccine: At 4 °C, the nano vaccine G0-P was prepared by combining OVA (chicken egg white albumin) solution with nano adjuvants in different proportions. D -Lys 50 -mPEG2k / OVA, in which G0-P D -Lys 50 -The mass ratios of mPEG2k to OVA were 1:1, 3:1, 5:1, 7:1, and 9:1.

[0101] Characterization

[0102] The present invention performs nuclear magnetic resonance hydrogen spectroscopy on the product obtained in Example 1 ( 1 H NMR) and infrared spectroscopy (FT-IR), such as Figure 2 A and 2B. Wherein, different grafting ratios G0-P in Example 1 D -Lys 50 -mPEG2k 1 H NMR spectrum, such as Figure 2 As shown in A, it is proved that G0-P D -Lys 50 -The successful synthesis of mPEG2k; G0-P in Example 1 D -Lys 50 And different grafting ratios G0-P D -Lys 50 -FT-IR spectrum of mPEG2k Figure 2 As shown in B, it proves that mPEG2k-COOH has been successfully grafted onto G0-P D -Lys 50 .

[0103] The present invention took 50 μL of the nanoadjuvant and nanovaccine obtained in Example 2 and dripped them onto a special copper grid. After air-drying in a cool place, the particle size of the nanoadjuvant and nanovaccine was tested by transmission electron microscopy (TEM). At the same time, the present invention took 1 mL of the nanoadjuvant and nanovaccine solution obtained in Example 2 and placed it in a quartz dish for dynamic light scattering (DLS) testing to obtain the hydrated particle size and surface potential of the nanoadjuvant and nanovaccine. The test results are as follows: Figure 3 As shown in A~G.

[0104] Depend on Figure 3 It can be seen that the present invention has successfully synthesized nano-adjuvants and nano-vaccines with uniform particle size within 10-100 nm. Figure 3 The nanoadjuvant in the D -Lys 50 -mPEG2k(4.9%), G0-P D -Lys 50 -mPEG2k (9.8%) or G0-P D -Lys 50 -mPEG2k (14.8%), nanovaccine is expressed as G0-P D -Lys 50 -mPEG2k(4.9%) / OVA, G0-P D -Lys 50 -mPEG2k (9.8%) / OVA or G0-P D -Lys 50 -mPEG2k(14.8%) / OVA.

[0105] Cytotoxicity assay

[0106] In order to explore whether the cytotoxicity of different nanovaccines to DC is too strong, the present invention uses CCK8 kit to detect the cytotoxicity of nanovaccines. First, the DC2.4 cell line was cultured. The purchased DC2.4 cell cryopreservation solution was prepared into a single cell suspension and then cultured in RPMI 1640 medium. 10% heat-inactivated fetal bovine serum and 1% penicillin-streptomycin solution were added to the medium. The adherent cells were collected in the second week for subsequent experiments. 5×10 cells were added to each well in a 96-well plate. 3 DC2.4 cells were inoculated at a density of 10 cells and different concentrations of nanoadjuvants (G0-P D -Lys 50 -mPEG2k(4.9%), G0-P D -Lys 50 -mPEG2k(9.8%), G0-P D -Lys 50 -mPEG2k(14.8%), G0-P D -Lys 50 -mPEG1000(5%)、G0-P D -Lys 50 -mPEG750(5%), G0-P D -Lys 50 ) solution, and a PBS control group was set up at the same time. After 24 hours of incubation, 10 μL of CCK8 reagent was added to each well and incubated for another 2 hours. The absorbance of each well was then measured at a wavelength of 450 nm using a microplate reader. By comparing the absorbance of each group with that of the blank control group, the cell survival rate was calculated to evaluate the cytotoxicity of different nanovaccines. Figure 4 The results showed that the cell survival rate of the nano vaccine grafted with 2000 molecular weight polyethylene glycol was higher than that of the other experimental groups, indicating that G0-P D -Lys 50 -mPEG2k(4.9%), G0-P D -Lys 50 -mPEG2k (9.8%) or G0-P D -Lys 50 -mPEG2k (14.8%) has low toxicity to DC2.4 cells and good biocompatibility, providing a reliable basis for subsequent animal experiments and clinical applications.

[0107] OVA drug loading rate test

[0108] In order to determine the drug loading efficiency of different nano-vaccines loaded with OVA, the present invention uses a BSA protein loading kit to test it, and the specific steps are as follows:

[0109] First, a certain amount of nanoadjuvants (G0-P D -Lys 50 -mPEG2k(4.9%), G0-P D -Lys 50 -mPEG2k(9.8%), G0-P D -Lys 50 -mPEG2k (14.8%)) and dispersed it in an appropriate amount of PBS buffer to ensure that the adjuvant was evenly dispersed, and finally obtained nanoadjuvant dispersion solutions with concentrations of 1, 1.2, and 1.4 mg / mL. D -Lys 50 -mPEG2k(4.9%), G0-P D -Lys 50 -mPEG2k(9.8%), G0-P D -Lys 50 -mPEG2k (14.8%):OVA mass ratio = 1:1, 3:1, 5:1, 7:1, 9:1, OVA antigen solution was added to the nanoadjuvant dispersion solution, and stirred under mild conditions for 0.5 h to ensure that the OVA antigen was fully loaded on the nanoadjuvant.

[0110] Then, using the BSA protein loading kit from Beyotime, follow the instructions to test the loaded solution. By measuring the concentration of unloaded OVA antigen, the amount of OVA actually loaded on the nanovaccine can be calculated, thereby obtaining the drug loading efficiency. The calculation formula is as follows:

[0111] Drug loading efficiency = 1 - the mass of free OVA in the supernatant of the loaded solution / the mass of total OVA.

[0112] The results are as follows Figure 5 As shown, it can be seen that the G0-P prepared by the present invention D -Lys 50 -mPEG2k(4.9%), G0-P D -Lys 50 -mPEG2k(9.8%), G0-P D -Lys 50 When the mass ratio of -mPEG2k (14.8%) to OVA was 5:1, the three nanovaccines obtained, namely, G0-P D -Lys 50 -mPEG2k(4.9%) / OVA, G0-P D -Lys 50 -mPEG2k(9.8%) / OVA, G0-P D -Lys50 -mPEG2k (14.8%) / OVA has a similar drug loading efficiency, which can make the injection amount of vaccine in subsequent experiments approximately the same, providing strong support for subsequent vaccine preparation and application.

[0113] Functional Verification

[0114] The bone marrow-derived dendritic cell (BMDC) activation experiment verified that the vaccine antigen promoted the maturation and activation of immune cells. The specific steps are as follows:

[0115] Extraction and culture of BMDC: BMDC were isolated from the bone marrow cells of 6-8 week old C57BL / 6N female mice. The femur and tibia of the mice were dissected and the bone marrow was collected. The single cell suspension was prepared and cultured in RPMI 1640 medium supplemented with 10% heat-inactivated fetal bovine serum, 1% penicillin-streptomycin solution, 20 ng / mL GM-CSF and 10 ng / mL IL-4. Non-adherent cells and loosely adherent cells on the 7th day of differentiation were collected for subsequent studies. In a 24-well plate (6 columns, 4 wells per column), 200,000 BMDC were added to each well, and then G0-P was added to each well of the 3 columns in sequence. D -Lys 50 -mPEG2k(4.9%) / OVA, G0-P D -Lys 50 -mPEG2k(9.8%) / OVA, G0-P D -Lys 50 -mPEG2k (14.8%) / OVA nanovaccine was added to the cells, resulting in a final concentration of 4 μg / mL. Simultaneously, PBS and OVA solutions of the same concentration were added to the remaining two columns of wells as controls, designated the PBS and OVA groups. The 24-well plates were placed in a 37°C incubator for 24 hours. Cell supernatants were collected, and a portion was incubated with anti-CD11c, anti-CD80, and anti-MHC-II for 30 minutes at 4°C to analyze BMDC activation and antigen cross-presentation. Flow cytometric data were acquired on a BD-FACS Celesta and analyzed using FlowJo software. Other supernatants were analyzed by enzyme-linked immunosorbent assay (ELISA) to measure TNF-α and IL-6 levels.

[0116] The experimental results are as follows Figure 6 As shown in the figure, BMDC activation markers CD80 and MHC-II increased, proving that the nanovaccine activated DC cells and caused antigen cross-presentation by DC cells. Among them, the nanovaccine prepared by mixing the material with a 5% polyethylene glycol grafting ratio and OVA had the strongest effect on DC cell activation and antigen cross-presentation.

[0117] The present invention also verifies the process of nano-vaccine migration and retention to lymph nodes in vivo through in vivo imaging, proving that nano-vaccine can effectively migrate to lymph nodes ( Figure 7 ). The verification process is as follows:

[0118] OVA and the purchased experimental fluorescent dye Cy5.5-NHS were mixed at a molar ratio of 50:1 and stirred for 24 h. The mixture was then dialyzed for 3 days using a dialysis bag with a cut-off molecular weight of 3500 Da. After lyophilization, OVA-Cy5.5 was obtained for subsequent experiments. In the present invention, 6-8 week old C57BL / 6N female mice weighing 17-22 g were used as experimental subjects. At 0 h, 40 μL of PBS, OVA-Cy5.5, G0-P were injected into the footpad of the mice. D -Lys 50 / OVA-Cy5.5 nanovaccine (G0-PDLys 50 The mass ratio of G0-P to OVA-Cy5.5 is 5:1), D -Lys 50 -mPEG2k(4.9%) / OVA-Cy5.5, G0-P D -Lys 50 -mPEG2k(9.8%) / OVA-Cy5.5, G0-P D -Lys 50 -mPEG2k(14.8%) / OVA-Cy5.5 nanovaccine(G0-P D -Lys 50 -mPEG2k(4.9%), G0-P D -Lys 50 -mPEG2k(9.8%), G0-P D -Lys 50 -mPEG2k (14.8%) and OVA-Cy5.5 mass ratio was 5:1), OVA-Cy5.5, nano vaccine injection dose was 20 μg per mouse. Subsequently, at 6, 12, and 24 hours, the distribution of nano vaccine in mice was photographed and data processed using an optical living imager and Living Image software. Figure 7 The experimental results showed that the nano vaccine gradually migrated from the mouse foot pad to the mouse inguinal lymph node over time, reaching a peak at 12 hours and then gradually metabolized out of the body. The nano vaccine prepared by mixing the material with a polyethylene glycol grafting rate of 5% with OVA (i.e., G0-P D -Lys 50-mPEG2k(4.9%) / OVA-Cy5.5) has better migration effect in the body and longer residence time in lymph nodes than other vaccines.

[0119] In this study, 6-8 week-old C57BL / 6N female mice weighing 17-22 g were used as experimental subjects to prepare a B16-OVA tumor model. After the mouse model was successfully constructed, B16-OVA cells (2.0×10 5 Cells) were subcutaneously inoculated into the left dorsal subcutaneous tissue of 6-8 week old female C57BL / 6N mice. On days 4, 7, 11, and 14, 200 μL of PBS, OVA, G0-P D -Lys 50 -mPEG2k(4.9%) / OVA nanovaccine(G0-P D -Lys 50 -mPEG2k (4.9%) and OVA mass ratio of 5:1) and G0-P D -Lys 50 / OVA nano vaccine (G0-P D -Lys 50 The mice were subcutaneously injected with 100 μg of OVA and nanovaccine (at a mass ratio of 5:1 to OVA) into the right dorsal surface of the mice according to the group. The weight and tumor volume of the mice were then checked every other day. When the tumor volume reached 2000 mm 3 When the skin was severely ulcerated, the mice were killed. Figure 8 .Depend on Figure 8 The results show that injection of G0-P D -Lys50-mPEG2k (4.9%) / OVA nanovaccine can more effectively inhibit tumor growth, with obvious advantages.

[0120] The above description of the disclosed embodiments will enable one skilled in the art to implement or use the present invention. Various modifications to these embodiments will be readily apparent to one skilled in the art, and the general principles defined herein may be implemented in other embodiments without departing from the spirit or scope of the present invention. Therefore, the present invention is not limited to the embodiments shown herein but is to be construed in the widest manner consistent with the principles and novel features disclosed herein.

Claims

1. A polyethylene glycol-modified polylysine, characterized in that: The invention comprises a dendrimer initiator and polylysine connected by an amide bond, wherein polyethylene glycol is grafted on the side chain of the polylysine; The dendrimer initiator is formed by the loss of one hydrogen atom from the terminal amino group of the dendrimer, wherein the dendrimer comprises polyamide-amine; the generation number of the polyamide-amine is 0 to 5; The polyethylene glycol-modified polylysine has the structural formula shown in the following formula I: Formula I; Where x = any number from 2 to 8, y = any number from 42 to 48, and x + y = n = 50.

2. The polyethylene glycol-modified polylysine according to claim 1, characterized in that The grafting rate of the polyethylene glycol is 5-15%.

3. The polyethylene glycol-modified polylysine according to claim 1, characterized in that The polyethylene glycol-modified polylysine is spherical and has a particle size of 10 to 100 nm.

4. A method for preparing polyethylene glycol-modified polylysine according to any one of claims 1 to 3, characterized in that: The following steps are involved: S1: mixing the amino-protected lysine-N-internal carboxylic anhydride with the dendrimer and performing a ring-opening polymerization reaction, and then deprotecting to obtain an intermediate; S2: reacting the intermediate with mPEG-COOH to obtain polyethylene glycol-modified polylysine.

5. The preparation method according to claim 4, characterized in that The temperature of the ring-opening polymerization reaction in step S1 is 10-30° C. and the time is 0.5-6 h; The reaction temperature in step S2 is 10-30° C. and the reaction time is 1-7 days.

6. The preparation method according to claim 4, characterized in that The molar ratio of the amino-protected lysine-N-internal carboxylic acid anhydride to the dendritic polymer is (10-200):1; The molar ratio of the intermediate to mPEG-COOH is (6-20):

1.

7. A nano vaccine, characterized in that: Obtained by complexing the polyethylene glycol-modified polylysine according to any one of claims 1 to 3, the polyethylene glycol-modified polylysine prepared by the preparation method according to any one of claims 4 to 6, or a nanoadjuvant with an antigen; The nanoadjuvant comprises the polyethylene glycol-modified polylysine and a solvent according to any one of claims 1 to 3, or the polyethylene glycol-modified polylysine prepared by the preparation method according to any one of claims 4 to 6, and a solvent.

8. The nanovaccine according to claim 7, characterized in that The solvent is selected from water or PBS buffer solution; The concentration of the nanoadjuvant is 0.1~5 mg / mL; The mass ratio of the antigen to polyethylene glycol-modified polylysine is 1:(1-10); The antigen includes chicken ovalbumin, tumor extract or tumor cell membrane extract.

9. Use of the polyethylene glycol-modified polylysine according to any one of claims 1 to 3, the polyethylene glycol-modified polylysine prepared by the preparation method according to any one of claims 4 to 6, or the nanovaccine according to claim 7 or 8 in the preparation of a vaccine for preventing tumors; The tumor is melanoma.

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