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

Abstract

The invention provides polyethylene glycol modified polylysine, a preparation method of the polyethylene glycol modified polylysine, a nano vaccine and application of the nano vaccine. The polyethylene glycol modified polylysine comprises a dendritic polymer initiator and chiral polylysine which are connected through an amido bond. The polyethylene glycol is grafted on the side chain of the polylysine, so that the charge density can be adjusted, the migration effect of the nanoparticles to lymph nodes is enhanced, and finally, the nano vaccine is prepared by combining the electrostatic adsorption effect with an antigen. Through research, the nano vaccine obtained after antigen compounding has the functions of promoting antigen presenting cells (APCs) to endocytosis and migrating to lymph nodes to activate immune cells in the lymph nodes, can stimulate dendritic cells (DC cells) to mature and release inflammation-related cytokines, and promotes antigen cross presentation of the DC cells in vivo; and a strong antigen-specific immunoreaction is initiated.

Description

Technical Field

[0001] The invention belongs to the technical field of biomedicine, 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 is an effective method for treating cancer. Generally, after subcutaneous or intramuscular injection of tumor vaccines, the vaccine passively migrates through intercellular spaces and lymphatic vessels to reach the lymph nodes, where it will be taken up by antigen-presenting cells and delivered to the lymph nodes, thereby activating the immune cells in the lymph nodes. After maturation, the T lymphocytes will migrate and penetrate into the tumor site, attack tumor cells, and cause tumor cell death. During this period, the substances released by tumor cells will act as antigens and continue to trigger immune responses in the body. Therefore, in the treatment of tumor vaccines, lymph nodes, as the center of differentiation and maturation of immune cells, are critical to the treatment effect.

[0003] At present, in order for tumor vaccines to reach lymph nodes better, it is necessary to break through four barriers of lymph nodes. Barrier 1: The surrounding interstitial cells block the passive transport of tumor vaccines to lymph nodes; Barrier 2: The afferent lymphatic vessels have a small inner diameter, usually only allowing substances around 10 to 100 nm to pass through; Barrier 3: Macrophages in the subcutaneous sinus area of ​​the lymph nodes can clear larger particles in the lymph fluid; Barrier 4: The very small inner diameter of the catheter poses a challenge to reaching the deep areas of the lymph nodes. It can be seen that only by breaking through the above four major physiological barriers of the lymph nodes can tumor vaccines effectively activate the body's own immune ability.

[0004] At present, studies have shown that by changing the size of the material during the migration process, the material can better pass through the lymphatic vessels of the lymph nodes, or by prodrug modification, the material can be specifically aggregated in the lymph node sinus area, or by changing the surface potential, hydrophilicity and other properties of the material to improve the migration efficiency of the material to the lymph nodes. However, although the above methods effectively improve the migration efficiency of the material to the lymph nodes, there are still the following problems: the material has poor movement effect in the intercellular space and low passive transport efficiency to the lymph nodes. Summary of the invention

[0005] In view of this, the object of the present invention is to provide a polyethylene glycol-modified polylysine and a preparation method thereof, a nano vaccine and application thereof. The nano vaccine has a better efficiency of 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 the terminal amino group of a dendrimer losing one hydrogen atom, and the dendrimer comprises polyamide-amine; the generation number of the polyamide-amine is 0 to 5 generations.

[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 a structural formula as shown in the following formula I:

[0013] Formula I;

[0014] Among them, 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 its particle size is 10-100 nm.

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

[0017] S1: amino-protected lysine- N - The internal carboxylic acid anhydride is mixed with the dendritic polymer and undergoes a ring-opening polymerization reaction, and after deprotection, an intermediate is obtained;

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

[0019] Preferably, the temperature of the ring-opening polymerization reaction in step S1 is 10-30° C., and the time is 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-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 nano adjuvant, comprising the polyethylene glycol-modified polylysine mentioned above, 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 nano adjuvant 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 the polylysine as a substrate to prepare a nano adjuvant and a nano vaccine with an immune activation function. The nano vaccine has an immunomodulatory function and a function of activating the body's own immunity, and can be used to prepare a vaccine for preventing tumors. The polyethylene glycol-modified polylysine in the present invention is a nanoparticle, comprising a dendritic polymer initiator and polylysine connected by an amide bond. Among them, the presence of the dendritic polymer initiator can regulate the topological structure of the nano adjuvant, and the presence of the chiral lysine structural unit can regulate the ability of the nano adjuvant to activate immune cells. The present invention further grafts polyethylene glycol on the side chain of polylysine to adjust the charge density, enhance the effect and transport efficiency of the passive migration of nanoparticles to lymph nodes, and finally modify the antigen through electrostatic adsorption to further enhance the antigen cross-presentation of the nanoparticles at 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 diagram of mPEG2k nanoadjuvant before and after OVA antigen complex;

[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 -Particle size distribution of mPEG2k (14.8%) / OVA, 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 the cytotoxicity experiment 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 - Result 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 the cytotoxicity experiment 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 nano-vaccine 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~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 Animal in vivo imaging of 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 / OVA group's treatment effect on B16 tumor. DETAILED DESCRIPTION

[0051] The technical solution of the present invention will be clearly and completely described below in conjunction with the embodiments of the present invention. Obviously, the described embodiments 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 creative work are within the scope of protection of the present invention.

[0052] In view of the problems in the prior art that tumor vaccines have poor mobility in the interstitial matrix and low passive transport efficiency to lymph nodes, the present invention provides a polyethylene glycol-modified polylysine, which includes a dendrimer initiator and polylysine connected by amide bonds.

[0053] In the present invention, the presence of the dendritic polymer initiator can adjust the dispersibility of the nanoparticles in the solution, which is generally formed by the end amino group of the dendritic polymer losing one hydrogen atom. The present invention preferably includes polyamide-amine (PAMAM for short), which has the advantages of low price and more surface reactive groups compared to other dendritic polymers, such as polyethyleneimine. In the present invention, the generation of the polyamide-amine is preferably 0 to 5 generations, such as 0, 1, 2, 3, 4 and 5 generations, more preferably 0 generations, so that the nanoadjuvant can be more stable in PBS or water. That is, in some embodiments of the present invention, the dendritic polymer is preferably PAMAM-GO.

[0054] In the present invention, the presence of polylysine can regulate and activate related 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, which can make the prepared polyethylene glycol-modified polylysine better at promoting the antigen presentation function of immune cells compared with achiral and chiral ("L") lysine.

[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 has an influence on the size of the nanoparticles. Therefore, the molecular weight of the polyethylene glycol is preferably 750-2000 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 it will cause the material to 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] Among them, 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 can be applied to other values ​​of x and y.

[0062] It should be noted that the number of lysine in the structural unit of polylysine has an impact on 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 is spherical in appearance and has 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 enumeration 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 - The internal carboxylic acid anhydride is mixed with the dendritic polymer and undergoes a ring-opening polymerization reaction, 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 -The internal carboxylic acid anhydride is mixed with the dendritic polymer and undergoes a ring-opening polymerization reaction, 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 anhydride (abbreviated as: D-Lys NCA), more preferably D-lysine protected by benzyloxycarbonyl- N -Internal carboxylic anhydride (abbreviation: Cbz- D -Lys NCA), the Cbz- D The molar ratio of Cbz-NCA to dendritic polymer is (10-200):1, preferably (30-100):1, and more preferably 50:1. D -Lys NCA and the dendritic polymer are mixed in the above ratio and undergo a ring-opening polymerization reaction. 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, the temperature is 10-30 ° C, preferably 15-25 ° C; the time is 0.5-6 h, preferably 1-5 h, and more preferably 2-4 h. After the ring-opening reaction is completed, deprotection is carried out to obtain an intermediate. The present invention has no particular limitation on the means of deprotection, and it 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 h. After the reaction is complete, the product is precipitated through ice petroleum ether and the crude product is obtained by suction filtration. Further, the crude product is dissolved in cold ethyl acetate and transferred to a separatory funnel, and 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 h. 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 is added, preferably anhydrous dichloromethane, to stir and dissolve. PAMAM-G0 initiator has 4 primary amine groups. PAMAM-G0 initiator is added in the required monomer / initiator ratio and the reaction is carried out at 10-30°C for 0.5-6 h. After the reaction is completed, the mixture is precipitated with ice-cold anhydrous ether and centrifuged to obtain undeprotected G0-P(Cbz- D -Lys 50 ) (i.e., in Cbz- D -Lys NCA in the presence of PAMAM-G0 initiator, the product obtained by ring-opening polymerization). D -Lys 50To 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 h. 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 (3 times, 50 mL each time) of anhydrous ether to remove iodobenzyl. Transfer the obtained colorless aqueous solution to a dialysis bag with a molecular weight cutoff of 3500-5000 Da and dialyze with deionized water for 3-5 days. G0-P after deprotection D -Lys 50 After freeze-drying, it 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] Illustratively, 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 it and react at room temperature for 1 to 5 days, preferably 3 days. The resulting product is dialyzed for 3 to 5 days using a dialysis bag with a molecular weight cutoff of 3500 to 5000 Da. The dialysis water is changed 3 to 5 times a day. The solution is freeze-dried using a freeze dryer to obtain a white solid, i.e., 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 mPEG2k is fully dissolved. After dissolution, add succinic anhydride to the flask and react for 1 to 5 days, preferably 3 days. After the reaction, precipitate the product with anhydrous ether. The obtained crude product is dissolved in water and poured into a dialysis bag with a molecular weight cutoff of 1000 to 2000 Da for 3 to 5 days. Freeze-dry the solution with 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 preparing 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 compounding the polyethylene glycol-modified polylysine or nano adjuvant with an antigen.

[0084] In the present invention, the antigen may 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 egg white albumin as an example, the present invention explores the effects of different mass ratios of chicken egg white albumin to polyethylene glycol-modified polylysine on the loading rate of chicken egg white albumin in polyethylene glycol-modified polylysine with polyethylene glycol grafting rates of 5%, 10% and 15%, and finds that when the mass ratio of the two is 1:5, the chicken egg white albumin loading rates of the polyethylene glycol-modified polylysine obtained at the above three grafting rates are not much different. Therefore, the preferred mass ratio of chicken egg white albumin 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 in the present invention has the function of promoting antigen presenting cell endocytosis and migration to lymph nodes to activate immune cells in lymph nodes, and can stimulate dendritic cells (DC cells) to mature and release inflammation-related cytokines, such as IL-6, IL-12, IFN-γ and TNF-α, etc., 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, so that the vaccine can better gather at the lymph nodes, thereby promoting immune cell activation, maturation and differentiation, and solving the problem that other vaccines in the research field are easily retained at the injection site and have poor immunoregulatory ability.

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

[0089] The tumors include but are not limited to melanoma (B16) and Lewis lung carcinoma (LLC).

[0090] To further illustrate the present invention, the following examples are used to describe the invention in detail. 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, with a model of 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 is added to a 200 mL round-bottom flask. Then, 40 mL of dichloromethane and 20 mL of pyridine are added and stirred until mPEG2k is fully dissolved. After dissolution, 16 g of succinic anhydride is added to the flask and reacted for 3 days. After the reaction, the product is precipitated with 200 mL of anhydrous ether. The obtained crude product is dissolved in water and poured into a dialysis bag with a molecular weight cutoff of 1000 Da for 5 days. The solution is freeze-dried with a freeze dryer to obtain the pure product mPEG2k-COOH.

[0093] Meanwhile, 18 g Cbz- D -Lys and 9 g triphosgene were reacted at 55 °C for 2 h. After the reaction was complete, the product was precipitated by ice petroleum ether and the crude product was obtained by suction filtration. Further, the crude product was dissolved in cold ethyl acetate and transferred to a separatory funnel, and 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 Figure 1 A.

[0094] (2) Preparation of G0-P D -Lys 50 , the synthetic route is as follows Figure 1 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 10 mL of anhydrous dichloromethane was added for stirring and dissolution. PAMAM-G0 initiator has 4 primary amine groups. PAMAM-G0 initiator (1.68 mg, 8 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, it was precipitated with ice-cold anhydrous ether and centrifuged to obtain the undeprotected G0-P(Cbz- D -Lys) 50 Weigh the undeprotected dendritic 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, saturated sodium bicarbonate (NaHCO3) solution (4 mL) and deionized water (4 mL) were added to dissolve the residue, and 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 obtained colorless aqueous solution was transferred to a dialysis bag with a molecular weight cutoff of 3500 Da and dialyzed with deionized water for 3 days. The deprotected G0-P D -Lys 50 After freeze-drying, it is 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 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 reacted at room temperature for 3 days. The obtained product was dialyzed for 5 days with 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 with a freeze dryer, and the white solids obtained were G0-P with a grafting ratio of 5%, 10%, and 15% of mPEG2k-COOH. 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 Nanoadjuvant and Nanovaccine

[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 subjected to ultrasonic treatment (the ultrasonic temperature was 25°C, the time was 30 min, and the power was 0.45 KW), and the nanoadjuvants G0-P were obtained respectively. 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, where 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 spectrum ( 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 -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 takes 50 μL of the nanoadjuvant and nanovaccine obtained in Example 2 and drips them onto a special copper mesh. After air-drying in a cool place, the particle size of the nanoadjuvant and nanovaccine is tested by transmission electron microscopy (TEM). At the same time, the present invention takes 1 mL of the nanoadjuvant and nanovaccine solution obtained in Example 2 and puts them into a quartz dish for dynamic light scattering (DLS) test 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 successfully synthesized nano-adjuvants and nano-vaccines with uniform particle sizes 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 denoted 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, and 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, and the adherent cells in the second week were collected for subsequent experiments. 5×10 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 set up a PBS control group. After 24 hours of incubation, 10 μL of CCK8 reagent was added to each well, and the incubation continued for 2 hours. Then, the absorbance value of each well was measured at a wavelength of 450 nm using an enzyme reader. By comparing the absorbance value 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 is evenly dispersed, and finally obtained a nanoadjuvant dispersion solution with a concentration 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 allow the OVA antigen to be fully loaded on the nanoadjuvant.

[0110] Then, use the BSA protein loading kit of Beyotime and follow the operating steps in the manual 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 loading 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 the 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. After preparing into a single cell suspension, it was cultured in RPMI 1640 medium and 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 BMDCs 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 make the final concentration of the nanovaccine 4 μg / mL. At the same time, PBS solution and OVA solution of the same concentration were used as the control group and added to the remaining 2 columns of wells, referred to as the PBS group and the OVA group. The 24-well plate was placed in a 37°C constant temperature incubator for 24 hours. The cell supernatant was collected, and a portion was incubated with anti-CD11c, anti-CD80, and anti-MHC-Ⅱ at 4°C for 30 minutes to analyze the activation of BMDC and the effect of antigen cross-presentation. The samples were used to obtain flow data on a flow cytometer (BD-FACS Celesta) and analyzed by FlowJo software; another portion of the supernatant was analyzed by enzyme-linked immunosorbent assay (ELISA) to measure the levels of cytokines TNF-α and IL-6.

[0116] The experimental results are as follows Figure 6 As shown, the BMDC cell activation markers CD80 and MHC-Ⅱ increased, proving that the nanovaccine caused DC cell activation and antigen cross-presentation of DC cells. Among them, the nanovaccine prepared by mixing the material with a polyethylene glycol grafting ratio of 5% 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 and stirred at a molar ratio of 50:1 for 24 h, and then dialyzed for 3 days using a dialysis bag with a cut-off molecular weight of 3500 Da. After freeze-drying, OVA-Cy5.5 was obtained for subsequent experiments. The present invention used C57BL / 6N female mice aged 6 to 8 weeks and weighing 17 to 22 g as experimental subjects. 40 μL of PBS, OVA-Cy5.5, G0-P were injected into the foot pad of the mice at 0 h. 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 OVA-Cy5.5 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 nanovaccine 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 nanovaccine 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 vivo and longer residence time in lymph nodes than other vaccines.

[0119] In the present invention, C57BL / 6N female mice aged 6 to 8 weeks and weighing 17 to 22 g were used as experimental subjects to prepare a B16-OVA tumor model. On the basis of the successful construction of the mouse model, B16-OVA cells (2.0×10 5 100 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 weight ratio of OVA to OVA was 5:1) and the amount of OVA and nano-vaccine injected was 100 μg OVA per mouse. The weight of the mice and the volume of the tumor 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 enables those skilled in the art to implement or use the present invention. Various modifications to these embodiments will be apparent to those 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 will not be limited to the embodiments shown herein, but rather to the widest scope consistent with the principles and novel features disclosed herein.

Claims

1. A polyethylene glycol-modified polylysine, characterized in that: It comprises a dendrimer initiator and polylysine connected by amide bonds, wherein polyethylene glycol is grafted on the side chain of the polylysine; The dendritic polymer initiator is formed when the terminal amino group of the dendritic polymer loses one hydrogen atom. The dendritic polymer includes polyamide-amine; the generation number of the polyamide-amine is 0 to 5.

2. The polyethylene glycol-modified polylysine according to claim 1, characterized in that The polyethylene glycol-modified polylysine has a structural formula as shown in the following formula I: Formula I; Among them, x=any number from 2 to 8, y=any number from 42 to 48, and x+y=n=50.

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

4. 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.

5. A method for preparing polyethylene glycol-modified polylysine according to any one of claims 1 to 4, characterized in that: The following steps are involved: S1: amino-protected lysine- N - The internal carboxylic acid anhydride is mixed with the dendritic polymer and undergoes a ring-opening polymerization reaction, and after deprotection, an intermediate is obtained; S2: reacting the intermediate with mPEG-COOH to obtain polyethylene glycol-modified polylysine.

6. The preparation method according to claim 5, 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.

7. The preparation method according to claim 5, characterized in that: The amino group is protected lysine- N - the molar ratio of the internal carboxylic anhydride to the dendritic polymer is (10-200):1; The molar ratio of the intermediate to mPEG-COOH is (6-20):

1.

8. A nano vaccine, characterized in that: The polylysine modified with polyethylene glycol according to any one of claims 1 to 4, the polylysine modified with polyethylene glycol prepared by the preparation method according to any one of claims 5 to 7, or the nano-adjuvant is complexed with an antigen; The nanoadjuvant comprises the polyethylene glycol-modified polylysine according to any one of claims 1 to 4, the polyethylene glycol-modified polylysine prepared by the preparation method according to any one of claims 5 to 7, and a solvent.

9. The nanovaccine according to claim 8, 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.

10. Use of the polyethylene glycol-modified polylysine according to any one of claims 1 to 4, the polyethylene glycol-modified polylysine prepared by the preparation method according to any one of claims 5 to 7, and the nanovaccine according to claim 8 or 9 in the preparation of a vaccine for preventing tumors; The tumor includes, but is not limited to, melanoma or Lewis lung carcinoma.

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