Responsive dendritic polyamino acid modified polyurethane as well as preparation method and application thereof

Abstract

The invention relates to the technical field of polymer chemistry, in particular to responsive dendritic polyamino acid modified polyurethane as well as a preparation method and application thereof. The responsive dendritic polyamino acid modified polyurethane has a structure as shown in a formula I which is described in the specification. The responsive dendritic polyamino acid modified polyurethane provided by the invention can specifically expose electropositive dendritic polylysine in a unique acidic environment of tumor tissues, trigger immunogenic death of tumor cells, release tumor antigens and comprehensively activate anti-tumor immune response. Meanwhile, the positive electricity dendritic polylysine causes the rise of reactive oxygen species (ROS) of tumor cells, a diselenide bond in polyurethane is oxidized into selenic acid, the expression of immune checkpoints of the tumor cells is reduced, the immunosuppression microenvironment of tumor tissues is reversed, and collaborative treatment of immune activation and tumor immunosuppression microenvironment reversal is realized. And a better tumor immunotherapy effect is achieved.

Description

Technical Field

[0001] The present invention relates to the technical field of polymer chemistry, and in particular to a responsive dendritic polyamino acid-modified polyurethane, a preparation method thereof, and an application thereof. Background Art

[0002] Immunotherapy is an emerging strategy for tumor treatment. It works by activating specific anti-tumor immune responses or reversing the tumor immunosuppressive microenvironment to enhance the body's immune system's clearance of tumors.

[0003] Synergistic regulation of immune activation and reversal of the tumor's immunosuppressive microenvironment is an important approach to overcome the low response rate of immunotherapy. Currently, this synergistic immune activation is mainly achieved through the coordinated administration of multiple drugs, but complex drug delivery systems and poor synergistic effects limit its clinical promotion and application. Summary of the Invention

[0004] In view of this, the technical problem to be solved by the present invention is to provide a responsive dendritic polyamino acid-modified polyurethane and its preparation method and application, so as to achieve synergistic treatment of immune activation and reversal of tumor immunosuppressive microenvironment.

[0005] The present invention provides a responsive dendritic polyamino acid-modified polyurethane having a structure shown in Formula I:

[0006]

[0007] Formula I

[0008]

[0009] m is any integer selected from 1 to 50;

[0010] n is any integer selected from 1 to 100;

[0011] p and q are independently selected from any integers ranging from 1 to 500.

[0012] The present invention provides a method for preparing the above-mentioned responsive dendritic polyamino acid-modified polyurethane, comprising the following steps:

[0013] The dendritic polyamino acid represented by formula I-a and the selenium-containing polyurethane represented by formula I-b undergo a click reaction to obtain a dendritic polyamino acid-modified polyurethane represented by formula I-c;

[0014] The dendritic polyamino acid-modified polyurethane shown in formula I-c is subjected to a ring-opening reaction with dimethylmaleic anhydride or cis-aconitic anhydride to obtain a responsive dendritic polyamino acid-modified polyurethane shown in formula I-d;

[0015]

[0016] Formula I-a;

[0017]

[0018] Formula I-b;

[0019]

[0020] Formula I-c.

[0021]

[0022] Formula I-d;

[0023]

[0024] m is any integer selected from 1 to 50;

[0025] n is any integer selected from 1 to 100;

[0026] p and q are independently selected from any integers ranging from 1 to 500.

[0027] Optionally, the dendritic polyamino acid represented by formula I-a is prepared according to the following steps:

[0028] Di-tert-butyl ((10S, 13S, 20S)-10-(tert-butoxycarbonyl)amino)-2,2-dimethyl-4,11,19-trioxo-13-(prop-2-yn-1-ylcarbamoyl)-3-oxa-5,12,18-triazatetradioxane-20,24-diyl) dicarbamate and 2,6-di-tert-butoxycarbonylaminocaproic acid are reacted to obtain tetra-tert-butyl ((10R, 1 0'R,13R,13'R,20R,20'R)-((R)-6-oxo-6-(prop-2-yn-1-ylamino)hexane-1,5-diyl)bis(azadiyl)bis(carbonyl))bis(10-(tert-butoxycarbonyl)amino)-2,2-dimethyl-4,11,19-trioxo-3-oxa-5,12,18-trioxatetraoxane-13,20,24-triyl))tetracarbamate.

[0029] Optionally, the di-tert-butyl ((10S, 13S, 20S)-10-(tert-butoxycarbonyl)amino)-2,2-dimethyl-4,11,19-trioxo-13-(prop-2-yn-1-ylcarbamoyl)-3-oxa-5,12,18-triazatetradioxane-20,24-diyl) dicarbamate is prepared according to the following method:

[0030] Di-tert-butyl (6-oxo-6-(prop-2-yn-1-ylamino)hexane-1,5-diyl) (S)-dicarbamate and 2,6-di-tert-butoxycarbonylaminohexanoic acid are reacted to obtain di-tert-butyl ((10S,13S,20S)-10-(tert-butoxycarbonyl)amino)-2,2-dimethyl-4,11,19-trioxo-13-(prop-2-yn-1-ylcarbamoyl)-3-oxa-5,12,18-triazatetradioxane-20,24-diyl) dicarbamate.

[0031] Optionally, the di-tert-butyl (6-oxo-6-(prop-2-yn-1-ylamino)hexane-1,5-diyl) (S)-dicarbamate is prepared according to the following method:

[0032] 2,6-di-tert-butyloxycarbonylaminohexanoic acid and propargylamine react to obtain di-tert-butyl (6-oxo-6-(prop-2-yn-1-ylamino)hexane-1,5-diyl) (S)-dicarbamate.

[0033] Optionally, the selenium-containing polyurethane represented by formula I-b is prepared according to the following method:

[0034] 11,11'-Diselenyldiylbis(undecanol-1-ol) is reacted with ethyl lysine diisocyanate and azidoethanol in sequence to obtain a selenium-containing polyurethane as shown in formula I-b.

[0035] Optionally, the mass ratio of the dendritic polyamino acid represented by formula I-a to the selenium-containing polyurethane represented by formula I-b is 1:(0.1-10);

[0036] Pentamethyldiethylenetriamine and CuBr are added during the click reaction;

[0037] The solvent for the click reaction is DMF;

[0038] The temperature of the click reaction is 30-60° C., and the time of the click reaction is 20-30 h.

[0039] The present invention provides a responsive dendritic polyamino acid-modified polyurethane nanoparticle, which is prepared from the above-mentioned responsive dendritic polyamino acid-modified polyurethane.

[0040] Optionally, the nanoparticles are prepared from the above-mentioned responsive dendritic polyamino acid-modified polyurethane and the dendritic polyamino acid-modified polyurethane shown in formula I-c;

[0041]

[0042] Formula I-c.

[0043] The present invention provides the use of the above-mentioned responsive dendritic polyamino acid-modified polyurethane or the above-mentioned responsive dendritic polyamino acid-modified polyurethane nanoparticles in the preparation of tumor immunotherapy reagents.

[0044] Compared to the prior art, the present invention provides a responsive dendritic polyamino acid-modified polyurethane having the structure shown in Formula I. The responsive dendritic polyamino acid-modified polyurethane provided by the present invention can specifically expose positively charged dendritic polylysine in the unique acidic environment of tumor tissue, triggering immunogenic cell death in tumor cells, releasing tumor antigens, and fully activating the anti-tumor immune response. Simultaneously, the positively charged dendritic polylysine increases reactive oxygen species (ROS) in tumor cells, oxidizing the diselenide bonds in the polyurethane to selenic acid, reducing tumor cell immune checkpoint expression and reversing the immunosuppressive microenvironment of tumor tissue. This achieves synergistic immune activation and reversal of the tumor immunosuppressive microenvironment, resulting in improved tumor immunotherapy efficacy. BRIEF DESCRIPTION OF THE DRAWINGS

[0045] Figure 1 This is the H NMR spectrum of selenium-containing polyurethane;

[0046] Figure 2 This is the H NMR spectrum of polyurethane modified with dendrimer polyamino acid;

[0047] Figure 3 The H NMR spectrum of polyurethane modified with responsive dendritic polyamino acid;

[0048] Figure 4 Transmission electron microscopy image of the prepared responsive dendritic polyamino acid modified polyurethane nanoparticles;

[0049] Figure 5 Figure 1 is the result of cell apoptosis;

[0050] Figure 6 This is the result of cell CRT flow cytometry;

[0051] Figure 7 This is the flow cytometry result of cellular immune checkpoints;

[0052] Figure 8 Tumor volume growth graph. DETAILED DESCRIPTION

[0053] The present invention provides a responsive dendritic polyamino acid-modified polyurethane having a structure shown in Formula I:

[0054]

[0055] Formula I

[0056]

[0057] m is any integer selected from 1 to 50; preferably any integer from 1 to 20; more preferably any integer from 1 to 15, specifically 5, 10, 11, 12, 13, 14 or 15.

[0058] n is any integer selected from 1 to 100; preferably any integer from 1 to 20; more preferably any integer from 1 to 15, specifically 5, 10, 11, 12, 13, 14 or 15.

[0059] p and q are independently selected from any integers ranging from 1 to 500.

[0060] Indicates the connection location.

[0061] Among them, the optional structure of R2 is not distinguished between left and right, and the two connecting bonds can be connected to the mother core at will.

[0062] The present invention provides a method for preparing the above-mentioned responsive dendritic polyamino acid-modified polyurethane, comprising the following steps:

[0063] The dendritic polyamino acid represented by formula I-a and the selenium-containing polyurethane represented by formula I-b undergo a click reaction to obtain a dendritic polyamino acid-modified polyurethane represented by formula I-c;

[0064] The dendritic polyamino acid-modified polyurethane shown in formula I-c is subjected to a ring-opening reaction with dimethylmaleic anhydride or cis-aconitic anhydride to obtain a responsive dendritic polyamino acid-modified polyurethane shown in formula I-d;

[0065]

[0066] Formula I-a;

[0067]

[0068] Formula I-b;

[0069]

[0070] Formula I-c

[0071]

[0072] Formula I-d;

[0073]

[0074] m is independently selected from any integer from 1 to 50;

[0075] n is independently selected from any integer from 1 to 100.

[0076] p and q are independently selected from any integers ranging from 1 to 500.

[0077] The equation for the above reaction is as follows:

[0078]

[0079]

[0080] The equation for the reaction of Formula I-c with cis-aconitic anhydride is similar to the above equation.

[0081] The click reaction between the dendritic polyamino acid represented by formula Ⅰ-a and the selenium-containing polyurethane represented by formula Ⅰ-b is a CuAAC reaction.

[0082] The mass ratio of the dendritic polyamino acid represented by formula I-a to the selenium-containing polyurethane represented by formula I-b is preferably 1:(0.1-10), more preferably 1:(0.5-5), and even more preferably 1:(0.5-2).

[0083] Pentamethyldiethylenetriamine and CuBr are added during the reaction.

[0084] The solvent for the reaction is preferably DMF.

[0085] The reaction temperature is preferably 30-60° C., and the reaction time is preferably 20-30 h.

[0086] After the reaction is completed, preferably, the product is dialyzed and freeze-dried to obtain the product.

[0087] Preferably, the dendritic polyamino acid represented by the above formula I-a is prepared according to the following steps:

[0088] Di-tert-butyl ((10S, 13S, 20S)-10-(tert-butoxycarbonyl)amino)-2,2-dimethyl-4,11,19-trioxo-13-(prop-2-yn-1-ylcarbamoyl)-3-oxa-5,12,18-triazatetradioxane-20,24-diyl) dicarbamate and 2,6-di-tert-butoxycarbonylaminocaproic acid are reacted to obtain tetra-tert-butyl ((10R, 1 0'R,13R,13'R,20R,20'R)-((R)-6-oxo-6-(prop-2-yn-1-ylamino)hexane-1,5-diyl)bis(azadiyl)bis(carbonyl))bis(10-(tert-butoxycarbonyl)amino)-2,2-dimethyl-4,11,19-trioxo-3-oxa-5,12,18-trioxatetraoxane-13,20,24-triyl))tetracarbamate.

[0089] The solvent for the reaction is preferably dichloromethane.

[0090] The above reaction is preferably carried out under the action of N-hydroxysuccinimide or N,N'-dicyclohexylcarbodiimide.

[0091] The mass ratio of the di-tert-butyl ((10S,13S,20S)-10-(tert-butoxycarbonyl)amino)-2,2-dimethyl-4,11,19-trioxo-13-(prop-2-yn-1-ylcarbamoyl)-3-oxa-5,12,18-triazatetradioxane-20,24-diyl) dicarbamate to 2,6-di-tert-butoxycarbonylaminohexanoic acid is preferably 1:(1-10), more preferably 1:(1-5), and even more preferably 1:(2-3).

[0092] Preferably, the di-tert-butyl ((10S, 13S, 20S)-10-(tert-butoxycarbonyl)amino)-2,2-dimethyl-4,11,19-trioxo-13-(prop-2-yn-1-ylcarbamoyl)-3-oxa-5,12,18-triazatetradioxane-20,24-diyl) dicarbamate is prepared according to the following method:

[0093] Di-tert-butyl (6-oxo-6-(prop-2-yn-1-ylamino)hexane-1,5-diyl) (S)-dicarbamate and 2,6-di-tert-butoxycarbonylaminohexanoic acid are reacted to obtain di-tert-butyl ((10S,13S,20S)-10-(tert-butoxycarbonyl)amino)-2,2-dimethyl-4,11,19-trioxo-13-(prop-2-yn-1-ylcarbamoyl)-3-oxa-5,12,18-triazatetradioxane-20,24-diyl) dicarbamate.

[0094] The solvent for the reaction is preferably dichloromethane.

[0095] The above reaction is preferably carried out under the action of N-hydroxysuccinimide or N,N'-dicyclohexylcarbodiimide.

[0096] The mass ratio of the di-tert-butyl (6-oxo-6-(prop-2-yn-1-ylamino)hexane-1,5-diyl) (S)-dicarbamate to 2,6-di-tert-butoxycarbonylaminohexanoic acid is preferably 1:(1-10), more preferably 1:(1-5), and even more preferably 1:(2-3).

[0097] Preferably, the di-tert-butyl (6-oxo-6-(prop-2-yn-1-ylamino)hexane-1,5-diyl) (S)-dicarbamate is prepared according to the following method:

[0098] 2,6-di-tert-butyloxycarbonylaminohexanoic acid and propargylamine react to obtain di-tert-butyl (6-oxo-6-(prop-2-yn-1-ylamino)hexane-1,5-diyl) (S)-dicarbamate.

[0099] The solvent for the reaction is preferably dichloromethane.

[0100] The above reaction is preferably carried out under the action of N-hydroxysuccinimide or N,N'-dicyclohexylcarbodiimide.

[0101] The mass volume ratio of the 2,6-di-tert-butoxycarbonylaminocaproic acid and propargylamine is 1 g: (0.01-1) mL, more preferably 1 g: (0.05-0.5) mL, and even more preferably 1 g: (0.1-0.3) mL.

[0102] Preferably, the selenium-containing polyurethane represented by formula I-b is prepared according to the following method:

[0103] 11,11'-Diselenyldiylbis(undecanol-1-ol) is reacted with ethyl lysine diisocyanate and azidoethanol in sequence to obtain a selenium-containing polyurethane as shown in formula I-b.

[0104] The equation for the above reaction is as follows:

[0105]

[0106] The 11,11'-diselenyldiylbis(undecanol-1-ol) is first reacted with ethyl lysine diisocyanate to obtain the following intermediate:

[0107]

[0108] The solvent for the reaction is preferably N,N-dimethylacetamide (DMAc).

[0109] The reaction is preferably carried out under the catalysis of dibutyltin dilaurate.

[0110] The reaction temperature is preferably 30-80° C., more preferably 40-70° C. The reaction time is preferably 0.5-1.5 h, more preferably 1-1.5 h.

[0111] The intermediate reacts with azidoethanol to obtain the selenium-containing polyurethane shown in formula I-b.

[0112] The reaction temperature is preferably 30-80° C., more preferably 40-70° C. The reaction time is preferably 0.5-1.5 h, more preferably 1-1.5 h.

[0113] After the reaction is completed, the selenium-containing polyurethane shown in formula Ⅰ-b can be obtained by precipitation with anhydrous ether and drying.

[0114] The present invention provides a responsive dendritic polyamino acid-modified polyurethane nanoparticle, which is prepared from the above-mentioned responsive dendritic polyamino acid-modified polyurethane.

[0115] The present invention has no particular limitation on the preparation method of the nanoparticles, and may be any general method for preparing nanoparticles from polymers well known to those skilled in the art, including but not limited to a microemulsion method, a solvent evaporation method, a solvent diffusion method, and the like.

[0116] Optionally, the raw materials for preparing the nanoparticles include the above-mentioned responsive dendritic polyamino acid-modified polyurethane and the dendritic polyamino acid-modified polyurethane shown in Formula I-c.

[0117] Simultaneously adding the polyurethane modified with the dendritic polyamino acid shown in formula I-c can make the surface of the nanoparticles electrically neutral and facilitate subsequent modifications, such as the connection of fluorescent molecules.

[0118] The nanoparticles provided by the present invention can induce immunogenic cell death and downregulate immune checkpoints in tumor cells. Under acidic conditions, the nanoparticles hydrolyze anhydrides, exposing positively charged amino groups that lead to immunogenic cell death, release tumor antigens, and fully activate anti-tumor immune responses. Simultaneously, the diselenide bonds in the polyurethane are oxidized to selenoic acid, which reduces the expression of immune checkpoints in tumor cells, reverses the immunosuppressive microenvironment, and synergistically enhances tumor immunotherapy.

[0119] Based on this, the present invention provides the use of the above-mentioned responsive dendritic polyamino acid-modified polyurethane or the above-mentioned responsive dendritic polyamino acid-modified polyurethane nanoparticles in the preparation of tumor immunotherapy reagents.

[0120] To further illustrate the present invention, the following is a detailed description of the embodiments. However, it should be understood that these descriptions are only for the purpose of further illustrating the features and advantages of the present invention, rather than for limiting the scope of the invention.

[0121] All raw materials of the present invention are not particularly limited in their sources and can be purchased from the market or prepared according to conventional methods well known to those skilled in the art.

[0122] Example 1

[0123] 1.1 Preparation of di-tert-butyl (6-oxo-6-(prop-2-yn-1-ylamino)hexane-1,5-diyl) (S)-dicarbamate

[0124] To a 500 mL three-necked flask, add 12.5 g of 2,6-di-tert-butoxycarbonylaminocaproic acid and 200 mL of anhydrous dichloromethane (DCM) under nitrogen bubbling. Transfer to an ice bath under nitrogen and stir for 30 min. Then, add 3.8 g of N-hydroxysuccinimide and 6.9 g of N,N'-dicyclohexylcarbodiimide. Stir for 10 min and transfer to room temperature. Add 2.28 mL of propargylamine and dropwise add 4.62 mL of triethylamine. Seal the reaction flask and allow to react at room temperature for 24 h. After completion of the reaction, filter the flask and wash with saturated NaHCO₃ solution (30 mL x 3), saturated NaHSO₄ solution (30 mL x 3), and saturated NaCl solution (30 mL x 3). Dry over anhydrous MgSO₄, filter, concentrate under reduced pressure, and separate by column chromatography using a 1:2 ratio of ethyl acetate to n-hexane. Dry under vacuum to obtain a white solid with a purity of 99% and a yield of 83%.

[0125] 1.2 Preparation of di-tert-butyl ((10S,13S,20S)-10-(tert-butoxycarbonyl)amino)-2,2-dimethyl-4,11,19-trioxo-13-(prop-2-yn-1-ylcarbamoyl)-3-oxa-5,12,18-triazatetradioxane-20,24-diyl) dicarbamate

[0126] To a 200 mL round-bottom flask, add 3.8 g of di-tert-butyl (6-oxo-6-(prop-2-yn-1-ylamino)hexane-1,5-diyl)(S)-dicarbamate and dissolve in 50 mL of anhydrous DCM. Stir in an ice bath for 30 min, then add 30 mL of trifluoroacetic acid dropwise. Stir for 10 min, and then react at room temperature for 4 h. Remove the solvent under reduced pressure, precipitate three times with 50 mL of anhydrous ether, and dry in vacuo to obtain a white solid. To a 500 mL three-necked flask, add 10.4 g of 2,6-di-tert-butyloxycarbonylaminohexanoic acid and 200 mL of anhydrous dichloromethane (DCM) under nitrogen sparging. Transfer to an ice bath under nitrogen, stir for 30 min, then add 3.5 g of N-hydroxysuccinimide and 6.2 g of N,N'-dicyclohexylcarbodiimide. Stir for 10 min, and then transfer to room temperature. The dried solid dissolved in 50 mL of anhydrous DCM was added, followed by the dropwise addition of 4.17 mL of triethylamine, and the reaction flask was sealed. The reaction was allowed to react at room temperature for 24 h. After completion of the reaction, the mixture was filtered and washed sequentially with saturated NaHCO₃ solution (30 mL x 3), saturated NaHSO₄ solution (30 mL x 3), and saturated NaCl solution (30 mL x 3). Drying was performed over anhydrous MgSO₄, filtered, and concentrated under reduced pressure. The product was separated by column chromatography using a 4:1 ratio of ethyl acetate to n-hexane as the eluent and dried under vacuum to yield a white solid with a purity of 99% and a yield of 80%.

[0127] 1.3 Preparation of Tetra-tert-butyl ((10R, 10'R, 13R, 13'R, 20R, 20'R)-((R)-6-oxo-6-(prop-2-yn-1-ylamino)hexane-1,5-diyl)bis(azadiyl))bis(carbonyl))bis(10-(tert-butoxycarbonyl)amino)-2,2-dimethyl-4,11,19-trioxo-3-oxa-5,12,18-trioxatetraoxane-13,20,24-triyl))tetracarbamate

[0128] To a 200 mL round-bottom flask, add 4.2 g of di-tert-butyl ((10S,13S,20S)-10-(tert-butoxycarbonyl)amino)-2,2-dimethyl-4,11,19-trioxo-13-(prop-2-yn-1-ylcarbamoyl)-3-oxa-5,12,18-triazatetradioxane-20,24-diyl) dicarbamate and dissolve in 50 mL of anhydrous DCM. Stir in an ice bath for 30 minutes, then add 30 mL of trifluoroacetic acid dropwise. Stir for 10 minutes, then react at room temperature for 4 hours. Remove the solvent under reduced pressure, precipitate three times with 50 mL of anhydrous ether, and dry in vacuo to obtain a white solid. To a 500 mL three-necked flask, add 10.4 g of 2,6-di-tert-butoxycarbonylaminohexanoic acid and 200 mL of anhydrous dichloromethane (DCM) and sparge with nitrogen. Under nitrogen, transfer to an ice bath and stir for 30 minutes. Then, add 3.5 g of N-hydroxysuccinimide and 6.2 g of N,N'-dicyclohexylcarbodiimide. Stir for 10 minutes and then transfer to room temperature. Add the dried solid dissolved in 50 mL of anhydrous DCM, add 4.17 mL of triethylamine dropwise, and seal the reaction flask. Allow to react at room temperature for 24 hours. After completion of the reaction, filter and wash sequentially with saturated NaHCO₃ solution (30 mL x 3), saturated NaHSO₄ solution (30 mL x 3), and saturated NaCl solution (30 mL x 3). Dry over anhydrous MgSO₄, filter, concentrate under reduced pressure, and separate by column chromatography using a 1:10 ratio of methanol to dichloromethane. Dry under vacuum to obtain a white solid with a purity of 99% and a yield of 74%.

[0129] 1.4 Preparation of 11,11'-Diselenyldiylbis(undecanol-1-ol)

[0130] To a 250 mL three-necked flask, add 1.5 g of NaBH₄ and place in an ice bath. Add 40 mL of pre-cooled deionized water and stir for 30 minutes. Then, add 3.2 g of selenium powder in small portions, causing bubbles to form. After the bubbles disappear, sparge with nitrogen. Transfer the mixture to an oil bath and heat to 50°C under nitrogen for 1 hour. To a 200 mL round-bottom flask, add 95 mL of tetrahydrofuran and 10.3 g of 11-bromodecanol. Stir to dissolve, then sparge with nitrogen for 30 minutes. The 11-bromodecanol solution is added to the three-necked flask via syringe and allowed to react at 50°C for 24 hours. Dilute the reaction mixture with 300 mL of ethyl acetate, wash with saturated NaCl solution (30 mL x 3), dry over anhydrous MgSO₄, filter, and concentrate under reduced pressure. Separate the mixture by column chromatography using a 1:2 ratio of ethyl acetate to dichloromethane and dry under vacuum to obtain a yellow solid with a purity of 99% and a yield of 77%.

[0131] 1.5 Preparation of Selenium-Containing Polyurethane

[0132] To a 250 mL three-necked flask, add 3.3 g of 11,11'-diselenediylbis(undecanol-1-ol), 4.5 g of ethyl lysine diisocyanate, and 20 mL of N,N-dimethylacetamide. Stir and sparge with nitrogen. Add 41 mg of dibutyltin dilaurate and heat to 65°C for 1 h. Add azidoethanol and react at 65°C for 1 h. Precipitate three times with 200 mL of anhydrous ether and dry in vacuo to obtain a yellow solid. The product has a purity of 99% and a yield of 93%. Figure 1 This is the hydrogen nuclear magnetic resonance spectrum of selenium-containing polyurethane.

[0133] The reaction equation is as follows:

[0134]

[0135] m=11, n=13.

[0136] 1.6 Preparation of dendrimer polyamino acid-modified polyurethanes

[0137] To a 100 mL flask, 1.0 g of selenium-containing polyurethane, 0.9 g of tetra-tert-butyl ((10R,10'R,13R,13'R,20R,20'R)-((R)-6-oxo-6-(prop-2-yn-1-ylamino)hexane-1,5-diyl)bis(azadiyl)bis(carbonyl)bis(10-(tert-butoxycarbonyl)amino)-2,2-dimethyl-4,11,19-trioxo-3-oxa-5,12,18-trioxatetraoxane-13,20,24-triyl)tetracarbamate, and 20 mL of anhydrous DMF were added and stirred to dissolve. 334 μL of pentamethyldiethylenetriamine and 57 mg of CuBr were added and reacted at 45°C for 24 h. The solution was dialyzed and lyophilized. Place the lyophilized product in a 100 mL flask and dissolve it in 20 mL of anhydrous DCM. Stir in an ice bath for 30 minutes, then add 10 mL of trifluoroacetic acid dropwise. Stir for 10 minutes, then react at room temperature for 4 hours. Remove the solvent under reduced pressure, precipitate three times with 300 mL of anhydrous ether, and dry in vacuo to obtain a yellow solid. The purity is 99%, and the yield is 95%. Figure 2 This is the H NMR spectrum of polyurethane modified with dendrimer polyamino acid.

[0138] The reaction equation is as follows:

[0139]

[0140] m=11, n=13.

[0141] 1.7 Preparation of Responsive Dendritic Polyamino Acid-Modified Polyurethane

[0142] To a 100 mL flask, add 440 mg of dendrimer-modified polyurethane (PDA) and dissolve it in 10 mL of anhydrous DMF. Then, add 400 mg of dimethylmaleic anhydride and 27 μL of triethylamine. The mixture was reacted at 25°C for 24 h. The mixture was precipitated three times with 100 mL of anhydrous ether and dried under vacuum to obtain a yellow solid with a purity of 99% and a yield of 93%. Figure 3 H NMR spectrum of polyurethane modified with responsive dendritic polyamino acid.

[0143] The reaction equation is as follows:

[0144]

[0145]

[0146] m=11, n=13.

[0147] 1.8 Preparation of Responsive Dendritic Polyamino Acid-Modified Polyurethane Nanoparticles

[0148] Dissolve 20.0 mg of polyurethane modified with dendritic polyamino acids and 20.0 mg of polyurethane modified with responsive dendritic polyamino acids in 20 mL of DMF, then mix. Add 2 mL of the mixed solution dropwise to a stirred 6 mL of PBS (pH 7.4). Responsive polyurethane modified with dendritic polyamino acids nanoparticles were obtained after dialysis against PBS (pH 7.4). Figure 4 Transmission electron microscopy image of the prepared responsive dendritic polyamino acid modified polyurethane nanoparticles.

[0149] Depend on Figure 4 It can be seen that spherical nanoparticles with a particle size of about 50 nm were prepared.

[0150] 1.9 Nanoparticles induce tumor cell apoptosis

[0151] 100,000 B16 tumor cells were seeded in a 12-well plate. After 12 hours, the nanoparticles were diluted to 20 μg / mL in 1640 complete medium (pH 6.8). The supernatant was discarded, and 1 mL of nanoparticles was added and incubated for 24 hours. PBS served as a control group. Cell apoptosis was detected using the Annexin V-FITC / PI apoptosis detection kit. Figure 5 The results of cell apoptosis are shown in Figure 2.

[0152] Depend on Figure 5 It can be seen that nanoparticles can induce tumor cell apoptosis.

[0153] 1.10 Nanoparticles induce immunogenic death of tumor cells

[0154] 100,000 B16 tumor cells were seeded in a 12-well plate. After 12 hours, the nanoparticles were diluted to 20 μg / mL in 1640 complete medium (pH 6.8). The supernatant was discarded, and 1 mL of nanoparticles was added and incubated for 24 hours. PBS served as a control group. Cells were harvested and CRT externalization was detected by flow cytometry. Figure 6 This is the result of cell CRT flow cytometry.

[0155] Depend on Figure 6 It can be seen that nanoparticles can induce CRT eversion in tumor cells.

[0156] 1.11 Nanoparticles induce downregulation of immune checkpoints in tumor cells

[0157] 80,000 B16 tumor cells were seeded in a 12-well plate. 12 hours later, IFN-γ (20 ng / mL) was added and cultured for 24 hours. Nanoparticles were diluted to 20 μg / mL in 1640 complete medium (pH 6.8). The supernatant was discarded from the plate, and 1 mL of nanoparticles was added and cultured for 24 hours. PBS served as a control group. Cells were harvested and immune checkpoint markers were analyzed by flow cytometry. Figure 7 This is the flow cytometry result of cellular immune checkpoints.

[0158] Depend on Figure 7 It can be seen that nanoparticles can induce downregulation of immune checkpoints in tumor cells.

[0159] 1.12 Evaluation of Tumor Treatment Effectiveness

[0160] One million B16 tumor cells were inoculated subcutaneously in C57BL / 6 mice. After 7 days, the tumor volume was approximately 100 mm. 3 100 μL of 4 mg / mL nanoparticles were injected via the tail vein every three days. PBS served as a control group. Tumor volumes were recorded every two days. Figure 8 Tumor volume growth graph.

[0161] Depend on Figure 8 It can be seen that nanoparticles can significantly inhibit tumor growth.

[0162] The above embodiments are only intended to help understand the method and core concept of the present invention. It should be noted that, without departing from the principles of the present invention, a number of improvements and modifications may be made to the present invention by those skilled in the art, and such improvements and modifications also fall within the scope of protection of the claims of the present invention.

Claims

1. A responsive dendritic polyamino acid-modified polyurethane having the structure shown in Formula I: Formula I m is any integer selected from 1 to 50; n is any integer selected from 1 to 100; p and q are independently selected from any integers of 1 to 500.

2. The method for preparing the responsive dendritic polyamino acid-modified polyurethane according to claim 1, comprising the following steps: The dendritic polyamino acid represented by formula I-a and the selenium-containing polyurethane represented by formula I-b undergo a click reaction to obtain a dendritic polyamino acid-modified polyurethane represented by formula I-c; The dendritic polyamino acid-modified polyurethane shown in formula I-c is subjected to a ring-opening reaction with dimethylmaleic anhydride or cis-aconitic anhydride to obtain a responsive dendritic polyamino acid-modified polyurethane shown in formula I-d; Formula I-a; Formula I-b; Formula I-c Formula I-d; m is any integer selected from 1 to 50; n is any integer selected from 1 to 100; p and q are independently selected from any integers of 1 to 500.

3. The preparation method according to claim 2, characterized in that The dendritic polyamino acid represented by formula Ⅰ-a is prepared according to the following steps: Di-tert-butyl ((10S, 13S, 20S)-10-(tert-butoxycarbonyl)amino)-2,2-dimethyl-4,11,19-trioxo-13-(prop-2-yn-1-ylcarbamoyl)-3-oxa-5,12,18-triazatetradioxane-20,24-diyl) dicarbamate and 2,6-di-tert-butoxycarbonylaminocaproic acid are reacted to obtain tetra-tert-butyl ((10R, 1 0'R,13R,13'R,20R,20'R)-((R)-6-oxo-6-(prop-2-yn-1-ylamino)hexane-1,5-diyl)bis(azadiyl)bis(carbonyl))bis(10-(tert-butoxycarbonyl)amino)-2,2-dimethyl-4,11,19-trioxo-3-oxa-5,12,18-trioxatetraoxane-13,20,24-triyl))tetracarbamate.

4. The preparation method according to claim 3, characterized in that The di-tert-butyl ((10S, 13S, 20S)-10-(tert-butoxycarbonyl)amino)-2,2-dimethyl-4,11,19-trioxo-13-(prop-2-yn-1-ylcarbamoyl)-3-oxa-5,12,18-triazatetradioxane-20,24-diyl) dicarbamate was prepared according to the following method: Di-tert-butyl (6-oxo-6-(prop-2-yn-1-ylamino)hexane-1,5-diyl) (S)-dicarbamate and 2,6-di-tert-butoxycarbonylaminohexanoic acid are reacted to obtain di-tert-butyl ((10S,13S,20S)-10-(tert-butoxycarbonyl)amino)-2,2-dimethyl-4,11,19-trioxo-13-(prop-2-yn-1-ylcarbamoyl)-3-oxa-5,12,18-triazatetradioxane-20,24-diyl) dicarbamate.

5. The preparation method according to claim 4, characterized in that The di-tert-butyl (6-oxo-6-(prop-2-yn-1-ylamino)hexane-1,5-diyl) (S)-dicarbamate was prepared according to the following method: 2,6-di-tert-butyloxycarbonylaminohexanoic acid and propargylamine react to obtain di-tert-butyl (6-oxo-6-(prop-2-yn-1-ylamino)hexane-1,5-diyl) (S)-dicarbamate.

6. The preparation method according to claim 2, characterized in that The selenium-containing polyurethane represented by formula I-b is prepared according to the following method: 11,11'-Diselenyldiylbis(undecanol-1-ol) is reacted with ethyl lysine diisocyanate and azidoethanol in sequence to obtain a selenium-containing polyurethane as shown in formula I-b.

7. The preparation method according to claim 2, characterized in that The mass ratio of the dendritic polyamino acid represented by formula I-a to the selenium-containing polyurethane represented by formula I-b is 1:(0.1-10); Pentamethyldiethylenetriamine and CuBr are added during the click reaction; The solvent for the click reaction is DMF; The temperature of the click reaction is 30-60° C., and the time of the click reaction is 20-30 h.

8. A responsive dendritic polyamino acid-modified polyurethane nanoparticle, characterized in that: The polyurethane is prepared from the responsive dendritic polyamino acid modified polyurethane according to claim 1.

9. The responsive dendritic polyamino acid-modified polyurethane nanoparticles according to claim 8, characterized in that: The nanoparticles are prepared from the responsive dendritic polyamino acid-modified polyurethane according to claim 1 and the dendritic polyamino acid-modified polyurethane shown in formula I-c; Formula I-c.

10. Use of the responsive dendritic polyamino acid-modified polyurethane according to claim 1 or the responsive dendritic polyamino acid-modified polyurethane nanoparticles according to any one of claims 8 to 9 in the preparation of a tumor immunotherapy agent.

Images