Responsive dendrimeric polyamino acid modified polyurethane, and preparation method and application thereof

Abstract

The present application relates to the technical field of polymer chemistry, and particularly relates to a responsive dendritic polyamino acid modified polyurethane and a preparation method and application thereof.The responsive dendritic polyamino acid modified polyurethane has a structure shown in formula I.The responsive dendritic polyamino acid modified polyurethane provided by the present application can specifically expose positive dendritic polylysine in the unique acidic environment of tumor tissue, trigger immunogenic death of tumor cells, release tumor antigens, and comprehensively activate an anti-tumor immune response.Meanwhile, the positive dendritic polylysine causes an increase in reactive oxygen species (ROS) of tumor cells, oxidizes a diselenium bond in the polyurethane into selenic acid, reduces the expression of immune checkpoints of tumor cells, reverses the immunosuppressive microenvironment of tumor tissue, realizes synergistic treatment of immune activation and reversal of the tumor immunosuppressive microenvironment, and realizes a better tumor immunotherapy effect.

Description

Technical Field

[0001] This invention relates to the field of polymer chemistry, and more particularly to a responsive dendritic polyamino acid-modified polyurethane, its preparation method, and its application. Background Technology

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

[0003] The synergistic regulation of immune activation and reversal of the tumor immunosuppressive microenvironment is an important way to overcome the low response rate of immunotherapy. Currently, the synergistic immune activation is mainly achieved through the synergistic administration of multiple drugs, but the complex drug delivery system and poor synergistic effect 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 polyurethane modified with amino acids, its preparation method and application, which realizes synergistic therapy of immune activation and reversal of tumor immunosuppressive microenvironment.

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

[0006]

[0007] Formula I

[0008]

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

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

[0011] p and q are independent integers selected from 1 to 500.

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

[0013] The dendritic polyamino acid shown in Formula I-a and the selenium-containing polyurethane shown in Formula I-b are subjected to a click reaction to obtain the dendritic polyamino acid-modified polyurethane shown in Formula I-c.

[0014] The dendritic polyamino acid-modified polyurethane shown in Formula I-c undergoes a ring-opening reaction with dimethyl maleic anhydride or cis aconitic anhydride to obtain the 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 selected from any integer from 1 to 50;

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

[0026] p and q are independent integers selected 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-triazatetradecane-20,24-diyl)dicarbamate and 2,6-di-tert-butoxycarbonylaminohexanoate are reacted to give tetra-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-triazatetradecane-20,24-diyl)dicarbamate and 2,6-di-tert-butoxycarbonylaminohexanoate as shown in Formula I-a. 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-triazatetradiane-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-butoxycarbonylaminohexanoate are reacted to give 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-triazatetradecane-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-butoxycarbonylaminohexanoic acid reacts with propyneamine to give 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'-diselenodimethylbis(undecanol-1-ol) was reacted sequentially with ethyl lysine diisocyanate and azidoethanol to obtain the selenium-containing polyurethane 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] The click reaction process includes the addition of pentamethyldiethylenetriamine and CuBr.

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

[0038] The temperature of the click reaction is 30~60℃, and the time of the click reaction is 20~30h.

[0039] This invention provides responsive dendritic polyamino acid-modified polyurethane nanoparticles, which are prepared from the aforementioned responsive dendritic polyamino acid-modified polyurethane.

[0040] Optionally, the nanoparticles are prepared from the above-described responsive dendritic polyamino acid-modified polyurethane and the dendritic polyamino acid-modified polyurethane shown in Formula I-c.

[0041]

[0042] Formula I-c.

[0043] This invention provides the application 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 with existing technologies, this invention provides a responsive dendritic polyamino acid-modified polyurethane with the structure shown in Formula I. The responsive dendritic polyamino acid-modified polyurethane provided by this invention can specifically expose positively charged dendritic polylysine in the unique acidic environment of tumor tissue, inducing immunogenic death of tumor cells, releasing tumor antigens, and comprehensively activating the anti-tumor immune response. Simultaneously, the positively charged dendritic polylysine leads to an increase in reactive oxygen species (ROS) in tumor cells, oxidizing the diselenyl bonds in the polyurethane to selenic acid, reducing the expression of immune checkpoints in tumor cells, reversing the immunosuppressive microenvironment of tumor tissue, achieving synergistic therapy of immune activation and reversal of the tumor immunosuppressive microenvironment, and achieving better tumor immunotherapy effects. Attached Figure Description

[0045] Figure 1 The hydrogen nuclear magnetic resonance spectrum of selenium-containing polyurethane;

[0046] Figure 2 The 1H NMR spectrum of dendritic polyamino acid-modified polyurethane.

[0047] Figure 3 The proton NMR spectrum of a responsive dendritic polyamino acid-modified polyurethane.

[0048] Figure 4 Transmission electron microscope image of the prepared responsive dendritic polyamino acid-modified polyurethane nanoparticles.

[0049] Figure 5 This is a diagram showing the results of cell apoptosis.

[0050] Figure 6 This is a graph showing the results of CRT flow cytometry for cells;

[0051] Figure 7 Flow cytometry results for cellular immune checkpoints;

[0052] Figure 8 This is a graph showing the growth of tumor volume. Detailed Implementation

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

[0054]

[0055] Formula I

[0056]

[0057] m is selected from any integer 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 selected from any integer from 1 to 100; preferably, it is any integer from 1 to 20; more preferably, it is any integer from 1 to 15, specifically 5, 10, 11, 12, 13, 14 or 15.

[0059] p and q are independent integers selected from 1 to 500.

[0060] Indicates the connection location.

[0061] In R2, the optional structure does not distinguish between left and right, and the two connection keys can be arbitrarily connected to the mother core.

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

[0063] The dendritic polyamino acid shown in Formula I-a and the selenium-containing polyurethane shown in Formula I-b are subjected to a click reaction to obtain the dendritic polyamino acid-modified polyurethane shown in Formula I-c.

[0064] The dendritic polyamino acid-modified polyurethane shown in Formula I-c undergoes a ring-opening reaction with dimethyl maleic anhydride or cis aconitic anhydride to obtain the 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 an independent integer selected from 1 to 50;

[0075] n is an independent integer selected from 1 to 100.

[0076] p and q are independent integers selected from 1 to 500.

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

[0078]

[0079]

[0080] The equations for the reaction of Formula I-c and cis-aconitine are similar to those above.

[0081] The click reaction between the dendritic polyamino acid shown in Formula I-a and the selenium-containing polyurethane shown in 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] The reaction process involves the addition of pentamethyldiethylenetriamine and CuBr.

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

[0085] The preferred temperature for the reaction is 30-60°C, and the preferred reaction time is 20-30 hours.

[0086] After the reaction is complete, the product is preferably obtained by lyophilization via dialyzing.

[0087] Preferably, the dendritic polyamino acid represented by Formula I-a above 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-triazatetradecane-20,24-diyl)dicarbamate and 2,6-di-tert-butoxycarbonylaminohexanoate are reacted to give tetra-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-triazatetradecane-20,24-diyl)dicarbamate and 2,6-di-tert-butoxycarbonylaminohexanoate as shown in Formula I-a. 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 and N,N'-dicyclohexylcarbodiimide.

[0091] The mass ratio 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-triazatetradialkyl-20,24-diyl)dicarbamate and 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-triazatetradiane-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-butoxycarbonylaminohexanoate are reacted to give 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-triazatetradecane-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 and N,N'-dicyclohexylcarbodiimide.

[0096] The mass ratio of di-tert-butyl(6-oxo-6-(prop-2-yn-1-ylamino)hexane-1,5-diyl)(S)-dicarbamate and 2,6-di-tert-butyloxycarbonylaminohexanoic 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-butoxycarbonylaminohexanoic acid reacts with propyneamine to give 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 and N,N'-dicyclohexylcarbodiimide.

[0101] The mass-to-volume ratio of 2,6-di-tert-butoxycarbonylaminohexanoic acid and propyneamine 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'-diselenodimethylbis(undecanol-1-ol) was reacted sequentially with ethyl lysine diisocyanate and azidoethanol to obtain the selenium-containing polyurethane shown in Formula I-b.

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

[0105]

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

[0107]

[0108] The preferred solvent for the reaction is 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℃, more preferably 40~70℃. The reaction time is preferably 0.5~1.5h, more preferably 1~1.5h.

[0111] The intermediate reacts with azide ethanol to obtain the selenium-containing polyurethane shown in Formula I-b.

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

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

[0114] This invention provides responsive dendritic polyamino acid-modified polyurethane nanoparticles, which are prepared from the aforementioned responsive dendritic polyamino acid-modified polyurethane.

[0115] The present invention does not specifically limit the preparation method of the above-mentioned nanoparticles. It can be any general polymer preparation method known to those skilled in the art, including but not limited to microemulsion method, solvent evaporation method, solvent diffusion method, etc.

[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 dendritic polyamino acid-modified polyurethane as shown in Formula I-c can make the surface of nanoparticles electrically neutral and facilitate subsequent modifications, such as connecting fluorescent molecules.

[0118] The nanoparticles provided by this invention can induce immunogenic death of tumor cells and downregulation of immune checkpoints. These nanoparticles can hydrolyze acid anhydrides under acidic conditions, exposing positively charged amino groups that lead to immunogenic death of tumor cells, releasing tumor antigens and comprehensively activating the anti-tumor immune response. Simultaneously, the diselenyl bonds in the polyurethane are oxidized to selenic acid, reducing the expression of immune checkpoints in tumor cells, reversing the immunosuppressive microenvironment, and synergistically enhancing tumor immunotherapy.

[0119] Based on this, the present invention provides the application 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, a detailed description is provided below with reference to embodiments. However, it should be understood that these descriptions are merely for further illustrating the features and advantages of the present invention, and not for limiting the scope of the claims.

[0121] There are no particular restrictions on the source of any raw materials used in this invention; they can be purchased from the market or prepared using conventional methods 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] 12.5 g of 2,6-di-tert-butoxycarbonylaminohexanoic acid and 200 mL of anhydrous dichloromethane (DCM) were added to a 500 mL three-necked flask, and the mixture was bubbled with nitrogen. Under nitrogen purging, the flask was transferred to an ice bath and stirred for 30 min. Then, 3.8 g of N-hydroxysuccinimide and 6.9 g of N,N'-dicyclohexylcarbodiimide were added, and the mixture was stirred for 10 min before being transferred to room temperature. 2.28 mL of propyneamine was added, followed by dropwise addition of 4.62 mL of triethylamine. The reaction flask was then sealed. The reaction was allowed to proceed at room temperature for 24 h. After the reaction was complete, the mixture was filtered and washed successively with saturated NaHCO3 solution (30 mL x 3), saturated NaHSO4 solution (30 mL x 3), and saturated NaCl solution (30 mL x 3). The solution was dried over anhydrous MgSO4, filtered, concentrated under reduced pressure, and separated by column chromatography using ethyl acetate:n-hexane = 1:2 eluent. The solid was dried 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-triazatetradiane-20,24-diyl)dicarbamate

[0126] 3.8 g of di-tert-butyl(6-oxo-6-(prop-2-yn-1-ylamino)hexane-1,5-diyl)(S)-dicarbamate was added to a 200 mL round-bottom flask and dissolved in 50 mL of anhydrous DCM. The mixture was stirred in an ice bath for 30 min, followed by the addition of 30 mL of trifluoroacetic acid. After stirring for 10 min, the mixture was reacted at room temperature for 4 h. The solvent was removed under reduced pressure, and the solid was precipitated three times with 50 mL of anhydrous diethyl ether and dried under vacuum to obtain a white solid. 10.4 g of 2,6-di-tert-butoxycarbonylaminohexanoic acid and 200 mL of anhydrous dichloromethane (DCM) were added to a 500 mL three-necked flask, and the mixture was bubbled with nitrogen. Under nitrogen purging, the mixture was transferred to an ice bath and stirred for 30 min. Then, 3.5 g of N-hydroxysuccinimide and 6.2 g of N,N'-dicyclohexylcarbodiimide were added, and the mixture was stirred for 10 min before being transferred to room temperature. Add the dried solid dissolved in 50 mL of anhydrous DCM, then add 4.17 mL of triethylamine dropwise, and seal the reaction flask. Incubate at room temperature for 24 h. After the reaction is complete, filter and wash successively with saturated NaHCO3 solution (30 mL x 3), saturated NaHSO4 solution (30 mL x 3), and saturated NaCl solution (30 mL x 3). Dry under anhydrous MgSO4, filter, concentrate under reduced pressure, and separate by column chromatography with ethyl acetate:n-hexane = 4:1 eluent. Dry under vacuum to obtain a white solid. Purity: 99%, yield: 80%.

[0127] 1.3 Preparation of tetratert-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] 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-triazatetradecane-20,24-diyl)dicarbamate was added to a 200 mL round-bottom flask and dissolved in 50 mL of anhydrous DCM. The mixture was stirred in an ice bath for 30 min, followed by the addition of 30 mL of trifluoroacetic acid. After stirring for 10 min, the mixture was reacted at room temperature for 4 h. The solvent was removed under reduced pressure, and the solid was precipitated three times with 50 mL of anhydrous diethyl ether and dried under vacuum to obtain a white solid. 10.4 g of 2,6-di-tert-butoxycarbonylaminohexanoic acid and 200 mL of anhydrous dichloromethane (DCM) were added to a 500 mL three-necked flask, and the mixture was bubbled with nitrogen. Under nitrogen purging, the mixture was transferred to an ice bath and stirred for 30 min. Then, 3.5 g of N-hydroxysuccinimide and 6.2 g of N,N'-dicyclohexylcarbodiimide were added, and the mixture was stirred for 10 min before being transferred to room temperature. The dried solid dissolved in 50 mL of anhydrous DCM was added, followed by dropwise addition of 4.17 mL of triethylamine. The reaction flask was then sealed. The reaction was allowed to proceed at room temperature for 24 h. After the reaction was complete, the mixture was filtered and washed successively with saturated NaHCO3 solution (30 mL x 3), saturated NaHSO4 solution (30 mL x 3), and saturated NaCl solution (30 mL x 3). The solution was dried over anhydrous MgSO4, filtered, concentrated under reduced pressure, and separated by column chromatography using methanol:dichloromethane = 1:10 eluent. The solid was dried under vacuum to obtain a white solid with a purity of 99% and a yield of 74%.

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

[0130] Add 1.5 g NaBH4 to a 250 mL three-necked flask, place in an ice bath, add 40 mL of pre-cooled deionized water, stir for 30 min, and add 3.2 g selenium powder in small amounts several times, generating bubbles. After the bubbles disappear, bubble with nitrogen. Under nitrogen purging, transfer to an oil bath and heat to 50 °C, react for 1 h. Add 95 mL tetrahydrofuran and 10.3 g 11-bromoundecyl alcohol to a 200 mL round-bottom flask, stir to dissolve, and bubble with nitrogen for 30 min. Add the 11-bromoundecyl alcohol solution to the three-necked flask using a syringe, and react at 50 °C for 24 h. Dilute the reaction mixture with 300 mL ethyl acetate, wash with saturated NaCl solution (30 mL x 3), dry with anhydrous MgSO4, filter, and concentrate under reduced pressure. Separate by column chromatography using ethyl acetate:dichloromethane = 1:2 eluent, and dry under vacuum to obtain a yellow solid. Purity: 99%, yield: 77%.

[0131] 1.5 Preparation of selenium-containing polyurethane

[0132] 3.3 g of 11,11'-diselenodimethylbis(undecyl-1-ol), 4.5 g of ethyl lysine diisocyanate, and 20 mL of N,N-dimethylacetamide were added to a 250 mL three-necked flask, and the mixture was stirred and bubbled under nitrogen. 41 mg of dibutyltin dilaurate was added, and the mixture was heated to 65 °C and reacted for 1 h. Azide ethanol was added, and the mixture was reacted at 65 °C for 1 h. The mixture was precipitated three times with 200 mL of anhydrous diethyl ether and dried under vacuum to give a yellow solid. The purity was 99%, and the yield was 93%. Figure 1 This is the 1H NMR spectrum of selenium-containing polyurethane.

[0133] The reaction equation is as follows:

[0134]

[0135] m=11, n=13.

[0136] 1.6 Preparation of dendritic polyamino acid-modified polyurethane

[0137] In a 100 mL flask, 1.0 g of selenium-containing polyurethane, 0.9 g of tetratert-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 sequentially, and the mixture was stirred to dissolve. 334 μL of pentamethyldiethylenetriamine and 57 mg of CuBr were added, and the reaction was carried out at 45 °C for 24 h. The mixture was then dialyzed and lyophilized. The lyophilized product was added to a 100 mL flask and dissolved in 20 mL of anhydrous DCM. The mixture was stirred in an ice bath for 30 min, followed by dropwise addition of 10 mL of trifluoroacetic acid. After stirring for 10 min, the mixture was reacted at room temperature for 4 h. The solvent was removed under reduced pressure, and the product was precipitated three times with 300 mL of anhydrous diethyl ether. The precipitate was dried under vacuum to give a yellow solid. The purity was 99%, and the yield was 95%. Figure 2 The 1H NMR spectrum of dendritic polyamino acid-modified polyurethane.

[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] 440 mg of dendritic polyamino acid-modified polyurethane was added to a 100 mL flask, dissolved in 10 mL of anhydrous DMF, followed by the addition of 400 mg of dimethyl maleic anhydride and 27 μL of triethylamine. The mixture was reacted at 25 °C for 24 h. Precipitation was performed three times with 100 mL of anhydrous diethyl ether, and the solid was dried under vacuum to obtain a yellow solid. The purity was 99%, and the yield was 93%. Figure 3 The proton NMR spectrum of a responsive dendritic polyamino acid-modified polyurethane.

[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] 20.0 mg of dendritic polyamino acid-modified polyurethane and 20.0 mg of responsive dendritic polyamino acid-modified polyurethane were dissolved separately in 20 mL of DMF and mixed. 2 mL of the mixture was slowly added dropwise to 6 mL of stirred PBS (pH 7.4). After dialyzing with PBS (pH 7.4), responsive dendritic polyamino acid-modified polyurethane nanoparticles were obtained. Figure 4 Transmission electron microscope 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 approximately 50 nm were prepared.

[0150] 1.9 Nanoparticle-induced tumor cell apoptosis

[0151] 100,000 B16 tumor cells were seeded in 12-well plates. After 12 h, the nanoparticles were diluted to 20 μg / mL with 1640 complete medium (pH 6.8), the supernatant was discarded, and 1 mL of nanoparticles was added to each well. The plates were then incubated for 24 h. PBS was used as a control group. Apoptosis was detected using the Annexin V-FITC / PI apoptosis detection kit. Figure 5 This is a diagram showing the results of cell apoptosis.

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

[0153] 1.10 Nanoparticles induce immunogenic death in tumor cells

[0154] 100,000 B16 tumor cells were seeded in 12-well plates. After 12 h, the nanoparticles were diluted to 20 μg / mL with 1640 complete medium (pH 6.8), the supernatant was discarded, and 1 mL of nanoparticles was added. The cells were then cultured for 24 h. PBS was used as a control group. Cells were collected, and CRT eversion was detected by flow cytometry. Figure 6 This is a graph showing the results of CRT flow cytometry for cells.

[0155] Depend on Figure 6 It can be seen that nanoparticles can induce tumor cells to evert (CRT).

[0156] 1.11 Nanoparticles induce downregulation of tumor cell immune checkpoints

[0157] 80,000 B16 tumor cells were seeded in 12-well plates and cultured for 24 h after 12 h with IFN-γ (20 ng / mL). Nanoparticles were diluted to 20 μg / mL with 1640 complete medium (pH 6.8), the supernatant was discarded, 1 mL of nanoparticles was added, and the plates were cultured for 24 h. PBS was used as a control group. Cells were collected, and immune checkpoints were detected by flow cytometry. Figure 7 This is a flow cytometry result of cellular immune checkpoints.

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

[0159] 1.12 Evaluation of Tumor Treatment Efficacy

[0160] One million B16 tumor cells were subcutaneously inoculated into C57BL / 6 mice; after 7 days, the tumor volume was approximately 100 mm. 3 Mice were injected with 100 μL of nanoparticles at a concentration of 4 mg / mL via the tail vein every three days. PBS was used as a control group. Tumor volume was recorded every two days. Figure 8 This is a graph showing the growth of tumor volume.

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

[0162] The above description of the embodiments is only for the purpose of helping to understand the method and core ideas of the present invention. It should be noted that those skilled in the art can make several improvements and modifications to the present invention without departing from the principles of the present invention, and these improvements and modifications also fall within the protection scope of the claims of the present invention.

Claims

1. A responsive dendrimer polyamino acid modified polyurethane having the structure of Formula I: Formula I R1= Se; m is an integer selected from 1 to 50; n is an integer selected from 1 to 100.

2. A method of preparing the responsive dendrimer polyamino acid modified polyurethane of claim 1, comprising the steps of: performing a click reaction between a dendrimer polyamino acid of Formula I-a and a selenium-containing polyurethane of Formula I-b to obtain a dendrimer polyamino acid modified polyurethane of Formula I-c; performing an opening ring reaction between the dendrimer polyamino acid modified polyurethane of Formula I-c and dimethyl maleic anhydride or cis-aconitic anhydride to obtain a responsive dendrimer polyamino acid modified polyurethane of Formula I-d; Formula I-a; Formula I-b; Formula I-c Formula I-d; m is an integer selected from 1 to 50; n is an integer selected from 1 to 100.

3. The dendrimer polyamino acid of Formula I-a is prepared by the following steps: performing a reaction between 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-triazatetraoxan-20,24-diyl)dicarbamate and 2,6-di-tert-butoxycarbonylamino hexanoic acid to obtain 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(azanediyl))bis(carbonyl))bis(10-(tert-butoxycarbonyl)amino)-2,2-dimethyl-4,11,19-trioxo-3-oxa-5,12,18-trioxatetraoxan-13,20,24-triyl))tetraaminocarbamate. ； ； ； 4. 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-triazatetraoxan-20,24-diyl)dicarbamate is prepared by the following method: performing a reaction between di-tert-butyl (6-oxo-6-(prop-2-yn-1-ylamino)hexane-1,5-diyl)(S)-dicarbamate and 2,6-di-tert-butoxycarbonylamino hexanoic acid 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-triazatetraoxan-20,24-diyl)dicarbamate.

5. The di-tert-butyl (6-oxo-6-(prop-2-yn-1-ylamino)hexane-1,5-diyl)(S)-dicarbamate is prepared by the following method: ​ ​ ​ ​ ​ ​ ​ ； ​ ​ 3. The production method according to claim 2, characterized by, ​ ​ 4. The production method according to claim 3, characterized by, ​ ​ 5. The preparation method according to claim 4, characterized in that, ​ 2,6-di-tert-butoxycarbonylaminohexanoic acid and propargylamine are reacted 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 shown in formula I-b is prepared according to the following method: 11,11'-diselenolediyl bis (undecanol-1-ol) is sequentially reacted with lysine diisocyanate ethyl ester and azidoethanol to obtain the selenium-containing polyurethane shown in formula I-b.

7. The preparation method according to claim 2, characterized in that, The mass ratio of the dendritic polyamino acid shown in formula I-a and the selenium-containing polyurethane shown in formula I-b is 1: (0.1-10); Five-methyl diethylene triamine and CuBr are added in the click reaction process; The solvent of the click reaction is DMF; The temperature of the click reaction is 30-60℃, and the time of the click reaction is 20-30h.

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

9. The responsive dendrimeric polyamino acid-modified polyurethane nanoparticle of claim 8, wherein, The nanoparticles are prepared from the responsive dendritic polyamino acid modified polyurethane of claim 1 and the dendritic polyamino acid modified polyurethane shown in formula I-c; Formula I-c.

10. The use of the responsive dendritic polyamino acid modified polyurethane of claim 1 or the responsive dendritic polyamino acid modified polyurethane nanoparticles of any one of claims 8-9 in the preparation of a tumor immunotherapy reagent.

Images