Amphiphilic polyamino acid assembly as well as preparation method and application thereof

Abstract

The invention relates to an amphiphilic polyamino acid assembly as well as a preparation method and application thereof, and belongs to the technical field of macromolecular biological materials. In order to solve the technical problems in the prior art that the preparation process of polyamino acid is limited and the polymerization-induced self-assembly reaction is difficult to carry out due to the characteristic that N-carboxylic anhydride is easy to hydrolyze, the invention provides the preparation method of the amphiphilic polyamino acid assembly. Controlling the pH value in the reaction system; and hydrophilic macromolecular amino-terminated polyethylene glycol monomethyl ether is selected as an initiator. The preparation method provided by the invention is simple and easy to operate, the preparation cost is relatively low, the product purity is relatively high, the synthesis efficiency is relatively high, the product property is stable, the product structure is simple and easy to regulate and control, and the product has certain stereoregularity and can be applied to industrial large-scale preparation of the polyamino acid assembly; and the material can be used as a drug release carrier to be applied to biomedical materials.

Description

Technical Field

[0001] This invention belongs to the field of polymer biomaterials technology, and relates to a method for preparing polyamino acid assemblies, as well as a polyamino acid assembly and its application. Background Technology

[0002] Polyamino acids are high-molecular-weight biomaterials with excellent biocompatibility, biodegradability, and environmental friendliness, and are widely used in industry, agriculture, national defense, and daily life, becoming a research hotspot in the field of materials worldwide. If polyamino acids are amphiphilic, they can self-assemble into nanoscale micelle particles, which can be applied in the biomedical field as drug delivery carriers. The self-assembly of amphiphilic polymers is an efficient strategy for designing nanomaterials with unique properties. Chemically synthesized polyamino acids are a new class of biomedical materials; therefore, the precise design of amphiphilic polyamino acid block copolymers is of great significance.

[0003] Currently, the most economical and efficient method for preparing polyamino acids is a one-step preparation method based on the ring-opening polymerization of N-carboxylic anhydride (NCA) monomers. However, the NCA polymerization reaction is subject to many limitations, such as cumbersome monomer purification steps, sensitivity to air humidity, and the use of toxic organic solvents. Therefore, this synthesis method requires relatively complex steps, resulting in a low yield of the target product, which is not conducive to large-scale production and application. Furthermore, the use of toxic organic solvents, if not thoroughly removed in the later stages, will hinder further application in medical materials.

[0004] Polymerization-induced self-assembly (PISA) is a simple and rapid method for preparing polyamino acid assemblies. PISA can achieve the goal of self-assembling of amphiphilic polymer chains into nanostructures during in-situ growth. However, due to the easy hydrolysis of NCA, it is difficult to carry out ring-opening polymerization of NCA in aqueous phase using PISA.

[0005] Those skilled in the art have long sought to solve the technical problems in traditional polyamino acid preparation methods, such as cumbersome synthesis steps, high environmental requirements, use of toxic organic solvents, and the difficulty in PISA reactions due to the easy hydrolysis of NCA. Summary of the Invention

[0006] To address the limitations in the preparation process of polyamino acids in existing technologies, and the difficulty in initiating polymerization-induced self-assembly reactions due to the easy hydrolysis of N-carboxylic anhydrides, this invention provides an amphiphilic polyamino acid assembly, its preparation method, and its applications.

[0007] One objective of this invention is to provide a method for preparing amphiphilic polyamino acid assemblies, the method comprising the following steps:

[0008] S1: The amino acid was reacted with triphosgene under a nitrogen atmosphere, and then recrystallized from tetrahydrofuran and n-hexane to obtain the amino acid N-carboxylic acid anhydride.

[0009] S2: The amino acid N-carboxylic acid anhydride obtained in S1 and the initiator are polymerized in an alkaline aqueous solution in a certain proportion to obtain a polyamino acid assembly.

[0010] In a preferred embodiment of the present invention, the structural formula of the amino acid in S1 is:

[0011]

[0012] In the structural formula, R is any one of methyl, benzyl, isopropyl, n-butyl, isobutyl, indolemethyl, 2-methylthioethyl, 3-benzylpropionate or 4-benzyloxybutyramide; m is any one of 45, 113 or 227; n is any one of 10-120.

[0013] In a preferred embodiment of the present invention, the molar ratio of the amino acid to triphosgene in S1 is 1:0.4.

[0014] In a preferred embodiment of the present invention, the reaction solvent in S1 is tetrahydrofuran, the reaction temperature is 55°C, and the reaction time is 2-3 hours.

[0015] In a preferred embodiment of the present invention, the molar ratio of the amino acid N-carboxylic acid anhydride and the initiator in S2 is 1:(10-120).

[0016] In a preferred embodiment of the present invention, the initiator in S2 is an amino-terminated polyethylene glycol monomethyl ether, and the molecular weight of the amino-terminated polyethylene glycol monomethyl ether is 2000, 5000 or 10000.

[0017] In a preferred embodiment of the present invention, the alkaline aqueous solution in S2 is a 50 mM sodium bicarbonate aqueous solution, and the pH value of the sodium bicarbonate aqueous solution is 8.5.

[0018] In a preferred embodiment of the present invention, the polymerization reaction described in S2 is carried out in a cleaning ultrasonic machine, the reaction temperature of the polymerization reaction is 0°C, and the reaction time of the polymerization reaction is 0.5-2h.

[0019] The second objective of this invention is to provide an amphiphilic polyamino acid assembly, which is obtained by the above-described preparation method.

[0020] A third objective of this invention is to provide the application of the above-mentioned amphiphilic polyamino acid assemblies in biomedical materials, wherein the application refers to using the amphiphilic polyamino acid assemblies as a carrier for drug release.

[0021] The beneficial effects of this invention are:

[0022] Because NCA is easily hydrolyzed, the ring-opening polymerization of NCA monomers in existing technologies is often carried out in organic solvents (such as chloroform or dichloromethane), making it difficult to prepare polyamino acid assemblies using PISA.

[0023] The present invention provides a method for preparing amphiphilic polyamino acid assemblies with high stereoregularity. On the one hand, by conducting the polymerization reaction in a cleaning ultrasonic machine and controlling the pH of the reaction system at 8.5 using a sodium bicarbonate aqueous solution, the nucleophilic attack rate associated with the amino group is ensured to be greater than the hydrolysis rate. On the other hand, by selecting hydrophilic macromolecular terminal amino polyethylene glycol monomethyl ether as an initiator, micelles from the amphiphilic block copolymer protect the NCA monomer in the early stages of polymerization, preventing unreacted NCA monomers from being destroyed by the aqueous solvent during polymerization. Through these technical means, the present invention overcomes the technical problems of easy hydrolysis of NCA monomers during ring-opening polymerization and the difficulty in preparing polyamino acid assemblies using PISA.

[0024] Compared with traditional methods, the method for preparing amphiphilic polyamino acid assemblies provided by this invention has simpler and easier synthesis steps, lower requirements for environmental conditions, does not use toxic organic solvents, reduces the work of removing toxic organic solvents in the later stage, lowers the preparation cost, and has higher synthesis efficiency. In addition, the prepared amphiphilic polyamino acid assemblies have the advantages of high product purity, stable product properties, simple product structure, and easy control.

[0025] The method for preparing amphiphilic polyamino acid assemblies provided by this invention can be applied to the large-scale industrial preparation of polyamino acid assemblies. The prepared polyamino acid assemblies have the characteristics of being amphiphilic and having high stereoregularity. The polymer-constructed assemblies can be used as drug release carriers in biomedical materials, and have broad potential application and research value. Attached Figure Description

[0026] Figure 1 This is a structural diagram of the poly(ε-benzyloxycarbonyllysine) assembly in Example 1;

[0027] Figure 2 The poly-ε-benzyloxycarbonyl lysine assembly in Example 1 1 H NMR spectrum;

[0028] Figure 3Homonuclear decoupling of the poly-ε-benzyloxycarbonyl lysine-assembly in Example 1 1 H NMR spectrum;

[0029] Figure 4 This is a TEM image of the poly(ε-benzyloxycarbonyl lysine) assembly in Example 1;

[0030] Figure 5 This is a structural diagram of the polyglutamic acid benzyl ester assembly in Example 2;

[0031] Figure 6 The polyglutamic acid benzyl ester assembly in Example 2 1 H NMR spectrum;

[0032] Figure 7 This is a TEM image of the polyglutamic acid benzyl ester assembly in Example 2;

[0033] Figure 8 This is a structural diagram of the polyphenylalanine-assembly in Example 3;

[0034] Figure 9 The polyphenylalanine assembly in Example 3 1 H NMR spectrum;

[0035] Figure 10 Homonuclear decoupling of the polyphenylalanine assembly in Example 3 1 H NMR spectrum;

[0036] Figure 11 This is a TEM image of the polyphenylalanine assembly in Example 3;

[0037] Figure 12 This is a structural diagram of the polyalanine assembly in Example 4;

[0038] Figure 13 This is a structural diagram of the polyvaline assembly in Example 5;

[0039] Figure 14 This is a structural diagram of the polyleucine assembly in Example 6;

[0040] Figure 15 This is a structural diagram of the polyisoleucine assembly in Example 7;

[0041] Figure 16 This is a structural diagram of the polymethionine assembly in Example 8;

[0042] Figure 17 This is a structural diagram of the polytryptophan assembly in Example 9. Detailed Implementation

[0043] Those skilled in the art can refer to the content of this document and appropriately improve the process parameters to achieve the desired results. It should be particularly noted that all similar substitutions and modifications are obvious to those skilled in the art and are considered to be included in this invention. The methods and applications of this invention have been described through preferred embodiments, and those skilled in the art can obviously make modifications or appropriate alterations and combinations to the methods and applications described herein without departing from the content and scope of this invention to implement and apply the technology of this invention.

[0044] To make the objectives, technical solutions, and advantages of this invention clearer, the invention will be further described in detail below with reference to specific embodiments and accompanying drawings. Unless otherwise specified, the experimental methods used in the following embodiments are conventional methods, and the materials, reagents, methods, and instruments used are all conventional materials, reagents, methods, and instruments in the art, and can be obtained commercially by those skilled in the art.

[0045] The methods for determining the ring-opening polymerization conversion rate and number-average molecular weight described in the following examples are as follows: 1 ¹H NMR was performed using a Bruker 500MHz instrument with CDCl₃ as the solvent at a test temperature of 25°C. Molecular weight distribution was determined by gel permeation chromatography using a Waters e2695 separation unit and a Waters 2414 refractive index detector, with DMF as the solvent and a flow rate of 1 mL / min. -1 The test temperature was 50℃.

[0046] Example 1: Preparation of poly(ε-benzyloxycarbonyllysine) assemblies

[0047] S1: First, add ε-benzyloxycarbonyl lysine (6.0 g, 21.4 mmol) to a 500 mL round-bottom flask, then add 60 mL of anhydrous tetrahydrofuran solvent, and then add triphosgene (2.5 g, 8.6 mmol) to obtain a mixed solution;

[0048] S2: The mixed solution obtained in S1 was stirred at 50°C under a nitrogen atmosphere for 3 hours, then the tetrahydrofuran solvent was removed by rotary evaporation, and then dissolved in 30 mL of tetrahydrofuran. The solution was then recrystallized in n-hexane, sealed after recrystallization, and placed in a -20°C refrigerator for 24 hours. The above steps were repeated 3 times to obtain ε-benzyloxycarbonyllysine-N-carboxylic anhydride (ZLL-NCA) white solid.

[0049] S3: Weigh 16.3 mg (0.0033 mmol) of amino-terminated polyethylene glycol monomethyl ether, add 1126 μL of prepared sodium bicarbonate aqueous solution to dissolve it, and obtain the initiator solution;

[0050] S4: Weigh 40 mg (0.13 mmol) of ε-benzyloxycarbonyl lysine-N-carboxylic anhydride obtained in S2 and place it in a 10 mL centrifuge tube. Then, under ice-water mixture conditions, transfer the initiator solution prepared in S3 to the centrifuge tube for reaction. The pH of the reaction system is 8.5. Then place the centrifuge tube in an ultrasonic cleaner and stir continuously for 90 min to obtain the reaction solution.

[0051] S5: The reaction solution obtained in S4 was transferred to a 3.5 kDa dialysis membrane and dialyzed in deionized water for 3 days to obtain a dispersion. The dispersion was then freeze-dried to obtain a white powder of poly(ε-benzyloxycarbonyl lysine) assembly. The structural formula of the polyamino acid assembly is as follows: Figure 1 As shown.

[0052] This embodiment performs relevant tests on the obtained ε-benzyloxycarbonyllysine-N-carboxylic anhydride or polyε-benzyloxycarbonyllysine assembly. The results show that the yield of ε-benzyloxycarbonyllysine-N-carboxylic anhydride obtained in S2 of this embodiment is 5.18 g, with a yield of 80%; 1H NMR spectrum. 1 H NMR (400MHz, DMSO-d6) δ9.08 (s, 1H), δ7.40-7.28 (m, 5H) 7.26 (t, J=6.1Hz, 1H) 5.00 (s, 2H) 4 .43 (dd, J=7.4, 5.1Hz, 1H) 2.99 (dt, J=6.1, 6.1Hz, 2H) 1.80-1.58 (m, 1H), 1.48-1.22 (m, 4H).

[0053] The yield of the poly(ε-benzyloxycarbonyllysine) assembly obtained in Example S5 was 81%; 1H NMR spectrum. 1 ¹H NMR (500MHz, CDCl₃) δ 7.40–7.28 (m, 5H), 5.1 (t, 2H), 4.45 (t, 1H), 3.25 (t, 1H), 1.85–1.25 (m, 6H), results as follows Figure 2 As shown; 1H NMR spectrum 1 The number-average molecular weight was 15480 g / mol as determined by 1H NMR, and the number-average degree of polymerization was 40. The molecular weight distribution was 1.20 as determined by gel permeation chromatography. Homonuclear decoupled 1H NMR spectrum... 1 The H NMR results are as follows Figure 3 As shown, its P m A value of 0.72 is shown in the TEM image of the ε-benzyloxycarbonyl lysine-polyamino acid assembly, as follows. Figure 4 As shown.

[0054] Example 2: Preparation of polyglutamic acid benzyl ester assemblies

[0055] S1: First, add benzyl glutamate (6.0 g, 25.3 mmol) to a 500 mL round-bottom flask, then add 60 mL of anhydrous tetrahydrofuran solvent, and then add triphosgene (3 g, 10.1 mmol) to obtain a mixed solution;

[0056] S2: The mixed solution obtained in S1 was stirred at 50°C under a nitrogen atmosphere for 3 hours, then the tetrahydrofuran solvent was removed by rotary evaporation, and then dissolved in 30 mL of tetrahydrofuran. The solution was then recrystallized in n-hexane, sealed after recrystallization, and placed in a -20°C refrigerator for 24 hours. The above steps were repeated 3 times to obtain a white solid of benzyl glutamate-N-carboxylic anhydride (BLG-NCA).

[0057] S3: Weigh 19.0 mg (0.0038 mmol) of amino-terminated polyethylene glycol monomethyl ether, add 1180 μL of prepared sodium bicarbonate aqueous solution to dissolve it, and obtain the initiator solution;

[0058] S4: Weigh 40 mg (0.15 mmol) of benzyl glutamate-N-carboxylic anhydride obtained in S2 and place it in a 10 mL centrifuge tube. Under ice-water mixture conditions, transfer the initiator solution prepared in S3 to the centrifuge tube for reaction. The pH of the reaction system is 8.5. Then place the centrifuge tube in an ultrasonic cleaner and stir continuously for 90 min to obtain the reaction solution.

[0059] S5: The reaction solution obtained in S4 is transferred to a 3.5 kDa dialysis membrane and dialyzed in deionized water for 3 days to obtain a dispersion. The dispersion is then freeze-dried to obtain a white powder of polyglutamic acid benzyl ester assembly. The structural formula of the polyamino acid assembly is as follows: Figure 5 As shown.

[0060] This embodiment performs relevant tests on the obtained benzyl glutamate-N-carboxylic anhydride or polybenzyl glutamate assembly. The results show that the yield of benzyl glutamate-N-carboxylic anhydride obtained in S2 of this embodiment is 5.58 g, with a yield of 84%; 1H NMR spectroscopy... 1 ¹H NMR (400MHz, CDCl₃) δ 7.42–7.31 (m, 5H), 6.70 (br, 1H), 5.13 (s, 2H), 4.38 (ddd, J = 6.7, 5.4, 1.0 Hz, 1H), 2.59 (t, J = 6.9 Hz, 1H), 2.32–2.21 (m, 1H), 2.18–2.06 (m, 1H). The results are as follows: Figure 6 As shown; the yield of the polyglutamic acid benzyl ester assembly obtained in S5 of this embodiment was 78%. TEM image of the polyglutamic acid benzyl ester assembly is shown below. Figure 7 As shown.

[0061] Example 3: Preparation of polyphenylalanine assemblies

[0062] S1: In a 500 mL round-bottom flask, first add phenylalanine (6.0 g, 36.4 mmol), then add 60 mL of anhydrous tetrahydrofuran solvent, and then add triphosgene (4.32 g, 14.5 mmol) to obtain a mixed solution;

[0063] S2: The mixed solution obtained in S1 was stirred at 50°C under a nitrogen atmosphere for 3 hours, then the tetrahydrofuran solvent was removed by rotary evaporation, and then dissolved in 30 mL of tetrahydrofuran. The solution was then recrystallized in n-hexane, sealed after recrystallization, and placed in a -20°C refrigerator for 24 hours. The above steps were repeated 3 times to obtain a white solid of phenylalanine-N-carboxylic anhydride (Phe-NCA).

[0064] S3: Weigh 26.2 mg (0.0052 mmol) of amino-terminated polyethylene glycol monomethyl ether, add 1324 μL of prepared sodium bicarbonate aqueous solution to dissolve it, and obtain the initiator solution;

[0065] S4: Weigh 40 mg (0.21 mmol) of phenylalanine-N-carboxylic anhydride obtained in S2 and place it in a 10 mL centrifuge tube. Then, under ice-water mixture conditions, transfer the initiator solution prepared in S3 to the centrifuge tube for reaction. The pH of the reaction system is 8.5. Then place the centrifuge tube in an ultrasonic cleaner and stir continuously for 90 min to obtain the reaction solution.

[0066] S5: The reaction solution obtained in S4 is transferred to a 3.5 kDa dialysis membrane and dialyzed in deionized water for 3 days to obtain a dispersion. The dispersion is then freeze-dried to obtain a white powder of polyphenylalanine assembly. The structural formula of the polyamino acid assembly is as follows: Figure 8 As shown.

[0067] In this embodiment, the obtained phenylalanine-N-carboxylic anhydride or polyphenylalanine assembly was subjected to relevant tests. The results showed that the yield of phenylalanine-N-carboxylic anhydride obtained in S2 of this embodiment was 5.77 g, with a yield of 83%; 1H NMR spectrum... 1 HNMR (400MHz, CDCl3) δ7.38-7.27 (m, 3H), 7.17 (dd, J=7.7, 1.8Hz, 2H), 6.48 (br, 1H), 4.53 (ddd, J=7.8, 4.3, 1.0Hz, 1H), 3.24 (dd, J=14.2, 4.3Hz, 1H), 3.01 (dd, J=14.2, 7.8Hz, 1H), such as Figure 9 As shown.

[0068] The polyphenylalanine assembly obtained in Example S5 had a yield of 76%; 1H NMR spectrum. 11H NMR (500MHz, CDCl3) δ 7.28–7.20 (m, 5H), 4.65 (t, 1H), 2.85 (t, 2H), 1H NMR spectrum 1 The number-average molecular weight was 10880 g / mol as determined by 1H NMR, and the number-average degree of polymerization was 40. The molecular weight distribution was 1.12 as determined by gel permeation chromatography. Homonuclear decoupled 1H NMR spectrum... 1 The H NMR results are as follows Figure 10 As shown, its P m A value of 0.74, TEM image of phenylalanine polyamino acid assembly, as shown. Figure 11 As shown.

[0069] Example 4: Preparation of polyalanine assemblies

[0070] S1: First, add alanine (6.0 g, 67.4 mmol) to a 500 mL round-bottom flask, then add 60 mL of anhydrous tetrahydrofuran solvent, and then add triphosgene (8.0 g, 26.9 mmol) to obtain a mixed solution;

[0071] S2: The mixed solution obtained in S1 was stirred at 50°C under a nitrogen atmosphere for 3 hours, then the tetrahydrofuran solvent was removed by rotary evaporation, and then dissolved in 30 mL of tetrahydrofuran. The solution was then recrystallized in n-hexane, sealed after recrystallization, and placed in a -20°C refrigerator for 24 hours. The above steps were repeated 3 times to obtain a white solid of alanine-N-carboxylic anhydride (Ala-NCA).

[0072] S3: Weigh 43.5 mg (0.0087 mmol) of amino-terminated polyethylene glycol monomethyl ether, add 1670 μL of prepared sodium bicarbonate aqueous solution to dissolve it, and obtain the initiator solution;

[0073] S4: Weigh 40 mg (0.21 mmol) of alanine-N-carboxylic anhydride obtained in S2 and place it in a 10 mL centrifuge tube. Under ice-water mixture conditions, transfer the initiator solution prepared in S3 to the centrifuge tube for reaction. The pH of the reaction system is 8.5. Then place the centrifuge tube in an ultrasonic cleaner and stir continuously for 90 min to obtain the reaction solution.

[0074] S5: The reaction solution obtained in S4 is transferred to a 3.5 kDa dialysis membrane and dialyzed in deionized water for 3 days to obtain a dispersion. The dispersion is then freeze-dried to obtain a white powder of polyalanine assembly. The structural formula of the polyamino acid assembly is as follows: Figure 12 As shown.

[0075] In this embodiment, the alanine-N-carboxylic anhydride or polyalanine assembly obtained above was subjected to relevant tests. The results showed that the yield of alanine-N-carboxylic anhydride obtained in S2 of this embodiment was 4.93 g, with a yield of 64%; 1H NMR spectrum.1 ¹H NMR (400MHz, CDCl₃) δ 6.76 (br, 1H) 4.42 (qd, J = 7.0, 1.0Hz, 1H) 1.56 (d, J = 7.0Hz, 3H); The yield of the polyalanine assembly obtained in S5 of this example was 68%.

[0076] Example 5: Preparation of polyvaline assemblies

[0077] S1: First, add alanine (6.0 g, 51.3 mmol) to a 500 mL round-bottom flask, then add 60 mL of anhydrous tetrahydrofuran solvent, and then add triphosgene (6.09 g, 20.5 mmol) to obtain a mixed solution;

[0078] S2: The mixed solution obtained in S1 was stirred at 50°C under a nitrogen atmosphere for 3 hours, then the tetrahydrofuran solvent was removed by rotary evaporation, and then dissolved in 30 mL of tetrahydrofuran. The solution was then recrystallized in n-hexane, sealed after recrystallization, and placed in a -20°C refrigerator for 24 hours. The above steps were repeated 3 times to obtain a white solid of valine-N-carboxylic anhydride (Val-NCA).

[0079] S3: Weigh 35.0 mg (0.0070 mmol) of amino-terminated polyethylene glycol monomethyl ether, add 1436 μL of prepared sodium bicarbonate aqueous solution to dissolve it, and obtain the initiator solution;

[0080] S4: Weigh 40 mg (0.28 mmol) of valine-N-carboxylic anhydride obtained in S2 and place it in a 10 mL centrifuge tube. Under ice-water mixture conditions, transfer the initiator solution prepared in S3 to the centrifuge tube for reaction. The pH of the reaction system is 8.5. Then place the centrifuge tube in an ultrasonic cleaner and stir continuously for 90 min to obtain the reaction solution.

[0081] S5: The reaction solution obtained in S4 is transferred to a 3.5 kDa dialysis membrane and dialyzed in deionized water for 3 days to obtain a dispersion. The dispersion is then freeze-dried to obtain a white powder of polyvaline assembly. The structural formula of the polyamino acid assembly is as follows: Figure 13 As shown.

[0082] This embodiment performs relevant tests on the valine-N-carboxylic anhydride / polyvaline assembly obtained above. The results show that the yield of valine-N-carboxylic anhydride obtained in S2 of this embodiment is 5.34 g, with a yield of 72%; 1H NMR spectrum. 1H NMR (400MHz, CDCl3) δ6.85 (br, 1H), 4.22 (dd, J=4.2, 1.0Hz, 1H), 2.25 (heptd, J=6.9, 4.2Hz, 1H), 1.08 (d, J=6.9Hz, 3H), 1.03 (d, J=6.9Hz, 3H).

[0083] Example 6: Preparation of polyleucine assemblies

[0084] S1: In a 500 mL round-bottom flask, first add leucine (6.0 g, 45.7 mmol), then add 60 mL of anhydrous tetrahydrofuran solvent, and then add triphosgene (5.43 g, 18.3 mmol) to obtain a mixed solution;

[0085] S2: The mixed solution obtained in S1 was stirred at 50°C under a nitrogen atmosphere for 3 hours, then the tetrahydrofuran solvent was removed by rotary evaporation, and then dissolved in 30 mL of tetrahydrofuran. The solution was then recrystallized in n-hexane, sealed after recrystallization, and placed in a -20°C refrigerator for 24 hours. The above steps were repeated 3 times to obtain a white solid of leucine-N-carboxylic anhydride (Leu-NCA).

[0086] S3: Weigh 31.8 mg (0.0064 mmol) of amino-terminated polyethylene glycol monomethyl ether, add 1436 μL of prepared sodium bicarbonate aqueous solution to dissolve it, and obtain the initiator solution;

[0087] S4: Weigh 40 mg (0.25 mmol) of leucine-N-carboxylic anhydride obtained in S2 and place it in a 10 mL centrifuge tube. Then, under ice-water mixture conditions, transfer the initiator solution prepared in S3 to the centrifuge tube for reaction. The pH of the reaction system is 8.5. Then place the centrifuge tube in an ultrasonic cleaner and stir continuously for 90 min to obtain the reaction solution.

[0088] S5: The reaction solution obtained in S4 is transferred to a 3.5 kDa dialysis membrane and dialyzed in deionized water for 3 days to obtain a dispersion. The dispersion is then freeze-dried to obtain a white powder of polyleucine assembly. The structural formula of the above leucine-polyamino acid assembly is as follows: Figure 14 As shown.

[0089] In this embodiment, the obtained leucine-N-carboxylic anhydride or polyleucine assembly was subjected to relevant tests. The results showed that the yield of leucine-N-carboxylic anhydride obtained in S2 of this embodiment was 4.31 g, with a yield of 60%; 1H NMR spectrum. 1H NMR (400MHz, DMSO-d6) δ9.13 (br, 1H), 4.45 (dd, J=8.7, 5.5Hz, 1H), 1.81-1.65 (m, 1H), 1.65-1.49 (m, 2H), 0.92-0.85 (m, 6H).

[0090] Example 7: Preparation of polyisoleucine assemblies

[0091] S1: In a 500 mL round-bottom flask, first add isoleucine (6.0 g, 45.7 mmol), then add 60 mL of anhydrous tetrahydrofuran solvent, and then add triphosgene (5.43 g, 18.3 mmol) to obtain a mixed solution;

[0092] S2: The mixed solution obtained in S1 was stirred at 50°C under a nitrogen atmosphere for 3 hours, then the tetrahydrofuran solvent was removed by rotary evaporation, and then dissolved in 30 mL of tetrahydrofuran. The solution was then recrystallized in n-hexane, sealed after recrystallization, and placed in a -20°C refrigerator for 24 hours. The above steps were repeated 3 times to obtain isoleucine-N-carboxylic anhydride (Ile-NCA) white solid.

[0093] S3: Weigh 31.8 mg (0.0064 mmol) of amino-terminated polyethylene glycol monomethyl ether, add 1436 μL of prepared sodium bicarbonate aqueous solution to dissolve it, and obtain the initiator solution;

[0094] S4: Weigh 40 mg (0.25 mmol) of isoleucine-N-carboxylic anhydride obtained in S2 and place it in a 10 mL centrifuge tube. Then, under ice-water mixture conditions, transfer the initiator solution prepared in S3 to the centrifuge tube for reaction. The pH of the reaction system is 8.5. Then place the centrifuge tube in an ultrasonic cleaner and stir continuously for 90 min to obtain the reaction solution.

[0095] S5: The reaction solution obtained in S4 is transferred to a 3.5 kDa dialysis membrane and dialyzed in deionized water for 3 days to obtain a dispersion. The dispersion is then freeze-dried to obtain a white powder of poly(isoleucine) assembly. The structural formula of the isoleucine-polyamino acid assembly is as follows: Figure 15 As shown.

[0096] In this embodiment, the isoleucine-N-carboxylic anhydride or polyisoleucine assembly obtained above was subjected to relevant tests. The results showed that the yield of isoleucine-N-carboxylic anhydride obtained in S2 of this embodiment was 4.31 g, with a yield of 60%; 1H NMR spectrum. 1HNMR (DMSO-d6, 400MHz, ppm) δ0.86 (t, 3H, J=9.0Hz), 0.92 (d, 3H, J=7.0Hz), 1.28 (m, 2H), 1.79 (s, 1H), 4.39, (s, 1H), 9.09 (br, 1H).

[0097] Example 8: Preparation of polymethionine assemblies

[0098] S1: First, add methionine (6.0 g, 40.2 mmol) to a 500 mL round-bottom flask, then add 60 mL of anhydrous tetrahydrofuran solvent, and then add triphosgene (4.78 g, 16.1 mmol) to obtain a mixed solution;

[0099] S2: The mixed solution obtained in S1 was stirred at 50°C under a nitrogen atmosphere for 3 hours, then the tetrahydrofuran solvent was removed by rotary evaporation, and then dissolved in 30 mL of tetrahydrofuran. The solution was then recrystallized in n-hexane, sealed after recrystallization, and placed in a -20°C refrigerator for 24 hours. The above steps were repeated 3 times to obtain a yellow oily substance of methionine-N-carboxylic anhydride (Met-NCA).

[0100] S3: Weigh 28.5 mg (0.0057 mmol) of amino-terminated polyethylene glycol monomethyl ether, add 1371 μL of prepared sodium bicarbonate aqueous solution to dissolve it, and obtain the initiator solution;

[0101] S4: Weigh 40 mg (0.23 mmol) of methionine-N-carboxylic anhydride obtained in S2 and place it in a 10 mL centrifuge tube. Then, under ice-water mixture conditions, transfer the initiator solution prepared in S3 to the centrifuge tube for reaction. The pH of the reaction system is 8.5. Then place the centrifuge tube in an ultrasonic cleaner and stir continuously for 90 min to obtain the reaction solution.

[0102] S5: The reaction solution obtained in S4 is transferred to a 3.5 kDa dialysis membrane and dialyzed in deionized water for 3 days to obtain a dispersion. The dispersion is then freeze-dried to obtain a polymethionine assembly powder. The structural formula of the methionine-polyamino acid assembly is as follows: Figure 16 As shown.

[0103] In this embodiment, the obtained methionine-N-carboxylic anhydride or polymethionine assembly was subjected to relevant tests. The results showed that the yield of methionine-N-carboxylic anhydride obtained in S2 of this embodiment was 4.2 g, with a yield of 60%; 1H NMR spectrum. 1HNMR (400MHz, CDCl3) δ7.06 (br, 1H), 4.51 (ddd, J=7.4, 5.0, 1.1Hz, 1H), 2.67 (t, J =6.6Hz, 2H), 2.25 (dtd, J=14.6, 6.6, 5.0Hz, 1H), 2.17-2.01 (m, 1H), 2.09 (s, 3H).

[0104] Example 9: Preparation of polytryptophan assemblies

[0105] S1: In a 500 mL round-bottom flask, first add tryptophan (6.0 g, 29.3 mmol), then add 60 mL of anhydrous tetrahydrofuran solvent, and then add triphosgene (3.49 g, 11.8 mmol) to obtain a mixed solution;

[0106] S2: The mixed solution obtained in S1 was stirred at 50°C under a nitrogen atmosphere for 3 hours, then the tetrahydrofuran solvent was removed by rotary evaporation, and then dissolved in 30 mL of tetrahydrofuran. The solution was then recrystallized in n-hexane, sealed after recrystallization, and placed in a -20°C refrigerator for 24 hours. The above steps were repeated 3 times to obtain tryptophan-N-carboxylic anhydride (Trp-NCA) white needle-like solid.

[0107] S3: Weigh 21.7 mg (0.0043 mmol) of amino-terminated polyethylene glycol monomethyl ether, add 1234 μL of prepared sodium bicarbonate aqueous solution to dissolve it, and obtain the initiator solution;

[0108] S4: Weigh 40 mg (0.17 mmol) of tryptophan-N-carboxylic anhydride obtained in S2 and place it in a 10 mL centrifuge tube. Then, under ice-water mixture conditions, transfer the initiator solution prepared in S3 to the centrifuge tube for reaction. The pH of the reaction system is 8.5. Then place the centrifuge tube in an ultrasonic cleaner and stir continuously for 90 min to obtain the reaction solution.

[0109] S5: The reaction solution obtained in S4 is transferred to a 3.5 kDa dialysis membrane and dialyzed in deionized water for 3 days to obtain a dispersion. The dispersion is then freeze-dried to obtain a polytryptophan-assembly powder. The structural formula of the above tryptophan-polyamino acid assembly is as follows: Figure 17 As shown.

[0110] In this embodiment, relevant tests were performed on the tryptophan-N-carboxylic anhydride or polytryptophan assembly obtained above. The results showed that the yield of tryptophan-N-carboxylic anhydride obtained in S2 of this embodiment was 6g, with a yield of 90%; 1H NMR spectrum. 1¹H NMR (400MHz, DMSO-d6) δ 11.00 (d, J = 2.5Hz, 1H), 9.09 (s, 1H), 7.55 (d, J = 7.9Hz, 1H), 7.36 (d, J = 8.1Hz, 1H), 7.15 (d, J = 2.5Hz, 1H), 7.13–7.05 (m, 1H), 7.05–6.96 (m, 1H), 4.78 (t, J = 5.0Hz, 1H), 3.22 (dd, J = 15.0, 5.0Hz, 1H), 3.14 (dd, J = 15.0, 5.0Hz, 1H); The polytryptophan assembly obtained in S5 of this example had a yield of 82%.

[0111] The contents not described in detail in this specification are well-known to those skilled in the art. Although the present invention has been disclosed above with reference to preferred embodiments, it is not intended to limit the invention. Anyone skilled in the art can make various modifications and alterations without departing from the spirit and scope of the invention. Therefore, the scope of protection of the present invention should be determined by the claims.

Claims

1. A method for preparing an amphiphilic polyamino acid assembly, characterized in that, The preparation method includes the following steps: S1: The amino acid was reacted with triphosgene under a nitrogen atmosphere, and then recrystallized from tetrahydrofuran and n-hexane to obtain the amino acid N-carboxylic acid anhydride. S2: The amino acid N-carboxylic acid anhydride obtained in S1 and the initiator are polymerized in an alkaline aqueous solution in a certain proportion to obtain a polyamino acid assembly.

2. The preparation method according to claim 1, characterized in that, The structural formula of the amino acid described in S1 is: In the structural formula, R is any one of methyl, benzyl, isopropyl, n-butyl, isobutyl, indolemethyl, 2-methylthioethyl, 3-benzylpropionate or 4-benzyloxybutyramide; m is any one of 45, 113 or 227; n is any one of 10-120.

3. The preparation method according to claim 1, characterized in that, The molar ratio of the amino acid to triphosgene in S1 is 1:0.

4.

4. The preparation method according to claim 1, characterized in that, The reaction solvent for the reaction described in S1 is tetrahydrofuran, the reaction temperature is 55℃, and the reaction time is 2-3h.

5. The preparation method according to claim 1, characterized in that, The molar ratio of the amino acid N-carboxylic acid anhydride and the initiator in S2 is 1:(10-120).

6. The preparation method according to claim 1, characterized in that, The initiator mentioned in S2 is amino-terminated polyethylene glycol monomethyl ether, and the molecular weight of the amino-terminated polyethylene glycol monomethyl ether is 2000, 5000 or 10000.

7. The preparation method according to claim 1, characterized in that, The alkaline aqueous solution mentioned in S2 is a 50mM sodium bicarbonate aqueous solution with a pH of 8.

5.

8. The preparation method according to claim 1, characterized in that, The polymerization reaction described in S2 is carried out in a cleaning ultrasonic machine, the reaction temperature is 0°C, and the reaction time is 0.5-2h.

9. An amphiphilic polyamino acid assembly, characterized in that, The amphiphilic polyamino acid assembly is obtained by the preparation method described in any one of claims 1 to 8.

10. The application of the amphiphilic polyamino acid assembly according to claim 9 in biomedical materials, characterized in that, The application refers to using amphiphilic polyamino acid assemblies as carriers for drug release.

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