Modified polypeptide as well as preparation method and application thereof

Abstract

The invention discloses a modified polypeptide as well as a preparation method and application thereof, the preparation method comprises the following steps: on the basis of solid-phase synthesis of RADA16, 1-hydroxybenzotriazole and benzotriazole-N, N, N ', N'-tetramethylurea hexafluorophosphate are adopted as condensing agents, N, N-diisopropylethylamine-containing N, N-diisopropylethylamine is added into the condensing agents, and a reaction is carried out for 2-4 hours to obtain the modified polypeptide. And adding amino-terminated or carboxyl-terminated polyethylene glycol or amino-containing chitosan oligosaccharide into N, N-dimethylformamide for condensation, cutting polypeptide from the resin after the reaction is completed, precipitating, washing, re-dissolving and freeze-drying to obtain the polypeptide. The polypeptide provided by the invention has the characteristic of forming hydrogel through self-assembly in a specific medium, and the gel forming time of the polypeptide is within 21-60 seconds and is obviously faster than that of RADA peptide (gel forming in 150 seconds) under the same condition; the polypeptide modified by the polyethylene glycol or the chitosan oligosaccharide can spontaneously form gel after being injected into the blood vessel so as to embolize the blood vessel, and the embolism is long in stabilization time and stable, and meets the actual clinical requirements.

Description

Technical Field

[0001] The present invention relates to the field of medical biomaterials, and in particular to a polyethylene glycol or chitosan oligosaccharide modified polypeptide and a preparation method thereof, as well as application of the polypeptide in preparing tumor vascular embolization hydrogel. Background Art

[0002] Transcatheter arterial embolization is an important interventional therapy. With the assistance and monitoring of medical imaging equipment (digital subtraction angiography, computed tomography, magnetic resonance imaging, etc.), embolic agents are injected into the blood vessels supplying the diseased organs through intra-arterial catheters, resulting in vascular occlusion and interruption of blood supply, thereby playing a role in treating tumors, vascular lesions and hemorrhagic diseases. Embolization therapy has the characteristics of visualization, localization, minimal invasion, high efficacy and low toxicity and side effects, and occupies an increasingly important position in clinical diagnosis and treatment.

[0003] As a key factor in transcatheter arterial embolization, embolic agents should have the following characteristics: 1. Non-toxic, non-carcinogenic and non-teratogenic; 2. Good tissue compatibility; 3. Easy to inject into the catheter, safe, convenient and non-sticky; 4. Controllable vascular embolization time; 5. Rapid, complete and effective embolization; 6. Visualizable under X-ray. Currently, the commonly used embolic agents in clinical practice mainly include 1. Spring coils, which are radiopaque and can be precisely positioned and released, but their general applicability is low and embolization is incomplete; 2. Gelatin sponge, which is an animal protein, easy to use and degradable in the body, but its embolization time is uncertain and prone to serious complications; 3. Polyvinyl alcohol particles, which have better embolic strength but uneven particle size and irregular shape, and are easy to block the catheter; 4. Iodized oil, which is the most widely used embolic agent in clinical practice, has excellent X-ray imaging capabilities, good fluidity and certain drug loading, but its stability is poor and the embolic strength is low. Therefore, there is still an urgent need for new embolic agents with excellent and reliable performance in clinical practice.

[0004] With the development of bioengineering and peptide synthesis technology, a class of peptides with self-assembling properties can be obtained through rational design strategies by utilizing the effects of different side chains on the physicochemical properties of amino acids. Self-assembling peptides can self-assemble into various nanostructures and even supramolecular structures under appropriate conditions through various reversible non-covalent interactions, such as hydrophobic interaction, π-π stacking, electrostatic interaction, hydrogen bonding and van der Waals forces. Among them, RADA16 peptide is a short peptide composed of arginine (R), aspartic acid (D) and alanine (A) connected in the RADA sequence. Under specific conditions, it can spontaneously form a β-pleated sheet structure, further self-assemble to form a hydrogel, and then can be used as a vascular embolic agent for interventional therapy. Summary of the invention

[0005] The technical problem to be solved by the present invention is to provide a modified polypeptide, a preparation method and application thereof in view of the deficiencies in the above-mentioned prior art.

[0006] In order to solve the above technical problems, the technical solution adopted by the present invention is: in the first aspect of the present invention, a modified polypeptide is provided, which is a polyethylene glycol or chitosan oligosaccharide modified polypeptide, which is composed of polyethylene glycol or chitosan oligosaccharide and RADA16 polypeptide covalently linked, and has the following general structural formula:

[0007]

[0008] Wherein m, n, o are the degree of polymerization, 0≤m≤44, 0≤n≤44, 0≤o≤30; the carbon end or nitrogen end of the RADA16 peptide is condensed and connected with polyethylene glycol or chitosan oligosaccharide.

[0009] The modified polypeptide is solid at room temperature, and its pure aqueous solution has good fluidity. When dissolved in a specific liquid environment, it can form a hydrogel with a certain mechanical strength. The specific liquid environment includes physiological saline, blood and body fluids.

[0010] Preferably, the polyethylene glycol-modified polypeptide is obtained by an acid-amine condensation reaction between terminal amino- or carboxyl-terminated polyethylene glycol and a RADA16 polypeptide.

[0011] Preferably, the chitosan oligosaccharide-modified polypeptide is obtained by acid-amine condensation reaction of chitosan oligosaccharide and RADA16 polypeptide.

[0012] The second aspect of the present invention provides a method for preparing the modified polypeptide as described above, comprising the following steps:

[0013] Based on the solid phase synthesis of RADA16, 1-hydroxybenzotriazole (HOBT) and benzotriazole-N,N,N',N'-tetramethyluronium hexafluorophosphate (HBTU) are used as condensation agents, and the modified components are added to N,N-dimethylformamide (DMF) containing N,N-diisopropylethylamine (DIEA) for condensation. After the reaction is completed, the peptide is cut from the resin, precipitated and washed, and then redissolved and freeze-dried to obtain a modified peptide freeze-dried powder;

[0014] Among them, the modification component is terminal amino- or terminal carboxyl-terminated polyethylene glycol, or chitosan oligosaccharide containing amino groups is added; when the modification component is terminal amino- or terminal carboxyl-terminated polyethylene glycol, a polyethylene glycol-modified polypeptide is obtained, and when the modification component is chitosan oligosaccharide containing amino groups, a chitosan oligosaccharide-modified polypeptide is obtained.

[0015] Preferably, the polyethylene glycol-modified polypeptide is prepared by the following method:

[0016] The RADA16 polypeptide is solid-phase synthesized. The RADA16 polypeptide is attached to a resin. The resin to which the RADA16 polypeptide is attached and 1-hydroxybenzotriazole and benzotriazole-N,N,N',N'-tetramethyluronium hexafluorophosphate are added to N,N-dimethylformamide (DMF) containing N,N-diisopropylethylamine, and then terminal amino or terminal carboxyl polyethylene glycol is added to react at room temperature. After the reaction is completed, the polypeptide is cut from the resin, precipitated and washed, and then redissolved and freeze-dried to obtain a polyethylene glycol-modified polypeptide freeze-dried powder.

[0017] Preferably, the molar ratio of polyethylene glycol to RADA16 polypeptide is 1-3:1; and the reaction time is 0.5-2 hours.

[0018] Preferably, the chitosan oligosaccharide-modified polypeptide is prepared by the following method:

[0019] The RADA16 polypeptide is solid-phase synthesized. The RADA16 polypeptide is attached to a resin. The resin to which the RADA16 polypeptide is attached and 1-hydroxybenzotriazole and benzotriazole-N,N,N',N'-tetramethyluronium hexafluorophosphate are added to N,N-dimethylformamide (DMF) containing N,N-diisopropylethylamine, and then chitosan oligosaccharides containing amino groups are added to react at room temperature. After the reaction is completed, the polypeptide is cut from the resin, precipitated and washed, and then redissolved and freeze-dried to obtain chitosan oligosaccharide-modified polypeptide freeze-dried powder.

[0020] Preferably, the molar ratio of chitosan oligosaccharide to RADA16 peptide is 0.5-2:1; and the reaction time is 0.5-2 hours.

[0021] Preferably, the molar ratio of 1-hydroxybenzotriazole to RADA16 polypeptide is 2-3:1; the molar ratio of benzotriazole-N,N,N',N'-tetramethyluronium hexafluorophosphate to RADA16 polypeptide is 2-3:1; the molar ratio of N,N-diisopropylethylamine to RADA16 polypeptide is 4-6:1;

[0022] With the mass unit being mg and the volume unit being mL, the ratio of the mass of RADA16 polypeptide to the volume of N,N-dimethylformamide is 2:1-8:1.

[0023] The third aspect of the present invention provides a use of the modified polypeptide as described above in the preparation of a hydrogel for tumor vascular embolization.

[0024] The beneficial effects of the present invention are:

[0025] The present invention provides a polypeptide modified with polyethylene glycol or chitosan oligosaccharide, which has the property of forming a hydrogel by self-assembly in a specific medium, and the gelation time thereof is within 21-60 seconds, which is significantly faster than RADA peptide (gelation in 150 seconds) under the same conditions; after being injected into a blood vessel, the polypeptide modified with polyethylene glycol or chitosan oligosaccharide can spontaneously gel and thus embolize the blood vessel, and the embolism is stable for a long time and is stable, which meets the actual clinical needs. BRIEF DESCRIPTION OF THE DRAWINGS

[0026] Figure 1 H NMR spectra of RADA16, peptide P2, and peptide P3;

[0027] Figure 2 The morphology of the polyethylene glycol-modified polypeptide P3 in different liquid media;

[0028] Figure 3 DSA images before and after renal artery embolization with different concentrations of peptide P1 and peptide P2. DETAILED DESCRIPTION

[0029] The present invention is further described in detail below in conjunction with embodiments so that those skilled in the art can implement the invention with reference to the description.

[0030] It should be understood that the terms such as “having”, “including” and “comprising” used herein do not exclude the existence or addition of one or more other elements or combinations thereof.

[0031] The test methods used in the following examples are conventional methods unless otherwise specified. The materials and reagents used in the following examples are all commercially available unless otherwise specified. In the following examples, if no specific conditions are specified, the experiments were carried out under conventional conditions or conditions recommended by the manufacturer. The reagents or instruments used, if the manufacturer is not specified, are all conventional products that can be purchased commercially.

[0032] The sequence of the RADA16 peptide in the following examples is: RADARADARADARADARADA (SEQ ID NO. 1).

[0033] Example 1

[0034] 1. Preparation and physicochemical properties of polyethylene glycol-modified peptides

[0035] This example provides a polyethylene glycol-modified polypeptide: Ac-RADARADARADARADA-PEG C 69 H 122 N 28 O 27The preparation method of (denoted as P1) utilizes the condensation reaction between the amino group with strong reactivity in the terminal carboxyl polyethylene glycol and the carboxyl group of the RADA peptide, and the specific steps are as follows:

[0036] On the basis of using CTC-Resin resin to solid-phase synthesize 200 mg of RADA16 polypeptide from carbon end to nitrogen end according to the amino acid sequence, a liquid phase synthesis method was further used to condense polyethylene glycol with the carboxyl group of the carbon end of RADA16. 47.3 mg of 1-hydroxybenzotriazole and 88.6 mg of benzotriazole-N,N,N',N'-tetramethyluronium hexafluorophosphate were weighed as condensation agents. 31 mg of terminal amino ethylene glycol was added to 50 mL of N,N-dimethylformamide containing 200 mg of RADA16 polypeptide and 90.6 mg of N,N-diisopropylethylamine for condensation. The reaction was carried out for 2 hours. After the reaction was completed, ether was added, precipitated and washed, and freeze-dried to obtain a polyethylene glycol-modified polypeptide freeze-dried powder with a molecular weight of 1788.98.

[0037] The gelation time of the polyethylene glycol-modified polypeptide in normal saline was determined by the vial inversion method. 100 mg of the polyethylene glycol-modified polypeptide was weighed and added to a vial containing 2 mL of normal saline. The vial was placed in an ultrasonic instrument at 30°C and inverted every 3 seconds. The gelation time of the polyethylene glycol-modified polypeptide was measured to be 60 seconds.

[0038] 2. Study on the blocking effect of polyethylene glycol-modified peptides on rabbit renal artery blood flow

[0039] Under the guidance of digital subtraction angiography (DSA), the blocking effect of polyethylene glycol-modified peptides on the blood flow in rabbit renal arteries was studied. First, the experimental rabbits were anesthetized by pentobarbital injection through the ear vein. The skin was prepared and disinfected in the right femoral artery area, and the skin was longitudinally incised along the course of the right femoral artery of the rabbit to expose and bluntly separate the right femoral artery sheath layer by layer. Subsequently, a 4-Fr sheath (Cook, Bloomington, USA) was directly inserted into the artery, and under the guidance of digital subtraction angiography (DSA, AlluraXper, Philips Healthcare, Nederland), a 2.7F microcatheter (Terumo, Tokyo, Japan) was selectively introduced into the corresponding right renal artery. Before renal artery embolization, iohexol was injected through the microcatheter for renal artery angiography. Subsequently, the mixed suspension of peptides and iohexol was carefully injected into the right renal artery. After the embolization was completed, arteriography was performed on the embolized renal artery to confirm that there was no blood flow to the kidney. Two weeks after embolization, volume reconstruction was performed using electronic images (DICOM) to evaluate the embolization efficacy of the peptide.

[0040] The in vivo embolization effect of peptides was studied using a rabbit kidney model. The results showed that peptide P1 could be easily injected into the renal artery through a microcatheter under the guidance of DSA. The right renal blood vessels were clearly visible before peptide injection. In contrast, the right renal blood vessel network disappeared after peptide injection, which was due to the inability of iohexol contrast agent to diffuse into the embolized renal artery. This result also revealed that peptide P1 can block the blood vessels of the kidney, such as the attached Figure 3 shown.

[0041] 3. Evaluation of regional blood perfusion recovery after polyethylene glycol-modified peptide embolization

[0042] To evaluate the long-term in vivo embolization efficiency of the polypeptide and the recovery of blood perfusion in the embolized area after the use of the polypeptide, CT scanning and volume reconstruction were performed on the experimental rabbits 14 days after embolization in Example 4. In the polypeptide (P1) group, both left and right renal arteries were clearly visualized ( Figure 3 Based on this result, the inventors subsequently evaluated the effect of high-concentration peptide on the right renal artery embolization. In the peptide (P1) group, both left and right renal arteries were still clearly visualized ( Figure 3 ).

[0043] Example 2

[0044] 1. Preparation and physicochemical properties of polyethylene glycol-modified peptides

[0045] This example provides a polyethylene glycol-modified polypeptide: PEG-RADARADARADARAD A-NH2C 70 H 121 N 29 O 27 The preparation of (P2) utilizes the condensation reaction between the carboxyl group with strong reactivity in the terminal carboxyl polyethylene glycol and the amino group of the RADA peptide. On the basis of using Rink Amide resin to solid-phase synthesize 200 mg of RADA16 polypeptide from the carbon end to the nitrogen end according to the amino acid sequence, 47.3 mg of 1-hydroxybenzotriazole and 88.6 mg of benzotriazole-N,N,N',N'-tetramethyluronium hexafluorophosphate were weighed as condensation agents, and 24.5 mg of terminal carboxyl polyethylene glycol was added to 50 mL of N,N-dimethylformamide containing 200 mg of RADA16 polypeptide and 90.6 mg of N,N-diisopropylethylamine for condensation, and the reaction was carried out for 2 hours. After the reaction is completed, trifluoroacetic acid is added to cut the polypeptide from the resin, and ether is added to the filtrate after filtration, precipitated and washed, and freeze-dried to obtain a polyethylene glycol-modified polypeptide freeze-dried powder with a molecular weight of 1800.94; its nuclear magnetic resonance hydrogen spectrum is as shown Figure 1 As shown in the figure, the characteristic proton peak of ethylene glycol (=3.55) appears in the polypeptide modified with polyethylene glycol, indicating the successful modification with polyethylene glycol.

[0046] The gelation time of the polyethylene glycol-modified polypeptide in normal saline was determined by the vial inversion method. 100 mg of the polyethylene glycol-modified polypeptide was weighed and added to a vial containing 2 mL of normal saline. The vial was placed in an ultrasonicator at 30°C and inverted every 3 seconds. The gelation time of the polyethylene glycol-modified polypeptide was measured to be 57 seconds.

[0047] 2. Study on the blocking effect of polyethylene glycol-modified peptides on rabbit renal artery blood flow

[0048] Under the guidance of digital subtraction angiography (DSA), the blocking effect of polyethylene glycol-modified peptides on the blood flow in rabbit renal arteries was studied. First, the experimental rabbits were anesthetized by pentobarbital injection through the ear vein. The skin was prepared and disinfected in the right femoral artery area, and the skin was longitudinally incised along the course of the right femoral artery of the rabbit to expose and bluntly separate the right femoral artery sheath layer by layer. Subsequently, a 4-Fr sheath (Cook, Bloomington, USA) was directly inserted into the artery, and under the guidance of digital subtraction angiography (DSA, AlluraXper, Philips Healthcare, Nederland), a 2.7F microcatheter (Terumo, Tokyo, Japan) was selectively introduced into the corresponding right renal artery. Before renal artery embolization, iohexol was injected through the microcatheter for renal artery angiography. Subsequently, the mixed suspension of peptides and iohexol was carefully injected into the right renal artery. After the embolization was completed, arteriography was performed on the embolized renal artery to confirm that there was no blood flow to the kidney. Two weeks after embolization, volume reconstruction was performed using electronic images (DICOM) to evaluate the embolization efficacy of the peptide.

[0049] The in vivo embolization effect of the peptide was studied using a rabbit kidney model. The results showed that the peptide could be easily injected into the renal artery through a microcatheter under the guidance of DSA. The right renal blood vessels were clearly visible before the peptide injection. In contrast, the right renal vascular network disappeared after the peptide injection. This was because the iohexol contrast agent could not diffuse into the embolized renal artery. This result also revealed that the peptide P2 can completely block the blood vessels of the kidney, as shown in the attached Figure 3 shown.

[0050] 3. Evaluation of regional blood perfusion recovery after polyethylene glycol-modified peptide embolization

[0051] To evaluate the long-term in vivo embolization efficiency of the polypeptide and the recovery of blood perfusion in the embolized area after the use of the polypeptide, CT scanning and volume reconstruction were performed on the experimental rabbits 14 days after embolization in Example 4. Peptide P2 renal artery angiography showed that the left renal artery was clearly visualized, the distal end of the right renal artery was not visualized, but the main trunk of the right renal artery was not completely blocked ( Figure 3 Based on this result, the inventors subsequently evaluated the embolization effect of the high-concentration peptide in the right renal artery. The volume reconstruction image shows that the right renal artery in the peptide (P2) group is clearly visualized ( Figure 3), the right kidney was not visualized at the distal end of the rabbit renal artery, and the main trunk was completely embolized. Therefore, the embolization efficiency of polypeptide P2 was better.

[0052] Example 3

[0053] 1. Preparation and physicochemical properties of polyethylene glycol-modified peptides

[0054] This example provides a polyethylene glycol-modified polypeptide: MPEG 2000 -RADARADARADA RADA-NH2C 156 H 293 N 29 O 70 The preparation of (P3) utilizes the condensation reaction between the amino group with strong reactivity in the terminal carboxyl polyethylene glycol and the amino group of the RADA peptide. On the basis of using Rink Amide resin to solid-phase synthesize 200 mg of RADA16 polypeptide from the carbon end to the nitrogen end according to the amino acid sequence, 47.3 mg of 1-hydroxybenzotriazole and 88.6 mg of benzotriazole-N,N,N',N'-tetramethyluronium hexafluorophosphate were weighed as condensation agents, and 700 mg of terminal carboxyl polyethylene glycol with an average molecular weight of 2000 was added to 50 mL of N,N-dimethylformamide containing 200 mg of RADA16 polypeptide and 90.6 mg of N,N-diisopropylethylamine for condensation, and the reaction was carried out for 2 hours. After the reaction is completed, trifluoroacetic acid is added to cut the polypeptide from the resin, and ether is added to the filtrate after filtration, precipitated and washed, and freeze-dried to obtain a polyethylene glycol-modified polypeptide freeze-dried powder with a molecular weight of 3695.25; its nuclear magnetic resonance hydrogen spectrum is as shown Figure 1 As shown in the figure, multiple characteristic proton peaks of ethylene glycol (-3.6) appeared in the polyethylene glycol-modified polypeptide, indicating the successful modification of polyethylene glycol.

[0055] The gelation time of the polyethylene glycol-modified polypeptide in normal saline was determined by the vial inversion method. 100 mg of the polyethylene glycol-modified polypeptide was weighed and added to a vial containing 2 mL of normal saline. The vial was placed in an ultrasonic instrument at 30°C and inverted every 3 seconds. The gelation time of the polyethylene glycol-modified polypeptide was measured to be 21 seconds.

[0056] Embodiment 4:

[0057] 1. Preparation and physicochemical properties of polyethylene glycol-modified peptides

[0058] This example provides a polyethylene glycol-modified polypeptide: MPEG 1000 -RADARADARADA RADA-NH2C 112 H 205 N 29 O 48Preparation, using the condensation reaction between the amino group with strong reactivity in the terminal carboxyl polyethylene glycol and the amino group of the RADA peptide. On the basis of using Rink Amide resin to solid-phase synthesize 200 mg of RADA16 polypeptide from the carbon end to the nitrogen end according to the amino acid sequence, 47.3 mg of 1-hydroxybenzotriazole and 88.6 mg of benzotriazole-N,N,N',N'-tetramethyluronium hexafluorophosphate were weighed as condensation agents, and 350 mg of terminal carboxyl polyethylene glycol with an average molecular weight of 1000 was added to 50 mL of N,N-dimethylformamide containing 200 mg of RADA16 polypeptide and 90.6 mg of N,N-diisopropylethylamine for condensation, and the reaction was carried out for 2 hours. After the reaction was completed, trifluoroacetic acid was added to cut the polypeptide from the resin, and ether was added to the filtrate after filtration, precipitated and washed, and freeze-dried to obtain a polyethylene glycol-modified polypeptide freeze-dried powder with a molecular weight of 2726.10.

[0059] The gelation time of the polyethylene glycol-modified polypeptide in normal saline was determined by the vial inversion method. 100 mg of the polyethylene glycol-modified polypeptide was weighed and added to a vial containing 2 mL of normal saline. The vial was placed in an ultrasonic instrument at 30°C and inverted every 3 seconds. The gelation time of the polyethylene glycol-modified polypeptide was measured to be 30 seconds.

[0060] Example 5

[0061] 1. Preparation and physicochemical properties of polyethylene glycol-modified peptides

[0062] This example provides a polyethylene glycol-modified polypeptide: MPEG 600 -RADARADARADA RADA-NH2C 94 H 169 N 29 O 39 Preparation, using the condensation reaction between the amino group with strong reactivity in the terminal carboxyl polyethylene glycol and the amino group of the RADA peptide. Based on the solid phase synthesis of 200 mg of RADA16 polypeptide from the carbon end to the nitrogen end using Rink Amide resin according to the amino acid sequence, 47.3 mg of 1-hydroxybenzotriazole and 88.6 mg of benzotriazole-N,N,N',N'-tetramethyluronium hexafluorophosphate were weighed as condensation agents, and 210 mg of terminal carboxyl polyethylene glycol with an average molecular weight of 600 was added to 50 mL of N,N-dimethylformamide containing 200 mg of RADA16 polypeptide and 90.6 mg of N,N-diisopropylethylamine for condensation, and the reaction was carried out for 2 hours. After the reaction was completed, trifluoroacetic acid was added to cut the polypeptide from the resin, and ether was added to the filtrate after filtration, precipitated and washed, and freeze-dried to obtain a polyethylene glycol-modified polypeptide freeze-dried powder with a molecular weight of 2329.60.

[0063] The gelation time of the polyethylene glycol-modified polypeptide in normal saline was determined by the vial inversion method. 100 mg of the polyethylene glycol-modified polypeptide was weighed and added to a vial containing 2 mL of normal saline. The vial was placed in an ultrasonic instrument at 30°C and inverted every 3 seconds. The gelation time of the polyethylene glycol-modified polypeptide was measured to be 36 seconds.

[0064] Embodiment 6:

[0065] 1. Preparation and physicochemical properties of polyethylene glycol-modified peptides

[0066] This example provides a polyethylene glycol-modified polypeptide: MPEG 400 -RADARADARADA RADA-NH2C 86 H 153 N 29 O 35 Preparation, using the condensation reaction between the amino group with strong reactivity in the terminal carboxyl polyethylene glycol and the amino group of the RADA peptide. Based on the solid phase synthesis of 200 mg of RADA16 polypeptide from the carbon end to the nitrogen end using Rink Amide resin according to the amino acid sequence, 47.3 mg of 1-hydroxybenzotriazole and 88.6 mg of benzotriazole-N,N,N',N'-tetramethyluronium hexafluorophosphate were weighed as condensation agents, and 140 mg of terminal carboxyl polyethylene glycol with an average molecular weight of 400 was added to 50 mL of N,N-dimethylformamide containing 200 mg of RADA16 polypeptide and 90.6 mg of N,N-diisopropylethylamine for condensation, and the reaction was carried out for 2 hours. After the reaction was completed, trifluoroacetic acid was added to cut the polypeptide from the resin, and ether was added to the filtrate after filtration, precipitated and washed, and freeze-dried to obtain a polyethylene glycol-modified polypeptide freeze-dried powder with a molecular weight of 2153.40.

[0067] The gelation time of the polyethylene glycol-modified polypeptide in normal saline was determined by the vial inversion method. 100 mg of the polyethylene glycol-modified polypeptide was weighed and added to a vial containing 2 mL of normal saline. The vial was placed in an ultrasonic instrument at 30°C and inverted every 3 seconds. The gelation time of the polyethylene glycol-modified polypeptide was measured to be 42 seconds.

[0068] Example 7

[0069] 1. Preparation and physicochemical properties of polyethylene glycol-modified peptides

[0070] This example provides a polyethylene glycol-modified polypeptide: Ac-RADARADARADARADA-MPEG 400 C 84 H 148 N 28 O 34Preparation, using the condensation reaction between the amino group with strong reactivity in the terminal amino polyethylene glycol and the carboxyl group of the RADA peptide. Based on the solid phase synthesis of 200 mg of RADA16 polypeptide from the carbon end to the nitrogen end using CTC-Resin resin according to the amino acid sequence, the polyethylene glycol was further condensed with the carboxyl group of the carbon end of RADA16 by a liquid phase synthesis method, 47.3 mg of 1-hydroxybenzotriazole and 88.6 mg of benzotriazole-N,N,N',N'-tetramethyluronium hexafluorophosphate were weighed as condensation agents, and 150 mg of terminal amino ethylene glycol with an average molecular weight of 400 was added to 50 mL of N,N-dimethylformamide containing 200 mg of RADA16 polypeptide and 90.6 mg of N,N-diisopropylethylamine for condensation, and the reaction was carried out for 2 hours. After the reaction was completed, ether was added, precipitated and washed, and freeze-dried to obtain a polyethylene glycol-modified polypeptide freeze-dried powder with a molecular weight of 2094.21.

[0071] The gelation time of the polyethylene glycol-modified polypeptide in normal saline was determined by the vial inversion method. 100 mg of the polyethylene glycol-modified polypeptide was weighed and added to a vial containing 2 mL of normal saline. The vial was placed in an ultrasonic instrument at 30°C and inverted every 3 seconds. The gelation time of the polyethylene glycol-modified polypeptide was measured to be 48 seconds.

[0072] Example 8

[0073] 1. Preparation and physicochemical properties of polyethylene glycol-modified peptides

[0074] This example provides a polyethylene glycol-modified polypeptide: MPEG 200 -RADARADARADA RADA-NH2C 76 H 133 N 29 O 30 Preparation, using the condensation reaction between the amino group with strong reactivity in the terminal carboxyl polyethylene glycol and the amino group of the RADA peptide. Based on the solid phase synthesis of 200 mg of RADA16 polypeptide from the carbon end to the nitrogen end using Rink Amide resin according to the amino acid sequence, 47.3 mg of 1-hydroxybenzotriazole and 88.6 mg of benzotriazole-N,N,N',N'-tetramethyluronium hexafluorophosphate were weighed as condensation agents, and 70 mg of terminal carboxyl polyethylene glycol with an average molecular weight of 200 was added to 50 mL of N,N-dimethylformamide containing 200 mg of RADA16 polypeptide and 90.6 mg of N,N-diisopropylethylamine for condensation, and the reaction was carried out for 2 hours. After the reaction was completed, trifluoroacetic acid was added to cut the polypeptide from the resin, and ether was added to the filtrate after filtration, precipitated and washed, and freeze-dried to obtain a polyethylene glycol-modified polypeptide freeze-dried powder with a molecular weight of 1933.12.

[0075] The gelation time of the polyethylene glycol-modified polypeptide in normal saline was determined by the vial inversion method. 100 mg of the polyethylene glycol-modified polypeptide was weighed and added to a vial containing 2 mL of normal saline. The vial was placed in an ultrasonic instrument at 30°C and inverted every 3 seconds. The gelation time of the polyethylene glycol-modified polypeptide was measured to be 48 seconds.

[0076] Example 9

[0077] 1. Preparation and physicochemical properties of polyethylene glycol-modified peptides

[0078] This example provides a polyethylene glycol-modified polypeptide: Ac-RADARADARADARADA-MPEG 200 C 76 H 132 N 28 O 30 Preparation, using the condensation reaction between the carboxyl group with strong reactivity in the terminal amino polyethylene glycol and the carboxyl group of the RADA peptide. Based on the solid phase synthesis of 200 mg of RADA16 polypeptide from the carbon end to the nitrogen end using CTC-Resin resin according to the amino acid sequence, the polyethylene glycol was further condensed with the carboxyl group of the carbon end of RADA16 by a liquid phase synthesis method, 47.3 mg of 1-hydroxybenzotriazole and 88.6 mg of benzotriazole-N,N,N',N'-tetramethyluronium hexafluorophosphate were weighed as condensation agents, and 75 mg of terminal amino ethylene glycol with an average molecular weight of 200 was added to 50 mL of N,N-dimethylformamide containing 200 mg of RADA16 polypeptide and 90.6 mg of N,N-diisopropylethylamine for condensation, and the reaction was carried out for 2 hours. After the reaction was completed, ether was added, precipitated and washed, and freeze-dried to obtain a polyethylene glycol-modified polypeptide freeze-dried powder with a molecular weight of 1918.06.

[0079] The gelation time of the polyethylene glycol-modified polypeptide in normal saline was determined by the vial inversion method. 100 mg of the polyethylene glycol-modified polypeptide was weighed and added to a vial containing 2 mL of normal saline. The vial was placed in an ultrasonic instrument at 30°C and inverted every 3 seconds. The gelation time of the polyethylene glycol-modified polypeptide was measured to be 51 seconds.

[0080] Example 10

[0081] 1. Preparation and physicochemical properties of polyethylene glycol-modified peptides

[0082] This example provides a polyethylene glycol-modified polypeptide: MPEG 200 -RADARADARADA RADA-MPEG 200 C 85 H 151 N 29 O 35Preparation, using the condensation reaction between the amino group with strong reactivity in the terminal amino polyethylene glycol and the carboxyl group of the RADA peptide, and between the carboxyl group with strong reactivity in the terminal carboxyl polyethylene glycol and the amino group of the RADA peptide. Based on the solid phase synthesis of 300 mg of RADA16 polypeptide from the carbon end to the nitrogen end according to the amino acid sequence using CTC-Resin resin, 62.1 mg of 1-hydroxybenzotriazole and 132.5 mg of benzotriazole-N,N,N',N'-tetramethyluronium hexafluorophosphate were weighed as condensation agents, and 105 mg of terminal carboxyl ethylene glycol with an average molecular weight of 200 was added to 75 mL of N,N-dimethylformamide containing 136.2 mg of N,N-diisopropylethylamine for condensation, and the reaction was carried out for 2 hours. After the reaction is completed, trifluoroacetic acid is added to cut the polypeptide from the resin, and ether is added to the filtrate after filtration, precipitated and washed, and lyophilized to obtain a lyophilized powder of a polypeptide modified with polyethylene glycol at the nitrogen end. The polyethylene glycol is further condensed with the carboxyl group at the carbon end of RADA16 by a liquid phase synthesis method. 47.3 mg of 1-hydroxybenzotriazole and 88.6 mg of benzotriazole-N,N,N',N'-tetramethyluronium hexafluorophosphate are weighed as condensation agents. 70 mg of terminal amino polyethylene glycol with an average molecular weight of 200 and 75 mg of terminal carboxyl polyethylene glycol with an average molecular weight of 200 are added to 50 mL of N,N-dimethylformamide containing 300 mg of a polypeptide modified with polyethylene glycol at the nitrogen end and 90.6 mg of N,N-diisopropylethylamine for condensation. The reaction is carried out for 2 hours. After the reaction is completed, ether is added, precipitated and washed, and lyophilized to obtain a lyophilized powder of a polypeptide modified with polyethylene glycol with a molecular weight of 2139.40.

[0083] The gelation time of the polyethylene glycol-modified polypeptide in normal saline was determined by the vial inversion method. 100 mg of the polyethylene glycol-modified polypeptide was weighed and added to a vial containing 2 mL of normal saline. The vial was placed in an ultrasonic instrument at 30°C and inverted every 3 seconds. The gelation time of the polyethylene glycol-modified polypeptide was measured to be 39 seconds.

[0084] Embodiment 11

[0085] This example provides a chitosan oligosaccharide modified polypeptide: Ac-RADARADARADARADA-Chitosan, which has the following structure:

[0086]

[0087] The chitosan oligosaccharide modified peptide was synthesized by the condensation reaction between the abundant amino groups in the chitosan oligosaccharide structure and the carboxyl groups of the RADA peptide. Based on the solid phase synthesis of 20 mg of RADA16 peptide from the carbon end to the nitrogen end using CTC-Resin resin according to the amino acid sequence, the chitosan oligosaccharide was further condensed with the carboxyl group of the carbon end of RADA16 by a liquid phase synthesis method. 4.73 mg of 1-hydroxybenzotriazole and 8.86 mg of benzotriazole-N,N,N',N'-tetramethyluronium hexafluorophosphate were weighed as condensation agents, and chitosan oligosaccharide (the molar ratio of RADA peptide to chitosan oligosaccharide was 2:1, the average molecular weight of chitosan oligosaccharide was 5000, and the degree of polymerization was 2-30) was added to 5 mL of N,N-dimethylformamide containing 20 mg of RADA16 polypeptide and 9.06 mg of N,N-diisopropylethylamine for condensation for 2-5 hours. After the reaction was completed, trifluoroacetic acid was added to cut the polypeptide from the resin, and ether was added to the filtrate after filtration, precipitated and washed, and freeze-dried to obtain a polyethylene glycol-modified polypeptide coupled with chitosan oligosaccharide. The average molecular weight was 56,000 as determined by gel permeation chromatography.

[0088] Example 12

[0089] This embodiment provides a preparation of a polyethylene glycol-modified polypeptide coupled with chitosan oligosaccharide: Ac-RADARADAR ADARADA-Chitosan, which has the following structure:

[0090]

[0091] The chitosan oligosaccharide modified peptide was synthesized by condensation reaction between the abundant amino groups in the chitosan oligosaccharide structure and the carboxyl groups of the RADA peptide. Based on the solid phase synthesis of 20 mg of RADA16 peptide from the carbon end to the nitrogen end using CTC-Resin resin according to the amino acid sequence, the chitosan oligosaccharide was further condensed with the carboxyl group of the carbon end of RADA16 by liquid phase synthesis method. 4.73 mg of 1-hydroxybenzotriazole and 8.86 mg of benzotriazole-N,N,N',N'-tetramethyluronium hexafluorophosphate were weighed as condensation agents, and chitosan oligosaccharide (the molar ratio of RADA peptide to chitosan oligosaccharide was 1:1, and the average molecular weight of chitosan oligosaccharide was 5000) was added to 5 mL of N,N-dimethylformamide containing 20 mg of RADA16 polypeptide and 9.06 mg of N,N-diisopropylethylamine for condensation for 2-5 hours. After the reaction was completed, trifluoroacetic acid was added to cut the polypeptide from the resin, and ether was added to the filtrate after filtration, precipitated and washed, and freeze-dried to obtain the polyethylene glycol-modified polypeptide coupled with chitosan oligosaccharide. The average molecular weight was 30,000 as determined by gel permeation chromatography.

[0092] Although the embodiments of the present invention have been disclosed as above, they are not limited to the applications listed in the specification and the implementation modes. They can be fully applied to various fields suitable for the present invention. For those familiar with the art, additional modifications can be easily implemented. Therefore, without departing from the general concept defined by the claims and the scope of equivalents, the present invention is not limited to specific details.

Claims

1. A modified polypeptide, characterized in that: It is a polypeptide modified by polyethylene glycol or chitosan oligosaccharide, which is composed of polyethylene glycol or chitosan oligosaccharide and RADA16 polypeptide covalently linked, and has the following general structural formula: Wherein m, n, o are the degree of polymerization, 0≤m≤44, 0≤n≤44, 0≤o≤30; the carbon end or nitrogen end of the RADA16 peptide is condensed and connected with polyethylene glycol or chitosan oligosaccharide.

2. The modified polypeptide according to claim 1, characterized in that in, The polyethylene glycol-modified polypeptide is obtained by an acid-amine condensation reaction between terminal amino- or carboxyl-terminated polyethylene glycol and a RADA16 polypeptide.

3. The modified polypeptide according to claim 1, characterized in that in, The chitosan oligosaccharide-modified polypeptide is obtained by acid-amine condensation reaction of chitosan oligosaccharide and RADA16 polypeptide.

4. A method for preparing a modified polypeptide according to any one of claims 1 to 3, characterized in that: The following steps are involved: Based on the solid phase synthesis of RADA16, 1-hydroxybenzotriazole (HOBT) and benzotriazole-N,N,N',N'-tetramethyluronium hexafluorophosphate (HBTU) are used as condensation agents, and the modified components are added to N,N-dimethylformamide (DMF) containing N,N-diisopropylethylamine (DIEA) for condensation. After the reaction is completed, the peptide is cut from the resin, precipitated and washed, and then redissolved and freeze-dried to obtain a modified peptide freeze-dried powder; Among them, the modification component is terminal amino- or terminal carboxyl-terminated polyethylene glycol, or chitosan oligosaccharide containing amino groups is added; when the modification component is terminal amino- or terminal carboxyl-terminated polyethylene glycol, a polyethylene glycol-modified polypeptide is obtained, and when the modification component is chitosan oligosaccharide containing amino groups, a chitosan oligosaccharide-modified polypeptide is obtained.

5. The method for preparing a modified polypeptide according to claim 4, characterized in that: The polyethylene glycol-modified peptide was prepared by the following method: On the basis of solid phase synthesis of RADA16 polypeptide, 1-hydroxybenzotriazole and benzotriazole-N,N,N',N'-tetramethyluronium hexafluorophosphate are used as condensation agents, and terminal amino or terminal carboxyl polyethylene glycol is added to N,N-dimethylformamide containing RADA16 polypeptide and N,N-diisopropylethylamine, and the condensation reaction is carried out at room temperature. After the reaction is completed, the polypeptide is cut from the resin, precipitated and washed, and then redissolved and freeze-dried to obtain polyethylene glycol-modified polypeptide freeze-dried powder.

6. The method for preparing a modified polypeptide according to claim 5, characterized in that: in, The molar ratio of polyethylene glycol to RADA16 polypeptide is 1-3:1; and the reaction time is 0.5-2 hours.

7. The method for preparing a modified polypeptide according to claim 4, characterized in that: The chitosan oligosaccharide modified polypeptide was prepared by the following method: Based on the solid phase synthesis of RADA16 polypeptide, 1-hydroxybenzotriazole and benzotriazole-N,N,N',N'-tetramethyluronium hexafluorophosphate are used as condensation agents, and amino-containing chitosan oligosaccharides are added to N,N-dimethylformamide containing RADA16 polypeptide and N,N-diisopropylethylamine. The condensation reaction is carried out at room temperature. After the reaction is completed, the polypeptide is cut from the resin, precipitated and washed, and then redissolved and freeze-dried to obtain chitosan oligosaccharide-modified polypeptide freeze-dried powder.

8. The method for preparing a modified polypeptide according to claim 7, characterized in that: in, The molar ratio of chitosan oligosaccharide to RADA16 peptide is 0.5-2:1; and the reaction time is 0.5-2 hours.

9. The method for preparing a modified polypeptide according to claim 5 or 7, characterized in that: in, The molar ratio of 1-hydroxybenzotriazole to RADA16 polypeptide is 2-3:1; the molar ratio of benzotriazole-N,N,N',N'-tetramethyluronium hexafluorophosphate to RADA16 polypeptide is 2-3:1; the molar ratio of N,N-diisopropylethylamine to RADA16 polypeptide is 4-6:1; With the mass unit being mg and the volume unit being mL, the ratio of the mass of RADA16 polypeptide to the volume of N,N-dimethylformamide is 2:1-8:

1.

10. Use of the modified polypeptide according to any one of claims 1 to 3 in preparing a hydrogel for tumor vascular embolization.

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