CN107261124B - Preparation method and application of angiogenesis inhibiting peptide and hyaluronic acid modifier thereof - Google Patents
Preparation method and application of angiogenesis inhibiting peptide and hyaluronic acid modifier thereof Download PDFInfo
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Abstract
The invention discloses a preparation method and application of a new angiogenesis inhibiting peptide and a hyaluronic acid modifier thereof, wherein the amino acid sequence of the ES-2-AF peptide is shown as SEQ ID NO.1 in a sequence table; the hyaluronic acid modifier of the ES-2-AF peptide is formed by combining amino of the ES-2-AF peptide and carboxyl on hyaluronic acid molecules through amido bonds, and the structural formula is as follows: HA-CO-NH- (ES-2-AF) n. The ES-2-AF peptide of the invention retains or even enhances the ability to inhibit angiogenesis compared to Anti-flt1 peptide, ES2 peptide. Compared with the ES-2-AF peptide, the hyaluronic acid modified substance of the ES-2-AF peptide retains the anti-angiogenesis and anti-tumor activity of the ES-2-AF peptide, integrates the anti-angiogenesis and other characteristics of macromolecular hyaluronic acid, and has the characteristics of higher stability, stronger hydrophilicity, stronger targeting property and the like, so that the hyaluronic acid modified substance has better use effect and application value.
Description
Technical Field
The invention relates to the technical field of biological medicines, in particular to a preparation method and application of a angiogenesis inhibiting peptide and a hyaluronic acid modifier thereof.
Background
Endostatin (ES) is a 20KDa polypeptide isolated from vascular endotheliomas and is identified as the C-terminal portion of collagen XVIII. Endostatin can directly target endothelial cells near tumors without significant toxicity to normal cells. It can also inhibit endothelial cell proliferation and migration, and induce apoptosis. Its anti-angiogenic activity is achieved by modulating the expression of the vascular endothelial growth factor VEGF. ES-2(IVRRADRAAVP) is a short peptide segment with significant anti-angiogenesis and anti-tumor activities in ES structure, and is easier to enter into cells and to obtain and reform, but like ES, ES-2 also has the disadvantages of short half-life in vivo and poor stability, and the disadvantages greatly limit the further application of ES-2.
The Anti-flt1 peptide (GGNQWFI) specifically binds to VEGFR1(fms-like tyrosine kinase-1orFlt1), thereby acting as an antagonist to prevent VEGFR1 binding to VEGFA, VEGFB, placental growth factor PIGF, and the like. Unlike other monoclonal antibodies or RNA antagonists of VEGFR1, the Anti-flt1 peptide is a short, non-immunogenic peptide that is less expensive to synthesize. However, the Anti-flt1 peptide has poor water solubility compared with other polypeptides such as ES-2, which greatly affects the exertion of its effect.
In conclusion, the existing polypeptide angiogenesis inhibitor has the problems of short half-life period in vivo, poor stability and the like, and the application of the polypeptide drug in the preparation of the angiogenesis inhibitor is limited. Therefore, there is an urgent need in the art to develop a novel polypeptide neovascular inhibitor.
Disclosure of Invention
In view of the above-mentioned deficiencies of the prior art, the present invention aims to provide an ES-2-AF peptide which is improved in activity, solubility, stability and half-life and has a significant anti-angiogenesis effect.
Another object of the present invention is to provide a hyaluronic acid-modified ES-2-AF peptide.
In order to achieve the purpose, the invention adopts the following technical scheme:
the first aspect of the invention provides an ES-2-AF peptide, the amino acid sequence of which is shown as SEQ ID NO.1 in the sequence table, and the amino acid sequence is as follows:
ES-2-AF peptides: IVRRADRAAVPGGGGGGNQWFI, respectively; (SEQ ID NO. 1).
The Anti-flt1 peptide and the ES-2 peptide are fused together, and the obtained ES-2-AF peptide is well improved in the aspects of activity, solubility, stability, half-life period and the like.
The ES-2-AF peptide is subjected to Fmoc solid phase synthesis, is purified by high performance liquid chromatography, is confirmed in structure by means of mass spectrometry, high performance liquid chromatography and the like, and has the purity of over 95 percent.
In a second aspect of the present invention, there is provided a hyaluronic acid modification of ES-2-AF peptide, wherein the amino group of the ES-2-AF peptide is bonded to the carboxyl group of the hyaluronic acid molecule through an amide bond, and the structural formula is as follows:
HA-CO-NH-(ES-2-AF)n；
wherein n is 30 to 60; ES-2-AF HAs a molecular weight of 2197.5Da and HA HAs a molecular weight of 240000 Da.
The supply amount of ES-2-AF (i.e. the value of n in the structural formula) affects the degree of binding to Hyaluronic Acid (HA), and the present invention considers the supply amount of ES-2-AF optimally, considering the influence on HA spatial conformation and the degree of binding between ES-2-AF and hyaluronic acid, preferably n is 60.
In a third aspect of the present invention, there is provided a method for producing a hyaluronic acid-modified ES-2-AF peptide.
The preparation method of the hyaluronic acid modifier of the ES-2-AF peptide provided by the invention comprises the following steps:
(1) preparing HA-TBA conjugate, improving the solubility of HA, and enabling the HA to be dissolved in an organic solvent;
(2) dissolving HA-TBA conjugate in organic solvent, adding a kat condensing agent to activate carboxyl of HA; then mixing the HA-TBA, the ES-2-AF peptide and N, N-Diisopropylethylamine (DIPEA) after activating carboxyl, reacting for 24h, and adding NaOH solution; then adjusting the pH value of the reaction system to be reduced to 3.0 to stop the reaction; and then, the pH value of the reaction system is increased to 7.0, and the reaction product is dialyzed and dried to obtain the hyaluronic acid modifier of the ES-2-AF peptide.
In the step (1), the HA-TBA conjugate is prepared by the following steps: adding excessive tetrabutyl ammonium hydroxide (TBA-OH) into Dowex ion exchange resin, uniformly mixing to obtain Dowex-TBA resin, and filtering to remove supernatant; dissolving sodium hyaluronate NaHA in water, pouring into prepared Dowex-TBA resin, uniformly mixing, filtering to remove Dowex resin to obtain a clear HA-TBA solution, and freeze-drying to obtain the HA-TBA conjugate.
In the step (2), the organic solvent is DMSO.
In the step (2), the molar ratio of HA-TBA to ES-2-AF is 1 (4-100).
In the step (2), the concentration of the NaOH solution is 1M.
In the step (2), 1M HCl is adopted to adjust the pH value to be 3.0; the pH was raised to 7.0 with 1M NaOH.
In the step (2), the method for dialyzing the reaction product comprises the following steps: the reaction product was poured into a pre-treated dialysis bag (10kDa) and dialyzed against NaCl solution, 25% ethanol and water, respectively.
The invention improves the solubility of HA by preparing the HA-TBA conjugate, so that the HA which is insoluble in an organic solvent can be dissolved in DMSO. Excess BOP is sufficient to activate the carboxyl group of HA to react with the amino group of the peptide chain to form an amide bond. The Katt condensing agent is a common method in protein and peptide chain reaction, but factors such as reaction temperature, pH of a reaction system, reaction time and the like have great influence on reaction efficiency. According to the invention, the hyaluronic acid ES-2-AF peptide modifier with increased half-life period and stronger activity is successfully prepared by optimizing the conditions of the supply amount of the Kate condensing agent, the reaction temperature, the pH value of a reaction system, the reaction time and the like.
In a fourth aspect, the invention provides the use of the ES-2-AF peptide and/or the hyaluronic acid modification of the ES-2-AF peptide in the preparation of a medicament for inhibiting angiogenesis.
Furthermore, the invention also provides application of the ES-2-AF peptide and/or the hyaluronic acid modification compound of the ES-2-AF peptide in preparing a treatment drug and/or an anti-tumor drug for treating angiogenesis diseases.
The accompanying angiogenesis diseases comprise diabetic retinopathy, age-related macular degeneration, arthritis and the like.
The invention has the beneficial effects that:
(1) the hyaluronic acid-ES-2-AF peptide conjugates with different degrees of conjugation can be prepared by adjusting conditions such as the supply amount of the ES-2-AF peptide, the reaction temperature, the pH of the reaction system, the reaction time and the like.
(2) Compared with Anti-flt1 and ES-2 peptide, the ES-2-AF peptide prepared by the invention has better Anti-angiogenesis effects at the molecular level and the cell level than the Anti-angiogenesis effects of the Anti-angiogenesis peptides.
(3) Compared with ES-2-AF peptide, the ES-2-AF peptide hyaluronic acid modifier prepared by the invention has higher stability and stronger biological activity.
Drawings
The accompanying drawings, which are incorporated in and constitute a part of this application, illustrate embodiments of the application and, together with the description, serve to explain the application and are not intended to limit the application.
FIG. 1: nuclear magnetic resonance of ES-2-AF peptide hyaluronic acid modifier1H, map;
FIG. 2: the inhibition effect of Anti-flt1, ES-2 and ES-2-AF on endothelial cell proliferation;
FIG. 3: Anti-Flt1, ES-2-AF can inhibit the binding of VEGF and its receptor VEGFR1 (Flt-1);
FIG. 4: ES-2-AF, HA & ES-2-AF, HA-ES-2-AF inhibit the proliferation of endothelial cells;
FIG. 5: ES-2-AF, HA & ES-2-AF, HA-ES-2-AF inhibit the binding of VEGF and its receptor VEGFR1 (Flt-1);
FIG. 6: ES-2-AF, HA & ES-2-AF, and HA-ES-2-AF endothelial cell metastasis.
Detailed Description
It should be noted that the following detailed description is exemplary and is intended to provide further explanation of the disclosure. Unless defined otherwise, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this application belongs.
It is noted that the terminology used herein is for the purpose of describing particular embodiments only and is not intended to be limiting of example embodiments according to the present application. As used herein, the singular forms "a", "an" and "the" are intended to include the plural forms as well, and it should be understood that when the terms "comprises" and/or "comprising" are used in this specification, they specify the presence of stated features, steps, operations, devices, components, and/or combinations thereof, unless the context clearly indicates otherwise.
As introduced in the background art, most of the existing polypeptide neovascular inhibitors have the problems of short half-life in vivo, poor stability, poor water solubility and the like. Based on the above, the invention provides a new angiogenesis inhibiting peptide and a hyaluronic acid modifier thereof.
In one embodiment of the present application, there is provided an ES-2-AF peptide having an isoelectric point of 11.54 and the following amino acid sequence:
ES-2-AF peptides: IVRRADRAAVPGGGGGGNQWFI are provided.
Since Anti-flt1 and ES-2 are both similar in activity and many different, the difficulty in fusing Anti-flt1 and ES-2 is: how to ensure that the advantages of Anti-flt1 and ES-2 are fused in the same peptide, and overcome the inherent disadvantages of each polypeptide before fusion. The fusion of Anti-flt1 and ES-2 is realized by designing a linker (GGGG), and the fused ES-2-AF peptide has better bioactivity, a more definite action mechanism, better stability and longer half-life.
In another embodiment of the present application, there is provided a hyaluronic acid modification of ES-2-AF peptide, wherein the amino group of ES-2-AF peptide is bonded to the carboxyl group of hyaluronic acid molecule through an amide bond, and the structure formula is as follows:
HA-CO-NH-(ES-2-AF)n；
wherein n is 30 to 60; ES-2-AF HAs a molecular weight of 2197.5Da and HA HAs a molecular weight of 240000 Da.
Compared with the ES-2-AF peptide, the hyaluronic acid modified substance of the ES-2-AF peptide retains the anti-angiogenesis and anti-tumor activity of the ES-2-AF peptide, integrates the anti-angiogenesis and other characteristics of macromolecular hyaluronic acid, and has the characteristics of higher stability, stronger hydrophilicity, stronger targeting property and the like, so that the hyaluronic acid modified substance has better use effect and application value.
In order to make the technical solutions of the present application more clearly understood by those skilled in the art, the technical solutions of the present application will be described in detail below with reference to specific embodiments.
The test materials used in the examples of the present invention are all conventional in the art and commercially available.
Example 1: preparation of ES-2-AF peptides
The molecular weight of the ES-2-AF peptide is 2197.5Da, Fmoc solid phase synthesis is adopted, high performance liquid phase purification is carried out, and the structure confirmation is carried out by means of mass spectrometry, high performance liquid chromatography and the like, wherein the purity can reach more than 95%.
Example 2: preparation of ES-2-AF peptide hyaluronic acid modifier (HA-ES-2-AF)
The preparation steps are as follows:
(1) dowex ion exchange resin was washed with water, and an excess of tetrabutylammonium hydroxide TBA-OH (24.5ml) was added and mixed well. Dowex-TBA resin was obtained and the supernatant was removed by filtration. Sodium hyaluronate NaHA (1g) was dissolved in water and poured into prepared Dowex-TBA resin (10 g). After mixing for 3h, filter with a 0.45 μm syringe filter to remove Dowex resin to give a clear HA-TBA solution, and lyophilize for 3 days.
(2) Respectively dissolving the HA-TBA and the ES-2-AF peptide prepared in the step (1) in DMSO. After HA-TBA was fully dissolved, an excess amount of Kate's condensing agent BOP was added to activate the HA carboxyl group, and mixed well for 30 min. Then, the HA-TBA solution, the peptide solution and an equimolar amount of DIPEA are mixed and dissolved in DMSO, and after 24 hours of reaction, an equal volume of 1M NaOH aqueous solution is added. The reaction was stopped by lowering the pH to 3.0 with 1M HCl and then raising the pH to 7.0 with 1M NaOH. The reaction product was poured into a pre-treated dialysis bag (10kDa) and dialyzed against a large amount of NaCl solution, 25% ethanol, water. Freeze-drying for 3 days.
By using1The structure is identified by H nuclear magnetic resonance, the result is shown in figure 1, and the result shows that hyaluronic acid successfully modifies ES-2-AF peptide, and the average connection rate is 93.75%.
Test example 1: comparison of the Anti-cell-proliferation Effect of ES-2-AF peptide with that of Anti-flt1 and ES-2 peptide
The experimental procedure was as follows:
(1) test drugs: the Anti-flt1 peptide and the ES-2 peptide were identical to the ES-2-AF peptide prepared in example 1, and the peptide concentrations in the three groups of drugs were identical.
(2) The test method comprises the following steps:
collecting human umbilical vein endothelial cell EAhy926 at logarithmic phase, adjusting cell suspension concentration to 7000/well, dividing into 96-well plates, placing 200 μ L (10% FBS-containing DMEM medium) per well, and standing at 37 deg.C and 5% CO2Culturing in a constant temperature incubator to make the cells adhere to the wall; adding test drugs with different concentration gradients, and continuously culturing for 48h, wherein each drug has 5 multiple holes; adding 20 mu L of MTT solution into each well, continuing to culture for 4h at 37 ℃, terminating the culture, measuring the absorbance (OD) value of each well at the wavelength of 490nm by using an enzyme-linked immunosorbent assay (ELISA) detector, and calculating the inhibition rate: inhibition rate is 1- [ (experimental-blank)/(control-blank). The experiment was repeated three times and the mean value was taken.
The results of the Anti-endothelial cell proliferation assay are shown in FIG. 2, and it can be seen from FIG. 2 that the Anti-endothelial cell proliferation effect of the ES-2-AF peptide is generally higher than that of the Anti-flt1 peptide and the ES-2 peptide after 48 hours of the effect of the Anti-flt1 peptide, the ES-2 peptide and the ES-2-AF peptide in different concentration gradients.
Test example 2: in vitro test (ELISA method) for the ability of Anti-Flt1, ES-2-AF peptides to inhibit the binding of VEGF and its receptor VEGFR1(Flt-1)
(1) Test drugs: the Anti-flt1 peptide and the ES-2 peptide were identical to the ES-2-AF peptide prepared in example 1, and the peptide concentrations in the three groups of drugs were identical.
(2) The test method comprises the following steps:
VEGF165dissolved in PBS to make 0.5. mu.g/mL, and applied to a 96-well plate at 50. mu.L/well overnight at 4 ℃. Phosphate buffered saline PBS plates were washed with 300 μ L x 3 times for 5 minutes each. 3 wt% BSA in PBS 250. mu.L/well, blocked for 2 hours at 37 ℃. Phosphate Buffered Saline (PBS) was washed, 300. mu.L.3 times,each time for 5 minutes. Adding the sample to be tested. Each concentration was 3 replicates. Incubate at room temperature for 1 hour. Positive control: 500ng/mL Flt-Fc in BSA 1 wt% in PBS. Experimental groups: 500ng/mL Flt-Fc + Anti-Flt1 group, ES-2-AF group, peptide concentrations in all three groups were 5, 25, 50, 100, 200. mu.g/mL. PBS containing 0.05 wt% Tween20 was washed 300 μ L by 3 times for 5 minutes each. A secondary antibody anti-human IgG-HRP-Fc in 0.3 wt% BSA in PBS (1:1000, 50. mu.L) was added and incubated at room temperature for 1 hour. PBS containing 0.05 wt% Tween20 was washed 300 μ L x 2 times for 5min each, then PBS was washed 300 μ L x 1 times for 5 min. The water is sucked up by tapping on the water-absorbing cloth. And (3) incubating the TMB color development solution with the light shielding capacity of 200 mu L per well, and testing the TMB color development solution by an enzyme immunoassay instrument at 450nm after 10 minutes.
The results are shown in FIG. 3, and when the concentration is more than 25 μ g/mL, the ability of ES-2-AF to inhibit the combination of VEGF and the receptor thereof is better than that of Anti-flt1 and ES-2 peptide.
Test example 3: the comparison experiment process of the cell proliferation resisting effect of the ES-2-AF peptide hyaluronic acid modifier and the ES-2-AF peptide is as follows:
(1) test drugs: the hyaluronic acid-modified ES-2-AF peptide prepared in example 2 (HA-ES-2-AF), the ES-2-AF peptide prepared in example 1, and a mixture of the ES-2-AF peptide and hyaluronic acid in a binding ratio (HA & ES-2-AF) were used, and the peptide concentrations in the three groups of drugs were identical.
(2) The test method comprises the following steps:
collecting human umbilical vein endothelial cell EAhy926 at logarithmic phase, adjusting cell suspension concentration to 7000/well, dividing into 96-well plates, placing 200 μ L (10% FBS-containing DMEM medium) per well, and standing at 37 deg.C and 5% CO2Culturing in a constant temperature incubator to make the cells adhere to the wall; adding test drugs with different concentration gradients, and continuously culturing for 48h, wherein each drug has 5 multiple holes; adding 20 mu L of MTT solution into each well, continuing to culture for 4h at 37 ℃, terminating the culture, measuring the absorbance (OD) value of each well at the wavelength of 490nm by using an enzyme-linked immunosorbent assay (ELISA) detector, and calculating the inhibition rate: inhibition rate is 1- [ (experimental-blank)/(control-blank). The experiment was repeated three times and the mean value was taken.
The result of the anti-endothelial cell proliferation test is shown in fig. 4, and it can be seen from fig. 4 that after the ES-2-AF peptide hyaluronic acid modification compound and the ES-2-AF peptide with different concentration gradients act for 48 hours, the anti-endothelial cell proliferation effect of the ES-2-AF peptide hyaluronic acid modification compound is obviously stronger than that of the ES-2-AF peptide with the same peptide concentration and the mixture of the ES-2-AF peptide and the ES-2-AF peptide with the same peptide concentration.
Test example 4: in vitro assay for the binding Capacity of HA-ES-2-AF conjugates to inhibit VEGF and its receptor VEGFR1(Flt-1) (ELISA method)
(1) Test drugs: the hyaluronic acid-modified ES-2-AF peptide prepared in example 2 (HA-ES-2-AF), the ES-2-AF peptide prepared in example 1, and a mixture of the ES-2-AF peptide and hyaluronic acid in a binding ratio (HA & ES-2-AF) were used, and the peptide concentrations in the three groups of drugs were identical.
(2) The test method comprises the following steps:
VEGF165dissolved in PBS to make 0.5. mu.g/mL, and applied to a 96-well plate at 50. mu.L/well overnight at 4 ℃. Phosphate buffered saline PBS plates were washed with 300 μ L x 3 times for 5 minutes each. 3 wt% BSA in PBS 250. mu.L/well, blocked for 2 hours at 37 ℃. Phosphate buffered saline PBS plates were washed with 300 μ L x 3 times for 5 minutes each. Adding the sample to be tested. Each concentration was 3 replicates. Incubate at room temperature for 1 hour. Positive control: 500ng/mL Flt-Fc in BSA 1 wt% in PBS. Experimental groups: 500ng/mL Flt-Fc + ES-2-AF group, HA&ES-2-AF group and HA-ES-2-AF group, and the peptide concentration in the three groups is 5, 25, 50, 100 and 200 mug/mL. PBS containing 0.05 wt% Tween20 was washed 300 μ L by 3 times for 5 minutes each. A secondary antibody anti-human IgG-HRP-Fc in 0.3 wt% BSA in PBS (1:1000, 50. mu.L) was added and incubated at room temperature for 1 hour. PBS containing 0.05 wt% Tween20 was washed 300 μ L x 2 times for 5min each, then PBS was washed 300 μ L x 1 times for 5 min. The water is sucked up by tapping on the water-absorbing cloth. And (3) incubating the TMB color development solution with the light shielding capacity of 200 mu L per well, and testing the TMB color development solution by an enzyme immunoassay instrument at 450nm after 10 minutes.
The results are shown in FIG. 5, and the inhibitory effects of the ES-2-AF peptide, HA & ES-2-AF mixture, and HA-ES-2-AF conjugate all increased in a concentration-dependent manner. And when the peptide concentration is higher than 25 mu g/mL, the inhibition effect of the mixture group is better than that of the ES-2-AF peptide, and the inhibition effect of the HA-ES-2-AF conjugate group is the most obvious of the three.
Test example 5: transwell cell assay for inhibition of cell migration
(1) Test drugs: the hyaluronic acid-modified ES-2-AF peptide prepared in example 2, the ES-2-AF peptide prepared in example 1, and a mixture of the ES-2-AF peptide and hyaluronic acid in a binding ratio were used, and the peptide concentrations in the three groups of drugs were consistent.
(2) The test method comprises the following steps:
the sterile pipette tips were placed in a refrigerator at 4 ℃ and refrigerated overnight. The Matrigel gel was transferred to a 4 ℃ freezer and thawed for about 40 min. Matrigel gel was diluted 1:6 with FBS-free antibiotic-free DMEM, 50 μ l was added to each well of the upper layer of the Transwell chamber, incubated in an incubator for 1h, the Transwell chamber was removed, the non-coagulated liquid in the upper chamber was aspirated, and the chamber was washed once with DMEM, a sterile medium. The chamber was inverted, and FN (fibronectin) 10. mu.l was uniformly applied to the lower layer of the semipermeable membrane, and air-dried in a clean bench. Add 600. mu.l (containing DMEM complete medium) to each well of 24-well plate, put the chamber therein; endothelial cells EAhy926 at logarithmic growth phase were collected, cell suspension concentration was adjusted to 5 × 104/well, 100 μ l of cells were seeded in the upper chamber of the Transwell chamber, drug-treated cells were added, incubated in the incubator for 24h, the chamber was removed, the upper medium was aspirated, Matrigel and cells that did not cross the membrane were wiped off with a cotton swab, and washed once with PBS. The chamber was fixed in fixative (methanol: glacial acetic acid ═ 3:1) for 25min, washed once with PBS, stained with 0.1% crystal violet stain for 25min, washed twice with PBS, air dried, photographed under an inverted fluorescence microscope, cells stained through Matrigel gel to the lower layer of the chamber were counted and a statistical plot was made. The experiment was repeated 5 times and the mean value was taken.
The experimental result is shown in figure 6, and each medicine adding group has obvious effect of inhibiting the migration of endothelial cells compared with a control group. The least number of cells migrated in the HA-ES-2-AF conjugate group compared to the ES-2-AF peptide and HA & ES-2-AF mixture indicates that HA-ES-2-AF inhibits cell migration most strongly.
The above description is only a preferred embodiment of the present application and is not intended to limit the present application, and various modifications and changes may be made by those skilled in the art. Any modification, equivalent replacement, improvement and the like made within the spirit and principle of the present application shall be included in the protection scope of the present application.
SEQUENCE LISTING
<110> Shandong university
<120> preparation methods and applications of neoangiogenesis inhibitory peptide and hyaluronic acid modifier thereof
<130>
<160>1
<170>PatentIn version 3.3
<210>1
<211>22
<212>PRT
<213> Artificial Synthesis
<400>1
Ile Val Arg Arg Ala Asp Arg Ala Ala Val Pro Gly Gly Gly Gly Gly
1 5 10 15
Gly Asn Gln Trp Phe Ile
20
Claims (10)
1. An ES-2-AF peptide is characterized in that the amino acid sequence is shown as SEQ ID NO.1 in a sequence table.
2. A hyaluronic acid-modified ES-2-AF peptide, which is obtained by bonding an amino group of the ES-2-AF peptide of claim 1 with a carboxyl group of a hyaluronic acid molecule through an amide bond, and has the following structural formula:
HA-CO-NH-(ES-2-AF)n；
wherein n is 30 to 60.
3. The hyaluronic acid modification of ES-2-AF peptide according to claim 2, wherein n is 60.
4. The method for preparing a hyaluronic acid modification of an ES-2-AF peptide according to claim 2, comprising the steps of:
(1) preparing HA-TBA conjugate, improving the solubility of HA, and enabling the HA to be dissolved in an organic solvent;
(2) dissolving HA-TBA conjugate in organic solvent, adding a kat condensing agent to activate carboxyl of HA; then mixing the HA-TBA, the ES-2-AF peptide and the DIPEA after activating carboxyl, reacting for 24 hours, and adding NaOH solution; then adjusting the pH value of the reaction system to be reduced to 3.0 to stop the reaction; and then, the pH value of the reaction system is increased to 7.0, and the reaction product is dialyzed and dried to obtain the hyaluronic acid modifier of the ES-2-AF peptide.
5. The method of claim 4, wherein in step (1), the HA-TBA conjugate is prepared by: adding excessive tetrabutylammonium hydroxide into Dowex ion exchange resin, uniformly mixing to obtain Dowex-TBA resin, and filtering to remove supernatant; dissolving sodium hyaluronate NaHA in water, pouring into prepared Dowex-TBA resin, uniformly mixing, filtering to remove Dowex resin to obtain a clear HA-TBA solution, and freeze-drying to obtain the HA-TBA conjugate.
6. The method according to claim 4, wherein in the step (2), the molar ratio of HA-TBA to ES-2-AF is 1 (4-100).
7. The method according to claim 4, wherein in the step (2), the pH is adjusted to 3.0 with 1M HCl; the pH was raised to 7.0 with 1M NaOH.
8. The method according to claim 4, wherein in the step (2), the reaction product is dialyzed by: the reaction product was poured into a pretreated dialysis bag and dialyzed against NaCl solution, 25% ethanol and water, respectively.
9. Use of an ES-2-AF peptide according to claim 1 and/or a hyaluronan modification of an ES-2-AF peptide according to claim 2 for the preparation of a medicament for inhibiting angiogenesis.
10. Use of the ES-2-AF peptide of claim 1 and/or the hyaluronan modification of the ES-2-AF peptide of claim 2 for the preparation of a therapeutic and/or anti-tumor drug associated with a neoangiogenic disease.
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| CN201710432512.9A CN107261124B (en) | 2017-06-09 | 2017-06-09 | Preparation method and application of angiogenesis inhibiting peptide and hyaluronic acid modifier thereof |
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| CN201710432512.9A CN107261124B (en) | 2017-06-09 | 2017-06-09 | Preparation method and application of angiogenesis inhibiting peptide and hyaluronic acid modifier thereof |
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| CN107261124B true CN107261124B (en) | 2020-05-22 |
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| CN110358799B (en) * | 2019-07-26 | 2021-02-26 | 山东大学 | Method for site-directed modification of polypeptide based on enzyme-catalyzed hyaluronic acid |
| CN115611967A (en) * | 2019-10-11 | 2023-01-17 | 润辉生物技术（威海）有限公司 | Hyaluronan oligopeptide and preparation and application methods thereof |
| CN112618714B (en) * | 2020-11-30 | 2022-02-22 | 四川大学 | Anti-FLT1 polypeptide mediated synthetic gold cluster and preparation method and application thereof |
| CN113648427B (en) * | 2021-08-20 | 2023-07-28 | 山东大学 | Hyaluronic acid-ES 2-AF peptide conjugate, and preparation method and application thereof |
| CN117050146A (en) * | 2023-10-11 | 2023-11-14 | 杭州湃肽生化科技有限公司 | Hyaluronic acid modified cosmetic peptide, preparation method and application thereof |
| CN118063557A (en) * | 2023-10-11 | 2024-05-24 | 杭州湃肽生化科技有限公司 | Hyaluronic acid modifier of cosmetic peptide and application thereof |
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