CN113061180A

CN113061180A - Conjugate of ES2 peptide without anticoagulation activity heparin modification and combination with paclitaxel, preparation method and application thereof

Info

Publication number: CN113061180A
Application number: CN202110362873.7A
Authority: CN
Inventors: 谭海宁; 孙凤; 侯慧文; 王洁; 唐雯; 李妍; 符家爱; 卢鲁
Original assignee: Shandong University
Current assignee: Shandong University
Priority date: 2021-04-02
Filing date: 2021-04-02
Publication date: 2021-07-02

Abstract

The invention provides a conjugate of ES2 peptide without anticoagulation activity heparinization modification and combined with paclitaxel, a preparation method and application thereof, belonging to the technical field of biological medicines. The invention can prepare PTX-anticoagulant-free active heparin-CYS-ES 2 peptide conjugates with different binding degrees by controlling related process conditions. Compared with ES2 peptide, the prepared conjugate of ES2 peptide modified by heparinization without anticoagulation activity and combined with paclitaxel retains the anti-angiogenesis and anti-tumor activities of ES2 peptide, integrates the anti-tumor activities of heparin and paclitaxel, and has the characteristics of higher stability, hydrophilicity, targeting property and the like, so that the conjugate has better use effect and application value.

Description

Conjugate of ES2 peptide without anticoagulation activity heparin modification and combination with paclitaxel, preparation method and application thereof

Technical Field

The invention belongs to the technical field of biological medicines, and particularly relates to a conjugate of ES2 peptide without anticoagulation activity heparin modification and combined with paclitaxel, and a preparation method and application thereof.

Background

The information in this background section is only for enhancement of understanding of the general background of the invention and is not necessarily to be construed as an admission or any form of suggestion that this information forms the prior art that is already known to a person of ordinary skill in the art.

Endostatin (ES) is one of the most effective endogenous angiogenesis inhibitors at present, can effectively inhibit the pathological angiogenesis process in the body, has broad-spectrum anti-tumor capability and has no obvious toxic effect. Through years of efforts of domestic and foreign research scholars, ES is found to have unique anti-angiogenesis and anti-tumor activity.

In 2005, 9 months, the national food and drug administration approved domestic new recombinant human endostatin (Endostar, engdu) to be marketed in China, and used in combination with chemotherapeutic drugs for the treatment of advanced non-small cell lung cancer. ES2(IVRRADRAAVP, SEQ ID NO.1) is a polypeptide sequence of 11 amino acids in ES, including ESAmino acid 60-70 sequence and three surface exposed arginine residues Arg⁶²，Arg⁶³And Arg⁶⁶. ES2 has significant anti-angiogenic and anti-tumor activity, is easier to access the interior of blood vessels and is more accessible and remodelable. However, ES2 has the disadvantages of short in vivo half-life and poor stability, and its application is greatly limited.

Disclosure of Invention

Aiming at the defects of the prior art, the invention provides a conjugate of ES2 peptide, which is heparin-modified without anticoagulation activity and combined with paclitaxel, and a preparation method and application thereof. The invention successfully prepares the heparin-cystamine-ES 2 peptide conjugate without anticoagulation activity and/or the paclitaxel-cystamine-ES 2 peptide conjugate without anticoagulation activity with different conjugation degrees by optimizing and controlling the relevant parameters and conditions of the reaction, and the conjugates have good stability and bioactivity and thus have good value of practical application.

In order to achieve the technical purpose, the technical scheme adopted by the invention is as follows:

the invention provides a heparin modified product without anticoagulation activity of ES2 peptide, which is prepared by linking amino in cystamine with carboxyl in anticoagulant-free heparin (GSHP) through amidation reaction and then binding carboxyl of ES2 peptide with free amino in cystamine through amidation reaction, wherein the structural formula is as follows:

GSHP～CYS-(ES2)n；

wherein n is 1 to 10; the molecular weight of ES2 is 1223Da, and the molecular weight of GSHP is 3000-8000 Da.

Further, the heparin modification product without anticoagulation activity of the ES2 peptide can be combined with paclitaxel, specifically, the amino group on cystamine is connected with carboxyl on glycol-cleaved heparin (GSHP) through an amido bond, the hydroxyl group of paclitaxel is connected with the carboxyl in GSHP through an ester bond, and the carboxyl of ES2 peptide is connected with free amino on cystamine through an amido bond, and the structural formula is as follows:

(PTX)n₁-GSHP～CYS-(ES2)n₂；

in the formula, n₁＝1～5，n₂1-10, ES2 molecular weight of 1223Da, the isoelectric point is 10.42, the molecular weight of GSHP is 1000 Da-8000 Da, and the anticoagulation activity is not higher than 40%.

Preferably, said n₁＝2，n₂10, the molecular weight is 3000-8000Da, and the anticoagulant activity is not higher than 20%.

In a second aspect of the present invention, there is provided a method for preparing a conjugate of the aforementioned ES2 peptide modified by heparinization without anticoagulation activity in combination with paclitaxel, comprising:

(1) when the heparin modified substance without anticoagulation activity of the ES2 peptide is GSHP-CYS-ES 2, the preparation method comprises the following steps:

s1, preparation of GSHP-CYS conjugates: dissolving GSHP in water, adding EDCI and NHS as catalysts to activate carboxyl in the GSHP, adjusting pH to alkalescence after activation is finished, slowly adding CYS solution to react, and purifying reaction products after reaction is finished to obtain a GSHP-CYS combination; CYS is used as an intermediate connector to connect ES2 and GSHP, so that ES2 can be released from GSHP-CYS-ES 2 conjugate in a tumor microenvironment to be in a free state and quickly reach endothelial cells around the tumor to play an anti-angiogenesis role.

S2, preparation of GSHP-CYS-ES 2 conjugate: dissolving ES2 in water, adding EDCI and NHS as catalysts, activating, slowly adding the GSHP-CYS solution for continuous reaction, and after the reaction is finished, purifying the reaction product to obtain the GSHP-CYS-ES 2 conjugate.

(2) When the conjugate of the ES2 peptide without anticoagulation activity heparin modification and paclitaxel is PTX-GSHP-CYS-ES 2, the preparation method comprises the following steps:

s1, preparation of GSHP-CYS conjugates: dissolving GSHP in water, adding EDCI and NHS as catalysts to activate carboxyl in the GSHP, adjusting pH to alkalescence after activation is finished, slowly adding CYS solution to react, and purifying reaction products after reaction is finished to obtain a GSHP-CYS combination; CYS is used as an intermediate connector to connect ES2 and GSHP, so that ES2 can be released from PTX-GSHP-CYS-ES 2 conjugates in a tumor microenvironment to be in a free state and quickly reach endothelial cells around the tumor to play an anti-angiogenesis role.

S2, preparation of GSHP-CYS-TBA conjugates: dissolving the GSHP-CYS in double distilled water, adding Dowex ion exchange resin, stirring, filtering to remove the resin, adjusting the pH value of the GSHP-CYS solution to be neutral or alkalescent by using TBA solution, and freeze-drying to obtain the GSHP-CYS-TBA conjugate. The addition of TBA can improve the solubility of GSHP-CYS in organic solvent, so that GSHP-CYS can be dissolved in the organic solvent.

S3, preparation of PTX-GSHP-CYS conjugates: dissolving the GSHP-CYS-TBA in an organic solvent, adding DCC and DMAP as catalysts to perform catalytic reaction, slowly adding the PTX solution to continue the reaction, and after the reaction is finished, purifying the reaction product to obtain the PTX-GSHP-CYS combination.

S4, preparation of PTX-GSHP-CYS-ES 2 conjugate: dissolving ES2 in water, adding EDCI and NHS as catalysts to activate carboxyl, slowly adding the PTX-GSHP-CYS solution to continue reaction after activation, and after the reaction is finished, purifying the reaction product to obtain the PTX-GSHP-CYS-ES 2 conjugate.

In the step S1, adjusting the pH to be alkalescent specifically by adding an alkali solution (such as NaOH) to adjust the pH to about 7.40; after the CYS solution is added, the reaction time is controlled to be 8-15 h, preferably 10 h; the reaction product purification step includes dialysis and drying steps.

In a third aspect of the present invention, there is provided a use of the conjugates of the ES2 peptide without heparinization modification and paclitaxel in combination for preparing anti-angiogenesis-related diseases and/or anti-tumor drugs.

Wherein, the angiogenesis related diseases include but are not limited to diabetic retinopathy, age-related macular degeneration, arthritis and the like;

such tumors include, but are not limited to, melanoma, breast, lung, colon, ovarian, renal tumors, and the like.

In a fourth aspect of the present invention, there is provided a pharmaceutical and/or antineoplastic agent against angiogenesis-related diseases, said agent comprising a non-anticoagulant heparin-modified ES2 peptide as defined above in combination with a paclitaxel conjugate, and further comprising at least one or more pharmaceutically or dietetically acceptable excipients. The adjuvants can be solid or liquid.

Compared with the prior art, one or more technical schemes have the following beneficial technical effects:

(1) according to the technical scheme, PTX-anticoagulant-free active heparin-CYS-ES 2 peptide conjugates with different binding degrees can be prepared by controlling the supply amount of CYS, ES2 peptide and PTX, the pH value of a reaction system, the reaction time and other conditions.

(2) Compared with ES2 peptide, the GSHP-CYS-ES 2/PTX-GSHP-CYS-ES 2 conjugate prepared by the technical scheme has higher stability and stronger biological activity, thereby having good practical application value.

Drawings

The accompanying drawings, which are incorporated in and constitute a part of this specification, are included to provide a further understanding of the invention, and are incorporated in and constitute a part of this specification, illustrate exemplary embodiments of the invention and together with the description serve to explain the invention and not to limit the invention.

FIG. 1: nuclear magnetic resonance hydrogen spectrum of GSHP-CYS-ES 2 conjugate;

FIG. 2: nuclear magnetic resonance hydrogen spectrum of PTX-GSHP-CYS-ES 2 conjugate;

FIG. 3: inhibition of endothelial cell proliferation by ES2, GSHP-CYS-ES 2 and PTX-GSHP-CYS-ES 2 conjugates;

FIG. 4: the ES2, GSHP-CYS-ES 2 and PTX-GSHP-CYS-ES 2 conjugates have the effect of inhibiting the proliferation of melanoma cells;

FIG. 5: the combination of ES2, GSHP-CYS-ES 2 and PTX-GSHP-CYS-ES 2 has the function of inhibiting the migration of endothelial cells;

FIG. 6: inhibition of endothelial cell lumen formation by ES2, GSHP-CYS-ES 2 and PTX-GSHP-CYS-ES 2 conjugates;

FIG. 7: the inhibition effect of ES2, GSHP-CYS-ES 2 and PTX-GSHP-CYS-ES 2 combination on the invasion of melanoma cells;

FIG. 8: GSHP-CYS-ES 2 and PTX-GSHP-CYS-ES 2 conjugates were evaluated for environmentally responsive release behavior.

Detailed Description

It is to be understood that the following detailed description is exemplary and is intended to provide further explanation of the invention as claimed. 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 invention 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 exemplary embodiments according to the invention. 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. It is to be understood that the scope of the invention is not to be limited to the specific embodiments described below; it is also to be understood that the terminology used in the examples is for the purpose of describing particular embodiments only, and is not intended to limit the scope of the present invention.

Unless otherwise indicated, the term "anticoagulant activity": the ability to inhibit certain coagulation factors in the blood, preventing blood coagulation; the term "glycol-cleaved heparin": heparin derivatives obtained by breaking chemical bonds of ortho-hydroxyl of non-sulfated uronic acid in heparin structure.

As described above, ES2 peptide (IVRRADRAAVP, SEQ ID NO.1) has significant anti-angiogenic and anti-tumor activities, is easier to access the interior of blood vessels, and is more accessible and remodelable. However, ES2 has the disadvantages of short in vivo half-life and poor stability, and its application is greatly limited.

Meanwhile, in the process of generating and developing the tumor, the formation of new blood vessels can provide nutrient substances for the growth of tumor tissues, and if the generation and development of the tumor are required to be inhibited, the nutrient supply is cut off, namely the formation of the new blood vessels is inhibited, and finally the purpose of starving the tumor is achieved. Diabetic Retinopathy (DR) is one of the common complications of diabetes. Studies have shown that retinal neovascularization is one of the major pathogenic causes of DR disease, and thus inhibition of neovascularization is effective in inhibiting DR.

Therefore, the invention also aims to provide the application of the ES2 peptide conjugate which is modified by heparin without anticoagulation activity and combined with paclitaxel in preparing anti-angiogenesis or anti-tumor drugs.

Specifically, in a typical embodiment of the present invention, a heparin modified product without anticoagulation activity of ES2 peptide is provided, which is formed by linking amino group in cystamine with carboxyl group in heparin without anticoagulation activity through amidation reaction, and then binding carboxyl group in ES2 peptide with free amino group in cystamine through amidation reaction, and the structural formula is as follows:

GSHP～CYS-(ES2)n；

Compared with ES2 peptide, the heparin modification without anticoagulation activity of the ES2 peptide enhances the anti-angiogenesis and anti-tumor activity of the ES2 peptide, integrates the targeting property and anti-tumor property of heparin molecules, and enables the obtained conjugate to have stronger biological activity, higher targeting property and stronger stability, thereby showing better application potential.

In another embodiment of the present invention, the heparin-modified product without anticoagulation activity of ES2 peptide can be combined with paclitaxel, specifically, it is formed by connecting amino group on cystamine with carboxyl group on glycol-cleaved heparin (GSHP) through amide bond, hydroxyl group of paclitaxel is connected with carboxyl group in GSHP through ester bond, and carboxyl group of ES2 peptide is connected with free amino group on cystamine through amide bond, and the structural formula is as follows:

(PTX)n₁-GSHP～CYS-(ES2)n₂；

in the formula, n₁＝1～5，n₂1-10, ES2 has molecular weight of 1223Da, isoelectric point of 10.42, GSHP has molecular weight of 1000 Da-8000 Da, and anticoagulant activity is not higher than 40%.

Compared with GSHP-CYS-ES 2 and ES2 peptides, the conjugate of the ES2 peptide without anticoagulation activity heparin modification combined paclitaxel has stronger anti-angiogenesis activity and anti-tumor activity, integrates the targeting property and anti-tumor activity of heparin molecules in tumor tissues and the anti-tumor activity of paclitaxel, ensures that the obtained conjugate has stronger anti-tumor effect, more stable structure and stronger hydrophilicity, and thus shows better use effect and application value in the anti-tumor field.

The molecular weight of the glycol-broken heparin influences the activity of the ES2 peptide, and if the molecular weight of the glycol-broken heparin is too large, certain steric hindrance and conformational change are generated, so that the activity of the micromolecule ES2 peptide is reduced; however, the molecular weight of the glycol-cleaved heparin should not be too low, and a certain amount of carboxyl groups should be ensured. The invention comprehensively considers the combination degree of ES2 peptide and paclitaxel and the biological activity of the conjugate, preferably the molecular weight of GSHP is 3000-8000Da, and the anticoagulant activity of the conjugate is reduced while the good activity of ES2 peptide is kept.

The supply amount of Cystamine (CYS) influences the combination degree of cystamine and GSHP, the invention optimizes the supply amount of CYS and comprehensively considers the influence on the spatial conformation of GSHP, the combination degree of paclitaxel and GSHP and the combination degree of ES2 and GSHP, and the supply amount of cystamine preferably accounts for 80 percent of the disaccharide unit of GSHP.

Amount of ES2 peptide supplied (i.e., n in the above structural formula)₂) The invention optimizes and considers the supply amount of ES2, and comprehensively considers the influence on GSHP space conformation, the binding degree of paclitaxel and chondroitin sulfate and the binding degree of ES2 and chondroitin sulfate, preferably n₂＝10。

The amount of Paclitaxel (PTX) (i.e., n in the above formula)₁) Influence on the binding degree of the PTX and the optimal consideration of the supply amount of the PTX, the combination of the influence on the spatial conformation of the GSHP, the binding degree of the taxol and the GSHP and the binding degree of the ES2 and the GSHP, preferably n₁＝2。

In another embodiment of the present invention, there is provided a method for preparing a heparin-modified product of the ES2 peptide without anticoagulation activity, comprising:

In the step S2, the activation time is controlled to be 10-60min, preferably 45 min; slowly adding the GSHP-CYS solution to continue reacting, wherein the reaction time is controlled to be 12-48 h, preferably 24 h; the reaction product purification step comprises dialysis and drying.

In the step S2, the organic solvent may be dichloromethane or DMSO; adding Dowex ion exchange resin, and then stirring, wherein the stirring time is controlled to be 8-10 h, preferably 10 h; the concentration of the TBA solution is controlled to be 10-30% (preferably 20%); and (3) using TBA solution to adjust the pH of the GSHP-CYS solution to be neutral or alkalescent, specifically using 20% TBA solution to adjust the pH of the GSHP-CYS solution to be 7.07.

In the step S3, the catalytic reaction is controlled for 10-60min, preferably 45 min; after the PTX solution is added, the reaction time is controlled to be 24-72 h, preferably 48 h; the reaction product purification step comprises dialysis and drying;

more specifically, the dialysis method of the reaction product is as follows: the reaction product was poured into a pre-treated dialysis bag, dialyzed against DMSO for one day and then against water.

In the step S4, the activation time is controlled to be 10-60min, preferably 45 min; slowly adding the PTX-GSHP-CYS solution to continue reacting, wherein the reaction time is controlled to be 12-48 h, and preferably 24 h; the reaction product purification step comprises dialysis and drying.

The invention makes CYS become an intermediate connector of GSHP and ES2 by preparing GSHP-CYS combination. CYS has a disulfide bond that can be cleaved around the tumor, leaving ES2 free from the GSHP sugar chain.

The invention changes the solubility of the GSHP by preparing the GSHP-CYS-TBA combination, so that the GSHP which is not dissolved in an organic solvent can be dissolved in dichloromethane.

The invention can change the solubility of the paclitaxel by preparing the PTX-GSHP-CYS conjugate, so that the paclitaxel conjugate is dissolved in double distilled water.

The invention successfully prepares the conjugate PTX-GSHP-CYS-ES 2 of ES2 peptide without anticoagulation activity, which has longer half-life and stronger activity, and combines with paclitaxel by optimizing the conditions of catalyst supply, pH of a reaction system, reaction time and the like.

In another embodiment of the present invention, there is provided a use of the conjugates of the ES2 peptide without heparinization modification and paclitaxel in combination for preparing anti-angiogenesis-related diseases and/or anti-tumor drugs.

In another embodiment of the present invention, there is provided a pharmaceutical and/or antineoplastic agent for treating angiogenesis-related diseases, wherein the pharmaceutical and/or antineoplastic agent comprises the heparin modification product without anticoagulation activity of the ES2 peptide, and further comprises at least one or more pharmaceutically or dietetically acceptable excipients. The adjuvants can be solid or liquid.

In still another embodiment of the present invention, the drug is a solid oral preparation, a liquid oral preparation or an injection.

In yet another embodiment of the present invention, the pharmaceutical dosage form is injectable implants, emulsions, liposomes, microcapsules, microspheres, nanoparticles, and the like.

The invention is further illustrated by the following examples, which are not to be construed as limiting the invention thereto. It should be understood that these examples are for illustrative purposes only and are not intended to limit the scope of the present invention.

Example 1: preparation of heparin modified products (GSHP-CYS-ES 2) without anticoagulation activity of ES2 peptide

The preparation steps are as follows:

(1) dissolving a proper amount of GSHP in double distilled water to obtain a GSHP solution, and then adding EDCI and NHS catalysts into the solution in a ratio of 3: 2, mixing uniformly, slowly stirring at room temperature, and adjusting the pH value to 7.41 after stirring. Dissolving cystamine by using double distilled water, dropwise adding the cystamine solution into the GSHP solution, and slowly stirring for reaction for 10 hours. After the reaction was completed, the reaction solution was transferred to a dialysis bag having MWCO of 1000Da and dialyzed with double distilled water for two days to remove impurities. And after dialysis is finished, collecting reaction liquid, and putting the reaction liquid into a freeze dryer for freeze drying to obtain the GSHP-CYS combination.

(2) ES2 short peptide (synthesized by solid phase synthesis) was dissolved in double distilled water to obtain ES2 solution. EDCI and NHS were added separately and activated with slow stirring for 45 min. Dissolving a proper amount of GSHP-CYS in double distilled water, dropwise adding the solution into ES2, and reacting for 24h at room temperature. After completion of the reaction, the mixture was dialyzed against double distilled water in a dialysis bag having MWCO of 1000Da for two days to remove impurities. And after the dialysis is finished, collecting reaction liquid, and putting the reaction liquid into a freeze dryer for freeze drying to obtain the GSHP-CYS-ES 2 conjugate.

Example 2: preparation of heparin-modified ES2 peptide without anticoagulation Activity in combination with conjugate of paclitaxel (PTX-GSHP-CYS-ES 2)

The preparation steps are as follows:

(2) Dissolving a proper amount of GSHP-CYS samples in double distilled water, and adding the same amount of GSHP-CYS samples into the double distilled water

50WX8-400 ion exchange resin, and slowly stirring for about 9h at room temperature. After the reaction is finished, filtering the reaction solution by using a 0.45-micron filter membrane to remove resin, adjusting the pH to about 7.07 by using 20% TBA solution, and freeze-drying in a freeze dryer to obtain GSHP-CYS-TBA conjugate.

(3) Dissolving a proper amount of GSHP-CYS-TBA by using dichloromethane, respectively adding DCC and DMAP into the solution as catalysts, oscillating and uniformly mixing, and activating for 45 min. After completion of the activation, PTX was added to the reaction solution, and the reaction was continued for 48 hours. After the reaction is finished, putting the reaction solution into a dialysis bag with MWCO of 1000Da, dialyzing for 24h by DMSO, then changing water for dialysis for three days, and removing impurities. After dialysis, collecting reaction liquid, and freeze-drying in a freeze-dryer to obtain the PTX-GSHP-CYS conjugate.

(4) An appropriate amount of ES2 was dissolved in double distilled water to obtain an ES2 solution, to which EDCI and NHS were added as a carboxyl catalyst and activated for 45 min. Then, PTX-GSHP-CYS is dissolved in double distilled water, and is dropwise added into the reaction liquid, and the mixture is slowly stirred for 24 hours at room temperature. After completion of the reaction, the reaction solution was transferred to a dialysis bag with MWCO of 5000Da and dialyzed against double distilled water for two days, with water being changed every 4 hours, to remove unreacted ES 2. After dialysis, the reaction solution was collected and lyophilized in a freeze-dryer to obtain PTX-GSHP-CYS-ES 2 conjugate.

By using¹H NMR analysis shows that GSHP-CYS-ES 2/PTX-GSHP-CYS-ES 2 structure, and as a result, GSHP-CYS-ES 2/PTX-GSHP-CYS-ES 2 conjugate is successfully prepared as shown in figure 1 and figure 2.

Experimental example 1: comparison of the inhibitory Effect of ES2 peptide, GSHP-CYS-ES 2 and PTX-GSHP-CYS-ES 2 conjugates on endothelial cell proliferation

The experimental procedure was as follows:

(1) experimental drugs: the ES2 peptide, GSHP-CYS-ES 2 conjugate prepared in example 1, and PTX-GSHP-CYS-ES 2 conjugate prepared in example 2 were combined at the same concentration of ES2 peptide in the three groups of drugs.

(2) The experimental method comprises the following steps: EAhy926 cells grown in log phase were collected, the cell suspension was adjusted to the appropriate concentration and grown at 1X 10 per well⁴The individual cells were seeded in a 96-well plate, placed in a carbon dioxide incubator, and the 96-well plate was cultured overnight at 37 ℃ until the cells were adherent. Then adding ES2, PTX-GSHP-CYS-ES 2 and GSHP-CYS-ES 2 drugs respectively, wherein the drug concentrations are respectively 5 mug/mL, 25 mug/mL, 50 mug/mL, 75 mug/mL and 125 mug/mL (the concentration is based on the ES2 concentration), and 8 compound wells are arranged for each drug. Wells containing DMEM medium alone were set as a blank control, and wells containing cells without drug-containing medium were set as a negative control. Placing a 96-well plate in a carbon dioxide incubator for incubation for 48h, discarding the culture medium under the condition of keeping out of the sun, adding a CCK-8 solution into the 96-well plate, wherein each well is 10 mu L, placing the 96-well plate in the incubator, taking out the culture medium after the culture medium is changed into orange, detecting the OD value of each well under the condition of 450nm wavelength by using a microplate reader, and calculating the cell inhibition rate according to the following calculation formula: inhibition rate ═ 1- [ (experimental group-blank control group)/(negative control group-blank control group)]]×100％。

The results of the experiments on the inhibition of endothelial cell proliferation are shown in FIG. 3. As can be seen from the figure, the three groups of drugs have obvious inhibition effect on the proliferation of EAhy926 cells, and the inhibition rate is increased in a dose-dependent manner with the increase of the peptide concentration. It was also found that PTX-GSHP-CYS-ES 2 showed better inhibition than the other two groups with increasing concentration.

Experimental example 2: comparison of the inhibitory Effect of ES2 peptide, GSHP-CYS-ES 2 and PTX-GSHP-CYS-ES 2 conjugates on the proliferation of B16F10 hyper-metastatic melanoma cells

The experimental procedure was as follows:

(2) The experimental method comprises the following steps: B16F10 cells grown in log phase were collected, the cell suspension was adjusted to an appropriate concentration, and seeded into 96-well plates at 1 × 104 cells per well, placed in a carbon dioxide incubator, and the 96-well plates were cultured overnight at 37 ℃ until the cells attached to the wall. Then adding ES2, PTX-GSHP-CYS-ES 2 and GSHP-CYS-ES 2 drugs respectively, wherein the drug concentrations are respectively 5 mug/mL, 25 mug/mL, 50 mug/mL, 75 mug/mL and 125 mug/mL (the concentration is based on the ES2 concentration), and 8 compound wells are arranged for each drug. Wells containing DMEM medium alone were set as a blank control, and wells containing cells without drug-containing medium were set as a negative control. Placing a 96-well plate in a carbon dioxide incubator for incubation for 48h, discarding the culture medium under the condition of keeping out of the sun, adding a CCK-8 solution into the 96-well plate, wherein each well is 10 mu L, placing the 96-well plate in the incubator, taking out the culture medium after the culture medium is changed into orange, detecting the OD value of each well under the condition of 450nm wavelength by using a microplate reader, and calculating the cell inhibition rate according to the following calculation formula: the inhibition rate was [1- [ (experimental group-blank group)/(negative control group-blank group) ] ] × 100%.

The results of the experiment for inhibiting the proliferation of melanoma cells are shown in FIG. 4. As can be seen from the figure, the ES2 group of drugs has no great influence on the proliferation of B16F10 cells, while the GSHP-CYS-ES 2 and the PTX-GSHP-CYS-ES 2 group of drugs have obvious inhibition effect on the B16F10 cells, and the inhibition rate is increased in a dose-dependent manner along with the increase of the concentration. Meanwhile, PTX-GSHP-CYS-ES 2 is found to show better inhibition effect than the GSHP-CYS-ES 2 group along with the increase of the concentration.

Experimental example 3: comparison of the inhibition of endothelial cell migration by ES2 peptide, GSHP-CYS-ES 2 and PTX-GSHP-CYS-ES 2 conjugates

The experimental procedure was as follows:

(2) The experimental method comprises the following steps: placing 20cm ruler into a clean bench, ultraviolet irradiating for 30min, and placing the yellow gun head in a refrigerator at 4 deg.C for overnight use. Collecting EAhy926 cells growing in logarithmic phase, and makingThe cell forming suspension is inoculated in a 6-well plate, and the inoculation amount is 5 multiplied by 10⁴Perwell, 2mL DMEM medium was added to each well, and 6-well plates were placed at 37 ℃ with 5% CO₂Culturing in a cell culture box until the cells adhere to the wall. The medium was discarded, PBS washed to remove floating dead cells, the cell surface was scratched with a yellow tip using a sterile ruler as a comparison to obtain a straight line without cells, and this operation was repeated three times per well to obtain three parallel straight lines. PBS was rinsed again to remove floating cells. 2mL of serum-free medium containing ES2, PTX-GSHP-CYS-ES 2 and GSHP-CYS-ES 2 were added to each of the three drugs, each at 25. mu.g/mL, 75. mu.g/mL and 125. mu.g/mL (the concentrations of the drugs were based on the concentration of ES2), PBS was used as a negative control, and three replicates of each drug were performed. At 37 deg.C, 5% CO₂Incubate 6-well plates in the cell incubator for 48 h. The 6-well plate after completion of incubation was photographed with an inverted fluorescence microscope, and the number of migrated cells was counted. The experiment was repeated 5 times to obtain an average value. The results of the experiments on the inhibition of endothelial cell migration are shown in FIG. 5. As can be seen from the figure, GSHP-CYS-ES 2 and PTX-GSHP-CYS-ES 2 both significantly reduced the migration of cells compared to ES2, but PTX-GSHP-CYS-ES 2 had a stronger ability to inhibit the migration of endothelial cells.

Experimental example 4: comparison of the inhibitory Effect of ES2 peptide, GSHP-CYS-ES 2 and PTX-GSHP-CYS-ES 2 conjugates on endothelial cell luminal formation

The experimental procedure was as follows:

(2) The experimental method comprises the following steps: the sterile yellow tip and 48-well plate were pre-cooled overnight in a 4 ℃ freezer and the Matrigel gel was placed in a 4 ℃ freezer to completely melt. The melted Matrigel gel was added to a pre-cooled 48-well plate (80. mu.L/well), and the 48-well plate was placed in a 4 ℃ freezer for 30min to remove air bubbles and to flatten the Matrigel. Then placing the plate into a cell culture box for incubation, collecting EAhy926 cells in a logarithmic growth phase after the Matrigel gel is solidified, and adjusting the concentration of the cell suspension to 8 multiplied by 10⁴Each well was added to a matrigel-laid plate. Respectively adding ES2, PTX-GSHP-CYS-ES 2 and GSHP-CYS-ES 2 serum-free medium containing three drugs with concentrations of 25. mu.g/mL, 75. mu.g/mL and 125. mu.g/mL (each concentration is based on the concentration of ES2), and bFGF with a final concentration of 5ng/mL is added to each well, and 3 duplicate wells are provided for each drug. And (3) placing the 48-hole plate into a constant-temperature cell culture box for 6-8h, and then taking a picture by using an inverted fluorescence microscope to observe the generation condition of the cellular vascular network. The experiment was repeated 5 times and the mean value was taken.

The results of the inhibition lumen formation experiments are shown in fig. 6. As can be seen from the figure, the cell treated with PTX-GSHP-CYS-ES 2 at a concentration of 25-125 μ g/mL can significantly inhibit the ability of EAhy926 to form lumen, and at the same concentration, the ability to inhibit lumen formation is enhanced compared with ES2 and GSHP-CYS-ES 2. It can also be seen that the GSHP-CYS-ES 2 conjugate inhibited endothelial cell luminal formation slightly more strongly than ES2 under the same conditions.

Experimental example 5: comparison of the effects of ES2 peptide, GSHP-CYS-ES 2 and PTX-GSHP-CYS-ES 2 conjugates on inhibition of melanoma cell invasion

The experimental procedure was as follows:

(2) The experimental method comprises the following steps: the pipette tip is placed in a refrigerator at 4 ℃ for pre-cooling, and the Matrigel gel is placed in the refrigerator at 4 ℃ until the Matrigel is completely melted. The thawed Matrigel gel was diluted in serum-free 1640 medium at a dilution ratio of 1: 6. 50 μ L of diluted matrigel was added to the upper layer of each of the Transwell chambers in the 24-well plate, placed in a cell incubator and incubated for 40min until the matrigel was completely solidified, the Transwell chambers were removed and washed once with serum-free medium. Then, the chamber was inverted, fibronectin FN was applied to the lower layer of the semipermeable membrane of the chamber at 10. mu.L/chamber, and the chamber was air-dried in a clean bench after the application was completed. Each well of the 24-well plate was filled with 600. mu.L of 1640 medium, and the chamber was placed therein, respectively. Collecting logarithmic phase-grown B16F10 cells, making into cell suspension, and inoculating 100 μ L of cells at an inoculum size of 5 × 10⁴One hole is inoculated in the upper chamber of the Transwell chamber, and the three drugs including ES2, GSHP-CYS-ES 2 and PTX-GSHP-CYS-ES 2 are added according to the final concentrationCells were treated at 25. mu.g/mL, 75. mu.g/mL, 125. mu.g/mL (concentrations are based on ES2), and 3 duplicate wells were provided for each drug. The 24-well plate was incubated at 37 ℃ in a 5% CO2 cell incubator for 24 h. After incubation was complete the chamber was removed, the upper layer of medium was discarded, Matrigel gel and some cells that did not cross the membrane were gently wiped off with a cotton swab and rinsed in PBS for 2 min. Then the chamber was fixed in a fixative for 35min, rinsed 1 time with PBS, stained with 0.1% crystal violet stain for 40min, washed twice with PBS, air dried and photographed under an inverted fluorescence microscope. The experiment was repeated 5 times and the mean value was taken.

The results of experiments on inhibition of melanoma cell invasion are shown in FIG. 7. As can be seen, after 24h of incubation under the same conditions, the number of cells migrating in the PTX-GSHP-CYS-ES 2 group was significantly reduced compared to the ES2 and GSHP-CYS-ES 2 groups, and it was found that the addition of PTX significantly improved the ability of the entire conjugate to resist tumor cell invasion.

Experimental example 6: evaluation of environmental responsive Release behavior of ES2 peptides, GSHP-CYS-ES 2 and PTX-GSHP-CYS-ES 2 conjugates

The experimental procedure was as follows:

(2) The experimental method comprises the following steps: in order to verify whether the synthesized PTX-GSHP-CYS-ES 2 has responsiveness in Glutathione (GSH) environment, the accumulated release amount of ES2 in the two drugs is detected by an ultraviolet spectrophotometry. 2mg/mL PTX-GSHP-CYS-ES 2 and 1mL of GSHP-CYS-ES 2 drugs were respectively put in 8mL PBS buffer solutions containing GSH with different concentrations (30.65mg/mL, 12.26mg/mL, 6.13mg/mL, 1.23 μ g/mL) and without GSH for in vitro simulated disulfide bond degradation experiments. Each solution was incubated on a shaker at 37 ℃ and then 1mL of the drug was taken at various time points (0.5h, 4h, 8h, 24h, 48h) and the UV value of ES2 in the collected drug was determined spectrophotometrically.

The evaluation results of the environmentally-responsive release behavior of the conjugate are shown in FIG. 8. As can be seen from the figure, the two combinations of PTX-GSHP-CYS-ES 2 and GSHP-CYS-ES 2 have better redox responsiveness; it was also found that at a concentration of 1.23. mu.g/mL (2. mu.M), cleavage of the disulfide bonds did not occur substantially; the UV absorption of ES2 was similar in both groups at concentrations of 12.26mg/mL (20mM) and 6.13mg/mL (10 mM); whereas, at a concentration of 30.65mg/mL (20nM), ES2 showed a smaller difference in UV from the previous two groups. The disulfide bonds were mainly concentrated at a GSH concentration of 6.13mg/mL (10mM) and the concentration was similar to that in tumor cells, indicating a better intracellular responsive release of both drugs.

It should be noted that the above examples are only used to illustrate the technical solutions of the present invention and not to limit them. Although the present invention has been described in detail with reference to the examples given, those skilled in the art can modify the technical solution of the present invention as needed or equivalent substitutions without departing from the spirit and scope of the technical solution of the present invention.

SEQUENCE LISTING

<110> Shandong university

<120> conjugate of ES2 peptide, which is heparin modified without anticoagulation activity and combined with paclitaxel, and preparation method and application thereof

<130>

<160> 1

<170> PatentIn version 3.3

<210> 1

<211> 11

<212> PRT

<213> ES2 polypeptide sequence

<400> 1

Ile Val Arg Arg Ala Asp Arg Ala Ala Val Pro

1 5 10

Claims

1. The heparin modification product without anticoagulation activity of ES2 peptide is characterized in that the heparin modification product is formed by connecting amino in cystamine with carboxyl in heparin without anticoagulation activity through amidation reaction and then combining carboxyl of ES2 peptide with free amino in cystamine through amidation reaction, and the structural formula is as follows:

GSHP～CYS-(ES2)n；

wherein n is 1 to 10; the molecular weight of GSHP is 3000-8000 Da.

2. The heparin-modified product without anticoagulation activity of ES2 peptide according to claim 1, wherein the heparin-modified product without anticoagulation activity of ES2 peptide is further modified with paclitaxel, i.e., heparin-modified product without anticoagulation activity of ES2 peptide, and combined with a conjugate of paclitaxel, specifically, the heparin-modified product is formed by linking amino group of cystamine with carboxyl group of glycol-cleaved heparin through amide bond, linking hydroxyl group of paclitaxel with carboxyl group of GSHP through ester bond, and linking carboxyl group of ES2 peptide with free amino group of cystamine through amide bond, and the structural formula is as follows:

(PTX)n₁-GSHP～CYS-(ES2)n₂；

in the formula, n₁＝1～5，n₂1-10, the molecular weight of GSHP is 1000 Da-8000 Da, and the anticoagulation activity is not higher than 40%;

3. The method for producing an ES2 peptide heparin-modified product without anticoagulation activity according to claim 1, which comprises:

s1, preparation of GSHP-CYS conjugates: dissolving GSHP in water, adding EDCI and NHS as catalysts to activate carboxyl in the GSHP, adjusting pH to alkalescence after activation is finished, slowly adding CYS solution to react, and purifying reaction products after reaction is finished to obtain a GSHP-CYS combination;

4. The method according to claim 3, wherein the step S1, the pH adjustment is performed by adding alkali solution to adjust the pH to be slightly alkaline to about 7.40; after the CYS solution is added, the reaction time is controlled to be 8-15 h, preferably 10 h; the reaction product purification step comprises dialysis and drying steps; or the like, or, alternatively,

in the step S2, the activation time is controlled to be 10-60min, preferably 45 min; slowly adding GSHP-CYS solution to continue reacting, wherein the reaction time is controlled to be 12-48 h, preferably 24 h; the reaction product purification step comprises dialysis and drying.

5. The method of claim 2 for preparing conjugates of ES2 peptide modified by heparinization without anticoagulant activity in combination with paclitaxel, comprising:

s2, preparation of GSHP-CYS-TBA conjugates: dissolving the GSHP-CYS in double distilled water, adding Dowex ion exchange resin, stirring, filtering to remove the resin, adjusting the pH value of the GSHP-CYS solution to be neutral or alkalescent by using a TBA solution, and freeze-drying to obtain a GSHP-CYS-TBA conjugate;

s3, preparation of PTX-GSHP-CYS conjugates: dissolving GSHP-CYS-TBA in an organic solvent, adding DCC and DMAP as catalysts to perform catalytic reaction, slowly adding a PTX solution to continue the reaction, and after the reaction is finished, purifying reaction products to obtain a PTX-GSHP-CYS combination;

s4, preparation of PTX-GSHP-CYS-ES 2 conjugate: the preparation method comprises the following steps: dissolving ES2 in water, adding EDCI and NHS as catalysts to activate carboxyl, slowly adding the PTX-GSHP-CYS solution to continue reaction after activation, and after the reaction is finished, purifying the reaction product to obtain the PTX-GSHP-CYS-ES 2 conjugate.

6. The method according to claim 5, wherein the step S1, the pH adjustment is performed by adding alkali solution to adjust the pH to be slightly alkaline to about 7.40; after the CYS solution is added, the reaction time is controlled to be 8-15 h, preferably 10 h; the reaction product purification step comprises dialysis and drying steps; or the like, or, alternatively,

in the step S2, the organic solvent includes dichloromethane or DMSO; adding Dowex ion exchange resin and stirring, wherein the stirring time is controlled to be 8-10 h; the concentration of the TBA solution is controlled to be 10-30% (preferably 20%); and (3) using TBA solution to adjust the pH of the GSHP-CYS solution to be neutral or alkalescent, specifically using 20% TBA solution to adjust the pH of the GSHP-CYS solution to be 7.07.

7. The method according to claim 5, wherein in step S3, the catalytic reaction is controlled for 10-60min, preferably 45 min; after the PTX solution is added, the reaction time is controlled to be 24-72 h, preferably 48 h; the reaction product purification step comprises dialysis and drying;

preferably, the dialysis method of the reaction product is: the reaction product was poured into a pre-treated dialysis bag, dialyzed against DMSO for one day and then against water.

8. The method according to claim 5, wherein in step S4, the activation time is controlled to 10-60min, preferably 45 min; slowly adding the PTX-GSHP-CYS solution to continue reacting, wherein the reaction time is controlled to be 12-48 h, and preferably 24 h; the reaction product purification step comprises dialysis and drying.

9. Use of a conjugate of the heparin modification without anticoagulation activity of the ES2 peptide according to claim 1 or the heparin modification without anticoagulation activity of the ES2 peptide according to claim 2 in combination with paclitaxel for preparing a medicament and/or an antitumor medicament for treating a disease associated with angiogenesis;

preferably, the angiogenesis-related diseases include diabetic retinopathy, age-related macular degeneration and arthritis;

the tumor includes melanoma, breast tumor, lung tumor, colon tumor, ovarian tumor, and kidney tumor.

10. A drug and/or antitumor agent against a disease associated with angiogenesis, which comprises a heparin-modified non-anticoagulation activity modification product of the ES2 peptide according to claim 1 or a heparin-modified non-anticoagulation activity product of the ES2 peptide according to claim 2 in combination with a conjugate of paclitaxel, and further comprises at least one or more pharmaceutically or dietetically acceptable excipients.