Sulfonated redv and synthesis method and application thereof

Abstract

The application relates to sulfonated REDV and a synthesis method and application thereof, and belongs to the technical field of biomaterial synthesis. The synthesis method of the sulfonated REDV comprises the following steps: dissolving REDV in an organic solvent I to obtain a REDV solution, dissolving a sulfonating reagent in an organic solvent II to obtain a sulfonating reagent solution; adding the sulfonating reagent solution into the REDV solution to perform a sulfonation reaction and obtain a mixed solution; adjusting the pH of the mixed solution to be alkaline, then adding a precipitant to obtain flocculent precipitates, and the flocculent precipitates are purified by dialysis and freeze-dried to obtain the sulfonated REDV. The synthesis method of the sulfonated REDV is clear in steps, the reaction condition is mild and controllable, the method is easy to operate, and the method is suitable for laboratory and large-scale production.

Description

Technical Field

[0001] This invention belongs to the field of biomaterial synthesis technology, specifically relating to a sulfonated REDV, its synthesis method, and its application. Background Technology

[0002] REDV peptide (Arg-Glu-Asp-Val), as an endothelial cell-specific adhesion peptide, can specifically bind to α4β1 integrin on the surface of vascular endothelial cells, and has important application value in promoting endothelialization of material surfaces. However, natural REDV has the following limitations: poor biological stability, easily digested by enzymes in vivo; and limited ability to bind to material surfaces, limiting its long-term effect in coatings.

[0003] Sulfonation modification can endow peptides with new physicochemical properties. The negative charge introduced into the peptide molecule by the sulfonic acid group generates an intermolecular charge repulsion effect, allowing the peptide to remain dispersed even at low concentrations and improving the uniformity of substrate adsorption during coating. Simultaneously, the hydrophilicity of the sulfonic acid group improves the peptide's water solubility and resistance to enzymatic degradation. Traditional chemical sulfonation methods utilize sulfonating agents to react with the hydroxyl groups of tyrosine (Tyr), serine (Ser), or threonine (Thr) residues in the peptide chain to form sulfate esters, or with the amino group of lysine (Lys) to form sulfonamides. However, traditional sulfonating agents, such as concentrated sulfuric acid, operate under harsh conditions, easily leading to peptide structure destruction or side reactions. Furthermore, REDV has a small molecular weight and lacks specific reaction sites; traditional sulfonating agents easily attack peptide bonds or generate non-specific substitutions, resulting in complex product composition that is difficult to separate and purify, hindering large-scale production. Therefore, there is an urgent need to develop an efficient and stable sulfonated REDV synthesis process. Summary of the Invention

[0004] The first objective of this invention is to provide a method for synthesizing sulfonated REDV, which can efficiently and controllably prepare sulfonated REDV.

[0005] A second objective of this invention is to provide a sulfonated REDV.

[0006] A third objective of this invention is to provide an application of sulfonated REDV.

[0007] To achieve the above objectives, the technical solution adopted by the present invention is as follows: A method for synthesizing sulfonated REDV includes the following steps: dissolving REDV in organic solvent one to obtain a REDV solution, dissolving a sulfonating reagent in organic solvent two to obtain a sulfonating reagent solution; adding the sulfonating reagent solution to the REDV solution to carry out a sulfonation reaction to obtain a mixed solution; adjusting the pH of the mixed solution to alkaline; subsequently adding a precipitant to obtain a flocculent precipitate; purifying the flocculent precipitate by dialysis and freeze-drying to obtain the final product.

[0008] Furthermore, the molar ratio of REDV to sulfonating agent is 1:5 to 10; the concentration of REDV solution is 2 to 5 mg / mL; and the concentration of sulfonating agent solution is 7 to 15.5 mg / mL.

[0009] Furthermore, the sulfonating agent is a sulfur trioxide pyridine complex; both organic solvent one and organic solvent two are dimethyl sulfoxide.

[0010] Furthermore, the sulfonation reaction is carried out at a temperature of 20–26 °C for 1–3 h.

[0011] Furthermore, the pH of the mixed solution is adjusted to an alkaline pH of 8-9.

[0012] Furthermore, the precipitant is acetone, and the volume ratio of the mixed solution to acetone is 1:2 to 5.

[0013] Furthermore, the dialysis purification time is 30–60 h.

[0014] A sulfonated REDV is prepared using the above-described synthetic method for sulfonated REDV; the reaction formula for sulfonated REDV is shown in Formula I: Formula I.

[0015] Application of sulfonated REDV in cell culture additives, drug carriers or biomaterial surface active coatings.

[0016] The beneficial effects of this invention are: The N-terminal amino group of the arginine residue in REDV reacts with a sulfur trioxide-pyridine complex to generate sulfonated REDV. In the sulfonation reaction, the sulfur trioxide-pyridine complex acts as a mild sulfonating agent. The resulting electrophilic sulfonated group dissociates and undergoes a nucleophilic substitution reaction with the amino group of the arginine residue in REDV, introducing a sulfonic acid group into REDV, thus yielding sulfonated REDV. This invention controls the degree of sulfonation by adjusting temperature and time, utilizing the strong nucleophilicity of the arginine amino group to achieve sulfonation. Controlling temperature and pH conditions ensures the smooth progress of the reaction and the stability of the sulfonated REDV product.

[0017] The sulfonated REDV synthesis method of this invention has clearly defined steps, mild and controllable reaction conditions, and is easy to operate, making it suitable for laboratory and large-scale production. The sulfonated REDV synthesized by the method of this invention has a well-defined chemical structure. The introduction of sulfonic acid groups endows sulfonated REDV with unique physicochemical properties and biological activity, and improves the stability of the sulfonated REDV product.

[0018] The sulfonated REDV synthesized in this invention has better endothelial cell adhesion, proliferation characteristics and anti-proliferative effects compared with the unsulfonated REDV, and is expected to be applied in potential biomedical fields such as vascular stent coatings. Attached Figure Description

[0019] Figure 1 Flowchart for the preparation of sulfonated REDV; Figure 2 FTIR spectra of unsulfonated REDV and sulfonated REDV from Examples 1-3; Figure 3 XPS full spectra of unsulfonated REDV and sulfonated REDV from Examples 1-3; Figure 4 Fine sulfur element spectra of unsulfonated REDV and sulfonated REDV of Examples 1-3; Figure 5 The CCK-8 absorbance values ​​of human umbilical vein endothelial cells in each group; Figure 6 The absorbance of NO in human umbilical vein endothelial cells in each group is represented. Figure 7 Fluorescent staining images of human umbilical vein endothelial cells in each group; Figure 8 Apoptosis staining pattern of human umbilical vein endothelial cells in each group; Figure 9 Fluorescent staining images of smooth muscle cells in each group; Figure 10 The results of fluorescence staining of specific factors in smooth muscle cells in each group and the fluorescence intensity of Ipwin32 three-dimensional imaging. Detailed Implementation

[0020] The present invention will be further described below with reference to the embodiments and accompanying drawings.

[0021] The amino acid sequence of REDV is arginine-glutamic acid-aspartic acid-valine (Arg-Glu-Asp-Val), and its molecular weight is 517 g / mol.

[0022] Example 1 The method for synthesizing sulfonated REDV in Example 1 includes the following steps: S1: Add 100 mg of REDV to 50 mL of dimethyl sulfoxide (DMSO) and stir magnetically at 20 °C until REDV is completely dissolved to obtain a REDV solution; add 154 mg of sulfur trioxide pyridine complex (SO3-Py) to 20 mL of DMSO and stir magnetically at 20 °C until SO3-Py is completely dissolved to obtain an SO3-Py solution. Slowly add the SO3-Py solution to the REDV solution using a dropper, and carry out the sulfonation reaction by magnetic stirring at 20 °C for 1 h to obtain a mixed solution.

[0023] S2: After the sulfonation reaction is completed, 1 mol / L NaOH anhydrous ethanol solution is added to the mixed solution to adjust the pH of the mixed solution to 8. The mixed solution is washed multiple times with 2 times the volume of acetone to obtain a white flocculent precipitate. The white flocculent precipitate is dialyzed in ultrapure water for 2 days, and the dialysate is changed every 10-15 h. After dialysis purification, it is freeze-dried to obtain powdered sulfonated REDV. The sulfonated REDV of Example 1 is named S-REDV-1.

[0024] The sulfonated REDV of Example 1 was prepared according to the synthesis method of the sulfonated REDV of Example 1.

[0025] Example 2 The method for synthesizing sulfonated REDV in Example 2 includes the following steps: S1: Add 200 mg of REDV to 40 mL of DMSO and stir magnetically at 26 °C until REDV is completely dissolved to obtain a REDV solution; add 615 mg of SO3-Py to 40 mL of DMSO and stir magnetically at 26 °C until SO3-Py is completely dissolved to obtain an SO3-Py solution. Slowly add the SO3-Py solution to the REDV solution using a dropper, and stir magnetically at 26 °C for 2 h to carry out the sulfonation reaction to obtain a mixed solution.

[0026] S2: After the sulfonation reaction was completed, 1 mol / L NaOH anhydrous ethanol solution was added to the mixed solution to adjust the pH of the mixed solution to 8.5. Three times the volume of acetone was added to wash the mixed solution several times to obtain a white flocculent precipitate. The white flocculent precipitate was dialyzed in ultrapure water for 2 days, and the dialysate was changed every 10-15 hours. After dialysis purification, it was freeze-dried to obtain powdered sulfonated REDV. The sulfonated REDV of Example 2 was named S-REDV-2.

[0027] The sulfonated REDV of Example 2 was prepared according to the synthesis method of the sulfonated REDV of Example 2.

[0028] Example 3 The synthesis method of sulfonated REDV in Example 3 includes the following steps: S1: Add 300 mg of REDV to 60 mL of DMSO and stir magnetically at 20 °C until REDV is completely dissolved to obtain a REDV solution; add 922.5 mg of SO3-Py to 60 mL of DMSO and stir magnetically at 20 °C until SO3-Py is completely dissolved to obtain an SO3-Py solution. Slowly add the SO3-Py solution to the REDV solution using a dropper, and stir magnetically at 20 °C for 3 hours to carry out the sulfonation reaction to obtain a mixed solution.

[0029] S2: After the sulfonation reaction is completed, 1 mol / L NaOH anhydrous ethanol solution is added to the mixed solution to adjust the pH of the mixed solution to 9. Five times the volume of acetone is added to wash the mixed solution several times to obtain a white flocculent precipitate. The white flocculent precipitate is dialyzed in ultrapure water for 2 days, and the dialysate is changed every 10-15 hours. After dialysis purification, it is freeze-dried to obtain powdered sulfonated REDV. The sulfonated REDV in Example 3 is named S-REDV-3.

[0030] The sulfonated REDV of Example 3 was prepared according to the synthesis method of the sulfonated REDV of Example 3.

[0031] from Figure 2 It can be seen that, compared with REDV that has not undergone sulfonation, the sulfonated REDV in Examples 1-3 has a higher growth rate at 1350 cm⁻¹. -1 and 1120~1130 cm -1 The presence of a new characteristic peak for S=O in the sulfonic acid group at all locations indicates that the sulfonated REDV in Examples 1-3 has successfully introduced sulfonic acid groups. (1350 cm⁻¹) -1 The peak at 1120–1130 cm⁻¹ is a peak of S=O antisymmetric stretching. -1 The peak at 620 cm⁻¹ is a peak resulting from S=O symmetric stretching. -1 The new peak appearing at this location may correspond to the bending vibration of the SO bond in the sulfonic acid group. (The peak appears at 1600–1700 cm⁻¹.) -1 The retained peaks all indicate the retention of peptide bonds, confirming the integrity of the sulfonated REDV polypeptide structure in Examples 1-3.

[0032] from Figure 3 It can be seen that the sulfonated REDV in Examples 1-3 all showed a Na 1s peak. This is because after the sulfonation reaction and pH adjustment, Na... + It combines with carboxyl and sulfonic acid groups to form sodium carboxylate and sodium sulfonate.

[0033] from Figure 3 It can be seen that obvious S 2p absorption peaks appeared on the surface of the sulfonated REDV in Examples 1-3, indicating that the sulfonic acid groups were successfully introduced into the sulfonated REDV in Examples 1-3. Figure 4It can be seen that the positions of the S 2p peaks in the sulfonated REDV of Examples 1-3 are significantly different, at 168.38 eV, 168.26 eV, and 168.32 eV respectively, indicating that the chemical environment of the S element in the sulfonated REDV of Examples 1-3 is quite different. This may be because the content of sulfonic acid groups in the sulfonated REDV of Examples 1-3 is significantly different. As the sulfonation reaction time increases, the peak area of ​​the S element also increases, indicating that the S content of the sulfonated REDV of Examples 1-3 gradually increases.

[0034] Table 1. Sulfonated REDV and relative S content in REDV in Examples 1-3

[0035] Application Example 1 I. Endothelial cell proliferation Blank control group: Endothelial cell culture medium + endothelial cells + CCK-8; REDV group: Endothelial cell culture medium + endothelial cells + REDV + CCK-8; S-REDV-1 group: Endothelial cell culture medium + endothelial cells + S-REDV-1 + CCK-8; S-REDV-2 group: Endothelial cell culture medium + endothelial cells + S-REDV-2 + CCK-8; S-REDV-3 group: Endothelial cell culture medium + endothelial cells + S-REDV-3 + CCK-8.

[0036] The number of viable cells was determined using the CCK-8 reagent. The absorbance of the cell culture supernatant at 450 nm was measured using a microplate reader. Figure 5 As can be seen, after 1 day of endothelial cell culture, there were no significant differences in the absorbance values ​​of the cell culture supernatant among the blank control group, REDV group, S-REDV-1 group, S-REDV-2 group, and S-REDV-3 group. After 3 days of endothelial cell culture, the absorbance values ​​of the cell culture supernatant in the S-REDV-1 group, S-REDV-2 group, and S-REDV-3 group were all higher than those in the blank control group and REDV group, showing a gradually increasing trend. Therefore, it is evident that the sulfonated REDV in Examples 1-3 can significantly promote endothelial cell proliferation.

[0037] II. Release of the functional factor NO NO released by endothelial cells can inhibit platelet aggregation, suppress smooth muscle cell proliferation, and promote vasodilation, making it a key signaling molecule for maintaining vascular homeostasis. The NO release levels from endothelial cells on days 1 and 3 in the blank control group, REDV group, S-REDV-1 group, S-REDV-2 group, and S-REDV-3 group were measured using Griess reagent. Figure 6 As shown. By Figure 6As can be seen, after 1 day of endothelial cell culture, there were no significant differences in the absorbance values ​​of the supernatant of the cell culture medium among the blank control group, REDV group, S-REDV-1 group, S-REDV-2 group, and S-REDV-3 group. After 3 days of endothelial cell culture, the absorbance values ​​of the REDV group, S-REDV-1 group, S-REDV-2 group, and S-REDV-3 group were all higher than those of the blank control group, while the absorbance value of the S-REDV-1 group was slightly lower than that of the REDV group. This may be because the amount of sulfonic acid groups introduced into S-REDV-1 in Example 1 was relatively low, and the surface charge and conformation of REDV were in a transitional state, resulting in a slightly lower NO secretion level in endothelial cells than in the REDV group. However, due to the increased adsorption by endothelial cells, the proliferation capacity of endothelial cells still showed an upward trend. Therefore, the sulfonated REDV in Examples 1-3 can significantly promote the release of the functional factor NO from endothelial cells and promote the differentiation of endothelial cells towards functional maturity.

[0038] III. Fluorescent staining analysis of endothelial cell morphology Depend on Figure 7 It can be seen that after 1 day of endothelial cell culture, most endothelial cells in the blank control group showed a shrunken state and poor endothelial cell spreading, while some endothelial cells in the REDV group, S-REDV-1 group, S-REDV-2 group and S-REDV-3 group showed better spreading. After 3 days of endothelial cell culture, the endothelial cells in the blank control group showed obvious shrunkenness, while the number of endothelial cells adhering in the REDV group, S-REDV-1 group, S-REDV-2 group and S-REDV-3 group increased significantly. Moreover, the S-REDV-1 group, S-REDV-2 group and S-REDV-3 group all showed a large number of adhering cells, a large spreading area and a good cytoskeleton.

[0039] IV. AO / EB reagent staining analysis of endothelial cells from Figure 8 It can be seen that after 1 day of endothelial cell culture, except for a small number of apoptotic endothelial cells in the blank control group, most endothelial cells in the REDV group, S-REDV-1 group, S-REDV-2 group, and S-REDV-3 group were in a viable state. After 3 days of endothelial cell culture, the number of apoptotic endothelial cells in the blank control group increased significantly, and the degree of apoptosis was more severe. The number of endothelial cells in the REDV group, S-REDV-1 group, S-REDV-2 group, and S-REDV-3 group was significantly increased compared with the blank control group. Moreover, no significant apoptosis was observed in the endothelial cells of the S-REDV-1 group, S-REDV-2 group, and S-REDV-3 group compared with the blank control group and the REDV group. Therefore, it can be seen that the sulfonated REDV in Examples 1-3 is beneficial to promoting the activation of endothelial cells.

[0040] Application Example 2 Smooth muscle cell morphology and fluorescence staining analysis Blank control group: smooth muscle cell culture medium + smooth muscle cells; REDV group: smooth muscle cell culture medium + smooth muscle cells + REDV; S-REDV-1 group: smooth muscle cell culture medium + smooth muscle cells + S-REDV-1; S-REDV-2 group: smooth muscle cell culture medium + smooth muscle cells + S-REDV-2; S-REDV-3 group: smooth muscle cell culture medium + smooth muscle cells + S-REDV-3.

[0041] Depend on Figure 9 It was found that after one day of smooth muscle cell culture, the number of adherent smooth muscle cells in the REDV group, S-REDV-1 group, S-REDV-2 group, and S-REDV-3 group was significantly less than that in the blank control group. After three days of smooth muscle cell culture, the number of adherent smooth muscle cells in all groups increased. The number of adherent smooth muscle cells in the S-REDV-1 group, S-REDV-2 group, and S-REDV-3 group was lower, and the cells were more relaxed, without apoptosis or necrosis. This result indicates that sulfonated REDV has no cytotoxicity to smooth muscle cells, and the decrease in the number of smooth muscle cells caused by sulfonated REDV is not caused by killing, but rather by the fact that its surface does not support non-specific adhesion and excessive proliferation of smooth muscle cells.

[0042] Contractile smooth muscle cells play an indispensable role in the repair and regeneration of vascular endothelium through multiple mechanisms. Figure 10 The images show the fluorescence staining results and fluorescence intensity maps of smooth muscle cells in each group at 24 h. Figure 10 It can be seen that the fluorescence expression intensity of α-SMA factor in smooth muscle cells of the S-REDV-1 group, S-REDV-2 group and S-REDV-3 group was significantly higher than that of the blank control group. This indicates that sulfonated REDV is beneficial to maintaining the contractile phenotype of smooth muscle cells and inhibiting the transformation of smooth muscle cells into the pathological synthetic phenotype. This proves that the sulfonated REDV synthesized in this invention has better anti-proliferative effects.

[0043] The above results indicate that REDV and sulfonated REDV do not mediate the specific adhesion and activation proliferation of smooth muscle cells. REDV and sulfonated REDV can effectively inhibit the non-specific adhesion and excessive proliferation of smooth muscle cells without producing cytotoxicity, and are beneficial in maintaining the physiological contractile phenotype of smooth muscle cells, reducing the risk of restenosis caused by excessive proliferation of synthetic smooth muscle cells, and can be combined with the specific promoting effect of endothelial cells.

[0044] Other embodiments of this disclosure will readily occur to those skilled in the art upon consideration of the specification and practice of the disclosure herein. This disclosure is intended to cover any variations, uses, or adaptations of this disclosure that follow the general principles of this disclosure and include common knowledge or customary techniques in the art not disclosed herein. The specification and examples are to be considered exemplary only, and the true scope and spirit of this disclosure are indicated by the claims.

[0045] The above description is merely a preferred embodiment of the present invention and is not intended to limit the present invention. The scope of patent protection of the present invention shall be determined by the claims. Similarly, any equivalent structural changes made based on the content of the present invention's specification shall also be included within the scope of protection of the present invention.

Claims

1. A method for synthesizing sulfonated REDV, characterized in that, Includes the following steps: REDV was dissolved in organic solvent one to obtain a REDV solution, and the sulfonating agent was dissolved in organic solvent two to obtain a sulfonating agent solution; The sulfonating reagent solution was added to the REDV solution to carry out the sulfonation reaction to obtain a mixed solution. The pH of the mixed solution was adjusted to alkaline, and then a precipitating agent was added to obtain a flocculent precipitate. The flocculent precipitate was purified by dialysis and freeze-dried to obtain the final product.

2. The method for synthesizing sulfonated REDV according to claim 1, characterized in that, The molar ratio of REDV to sulfonating reagent is 1:5 to 10; the concentration of REDV solution is 2 to 5 mg / mL; and the concentration of sulfonating reagent solution is 7 to 15.5 mg / mL.

3. The method for synthesizing sulfonated REDV according to claim 1, characterized in that, The sulfonating agent is a sulfur trioxide pyridine complex; both organic solvent one and organic solvent two are dimethyl sulfoxide.

4. The method for synthesizing sulfonated REDV according to claim 1, characterized in that, The sulfonation reaction is carried out at a temperature of 20–26 °C for 1–3 h.

5. The method for synthesizing sulfonated REDV according to claim 1, characterized in that, The pH of the mixed solution is adjusted to an alkaline pH of 8-9.

6. The method for synthesizing sulfonated REDV according to claim 1, characterized in that, The precipitant is acetone, and the volume ratio of the mixed solution to acetone is 1:2 to 5.

7. The method for synthesizing sulfonated REDV according to claim 1, characterized in that, The dialysis purification time is 30–60 h.

8. A sulfonated REDV, characterized in that, It was prepared by the synthetic method of sulfonated REDV as described in any one of claims 1 to 7.

9. The application of sulfonated REDV as described in claim 8 in cell culture additives, drug carriers, or biomaterial surface active coatings.

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