Preparation method of CS-Pr / Odex / EGCG composite hydrogel and product

Abstract

The invention belongs to the technical field of hydrogel preparation, and particularly discloses a preparation method of CS-Pr / Odex / EGCG composite hydrogel, the hydrogel is prepared by compounding proline modified chitosan CS-Pr, oxidized dextran and EGCG, so that the prepared hydrogel not only has good injectable performance, wound healing performance and biocompatibility, but also has good biocompatibility, and can be used for preparing the hydrogel. And excellent anti-oxidation and anti-bacterial capabilities are also realized.

Description

Technical Field

[0001] The invention belongs to the technical field of composite hydrogel preparation, and specifically relates to a preparation method and product of a CS-Pr / Odex / EGCG composite hydrogel. Background Art

[0002] Wound healing includes four stages: hemostasis, inflammatory response, proliferation and remodeling, and involves different cells, including inflammatory cells, platelets (such as neutrophils and monocytes), immune cells and endothelial cells. At the same time, the wound healing process (especially full-thickness wounds and diabetic wounds) usually takes a long time to repair, thus increasing the risk of aggravating wound infection, which may lead to ulcers, necrosis and even life-threatening complications. Therefore, it is necessary to design and synthesize wound dressings with antibacterial and wound healing functions. Summary of the invention

[0003] The purpose of the present invention is to provide a method for preparing a CS-Pr / Odex / EGCG composite hydrogel, and providing a composite hydrogel product obtained by the preparation method is the second purpose of the present invention.

[0004] In order to achieve the above-mentioned object of the invention, the present invention adopts the following technical scheme:

[0005] The present invention discloses a method for preparing a CS-Pr / Odex / EGCG composite hydrogel, comprising the following steps:

[0006] 1) dissolving 1.0-4.0 g of chitosan in 180-220 mL of acetic acid solution to obtain a chitosan solution; adding 1.6-2.0 g of proline, 2.0-2.4 g of NHS and 3.2-4.0 g of EDCI to 20-30 mL of a buffer solution, and stirring at room temperature for 25-40 minutes to obtain a proline mixed solution; adding the proline mixed solution to the chitosan solution and stirring for 10-14 hours, dialyzing and freeze-drying to obtain proline-modified chitosan CS-Pr;

[0007] 2) Dissolve 2.0-4.0 g of dextran in 45-55 mL of deionized water, add 3.2-3.6 g of sodium periodate, stir at room temperature in the dark for 10-14 hours, dialyze in deionized water, and freeze-dry to obtain Odex;

[0008] 3) Dissolve 0.04-0.06 g CS-Pr in 1 ml PBS solution to obtain a 4-6% CS-Pr solution; dissolve 0.1-0.4 g Odex in 1 mL PBS solution to obtain a 10-40% Odex solution, add 0.01-0.03 g EGCG to the Odex solution to obtain an EGCG / Odex solution, mix equal volumes of EGCG / Odex solution and CS-Pr solution at room temperature, and let stand at 37°C to obtain a composite hydrogel.

[0009] As further preferred embodiment of the present invention, in step 1), the mass concentration of the acetic acid solution is 1-3%; the buffer solution is a MES buffer solution, pH=5.0; the dialyzed solution is performed with distilled water for 4-6 days, and the MWCO of the dialysis membrane is 3.5 kDa.

[0010] As a further preferred embodiment of the present invention, the dialysis is performed again for 2-4 days using a 7000Da molecular weight cut-off dialysis membrane and distilled water.

[0011] As a further preferred embodiment of the present invention, in step 3), the pH of the PBS solution is 7.4.

[0012] As a further preferred embodiment of the present invention, in step 3), the standing time is 3-4 minutes.

[0013] The invention also discloses a CS-Pr / Odex / EGCG composite hydrogel prepared by the preparation method.

[0014] Compared with the prior art, the present invention has the following technical effects:

[0015] The present invention develops an injectable composite hydrogel, which is prepared by using proline-functionalized chitosan and oxidized dextran through Schiff base reaction. In addition, EGCG is incorporated into the composite hydrogel, so that the prepared hydrogel has excellent antioxidant and antibacterial capabilities. BRIEF DESCRIPTION OF THE DRAWINGS

[0016] Figure 1 Schematic diagram of the reaction process of the hydrogel of the present invention; wherein (a) proline-modified chitosan CS-Pr; (b) the synthesis method of oxidized dextran (Odex); (c) a schematic diagram of preparing a composite hydrogel from Odex, CS-Pr and EGCG;

[0017] Figure 2 Odex (a) and CS-Pr (c) 1 H NMR spectra; (b) FT-IR spectra of Odex and dextran; (d) FT-IR spectra of CS and CS-Pr;

[0018] Figure 3The rheological properties of the hydrogel of the present invention; (a) time scan, (b) strain scan, (c-e) the relationship between the viscosity and shear rate of Gel 1-Gel 3, the inset of Figure ce shows that the prepared hydrogel can be injected by a syringe; (f) the relationship between the time and G'&G" of Gel 1; (g) is a dynamic photo of the self-healing characteristics of Gel 1; (h-i) the relationship between the time and G'&G" of Gel 2 and Gel 3;

[0019] Figure 4 (a) SEM image of hydrogel Gel 1-3; (b) pore size distribution of hydrogel Gel 1-3; (c) swelling ratio results of hydrogel Gel 1-3;

[0020] Figure 5 (a) Release curves of EGCG from Gel 2 and Gel 3. (b) ABTS removal effect of composite hydrogel. (c) Bacterial killing rate of hydrogel against Escherichia coli and Staphylococcus aureus (12 h) and (d) images of hydrogel co-cultured with Staphylococcus aureus and Escherichia coli, respectively (12 h);

[0021] Figure 6 Live / dead staining of NIH 3T3 cells co-cultured with hydrogel extracts after 1, 3, and 5 days. Blue: cells stained with DAPI. Green: cells stained with phalloidin-FITC. Scale bar: 75 μm. DETAILED DESCRIPTION

[0022] The present invention is further described in detail below with reference to the accompanying drawings. It should be noted that in the present invention, room temperature refers to 20-30°C, which can achieve the effects of the present invention. For the following embodiments of the present invention, the room temperature range is controlled between 25-28°C.

[0023] Example 1 EGCG-free composite hydrogel and its preparation

[0024] This embodiment discloses a method for preparing a CS-Pr / Odex composite hydrogel. Figure 1 As shown, the following steps are included:

[0025] 1) Dissolve 2.0 g of chitosan in 200 mL of 1% acetic acid solution to obtain a chitosan solution; add 1.8 g of proline, 2.2 g of NHS and 3.6 g of EDCI to 25 mL of MES buffer solution (pH = 5.0), and stir at room temperature for 30 minutes to obtain a proline mixed solution; add the proline mixed solution to the chitosan solution and stir for 12 hours, then dialyze with distilled water for 5 days (MWCO = 3.5 kDa), and freeze-dry at -80°C to obtain proline-modified chitosan CS-Pr;

[0026] 2) Dissolve 4.0 g of dextran in 50 mL of PBS buffer solution (pH = 7.2), add 3.6 g of sodium periodate and mix, react the resulting mixture at room temperature in the dark for 12 hours, dialyze with distilled water using a 7000 Da molecular weight cutoff dialysis membrane for 3 days, and freeze-dry at -80°C to obtain oxidized dextran Odex;

[0027] 3) 0.04 g CS-Pr was dissolved in 1 ml PBS solution (pH = 7.4) to obtain a 4% CS-Pr solution; 0.1 g ODEX was dissolved in 1 mL PBS solution to obtain a 10% Odex solution, and equal volumes of the Odex solution and the CS-Pr solution were mixed at room temperature and allowed to stand at 37°C for 3 minutes to obtain a composite hydrogel, which was recorded as Gel 1.

[0028] Example 2 Composite hydrogel containing 1% EGCG and its preparation

[0029] This embodiment discloses a method for preparing a CS-Pr / Odex / EGCG composite hydrogel, comprising the following steps:

[0030] 1) Dissolve 2.0 g of chitosan in 200 mL of 1% acetic acid solution to obtain a chitosan solution; add 1.8 g of proline, 2.2 g of NHS and 3.6 g of EDCI to 25 mL of MES buffer solution (pH = 5.0), and stir at room temperature for 30 minutes to obtain a proline mixed solution; add the proline mixed solution to the chitosan solution and stir for 12 hours, then dialyze with distilled water for 5 days (MWCO = 3.5 kDa), and freeze-dry at -80°C to obtain proline-modified chitosan CS-Pr;

[0031] 2) Dissolve 4.0 g of dextran in 50 mL of PBS buffer solution (pH = 7.2), add 3.6 g of sodium periodate and mix, react the resulting mixture at room temperature in the dark for 12 hours, dialyze with distilled water using a 7000 Da molecular weight cutoff dialysis membrane for 3 days, and freeze-dry at -80°C to obtain oxidized dextran Odex;

[0032] 3) 0.04 g CS-Pr was dissolved in 1 ml PBS solution (pH = 7.4) to obtain a 4% CS-Pr solution; 0.1 g ODEX was dissolved in 1 mL PBS solution to obtain a 10% Odex solution; 0.01 g EGCG was added to the Odex solution to obtain EGCG / Odex solutions, and equal volumes of EGCG / Odex solution and CS-Pr solution were mixed at room temperature and allowed to stand for 3 minutes to obtain a composite hydrogel named Gel2.

[0033] Example 3 Composite hydrogel containing 3% EGCG and its preparation

[0034] This embodiment discloses a method for preparing a CS-Pr / Odex / EGCG composite hydrogel, comprising the following steps:

[0035] 1) Dissolve 2.0 g of chitosan in 200 mL of 1% acetic acid solution to obtain a chitosan solution; add 1.8 g of proline, 2.2 g of NHS and 3.6 g of EDCI to 25 mL of MES buffer solution (pH = 5.0), and stir at room temperature for 30 minutes to obtain a proline mixed solution; add the proline mixed solution to the chitosan solution and stir for 12 hours, then dialyze with distilled water for 5 days (MWCO = 3.5 kDa), and freeze-dry at -80°C to obtain proline-modified chitosan CS-Pr;

[0036] 2) 4.0 g of dextran was dissolved in 50 mL of PBS buffer solution (pH = 7.2), and 3.6 g of sodium periodate was added and mixed. The resulting mixture was reacted at room temperature in the dark for 12 hours, dialyzed with distilled water using a 7000 Da molecular weight cutoff dialysis membrane for 3 days, and freeze-dried at -80°C to obtain oxidized dextran Odex;

[0037] 3) 0.04 g CS-Pr was dissolved in 1 ml PBS solution (pH = 7.4) to obtain a 4% CS-Pr solution; 0.1 g ODEX was dissolved in 1 mL PBS solution to obtain a 10% Odex solution; 0.03 g EGCG was added to the Odex solution to obtain EGCG / Odex solutions, and equal volumes of EGCG / Odex solution and CS-Pr solution were mixed at room temperature and allowed to stand for 3 minutes to obtain a composite hydrogel named Gel3.

[0038] In other embodiments of preparing CS-Pr / Odex / EGCG composite hydrogel, the preparation steps can achieve the effect of the present invention if they meet the following conditions:

[0039] 1) dissolving 1.0-4.0g chitosan in 180-220mL acetic acid solution with a mass concentration of 1-3% to obtain a chitosan solution; adding 1.6-2.0g proline, 2.0-2.4g NHS and 3.2-4.0g EDCI to 20-30mL MES buffer solution with pH=5.0, stirring at room temperature for 25-40 minutes to obtain a proline mixed solution; adding the proline mixed solution to the chitosan solution and stirring for 10-14 hours, dialyzing with distilled water for 4-6 days (MWCO of the dialysis membrane=3.5kDa), and freeze-drying at -80°C to obtain proline-modified chitosan CS-Pr;

[0040] 2) Dissolve 2.0-4.0 g of dextran in 45-55 mL of deionized water, add 3.2-3.6 g of sodium periodate, stir at room temperature in the dark for 10-14 hours, dialyze again with distilled water using a 7000 Da molecular weight cutoff dialysis membrane for 2-4 days, and freeze-dry at -80°C to obtain Odex;

[0041] 3) Dissolve 0.04-0.06 g CS-Pr in 1 ml PBS solution to obtain a 4-6% CS-Pr solution; dissolve 0.1-0.4 g Odex in 1 mL pH = 7.4 PBS solution to obtain a 10-40% Odex solution, add 0.01-0.03 g EGCG to the Odex solution to obtain an EGCG / Odex solution, mix equal volumes of EGCG / Odex solution and CS-Pr solution at room temperature, and let stand at 37°C for 3-4 minutes to obtain a composite hydrogel.

[0042] Experimental Example 1 1 H NMR and FT-IR testing

[0043] CS-Pr and Odex prepared in Example 1 were respectively 1 H NMR test. Results are shown in Figure 2 shown.

[0044] like Figure 2 As shown in a, compared with the NMR spectrum of dextran, several new peaks in the range of 5.0 to 5.5 ppm are attributed to the hemiacetal group of Odex. Figure 2 In b, compared with dextran, the FT-IR spectrum of Odex shows a -1 There is a characteristic peak at , which is attributed to the stretching vibration peak of the aldehyde group. 1 In the H NMR spectrum, the chemical shifts at 3.07 and 3.64-3.82 ppm are attributed to the H protons of chitosan [H1] and [H2]-[H5], respectively. Compared with the CS spectrum, the new peaks at 1.86, 3.36, and 3.51 ppm correspond to the H protons of proline. Figure 2 As shown in d, in the infrared spectrum of chitosan, at 1625 cm -1 ,1523cm -1 ,1415cm -1 and 1383cm -1 The absorption peaks at 1150 cm-1 are attributed to the C=O stretching, NH bending, CH bending and -OH bending vibrations of the amide I band. -1 and 1065cm -1 The peaks at belong to (COC) antisymmetric stretching and CO stretching, respectively. Figure 2 In Figure d, it can be seen that, except for the changes in the intensity of several peaks, most of the peaks in the CS-Pr spectrum overlap with those of chitosan. For example, the absorption peaks of C=O stretching and NH bending shift from 1625 cm -1 and 1523cm -1 Shifted to 1610cm -1 and 1538cm -1 In addition, 3088cm -1The absorption peak at is attributed to the stretching vibration of the proline NH group. 1 H NMR and FT-IR results show that CS-Pr and Odex are successfully prepared by the method of the present invention.

[0045] Experimental Example 2 Rheological Analysis of Hydrogel

[0046] The rheological properties of the composite hydrogel Gel 1-3 prepared in Examples 1-3 were characterized by a rheometer. The results are as follows: Figure 3 shown.

[0047] Figure 3 In a, during the time scan, the storage modulus (G′) and loss modulus (G″) increased with time, and the value of G′ was greater than G″, indicating gel formation. In addition, with the gradual increase of EGCG, the value of G′ increased, which was attributed to the formation of new chemical bonds between EGCG and chitosan, which strengthened the network structure of the hydrogel. When subjected to strain scanning, G″ remained relatively stable in the linear viscoelastic state ( Figure 3 b) As the strain continues to increase, G′ gradually decreases until a certain point, where G″ is greater than G′, indicating the collapse of the hydrogel structure. Figure 3 In c-3d, the viscosity of the hydrogel increases as the shear rate increases from 1 s -1 Increase to 100s -1 The shear thinning ability of the hydrogel is shown by the fact that the hydrogel can be directly injected to completely cover the irregularly shaped wound site. In addition, when used in medical applications, the structure of the hydrogel will inevitably be destroyed or deformed by external forces, thereby compromising the therapeutic effect. Therefore, the hydrogel with self-healing properties can restore its network structure through reversible chemical bonds, thereby extending its service life. In the present invention, a step strain test is used to evaluate the rheological recovery behavior of the hydrogel ( Figure 3 f~g). Taking Gel 1 as an example, at 1% strain, G′ is greater than G″, indicating that the gel network is formed. At high strain (1000%), G″ is greater than G′, indicating that the hydrogel network structure is destroyed. When the strain is switched to low strain (1%), G′ is greater than G″ during the test, which means the recovery of the network structure in the hydrogel. Figure 3 h is the macroscopic self-healing test result of Gel 1, which shows that CS-Pr / Odex composite hydrogel has good self-healing performance.

[0048] Experimental Example 3 SEM, swelling properties, and antibacterial and antioxidant properties of hydrogels

[0049] The hydrogels Gel 1-3 of the present invention were subjected to SEM test, pore size distribution analysis, swelling performance, and antibacterial and antioxidant performance tests. The swelling ratio experiment was as follows: the freeze-dried hydrogel was weighed (W0) and then immersed in PBS buffer at room temperature. Then, the hydrogel was taken out at different time intervals, and the water on the surface of the hydrogel was removed using filter paper and weighed (W t ). Finally, the swelling ratio was calculated by the following equation:

[0050]

[0051] The test results are as follows Figure 4 and Figure 5 shown.

[0052] Figure 4 (a) SEM image of hydrogel Gel 1-3; (b) pore size distribution of hydrogel Gel 1-3; (c) swelling ratio results of hydrogel Gel 1-3. As shown in 4a, the prepared hydrogel has an uninterrupted three-dimensional porous network, which is conducive to water absorption, drug release, nutrient transport and cell migration. Figure 4 b The results showed that the average pore size of the hydrogel decreased with the increase of EGCG. Figure 4 In b, the pore size of Gel 1 is 207.88±59.99 μm, while the pore sizes of Gel 2 and Gel 3 are 195.73±56.41 μm and 157.75±56.41 μm, respectively. In addition, the present invention also studies the swelling behavior of the hydrogel at 37°C and physiological pH (7.2). Figure 4 c It was found that Gel 1 showed a swelling percentage of 1000%. Meanwhile, the swelling percentages of Gel 2 and Gel 3 were 950% and 800%, respectively. These results indicate that the composite hydrogel has a significant water absorption capacity to maintain a moist environment at the wound site, thereby promoting wound healing. In addition, the interpenetrating structure and excellent water absorption capacity are conducive to the release of EGCG in the composite hydrogel. Figure 5 As shown in a, Gel 2 exhibited sustained release behavior, with a cumulative release rate of 18% within 6 h, while the cumulative release rate of Gel 3 was 43%. These results indicate that the composite hydrogel exhibited sustained release behavior without significant burst release, which contributed to the effective release of EGCG at the wound site during application.

[0053] The antioxidant and antibacterial properties of hydrogels Figure 5As shown in Figure b, the antioxidant capacity of the composite hydrogel increases with the increase of EGCG. The ABTS scavenging rate of Gel 1 is 47%, while the ABTS scavenging rates of Gel 2 and Gel 3 are 96% and 96%, respectively. This shows that the composite hydrogel of the present invention has good free radical scavenging ability and antioxidant capacity. The antibacterial activity of the hydrogel against Gram-negative Escherichia coli and Staphylococcus aureus is shown in Figure 2. Figure 5 As shown in Figure c, the bacterial killing rates of Gel 1, Gel 2, and Gel 3 against Escherichia coli were 37%, 42%, and 58%, respectively. In addition, the killing rates of Gel 1, Gel 2, and Gel 3 against Staphylococcus aureus were 52%, 76%, and 81%, respectively. Therefore, these data indicate that the antibacterial and antioxidant activities of the hydrogels increased with the addition of EGCG, which can effectively reduce ROS levels and prohibit bacterial invasion during the wound healing process.

[0054] Experimental Example 4 Compatibility test of the hydrogel of the present invention

[0055] The present invention evaluates the cell compatibility of the hydrogel by co-culturing with NIH 3T3 cells, and measures the cell activity by the standard CCK-8 method. After culturing on the hydrogel surface for 1, 3, and 5 days, the cell activity of NIH 3T3 cells gradually increases with the increase of culture time. In addition, the present invention also observes the morphology of NIH 3T3 cells co-cultured with the gel extract for 1, 3, and 5 days by the live / dead staining method. Figure 6 It was shown that living cells specifically labeled with bright blue fluorescence were observed in all hydrogel groups and increased with the extension of incubation time. The results showed that the hydrogel prepared by the present invention was cell-compatible in vitro.

[0056] The above is only a preferred embodiment of the present invention, and does not limit the present invention in other forms. Any person skilled in the art can use the above disclosed technical content to change or modify it into an equivalent embodiment with equivalent changes. However, any simple modification, equivalent change and modification made to the above embodiment according to the technical essence of the present invention without departing from the technical solution of the present invention still falls within the protection scope of the technical solution of the present invention.

[0057] The above embodiments provided by the present invention are only preferred embodiments of the present invention, and are not intended to limit the present invention in other forms. Any person skilled in the art can use the above disclosed technical contents to change or modify them into equivalent embodiments with equivalent changes. However, any simple modification, equivalent change and modification made to the above embodiments according to the technical essence of the present invention without departing from the technical solution of the present invention still falls within the protection scope of the technical solution of the present invention.

Claims

1. A method for preparing a CS-Pr / Odex / EGCG composite hydrogel, characterized in that: The following steps are involved: 1) dissolving 1.0-4.0 g of chitosan in 180-220 mL of acetic acid solution to obtain a chitosan solution; adding 1.6-2.0 g of proline, 2.0-2.4 g of NHS and 3.2-4.0 g of EDCI to 20-30 mL of a buffer solution, and stirring at room temperature for 25-40 minutes to obtain a proline mixed solution; adding the proline mixed solution to the chitosan solution and stirring for 10-14 hours, dialyzing and freeze-drying to obtain proline-modified chitosan CS-Pr; 2) Dissolve 2.0-4.0 g of dextran in 45-55 mL of deionized water, add 3.2-3.6 g of sodium periodate, stir at room temperature in the dark for 10-14 hours, dialyze in deionized water, and freeze-dry to obtain Odex; 3) Dissolve 0.04-0.06 g CS-Pr in 1 ml PBS solution to obtain a 4-6% CS-Pr solution; 0.1-0.4 g Odex was dissolved in 1 mL PBS solution to obtain a 10-40% Odex solution, 0.01-0.03 g EGCG was added to the Odex solution to obtain an EGCG / Odex solution, and equal volumes of the EGCG / Odex solution and the CS-Pr solution were mixed at room temperature and allowed to stand at 37° C. to obtain a composite hydrogel.

2. The method for preparing the CS-Pr / Odex / EGCG composite hydrogel according to claim 1, characterized in that: In step 1), the mass concentration of the acetic acid solution is 1-3%; the buffer solution is MES buffer solution, pH=5.0; the dialyzed solution is dialyzed with distilled water for 4-6 days, and the MWCO of the dialysis membrane is 3.5 kDa.

3. The method for preparing the CS-Pr / Odex / EGCG composite hydrogel according to claim 1, characterized in that: In step 2), the mixture is dialyzed again with distilled water using a 7000Da molecular weight cutoff dialysis membrane for 2-4 days.

4. The method for preparing the CS-Pr / Odex / EGCG composite hydrogel according to claim 1, characterized in that: In step 3), the pH of the PBS solution is 7.

4.

5. The method for preparing the CS-Pr / Odex / EGCG composite hydrogel according to claim 1, characterized in that: In step 3), the standing time is 3-4 minutes.

6. The CS-Pr / Odex / EGCG composite hydrogel prepared by the preparation method according to any one of claims 1 to 5.

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