Preparation method and application of metal ion-containing coordination gel

Abstract

The present invention discloses a preparation method and application of a metal ion coordination gel, wherein the gel material comprises: H-(RADA)4 and (RADA)4 gel stock solution, through histidine (His) and Cu 2+ The resulting complex solution, H-(RADA)4-Cu, is then mixed evenly with the (RADA)4 solution and added to a PBS buffer solution to form a stable gel. This gel exhibits excellent biocompatibility, promotes corneal epithelial cell repair, inhibits the growth of Staphylococcus aureus (S. aureus) and Escherichia coli (E. coli), and is effective in treating keratitis.

Description

Technical Field

[0001] The present invention relates to the technical field of biomaterial research and development and application, and in particular to a preparation method and application of a coordination gel containing metal ions. Background Art

[0002] Bacterial and fungal keratitis are often accompanied by corneal damage, which can cause eye inflammation, slowing wound healing and forming chronic wounds, leading to a variety of eye diseases. Severe cases may cause corneal perforation or even blindness. Therefore, when bacterial or fungal keratitis is diagnosed, a fast and effective treatment method is particularly important for controlling a series of eye diseases caused by infection. Currently, the main clinical treatments for early bacterial and fungal keratitis include systemic administration in the form of capsules, tablets or intravenous injections, intraocular administration in the form of intraocular injections, and topical administration in the form of eye drops. The common problems of these three methods are low bioavailability, high ocular surface toxicity, and slow epithelial repair.

[0003] Therefore, in situ gel drug delivery is a new and effective method for the early treatment of bacterial and fungal keratitis. Summary of the Invention

[0004] In order to solve the above problems, the present invention provides a preparation method and application of a coordination gel containing metal ions.

[0005] The technical solution adopted in the present invention is:

[0006] A preparation method and application of a coordination gel containing metal ions, comprising the following steps:

[0007] S1. Prepare CuCl2 solution, H-(RADA)4 and (RADA)4 gel stock solutions respectively and set aside;

[0008] S2, taking CuCl2 solution and mixing it with H-(RADA)4 gel stock solution to obtain H-(RADA)4-Cu gel solution;

[0009] S3, diluting the H-(RADA)4-Cu gel solution with a (RADA)4 solution, wherein the ratio of the H-(RADA)4-Cu solution to the (RADA)4 solution is 1:1 to 1:4, and this amount ensures that the final gel has good bactericidal properties and biocompatibility;

[0010] S4, mixing the gel solution obtained in S3 with a buffer solution containing salt ions, and letting it stand at 4°C for 24 hours to obtain H-(RADA)4-Cu gel;

[0011] S5. After the reaction, the gel is rinsed three times to obtain a gel containing metal ion coordination.

[0012] Furthermore, the (RADA)4 has an amino acid sequence of SEQ ID NO: 1.

[0013] Furthermore, the H-(RADA)4 has an amino acid sequence of SEQ ID NO: 2.

[0014] Furthermore, the concentration of the CuCl2 solution is 9 mg / mL, which ensures the complete dissolution of CuCl2. The solvent is double-distilled deionized water, which does not contain any other ions, ensuring that the finally formed gel has a simple composition.

[0015] Furthermore, the concentration of the H-(RADA)4 solution is 10 mg / mL, which ensures that the H-(RADA)4 lyophilized powder is completely dissolved and has a stable gel network structure.

[0016] Furthermore, the concentration of the (RADA)4 solution is 10 mg / mL, which ensures that the (RADA)4 lyophilized powder is completely dissolved and has a stable gel network structure.

[0017] Furthermore, in step S2, the ratio of H-(RADA)4 solution to CuCl2 solution is 100:4.9, which ensures that histidine (His) and Cu 2 + Completely coordinated at a ratio of 2:1 to form a stable gel containing metal ion complexes with good bactericidal properties.

[0018] Furthermore, in step S4, the ratio of the amount of the gel solution obtained in S3 to the amount of the buffer solution containing salt ions is 1:1.

[0019] Furthermore, the metal ion-containing coordination gel obtained by the preparation method is used in drug carriers.

[0020] The beneficial effects of the present invention are:

[0021] 1. The metal ion-coordinated gel material provided by the present invention is a new material for treating bacterial and fungal keratitis. 2 + coordinated with (RADA)4 and H-(RADA)4 polypeptides to form a fibrous porous network structure. 2 The distribution of + is consistent with the fiber structure of the gel, so that the hydrogel has the effect of inhibiting the growth of Staphylococcus aureus (S. aureus) and Escherichia coli (E. coli) and promoting the repair of corneal epithelial cells.

[0022] 2. The metal ion-coordinated gel material provided by the present invention has excellent stability and is effective in treating bacterial and fungal keratitis. It can effectively address issues such as low drug permeability to the ocular surface, the growth of drug-resistant bacteria caused by frequent drug administration, and delayed corneal epithelial cell repair. BRIEF DESCRIPTION OF THE DRAWINGS

[0023] In order to more clearly illustrate the technical solutions of the embodiments of the present invention, the drawings of the embodiments will be briefly introduced below. Obviously, the drawings in the following description only relate to some embodiments of the present invention, but are not intended to limit the present invention.

[0024] Figure 1 is the structural formula of the H-(RADA)4-Cu gel of Example 3 of the present invention;

[0025] Figure 2 The microstructure of H-(RADA)4 and H-(RADA)4-Cu gels according to Example 3 of the present invention under a scanning electron microscope (SEM) is shown;

[0026] Figure 3 This is an infrared image of the gel of Example 3 of the present invention;

[0027] Figure 4 This is the XPS graph of the gel of Example 3 of the present invention;

[0028] Figure 5 This is the UV image of the gel of Example 3 of the present invention;

[0029] Figure 6 The results of corneal epithelial cell culture using the gel of Example 3 of the present invention are as follows;

[0030] Figure 7 The results of Staphylococcus aureus (S. aureus) and Escherichia coli (E. coli) culture on the gel of Example 3 of the present invention; DETAILED DESCRIPTION

[0031] To make the purpose, technical solutions, and advantages of the embodiments of the present invention more clear, the technical solutions of the embodiments of the present invention will be clearly and completely described below in conjunction with the accompanying drawings of the embodiments of the present invention. Obviously, the described embodiments are part of the embodiments of the present invention, not all of the embodiments. Based on the described embodiments of the present invention, all other embodiments obtained by ordinary technicians in this field without creative work are within the scope of protection of the present invention.

[0032] The present invention will be further described below with reference to the accompanying drawings and examples.

[0033] Preparation method of the eye gel of the present invention:

[0034] S1. Prepare 9 mg / mL CuCl2 solution, 10 mg / mL H-(RADA)4 and (RADA)4 gel stock solutions respectively and set aside.

[0035] S2. Take the above CuCl2 solution and mix it evenly with the H-(RADA)4 gel stock solution at a ratio of 4.9uL:100uL to obtain H-(RADA)4-Cu gel solution.

[0036] S3. Take (RADA)4 solution to dilute the H-(RADA)4-Cu gel solution.

[0037] S4. Mix the gel solution obtained in S3 with a buffer solution containing salt ions. Preferably, the buffer solution containing salt ions used in the present invention is PBS. Let it stand at 4° C. for 24 hours to obtain H-(RADA) 4 -Cu gel.

[0038] S5. After the reaction, the gel is rinsed three times to obtain an ophthalmic gel containing metal ion coordination.

[0039] The (RADA)4 has an amino acid sequence of SEQ ID NO: 1, and the H-(RADA)4 has an amino acid sequence of SEQ ID NO: 2-11. In this embodiment, the H-(RADA)4 selected has an amino acid sequence of SEQ ID NO: 2.

[0040] Example 1

[0041] S1. Prepare 9 mg / mL CuCl2 solution, 10 mg / mL H-(RADA)4 and (RADA)4 gel stock solutions respectively and set aside.

[0042] S2. Take the above CuCl2 solution and mix it evenly with the H-(RADA)4 gel stock solution at a ratio of 4.9uL:100uL to obtain H-(RADA)4-Cu gel solution.

[0043] S3. Take 50uL H-(RADA)4-Cu gel solution and mix it with 50uL (RADA)4 solution and mix them evenly.

[0044] S4. Mix 100 uL of the above-mentioned uniformly mixed (RADA) 4 solution and H-(RADA) 4 -Cu gel solution with 100 uL of PBS solution, and let it stand at 4° C. for 24 hours to obtain H-(RADA) 4 -Cu gel.

[0045] S5. After the reaction, the gel was gently washed three times with PBS solution and sterilized by ultraviolet irradiation to obtain a gel containing metal ion coordination.

[0046] Example 2

[0047] S1. Prepare 9 mg / mL CuCl2 solution, 10 mg / mL H-(RADA)4 and (RADA)4 gel stock solutions respectively and set aside.

[0048] S2. Take the above CuCl2 solution and mix it evenly with the H-(RADA)4 gel stock solution at a ratio of 4.9uL:100uL to obtain H-(RADA)4-Cu gel solution.

[0049] S3. Take 20uL H-(RADA)4-Cu gel solution and mix it with 60uL (RADA)4 solution and mix well.

[0050] S4. Evenly mix the above 80 uL of the evenly mixed (RADA) 4 solution and H-(RADA) 4 -Cu gel solution with 80 uL of PBS solution, and let it stand at 4° C. for 24 hours to obtain H-(RADA) 4 -Cu gel.

[0051] S5. After the reaction, the gel was gently washed three times with PBS solution and sterilized by ultraviolet irradiation to obtain a gel containing metal ion coordination.

[0052] Example 3

[0053] S1. Prepare 9 mg / mL CuCl2 solution, 10 mg / mL H-(RADA)4 and (RADA)4 gel stock solutions respectively and set aside.

[0054] S2. Take the above CuCl2 solution and mix it evenly with the H-(RADA)4 gel stock solution at a ratio of 4.9uL:100uL to obtain H-(RADA)4-Cu gel solution.

[0055] S3. Take 20uL H-(RADA)4-Cu gel solution and mix it with 80uL (RADA)4 solution and mix well.

[0056] S4. Mix 100 uL of the above-mentioned uniformly mixed (RADA) 4 solution and H-(RADA) 4 -Cu gel solution with 100 uL of PBS solution and let it stand at 4° C. for 24 hours to obtain H-(RADA) 4 -Cu gel.

[0057] S5. After the reaction, the gel was gently washed three times with PBS solution and sterilized by ultraviolet irradiation to obtain a gel containing metal ion coordination.

[0058] Test Example 1

[0059] The metal ion-coordinated gel material of Example 3 was selected and observed using a scanning electron microscope (SEM). Figure 2 It can be observed that the microstructure of the gel is a fibrous porous network structure, and Cu 2The distribution of + is consistent with the fiber structure of the gel and is not randomly distributed, indicating that Cu 2 + is not randomly mixed in the gel, but undergoes cross-linking reaction with the gel, so Cu 2 The presence of + gives the gel an antibacterial effect.

[0060] Test Example 2

[0061] The metal ion coordinated gel material of Example 3 was selected and subjected to X-ray photoelectron spectroscopy (XPS) and Fourier transform infrared testing. Figure 3 and Figure 4 It can be seen that Cu 2 +Successfully combined with H-(RADA)4 gel to form a stable metal ion coordination gel.

[0062] Test Example 3

[0063] The metal ion coordinated gel material of Example 3 was selected and its visible light photometry was measured using an ultraviolet spectrophotometer. Figure 5 It can be seen that the gel has good light transmittance.

[0064] Test Example 4

[0065] The metal ion-coordinated gel material of Example 3 was selected and subjected to a corneal epithelial cell culture test. The corneal epithelial cell concentration was 2.5×10 4 A cell suspension of 100 cells / ml was added dropwise to a 96-well plate for co-culture with the sample. After 1 day, the culture medium in the plate was discarded, washed once with PBS, and 220uL of the prepared culture medium containing CCK-8 (cck-8: culture medium = 1:9) was added to each well. The plate was then incubated at 37°C for 2 hours. After 2 hours, the culture medium in the plate was transferred to a new 96-well plate and the absorbance was measured at an excitation wavelength of 450nm. The cell-seeded sample was washed three times with physiological saline, fixed with 2.5% glutaraldehyde solution for 4 hours, and stained with rhodamine. The samples were observed under a fluorescence microscope and photographed for statistics.

[0066] Depend on Figure 6 It can be seen that the experimental results show that the gel material containing metal ion coordination has good biocompatibility and can effectively promote the proliferation of corneal epithelial cells.

[0067] Test Example 5

[0068] The metal ion-coordinated gel material of Example 3 was selected and subjected to a Staphylococcus aureus culture test. The bacterial concentration was 1×10 6A bacterial suspension containing 100 cfu / mL of sample was added dropwise to a 24-well plate and co-cultured with the sample. Each well contained 100 μL of sample and 1 mL of bacterial suspension and incubated in a 37°C incubator for 1 day. After 1 day, the bacterial suspension was removed from the plate and diluted 10-fold in five gradients. The dilutions were spread onto solid culture medium and incubated overnight in a 37°C incubator to monitor bacterial growth.

[0069] Depend on Figure 7 It can be seen that the experimental results show that the gel containing metal ion coordination has good antibacterial properties and can effectively inhibit the growth of Staphylococcus aureus.

[0070] Test Example 6

[0071] The gel material containing metal ion coordination of Example 3 was selected and subjected to E. coli culture test. The bacterial concentration was 1×10 6 A bacterial suspension containing 100 cfu / mL of sample was added dropwise to a 24-well plate and co-cultured with the sample. Each well contained 100 μL of sample and 1 mL of bacterial suspension and incubated in a 37°C incubator for 1 day. After 1 day, the bacterial suspension was removed from the plate and diluted 10-fold in five gradients. The dilutions were spread onto solid culture medium and incubated overnight in a 37°C incubator to monitor bacterial growth.

[0072] Depend on Figure 7 It can be seen that the experimental results show that the gel material containing metal ion coordination has good antibacterial properties and can effectively inhibit the growth of Escherichia coli.

[0073] The sequence listing is as follows:

[0074]

[0075]

[0076]

[0077]

[0078] The above description is merely a preferred embodiment of the present invention and does not constitute any form of limitation to the present invention. Although the present invention has been disclosed as a preferred embodiment as above, it is not intended to limit the present invention. Any technician familiar with this profession can make some changes or modifications to equivalent embodiments of the technical contents disclosed above without departing from the scope of the technical solution of the present invention. However, any simple modifications, equivalent changes and modifications made to the above embodiments based on the technical essence of the present invention without departing from the content of the technical solution of the present invention are still within the scope of the technical solution of the present invention.

Claims

1. A method for preparing a metal ion coordination gel, characterized in that: The steps include: S1. Prepare 9 mg / mL CuCl2 solution, 10 mg / mL H-(RADA)4 gel stock solution, and 10 mg / mL (RADA)4 gel stock solution respectively, and set aside; the (RADA)4 has the amino acid sequence shown in SEQ ID NO: 1; the H-(RADA)4 has the amino acid sequence shown in SEQ ID NO: 2 or SEQ ID NO: 7; S2, taking CuCl2 solution and H-(RADA)4 gel stock solution in a volume ratio of 100:4.9 and mixing them evenly to obtain H-(RADA)4-Cu gel solution; S3, taking the (RADA)4 gel stock solution to dilute the H-(RADA)4-Cu gel solution to obtain a gel mixed solution; the amount ratio of the H-(RADA)4-Cu gel solution to the (RADA)4 gel stock solution is 1:1 to 1:4; S4, mixing the gel mixture solution obtained in S3 with a buffer solution containing salt ions at a volume ratio of 1:1, and allowing to stand at 4° C. for 24 hours to form an H-(RADA)4-Cu gel having a fibrous porous network structure; S5. After the reaction, the gel is rinsed three times to obtain a gel containing metal ion coordination.

2. Use of the metal ion-containing coordination gel obtained by the preparation method according to claim 1 in preparing a drug for treating keratitis, wherein the metal ion-containing coordination gel is used as a drug carrier during the drug preparation process.

Images