Application of natural carrier-free micromolecular hydrogel as antibacterial, anti-inflammatory and angiogenesis promoting medicine

Abstract

The invention provides a structure, preparation and application of a supramolecular hydrogel which is formed by self-assembly of two single components, namely alpha-D-pentagalloylgalactose and alpha-D-pentagalloylmannose, in water under the condition of not being subjected to any structural modification and pretreatment, as well as a preparation method and application of the supramolecular hydrogel. According to the alpha-D-pentagalloyl galactose and alpha-D-pentagalloyl mannose supramolecular hydrogel, the hydrogel can be formed under the condition that no gelator is used, and the hydrogel has excellent gel properties such as injectability, heat sensitivity, self-healing property and stability. Meanwhile, the two supramolecular hydrogels both have good immunoregulation, anti-inflammatory and antioxidant activities, are both superior to clinical first-line drugs (dexamethasone and ascorbic acid), and also show good angiogenesis promoting ability. A large number of tests show that the supramolecular hydrogel has good biological safety, and clinical medication safety can be further guaranteed.

Description

Technical Field

[0001] This invention relates to a method for preparing α-D-pentagalloylgalactose and α-D-pentagalloylmannose hydrogels and their applications as antibacterial, immunomodulatory, angiogenic, anti-inflammatory, and antioxidant drugs. These hydrogels exhibit good inhibitory effects against clinically common methicillin-resistant Staphylococcus aureus (MRSA) and Escherichia coli, demonstrating broad-spectrum antibacterial activity. They also exhibit good immunomodulatory effects, with good anti-inflammatory, angiogenic, and antioxidant activities. Furthermore, they demonstrate good biocompatibility, further ensuring safe clinical use. In addition, they can be prepared into hydrogels for use as drug delivery carriers, belonging to the field of medicinal chemistry. Background Technology

[0002] Hydrogels, due to their unique three-dimensional porous network and hydrophilic structure, high water content, excellent biocompatibility, and mechanical properties similar to human soft tissue, have become one of the core materials in the field of biomedical engineering. Over the past few decades, hydrogels have been widely used in tissue engineering scaffolds, drug delivery systems, wound dressings, and coatings for implantable medical devices. However, previously reported hydrogels are mostly based on high molecular weight polymers, and their application in complex physiological environments still faces multiple challenges, including poor stability, functional limitations, and difficulties in clinical translation. With the rapid development of supramolecular self-assembly technology, fundamental research and technological innovation in next-generation biomedical hydrogels have been driven.

[0003] Compared to traditional hydrogels, single-molecule self-assembled supramolecular hydrogels spontaneously form three-dimensional network structures through non-covalent interactions (hydrogen bonds, π-π stacking, hydrophobic interactions, etc.), exhibiting high biocompatibility and metabolic safety, dynamic reversibility and stimulus responsiveness, molecular structural simplicity and functional programmability. They hold immense potential for biomedical applications such as drug delivery, wound healing, and tissue engineering. Despite the significant potential of single-molecule self-assembled hydrogels, designing them using bioactive small molecule compounds remains a formidable challenge. The self-assembly of bioactive small molecules primarily depends on a favorable ratio of hydrophilic and hydrophobic groups within the molecular structure and their perfect equilibrium in water. Subtle structural variations, including intermolecular interactions, chirality, and spatial molecular arrangement, can lead to significant differences in assembly capabilities.

[0004] Based on the previous discovery that the small molecule β-D-pentagalloglucopyranose can self-assemble to form supramolecular hydrogels (application number: 202410062761.3), this invention synthesized several galloyl glycoside compounds with very similar structures, including 10 galloyl glycosides such as α / β-D-pentagalloglucopyranose, α / β-D-pentagallomannose, α / β-D-pentagallomannose, α / β-L-tetragallorhamnose, and α / β-L-tetragallomannose. This invention found that only α-D-pentagalloglucopyranose and α-D-pentagallomannose can self-assemble to form supramolecular hydrogels, while their epiomers β-D-pentagalloglucopyranose and β-D-pentagallomannose cannot self-assemble to form hydrogels in water. Furthermore, α / β-D-pentagaloylalose, which shares the same hexacarbon aldose structure, and α / β-L-tetragaloylrhamnose and α / β-L-tetragaloylfurose, which have similar structures among methyl pentoses, cannot self-assemble to form supramolecular hydrogels. The prepared α-D-pentagaloylgalactose and α-D-pentagaloylmannose hydrogels both exhibit excellent gel properties such as injectability, thermosensitivity, self-healing, and stability. Simultaneously, both supramolecular hydrogels possess good immunomodulatory, anti-inflammatory, angiogenesis-promoting, and antioxidant bioactivities, and also demonstrate good biocompatibility. Therefore, this invention not only enriches the existing small molecule hydrogel library but also provides new ideas for the development of biomedical hydrogels based on small molecule compounds. Summary of the Invention

[0005] Based on supramolecular chemistry techniques, the inventors innovatively discovered that α-D-pentagalloylgalactose and α-D-pentagalloylmannose can self-assemble into hydrogels in deionized water through a simple heating-cooling process without the addition of any gelling agents. However, their epiomers, β-D-pentagalloylgalactose and β-D-pentagalloylmannose, cannot self-assemble into hydrogels in water. Furthermore, α / β-D-pentagalloylalose, a hexacarbon aldose with the same structure, and α / β-L-tetragalloylrhamnose and α / β-L-tetragalloylfusose, two configurations of structurally similar methyl pentoses, cannot self-assemble into supramolecular hydrogels. The prepared α-D-pentagalloylgalactose and α-D-pentagalloylmannose supramolecular hydrogels both possess excellent gelling properties such as injectability, thermosensitivity, self-healing, and stability. Both supramolecular hydrogels exhibit excellent immunomodulatory, anti-inflammatory, angiogenesis-promoting, and antioxidant bioactivities, and also demonstrate good biocompatibility, making them ideal biomedical hydrogels for clinical application. This invention not only enriches the existing small molecule hydrogel library but also provides new ideas for the development of biomedical hydrogels based on small molecule compounds.

[0006] One of the objectives of this invention is to provide α-D-pentagalloylgalactose and α-D-pentagalloylmannose supramolecular hydrogels.

[0007] A second objective of this invention is to provide a method for preparing α-D-pentagalloylgalactose and α-D-pentagalloylmannose supramolecular hydrogels.

[0008] A third objective of this invention is to provide excellent gel material properties of α-D-pentagalloylgalactose and α-D-pentagalloylmannose supramolecular hydrogels.

[0009] The fourth objective of this invention is to provide the application of α-D-pentagalloylgalactose and α-D-pentagalloylmannose supramolecular hydrogels in the field of antibacterial applications.

[0010] The fifth objective of this invention is to provide the application of α-D-pentagalloylgalactose and α-D-pentagalloylmannose supramolecular hydrogels in the field of inhibiting bacterial biofilm formation.

[0011] The sixth objective of this invention is to provide the application of α-D-pentagalloylgalactose and α-D-pentagalloylmannose supramolecular hydrogels in the fields of anti-inflammatory and immunomodulatory effects.

[0012] The seventh objective of this invention is to provide the application of α-D-pentagalloylgalactose and α-D-pentagalloylmannose supramolecular hydrogels in promoting angiogenesis.

[0013] The eighth objective of this invention is to provide the application of α-D-pentagalloylgalactose and α-D-pentagalloylmannose supramolecular hydrogels in the field of antioxidants.

[0014] The ninth objective of this invention is to provide α-D-pentagalloylgalactose and α-D-pentagalloylmannose supramolecular hydrogels as biomedical materials for use in assembly engineering and disease treatment.

[0015] The tenth objective of this invention is to provide the application of α-D-pentagalloylgalactose and α-D-pentagalloylmannose supramolecular hydrogels as drug delivery materials.

[0016] To achieve this objective, the present invention adopts the following technical solution:

[0017] 1. Ten galloyl glycosides, including α / β-D-pentagalloylgalactose, α / β-D-pentagalloylmannose, α / β-D-pentagalloylaloose, α / β-L-tetragalloylrhamnose, and α / β-L-tetragalloylfusose, were successfully synthesized by synthetic methods, and their structures were confirmed by 1H NMR spectroscopy and high-resolution mass spectrometry.

[0018] 2. The ability of synthesized galloyl glycosides to self-assemble into hydrogels was determined by exploring different conditions.

[0019] (1) Weigh different masses of galloyl glycosides, suspend them in water, and heat and stir until homogeneous.

[0020] (2) Let the galloyl glycoside aqueous solution prepared in step (1) stand and cool.

[0021] Preferably, the galloyl glycoside concentration in step (1) is 5 mg / mL, 10 mg / mL, 15 mg / mL, 20 mg / mL, 40 mg / mL, 80 mg / mL, or 100 mg / mL.

[0022] Preferably, the water used in step (1) is deionized water or ultrapure water.

[0023] Preferably, the heating temperature in step (1) is 40-100℃, for example 40℃, 50℃, 60℃, 80℃, 100℃.

[0024] Preferably, the stirring speed in step (1) is 0-1500 rpm, for example 100 rpm, 200 rpm, 300 rpm, 400 rpm, 500 rpm, 600 rpm, 700 rpm, 800 rpm, 900 rpm, 1000 rpm or 1500 rpm.

[0025] Preferably, the static cooling conditions in step (2) are room temperature or in a 4°C refrigerator.

[0026] Preferably, the galloyl glycosides that can self-assemble into hydrogels after being left to stand and cool in step (2) are α-D-pentagalloylgalactose and α-D-pentagalloylmannose.

[0027] 3. Rheological studies were conducted on the supramolecular hydrogels of α-D-pentagalloylgalactose and α-D-pentagalloylmannose.

[0028] The main research steps are as follows:

[0029] The gel material properties of α-D-pentagalloylgalactose and α-D-pentagalloylmannose supramolecular hydrogels were characterized by frequency scanning and amplitude scanning.

[0030] 4. The antibacterial effect was evaluated by measuring the MIC values ​​of different bacterial species; the effect on the micromorphology of methicillin-resistant Staphylococcus aureus and Escherichia coli was observed by scanning electron microscopy.

[0031] The antibacterial evaluation of α-D-pentagalloylgalactose and α-D-pentagalloylmannose hydrogels included the following steps:

[0032] (1) The MIC values ​​of α-D-pentagalloylgalactose and α-D-pentagalloylmannose supramolecular hydrogels against Staphylococcus aureus, Escherichia coli, Bacillus subtilis, Enterococcus faecalis and methicillin-resistant Staphylococcus aureus were determined by broth dilution method.

[0033] (2) Scanning electron microscopy was used to observe the morphological effects of 1 / 3 MIC drug concentration on methicillin-resistant Staphylococcus aureus and Escherichia coli.

[0034] 5. The inhibitory effect of XTT on the formation of methicillin-resistant Staphylococcus aureus biofilm was evaluated by XTT quantitative assay; the inhibitory effect on the formation of methicillin-resistant Staphylococcus aureus biofilm was further verified by scanning electron microscopy and laser confocal microscopy characterization.

[0035] The main research steps are as follows:

[0036] Different concentrations of α-D-pentagalloylgalactose and α-D-pentagalloylmannose hydrogels were co-cultured with methicillin-resistant Staphylococcus aureus (MRSA) in a high-glucose medium for 24 hours. The medium was then gently aspirated with a syringe, and the free bacteria were washed with PBS. XTT dye was added, and the mixture was incubated in the dark for 2 hours. The absorbance was then measured at 450 nm.

[0037] 6. The antioxidant effect was evaluated by measuring the scavenging rate of different free radicals.

[0038] The evaluation of the antioxidant effects of α-D-pentagalloylgalactose and α-D-pentagalloylmannose hydrogels included the following steps:

[0039] (1) Prepare a 200 μM DPPH stock solution, then add different concentrations of α-D-pentagalloylgalactose and α-D-pentagalloylmannose supramolecular hydrogels, incubate in the dark for 30 minutes, and then measure the absorbance at 517 nm.

[0040] (2) ABTS (7mM) solution was reacted with potassium persulfate (K2S2O8, 2.45mM) at room temperature in the dark for 16h to generate ABTS·+. Then, an appropriate concentration of ABTS·+ solution was reacted with α-D-pentagalloylgalactose and α-D-pentagalloylmannose hydrogels in the dark for 10min. Finally, the absorbance at 734nm was measured.

[0041] 7. The anti-inflammatory effects of different pro-inflammatory factors in various inflammatory models were evaluated by measuring the inhibition rates of different pro-inflammatory factors in different cell lines.

[0042] The main research steps are as follows:

[0043] Inflammation models were established by inducing mouse RAW 264.7 macrophages, human normal chondrocytes c28 / i2, and human normal colonic epithelial cells NCM460 using lipopolysaccharide (LPS). Different concentrations of α-D-pentagalloylgalactose and α-D-pentagalloylmannose supramolecular hydrogels were then added and incubated for 24 hours. The effects of α-D-pentagalloylgalactose and α-D-pentagalloylmannose supramolecular hydrogels on inflammatory factors produced by LPS-induced macrophages were measured to evaluate their anti-inflammatory activity.

[0044] 8. The immunomodulatory effects of RAW 264.7 macrophages were evaluated by measuring their phenotype.

[0045] The main research steps are as follows:

[0046] An inflammation model was established by inducing mouse macrophage RAW 264.7 cells with lipopolysaccharide (LPS). After incubation for 24 hours with α-D-pentagalloylgalactose and α-D-pentagalloylmannose supramolecular hydrogels, the phenotypes of M1 and M2 macrophages were quantified by immunofluorescence staining to evaluate their immunomodulatory effects.

[0047] 9. Assess the ability of human umbilical vein endothelial cells (HUVECs) to promote angiogenesis by measuring angiogenesis.

[0048] The main research steps are as follows: RAW 264.7 cells were cultured at a rate of 2 × 10⁻⁶. 5 HUVECs were seeded at a density of 1.5 × 10⁶ cells / well in 6-well plates and co-incubated with α-D-pentagalloylgalactose and α-D-pentagalloylmannose supramolecular hydrogels for 24 h. The supernatant was collected, and the suspended cells were removed by centrifugation. Subsequently, HUVECs were seeded into 24-well plates pre-seeded with Matrigel matrix at a cell density of 1.5 × 10⁶ cells / well. 4 / well, add the collected cell supernatant to co-incubate with it. Finally, images of angiogenesis are taken at different time points and analyzed.

[0049] 10. Safety was evaluated through cell safety experiments, in vitro hemolysis experiments, skin irritation experiments, and pathological sections of mouse liver, heart, spleen, lungs, and kidneys.

[0050] (1) The MTT assay was used to assess the survival rate of human immortalized epidermal cells (HaCat) and mouse fibroblasts (L929) after 24 hours of culture in hydrogels containing α-D-pentagalloylgalactose and α-D-pentagalloylmannose, and to evaluate the cytotoxicity of the drugs.

[0051] (2) Apply α-D-pentagalloylgalactose and α-D-pentagalloylmannose supramolecular hydrogels to the skin surface of mice. After 24 hours, remove the skin and compare the skin pathology of normal mice with that of mice treated with the drug to evaluate the skin irritation of the drug.

[0052] (3) Take the heart, liver, spleen, lung and kidney tissues of mice that have been treated for 12 days, compare the pathological condition of the tissues of normal mice and mice in the treatment group, and evaluate the in vivo safety of the drug.

[0053] The specific research results of this invention are described below:

[0054] I. Formation of Hydrogels

[0055] The inventors discovered that the minimum gelling concentrations for α-D-pentagalloylgalactose and α-D-pentagalloylmannose are 20 mg / mL and 10 mg / mL, respectively, while their epiomers β-D-pentagalloylgalactose and β-D-pentagalloylmannose cannot self-assemble into hydrogels in water. Furthermore, α / β-D-pentagalloylalose, a hexacarbon aldose with the same structure, and α / β-L-tetragalloylrhamnose and α / β-L-tetragalloylfusose, both structurally similar methyl pentoses, cannot self-assemble into supramolecular hydrogels.

[0056] In summary, the formation of galloyl glycoside small molecule hydrogels is unpredictable and requires comprehensive consideration of factors such as the compound's structure, chirality, steric hindrance, and concentration. Furthermore, the supramolecular hydrogel compound of this invention possesses excellent gel properties such as injectability, thermosensitivity, self-healing ability, and stability, and exhibits good rheological properties; its storage modulus is greater than its loss modulus under changes in frequency and shear stress.

[0057] II. The antibacterial, immunomodulatory, angiogenesis-promoting, antioxidant, and wound-healing properties of hydrogels

[0058] In in vitro experiments, the antibacterial effect of β-D-pentagalloglucopyranosylglucose supramolecular hydrogel is shown in Table 1 of Case 5; the antioxidant effect is shown in Tables 2 and 3 of Case 8; and the anti-inflammatory effect is shown in Tables 4-6 of Case 9. This supramolecular hydrogel exhibits selective antibacterial activity, selectively killing pathogenic bacteria such as methicillin-resistant Staphylococcus aureus (MRSA) without affecting the activity of beneficial intestinal bacteria such as Bacillus subtilis and Enterococcus faecalis. Scanning electron microscopy results also showed that α-D-pentagalloglucopyranosylgalactose and α-D-pentagalloglucopyranosylmannose supramolecular hydrogels can disrupt bacterial cell wall structures, promote bacterial cell deformation and lysis, and thus significantly inhibit biofilm formation. Furthermore, this supramolecular hydrogel also exhibits good anti-inflammatory and antioxidant activities, both superior to first-line clinical drugs (dexamethasone and ascorbic acid), and also promotes angiogenesis.

[0059] In in vivo animal experiments, during the 12-day treatment period, the skin regeneration rate of the α-D-pentagalloylgalactose and α-D-pentagalloylmannose supramolecular hydrogel groups was significantly higher than that of the methicillin-resistant Staphylococcus aureus (MRSA) group and the control group at different time points. On day 6, the α-D-pentagalloylgalactose and α-D-pentagalloylmannose supramolecular hydrogel groups showed lower expression of inflammatory factors and higher expression of pro-angiogenic factors. Histopathological sections after 12 days showed that the wounds of the α-D-pentagalloylgalactose and α-D-pentagalloylmannose supramolecular hydrogel groups healed well, with a clear distinction between the epidermis and dermis, more hair follicle growth, fewer inflammatory factors, and more collagen fiber deposition; while the skin of the MRSA group was still healing, with visible granulation tissue, no hair follicle recovery, more inflammatory factors, and less collagen fiber deposition.

[0060] Based on the above, it can be seen that, compared with the prior art, the present invention has the following advantages:

[0061] In this invention, α-D-pentagalloylgalactose and α-D-pentagalloylmannose can directly self-assemble into supramolecular hydrogels without the need for any gelling agents. Directly utilizing small molecule components to self-assemble into hydrogels allows for the direct combination of traditional drug molecules and biomedical functional materials, avoiding the complexity of traditional polymer hydrogel preparation processes, as well as their poor drug loading capacity and potential high toxicity.

[0062] This invention not only enriches the existing small molecule hydrogel library, but also provides new ideas for the development of biomedical hydrogels based on small molecule compounds. Attached Figure Description

[0063] Figure 1 The α-D-pentagalloylgalactose and α-D-pentagalloylmannose supramolecular hydrogels prepared in Example 2 of this invention are shown.

[0064] Figure 2 The gel material properties of the α-D-pentagalloylgalactose and α-D-pentagalloylmannose supramolecular hydrogels prepared in Example 3 of this invention are described.

[0065] Figure 3 The diagram shows the effect of the α-D-pentagalloylgalactose and α-D-pentagalloylmannose supramolecular hydrogels obtained in Example 6 of this invention on the microstructure of methicillin-resistant Staphylococcus aureus.

[0066] Figure 4Electron micrograph of the effect of the α-D-pentagalloylgalactose and α-D-pentagalloylmannose supramolecular hydrogel obtained in Example 7 of the present invention on the inhibition of biofilm formation by methicillin-resistant Staphylococcus aureus.

[0067] Figure 5 The results of regulating macrophage phenotype with α-D-pentagalloylmannose supramolecular hydrogel obtained in Example 10 of this invention.

[0068] Figure 6 This is the result of the α-D-pentagalloylmannose supramolecular hydrogel obtained in Example 11 of the present invention promoting angiogenesis.

[0069] Figure 7 This is the macroscopic result of the α-D-pentagalloylmannose supramolecular hydrogel obtained in Example 12 of the present invention promoting wound healing.

[0070] Figure 8 This is an immunofluorescence image of inflammatory factors in mouse skin wounds in Example 12 of the present invention. Detailed Implementation

[0071] The following embodiments are intended to further illustrate the present invention. Those skilled in the art should understand that the embodiments are merely illustrative of the invention and should not be considered as specific limitations thereof. Those skilled in the art should understand that any improvements to the present invention, equivalent substitutions of the raw materials used, additions of auxiliary components, and selection of specific methods, etc., all fall within the protection and disclosure scope of the present invention.

[0072] Example 1

[0073] Based on the reported synthetic method for α / β-D-pentagalloyl glucose (J Med Chem 2006; 49:2829-37), α / β-D-pentagalloylgalactose, α / β-D-pentagalloylmannose, α / β-D-pentagalloylallose, α / β-L-tetragalloylrhamnose, and α / β-L-tetragalloylfusose (10-galloyl glycosides) were successfully synthesized through optimization of the synthetic method. Their structures were determined by 1H NMR and high-resolution mass spectrometry. The coupling constants of H on the second carbon of the α-D and β-L-type sugar rings were 2–4, and those of the α-L and β-D-type sugar rings were 6–10. The results are as follows:

[0074] α-D-Pentalgalogalactose: 1H NMR (400MHz, DMSO-d6) δ7.11(s,2H),7.02(s,2H),6.89(s,2H),6.79(s,2H),6.77(s,2H),6.67(d,J=3.5Hz,1H),5.88(d,J=3.6Hz,1H),5.83( t,J=7.0Hz,1H),5.65(dd,J=10.8,3.6Hz,1H),4.80(t,J=6.9Hz,1H),4.29(dd,J=11.2,6.5Hz,1H),4.19(dd,J=11.1,6.8Hz,1H).HRMS(ESI)(C 41 H 32 O 26 ), m / z calculated[M+NH4] + 958.1520, measured[M+NH4] + :958.1511 (-0.94ppm).

[0075] β-D-pentagalloylgalactose: 1 H NMR (400MHz, DMSO-d6) δ9.22(s,15H),7.01(s,2H),6.95(s,2H),6.92(s,2H),6.84(s,2H),6.74(s,2H),6.31(d,J=8.3Hz,1H ),5.79(dd,J=10.5,3.4Hz,1H),5.73–5.65(m,2H),4.79(t,J=6.7Hz,1H),4.25(ddd,J=31.4,11.4,6.4Hz,2H).HRMS(ESI)(C 41 H 32 O 26 ), m / z calculated[M+NH4] + 958.1520, measured[M+NH4] + 958.1515 (-0.52ppm).

[0076] α-D-Pentalgalomannose: 1H NMR (400MHz, DMSO-d6) δ9.38–8.99(m,15H),7.05(s,2H),6.95(s,2H),6.93(s,2H),6.84(s,2H),6.75(s,2H),6. 48(s,1H),5.83–5.76(m,2H),5.50(t,J=2.8Hz,1H),4.49–4.37(m,2H),4.30(dd,J=2.7,2.3Hz,1H).HRMS(ESI)(C 41 H 32 O 26 ), m / z calculated[M+NH4] + 958.1520, measured[M+NH4] + 958.1514 (-0.63ppm).

[0077] β-D-pentagallomannose: 1 H NMR (400MHz, DMSO-d6) δ9.60–8.86(m,15H),7.13(s,2H),7.02(s,2H),6.96(s,2H),6.92(s,2H),6.78(s,2H),6.32(d,J=8.9Hz,1H),5.71( dd,J=10.2,3.2Hz,1H),5.66(d,J=8.7Hz,1H),5.63–5.59(m,1H),4.44(dt,J=26.7,12.4Hz,2H),4.34(dd,J=12.2,7.4Hz,1H).HRMS(ESI)(C 41 H 32 O 26 ), m / z calculated[M+NH4] + 958.1520, measured[M+NH4] + 958.1513 (-0.73ppm).

[0078] α-D-pentagaloylolose: 1H NMR (400MHz, DMSO-d6) δ9.48–8.73(m,15H),7.10(s,2H),7.03(s,2H),6.98(s,2H),6.88(s,2H),6.75(s,2H),6.66(d,J=3.6Hz,1H),6. 32(d,J=3.2Hz,1H),5.86(d,J=8.4Hz,1H),5.59–5.43(m,1H),4.64(dt,J=22.4,12.4Hz,2H),4.48(dd,J=12.2,7.4Hz,1H).HRMS(ESI)(C 41 H 32 O 26 ), m / z calculated[M+NH4] + 958.1520, measured[M+NH4] + 958.1516 (-0.42ppm)

[0079] β-D-pentagaloylolose: 1 H NMR(400MHz,DMSO-d6)δ9.48–8.73(m,15H),6.79(s,2H),6.72(s,2H),6.66(s,2H),6.56(s,2H),6.52(s,2H),6.38 (d,J=8.3Hz,1H),5.96(t,J=9.7Hz,1H),5.47–5.39(m,2H),4.60(dt,J=10.3,3.2Hz,1H),4.31(s,2H).HRMS(ESI)(C 41 H 32 O 26 ), m / z calculated[M+Na + 963.1074, measured[M+Na] + 963.1054 (-2.07ppm).

[0080] α-L-Tetragalloylrhamnose: 1 H NMR (400MHz, DMSO-d6) δ9.56–8.72(m,12H),6.88(s,2H),6.82(s,2H),6.71(s,2H),6.59(s,2H),6.06(d,J=6.8Hz,1H),5. 98(d,J=8.4Hz,1H),5.63(d,J=7.2Hz,1H),5.29(d,J=7.2Hz,1H),4.84–4.69(m,1H),3.31(d,J=11.2Hz,3H).HRMS(ESI)(C41 H 32 O 26 ),m / z calculated[M+H] + 773.1195, measured[M+H] + 773.1120 (0.65ppm).

[0081] β-L-tetragalloylrhamnose: 1 H NMR (400MHz, DMSO-d6) δ9.50–8.66(m,12H),6.77(s,2H),6.71(s,2H),6.66(s,2H),6.59(s,2H),6.24(d,J=2.6Hz,1H),6. 16(t,J=2.8Hz,1H),5.84(t,J=3.2Hz,1H),5.48(t,J=2.8Hz,1H),5.44–5.29(m,1H),3.33(d,J=11.2Hz,3H).HRMS(ESI)(C 41 H 32 O 26 ),m / z calculated[M+H] + 773.1195, measured[M+H] + 773.1186 (-1.16ppm).

[0082] α-L-tetragallopose: 1 H NMR (400MHz, DMSO-d6) δ9.56–8.72(m,12H),6.88(s,2H),6.82(s,2H),6.71(s,2H),6.59(s,2H),6.36(d,J=7 .8Hz,1H),6.09(d,J=8.4Hz,1H),5.63–5.29(m,2H),4.64–4.59(m,1H),3.31(d,J=11.2Hz,3H).HRMS(ESI)(C 41 H 32 O 26 ),m / z calculated[M+H] + 773.1195, measured[M+H] + 773.1189 (-0.77ppm).

[0083] β-L-tetragallofusaccharide: 1H NMR (400MHz, DMSO-d6) δ9.52–8.69(m,12H),6.83(s,2H),6.76(s,2H),6.68(s,2H),6.57(s,2H),6.28(d,J=2 .6Hz,1H),6.12(t,J=2.6Hz,1H),5.92–5.48(m,2H),4.44–4.29(m,1H),3.31(d,J=11.2Hz,3H).HRMS(ESI)(C 41 H 32 O 26 ),m / z calculated[M+H] + 773.1195, measured[M+H] + 773.1198 (0.38ppm).

[0084] Example 2

[0085] Different masses of α / β-D-pentagalloylgalactose, α / β-D-pentagalloylmannose, α / β-D-pentagalloylallose, α / β-L-tetragalloylrhamnose, and α / β-L-tetragalloylfurose were weighed and dissolved by heating, then allowed to cool at 4°C to observe whether self-assembled hydrogels could be formed. It was found that the minimum gelling concentrations of α-D-pentagalloylgalactose and α-D-pentagalloylmannose were 20 mg / mL and 10 mg / mL, respectively, while their epimers β-D-pentagalloylgalactose and β-D-pentagalloylmannose could not self-assemble into hydrogels in water. Furthermore, α / β-D-pentagaloylolose, which shares the same hexacarbon aldose structure, and α / β-L-tetragaloylrhamnose and α / β-L-tetragaloylfose, both structurally similar methyl pentoses, cannot self-assemble to form supramolecular hydrogels. This demonstrates that whether monomers with different structures can form hydrogels is unpredictable, involving chance and uncertainty, and requires comprehensive consideration of factors such as the compound's structure, chirality, steric hindrance, and concentration.

[0086] Example 3

[0087] The supramolecular hydrogel obtained in Example 2 was further characterized in terms of its gel material properties, as follows:

[0088] The prepared α-D-pentagalloylgalactose and α-D-pentagalloylmannose supramolecular hydrogels were placed at room temperature and observed at intervals to determine their stability. The prepared hydrogels were heated to dissolve them and then allowed to cool at room temperature to observe whether they could self-assemble into hydrogels again, thus determining their thermosensitivity. The prepared hydrogels were shaken to disrupt their structure and then allowed to stand at room temperature to observe whether they could recover into hydrogels, thus determining their self-healing properties. The prepared hydrogels were added to a syringe and then gently ejected to determine their injectability.

[0089] The results showed that both α-D-pentagalloylgalactose and α-D-pentagalloylmannose supramolecular hydrogels remained intact for at least 120 days at room temperature, exhibiting good stability. After heating and then cooling to room temperature, they self-assembled to form hydrogels again, indicating their thermosensitivity. After shaking and then allowing to stand, they recovered to form hydrogels, demonstrating their self-healing properties. The hydrogels could be easily ejected with a syringe, indicating their injectability. These results demonstrate that α-D-pentagalloylgalactose and α-D-pentagalloylmannose supramolecular hydrogels possess excellent gel properties, including good stability, thermosensitivity, self-healing, and injectability, making them ideal biomedical materials for clinical application.

[0090] Example 4

[0091] In this embodiment, the rheological tests of the α-D-pentagalloylgalactose and α-D-pentagalloylmannose supramolecular hydrogels prepared in Example 2 were performed using the following methods:

[0092] α-D-pentagalloylgalactose and α-D-pentagalloylmannose supramolecular hydrogels were placed on a rheometer measuring plate with a gap of 5 mm and a temperature of 25℃. Frequency scanning: the strain was constant at 0.1%, and the measurement frequency range was 0.1 Hz–10 Hz. Amplitude scanning: the frequency was constant at 1 Hz, and the strain variation range was 0.001%–10%, obtaining the storage modulus (G′) and loss modulus (G″) as a function of strain and frequency. Under varying frequency and shear stress, the storage modulus was greater than the loss modulus, indicating excellent rheological properties.

[0093] Example 5

[0094] In this embodiment, the antibacterial activity of the β-D-pentagalloglucopyranoside hydrogel prepared in Example 2 was determined, and the method is as follows:

[0095] The inhibitory effects of the hydrogel on pathogenic Staphylococcus aureus, opportunistic pathogenic Escherichia coli, probiotics Bacillus subtilis and Enterococcus faecalis, and clinically isolated methicillin-resistant Staphylococcus aureus were observed using a turbidimetric assay. Staphylococcus aureus, Escherichia coli, Bacillus subtilis, and Enterococcus faecalis were all obtained from the School of Life Sciences, Beijing University of Chinese Medicine. Methicillin-resistant Staphylococcus aureus was obtained from Mingzhou Biotechnology Co., Ltd. (sample number: ATCC43300).

[0096] Bacterial resuscitation and stock preparation: Bacteria were dispersed in LB medium and cultured overnight at 37°C and 200 rpm by rotation. Then, a stock solution of 2 × 10⁻⁶ bacteria was prepared using LB medium. 6 The bacterial stock solution with CFU / mL was counted using the plate count method.

[0097] The minimum inhibitory concentrations (MICs) of α-D-pentagalloylgalactose and α-D-pentagalloylmannose supramolecular hydrogels were determined using a dilution method. In 48-well plates, 1 mL of hydrogel solutions with concentrations of 150, 125, 100, 75, 50, and 37.5 μmol / L were prepared using LB medium. Subsequently, 20 μL of bacterial stock solution was added to each solution, and the plates were incubated at 37°C, 5% CO2, and saturated humidity for 16 hours. The OD values ​​were measured at 600 nm using a microplate reader. The experiment was repeated three times, and the concentration at which bacterial survival was greater than 80% was defined as the minimum inhibitory concentration (MIC) for that sample. The control group (no drug, no bacteria) and the control group (with bacteria, no drug) were defined as the control group. Bacterial survival rate (%) = (sample group absorbance - control group absorbance) / (control group absorbance - control group absorbance) × 100%.

[0098] The specific results are shown in Table 1.

[0099] Table 1: Antibacterial effects of the hydrogel prepared in this invention against different bacteria

[0100]

[0101]

[0102] The results showed that the supramolecular hydrogels based on α-D-pentagalloylgalactose and α-D-pentagalloylmannose exhibited strong antibacterial activity against most pathogenic bacteria, which was stronger than that of their monomers. At the same time, they showed weak inhibitory activity against the probiotics Bacillus subtilis and Enterococcus faecalis in the intestine, but still showed excellent sensitivity against clinically isolated methicillin-resistant Staphylococcus aureus, which has value for further research and clinical development.

[0103] Example 6

[0104] In this example, the effects of the α-D-pentagalloylgalactose and α-D-pentagalloylmannose supramolecular hydrogels prepared in Example 2 on the microstructure of bacteria are investigated using the following method:

[0105] Bacteria were co-incubated with supramolecular hydrogels containing 1 / 3 MIC concentrations of α-D-pentagalloylgalactose and α-D-pentagalloylmannose for 6 hours. After centrifugation, the bacteria were fixed with 2.5% glutaraldehyde for 4 hours, eluted with a gradient of ethanol, and finally imaged using scanning electron microscopy. The results showed that the self-assembled supramolecular hydrogels could induce bacterial cell wall shrinkage and even rupture.

[0106] Example 7

[0107] In this embodiment, the in vitro bacterial biofilm inhibition rate of the α-D-pentagalloylgalactose and α-D-pentagalloylmannose supramolecular hydrogels prepared in Example 2 was detected by the following method:

[0108] First, the bacterial suspension was injected into 96-well plates, followed by the addition of hydrogel samples of varying concentrations. All samples were incubated at 37°C for 24 hours. After 24 hours, the supernatant was aspirated, revealing bacterial biofilms adhering to the bottom. The biofilms were washed three times with PBS buffer, and 200 μL of XTT staining solution was added to each well. The plates were then incubated at 37°C for 2 hours, and the absorbance was measured at 490 nm. Blank culture medium was used as a solvent control, and culture medium with added bacteria served as a blank bacterial control. The inhibition rate of the samples on the biofilm was calculated using the following formula.

[0109] Inhibition rate (%) = (1 - (OD sample - OD solvent) / (OD blank bacteria - OD solvent)) × 100%. Furthermore, 5mm × 5mm samples were placed on identical silicon wafers into 24-well plates, bacterial suspension was added, and α-D-pentagalloylgalactose and α-D-pentagalloylmannose supramolecular hydrogels were added respectively for treatment, followed by incubation at 37°C for 24 hours. The biofilms were washed three times with PBS buffer and fixed with 2.5% glutaraldehyde at 37°C for 4 hours. Subsequently, the samples were dehydrated using a gradient of ethanol solutions with gradually increasing concentrations (30%, 50%, 70%, 80%, 90%, 95%, 100%) and analyzed by scanning electron microscopy (FESEM).

[0110] Example 8

[0111] In this embodiment, the antioxidant activity of the α-D-pentagalloylgalactose and α-D-pentagalloylmannose supramolecular hydrogels prepared in Example 2 was determined, and the method is as follows:

[0112] (1) Prepare a 0.2 mmol / L DPPH solution with anhydrous ethanol and store it in the dark. Then prepare test samples of different concentrations with anhydrous ethanol. Add 2 mL of the test sample solution and 2 mL of DPPH solution to the same test tube, shake well, and let it stand at room temperature in the dark for 30 minutes before measuring its absorbance A at 520 nm. sample Simultaneously, the absorbance A0 of a mixture of 2 mL DPPH solution and 2 mL anhydrous ethanol was measured. Finally, the absorbance was calculated using the formula:

[0113] DPPH clearance percentage = (A0 - A) sample ) / A0*100%

[0114] The specific results are shown in Table 2.

[0115] Table 2: Scavenging rate of DPPH by α-D-pentagalloylgalactose supramolecular hydrogel

[0116]

[0117]

[0118] (2) ABTS (7 mM) solution was reacted with potassium persulfate (K2S2O8, 2.45 mM) at room temperature in the dark for 16 h to generate ABTS·+. Then, an appropriate concentration of ABTS·+ solution was reacted with α-D-pentagalloylmannose hydrogel in the dark for 10 min. Finally, the absorbance at 734 nm was measured. The results were then calculated using the formula:

[0119] ABTS·+ clearance percentage = A0 - (A sample -A x0 ) / A0x100%

[0120] Where A0 represents the absorbance of the blank control group, A sample A represents the absorbance of the sample group. x0 The absorbance is without the sample. See Table 3 for specific results.

[0121] Table 3: Scavenging rate of ABTS·+ by α-D-pentagalloylmannose supramolecular hydrogel

[0122]

[0123] The results showed that the antioxidant activity of supramolecular hydrogels was significantly enhanced compared to ascorbic acid, and they have value for further research and clinical development.

[0124] Example 9

[0125] In this embodiment, the anti-inflammatory activity of the supramolecular hydrogel prepared in Example 2 was evaluated using different cell lines, and the methods are as follows:

[0126] 1. Using LPS-induced mouse macrophage RAW 264.7 as a model, the effects of the prepared supramolecular hydrogel on inflammatory factors (TNF-α, IL-1β, IL-6, and NO) produced by LPS-induced macrophages were determined using an ELISA kit to evaluate its anti-inflammatory activity. Specific results are shown in Table 4.

[0127] Table 4: Effects of supramolecular hydrogels on inflammatory factors in mouse macrophage RAW 264.7 cells.

[0128]

[0129] 2. Using LPS-induced normal human chondrocytes c28 / i2 as a model, the effects of supramolecular hydrogel on inflammatory factors (TNF-α, IL-1β, IL-6, and PGE-2) produced by LPS-induced macrophages were measured using an ELISA kit to evaluate its anti-arthritis activity. Specific results are shown in Table 5.

[0130] Table 5: Effects of supramolecular hydrogels on C28 / I2 inflammatory factors in normal human chondrocytes

[0131]

[0132] 3. Using LPS-induced normal human colonic epithelial cells NCM460 as a model, the effects of supramolecular hydrogel on inflammatory factors (TNF-α, IL-1β, IL-6, and NO) produced by LPS-induced macrophages were measured using an ELISA kit to evaluate its anti-arthritis activity. Specific results are shown in Table 6.

[0133] Table 6: Effects of supramolecular hydrogels on inflammatory factors in NCM460 cells of normal human colonic epithelial cells

[0134]

[0135]

[0136] The above results indicate that supramolecular hydrogels significantly inhibit inflammatory factors induced by LPS in mouse RAW 264.7 macrophages, human normal chondrocytes c28 / i2, and human normal colonic epithelial cells NCM460, demonstrating strong anti-inflammatory activity and possessing value for further research and clinical development.

[0137] Example 10

[0138] In this embodiment, the α-D-pentagalloylgalactose and α-D-pentagalloylmannose supramolecular hydrogels prepared in Example 2 were used to evaluate the regulation of macrophage phenotypes. The method is as follows:

[0139] The expression of surface markers (iNOS and CD206) on M1 and M2 was detected by immunofluorescence staining. Simply put, the hydrogel was added to a confocal culture dish and sterilized with ethanol and ultraviolet light. 1×10⁻⁶ 6 RAW 264.7 macrophages were seeded on sterile hydrogels and incubated for 48 hours. Then, the macrophages were stimulated with 100 ng / mL LPS and 20 ng / mL IFN-γ for 24 hours to systematize the inflammatory microenvironment. Immunofluorescence staining was then performed, followed by imaging under a laser confocal microscope. The results showed that the CD206 (M2 macrophage) marker in the β-pentagalloglucopyranoside supramolecular hydrogel group was significantly higher than that in the control and model groups, while the iNOS (M1 macrophage) marker was significantly lower than that in the control and model groups. This indicates that both α-D-pentagalloglucopyranoside and α-D-pentagalloglucopyranoside supramolecular hydrogels can effectively promote the conversion of RAW 264.7 macrophages to the M2 phenotype and simultaneously inhibit the LPS-induced conversion of macrophages to the M1 phenotype.

[0140] Example 11

[0141] In this embodiment, the α-D-pentagalloylgalactose and α-D-pentagalloylmannose supramolecular hydrogels prepared in Example 2 were used to evaluate the regulation of macrophage phenotypes. The method is as follows:

[0142] RAW 264.7 cells were cultured at a rate of 2 × 10⁻⁶. 5 HUVECs were seeded at a density of 1.5 × 10⁶ cells / well in 6-well plates and co-incubated with α-D-pentagalloylgalactose and α-D-pentagalloylmannose supramolecular hydrogels for 24 h. The supernatant was collected, and the suspended cells were removed by centrifugation. Subsequently, HUVECs were seeded into 24-well plates pre-seeded with Matrigel matrix at a cell density of 1.5 × 10⁶ cells / well. 4 / well, and the collected cell supernatant was added and co-incubated. Finally, angiogenesis was photographed at different time points, and statistical analysis was performed.

[0143] Example 12

[0144] In this embodiment, the in vivo pharmacodynamics of the α-D-pentagalloylgalactose and α-D-pentagalloylmannose supramolecular hydrogels prepared in Example 2 were evaluated using the following methods:

[0145] Female Balb / c mice aged 6-8 weeks and weighing 16-18g were used as experimental animals. The fur on the backs of the mice was shaved with electric clippers, and the mice were anesthetized with pentobarbital. Once the mice became lethargic and their breathing became deep and slow, the model was established. The skin of the shaved area on the back of the mice was disinfected by wiping it with cotton balls soaked in 75% alcohol. After the alcohol had completely evaporated and the skin had dried, 8mm circular holes were punched in the backs of the mice to define the shape, and then 8mm diameter circular pieces of skin were cut off with scissors. The mice were then randomly divided into four groups: a control group, a drug treatment group (Hydrogel), a model group (MRSA), and a treatment group (MRSA + Hydrogel). Control group: no bacterial infection treatment; Hydrogel: hydrogel only, no bacterial infection treatment; MRSA: bacterial infection treatment, no treatment; MRSA + Hydrogel: MRSA bacterial infection was first performed on mouse wounds, followed by treatment with α-D-pentagalloylgalactose and α-D-pentagalloylmannose supramolecular hydrogels. Eight mice (n=8) were used as parallel controls in each group, and the treatment period was 12 days. Wound size was recorded every 3 days, and wound healing rate was calculated. It was found that both α-D-pentagalloylgalactose and α-D-pentagalloylmannose supramolecular hydrogels effectively promoted wound healing.

[0146] After the 12-day treatment cycle, the mice were euthanized by cervical dislocation. The skin on the back of the mice was cut off with sterile surgical scissors that had been autoclaved, and the wounds were flattened and fixed in paraformaldehyde fixative.

[0147] Twelve days later, histopathological sections showed that the tissue sections of the α-D-pentagalloylgalactose and α-D-pentagalloylmannose supramolecular hydrogel groups showed good recovery, with clear separation of the epidermal and dermal layers, abundant hair follicle growth, fewer inflammatory cells, and more collagen fiber deposition. In contrast, the skin of the MRSA group was still healing, with visible granulation tissue, no hair follicle recovery, more inflammatory cells, and less collagen fiber deposition. On day 6 of drug administration, the CD206 (M2 macrophage) markers in the α-D-pentagalloylgalactose and α-D-pentagalloylmannose supramolecular hydrogel groups were significantly higher than those in the control and model groups, while the iNOS (M1 macrophage) markers were significantly lower. Furthermore, the levels of inflammatory factors TNF-α and IL-6 were significantly lower in the α-D-pentagalloylgalactose and α-D-pentagalloylmannose supramolecular hydrogel groups, while the levels of the angiogenic factor VEGF were higher. The results indicate that both α-D-pentagalloylgalactose and α-D-pentagalloylmannose supramolecular hydrogels can effectively inhibit the normal growth of bacteria in vivo, promote the conversion of macrophages to the M2 phenotype, and exhibit significant anti-inflammatory capabilities. They also have good ability to promote angiogenesis and synergistically accelerate wound healing.

[0148] Example 13

[0149] In this example, the safety of the supramolecular hydrogel prepared in Example 2 was evaluated. The method was as follows: HaCat and L929 cells were treated with a drug in the range of 37.5–200 μM for 24 h. Then, 20 μL of MTT solution was added to the cells in a 96-well plate, and incubation continued for 4 h. Subsequently, 150 μL of DMSO was added to dissolve the cells, and the absorbance was measured at 490 nm. Cell viability was calculated using the following formula:

[0150] Cell viability (%) = (OD) 给药组 -OD 空白组 ) / (OD 正常组 -OD 空白组 )×100%

[0151] The results showed that the cell survival rate of different concentration groups was greater than 80% after 24 hours of incubation, indicating that α-D-pentagalloylgalactose and α-D-pentagalloylmannose supramolecular hydrogels had no significant cytotoxicity.

[0152] Fresh rat blood was used to determine the in vitro hemolysis effect of the samples. First, red blood cells were collected by centrifugation at 3000 rpm for 15 minutes and washed three times with physiological saline. Then, 3 mL of the centrifuged red blood cells were mixed with 11 mL of physiological saline for storage and dispersion. α-D-pentagalloylgalactose and α-D-pentagalloylmannose supramolecular hydrogels were diluted with physiological saline to their respective concentrations. Then, 1 mL of the test solution was mixed with 100 μL of red blood cell stock solution to obtain a 4% red blood cell solution, which was incubated at 37°C for 4 hours. Afterward, the solution was centrifuged at 3000 rpm for 15 minutes, and the absorbance of the supernatant was measured at 570 nm using a microplate reader. Deionized water was used as the positive control, and physiological saline was used as the negative control. The hemolysis rate was calculated using the following formula:

[0153] Hemolysis rate (%) = (A 给药组 -A PBS组 ) / (A 去离子水组 -A PBS组 )×100%

[0154] Hemolysis tests showed that α-D-pentagalloylgalactose and α-D-pentagalloylmannose supramolecular hydrogels had no obvious hemolytic properties. Even at a concentration as high as 600 μM, the hemolysis rate was still lower than the internationally recognized standard of 5%.

[0155] α-D-pentagalloylgalactose and α-D-pentagalloylmannose supramolecular hydrogels were applied to the skin of mice. After 24 hours, the skin was harvested, fixed in paraformaldehyde fixative, and then sectioned. The skin pathology of normal mice and treated mice was compared to evaluate the skin irritation of the drugs. No obvious redness, swelling, or inflammatory cells were observed in the skin of the treated mice, which was similar to normal skin, suggesting that α-D-pentagalloylgalactose and α-D-pentagalloylmannose supramolecular hydrogels have no skin irritation.

[0156] After a 12-day treatment cycle, mice were euthanized by cervical dislocation. The hearts, livers, spleens, lungs, and kidneys of the mice were dissected and fixed in paraformaldehyde fixative. Histological sections were then prepared. The heart, liver, spleen, lungs, and kidneys of the treated group were similar to those of the normal group, with no pathological changes observed, indicating that the α-D-pentagalloylgalactose and α-D-pentagalloylmannose supramolecular hydrogels have good safety profiles.

[0157] Example 14

[0158] In this embodiment, the supramolecular hydrogel prepared in Example 2 is evaluated as a drug delivery carrier, and the method is as follows:

[0159] Antibacterial drugs (berberine hydrochloride, sanguinarine hydrochloride, and penicillin sodium, etc.), anti-inflammatory drugs (aspirin, dexamethasone, methotrexate, etc.), and antitumor drugs (doxorubicin, camptothecin, and cantharidin) were dissolved together with single molecules of α-D-pentagalloylgalactose and α-D-pentagalloylmannose upon heating. It was found that α-D-pentagalloylgalactose and α-D-pentagalloylmannose effectively promoted the solubility of the aforementioned drugs. After standing and cooling, a stable hydrogel was formed. This indicates that the supramolecular hydrogels of α-D-pentagalloylgalactose and α-D-pentagalloylmannose can serve as drug delivery carriers with good stability.

Claims

1. A carrier-free self-assembling hydrogel, the structure of which is shown in Formula 1: in, R can be -H or -O-galloyl; meanwhile, the galloyl group and the R group substituent on the sugar ring can be independently located on or under the sugar ring in the Haworth formula.

2. The preferred structure of the self-assembled hydrogel compound as described in claim 1 is shown in Figures 1-2:

3. The carrier-free self-assembling hydrogel as described in claim 1 or 2, characterized in that, The hydrogel is obtained solely from α-D-pentagalloylgalactose or α-D-pentagalloylmannose by heating and cooling in water.

4. The use of the self-assembled carrier-free hydrogel as described in any one of claims 1, 2 or 3 in the preparation of antibacterial, anti-inflammatory, immunomodulatory, antioxidant or angiogenesis-promoting drugs.

5. The application as described in claim 4, characterized in that, The antimicrobial drug is used for external human bacterial infections, internal bacterial infections, bacterial diarrhea, skin wound infections, or for infections caused by Staphylococcus aureus, Escherichia coli, methicillin-resistant Staphylococcus aureus, or the bacteria causing these infections.

6. The application as described in claim 4, characterized in that, The anti-inflammatory drug is used for inflammatory responses following tissue damage, osteoarthritis, rheumatoid arthritis, or ulcerative colitis.

7. The application as described in claim 4, characterized in that... Application in the preparation of drugs that inhibit bacterial biofilm.

8. The application as described in claim 4, characterized in that, The angiogenesis-promoting drugs are used in tissue repair and regeneration therapies related to angiogenesis.

9. The use of the carrier-free self-assembly hydrogel as a drug delivery carrier according to any one of claims 1-3.

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