Multi-network metal polyphenol nanogel loaded with anti-inflammatory active components as well as preparation method and application of multi-network metal polyphenol nanogel

Abstract

The invention relates to a multi-network metal polyphenol nanogel loaded with anti-inflammatory active components and a preparation method and application thereof, the nanogel is formed by aldehyde-terminated polyethylene glycol and an amino-containing macromolecular material, then a polyphenol compound and an anti-inflammatory active material are added, finally a salt solution containing metal cations is added, and the anti-inflammatory active component loaded multi-network metal polyphenol nanogel is obtained. The multi-network metal polyphenol nanogel is formed. The tissue adhesion of the nanogel is synergistically enhanced through a double dynamic bond cross-linked network of a polyphenol compound-metal cation coordination bond and a Schiff base bond, and long-acting retention on the ocular surface is realized. And an anti-inflammatory active material is loaded, so that the expression of ocular surface inflammatory factors can be inhibited, ocular surface repair can be promoted, vicious circle of inflammation, oxidative stress and tear film instability in the xerophthalmia can be accurately blocked, and multiple effects are integrated. The multi-network metal polyphenol nanogel can be used for preparing a liquid medicine for treating ocular surface inflammatory diseases and bacterial infectious diseases, and is applied to eyes in a flowing aqueous solution form in a dripping point-to-eye manner.

Description

TECHNICAL FIELD

[0001] The application belongs to the technical field of biological medicine, and relates to a nanogel for relieving dry eye, in particular to a multi-network metal polyphenol nanogel loaded with anti-inflammatory active ingredients and a preparation method and application thereof. BACKGROUND

[0002] Dry eye is a common ocular surface disease, and its core pathological mechanism is tear film instability on the ocular surface, insufficient tear secretion or excessive tear evaporation, which further causes ocular surface inflammation, oxidative stress damage, and finally leads to ocular surface epithelial cell damage, vision loss and other serious consequences. At present, the main clinical treatments for dry eye include artificial tears, anti-inflammatory drugs, glucocorticoids and the like, but all have obvious defects: (1) Short retention time and low bioavailability: Most of the drugs in artificial tears and conventional eye drops will be discharged out of the body with tears in a short time due to the limitation of the corneal barrier and the flushing effect of tears, and frequent use is required, which is poor in patient compliance, low in bioavailability, and seriously affects the treatment effect.

[0003] (2) Single efficacy: Artificial tears can only play a short-term moisturizing role and cannot inhibit ocular surface inflammation and oxidative stress; although anti-inflammatory drugs can relieve inflammation, they lack moisturizing and antioxidant ability and are difficult to break the vicious cycle of dry eye.

[0004] (3) Safety risk: In order to prolong the shelf life, preservatives and other auxiliary materials are added in addition to active molecules, and the accumulation of preservatives in the eye tissue increases with frequent use of eye drops, which has a toxic effect on the corneal epithelium, leading to corneal epithelial damage, aggravation of dry eye symptoms and other adverse reactions. Long-term use of glucocorticoids can easily cause side effects such as elevated intraocular pressure and cataracts, and is limited in clinical application.

[0005] In summary, there is an urgent need to develop a material with long-acting ocular surface retention, anti-inflammatory, antioxidant and moisturizing multi-effect combined characteristics to break the vicious cycle of dry eye. SUMMARY

[0006] In view of the deficiencies of the prior art, the application provides a multi-network metal polyphenol nanogel loaded with anti-inflammatory active ingredients and a preparation method and application thereof. Aldehyde group-terminated polyethylene glycol is used to form a nanogel with a macromolecular material with an amino group, polyphenol and metal cations are added to improve the ocular surface bioadhesion and achieve long-acting retention on the ocular surface; the anti-inflammatory active material is loaded to promote ocular surface repair and precisely block the vicious cycle of inflammation, oxidative stress and tear film instability in dry eye, and multi-effect combination is achieved.

[0007] A multi-network metal polyphenol nanogel loaded with anti-inflammatory active ingredients comprises a nanogel network, a polyphenol compound with antioxidant activity, metal cations and an anti-inflammatory active ingredient.

[0008] The nanogel network is crosslinked by Schiff base reaction between aldehyde-terminated polyethylene glycol and amino-bearing macromolecular material polymer.

[0009] As preferred, the polyphenolic compound is one or more of tannic acid, tea polyphenol, gallic acid, ellagic acid or chlorogenic acid.

[0010] As preferred, the metal ion is one or more of Zn 2+ , Fe 3+ , Cu 2+ .

[0011] As preferred, the anti-inflammatory active ingredient is berberine.

[0012] As preferred, the amino-bearing macromolecular material is carboxymethyl chitosan.

[0013] As preferred, the mass percentage of the polyphenolic compound is 10% to 30%, the mass percentage of the metal cation is 5% to 20%, the mass percentage of the carboxymethyl chitosan is 20% to 40%, the mass percentage of the aldehyde-terminated polyethylene glycol is 20% to 40%, and the mass percentage of the anti-inflammatory active ingredient is 0.2% to 5%.

[0014] A preparation method of a multi-network metal polyphenol nanogel loaded with an anti-inflammatory active ingredient, comprising the following specific steps: Step 1: Preparation of nanogel network An amino-bearing macromolecular material is added to deionized water to prepare solution A. Aldehyde-terminated polyethylene glycol is dissolved in an ethanol solution to prepare solution B. Solution A and B are added to 2 mL of deionized water for mixing, so that the mass ratio of the amino-bearing macromolecular material to the aldehyde-terminated polyethylene glycol is 1:1, and Schiff base reaction occurs between them to crosslink and form a nanogel network.

[0015] Step 2: Loading of active ingredient A polyphenolic compound is dissolved in deionized water to prepare solution C. An anti-inflammatory active ingredient is dissolved in dimethyl sulfoxide to prepare solution D. Solution C and solution D are sequentially and slowly added to 5 mL of deionized water and stirred uniformly, and the mass ratio of the polyphenolic compound to the anti-inflammatory active ingredient is 1: (0.02 to 0.2). The nanogel network obtained in step 1 is further added, and stirring is continued to form a polyphenol nanogel network loaded with an anti-inflammatory active ingredient.

[0016] Step 3: Construction of metal polyphenol multi-network nanogel The metal salt aqueous solution is configured, slowly added into the polyphenol nanogel network loaded with anti-inflammatory active ingredients, the mass ratio of metal cation to polyphenol compound is 1: (0.5-2), the pH of the system is adjusted to 6.5-7.5, and uniform and transparent metal polyphenol multi-network nanogel is formed under continuous stirring.

[0017] The application of the multi-network metal polyphenol nanogel loaded with anti-inflammatory active ingredients in the preparation of a liquid medicine for treating ocular surface inflammatory diseases and bacterial infectious diseases, the multi-network metal polyphenol nanogel loaded with anti-inflammatory active ingredients is applied to the eye in the form of a flowing aqueous solution through a drop point eye, and diseases such as dry eye, bacterial infectious keratitis, dry eye related conjunctivitis, and blepharitis are treated.

[0018] The application has the following beneficial effects: 1. Long-acting ocular surface retention: the tissue adhesion of the nanogel is synergistically enhanced by the double dynamic bond crosslinking network of the polyphenol compound-metal cation coordination bond and the Schiff base bond, the nanogel can enter the corneal epithelial cells within 1 minute, the concentration of the drug on the ocular surface is improved, and the curative effect is enhanced.

[0019] 2. Multi-effect in one breaks vicious cycle: the anti-inflammatory active molecules can inhibit the expression of ocular surface inflammatory factors such as TNF-alpha, IL-6 and IL-1 beta, reduce the inflammatory reaction of the ocular surface, and at the same time, cooperate with the polyphenol compound to remove active oxygen on the ocular surface, reduce oxidative stress damage, and protect the ocular surface epithelial cells; the nanogel network has a moisturizing and lubricating effect, can form a persistent hydration film on the ocular surface, maintain the stability of the tear film, alleviate the problem of too fast tear evaporation, and further promote the repair of the ocular surface, thereby fundamentally breaking the vicious cycle of dry eye and efficiently treating dry eye through the multi-effect in one method.

[0020] 3. Stability and good safety: the Schiff base crosslinking of the amino-containing macromolecular material and the aldehyde-terminated polyethylene glycol, and the coordination of the polyphenol compound and the metal cation form the metal polyphenol nanogel through secondary crosslinking, and the multi-crosslinking network structure endows the nanogel with excellent stability and mechanical properties, can be stored for a long time without aggregation, and can continuously and stably release the drug in the complex physiological environment of the ocular surface. In addition, the polyphenol compound and the metal cation in the metal polyphenol nanogel have good antibacterial properties, can inhibit the growth of microorganisms during long-term storage, do not need to add additional preservatives, avoid the toxic side effects caused by the addition of preservatives during long-term storage, have better biological safety, and can be used for a long time.

[0021] 4. Simple preparation process: no complex equipment is needed, the reaction conditions are mild, and the process is suitable for industrial production. BRIEF DESCRIPTION OF DRAWINGS

[0022] Figure 1 Transmission electron microscopy images of MPN-NGs prepared for Comparative Example 1 and BBR@MPN-NGs prepared for Example 1.

[0023] Figure 2 Hydrated particle size of MPN-NGs prepared for Comparative Example 1 and BBR@MPN-NGs prepared for Example 1.

[0024] Figure 3 Results of ultraviolet absorption spectrum determination of MPN-NGs, BBR@MPN-NGs and berberine.

[0025] Figure 4 Results of changes in hydrated particle size and PDI of BBR@MPN-NGs in Test Example 1 after being placed at 4℃ for different time.

[0026] Figure 5 Comparison of DPPH radical scavenging efficiency of BBR@MPN-NGs in Test Example 1 before and after being placed at 4℃ for 1 year.

[0027] Figure 6 Results of antibacterial performance verification of BBR@MPN-NGs in Test Example 2.

[0028] Figure 7 Results of fluorescence test of different materials after being incubated with corneal epithelial cells for 1 minute in Test Example 3.

[0029] Figure 8 DCF fluorescence images of ROS production after different materials were incubated with corneal epithelial cells in Test Example 4.

[0030] Figure 9 Statistics of apoptosis of corneal epithelial cells caused by inhibition of H2O2 oxidative damage after different materials were added in Test Example 5.

[0031] Figure 10 qPCR detection of inflammatory factors IL-1β, IL-6 and MMP-9 in corneal epithelial cells after different materials were added in Test Example 5.

[0032] Figure 11 Pictures of ocular surface fluorescein sodium after different materials treated severe dry eye in mice in Test Example 6.

[0033] Figure 12 Detection results of tear secretion and tear film break-up time after different materials treated severe dry eye in mice in Test Example 6.

[0034] Wherein, the number of "*" is used to represent the significance difference p value between statistics, "*" represents "p<0.05", "**" represents "p<0.01", "***" represents "p<0.001", and "****" represents "p<0.0001". DETAILED DESCRIPTION

[0035] The application will be further explained in conjunction with the accompanying drawings; Comparative Example 1 In this embodiment, a multi-network metal polyphenol nanogel without loading anti-inflammatory active molecules is prepared as a comparison, and the specific steps are as follows: Step 1: Preparation of nanogel network Carboxymethyl chitosan is dissolved in deionized water at 60-80°C to prepare solution A with a concentration of 15 mg / mL. Aldehyde-terminated polyethylene glycol is dissolved in ethanol to prepare solution B with a concentration of 22 mg / mL. Solution A and B are added to 2 mL of deionized water and mixed, and the mass ratio of carboxymethyl chitosan to aldehyde-terminated polyethylene glycol is controlled to be 1:1. The mixture is incubated at 37°C for 30 minutes to undergo Schiff base reaction and crosslinking to form a nanogel network.

[0036] Step 2: Loading tannic acid Take 160 μL of tannic acid aqueous solution with a concentration of 20 mg / mL, slowly add it to 5 mL of deionized water, and stir uniformly at a speed of 500 rpm. Then slowly add the nanogel network obtained in step 1, continue to stir at 37°C and a stirring speed of 500 rpm for 10 minutes. Hydrogen bonding occurs between tannic acid and polyethylene glycol to form a polyphenol nanogel network.

[0037] Step 3: Construction of metal polyphenol multi-network nanogel Take 200 μL of zinc nitrate hexahydrate solution with a concentration of 10 mg / mL, quickly add it to the polyphenol nanogel network solution obtained in step 2, adjust the pH of the system to 7.0 with 0.1 M sodium hydroxide solution, and stir at room temperature for 10 minutes. A milky white, uniform and transparent solution is obtained, indicating that the metal polyphenol multi-network nanogel (MPN-NGs) has been successfully formed.

[0038] The metal polyphenol multi-network nanogel (MPN-NGs) is centrifuged and washed with deionized water, repeated 3 times to remove free substances. The white precipitate at the bottom is resuspended with deionized water and stored at 4°C for long-term preservation. The centrifugation conditions are 4°C, 10000 rpm, and 15 min each time.

[0039] Example 1

[0040] This embodiment provides a method for preparing a multi-network metal polyphenol nanogel loaded with anti-inflammatory active ingredients, the specific steps of which are as follows: Step 1: Preparation of nanogel networks Carboxymethyl chitosan was dissolved in deionized water at 60–80 °C to prepare solution A with a concentration of 15 mg / mL. Aldehyde-terminated polyethylene glycol was prepared into an ethanol solution B with a concentration of 22 mg / mL. Solutions A and B were added to 2 mL of deionized water and mixed, maintaining a mass ratio of carboxymethyl chitosan to aldehyde-terminated polyethylene glycol of 1:1. The mixture was incubated at 37 °C for 30 minutes to induce a Schiff base reaction, resulting in cross-linking and the formation of a nanogel network.

[0041] Step 2: Loading berberine and tannic acid A 20 mg / mL aqueous solution of tannic acid and a 3 mg / mL solution of berberine (BBR) dimethyl sulfoxide were slowly added to 5 mL of deionized water, maintaining a tannic acid to berberine mass ratio of 1:0.05. The mixture was stirred at 500 rpm until homogeneous. Subsequently, the nanogel network obtained in step 1 was slowly added dropwise, and the mixture was stirred for another 10 minutes at 37°C and 500 rpm to form a polyphenol nanogel network loaded with berberine.

[0042] Step 3: Constructing a metal polyphenol multi-network nanogel Take 200 μL of zinc nitrate hexahydrate solution with a concentration of 10 mg / mL and quickly add it to the polyphenol nanogel network solution loaded with berberine obtained in step 2. Adjust the pH of the system to 7.0 with 0.1M sodium hydroxide solution and stir for 10 minutes at room temperature to obtain a yellow, uniform, and transparent solution, indicating that the metal polyphenol multi-network nanogel loaded with berberine (BBR@MPN-NGs) was successfully formed.

[0043] The berberine-loaded metal polyphenol multi-network nanogel (BBR@MPN-NGs) was centrifuged and washed with deionized water, repeated three times. The yellow precipitate at the bottom was resuspended in deionized water and stored at 4°C for long-term preservation. The centrifugation conditions were 4°C, 10,000 rpm, and 15 min each time.

[0044] The metal polyphenol multi-network nanogels (MPN-NGs) prepared in Comparative Example 1 and Example 1, and the metal polyphenol multi-network nanogel loaded with berberine (BBR@MPN-NGs) were observed under transmission electron microscopy. Figure 1 As shown, both nanogels have spherical structures. The hydrated particle size of both was measured, as follows: Figure 2 As shown, the particle size of MPN-NGs is about 200 nm, while that of BBR@MPN-NGs is about 220 nm, indicating that the nanogel prepared in Example 1 successfully achieved berberine loading.

[0045] The UV absorption spectrum of BBR, BBR@MPN-NGs, MPN-NGs was measured, and the results are shown in FIG. 1. As shown, BBR has characteristic absorption peaks at 346 nm and 421 nm, MPN-NGs has no characteristic absorption peaks at this point, and BBR@MPN-NGs also has a wide absorption peak at 346 nm and 421 nm, proving that BBR is successfully loaded into the multi-network nanogel. Figure 3

[0046] Example 2

[0047] The present embodiment provides a preparation method of a multi-network metal polyphenol nanogel loaded with an anti-inflammatory active ingredient, and the specific steps are as follows: Step 1: Preparation of nanogel network Dissolve chitosan in 1% acetic acid solution to prepare solution A with a concentration of 15 mg / mL. Prepare aldehyde-terminated polyethylene glycol in ethanol solution B with a concentration of 22 mg / mL. Mix solutions A and B in 1 mL of deionized water, control the mass ratio of carboxymethyl chitosan and aldehyde-terminated polyethylene glycol to be 1:1, and incubate at 37°C for 30 minutes to undergo Schiff base reaction and crosslinking to form a nanogel network.

[0048] Step 2: Construction of metal polyphenol network, loading berberine and tea polyphenols Dissolve tea polyphenols (EGCG) in 50% ethanol solution with a concentration of 20 mg / mL, and slowly add the tea polyphenol solution and 3 mg / mL berberine (BBR) dimethyl sulfoxide solution to 1 mL of deionized water, control the mass ratio of tea polyphenols and berberine to be 1:0.125, and stir uniformly at a stirring rate of 500 rpm. Then add 40 μL of 10 mg / mL zinc nitrate hexahydrate solution, adjust the pH of the system to 7.0 with 0.1 M sodium hydroxide solution, and continue stirring at 37°C and a stirring rate of 500 rpm for 10 minutes to form a metal polyphenol network loaded with berberine.

[0049] Step 3: Construction of metal polyphenol multi-network nanogel Slowly add the metal polyphenol network loaded with berberine obtained in step 2 to the nanogel network solution obtained in step 1 drop by drop, stir at room temperature for 10 minutes, and obtain a yellow, uniform, and transparent solution, indicating that the metal polyphenol multi-network nanogel loaded with berberine is successfully formed.

[0050] Centrifuge the metal polyphenol multi-network nanogel loaded with berberine and wash with deionized water, repeat 3 times, resuspend the yellow precipitate at the bottom with deionized water, and store at 4°C for long-term preservation. The centrifugation conditions are 4°C, 10000 rpm, and 15 min each time. ​

[0051] Example 3

[0052] The present embodiment provides a method for preparing a multi-network metal polyphenol nanogel loaded with anti-inflammatory active ingredients, and the specific steps are as follows: Step 1: Preparation of nanogel network Carboxymethyl chitosan was dissolved in deionized water at 60-80°C to prepare solution A with a concentration of 15 mg / mL. Aldehyde-terminated polyethylene glycol was configured into solution B with a concentration of 22 mg / mL in ethanol. Solutions A and B were mixed in 2 mL of deionized water, and the mass ratio of carboxymethyl chitosan to aldehyde-terminated polyethylene glycol was controlled at 1:1. The mixture was incubated at 37°C for 30 minutes to undergo Schiff base reaction and crosslinking to form a nanogel network.

[0053] Step 2: Loading of rhein and tannic acid A tannic acid aqueous solution with a concentration of 20 mg / mL and a rhein dimethyl sulfoxide solution with a concentration of 5 mg / mL were slowly added to 5 mL of deionized water, and the mass ratio of tannic acid to berberine was controlled at 1:0.15. The mixture was stirred uniformly at a speed of 500 rpm. Subsequently, the nanogel network obtained in step 1 was slowly added dropwise, and the stirring was continued at 37°C and a stirring speed of 500 rpm for 10 minutes to form a polyphenol nanogel network loaded with rhein.

[0054] Step 3: Construction of metal polyphenol multi-network nanogel 200 μL of a zinc nitrate hexahydrate solution with a concentration of 10 mg / mL was quickly added to the polyphenol nanogel network loaded with rhein obtained in step 2. The pH of the system was adjusted to 7.0 with a 0.1M sodium hydroxide solution, and the mixture was stirred at room temperature for 10 minutes to obtain a yellow, uniform and transparent solution, indicating that the metal polyphenol multi-network nanogel loaded with rhein was successfully formed. The metal polyphenol multi-network nanogel loaded with rhein was centrifuged and washed with deionized water, and the process was repeated three times. The yellow precipitate at the bottom was resuspended in deionized water and stored at 4°C for long-term preservation. The centrifugation conditions were 4°C, 10000 rpm and 15 min.

[0055] Test Example 1 The present test example tests the stability of the metal polyphenol multi-network nanogel loaded with berberine (BBR@MPN-NGs), and the specific steps are as follows: The BBR@MPN-NGs prepared in Example 1 were stored at 4°C, and a portion was taken out every month. The sample was diluted with ultrapure water to a concentration of 100 μg / mL, and then added to a micro-volume measuring cell. The particle size and polydispersity index of the sample were detected using a dynamic light scattering particle size analyzer, and the results are shown in Table 1. Figure 4As shown, the particle size of BBR@MPN-NGs remained stable at around 220 nm over a one-year period, without any significant changes.

[0056] Following the method in Example 1, a series of metal polyphenol multi-network nanogel solutions loaded with berberine were prepared with tannin concentrations of 1 μg / mL, 2.5 μg / mL, 5 μg / mL and 10 μg / mL.

[0057] Weigh 2 mg of DPPH (1,1-diphenyl-2-trinitrophenylhydrazine) and dissolve it in 50 mL of anhydrous ethanol. Mix the DPPH solution with an equal volume of the above-mentioned nanogel solutions with different tannic acid concentrations, incubate at 37°C in the dark for 30 min, and measure the absorbance at 517 nm to calculate the scavenging efficiency of the nanogel on DPPH free radicals. After storing the nanogel solutions with different tannic acid concentrations for 1 year, repeat the experiment to calculate the scavenging efficiency of the nanogel on DPPH free radicals. The results are as follows: Figure 5 As shown, there was no difference in the scavenging efficiency of the nanogel for DPPH free radicals before and after one year of storage, proving that the metal polyphenol multi-network nanogel loaded with berberine has excellent stability.

[0058] Test Example 2 This test example demonstrates the corrosion resistance of the berberine-loaded metal polyphenol multi-network nanogel (BBR@MPN-NGs). Specifically: Single Escherichia coli colonies were inoculated into LB medium at 37°C, shaken overnight, diluted with medium, and cultured to the logarithmic growth phase. The E. coli were then quantitatively diluted to 1×10⁻⁶ using UV light. 6 Add CFU / mL of berberine-loaded multi-network metal polyphenol nanogel or an equal volume of deionized water to the bacterial culture at a tannic acid concentration of 4 mg / mL. Incubate with the bacterial culture for 12 hours, then place the culture in solid LB medium and incubate overnight. Observe colony formation. Figure 6 As shown, the E. coli group with only deionized water solution grew a large number of colonies, while no colonies were generated after adding BBR@MPN-NGs, with an antibacterial effect of 99.99%, proving that the multi-network metal polyphenol nanogel loaded with berberine has excellent antibacterial properties.

[0059] Test Example 3 This test example demonstrates the bioadhesion properties of the berberine-loaded metal polyphenol multi-network nanogel (BBR@MPN-NGs). Specifically: ①Preparation of liposomes (C6@lipid) encapsulating fluorescent small molecule coumarin 6.

[0060] ② Following the method in Example 1, a metal polyphenol multi-network nanogel (C6@MPN-NGs(CHO)) loaded with fluorescent small molecule coumarin 6 was prepared.

[0061] ③ Replace the aldehyde-terminated polyethylene glycol with methoxy polyethylene glycol to prepare a Schiff base-free nanogel (C6@MPN-NGs(OCH3)) loaded with the fluorescent small molecule coumarin 6.

[0062] Logarithmic growth phase corneal epithelial cells were seeded at a rate of 10,000 cells / well in 96-well plates and incubated overnight for adhesion. The old culture medium was discarded, and the three types of nanoparticles (containing C6 fixed at a concentration of 0.1 μg / mL) diluted with complete culture medium were added. The nanoparticles were incubated with corneal epithelial cells for 1 minute, washed three times with PBS, and the fluorescence of the fluorescent molecule coumarin 6 in the cells was observed under a fluorescence microscope. The results are as follows: Figure 7 As shown, compared with C6@lipid and C6@MPN-NGs(OCH3), C6@MPN-NGs(CHO) exhibits the strongest fluorescence due to the synergistic effect of tannic acid and Schiff base bonds, indicating the strongest adhesion to cells.

[0063] Test Example 4 This test example examines the antioxidant properties of the berberine-loaded metal polyphenol multi-network nanogel (BBR@MPN-NGs). Specifically: Corneal epithelial cells were seeded at 10,000 cells / well in 96-well plates and incubated overnight for adhesion. Then, the following were added: ① A complete culture medium free of H2O2, berberine, and metal polyphenol multi-network nanogels; ② H2O2 with a concentration of 300 μM; ③ A mixture of 300 μM H2O2 and 12.5 μg / mL tannic acid metal polyphenol multi-network nanogels MPN-NGs prepared according to Comparative Example 1. ④ A mixed liquid of 300 μM H2O2 and 3.5 μM berberine; ⑤ A mixture of 300 μM H2O2 and BBR@MPN-NGs metal polyphenol multi-network nanogels loaded with berberine, prepared according to Example 1, with a tannic acid concentration of 12.5 μg / mL and a berberine concentration of 3.5 μM. After incubation for 2 hours, DCFH-DA was incubated in serum-free medium at 7°C in the dark for 30 minutes. Fluorescence images were then acquired under a fluorescence microscope, as shown below. Figure 8 As shown in the figure, with a scale bar of 50 μm, it can be seen that the ROS produced by the BBR@MPN-NGs group cells is significantly reduced.

[0064] Corneal epithelial cells were seeded and incubated in the same manner, and after incubation for 2 hours with four different materials, MTT was added to compare the rescue effects of different materials on H2O2-induced oxidative damage. Figure 9 As shown, tannic acid in the MPN-NGs group has antioxidant activity, and berberine in the berberine group has anti-inflammatory activity, both of which alleviated cell apoptosis caused by H2O2 oxidative damage to a certain extent. However, compared with MPN-NGs and free berberine, the BBR@MPN-NGs group synergistically exerted the anti-inflammatory and antioxidant effects of both, and its ability to rescue H2O2-induced oxidative damage to cells was the strongest, showing the best anti-apoptotic effect. This proves that the metal polyphenol multi-network nanogel loaded with berberine has strong anti-inflammatory and antioxidant activity.

[0065] Test Example 5 This test example examines the anti-inflammatory properties of the berberine-loaded metal polyphenol multi-network nanogel (BBR@MPN-NGs). Specifically: An osmotic pressure of 500 mOs / mL was used in corneal epithelial cells. -1 Hypertonic medium was used to induce the production of excessive reactive oxygen species, and then the cells were cultured overnight in 12-well plates. Subsequently, the following were added to the hypertonic medium: BBR concentration of 3.5 μM in nanogel and tannic acid concentration of 12.5 μg / mL.

[0066] ① A complete culture medium without hypertonic medium, berberine, or metal polyphenol multi-network nanogel was used as the control group; ② A culture medium containing 70 mM NaCl simulates a hyperosmotic environment (HS) with an osmotic pressure of 450 mOsm; ③ Berberine at a concentration of 3.5 μM; ④ The metal polyphenol multi-network nanogel MPN-NGs with a tannic acid concentration of 12.5 μg / mL prepared in Comparative Example 1; ⑤ The metal polyphenol multi-network nanogel BBR@MPN-NGs loaded with berberine, with a tannic acid concentration of 12.5 μg / mL and a berberine concentration of 3.5 μM, prepared according to Example 1; After 24 hours of incubation, RNA and protein were extracted and subjected to qPCR detection, such as... Figure 10 As shown, the transcriptional levels of inflammatory factors IL-1β, IL-6 and MMP-9 in corneal epithelial cells stimulated with hypertonic medium were significantly increased. Compared with BBR and MPN-NGs, BBR@MPN-NGs showed superior anti-inflammatory effects.

[0067] Test Example 6 This embodiment uses the berberine-loaded metal polyphenol multi-network nanogel (BBR@MPN-NGs) to prepare a liquid drug for treating dry eye syndrome, and tests its therapeutic effect on moderate to severe dry eye syndrome: (1) Ocular surface fluorescein sodium score: A benzalkonium chloride (BAC)-induced dry eye model was used in C57BL / 6 mice. For 7 consecutive days, 0.2% BAC eye drops were instilled into the right eye of each mouse. After successful modeling, the mice were treated with 5 μL of PBS, BBR, MPN-NGs and BBR@MPN-NGs eye drops at 9 am and 9 pm each time. The concentration of BBR was 200 μM and the concentration of tannic acid was 700 μg / mL.

[0068] Four days after treatment, the therapeutic effect was verified using clinical assessment methods for dry eye syndrome. Fluorescein sodium solution was instilled into the lower conjunctival sac and eyelids of mice, and fluorescein-stained images were captured under cobalt blue light using a slit-lamp microscope to assess the formation of corneal epithelial cell lesions. Figure 11 As shown, the PBS group showed deeper fluorescein staining, which persisted until day 4. Compared with MPN-NGs and BBR, mice using BBR@MPN-NGs eye drops showed significantly lighter fluorescein staining on day 2, and by day 5, the corneas of the mice were completely free of fluorescein staining, demonstrating that BBR@MPN-NGs has excellent therapeutic effects on BAC-induced moderate to severe dry eye syndrome.

[0069] (2) Moisturizing and Tear Film Stability Test: Tear secretion and tear film breakup time were measured after treatment to assess tear film stability. A phenol red cotton thread was placed on the lower eyelid conjunctiva at a distance of 1 / 3 from the outer canthus for 15 seconds, and the length of the wetted portion of the thread was measured with calipers to measure tear secretion. Under cobalt blue light, a slit-lamp imaging system was used for observation. Timing was stopped when the first tear film breakup point appeared, and the tear film breakup time was recorded to assess tear film stability. Figure 12 As shown, mice treated with PBS alone had a phenol red cotton thread length of less than 2 mm for tear secretion. Compared to MPN-NGs and BBR, mice treated with BBR@MPN-NGs eye drops had a phenol red cotton thread length exceeding 5 mm on day 4, similar to normal mice, demonstrating that BBR@MPN-NGs can effectively treat dry eye and increase tear secretion in mice. Furthermore, BBR@MPN-NGs also effectively prolonged tear film breakup time in mice, significantly better than the control group, indicating that BBR@MPN-NGs can effectively moisturize and maintain tear film stability.

Claims

1. A multi-network metal polyphenol nanogel loaded with an anti-inflammatory active ingredient, characterized in that: The nanogel network, the polyphenol compound with antioxidant activity, the metal cation and the anti-inflammatory active ingredient are included. The nanogel network is formed by cross-linking aldehyde-terminated polyethylene glycol and the amino-containing macromolecular material polymer through Schiff base reaction.

2. The multi-network metal polyphenol nanogel loaded with an anti-inflammatory active ingredient as claimed in claim 1, wherein: The polyphenol compound is one or more of tannic acid, tea polyphenol, gallic acid, ellagic acid or chlorogenic acid.

3. The multi-network metal polyphenol nanogel loaded with an anti-inflammatory active ingredient as claimed in claim 1, wherein: the metal cation is one or more of Zn 2+ , Fe 3+ , Cu 2+ .

4. The multi-network metal polyphenol nanogel loaded with an anti-inflammatory active ingredient as claimed in claim 1, wherein: The anti-inflammatory active ingredient is berberine.

5. The multi-network metal polyphenol nanogel loaded with an anti-inflammatory active ingredient as claimed in claim 1, wherein: The amino-containing macromolecular material is carboxymethyl chitosan.

6. The multi-network metal polyphenol nanogel loaded with an anti-inflammatory active ingredient as claimed in claim 1, wherein: The mass percentage of the polyphenol compound is 10-30%, the mass percentage of the metal cation is 5-20%, the mass percentage of the carboxymethyl chitosan is 20-40%, the mass percentage of the aldehyde-terminated polyethylene glycol is 20-40%, and the mass percentage of the anti-inflammatory active ingredient is 0.2-5%.

7. The method of claim 1-6, wherein the preparation of the multi-network metal polyphenol nanogel loaded with anti-inflammatory active ingredients is characterized by: The solution A of the amino-containing macromolecular material is mixed with the solution B of the aldehyde-terminated polyethylene glycol to make the amino-containing macromolecular material and the aldehyde-terminated polyethylene glycol react through Schiff base reaction to cross-link and form the nanogel network; the polyphenol compound and the anti-inflammatory active ingredient are added to the obtained nanogel network to form the polyphenol nanogel network loaded with the anti-inflammatory active ingredient; and finally, the metal salt aqueous solution is added to form the uniform and transparent metal polyphenol multi-network nanogel.

8. The method for preparing a multi-network metal polyphenol nanogel loaded with anti-inflammatory active ingredients as described in claim 7, characterized in that: The specific steps are as follows: Step 1: The amino-containing macromolecular material is added to deionized water to prepare the solution A with a concentration of 15 mg / mL; the aldehyde-terminated polyethylene glycol is dissolved in an ethanol solution to prepare the solution B with a concentration of 22 mg / mL; the solutions A and B are added to 2 mL of deionized water to mix, so that the mass ratio of the amino-containing macromolecular material to the aldehyde-terminated polyethylene glycol is 1:1, and the nanogel network is cross-linked; Step 2: The polyphenol compound is dissolved in deionized water to prepare the solution C with a concentration of 20 mg / mL; the anti-inflammatory active ingredient is dissolved in dimethyl sulfoxide to prepare the solution D with a concentration of 3 mg / mL; the solutions C and D are sequentially and slowly added to 5 mL of deionized water and stirred uniformly, and the mass ratio of the polyphenol compound to the anti-inflammatory active ingredient is 1:(0.02-0.2); and then the nanogel network obtained in step 1 is added, and the stirring is continued to form the polyphenol nanogel network loaded with the anti-inflammatory active ingredient; Step 3: The metal salt aqueous solution is prepared, slowly added to the polyphenol nanogel network loaded with the anti-inflammatory active ingredient, so that the mass ratio of the metal cation to the polyphenol compound is 1:(0.5-2), and the pH of the system is adjusted to 6.5-7.5, and the stirring is continued to form the uniform and transparent metal polyphenol multi-network nanogel.

9. The use of the multi-network metal polyphenol nanogel loaded with the anti-inflammatory active ingredient according to any one of claims 1-6 in the preparation of a liquid medicine for treating ocular surface inflammatory diseases and bacterial infectious diseases.

10. The use of the multi-network metal polyphenol nanogel loaded with the anti-inflammatory active ingredient according to any one of claims 1-6 in the preparation of a liquid medicine for treating dry eye, bacterial infectious keratoconjunctivitis, dry eye associated conjunctivitis and blepharitis.

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