Preparation of light-operated adhesion anti-oxidation, anti-inflammatory and anti-fibrosis hydrogel implant and application of light-operated adhesion anti-oxidation, anti-inflammatory and anti-fibrosis hydrogel implant in resisting glaucoma postoperative scars

Abstract

The invention discloses preparation of a light-operated adhesion anti-oxidation, anti-inflammatory and anti-fibrosis hydrogel implant and application of the light-operated adhesion anti-oxidation, anti-inflammatory and anti-fibrosis hydrogel implant in resisting glaucoma postoperative scars. The hydrogel disclosed by the invention is composed of a carboxymethyl chitosan derivative formed by modifying carboxymethyl chitosan with phenylboronic acid and o-nitrobenzyl alcohol respectively, and a polyphenol anti-inflammatory and antioxidant drug, wherein the phenylboronic acid modified carboxymethyl chitosan derivative and the polyphenol drug form a phenylboronic acid ester bond with active oxygen response; the o-nitrobenzyl alcohol modified carboxymethyl chitosan derivative is triggered by ultraviolet light and can form a dynamic covalent imine bond with an amino group on a tissue or a polymer. The hydrogel provided by the invention can enhance the adhesion to conjunctiva through light triggering at a glaucoma filtration wound part, and the viscous implant can realize an anti-fiber effect. In addition, the anti-inflammatory and anti-oxidation medicine loaded in the hydrogel can respond to wound active oxygen release, and postoperative wound oxidative stress and inflammatory response are relieved.

Description

Technical Field

[0001] The present invention belongs to the technical field of biomedical materials, and relates to the preparation of a conjunctival light-controlled adhesion antioxidant, anti-inflammatory and anti-fibrotic hydrogel and its application in preventing scar after glaucoma surgery. Background Art

[0002] Glaucoma refers to visual dysfunction caused by pathological high intraocular pressure or insufficient perfusion pressure. The normal intraocular pressure is between 10 and 21 mmHg. If it is between 21 and 24 mmHg, it is glaucoma.

[0003] Intraocular pressure (IOP, normal range 10 - 21 mm Hg) is regulated by the balance between aqueous humor secretion by the ciliary body in the posterior chamber and aqueous humor drainage through the anterior chamber angle, via the trabecular meshwork and Schlemm's canal, or through the scleral outflow pathway that enters the scleral interface at the iris root. Elevated IOP is the most important risk factor for glaucoma, and treatment to reduce IOP is based on topical medications, laser therapy, and surgical intervention. Topical medications have the problem of low bioavailability. In addition, frequent dosing can also lead to poor patient compliance; the amplitude of IOP reduction by laser is limited. If neither of the above two methods can reduce IOP to the target IOP, surgical intervention is required. The most widely used surgery clinically is trabeculectomy, and its basic principle is to drain aqueous humor into the subconjunctival space. However, scar formation in the subconjunctiva often leads to surgical failure, and the surgical failure rate caused by scarring of the postoperative filtration opening still reaches 15% - 35%. The main reason for scarring after glaucoma surgery is excessive wound healing in the filtration bleb area, resulting in fibrotic proliferation that blocks the filtration pathway. Currently, the main means of preventing scarring after trabeculectomy clinically are medical treatments, such as antimetabolic drugs, combined with auxiliary means such as using barrier materials, improving surgical techniques, and correct postoperative massage. Antimetabolic drugs inhibit fibroblast proliferation by interfering with DNA replication, such as mitomycin C and 5-fluorouracil, but they can cause a series of serious complications, such as low IOP, bleb leakage, accelerated progression of cataract, choroidal effusion, and hemorrhage. Barrier materials include biodegradable collagen matrices, hyaluronic acid gels, cross-linked sodium hyaluronate bioadhesives, amniotic membranes, bio-visiosheets, etc. The Ologen implant, a disk-shaped biodegradable collagen matrix derived from pigs, can be used to prevent excessive scar formation after glaucoma filtration surgery. However, this implant has no antifibrotic properties, has a lower success rate compared to mitomycin C, and has a risk of adding ocular inflammation. Healaflow is a non-pyrogenic, viscoelastic, colorless, transparent, large-molecular-weight isotonic colloidal solution composed of non-animal-derived reticular sodium hyaluronate, used for antifibrosis after trabeculectomy to improve the survival rate of filtration blebs, but complications such as shallow anterior chamber still occur. Intraoperative implantation of the cross-linked sodium hyaluronate bioadhesive SK can reduce postoperative scarring, but the indication of SK bioadhesive is narrow, the surgical difficulty is high, and the price is expensive. Amniotic membrane transplantation can maintain the physiological state of the bleb while preserving the integrity of the overlying conjunctiva. When combined with trabeculectomy, it can significantly improve the surgical success rate and show advantages such as stable IOP control and low postoperative complication incidence. Another biological barrier material - bio-visiosheet, is a membrane extracted from bovine tendon, and its main components are type I collagen and collagen glucosamine polymer. This material improves the surgical efficacy by inhibiting postoperative scar formation. However, both amniotic membranes and bio-visiosheets of biological origin face problems such as difficult material acquisition and potential biogenicity.Research scholars at home and abroad mainly focus on improving the efficacy of drug treatment and enhancing the barrier performance in the prevention and treatment of postoperative scarring after trabeculectomy. Starting from the molecular mechanism of scarring, they regulate the dynamic balance of wound healing, aqueous humor drainage, and fibrosis. However, while improving the gel barrier performance and operability, there are relatively few studies on deeply clarifying the mechanism of postoperative scarring after glaucoma filtration surgery and "intelligently responding" to prevent and control it from the perspective of the disease's occurrence and development mechanism.

[0004] Currently, there is no means or drug that can perfectly inhibit postoperative scarring without causing serious complications. Therefore, developing a drug or means with good biosecurity to inhibit postoperative scarring, improving the success rate of glaucoma filtration surgery, and thus improving the quality of life of patients is an urgent problem to be solved clinically. Combining the current treatment status of postoperative scarring and deeply clarifying its disease mechanism, this study not only improves the gel barrier performance and biocompatibility but also provides new ideas for the treatment means and treatment mode of preventing postoperative scarring by preventing and controlling it from the perspective of the disease's occurrence and development mechanism. Summary of the Invention

[0005] Based on the above situation, the present invention aims to provide a preparation method and application of an anti-scar multifunctional hydrogel for glaucoma filtration surgery. The hydrogel is prepared by crosslinking with dynamic imine bonds and borate ester bonds, and can adhere to the subconjunctival wound site of the filtering bleb through ultraviolet light triggering; the antioxidant, anti-inflammatory, and anti-fibrotic drugs loaded by the borate ester bonds in the hydrogel can respond to the release of reactive oxygen species in the wound microenvironment, intelligently relieve postoperative oxidative stress and inflammatory responses, effectively inhibit the colonization of inflammatory cells at the injury site, and can inhibit the transformation of colon fibroblasts into myofibroblasts, effectively alleviating the subconjunctival fibrosis reaction.

[0006] To achieve the above object, the technical solution adopted by the present invention is:

[0007] The preparation of a conjunctival photo-controlled adhesion antioxidant, anti-inflammatory, and anti-fibrotic hydrogel, characterized in that it consists of two carboxymethyl chitosan derivatives (CS-NB, CS-PBA) and a polyphenol drug. The structural feature of CS-NB is that the amino group of CS is covalently modified with o-nitrobenzyl alcohol, and the structural feature of CS-PBA is that the amino group of CS is covalently modified with 3-fluorobenzeneboronic acid. After mixing CS-NB, CS-PBA, and the polyphenol drug, the polyphenol drug can form dynamic borate ester bonds with the benzeneboronic acid in CS-PBA. After in-situ ultraviolet light irradiation, CS-NB becomes CS-ONB, and the aldehyde group then reacts with the amino group on CS or the amino group on the tissue to form dynamic covalent amide bonds, and the nitroso group reacts with the sulfhydryl group on the tissue to generate S-N bonds, realizing the synchronous improvement of in-situ adhesion of the conjunctiva and mechanical properties.

[0008] x, y, and z are natural numbers, and x + y + z is a natural number between 20 and 200.

[0009]

[0010] The polyphenol antioxidant, anti-inflammatory, and anti-fibrotic drug is one of epigallocatechin gallate, epicatechin gallate, catechin gallate, and tannic acid; the grafting rate of NB in CS-NB is 2% - 20%, and the grafting rate of PBA in CS-PBA is 2% - 20%. The concentration of drug D is 0.8% - 5%; the wavelength of the ultraviolet light is 365 nm - 405 nm, and the power is 10 w - 30 w. The mixing method of CS-NB, CS-PBA, and D is mixing before use, where the volume ratio of CS-NB to CS-PBA is 2:1, 1:1, and 1:2, and the volume of drug D accounts for 1 / 10 of the total volume after mixing.

[0011] The present invention provides a method for preparing a conjunctival light-controlled adhesion antioxidant, anti-inflammatory, and anti-fibrotic hydrogel, comprising the following steps:

[0012] Step 1, synthesize the ultraviolet light-sensitive small molecule NB;

[0013]

[0014] Step 2, use carboxymethyl chitosan CS and NB as raw materials to synthesize CS-NB through an amide condensation reaction;

[0015]

[0016] m and n are natural numbers, and m + n is a natural number between 20 and 200; x, y, and z are natural numbers, and x + y + z is a natural number between 20 and 200.

[0017] Step 3, use carboxymethyl chitosan CS and PBA as raw materials to synthesize CS-PBA through an amide condensation reaction;

[0018]

[0019] m and n are natural numbers, and m + n is a natural number between 20 and 200; x, y, and z are natural numbers, and x + y + z is a natural number between 20 and 200.

[0020] Step 4, dissolve CS-NB, CS-PBA, and drug D in an aqueous medium to form solutions (pH = 7.4), and then mix the three;

[0021] Step 5: Inject the hydrogel obtained by mixing in Step 4 into the subconjunctival area of glaucoma filtration surgery. After fully spreading it flat, trigger it with ultraviolet light to obtain an antioxidant, anti-inflammatory, and anti-fibrotic hydrogel implant.

[0022] In some embodiments, the concentration of CS-NB is 1.25% - 5%; the concentration of CS-PBA is 2.5% - 7.5%; the concentration of drug D is 0.8% - 5%; the wavelength of the ultraviolet light is 365nm - 405nm, and the power is 10w - 30w. The ultraviolet light irradiation time is 10 - 30s. The mixing method is to mix CS-NB, CS-PBA, and drug D before application, and irradiate with ultraviolet light in situ after injection into the subconjunctiva.

[0023] Another object of the present invention is to provide the use of the above-mentioned light-controlled adhesion antioxidant, anti-inflammatory, and anti-fibrotic hydrogel in preventing scarring after glaucoma surgery.

[0024] In some embodiments, the glaucoma surgery includes one or more of trabeculectomy and non-penetrating trabecular surgery.

[0025] Compared with the prior art, the present invention has the following beneficial effects:

[0026] (1) The hydrogel provided by the present invention can adhere in situ to the traumatized conjunctival site, effectively inhibiting the colonization and proliferation of inflammatory cells; the hydrogel can intelligently respond to the reactive oxygen species level generated by the surgical trauma microenvironment after glaucoma surgery, and release natural polyphenol antioxidant, anti-inflammatory, and anti-fibrotic drugs as needed to scavenge reactive oxygen species, effectively alleviating postoperative oxidative stress, inflammatory response, and fibroblast proliferation reaction; it overcomes the limitations of the need for repeated drug administration and the difficulty in determining the drug dosage when using drugs alone, also overcomes the problem of uncontrollable release of drugs encapsulated in the gel, and further overcomes the problem that the existing gel materials in clinical practice only play an auxiliary therapeutic role and do not prevent scarring from the mechanism of scar formation.

[0027] (2) After glaucoma filtration surgery, fibroblasts at the filtration bleb site are activated under the stimulation of inflammation or injury, and then transformed into synthetically active myofibroblasts. These myofibroblasts can proliferate rapidly and simultaneously synthesize and secrete a large amount of extracellular matrix (ECM) components, such as collagen (Col-1). The excessive deposition and cross-linking of these ECMs are the main causes of scarring. The hydrogel provided by the present invention can slowly release anti-fibrotic polyphenol drugs, reduce the activation of fibroblasts, and effectively inhibit the fibrotic reaction.

[0028] (3) Reactive oxygen species usually increase in the postoperative traumatic microenvironment. The hydrogel crosslinks polyphenolic drugs through borate ester bonds, which are ROS-sensitive chemical groups. Under normal physiological conditions, the polyphenolic drugs are stably encapsulated in the material; while at the inflammatory site (where the ROS concentration is relatively high), the borate ester bonds respond and break, releasing the polyphenolic drugs, thus achieving targeted therapy.

[0029] (4) The hydrogel provided by the present invention can adhere in situ at the traumatic conjunctiva site, effectively cover the tissue wound surface, has good mechanical strength, and can effectively exert antioxidant, anti-inflammatory and anti-fibrotic functions during the critical period (within 14 days) of scar formation after glaucoma surgery. Description of the Drawings

[0030] The following further elaborates on the specific implementation manners of the present invention in conjunction with the drawings, where

[0031] Figure 1 are the NMR spectrum (A) of NB and the UV spectra (B) obtained by UV irradiation at different times in Example 1.

[0032] Figure 2 are the NMR spectrum (A) of CS-NB and the UV spectrum (B) of CS-NB in Example 2.

[0033] Figure 3 are the NMR spectrum (A) of CS-PBA and the UV spectrum (B) of CS-PBA in Example 3.

[0034] Figure 4 are the adhesiveness (A), injectability (B), self-healing macroscopic view (C) of the PNEU gel and the adhesion strength of the gel to the conjunctiva (D) in Example 4.

[0035] Figure 5 is the infrared spectrum of the PNEU gel in Example 4.

[0036] Figure 6 are the strain sweep curves (frequency 5 Hz, strain range 0.1%-100%) and time sweep curves (B) (frequency 5 Hz, strain 10%, time 3 min) of the PNU, PNE, and PNEU hydrogels in Comparative Example 1.

[0037] Figure 7 are the cumulative release curves of the PNU, PNE, and PNEU hydrogels in PBS (pH 7.4) buffer containing different concentrations of H2O2 (0, 200 μM, 1 mM, 5 mM) in Example 5.

[0038] Figure 8 are the abilities of the PNU, PNE, and PNEU hydrogels to scavenge (A) DPPH and (B) ·OH in Example 6.

[0039] Figure 9 NO levels (A) and relative expression levels of iNOS mRNA (B) after incubation of PNU, PNE, and PNEU hydrogel extracts with RAW 264.7 cells in Example 7.

[0040] Figure 10 Relative expression levels of α-SMA and Col-1 mRNA (A, B) after incubation of PNU, PNE, and PNEU hydrogel extracts with RCFs cells in Example 7.

[0041] Figure 11 Observation results of filtering blebs (A), intraocular pressure changes (B), survival curves of filtering blebs (C), and hematoxylin-eosin staining results (D) at 28 days after surgery in Example 8. Detailed implementation manners

[0042] The preferred embodiments of the present invention will be described in detail below with reference to the accompanying drawings. It should be understood that the preferred embodiments described herein are only for the purpose of illustrating and explaining the present invention, and are not intended to limit the present invention.

[0043] The raw materials and equipment used in the present invention are all known products and are obtained by purchasing commercially available products.

[0044] Example 1: Synthesis of ultraviolet light-sensitive NB small molecules:

[0045] Step 1: Vanillin (4.45 g, 29.25 mmol), methyl 4-bromobutyrate (4.95 g, 27.5 mmol), and potassium carbonate (5.10 g, 36.90 mmol) were dissolved in 20 mL of DMF and stirred at room temperature for 24 h. The reaction solution was poured into 300 mL of ice water, allowed to stand and precipitate at 0 °C for 15 min, filtered, and the filter cake was washed with water. The crude product was dissolved in dichloromethane, dried over anhydrous magnesium sulfate, and concentrated under reduced pressure to obtain a white solid intermediate 2. Spin-dry

[0046] Step 2: Intermediate 2 (4.70 g, 18.65 mmol) was slowly added to a mixture of potassium nitrate / trifluoroacetic acid (20 mL) at 0 °C, and the reaction was carried out at 0 °C for 1 h. After the reaction was completed, the mixture was concentrated under reduced pressure to obtain a brownish-yellow oily product, which was redissolved in ethyl acetate and extracted 3 times with saturated NaCl aqueous solution. The organic layers were combined and concentrated under reduced pressure to obtain a pale yellow oily intermediate 3.

[0047] Step 3: At 0 °C, sodium borohydride (2.0 g, 52.97 mmol) was slowly added to a mixed solution of intermediate 3 (3.85 g, 12.95 mmol) in ethanol / tetrahydrofuran (1:1 v / v, 50 mL), and the reaction was carried out for 3 h. After concentration under reduced pressure, the resulting product was redissolved in a mixed solution of water / dichloromethane (1:1 v / v, 100 mL). The aqueous phase was extracted twice with dichloromethane (50 mL each time), and the combined organic layers were dried over anhydrous magnesium sulfate. After concentration under reduced pressure, 100 mL of methanol and 50 mg of p-toluenesulfonic acid were added to the residue, and stirring was continued at room temperature for 12 h. After completion, the solvent was removed by concentration under reduced pressure, and the residue was redissolved in a mixed solution of water / dichloromethane (1:1 v / v, 50 mL). The aqueous phase was extracted twice with dichloromethane (25 mL each time), and the combined organic layers were dried over anhydrous magnesium sulfate. After concentration under reduced pressure, a crude yellow solid was obtained. It was purified by silica gel column chromatography (using hexane / ethyl acetate = 1:1 as the eluent) to obtain a pale yellow powder of intermediate 4.

[0048] Step 4: Intermediate 4 (1.5 g, 5.4 mmol) and NaOH (1 g, 25.00 mmol) were dissolved in tetrahydrofuran / water (1:4 v / v, a total of 20 mL), and the reaction was carried out for 2 h. The pH was adjusted to 5 with 0.1 mmol of hydrochloric acid, and a pale yellow precipitate of intermediate 5 was precipitated. After suction filtration, the filter cake was redissolved in a mixed solution of dichloromethane / methanol (1:1 v / v, a total of 20 mL), and concentrated under reduced pressure to obtain a light yellow powder of NB. The product was characterized by 1H NMR and verified for its UV response function.

[0049] The synthesis reaction formula of NB is as follows:

[0050]

[0051] The experimental results show that ( Figure 1 A), 7.67 ppm and 7.38 ppm are the chemical shifts of two hydrogen atoms of the benzene ring respectively, 4.82 ppm is the chemical shift of two hydrogen atoms of the methylene group on the hydroxyethyl group, 4.07 ppm, 2.40 ppm, and 1.96 ppm are the chemical shifts of two hydrogen atoms of the three methylene groups on butyric acid respectively, and 3.07 ppm is the chemical shift of three hydrogen atoms of the methyl group on the methoxy group. Figure 1 B The results show that after the NB solution was irradiated with UV, it changed from clear and transparent to light yellow, indicating that the chemical structure might have changed. The UV response mechanism of NB was investigated using UV-Vis, and the results are as Figure 1-5As shown, it can be seen that after NB is irradiated with UV, the absorbance intensity of the characteristic absorption peak changes significantly. A new absorption peak appears at 281 nm, which may be the characteristic absorption wavelength generated by the n→π* transition of the aldehyde group (=O), and the intensity of the absorption peak gradually increases with the prolongation of the irradiation time. The absorption peak at 376 nm redshifts. It may be that the nitro group (-NO2) connected to the benzene ring is triggered by UV to turn into a nitroso group (-NO). This change leads to an increase in the conjugated system, resulting in π→π* transition. The system undergoes a hyperchromic effect, the energy of electron transition decreases and the transition probability increases, so it is easier to undergo transition. The characteristic absorption peak at 243 nm represents the π→π* transition of the benzene ring, and the absorption peak at 218 nm may originate from the π→π* transition of the unsaturated structure in the sample.

[0052] Example 2: Synthesis of hydrogel precursor material CS-NB:

[0053] Weigh NB (96.8 mg) and dissolve it in 10 mL of dimethyl sulfoxide, then add 1-(3-dimethylaminopropyl)-3-ethylcarbodiimide hydrochloride (EDCI, 52.68 mg) and N-hydroxysuccinimide (NHS, 39.01 mg), and stir at room temperature for 2 h. Weigh 500 mg of carboxymethyl chitosan (CS) and dissolve it in 100 mL of deionized water, stir at room temperature until completely dissolved, and then add the above-activated reaction solution dropwise and stir at room temperature for 24 h. After the reaction is completed, dialyze the reaction solution with 95% ethanol and pure water at a ratio of 1:3 for one day, and continue to dialyze with pure water for two days. The dialysate is freeze-dried to obtain a yellow-brown product. Perform 1 1H NMR analysis and UV analysis on this product, and use a UV spectrophotometer to calculate the grafting rate.

[0054] The synthesis reaction formula for obtaining CS-NB by grafting o-nitrobenzyl alcohol onto carboxymethyl chitosan is as follows:

[0055]

[0056] The experimental results show ( Figure 2 ) that the characteristic absorption peak of NB appears in CS-NB. The chemical shifts of two hydrogen atoms on the benzene ring of NB are 7.25 ppm and 7.74 ppm respectively, indicating that NB is successfully grafted onto CS. The characteristic absorption peaks of NB are 218 nm, 243 nm, and 350 nm. 218 nm is the conjugation of the benzene ring and the nitro group, 243 nm is the characteristic absorption of the benzene ring, and 350 nm is the n-→π* transition of the nitro group. The corresponding characteristic absorption wavelengths appear in CS-NB, indicating that NB is successfully grafted onto CS. Using 243 nm as the characteristic absorption wavelength, a standard curve of NB is made, and finally the grafting rate of the polymer CS-NB is calculated to be 2% - 20%.

[0057] Example 3: Synthesis of hydrogel precursor material CS-PBA:

[0058] Weigh PBA (62.45 mg) and dissolve it in 3 mL of dimethyl sulfoxide, then add EDCl (52.68 mg) and NHS (39.01 mg), and stir at room temperature for 2 h. Weigh 500 mg of CS and dissolve it in 100 mL of deionized water, stir at room temperature until completely dissolved, and then add the above reaction solution after activation dropwise, and stir at room temperature for 24 h. After the reaction is completed, dialyze the reaction solution with 95% ethanol and pure water at a ratio of 1:3 for one day, and continue to dialyze with pure water for two days. Freeze-dry the dialysis solution to obtain a white product. Perform 1 1H NMR analysis and UV analysis on this product, and calculate the grafting rate using a UV spectrophotometer.

[0059] The synthesis reaction formula for obtaining CS-PBA by grafting phenylboronic acid onto carboxymethyl chitosan is as follows:

[0060]

[0061] The experimental results show ( Figure 3 ) that the characteristic absorption peaks of PBA are 7.53 ppm, 7.61 ppm, and 7.66 ppm respectively. The characteristic absorption peaks of PBA appear in CS-PBA, indicating that PBA has been successfully grafted onto CS. The UV results show that the characteristic absorption wavelengths of PBA appear in CS-PBA, namely 280 nm and 232 nm. Among them, the n→π* transition of the carboxyl group occurs at 280 nm, and the characteristic absorption peak of the π-π* of the benzene ring occurs at 232 nm. Using 232 nm as the characteristic absorption wavelength, a standard curve of PBA is made, and finally the grafting rate of the polymer CS-PBA is calculated to be 2% - 20%.

[0062] Example 4: Synthesis and characterization of PNEU hydrogel:

[0063] Dissolve 12.5 mg, 25 mg, and 50 mg of CS-NB in 1 mL of pH 7.4 PBS buffer solution to prepare 1.25 wt%, 2.5 wt%, and 5 wt% solutions. Dissolve 25 mg, 50 mg, and 75 mg of CS-PBA in 1 mL of pH 7.4 PBS buffer solution to prepare 2.5 wt%, 5 wt%, and 7.5 wt% solutions. Dissolve 8 mg, 20 mg, 30 mg, and 50 mg of epigallocatechin gallate (EGCG) in 1 mL of pH 7.4 PBS buffer solution to prepare 0.8 wt%, 2 wt%, 3 wt%, and 5 wt% solutions. Mix the corresponding concentration CS-NB and CS-PBA solutions (CS-NB + CS-PBA = 1.25 wt% + 2.5 wt%, 2.5 wt% + 5 wt%, 5 wt% + 7.5 wt%) according to the volume ratio (CS-NB:CS-PBA) of 2:1, 1:1, and 1:2 to prepare hydrogels with different ratios, and then mix in 0.1 of the total volume of the EGCG solution. After the above solutions are mixed evenly, trigger with ultraviolet light to obtain PNEU gel. Prepare the other two control gels PNU (without EGCG) and PNE (without ultraviolet light trigger) in the same way.

[0064] According to the same method, use epicatechin gallate, catechin gallate, and tannic acid to replace EGCG to prepare different gel materials.

[0065] Mix the mixed solution of 2.5% CS-NB, 5% CS-PBA, and 5% polyphenol drug evenly and inject it subconjunctivally, and then trigger with ultraviolet light through the conjunctiva. It can quickly adhere in situ under the conjunctiva. Use a rotational rheometer to investigate the adhesion strength of the gel to the conjunctiva. Extrude 2.5% CS-NB, 5% CS-PBA, and 5% polyphenol drug with a syringe to investigate the injectability of the hydrogel before ultraviolet light triggering. Use tools to investigate the macroscopic self-healing properties of the hydrogel. Prepare a mixed solution of 2.5% CS-NB and 5% CS-PBA in a volume ratio of 1:1, and then mix in 0.1 volume ratio of 5% polyphenol drug solution. Trigger with ultraviolet light for 10 s, and then freeze-dry the hydrogel. Use a Fourier transform infrared spectrometer to investigate the chemical bonds of the freeze-dried hydrogel.

[0066] The experimental results show ( Figure 4 ), that the PNEU hydrogel can effectively adhere under the rabbit eye conjunctiva (A), and compared with the PNU and PNE gels, the PNEU gel has the strongest adhesion strength, which is 3.58 kPa (D). This gel has good injectability and self-healing properties (B, C). Investigate the gelation mechanism of PNEU by infrared spectroscopy ( Figure 5 ). Characteristic absorption peaks of epigallocatechin gallate (EGCG) and borate ester bonds appear in PNE and PNEU, including carboxymethyl chitosan CS at 3323 cm-1 The strong and broad peak at this position is the stretching vibration peak of O-H and N-H, and at 2900 cm -1 and at 1323 cm -1 are respectively the stretching vibration peak and the bending vibration absorption peak of C-H. The absorption peak around 1057 cm -1 is the characteristic absorption peak of the ether bond formed by primary alcohol, and the absorption peaks around 1586 cm -1 and 1408 cm -1 are respectively the asymmetric and symmetric stretching vibration peaks of the carboxyl group, indicating the presence of the carboxyl group. The characteristic absorption of EGCG has multiple absorption peaks in the range of 1000 - 1600 cm -1 , and these peaks are related to the in-plane bending vibration of the benzene ring. The enhancement at 1323 cm -1 indicates the vibration of the boric acid group (B-O) of the phenylboronic acid bond. Therefore, the PNEU hydrogel forms a double-crosslinked hydrogel by forming borate bonds and imine bonds.

[0067] Comparative Example 1: Comparison of the mechanical strength between the PNEU hydrogel and the control hydrogel:

[0068] First, mix the 2.5 wt% CS-NB precursor solution and the 5 wt% CS-PBA precursor solution in a volume ratio of 1:1, then add EGCG and mix evenly, and finally perform ultraviolet light irradiation to investigate the rheological properties of the formed PNEU hydrogel. Prepare two single-crosslinked hydrogels, PNU and PNE, as controls according to the same method. The basic parameters of the rheometer are set as follows: the measuring fixture is a cone plate CP20, the gap is 0.5 mm, the test temperature is 37 °C, and the air pressure is 5 bar. The fixed frequency is 5 Hz, and a strain sweep test is performed in the range of 0.1% - 100%; the fixed frequency is 5 Hz, the fixed strain is 1% (within the linear viscoelastic region), and a 3-min time sweep is performed on different gels.

[0069] The experimental results show ( Figure 6 ): For each group of hydrogels, G′ is greater than G″ in the strain range of 1% - 100%, and in the strain range of 1% - 20%, the values remain basically stable, indicating that each group of hydrogels exhibits stable characteristics in the low-strain region. The G′ of PNEU is greater than that of PNU and PNE, and the tan δ value of PNEU is about 0.1, which is the lowest, indicating that it has a higher G′ and a lower G″ to ensure that the material remains in the gel state under low stress ( Figure 6 A). The experimental results show ( Figure 6 B) that the PNEU gel after ultraviolet light triggering has a higher modulus, indicating that chemical bond crosslinking has occurred inside the gel. From the ultraviolet light triggering mechanism of NB small molecules, it can be inferred that more imine bonds are formed inside the PNEU gel after ultraviolet light triggering.

[0070] Comparative Example 2: Comparison of the ability of PNEU hydrogel and control hydrogel to scavenge reactive oxygen species in vitro:

[0071] The rheological properties of the PNEU hydrogel formed by mixing 2.5 wt% CS-NB precursor solution and 5 wt% CS-PBA precursor solution in a volume ratio of 1:1, then adding EGCG and mixing evenly, and finally performing ultraviolet light irradiation. Two single-crosslinked hydrogels, PNU and PNE, were prepared as controls according to the same method.

[0072] Investigate the ability of the hydrogel to scavenge the nitrogen free radical DPPH:

[0073] PNU, PNE, and PNEU hydrogels with different dry weights (2 mg, 4 mg, 8 mg, 16 mg) were immersed in 2 mL of DPPH methanol solution and incubated in the dark at room temperature for 30 min. The absorbance at a wavelength of 517 nm was measured using a microplate reader. The calculation formula for the relative scavenging rate of DPPH is:

[0074]

[0075] Among them, the sample group represents the absorbance of the hydrogel + DPPH, the blank group represents the absorbance of the hydrogel, and the control group represents the absorbance of DPPH.

[0076] The experimental results show ( Figure 7 A): The hydrogel of PNU without EGCG can scavenge DPPH to a certain extent and shows a mass-dependent trend, which may be due to the consumption of DPPH by the phenylboronic acid structure in the hydrogel. While the PNE and PNEU hydrogels can effectively scavenge DPPH at each mass, indicating that the loaded EGCG has a strong ability to scavenge DPPH.

[0077] Investigate the ability of the hydrogel to scavenge ·OH: First, prepare 2.25 mM FeCl2 aqueous solution, 2.25 mM H2O2 aqueous solution, and 2.25 mM salicylic acid ethanol solution. PNU, PNE, and PNEU hydrogels with different dry weights (1.875 mg, 3.75 mg, 7.5 mg, 15 mg) were immersed in the Fenton solution prepared by mixing 1 mL of 2.25 mM FeCl2 aqueous solution and 1 mL of 2.25 mM H2O2 aqueous solution, then 1 mL of 2.25 mM salicylic acid ethanol solution was added, and incubated in the dark at room temperature for 15 min. Then the absorbance at a wavelength of 510 nm was measured using a UV spectrophotometer. The calculation formula for the relative scavenging rate of ·OH is:

[0078]

[0079] Among them, the sample group represents the absorbance of hydrogel + Fenton's reagent + salicylic acid, the blank group represents the absorbance of hydrogel + salicylic acid, and the control group represents the absorbance of Fenton's reagent + salicylic acid.

[0080] The experimental results show that ( Figure 7 B): The hydrogel of PNU without EGCG can scavenge ·OH to a certain extent and shows a mass-dependent trend, which may be due to the consumption of ·OH by the phenylboronic acid structure in the hydrogel. The PNE and PNEU hydrogels can effectively scavenge ·OH at each mass, indicating that the loaded EGCG has a strong scavenging ability for DPPH. However, due to the denser hydrogel of PNEU and the slow release of EGCG, the scavenging effect is between that of PNU and PNE. As the mass increases, the ·OH scavenging rate also gradually increases.

[0081] Example 5: Investigation of the in vitro reactive oxygen species-responsive drug release behavior of the hydrogel:

[0082] Seal the PNEU hydrogel in a dialysis bag with MwCO 3500Da and immerse it in 30 mL of PBS (pH 7.4) containing different

[0083] concentrations of H2O2 (0, 200 μM, 1 mM, 5 mM) respectively, and oscillate at a constant temperature of 37°C. At specific time points, 3 mL of the release medium was taken and 3 mL of fresh release medium was added. The concentration of EGCG in the release medium was detected using a UV-visible spectrophotometer at a detection wavelength of 207 nm, and the cumulative release rate was further calculated.

[0084] The experimental results show that ( Figure 8 ), in the pH 7.4 PBS solution, the cumulative release of EGCG from the hydrogel within 11 days is less than 40%. When containing 200 μM H2O2, the cumulative release rate of EGCG is about 60%. When containing 1 mM H2O2, the cumulative release rate of EGCG is about 75%, and when the H2O2 concentration in the release medium is increased to 5 mM, the cumulative release rate is close to 90%. The above results indicate that when the release medium contains H2O2, the release rate of EGCG is significantly accelerated and increases with the increase of the H2O2 concentration.

[0085] Example 6: Investigation of the anti-inflammatory ability of the hydrogel and the control gel:

[0086] Seed RAW 264.7 cells into a 12-well plate at a density of 1×10 5 cells / well. After the cells adhere, add LPS to the M1 group and each experimental group to a final concentration of 1 μg / mL, and add IL-4 to the M2 group to a final concentration of 40 ng / mL.

[0087] The M0 group was not treated and incubated for another 12 h. After the incubation, it was washed twice with PBS. The experimental groups were respectively added with the hydrogel extracts of PNU, PNE and PNEU and incubated for 24 h. The M0, M1 and M2 groups were added with blank culture medium. After the incubation, the incubated cell culture medium was collected and the content of NO in it was measured. At the same time, the relative mRNA expression levels of the macrophage marker iNOS in each group were examined by RT-PCR method.

[0088] The experimental results showed ( Figure 9 ), compared with the cells in the M0 group, the NO in the culture medium of macrophages stimulated by LPS increased significantly, and the M1-type macrophage marker iNOS increased significantly. After adding different hydrogel extracts, it was found that the PNE and PNEU extracts could down-regulate the amount of NO produced by LPS stimulation. The RT-PCR results showed that the relative mRNA expression levels of iNOS also decreased significantly, and the down-regulation degree of PNE was stronger than that of PNEU. There was no significant difference in the expression levels of NO and iNOS mRNA in the PNU extract group compared with the LPS stimulation group.

[0089] Example 7: Investigation of the anti-fibrotic ability of the hydrogel and the control gel:

[0090] Firstly, primary conjunctival fibroblasts RCFs were isolated and extracted from fresh rabbit eyes. The RCFs were seeded in 6-well plates at a density of 1×10 5 cells / well. After the cells adhered, the experimental groups were respectively added with the hydrogel extracts of PNU, PNE and PNEU, and then TGF-β1 was added to make the final concentration 10 μg / mL. The blank group was given the culture medium, and the model group was given the culture medium and TGF-β1. After incubation for 24 h, the relative mRNA expression levels of the fibrosis markers α-SMA and Col-1 in each group were examined by RT-PCR.

[0091] The experimental results showed ( Figure 10 ), both the PNE and PNEU groups could significantly down-regulate the mRNA expression levels of fibrosis-related α-SMA and Col-1. The down-regulation of α-SMA in the PNU group was not very obvious, but it could still significantly down-regulate the mRNA expression of Col-1. The above results indicated that the PNEU group could significantly down-regulate the relative mRNA expression levels of fibrosis-related α-SMA and Col-1.

[0092] Example 8: Investigation of the prevention and treatment of glaucoma postoperative scar by PNEU hydrogel

[0093] Model construction of scar after rabbit eye surgery in New Zealand rabbits: The rabbits were anesthetized with 3% sodium pentobarbital (1 mL / kg) via the marginal ear vein. Anesthesia could be supplemented as appropriate during the operation. The operative eye was disinfected with iodophor, and topical anesthesia was given with 0.5% oxybuprocaine hydrochloride eye drops. The eye was washed with sterile normal saline. A conjunctival flap based on the fornix was made along the corneal limbus above the operative eye, and the conjunctiva was bluntly dissected to expose the sclera of 10×10 mm size. A 24G intravenous indwelling needle was inserted into the anterior chamber from a position 2 mm away from the corneoscleral limbus at the 11:00-13:00 position until it exceeded the iris pupil margin. The guide wire was removed, and aqueous humor outflow was visible. The excess catheter was cut off 2 mm away from the puncture site. The conjunctiva was sutured watertight with 10.0 suture. The suture tension should not be too large to avoid affecting aqueous humor drainage. The blank group did not undergo surgery and only observed the right eye. The Model group was directly sutured. The PNEU gel preparation group subconjunctivally injected a mixed solution of precursor and polyphenol drugs with a volume of 100 μL using a 1 mL syringe after suturing the conjunctiva. After suturing the conjunctiva, ultraviolet light was irradiated for 10 s to form a conjunctival adhesion hydrogel. After the operation, tobramycin eye drops were instilled. After the animals woke up, they were put back into the cage. Tobramycin eye drops were instilled twice a day for 7 consecutive days after the operation.

[0094] At 28 days after the operation, intraocular pressure (IOP) was measured. The intraocular pressure of New Zealand rabbits was measured using a Suzhou Liuliu Vision indentation tonometer VTYZ7A. The morphology of the postoperative filtration bleb was recorded using a digital camera and a slit lamp.

[0095] After the observation, the New Zealand rabbits were sacrificed by rapid intravenous injection of an overdose of 3% sodium pentobarbital (100 mg / kg) via the marginal ear vein. The diseased part of the conjunctiva was separated for subsequent pathological and molecular biological evaluation.

[0096] The experimental results showed ( Figure 11 A, C), obvious elevated, slightly white, and diffuse cystic-like filtration blebs were formed in all operative eyes. The depth of the central anterior chamber was significantly shallowed on the 7th day after the operation, and the conjunctiva was moderately congested. In the Model group, the filtration bleb began to shrink on the 14th day after the operation, and the depth of the central anterior chamber returned to the preoperative level. The filtration blebs became flat and disappeared successively on the 14th day after the operation. In the PNEU group, the hydrogel implant could still be observed on the 14th day after the operation. The area of the filtration bleb slightly decreased on the 21st day after the operation, but local elevation could be observed around the drainage tube, and a cystic structure existed on the 28th day, indicating that the filtration bleb existed for a longer time. In summary, the filtration bleb after PNEU treatment presented a more diffuse appearance, and the conjunctival congestion was relieved.

[0097] The experimental results showed ( Figure 11B) It can be observed from the measured intraocular pressure before and after the operation that there was no statistically significant difference in the intraocular pressure among groups before modeling (P = 0.819). The intraocular pressure of each operative eye decreased significantly 4 days after the operation compared with that before the operation. The intraocular pressure of the Model group reached the lowest value on the 4th day, which was 9.487 ± 0.144 mmHg, while that of the PNEU group was 10.604 ± 1.319 mmHg. The intraocular pressure of the Model group returned to the normal value 10 days after the operation, while that of the PNEU group continued to decrease, showing a statistical difference from the Model group. After that, the intraocular pressure of each group was basically stable. In summary, the PNEU group could maintain low intraocular pressure after the operation.

[0098] The experimental results showed ( Figure 11 D) The results of hematoxylin-eosin (H&E) staining showed that 28 days after the operation, a large number of inflammatory cells, fibroblasts, new blood vessels and collagen fibers infiltrated in the conjunctiva of the Model group, and the arrangement of collagen connective tissue was disordered. While only a small amount of inflammatory cells, fibroblasts and collagen fibers were seen in the PNEU group, which was similar to the normal conjunctival structure before the operation. The above results indicate that implanting PNEU hydrogel in the operative eye can reduce the inflammatory reaction and fibrosis reaction, and has the potential to resist scarring after glaucoma surgery.

Claims

1. Preparation of a conjunctival light-controlled adhesion antioxidant, anti-inflammatory and anti-fibrotic hydrogel, characterized in that, It consists of two gel precursor materials, carboxymethyl chitosan (CS) derivatives CS-PBA and CS-NB, and a polyphenol drug. The polyphenol drug is cross-linked with CS-PBA through borate ester bonds. CS-NB generates aldehyde groups under the triggering of ultraviolet light passing through the conjunctiva, and then bonds with the amino groups on the subconjunctival tissue or CS through amide bonds, achieving synchronous improvement of gel in-situ adhesion to the conjunctiva and mechanical properties.

2. Preparation of the conjunctival light-controlled adhesion antioxidant, anti-inflammatory and anti-fibrotic hydrogel according to claim 1, characterized in that CS-NB( where x, y, and z are natural numbers and x + y + z is a natural number from 20 to 200) is transformed into CS-ONB( where x, y, and z are natural numbers and x + y + z is a natural number from 20 to 200) upon ultraviolet light triggering. The aldehyde group in CS-ONB undergoes a covalent reaction with the amino group of CS or the subconjunctival tissue to form an amide bond, and the nitroso group reacts with the sulfhydryl group on the tissue to generate an S-N bond, achieving simultaneous improvement in in-situ adhesion of the gel conjunctiva and mechanical properties.

3. Preparation of the conjunctival light-controlled adhesion antioxidant, anti-inflammatory and anti-fibrotic hydrogel according to claim 1, characterized in that, The described CS-PBA ( where x, y, and z are natural numbers and x + y + z is a natural number from 20 to 200) has a structural feature that 4-carboxy-3-fluorophenylboronic acid (PBA, ) is covalently modified on a part of the amino groups of carboxymethyl chitosan; the described CS-NB ( where x, y, and z are natural numbers and x + y + z is a natural number from 20 to 200) has a structural feature that 4-(4-hydroxymethyl-2-methoxy-5-nitrophenoxy)butyric acid (NB, ) is covalently modified on a part of the amino groups of carboxymethyl chitosan; after the CS-PBA solution and the CS-NB solution are mixed evenly, an antioxidant, anti-inflammatory, and anti-fibrotic drug containing polyphenols is further mixed. Polyphenolic drugs react with the phenylboronic acid of CS-PBA to form a reactive oxygen species-responsive phenylboronate ester bond, and then ultraviolet light is used to trigger the transformation of CS-NB( ) into CS-ONB( ). CS-ONB reacts with the amino groups on carboxymethyl chitosan to form a dynamic covalent hydrazone bond, and a double-crosslinked reactive oxygen species-responsive anti-inflammatory, antioxidant, and anti-fibrotic hydrogel is prepared.

4. Preparation of a conjunctival light-controlled adhesion antioxidant, anti-inflammatory and anti-fibrotic hydrogel according to claim 3, characterized in that, The synthesis of NB includes the following steps: Step 1, 4-hydroxy-3-methoxybenzaldehyde (vanillin) undergoes bromination reaction to obtain 4-(4-formyl-2-methoxyphenoxy) butyrate; Step 2, the benzene ring of the reactant in the previous step is subjected to nitration substitution to obtain 4-(4-formyl 2-methoxy-5-nitrophenoxy) butyrate; Step 3, the aldehyde group in the reaction of the previous step is reduced to a hydroxyl group to obtain 4-(4-hydroxymethyl-2-methoxy-5-nitrophenoxy) butyrate; Step 4, the product obtained in Step 3 is de-methylated to obtain 4-(4-hydroxymethyl-2-methoxy-5-nitrophenoxy) butyric acid (NB).

5. Preparation of a conjunctival light-controlled adhesion antioxidant, anti-inflammatory and anti-fibrotic hydrogel according to claim 3, characterized in that, The preparation of CS-NB includes the following steps: Step 1, synthesize the ultraviolet light-sensitive small molecule NB; Step 2, use carboxymethyl chitosan CS and NB as raw materials to synthesize CS-NB through amide condensation reaction; Step 3, use carboxymethyl chitosan CS and PBA as raw materials to synthesize CS-PBA through amide condensation reaction; Step 4, dissolve CS-NB, CS-PBA and polyphenol drugs in an aqueous medium to make solutions (pH = 7.4), and then mix the three; Step 5, inject the mixture obtained in Step 4 into the subconjunctival site of glaucoma filtration surgery, and after spreading it evenly, trigger it with ultraviolet light to obtain an antioxidant, anti-inflammatory and anti-fibrotic conjunctival adhesion hydrogel.

6. The preparation of a photo-controlled adhesion antioxidant, anti-inflammatory and anti-fibrotic hydrogel implant according to claim 3, characterized in that, The grafting rate of NB in CS-NB is 2% to 20%; the grafting rate of PBA in CS-PBA is 2% to 20%; the antioxidant, anti-inflammatory and anti-fibrotic drug containing polyphenols is epigallocatechin gallate (EGCG, ), epicatechin gallate (ECG, ), catechin gallate (CG, ), tannic acid (TA, ), or one of them.

7. Preparation of a conjunctival light-controlled adhesion antioxidant, anti-inflammatory and anti-fibrotic hydrogel according to claim 3, characterized in that, The concentration of the CS-NB is 1.25% - 5%; the concentration of the CS-PBA is 2.5% - 7.5%; the concentration of the polyphenol drug is 0.8% - 5%; the wavelength of the ultraviolet light is 365 - 405 nm, and the power is 10 w - 30 w; the mixing method of the CS-NB, CS-PBA and polyphenol drugs is mixing before use, where the volume ratio of CS-NB and CS-PBA is 2:1, 1:1 and 1:2, and the volume of the polyphenol drug accounts for 1 / 10 of the total volume after mixing; the triggering timing of the ultraviolet light is after the precursor mixture is injected subconjunctivally and contacts the conjunctiva, and the conjunctival site is irradiated with ultraviolet light for 10 s - 30 s.

8. Preparation of a conjunctival light-controlled adhesion antioxidant, anti-inflammatory and anti-fibrotic hydrogel according to claim 3, characterized in that, The hydrogel can intelligently respond to the reactive oxygen level generated by the surgical trauma microenvironment after glaucoma surgery, release polyphenol drugs as needed, scavenge reactive oxygen, and effectively relieve postoperative oxidative stress, inflammatory response and fibroblast proliferation response.

9. The application of the preparation of a conjunctival light-controlled adhesion antioxidant, anti-inflammatory and anti-fibrotic hydrogel implant and anti-glaucoma postoperative scar according to claims 1 - 7, wherein the glaucoma postoperative scar surgery includes glaucoma filtration surgeries such as trabeculectomy and non-penetrating trabecular surgery.

Images