Calycosin-7-glucoside PLGA hydrogel applied to glaucoma surgery

Abstract

The invention discloses a calycosin-7-glucoside PLGA hydrogel applied to glaucoma surgery, and relates to the technical field of biological medicine, and the calycosin-7-glucoside PLGA hydrogel is prepared from the following components by weight: 1-5 parts of calycosin-7-glucoside, 20-40 parts of PLGA, 0.1-3 parts of an emulsion stabilizer, 3-8 parts of a hydrogel matrix, and 3-8 parts of a cross-linking agent. Through the construction of a dual slow-release system, the sustained release time is long, and the peak period of scar hyperplasia after operation can be covered; the semi-solid gel is attached to the sclera flap in a form, so that the adhesiveness of the surgical site is improved, and the side effects of the whole body and the secondary surgical rate can be reduced; the biocompatibility is excellent, no obvious toxicity or stimulation exists, and complications (such as low intraocular pressure and inflammation) of traditional anti-metabolic drugs can be avoided.

Description

Technical Field

[0001] This invention relates to the field of biomedical technology, and in particular to a PLGA hydrogel for use in glaucoma surgery. Background Technology

[0002] Glaucoma is the leading cause of irreversible blindness worldwide, and surgical interventions (such as trabeculectomy) are one of the main treatments for intermediate-to-late-stage glaucoma. However, postoperative scarring of the filtration tract is the most common cause of surgical failure, leading to increased intraocular pressure (IOP) and requiring secondary intervention. As of 2025, antimetabolites such as mitomycin C (MMC) and 5-fluorouracil (5-FU) are commonly used clinically to inhibit fibroblast proliferation and scarring, but these drugs have significant drawbacks: high toxicity, potentially causing complications such as hypotension, infection, hemorrhage, intraocular inflammation, vision loss, and cataract formation. Furthermore, traditional eye drops or oral administration methods have low bioavailability (<5%), are easily affected by the tear film and corneal barrier, and cannot maintain high local drug concentrations. Verbena isoflavone glycosides, as a natural anti-inflammatory, antioxidant, and anti-fibrotic compound, have the potential to inhibit postoperative scarring, but their hydrophobicity and low solubility limit their direct application. Therefore, there is an urgent need for a biocompatible, sustained-release, and highly efficient local delivery system to improve the success rate of glaucoma surgery and reduce the risk of complications. Summary of the Invention

[0003] To address the aforementioned technical problems, this invention provides a PLGA hydrogel containing vernix isoflavone glycosides for use during glaucoma surgery. The specific technical solution is as follows: A hydrogel containing vernix isoflavone glycoside (PLGA) for use in glaucoma surgery is composed of the following components by weight: 1-5 parts vernix isoflavone glycoside, 20-40 parts PLGA, 0.1-3 parts emulsifying stabilizer, 3-8 parts hydrogel matrix, and 3-8 parts crosslinking agent.

[0004] Preferably, the emulsifying stabilizer is selected from at least one of polyvinyl alcohol, poloxamer, carbomer, or tocopheryl polyethylene glycol succinate; and / or the hydrogel matrix is ​​selected from at least one of carboxymethyl chitosan, sodium helium oleate, sodium alginate, polyethylene glycol, oxidized gellan gum, and methacryloyl hydroxypropyl methylcellulose; and / or the crosslinking agent is selected from at least one of oxidized dextran, oxidized helium oleate, oxidized sodium alginate, dialdehyde starch, disulfide-containing oxidized dextran, and dialdehyde polyethylene glycol.

[0005] Preferably, the verbascoflavonoid glycoside PLGA hydrogel is prepared by the following steps: a. Dissolve verbascoside and PLGA in an organic phase, emulsify with an emulsion stabilizer in an aqueous phase to form a W / O / W complex emulsion, evaporate the organic solvent and centrifuge to collect the drug-loaded PLGA nanoparticles, wherein the mass ratio of verbascoside to PLGA is 1:(10~20). b. Disperse the drug-loaded PLGA nanoparticles in a hydrogel matrix mixture, mix them with a crosslinking agent solution at a volume ratio of (0.5~2):1, react to form a hydrogel, and then sterilize.

[0006] Preferably, the oxidized gellan gum is prepared by the following steps: Dissolve gellan gum in ultrapure water to prepare a solution with a concentration of 0.5~2wt%; Add sodium periodate as an oxidant and stir at room temperature in the dark for 24-72 hours, wherein the molar ratio of sodium periodate to gellan gum is (0.3-1.5):1; Excess oxidant is quenched with ethylene glycol, the amount of which added is 1 to 2 times the molar amount of sodium periodate; Purify by dialysis with a dialysis bag for 2-5 days, changing the water 2-4 times a day; Pre-cooled to -20~-80℃ and freeze-dried to obtain oxidized gellan gum powder.

[0007] Preferably, the methacrylamide hydroxypropyl methylcellulose is prepared by the following steps: Hydroxypropyl methylcellulose was dissolved in dimethyl sulfoxide to prepare a solution with a concentration of 2-5 wt%. Add methacryloyl anhydride to adjust the pH to 7-10, wherein the molar ratio of methacryloyl anhydride to hydroxypropyl methylcellulose is (0.1-0.5):1; React at 40~70℃ for 2~6 hours; Precipitate the product with diethyl ether and collect it by centrifugation; Purify using dialysis bags for 1-3 days, then freeze-dry to obtain powder.

[0008] Preferably, the disulfide-containing oxidized dextran is prepared by the following steps: Dissolve dextran in ultrapure water to prepare a solution with a concentration of 40~80 mg / ml; Add sodium periodate as an oxidant and stir at room temperature in the dark for 12-36 hours, wherein the molar ratio of sodium periodate to dextran is (0.1-0.5):1; Excess oxidant was quenched with ethylene glycol, purified by dialysis for 2-4 days with water changes 2-3 times daily, and then freeze-dried to obtain oxidized dextran. Dissolve the above-mentioned oxidized dextran in water to prepare a solution with a concentration of 0.2~1wt%; Cystamine was added, and the pH was adjusted to 4-7 under EDC / NHS catalysis. The reaction was carried out at room temperature for 8-18 hours, with the mass ratio of cystamine to oxidized dextran being (0.5-2):5. Purify using dialysis bags for 1-3 days, then pre-freeze-dry to obtain powder.

[0009] Preferably, the dialdehyde polyethylene glycol is prepared by the following steps: Dissolve polyethylene glycol in methanol to prepare a solution with a concentration of 0.5~2wt%; Add sodium periodate as an oxidant and stir at room temperature in the dark for 24-48 hours, wherein the mass ratio of sodium periodate to polyethylene glycol is (0.1-0.5):1; Excess oxidant was quenched with ethylene glycol. Dialysis purification for 1-3 days, with solvent changed 2-4 times daily; Freeze-drying yields a powder.

[0010] Preferably, step a specifically includes the following sub-steps: a1. Dissolve verbascoside in dimethyl sulfoxide to prepare a solution with a concentration of 50-150 mg / ml; dissolve PLGA in dichloromethane to prepare a solution with a concentration of 20-40 mg / ml; a2. Mix the two solutions mentioned above at a mass ratio of verbenacisin to PLGA of 1:(10~20) to obtain the organic phase; a3. Dissolve the emulsifying stabilizer in water to prepare an aqueous phase with a concentration of 0.5~2% w / v; mix the organic phase with part of the aqueous phase and use an ultrasonic cell disruptor to form a proemulsion; a4. Slowly drip the colostrum into the remaining aqueous phase and use a water bath to sonicate to form a W / O / W double emulsion; a5. Place the double emulsion at room temperature and stir magnetically to evaporate the organic solvent; a6. Centrifuge to collect the precipitate and obtain drug-loaded PLGA nanoparticles.

[0011] Preferably, step b specifically includes the following sub-steps: b1. Disperse the hydrogel matrix in water to prepare a hydrogel matrix mixture with a concentration of 3~8wt%; b2. Disperse the drug-loaded PLGA nanoparticles in a hydrogel matrix mixture and blow them evenly to form a suspension; b3. Dissolve the crosslinking agent in water to prepare a crosslinking agent solution with a concentration of 3~8wt%; b4. Mix the suspension and crosslinking agent solution thoroughly at a volume ratio of (0.5~2):1, and react to form a hydrogel; b5. The hydrogel was sterilized to obtain verbascoside PLGA hydrogel.

[0012] Preferably, in step a3, the ultrasonic cell disruptor has a power of 50-200W and an ultrasonic time of 1-5 min; in step a4, the water bath ultrasonic temperature is 20-40℃, the power is 100-300W, and the time is 5-15 min; in step a5, the magnetic stirring time is 8-24 h; in step a6, the centrifugation speed is 10000-20000 rpm, and the time is 10-30 min; in step b4, the reaction time after mixing is 10-60 min; and in step b5, sterilization is performed using electron beam sterilization.

[0013] The PLGA hydrogel containing cisplatin and isoflavone glycosides used in glaucoma surgery provided by this invention has the following beneficial effects: 1. Through the construction of a dual sustained-release system, the release duration is long and can cover the peak period of postoperative scar hyperplasia.

[0014] 2. The semi-solid gel shape fits the scleral flap, improving adhesion at the surgical site and reducing systemic side effects and the rate of reoperation.

[0015] 3. It has excellent biocompatibility, no obvious toxicity or irritation, and can avoid the complications of traditional antimetabolites (such as low intraocular pressure and inflammation). Attached Figure Description

[0016] To more clearly illustrate the technical solutions in the embodiments of the present invention or the prior art, the accompanying drawings used in the description of the embodiments or the prior art will be briefly introduced below.

[0017] Figure 1 This is a transmission electron microscope image of the PLGA hydrogel of verbascoside provided in Example 1 of the present invention. Figure 2 This is a scanning electron microscope image of the PLGA hydrogel of verbascoside provided in Example 1 of the present invention; Figure 3 The particle size measurement diagram of the verbascoside PLGA hydrogel provided in Example 1 of the present invention in different environments; Figure 4 This is a standard curve diagram of verbascoside PLGA hydrogel provided in Example 1 of the present invention; Figure 5 This is a graph showing the swelling rate measurement of the verbascoside PLGA hydrogel provided in Example 1 of this invention. Figure 6 This is a rheological detection diagram of the PLGA hydrogel of verbascoside provided in Example 1 of the present invention. Detailed Implementation

[0018] To enable those skilled in the art to better understand the technical solution of the present invention, the present invention will be described in detail below with reference to the accompanying drawings. The description in this part is only exemplary and explanatory, and should not be used to limit the scope of protection of the present invention in any way.

[0019] This embodiment provides a PLGA hydrogel for use in glaucoma surgery, which is composed of the following components by weight: 1-5 parts of PLGA, 20-40 parts of PLGA, 0.1-3 parts of emulsifying stabilizer, 3-8 parts of hydrogel matrix, and 3-8 parts of crosslinking agent.

[0020] In this embodiment, hydrophobic vernix isoflavone glycosides are encapsulated using PLGA nanoparticles (50-1000 nm in diameter) as the core carrier. The biodegradability of PLGA (degrading into lactic acid and glycolic acid, which are safely eliminated via the TCA cycle) enables controlled release in the later stages. A three-dimensional network is formed between the hydrogel matrix and the cross-linking agent through a Schiff base reaction, providing initial dissolution and release, as well as mechanical support, ensuring the maintenance of a high local concentration of the drug within the eye. An emulsifying stabilizer optimizes the dispersibility of the nanoparticles, preventing aggregation. The overall proportions are designed to match the scar cycle, inhibiting fibroblast proliferation and collagen deposition, thus achieving targeted anti-fibrotic therapy. The vernix isoflavone glycoside PLGA hydrogel provided in this embodiment can not only be used to inhibit scar formation in the filtration tract after glaucoma surgery but can also be extended to other ophthalmic anti-fibrotic surgeries.

[0021] The verbascoside PLGA hydrogel used in glaucoma surgery provided in this embodiment has the following beneficial effects: By constructing a dual sustained-release system, the release duration is long enough to cover the peak period of postoperative scar hyperplasia.

[0022] The semi-solid gel form fits the scleral flap, improving adhesion at the surgical site and reducing systemic side effects and the rate of reoperation.

[0023] It has excellent biocompatibility, no obvious toxicity or irritation, and can avoid the complications of traditional antimetabolites (such as low intraocular pressure and inflammation).

[0024] Further, the emulsifying stabilizer is selected from at least one of polyvinyl alcohol, poloxamer, carbomer, or tocopheryl polyethylene glycol succinate; and / or the hydrogel matrix is ​​selected from at least one of carboxymethyl chitosan, sodium helium oleate, sodium alginate, polyethylene glycol, oxidized gellan gum, or methacryloyl hydroxypropyl methylcellulose; and / or the crosslinking agent is selected from at least one of oxidized dextran, oxidized helium oleate, oxidized sodium alginate, dialdehyde starch, disulfide-containing oxidized dextran, or dialdehyde polyethylene glycol.

[0025] The emulsifying stabilizer reduces interfacial tension through surface activity, ensuring the stability of the W / O / W emulsion and forming uniform nanoparticles. The hydrogel matrix provides a hydrophilic network, promoting cell adhesion and hydration. The crosslinking agent forms dynamic bonds via Schiff base reaction, achieving pH / GSH-responsive degradation. This combination leverages the multifunctionality of the materials to synergistically control drug diffusion and degradation release.

[0026] Beneficially, the introduction of a variety of ophthalmic compatible materials significantly broadens the flexibility of formulation; and improves biocompatibility and adhesion, reduces ocular surface irritation, and is suitable for different patients with different ocular physiological conditions; and optimizes drug loading and release curves, extending shelf life.

[0027] Furthermore, the verbascoside PLGA hydrogel was prepared via the following steps: Versicolor isoflavone glycoside and PLGA are dissolved in an organic phase and emulsified with an emulsion stabilizer in an aqueous phase to form a W / O / W complex emulsion. After the organic solvent is evaporated, the drug-loaded PLGA nanoparticles are collected by centrifugation, wherein the mass ratio of versicolor isoflavone glycoside to PLGA is 1:(10~20).

[0028] Drug-loaded PLGA nanoparticles were dispersed in a hydrogel matrix mixture and mixed with a crosslinking agent solution at a volume ratio of (0.5~2):1. After the reaction formed a hydrogel, the mixture was sterilized.

[0029] In step a, a W / O / W double emulsion solvent evaporation method is used, where the organic phase forms a stable primary emulsion in the aqueous phase of the emulsion stabilizer. After the dichloromethane evaporates, the drug is precipitated and encapsulated, ensuring high loading and sustained-release curve matching the scar peak. In step b, nanoparticles are dispersed in a hydrogel matrix mixture and mixed with a crosslinking agent. The Schiff base reaction (amino-aldehyde condensation) rapidly crosslinks to form a network, achieving dual release (initial gel dissolution + later PLGA degradation). This method utilizes the hydrophilic / hydrophobic balance of the material and the pH neutral environment to ensure biosafety and obtains ideal morphology and efficacy without complex equipment.

[0030] Beneficially, this embodiment achieves efficient and repeatable production through a two-step process, ensuring uniform nanoparticle size, avoiding ocular surface irritation and promoting corneal absorption; the resulting gel has strong stability, low swelling rate, and excellent rheological properties, which can improve the overall surgical efficacy and reduce the risk of postoperative filtration channel failure.

[0031] Furthermore, oxidized gellan gum is prepared through the following steps: Dissolve gellan gum in ultrapure water to prepare a solution with a concentration of 0.5~2wt%.

[0032] Add sodium periodate as an oxidant and stir at room temperature in the dark for 24-72 hours, wherein the molar ratio of sodium periodate to gellan gum is (0.3-1.5):1.

[0033] Excess oxidant is quenched with ethylene glycol, the amount of which is 1 to 2 times the molar amount of sodium periodate.

[0034] Purify by dialysis with a dialysis bag for 2-5 days, changing the water 2-4 times a day.

[0035] Pre-cooled to -20~-80℃ and freeze-dried to obtain oxidized gellan gum powder.

[0036] In this process, gellan gum is oxidized with sodium periodate to produce aldehyde groups, which are then quenched with ethylene glycol and dialyzed to remove byproducts. Lyophilization preserves the activity. This process introduces Schiff base crosslinking sites, forming a pH-sensitive network that allows for slow hydration / dissolution under intraocular physiological conditions (pH 7.4), promoting drug diffusion.

[0037] Furthermore, methacrylamide-modified hydroxypropyl methylcellulose is prepared by the following steps: Hydroxypropyl methylcellulose was dissolved in dimethyl sulfoxide to prepare a solution with a concentration of 2-5 wt%.

[0038] Add methacryloyl anhydride to adjust the pH to 7-10, wherein the molar ratio of methacryloyl anhydride to hydroxypropyl methylcellulose is (0.1-0.5):1.

[0039] React at 40~70℃ for 2~6 hours.

[0040] The product was precipitated with diethyl ether and collected by centrifugation.

[0041] Purify using dialysis bags for 1-3 days, then freeze-dry to obtain powder.

[0042] Hydroxypropyl methylcellulose (HPMC) is esterified with methacryloyl anhydride to introduce double bonds, and the reaction is promoted by heating at pH 7-10. Unreacted substances are removed by precipitation / dialysis purification. The resulting material forms a light / thermal responsive network, which is synergistically crosslinked with Schiff base to improve rheological properties. High hydration promotes drug diffusion, and the intraocular environment triggers degradation and release, avoiding burst release.

[0043] Furthermore, the disulfide-containing oxidized dextran is prepared by the following steps: Dissolve dextran in ultrapure water to prepare a solution with a concentration of 40-80 mg / ml.

[0044] Add sodium periodate as an oxidant and stir at room temperature in the dark for 12-36 hours, wherein the molar ratio of sodium periodate to dextran is (0.1-0.5):1.

[0045] Excess oxidant was quenched with ethylene glycol, purified by dialysis for 2-4 days, with water changed 2-3 times daily, and then lyophilized to obtain oxidized dextran.

[0046] Dissolve the above-mentioned oxidized dextran in water to prepare a solution with a concentration of 0.2~1wt%.

[0047] Cystamine was added, and the pH was adjusted to 4-7 under EDC / NHS catalysis. The reaction was carried out at room temperature for 8-18 hours, with the mass ratio of cystamine to oxidized dextran being (0.5-2):5.

[0048] Purify using dialysis bags for 1-3 days, then pre-freeze-dry to obtain powder.

[0049] In this process, dextran is first oxidized to generate an aldehyde group, which is then quenched / lyophilized and then coupled with cystamine (EDC / NHS) to form a disulfide bond, followed by dialysis purification. This double bond structure (Schiff base + disulfide) enables pH / GSH dual-response degradation. Dynamic equilibrium promotes self-healing, and the control of porosity achieves a zero-order release curve.

[0050] Furthermore, dialdehyde polyethylene glycol is prepared by the following steps: Dissolve polyethylene glycol in methanol to prepare a solution with a concentration of 0.5~2wt%.

[0051] Add sodium periodate as an oxidant and stir at room temperature in the dark for 24-48 hours, wherein the mass ratio of sodium periodate to polyethylene glycol is (0.1-0.5):1.

[0052] Excess oxidant was quenched with ethylene glycol.

[0053] Dialysis purification takes 1-3 days, with solvent changed 2-4 times daily.

[0054] Freeze-drying yields a powder.

[0055] In this process, the polyethylene glycol (PEG) end groups are oxidized to dialdehyde by sodium periodate, salts are removed by quenching / dialysis, and the structure is stabilized by freeze drying. The crosslinking agent forms a Schiff base network with the amino matrix, and the hydrophilic chain of PEG provides high water content to promote diffusion. Photo / enzyme sensitivity triggers controlled degradation in the eye to ensure gentle release and tissue integration.

[0056] Furthermore, step a specifically includes the following sub-steps: a1. Dissolve verbascoside in dimethyl sulfoxide to prepare a solution with a concentration of 50~150 mg / ml; dissolve PLGA in dichloromethane to prepare a solution with a concentration of 20~40 mg / ml.

[0057] a2. Mix the two solutions above at a mass ratio of verbascoside to PLGA of 1:(10~20) to obtain the organic phase.

[0058] a3. Dissolve the emulsifying stabilizer in water to prepare an aqueous phase with a concentration of 0.5~2% w / v; mix the organic phase with part of the aqueous phase and use an ultrasonic cell disruptor to form a colostrum.

[0059] a4. Slowly drip the colostrum into the remaining aqueous phase and sonicate in a water bath to form a W / O / W double emulsion.

[0060] a5. Place the double emulsion at room temperature and stir magnetically to evaporate the organic solvent.

[0061] a6. Centrifuge to collect the precipitate and obtain drug-loaded PLGA nanoparticles.

[0062] Among them, the organic phase ratio of a1 to a2 ensures uniform drug loading; a3 to a4 ultrasonic emulsification reduces interfacial energy and forms a stable complex emulsion (W / O / W structure encapsulates hydrophobic drugs); a5 volatilizes dichloromethane to solidify particles; a6 centrifuges for purification; this method utilizes solvent evaporation to induce phase separation, and PLGA polymer chains entangle to form a dense shell, controlling the degradation rate to match the scar cycle.

[0063] Furthermore, step b specifically includes the following sub-steps: b1. Disperse the hydrogel matrix in water to prepare a hydrogel matrix mixture with a concentration of 3~8wt%.

[0064] b2. Disperse the drug-loaded PLGA nanoparticles in a hydrogel matrix mixture and blow them evenly to form a suspension.

[0065] b3. Dissolve the crosslinking agent in water to prepare a crosslinking agent solution with a concentration of 3~8wt%.

[0066] b4. Mix the suspension and crosslinking agent solution thoroughly at a volume ratio of (0.5~2):1, and react to form a hydrogel.

[0067] b5. The hydrogel was sterilized to obtain verbascoside PLGA hydrogel.

[0068] Among them, the matrix mixture of b1~b2 provides a uniform medium and avoids agglomeration by blowing; the crosslinking agent mixture of b3~b4 triggers the Schiff reaction (aldehyde-amine condensation, rapid gelation at neutral pH); the electron beam sterilization of b5 destroys microbial DNA; this process forms an interpenetrating network, with nanoparticles embedded in the matrix, synergistically dissolving / degrading and releasing, and optimizing rheology (semi-solid adhesion to the sclera).

[0069] Further, in step a3, the ultrasonic cell disruptor has a power of 50~200W and an ultrasonic time of 1~5min; in step a4, the water bath ultrasonic temperature is 20~40℃, the power is 100~300W, and the time is 5~15min; in step a5, the magnetic stirring time is 8~24h; in step a6, the centrifugation speed is 10000~20000rpm, and the time is 10~30min; in step b4, the reaction time after mixing is 10~60min; in step b5, sterilization is performed using electron beam sterilization.

[0070] Specific embodiments are provided below. These embodiments are intended to enable those skilled in the art to more fully understand the present invention, but do not limit the present invention in any way.

[0071] Example 1 Dissolve 1.218 g of sodium periodate in 10 mL of ultrapure water. Slowly add 590 mg of dextran at room temperature (25 °C) and stir magnetically (300 rpm) in the dark. After reacting for 24 h, add 0.5 mL of ethylene glycol to quench excess oxidant and continue stirring for 30 min. Transfer to a dialysis bag and place in 500 mL of ultrapure water. Stir at 4 °C in the dark for 48 h, changing the water twice during this period. Store the dialysate at -80 °C and pre-freeze for 2 h, then freeze-dry for 48 h to obtain approximately 460 mg of light yellow oxidized dextran (Odex) powder.

[0072] Weigh 2 mg of verbascoside and dissolve it in 0.02 mL of DMSO. Weigh 30 mg of PLGA and dissolve it in 1 mL of DCM, mixing to obtain approximately 1.02 mL of organic phase. Take 0.5 mL of 1% PVA aqueous solution (50 mg PVA dissolved in 5 mL of water at 25℃, stirred for 2 h), add it to the organic phase, and emulsify using an ultrasonic cell disruptor (100 W power) for 5 min to obtain the proemulsion. Slowly add the proemulsion dropwise to 2 mL of 1% PVA aqueous solution (dropping rate 0.1 mL / min), place in a water bath (temperature 30℃) and sonicate (power 150 W power) for 10 min to obtain approximately 2.5 mL of W / O / W double emulsion. Place the double emulsion in a magnetic stirrer (300 rpm, room temperature 25℃) and stir overnight (16 h) to evaporate the DCM (monitor solvent residue <0.1%). Transfer to a centrifuge tube, centrifuge at 4°C and 14,800 rpm for 20 min, discard the supernatant, resuspend the precipitate in 2 mL of ultrapure water, repeat centrifugation twice for purification, and obtain approximately 1 mL of drug-loaded PLGA nanoparticle suspension.

[0073] Weigh 250 mg of CMCS and dissolve it in 5 mL of ultrapure water (adjust pH to 7.0 with 0.1 M HCl and stir at room temperature for 3 h until completely dissolved). Weigh 460 mg of oxidized dextran powder and dissolve it in 9.2 mL of ultrapure water (stir at room temperature for 1 h). Add 1 mL of PLGA nanoparticle suspension to 5 mL of CMCS solution and vortex mix (2000 rpm) for 2 min to obtain 6 mL of homogeneous suspension. Mix the suspension and oxidized dextran solution at a volume ratio of 1:1 (3 mL:3 mL) and allow to stand at room temperature (25 °C) for 30 min (monitor gelation, G' > G'' for preliminary rheological testing). Transfer to a sterilizer and sterilize with electron beam (25 kGy, 5 min) to obtain the final gel (approximately 6 mL, semi-solid, light yellow).

[0074] Particle size and PDI were measured using dynamic light scattering (laser wavelength 633 nm, temperature 25℃). 0.1 mL of nanoparticle suspension was diluted in 1 mL of water and ultrasonically dispersed for 5 min. Measurements were performed three times. The environment included FBS, H2O, and PBS. Changes were observed from 0 to 24 h. The measurement results are shown below. Figure 3 As shown.

[0075] Encapsulation efficiency (EE%) was determined by HPLC. The column was a C18 column (4.6 × 250 mm, 5 μm); mobile phase: acetonitrile:water = 60:35, flow rate 1.0 mL / min, detection wavelength 280 nm, column temperature 30℃. Free drug concentration was determined from the supernatant after centrifugation (14800 rpm, 20 min). Total drug = free drug + precipitated drug (dissolved in DMSO before measurement). EE% = (total drug - free drug) / total drug × 100%. Standard curve: y = 0.0418x + 0.0331, R² 2 =0.9988.

[0076] In vitro release profiles were determined by dialysis (PBS pH 7.4, 37°C, containing 0.5% Tween 80). 0.5 g of gel was placed in a dialysis bag and immersed in 50 mL of PBS, then shaken (100 rpm, 37°C). At regular intervals (1, 3, 7, 14, and 21 days), 1 mL of the release solution was collected (drug concentration determined by HPLC), and an equal volume of PBS was added. Cumulative release % = (released drug / total drug) × 100%.

[0077] The swelling percentage (SR%) was determined by gravimetric method. An initial wet gel (W0 ≈ 0.2 g) was immersed in PBS (pH 7.4, 37℃) at regular intervals (0.5, 1–7 h), then removed, the surface water was blotted dry with filter paper, and the weight (Wt) was calculated. SR% = (Wt - W0) / W0 × 100%. Triple parallel tests were performed. The test results are as follows: Figure 5 As shown.

[0078] Rheological properties were measured using a rotational rheometer (parallel plates, diameter 25 mm, gap 1 mm, temperature 37℃). In oscillation mode (frequency 1 Hz, strain 1%), storage modulus G' and loss modulus G'' were measured, and gelation time (G'=G'' crossover point) and equilibrium value (5% Odex concentration) were monitored. Test results are as follows: Figure 6 As shown.

[0079] Cytotoxicity was determined using the MTT assay. Rabbit corneal epithelial cells (SIRC, ATCC CCL-60) were used in 96-well plates at a density of 5 × 10⁶ cells / well. 3 Cells / well. Take gel extraction buffer (0.1 g gel soaked in PBS for 24 h, diluted 1:10) and treat for 24 / 48 h, add MTT (5 mg / mL, 4 h), dissolve in DMSO, measure absorbance with a microplate reader (570 nm), cell viability % = (sample OD / control OD) × 100%.

[0080] The test data is shown in the table below: Example 2 Dissolve 100 mg of gellan gum in 10 mL of ultrapure water. Slowly add 0.456 g of sodium periodate at room temperature (25°C) and stir magnetically (300 rpm) in the dark. After reacting for 48 h, add 0.25 mL of ethylene glycol to quench excess oxidant and continue stirring for 30 min. Transfer to a dialysis bag and place in 500 mL of ultrapure water. Stir at 4°C in the dark for 72 h, changing the water 3 times daily. Pre-freeze the dialysate at -50°C for 2 h and freeze-dry for 48 h to obtain approximately 82 mg of light yellow OGG powder.

[0081] Dissolve 1.218 g of sodium periodate in 10 mL of ultrapure water. Slowly add 590 mg of dextran at room temperature (25 °C) with magnetic stirring (300 rpm) in the dark. After reacting for 24 h, add 0.25 mL of ethylene glycol to quench excess oxidant and continue stirring for 30 min. Transfer to a dialysis bag and place in 500 mL of ultrapure water. Stir at 4 °C in the dark for 48 h, changing the water twice during this period. Store the dialysate at -80 °C and pre-freeze for 2 h, then freeze-dry for 48 h to obtain approximately 460 mg of light yellow Odex powder.

[0082] 2 mg of verbascoside was dissolved in 0.02 mL of DMSO, and 30 mg of PLGA was dissolved in 1 mL of DCM. The two solutions were mixed at a mass ratio of 2:30 to obtain 1.02 mL of organic phase. 0.5 mL of a 1% PVA aqueous solution (50 mg PVA dissolved in 5 mL of water at 25°C, stirred for 2 h) was added to the organic phase, and the mixture was emulsified for 5 min using an ultrasonic cell disruptor (100 W) to obtain the primary emulsion. The primary emulsion was slowly added dropwise to 2 mL of a 1% PVA aqueous solution (0.1 mL / min), and the mixture was ultrasonicated (150 W) in a water bath (30°C) for 10 min to obtain approximately 2.5 mL of secondary emulsion. The secondary emulsion was stirred overnight (16 h) using a magnetic stirrer (300 rpm, 25°C) to evaporate the DCM (solvent residue was monitored to be <0.1%). Transfer to a centrifuge tube, centrifuge at 4℃ and 14800 rpm for 20 min, discard the supernatant, resuspend the precipitate in 2 mL of ultrapure water, repeat centrifugation twice for purification, and obtain about 1 mL of drug-loaded PLGA nanoparticle suspension.

[0083] Weigh 82 mg of OGG powder and dissolve it in 1.64 mL of ultrapure water (adjust pH to 7.0, stir at room temperature for 3 h until completely dissolved). Weigh 460 mg of Odex powder and dissolve it in 9.2 mL of ultrapure water (stir at room temperature for 1 h). Add 1 mL of PLGA nanoparticle suspension to the OGG solution (1.64 mL), vortex (2000 rpm) for 2 min to obtain a homogeneous suspension (total volume 2.64 mL). Mix the suspension and Odex solution at a volume ratio of 1:1 (1.32 mL:1.32 mL), and allow to stand at room temperature (25 °C) for 30 min (monitor gelation, G' > G'' for preliminary rheological testing). Transfer to a sterilizer and sterilize with electron beam (25 kGy, 5 min) to obtain the final gel (approximately 2.64 mL, semi-solid, transparent and slightly yellow).

[0084] The test items (encapsulation efficiency, in vitro release curve, swelling ratio, rheological properties) and test methods are the same as in Example 1. The test data are shown in the table below: Example 3 Dissolve 150 mg of HPMC in 5 mL of DMSO. Slowly add 0.045 g of methacryloyl anhydride at room temperature (25°C), and adjust the pH to 8.5 with 0.1 mL of triethylamine. Stir magnetically (300 rpm) in the dark. React in a 55°C water bath for 4 h. After the reaction is complete, cool to room temperature and slowly add dropwise to 50 mL of cold diethyl ether to precipitate. Continue stirring for 30 min. Centrifuge at 5000 rpm for 15 min at 4°C, collect the precipitate, resuspend it in 10 mL of DMSO, and repeat the precipitation / centrifugation twice. Transfer to a dialysis bag, place in 500 mL of ultrapure water, and stir at 4°C in the dark for 48 h, changing the water twice daily. Pre-freeze the dialysate at -60°C for 2 h, and freeze-dry for 48 h to obtain approximately 132 mg of white MA-HPMC powder.

[0085] Dissolve 1.218 g of sodium periodate in 10 mL of ultrapure water. Slowly add 590 mg of dextran at room temperature (25 °C) and stir magnetically (300 rpm) in the dark. After reacting for 24 h, add 0.5 mL of ethylene glycol to quench excess oxidant and continue stirring for 30 min. Transfer to a dialysis bag and place in 500 mL of ultrapure water. Stir at 4 °C in the dark for 48 h, changing the water twice during this period. Store the dialysate at -80 °C and pre-freeze for 2 h, then freeze-dry for 48 h to obtain approximately 460 mg of light yellow Odex powder.

[0086] 2 mg of verbascoside was dissolved in 0.02 mL of DMSO, and 30 mg of PLGA was dissolved in 1 mL of DCM. The two solutions were mixed at a mass ratio of 2:30 to obtain approximately 1.02 mL of organic phase. 0.5 mL of a 1% PVA aqueous solution (50 mg of PVA dissolved in 5 mL of water at 25°C, stirred for 2 h) was added to the organic phase, and the mixture was emulsified for 5 min using an ultrasonic cell disruptor (100 W) to obtain a primary emulsion. The primary emulsion was slowly added dropwise to 2 mL of a 1% PVA aqueous solution (0.1 mL / min), and the mixture was ultrasonicated (150 W) in a water bath (30°C) for 10 min to obtain approximately 2.5 mL of secondary emulsion. The secondary emulsion was stirred overnight (16 h) using a magnetic stirrer (300 rpm, 25°C) to evaporate the DCM (solvent residue was monitored to be <0.1%). Transfer to a centrifuge tube, centrifuge at 4℃ and 14800 rpm for 20 min, discard the supernatant, resuspend the precipitate in 2 mL of ultrapure water, repeat centrifugation twice for purification, and obtain about 1 mL of drug-loaded PLGA nanoparticle suspension.

[0087] Weigh 132 mg of MA-HPMC powder and dissolve it in 2.64 mL of ultrapure water (adjust pH to 7.0, stir at room temperature for 3 h until completely dissolved). Weigh 460 mg of Odex powder and dissolve it in 9.2 mL of ultrapure water (stir at room temperature for 1 h). Add 1 mL of PLGA nanoparticle suspension to the MA-HPMC solution (2.64 mL), vortex (2000 rpm) for 2 min to obtain a homogeneous suspension (total volume 3.64 mL). Mix the suspension and Odex solution at a volume ratio of 1:1 (1.82 mL:1.82 mL), and allow to stand at room temperature (25 °C) for 30 min (monitor gelation, G' > G'' for preliminary rheological testing). Transfer to a sterilizer and sterilize with electron beam (25 kGy, 5 min) to obtain the final gel (total volume approximately 3.64 mL, semi-solid, translucent light yellow).

[0088] The test items and methods are the same as in Example 2, and the test data are shown in the table below: Example 4 Dissolve 1.218 g of sodium periodate in 10 mL of ultrapure water. Slowly add 590 mg of dextran at room temperature (25°C) and stir magnetically (300 rpm) in the dark for 18 h to obtain an Odex intermediate solution. Add 0.5 mL of ethylene glycol to quench excess oxidant and continue stirring for 30 min. Transfer to a dialysis bag and place in 500 mL of ultrapure water. Stir at 4°C in the dark for 36 h, changing the water twice daily. After storing the dialysate at -80°C, pre-freeze for 2 h and freeze-dry for 48 h to obtain approximately 460 mg of Odex powder. Dissolve 460 mg of Odex powder in 9.2 mL of ultrapure water, add 92 mg of cystamine, 150 mg of EDC, and 75 mg of NHS, adjust the pH to 5.5, and stir magnetically (300 rpm) at room temperature in the dark for 12 h. The solution was transferred to a dialysis bag and placed in 500 mL of ultrapure water. It was stirred at 4°C in the dark for 48 hours, with the water changed 3 times daily. The dialysate was pre-frozen at -60°C for 2 hours and then lyophilized for 48 hours to obtain approximately 345 mg of Dex-SS-Odex powder.

[0089] 2 mg of verbascoside was dissolved in 0.02 mL of DMSO, and 30 mg of PLGA was dissolved in 1 mL of DCM. The two solutions were mixed at a mass ratio of 2:30 to obtain approximately 1.02 mL of organic phase. 0.5 mL of a 1% PVA aqueous solution (50 mg of PVA dissolved in 5 mL of water at 25°C, stirred for 2 h) was added to the organic phase, and the mixture was emulsified for 5 min using an ultrasonic cell disruptor (100 W) to obtain a primary emulsion. The primary emulsion was slowly added dropwise to 2 mL of a 1% PVA aqueous solution (0.1 mL / min), and the mixture was ultrasonicated (150 W) in a water bath (30°C) for 10 min to obtain approximately 2.5 mL of secondary emulsion. The secondary emulsion was stirred overnight (16 h) using a magnetic stirrer (300 rpm, 25°C) to evaporate the DCM (solvent residue was monitored to be <0.1%). Transfer to a centrifuge tube, centrifuge at 4℃ and 14800 rpm for 20 min, discard the supernatant, resuspend the precipitate in 2 mL of ultrapure water, repeat centrifugation twice for purification, and obtain about 1 mL of drug-loaded PLGA nanoparticle suspension.

[0090] Weigh 250 mg of CMCS and dissolve it in 5 mL of ultrapure water (adjust pH to 7.0, stir at room temperature for 3 h until completely dissolved). Weigh 345 mg of Dex-SS-Odex powder and dissolve it in 6.9 mL of ultrapure water (stir at room temperature for 1 h). Add 1 mL of PLGA nanoparticle suspension to 5 mL of CMCS solution and vortex (2000 rpm) for 2 min to obtain a homogeneous suspension (6 mL). Mix the suspension with the Dex-SS-Odex solution at a volume ratio of 1:1 (3 mL:3 mL) and allow to stand at room temperature (25 °C) for 30 min (monitor gelation, G' > G'' for preliminary rheological testing). Transfer to a sterilizer and sterilize with electron beam (25 kGy, 5 min) to obtain the final gel (approximately 6 mL, semi-solid, light yellow).

[0091] The test items (encapsulation efficiency, swelling rate, rheological properties) and test methods are the same as in Example 1; the in vitro release curve test item (10 mM GSH PBS) is added, and the test procedure for this test item is the same as in Example 1. The test data are shown in the table below: Example 5 Dissolve 1 g of PEG-diol in 100 mL of methanol, and slowly add 0.3 g of sodium periodate at room temperature (25°C). Stir magnetically (300 rpm) in the dark for 36 h. Add 0.5 mL of ethylene glycol to quench excess oxidant, and continue stirring for 30 min. Transfer to a dialysis bag and place in 500 mL of methanol / water mixture (1:1 v / v). Stir at 4°C in the dark for 48 h, changing the solvent three times daily. Pre-freeze the dialysate at -70°C for 2 h, and then freeze-dry for 48 h to obtain approximately 810 mg of white PEG-CHO powder.

[0092] 2 mg of verbascoside was dissolved in 0.02 mL of DMSO, and 30 mg of PLGA was dissolved in 1 mL of DCM. The two solutions were mixed at a mass ratio of 2:30 to obtain approximately 1.02 mL of organic phase. 0.5 mL of a 1% PVA aqueous solution (50 mg of PVA dissolved in 5 mL of water at 25°C, stirred for 2 h) was added to the organic phase, and the mixture was emulsified for 5 min using an ultrasonic cell disruptor (100 W) to obtain a primary emulsion. The primary emulsion was slowly added dropwise to 2 mL of a 1% PVA aqueous solution (0.1 mL / min), and the mixture was ultrasonicated (150 W) in a water bath (30°C) for 10 min to obtain approximately 2.5 mL of secondary emulsion. The secondary emulsion was stirred overnight (16 h) using a magnetic stirrer (300 rpm, 25°C) to evaporate the DCM (solvent residue was monitored to be <0.1%). Transfer to a centrifuge tube, centrifuge at 4℃ and 14800 rpm for 20 min, discard the supernatant, resuspend the precipitate in 2 mL of ultrapure water, repeat centrifugation twice for purification, and obtain about 1 mL of drug-loaded PLGA nanoparticle suspension.

[0093] Weigh 250 mg of CMCS and dissolve it in 5 mL of ultrapure water (adjust pH to 7.0, stir at room temperature for 3 h until completely dissolved). Weigh 810 mg of PEG-CHO powder and dissolve it in 16.2 mL of ultrapure water (stir at room temperature for 1 h). Add 1 mL of PLGA nanoparticle suspension to 5 mL of CMCS solution and vortex (2000 rpm) for 2 min to obtain 6 mL of homogeneous suspension. Mix the suspension with PEG-CHO solution at a volume ratio of 1:1 (3 mL:3 mL) and allow to stand at room temperature (25 °C) for 30 min (monitor gelation, G' > G'' for preliminary rheological testing). Transfer to a sterilizer and sterilize with electron beam (25 kGy, 5 min) to obtain the final gel (approximately 6 mL, semi-solid, highly transparent light yellow).

[0094] The test items and methods are the same as in Example 2, and the test data are shown in the table below: This article uses specific examples to illustrate the principles and implementation methods of the present invention. The descriptions of the above embodiments are only for the purpose of helping to understand the method and core ideas of the present invention. The above are merely preferred embodiments of the present invention, but the scope of protection of the present invention is not limited thereto. Any equivalent substitutions or modifications made by those skilled in the art within the technical scope disclosed in the present invention, based on the technical solution and inventive concept of the present invention, should be within the scope of protection of the present invention.

Claims

1. A hydrogel containing cisplatin (PLGA) and isoflavone glycosides used in glaucoma surgery, characterized in that... The product is made from the following components in parts by weight: 1-5 parts of verbascoside, 20-40 parts of PLGA, 0.1-3 parts of emulsifying stabilizer, 3-8 parts of hydrogel matrix, and 3-8 parts of crosslinking agent.

2. The PLGA hydrogel containing versicolor isoflavone glycosides used in glaucoma surgery according to claim 1, characterized in that: The emulsifying stabilizer is selected from at least one of polyvinyl alcohol, poloxamer, carbomer, or tocopherol polyethylene glycol succinate. And / or the hydrogel matrix is ​​selected from at least one of carboxymethyl chitosan, sodium helium ether, sodium alginate, polyethylene glycol, oxidized gellan gum, and methacryloyl hydroxypropyl methylcellulose; The crosslinking agent is selected from at least one of oxidized dextran, oxidized helium acid, oxidized sodium alginate, dialdehyde starch, disulfide-containing oxidized dextran, and dialdehyde polyethylene glycol.

3. The cyclosporine glycoside PLGA hydrogel used in glaucoma surgery according to claim 1, characterized in that, The verbascoflavonoid PLGA hydrogel was prepared by the following steps: Versicolor isoflavone glycoside and PLGA are dissolved in an organic phase and emulsified with an emulsion stabilizer in an aqueous phase to form a W / O / W complex emulsion. After the organic solvent is evaporated, the drug-loaded PLGA nanoparticles are collected by centrifugation, wherein the mass ratio of versicolor isoflavone glycoside to PLGA is 1:(10~20). The drug-loaded PLGA nanoparticles were dispersed in a hydrogel matrix mixture and mixed with a crosslinking agent solution at a volume ratio of (0.5~2):

1. After the reaction formed a hydrogel, the mixture was sterilized.

4. The cyclosporine glycoside PLGA hydrogel used in glaucoma surgery according to claim 2, characterized in that, The oxidized gellan gum is prepared by the following steps: Dissolve gellan gum in ultrapure water to prepare a solution with a concentration of 0.5~2wt%; Add sodium periodate as an oxidant and stir at room temperature in the dark for 24-72 hours, wherein the molar ratio of sodium periodate to gellan gum is (0.3-1.5):1; Excess oxidant is quenched with ethylene glycol, the amount of which added is 1 to 2 times the molar amount of sodium periodate; Purify by dialysis with a dialysis bag for 2-5 days, changing the water 2-4 times a day; Pre-cooled to -20~-80℃ and freeze-dried to obtain oxidized gellan gum powder.

5. The PLGA hydrogel containing versicolor isoflavone glycosides used in glaucoma surgery according to claim 2, characterized in that, The methacrylamide hydroxypropyl methylcellulose was prepared by the following steps: Hydroxypropyl methylcellulose was dissolved in dimethyl sulfoxide to prepare a solution with a concentration of 2-5 wt%. Add methacryloyl anhydride to adjust the pH to 7-10, wherein the molar ratio of methacryloyl anhydride to hydroxypropyl methylcellulose is (0.1-0.5):1; React at 40~70℃ for 2~6 hours; Precipitate the product with diethyl ether and collect it by centrifugation; Purify using dialysis bags for 1-3 days, then freeze-dry to obtain powder.

6. The PLGA hydrogel containing vernix isoflavone glycosides used in glaucoma surgery according to claim 2, characterized in that, The disulfide-containing oxidized dextran was prepared by the following steps: Dissolve dextran in ultrapure water to prepare a solution with a concentration of 40~80 mg / ml; Add sodium periodate as an oxidant and stir at room temperature in the dark for 12-36 hours, wherein the molar ratio of sodium periodate to dextran is (0.1-0.5):1; Excess oxidant was quenched with ethylene glycol, purified by dialysis for 2-4 days with water changes 2-3 times daily, and then freeze-dried to obtain oxidized dextran. Dissolve the above-mentioned oxidized dextran in water to prepare a solution with a concentration of 0.2~1wt%; Cystamine was added, and the pH was adjusted to 4-7 under EDC / NHS catalysis. The reaction was carried out at room temperature for 8-18 hours, with the mass ratio of cystamine to oxidized dextran being (0.5-2):

5. Purify using dialysis bags for 1-3 days, then pre-freeze-dry to obtain powder.

7. The PLGA hydrogel containing versicolor isoflavone glycosides used in glaucoma surgery according to claim 2, characterized in that, The dialdehyde-based polyethylene glycol is prepared by the following steps: Dissolve polyethylene glycol in methanol to prepare a solution with a concentration of 0.5~2wt%; Add sodium periodate as an oxidant and stir at room temperature in the dark for 24-48 hours, wherein the mass ratio of sodium periodate to polyethylene glycol is (0.1-0.5):1; Excess oxidant was quenched with ethylene glycol. Dialysis purification for 1-3 days, with solvent changed 2-4 times daily; Freeze-drying yields a powder.

8. The PLGA hydrogel containing versicolor isoflavone glycosides used in glaucoma surgery according to claim 3, characterized in that, Step a specifically includes the following sub-steps: a1) Dissolve verbenaflavonoid glycoside in dimethyl sulfoxide to prepare a solution with a concentration of 50~150mg / ml; dissolve PLGA in dichloromethane to prepare a solution with a concentration of 20~40mg / ml; a2) Mix the two solutions above at a mass ratio of verbenacisin to PLGA of 1:(10~20) to obtain an organic phase; a3) Dissolve the emulsifying stabilizer in water to prepare an aqueous phase with a concentration of 0.5~2% w / v; mix the organic phase with part of the aqueous phase and use an ultrasonic cell disruptor to form a primary emulsion; a4) Slowly drip the colostrum into the remaining aqueous phase and use ultrasonic bath to form a W / O / W double emulsion; a5) Place the double emulsion at room temperature and stir magnetically to evaporate the organic solvent; a6) Centrifuge to collect the precipitate and obtain drug-loaded PLGA nanoparticles.

9. The cyclosporine glycoside PLGA hydrogel used in glaucoma surgery according to claim 8, characterized in that, Step b specifically includes the following sub-steps: b1) Disperse the hydrogel matrix in water to prepare a hydrogel matrix mixture with a concentration of 3~8wt%; b2) Disperse the drug-loaded PLGA nanoparticles in a hydrogel matrix mixture and blow them evenly to form a suspension; b3) Dissolve the crosslinking agent in water to prepare a crosslinking agent solution with a concentration of 3~8wt%; b4) Mix the suspension and crosslinking agent solution thoroughly at a volume ratio of (0.5~2):1, and react to form a hydrogel; b5) The hydrogel was sterilized to obtain verbascoside PLGA hydrogel.

10. The PLGA hydrogel containing versicolor isoflavone glycosides used in glaucoma surgery according to claim 9, characterized in that: In step a3, the ultrasonic cell disruptor has a power of 50~200W and an ultrasonic time of 1~5min; In step a4, the water bath ultrasonic temperature is 20~40℃, the power is 100~300W, and the time is 5~15min; In step a5, the magnetic stirring time is 8~24h; In step a6, the centrifugation speed is 10,000~20,000 rpm and the time is 10~30 min; In step b4, the reaction time after mixing is 10~60 min; In step b5, sterilization is performed using electron beam sterilization.

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