Sequential controlled release dual-drug loaded injectable hydrogel and preparation method and application thereof

Abstract

The invention belongs to the technical field of injectable drugs, and relates to a sequential controlled-release dual-drug loaded injectable hydrogel, and a preparation method and application thereof. A cross-linked γ-cyclodextrin metal organic framework loaded with a first drug and chitosan nanoparticles loaded with a second drug and coated with hyaluronic acid and polyvinyl pyrrolidone from the inside to the outside are dispersed in an aldehyded β-cyclodextrin solution loaded with the first drug to form a first dispersion; the amino hyaluronic acid is dispersed in a PBS buffer solution to form a second dispersion; then the first dispersion and the second dispersion are mixed in equal volumes, and the sequential controlled-release dual-drug loaded injectable hydrogel is prepared through a Schiff base reaction; the preparation method has a scientific principle, simple operation, low cost, and is widely applicable to drugs; as an injectable drug technology, it has great application potential in the preparation of any drug carrier that needs to control the spatiotemporal specific release of different drugs, and is suitable for large-scale promotion.

Description

Technical field:

[0001] The invention belongs to the technical field of injectable drugs, and in particular to a sequential controlled-release dual-drug loaded injectable hydrogel and a preparation method and application thereof. Background technology:

[0002] A single drug is not sufficient to treat certain diseases (such as tumors, glaucoma, retinal diseases, etc.). The combination of two or more drugs has become a key strategy to improve clinical treatment effects through synergistic effects. Usually, the strategy of sequential drug controlled release is to achieve spatiotemporal specific loading through microspheres, nanomolecular cages, macromolecular prodrugs, supramolecular hydrogels, etc.

[0003] Acute retinal necrosis (ARN) is an ophthalmic disease that requires the use of both antiviral and anti-inflammatory drugs during clinical treatment. The clinical treatment plan is to use antiviral drugs first in the early stage of treatment, and then add an anti-inflammatory drug 24-48 hours later to achieve better therapeutic effects. The time of starting to use anti-inflammatory drugs is critical. If it is too early, it will enhance viral replication and lead to the rapid development of ARN; if it is too late, it will not achieve a good anti-inflammatory effect. Ganciclovir (GCV) is a clinically used viral DNA polymerase inhibitor with anti-cytomegalovirus and herpes simplex virus activity. However, due to its short half-life, GCV should be injected into the vitreous 2-3 times a week to achieve therapeutic effects, and the risk of traumatic blindness is high. Dexamethasone sodium phosphate (DEX) is a highly effective and long-lasting glucocorticoid anti-inflammatory drug. The clinical treatment plan is to administer DEX within 24-48 hours after the start of GCV antiviral treatment. The two work together to treat ARN. The time point when DEX starts to be used for treatment is crucial, and no DEX should appear in the lesion before this time point.

[0004] Natural polysaccharide injectable hydrogels are based on cross-linked three-dimensional networks, retain a large amount of water and can be formed in situ. They can be applied to various aspects of the biomedical field, such as the delivery of drugs, cells and other bioactive substances. In the field of drug delivery, compared with systemic administration, drug-loaded injectable hydrogels achieve targeted therapy by injection into the lesion site, and they also show sustained drug release behavior, reducing the frequency of external administration.

[0005] Metal-organic frameworks (MOFs) are porous crystalline materials assembled by metal ions or clusters and organic ligands through coordination bonds. These structures have a simple synthesis method, large specific surface area, high porosity, controllable pore structure and strong encapsulation ability for guest molecules, making them promising in drug delivery applications. However, traditional MOFs materials show obvious biological toxicity, which limits their application in the biomedical field. Cyclodextrin has good biocompatibility and structural strength and can be used to synthesize cyclodextrin-based metal-organic frameworks, but it needs to be modified to improve its water stability.

[0006] Chitosan nanoparticles have received extensive attention as drug carriers in recent years, and hyaluronic acid (HA) can improve the stability of particles in serum by covering the surface of nanoparticles. Therefore, it can be used to coat chitosan nanoparticles to prolong the retention time of drugs in the body and improve their bioavailability. For example, Mohd Abul Kalam prepared chitosan-sodium tripolyphosphate nanoparticles (CS-NPs) using the ion gel method and coated CS-NPs with HA (HA-CS-NPs), which made the nanoparticles more dispersed and improved their adhesion. In the in vitro release study, the rapid release phase of DEX coated by CS-NPs and HA lasted for up to 2 hours. In this rapid phase, about 40-50% of DEX and about 35% of DEX were released from CS-NPs and HA-CS-NPs, respectively. In addition, both CS-NPs and HA-CS-NPs released more than 75% of DEX within 12 hours. In another study, Lisna Meylina et al. prepared a hyaluronic acid-coated chitosan nanoparticle loaded with α-mangiferin (AM) (AM-CS / HA). The release of AM from AM-CS and AM-CS / HA was 11% and 8% in the first hour, respectively, and then continued to release for 96 hours, showing a certain sustained release effect. The total amount of AM released from AM-CS and AM-CS / HA was about 66% and 60%, respectively. Therefore, even if HA is used to coat CS-NPs, the release of drugs can be delayed, but it cannot solve the problem of the initial burst release of drugs and the long-term sustained release of drugs in the subsequent stages. In addition, FaribaNokhodi et al. synthesized a targeted drug delivery system of hyaluronic acid-coated chitosan nanoparticles (HA-CS-NPs) loaded with tamoxifen (TMX). Within 48 hours, the TMX release rates of CS-NPs and HA-CS-NPs in PBS were approximately 40% and 20%, respectively. Compared with CS-NPs, the TMX release rate in HA-CS-NPs was delayed by approximately 50%. In addition, compared with CS-NPs, the TMX release rate of HA-CS-NPs in PBS was approximately 15% lower within 5 days. Although these systems have achieved sustained drug release to a certain extent, none of the measures have been able to achieve a sequential release time interval of 24-48 hours between one of the encapsulated drugs and another drug, that is, when one of the drugs is continuously released, the other drug is not released at all within the first 24-48 hours. And overall, the sustained and controlled release duration of these drug delivery systems is obviously insufficient.

[0007] Chinese patents CN115400071A and CN108434093A disclose injectable hydrogels for controlled release of water-insoluble intraocular drugs and their preparation methods, which only load the water-insoluble drug voriconazole into the molecular cage of aldehyde- or linear cyclodextrin, and do not involve the loading of other nanoparticles for the second drug and the sequential drug release technology; Chinese patents CN117582546A, CN106117547B and CN114601958B only involve drug carrier injectable hydrogel technology, and do not reflect the related technology of dual-drug loaded nanocomposite injectable hydrogel and sequential drug long-term controlled release. Therefore, in view of the problems existing in the prior art: on the one hand, there is no suitable structural design to make the initial release amount of a certain drug in the system zero, and start to release after 24-48 hours; on the other hand, although the drug carrier hydrogel can also achieve a certain degree of sustained release, the initial burst release phenomenon is serious and the sustained release time is short. It is necessary to further invent a sequential drug controlled release injectable hydrogel carrier technology to achieve the temporal and spatial specificity, better drug controlled release performance and longer drug controlled release time required for the treatment of such diseases to meet clinical needs. Summary of the invention:

[0008] The invention aims to provide a sequential controlled release dual-drug loaded injectable hydrogel and a preparation method and application thereof, aiming at the problems that the prior art cannot realize the sequential release of dual drugs, cannot overcome the burst release and has a short controlled release time. The invention comprises the following steps: a cross-linked γ-cyclodextrin metal organic framework loaded with a first drug and chitosan nanoparticles loaded with a second drug and coated with hyaluronic acid and polyvinyl pyrrolidone from the inside to the outside in sequence are dispersed in a PBS buffer solution of aldehyded β-cyclodextrin loaded with the first drug to form a first dispersion; aminoated hyaluronic acid is dispersed in a PBS buffer solution to form a second dispersion; then the first dispersion and the second dispersion are mixed in equal volumes, and a sequential controlled release dual-drug loaded injectable hydrogel is prepared through a Schiff base reaction, which can realize the interval sequential release of two drugs, the quasi-zero-order controlled release of the first drug and the long-acting controlled release of the overall dual drugs.

[0009] In order to achieve the above object, the present invention provides a method for preparing a sequential controlled release dual-drug loaded injectable hydrogel, wherein a cross-linked γ-cyclodextrin metal organic framework loaded with a first drug and chitosan nanoparticles loaded with a second drug and coated with hyaluronic acid and polyvinyl pyrrolidone from the inside to the outside are dispersed in a PBS buffer solution loaded with aldehyded β-cyclodextrin of the first drug to form a first dispersion; amino hyaluronic acid is dispersed in the PBS buffer solution to form a second dispersion; then the first dispersion and the second dispersion are mixed in equal volumes, and a sequential controlled release dual-drug loaded injectable hydrogel is prepared by a Schiff base reaction, which specifically comprises the following steps:

[0010] Step 1: preparing cross-linked γ-cyclodextrin metal organic framework nanoparticles (GCV-CL-MOF) loaded with the first drug, the specific steps are as follows:

[0011] (1) Preparation of γ-CD-MOF nanoparticles: γ-cyclodextrin and potassium hydroxide (KOH) were dissolved in deionized water, sonicated, and insoluble matter was removed by filtration with a filter membrane. Methanol was then added, mixed evenly, and heated and stirred in a constant temperature water bath at 50°C until the liquid changed from turbid to clear; then, polyethylene glycol 20000 and methanol were quickly added and precipitation of crystalline materials was induced in cold water, followed by centrifugation, washing, and drying. The solid particles were soaked in dichloromethane (DCM) for three days, centrifuged, and vacuum dried to constant weight to obtain γ-cyclodextrin metal organic framework nanoparticles (γ-CD-MOF);

[0012] (2) Preparation of cross-linked γ-CD-MOF (CL-MOF): γ-CD-MOF and diphenyl carbonate (DPC) were added to a mixed solution of dimethylformamide (DMF) and triethanolamine (TEA), stirred at a constant temperature of 60-100°C for 30-60 hours, cooled to room temperature, ethanol was added to quench the reaction, washed, and vacuum dried to constant weight to obtain CL-MOF;

[0013] (3) preparing a cross-linked γ-CD-MOF loaded with a first drug: mixing the first drug and CL-MOF in ethanol, magnetically stirring in a constant temperature water bath for 10-40 h, centrifuging, washing, removing the unloaded first drug, and vacuum drying the precipitate to constant weight to obtain a cross-linked γ-CD-MOF loaded with the first drug (GCV-CL-MOF), wherein the mass volume ratio of the first drug, the cross-linked γ-cyclodextrin metal organic framework and the ethanol is 10-15 mg:50 mg:10 mL;

[0014] Step 2: preparing an aldehyde-modified β-cyclodextrin solution containing the first drug

[0015] β-cyclodextrin was added to deionized water, heated and stirred to dissolve, cooled to room temperature, and then sodium periodate (NaIO 4 ) was stirred in dark for 1-5h, ethylene glycol was added and stirred for 0.5-4h, the product was dialyzed with a dialysis bag (MW 500) and deionized water for 1-2 days, and freeze-dried to obtain aldehyded β-cyclodextrin (ACD);

[0016] ACD is dissolved in a PBS solution to obtain a 40 mg / mL ACD solution, and then the first drug is dispersed into the cyclodextrin molecular cage in the ACD by ultrasonic dispersion to obtain an ACD solution loaded with the first drug, wherein the concentration of aldehyded β-cyclodextrin in the solution is 40 mg / mL, and the concentration of the first drug is 0-0.6 mg / mL;

[0017] Step 3: preparing chitosan nanoparticles loaded with a second drug and coated with hyaluronic acid and polyvinyl pyrrolidone in sequence from the inside to the outside, the specific steps are as follows:

[0018] (1) Preparation of chitosan nanoparticles (CS-NPs) loaded with a second drug: chitosan was dissolved in an aqueous acetic acid solution to obtain a chitosan acetic acid solution with a concentration of 0.6 mg / mL, and the second drug was then added to the chitosan acetic acid solution; sodium tripolyphosphate solution with a pH of 7.4 was then stirred at room temperature at 1.5 mL min -1 The chitosan acetic acid solution containing the second drug is added at a rate of 1000 μg / min to the chitosan acetic acid solution containing the second drug, and stirred for 0.5-4 h; freeze-dried to obtain chitosan nanoparticles loaded with the second drug;

[0019] (2) preparing CS-NPs coated with hyaluronic acid (HA) and loaded with a second drug: dispersing the chitosan nanoparticles loaded with the second drug prepared in step (1) in an acetic acid buffer; then, adding the acetic acid buffer containing hyaluronic acid, stirring for 20-60 minutes, and freeze-drying to obtain HA-coated chitosan nanoparticles loaded with a second drug;

[0020] (3) Preparing hyaluronic acid-coated CS-NPs loaded with a second drug coated with polyvinyl pyrrolidone (PVP): dispersing the hyaluronic acid-coated chitosan nanoparticles loaded with a second drug prepared in step (2) in an acetic acid buffer; then, adding an acetic acid buffer containing 0.5 mg / mL PVP, stirring for 20-60 min, and freeze-drying to obtain chitosan nanoparticles loaded with a second drug (PHDCNPs) coated with hyaluronic acid and polyvinyl pyrrolidone in sequence from the inside to the outside;

[0021] Step 4: Preparation of sequential controlled release dual-drug loaded injectable hydrogel, the specific steps are as follows:

[0022] (1) preparing a first dispersion: dispersing the cross-linked γ-cyclodextrin metal organic framework nanoparticles loaded with the first drug prepared in step 1 and the chitosan nanoparticles loaded with the second drug coated with hyaluronic acid and polyvinyl pyrrolidone from the inside to the outside in sequence prepared in step 3 into the aldehyde-modified β-cyclodextrin solution loaded with the first drug prepared in step 2 to obtain a first dispersion;

[0023] (2) preparing the second dispersion: first prepare amino hyaluronic acid (NHA), dissolve HA in distilled water, add ethylenediamine hydrochloride and 4-dimethylaminopyridine (DAMP) (catalyst) and mix well; after mixing well, add 1-ethyl-(3-dimethylaminopropyl)carbodiimide (EDC) and N-hydroxysuccinimide (NHS), then add NaOH solution to adjust the pH to 4.5-5.5, continue stirring and reacting for 2 days, during which the pH is maintained in the range of 4.5-5.5, and after 2 days, adjust the pH to 7 with NaOH solution to stop the reaction; then dialyze with a dialysis bag (MW 14000) and distilled water for 3-5 days, then take it out and freeze-dry it, and the freeze-dried solid is NHA; add NHA to PBS solution to prepare a second dispersion with an NHA concentration of 50 mg / mL;

[0024] (3) Preparation of sequential controlled release dual drug-loaded injectable hydrogel: The first dispersion and the second dispersion are evenly mixed, and cross-linked by Schiff base reaction between aldehyde groups and amino groups to obtain a sequential controlled release dual drug-loaded injectable hydrogel.

[0025] The first drug of the present invention is a water-insoluble drug, and the second drug is a water-soluble drug.

[0026] The first drug of the present invention is ganciclovir, and the second drug is dexamethasone sodium phosphate. The concentration of the phosphate buffer solution PBS of the present invention is 0.01M, and the pH is 7.4.

[0027] The concentration of the acetic acid buffer of the present invention is 0.1M and the pH is 5.0.

[0028] In step 1 (2) of the present invention, the amount of ethanol added is twice the volume of the reaction solution.

[0029] The drug loading rate (by mass) of the cross-linked γ-CD-MOF (GCV-CL-MOF) loaded with the first drug in step 1 (3) of the present invention is 18.4±0.6%. The appropriate amount of GCV-CL-MOF can be added by calculation according to the required drug concentration in the hydrogel system.

[0030] The first dispersion of the present invention contains 326.0mg to 652.0mg of cross-linked γ-cyclodextrin metal organic framework nanoparticles loaded with a first drug and 464.0mg of chitosan nanoparticles loaded with a second drug and coated with hyaluronic acid and polyvinyl pyrrolidone in sequence from inside to outside per 100.0mL.

[0031] The drug loading rate (by mass) of the aldehyded β-cyclodextrin (ACD) in step 2 of the present invention is 9.56±0.29%. As ACD has dual functions of loading drugs and acting as a cross-linking agent, the amount of ACD added to the hydrogel system mainly depends on the preparation of the hydrogel body. If the added drug cannot be completely loaded by the cyclodextrin molecular cage in the ACD, then the excess drug will be directly dispersed into the water contained in the hydrogel body.

[0032] In step 1 and step 2 of the present invention, the first drug is loaded into CL-MOF and ACD, both of which utilize the adsorption effect of the micro-nano pores of the two nanostructures and a certain hydrogen bond interaction force, and the sustained release of the first drug is achieved through the sequential destruction of the two structures during the degradation process.

[0033] The preparation principle in step three of the present invention includes forming chitosan nanoparticles through an ionic crosslinking reaction between sodium tripolyphosphate and chitosan; the molecular chain of hyaluronic acid carries a negative charge, and the amino group on the chitosan molecular chain carries a positive charge, and a charge interaction force is formed between the two, thereby forming a coating on the nano-chitosan particles; PVP has good film-forming ability and biocompatibility, and the lone pair electrons of oxygen in HA and PVP molecules and adjacent hydrogen atoms can form hydrogen bonds, so PVP can coat the chitosan nanoparticles coated with hyaluronic acid again, and the sequential release of the second drug and the first drug with an interval of 24 hours and the sustained release of the two drugs after 24 hours are achieved through double-layer coating.

[0034] The drug loading rate of the chitosan nanoparticles loaded with the second drug in step three of the present invention is 19.01±0.95%, and the drug loading rate of the chitosan nanoparticles loaded with the second drug coated with hyaluronic acid and polyvinyl pyrrolidone from the inside to the outside (PHDCNPs) is 17.28±0.51%. PHDCNPs can be added in an appropriate amount by calculation according to the required drug concentration in the hydrogel system.

[0035] In step 4 of the present invention, the molecular weight of hyaluronic acid (HA) is 100,000-200,000, the amount of ethylenediamine hydrochloride added is 50 times the molar number of HA repeating units, the amount of 4-dimethylaminopyridine (DAMP) added is 30 times the molar number of HA repeating units, and the amounts of 1-ethyl-(3-dimethylaminopropyl)carbodiimide (EDC) and N-hydroxysuccinimide (NHS) added are 25 times the molar number of HA repeating units.

[0036] In step 4 of the present invention, the mixing volume ratio of the first dispersion liquid and the second dispersion liquid is 1:1.

[0037] The present invention also provides a sequential controlled release dual-drug loaded injectable hydrogel prepared by the above preparation method, which realizes the sequential release of a first drug and a second drug, wherein the first drug can be released in zero order, and the second drug begins to be released 24 hours after the first drug is released, and the sustained-controlled release time of the two drugs is greater than 20 days.

[0038] The present invention also provides the use of the sequential controlled release dual-drug loaded injectable hydrogel in preparing a drug for treating acute retinal necrosis, wherein the first drug is ganciclovir, the second drug is dexamethasone sodium phosphate, ganciclovir can be released in zero order, and the burst release phenomenon is not obvious, and dexamethasone sodium phosphate starts to be released 24 hours after the release of ganciclovir, and the drug controlled release time is greater than 20 days.

[0039] When the first drug of the present invention is ganciclovir and the second drug is dexamethasone sodium phosphate, the sequential controlled release dual-drug loaded injectable hydrogel prepared by the preparation method of the present invention can be used as a drug for treating acute retinal necrosis. By using the preparation method of the present invention, the specific components of the first drug and the second drug can be selected according to actual conditions to prepare drugs for treating other diseases, thereby realizing the sequential controlled release of the dual drugs.

[0040] Compared with the prior art, the present invention has the following beneficial effects:

[0041] (1) The sequential controlled release dual-drug loaded injectable hydrogel prepared by the present invention realizes the sequential controlled release of the first drug and the second drug within a time interval of 24h-48h, and no second drug is released in the first 24h.

[0042] (2) The sequential controlled release dual-drug loaded injectable hydrogel prepared by the present invention overcomes the initial drug burst release phenomenon of the first drug and achieves a quasi-zero-order release of the first drug.

[0043] (3) The sequential controlled release dual-drug loaded injectable hydrogel prepared by the present invention achieves long-term controlled release of dual drugs sequentially for >20 days.

[0044] In summary, the preparation method of the sequential controlled-release dual-drug loaded injectable hydrogel of the present invention has a scientific principle, simple operation, low cost, and a wide range of applicable drugs; as an injectable drug technology, it has great application potential in the preparation of any drug carrier that needs to control the spatiotemporal specific release of different drugs, and is suitable for large-scale promotion. Description of the drawings:

[0045] Figure 1 This is a schematic diagram of the preparation principle of the sequential controlled release dual-drug loaded injectable hydrogel according to Example 1 of the present invention.

[0046] Figure 2Schematic diagram of the chemical reaction for preparing a sequential controlled release dual drug loaded injectable hydrogel according to Example 1 of the present invention, wherein a is the formation process of the CL-MOF structure, and the C=O bond in DPC replaces K + , forming a more stable CL-MOF structure; b is the double-layer coating process of HA and PVP on CS-NPs mainly through charge interaction and hydrogen bonding to form PHDCNPs, and c is the process of forming hydrogel through Schiff base reaction.

[0047] Figure 3 SEM and TEM images of γ-CD-MOF, CL-MOF and CS-NPs, HA-CS-NPs and PVP-HA-CS-NPs synthesized in the present invention, wherein a is γ-CD-MOF and CL-MOF; b is CS-NPs, HA-CS-NPs and PVP-HA-CS-NPs.

[0048] Figure 4 This is the infrared spectrum of the sequential controlled release dual-drug loaded injectable hydrogel of Example 1 of the present invention.

[0049] Figure 5 This is a SEM image of the sequential controlled release dual-drug loaded injectable hydrogel according to Example 1 of the present invention, wherein the scale of a is 500 μm, and the scale of b is 10 μm.

[0050] Figure 6 This is a diagram of the gelation process of the sequential controlled release dual-drug loaded injectable hydrogel according to Example 1 of the present invention.

[0051] Figure 7 This is a schematic diagram of the rheological properties experimental results of the sequential controlled release dual-drug loaded injectable hydrogel of Example 1 of the present invention, wherein a is the change of the storage modulus (G') and loss modulus (G") of the hydrogel sample, and b is the change of G' and G" when the angular frequency is 0.1-100 rad / s.

[0052] Figure 8 This is a schematic diagram of the injectability performance experimental results of the sequential controlled release dual-drug loaded injectable hydrogel according to Example 1 of the present invention, wherein a is the change of the viscosity of the hydrogel with the shear rate, and b is the injectability experiment.

[0053] Fig. 9 This is a curve showing the change in swelling rate over time of the sequential controlled release dual-drug loaded injectable hydrogel according to Example 1 of the present invention.

[0054] Fig.10 This is a schematic diagram of the degradation performance experimental results of the sequential controlled release dual-drug loaded injectable hydrogel according to Example 1 of the present invention.

[0055] Fig.11This is a graph showing the drug controlled release performance experimental data of the sequential controlled release dual-drug loaded injectable hydrogels of Examples 1-5 of the present invention, wherein a is the drug release curves of GCV and DEX in Example 1, wherein the start release time of DEX is 1 day after the release of GCV, and b is the drug release curves of GCV in Examples 2-5.

[0056] Fig.12 This is the GCV controlled release fitting curve of the sequential controlled release dual-drug loaded injectable hydrogel of Examples 1-5 of the present invention, a is Example 1, b is Example 2, c is Example 3, d is Example 4, and e is Example 5.

[0057] Fig.13 These are the in vitro cytocompatibility test results of the sequential controlled release dual-drug loaded injectable hydrogel according to Example 1 of the present invention, wherein a is the live / dead cell staining diagram, and b is the cell survival rate.

[0058] Fig.14 These are the results of the animal in vivo safety experiment of the sequential controlled release dual-drug loaded injectable hydrogel of Example 1 of the present invention, wherein a is fundus photography, b is OCT test, and c is a tissue section image after H&E staining.

[0059] Fig.15 These are the in vitro antiviral effectiveness experimental results of the sequential controlled release dual-drug loaded injectable hydrogel according to Example 1 of the present invention.

[0060] Fig.16 The present invention relates to the in vivo antiviral effectiveness experimental results of the sequential controlled release dual-drug loaded injectable hydrogel of Example 1, wherein a is a timeline diagram of Example 1 for treating intraocular HSV-1 infection, b is the retinal morphology of rabbits in different treatment groups observed on the 1st, 3rd, 5th and 7th day after infection, c is the vital signs and slit lamp photos of the different treatment groups on the 14th day after infection, d and e are the retinal inflammation scores and vitreous opacity scores of 6 HSV-1 infected rabbits on the 1st, 3rd, 5th and 7th day after infection, respectively, f is the expression levels of IL-1β, IL-6 and TNF-α in the aqueous humor of each group detected on the 7th day after infection (n=3), and g is the H&E staining picture of the cornea and retina of rabbits in different treatment groups 14 days after infection. Specific implementation method:

[0061] The present invention will be further described in detail below through specific embodiments in conjunction with the accompanying drawings.

[0062] Embodiment 1:

[0063] This embodiment relates to a method for preparing a sequential controlled release dual-drug loaded injectable hydrogel, and the specific steps are as follows:

[0064] Step 1: Preparation of cross-linked γ-cyclodextrin metal organic framework nanoparticles (GCV-CL-MOF) loaded with ganciclovir, the specific steps are as follows:

[0065] (1) Preparation of γ-CD-MOF nanoparticles: 3.24 g of γ-cyclodextrin (γ-CD) and 1.12 g of potassium hydroxide (KOH) were dissolved in 100 mL of deionized water, ultrasonicated for 30 minutes, and filtered to remove insoluble matter. Then, 60 mL of methanol was added, mixed evenly, and heated and stirred in a constant temperature water bath at 50°C until the liquid changed from turbid to clear; then, 1.28 g of polyethylene glycol 20000 (PEG 20000) and 160 mL of methanol were quickly added and crystal precipitation was induced in cold water. After 12 hours, MOF particles were collected by centrifugation, and then the above samples were washed with 30 mL of ethanol and methanol respectively and centrifuged three times. The precipitate was soaked in dichloromethane (DCM) for three days, and then centrifuged and dried in a vacuum oven at 50°C to constant weight to obtain γ-CD-MOF;

[0066] (2) Preparation of CL-MOF: 422.4 mg of γ-CD-MOF and 385.6 mg of diphenyl carbonate (DPC) were added to a mixed solution of 20 mL of dimethylformamide (DMF) and 0.45 mL of triethanolamine (TEA), and reacted with magnetic stirring at 80°C in a constant temperature oil bath for 48 hours. After the reaction was completed, the mixture was cooled to room temperature and 2 volumes of ethanol were added to quench the reaction. The precipitate was then washed thoroughly with water and ethanol, and the precipitate was vacuum dried at 50°C to constant weight to obtain CL-MOF.

[0067] (3) Preparation of CL-MOF loaded with ganciclovir (GCV): 20 mg of GCV and 100 mg of CL-MOF were mixed in 20 mL of ethanol, magnetically stirred in a constant temperature water bath at 35°C for 24 h, and then centrifuged to obtain a precipitate. The precipitate was washed three times with 15 mL of DMF to remove unloaded GCV; the precipitate was vacuum dried at 50°C to constant weight to obtain GCV-CL-MOF nanoparticles;

[0068] Step 2: Preparation of aldehyde-modified β-cyclodextrin solution (GCV-Gel) loaded with ganciclovir

[0069] Take 12g of β-cyclodextrin and add it to 200mL of distilled water. Stir it in a 60℃ water bath until it dissolves. Take it out and cool it. Then add 6.8g of sodium periodate (NaIO 4) was subjected to light-proof magnetic stirring for 3 h, and then 4 mL of ethylene glycol was added and stirred for 2 h; taken out, dialyzed with a MW500 dialysis bag and distilled water for 1-2 days, and then taken out and placed in a beaker for freeze drying to obtain aldehyded β-cyclodextrin (ACD); 40 mg of aldehyded β-cyclodextrin was dissolved in 1 mL of phosphate buffered saline (PBS) to obtain an aldehyded β-cyclodextrin solution with a concentration of 40 mg / mL, and then 0.2 mg of ganciclovir was dispersed into the above aldehyded β-cyclodextrin solution by ultrasonic dispersion to obtain an aldehyded β-cyclodextrin solution loaded with ganciclovir;

[0070] Step 3: preparing chitosan nanoparticles loaded with a second drug and coated with hyaluronic acid and polyvinyl pyrrolidone in sequence from the inside to the outside, the specific steps are as follows:

[0071] (1) Preparation of chitosan nanoparticles loaded with dexamethasone sodium phosphate: First, 28.8 mg of CS (chitosan) was dissolved in 48 mL of acetic acid aqueous solution (1%, v / v) to prepare a CS solution with a concentration of 0.6 mg / mL; secondly, 10 mg of DEX (dexamethasone sodium phosphate) was added to the above CS solution to obtain a DEX-CS solution; then, the mixture was incubated at room temperature at 1.5 mL min -1 24 mL of 0.4 mg / mL sodium tripolyphosphate (TPP) solution was added to the above DEX-CS solution at a rate of , and stirred at 500 rpm for 120 minutes using a magnetic stirrer; finally, the stirred solution was freeze-dried to obtain chitosan nanoparticles loaded with dexamethasone sodium phosphate (DEX-CS-NPs), which were stored at 4°C for further use;

[0072] (2) Hyaluronic acid coating: 20 mg of DEX-CS-NPs were dispersed in 2.0 mL of acetic acid buffer; then, 2.0 mL of acetic acid buffer containing HA (hyaluronic acid) (HA concentration was 0.5 mg / mL) was added, and magnetic stirring was performed at a speed of 1000 rpm for 45 minutes to obtain a dispersion solution; finally, the dispersion solution was filtered through a filter with a pore size of 0.45 μm and freeze-dried to obtain hyaluronic acid-coated chitosan nanoparticles loaded with dexamethasone sodium phosphate (HA-DEX-CS-NPs);

[0073] (3) Coating with polyvinyl pyrrolidone: 20 mg of HA-DEX-CS-NPs was dispersed in 2.0 mL of acetic acid buffer; then, 2.0 mL of acetic acid buffer containing PVP (polyvinyl pyrrolidone) (the concentration of PVP was 0.5 mg / mL) was added, and magnetic stirring was performed at a speed of 1000 rpm for 45 min; finally, the stirred solution was filtered through a filter with a pore size of 0.45 μm and freeze-dried to obtain chitosan nanoparticles (PHDCNPs) loaded with a second drug, which were coated with hyaluronic acid and polyvinyl pyrrolidone from the inside to the outside.

[0074] Step 4: Preparation of sequential controlled release dual-drug loaded injectable hydrogel, the specific steps are as follows:

[0075] (1) Preparing a first dispersion: 5.43 mg of the cross-linked γ-cyclodextrin metal organic framework nanoparticles loaded with ganciclovir prepared in step 1 and 4.64 mg of the chitosan nanoparticles loaded with the second drug and coated with hyaluronic acid and polyvinyl pyrrolidone from the inside to the outside in sequence prepared in step 3 are co-dispersed in 1 mL of the aldehyde-modified β-cyclodextrin solution loaded with 0.2 mg of ganciclovir prepared in step 2 to obtain a first dispersion;

[0076] (2) Preparation of the second dispersion: First, prepare amino hyaluronic acid (NHA): take 0.5g hyaluronic acid (HA) (molecular weight of 100,000-200,000) and dissolve it in 100mL distilled water. After it is completely dissolved, add 8.37g ethylenediamine hydrochloride and 0.05g 4-dimethylaminopyridine (DAMP) and mix them evenly; after mixing, add 6g 1-ethyl-(3-dimethylaminopropyl) carbodiimide (EDC) and 3.7g N-hydroxysuccinimide (NHS), then add NaOH solution to adjust the pH to 5, continue stirring and reacting for two days, during which the pH is maintained at 5, and after two days, adjust the pH to 7 with NaOH to stop the reaction; then dialyze with a dialysis bag (MW 14000) and deionized water for 3-5 days, freeze-dry, and obtain amino hyaluronic acid; add 50mg of amino hyaluronic acid into 1mL PBS solution to prepare a second dispersion with an NHA concentration of 50mg / mL;

[0077] (3) Preparation of dual-drug loaded nanocomposite injectable hydrogel: The first dispersion and the second dispersion were uniformly mixed in a volume ratio of 1:1, and cross-linked by Schiff base reaction between aldehyde groups and amino groups to prepare dual-drug loaded nanocomposite injectable hydrogel. The concentrations of GCV and DEX in the hydrogel were 0.6 mg / mL and 0.4 mg / mL, respectively, denoted as PHDCNPs / CL-MOF-0.5 / Gel-0.1, Gel-0.1 represents that the concentration of GCV loaded in the aldehyde-modified β-cyclodextrin cavity structure in the whole hydrogel system is 0.1 mg / mL; CL-MOF-0.5 represents that the concentration of GCV loaded in CL-MOF in the whole hydrogel system is 0.5 mg / mL.

[0078] The concentration of the phosphate buffer described in this example is 0.01 mol / L, and the pH is 7.4.

[0079] The concentration of the acetic acid buffer is 0.1 mol / L, and the pH is 5.0.

[0080] The schematic diagram of the preparation principle of the sequential controlled release dual-drug loaded injectable hydrogel (hereinafter referred to as injectable hydrogel) involved in this embodiment and the schematic diagram of the structural principle of the composite hydrogel loaded with two drugs, ganciclovir and dexamethasone sodium phosphate, are shown in FIG. Figure 1 and 2 As shown. First, ganciclovir is loaded into a cross-linked γ-cyclodextrin metal organic framework. Secondly, chitosan nanoparticles loaded with dexamethasone sodium phosphate are synthesized by ionic crosslinking of chitosan and sodium tripolyphosphate. The mechanism is that sodium tripolyphosphate exists in the main body of chitosan nanoparticles, bridging the positive charge of the chitosan chain, and then the negatively charged hyaluronic acid combines with the positively charged chitosan on the surface of the nanoparticles by electrostatic interaction, and is further coated on the surface of the chitosan nanoparticles; in addition, since the nitrogen atom on the pyrrolidone ring in polyvinyl pyrrolidone has a certain electrophilicity, it can form a hydrogen bond complex with an organic molecule containing a hydrogen bond donor group. The organic molecule is a hydroxyl compound, a carboxylic acid derivative, an amine and its derivatives, etc.; therefore, the chitosan nanoparticles coated with hyaluronic acid are further coated with polyvinyl pyrrolidone to achieve the purpose of releasing dexamethasone sodium phosphate after 24-48 hours. Finally, PHDCNPs, GCV-Gel and GCV-CL-MOF were embedded in the hydrogel formed by the Schiff base reaction of aldehyde and amino groups to prepare the nanocomposite hydrogel, in which the PVP on the surface of PHDCNPs also produced hydrogen bonding interactions with the hydrogel matrix.

[0081] Embodiment 2:

[0082] This embodiment relates to a method for preparing a dual-drug loaded nanocomposite injectable hydrogel, and the specific steps are as follows:

[0083] Step 1: preparing cross-linked γ-cyclodextrin metal organic framework nanoparticles (GCV-CL-MOF) loaded with ganciclovir, the specific steps are the same as those in Example 1;

[0084] Step 2, preparing an aldehyde-modified β-cyclodextrin solution loaded with ganciclovir: Different from Example 1, the amount of GCV added was 0 mg, and the other steps were the same, that is, a pure ACD PBS solution without GCV was prepared for subsequent cross-linking of the main hydrogel;

[0085] Step 3: preparing chitosan nanoparticles loaded with a second drug and coated with hyaluronic acid and polyvinyl pyrrolidone in sequence from the inside to the outside, the specific steps are the same as those in Example 1;

[0086] Step 4. Preparation of dual-drug loaded nanocomposite injectable hydrogel: Different from Example 1, the amount of GCV-CL-MOF used is 6.52 mg, and the other steps are the same, that is, the dual-drug loaded nanocomposite injectable hydrogel is recorded as PHDCNPs / CL-MOF-0.6 / Gel, CL-MOF-0.6 represents that the concentration of GCV loaded in CL-MOF in the entire hydrogel system is 0.6 mg / mL.

[0087] Embodiment 3:

[0088] This embodiment relates to a method for preparing a sequential controlled release dual-drug loaded injectable hydrogel, and the specific steps are as follows:

[0089] Step 1: preparing cross-linked γ-cyclodextrin metal organic framework nanoparticles (GCV-CL-MOF) loaded with ganciclovir, the specific steps are the same as those in Example 1;

[0090] Step 2, preparing an aldehyde-modified β-cyclodextrin solution loaded with ganciclovir: different from Example 1, the amount of GCV added was 0.4 mg, and the other steps were the same;

[0091] Step 3: preparing chitosan nanoparticles loaded with a second drug and coated with hyaluronic acid and polyvinyl pyrrolidone in sequence from the inside to the outside, the specific steps are the same as those in Example 1;

[0092] Step 4. Preparation of sequential controlled release dual-drug loaded injectable hydrogel: Different from Example 1, the dosage of GCV-CL-MOF is 4.35 mg, and the other steps are the same, that is, the prepared sequential controlled release dual-drug loaded injectable hydrogel is recorded as PHDCNPs / CL-MOF-0.4 / Gel-0.2, Gel-0.2 represents that the concentration of GCV loaded in the ACD cavity structure in the entire hydrogel system is 0.2 mg / mL; CL-MOF-0.4 represents that the concentration of GCV loaded in CL-MOF in the entire hydrogel system is 0.4 mg / mL.

[0093] Embodiment 4:

[0094] This embodiment relates to a method for preparing a sequential controlled release dual-drug loaded injectable hydrogel, and the specific steps are as follows:

[0095] Step 1: preparing cross-linked γ-cyclodextrin metal organic framework nanoparticles (GCV-CL-MOF) loaded with ganciclovir, the specific steps are the same as those in Example 1;

[0096] Step 2, preparing an aldehyde-modified β-cyclodextrin solution loaded with ganciclovir: different from Example 1, the amount of GCV added was 0.6 mg, and the other steps were the same;

[0097] Step 3: preparing chitosan nanoparticles loaded with a second drug and coated with hyaluronic acid and polyvinyl pyrrolidone in sequence from the inside to the outside, the specific steps are the same as those in Example 1;

[0098] Step 4. Preparation of sequential controlled release dual-drug loaded injectable hydrogel: Different from Example 1, the dosage of GCV-CL-MOF is 3.26 mg, and the other steps are the same, that is, the prepared sequential controlled release dual-drug loaded injectable hydrogel is recorded as PHDCNPs / CL-MOF-0.3 / Gel-0.3, Gel-0.3 represents that the concentration of GCV loaded in the ACD cavity structure in the entire hydrogel system is 0.3 mg / mL; CL-MOF-0.3 represents that the concentration of GCV loaded in CL-MOF in the entire hydrogel system is 0.3 mg / mL.

[0099] Embodiment 5:

[0100] This embodiment relates to a method for preparing a sequential controlled release dual-drug loaded injectable hydrogel, and the specific steps are as follows:

[0101] Step 1, preparing an aldehyde-modified β-cyclodextrin solution loaded with ganciclovir: different from Example 1, the amount of GCV added was 1.2 mg, and the other steps were the same;

[0102] Step 2: preparing chitosan nanoparticles loaded with a second drug and coated with hyaluronic acid and polyvinyl pyrrolidone in sequence from the inside to the outside, the specific steps are the same as those in Example 1;

[0103] Step 3. Preparation of sequential controlled release dual-drug loaded injectable hydrogel: The difference from Example 1 is that when preparing the first dispersion, 4.64 mg of chitosan nanoparticles loaded with the second drug and coated with hyaluronic acid and polyvinyl pyrrolidone from the inside to the outside prepared in step 2 are dispersed into 1 mL of the aldehyde-modified β-cyclodextrin solution loaded with ganciclovir prepared in step 1 to obtain a first dispersion; then the first dispersion and the second dispersion are mixed evenly, and the obtained sequential controlled release dual-drug loaded injectable hydrogel is recorded as PHDCNPs / Gel-0.6, where Gel-0.6 represents that the concentration of GCV loaded in the ACD cavity structure in the entire hydrogel system is 0.6 mg / mL.

[0104] The difference between this embodiment and embodiment 1 is that no GCV-loaded CL-MOF nanoparticles are added to this system, and all 1.2 mg of GCV is loaded into the ACD cyclodextrin molecular cage and cross-linked into the main gel.

[0105] Embodiment 6:

[0106] In this example, the sequential controlled release dual-drug loaded injectable hydrogels prepared in Examples 1-5 were characterized and their performance tested.

[0107] Test Example 1:

[0108] The γ-CD-MOF, CL-MOF, CS-NPs, HA-CS-NPs and PVP-HA-CS-NPs synthesized in Example 1 were characterized by SEM and TEM morphology, and the results are as follows: Figure 3 shown.

[0109] Figure 3 The upper two images in a are SEM images of γ-CD-MOF and CL-MOF, and the lower two are locally enlarged TEM images; Figure 3 b is the TEM image of CS-NPs, HA-CS-NPs and PVP-HA-CS-NPs, and the three images below are partial enlargements of the three images above. Figure 3 It can be clearly seen that γ-CD-MOF and CL-MOF have regular shapes, and CS-NPs have achieved double-layer coating of HA and PVP.

[0110] Test Example 2:

[0111] This test involves the characterization of the sequential controlled release dual-drug loaded injectable hydrogels prepared in Examples 1-5, and Example 1 is used as a representative to perform infrared spectroscopy characterization. Figure 4 The infrared spectra of GCV-Gel and PHDCNPs / CL-MOF-0.5 / Gel-0.1 were at 1632 cm -1 and 1634cm -1A carbon-nitrogen bond (C=N) appeared at the hydrogel, indicating the formation of a Schiff base bond, which shows that the compounding of drug-loaded nanoparticles into the hydrogel does not affect the formation of the cross-linked structure of the hydrogel.

[0112] Test Example 3:

[0113] In order to study the microscopic morphology of the hydrogel, the SEM image of the injectable hydrogel of Example 1 was tested, and the results are as follows: Figure 5 As shown. Figure 5 a It can be observed that the injectable hydrogel has a pore size of 66.45 ± 21.85 μm. Figure 5 In b, it can be clearly seen that PHDCNPs and GCV-CL-MOF are suspended in the hydrogel in the form of spherical and cubic structures, respectively.

[0114] Test Example 4:

[0115] In order to observe the gelation process of the hydrogel more intuitively, the gelation process of the Schiff base reaction was observed by the vial inversion method at room temperature, as shown in Figure 6 shown.

[0116] Test Example 5:

[0117] The rheological properties of the injectable hydrogel of Example 1 were tested, and the results were as follows: Figure 7-8 As shown, from Figure 7 a It can be observed that the gel point of the injectable hydrogel is around 44s (<180s), which meets the gelation time range required by clinical practice; Figure 7 b It can be observed that in the range of angular frequency of 0.1-100 rad / s, G' is always greater than G", indicating that a sol-gel transition has been formed. Figure 8 a It can be observed that the viscosity of the injectable hydrogel gradually decreases with the increase of shear rate, which lays the rheological foundation for the injectability of the hydrogel. Figure 8 b illustrates that the hydrogel has shear thinning properties and is injectable.

[0118] Test Example 6:

[0119] This test involves the controlled release of drugs in sequential controlled release dual-drug loaded injectable hydrogels, which is affected by the degradation behavior of the hydrogel after the hydrogel reaches swelling equilibrium. Taking the injectable hydrogel of Example 1 as an example, the results are as follows: Figure 9-10 As shown, the injectable hydrogel can reach swelling equilibrium in about 10 hours, and the swelling equilibrium ratio is 28.7±1.4% (<50%), which meets the hydrogel swelling rate range required by clinical requirements. The degradation rate of the injectable hydrogel reaches 78.4±2.4% within 20 days.

[0120] The injectable hydrogels of Examples 1-5 were subjected to drug controlled release experiments. The results are as follows: Fig.11 shown. Fig.11 a is the release curve of the two drugs GCV and DEX in the injectable hydrogel of Example 1. It can be seen that the second drug DEX begins to be released 24 hours after the release of GCV; the release amount of GCV drug is 73.9±2.2%. Fig.11 b is the GCV release curve of the injectable hydrogel of Example 2-5. It can be seen that the drug release amounts of the injectable hydrogel of Example 2-5 are 71.4±2.1%, 76.5±2.3%, 75.0±2.3% and 81.5±2.4%, respectively. It can be seen that the GCV drug release rate of Examples 1-5 is close to the degradation rate of the injectable hydrogel, which is also in line with the sustained-release drug mechanism of drug-loaded hydrogels, because the drug release behavior of drug-loaded hydrogels is inevitably affected by the degradation of the hydrogel skeleton, and its degradation rate is much lower than that of the reported drugs, which is suitable for long-term drug controlled release. At the same time, Fig.12 As for the GCV controlled release fitting curves of the sequential controlled release dual-drug loaded injectable composite hydrogels of Examples 1-5 of the present invention, it can be seen that Example 1 can achieve quasi-zero-order release of GCV drug controlled release.

[0121] Embodiment 7:

[0122] This example relates to in vitro and in vivo safety and efficacy experiments of the composite hydrogel prepared in Example 1.

[0123] 1. In vitro safety experiment:

[0124] The PHDCNPs / CL-MOF-0.5 / Gel-0.1 prepared in Example 1 was subjected to a cytotoxicity test. A cell culture medium (DMEM / F12) solution containing 10% fetal bovine serum (FBS) and 1% antibiotics / antimycotics was used to culture the cells in a flask at 37°C and 5% CO. 2 Human umbilical vein endothelial cells (HUVECs) were cultured in an environment. The original culture medium of the experimental group was removed, and 1000 μL of hydrogel extract was added to the experimental group, and 1000 μL of fresh cell culture medium was added to the control group. HUVECs were evaluated using a live / dead kit. Live cells and dead cells were stained green and red, respectively, and images were captured by laser scanning confocal microscopy; 100 μL of hydrogel extract was added to the 96-well plate in the experimental group. 100 μL of fresh cell culture medium was added to the control group, and cell culture medium was only added to the wells without HUVECs in the blank group. After incubation for 24 hours, 10 μL of CCK-8 solution was added to the wells, and then transferred to a cell culture incubator for 2 hours. The absorbance was measured at 450 nm using a microplate reader to calculate the cell viability, and the results are shown in Fig.13 shown.

[0125] from Fig.13As can be seen from a, a large number of HUVECs (living cells) with green fluorescence were observed in the experimental group in the entire field of view, and only a small number of scattered red dots (dead cells) were present in the fluorescent image. Fig.13 b It can be seen that the in vitro viability of HUVECs in the control group and Gel-0.1 / CL-MOF-0.5 / PNPs group was 97.0% and 98.4%, respectively, indicating that the injectable hydrogel was not only non-cytotoxic compared with the control group, but also did not interfere with cell growth or differentiation. Therefore, PHDCNPs / CL-MOF-0.5 / Gel-0.1 hydrogel exhibited ideal cell compatibility.

[0126] 2. In vivo safety test:

[0127] This example involves testing the in vivo safety of the PHDCNPs / CL-MOF-0.5 / Gel-0.1 sequential controlled release dual drug loaded injectable hydrogel prepared in Example 1. The specific testing method and results are as follows:

[0128] Experimental procedures: Healthy New Zealand white rabbits (male, 3-4 months old, weighing 2.0-2.5 kg, 10 rabbits) were generally anesthetized with 3% sodium pentobarbital (50.0 mg / kg), supplemented with topical anesthesia with hydrochloric acid eye drops. Use a 1 mL syringe to inject 0.04 mL of Example 1 PHDCNPs / CL-MOF-0.5 / Gel-0.1 or PBS (control group) into the rabbit's vitreous through the scleral margin 1.5 mm away from the edge. Antibiotic ointment was used for postoperative treatment to prevent bacterial infection. 14 days after surgery, slit lamp examination, fundus photography, OCT, and H&E staining were used to evaluate the effects of Example 1 or PBS on rabbit eyes. The results are as follows: Fig.14 As shown. Fig.14 It can be seen that compared with the control group, in the PHDCNPs / CL-MOF-0.5 / Gel-0.1 group of Example 1, the rabbit conjunctiva had no obvious congestion or swelling, the cornea was transparent, no inflammation was observed in the anterior chamber, the lens had no obvious opacity, the vitreous was clear, the retinal blood vessels were normal, and no obvious abnormalities were observed ( Fig.14 a). Further examination with OCT showed no significant changes in the cornea and retina compared with the control group ( Fig.14 b). H&E staining results showed that there was no obvious inflammatory reaction in the corneal tissue of both groups ( Fig.14 c).

[0129] 3. In vitro effectiveness test:

[0130] The immunofluorescence assay was used to determine the anti-herpes simplex virus type Ⅰ (HSV-1, McKrae strain, 1×10 8 PFU / mL) effectiveness. The cells were divided into four groups: a blank experimental group, a virus control group, an injectable hydrogel treatment group of Example 1, and a hydrogel treatment group without DEX (the only difference from Example 1 is that it does not contain DEX), and then cultured in 2% FBS medium for 24 hours. The cells were then fixed with 10% paraformaldehyde, permeabilized with 0.1% Triton-X, labeled with a primary antibody, and labeled with a FITC-conjugated secondary antibody when necessary. Observation was performed using a confocal microscope, a 63╳ oil objective, and the pinhole was set to 1 ventilation block. Cell surface proteins were labeled with antibodies before fixation, and fluorescence was quantified using ImageJ software. By using primer sets for HSV1-GB, HSV1-GK, and HSV1-ICP0, the gene expression levels compared with the control group were analyzed using the comparative Ct method 2^(-ΔΔCt) to analyze the expression results, and GAPDH was used as the internal reference gene needle for normalization calculation. The effects of the extracts of the hydrogel without DEX on the first and second days (named NO-DEX-1 and NO-DEX-2) and the extracts of the injectable hydrogel of Example 1 on the first and second days (named DEX-1 and DEX-2) on viral infection were compared, and the HSV-1 group was used as the control group. The results are shown in FIG. Fig.15 shown.

[0131] from Fig.15 It can be seen that the virus titers of NO-DEX-1, NO-DEX-2, DEX-1 and DEX-2 groups decreased significantly (P<0.0001), indicating that these four groups have an inactivation effect on HSV-1 infection, and when the extract of the first day after the injectable hydrogel of Example 1 is added to the system, since DEX has not been released, the antiviral effect is similar to that of NO-DEX-1 (the number of viruses remaining is less than 200); the extract of the second day after the injectable hydrogel of Example 1 is added, the antiviral effect of HSV-1 is better than that of NO-DEX-2 without DEX, because DEX in the injectable hydrogel of Example 1 has begun to be released at this time, which can assist GCV in antiviral treatment. It shows that the overall antiviral effect of the injectable hydrogel of Example 1 is significant, and the interval release of DEX and GCV, the synergistic treatment scheme of the two is effective.

[0132] IV. In vivo effectiveness test:

[0133] A rabbit model of ocular viral infection was established to test the anti-HSV-1 effectiveness of the injectable hydrogel prepared in Example 1 in animals. Twelve New Zealand white rabbits were randomly divided into a control group and an injectable hydrogel group prepared in Example 1 PHDCNPs / CL-MOF-0.5 / Gel-0.1. Anesthesia was induced with 3% sodium pentobarbital (50.0 mg / kg), supplemented with hydrochloric acid eye drops for surface anesthesia. A 1 mL syringe was used to inject 0.01 mL of HSV-1 (1×10 6 PFU / mL). Antibiotic ointment was applied after surgery to prevent bacterial infection. Anesthesia was performed in the same way on the first day after surgery, and then 0.04 mL of PBS or the injectable hydrogel of Example 1 was administered according to the grouping. Fundus photography was performed 1, 3, 5, and 7 days after infection, and the evaluation was performed according to the standard scoring criteria for retinal inflammation and international vitreous opacity. Aqueous humor specimens were collected on the 7th day, and the viral load was detected by the TCID50 method, and the levels of inflammatory factors (IL-6, TNF-α, IL1-β) were detected by ELISA. Vital signs were observed from the 7th to the 14th day. The morphology of the eyeballs of each group was observed under a slit lamp microscope on the 14th day after modeling, and then the experimental rabbits were killed, and the eyeballs were removed for H&E staining for histological analysis. The results are shown in Fig.16 shown.

[0134] Fig.16 a Demonstrated the feasibility of using sustained-release materials in intravitreal antiviral therapy. Fig.16 b It can be seen that on the first day of HSV-1 infection, all groups showed mild vitreous opacity with or without optic disc edema; on the third day, the control group had obvious vitreous opacity, accompanied by extensive hemorrhage and necrotic foci around the optic disc, and the injectable hydrogel group of Example 1 had obvious vitreous opacity, but no hemorrhage or necrotic foci were found. This indicates that the release of GCV loaded in the hydrogel can better inhibit the proliferation of HSV-1. Over time, the necrotic lesions in the control group gradually increased until the vitreous opacity worsened and the retina was no longer visible, while no retinal inflammatory lesions were found in the Example 1 group, and the vitreous opacity gradually alleviated. Further evaluation by retinal inflammation score ( Fig.16 d) showed that compared with the control group, the Example 1 group showed better antiviral effect, with statistically significant differences. This confirms that the GCV loaded in the injectable hydrogel of Example 1 has a good antiviral effect, and the delayed release of DEX does not cause immunosuppression in the body, leading to viral proliferation. Evaluation based on the International Vitreous Opacity Score ( Fig.16 e) showed that compared with the control group, the vitreous opacity of the Example 1 group was reduced more significantly, which was statistically significant, indicating that the Example 1 can continuously release DEX to reduce inflammation, thereby reducing vitreous opacity. In order to further study the anti-inflammatory effect of Example 1, aqueous humor was extracted on the 7th day after infection to detect inflammatory factors. Fig.16 As shown in Figure 5, compared with the control group, the levels of inflammatory factors IL-1β, IL-6, and TNF-α in the Example 1 group were reduced, verifying its anti-inflammatory effect. Fig.16 As shown in Figure c, during the observation from the 7th to the 14th day, the experimental rabbits in the control group developed symptoms of viral encephalitis, such as nystagmus, drooling, mandibular spasm, unsteady standing, and angular arching, leading to death, with a mortality rate of 66.67% (4 / 6). This may be due to the fact that HSV-1 crosses the connection between the eye and brain immune systems, leading to viral encephalitis. In contrast, the Example 1 group did not show similar symptoms leading to death, indicating that PHDCNPs / CL-MOF-0.5 / Gel-0.1 can fight viruses and reduce mortality. On the 14th day, the anterior segment of the experimental rabbits was photographed. The control group had obvious conjunctival congestion, abundant purulent secretions, and obvious iris congestion; while PHDCNPs / CL-MOF-0.5 / Gel-0.1 had no obvious conjunctival and iris abnormalities, further confirming the antiviral and anti-inflammatory effects of PHDCNPs / CL-MOF-0.5 / Gel-0.1. H&E staining ( Fig.16 g) showed that the rats in the control group had severe choroidal edema, cystic necrosis, unclear retinal tissue layers, and extensive inflammatory cell infiltration; compared with the control group, the retinal layers of the PHDCNPs / CL-MOF-0.5 / Gel-0.1 group were clear, the vitreous opacity was milder, and the inflammatory cell infiltration was less.

Claims

1. A method for preparing a sequential controlled release dual-drug loaded injectable hydrogel, characterized in that: The following steps are involved: A cross-linked γ-cyclodextrin metal organic framework loaded with a first drug and chitosan nanoparticles loaded with a second drug and coated with hyaluronic acid and polyvinyl pyrrolidone from the inside out are dispersed in an aldehyde-modified β-cyclodextrin solution loaded with the first drug to form a first dispersion; amino-modified hyaluronic acid is dispersed in a PBS buffer solution to form a second dispersion; the first dispersion and the second dispersion are then mixed to prepare a sequential controlled release dual-drug loaded injectable hydrogel through a Schiff base reaction; The preparation method of the chitosan nanoparticles loaded with the second drug and coated with hyaluronic acid and polyvinyl pyrrolidone in sequence from the inside to the outside is as follows: the second drug is added to a chitosan acetic acid solution, and then a sodium tripolyphosphate solution is added, and freeze-dried to prepare chitosan nanoparticles loaded with the second drug; the above product is dispersed in an acetic acid buffer, and then an acetic acid buffer containing hyaluronic acid is added, and freeze-dried to obtain chitosan nanoparticles coated with hyaluronic acid; then the chitosan nanoparticles coated with hyaluronic acid are dispersed in an acetic acid buffer, and then an acetic acid buffer containing polyvinyl pyrrolidone is added, and freeze-dried to obtain chitosan nanoparticles loaded with the second drug and coated with hyaluronic acid and polyvinyl pyrrolidone in sequence from the inside to the outside.

2. The method for preparing the sequential controlled release dual-drug loaded injectable hydrogel according to claim 1, characterized in that: The preparation method of the cross-linked γ-cyclodextrin metal organic framework carrying the first drug is as follows: adding the γ-cyclodextrin metal organic framework material and diphenyl carbonate into a mixed solution of dimethylformamide and triethanolamine, stirring and reacting at a constant temperature of 60-100°C for 30-60 hours, then cooling to room temperature, washing the precipitate, and vacuum drying to obtain the cross-linked γ-cyclodextrin metal organic framework material; then dispersing the first drug and the cross-linked γ-cyclodextrin metal organic framework in ethanol, stirring for 10-40 hours, centrifuging, washing, and drying to obtain the cross-linked γ-cyclodextrin metal organic framework material carrying the first drug.

3. The method for preparing the sequential controlled release dual-drug loaded injectable hydrogel according to claim 1, characterized in that: The preparation method of the aldehyded β-cyclodextrin solution loaded with the first drug is as follows: dissolving the aldehyded β-cyclodextrin in a PBS solution to prepare the aldehyded β-cyclodextrin solution, and dispersing the first drug into the aldehyded β-cyclodextrin solution to obtain the aldehyded β-cyclodextrin solution loaded with the first drug.

4. The method for preparing the sequential controlled release dual-drug loaded injectable hydrogel according to claim 1, characterized in that: In the aldehyde-modified β-cyclodextrin solution loaded with the first drug, the concentration of aldehyde-modified β-cyclodextrin is 40 mg / mL; and the concentration of amino-modified hyaluronic acid in the second dispersion is 50 mg / mL.

5. The method for preparing the sequential controlled release dual-drug loaded injectable hydrogel according to claim 1, characterized in that: The volume ratio of the first dispersion liquid to the second dispersion liquid is 1:

1.

6. The method for preparing the sequential controlled release dual-drug loaded injectable hydrogel according to claim 1, characterized in that: The first drug is a water-insoluble drug, and the second drug is a water-soluble drug.

7. The method for preparing the sequential controlled release dual-drug loaded injectable hydrogel according to claim 1, characterized in that: The pH of the sodium tripolyphosphate solution was 7.4 and the -1 The rate was added to the chitosan acetic acid solution.

8. A sequential controlled-release dual-drug loaded injectable hydrogel obtained by the preparation method according to any one of claims 1 to 7.

9. Use of the sequential controlled release dual-drug loaded injectable hydrogel according to claim 8 in the preparation of a drug for treating acute retinal necrosis, wherein the first drug is ganciclovir, and the second drug is dexamethasone sodium phosphate, ganciclovir can be released in zero order, and dexamethasone sodium phosphate begins to be released 24 hours after the first drug is released, and the drug controlled release time is greater than 20 days.

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