Controllable in-situ bionic hydrogel system and application thereof in preparation of ophthalmic medicine

Abstract

The invention relates to a controllable in-situ bionic hydrogel system and application thereof in preparation of ophthalmic drugs. The in-situ bionic hydrogel system disclosed by the invention comprises 4r-PEG (Polyethylene Glycol) coated DMPE (Dimethylolpropane Polyethylene)-PEG (Polyethylene Glycol) hydrogel and / or DMPE-PEG coated DMPE-PEG hydrogel, the 4r-PEG (at) DMPE-PEG hydrogel comprises a 4r-PEG nano particle and a DMPE-PEG nano particle, wherein the 4r-PEG nano particle and the DMPE-PEG nano particle are connected with a maleimide group through a sulfydryl group; the DMPE-PEG coated DMPE-PEG hydrogel is prepared from DMPE-PEG nano particles which are connected with a maleimide group through a sulfydryl group. The DMPE-PEG-coated DMPE-PEG hydrogel can be used for bionic artificial vitreous bodies, can simulate the physical properties of real vitreous bodies, and provides a better treatment scheme for rehabilitation after vitrectomy. Meanwhile, the 4r-PEG-coated DMPE-PEG hydrogel is used for bionic artificial tear films, and a new treatment means is provided for ocular surface diseases such as xerophthalmia. The hydrogel system provided by the invention has good biological safety and controllable swelling ratio, can load drugs with various properties, and provides a novel drug delivery system for eye disease treatment.

Description

Technical Field

[0001] The invention relates to the technical field of biomaterials, and in particular to an adjustable in-situ bionic hydrogel system and application thereof in preparing ophthalmic drugs. Background Art

[0002] As one of the commonly used ophthalmic drug dosage forms, traditional ophthalmic gels are mostly semi-solid and have a certain viscosity. They are widely used in the treatment of ocular surface-related diseases such as dry eye, keratitis, and ocular surface damage. However, its own characteristics also bring some limitations. Due to its viscosity, after being dripped into the eye, it will temporarily interfere with vision, causing blurred vision and inconvenience to patients. Compared with eye drops, ophthalmic gels are sticky in texture, and patients are prone to foreign body sensation when using them, and it is relatively difficult to clean up the residue after use. In addition, traditional ophthalmic gels are often difficult to achieve uniform distribution on the ocular surface, and it is easy for the drug to not be evenly dispersed and released, which may have a certain impact on the treatment effect. The above-mentioned factors have limited the widespread clinical application of ophthalmic gels to a certain extent. For example, for patients with dry eye, artificial tears have defects such as a single ingredient, inability to replace natural tears, short ocular surface retention time, and fast loss. Given that ophthalmic gels can easily cause blurred vision in users, when treating dry eyes in clinical practice, commonly used ophthalmic gels such as carbomer ophthalmic gel and vitamin A palmitate ophthalmic gel are recommended to be used before bedtime to minimize their impact on daily vision. In addition, at this stage, ophthalmic gels have the limitation of being difficult to load hydrophobic drugs. Clinically, a strategy of combining ophthalmic gels with eye drops is often used to construct a multi-pathway treatment model to deal with dry eyes. Therefore, the development of new ophthalmic gels with excellent performance, easy use, and the ability to effectively load multiple drugs has become an urgent problem to be solved. This is of great significance for improving the treatment effect of ocular surface diseases and expanding the application range of ophthalmic preparations.

[0003] Due to the shortcomings of existing eye gels, ocular surface-related diseases urgently need the development of new eye gels to meet the treatment needs. Similarly, fundus-related diseases also have an urgent need for new eye gels during treatment. Taking vitrectomy treatment-related diseases as an example, a bionic vitreous material that can be used for filling is urgently needed after surgery to promote the patient's recovery process. In clinical practice, expandable gas or silicone oil is usually used for vitreous filling. However, expandable gas is absorbed quickly in the eye, usually most of it will be absorbed within 1-2 days, and its effective time for pressing the retina is short. In addition, small droplets of silicone oil after emulsification of silicone oil will cause inflammatory reactions in the eye and affect the normal physiological functions of intraocular tissues. And silicone oil usually requires additional removal surgery 3-6 months after surgery, which increases the pain and economic burden of patients, and the secondary surgery itself also has certain risks, such as infection and retinal detachment again. In short, the development of new eye gels is of vital significance to improving the treatment effects of ocular surface and fundus diseases and promoting the advancement of ocular disease treatment technology. Summary of the invention

[0004] In order to solve the above technical problems, the present invention provides an adjustable in-situ biomimetic hydrogel system and its application in the preparation of ophthalmic drugs. The present invention aims to develop a hydrogel system that utilizes bioorthogonal reaction or click chemistry technology to have in-situ gelation ability and whose physical properties can be precisely controlled. The present invention is based on the fast-reacting Michael addition reaction, that is, the reaction of thiol and maleimide to promote the polymer 4r-PEG (four-arm PEG) and DMPE-PEG to prepare in-situ biomimetic hydrogel, which has good biosafety, adjustable physical structure, controllable swelling rate, and can load drugs of various properties. In addition, the addition reaction of thiol and maleimide is mild, which is suitable for in-situ polymerization reaction of organisms.

[0005] The in-situ biomimetic hydrogel system of the present invention shows unique adaptability and application advantages in the field of ocular drug delivery, and is expected to become a key component of a new drug delivery system in the treatment of ocular diseases. The present invention designs two PEG-based nanoparticles, namely four-arm PEG nanoparticles (i.e., 4r-PEG NPs) and DMPE-PEG nanoparticles (i.e., DMPE-PEGNPs), and the outer layer of the nanoparticles carries SH or MAL groups. Through the addition reaction between SH and MAL groups, the nanoparticles are combined in pairs to form three new hydrogels with different properties in the tissue in situ (named 4r-PEG@4r-PEG, 4r-PEG@DMPE-PEG and DMPE-PEG@DMPE-PEG, respectively). The above-mentioned hydrogels have physical morphologies, drug loading ranges and pharmacokinetics of different properties. Among them, DMPE-PEG@DMPE-PEG hydrogels can be used for artificial biomimetic vitreous, and 4r-PEG@DMPE-PEG hydrogels can be used for biomimetic artificial tear film.

[0006] The present invention is achieved through the following technical solutions:

[0007] The first object of the present invention is to provide an adjustable in situ biomimetic hydrogel system, wherein the in situ biomimetic hydrogel system comprises 4r-PEG@DMPE-PEG hydrogel and / or DMPE-PEG@DMPE-PEG hydrogel;

[0008] The 4r-PEG@DMPE-PEG hydrogel includes 4r-PEG nanoparticles and DMPE-PEG nanoparticles connected via thiol groups and maleimide groups;

[0009] The DMPE-PEG@DMPE-PEG hydrogel includes DMPE-PEG nanoparticles connected to maleimide groups via thiol groups.

[0010] In one embodiment of the present invention, the mass ratio of 4r-PEG nanoparticles to DMPE-PEG nanoparticles in the 4r-PEG@DMPE-PEG hydrogel is 8 to 10:10.

[0011] The second object of the present invention is to provide a method for preparing the controllable in-situ biomimetic hydrogel system, comprising the following steps:

[0012] (1) preparing a 4r-PEG nanoparticle solution containing a thiol group and a 4r-PEG nanoparticle solution containing a maleimide group respectively;

[0013] (2) preparing a DMPE-PEG nanoparticle solution containing a thiol group and a DMPE-PEG nanoparticle solution containing a maleimide group, respectively;

[0014] (3) subjecting any two of the nanoparticle solutions obtained in step (1) and step (2) to an addition reaction to obtain an adjustable in-situ bionic hydrogel system.

[0015] The in-situ biomimetic hydrogel system of the present invention includes 4r-PEG@DMPE-PEG hydrogel and / or DMPE-PEG@DMPE-PEG hydrogel, and the preparation steps are as follows:

[0016] The 4r-PEG@DMPE-PEG hydrogel of the present invention is obtained by the following preparation method:

[0017] 1) preparing 4r-PEG nanoparticle solutions containing thiol groups or maleimide groups respectively;

[0018] 2) preparing DMPE-PEG nanoparticle solutions containing thiol groups or maleimide groups respectively;

[0019] 3) The 4r-PEG nanoparticle solution prepared in step 1) and the DMPE-PEG nanoparticle solution prepared in step 2) are mixed and reacted to obtain the 4r-PEG@DMPE-PEG hydrogel.

[0020] In one embodiment of the present invention, the 4r-PEG nanoparticle solution containing a thiol group or a maleimide group is obtained by dispersing and dissolving 4r-PEG containing a thiol group or a maleimide group in a buffer.

[0021] In one embodiment of the present invention, the buffer is selected from one or more of phosphate buffer, Tris-HCl buffer, borate buffer, HEPES buffer and acetate buffer;

[0022] In one embodiment of the present invention, the DMPE-PEG nanoparticle solution is obtained by dissolving DMPE-PEG containing a thiol group or a maleimide group in a good solvent to obtain an organic phase layer; adding an aqueous phase for ultrasonic dispersion, and removing the organic phase.

[0023] In one embodiment of the present invention, the good solvent is selected from one or more of chloroform, ether, ethanol, propanol and n-hexane.

[0024] In one embodiment of the present invention, the aqueous phase is selected from one or more of phosphate buffer, Tris-HCl buffer, borate buffer, HEPES buffer and acetate buffer.

[0025] The DMPE-PEG@DMPE-PEG hydrogel of the present invention is prepared by the following method:

[0026] The DMPE-PEG nanoparticle solution containing a thiol group and the DMPE-PEG nanoparticle solution containing a maleimide group are mixed and subjected to addition reaction to obtain the DMPE-PEG@DMPE-PEG hydrogel.

[0027] In one embodiment of the present invention, the DMPE-PEG nanoparticle solution is obtained by dissolving DMPE-PEG containing a thiol group or a maleimide group in a good solvent to obtain an organic phase layer; adding an aqueous phase for ultrasonic dispersion, and removing the organic phase.

[0028] In one embodiment of the present invention, the good solvent is selected from one or more of chloroform, ether, ethanol, propanol and n-hexane.

[0029] In one embodiment of the present invention, the aqueous phase is selected from one or more of phosphate buffer, Tris-HCl buffer, borate buffer, HEPES buffer and acetate buffer.

[0030] The third object of the present invention is to provide the application of the in-situ bionic hydrogel system in the preparation of bionic artificial vitreous, which can be used to treat related diseases requiring vitrectomy, such as bacterial endophthalmitis, etc., wherein the bacteria is Staphylococcus aureus.

[0031] In one embodiment of the present invention, the in situ biomimetic hydrogel system includes DMPE-PEG@DMPE-PEG hydrogel.

[0032] The fourth object of the present invention is to provide the use of the in situ bionic hydrogel system in the preparation of a bionic artificial tear film, which can be used to treat ocular surface-related diseases, such as dry eye.

[0033] In one embodiment of the present invention, the in situ biomimetic hydrogel system includes 4r-PEG@DMPE-PEG hydrogel.

[0034] In one embodiment of the present invention, the concentration of the 4r-PEG@DMPE-PEG hydrogel is ≤20 mg / mL.

[0035] The fifth object of the present invention is to provide the use of the in-situ bionic hydrogel system in the preparation of drugs for treating eye diseases.

[0036] In one embodiment of the present invention, the eye disease includes one or more of bacterial endophthalmitis, dry eye, conjunctivitis, dacryocystitis, scleritis, uveitis, retinal detachment, retinal vasculopathy, retinoblastoma, choroidal melanoma and optic neuritis.

[0037] The sixth object of the present invention is to provide an ocular drug preparation, comprising a drug carrier and a drug; the drug carrier is the in-situ bionic hydrogel system.

[0038] In one embodiment of the present invention, the drug is selected from one or more of anti-infective drugs, anti-allergic drugs, anti-angiogenic drugs and anti-inflammatory drugs.

[0039] In one embodiment of the present invention, the anti-infective drugs include levofloxacin, vancomycin, tobramycin, acyclovir, ganciclovir, etc.; the anti-allergic drugs include olopatadine, azelastine, etc.; the anti-angiogenic drugs include ranibizumab, etc.; the anti-inflammatory drugs include cyclosporine A, etc.

[0040] The above technical solution of the present invention has the following advantages compared with the prior art:

[0041] 1. Novel drug delivery system: The present invention has developed a hydrogel system with in situ gelation ability and precisely controllable physical properties. This hydrogel system is based on 4r-PEG and DMPE-PEG nanoparticles, has good biosafety and controllable swelling rate, and can load drugs of various properties, providing a new drug delivery system for the treatment of eye diseases.

[0042] 2. Improve patient experience: Traditional eye gels are inconvenient to use and can easily cause blurred vision. The hydrogel system of the present invention reduces interference with patient vision by improving physical properties and provides a more comfortable user experience.

[0043] 3. Improve therapeutic effect: The hydrogel system can achieve uniform dispersion and release of drugs, improve the therapeutic effect of ocular surface diseases, and help improve the clinical effect of the treatment of ocular surface and fundus diseases.

[0044] 4. Application of bionic materials: The DMPE-PEG@DMPE-PEG hydrogel mentioned in the present invention can be used to mimic artificial vitreous, which can simulate the physical properties of real vitreous and provide a better treatment plan for rehabilitation after vitrectomy. At the same time, 4r-PEG@DMPE-PEG hydrogel is used to mimic artificial tear film, providing a new treatment for ocular surface diseases such as dry eye.

[0045] 5. Improvement of drug sustained release and treatment efficiency: The hydrogel system of the present invention exhibits good drug sustained release behavior and can maintain the concentration of the drug in the eye for a long time, thereby improving the treatment efficiency, reducing the frequency of medication for patients, and reducing the side effects of the drug. BRIEF DESCRIPTION OF THE DRAWINGS

[0046] In order to make the content of the present invention more clearly understood, the present invention is further described in detail below according to specific embodiments of the present invention in conjunction with the accompanying drawings, wherein:

[0047] Figure 1 The particle size distribution diagram of 4r-PEG nanoparticles and DMPE-PEG nanoparticles with SH or MAL groups on the surface (a) and the schematic diagram of the preparation of three hydrogels (4r-PEG@4r-PEG, 4r-PEG@DMPE-PEG, DMPE-PEG@DMPE-PEG);

[0048] Figure 2 The rheological behavior of the above hydrogels with different concentrations was observed over time by rheometer; and the microscopic morphology of the three hydrogels of 4r-PEG@4r-PEG, 4r-PEG@DMPE-PEG and DMPE-PEG@DMPE-PEG at different concentrations (10 mg / mL, 20 mg / mL, 30 mg / mL) was observed by SEM;

[0049] Figure 3 The results of evaluating the drug release behavior after encapsulating drugs of different properties using a hydrogel system with a concentration of 20 mg / mL;

[0050] Figure 4 are the physical properties of fresh porcine vitreous and the physical properties of DMPE-PEG@DMPE-PEG hydrogel;

[0051] Figure 5 It is a DMPE-PEG@DMPE-PEG bionic artificial vitreous sustained release experiment;

[0052] Figure 6 It is a flow chart and grouping diagram of DMPE-PEG@DMPE-PEG bionic artificial vitreous filling for the treatment of bacterial endophthalmitis;

[0053] Figure 7 These are pictures of mouse eyeballs taken with a slit lamp at days 0, 7, 14, 30, and 60 during an experiment using DMPE-PEG@DMPE-PEG bionic artificial vitreous to fill bacterial endophthalmitis.

[0054] Figure 8 On the 14th day of treatment, 1 μL of vitreous fluid was extracted from each group of mice and diluted with 1×10 3 After culturing on the bacterial culture plate for 24 hours, the bacterial density statistics were obtained by counting the colonies on the culture plate.

[0055] Fig. 9 This is the immunofluorescence staining of the distribution of ZO-1, Occludin, NFκB and COX-2 proteins in the retina after DMPE-PEG@DMPE-PEG bionic artificial vitreous filling for the treatment of bacterial endophthalmitis;

[0056] Fig.10 is the changes in inflammatory cells and inflammatory factors in the retina after different treatments;

[0057] Fig.11 It is a schematic diagram of the preparation process of the artificial tear film hydrogel of the present invention;

[0058] Fig.12 It is the distribution of artificial tear film of different concentrations on the ocular surface;

[0059] Fig.13 is the ocular surface pharmacokinetics of the artificial tear film of the present invention loaded with different drugs, wherein a is a hydrophobic small molecule drug, b is a hydrophilic small molecule drug, c is a macromolecular drug, and d is the ocular surface retention of the artificial tear film itself;

[0060] Fig.14It is the ability of the artificial tear film of the present invention to remove ROS and protect HCEC in a hypertonic environment, wherein a is a representative image of flow cytometry, b is a corresponding statistical graph; c is a representative image of HCECs live and dead staining;

[0061] Fig.15 It is the effect of the artificial tear film of the present invention in inhibiting inflammation, wherein a is a representative flow image, and b is a corresponding statistical graph;

[0062] Fig.16 is the corneal transparency and corneal damage degree of mice after different treatments of the present invention; wherein a is corneal transparency, and b is corneal damage degree;

[0063] Fig.17 The in vitro corneal transparency and tear secretion of mice after different treatments of the present invention; wherein a is the in vitro corneal transparency, and b is the tear secretion;

[0064] Fig.18 1 is a graph showing the changes in immune cells in the anterior segment of the eye of mice after different treatments of the present invention, wherein a is a representative image of M1 type flow cytometry, and b is a corresponding statistical graph; c is a representative image of M2 type flow cytometry, and d is a corresponding statistical graph;

[0065] Fig.19 It is the quantitative situation of inflammatory factors in the anterior segment of the eye of the mouse after different treatments of the present invention, wherein a is the quantitative situation of TNFα in the anterior segment of the eye of the mouse, b is the quantitative situation of IL-1β in the anterior segment of the eye of the mouse, and c is the quantitative situation of IL-6 in the anterior segment of the eye of the mouse. DETAILED DESCRIPTION

[0066] The present invention is further described below in conjunction with the accompanying drawings and specific embodiments so that those skilled in the art can better understand the present invention and implement it, but the embodiments are not intended to limit the present invention.

[0067] The first aspect of the present invention provides two PEG-based nanoparticles, namely four-arm PEG nanoparticles (i.e., 4r-PEG NPs) and DMPE-PEG nanoparticles (i.e., DMPE-PEG NPs). The outer layer of the nanoparticles carries SH or MAL groups. Through the addition reaction between the SH and MAL groups, the nanoparticles can be combined in pairs to form three new hydrogels with different properties in situ in the tissue (named 4r-PEG@4r-PEG, 4r-PEG@DMPE-PEG and DMPE-PEG@DMPE-PEG, respectively).

[0068] The present invention shows different physical properties of three hydrogels with different components, including physical structure, expansion rate, elastic modulus, light transmittance, refractive index and pH. Under the influence of different properties, these three hydrogels have different drug loading ranges, including hydrophilic and hydrophobic small molecule drugs and macromolecular drugs. In addition, these three hydrogels have different sustained release behaviors after encapsulating drugs of different properties.

[0069] The second aspect of the present invention utilizes one of the hydrogels provided in the first aspect, namely DMPE-PEG@DMPE-PEG, and by comparing the physical properties with the vitreous components in the real eyeball, it is found that it can be used as a vitreous substitute, as a bionic artificial vitreous. In addition, this bionic hydrogel also has a wide drug loading spectrum, providing a convenient drug delivery method for intraocular diseases that require vitrectomy, such as the treatment of bacterial endophthalmitis. In the treatment of the endophthalmitis model of Staphylococcus aureus infection in mice, this method significantly improved the development of endophthalmitis in mice, inhibited the spread of bacteria, protected retinal damage, maintained vision, and achieved excellent therapeutic effects.

[0070] The third aspect of the present invention utilizes one of the hydrogels provided in the first aspect, namely 4r-PEG@DMPE-PEG, and selects the optimal bionic artificial tear film hydrogel by screening two nanoparticle eye drops of different concentrations and dripping them on the ocular surface respectively, and the uniformity, thickness, transmittance and other physical properties of the formed hydrogel film. In addition, this bionic artificial tear film has a wide drug loading spectrum, which provides an innovative treatment strategy and a convenient administration method for the continuous treatment of ocular surface-related diseases, such as dry eye treatment. In the treatment of dry eye models in mice, this method significantly improved the transparency of the mouse cornea, alleviated the degree of apoptosis of mouse corneal / conjunctival cells, increased the tear secretion of mice, and significantly improved dry eye indicators, achieving good therapeutic effects.

[0071] Furthermore, the raw materials of the four-arm PEG nanoparticles (i.e., 4r-PEG NPs) include 4r-PEG 5000 -SH and 4r-PEG 5000 -MAL two polymer materials, the mass ratio of which is 1 to 10:10 for preparing nanoparticles, preferably 10:2 for preparing 4r-PEG NPs with SH on the surface; on the contrary, 2:10 for preparing 4r-PEG NPs with MAL on the surface. The total mass concentration of the nanoparticles is 10 to 30 mg / mL.

[0072] Further, the raw material of the DMPE-PEG nanoparticles (i.e., DMPE-PEG NPs) is composed of a single polymer material (DMPE-PEG 5000 -SH or DMPE-PEG 5000-MAL) was prepared by ultrasonic dispersion, wherein the ultrasonic power was 150kHz and the ultrasonic duration was 20 minutes. The total mass concentration of the nanoparticles was 10-30 mg / mL.

[0073] Furthermore, the 4r-PEG@4r-PEG hydrogel is prepared from 4r-PEG NPs with SH and MAL on the surface. When the nanoparticles are at the same mass concentration, the mass ratio of the gel is 4 to 10:10, preferably 10:10.

[0074] Furthermore, the DMPE-PEG@DMPE-PEG hydrogel is prepared from DMPE-PEG NPs with SH or MAL on the surface. When the nanoparticles are at the same mass concentration, the mass ratio range of the gel is 8 to 10:10, preferably 10:10.

[0075] Furthermore, the 4r-PEG@DMPE-PEG hydrogel is prepared from DMPE-PEG NPs with SH on the surface and 4r-PEG NPs with MAL on the surface. When the nanoparticles are at the same mass concentration, the mass ratio range of the gel is 8 to 10:10, preferably 10:10.

[0076] Furthermore, the solution of the hydrogel is a 1×PBS aqueous solution.

[0077] Unless otherwise specified, the experimental methods used in the following examples are all conventional methods, and the materials, reagents, etc. used are all available from commercial sources unless otherwise specified.

[0078] 4r-PEG used in the examples of the present invention 5000 -MAL, 4r-PEG 5000 -SH, DMPE-PEG 5000 -MAL and DMPE-PEG 5000 -SH were purchased from Xi'an Ruixi Biotechnology Co., Ltd.; mice were purchased from Changzhou Cavens Experimental Animal Co., Ltd.

[0079] Example 1: Preparation of hydrogels based on PEG nanoparticles and 4r-PEG@4r-PEG, 4r-PEG@DMPE-PEG and DMPE-PEG@DMPE-PEG.

[0080] Weigh 4r-PEG 5000 -MAL and 4r-PEG 5000The polymer raw materials of -SH were dispersed and dissolved in PBS solution (pH 7.4, 0.01M) to obtain two polymer solutions with a mother solution of 60 mg / mL. They were mixed in a mass ratio of 2:8 to form 4r-PEG nanoparticles with SH and MAL groups on the surface at a concentration of 20 mg / mL.

[0081] Weigh DMPE-PEG 5000 -MAL and DMPE-PEG 5000 -SH polymer raw materials were dissolved in chloroform to obtain an organic phase layer at a concentration of 40 mg / mL, and then a turbid solution was prepared by adding an aqueous phase twice the volume of the organic phase, namely a PBS aqueous solution (pH 7.4, 0.01 M), and then an ultrasonic device, namely a KQ-100KDB ultrasonic bath (150 kHz, 20 min) was used in a 0°C environment to make the turbid solution dispersed evenly. After the ultrasonication, the liquid was immediately transferred to a magnetic stirrer and rotated overnight at a speed of 1000 r / min until the organic phase was completely volatilized, and 20 mg / mL of DMPE-PEG nanoparticles with SH and MAL groups on the surface were obtained.

[0082] The four nanoparticles obtained above were combined in any two in a ratio of 1:1 to obtain three types of hydrogels, namely 4r-PEG@4r-PEG hydrogel, 4r-PEG@DMPE-PEG hydrogel and DMPE-PEG@DMPE-PEG hydrogel.

[0083] Example 2: Standards based on PEG nanoparticles and 4r-PEG@4r-PEG, 4r-PEG@DMPE-PEG and DMPE-PEG@DMPE-PEG hydrogels.

[0084] The particle sizes of the four nanoparticles were obtained by Nano-ZS90-Zeta particle size analyzer. SEM images were obtained using a ZeissGemini 500 scanning electron microscope. The rheological behavior was measured using a Thermo Fisher HAAKE Mars40 with a P20 Ti parallel plate at 25 °C. The absorption curves were recorded using a Jasco V-750 spectrophotometer. The results are shown in Figure 1-2 .

[0085] Figure 1 a in the figure is the particle size distribution diagram of 4r-PEG nanoparticles with SH or MAL groups on the surface and DMPE-PEG nanoparticles with SH or MAL groups on the surface. Figure 1 Figure b is a schematic diagram of the preparation of three hydrogels: 4r-PEG@4r-PEG, 4r-PEG@DMPE-PEG and DMPE-PEG@DMPE-PEG.

[0086] Figure 2 The rheological behavior of the above hydrogels with different concentrations was observed over time by rheometer; and the microscopic morphology of the hydrogels was observed by SEM at different concentrations of 4r-PEG@4r-PEG, 4r-PEG@DMPE-PEG and DMPE-PEG@DMPE-PEG at different concentrations, i.e., 10 mg / mL, 20 mg / mL, and 30 mg / mL. At the same concentration, the storage modulus and loss modulus of the three hydrogels are 4r-PEG@4r-PEG<DMPE-PEG@DMPE-PEG<4r-PEG@DMPE-PEG. And all hydrogels will increase their storage modulus and loss modulus with increasing concentration, proving that the hydrogels formed by the four nanoparticles with different concentrations have different properties, covering most application scenarios. The SEM image shows the internal structure and density of the hydrogel. The three different hydrogels have different internal structures, and consistent with the rheological results, the density inside the hydrogel increases with increasing concentration. It is conducive to the selection of suitable hydrogels according to different application scenarios in the later stage.

[0087] Example 3: Drug release behavior of 4r-PEG@4r-PEG, 4r-PEG@DMPE-PEG and DMPE-PEG@DMPE-PEG hydrogels.

[0088] The drug release behaviors of various properties of 4r-PEG@4r-PEG, 4r-PEG@DMPE-PEG and DMPE-PEG@DMPE-PEG hydrogels were measured using a transwell system (Corning, catalog number 3422) at 37°C to simulate a physiological environment. The transwell system is divided into a loading pool and a receiving pool. 200 μL of the hydrogel system is added to the loading pool, and 1 mL of PBS receiving solution containing 1% Tween 20 is added to the receiving pool. Each time a sample is collected from the receiving solution, it is necessary to replenish the corresponding fresh receiving solution to ensure the drug leakage condition. Representative drugs of different properties are: the representative drug of fat-soluble drugs is cyanine dye Cy5.5; the representative drug of protein drugs is bovine serum albumin BSA labeled with Cy5.5; the representative drug of water-soluble drugs is fluorescein 5-isothiocyanate dye FITC. The samples collected at different times were measured by fluorescence spectroscopy using a HORIBAFL3 fluorescence spectrometer to measure the drug fluorescence concentration and calculate the cumulative transmittance using the following formula. The results are shown in Figure 3 .

[0089] Cumulative permeability = [R n ×V 1 +(R n-1 +R n-2 +Rn-3 …+R 1 )×V 2 ]×100% / R all ×V 3 , where R n is the fluorescence value of the receptor solution collected n times, R n-1 is the fluorescence value of the receptor solution collected n-1 times, R 1 is the fluorescence value of the receptor solution collected once, R all is the fluorescence value of the sample in the donor chamber, V 1 is the volume of the total receptor solution (the volume of the receptor chamber is 1 mL), V 2 is the volume of the collected solution (the collected solution is 0.2 mL each time), V 3 is the volume of the sample solution in the donor chamber (the total sample solution is 0.2 mL).

[0090] Figure 3 The results are the results of evaluating the drug release behavior after encapsulating drugs of different properties using a hydrogel system with a concentration of 20 mg / mL. Figure 3 In a, the sustained release behavior of the fat-soluble drug cyanine dye Cy5.5 in hydrogels of different components at a concentration of 0.1 mg / mL is compared. Among them, the micelle group is DMPE-PEG nanoparticles encapsulating Cy5.5, and does not gel with another component; while 4r-PEG@4r-PEG cannot encapsulate fat-soluble drugs. In the fat-soluble drug sustained release experiment, samples in the receiving pool were collected at 0, 2, 3, 4, 5, 6, 8, 10, 15, 18, 25 and 28 days, the concentration was detected and the cumulative release rate was calculated. Figure 3 In b, the sustained release behavior of protein drugs labeled with Cy5.5 in hydrogels of different components at a concentration of 5 mg / mL is compared, wherein the free drug is a protein drug dissolved in a PBS aqueous solution. In the protein drug sustained release experiment, samples in the receiving pool were collected at 0, 1, 2, 3, 4, 5, 6, 7, 8 and 9 days, the concentration was detected and the cumulative release rate was calculated. Figure 3 Figure c compares the sustained release behavior of the water-soluble drug fluorescein 5-isothiocyanate dye FITC in hydrogels of different components at a concentration of 0.5 mg / mL, where the free drug is FITC dissolved in a PBS aqueous solution. In the water-soluble drug sustained release experiment, samples in the receiving pool were collected at 0, 6, 10, 20, 48, 72, 96, 120 and 144 hours, the concentration was detected and the cumulative release rate was calculated.

[0091] The above results show that the physical properties of 4r-PEG@4r-PEG and DMPE-PEG@DMPE-PEG can simulate artificial vitreous, and the physical properties of 4r-PEG@DMPE-PEG can simulate artificial tear film. Among them, DMPE-PEG@DMPE-PEG and 4r-PEG@DMPE-PEG have a broader drug loading function than 4r-PEG@4r-PEG, and can not only encapsulate water-soluble drugs and protein drugs, but also load fat-soluble drugs.

[0092] Example 4: DMPE-PEG@DMPE-PEG hydrogel has the physical properties of bionic artificial vitreous.

[0093] Fresh porcine vitreous was collected and the absorbance and transmittance of porcine vitreous and DMPE-PEG@DMPE-PEG hydrogel were measured using a PerkinElmer UV-Vis spectrophotometer. The rheological behavior of porcine vitreous was measured using a Thermo Fisher HAAKE Mars40 rheometer and a P20 Ti parallel plate at 25°C. Figure 4 .

[0094] Figure 4 The physical properties of fresh porcine vitreous are similar to those of DMPE-PEG@DMPE-PEG hydrogel. The results show that DMPE-PEG@DMPE-PEG hydrogel has similar physical properties to porcine vitreous and can be used as a bionic artificial vitreous as a vitreous substitute.

[0095] Example 5: Drug release behavior of DMPE-PEG@DMPE-PEG bionic artificial vitreous.

[0096] The Lumina small animal in vivo fluorescence imaging system was used to continuously monitor the mice with artificial vitreous filled with Cy5.5-DMPE-PEG micelles or DMPE-PEG@DMPE-PEG containing Cy5.5 after vitrectomy. HPLC was also used to continuously monitor the sustained release of artificial vitreous encapsulated with dexamethasone and vancomycin in vitro. The results are shown in Figure 5 .

[0097] Figure 5This is a DMPE-PEG@DMPE-PEG bionic artificial vitreous in vivo sustained release experiment. First, mice underwent vitrectomy with the help of a SMZ800N stereo microscope, and 1.5 μL of Cy5.5-DMPE-PEG micelles containing SH and MAL were injected with a Hamilton microinjection needle to achieve the function of in situ formation of hydrogels in the mouse vitreous cavity. The control group was Cy5.5-DMPE-PEG micelles containing SH alone. Fluorescence imaging of the mouse eyeballs was performed on days 0, 1, 3, 10, 18, 24, and 30, respectively. The total fluorescence intensity of the eyeballs at each time point was counted. The results showed that from the third day to the 30th day, the intraocular fluorescence intensity of the DMPE-PEG@DMPE-PEG bionic artificial vitreous was significantly higher than that of the micelle group. This shows that in addition to filling the intraocular vitreous cavity and preventing collapse to maintain vision, artificial vitreous also provides a broad drug loading spectrum and excellent drug release curve for the treatment of intraocular related diseases.

[0098] Example 6: DMPE-PEG@DMPE-PEG bionic artificial vitreous filling for the treatment of bacterial endophthalmitis.

[0099] First, S. Aureus bacteria (1×10 3 CFU / eye), i.e., Staphylococcus aureus. After three days, the vitreous opacity was observed by Diagnosys slit lamp to screen out mice with successful establishment of bacterial endophthalmitis. The day when bacterial endophthalmitis was successfully established in mice was set as day 0, and vitrectomy was subsequently performed. After surgery, the mice were randomly divided into five groups and filled with the following different vitreous substitutes, namely, blank artificial vitreous (DMPE-PEG@DMPE-PEG bionic artificial vitreous), dexamethasone-vancomycin micelles, dexamethasone artificial vitreous (loaded with dexamethasone DMPE-PEG@DMPE-PEG bionic artificial vitreous), vancomycin artificial vitreous (loaded with vancomycin DMPE-PEG@DMPE-PEG bionic artificial vitreous) and dexamethasone-vancomycin artificial glass (loaded with dexamethasone-vancomycin DMPE-PEG@DMPE-PEG bionic artificial vitreous). Subsequently, the vitreous opacity was observed by slit lamp, and the bacterial density in the mouse eyes was evaluated on the 14th day. The results are shown in Figure 6-8 .

[0100] Figure 6 This is a flow chart and grouping diagram of DMPE-PEG@DMPE-PEG bionic artificial vitreous filling for the treatment of bacterial endophthalmitis.

[0101] Figure 7The following are pictures of mouse eyeballs taken with a slit lamp at days 0, 7, 14, 30, and 60 during an experiment using DMPE-PEG@DMPE-PEG bionic vitreous filling to treat bacterial endophthalmitis. The pictures were not taken because the mouse eyeballs were ruptured.

[0102] Figure 8 On the 14th day of treatment, 1 μL of vitreous fluid was extracted from each group of mice and diluted with 1×10 3 The bacterial density was then statistically analyzed by counting the colonies on the bacterial culture plate after culturing for 24 hours. The results showed that the dexamethasone-vancomycin artificial vitreous not only greatly prolonged the eyeball retention rate of mice with bacterial endophthalmitis, but also effectively inhibited the reproduction of bacteria in the vitreous.

[0103] Example 7: Tissue evaluation and intraocular inflammation evaluation of DMPE-PEG@DMPE-PEG bionic artificial vitreous filling for the treatment of bacterial endophthalmitis.

[0104] This example explores the tissue changes and inflammatory changes of bacterial uveitis after biomimetic vitreous filling vitrectomy. The modeling method and treatment method are detailed in Example 6. The eyeballs of mice in each group were collected on the 14th day for immunofluorescence staining evaluation and ELISA inflammatory factor testing. The results are shown in Figure 9-10 .

[0105] The mouse eyeballs were fixed with FACS eyeball fixative for 1 hour, and then fixed with OCT thermosensitive glue at -80°C. The mouse eyeballs were cut into 8 μm thick slices using a Leica cryostat and adsorbed on a glass slide, followed by blocking with 5% BSA for 30 minutes. Next, the tissue slices were incubated with rabbit anti-mouse ZO-1 (Abcam, catalog number ab186271) and rat anti-mouse Occludin (Abcam, catalog number ab121123). In addition, rabbit anti-mouse NFκB (Abcam, catalog number ab221233) and rat anti-mouse COX-2 (Abcam, catalog number ab817261) primary antibodies were incubated at a dilution of 1:800 at 4°C overnight (NFκB and COX-2 dilutions contained 0.1% TritonX-100 to facilitate membrane permeabilization). Subsequently, the cells were washed with PBS and incubated with Alexa Fluor. 488-labeled goat anti-rabbit IgG H&L (Abcam, catalog number ab150077) secondary antibody and goat anti-rat IgG H&L (Abcam, catalog number ab158721) were incubated at room temperature for 1 hour at a dilution of 1:400 and stained with 4,6-diamidino-2-phenylindole (DAPI) for 10 minutes. Finally, the slides were mounted and imaged by confocal microscopy (Zeiss LSM 800). The results are shown in Fig. 9 .

[0106] Fig. 9 This is an immunofluorescence staining of the distribution of ZO-1, Occludin, NFκB and COX-2 proteins in the retina after DMPE-PEG@DMPE-PEG bionic artificial vitreous filling for the treatment of bacterial endophthalmitis. Among them, the intercellular tight junctions of the retina of mice that underwent vitrectomy alone were completely destroyed, and the NFκB and COX-2 inflammatory pathways were upregulated, indicating that simple vitrectomy cannot cure bacterial uveitis and still has recurrence. The DMPE-PEG@DMPE-PEG bionic artificial vitreous filling with long-term sustained release of dexamethasone, which has anti-inflammatory properties, and vancomycin, which inhibits bacterial reproduction, showed good intercellular tight junctions in the back of the mouse eyeball after surgery, protecting the retinal barrier function from bacteria and immune cells. In addition, these two inflammatory pathways were greatly inhibited, and the antibacterial and anti-inflammatory effects were combined to protect the retina to the greatest extent.

[0107] In addition, the changes in inflammatory cells in bacterial endophthalmitis induced by vitreous fillings of different components were quantitatively determined by flow cytometry. The eyeballs of mice in each group were collected, retinal cells were extracted, and the cells were collected into Eppendorf tubes by centrifugation (1200 rpm, 3 minutes), fixed with 0.25% formaldehyde for 10 minutes, and blocked with anti-CD16 / 32 (BioLegend, 101302) antibodies. Next, anti-mouse CD80 (Abcam, catalog number ab92987), anti-mouse Ly6G (Abcam, catalog number ab787111), anti-mouse CD11b (Abcam, catalog number ab871019), anti-mouse Foxp3 (Abcam, catalog number ab396410), anti-mouse CD4 (Abcam, catalog number ab100412) and anti-mouse CD3 (Abcam, catalog number ab100204) primary antibodies were incubated at room temperature for 1 hour at a dilution of 1:500. Then, the cells were washed with PBS and incubated with Alexa Fluor The secondary antibody labeled with 488 goat anti-rabbit IgG H&L (Abcam, catalog number ab150077) was incubated at room temperature for 1 hour at a dilution of 1:400. Finally, the retinal cells of each sample were tested using a BD Accuri C6 plus flow cytometer and analyzed using FlowJo software (Ver.10.0.7). The results are shown in Fig.10 ac. In addition, the supernatant of retinal cells after centrifugation was collected, and the inflammatory factors in the supernatant were detected using mouse IL-6 (eBioscience, catalog number 88-7291-88) and TNFα ELISA kits (eBioscience, catalog number 88-7224-88). The results are shown in Fig.10 de.

[0108] Fig.10 The changes of inflammatory cells and inflammatory factors in the retina after different treatments. Fig.10 As shown in Figures ac, after the DMPE-PEG@DMPE-PEG bionic artificial vitreous filling surgery, which has the function of long-term sustained release of anti-inflammatory dexamethasone and vancomycin that inhibits bacterial growth, the inflammatory cells and inflammatory factors in the mouse retina decreased. This experimental result further proves the effectiveness of DMPE-PEG@DMPE-PEG as an artificial vitreous filler, and under this premise, the wide drug loading capacity of DMPE-PEG@DMPE-PEG artificial vitreous gives the possibility of treating more fundus diseases.

[0109] Example 15: 4r-PEG@DMPE-PEG hydrogel has the physical properties of bionic artificial tear film.

[0110] The artificial tear film hydrogel was obtained by mixing 4r-PEGNPs and DMPE-NPs in a ratio of 1:1. The hydrogel cleverly simulated the three-layer structure of the tear film from the inside to the outside (mucin layer, middle aqueous layer, lipid layer), which helps dry eye patients reshape a healthy tear film environment and has great significance for the treatment of dry eye. The schematic diagram of the preparation process of the bionic artificial tear film hydrogel in the present invention is shown in Fig.11 .

[0111] Pre-gels with concentrations of 10 mg / mL, 20 mg / mL, and 30 mg / mL were prepared according to the previous method. First, 2 μL of 4r-PEG nanoparticles carrying MAL groups on the surface were dropped on the mouse ocular surface, and then 2 μL of DMPE-PEG nanoparticles carrying SH groups on the surface were dropped on the ocular surface. The addition of SH and MAL groups was used to form artificial tear film hydrogel in situ on the mouse ocular surface. Optical coherence tomography (OCT) was then used to investigate the distribution of artificial tear film hydrogels of different concentrations on the ocular surface. The results are shown in Figure 2. Fig.12 .

[0112] Fig.12 The distribution of 4r-PEG@DMPE-PEG hydrogels of different concentrations on the ocular surface was studied. The results showed that with increasing concentration, the storage modulus and loss modulus of the artificial tear film hydrogel increased. When the concentration was higher than 20 mg / mL, the artificial tear film hydrogel layer was too thick and easily fell off by blinking. When the concentration was less than or equal to 20 mg / mL, a uniform thin film could be formed on the ocular surface, which could be used as a substitute for the tear film.

[0113] Example 16: Ocular surface pharmacokinetics of 4r-PEG@DMPE-PEG bionic artificial tear film hydrogel.

[0114] The 4r-PEG@DMPE-PEG artificial tear film hydrogel prepared by the present invention is a multifunctional hydrogel that can simulate the mucus layer, water layer, and lipid layer, and has the characteristics of wide drug loading range and wide application range. Among them, the 4r-PEG nanoparticles carrying the MAL group and the DMPE-PEG nanoparticles carrying the SH group can be loaded with hydrophilic, macromolecular and hydrophobic drugs respectively. In order to better observe the release behavior of drug molecules of different properties in the hydrogel locally on the ocular surface, the present invention uses Cy5.5 to simulate the release behavior of hydrophobic small molecules, rhodamine B (RhoB) to simulate the release behavior of hydrophilic small molecules, and BSA-Cy5.5 to simulate the release behavior of macromolecules, and the corresponding free molecules are used as controls to compare the sustained release ability of the artificial tear film hydrogel, wherein the free control of the hydrophobic small molecules is Cy5.5-DMPE-PEG nanoparticles. The same volume (4 μL) of free drug and drug-loaded 4r-PEG@DMPE-PEG artificial tear film hydrogel was dropped onto the mouse ocular surface, and the mouse ocular surface was washed with PBS every 15 minutes to simulate the conditions of natural tears flushing the ocular surface. On this basis, the small animal in vivo fluorescence imaging system was used to investigate the retention of fluorescent molecules of different properties on the ocular surface. In addition, in order to investigate the retention of lipid components in the artificial tear film hydrogel on the ocular surface, the DMPE-PEG component in the artificial tear film hydrogel was labeled with DIR, and the ocular surface retention of the artificial tear film hydrogel at different times was recorded according to the previous method. The results are shown in Fig.13 .

[0115] Fig.13 The ocular surface pharmacokinetics of 4r-PEG@DMPE-PEG artificial tear film loaded with different drugs, among which Fig.13 a in is a hydrophobic small molecule drug, Fig.13 b in the formula is a hydrophilic small molecule drug. Fig.13 The c in is a macromolecular drug. Fig.13d in the figure is the retention of DMPE-PEG, a component of the artificial tear film, on the ocular surface. The results show that compared with hydrophobic small molecules that are metabolized in micelles for nearly 6 hours on the ocular surface, 4r-PEG@DMPE-PEG artificial tear film hydrogels maintain the fluorescence of Cy5.5 on the ocular surface for up to 24 hours; compared with hydrophilic small molecule free drugs that only maintain an effective dose on the ocular surface for 30 minutes, 4r-PEG@DMPE-PEG artificial tear film hydrogels maintain the fluorescence intensity of RhoB on the ocular surface for up to 6 hours; compared with large molecule free drugs that maintain an effective dose on the ocular surface for nearly 4 hours, 4r-PEG@DMPE-PEG artificial tear film hydrogels maintain the fluorescence intensity of BSA-Cy5.5 on the ocular surface for up to 10 hours; compared with DIR-DMPE-PEG that only maintains on the ocular surface for 6 hours, the DIR fluorescence signal in 4r-PEG@DMPE-PEG artificial tear film hydrogels is maintained for up to 24 hours. The above results indicate that artificial tear film hydrogel can effectively persist on the ocular surface for at least 24 hours. In addition, it can load and sustainably release multi-property drugs including lipid-soluble, water-soluble, and protein drugs.

[0116] Example 17: Functional verification of 4r-PEG@DMPE-PEG bionic artificial tear film in removing ROS and repairing corneal epithelial cells.

[0117] The present invention uses corneal epithelial cells (HCECs) to investigate the ability of 4r-PEG@DMPE-PEG artificial tear film loaded with cyclosporine A (CsA) to remove ROS and fight inflammation. First, in order to simulate the hyperosmotic (HS) environment of the ocular surface of patients with dry eye, an appropriate amount of NaCl was added to the high-glucose culture medium used as the cell culture medium to make the osmotic pressure of the final solution reach 600mOsm / L. At the same time, 200μL of 4r-PEG@DMPE-PEG artificial tear film hydrogel loaded with CsA or simple CsA nanoparticles were added to the Transwell and incubated with HCECs cells for 12h. After 12h, the cells were digested with 0.25% trypsin, transferred to a 1.5mL ep tube, centrifuged at 1000rpm for 3 minutes, the upper culture medium was discarded, and the cells were resuspended in 200μL of PBS. Add the cell permeable reactive oxygen species probe DCFH-DA (MCE, catalog number 4091-99-0, working concentration 10 μM), incubate at room temperature in the dark for 30 minutes, and then use Fongcyte flow cytometry to analyze the level of intracellular ROS under different material treatments. Fig.14At the same time, the present invention also uses the live / dead cell double staining kit CalceinAM / PI (Sigma, catalog number 92210) to perform live / dead staining on HCECs treated under the above conditions, adding CalceinAM (working concentration of 2 μM) and PI (working concentration of 4.5 μM), incubating at room temperature in the dark for 15-20 minutes, and imaging using a Zeiss confocal microscope (Zeiss LSM 800). The results are shown in Fig.14 c in.

[0118] Fig.14 The 4r-PEG@DMPE-PEG bionic artificial tear film of the present invention is capable of removing ROS and protecting HCEC in a hypertonic environment, wherein Fig.14 a in the figure is a representative image of flow. Fig.14 b in is the corresponding statistical graph; Fig.14 The c in the figure is a representative image of HCECs live and dead staining. The results show that the 4r-PEG@DMPE-PEG bionic artificial tear film of the present invention has the ability to remove ROS and protect and repair corneal epithelial cells.

[0119] Example 18: Verification of the anti-inflammatory function of 4r-PEG@DMPE-PEG bionic artificial tear film.

[0120] In order to investigate the anti-inflammatory ability of 4r-PEG@DMPE-PEG bionic artificial tear film loaded with CsA, the present invention cultured RAW264.7 cells in a 24-well plate, added lipopolysaccharide LPS (working concentration of 100 ng / mL) to stimulate RAW 264.7 polarization, and added artificial tear film (200 μL), CsA (final concentration of 5 μg / mL), and CsA artificial tear film (200 μL) at the same time. After incubation for 24 hours, the cells were blown off, centrifuged at 1000 rpm for 3 minutes, the culture medium was discarded, and the cells were resuspended in 200 μL PBS, PE-CD80 and APC-CD206 were added to stain M1 and M2 phenotypes respectively, and the proportion of cells with different phenotypes was analyzed by Fongcyte flow cytometry after incubation at room temperature and in the dark for 30 minutes, so as to investigate the anti-inflammatory ability of artificial tear film and CsA artificial tear film. The results are shown in Fig.15 .

[0121] Fig.15 The effect of 4r-PEG@DMPE-PEG artificial tear film loaded with CsA on inhibiting inflammation, where a is a representative flow cytometry image and b is the corresponding statistical graph. The results show that 4r-PEG@DMPE-PEG bionic artificial tear film loaded with CsA can inhibit inflammation by increasing the proportion of M2 macrophages.

[0122] Example 19: Therapeutic effect of 4r-PEG@DMPE-PEG bionic artificial tear film loaded with CsA in dry eye.

[0123] In order to investigate the therapeutic effect of artificial tear film and CsA artificial tear film in dry eye, a dry eye model was established in mice. First, 0.4% benzalkonium chloride (BAC) solution was used for eye drops for one week, twice a day. After one week, the mouse dry eye model was successfully established. In order to maintain the dry eye symptoms of mice, 0.2% BAC solution was continued to be used for one week, and the frequency was reduced to once a day. At the same time, sea dew (a commercially available drug for the treatment of dry eye), artificial tear film (4r-PEG@DMPE-PEG bionic artificial tear film), CsA, and CsA artificial tear film (4r-PEG@DMPE-PEG bionic artificial tear film loaded with CsA) were dripped every day for treatment. At different time periods of treatment, the corneal transparency of different groups was examined by slit lamp, and the degree of corneal damage of different groups was examined by sodium fluorescein staining. At different time periods of treatment, commercially available phenol red cotton thread was placed in the conjunctival sac of mice, and the timer was used to count for 30s. The part wetted by tears appeared red, and the length of the red cotton thread was measured with a ruler, which was the amount of tear secretion. After the treatment, the mice were euthanized, their corneas were collected and placed on a paper printed with the word "DED" to examine the corneal transparency of the mice in different groups. Figure 16-17 .

[0124] Fig.16 The corneal transparency and corneal damage degree of mice after different treatments of the present invention; a is corneal transparency and b is corneal damage degree. The results show that 4r-PEG@DMPE-PEG bionic artificial tear film can effectively restore corneal transparency and alleviate corneal damage caused by dry eye to a certain extent. In comparison, 4r-PEG@DMPE-PEG bionic artificial tear film loaded with CsA shows better effect in repairing corneal damage.

[0125] Fig.17 The results show that the 4r-PEG@DMPE-PEG bionic artificial tear film can effectively restore corneal transparency, increase tear secretion, and slow down the decrease in tear secretion caused by dry eye. In contrast, the 4r-PEG@DMPE-PEG bionic artificial tear film loaded with CsA showed better wetting of the ocular surface and increased tear secretion.

[0126] Example 20: Immune and inflammatory evaluation of 4r-PEG@DMPE-PEG bionic artificial tear film loaded with CsA in the treatment of dry eye.

[0127] (1) Immune evaluation: The presence of immune cells in the anterior eye tissue of mice was further studied using a Fongcyte flow cytometer. Anterior eye tissues of mice in different groups were collected on the 8th day after treatment and homogenized into a single cell suspension in cold FACS buffer solution (1% FBS in PBS), and stained with the following antibodies according to the manufacturer's instructions: PE anti-mouse F4 / 80 (BioLegend, catalog number 111603), APC anti-mouse CD86 (BioLegend, catalog number 159215), APC-CD206 (BioLegend, catalog number 321110). Analysis was performed using a Fongcyte flow cytometer and analyzed by FlowJo software (version 10.8.1). Results are shown in Fig.18 .

[0128] Fig.18 It is a result diagram of the changes in immune cells in the anterior segment of the eyes of mice after different treatments of the present invention, wherein a is a representative image of M1 flow cytometry, and b is the corresponding statistical graph; c is a representative image of M2 flow cytometry, and d is the corresponding statistical graph. The results show that the proportion of M1 macrophages and M2 macrophages in the anterior segment of the eyes of mice with dry eye disease increased significantly, indicating that dry eye disease is accompanied by ocular surface inflammation and infiltration of inflammation-related cells. Among all groups, 4r-PEG@DMPE-PEG loaded with CsA reduced the proportion of M1 macrophages and M2 macrophages to the minimum, and the proportion of M1 macrophages and M2 macrophages under 4r-PEG@DMPE-PEG treatment was slightly higher than that of the bionic artificial tear film loaded with CsA.

[0129] (2) Detection of inflammatory factors: In addition, after mincing the anterior segment tissue of the mouse eyes, the supernatant was collected and analyzed for inflammatory factors using an enzyme-linked immunosorbent assay. The mouse ELISA kit (Beyotime, catalog number PH406) was used for measurement according to the manufacturer's instructions. First, the ELISA adsorption plate was coated with the Capture antibody, 100 μL / well, and incubated overnight at 4°C. The next day, it was washed three times with PBST washing solution, then blocked with Diulent and incubated at room temperature for 1 hour. Samples and standards were added, 100 μL / well, and incubated at room temperature for 2 hours. Then, it was washed three times with PBST washing solution, and the Detection antibody 100 μL / well was added and incubated at room temperature for 1 hour. Washed three times with PBST washing solution, 100 μL / well of HRP was added, and incubated at room temperature for 30 minutes. After washing three times with PBST washing solution, TMB solution was added. After incubation at room temperature for 15 minutes, the stop solution was added, and the OD values ​​of 450nm and 570nm were measured using an enzyme reader. Results are shown in Fig.19 .

[0130] Fig.19It is the quantitative situation of inflammatory factors in the anterior segment of the eye of the mouse after different treatments of the present invention, wherein a is the quantitative situation of TNFα in the anterior segment of the eye of the mouse, b is the quantitative situation of IL-1β in the anterior segment of the eye of the mouse, and c is the quantitative situation of IL-6 in the anterior segment of the eye of the mouse. The results show that 4r-PEG@DMPE-PEG loaded with CsA can significantly reduce the inflammatory-related factors TNFα, IL-1β and IL-6, and inhibit the ocular surface inflammatory microenvironment. In addition, the 4r-PEG@DMPE-PEG bionic artificial tear film can also indirectly inhibit inflammation by moistening the ocular surface and alleviating ocular surface hyperosmosis.

[0131] Obviously, the above embodiments are merely examples for clear explanation and are not intended to limit the implementation methods. For those skilled in the art, other different forms of changes or modifications can be made based on the above description. It is not necessary and impossible to list all the implementation methods here. The obvious changes or modifications derived from these are still within the protection scope of the invention.

Claims

1. A controllable in-situ biomimetic hydrogel system, characterized in that: The in situ biomimetic hydrogel system includes 4r-PEG@DMPE-PEG hydrogel and / or DMPE-PEG@DMPE-PEG hydrogel; The 4r-PEG@DMPE-PEG hydrogel includes 4r-PEG nanoparticles and DMPE-PEG nanoparticles connected via thiol groups and maleimide groups; The DMPE-PEG@DMPE-PEG hydrogel includes DMPE-PEG nanoparticles connected to maleimide groups via thiol groups.

2. The in situ biomimetic hydrogel system according to claim 1, characterized in that: The mass ratio of 4r-PEG nanoparticles to DMPE-PEG nanoparticles in the 4r-PEG@DMPE-PEG hydrogel is 8 to 10:

10.

3. The method for preparing the controllable in-situ biomimetic hydrogel system according to claim 1 or 2, characterized in that: The following steps are involved: (1) preparing a 4r-PEG nanoparticle solution containing a thiol group and a 4r-PEG nanoparticle solution containing a maleimide group respectively; (2) preparing a DMPE-PEG nanoparticle solution containing a thiol group and a DMPE-PEG nanoparticle solution containing a maleimide group, respectively; (3) subjecting any two of the nanoparticle solutions obtained in step (1) and step (2) to an addition reaction to obtain an adjustable in-situ bionic hydrogel system.

4. Use of the in-situ bionic hydrogel system described in claim 1 or 2 in preparing bionic artificial vitreous, characterized in that: The in-situ biomimetic hydrogel system includes DMPE-PEG@DMPE-PEG hydrogel.

5. Use of the in situ bionic hydrogel system described in claim 1 or 2 in preparing a bionic artificial tear film, characterized in that: The in situ biomimetic hydrogel system includes 4r-PEG@DMPE-PEG hydrogel.

6. The use according to claim 5, characterized in that: The concentration of the 4r-PEG@DMPE-PEG hydrogel is ≤20 mg / mL.

7. Use of the in-situ bionic hydrogel system described in claim 1 or 2 in the preparation of drugs for treating eye diseases.

8. The use according to claim 7, characterized in that: The ocular disease includes one or more of bacterial endophthalmitis, dry eye, conjunctivitis, dacryocystitis, scleritis, uveitis, retinal detachment, retinal vasculopathy, retinoblastoma, choroidal melanoma and optic neuritis.

9. An ocular pharmaceutical preparation, characterized in that: It comprises a drug carrier and a drug; the drug carrier is the in-situ bionic hydrogel system described in claim 1 or 2.

10. The ocular pharmaceutical preparation according to claim 9, characterized in that The drug is selected from one or more of anti-infective drugs, anti-allergic drugs, anti-angiogenic drugs and anti-inflammatory drugs.

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