Injectable hama hydrogel microspheres loaded with exosomes and methods of making and using the same
By encapsulating exosomes with HAMA hydrogel microspheres, the problems of rapid clearance and short retention time of exosomes in the eye were solved, achieving stable encapsulation and slow release of exosomes, thus improving the efficacy of glaucoma treatment.
Patent Information
- Authority / Receiving Office
- CN · China
- Patent Type
- Applications(China)
- Current Assignee / Owner
- SHENZHEN EYE HOSPITAL
- Filing Date
- 2026-05-13
- Publication Date
- 2026-07-31
AI Technical Summary
Existing technologies for intraocular applications of exosomes suffer from problems such as rapid diffusion and clearance, short local retention time, and the need for frequent administration. Furthermore, existing hydrogel systems cannot achieve controllable microsphere size and optimized dispersibility, making it difficult to achieve stable encapsulation, sustained release, and improved RGC delivery efficiency of exosomes.
Using HAMA hydrogel microspheres as a carrier, a three-dimensional network structure is formed through cross-linking to encapsulate exosomes, achieving stable encapsulation and slow release of exosomes in the vitreous cavity, reducing the frequency of drug administration, and improving local retention time and delivery efficiency.
It achieves long-term retention and sustained release of exosomes in the vitreous cavity, reduces the frequency of administration, lowers the risk of intraocular infection and inflammation, improves patient compliance, and significantly improves the protective effect on retinal ganglion cells.
Smart Images
Figure CN122478879A_ABST
Abstract
Description
Technical Field
[0001] This application relates to the fields of biomedical materials and drug delivery technology, and in particular to an injectable HAMA sustained-release hydrogel microsphere loaded with exosomes, its preparation method, and its application in the preparation of glaucoma treatment drugs. Background Technology
[0002] Glaucoma is a blinding eye disease characterized by progressive damage to retinal ganglion cells (RGCs) and their axons. Its course is insidious, the damage is irreversible, and in severe cases, it can lead to permanent visual loss. Current clinical treatment for glaucoma primarily focuses on lowering intraocular pressure, including topical eye drops, laser therapy, and surgery. However, numerous studies and clinical practice have shown that controlling intraocular pressure alone cannot completely prevent the continued loss of RGCs and the deterioration of visual function. Therefore, treatment methods based on neuroprotective strategies have become an important direction for development in the field of glaucoma prevention and treatment.
[0003] Exosomes, as nanoscale vesicles secreted by cells, possess excellent biocompatibility, low immunogenicity, and the ability to carry various bioactive molecules such as proteins, nucleic acids, and lipids, showing potential application value in anti-inflammation, anti-apoptosis, neuroprotection, and tissue repair. However, to date, there are few reports on the use of exosomes for the intervention of glaucoma-related optic nerve damage. This is because the intraocular application of free exosomes has significant limitations: firstly, the intravitreal fluid environment allows for rapid diffusion and clearance, resulting in a short local retention time and difficulty in maintaining a stable and effective concentration; secondly, to achieve sustained therapeutic effects, repeated administration is often required, but frequent intravitreal injections may increase the risks of intraocular inflammation, infection, and retinal damage, and are also detrimental to patient compliance and clinical translation.
[0004] In existing technologies, studies have proposed combining exosomes with intraocular injectable hydrogel systems to improve optic nerve damage associated with glaucoma. For example, patent CN117338700A / B discloses a functional hydrogel comprising a chitosan-based hydrogel matrix, active ingredient A, and bone marrow mesenchymal stem cell-derived exosomes, which can be used as an intravitreal injection formulation for optic nerve protection. Another patent, CN117717566A, discloses miR22 or miR22-overexpressing MSC exosomes for the treatment of eye diseases, exerting RGC protective effects by inhibiting inflammation / apoptosis-related factors.
[0005] However, the existing technologies still have the following shortcomings: First, most exosome-related solutions focus on the biological effects of exosomes themselves, lacking specialized carrier designs for rapid intraocular clearance, insufficient local retention, and long-term delivery requirements; Second, the existing technology (CN117338700A) uses chitosan-based monolithic functional hydrogels, which cannot achieve controllable microsphere size and optimized dispersibility; Third, the existing technologies have not fully solved the problems of stable encapsulation and continuous release of exosomes in the vitreous cavity, reducing the frequency of administration, and improving the local delivery efficiency of RGCs while maintaining good injectability.
[0006] In summary, developing a novel hydrogel microsphere system that combines injectability, good intraocular compatibility, exosome protection, and sustained-release properties for the treatment of glaucoma-related RGC damage would have significant research value and promising application prospects. Summary of the Invention
[0007] Terminology Explanation:
[0008] HAMA: methacryloyl hyaluronic acid.
[0009] RGC: Retinal ganglion cells;
[0010] Exo: exosome;
[0011] Exo@HAMA: Exosomes@HAMA microspheres.
[0012] To address the aforementioned problems in existing technologies, and considering the shortcomings of current glaucoma treatments, which primarily rely on intraocular pressure-lowering methods, lack sustained and effective protection of retinal ganglion cells, and suffer from short retention time, rapid clearance, limited duration of action, and the need for repeated administration of exosomes after intraocular delivery, this application proposes a novel injectable HAMA sustained-release hydrogel microsphere loaded with exosomes and its preparation method. This invention constructs a microspherical intraocular delivery carrier based on HAMA, achieving stable encapsulation and sustained release of exosomes within the vitreous cavity, prolonging the local retention time of exosomes in the eye, improving their delivery efficiency in the retina, especially at retinal ganglion cells, reducing the frequency of administration, improving the duration of drug action, and enhancing the therapeutic effect of glaucoma based on optic nerve protection and visual function improvement mechanisms, while ensuring good biocompatibility and injection suitability.
[0013] Our research group has found through long-term studies that while exosomes alone have a certain neuroprotective effect when used to intervene in glaucoma-related retinal nerve damage, they still suffer from problems such as rapid diffusion within the vitreous cavity, rapid metabolic clearance, short local action time, and insufficient maintenance of efficacy. To address these shortcomings, this invention encapsulates and disperses exosomes within a HAMA hydrogel microsphere matrix. This design not only protects the exosomes but also allows for slow release and long-term retention through the microsphere matrix, thereby achieving long-term therapeutic goals (protecting retinal ganglion cells) and reducing the frequency of administration, as well as the toxic side effects and poor patient compliance associated with repeated intravitreal administration.
[0014] To achieve the above objectives, the present invention provides the following technical solution:
[0015] In a first aspect, the present invention provides an injectable HAMA sustained-release hydrogel microsphere loaded with exosomes. The hydrogel microsphere uses HAMA (methacryloyl hyaluronic acid) as a matrix, which is cross-linked to form a three-dimensional network structure and encapsulates a pharmaceutically effective amount of exosomes for local sustained-release delivery of exosomes after intraocular injection.
[0016] In this invention, the HAMA hydrogel microsphere matrix is the key to achieving sustained delivery of exosomes and long-lasting local effects in the eye. The HAMA is prepared by methacrylylation of hyaluronic acid, which combines the good biocompatibility of hyaluronic acid with the crosslinking gel properties imparted by the methacrylyl group.
[0017] Furthermore, in the injectable HAMA sustained-release hydrogel microspheres loaded with exosomes of the present invention, the mass concentration of HAMA is 4% to 6% (w / v), preferably 4% (w / v).
[0018] Furthermore, in the injectable HAMA sustained-release hydrogel microspheres loaded with exosomes of the present invention, the exosomes are preferably derived from umbilical cord blood, with a particle size of 60-100 nm, more preferably 70-80 nm, and a concentration of 10^6 μm based on particle concentration. 10 p / ml ~10^ 12 p / ml, preferably 10^ 11 p / ml, the exosomes are dispersed in HAMA precursor solution and are uniformly embedded in the interior of the microspheres or in the pore network during the microsphere forming and cross-linking process.
[0019] Furthermore, the injectable HAMA sustained-release hydrogel microspheres loaded with exosomes of the present invention may also contain a photoinitiator; preferably, the photoinitiator is Irgacure 2959, LAP or a combination thereof; the concentration of the photoinitiator is preferably 0.25% (w / v).
[0020] Furthermore, the injectable HAMA sustained-release hydrogel microspheres loaded with exosomes of the present invention are microspheres that can be administered via intravitreal injection. They can pass smoothly through the injection needle under the action of injection shear force and maintain the structural integrity of the microspheres after injection.
[0021] Secondly, the present invention provides a method for preparing injectable HAMA sustained-release hydrogel microspheres loaded with exosomes, comprising the following steps:
[0022] S1. Dissolve HAMA in buffer solution to prepare HAMA precursor solution, and add photoinitiator to form aqueous phase;
[0023] S2. Add the exosomes to the aqueous phase and mix well to obtain a HAMA mixed precursor solution containing exosomes;
[0024] S3. The mixed precursor liquid is processed into droplets using a microfluidic device to form droplets;
[0025] S4. The droplets are photocrosslinked and cured to obtain HAMA sustained-release hydrogel microspheres loaded with exosomes;
[0026] S5. Wash, separate and preserve the obtained hydrogel microspheres.
[0027] Furthermore, in the preparation method of the present invention, the microfluidic device droplet processing in step S3 adopts an oil-in-water system, the aqueous phase is a HAMA mixed precursor liquid containing exosomes, and the oil phase contains HPE7500 fluorinated oil and 2% surfactant.
[0028] Furthermore, in the preparation method of the present invention, the photocrosslinking curing in step S4 is performed by irradiation with ultraviolet light or visible light, and the crosslinking time is 2 to 3 minutes, preferably 2 minutes.
[0029] Furthermore, in the preparation method of the present invention, the step of obtaining the exosomes includes: obtaining an umbilical cord blood sample or its separated components, and obtaining exosomes by centrifugation, filtration, separation and resuspension of the precipitate; or, culturing exosome donor cells, harvesting the cell supernatant, and obtaining exosomes by centrifugation, filtration, separation and resuspension of the precipitate.
[0030] Thirdly, the present invention provides the application of the injectable HAMA sustained-release hydrogel microspheres loaded with exosomes in the preparation of glaucoma treatment drugs with retinal ganglion cell protection effects.
[0031] Our research group's findings indicate that a combined delivery system based on exosomes and HAMA hydrogel microspheres can improve the stability and local utilization efficiency of exosomes in the eye by utilizing the microsphere matrix for encapsulation protection and sustained-release regulation. In a mouse model of retinal ischemia-reperfusion injury, this system exhibits significant RGC protection, demonstrating its potential application in the protective treatment of glaucoma-related optic nerves.
[0032] Therefore, the injectable HAMA sustained-release hydrogel microspheres loaded with exosomes of the present invention can be used to prepare intraocular injection drugs that protect retinal ganglion cells and improve retinal ischemia-reperfusion injury.
[0033] Fourthly, the present invention provides a pharmaceutical composition for treating glaucoma, comprising the injectable HAMA sustained-release hydrogel microspheres loaded with exosomes, and pharmaceutically acceptable excipients; and the pharmaceutical composition is an intraocular injection formulation, preferably an intravitreal injection formulation.
[0034] Furthermore, based on different intraocular environments and treatment needs, parameters such as particle size, drug loading, material concentration, crosslinking density, degradation rate, and injection volume of the injectable HAMA sustained-release hydrogel microspheres loaded with exosomes of the present invention can be dynamically adjusted to obtain the best therapeutic effect.
[0035] In summary, compared with the prior art, the present invention has the following technical advantages:
[0036] (1) The injectable HAMA sustained-release hydrogel microspheres provided by the present invention use HAMA as the microsphere matrix and combine it with exosomes to construct an intraocular sustained-release delivery system. Compared with existing PLGA microspheres / implants, non-degradable intraocular implants and other complex composite hydrogel delivery systems, the system described in the present invention can achieve effective encapsulation of exosomes, long-term local retention and continuous release in the intraocular region while maintaining good intraocular biocompatibility and injectability, and is conducive to maintaining the biological activity of exosomes, thereby providing a new treatment approach for optic nerve protection and visual function improvement in glaucoma.
[0037] (2) Thanks to the encapsulation and sustained release effect of the HAMA sustained-release hydrogel microspheres on exosomes, the present invention can overcome the defects of short intraocular retention time of free exosomes, insufficient maintenance of local effective concentration and limited duration of efficacy to a certain extent, thereby reducing the frequency of intraocular administration in glaucoma treatment, reducing the potential risks of infection, inflammation and intraocular pressure fluctuation caused by repeated administration, and improving patient compliance.
[0038] (3) Compared with existing exosome-hydrogel ophthalmic delivery strategies, the material composition of the system of the present invention is relatively simple, the preparation path is clear, and the microsphere particle size is easy to control, which is more conducive to achieving standardized preparation and clinical translation application.
[0039] (4) The present invention uses microfluidic technology to prepare HAMA hydrogel microspheres, which can achieve good particle size uniformity, batch-to-batch repeatability and exosome loading efficiency, providing a reliable technical guarantee for long-term sustained-release delivery in the eye.
[0040] Other features and advantages of this application will be set forth in detail in the following description, or will become apparent through the implementation of the relevant technical solutions of this application. The objectives and other advantages of this application can be achieved through the technical features and means explicitly pointed out in the description, claims, and drawings, and will be obtained through the implementation of these technical contents. Attached Figure Description
[0041] To more clearly illustrate the technical solution of this application, the accompanying drawings involved in the description of this invention will be briefly introduced below. It should be noted that the drawings only show some embodiments of the invention. For those skilled in the art, other related drawings can be derived from these drawings without creative effort.
[0042] Figure 1 This is a schematic diagram of the characterization results of exosomes in this invention, including transmission electron microscopy morphology diagram, particle size distribution diagram, and exosome marker protein detection results.
[0043] Figure 2 Bright-field morphology, fluorescence tracer image, and scanning electron microscope morphology image of the HAMA sustained-release hydrogel microspheres loaded with exosomes according to the present invention.
[0044] Figure 3 This is a schematic diagram showing the injectability and particle size distribution of the HAMA sustained-release hydrogel microspheres loaded with exosomes according to the present invention.
[0045] Figure 4 The images show the in vitro release curves and degradation results of the HAMA sustained-release hydrogel microspheres loaded with exosomes according to the present invention.
[0046] Figure 5 This is a schematic diagram of the intraocular safety evaluation results after intravitreal injection of HAMA sustained-release hydrogel microspheres of the present invention, including body weight, intraocular pressure, fundus, OCT, ERG, HE staining, etc.
[0047] Figure 6This diagram illustrates the structural protective effect of HAMA sustained-release hydrogel microspheres loaded with exosomes in a glaucoma-related retinal injury model. The results include: OCT imaging results; whole-eye H&E staining images (scale bar = 500 μm); high-magnification retinal H&E staining images (scale bar = 50 μm); RBPMS immunofluorescence staining images (scale bar = 50 μm); TUNEL staining images (scale bar = 50 μm); and quantitative analysis results of overall retinal thickness, inner plexus layer (IPL) thickness, number of RGCs per unit length of retina, number of TUNEL-positive cells per unit length of retina, and RGC density per unit area. Detailed Implementation
[0048] To make the objectives, technical solutions, and advantages of this application clearer, the technical solutions of this application will be clearly and completely described below in conjunction with specific embodiments. Obviously, the described embodiments are only some embodiments of this application, and this application can also be implemented or applied through other different specific implementation methods. The details in this specification can also be modified or changed based on different viewpoints and applications.
[0049] At the same time, it should be understood that the scope of protection of this application is not limited to the specific implementation schemes described below; it should also be understood that the terminology used in the embodiments of this application is for describing specific implementation schemes, and not for limiting the scope of protection of this application.
[0050] When numerical ranges are given in the embodiments, it should be understood that, unless otherwise stated in this application, both endpoints of each numerical range and any value between the two endpoints may be selected. Unless otherwise defined, all technical terms used in this application have the same meaning as commonly understood by those skilled in the art. In addition to the specific methods, equipment, and materials used in the embodiments, based on the knowledge of those skilled in the art and the description in this application, any prior art methods, equipment, and materials similar to or equivalent to those described, used, and materials in the embodiments of this application may be used to implement this application. In this application, unless otherwise specified, all parts and percentages are in units of weight, and unless otherwise specified, all instruments and reagents are commercially available or commonly used in the industry. Unless otherwise specified, the experimental methods and operations in the following embodiments are conventional experimental methods and operations in the art.
[0051] An injectable HAMA sustained-release hydrogel microsphere loaded with exosomes
[0052] (I) Preparation method
[0053] 1. Extraction and characterization of exosomes
[0054] Exosome-derived samples were obtained, and cells and cell debris were removed by low-speed centrifugation. The supernatant was collected and further purified by filtration, ultracentrifugation, or ultrafiltration to obtain exosome precipitate. The precipitate was resuspended in PBS to obtain an exosome suspension.
[0055] The extracted exosomes were characterized by observing their morphology using transmission electron microscopy, detecting their particle size distribution using particle size analysis, and detecting the expression of exosome marker proteins using Western blot.
[0056] like Figure 1 As shown, the exosomes extracted in this invention exhibit a typical vesicle-like structure, with particle sizes mainly distributed between 30 and 200 nm, indicating that the extracted product possesses typical characteristics of exosomes.
[0057] 2. Construction of HAMA sustained-release hydrogel microspheres and their exosome-loaded system
[0058] Dissolve HAMA in sterile PBS to prepare a 4 w / v % HAMA precursor solution, add photoinitiator, mix well and set aside.
[0059] The exosomes obtained in the previous step were added to the HAMA precursor solution and gently mixed to obtain a HAMA mixed precursor solution containing exosomes. In the following examples, unless otherwise specified, the HAMA sustained-release hydrogel microspheres loaded with exosomes refer to HAMA concentrations of 4 w / v% and exosome concentrations of 10^9%. 11 Microsphere system with particles / mL.
[0060] The above-mentioned HAMA mixed precursor containing exosomes was used as the aqueous phase, and the mixture of HFE7500 fluorinated oil and 2% surfactant was used as the oil phase. Droplet precursors were prepared by microfluidic droplet method.
[0061] The formed droplets were cross-linked and cured by ultraviolet light irradiation, preferably for 2 minutes, to obtain HAMA sustained-release hydrogel microspheres. The surfactant was then removed using a demulsifier, followed by repeated washing with PBS to remove residual oil phase, thus obtaining an injectable HAMA sustained-release hydrogel microsphere-loaded exosome system. (II) Specific Implementation Examples
[0063] Example 1: Morphology, encapsulation characteristics and injectability evaluation of HAMA sustained-release hydrogel microspheres loaded with exosomes
[0064] The HAMA sustained-release hydrogel microspheres prepared by the aforementioned method were observed under bright-field microscopy, fluorescence microscopy, and scanning electron microscopy to evaluate their morphology and exosome encapsulation. To verify whether the exosomes were successfully encapsulated within the microspheres, the exosomes were first fluorescently labeled, then mixed with the HAMA precursor solution, and microspheres were prepared using a microfluidic method. The fluorescence signal distribution was then observed under a fluorescence microscope. The resulting microspheres were also lyophilized and their microstructure was observed under a scanning electron microscope.
[0065] Meanwhile, the prepared HAMA sustained-release hydrogel microspheres were loaded into a microsyringe and injected through a fine needle to evaluate their injectability; and the particle size of the microspheres was measured and statistically analyzed.
[0066] like Figure 2 As shown, A: The HAMA sustained-release hydrogel microspheres loaded with exosomes provided by this invention are regular spherical in shape and have a relatively uniform particle size under a bright-field microscope; B: Under a fluorescence microscope, fluorescence signals are uniformly distributed inside the microspheres, indicating that the exosomes are successfully encapsulated in the HAMA microspheres; C: Observed by a scanning electron microscope, the lyophilized HAMA microspheres are relatively regular spherical in shape and the surface of the microspheres is smooth.
[0067] like Figure 3 As shown, A: The HAMA sustained-release hydrogel microspheres loaded with exosomes provided by the present invention can be smoothly injected through a microsyringe, exhibiting good injectability; B: The microsphere particle size distribution is relatively concentrated, indicating that the HAMA sustained-release hydrogel microspheres prepared by the method of the present invention have good size uniformity.
[0068] Example 2: Evaluation of the in vitro sustained-release effect of HAMA sustained-release hydrogel microspheres loaded with exosomes on exosomes and the degradation of the microspheres.
[0069] The HAMA sustained-release hydrogel microspheres loaded with exosomes prepared in Example 1 were placed in PBS, and the release solution was collected at preset time points, while an equal volume of fresh PBS was added. The cumulative release curve was plotted using a protein quantification method.
[0070] Meanwhile, the degradation of the microspheres during the release process was observed and analyzed to evaluate the relationship between their sustained-release performance and material degradation.
[0071] like Figure 4 As shown, HAMA sustained-release hydrogel microspheres loaded with exosomes can continuously release exosomes over a relatively long period of time, while the microspheres gradually degrade over time. The results indicate that the microsphere system described in this invention can achieve sustained release of exosomes, and further promote exosome release through material degradation, thereby significantly improving the duration of exosome action.
[0072] Example 3: Intraocular safety evaluation after intravitreal injection of HAMA sustained-release hydrogel microspheres
[0073] Animal experiments: Healthy C57BL / 6J mice were used and injected intravitreally with the HAMA sustained-release hydrogel microspheres prepared in Example 1. Before injection, mice were general anesthetized, and topical anesthetic and mydriatic drugs were applied locally. After ocular disinfection, 0.5 μL of the microsphere suspension was slowly injected into the vitreous cavity through the limbus using a 33G needle microsyringe under a microscope. The control group received the same volume of solvent.
[0074] After the injection, relevant safety tests were conducted in the second and fourth weeks, including intraocular pressure measurement, fundus observation, OCT examination, ERG test and HE staining.
[0075] like Figure 5 As shown, after intraocular injection of HAMA sustained-release hydrogel microspheres, no significant abnormalities were observed in intraocular pressure, fundus morphology, retinal laminar structure, ERG reaction, and HE staining results between the experimental group and the control group, suggesting that the HAMA sustained-release hydrogel microspheres described in this invention have good intraocular biocompatibility.
[0076] Example 4: Structural protection of HAMA sustained-release hydrogel microspheres loaded with exosomes in a glaucoma-related retinal injury model.
[0077] A mouse model of glaucoma-related retinal injury was established using a retinal ischemia-reperfusion injury model. After modeling, mice in each group were administered intravitreal injections at predetermined time points.
[0078] The specific groups included: normal control group; model group + PBS; free exosome group; and HAMA sustained-release hydrogel microsphere group loaded with exosomes.
[0079] After the intervention, eyeballs from mice in each group were harvested and retinal slides or sections were prepared. Immunofluorescence staining with RBPMS antibody was used to assess retinal ganglion cell viability; TUNEL staining was used to detect retinal cell apoptosis; and retinal thickness was analyzed using OCT or tissue sections to evaluate the degree of retinal structural damage.
[0080] like Figure 6 As shown, compared with the model group, the number of RBPMS positive cells in the HAMA sustained-release hydrogel microsphere group loaded with exosomes was significantly increased, the TUNEL positive signal was significantly reduced, and the loss of retinal thickness was improved, indicating that the sustained-release delivery system constructed in this invention has a significant structural protective effect on glaucoma-related retinal damage; and its protective effect is better than that of the free exosome group, suggesting that sustained-release delivery can enhance the in vivo therapeutic effect of exosomes.
[0081] The above description is merely a preferred embodiment of this application and is not intended to limit this application in any way. Any person skilled in the art can make some modifications or alterations to the above-disclosed technical content to obtain equivalent embodiments without departing from the scope of the technical solution of this application. Any simple modifications, equivalent changes and alterations made to the above embodiments based on the technical essence of this application without departing from the content of the technical solution of this application should be included within the scope of protection of the claims of this application.
Claims
1. An injectable HAMA sustained-release hydrogel microsphere loaded with exosomes, characterized in that, The hydrogel microspheres are based on HAMA and cross-linked to form a three-dimensional network structure, and encapsulate a pharmaceutically effective amount of exosomes for local sustained-release delivery of exosomes after intraocular injection.
2. The injectable HAMA sustained-release hydrogel microspheres loaded with exosomes according to claim 1, characterized in that, The mass concentration of HAMA is 4% to 6% (w / v); the particle size of the exosomes is 60 to 100 nm.
3. The injectable HAMA sustained-release hydrogel microspheres loaded with exosomes according to claim 1, characterized in that, The exosomes are dispersed in HAMA precursor solution and are uniformly embedded inside the microspheres or in the pore network during the microsphere molding and cross-linking process.
4. The injectable HAMA sustained-release hydrogel microspheres loaded with exosomes according to claim 1, characterized in that, The hydrogel microspheres are microspheres that can be administered via intravitreal injection. They can pass smoothly through the injection needle under the action of injection shear force and maintain the structural integrity of the microspheres after injection.
5. A method for preparing injectable HAMA sustained-release hydrogel microspheres loaded with exosomes as described in any one of claims 1 to 4, characterized in that, Includes the following steps: S1. Dissolve HAMA in buffer solution to prepare HAMA precursor solution, and add photoinitiator to form aqueous phase; S2. Add the exosomes to the aqueous phase and mix well to obtain a HAMA mixed precursor solution containing exosomes; S3. The mixed precursor liquid is processed into droplets using a microfluidic device to form droplets; S4. The droplets are photocrosslinked and cured to obtain HAMA sustained-release hydrogel microspheres loaded with exosomes; S5. Wash, separate and preserve the obtained hydrogel microspheres.
6. The preparation method according to claim 5, characterized in that, The microfluidic device droplet forming process described in step S3 uses an oil-in-water system, with the oil phase containing HPE7500 fluorinated oil and 2% surfactant.
7. The preparation method according to claim 5, characterized in that, The photocrosslinking curing in step S4 is performed by irradiation with ultraviolet light or visible light, and the crosslinking time is 2 to 3 minutes.
8. The use of injectable HAMA sustained-release hydrogel microspheres loaded with exosomes according to any one of claims 1 to 4 in the preparation of glaucoma treatment drugs.
9. The application according to claim 8, characterized in that, The injectable HAMA sustained-release hydrogel microspheres loaded with exosomes are used to protect retinal ganglion cells and improve retinal ischemia-induced damage.
10. A pharmaceutical composition for treating glaucoma, characterized in that, The pharmaceutical composition comprises injectable HAMA sustained-release hydrogel microspheres loaded with exosomes as described in any one of claims 1 to 4, and pharmaceutically acceptable excipients; and the pharmaceutical composition is a formulation for intravitreal injection.