Hydrogel sustained-release system loaded with 3D culture MSC source exosome and preparation method and application of hydrogel sustained-release system

Abstract

The invention discloses a hydrogel sustained-release system loaded with a 3D cultured MSC source exosome and a preparation method and application thereof, and belongs to the technical field of biomedicine, the exosome is the 3D cultured MSC source exosome, MSC comprises bone marrow MSC, and hydrogel comprises GelMA. According to the invention, the methacrylated gelatin (GelMA) hydrogel is used as a carrier to load the 3D culture MSC source exosome, and an anti-inflammatory and repair-promoting exosome-hydrogel sustained release system is successfully established and is applied to treatment of corneal injury repair. Researches prove that the 3D cultured MSC exosome can relieve inflammatory response and stromal scar formation after corneal injury and promote corneal epithelium regeneration. The hydrogel is used as a carrier, so that continuous slow release of the functional exosome on the surface of the cornea is realized, the action time of the functional exosome is prolonged, the treatment effect of the functional exosome is improved, corneal stroma regeneration can be promoted, and the repair of corneal injury is comprehensively promoted.

Description

Technical Field

[0001] The present invention relates to the field of biomedical technology, and in particular to a hydrogel sustained-release system loaded with 3D cultured MSC-derived exosomes, and a preparation method and application thereof. Background Art

[0002] The cornea is the outermost layer of the eye and an important part of the visual system. It also protects the intraocular structures and tissues from the external environment and pathogens. Corneal injuries, especially scalds and chemical burns of the cornea, usually lead to limbal stem cell decompensation (LSCD) and deep damage to the corneal stroma. Such patients cannot be treated with traditional donor corneal transplantation, but require special stem cell transplantation to reconstruct the ocular surface function. At present, the clinical treatment of corneal stroma damage with LSCD mostly uses corneal transplantation combined with annular limbal transplantation or corneal transplantation combined with cultured limbal stem cell (LSCs) transplantation. However, there are limitations such as shortage of donor materials, high postoperative transplant rejection rate, and poor prognosis. Clinical treatment is difficult and the social and family burden is serious. It is a major clinical scientific problem that needs to be solved urgently.

[0003] Hydrogel is a three-dimensional (3D) network polymer that can retain a large amount of water or biological fluids, allowing oxygen, water and glucose to diffuse through the network to promote wound healing. The application of corneal hydrogel has largely alleviated the shortage of corneal donor materials. Stem cell transplantation for LSCD patients is usually successful in the short to medium term, but the effect seems to be less ideal in the long term. The reasons for failure may be affected by factors such as disease stage, etiology, coexisting ocular diseases, concomitant systemic diseases, stem cell transplantation method and source (autologous, allogeneic). Stem cell therapy combined with 3D hydrogel bioscaffolds has great potential in replacing donor tissue for transplantation or improving wound healing. However, stem cell transplantation for tissue regeneration faces major challenges such as tumorigenicity, pro-inflammatory cytotoxicity and host rejection.

[0004] With the continuous development of stem cell and regenerative medicine technology, a large number of studies have shown that mesenchymal stem cells (MSC) have the ability of self-renewal and multidirectional differentiation. Under certain conditions, they can differentiate into corneal epithelial cells, corneal stromal cells and endothelial cells to repair damaged corneas. The cytokines secreted by MSC have the functions of immunoregulation, promoting repair and anti-angiogenesis, bringing new hope to the repair of corneal damage. Further studies have found that the therapeutic effect of MSC is mainly attributed to its paracrine exosomes. Exosomes are nanoscale bioactive vesicles with a diameter of 40-160nm, which can transfer bioactive molecules such as DNA, mRNA, miRNA, proteins and lipids from donor cells to recipient cells, realize cell-to-cell communication, and participate in the regulation of different physiological activities. Stem cell-derived exosomes retain the characteristics of stem cells, including promoting tissue repair and regeneration and immunoregulation. Compared with stem cells, exosomes also have the following advantages: (1) low immunogenicity, avoiding the occurrence of immune rejection reactions; (2) high stability and easy storage; (3) exosome production can be achieved by changing culture conditions; (4) exosome function can be changed by changing the cell environment.

[0005] However, the current research on MSC-derived exosomes for corneal injury repair is limited to 2D exosomes. However, traditional 2D culture is difficult to simulate the microenvironment in which stem cells survive, which inevitably leads to the loss of stemness of MSCs, and has limitations such as low exosome production and low biological activity, which greatly limits its therapeutic effect. Cell-cell and cell-matrix interactions in 3D culture can better simulate the original microenvironment in which cells survive, and play a key role in cell adhesion, proliferation, migration and differentiation, and paracrine function regulation. So far, there has been no research report on the use of 3D cultured MSC-derived exosomes for corneal injury repair treatment. In addition, the methods for exosome treatment of corneal injury repair are mostly eye drops or subconjunctival injections, which have the limitations of low drug utilization, reduced therapeutic effect, and secondary damage, and repeated subconjunctival injections have the risk of bleeding and infection. Therefore, it is necessary to provide a method for preparing 3D cultured MSC exosomes, and then release them through a simple, non-invasive and effective hydrogel system. More importantly, when corneal damage involves the corneal stroma, the exosome-hydrogel sustained-release system in the form of simple eye drops is often unsatisfactory due to the lack of sufficient mechanical properties. Therefore, the selected hydrogel carrier should not only play the role of sustained release of exosomes, but also have the function of corneal scaffold to promote corneal stroma regeneration. Comprehensive treatment of corneal damage repair from the perspective of reducing inflammatory response, promoting corneal epithelial repair and stroma regeneration, and reducing stroma scarring is of great significance. Summary of the invention

[0006] The purpose of the present invention is to provide a hydrogel sustained-release system loaded with 3D cultured MSC-derived exosomes and a preparation method and application thereof, so as to solve the problems existing in the above-mentioned prior art. The hydrogel sustained-release system loaded with 3D cultured MSC-derived exosomes can comprehensively promote the repair of corneal damage, enrich the current means of treating corneal damage, and provide new ideas for the treatment of corneal damage-related diseases, and has good clinical application value.

[0007] To achieve the above object, the present invention provides the following solutions:

[0008] The present invention provides a hydrogel sustained-release system loaded with exosomes, characterized in that the exosomes are exosomes derived from 3D cultured MSCs;

[0009] The MSCs include bone marrow MSCs; and the hydrogel includes GelMA.

[0010] The present invention also provides a method for preparing the hydrogel sustained-release system loaded with 3D cultured MSC exosomes, comprising the following steps:

[0011] Heat GelMA until it is completely dissolved, add photoinitiator LAP to prepare GelMA hydrogel precursor solution;

[0012] The 3D cultured MSCs were centrifuged multiple times, and the precipitates were collected to obtain the 3D cultured MSC-derived exosomes;

[0013] Adding the exosomes to the GelMA hydrogel precursor solution to obtain an exosome-hydrogel mixed solution;

[0014] The exosome-hydrogel mixed solution is placed under ultraviolet light for cross-linking to form the exosome-loaded hydrogel sustained-release system.

[0015] Furthermore, the mass concentration of the GelMA hydrogel precursor solution is 10%;

[0016] The MSCs include bone marrow MSCs; the multiple centrifugations are: 4°C, 300g for the first centrifugation for 10 min, collecting the supernatant; 4°C, 2000g for the second centrifugation for 10 min, collecting the supernatant; 4°C, 10000g for the third centrifugation for 30 min, collecting the supernatant; 4°C, 100000g for the fourth centrifugation for 90 min, collecting the precipitate.

[0017] Furthermore, 60 μg of 3D cultured MSC-derived exosomes were loaded into each 1 mL of the hydrogel sustained-release system.

[0018] Furthermore, the 3D cultured MSCs are prepared by the following steps:

[0019] MSCs were inoculated into DMEM / F12 medium containing 10% fetal bovine serum and 1% penicillin-streptomycin for culture and passage to obtain 2D cultured MSCs;

[0020] Heat GelMA until it is completely dissolved, add photoinitiator LAP to prepare a GelMA hydrogel precursor solution with a mass concentration of 5%;

[0021] The 2D cultured MSCs were resuspended in GelMA hydrogel precursor solution to form bio-ink, which was then cured by UV light and added to complete culture medium to obtain 3D cultured MSCs.

[0022] Furthermore, the culture condition is constant temperature culture at 37°C.

[0023] Furthermore, the density of 2D cultured MSCs in the bio-ink is 1.0×10 6 / mL-1.0×10 7 / mL.

[0024] The present invention also provides application of the hydrogel sustained-release system in preparing medicine for treating corneal damage.

[0025] Furthermore, the hydrogel sustained-release system loaded with 3D cultured MSC exosomes plays a role in treating corneal damage by promoting corneal epithelial regeneration and matrix transparency repair, reducing matrix edema and alleviating inflammatory response.

[0026] Furthermore, the hydrogel sustained-release system loaded with 3D cultured MSC exosomes plays a role in treating corneal damage by reducing the expression level of pro-inflammatory related factors and increasing the expression level of anti-inflammatory related factors and pro-repair related factors.

[0027] The present invention discloses the following technical effects:

[0028] The inventors' previous studies have found that compared with traditional 2D cell culture, cell-cell and cell-matrix interactions in 3D culture can better simulate the original microenvironment for cell survival, and play a key role in cell adhesion, proliferation, migration and differentiation, and paracrine function regulation. 3D culture of MSC can significantly increase the production and biological activity of exosomes. In addition, hydrogel is a 3D network polymer that can retain a large amount of water or biological fluids, allowing oxygen, water and glucose to diffuse through the network to promote wound healing. Moreover, it can also be used as a carrier for sustained release of functional drugs, molecules and cells to increase the frequency of drug utilization. The construction of a sustained release system by hydrogel-loaded exosomes can prolong the local action time of exosomes and promote corneal damage repair.

[0029] The present invention first discovered that compared with exosomes derived from 2D cultured MSC, exosomes derived from 3D cultured MSC can more effectively inhibit ocular surface inflammatory response, maintain LSCs dryness, promote corneal epithelial regeneration, reduce corneal stroma turbidity, and thus promote the repair of corneal damage. Using GelMA hydrogel as a carrier to load 3D cultured MSC-derived exosomes, an anti-inflammatory and pro-repair exosome-hydrogel sustained-release system was successfully established, which not only achieved the sustained sustained release of functional exosomes on the corneal surface, prolonged its action time, and improved its therapeutic effect, but also promoted corneal stroma regeneration, thereby comprehensively promoting the repair of corneal damage, enriching the current means of treating corneal damage, and providing new ideas for the treatment of corneal damage-related diseases, with good clinical application value. BRIEF DESCRIPTION OF THE DRAWINGS

[0030] In order to more clearly illustrate the embodiments of the present invention or the technical solutions in the prior art, the drawings required for use in the embodiments will be briefly introduced below. Obviously, the drawings described below are only some embodiments of the present invention. For ordinary technicians in this field, other drawings can be obtained based on these drawings without paying creative work.

[0031] Figure 1 Flow chart for preparing the sustained-release system of GelMA hydrogel loaded with 3D cultured MSC-derived exosomes;

[0032] Figure 2 The morphology of 2D and 3D cultured MSCs observed under an optical microscope (A) and live / dead cell staining (B);

[0033] Figure 3 Immunofluorescence staining of stem cell markers Sox2 (A) and Nanog (B) in 2D and 3D cultured MSCs;

[0034] Figure 4 Schematic diagram of the extraction of exosomes from 2D and 3D cultured MSCs;

[0035] Figure 5 The morphology of 2D and 3D cultured MSC-derived exosomes was observed using transmission electron microscopy;

[0036] Figure 6 To detect the expression of surface marker proteins CD9 and CD81 of exosomes derived from 2D and 3D cultured MSCs using Western blotting technology;

[0037] Figure 7 To detect the particle size distribution and concentration of exosomes derived from 2D and 3D cultured MSCs using nanoparticle tracing technology;

[0038] A: Particle size distribution of MSC exosomes cultured in 2D; B: Particle size distribution of MSC exosomes cultured in 3D; C: 2D and

[0039] Concentration diagram of 3D cultured MSC exosomes;

[0040] Figure 8 PKH-67 fluorescence staining of corneal epithelial cells (A) and stromal cells (B) for the internalization of 2D and 3D cultured MSC-derived exosomes;

[0041] Fig. 9 Immunofluorescence staining of MSC exosomes on the protein expression of limbal epithelial stem cells (A) and stromal cells (B);

[0042] Fig.10 The miRNA sequencing analysis of exosomes derived from 2D and 3D cultured MSCs; A: heat map B: volcano map;

[0043] Fig.11 Transparency of GelMA hydrogel loaded with 2D and 3D exosomes (A) and transmittance of GelMA hydrogel (B);

[0044] Fig.12 Scanning electron microscopy images of GelMA hydrogels loaded with 2D (A) and 3D (B) cultured MSC-derived exosomes;

[0045] Fig.13 The in vitro sustained release behavior diagram of the GelMA hydrogel sustained release system loaded with 2D and 3D cultured MSC-derived exosomes;

[0046] Fig.14 Biocompatibility test of co-cultured corneal epithelial and stromal cells with GelMA hydrogel sustained-release system loaded with 3D cultured MSC-derived exosomes; A: live / dead cell staining; B: corneal epithelial cell survival rate; C: corneal stromal cell survival rate;

[0047] Fig.15 The effect of GelMA hydrogel sustained-release system loaded with 3D cultured MSC-derived exosomes on promoting corneal damage repair; A: slit lamp microscopy and sodium fluorescein staining; B: statistical graph of corneal epithelial healing speed; C: statistical graph of corneal thickness;

[0048] Fig.16 Real-time PCR detection results of GelMA hydrogel sustained-release system loaded with 3D cultured MSC-derived exosomes to promote corneal damage repair; A: CD86; B: IL-1β; C: CD206; D: IL-10; E: PDGF; F: TGF-β1. DETAILED DESCRIPTION

[0049] Various exemplary embodiments of the present invention will now be described in detail. This detailed description should not be considered as limiting the present invention, but should be understood as a more detailed description of certain aspects, features, and embodiments of the present invention.

[0050] It should be understood that the terms described in the present invention are only for describing a particular embodiment and are not intended to limit the present invention. In addition, for the numerical range in the present invention, it should be understood that each intermediate value between the upper and lower limits of the scope is also specifically disclosed. The intermediate value in any stated value or stated range, and each smaller range between any other stated value or intermediate value in the described range is also included in the present invention. The upper and lower limits of these smaller ranges can be independently included or excluded in the scope.

[0051] Unless otherwise indicated, all technical and scientific terms used herein have the same meanings as those generally understood by those skilled in the art. Although the present invention describes only preferred methods and materials, any methods and materials similar or equivalent to those described herein may also be used in the implementation or testing of the present invention. All documents mentioned in this specification are incorporated by reference to disclose and describe the methods and / or materials associated with the documents. In the event of a conflict with any incorporated document, the content of this specification shall prevail.

[0052] It will be apparent to those skilled in the art that various modifications and variations may be made to the specific embodiments of the present invention description without departing from the scope or spirit of the present invention. Other embodiments derived from the present invention description will be apparent to those skilled in the art. The present invention description and examples are exemplary only.

[0053] The words “include,” “including,” “have,” “contain,” etc. used in this document are open-ended terms, meaning including but not limited to.

[0054] Human bone marrow MSCs were purchased from Saiye Biotechnology Co., Ltd.

[0055] Lyophilized GelMA: It is a gelatin derivative obtained by the reaction of gelatin and methacrylic anhydride, purchased from Suzhou Yongqinquan Intelligent Equipment Co., Ltd., item number EFL-GM-60.

[0056] Photoinitiator LAP and cell curing ring were purchased from Suzhou Yongqinquan Intelligent Equipment Co., Ltd.

[0057] Example 1

[0058] 1. Cultivation of MSCs

[0059] 1.1 2D culture of MSCs

[0060] The purchased bone marrow MSC cells were cultured in a 37°C constant temperature incubator with DMEM / F12 medium containing 10% fetal bovine serum and 1% penicillin-streptomycin, and the medium was changed every 48 hours. When the cell density reached 80%, the cells were subcultured. The MSCs after the third passage (P3) were used for subsequent studies.

[0061] 1.2 3D culture of MSCs

[0062] The freeze-dried GelMA was heated in a water bath at 50-60°C until completely dissolved, and 0.25% of the photoinitiator LAP was added to prepare a 5% (w / v) concentration of GelMA hydrogel precursor solution. The MSCs that were cultured in 2D and passed to the third generation were collected and resuspended in a 5% (w / v) concentration of GelMA hydrogel precursor solution to form a bio-ink. The cell density was 1.0×10 6 / mL-1.0×10 7 / mL. The bio-ink was added to the curing ring, UV-cured for 10 seconds, and then complete culture medium was added and placed in a 37°C constant temperature incubator for culture.

[0063] 2. Detection of MSC viability and stemness markers

[0064] 2.1 Survivability

[0065] The viability of MSCs was evaluated by staining with a live / dead cell staining kit (Abcam, USA). The prepared live / dead cell staining solution was added to 2D and 3D cultured MSCs respectively and incubated at room temperature for 10 min. Photos were taken under a laser confocal microscope. The results showed that under 3D culture conditions, MSCs were evenly distributed in the GelMA hydrogel, and almost no dead cells were detected ( Figure 2 ).

[0066] 2.2 Dry markers

[0067] Conventional immunofluorescence staining techniques were used to detect MSC stemness markers SOX2 and Nanog.

[0068] 2D and 3D cultured MSCs were fixed with 4% paraformaldehyde, blocked with 5% fetal bovine serum (BSA) at room temperature for 1 hour, incubated with SOX2 and Nanog antibodies at 4°C overnight, and then added with corresponding secondary antibodies. They were incubated at room temperature and away from light for 1 hour, and anti-fluorescence quencher containing DAPI was added. Finally, the images were taken under a laser confocal microscope. The experimental results showed that compared with 2D cultured MSCs, the fluorescence intensity of MSC stemness markers SOX2 and Nanog in 3D culture was enhanced ( Figure 3 ).

[0069] 3. Collection of MSC exosomes

[0070] When MSC is passaged to the 4th to 5th generation and the growth covers 70% to 80% of the bottom of the bottle, replace the exosome-free serum and continue to culture for 24-72 hours, and collect the culture medium. The obtained culture medium is separated from the exosomes by a conventional ultracentrifuge, specifically 4°C, 300g centrifugation for 10 minutes, and the supernatant is collected to remove dead cells; 4°C, 2000g centrifugation for 10 minutes, and the supernatant is collected to remove cell debris; 4°C, 10000g centrifugation for 30 minutes, and the supernatant is collected to remove microvesicles; 4°C, 100000g centrifugation for 90 minutes, and the precipitate is collected as exosomes. The exosomes are resuspended in PBS twice and filtered through a 0.22μm filter. The obtained exosome suspension is stored in a -80°C refrigerator for later use ( Figure 4 ). Exosomes derived from 2D cultured MSCs (abbreviated as 2D exosomes, 2D-Exo) and exosomes derived from 3D cultured MSCs (abbreviated as 3D exosomes, 3D-Exo) were obtained.

[0071] 4. Observation of MSC exosome morphology

[0072] 5 μL of diluted exosome suspension was added to the copper mesh, and after standing and air-drying at room temperature, 20 μL of 1% phosphotungstic acid dye solution was added to the surface of the copper mesh, and it was allowed to stand at room temperature for 2 minutes of negative staining. After the negative staining was completed, the dye solution was absorbed with filter paper, and the surface of the copper mesh was washed with PBS solution. After the filter paper was dried, the copper mesh was allowed to stand and air-dry at room temperature. Finally, the copper mesh was placed in the sample chamber of the transmission electron microscope and photographed for observation. The results showed that MSC exosomes were disc-shaped vesicles with bilateral depressions, and the double-layer vesicle structure ( Figure 5 ).

[0073] 5. Identification of MSC exosome marker proteins

[0074] Western blotting was used to detect the expression of specific marker proteins on the surface of MSC exosomes. The exosome suspension was taken and added with RIPA cell lysis buffer. The protein concentration was quantified according to the BCA protein quantification kit. Conventional Western blotting was used to detect the expression of CD9 and CD81, and MSC was used as a control. The results showed that compared with MSC, MSC exosomes highly expressed CD9 and CD81 ( Figure 6 ).

[0075] 6. Analysis of MSC exosome size and concentration

[0076] The particle size distribution and particle concentration of MSC exosomes were detected by nanoparticle tracking analysis (NTA). The sample was diluted with 1×PBS and added to the sample pool, and the real-time dynamic image of exosome particles was observed on the computer screen. The NTA results showed that the diameter of exosomes was between 40-160nm, and the concentration of exosomes from 3D cultured MSCs was 1.3 times that of exosomes from 2D cultured MSCs ( Figure 7 ).

[0077] 7. Endocytosis of MSC exosomes by corneal epithelial cells and stromal cells

[0078] PKH-67 fluorescent staining kit was used to label MSC exosomes according to the method provided in the instruction manual. Then the labeled 2D and 3D exosomes (60ug) were co-cultured with corneal epithelial cells and stromal cells for 24h, respectively. The images were taken under a laser confocal microscope. The experimental results showed that compared with 2D exosomes, 3D exosomes were more easily taken up and phagocytosed by corneal epithelial and stromal cells ( Figure 8 )

[0079] 8. Effects of MSC exosomes on protein expression of limbal stem cells (LSCs) and stromal cells

[0080] Conventional immunofluorescence staining techniques were used to detect the expression of LSCs and stromal cell-related protein markers.

[0081] 2D and 3D exosomes (60ug) were co-cultured with limbal epithelial stem cells and stromal cells for 24 hours, fixed with 4% paraformaldehyde, blocked with 5% BSA at room temperature for 1 hour, incubated with CK3, CK12, P63 (LSCs markers) and DCN, LUM, α-SMA (corneal stromal cell markers) antibodies at 4°C overnight, added with corresponding secondary antibodies, incubated at room temperature and away from light for 1 hour, added with anti-fluorescence quencher containing DAPI, and finally photographed under a laser confocal microscope. The experimental results showed that compared with 2D exosomes, 3D exosomes can better maintain the activity of LSCs stem cells, promote the expression of corneal proteoglycan markers, and inhibit the expression of scar markers ( Fig. 9 ).

[0082] 9. MSC exosome miRNA sequencing analysis

[0083] Total exosome RNA was extracted using TRlzol reagent according to the method provided in the instructions. The total amount and integrity of RNA were evaluated using Nanodrop 2000 (Thermo Fisher Scientific Inc., USA) and Agilent 2100 Bioanalyzer (Agilent Technology, USA), respectively. 1 μg of total RNA was taken from each sample, and the small RNA library was constructed using the NEB Next Small RNA Library Prep Set for Illumina kit (Cat. No. NEB # E7330S, NEB, USA). Subsequently, agarose gel electrophoresis was performed to separate and purify the 140-160 bp PCR products as small RNA libraries. After the library quality was qualified by the Agilent 2100 Bioanalyzer, it was sequenced using the Illumina Novasek 6000 platform to generate 150 bp double-end sequences, and the data were analyzed using software programs. The results showed that compared with 2D exosomes, the contents of 28 miRNAs in 3D exosomes changed, of which 17 were upregulated and 11 were downregulated ( Fig.10 ).

[0084] Based on the above results, 3D cultured MSC-derived exosomes are significantly superior to 2D exosomes in all aspects.

[0085] Example 2 Construction of exosome-loaded GelMA hydrogel sustained-release system

[0086] Process such as Figure 1 As shown, the freeze-dried GelMA was heated in a water bath at 50-60°C until completely dissolved, and 0.25% of the photoinitiator LAP was added to prepare a 10% (w / v) concentration of GelMA hydrogel precursor solution. When the temperature was lowered to room temperature, the exosomes from the 2D cultured MSC source and the 3D cultured MSC source extracted in Example 1 were added respectively, so that each 1mL of GelMA hydrogel contained 60μg of exosomes; then the exosome-hydrogel mixed solution was placed under ultraviolet light for 30s to crosslink into a gel, forming a 2D exosome-loaded GelMA hydrogel sustained-release system and a 3D exosome-loaded GelMA hydrogel sustained-release system.

[0087] 1. Detect transparency and transmittance

[0088] The GelMA hydrogel loaded with exosomes was prepared into a corneal shape with a diameter of about 10mm and a thickness of about 1000mm, and placed on a Cornea with black text on a white background for observation and photography; the GelMA hydrogel loaded with 2D and 3D exosomes was prepared into a corneal shape with a diameter of about 10mm and a thickness of about 1000mm, and fixed in the sample compartment of the UV spectrophotometer. The transmittance of the sample in the wavelength range of 300-800nm ​​was tested, with a detection point every 50nm wavelength, and pure water was used as a control. The results showed that the GelMA hydrogel loaded with exosomes had good transparency and transmittance ( Fig.11 ).

[0089] 2. Scanning electron microscopy observation of hydrogel successfully loaded with exosomes

[0090] Take an appropriate amount of freeze-dried GelMA hydrogel loaded with 2D and 3D exosomes, spray gold and perform electron microscopy scanning. The results show that the exosomes are evenly distributed in the gaps of the hydrogel ( Fig.12 ).

[0091] 3. Determination of in vitro drug release behavior

[0092] In order to determine the release of exosomes in the exosome-loaded GelMA hydrogel sustained-release system, 200 μL of 2D and 3D exosome-loaded hydrogels were immersed in 1 mL PBS and incubated in a 37°C incubator for 14 days. All PBS solutions were collected at a specific time each day and stored in a 4°C refrigerator. Fresh PBS was then added to the hydrogel. The exosomes in the collected PBS solution were quantified using a BCA protein quantification kit to evaluate the release of exosomes. The results showed that more than 50% of the exosomes were continuously released from the hydrogel within 5 days ( Fig.13 ).

[0093] 4. Effect of GelMA hydrogel on the biocompatibility of corneal epithelial and stromal cells

[0094] The biocompatibility of 2D exosome-loaded and 3D exosome-loaded GelMA hydrogels on corneal epithelial (HCEC) and stromal cells (HCSC) was evaluated using a live / dead cell staining kit (Abcam, USA). The corneal epithelial and stromal cells were co-cultured with GelMA hydrogels for 24 hours, and the prepared live / dead cell staining working solution was added to the above co-cultured two types of cells and incubated at room temperature for 10 minutes. Photos were taken under a laser confocal microscope. The results showed that GelMA has good biocompatibility, and the survival rates of the two types of cells in 2D exosome-loaded and 3D exosome-loaded GelMA hydrogels were both higher than 90%, and almost no dead cells were detected ( Fig.14 ).

[0095] Example 3 Animal Experiment

[0096] Construction of rabbit corneal injury model: The corneal epithelial cells of New Zealand white rabbits were scraped off with an epithelial knife, and 1 / 2 circle of limbal tissue at the junction of cornea and sclera was peeled off to construct a partial LSCD model; a 1 / 2 full corneal thickness matrix defect model was prepared using a 3 mm trephine drill and a lamellar knife.

[0097] Experimental design: Rabbits (source: purchased from Jinan Xilingjiao Breeding Center, n = 12) were randomly divided into 4 groups: control group (no hydrogel and exosome treatment after modeling), Hydrogel group (hydrogel after modeling), Hydrogel + 2D-Exo group (hydrogel loaded with 2D exosomes after modeling), Hydrogel + 3D-Exo group (hydrogel loaded with 3D exosomes after modeling). Hydrogel or hydrogel loaded with exosomes was dripped into the corneal stromal defect of the corresponding rabbits, and slit lamp microscopy and sodium fluorescein staining were used at different time points after surgery to verify the therapeutic effect of the hydrogel sustained-release system.

[0098] Evaluation of corneal injury repair effect by slit lamp microscopy and sodium fluorescein staining: Slit lamp microscopy and sodium fluorescein staining were used to observe corneal epithelial injury repair, stromal edema, and stromal transparency of rabbits in each group at 1, 4, 7, 10, and 13 days after modeling. The results showed that compared with the control group, Hydrogel group, and Hydrogel+2D-Exo group, the Hydrogel+3D-Exo group could significantly promote corneal epithelial regeneration and stromal transparency repair, reduce stromal edema, and alleviate inflammatory response ( Fig.15 ).

[0099] Real-time PCR (RT-PCR) detection to evaluate the corneal damage repair effect: rabbits were killed 4 and 14 days after modeling to obtain corneas, RNA was extracted according to the instructions of the kit, reverse transcribed into DNA and real-time quantitative PCR was performed, and the program parameters were set as follows: 95℃, 5min→95℃, 10s; 60℃, 30s (repeated 40 times)→95℃, 15s; 60℃, 60s; 95℃, 15s. Copy data and analyze: GAPDH was selected as the internal reference, and 2 -ΔΔCT The RT-PCR results were analyzed and compared. The experimental results showed that compared with the control group, Hydrogel group, and Hydrogel+2D-Exo group, the Hydrogel+3D-Exo group could significantly reduce the expression levels of pro-inflammatory factors CD86 and IL-1β, and increase the expression levels of anti-inflammatory factors CD206 and IL-10 and pro-repair factors PDGF and TGF-β1 ( Fig.16 ).

[0100] Among them, the use of III RT SuperMix for qPCR (+gDNAwiper) kit was used for reverse transcription. The reverse transcription system is shown in Table 1.

[0101] Table 1 Reverse transcription system

[0102]

[0103] The PCR reaction was performed using the ChamQ Universal SYBR qPCR Master Mix kit. The reaction system is shown in Table 2.

[0104] Table 2 PCR reaction system

[0105]

[0106]

[0107] The target genes to be detected and their primers are shown in Table 3.

[0108] Table 3 Primer sequences of each gene

[0109]

[0110] The embodiments described above are only descriptions of the preferred modes of the present invention, and are not intended to limit the scope of the present invention. Without departing from the design spirit of the present invention, various modifications and improvements made to the technical solutions of the present invention by ordinary technicians in this field should all fall within the protection scope determined by the claims of the present invention.

Claims

1. A hydrogel sustained-release system loaded with exosomes, characterized in that: The exosomes are exosomes derived from 3D cultured MSCs; The MSCs include bone marrow MSCs; and the hydrogel includes GelMA.

2. The method for preparing the hydrogel sustained-release system according to claim 1, characterized in that: The following steps are involved: Heat GelMA until it is completely dissolved, add photoinitiator LAP to prepare GelMA hydrogel precursor solution; The 3D cultured MSCs were centrifuged multiple times, and the precipitates were collected to obtain the 3D cultured MSC-derived exosomes; Adding the exosomes to the GelMA hydrogel precursor solution to obtain an exosome-hydrogel mixed solution; The exosome-hydrogel mixed solution is placed under ultraviolet light for cross-linking to form the exosome-loaded hydrogel sustained-release system.

3. The preparation method according to claim 2, characterized in that: The mass concentration of the GelMA hydrogel precursor solution is 10%; The MSCs include bone marrow MSCs; the multiple centrifugations are: 4°C, 300g for the first centrifugation for 10 min, collecting the supernatant; 4°C, 2000g for the second centrifugation for 10 min, collecting the supernatant; 4°C, 10000g for the third centrifugation for 30 min, collecting the supernatant; 4°C, 100000g for the fourth centrifugation for 90 min, collecting the precipitate.

4. The preparation method according to claim 2, characterized in that: Each 1 mL of the hydrogel sustained-release system was loaded with 60 μg of 3D cultured MSC-derived exosomes.

5. The preparation method according to claim 2, characterized in that: The 3D cultured MSCs were prepared by the following steps: MSCs were inoculated into DMEM / F12 medium containing 10% fetal bovine serum and 1% penicillin-streptomycin for culture and passage to obtain 2D cultured MSCs; Heat GelMA until it is completely dissolved, add photoinitiator LAP to prepare a GelMA hydrogel precursor solution with a mass concentration of 5%; The 2D cultured MSCs were resuspended in GelMA hydrogel precursor solution to form bio-ink, which was then cured by UV light and added to complete culture medium to obtain 3D cultured MSCs.

6. The preparation method according to claim 5, characterized in that: The culture condition is 37°C constant temperature culture.

7. The preparation method according to claim 5, characterized in that: The density of 2D cultured MSCs in the bio-ink was 1.0×10 6 / mL-1.0×10 7 / mL.

8. Use of the hydrogel sustained-release system according to claim 1 in preparing a drug for treating corneal damage.

9. The use according to claim 8, characterized in that The hydrogel sustained-release system plays a role in treating corneal damage by promoting corneal epithelial regeneration and matrix transparency repair, reducing matrix edema and reducing inflammatory response.

10. The use according to claim 8, characterized in that The hydrogel sustained-release system plays a role in treating corneal damage by reducing the expression level of pro-inflammatory related factors and increasing the expression level of anti-inflammatory related factors and pro-repair related factors.

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