Hydrogel containing pilose antler mesenchymal stem cell exosome and preparation method and application thereof

Abstract

This invention relates to the field of medical materials technology, providing a hydrogel containing antler mesenchymal stem cell exosomes, its preparation method, and applications. The method includes the following steps: preparation of antler mesenchymal stem cell exosomes; preparation of PVP-HA composite hydrogel; uniformly mixing AMSC-Exos with a hydrogel precursor solution, and forming a hydrogel containing antler mesenchymal stem cell exosomes through physical embedding and non-covalent interactions. This invention utilizes the three-dimensional porous network structure of the hydrogel to achieve long-term sustained release of exosomes, protecting them from rapid degradation; it leverages the unique regenerative advantages of antler-derived exosomes to synergistically promote angiogenesis, inhibit inflammatory responses, and regulate macrophage polarization; it promotes angiogenesis through the miR-486-5p / FGF9 axis, achieving efficient repair of chronic diabetic wounds; and it provides a novel, efficient, safe, and clinically translational strategy for the treatment of chronic diabetic skin lesions.

Description

Technical Field

[0001] This invention belongs to the field of medical materials technology, and particularly relates to hydrogels containing deer antler mesenchymal stem cell exosomes, their preparation methods, and applications. Background Technology

[0002] The fundamental reason why diabetic wounds are difficult to heal lies in the complex pathophysiological mechanisms. Studies have shown that oxidative stress, abnormal inflammatory response, slow vascular epithelial repair, fibroblast dysfunction, and abnormal accumulation of pro-inflammatory macrophages are the main reasons why diabetic wounds are difficult to heal.

[0003] Currently, conventional treatments for diabetic skin lesions mainly include debridement, dressing, lesion decompression, anti-infection, peripheral vascular disease management, and blood glucose control. However, these methods mainly focus on relieving symptoms and do not fundamentally intervene in the molecular mechanisms of the damage, thus their efficacy is limited.

[0004] In recent years, exosomes derived from mesenchymal stem cells have shown great potential in cell-free therapy. Exosomes are nanoscale lipid bilayer membrane vesicles with a diameter of about 30-150 nm, rich in a variety of bioactive molecules (such as mRNA, miRNA, proteins and growth factors), which can deliver the functional substances they carry to target cells and regulate skin cell migration, proliferation and collagen secretion.

[0005] Antler is the only organ in mammals that can regenerate completely. Its regeneration process depends on the high proliferation and differentiation potential of antler stem cells. Therefore, exosomes derived from antler mesenchymal stem cells have significant potential in promoting skin wound healing. However, exosomes have limited efficacy in their natural state and are easily degraded, making them difficult to treat complex chronic diseases such as diabetic wounds.

[0006] Hydrogels, as ideal delivery carriers, can absorb tissue exudate and maintain a moist wound environment. Their three-dimensional network structure can effectively isolate pathogenic microorganisms. Studies have shown that hydrogels loaded with exosomes can effectively promote angiogenesis in wounds and reduce inflammatory responses in diabetic wounds. However, research on the combined use of deer antler mesenchymal stem cell exosomes and hydrogels for the treatment of chronic diabetic wounds has not yet been reported. Summary of the Invention

[0007] The purpose of this invention is to provide a hydrogel containing deer antler mesenchymal stem cell exosomes, its preparation method, and its application, in order to solve the problems mentioned in the background art.

[0008] To achieve the above objectives, the present invention provides the following technical solution:

[0009] On one hand, the present invention provides a method for preparing a hydrogel containing exosomes of deer antler mesenchymal stem cells, comprising the following steps:

[0010] S1. Preparation of exosomes from deer antler mesenchymal stem cells: Deer antler mesenchymal stem cells of the 3rd to 5th generation were taken, and after culture, the cell culture supernatant was collected. The cells were centrifuged to remove dead cells and debris, then centrifuged at high speed to remove large vesicles, and finally centrifuged at ultraspeed. The precipitate obtained was exosome AMSC-Exos.

[0011] Preparation of S2, PVP-HA composite hydrogel: Povidone was added to purified water to prepare PVP solution; sodium hyaluronate and polyethylene glycol 400 were added and stirred until completely dissolved to obtain hydrogel precursor solution;

[0012] S3. Construction of hydrogel containing antler mesenchymal stem cell exosomes: The AMSC-Exos prepared in S1 was uniformly mixed with the hydrogel precursor solution prepared in S2. The homogeneous solution was encapsulated in a mold and cross-linked by electron accelerator irradiation to form a hydrogel containing antler mesenchymal stem cell exosomes.

[0013] On the other hand, the present invention also provides a hydrogel containing deer antler mesenchymal stem cell exosomes, which is prepared by the above-described preparation method.

[0014] On the other hand, the present invention also provides the application of the above-mentioned hydrogel containing deer antler mesenchymal stem cell exosomes in the preparation of drugs or dressings for treating chronic skin lesions of diabetes.

[0015] Compared with the prior art, the specific beneficial effects of the present invention are as follows:

[0016] 1. High biocompatibility and safety: The hydrogel materials (PVP, HA, PEG400) have good biocompatibility. Electron beam irradiation crosslinking avoids the potential toxicity of chemical crosslinking agents. Zeta potential analysis (-21~-23mV) confirms the excellent colloidal stability of the composite system.

[0017] 2. Significant long-lasting sustained-release effect: In vitro release experiments show that, compared with the burst release of 80-95% of free exosomes within 1 day, the present invention can extend the release period of exosomes to more than 10 days, achieving continuous and stable release;

[0018] 3. Synergistic enhancement of in vitro repair function:

[0019] Immunomodulation: It can efficiently induce macrophage polarization from M1 to M2, reducing the proportion of M1 cells from 41-45% to 9-14% and increasing the proportion of M2 cells from 18-26% to 69-75%;

[0020] Pro-angiogenesis: It can significantly promote the proliferation, migration and lumen formation of HUVECs;

[0021] 4. Excellent in vivo effect on promoting diabetic wound healing:

[0022] Accelerated healing: In a diabetic mouse model, the AMSC-Exos@Gel treatment group showed the fastest wound healing, with the wounds almost completely closed by day 14.

[0023] Improved histological morphology: HE and Masson staining showed that the skin structure of the treatment group was basically reconstructed, the collagen fibers in the dermis were densely arranged, the hair follicle structure was abundant, and the inflammatory infiltration was significantly reduced;

[0024] Promoting angiogenesis: Immunohistochemistry showed significantly increased expression of CD31, Ki67, VEGF, and α-SMA;

[0025] Regulation of the immune microenvironment: CD86 expression decreased, CD206 expression increased, and the ratio of type I / III collagen tended to be within the physiological range. Attached Figure Description

[0026] Figure 1 This is a schematic diagram illustrating the preparation of a hydrogel containing deer antler mesenchymal stem cell exosomes and its mechanism for promoting the healing of diabetic wounds, as provided in an embodiment of the present invention; A is the exosome isolation process, B is the preparation of the hydrogel loaded with exosomes, and C is the mechanism by which the hydrogel loaded with AMSC-exos promotes the healing of chronic diabetic wounds.

[0027] Figure 2 The following are the results of exosome identification provided in the embodiments of the present invention: A is a transmission electron microscope image of exosomes, B is a nanoparticle tracking analysis of exosome particle size distribution, C is a Coomassie brilliant blue staining image of exosomes, D is a WB image of exosomes, and E is a confocal image of exosome uptake by HUVEC cells. Scale bar = 50 μm.

[0028] Figure 3 Characterization results of the hydrogel provided in the embodiments of the present invention; A is a scanning electron microscope image of the hydrogel and the hydrogel loaded with exosomes; B is a confocal laser scanning microscope image showing that PKH-26 labeled exosomes (red) are uniformly distributed in three-dimensional space within the hydrogel; C is a Fourier transform infrared spectrum; D is the change in Zeta potential before and after loading exosomes onto the hydrogel; E is the release curve of exosomes in the gel hydrogel.

[0029] Figure 4The hydrogel provided in this embodiment of the invention reduces the inflammatory response by inducing RAW 264.7 cells to polarize from the M1 phenotype to the M2 phenotype; A and C are the expression levels (red / green fluorescence) of the M1 macrophage marker iNOS (C) and the M2 macrophage marker Arg-1 (A) after 12 hours of incubation under different conditions, as detected by immunofluorescence staining (scale bar = 250 μm); B is the quantitative analysis of Arg-1 fluorescence intensity; D is the quantitative analysis of iNOS fluorescence intensity; EG shows the effect of H2O2 stimulation and different treatments on the polarization of M1 and M2 macrophages; E is the flow cytometry scatter plot of M1 type (F4 / 80+ CD86+) macrophages, and F is the F4 / 80 ratio of each group. + CD86 + The statistical results of absolute cell counts, G represents M2 type (F4 / 80) in macrophages. + CD206 + Flow cytometry scatter plot; H represents F4 / 80 for each group. + CD206 + Statistical results of absolute cell count; I and J represent the secretion levels of anti-inflammatory factor IL-10 (I) and pro-inflammatory factor TNF-α (J) in the culture supernatant of macrophages in each group in vitro, as detected by ELISA (*p < 0.05, **p < 0.01 ***p < 0.001, ****p < 0.0001).

[0030] Figure 5 The following are the experimental results of promoting angiogenesis provided in the embodiments of the present invention: A is the cell scratch assay to evaluate the proliferation of HUVECs, C is the Transwell assay to evaluate the migration of HUVECs, E is the evaluation of the lumen-forming ability of HUVECs, B is the quantitative statistics of proliferation rate, D is the quantitative statistics of the number of migrating cells, and FH is the quantitative statistics of lumen length and number of branch points. Scale bar: 100 μm (*p < 0.05, **p < 0.01 ***p < 0.001, ****p < 0.0001).

[0031] Figure 6 The following are the results of an in vivo wound healing experiment provided in this embodiment of the invention: A is a diagram of the skin wound healing process of normal mice and diabetic mice after different drug treatments; B is a diagram of the wound area reduction trajectory of each group; C is a comparison of the closure rate of diabetic wounds among the groups; D is HE staining of mouse skin tissue in each group 14 days after treatment (yellow arrow: new epidermis; red arrow: dermis; black arrow: inflammatory cells; green arrow: hair follicles); E is Masson staining of mouse skin tissue 14 days after treatment, scale bar: 100 μm.

[0032] Figure 7 The following are the results of angiogenesis and cell proliferation provided in the embodiments of the present invention; A is a representative immunohistochemical image of CD31, Ki67, VEGF and α-SMA expression in regenerated skin tissue of diabetic mice 14 days after skin injury in the normal group and each treatment group; B and C are quantitative analyses of the expression areas of CD31, Ki67, VEGF and α-SMA, respectively. Scale bar: 100 μm (*p < 0.05, **p < 0.01, ***p < 0.001, ****p < 0.0001).

[0033] Figure 8 The following are the results of the mechanism verification of miR-486-5p targeting FGF9 provided in the embodiments of the present invention: A is the potential target gene of miR-486-5p analyzed by three independent online databases (TargetScan, Miranda, and miR Tarbase); B is the predicted binding sequence of miR-486-5p in the FGF9 transcript; C is the expression level of FGF9 in AMSCs-exo detected by qRT-PCR; D is the verification that FGF9 is a direct target of miR-486-5p by dual-luciferase reporter gene assay; E is the expression of FGF9 in HUVECs after transfection with miR-486-5p inhibitor detected by qRT-PCR; F is the lumen formation ability of HUVECs transfected with inhibitor or siRNA; GH is the quantitative analysis of total lumen length and branching point number; all data are expressed as mean ± standard deviation. Statistical significance: p < 0.05, p < 0.01, p < 0.05. 0.001; ns, no significant difference. Detailed Implementation

[0034] To make the objectives, technical solutions, and advantages of this invention clearer, the invention will be further described in detail below with reference to the accompanying drawings and embodiments. It should be understood that the specific embodiments described herein are merely illustrative and not intended to limit the invention.

[0035] The specific implementation of the present invention will be described in detail below with reference to specific embodiments.

[0036] Example 1: A hydrogel AMSC-Exos@Gel containing deer antler mesenchymal stem cell exosomes, such as... Figure 1 As shown, its preparation method includes the following steps:

[0037] S1. Preparation and identification of exosomes from deer antler mesenchymal stem cells:

[0038] 3rd-5th generation deer antler mesenchymal stem cells (AMSCs) were cultured for 48 hours. The cell culture supernatant was collected and centrifuged at 300g for 15 minutes at 4℃ to remove cell pellet, centrifuged at 1000g for 20 minutes to remove dead cells and debris, centrifuged at 10000g for 30 minutes to remove large vesicles, and finally centrifuged at 100000g for 90 minutes. The pellet was the exosome AMSC-Exos. The collected exosomes were resuspended in PBS and stored at -80℃ for later use.

[0039] The obtained exosomes were identified, and the results are as follows: Figure 2 As shown, specifically:

[0040] Transmission electron microscopy: Exosome morphology was observed, showing a typical cup shape with intact vesicle membranes. Figure 2 (A)

[0041] Nanoparticle tracking analysis: Exosome particle size was measured, concentrated at 55.1 ± 12.1 nm ( Figure 2 (Middle B)

[0042] Western blot: Detected the expression of exosome surface markers CD9, CD63, and TSG101; Calnexin-negative markers were not expressed. Figure 2 (D)

[0043] Cellular uptake assay: PKH-26-labeled exosomes were co-incubated with HUVECs, and uptake was observed using a laser confocal microscope. Figure 2 (E)

[0044] Preparation of S2, PVP-HA composite hydrogel:

[0045] Add povidone (PVP) to purified water to prepare 200 mL of 0.6% (w / v) PVP solution;

[0046] Add 0.5g sodium hyaluronate (HA) and 0.4mL of 0.2% (w / v) polyethylene glycol 400 (PEG400), stir for 3-4 hours until completely dissolved to obtain a hydrogel precursor solution;

[0047] The homogeneous solution was encapsulated in a mold and irradiated with an electron accelerator at a dose of 10-30 kGy to complete cross-linking and obtain PVP-HA composite hydrogel.

[0048] The reaction mechanism of the PVP-HA composite hydrogel is as follows: EB radiation initiates water radiolysis to generate active particles such as hydroxyl radicals (·OH), which react with the PVP molecular chain to generate PVP radicals, initiating covalent coupling between PVP molecules to form a three-dimensional network. At the same time, the radicals react with the active sites of the HA molecular chain to achieve radiation grafting of HA. PEG400 forms hydrogen bonds with the amide groups of PVP and the carboxyl / hydroxyl groups of HA through the hydroxyl groups at both ends, forming non-covalent crosslinks and enhancing the network stability.

[0049] S3. Construction of exosome-loaded hydrogel (AMSC-Exos@Gel):

[0050] The AMSC-Exos prepared in S1 and the hydrogel precursor solution prepared in S2 were uniformly mixed at a volume ratio of 1:9-1:19 to achieve a final concentration of 50 μg / mL of exosomes. The homogeneous solution was encapsulated in a mold and irradiated with an electron accelerator at a dose of 10-30 kGy to complete cross-linking. The exosomes were uniformly fixed in the hydrogel network through physical embedding (three-dimensional network trapping) and non-covalent interactions such as hydrogen bonding and electrostatic attraction to obtain AMSC-Exos@Gel.

[0051] The hydrogel was characterized, and the results are as follows: Figure 3 As shown, specifically:

[0052] Scanning electron microscopy: Observation of the microstructure of the hydrogel reveals an interconnected multilayer porous network structure within it. Figure 3 (A)

[0053] Fourier transform infrared spectroscopy: Analysis of non-covalent interactions between exosomes and hydrogel framework ( Figure 3 (C)

[0054] Zeta potential analysis: Detection of potential changes after hydrogel and exosome loading ( Figure 3 (D)

[0055] In vitro release assay: Evaluation of the release behavior of free exosomes and hydrogel-loaded exosomes ( Figure 3 (E).

[0056] Example 2: Effects of hydrogels loaded with AMSC-exos exosomes on cell proliferation, migration, and lumen formation:

[0057] Cellular uptake experiment: AMSC-Exos were co-incubated with HUVECs, and a control group was set up. The uptake of exosomes by HUVECs was observed by laser confocal microscopy.

[0058] Cell proliferation assay: The effects of H2O2, Gel, AMSC-Exos and AMSC-Exos@Gel on HUVEC proliferation were evaluated using the CCK-8 assay and EdU staining method.

[0059] Cell migration assays: The effects of H2O2, gel, AMSC-Exos and AMSC-Exos@Gel on the migration ability of HUVECs were evaluated using cell scratch assays and Transwell migration assays.

[0060] Lumen formation experiment: The effects of H2O2, Gel, AMSC-Exos and AMSC-Exos@Gel on the lumen formation ability of HUVECs were evaluated using the Matrigel matrix gel experiment;

[0061] Macrophage polarization assay: RAW264.7 cells were used to establish an oxidative stress model induced by H2O2. Different treatments were then administered, and the expression of iNOS (M1-labeled) and Arg-1 (M2-labeled) was detected by immunofluorescence. Flow cytometry was used to detect F4 / 80... + CD86 + (M1) and F4 / 80 + CD206 + (M2) cell proportion; ELISA detection of IL-10 and TNF-α levels;

[0062] The results are as follows Figure 4 As shown, the AMSC-Exos@Gel prepared in this embodiment of the invention induces macrophages to polarize from the M1 phenotype to the M2 phenotype, reduces the inflammatory burden, and accelerates wound healing in diabetic mice.

[0063] like Figure 5 As shown in the figure, the AMSC-Exos@Gel prepared in the embodiments of the present invention significantly promotes the proliferation, migration and lumen formation of HUVECs.

[0064] Example 3: Application of AMSC-Exos@Gel in a full-thickness skin wound model in diabetic mice:

[0065] Establishment of a diabetic mouse model: Male C57BL / 6J mice aged 6-8 weeks were fed a high-sugar, high-fat diet and STZ (35 mg / kg, intraperitoneal injection for two consecutive days) to establish a type 2 diabetes model. A random blood glucose level ≥16.8 mM was considered a successful model.

[0066] Preparation of a full-thickness skin wound model in diabetic mice: A 1×1 cm full-thickness skin excision wound was made on the back of diabetic mice. The mice were randomly divided into 5 groups: normal control group (NC), diabetic model group (DC), hydrogel group (Gel), deer antler exosome group (AMSC-exos), and deer antler exosome + hydrogel group (AMSC-exos@Gel). Treatment was performed on days 0, 5, and 10 after injury.

[0067] Wound healing assessment: The wound was photographed and recorded on days 0, 5, 10, and 15, and the wound healing rate was calculated;

[0068] Histological and immunohistochemical examination: Mice were sacrificed on day 14, and wound tissue was collected for H&E staining, Masson staining, immunohistochemical staining (CD31, Ki67, VEGF, α-SMA) and immunofluorescence staining (CD86, CD206, Collagen I / III).

[0069] The results are as follows Figure 6 , 7 As shown, the wound healing rate in the AMSC-exos@Gel treatment group was significantly improved, and the wounds were basically closed by day 14. H&E staining showed that the skin structure was basically reconstructed and the hair follicles were abundant. Masson staining showed that collagen deposition was increased and arranged in an orderly manner. Immunohistochemistry showed that the expression of CD31, Ki67, VEGF, and α-SMA was significantly increased. Immunofluorescence showed that the expression of CD86 was decreased and the expression of CD206 was increased, and the ratio of type I / III collagen tended to be within the physiological range.

[0070] Example 4: Verification of the molecular mechanism by which miR-486-5p targets FGF9:

[0071] qRT-PCR detection of miR-486-5p expression: Total RNA was extracted from AMSCs, AMSC-exos and HUVECs, and the expression level of miR-486-5p was detected by stem-loop qRT-PCR, with U6 as an internal control;

[0072] Target gene prediction and dual-luciferase reporter gene experiment: The target genes of miR-486-5p were predicted by three databases: TargetScan, miRanda and miRTarBase. FGF9 was screened out, and FGF9 3'-UTR wild-type and mutant luciferase reporter plasmids were constructed. They were co-transfected with miR-486-5p mimic into 293T cells, and luciferase activity was detected.

[0073] miRNA function verification: transfect miR-486-5p mimic or inhibitor into HUVECs and detect changes in FGF9 mRNA and protein expression;

[0074] Functional rescue experiment: Groups were set up: miR-486-5p mimic group, miR-486-5p mimic+FGF9 overexpression group, and control group to detect the lumen formation ability of HUVECs;

[0075] The results are as follows Figure 8 As shown, miR-486-5p is highly expressed in AMSC-exos; dual-luciferase assay confirmed that miR-486-5p directly binds to FGF9 3′-UTR; functional rescue assay confirmed that FGF9 overexpression can reverse the angiogenic effect of miR-486-5p mimic.

[0076] The above description is only a preferred embodiment of the present invention and is not intended to limit the present invention. Any modifications, equivalent substitutions, and improvements made within the spirit and principles of the present invention should be included within the protection scope of the present invention.

Claims

1. A method for preparing a hydrogel containing Cervus elaphus mesenchymal stem cell exosomes, characterized by, Includes the following steps: S1. Preparation of exosomes from deer antler mesenchymal stem cells: Deer antler mesenchymal stem cells of the 3rd to 5th generation were taken, and after culture, the cell culture supernatant was collected. The cells were centrifuged to remove dead cells and debris, then centrifuged at high speed to remove large vesicles, and finally centrifuged at ultraspeed. The precipitate obtained was exosome AMSC-Exos. Preparation of S2, PVP-HA composite hydrogel: Povidone was added to purified water to prepare PVP solution; sodium hyaluronate and polyethylene glycol 400 were added and stirred until completely dissolved to obtain hydrogel precursor solution; S3. Construction of hydrogel containing antler mesenchymal stem cell exosomes: The AMSC-Exos prepared in S1 was uniformly mixed with the hydrogel precursor solution prepared in S2. The homogeneous solution was encapsulated in a mold and cross-linked by electron accelerator irradiation to form a hydrogel containing antler mesenchymal stem cell exosomes.

2. The method of claim 1, wherein the preparation of the hydrogel containing the Cervus elaphus mesenchymal stem cell exosome is characterized by, In S1, the centrifugation process to remove dead cells and debris specifically involves centrifuging at 300g for 15 minutes at 4°C to remove cell precipitate, followed by centrifugation at 1000g for 20 minutes to remove dead cells and debris.

3. The method for preparing a hydrogel containing deer antler mesenchymal stem cell exosomes according to claim 1, characterized in that, In S1, the process of removing large vesicles by high-speed centrifugation specifically involves centrifuging at 10000g for 20 minutes.

4. The method for preparing a hydrogel containing deer antler mesenchymal stem cell exosomes according to claim 1, characterized in that, In S1, the ultracentrifugation process specifically involves ultracentrifuging at 100,000g for 90 minutes.

5. The method for preparing a hydrogel containing deer antler mesenchymal stem cell exosomes according to claim 1, characterized in that, In S2, the concentration of the PVP solution is 0.6% w / v, and the concentration of the polyethylene glycol 400 is 0.2% w / v.

6. The method for preparing a hydrogel containing deer antler mesenchymal stem cell exosomes according to claim 1, characterized in that, In S3, the irradiation dose is 10-30 kGy.

7. A hydrogel containing exosomes of deer antler mesenchymal stem cells, characterized in that, It is prepared using the preparation method described in any one of claims 1-6.

8. The use of a hydrogel containing deer antler mesenchymal stem cell exosomes as described in claim 7 in the preparation of a medicament or dressing for treating chronic skin lesions of diabetes.

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