Stem cell exosome composition and application thereof in treating diabetic complications

Abstract

The invention discloses a targeting stem cell exosome composition and application thereof in treating diabetic foot ulcer. According to the composition, a mesenchymal stem cell exosome is modified through genetic engineering, specific targeting fusion polypeptide is expressed on the surface of the mesenchymal stem cell exosome, anti-inflammatory miR-146a and a pro-angiogenic factor VEGF are loaded, and then the mesenchymal stem cell exosome is compounded with temperature-sensitive hyaluronic acid hydrogel to form a sustained release system. The sequence of the fusion polypeptide is as shown in SEQ ID NO: 1, and the targeting of exosomes to diabetic inflammation parts is remarkably improved; the stability of miR-146a is enhanced through chemical modification, and the miR-146a cooperates with VEGF to inhibit inflammation and promote revascularization. In-vitro experiments show that the composition reduces the levels of TNF-alpha and IL-6 by 85% and 88% respectively, and the cell migration rate reaches 99.3%; in-vivo experiments show that the wound healing rate of diabetic rats reaches 99.6% on the 14th day, and the collagen deposition area proportion is increased to 77.5%. The invention has targeting property, function synergy and slow release performance, and provides an efficient treatment strategy for diabetic foot ulcer.

Description

Technical Field

[0001] The present invention belongs to the technical field of biomedicine, and particularly relates to a stem cell exosome composition and its application in the treatment of diabetic complications. Background Art

[0002] Diabetic Foot Ulcer (DFU) is one of the most common and highly disabling serious complications in diabetic patients, and its occurrence is closely related to the multi-factor synergistic damage caused by long-term hyperglycemic state. The hyperglycemic microenvironment can induce the body to produce a large amount of reactive oxygen species (ROS), causing oxidative stress response; at the same time, it promotes the over-release of inflammatory factors (such as TNF-α, IL-6), triggering a persistent chronic inflammatory response. In addition, high glucose can also damage the function of vascular endothelial cells, inhibit neovascularization, exacerbate local hypoxia and nutritional supply disorders; the accelerated degradation of the extracellular matrix (ECM) will also significantly interfere with cell migration and tissue reconstruction processes. The interaction of these factors makes the DFU wound in a non-healing state for a long time.

[0003] Existing clinical treatment methods such as debridement, antibiotic treatment, growth factor dressings, hyperbaric oxygen therapy, etc., although can relieve the condition to a certain extent, still generally have problems such as unstable curative effect, long healing cycle, and easy recurrence. Especially most of these methods have a single effect and are difficult to comprehensively regulate the complex pathological microenvironment of DFU, so the problem of limited wound tissue regeneration cannot be fundamentally solved. In recent years, with the development of regenerative medicine and extracellular therapy technologies, mesenchymal stem cell-derived exosomes (MSC-Exos) have become an emerging direction for DFU treatment because they naturally carry a variety of bioactive molecules (such as miRNA, proteins, lipids).

[0004] MSC-Exos have low immunogenicity, good tissue permeability, and controllable molecular loading capacity, and can achieve functions such as immunomodulation, angiogenesis promotion, and anti-inflammatory repair of target tissues in a cell-free treatment mode, with good application prospects. However, natural exosomes still face the following core problems in DFU treatment: First, they lack tissue specificity, are easily cleared by the mononuclear-macrophage system, and are difficult to effectively accumulate in the inflammatory wound; second, they have a single function and cannot simultaneously cope with oxidative stress, immune imbalance, and vascular damage induced by high glucose; third, they have poor stability, stay in the wound locally for a short time, and need to be repeatedly administered to maintain the treatment effect, increasing the treatment cost and patient pain.

[0005] In response to the above problems, current research has attempted to improve the therapeutic performance of exosomes through engineering means, such as chemical modification (e.g., PEGylation) to extend the circulation time of exosomes in vivo, physical encapsulation (e.g., thermosensitive hydrogels) to enhance their retention ability at the wound site, and molecular engineering means (e.g., CXCR4 gene overexpression) to enhance their responsiveness to inflammatory chemokines. Most of the current research on fusion polypeptides focuses on tumor targeting (such as RGD peptides, etc.), and there is a lack of specific recognition peptides for the inflammatory microenvironment of DFU and the design of their delivery systems, which urgently needs innovative breakthroughs. Summary of the Invention

[0006] The present invention provides a targeted stem cell exosome composition, which is achieved through the following technical solutions:

[0007] 1. Preparation of gene-engineered targeted exosomes: Design the fusion polypeptide CRKDKC-GGGGS×3-CD63 (SEQ ID NO:1), and transfect mesenchymal stem cells through lentivirus to stably express the polypeptide targeting ICAM-1 (binding affinity KD = 12.3 nM) on the surface of exosomes; the exosomes are purified by ultracentrifugation, with a particle size of 122.5 ± 8.3 nm, and the marker proteins CD63 and Alix are positive.

[0008] 2. Co-loading of bifunctional molecules: Chemically modified miR-146a-5p (2'-O-methylation, phosphorothioate bond and cholesterol conjugation) and VEGF mRNA are co-loaded at a molar ratio of 1:1, and the loading efficiency reaches 1.2×10 4 copies / μg exosome protein; the stability of miR-146a in serum is increased to 87.2% after modification (18.7% in the unmodified group), and the efficiency of targeted inhibition of IRAK1 reaches 78%.

[0009] 3. Sustained-release hydrogel composite system: Thermosensitive HA-Gel (4% HA-MA, 0.5% LAP) loads exosomes through ultraviolet cross-linking, and the cumulative release rate in 72 hours is 82.3%; the gel has injectability, adheres to the wound surface and prolongs the activity of exosomes.

[0010] Therefore, the beneficial effects of the present invention are summarized as follows:

[0011] 1. Precise targeting: The fusion polypeptide CRKDKC increases the binding rate of exosomes to ICAM-1 high-expressing endothelial cells by 3.5 times, significantly improving the drug delivery efficiency.

[0012] 2. Anti-inflammatory and pro-angiogenic synergy: miR-146a inhibits the NF-κB pathway, reducing the levels of TNF-α and IL-6 to 48.0 ± 2.0 pg / mL and 35.0 ± 1.0 pg / mL, respectively (321.7 ± 6.5 pg / mL and 295.0 ± 5.7 pg / mL in the control group).

[0013] 3. The expression of VEGF protein is upregulated by 5.2-fold, promoting tube formation of HUVECs (total length 3,780 ± 57 μm vs. 1,120 ± 57 μm in the control group).

[0014] 4. Efficient wound repair: In vitro scratch assay shows that the cell migration rate reaches 99.3%; in the diabetic rat model, the wound healing rate reaches 99.6% on the 14th day, and the collagen deposition area accounts for 77.5% (22.1% in the control group).

[0015] 5. Sustained release and safety: HA-Gel extends the retention time of exosomes to 72 hours, with a cell survival rate > 95% and no liver and kidney toxicity.

[0016] The above results indicate that the present invention combines a target exosome delivery system, functionalized miRNA, and a hydrogel complex to form a novel drug delivery platform with broad application prospects. Description of the Drawings

[0017] Figure 1 Results of the physicochemical characterization and target verification of exosomes, where A is the particle size distribution histogram, B is the Western blot result of exosome marker proteins, and C is the result of target binding rate verification (flow cytometry). Detailed Embodiments

[0018] Unless otherwise defined, all technical and scientific terms used herein have the same meaning as commonly understood by those skilled in the technical field to which this invention belongs. The terms used in the description of the present invention herein are only for the purpose of describing specific embodiments and are not intended to limit the present invention. The term "and / or" used herein includes any and all combinations of one or more of the related listed items.

[0019] Unless otherwise specified, the reagents, methods, and equipment used in the present invention are conventional reagents, methods, and equipment in this technical field. Unless otherwise specified, the reagents and materials used in the following examples are all commercially available.

[0020] Example 1: Construction of a Targeted Fusion Polypeptide and Preparation of Genetically Engineered Exosomes

[0021] 1. Targeted fusion polypeptide design: To enhance the tissue targeting ability of exosomes in the wound area of diabetic foot ulcer (DFU), a targeted fusion polypeptide that can be anchored to the exosome membrane structure was designed. This fusion polypeptide consists of three functional domains:

[0022] (1) Target recognition domain: The cyclic polypeptide sequence CRKDKC (Cys-Arg-Lys-Asp-Lys-Cys) with high affinity for binding to the cell adhesion molecule ICAM-1 was selected and formed a stable cyclic conformation through an intramolecular disulfide bond. The binding affinity (Kd) of this peptide segment to ICAM-1 is 12.3 nM (2-fold higher than the affinity without the cyclic structure), providing a good biological targeting basis.

[0023] (2) Flexible linker peptide: To avoid steric hindrance between domains, a triple-repeat flexible linker peptide (GGGGS)×3 was inserted, which helps the fusion polypeptide form a reasonable spatial conformation on the membrane surface and maintain the functional exposure of the targeting domain.

[0024] (3) Membrane anchoring domain: To achieve stable expression of the polypeptide on the exosome membrane surface, the human C-terminal transmembrane region of the exosome membrane protein CD63 (amino acid sequence: LLVGVALLLGIVLGLVAMY) was selected as the anchoring fragment, which can effectively anchor the fusion polypeptide to the exosome membrane surface.

[0025] (4) The complete amino acid sequence of the fusion polypeptide is as follows (SEQ ID NO:1). This sequence was verified by online structure prediction and simulation to have a stable cyclic structure, reasonable spatial layout, which is beneficial for target receptor binding and does not affect the localization function of CD63 in the membrane structure.

[0026] 2. Lentiviral vector construction and virus packaging

[0027] (1) To achieve stable expression of the fusion polypeptide in stem cells, the DNA fragment encoding the complete fusion polypeptide sequence (SEQ ID NO:1) was cloned into the multiple cloning site of the lentiviral expression vector pLVX-IRES-Puro, and the CRKDKC targeting domain was inserted after the CD63 coding sequence to construct the recombinant plasmid pLVX-CD63-CRKDKC. After the obtained recombinant plasmid was confirmed by sequencing to have no mutations, it was co-transfected with the helper plasmids psPAX2 and pMD2.G into HEK293T cells, and virus packaging was carried out by the calcium phosphate co-precipitation method or the liposome method.

[0028] (2) The virus supernatant was collected 48 hours after transfection, filtered through a 0.45 μm filter membrane and then concentrated by ultracentrifugation. The virus titer was determined by qPCR method or limited dilution infection method, and finally a virus titer of 1×10 8Highly efficient lentivirus solution at TU / mL for subsequent cell transfection.

[0029] 3. Transduction of hUC-MSCs and preparation of targeted exosomes

[0030] (1) Transfect the above-packaged lentivirus into human umbilical cord mesenchymal stem cells (hUC-MSCs), and add polycation (Polybrene, 8 μg / mL) during the transfection process to improve the infection efficiency. Replace with fresh culture medium 24 hours after transfection, and add puromycin (2 μg / mL) for screening 48 hours later. A cell line stably expressing the targeted fusion polypeptide can be obtained after about 5 - 7 days.

[0031] (2) Exosomes were collected using the classical ultracentrifugation method. The brief process is as follows: After collecting the cell culture supernatant, remove cells by centrifugation at 300×g (10 min), remove cell debris at 2,000×g (20 min), and remove large particles at 10,000×g (30 min) in sequence; then precipitate exosome particles by ultracentrifugation at 100,000×g, 4°C for 2 hours. The precipitate was resuspended with PBS and then ultracentrifuged again for purification, and finally, exosomes stably expressing the fusion polypeptide (named Exo-CRKDKC) were obtained.

[0032] 4. Physicochemical characterization and targeting verification of exosomes

[0033] (1) Detection of particle size distribution: The particle size of Exo-CRKDKC was detected using a nanoparticle tracking analyzer (NanoSight NS300). The results showed that the average particle size of exosomes was 122.5 ± 8.3 nm, showing a typical spherical distribution, which was in line with the characteristics of exosomes ( Figure 1 A).

[0034] (2) Detection of marker proteins: Exosome markers were detected by Western blot. The results showed that both CD63 and Alix were positively expressed in Exo-CRKDKC, confirming that it was a typical exosome derived from hUC-MSCs ( Figure 1 B).

[0035] (3) Targeting function verification: An ICAM-1 overexpressing human umbilical vein endothelial cell (HUVECs) model was constructed, and the binding ability of Exo-CRKDKC was analyzed by flow cytometry. The results showed that compared with the blank control exosomes, the binding rate of Exo-CRKDKC to ICAM-1 highly expressing cells was significantly increased, with an average increase of about 3.5 times ( Figure 1 C), verifying its good targeting and cell recognition ability.

[0036] Example 2: Design of miR-146a and functionalized exosome loading and hydrogel composite

[0037] 1. Selection and sequence description of miR-146a-5p: miR-146a is one of the key regulatory miRNAs in the human body and has two mature forms: miR-146a-5p and miR-146a-3p. The literature shows that miR-146a-5p is the main functional form, which can effectively target key signaling molecules such as IRAK1 (Interleukin-1 receptor-associated kinase 1) and TRAF6 (TNF receptor-associated factor 6) in the inflammatory signaling pathway, regulate the downstream NF-κB pathway, and thus inhibit the overexpression of inflammatory factors. In the present invention, miR-146a-5p is selected, and its mature sequence is: 5'-UGAGAACUGAAUUCCAUGGGUU-3'.

[0038] 2. Chemical modification strategy of miR-146a: To improve the stability, bioavailability and loading efficiency of miRNAs in the in vitro and in vivo environments, the following multi-site chemical modification method is adopted. The modified miR-146a is named miR-146amimics:

[0039] (1) 2'-O-methylation modification (2'-OMe): This modification is introduced at the 2nd, 8th and 13th bases to block the RNase recognition site and enhance the sequence stability;

[0040] (2) Phosphorothioate bond modification (Phosphorothioate, PS): Replace the phosphate backbone of the three nucleotides at both ends with PS bonds to effectively extend the nucleic acid half-life;

[0041] (3) Cholesterol molecule conjugation: Cholesterol (Chol) is introduced at the 3' end through TEG (triethylene glycol) connection to improve the cell membrane penetration ability and promote exosome loading.

[0042] (4) miR-146amimics sequence: 5'-UG(2'-OMe)AGAACUG(PS)AAUU(2'-OMe)CCAUGGGUU-Chol-TEG-3'.

[0043] 3. Verification of chemical modification effects: To verify the modification effects, this study conducted experimental analyses from two aspects: stability and targeting function:

[0044] (1) Stability analysis: The modified and unmodified miR-146a were incubated in a medium containing 10% fetal bovine serum at 37°C for 24 hours. The qPCR detection results showed that the residual rate of the modified miRNA was 87.2 ± 3.4%, while that of the unmodified group decreased to 18.7 ± 2.9%;

[0045] (2) Functional verification: The dual-luciferase reporter system was used to detect the inhibitory activity of miR-146a on the 3'-UTR of IRAK1. The results showed that the inhibition efficiency of the modified group was 78 ± 5%, which was significantly higher than 65 ± 4% of the unmodified group.

[0046] 4. Loading process and quantitative analysis for exosome delivery

[0047] (1) Loading method: miR-146a mimics and VEGF mRNA (GenBank: AF024710.1) were mixed at a molar ratio of 1:1 and then loaded into the engineered exosomes Exo-CRKDKC by electroporation technology, denoted as Exo-CRKDKC-146a. The electroporation parameters were: voltage 400V, capacitance 150 μF.

[0048] (2) Detection of loading efficiency: Real-time quantitative PCR (qPCR) was used to analyze the miRNA loading amount. The results showed that an average of 1.2×10 4 copies of miR-146a and 9.8×10 3 VEGF mRNA could be detected per microgram of Exo-CRKDKC-146a protein, indicating that this loading method had good loading capacity and consistency.

[0049] 5. Preparation and performance evaluation of HA-Gel composite preparation

[0050] 5.1 Material composition and ratio optimization: To construct an exosome delivery platform with good biocompatibility, gel-forming property, and drug sustained-release performance, the present invention selected methacrylated hyaluronic acid (HA-MA) as the hydrogel skeleton and cooperated with the photoinitiator LAP (Lithium phenyl-2,4,6-trimethylbenzoylphosphinate) for ultraviolet cross-linking to form a composite hydrogel.

[0051] (1) Mass fraction of HA-MA: 4 wt%, dissolved in sterile PBS;

[0052] (2) Concentration of photoinitiator LAP: 0.5 wt%, formulated in the HA-MA solution;

[0053] (3) Naming of the composite carrier: HA-Gel.

[0054] 5.2 Exosome loading and gel cross-linking conditions: The pre-prepared and quantified Exo-CRKDKC-146a (1×10^10 particles / mL) was slowly added to the HA-MA / LAP prepolymer solution, gently mixed, and then immediately subjected to ultraviolet cross-linking for solidification and molding; the ultraviolet irradiation wavelength was 365 nm, and the light intensity was 5 mW / cm 2, irradiation time was 60 seconds; the results showed that it could form a transparent gel with uniform texture and dense structure, having good local adhesiveness and injectability, named HA-Gel@Exo-CRKDKC-146a.

[0055] 5.3 Sustained-release performance analysis: In vitro release experiments were used to evaluate the sustained-release ability of HA-Gel@Exo-CRKDKC-146a:

[0056] (1) Experimental conditions: Incubated at a constant temperature of 37 °C in PBS buffer.

[0057] (2) Sampling time points: 1 h, 6 h, 12 h, 24 h, 48 h, 72 h;

[0058] (3) Detection method: Nanosight NTA technology was used to detect the number of released exosome particles.

[0059] (4) Release curve analysis: The results showed that the preparation presented a rapid release stage in the first 24 hours, and the release rate gradually slowed down after 48 - 72 hours, and finally the cumulative release rate reached 82.3% ± 3.1% at 72 hours.

[0060] (5) This sustained-release behavior indicated that the HA-Gel carrier system could continuously deliver Exo-CRKDKC-146a locally, providing a stable treatment window for wound repair, etc.

[0061] Example 3: In vitro verification test

[0062] 1. Anti-inflammatory effect evaluation

[0063] 1.1 Materials and methods

[0064] 1.1.1 Cell culture: Human skin fibroblasts (HSF), cell passages 3 - 6. They were cultured in a medium (high-glucose DMEM (glucose concentration 25 mM) + 10% FBS + 1% P / S) in a constant temperature incubator at 37 °C and 5% CO2.

[0065] 1.1.2 Model establishment: HSF cells were seeded in 6-well plates at a density of 2×10 5 cells / well. After the cells adhered, the high-glucose medium was changed and the cells were cultured for another 48 hours to establish a high-glucose inflammation model.

[0066] 1.1.3 Grouping design and treatment, the cells were divided into 5 groups, with 3 parallel wells in each group (n = 3):

[0067] Table 1 Experimental grouping

[0068]

[0069] Continue to culture for 24 hours after treatment.

[0070] 1.1.4 ELISA assay: Collect the culture supernatant and centrifuge (1,500 rpm, 5 min) to remove cell debris. Operate according to the instructions of the TNF-α and IL-6 ELISA kits. Read the absorbance at 450 nm and calculate the concentration by standard curve quantification. The results are shown in Table 2. The Exo group, Exo-CRKDKC group, Exo-CRKDKC+HA-Gel group, and Exo-CRKDKC-146a+HA-Gel group could all significantly reduce the levels of inflammatory factors TNF-α and IL-6. The Exo-CRKDKC-146a+HA-Gel group had the largest reduction (TNF-α decreased by 85%, IL-6 decreased by 88%).

[0071] Table 2 Statistical results of ELISA assay (pg / mL)

[0072]

[0073] 1.1.5 Scratch assay: After 24 hours of cell treatment, use a 200 μL pipette tip to draw a straight line in the center of the cell monolayer. Rinse with PBS to remove floating cells. Add the corresponding treatment and culture for 24 hours. Take scratch images at 0 hour and 24 hours, and use ImageJ to measure the healing area. The results are shown in Table 3. All treatment groups could significantly promote the migration of HSF. The migration rate of the Exo-CRKDKC-146a+HA-Gel group was close to complete closure (99.3%, P<0.001 vs Exo-CRKDKC+HA-Gel group).

[0074] Table 3 Results of scratch assay

[0075]

[0076] 2. Evaluation of angiogenesis ability

[0077] 2.1 Materials and methods

[0078] 2.1.1 HUVECs tube formation assay: Seed HUVECs (human umbilical vein endothelial cells) in a 96-well plate pre-coated with Matrigel (2×10 4 cells / well). Add the exosome treatment solutions of each group (final concentration 1×10 10 particles / mL). After culturing for 6 hours, randomly take 5 fields of view with a microscope. Use ImageJ to measure the total tube length of each well. The results are shown in Table 4. The Exo-CRKDKC-146a+HA-Gel group significantly enhanced the tube formation ability of HUVECs, and the total tube length increased by about 3.4 times compared with the control group (P<0.001).

[0079] Table 4 Results of tube formation assay of HUVECs (μm)

[0080]

[0081] 2.1.2 Detection of VEGF by Western blot: Total proteins were extracted by cell lysis and quantified by BCA method.

[0082] Separated by SDS-PAGE, after transferring the membrane, incubated with anti-VEGF (1:1000) and β-actin (1:5000) primary antibodies respectively. The ratio of gray value (VEGF / β-actin) was analyzed by normalization. The results are shown in Table 5. The expression level of VEGF protein reached the highest level in the Exo-CRKDKC-146a+HA-Gel group (5.2 times that of the control group).

[0083] Table 5 Results of Western blot gray analysis (VEGF / β-actin)

[0084]

[0085] The above results indicate that miR-146a-loaded CRKDKC-modified exosomes and HA hydrogel synergistically enhanced the pro-angiogenic effect. Exo-CRKDKC-146a+HA-Gel can significantly inhibit the high-glucose-induced inflammatory response, promote cell migration and angiogenesis, and show good potential for tissue repair.

[0086] Example 4: In vivo verification test

[0087] 1. Animal modeling and grouping

[0088] 1.1 Establishment of animal model

[0089] (1) Animal selection: Male SD rats aged 8-10 weeks and weighing 200-250 g were selected.

[0090] (2) Diabetes modeling: STZ (Sigma-Aldrich, 55 mg / kg) was dissolved in 0.1 M sodium citrate buffer (pH 4.5) and injected intraperitoneally after fasting for 12 hours. After confirming the successful establishment of the diabetes model, blood glucose was measured by tail vein blood sampling, and the blood glucose was stably higher than 16.7 mmol / L.

[0091] (3) Animal grouping: Before the experiment, according to the blood glucose level (>16.7 mmol / L), the animals were randomly divided into the following groups, with 6 animals in each group: control group (PBS dropwise), Exo group (100 μg of ordinary exosomes), Exo-CRKDKC group (100 μg of Exo-CRKDKC exosomes), Exo-CRKDKC+HA-Gel group (100 μg of Exo-CRKDKC exosomes + HA hydrogel), Exo-CRKDKC-146a+HA-Gel group (100 μg of Exo-CRKDKC-146a exosomes + HA hydrogel).

[0092] 1.2 Wound preparation

[0093] (1) Preoperative preparation: Anesthesia: Anesthetized with isoflurane (2% inhalation), and the depth of anesthesia was monitored to ensure that the animals had no response.

[0094] (2) Wound preparation: A full-thickness skin defect was prepared on the back of the rats using a circular punch with a diameter of 1 cm, penetrating the dermis layer deeply without involving the muscle layer, ensuring that the wound surface was uniform and not sutured around the wound.

[0095] 2. Analysis of wound healing rate

[0096] 2.1 Calculation of wound healing rate: The experimental observation times were day 0, day 7, and day 14 respectively, and the wound area was measured each time. The wound surface was photographed with standardized photos (with the same light and angle), and then the wound area was measured using ImageJ software. The formula for the healing rate is:

[0097]

[0098] 2.2 The experimental results are shown in Table 6.

[0099] (1) On day 7: The Exo-CRKDKC-146a+HA-Gel group showed a healing rate of 85.8%, which was significantly higher than that of the control group (28.3%) and the Exo group (44.2%). The therapeutic effect of this group was significantly better than that of other groups, indicating that the treatment of CRKDKC-modified exosomes combined with miR-146a in HA hydrogel has the effect of accelerating wound healing.

[0100] (2) On day 14: The Exo-CRKDKC-146a+HA-Gel group was almost completely healed (99.6%), and its healing rate was significantly higher than that of other groups, especially the control group (45%), showing the potential of this treatment strategy in the repair of diabetic foot ulcers.

[0101] Table 6 Summary of wound healing rate results

[0102]

[0103] 3. Histological analysis

[0104] 3.1 HE staining: Fix the wound tissue with 4% paraformaldehyde, embed it in paraffin, section (5 μm), and perform HE staining. Observe the integrity of epidermal regeneration, the density of dermal structure, and the infiltration of inflammatory cells.

[0105] 3.2 Masson staining (collagen deposition): Perform Masson trichrome staining on the skin wound specimens, and observe the distribution of collagen fibers. The amount of collagen deposition is semi-quantitatively analyzed by ImageJ software to calculate the proportion of the area of blue staining to the total area.

[0106] 3.3 The experimental results are shown in Table 7. The collagen deposition in the control group was the least (22.1%), indicating that the wound healed slowly and there was insufficient collagen deposition without treatment; the group of Exo-CRKDKC-146a + HA-Gel had the highest proportion of collagen deposition (77.5%), indicating that the strategy of exosomes combined with HA hydrogel and miR-146a could promote collagen deposition and accelerate wound healing.

[0107] Table 7 Analysis of histological experimental results

[0108]

[0109] 4. Summary and discussion

[0110] 4.1 The wound healing effect of the Exo-CRKDKC-146a + HA-Gel group was the most significant, with the highest levels of epidermal regeneration and collagen deposition. The loading of miR-146a gave this treatment strategy significant advantages in reducing inflammation, promoting angiogenesis, and collagen deposition.

[0111] 4.2 Discussion on the mechanism of action: miR-146a may downregulate the inflammatory pathway by targeting IRAK1, reducing the local inflammatory response at the wound, thereby promoting wound healing. The combination of Exo-CRKDKC and HA hydrogel not only improves the stability and targeting of exosomes but also provides an effective carrier system for treatment.

[0112] 4.3 Clinical significance: This study provides new ideas for the treatment of diabetic foot ulcers, especially the delivery of miR-146a through a non-viral vector (exosomes + HA hydrogel), which is both safe and effective and has great potential for clinical application.

[0113] The above embodiments are preferred embodiments of the present invention, but the embodiments of the present invention are not limited to the above embodiments. Any other changes, modifications, substitutions, combinations, and simplifications made without departing from the spirit and principle of the present invention shall be equivalent replacement methods and are all included in the protection scope of the present invention.

Claims

1. A targeted stem cell exosome composition, characterized in that, The composition comprises: (1) Genetically engineered mesenchymal stem cell exosomes that express a fusion polypeptide shown in SEQ ID NO: 1 on their surface, and the fusion polypeptide consists of a cyclic polypeptide CRKDKC targeting ICAM-1, a flexible linker peptide, and a CD63 transmembrane region; (2) Anti-inflammatory miR-146a mimics and angiogenic factor VEGF mRNA loaded in the exosomes; (3) A thermosensitive hyaluronic acid hydrogel complexed with the exosomes to form a sustained-release system.

2. The composition according to claim 1, wherein The miR-146a mimics is obtained by modifying miR-146a-5p with 2'-O-methylation, phosphorothioate bond, and cholesterol conjugation, and the sequence of the miR-146a mimics is: 5'-UG(2'-OMe)AGAACUG(PS)AAUU(2'-OMe)CCAUGGGUU-Chol-TEG-3'.

3. The composition according to claim 1, wherein The VEGF mRNA is mRNA encoding vascular endothelial growth factor, and it is co-loaded in the exosomes with miR-146a mimics at a molar ratio of 1:

1.

4. The composition according to claim 1, wherein The thermosensitive hyaluronic acid hydrogel consists of 4% methacrylated hyaluronic acid and 0.5% photoinitiator LAP, and is formed by ultraviolet cross-linking.

5. A method for preparing the composition according to claim 1, characterized in that, The method comprises the following steps: (1) Construct a lentiviral vector encoding SEQ ID NO: 1, transfect mesenchymal stem cells and screen for stable strains, and isolate exosomes by ultracentrifugation to obtain Exo-CRKDKC; (2) Mix miR-146a mimics and VEGF mRNA at a molar ratio of 1:1, and co-load them onto Exo-CRKDKC prepared in step (1) by electroporation to obtain Exo-CRKDKC-146a; (3) Mix Exo-CRKDKC-146a prepared in step (2) with the HA-MA / LAP prepolymer, and form a HA-Gel composite preparation by ultraviolet cross-linking to obtain HA-Gel@Exo-CRKDKC-146a.

6. Use of the composition according to any one of claims 1-5 in the preparation of a drug for treating diabetic foot ulcers.

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