A targeted exosome-hydrogel formulation, and methods of making and using the same
Patent Information
- Application Number
- CN202611034172.X
- Authority / Receiving Office
- CN · China
- Patent Type
- Applications(China)
- Current Assignee / Owner
- Filing Date
- 2026-07-13
- Publication Date
- 2026-10-30
AI Technical Summary
[0036]1.本申请通过采用分步加料联合梯度pH调控工艺配制功能化温敏水凝胶前体，先调节体系pH至6.5再加入透明质酸钠、最后微量添加氯化锶，从根源规避壳聚糖与透明质酸钠的聚电解质絮凝、锶离子与磷酸根形成微晶沉淀两大配伍缺陷，实现壳聚糖、透明质酸钠、氯化锶、β-甘油磷酸钠四种功能组分稳定共存，得到澄清可注射的凝胶基质，解决传统多功能凝胶体系易浑浊、堵针与力学不均的行业难题。
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Abstract
Description
Technical Field
[0001] This application relates to the fields of biomedical materials and drug delivery technology, and in particular to a targeted exosome-hydrogel formulation, its preparation method, and its application. Background Technology
[0002] Articular cartilage is a special connective tissue without blood vessels, nerves, or lymphatic tissue, and its self-repair capacity is extremely limited. In pathological conditions such as osteoarthritis, traumatic articular cartilage defects, and meniscus injuries, a large amount of type II collagen, the main component of the cartilage matrix, is exposed. The lesion site is also accompanied by high levels of reactive oxygen species and persistent inflammatory factor infiltration, forming a vicious cycle of oxidative stress and inflammation. This further inhibits the proliferation and phenotypic maintenance of chondrocytes, accelerates the degradation of proteoglycans and collagen, and ultimately leads to loss of joint function. Current clinical treatments include physical therapy, nonsteroidal anti-inflammatory drugs, and intra-articular injection of sodium hyaluronate or glucocorticoids, but these mainly relieve symptoms and are difficult to achieve structural regeneration and repair of the damaged tissue.
[0003] Mesenchymal stem cell-derived exosomes are extracellular vesicles with a diameter of approximately 30–150 nm secreted by cells. They naturally carry a variety of repair-promoting active molecules such as proteins, mRNA, and microRNA. They have advantages such as low immunogenicity, strong penetrability, and the ability to cross biological barriers, showing great potential in the field of cartilage repair. However, after free exosomes are injected into the joint cavity, they are rapidly diluted by the synovial fluid and lost through the circulation and metabolism of the synovial fluid. Their residence time in the body is extremely short, and the effective concentration at the lesion site is low, which greatly limits their therapeutic effect.
[0004] To prolong the retention time of exosomes in the joint cavity, researchers attempted to encapsulate exosomes in thermosensitive injectable hydrogels. Utilizing the sol-gel transition property of hydrogels at body temperature, a local drug reservoir was formed, delaying exosome release. The chitosan / β-glycerophosphate system is one of the most classic thermosensitive hydrogels, possessing good biocompatibility and biodegradability. However, this system only has simple physical encapsulation and passive sustained-release functions; it cannot endow exosomes with active targeting recognition capabilities, nor can it improve the high oxidative stress and inflammatory microenvironment at the lesion site. After release from the gel, exosomes still diffuse randomly and are easily damaged by oxidative attack, resulting in a significant reduction in actual repair efficacy.
[0005] To achieve active targeted delivery of exosomes, genetic engineering is currently used to modify donor cells so that the surface of the exosome membrane they secrete expresses target peptides or proteins. However, this method has a long cell culture cycle, the transfection efficiency is greatly affected by cell passage and state, the expression level of target molecules fluctuates significantly between batches, and plasmid vectors also pose potential biosafety risks of genome integration. The overall process is complex and not conducive to standardization and large-scale production. If a simple physical adsorption method is used to fix the target molecules on the surface of exosomes, the binding force is weak, and they are easily detached under the continuous flushing of synovial fluid and protein competition in vivo, making it difficult to truly achieve lesion enrichment.
[0006] More importantly, the following prominent defects in multi-component compatibility have not yet been recognized and resolved in the existing technology: When attempting to compound sodium hyaluronate and strontium chloride to impart additional lubricating, anti-inflammatory, antioxidant and cartilage regeneration properties to hydrogels, the direct mixing of cationic chitosan and anionic high-molecular-weight sodium hyaluronate will produce polyelectrolyte flocculation. The phosphate ions produced by the hydrolysis of β-glycerophosphate are easy to form microcrystalline precipitates with strontium ions, causing the system to become turbid and the needle to become clogged. The existing system cannot simultaneously accommodate four types of functional components.
[0007] In addition, traditional formulations are all liquids that are prepared and used immediately, and the strict cold chain limits their use in primary care clinics. Existing technologies lack an integrated solution that takes into account targeting, sustained release, microenvironment regulation, and long-term stable storage. Summary of the Invention
[0008] Therefore, it is necessary to provide a targeted exosome-hydrogel formulation, its preparation method, and its application to address the aforementioned technical problems.
[0009] This application provides a targeted exosome-hydrogel formulation with the following technical solution: it comprises targeted exosomes and a functionalized thermosensitive hydrogel precursor solution. The targeted exosomes are exosomes derived from P3-P6 generation human adipose-derived mesenchymal stem cells. The membrane surface is modified with phospholipid anchoring via DSPE-PEG2000-WYRGRLC conjugate, and the internal structure is loaded with miR-140-5p mimics. The targeted exosomes have a particle size of 100-130 nm, a Zeta potential of -12 to -8 mV, a WYRGRLC-modified molecule density of 200-500 molecules on the surface of a single exosome, and a miR-140-5p encapsulation efficiency of ≥35%.
[0010] The final concentrations of each component in the functionalized thermosensitive hydrogel precursor solution are as follows: chitosan 1.8%–2.2% w / v, sodium β-glycerophosphate 5%–8% w / v, sodium hyaluronate 0.2%–0.5% w / v, strontium chloride 0.05–0.1 mmol / L, and the pH of the system is 7.0–7.2; the degree of deacetylation of the chitosan is ≥85%, and the molecular weight is 200–400 kDa; the molecular weight of the sodium hyaluronate is 1000–1500 kDa.
[0011] The functionalized thermosensitive hydrogel precursor solution was prepared by a stepwise feeding and gradient pH control process. Specifically, under an ice bath at 4°C and stirring, chitosan solution and sodium β-glycerophosphate solution were mixed. First, alkali was slowly added to adjust the pH of the system to 6.5. Then, sodium hyaluronate was added and fully dissolved. Finally, strontium chloride was added and the final pH was adjusted to obtain a homogeneous solution without polyelectrolyte flocculation and phosphate precipitation.
[0012] The formulation is a flowable, low-viscosity liquid at 4–25°C and can be passed through a 26G injection needle to form an in-situ gel with a storage modulus G' ≥ 500 Pa at 35–39°C for 5–8 minutes. It exists as a liquid injection solution or as a lyophilized powder injection obtained by freeze-drying after the final addition of 5% w / v trehalose.
[0013] By adopting the above technical solution, using a step-by-step feeding process combined with a gradient pH 6.5 pretreatment process, the flocculation and precipitation reactions between cationic chitosan and anionic sodium hyaluronate, and between strontium ions and phosphates are avoided, achieving stable coexistence of four functional excipients. Exosomes are modified by phospholipid anchoring, without the need for genetic modification, resulting in higher biocompatibility. Combined with low-damage electroporation loading miR-140-5p, the cartilage repair activity is enhanced. The temperature-sensitive gel can form a drug reservoir in situ in the joint cavity, prolonging the retention time of exosome lesions. At the same time, it is available in both liquid and lyophilized forms. The lyophilized powder does not require an ultra-low temperature cold chain, making it suitable for clinical use at the grassroots level.
[0014] Preferably, the freeze-dried powder injection has a stability period of not less than 12 months when stored at 2-8°C in the dark, and after reconstitution with sterile water for injection, its gelling properties and exosome targeting activity are not significantly different from those of the fresh liquid formulation.
[0015] By adopting the above technical solutions, freeze-dried dosage forms can significantly relax the requirements for storage and transportation temperatures, reduce transportation and storage costs, and ensure that the performance of the formulation does not degrade after long-term storage, thus expanding the scope of application.
[0016] Preferably, the formulation can continuously release exosomes in vitro for 15 days, and the gel has an anti-enzymatic degradation cycle of not less than 28 days in vivo.
[0017] By adopting the above technical solution, the active components of exosomes can be released in a long-term and sustained manner, continuously regulating the inflammatory and oxidative microenvironment of lesions, prolonging the repair cycle, and reducing the frequency of repeated injections.
[0018] This application provides a method for preparing a targeted exosome-hydrogel formulation, comprising the following steps:
[0019] S1: Collect exosome-free culture supernatant of P3-P6 generation human adipose mesenchymal stem cells, purify exosomes by multi-stage differential centrifugation combined with ultracentrifugation, quality control, particle size main peak 90-130nm, positive for CD9, CD63, and TSG101, endotoxin ≤0.1EU / μg protein;
[0020] S2: Electroporation buffer consisted of 1.15 mM KH2PO4, 25 mM KCl, and 21% OptiPrep. The mass ratio of total exosome protein to miR-140-5p mimic was 20:1. The membrane was repaired by a single pulse electroporation at 420 V and 150 μF, followed by incubation on ice at 37 °C. Free nucleic acids were removed by RNase A, and the drug-loaded exosomes were obtained by ultracentrifugation.
[0021] S3: WYRGRLC peptide and DSPE-PEG2000-maleimide were reacted in a molar ratio of 1.2:1 in deoxyriboPBS at pH 6.8 in the dark, followed by lyophilization by dialyzing, with a coupling efficiency ≥90%.
[0022] S4: The total protein to conjugate mass ratio of drug-loaded exosomes was 20:1. Incubate at 37℃ for 2 h, centrifuge at 3000×g for 15 min in a 100 kDa ultrafiltration tube, wash 3 times with sterile PBS, and the modification positivity rate was ≥85%.
[0023] S5: Add chitosan and sodium β-glycerophosphate in steps in an ice bath at 4℃, gradually adjust the pH to 6.5, then add sodium hyaluronate, and finally add strontium chloride to adjust the final pH to 7.0-7.2;
[0024] S6: Mix at 4-10℃ to obtain a liquid injection solution, or add trehalose at the end and freeze-dry to prepare a freeze-dried powder injection.
[0025] Preferably, in step S3, the coupling material is sealed and stored at -20°C in the dark.
[0026] By adopting the above technical solutions, the storage stability of the targeted conjugate is ensured, and the efficiency of exosome targeted modification is avoided due to the hydrolysis of active groups.
[0027] Preferably, in step S4, ultrafiltration washing is performed three times to completely remove unbound free target conjugates.
[0028] By adopting the above technical solution, the competitive interference of free peptides on cartilage recognition in vivo is eliminated, ensuring the enrichment efficiency of targeted exosome lesions.
[0029] Preferably, in step S6, the freeze-drying parameters are: pre-freezing at -45℃ for 4 hours, primary drying at -20℃ for 12 hours, and secondary drying at 25℃ for 6 hours.
[0030] By adopting the above technical solution, the freeze-drying curve can protect the biological activity of exosomes to the greatest extent, and there are no significant changes in particle size, potential and targeting ability after freeze-drying and rehydration.
[0031] This application also protects the use of the above-mentioned targeted exosome-hydrogel formulation in the preparation of drugs for the treatment of osteoarthritis.
[0032] Preferably, the drug is an intra-articular injection, and the indications are selected from osteoarthritis, traumatic articular cartilage defects, meniscus injuries, and knee synovitis.
[0033] Preferably, the intra-articular injection site is the diseased joint cavity of the human knee or ankle joint.
[0034] By adopting the above technical solutions, the clinical administration scenarios and applicable lesion sites of the formulation are limited, the boundaries of the protective use of the drug are clarified, and the local minimally invasive drug administration needs of diseases such as bone and joint cartilage damage are met.
[0035] In summary, this application includes the following beneficial technical effects:
[0036] 1. This application employs a stepwise feeding process combined with gradient pH control to formulate a functionalized thermosensitive hydrogel precursor. First, the pH of the system is adjusted to 6.5 before adding sodium hyaluronate, and finally, a trace amount of strontium chloride is added. This process avoids two major compatibility defects at the source: polyelectrolyte flocculation between chitosan and sodium hyaluronate, and microcrystalline precipitation formed by strontium ions and phosphate ions. It achieves stable coexistence of four functional components: chitosan, sodium hyaluronate, strontium chloride, and sodium β-glycerophosphate, resulting in a clear and injectable gel matrix. This solves the industry problems of easy turbidity, needle clogging, and mechanical inhomogeneity in traditional multifunctional gel systems.
[0037] 2. This application uses in vitro DSPE-PEG phospholipid-anchored modified exosomes to replace genetically engineered donor cells. It does not require plasmid transfection and has no safety risks associated with genome integration. The entire modification process is mild and time-saving. With a limited optimal material ratio of 20:1, a positive rate of over 85% for targeted modification can be achieved. The high modification density can alleviate the problem of target peptide detachment caused by competitive protein replacement in synovial fluid, ensuring that exosomes continue to have the ability to recognize type II collagen in the joint cavity.
[0038] 3. This application optimizes the 20:1 low nucleic acid ratio electroporation drug delivery system, and combines it with matching buffer and electroporation parameters to significantly reduce the risk of exosome vesicle rupture and free nucleic acid leakage caused by electroporation, reduce transient inflammatory stimulation in the joint cavity, and fully preserve the original active components such as exosome proteins and growth factors while ensuring a miR-140-5p encapsulation rate of ≥35%. This enables endogenous active substances and exogenous microRNA to synergistically promote cartilage matrix synthesis.
[0039] 4. This application adopts a freeze-drying preparation method with the final addition of trehalose, without mixing in a protective agent during the gel preparation stage, thus avoiding interference from seaweed with the sol-gel phase transition rate and gel mechanical strength; the resulting freeze-dried powder injection can be stably stored at 2-8℃ for 12 months, and after reconstitution, its gelation, targeting, and drug loading properties are no different from those of the fresh liquid formulation, breaking the limitations of ultra-low temperature cold chain storage and transportation of exosome formulations, and is suitable for flexible use scenarios in primary clinics and emergency departments.
[0040] 5. This application constructs a triple synergistic repair system of targeted recognition, physical sustained release, and lesion microenvironment regulation. After injection into the joint cavity, the thermosensitive gel forms a drug reservoir in situ, continuously releasing active exosomes for 15 days. The in vivo gel anti-degradation cycle can reach 28 days. Strontium ions in the matrix remove excess reactive oxygen species in the lesion, and sodium hyaluronate replenishes the synovial fluid and buffers the lactic acid produced by chitosan degradation, jointly alleviating the oxidative inflammatory microenvironment and significantly improving the in vivo retention time of exosomes. Compared with single exosomes or ordinary hydrogels, the cartilage repair and degeneration inhibition effects are significantly improved.
[0041] 6. This application sets up a complete set of quantitative quality control standards for raw material generation, exosome physicochemical indicators, targeted modification density, nucleic acid encapsulation rate, and gel rheological parameters. The process parameters throughout the entire process are clear and controllable, with strong batch repeatability. It also provides two dosage forms: liquid injection and lyophilized powder injection. The administration is limited to local puncture injection in the joint cavity of knee and ankle joint lesions. The process has a high degree of standardization and meets the requirements for registration and large-scale industrial production of biopharmaceutical-device combination products. Attached Figure Description
[0042] Figure 1 This is a schematic diagram of the preparation method of this application. Detailed Implementation
[0043] To make the objectives, technical solutions, and advantages of this application clearer, the following detailed description is provided in conjunction with the accompanying drawings and embodiments. It should be understood that the specific embodiments described herein are merely illustrative and not intended to limit the scope of this application.
[0044] like Figure 1 As shown in Example 1: Standard formulation of liquid targeted exosome-hydrogel preparation, the specific preparation steps are as follows:
[0045] Step 1: P4 generation human adipose-derived mesenchymal stem cells were cultured and the exosome-free culture supernatant was collected. The purified exosomes were obtained by multi-stage differential centrifugation, two rounds of ultracentrifugation, and filtration ultrafiltration. The main peak particle size was 106 nm. The expression of CD9, CD63, and TSG101 was positive. The endotoxin was 0.07 EU / μg, and the mycoplasma was negative.
[0046] Step 2: Using a protein / nucleic acid mass ratio of 20:1 and electroporation parameters of 420V / 150μF, miR-140-5p was loaded, followed by ice bath retrieval, RNase A purification, and ultra-fast purification, resulting in a nucleic acid encapsulation efficiency of 41%.
[0047] Step 3: The WYRGRLC-Cys peptide and DSPE-PEG2000-MAL were reacted in the dark at a molar ratio of 1.2:1, followed by lyophilization by dialyzing. The coupling efficiency was 92.7%.
[0048] Step 4: The drug-loaded exosomes and conjugates were incubated at a mass ratio of 20:1 at 37°C for 2 hours, followed by 3 washes with 100kDa ultrafiltration. The positive rate of targeted modification was 89.2%.
[0049] Step 5: Add materials in stages at 4℃ ice bath and adjust pH to 6.5 in a gradient manner to prepare a standard hydrogel precursor. The final concentrations of each component are: chitosan 2.0% w / v, sodium β-glycerophosphate 6% w / v, sodium hyaluronate 0.3% w / v, and strontium chloride 0.08 mmol / L. The system is clear and free of flocculation and precipitation.
[0050] Step 6: Mix the targeted exosomes and precursor fluid at 6℃ with equal volumes, and aseptically dispense to obtain a liquid injection solution; it can pass through a 26G needle at 20℃, and completely gels at 37℃ for 7 minutes, with a storage modulus of 560Pa.
[0051] Example 2: Standard ratio lyophilized powder injection, the specific preparation steps are as follows:
[0052] The procedures for exosome extraction, drug loading, targeted modification, and hydrogel preparation were completely consistent with those in Example 1.
[0053] After low-temperature mixing, 5% w / v trehalose was added at the end, and the mixture was dispensed into penicillin semi-stoppered containers. The freeze-drying procedure was as follows: pre-freeze at -45℃ for 4 hours, dry once at -20℃ for 12 hours, and dry twice at 25℃ for 6 hours. After sealing, the mixture was stored at 2-8℃ away from light. After 12 months of storage, the mixture was reconstituted, and the gelation and targeting performance of the formulation showed no significant attenuation.
[0054] Example 3: Low-concentration liquid formulation, the specific preparation steps are as follows:
[0055] The final concentrations of each component in the hydrogel precursor were adjusted to: chitosan 1.8% w / v, sodium β-glycerophosphate 5% w / v, sodium hyaluronate 0.2% w / v, and strontium chloride 0.05 mmol / L; the remaining cell, drug loading, and targeted modification processes were the same as in the previous example; gelation was performed at 37°C for 8 minutes, resulting in lower viscosity, suitable for mild cartilage facet joint injections.
[0056] Example 4: High-concentration liquid formulation, the specific preparation steps are as follows:
[0057] The final concentrations of each component in the hydrogel precursor were adjusted to 2.2% w / v chitosan, 8% w / v sodium β-glycerophosphate, 0.5% w / v sodium hyaluronate, and 0.1 mmol / L strontium chloride. The system was prepared using a stepwise gradient pH process and remained turbid. It gelled rapidly at 37℃ for 5 min, resulting in higher mechanical strength and making it suitable for severe knee osteoarthritis.
[0058] Example 5: Targeted coupling molar ratio control sample, the specific preparation steps are as follows:
[0059] The only difference was that the molar ratio of the peptide to DSPE-PEG2000-MAL was adjusted to 1:1, while the other reaction and purification conditions were the same as in Example 1; the final coupling efficiency was only 82.1%, and the cartilage-targeting fluorescence intensity decreased significantly.
[0060] Example 6: Electroporation parameter control sample, the specific preparation steps are as follows:
[0061] Only the electroporation capacitor was adjusted to 125μF, while the other buffer and material ratios remained unchanged; the encapsulation efficiency of miR-140-5p decreased to 32.3%, and the drug loading effect was inferior to that of Example 1.
[0062] Example 7: Targeted modification ratio control sample, the specific preparation steps are as follows:
[0063] The mass ratio of drug-loaded exosomes to conjugates was adjusted to 40:1, and the mixture was incubated at 37°C for 2 hours. The positive rate of the modified exosomes was only 61.5% by flow cytometry, and the lesion enrichment ability was significantly reduced.
[0064] Comparative Example 1: Gel preparation without gradient pH adjustment in a one-step feeding process. The specific preparation steps are as follows:
[0065] Chitosan and sodium hyaluronate were mixed directly during preparation without adjusting the pH to 6.5 beforehand; the system instantly produced a large amount of white flocculent matter, the solution became turbid, and it could not pass through the injection needle.
[0066] Comparative Example 2: Strontium chloride was added in advance to prepare the gel. The specific preparation steps are as follows:
[0067] First, strontium chloride was added to the chitosan solution, followed by the dropwise addition of sodium β-glycerophosphate. After the system was allowed to stand, white strontium phosphate microcrystals precipitated, posing a risk of injection needle blockage.
[0068] Comparative Example 3: Seaweed was mixed into the gel matrix in advance. The specific preparation steps are as follows:
[0069] 5% trehalose was added during the hydrogel preparation stage, followed by mixing with targeted exosomes; it took 12 minutes at 37°C to slowly gel, and the gel storage modulus was only 320 Pa, which did not meet the requirements for long-term retention in terms of mechanical properties.
[0070] This application provides a targeted exosome-hydrogel formulation, its preparation method, and its application. By optimizing the hydrogel compounding process, exosome targeted modification methods, and formulation storage dosage form, it solves the problems of existing bone and joint repair formulations, such as compatibility conflicts, poor targeted safety, short retention time, and harsh storage and transportation conditions, and has good prospects for clinical translation and industrialization.
Claims
1. A targeted exosome-hydrogel formulation, characterized in that: The product comprises a targeted exosome and a functionalized thermosensitive hydrogel precursor solution. The targeted exosomes are exosomes derived from P3-P6 generation human adipose-derived mesenchymal stem cells. The membrane surface is modified with phospholipid anchoring via DSPE-PEG2000-WYRGRLC conjugate, and the internal structure is loaded with miR-140-5p mimics. The targeted exosomes have a particle size of 100-130 nm, a Zeta potential of -12 to -8 mV, a WYRGRLC-modified molecule density of 200-500 molecules on the surface of a single exosome, and a miR-140-5p encapsulation efficiency of ≥35%. The final concentrations of each component in the functionalized thermosensitive hydrogel precursor solution are as follows: chitosan 1.8%–2.2% w / v, sodium β-glycerophosphate 5%–8% w / v, sodium hyaluronate 0.2%–0.5% w / v, strontium chloride 0.05–0.1 mmol / L, and the pH of the system is 7.0–7.2; the degree of deacetylation of the chitosan is ≥85%, and the molecular weight is 200–400 kDa; the molecular weight of the sodium hyaluronate is 1000–1500 kDa. The functionalized thermosensitive hydrogel precursor solution was prepared by a stepwise feeding and gradient pH control process. Specifically, under an ice bath at 4°C and stirring, chitosan solution and sodium β-glycerophosphate solution were mixed. First, alkali was slowly added to adjust the pH of the system to 6.
5. Then, sodium hyaluronate was added and fully dissolved. Finally, strontium chloride was added and the final pH was adjusted to obtain a homogeneous solution without polyelectrolyte flocculation and phosphate precipitation. The formulation is a flowable, low-viscosity liquid at 4–25°C and can be passed through a 26G injection needle to form an in-situ gel with a storage modulus G' ≥ 500 Pa at 35–39°C for 5–8 minutes. It exists as a liquid injection solution or as a lyophilized powder injection obtained by freeze-drying after the final addition of 5% w / v trehalose.
2. The targeted exosome-hydrogel formulation according to claim 1, characterized in that: The lyophilized powder injection has a stability period of not less than 12 months when stored at 2–8°C in the dark. After reconstitution with sterile water for injection, its gel-forming properties and exosome targeting activity are not significantly different from those of the fresh liquid formulation.
3. The targeted exosome-hydrogel formulation according to claim 1, characterized in that: The formulation can continuously release exosomes in vitro for 15 days, and the gel has an anti-enzymatic degradation cycle of no less than 28 days in vivo.
4. A method for preparing a targeted exosome-hydrogel formulation, wherein the targeted exosome-hydrogel formulation according to any one of claims 1 to 3 is characterized in that, Includes the following steps: S1: Collect exosome-free culture supernatant of P3-P6 generation human adipose mesenchymal stem cells, purify exosomes by multi-stage differential centrifugation combined with ultracentrifugation, quality control, particle size main peak 90-130nm, positive for CD9, CD63, and TSG101, endotoxin ≤0.1EU / μg protein; S2: Electroporation buffer consisted of 1.15 mM KH2PO4, 25 mM KCl, and 21% OptiPrep. The mass ratio of total exosome protein to miR-140-5p mimic was 20:
1. The membrane was repaired by a single pulse electroporation at 420 V and 150 μF, followed by incubation on ice at 37 °C. Free nucleic acids were removed by RNase A, and the drug-loaded exosomes were obtained by ultracentrifugation. S3: WYRGRLC peptide and DSPE-PEG2000-maleimide were reacted in a molar ratio of 1.2:1 in deoxyriboPBS at pH 6.8 in the dark, followed by lyophilization by dialyzing, with a coupling efficiency ≥90%. S4: The total protein to conjugate mass ratio of drug-loaded exosomes was 20:
1. Incubate at 37℃ for 2 h, centrifuge at 3000×g for 15 min in a 100 kDa ultrafiltration tube, wash 3 times with sterile PBS, and the modification positivity rate was ≥85%. S5: Add chitosan and sodium β-glycerophosphate in steps in an ice bath at 4℃, gradually adjust the pH to 6.5, then add sodium hyaluronate, and finally add strontium chloride to adjust the final pH to 7.0-7.2; S6: Mix at 4-10℃ to obtain a liquid injection solution, or add trehalose at the end and freeze-dry to prepare a freeze-dried powder injection.
5. The method for preparing a targeted exosome-hydrogel formulation according to claim 4, characterized in that: In step S3, the coupling material is sealed and stored at -20°C in the dark.
6. The method for preparing a targeted exosome-hydrogel formulation according to claim 4, characterized in that: In step S4, ultrafiltration washing is performed three times to completely remove unbound free target conjugates.
7. The method for preparing a targeted exosome-hydrogel formulation according to claim 4, characterized in that: In step S6, the freeze-drying parameters are: pre-freezing at -45℃ for 4 hours, drying at -20℃ for 12 hours, and drying at 25℃ for 6 hours.
8. The use of the targeted exosome-hydrogel formulation according to any one of claims 1 to 3 in the preparation of a medicament for treating osteoarthritis.
9. The application according to claim 8, characterized in that: The drug is an intra-articular injection, and its indications are selected from osteoarthritis, traumatic articular cartilage defects, meniscus injuries, and knee synovitis.
10. The application according to claim 9, characterized in that: The intra-articular injection site is the diseased joint cavity of the human knee or ankle joint.