Targeting exosome-hydrogel preparation as well as preparation method and application thereof

Abstract

The invention discloses a targeted exosome-hydrogel preparation as well as a preparation method and application thereof, and belongs to the technical field of biological medicines. The preparation is composed of an umbilical cord mesenchymal stem cell exosome which is subjected to targeting modification by cartilage affinity peptide, therapeutic nucleic acid loaded in the umbilical cord mesenchymal stem cell exosome, and PLGA-PEG-PLGA temperature-sensitive hydrogel encapsulating the carrier. The preparation method comprises the following steps: efficiently preparing an active exosome by combining three-dimensional dynamic hypoxia culture with TSG-6 agonist stimulation; rapid and mild separation is carried out by using a micro-fluidic chip; carrying out CAP peptide targeted modification through chemical coupling; and finally, mixing the drug-loaded exosome with the pH response type hydrogel precursor solution to further prepare freeze-dried powder. Through combination of targeted modification and the intelligent hydrogel carrier, the enrichment retention capacity of the exosome in the articular cartilage and the response release efficiency of the exosome in a lesion environment are remarkably improved, collaborative treatment of anti-inflammation and cartilage regeneration promotion is achieved, and the preparation is good in stability and lasting in effect.

Description

Technical Field

[0001] This invention relates to the field of biomedical technology, specifically to a targeted exosome-hydrogel formulation, its preparation method, and its application. Background Technology

[0002] Osteoarthritis is a panarticular disease characterized by degenerative changes in articular cartilage, inflammation of the synovium, and sclerosis of the subchondral bone. It is one of the leading causes of pain and disability worldwide. Due to the avascular, nerveless, and lymphatic-free distribution of cartilage tissue, its self-repair and regeneration capabilities are extremely limited.

[0003] Currently, clinical treatment strategies for osteoarthritis mainly focus on symptom relief, including nonsteroidal anti-inflammatory drugs (NSAIDs), intra-articular injections of hyaluronic acid, or corticosteroids. However, these methods cannot reverse cartilage damage, and long-term use can lead to side effects. In recent years, regenerative therapies based on mesenchymal stem cells and their secreted exosomes have shown great potential. Exosomes, as key carriers of intercellular communication, can deliver active substances such as proteins and nucleic acids, regulate the biological functions of recipient cells, and have advantages such as low immunogenicity and good stability.

[0004] However, applying exosome therapy to the clinical treatment of osteoarthritis still faces three major technical bottlenecks:

[0005] Poor targeting: Free exosomes injected into the joint cavity are easily cleared by the synovial tissue, resulting in a short retention time in the joint cavity (usually less than 12 hours) and difficulty in effectively penetrating the dense cartilage matrix to reach the lesion site, leading to low cartilage targeting efficiency.

[0006] Insufficient stability: Exosomes exhibit rapid activity decay at room temperature, and conventional cryopreservation processes can easily lead to membrane rupture, requiring stringent storage and transportation conditions.

[0007] Single treatment modality: Most existing technologies only load a single type of active molecule, such as anti-inflammatory factors or pro-anabolism factors, which are not easy to simultaneously regulate the inflammatory response and cartilage regeneration and repair that coexist in the process of osteoarthritis, making it difficult to achieve synergistic treatment.

[0008] Therefore, there is an urgent need in this field to develop a novel exosome delivery system that can actively target cartilage, intelligently release at the lesion site, and synergistically exert both anti-inflammatory and regenerative functions. Summary of the Invention

[0009] The purpose of this invention is to provide a targeted exosome-hydrogel formulation, its preparation method, and its application, in order to solve the problems mentioned in the background art.

[0010] To achieve the above objectives, the present invention adopts the following technical solution:

[0011] In a first aspect, the present invention provides a targeted exosome-hydrogel formulation comprising the following components:

[0012] (a) Exosomes derived from umbilical cord mesenchymal stem cells, wherein the surface of the exosomes is covalently linked to a chondroitin peptide, the amino acid sequence of which is CGGRKNDRKDN;

[0013] (b) An active ingredient loaded within the exosome, the active ingredient comprising miR-140-5p and siRNA targeting TGF-β1;

[0014] (c) pH-responsive fluorescent probes whose excitation threshold is activated at pH ≤ 6.5;

[0015] (d) An injectable thermosensitive hydrogel carrier for encapsulating the exosomes, wherein the hydrogel carrier is a PLGA-PEG-PLGA triblock copolymer.

[0016] Preferably, the exosomes are prepared using a three-dimensional dynamic culture system, and the preparation method is as follows:

[0017] Three-dimensional dynamic culture was carried out in a rotating bioreactor under hypoxic conditions with an oxygen concentration of 5% at a rotation speed of 8 rpm.

[0018] During the culture process, a TSG-6 agonist at a concentration of 5-15 μM was added, and the culture time was 72 hours.

[0019] After culture, exosomes were sorted using a microfluidic chip. The surface of the microfluidic chip was modified with CD63 and CD81 nanoprobes, and the sorting flow rate was controlled at 50 μL / min.

[0020] Preferably, the microfluidic chip has a channel width of 50 μm ± 5 μm and a nanoprobe density of 200–250 nanoprobes / μm. 2 .

[0021] Preferably, the chondroitin affinity peptide is coupled to the DSPE-PEG2000 lipid molecules on the exosome membrane via an EDC / NHS chemical cross-linking method, and its binding constant Ka with chondroitin sulfate is not less than 1.8 × 10⁻⁶. 7 M -1 .

[0022] Preferably, the encapsulation efficiency of miR-140-5p and the siRNA targeting TGF-β1 is not less than 92.4%, and the siRNA targets the mRNA sequence of TGF-β1.

[0023] Preferably, the hydrogel carrier can achieve pH-responsive release in the acidic environment of the joint cavity in osteoarthritis. In an environment of pH 6.2–6.5, the exosome release rate is more than 2.3 times faster than in a neutral environment, and it can be stably stored at 25°C for no less than 7 days.

[0024] Secondly, the present invention provides a method for preparing the above-mentioned targeted exosome-hydrogel formulation, comprising the following steps:

[0025] S1. Umbilical cord mesenchymal stem cells were cultured in a hypoxic environment and a rotating bioreactor, and TSG-6 agonist was added. The cell supernatant was then collected.

[0026] S2. Exosomes are obtained by sorting from cell supernatant using a microfluidic chip;

[0027] S3. Cartilage affinity peptides were covalently linked to the surface of the resulting exosomes via an EDC / NHS chemical cross-linking method.

[0028] S4. The targeted exosomes obtained in the above steps are co-incubated with a pH-responsive fluorescent probe and a solution loaded with miR-140-5p and siRNA to achieve loading of active ingredients.

[0029] S5. Mix the exosomes obtained in step S4 with the PLGA-PEG-PLGA hydrogel precursor solution to form a composite formulation;

[0030] S6. The compound preparation is subjected to gradient cooling freeze-drying treatment, and a freeze-drying protectant containing 10% trehalose is added to prepare a freeze-dried powder preparation.

[0031] Preferably, the gradient cooling freeze-drying process includes:

[0032] Cool to -80℃ at a rate of 1℃ / min;

[0033] Add a freeze-drying protectant at -80℃;

[0034] After lyophilization and reconstitution, the resulting exosome particle size distribution (PDI) was <0.15.

[0035] Thirdly, the present invention provides the use of the above-mentioned targeted exosome-hydrogel formulation in the preparation of a medicament for treating osteoarthritis.

[0036] Preferably, the drug is administered via intra-articular injection, with a duration of efficacy of not less than 14 days after a single injection, and can significantly reduce the IL-1β level in the synovial fluid to below 78.4 pg / mL.

[0037] Compared with the prior art, the beneficial effects of the present invention are:

[0038] This invention combines three-dimensional dynamic culture, hypoxia-induced environment, and TSG-6 agonist stimulation to form a synergistic culture system that can significantly activate the paracrine function of umbilical cord mesenchymal stem cells, thereby revolutionarily increasing the basic production of exosomes and enhancing their intrinsic biological activity.

[0039] This invention uses microfluidic chip technology to replace the traditional ultracentrifugation method and utilizes specific nanoprobes to achieve precise capture of exosomes. This not only greatly shortens the separation time and improves the separation efficiency, but also avoids the potential damage to the vesicle structure caused by the strong shear force generated during ultracentrifugation, ensuring the integrity and uniformity of the obtained exosomes.

[0040] This invention modifies the surface of exosomes with novel chondroitin affinity peptides through chemical coupling, enabling them to specifically recognize and tightly bind to the main components in the extracellular matrix of chondrocytes. This enhances the enrichment and retention of exosomes in diseased cartilage tissue, solving the core problems of short retention time of free exosomes in the joint cavity and low cartilage penetration rate, and achieving precise drug delivery.

[0041] This invention simultaneously loads miRNA with cartilage regeneration function and siRNA with anti-inflammatory function inside exosomes, realizing synergistic intervention on two key pathological links of osteoarthritis, namely "inflammatory environment" and "regenerative repair", in the same delivery system. Furthermore, by encapsulating the exosomes in a pH-responsive thermosensitive hydrogel, an "intelligent" controlled release mode is achieved, which allows for slow release of the drug under normal physiological conditions and rapid release under the acidic microenvironment of osteoarthritis, significantly improving the timeliness and specificity of treatment.

[0042] This invention, through an optimized lyophilization protectant formulation and gradient cooling process, successfully prepares a stable lyophilized powder from a liquid composite formulation, significantly improving the storage and transportation stability of exosomes at room temperature and overcoming their absolute dependence on cold chain transportation. At the same time, a single intra-articular injection can achieve long-term treatment, effectively prolonging the duration of drug action and reducing the frequency of drug administration for patients. Attached Figure Description

[0043] Figure 1 This is a schematic diagram of the microfluidic exosome sorting chip of the present invention;

[0044] Figure 2 This is a flowchart of the three-dimensional hypoxic dynamic culture preparation process of exosomes according to the present invention;

[0045] Figure 3 This is a pH-responsive exosome release curve of the present invention;

[0046] Figure 4 This is a schematic diagram of the freeze-drying and reconstitution process of the present invention. Detailed Implementation

[0047] To further explain the technical solution of the present invention, a detailed description is provided below through specific embodiments.

[0048] Please see Figures 1-4 :

[0049] First aspect: The present invention provides a targeted exosome-hydrogel formulation, comprising the following components:

[0050] (a) Exosomes derived from umbilical cord mesenchymal stem cells (UC-MSCs), wherein the surface of the exosomes is covalently linked to a cartilage-affinity peptide (CAP), the amino acid sequence of which is CGGRKNDRKDN;

[0051] (b) An active ingredient loaded within the exosome, the active ingredient comprising miR-140-5p and siRNA targeting TGF-β1;

[0052] (c) pH-responsive fluorescent probes, whose excitation threshold is activated at pH ≤ 6.5, are used for tracking and as release switches;

[0053] (d) An injectable thermosensitive hydrogel carrier for encapsulating the exosomes, wherein the hydrogel carrier is a PLGA-PEG-PLGA triblock copolymer.

[0054] The exosomes were prepared using a three-dimensional dynamic culture system as follows: under hypoxic conditions (5% oxygen concentration), three-dimensional dynamic culture was carried out in a rotating bioreactor at a rotation speed of 8 rpm; during the culture process, a TSG-6 agonist at a concentration of 5-15 μM (preferably 10 μM) was added, and the culture time was 72 hours; after the culture was completed, exosomes were sorted using a microfluidic chip, the surface of which was modified with CD63 and CD81 nanoprobes, and the sorting flow rate was controlled at 50 μL / min.

[0055] Furthermore, the microfluidic chip has a channel width of 50 μm ± 5 μm and a nanoprobe density of 200–250 nanoprobes / μm. 2 .

[0056] The chondroitin affinity peptide is coupled to the DSPE-PEG2000 lipid molecules on the exosome membrane via an EDC / NHS chemical cross-linking method. This modification ensures that the binding constant Ka between the exosome and chondroitin sulfate is not less than 1.8 × 10⁻⁶. 7 M -1 The levels were significantly higher than those of unmodified exosomes (Ka≈3.2×10⁻⁶). 5 M -1 ).

[0057] The encapsulation efficiency of miR-140-5p and the siRNA targeting TGF-β1 in exosomes is no less than 92.4%, which is much higher than that of the single-load system (about 65-75%), and the siRNA targets the mRNA sequence of TGF-β1.

[0058] The hydrogel carrier can achieve pH-responsive release in the acidic environment (pH 6.2–6.5) of the joint cavity in osteoarthritis, and the exosome release rate is more than 2.3 times faster than in a neutral environment (pH 7.4). Furthermore, the formulation can be stably stored at 25°C for no less than 7 days after lyophilization.

[0059] Secondly, this invention provides a method for preparing the above-mentioned targeted exosome-hydrogel formulation, comprising the following steps:

[0060] S1. Umbilical cord mesenchymal stem cells were cultured in a hypoxic environment and a rotating bioreactor, and TSG-6 agonist was added. The cell supernatant was then collected.

[0061] S2. High-purity exosomes are obtained by sorting from cell supernatant using a microfluidic chip;

[0062] S3. Cartilage affinity peptide (CAP) is covalently linked to the surface of the obtained exosomes by EDC / NHS chemical cross-linking method to obtain targeted exosomes;

[0063] S4. The targeted exosomes obtained in the above steps are co-incubated with a pH-responsive fluorescent probe and a solution loaded with miR-140-5p and siRNA to achieve loading of active ingredients.

[0064] S5. Mix the exosomes obtained in step S4 with the PLGA-PEG-PLGA hydrogel precursor solution to form an injectable composite formulation.

[0065] S6. The compound preparation is subjected to gradient cooling freeze-drying treatment, and a freeze-drying protectant containing 10% trehalose is added to prepare a freeze-dried powder preparation that is easy to store and transport.

[0066] The gradient cooling freeze-drying process involves cooling the temperature to -80°C at a rate of 1°C / min, and adding a freeze-drying protectant at this temperature. After the freeze-dried powder is reconstituted, the resulting exosomes have a polydispersity index (PDI) of <0.15, indicating that they have extremely high uniformity and stability.

[0067] Thirdly, this invention provides the use of the above-mentioned targeted exosome-hydrogel formulation in the preparation of a medicament for treating osteoarthritis.

[0068] The drug is administered via intra-articular injection; in animal experiments using osteoarthritis models, the therapeutic effect lasts for at least 14 days after a single injection, and it can significantly reduce the level of the inflammatory factor IL-1β in the synovial fluid to below 78.4 pg / mL.

[0069] Example 1: Efficient preparation and sorting of exosomes:

[0070] 1. Cell culture: Take 3rd-5th generation umbilical cord mesenchymal stem cells and resuspend them in a special culture medium.

[0071] 2. Three-dimensional dynamic hypoxia culture: The cell suspension was transferred to a rotary bioreactor and the rotation speed was set to 8 rpm. The reactor was placed in a hypoxia incubator containing 5% oxygen at 37°C. TSG-6 agonist was added to the culture medium and the concentration gradients were set to 5 μM, 10 μM and 15 μM, respectively, and the culture was carried out for 72 hours.

[0072] 3. Supernatant collection: After the culture is completed, the cell supernatant is collected, centrifuged at 300g for 10 minutes to remove cell debris, and then centrifuged at 2000g for 20 minutes for further clarification.

[0073] 4. Microfluidic sorting: The clarified supernatant was pumped into a microfluidic chip (channel width 50 μm) with CD63 / CD81 nanoantibodies on the surface at a flow rate of 50 μL / min for sorting; the exosome suspension at the outlet was collected.

[0074] 5. Results: Nanoparticle tracking analysis (NTA) and protein quantification showed that the 10 μM TSG-6 agonist group had the highest exosome production, reaching 6.7 × 10⁻⁶. 10 Particles / 10 6 The cells were found to have the highest TSG-6 protein expression level (486 pg / μg protein) as confirmed by ELISA.

[0075] Example 2, Targeted Modification and Drug Loading of Exosomes:

[0076] 1. CAP peptide conjugation: Take 1 mg of the exosomes obtained in Example 1 and activate the carboxyl groups on their membranes using an EDC / NHS activation system; then add excess DSPE-PEG2000-CAP (sequence: CGGRKNDRKDN) molecules and react at room temperature in the dark for 4 hours; after the reaction is complete, remove unreacted conjugates by ultrafiltration centrifugation.

[0077] 2. Drug loading: The above-mentioned CAP-modified exosomes were co-loaded with miR-140-5p mimics and siRNA targeting TGF-β1 by electroporation; the encapsulation efficiency was determined by HPLC to be 92.4% ± 3.7%.

[0078] 3. Targeting capability verification: The binding constant Ka of the modified exosomes and immobilized chondroitin sulfate was determined by surface plasmon resonance (SPR) technology. The results showed that the Ka value reached 1.8 × 10⁻⁶. 7 M -1 This demonstrates that its ability to bind to the cartilage matrix is ​​significantly enhanced.

[0079] Example 3, Preparation and characterization of the compound formulation:

[0080] 1. Hydrogel composite: The targeted and drug-loaded exosomes obtained in Example 2 were mixed with the hydrogel precursor solution of PLGA-PEG-PLGA triblock copolymer (dissolved in PBS, concentration of 20% w / v) at a volume ratio of 1:5 and gently blown to homogenize to obtain an injectable composite formulation.

[0081] 2. pH-responsive release test: The compound formulation was placed in a Transwell chamber and immersed in release media at pH 7.4 and pH 6.5, respectively, and shaken at 37°C. Samples were taken at predetermined time points, and the cumulative release rate of exosomes was calculated by detecting the intensity of the fluorescent probe in the media. The results are as follows: Figure 3 As shown, at 72 hours, the release rate of the pH 6.5 group was 2.3 times that of the pH 7.4 group.

[0082] 3. Freeze-drying preparation: The above-mentioned compound preparation was programmed to cool to -80℃ at a rate of 1℃ / min, and a freeze-drying protectant containing 10% trehalose was quickly added when the temperature reached -80℃, followed by freeze-drying; after reconstitution, the obtained freeze-dried powder was detected by dynamic light scattering (DLS), and its particle size polydispersity index (PDI) was 0.12, indicating that the reconstituted powder had good dispersibility and high vesicle integrity.

[0083] Example 4, in vivo pharmacodynamic evaluation in animals:

[0084] 1. Establishment of osteoarthritis model: New Zealand white rabbits were selected, and an anterior cruciate ligament transection was used to induce the establishment of a knee osteoarthritis model.

[0085] 2. Grouping and administration: Animals that successfully developed the model were randomly divided into three groups: (1) model control group (injected with PBS); (2) positive control group (injected with commercial hyaluronic acid preparation); (3) the preparation group of the present invention (injected with the lyophilized powder reconstituted solution prepared in Example 3). All groups were given a single intra-articular injection.

[0086] 3. Efficacy evaluation:

[0087] MRI imaging analysis: Knee joint MRI scans were performed on day 14 after injection to assess the area of ​​cartilage defects, cartilage thickness, and signal intensity. The results showed that the cartilage surface of the formulation group of this invention was more intact, and the area of ​​cartilage defects was significantly smaller than that of the model control group and the positive control group.

[0088] Inflammatory factor detection: Joint fluid was collected and IL-1β levels were detected using an ELISA kit; the concentration of IL-1β in the joint fluid of the preparation group of the present invention was significantly lower than that in the model control group and the positive control group, indicating that the preparation of the present invention has an effective anti-inflammatory effect.

[0089] Histological analysis: Joint tissues from euthanized animals were subjected to Safranin-O and COL2A1 immunohistochemical staining. The results showed that the cartilage surface of the preparation group of this invention was smoother, the chondrocytes were arranged in an orderly manner, and the area and intensity of the COL2A1 positive staining area were significantly higher than those of the other two groups, indicating that the cartilage synthesis and metabolism were active.

[0090] The above embodiments fully demonstrate that the targeted exosome-hydrogel formulation and its preparation method provided by the present invention have significant technological advancements and excellent therapeutic effects.

[0091] Finally, it should be noted that the above embodiments are only used to illustrate the technical solutions of the present invention and are not intended to limit it. Although the present invention has been described in detail with reference to preferred embodiments, those skilled in the art should understand that modifications or equivalent substitutions can be made to the technical solutions of the present invention without departing from the spirit and scope of the present invention, and all such modifications and substitutions should be covered within the scope of the claims of the present invention. For example, the active ingredient miR-140-5p can be replaced by its homologous sequence miR-140-3p or other miRNAs with similar cartilage-protective functions; the hydrogel carrier can also be replaced by other biocompatible polymer materials with temperature-sensitive or pH-responsive properties.

Claims

1. A targeted exosome-hydrogel formulation, characterized in that, Includes the following components: (a) Exosomes derived from umbilical cord mesenchymal stem cells, wherein the surface of the exosomes is covalently linked to a chondroitin peptide, the amino acid sequence of which is CGGRKNDRKDN; (b) An active ingredient loaded within the exosome, the active ingredient comprising miR-140-5p and siRNA targeting TGF-β1; (c) pH-responsive fluorescent probes whose excitation threshold is activated at pH ≤ 6.5; (d) An injectable thermosensitive hydrogel carrier for encapsulating the exosomes, wherein the hydrogel carrier is a PLGA-PEG-PLGA triblock copolymer.

2. The targeted exosome-hydrogel formulation according to claim 1, characterized in that, The exosomes were prepared using a three-dimensional dynamic culture system, and the preparation method is as follows: Three-dimensional dynamic culture was carried out in a rotating bioreactor under hypoxic conditions with an oxygen concentration of 5% at a rotation speed of 8 rpm. During the culture process, a TSG-6 agonist at a concentration of 5-15 μM was added, and the culture time was 72 hours. After culture, exosomes were sorted using a microfluidic chip. The surface of the microfluidic chip was modified with CD63 and CD81 nanoprobes, and the sorting flow rate was controlled at 50 μL / min.

3. The targeted exosome-hydrogel formulation according to claim 2, characterized in that, The microfluidic chip has a channel width of 50 μm ± 5 μm and a nanoprobe density of 200–250 probes / μm. 2 .

4. The targeted exosome-hydrogel formulation according to claim 1, characterized in that, The chondroitin affinity peptide is coupled to the DSPE-PEG2000 lipid molecules on the exosome membrane via an EDC / NHS chemical cross-linking method, and its binding constant Ka with chondroitin sulfate is not less than 1.8 × 10⁻⁶. 7 M -1 .

5. The targeted exosome-hydrogel formulation according to claim 1, characterized in that, The encapsulation efficiency of miR-140-5p and the siRNA targeting TGF-β1 is not less than 92.4%, and the siRNA targets the mRNA sequence of TGF-β1.

6. The targeted exosome-hydrogel formulation according to claim 1, characterized in that, The hydrogel carrier can achieve pH-responsive release in the acidic environment of the joint cavity in osteoarthritis. In an environment of pH 6.2–6.5, the exosome release rate is more than 2.3 times faster than in a neutral environment, and it can be stably stored at 25°C for no less than 7 days.

7. A method for preparing a targeted exosome-hydrogel formulation as described in any one of claims 1-6, characterized in that, Includes the following steps: S1. Umbilical cord mesenchymal stem cells were cultured in a hypoxic environment and a rotating bioreactor, and TSG-6 agonist was added. The cell supernatant was then collected. S2. Exosomes are obtained by sorting from cell supernatant using a microfluidic chip; S3. Cartilage affinity peptides were covalently linked to the surface of the resulting exosomes via an EDC / NHS chemical cross-linking method. S4. The targeted exosomes obtained in the above steps are co-incubated with a pH-responsive fluorescent probe and a solution loaded with miR-140-5p and siRNA to achieve loading of active ingredients. S5. Mix the exosomes obtained in step S4 with the PLGA-PEG-PLGA hydrogel precursor solution to form a composite formulation; S6. The compound preparation is subjected to gradient cooling freeze-drying treatment, and a freeze-drying protectant containing 10% trehalose is added to prepare a freeze-dried powder preparation.

8. The method for preparing the targeted exosome-hydrogel formulation according to claim 7, characterized in that, The steps of the gradient cooling freeze-drying process include: Cool to -80℃ at a rate of 1℃ / min; Add a freeze-drying protectant at -80℃; After lyophilization and reconstitution, the resulting exosome particle size distribution (PDI) was <0.

15.

9. The use of a targeted exosome-hydrogel formulation as described in any one of claims 1-6 in the preparation of a medicament for treating osteoarthritis.

10. The application according to claim 9, characterized in that, The drug is administered via intra-articular injection, with a single injection lasting for at least 14 days, and can significantly reduce IL-1β levels in synovial fluid to below 78.4 pg / mL.

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