A dual-exosome time-release hydrogel formulation, its preparation method and application in treating osteoarthritis

Abstract

The application discloses a kind of double exosome time sequence release hydrogel preparation and its preparation method and application.The preparation includes: (1) derived from inflammatory factor (TNF-alpha+IL-1 beta) and hypoxia pretreatment mesenchymal stem cell exosome (anti-inflammatory type);(2) derived from the chondrocyte-like cell exosome (repair type) differentiated by TGF-beta3 start and enhanced by GDF-5;(3) time sequence release carrier constituted by fast and slow degradation hydrogel.The application realizes early release anti-inflammatory exosome to inhibit synovial inflammation by once injection, removes senile chondrocyte, releases repair exosome to promote cartilage matrix regeneration in later period, and is synergistically treated osteoarthritis.The application avoids the risk of gene modified living cell reinfusion, has the advantages of simple preparation, high safety, remarkable curative effect and the like, and has good clinical conversion prospect.

Description

[Background Technology]

[0001] Osteoarthritis (OA) is an age-related disease characterized by articular cartilage degeneration, synovial inflammation, and subchondral bone remodeling. Existing therapies (such as NSAIDs and hyaluronic acid injections) only relieve symptoms and cannot reverse cartilage damage. Cell therapies (such as MSC injections) have issues such as heterogeneity, tumorigenesis risk, and immune rejection. Exosomes, as cell-free therapeutic carriers, have advantages such as low immunogenicity and high stability; however, existing single-exosome strategies struggle to simultaneously achieve the synergistic effects of early anti-inflammatory effects and late-stage cartilage repair. Furthermore, ordinary hydrogels cannot achieve programmed release of different exosomes. Therefore, developing a safe and controllable exosome delivery system that can mimic the timing requirements of cartilage repair has significant clinical value. [Summary of the Invention] Purpose of the invention

[0002] A dual-exosome sequential release hydrogel formulation is provided, which releases anti-inflammatory exosomes and repair-promoting exosomes sequentially through a single intra-articular injection, achieving synergistic treatment of OA. Technical solution

[0003] A dual exosome time-release hydrogel formulation, characterized in that it comprises: First exosome (anti-inflammatory type): derived from mesenchymal stem cells (MSCs) pretreated with a combination of inflammatory factors and / or physical hypoxia. Second exosome (repair type): derived from chondrocytes that have undergone chondrogenic induction and GDF-5 functional enhancement; Hydrogel carrier: containing at least two injectable hydrogel materials with different degradation rates, wherein the first exosome is encapsulated in a fast-degrading phase and the second exosome is encapsulated in a slow-degrading phase, or adopts a core-shell structure (fast-degrading outer layer + slow-degrading inner core).

[0004] Specific technical features 1. Preparation of the first exosome Seed cells: MSCs derived from human umbilical cord, fat, or bone marrow.

[0005] Pretreatment regimen: TNF-α + IL-1β combined stimulation at a concentration of 1-50 ng / mL each for 12-48 hours; alternatively, hypoxic culture (1-5% O2) for 24-72 hours can be combined.

[0006] Exosome extraction: ultracentrifugation or tangential flow filtration, particle size 30-150 nm, positive for surface markers CD9 / CD63 / CD81.

[0007] Functional characteristics: High expression of miR-146a, TSG-6, and IL-10, and low expression of pro-inflammatory factors.

[0008] 2. Preparation of the second exosome Step 1: MSCs are cultured in a chondrogenic induction solution (containing TGF-β3 5-20 ng / mL, BMP-2 10-50 ng / mL, dexamethasone, proline, etc.) for 7-14 days to form chondrogenic precursor cells.

[0009] Step 2: Add GDF-5 10-50 ng / mL to the above culture system and continue culturing for 7-14 days to obtain mature chondrocytes.

[0010] Exosome extraction: Same as above.

[0011] Functional characteristics: High expression of miR-140, COL2A1, ACAN, and SOX9.

[0012] 3. Time-release hydrogel Rapidly degradable phase materials: gelatin-hydroxyphenylpropionic acid (Gel-HPA) or thiolated hyaluronic acid (HA-SH), degradation time 3-7 days.

[0013] Slow-degrading phase materials: calcium alginate, polyethylene glycol diacrylate (PEGDA) or methacrylamide gelatin (GelMA), with a degradation time of 14-28 days.

[0014] Formulation: Physically mixed and then injected (biphasic gel) or pre-formed into a core-shell microsphere suspension (core: slow degradation + second exosome, shell: fast degradation + first exosome).

[0015] After injection: the first exosome releases early to inhibit synovitis and clear senescent chondrocytes; the second exosome releases late to promote cartilage matrix regeneration.

[0016] Preferred Implementation First exosome preparation: Pretreatment with TNF-α 20 ng / mL + IL-1β 20 ng / mL for 24 hours, followed by culture under hypoxia (2% O2).

[0017] Second exosome preparation: TGF-β3 10 ng / mL + BMP-2 20 ng / mL to initiate differentiation for 10 days, then add GDF-5 25 ng / mL to continue induction for 10 days.

[0018] Hydrogel: The fast-degrading phase is 1.5% Gel-HPA, and the slow-degrading phase is 2% calcium alginate + 0.5% GelMA; the two exosomes are each at 10¹ 0 Particle / mL concentration encapsulation. Beneficial effects

[0019] Synergistic effect: Dual exosomes target inflammation and regeneration respectively, overcoming the limitations of single-factor efficacy.

[0020] Timing matching: The hydrogel degradation rate is consistent with the pathological repair process (anti-inflammatory → repair), reducing the number of injections and improving patient compliance.

[0021] Safe and non-genetically modified: All exosomes are derived from pretreated or directed-differentiated cells, with no viral vectors and no risk of genetic modification.

[0022] Anti-aging mechanism: By downregulating p16INK4a in senescent chondrocytes through miRNAs and proteins carried by exosomes, the SIRT1 pathway is activated, thereby intervening in the progression of OA from the root.

Detailed Implementation Methods

[0023] Human umbilical cord MSCs were cultured until 80% fusion, then replaced with DMEM / F12 containing 20 ng / mL TNF-α and 20 ng / mL IL-1β, and treated in a 2% O2 hypoxic incubator for 24 hours.

[0024] Collect the supernatant, remove cell debris by 300g×10min, 2000g×20min, and 10000g×30min, and obtain exosome precipitate by ultracentrifugation at 120000g×70min, then resuspend in PBS.

[0025] NTA particle size analysis: average particle size 85 nm; Western blot: CD63 and TSG-6 positive; qPCR: miR-146a increased 8-fold compared with untreated MSC-Exo. Example 2: Preparation of the second exosome

[0026] MSCs were cultured in microspheres and induced for 10 days using chondrogenic induction solution (DMEM high glucose + ITS + 10 ng / mL TGF-β3 + 20 ng / mL BMP-2 + 0.1 μM dexamethasone + 50 μg / mL proline).

[0027] Replace with the same basal solution containing 25 ng / mL GDF-5 and continue induction for 10 days.

[0028] Collect the supernatant and extract exosomes using the same method. ELISA detection: COL2A1 content was 6 times that of the unstimulated group; qPCR: miR-140 increased 12-fold. Example 3: Construction of time-series hydrogels and animal experiments

[0029] Rapidly degradable gel: 1.5% Gel-HPA dissolved in PBS, with the first exosome added (1×10¹). 0(particles / mL) and horseradish peroxidase / H2O2 crosslinking.

[0030] Slow-degrading gel: 2% sodium alginate + 0.5% GelMA were mixed with second exosomes and core-shell microspheres (core diameter 50 μm, shell thickness 10 μm) were prepared by microfluidic method and then cross-linked with Ca²⁺.

[0031] Animal model: An OA model was established in 8-week-old SD rats by ACLT in the right knee. After 4 weeks, the rats were divided into groups (n=10): PBS, single anti-inflammatory exosome gel, single repair exosome gel, dual exosome mixed gel (without time sequence), and dual exosome time sequence gel (this invention).

[0032] Injection volume 50 μL, containing a total exosome count of 2 × 10¹ 0 Granules. The animals were sacrificed 8 weeks later, and the knee joint sections were stained (Safranin O-Fix Green, Toluidine Blue, COL2A1 immunohistochemistry).

[0033] Results: The OARSI score of the time-series gel group (2.1±0.5) was significantly lower than that of the dual exosome mixed group (4.5±0.8) and the PBS group (6.8±0.9); immunohistochemistry showed that COL2A1 recovered to near normal levels; TUNEL staining showed that the number of senescent chondrocytes (p16INK4a⁺) decreased by 70%. Example 4: Safety Evaluation

[0034] IL-6 and TNF-α levels in rat synovial fluid were measured at 1, 3, and 7 days after intra-articular injection, and no increase in acute inflammation was observed.

[0035] HE staining of major organs showed no abnormalities.

[0036] Blood biochemistry (ALT, AST, Cr, BUN) is normal.

Claims

1. A dual exosome time-release hydrogel formulation, characterized in that, Include: The first exosome is derived from mesenchymal stem cells pretreated with inflammatory factors and / or hypoxia; The second exosome is derived from chondrocytes that have undergone chondrogenic induction and GDF-5 treatment; The hydrogel carrier is composed of at least two biomaterials with different degradation rates, wherein the first exosome is loaded in the fast degradation phase and the second exosome is loaded in the slow degradation phase.

2. The formulation according to claim 1, characterized in that, The inflammatory factors are a combination of TNF-α and IL-1β, each at a concentration of 1-50 ng / mL, and the pretreatment time is 12-48 hours.

3. The formulation according to claim 1, characterized in that, The hypoxic pretreatment involves incubation with 1-5% O2 for 24-72 hours.

4. The formulation according to claim 1, characterized in that, In the preparation of the second exosome, chondrogenic induction was performed using TGF-β3 and / or BMP-2, and GDF-5 treatment was performed at a concentration of 10-50 ng / mL.

5. The formulation according to claim 1, characterized in that, The rapid degradation phase of the hydrogel has a degradation time of 3-7 days, and the slow degradation phase has a degradation time of 14-28 days.

6. The formulation according to claim 1, characterized in that, The hydrogel is a core-shell structured microsphere or a biphase physical hybrid gel.

7. The formulation according to claim 1, characterized in that, The formulation is used for a single intra-articular injection to treat osteoarthritis.

8. A method for preparing the formulation according to any one of claims 1-7, comprising: Step a: Pre-treat MSCs with inflammatory factors and / or hypoxia, collect the supernatant and extract the first exosome; Step b: MSCs were treated sequentially with chondrogenesis induction solution and GDF-5 to differentiate into chondrocytes, and the supernatant was collected to extract the second exosomes; Step c: Mix the first exosome with the fast-degrading hydrogel precursor, and mix the second exosome with the slow-degrading hydrogel precursor to form an injectable formulation.

9. The method according to claim 8, characterized in that, In step c, the combination method is physical blending or preparation of core-shell microspheres.

10. Use of the formulation according to any one of claims 1-7 in the preparation of a medicament for treating osteoarthritis.