Knee bone joint targeting stem cell exosome composition as well as preparation method and application thereof

Abstract

The invention discloses a knee joint targeting stem cell exosome composition as well as a preparation method and application thereof, and relates to the technical field of biological medicines. The composition comprises a core-shell structure consisting of a stem cell exosome and a targeting stabilized functional macromolecule coating the surface of the stem cell exosome, the targeting stabilizing functional macromolecule is formed by connecting a biocompatible polysaccharide skeleton, a membrane anchoring hydrophobic group and a knee joint focus targeting ligand through covalent bonds. Through integrated molecular design, a stabilizing function and a targeting function are integrated on the same macromolecular carrier, and the macromolecular carrier is worn on the surface of the exosome through a self-assembly technology, so that the technical contradiction that stability and targeting cannot be achieved at the same time in an existing stem cell exosome technology is solved.

Description

Technical Field

[0001] The present invention relates to the field of biomedicine technology, and in particular to a knee joint-targeted stem cell exosome composition, a preparation method, and an application thereof. Background Art

[0002] In modern biomedicine, with the deepening advancement of regenerative medicine and targeted therapy, exosomes, nanoscale vesicles derived from specific cell types, have become a key vehicle for intercellular communication and are a key focus of research and development for novel therapeutic strategies. Mesenchymal stem cell-derived exosomes, rich in growth factors, cytokines, and nucleic acids, possess inherently low immunogenicity, and possess the ability to cross biological barriers. These exosomes offer unique advantages in promoting tissue repair and regulating the local inflammatory microenvironment. In the treatment of degenerative diseases such as knee osteoarthritis (KOA), exosomes can be administered intra-articularly, offering a promising new therapeutic approach for inhibiting chondrocyte apoptosis, promoting cartilage matrix synthesis, and alleviating synovial inflammation.

[0003] However, efficiently and stably translating the theoretical therapeutic potential of stem cell exosomes into clinically applicable formulations still faces a series of severe technical challenges. The primary obstacle is the inherent structural fragility of stem cell exosomes. Their phospholipid bilayer membrane is highly susceptible to temperature fluctuations, pH changes, osmotic pressure, and mechanical shear forces in vitro, leading to rupture, aggregation, or degradation, resulting in a rapid loss of biological activity and, consequently, therapeutic value.

[0004] To overcome the inherent instability of stem cell exosomes, existing research focuses on improving the storage and transportation stability of exosome preparations. For example, the technical solution disclosed in the patent application with publication number CN116897922A addresses this problem by constructing a physical protective layer. This solution utilizes polysaccharide biomacromolecules such as sodium alginate and pectin to form a network-like microscopic gel structure in a physiological environment such as phosphate buffer, gently encapsulating the exosomes. This strategy effectively slows the Brownian motion and mutual collisions of exosomes in the liquid state through steric hindrance and the binding of water molecules, forming a physical barrier to resist external environmental stress, thereby significantly delaying the disintegration of the exosome structure and the attenuation of biological activity within a specific time. For example, the technical solution disclosed in the patent application with publication number CN115968867A focuses on achieving long-term stable preservation of exosomes through a freeze-drying process. The core of this solution lies in the design and optimization of a freeze-drying protectant formula containing ingredients such as trehalose and mannitol. During the process of water sublimation, these protectant molecules can replace water molecules to form hydrogen bonds with the phospholipid heads on the surface of the exosome membrane, constructing an amorphous glassy matrix, thereby maintaining the integrity of its membrane structure and ensuring that its biological function can be restored after re-dissolution.

[0005] While the two aforementioned technical solutions can, to a certain extent, address the inherent instability of stem cell exosomes, with the increasing demand for precise treatment of localized diseases like knee osteoarthritis, we have come to realize that the technical approach of prioritizing physical stability as a design goal has, at a deeper level, created and solidified a new, more insidious technical contradiction: the inherent incompatibility and process conflict between achieving stability and subsequent functional modifications (especially targeted modification). This stems from the fundamental mechanism by which both the external coating layer formed by polysaccharide macromolecules and the vitrified matrix formed by lyoprotectants function to create a relatively "inert" and "enclosed" protective microenvironment for the fragile exosomes. While this protective shell effectively isolates the exosomes from external physical and chemical destructive factors, it also inevitably isolates the exosomes from the external environment, thereby shielding or occupying key protein sites on the exosome membrane. These protein sites are precisely the key "anchor points" for targeted molecular modification, such as the attachment of ligands targeting knee chondrocytes or synovial cells through chemical conjugation or physical adsorption. Therefore, after such stabilization treatment, the surface of exosomes becomes difficult to efficiently and controllably chemically functionalize. Forced modification not only results in low reaction efficiency but also risks destroying the hard-earned stabilized structure. This contradiction is further reflected in the inherent conflicts within the entire preparation process. If targeted modification is performed on "naked" exosomes before stabilization treatment, the targeting ligands introduced during the modification process may interfere with the effective interaction of subsequent stabilizers (such as polysaccharides or lyoprotectants) with the exosome surface, thereby weakening the stability of the final formulation. More critically, the modified functionalized exosomes remain exposed to the risk of instability before stabilization treatment. Conversely, if stabilization treatment is performed first before targeted modification, as mentioned above, the strong protective layer becomes an insurmountable obstacle to subsequent modification reactions. This creates a dilemma for the entire preparation process: stability and functionality are often difficult to achieve simultaneously within the existing technical framework. This "protective barrier" constructed to solve storage and transportation problems has inadvertently become a "technical barrier" to achieving precise delivery and efficient treatment, greatly limiting the potential of exosomes to achieve lesion enrichment and maximize therapeutic efficacy in the treatment of knee osteoarthritis.

[0006] Therefore, how to break through the limitations of the mutual constraints between stability construction and targeted functionalization in existing technologies has become a key challenge currently faced by technical personnel in this field and a technical problem that needs to be solved urgently. Summary of the Invention

[0007] The present invention provides a knee joint-targeted stem cell exosome composition and a preparation method thereof. The composition, through an integrated molecular design, integrates a stabilization module and a targeting module into a single functional macromolecule, and utilizes the non-covalent self-assembly of the macromolecule and the exosomes to simultaneously achieve efficient protection of the exosomes and precise targeting function, thereby overcoming the inherent process conflicts and performance bottlenecks between the physicochemical stability construction and targeted functional modification of stem cell exosome preparations in the prior art.

[0008] The technical solution adopted in the present invention is as follows: One of the objectives of the present invention is to provide a knee joint targeted stem cell exosome composition, which comprises a core-shell structure consisting of stem cell exosomes and targeted stabilizing functional macromolecules coated on the surface of the stem cell exosomes; the targeted stabilizing functional macromolecules are composed of a biocompatible polysaccharide skeleton, a membrane-anchored hydrophobic group and a knee joint lesion targeting ligand connected by covalent bonds, and the targeted stabilizing functional macromolecules are coated on the surface of the stem cell exosomes through membrane-anchored hydrophobic groups that can be inserted into the phospholipid bilayer membrane of the stem cell exosomes.

[0009] Through this structural design, the targeted stabilizing functional macromolecule forms a sophisticated molecular structure: a biocompatible polysaccharide backbone covalently linked to multiple hydrophobic groups acting as "anchors" and multiple targeting ligands acting as "navigators." When the targeted stabilizing functional macromolecule is mixed with stem cell exosomes in an aqueous environment, the long hydrophobic chains acting as hydrophobic groups spontaneously insert into the exosome's phospholipid bilayer membrane, like an anchor dropped to the seabed, firmly "anchoring" the entire macromolecule to the exosome surface. This process forms a core-shell functionalized exosome complex, with the stem cell exosomes as the "core" and the polymeric layer formed by the targeted stabilizing functional macromolecule as the "shell." This core-shell structure imparts unique synergistic advantages to the complex. First, the shell forms a highly hydrated physical barrier in the aqueous environment. Through steric hindrance and the bound water layer, it effectively prevents aggregation of exosomes and protects them from mechanical shear and osmotic pressure fluctuations, thereby significantly enhancing their stability in the liquid state. Secondly, the targeting ligand is located in the outermost layer of the entire core-shell structure, and its spatial conformation is unhindered, so it can be fully exposed and efficiently bind to the target in the joint cavity.

[0010] Furthermore, the stem cell exosomes are derived from mesenchymal stem cells, and the stem cell exosomes have a double-membrane vesicle structure, and their particle size distribution range is 50~150 nm.

[0011] Further, the biocompatible polysaccharide skeleton is hyaluronic acid or a pharmaceutically acceptable salt thereof, and the weight average molecular weight is 100-300 kDa, which can ensure the formation of an effective protective layer while having suitable tissue permeability.

[0012] Further, the membrane-anchoring hydrophobic group is phosphatidylethanolamine or a derivative thereof, and the mass ratio of the biocompatible polysaccharide skeleton to the membrane-anchoring hydrophobic group is 5-20:1.

[0013] Further, the phosphatidylethanolamine is 1,2-distearoyl-SN-glycero-3-phosphoethanolamine (DSPE), and in the embodiment in which hyaluronic acid is used as the biocompatible polysaccharide skeleton, the 1,2-distearoyl-SN-glycero-3-phosphoethanolamine is covalently connected to the carboxyl group on the hyaluronic acid skeleton via an amide bond through its amino group.

[0014] Further, the knee joint lesion targeting ligand is a type II collagen targeting polypeptide (CBP, sequence WYRGRL), and the mass ratio of the biocompatible polysaccharide skeleton to the knee joint lesion targeting ligand is 5-20:1. Type II collagen is the main component of articular cartilage matrix, and its structure is exposed when the cartilage is damaged. The selection of the type II collagen targeting polypeptide as the knee joint lesion targeting ligand can achieve precise anchoring and enrichment in the lesion area. In the embodiment in which hyaluronic acid is used as the biocompatible polysaccharide skeleton, the WYRGRL is covalently connected to the carboxyl group on the hyaluronic acid skeleton via an amide bond through its terminal amino group.

[0015] Further, the composition is a white or white-like lyophilized powder injection, and the components further include a cryoprotectant.

[0016] Further, the cryoprotectant is trehalose. In the embodiment in which hyaluronic acid is used as the biocompatible polysaccharide skeleton, during the freeze-drying process, the hyaluronic acid skeleton coated on the surface of the exosome acts as a macromolecular protective agent, and cooperates with trehalose as a small molecular protective agent to form a stable amorphous glassy matrix after water sublimation, thereby maintaining the integrity of the exosome membrane structure and ensuring that the exosome can completely recover the biological activity after reconstitution.

[0017] The second object of the present application is to provide a preparation method matched with the above-mentioned composition, which comprises the following steps: (1) Preparation of targeted stabilizing functional macromolecules: Dissolve the biocompatible polysaccharide skeleton in MES buffer solution, add N-(3-dimethylaminopropyl)-N'-ethylcarbodiimide hydrochloride (EDC) and N-hydroxysuccinimide (NHS) to perform carboxyl activation reaction, then add membrane-anchored hydrophobic groups and knee joint lesion targeting ligands step by step or simultaneously to perform coupling reaction. After the reaction, remove unreacted small molecules by ultrafiltration or dialysis, purify and freeze-dry to obtain pure targeted stabilizing functional macromolecule powder; (2) Preparation of functionalized exosome composition: The targeted stabilized functional macromolecule powder prepared in step (1) is dissolved in PBS buffer to obtain a clear solution, and the solution is mixed with high-purity stem cell exosomes, wherein the mass ratio of the targeted stabilized functional macromolecule to the stem cell exosomes is in the range of 5-10:1; the obtained mixture is incubated at 25-37°C for 0.5-2h, during which gentle shaking is performed to promote the membrane-anchored hydrophobic groups to fully insert into the exosome membrane, thereby completing the self-assembly process of the functionalized exosome composition and obtaining a functionalized exosome composition suspension; (3) Lyophilization: Add a cryoprotectant with a final concentration of 2-10% to the functionalized exosome composition suspension and gently mix. Sterile filter the final mixed solution and dispense it into vials. Place the vials in a freeze dryer to freeze and obtain a white or off-white lyophilized powder injection.

[0018] The third object of the present invention is to disclose the use of the composition in the preparation of an intra-articular injection medicament for preventing and / or treating knee osteoarthritis in mammals.

[0019] In summary, compared with the prior art, the present invention has the following advantages and beneficial effects: 1. This invention integrates stabilization and targeting functions into a single macromolecular carrier through integrated molecular design and "applies" it to the exosome surface via self-assembly technology, resolving the technical contradiction in existing stem cell exosome technologies, which often require both stability and targeting. This strategy fundamentally avoids the shielding effect of the stabilizer layer on the active sites on the exosome surface, as is common in existing technologies, and also circumvents interference of the targeting ligand on the stabilizer during the modification-before-stabilization process.

[0020] 2. The core assembly process of the present invention relies on a spontaneous process driven by hydrophobic interactions, which does not require harsh chemical reaction conditions, avoids damage to the biological activity of exosomes, and separates the complex chemical modification steps from the fragile biological vesicle preparation. After both are completed, gentle physical assembly is performed, which provides high process controllability and reproducibility.

[0021] 3. The coated shell layer of the present invention not only plays a steric stabilizing role in the liquid preparation, but also acts as a macromolecular skeleton during the freeze-drying process, forming a dense vitrified matrix with the small molecule cryoprotectant, which greatly protects the integrity of the exosome membrane structure, enabling it to be stored stably for a long time and maintain high biological activity after reconstitution.

[0022] 4. The composition provided by the present invention has a targeting ligand located in the outermost layer of its core-shell structure, which can mediate the efficient binding of the composition to type II collagen exposed at the cartilage damage site after injection into the joint cavity, thereby "anchoring" the exosomes loaded with therapeutic factors at the lesion site, prolonging their effective action time in the joint cavity, reducing the risk of rapid clearance by synovial fluid, and thereby improving local drug concentration and therapeutic efficiency.

[0023] 5. The composition provided by the present invention has stem cell exosomes as the core and hyaluronic acid modified with membrane-anchored hydrophobic groups and knee joint lesion-targeting ligands as the shell. After it is injected into the lesion site, the hyaluronic acid shell first relieves inflammation through lubrication, and then gradually degrades and releases exosomes to achieve long-term repair. The two form a synergistic effect in the time dimension. BRIEF DESCRIPTION OF THE DRAWINGS

[0024] Figure 1 Schematic diagram of the preparation process of the core-shell structure involved in Example 1. DETAILED DESCRIPTION

[0025] The present invention will be described in detail below in conjunction with specific embodiments and examples, and the advantages and various effects of the present invention will be more clearly presented. It should be understood by those skilled in the art that these specific embodiments and examples are for illustrating the present invention, rather than for limiting the present invention.

[0026] Throughout this specification, unless otherwise specified, the terms used herein should be understood as having the same meaning as commonly used in the art. Therefore, unless otherwise defined, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this invention belongs. In the event of any conflict, the present specification shall take precedence.

[0027] Unless otherwise specified, various raw materials, reagents, instruments and equipment used in the present invention can be purchased from the market or prepared by existing methods.

[0028] The present invention provides a knee joint-targeted stem cell exosome composition, which comprises a core-shell structure consisting of stem cell exosomes and a targeted stabilizing functional macromolecule coated on the surface of the stem cell exosomes; the targeted stabilizing functional macromolecule is composed of a biocompatible polysaccharide skeleton, a membrane-anchored hydrophobic group, and a knee joint lesion-targeting ligand connected by covalent bonds; the targeted stabilizing functional macromolecule is coated on the surface of the stem cell exosomes via a membrane-anchored hydrophobic group that can be inserted into the phospholipid bilayer membrane of the stem cell exosomes.

[0029] Among them, the stem cell exosomes used in the present invention are derived from mesenchymal stem cells and have a double-layer membrane vesicle structure (see Figure 1 The stem cell exosome structure in the present invention is purified by density gradient centrifugation. The stem cell exosomes used in the present invention are high-purity exosomes with a particle size distribution range of 50 to 150 nm, and the detection results of their surface marker proteins are CD9 positive, CD63 positive, CD81 positive, and calnexin negative.

[0030] In order to more specifically illustrate the technical effects of the present invention, specific examples and comparative examples are provided below for exosome suspensions produced in the same batch (with an average particle size of approximately 115 nm).

[0031] Example 1 This embodiment provides a lyophilized powder injection of a knee joint-targeted stem cell exosome composition, the preparation process of which includes three steps: preparation of a targeted stabilized functional macromolecule, preparation of a functionalized exosome composition (core-shell structure), and lyophilization. The process of preparing the targeted stabilized functional macromolecule and the functionalized exosome composition is as follows: Figure 1 As shown, during the preparation process, the biocompatible polysaccharide skeleton is hyaluronic acid (HA) with a weight-average molecular weight of 200 kDa; the membrane-anchoring hydrophobic group is 1,2-distearoyl-SN-glycero-3-phosphoethanolamine (DSPE), and the knee joint lesion targeting ligand is type II collagen targeting polypeptide (CBP, sequence is WYRGRL).

[0032] The specific preparation process and related parameters are: The first part is the preparation of the targeted stabilizing functional macromolecule, namely CBP-HA(200k)-DSPE. The process is divided into two steps.

[0033] First, the HA-DSPE intermediate was synthesized: 300 mg of sodium hyaluronate was accurately weighed and dissolved in 100 mL of 0.1 M MES buffer (pH 5.5). Stirring was continued until complete dissolution formed a clear solution. 44 mg of EDC and 26 mg of NHS were added to this solution and stirred at room temperature for 2 hours to fully activate the carboxyl groups on the hyaluronic acid. Separately, 30 mg of DSPE-PEG2000-NH2 was accurately weighed and dissolved in 5 mL of MES buffer. This solution was then slowly added dropwise to the activated hyaluronic acid solution. The pH of the reaction system was adjusted to 7.0-7.5 with dilute NaOH solution, and the reaction was stirred at room temperature for 24 hours. After the reaction, the resulting reaction mixture was transferred to a 100 kDa dialysis bag and dialyzed against deionized water for a total of 72 hours, with fresh deionized water replaced every 8 hours. After dialysis, the liquid in the dialysis bag was collected and freeze-dried using a freeze dryer, yielding a white, flocculent solid, the HA-DSPE intermediate. Proton nuclear magnetic resonance spectroscopy confirmed that DSPE had been successfully grafted onto the hyaluronic acid backbone, with a grafting efficiency (the molar ratio of DSPE molecules to HA disaccharide repeating units) of approximately 2%.

[0034] Next, the final product, CBP-HA-DSPE, was synthesized: 150 mg of the HA-DSPE intermediate prepared above was accurately weighed and dissolved in 50 mL of 0.1 M MES buffer (pH 5.5). 20 mg of EDC and 12 mg of NHS were added to the solution, and the mixture was activated at room temperature for 2 hours with stirring. Separately, 15 mg of the type II collagen-targeting peptide CBP (purchased from a distributor) was accurately weighed and dissolved in 2 mL of MES buffer. This peptide solution was then added to the activated HA-DSPE solution. The pH of the reaction system was adjusted to 7.0-7.5, and the reaction was continued at room temperature for 24 hours. After completion of the reaction, the product was purified using the same method as the previous step, using a dialysis bag with a molecular weight cutoff of 100 kDa. The purified solution was then freeze-dried to obtain the final, targeted, stabilized functional macromolecule, CBP-HA-DSPE. Elemental analysis determined the nitrogen content, and the calculated CBP grafting efficiency was approximately 1.5%.

[0035] Second part, self-assembly of functionalized exosome complex (CBP-HA(200k)-DSPE / Exo). CBP-HA-DSPE lyophilized powder prepared in the first part was dissolved in PBS buffer to prepare a solution with a concentration of 10 mg / mL. 5 mL of the solution (containing 50 mg of functional macromolecule) was mixed with 4 mL of exosome suspension (with a protein concentration of 2.5 mg / mL, containing 10 mg of exosome protein) in a sterile centrifuge tube, so that the mass ratio of CBP-HA-DSPE to total protein of exosome was exactly 5:1. The mixture was incubated in a constant temperature shaker at 37°C for 1 hour at a speed of 100 rpm to complete the self-assembly process.

[0036] Third part, preparation of lyophilized powder injection. To the above prepared complex suspension, trehalose powder was added, and the mixture was gently inverted to mix well to completely dissolve the trehalose, and the final concentration of trehalose was 5% (w / v). The final solution was filtered through a 0.22 μm sterile filter, and then accurately dispensed into 2 mL sterile vials at a volume of 1 mL per vial. The dispensed vials were half-tightened, and then quickly transferred to a freeze-drying machine. The freeze-drying program was set and executed: first pre-freeze at a plate temperature of -40°C for 3 hours; then the vacuum degree was pulled to 10 Pa, and the plate temperature was maintained at -25°C for primary drying for 24 hours; then the plate temperature was linearly increased to 25°C within 2 hours, and the vacuum degree was further reduced to 5 Pa for secondary drying for 12 hours. After the program ended, the vacuum was released under nitrogen protection, and the tightness was completed to obtain white, loose and porous lyophilized powder injection, which was the final product composition 1 of the present example.

[0037] Example 2 This example aims to investigate the effect of the molecular weight of the hyaluronic acid backbone on the properties of the composition. Four experiments were conducted, including two experimental groups and two control groups, according to the preparation process that was essentially the same as that in Example 1. The four experiments differed from Example 1 in that, based on Example 1, only the weight-average molecular weight of the first portion of sodium hyaluronate was replaced with 50 kDa, 100 kDa, 300 kDa, and 400 kDa, respectively. The molecular weight cutoffs of the dialysis bags during dialysis were 30 kDa, 80 kDa, 100 kDa, 100 kDa, and 100 kDa, respectively. kDa, CBP-HA(50k)-DSPE, CBP-HA(100k)-DSPE, CBP-HA(300k)-DSPE and CBP-HA(400k)-DSPE were prepared respectively, and the types and amounts of all other raw materials, reaction conditions and purification methods were consistent with those in Example 1, and the corresponding lyophilized powder injections were finally obtained, which were marked as composition 2 (CBP-HA(50k)-DSPE / Exo), composition 3 (CBP-HA(100k)-DSPE / Exo), composition 4 (CBP-HA(300k)-DSPE / Exo) and composition 5 (CBP-HA(400k)-DSPE / Exo).

[0038] Example 3 This example investigates the effect of targeting ligand grafting density on the functionality of the composition. The preparation process was essentially identical to that of Example 1. Two sets of experiments were designed. The differences between these two sets of experiments and Example 1 were as follows: in the first step, preparing the targeted, stabilized functional macromolecule, the CBP grafting reaction was performed using 10 mg and 30 mg of the type II collagen targeting peptide (CBP), respectively. All other raw material types and amounts, reaction conditions, and purification methods remained consistent with those of Example 1. Elemental analysis revealed that the CBP grafting ratios on the prepared CBP-HA-DSPE were approximately 1.0% and 3.0%, respectively. The resulting lyophilized powder injections were Composition 6 (CBP grafting ratio approximately 1.0%) and Composition 7 (CBP grafting ratio approximately 2.8%).

[0039] Example 4 This example investigates the effect of hydrophobic group grafting density on the functionality of the composition. The preparation process was essentially identical to that of Example 1. Two sets of experiments were designed. The differences between these two sets of experiments and Example 1 were as follows: in the first step, preparing the targeted, stabilized functional macromolecule, the DSPE-PEG2000-NH2 loading was changed to 20 mg and 40 mg, respectively, during the hydrophobic group grafting reaction. All other raw material types and amounts, reaction conditions, and purification methods remained consistent with those of Example 1. Elemental analysis revealed that the DSPE grafting ratios on the prepared CBP-HA-DSPE were approximately 1.1% and 2.5%, respectively. The resulting lyophilized powders for injection were Composition 8 (DSPE grafting ratio approximately 1.1%) and Composition 9 (DSPE grafting ratio approximately 2.5%), demonstrating that the relationship between the hydrophobic group grafting ratio and the DSPE-PEG2000-NH2 loading is not linear.

[0040] Example 5 This example investigated the effect of the mass ratio of a targeted, stabilized functional macromolecule to stem cell exosomes on the composition's function. The preparation process was essentially identical to that of Example 1. Two sets of experiments were designed. These differed from Example 1 in that, during the second self-assembly step (CBP-HA(200k)-DSPE / Exo), the mass ratios of CBP-HA-DSPE to total exosome protein were changed to 3:1 and 10:1, respectively. The types and amounts of all other raw materials, reaction conditions, and purification methods remained consistent with those of Example 1. The resulting lyophilized powder injections were Composition 10 (CBP-HA-DSPE to total exosome protein mass ratio of 3:1) and Composition 11 (CBP-HA-DSPE to total exosome protein mass ratio of 10:1).

[0041] Comparative Example 1 To compare the effects, this comparative example prepared an exosome preparation that was not coated with the functional macromolecule described herein. Specifically, an equivalent amount of exosome suspension (4 mL, containing 10 mg of exosome protein) as in Example 1 was directly added with trehalose powder to a final concentration of 5% (w / v). After gentle mixing, the preparation was sterile filtered, packaged, and freeze-dried using the same procedures as in Example 1. This preparation, containing only exosomes and trehalose as a cryoprotectant, was designated Composition 12.

[0042] Comparative Example 2 This comparative example demonstrates a stabilization-first, then targeted modification approach. First, an HA-DSPE intermediate without a targeting ligand was prepared according to the method of Example 1. This intermediate was then self-assembled with exosomes to form a stabilized HA-DSPE / Exo complex, following the self-assembly method of Example 1. Next, a "post-modification" chemical reaction was attempted in this stabilized complex suspension, involving the addition of an EDC / NHS activator and a CBP peptide. Following the reaction, unreacted CBP and small molecules were removed by ultrafiltration. Trehalose was then added to the purified suspension to a concentration of 5% (w / v). Finally, freeze-drying was performed as described in Example 1 to yield Composition 13.

[0043] Comparative Example 3 This comparative example experimented with a technical approach that involved targeted modification followed by stabilization. Specifically, EDC / NHS was added to an equivalent amount of purified exosome suspension as in Example 1 to activate the exposed carboxyl groups on the exosome's own membrane proteins. CBP polypeptide was then added for coupling. After the reaction, the mixture was washed several times by ultracentrifugation to remove unreacted CBP and small molecules. Next, the fully functional macromolecule CBP-HA-DSPE prepared in Example 1 was added to this directly chemically modified CBP-Exo, along with trehalose. Mixing and stabilization were attempted, and finally, freeze-drying was performed in the same manner as in Example 1 to obtain Composition 14.

[0044] Experimental Example 1 In this experimental example, the physicochemical properties, in vitro stability and targeting of the freeze-dried powder injections prepared in the above examples and comparative examples were evaluated. Three parallel experiments were set up for each evaluation experiment, and the experimental results were averaged.

[0045] First, the physicochemical properties were characterized. Each group of lyophilized powder injections prepared in Examples 1-5 and Comparative Examples 1-3 was reconstituted with an equal volume of PBS and gently shaken until completely dissolved. Dynamic light scattering (DLS) was used to measure the hydrated particle size and polydispersity index (PDI) of each group of samples after reconstitution. A smaller PDI value indicates a more uniform particle size distribution. The results are summarized in Table 1.

[0046] Table 1 Characterization results of physicochemical properties of each group of compositions

[0047] The data in Table 1 show that the average hydrated particle size of the nanoparticle suspensions formed after reconstitution of the aforementioned lyophilized powder injections coated with a hyaluronic acid hydration layer increased compared to that of naked exosomes (approximately 115 nm), consistent with the expected coating of the hyaluronic acid hydration layer. Furthermore, the PDI values ​​of Compositions 1, 2-4, 6-9, 11, and 13 were all significantly below 0.3, indicating that the corresponding CBP-HA-DSPE or HA-DSPE coating layer effectively prevented irreversible aggregation of exosomes during the lyophilization and reconstitution processes. Furthermore, the molecular weight of the hyaluronic acid backbone and the amount of coating layer used significantly influenced the PDI value after reconstitution. A hyaluronic acid backbone with an excessively large molecular weight may impair self-assembly and thus the coating effect due to steric hindrance. A hyaluronic acid backbone with an excessively large molecular weight may impair self-assembly and, consequently, the coating effect. A CBP-HA-DSPE coating with an inadequate amount may not completely coat the stem cell exosomes, leading to some aggregation. The targeting ligand grafting density and the hydrophobic group grafting density had relatively little effect on the PDI value after reconstitution. In contrast, the formulation in Comparative Example 1 (Composition 12) exhibited severe aggregation after reconstitution, resulting in a large average particle size and an extremely high PDI value, indicating that small molecule cryoprotectants alone are insufficient to fully protect exosomes. While the particle size of Comparative Example 2 (Composition 13) was controllable, subsequent targeted function testing yielded poor results. Comparative Example 3 (Composition 14), due to direct chemical modification of the exosome surface, compromised the stability of the exosome membrane. This resulted in partial aggregation prior to subsequent stabilization treatment, resulting in uneven particle size and poor stability after reconstitution.

[0048] Next, we conducted an accelerated stability test. Each group of freeze-dried powder injection samples prepared in Examples 1-4 and Comparative Examples 1-3 were placed in a 25°C incubator for a three-month accelerated stability study. Samples were taken at 0, 1, 2, and 3 months, and the changes in PDI values ​​were measured after reconstitution with PBS. The results are shown in Table 2.

[0049] Table 2 PDI values ​​of the compositions in each group at 0, 1, 2, and 3 months

[0050] As shown in Table 2, even after being stored at 25°C for three months under accelerated conditions, the PDI values ​​of Compositions 1, 3-4, 6-9, 11, and 13 only increased slightly, demonstrating excellent long-term storage stability. Composition 2, while exhibiting a PDI value below 0.3, exhibited relatively poor stability, and the remaining compositions exhibited suboptimal stability. Composition 10 may have been affected by the inability of CBP-HA-DSPE to fully encapsulate the exosomes, affecting its stability. The results for Comparative Example 3 (Composition 14) were even closer to those of Comparative Example 1 (Composition 12), indicating that the PDI values ​​of both unprotected exosome preparations and those whose structures were destroyed before protection increased rapidly over time, indicating that the nanoparticles underwent continuous degradation and aggregation.

[0051] Finally, the targeting evaluation was performed. In order to verify the targeting function of the composition of the present invention, an in vitro binding experiment was performed. The bottom of the wells of a 96-well enzyme-labeled plate was coated with a type II collagen targeting peptide solution (10 μg / mL) at 4°C overnight. The next day, non-specific binding sites were blocked with a blocking solution containing bovine serum albumin. The preparations of Examples 1 to 5 and Comparative Examples 1 to 3 were fluorescently labeled with the near-infrared fluorescent dye DiR, and after redissolution, the concentration of exosome protein was adjusted to the same, added to the coated wells, and incubated at 37°C for 2 hours. After the incubation, the cells were washed several times with a washing solution to remove unbound samples, and then the relative fluorescence intensity (RFU) of the samples retained in each well due to binding was detected using a fluorescence microplate reader. The results are shown in Table 3.

[0052] Table 3 Relative fluorescence intensity measurement results of each group of compositions

[0053] The experimental results in Table 3 strongly demonstrate the superior targeting properties of the compositions of the present invention. Compositions 1, 2-4, 6-9, and 11 all exhibited strong binding to type II collagen-coated wells, with Composition 7, which had a higher density of targeting peptides, exhibiting even stronger binding. The binding fluorescence signals of Comparative Examples 1 and 2 were very weak, close to background values, indicating that they lacked targeting ability. While Comparative Example 3 exhibited some targeted binding, its binding efficiency was significantly lower than that of the examples of the present invention. This may be due to the damage to the conformation and activity of the exosome surface proteins caused by direct chemical modification, as well as its poor physical stability, which limited its effectiveness.

[0054] Experimental Example 2 In this experimental example, the in vivo therapeutic effect of the lyophilized powder injection prepared by the present invention was evaluated in a rat knee osteoarthritis model. Compositions 1 and 7 with better stability and targeting effects in Experimental Example 1 were taken as the experimental group, normal saline was used as the blank control group, and compositions 12 to 14 and the targeted, stabilized functional macromolecule CBP-HA-DSPE and stem cell exosome mixture prepared in Example 1 were used as the control group. As a non-core-shell self-assembly control group, CBP-HA-DSPE and stem cell exosomes were mixed at a low temperature of 0 to 5°C and used in the experiment. The mass ratio of CBP-HA-DSPE to stem cell exosomes was 5:1.

[0055] The specific experimental procedure for this example was as follows: Healthy male Sprague-Dawley rats were surgically induced into the right hind knee joint via transection of the anterior cruciate ligament and partial resection of the medial meniscus (Hulth method). After four weeks of postoperative maintenance, the rats were randomly divided into seven treatment groups, each with eight rats: (A) saline control group; (B) Composition 1 experimental group; (C) Composition 7 experimental group; (D) Composition 12 experimental group; (E) Composition 13 experimental group; (F) Composition 14 experimental group; and (G) non-core-shell self-assembly control group. Each group received drug via intra-articular injection once weekly for four consecutive weeks, with 20 μL of the corresponding formulation's reconstituted solution (containing an equal amount of exosome protein). Following the final dose, the rats were maintained for an additional four weeks. After the final dose, all rats were sacrificed, and the right knee joints were harvested for subsequent histological analysis. After decalcification and paraffin embedding, sagittal sections of the knee joint specimens were prepared, and the degree of cartilage damage was semiquantitatively scored according to the Osteoarthritis Research Society International (OARSI) scoring system. The results are shown in Table 4. Higher scores indicate more severe damage.

[0056] Table 4 OARSI score results of each experimental group

[0057] The experimental results in Table 4 demonstrate that the targeted composition of the present invention has excellent in vivo therapeutic effects. The saline group, serving as the control group, had the highest OARSI score, demonstrating the successful modeling of this experimental example. Compositions with poor in vitro stability and targeting had relatively high OARSI scores, while compositions with good in vitro stability and targeting had low OARSI scores. This result strongly demonstrates that the knee joint-targeted stem cell-exosome composition provided by the present invention, through its sophisticated core-shell structure, not only ensures the stability of the exosomes in vivo, but also, through the guidance of the targeting ligand, can efficiently deliver and anchor the exosomes with therapeutic potential in areas of cartilage lesions, thereby exerting a powerful cartilage protection and repair effect. Its therapeutic effect is far superior to that of non-targeted stable formulations and unstable exosome formulations. The therapeutic effect of the non-core-shell self-assembly control group was only better than that of Comparative Example 1 and worse than that of Comparative Example 2 / 3, indicating that the in vivo therapeutic effect of the targeted composition is not solely due to the component design but also related to the specific core-shell structure design.

[0058] Finally, it should be noted that the terms "comprises," "comprising," or any other variations thereof are intended to encompass non-exclusive inclusion, such that a process, method, article, or apparatus comprising a list of elements includes not only those elements but also other elements not expressly listed, or elements inherent to such process, method, article, or apparatus. Although preferred embodiments of the present invention have been described, additional changes and modifications may be made to these embodiments by those skilled in the art once the basic inventive concepts are known. Therefore, the appended claims are intended to be interpreted as including the preferred embodiments and all changes and modifications that fall within the scope of the invention.

[0059] The above-described embodiments merely represent specific implementation methods of the present application. While the descriptions are relatively specific and detailed, they should not be construed as limiting the scope of protection of the present application. It should be noted that a person skilled in the art would be able to make numerous variations and improvements without departing from the technical concept of the present application, and all such variations and improvements fall within the scope of protection of the present application.

Claims

1. A knee joint-targeted stem cell exosome composition, characterized in that: The composition comprises a core-shell structure consisting of stem cell exosomes and targeted stabilizing functional macromolecules coated on the surface of the stem cell exosomes; the targeted stabilizing functional macromolecules are composed of a biocompatible polysaccharide skeleton, a membrane-anchored hydrophobic group and a knee joint lesion-targeting ligand connected by covalent bonds; the targeted stabilizing functional macromolecules are coated on the surface of the stem cell exosomes through membrane-anchored hydrophobic groups that can be inserted into the phospholipid bimolecular membrane of the stem cell exosomes.

2. The composition according to claim 1, wherein The stem cell exosomes are derived from mesenchymal stem cells, have a double-membrane vesicle structure, and have a particle size distribution range of 50-150 nm.

3. The composition according to claim 1, wherein The biocompatible polysaccharide skeleton is hyaluronic acid or a pharmaceutically acceptable salt thereof, and has a weight average molecular weight of 100-300 kDa.

4. The composition according to claim 1, wherein The membrane-anchoring hydrophobic group is phosphatidylethanolamine or a derivative thereof, and the mass ratio of the biocompatible polysaccharide skeleton to the membrane-anchoring hydrophobic group is 5-20:

1.

5. The composition according to claim 4, wherein The phosphatidylethanolamine is 1,2-distearoyl-SN-glycerol-3-phosphatidylethanolamine.

6. The composition according to claim 1, wherein The knee joint lesion targeting ligand is a type II collagen targeting polypeptide, and the mass ratio of the biocompatible polysaccharide skeleton to the knee joint lesion targeting ligand is 5-20:

1.

7. The composition according to claim 1, wherein The composition is a white or off-white freeze-dried powder injection, and its components also include a cryoprotectant.

8. The composition according to claim 7, wherein The cryoprotectant is trehalose.

9. The method for preparing the composition according to any one of claims 7 or 8, wherein: The steps include: (1) Preparation of targeted stabilizing functional macromolecules: dissolve the biocompatible polysaccharide skeleton in MES buffer solution, add N-(3-dimethylaminopropyl)-N'-ethylcarbodiimide hydrochloride and N-hydroxysuccinimide to carry out carboxyl activation reaction, then add membrane anchoring hydrophobic groups and knee joint lesion targeting ligands step by step or simultaneously to carry out coupling reaction. After the reaction is completed, remove unreacted small molecules by ultrafiltration or dialysis, purify and freeze-dry to obtain pure targeted stabilizing functional macromolecule powder; (2) Preparation of functionalized exosome composition: The targeted stabilized functional macromolecule powder prepared in step (1) is dissolved in PBS buffer to obtain a clear solution, and the solution is mixed with high-purity stem cell exosomes, wherein the mass ratio of the targeted stabilized functional macromolecule to the stem cell exosomes is in the range of 5-10:1; the obtained mixture is incubated at 25-37°C for 0.5-2h, during which gentle shaking is performed to promote the membrane-anchored hydrophobic groups to fully insert into the exosome membrane, thereby completing the self-assembly process of the functionalized exosome composition and obtaining a functionalized exosome composition suspension; (3) Lyophilization: Add a cryoprotectant with a final concentration of 2-10% to the functionalized exosome composition suspension and gently mix. Sterile filter the final mixed solution and dispense it into vials. Place the vials in a freeze dryer to freeze and obtain a white or off-white lyophilized powder injection.

10. Use of the composition according to any one of claims 1 to 8 in the preparation of a medicament for intra-articular injection for preventing and / or treating knee osteoarthritis in mammals.

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