A knee osteoarthritic targeting stem cell exosome composition, and a preparation method and application thereof

Abstract

The application discloses a kind of knee joint targeting stem cell exosome compositions and preparation method and application thereof, it is related to biological medicine technical field.The composition includes the core-shell structure that is made of stem cell exosome and the targeting stable functional macromolecule that is coated on the surface of stem cell exosome;The targeting stable functional macromolecule is made of biocompatible polysaccharide skeleton, membrane anchoring hydrophobic group and knee joint lesion targeting ligand by covalent bond connection composition.The application is integrated in the same macromolecule carrier by integrated molecular design, and by self-assembly technology, it is " dressed " on the surface of exosome, solves the technical contradiction that stability and targeting cannot be obtained in existing stem cell exosome technology.

Description

TECHNICAL FIELD

[0001] The present application relates to the technical field of biological medicine, in particular to a knee joint targeting stem cell exosome composition and a preparation method and application thereof. BACKGROUND

[0002] In the field of modern biological medicine, with the continuous deepening of the concept of regenerative medicine and targeted therapy, nanoscale vesicles derived from specific cell types, exosomes, as key carriers for intercellular information transmission, have gradually become the focus of research and development of new treatment strategies. Among them, mesenchymal stem cell-derived exosomes, due to their rich growth factors, cytokines and nucleic acid substances, and their natural low immunogenicity and ability to cross biological barriers, have unique advantages in promoting tissue repair and regulating local inflammatory microenvironment, especially in the treatment of degenerative diseases such as knee osteoarthritis (KOA), stem cell exosomes can be administered intra-articularly to provide a promising new treatment approach for inhibiting chondrocyte apoptosis, promoting cartilage matrix synthesis, and relieving synovial inflammation.

[0003] However, the efficient and stable transformation of the theoretical therapeutic potential of stem cell exosomes into a clinical application preparation still faces a series of severe technical challenges. Among them, the fragility of the structure of stem cell exosomes is the primary obstacle. The membrane structure composed of phospholipid bilayers is extremely susceptible to temperature fluctuations, pH changes, osmotic pressure, and mechanical shear forces in the in vitro environment, and is easily ruptured, aggregated or degraded, resulting in rapid loss of biological activity and thus loss of therapeutic value.

[0004] In order to overcome the inherent instability of stem cell exosomes, the research focus of the prior art is generally concentrated on improving the storage and transportation stability of exosome preparations. For example, the technical solution disclosed in patent application No. CN116897922A is to solve this problem by constructing a physical protective layer. This scheme uses polysaccharide biomacromolecules such as sodium alginate and pectin to form a network-like microgel structure in a physiological environment such as phosphate buffer, which gently embeds the exosomes. This strategy effectively slows down the Brownian motion and mutual collision of exosomes in a liquid state through steric hindrance and water molecule binding, and forms a physical barrier against external environmental stress, thereby significantly delaying the disintegration of the exosome structure and the decay of biological activity within a certain period of time. For example, the technical solution disclosed in patent application No. CN115968867A focuses on achieving long-term stable storage of exosomes through freeze-drying process. The core of this scheme is to design and optimize a freeze-drying protectant formula containing trehalose, mannitol and other components. During the process of water sublimation, these protectant molecules can replace water molecules and form hydrogen bonds with the phospholipid head of the exosome membrane surface to construct an amorphous glassy matrix, thereby maintaining the integrity of its membrane structure and ensuring the recovery of its biological function after reconstitution.

[0005] Although the above two technical solutions can solve the inherent instability problem of stem cell exosomes to some extent, as the requirement for the precision of treatment of local lesions such as knee osteoarthritis continues to improve, it is gradually realized that the technical path with the primary design goal of maximizing the physical stability has inadvertently created and solidified a new and more hidden technical contradiction: there is an inherent incompatibility and process conflict between the construction of stability and subsequent functional modification, especially targeted modification. The reason is that whether it is the external coating layer formed by polysaccharide macromolecules or the glassy matrix formed by lyophilization protectants, the fundamental mechanism is to build a relatively "inert" and "closed" external protective microenvironment for the fragile exosomes. This protective shell not only effectively isolates the exosomes from the external environment, but also shields or occupies the key protein sites on the surface of the exosome membrane. These protein sites are the key "anchor points" for targeted molecular modification, such as connecting targeted ligands for knee joint chondrocytes or synoviocytes through chemical coupling or physical adsorption. Therefore, after such stabilization treatment, the surface of the exosome becomes difficult to perform efficient and controllable chemical functionalization modification. If forced to modify, not only is the reaction efficiency low, but it is also likely to destroy the hard-earned stabilized structure. This contradiction is further reflected in the internal conflict of the entire preparation process. If the "naked" exosomes are first modified with a target, and then stabilized, the target ligand introduced during the modification process may interfere with the effective action of the stabilizer (such as polysaccharides or lyophilization protectants) on the surface of the exosome, thereby weakening the stability of the final preparation. More critically, the functionalized exosomes after modification are still exposed to the risk of instability before completing the stabilization treatment. Conversely, if the stabilization is performed first and then the targeted modification is attempted, as mentioned above, the solid protective layer will become an insurmountable obstacle to subsequent modification reactions. This has led the entire preparation process to a dilemma of "losing one to gain the other", that is, stability and functionality are often difficult to achieve under the existing technical framework. This "protective barrier" built to solve the problem of storage and transportation has inadvertently become a "technical barrier" for achieving precise delivery and efficient treatment, greatly limiting the potential of exosomes in achieving lesion enrichment and maximizing efficacy in the treatment of knee osteoarthritis.

[0006] Therefore, how to break through the limitations of the existing technology of stability construction and targeted functionalization has become a key challenge and technical problem to be solved for those skilled in the art. SUMMARY

[0007] The application provides a knee joint-targeting stem cell exosome composition and a preparation method thereof.

[0008] The technical scheme adopted by the application is as follows:

[0009] One of the purposes of the application is to provide a knee joint-targeting stem cell exosome composition, which comprises a core-shell structure composed of stem cell exosomes and targeting and stabilizing functional macromolecules coated on the surface of the stem cell exosomes; the targeting and stabilizing functional macromolecules are composed of a biocompatible polysaccharide skeleton, a membrane-anchored hydrophobic group and a knee joint lesion-targeting ligand and are connected by covalent bonds; the targeting and stabilizing functional macromolecules are coated on the surface of the stem cell exosomes through the membrane-anchored hydrophobic group capable of inserting into the phospholipid bilayer membrane of the stem cell exosomes.

[0010] Through the above structural design, the targeting and stabilizing functional macromolecules form a precise molecular structure: the biocompatible polysaccharide skeleton is covalently connected with multiple hydrophobic groups as “anchors” and multiple targeting ligands as “navigation”. When the targeting and stabilizing functional macromolecules are mixed with stem cell exosomes in an aqueous environment, the hydrophobic long chains as hydrophobic groups spontaneously insert into the phospholipid bilayer membrane of the exosomes, like throwing an anchor into the seabed, firmly “anchoring” the entire macromolecule on the surface of the exosomes, thereby forming a functional exosome complex with a core-shell structure in the process, in which the stem cell exosomes are “nuclei” and the polymer layer formed by the targeting and stabilizing functional macromolecules is “shells”. This core-shell structure gives the composition unique synergistic advantages. First, the shell layer forms a highly hydrated physical barrier in an aqueous environment, effectively preventing the aggregation of exosomes and protecting them from mechanical shear force and osmotic pressure changes through steric hindrance effect and bound water layer, thereby greatly improving their stability in a liquid state. Second, the targeting ligands are located on the outermost layer of the entire core-shell structure, and their spatial conformation is not hindered, which can be completely exposed and efficiently combined with the target points in the joint cavity.

[0011] Further, the stem cell exosomes are derived from mesenchymal stem cells, and the stem cell exosomes have a double-membrane vesicle structure with a particle size distribution range of 50-150 nm.

[0012] 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.

[0013] 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.

[0014] Further, the phosphatidylethanolamine is 1,2-distearyl-SN-glycero-3-phosphoethanolamine (DSPE), and in the embodiment in which hyaluronic acid is used as the biocompatible polysaccharide skeleton, the 1,2-distearyl-SN-glycero-3-phosphoethanolamine is covalently connected to the carboxyl group on the hyaluronic acid skeleton via an amide bond through its amino group.

[0015] 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.

[0016] Further, the composition is a white or white-like lyophilized powder injection, and the components further include a cryoprotectant.

[0017] 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.

[0018] The second object of the present application is to provide a preparation method matched with the above-mentioned composition, which comprises the following steps:

[0019] (1) Preparation of targeted and stabilized functional macromolecules: Dissolve the biocompatible polysaccharide skeleton in the MES buffer solution, add N-(3-dimethylamino propyl)-N'-ethyl carbodiimide hydrochloride (EDC) and N-hydroxysuccinimide (NHS) to carry out carboxyl activation reaction, then add the membrane anchoring hydrophobic group and the knee joint lesion targeting ligand step by step or synchronously to carry out coupling reaction, after the reaction is completed, remove the unreacted small molecule substances by ultrafiltration or dialysis method, freeze-dry after purification to obtain pure targeted and stabilized functional macromolecule powder;

[0020] (2) Preparation of functionalized exosome composition: Dissolve the targeted and stabilized functional macromolecule powder prepared in step (1) in PBS buffer to obtain a clear solution, mix the solution with high-purity stem cell exosomes, wherein the mass ratio of the targeted and stabilized functional macromolecule and the stem cell exosome ranges from 5 to 10:1; incubate the obtained mixed solution at 25-37 DEG C for 0.5-2h, and carry out gentle shaking during the incubation to promote the membrane anchoring hydrophobic group to fully insert into the exosome membrane, so as to complete the self-assembly process of the functionalized exosome composition, and obtain a functionalized exosome composition suspension;

[0021] (3) Freeze-drying: Add a cryoprotectant with a final concentration of 2-10% to the functionalized exosome composition suspension and mix gently, sterile filter the final mixed solution, and distribute it to a Schlenk bottle; freeze the Schlenk bottle in a freeze-drying machine to obtain a white or white-like freeze-dried powder injection.

[0022] The third object of the application is to disclose the use of the composition in the preparation of a medicine for intra-articular injection for preventing and / or treating knee osteoarthritis in mammals.

[0023] In summary, compared with the prior art, the present application has the following advantages and benefits:

[0024] 1. The present application integrates the stabilization function and the targeting function in the same macromolecular carrier through integrated molecular design, and "wears" them on the surface of the exosome through self-assembly technology, which solves the technical contradiction between stability and targeting in the existing stem cell exosome technology. The above-mentioned strategy fundamentally avoids the shielding effect of the stabilizer layer on the active sites on the surface of the exosome in the prior art, and also avoids the interference of the targeting ligand on the action of the stabilizer in the process of modification first and then stabilization.

[0025] 2. The core assembly process of the present application relies on a spontaneous process driven by hydrophobic interaction, which does not require harsh chemical reaction conditions, avoids damage to the biological activity of the exosome, and separates the complex chemical modification steps from the fragile biological vesicle preparation. After both are completed, gentle physical assembly is carried out, and the process controllability and reproducibility are high.

[0026] 3、The coated shell not only plays a space stabilizing role in the liquid preparation, but also acts as a macromolecular skeleton in the freeze-drying process, forming a dense glassy matrix with small molecule cryoprotectants, greatly protecting the integrity of the exosome membrane structure, enabling long-term stable storage and maintaining high bioactivity after reconstitution.

[0027] 4、The targeting ligand located at the outermost layer of the core-shell structure of the composition provided by the application can mediate the efficient combination of the composition with the exposed type II collagen at the cartilage damage site after injection into the joint cavity, thereby "anchoring" the exosome loaded with therapeutic factors at the lesion site, prolonging the effective action time in the joint cavity, reducing the risk of rapid clearance by synovial fluid, and thereby improving the local drug concentration and treatment efficiency.

[0028] 5、The composition provided by the application takes stem cell exosomes as the inner core and takes membrane-anchored hydrophobic groups and hyaluronic acid modified with knee joint lesion targeting ligands as the outer shell, and after injection into the lesion site, the hyaluronic acid shell first relieves inflammation through lubrication, and then gradually degrades to release exosomes, realizing long-term repair, and the two form a synergy in the time dimension. BRIEF DESCRIPTION OF DRAWINGS

[0029] Figure 1 The figure is a schematic diagram of the preparation process of the core-shell structure involved in Example 1. DETAILED DESCRIPTION

[0030] The advantages and various effects of the present application will be more clearly presented from the specific embodiments and examples below. Those skilled in the art should understand that these specific embodiments and examples are used to illustrate the present application, not to limit the present application.

[0031] Throughout the specification, unless otherwise specifically indicated, the terms used herein are to be understood as having the meanings commonly used in the art. Therefore, unless otherwise defined, all technical and scientific terms used herein have the same meaning as generally understood by those skilled in the art to which the present application belongs. If there is a conflict, the present specification takes precedence.

[0032] Unless otherwise specifically stated, various raw materials, reagents, instruments and equipment used in the present application can be purchased from the market or can be prepared by existing methods.

[0033] The present application provides a knee joint targeting stem cell exosome composition, which comprises a core-shell structure composed of stem cell exosomes and targeting stabilizing functional macromolecules coated on the surface of the stem cell exosomes; the targeting stabilizing functional macromolecules are composed of a biocompatible polysaccharide skeleton, a membrane-anchored hydrophobic group and a knee joint lesion targeting ligand connected by a covalent bond, and the targeting stabilizing functional macromolecules are coated on the surface of the stem cell exosomes through the membrane-anchored hydrophobic group capable of inserting into the phospholipid bilayer membrane of the stem cell exosomes.

[0034] Among them, the stem cell exosomes used in the present application are derived from mesenchymal stem cells and have a double-membrane vesicle structure (see Figure 1 The purification method of the stem cell exosomes is density gradient centrifugation, the stem cell exosomes used in the present application are high-purity exosomes with a particle size distribution range of 50-150 nm, and the detection results of the surface marker proteins thereof are CD9 positive, CD63 positive, CD81 positive and calcium binding protein negative.

[0035] In order to more specifically illustrate the technical effect of the present application, the following specific examples and comparative examples are carried out for the exosome suspension (average particle size about 115 nm) produced in the same batch.

[0036] Example 1

[0037] The present example provides a freeze-dried powder injection of a knee joint targeting stem cell exosome composition, and the preparation process thereof includes three steps of preparation of targeting stabilizing functional macromolecules, preparation of functionalized exosome composition (core-shell structure) and freeze-drying, wherein the preparation process of the targeting stabilizing functional macromolecules and the preparation process of the functionalized exosome composition are as shown in Figure 1 In the preparation process, the biocompatible polysaccharide skeleton is hyaluronic acid (HA) with a weight average molecular weight of 200 kDa; the membrane-anchored hydrophobic group is 1,2-distearoyl-SN-glycero-3-phosphoethanolamine (DSPE), and the knee joint lesion targeting ligand is type II collagen targeting peptide (CBP, sequence WYRGRL).

[0038] The specific preparation process and related parameters are as follows:

[0039] The first part is the preparation of the targeting stabilizing functional macromolecule, i.e. CBP-HA(200k)-DSPE. The process is synthesized in two steps.

[0040] First, the synthesis of HA-DSPE intermediate was carried out: 300 mg of sodium hyaluronate was accurately weighed and dissolved in 100 mL of 0.1 M MES buffer (pH 5.5) with continuous stirring until completely dissolved to form a clear solution, 44 mg of EDC and 26 mg of NHS were added to the solution, and the reaction was stirred at room temperature for 2 hours to fully activate the carboxyl groups on the hyaluronic acid. In addition, 30 mg of DSPE-PEG2000-NH2 was accurately weighed and dissolved in 5 mL of MES buffer, and then the solution was slowly added dropwise to the above activated hyaluronic acid solution. The pH of the reaction system was adjusted to 7.0-7.5 using dilute NaOH solution, and the reaction was continuously stirred at room temperature for 24 hours. After the reaction was completed, the obtained reaction mixture was transferred to a dialysis bag with a molecular weight cutoff of 100 kDa, and dialysis was carried out using deionized water as the external dialysis liquid, with a total dialysis time of 72 hours, during which the fresh deionized water was replaced every 8 hours. After dialysis was completed, the liquid in the dialysis bag was collected and freeze-dried using a freeze-dryer, and finally a white flocculent solid was obtained, which was the HA-DSPE intermediate. Nuclear magnetic resonance hydrogen spectrum analysis confirmed that DSPE had been successfully grafted onto the hyaluronic acid backbone, and the grafting rate (molar ratio of DSPE molecules to HA disaccharide repeat units) was about 2%.

[0041] Next, the synthesis of the final product CBP-HA-DSPE was carried out: 150 mg of the above prepared HA-DSPE intermediate 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 reaction was stirred at room temperature for 2 hours to activate. In addition, 15 mg of type II collagen targeting peptide CBP (purchased) was accurately weighed and dissolved in 2 mL of MES buffer, and then the peptide solution was 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 the reaction was completed, the same purification method as in the previous step was used, i.e., the purified solution was dialyzed in a dialysis bag with a molecular weight cutoff of 100 kDa, and then freeze-dried to obtain the final targeting and stabilizing functional macromolecule CBP-HA-DSPE. The nitrogen content was determined by elemental analysis, and the grafting rate of CBP was calculated to be about 1.5%.

[0042] Second part, self-assembly of functionalized exosome complex (CBP-HA(200k)-DSPE / Exo). The 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.

[0043] 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 and the tightness was completed under nitrogen protection, and white loose and porous lyophilized powder injection was obtained, which was the final product composition 1 of the present embodiment.

[0044] Example 2

[0045] The present example aims to investigate the effect of the molecular weight of the hyaluronic acid backbone on the properties of the composition. Two experimental groups and two control groups were designed according to the same preparation process as in Example 1. The difference between the four experiments and Example 1 is that, on the basis of Example 1, only the weight average molecular weight of the first part of sodium hyaluronate is replaced by 50 kDa, 100 kDa, 300 kDa and 400 kDa, respectively, and the dialysis bag cut-off molecular weight during dialysis is 30 kDa, 80 kDa, 100 kDa and 100 kDa, respectively. CBP-HA(50k)-DSPE, CBP-HA(100k)-DSPE, CBP-HA(300k)-DSPE and CBP-HA(400k)-DSPE are prepared, respectively. The types and amounts of all other raw materials, reaction conditions, purification methods, etc. are the same as in Example 1. The final lyophilized powder injection is labeled 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).

[0046] Example 3

[0047] The present example aims to investigate the effect of the grafting density of the targeting ligand on the function of the composition. The preparation process is basically the same as in Example 1. Two experiments were designed. The difference between the two experiments and Example 1 is that, in the preparation step of the first part of the targeting and stabilizing functional macromolecule, the amount of CBP grafted with type II collagen targeting peptide (CBP) is replaced by 10 mg and 30 mg, respectively. The types and amounts of all other raw materials, reaction conditions, purification methods, etc. are the same as in Example 1. The grafting rate of CBP on CBP-HA-DSPE is about 1.0% and 3.0%, respectively, as determined by elemental analysis. The final lyophilized powder injection is Composition 6 (CBP grafting rate about 1.0%) and Composition 7 (CBP grafting rate about 2.8%).

[0048] Example 4

[0049] This example aims to investigate the effect of hydrophobic group grafting density on the function of the composition. The preparation process is basically the same as that of Example 1. Two groups of experiments are designed, and the difference between the two groups of experiments and Example 1 is that in the preparation process of the first part of the targeted stabilizing functional macromolecule, the amount of DSPE-PEG2000-NH2 is replaced by 20 mg and 40 mg respectively when the hydrophobic group grafting reaction is carried out. The types and amounts of all other raw materials, reaction conditions, purification methods, etc. are the same as those of Example 1. The grafting rate of DSPE on the prepared CBP-HA-DSPE is about 1.1% and 2.5% respectively as determined by elemental analysis. The corresponding freeze-dried powder injections obtained are composition 8 (DSPE grafting rate about 1.1%) and composition 9 (DSPE grafting rate about 2.5%), and it can be seen that the relationship between the hydrophobic group grafting rate and the amount of DSPE-PEG2000-NH2 is not linear.

[0050] Example 5

[0051] This example aims to investigate the effect of the mass ratio of targeted stabilizing functional macromolecule and stem cell exosome on the function of the composition. The preparation process is basically the same as that of Example 1. Two groups of experiments are designed, and the difference between the two groups of experiments and Example 1 is that in the self-assembly process of the second part (CBP-HA(200k)-DSPE / Exo), the mass ratio of CBP-HA-DSPE to total protein of exosome is replaced by 3:1 and 10:1 respectively, and the types and amounts of all other raw materials, reaction conditions, purification methods, etc. are the same as those of Example 1. The corresponding freeze-dried powder injections obtained are composition 10 (mass ratio of CBP-HA-DSPE to total protein of exosome 3:1) and composition 11 (mass ratio of CBP-HA-DSPE to total protein of exosome 10:1).

[0052] Comparative Example 1

[0053] In order to compare the effects, this comparative example prepared an exosome preparation without being coated with the functional macromolecule described in the present application. The specific operation is as follows: take the same amount of exosome suspension (4 mL, containing 10 mg of exosome protein) as Example 1, directly add trehalose powder to it to make its final concentration reach 5% (w / v), gently mix, and then carry out the same sterile filtration, sub-packaging and freeze-drying process as Example 1. This preparation only contains exosomes and trehalose as a cryoprotectant, and is recorded as composition 12.

[0054] Comparative Example 2

[0055] The comparative experiment tests the technical path of first stabilization and then targeted modification. First, the HA-DSPE intermediate without a targeting ligand is prepared according to the method of Example 1, and is self-assembled with exosomes to form a HA-DSPE / Exo stabilized complex according to the self-assembly method of Example 1. Then, the "post-modification" chemical reaction is attempted, i.e., EDC / NHS activators and CBP polypeptides are added to the stabilized complex suspension. After the reaction is completed, the unreacted CBP and small molecules are removed by ultrafiltration, and then trehalose is added to the purified suspension to a concentration of 5% (w / v). Finally, the same freeze-drying as in Example 1 is performed to obtain composition 13.

[0056] Comparative Example 3

[0057] The comparative experiment tests the technical path of first stabilization and then targeted modification. First, the HA-DSPE intermediate without a targeting ligand is prepared according to the method of Example 1, and is self-assembled with exosomes to form a HA-DSPE / Exo stabilized complex according to the self-assembly method of Example 1. Then, the "post-modification" chemical reaction is attempted, i.e., EDC / NHS activators and CBP polypeptides are added to the stabilized complex suspension. After the reaction is completed, the unreacted CBP and small molecules are removed by ultrafiltration, and then trehalose is added to the purified suspension to a concentration of 5% (w / v). Finally, the same freeze-drying as in Example 1 is performed to obtain composition 13.

[0058] Experimental Example 1

[0059] In this experimental example, the freeze-dried powders prepared in the above examples and comparative examples are evaluated for physicochemical properties, in vitro stability, and targeting. Each evaluation experiment is set up with 3 parallel experiments, and the experimental results are averaged.

[0060] First, the physicochemical properties are characterized. The freeze-dried powders of each group prepared in Examples 1-5 and Comparative Examples 1-3 are reconstituted with an equal volume of PBS and shaken gently until completely dissolved. Dynamic light scattering (DLS) is used to measure the hydrated particle size and polydispersity index (PDI) of each group of samples after reconstitution. The smaller the PDI value, the more uniform the particle size distribution. The results are summarized in Table 1.

[0061] Table 1 Physicochemical property characterization results of each group of compositions

[0062]

[0063] The data in Table 1 show that the average hydrated particle size of the nanoparticle suspensions formed after reconstitution of the lyophilized powder injections of each of the above groups coated with a hyaluronic acid hydration layer is increased compared to that of the naked exosomes (about 115 nm), which is consistent with the expected coating of the hyaluronic acid hydration layer, and the PDI values of Composition 1, Composition 2-4, Composition 6-9, Composition 11, and Composition 13 are all significantly lower than 0.3, indicating that the CBP-HA-DSPE or HA-DSPE coating layer effectively prevents the irreversible aggregation of exosomes during lyophilization and reconstitution, and the molecular weight of the hyaluronic acid backbone and the amount of the coating layer used both have a significant impact on the PDI value after reconstitution. A hyaluronic acid backbone with too large a molecular weight may affect self-assembly due to steric hindrance, thereby affecting the coating effect, and too little CBP-HA-DSPE coating layer may not be able to completely coat the stem cell exosomes, resulting in some aggregation. In contrast, the formulation of Comparative Example 1 (Composition 12) showed severe aggregation after reconstitution, with a large average particle size and a very high PDI value, indicating that a small molecule cryoprotectant alone is not enough to completely protect the exosomes. Although the particle size of Comparative Example 2 (Composition 13) is controllable, the subsequent targeted functional test results are not good. Comparative Example 3 (Composition 14) directly chemically modifies the surface of the exosomes, which damages the stability of the exosome membrane, and some aggregation occurs before subsequent stabilization treatment, resulting in uneven particle size and poor stability after reconstitution.

[0064] Secondly, accelerated stability testing. The lyophilized powder injection samples prepared in Examples 1-4 and Comparative Examples 1-3 were placed in a constant temperature incubator at 25°C for 3 months of accelerated stability testing. At time points of 0, 1, 2, and 3 months, samples were taken and reconstituted with PBS, and the changes in PDI values were measured, as shown in Table 2.

[0065] Table 2 PDI value measurement results of each group of compositions at time points of 0, 1, 2, and 3 months

[0066]

[0067] From the results of Table 2, it can be seen that the PDI values of Composition 1, Composition 3-4, Composition 6-9, Composition 11 and Composition 13 only have a slight increase even after 3 months of storage under accelerated conditions at 25°C, showing excellent long-term storage stability. Although the PDI value of Composition 2 is less than 0.3, its stability is relatively poor. The stability of the remaining compositions is not ideal, and the stability of Composition 10 may be affected because CBP-HA-DSPE cannot completely coat the exosomes. The results of Comparative Example 3 (Composition 14) are similar to the stability of Comparative Example 1 (Composition 12), indicating that whether the exosome preparation is unprotected or the structure has been destroyed before protection, the PDI value increases rapidly over time, and the nanoparticles continue to degrade and aggregate.

[0068] Finally, the targeting evaluation. To verify the targeting function of the composition of the present application, in vitro binding experiments were carried out. 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 1-5 and Comparative Examples 1-3 were fluorescently labeled with near-infrared fluorescent dye DiR, and after reconstitution, the exosome protein concentration was adjusted to be the same. The coated wells were added to the wells, incubated at 37°C for 2 hours. After incubation, the wells were washed several times with 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 enzyme-labeled instrument. The results are shown in Table 3.

[0069] Table 3 Relative fluorescence intensity determination results of compositions in each group

[0070]

[0071] The experimental results of Table 3 strongly demonstrate the superior targeting of the composition of the present application. Composition 1, Composition 2-4, Composition 6-9 and Composition 11 all exhibit very strong binding ability to type II collagen-coated wells, and Composition 7 with a higher density of targeting peptides has stronger binding ability. The binding fluorescence signal of Comparative Example 1 and Comparative Example 2 is very weak and close to the background value, indicating that they do not have targeting ability. Although Comparative Example 3 shows a certain degree of targeting binding, its binding efficiency is much lower than that of the examples of the present application, which may be due to the damage to the conformation and activity of the exosome surface protein caused by direct chemical modification, and its poor physical stability limits its effectiveness.

[0072] Experimental Example 2

[0073] The freeze-dried powder injection prepared in the present application is further evaluated for in-vivo treatment effect on a rat knee osteoarthritis model in the present experimental example. Composition 1 and composition 7 having good stability and targeting effect in experimental example 1 are taken as experimental groups, normal saline is taken as a blank control group, and composition 12-14 and the mixture of targeted and stable functional macromolecule CBP-HA-DSPE and stem cell exosome prepared in experimental example 1 are taken as control groups, and as non-core-shell self-assembly control groups. CBP-HA-DSPE and stem cell exosome are mixed at 0-5°C and then used for experiment, and the mass ratio of CBP-HA-DSPE and stem cell exosome is 5:1.

[0074] The specific experimental process of the present experimental example is as follows: healthy male SD rats are selected, and a KOA model is induced in the right hind knee joint by surgical operation method (cutting the anterior cruciate ligament and partially resecting the medial meniscus, i.e. Hulth method). After surgery, the rats are raised for 4 weeks, and after the model is stable, the successful model rats are randomly divided into 7 treatment groups, 8 rats in each group, which are respectively: (A) normal saline blank 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; (G) non-core-shell self-assembly control group. Each group is administered by intra-articular injection, once a week, 20 μL of the corresponding preparation reconstituted solution (containing equal amount of exosome protein) is injected each time, and the administration is continued for 4 weeks. After the last administration, all rats are raised for another 4 weeks, and then sacrificed, and the right knee joint is taken for subsequent histological analysis. After decalcification and paraffin embedding, sagittal section of the knee joint specimen is prepared, and the degree of cartilage damage is semi-quantitatively scored according to the scoring system of the International Osteoarthritis Research Association (OARSI), and the results are shown in Table 4. The higher the score, the more serious the damage.

[0075] Table 4 OARSI scoring results of each group experiment

[0076]

[0077] The experimental results of Table 4 show that the targeting composition of the present application has excellent in vivo therapeutic effect, the saline group as a control group has the highest OARSI score, proving the success of the modeling of the experimental example, the OARSI of the composition with poor in vitro stability and targeting is relatively high, on the contrary, the OARSI of the composition with good in vitro stability and targeting is low, this result strongly proves that the knee joint targeting stem cell exosome composition provided by the present application, through its ingenious core-shell structure, not only ensures the stability of the exosome in vivo, but also can efficiently deliver and anchor the exosome with therapeutic potential in the cartilage lesion area through the guiding effect of the targeting ligand, thereby exerting strong cartilage protection and repair effect, its therapeutic effect is much better than that of the non-targeting stable preparation and the unstable exosome preparation; the therapeutic effect of the non-core-shell self-assembly control group is only better than that of Comparative Example 1 and worse than that of Comparative Examples 2 / 3, which shows that the in vivo therapeutic effect of the targeting composition is not only because of the component design, but also related to the specific core-shell structure design.

[0078] Finally, it should also be noted that the terms "comprising", "including", or any other variant thereof are intended to cover non-exclusive inclusion, so that the process, method, article or equipment including a series of elements not only includes those elements, but also includes other elements not explicitly listed or inherent to such process, method, article or equipment. Although the preferred embodiments of the present application have been described, those skilled in the art can make additional changes and modifications to the embodiments once they know the basic inventive concept. Therefore, the appended claims are intended to be interpreted as including the preferred embodiments and all changes and modifications falling within the scope of the present application.

[0079] The above-described embodiments only express the specific implementation of the present application, which is described in detail and specifically, but should not be understood as limiting the protection scope of the present application. It should be noted that for those skilled in the art, without departing from the technical concept of the present application, a number of modifications and improvements can be made, which are all within the protection scope of the present application.

Claims

1. A knee osteoarticular targeting stem cell exosome composition, characterized in that, The composition comprises a core-shell structure composed of stem cell exosomes and targeting stabilizing functional macromolecules coated on the surface of stem cell exosomes; the targeting 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 targeting stabilizing functional macromolecules are coated on the surface of stem cell exosomes through the membrane-anchored hydrophobic group capable of inserting into the phospholipid bilayer membrane of stem cell exosomes; the biocompatible polysaccharide skeleton is hyaluronic acid or a pharmaceutically acceptable salt thereof, and the weight average molecular weight is 100-300 kDa; the membrane-anchored hydrophobic group is 1,2-distearoyl-SN-glycero-3-phosphoethanolamine, and the mass ratio of the biocompatible polysaccharide skeleton to the membrane-anchored hydrophobic group is 5-20:1; the knee joint lesion targeting ligand is a type II collagen targeting polypeptide CBP, and the amino acid sequence is WYRGRL, and the mass ratio of the biocompatible polysaccharide skeleton to the knee joint lesion targeting ligand is 5-20:

1.

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

3. The composition of claim 1, wherein The composition is a white or white-like lyophilized powder injection, and the components further include a cryoprotectant.

4. The composition of claim 3, wherein The cryoprotectant is trehalose.

5. A process for the preparation of a composition according to any one of claims 3 or 4, characterised in that, The method comprises the following steps: (1) Preparation of targeting stabilizing functional macromolecules: dissolve the biocompatible polysaccharide skeleton in the MES buffer solution, add N-(3-dimethylaminopropyl)-N'-ethyl carbodiimide hydrochloride and N-hydroxysuccinimide for carboxyl activation reaction, then add the membrane-anchored hydrophobic group and the knee joint lesion targeting ligand step by step or synchronously for coupling reaction, after the reaction is completed, remove the unreacted small molecule substances by ultrafiltration or dialysis, and then freeze-dry after purification to obtain pure targeting stabilizing functional macromolecule powder; (2) Preparation of functionalized exosome composition: dissolve the targeting stabilizing functional macromolecule powder prepared in step (1) in PBS buffer to obtain a clear solution, mix the solution with high-purity stem cell exosomes, wherein the mass ratio of the targeting stabilizing functional macromolecule to the stem cell exosome is 5-10:1; incubate the obtained mixed solution at 25-37°C for 0.5-2h, and perform gentle shaking during the incubation to promote the membrane-anchored hydrophobic group to fully insert into the exosome membrane, so as to complete the self-assembly process of the functionalized exosome composition, and obtain a functionalized exosome composition suspension; (3) Freeze-drying: add a cryoprotectant with a final concentration of 2-10% to the functionalized exosome composition suspension, and mix gently, sterilize the final mixed solution, and then divide and pack into a Schlenk bottle; freeze the Schlenk bottle in a freeze dryer to obtain a white or white-like lyophilized powder injection.

6. Use of the composition of any one of claims 1-4 in the preparation of a medicine for intra-articular injection for preventing and / or treating knee osteoarthritis of a mammal.

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