Preparation method and application of amniotic fluid active complex

Abstract

The invention belongs to the technical field of biological medicine, and particularly relates to a preparation method and application of an amniotic fluid active complex. According to the method, inherent physical and chemical differences of different components in amniotic fluid are utilized, through a series of physical steps, the components are firstly and mildly grouped and enriched according to categories, and then the components are guided to be self-assembled again under controllable conditions to form a composite particle with a compact structure. The natural aggregation state of active ingredients in a living body before secretion or action is simulated, so that the multi-element active ingredients which naturally coexist and interact in amniotic fluid are efficiently captured and transferred as a complete functional unit, the natural ingredient spectrum is not damaged, and the original advanced structure and function are not lost. The invention provides application of an amniotic fluid active complex (RSC) in treating or preventing joint diseases or symptoms. The RSC has obvious effects in promoting cartilage matrix synthesis, inhibiting moderate and mild osteoarthritis and relieving pain.

Description

Technical Field

[0001] This invention belongs to the field of biomedical technology, and specifically relates to a method for preparing an amniotic fluid active complex and its application. Background Technology

[0002] Amniotic fluid (AF) is the fluid that fills the amniotic cavity during pregnancy. It primarily originates from: ① early pregnancy, formed by maternal serum permeating through the amnion; ② later pregnancy, with fetal urine becoming the main source, supplemented by fetal respiratory and skin secretions. Its composition changes dynamically with gestational age, reaching approximately 800-1000 ml at delivery, making it an important source of perinatal biological resources. AF typically contains a mixture of growth factors, pro-inflammatory cytokines, and anti-inflammatory cytokines, as well as various macromolecules, including carbohydrates, proteins and peptides, lipids, lactate, pyruvate, electrolytes, enzymes, and hormones.

[0003] Amniotic fluid, as a unique biological resource, has long been considered medical waste during childbirth, and its value has not been recognized. The therapeutic potential of amniotic fluid stems from the diversity and systemic nature of its endogenous active components. These components, including extracellular vesicles, growth factors, cytokines, and functional nucleic acids, do not exist in isolation in the body, but rather form a functional network through precise interactions, jointly regulating tissue repair and regeneration.

[0004] The current technical approach to obtaining bioactive substances from perinatal tissues presents a fundamental dilemma in its concept: (1) "Separation Paradox": Purification techniques, represented by ultracentrifugation and size exclusion chromatography, are based on the principle of "separation for purity". For example, to obtain high-purity exosomes, it is necessary to discard a large number of coexisting soluble proteins (such as cytokines) and nucleic acids with clear biological activity in the supernatant. This approach sacrifices the "diversity" of components, destroys the natural compositional profile, and the single component obtained is difficult to reproduce the complex regulatory functions inherent in tissues.

[0005] (2) "Destruction Paradox": Extraction techniques, represented by enzymatic hydrolysis and acid / alkali treatment, are based on the principle of "destruction for acquisition." Although this method can obtain various small peptides or nucleotides, it completely destroys the membrane structure of vesicles, the tertiary / quaternary structure of proteins, and the natural interaction interfaces between components by breaking chemical bonds. This path sacrifices the "natural conformation" and "interactions" of the components, and the resulting products are molecular fragments that have lost their original higher-order structures and functions.

[0006] Therefore, how to efficiently capture and transfer the naturally coexisting and interacting multi-active components in perinatal tissues as a complete functional unit without separation or damage is a bottleneck restricting the use of perinatal tissues and their derivatives for treatment, and is also a current pain point in the industry. Summary of the Invention

[0007] The present invention aims to provide a novel purification pathway—namely, to prepare an "active complex" that retains the natural characteristics and functional network relationships of amniotic fluid to the greatest extent possible, and to demonstrate its superior synergistic therapeutic effect compared to existing technology products.

[0008] The objective of this invention is achieved through the following technical solution: In a first aspect, the present invention provides a method for preparing an amniotic fluid bioactive complex, comprising the following steps: a) Collect amniotic fluid from healthy donors and centrifuge at 10,000-15,000×g for 30-50 min under the first centrifugation conditions to obtain supernatant (U1) and precipitate (S1). The precipitate (S1) is resuspended for the first time in a first buffer containing divalent cations to obtain the structure component SC for later use. b) The supernatant (U1) obtained in step a is concentrated by ultrafiltration with a pressure of 20-40 kDa to obtain the signal component SS (Signal Substance) for later use; c) Mix SC and SS completely, and centrifuge at 40,000-60,000×g for 100-120 min under the second centrifugation conditions. The precipitate is then resuspended a second time with the second buffer solution, which is the amniotic fluid active complex RSC (Re-Structured Complex).

[0009] In some specific embodiments of the invention, the amniotic fluid active complex is prepared from sterile human amniotic fluid obtained from pregnant women. Suitable sources, such as amniotic fluid (AF), include AF obtained from patients undergoing amniocentesis, patients undergoing cesarean section, and patients undergoing normal delivery (using specially designed containers to collect the fluid after rupture of membranes).

[0010] In some specific embodiments of the present invention, the preparation method is carried out at a temperature of 4°C. Further, the first buffer and the second buffer are Tris-HCl buffer and / or PBS buffer. Further, the pH of the Tris-HCl buffer is 7.2-7.4. Further, the volume of the second buffer is 1 ml.

[0011] In some specific embodiments of the present invention, the amniotic fluid needs to be pre-centrifuged to remove cell debris before centrifugation. Furthermore, the pre-centrifugation conditions are: centrifugation at 300-500×g for 8-15 min.

[0012] In some specific embodiments of the present invention, the divalent cation is Ca²⁺. + and / or Mg² + Furthermore, the concentration of the divalent cation is 0.5-2.0 mM.

[0013] In some specific embodiments of the present invention, the amniotic fluid active complex needs to be filtered through 0.22 μm. Further, the filtration can be performed before the first centrifugation in step a or after the second resuspension in step c.

[0014] In a second aspect, the present invention provides an amniotic fluid bioactive complex, which is prepared by the aforementioned preparation method. Furthermore, the amniotic fluid bioactive complex exhibits a composite structure with vesicles as the core and amorphous material surrounding them under a transmission electron microscope.

[0015] In some embodiments of the present invention, the particle size range of the amniotic fluid active complex is 50 nm-200 nm. Further, the amniotic fluid active complex includes surface-binding proteins, cytokines, and functional nucleic acids. Further, the surface-binding proteins include at least one of CD9, CD63, CD81, CD326, CD133, and CD14. The cytokines include at least one of angiopoietin, B-lymphocyte chemokine, epidermal growth factor, fibroblast growth factor 6, granulocyte chemoattractant protein-2, insulin-like growth factor binding protein 1, and insulin-like growth factor binding protein 2. The functional nucleic acid is miRNA. Further, the miRNA includes at least one of hsa-let-7b, hsa-mir-200c, hsa-mir-30d, hsa-mir-125a, hsa-mir-483, hsa-mir-34c, and hsa-mir-200a.

[0016] In a third aspect, the present invention provides the use of the aforementioned amniotic fluid active complex in the preparation of a medicament for treating or preventing joint diseases or conditions, wherein the joint disease or condition is osteoarthritis.

[0017] In some embodiments of the invention, the amount of therapeutic agent is typically determined by the tissue to be treated. Those skilled in the art can readily determine the specific dosage. See Ansel, Howard C. et al. Pharmaceutical dosage forms and delivery systems (6 thed.) Williams and Wilkins, Malvern, Pa. (1995).

[0018] Concentration and dosage (the amount of formulation per day over a period of time) will vary depending on the condition being treated, the severity of the condition, and whether other therapeutic, preventative, or diagnostic agents are included. As illustrated in the examples, the appropriate amount is determined on an individual basis and by measuring the response to treatment over time.

[0019] In a fourth aspect, the present invention provides a pharmaceutical composition comprising the aforementioned amniotic fluid active complex, further comprising pharmaceutically acceptable excipients, further comprising the pharmaceutical composition used alone or in combination with other drugs, further comprising the pharmaceutical composition for treating or preventing joint diseases or conditions, further comprising osteoarthritis.

[0020] In some specific embodiments of the present invention, the dosage and administration method of the combined treatment are as follows: Similar considerations apply to the treatment of joint injuries, where variables include, for example, joint size, the severity of the injury to be treated, the purpose of treatment, and age-related factors. This is in comparison to long-term low-dose surgery.

[0021] The standard regimen is once daily, once every two days, once weekly, once every two weeks, once monthly, or more, depending on the indication and the severity of the injury or inflammation. A series of three injections at longer intervals may be administered. The frequency or dosage may be reduced over time.

[0022] In a fifth aspect, the present invention provides a method for promoting cartilage repair and / or regeneration in a subject by applying the aforementioned amniotic fluid active complex or the aforementioned pharmaceutical composition to a desired site or vicinity, further, the desired site being the surface cartilage of a joint, further, the surface cartilage being the articular cartilage of the femur, tibia, and / or patella, and further, the subject suffering from osteoarthritis.

[0023] The technical solution provided by this invention has the following technical contributions: (1) This invention utilizes the inherent physicochemical differences (such as size, density, and solubility) of different components in amniotic fluid. Through a series of physical steps, these components are first gently "grouped and enriched" according to their categories, and then guided to "self-assemble" into a tightly structured composite particle under controlled conditions. This simulates the natural aggregation state of active ingredients in organisms before secretion or action, thereby efficiently capturing and transferring the naturally coexisting and interacting multiple active ingredients in amniotic fluid as a complete functional unit, without destroying the natural component spectrum or losing the original advanced structure and function.

[0024] (2) This invention innovatively integrates two completely different purification methods, which can obtain the common advantages of the two traditional methods. It has both extremely high raw material utilization efficiency and extremely high effective component concentration, realizing the "full component utilization" (without waste) of amniotic fluid active components. The raw material utilization rate is much higher than that of conventional ultracentrifugation and ultrafiltration membrane treatment, and the quality of the RSC final product is also significantly higher than that of existing amniotic fluid products.

[0025] (3) The first centrifugation condition of this invention is based on differential capture of size and density. The purpose is not simply to remove impurities, but to actively capture a class of key components that serve as the "structural framework" of subsequent complexes. The precipitate is rich in microvesicles, apoptotic bodies, and growth factor complexes that bind to extracellular matrix fragments (such as hyaluronic acid networks and fibrin fragments) through hydrophobic interactions or ionic bonds. Using Ca²⁺... + / Mg² + The precipitate was resuspended in a buffer solution to obtain the structural component (SC). The introduction of divalent cations was intended to stabilize the matrix structure and bridge the negatively charged molecules.

[0026] (4) The ultrafiltration membrane used in this invention is based on the concentration of soluble components by molecular weight cutoff (MWCO). A gentle tangential flow filtration is performed using an ultrafiltration membrane with a MWCO of 30-40 kDa to concentrate the soluble components to 5-15% of their original volume. A MWCO of 30-40 kDa is sufficient to retain most functional proteins (such as albumin, apolipoproteins, and most cytokines) and all extracellular vesicles, while allowing small molecule metabolites, inorganic salts, and other inactive impurities to pass through. The resulting concentrate is called the signal component (SS), which is rich in soluble active proteins, nucleic acids, and small exosomes.

[0027] (5) This invention enables the guided self-assembly of structural components (SC) and signaling components (SS) based on physicochemical interactions. Under specific second centrifugation conditions and mediated by previously introduced divalent cations, the "backbone units" in SC and the "signaling units" in SS undergo directional physical binding, including but not limited to: vesicle membrane fusion or adhesion, protein adsorption onto the vesicle surface via electrostatic or ligand-receptor interactions, and nucleic acid encapsulation or adsorption. This process forms a novel, structurally irreversible composite entity.

[0028] (6) This invention provides the application of amniotic fluid active complex (RSC) in the treatment or prevention of joint diseases or conditions, preferably osteoarthritis. In existing animal models, RSC has shown significant effects in promoting cartilage matrix synthesis, inhibiting mild to moderate osteoarthritis, and relieving pain. This invention provides amniotic fluid active complex (RSC) as a "ready-to-use" functional unit, which doubles the therapeutic effect and can reduce the clinical dosage and frequency of administration.

[0029] (7) Compared with the prior art, the present invention not only solves the long-standing technical bottlenecks such as the "separation paradox" and the "destruction paradox", but its preparation process is entirely a physical process, which is easy to scale up and produce in compliance with regulations. The performance of the RSC final product is significantly improved compared with the existing amniotic fluid preparations, and it can bring multiple technical effects such as the reuse of medical waste resources, and has a clear prospect for transformation. Attached Figure Description

[0030] Figure 1 Electron micrograph of the amniotic fluid active complex prepared in this invention. Figure 1 Observations were conducted using different magnifications. Figure 1 The left side is 100,000×, Figure 1 On the right, at 500,000×, standard vesicle structures are visible.

[0031] Figure 2 The transmission electron microscope (TEM) distribution of the amniotic fluid bioactive complex prepared in this invention is shown. The TEM distribution range of the amniotic fluid bioactive complex is 50-200 nm, with the particle size concentrated between 50-150 nm.

[0032] Figure 3 The nanoparticle tracking analysis (NTA) distribution of the amniotic fluid bioactive complex prepared in this invention is shown. The NTA distribution range of the amniotic fluid bioactive complex is 50-250 nm, with the particle size concentrated between 50-150 nm.

[0033] Figure 4 The median strength of the protein in the amniotic fluid active complex prepared in this invention. Figure 4The study showed that RSCs contain exosome markers (CD63, CD9, CD81, etc.), key extracellular matrix proteins (Decorin, Lumican, etc.), and active factors with cartilage repair and anti-inflammatory functions (TGF-β1, FGF2, IL-1RA, etc.).

[0034] Figure 5 The miRNAs of the amniotic fluid active complex prepared in this invention were sequenced to determine their types and quantities. Figure 5 The RSC shows that it contains different numbers of miRNAs, including hsa-let-7b, hsa-mir-200c, hsa-mir-30d, hsa-mir-125a, hsa-mir-483, hsa-mir-34c, and hsa-mir-200a.

[0035] Figure 6 This is a comparison of the protein content of the final product under the first centrifugation conditions in Example 3: centrifugation at 12000×g for 25 min, 40 min, 50 min, and 60 min. (Note: Each example will be performed multiple times. The upper and lower limits are the highest and lowest values, and the column height shows the average value. Other bar charts in this invention are represented in the same way.) Figure 7 The comparison of vesicle content in the final product under the first centrifugation conditions in Example 3 was conducted at 12000×g for 25 min, 40 min, 50 min, and 60 min.

[0036] Figure 8 The protein count in Example 4 is calculated under the following first centrifugation conditions: centrifugation at 10,000×g, 12,000×g, and 15,000×g for 40 min.

[0037] Figure 9 The number of vesicles in Example 4 is calculated under the following first centrifugation conditions: 10,000×g, 12,000×g and 15,000×g for 40 min.

[0038] Figure 10 The number of vesicles obtained by resuspending the second suspension in Example 5 using Tris-HCl buffers with 0.5 nM, 1.0 nM, 1.5 nM, and 2.0 nM CaCl2, respectively.

[0039] Figure 11 This is a comparison of the protein content in the final products of Examples 1 and 6.

[0040] Figure 12 The second centrifugation conditions in Example 7 were as follows: the number of assembled vesicles was determined by centrifugation speeds of 40,000×g, 50,000×g, and 60,000×g, respectively.

[0041] Figure 13 The protein content of the final products AF, BF, and CF in Example 8 is compared.

[0042] Figure 14 This is a comparison of the vesicle content of the final products AF, BF, and CF in Example 8. Detailed Implementation

[0043] The technical solutions in the embodiments of the present invention will be clearly and completely described below. Obviously, the described embodiments are only some embodiments of the present invention, and not all embodiments. Based on the embodiments of the present invention, all other embodiments obtained by those skilled in the art without creative effort are within the scope of protection of the present invention.

[0044] Unless otherwise specified, experimental methods in the following examples were performed under standard conditions, such as those described in Sambrook et al., Molecular Cloning: A Laboratory Manual (New York: Cold Spring Harbor Laboratory Press, 1989), or as recommended by the manufacturer. All commonly used reagents used in the examples are commercially available products.

[0045] 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 pertains. The terminology used in this specification is for the purpose of describing particular embodiments only and is not intended to be limiting of the invention. The term "and / or" as used herein includes any and all combinations of one or more of the associated listed items.

[0046] In the following embodiments, unless otherwise specified, the terms used are explained as follows: In this disclosure, unless otherwise specified, the term "perinatal tissues" specifically refers to amniotic fluid, which can be collected as waste after delivery (without ethical controversy) and is characterized by its abundant sources and diverse bioactive components.

[0047] In this disclosure, unless otherwise specified, the term "ultrafiltration membrane" refers to a pressure-driven semi-permeable membrane with a pore size typically ranging from 1 to 100 nm and a molecular weight cutoff (MWCO) ranging from 1 to 1000 kDa, whose separation mechanism is a combination of molecular sieve effect and charge effect.

[0048] In this disclosure, unless otherwise specified, the core of the term "MWCO hollow fiber column tangential flow filtration (TFF)" is hundreds of hollow fiber membrane filaments, with an inner diameter typically of 0.5–2 mm. The feed liquid flows tangentially inside the membrane filaments, rather than perpendicularly to the membrane surface. As the feed liquid flows within the membrane filaments at a certain flow rate, driven by transmembrane pressure (TMP), small molecule components (permeate) pass through the membrane wall and are collected, while large molecule components (retentate) are retained by the membrane and flow out with the mainstream direction of the feed liquid, and can be recycled back to the feed tank.

[0049] In this disclosure, unless otherwise specified, the term "exosome" refers to small membrane vesicles (30-150 nm) containing complex RNA and proteins; currently, it specifically refers to disc-shaped vesicles with a diameter of 40-100 nm. Exosomes were first discovered in sheep reticulocytes in 1983, and Johnstone named them "exosome" in 1987. Various cell types can secrete exosomes under both normal and pathological conditions. They mainly originate from multivesicular bodies formed by the invagination of intracellular lysosomal microparticles, which are released into the extracellular matrix after the outer membrane of the multivesicular body fuses with the cell membrane.

[0050] In this disclosure, unless otherwise specified, the term "exosome marker" refers to a class of molecules used to specifically identify and characterize exosomes, primarily distinguishing them from other extracellular vesicles or non-vesicle structures. Currently recognized transmembrane protein families (such as CD9, CD63, and CD81) and cytoplasmic proteins (such as TSG101 and Alix) are commonly used exosome markers, but their expression may vary depending on cell origin or physiological state.

[0051] In this disclosure, unless otherwise specified, the term "Decorin" refers to a secreted proteoglycan belonging to the leucine-rich low molecular weight proteoglycan (SLRP) family. Decorin regulates the biological activity of the extracellular collagen matrix and matrix-associated growth factors FGF, GDF-8 / Myostatin, TGF-beta, and WISP-1. It also binds to and activates EGF receptors, ErbB4, and IGF-1 receptors. Decorin promotes myoblast differentiation, supports angiogenesis, and inhibits tumor development.

[0052] In this disclosure, unless otherwise specified, the term "lumican" refers to a member of the leucine-rich small proteoglycan (SLRP) family that regulates cell migration and proliferation, is expressed in vascular SMCs, and is variable in glycosylation after translation. The important structural roles of lumican are attributed to its ability to assemble tissue cells, regulate collagen fiber production, stabilize collagen fibers, and limit the diameter of collagen fibers in the cornea, skin, and wounds.

[0053] In this disclosure, unless otherwise specified, the term "average particle size" or "particle size" generally refers to the statistical average particle size (diameter) of particles in a swarm. The diameter of a fundamentally spherical particle may refer to its physical or hydrodynamic diameter. The diameter of a non-spherical particle may preferably refer to its hydrodynamic diameter. As used herein, the diameter of a non-spherical particle may refer to the maximum linear distance between two points on the particle surface. The average particle size can be measured using methods known in the art, such as dynamic light scattering.

[0054] In this disclosure, unless otherwise specified, the term "combination" refers to the use of more than one therapeutic agent. The use of the term "combination" does not limit the order in which the therapeutic agents are administered to the subject.

[0055] In this disclosure, unless otherwise specified, the terms “treating” or “treatment” mean alleviating, reducing, ameliorating, relieving, or controlling one or more clinical signs of a disease or disorder, and lowering, stopping, or reversing the progression of the severity of a condition or symptom that is being treated.

[0056] In this disclosure, unless otherwise specified, pharmaceutical products may be manufactured using techniques known to those skilled in the art into a dosage form suitable for parenterally administration, including, but not limited to: injections [e.g., sterile aqueous solutions or dispersions], sterile powders, tablets, troche, lozenges, pills, capsules, dispersible powders or granules, solutions, suspensions, emulsions, syrups, elixirs, slurries, and the like.

[0057] In this disclosure, unless otherwise specified, pharmaceutical products according to the present invention can be administered via a parenteral route selected from the group consisting of: intraperitoneal injection, subcutaneous injection, intraepidermal injection, intradermal injection, intramuscular injection, intravenous injection, and intralesional injection. In this disclosure, unless otherwise specified, pharmaceutically acceptable excipients preferably include one or more of fillers, disintegrants, binders, lubricants, flavoring agents, coating agents, surfactants, preservatives, antioxidants, sustained-release materials, colorants, solvents, and suspending agents.

[0058] In this invention, the filler preferably includes one or more of the following: lactose, microcrystalline cellulose, pregelatinized starch, starch, dextrin, mannitol, dicalcium phosphate, cellulose, sucrose, glucose, sorbitol, and xylitol.

[0059] In this invention, the disintegrant preferably includes one or more of the following: crospovidone, sodium carboxymethyl starch, low-substituted hydroxypropyl cellulose, hydroxypropyl methyl cellulose, and ethyl cellulose.

[0060] In this invention, the adhesive preferably includes one or more of the following: povidone, hydroxypropyl cellulose, methylcellulose, sodium hydroxymethylcellulose, hydroxypropyl methylcellulose, ethylcellulose, polyvinylpyrrolidone, and gelatin.

[0061] In this invention, the lubricant preferably includes one or more of the following: micronized silica gel, magnesium stearate, zinc stearate, calcium stearate, stearic acid, hydrogenated vegetable oil, palmitic acid, talc, polyethylene glycol, sodium dodecyl sulfate, and magnesium dodecyl sulfate.

[0062] In this invention, the flavoring agent preferably includes flavorings, sucrose, aspartame, sucralose, simple syrup, glycerin, etc. It contains one or more of the following: sorbitol, mannitol, steviol glycoside, sodium saccharin, and citric acid.

[0063] In this invention, the coating agent preferably includes one or more of hydroxypropyl methylcellulose, polyvinyl alcohol, cellulose acetate phthalate, acrylic resin, sucrose, talc, and ethyl cellulose.

[0064] In this invention, the surfactant preferably includes one or more of polysorbate, poloxamer, sodium dodecyl sulfate, and benzalkonium chloride.

[0065] In this invention, the preservative preferably includes one or more of sodium benzoate, benzoic acid, methylparaben, ethylparaben, propylparaben, benzalkonium chloride, benzalkonium bromide, ethanol, phenethyl alcohol, and potassium sorbate.

[0066] In this invention, the antioxidant preferably includes one or more of sodium sulfite, sodium bisulfite, ascorbic acid, cysteine, vitamin E, tert-butyl-p-hydroxyanisole, tert-butyl-hydroxytoluene, disodium EDTA and citric acid.

[0067] In this invention, the sustained-release material preferably includes hydroxypropyl methylcellulose, sodium carboxymethyl cellulose, ethyl cellulose, acrylic resin, cellulose acetate phthalate, polylactic acid, and polylactic acid. One or more of the glycolic acid copolymers.

[0068] In this invention, the colorant preferably includes natural pigments and / or synthetic pigments; the natural pigments preferably include sodium copper chlorophyllin and / or caramel color; the synthetic pigments preferably include one or more of titanium dioxide, iron oxide red, iron oxide yellow, iron oxide black, indigo and lemon yellow.

[0069] In this invention, the solvent preferably includes one or more of purified water, water for injection, ethanol, propylene glycol, and glycerol.

[0070] In this invention, the suspending agent preferably includes one or more of xanthan gum, gum arabic, carbomer, and sodium carboxymethyl cellulose.

[0071] The selection and quantity of these reagents fall within the scope of professional competence and routine techniques of those skilled in this art. The following is an explanation using specific examples. Example 1: Preparation of Amniotic Fluid Active Complex (RSC) 1. Collect 150mL of amniotic fluid from a healthy donor.

[0072] 2. At 4℃, under pre-centrifugation conditions (400×g for 10 min), obtain supernatant (U0) and precipitate (S0). Remove the precipitate (S0) containing cell debris and keep the supernatant (U0) for later use.

[0073] 3. The supernatant (U0) obtained in step 2 was centrifuged at 4°C under the first centrifugation conditions (12,000×g for 40 min) to obtain supernatant (U1) and precipitate (S1). The precipitate (S1) was resuspended for the first time with 2 mL of Tris-HCl buffer (pH 7.4) containing 1.5 mM to obtain SC.

[0074] 4. The supernatant (U1) obtained in step 3 was filtered through a 30 kDa MWCO hollow fiber column tangential flow filter (TMP: 0.3 Bar, linear flow rate 2.5 m / s, polyethersulfone (PES) membrane) and concentrated to 8 mL to obtain SS.

[0075] 5. Mix SC and SS completely, and collect the precipitate (S2) at 4°C under the second centrifugation conditions (50,000×g for 100 min).

[0076] 6. The precipitate (S2) was resuspended a second time with 1 mL of PBS and filtered through a 0.22 μm filter to obtain the final product RSC.

[0077] Example 2: Performance Testing of RSC Multiple batches of amniotic fluid active complex (RSC) were obtained according to the method in Example 1, and the following tests were performed: (1) Sterility and endotoxin tests: Randomly select 10% of the total batch volume for a 14-day sterility test. Endotoxin testing is conducted according to USP. <85> The sterility test was performed according to the guidelines and USP. <71> The procedure was followed. Testing revealed that the endotoxin level of the RSCs prepared according to this invention was below 3 EU / mL, and all samples were sterile.

[0078] (2) Morphological observation by transmission electron microscopy: After negative staining, RSC samples were observed under a transmission electron microscope. The results are as follows: Figure 1 As shown, observations were performed using different magnifications. Figure 1 The left side is 100,000×. Figure 1 On the right, at 500,000×, standard vesicle structures are visible. Figure 1 Numerous vesicle structures of varying sizes are visible, with their membrane structures encapsulated or adsorbed with amorphous, cloud-like substances of uneven electron density, forming a stark contrast to the smooth, pure exosome morphology.

[0079] The TEM distribution range of RSC is 50-200 nm, with the particle size concentrated between 50-150 nm. Figure 2 ).

[0080] (3) Particle size distribution of RSC: RSC was analyzed using a Nanosight NS300 (Malvern Panalytical) instrument. The results showed that the nanoparticle concentration in the final product was reproducible, with an average concentration of 2.2 × 10⁻⁶. 10 Particles / mL, average pattern particle size 125 nm.

[0081] The NTA distribution range of RSC is 50-250 nm, with the particle size concentrated between 50-150 nm. Figure 3 ).

[0082] (4) Total soluble protein content of RSC: The total protein concentration of each batch of the complex composition was determined using the Bradford method to confirm the consistency of the concentration. The specific analytical procedure is as follows: First, take equal volumes of the sample and standard solution and add them separately to test tubes containing Bradford's reagent, ensuring that the final volumes of each tube are consistent. After gently mixing, incubate at room temperature in the dark for 5-10 minutes to allow the dye to fully bind to the protein and develop color. Then, measure the absorbance of each tube at 595 nm using a spectrophotometer. Finally, plot a standard curve based on the absorbance values ​​of the known concentrations of the standards, and substitute the sample absorbance values ​​into the curve equation to calculate the protein concentration.

[0083] The total soluble protein content of the product was confirmed to be repeatable by routine batch testing using this method, with an average value of 18.5 mg ± 1.2 mg.

[0084] (5) Median strength of proteins with RSC: Samples were labeled with Cy3 fluorescent dye and hybridized with a RayBiotech antibody chip at 4°C for 12 hours. After washing, images were acquired using a GenePix 4000B microarray scanner (PMT gain set to 600, excitation wavelength 532 nm, pixel resolution 10 μm). Spots were identified and median fluorescence intensity was read using GenePix Pro 7.0 software. After subtracting local background, the relative expression level of the target protein was calculated by normalizing the signal intensity of all internal control protein spots to the median.

[0085] The results are as follows Figure 4 As shown, we found exosome markers (CD63, CD9, CD81, etc.), key extracellular matrix proteins (Decorin, Lumican, etc.), and active factors with cartilage repair and anti-inflammatory functions (TGF-β1, FGF2, IL-1RA, etc.) in the proteins of RSC. Based on other published papers, the effectiveness of these components in promoting chondrocyte proliferation and matrix synthesis, inhibiting joint inflammation, and regulating extracellular matrix homeostasis has been confirmed, thus potentially producing a positive therapeutic effect on osteoarthritis.

[0086] (7) Functional nucleic acid assay of RSC: The specific steps are as follows: 1. Take RSC, extract nucleic acid, and process it through fragmentation and adapter addition (using Thermo Fisher nucleic acid extractor and Covaris disruptor).

[0087] 2. Sequencing: The library is loaded into the sequencer for sequencing while synthesizing (Illumina NovaSeq).

[0088] 3. Data Analysis: The data from the anatomical unit were compared using the Illumina DRAGEN bioinformatics platform.

[0089] The results are as follows Figure 5 As shown, RSC detected various seed sequences and their quantities, indicating the corresponding miRNAs and their amounts, including hsa-let-7b, hsa-mir-200c, hsa-mir-30d, hsa-mir-125a, hsa-mir-483, hsa-mir-34c, and hsa-mir-200a. These miRNAs are all related to cellular regulation and immune expression, and according to other published papers, their presence has a positive effect on the treatment of osteoarthritis.

[0090] (8) Preservation: For short-term storage (within 30 days) at 4°C, or long-term storage (1 month to 6 months) at -20°C or long-term storage (6 months to 5 years) at -80°C. When needed, use directly at 4°C, or remove from -20°C or -80°C and place at 4°C to thaw. Samples should be stored under stable conditions, avoiding repeated freeze-thaw cycles.

[0091] Example 3: Preparation of RSC Unlike Example 1, the first centrifugation conditions in this example are: centrifugation at 12000×g for 25 min, 40 min, 50 min, and 60 min.

[0092] The results of this embodiment are as follows: Figure 6-7 As shown, the results indicate that 40 minutes is the optimal time for preparing structural components (SCs) at a centrifugal force of 12,000 × g. This optimal time achieves the best balance between maximizing the recovery of intact vesicles and specifically enriching the target active protein complex, thus ensuring that the final product RSCs have the highest degree of functional integration (CSPR) and bioactivity. Shortening the centrifugation time to 25 minutes leads to incomplete sedimentation; extending it to 60 minutes, while increasing the total protein precipitation, may sacrifice vesicle integrity and component specificity, resulting in a decrease in the quality of the final product.

[0093] Example 4: Preparation of RSC Unlike Example 1, the first centrifugation conditions in this example are: centrifugation at 10,000×g, 12,000×g and 15,000×g for 40 min.

[0094] The results of this embodiment are as follows: Figure 8-9 As shown, the results indicate that the number of vesicles obtained varies under different centrifugal forces, suggesting that higher centrifugation speeds can more effectively precipitate a greater number of vesicles. The experimental data also show that the marginal benefit of further increasing the centrifugal force is limited; therefore, considering equipment wear and cost, further increasing the centrifugal force is unnecessary. This embodiment also confirms that 10,000-15,000 × g is the effective range.

[0095] Example 5: Preparation of RSC Unlike Example 1, in this example, the first resuspension in step 3 was performed using Tris-HCl buffer solutions of 0.5 nM, 1.0 nM, 1.5 nM, and 2.0 nM CaCl2, respectively.

[0096] The results of this embodiment are as follows: Figure 10 As shown, the number of vesicles obtained with different concentrations of divalent calcium ions varies, with concentrations ranging from 0.5 to 2.0 mM. + This is the effective range.

[0097] Example 6: Preparation of RSC Unlike Example 1, this example uses a 50 kDa filter.

[0098] The results of this embodiment are as follows: Figure 11 As shown, when using 50 kDa for ultrafiltration, the protein retention shows a decreasing trend compared to using a molecular weight cutoff (MWCO) of 30 kDa (Example 1). The MWCO of the hollow fiber column needs to be selected according to the molecular weight of the target product, usually 1 / 3–1 / 2 of the target product molecular weight, so an ultrafiltration membrane of 20–40 kDa is an effective range.

[0099] Example 7: Preparation of RSC Unlike Example 1, the second centrifugation conditions in this embodiment are: self-assembly is performed using centrifugation speeds of 40,000×g, 50,000×g, and 60,000×g, respectively.

[0100] The results of this embodiment are as follows: Figure 12 As shown, different centrifugation speeds yield different numbers of vesicles, with 40,000-60,000×g centrifugation force being the effective range, and 50,000×g yielding the highest number of vesicles.

[0101] Example 8: Comparison of product performance using different preparation methods 1. 450 mL of amniotic fluid was collected from a healthy donor. The acquisition of the amniotic fluid and related research were approved by the relevant ethics committee and conducted with the informed consent of the donor. The amniotic fluid was randomly divided into 3 portions, each containing 150 mL, namely Sample A, Sample B, and Sample C.

[0102] 2. Sample A was centrifuged at 4℃ under pre-centrifugation conditions (400×g for 10 min) to obtain supernatant A (U0) and precipitate A (S0). Supernatant A (U0) was filtered through a 0.22 μm filter at 4℃ and centrifuged under the first centrifugation conditions (12,000×g for 40 min) to obtain supernatant A (U1) and precipitate A (S1). Precipitate A (S1) was resuspended for the first time in 2 mL of Tris-HCl buffer (pH 7.4) containing 1.5 mM CaCl2 to obtain A-SC.

[0103] 3. Collect the precipitate of A-SC at 4℃ under the second centrifugation conditions (50,000×g for 100 min). Resuspend the precipitate a second time with 1 mL of PBS to obtain product AF.

[0104] 4. Sample B was centrifuged at 4℃ under pre-centrifugation conditions (400×g for 10 min) to obtain supernatant B (U0) and precipitate B (S0). Supernatant B (U0) was filtered at 4℃ using a 0.22μm filter, and then filtered through a 30 kDa MWCO hollow fiber column for tangential flow filtration. The solution was concentrated to 8 mL to obtain product BF.

[0105] 5. Process sample C completely according to the steps in Example 1 to obtain product CF.

[0106] 6. Compare the protein content and vesicle content in AF, BF, and CF. The results are as follows: Figure 13 and 14 It can be seen that the protein content and vesicle content in CF are better than those in AF and BF. That is, the product obtained by the RSC preparation method provided by the present invention is superior to the product obtained by traditional single centrifugation (AF) or single ultrafiltration (BF).

[0107] Example 9: Animal Experiment 1. Experimental materials: 80 osteoarthritis model rats, weighing between 200g and 250g, were purchased from TACONICBIOSCIENCES.

[0108] 2. Experimental grouping and treatment: (1) Control group: 40 osteoarthritis model rats were injected with 35 μL of physiological saline into the joint.

[0109] (2) Treatment group: 40 osteoarthritis model rats were injected intra-articularly with 35 μL of RSC prepared according to Example 1.

[0110] 3. Evaluation indicators and time points: (1) Pain assessment: Animal pain assessment was conducted before injection, on day 15, day 30 and day 90 after injection using an animal pain rating scale modified based on the Wagner pain rating scale.

[0111] (2) Joint function assessment: Assessment was conducted through walking speed, standing time and forced swimming test (recording swimming time), at the same time points as above.

[0112] (3) Scoring criteria (0–4 points): 0 points: No pain behavior, free movement, normal social and exploratory behavior.

[0113] 1 point: Mild pain, occasional limping, and occasional licking or biting of the injured area.

[0114] 2 points: Moderate pain, significant limping, and reduced activity.

[0115] 3 points: Severe pain, severe limping, or refusal to bear weight.

[0116] 4 points: Extreme pain, complete refusal to move.

[0117] 4. Experimental Results: Table 1. Comparison of pain scores between the treatment group and the control group (mean ± standard deviation) As shown in Table 1, injection of amniotic fluid active complex (RSC) can significantly alleviate osteoarthritis pain induced by meniscus tear in rats.

[0118] Table 2 Comparison of walking speed between the treatment group and the control group (m / min, mean ± standard deviation) As shown in Table 2, the walking speed of the treatment group was significantly higher than the baseline from day 30 and continued to increase over time, while the control group showed no significant change, indicating that the exercise capacity of the rats in the treatment group was substantially improved.

[0119] Table 3. Comparison of standing time between the treatment group and the control group (s, mean ± standard deviation) As shown in Table 3, the standing time in the treatment group was significantly prolonged from day 30, reflecting an enhanced joint weight-bearing capacity, while the control group showed no significant change.

[0120] Table 4 Comparison of forced swimming time between the treatment group and the control group (s, mean ± standard deviation) As shown in Table 4, the swimming time in the treatment group was significantly longer than that in the control group on day 30, indicating that the pain tolerance was significantly improved, physical fitness was restored, and pain was reduced.

[0121] In summary, injection of the amniotic fluid active complex (RSC) prepared in this invention can significantly improve the symptoms of osteoarthritis induced by meniscus tears in rats.

[0122] The above description is merely a preferred embodiment of this application and is not intended to limit this application. Any modifications, equivalent substitutions, improvements, etc., made within the spirit and principles of this application should be included within the scope of protection of this application.

Claims

1. A method for preparing an amniotic fluid active complex, characterized in that, Includes the following steps: a) Collect amniotic fluid from healthy donors and centrifuge at 10,000-15,000×g for 30-50 min under the first centrifugation conditions to obtain supernatant (U1) and precipitate (S1). The precipitate (S1) is resuspended for the first time in a first buffer containing divalent cations to obtain the structure component SC for later use. b) The supernatant (U1) obtained in step a is concentrated by ultrafiltration membrane with a pressure of 20-40 kDa to obtain the signal component SS (SignalSubstance) for later use; c) Mix SC and SS completely, and centrifuge at 40,000-60,000×g for 100-120 min under the second centrifugation conditions. The precipitate is then resuspended a second time with the second buffer solution, which is the amniotic fluid active complex RSC (Re-Structured Complex).

2. The preparation method according to claim 1, characterized in that, The preparation method is performed at a temperature of 4°C. Furthermore, the first and second buffer solutions are Tris-HCl buffer and / or PBS buffer. Furthermore, the pH of the Tris-HCl buffer is 7.2-7.

4. Furthermore, the volume of the second buffer solution is 1 ml.

3. The preparation method according to claim 1, characterized in that, The amniotic fluid needs to be pre-centrifuged to remove cell debris before centrifugation. The pre-centrifugation conditions are: centrifugation at 300-500×g for 8-15 min.

4. The preparation method according to claim 1, characterized in that, The divalent cation is Ca²⁺. + and / or Mg² + Furthermore, the concentration of the divalent cation is 0.5-2.0 mM.

5. The preparation method according to claim 1, characterized in that, The amniotic fluid active complex needs to be filtered through 0.22 μm. Further, the filtration can be performed before the first centrifugation in step a or after the second resuspension in step c.

6. An amniotic fluid bioactive complex, characterized in that, The amniotic fluid active complex is prepared by the preparation method according to any one of claims 1-5. Furthermore, the amniotic fluid active complex exhibits a composite structure with vesicles as the core and amorphous substances surrounding them under a transmission electron microscope.

7. The amniotic fluid active complex according to claim 6, characterized in that, The amniotic fluid bioactive complex has a particle size range of 50 nm to 200 nm. Further, the amniotic fluid bioactive complex includes surface-binding proteins, cytokines, and functional nucleic acids. Further, the surface-binding proteins include at least one of CD9, CD63, CD81, CD326, CD133, and CD14. The cytokines include at least one of angiopoietin, B-lymphocyte chemokine, epidermal growth factor, fibroblast growth factor 6, granulocyte chemoattractant protein-2, insulin-like growth factor binding protein 1, and insulin-like growth factor binding protein 2. The functional nucleic acid is a miRNA. Further, the miRNA includes at least one of hsa-let-7b, hsa-mir-200c, hsa-mir-30d, hsa-mir-125a, hsa-mir-483, hsa-mir-34c, and hsa-mir-200a.

8. The use of the amniotic fluid active complex of claim 6 or 7 in the preparation of a medicament for treating or preventing joint diseases or conditions, further wherein the joint disease or condition is osteoarthritis.

9. A pharmaceutical composition, characterized in that, It comprises the amniotic fluid active complex as described in claim 6 or 7, further comprising pharmaceutically acceptable excipients, further comprising the pharmaceutical composition alone or in combination with other drugs, further comprising the pharmaceutical composition for the treatment or prevention of joint diseases or conditions, further comprising the joint disease or condition being osteoarthritis.

10. A method for promoting cartilage repair and / or regeneration in a subject, characterized in that, The amniotic fluid active complex of claim 6 or 7 or the pharmaceutical composition of claim 9 is applied to the desired site or vicinity, further, the desired site being the surface cartilage of a joint, further, the surface cartilage being the articular cartilage of the femur, tibia and / or patella, further, the subject suffering from osteoarthritis.

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