Pharmaceutical suspension composition for ameliorating osteoarticular diseases and method for preparing same

Abstract

The invention relates to the field of biological medicine, and discloses a medicine suspension composition for improving osteoarticular diseases and a preparation method thereof. In the embodiment 1, the composition comprises sterile normal saline and umbilical cord mesenchymal stem cell-loaded modified gelatin microspheres with the volume fraction of 50%; the microspheres are prepared from phenylboronic acid-sulfobetaine bifunctional modified gelatin, 0.2 M trehalose, a photoinitiator and stem cells through a microfluidic technology. The modified gelatin is grafted with 3-carboxyphenylboronic acid through an amido bond, and is grafted with a sulfydryl-containing sulphobetaine derivative through Michael addition; the cartilage anchoring and active oxygen removal are realized by utilizing phenylboronic acid groups, agglomeration is prevented by utilizing a sulphobetaine hydration layer, cells are protected by matching with trehalose, and the cartilage hydrogel has high dispersity, high survival rate and excellent tissue adhesion.

Description

Technical Field

[0001] This invention relates to the field of biomedicine and discloses a drug suspension composition for improving osteoarthritis and its preparation method. Background Technology

[0002] With the increasing demand for treatment of osteoarthritis, the treatment environment for intra-articular local drug delivery is becoming increasingly complex, often facing extreme physiological conditions such as a high-level reactive oxygen microenvironment and continuous mechanical friction within the joint cavity. This complexity poses a severe challenge to the dispersion stability of drug delivery systems and the maintenance of the bioactivity of loaded cells. Currently, existing drugs for improving osteoarthritis usually rely on simple physical blending techniques to prepare stem cell suspensions, or directly encapsulate them using unmodified natural polymer materials as carriers. This traditional method mainly focuses on the physical filling or short-term lubrication of damaged tissues, and lacks effective construction of the microscopic hydration characteristics and antioxidant protection mechanisms of the drug carrier surface. However, this single-dimensional formulation strategy has significant limitations. When encountering the complex pathological environment within the joint cavity and the low-temperature conditions of long-term storage, due to the lack of an effective repulsive force layer and chemical anchoring points, the suspension system often fails to take into account cell protection in order to maintain the basic physical morphology, resulting in severe non-specific aggregation and sedimentation between microspheres. This passive physical mixing not only easily leads to needle blockage or uneven dosage during injection, but also causes the loaded stem cells to have the risk of latent inactivation and apoptosis that are difficult to be detected in time by conventional detection methods due to the lack of reactive oxygen scavenging ability and cryoprotection barrier. Furthermore, it is impossible to make a dynamic trade-off between the physical flowability and tissue retention capacity of the formulation. Therefore, how to effectively detect and avoid the risks of structural damage and loss of cell activity caused by oxidative stress, mechanical shearing and freezing damage while ensuring that the drug suspension composition has excellent dispersibility and injection performance has become an urgent problem to be solved in this field. Summary of the Invention

[0003] The purpose of this invention is to provide a drug suspension composition for improving osteoarthritis and its preparation method, aiming to solve the problems of drug loss, cell inactivation, and non-specific aggregation of existing intra-articular injection formulations in complex pathological environments due to the lack of effective anchoring sites and antioxidant protection mechanisms. Specifically, the technical solution of this invention includes the following steps: The pharmaceutical composition comprises modified gelatin microspheres carrying umbilical cord mesenchymal stem cells and a dispersing solvent; the modified gelatin microspheres carrying umbilical cord mesenchymal stem cells are dispersed in the dispersing solvent, and the volume fraction of the microspheres in the suspension is 45%–55%; Modified gelatin microspheres carrying umbilical cord mesenchymal stem cells were prepared using microfluidic technology from an inner phase raw material comprising the following concentrations: 5% w / v phenylboronic acid-sulfobetaine bifunctional modified gelatin, 0.1 M–0.5 M trehalose, 0.5% w / v photoinitiator, and a concentration of [missing information - likely a specific concentration]. Umbilical cord mesenchymal stem cells; The product obtained by phenylboronic acid-sulfobetaine bifunctional modified gelatin is a gelatin skeleton modified with methacryloyl groups by methacrylic anhydride modification, then grafting 3-carboxyphenylboronic acid with the remaining amino groups via amide bonds, and grafting a sulfobetaine derivative containing thiol groups with some of the methacryloyl groups via Michael addition reaction. Umbilical cord mesenchymal stem cells are encapsulated within a cross-linked network formed by the phenylboronic acid-sulfobetaine bifunctional modified gelatin.

[0004] Preferably, the preparation method of the phenylboronic acid-sulfobetaine bifunctional modified gelatin includes the following steps: Step 1, Preparation of phenylboronic acid modified methacrylamide gelatin: Dissolve gelatin in sodium bicarbonate buffer at pH 9.0, stir and dissolve at a constant temperature of 45℃~55℃, add methacrylic anhydride dropwise, react for 1h~3h, purify by dialysis and freeze-dry to obtain methacrylamide gelatin; Dissolve 3-carboxyphenylboronic acid in MES buffer at pH 5.5, add activator and activate for 20min~40min, dropwise add to PBS solution at pH 7.4 containing methacrylamide gelatin, react at room temperature in the dark for 20h~28h, purify by dialysis and freeze-dry to obtain phenylboronic acid modified methacrylamide gelatin; Step 2, Preparation of phenylboronic acid-sulfobetaine bifunctional modified gelatin: The phenylboronic acid modified methacrylamide gelatin obtained in Step 1 was dissolved in PBS at pH 8.0, and nitrogen gas was introduced for deoxygenation treatment for 20 min to 40 min. A sulfobetaine derivative containing thiol groups and a catalyst were added, and a Michael addition reaction was carried out under closed conditions at 35℃ to 40℃ for 10 h to 14 h. The reaction solution was purified by dialysis and freeze-dried to obtain phenylboronic acid-sulfobetaine bifunctional modified gelatin.

[0005] Preferably, the method for preparing the modified gelatin microspheres carrying umbilical cord mesenchymal stem cells includes the following steps: Step 1, Preparation of the inner phase: Dissolve the phenylboronic acid-sulfobetaine bifunctional modified gelatin in PBS, add trehalose and photoinitiator, mix well and then filter under sterile conditions. Add umbilical cord mesenchymal stem cells and gently resuspend to obtain the inner phase mixture. Step 2, preparation of external phase: Take mineral oil, add 1.5% to 2.5% by volume of surfactant Span-80, mix well to obtain external phase oil solution; Step 3, Microfluidic Pellet Formation and Solidification: The inner phase mixture and the outer phase oil are separately introduced into the flow-focusing microfluidic chip. A capillary count monitoring mechanism is introduced to fine-tune the outer phase flow rate based on the viscosity fluctuation of the inner phase, so that the diameter of the generated droplets meets the requirements. The relationship is controlled at 130μm. Within a 170μm range, as the droplets flow through the outlet, they are irradiated with visible light at a wavelength of 405nm for in-situ photocuring, and the cured microspheres are collected. Step 4, washing and collection: Add demulsifier to the collected microspheres, centrifuge to remove the upper oil phase, rinse the precipitate with PBS buffer containing bovine serum albumin, centrifuge to collect, and obtain modified gelatin microspheres carrying umbilical cord mesenchymal stem cells.

[0006] Preferably, the ratio of gelatin to sodium bicarbonate buffer in step 1 is 1 g:(8 mL to 12 mL), and the dropping rate of methacrylic anhydride is 0.3 mL / min to 0.7 mL / min; The activator is 1-(3-dimethylaminopropyl)-3-ethylcarbodiimide hydrochloride and N-hydroxysuccinimide.

[0007] Preferably, the sulfobetaine derivative containing a thiol group in step 2 is a thiolized product of the inner salt of 3-((2-(methacrylamido)ethyl)dimethylammonium)propane-1-sulfonic acid; and the catalyst is triethylamine.

[0008] Preferably, in step 3, the internal phase flow rate is 0.4 mL / h to 0.6 mL / h, and the external phase flow rate is 3.5 mL / h to 4.5 mL / h; the light intensity of the in-situ photocuring irradiation is 15 mW / cm² to 25 mW / cm², and the irradiation time is 20 s to 40 s.

[0009] Preferably, the demulsifier in step 4 is 1H,1H,2H,2H-perfluoro-1-octanol; the centrifugation speed is 1000 r / min to 2000 r / min, and the centrifugation time is 3 min to 5 min.

[0010] Preferably, the dispersing solvent is sterile physiological saline or low-viscosity pharmaceutical buffer; the concentration of trehalose in the internal phase raw material is preferably 0.2M.

[0011] A method for preparing a drug suspension composition for improving osteoarthritis includes: preparing modified gelatin microspheres carrying umbilical cord mesenchymal stem cells according to the method; adding the microspheres to a dispersion solvent by volume fraction; gently mixing them evenly under sterile conditions; and dispersing the drug suspension composition by utilizing the repulsive force provided by the sulfobetaine hydration layer on the surface of the modified gelatin microspheres.

[0012] Compared with the prior art, the present invention has the following beneficial effects: 1. This invention uses phenylboronic acid-sulfobetaine bifunctional modified gelatin as the microsphere matrix material, endowing the drug carrier with multiple key functions; the phenylboronic acid group not only provides a chemical anchoring point for the cartilage matrix, increasing the drug's retention time in the joint cavity, but also acts as a sacrificial target for reactive oxygen species, effectively clearing excess free radicals at the lesion site and improving the inflammatory microenvironment; at the same time, the sulfobetaine group constructs a strong hydration layer on the surface of the microspheres, and utilizes the electrostatic repulsion of zwitterions to effectively prevent the irreversible aggregation of microspheres in the static or in vivo environment, significantly improving the dispersion stability and lubrication performance of the suspension; 2. This invention introduces a specific concentration of trehalose into the inner phase of microspheres, which synergistically protects the cells with the hydrophilic network of modified gelatin. During freezing and thawing, trehalose can effectively replace water molecules to form hydrogen bonds with cell membranes and proteins, preventing mechanical damage to cells from ice crystals and protecting the hydration structure of sulfobetaine. This dual protection mechanism ensures the high survival rate and metabolic activity of umbilical cord mesenchymal stem cells after freeze-drying or deep cryogenic storage. Furthermore, the drug composition can be restored to uniform dispersion by gentle shaking after standing for 24 hours, solving the problem of poor storage and transportation stability of traditional cell preparations. 3. This invention utilizes microfluidic technology combined with in-situ photopolymerization to prepare cell-loaded microspheres. By controlling the ratio of internal and external phase flow rates and illumination parameters, the microsphere particle size can be controlled within a certain range. Precise control and uniform distribution within the range; the process uses a mild photoinitiation system and low light intensity to minimize damage to stem cell activity during preparation; the resulting microspheres have excellent shear-thinning properties, and can be smoothly injected through a fine needle even at high volume fractions, which ensures high cell loading per injection, reduces injection thrust in clinical operations, and improves patient compliance. 4. The drug suspension composition provided by this invention constructs a microenvironment suitable for stem cell survival and function; the moderate cross-linking of the modified gelatin network structure ensures the structural integrity of the microspheres in the joint cavity and facilitates the exchange of nutrients; the phenylboronic acid groups form dynamic covalent bonds with the diol structure on the cartilage surface, realizing the targeted adhesion and enrichment of microspheres at the cartilage defect site; combined with the hydrated lubricating layer on the surface of the microspheres, this composition provides biological therapy while playing a physical friction-reducing role, and can maintain a high cell survival rate in the simulated oxidative environment of the joint cavity, achieving a comprehensive therapeutic effect of anti-inflammation, lubrication and cartilage repair. Attached Figure Description

[0013] To more clearly illustrate the technical solutions in the embodiments of the present invention or the prior art, the drawings used in the description of the embodiments or the prior art will be briefly introduced below. Obviously, the drawings described below are only some embodiments of the present invention. For those skilled in the art, other drawings can be obtained based on these drawings without creative effort. Figure 1 This is a flowchart of a method for preparing a drug suspension for improving osteoarthritis according to the present invention. Detailed Implementation

[0014] The technical solutions of the present invention will be clearly and completely described below with reference to the accompanying drawings of the embodiments of the present invention. Obviously, the described embodiments are only some embodiments of the present invention, and not all embodiments. All other embodiments obtained by those skilled in the art based on the embodiments of the present invention without creative effort are within the scope of protection of the present invention.

[0015] Example 1: This embodiment provides a drug suspension composition for improving osteoarthritis. The drug suspension composition consists of modified gelatin microspheres loaded with umbilical cord mesenchymal stem cells and a dispersing solvent, wherein the volume fraction of the microspheres in the suspension is set to 50%. The modified gelatin microspheres loaded with umbilical cord mesenchymal stem cells are dispersed in sterile physiological saline as the dispersing solvent. The modified gelatin microspheres loaded with umbilical cord mesenchymal stem cells are prepared from internal phase raw materials using microfluidic technology. The internal phase raw materials include: 5% w / v phenylboronic acid-sulfobetaine bifunctional modified gelatin, 0.2M trehalose, 0.5% w / v photoinitiator LAP, and a concentration of [missing information - likely a specific concentration]. Umbilical cord mesenchymal stem cells; In the preparation of phenylboronic acid-sulfobetaine bifunctional modified gelatin, phenylboronic acid modified methacrylamide gelatin was prepared as follows: gelatin was dissolved in sodium bicarbonate buffer at pH 9.0 at a ratio of 1g:10mL, and stirred at 50℃. Methacrylic anhydride was added dropwise at a rate of 0.5mL / min, and the reaction was carried out for 2h. After dialyzing and lyophilization, methacrylamide gelatin was obtained. 3-Carboxyphenylboronic acid was dissolved in MES buffer at pH 5.5, and activators, namely EDC and NHS, were added for 30min. The activators were added dropwise to PBS solution at pH 7.4 containing methacrylamide gelatin, and the reaction was carried out at room temperature in the dark for 24h. After dialyzing and lyophilization, phenylboronic acid modified methacrylamide gelatin was obtained. Preparation of phenylboronic acid-sulfobetaine bifunctional modified gelatin: phenylboronic acid-modified methacrylamide gelatin was dissolved in PBS at pH 8.0, and nitrogen gas was purged for 30 min to remove oxygen. A sulfobetaine derivative containing thiol (the thiolized product of the inner salt of 3-((2-(methacrylamido)ethyl)dimethylammono)propane-1-sulfonic acid) and triethylamine as catalyst were added. Michael addition reaction was carried out at 37℃ under closed conditions for 12 h. The final product was obtained by dialysis and lyophilization. The preparation method of modified gelatin microspheres carrying umbilical cord mesenchymal stem cells is as follows: Figure 1 As shown, specifically: when preparing the inner phase, phenylboronic acid-sulfobetaine bifunctional modified gelatin was dissolved in PBS, 0.2M trehalose and a photoinitiator were added, and after sterile filtration, umbilical cord mesenchymal stem cells were added for resuspension; when preparing the outer phase, mineral oil was added to 2.0% by volume of Span-80; during the microfluidic sphere formation and solidification stage, the inner phase flow rate was 0.5 mL / h, and the outer phase flow rate was 4.0 mL / h. A capillary number monitoring mechanism was introduced, and the outer phase flow rate was finely adjusted to control the droplet diameter at 150 μm. The droplets were then irradiated in situ with light at a wavelength of 405 nm and an intensity of 20 mW / cm² for 30 s at the outlet; after demulsification with 1H,1H,2H,2H-perfluoro-1-octanol, centrifugation at 1500 r / min for 4 min, and washing with PBS containing bovine serum albumin, microspheres were obtained; the microspheres were added to the dispersion solvent at a volume fraction of 50% and gently mixed evenly; In this embodiment, the phenylboronic acid-sulfobetaine bifunctional modified gelatin utilizes the phenylboronic acid group to provide a chemical anchoring site for the cartilage matrix, while also serving as a sacrificial target for reactive oxygen species; a strong hydration layer is constructed using the sulfobetaine group; trehalose at a concentration of 0.2M effectively protects the hydration structure and cell activity of sulfobetaine during freezing and thawing; the drug composition did not exhibit irreversible hard aggregation after standing for 24 hours, and recovered uniform dispersion with gentle shaking; and cell viability remained at a high level under simulated joint cavity oxidation conditions.

[0016] Example 2: This embodiment provides a drug suspension composition for improving osteoarthritis; the drug suspension composition consists of modified gelatin microspheres carrying umbilical cord mesenchymal stem cells and a dispersing solvent, wherein the volume fraction of the microspheres in the suspension is set to 45%; the modified gelatin microspheres carrying umbilical cord mesenchymal stem cells are dispersed in a low-viscosity pharmaceutical buffer as the dispersing solvent; the modified gelatin microspheres carrying umbilical cord mesenchymal stem cells are prepared from internal phase raw materials using microfluidic technology, the internal phase raw materials including: 5% w / v phenylboronic acid-sulfobetaine bifunctional modified gelatin, 0.1M trehalose, 0.5% w / v photoinitiator, and a concentration of [missing information]. Umbilical cord mesenchymal stem cells; The preparation parameters for phenylboronic acid-sulfobetaine bifunctional modified gelatin were adjusted as follows: For the preparation of phenylboronic acid-modified methacrylamide gelatin, the ratio of gelatin to sodium bicarbonate buffer was 1 g:8 mL, the reaction temperature was 45℃, the methacrylic anhydride dropping rate was 0.3 mL / min, and the reaction time was 1 h; 3-carboxyphenylboronic acid was activated for 20 min, and the reaction time was 20 h; For the preparation of phenylboronic acid-sulfobetaine bifunctional modified gelatin, oxygen was removed for 20 min, the Michael addition reaction temperature was 35℃, and the reaction time was 10 h. The preparation parameters for modified gelatin microspheres carrying umbilical cord mesenchymal stem cells were adjusted as follows: external phase surfactant volume fraction 1.5%; microfluidic internal phase flow rate 0.4 mL / h, external phase flow rate 3.5 mL / h, droplet diameter controlled at 130 μm; in-situ photocuring light intensity 15 mW / cm², irradiation time 40 s; centrifugation speed 1000 r / min, time 3 min during washing; In this embodiment, the lower microsphere volume fraction of 45% and the smaller microsphere particle size of 130 μm endow the drug composition with lower injection thrust, making it suitable for fine needle injection; although the trehalose concentration of 0.1 M is at the lower limit, the hydrophilic network of the phenylboronic acid-sulfobetaine bifunctional modified gelatin still maintains basic cell protection; the milder photocuring conditions of 15 mW / cm² reduce the potential impact of free radicals on cells, resulting in excellent cell viability of the drug composition in the early stages of preparation.

[0017] Example 3: This embodiment provides a drug suspension composition for improving osteoarthritis; the drug suspension composition consists of modified gelatin microspheres carrying umbilical cord mesenchymal stem cells and a dispersing solvent, wherein the volume fraction of the microspheres in the suspension is set to 55%; the modified gelatin microspheres carrying umbilical cord mesenchymal stem cells are dispersed in sterile physiological saline as the dispersing solvent; the modified gelatin microspheres carrying umbilical cord mesenchymal stem cells are prepared from internal phase raw materials using microfluidic technology, the internal phase raw materials include: 5% w / v phenylboronic acid-sulfobetaine bifunctional modified gelatin, 0.5M trehalose, 0.5% w / v photoinitiator, and a concentration of [missing information]. Umbilical cord mesenchymal stem cells; The preparation parameters for phenylboronic acid-sulfobetaine bifunctional modified gelatin were adjusted as follows: For the preparation of phenylboronic acid-modified methacrylamide gelatin, the ratio of gelatin to sodium bicarbonate buffer was 1 g:12 mL, the reaction temperature was 55℃, the methacrylic anhydride dropping rate was 0.7 mL / min, and the reaction time was 3 h; 3-carboxyphenylboronic acid was activated for 40 min, and the reaction time was 28 h; For the preparation of phenylboronic acid-sulfobetaine bifunctional modified gelatin, oxygen was removed for 40 min, the Michael addition reaction temperature was 40℃, and the reaction time was 14 h. The preparation parameters for modified gelatin microspheres carrying umbilical cord mesenchymal stem cells were adjusted as follows: external phase surfactant volume fraction 2.5%; microfluidic internal phase flow rate 0.6 mL / h, external phase flow rate 4.5 mL / h, droplet diameter controlled at 170 μm; in-situ photocuring light intensity 25 mW / cm², irradiation time 20 s; centrifugation speed 2000 r / min, time 5 min during washing; In this embodiment, a higher microsphere volume fraction of 55% and cell concentration were used. The therapeutic dose per unit volume was significantly increased; the high concentration of 0.5M trehalose provided enhanced cryoprotection, ensuring a high recovery rate after storage at extremely low temperatures; although the high volume fraction increased viscosity, the drug composition still maintained good shear-thinning properties due to the strong electrostatic repulsion and hydration lubrication provided by the sulfobetaine groups on the surface of the phenylboronic acid-sulfobetaine bifunctionalized gelatin, enabling it to be smoothly injected through a syringe and achieve high-density coverage at the lesion site.

[0018] Example 4: This embodiment provides a drug suspension composition for improving osteoarthritis; the drug suspension composition consists of modified gelatin microspheres carrying umbilical cord mesenchymal stem cells and a dispersing solvent, wherein the volume fraction of the microspheres in the suspension is set to 48%; the modified gelatin microspheres carrying umbilical cord mesenchymal stem cells are dispersed in a low-viscosity pharmaceutical buffer; the modified gelatin microspheres carrying umbilical cord mesenchymal stem cells are prepared from internal phase raw materials using microfluidic technology, the internal phase raw materials including: 5% w / v phenylboronic acid-sulfobetaine bifunctional modified gelatin, 0.3M trehalose, 0.5% w / v photoinitiator, and a concentration of... Umbilical cord mesenchymal stem cells; The preparation process parameters for phenylboronic acid-sulfobetaine bifunctional modified gelatin and microspheres are between those of Example 1 and Example 2: methacrylation reaction temperature 50℃, reaction time 2h; Michael addition reaction temperature 37℃, reaction time 12h; during microfluidic sphere formation, the internal phase flow rate is 0.45mL / h, the external phase flow rate is 3.8mL / h, and the droplet diameter is controlled at 140μm; the in-situ photocuring light intensity is 18mW / cm², and the irradiation time is 35s. In this embodiment, by adjusting the trehalose concentration to 0.3M and the appropriate cell loading, the drug composition exhibits good comprehensive performance in balancing cell metabolic activity and resisting cryopreservation damage; the crosslinking density of the phenylboronic acid-sulfobetaine bifunctional modified gelatin network structure is moderate, which not only ensures the structural integrity of the microspheres in the joint cavity, but also facilitates the contact between the phenylboronic acid groups and the cartilage surface, thus achieving effective tissue adhesion and reactive oxygen species scavenging.

[0019] Example 5: This embodiment provides a drug suspension composition for improving osteoarthritis; the drug suspension composition consists of modified gelatin microspheres carrying umbilical cord mesenchymal stem cells and a dispersing solvent, wherein the volume fraction of the microspheres in the suspension is set to 52%; the modified gelatin microspheres carrying umbilical cord mesenchymal stem cells are dispersed in sterile physiological saline; the modified gelatin microspheres carrying umbilical cord mesenchymal stem cells are prepared from internal phase raw materials using microfluidic technology, the internal phase raw materials include: 5% w / v phenylboronic acid-sulfobetaine bifunctional modified gelatin, 0.4M trehalose, 0.5% w / v photoinitiator, and a concentration of [missing information]. Umbilical cord mesenchymal stem cells; The preparation process parameters of phenylboronic acid-sulfobetaine bifunctional modified gelatin and microspheres are between those of Example 1 and Example 3; during microfluidic sphere formation, the internal phase flow rate is 0.55 mL / h, the external phase flow rate is 4.2 mL / h, and the droplet diameter is controlled at 160 μm; the in-situ photocuring light intensity is 22 mW / cm², and the irradiation time is 25 s; In this embodiment, the 0.4M trehalose concentration, in synergy with the phenylboronic acid-sulfobetaine bifunctional modified gelatin, effectively prevents structural damage and aggregation of microspheres due to ice crystal formation under freeze-drying or deep cryogenic storage conditions. The 52% volume fraction ensures high bioavailability while maintaining the fluidity of the suspension through the zwitterionic hydration layer on the surface of the microspheres, resulting in good injection feel and uniform dispersion of the drug composition in clinical operations.

[0020] In Examples 1-5 and Comparative Examples 1-4, the gelatin was from Shanghai Aladdin Biochemical Technology Co., Ltd., type A (acid extraction), CAS No.: 9000-70-8; the methacrylic anhydride was from Shanghai Maclean Biochemical Technology Co., Ltd., with a purity of 94% and containing 2000 ppm. 2,6-Di-tert-butyl-p-cresol stabilizer, CAS No.: 760-93-0; 3-Carboxyphenylboronic acid from Shanghai Aladdin Biochemical Technology Co., Ltd., purity 97%, CAS No.: 25487-66-5; Trehalose from Sigma-Aldrich, biotechnology grade, purity ≥99%, CAS No.: 6138-23-4; Photoinitiator LAP (phenyl-2,4,6-trimethylbenzoylphosphine lithium) from Suzhou Kanan Biotechnology Co., Ltd., purity ≥98%, CAS No.: 85073-19-4; 1-(3-dimethylaminopropyl)-3-ethylcarbodiimide hydrochloride (EDC) and N-hydroxysuccinimide (NHS) are both from Shanghai Aladdin Biochemical Technology Co., Ltd. Aladdin Biochemical Technology Co., Ltd., purity 98%; surfactant Span-80 from Sinopharm Chemical Reagent Co., Ltd., chemically pure, CAS No.: 1338-43-8; 1H,1H,2H,2H-perfluoro-1-octanol from Shanghai Maclean Biochemical Technology Co., Ltd., purity 97%, CAS No.: 647-42-7; sulfobetaine derivative raw material from Henan Lezhi Chemical Technology Co., Ltd., purity 98%; umbilical cord mesenchymal stem cells purchased from Cyagen (Suzhou) Biotechnology Co., Ltd., catalog number: HUXUC-01001; bovine serum albumin (BSA) from Beijing Solarbio Science & Technology Co., Ltd., catalog number: A8020; other reagents are commercially available analytical grade products.

[0021] Comparative Example 1: This comparative example provides a drug suspension composition that differs from Example 1 in that: when preparing the modified gelatin, the phenylboronic acid-sulfobetaine bifunctional modification in step 2 was not performed, and only the phenylboronic acid-modified methacrylamide gelatin obtained in step 1 was used as the microsphere raw material; the remaining preparation steps and parameters are consistent with those in Example 1; due to the lack of sulfobetaine groups, the surface of the microspheres lacks a strong hydration layer.

[0022] Comparative Example 2: This comparative example provides a drug suspension composition that differs from Example 1 in that: when preparing the modified gelatin, 3-carboxyphenylboronic acid was not grafted, and sulfobetaine derivatives were grafted only onto the methacrylated gelatin; the remaining preparation steps and parameters are consistent with those of Example 1; due to the lack of phenylboronic acid groups, the microspheres lack reactive oxygen species responsiveness and active adhesion sites.

[0023] Comparative Example 3: This comparative example provides a drug suspension composition that differs from Example 1 in that: no trehalose is added to the internal phase raw material, while the remaining components and preparation method are completely consistent with Example 1; the performance after freezing and thawing is investigated in the absence of trehalose.

[0024] Comparative Example 4: This comparative example provides a drug suspension composition that differs from Example 1 in that it uses a traditional physical blending method to directly mix 3-carboxyphenylboronic acid and sulfobetaine into the gelatin solution, rather than through chemical bonding, i.e., amide bonds and Michael addition grafting onto the gelatin backbone; the remaining preparation steps and parameters are the same as in Example 1.

[0025] To verify the beneficial effects of the technical solution of the present invention, the drug suspension compositions prepared in Examples 1-5 and Comparative Examples 1-4 were subjected to performance tests. The test items included: 1. Physical stability: After the suspension was allowed to stand for 24 hours, the ratio of the sedimentation layer height to the total height was measured. The closer the F value was to 1, the better the suspension stability. 2. Antioxidant capacity: After culturing in 200 μM H2O2 environment for 48 hours, the cell viability was detected. 3. Tissue adhesion: After flushing in an isolated cartilage defect model, the retention percentage of microspheres was calculated. 4. Resuscitation and dispersibility: After freezing and thawing at -80℃, the presence of hard agglomerates was observed, and the morphology and dispersion of microspheres were observed under a microscope.

[0026] The test results are shown in the table below. Results analysis: As shown in the table, the drug suspension compositions prepared in Examples 1-5 all performed excellently in all indicators.

[0027] Comparing Example 1 with Comparative Example 1: Comparative Example 1 lacks the modification of sulfobetaine, and the microspheres lack the electrostatic repulsion provided by the strong hydration layer, resulting in a significant decrease in physical stability, i.e., an F value of only 0.35, and severe aggregation after recovery; This indicates that in the phenylboronic acid-sulfobetaine bifunctional modified gelatin, the sulfobetaine introduced by Michael addition is the key to achieving zero aggregation and high dispersibility; Comparative Example 1 and Comparative Example 2: Comparative Example 2 lacks phenylboronic acid groups. Although it has good dispersibility, its cell survival rate is significantly reduced under oxidative stress and its retention rate on the cartilage surface is extremely low. This confirms that the phenylboronic acid group plays a dual role as a sacrificial target for reactive oxygen species and a tissue adhesion anchor, which is the core of the composition to achieve therapeutic effects. Comparative Example 1 and Comparative Example 3: Comparative Example 3 did not add trehalose, which resulted in some hard agglomeration after freezing and thawing, and a decrease in cell viability; this indicates that the concentration of trehalose plays an important role in maintaining the structural integrity of microspheres at low temperature and protecting cell activity. Compared with Comparative Example 1 and Comparative Example 4, the physical mixing group showed unstable performance in all aspects, indicating that a specific chemical grafting path is crucial for constructing a stable polymer network structure, and chemical bonding ensures the long-term effect of functional groups under physiological conditions. In summary, this invention, by using phenylboronic acid-sulfobetaine bifunctional modified gelatin as a carrier material, combined with trehalose and specific microfluidic technology, successfully solves the technical problems of traditional stem cell suspensions being prone to sedimentation, aggregation, low survival rate at lesions, and poor retention capacity, and provides a drug suspension composition for improving osteoarthritis with good clinical application prospects. The above description is merely a preferred embodiment of the present invention and is not intended to limit the present invention. Although the present invention has been described in detail with reference to the foregoing embodiments, those skilled in the art can still modify the technical solutions described in the foregoing embodiments or make equivalent substitutions for some of the technical features. Any modifications, equivalent substitutions, improvements, etc., made within the spirit and principles of the present invention should be included within the protection scope of the present invention. It should be noted that, in this document, the terms include, encompass, or any other variations thereof are intended to cover non-exclusive inclusion, such that a process, method, article, or apparatus that includes a list of elements includes not only those elements but also other elements not expressly listed, or elements inherent to such a process, method, article, or apparatus; and, without further limitation, an element defined by the phrase including a… does not exclude the presence of other identical elements in the process, method, article, or apparatus that includes said element. In the description of this invention, it should be understood that the terms first, second, etc. are used only for distinguishing descriptions and should not be construed as indicating or implying relative importance; In the description of this invention, unless otherwise stated, "multiple" means two or more. In the description of this invention, "several" means one or more, and "a large number" means two or more. In the description of this specification, references to the terms "an embodiment," "some embodiments," "example," "specific example," or "some examples," etc., indicate that a specific feature, structure, material, or characteristic described in connection with that embodiment or example is included in at least one embodiment or example of the present invention; in this specification, the illustrative expressions of the above terms do not necessarily refer to the same embodiment or example; moreover, the specific features, structures, materials, or characteristics described may be combined in any suitable manner in one or more embodiments or examples; Although embodiments of the invention have been shown and described, those skilled in the art will understand that various changes, modifications, substitutions and alterations can be made to these embodiments without departing from the principles and spirit of the invention, the scope of which is defined by equivalents.

[0028] The above are merely preferred embodiments of the present invention and are not intended to limit the present invention in any other way. Any person skilled in the art may make changes or modifications to the above-disclosed technical content to create equivalent embodiments that can be applied to other fields. However, any simple modifications, equivalent changes, and modifications made to the above embodiments based on the technical essence of the present invention without departing from the scope of the present invention shall still fall within the protection scope of the present invention.

Claims

1. A drug suspension composition for improving osteoarthritis, characterized in that, The pharmaceutical composition comprises modified gelatin microspheres carrying umbilical cord mesenchymal stem cells and a dispersing solvent; Modified gelatin microspheres carrying umbilical cord mesenchymal stem cells are dispersed in the dispersion solvent, and the volume fraction of the microspheres in the suspension is 45%–55%. Modified gelatin microspheres carrying umbilical cord mesenchymal stem cells were prepared using microfluidic technology from an inner phase raw material comprising the following concentrations: 5% w / v phenylboronic acid-sulfobetaine bifunctional modified gelatin, 0.1 M–0.5 M trehalose, 0.5% w / v photoinitiator, and a concentration of [missing information - likely a specific concentration]. ~ Umbilical cord mesenchymal stem cells; The product obtained by phenylboronic acid-sulfobetaine bifunctional modified gelatin is a gelatin skeleton modified with methacryloyl groups by introducing methacrylic anhydride, using the remaining amino groups to graft 3-carboxyphenylboronic acid through amide bonds, and using some of the methacryloyl groups to graft sulfobetaine derivatives containing mercapto groups through Michael addition reaction. Umbilical cord mesenchymal stem cells are encapsulated within a cross-linked network formed by the phenylboronic acid-sulfobetaine bifunctional modified gelatin.

2. The drug suspension composition for improving osteoarthritis according to claim 1, characterized in that, The preparation method of the phenylboronic acid-sulfobetaine bifunctional modified gelatin includes the following steps: Step 1: Preparation of phenylboronic acid modified methacrylamide gelatin: Dissolve gelatin in sodium bicarbonate buffer at pH 9.0, stir and dissolve at a constant temperature of 45℃~55℃, add methacrylic anhydride dropwise, react for 1h~3h, purify by dialysis and freeze-dry to obtain methacrylamide gelatin; Dissolve 3-carboxyphenylboronic acid in MES buffer at pH 5.5, add activator and activate for 20min~40min, dropwise add to PBS solution at pH 7.4 containing methacrylamide gelatin, react at room temperature in the dark for 20h~28h, purify by dialysis and freeze-dry to obtain phenylboronic acid modified methacrylamide gelatin; Step 2: Preparation of phenylboronic acid-sulfobetaine bifunctional modified gelatin: The phenylboronic acid modified methacrylamide gelatin obtained in Step 1 was dissolved in PBS at pH 8.0, and nitrogen gas was introduced for deoxygenation treatment for 20 min to 40 min. A sulfobetaine derivative containing thiol groups and a catalyst were added, and a Michael addition reaction was carried out under closed conditions at 35℃ to 40℃ for 10 h to 14 h. The reaction solution was purified by dialysis and freeze-dried to obtain phenylboronic acid-sulfobetaine bifunctional modified gelatin.

3. The drug suspension composition for improving osteoarthritis according to claim 1, characterized in that, The method for preparing the modified gelatin microspheres carrying umbilical cord mesenchymal stem cells includes the following steps: Step 1, Preparation of the inner phase: Dissolve the phenylboronic acid-sulfobetaine bifunctional modified gelatin in PBS, add trehalose and photoinitiator, mix well and then filter under sterile conditions. Add umbilical cord mesenchymal stem cells and gently resuspend to obtain the inner phase mixture. Step 2, Preparation of external phase: Take mineral oil, add 1.5% to 2.5% by volume of surfactant Span-80, mix well to obtain external phase oil solution; Step 3, Microfluidic Pellet Formation and Solidification: The inner phase mixture and the outer phase oil are separately introduced into the flow-focusing microfluidic chip. A capillary count monitoring mechanism is introduced to fine-tune the outer phase flow rate based on the viscosity fluctuation of the inner phase, so that the diameter of the generated droplets meets the requirements. The relationship between the droplets and the microspheres was controlled within the range of 130μm to 170μm. When the droplets flowed through the outlet, they were irradiated with visible light at a wavelength of 405nm for in-situ photocuring, and the cured microspheres were collected. Step 4, washing and collection: Add demulsifier to the collected microspheres, centrifuge to remove the upper oil phase, rinse the precipitate with PBS buffer containing bovine serum albumin, centrifuge to collect, and obtain modified gelatin microspheres carrying umbilical cord mesenchymal stem cells.

4. The drug suspension composition for improving osteoarthritis according to claim 2, characterized in that, In step 1, the ratio of gelatin to sodium bicarbonate buffer is 1 g:(8 mL to 12 mL), the dropping rate of methacrylic anhydride is 0.3 mL / min to 0.7 mL / min, and the activator is 1-(3-dimethylaminopropyl)-3-ethylcarbodiimide hydrochloride and N-hydroxysuccinimide.

5. The drug suspension composition for improving osteoarthritis according to claim 2, characterized in that, The sulfobetaine derivative containing a thiol group mentioned in step 2 is the thiolized product of the inner salt of 3-((2-(methacrylamido)ethyl)dimethylammonium)propane-1-sulfonic acid; the catalyst is triethylamine.

6. The drug suspension composition for improving osteoarthritis according to claim 3, characterized in that, In step 3, the internal phase flow rate is 0.4 mL / h to 0.6 mL / h, and the external phase flow rate is 3.5 mL / h to 4.5 mL / h; the light intensity of the in-situ photocuring irradiation is 15 mW / cm² to 25 mW / cm², and the irradiation time is 20 s to 40 s.

7. The drug suspension composition for improving osteoarthritis according to claim 3, characterized in that, The demulsifier mentioned in step 4 is 1H,1H,2H,2H-perfluoro-1-octanol; the centrifugation speed is 1000 r / min to 2000 r / min, and the centrifugation time is 3 min to 5 min.

8. The drug suspension composition for improving osteoarthritis according to claim 1, characterized in that, The dispersion solvent is sterile physiological saline or low-viscosity pharmaceutical buffer; the concentration of trehalose in the internal phase raw material is preferably 0.2M.

9. A method for preparing a drug suspension composition for improving osteoarthritis as described in claim 1, characterized in that, The preparation method includes the following steps: modified gelatin microspheres carrying umbilical cord mesenchymal stem cells are prepared according to the method; the microspheres are added to the dispersion solvent according to the volume fraction; they are gently mixed evenly under sterile conditions; and dispersion is achieved by utilizing the repulsive force provided by the sulfobetaine hydration layer on the surface of the modified gelatin microspheres to obtain a drug suspension composition.

Images