Co-loaded dexamethasone sodium phosphate and type i collagen microspheres and preparation method thereof

Abstract

The application discloses a kind of co-loading dexamethasone sodium phosphate and type I collagen microspheres and preparation method thereof, comprising the following steps: respectively dexamethasone sodium phosphate and type I collagen are dissolved in water to obtain aqueous solution;Carrier material is dissolved in organic solvent to obtain oil phase solution;Aqueous solution is added to oil phase solution, and mixed to obtain W / O initial milk;W / O initial milk is added to the first PVA solution, and mixed to obtain W / O / W complex milk;W / O / W complex milk is transferred to the second PVA solution, solidification forming, freeze-drying treatment.The preparation conditions of the application are optimized initial milk and complex milk, and the method of more gentle homogenate and ultrasound is used, and the damage to the biological activity of type I collagen is small, and the organic solvent of oil phase is replaced by less toxic and more moderate polarity ethyl acetate, which can reduce the water phase migration of two active ingredients in the preparation process, and can significantly improve the clinical safety of preparation while ensuring the forming quality of microspheres.

Description

Technical Field

[0001] This invention relates to the field of drug-loaded microsphere technology, specifically to a microsphere co-loaded with dexamethasone sodium phosphate and type I collagen, and its preparation method. Background Technology

[0002] Knee osteoarthritis (KOA) is a chronic degenerative disease characterized by degenerative changes in articular cartilage, chronic inflammation of the synovium, cartilage damage, subchondral bone remodeling, and impaired joint function. It is most common in middle-aged and elderly people, but in recent years, due to factors such as sports injuries and obesity, it has shown a trend of affecting younger people and has become one of the leading causes of disability worldwide. The disease has a long and progressive course, with typical clinical manifestations including joint pain, swelling, stiffness, and limited mobility, severely impacting patients' quality of life and placing a heavy burden on the social healthcare system.

[0003] Currently, routine clinical treatment for KOA mainly involves oral nonsteroidal anti-inflammatory drugs (NSAIDs), intra-articular injection of glucocorticoids, physical therapy, and end-stage joint replacement. However, existing regimens have significant limitations: systemic oral drugs have low bioavailability and long-term use can easily cause gastrointestinal, liver, and kidney damage; intra-articular injection of free drugs results in large fluctuations in local drug concentration and a short half-life, requiring repeated administration and increasing the risk of joint infection; traditional treatments only focus on short-term anti-inflammatory and analgesic effects, lacking repair effects on damaged cartilage and failing to reverse the pathological process of KOA. Therefore, developing a long-acting local delivery system that can simultaneously achieve anti-inflammatory and repair-promoting effects and precisely release active ingredients has become an important direction for the research and development of new targeted therapies for KOA.

[0004] Dexamethasone sodium phosphate is a widely used water-soluble glucocorticoid in clinical practice. It has potent anti-inflammatory, analgesic, anti-allergic, and immunosuppressive activities, and plays an important role in the treatment of inflammatory diseases such as osteoarthritis and soft tissue injuries. However, its free form has a short half-life and rapid clearance rate in the body, requiring frequent administration to maintain an effective local concentration. This can easily cause local tissue irritation and systemic adverse reactions, resulting in significant limitations in its clinical application.

[0005] Type I collagen possesses excellent biocompatibility, biodegradability, and cell adhesion activity. It can assist cell adhesion, proliferation, and differentiation by providing an extracellular scaffold, demonstrating significant regenerative potential in the repair of bone, cartilage, and soft tissue injuries. However, as a large-molecule active substance, type I collagen has poor stability and is easily degraded rapidly by proteases in the body. After being administered in its free form, it is difficult to maintain its biological activity at the injury site for a long period. When used alone, its repair-promoting effect is limited and cannot meet the long-term needs of tissue repair.

[0006] Therefore, developing a long-acting delivery system co-loaded with dexamethasone sodium phosphate and type I collagen can synergize the anti-inflammatory and analgesic pharmacological activity of dexamethasone sodium phosphate with the physical adhesion and repair-promoting properties of type I collagen. This allows the microspheres to adhere to the surface of damaged cartilage in the joint cavity, forming a protective physical barrier. This reduces the contact between damaged cartilage and inflammatory factors in the joint cavity, thereby reducing further cartilage damage and achieving a "drug-device synergy" effect. This is a new strategy that breaks through the limitations of existing treatment options.

[0007] Long-acting injectable microsphere formulations, by maintaining effective drug concentrations at the injection site for an extended period after a single local administration, significantly reduce dosing frequency and improve patient compliance, and have become an important technological platform for the long-acting delivery of peptides, proteins, and small molecule drugs. Biodegradable polymers such as PLGA (polylactic-co-glycolic acid copolymer) have been approved by the FDA for the development of various long-acting microsphere formulations due to their good biocompatibility, adjustable degradation rate, and drug loading capacity, such as leuprorelin microspheres, triptorelin microspheres, and risperidone microspheres, fully validating the clinical translational feasibility of this technology.

[0008] W / O / W double emulsion is currently the mainstream method for preparing water-soluble drug microspheres. However, type I collagen is easily denatured during double emulsion preparation due to the influence of organic solvents, shear forces, and interfacial tension, leading to reduced bioactivity and affecting its adhesion-promoting and repair functions. In addition, research on the combined application of dexamethasone sodium phosphate and type I collagen has mostly focused on dosage forms such as hydrogels and composite scaffolds. Although such formulations can achieve co-loading of the two components, there are still many technical bottlenecks: the degradation rate of collagen-based hydrogels is too fast, which can easily lead to a large burst release of the drug in the early stage, but insufficient effective concentration in the later stage, making it difficult to match the dual-stage treatment needs of inflammation suppression and tissue repair; although composite scaffolds can increase drug loading, the preparation process is complex, large-scale production is difficult, and the release behavior of the two active ingredients is difficult to independently control, making it impossible to achieve the time-sequential release of rapid anti-inflammatory effect during the inflammatory phase and continuous regeneration promotion during the repair phase. In addition, existing microsphere preparation processes generally use dichloromethane as an oil phase organic solvent. Dichloromethane is a Class II halogenated toxic solvent, and excessive residues can lead to neurotoxicity and potential carcinogenic risks. Pharmacopoes of various countries strictly control its residue limits. Summary of the Invention

[0009] This invention addresses the shortcomings of existing technologies by providing a method for preparing microspheres co-loaded with dexamethasone sodium phosphate and type I collagen. It optimizes the preparation conditions of the colostrum and re-emulsion, employs gentler homogenization and sonication methods, minimizes damage to the bioactivity of type I collagen, and replaces the oil-phase organic solvent with ethyl acetate, which is less toxic and has a milder polarity. This reduces the migration of the two active ingredients in the aqueous phase during the preparation process and significantly improves the clinical safety of the formulation while ensuring the quality of microsphere formation.

[0010] To address the aforementioned technical problems, the first aspect of this invention provides a method for preparing co-loaded dexamethasone sodium phosphate and type I collagen microspheres, comprising the following steps:

[0011] S1. Dissolve dexamethasone sodium phosphate and type I collagen in water to obtain an aqueous solution; dissolve the carrier material in an organic solvent to obtain an oil solution.

[0012] S2. Add the aqueous phase solution to the oil phase solution and mix to obtain a W / O primary emulsion;

[0013] S3. Add the W / O pre-emulsion to the first PVA solution and mix to obtain a W / O / W double emulsion;

[0014] S4. The W / O / W double emulsion is transferred to the second PVA solution, solidified and lyophilized in a freeze-drying protectant solution to obtain the co-loaded dexamethasone sodium phosphate and type I collagen microspheres.

[0015] This invention employs optimized preparation conditions for colostrum and re-emulsion, using gentler homogenization and ultrasonic methods. This minimizes damage to the bioactivity of type I collagen, resulting in drug-loaded polymer microspheres that are perfectly spherical with uniform particle size, good drug loading, and a sustained-release period of over twenty days without any burst release.

[0016] This invention replaces the oil-phase organic solvent with ethyl acetate, which is less toxic and has a milder polarity. This reduces the migration of the two active ingredients into the aqueous phase during the preparation process and significantly improves the clinical safety of the formulation while ensuring the quality of microsphere molding.

[0017] Furthermore, in S1, the concentration of dexamethasone sodium phosphate dissolved in water is 10-20 mg / mL, and the concentration of type I collagen dissolved in water is 15-30 mg / mL.

[0018] Furthermore, in S1, the concentration of the carrier material dissolved in the organic solvent is 20-100 mg / mL, and the organic solvent is ethyl acetate.

[0019] Furthermore, in S1, the carrier material is selected from polylactic acid and / or polylactic acid-glycolic acid copolymer;

[0020] The molecular weight of the polylactic acid is 40K-60K, and the molecular weight of the polylactic acid-glycolic acid copolymer is 20K-107K.

[0021] The molar ratio of polylactic acid to glycolic acid in the polylactic acid-glycolic acid copolymer is (85:15) to (50:50).

[0022] Furthermore, in S2 and S3, the mixing process specifically involves: vortexing for 8-15 seconds, followed by homogenization. Preferably, the homogenization speed is 8000-12000 rpm, and the time is 20-60 seconds.

[0023] Furthermore, in S3, the concentration of the first PVA solution is 0.8-1.0 wt%.

[0024] Furthermore, in S4, the concentration of the second PVA solution is 4-10 mg / mL.

[0025] Further, in S4, the curing and molding process specifically involves: magnetic stirring to evaporate the solvent, wherein the magnetic stirring speed is 200-400 rpm and the time is 3-6 hours. Preferably, after curing and molding, the cured microspheres are collected by centrifugation at a speed of 2000-3000 rpm for 15-20 minutes.

[0026] Furthermore, in S4, the lyophilization protectant is selected from trehalose and / or sorbitol, and the concentration of the lyophilization protectant in the lyophilization protectant solution is 10-50 mg / mL.

[0027] The second aspect of the present invention provides co-loaded dexamethasone sodium phosphate and type I collagen microspheres prepared by the preparation method described in the first aspect.

[0028] The beneficial effects of this invention are:

[0029] This invention employs a W / O / W double emulsion method, optimizing the preparation conditions of the colostrum and double emulsion. The entire process utilizes gentler homogenization and ultrasonic methods to prevent drug damage. By optimizing and adjusting the preparation parameters, the particle size distribution and pore structure of the microspheres are precisely controlled, improving the drug encapsulation efficiency and reducing drug adsorption on the microsphere surface, thereby reducing the burst release effect.

[0030] This invention achieves an encapsulation rate of approximately 70% for both dexamethasone sodium phosphate and type I collagen, with a low burst release rate (first-day release rate controllable within the range of 1.47-7.43%), stable and sustained drug release rate, and a sustained-release period of up to twenty days. Furthermore, the microspheres have smooth surfaces, intact shapes, uniform particle size distribution, and minimal batch-to-batch variation. This method enables long-term synergistic release of the two drugs, leveraging the potent anti-inflammatory pharmacological effects of dexamethasone sodium phosphate while utilizing the physical adhesion properties of type I collagen to allow the microspheres to adhere to the surface of damaged cartilage, achieving a synergistic therapeutic effect. It is particularly suitable for the treatment of osteoarthritis and possesses clear clinical application value. Attached Figure Description

[0031] To more clearly illustrate the technical solution of the present invention, the drawings used in the embodiments will be briefly introduced below. Obviously, the drawings described below are only embodiments of the present invention. For those skilled in the art, other drawings can be obtained based on these drawings without creative effort.

[0032] Figure 1 This is a schematic diagram of the preparation method of the co-loaded dexamethasone sodium phosphate and type I collagen microspheres of the present invention;

[0033] Figure 2 In the image, A and B represent the particle size and potential of the microspheres 1 obtained in Example 1 of this invention, respectively, and C is a SEM image.

[0034] Figure 3 In the figures, A and B are the particle size and potential of the microspheres 2 obtained in Example 2 of the present invention, respectively, and C and D are SEM images;

[0035] Figure 4 In the image, A and B represent the particle size and potential of the microspheres 3 obtained in Example 2 of this invention, respectively, and C is a SEM image.

[0036] Figure 5 In the image, A and B represent the particle size and potential of the microspheres 4 obtained in Example 2 of this invention, respectively, and C is a SEM image.

[0037] Figure 6 In the image, A and B represent the particle size and potential of the microspheres 5 obtained in Example 2 of this invention, respectively, and C is a SEM image.

[0038] Figure 7 This is the in vitro cumulative drug release curve of microsphere 1 PLGA (50:50, 20 K) obtained in Example 1 of the present invention;

[0039] Figure 8 This is the in vitro cumulative drug release curve of microspheres 2 PLGA (50:50, 30 K) obtained in Example 2 of the present invention;

[0040] Figure 9 This is the in vitro cumulative drug release curve of microspheres 3 PLGA (85:15, 50-75 K) obtained in Example 2 of the present invention;

[0041] Figure 10 This is the in vitro cumulative drug release curve of microspheres 4 PLGA (75:25, 66-107 K) obtained in Example 2 of the present invention;

[0042] Figure 11 This is the in vitro cumulative drug release curve of microspheres 5 PLA (50 K) obtained in Example 2 of the present invention. Detailed Implementation

[0043] The technical solution of the present invention will be clearly and completely described below with reference to specific embodiments. 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] Example 1

[0045] This embodiment relates to a method for preparing co-loaded dexamethasone sodium phosphate and type I collagen microspheres, referencing... Figure 1 It includes the following steps:

[0046] (1) Prepare a 30 mg / mL aqueous solution of type I collagen and a 20 mg / mL aqueous solution of dexamethasone sodium phosphate. Sterilize the solvent by passing ultrapure water through a 0.22 μm filter membrane to obtain an aqueous solution. Dissolve polylactic acid-glycolic acid copolymer PLGA (50:50, 20 K) in ethyl acetate solution to obtain an organic solution of 20 mg / mL, which is used as the oil phase solution.

[0047] (2) Add the aqueous phase solution (0.4 mL of 30 mg / mL type I collagen aqueous solution and 0.4 mL of 20 mg / mL dexamethasone sodium phosphate aqueous solution) to the oil phase solution (1 mL of PLGA in ethyl acetate solution), vortex mix for 10 s, and then stir with a homogenizer at speed 2 (10000 rpm) for 40 s to obtain W / O colostrum;

[0048] (3) Add the W / O pre-emulsion to 10 mL of pre-cooled 1.0 wt% PVA aqueous solution, vortex mix for 10 s, and then stir for 20 s using a homogenizer at speed 2 (10000 rpm) to obtain W / O / W double emulsion;

[0049] (4) Transfer the W / O / W double emulsion to 50 mL of PVA (5 mg / mL, 0.5 w / v% aqueous solution) that is being stirred at 300 rpm, and continue stirring at 300 rpm for 3 h to evaporate the organic solvent ethyl acetate and solidify the microspheres;

[0050] (5) After curing for 3 h, centrifuge at 2000 rpm for 15 min, wash the microspheres 2-3 times with 10 mL of deionized water, centrifuge at 2000 rpm for 15 min after each washing, and collect the cured microspheres.

[0051] (6) Resuspend the microspheres in 1 mL of 10 mg / mL (1% w / v) sorbitol aqueous solution; freeze-dry them for two days using a four-ring freeze dryer, and store the freeze-dried microspheres in a -80℃ freezer for later use. This microsphere is designated as microsphere 1.

[0052] The microspheres 1 obtained in this embodiment were redissolved in ultrapure water, and the particle size and potential of the microspheres were detected using a laser particle size analyzer. The results are as follows. Figure 2 As shown in Figure AB, the microspheres have a particle size of approximately 12.95 μm and a span of approximately 1.44. Figure 2 Image C is a SEM image of the microspheres. As can be seen, the microspheres are perfectly spherical with smooth surfaces, relatively uniform particle size, and no overall adhesion.

[0053] Accurately weigh 15 mg of the co-loaded dexamethasone sodium phosphate and type I collagen microspheres prepared in this example, add 1 mL of lysis buffer (9.5 mL H2O, 450 μL Tween-20, 50 μL 10M NaOH) to thoroughly suspend the microspheres, and incubate in a 90℃ water bath for 2 h. Centrifuge at 15000 rpm for 20 min, collect the supernatant, and use a microBCA kit to detect the protein concentration. Then calculate the drug loading and encapsulation efficiency in the microspheres. The experimental results show that the drug loading of type I collagen in the co-loaded dexamethasone sodium phosphate and type I collagen microspheres prepared in Example 1 is approximately 20.14%, and the encapsulation efficiency is approximately 60.59%.

[0054] Accurately weigh the co-loaded dexamethasone sodium phosphate and type I collagen microspheres prepared in this example (approximately 15 mg per portion, three portions), add 1 mL (approximately 3 times the volume) of acetonitrile, and dissolve by sonication or vortexing. After high-speed centrifugation (15000 rpm, 20 min, 4℃), collect the supernatant and determine the total concentration W of DSP in the supernatant using a UV spectrophotometer. Separately, take an appropriate amount of lyophilized microspheres (approximately 15 mg per portion, three portions), add 1 mL (approximately 3 times the volume) of ultrapure water, and dissolve by sonication or vortexing. After high-speed centrifugation under the above conditions, collect the supernatant and determine the free DSP concentration W0 in the supernatant using a UV spectrophotometer. W1 is the carrier concentration. Based on this, calculate the drug loading and encapsulation efficiency of dexamethasone sodium phosphate in the microspheres. The results showed that the drug loading of dexamethasone sodium phosphate in the co-loaded dexamethasone sodium phosphate and type I collagen microspheres prepared in Example 1 was about 3.32%, and the encapsulation efficiency was about 67.85%.

[0055] Example 2

[0056] The difference between this embodiment and Embodiment 1 is that the carrier material in the oil phase solution was adjusted and divided into four groups: PLGA (50:50, 30 K), PLGA (85:15, 50-75 K), PLGA (75:25, 66-107 K), and PLA (50 K). The resulting microspheres were numbered 2-5 respectively.

[0057] In this embodiment, the co-loaded dexamethasone sodium phosphate and type I collagen microspheres were redissolved in ultrapure water. The particle size and potential of the microspheres were measured using a laser particle size analyzer. The particle size and potential distribution results of each microsphere are shown in the figure. Figure 3-6 Figures A and B show the specific particle size, span, and potential of each microsphere, as shown in Table 1.

[0058] Table 1

[0059]

[0060] Table 1 shows that the size of the microspheres co-loaded with dexamethasone sodium phosphate and type I collagen is related to the polymer carrier material. Under the same conditions, when different polymers were used as carriers to prepare microspheres, the microspheres prepared with PLGA as the carrier material had a larger particle size than those prepared with PLA as the carrier material. Furthermore, regardless of whether PLGA or PLA was used as the carrier material, all microspheres co-loaded with dexamethasone sodium phosphate and type I collagen carried a negative charge. It has been reported that glycosaminoglycans in the extracellular matrix of chondrocytes carry a negative charge. During cartilage injury, the reduction of glycosaminoglycan content leads to the disruption of cartilage structural integrity; therefore, the local negative potential decreases, which is conducive to the aggregation of negatively charged microspheres at the injury site.

[0061] Figure 3 Chinese CD, and Figure 4-6 In the middle C, there are SEM images of microspheres 2-5. It can be seen from the image that, except for PLGA (50:50, 30K), the other microspheres are all perfectly spherical with smooth surfaces, relatively uniform particle size, and no overall adhesion. However, the microspheres prepared by the polymer carrier material PLGA (50:50, 30 K) are spherical, but their surfaces have a network structure with more pores.

[0062] Following the method in Example 1, the drug loading and encapsulation efficiency of microspheres 2-5 were tested. The experimental results are shown in Table 2.

[0063] Table 2

[0064]

[0065] Table 2 shows that the drug loading and encapsulation efficiency of microspheres co-loaded with dexamethasone sodium phosphate and type I collagen are related to the type of polymeric carrier material and the type of drug encapsulated. For dexamethasone sodium phosphate, microspheres prepared with PLGA as the carrier material have higher drug loading and encapsulation efficiency than those prepared with PLA as the carrier material. However, for type I collagen, the drug loading and encapsulation efficiency of each microsphere are not significantly different regardless of whether PLGA or PLA is used as the carrier material.

[0066] Comparative Example 1

[0067] This comparative example involves a method for preparing co-loaded dexamethasone sodium phosphate and type I collagen microspheres. The difference from Example 1 lies in the homogenization conditions, specifically including the following steps:

[0068] (1) Prepare a 30 mg / mL type I collagen solution and a 20 mg / mL dexamethasone sodium phosphate solution. Sterilize the solution by passing the solvent ultrapure water through a 0.22 μm filter membrane to obtain an aqueous phase solution. Dissolve PLGA (50:50, 20 K) in ethyl acetate solution to obtain an organic solution of 20 mg / mL (2% w / v) as the oil phase solution.

[0069] (2) Add the aqueous phase solution (0.4 mL 30 mg / mL type I collagen solution and 0.4 mL 20 mg / mL dexamethasone sodium phosphate solution) to the oil phase solution (1 mL PLGA ethyl acetate solution) and homogenize for 40 s at speed 2 (10000 rpm) to obtain W / O colostrum;

[0070] (3) Add the W / O colostrum to 10 mL of pre-cooled 1.0wt% PVA solution, vortex mix for 10 s, and then use a homogenizer at speeds 2 (10000 rpm), 4 (15000 rpm), and 6 (20000 rpm) respectively (the resulting microspheres are recorded as 6-8) for 180 s to obtain W / O / W double emulsion;

[0071] (4) Transfer the W / O / W double emulsion to 50 mL of PVA (0.5wt%) aqueous solution that is being stirred at 300 rpm, and continue stirring at 300 rpm for 3 h to evaporate the organic solvent ethyl acetate and solidify the microspheres;

[0072] (5) After curing for 3 h, centrifuge at 2000 rpm for 15 min, wash the microspheres 2-3 times with 10 mL of deionized water, centrifuge at 3000 rpm for 15 min after each washing, and collect the cured microspheres.

[0073] (6) The microspheres were resuspended in 1 mL of 10 mg / mL sorbitol solution and freeze-dried for two days using a four-ring freeze dryer. The freeze-dried microspheres were stored in a -80℃ freezer for later use and were designated as microspheres 6-8.

[0074] The co-loaded dexamethasone sodium phosphate and type I collagen microspheres obtained in this comparative example were redissolved in ultrapure water. The microsphere particle size was measured using a laser particle size analyzer. The particle sizes of microspheres 6-8 were 10.48, 7.88, and 6.06 μm, respectively, and the spans of microspheres 6-8 were 2.94, 2.48, and 2.23 μm, respectively, which were all relatively large. This indicates that the microsphere particle size distribution is not uniform. This is because if the homogenization time is too long, the probability of deformation and breakage due to collision between droplets increases, which damages the shape of the formed microspheres and makes the spherical shape uneven. In addition, if the homogenization setting is too high, the shear force is too large, and the emulsion system will form turbulence, which will make the force on the droplets uneven, resulting in uneven microsphere particle size distribution.

[0075] Comparative Example 2

[0076] This comparative example relates to a method for preparing co-loaded dexamethasone sodium phosphate and type I collagen microspheres, where the homogenization process is replaced with ultrasonic treatment, specifically including the following steps:

[0077] (1) Prepare a 30 mg / mL type I collagen solution and a 20 mg / mL dexamethasone sodium phosphate solution. Sterilize the solution by passing the solvent ultrapure water through a 0.22 μm filter membrane to obtain an aqueous phase solution. Dissolve PLGA (50:50, 20 K) in ethyl acetate solution to obtain an organic solution of 20 mg / mL (2% w / v) as the oil phase solution.

[0078] (2) Add the aqueous phase solution (0.4 mL 30 mg / mL type I collagen solution and 0.4 mL 20 mg / mL dexamethasone sodium phosphate solution) to the oil phase solution (1 mL PLGA in dichloromethane solution), vortex premix for 10 s, and then sonicate at 50 W ultrasonic power for 60 s to obtain W / O colostrum;

[0079] (3) Add the W / O colostrum to 10 mL of pre-cooled 1.0wt% PVA aqueous solution, vortex mix for 10 s, and then ultrasonically emulsify with 50 W ultrasonic power for 60 s to obtain W / O / W double emulsion.

[0080] (4) Transfer the W / O / W double emulsion to 50 mL of 5 mg / mL PVA (0.5% w / v) solution that is being stirred at 350 rpm, and continue stirring at 350 rpm for 3 h to evaporate the organic solvent ethyl acetate and solidify the microspheres;

[0081] (5) After curing for 3 h, centrifuge at 2000 rpm for 15 min, wash the microspheres 2-3 times with 10 mL of deionized water, centrifuge at 3000 rpm for 15 min after each washing, and collect the cured microspheres.

[0082] (6) The microspheres were resuspended in 1 mL of 10 mg / mL (1% w / v) trehalose solution and freeze-dried for two days using a four-ring freeze dryer. The freeze-dried microspheres were stored in a -80℃ freezer for later use and were designated as microsphere 9.

[0083] The co-loaded dexamethasone sodium phosphate and type I collagen microspheres of the comparative example were redissolved in ultrapure water. The microsphere particle size was detected by laser particle size analyzer. The results showed that the microsphere particle size was 0.64 μm, but the span was large and the particle size distribution was uneven. This may be because the ultrasound only forms a high-intensity cavitation region at the tip of the probe, and the shear energy is uneven throughout the entire domain. It is easy to generate both ultrafine droplets and large, unbroken oil droplets at the same time, which leads to the uneven distribution of the microsphere particle size.

[0084] Test Example: Detection of in vitro drug release curves for dexamethasone sodium phosphate co-loaded with type I collagen microspheres.

[0085] The release of dexamethasone sodium phosphate and type I collagen from microspheres was tested in a phosphate-buffered saline solution system (pH 7.4 PBS simulated the physiological conditions of normal synovial fluid in the joint cavity, and pH 6.5 PBS simulated the slightly acidic environment of synovial fluid in the joint cavity during inflammation). In Examples 1-2, two portions of each polymer microsphere (1-5) were weighed (approximately 60 mg each) and placed in two 10 mL EP tubes. Each tube was dissolved in 7.2 mL of PBS at different pH values, then divided into six equal portions of 1.2 mL each, placed in 2 mL EP tubes, and incubated at 37°C and 100 rpm in a constant temperature shaker. At each time point (days 1, 3, 7, 10, 14, and 21), the polymer microspheres in the EP tubes were centrifuged at 8000 rpm for 5 min. The supernatant was collected, and the contents of DSP and COLⅠ were determined using UV spectroscopy and BCA protein quantification methods to calculate the cumulative in vitro drug release (results are shown in [link to results]). Figure 7-11 The cumulative drug release of each microsphere in vitro is shown in Table 3.

[0086] Table 3

[0087]

[0088] The results showed that none of the microspheres exhibited burst release. Under the same preparation conditions, the cumulative in vitro release of each microsphere was measured under the same conditions. The cumulative in vitro release rate of the drug-loaded polymer microspheres prepared with PLA as the carrier was greater than that of the drug-loaded polymer microspheres prepared with PLGA as the carrier material on day 21.

[0089] The present invention has been described in detail above with reference to specific embodiments and exemplary examples; however, these descriptions should not be construed as limiting the present invention. Those skilled in the art will understand that various equivalent substitutions, modifications, or improvements can be made to the technical solutions and embodiments of the present invention without departing from the spirit and scope of the invention, and all such modifications and improvements fall within the scope of the present invention. The scope of protection of the present invention is defined by the appended claims.

Claims

1. A method for preparing microspheres co-loaded with dexamethasone sodium phosphate and type I collagen, characterized in that, Includes the following steps: S1. Dissolve dexamethasone sodium phosphate and type I collagen in water to obtain an aqueous solution; dissolve the carrier material in an organic solvent to obtain an oil solution. S2. Add the aqueous phase solution to the oil phase solution and mix to obtain a W / O primary emulsion; S3. Add the W / O pre-emulsion to the first PVA solution and mix to obtain a W / O / W double emulsion; S4. The W / O / W double emulsion is transferred to the second PVA solution, solidified and lyophilized in a freeze-drying protectant solution to obtain the co-loaded dexamethasone sodium phosphate and type I collagen microspheres.

2. The preparation method of co-loaded dexamethasone sodium phosphate and type I collagen microspheres as described in claim 1, characterized in that, In S1, the concentration of dexamethasone sodium phosphate dissolved in water is 10-20 mg / mL, and the concentration of type I collagen dissolved in water is 15-30 mg / mL.

3. The preparation method of co-loaded dexamethasone sodium phosphate and type I collagen microspheres as described in claim 1, characterized in that, In S1, the concentration of the carrier material dissolved in the organic solvent is 20-100 mg / mL, and the organic solvent is ethyl acetate.

4. The preparation method of co-loaded dexamethasone sodium phosphate and type I collagen microspheres as described in claim 1, characterized in that, In S1, the carrier material is selected from polylactic acid and / or polylactic acid-glycolic acid copolymer; The molecular weight of the polylactic acid is 40K-60K, and the molecular weight of the polylactic acid-glycolic acid copolymer is 20K-107K. The molar ratio of polylactic acid to glycolic acid in the polylactic acid-glycolic acid copolymer is (85:15) to (50:50).

5. The preparation method of co-loaded dexamethasone sodium phosphate and type I collagen microspheres as described in claim 1, characterized in that, In S2 and S3, the mixing process specifically involves: vortexing for 8-15 seconds, followed by homogenization.

6. The preparation method of co-loaded dexamethasone sodium phosphate and type I collagen microspheres as described in claim 1, characterized in that, In S3, the concentration of the first PVA solution is 0.8-1.0 wt%.

7. The preparation method of co-loaded dexamethasone sodium phosphate and type I collagen microspheres as described in claim 1, characterized in that, In S4, the concentration of the second PVA solution is 4-10 mg / mL.

8. The method for preparing co-loaded dexamethasone sodium phosphate and type I collagen microspheres as described in claim 1, characterized in that, In S4, the curing and molding process specifically involves: magnetic stirring to evaporate the solvent, wherein the magnetic stirring speed is 200-400 rpm and the time is 3-6 hours.

9. The method for preparing co-loaded dexamethasone sodium phosphate and type I collagen microspheres as described in claim 1, characterized in that, In S4, the lyophilization protectant is selected from trehalose and / or sorbitol, and the concentration of the lyophilization protectant in the lyophilization protectant solution is 10-50 mg / mL.

10. A co-loaded dexamethasone sodium phosphate and type I collagen microsphere prepared by the preparation method according to any one of claims 1-9.

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