Collagen complex injection and preparation method thereof

Abstract

The application discloses a kind of by degradable polyester microspheres and collagen composition injection and preparation method thereof, belong to medical biomaterial and medical beauty injection preparation technical field.The application is mixed with the copolymerization of poly (L-lactide-epsilon-caprolactone) microspheres (PLCL MS) and the collagen protein gel solution of salting-out purification processing, and the composite injection is prepared by irradiation sterilization.The injection has the advantages of excellent biocompatibility of collagen protein, soft tissue fitting and cell repair activity, and long-acting mechanical support of copolymer microspheres, controllable degradation, can be widely used in subcutaneous depression correction, face soft tissue filling, skin minimally invasive wound repair and the like, and its preparation process parameter is controllable, and repeatability is strong, and preparation is uniform and stable, and injection is good, is suitable for industrial scale production.

Description

Technical Field

[0001] This invention relates to the field of collagen injection preparation technology, specifically to a composite injection composed of biodegradable polyester microspheres (PLCL MS) and collagen and its preparation method. This composite injection is mainly used in clinical fields such as medical aesthetic soft tissue filling, skin defect repair, subcutaneous depression correction, and minimally invasive wound repair. Background Technology

[0002] Collagen is a core functional protein in human connective tissue, possessing excellent biocompatibility, low immunogenicity, good cell adhesion, and tissue regeneration induction capabilities, making it one of the core raw materials for medical injectable preparations. Traditional single-protein collagen injections are soft in texture and have high tissue conformity, but they have significant technical shortcomings: First, they are rapidly metabolized and enzymatically digested in vivo, resulting in a short duration of shaping and support effects, requiring repeated injections, increasing treatment costs and physical burden on patients; second, the purity of commercially available collagen raw materials varies, containing impurities such as other proteins, lipids, and small molecules, which cannot be completely removed by conventional centrifugation and filtration purification methods, easily causing mild inflammation and allergic reactions, thus limiting biocompatibility; third, their mechanical support strength is insufficient, resulting in poor shaping effects for deep subcutaneous depressions and soft tissue defects, and they cannot achieve long-term shaping.

[0003] To address the issue of insufficient long-lasting effects in collagen formulations, existing technologies often employ compounding and modification of collagen with single polyester microspheres such as polylactic acid (PLA) and polycaprolactone (PCL). PLA microspheres offer high mechanical strength and good support, but are relatively hard and lack flexibility, resulting in poor compatibility with human soft tissues and a tendency to cause a foreign body sensation. PCL microspheres, on the other hand, exhibit excellent flexibility and biocompatibility, but are extremely hydrophobic and have poor suspension stability. Furthermore, there is a conflict in sterilization processes between polyester microsphere and collagen compound injections: collagen, being a protein-based biomolecule, is difficult to withstand moist heat sterilization and conventional irradiation sterilization, requiring aseptic filling; while polyester microspheres often undergo EO or irradiation sterilization, making it impossible for them to share the same terminal sterilization method. Both must rely on aseptic filling in a Class A environment, which is challenging and costly to maintain aseptic performance. Summary of the Invention

[0004] To address the technical problems of existing collagen injections, such as rapid degradation, poor shape retention, and insufficient stability, as well as the weak adaptability, strong hydrophobicity, and insufficient formulation stability of traditional polyester microspheres, this invention provides a collagen composite injection and its preparation method. It uses controllable degradation PLCL MS and salt-precipitated purified collagen as components, which can achieve the technical effects of long-lasting shape retention, safety and stability, and strong biocompatibility, making it suitable for various clinical soft tissue repair and filling scenarios.

[0005] To achieve the above objectives, the present invention adopts the following technical solution: One of the objectives of this invention is to protect a collagen complex injection, which, by weight percentage, comprises the following components in weight percentage: 2.0% to 5.0% purified collagen, 18.0% to 50.0% biodegradable polyester microspheres, and 45.0% to 80.0% medical injectable phosphate buffer.

[0006] Preferably, the composite injection consists of 3.5% purified collagen, 35% biodegradable polyester microspheres, and 61.5% medical injectable phosphate buffer.

[0007] Furthermore, the purified collagen is obtained by dialysis refolding of type I collagen after salting out with saturated sodium chloride.

[0008] Furthermore, the biodegradable polyester microspheres are copolymerized from L-lactide and ε-caprolactone in a molar ratio of 7:3 to 9:1, with a particle size of 20 to 45 μm, regular shape, and smooth surface. Preferably, the molar ratio of L-lactide to ε-caprolactone is 5:5.

[0009] Furthermore, the medical injectable phosphate buffer has a pH of 7.2-7.4, an osmotic pressure of 280-320 mOsm / L, is sterile and pyrogen-free, and meets the medical injectable grade standard.

[0010] A second objective of this invention is to protect the preparation method of the collagen complex injection, which includes the following steps: (1) Copolymerization to form biodegradable polyester microspheres; (2) Type I collagen was salted out with saturated sodium chloride and dialyzed to refold it into purified collagen. Then it was mixed with medical injection phosphate buffer and homogenized to prepare a highly active collagen gel solution. (3) The collagen gel solution and biodegradable polyester microspheres are thoroughly mixed and uniformly, and then sterilized by Co-60 γ-ray radiation after bubble-free filling to obtain the finished product.

[0011] Furthermore, the synthesis of biodegradable polyester microspheres in step (1) includes the following steps: i) Copolymerization: Weigh L-lactide and ε-caprolactone monomers according to the proportion, place them in an anhydrous and dry polymerization reactor, and add 0.05%~0.1% of tin isooctanoate catalyst by total monomer mass. After purging the reactor with high-purity nitrogen three times to replace the air in the reactor, seal the reaction system; heat to 130~150 ℃, and polymerize by stirring at a constant temperature for 24~48 h to obtain crude copolymer; dissolve the obtained crude copolymer in chloroform and then add anhydrous ethanol for precipitation purification. The obtained precipitate is dried under vacuum for 24~48 h to obtain high-purity PLCL copolymer; ii) Emulsification and pelletizing: Dissolve 4.0~8.0 g of PLCL copolymer in 100~200 mL of chloroform to obtain an oil phase solution; dissolve 10.0~150.0 g of polyvinyl alcohol in 1.0~5.0 L of sterile deionized water to prepare an aqueous emulsion with a mass concentration of 1.0%~3.0%; slowly add the oil phase solution dropwise to the aqueous emulsion under high-speed stirring (stirring speed 500~2000 r / min), and emulsify at room temperature for 1~2 h to form a stable and uniform oil-in-water emulsion; iii) Solidification and sieving: The oil-in-water emulsion was continuously stirred at 500 r / min for 24-48 h to allow the chloroform to completely evaporate and the microspheres to fully solidify. The microsphere precipitate was then collected by centrifugation at 4℃ and washed 3-5 times with sterile deionized water to remove residual polyvinyl alcohol. The microspheres were then sieved through 20 μm and 45 μm standard sieves. The obtained microspheres were then freeze-dried under vacuum and sealed for low-temperature storage.

[0012] Furthermore, the preparation of purified collagen in step (2) includes the following steps: a) Salting out purification: Type I collagen was fully dissolved in 0.3 vol% malonic acid aqueous solution to form a collagen solution. Then, it was slowly added to a saturated sodium chloride solution and allowed to stand at 4°C for 6-24 hours for salting out. The collagen precipitate was collected by centrifugation at 4°C. The salting out-centrifugation operation was repeated twice to achieve deep purification. b) Dialysis refolding: The collagen precipitate collected after salting out is resuspended in medical injectable phosphate buffer and dialyzed for 24 h at 4 ℃ using a dialysis bag with a molecular weight cutoff of 8000~14000 Da (the dialysate is changed every 6 h during this period to remove sodium chloride residue).

[0013] This invention utilizes poly(L-lactide-ε-caprolactone) microspheres (PLCL MS) combined with collagen salting-out purification and thorough homogenization to prepare a composite injectable formulation exhibiting high biocompatibility, long-lasting shape retention, and high stability. PLCL is a random copolymer of L-lactide and ε-caprolactone. Compared to homopolymers of polylactic acid and polycaprolactone, its copolymer structure disrupts the regularity of the polymer chains, significantly reducing the crystallinity of the material and improving surface wettability. Its hydrophobicity is lower than that of PCL and PLLA, which improves the dispersion performance of microspheres in aqueous media and reduces microsphere aggregation and sedimentation. Simultaneously, it combines the rigidity of polylactic acid microspheres with the flexibility of polycaprolactone microspheres, possessing excellent mechanical properties and biocompatibility. Furthermore, its degradation rate can be controlled by the copolymerization ratio to meet application requirements that match degradation behavior. After proper homogenization, collagen did not separate into layers after irradiation sterilization, and the microspheres remained uniformly suspended, allowing for smooth injection of the compound injection. This solved the problem that existing compound injections could not use the same sterilization method.

[0014] The beneficial effects of this invention are: (1) Controllable copolymerization of PLCL MS with excellent adaptability of degradation rate and mechanical strength performance: By controlling the copolymerization ratio of L-lactide and ε-caprolactone, this invention takes into account both the mechanical support strength of polylactic acid and the flexibility of polycaprolactone. The microspheres have uniform particle size and smooth surface, which not only solves the problems of single polyester microspheres being too hard and easy to leave residues, but also reduces hydrophobicity, improves the suspension stability in collagen, avoids formulation stratification and needle blockage, achieves long-term shaping, and has no long-term foreign matter residues.

[0015] (2) Collagen salting out purification, greatly improving biosafety: The present invention uses saturated salting out process to replace traditional simple filtration purification, which can effectively remove impurities, lipids and small molecule allergens from crude collagen; at the same time, salting out treatment can stabilize the triple helix structure of collagen, inhibit protein denaturation, and greatly improve the storage stability and in vivo bioactivity of the preparation.

[0016] (3) Appropriate homogenization treatment to achieve irradiation sterilization: The present invention uses low-temperature homogenization treatment to treat the collagen purified by salting out. Unlike the phenomenon that unhomogenized collagen solution is prone to aggregation after irradiation, the homogenized injection can still retain homogeneity after irradiation sterilization, and the injection is smooth, realizing the possibility of irradiation sterilization of compound injections.

[0017] (4) The compound system has synergistic effect and its comprehensive performance far exceeds that of the single formulation: The compound injection of the present invention combines the excellent tissue adhesion and cell compatibility of collagen with the long-term support of PLCL MS, and the controllable adaptation of degradation rate and mechanical properties. It not only makes up for the shortcomings of pure collagen injections that are easy to degrade and have poor plasticity, but also solves the problems of weak bio-adhesion and strong foreign body sensation of single polyester microspheres.

[0018] (5) Standardized process, mass production, and strong clinical applicability: The process parameters of the entire process, such as copolymerization, collagen purification, compound filling, etc., adopted in this invention are controllable and highly repeatable. No complex equipment is required, making it suitable for industrial-scale production. The finished product is sterile, pyrogen-free, and has good stability. It has excellent injection smoothness, is suitable for minimally invasive injection clinical operations, and has a wide range of applicable scenarios. Attached Figure Description

[0019] Figure 1 The results are the GPC molecular weight test results of the PLCL MS obtained in Example 1.

[0020] Figure 2 The image shows the SEM microstructure of the PLCL MS obtained in Example 1.

[0021] Figure 3 The images show sample images of the composite injections obtained in Example 1 and Comparative Example 2.

[0022] Figure 4The results are the cytotoxicity test results of the injections obtained in Examples 1-3. Detailed Implementation

[0023] A collagen complex injection, based on a total weight percentage of 100%, comprises the following components in weight percentage: purified collagen 2.0%~5.0%, poly(L-lactide-ε-caprolactone) microspheres (PLCL MS) 18.0%~50.0%, and medical injectable phosphate buffer 45.0%~80.0%.

[0024] The preparation of the collagen complex injection includes the following steps: Step 1: Copolymerization of PLCL MS (1) Copolymerization of PLCL: Weigh L-lactide and ε-caprolactone monomers in a molar ratio of 7:3 to 9:1, place them in an anhydrous and dry polymerization reactor, and add 0.05% to 0.1% of the total mass of the monomers of tin isooctanoate catalyst. After purging the reactor with high-purity nitrogen three times, seal the reaction system. Heat to 130 to 150 °C and polymerize by stirring at a constant temperature for 24 to 48 h to obtain crude copolymer. Dissolve the obtained crude copolymer in chloroform and then add anhydrous ethanol for precipitation and purification. Dry the precipitate under vacuum for 24 to 48 h to obtain high-purity PLCL copolymer. (2) Emulsification and pelletizing: Dissolve 4.0~8.0 g of PLCL copolymer in 100~200 mL of chloroform to obtain an oil phase solution; dissolve 10.0~150.0 g of polyvinyl alcohol (PVA) in 1.0~5.0 L of sterile deionized water to prepare an aqueous emulsion with a mass concentration of 1.0%~3.0%; slowly add the oil phase solution dropwise to the aqueous emulsion stirred at high speed (stirring speed 500~2000 r / min), emulsify at room temperature for 1~2 h to form a stable and uniform oil-in-water emulsion; (3) Solidification and sieving: The oil-in-water emulsion was continuously stirred at 500 r / min for 24-48 h to allow the chloroform to evaporate completely and the microspheres to solidify fully; then the microsphere precipitate was collected by centrifugation at 4℃ and washed 3-5 times with sterile deionized water to remove residual polyvinyl alcohol, and then sieved through 20 μm and 45 μm standard sieves in sequence. The resulting polyester microspheres with a particle size of 20-45 μm were freeze-dried under vacuum and then sealed and stored at low temperature.

[0025] Step 2: Salting out and purifying collagen (4) Salting out purification: Type I collagen is fully dissolved in 0.3 vol% malonic acid solution to form a collagen solution, and then slowly added to saturated sodium chloride solution. After standing at 4℃ for 6~24 h for salting out, the collagen precipitate is collected by centrifugation at 4℃. Repeat the salting out-centrifugation operation twice to achieve deep purification. (5) Dialysis rehydration: The collagen precipitate collected after salting out was resuspended in medical injectable phosphate buffer, placed in a dialysis bag with a molecular weight cutoff of 8000~14000 Da, and dialyzed at 4 ℃ for 24 h (the dialysate was changed every 6 h to remove sodium chloride residue); the collagen content of the dialyzed collagen solution was tested, and then medical injectable phosphate buffer was added in proportion for rehydration. (6) Homogenization: Place the solution prepared in step (5) in a homogenizer and homogenize it at 4 ℃ under low temperature and high pressure for 1-5 times to obtain collagen gel. Store it at 4 ℃ for later use.

[0026] Step 3: Compound molding (7) Uniform mixing: Add the obtained polyester microspheres into the collagen gel in proportion, stir thoroughly to make the microspheres uniformly dispersed without agglomeration and stratification, and let stand to remove bubbles; (8) Filling and sterilization: The compound solution is filled into a medical injection needle tube, the tube is sealed, and sterilized by Co-60 γ-ray radiation to obtain the finished injection product, which is stored at low temperature and protected from light.

[0027] To make the content of this invention easier to understand, the technical solution of this invention will be further described below with reference to specific embodiments, but this invention is not limited thereto.

[0028] The medical injectable phosphate buffer used has a pH of 7.2~7.4, an osmotic pressure of 280~320 mOsm / L, is sterile and pyrogen-free, and meets the medical injectable grade standard.

[0029] Different ratios of L-lactide and ε-caprolactone are only used to adjust the degradation rate of microspheres and do not affect the microsphere molding. Therefore, in the examples, the molar ratio of L-lactide to ε-caprolactone is uniformly 5:5. Example 1

[0030] A collagen complex injection, by weight percentage, comprises 3.5% purified collagen, 35.0% PLCLMS, and 61.5% medical injectable phosphate buffer; its preparation steps are as follows: 1. Preparation of PLCL MS: 1.5 g of PLCL copolymer was dissolved in 37.5 mL of chloroform to prepare an oil phase; 7.5 g of PVA was dissolved in 375 mL of sterile deionized water to prepare an aqueous emulsion with a mass concentration of 2.0%; the oil phase was slowly added dropwise to the aqueous emulsion and emulsified at 1000 r / min at room temperature for 90 min to form a stable emulsion. Then, the mixture was continuously stirred at 500 r / min for 48 h to evaporate the solvent and solidify the microspheres. The microspheres were then collected by centrifugation at 8000 r / min for 10 min at 4 °C. After washing four times with sterile deionized water, the microspheres were sieved through 20 μm and 45 μm standard sieves to obtain polyester microspheres with a particle size of 20~45 μm. The microspheres were then freeze-dried under vacuum for 12 h and then sealed and stored at low temperature.

[0031] 2. Salting-out purification of collagen: Type I collagen was fully dissolved in 0.3 vol% malonic acid solution to form a collagen solution. Then, the collagen solution was slowly added to a saturated sodium chloride solution. After salting out at 4°C for 12 h, the collagen precipitate was collected by centrifugation at 4°C. The salting-out-centrifugation process was repeated twice. The collagen precipitate after salting out was then resuspended in medical injectable phosphate buffer and placed in a dialysis bag with a molecular weight cutoff of 8000~14000 Da. Dialysis was performed at 4°C for 24 h (the dialysis solution was changed every 6 h to remove residual sodium chloride). The collagen content of the dialysis collagen solution was measured. Then, medical injectable phosphate buffer was added in proportion for reconstitution and homogenized three times under low temperature and high pressure to obtain collagen gel.

[0032] 3. Compound filling: The obtained PLCL MS is added to the collagen gel in proportion and stirred thoroughly for 5 hours to ensure that the microspheres are evenly dispersed and do not agglomerate or separate into layers. After standing, the mixture is degassed. After filling and sealing, it is sterilized by Co-60 γ-ray radiation to obtain the finished injection product, which is then stored at low temperature and protected from light. Example 2

[0033] A collagen complex injection, by weight percentage, comprises 3.5% purified collagen, 18.0% PLCLMS, and 78.5% medical injectable phosphate buffer, and its preparation steps are the same as in Example 1. Example 3

[0034] A collagen complex injection, by weight percentage, comprises 3.5% purified collagen, 45.0% PLCLMS, and 51.5% medical injectable phosphate buffer, and its preparation steps are the same as in Example 1. Example 4

[0035] A collagen complex injection, by weight percentage, comprises 5.0% purified collagen, 35.0% PLCLMS, and 60.0% medical injectable phosphate buffer, and its preparation steps are the same as in Example 1.

[0036] Comparative Example 1 Step 2: After mixing the collagen solution with medical injectable phosphate buffer, do not homogenize. The proportions and preparation steps of other components are the same as in Example 1.

[0037] Comparative Example 2 Step 2 involves mixing the collagen solution with medical injectable phosphate buffer and homogenizing it only once. The proportions and preparation steps of other components are the same as in Example 1.

[0038] Performance test Storage stability, biosafety, and injection performance of the injectable formulations obtained in each embodiment and comparative example were tested, and the results are as follows: 1. Storage stability: The injections prepared in Examples 1-4 were stored at 4 ℃ for 3 months and remained milky white, without stratification, precipitation, protein denaturation, or microsphere aggregation. The injections obtained in Comparative Examples 1 and 2 showed protein precipitation, microsphere aggregation, and stratification after irradiation sterilization.

[0039] 2. Biosafety: All samples showed a cytotoxicity level of 0, indicating no cytotoxicity.

[0040] 3. Injection performance: The injections prepared in Examples 1 and 2 have excellent flowability, with no needle blockage or injection resistance; the injection prepared in Example 3 has a higher microsphere concentration, resulting in greater injection pushing force; the injection prepared in Example 4 has a higher collagen concentration, and the collagen is not stable enough after irradiation sterilization, resulting in insufficient injection smoothness; the injections prepared in Comparative Examples 1 and 2 show collagen denaturation, precipitation, and aggregation, causing needle blockage.

[0041] In summary, the formulation prepared in Example 1 is significantly superior to the comparative example and other examples in terms of overall performance, including stability, safety, long-lasting plasticity, and injection experience.

[0042] The above description is only a preferred embodiment of the present invention. All equivalent changes and modifications made within the scope of the claims of the present invention should be included in the scope of the present invention.

Claims

1. A collagen complex injection, characterized in that, The compound injection is composed of the following components in weight percentages, which together constitute 100% of the total weight percentage: 2.0% to 5.0% purified collagen, 18.0% to 50.0% biodegradable polyester microspheres, and 45.0% to 80.0% medical injectable phosphate buffer.

2. The collagen complex injection according to claim 1, characterized in that, The compound injection consists of 3.5% purified collagen, 35% biodegradable polyester microspheres, and 61.5% medical injectable phosphate buffer.

3. The collagen complex injection according to claim 1, characterized in that, The purified collagen was prepared by dialysis refolding of type I collagen after salting out with saturated sodium chloride.

4. The collagen complex injection according to claim 1 or 3, characterized in that, The preparation of the purified collagen includes the following steps: a) Salting out purification: Type I collagen was fully dissolved in 0.3 vol% malonic acid solution to form a collagen solution, which was then slowly added to a saturated sodium chloride solution. After salting out at 4°C for 6-24 hours, the collagen precipitate was collected by low-temperature centrifugation. The salting out-centrifugation operation was repeated twice to achieve deep purification. b) Dialysis refolding: The collagen precipitate collected after salting out is resuspended in medical injectable phosphate buffer and dialyzed for 24 h at 4 ℃ using a dialysis bag with a molecular weight cutoff of 8000~14000 Da.

5. The collagen complex injection according to claim 1, characterized in that, The biodegradable polyester microspheres are copolymerized from L-lactide and ε-caprolactone in a molar ratio of 7:3 to 9:1, and have a particle size of 20 to 45 μm.

6. The collagen complex injection according to claim 1 or 5, characterized in that, The synthesis of the biodegradable polyester microspheres includes the following steps: i) Copolymerization: Weigh L-lactide and ε-caprolactone monomers according to the proportion, place them in an anhydrous and dry polymerization reactor, and add 0.05%~0.1% of tin isooctanoate catalyst by total monomer mass. After purging the reactor with high-purity nitrogen three times, seal the reaction system; heat to 130~150 ℃, and polymerize by stirring at a constant temperature for 24~48 h to obtain crude copolymer; dissolve the obtained crude copolymer in chloroform and then add anhydrous ethanol for precipitation purification. The obtained precipitate is dried under vacuum to obtain high-purity PLCL copolymer; ii) Emulsification and pelletizing: 4.0~8.0 g of PLCL copolymer is fully dissolved in chloroform to obtain an oil phase solution; 10.0~150.0 g of polyvinyl alcohol is dissolved in sterile deionized water to prepare an aqueous emulsion with a mass concentration of 1.0%~3.0%; the oil phase solution is slowly added dropwise to the aqueous emulsion being stirred at high speed, and emulsified at room temperature for 1~2 h to form a stable and uniform oil-in-water emulsion; iii) Solidification and sieving: The oil-in-water emulsion is continuously stirred for 24-48 h to allow the chloroform to completely evaporate and the microspheres to fully solidify. Then, the microsphere precipitate is collected by centrifugation and washed 3-5 times with sterile deionized water to remove residual polyvinyl alcohol. The microspheres are then sieved through 20 μm and 45 μm standard sieves in sequence. The obtained microspheres are then freeze-dried under vacuum and sealed for low-temperature storage.

7. The collagen complex injection according to claim 1, characterized in that, The medical injectable phosphate buffer has a pH of 7.2-7.4, an osmotic pressure of 280-320 mOsm / L, and is sterile and pyrogen-free.

8. A method for preparing the collagen complex injection as described in claim 1, characterized in that, Includes the following steps: (1) The purified collagen was mixed with medical injection phosphate buffer and homogenized to prepare a highly active collagen gel solution; (2) The collagen gel solution and biodegradable polyester microspheres are thoroughly mixed and uniformly, and then sterilized by Co-60 γ-ray radiation after bubble-free filling to obtain the finished product.

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