Polyamino acid / chitosan porous microspheres with antibacterial and osteoinductive spatiotemporal effects and a preparation method thereof

Abstract

The present application belongs to the technical field of tissue engineering, and particularly relates to a polyamino acid / chitosan porous microsphere with antibacterial and osteogenesis promoting spatiotemporal effects and a preparation method thereof. The present application respectively prepares biomacromolecules with osteogenesis promoting activity and antibacterial activity, and sequentially assembles the osteogenesis promoting layer and the antibacterial layer on the surface of the chitosan porous microsphere by using a layer-by-layer assembly method, to form a porous microsphere system with a spatial layered structure. This design can inhibit bacterial growth in the early stage, and promote the behavior of osteogenesis related cells in the later stage, so as to realize the temporal regulation of the antibacterial and osteogenesis functions.

Description

Technical Field

[0001] This invention belongs to the field of tissue engineering technology, specifically relating to a polyamino acid / chitosan porous microsphere with antibacterial and osteopromoting spatiotemporal effects and its preparation method. Background Technology

[0002] Tumors, trauma, necrosis, congenital malformations, and a range of other causes often lead to bone defects. Influenced by factors such as population aging and changes in exercise patterns, the number of patients with bone defects is expected to continue to increase. Repairing irregularly shaped and highly infectious open or infected bone defects presents a significant challenge for orthopedics. Autologous or allogeneic bone, due to its excellent osteoconduction, osteoinduction, and osteogenic effects, is considered the "gold standard" for clinical bone defect repair, but its use is severely limited by donor shortages or immune rejection. Because bone injuries are often irregularly shaped, bone graft filling is extremely difficult, resulting in poor conformation. Furthermore, implants cannot be delivered through minimally invasive surgery with minimal trauma, bleeding, and rapid recovery, especially for infected bone defects where bacteria easily adhere to the graft surface, lacking anti-infection capabilities. Porous microspheres, due to their large specific surface area, good stability, and strong surface permeability, are widely used in bone tissue repair. Developing porous microspheres with good biocompatibility, bone function promotion, and antibacterial functions has significant clinical implications.

[0003] Existing materials used to prepare porous microspheres for bone repair mainly fall into three categories: natural polymers, synthetic polymers, and inorganic materials. Among them, biodegradable synthetic polymers have become a research hotspot in bone repair materials due to their excellent biocompatibility, lack of immunogenicity, and the ability to precisely control their mechanical properties and degradation rates, thus matching the pace of bone tissue regeneration. However, currently reported porous microspheres made of synthetic polymers have limited functions and lack osteogenic induction activity and antibacterial properties. They typically require loading small molecule drugs or inorganic nanoparticles to impart repair functions. However, such physical loading systems generally suffer from severe drug burst release, which not only fails to achieve long-term therapeutic effects but also easily induces side effects, significantly impacting repair efficacy and clinical safety.

[0004] Taurine is an amphoteric sulfonic acid. Studies have shown that it can promote osteogenic differentiation of bone marrow stem cells, induce osteogenic growth factor expression, and inhibit osteoclast formation, thereby effectively promoting bone tissue regeneration. Currently reported taurine is mostly ingested in small molecule form to promote osteoogenesis. Patent CN112493491A discloses a supplement prepared using magnesium taurate as a raw material to improve bone health. Patent JP2003171250A uses a combination of taurine and *Cotinus corydalis* extract as active ingredients to effectively prevent bone resorption and prevent and treat periodontitis. Currently, most studies on taurine promoting bone regeneration achieve its function through doping. However, taurine has a low molecular weight and strong hydrophilicity; introduction through physical encapsulation or surface adsorption alone is prone to rapid release problems.

[0005] Furthermore, bacterial infection is very common in the clinical treatment of bone defect repair. Surgical instruments, surgical environment, and human factors often lead to infection at the bone defect site, severely impairing bone tissue regeneration. Currently, functional bone tissue repair materials mainly load drugs through coating, adsorption, and mixing, which also suffers from problems such as burst release of active substances and difficulty in maintaining antibacterial effects. Although the natural biopolymer chitosan (CS) has broad antibacterial efficacy, its low solubility in water limits its antibacterial application. Patent CN113336872A discloses that α-aminophosphonates and their derivatives can be used as enzyme inhibitors and antibacterial agents. Introducing them into the CS molecular chain is expected to improve solubility and further enhance the antibacterial properties of the material. However, research on phosphonic acid group modification of CS for antibacterial repair of bone defects is still limited, and this area has the value for further exploration.

[0006] During bone defect repair, there is a close temporal relationship between bacterial infection and bone tissue regeneration. Bacterial adhesion and biofilm formation at the defect site can sustainably induce an inflammatory response, promoting the upregulation of inflammatory factors such as tumor necrosis factor-α (TNF-α) and interleukin-1β (IL-1β), thereby disrupting the dynamic balance between osteogenic and osteoclast processes and inhibiting bone regeneration. Current treatment strategies often focus on a single function and are insufficient to achieve synergistic regulation of both antibacterial and osteogenic processes. Summary of the Invention

[0007] The purpose of this invention is to overcome the defects of the prior art and provide a polyamino acid / chitosan porous microsphere with antibacterial and osteopromoting spatiotemporal effects, as well as a method for its preparation.

[0008] The objective of this invention can be achieved through the following technical solutions: A polyamino acid / chitosan porous microsphere with antibacterial and osteopromoting spatiotemporal effects is disclosed. The porous microsphere has a three-layer composite structure: an inner layer of chitosan porous microspheres, a middle layer of osteopromoting layer, and an outer layer of antibacterial layer; structurally, it achieves antibacterial effect followed by osteopromoting effect. The bone-promoting layer is composed of taurine-grafted polyamino acids (PLGT).

[0009] Furthermore, the inner layer of chitosan porous microspheres has a diameter of 250-350 micrometers, an average pore size of 25-45 micrometers, and a porosity of ≥85%. The viscosity-average molecular weight of the chitosan is 10,000 to 50,000.

[0010] Furthermore, the taurine-grafted polyamino acid is formed by an amidation covalent grafting reaction between the amino group (-NH2) in taurine and the carboxyl group (-COOH) in the polyamino acid, with a grafting rate of 10%~20%. The polyamino acid is poly(L-glutamic acid) or poly(L-aspartic acid), with a number-average molecular weight of 20,000 to 40,000.

[0011] Furthermore, the antibacterial layer is composed of phosphonated chitosan; the degree of phosphonation of the phosphonated chitosan is 10% to 30%.

[0012] The present invention also provides a method for preparing polyamino acid / chitosan porous microspheres with antibacterial and osteopromoting spatiotemporal effects as described in any of the preceding claims, comprising the steps of: S1. Preparation of the inner layer: Chitosan aqueous solution and Span80 petroleum ether solution were mixed, and after emulsification, water-in-oil (W / O) droplets were prepared. After freeze crystallization, stable chitosan porous microspheres were obtained by freeze drying and treatment with sodium hydroxide ethanol solution. S2, Preparation of the middle layer: Taurine and polyamino acids are dissolved in an alkaline aqueous solution, a condensing agent is added and reacted, unreacted taurine and condensing agent are removed by dialysis, and taurine-grafted polyamino acids are obtained by freeze drying; S3. Preparation of the outer layer: Chitosan was dissolved in an acidic aqueous solution, and an aqueous solution of phosphorous acid and formaldehyde was added to carry out the Mannich reaction. After dialysis and freeze drying, phosphonated chitosan (NMPC) with antibacterial properties was obtained. S4. Preparation of polyamino acid / chitosan porous microspheres: The taurine-grafted polyamino acid obtained in step S2 and the phosphonated chitosan obtained in step S3 are dissolved in water to obtain an aqueous solution of taurine-grafted polyamino acid and an aqueous solution of phosphonated chitosan. The chitosan porous microspheres obtained in step S1 are immersed in the aqueous solution of taurine-grafted polyamino acid, washed, freeze-dried, and then immersed in the aqueous solution of phosphonated chitosan. After washing and freeze-drying, the polyamino acid / chitosan porous microspheres with antibacterial and osteogenic spatiotemporal effects are obtained.

[0013] Further, in step S1, the degree of deacetylation of the chitosan is ≥85%, and the concentration of the chitosan aqueous solution is 1~2 w / v; the chitosan aqueous solution is prepared by dissolving chitosan in a 0.5~2 v / v% acetic acid aqueous solution; the concentration of the sodium hydroxide ethanol solution is 0.5~2 w / v; and the volume concentration of Span80 in the Span80 petroleum ether solution is 5~15%.

[0014] Further, in step S2, the alkaline aqueous solution is a 0.1~0.5 M sodium hydroxide aqueous solution; the condensing agent is 4-(4,6-dimethoxytriazine-2-yl)-4-methylmorpholine hydrochloride; the molar ratio of the polyamino acid, taurine and the condensing agent is 5:(0.5~1.5):(0.5~1.5); the reaction temperature is 25~40℃, and the reaction time is 8~24 h.

[0015] Further, in step S3, the acidic aqueous solution is a 0.5~1.5 v / v% acetic acid aqueous solution; the concentration of the phosphorous acid aqueous solution is 10~15 w / v; the concentration of the formaldehyde aqueous solution is 37 w / w; the reaction steps are as follows: heat the chitosan aqueous solution to 50~65℃, add the phosphorous acid aqueous solution dropwise, then heat to 68~80℃, add the formaldehyde aqueous solution dropwise, and react at 68~80℃ for 8~24 h; wherein the molar ratio of chitosan, formaldehyde, and phosphorous acid is 10:(1~5):(1~6).

[0016] Further, in step S4, the concentration of the taurine-grafted polyamino acid aqueous solution is 0.1 mg / mL, and the pH is adjusted to 5.5-5.9 using sodium hydroxide aqueous solution; The concentration of the phosphonated chitosan aqueous solution was 0.1 mg / mL, and the pH was adjusted to 5.5-5.9 using sodium hydroxide aqueous solution. The ratio of the number of moles of amino groups (-NH2) in the chitosan porous microspheres to the total number of moles of carboxyl groups (-COOH) and sulfonic acid groups (-SO3H) in the taurine-grafted polyamino acids is 1:(1~5). The total molar number of carboxyl groups (-COOH) and sulfonic acid groups (-SO3H) in the taurine-grafted polyamino acid is in the ratio of the molar number of amino groups (-NH2) in the phosphonic chitosan to 1:(1~5). The soaking time for both soaking sessions was 8 to 24 hours.

[0017] Furthermore, in step S4, the water is rinsed three times with deionized water after each soaking; the freeze-drying is carried out at -40~-60℃.

[0018] Compared with the prior art, the present invention has the following beneficial effects: This invention proposes a polyamino acid / chitosan porous microsphere with antibacterial and osteogenic spatiotemporal effects and its preparation method. To mimic the compositional characteristics of polysaccharides and peptides in the extracellular matrix, biomolecules with osteogenic and antibacterial activities are synthesized based on polyamino acids and chitosan, respectively. Using chitosan porous microspheres as a matrix, a layer-by-layer assembly method is employed to sequentially assemble a bone function layer and an antibacterial function layer on the surface, forming a porous microsphere system with a spatially layered structure. This system avoids the problems of burst release, short duration of action, and single function associated with traditional small-molecule drugs. The outer antibacterial layer inhibits bacterial growth in the early stages, while the inner osteogenic layer promotes bone regeneration in the later stages, thus achieving temporal regulation of antibacterial and osteogenic functions. The resulting microspheres have a porosity ≥85%, exhibit antibacterial activity within 24 hours, and begin to promote bone regeneration after 5 days. This provides a new approach for the treatment of bone defects.

[0019] This invention constructs a polyamino acid / chitosan porous microsphere system based on functional biomacromolecules. Spatially, layer-by-layer assembly achieves a functional distribution of an outer antibacterial layer and an inner osteogenic inner layer. Temporally, the sequential assembly order enables synergistic regulation of early infection inhibition and later osteogenic promotion. This system provides a novel material design strategy for the repair of infected bone defects. Attached Figure Description

[0020] Figure 1 Characterization results of the chitosan porous microspheres prepared in Example 6 of the present invention: (a) Scanning electron microscope image and energy spectrum of C, O and N elemental distribution (scale bar is 50 μm); (b) Pore size distribution map; Figure 2 Characterization results of the assembled taurine-grafted polyamino acid porous microspheres prepared in Example 6 of the present invention: (a) Scanning electron microscope image and energy spectrum of C, O, and S elemental distribution (scale bar is 50 μm); (b) Pore size distribution map; Figure 3 The characterization results of the polyamino acid / chitosan porous microspheres finally obtained in Example 6 of the present invention are as follows: (a) Scanning electron microscope image and energy spectrum of C, O, and P elements (scale bar is 50 μm); (b) Pore size distribution map; Figure 4 The antibacterial performance test results of the polyamino acid / chitosan porous microspheres prepared in Example 6 of the present invention are as follows: (a) OD of polyamino acid / chitosan porous microspheres with different concentrations after co-culturing with Escherichia coli (E. coli) for 6 h and 24 h. 600 (a) OD values; (b) OD values ​​of polyamino acid / chitosan porous microspheres with different concentrations after co-culturing with Staphylococcus aureus for 6 h and 24 h. 600(c) Scanning electron microscope images (scale bar 1 μm) of polyamino acid / chitosan porous microspheres with a mass concentration of 5 mg / mL after co-culturing with the two bacteria mentioned above for 24 h. Figure 5 This is a Micro-CT three-dimensional reconstruction image of the femoral repair morphology 6 weeks after implantation of the polyamino acid / chitosan porous microspheres prepared in Example 6 of the present invention. Figure 6 Scanning electron microscope images of the polyamino acid / chitosan porous microspheres prepared in Example 6 of the present invention: (a) Overall morphology of the polyamino acid / chitosan porous microspheres (scale bar 50 μm); (b) Detail of the internal structure of the pores (scale bar 10 μm). Detailed Implementation

[0021] The present invention will now be described in detail with reference to the accompanying drawings and specific embodiments. These embodiments are based on the technical solution of the present invention and provide detailed implementation methods and specific operating procedures. However, the scope of protection of the present invention is not limited to the following embodiments.

[0022] Unless otherwise specified, all raw materials used in this invention are commercially available products.

[0023] Example 1 A porous polyamino acid / chitosan microsphere with antibacterial and osteopromoting spatiotemporal effects is prepared by the following steps: S1. Construction of the inner layer: Prepare a 1.25 w / v% chitosan aqueous solution (solvent is 1 v / v% acetic acid aqueous solution) and a 10% Span80 petroleum ether solution; add 4 mL of chitosan aqueous solution to 40 mL of Span80 petroleum ether solution, emulsify thoroughly, pour into 400 mL of -20℃ petroleum ether, let stand, decant the supernatant, and freeze-dry; then treat with 1 w / v% sodium hydroxide ethanol solution, wash with deionized water, and freeze-dry to obtain chitosan porous microspheres.

[0024] S2. Construction of the Intermediate Layer: Preparation of an intermediate layer material with bone regeneration-promoting properties. Poly(L-glutamic acid) (Mn: 20000) and taurine were added to a 0.1 M sodium hydroxide aqueous solution, followed by the addition of a condensing agent, 4-(4,6-dimethoxytriazine-2-yl)-4-methylmorpholine hydrochloride. The molar ratio of poly(L-glutamic acid), taurine, and condensing agent was 5:1:1. The mixture was stirred to ensure uniform dispersion, and the reaction was carried out at 25°C under sealed conditions for 24 hours. After the reaction was completed, the mixture was dialyzed in deionized water for 3 days to completely remove residual condensing agent and taurine. The resulting product was freeze-dried to obtain taurine-grafted polyamino acid (grafting rate 16%).

[0025] S3. Construction of the outer layer: A 5 w / v% chitosan aqueous solution (solvent: 1 v / v% acetic acid aqueous solution) was heated to 60°C, and a 10 w / v% phosphorous acid aqueous solution was added dropwise. The temperature was then raised to 70°C, and a 37 w / w% formaldehyde aqueous solution was added dropwise. The reaction was carried out at 70°C for 8 hours. The molar ratio of chitosan, formaldehyde, and phosphorous acid was 10:1.7:5.1. After the reaction was completed, the mixture was cooled and dialyzed in deionized water for 3 days to completely remove residual acetic acid, formaldehyde, and phosphorous acid. The mixture was then freeze-dried to obtain phosphonated chitosan (phosphonation degree of 18%).

[0026] S4. Construction of polyamino acid / chitosan porous microspheres: The chitosan porous microspheres obtained in step S1 were soaked in a 0.1 mg / mL taurine-grafted polyamino acid aqueous solution. The pH was adjusted to 5.5 using a 0.1 M sodium hydroxide aqueous solution. The molar ratio of -NH2 in chitosan to -COOH and -SO3H groups in polyamino acid was 1:1. After soaking for 12 hours, the microspheres were washed three times with deionized water and then freeze-dried to obtain assembled taurine-grafted polyamino acid porous microspheres. Prepare a 0.1 mg / mL phosphonated chitosan aqueous solution, adjust the pH to 5.5 with 0.1 M sodium hydroxide aqueous solution, and immerse the assembled taurine-grafted polyamino acid porous microspheres in this aqueous solution. Control the ratio of the total molar number of -COOH and -SO3H groups in the polyamino acid to the molar number of -NH2 groups in the phosphonated chitosan to be 1:1. Immerse for 12 hours, wash three times with deionized water, and freeze-dry at -50℃ to obtain polyamino acid / chitosan porous microspheres with antibacterial and osteopromoting spatiotemporal effects.

[0027] The obtained polyamino acid / chitosan porous microspheres have a diameter of 278.2 micrometers, an average pore size of 35 micrometers, and a porosity of over 85%.

[0028] Example 2 A porous polyamino acid / chitosan microsphere with antibacterial and osteopromoting spatiotemporal effects is prepared by the following steps: S1. Construction of the inner layer: Prepare a 1.25 w / v% chitosan aqueous solution (solvent is 1 v / v% acetic acid aqueous solution) and a 10% Span80 petroleum ether solution; add 4 mL of chitosan aqueous solution to 40 mL of Span80 petroleum ether solution, emulsify thoroughly, pour into 400 mL of -20℃ petroleum ether, let stand, decant the supernatant, and freeze-dry; then treat with 1 w / v% sodium hydroxide ethanol solution, wash with deionized water, and freeze-dry to obtain chitosan porous microspheres.

[0029] S2. Construction of the Intermediate Layer: Preparation of an intermediate layer material with bone regeneration-promoting properties. Poly(L-glutamic acid) (Mn: 20000) and taurine were added to a 0.1 M sodium hydroxide aqueous solution, followed by the addition of a condensing agent, 4-(4,6-dimethoxytriazine-2-yl)-4-methylmorpholine hydrochloride. The molar ratio of poly(L-glutamic acid), taurine, and condensing agent was 5:1:1. The mixture was stirred to ensure uniform dispersion, and the reaction was carried out at 25°C under sealed conditions for 24 hours. After the reaction was completed, the mixture was dialyzed in deionized water for 3 days to completely remove residual condensing agent and taurine. The resulting product was freeze-dried to obtain taurine-grafted polyamino acid (grafting rate 16%).

[0030] S3. Construction of the outer layer: A 5 w / v% chitosan aqueous solution (solvent: 1 v / v% acetic acid aqueous solution) was heated to 60°C, and a 10 w / v% phosphorous acid aqueous solution was added dropwise. The temperature was then raised to 70°C, and a 37 w / w% formaldehyde aqueous solution was added dropwise. The reaction was carried out at 70°C for 8 hours. The molar ratio of chitosan, formaldehyde, and phosphorous acid was 10:1.7:5.1. After the reaction was completed, the mixture was cooled and dialyzed in deionized water for 3 days to completely remove residual acetic acid, formaldehyde, and phosphorous acid. The mixture was then freeze-dried to obtain phosphonated chitosan (phosphonation degree of 18%).

[0031] S4. Construction of polyamino acid / chitosan porous microspheres: The chitosan porous microspheres obtained in step S1 were soaked in a 0.1 mg / mL taurine-grafted polyamino acid aqueous solution. The pH was adjusted to 5.7 using a 0.1 M sodium hydroxide aqueous solution. The molar ratio of -NH2 in chitosan to -COOH and -SO3H groups in polyamino acid was 1:1. After soaking for 12 hours, the microspheres were washed three times with deionized water and then freeze-dried to obtain assembled taurine-grafted polyamino acid porous microspheres. Prepare a 0.1 mg / mL phosphonated chitosan aqueous solution, adjust the pH to 5.7 with 0.1 M sodium hydroxide aqueous solution, and immerse the above-assembled taurine-grafted polyamino acid porous microspheres in this aqueous solution. Control the ratio of the total molar number of -COOH and -SO3H groups in the polyamino acid to the molar number of -NH2 groups in the phosphonated chitosan to be 1:1. Immerse for 12 hours, wash three times with deionized water, and freeze-dry at -50℃ to obtain polyamino acid / chitosan porous microspheres with antibacterial and osteopromoting spatiotemporal effects.

[0032] The obtained polyamino acid / chitosan porous microspheres have a diameter of 283.2 micrometers, an average pore size of 34 micrometers, and a porosity of over 85%.

[0033] Example 3 A porous polyamino acid / chitosan microsphere with antibacterial and osteopromoting spatiotemporal effects is prepared by the following steps: S1. Construction of the inner layer: Prepare a 1.25 w / v% chitosan aqueous solution (solvent is 1 v / v% acetic acid aqueous solution) and a 10% Span80 petroleum ether solution; add 4 mL of chitosan aqueous solution to 40 mL of Span80 petroleum ether solution, emulsify thoroughly, pour into 400 mL of -20℃ petroleum ether, let stand, decant the supernatant, and freeze-dry; then treat with 1 w / v% sodium hydroxide ethanol solution, wash with deionized water, and freeze-dry to obtain chitosan porous microspheres.

[0034] S2. Construction of the Intermediate Layer: Preparation of an intermediate layer material with bone regeneration-promoting properties. Poly(L-glutamic acid) (Mn: 20000) and taurine were added to a 0.1 M sodium hydroxide aqueous solution, followed by the addition of a condensing agent, 4-(4,6-dimethoxytriazine-2-yl)-4-methylmorpholine hydrochloride. The molar ratio of poly(L-glutamic acid), taurine, and condensing agent was 5:1:1. The mixture was stirred to ensure uniform dispersion, and the reaction was carried out at 25°C under sealed conditions for 24 hours. After the reaction was completed, the mixture was dialyzed in deionized water for 3 days to completely remove residual condensing agent and taurine. The resulting product was freeze-dried to obtain taurine-grafted polyamino acid (grafting rate 16%).

[0035] S3. Construction of the outer layer: A 5 w / v% chitosan aqueous solution (solvent: 1 v / v% acetic acid aqueous solution) was heated to 60°C, and a 10 w / v% phosphorous acid aqueous solution was added dropwise. The temperature was then raised to 70°C, and a 37 w / w% formaldehyde aqueous solution was added dropwise. The reaction was carried out at 70°C for 8 hours. The molar ratio of chitosan, formaldehyde, and phosphorous acid was 10:1.7:5.1. After the reaction was completed, the mixture was cooled and dialyzed in deionized water for 3 days to completely remove residual acetic acid, formaldehyde, and phosphorous acid. The mixture was then freeze-dried to obtain phosphonated chitosan (phosphonation degree of 18%).

[0036] S4. Construction of polyamino acid / chitosan porous microspheres: The chitosan porous microspheres obtained in step S1 were soaked in a 0.1 mg / mL taurine-grafted polyamino acid aqueous solution. The pH was adjusted to 5.9 using a 0.1 M sodium hydroxide aqueous solution. The molar ratio of -NH2 in chitosan to -COOH and -SO3H groups in polyamino acid was 1:1. After soaking for 12 hours, the microspheres were washed three times with deionized water and then freeze-dried to obtain assembled taurine-grafted polyamino acid porous microspheres. Prepare a 0.1 mg / mL phosphonated chitosan aqueous solution, adjust the pH to 5.9 with 0.1 M sodium hydroxide aqueous solution, and immerse the assembled taurine-grafted polyamino acid porous microspheres in this aqueous solution. Control the ratio of the total molar number of -COOH and -SO3H groups in the polyamino acid to the molar number of -NH2 groups in the phosphonated chitosan to be 1:1. Immerse for 12 hours, wash three times with deionized water, and freeze-dry at -50℃ to obtain polyamino acid / chitosan porous microspheres with antibacterial and osteopromoting spatiotemporal effects.

[0037] The obtained polyamino acid / chitosan porous microspheres have a diameter of 278.6 micrometers, an average pore size of 34.5 micrometers, and a porosity of over 85%.

[0038] Example 4 A porous polyamino acid / chitosan microsphere with antibacterial and osteopromoting spatiotemporal effects is prepared by the following steps: S1. Construction of the inner layer: Prepare a 1.25 w / v% chitosan aqueous solution (solvent is 1 v / v% acetic acid aqueous solution) and a 10% Span80 petroleum ether solution; add 4 mL of chitosan aqueous solution to 40 mL of Span80 petroleum ether solution, emulsify thoroughly, pour into 400 mL of -20℃ petroleum ether, let stand, decant the supernatant, and freeze-dry; then treat with 1 w / v% sodium hydroxide ethanol solution, wash with deionized water, and freeze-dry to obtain chitosan porous microspheres.

[0039] S2. Construction of the Intermediate Layer: Preparation of an intermediate layer material with bone regeneration-promoting properties. Poly(L-glutamic acid) (Mn: 20000) and taurine were added to a 0.1 M sodium hydroxide aqueous solution, followed by the addition of a condensing agent, 4-(4,6-dimethoxytriazine-2-yl)-4-methylmorpholine hydrochloride. The molar ratio of poly(L-glutamic acid), taurine, and condensing agent was 5:1:1. The mixture was stirred to ensure uniform dispersion, and the reaction was carried out at 25°C under sealed conditions for 24 hours. After the reaction was completed, the mixture was dialyzed in deionized water for 3 days to completely remove residual condensing agent and taurine. The resulting product was freeze-dried to obtain taurine-grafted polyamino acid (grafting rate 16%).

[0040] S3. Construction of the outer layer: A 5 w / v% chitosan aqueous solution (solvent: 1 v / v% acetic acid aqueous solution) was heated to 60°C, and a 10 w / v% phosphorous acid aqueous solution was added dropwise. The temperature was then raised to 70°C, and a 37 w / w% formaldehyde aqueous solution was added dropwise. The reaction was carried out at 70°C for 8 hours. The molar ratio of chitosan, formaldehyde, and phosphorous acid was 10:1.7:5.1. After the reaction was completed, the mixture was cooled and dialyzed in deionized water for 3 days to completely remove residual acetic acid, formaldehyde, and phosphorous acid. The mixture was then freeze-dried to obtain phosphonated chitosan (phosphonation degree of 18%).

[0041] S4. Construction of polyamino acid / chitosan porous microspheres: The chitosan porous microspheres obtained in step S1 were soaked in a 0.1 mg / mL taurine-grafted polyamino acid aqueous solution. The pH was adjusted to 5.7 using a 0.1 M sodium hydroxide aqueous solution. The molar ratio of -NH2 in chitosan to -COOH and -SO3H groups in polyamino acid was 1:2. After soaking for 12 hours, the microspheres were washed three times with deionized water and then freeze-dried to obtain assembled taurine-grafted polyamino acid porous microspheres. Prepare a 0.1 mg / mL phosphonated chitosan aqueous solution, adjust the pH to 5.7 with 0.1 M sodium hydroxide aqueous solution, and immerse the above-assembled taurine-grafted polyamino acid porous microspheres in this aqueous solution. Control the ratio of the total molar number of -COOH and -SO3H groups in the polyamino acid to the molar number of -NH2 groups in the phosphonated chitosan to be 1:2. Immerse for 12 hours, wash three times with deionized water, and freeze-dry at -50℃ to obtain polyamino acid / chitosan porous microspheres with antibacterial and osteopromoting spatiotemporal effects.

[0042] The obtained polyamino acid / chitosan porous microspheres have a diameter of 290.1 ​​micrometers, an average pore size of 33.4 micrometers, and a porosity of over 85%.

[0043] Example 5 A porous polyamino acid / chitosan microsphere with antibacterial and osteopromoting spatiotemporal effects is prepared by the following steps: S1. Construction of the inner layer: Prepare a 1.25 w / v% chitosan aqueous solution (solvent is 1 v / v% acetic acid aqueous solution) and a 10% Span80 petroleum ether solution; add 4 mL of chitosan aqueous solution to 40 mL of Span80 petroleum ether solution, emulsify thoroughly, pour into 400 mL of -20℃ petroleum ether, let stand, decant the supernatant, and freeze-dry; then treat with 1 w / v% sodium hydroxide ethanol solution, wash with deionized water, and freeze-dry to obtain chitosan porous microspheres.

[0044] S2. Construction of the Intermediate Layer: Preparation of an intermediate layer material with bone regeneration-promoting properties. Poly(L-glutamic acid) (Mn: 20000) and taurine were added to a 0.1 M sodium hydroxide aqueous solution, followed by the addition of a condensing agent, 4-(4,6-dimethoxytriazine-2-yl)-4-methylmorpholine hydrochloride. The molar ratio of poly(L-glutamic acid), taurine, and condensing agent was 5:1:1. The mixture was stirred to ensure uniform dispersion, and the reaction was carried out at 25°C under sealed conditions for 24 hours. After the reaction was completed, the mixture was dialyzed in deionized water for 3 days to completely remove residual condensing agent and taurine. The resulting product was freeze-dried to obtain taurine-grafted polyamino acid (grafting rate 16%).

[0045] S3. Construction of the outer layer: A 5 w / v% chitosan aqueous solution (solvent: 1 v / v% acetic acid aqueous solution) was heated to 60°C, and a 10 w / v% phosphorous acid aqueous solution was added dropwise. The temperature was then raised to 70°C, and a 37 w / w% formaldehyde aqueous solution was added dropwise. The reaction was carried out at 70°C for 8 hours. The molar ratio of chitosan, formaldehyde, and phosphorous acid was 10:1.7:5.1. After the reaction was completed, the mixture was cooled and dialyzed in deionized water for 3 days to completely remove residual acetic acid, formaldehyde, and phosphorous acid. The mixture was then freeze-dried to obtain phosphonated chitosan (phosphonation degree of 18%).

[0046] S4. Construction of polyamino acid / chitosan porous microspheres: The chitosan porous microspheres obtained in step S1 were soaked in a 0.1 mg / mL taurine-grafted polyamino acid aqueous solution. The pH was adjusted to 5.7 using a 0.1 M sodium hydroxide aqueous solution. The molar ratio of -NH2 in chitosan to -COOH and -SO3H groups in polyamino acid was 1:3. After soaking for 12 hours, the microspheres were washed three times with deionized water and then freeze-dried to obtain assembled taurine-grafted polyamino acid porous microspheres. Prepare a 0.1 mg / mL phosphonated chitosan aqueous solution, adjust the pH to 5.7 with 0.1 M sodium hydroxide aqueous solution, and immerse the assembled taurine-grafted polyamino acid porous microspheres in this aqueous solution. Control the ratio of the total molar number of -COOH and -SO3H groups in the polyamino acid to the molar number of -NH2 groups in the phosphonated chitosan to be 1:3. Immerse for 12 hours, wash three times with deionized water, and freeze-dry at -50℃ to obtain polyamino acid / chitosan porous microspheres with antibacterial and osteopromoting spatiotemporal effects.

[0047] The obtained polyamino acid / chitosan porous microspheres have a diameter of 294.3 micrometers, an average pore size of 32 micrometers, and a porosity of over 85%.

[0048] Example 6 A porous polyamino acid / chitosan microsphere with antibacterial and osteopromoting spatiotemporal effects is prepared by the following steps: S1. Construction of the inner layer: Prepare a 1.25 w / v% chitosan aqueous solution (solvent is 1 v / v% acetic acid aqueous solution) and a 10% Span80 petroleum ether solution; add 4 mL of chitosan aqueous solution to 40 mL of Span80 petroleum ether solution, emulsify thoroughly, pour into 400 mL of -20℃ petroleum ether, let stand, decant the supernatant, and freeze-dry; then treat with 1 w / v% sodium hydroxide ethanol solution, wash with deionized water, and freeze-dry to obtain chitosan porous microspheres.

[0049] S2. Construction of the Intermediate Layer: Preparation of an intermediate layer material with bone regeneration-promoting properties. Poly(L-glutamic acid) (Mn: 20000) and taurine were added to a 0.1 M sodium hydroxide aqueous solution, followed by the addition of a condensing agent, 4-(4,6-dimethoxytriazine-2-yl)-4-methylmorpholine hydrochloride. The molar ratio of poly(L-glutamic acid), taurine, and condensing agent was 5:1:1. The mixture was stirred to ensure uniform dispersion, and the reaction was carried out at 25°C under sealed conditions for 24 hours. After the reaction was completed, the mixture was dialyzed in deionized water for 3 days to completely remove residual condensing agent and taurine. The resulting product was freeze-dried to obtain taurine-grafted polyamino acid (grafting rate 16%).

[0050] S3. Construction of the outer layer: A 5 w / v% chitosan aqueous solution (solvent: 1 v / v% acetic acid aqueous solution) was heated to 60°C, and a 10 w / v% phosphorous acid aqueous solution was added dropwise. The temperature was then raised to 70°C, and a 37 w / w% formaldehyde aqueous solution was added dropwise. The reaction was carried out at 70°C for 8 hours. The molar ratio of chitosan, formaldehyde, and phosphorous acid was 10:1.7:5.1. After the reaction was completed, the mixture was cooled and dialyzed in deionized water for 3 days to completely remove residual acetic acid, formaldehyde, and phosphorous acid. The mixture was then freeze-dried to obtain phosphonated chitosan (phosphonation degree of 18%).

[0051] S4. Construction of polyamino acid / chitosan porous microspheres: The chitosan porous microspheres obtained in step S1 were soaked in a 0.1 mg / mL taurine-grafted polyamino acid aqueous solution. The pH was adjusted to 5.7 using a 0.1 M sodium hydroxide aqueous solution. The molar ratio of -NH2 in chitosan to -COOH and -SO3H groups in polyamino acid was 1:4. After soaking for 12 hours, the microspheres were washed three times with deionized water and then freeze-dried to obtain assembled taurine-grafted polyamino acid porous microspheres. Prepare a 0.1 mg / mL phosphonated chitosan aqueous solution, adjust the pH to 5.7 with 0.1 M sodium hydroxide aqueous solution, and immerse the above-assembled taurine-grafted polyamino acid porous microspheres in this aqueous solution. Control the ratio of the total molar number of -COOH and -SO3H groups in the polyamino acid to the molar number of -NH2 groups in the phosphonated chitosan to be 1:4. Immerse for 12 hours, wash three times with deionized water, and freeze-dry at -50℃ to obtain polyamino acid / chitosan porous microspheres with antibacterial and osteopromoting spatiotemporal effects.

[0052] The microstructure and elemental distribution of the samples were observed using scanning electron microscopy combined with energy-dispersive X-ray spectroscopy (EDS), and the pore size distribution of the samples was statistically analyzed using ImageJ to analyze the microstructural characteristics of the materials. Figure 1 Characterization results of the chitosan porous microspheres prepared in this embodiment: (a) Scanning electron microscope image and energy dispersive spectroscopy (EDS) spectrum of C, O, and N elements (scale bar: 50 μm); (b) Pore size distribution diagram. As shown in the figure, the chitosan matrix microspheres are well-formed and have a continuous porous structure. The C, O, and N elements are evenly distributed on the matrix surface, and the pore size distribution range is reasonable, which can provide a stable structural basis and sufficient reaction sites for assembly modification.

[0053] Figure 2Characterization results of the assembled taurine-grafted polyamino acid porous microspheres prepared in this embodiment: (a) Scanning electron microscope image and energy dispersive spectroscopy (EDS) spectrum of C, O, and S elements (scale bar: 50 μm); (b) Pore size distribution map. The results show that after introducing taurine-grafted polyamino acids, the microspheres still maintain a regular spherical shape and porous morphology, and no structural collapse occurs; the elemental distribution characteristics show significant changes, proving that the taurine-grafted polyamino acids have been successfully bound to the surface of chitosan microspheres, and the modification effect is good.

[0054] Figure 3 Characterization results of the polyamino acid / chitosan porous microspheres prepared in this embodiment: (a) Scanning electron microscope image and energy dispersive spectroscopy (EDS) of C, O, and P elements (scale bar: 50 μm); (b) Pore size distribution map. After phosphonate-modified chitosan assembly, the overall morphology and pore structure of the microspheres remained stable, while the elemental composition was further altered, confirming that phosphonate-modified chitosan was successfully loaded onto the microspheres. The target composite porous microspheres were successfully prepared, and their pore structure parameters met the requirements for use as bone repair scaffold materials. Testing showed that the obtained polyamino acid / chitosan porous microspheres had a diameter of 296.7 μm, an average pore size of 31.0 μm, and a porosity exceeding 85%. All pore structure parameters fully met the requirements for use as bone repair scaffold materials.

[0055] OD of bacterial culture medium was measured using an enzyme-linked immunosorbent assay (ELISA) reader. 600 The antibacterial ability of the material was evaluated, and the morphological changes of bacteria were observed using scanning electron microscopy. Escherichia coli and Staphylococcus aureus were selected as test species. Figure 4 The antibacterial performance test results of the polyamino acid / chitosan porous microspheres prepared in this embodiment are as follows: (a) OD of polyamino acid / chitosan porous microspheres with different concentrations after co-culturing with Escherichia coli for 6 h and 24 h. 600 (a) OD values; (b) OD values ​​of different concentrations of polyamino acid / chitosan porous microspheres after co-culturing with Staphylococcus aureus for 6 h and 24 h. 600 (c) Scanning electron microscope images (scale bar 1 μm) of polyamino acid / chitosan porous microspheres with a mass concentration of 5 mg / mL after co-culturing with the two bacteria mentioned above for 24 h. The test results show that the composite microspheres can significantly inhibit the growth and reproduction of the two pathogenic bacteria, and the antibacterial effect increases with the increase of material concentration; microscopic morphological observation shows that the bacterial structure is obviously damaged, indicating that the material has excellent broad-spectrum antibacterial properties.

[0056] An animal femoral defect model was constructed, and polyamino acid / chitosan porous microspheres were implanted and fed for 6 weeks. Micro-CT was used for scanning and three-dimensional reconstruction to evaluate the material's osteogenic capacity in vivo. Figure 5This image shows a Micro-CT 3D reconstruction of the femoral bone repair morphology 6 weeks after implantation of the polyamino acid / chitosan porous microspheres prepared in this embodiment. As can be seen from the image, there is sufficient new bone tissue in the femoral defect area, and the osseointegration effect is excellent, demonstrating that the composite microspheres have good biocompatibility, can effectively induce bone regeneration, and possess excellent in vivo osteogenic properties.

[0057] Figure 6 Scanning electron microscope images of the polyamino acid / chitosan porous microspheres prepared in this embodiment: (a) Overall morphology of the polyamino acid / chitosan porous microspheres (scale bar 50 μm); (b) Detail of the internal structure of the pores (scale bar 10 μm). The images show that the finished microspheres have uniform particle size and good sphericity, with interconnected internal pores and a rough pore wall structure. This microstructure can provide a suitable environment for cell adhesion, proliferation, and the transport of nutrients and metabolites, further ensuring the bone repair application effect of the material.

[0058] Example 7 A porous polyamino acid / chitosan microsphere with antibacterial and osteopromoting spatiotemporal effects is prepared by the following steps: S1. Construction of the inner layer: Prepare a 1.25 w / v% chitosan aqueous solution (solvent is 1 v / v% acetic acid aqueous solution) and a 10% Span80 petroleum ether solution; add 4 mL of chitosan aqueous solution to 40 mL of Span80 petroleum ether solution, emulsify thoroughly, pour into 400 mL of -20℃ petroleum ether, let stand, decant the supernatant, and freeze-dry; then treat with 1 w / v% sodium hydroxide ethanol solution, wash with deionized water, and freeze-dry to obtain chitosan porous microspheres.

[0059] S2. Construction of the Intermediate Layer: Preparation of an intermediate layer material with bone regeneration-promoting properties. Poly(L-glutamic acid) (Mn: 20000) and taurine were added to a 0.1 M sodium hydroxide aqueous solution, followed by the addition of a condensing agent, 4-(4,6-dimethoxytriazine-2-yl)-4-methylmorpholine hydrochloride. The molar ratio of poly(L-glutamic acid), taurine, and condensing agent was 5:1:1. The mixture was stirred to ensure uniform dispersion, and the reaction was carried out at 25°C under sealed conditions for 24 hours. After the reaction was completed, the mixture was dialyzed in deionized water for 3 days to completely remove residual condensing agent and taurine. The resulting product was freeze-dried to obtain taurine-grafted polyamino acid (grafting rate 16%).

[0060] S3. Construction of the outer layer: A 5 w / v% chitosan aqueous solution (solvent: 1 v / v% acetic acid aqueous solution) was heated to 60°C, and a 10 w / v% phosphorous acid aqueous solution was added dropwise. The temperature was then raised to 70°C, and a 37 w / w% formaldehyde aqueous solution was added dropwise. The reaction was carried out at 70°C for 8 hours. The molar ratio of chitosan, formaldehyde, and phosphorous acid was 10:1.7:5.1. After the reaction was completed, the mixture was cooled and dialyzed in deionized water for 3 days to completely remove residual acetic acid, formaldehyde, and phosphorous acid. The mixture was then freeze-dried to obtain phosphonated chitosan (phosphonation degree of 18%).

[0061] S4. Construction of polyamino acid / chitosan porous microspheres: The chitosan porous microspheres obtained in step S1 were soaked in a 0.1 mg / mL taurine-grafted polyamino acid aqueous solution. The pH was adjusted to 5.7 using a 0.1 M sodium hydroxide aqueous solution. The molar ratio of -NH2 in chitosan to -COOH and -SO3H groups in polyamino acid was 1:5. After soaking for 12 hours, the microspheres were washed three times with deionized water and then freeze-dried to obtain assembled taurine-grafted polyamino acid porous microspheres. Prepare a 0.1 mg / mL phosphonated chitosan aqueous solution, adjust the pH to 5.7 with 0.1 M sodium hydroxide aqueous solution, and immerse the assembled taurine-grafted polyamino acid porous microspheres in this aqueous solution. Control the ratio of the total molar number of -COOH and -SO3H groups in the polyamino acid to the molar number of -NH2 groups in the phosphonated chitosan to be 1:5. Immerse for 12 hours, wash three times with deionized water, and freeze-dry at -50℃ to obtain polyamino acid / chitosan porous microspheres with antibacterial and osteopromoting spatiotemporal effects.

[0062] The obtained polyamino acid / chitosan porous microspheres have a diameter of 295.8 micrometers, an average pore size of 31 micrometers, and a porosity of over 85%.

[0063] The above description is merely a preferred embodiment of the present invention and is not intended to limit the invention. Various modifications and variations can be made to the present invention by those skilled in the art. Any modifications, equivalent substitutions, improvements, etc., made within the spirit and principles of the present invention should be included within the scope of protection of the present invention.

Claims

1. A porous polyamino acid / chitosan microsphere with antibacterial and osteopromoting spatiotemporal effects, characterized in that, The porous microspheres have a three-layer composite structure: the inner layer is a chitosan porous microsphere, the middle layer is a bone-promoting layer, and the outer layer is an antibacterial layer. The bone-promoting layer is composed of taurine-grafted polyamino acids.

2. The polyamino acid / chitosan porous microspheres with antibacterial and osteopromoting spatiotemporal effects according to claim 1, characterized in that, The inner layer of chitosan porous microspheres has a diameter of 250-350 micrometers, an average pore size of 25-45 micrometers, and a porosity of ≥85%. The viscosity-average molecular weight of the chitosan is 10,000 to 50,000.

3. The polyamino acid / chitosan porous microspheres with antibacterial and osteopromoting spatiotemporal effects according to claim 1, characterized in that, The taurine-grafted polyamino acid is formed by a covalent amidation reaction between the amino group of taurine and the carboxyl group of the polyamino acid, with a grafting rate of 10% to 20%. The polyamino acid is poly(L-glutamic acid) or poly(L-aspartic acid), with a number-average molecular weight of 20,000 to 40,000.

4. The polyamino acid / chitosan porous microspheres with antibacterial and osteopromoting spatiotemporal effects according to claim 1, characterized in that, The antibacterial layer is composed of phosphonated chitosan; the degree of phosphonation of the phosphonated chitosan is 10% to 30%.

5. A method for preparing polyamino acid / chitosan porous microspheres with antibacterial and osteopromoting spatiotemporal effects as described in any one of claims 1 to 4, characterized in that, Including the following steps: S1. Preparation of the inner layer: Chitosan aqueous solution and Span80 petroleum ether solution were mixed, emulsified and then frozen to crystallize. Chitosan porous microspheres were obtained by freeze drying and treatment with sodium hydroxide ethanol solution. S2, Preparation of the middle layer: Taurine and polyamino acids are dissolved in an alkaline aqueous solution, a condensing agent is added and reacted, and the taurine-grafted polyamino acids are obtained by dialysis and freeze-drying. S3. Preparation of the outer layer: Chitosan was dissolved in an acidic aqueous solution, and an aqueous solution of phosphorous acid and formaldehyde was added to carry out the Mannich reaction. After dialysis and freeze drying, phosphonated chitosan was obtained. S4. Preparation of polyamino acid / chitosan porous microspheres: The taurine-grafted polyamino acid obtained in step S2 and the phosphonated chitosan obtained in step S3 are dissolved in water to obtain an aqueous solution of taurine-grafted polyamino acid and an aqueous solution of phosphonated chitosan. The chitosan porous microspheres obtained in step S1 are immersed in the aqueous solution of taurine-grafted polyamino acid, washed, freeze-dried, and then immersed in the aqueous solution of phosphonated chitosan. After washing and freeze-drying, the polyamino acid / chitosan porous microspheres with antibacterial and osteogenic spatiotemporal effects are obtained.

6. The method for preparing polyamino acid / chitosan porous microspheres with antibacterial and osteopromoting spatiotemporal effects according to claim 5, characterized in that, In step S1, the degree of deacetylation of the chitosan is ≥85%, and the concentration of the chitosan aqueous solution is 1~2 w / v; the chitosan aqueous solution is prepared by dissolving chitosan in 0.5~2 v / v% acetic acid aqueous solution; the concentration of the sodium hydroxide ethanol solution is 0.5~2 w / v; and the volume concentration of Span80 in the Span80 petroleum ether solution is 5~15%.

7. The method for preparing polyamino acid / chitosan porous microspheres with antibacterial and osteopromoting spatiotemporal effects according to claim 5, characterized in that, In step S2, the alkaline aqueous solution is a 0.1~0.5 M sodium hydroxide aqueous solution; the condensing agent is 4-(4,6-dimethoxytriazine-2-yl)-4-methylmorpholine hydrochloride; the molar ratio of the polyamino acid, taurine and the condensing agent is 5:(0.5~1.5):(0.5~1.5); the reaction temperature is 25~40℃ and the reaction time is 8~24 h.

8. The method for preparing polyamino acid / chitosan porous microspheres with antibacterial and osteopromoting spatiotemporal effects according to claim 5, characterized in that, In step S3, the acidic aqueous solution is a 0.5~1.5 v / v% acetic acid aqueous solution; the concentration of the phosphorous acid aqueous solution is 10~15 w / v; the concentration of the formaldehyde aqueous solution is 37 w / w; the reaction steps are as follows: heat the chitosan aqueous solution to 50~65℃, add the phosphorous acid aqueous solution dropwise, then heat to 68~80℃, add the formaldehyde aqueous solution dropwise, and react at 68~80℃ for 8~24 h; wherein the molar ratio of chitosan, formaldehyde, and phosphorous acid is 10:(1~5):(1~6).

9. The method for preparing polyamino acid / chitosan porous microspheres with antibacterial and osteopromoting spatiotemporal effects according to claim 5, characterized in that, In step S4, the concentration of the taurine-grafted polyamino acid aqueous solution is 0.1 mg / mL, and the pH is adjusted to 5.5-5.9 using sodium hydroxide aqueous solution; The concentration of the phosphonated chitosan aqueous solution was 0.1 mg / mL, and the pH was adjusted to 5.5-5.9 using 0.1 M sodium hydroxide aqueous solution. The ratio of the number of moles of amino groups in the chitosan porous microspheres to the total number of moles of carboxyl and sulfonic acid groups in the taurine-grafted polyamino acids is 1:(1~5). The ratio of the total molar number of carboxyl and sulfonic acid groups in the taurine-grafted polyamino acid to the molar number of amino groups in the phosphonic chitosan is 1:(1~5). The soaking time for both soaking sessions was 8 to 24 hours.

10. The method for preparing polyamino acid / chitosan porous microspheres with antibacterial and osteopromoting spatiotemporal effects according to claim 5, characterized in that, In step S4, the water is rinsed three times with deionized water after each soaking; the freeze-drying is carried out at -40~-60℃.

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