Composite hemostatic microspheres and a method for preparing the same

Abstract

The application discloses a kind of composite hemostatic microspheres and preparation method thereof, belong to the field of biomedical materials.The hemostatic microspheres are composed of polysaccharide polymer, inorganic nanoparticles and silk fibroin, the mixing ratio of raw materials is accurately regulated using microfluidic, monodisperse microspheres with hemostatic function are produced in batches by centrifugal spraying, and the particle size of microspheres is uniform and controllable.The polysaccharide polymer gives the microspheres rapid water absorption and film forming properties, the inorganic nanoparticles activate blood coagulation factors by contact, and the silk fibroin promotes wound healing.The application utilizes the double shearing action of microfluidic chip flow channel and centrifugal spraying system flow channel to achieve uniform distribution of inorganic nanoparticles in the superficial layer of microspheres, and the hemostatic effect is remarkable;The film forming property is good, and the risk of distal embolism can be avoided;And it has anti-inflammatory and healing functions, and is suitable for rapid hemostasis of body surface wounds and organ surfaces.

Description

Technical Field

[0001] This invention relates to the field of biomedical materials, specifically to a composite hemostatic microsphere and its preparation method. Background Technology

[0002] Surgical procedures and uncontrollable traumatic bleeding pose a core challenge in clinical emergency care and postoperative management. Rapid and effective hemostasis is crucial for reducing patient mortality and improving prognosis. Especially in battlefield emergency care, traffic accidents, and complex surgical procedures, uncontrollable bleeding is the leading cause of death among injured individuals. Therefore, developing safe, efficient, and easy-to-use hemostatic materials has always been a hot topic and a significant challenge in the field of biomedical materials.

[0003] Polysaccharide polymer hemostatic materials have attracted widespread attention due to their advantages such as wide availability, low cost, biodegradability, and lack of immunogenicity. Currently, the most typical polysaccharide hemostatic product on the market is Arista hemostatic powder produced by Medafor in the United States. This product is made by purifying plant starch, removing proteins, and then undergoing a 28-day emulsification and cross-linking process to generate polysaccharide spherical particles with a particle size of approximately 100 μm and a surface covered with micropores. It is mainly suitable for controlling oozing from superficial wounds. However, clinical practice and research have shown that Arista hemostatic powder and similar products still have many shortcomings. These mainly manifest in a single hemostatic mechanism and poor efficacy against active bleeding from the body surface. The simple physical water absorption and concentration mechanism is effective for capillary bleeding and small venous bleeding, but for larger volumes of active bleeding, the gel formed is easily washed away by the blood flow, significantly reducing hemostatic efficacy.

[0004] Literature CN122124306A discloses a kaolin-based multifunctional hemostatic microsphere, which combines a kaolin-calcium peroxide composite material with sodium alginate. The hemostatic performance is enhanced by utilizing the contact-activated coagulation activity of kaolin and the synergistic effect of calcium ion precipitation, showing improved hemostatic efficacy compared to single polysaccharide hemostatic materials. Constructing composite microspheres with multi-mechanism synergistic hemostasis by introducing components with active coagulation functions, such as inorganic nanoparticles like kaolin, halloysite, and diatomaceous earth, has become a research hotspot. However, in existing technologies, simply mixing inorganic nanoparticles with polysaccharide polymers to form microspheres results in the inorganic nanoparticles being encapsulated inside the microsphere, preventing sufficient contact with blood and significantly reducing coagulation activation efficiency. The ideal solution would be to adsorb inorganic nanoparticles onto the surface and near-surface layer of the microsphere body, but current preparation processes struggle to achieve this structural design.

[0005] Literature CN117603496A discloses a chitosan microsphere and its preparation method. Porous chitosan hemostatic microspheres are prepared using an airflow-assisted extrusion method combined with tert-butanol displacement freeze-drying, achieving improved hemostatic performance with a simplified process and low toxicity. However, this method uses chitosan as the single active ingredient, and the hemostatic mechanism mainly relies on the electrostatic adsorption of erythrocytes and platelets by the positive charge of chitosan itself. No active components promoting tissue repair are introduced, and there is a lack of active intervention in the wound healing process after hemostasis. Silk fibroin, a natural polymer extracted from silkworm cocoons, possesses excellent biocompatibility, controllable degradation properties, and the ability to promote cell adhesion and proliferation. It can accelerate fibroblast migration and collagen deposition and has certain anti-inflammatory effects, positively promoting wound healing. Simply adding silk fibroin to hemostatic microspheres will result in rapid degradation and ineffectiveness if it is distributed on the surface of the microspheres; if it is distributed inside, its release behavior needs to be controlled. Therefore, introducing silk fibroin into hemostatic microspheres and distributing it inside the microspheres to exert a continuous effect is an effective way to solve the above problems. However, existing technologies lack precise control over the spatial distribution of silk fibroin.

[0006] Besides the aforementioned product-level shortcomings, existing preparation technologies themselves also have significant defects. Literature CN120189545A discloses a gelatin hemostatic microsphere method, which relies on the shear force of mechanical stirring to control particle size. The droplet size in the emulsion system is affected by multiple factors such as stirring speed, temperature, and emulsifier concentration, resulting in limited precision in particle size control and making it difficult to achieve stable preparation of highly monodisperse microspheres. In recent years, microfluidic technology has received widespread attention due to its advantage in preparing highly monodisperse microspheres; however, the throughput of a single microfluidic technology is low, making it difficult to meet the needs of mass production. Centrifugal spraying technology can achieve rapid production, but its use alone makes it difficult to precisely control the microsphere particle size. Therefore, combining microfluidics with centrifugal spraying, utilizing the dual shearing action of the microfluidic chip channel and the centrifugal spray channel, holds promise for achieving a unified approach to precise microsphere particle size control and mass production; however, such methods are currently rarely reported.

[0007] In summary, the existing technologies have the following technical problems: (1) Commercially available hemostatic products have a single hemostatic mechanism and are not effective for active bleeding; (2) Inorganic nanoparticles are easily encapsulated inside after simple mixing, making it difficult to achieve their distribution on the surface and near-surface layer of microspheres; (3) Existing hemostatic materials lack the function of promoting healing, making it difficult to effectively connect the tissue repair process after hemostasis; (4) The microspheres prepared by traditional methods have uneven particle size (CV is usually >20%), poor batch stability, and cannot stably achieve the requirement of uniform particle size; (5) Single microfluidic throughput is low, single centrifugal spray particle size control is poor, and there is a lack of technical solutions that combine high monodispersity and mass production capability.

[0008] Therefore, how to solve the problems of uneven distribution and easy encapsulation by polymers from the perspective of material composite structure design while retaining the high coagulation activity of inorganic nanoparticles, and how to achieve highly uniform and controllable microsphere size and stable mass production through innovative preparation processes, while taking into account the tissue repair needs in the wound healing process, has become a technical bottleneck that urgently needs to be overcome in this field. Summary of the Invention

[0009] This invention addresses the shortcomings of existing technologies by providing a composite hemostatic microsphere and its preparation method.

[0010] The composite hemostatic microspheres comprise: polysaccharide polymers, inorganic nanoparticles, and silk fibroin, with a particle size of 5-50 μm and a particle size variation coefficient ≤5%; The polysaccharide polymer forms the microsphere framework, the inorganic nanoparticles are distributed on the surface and near-surface layer of the microsphere body, and the silk fibroin is distributed inside the microsphere.

[0011] Preferably, the polysaccharide polymer is 40-60 parts, the inorganic nanoparticles are 1-10 parts, and the silk fibroin is 20-40 parts.

[0012] Preferably, the polysaccharide polymer is one or a combination of two or more of lotus root starch, potato starch, ethyl cellulose, and oxidized cellulose.

[0013] Preferably, the inorganic nanoparticles are one or more of kaolin, halloysite, or diatomaceous bio-silica; the particle size of the inorganic nanoparticles is 200-800 nm.

[0014] The preparation method of the composite hemostatic microspheres includes the following steps: (1) Prepare silk fibroin solution A and polysaccharide polymer and inorganic nanoparticle mixed solution B; (2) Solution A and solution B are pumped into a Y-type microfluidic chip to precisely control the solution ratio, thus obtaining solution C; (3) Simultaneously, solution C is directly connected to the centrifugal spray system for batch preparation to obtain composite hemostatic microspheres.

[0015] Preferably, in step (1), the concentration of silk fibroin in solution A is 2-8%, and the solvent is one or more of formic acid, ethanol / water / calcium chloride solution or ionic liquid; the concentration of polysaccharide polymer in solution B is 2%-8%, and the solvent is one or more of ethanol, alkaline aqueous solution or ionic liquid.

[0016] Preferably, in step (2), the inner diameter of the Y-type chip channel is 100-300 μm, the flow rate of solution A is 100-400 μL / min, and the flow rate of solution B is 20-600 μL / min.

[0017] Preferably, in step (3), the centrifugal spray speed is 2000-4000 rpm and the spinneret orifice is 25-27 G.

[0018] The beneficial effects of this invention are: (1) Significantly improved hemostatic effect. Polysaccharide polymers endow microspheres with rapid water absorption and film-forming properties, achieving physical concentration hemostasis; inorganic nanoparticles adsorb onto the surface and near-surface layer of microspheres, activating coagulation factors through contact and initiating the intrinsic coagulation pathway. The synergistic effect of the two significantly shortens the in vitro coagulation time, resulting in rapid hemostasis for active bleeding on the body surface.

[0019] (2) The microfluidic ratio is accurate, the centrifugal spray system has high output, the resulting microspheres have uniform particle size (5-50 μm, CV≤5%), the product quality is stable, and the batch-to-batch repeatability is good, avoiding the problem of unstable hemostatic effect caused by the wide particle size distribution of traditional methods.

[0020] (3) Under the shearing action of the flow channel in the microfluidic chip and the shearing action of the centrifugal spray flow channel, the particulate matter is concentrated on the surface of the microsphere, and the inorganic nanoparticles are effectively exposed on the surface of the microsphere and the near-surface layer, resulting in high contact activation efficiency and avoiding the problem of inorganic particles being encapsulated and deactivated due to traditional simple mixing.

[0021] (4) Due to its high molecular weight, silk fibroin is distributed inside the microspheres under shearing action. As the microspheres absorb water, expand and break down, they gradually exert anti-inflammatory and healing effects. When applied to the mid-to-late stages of wound management, it can accelerate wound repair. Attached Figure Description

[0022] To more clearly illustrate the technical solutions in the embodiments of the present invention or the prior art, the drawings used in the description of the embodiments or the prior art will be briefly introduced below. Obviously, the drawings described below are only some embodiments of the present invention. For those skilled in the art, other drawings can be obtained based on these drawings without creative effort.

[0023] Figure 1 Flowchart of the preparation process for composite hemostatic microspheres; Figure 2 This is a scanning electron microscope (SEM) image of the composite hemostatic microspheres in Example 1; Figure 3 The relative cell viability of L929 cells after culturing in the culture medium containing the composite hemostatic microspheres of Example 1 for 72 h; Figure 4 Cell viability / dead color images after culturing L929 cells in the culture medium containing the composite hemostatic microspheres from Example 1 for 72 h; Figure 5The hemostatic effect of the composite hemostatic microspheres in the rabbit ear bleeding model in Example 1 within 120 seconds; Figure 6 The healing effect of the composite hemostatic microspheres in the rat wound model in Example 1 within 7 days is shown. Detailed Implementation

[0024] To facilitate understanding by those skilled in the art, the present invention will be further described below with reference to embodiments and accompanying drawings. The content mentioned in the embodiments is not intended to limit the present invention.

[0025] like Figure 1 As shown, the present invention provides a method for preparing composite hemostatic microspheres, comprising the following steps: Step (1): Prepare silk fibroin solution A and a mixed solution B of polysaccharide polymer and inorganic nanoparticles; Step (2): Pump solutions A and B into a Y-type microfluidic chip to precisely control the solution ratio, thus obtaining solution C; Step (3): Simultaneously connect solution C directly to the centrifugal spray system for batch preparation to obtain composite hemostatic microspheres.

[0026] Example 1: Take 40 parts lotus root starch, 1 part kaolin, and 20 parts silk fibroin. First, prepare silk fibroin solution A (concentration 2%, solvent: formic acid) and a mixed solution B (lotus root starch concentration 2%, solvent: alkaline aqueous solution) of lotus root starch and kaolin particles (particle size 500 nm). Pump solution A at 100 μL / min and solution B at 20 μL / min into a Y-type microfluidic chip (channel inner diameter 100 μm) for ratio control and initial shearing to obtain solution C. Connect the tubing containing solution C directly to a centrifugal spray system (speed 2000 rpm, needle specification 27 G) for secondary shearing and shaping to obtain composite hemostatic microspheres. The average particle size is 15 μm, CV=4.5%, water absorption ratio is 32-fold, the relative cell viability of L929 cells after 72 hours of culture is approximately 98%, the hemostasis time in the rabbit ear hemorrhage model is 165±4 seconds, and the bacterial endotoxin is <0.05 EU.

[0027] The morphology and performance of the composite hemostatic microspheres prepared in this embodiment were characterized and evaluated. Scanning electron microscopy observations as follows Figure 2 As shown, the microspheres are regularly spherical, and the kaolin nanoparticles are uniformly distributed on the surface and near-surface layer of the microspheres. This indicates that the present invention successfully achieves the enrichment of inorganic nanoparticles in the shallow layer of the microspheres through the dual shearing action of microfluidic chip and centrifugal spray, avoiding the particles being completely encapsulated inside the microspheres and improving the coagulation activation efficiency.

[0028] Cell compatibility evaluation: The microspheres prepared in this example were added to L929 cell culture medium at different concentrations (0.4, 0.8, 1.6, 3.2, 6.4 mg / mL) and cultured for 72 h. CCK-8 assay results showed that the relative cell viability of each concentration group was above 95%. Figure 3 As shown; live / dead staining images of L929 cells cultured in 6.4 mg / mL microsphere medium for 72 h show that live cells (green fluorescence) are absolutely dominant, and dead cells (red fluorescence) are very few, as shown. Figure 4 As shown, the microspheres of the present invention are non-cytotoxic and have good biosafety.

[0029] Evaluation of hemostatic effect: A rabbit ear bleeding model was used for hemostasis experiments. The wound results within 120 seconds after drug application were as follows: Figure 5 As shown, the microsphere group of this invention formed a stable blood clot on the wound surface within 120 seconds. After complete timing and statistical analysis, the average time to complete hemostasis was 165±4 seconds, while the average hemostasis time in the blank control group under the same conditions exceeded 300 seconds without complete hemostasis. The hemostatic effect of the microsphere group was significantly better than that of the blank control group. These results indicate that the rapid water absorption and film formation of the polysaccharide polymer, combined with the contact activation of coagulation by kaolin, has a significant synergistic hemostatic effect.

[0030] Evaluation of healing promotion effect: The rat full-thickness skin wound model was used to evaluate the healing promotion effect. The wound results on days 1, 3, and 7 postoperatively are as follows: Figure 6 As shown, the wound healing rate in the microsphere treatment group was significantly higher than that in the blank control group on the 7th postoperative day, with obvious wound contraction and complete new epithelial coverage. On the 7th postoperative day, wound tissue was taken for HE staining. The results showed that the treatment group had abundant new blood vessels, neatly arranged collagen fibers, and less inflammatory cell infiltration, confirming that the silk fibroin distributed inside the microspheres was continuously released after hemostasis, exerting anti-inflammatory and tissue repair-promoting effects.

[0031] Example 2: Take 50 parts of potato starch, 5.5 parts of halloysite (particle size 200 nm), and 30 parts of silk fibroin. First, prepare a 5% silk fibroin solution A (solvent: ethanol / water / calcium chloride solution) and a mixed solution B of potato starch and halloysite (potato starch concentration 5%, solvent: ionic liquid). Pump solution A at 250 μL / min and solution B at 310 μL / min into a Y-type microfluidic chip (inner diameter 200 μm) for initial shearing to obtain solution C. Connect the tubing containing solution C directly to a centrifugal spray system (3000 rpm, needle specification 26 G) for secondary shearing and shaping to obtain composite hemostatic microspheres. The average particle size is 25 μm, CV = 4.8%, water absorption ratio is 30-fold, the relative cell viability of L929 cells after 72 hours of culture is approximately 97%, the hemostasis time in a rabbit ear hemorrhage model is 135 ± 3 seconds, and the bacterial endotoxin is <0.05 EU.

[0032] Example 3: Take 60 parts of a 1:1 mixture of ethyl cellulose and oxidized cellulose, 10 parts of a 1:1 mixture of halloysite and diatomaceous earth silica (800 nm particle size), and 40 parts of silk fibroin. Prepare silk fibroin solution A (8% concentration, ionic liquid composite solvent) and a mixed solution B of ethyl cellulose / oxidized cellulose and halloysite / diatomaceous earth silica (8% polysaccharide concentration, ethanol / alkaline aqueous solution). Pump solution A at 400 μL / min and solution B at 600 μL / min into a Y-type microfluidic chip (300 μm inner diameter) for initial shearing to obtain solution C. Connect the tubing containing solution C directly to a centrifugal spray system (4000 rpm, 25 G needle size) for secondary shearing and shaping to obtain composite hemostatic microspheres. Its average particle size is 50 μm, CV=4.2%, water absorption ratio is 27 times, the relative cell viability of L929 cells after 72 hours of culture is about 95%, the hemostasis time of rabbit ear hemorrhage model is 93±4 seconds, and the bacterial endotoxin is <0.05EU.

[0033] Example 4: Take 50 parts of lotus root starch, 5.5 parts of diatomaceous earth (particle size 500 nm), and 30 parts of silk fibroin. First, prepare a 5% silk fibroin solution A (solvent: formic acid) and a mixed solution B of lotus root starch and diatomaceous earth (polysaccharide concentration 5%, solvent: ethanol / alkaline aqueous solution). Pump solution A at 250 μL / min and solution B at 310 μL / min into a Y-type microfluidic chip. By adjusting the inner diameter of the Y-type microfluidic chip (100 μm, 200 μm, 300 μm) and the flow rate ratio, composite hemostatic microspheres with average particle sizes of 5 μm, 25 μm, and 50 μm are prepared respectively. Among them, the 5 μm group has a water absorption ratio of 35 times, and the relative cell viability of L929 cells after 72 hours of culture is approximately 96.5%, and the hemostasis time in the rabbit ear hemorrhage model is 108±3 seconds. The 25 μm group showed the best overall performance with a water absorption rate of 30-fold, a relative cell viability of approximately 94.8% for L929 cells after 72 hours of culture, and a hemostasis time of 87±3 seconds in the rabbit ear hemorrhage model. The 50 μm group showed a water absorption rate of 27-fold, a relative cell viability of approximately 94.5% for L929 cells after 72 hours of culture, and a hemostasis time of 81±3 seconds in the rabbit ear hemorrhage model. All groups had a CV (volume variation) of ≤4.5% for the hemostatic microspheres, indicating uniform and controllable particle size.

[0034] The above embodiments are preferred implementations of the present invention. In addition, the present invention can be implemented in other ways. Any obvious substitutions without departing from the concept of the present technical solution are within the protection scope of the present invention.

[0035] To facilitate understanding by those skilled in the art of the improvements of this invention over the prior art, some of the accompanying drawings and descriptions have been simplified, and for clarity, some other elements have been omitted from this application. Those skilled in the art should realize that these omitted elements may also constitute the content of this invention.

Claims

1. A composite hemostatic microsphere, characterized in that, The composite hemostatic microspheres comprise: polysaccharide polymers, inorganic nanoparticles, and silk fibroin, with a particle size of 5-50 μm and a particle size variation coefficient ≤5%; The polysaccharide polymer forms the microsphere framework, the inorganic nanoparticles are distributed on the surface and near-surface layer of the microsphere body, and the silk fibroin is distributed inside the microsphere.

2. The composite hemostatic microspheres according to claim 1, characterized in that, The polysaccharide polymer is 40-60 parts, the inorganic nanoparticles are 1-10 parts, and the silk fibroin is 20-40 parts.

3. The composite hemostatic microspheres according to claim 1, characterized in that, The polysaccharide polymer is one or more of lotus root starch, potato starch, ethyl cellulose, and oxidized cellulose.

4. The composite hemostatic microspheres according to claim 1, characterized in that, The inorganic nanoparticles are one or more of kaolin, halloysite, or diatomaceous bio-silica; the particle size of the inorganic nanoparticles is 200-800 nm.

5. A method for preparing composite hemostatic microspheres according to any one of claims 1-4, characterized in that, Includes the following steps: Step (1): Prepare silk fibroin solution A and a mixed solution B of polysaccharide polymer and inorganic nanoparticles; Step (2): Pump solutions A and B into a Y-type microfluidic chip to precisely control the solution ratio, thus obtaining solution C; Step (3): Simultaneously connect solution C directly to the centrifugal spray system for batch preparation to obtain composite hemostatic microspheres.

6. The method for preparing the composite hemostatic microspheres according to claim 5, characterized in that, In step (1), the concentration of silk fibroin in solution A is 2-8%, and the solvent is one or more of formic acid, ethanol / water / calcium chloride solution or ionic liquid.

7. The method for preparing the composite hemostatic microspheres according to claim 5, characterized in that, In step (1), the concentration of polysaccharide polymer in solution B is 2-8%, and the solvent is one or more of ethanol, alkaline aqueous solution or ionic liquid.

8. The method for preparing the composite hemostatic microspheres according to claim 5, characterized in that, In step (2), the inner diameter of the Y-type chip channel is 100-300 μm, the flow rate of solution A is 100-400 μL / min, and the flow rate of solution B is 20-600 μL / min.

9. The method for preparing the composite hemostatic microspheres according to claim 5, characterized in that, In step (3), the centrifugal spray speed is 2000-4000 rpm and the spinneret orifice is 25-27 G.

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