Doxorubicin-loaded anti-tumor hemostatic microspheres and preparation and use thereof

Abstract

The present application relates to providing a doxorubicin-loaded anti-tumor hemostatic microsphere constructed by a simple, safe and low-cost method and its preparation and application, the doxorubicin-loaded anti-tumor hemostatic microsphere comprises alginate, hemostatic component A, doxorubicin or doxorubicin liposome, crosslinking agent, the hemostatic component A is selected from one or more of carboxymethyl chitosan, hyaluronic acid, collagen, silk fibroin, solve the practical pain point problems such as easy bleeding in tumor resection surgery, large toxicity of chemotherapeutic drugs, large drug dose, frequent drug administration, etc., realize rapid hemostasis in operation, postoperative precise targeted drug delivery, reduce toxic side effects, long-acting drug sustained release, at the same time, all components of the system are safe and reliable, and have good biocompatibility.

Description

Technical Field

[0001] This invention belongs to the field of pharmaceutical technology, specifically relating to an antitumor hemostatic microsphere loaded with doxorubicin, its preparation and application. Background Technology

[0002] Although oncology has undergone rapid development over the past few decades, giving rise to emerging anti-tumor therapies such as immunotherapy, hormone therapy, and gene therapy, traditional surgical resection combined with chemotherapy remains the most effective means of combating solid tumors. However, problems such as easy bleeding during surgery, high recurrence and metastasis rates of local tumors after surgery, and poor postoperative patient health making systemic chemotherapy unsuitable limit its actual efficacy. Residual tumor cells and changes in the tumor microenvironment caused by surgical trauma can stimulate the rampant proliferation and metastasis of residual tumor cells. Therefore, there is an urgent need to develop a treatment plan that can quickly stop bleeding during tumor resection surgery, eliminate residual tumor cells at the surgical site after surgery, isolate the microenvironment to inhibit tumor recurrence, minimize the risk of cancer cell metastasis, and buy valuable time for the chemotherapy "window period."

[0003] In recent years, the use of nanoparticles to improve drug solubility, enhance drug stability, and target drug delivery has attracted much attention in the field of drug delivery, especially liposome nanovesicles. For example, Chinese patent CN111407742B discloses an anti-tumor nanoparticle, its preparation method, and its application. This nanoparticle can accumulate at the tumor site and release its load through cell vesicle disintegration, allowing it to penetrate deeper into the tumor for treatment. In this technology, large-diameter cell vesicles have cavities for loading loads, including photothermal quantum dots. The cell vesicles act as carriers, prolonging their in vivo circulation time and accumulating at the tumor site. The loads within their cavities also accumulate at the tumor site. Applying photostimulation to the anti-tumor nanoparticles causes the cell vesicles to disintegrate, releasing small-diameter loads. These small-diameter loads can penetrate deeper into the tumor for treatment, and the photothermal quantum dots have photothermal therapeutic effects.

[0004] Chinese patent CN109481418A discloses an anti-tumor nanoparticle comprising a core, a coating layer encapsulating the core, and an immune layer encapsulating the coating layer. The core includes a photosensitizer, which has the function of image-guided photodynamic therapy. The coating layer includes a high-molecular-weight organic compound, which is coated around the core to form nanoparticles, allowing for controllable particle size. The synthesized nanoparticles have a particle size range that conforms to the enhanced permeability and retention effect, resulting in higher stability. The immune layer includes an immune adjuvant, which, when coated around the nanoparticles, enables the nanoparticles to actively target specific areas. Furthermore, the immune adjuvant also has the function of inducing an immune response. The anti-tumor nanoparticles obtained by encapsulating nanoparticles with an immune adjuvant can achieve a combined therapeutic effect of photodynamic therapy and immunotherapy. They exhibit selectivity and controllability in targeting tissues and the degree of damage, reducing damage to normal tissues and making them safer. Simultaneously, they treat tumors by enhancing the patient's own immunity, and are characterized by rapid response, high efficiency, no side effects, long-lasting efficacy, stability, and an important role in preventing postoperative recurrence.

[0005] Doxorubicin liposome nanoparticles (e.g., Doxil®, DOX) have been widely used in the treatment of ovarian cancer, breast cancer, bone cancer, lung cancer, brain cancer, leukemia, and AIDS since their FDA approval. The liposomes are controlled to a particle size between 85 and 100 nanometers, allowing them to accumulate and remain in tumor tissues via the tumor EPR effect (they can only penetrate the intercellular spaces of tumor vascular endothelial cells, 100-400 nm). This significantly improves the actual efficacy of doxorubicin and reduces toxic side effects. However, doxorubicin liposomes still exhibit significant hematologic and cardiotoxicity. Firstly, because doxorubicin liposome formulations are administered intravenously, the drug inevitably enters normal cells and tissues, producing non-targeted toxic side effects. Secondly, because doxorubicin liposomes are a potent chemotherapeutic drug, they are rarely used clinically as an option for in situ local treatment. These issues limit the actual anti-tumor efficacy of doxorubicin liposomes; therefore, more rational and scientific management of their use is still needed. Summary of the Invention

[0006] The technical problem to be solved by this invention is to overcome the shortcomings of the prior art and provide a doxorubicin-loaded antitumor hemostatic microsphere, its preparation and application, which can be constructed in a simple, safe and low-cost manner. This invention solves the practical problems of easy bleeding, high toxicity of chemotherapy drugs, large dosage and frequent administration during tumor resection surgery, and achieves rapid hemostasis during surgery, precise targeted drug administration after surgery, reduced toxic side effects and long-acting drug release. At the same time, all components of this system are safe and reliable and have good biocompatibility.

[0007] To solve the above technical problems, the technical solution adopted by the present invention is as follows:

[0008] An antitumor hemostatic microsphere loaded with doxorubicin comprises alginate, hemostatic component A, doxorubicin or doxorubicin liposomes, and a cross-linking agent. Hemostatic component A is selected from one or more of carboxymethyl chitosan, hyaluronic acid, collagen, and silk fibroin.

[0009] Preferably, the mass ratio of alginate to hemostatic component A to doxorubicin or doxorubicin liposomes is 0.1-5:1000-10:1000-10, more preferably 0.5-5:100-10:100-10, and even more preferably 1-5:50-25:50-25.

[0010] Preferably, the average particle size of the microspheres is 200-2000 μm, and more preferably 200-800 μm.

[0011] Preferably, the alginate is selected from sodium alginate;

[0012] Preferably, the crosslinking agent is selected from those containing Ca. 2+ Crosslinking agent, Ba-containing 2+ Crosslinking agent, Cu-containing 2+ One or more of crosslinking agents or glutaraldehyde.

[0013] To solve the above technical problems, another technical solution adopted by the present invention is:

[0014] A method for preparing doxorubicin-loaded antitumor hemostatic microspheres as described above includes the following steps:

[0015] (1) Preparation of matrix solution: Alginate solution and hemostatic component A solution were prepared separately with purified water. The two solutions were mixed evenly to obtain matrix solution;

[0016] (2) Mixing the drug with the matrix solution: Mix the matrix solution obtained in step (1) with the doxorubicin drug solution evenly, transfer the mixed solution into a sterile syringe, let it stand, and remove air bubbles;

[0017] (3) Cross-linking to prepare microspheres: The syringe is placed in a micro-injection pump, and the mixed solution is slowly injected into the cross-linking agent solution. The microspheres are fully cross-linked by continuous magnetic stirring. The obtained microsphere suspension is allowed to stand, the supernatant is discarded, and the microspheres are washed with sterile pure water and collected.

[0018] Preferably, in step (1), the mass percentage concentration of alginate is 0.1-15%, and the mass percentage concentration of hemostatic component A is 0.1-15%.

[0019] Preferably, in step (1), the alginate solution and the hemostatic component A solution are mixed at a volume ratio of 1-10:0.1-10, more preferably at a ratio of 1:1.

[0020] Preferably, in step (2), the volume ratio of the matrix solution to the doxorubicin solution is 50-1:1;

[0021] Preferably, the doxorubicin solution is selected from doxorubicin liposomes or doxorubicin aqueous solution;

[0022] Preferably, the concentration of doxorubicin in the aqueous solution is 2 mg / mL;

[0023] Preferably, the concentration of doxorubicin liposomes is 2 mg / mL.

[0024] Preferably, in step (3), the syringe needle diameter is 0.001-0.16 mm, the distance between the needle and the crosslinking agent liquid surface is 3-8 cm, and the injection speed is 2-7 mL / min;

[0025] Preferably, the crosslinking agent solution is a CaCl2 solution, and the weight percentage of CaCl2 is 0.5-15%.

[0026] Preferably, the process further includes step (4) freeze drying: adding sufficient liquid nitrogen to fully freeze the microspheres and then completely drying the sample using a freeze dryer to obtain the final product.

[0027] To solve the above technical problems, another technical solution adopted by the present invention is:

[0028] The application of doxorubicin-loaded antitumor hemostatic microspheres as described above in the preparation of antitumor drugs and / or hemostatic drugs.

[0029] This invention also discloses the application effects of the above-mentioned hemostatic microsphere product in experiments such as hemostasis of SD rat tail truncation, hemostasis of SD rat liver injury, and human pancreatic cancer fluorescent cell line PANc-l-luc.

[0030] The mechanism of action of this invention is as follows: Doxorubicin-containing hemostatic microspheres, after freeze-drying, possess micron-sized pores and a loose, porous interior, exhibiting extremely high water absorption and swelling ratios. They are successfully loaded with doxorubicin, achieving high drug loading and encapsulation efficiency (greater than 95%). In a simulated solid tumor resection model, the high water absorption and swelling properties and strong cell adhesion of sodium alginate and carboxymethyl chitosan are utilized to rapidly accumulate blood cells and coagulation factors, forming a physical embolism to reduce blood loss. Simultaneously, Ca2+ is released... 2+ It enters the small tissue gaps and body fluids to accelerate the coagulation cascade reaction to form a stable thrombus clot for rapid hemostasis. Then, it uses the fibrinolytic system to slowly degrade the thrombus clot and the hydrolytic enzyme system to degrade the gel matrix, slowly releasing doxorubicin to directly deliver anti-tumor drugs to the remaining tumor tissue in situ. It has a cytotoxic killing effect on residual tumor cells and forms a physical barrier to prevent residual tumor cells from metastasizing to other organs and blood vessels.

[0031] Due to the adoption of the above technical solutions, the present invention has the following advantages compared with the prior art:

[0032] 1. This invention constructs drug-loaded hemostatic microspheres of doxorubicin / sodium alginate / carboxymethyl chitosan in a simple, safe and low-cost manner. All components of this system are safe and reliable and have good biocompatibility.

[0033] 2. This invention achieves good preparation results through a simple, green, and low-toxicity preparation process, with high drug loading capacity, high drug encapsulation efficiency, no introduction of other chemical reagents, and safe use;

[0034] 3. The hemostatic microspheres of the present invention have a short hemostatic time, and coagulation is completed within 1 minute in both the SD rat half-tail model and the liver injury model;

[0035] 4. This invention enables precise targeted drug delivery after surgery, resulting in potent anti-tumor effects, reduced non-targeted toxicity of chemotherapy drugs, and decreased frequency and dosage of drug administration;

[0036] 5. This invention is easy to use; it can be applied directly to the wound after surgery and provides long-lasting drug release.

[0037] 6. This invention is easy to store and has a long shelf life;

[0038] 7. The sodium alginate and carboxymethyl chitosan in the hemostatic microspheres of this invention are hydrolyzable, have good degradation performance, and are non-immunogenic. Attached Figure Description

[0039] Figure 1 Scanning electron microscope images of the antitumor hemostatic microsphere products in Examples 1-5;

[0040] Figure 2 The graphs shown in Examples 1-4 depict the drug loading, encapsulation efficiency, in vitro sustained release, swelling, and degradation of the antitumor hemostatic microsphere products, where: a) drug loading; b) encapsulation efficiency; c) swelling rate; d) degradation rate; e) in vitro release curve; f) drug release curve within 24 hours.

[0041] Figure 3 Figures showing the in vitro hemostatic performance evaluation of the antitumor hemostatic microspheres in Examples 1-4 (****: P <0.0001), where: a) whole blood inversion experiment result image; b) thrombus clot scanning electron microscope image; c) platelet aggregation rate image;

[0042] Figure 4 The following are graphs evaluating the hemostatic performance of the antitumor hemostatic microspheres in the SD rat semi-tail model of Examples 1-4 (**: P<0.01, ***: P<0.001), where: a) tail wound; b) clotting time; c) bleeding volume.

[0043] Figure 5 The following are the hemostatic performance evaluation figures for the antitumor hemostatic microsphere products in the SD rat liver injury model of Examples 1-4 (*: P<0.05, ***: P<0.001, ****: P<0.0001), where: a: 1. Preoperative figure; 2. Intraoperative figure; 3. Postoperative figure; b. Coagulation time figure; c. Bleeding volume figure;

[0044] Figure 6 The images shown are evaluation diagrams of the antitumor performance of the antitumor hemostatic microspheres in nude mouse models in Examples 1-2, where: a) tumor anatomy diagram after 21 days (× represents no tumor); b) tumor HE staining diagram; c) tumor mass diagram; d) nude mouse weight monitoring diagram; e) in vivo tumor cell imaging diagram; f) in vivo imaging fluorescence quantitative analysis diagram.

[0045] Figure 7 Scanning electron microscope images of the antitumor hemostatic microsphere products in Examples 6-8;

[0046] Figure 8 The graphs show the whole blood coagulation performance evaluation of the antitumor hemostatic microsphere products in Examples 5-8. Detailed Implementation

[0047] To make the technical solution and beneficial effects of the present invention more apparent and understandable, a detailed description is provided below by listing specific embodiments. It should be understood that these embodiments are for illustrative purposes only and are not intended to limit the scope of the invention. Any product identical or similar to the present invention, derived by any person under the guidance of the present invention or by combining the features of the present invention with other prior art, falls within the protection scope of the present invention.

[0048] Unless otherwise specified, the techniques or conditions described in the following embodiments are generally performed in accordance with conventional techniques or conditions described in the literature in this field, or in accordance with the product manual and the manufacturer's recommendations.

[0049] Unless otherwise specified, all starting materials, ingredients and reagents are commercially available or synthesized according to known methods.

[0050] The present invention discloses an antitumor hemostatic microsphere loaded with doxorubicin, comprising alginate, hemostatic component A, doxorubicin or doxorubicin liposomes, and a cross-linking agent. Hemostatic component A is selected from one or more of carboxymethyl chitosan, hyaluronic acid, collagen, and silk fibroin.

[0051] In some specific embodiments, the mass ratio of alginate to hemostatic component A to doxorubicin or doxorubicin liposomes is 0.1-5:1000-10:1000-10, preferably 0.5-5:100-10:100-10, and more preferably 1-5:50-25:50-25.

[0052] In one specific embodiment, the mass ratio of alginate to hemostatic component A to doxorubicin is 1:50:50.

[0053] In one specific embodiment, the mass ratio of alginate to hemostatic component A to doxorubicin is 1:25:25.

[0054] In one specific embodiment, the mass ratio of alginate to hemostatic component A to doxorubicin liposomes is 1:50:50.

[0055] In one specific embodiment, the mass ratio of alginate to hemostatic component A to doxorubicin liposomes is 1:25:25.

[0056] In some specific embodiments, the average particle size of the microspheres is 200-2000 μm, preferably 200-800 μm, and more preferably 400-600 μm.

[0057] In a specific embodiment, the average particle size of the microspheres can be 400 μm, 500 μm, or 600 μm.

[0058] In some specific embodiments, the alginate is selected from sodium alginate (SA). Sodium alginate (SA) is a natural linear anionic polysaccharide extracted from brown algae. It possesses excellent biocompatibility, is non-toxic, non-immunogenic, easily gelled, and readily available, and is considered an excellent hemostatic polymeric biomaterial, a safe material approved for use by the FDA. The α-L-glucuronic acid residues of alginate can react with polyvalent cations (Ca... 2+ It is the most common ion) covalently cross-linked to form an egg-box conformation hydrogel. In the field of hemostasis, alginate / Ca 2+ The cross-linked system exhibits excellent coagulation properties. The mechanism includes (1) the system can release Ca through controlled release. 2+ (1) This ion is a cofactor for platelet activation and several coagulation cascade reactions; (2) Alginate-based wound dressings have excellent water absorption and swelling capacity. When in contact with blood, they can gather a large number of blood cells in the blood, seal capillaries and small blood vessels, physically compress the bleeding wound, and accelerate the formation of thrombi.

[0059] In some specific embodiments, hemostatic component A is selected from one or more of carboxymethyl chitosan, hyaluronic acid, collagen, and silk fibroin, preferably carboxymethyl chitosan.

[0060] Chitosan (Cs) and its derivatives are among the most studied polysaccharides in the field of biological hemostatic materials. Due to their antibacterial, hemostatic, analgesic, and biocompatibility properties, they are widely used in surgery, drug delivery, and tissue engineering. Regarding its coagulation mechanism, firstly, because it contains positively charged amino groups, it can attract and accumulate negatively charged blood cells. When in contact with red blood cells, it exposes phosphatidylserinease on the surface of red blood cells, thereby activating the inherent coagulation pathway. Secondly, after contact with blood, chitosan absorbs platelets and plasma proteins (including fibrinogen). Subsequently, the platelets adhering to the chitosan interact with fibrinogen, leading to intracellular calcium... 2+ Increased concentration stimulates platelet activation, initiating the intrinsic coagulation cascade. In a specific embodiment, carboxymethyl chitosan is used in this invention.

[0061] In some specific embodiments, the crosslinking agent is selected from those containing Ca. 2+ Crosslinking agent, Ba-containing 2+ Crosslinking agent, Cu-containing 2+ One or more of crosslinking agents or glutaraldehyde.

[0062] In a specific embodiment, Ca is included. 2+ The crosslinking agent is selected from CaCl2.

[0063] In a specific embodiment, Cu 2+ The crosslinking agent is selected from CuSO4.

[0064] In a specific embodiment, containing Ba 2+ The crosslinking agent is selected from BaCl2.

[0065] The preparation method of the doxorubicin-loaded antitumor hemostatic microspheres as described above includes the following steps:

[0066] (1) Preparation of matrix solution: Alginate solution and hemostatic component A solution were prepared separately with purified water. The two solutions were mixed evenly to obtain matrix solution;

[0067] (2) Mixing the drug with the matrix solution: Mix the matrix solution obtained in step (1) with the doxorubicin drug solution evenly, transfer the mixed solution into a sterile syringe, let it stand, and remove air bubbles;

[0068] (3) Cross-linking to prepare microspheres: The syringe is placed in a micro-injection pump, and the mixed solution is slowly injected into the cross-linking agent solution. The microspheres are fully cross-linked by continuous magnetic stirring. The obtained microsphere suspension is allowed to stand, the supernatant is discarded, and the microspheres are washed with sterile pure water and collected.

[0069] In some specific embodiments, the mass percentage concentration of alginate in step (1) is 0.1-15%, and can also be 0.1-12%, 0.1-10%, 0.1-8%, 0.1-5%, or more specifically 0.5%, 1%, 2%, 3%, 4%, 5%.

[0070] In some specific embodiments, the mass percentage concentration of hemostatic component A in step (1) is 0.1-15%, and can also be 0.1-12%, 0.1-10%, 0.1-8%, 0.1-5%, or more specifically 0.5%, 1%, 2%, 3%, 4%, 5%.

[0071] In a specific embodiment, the hemostatic component A in step (1) is selected from carboxymethyl chitosan. The mass percentage concentration of carboxymethyl chitosan is 0.1-15%, and can also be 0.1-12%, 0.1-10%, 0.1-8%, 0.1-5%, or more specifically 0.5%, 1%, 2%, 3%, 4%, 5%.

[0072] In a specific embodiment, the mass percentage concentration of alginate in step (1) is 2%.

[0073] In a specific embodiment, the mass percentage concentration of hemostatic component A in step (1) is 2%.

[0074] In some specific embodiments, in step (1), the alginate solution and the hemostatic component A solution are mixed at a volume ratio of 1-10:0.1-10, or 1-5:0.1-5, 1-3:0.1-5, 1-3:0.1-3, or more preferably 1:1.

[0075] In some specific embodiments, the volume ratio of the matrix solution to the doxorubicin solution in step (2) is 50-1:1, and can also be 20-1:1, 15-1:1, 12-5:1, 10-5:1, and more specifically 5:1, 6:1, 7:1, 8:1, 9:1, 10:1.

[0076] In some specific embodiments, the doxorubicin solution is selected from doxorubicin liposomes or doxorubicin aqueous solution.

[0077] In some specific embodiments, the concentration of doxorubicin in the aqueous solution is 2 mg / mL.

[0078] In some specific embodiments, the concentration of doxorubicin liposomes is 2 mg / mL.

[0079] In some specific embodiments, the needle diameter of the syringe in step (3) is 0.001-0.16 mm, preferably 0.16 mm, the distance between the needle and the surface of the crosslinking agent is 3-8 cm, and the injection speed is 2-7 mL / min.

[0080] In a specific embodiment, in step (3), the distance between the needle and the surface of the crosslinking agent is 5 cm, and the injection speed is 4.5 mL / min.

[0081] In some specific embodiments, the crosslinking agent is selected from those containing Ca. 2+ Crosslinking agent, Ba-containing 2+ Crosslinking agent, Cu-containing 2+ One or more of crosslinking agents or glutaraldehyde.

[0082] In a specific embodiment, Ca is included. 2+ The crosslinking agent is selected from CaCl2.

[0083] In a specific embodiment, Cu 2+ The crosslinking agent is selected from CuSO4.

[0084] In a specific embodiment, containing Ba 2+ The crosslinking agent is selected from BaCl2.

[0085] In a specific embodiment, the crosslinking agent solution is a CaCl2 solution, and the weight percentage of CaCl2 is 0.5-15%, or can be 0.5-12%, 0.5-10%, 2-8%, preferably 5%.

[0086] In some specific embodiments, step (4) freeze-drying is also included: adding sufficient liquid nitrogen to fully freeze the microspheres and then completely drying the sample using a freeze dryer to obtain the final product.

[0087] In some specific embodiments, the freeze-dried microspheres were placed in brown sample vials, sealed, and stored at -20 °C.

[0088] The application of doxorubicin-loaded antitumor hemostatic microspheres as described above in the preparation of antitumor drugs and / or hemostatic drugs.

[0089] Example 1

[0090] (1) Preparation of matrix solution: Prepare a 2% sodium alginate solution and a 2% carboxymethyl chitosan solution with pure water respectively. Mix the two solutions at a volume ratio of 1:1 to obtain the matrix solution for later use.

[0091] (2) Mixing the drug with the matrix solution: The matrix solution and doxorubicin liposome (Doxil®, 2 mg / mL) solution were then mixed at a volume ratio of 10:1 (SCs-lip-1). The mixture was then transferred to a sterile syringe and placed in a 4 ℃ refrigerator for 2 h to remove air bubbles.

[0092] (3) Cross-linking microspheres: The syringe (needle diameter 0.16 mm) was placed in the micro-injection pump, and the mixed solution was slowly injected into 50 mL of 5% CaCl2 solution. The needle was 5 cm away from the surface of the cross-linking agent. The injection speed was 4.5 mL / min. The cross-linking was carried out by magnetic stirring for 10 min to ensure that the microspheres were fully cross-linked. The obtained microsphere suspension was allowed to stand for 5 min, the supernatant was discarded, and 50 mL of sterile pure water was added to wash 3 times. The microspheres were then collected.

[0093] (4) Add sufficient liquid nitrogen to freeze the microspheres thoroughly, and dry the sample completely using a freeze dryer. Place the treated microspheres in a brown sample bottle, seal it, and store it at -20 ℃.

[0094] Example 2

[0095] (1) Preparation of matrix solution: Prepare a 2% sodium alginate solution and a 2% carboxymethyl chitosan solution with pure water respectively. Mix the two solutions at a volume ratio of 1:1 to obtain the matrix solution for later use.

[0096] (2) Mixing the drug with the matrix solution: The matrix solution and doxorubicin liposome (Doxil®, 2 mg / mL) solution were then mixed evenly at a volume ratio of 5:1 (SCs-lip-2). The mixture was then transferred to a sterile syringe and placed in a 4 ℃ refrigerator for 2 h to remove air bubbles.

[0097] (3) Cross-linking microspheres: The syringe (needle diameter 0.16 mm) was placed in the micro-injection pump, and the mixed solution was slowly injected into 50 mL of 5% CaCl2 solution. The needle was 5 cm away from the surface of the cross-linking agent. The injection speed was 4.5 mL / min. The cross-linking was carried out by magnetic stirring for 10 min to ensure that the microspheres were fully cross-linked. The obtained microsphere suspension was allowed to stand for 5 min, the supernatant was discarded, and 50 mL of sterile pure water was added to wash 3 times. The microspheres were then collected.

[0098] (4) Add sufficient liquid nitrogen to freeze the microspheres thoroughly, and dry the sample completely using a freeze dryer. Place the treated microspheres in a brown sample bottle, seal it, and store it at -20 ℃.

[0099] Example 3

[0100] (1) Preparation of matrix solution: Prepare a 2% sodium alginate solution and a 2% carboxymethyl chitosan solution with pure water respectively. Mix the two solutions at a volume ratio of 1:1 to obtain the matrix solution for later use.

[0101] (2) Mixing the drug with the matrix solution: Then mix the above matrix solution with the 2 mg / mL doxorubicin aqueous solution at a volume ratio of 10:1 (numbered SCs-DOX-1), then transfer the mixed solution into a sterile syringe and place it in a 4 ℃ refrigerator for 2 h to remove air bubbles;

[0102] (3) Cross-linking microspheres: The syringe (needle diameter 0.16 mm) was placed in the micro-injection pump, and the mixed solution was slowly injected into 50 mL of 5% CaCl2 solution. The needle was 5 cm away from the surface of the cross-linking agent. The injection speed was 4.5 mL / min. The cross-linking was carried out by magnetic stirring for 10 min to ensure that the microspheres were fully cross-linked. The obtained microsphere suspension was allowed to stand for 5 min, the supernatant was discarded, and 50 mL of sterile pure water was added to wash 3 times. The microspheres were then collected.

[0103] (4) Add sufficient liquid nitrogen to freeze the microspheres thoroughly, and dry the sample completely using a freeze dryer. Place the treated microspheres in a brown sample bottle, seal it, and store it at -20 ℃.

[0104] Example 4

[0105] (1) Preparation of matrix solution: Prepare a 2% sodium alginate solution and a 2% carboxymethyl chitosan solution with pure water respectively. Mix the two solutions at a volume ratio of 1:1 to obtain the matrix solution for later use.

[0106] (2) Mixing the drug with the matrix solution: Then mix the above matrix solution with 2 mg / mL doxorubicin aqueous solution at a volume ratio of 5:1 (SCs-DOX-2). Then transfer the mixed solution into a sterile syringe and place it in a 4 ℃ refrigerator for 2 h to remove air bubbles.

[0107] (3) Cross-linking microspheres: The syringe (needle diameter 0.16 mm) was placed in the micro-injection pump, and the mixed solution was slowly injected into 50 mL of 5% CaCl2 solution. The needle was 5 cm away from the surface of the cross-linking agent. The injection speed was 4.5 mL / min. The cross-linking was carried out by magnetic stirring for 10 min to ensure that the microspheres were fully cross-linked. The obtained microsphere suspension was allowed to stand for 5 min, the supernatant was discarded, and 50 mL of sterile pure water was added to wash 3 times. The microspheres were then collected.

[0108] (4) Add sufficient liquid nitrogen to freeze the microspheres thoroughly, and dry the sample completely using a freeze dryer. Place the treated microspheres in a brown sample bottle, seal it, and store it at -20 ℃.

[0109] Example 5

[0110] (1) Preparation of matrix solution: Prepare a 2% sodium alginate solution and a 2% carboxymethyl chitosan solution with pure water respectively. Mix the two solutions at a volume ratio of 1:1 to obtain the matrix solution for later use.

[0111] (2) Mixing the drug with the matrix solution: The matrix solution and doxorubicin liposome (Doxil®, 2 mg / mL) solution were then mixed at a volume ratio of 10:1 (SCs-lip-3). The mixture was then transferred to a sterile syringe and placed in a 4 ℃ refrigerator for 2 h to remove air bubbles.

[0112] (3) Cross-linking microspheres: The syringe (needle diameter 0.16 mm) was placed in the micro-injection pump, and the mixed solution was slowly injected into 50 mL of 5% CaCl2 solution. The needle was 5 cm away from the surface of the cross-linking agent. The injection speed was 4.5 mL / min. The cross-linking was carried out by magnetic stirring for 10 min to ensure that the microspheres were fully cross-linked. The obtained microsphere suspension was allowed to stand for 5 min, the supernatant was discarded, and 50 mL of sterile pure water was added to wash 3 times. The microspheres were then collected.

[0113] (4) Place the microspheres in a -80 ℃ refrigerator to pre-cool overnight, and then completely dry the sample using a freeze dryer. Place the treated microspheres in a brown sample bottle, seal it, and store it at -20 ℃.

[0114] Example 6

[0115] (1) Preparation of matrix solution: Prepare a 2% sodium alginate solution and a 2% hyaluronic acid solution by using pure water. Mix the two solutions at a volume ratio of 1:1 to obtain the matrix solution for later use.

[0116] (2) Mixing the drug with the matrix solution: Then mix the above matrix solution with the doxorubicin liposome (Doxil®, 2 mg / mL) solution at a volume ratio of 5:1 (number SH-lip-2). Then transfer the mixed solution into a sterile syringe and place it in a 4 ℃ refrigerator for 2 h to remove air bubbles.

[0117] (3) Cross-linking microspheres: The syringe (needle diameter 0.16 mm) was placed in the micro-injection pump, and the mixed solution was slowly injected into 50 mL of 5% CaCl2 solution. The needle was 5 cm away from the surface of the cross-linking agent. The injection speed was 4.5 mL / min. The cross-linking was carried out by magnetic stirring for 10 min to ensure that the microspheres were fully cross-linked. The obtained microsphere suspension was allowed to stand for 5 min, the supernatant was discarded, and 50 mL of sterile pure water was added to wash 3 times. The microspheres were then collected.

[0118] (4) Add sufficient liquid nitrogen to freeze the microspheres thoroughly, and dry the sample completely using a freeze dryer. Place the treated microspheres in a brown sample bottle, seal it, and store it at -20 ℃.

[0119] Example 7

[0120] (1) Preparation of matrix solution: Prepare a 2% sodium alginate solution and a 2% collagen solution by using purified water. Mix the two solutions at a volume ratio of 1:1 to obtain the matrix solution for later use.

[0121] (2) Mixing the drug with the matrix solution: The matrix solution and doxorubicin liposome (Doxil®, 2 mg / mL) solution were then mixed evenly at a volume ratio of 5:1 (numbered SCol-lip-2). The mixture was then transferred to a sterile syringe and placed in a 4 ℃ refrigerator for 2 h to remove air bubbles.

[0122] (3) Cross-linking microspheres: The syringe (needle diameter 0.16 mm) was placed in the micro-injection pump, and the mixed solution was slowly injected into 50 mL of 5% CaCl2 solution. The needle was 5 cm away from the surface of the cross-linking agent. The injection speed was 4.5 mL / min. The cross-linking was carried out by magnetic stirring for 10 min to ensure that the microspheres were fully cross-linked. The obtained microsphere suspension was allowed to stand for 5 min, the supernatant was discarded, and 50 mL of sterile pure water was added to wash 3 times. The microspheres were then collected.

[0123] (4) Add sufficient liquid nitrogen to freeze the microspheres thoroughly, and dry the sample completely using a freeze dryer. Place the treated microspheres in a brown sample bottle, seal it, and store it at -20 ℃.

[0124] Example 8

[0125] (1) Preparation of matrix solution: Prepare a 2% sodium alginate solution and a 2% silk fibroin solution with pure water respectively. Mix the two solutions at a volume ratio of 1:1 to obtain the matrix solution for later use.

[0126] (2) Mixing the drug with the matrix solution: The matrix solution and doxorubicin liposome (Doxil®, 2 mg / mL) solution were then mixed evenly at a volume ratio of 5:1 (numbered SSF-lip-2). The mixture was then transferred to a sterile syringe and placed in a 4 ℃ refrigerator for 2 h to remove air bubbles.

[0127] (3) Cross-linking microspheres: The syringe (needle diameter 0.16 mm) was placed in the micro-injection pump, and the mixed solution was slowly injected into 50 mL of 5% CaCl2 solution. The needle was 5 cm away from the surface of the cross-linking agent. The injection speed was 4.5 mL / min. The cross-linking was carried out by magnetic stirring for 10 min to ensure that the microspheres were fully cross-linked. The obtained microsphere suspension was allowed to stand for 5 min, the supernatant was discarded, and 50 mL of sterile pure water was added to wash 3 times. The microspheres were then collected.

[0128] (4) Add sufficient liquid nitrogen to freeze the microspheres thoroughly, and dry the sample completely using a freeze dryer. Place the treated microspheres in a brown sample bottle, seal it, and store it at -20 ℃.

[0129] Test case

[0130] 1. Scanning electron microscopy analysis of microspheres

[0131] Objective: To observe the microstructure characteristics of the microsphere products and substrate materials (SCs) in Examples 1-5.

[0132] Methods: To observe the surface morphology of hemostatic microspheres, the surface of the microsphere samples was coated with gold and then scanned using a scanning electron microscope (Hitachi S-4800, Japan). The results are shown in the attached figure. Figure 1 As shown.

[0133] Results: (attached) Figure 1 The microspheres, in their dry state, are approximately 1 mm in diameter, with a wrinkled surface and a loose, porous network structure inside. This structure significantly increases the specific surface area and water absorption swelling ratio of the microspheres. At 50,000x magnification, liposome particles (approximately 100 nm) are clearly visible attached to the microsphere surface. Due to the high cross-linking density and good mechanical properties, the microspheres maintain a good spherical morphology during freeze-drying without collapse. Microspheres pre-cooled with liquid nitrogen retain their morphology better after freeze-drying, while samples pre-cooled at -80℃ show severe collapse and fail to maintain their normal morphology.

[0134] 2. Drug loading and encapsulation, in vitro sustained release, swelling and degradation analysis

[0135] Objective: To determine the drug loading capacity, drug release behavior, water absorption swelling and degradation properties of the microsphere products in Examples 1-4.

[0136] Methods: Determination of drug loading and encapsulation efficiency: Standard curves were plotted according to Part II of the 2015 edition of the Chinese Pharmacopoeia. Appropriate amounts of doxorubicin standard were diluted with methanol to concentrations of 0, 10, 20, 30, 40, and 50 µg / mL. OD values ​​were measured using a UV spectrophotometer. 478 (n = 3). Weigh 2 mg of the sample to be tested, add 2 mL of 1×PBS buffer (pH = 7.4), and allow it to swell completely at 37°C. Then centrifuge at 12000 rpm for 10 min, and take the supernatant to measure OD. 478 Each sample was tested in triplicate to calculate drug loading and encapsulation efficiency. The results are shown in the attached figure. Figure 2 As shown in a and b.

[0137] Drug loading EE% = Drug content / Total material mass × 100%

[0138] Encapsulation efficiency EF% = (Drug content / Theoretical drug amount) × 100%

[0139] In vitro sustained release: To simulate the drug release model of hemostatic microspheres in vitro, 10 mg of microspheres were co-incubated with 4 mL of PBS solution (pH = 7.2) at 37°C. 100 µL of supernatant was collected at 0 h, 0.5 h, 2 h, 6 h, 24 h, 48 h, 72 h, 96 h, and 120 h (with an equal volume of fresh PBS added) and stored at -20°C. A 10 µg / mL DOX (doxorubicin) standard solution was prepared with methanol, and a standard curve was plotted at injection volumes of 20, 15, 10, 5, and 0 µL. The supernatant was collected, dissolved at room temperature, and 200 µL of chromatographic grade methanol was added. The mixture was vortexed for 5 min and then centrifuged at 12000 rpm for 10 min. 100 µL was transferred to a sample vial for high-performance liquid chromatography (HPLC) to determine the doxorubicin content in the sample solution. The results are shown in the attached figure. Figure 2 As shown in e and f.

[0140] Liquid chromatography conditions: Instrument model (Agilent 1100 Series), chromatographic column (Agilent Zorbax300SB C) 18 The sample was prepared using a 250 mm × 4.6 mm, 5 mm fluorescence detector (FLD G1321A). The mobile phase consisted of 32.5% acetonitrile, 67.4% water, and 0.1% triethylamine, adjusted to pH 3 with phosphoric acid. After ultrasonic agitation, the sample was filtered through a 0.22 µm filter membrane. The injection flow rate was set to 1 mL / min, the temperature to 37 °C, and the excitation and receiving wavelengths to 480 nm and 560 nm, respectively.

[0141] Swelling and Degradation: To investigate the water absorption and swelling capacity of the antitumor hemostatic microspheres, 2 mg of microspheres were weighed, and the weight of the EP tube (W0) and the weight of the EP tube containing the microspheres (W1) were recorded. The microspheres were incubated in 2 mL of PBS solution (pH = 7.2) at 37°C for 1 h, 2 h, 6 h, 24 h, 48 h, and 72 h. After centrifugation at 12000 rpm for 2 min to remove excess liquid, the wet weight (W2) was recorded. Fresh 2 mL of PBS solution was added after each recording. The swelling rate was calculated using the following formula, and the results are shown in the appendix. Figure 2 As shown in c:

[0142] Swelling rate (%) = (W2−W1) / (W1−W0)×100%

[0143] To simulate the degradation of microspheres in vivo, 2 mg of microspheres were weighed and added to 2 mL of PBS for complete swelling. The microspheres were then centrifuged to remove the supernatant, and the initial wet weight (W0) was recorded. Subsequently, the microspheres were incubated at 37°C in PBS (pH = 7.2) containing 1.5 μg / mL lysozyme. Finally, at 0 h, 5 h, 10 h, and 15 h, the microspheres were centrifuged, the supernatant was removed, and the wet weight (W1) was recorded. The biodegradation rate was calculated using the following formula, and the results are attached. Figure 2 As shown in d:

[0144] Degradation rate (%) = (W0 − W1) / W0 × 100%

[0145] Results: (attached) Figure 2 a and b indicate that the drug loading of SCs-lip-1 is around 1%, while that of SCs-lip-2 is around 2%, showing a good dose-response relationship consistent with theoretical dose differences. Meanwhile, the drug loading and encapsulation efficiency of the SCs-lip series microspheres are significantly higher than those of the free dose group SCs-DOX, suggesting that liposome-loaded doxorubicin can better bind to the SA / CMCs composite matrix, forming a more stable system.

[0146] Appendix Figure 2Figures e and f show that, in sufficient PBS buffer, all samples reached their peak release around 24 hours, followed by a slow decline (possibly due to the instability and slow degradation of DOX under alkaline conditions). Notably, the SCs-lip drug-loaded microspheres exhibited a typical biphasic release behavior (fast initially, then slow). This release model can provide good therapeutic effect in a short time and maintain the effective concentration through prolonged drug release. However, this short-term burst release behavior is not always ideal; excessive release can lead to drug degradation and short drug retention time, resulting in decreased efficacy. Therefore, the applicant compared the cumulative drug release during the rapid release phase (0-24 h) with the theoretical maximum release. The results showed that the cumulative drug content during the rapid release phase was consistently around 20%. This is a reasonable result, ensuring both rapid drug release for therapeutic effect and preventing short-term sustained release due to excessive burst release. These results demonstrate the excellent sustained-release performance of the SCs-lip series of hemostatic microspheres.

[0147] Appendix Figure 2 c shows that all samples reached their maximum swelling ratio in approximately 6 hours. Notably, the substrate material group (SCs) rapidly reached its maximum swelling ratio of 69.8 ± 14.1 within 1 hour before rapidly degrading. This is likely due to the extremely low cross-linking density of carboxymethyl chitosan, which continuously dissolved in the changing buffer system, leading to a rapid decrease in mass. SCs-lip-2 exhibited the best water absorption capacity among all material groups, achieving an impressive maximum swelling ratio of 74.2 ± 11.2 times. This strong water absorption capacity ensures hemostatic performance, enabling rapid absorption and aggregation of blood cells and clotting factors, thus promoting thrombus formation.

[0148] After reaching maximum swelling ratio, the degradation behavior of the microspheres in vivo was simulated. Since the human body does not possess a specific enzyme for alginate degradation under natural conditions, it can, however, utilize Ca... 2+ Lysozyme replaces monovalent cations in body fluids, disrupting their cross-linked systems and thus achieving degradation. Therefore, lysozyme was introduced into the PBS buffer system to mimic its degradation behavior by acidic polysaccharide-degrading enzymes in vivo. Figure 2 The results showed that all samples degraded slowly within 15 hours, with a maximum degradation rate of only about 20%. This slow degradation is beneficial for the slow release of the drug, prolongs the drug residence time, and reduces the risks associated with sudden drug release.

[0149] 3. In vitro coagulation analysis

[0150] Objective: To investigate the coagulation and blood cell enrichment properties of the hemostatic microspheres in Examples 1-4 in vitro.

[0151] Methods: Whole blood inversion test: Weigh 5 mg of hemostatic material (3 portions) into 1.5 mL Eppendorf tubes (using commercially available hemostatic microspheres CELOX as a positive control), add 500 µL of rabbit anticoagulated blood, vortex for 5 s, and invert the tubes every 100 s to observe the blood flow. BK is the blank control group. If the blood is flowable, reset the tubing until the blood is completely coagulated or the time exceeds 600 s. Record the time required for complete coagulation. Results are attached. Figure 3 As shown in a.

[0152] Blood cell adsorption capacity: Thrombi collected after the above inverted tube experiment (30 min later) were washed twice with 2 mL of sterile PBS to remove unadhered blood cells, and fixed with 0.5 mL of 2% glutaraldehyde (electron microscopy fixative) for 2 h. The thrombus samples were then removed and dehydrated using a gradient of 2 mL ethanol (20%, 50%, 70%, 100%, 12 h for each step, 4°C). After dehydration, the samples were air-dried overnight at room temperature, cut into small pieces, fixed onto a silicon electron microscope slide, sputter-coated with gold, and the adsorption of blood cells was observed using a scanning electron microscope. The results are shown in the attached figure. Figure 3 As shown in b.

[0153] Platelet aggregation: 15 mL of New Zealand rabbit whole blood (purchased from the Animal Experiment Center of Hangzhou Normal University) was centrifuged three times at 800 rpm for 8 min in a 15 mL centrifuge tube, collecting the platelet-rich plasma (PRP) supernatant. Then, 2.5 mg of hemostatic sample (three replicates per sample) was mixed with 0.5 mL of rabbit platelet suspension (RPR) (CELOX was used as a positive control). After incubation for 1, 3, and 5 minutes, 10 µL of supernatant was collected from each tube, diluted, and the number of platelets was counted using an inverted fluorescence microscope. The pre-aggregation platelet count (BA) was used as a blank control. The platelet aggregation rate (AGR) was calculated using the following formula, and the results are attached. Figure 3 As shown in c:

[0154] AGR = (BA - AA) / AA × 100% (BA: platelet count before aggregation; AA: platelet count after aggregation)

[0155] Results: (attached) Figure 3 The results showed that the liposome-loaded microspheres (SCs-lip) exhibited excellent coagulation ability, forming stable thrombus clots within 100 s. The free DOX group (SCs-DOX) and the substrate material group (SCs) also formed stable thrombus clots within 200 s. This may be due in part to the rapid release of a large amount of Ca2+ from the liposome-loaded microspheres upon contact with blood. 2+It accelerates blood clotting, thanks in part to the freeze-drying process and the high swelling and water absorption rate resulting from its high specific surface area. However, commercially available CELOX does not provide sufficient clotting performance at a 5 mg dose, and it still cannot form a stable thrombus clot after prolonged standing (500 s).

[0156] Further scanning electron microscopy imaging of the thrombus clot was performed, with attached... Figure 3 b shows that a large number of blood cells are adsorbed on the surface of the hemostatic material, and red blood cells (disc-shaped) and activated platelets (stellate-shaped) can be clearly seen.

[0157] Appendix Figure 3 c shows that although the platelet aggregation experiment showed no significant difference in platelet enrichment rate among all material groups, all were significantly different from the positive control CELOX group (P < 0.0001). In summary, these results indicate that the SCs-lip series hemostatic microspheres accelerate blood solidification by enriching blood cells and coagulation factors through high water absorption and swelling ratio.

[0158] 4. Analysis of the SD rat hemostasis model with partial tail truncation

[0159] Objective: To evaluate the coagulation performance of the microsphere products from Examples 1-4 in SD rat tail vein and artery models with high blood flow and high blood pressure.

[0160] Methods: Forty-two male SD rats (200-250 g) were randomly divided into 7 groups. Dry gauze was used as a blank control (BK), and commercially available hemostatic microspheres CELOX were used as a positive control. The rats were anesthetized by intraperitoneal injection of pentobarbital (30 mg / kg). After scalpel cutting off half of the rat's tail, the tail was allowed to bleed normally for 15 seconds. The tail was then immersed in a 5 mL EP tube containing 20 mg of pre-weighed hemostatic material, and the wound was covered with minimal pressure. Clotting time (s) and blood loss (g) were recorded during hemostasis. The results are shown in the attached figure. Figure 4 As shown. All rats were euthanized at the end of the experiment in accordance with animal ethics requirements.

[0161] Results: The rat tail is rich in tail veins and one tail artery, and is therefore often used to evaluate the hemostatic performance of hemostatic materials. Due to the presence of arterial and venous pressures, the tail-cuneation model is a significant test of the adhesion and rapid hemostatic action of hemostatic materials. (Appendix) Figure 4The results showed that, at the same mass (20 mg), the liposome-containing hemostatic microspheres SCs-lip-1 and SCs-lip-2 exhibited exceptionally good coagulation properties. After normal bleeding from the wound, immersion in the hemostatic microspheres SCs-DOX-2, SCs-lip-1, and SCs-lip-2 resulted in complete hemostasis within 50 seconds, significantly superior to the blank control group (*** P < 0.001) and the positive control group (assessment criterion: no further bleeding within 20 seconds), with less blood loss. The SCs-lip-2 group showed significantly lower blood loss and clotting time compared to the commercially available hemostatic microsphere CELOX.

[0162] 5. Analysis of a hemostasis model of liver injury in SD rats

[0163] Objective: To evaluate the coagulation properties of the microsphere products from Examples 1-4 in a rat model of liver injury in SD rats.

[0164] Methods: Forty-two male SD rats were randomly divided into seven groups of six each. Dry gauze was used as a blank control (BK), and commercially available hemostatic microspheres CELOX were used as a positive control. After anesthetizing the rats with pentobarbital (30 mg / kg), the skin was incised along the midline of the abdomen to expose the liver, and the wound was secured with a mouth gag. Excess fluid on the surface of the liver was then wiped away with clean, sterile gauze. A 1cm × 1cm × 0.5cm cross-shaped incision was then made in the left lobe of the liver using a sterile scalpel. After wiping away any oozing blood with sterile cotton, 10 mg of microspheres was quickly administered for hemostasis. Bleeding time, preoperative gauze weight (W0), and postoperative gauze weight (W1) were recorded. Results are attached. Figure 5 As shown in the image. Finally, the abdominal wound was sutured shut, and all rats were euthanized by injecting an overdose of anesthetic at the end of the experiment.

[0165] Results: The liver is the body's blood reservoir, containing a large amount of blood, and is therefore often used in coagulation models. Immediately after creating a cross-shaped wound, 10 mg of hemostatic material was administered, and clotting time and blood loss were recorded. Figure 5 The results showed significant differences between the commercially available positive control CELOX group and the substrate material SCs group and the control group (gauze) (P < 0.001), while there were highly significant differences between the two SCs-lip groups and the blank control group (P < 0.0001). SCs-lip-1 and SCs-lip-2 exhibited excellent coagulation performance in both clotting time and blood loss. The SCs-lip series of hemostatic microspheres not only rapidly absorb water and swell, enriching blood cells to form physical emboli, but also release Ca2+. 2+ It enters bodily fluids and then seeps into tiny wound crevices, accelerating blood clotting.

[0166] 6. Anti-tumor performance analysis

[0167] Objective: To evaluate the antitumor performance of the antitumor hemostatic microspheres from Examples 1-2 during tumor resection surgery.

[0168] Methods: Human pancreatic cancer cells PANC-1-luc were seeded at a 30% inoculum in 1640 medium containing 10% FBS and 1% penicillin and streptomycin. The cells were cultured at 90% confluence in a 5% CO2 incubator at 37°C and then passaged for expansion. Once the desired cell count was reached, all cells were digested with trypsin, centrifuged, and collected into 50 mL sterile centrifuge tubes. The cells were washed three times with sterile saline to remove residual serum and other contaminants, and finally resuspended in saline. The cells were then divided into 1×10⁻⁶ cells / tubes. 7 200 μL / cell was subcutaneously injected into the axilla of nude mice. The tumor was allowed to grow to approximately 200 mm in size. 3 Nude mice were anesthetized with Sertazone 50 (50 mg / kg) to simulate tumor resection surgery, leaving approximately 100 mm of tumor tissue intact. 3 Tumors. After treatment with 5 mg of hemostatic microspheres (positive control group treated with an equivalent dose of doxorubicin in SCs-lip-2 via tail vein injection; nude mice undergoing the same surgical procedure without any treatment served as blank control (BK)), the wound was sutured and ampicillin was administered to prevent infection. At specified time intervals, nude mice were intraperitoneally injected with a luminescent substrate (luciferin). Tumor size and density were assessed by detecting the fluorescence intensity of tumor cells using in vivo imaging. Results are shown in the attached figure. Figure 6 As shown.

[0169] Results: In a simulated solid tumor resection surgery experiment, 5 mg of antitumor hemostatic microspheres were added to the tumor resection site. An equivalent dose of Doxil® was injected intravenously into nude mice as a positive control. Nude mice undergoing the same surgical procedure without any treatment served as a blank control. This was used to evaluate the antitumor performance of SCs-lip hemostatic microspheres. Figure 6 The results showed that after the 21-day experimental period, the tumors in the SCs-lip-1 and SCs-lip-2 treatment groups were significantly smaller than those in the blank control group and the positive control group. In particular, one nude mouse in each of the SCs-lip-1 and SCs-lip-2 treatment groups had no visible residual tumor tissue, and the average tumor mass was also significantly less than that in the control group.

[0170] Quantitative analysis of tumor cell viability in tissues was performed using in vivo imaging. Results showed that one day after surgery, SCs-lip hemostatic microspheres loaded with doxorubicin liposomes exhibited high local concentrations of liposomes, leading to a rapid and significant decrease in the viability of remaining tumor cells, which expressed only a small amount of live-cell fluorescence. Over time, the viable tumor cells in both experimental groups showed slow growth, indicating that the liposomes loaded with SCs-lip were slowly released over a long period, demonstrating a long-term tumor-inhibiting effect. In contrast, the blank control group experienced rapid tumor cell proliferation post-surgery. During the 21-day experimental period, the relative tumor inhibition rate of SCs-lip antitumor hemostatic microspheres was over 95% compared to the blank control group, indicating that SCs-lip can enhance the retention time of doxorubicin in tumor tissue and exhibit a long-lasting and potent antitumor effect.

[0171] 7. Scanning electron microscopy analysis

[0172] Objective: To observe the microstructure of the products in Examples 6-8

[0173] Methods: To observe the surface morphology of hemostatic microspheres, the surface of the microsphere samples was coated with gold and then scanned using a scanning electron microscope (Hitachi S-4800, Japan). The results are shown in the attached figure. Figure 7 As shown.

[0174] Results: Attached Figure 7 The results showed that all samples exhibited a complete ellipsoidal shape with a rough, wrinkled surface, which facilitates blood cell adhesion and promotes coagulation. However, the sodium alginate / silk fibroin composite microspheres had weak mechanical properties, and their spherical shape collapsed and ruptured after freeze-drying.

[0175] 8. In vitro coagulation analysis

[0176] Objective: To evaluate the whole blood coagulation properties of the products in Examples 5-8 in vitro.

[0177] Methods: Whole blood inversion test: Weigh 5 mg of hemostatic material (3 portions) into 1.5 mL Eppendorf tubes (with no treatment as a blank control, BK) and add 500 µL of rabbit anticoagulated blood. Vortex for 5 s. Invert the tubes every 100 s and observe the blood flow. If the blood is still flowing, reset the tubing until the blood is completely coagulated or the time exceeds 600 s. Record the time required for complete coagulation.

[0178] Results: (attached) Figure 8The results showed that the tested samples SCs-lip-3 and SH-lip-2 exhibited excellent coagulation ability, forming stable thrombus clots within 100 seconds. SCl-lip-2 and SSF-lip-2, on the other hand, showed good coagulation ability, forming stable thrombus clots within 300 seconds. Therefore, SCs-lip-3 and SH-lip-2 demonstrated superior coagulation ability compared to SCl-lip-2 and SSF-lip-2.

[0179] In summary, as shown in the appendix Figure 3 The results showed that both SCs-lip groups of microspheres could form stable thrombus clots within 100 s. The alginate / chitosan-free DOX group and the substrate material group (SCs) also formed stable thrombus clots within 200 s. This was partly due to the DOX-loaded liposome microspheres in Ca... 2+ The cross-linked system forms a more stable system, and the microspheres contain a higher concentration, causing the microspheres to rapidly release a large amount of Ca upon contact with blood. 2+ Accelerated coagulation is also attributed to the freeze-drying process and the high swelling and water absorption rate resulting from the high specific surface area. Further scanning electron microscopy imaging of the thrombus clot revealed a large number of blood cells adsorbed on the surface of the hemostatic material, with red blood cells (disc-shaped) and activated platelets (stellate-shaped) clearly visible. Platelet aggregation experiments showed no significant difference in platelet enrichment between the SCs series microspheres and the positive control CELOX group (P<0.0001). In summary, the SCs series hemostatic microspheres enrich blood cells and coagulation factors and release Ca through high water absorption and swelling rate. 2+ It accelerates blood solidification. The SCs-lip series microspheres, due to their more complete morphology and higher Ca concentration, [are effective in this regard]. 2+ It exhibits excellent hemostasis speed.

[0180] This invention utilizes sodium alginate, carboxymethyl chitosan, and doxorubicin liposomes as raw materials, and calcium chloride as a green crosslinking agent to prepare biodegradable composite antitumor hemostatic microspheres. The aim is to achieve rapid hemostasis during tumor resection surgery and, postoperatively, to serve as a "warehouse" for long-acting, sustained-release antitumor drugs. This invention leverages the high water absorption and swelling ratio, strong cell adhesion, and calcium chloride properties of sodium alginate and carboxymethyl chitosan. 2+ The coagulation cascade reaction plays a role in rapid hemostasis during tumor resection surgery. Furthermore, the swollen gel-liposome system can be used postoperatively for precise in-situ drug delivery and sustained release of anti-tumor drugs, thereby achieving rapid hemostasis during tumor resection surgery and local sustained release of anti-tumor drugs after surgery to inhibit tumor recurrence and metastasis.

[0181] It should be understood that the above embodiments are exemplary and are not intended to encompass all possible implementations included in the claims. Various modifications and changes can be made to the above embodiments without departing from the scope of this disclosure. Similarly, the various technical features of the above embodiments can be arbitrarily combined to form other embodiments of the present invention that may not be explicitly described. Therefore, the above embodiments only illustrate several implementations of the present invention and do not limit the scope of protection of this patent.

Claims

1. A doxorubicin-loaded antitumor hemostatic microsphere, characterized in that, It contains sodium alginate, hemostatic component A, doxorubicin liposomes, and a cross-linking agent, wherein the hemostatic component A is selected from carboxymethyl chitosan; The crosslinking agent is selected from Ca 2+ Crosslinking agent; The mass ratio of sodium alginate to hemostatic component A to doxorubicin liposomes is 1-5:50-25:50-25.

2. The doxorubicin-loaded antitumor hemostatic microspheres according to claim 1, characterized in that, The average particle size of the microspheres is 200-2000 μm.

3. The doxorubicin-loaded antitumor hemostatic microspheres according to claim 1 or 2, characterized in that, The average particle size of the microspheres is 200-800 μm.

4. A method for preparing doxorubicin-loaded antitumor hemostatic microspheres as described in any one of claims 1 to 3, characterized in that, Includes the following steps: (1) Preparation of matrix solution: Alginate solution and hemostatic component A solution were prepared separately with purified water. The two solutions were mixed evenly to obtain matrix solution; (2) Mixing the drug with the matrix solution: Mix the matrix solution obtained in step (1) with doxorubicin liposomes evenly, transfer the mixed solution into a sterile syringe, let it stand, and remove air bubbles; (3) Cross-linking to prepare microspheres: The syringe is placed in a micro-injection pump, and the mixed solution is slowly injected into the cross-linking agent solution. The microspheres are fully cross-linked by continuous magnetic stirring. The obtained microsphere suspension is allowed to stand, the supernatant is discarded, and the microspheres are collected by washing with sterile pure water.

5. The method for preparing doxorubicin-loaded antitumor hemostatic microspheres according to claim 4, characterized in that, In step (1), the mass percentage concentration of alginate is 0.1-15%, and the mass percentage concentration of hemostatic component A is 0.1-15%.

6. The method for preparing doxorubicin-loaded antitumor hemostatic microspheres according to claim 4, characterized in that, In step (1), the alginate solution and the hemostatic component A solution are mixed at a volume ratio of 1-10:0.1-10.

7. The method for preparing doxorubicin-loaded antitumor hemostatic microspheres according to claim 6, characterized in that, In step (1), the alginate solution and the hemostatic component A solution are mixed at a volume ratio of 1:

1.

8. The method for preparing doxorubicin-loaded antitumor hemostatic microspheres according to claim 4, characterized in that, In step (2), the volume ratio of the matrix solution to doxorubicin liposomes is 50-1:

1.

9. The method for preparing doxorubicin-loaded antitumor hemostatic microspheres according to claim 4, characterized in that, The concentration of doxorubicin liposomes was 2 mg / mL.

10. The method for preparing doxorubicin-loaded antitumor hemostatic microspheres according to claim 4, characterized in that, In step (3), the syringe needle diameter is 0.001-0.16 mm, the distance between the needle and the crosslinking agent liquid surface is 3-8 cm, and the injection speed is 2-7 mL / min.

11. The method for preparing doxorubicin-loaded antitumor hemostatic microspheres according to claim 4, characterized in that, The crosslinking agent solution is a CaCl2 solution, and the weight percentage of CaCl2 is 0.5-15%.

12. The method for preparing doxorubicin-loaded antitumor hemostatic microspheres according to claim 4, characterized in that, It also includes step (4) freeze drying: add sufficient liquid nitrogen to freeze the microspheres fully, and then use a freeze dryer to completely dry the sample to obtain the final product.

13. The use of doxorubicin-loaded antitumor hemostatic microspheres as described in any one of claims 1 to 3 in the preparation of antitumor drugs and / or hemostatic drugs.

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