Porous microsphere as well as preparation method and application thereof

Abstract

The invention relates to the technical field of biological medicines, and particularly discloses a porous microsphere as well as a preparation method and application thereof. Comprising the following steps: (1) weighing alginate and dissolving the alginate in a solvent to obtain an alginate solution; (2) preparing an ethanol solution containing calcium ions as a stationary liquid, and performing first freezing treatment to obtain a frozen stationary liquid; (3) dispersing the alginate solution obtained in the step (1) into liquid drops under the action of an electric field, and performing second freezing treatment to obtain ice beads; (4) adding the ice beads in the step (3) into the stationary liquid subjected to freezing treatment in the step (2), stirring and standing to obtain semi-finished microspheres; and (5) washing and collecting the semi-finished microspheres obtained in the step (4), and freeze-drying to obtain the porous microspheres. The calcium alginate microspheres which are controllable in particle size and are of a porous structure are efficiently prepared, and the calcium alginate microspheres have good biocompatibility, good liquid absorption performance and excellent hemostasis capacity.

Description

Technical Field

[0001] The present invention relates to the field of biomedical technologies, and particularly to a porous microsphere, a preparation method thereof, and an application thereof. Background Art

[0002] Biocompatible porous microspheres have been widely used in biomedical fields such as cell culture and carriers, drug delivery systems, tissue engineering, adsorption, wound hemostasis, and tissue reconstruction due to their excellent biocompatibility, large pore structure, and adjustable physical and chemical properties. Among the commonly used microsphere preparation methods at present, the emulsification method requires a large amount of organic solvents such as oil phase and emulsifier, and there may be problems of solvent residues, which are difficult to meet the requirements of green and environmental protection preparation; the supercritical fluid method has high equipment requirements, complex processes, and high costs, which limits its application; the gas foaming method and spray drying method can lead to uneven pore distribution and affect the performance consistency of microspheres.

[0003] The electrostatic dispersion method is a simple method commonly used for the preparation of gel microspheres and can relatively accurately control the particle size. Taking alginate microspheres as an example, an alginate sodium solution is dispersed under the action of a high-voltage electric field and dropped into an aqueous solution rich in calcium ions to form gel microspheres by solidification. However, after the alginate gel microspheres prepared by this method are freeze-dried, it is difficult to directly form the required microporous structure, which limits its application in the preparation of porous microspheres.

[0004] Generally, porous microspheres can be applied to fields such as wound hemostasis. Severe bleeding caused by trauma seriously endangers life, and timely and effective hemostasis is crucial for saving lives. Traditional hemostasis methods, such as ligation, suture, electrocoagulation, absorbable hemostatic clips, and tourniquets, rely on physical compression hemostasis and can cause tissue adhesion and secondary injuries. In contrast, hemostatic powders are simple to use and only need to be sprinkled on the bleeding site to quickly play a hemostatic role, avoiding complex operations. However, when the current microspheres are applied to the field of wound hemostasis, the microspheres are implanted into the body at the wound. After entering the body, due to poor compatibility, they cannot be completely degraded; and the current microspheres generally have a high hemolysis rate and do not have good blood compatibility, further leading to a decrease in their coagulation ability; at the same time, due to still existing deficiencies in the structure of the current microspheres, when hemostasis is performed, red blood cells and platelets cannot be adhered to their surfaces, so that blood cannot be quickly absorbed and blood cells cannot be enriched, and the coagulation ability is poor.

[0005] Therefore, it is necessary to provide a porous microsphere, a preparation method thereof, and an application thereof, so that the porous microsphere has good cell compatibility, high liquid absorption capacity, blood coagulation promotion and hemostasis ability. Summary of the Invention

[0006] The present invention aims to solve at least one of the technical problems existing in the above-mentioned prior art. For this purpose, the present invention provides a porous microsphere, a preparation method thereof and an application thereof, so that the porous microsphere has good cell compatibility, high liquid absorption capacity, blood coagulation promotion and hemostasis ability.

[0007] The first aspect of the present invention provides a preparation method of a porous microsphere.

[0008] Specifically, a preparation method of a porous microsphere includes the following steps:

[0009] (1) Weigh alginate and dissolve it in a solvent to obtain an alginate solution;

[0010] (2) Prepare an ethanol solution containing calcium ions as a fixing solution and perform a first freezing treatment to obtain the fixed solution after freezing treatment;

[0011] (3) Under the action of an electric field, disperse the alginate solution in step (1) into droplets and perform a second freezing treatment to obtain ice beads;

[0012] (4) Add the ice beads in step (3) to the fixed solution after freezing treatment in step (2), stir and let stand to obtain semi-finished microspheres;

[0013] (5) Rinse and collect the semi-finished microspheres in step (4), and freeze-dry to obtain the porous microspheres.

[0014] Preferably, in step (1), the mass ratio of the alginate to the solvent is 0.001 - 0.1:1.

[0015] More preferably, in step (1), the mass ratio of the alginate to the solvent is 0.01 - 0.1:1.

[0016] Even more preferably, in step (1), the mass ratio of the alginate to the solvent is 0.01 - 0.05:1.

[0017] Preferably, the alginate includes sodium alginate.

[0018] Preferably, in step (2), the components of the ethanol solution containing calcium ions include calcium chloride, water and ethanol;

[0019] The mass ratio of the calcium chloride, water and ethanol is 0.001 - 0.2:0.01 - 0.2:1.

[0020] More preferably, the mass ratio of the calcium chloride, water and ethanol is 0.005 - 0.01:0.05 - 0.1:1.

[0021] Even more preferably, the mass ratio of the calcium chloride, water and ethanol is 0.008:0.08:1.

[0022] Preferably, in step (3), the voltage of the electric field action is 2 to 30 kV.

[0023] More preferably, in step (3), the voltage of the electric field action is 2 to 25 kV.

[0024] Even more preferably, in step (3), the voltage of the electric field action is 2 to 20 kV.

[0025] Preferably, in step (3), the temperature of the second freezing treatment is -160 to -196 °C, and the time is 5 to 30 min.

[0026] More preferably, in step (3), the temperature of the second freezing treatment is -195 to -196 °C, and the time is 5 to 30 min.

[0027] Even more preferably, in step (3), the temperature of the second freezing treatment is -196 °C, and the time is 5 to 30 min.

[0028] Preferably, in step (2), the temperature of the first freezing treatment is -20 to -100 °C.

[0029] More preferably, in step (2), the temperature of the first freezing treatment is -20 to -90 °C.

[0030] Even more preferably, in step (2), the temperature of the first freezing treatment is -20 to -80 °C.

[0031] Preferably, in step (4), the mass ratio of the ice beads to the fixed liquid after the freezing treatment is 0.01 to 0.8:1.

[0032] The second aspect of the present invention provides a porous microsphere.

[0033] Specifically, the porous microsphere is provided by the first aspect of the present invention;

[0034] The surface of the porous microsphere is a porous connected network structure;

[0035] The particle size of the porous microsphere is 60 to 1600 μm;

[0036] The pore size of the porous microsphere is 0.1 to 80 μm.

[0037] Preferably, the particle size of the porous microsphere is 65 to 1500 μm.

[0038] More preferably, the particle size of the porous microsphere is 70 to 1450 μm.

[0039] Preferably, the pore size of the porous microspheres is 0.5 - 78 μm.

[0040] More preferably, the pore size of the porous microspheres is 0.5 - 75 μm.

[0041] Preferably, the porosity of the porous microspheres is 70 - 95%.

[0042] More preferably, the porosity of the porous microspheres is 71 - 94%.

[0043] Even more preferably, the porosity of the porous microspheres is 72 - 93%.

[0044] The third aspect of the present invention provides an application of the porous microspheres in the preparation of a hemostatic agent.

[0045] Compared with the prior art, the beneficial effects of the present invention are as follows:

[0046] The present invention efficiently prepares calcium alginate microspheres with controllable particle size and a porous structure, while significantly reducing the use of organic solvents and avoiding adverse effects on the environment. The prepared porous microspheres have good biocompatibility, can be completely degraded after being implanted into the human body, and the degradation products are non-toxic and can be excreted from the body with human metabolism. The microspheres also have good liquid absorption performance and excellent hemostatic ability. The prepared microspheres have simple composition and wide sources, which is conducive to their clinical approval and translational application. BRIEF DESCRIPTION OF THE DRAWINGS

[0047] Figure 1 It is the SEM image of AlgMS prepared in Examples 1 - 5 of the present invention;

[0048] Figure 2 It is the comparison chart of the liquid absorption rate of AlgMS prepared in Examples 1 - 3 of the present invention, Celox and YB hemostatic powder;

[0049] Figure 3 It is the coagulation ability test chart of AlgMS prepared in Example 1 and Comparative Examples 1 - 6 of the present invention;

[0050] Figure 4 It is the in vitro blood and cell compatibility test chart of AlgMS prepared in Example 1 of the present invention;

[0051] Figure 5 It is the evaluation of the hemostatic effect of AlgMS prepared in Examples 1 - 3 of the present invention in the rat partial liver resection model. DETAILED DESCRIPTION OF THE EMBODIMENTS

[0052] In order to make the technical solutions described in the present invention clearer and more understandable to those skilled in the art, the following examples are listed for illustration. It should be noted that the following examples do not limit the scope of protection required by the present invention.

[0053] Unless otherwise specified, the raw materials, reagents or devices used in the following examples can be obtained from conventional commercial channels or can be obtained by existing known methods.

[0054] SEM test: The prepared porous microspheres (AlgMS) were adhered to the conductive adhesive, and the surface morphology of AlgMS was observed using a scanning electron microscope.

[0055] Example 1

[0056] A porous microsphere and a preparation method thereof.

[0057] AlgMS-1 was in the form of circular microspheres. After further magnification, it was observed that the surface of AlgMS-1 presented a porous interconnected network structure (as shown in a) in Figure 1 . The particle size of the microspheres was 463.2 ± 72.1 μm, the pore size was 27.4 ± 13.2 μm, and the porosity was 84%.

[0058] The steps of the preparation method are as follows:

[0059] Sodium alginate was dissolved in deionized water at a ratio of 0.015:1 and stirred overnight. A fixing solution was prepared by mixing calcium chloride, water, and absolute ethanol at a mass ratio of 0.008:0.08:1 and stored in a -80 °C refrigerator for the first freeze storage. A 26G needle was connected to a syringe filled with the sodium alginate solution and installed on an electrostatic dispersion system. An iron ring was grounded and installed 2 cm below the needle. With a voltage of 8 kV and an injection speed of 2000 μL / min, the sodium alginate solution was dispersed into -196 °C liquid nitrogen for the second freezing treatment to form ice beads by rapid quenching, and then poured into the low-temperature fixing solution. 200 mL of ice beads were poured into 400 mL of the fixing solution. The microspheres in the fixing solution were warmed to room temperature, then filtered through a sieve and washed five times with pure water, and finally freeze-dried for 48 h to obtain AlgMS-1.

[0060] Example 2

[0061] A porous microsphere and a preparation method thereof.

[0062] AlgMS-2 was in the form of circular microspheres. After further magnification, it was observed that the surface of AlgMS-2 presented a porous interconnected network structure (as shown in b) in Figure 1 . The particle size of the microspheres was 487.6 ± 65.7 μm, the pore size was 61.46 ± 11.6 μm, and the porosity was 81%.

[0063] The steps of the preparation method are as follows:

[0064] Sodium alginate was dissolved in deionized water at a ratio of 0.015:1 and stirred overnight. A fixing solution was prepared by mixing calcium chloride, water, and absolute ethanol at a mass ratio of 0.008:0.08:1 and stored in a refrigerator at -20 °C for the first freezing storage. A 26G needle was connected to a syringe filled with the sodium alginate solution and installed on an electrostatic dispersion system. An iron ring was grounded and installed 2 cm below the needle. With a voltage of 8 kV and an injection speed of 2000 μL / min, the sodium alginate solution was dispersed into liquid nitrogen at -196 °C for the second freezing treatment to form ice beads by rapid quenching. The ice beads were maintained at a temperature of -20 °C and then poured into the low-temperature fixing solution. 200 mL of ice beads were poured into 400 mL of the fixing solution. The microspheres in the fixing solution were warmed to room temperature, then filtered through a sieve and washed five times with pure water, and finally freeze-dried for 48 h to obtain AlgMS-2.

[0065] Example 3

[0066] A porous microsphere and its preparation method.

[0067] AlgMS-3 was in the form of round microspheres. After further magnification, it was observed that the surface of AlgMS-3 presented a porous connected network structure (as shown in Figure 1 c). The particle size of the microspheres was 714.8 ± 58.1 μm, the pore size was 8.7 ± 6.2 μm, and the porosity was 72%.

[0068] The steps of the preparation method are as follows:

[0069] The difference from Example 1 was that the mass ratio of sodium alginate to deionized water was 0.04:1.

[0070] Example 4

[0071] A porous microsphere and its preparation method.

[0072] AlgMS-4 was in the form of round microspheres. After further magnification, it was observed that the surface of AlgMS-4 presented a porous connected network structure (as shown in Figure 1 d). The particle size of the microspheres was 218.4 ± 46.9 μm, the pore size was 15.3 ± 3.7 μm, and the porosity was 93%.

[0073] The steps of the preparation method are as follows:

[0074] The difference from Example 1 was that the mass ratio of sodium alginate to deionized water was 0.01:1

[0075] Example 5

[0076] A porous microsphere and its preparation method.

[0077] AlgMS-5 is in the form of spherical microspheres. After further magnification, a porous interconnected network structure is observed on the surface of AlgMS-5 (as shown in Figure 1 e). The particle size of the microspheres is 82.2 ± 10.8 μm, the pore size is 2.1 ± 1.3 μm, and the porosity is 83%.

[0078] The steps of the preparation method are as follows:

[0079] It is different from Example 1 in that the voltage is maintained at 20 kV.

[0080] Example 6

[0081] A porous microsphere and a preparation method thereof.

[0082] AlgMS-6 is in the form of spherical microspheres. After further magnification, a porous interconnected network structure is observed on the surface of AlgMS-6 (as shown in Figure 1 f). The particle size of the microspheres is 1063.2 ± 338.5 μm, the pore size is 42.4 ± 25.5 μm, and the porosity is 84%.

[0083] The steps of the preparation method are as follows:

[0084] It is different from Example 1 in that the voltage is maintained at 2 kV.

[0085] Comparative Example 1

[0086] A commercial Celox hemostatic powder.

[0087] The Celox hemostatic powder was purchased from Medtrade Products Limited in the UK.

[0088] Comparative Example 2

[0089] A commercial Yunnan Baiyao (YB) hemostatic powder.

[0090] The YB hemostatic powder was purchased from Yunnan Baiyao Group Co., Ltd. in China.

[0091] Comparative Example 3

[0092] It is different from Example 1 in that by adjusting the second freezing temperature, AlgMS (labeled as AlgMS / X / Y) is prepared, where X and Y represent the first freezing temperature and the second freezing temperature respectively. The sample prepared at the second freezing temperature of -150 °C is denoted as AlgMS / -80 / -150.

[0093] Comparative Example 4

[0094] The difference from Example 1 is that by adjusting the second freezing temperature, AlgMS is prepared (denoted as AlgMS / X / Y), where X and Y represent the first freezing temperature and the second freezing temperature, respectively. The sample prepared at the second freezing temperature of -80°C is denoted as AlgMS / -80 / -80.

[0095] Comparative Example 5

[0096] The difference from Example 2 is that by adjusting the second freezing temperature, AlgMS is prepared (denoted as AlgMS / X / Y), where X and Y represent the first freezing temperature and the second freezing temperature, respectively. The sample prepared at the second freezing temperature of -80°C is denoted as AlgMS / -20 / -80.

[0097] Comparative Example 6

[0098] The difference from Example 2 is that by adjusting the second freezing temperature, AlgMS is prepared (denoted as AlgMS / X / Y), where X and Y represent the first freezing temperature and the second freezing temperature, respectively. The sample prepared at the second freezing temperature of -80°C is denoted as AlgMS / -20 / -20.

[0099] Performance test:

[0100] 1. Liquid absorption rate test: To evaluate the liquid absorption performance of the materials, AlgMS, Celox, and YB hemostatic powder were accurately weighed (denoted as W 0 ), and placed on a 120-mesh nylon screen. Subsequently, the screen was completely immersed in pure water, whole blood, or phosphate buffer solution (PBS), taken out after 30 seconds, and the excess liquid on the surface was removed with absorbent paper and weighed again (denoted as W t ). The liquid absorption capacity of the material was calculated according to the formula Liquid absorption(%) = W t / W 0 ×100% (g / g) to evaluate its liquid absorption capacity in different liquid environments.

[0101] 2. In vitro coagulation test: The in vitro coagulation test aims to explore the blood coagulation effect of the materials on the materials. First, the coagulation ability was restored by adding 0.1M calcium chloride solution (40 μL) to fresh rat whole blood treated with sodium citrate (360 μL). Then, AlgMS, Celox, and YB hemostatic powder materials (10 mg) were placed in plastic centrifuge tubes respectively, and 50 μL of the above rat whole blood was dropped in. After reaching the preset time point, 2 mL of deionized water was added and gently pipetted to disperse the uncoagulated blood. 100 μL of the mixed solution was taken into a 96-well plate, and the absorbance was measured at a wavelength of 540 nm using an enzyme-linked immunosorbent assay (ELISA) reader, and the blood coagulation index (BCI) was calculated to quantify the in vitro coagulation efficacy of the materials, where a lower BCI indicates a higher coagulation ability.

[0102] 3. Blood compatibility test: Samples with different concentrations (10 and 30 mg / mL) were added to the PBS suspension containing 5% RBC, and after incubation at 37 °C for 1 h, centrifugation was performed; 0.1% Triton X-100 and PBS were used as positive and negative controls respectively. The absorbance of the supernatant at 540 nm was measured by an enzyme-linked immunosorbent assay (ELISA) reader, the hemolysis rate was calculated, and the degree of damage to red blood cells and blood compatibility of the material were evaluated.

[0103] 4. Cytotoxicity test: The NIH-3T3 cell line was used for the cytotoxicity test. AlgMS microspheres (60 mg) were soaked in a specific culture medium for 24 h to prepare an extract, which was filtered through a filter membrane and then replaced the original culture medium in a 96-well plate. The control group was added with fresh culture medium. The cell viability was detected by the CCK8 method at preset time points (24, 48, 72 h) to evaluate the effect of the material on cell growth.

[0104] 5. Hemostasis of the rat liver lobectomy wound model: Rats aged 14 - 15 weeks were selected. After anesthesia with isoflurane, the liver was exposed through an abdominal incision, and filter paper was placed below to collect bleeding. A truncated incision was made in the liver lobe to induce bleeding. After 5 seconds of free bleeding, 100 mg of different hemostatic powders were immediately applied to the wound, and the blank group was not treated with anything. The blood loss was calculated by weighing the filter paper, and the bleeding time was recorded to evaluate the in vivo hemostatic effect of each hemostatic material.

[0105] As Figure 2 shown, the liquid absorption performance of the AlgMS prepared in Examples 1 - 3 and the commercial hemostatic powder products of Comparative Examples 1 - 2 was tested and compared. YB had only an absorption rate of about 2.5 times, and Celox had an absorption rate of 4.5 times. Due to the porous structure, the absorption rate of the AlgMS prepared in the present invention could reach 24 times its own weight, which was much higher than that of commercial products.

[0106] As Figure 3 shown in (a), taking AlgMS-1 prepared in Example 1 and AlgMS prepared by changing the freezing temperature in Comparative Examples 3 - 6 as examples, the effect of different freezing temperatures on the in vitro blood coagulation ability of AlgMS was evaluated by the blood coagulation index (BCI) after 60 s of co-incubation with whole blood. As the first and second freezing temperatures increased, the BCI value of AlgMS at the 60 s incubation time increased, indicating that AlgMS prepared at a lower freezing temperature had a stronger blood coagulation promoting ability. As Figure 3As shown in (b) of [Example 1], taking AlgMS prepared in Example 1 as an example, the in vitro blood coagulation ability of AlgMS was evaluated by the coagulation time after co-incubation with whole blood, and YB and Celox were used as controls. The blank group began to form blood clots at about 7 min. After co-incubation with YB and Celox, stable blood clots were formed within 5 min, indicating that they have certain procoagulant abilities. Since AlgMS contains calcium ions and a porous capillary structure, it can rapidly absorb blood, enrich blood cells, and accelerate blood coagulation, thus significantly shortening the coagulation time to 2 min. As Figure 3 shown in (b) of [Example 1], the procoagulant performance was further quantitatively evaluated by measuring BCI. The BCI value of the AlgMS group was significantly lower than that of YB and Celox within the first 300 s, indicating that AlgMS has excellent blood coagulation ability compared with commercial products. As Figure 3 shown in (c) of [Example 1], the sample of AlgMS co-incubated with blood for 2 min in Example 1 was characterized by SEM. The electron micrograph showed that a large number of red blood cells and platelets adhered to the surface of AlgMS, and an obvious fibrin network formation could be observed, indicating that AlgMS successfully activated the blood coagulation ability.

[0107] As Figure 4 shown in (a) of [Example 1], the in vitro blood compatibility of AlgMS was characterized. It can be seen that the hemolysis rate of AlgMS is lower than the use standard of 5%, indicating that AlgMS has excellent blood compatibility and can be used as a blood contact material. As Figure 4 shown in (b) of [Example 1], 3T3 mouse fibroblasts were co-cultured with the AlgMS leaching solution, and its in vitro cell compatibility was evaluated by the CCK8 experiment. The experimental results showed that the cell viability of the AlgMS group was comparable to that of the control group, both higher than 90%, indicating that AlgMS has very good cell safety.

[0108] As Figure 5 shown, the performance of the AlgMS microsphere hemostatic powder prepared in Examples 1 to 3 and the commercial product in Comparative Example 1 were compared and tested using a partial liver resection model of SD rats. The Celox group achieved hemostasis within about 120 s, and the average blood loss was about 570 mg. In contrast, the blood loss of the AlgMS-1 group was only about 390 mg, and hemostasis was successfully achieved within about 92 s, significantly superior to the commercial Celox hemostatic powder.

[0109] The preferred specific embodiments of the present invention have been described in detail above. It should be understood that those of ordinary skill in the art can make many modifications and variations according to the concept of the present invention without creative work. Therefore, any technical solutions obtained by those skilled in the art in the technical field of the present invention through logical analysis, reasoning or limited experiments based on the concept of the present invention on the basis of the prior art, such as any modifications, equivalent replacements, improvements, etc., shall fall within the protection scope determined by the claims.

Claims

1. A method for preparing porous microspheres, characterized in that: The following steps are involved: (1) weighing alginate and dissolving it in a solvent to obtain an alginate solution; (2) preparing an ethanol solution containing calcium ions as a fixing solution, and performing a first freezing treatment to obtain a fixing solution after freezing treatment; (3) under the action of an electric field, dispersing the alginate solution in step (1) into droplets, and performing a second freezing treatment to obtain ice beads; (4) adding the ice beads described in step (3) to the fixing solution after the freezing treatment described in step (2), stirring, and standing to obtain semi-finished microspheres; (5) Rinse and collect the semi-finished microspheres of step (4), and freeze-dry them to obtain the porous microspheres.

2. The preparation method according to claim 1, characterized in that: In step (1), the mass ratio of the alginate to the solvent is 0.001 to 0.1:

1.

3. The preparation method according to claim 1, characterized in that: In step (2), the components of the ethanol solution containing calcium ions include calcium chloride, water and ethanol; The mass ratio of the calcium chloride, water and ethanol is 0.001-0.2:0.01-0.2:

1.

4. The preparation method according to claim 1, characterized in that: In step (3), the voltage corresponding to the electric field action is 2 to 30 kV.

5. The preparation method according to claim 3, characterized in that: In step (3), the temperature of the second freezing treatment is -160 to -196°C, and the time is 5 to 30 minutes.

6. The preparation method according to claim 3, characterized in that: In step (2), the temperature of the first freezing treatment is -20 to -100°C.

7. The preparation method according to claim 3, characterized in that: In step (4), the mass ratio of the ice beads to the frozen fixative is 0.01 to 0.8:

1.

8. A porous microsphere, characterized in that: The porous microspheres are prepared by the preparation method according to any one of claims 1 to 7; The surface of the porous microspheres is a porous interconnected network structure; The particle size of the porous microspheres is 60 to 1600 μm; The pore size of the porous microspheres is 2 to 80 μm.

9. The porous microsphere according to claim 8, characterized in that: The porosity of the porous microspheres is 70-95%.

10. Use of the porous microspheres according to any one of claims 8 to 9 in the preparation of hemostatic agents.

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