Anti-inflammatory hemostatic composite gel microspheres and preparation method thereof

Abstract

The application belongs to the technical field of biomedical materials, and provides an anti-inflammatory hemostatic composite gel microsphere and a preparation method and application thereof, and comprises the following steps: mixing sodium alginate and baicalin to be dissolved in deionized water to obtain a mixed solution, spraying the mixed solution into a calcium chloride solution by using electrostatic spraying, and forming gel microspheres through ion cross-linking; rinsing the gel microspheres with a calcium chloride solution, and then immersing the gel microspheres in a chitosan acetic acid solution to obtain chitosan-coated gel microspheres; placing the gel microspheres in a copper sulfate solution, and performing low-temperature calcium-copper ion exchange to obtain baicalin / copper alginate composite gel microspheres; and filtering, washing and freeze-drying the composite gel microspheres. Through the electrostatic spraying technology combined with ion cross-linking, the application realizes accurate control of the size and morphology of the microspheres, has a significant bacteriostatic effect on escherichia coli and staphylococcus aureus, shortens the in-vitro blood clotting time by 30%, has low cytotoxicity, and has good biocompatibility.

Description

TECHNICAL FIELD

[0001] The present application belongs to the technical field of biomedical materials, and particularly relates to an anti-inflammatory hemostatic composite gel microsphere and a preparation method thereof. BACKGROUND

[0002] Sodium alginate is often used to prepare gel microspheres due to its good biocompatibility and cross-linking gelation characteristics. However, single sodium alginate microspheres have limited functions, and often need to introduce functional components to enhance their biological activity. Baicalin is a natural flavonoid compound with significant anti-inflammatory and antioxidant properties, but its poor water solubility and low stability limit its application. Copper ions have broad-spectrum antibacterial properties and can promote angiogenesis and wound healing.

[0003] There is no technical solution in the prior art to simultaneously integrate baicalin and copper ions into sodium alginate-based microspheres to prepare composite gel microspheres with multiple biological functions. SUMMARY

[0004] The purpose of the embodiment of the present application is to provide a preparation method of an anti-inflammatory hemostatic composite gel microsphere, aiming to solve the problems raised in the background art.

[0005] The embodiment of the present application is implemented as follows: a preparation method of an anti-inflammatory hemostatic composite gel microsphere, comprising the following steps:

[0006] Ion cross-linking: mixing sodium alginate and baicalin in deionized water to obtain a mixed solution, using electrostatic spinning electrospray to spray the mixed solution into a calcium chloride solution, and forming gel microspheres through ion cross-linking;

[0007] Drug encapsulation: rinsing the gel microspheres with a calcium chloride solution, and then immersing them in a chitosan acetic acid solution to obtain chitosan-encapsulated baicalin / calcium alginate gel microspheres;

[0008] Ion exchange: placing the chitosan-encapsulated baicalin / calcium alginate gel microspheres in a copper sulfate solution for low-temperature calcium-copper ion exchange to obtain baicalin / copper alginate composite gel microspheres;

[0009] Post-processing: filtering, washing and freeze-drying the baicalin / copper alginate composite gel microspheres to obtain anti-inflammatory hemostatic composite gel microspheres.

[0010] Another purpose of the embodiment of the present application is to provide an anti-inflammatory hemostatic composite gel microsphere prepared by the above preparation method.

[0011] Another purpose of the embodiment of the present application is to provide an application of the anti-inflammatory hemostatic composite gel microsphere in preparing a wound dressing, a hemostatic material or a drug release carrier.

[0012] The embodiment of the present application can realize accurate control of the size and morphology of microspheres by combining ion crosslinking with electrospraying technology, the microspheres have a porous structure and the diameter of the microspheres is adjustable in the range of 50-500 μm; the chitosan layer further improves the encapsulation rate and sustained-release performance of baicalin; the baicalin endows the gel microspheres with excellent antibacterial property, antioxidant property and hemostatic capacity; the introduction of copper ions synergistically enhances the antibacterial and wound healing-promoting capacity of the gel microspheres;

[0013] The composite gel microspheres prepared in the embodiment of the present application have remarkable bacteriostatic effect on E. coli and S. aureus, the in vitro blood clotting time is shortened by 30%, the cytotoxicity is low, the composite gel microspheres have good biocompatibility, and have wide application prospects in the fields of wound dressings, tissue repair and drug sustained release. BRIEF DESCRIPTION OF DRAWINGS

[0014] Figure 1 The micro-morphology structure diagram provided for the embodiment of the present application, (a is a picture of the composite gel microspheres before freeze-drying, and b is a picture of the freeze-dried composite gel microspheres when swelling and releasing drugs in PBS buffer);

[0015] Figure 2 The infrared spectrum diagram of the samples at each stage provided for the embodiment of the present application;

[0016] Figure 3 The particle size distribution diagram provided for the embodiment of the present application;

[0017] Figure 4 The antioxidant data diagram provided for the embodiment of the present application;

[0018] Figure 5 The procoagulant effect diagram provided for the embodiment of the present application;

[0019] Figure 6 The baicalin standard curve diagram provided for the embodiment of the present application;

[0020] Figure 7 The drug release diagram provided for the embodiment of the present application;

[0021] Figure 8 The bacteriostatic effect diagram provided for the embodiment of the present application (a is a bacteriostatic circle diameter diagram for S. aureus, and b is a minimum bacteriostatic concentration diagram for E. coli). DETAILED DESCRIPTION

[0022] In order to make the purpose, technical scheme and advantages of the present application clearer, the present application is further described in detail below with reference to the drawings and embodiments. It should be understood that the specific embodiments described herein are only used to explain the present application, and are not used to limit the present application.

[0023] The sodium alginate in the embodiment of the application is purchased from McKin reagent, the model number is S817372-500 g; the baicalin is purchased from Luo En reagent, the model number is R005094-1 g; the escherichia coli and staphylococcus aureus are purchased from Qingdao Haibo Biotechnology Co., Ltd., and other reagents, materials and equipment are commercially available sources unless otherwise specified; the experimental methods are conventional experimental methods in the art unless otherwise specified.

[0024] The specific implementation of the application is described in detail below in combination with specific embodiments.

[0025] Embodiment 1, an anti-inflammatory hemostatic composite gel microsphere, a preparation method thereof comprises the following steps:

[0026] (1) mixing and dissolving sodium alginate (SA) and baicalin (BA) in deionized water at a mass ratio of 10:1 to obtain a mixed solution;

[0027] (2) stirring the mixed solution at a speed of 750 rpm for 4 h to make it fully dissolved;

[0028] (3) sucking the sodium alginate / baicalin mixed solution into a syringe and connecting it to an electrospinning instrument, and making the solution sprayed from the needle by an external electric field force of 10 kV and a syringe pushing force of 10 mL / h, and dropping into a calcium chloride solution with a concentration of 0.1 mol / L to form gel microspheres;

[0029] (4) placing the calcium alginate gel microspheres loaded with baicalin in a 0.1 mol / L calcium chloride solution for 2 h (BA-SA);

[0030] (5) fishing out the gel microspheres, rinsing them with a 0.1 mol / L calcium chloride solution stored at 4 ℃, and then immersing them in a 1 wt% chitosan acetic acid solution, and performing drug encapsulation at 6 ℃ and a rotation speed of 100 rpm for 1 h;

[0031] (6) filtering the chitosan-encapsulated gel microspheres and placing them in a 0.5 mol / L copper sulfate solution for low-temperature "calcium-copper" ion exchange for 6 h, and then filtering and freeze-drying to obtain anti-inflammatory hemostatic composite gel microspheres (BA-SA-Cu).

[0032] Embodiment 2, compared with embodiment 1, the only difference is that the mass ratio of sodium alginate to baicalin is adjusted to 20:1, the concentration of chitosan acetic acid solution is 2 wt%, the encapsulation time is 30 min, the concentration of copper sulfate is 0.2 mol / L, and the rest of the steps are the same as embodiment 1.

[0033] Morphology and size analysis:

[0034] 1. The gel microspheres prepared in Example 2 were taken with a rubber bulb dropper and placed under an electron microscope to observe the spherical morphology, and the results are shown in Figure 1 , wherein a is a picture of the gel microspheres before freeze-drying, and b is a picture of the freeze-dried gel microspheres when they swell and release drugs in a PBS buffer;

[0035] 2. Infrared spectrum analysis was performed on baicalin (BA), sodium alginate (SA), the baicalin / alginate calcium microspheres (BA-SA) obtained in step (4) of Example 2 before ion exchange, and the baicalin / alginate copper microspheres (BA-SA-Cu) obtained in step (6) of Example 2 after ion exchange, in the wavelength range of 4000-400 nm with a resolution of 4 nm, and the results are shown in Figure 2 ;

[0036] 3. The baicalin / alginate calcium microspheres (BA-SA) obtained in step (4) of Example 2 before ion exchange and the baicalin / alginate copper microspheres (BA-SA-Cu) obtained in step (6) of Example 2 after ion exchange were dispersed in deionized water, and the particle size distribution of the samples was determined by a laser particle size analyzer, and the results are shown in Figure 3 . It can be seen that the replacement of copper ions hardly affects the particle size of the gel microspheres, and the particle size is still mostly between 125-225 μm;

[0037] Antioxidant performance analysis:

[0038] 39.429 mg of 1,1-diphenyl-2-trinitrobenzene hydrazine (DPPH) powder was dissolved in 1 L of anhydrous ethanol, and the absorbance was measured at 517 nm by a UV spectrophotometer to be Ab; different amounts of the gel microspheres prepared in Example 2 were weighed and soaked in anhydrous ethanol solution to obtain extraction solutions with concentrations of 100 μg / mL, 50 μg / mL, 25 μg / mL, 10 μg / mL and 5 μg / mL, respectively; the extraction solution and the DPPH solution were mixed in equal volumes, and after being mixed thoroughly in the dark for 1 h, the absorbance was measured at 517 nm by a UV spectrophotometer to be As; the blank control group was a mixture of anhydrous ethanol and DPPH solution in equal volumes; the antioxidant capacity can be calculated as follows:

[0039] ;

[0040] The results are shown in Figure 4 . Since copper ions can catalyze the generation of hydroxyl groups, when the sample concentration is low, its antioxidant effect is not as good as that of BA and BA-SA; but when the concentration reaches 50 μg / mL, the antioxidant effect of baicalin can completely cover the pro-oxidation effect of copper ions itself, and finally achieve the same antioxidant effect as BA and BA-SA.

[0041] Coagulation-promoting effect analysis:

[0042] The anticoagulant sodium citrate in the anticoagulated chicken blood was removed by using a 0.9 wt% calcium chloride solution; gel microspheres prepared in Example 2 were placed in a certain amount of PBS solution to obtain a sample solution with a concentration of 100-500 μg / mL; the sample solution and the chicken blood were mixed in a sample tube in equal volumes; the blank control group was a mixture of PBS solution and chicken blood in equal volumes; the sample tube was tilted every five minutes, and whether the liquid surface was tilted, i.e., whether the blood was coagulated, was observed until the blood in the sample tube stopped flowing, and the coagulation time was recorded;

[0043] The results are shown in Table 1. Figure 5 As shown in Table 1, this is because copper ions are cofactors for multiple coagulation factors, which help the formation of thrombin, and when baicalin and copper ions act synergistically, they can better and faster promote blood coagulation and achieve coagulation effect.

[0044] Drug release analysis:

[0045] Baicalin was dissolved in PBS solution, and full-wavelength scanning was performed at 278 nm by using a UV-visible spectrophotometer. Standard curves were drawn using baicalin solutions with concentrations of 10 μg / mL, 20 μg / mL, 30 μg / mL, 40 μg / mL, and 50 μg / mL, respectively, as shown in Table 2. Figure 6 As shown in Table 2, there is a good linear relationship between the absorbance of baicalin in the range of 10-50 μg / mL and its mass concentration, and the standard curve equation is y=0.051x-0.0099, R 2 =0.9998 (y represents the absorbance at 278 nm, and x represents the concentration); the gel microspheres prepared in Example 2 were ultrasonically treated in a certain amount of PBS solution for 30 minutes, then centrifuged at 1500 rpm for 10 minutes, and the supernatant was measured for absorbance at 278 nm by using a UV spectrophotometer. The actual total amount of drugs loaded in the microsphere particles was calculated by using the above standard curve equation, and was recorded as .

[0046] The gel microspheres prepared in Example 2 were placed in a certain amount of PBS solution, and in vitro drug release experiments were performed on a 37 ℃ shaking table. The drug release amount at different times was recorded, the absorbance of the solution was measured at 278 nm by using a UV spectrophotometer, and the drug release amount was calculated by using the standard curve equation, and was recorded as , n is the number of time nodes:

[0047] ;

[0048] wherein, is the total mass of the drug released from the carrier at the nth time node; Cn is the sample concentration measured at the nth time point; V is the total volume of the drug release system; S is the total amount of drug contained in all the samples taken from the first time node to the nth time point;

[0049] The cumulative drug release rate is calculated as follows:

[0050]

[0051] The test results are shown in Table 1, and the cumulative release rate of baicalin at 6 h is as high as 90% or more. Figure 7 Bacteriostatic effect analysis:

[0052] 1. The bacteriostatic circle diameter test method comprises the following steps:

[0053] Preparation of bacterial suspension: the staphylococcus aureus strain preserved on a slope is continuously subcultured;

[0054] Preparation of solid culture medium: 12.5 g of LB broth, 8.5 g of agar powder, 500 mL of distilled water;

[0055] Sample pretreatment: the composite gel microsphere particles (BA-SA-Cu) prepared in Example 2 are placed in a PBS solution to allow the drug to be completely released, obtaining a baicalin solution with a concentration of 50 mg / mL, sterile filter paper is cut into a disc with a diameter of 5 mm, and the sample solution and the sterile disc are periodically placed under ultraviolet light for ultraviolet sterilization;

[0056] The pipette gun is used to suck 120 μL of bacterial suspension with a concentration of 10 7 CFU / mL, which is uniformly coated on the surface of the solid culture medium with a coating rod, the surface dish is covered and placed at room temperature for 5 min. Two discs (both of which are added with 20 μL of sample solution) are placed on each bacterial plate, the centers of the discs are separated by more than 25 mm, and the edges of the plate are separated by more than 15 mm; placed in a 37℃ constant temperature incubator, cultured for 12 h, and the results were observed. The bacteriostatic circle diameter of the sample on the staphylococcus aureus strain was measured with a vernier caliper and recorded, and the experiment was repeated 3 times to ensure the stability of the antibacterial performance of the sample;

[0057] The test results are shown in Table 1, and the cumulative release rate of baicalin at 6 h is as high as 90% or more.

[0058] Figure 8 As shown in Table 1, it can be seen that the bacteriostatic circle diameter of the composite gel microspheres prepared in the embodiment of the present application on the staphylococcus aureus is 12 mm, indicating that the staphylococcus aureus has good bacteriostatic effect.

[0059] 2. The minimum bacteriostatic concentration test method comprises the following steps:

[0060] ​​Preparation of liquid medium: 3.125 g LB broth, 125 mL distilled water

[0061] Take 10 sterilized test tubes, and add 3 mL of baicalin solution with concentrations of 50.00 mg / mL, 25.00 mg / mL, 12.50 mg / mL, 6.25 mg / mL, 3.13 mg / mL, 1.56 mg / mL, 0.78 mg / mL, 0.39 mg / mL, 0.195 mg / mL and 0.0975 mg / mL respectively into the test tubes, and then add 0.1 mL of E. coli liquid (concentration of 10 7 CFU / mL) into each test tube, and then culture at 37 ℃ for one day; repeat the experiment for 3 times, and take the average value; when there is no turbidity, no precipitation, no surface growth and any other phenomenon under visual observation, the concentration of the baicalin solution at this time is the minimum inhibitory concentration (MIC);

[0062] The results are shown in Table b. Figure 8

[0063] In summary, the anti-inflammatory hemostatic composite gel microspheres prepared by the method of "electrospraying-ion crosslinking-packaging-ion exchange" have uniform particle size distribution, have significant antioxidant, antibacterial and procoagulant effects, and have wide application prospects in the fields of biological medicine and materials.

[0064] The above only describes the preferred embodiments of the present application and should not be used to limit the present application, and any modification, equivalent replacement and improvement made within the spirit and principle of the present application should be included in the protection scope of the present application.​

Claims

1. A method of preparing an anti-inflammatory hemostatic composite gel microspheres, characterized by, The method comprises the following steps: ion crosslinking: mixing sodium alginate and baicalin in deionized water to obtain a mixed solution, using electrostatic spinning electrospray to spray the mixed solution into a calcium chloride solution, and forming a gel microsphere by ion crosslinking; drug encapsulation: rinsing the gel microsphere with a calcium chloride solution, and then immersing it in a chitosan acetic acid solution to obtain a chitosan-encapsulated baicalin / calcium alginate gel microsphere; ion exchange: placing the chitosan-encapsulated baicalin / calcium alginate gel microsphere in a copper sulfate solution to perform low-temperature calcium-copper ion exchange, and obtaining a baicalin / copper alginate composite gel microsphere; post-treatment: filtering, washing and freeze-drying the baicalin / copper alginate composite gel microsphere to obtain an anti-inflammatory hemostatic composite gel microsphere.

2. The method of claim 1, wherein the anti-inflammatory hemostatic composite gel microspheres are prepared by the steps of: In the step of mixing sodium alginate and baicalin in deionized water to obtain a mixed solution, the mass ratio of sodium alginate to baicalin is 10-100:

1.

3. The method of claim 1, wherein the anti-inflammatory hemostatic composite gel microspheres are prepared by the steps of: In the step of using electrostatic spinning electrospray to spray the mixed solution into a calcium chloride solution, the electrospinning voltage is 5-20 kV, the propelling speed is 5-20 mL / h, the calcium chloride concentration is 0.1-2 mol / L, and the standing time is 1-10 h.

4. The method of claim 1, wherein the anti-inflammatory hemostatic composite gel microspheres are prepared by the steps of: In the step of rinsing the gel microsphere with a calcium chloride solution, and then immersing it in a chitosan acetic acid solution, the mass fraction of the chitosan acetic acid solution is 0.5-3 wt%, the pH is 5-6, the immersion temperature is 2-20 ℃, and the immersion time is 5-150 min.

5. The method of claim 1, wherein the anti-inflammatory hemostatic composite gel microspheres are prepared by the steps of: In the step of placing the chitosan-encapsulated baicalin / calcium alginate gel microsphere in a copper sulfate solution to perform low-temperature calcium-copper ion exchange, the copper sulfate solution concentration is 0.1-2 mol / L, the low-temperature calcium-copper ion exchange temperature is 2-20 ℃, and the exchange time is 0.5-5 h.

6. The method of claim 1, wherein the anti-inflammatory hemostatic composite gel microspheres are prepared by the steps of: In the step of filtering, washing and freeze-drying the baicalin / copper alginate composite gel microsphere, the freeze-drying temperature is -20~-40 ℃, and the freeze-drying time is 2-10 h.

7. An anti-inflammatory hemostatic composite gel microsphere, characterized in that, The anti-inflammatory hemostatic composite gel microsphere is prepared by the preparation method of any one of claims 1-6.

8. Use of the anti-inflammatory hemostatic composite gel microsphere according to claim 7 in the preparation of a wound dressing, a hemostatic material or a drug sustained-release carrier.

Images