CN113087962B

CN113087962B - Preparation method of anti-oxidation high-liquid-absorption-rate hemostatic sponge

Info

Publication number: CN113087962B
Application number: CN202110414960.2A
Authority: CN
Inventors: 徐福建; 胡杨; 缪腾飞
Original assignee: Beijing University of Chemical Technology
Current assignee: Beijing University of Chemical Technology
Priority date: 2021-04-17
Filing date: 2021-04-17
Publication date: 2022-04-29
Anticipated expiration: 2041-04-17
Also published as: CN113087962A

Abstract

The invention discloses a preparation method of an antioxidant high-liquid-absorption-ratio hemostatic sponge, which comprises the steps of preparing an acetic acid solution of chitosan, dissolving gelatin in hot water, stirring at a low speed for mixing and crosslinking, increasing the rotating speed for high-speed foaming, and stopping foaming until uniform and stable foaming is obtained; pouring the foaming solution into a mold, freezing and drying at low temperature to obtain foaming sponge, soaking the foaming sponge in an absolute ethyl alcohol solution to wash acetic acid, and performing vacuum drying; the mass ratio of the gelatin to the chitosan is 1: (1.5-10); preparing an alkaline solution of natural polyphenol, wherein the concentration is 1-10 mg/mL, adjusting the pH value to 8-10, soaking the foamed sponge after vacuum drying in the alkaline solution of natural polyphenol for crosslinking, taking out the foamed sponge, washing with deionized water, freezing the foamed sponge at a low temperature after washing, and performing secondary freeze drying. The high-efficiency hemostatic material with excellent hemostatic performance, good biocompatibility and good oxidation resistance is obtained.

Description

Preparation method of anti-oxidation high-liquid-absorption-rate hemostatic sponge

Technical Field

The invention belongs to the field of medical hemostatic materials, and relates to a preparation method of an antioxidant high-liquid-absorption-rate hemostatic sponge.

Background

Uncontrolled bleeding is a life threat in the life of people at present and is one of the challenges faced by emergency personnel in hospitals, so that the preparation of the material with safe and efficient hemostatic performance has strong practical significance. Gelatin has the advantages of low antigenicity, good biocompatibility, good biodegradability and relatively low cost, and is widely used as a hemostatic material, however, the mechanical strength of gelatin products is poor, and the strength of gelatin products needs to be enhanced by compounding with other materials with high mechanical strength or by crosslinking. The common gelatin sponges in the market, represented by Jinling sponges, are usually crosslinked by micromolecular aldehydes such as formaldehyde, glutaraldehyde and the like, the liquid absorption is slow, the hemostatic performance is common, and the aldehydes have certain biological toxicity, so that the development of a gelatin-based hemostatic material with high-efficiency liquid absorption, rapid hemostasis and higher biological safety is very urgent.

The natural polyphenol is a general name of plant secondary metabolites which are characterized by taking phenol as a basic skeleton and polyhydroxy substitution on a benzene ring, and has multiple functions of resisting oxidation, tumors and the like, wounds are always exposed to risks of infection, inflammation and the like in a healing stage, so the antioxidant property of the polyphenol has potential application in the aspect of weakening the inflammatory response of an organism. Lan and the like are crosslinked by adding tannic acid in the mixing and stirring process of chitosan and gelatin, so that the strength of the sponge is improved, and the hemostatic performance of the sponge is further improved [ Colloids and Surfaces B: Biointerfaces 136(2015) 1026-; furthermore, the effect of the speed of rotation of the stirring process on the properties of the gelatin-based sponge is not of concern.

Disclosure of Invention

In view of this, the invention provides a preparation method of an antioxidant high-liquid-absorption-rate hemostatic sponge. The invention specifically provides the following technical scheme:

a preparation method of an antioxidant high-liquid-absorption-rate hemostatic sponge comprises the following steps:

1) preparing acetic acid solution of chitosan, dissolving gelatin in hot water, adding the solution, stirring at low speed for mixing and crosslinking, increasing the rotation speed after a period of time, and foaming at high speed until uniform and stable foam is obtained, and stopping foaming; pouring the foaming solution into a mold, freezing and drying at low temperature to obtain foaming sponge, soaking the foaming sponge in an absolute ethyl alcohol solution to wash acetic acid, and then carrying out vacuum drying; the mass ratio of the gelatin to the chitosan is 1: (1.5-10);

2) preparing an alkaline solution of natural polyphenol, wherein the concentration is 1-10 mg/mL, adjusting the pH value to 8-10, soaking the foamed sponge subjected to vacuum drying in the step 1) in the alkaline solution of natural polyphenol for crosslinking, taking out the foamed sponge, washing with deionized water, freezing the foamed sponge at a low temperature after washing, and performing secondary freeze drying.

Further, the temperature of the low-speed stirring in the step 1) is 30-55 ℃.

Further, the concentration of the mixed solution of the chitosan and the gelatin in the step 1) is 10-100 mg/mL, the rotation speed of low-speed stirring is 100-600rpm, and the crosslinking time is 5-120 min.

Further, the deacetylation degree of the chitosan in the step 1) is 75-95%, and the number average molecular weight is 2-100 ten thousand.

Further, the power of the gelatin in the step 1) is 100-300ps, and the number average molecular weight is 5-100 ten thousand.

Further, the high-speed foaming time in the step 1) is 10min-120min, and the rotation speed of the high-speed foaming is 700-1800 rpm.

Further, the mass ratio of the gelatin to the chitosan in the step 1) is 1:9-3: 7.

Further, the natural polyphenol in the step 2) is one or more of tea polyphenol, procyanidine, tannic acid and ellagic acid.

Further, the concentration in the step 2) is 2-4 mg/mL.

Further, the crosslinking time in the step 2) is 10-200 min.

The invention has the beneficial effects that: the composite hemostatic sponge is prepared by mixing and reacting chitosan/gelatin crosslinked by natural polyphenol, the solution can be foamed under stirring by controlling the high-speed stirring speed and time and the ratio and concentration of the chitosan and the gelatin, and the sponge formed by freeze-drying after foaming is softer after imbibing liquid, is easy to attach to a wound surface and is beneficial to hemostasis. And natural polyphenol is added in the second step, on one hand, phenolic hydroxyl under the alkaline condition is selected to carry out Schiff base crosslinking reaction with amino in gelatin/chitosan, so that the prepared sponge forms a more stable crosslinked network structure, the liquid absorption rate of the gelatin sponge is greatly enhanced, and the mechanical strength of the sponge is greatly enhanced. On the other hand, a large amount of natural polyphenol introduced in the second step can also provide the anti-oxidation performance of the hemostatic sponge, so that the anti-oxidation and anti-inflammation of the wound are facilitated, and the wound healing is further promoted.

The natural polyphenol/gelatin/chitosan composite foaming hemostatic sponge is an organic combination of plant polyphenol substances, bio-based collagen degradation products and polysaccharide, the mixing and crosslinking of the plant polyphenol substances, the bio-based collagen degradation products and the polysaccharide can not only give full play to the high liquid absorption rate of a gelatin foaming system, but also improve the poor mechanical strength of a gelatin product (ACS appl. Mater. interfaces 9(2017) 29457-29468), and the hemostatic performance of the sponge can be further enhanced by adding electropositive chitosan; the natural polyphenol has antioxidant performance, so that the prepared natural polyphenol/gelatin/chitosan composite foaming hemostatic sponge prevents and promotes wound inflammation after hemostasis, and accelerates wound healing. The high-imbibition-rate antioxidant composite hemostatic sponge prepared by the method is a high-efficiency hemostatic material with excellent hemostatic performance, good biocompatibility and good antioxidant performance, and is expected to be applied to clinic.

Detailed Description

The following describes in detail preferred embodiments of the present invention.

Example 1

1) Weighing 1g of gelatin and dissolving in 50mL of deionized water; 1g of chitosan was weighed and dissolved in 50mL of an acetic acid/water mixed solution. Mixing gelatin and chitosan solution at a mass ratio of 1:9 under the condition of 40 ℃ water bath, stirring at a low speed of 200rpm for 10min for uniformly mixing and crosslinking, then increasing the rotation speed to 1100rpm for foaming, pouring into a culture dish after foaming for 35min, freezing at a low temperature, and then freeze-drying. Washing the frozen and dried sponge with ethanol to obtain acetic acid, washing for several times, and vacuum drying.

2) Dissolving tannic acid in Tris buffer (pH 9) at a concentration of 5mg/mL, soaking the obtained sponge in tannic acid for 15min, taking out the sponge, washing with deionized water for three times, freezing at low temperature, and lyophilizing twice.

Example 2

1) Weighing 1g of gelatin and dissolving in 50mL of deionized water; 1g of chitosan was weighed and dissolved in 50mL of an acetic acid/water mixed solution. Mixing gelatin and chitosan solution at a mass ratio of 3:7 under the condition of 40 ℃ water bath, stirring at a low speed of 200rpm for 15min for uniformly mixing and crosslinking, then increasing the rotation speed to 1300rpm for foaming, pouring into a culture dish after foaming for 25min, freezing at a low temperature, and then freeze-drying. Washing the frozen and dried sponge with ethanol to obtain acetic acid, washing for several times, and vacuum drying.

2) Dissolving procyanidin in Tris buffer (pH 8.5) with concentration of 3mg/mL, soaking the obtained sponge in the solution for 0min, taking out the sponge, washing with deionized water for three times, freezing at low temperature, and lyophilizing twice.

Comparative example 1

Weighing 1g of gelatin and dissolving in 50mL of deionized water; weighing 1g of chitosan, dissolving in 50mL of acetic acid/water mixed solution to prepare 20mg/mL solution, pouring the solution into a culture dish, mixing the solution in a mass ratio of 4:6 under the condition of 40-DEG water bath, stirring at 600rpm at a low speed for 30min, uniformly mixing and crosslinking, directly freezing at a low temperature, and then freeze-drying. Washing the frozen and dried sponge with ethanol to obtain acetic acid, washing for several times, and vacuum drying.

Comparative example 2

1) Weighing 1g of gelatin and dissolving in 50mL of deionized water; weighing 1g of chitosan, and dissolving in 50mL of acetic acid/water mixed solution to prepare 20mg/mL solutions. Mixing the solutions in a mass ratio of 3:7 under the condition of 40 ℃ water bath, stirring at a low speed of 600rpm for 60min for uniformly mixing and crosslinking, then increasing the rotation speed to 2000rpm for foaming, pouring into a culture dish after foaming for 65min, freezing at a low temperature, and then freeze-drying. Washing the frozen and dried sponge with ethanol to obtain acetic acid, washing for several times, and vacuum drying.

2) Dissolving procyanidin in Tris buffer (pH 8.5) at a concentration of 3mg/mL, collecting the obtained sponge solution for 15min, washing with deionized water for three times, freezing at low temperature, and lyophilizing twice.

Comparative example 3

1) Weighing 1g of gelatin and dissolving in 50mL of deionized water; weighing 1g of chitosan, and dissolving in 50mL of acetic acid/water mixed solution to prepare 20mg/mL solutions. Mixing gelatin and chitosan solution at a mass ratio of 6:4 under the condition of 40 ℃ water bath, stirring at a low speed of 600rpm for 60min for uniformly mixing and crosslinking, then increasing the rotation speed to 1300rpm for foaming, pouring into a culture dish after foaming for 65min, freezing at a low temperature, and then freeze-drying. Washing the frozen and dried sponge with ethanol to obtain acetic acid, washing for several times, and vacuum drying.

2) Dissolving tannic acid in deionized water at a concentration of 3mg/mL, soaking the obtained sponge in the solution for 30min, taking out the sponge, washing with deionized water for three times, freezing at low temperature, and lyophilizing for two times.

Comparative example 4

1) Weighing 1g of gelatin and dissolving in 50mL of deionized water; weighing 1g of chitosan, and dissolving in 50mL of acetic acid/water mixed solution to prepare 20mg/mL solutions. Mixing gelatin and chitosan solution at a mass ratio of 5:5 under the condition of 40 ℃ water bath, stirring at a low speed of 600rpm for 45min for uniformly mixing and crosslinking, then increasing the rotation speed to 1300rpm for foaming, pouring into a culture dish after foaming for 35min, freezing at a low temperature, and then freeze-drying. Washing the frozen and dried sponge with ethanol to obtain acetic acid, washing for several times, and vacuum drying.

2) Dissolving tannic acid in deionized water with the concentration of 0.1mg/mL, soaking the obtained sponge in the solution for 30min, taking out the sponge, washing with the deionized water for three times, freezing at low temperature, and freeze-drying for two times.

Comparative example 5

Weighing 1g of gelatin and dissolving in 50mL of deionized water; weighing 1g of chitosan, and dissolving in 50mL of acetic acid/water mixed solution to prepare 20mg/mL solutions. Mixing the solutions in a mass ratio of 5:5 under the condition of 40 ℃ water bath, simultaneously adding 5mL of prepared 10mg/mL procyanidine solution, then stirring at a low speed of 600rpm for 60min for uniform mixing and crosslinking, then increasing the rotation speed to 1300rpm for foaming, pouring the foamed mixture into a culture dish after 65min for foaming, freezing at a low temperature, and then freeze-drying. Washing the frozen and dried sponge with ethanol to obtain acetic acid, washing for several times, and vacuum drying.

Comparative example 6

Absorbable gelatin sponge of Jinling sold in market

Test example 1

The antioxidant high-absorbency hemostatic sponges prepared in examples 1-2 and the gelatin hemostatic sponges of comparative examples 1-6 were subjected to a 30-second internal-imbibition performance comparison experiment. The detection method comprises the following steps: firstly, cutting a sponge with the size of 2cm x 2cm and a culture dish, weighing the sponge, recording the weight as m0, adding deionized water with the weight about 40 times that of the test material, starting timing at the moment, clamping one corner or one end of a sample by using forceps after 30 seconds, hanging for 30 seconds, and weighing, wherein the weight is recorded as m 1. The absorbency of the sponge within 30s (spinning Ratio, SR) can be calculated according to the following equation

Wherein SR is the liquid absorption rate of the sponge, m0 is the dry weight of the sponge, and m1 is the equilibrium weight of the sponge after liquid absorption. Three replicates were set up for each set of experiments and the mean and variance were calculated.

The hemostasis times for the examples and comparative examples are shown in table 1.

TABLE 1 results of liquid suction test of each of examples and comparative examples

Grouping	Multiple of liquid absorption
		Experimental example 1	37±3
Experimental example 2	42±2
		Comparative example 1	32±3
Comparative example 2	18±6
		Comparative example 3	24±3
Comparative example 4	25±3
		Comparative example 5	24±4
Comparative example 6	3±1

Test example 2

The prepared anti-oxidation high-imbibition-ratio hemostatic sponge and the gelatin hemostatic sponges of comparative examples 1-6 are subjected to a hemostatic effect comparison experiment.

The detection method comprises the following steps: cutting sponge into 2mg pieces, placing into 2mL centrifuge tube, adding 100 μ L of the above rat anticoagulated whole blood dropwise onto the sample surface, and immediately adding 10 μ L of prepared 0.2M CaCl₂The solution was added to the blood, at which time a timer was started (the experiment was kept at a constant temperature of 37 ℃), and 2min later the uncoagulated blood cells were immediately disrupted with 10mL of deionized water, at which time all solutions were incubated for a further 3min at 37 ℃. After 3min, 100. mu.L of the supernatant was pipetted into a 96-well plate and the absorbance at 545nm was read with a microplate reader. The negative control group for this experiment was an empty centrifuge tube (surface component was polypropylene) and the positive control group was 100 μ L of whole blood in the centrifuge tube that was directly disrupted with 10mL of deionized water (we default to complete rupture of blood cells in this case). The Blood Coagulation Index (BCI) can be calculated by the formulas 2-4

Wherein BCI is the material blood coagulation index, OD_samThe absorbance value, OD, of the sample at a wavelength of 545nm_negAbsorbance value at 545nm of negative control group, OD_posThe absorbance value of the positive control group at the wavelength of 545nm is shown.

The lower the BCI value, the better the coagulation effect of the material, and the worse the coagulation performance.

TABLE 2 hemostatic test results for each of the examples and comparative examples

Grouping	BCI(％)
		Experimental example 1	45±2
Experimental example 2	30±3
		Comparative example 1	60±3
Comparative example 2	56±1
		Comparative example 3	54±2
Comparative example 4	55±4
		Comparative example 5	50±1
Comparative example 6	63±2

Test example 3

The anti-oxidation high-imbibition-ratio hemostatic sponge prepared by the invention and the gelatin hemostatic sponges of comparative examples 1 and 4 are subjected to an anti-oxidation performance comparison experiment.

The detection method comprises the following steps: this experiment needs to be performed completely protected from light: first, a PBS (pH 7.4, 25mM) solution containing 20. mu.M of vitamin B2, 12.5mM of DL-methionine and 75. mu.M of nitrosotetrazole blue was prepared, mixed with a sponge powder (sponge dispersion concentration: 2mg/mL), and irradiated with ultraviolet light for 15 min. The absorbance (560nm) of the mixture after irradiation is a positive control (A)_p) Negative control before irradiation (A)_n) The absorbance of the sponge powder-containing mixture after irradiation was (A)₀) The scavenging efficiency of superoxide radicals was calculated.

O₂·scavenging rate(％)＝1-(A₀-An/Ap-An)×100％％ (3-3)

The higher the scavenging rate of the material to superoxide radicals, the better the oxidation resistance of the material is represented.

TABLE 3 hemostatic test results for each of the examples and comparative examples

Grouping	Superoxide radical clearance (%)
		Experimental example 1	46±3
Experimental example 2	50±4
		Comparative example 1	10±2
Comparative example 4	21±2

As can be seen from tables 1 to 3, examples 1 and 2 of the present invention have far higher imbibition factors than comparative examples 1 to 6, lower blood coagulation indexes than comparative examples 1 to 6, and higher superoxide radical clearance than comparative examples 1 and 4 for the following reasons:

in the embodiment 1 and the embodiment 2 of the invention, the performance of the composite sponge in the first step is regulated and controlled by controlling the composite proportion of chitosan and gelatin and the foaming rotating speed, and the performance of the composite sponge in the second step is regulated and controlled by controlling the pH of polyphenol crosslinking and the amount of crosslinked polyphenol so that a proper amount of polyphenol is crosslinked and loaded on the sponge, thereby improving the liquid absorption rate and the hemostatic performance and having better antioxidant performance.

Comparative example 1 no polyphenol was added, the mechanical strength of the sponge was low due to no crosslinking of polyphenol, and thus there was a problem that the sponge collapsed after imbibing, the imbibing rate was decreased, and the hemostatic property of the sponge was poor and the oxidation resistance was poor due to no loading of polyphenol.

Comparative example 2 the foaming rotational speed was too high, excessive foaming occurred, and the mechanical strength of the sponge itself was lowered, so that there was a problem that the sponge collapsed after liquid absorption, resulting in a decrease in liquid absorption rate.

Comparative example 3 gelatin content is high for the cross-linking is not enough, and the foaming is more, and the cell grow becomes more, makes the mechanicalness of sponge worsen, because do not control alkaline reaction condition and carry out secondary crosslinking, makes only a small part polyphenol load on the sponge with the hydrogen bond form, can not effectively improve its mechanical strength, therefore can produce the problem that the sponge sinks after the imbibition, leads to the imbibition multiplying power to descend, because the load of few polyphenol leads to the hemostatic performance of sponge poor.

Comparative example 4 is that the soaking concentration of polyphenol is less than 1mg/mL, the gelatin content is high, the polyphenol loading is low, the imbibition multiple is low, the hemostatic performance is poor, and the oxidation resistance is poor.

The comparative example 5 is one-step mixing, the chitosan solution is acidic, and the phenolic hydroxyl group on the polyphenol, the gelatin and the chitosan can only carry out physical crosslinking of hydrogen bonds under the acidic condition, so that the liquid absorption multiple is low, and the hemostatic performance is poor.

Finally, it is noted that the above-mentioned preferred embodiments illustrate rather than limit the invention, and that, although the invention has been described in detail with reference to the above-mentioned preferred embodiments, it will be understood by those skilled in the art that various changes in form and detail may be made therein without departing from the scope of the invention as defined by the appended claims.

Claims

1. A preparation method of an antioxidant high-liquid-absorption-rate hemostatic sponge is characterized by comprising the following steps:

1) preparing acetic acid solution of chitosan, dissolving gelatin in hot water, adding the solution, stirring at low speed for mixing and crosslinking, increasing the rotation speed after a period of time, and foaming at high speed until uniform and stable foam is obtained, and stopping foaming; pouring the foaming solution into a mold, freezing and drying at low temperature to obtain foaming sponge, soaking the foaming sponge in an absolute ethyl alcohol solution to wash acetic acid, and then carrying out vacuum drying; the mass ratio of the gelatin to the chitosan is 1: (1.5-10); the concentration of the mixed solution of the chitosan and the gelatin is 10-100 mg/mL, the rotation speed of the low-speed stirring is 100-600rpm, and the crosslinking time is 5-120 min; the high-speed foaming time is 10min-120min, and the rotating speed of the high-speed foaming is 700 plus 1800 rpm;

2) preparing an alkaline solution of natural polyphenol, wherein the concentration is 1-10 mg/mL, the pH is adjusted to 8-10, the foamed sponge dried in the vacuum in the step 1) is soaked in the alkaline solution of the natural polyphenol for crosslinking, then the foamed sponge is taken out and washed by deionized water, and the foamed sponge is frozen at a low temperature after washing and is subjected to secondary freeze drying; the natural polyphenol is one or more of tea polyphenol, procyanidine, tannic acid and ellagic acid.

2. The method for preparing an antioxidant high-absorbency hemostatic sponge as claimed in claim 1, wherein the temperature of the low speed stirring in step 1) is 30-55 ℃.

3. The method for preparing an antioxidant high-absorbency hemostatic sponge as claimed in claim 1, wherein the chitosan in step 1) has a deacetylation degree of 75-95% and a number average molecular weight of 2-100 ten thousand.

4. The preparation method of the anti-oxidation high-liquid-absorption-rate hemostatic sponge as claimed in claim 1, wherein the mass ratio of the gelatin to the chitosan in the step 1) is 1 (2-5).

5. The method for preparing an antioxidant high-absorbency hemostatic sponge according to claim 1, wherein the concentration in step 2) is 2-4 mg/mL.

6. The method for preparing an antioxidant high-absorbency hemostatic sponge as claimed in claim 1, wherein the time for cross-linking in step 2) is 10-200 min.