Preparation method of antioxidant procoagulant bifunctional sponge
By preparing an antioxidant and procoagulant sponge with a Janus structure, the problems of limited hemostatic performance and lack of antioxidant function in existing hemostatic products have been solved, achieving the dual effects of efficient hemostasis and antioxidation.
Patent Information
- Authority / Receiving Office
- CN · China
- Patent Type
- Patents(China)
- Current Assignee / Owner
- BEIJING UNIV OF CHEM TECH
- Filing Date
- 2023-12-22
- Publication Date
- 2026-05-29
AI Technical Summary
Existing hemostatic products based on polysaccharides and biologically derived proteins have simple structures and limited hemostatic properties, resulting in their clinical indications being limited to minor bleeding. Furthermore, commercially available gelatin sponges lack antioxidant properties.
A coagulant nanoparticle dispersion was prepared by dissolving zein in an ethanol solution of a specific concentration and then crosslinking it with gelatin sponge to form an antioxidant and coagulant bifunctional sponge. The gelatin sponge with a Janus structure incorporates zein coagulant nanoparticles at its bottom.
The prepared sponge significantly improves hemostasis efficiency and antioxidant properties, avoids the risk of intravascular thrombosis, promotes wound anti-inflammation, and has a porosity similar to that of ordinary gelatin sponge, thus synergistically improving the hemostasis effect.
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Figure CN117860955B_ABST
Abstract
Description
Technical Field
[0001] This invention belongs to the field of medical materials and relates to a method for preparing an antioxidant and procoagulant dual-function sponge. Background Technology
[0002] Natural polymers, such as bio-derived polysaccharides and proteins, have been widely used in medical materials due to their high biocompatibility, unique bioactivity, and abundant material sources. Despite their good clinical safety profile, commercially available hemostatic products based on polysaccharides (such as Arista powder, Surgicel hemostatic devices, and gelatin sponges) have limited clinical indications for minor bleeding due to their simple structure and limited hemostatic properties. Bio-derived proteins (or peptides) have more complex structures than polysaccharides, further enriched by protein (self)assembly processes, leading to various types of protein-cell and / or protein-protein interactions. Upon interaction with blood, they have the potential to exert medical functions such as procoagulant or anticoagulant effects. Recently, zein has been considered a promising natural polymer due to its amphiphilicity, unique solubility, and abundant corn source, offering broad potential for development and application in protein-based materials. Summary of the Invention
[0003] In view of this, the present invention provides a method for preparing an antioxidant and procoagulant dual-function sponge. Specifically, the present invention provides the following technical solution:
[0004] A method for preparing an antioxidant and procoagulant bifunctional sponge, comprising the following steps:
[0005] 1) Prepare an ethanol solution, add zein to dissolve it in the ethanol solution, add an equal volume of deionized water to reverse precipitation and ultrasonically disperse, remove half the volume of solution from the dispersion by rotary evaporation, add glutaraldehyde aqueous solution to crosslink, and obtain a crosslinked procoagulant nanoparticle dispersion.
[0006] 2) Dissolve gelatin in hot water, stir at low speed until dissolved, add glutaraldehyde, increase the speed to carry out chemical cross-linking, and foam at high speed until uniform and stable foam is obtained, then stop foaming to obtain foamed gelatin solution.
[0007] 3) Pour the foamed gelatin solution obtained in step 2) into the cross-linked procoagulant nanoparticle dispersion obtained in step 1), freeze-dry, and obtain an antioxidant / procoagulant bifunctional sponge.
[0008] The volume fraction of the ethanol solution in step 1) is 60-70%;
[0009] The concentration of the zein solution mentioned in step 1) is 10–50 mg / mL;
[0010] The volume ratio of the cross-linked procoagulant rice granule dispersion and the gelatin solution in step 3) is 1:10 to 1:90.
[0011] Furthermore, the concentration of the ethanol solution in step 1) is 64-66%.
[0012] Furthermore, the volume fraction of the glutaraldehyde aqueous solution in step 1) is 0.25%.
[0013] Furthermore, the crosslinking temperature described in step 1) is 30–40°C.
[0014] Furthermore, in step 3), the volume ratio of the cross-linked procoagulant rice granule dispersion to the gelatin solution is 1:15-1:25.
[0015] Furthermore, in step 2), the stirring speed of the low-speed stirring is 50-400 rpm and the temperature is 30-50℃.
[0016] Furthermore, the chemical cross-linking time in step 2) is 10-60 min.
[0017] Furthermore, the gelatin mentioned in step 2) is type A, derived from pigskin, and has a gel strength of 250-300g Bloom.
[0018] Furthermore, the high-speed foaming time in step 2) is 10-60 minutes, and the high-speed foaming speed is 800-1200 rpm.
[0019] The beneficial effects of this invention are as follows: This invention uses zein dissolved in a specific concentration of ethanol solution to prepare a coagulant nanoparticle dispersion via a one-step reverse precipitation method. Then, foamed gelatin sponge is added, and the volume ratio of the coagulant nanoparticle dispersion to the gelatin sponge is controlled to prepare an antioxidant and coagulant-promoting dual-function sponge. This sponge can effectively fix the coagulant nanoparticles, avoiding the risk of intravascular thrombosis. The bottom of the foamed gelatin sponge incorporates zein coagulant nanoparticles, exhibiting a distinct Janus structure. The Janus structure of the sponge with embedded coagulant nanoparticles has almost the same porosity as the gelatin sponge, which can synergistically improve hemostatic efficiency. Natural zein contains hydrophobic amino acids with certain antioxidant properties, which can promote wound anti-inflammatory effects. Attached Figure Description
[0020] To make the objectives, technical solutions, and beneficial effects of this invention clearer, the following figures are provided.
[0021] Figure 1 The images show scanning electron microscope (SEM) images of the surface, cross-section, and bottom of the sponge samples prepared in Example 1, Comparative Example 2, and Comparative Example 4. Detailed Implementation
[0022] The preferred embodiments of the present invention will now be described in detail with reference to the accompanying drawings.
[0023] Example 1
[0024] 1) Prepare 20 mL of 65% ethanol-water solution. Weigh 500 mg of zein and dissolve it in the prepared ethanol solution. Shake to dissolve completely. Add 20 mL of water to the dissolved zein solution. Sonicate the nanoparticle dispersion formed by the back precipitation for 5 min. Remove half of the solvent in the system by rotary evaporation (40) (remove 20 mL of solution by rotary evaporation) to obtain the nanoparticle dispersion. Then add 0.25% (volume ratio) of glutaraldehyde for crosslinking for 30 min. Weigh 3.5 mL of the crosslinked nanoparticle dispersion at the bottom of the petri dish.
[0025] 2) Weigh 10g of gelatin and add it to 200mL of deionized water at 37°C. Stir at 300rpm for 5 minutes to completely dissolve the gelatin. Then add 0.25% (V) 戊二醛 :V 去离子水 After adding glutaraldehyde (50% concentration solution, ~5.6mol / L) to the solution, the mixture was allowed to self-foam at 1000rpm for 30min at 37°C.
[0026] 3) Immediately pour the foamed gelatin prepared in step 2) into a culture dish covering the cross-linked nanoparticle dispersion in step 1), with 3.5 mL of cross-linked nanoparticle dispersion and 66.5 mL of cross-linked foamed gelatin solution. After freeze-drying, an antioxidant / procoagulant bifunctional sponge is obtained, named J-ZGS.
[0027] Comparative Example 1
[0028] 1) Prepare 20 mL of 75% ethanol-water solution. Weigh 500 mg of zein and dissolve it in the prepared ethanol solution. Shake to dissolve completely. Then add 20 mL of water to the dissolved zein solution. Sonicate the nanoparticle dispersion formed by the back precipitation for 5 min. Remove half of the solvent in the system by rotary evaporation (40) (remove 20 mL of solution by rotary evaporation) to obtain the nanoparticle dispersion. Then add 0.25% (volume ratio) of glutaraldehyde for crosslinking for 30 min. Weigh 3.5 mL of the crosslinked nanoparticle dispersion at the bottom of the petri dish.
[0029] 2) Weigh 10g of gelatin and add it to 200mL of deionized water at 37°C. Stir at 300rpm for 5min to completely dissolve the gelatin. Then add 0.25% (V glutaraldehyde:V deionized water) of glutaraldehyde (50% concentration solution, ~5.6mol / L) and allow it to self-foam at 1000rpm for 30min at 37°C.
[0030] 3) Immediately pour the foamed gelatin prepared in step 2) into a petri dish covering the cross-linked nanoparticle dispersion from step 1), using 3.5 mL of the cross-linked nanoparticle dispersion and 66.5 mL of the cross-linked foamed gelatin solution. After freeze-drying, the resulting sponge is named JP. 75 .
[0031] Comparative Example 2
[0032] Weigh 10g of gelatin and add it to 200mL of deionized water at 37°C. Stir at 300rpm for 5 minutes until the gelatin is completely dissolved. Then add 0.25% (V) 戊二醛 :V 去离子水 The gelatin was prepared by adding glutaraldehyde (50% solution, ~5.6 mol / L) to a culture dish and then allowing it to foam at 1000 rpm for 30 min at 37°C. The foamed gelatin was immediately poured onto the culture dish and freeze-dried to obtain a gelatin sponge, named S-GS.
[0033] This preparation system does not contain zein nanoparticles.
[0034] Comparative Example 3
[0035] 1) Prepare 20 mL of 85% ethanol-water solution. Weigh 500 mg of zein and dissolve it in the prepared ethanol solution. Shake to dissolve completely. Then add 20 mL of water to the dissolved zein solution. Sonicate the nanoparticle dispersion formed by the back precipitation for 5 min. Remove half of the solvent in the system by rotary evaporation (40) (remove 20 mL of solution by rotary evaporation) to obtain the nanoparticle dispersion. Then add 0.25% (volume ratio) of glutaraldehyde for crosslinking for 30 min. Weigh 3.5 mL of the crosslinked nanoparticle dispersion at the bottom of the petri dish.
[0036] 2) Weigh 10g of gelatin and add it to 200mL of deionized water at 37°C. Stir at 300rpm for 5 minutes to completely dissolve the gelatin. Then add 0.25% (V) 戊二醛 :V 去离子水 After adding glutaraldehyde (50% concentration solution, ~5.6mol / L) to the solution, the mixture was allowed to self-foam at 1000rpm for 30min at 37°C.
[0037] 3) Immediately pour the foamed gelatin prepared in step 2) into a petri dish covering the cross-linked nanoparticle dispersion from step 1), using 3.5 mL of the cross-linked nanoparticle dispersion and 66.5 mL of the cross-linked foamed gelatin solution. After freeze-drying, a sponge is obtained, named JP. 85 .
[0038] Comparative Example 4
[0039] The commercially available Jinling absorbable gelatin sponge is named C-GS.
[0040] Test Example 1: Electron Microscopy Test
[0041] The sponge samples prepared in Example 1, Comparative Example 2, and Comparative Example 4 were structurally characterized. Figure 1 As shown.
[0042] Detection method: The sponge samples prepared in Example 1, Comparative Example 2 and Comparative Example 4 were rapidly quenched / frozen in liquid nitrogen, and the samples were cut off and then attached to the SEM sample stage to observe the surface, cross-section and bottom morphology of the samples. Figure 1 The images show scanning electron microscope (SEM) images of the surface, cross-section, and bottom of the sponge samples prepared in Example 1, Comparative Example 2, and Comparative Example 4.
[0043] As can be seen from the SEM surface structure images, the morphology of Comparative Example 4 sponge C-GS is a uniform lamellar structure, while Comparative Example 2 sponge S-GS, due to the freeze-drying of the foamed gelatin solution, exhibits a uniform three-dimensional network structure with high porosity. The surface of Example 1 sponge J-ZGS, after freeze-drying of the foamed gelatin solution, exhibits a uniform three-dimensional network structure with high porosity.
[0044] As can be seen from the SEM cross-sectional structure image, the sponge J-ZGS in Example 1 and the sponge S-GS in Comparative Example 2 exhibit a distinct Janus internal morphology due to the incorporation of nanoparticle dispersion at the bottom of the foamed gelatin solution.
[0045] As can be seen from the SEM bottom structure image, the sponge J-ZGS of Example 1 has a denser structure than the sponge S-GS of Comparative Example 2 due to the double cross-linking of gelatin chains / zein nanoparticles.
[0046] Test Example 2: Porosity Test
[0047] The porosity of the sponge samples prepared in Example 1, Comparative Example 2, and Comparative Example 4 was tested.
[0048] Detection method: The volumes of the sponge samples prepared in Example 1, Comparative Example 2, and Comparative Example 4 were measured and weighed. They were then immersed in a certain amount of ethanol for 30 minutes. The immersed sponges were removed and weighed again. The porosity (P) calculation formula is as follows:
[0049]
[0050] Where W2 and W1 represent the weight of the sponge before and after immersion in alcohol, respectively. V0 is the volume of the sponge sample, and ρ is the density of ethanol (0.785 g / cm³). 3 ).
[0051] Table 1. Porosity results of each embodiment and comparative sample.
[0052]
[0053]
[0054] The porosity of C-GS, S-GS, and J-ZGS was determined by the ethanol replacement method, and it was found that the three had similar porosities, which is consistent with... Figure 1 Consistent with SEM observations, the three-dimensional gelatin network exhibits uniform cross-linking. The Janus-structured sponge with embedded coagulant nanoparticles possesses almost the same porosity as the gelatin sponge (Comparative Example 2 without particles), which can synergistically improve hemostatic efficiency.
[0055] Test Example 3 Antioxidant Test
[0056] Natural zein contains hydrophobic amino acids. When its nanoparticle dispersion is added to gelatin sponges, it imparts certain antioxidant properties, promoting wound healing. Antioxidant performance was compared between sponges from Example 1 and Comparative Examples 1-4. Detection method: A 0.1 mM methanol solution of 1,1-diphenyl-2-trinitrophenylhydrazine (DPPH) was prepared; 3 mg of sponge was weighed into a 2 mL centrifuge tube, then 1.5 mL of DPPH solution was added. After incubation for 30 min, 100 μL was taken, and the absorbance was read at 517 nm to calculate the DPPH free radical scavenging rate.
[0057] DPPH clearance rate (%) = 1 - (A0 / A s )×100% (1)
[0058] The blank DPPH methanol solution is denoted as A0, and the sample is A. s .
[0059] Table 2. DPPH radical scavenging experimental results for each embodiment and comparative sample.
[0060] Grouping DPPH removal rate (%) Example 1 21.3±1.1 Comparative Example 1 18.2±1.0 Comparative Example 2 15.5±1.2 Comparative Example 3 16.4±2.8 Comparative Example 4 9.5±3.1
[0061] As can be seen from Table 2, Example 1 has the strongest ability to scavenge DPPH free radicals, and its free radical scavenging rate is significantly higher than that of the commercially available C-GS sponge in Comparative Example 4.
[0062] Comparative Example 1 showed a lower DPPH removal rate because it formed nanoparticle dispersions with different surface properties in a 75% ethanol solution, due to the different nanoparticle structures.
[0063] Comparative Example 2 had a lower DPPH removal rate because it did not contain a nanoparticle dispersion.
[0064] Comparative Example 3 shows that the formation of nanoparticle dispersions with different surface properties in 85% ethanol solution is due to the different nanoparticle structures, resulting in a lower DPPH removal rate.
[0065] Comparative Example 4 shows that commercially available C-GS sponges have a lower DPPH removal rate because they do not contain nanoparticle dispersion.
[0066] Therefore, it can be seen that the volume ratio of ethanol to water can regulate the structure of zein nanoparticles, thereby leading to differences in their free radical scavenging performance. The nanoparticle structure produced by regulating the ratio of ethanol to water at 65% has the best ability to scavenge DPPH free radicals.
[0067] Test Example 4: Hemostatic Performance Test
[0068] A comparative experiment was conducted to compare the hemostatic effects of the sponges in Example 1 and Comparative Examples 1-4.
[0069] Detection method: Accurately weigh 5 mg of sample into a 1.5 mL centrifuge tube, add 50 μL of anticoagulated whole blood, then add 5 μL of prepared 0.2 M CaCl2, incubate at 37°C for 3 min, then add 5 mL of pre-incubated deionized water at 37°C, and continue lysing uncoagulated red blood cells at 37°C. Transfer 100 μL of the liquid to a 96-well plate and read the absorbance at 545 nm using a microplate reader. The negative control group was an empty centrifuge tube (surface component: polypropylene), and the positive control group was 50 μL of whole blood in a centrifuge tube directly lysed with 5 mL of deionized water. The coagulation index (BCI) can be calculated using formula 2-4.
[0070]
[0071] Wherein, BCI is the sample coagulation index, and OD... s The absorbance value of the sample at a wavelength of 545 nm is OD. n The absorbance value of the negative control group at a wavelength of 545 nm is OD. p The absorbance value of the positive control group at a wavelength of 545 nm is shown.
[0072] A lower BCI value indicates a lower proportion of red blood cells not included in the blood clot after the sample comes into contact with blood, suggesting better coagulation performance. Conversely, a higher BCI value indicates poorer coagulation performance.
[0073] Table 3 Results of hemostatic performance test
[0074] Grouping BCI (%) Experimental Example 1 30.4±0.2 Comparative Example 1 42.4±1.2 Comparative Example 2 43.2±0.6 Comparative Example 3 54.0±0.3 Comparative Example 4 71.7±0.8
[0075] As can be seen from Table 3, the BCI value of J-ZGS in Example 1 is significantly lower than that of the commercially available C-GS sponge in Comparative Example 4, indicating that the former's in vitro hemostatic performance is significantly better than the latter.
[0076] The BCI value of J-ZGS in Example 1 was significantly lower than that of S-GS in Comparative Example 2, indicating that the addition of zein nanoparticle dispersion can significantly enhance the hemostatic performance of medical sponge.
[0077] Comparative Examples 1 and 3 show that the zein nanoparticle structure differs from that of Example 1 due to an unsuitable volume fraction of ethanol, and their in vitro coagulation effect is not significantly improved.
[0078] Finally, it should be noted that the above preferred embodiments are only used to illustrate the technical solutions of the present invention and are not intended to limit it. Although the present invention has been described in detail through the above preferred embodiments, those skilled in the art should understand that various changes can be made to it in form and detail without departing from the scope defined by the claims of the present invention.
Claims
1. A method for preparing an antioxidant and procoagulant bifunctional sponge, characterized in that, The preparation steps are as follows: 1) Prepare an ethanol solution, add zein to dissolve it in the ethanol solution, add an equal volume of deionized water to reverse precipitation and ultrasonically disperse, remove half the volume of solution from the dispersion by rotary evaporation, add glutaraldehyde aqueous solution to crosslink, and obtain a crosslinked procoagulant nanoparticle dispersion. 2) Dissolve gelatin in hot water, stir at low speed until dissolved, add glutaraldehyde, increase the speed to carry out chemical cross-linking, and foam at high speed until uniform and stable foam is obtained, then stop foaming to obtain foamed gelatin solution. 3) Pour the foamed gelatin solution obtained in step 2) into the cross-linked procoagulant nanoparticle dispersion obtained in step 1), freeze-dry, and obtain an antioxidant / procoagulant bifunctional sponge. The volume fraction of the ethanol solution in step 1) is 60-70%; The concentration of the zein solution mentioned in step 1) is 10–50 mg / mL; The volume ratio of the cross-linked procoagulant rice granule dispersion and the gelatin solution in step 3) is 1:10 to 1:
90.
2. The method for preparing an antioxidant and procoagulant dual-function sponge according to claim 1, characterized in that, The concentration of the ethanol solution mentioned in step 1) is 64-66%.
3. The method for preparing an antioxidant and procoagulant dual-function sponge according to claim 1, characterized in that, The volume fraction of the glutaraldehyde aqueous solution in step 1) is 0.25%.
4. The method for preparing an antioxidant and procoagulant dual-function sponge according to claim 1, characterized in that, The cross-linking temperature described in step 1) is 30–40°C.
5. The method for preparing an antioxidant and procoagulant dual-function sponge according to claim 1, characterized in that, The volume ratio of the cross-linked procoagulant rice granule dispersion and the gelatin solution in step 3) is 1:15-1:
25.
6. The method for preparing an antioxidant and procoagulant sponge according to claim 1, characterized in that, Step 2) The stirring speed for low-speed stirring is 50-400 rpm and the temperature is 30-50℃.
7. The method for preparing an antioxidant and procoagulant dual-function sponge according to claim 1, characterized in that, The chemical cross-linking time in step 2) is 10-60 min.
8. The method for preparing an antioxidant and procoagulant dual-function sponge according to claim 1, characterized in that, The gelatin mentioned in step 2) is type A, derived from pigskin, with a gel strength of 250-300g Bloom.
9. The method for preparing an antioxidant and procoagulant dual-function sponge according to claim 1, characterized in that, The high-speed foaming time mentioned in step 2) is 10min-60min, and the high-speed foaming speed is 800-1200rpm.