CN111116973B

CN111116973B - Preparation method and application of polyvinyl alcohol hemostatic porous material with high liquid absorption and high expansion performance and active hemostatic function

Info

Publication number: CN111116973B
Application number: CN201911342206.1A
Authority: CN
Inventors: 石长灿; 杨啸; 刘雯; 席光辉; 李徐坚; 季志孝
Original assignee: Wenzhou Research Institute Of Chinese Academy Of Sciences Wenzhou Institute Of Biomaterials And Engineering
Current assignee: Anhui Zhongke Maide Medical Technology Co ltd; Wenzhou Research Institute Of Guoke Wenzhou Institute Of Biomaterials And Engineering
Priority date: 2019-12-24
Filing date: 2019-12-24
Publication date: 2021-05-07
Anticipated expiration: 2039-12-24
Also published as: CN111116973A

Abstract

The invention relates to a preparation method and application of a polyvinyl alcohol hemostatic porous material with high imbibition and high expansion performance and an active hemostatic function, the hemostatic sponge prepared by the invention can be used for hemostasis of wounds penetrating through emergency sites and irregular incompressible wounds, the prepared polyvinyl alcohol hemostatic sponge does not adopt a traditional aldehyde crosslinking mode, and a large amount of concentrated acid catalysts are not added, so that the complicated cleaning step of post-treatment is avoided, the biological safety of a final product is greatly improved, and the prepared sponge has high imbibition capacity and expansion performance, can absorb moisture in blood in large quantity so as to concentrate blood coagulation components after being contacted with the blood, and can expand rapidly in the imbibition process to achieve the effect of compression hemostasis; meanwhile, the addition of the active hemostatic preparation can better improve the blood coagulation effect of the sponge, and the sponge has good biocompatibility, high imbibition capability and expansion performance, excellent hemostatic effect and the like, and has wide clinical application prospect.

Description

Preparation method and application of polyvinyl alcohol hemostatic porous material with high liquid absorption and high expansion performance and active hemostatic function

Technical Field

The invention specifically relates to the technical field of medical instruments, and particularly relates to a preparation method and application of a polyvinyl alcohol hemostatic porous material with high liquid absorption, high expansion and active hemostatic functions.

Background

In the first aid scene, the probability of injuring special parts of the wounded body, such as limb joints or joint junctions (joints, armpits, groin, subcortical cavities and the like) is high, the blood flow of the parts is rich, the major artery is broken when the parts are seriously injured, pulsatile or jet major hemorrhage is formed, and deep, irregular and incompressible wound major hemorrhage caused by small-caliber firearms or explosion in battlefields, traffic accidents and daily life is also existed. This type of bleeding is often uncontrollable with traditional hemostasis. Therefore, effectively addressing this type of bleeding is likely to be an important goal in the next development of wound resuscitation.

The high-expansibility hemostatic sponge is a hemostatic product with a porous structure, high water absorption capacity and quick expansibility. The sponge can quickly absorb blood to concentrate components such as platelets, red blood cells, blood coagulation proteins and the like so as to improve the blood coagulation capability and accelerate hemostasis. But it expands more than 10 times in volume when it encounters body fluid or blood, compared to the existing hemostatic sponges on the market. Thus, sufficient compression force can be generated to assist hemostasis while absorbing wound bleeding.

Polyvinyl alcohol is a synthetic, non-toxic, non-immunogenic and well biocompatible polymer that has been widely used in many biomedical applications. In recent years, there have been many reports of processes for preparing high-expansion polyvinyl alcohol sponges in the published patents. And the sponges with the porous structure have excellent liquid absorption capacity and rapid expansion capacity, and have obvious advantages and application prospects compared with the traditional gauze and cotton hemostatic materials. However, these polyvinyl alcohol expanded sponges generally use formaldehyde or glutaraldehyde as a crosslinking agent during the preparation process, and a large amount of acid as a catalyst, which may complicate the process of removing the residual agent during the post-treatment and even cause toxic side effects on the final product. In addition, the single polyvinyl alcohol sponge can accelerate hemostasis only by means of self liquid absorption and compression capacity, and the cell adhesion function and the active hemostasis effect are lacked.

Disclosure of Invention

In order to solve the problems in the prior art, the invention provides a preparation method and application of a polyvinyl alcohol hemostatic porous material with high liquid absorption, high expansion performance and an active hemostatic function, and the polyvinyl alcohol hemostatic porous material has the advantages of high biological safety, strong liquid absorption capacity, high expansion performance and no need of adding an aldehyde crosslinking agent and a concentrated acid catalyst.

The technical solution adopted by the invention is as follows: a preparation method of a polyvinyl alcohol hemostatic porous material with high liquid absorption and high expansion performance and an active hemostatic function comprises the following steps:

(1) graft-modified polyvinyl alcohol: dispersing polyvinyl alcohol into dimethyl sulfoxide to prepare suspension with the mass fraction of 5% -20%; heating to 50-150 deg.C, stirring for 1-3h, and cooling to room temperature; adding a reaction reagent containing carbon-carbon unsaturated double bonds, and continuously stirring for reaction for 24-48 h; dialyzing in dialysis bag for 3 days, and freeze drying;

(2) and (3) crosslinking reaction: dispersing the obtained product in distilled water to prepare a solution with the mass fraction of 20-30%; adding 0.1-1% of photoinitiator, 10-50% of dimercapto cross-linking agent, 1-10% of polymer with active hemostasis function and 0.1-0.5% of foaming agent into the product, violently stirring for 30min at the rotating speed of 1500rpm for foaming, and after the foamed material is irradiated by ultraviolet light, washing for 3-5 times by using distilled water to obtain a porous gelatinous product;

(3) and placing the obtained porous gel-like product in a compression mould for compression, freezing and forming and freeze drying to obtain the polyvinyl alcohol hemostatic porous material with high liquid absorption, high expansion performance and active hemostatic function.

The addition amount of the reaction reagent containing carbon-carbon unsaturated double bonds in the step (1) is 30-70% of the mass of the added polyvinyl alcohol.

The reaction reagent containing carbon-carbon unsaturated double bonds in the step (1) is one or more of acrylic acid, acrylic anhydride, methacrylic anhydride, nadic anhydride, maleic anhydride, acryloyl chloride and methacryloyl chloride.

The dialysis bag in the step (1) has a molecular weight cutoff of 3500.

The illumination intensity of the ultraviolet light irradiation in the step (2) is 60 mu W/cm²The irradiation was 300 s.

The polyvinyl alcohol is at least one of types 2099, 2088, 2488, 1788, 0588, 2092, 1792, 2699, 2499 and 1799.

The photoinitiator is at least one of photoinitiator 184, photoinitiator 1173, photoinitiator 2959, photoinitiator 369 and photoinitiator 784.

The dimercapto cross-linking agent is at least one of dithiothreitol and mercapto-polyethylene glycol-mercapto with the weight-average molecular weight of 400-10000.

The polymer with the active hemostasis function is at least one of chitosan, sodium alginate, gelatin, thrombin, fibrin, kaolin, montmorillonite and bioactive glass.

The application of porous hemostatic material in preparing hemostatic material for war wound, wound emergency, perioperative period or wound healing.

The invention has the beneficial effects that: the invention provides a preparation method and application of a polyvinyl alcohol hemostatic porous material with high liquid absorption and high expansion performance and an active hemostatic function, wherein polyvinyl alcohol is subjected to graft modification, a polymer with double mercapto groups functionalized is used as a cross-linking agent, a light spot chemical technique is used for carrying out cross-linking reaction on a system, and finally, a modified polyvinyl alcohol hemostatic sponge with high safety, strong liquid absorption capacity and high expansion performance is obtained through compression treatment and freeze drying; on the other hand, the sponge after imbibing can be rapidly expanded and filled into the whole wound, and the effect of compression hemostasis is generated on the bleeding site. In addition, a material with an active hemostatic function is added into the sponge, so that better hemostatic capacity is given to the sponge. The invention thoroughly solves the problems of complicated post-treatment process and potential safety of the traditional aldehyde crosslinked polyvinyl alcohol hemostatic sponge, and endows the sponge with the efficacy of active hemostasis.

Drawings

FIG. 1 is an SEM photograph of the polyvinyl alcohol sponges of examples 1-4.

FIG. 2 shows the statistics of the maximum water absorption test for polyvinyl alcohol sponges and commercially available sponges.

FIG. 3 shows the swelling ratio statistics of polyvinyl alcohol sponges and commercially available sponges.

FIG. 4 is a photograph showing the whole blood coagulation test of polyvinyl alcohol sponge and commercially available sponge (A), and the statistics of the whole blood coagulation index of polyvinyl alcohol sponge and commercially available sponge (B).

FIG. 5.A is the experimental photograph of the in vitro coagulation time of the polyvinyl alcohol sponge and the commercially available sponge, 1: blank, 2: comparative examples 1, 3: comparative examples 2, 4: comparative examples 3, 5: examples 1, 6: examples 2, 7: examples 3, 8: example 4, (B), in vitro clotting time statistics for polyvinyl alcohol sponges and commercially available sponges.

Fig. 6 is a photograph (a) of the experimental process of the polyvinyl alcohol sponge in the liver penetrating injury model, a blood bleeding time statistic (B) of the polyvinyl alcohol sponge and the commercially available sponge in the liver penetrating injury model, and a blood bleeding amount statistic (C) of the polyvinyl alcohol sponge and the commercially available sponge in the liver penetrating injury model.

FIG. 7 shows the result of cytotoxicity analysis of polyvinyl alcohol sponges.

Detailed Description

The present invention is further illustrated by the following specific examples, which are provided to enable those skilled in the art to better understand the present invention, but are not intended to limit the present invention.

Example 1

A preparation method of a polyvinyl alcohol hemostatic sponge with high liquid absorption and high expansion performance and active hemostatic function comprises the following steps:

(1) dispersing polyvinyl alcohol 2099 into dimethyl sulfoxide to prepare suspension with the mass fraction of 10%; heating to 100 ℃, stirring for 2h, and cooling to room temperature; adding norbornene dianhydride with the mass of 50% of that of the polysaccharide, and continuously stirring for reaction for 24 hours; placing in dialysis bag with molecular weight cutoff of 3500, dialyzing for 3 days, and freeze drying;

(2) dispersing the product obtained in the step (1) in distilled water to prepare a solution with the mass fraction of 20%; adding 0.5% of photoinitiator 2959, 20% of sulfhydryl-polyethylene glycol-sulfhydryl with weight average molecular weight of 2000, 1% of gelatin, 0.5% of sodium dodecyl sulfate, stirring vigorously at 1500rpm for 30min for foaming, wherein the illumination intensity of the foamed material is 60 μ W/cm²Irradiating with ultraviolet light for 300s, and washing with distilled water for 3 times to obtain porous gel product;

(3) and (3) placing the porous gelatinous product in a compression mould for compression, freezing and forming and freeze drying to obtain the polyvinyl alcohol hemostatic sponge with high liquid absorption, high expansion performance and active hemostatic function.

Example 2

(1) dispersing polyvinyl alcohol 2699 into dimethyl sulfoxide to prepare suspension with the mass fraction of 10%; heating to 100 ℃, stirring for 2h, and cooling to room temperature; adding norbornene dianhydride with the mass of 50% of that of the polysaccharide, and continuously stirring for reaction for 24 hours; placing in dialysis bag with molecular weight cutoff of 3500, dialyzing for 3 days, and freeze drying;

(2) dispersing the product obtained in the step (1) in distilled water to prepare a solution with the mass fraction of 30%; adding 0.7 percent of photoinitiator 2959 of the product obtained in the step (1), 20 percent of sulfhydryl-polyethylene glycol-sulfhydryl with the weight average molecular weight of 2000, 1 percent of gelatin, 0.5 percent of sodium dodecyl sulfate, and vigorously stirring for 30min at the rotating speed of 1500rpm for foaming, wherein the illumination intensity of the foamed material is 60 mu W/cm²Irradiating with ultraviolet light for 300s, and washing with distilled water for 3 times to obtain porous gel product;

Example 3

(1) dispersing polyvinyl alcohol 2699 into dimethyl sulfoxide to prepare a suspension with the mass fraction of 20%; heating to 100 ℃, stirring for 2h, and cooling to room temperature; adding norbornene dianhydride with the mass of 50% of that of the polysaccharide, and continuously stirring for reaction for 24 hours; placing in dialysis bag with molecular weight cutoff of 3500, dialyzing for 3 days, and freeze drying;

(2) dispersing the product obtained in the step (1) in distilled water to prepare a solution with the mass fraction of 30%; adding 0.5% of photoinitiator 2959, 20% of sulfhydryl-polyethylene glycol-sulfhydryl with weight average molecular weight of 2000, 5% of gelatin, 0.5% of sodium dodecyl sulfate, stirring vigorously at 1500rpm for 30min for foaming, wherein the illumination intensity of the foamed material is 60 μ W/cm²Irradiating with ultraviolet light for 300s, and washing with distilled water for 3 times to obtain porous gelA product in the form of a paste;

Experiments prove that the polyvinyl alcohol with the model number of 2699 in the embodiment is replaced by the polyvinyl alcohol with the model numbers of 2088, 2488, 1788, 0588, 2092, 1792, 2499 and 1799, and other polyvinyl alcohol hemostatic sponges with high liquid absorption and high expansion performance and active hemostatic function are respectively prepared in the same way as the embodiment.

Experiments prove that acrylic anhydride, methacrylic anhydride, acryloyl chloride or methacryloyl chloride are respectively used for replacing the nadic anhydride in the embodiment, and other polyvinyl alcohol hemostatic sponges with high liquid absorption, high expansion performance and active hemostatic function are respectively prepared in the same way as the embodiment.

Experiments prove that the chitosan, the sodium alginate, the thrombin, the kaolin or the bioactive glass is used for replacing the gelatin in the embodiment, and other polyvinyl alcohol hemostatic sponges which have high liquid absorption, high expansion performance and active hemostatic function are respectively prepared in the same manner as the embodiment.

Experiments prove that the polyvinyl alcohol hemostatic sponge with high liquid absorption and high expansion performance and active hemostatic function is prepared by using fatty alcohol-polyoxyethylene ether sodium sulfate or rosin soap foaming agent instead of the lauryl sodium sulfate in the embodiment.

Example 4

(2) dispersing the product obtained in the step (1) into distilled water to prepare the product with the mass fraction ofA 30% solution; adding a photoinitiator 1173 accounting for 0.5 percent of the mass of the product obtained in the step (1), 20 percent of sulfhydryl-polyethylene glycol-sulfhydryl with the weight average molecular weight of 2000, 10 percent of gelatin, 0.5 percent of sodium dodecyl sulfate, and vigorously stirring for 30min at the rotating speed of 1500rpm for foaming, wherein the illumination intensity of the foamed material is 60 mu W/cm²Irradiating with ultraviolet light for 300s, and washing with distilled water for 3 times to obtain porous gel product;

Comparative examples 1 to 3

The medical PVA cotton sold in the market is taken as a comparative example 1, the kuaikang hemostatic sponge is taken as a comparative example 2, and the Xiangen absorbent gelatin sponge is taken as a comparative example 3.

Experimental example 1

The porous gelatinous products obtained in step (2) of examples 1, 2, 3, and 4 were lyophilized and observed by scanning electron microscopy. FIG. 1 shows a microstructure of a polyvinyl alcohol sponge according to various examples.

Experimental example 2

The maximum water absorption capacity of the high absorbency, high expansion polyvinyl alcohol hemostatic sponges with active hemostatic function of examples 1, 2, 3, 4 and the sponges of comparative examples 1, 2, 3 were tested. The specific experimental steps are as follows: a portion of the sponge was removed and weighed, and recordedW ₀. Completely immersing the sponge in distilled water for 30min, taking out, weighing again and recordingW ₁. By the formula M (%) = (W ₁-W ₀）/ W ₀The maximum water absorption of the sponge was calculated. The experimental result is shown in fig. 2, the water absorption capacity of the polyvinyl alcohol sponge can reach more than 30-45 times of the self weight, which is obviously superior to the sponge in the comparative example, and the material in the example 1 has the maximum water absorption rate which can reach 4600%.

Experimental example 3

Polyvinyl alcohol hemostatic sponges with active hemostatic function for high imbibition and high swelling performance in examples 1, 2, 3 and 4 andthe maximum expansion ratios of the sponges of ratios 1, 2 and 3 were tested. The specific experimental steps are as follows: record the initial length of the sponge after compression asL ₀Completely immersing the sponge in distilled water, and recording the length of the sponge after expansion after the sponge is completely expandedL ₁. By the formula P (%) =: (L ₁-L ₀）/ L ₀The maximum expansion of the sponge was calculated. The experimental results are shown in fig. 3, the swelling ratio of the polyvinyl alcohol sponge can reach over 9-12 times of the original swelling ratio, and the sponge of the comparative example has almost no swelling performance. The material in example 1 has the maximum expansion ratio which can reach 12 times.

Experimental example 4

The whole blood coagulation index of the polyvinyl alcohol hemostatic sponges with active hemostatic function of high imbibition and high expansion properties of examples 1, 2, 3, 4 and the sponges of comparative examples 1, 2, 3 were tested. The specific experimental steps are as follows: 20mg of the sample is placed in a surface dish, cultured at 37 ℃ for 5min, 200. mu.l of anticoagulated blood is slowly dripped on the surface of the sample, 20. mu.l of 0.2mol of calcium chloride is added, and the culture is continued at 37 ℃ for 5 min. After that, 25ml of distilled water was carefully added to the petri dish (as far as possible without destroying the formed blood clots) and cultured in a shaker with shaking at 30rpm for 10min, the red blood cells without cross-linking were washed away by the distilled water, the absorbance of the resulting hemoglobin solution was measured as a at 540nm, the absorbance of the anticoagulated blood in deionized water was used as reference B, and the formula for calculating the blood coagulation index was: . As shown in FIG. 5, the results show that the polyvinyl alcohol hemostatic sponges of examples 1-4 can fix red blood cells in blood better than the sponges of comparative examples 1-3, thereby facilitating blood coagulation.

Experimental example 5

The in vitro clotting times of the high imbibition, high swelling properties polyvinyl alcohol hemostatic sponges with active hemostatic function of examples 1, 2, 3, 4 and the sponges of comparative examples 1, 2, 3 were tested. The specific experimental steps are as follows: 20mg of sample is placed in a surface dish, cultured for 5min at 37 ℃, 200 mu l of anticoagulated blood is slowly dripped on the surface of the sample, 20 mu l of 0.2mol of calcium chloride is added, timing is started, and the blood flowing condition is observed. After the blood flow was observed to stop, the timer was ended and the time t was recorded. The results show that the polyvinyl alcohol hemostatic sponges of examples 1-4 and the sponges of comparative examples 1-3 both reduced clotting times compared to the blank, wherein the effect of examples 3 and 4 was significant and the clotting times could be reduced to 200 s. Indicating that the addition of a sufficient amount of the active coagulation preparation is effective in shortening the clotting time.

Experimental example 6

The high imbibition and high swelling properties of the polyvinyl alcohol hemostatic sponges with active hemostatic function of examples 1, 2, 3, 4 and the sponges of comparative examples 1, 2, 3 were evaluated for their hemostatic capabilities in vivo. The experiment was performed using the SD rat liver penetrating wound model. The specific experimental steps are as follows: anesthetized by intraperitoneal injection of aqueous chloral hydrate and the abdominal hairs were shaved off, opened in the abdomen to expose the liver, and weighed filter paper was placed under the liver. Perforating the liver by using a perforator with the inner diameter of 3cm, plugging the compressed sponge into a wound, observing bleeding until blood does not flow, and counting bleeding time and bleeding amount. As shown in FIG. 7, the experimental results of the liver penetrating injury bleeding model show that the polyvinyl alcohol hemostatic sponges of examples 1-4 can effectively shorten the hemostatic time and reduce the bleeding amount compared with the sponges of comparative examples 1-3, and the sponges of example 3 have the best in vivo hemostatic ability.

Experimental example 7

The cytotoxicity test analysis is carried out on the polyvinyl alcohol hemostatic sponges with high imbibition and high expansion performance and active hemostatic function in the examples 1, 2, 3 and 4. The specific experimental steps are as follows: the sponge was dispersed in ultrapure water and a suspension of 400. mu.g/ml concentration was prepared. Taking 100 μ l of 1 × 10⁴One/ml cell suspension was added to 96-well plates and incubated for 24 h. Then, CCK-8 solution was added to each well plate and incubation continued for 2 h. Finally, the absorbance of the solution in the well plate was measured at 450 nm. As shown in figure 4, cytotoxicity experiments show that the polyvinyl alcohol hemostatic sponges in examples 1-4 have no obvious toxic or side effect on L929 cells.

Conclusion

The sponge prepared by the invention can quickly absorb water in blood, on one hand, a great amount of blood coagulation components are enriched, and the blood coagulation process is accelerated; on the other hand, the sponge after imbibing can be rapidly expanded and filled into the whole wound, and the effect of compression hemostasis is generated on the bleeding site. In addition, a material with an active hemostatic function is added into the sponge, so that better hemostatic capacity is given to the sponge. The invention thoroughly solves the problems of complicated post-treatment process and potential safety of the traditional aldehyde crosslinked polyvinyl alcohol hemostatic sponge, and endows the sponge with the efficacy of active hemostasis.

The skilled person should understand that: although the invention has been described in terms of the above specific embodiments, the inventive concept is not limited thereto and any modification applying the inventive concept is intended to be included within the scope of the patent claims.

The above description is only a preferred embodiment of the present invention, and the protection scope of the present invention is not limited to the above embodiments, and all technical solutions belonging to the idea of the present invention belong to the protection scope of the present invention. It should be noted that modifications and embellishments within the scope of the invention may occur to those skilled in the art without departing from the principle of the invention, and are considered to be within the scope of the invention.

Claims

1. A preparation method of a polyvinyl alcohol hemostatic porous material with high liquid absorption and high expansion performance and an active hemostatic function is characterized by comprising the following steps:

wherein the reaction reagent containing carbon-carbon unsaturated double bonds is one or more of acrylic acid, acrylic anhydride, methacrylic anhydride, nadic anhydride, maleic anhydride, acryloyl chloride and methacryloyl chloride;

2. The preparation method according to claim 1, wherein the amount of the reaction reagent containing carbon-carbon unsaturated double bonds added in step (1) is 30-70% of the mass of the added polyvinyl alcohol.

3. The method of claim 1, wherein the dialysis bag of step (1) has a molecular weight cut-off of 3500.

4. The method according to claim 1, wherein the ultraviolet light is irradiated in the step (2) at an intensity of 60 μ W/cm²The irradiation was 300 s.

5. The method as claimed in claim 1, wherein the polyvinyl alcohol is at least one of type 2099, 2088, 2488, 1788, 0588, 2092, 1792, 2699, 2499 and 1799.

6. The method of claim 1, wherein the photoinitiator is at least one of photoinitiator 184, photoinitiator 1173, photoinitiator 2959, photoinitiator 369, and photoinitiator 784.

7. The method as claimed in claim 1, wherein the dimercapto crosslinker is at least one of dithiothreitol and mercapto-polyethylene glycol-mercapto group with a weight average molecular weight of 400-.

8. The preparation method of claim 1, wherein the polymer with active hemostatic function is at least one of chitosan, sodium alginate, gelatin, thrombin, and fibrin.

9. Use of a polyvinyl alcohol hemostatic porous material prepared by the preparation method of any one of claims 1 to 8 as a hemostatic material for war wounds, emergency wounds, perioperative period, or wound healing.