CN112891610A

CN112891610A - Pig-derived fibrin-electrostatic spinning nanofiber antibacterial hemostatic patch and preparation method thereof

Info

Publication number: CN112891610A
Application number: CN202110096953.2A
Authority: CN
Inventors: 张景添; 刘璐; 张军东; 吴剑英
Original assignee: Shanghai Likangrui Bioengineering Co ltd
Current assignee: Shanghai Likangrui Bioengineering Co ltd
Priority date: 2021-01-25
Filing date: 2021-01-25
Publication date: 2021-06-04

Abstract

The invention relates to the technical field of medical hemostatic patches, in particular to a porcine fibrin-electrostatic spinning nanofiber antibacterial hemostatic patch and a preparation method thereof. The patch has excellent hemostatic effect, combines a fibrin thrombin hemostatic mechanism with the mechanical property of an antibacterial silk fibroin film, enhances the hemostatic effect, is natural and absorbable, improves the hemostatic effect, simultaneously combines fibrinogen and thrombin into one, changes four components into a single component, does not need preparation, can be used immediately after opening, and greatly simplifies the problems of complex use and long time consumption of the conventional fibrinogen adhesive; can be stored and transported at room temperature, and has obvious technical, cost and clinical advantages.

Description

Pig-derived fibrin-electrostatic spinning nanofiber antibacterial hemostatic patch and preparation method thereof

Technical Field

The invention relates to the field of medical hemostatic patches, in particular to a nanofiber antibacterial hemostatic patch.

Background

Bleeding is one of the most common problems in surgery and trauma, and an effective hemostatic method and good hemostatic material are particularly critical. The problems of difficult suture of clinical wound bleeding, postoperative suture and bleeding and the like often bring troubles to the completion of the operation and the postoperative recovery of patients, and the problem can be effectively solved by selecting proper hemostatic dressing.

At present, the hemostatic auxiliary materials for medical surgery are gelatin sponge and collagen sponge, and the latest hemostatic product fibrin adhesive is also applied to clinic. The medical surgical fibrin adhesive has good hemostasis, histocompatibility and adhesiveness, and forms a sticky gel fibrin clot by simulating the reaction process of the final stage of blood coagulation, thereby achieving the effects of hemostasis and adhesion.

When the fibrinogen and the thrombin contact with the bleeding wound surface, the fibrinogen is contacted with physiological liquid (such as blood, lymph or physiological saline), the fibrinogen is converted into fibrin monomer, the fibrin monomer is polymerized into fibrin clot, and the collagen matrix is tightly adhered to the wound surface, so that the purpose of stopping bleeding is achieved. Fibrin is then cross-linked by endogenous factor XIII, forming a strong, mechanically stable network with good adhesive properties, thus also providing a seal, thus providing a physical barrier to bleeding.

The silk fibroin belongs to natural high-molecular fiber, contains more amino acid with hydrophilic amino and carboxyl, and has better air permeability, water permeability and water absorption due to hydrophilic groups in a non-crystallization region. The silk fibroin has good biocompatibility, no adverse effect on adhesion proliferation, apoptosis and cell secretion factors of cells, no irritation, no toxicity, small inflammatory response, excellent chemical and physical properties, certain biodegradability and can be used as a wound dressing. The research of the application of silk fibroin in hemostatic materials is also well focused.

Disclosure of Invention

The invention aims to provide a pig-derived fibrin-electrostatic spinning nanofiber antibacterial hemostatic patch and a preparation method thereof. The novel hemostatic patch developed by the invention overcomes the defects of complex operation, high requirement on storage conditions, difficult use of multiple components and the like of the existing fibrin adhesive, and endows the fibrin adhesive with mechanical properties to promote adhesion and hemostatic effects. The patch has excellent hemostatic effect and certain mechanical property, is natural and absorbable, improves the hemostatic effect, simultaneously combines fibrinogen and thrombin into one, is not required to be prepared, can be used immediately after being opened, can be stored and transported at room temperature, and has obvious technical, cost and clinical advantages. .

The invention firstly prepares the blending suspension of the porcine fibrinogen and the thrombin, then sprays or soaks the blending suspension in the electrostatic spinning technology to prepare the surface of the silk fibroin nanofiber film, and then dries the silk fibroin nanofiber film to prepare the degradable and absorbable porcine fibrin-electrostatic spinning nanofiber antibacterial hemostatic patch which can be used for hemostasis.

In order to achieve the purpose, the invention provides the following technical scheme:

a preparation method of a pig-derived fibrin-electrostatic spinning nanofiber antibacterial hemostatic patch comprises the following steps:

(1) preparing an antibacterial regenerated silk fibroin electrostatic spinning membrane: dissolving a certain amount of silk fibroin in hexafluoroisopropanol to prepare a silk fibroin solution with a certain mass concentration for later use;

dissolving a certain amount of polyoxyethylene in trifluoroethanol to prepare a polyoxyethylene solution with a certain mass concentration for later use;

mixing the prepared silk fibroin solution and polyethylene oxide solution according to the volume ratio of 1: 2, adding nano silver with the mass concentration of 0.2%, magnetically stirring for 2 hours, standing and defoaming to obtain a spinning solution;

filling the spinning solution into an injector, and pushing by using an injection pump; the distance between the needle head and the receiving plate is 10cm, and the receiving electrode is an aluminum foil with the thickness of 15 multiplied by 15cm and is used for collecting the nano fibers; two poles of a high-voltage direct-current power supply are respectively connected with the injection steel needle and the receiving aluminum foil;

vacuum drying the prepared fiber support for 24h, steaming in a 75% alcohol steam pot for 1h, taking out, naturally drying, and sterilizing with ultraviolet or ethylene oxide;

(2) preparation of a blending suspension of porcine fibrinogen and thrombin: respectively dispersing the extracted porcine fibrinogen and thrombin in alcohol and/or ketone solution to obtain fibrinogen suspension and thrombin suspension, and uniformly mixing the fibrinogen suspension and the thrombin suspension to obtain porcine fibrinogen and thrombin blended suspension;

(3) preparing a pig-derived fibrin-electrostatic spinning nanofiber antibacterial hemostatic patch: and (3) adhering the porcine fibrinogen and thrombin blended suspension to the prepared antibacterial regenerated silk fibroin electrostatic spinning film by using a spraying technology, and drying to obtain the porcine fibrinogen-electrostatic spinning nanofiber antibacterial hemostatic patch.

Wherein the mass concentration range of the fibroin protein solution in the step (1) is 4-8%.

Wherein the mass concentration of the polyoxyethylene solution in the step (1) is 6-10%.

Wherein the parameters in the electrostatic spinning technology in the step (1) comprise that a spinning nozzle is a 20G-standard blunt-end stainless steel needle for injection, the flow rate of a solution is controlled to be about 0.5-1 mL/h by an injection pump, the voltage is set to be within the range of 25-30 kV, the distance between a needle head and a collecting plate is 10cm, the environmental temperature is controlled to be 25 +/-1 ℃, and the humidity is controlled to be 45 +/-5%.

Wherein, before the two components are blended in the step (2), the fibrinogen suspension comprises several or more of fibrinogen, sodium citrate, sodium chloride, albumin, arginine hydrochloride and riboflavin.

The fibrinogen suspension comprises 30-65% of fibrinogen, 6-15% of sodium citrate, 4-14% of sodium chloride, 11-28% of albumin, 12-47% of arginine hydrochloride, 2-5% of riboflavin and the balance of absolute ethyl alcohol.

Wherein, before the two components are blended in the step (2), the thrombin suspension comprises thrombin, calcium chloride and mannitol.

Wherein the concentration range of each component in the thrombin suspension is 500-1000I.U./mL thrombin, 4-7% calcium chloride, 20-40% mannitol, and the balance of absolute ethyl alcohol.

Wherein the blending suspension of the porcine fibrinogen and the thrombin in the step (2) contains fibrinogen and thrombin particles, and the Folk Ward average diameter of the particles is 30-120 mu m.

Wherein, the fibrinogen suspension in the step (2) is uniformly mixed by adopting a stirring and homogenizing method, and the temperature is controlled to be 2-10 ℃.

Wherein, the thrombin suspension in the step (2) is uniformly mixed by adopting a stirring and homogenizing method, and the temperature is controlled to be 2-10 ℃.

Wherein, the blending suspension of the porcine fibrinogen and the thrombin in the step (2) is uniformly mixed by adopting a stirring and homogenizing method, and the temperature is controlled to be 2-10 ℃.

Wherein, the alcohol and/or ketone solution in the step (2) is absolute ethyl alcohol, isobutyl alcohol, isopropanol or acetone.

Wherein the content of fibrinogen in the blending suspension of the porcine fibrinogen and the thrombin in the step (2) is 30-80 mg/mL.

Wherein the thrombin content in the blending suspension of the porcine fibrinogen and the thrombin in the step (2) is 500-1000 I.U./mL.

Wherein, the blended suspension needs to be uniformly distributed during spraying or soaking in the step (3), and the temperature is controlled to be 2-10 ℃; controlling the relative humidity of the atmosphere to be 60-85 percent; the coated carrier film is kept at said humidity for a period of at least 1-2 h.

Or, replacing the polyoxyethylene in the step (1) with natural macromolecular materials such as hyaluronic acid or chitosan.

The pig-derived fibrin-electrostatic spinning nanofiber antibacterial hemostatic patch prepared by the preparation method.

Compared with the prior art, the invention has the beneficial effects that:

(1) the invention discloses a novel degradable and absorbable pig-derived fibrin-electrostatic spinning nanofiber antibacterial hemostatic patch, and active ingredients of the patch are pig-derived fibrinogen and thrombin. The fibrinogen and the thrombin fibrin are interwoven into a net shape, which is beneficial to adsorbing red blood cells and platelets to form a blood clot; has good adhesiveness, can fill damaged wound surface, and is waterproof, air-proof and capable of preventing secondary blood seepage; promoting adhesion, promoting the growth of endothelial cells and fibroblasts of capillary vessels, and promoting the formation of granulation tissues; has good biocompatibility and can be completely degraded in vivo.

(2) The carrier is a silk fibroin electrostatic spinning nanofiber membrane which is a natural degradable and absorbable material, has high specific surface area and high porosity, good biocompatibility and good air permeability, has no adverse effect on adhesion proliferation, apoptosis and cell secreted factors of cells, and has no stimulation, no toxicity, small inflammatory response and excellent chemical and physical properties; contains silver ions and has certain antibacterial and anti-infection properties.

(3) The patch combines the hemostatic mechanism of fibrin thrombin with the mechanical property of antibacterial silk fibroin film, and enhances the hemostatic effect.

(4) The novel hemostatic patch developed by the invention combines fibrinogen and thrombin into one, does not need preparation, can be used immediately after being opened, and greatly simplifies the problems of complex use and long consumed time of the conventional fibrinogen adhesive; can be stored and transported at room temperature, and has obvious technical, cost and clinical advantages.

(5) Compared with the existing patch taking collagen as a carrier, the carrier has relatively thin thickness and higher adjustment precision in the preparation process, can be better attached to a wound, and can avoid tissue inflammation and use limitation caused by the thickness to a certain extent.

Detailed Description

The technical solutions in the embodiments of the present invention will be clearly and completely described below, and it is obvious that the described embodiments are only a part of the embodiments of the present invention, and not all embodiments. All other embodiments, which can be derived by a person skilled in the art from the embodiments given herein without making any creative effort, shall fall within the protection scope of the present invention.

Example 1

A pig source fibrin-electrostatic spinning nanofiber antibacterial hemostatic patch is prepared by the following steps:

(1) preparing an antibacterial regenerated silk fibroin electrostatic spinning film: weighing silk fibroin with the mass concentration of 4%, dissolving the silk fibroin in hexafluoroisopropanol, and preparing a silk fibroin solution with a certain mass concentration for later use;

weighing polyoxyethylene with the mass concentration of 10%, dissolving the polyoxyethylene in trifluoroethanol to prepare polyoxyethylene solution with the mass concentration of 10% for later use;

mixing the prepared silk fibroin solution and polyethylene oxide solution according to the volume ratio of 1: 2, adding nano silver according to the mass concentration of 0.1%, magnetically stirring for 2h, and standing for defoaming to obtain the spinning solution.

The spinning solution was filled into a 10mL plastic syringe (small amounts were added in portions and the remaining solution was further vortexed and oscillated) and a 20G gauge stainless steel needle for blunt tip injection was used as the nozzle. The flow rate of the solution is controlled to be about 0.6mL/h by an injection pump, the voltage is set to be in the range of 25-30 kV, the distance between a needle head and a collecting plate is 10cm, and the receiving electrode adopts an aluminum foil with the specification of 15 multiplied by 15cm to collect the nano fibers. The two poles of the high-voltage DC power supply are respectively connected with the injection steel needle and the receiving aluminum foil, so that the good grounding environment temperature is controlled to be 25 +/-1 ℃, and the humidity is controlled to be 45 +/-5%. The rest parameters are kept unchanged except that the solution flow rate and the voltage can be finely adjusted in the electrospinning process.

The prepared fiber support is cut into a certain shape after being dried for 24 hours in vacuum, washed by ultrapure water, soaked in 75% alcohol for 1 hour, taken out, naturally dried, and fumigated and disinfected by ethylene oxide for later use.

(2) Preparing 1800mL of fibrinogen absolute ethanol suspension according to 30% of fibrinogen, 6% of sodium citrate, 5% of sodium chloride, 18% of albumin, 30% of arginine hydrochloride and 2% of riboflavin, uniformly stirring at 4 ℃, storing for 10 hours at 4 ℃, and continuously stirring;

preparing 400mL of thrombin absolute ethanol suspension according to 500I.U./mL of thrombin, 4% of calcium chloride and 20% of mannitol, uniformly stirring at-20 ℃, storing for 10 hours at-20 ℃, and continuously stirring;

the two suspensions were stirred and mixed well at 4 ℃ and added with absolute ethanol to a final volume of 2500 mL.

(3) Removing the aluminum foil at the bottom layer of the prepared antibacterial silk fibroin film, placing the antibacterial silk fibroin film in a glass dish, and uniformly spraying the mixed suspension of fibrinogen and thrombin on the surface of the antibacterial silk fibroin film under the conditions of 4 ℃ and 75% of atmospheric relative humidity. And standing for 1-2 hours after the spraying is finished. Drying to obtain the pig-derived fibrin-electrostatic spinning nanofiber antibacterial hemostatic patch. Cutting into proper shape according to the requirement.

Example 2

The fibrinogen anhydrous ethanol suspension in step (2) of example 1 was adjusted to 40% fibrinogen, 10% sodium citrate, 6% sodium chloride, 11% albumin, 25% arginine hydrochloride, and 4% riboflavin. The amount of thrombin in the anhydrous ethanol suspension was adjusted to 750i.u./mL thrombin, 6% calcium chloride, and 30% mannitol. The remaining step parameters remain unchanged.

Example 3

The amounts of the components of the fibrinogen anhydrous ethanol suspension obtained in step (2) of example 1 were adjusted to 35% fibrinogen, 6% sodium citrate, 4% sodium chloride, 15% albumin, 30% arginine hydrochloride, and 5% riboflavin. The amount of the thrombin-containing anhydrous ethanol suspension was adjusted to 1000i.u./mL of thrombin, 7% of calcium chloride, and 40% of mannitol. The remaining step parameters remain unchanged.

To highlight the beneficial effects of the present invention, the following comparative experiments were performed:

10 adult SD male rats weighing 220 + -10 g were selected and purchased from Shanghai Slek laboratory animals Co., Ltd. The groups were randomly divided into 2 groups of 10 individuals.

Groups were set as control blank (commercially available porcine fibrin adhesive) and experimental (porcine fibrin-electrospun nanofiber antibacterial hemostatic patch of examples 1-3).

All animals were anesthetized using sodium barbiturate intraperitoneal injection at a dose of 20 mg/kg. Constructing a liver bleeding wound model: the incision is roof-shaped, enters the abdomen, enters the abdominal cavity layer by layer, exposes the left lobe of the liver, and cuts off part of the left lobe to form a wound surface with the length of 20mm and the depth of 2 mm.

Experimental groups: the patch is clamped by tweezers and applied on the bleeding wound surface, timing is started until bleeding stops, and the bleeding stopping time and the bleeding amount are recorded. After application, the patient should be pressed for 10 s.

Control group: the preparation method comprises the following steps: the main body case was taken out from the refrigerator and allowed to stand to room temperature. And (3) absorbing the marked amount of the main body gel dissolving solution by using a disposable sterile injector, injecting the marked amount of the main body gel dissolving solution into the main body gel freeze-dried powder, standing for 30-60 seconds, slightly shaking to completely dissolve the main body gel dissolving solution, standing for 1-2 minutes, and absorbing 0.045mL of the dissolved main body gel solution by using the disposable sterile injector for later use. And (3) sucking a marked amount of catalyst solution by using a disposable sterile syringe, injecting the catalyst solution into the catalyst freeze-dried powder, slightly shaking to completely dissolve the catalyst solution, standing for 1-2 minutes, sucking 0.045mL of the dissolved catalyst solution by using the disposable sterile syringe, and standing for later use. Respectively injecting the main body glue solution and the catalyst solution in the prepared disposable sterile syringe into the syringe of the double-cavity liquid pusher, checking whether the syringe is loose or not, and placing for later use. When in use, the bleeding wound surface is evenly instilled, timing is started until bleeding stops, and the hemostasis time and the bleeding amount are recorded.

The experimental results are as follows:

	blank control group	Example 1	Example 2	Example 3	T-test
						Hemostasis time(s)	166±24	101±18	89±16	93±19	P<0.01
Bleeding volume (g)	1.11±0.15	0.97±0.06	0.88±0.05	0.91±0.06	P<0.01

The result shows that compared with the porcine fibrin adhesive sold in the market at present, the porcine fibrin patch has the advantages that the hemostatic effect is obviously improved, and the problem that the wound is easy to disintegrate again can be effectively solved. In the operation process, the patch is simple and convenient to use and operate, saves time and has remarkable advantages.

Although embodiments of the present invention have been shown and described, it will be appreciated by those skilled in the art that changes, modifications, substitutions and alterations can be made in these embodiments without departing from the principles and spirit of the invention, the scope of which is defined in the appended claims and their equivalents.

Claims

1. A preparation method of a pig-derived fibrin-electrostatic spinning nanofiber antibacterial hemostatic patch is characterized by comprising the following steps:

2. The preparation method of the pig-derived fibrin-electrospun nanofiber antibacterial hemostatic patch according to claim 1, characterized in that: the mass concentration range of the fibroin solution in the step (1) is 4-8%.

3. The preparation method of the pig-derived fibrin-electrospun nanofiber antibacterial hemostatic patch according to claim 1, characterized in that: the mass concentration of the polyoxyethylene solution in the step (1) is 6-10%.

4. The preparation method of the pig-derived fibrin-electrospun nanofiber antibacterial hemostatic patch according to claim 1, characterized in that: the parameters in the electrostatic spinning technology in the step (1) comprise that a spinning nozzle is a 20G-standard blunt-end stainless steel needle for injection, the flow rate of a solution is controlled to be about 0.5-1 mL/h by an injection pump, the voltage is set to be within the range of 25-30 kV, the distance between a needle head and a collecting plate is 10cm, the ambient temperature is controlled to be 25 +/-1 ℃, and the humidity is controlled to be 45 +/-5%.

5. The preparation method of the pig-derived fibrin-electrospun nanofiber antibacterial hemostatic patch according to claim 1, characterized in that: before the two components are blended in the step (2), the fibrinogen suspension comprises one or more of fibrinogen, sodium citrate, sodium chloride, albumin, arginine hydrochloride and riboflavin.

6. The preparation method of the pig-derived fibrin-electrospun nanofiber antibacterial hemostatic patch according to claim 5, characterized in that: the fibrinogen suspension comprises 30-65% of fibrinogen, 6-15% of sodium citrate, 4-14% of sodium chloride, 11-28% of albumin, 12-47% of arginine hydrochloride, 2-5% of riboflavin and the balance of absolute ethyl alcohol.

7. The preparation method of the pig-derived fibrin-electrospun nanofiber antibacterial hemostatic patch according to claim 1, characterized in that: and (3) before the two components are blended in the step (2), the thrombin suspension comprises thrombin, calcium chloride and mannitol.

8. The preparation method of the pig-derived fibrin-electrospun nanofiber antibacterial hemostatic patch according to claim 7, characterized in that: the concentration range of each component in the thrombin suspension is 500-.

9. The preparation method of the pig-derived fibrin-electrospun nanofiber antibacterial hemostatic patch according to claim 1, characterized in that: the blending suspension of the porcine fibrinogen and the thrombin in the step (2) contains fibrinogen and thrombin particles, and the Folk Ward average diameter of the particles is 30-120 mu m.

10. The preparation method of the pig-derived fibrin-electrospun nanofiber antibacterial hemostatic patch according to claim 1, characterized in that: and (3) uniformly mixing the fibrinogen suspension in the step (2) by adopting a stirring and homogenizing method, wherein the temperature is controlled to be 2-10 ℃.

11. The preparation method of the pig-derived fibrin-electrospun nanofiber antibacterial hemostatic patch according to claim 1, characterized in that: and (3) uniformly mixing the thrombin suspension in the step (2) by adopting a stirring and homogenizing method, wherein the temperature is controlled to be 2-10 ℃.

12. The preparation method of the pig-derived fibrin-electrospun nanofiber antibacterial hemostatic patch according to claim 1, characterized in that: and (3) uniformly mixing the porcine fibrinogen and thrombin blended suspension in the step (2) by adopting a stirring and homogenizing method, wherein the temperature is controlled to be 2-10 ℃.

13. The preparation method of the pig-derived fibrin-electrospun nanofiber antibacterial hemostatic patch according to claim 1, characterized in that: the alcohol and/or ketone solution in the step (2) is absolute ethyl alcohol, isobutyl alcohol, isopropanol or acetone.

14. The preparation method of the pig-derived fibrin-electrospun nanofiber antibacterial hemostatic patch according to claim 1, characterized in that: the content of fibrinogen in the blending suspension of the porcine fibrinogen and the thrombin in the step (2) is 30-80 mg/mL.

15. The preparation method of the pig-derived fibrin-electrospun nanofiber antibacterial hemostatic patch according to claim 1, characterized in that: the content of the thrombin in the blending suspension of the porcine fibrinogen and the thrombin in the step (2) is 500-1000 I.U./mL.

16. The preparation method of the pig-derived fibrin-electrospun nanofiber antibacterial hemostatic patch according to claim 1, characterized in that: the blended suspension needs to be uniformly distributed during spraying or soaking in the step (3), and the temperature is controlled to be 2-10 ℃; controlling the relative humidity of the atmosphere to be 60-85 percent; the coated carrier film is kept at said humidity for a period of at least 1-2 h.

17. A preparation method of a pig-derived fibrin-electrostatic spinning nanofiber antibacterial hemostatic patch is characterized by comprising the following steps: the polyethylene oxide in step (1) of claim 1 may be replaced with hyaluronic acid or chitosan.

18. The pig-derived fibrin-electrospun nanofiber antibacterial hemostatic patch prepared by the preparation method of any one of claims 1-17.