CN116019968A - Albumin medical tissue adhesive and preparation method and application thereof - Google Patents

Abstract

The invention discloses a medical tissue adhesive containing recombinant human albumin and a preparation method thereof, belonging to the field of biomedical materials. The recombinant human albumin medical tissue adhesive is crosslinked and connected through the non-covalent effect of the recombinant human albumin and polyethylene glycol, and has stronger interface cohesive force and cohesive force; the preparation process is simple, has good biocompatibility and biodegradability, can play the roles of noninvasive adhesion and timely sealing, and is suitable for skin beauty repair and postoperative wound healing.

Description

Albumin medical tissue adhesive and preparation method and use

technical field

The invention relates to the field of novel biomaterials, in particular to an albumin medical tissue adhesive, a preparation method and application.

Background technique

Medical tissue adhesives have attracted extensive attention in the medical field due to their significant advantages such as easy operation, non-invasive bonding, instant sealing and hemostasis, and effective shortening of operation time, and have gradually become an effective auxiliary or replacement for traditional surgical sutures. means. At present, a variety of commercialized medical tissue adhesives based on various materials have been approved for the repair and regeneration of soft tissues such as the heart, blood vessels, and lungs. However, most of the existing commercially available medical tissue adhesives still face challenges such as insufficient adhesive strength, single function, expensive raw materials, poor biocompatibility, and potential safety hazards. The need for adhesives is extremely urgent.

Albumin is one of the most important proteins in animal plasma, which can maintain the body's nutrition and osmotic pressure. Currently, a variety of albumin-based medical tissue adhesives have been developed. BioGlue (CryoLife Inc.) is an FDA-approved albumin adhesive for vascular injury repair during open surgery on large vessels such as the aorta, femoral artery, and carotid artery. Bioglue is made of albumin and glutaraldehyde. In practice, the adhesive strength of the adhesive can be optimized by changing the ratio and concentration of the two components [Journal of Cardiac Surgery, 2003, 18(6): 500-503. ]. However, since albumin is usually extracted from animals, it may cause a series of allergic reactions and some blood-borne diseases, which has potential safety hazards and limits its wide application.

PEG-based medical tissue adhesives are mainly composed of chemically modified linear or branched PEG molecules [Advanced Materials, 2019, 31(49):e201905761; Biomacromolecules, 2018, 19(3):980-988]. Coseal glue (Cohesion Technologies, Inc.) is a PEG adhesive approved by the FDA for sealing vascular wounds. imidate modification, another PEG end group is modified by thiol. After the two are mixed through a double-barreled syringe, a chemical reaction occurs between the sulfhydryl group and the carbonyl group of the succinimidyl ester to form a cross-linked network [Journal of Biomedical Materials Research, 2001, 58(5):545-555]. Duraseal glue (Covidien Inc.), another FDA-approved PEG-based adhesive consisting of PEG ester and trilysine amine solution, is commonly used to seal incisional anastomoses after neurosurgery to prevent leakage of cerebrospinal fluid [Journal of Cardiac Surgery, 2003, 18(6):504-506]. PEG-based medical tissue adhesives have the advantages of fast curing rate, strong adhesion to biological surfaces, good biocompatibility, and almost no inflammatory response, but their equilibrium swelling rate is high (greater than 400%) It may cause severe compression on the surrounding nerves and blood vessels of the tissue; at the same time, the excessively swollen hydrogel will also lose part of its mechanical strength, and may not be able to maintain its own structural integrity in the later stage of wound repair [Chemical Society Reviews, 2015, 44(7): 1820-1835].

To sum up, at present, all kinds of medical tissue adhesives, including the commercialized adhesives that have been approved by the FDA, have more or less defects, such as insufficient bonding strength, poor viscoelasticity at the bonding site, and poor function. Single, potential security risks, etc. The current challenges in clinical application and scientific research mainly include: ①Most commercialized adhesives have a single function and cannot provide more auxiliary therapeutic effects while achieving wound adhesion, which limits their wide application in clinical medicine Application; ②Its own mechanical strength is not enough, and it is urgent to improve the elasticity, toughness and deformation resistance of the adhesive body while ensuring high-strength interface adhesion, so as to achieve firm adhesion in a high dynamic physiological environment; ③Complicated The rich blood and interstitial fluid in the physiological environment hinder the rapid, firm and lasting adhesion of the adhesive to the biological tissue, and it is extremely difficult to adhere to the wet tissue surface. Therefore, there is an urgent need to develop medical tissue adhesives with high mechanical strength, good bonding performance, and high biological safety.

Contents of the invention

Purpose of the invention: In order to provide albumin medical tissue adhesive with better effect and its preparation method and application, see the multiple substantive technical effects in the specific implementation part for the specific purpose.

In order to achieve the above object, the present invention takes the following technical solutions:

An albumin medical tissue adhesive, characterized in that the adhesive includes recombinant human albumin, polyethylene glycol or polyethylene glycol derivatives.

The further technical solution of the present invention is that the recombinant human albumin and polyethylene glycol are cross-linked through non-covalent interaction; the mass ratio of the recombinant human albumin to the polyethylene glycol is (35-25): (25-35); the non-covalent interaction is at least one of hydrogen bond interaction, hydrophobic interaction and van der Waals interaction.

The further technical solution of the present invention is that the recombinant human albumin is recombinant human serum albumin obtained by genetic engineering technology, recombinant human serum albumin or a mixture of the two.

The further technical solution of the present invention lies in that the genetic engineering technology refers to recombinant human serum albumin obtained by using Pichia pastoris, transgenic animal and plant heterologous expression system, recombinant human serum albumin or a mixture of the two.

The further technical solution of the present invention lies in that the ratio of the number of free sulfhydryl groups contained in the recombinant human albumin is not less than 80% of the number of free radicals in the whole.

The further technical solution of the present invention is that the molecular weight of the polyethylene glycol or polyethylene glycol derivative is 200-4000; the polyethylene glycol is one of PEG200, PEG400, PEG600, PEG800, PEG1000, PEG2000, PEG4000 or several combinations.

The method for preparing the albumin medical tissue adhesive described in any one of the above is characterized in that it comprises the following steps: (1) according to the composition described in any one of the above, the recombinant human albumin is dissolved to obtain recombinant human albumin solution; (2) adding the polyethylene glycol to the recombinant human albumin solution in step (1); (3) cooling to obtain the albumin medical tissue adhesive.

The further technical solution of the present invention lies in that in step (1), the dissolving temperature of the recombinant human albumin is <60°C; in step (3), the temperature of dissolving polyethylene glycol is <60°C.

Use of recombinant human albumin, polyethylene glycol or polyethylene glycol derivatives, or albumin medical tissue adhesives as described above in the preparation of medical repair materials or cosmetic repair materials.

The preparation method of albumin medical tissue adhesive is characterized in that, any one of the following methods is adopted:

The first:

(1) Weigh 35g of recombinant human albumin (rHSA) and dissolve it in 100mL of aqueous solution to prepare an rHSA aqueous solution with a mass volume concentration of 35%, and stir the mixed solution slowly at room temperature to form a uniform solution;

(2) Add 25 g of polyethylene glycol (PEG) to the above-mentioned 35% rHSA aqueous solution in batches under heating and stirring, and the heating temperature is less than 60° C. until the PEG is completely dissolved;

(3) cooling to room temperature to obtain rHSA-PEG binder;

The second type:

(1) Weigh 33g of recombinant human albumin (rHSA) and dissolve it in 100mL of aqueous solution to prepare an rHSA aqueous solution with a mass volume concentration of 33%, and stir the mixed solution slowly at room temperature to form a uniform solution;

(2) Add 27 g of polyethylene glycol (PEG) to the above 33% rHSA aqueous solution in batches under heating and stirring, and the heating temperature is less than 60° C. until the PEG is completely dissolved;

(3) cooling to room temperature to obtain rHSA-PEG binder;

The third type:

(1) Weigh 30 g of recombinant human albumin (rHSA) and dissolve it in 100 mL of aqueous solution to prepare an rHSA aqueous solution with a mass volume concentration of 30%, and stir the mixed solution slowly at room temperature to form a uniform solution;

(2) Add 30 g of polyethylene glycol (PEG) to the above-mentioned 30% rHSA aqueous solution in batches under heating and stirring, and the heating temperature is less than 60° C. until the PEG is completely dissolved;

(3) cooling to room temperature to obtain rHSA-PEG binder;

The fourth type:

(1) Weigh 27g of recombinant human albumin (rHSA) and dissolve it in 100mL of aqueous solution to prepare an rHSA aqueous solution with a mass volume concentration of 27%, and stir the mixed solution slowly at room temperature to form a uniform solution;

(2) Add 33 g of polyethylene glycol (PEG) to the above-mentioned 27% rHSA aqueous solution in batches under heating and stirring, and the heating temperature is less than 60° C. until the PEG is completely dissolved;

(3) cooling to room temperature to obtain rHSA-PEG binder;

The fifth type:

(1) Weigh 25g of recombinant human albumin (rHSA) and dissolve it in 100mL of aqueous solution to prepare an rHSA aqueous solution with a mass volume concentration of 25%, and stir the mixed solution slowly at room temperature to form a uniform solution;

(2) Add 35 g of polyethylene glycol (PEG) to the above-mentioned 25% rHSA aqueous solution in batches under heating and stirring, and the heating temperature is less than 60° C. until the PEG is completely dissolved;

(3) cooling to room temperature to obtain rHSA-PEG adhesive.

The present invention adopting the above technical scheme has the following beneficial effects compared with the prior art: 1. The albumin medical tissue adhesive of the present invention is cross-linked through physical non-covalent action, and the adhesive has strong interfacial bonding force and molecular cohesion. 2. The albumin medical tissue adhesive of the present invention has good biocompatibility and biodegradability. 3. The preparation process of the albumin medical tissue adhesive of the present invention is simple and can realize expanded production. 4. The albumin medical tissue adhesive of the present invention can be used for skin cosmetic repair and postoperative wound healing.

Detailed ways

In order to make the object, technical solution and technical effect of the present invention clearer, the present invention will be further described in detail below in conjunction with specific embodiments. It should be understood that the specific implementations described in this specification are only for explaining the present invention, not for limiting the present invention. The present invention will be described in further detail below in conjunction with specific embodiments.

The purpose of the present invention is to provide a medical tissue adhesive with high mechanical strength, good bonding performance and high biological safety and a preparation method thereof. The adhesive adopts albumin as a main raw material to improve the problem of low mechanical strength caused by excessive swelling of the polyethylene glycol adhesive during use. The adhesive uses recombinant human albumin prepared by genetic engineering technology, which contains a higher proportion of free sulfhydryl groups than the currently widely used blood-derived albumin, which can increase the degree of cross-linking with human tissues and itself during the use of the adhesive. Moreover, the use of recombinant human albumin can avoid potential safety hazards of infection with blood-borne diseases. The preparation process of the adhesive is simple in operation and controllable in quality.

A preparation method of the above-mentioned albumin medical tissue adhesive, comprising the steps of:

(1) According to the composition of the above-mentioned albumin medical tissue adhesive, the above-mentioned recombinant human albumin is dissolved to obtain a recombinant human albumin solution;

(2) adding the above-mentioned polyethylene glycol to the recombinant human albumin solution described in step (1);

(3) cooling at room temperature to obtain albumin medical tissue adhesive.

When adding polyethylene glycol in the above step (2), add it in batches; preferably, add polyethylene glycol in the above step (2) in 5-15 times; more preferably, add polyethylene glycol in the step (2) in 8-10 times.

Preferably, the temperature for dissolving polyethylene glycol in the above step (3) is <60°C.

An application of the above-mentioned albumin medical tissue adhesive in the preparation of medical repair materials.

The medical repair material is skin cosmetic repair material and postoperative wound healing material.

Example 1:

In this embodiment, a kind of albumin medical tissue adhesive is prepared, and the specific process is as follows:

(1) Weigh 35g of recombinant human albumin (rHSA) and dissolve it in 100mL of aqueous solution to prepare a rHSA aqueous solution with a mass volume concentration of 35%. The mixed solution is slowly stirred at room temperature to form a uniform solution.

(2) Add 25 g of polyethylene glycol (PEG) to the above-mentioned 35% rHSA aqueous solution in batches under heating and stirring, and the heating temperature is lower than 60° C. until the PEG is completely dissolved.

(3) cooling to room temperature to obtain rHSA-PEG adhesive.

Example 2:

In this embodiment, a kind of albumin medical tissue adhesive is prepared, and the specific process is as follows:

(1) Weigh 33g of recombinant human albumin (rHSA) and dissolve it in 100mL of aqueous solution to prepare a rHSA aqueous solution with a mass volume concentration of 33%. The mixed solution is slowly stirred at room temperature to form a uniform solution.

(2) Add 27 g of polyethylene glycol (PEG) to the 33% rHSA aqueous solution in batches under heating and stirring. The heating temperature is lower than 60° C. until the PEG is completely dissolved.

(3) cooling to room temperature to obtain rHSA-PEG adhesive.

Example 3:

In this embodiment, a kind of albumin medical tissue adhesive is prepared, and the specific process is as follows:

(1) Weigh 30 g of recombinant human albumin (rHSA) and dissolve it in 100 mL of aqueous solution to prepare a rHSA aqueous solution with a mass volume concentration of 30%. The mixed solution is slowly stirred at room temperature to form a uniform solution.

(2) Add 30 g of polyethylene glycol (PEG) to the above-mentioned 30% rHSA aqueous solution in batches under heating and stirring, and the heating temperature is lower than 60° C. until the PEG is completely dissolved.

(3) cooling to room temperature to obtain rHSA-PEG adhesive.

Example 4:

In this embodiment, a kind of albumin medical tissue adhesive is prepared, and the specific process is as follows:

(1) Weigh 27g of recombinant human albumin (rHSA) and dissolve it in 100mL of aqueous solution to prepare an rHSA aqueous solution with a mass volume concentration of 27%. The mixed solution is stirred slowly at room temperature to form a uniform solution.

(2) Add 33 g of polyethylene glycol (PEG) to the above-mentioned 27% rHSA aqueous solution in batches under heating and stirring, and the heating temperature is lower than 60° C. until the PEG is completely dissolved.

(3) cooling to room temperature to obtain rHSA-PEG adhesive.

Example 5:

In this embodiment, a kind of albumin medical tissue adhesive is prepared, and the specific process is as follows:

(1) Weigh 25g of recombinant human albumin (rHSA) and dissolve it in 100mL of aqueous solution to prepare an rHSA aqueous solution with a mass volume concentration of 25%. The mixed solution is slowly stirred at room temperature to form a uniform solution.

(2) Add 35 g of polyethylene glycol (PEG) to the above-mentioned 25% rHSA aqueous solution in batches under heating and stirring, and the heating temperature is lower than 60° C. until the PEG is completely dissolved.

(3) cooling to room temperature to obtain rHSA-PEG adhesive.

Comparative example 1:

This comparative example prepared a polyethylene glycol adhesive, the main difference from Example 1 is that no recombinant human albumin was added in this comparative example, the specific process is:

(1) Take 60g of polyethylene glycol (PEG) and dissolve it in 100mL of aqueous solution to prepare an aqueous solution of polyethylene glycol (PEG) with a mass volume concentration of 60%;

(2) The mixed solution is heated and stirred at a temperature lower than 60° C. to form a uniform solution.

(3) Cool to room temperature to obtain PEG adhesive.

Comparative example 2:

This comparative example prepared a polyethylene glycol adhesive, and the main difference from Example 1 is that blood-derived human serum albumin was used instead of recombinant human albumin in this comparative example. The specific process is as follows:

(1) Weigh 35g of human serum albumin (pHSA) and dissolve it in 100mL of aqueous solution to prepare an rHSA aqueous solution with a mass volume concentration of 35%. The mixed solution is slowly stirred at room temperature to form a uniform solution.

(2) Add 25 g of polyethylene glycol (PEG) to the above-mentioned 35% pHSA aqueous solution in batches under heating and stirring, and the heating temperature is lower than 60° C. until the PEG is completely dissolved.

(3) Cool to room temperature to obtain pHSA-PEG adhesive.

Example 6: Tensile Strength and Elongation

Take 5mL of the adhesive samples of Examples 1-5 and Comparative Examples 1-2, and apply them in a stainless steel tank with a length, width, and depth of 10cm×1cm×0.5cm. After it solidifies, carefully remove the adhesive strip from the stainless steel tank The inner peeling was taken out, and the tensile strength and elongation rate of the rubber strip were measured with a universal testing machine. The results are shown in Table 1.

Table 1 Adhesive tensile strength and elongation test results

From the results in Table 1, it can be seen that the tensile strength and elongation rate of the embodiment are significantly better than those of the control example, and have good flexibility, and the present invention further optimizes the combination of recombinant human albumin and polyethylene glycol. The dosage of fen can further improve the tensile strength and elongation of the adhesive.

Example 7: Determination of Adhesion

Take each 5mL of the adhesive samples of Examples 1-5 and Comparative Examples 1-2, apply them in a stainless steel circular groove with a diameter of 3cm and a depth of 0.5cm, and quickly put a stainless steel circle with a diameter of 1cm and a thickness of 0.1cm on the adhesive. piece, the center of the other side of the disc is fixed with a drawstring. Fix the round groove and the disc pull rope to the fixtures on both sides of the universal testing machine respectively, and measure the maximum adhesive force. The results are shown in Table 2.

Table 2 Adhesive maximum adhesion test results

As can be seen from the results in Table 2, the maximum adhesive force of the embodiment is obviously better than that of the comparative example. Recombinant human albumin and polyethylene glycol cross-link each other to form a gel, which is beneficial to increase the viscosity of the adhesive, thereby improving the adhesion between the adhesive and the wound surface.

Example 8: Adhesion test of small intestine anastomosis in rats

42 SD rats, divided into cyanoacrylate group (n-butyl α-cyanoacrylate) and test group (embodiment 1-5, comparative example 1-2) totally 7 groups, 6 in every group, male and female respectively Half. The small intestine was anesthetized by intraperitoneal injection of 10% chloral hydrate (about 0.35mL/100g), and the small intestine was subjected to traditional intestinal preparation. After the small intestine was cut with surgical scissors, it was butt-sutured for 2 stitches, and 0.5mL of adhesive was applied along the incision. A drainage tube was placed, the abdominal cavity was sutured, and feeding and observation continued. The drainage volume and drainage time were recorded. After 6 weeks, the healing was observed by dissection, and the pathological examination was performed. The results are shown in Table 3.

Table 3 Adhesion test of small intestine anastomosis in rats

Compared with the control group, the drainage time of the group of examples 1-5 was significantly reduced (P<0.01), and there was no intestinal adhesion after operation, and there were 1 case and 2 cases in the control group respectively. The embodiment group had no anastomotic leakage and no anastomotic stenosis, which indicated that the embodiment group achieved better therapeutic effect in intestinal anastomosis.

To sum up, the adhesive of the present invention has high adhesive force, can greatly reduce restenosis and adhesion, has no toxic side effects, has little stimulation to the human body, and is suitable for adhesive repair of blood vessels.

The basic principles, main features and advantages of the present invention have been shown and described above. Those skilled in the art should understand that the present invention is not limited by the above-mentioned embodiments, and what described in the above-mentioned embodiments and the description only illustrates the principle of the present invention, and the present invention also has various aspects without departing from the spirit and scope of the present invention. Variations and improvements all fall within the scope of the claimed protection.

Claims (10)

1. An albumin medical tissue adhesive, characterized in that the adhesive comprises recombinant human albumin and polyethylene glycol. 2. albumin medical tissue adhesive as claimed in claim 1, is characterized in that, described recombinant human albumin and polyethylene glycol are connected by non-covalent cross-linking; Described recombinant human albumin and described The mass ratio of polyethylene glycol is (25-35):(25-35); the non-covalent interaction is at least one of hydrogen bond interaction, hydrophobic interaction and van der Waals force interaction. 3. albumin medical tissue adhesive as claimed in claim 1, is characterized in that, described recombinant human albumin is the recombinant human serum albumin that utilizes genetic engineering technology to obtain, recombinant human serum albumin or the mixture of the two . 4. The albumin medical tissue adhesive according to claim 3, wherein the genetic engineering technology refers to the recombinant human albumin obtained by utilizing Pichia pastoris, transgenic animal and plant heterologous expression system. 5 . The albumin medical tissue adhesive according to claim 1 , wherein the proportion of the number of free sulfhydryl groups contained in the recombinant human albumin is not less than 80% of the number of free radicals in the whole. 6. albumin medical tissue adhesive as claimed in claim 1, is characterized in that, the molecular weight of described polyethylene glycol or polyethylene glycol derivative is 200-4000; Described polyethylene glycol is PEG200, One or more combinations of PEG400, PEG600, PEG800, PEG1000, PEG2000, PEG4000. 7. prepare the method for albumin medical tissue adhesive as described in any one of claim 1-6, it is characterized in that, comprise the steps: (1) according to the composition described in any one of claim 1-6, will The recombinant human albumin is dissolved with a concentration of 25-35% (m/v) to obtain a recombinant human albumin solution; (2) adding the polyethylene glycol to the recombinant human albumin in step (1) solution; (3) cooling to obtain albumin medical tissue adhesive. 8. The preparation method of albumin medical tissue adhesive as claimed in claim 7, characterized in that, in step (1), the dissolution temperature of the recombinant human albumin is <60°C; in step (3), the The temperature for dissolving polyethylene glycol is <60°C. 9. The use of recombinant human albumin, polyethylene glycol or the albumin medical tissue adhesive according to any one of claims 1-7 in the preparation of medical repair materials or cosmetic repair materials. 10. The preparation method of albumin medical tissue adhesive, characterized in that, any one of the following methods is adopted: The first: (1) Weigh 35g of recombinant human albumin (rHSA) and dissolve it in 100mL of aqueous solution to prepare an rHSA aqueous solution with a mass volume concentration of 35%, and stir the mixed solution slowly at room temperature to form a uniform solution; (2) Add 25 g of polyethylene glycol (PEG) to the above-mentioned 35% rHSA aqueous solution in batches under heating and stirring, and the heating temperature is less than 60° C. until the PEG is completely dissolved; (3) cooling to room temperature to obtain rHSA-PEG binder; The second type: (1) Weigh 33g of recombinant human albumin (rHSA) and dissolve it in 100mL of aqueous solution to prepare an rHSA aqueous solution with a mass volume concentration of 33%, and stir the mixed solution slowly at room temperature to form a uniform solution; (2) Add 27 g of polyethylene glycol (PEG) to the above 33% rHSA aqueous solution in batches under heating and stirring, and the heating temperature is less than 60° C. until the PEG is completely dissolved; (3) cooling to room temperature to obtain rHSA-PEG binder; The third type: (1) Weigh 30 g of recombinant human albumin (rHSA) and dissolve it in 100 mL of aqueous solution to prepare an rHSA aqueous solution with a mass volume concentration of 30%, and stir the mixed solution slowly at room temperature to form a uniform solution; (2) Add 30 g of polyethylene glycol (PEG) to the above-mentioned 30% rHSA aqueous solution in batches under heating and stirring, and the heating temperature is less than 60° C. until the PEG is completely dissolved; (3) cooling to room temperature to obtain rHSA-PEG binder; The fourth type: (1) Weigh 27g of recombinant human albumin (rHSA) and dissolve it in 100mL of aqueous solution to prepare an rHSA aqueous solution with a mass volume concentration of 27%, and stir the mixed solution slowly at room temperature to form a uniform solution; (2) Add 33 g of polyethylene glycol (PEG) to the above-mentioned 27% rHSA aqueous solution in batches under heating and stirring, and the heating temperature is less than 60° C. until the PEG is completely dissolved; (3) cooling to room temperature to obtain rHSA-PEG binder; The fifth type: (1) Weigh 25 g of recombinant human albumin (rHSA) and dissolve it in 100 mL of aqueous solution to prepare an rHSA aqueous solution with a mass volume concentration of 25%, and stir the mixed solution slowly at room temperature to form a uniform solution; (2) Add 35 g of polyethylene glycol (PEG) to the above-mentioned 25% rHSA aqueous solution in batches under heating and stirring, and the heating temperature is less than 60° C. until the PEG is completely dissolved; (3) Cool to room temperature to obtain rHSA-PEG adhesive.