CN112386737B - A composition for limiting spillage of bodily fluids at a wound site - Google Patents

Abstract

The present invention provides a composition for limiting body fluid spillage, which comprises protein and assembled peptide, wherein the protein accounts for 1%-75% of the total mass of the composition, and the assembled peptide accounts for 25%-99% of the total mass of the composition. The compositions can be used to limit spillage and spread of bodily fluids, such as to stop bleeding at a wound site. The composition of the present invention has superior mechanical properties and low cost, and is a good new type of hemostatic material.

Description

A composition for limiting spillage of bodily fluids at a wound site

technical field

The present invention belongs to the technical field of medical hemostatic materials, and in particular, the present invention relates to a composition capable of restricting the overflow of body fluids at a wound site, which comprises proteins and assembled peptides. Among them, proteins and assembled peptides limit the overflow and diffusion of body fluids by forming a multi-layered nano-network structure to build a physical barrier. The present invention further relates to the use of the composition for limiting the spillage of bodily fluids from a wound site, in particular for hemostasis of a clinical surgical incision or wound site.

Background technique

Accidental bleeding of blood vessels or organs is more common in life, and timely hemostasis can provide patients with more rescue time, which is crucial to saving patients' lives. Among many hemostatic agents, assembled peptide hemostatic agents have the advantages of quick effect, degradability, safety and non-toxicity. However, the cost of the existing assembled peptide hemostatic agents is generally high, and the mechanical properties are not ideal.

Therefore, in view of the problems existing in the prior art, the present invention provides a protein/assembled peptide composition that can limit the overflow of body fluids at the wound site, and restricts the wound by the combination of the assembled peptide and the protein forming a multi-layered nano-network structure barrier. The spillage and spread of body fluids. The mechanical properties and hemostatic effect of the composition are significantly improved while reducing the usage content of the assembled peptide and reducing the cost.

SUMMARY OF THE INVENTION

The present invention provides a composition for limiting body fluid spillage at a wound site, comprising protein and assembled peptide, wherein the protein accounts for 1%-75%, preferably 1%-50% of the total mass of the composition, and the assembled peptide accounts for the 25%-99% of the total mass of the composition, preferably 50%-99%.

The protein is one or more of a fibrillar protein or a globular protein with a β-sheet structure or with a potential to form a β-sheet structure, examples of the fibrillar protein are: silk protein, cohesin, porcine fiber Protein; the globular proteins are: fetal bovine serum albumin, human serum albumin, ovalbumin, albumin, transferrin, and lysozyme.

The assembled peptide can be one or more structural assembled peptides. The assembled peptide has a symmetrical structure of hydrophilic and hydrophobic, alternating positive and negative charges, and can be assembled in water to form a nanofiber with a β-sheet structure, and the number of amino acids is 12-20.

Examples of such assembled peptides are AcKAEAKAEAKAEAKAEANH ₂ (KAEA16) and AcRARAKAKADADAEAEANH ₂ (RKDA16), or a combination of the two, wherein K, A, E, D, R represent lysine, alanine, glutamic acid, Aspartic acid and arginine.

In the protein-assembled peptide composition of the present invention, the protein and the assembled peptide can be combined arbitrarily, and non-limiting examples thereof include: silk fibroin-KAEA16, porcine fibrin-KAEA16, cohesin-KAEA16, fetal bovine serum albumin- KAEA16, human serum protein-KAEA16, albumin-KAEA16, ovalbumin-KAEA16, transferrin-KAEA16, lysozyme-KAEA16; silk protein-RKDA16, porcine fibrin-RKDA16, cohesin-RKDA16, fetal bovine Serum protein-RKDA16, human serum protein-RKDA16, ovalbumin-RKDA16, albumin-RKDA16, transferrin-RKDA16, lysozyme-RKDA16, silk protein-KAEA16-RKDA16, porcine fibrin-KAEA16-RKDA16, mucin zonulin-KAEA16-RKDA16, transferrin-lysozyme-KAEA16, silk protein-human serum protein-RKDA16, silk protein-ovalbumin RKDA16, silk protein-porcine fibrin-KAEA16-RKDA16, cohesin-human Source serum protein-KAEA16-RKDA16, albumin-transferrin-KAEA16-RKDA16, transferrin-lysozyme-KAEA16-RKDA16, etc.

In the composition of the present invention, the protein and the assembled peptide can interact to form an interwoven multi-level nano-network structure, thereby forming a physical barrier that can limit the overflow of body fluids at the wound site, so as to achieve the purpose of blocking and hemostasis.

The barrier contains multiple interwoven multi-layered nanofibers with significantly improved mechanical properties and good hemostasis. For example, the protein-assembled peptide composition liquid in the present invention has a storage modulus of 100-400 times that of the assembled peptide liquid at the same concentration, and the hemostatic time is also shorter than that of a single-assembled peptide hemostatic agent. Hemostasis time is only 10 seconds. In addition, the introduction of cheap protein effectively reduces the amount of assembled peptides and reduces the cost of use.

In addition, the composition of the present invention has no obvious cytotoxicity, has good biocompatibility, and can be used safely.

The composition can be applied directly to the wound site in the form of a dry solid powder, or applied directly to the wound in the form of a tablet or wafer for limiting the overflow of bodily fluids, such as hemostasis. The compositions of the present invention can also be formulated to be applied directly to the wound site in liquid form, or transferred to a carrier to form a coating and then transferred to the wound site.

In the case of a liquid formulation, based on the total mass of the liquid formulation, the protein content is 0.5%-7.5%, preferably 0.5%-5%; the assembled peptide content is 0.5%-7.5%, preferably 1%-3%; water The content is 85%-99%, preferably 92%-98%.

The body fluid is blood, intestinal fluid, cerebrospinal fluid, tissue fluid or bile.

The composition of the present invention has a short onset time for limiting the overflow of body fluids at the wound site, and the hemostasis time is about 10s-63s.

The present invention further relates to the use of a composition according to the present invention as a material for limiting the escape of body fluids at a wound site, in particular as a hemostatic material at a clinical surgical incision site or a wound site.

Compared with the prior art, the composition of the present invention has the following advantages in restricting the overflow of body fluids at the wound site:

1. The use of proteins from a wide range of sources and low prices reduces the amount of assembled peptides and costs, and the composition has no obvious cytotoxicity and good biocompatibility.

2. The onset time is short, which can meet the clinical needs such as rapid hemostasis.

3. The barrier formed in use to restrict the overflow of body fluids at the wound site has significantly enhanced mechanical properties.

4. The degradation products of the composition are amino acids, which can provide nutrients for wound tissue in situ, thereby promoting wound healing.

Description of drawings

Figure 1 is an effect diagram of the composition liquid prepared in Comparative Example 4 after treating SD rat wounds for 30s.

Figure 2 is a graph showing the hemostatic effect of the composition prepared in Example 25 after treating SD rat tail wounds for 30s.

FIG. 3 is a rheology graph of the composition liquid prepared in Example 25. FIG.

FIG. 4 is a rheological graph of the liquid prepared in Comparative Example 4. FIG.

FIG. 5 is a rheology graph of the composition liquid prepared in Example 34. FIG.

FIG. 6 is a high-resolution transmission electron microscope picture of the sample prepared in Comparative Example 3. FIG.

FIG. 7 is a high-resolution transmission electron microscope picture of the sample prepared in Comparative Example 4. FIG.

8 is a high-resolution transmission electron microscope picture of the composition liquid prepared in Example 25.

FIG. 9 shows the results of the cytotoxicity test on L929 cells of the composition powders prepared in Example 3 and Example 7. FIG.

Detailed ways

The present invention will be clearly and completely described by the following examples. The silk protein used in the following examples of the present invention is hydrolyzed silk, purchased from Shanghai Yuanye Biotechnology Co., Ltd., with a purity of >90%. The fetal bovine serum albumin used was from Shanghai Gaoxin Chemical Glass Instrument Co., Ltd., and the purity was >90%. Ovalbumin was from McLean's reagent, biotech grade. The assembled peptides used were customized from Nanjing Peptide Industry Biotechnology Co., Ltd., and the purity was above 96%.

Examples 1-24

Preparation of protein-assembled peptide composition dry powder

According to the mass ratios in Table 1 (Examples 1-12) and Table 2 (Examples 13-24), the corresponding mass of protein and assembled peptide were weighed, dissolved in 10 mL of deionized water, and assisted by vortexing for 30 minutes. Above, it was left to stand overnight to fully dissolve. Then, the dry powder of the composition is obtained by lyophilization, which is stored at -20°C for later use.

Table 1

Table 2

Examples 25-45:

Preparation of Liquid Protein-Assembled Peptide Compositions

According to the proportions in Table 3, weigh the corresponding mass of protein and assembled peptides, add them into deionized water, dissolve them at 25°C with the aid of ultrasound, and leave them overnight to fully dissolve to obtain the composition liquid. storage for backup.

table 3

Comparative Examples 1-4

Comparative Example 1 and Comparative Example 2 were prepared according to the methods of Examples 1-24, except that Comparative Example 1 only used silk fibroin, and Comparative Example 2 only used KAEA16.

Comparative Example 3 and Comparative Example 4 were prepared according to the methods of Examples 25-45, except that Comparative Example 3 only used silk fibroin, and Comparative Example 4 only used KAEA16.

The specific ingredient distribution ratios are shown in Table 4.

Table 4

Effect test example: SD rat tail cutting hemostatic experiment

1. Tail-cutting hemostasis experiment of protein-assembled peptide composition dry powder on SD rats

Several groups of SD rats with an age of 3 weeks and a body weight of about 200 g were selected, with three rats in each group. Use a scalpel to cut a 2 mm deep wound at about a quarter of its tail (cut the tail vein), and quickly apply the dry powder (10 mg) of the protein-assembled peptide composition prepared in Example 1-24 to the wound site, and at the same time A timer was used to record the time required for the actual hemostasis process, and the average value was obtained to observe the bleeding state of the wound. Complete hemostasis was defined as the state where the blood at the wound site did not flow at all, and the hemostasis time was the time (seconds) from the application of the hemostatic material to complete hemostasis. The results are shown in Table 5.

table 5

As can be seen from Table 5, for the composition powders containing fibrous proteins (such as silk fibroin) (Examples 1-4 and 13-16), in a wide concentration range (protein concentration 1%-75%, assembled peptides Concentration 25%-99%), complete hemostasis within 21s, which is better than a single assembled peptide powder (Comparative Example 2). At the same time, the hemostatic effect of the globulin composition is related to its concentration. Hemostatic effect (hemostasis time <30s). When the content of globulin in the composition exceeds 20% to less than 50%, the bleeding can still be completely stopped within 60s. In the presence of relatively high concentrations of globulin (50%-75%) in the composition, the hemostasis time is relatively increased, but the longest does not exceed 63 seconds (Examples 7-8, 11-12, 19-20 ) to meet the needs of rapid hemostasis.

Also, when the composition contains one or more proteins and one or more assembled peptides (Examples 21-24), it still has better hemostatic effect.

2. Tail-cutting hemostasis experiment of protein-assembled peptide composition liquid on SD rats

The protein-assembled peptide composition liquids prepared in Examples 25-45 and the liquids prepared in Comparative Examples 3-4 were used to apply to the wound site. The results of hemostasis are shown in Table 6.

Table 6

It can be seen from the above results that when the protein content is 0.5%-7.5%, the assembled peptide content is 0.5%-7.5%, and the water content is 85%-99%, the composition liquid can complete hemostasis within 30 seconds, and its The effect is better than that of Comparative Example 3 and Comparative Example 4. Compared with the single assembled peptide hemostatic agent (Comparative Example 2), which was easily dripped at the wound site (Fig. 1), the composition of the present invention can form a stable gel-like barrier at the wound site (Fig. 2), which can better Fits on the wound site for better hemostasis. This stable fit should be related to the multi-network structure and enhanced mechanical properties of the protein-assembled peptide barrier.

3. Test of the liquid mechanical properties of the composition

The rheological properties of the liquids prepared in Example 25 (FIG. 3), Comparative Example 4 (FIG. 4) and Example 34 (FIG. 5) were tested using a microrheometer. It can be seen from Figure 3 and Figure 5 that the storage modulus (G') of the protein-assembled peptide composition liquid of the present invention is significantly higher than its loss modulus (G"), indicating that the composition liquid of the present invention has good In addition, compared with the assembled peptide liquid of Comparative Example 4, the composition of the present invention showed better mechanical properties, for example, the G' of the silk fibroin-KAEA16 composition liquid (Example 25) was about the same At the same concentration, the storage modulus of the KAEA16 liquid (Comparative Example 4) was 400 times that of the KAEA16 liquid (Comparative Example 4). Meanwhile, the storage modulus of the FBS-KAEA16 composition liquid (Example 34) was about the same concentration as the KAEA16 liquid (Comparative Example 4). 100 times (Fig. 5). Good mechanical properties help to form a stable physical barrier, thereby avoiding the diffusion of blood exudation due to barrier rupture.

4. Characterization of composition liquid micromorphology

The liquids (10 μL) prepared in Example 3, Comparative Example 4 and Example 25 were carefully applied to a 200-mesh copper mesh with a carbon film, negatively stained with 3wt% phosphotungstic acid, carefully washed with deionized water three times, and frozen. Dry. Under vacuum conditions, the microscopic morphology was observed using a 200kv field emission transmission electron microscope.

It can be seen that, unlike the nanofibrous structures formed by a single fibroin (Comparative Example 3) and an assembled peptide (Comparative Example 4) (Figures 6 and 7), the fibroin-assembled peptide composition (Example 25) can be intertwined A multi-level nano-network structure was formed (Fig. 8), and the fibroin ran through the entire gel skeleton, and the junction with KAEA16 formed a trunk-branch-like microstructure, which indicated that there should be a certain interaction between fibroin and KAEA16. In turn, a gel barrier with certain mechanical properties is formed, thereby restricting the flow of body fluids (such as blood).

5 Composition Cytotoxicity Experiment

得到相对于空白组的细胞活力。Select mouse fibroblasts (L929) as the experimental object, configure the compositions prepared in Example 3 and Example 7 into different concentrations with DMEM medium, and then directly add them into a 96-well plate containing adherent cells, and then use DMEM medium. Add a certain amount of DMEM medium to 200 μL. Each group was set in 5 parallel groups, and then placed in an incubator (37°C, 5% CO ₂ ) for 48 hours. After that, 50 μL MTT (3wt% DMSO solution) was added to the ultra-clean bench, and it was put into the incubator again for 24 h. After the medium was carefully aspirated, 200 μL DMSO was added to each well plate and shaken for 10 min. Use a microplate reader to test the absorbance of each well plate at 490 nm ( _{sample group} A), and test the absorbance of the blank group solution ( _{control group} A). Then use the formula: cell viability (%)=A _{sample group} /A _{control group} ×100%, calculate

Cell viability was obtained relative to the blank group.

The results showed ( FIG. 9 ) that different concentrations of protein-assembled peptide compositions did not show significant cytotoxicity to L929 cells, and even when the concentration was as high as 2 mg/mL, L929 cells still maintained more than 80% cell viability. This indicates that the protein-assembled peptide composition of the present invention has good biocompatibility and can be safely used as wound hemostatic material.

To sum up, it can be seen that the composition of the present invention can form an intertwined multi-level nano network structure, and there is not only a physical entanglement but also a certain interaction force between the protein skeleton and the nanofiber, and then the macroscopic formation has a certain mechanical force. Performance gel barrier for limiting fluid spillage from wound sites, such as hemostasis. Compared with a single assembled peptide liquid, the composition gel barrier has better mechanical properties, lower usage cost and better hemostatic effect. For example, the storage modulus of the liquid composition of the present invention can reach 100-400 times the storage modulus of the assembled peptide liquid of the same concentration, while the cost price is only about 1/2 to 1/3 of the latter, and the fastest hemostasis is achieved. The time is only 10s, so it has good practical clinical application significance.

Claims (11)

1. A composition for limiting body fluid overflow, which comprises a protein and an assembly peptide, wherein the protein accounts for 20% -75% of the total mass of the composition, the assembly peptide accounts for 25% -80% of the total mass of the composition, the assembly peptide is assembled in water to form nanofibers with beta-sheet structures, the number of amino acids is 12-20, and the assembly peptide is AcKAEAKAEAKAEANH ₂ Or Ackaeakaeakaeakaeanh ₂ And AcRAAKADADAAEAEANH ₂ Wherein K, A, E, D, R represents lysine, alanine, glutamic acid, aspartic acid and arginine, respectively; the protein comprises one or more of the following proteins:fibroin, mucin, porcine fibrin, fetal bovine or human serum protein, ovalbumin, albumin, transferrin, and lysozyme; wherein the protein and the assembly peptide form an interlaced multistage nano-network structure through interaction.

2. The composition according to claim 1, wherein the protein accounts for 20% -50% of the total mass of the composition; the assembled peptide accounts for 50-80% of the total mass of the composition.

3. The composition according to claim 1, wherein the composition is used in the form of a coating applied directly to the wound site in dry powder, tablet, liquid form or as other carrier.

4. The composition of claim 3, wherein the tablet is in the form of a wafer.

5. The composition according to claim 3, wherein when the composition is used in liquid form, the protein content is 0.5-7.5%, the assembled peptide content is 0.5-7.5%, and the balance is water, based on the total mass of the liquid formulation.

6. The composition according to claim 5, wherein when the composition is used in liquid form, the protein content is 0.5-5% based on the total mass of the liquid formulation; the content of the assembly peptide is 1-3%; the balance being water.

7. Use of a composition according to any preceding claim for the preparation of a material for limiting the egress of bodily fluids from a wound site.

8. Use according to claim 7, wherein the body fluid is blood, intestinal fluid, cerebrospinal fluid, interstitial fluid.

9. The use of claim 7, wherein the bodily fluid is bile.

10. Use of a composition according to any of the preceding claims 1-6 for the preparation of a wound site hemostatic material.

11. The use according to claim 10, wherein the composition is used for the preparation of a clinical surgical incision site hemostatic material.

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