CN112386737B

CN112386737B - A composition for limiting the exudation of body fluids from a wound site

Info

Publication number: CN112386737B
Application number: CN202011319422.7A
Authority: CN
Inventors: 朱新远; 金鑫; 赵鹏; 童刚生
Original assignee: Shanghai Jiaotong University
Current assignee: Shanghai Jiaotong University
Priority date: 2020-11-23
Filing date: 2020-11-23
Publication date: 2022-07-26
Anticipated expiration: 2040-11-23
Also published as: CN112386737A

Abstract

The invention provides a composition for limiting body fluid overflow, which comprises protein and assembly peptide, wherein the protein accounts for 1-75% of the total mass of the composition, and the assembly peptide accounts for 25-99% of the total mass of the composition. The composition can be used to limit the escape and spread of body fluids, such as hemostasis at wound sites. The composition has excellent mechanical property and lower cost, and is a good novel hemostatic material.

Description

Composition for limiting body fluid exudation from wound site

Technical Field

The invention belongs to the technical field of medical hemostatic materials, and particularly relates to a composition capable of limiting body fluid overflow of a wound part, which comprises protein and assembly peptide. Wherein the proteins and the assembly peptides form a multilayer nano-network structure to construct a physical barrier so as to limit the overflow and diffusion of body fluid. The invention further relates to the use of the composition for limiting the escape of body fluids at a wound site, in particular for hemostasis at a clinical surgical incision or wound site.

Background

Accidental bleeding of blood vessels or organs is common in life, and timely hemostasis can provide more rescue time for a patient, which is very important for saving the life of the patient. Among various hemostatic agents, the assembled peptide hemostatic agent has the advantages of quick effect, degradability, safety, no toxicity and the like. However, the cost of the existing assembled peptide hemostatic is generally high, and the mechanical property is not ideal.

Therefore, in view of the problems in the prior art, the invention provides a protein/assembly peptide composition capable of limiting the body fluid overflow of a wound part, wherein the overflow and the diffusion of the body fluid of the wound part are limited by forming a multi-layer nano-network structure barrier through the combination of the assembly peptide and the protein. The content of the assembled peptide is reduced, the cost is reduced, and meanwhile, the mechanical property and the hemostatic effect of the composition are obviously improved.

Disclosure of Invention

The invention provides a composition capable of limiting body fluid overflow of a wound site, which comprises protein and assembly peptide, wherein the protein accounts for 1% -75%, preferably 1% -50% of the total mass of the composition, and the assembly peptide accounts for 25% -99%, preferably 50% -99% of the total mass of the composition.

The protein is one or more of a fibrous protein or globular protein having a β -sheet structure or having a potential to form a β -sheet structure, examples of the fibrous protein being: fibroin, mucin, porcine fibrin; the globular protein is: bovine serum albumin, human serum protein, ovalbumin, albumin, transferrin and lysozyme.

The assembly peptide may be one or more structures of assembly peptides. The assembly peptide has a symmetrical structure with hydrophile and hydrophobicity and alternating positive and negative charges, can be assembled in water to form the nanofiber with a beta-folded structure, and has the amino acid number of 12-20.

An example of the assembly peptide is AcKAEAKAEAKAEAEANH ₂ (KAEA16) and AcRAAKAKADAAEANH ₂ (RKDA16), or a combination of the two, wherein K, A, E, D, R represents lysine, alanine, glutamic acid, aspartic acid and arginine, respectively.

In the protein-assembling peptide composition of the present invention, the protein and the assembling peptide may be combined in any combination, non-limiting examples of which are as follows: fibroin-KAEA 16, pig fibrin-KAEA 16, fibronectin-KAEA 16, fetal bovine serum albumin-KAEA 16, human serum albumin-KAEA 16, albumin-KAEA 16, ovalbumin-KAEA 16, transferrin-KAEA 16, lysozyme-KAEA 16; fibroin-RKDA 16, porcine fibrin-RKDA 16, fibronectin-RKDA 16, fetal bovine serum albumin-RKDA 16, human serum protein-RKDA 16, ovalbumin-RKDA 16, albumin-RKDA 16, transferrin-RKDA 16, lysozyme-RKDA 16, fibroin-KAEA 16-RKDA16, porcine fibrin-KAEA 16-RKDA16, fibronectin-KAEA 16-RKDA16, transferrin-lysozyme-KAEA 16, fibroin-human serum protein-RKDA 16, fibroin-ovalbumin RKDA16, fibroin-porcine fibrin-KAEA 16-RKDA16, fibronectin-human serum protein-KAEA 16-RKDA16, albumin-transferrin-KAEA 16-RKDA16, lysozyme-RKDA 16-RKDA16 and the like.

In the composition, the protein and the assembly peptide can form an interlaced multistage nano-network structure through interaction, so that a physical barrier capable of limiting body fluid at a wound part to overflow is formed, and the aim of blocking and stopping bleeding is fulfilled.

The barrier comprises a plurality of multi-layer nano fibers which are woven with each other, and has obviously improved mechanical properties and good hemostatic effect. For example, the storage modulus of the protein-assembly peptide composition liquid can reach 100-400 times of that of the assembly peptide liquid with the same concentration, the hemostasis time is shorter than that of a single assembly peptide hemostatic, and the fastest hemostasis time is only 10 seconds. In addition, the introduction of the cheap protein effectively reduces the dosage of the assembly peptide and the use cost.

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

The composition may be applied directly to the wound site in a dry solid powder form or applied directly to the wound site in the form of a tablet or wafer for limiting fluid egress, such as hemostasis. The compositions of the present invention may also be formulated in liquid form for application directly to the wound site, or may be transferred to a carrier to form a coating prior to transfer to the wound site.

In the case of liquid preparations, the protein content is from 0.5% to 7.5%, preferably from 0.5% to 5%, based on the total mass of the liquid preparation; the content of the assembly peptide is 0.5-7.5%, preferably 1-3%; the water content is 85% to 99%, preferably 92% to 98%.

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

The compositions of the invention have a short onset of action for limiting the escape of body fluids at the wound site, with a hemostasis time of about 10s to 63 s.

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

Compared with the prior art, the composition of the invention has the following advantages in the aspect of limiting the body fluid overflow of the wound site:

1. the protein with wide source and low price is used, the dosage of the assembly peptide is reduced, the cost is reduced, and the composition has no obvious cytotoxicity and good biocompatibility.

2. Short onset time and can meet clinical requirements such as rapid hemostasis.

3. The barrier formed in use for limiting the body fluid from escaping from the wound site has obviously enhanced mechanical properties.

4. The degradation product of the composition is amino acid which can provide nutrients for wound tissues in situ, thereby playing a role in promoting wound healing.

Drawings

FIG. 1 is a graph showing the effect of the composition prepared in comparative example 4 after treating wounds of SD rats for 30 s.

FIG. 2 is a graph showing the hemostatic effect of the composition prepared in example 25 after treating SD rat tail wound for 30 s.

FIG. 3 is a graph showing the rheology profile of the liquid of the composition prepared in example 25.

Fig. 4 is a graph of the rheology of the liquid prepared in comparative example 4.

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

Fig. 6 is a high-resolution transmission electron microscope picture of the sample prepared in comparative example 3.

Fig. 7 is a high-resolution transmission electron microscope picture of the sample prepared in comparative example 4.

FIG. 8 is a high-resolution TEM image of the liquid composition prepared in example 25.

Fig. 9 shows the results of cytotoxicity test of the composition powders prepared in example 3 and example 7 against L929 cells.

Detailed Description

The invention is now described more fully by way of example. The fibroin used in the following examples of the present invention was hydrolyzed silk, purchased from Shanghai-derived leaf Biotechnology Ltd, with a purity of > 90%. The fetal bovine serum albumin is from Shanghai Gaoshimi glass instruments, Inc., and has purity of more than 90%. Ovalbumin was from mclin reagent, biotechnology grade. The assembled peptide is customized from Nanjing peptide Biotechnology Co., Ltd, and the purity of the assembled peptide is over 96%.

Examples 1 to 24

Preparation of protein-assembled peptide composition dry powder

Weighing proteins and assembly peptides with corresponding mass according to the mass ratio of table 1 (examples 1-12) and table 2 (examples 13-24), dissolving in 10mL deionized water, dissolving for more than 30min in a vortex oscillation mode, and standing overnight to fully dissolve. Then the composition is lyophilized to obtain dry powder, and the dry powder is stored at-20 ℃ for later use.

TABLE 1

TABLE 2

Examples 25 to 45:

preparation of protein-assembling peptide composition liquid

Weighing proteins and assembly peptides with corresponding mass according to the proportion in the table 3, adding the proteins and the assembly peptides into deionized water, dissolving at 25 ℃ by using ultrasound as an auxiliary, standing overnight to fully dissolve the proteins and the assembly peptides to obtain composition liquid, and storing the composition liquid in a refrigerator at 4 ℃ for later use.

TABLE 3

Comparative examples 1 to 4

Comparative example 1 and comparative example 2 were prepared according to the methods of examples 1-24, except that comparative example 1 used only fibroin and comparative example 2 used only KAEA 16.

Comparative example 3 and comparative example 4 were prepared according to the methods of examples 25-45, except that comparative example 3 used only fibroin and comparative example 4 used only KAEA 16.

The specific composition ratios are shown in table 4.

TABLE 4

Effect test example: SD rat tail-cutting hemostasis experiment

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

SD rats aged 3 weeks and weighing about 200g were selected in several groups of three rats each. A 2mm deep wound (tail vein incision) was transected at about one quarter of the tail portion with a scalpel, and the protein-assembled peptide composition dry powder (10mg) prepared in examples 1 to 24 was rapidly applied to the wound site while recording the time required for the actual hemostatic process using a timer, and the mean value was taken and the blood extravasation state at the wound site was observed. Complete hemostasis is defined as the state where blood at the wound site is not flowing at all, and the hemostasis time is the time (seconds) taken from the application of the hemostatic material to complete hemostasis, and the results are shown in table 5.

TABLE 5

As can be seen from table 5, the composition powders (examples 1 to 4 and examples 13 to 16) containing fibrous proteins (e.g., fibroin) exhibited complete hemostasis within a wide concentration range (protein concentration 1% to 75%, assembled peptide concentration 25% to 99%) and within 21s, which was superior to the single assembled peptide powder (comparative example 2). Meanwhile, the hemostatic effect of the globular protein composition is related to the concentration of the globular protein composition, and the globular protein compositions with low concentration (1% -20%) (examples 5-6, examples 9-10, examples 17-18) have better hemostatic effect (hemostatic time is less than 30 s). When the content of the globular protein in the composition exceeds 20 percent to less than 50 percent, the complete hemostasis can still be realized within 60 s. The presence of relatively high concentrations of globular protein (50% -75%) in the composition increases the time to hemostasis, but not more than 63 seconds at the longest (examples 7-8, 11-12, 19-20), which meets the need for rapid hemostasis.

Also, when the composition comprises one or more proteins and one or more assembly peptides (examples 21-24), it still has a better hemostatic effect.

2. Tail-cutting hemostasis experiment of SD rat by protein-assembled peptide composition liquid

The protein-assembled peptide composition liquid prepared in examples 25 to 45 and the liquid prepared in comparative examples 3 to 4 were used and applied to the wound site. The hemostatic results are shown in table 6.

TABLE 6

From the above results, it can be seen that when the protein content is 0.5% to 7.5%, the assembly peptide content is 0.5% to 7.5%, and the water content is 85% to 99%, the composition liquid can complete hemostasis within 30 seconds, and the effect is superior to that of comparative example 3 and comparative example 4. Compared with the single assembled peptide hemostatic (comparative example 2) which is easy to drip at a wound site (fig. 1), the composition of the invention can form a stable gel-like barrier at the wound site (fig. 2), can be better attached to the wound site, and thus achieves better hemostatic effect. This stable fit should be associated with a multinetwork structure and enhanced mechanical properties of the protein-assembled peptide barrier.

3. Testing of the mechanical Properties of the composition liquid

The liquids prepared in example 25 (fig. 3), comparative example 4 (fig. 4) and example 34 (fig. 5) were tested for rheological properties using a micro-rheometer. As can be seen from fig. 3 and 5, the storage modulus (G') of the protein-assembled peptide composition liquid of the present invention is significantly higher than the loss modulus (G ″), indicating that the composition liquid of the present invention has good gel characteristics. In addition, the compositions of the present invention exhibited better mechanical properties than the assembled peptide liquid of comparative example 4, e.g. the G' of the fibroin-KAEA 16 composition liquid (example 25) was about 400 times that of KAEA16 liquid (comparative example 4) at the same concentration (fig. 4). Meanwhile, the storage modulus of the liquid of the fetal bovine serum albumin-KAEA 16 composition (example 34) was about 100 times that of the liquid of KAEA16 (comparative example 4) at the same concentration (FIG. 5). Good mechanical properties help to form a stable physical barrier to prevent diffusion of blood exudation due to barrier rupture.

4 characterization of composition liquid microtopography

The liquids (10 μ L) prepared in comparative example 3, comparative example 4 and example 25 were each carefully applied to a 200-mesh copper mesh with a carbon film, negatively stained with 3 wt% phosphotungstic acid, carefully washed three times with deionized water, and lyophilized. The microscopic morphology was observed under vacuum using a 200kv field emission transmission electron microscope.

It can be seen that unlike the nanofibrous structure (fig. 6 and 7) formed by single fibroin (comparative example 3) and assembled peptide (comparative example 4), the fibroin-assembled peptide composition (example 25) can be interwoven to form a multi-level nano-network structure (fig. 8), the fibroin penetrates the whole gel framework and forms a trunk-branch-like microstructure at the interface with KAEA16, which indicates that there should be some interaction between fibroin and KAEA16, which in turn forms a gel barrier with certain mechanical properties, thereby restricting the flow of body fluids (such as blood).

5 composition cytotoxicity assay

Mouse fibroblast (L929) is selected as an experimental object, the compositions prepared in the examples 3 and 7 are prepared into different concentrations by using a DMEM culture medium, and then the compositions are directly added into a 96-well plate containing adherent cells, and a certain amount of DMEM culture medium is added to the 96-well plate until the volume is 200 mu L. Each group was set up as 5 parallel groups, which were then placed in an incubator (37 ℃, 5% CO) ₂ ) The culture was carried out for 48 hours. Then 50 μ LMTT (3 wt% DMSO solution) was added to the clean bench and placed in the incubator again for 24h, after carefully blotting the medium, 200 μ l DMSO was added to each well plate and shaken for 10 min. The absorbance at 490nm of each well plate was measured using a microplate reader (A) _{Sample set} ) And the absorbance of the blank solution was measured (A) _{Control group} ). Then using the formula: cell activity (%) ═ A _{Sample set} /A _{Control group} X 100%, calculation

Cell viability was obtained relative to the blank.

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

From the above, it can be seen that the composition of the present invention can form an interlaced multistage nano-network structure, and not only physical entanglement but also certain interaction force exists between the protein skeleton and the nanofibers, so as to form a gel barrier with certain mechanical properties in a macroscopic view, so as to limit body fluid overflow at a wound site, such as hemostasis. Compared with a single assembly peptide liquid, the composition gel barrier has better mechanical property, lower use cost and better hemostatic effect. For example, the storage modulus of the liquid of the composition can reach 100-400 times of that of the liquid of the assembled peptide with the same concentration, the cost is only about 1/2-1/3 of the latter, and the fastest hemostasis time is only 10s, so that the composition has good practical clinical application significance.

Claims

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.