CN112891550A - Improved clot gel carrier and preparation method and application thereof - Google Patents
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- A61L26/00—Chemical aspects of, or use of materials for, wound dressings or bandages in liquid, gel or powder form
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- A61L27/00—Materials for grafts or prostheses or for coating grafts or prostheses
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- A61L27/00—Materials for grafts or prostheses or for coating grafts or prostheses
- A61L27/36—Materials for grafts or prostheses or for coating grafts or prostheses containing ingredients of undetermined constitution or reaction products thereof, e.g. transplant tissue, natural bone, extracellular matrix
- A61L27/3604—Materials for grafts or prostheses or for coating grafts or prostheses containing ingredients of undetermined constitution or reaction products thereof, e.g. transplant tissue, natural bone, extracellular matrix characterised by the human or animal origin of the biological material, e.g. hair, fascia, fish scales, silk, shellac, pericardium, pleura, renal tissue, amniotic membrane, parenchymal tissue, fetal tissue, muscle tissue, fat tissue, enamel
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Abstract
The invention discloses a preparation method of an improved clot gel carrier, which comprises the following steps: mixing blood and additive in container, reacting at 25-50 deg.C for 2-15 min; then, drying to obtain the improved blood clot gel carrier; wherein the additive is an accelerating component or a crosslinking component. The invention also discloses the improved clot gel carrier prepared by the method. The improved clot gel carrier has the characteristics of simple process, safety, no toxicity, stable structure, good biocompatibility, excellent mechanical property and the like.
Description
Technical Field
The invention relates to the technical field of medical biology, in particular to an improved clot gel carrier and a preparation method and application thereof.
Background
Blood is mainly composed of blood cells and plasma, which accounts for 55% of the blood content. The other 45% is mainly composed of red blood cells, white blood cells and platelets. Blood transports oxygen and nutrients in the body and removes cellular waste. Platelets in the blood can aggregate and coagulate, and when the body bleeds, the platelets aggregate together to form a clot, and the formed clot helps to stop bleeding and protect wound infection. This coagulation process involves activation, adhesion and aggregation of platelets, as well as deposition and maturation of fibrin. Soluble fibrinogen in plasma gradually converts to insoluble fibrin, which bridges the mass of red blood cells, causing the flowing blood to become a gel. The blood clot has the advantages of rich source, simple preparation, good biocompatibility and the like. It can be completely metabolized in the body without producing toxic by-products. In addition, the red blood cells in the gel also have the functions of participating in immune regulation and maintaining the body homeostasis. In recent years, blood clot hydrogels constructed from blood as a basic raw material have received great attention in the field of biomedical materials, and have a wide prospect in the fields of drug delivery carriers, stent materials, biomimetic smart materials, regenerative medicine and the like.
In the research and application of blood clots, the use of natural blood clots without modification is limited. However, the natural blood clots have certain limitations in mechanical strength, mechanical properties, adhesion, etc. The traditional blood clot gel has a long preparation process, needs to wait for blood to coagulate, and is greatly influenced by the ambient temperature. The drying time in the preparation of the clot gel needs to be accurately controlled, and the clot gel is easily not formed or embrittled due to overlong or overlong short time. In addition, although the clot gel has certain mechanical properties, the clot gel has a difference compared with the polymer hydrogel, has poor viscoelasticity, and is easy to deform and break. In order to expand the application range of the blood clot as a biomedical material, the improvement and modification of the blood clot are urgently needed to meet the requirement of practical application.
Disclosure of Invention
The invention aims to solve the technical problem of providing a preparation method of an improved clot gel carrier, and effectively solves the problems of long preparation time, insufficient viscoelasticity and toughness of clot gel by adding additives. The improved blood clot hydrogel has the characteristics of simple process, safety, no toxicity, stable structure, good biocompatibility, excellent mechanical property and the like.
In order to solve the technical problems, the invention provides the following technical scheme:
the invention provides a preparation method of an improved clot gel carrier, which comprises the following steps:
mixing blood and additive in container, reacting at 25-50 deg.C for 2-15 min; then, drying to obtain the improved blood clot gel carrier;
wherein the additive is an accelerating component or a crosslinking component.
Further, the additive comprises one or more of chitosan, polyamidoamine dendrimer, snake venom hemocoagulase, vitamin K, desmopressin acetate, factor viii, prothrombin complex, thrombin, human fibrinogen, aminomethylbenzoic acid, tranexamic acid, aprotinin, aminocaproic acid and gelatin sponge.
Further, the mass concentration of the additive is 1-30%.
Further, the additive is chitosan. Preferably, the chitosan mass concentration ranges from 1% to 10%, within which injectable gels can be formed.
Further, the blood is anticoagulated blood or fresh blood.
Further, the drying time is 0.5 to 2 hours.
Further, the drying pressure is 0.05-0.1Mpa, and the drying temperature is 37-45 ℃.
Further, the container is a sterile mold.
In a second aspect, the invention provides an improved clot gel carrier prepared by the method of the first aspect.
In a third aspect, the invention provides the use of the modified clot gel carrier of the second aspect as a drug delivery carrier, a wound dressing and a tissue engineering scaffold material.
The invention has the beneficial effects that:
(1) according to the invention, the safe and efficient procoagulant component or crosslinking component is added into the blood clot gel, so that the mechanical strength, viscoelasticity, stability and durability of the blood clot gel are improved.
(2) The improved blood clot gel has the advantages of low construction cost, easily obtained raw materials, simple process, and excellent biological safety and biocompatibility.
(3) The preparation process of the improved blood clot gel is simple, the reaction condition is mild, the performance of the prepared improved blood clot gel is controllable and adjustable by adjusting the proportional relation between the additive and the blood, the improved blood clot gel has the implantable property and the injectable property, the application range of the blood clot gel is expanded, and the improved blood clot gel can be used as a drug carrier and can also be used as a wound dressing and a tissue engineering scaffold material.
Drawings
FIG. 1 is a photograph of a natural clot gel (A) and a modified clot gel (B) of the invention;
FIG. 2 is a scanning electron micrograph of a natural clot gel (A) and a modified clot gel (B) of the present invention;
FIG. 3 is a diagram showing the coagulation process of the natural clot gel (A) and the modified clot gel (B) of the present invention;
FIG. 4 is a clotting temperature screen of the modified clot gel of the present invention;
FIG. 5 is a drying condition examination of the modified clot gel of the present invention;
FIG. 6 is a study of the in vivo stability and persistence of the modified clot gel of the present invention.
Detailed Description
The present invention is further described below in conjunction with the following figures and specific examples so that those skilled in the art may better understand the present invention and practice it, but the examples are not intended to limit the present invention.
Unless defined otherwise, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this invention belongs. The terminology used in the description of the invention herein is for the purpose of describing particular embodiments only and is not intended to be limiting of the invention. As used herein, the term "and/or" includes any and all combinations of one or more of the associated listed items.
In the following examples and comparative examples, the experimental methods used were conventional unless otherwise specified, and the materials, reagents and the like used were commercially available without otherwise specified.
Example 1: synthesis of improved clot gel
(1) 5mg of chitosan as an additive is weighed in advance and placed in a sterile mold;
(2) adding fresh blood extracted from the fundus venous plexus of the mouse into the sterile mold, and uniformly mixing;
(3) the sterile mold was reacted at 37 ℃ for 5 minutes;
(4) the sterile mold was then transferred to a vacuum oven and dried gently at 0.08Mpa, 37 ℃ for 0.5 hours to yield the modified clot gel carrier.
FIG. 1 is a photograph of a natural clot gel and a modified clot gel of the invention.
Example 2: characterization of modified clot gels
Washing the modified blood clot gel prepared in example 1 with isotonic phosphate buffer solution for 3-4 times, collecting blood clots, adding 4% paraformaldehyde for fixing, standing for 12 hours, fixing the fixed modified blood clot gel on a silicon chip, washing and dehydrating cells with 75%, 85%, 95% and 100% alcohol in sequence, maintaining each concentration gradient for 15 minutes, taking out a sample, naturally drying in air, spraying gold, and observing the treated modified blood clot gel with a scanning electron microscope.
FIG. 2 is a scanning electron micrograph of a natural clot gel and a modified clot gel of the present invention. As can be seen from the figure, in the modified clot gel, the blood cells are cross-linked to each other; in the native clot gel, however, the blood cells are not cross-linked to each other.
Example 3: prescription process primary screen for improved blood clot gel
Two representative procoagulant components, commercial chitosan and thrombin, were selected, and the control group, which was a natural non-additive clot, was used as a reference control group. Taking 8 serum bottles, respectively adding 5mg samples, taking the same amount of natural blood as a control group, taking two vials for each sample, taking mouse blood by a mouse fundus vein blood sampling method, adding 150 mu L of blood into each test tube, rapidly mixing the procoagulant component and the blood, and placing in a constant temperature heater at 37 ℃. Gently tilting the bottle 1 time every 5s until the blood no longer flows, i.e. the blood coagulates, recording the total coagulation time, calculating the coagulation time of each component, and repeating the in vitro coagulation experiment. The clotting times of the different samples were determined.
The whole blood coagulation is one of important methods for evaluating the coagulation performance of the material, and the preparation time of the blood clot improved by the additive is visually evaluated by measuring the coagulation time of the whole blood. The clotting process of the modified clot in this example is shown in FIG. 3. Uncoagulated blood, when the bottle is tilted, will flow along the inner wall. A fully coagulated blood clot will adhere firmly to the bottom of the tube. The whole blood coagulation time after adding different additives is shown in table 1, and it can be seen from the table that compared with the traditional natural blood clot gel, the blood clot coagulation time is greatly shortened after adding procoagulant additives or macromolecular cross-linking agents.
TABLE 1 evaluation of clotting of clot gels by different coagulants according to the invention
| Sample (I) | Chitosan | Thrombin | Without addition |
| Clotting time(s) | 132±9 | 124±12 | 225±5 |
| Morphology of | Normalization | Normalization | Softer, with serum deposition |
| Viscoelastic property | Good taste | Good taste | Is poor |
Example 4: improved clot gel coagulation temperature screening
Taking commercial chitosan as an example, 5mg of chitosan is weighed in advance and placed in a sterile mold, 150 μ L of fresh blood is added into the mold, the mixture is mixed evenly, the mixture is shaken gently at 4 ℃, 37 ℃ and 60 ℃ every 10s, the blood coagulation condition is observed, and the coagulation degree, the morphology and the viscoelasticity of the blood clot are evaluated after the reaction is carried out for 8 minutes, and the result is shown in fig. 4.
As can be seen from fig. 4, the lower the temperature, the longer the time for formation of the modified blood clot results. The improved blood clot has poor forming degree and appearance under the condition of low temperature in the same time. Under the environment of 37 ℃, the forming speed and the appearance of the chitosan modified blood clot gel are improved. The regular morphology is formed at high temperature, but the cell components are denatured at high temperature, the protein is inactivated, and the color of the blood clot is changed into brick red.
Example 5: drying condition screening of modified clot gels
The blood clot is used as a biological product, and the preparation and the preservation of the blood clot have certain technical properties. The natural blood clots, which are not dried, may separate out serum and affect the application. Therefore, to avoid the precipitation of serum and to affect the efficiency of drug loading at a later date, mild drying methods are used to "block" the precipitation of serum, and drying conditions have a significant impact on improving clot formation.
Taking commercial chitosan as an example, 5mg of chitosan was weighed in advance and placed in a mold, 150. mu.L of fresh blood was added thereto, mixed well, and placed in a constant temperature heater of 37 ℃. Repeating for 3 parts, drying in vacuum drying oven at 20 deg.C, 37 deg.C and 50 deg.C, and drying under 0.1MPa for one hour, and observing blood clot state. The results are shown in FIG. 5. As can be seen from the figure, the chitosan modified natural blood clot carrier prepared in a dry environment at 37 ℃ is optimal.
Example 6: in vivo stability and persistence studies of improved clot gels
An injectable modified blood clot gel is prepared by using commercial chitosan, a natural blood clot is used as a control group, about 200 mu l of the modified blood clot gel and the natural blood clot gel are injected subcutaneously into Balb/c mice, and the stability and degradation condition of the subcutaneous blood clot of the mice are observed every five days. As shown in fig. 6, the unmodified clot gel was dispersed from the distal injection site, and the modified clot gel was more stable and retained at the injection site for a longer period of time than the unmodified native clot gel.
The above-mentioned embodiments are merely preferred embodiments for fully illustrating the present invention, and the scope of the present invention is not limited thereto. The equivalent substitution or change made by the technical personnel in the technical field on the basis of the invention is all within the protection scope of the invention. The protection scope of the invention is subject to the claims.
Claims (10)
1. A preparation method of an improved clot gel carrier is characterized by comprising the following steps:
mixing blood and additive in container, reacting at 25-50 deg.C for 2-15 min; then, drying to obtain the improved blood clot gel carrier;
wherein the additive is an accelerating component or a crosslinking component.
2. The method of claim 1, wherein the additive comprises one or more of chitosan, polyamidoamine dendrimer, hemocoagulase, vitamin K, desmopressin acetate, factor viii, prothrombin complex, thrombin, human fibrinogen, aminomethylbenzoic acid, tranexamic acid, aprotinin, aminocaproic acid and gelatin sponge.
3. The method as claimed in claim 1, wherein the additive is present in a concentration of 1-30% by weight.
4. The method for preparing an improved clot gel carrier of claim 2, wherein the additive is chitosan, and the mass concentration of the chitosan is 1% -10%.
5. The method of claim 1, wherein the blood is anticoagulated blood or fresh blood.
6. The method of claim 1, wherein the drying time is 0.5-2 hours.
7. The method of claim 1, wherein the drying pressure is 0.05-0.1MPa and the drying temperature is 37-45 ℃.
8. The method of claim 1, wherein the container is a sterile mold.
9. An improved clot gel carrier prepared according to the method of any one of claims 1 to 8.
10. Use of the modified clot gel carrier of claim 9 as a drug delivery vehicle, a wound dressing and a tissue engineering scaffold material.
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| CN202110113440.8A CN112891550A (en) | 2021-01-27 | 2021-01-27 | Improved clot gel carrier and preparation method and application thereof |
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| CN202110113440.8A CN112891550A (en) | 2021-01-27 | 2021-01-27 | Improved clot gel carrier and preparation method and application thereof |
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| CN112891550A true CN112891550A (en) | 2021-06-04 |
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| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN111184684A (en) * | 2020-02-08 | 2020-05-22 | 苏州大学 | Erythrocyte gel delivery system and preparation method and application thereof |
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| Publication number | Priority date | Publication date | Assignee | Title |
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| CN111184684A (en) * | 2020-02-08 | 2020-05-22 | 苏州大学 | Erythrocyte gel delivery system and preparation method and application thereof |
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| 刘佳鑫等: "壳聚糖及其复合物在治疗下肢慢性溃疡中的研究进展", 《中国普通外科杂志》 * |
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