CN113262323A

CN113262323A - Tissue factor loaded calcium carbonate particle self-propelled hemostatic dressing

Info

Publication number: CN113262323A
Application number: CN202110543342.8A
Authority: CN
Inventors: 刘承琨; 石壮; 王小强; 黄方
Original assignee: China University of Petroleum East China
Current assignee: China University of Petroleum East China
Priority date: 2021-05-19
Filing date: 2021-05-19
Publication date: 2021-08-17

Abstract

The invention discloses a method for preparing and producing a hemostatic dressing embedded with tissue factors by taking calcium carbonate as a raw material, which comprises the following steps: (1) expressing and purifying in bacteria to obtain the tissue factor with bioactivity. (2) And (2) treating phospholipid and the tissue factor obtained in the step (1) to obtain a combination of the tissue factor and the phospholipid vesicle. (3) Adding the tissue factor-vesicle combination obtained in the step (2) into a calcium chloride solution, fully and uniformly mixing, adding a sodium carbonate solution, and performing post-treatment to obtain CaCO₃A particulate powder. (4) The CaCO obtained in the step (3)₃And uniformly mixing the particle powder and the acid powder to obtain the hemostatic dressing with self-propelling capability. Tissue factor is a protein with biological activity and high coagulation ability,can start the extrinsic pathway of human coagulation cascade to stop bleeding. The prepared hemostatic dressing is in a powdery form, has self-pushing capacity, can push tissue factors to the deep part of a wound, is suitable for irregular wounds with certain depth and the conventional dressing can not effectively stop bleeding, and has wide application environment. The tissue factor is added, so that the hemostatic dressing plays a more active role in wound management, the passive hemostatic effect of the traditional hemostatic dressing in wound management is replaced, the biocompatibility is better, the degradability is strong, and the risk of secondary damage to the wound can be reduced.

Description

Tissue factor loaded calcium carbonate particle self-propelled hemostatic dressing

Technical Field

The invention relates to a hemostatic dressing with pushing capacity, which is constructed by taking calcium carbonate particles as a carrier and loading tissue factors with blood coagulation bioactivity into the calcium carbonate particles.

Background

In clinical situations (e.g. surgery and dental surgery, severe epistaxis, postpartum hemorrhage and haemophilia) uncontrolled bleeding may occur, during which the first task is to effectively control bleeding, prevent complications such as hemorrhagic shock, etc. from occurring, and endanger life. The risks of uncontrolled bleeding can be avoided by rapid delivery of therapeutic agents (e.g., clotting agents, anti-fibrinolytic agents, or growth factors) to damaged vascular tissue. Because some irregular wounds have a certain depth and a certain distance between the bleeding source and the skin surface, effective delivery of the therapeutic agent to the bleeding source cannot be achieved by an injection system or surface dressing. These agents are inherently difficult to pass through the blood upstream to the bleeding source of the wound, unless they are supplied with a driving force for the flow upstream to stop bleeding.

In recent years, the development of the hemostatic dressing is very rapid, the market demand is higher and higher, and the construction of the efficient, cheap and easy-to-store hemostatic material is necessary. Patent CN210185843U discloses a compression hemostatic dressing; patent CN211723685U discloses a liquid-absorbing expandable hemostatic dressing bag; patent CN110522945A discloses a medical bio-gel hemostatic dressing; patent CN110975001A discloses a chitosan-cellulose composite hemostatic sponge; patent CN107296976A discloses a medical multifunctional hemostatic dressing. For example, the hemostatic dressing can not meet the conditions of deep hemostasis and small injury during clearing, so that in order to solve the problems, the tissue factor with bioactivity is loaded into calcium carbonate particles, and then the hemostatic dressing with driving force is constructed.

In recent years, scientists have invested a great deal of effort and financial resources in developing wound dressings with highly effective hemostatic capabilities. The current hemostatic dressings are various in types and forms, but most of the hemostatic dressings have some conditions which cannot be met by wounds. The calcium carbonate particles are selected mainly because of the conditions of low cost, mild reaction, bubble generation due to the reaction of the calcium carbonate particles with acid and the like, and through the characteristics, a system with self-propelling capability can be constructed, so that the tissue factor can reach a bleeding source to stop bleeding. The necessary material for realizing the self-propelling capability of the hemostatic dressing also needs tranexamic acid, which is a fibrinolytic agent with good tolerance and high application value and has potential in the hemostatic application.

As a hemostatic dressing, the efficient hemostatic ability is the most important condition, in addition, the small side effect and the biodegradability of the wound are also important reference factors, meanwhile, according to a reaction product between calcium carbonate and tranexamic acid, enough driving force can be provided for particles, the purpose of conveying tissue factors to a bleeding source for hemostasis can be achieved, and the threat of uncontrolled bleeding of irregular wounds to life safety is avoided.

Disclosure of Invention

The invention aims to construct a hemostatic dressing with a driving force, which can transport medicine to a wounded tissue with a certain depth for rapid hemostasis, and the dressing is used for loading a tissue factor with bioactivity into calcium carbonate particles. The invention provides a method for stopping bleeding of a wound by the hemostatic dressing.

In order to realize the technical scheme, the preparation method of the tissue factor loaded calcium carbonate particle hemostatic dressing specifically comprises the following steps:

(1) expressing and purifying in bacteria to obtain the tissue factor with bioactivity.

(2) And (2) treating phospholipid and the tissue factor obtained in the step (1) to obtain a combination of the tissue factor and the phospholipid vesicle.

(3) And (3) adding the tissue factor-vesicle combination obtained in the step (2) into a calcium chloride solution, fully and uniformly mixing, adding a sodium carbonate solution, and performing post-treatment to obtain calcium carbonate particle powder.

(4) And (4) uniformly mixing the calcium carbonate particle powder obtained in the step (3) with acid powder to obtain the hemostatic dressing with self-propelling capability.

The plasmid used in the step (1) of the invention is pET-22b, pET-28a, and the bacterium used is Escherichia coli, but is not limited to these.

The phospholipid vesicles in the step (2) of the invention are DOPC and DOPS, but are not limited to the two; the treatment process is ultrasonic or extrusion, but not limited to these two.

The post-treatment process in step (3) related to the present invention is centrifugation, suction filtration or drying, but is not limited to these.

The acid in step (4) of the present invention is tranexamic acid or citric acid, but not limited to these two.

Compared with the prior art, the invention has the following advantages: (1) the tissue factor has high coagulation capacity as a biological coagulation factor; (2) the prepared hemostatic dressing has certain driving force, and can meet the application of irregular wounds with certain depth; (3) the preparation process is simple, the use is simple and convenient, and the cost is low; (4) the dressing can be decomposed by reaction, and is easy to remove in the later stage, so that the risk of secondary damage is avoided.

Description of the drawings:

FIG. 1 is a schematic view of tissue factor-loaded calcium carbonate microparticles according to example 1 of the present invention;

FIG. 2 is an SEM representation of tissue factor-loaded calcium carbonate microparticles of example 1 in accordance with the present invention;

FIG. 3 is an infrared spectrum of tissue factor-loaded calcium carbonate microparticles according to example 1 of the present invention;

FIG. 4 is a representation of the procoagulant activity of tissue factor loaded into calcium carbonate microparticles according to example 1 of the present invention;

FIG. 5 shows the effect of in vitro coagulation of tissue factor-loaded calcium carbonate microparticles according to example 1 of the present invention;

FIG. 6 shows the blood coagulation effect of SD rat liver by calcium carbonate microparticles loaded with tissue factor in example 1 according to the present invention.

Detailed description of the invention

The invention is further illustrated by the following specific examples and the accompanying drawings of the specification.

Example 1:

(1) expression and purification of tissue factor

A plasmid carrying a tissue factor gene (pET-22b) was transformed into bacterial cells. The cells were grown to A at 37 ℃ in ampicillin-containing medium₆₀₀To 0.6-0.8. The cultures were then moved to 25 ℃ and induced overnight with isopropyl- β -D-1-thiogalactopyranoside. The culture was centrifuged to obtain a pellet, which was then suspended in Triton X-100 reagent. After lysis with a French press and centrifugation, the supernatant was collected in a new tube. Next, the expressed tissue factor was purified from the supernatant by Ni-affinity chromatography in the presence of Tween 80.

(2) Preparation of tissue factor-integrated liposomes

The mixture of DOPC and DOPS was dried in a glass vial and then dissolved in Tris-HCl containing deoxycholate and tissue factor. The sample was stirred with the biological beads for 90min at room temperature, and then a portion of the biological bead removal detergent (deoxycholate and tween 80 for lysis of tissue factor) was added. The supernatant of the lipidated tissue factor (or called tissue factor-liposome) is collected and stored. The fluorescein isothiocyanate dye is added during the mixing of deoxycholate and tissue factor, so that the dye is wrapped in the liposome.

(3) Preparation of tissue factor-loaded calcium carbonate microparticles

Preparing 1mL of CaCl with the final concentration of 33mM₂Adding tissue factor-liposome with final concentration of 6nM into the solution, stirring, mixing well, and rapidly adding 1mL of NaCO with final concentration of 33mM₃Adding CaCl₂And (4) quickly taking out turbid liquid and carrying out suction filtration to obtain the tissue factor-loaded calcium carbonate particles. As shown in fig. 2, SEM image of calcium carbonate microparticles.

(4) Infrared spectroscopic analysis

Mixing dried calcium carbonate microparticles with dried KBr at a ratio of about 1:100(m/m), thoroughly grinding under dry conditions, and compressing the uniformly mixed sample into tablets using a manual tablet press to obtainSufficient light transmittance is ensured, and the sample after tabletting is thin and transparent. Taking a KBr sheet as a background, scanning a sample by using a Fourier transform infrared spectrometer, wherein the scanning range is 4000-^-1Resolution 2cm^-1. As shown in figure 3, the crystal form characteristic peak of the calcium carbonate particles is obvious.

(5) Characterization of procoagulant activity,

The procoagulant activity of the tissue factor released by the calcium carbonate particles is measured by using a human tissue factor chromogenic activity measuring kit and compared with the newly prepared TF-liposome. Prior to measurement, all reagents in the kit were brought to room temperature, and 70. mu.L of Assay mixture (Assay dilution 50. mu.L, FVII 10. mu.L, FX 10. mu.L) was prepared, mixed with 10. mu.L of TF sample and incubated at 37 ℃ for 30 min. mu.L of FX substrate was then added to the mixture containing the TF sample and absorbance at 405nm was recorded every 5min, a change which is proportional to TF activity. As shown in FIG. 4, the absorption at 405nm increased continuously with time, indicating that the tissue factor-liposome is released from calcium carbonate particles, and still has good activity, which can promote the coagulation cascade.

(6) In vitro coagulation

Incubating fresh heart blood of New Zealand big ear rabbit at 37 deg.C for 10min, adding 25mM CaCl₂The solution recalcifies the blood. Adding the mixture of the calcium carbonate particles loaded with the tissue factor and the acid powder into the blood after recalcification, placing the blood at 37 ℃ for incubation, slightly inclining the centrifugal tube every 10s, and judging whether the blood in the centrifugal tube is coagulated or not according to the liquidity of the solution. As shown in fig. 5, the system exhibited good hemostatic ability, with approximately 6-fold shorter clotting times compared to the control experiment.

(7) Liver hemostasis of SD rat

After anesthesia by intraperitoneal injection (10%, v/v) of chloral hydrate into the rats, the rats were fixed on the plate and the abdomen was opened with surgical scissors to expose the liver. The right lobe of the liver was then incised with a scalpel into a wound about 0.5cm in length and about 0.3cm in depth. After the wound had bleeding freely for 5s without any treatment, the blood and serum were carefully swabbed with gauze. Hemostasis was then achieved with 4mg of tissue factor loaded calcium carbonate microparticles and the time to hemostasis was recorded. As shown in figure 6, the calcium carbonate particles loaded with tissue factor have strong hemostatic ability.

Example 2:

this example was the same as example 1 except that the final tissue factor concentration in step (3) was 3 nM.

Example 3:

this example was identical to example 1 except that the final tissue factor concentration in step (3) was 9 nM.

Example 4:

this example and example 1 except that in step (3), the final concentration was 55mM CaCl₂And NaCO₃The other steps are the same.

Tests show that the in vivo and in vitro coagulation time of the tissue factor-loaded calcium carbonate microparticles prepared in example 2, example 3 and example 4 is different from that of example 1, and other performances are similar to those of example 1.

Claims

1. A preparation method of a tissue factor-loaded hemostatic dressing constructed by calcium carbonate particles is characterized by comprising the following steps:

(3) Adding the tissue factor-vesicle combination obtained in the step (2) into a calcium chloride solution, fully and uniformly mixing, adding a sodium carbonate solution, and performing post-treatment to obtain CaCO₃A particulate powder.

(4) The CaCO obtained in the step (3)₃And uniformly mixing the particle powder and the acid powder to obtain the hemostatic dressing with self-propelling capability.

2. The method for preparing a tissue factor-loaded hemostatic dressing constructed from calcium carbonate microparticles according to claim 1, wherein the plasmid is one of pET-22b and pET-28a, and the bacteria is one of Escherichia coli competent cells C41, BL21 and DH5 α, but not limited thereto.

3. The method for preparing a tissue factor-loaded hemostatic dressing constructed from calcium carbonate microparticles according to claim 2, wherein the phospholipid is one or more of Phosphatidylcholine (PC), Phosphatidylserine (PS), Phosphatidylethanolamine (PE) and glycerophosphatidic acid (PA), but not limited thereto.

4. The method for preparing a tissue factor-loaded hemostatic dressing constructed from calcium carbonate microparticles as claimed in claim 3, wherein the acid powder is one of citric acid or tranexamic acid, but not limited to the two.

5. The method for preparing the tissue factor-loaded hemostatic dressing constructed by calcium carbonate microparticles according to claim 4, wherein the tissue factor gene is embedded into the plasmid pET-28a in step (1), and the plasmid carrying the tissue factor gene is transformed into competent cells BL 21.

6. The method for preparing a tissue factor-loaded hemostatic dressing constructed from calcium carbonate microparticles according to claim 5, wherein in step (2), the treatment process is one of ultrasound or extrusion, but not limited to these two methods.

7. The preparation method of the tissue factor-loaded hemostatic dressing constructed by calcium carbonate microparticles according to claim 5 or 6, wherein in the step (2), the ultrasonic temperature is 0-80 ℃, the ultrasonic time is 20-60 minutes, and the addition amount of the biological beads is 0.35-0.5 g.

8. The method for preparing the tissue factor-loaded hemostatic dressing constructed by calcium carbonate microparticles according to claim 7, wherein the ultrasonic temperature is 60 ℃, the ultrasonic time is 30min, the adding amount of the biological beads is 0.4g, and the specific process comprises the following steps: and (3) mixing the vesicles obtained in the step (2) with the tissue factor, adding 0.05g of biological beads, oscillating for 90min by using a shaking table, adding 0.35g of biological beads, oscillating for 90min, and sucking out the solution to obtain the tissue factor-phospholipid vesicle combination.

9. The method for preparing the tissue factor-loaded hemostatic dressing constructed by calcium carbonate microparticles according to claim 7 or 8, wherein the post-treatment process in step (3) is one of centrifugation or suction filtration, but not limited to the two.

10. The method of preparing a tissue factor-loaded hemostatic dressing constructed from calcium carbonate microparticles according to any of claims 1-8, wherein the hemostatic dressing is suitable for use in any wound form for hemostasis.