TWI424859B - Hemostatic dressing and method for forming the same - Google Patents

Description

Hemostatic dressing and preparation method thereof

The invention relates to a medical article and a preparation method thereof, and in particular to a hemostatic dressing and a preparation method thereof.

Hemostasis is one of the important protective mechanisms of the human body and is a complex and sophisticated system. The process of hemostasis is mainly dominated by three aspects, the coagulation factors in the blood vessel wall, platelets and plasma. According to the order of occurrence of hemostasis, it can be divided into primary hemostasis and secondary hemostasis. And the above two hemostatic effects are related to each other rather than individually independent.

The initial hemostasis mainly includes vasoconstriction and the formation of platelet plugs, which are completed by the interaction of platelets with the blood vessel wall. Subsequent hemostasis has the continuation of clotting factors, inducing a series of reactions. Finally, fibrin is produced, and the platelet plug is cross-entangled to form a stable blood clot to complete the hemostasis. It is known that human blood plasma contains a variety of blood coagulation factors. According to the theory of blood coagulation proposed by Paul Morawitz in 1905, calcium ion is known as one of the blood coagulation factors, and plays a very important role in the coagulation mechanism.

A conventional method for preparing a dressing from natural chitosan is mainly formed by dissolving chitosan as an organic weak acid and then crosslinking with a crosslinking agent. And because the surface of the traditional chitosan dressing has positively charged ions, the traditional chitosan dressing can attract platelets and achieve rapid hemostasis. However, the traditional chitosan dressing can only induce the initial hemostasis of the human body, so it is necessary to induce the subsequent hemostasis by the human body to complete the hemostasis.

In summary, one aspect of the present invention provides a hemostatic dressing and a method of preparing the same, such that the hemostatic dressing has the ability to bind to calcium ions. In the case of a large amount of bleeding in the wound, it can provide sufficient calcium ions, and induce initial hemostasis and subsequent hemostasis, thereby reducing the clotting time and reducing the shock caused by the blood loss caused by the blood loss.

According to an embodiment of the invention, a hemostatic dressing is formed by reacting at least chitosan, a crosslinking agent and a calcium ion solution. The crosslinker has at least two reactive ends for reacting with the amine group of chitosan, and at least one acidic functional group having a dissociation constant (Ka) greater than the amino group dissociation constant. The weight ratio of calcium ion solution to chitosan is 2-5.

According to another embodiment of the present invention, a method of preparing a hemostatic dressing comprises the following steps. A substrate stock solution is prepared, and the substrate stock solution is freeze-dried to a substrate. Wherein, the substrate stock solution comprises chitosan and a crosslinking agent. The crosslinker has at least two reactive ends for reacting with an amine group of chitosan and at least one acidic functional group having a dissociation constant greater than the amine dissociation constant. Next, the chitosan and the crosslinking agent in the substrate are polymerized. Finally, the calcium ions are combined with the acidic functional groups of the substrate.

Because traditional chitosan materials and calcium ions are positively charged, traditional chitosan materials cannot bind to calcium ions. Therefore, the chitosan needs to be acidified first, so that the chitosan material is negatively charged to bind to calcium ions. The acidification method of the traditional chitosan is mainly to freeze the chitosan solution in an alkaline environment, and then lyophilized with isopropanol, and then treated with 10 times of carboxylic acid of chitosan. This method is not only complicated but also requires a long reaction time.

In view of the above problems, the present invention provides a method for preparing a hemostatic dressing material, which can easily make chitosan the main structure of the hemostatic dressing material and calcium without the complicated acidification step of the conventional chitosan. Ion binding. The hemostatic dressing according to the present invention and a method of preparing the same will be described in detail below. The properties and efficacy analysis of the hemostatic dressing of the present invention are carried out in a variety of test methods.

Method for preparing hemostatic dressing material

A method for preparing a hemostatic dressing comprises the following steps. A substrate stock solution containing chitosan and a crosslinking agent is prepared, and the substrate stock solution is freeze-dried to a substrate. Next, the chitosan and the crosslinking agent in the substrate are polymerized. Finally, the calcium ions are combined with the substrate.

First, a substrate stock solution containing chitosan and a crosslinking agent is prepared. The crosslinker needs to have at least two reactive ends and at least one acidic functional group. The reactive end is used to react with the amine group of chitosan to carry out the crosslinking reaction. According to an embodiment of the invention, the reactive end is a carboxyl group.

The dissociation constant of the acidic functional group of the crosslinking agent needs to be greater than the amino group dissociation constant of the chitosan, so that the acidic functional group of the crosslinking agent can be dissociated negatively charged to bind to the calcium ion in the subsequent step. According to an embodiment of the invention, the acidic functional group is a carboxyl group. In general, the carboxyl group has a pKa value of 1.8 to 2.5, and the amine group has a pKa value of 8.7-10.7. The pKa value takes a negative exponent of the dissociation constant, so the smaller the pKa value, the larger the dissociation constant.

In addition, when the acidic functional group of the crosslinking agent is a carboxyl group, when the pH is greater than or equal to 6, the carboxyl group can be dissociated negatively, and the dissociation constant of the carboxyl group is greater than the amino group dissociation constant of chitosan. Therefore, when the pH is greater than or equal to 6, the carboxyl group dissociates to release H ⁺ , and the amine group (NH ₂ ) of chitosan becomes NH ₃ ⁺ , which is positively charged.

Since the carboxyl group can react not only with the amine group of chitosan, but also the chitosan is crosslinked, the carboxyl group can also serve as an acidic functional group in the crosslinking agent. Therefore, when the reactive terminal of the crosslinking agent and the acidic functional group are both carboxyl groups, the crosslinking agent is a polycarboxylic acid. For example, malic acid, citric acid or succinic acid. At this time, a polycarboxylic acid as a crosslinking agent can also be used to dissolve chitosan.

It is worth noting that the actual cross-linking reaction does not reach a complete reaction, and it is inevitable that some of the carboxyl groups will not react with the amine group, leaving a free carboxylate group and binding to calcium ions. Therefore, when the crosslinking agent used is a dicarboxylic acid, a part of the carboxyl groups of a part of the dicarboxylic acid molecule react with an amine group of a different chitosan molecule to cause crosslinking of the chitosan. On the other hand, the two carboxyl groups of the dicarboxylic acid molecule will have only one carboxyl group reacting with the amine group of the chitosan molecule, and the other carboxyl group will be dissociated to form the carboxylate group (COO ^- ). Therefore, when a dicarboxylic acid is used as a crosslinking agent, the dicarboxylic acid can also crosslink the chitosan at the same time and be negatively charged.

Next, the substrate stock solution is freeze-dried to a substrate. According to an embodiment of the invention, the substrate stock solution is placed at -20 ° C until the substrate stock solution freezes and solidifies. The water in the frozen substrate stock solution is removed by freeze drying to form a sponge-like substrate.

Next, the chitosan and the crosslinking agent in the substrate are polymerized. According to an embodiment of the invention, the polymerization reaction is carried out by reacting a carboxyl group of a crosslinking agent with an amine group of chitosan to form an amide bond. The temperature of the polymerization was 90 °C. The polymerization can be carried out in a vacuum or non-vacuum environment. Since the removal of water from the reaction product in a vacuum environment allows the reaction to proceed toward the product, the reaction time is shorter than in a non-vacuum environment. According to an embodiment of the invention, the polymerization time is about 6 hours under a vacuum reaction. Under the non-vacuum reaction, the polymerization time should be extended to 16-20 hours to ensure the reaction is complete.

Finally, the calcium ions are combined with the substrate. In particular, the calcium ions are combined with acidic functional groups in the substrate that are not reactive with the amine groups. The calcium ion has a concentration by weight of 2% to 5%. The calcium ion source may be a calcium chloride solution, a calcium oxide solution, a calcium hydroxide solution, a calcium carbonate solution, a calcium sulfate solution or a calcium lactate solution. Further, since the pH is greater than or equal to 6, the aforementioned acidic functional group can be dissociated negatively. Therefore, to ensure that all acidic functional groups are negatively charged, the calcium ion solution can be adjusted to a pH of 7 by adding a small amount of sodium hydroxide to increase the amount of calcium ions bound to the substrate.

According to an embodiment of the present invention, the polymerized substrate is placed in a 2% aqueous solution of calcium chloride, and the reaction is stirred for 30 minutes, and then the substrate is taken out and rinsed in deionized water for 30 minutes. The excess water in the substrate is removed by freeze drying to obtain a hemostatic dressing having calcium ions. The following figure shows the chemical structure of a substrate polymerized with chitosan and citric acid after binding to calcium ions.

In the longitudinal direction, the preparation method of the hemostatic dressing of the present invention utilizes a polycarboxylic acid to simultaneously dissolve and crosslink chitosan, and the product after cross-linking is positively charged (NH ₃ ⁺ ) and negatively charged ( COO ^- ). Thus, calcium ions may be COO ^- binding. The hemostatic dressing has the ability to bind to calcium ions.

Please refer to Table 1, which is a reactant composition and polymerization reaction conditions of various embodiments of the present invention. According to the above preparation method, each embodiment prepares a hemostatic dressing with different cross-linking agents and different concentrations of calcium ion solution,

Hemostatic dressing

According to the preparation method of the embodiment of the present invention, a hemostatic dressing material is provided, which is formed by reacting at least chitosan, a crosslinking agent and a calcium ion solution. The crosslinker has at least two reactive ends for reacting with the amine group of chitosan and an acidic functional group having a dissociation constant greater than the amine dissociation constant. The weight ratio of calcium ion solution to chitosan is 2-5. Such a hemostatic dressing comprises Examples 1-5 as shown in Table 1, but is not limited thereto.

Analysis of the nature and efficacy of hemostatic dressing

The properties of the foregoing Table 1 were analyzed for their properties and performance. It includes cytotoxicity test, functional group detection, actual calcium ion adsorption capacity, calcium ion release capacity, blood coagulation test simulating human plasma, rat tail hemostasis test, and mouse liver hemostasis test. The detailed methods and results of the various tests are described in the following paragraphs.

(1) Cytotoxicity test

The cytotoxicity test method is a contact cytotoxicity test of atopic dermatitis desensitized fabric according to ASTM F813-83 specification. The procedure of the test method was to culture L929 mouse fibroblast in a 6 cm cell culture dish. When the cells grow to a confluence, the sterilized hemostatic dressing is placed in the center of the plate. After the cells were further cultured for one day, tissue staining was performed using 2% crystal violet, and the range of staining was observed to determine whether or not the cells were cytotoxic.

In addition to the cytotoxicity of the various examples listed in Table 1 of the test, the cytotoxicity of a plurality of comparative examples was also tested. The preparation of the comparative examples was the same as in the previous examples except that the composition of the reactants used was different. Table 2 shows the results of the cytotoxicity test of each of the examples and the comparative examples, and the sample compositions of the respective examples and the comparative examples are listed.

According to the results shown in Table 2, it can be found that when the calcium chloride solution used is 5% or less, the prepared hemostatic dressing is not cytotoxic regardless of the crosslinking agent used. However, when the concentration of the calcium chloride solution used was 7%, the prepared hemostatic dressing material was already cytotoxic. It is shown that although calcium ions are coagulation factors, when the calcium ion concentration is too high, it is toxic to cells.

(2) Functional group detection

The dried potassium bromide (KBr) powder was mixed with the hemostatic dressing powder to be tested at a weight ratio of 1:60 (total weight of about 0.1 g). The powder was uniformly mixed with an agate mortar, poured into a circular (13 mm) press, and pressed into a transparent thin salt tablet at a pressure of 10,000 to 15,000 psi per square inch. The test piece used for the background test was a salt tablet pressed with pure potassium bromide powder, and the scanning range was 450-4000 cm ^-1 . Next, the infrared absorption spectrum of the hemostatic dressing sample (resolution 4 cm ^-1 , scanning 3 times) was measured using a Fourier transform infrared spectrometer (Perkin Elmer Spectrum RX1 FT-IR System, Buckinghamshire, UK). The results measured by different samples are subtracted from the background test values and plotted as a map.

The samples subjected to the functional group test included Example 4, Example 4-1, Example 5, and Comparative Example 1 (chitosan/acetic acid/5% calcium chloride solution) as shown in Table 1, Comparative Example 4 (chitosan). Fig. 1 is a schematic view showing the results of detection of functional groups of the hemostatic dressing of the present invention. Curves 100-500 represent the results of Examples 4-1, 4, 5, Comparative Example 1, and Comparative Example 4, respectively. In general, the C+O (stretch) of the carboxylate (COO ^- ) has an absorption front at 1550-1610 cm ^-1 and the CO absorption front at 1250-1300 cm ^-1 . The absorption front of C=O (stretch) of the indoleamine bond is about 1650 cm ^-1 . As can be seen from Fig. 1, the above-mentioned hemostatic dressing material has a C=O (stretch) of a carboxylate and an absorption peak of CO, and an absorption peak of a C=O (stretch) of a guanamine bond, so that the hemostatic dressing can be confirmed. It does have free citrate, and the chitosan does react with the crosslinker to form a guanamine bond.

(3) Actual calcium ion adsorption amount

The substrate formed by polymerization of chitosan and different cross-linking agents was tested by the following method, and the amount of calcium ions adsorbed by the substrate after reacting with different calcium ion solutions. After confirming the substrate after polymerization, calcium ions are surely bonded to the substrate after being immersed in the calcium ion solution.

The test method is to take 0.05 g of the substrate and place it in 50 ml of calcium chloride solution of different concentration and different pH values. The reaction was carried out for 30 minutes on a horizontal shaker. After the reaction is completed, the substrate is taken out, a certain amount of the remaining solution is taken, and the residual amount of calcium ions in the solution is analyzed by a flame ionization detector (A.A.). Then, the amount of calcium ions to be adsorbed is subtracted from the amount of calcium ions in the original calcium ion solution, and the amount of calcium ion adsorbed per unit weight of the substrate is estimated. Table 3 shows the actual calcium ion adsorption amounts of the respective examples and comparative examples.

According to the results shown in Table 3, the hemostatic dressing of the present invention is actually combined with calcium ions. The substrate of the same composition has a higher amount of calcium ions bound to the substrate with a higher concentration of the calcium ion solution for a certain period of time. However, as a result of Example 4 and Example 5, it was found that although the concentration of calcium ions used was the same, the amount of calcium ions bound to the substrate was extremely different. This is because citric acid is a tricarboxylic acid and malic acid is a dicarboxylic acid. When a tricarboxylic acid molecule is crosslinked with chitosan, it still has a free carboxylate. In addition, the polymerization reaction is incomplete, so that the tricarboxylic acid as a cross-linking hemostatic dressing material, the more free carboxylate can be combined with calcium ions. Further, from the results of Example 4 and Comparative Example 5, by adjusting the pH of the calcium ion solution to 7, the amount of calcium ions bonded to the substrate can be greatly increased.

(4) Calcium ion release ability

Take 0.1 g of the hemostatic dressing and place it in 50 ml of physiological saline. The reaction was placed on a horizontal shaker, and several solutions were taken at 5, 10, and 30 minutes, respectively, and the amount of calcium ions in the solution was analyzed by a flame ionization detector (Atomic Absorption; A.A.). In order to calculate the amount of calcium ion release per unit weight of hemostatic dressing at different times. Table 4 shows the amount of calcium ion released per unit weight (1 gram) of the hemostatic dressing of Example 4.

According to the results shown in Table 4, it was shown that the hemostatic dressing of the present invention does have the ability to release calcium ions under physiological conditions. And as time increases, the total release also increases.

(5) Simulating blood coagulation test of human plasma

Take 10 mg of hemostatic dressing in artificial plasma. After the reaction was carried out at 37 ° C for different time, the hemostatic dressing was taken out, and a commercially available test kit (coagulomemer) was used to determine the partially activated prothrombin time (APTT). Prothrombin Time (PT). Whether the hemostatic dressing has a hemostatic effect is judged by the coagulation time of the artificial plasma. The commercially available partial activation prothrombin time test kit contains Ci-Trol reagent, Actin FSL reagent and CaCl ₂ reagent, and the commercially available prothrombin time test kit contains Ci-Trol reagent and Innovin reagent, both of which are Dade Behring Marburg GmbH. It was manufactured and purchased from Sismei Sandong Instrument Co., Ltd. Table 5 shows the test results of APTT and PT of each of the examples and comparative examples.

According to the results shown in Table 5, it is shown that the hemostatic dressing of the present invention has a blood coagulation effect. Moreover, the clotting time required for the hemostatic dressing of the present invention is shorter than that of commercially available chitosan. Therefore, the coagulation effect of the hemostatic dressing which can release calcium ions is better than that of the commercially available chitosan.

(6) Rat tail hemostasis test

The test animals were ICR male rats purchased from the Animal Center of National Taiwan University Hospital. The animals weighed about 40 g during the test. The tail hemostasis test of three mice was performed in each of the examples and the comparative examples. The detailed test method is as follows.

After the rats were anesthetized with gas, the tail was excised one centimeter above the end of the tail. Take about 40 mg of hemostatic dressing and gently press on the tail section to stop bleeding. The cast material was removed every ten seconds to observe the hemostasis. And continue to use the same material without blood stains to lightly stop bleeding. Repeat this step until the hemostatic dressing is no longer stained with blood. This time point is the time required for blood. The hemostatic dressing after hemostasis is immediately weighed, and the blood volume of the dressing is calculated, and the amount of blood sucked by the dressing represents the amount of bleeding of the mouse. Table 6 shows the average hemostasis time and average blood draw amount for some representative examples and comparative examples.

According to the results shown in Table 6, it is shown that the hemostatic dressing of the present invention does have a coagulation effect on the living body. Moreover, the hemostatic dressing of the present invention can also significantly reduce the amount of bleeding of the organism during the hemostasis process.

(7) Mouse liver hemostasis test

Test animals were tested with the tail of the mouse to stop bleeding. The detailed method of the mouse liver hemostasis test is as follows. After anesthetizing the mice with a gas, the liver of the mouse was cut into a wound of about 0.5 cm. Cut the hemostatic dressing into about 1 square centimeter, directly pressurize the wound to stop bleeding, remove the hemostatic dressing every 10 seconds to observe the hemostasis state until the incision no longer seeps the blood. This time point is the hemostasis time required. Table 7 is the average hemostasis time for some representative examples and comparative examples.

According to the results shown in Table 7, it is shown that the hemostatic dressing of the present invention does have a coagulation effect when a large amount of bleeding occurs in a living body. Moreover, the hemostatic dressing of the present invention has a shorter hemostatic time than various commercially available hemostatic dressings.

As can be seen from the above tests, the hemostatic dressing of the present invention does have the ability to bind to calcium ions. When a large amount of wound bleeding occurs in a wound, sufficient calcium ions can be provided to reduce the clotting time and reduce the amount of bleeding.

100-500. . . curve

The above and other objects, features, advantages and embodiments of the present invention will become more apparent and understood.

Fig. 1 is a schematic view showing the results of detection of functional groups of the hemostatic dressing of the present invention.

100-500. . . curve

Claims (17)

A method for preparing a hemostatic dressing, the method comprising the steps of: preparing a substrate stock solution comprising a chitosan and a crosslinking agent, wherein the crosslinking agent has at least two reactive ends for An amine group reaction of the chitosan and an acidic functional group having a dissociation constant greater than the amino group dissociation constant; freeze-drying the substrate stock to form a substrate; polymerizing the chitosan in the substrate with the a crosslinking agent; and combining at least one calcium ion with the acidic functional group of the substrate. The preparation method according to claim 1, wherein the calcium ion has a concentration by weight of 2% to 5%. The production method according to claim 1, wherein the reactive terminal is a carboxyl group. The production method according to claim 1, wherein the acidic functional group is a carboxyl group. The production method according to claim 1, wherein the crosslinking agent is a polycarboxylic acid. The preparation method according to claim 5, wherein the polycarboxylic acid is malic acid, monocitric acid or monosuccinic acid. The preparation method according to claim 1, wherein the calcium ion is derived from a calcium chloride solution, a calcium oxide solution, a calcium hydroxide solution, a calcium carbonate solution, a calcium monosulfate solution or a calcium lactate solution. The preparation method according to claim 1, wherein the polymerization step is carried out in a vacuum at 90 ° C for about 6 hours. The preparation method according to claim 1, wherein the polymerization step is carried out in a non-vacuum atmosphere at 90 ° C for about 16 to 20 hours. The preparation method according to claim 1, further comprising adjusting the pH of the calcium ion solution to 7. A hemostatic dressing comprising at least the following components: a chitosan; a cross-linking agent having at least two reactive ends for reacting with an amine group of the chitosan, and a dissociation constant greater than One of the amino group dissociation constants is an acidic functional group; and a calcium ion solution, wherein the weight ratio of the calcium ion solution to the chitosan is 2-5. The hemostatic dressing of claim 11, wherein the reactive end is a carboxyl group. The hemostatic dressing of claim 11, wherein the acidic functional group is a carboxyl group. The hemostatic dressing of claim 11, wherein the crosslinking agent is a polycarboxylic acid. The hemostatic dressing of claim 14, wherein the polycarboxylic acid is malic acid, monocitric acid or monosuccinic acid. The hemostatic dressing according to claim 11, wherein the calcium ion solution is a calcium chloride solution, a calcium oxide solution, a calcium hydroxide solution, a calcium carbonate solution, a calcium monosulfate solution or a calcium lactate solution. The hemostatic dressing of claim 11, wherein the calcium ion solution has a pH of 7.