KR102744402B1 - Hemostatic agent with film and method for preparing thereof - Google Patents

Abstract

The present invention relates to a material for hemostasis in the form of a film having hydrophilicity, characterized in that the film surface is hydrophilized to have hydrophilicity and includes a porous structure, and a material for hemostasis in the form of a film having a film surface hydrophilized to have hydrophilicity and including a porous structure, in which plasma is loaded into the porous structure of the film, and a method for manufacturing the same. The polymer film having a leaf-leaf laminated porous structure according to the present invention exhibits excellent mechanical strength and therefore can sufficiently serve as a support during hemostasis, and since it has the advantage of a large surface area due to its unique porous structure, it is easy to load plasma containing platelets that play a role in coagulating blood, and the polymer film having a porous structure loaded with plasma can overcome the limitations of conventional materials for hemostasis and materials for hemostasis manufactured using polymers, and can be used as a more effective material for hemostasis.

Description

{HEMOSTATIC AGENT WITH FILM AND METHOD FOR PREPARING THEREOF}

The present invention relates to a film-shaped hemostatic material and a method for manufacturing the same, and more particularly, to a film-shaped hemostatic material having a hydrophilic surface treated to have hydrophilicity and including a porous structure, wherein plasma is loaded onto the film-shaped hemostatic material to coagulate bleeding more quickly, and a method for manufacturing the same.

In modern times, surgical operations to treat various tissue and organ damages are rapidly increasing through advanced modern medical technology. Minor abrasions, cuts, and wounds, or bleeding from simple surgical procedures can be sufficiently controlled by the body's hemostasis. However, in cases of excessive bleeding in emergency situations such as war injuries, major accidents, and surgical operations, if the amount of bleeding exceeds 40%, it can lead to death, so hemostatic treatment is essential.

Bleeding is the leading cause of preventable death in trauma patients, second only to cerebral hemorrhage. In the case of bleeding, blood can cause infection when exposed to the outside, and can complicate the surgical procedure by obstructing the surgical field of view. At this time, hemostatic agents play a role in stopping bleeding by promoting the blood coagulation system at the bleeding site or inhibiting the fibrinolytic system by participating in the blood clotting process.

Hemostasis methods can be broadly divided into mechanical methods, hot and cold methods, and chemical methods. Mechanical methods use devices such as gauze, sutures, clamps, and clips to suppress bleeding, and hot and cold methods include cold compresses that suppress bleeding by lowering the temperature, and vascular cauterization that generates heat using electricity to cauterize blood vessels. Chemical methods use astringents, blood coagulants, and local hemostatic agents. Recently, since blood components have a negative charge, many hemostatic agents have been developed that use positively charged polymer materials or add additives that have a hemostatic effect.

Among the hemostatic agents currently used in clinical practice, when hemostasis is achieved through packing using gauze or compressed sponges, it can cause pain due to the pressure during packing, so absorbable packing is being developed to supplement this. However, absorbable packings currently on the market have poor hemostatic effects, are sold at very high prices, and have low biocompatibility.

In addition, hydrophilic hemostatic agents are being developed to absorb a lot of blood in cases of heavy bleeding, allowing for a better field of vision by absorbing the blood. Hydrophilic hemostatic agents have the advantage of being able to absorb blood more quickly and accelerate the coagulation cascade, but they may unnecessarily absorb excessive blood, which may lead to blood loss, and if the blood coagulates with the hemostatic agent, there is a risk of secondary bleeding when the hemostatic agent is removed due to the strong adhesive force between the hemostatic agent and the blood.

In addition, products currently on the market, such as Quikclot, can cause a fever reaction when they come into contact with blood. This poses a problem in that there is a risk of necrosis of surrounding tissue due to fever.

Additives that enhance the hemostatic effect of hemostatic agents include fibrin, fibrinogen, thrombin, and thromboplastin, which are blood, plasma, and tissue components of the coagulation system; gelatin sponges that absorb blood and promote the precipitation of fibrin to promote blood clotting; and oxidized regenerated cellulose that reacts with hemoglobin to promote blood clotting.

Meanwhile, as a prior art technology, Japanese Patent Publication No. 24095731 proposes a bioabsorbable medical material having a two-layer sheet structure in which one side is formed of a bioabsorbable film layer and the other side is formed of a bioabsorbable porous layer, which has the effects of hemostasis and adhesion prevention by closely contacting a wound.

Also, Korean Patent No. 10-1832518 proposes a hemostatic porous composite sponge, a method for producing the sponge, a method for treating a wound, and a kit for producing a wound cover comprising a sponge and a buffer solution, the sponge comprising i) a matrix of a biomaterial, and ii) a single hydrophilic polymer component comprising an electrophilic reactive group, wherein the hydrophilic polymer component is a hydrophilic cross-linker, and i) and ii) are combined with each other such that the reactivity of the polymer component is maintained, wherein the polymer component is coated on the surface of the matrix of the biomaterial, or the matrix is impregnated with the polymer component, or the polymer component is coated on the surface of the matrix of the biomaterial and the matrix is impregnated with the polymer component.

Although many studies are being conducted to develop a hemostatic material that is absorbable but does not absorb excessive blood and improves the hemostatic effect while not causing secondary problems, it is still difficult to effectively show the hemostatic effect in clinical applications. Therefore, a manufacturing method with a simple manufacturing process that can overcome these limitations and improve functionality is needed.

Japanese Publication Patent 24095731 Korean Patent Registration No. 10-1832518

The purpose of the present invention is to provide a material for hemostasis in the form of a film having a porous structure of a leaf-leaf laminate, by manufacturing a polymer film having a hydrophilic structure and treating the surface thereof to make it hydrophilic; or a material for hemostasis in the form of a film capable of hemostasis at a bleeding site by loading plasma into the porous structure of the porous polymer film to which the hydrophilicity has been provided.

In addition, another object of the present invention is to provide a film-shaped hemostatic material having the hydrophilic property and a method for manufacturing a film-shaped hemostatic material loaded with plasma.

A material for hemostasis in the form of a film having hydrophilicity according to one embodiment of the present invention has a film surface that is hydrophilically treated to have hydrophilicity and may include a porous structure.

According to another embodiment of the present invention, a material for hemostasis in the form of a film loaded with plasma has a film surface that is hydrophilically treated to have hydrophilicity, and plasma can be loaded into the porous structure of the film including a porous structure.

In each of the above embodiments, it is preferable that the film has a porous structure of a layered leaf-like structure, such as a layer of fallen leaves, on its surface and from 100 to 150 μm therefrom, and has a non-porous structure from the depth to the opposite surface.

The material for hemostasis in the form of a film having hydrophilicity according to one embodiment of the present invention is characterized in that, even after hydrophilization treatment, the porous structure of a layered leaf-like layer, such as that in which fallen leaves are laminated and piled, is maintained on the surface and within 100 to 150 μm therefrom.

According to one embodiment of the present invention, the hydrophilic-treated film may have a moisture absorption rate of 80% or more after hydrophilic treatment.

According to one embodiment of the present invention, the film is preferably a biocompatible polymer having a melting point (mp) of 40 to 300°C and a crystallinity of 5 to 99%.

According to one embodiment of the present invention, the film preferably comprises at least one biocompatible polymer selected from the group consisting of polyethylene, polydioxanone, polycaprolactone, polypropylene, polylactic acid, polyglycolic acid, and polyphosphorester.

The film-shaped hemostatic material having the above hydrophilicity can absorb bleeding blood into the porous structure of the leaf-laminated layer during bleeding, and reduce the amount of bleeding through blood coagulation by the absorbed blood coming into contact with a large surface area, thereby causing a hemostatic effect.

According to one embodiment of the present invention, the plasma may be at least one selected from platelet-rich plasma (PRP) and platelet-poor plasma (PPP).

The above plasma can be adsorbed or coated on the porous structure of the above polymer film.

The above plasma may be loaded to promote blood clotting at the site of bleeding.

According to one embodiment of the present invention, the film-shaped hemostasis material loaded with plasma can absorb blood between the porous structures, and blood coagulation can be activated immediately when the absorbed blood meets the plasma loaded in the porous structure.

The method for manufacturing a hemostatic material in the form of a film having hydrophilicity of the present invention may include a step of manufacturing a two-dimensional polymer film using a biocompatible polymer having a melting point (mp) of 40 to 300°C and crystallinity, a step of introducing a porous structure into the manufactured two-dimensional polymer film, and a step of providing hydrophilicity by hydrophilizing the surface of the polymer film.

It is preferable that the above hydrophilic treatment be performed until the moisture absorption rate of the polymer film after the hydrophilic treatment becomes 80% or more.

In addition, the method for manufacturing a film-shaped material for hemostasis loaded with plasma of the present invention may include a step of manufacturing a two-dimensional polymer film using a biocompatible polymer having a melting point (mp) of 40 to 300°C and crystallinity, a step of introducing a porous structure into the manufactured two-dimensional polymer film, a step of imparting hydrophilicity to the surface of the polymer film by hydrophilization treatment, and a step of loading plasma into the porous structure of the hydrophilization-treated polymer film.

In each of the above manufacturing methods, the polymer film may be manufactured using a 1 to 50 wt% polymer solution, or may be manufactured by applying heat and pressure to the polymer.

The introduction of a porous structure into the above two-dimensional polymer film is preferably accomplished by applying heat while bringing the two-dimensional polymer film into contact with a poor solvent composed of one or more mixtures selected from among tetraglycol, 1-methyl-2-pyrrolidinone (NMP), triacetin, benzyl alcohol, and dimethyl sulfoxide (DMSO).

The conditions for applying heat when manufacturing a polymer film according to one embodiment of the present invention can be at a temperature of 40 to 300°C.

In addition, it is preferable that the plasma be loaded by impregnating the porous polymer film with plasma and then applying positive and negative pressures so that the plasma can be adsorbed on the surface and inside of the leaf-leaf laminate structure.

The polymer film having a leaf-laminated porous structure according to the present invention exhibits excellent mechanical strength, so that it can sufficiently serve as a support during hemostasis, and its structure also has a leaf-laminated porous structure formed to a certain depth and a smooth non-porous structure from the depth to the opposite surface, so that unnecessary excessive absorption of blood can be avoided when used as a material for hemostasis.

Therefore, in the present invention, the polymer film having a unique leaf-laminated porous structure can be effectively used as a material for hemostasis simply by imparting hydrophilicity to its surface.

In addition, the polymer film having a porous structure according to the present invention has the advantage of a large surface area due to its unique porous structure, making it easy to load plasma containing platelets that play a role in coagulating blood, and the polymer film having a porous structure loaded with plasma can be used as a more effective material for hemostasis, overcoming the limitations of conventional hemostasis materials and hemostasis materials manufactured using polymers.

As a result, the film-shaped hemostasis material according to the present invention has the effect of easily loading a large amount of plasma capable of hemostasis on its surface due to the large surface area formed through surface modification (hydrophilic treatment). In addition, the porous structure refers to empty space existing between the leaf-leaf laminate structures, and due to this structural characteristic, a sufficient amount of blood can be absorbed after hydrophilic treatment, so that it can be effectively used as a hemostasis material.

Figure 1 is a drawing showing a manufacturing process for imparting hydrophilicity to a porous polymer film having a leaf-laminated structure according to Example 1 of the present invention.
Figure 2 is a photograph comparing the difference in the degree of hydrophilization between the porous polymer film having a leaf-leaf laminate structure of Example 1 and the porous polymer film having a leaf-leaf laminate structure that has not been hydrophilized of Comparative Example 1.
Figure 3 is an SEM surface photograph of a porous polymer film having a leaf-leaf laminate structure of Example 1 and a porous polymer film having a leaf-leaf laminate structure that has not been hydrophilicized of Comparative Example 1.
Figure 4 is a drawing showing a manufacturing process for loading plasma onto a porous polymer film with a leaf-leaf laminate structure.
Figure 5 is a SEM surface photograph of a porous polymer film with a leaf-leaf laminate structure loaded with plasma of Example 2.
Figure 6 shows the results of a cytotoxicity test to confirm the effect of the extracts of Example 1 and Control Groups 1 to 3 on cells in the above Example 2.
Figure 7 shows the results of a hemolysis assay to confirm the degree of destruction of red blood cells by the extracts of Example 2 and Control Groups 2 to 4 and Example 1.
Figure 8 shows the results of a whole blood coagulation experiment to confirm the blood coagulation effect in vitro of Example 2 and Control Groups 1 to 3 and Example 1.
Figure 9 shows the results of comparing the amount of bleeding after inflicting a wound on the liver to confirm the blood coagulation effect in vivo in Example 2, Control Groups 1 to 3, and Example 1.

The present invention is described in more detail below.

The terminology used herein is for the purpose of describing particular embodiments only and is not intended to limit the invention.

As used herein, the singular forms include the plural forms unless the context clearly dictates otherwise. Also, when used herein, the words "comprise" and/or "comprising" specify the presence of stated features, numbers, steps, operations, elements, elements and/or groups thereof, but do not preclude the presence or addition of one or more other features, numbers, operations, elements, elements and/or groups thereof.

The present invention relates to a material for hemostasis in a film form and a method for manufacturing the same.

Specifically, the film-shaped hemostatic material according to the present invention includes a film-shaped hemostatic material having hydrophilicity; or a film-shaped hemostatic material loaded with plasma, and these are described in detail below.

1. Material for hemostasis in the form of a film with hydrophilic properties

A material for hemostasis in the form of a film having hydrophilicity according to one embodiment of the present invention is characterized in that the film surface is hydrophilically treated to have hydrophilicity and includes a porous structure.

The material for hemostasis in the form of a film having the hydrophilicity described above is first manufactured by manufacturing a polymer film having a porous structure, and then hydrophilizing its surface to make it hydrophilic. Specifically, it can be manufactured through the steps of manufacturing a two-dimensional polymer film using a biocompatible polymer having a melting point (mp) of 40 to 300°C and crystallinity, introducing a porous structure into the manufactured two-dimensional polymer film, and hydrophilizing the surface of the polymer film to impart hydrophilicity.

The polymer film having the above porous structure and the method for manufacturing the same are described in detail in the applicant's Korean patent application No. 10-2021-128063, and all contents of the above patent may be incorporated as is in the present invention.

A polymer film having a porous structure according to the present invention is characterized by introducing a porous structure into a polymer film manufactured using a biocompatible polymer having a melting point (mp) of 40 to 300°C and crystallinity.

In the present invention, a biocompatible polymer that is harmless to the human body and is used in the manufacture of a polymer film so that it can be used on human tissues and has a melting point (mp) of 40 to 300°C and a crystallinity of 5 to 99% can be preferably used.

If the melting point of the biocompatible polymer is lower than 40°C, the stability at room temperature or in the body is low, and if it exceeds 300°C, excessive energy consumption and polymer decomposition are induced during the surface treatment process for introducing a porous structure, which is not desirable.

In addition, the biocompatible polymer may have a crystallinity in the range of 5 to 99%, preferably 30 to 80%. However, if the crystallinity of the biocompatible polymer is too low, less than 5%, or amorphous, or if the crystallinity is high, exceeding 99%, there is a problem that a leaf-like laminated porous structure cannot be formed, which is not preferable.

The biocompatible polymer according to the present invention satisfying the melting point and crystallinity as described above may be at least one selected from the group consisting of polyethylene, polydioxanone, polycaprolactone, polypropylene, polylactic acid, polyglycolic acid, and polyphosphorester, and among these, polycaprolactone, polydioxanone, polyethylene, etc. may be preferably used.

The polymer film according to the present invention is prepared by first dissolving a biocompatible polymer having a melting point (mp) of 40 to 300°C and crystallinity in a solvent to prepare a polymer solution, and then using a known method such as casting, the polymer solution can be prepared in the form of a two-dimensional polymer film. The concentration of the polymer solution is preferably 1 to 50 wt% in terms of controlling the thickness of the film to be formed and ease of handling the solution during the process of preparing the film.

In addition, in the present invention, without using a solvent, each polymer can be melted by applying heat and pressure to it to produce a two-dimensional polymer film.

In the present invention, the term 'two-dimensional polymer film' refers to a non-porous solid phase having a two-dimensional thickness using the polymer, and is a different concept used to distinguish it from a three-dimensional structure manufactured using 3D printing, etc., as mentioned in Patent Document 3 previously applied for by the applicant of the present invention.

In addition, in the present invention, the manufactured polymer film can be treated in a simple manner to introduce a predetermined porous structure.

The introduction of the above porous structure can be accomplished by manufacturing the above polymer film, and then applying heat while bringing the polymer film, to which the porous structure is to be introduced, into contact with a poor solvent. Specifically, when heat is applied to the polymer film to melt it, a phenomenon occurs in which the molten polymer is slightly dissolved in the poor solvent it is in contact with, and when this is cooled, the dissolved portion is deposited again on the surface of the polymer, thereby forming a leaf-pile porous structure, such as a pile of fallen leaves.

The 'poor solvent' used for introducing the above porous structure means a solvent having a solubility such that the manufactured polymer film hardly dissolves at room temperature but dissolves at a specific temperature or higher, and in the present invention, at least one selected from tetraglycol, 1-methyl-2-pyrrolidinone (NMP), triacetin, benzyl alcohol, and dimethyl sulfoxide (DMSO), or a mixture of two or more thereof, may be used.

When applying heat to the above two-dimensional polymer film, a temperature of 40°C to 300°C, which is near the melting point of the polymers used, is preferable, and when a method of dispensing a solvent onto the surface of the polymer film so that the solvent can come into contact with the surface of the polymer film to which a porous structure is to be introduced is used, the heated portion of the polymer film can be melted and then cooled to form a porous structure.

In addition, when introducing a porous structure into the polymer film, if the porous structure is introduced only to a cross-section, it goes without saying that, regardless of which of the methods listed above is used, the remaining side must be well sealed so that the porous structure is not formed.

The porous structure formed in the polymer film of the present invention is characterized by the presence of numerous independent pores, while the numerous pores are connected to each other, and having a leaf-stacked structure like a pile of numerous fallen leaves. In addition, the leaf-stacked porous structure in which numerous pores are connected to each other like the present invention has a high surface area compared to a porous structure in which numerous independent pores are not connected to each other, and thus has advantageous properties in terms of carrying a large amount of physiologically active factors, inducing sustained release of these factors, and allowing cell attachment to the surface.

In addition, a portion of the polymer film to which a porous structure is not introduced may have a smooth non-porous (dense) structure. That is, depending on the intended use, a porous structure may be introduced only to a cross-section, and even in the case where a porous structure is introduced only to a cross-section, it is preferable that a porous structure such as leaf-layered porous structures in which fallen leaves are laminated and piled is present up to a depth of a portion of the cross-section (for example, the surface and 100 to 150 μm therefrom) based on the entire thickness, and a non-porous structure is present from the depth to the opposite side.

Such a porous structure can be introduced in the present invention when a specific melting point and crystallinity are satisfied. If the polymer material used has a non-crystallization or a melting point not within the range of 40 to 300°C, even if heat is applied while a poor solvent is in contact with a two-dimensional polymer film as described above, a leaf-leaf laminated porous structure is not formed.

In addition, according to one embodiment of the present invention, a polymer film manufactured using a biocompatible polymer having a melting point range and crystallinity according to the present invention, and a polymer film having a porous structure introduced into the polymer film, has a characteristic in that the rate of change in the tensile strength value is within ±30%. This is preferable in satisfying the condition for ensuring that the polymer film according to the present invention has properties that can be handled when applied to the body.

That is, in the present invention, since the mechanical properties of the polymer film before and after introducing the porous structure are maintained almost similarly, even when the porous structure is introduced on the surface, the properties of the polymer film are not significantly reduced due to the porous structure, but are maintained similarly, thereby increasing the surface area of the film.

In addition, a polymer film having porosity can be manufactured through the above process, and then its surface can be treated to provide hydrophilicity.

The surface hydrophilization treatment of the above porous polymer film can be performed using a method such as etching the film surface with a base solution or etching with a strong acid such as perchloric acid, and the method is not particularly limited as long as it is a method capable of imparting hydrophilicity to the manufactured porous polymer film.

However, in the present invention, only a method is possible in which, even after hydrophilic treatment of a porous polymer film, the porous structure of a layered leaf-like layer, such as a layer of fallen leaves, is maintained on the surface and within 100 to 150 ㎛ therefrom.

In addition, the hydrophilic-treated film must meet the condition that the moisture absorption rate after the hydrophilic treatment is 80% or higher, preferably 80 to 95%, because it can act as a material for hemostasis within this moisture absorption rate range.

The hemostatic material in the form of a film having hydrophilicity according to the present invention manufactured through this process is preferable because it can exhibit a hemostatic effect by absorbing bleeding blood into the porous structure of the leaf-laminated porous structure during bleeding and reducing the amount of bleeding through blood coagulation by the absorbed blood coming into contact with a large surface area.

2. Film-type hemostasis material loaded with plasma

According to another embodiment of the present invention, a material for hemostasis in the form of a film loaded with plasma is characterized in that the film surface is hydrophilically treated to have hydrophilicity, and plasma is loaded into the porous structure of the film including a porous structure.

That is, it is a structure in which a material for hemostasis in the form of a film having hydrophilicity as described in detail in 1 above is manufactured, and plasma is loaded into the porous structure of the film.

Specifically, the method for manufacturing a material for hemostasis in the form of a film loaded with plasma may include the steps of manufacturing a two-dimensional polymer film using a biocompatible polymer having a melting point (mp) of 40 to 300°C and crystallinity, a step of introducing a porous structure into the manufactured two-dimensional polymer film, a step of imparting hydrophilicity to the surface of the polymer film by hydrophilization treatment, and a step of loading plasma into the porous structure of the hydrophilization-treated polymer film.

That is, the polymer film having the porous structure is manufactured according to the method of the applicant's Korean patent application No. 10-2021-128063 as described in 1 above, and the hydrophilicity imparting process can be performed according to the process of 1 above, and the description up to the hydrophilicity imparting process is omitted below.

In this process, a final plasma-loaded film-type hemostasis material can be manufactured by only going through the step of loading plasma into the porous structure of the hydrophilic-treated polymer film.

It is preferable that the above plasma is at least one selected from PRP (Platelet-Rich Plasma) and PPP (Platelet-Poor Plasma), and the above plasma may be adsorbed or coated on the porous structure of the polymer film.

The loading of the plasma may be accomplished by, but is not particularly limited to, mixing a hydrophilic, manufactured, leaf-laminated porous polymer film with a certain volume of plasma, applying positive and negative pressures to store the film for a certain period of time so that the plasma can be adsorbed or coated on the surface and interior of the leaf-laminated porous structure, and then removing the residual plasma and freeze-drying.

The above plasma can be loaded to have the effect of promoting blood coagulation at the bleeding site, and the film-shaped hemostatic material loaded with the above plasma in the present invention is used as a hemostatic material by the effect of blood being absorbed between the porous structures, and blood coagulation being activated immediately when the absorbed blood meets the plasma loaded in the porous structure.

As a result, the film-shaped hemostasis material according to the present invention has the effect of easily enabling a large amount of plasma capable of performing a hemostatic action to be loaded onto its surface due to the large surface area formed through surface modification (hydrophilic treatment).

In addition, the porous structure refers to the empty space existing between the leaf-layered structures, and due to this structural characteristic, a sufficient amount of blood can be absorbed after hydrophilic treatment, so it can be effectively used as a material for hemostasis.

Hereinafter, preferred embodiments of the present invention will be described in detail. The following embodiments are intended only to illustrate the present invention, and the scope of the present invention should not be construed as being limited by these embodiments. In addition, although specific compounds are used as examples in the following embodiments, it is obvious to those skilled in the art that equivalents thereof can also be used to exhibit similar or equivalent effects.

Example 1: Preparation of a porous film having hydrophilicity

1) Manufacturing of PCL film having porous structure in cross section

Polycaprolactone, which is biocompatible and biodegradable, has crystallinity (crystallization degree 30 to 50%), and a melting point of 60℃, was mixed in a solvent, dichloromethane, at a ratio of 10 wt%, and stirred until a transparent solution became available. The transparent solution was dispensed into a glass petri dish and dried in a hood for 5 hours. After a white PCL film was formed, it was placed in a vacuum oven and dried for 24 hours to produce a PCL polymer film.

In order to introduce a porous structure into the above PCL polymer film, the manufactured PCL film was fixed using a Teflon mold, and then tetraglycol, as a poor solvent, was heated in an oil bath to adjust the temperature of the tetraglycol to 55°C. After that, 55°C tetraglycol was dispensed onto one surface of the PCL film fixed to the Teflon mold, and the film was heated while maintaining the temperature at 55°C for 1 minute, and then the PCL film was cooled at 4°C for 10 minutes, and then immersed in new tetraglycol at room temperature and then removed to wash away the remaining tetraglycol solvent between the porous structures created on the surface of the PCL film. Thereafter, distilled water was added in an excessive amount to completely wash away the remaining tetraglycol.

The washed PCL film was freeze-dried to produce a PCL porous film having a porous structure on its cross-section.

2) Hydrophilic treatment

Polycaprolactone, which exhibits biocompatibility and biodegradability, was hydrophilized to give it more hydrophilic properties for use as a hemostatic material.

For hydrophilization treatment, a porous polymer film with a leaf-leaf laminate structure and 1N NaOH were mixed, and then positive and negative pressures were applied to etch the surface of the polymer film. After that, the porous polymer film with a leaf-leaf laminate structure was immersed in an excessive amount of distilled water to completely wash away the remaining 1N NaOH, and then freeze-drying was performed (see Fig. 1 for hydrophilization treatment process).

Comparative Example 1: Preparation of a porous film that does not have hydrophilicity

In the above Example 1, a porous polymer film with a leaf-laminated structure that was not subjected to hydrophilic treatment in 2) was used as a comparative example.

Experimental Example 1: Confirmation of hydrophilicity according to presence or absence of hydrophilic treatment of porous polymer film

In order to confirm the degree of hydrophilicity according to the hydrophilic treatment of the porous film having the leaf-laminated structure manufactured according to the above Example 1 and Comparative Example 1, a small amount of ink was added to water so that it could be confirmed with the naked eye. Based on the width*length*height=12*12*0.3 mm of the porous polymer film having the leaf-laminated structure, 100 μL of a mixed solution of water and ink was dropped and the change process was observed with the naked eye, and the results are shown in Fig. 2 below.

Referring to Figure 2 below, in the case of a porous polymer film with a leaf-laminated structure that has not been hydrophilicized (Comparative Example 1), it can be confirmed that even after dropping a water droplet, the water droplet is not absorbed and remains in its initial state over time.

However, in the case of a porous polymer film with a laminated structure of fallen leaves that has been hydrophilicized (Example 1 above), it can be confirmed that water begins to be absorbed immediately after a water drop is dropped, and that a much larger amount of water is absorbed over time.

In addition, based on these results, the moisture absorption rate according to the weight change of the polymer film before and after the hydrophilic treatment was performed three times for each sample, and the results are shown in Table 1 below.

Weight (g) Moisture absorption rate (%) ^* Before hydrophilic treatment After hydrophilic treatment Comparative Example 1 0.0379 0.0388 2.37 0.0375 0.0381 1.60 0.0381 0.0394 3.41 Example 1 0.0388 0.0709 82.73 0.0377 0.0710 88.33 0.0374 0.0729 94.92 Moisture absorption rate (%) = (Weight after hydrophilic treatment - Weight before hydrophilic treatment) / Weight before hydrophilic treatment X 100

Referring to the results in Table 1 above, it can be confirmed that the weight difference of the samples of the porous polymer film with the leaf-laminated structure (Comparative Example 1) that was not hydrophilicized before and after the 60-second hydrophilic treatment is less than 0.0015 g, showing almost no difference. This can be confirmed that when expressed as a water absorption rate by weight difference, it is all less than 5%, showing that almost no water is absorbed.

On the other hand, in the samples of the porous film having hydrophilicity by being hydrophilized according to the above-described Example 1 of the present invention, the weight difference between the porous film having the leaf-laminated structure before the hydrophilic treatment and after the 60-second hydrophilic treatment was, on average, 0.03 g or more, and when this was expressed as a water absorption rate, it could be confirmed that it could be a minimum of 82% and a maximum of about 95%. This shows that the porous film having the leaf-laminated structure can sufficiently absorb water between the pores due to the hydrophilic treatment, and it can be seen as a result of the film according to the present invention maintaining the leaf-laminated porous structure in which fallen leaves are laminated and piled on the surface and to a certain depth therefrom even after the hydrophilic treatment.

Experimental Example 2: SEM structural confirmation of porous polymer film with or without hydrophilization treatment

The structure of the porous film having a leaf-leaf laminate structure manufactured according to the above Example 1 and Comparative Example 1 was observed using a scanning electron microscope (SEM), and the results are shown in Fig. 3.

Referring to FIG. 3 below, it can be confirmed that the structure of the porous polymer film having a leaf-laminated structure that has been hydrophilized (Example 1) is not damaged or deformed by the hydrophilization treatment when compared to the porous polymer film having a leaf-laminated structure that has not been hydrophilized (Comparative Example 1). This confirms that the porous structure of the leaf-laminated structure that has not been hydrophilized is maintained as is even if the hydrophilization treatment is performed.

Example 2: Material for hemostasis in the form of a film loaded with plasma

In order to load plasma onto the porous polymer film having a hydrophilic leaf-laminated structure manufactured according to the above Example 1, the film was cut into a size of width*length*height=12*12*0.3 mm, and 1 mL of PRP (platelet-rich plasma) was prepared based on this.

After mixing the porous polymer film with the above-mentioned size of the leaf-leaf laminate structure and the above-mentioned volume of plasma, positive and negative pressures were applied so that the plasma could be adsorbed on the surface and inside of the leaf-leaf laminate structure, and the film was stored at 4°C for 3 hours. Thereafter, the residual plasma was removed, and freeze-dried to manufacture a porous polymer film with the leaf-leaf laminate structure loaded with plasma (see Fig. 4, plasma loading process).

Control group 1: Control

This is an experimental group for comparison with the above Example 2, meaning that the cytotoxicity test was not affected by the extract.

In the in vitro whole blood coagulation experiment, it refers to a general petri dish, and in the in vivo hemorrhage analysis, it refers to the liver bleeding induced without any treatment.

Control group 2: Commercially available hemostasis material

The efficacy of the hemostasis material for the film-type plasma-loaded hemostasis of Example 2 according to the present invention was compared with that of a commercially available hemostatic gauze (Quikclot, manufactured by G-Medica, USA).

Control group 3: PCL polymer film without porous structure introduced

Polycaprolactone, which exhibits biocompatibility and biodegradability, was mixed in a 10 wt% ratio with dichloromethane, a solvent, and stirred until a transparent solution was formed. The transparent solution was dispensed into a glass petri dish, dried in a hood for 5 hours, and when a white polymer film was formed, it was placed in a vacuum oven and dried for 24 hours to manufacture.

The film of Control Group 3 was a polymer film without a leaf-laminated porous structure introduced, and was used to compare the hemostatic efficacy with that of Example 2.

Experimental Example 3: Structural Verification of a Porous Polymer Film with a Leaf-Laminated Structure After Plasma Loading

The structure of the porous polymer film with a plasma-loaded leaf-laminated structure manufactured according to the above Example 2 was observed using SEM, and the results are shown in Fig. 5.

Referring to the structure of the porous film of the fallen leaf laminated structure loaded with plasma according to the above Example 2 (Fig. 4), it can be confirmed that the plasma is loaded onto the undeformed structure when compared to the above Example 1 (Fig. 3).

Experimental Example 4: Cytotoxicity Test Confirmation of Porous Polymer Film with Leaf-Laminated Structure

In order to confirm the viability of cells of the porous polymer film having a leaf-leaf laminate structure loaded with plasma prepared according to the above Example 1 and Control Groups 1 to 3 and the above Example 2 and the control group, an extract was prepared and quantified using the Cell Counting Kit-8 (CCK-8) assay, and the results are shown in Figure 6 below.

First, for cytotoxicity test, the extracts of the porous polymer film with a leaf-leaf laminate structure loaded with plasma (the above Example 2), quikclot (control group 2), the polymer film without a porous structure introduced (control group 3), and the porous polymer film with a leaf-leaf laminate structure treated with hydrophilization (Example 1) were prepared. The samples for the extracts were prepared according to the standard of ISO 10993-5. The extracts were prepared by storing the samples in a cell culture medium for 24 hours, and filtering them through a 0.2 μm filter.

Cells were seeded so that the total number of cells seeded per well of a 6-well plate was 2* ¹⁰⁵ cells. Then, the volume of the 2* ¹⁰⁵ cell suspension was 2 mL and cultured for 24 hours in a 37℃, 5% _CO2 incubator to induce cell attachment. After 24 hours, the medium was replaced with the prepared lysate and cultured in the incubator for another 24 hours. After that, the cell culture medium was replaced with new medium and the CCK-8 assay was performed.

Referring to the following Figure 6, the control (control group 1) is an experimental group grown only in a simple cell culture medium without treating the extract, and can be viewed as the state of cells grown normally without any influence by the extract. Cytotoxicity was expressed as a % by relatively comparing quikclot (control group 2), a polymer film without a porous structure (control group 3), a porous polymer film with a leaf-leaf laminate structure that was hydrophilicized (the above Example 1), and a porous polymer film with a leaf-leaf laminate structure loaded with plasma (the above Example 2), with the control as the standard of 100.

In the case of Quikclot (control group 2), it was confirmed that the cytotoxicity results were similar to those of the control group, as it is a commercially available hemostatic agent that is already being used clinically.

In addition, in the case of the polymer film (control group 3) in which the porous structure was not introduced, it was confirmed that it did not exhibit cytotoxicity when compared to the control film manufactured using a biocompatible polymer.

In addition, in the case of the porous polymer film with the hydrophilic treatment of the leaf-leaf laminate structure, it was confirmed that it did not exhibit cytotoxicity when compared to the control, which confirmed that the hydrophilic treatment did not affect the cytotoxicity. In addition, in the case of the porous polymer film with the leaf-leaf laminate structure loaded with plasma, it was confirmed that the plasma-induced eluate did not exhibit cytotoxicity when compared to the control even when the plasma was loaded.

As a result, this can mean that no cytotoxicity occurs at all even when hydrophilic treatment is applied to the porous film with the leaf-laminated structure or plasma is loaded thereon.

Control group 4: Solution used for hemolysis assay

In the hemolysis assay, Triton-X was used as the negative control group that can hemolyze red blood cells the most, and PBS was used as the positive control group that causes little hemolysis of red blood cells in an environment similar to blood.

Experimental Example 5: Hemolysis assay of porous film with leaf-leaf laminate structure

The extracts of Example 2 and Control Groups 1 to 3 and Example 1 were prepared, and the effects of the extracts on red blood cells when in contact with blood were observed through a hemolysis assay, and the results are shown in Fig. 7.

The hemolysis assay effluent was prepared by dissolving 12*12 ^mm2 of each sample in 25 mL PBS and storing it at 37℃ for 72 hours. In addition, the blood was centrifuged at 3000 rpm for 20 minutes, the separated supernatant was removed, and the settled cells were washed twice with PBS to prepare a 2% erythrocyte suspension. Then, 10 mL of the effluent and 2 mL of the erythrocyte suspension were mixed and incubated in a 37℃ incubator for 3 hours. After 3 hours, the mixture was centrifuged at 3000 rpm for 10 minutes, and the supernatant was measured for absorbance at 540 nm.

Referring to Figure 7 below, when comparing the degree of hemolysis of red blood cells in the extracts of the negative control and positive control (control group 4), quikclot (control group 2), a polymer film without a porous structure (control group 3), a porous polymer film with a leaf-leaf laminate structure that has been hydrophilized (the above Example 1), and a porous polymer film with a leaf-leaf laminate structure loaded with plasma (the above Example 2), it was confirmed that there was almost no effect on red blood cells.

This was done by using PBS, which has a blood-like environment, as a positive control and setting its absorbance value as 0, and using Triton-X, which almost completely hemolyzes red blood cells when in contact with blood, as a negative control and setting its absorbance value as 100.

Through the attached photo, in the case of PBS, it can be confirmed that the red blood cells completely settle when centrifuged without hemolysis by the extract, and in the case of Triton-X, it can be confirmed that the red blood cells are completely hemolyzed and dispersed.

In the case of control group 3, it was confirmed that there were no components that hemolyzed red blood cells, very similar to the PBS environment.

In addition, in the case of the porous polymer film with a quikclot and hydrophilic treated leaf-leaf laminate structure and the porous polymer film with a plasma-loaded leaf-leaf laminate structure, it was shown that red blood cells were hemolyzed compared to the PBS environment, but this showed a value of less than about 0.2% compared to triton-X, which means that when the hemostatic material comes into contact with blood, the red blood cells in the blood are not hemolyzed and the blood clotting process is not interfered with, so it can play a role as a hemostatic material.

Experimental Example 6: Porous polymer film with a leaf-leaf laminate structure in vitro Confirmation of whole blood coagulation experiment

In order to confirm the degree of blood coagulation in vitro of the above Example 2 and Control Groups 1 to 3 and the above Example 1, a whole blood coagulation experiment was conducted, and the results are shown in Fig. 8.

For the whole blood coagulation experiment, quikclot (control group 2), a polymer film without a porous structure (control group 3), a porous polymer film with a hydrophilic treatment and a leaf-leaf laminate structure (the above Example 1), and a porous polymer film with a leaf-leaf laminate structure loaded with plasma (the above Example 2) were placed in a petri dish, 100 μL of blood was dropped, and it was stored in a 37°C incubator for 30 minutes. At this time, the blood contained an anticoagulant (blood 9: anticoagulant 1). After 30 minutes, 5 mL of distilled water, which is a storage solution that can burst red blood cells of uncoagulated blood, was added. Then, the distilled water was recovered and the absorbance was measured at 540 nm.

Referring to Figure 8 below, in cases where blood is simply placed on a petri dish and a polymer film (control group 3) without a porous structure introduced has no effect on hemostatic action on the blood, it was found that there was almost no effect on blood coagulation.

On the other hand, in the case of quikclot (control group 2), since it is a hemostatic agent sold commercially, it was shown to have a hemostatic effect by coagulating blood to some extent compared to the control (control group 1).

In the case of a porous polymer film with a hydrophilic treated leaf-laminated structure (Example 1 above), it was shown that a hemostatic effect could be exhibited compared to the control because a certain amount of blood could be absorbed by the polymer film with the leaf-laminated structure introduced.

In addition, in the case of the porous polymer film with a leaf-leaf laminate structure loaded with plasma (the above Example 2), it was confirmed that the ratio of non-coagulated red blood cells differed by about 10 times or more compared to the control. This may indicate that the porous polymer film with a leaf-leaf laminate structure loaded with plasma of the present invention can effectively induce blood coagulation.

Experimental Example 7: Porous polymer film with a leaf-leaf laminate structure in vivo Check hemorrhage analysis

In order to confirm the degree of blood coagulation in vivo in Example 2 and Control Groups 1 to 3 and Example 1, the amount of bleeding was confirmed through an animal experiment using rats, and the results are shown in Figure 9.

In order to confirm the amount of bleeding in relation to the hemostatic effect of the group in which no treatment was applied to the bleeding site in vivo (control group 1), quikclot (control group 2), a polymer film without a porous structure (control group 3), a porous polymer film with a leaf-leaf laminate structure that was hydrophilized (the above Example 1), and a porous polymer film with a leaf-leaf laminate structure loaded with plasma (the above Example 2), the rats were anesthetized and the abdominal wall was incised to expose the left lobe of the liver. A filter paper was placed under the liver, and parafilm was placed underneath it to prevent blood other than blood due to bleeding from getting on it. Thereafter, a 4 mm deep wound was made in the liver, and the amount of bleeding was observed for 3 minutes. The amount of blood bleeding onto the filter paper was measured by weight and quantified.

Referring to Figure 9 below, the group in which nothing was treated at the site of bleeding in the liver (control group 1) showed the largest amount of bleeding as an experimental group that similarly showed the bleeding process at the actual site of bleeding.

In the case of Quikclot (control group 2), it was confirmed that although it is a hemostatic agent sold on the market, it has the effect of absorbing blood in the early stage of bleeding, but its hemostatic effect during the 3-minute period of observing the amount of bleeding was not significantly superior. This confirmed that even absorbable hemostatic agents are products that can be effective as a material for hemostasis only when an external factor called pressure is applied.

In addition, in the case of the polymer film (control group 3) to which the porous structure was not introduced, the results were slightly different from the results in the whole blood coagulation experiment (Experimental Example 5), and it was confirmed that a hemostatic effect similar to that of quikclot was shown compared to the group that did not treat the bleeding site with anything. This was confirmed to be because, compared to the group that did not treat the bleeding site with anything, it physically formed a barrier at the bleeding site and prevented excessive blood flow, thereby accelerating the hemostasis process and showing a coagulation effect.

The porous polymer film with a hydrophilic treated leaf-laminated structure (the above Example 1) was found to have a slightly greater hemostatic effect compared to quikclot, a polymer film without a porous structure. This was because the porous film with a leaf-laminated structure was able to absorb bleeding blood into the film, and the large surface area promoted blood clotting, which reduced the amount of blood flowing out, thereby reducing the amount of bleeding.

In the case of a porous polymer film with a plasma-loaded leaf-laminated structure (Example 2 above), it was confirmed that a blood clotting efficacy of about 10 times higher was exhibited compared to a group in which nothing was treated on the bleeding site.

It was confirmed that the plasma loaded on the structural surface of the porous film with a leaf-leaf laminated structure loaded with plasma helps activate blood coagulation immediately when it comes into contact with blood, and the blood flowing out between the porous structures is absorbed, thereby exhibiting a more effective blood coagulation effect. Therefore, it could be expected that the porous polymer film with a leaf-leaf laminated structure loaded with plasma can be more effectively utilized as a material for hemostasis.

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Claims (19)

A material for hemostasis in the form of a film having hydrophilic properties, including a porous structure and having a hydrophilic surface treated to be hydrophilic.
The above film is polycaprolactone,
The above hydrophilic treatment is performed by mixing a base solution into a polycaprolactone film including the porous structure and applying positive and negative pressures to etch the surface of the polycaprolactone film. After the hydrophilic treatment, the water absorption rate of the polycaprolactone film is 80 to 95%.
A material for hemostasis in a film form having hydrophilicity, characterized in that at least one type of plasma selected from PRP (Platelet-Rich Plasma) and PPP (Platelet-Poor Plasma) is loaded by adsorption or coating on the porous structure of the polycaprolactone film. delete delete delete delete In paragraph 1,
The above polycaprolactone film is a material for hemostasis in the form of a film that is a hydrophilic biocompatible polymer with a melting point (mp) of 40 to 300°C and a crystallinity of 5 to 99%. delete In paragraph 1,
The material for hemostasis in the form of a film having the above hydrophilic properties is:
When bleeding occurs, the bleeding blood is absorbed into the porous structure of the leaf-layered layer, and
A hemostatic material in the form of a film having hydrophilic properties that can be used as a hemostasis material through a process in which the amount of bleeding is reduced by blood coagulation upon contact of absorbed blood with a large surface area. delete delete In paragraph 1,
The above plasma is a film-shaped hemostatic material having hydrophilic properties that promotes blood clotting at the site of bleeding. In paragraph 1,
The material for hemostasis in the form of a film loaded with the above plasma is:
Blood is absorbed between the porous structures,
A hemostatic material in the form of a film having hydrophilic properties, which is used as a hemostasis material through a process in which blood coagulation is activated immediately when absorbed blood meets plasma loaded in the porous structure. A step of manufacturing a two-dimensional film using polycaprolactone having a melting point (mp) of 40 to 300°C and a crystallinity of 5 to 99%.
A step of introducing a porous structure into the two-dimensional polycaprolactone film manufactured above,
A step of etching the surface of the polycaprolactone film by mixing a base solution and applying positive and negative pressures to the surface of the polycaprolactone film to make it hydrophilic, and imparting hydrophilicity until the water absorption rate of the polycaprolactone film becomes 80 to 95% after the hydrophilic treatment, and
A method for producing a material for hemostasis in the form of a film having hydrophilicity according to claim 1, comprising a step of loading plasma into the porous structure of the hydrophilic-treated polycaprolactone film. delete delete In Article 13,
The above polycaprolactone film is manufactured using a solution containing polycaprolactone in a concentration of 1 to 50 wt%, or
A manufacturing method comprising: applying heat at a temperature of 40 to 300°C to polycaprolactone. In Article 13,
A manufacturing method in which the introduction of a porous structure into the above-mentioned two-dimensional polycaprolactone film is carried out by applying heat while bringing the two-dimensional polycaprolactone film into contact with a poor solvent composed of one or more mixtures selected from tetraglycol, 1-methyl-2-pyrrolidinone (NMP), triacetin, benzyl alcohol, and dimethyl sulfoxide (DMSO). delete In Article 13,
The above plasma loading is a manufacturing method in which the polycaprolactone film is impregnated with plasma and then positive and negative pressures are applied so that plasma can be adsorbed on the surface and inside of the leaf-leaf laminate structure.