JP2020127705A - Heparin-immobilized binder, its manufacturing method, medical supply and its manufacturing method - Google Patents

Abstract

To provide a heparin-immobilized binder and its manufacturing method, capable of immobilizing heparin onto a polymer base material surface easily and safely; and to provide a medical supply containing the heparin-immobilized binder, and its manufacturing method.SOLUTION: There is provided a heparin-immobilized binder formed by introducing an alkyl group into polyamine, and having both cationic property and hydrophobicity. The polyamine is ε-polylysine, and comprises a partially amidated product in which a part of an amino group of ε-polylysine is amidated by dodecyl succinic anhydride. An introduction ratio of dodecyl succinic anhydride into the partially amidated product is 20 mass% or more and 40 mass% or less.SELECTED DRAWING: None

Description

The present invention relates to a heparin-immobilized binder and a method for producing the same, a medical device and a method for producing the same.

In a surgical operation called an open heart surgery for treating heart disease, the movement of the heart is temporarily stopped to surgically repair a defective part of the cardiovascular system. Due to blood circulation blockage, irreversible lesions progress in 3 to 4 minutes or longer in organs/tissues that are vulnerable to hypoxia. Therefore, the artificial heart-lung machine artificially guides the patient's blood (venous blood) to the outside of the body, and continues the extracorporeal circulation by continuously supplying oxygen to the living body of the patient whose blood circulation is blocked. In addition to the open heart surgery, the artificial heart-lung machine is also used for the purpose of assisting the function of the weakened heart called assisted circulation in the treatment of heart failure.
The artificial heart-lung machine is composed of three parts: an artificial lung, a blood pump, and a blood circuit.

However, the extracorporeal circulation causes various non-physiological changes to the living body and causes great invasion.
Therefore, in the development of an extracorporeal circulation device such as an artificial cardiopulmonary circuit, it is important to further reduce the invasion to the living body. Therefore, in consideration of the flow state of blood, the device has been devised from the viewpoint of structural design and material design while maintaining high functionality. Structural design has been devised in terms of reducing the blood contact area and the amount of blood filling the product. The material design has been devised in terms of surface treatment with a material having blood compatibility.

What is needed in the field of artificial organs are blood and histocompatible materials. Blood has the property of forming a thrombus when it comes into contact with a foreign substance such as an artifact other than normal vascular endothelial cells. Therefore, in an artificial organ that comes into contact with blood, a thrombus is formed on the surface of the artificial organ and the function is deteriorated. If the blood clot comes off and is washed away by the organs such as the brain and clogs blood vessels, fatal injury may occur.

There are several coagulation proteins in blood that are involved in blood coagulation. The plasma protein called fibrinogen undergoes partial cleavage of the molecule, and a large number of them associate to form a three-dimensional network called fibrin. Since this three-dimensional mesh is a gel insoluble in water, the whole blood cannot flow and coagulates. Heparin is an example of a drug that inhibits this blood coagulation reaction.

In the case of artificial materials, plasma proteins are adsorbed on the surface of the materials, and platelets adhere via the proteins. Platelets are small blood cells with no nucleus, and when they adhere to a certain surface, platelets pile up on top of each other, forming large aggregates that stop the flow of blood. When platelet aggregation occurs, a fibrin mesh is formed, and the aggregate is reinforced by the mesh to form a strong thrombus.

Heparin is known to be a drug for preventing blood coagulation and platelet adhesion. In addition, heparin has been attempted to be bound to various polymer materials to improve antithrombotic properties. Heparin has already been widely used clinically as a material for catheters and the like. Since heparin is gradually released from the surface of the material into the blood and disappears, attempts have been made to stably fix heparin for a long period of time.
Heparin is an anticoagulant that forms a complex with antithrombin III and activates it to inhibit the function of thrombin.

Heparin is an anionic polysaccharide as shown in the following chemical formula (1). That is, heparin is capable of binding and immobilizing a cationic material by electrostatic interaction. Further, it has also been attempted to immobilize heparin by a covalent bond to a material having a functional group that reacts with the functional group of heparin.

Research on a method for coating the surface of a polymer material with heparin has been advanced, and many materials have been developed. The method of coating heparin is mainly classified into an ionic bond type fixing method and a covalent bond type fixing method.

A method of fixing anionic heparin via a cationic binder material is called an ionic bond-type fixing method. It has been reported that the ionic bond-type fixation method has high anticoagulant properties, almost no thrombus is formed even after contact with blood for 30 minutes, and a clean state is maintained on the material surface (for example, Non-Patent Document 1 1 and 2). This suggests that heparin is immobilized on the surface of the polymer material at multiple points by immobilization by the ionic bond type immobilization method.

A method of chemically bonding to a polymer using a functional group of heparin is called a covalent bond fixing method. In the covalent immobilization method, it is known that inactivation of thrombin causes albumin (a kind of plasma protein) to be adsorbed on the material surface to adhere platelets (for example, see Non-Patent Document 3). .. This is because the amount of heparin immobilized is smaller than that of the ionic bond-type immobilization method, and the binding strength between a small amount of immobilized heparin and the polymer is strong, so that the degree of freedom of heparin is lost and the anticoagulant activity is exhibited. It suggests that it could not be done (for example, refer to Patent Document 1).

Anti-thrombotic properties of medical devices by fixing heparin and heparin-immobilized polymer on the surface of medical devices that come into contact with blood (eg, blood circuit tubes, angiography catheters, artificial lungs, arterial filters, etc.) Can be given. An organic cationic compound such as an ammonium salt or a phosphonium salt is bonded to the negative charge (sulfonic acid group or aminosulfonic acid group) of heparin to form a complex insoluble in water and soluble in an organic solvent.
When a medical device is coated with a highly hydrophobic compound such as an organic cation compound, heparin can be retained for a long time, but the anticoagulant effect of heparin is suppressed, and thrombus is likely to occur. On the other hand, when the hydrophilicity of the compound coated on the medical device is high, heparin is eluted in a short time, so that the antithrombotic property cannot be maintained for a long time. Therefore, the organic cation compound coated on the medical device needs to have an appropriate balance of hydrophilicity and hydrophobicity (see, for example, Patent Document 1).

In the current clinical setting, many devices that come into contact with blood are used. Although research and development on antithrombotic materials have been continued for many years, clear understanding of antithrombotic properties and development of sufficient antithrombotic materials have not been achieved. Currently, the most studied methods include forming a material surface that does not cause blood coagulation. That is, research on coating the surface of a polymer material with heparin and research on coating a polymer surface with a polymer are underway. The purpose of these studies is to prevent the blood coagulation reaction by coating the surface of the polymer material to suppress the adhesion of platelets and the adsorption of plasma proteins, which trigger the blood coagulation reaction.

Examples of the coating method include a blood-compatible coating technique in which a polymer material surface is coated with a 2-methacryloyloxyphenyl phosphochlorine (MPC) polymer or a poly(2-methoxyethyl acrylate) (PMEA) polymer. .. Another example is a method of suppressing adsorption of proteins by forming a high-density polymer brush such as polyethylene glycol on the surface of the polymer material. In addition, there is a method that utilizes the fact that heparan sulfate contained in the heparin molecule contributes to the anticoagulant reaction. Furthermore, since blood does not coagulate even when it comes into contact with vascular endothelial cells, there is a method of fixing vascular endothelial cells on the surface of a polymer material.

Japanese Patent No. 4273965

Surface Science, Vol. 32, No. 9, pp. 581-586, 2011, Surface Modification of Polymeric for Medical Devices, Makoto Onishi Y. Ito, M.; Shishido, Y. Imanishi;J. Biomed. Mater. Res 20 113 (1986) Polymer and Medicine, Kiichi Takemoto, Junzo Sunamoto, Mitsuru Akashi, p14-16

As mentioned above, heparin is the clinically most commonly used anticoagulant and is widely used for coating the surface of medical devices that come into contact with blood. Therefore, the technique of coating the surface of the polymer material with heparin is an essential technique for extracorporeal circulation. However, in the extracorporeal circulation using a blood circuit coated with heparin, reduction of the amount of heparin administered systemically and persistence of antithrombotic property during long-term circulation have not been sufficiently examined.

Therefore, there has been a demand for the development of a new technique capable of easily and safely immobilizing heparin on the surface of a polymer substrate.

The present invention has been made in view of the above circumstances, and a heparin-immobilized binder capable of immobilizing heparin on the surface of a polymer substrate easily and safely, a method for producing the same, a medical device including the heparin-immobilized binder and the same. It is intended to provide a manufacturing method.

The present invention has the following aspects.
[1] A heparin-immobilized binder having a cationic property and a hydrophobic property, which is obtained by introducing an alkyl group into polyamine.
[2] The heparin-immobilized binder according to [1], wherein the polyamine is ε-polylysine, and a part of the amino group of the ε-polylysine is a partially amidated product amidated with dodecylsuccinic anhydride.
[3] The heparin-immobilized binder according to [2], wherein the introduction rate of the dodecyl succinic anhydride in the partially amidated product is 20% by mass or more and 40% by mass or less.
[4] A medical device in which at least a part of the blood contact surface is coated with the heparin-immobilized binder according to any one of [1] to [3].
[5] The medical device according to [4], wherein at least one of heparin and a heparin derivative is fixed to at least a part of the blood contact surface via the heparin-immobilized binder.
[6] The medical device according to [4] or [5], wherein the medical device is a tube.
[7] The medical device according to [4] or [5], wherein the medical device is a reservoir.
[8] A method for producing a heparin-immobilized binder, comprising a step of reacting a polyamine with an acid anhydride to amidate a part of the amino groups of the polyamine with the acid anhydride.
[9] The method for producing a heparin-immobilized binder according to [8], wherein the polyamine is ε-polylysine, and the acid anhydride is dodecyl succinic anhydride.
[10] A step of dissolving the heparin-immobilized binder according to any one of [1] to [3] in a solvent to prepare a solution containing the heparin-immobilized binder, and at least one of blood contact surfaces of medical devices. A step of applying the solution to a portion and coating at least a part of the blood contact surface with the heparin-immobilized binder.
[11] The method for producing a medical device according to [10], which has a step of fixing at least one of heparin and a heparin derivative on at least a part of the blood contact surface via the heparin-immobilized binder.

According to the present invention, it is possible to provide a heparin-immobilized binder capable of immobilizing heparin on the surface of a polymer substrate easily and safely, a method for producing the same, a medical device containing the heparin-immobilized binder, and a method for producing the same.

1 is a cross-sectional view showing a schematic configuration of a medical device of the present embodiment and showing a polymer base material forming the medical device in a thickness direction. It is a figure which shows schematic structure of the artificial heart-lung machine to which the tube and reservoir of this embodiment are applied. It is a longitudinal cross-sectional view illustrating a schematic configuration of the reservoir of the present embodiment, showing a state in which a pressure sensor is not attached. It is a longitudinal cross-sectional view illustrating a schematic configuration of the reservoir of the present embodiment, and is a conceptual diagram illustrating a schematic configuration with a pressure sensor attached. Introduction rate of sites from DDSA prepared in Experimental Example 1 is a diagram showing the result of ^{1 H-NMR} measurement about 20 wt% partially amidated. It is a figure which shows the result of the < ¹ >H-NMR measurement regarding the partial amidation product which the introduction rate of the site derived from DDSA produced in Experimental example 1 is 30 mass %. Introduction rate of sites from DDSA prepared in Experimental Example 1 is a diagram showing the result of ^{1 H-NMR} measurement about 40 wt% partially amidated. It is a figure which shows the result of the < ¹ >H-NMR measurement regarding the partial amidation product which the introduction rate of the site|part derived from the hexanoic acid anhydride produced in Experimental example 2 is 20 mass %. Introduction rate of sites from hexanoic acid anhydride prepared in Experimental Example 2 is a diagram showing the result of ^{1 H-NMR} measurement about 30 wt% partially amidated. Introduction rate of sites from hexanoic acid anhydride prepared in Experimental Example 2 is a diagram showing the result of ^{1 H-NMR} measurement about 40 wt% partially amidated. It is a figure which shows the result of the < ¹ >H-NMR measurement regarding the partial amidation thing which made the introduction rate of the site|part derived from the lauric acid anhydride produced in Experimental example 3 into 20 mass %. It is a schematic diagram explaining a contact angle analysis method. In Experimental example 4, it is a figure which shows the analysis result of the contact angle of the surface of the silicone rubber tube sample coated with DDSA-PLL, and the surface of the silicone rubber tube sample coated with heparin. In Experimental example 4, it is a figure which shows the infrared absorption spectrum of the surface of the silicone rubber tube sample which coated the DDSA-PLL which made the introduction rate of the site|part derived from DDSA 20 mass %. In Experimental example 4, it is a figure which shows the infrared absorption spectrum of the surface of the silicone rubber tube sample which coated the DDSA-PLL which made the introduction rate of the part derived from DDSA 30 mass %. In Experimental example 4, it is a figure which shows the infrared absorption spectrum of the surface of the silicone rubber tube sample which coated the DDSA-PLL which made the introduction rate of the part derived from DDSA 40 mass %. In Experimental example 4, it is a figure which shows the analysis result of the surface of the silicone rubber tube sample which is not coated with DDSA-PLL by X-ray photoelectron spectroscopy. In Experimental example 4, it is a figure which shows the analysis result of the surface of the silicone rubber tube sample which is not coated with DDSA-PLL by X-ray photoelectron spectroscopy. In Experimental example 4, it is a figure which shows the analysis result of the surface of the silicone rubber tube sample which is not coated with DDSA-PLL by X-ray photoelectron spectroscopy. In Experimental example 4, it is a figure which shows the analysis result of the surface of the silicone rubber tube sample which is not coated with DDSA-PLL by X-ray photoelectron spectroscopy. In Experimental example 4, it is a figure which shows the analysis result by the X-ray photoelectron spectroscopy of the surface of the silicone rubber tube sample which coated the DDSA-PLL which made the introduction rate of the site derived from DDSA 5 mass %. In Experimental example 4, it is a figure which shows the analysis result by the X-ray photoelectron spectroscopy of the surface of the silicone rubber tube sample which coated the DDSA-PLL which made the introduction rate of the site derived from DDSA 5 mass %. In Experimental example 4, it is a figure which shows the analysis result by the X-ray photoelectron spectroscopy of the surface of the silicone rubber tube sample which coated the DDSA-PLL which made the introduction rate of the site derived from DDSA 5 mass %. In Experimental example 4, it is a figure which shows the analysis result by the X-ray photoelectron spectroscopy of the surface of the silicone rubber tube sample which coated the DDSA-PLL which made the introduction rate of the site derived from DDSA 5 mass %. In Experimental example 4, it is a figure which shows the analysis result by the X-ray photoelectron spectroscopy of the surface of the silicone rubber tube sample which coated the DDSA-PLL which made the introduction rate of the site derived from DDSA 20 mass %. In Experimental example 4, it is a figure which shows the analysis result by the X-ray photoelectron spectroscopy of the surface of the silicone rubber tube sample which coated the DDSA-PLL which made the introduction rate of the site derived from DDSA 20 mass %. In Experimental example 4, it is a figure which shows the analysis result by the X-ray photoelectron spectroscopy of the surface of the silicone rubber tube sample which coated the DDSA-PLL which made the introduction rate of the site derived from DDSA 20 mass %. In Experimental example 4, it is a figure which shows the analysis result by the X-ray photoelectron spectroscopy of the surface of the silicone rubber tube sample which coated the DDSA-PLL which made the introduction rate of the site derived from DDSA 20 mass %. In Experimental example 4, it is a figure which shows the analysis result by the X-ray photoelectron spectroscopy of the surface of the silicone rubber tube sample which carried out the introduction rate of the part derived from DDSA to 30 mass %. In Experimental example 4, it is a figure which shows the analysis result by the X-ray photoelectron spectroscopy of the surface of the silicone rubber tube sample which carried out the introduction rate of the part derived from DDSA to 30 mass %. In Experimental example 4, it is a figure which shows the analysis result by the X-ray photoelectron spectroscopy of the surface of the silicone rubber tube sample which carried out the introduction rate of the part derived from DDSA to 30 mass %. In Experimental example 4, it is a figure which shows the analysis result by the X-ray photoelectron spectroscopy of the surface of the silicone rubber tube sample which carried out the introduction rate of the part derived from DDSA to 30 mass %. In Experimental example 4, it is a figure which shows the analysis result by the X-ray photoelectron spectroscopy of the surface of the silicone rubber tube sample which coated the DDSA-PLL which made the introduction rate of the site derived from DDSA 40 mass %. In Experimental example 4, it is a figure which shows the analysis result by the X-ray photoelectron spectroscopy of the surface of the silicone rubber tube sample which coated the DDSA-PLL which made the introduction rate of the site derived from DDSA 40 mass %. In Experimental example 4, it is a figure which shows the analysis result by the X-ray photoelectron spectroscopy of the surface of the silicone rubber tube sample which coated the DDSA-PLL which made the introduction rate of the site derived from DDSA 40 mass %. In Experimental example 4, it is a figure which shows the analysis result by the X-ray photoelectron spectroscopy of the surface of the silicone rubber tube sample which coated the DDSA-PLL which made the introduction rate of the site derived from DDSA 40 mass %. FIG. 37 is a diagram summarizing the analysis results shown in FIGS. 17 to 36 in Experimental Example 4. In Experimental example 4, it is a figure which shows the analysis result by X-ray photoelectron spectroscopy of the surface of the silicone rubber tube sample which coated heparin, without going through DDSA-PLL. In Experimental example 4, it is a figure which shows the analysis result by X-ray photoelectron spectroscopy of the surface of the silicone rubber tube sample which coated heparin, without going through DDSA-PLL. In Experimental example 4, it is a figure which shows the analysis result by X-ray photoelectron spectroscopy of the surface of the silicone rubber tube sample which coated heparin, without going through DDSA-PLL. In Experimental example 4, it is a figure which shows the analysis result by X-ray photoelectron spectroscopy of the surface of the silicone rubber tube sample which coated heparin, without going through DDSA-PLL. In Experimental example 4, it is a figure which shows the analysis result by X-ray photoelectron spectroscopy of the surface of the silicone rubber tube sample which coated heparin, without going through DDSA-PLL. In Experimental example 4, it is a figure which shows the analysis result by X-ray photoelectron spectroscopy of the surface of the silicone rubber tube sample which made the introduction rate of the site|part derived from DDSA 5 mass %, and heparin was coated via DDSA-PLL. In Experimental example 4, it is a figure which shows the analysis result by X-ray photoelectron spectroscopy of the surface of the silicone rubber tube sample which made the introduction rate of the site|part derived from DDSA 5 mass %, and heparin was coated via DDSA-PLL. In Experimental example 4, it is a figure which shows the analysis result by X-ray photoelectron spectroscopy of the surface of the silicone rubber tube sample which made the introduction rate of the site|part derived from DDSA 5 mass %, and heparin was coated via DDSA-PLL. In Experimental example 4, it is a figure which shows the analysis result by X-ray photoelectron spectroscopy of the surface of the silicone rubber tube sample which made the introduction rate of the site|part derived from DDSA 5 mass %, and heparin was coated via DDSA-PLL. In Experimental example 4, it is a figure which shows the analysis result by X-ray photoelectron spectroscopy of the surface of the silicone rubber tube sample which made the introduction rate of the site|part derived from DDSA 5 mass %, and heparin was coated via DDSA-PLL. In Experimental example 4, it is a figure which shows the analysis result by the X-ray photoelectron spectroscopy of the surface of the silicone rubber tube sample which made the introduction rate of the site|part derived from DDSA 20 mass %, and heparin was coated via DDSA-PLL. In Experimental example 4, it is a figure which shows the analysis result by the X-ray photoelectron spectroscopy of the surface of the silicone rubber tube sample which made the introduction rate of the site|part derived from DDSA 20 mass %, and heparin was coated via DDSA-PLL. In Experimental example 4, it is a figure which shows the analysis result by the X-ray photoelectron spectroscopy of the surface of the silicone rubber tube sample which made the introduction rate of the site|part derived from DDSA 20 mass %, and heparin was coated via DDSA-PLL. In Experimental example 4, it is a figure which shows the analysis result by the X-ray photoelectron spectroscopy of the surface of the silicone rubber tube sample which made the introduction rate of the site|part derived from DDSA 20 mass %, and heparin was coated via DDSA-PLL. In Experimental example 4, it is a figure which shows the analysis result by the X-ray photoelectron spectroscopy of the surface of the silicone rubber tube sample which made the introduction rate of the site|part derived from DDSA 20 mass %, and heparin was coated via DDSA-PLL. In Experimental example 4, it is a figure which shows the analysis result by X-ray photoelectron spectroscopy of the surface of the silicone rubber tube sample which made the introduction rate of the site|part derived from DDSA into 30 mass %, and heparin was coated through DDSA-PLL. In Experimental example 4, it is a figure which shows the analysis result by X-ray photoelectron spectroscopy of the surface of the silicone rubber tube sample which made the introduction rate of the site|part derived from DDSA into 30 mass %, and heparin was coated through DDSA-PLL. In Experimental example 4, it is a figure which shows the analysis result by X-ray photoelectron spectroscopy of the surface of the silicone rubber tube sample which made the introduction rate of the site|part derived from DDSA into 30 mass %, and heparin was coated through DDSA-PLL. In Experimental example 4, it is a figure which shows the analysis result by X-ray photoelectron spectroscopy of the surface of the silicone rubber tube sample which made the introduction rate of the site|part derived from DDSA into 30 mass %, and heparin was coated through DDSA-PLL. In Experimental example 4, it is a figure which shows the analysis result by X-ray photoelectron spectroscopy of the surface of the silicone rubber tube sample which made the introduction rate of the site|part derived from DDSA into 30 mass %, and heparin was coated through DDSA-PLL. In Experimental example 4, it is a figure which shows the analysis result by the X-ray photoelectron spectroscopy of the surface of the silicone rubber tube sample which made the introduction rate of the part derived from DDSA 40 mass %, and heparin was coated through DDSA-PLL. In Experimental example 4, it is a figure which shows the analysis result by the X-ray photoelectron spectroscopy of the surface of the silicone rubber tube sample which made the introduction rate of the part derived from DDSA 40 mass %, and heparin was coated through DDSA-PLL. In Experimental example 4, it is a figure which shows the analysis result by the X-ray photoelectron spectroscopy of the surface of the silicone rubber tube sample which made the introduction rate of the part derived from DDSA 40 mass %, and heparin was coated through DDSA-PLL. In Experimental example 4, it is a figure which shows the analysis result by the X-ray photoelectron spectroscopy of the surface of the silicone rubber tube sample which made the introduction rate of the part derived from DDSA 40 mass %, and heparin was coated through DDSA-PLL. In Experimental example 4, it is a figure which shows the analysis result by the X-ray photoelectron spectroscopy of the surface of the silicone rubber tube sample which made the introduction rate of the part derived from DDSA 40 mass %, and heparin was coated through DDSA-PLL. FIG. 63 is a diagram summarizing the analysis results shown in FIGS. 38 to 62 in Experimental Example 4. In Experimental Example 4, ζ-potentials of the surface of the silicone rubber tube sample coated with DDSA-PLL, the surface of the silicone rubber tube sample coated with heparin, and the surface of the silicone rubber tube sample not coated with DDSA-PLL It is a figure which shows the result of having measured. In Experimental Example 5, it is a figure which shows the analysis result of the contact angle of the surface of the PVC tube sample coated with DDSA-PLL, and the surface of the PVC tube sample coated with heparin. In Experimental example 5, it is a figure which shows the infrared absorption spectrum of the surface of the PVC tube sample which is not coated with DDSA-PLL. In Experimental example 5, it is a figure which shows the infrared absorption spectrum of the surface of the PVC tube sample which coated the DDSA-PLL which made the introduction rate of the site|part derived from DDSA 20 mass %. In Experimental example 5, it is a figure which shows the infrared absorption spectrum of the surface of the PVC tube sample which coated the DDSA-PLL which made the introduction rate of the site|part derived from DDSA 30 mass %. In Experimental example 5, it is a figure which shows the infrared absorption spectrum of the surface of the PVC tube sample which coated the DDSA-PLL which made the introduction rate of the site|part derived from DDSA 40 mass %. It is a figure which overlaps and shows Drawing 66-Drawing 69. It is a photograph which shows a PVC sheet before being immersed in DMSO. It is a photograph showing a PVC sheet after being immersed in DMSO for 1 day. It is a figure which shows the infrared absorption spectrum of heparin. In Experimental Example 5, the infrared of the surface of the PVC tube sample coated with DDSA-PLL and the surface of the PVC tube sample coated with heparin via the DDSA-PLL in which the introduction rate of the site derived from DDSA was 20% by mass. It is a figure which shows an absorption spectrum. In Experimental Example 5, the infrared of the surface of the PVC tube sample coated with DDSA-PLL and the surface of the PVC tube sample coated with heparin via the DDSA-PLL in which the introduction rate of the site derived from DDSA was 30% by mass. It is a figure which shows an absorption spectrum. In Experimental Example 5, the surface of the SiPVC tube sample coated with DDSA-PLL having the introduction rate of the DDSA-derived site of 40% by mass, and the surface of the PVC tube sample coated with heparin via the DDSA-PLL It is a figure which shows an infrared absorption spectrum. In Experimental example 6, it is a figure which shows the infrared absorption spectrum of the surface of the silicone rubber tube sample which coated the DDSA-PLL which made the introduction rate of the site|part derived from DDSA 20 mass %. In Experimental example 6, it is a figure which shows the infrared absorption spectrum of the surface of the silicone rubber tube sample which coated the DDSA-PLL which made the introduction rate of the part derived from DDSA 30 mass %. In Experimental example 6, it is a figure which shows the infrared absorption spectrum of the surface of the silicone rubber tube sample which coated the DDSA-PLL which made the introduction rate of the site|part derived from DDSA 40 mass %. In Experimental Example 6, an infrared absorption spectrum of the surface of a silicone rubber tube sample coated with heparin via DDSA-PLL in which the introduction rate of the site derived from DDSA after circulating distilled water for 1 day was 20% by mass is shown. It is a figure. In Experimental Example 6, an infrared absorption spectrum of a surface of a silicone rubber tube sample coated with heparin via DDSA-PLL in which the introduction rate of a site derived from DDSA after circulating distilled water for 1 day was 30 mass% is shown. It is a figure. In Experimental Example 6, an infrared absorption spectrum of a surface of a silicone rubber tube sample coated with heparin via DDSA-PLL in which the introduction rate of a site derived from DDSA after circulating distilled water for 1 day was 40% by mass is shown. It is a figure. It is a photograph which shows the state which heated the DMSO solution containing DDSA-PLL. It is a photograph which shows the state where the temperature of the DMSO solution containing DDSA-PLL decreased.

Embodiments of a heparin-immobilized binder, a method for producing the same, a medical device and a method for producing the same according to the present invention will be described.
It should be noted that the present embodiment is specifically described for better understanding of the gist of the invention, and does not limit the present invention unless otherwise specified.

[Heparin-immobilized binder]
The heparin-immobilized binder of the present embodiment is obtained by introducing an alkyl group into polyamine and has both cationicity and hydrophobicity.

Examples of polyamines include ε-polylysine (PLL) represented by the following chemical formula (2), polyarginine, polyornithine, and polyallylamine. Of these, ε-polylysine is preferable because it has relatively low toxicity and is a cationic polymer.

In the above chemical formula (2), n is 10 or more and 100 or less.

The heparin-immobilized binder of the present embodiment is composed of a partial amidation product in which a part of the amino group of polyamine is amidated with an acid anhydride.
The acid anhydride is not particularly limited as long as it is a hydrophobic compound and can react with a polyamine to amidate a part of the amino group of the polyamine. For example, the following chemical formula Dodecylsuccinic anhydride (DDSA) represented by (3), 3,3-dimethylsuccinic anhydride, phthalic anhydride and the like can be mentioned. Among these, dodecyl succinic anhydride is preferable because of its high hydrophobicity.

In the heparin-immobilized binder of the present embodiment, the polyamine is PLL, and a part of the amino groups of the PLL is preferably a partially amidated product that is amidated with DDSA that is an acid anhydride.
Hereinafter, a partial amidation product in which a part of the amino group of ε-polylysine is amidated with dodecylsuccinic anhydride is abbreviated as DDSA-PLL. This DDSA-PLL is represented by the following chemical formula (4).

In the above chemical formula (4), m is 5 or more and 50 or less, and l is 50 or more and 95 or less.

The introduction rate of the site derived from the acid anhydride in the above partial amidated product (for example, DDSA-PLL) is preferably 20% by mass or more and 40% by mass or less.
When the introduction rate of the site derived from an acid anhydride is at least the above lower limit value, the hydrophobicity is sufficient and the adhesion to the polymer base material can be expected. On the other hand, when the introduction rate of the site derived from the acid anhydride is not more than the above upper limit value, the anionic property is maintained, so that the interaction with heparin can be expected.

The heparin-immobilized binder of the present embodiment is obtained by introducing a DDSA-derived alkyl group into a PLL and has both PLL-derived cationicity and DDSA-derived hydrophobicity. Therefore, the heparin-immobilized binder of the present embodiment can bond the sulfate group of heparin to the amino group derived from PLL by ionic bond. Moreover, since the heparin-immobilized binder of the present embodiment has hydrophobicity, it can be bound to the surface (blood contact surface) of the polymer base material (polymer material) by hydrophobic interaction. Therefore, according to the heparin-immobilized binder of the present embodiment, heparin can be easily and safely immobilized on the surface of the polymer substrate, and the antithrombogenicity can be maintained on the surface of the polymer substrate for a long time. be able to.

[Method for producing heparin-immobilized binder]
The method for producing the heparin-immobilized binder of the present embodiment is a step of reacting a polyamine and an acid anhydride to amidate a part of amino groups of the polyamine with an acid anhydride (hereinafter, referred to as “amidation step”). There is also.)

As the polyamine, as described above, for example, PLL or the like is used.

As the acid anhydride, for example, DDSA or the like is used as described above.

In the method for producing the heparin-immobilized binder of the present embodiment, for example, as shown in the following chemical formula (5), the DDSA-derived hydrophobic site is introduced into the PLL by reacting the PLL with DDSA.

In the method for producing a heparin-immobilized binder of the present embodiment, a step of dissolving a polyamine in a solvent to prepare a polyamine solution before the amidation step (hereinafter, also referred to as “polyamine solution preparation step”). It is preferable to have

The solvent is not particularly limited as long as it can dissolve the polyamine, and examples thereof include dimethyl sulfoxide (DMSO) and dimethylformamide (DMF).

The content of polyamine in the polyamine solution is preferably 1% by mass or more and 20% by mass or less, and more preferably 5% by mass or more and 15% by mass or less.

In the amidation step, an acid anhydride is added to the polyamine solution prepared in the polyamine solution preparation step to react the polyamine with the acid anhydride, and a part of the amino group of the polyamine is amidated with the acid anhydride. As a result, a partial amidated product is obtained in which a part of the amino group of polyamine is amidated with an acid anhydride.
The reaction temperature (the temperature of the mixture of the polyamine solution and the acid anhydride) in the amidation step is preferably 50° C. or higher and 100° C. or lower, and more preferably 60° C. or higher and 80° C. or lower.

The reaction time in the amidation step (the time for reacting the polyamine solution with the acid anhydride) is preferably 0.5 hours or more and 5 hours or less, and more preferably 1 hour or more and 3 hours or less.

The polyamine solution and the acid anhydride are mixed using a device such as a stirring blade, a magnetic stirrer, and an ultrasonic homogenizer, if necessary.

The amount of the acid anhydride added to the polyamine solution is preferably such that the introduction rate of the site derived from the acid anhydride in the partial amidation product (for example, DDSA-PLL) is 20% by mass or more and 40% by mass or less.

For example, when the polyamine is PLL and the acid anhydride is DDSA, the introduction rate of the DDSA-derived site in DDSA-PLL is A (mass %), and the concentration (content rate) of the PLL in the polyamine solution is B (mass %). )), the addition amount X(g) of DDSA to the polyamine solution is represented by the following mathematical expression (α).
X=(10×A×B×268.40)/128 (α)

In formula (α), 268.40 is the weight average molecular weight (Mw) of DDSA, and 128 is the weight average molecular weight (Mw) of the repeating unit of PLL.

The obtained partially amidated product is dissolved in the solvent that constitutes the polyamine solution to form a solution, and thus distilled water is mixed with the solution to precipitate and recover the partially amidated product (collection step).

According to the method for producing a heparin-immobilized binder of the present embodiment, by reacting a polyamine and an acid anhydride and amidating a part of an amino group of the polyamine with an acid anhydride, it is possible to easily convert a partially amidated product. The heparin-immobilized binder of the present embodiment can be obtained.

[Medical equipment]
The medical device of the present embodiment is one in which at least a part of the blood contact surface of a polymer base material (polymer material) used in the medical device is coated with the heparin-immobilized binder of the present embodiment.

FIG. 1 shows a schematic configuration of the medical device according to the present embodiment, and is a cross-sectional view in the thickness direction of a polymer base material forming the medical device.
As shown in FIG. 1, the medical device 10 of the present embodiment includes a polymer base material 11 and a heparin-immobilized binder 12 coated on at least a part of a blood contact surface (one surface) 11a of the polymer base material 11. Have.

The polymer base material 11 is not particularly limited as long as it is generally used for medical devices, and examples thereof include polyethylene, polypropylene, polyvinyl chloride, polycarbonate, silicone rubber and the like.

The blood contact surface 11a of the polymer base material 11 is a surface that comes into contact with blood when the polymer base material 11 is applied to a medical device.

As the heparin-immobilized binder 12, the heparin-immobilized binder of this embodiment is used.

Further, the medical device 10 of the present embodiment has the heparin and the derivative thereof represented by the above chemical formula (1) on at least a part of the blood contact surface 11 a of the polymer-based material 11 via the heparin-immobilized binder 12. At least one of the (heparin derivative) is preferably fixed.

Examples of the heparin derivative include heparan sulfate in which about 50% of the glucosamine residues constituting heparin represented by the above chemical formula (1) are not N-sulfated.

According to the medical device 10 of the present embodiment, since the heparin-immobilized binder 12 is provided on at least a part of the blood contact surface 11a of the polymer-based material 11, the blood-contact surface 11a of the blood contact surface 11a is disposed via the heparin-immobilized binder 12. At least one of heparin and a heparin derivative can be immobilized on at least a part. Further, according to the medical device 10 of the present embodiment, the blood contact surface 11a of the polymer-based material 11 is interposed via the heparin-immobilized binder 12 coated on at least a part of the blood contact surface 11a of the polymer-based material 11. Since at least one of heparin and a heparin derivative is fixed to at least a part of the above, the antithrombogenicity can be maintained on the blood contact surface 11a of the polymer-based material 11 for a long time.

Next, a specific example of the medical device according to this embodiment will be described.

<Tube, reservoir>
The medical device of this embodiment may be a tube. The tube which is the medical device of the present embodiment has a tube body made of a polymer base material, and a heparin-immobilized binder coated on at least a part of the inner wall surface (blood contact surface) of the tube body.

Moreover, the medical device of the present embodiment may be a reservoir. The reservoir which is the medical device of the present embodiment has a reservoir body made of a polymer base material and a heparin-immobilized binder coated on at least a part of the inner wall surface (blood contact surface) of the reservoir body.

The tube and the reservoir of the present embodiment are used, for example, in a blood circulation system.
FIG. 2 is a diagram showing a schematic configuration of an artificial heart-lung machine to which the tube and the reservoir of this embodiment are applied. FIG. 3 is a vertical cross-sectional view for explaining the schematic configuration of the reservoir of the present embodiment, and is a view showing a state in which the pressure sensor is not attached. FIG. 4 is a vertical cross-sectional view illustrating the schematic configuration of the reservoir of the present embodiment, and is a conceptual diagram illustrating the schematic configuration with the pressure sensor attached.

The blood circulation system will be described below.
During or after surgery such as heart surgery, the heart is stopped or brought into a state close to stop, so that a cardiopulmonary system and a blood circulation system for assisted circulation are widely used as necessary.

An artificial heart-lung machine (blood circulation system) 100 including an artificial heart-lung machine, for example, as shown in FIG. 2, a blood removal line 101, a reservoir 102, a blood line 103, and a first blood supply line (blood supply line). ) 104, artificial lung 105, second blood supply line (blood supply line) 106, blood removal sensor (blood removal measuring means) 111, roller pump (blood supply pump) 120, blood removal regulator A (flow rate adjusting means) 121 and a control unit 140 are provided.

The blood removal line 101, the reservoir 102, the blood line 103, the roller pump 120, the first blood supply line 104, the artificial lung 105, and the second blood supply line 106 are connected in this order, and the blood removal line 101 and the blood supply line 101 are connected. The patient (human body) P can be connected via the blood line 106.

Then, the blood that has been removed is temporarily stored in the reservoir 102, and the roller pump (blood pump) 120 causes the patient to pass through the first blood supply line 104, the artificial lung 105, and the second blood supply line 106. It is circulated to P.

One end of the blood removal line 101 is connectable to the patient P, and the blood received from the vein is transferred to the reservoir 102.
Further, the blood removal line 101 is provided with a sensor or the like (not shown) for monitoring the concentration of blood and the concentration of oxygen as necessary. The sensors and the like may be provided in the blood line 103 and the first blood supply line 104 instead of the blood removal line 101.

In addition, the blood removal line 101, the blood line 103, and the first blood supply line 104 are configured by, for example, the tube of this embodiment.
Further, the blood removal line 101 is provided with a blood removal regulator 121 and a blood removal volume sensor 111 in this order. The blood removal amount sensor 111 and the blood removal regulator 121 may be arranged in this order.

The reservoir 102 includes a reservoir body 102A, a head cap 102B attached to the upper portion of the reservoir body 102A, a partition plate 102C arranged inside the reservoir body 102A, a filter 102F, and a pressure sensor 102S. Blood removed and aspirated blood can be stored therein.
The reservoir 102 is composed of, for example, the reservoir of this embodiment.

As shown in FIG. 3, the reservoir body 102A is configured to have a protruding portion that partially extends downward, and the partition plate 102C and the filter 102F define a first storage chamber Q1 and a second storage chamber Q2. ing.
Further, on the outer surface of the reservoir main body 102A, a liquid level scale 102G that allows the blood stored in the reservoir 102 to be visually observed is displayed.

Further, the reservoir body 102A has an inflow port P1 through which blood flows in, and the inflow port P1 is connected to the blood removal line 101, and blood removed through the blood removal line 101 is stored in the first storage chamber. It is designed to flow into Q1.

Further, the blood sucked into the reservoir main body 102A through the suction blood inflow port P2 formed in the head cap 102B flows into the second storage chamber Q2 as necessary.

The blood flowing into the second storage chamber Q2 is guided to the filter 102F by the partition plate 102C, and the filter 102F filters the blood in the second storage chamber Q2 and moves to the first storage chamber Q1. ing.

The first storage chamber Q1 temporarily stores the blood flowing in via the inflow port P1 and the blood flowing in via the filter 102F, and via the outflow port P3 formed at the lower end of the protruding portion of the reservoir body 102A. Blood is allowed to flow to the blood line 103.

Further, the reservoir body 102A has a sensor port 102P for mounting the pressure sensor 102S on the side surface of the protruding portion.
The sensor port 102P is formed with a through hole 102H penetrating the inside and the outside of the protrusion formed below the reservoir body 102A. As shown in FIG. 4, the pressure sensor 102S is provided in the through hole 102H. The pressure sensor 102S is attached in a liquid-tight manner.

When the pressure sensor 102S is not used, as shown in FIG. 3, the plug 102T is liquid-tightly inserted into the through hole 102H instead of the pressure sensor 102S.
With such a configuration, whether or not to use the pressure sensor 102S can be easily and arbitrarily set.

The pressure sensor 102S has a structure in which the pressure detecting portion is covered with a resin film or the like configured to be able to transmit pressure. For example, the pressure sensor 102S is disposed below the liquid surface of the blood stored in the reservoir body 102A, It is configured to sense pressure directly via blood.
The pressure sensor 102S is arranged, for example, in the vicinity of the blood outlet P3, as shown in FIG.

The blood line 103 has the same structure as the blood removal line 101, and transfers blood received from the reservoir 102 to the roller pump 120.

The roller pump 120 includes, for example, a rotary roller and a tube formed outside of the rotary roller and made of a flexible resin, and the rotary roller rotates to squeeze the tube to suck and deliver blood. Thus, the blood stored in the reservoir 102 is sucked through the blood line 103 and is transferred to the artificial lung 105 through the first blood supply line 104.

Further, the roller pump 120 controls the rotation speed of the rotation roller by a rotation control signal output from the control unit 140, and sucks and sends blood in an amount corresponding to the rotation speed of the rotation roller.

The first blood supply line 104 has the same structure as the blood removal line 101, and transfers the blood sent from the roller pump 120 to the artificial lung 105.

The artificial lung 105 includes, for example, a hollow fiber membrane or a flat membrane having excellent gas permeability, and is configured to discharge carbon dioxide in blood and add oxygen.
The artificial lung 105 is integrally formed with, for example, a heat exchanger for adjusting the temperature of blood.

The second blood supply line 106 is adapted to receive carbon dioxide-exhausted blood and oxygenated blood from the artificial lung 105 and transfer it to the artery of the patient P.
The second blood supply line 106 is provided with a filter (not shown) for removing foreign matter in blood such as thrombus and bubbles.
The second blood supply line 106 has the same configuration as the blood removal line 101.

The blood removal regulator 121 is provided in the blood removal line 101, and includes, for example, a clamper 121A including a pair of clamp members, a servomotor (not shown) that operates the clamper 121A, and a blood removal regulator operation unit 121B. The operator manually operates the blood removal regulator operating unit 121B and adjusts the clamp amount (sandwiching amount) of the clamper 121A by the servo motor, thereby changing the cross-sectional area of the blood removal line 101 to remove the blood removal line 101. It is configured to regulate the amount of blood removal flowing through.

The blood removal amount sensor (blood removal amount measuring means) 111 is provided in the blood removal line 101, and, for example, an ultrasonic sensor that measures the flow velocity of blood by ultrasonic waves is used. Is sent to the control unit 140.

[Medical device manufacturing method]
In the method for producing a medical device of the present embodiment, the heparin-immobilized binder of the present embodiment is dissolved in a solvent to prepare a solution containing the heparin-immobilized binder (hereinafter also referred to as “binder solution”). (Hereinafter, also referred to as “binder solution preparation step”), and applying the solution to at least a part of the blood contact surface of the medical device, and applying the heparin to at least a part of the blood contact surface. And a step of coating the immobilized binder (hereinafter, also referred to as “coating step”).

In the binder solution preparation step, the solvent for dissolving the heparin-immobilized binder is not particularly limited as long as it can dissolve the heparin-immobilized binder, and examples thereof include dimethyl sulfoxide (DNSO) and the like.

The content of the heparin-immobilized binder in the binder solution is preferably 0.1% by mass or more and 10% by mass or less, and more preferably 0.5% by mass or more and 5% by mass or less.

The method of applying the binder solution to the blood contact surface of the medical device in the coating step is not particularly limited, and examples thereof include dissolving in a solvent and applying the solution, followed by washing with a solvent.

In the coating step, the amount of the binder solution applied to the blood contact surface of the medical device is not particularly limited, and is appropriately adjusted according to the size and volume of the medical device used for application.

The method for producing a medical device of the present embodiment is a step of fixing at least one of heparin and a heparin derivative on at least a part of the blood contact surface via a heparin-immobilized binder coated on the blood contact surface of the medical device (hereinafter, Sometimes referred to as "heparin immobilization step").

In the heparin immobilization step, the amount of at least one of heparin and heparin derivative immobilized on the blood contact surface of the medical device is not particularly limited, and is appropriately adjusted according to the size and volume of the medical device at the time of application. ..

According to the method for producing a medical device of the present embodiment, at least a part of the blood contact surface of the medical device, by applying a binder solution containing the heparin-immobilized binder of the previous embodiment, at least a part of the blood contact surface. By coating with the heparin-immobilized binder, it is possible to easily obtain a medical device in which at least a part of the blood contact surface is coated with the heparin-immobilized binder. Furthermore, it is possible to obtain a medical device in which at least a part of the blood contact surface is coated with heparin via a heparin-immobilized binder.

Hereinafter, the present invention will be described in more detail with reference to experimental examples, but the present invention is not limited to the following experimental examples.

[Experimental Example 1]
"Preparation of heparin-immobilized binder"
Ε-Polylysine (PLL) represented by the following chemical formula (2) was dissolved in dimethylsulfoxide (DMSO) to prepare a PLL solution having a PLL concentration of 10% by mass.

A predetermined amount of dodecyl succinic anhydride (DDSA) represented by the following chemical formula (3) is added to the PLL solution, and the PLL and DDSA are reacted at 100° C. for 2 hours to obtain the following chemical formula (5 ), a hydrophobic site derived from DDSA was introduced into the PLL to obtain a partial amidation product (DDSA-PLL) in which a part of the amino group of the PLL was amidated with DDSA.

A DDSA-PLL having an introduction rate of a site derived from DDSA of 20% by mass, 30% by mass or 40% by mass was produced.
When the introduction rate of the site derived from DDSA in DDSA-PLL is A (20% by mass, 30% by mass or 40% by mass) and the concentration (introduction rate) of PLL in the polyamine solution is B (% by mass), it is relative to the polyamine solution. The addition amount X(g) of DDSA was calculated from the following mathematical expression (a).
X=(10×A×B×268.40)/128 (a)

In formula (a), 268.40 is the weight average molecular weight (Mw) of DDSA, and 128 is the weight average molecular weight (Mw) of the repeating unit of PLL.
Table 1 shows the addition amount X(g) of DDSA when the introduction rate of the site derived from DDSA is 20% by mass, 30% by mass or 40% by mass.

The obtained partially amidated product was dissolved in a solvent (DMSO) constituting the PLL solution to form a solution, and thus distilled water was mixed with the solution to precipitate and recover the partially amidated product.

"Structural analysis of partially amidated compounds"
The obtained partially amidated product was freeze-dried to give a powder, and then the powder was dissolved in heavy dimethyl sulfoxide to prepare a DMSO solution of the partially amidated product. Using this DMSO solution, the nuclear magnetic resonance (NMR) spectrum ( ¹ H) of the partially amidated product was measured by a nuclear magnetic resonance apparatus (NMR, trade name: AVAVCEIII, manufactured by Bruker), and the molecular structure of the partially amidated product was measured. Was analyzed. The results are shown in FIGS.

Figure 5 is a graph showing the result of ¹ H-NMR measurement concerning partial amides product in which the rate of introduction of sites from DDSA 20 mass%. FIG. 6 is a diagram showing the results of ¹ H-NMR measurement on a partially amidated product in which the introduction rate of the site derived from DDSA was 30 mass %. FIG. 7: is a figure which shows the result of ¹ H-NMR measurement regarding the partial amidation product which made the introduction rate of the site|part derived from DDSA 40 mass %.

From FIGS. 5 to 7, it was confirmed that a predetermined amount of the DDSA-derived site could be introduced into the PLL. From the methyl proton intensity 1 of the spectra in FIGS. 5 to 7, the introduction rate (content rate) of the DDSA-derived site was calculated based on the following mathematical expression (b).
(Introduction rate (mass %) of the site derived from DDSA)=x/3×100 (b)
In the mathematical expression (b), x is the integral value of the methyl proton intensity 1, and 3 is the number of methyl protons.

When the introduction rate of the DDSA-derived site was calculated based on the mathematical expression (b), it was about 22 mass% in FIG. 5, and the target introduction rate (20 mass%) was obtained. Moreover, in FIG. 6, it was about 31 mass %, and the target introduction rate (30 mass %) was obtained. Moreover, in FIG. 7, it was about 41 mass %, and the target introduction rate (40 mass %) was obtained.

[Experimental Example 2]
"Preparation of heparin-immobilized binder"
A PLL solution having a PLL concentration of 10% by mass was prepared in the same manner as in Experimental Example 1.

By adding a predetermined amount of hexanoic anhydride (Hexanoic Anhydride) represented by the following chemical formula (6) to the PLL solution, and reacting the PLL and hexanoic anhydride at 100° C. for 2 hours, As shown in the chemical formula (7), a partial amidation product (PLL-Hexanoic Anhydride) in which a hydrophobic site derived from hexanoic anhydride is introduced into PLL, and a part of the amino group of PLL is amidated with hexanoic anhydride ) Got.

A PLL-Hexanoic Anhydride in which the introduction rate of the site derived from hexanoic anhydride was 20% by mass, 30% by mass or 40% by mass was prepared.

The total amount of the polyamine solution was 10 mL, the introduction rate of the site derived from hexanoic anhydride in the PLL-Hexanoic Anhydride was A2 (20% by mass, 30% by mass or 40% by mass), and the concentration (content rate) of the PLL in the polyamine solution was B2. In the case of (mass %), the addition amount X2 (g) of hexanoic anhydride to the polyamine solution was calculated from the following mathematical expression (c).
X2=(10×A1×B2×214.3)/128 (c)
In formula (c), 214.3 is the weight average molecular weight (Mw) of hexanoic anhydride, and 128 is the weight average molecular weight (Mw) of the repeating unit of PLL.

Table 2 shows the addition amount X(g) of hexanoic anhydride when the introduction rate of the site derived from hexanoic anhydride is 20% by mass, 30% by mass or 40% by mass.

The obtained partially amidated product was dissolved in a solvent (DMSO) constituting the PLL solution to form a solution, and thus the solution was mixed with methanol to precipitate and recover the partially amidated product.

"Structural analysis of partially amidated compounds"
The obtained partially amidated product was freeze-dried to give a powder, and then the powder was dissolved in heavy dimethyl sulfoxide to prepare a DMSO solution of the partially amidated product. Using this DMSO solution, the nuclear magnetic resonance (NMR) spectrum ( ¹ H) of the partially amidated product was measured by a nuclear magnetic resonance apparatus (NMR, trade name: AVAVCEIII, manufactured by Bruker), and the molecular structure of the partially amidated product was measured. Was analyzed. The results are shown in FIGS.

Figure 8 is a diagram showing the result of ^{1 H-NMR} measurement concerning partial amides product where the introduction rate of the sites from hexanoic acid anhydride and 20 wt%. Figure 9 is a diagram showing the result of ^{1 H-NMR} measurement concerning partial amides product in which the rate of introduction of sites from hexanoic acid anhydride 30 wt%. Figure 10 is a diagram showing the result of ^{1 H-NMR} measurement concerning partial amides product where the introduction rate of the sites from hexanoic acid anhydride and 40 wt%.

From FIGS. 8 to 10, it was confirmed that a predetermined amount of sites derived from hexanoic anhydride could not be introduced into the PLL. From the methyl proton intensity 1 in the spectra of FIGS. 8 to 10, the introduction rate of the site derived from hexanoic anhydride was calculated based on the following mathematical formula (d).
(Introduction rate of sites derived from hexanoic anhydride (mass %))=x/3×100 (d)
In the mathematical expression (d), x is the integral value of the methyl proton intensity 1, and 3 is the number of methyl protons.

When the introduction rate of the site derived from hexanoic anhydride was calculated based on the mathematical expression (d), it was 37.48% by mass in FIG. 8, which was a larger value than the target introduction rate (20% by mass). It is considered that this is because the by-product of the partial amidation product is not completely removed due to insufficient washing with methanol. Further, in FIG. 9, the introduction rate of the site derived from hexanoic anhydride calculated based on the mathematical expression (d) was 127.16% by mass, which was a numerical value larger than the target introduction rate (30% by mass). .. It is considered that this is because the by-product of the partial amidation product is not completely removed due to insufficient washing with methanol. Furthermore, in FIG. 10, the introduction rate of the site derived from hexanoic anhydride calculated based on the mathematical formula (d) was about 42.1% by mass, and the target introduction rate (40% by mass) was obtained.

[Experimental Example 3]
"Preparation of heparin-immobilized binder"
A PLL solution having a PLL concentration of 10% by mass was prepared in the same manner as in Experimental Example 1.

By adding a predetermined amount of lauric acid anhydride (Lauric Anhydride) represented by the following chemical formula (8) to the PLL solution, and reacting the PLL with lauric acid anhydride at 100° C. for 2 hours, As shown in the chemical formula (9), a partial amide compound (PLL-Lauric Anhydride) in which a hydrophobic moiety derived from lauric anhydride is introduced into PLL and a part of the amino group of PLL is amidated with lauric anhydride ) Got.

A PLL-Lauric Anhydride in which the introduction rate of the site derived from lauric anhydride was 20% by mass, 30% by mass or 40% by mass was prepared.

The total amount of the polyamine solution was 10 mL, the introduction rate of the site derived from lauric anhydride in PLL-Lauric Anhydride was A3 (20% by mass, 30% by mass or 40% by mass), and the concentration (content rate) of the PLL in the polyamine solution was B3. In the case of (mass %), the addition amount X3 (g) of lauric anhydride to the polyamine solution was calculated from the following mathematical expression (e).
X2=(10×A1×B2×3822.63)/128 (e)
In formula (e), 382.63 is the weight average molecular weight (Mw) of hexanoic anhydride, and 128 is the weight average molecular weight (Mw) of the repeating unit of PLL.

Table 3 shows the addition amount X(g) of lauric anhydride when the introduction rate of the site derived from lauric anhydride is 20% by mass, 30% by mass or 40% by mass.

The obtained partial amidated product was dissolved in the solvent (DMSO) constituting the PLL solution to form a solution. Therefore, 2-propanol was mixed with the solution to precipitate and recover the partially amidated product.

"Structural analysis of partially amidated compounds"
The obtained partially amidated product was freeze-dried to give a powder, and then the powder was dissolved in heavy dimethyl sulfoxide to prepare a DMSO solution of the partially amidated product. Using this DMSO solution, the nuclear magnetic resonance (NMR) spectrum ( ¹ H) of the partially amidated product was measured by a nuclear magnetic resonance apparatus (NMR, trade name: AVAVCEIII, manufactured by Bruker), and the molecular structure of the partially amidated product was measured. Was analyzed. The results are shown in Fig. 11.

FIG. 11: is a figure which shows the result of ¹ H-NMR measurement regarding the partial amidation product which made the introduction rate of the site|part derived from a lauric acid anhydride 20 mass %.
From FIG. 11, it was confirmed that a predetermined amount of the site derived from lauric anhydride could not be introduced into the PLL. Since the peaks are complicated, it is suggested that the reaction is unlikely to occur between PLL and lauric anhydride.

From the above results, it can be judged that the intended partial amidated product is obtained when hexanoic anhydride is used, but the synthesis of PLL and lauric anhydride is difficult. However, PLL-Hexanoic Anhydride has high hydrophobicity, and although it was tried to dissolve it in various solvents (water, methanol, ethanol, tetrahydrofuran (THF), DMSO, 2-propanol, etc.), it was found that it was soluble in these solvents. It was difficult. Therefore, it was determined that PLL-Hexanoic Anhydride was difficult to use in the subsequent experiments. After that, an experiment on immobilization on a polymer substrate was conducted using DDSA-PLL.

[Experimental Example 4]
"Preparation of silicone rubber tube sample"
The introduction rate of the site derived from DDSA obtained in Experimental Example 1 was 20% by mass, 30% by mass or 40% by mass of DDSA-PLL was dissolved in dimethyl sulfoxide (DMSO), and 1 w/v% of DDSA-PLL was obtained. A solution was prepared.

A silicone rubber tube (trade name: Silicone tube, manufactured by As One Co., Ltd.) was immersed in the obtained solution for 1 day, washed with a phosphate buffer solution (pH=7.0), dried, and DDSA was formed on the inner surface. -A silicone rubber tube sample coated with PLL was obtained.
Then, after adding a heparin aqueous solution having a heparin content of 1% by mass into a DDSA-PLL-coated silicone rubber tube sample, the sample was washed with a phosphate buffer solution (pH=7.0), and dried, A silicone rubber tube sample having an inner surface coated with heparin via DDSA-PLL was obtained.

"Measurement of contact angle"
With respect to the DDSA-PLL-coated silicone rubber tube sample and the heparin-coated silicone rubber tube sample, the contact angle on the inner surface of the silicone rubber tube sample was measured by the following procedure. Similarly, for comparison, the contact angle on the inner surface of the silicone rubber tube sample not coated with DDSA-PLL (untreated) and the contact angle on the inner surface of the silicone rubber tube sample directly coated with heparin. Was measured.

(Procedure 1) Software (product name: FAMAS, manufactured by Kyowa Interface Science Co., Ltd.) is started.
(Procedure 2) A silicone rubber tube sample coated with heparin is placed on an automatic contact angle meter (trade name: Dm-301, manufactured by Kyowa Interface Science Co., Ltd.) via DDSA-PLL.
(Procedure 3) Droplets (water droplets) having a liquid volume of 2.0 μL are prepared and dropped on the surface of the silicone rubber tube sample.

The contact angle of the dropped drop is measured using the software described above. The contact angle analysis method is as follows.

(Contact angle analysis method)
As the contact angle analysis method, the contact angle was analyzed using the width height method (θ/2 method).
FIG. 12 is a schematic diagram for explaining the contact angle analysis method.
As a result of the image processing, as shown in FIG. 12, the left and right end points of the droplet 300 dropped on the surface 200a of the silicone rubber tube sample 200 coated with heparin via DDSA-PLL (the surface 200a and the droplet 300 are The points 300a and 300b that contact each other and the vertex 300c of the droplet 300 are specified, and the radius (r) and the height (h) of the image of the droplet 300 are obtained.

The calculated value of the radius (r) and the value of the height (h) are substituted into the following formula (f) to calculate the contact angle θ. That is, according to the width height method (θ/2 method), the angle θ of the straight line α connecting the left and right end points 300a and 300b of the droplet 300 and the vertex 300c of the droplet 300 with respect to the surface 200a of the silicone rubber tube sample 200. _The contact angle θ can be obtained by obtaining ₁ and multiplying the angle θ ₁ by 2.
θ=2θ ₁ (f)

Results are shown in FIG.
FIG. 13 is a diagram showing the analysis results of the contact angles of the surface of the silicone rubber tube sample coated with DDSA-PLL and the surface of the silicone rubber tube sample coated with heparin in Experimental Example 4.

From FIG. 13, it was confirmed that the DDSA-PLL coating exhibited hydrophobicity with respect to an untreated silicone rubber tube (control). Further, it was confirmed that when heparin was coated via DDSA-PLL, the silicone rubber tube coated with DDSA-PLL exhibited hydrophilicity. Since the heparin molecule contains a sulfonic acid group, it is considered that it shows hydrophilicity.

"Fourier transform infrared spectroscopy (FT-IR) surface analysis"
The surface of the silicone rubber tube sample coated with DDSA-PLL has PLL-derived amino groups. When the surface of the silicone rubber tube sample is coated with DDSA-PLL, the spectrum is analyzed by Fourier transform infrared spectroscopy (FT-IR), and it is affected by NH stretching vibration in the region of 3300 cm ^{−1 to} 3030 cm ^−1. Shows absorption. Therefore, according to the following procedure, the surface of the silicone rubber tube sample coated with DDSA-PLL by ATR FT-IR using a Fourier transform infrared spectrophotometer (trade name: FT/IR-400, manufactured by JASCO Corporation) The infrared absorption spectrum of was measured.

(Procedure 11) A silicone rubber tube sample coated with DDSA-PLL is prepared.
(Procedure 12) After the surface of the infrared transmission prism is washed with ethanol, the infrared absorption spectrum of the surface of the silicone rubber tube sample coated with DDSA-PLL is measured by ATR FT-IR.
(Procedure 13) An infrared absorption spectrum is analyzed using analysis software (trade name: spectra manager, manufactured by JASCO Corporation).

The results are shown in FIGS.
FIG. 14 is a diagram showing an infrared absorption spectrum of the surface of a silicone rubber tube sample coated with DDSA-PLL in which the introduction rate of the site derived from DDSA is 20% by mass. FIG. 15 is a diagram showing an infrared absorption spectrum of the surface of a silicone rubber tube sample coated with DDSA-PLL in which the introduction rate of the site derived from DDSA was 30% by mass. FIG. 16 is a diagram showing an infrared absorption spectrum of the surface of a silicone rubber tube sample coated with DDSA-PLL in which the introduction rate of the site derived from DDSA was 40% by mass.

14 to 16, control is the infrared absorption spectrum of the surface of the silicone rubber tube sample not coated with DDSA-PLL.
From 14 to ^16, it was possible to confirm an absorption peak due to N-H stretching vibration in the region of 3000cm ^-1 ~3500cm -1. Therefore, it was confirmed that DDSA-PLL was immobilized on the surface of the silicone rubber tube sample.

"Surface analysis by X-ray photoelectron spectroscopy (XPS)"
(1) Surface Analysis of Silicone Rubber Tube Sample Coated with DDSA-PLL Using an X-ray photoelectron spectrometer (trade name: ESCALAB250Xi, manufactured by Thermo Fisher Scientific Co., Ltd.), the DDSA-PLL was coated by the following procedure. The element ratio was measured on the surface of the silicone rubber tube sample.

(Procedure 21) The sample is set in the X-ray photoelectron spectrometer by using tweezers so that the sample surface is not contaminated.
(Procedure 22) In surface analysis by X-ray photoelectron spectroscopy, the inside of the X-ray photoelectron spectrometer needs to be analyzed in an ultra-vacuum state, so the inside of the X-ray photoelectron spectrometer is decompressed for one day.
(Procedure 23) After decompressing the inside of the X-ray photoelectron spectrometer, the surface of the sample is irradiated with X-rays to perform qualitative/quantitative analysis of elements on the surface of the sample.
(Procedure 24) Peak separation was performed on the N1s and S2p spectra obtained by surface analysis by X-ray photoelectron spectroscopy, and the peak of the amino group derived from PLL was confirmed from each peak, and DDSA- on the surface of the silicone rubber tube sample was confirmed. Check if the PLL is locked.

The results are shown in FIGS.
17 to 20 are diagrams showing the results of analysis by X-ray photoelectron spectroscopy of the surface of a silicone rubber tube sample not coated with DDSA-PLL. 21 to 24 are diagrams showing the analysis results by X-ray photoelectron spectroscopy of the surface of the silicone rubber tube sample coated with DDSA-PLL in which the introduction rate of the DDSA-derived site was 5% by mass. 25 to 28 are diagrams showing the analysis results by X-ray photoelectron spectroscopy of the surface of the silicone rubber tube sample coated with DDSA-PLL in which the introduction rate of the site derived from DDSA was 20% by mass. 29 to 32 are diagrams showing the results of analysis by X-ray photoelectron spectroscopy on the surface of a silicone rubber tube sample coated with DDSA-PLL in which the introduction rate of the site derived from DDSA was 30% by mass. 33 to 36 are diagrams showing the results of analysis by X-ray photoelectron spectroscopy of the surface of a silicone rubber tube sample coated with DDSA-PLL in which the introduction rate of the site derived from DDSA was 40% by mass.

23, 27, 31 and 35, since the peak of the amino group derived from PLL was detected in the binding energy of 400 eV to 403 eV, DDSA-PLL could be immobilized on the surface of the silicone rubber tube sample. it is conceivable that.

Further, the analysis results shown in FIGS. 17 to 36 are collectively shown in FIG.
From FIG. 37, in the silicone rubber tube sample (control) not coated with DDSA-PLL and the silicone rubber tube sample coated with DDSA-PLL in which the introduction rate of the site derived from DDSA was 5% by mass, nitrogen (N) Although there is no significant change in the quantitative value, when the introduction rate of the site derived from DDSA is increased, the ratio of nitrogen (N) increases, and DDSA-PLL can be immobilized on the surface of the silicone rubber tube sample.

(2) Surface analysis of silicone rubber tube sample coated with heparin via DDSA-PLL Similar to the surface analysis of silicone rubber tube sample coated with DDSA-PLL, silicone rubber coated with heparin via DDSA-PLL The element ratio was measured on the surface of the tube sample.

The results are shown in FIGS. 38 to 62.
38 to 42 are diagrams showing the results of analysis by X-ray photoelectron spectroscopy of the surface of a silicone rubber tube sample coated with heparin, not via DDSA-PLL. 43 to 47 are diagrams showing the results of analysis by X-ray photoelectron spectroscopy of the surface of a silicone rubber tube sample coated with heparin via DDSA-PLL with the introduction rate of the site derived from DDSA being 5% by mass. .. 48 to 52 are diagrams showing the results of analysis by X-ray photoelectron spectroscopy of the surface of a silicone rubber tube sample coated with heparin via DDSA-PLL with the introduction rate of the site derived from DDSA being 20% by mass. .. 53 to 57 are diagrams showing the analysis results by X-ray photoelectron spectroscopy of the surface of a silicone rubber tube sample coated with heparin via DDSA-PLL with the introduction rate of the site derived from DDSA being 30% by mass. .. 58 to 62 are diagrams showing the results of analysis by X-ray photoelectron spectroscopy of the surface of a silicone rubber tube sample coated with heparin via DDSA-PLL with the introduction rate of the site derived from DDSA being 40% by mass. ..

From FIG. 41, the peak of the sulfonic acid group derived from heparin could not be confirmed, and it could not be confirmed that heparin was immobilized on the surface of the silicone rubber tube sample not coated with DDSA-PLL.

46, FIG. 51, FIG. 56 and FIG. 61, since the peak of the sulfonic acid group derived from heparin was detected when the binding energy was 168 eV to 171 eV, heparin was detected on the surface of the silicone rubber tube sample via DDSA-PLL. It is thought that it has been fixed.

Further, the analysis results shown in FIGS. 38 to 62 are collectively shown in FIG. 63.
From FIG. 63, in the silicone rubber tube sample obtained by coating the silicone rubber tube sample (control) not coated with DDSA-PLL with heparin, sulfur (S) of the sulfonic acid group could not be confirmed. It was confirmed that when the introduction rate of the site derived from DDSA was increased, the proportion of sulfur (S) in the sulfonic acid group increased, and the amount of heparin immobilized on the surface of the silicone rubber tube sample tended to increase.

"Zeta potential measurement"
Using a zeta potential measurement system (trade name: ELSZ-2, manufactured by Otsuka Electronics Co., Ltd.), the ζ potential was measured on the surface of the silicone rubber tube sample coated with heparin via DDSA-PLL by the following procedure.

<Gel coating>
(Procedure 31) A flat plate quartz cell is immersed in concentrated sulfuric acid for 15 to 30 minutes, and then, the flat plate quartz cell is thoroughly washed with secondary distilled water, further washed with methanol, and dried by nitrogen blowing.
(Procedure 32) A flat-plate quartz cell is fired at 160° C. for 8 hours to 10 hours.

(Procedure 33) In a 50 mL centrifuge tube, 50 mL of 60% 3-methacryloyloxypropyltriethoxysilane (MPTMS, trade name: T2676, CAS: 211242-29-0, manufactured by Tokyo Chemical Industry Co., Ltd.) was prepared, and the methanol solution was prepared. , 0.22 μm through an organic solvent resistant filter.

(Procedure 34) After cooling the flat plate quartz cell to around room temperature, 0.1 mol/L to 0.5 mol/L sodium hydroxide aqueous solution is injected into a 50 mL centrifuge tube, and the flat plate quartz cell is immersed therein to React for 5 hours. The plate quartz cell is thoroughly washed with secondary distilled water, further washed with methanol, and dried by nitrogen blow. Immediately thereafter, 60% 3-methacryloyloxypropyltriethoxysilane solution in methanol was poured over the surface of the flat plate quartz cell 10 times with a Pasteur pipette. Further, the flat plate quartz cell is immersed in a remaining 60% methanol solution of 3-methacryloyloxypropyltriethoxysilane, and reacted at 30° C. for 8 hours to 10 hours.

(Procedure 35) 1.05 g of acrylamide-HG (AAm, trade name: 011-08015, manufactured by Wako Pure Chemical Industries, Ltd.) and potassium peroxodisulfate (PPOS, trade name: 169-11891, Wako Pure Chemical Industries, Ltd.) 30 mg) is put into a 50 mL centrifuge tube, and AAm and PPOS are dissolved in 30 mL of secondary distilled water.

(Procedure 36) After the flat plate quartz cell immersed in the methanol solution is thoroughly washed with methanol, the flat plate quartz cell is alternately washed with secondary distilled water and methanol, and then the methanol is dried by nitrogen blow.

(Procedure 37) To an aqueous solution of AAm and PPOS, 30 μL of N,N,N′,N′-tetramethylethylenediamine (TEMED, trade name: 205-06313, manufactured by Wako Pure Chemical Industries, Ltd.) was added, and turned over. After stirring for 2 to 3 times, the flat plate quartz cell prepared in step 36 is immediately added to the aqueous solution, and left standing for 2 hours at room temperature.

(Procedure 38) After that, the flat plate quartz cell is thoroughly washed with secondary distilled water and then washed with 0.5 mol/L hydrochloric acid to remove excess AAm. The washing with 0.5 mol/L hydrochloric acid is performed twice with a volume of 25 mL each.

(Procedure 39) The flat plate quartz cell is thoroughly washed with secondary distilled water to remove hydrochloric acid, and then the flat plate quartz cell is immersed in secondary distilled water and temporarily stored until zeta potential measurement so as not to dry. ..

<Measurement preparation>
(Procedure 40) Prepare a 10 mmol/L sodium chloride aqueous solution.
(Procedure 41) The stirrer chip was placed in a container containing the aqueous sodium chloride solution prepared in step 40, and the stirrer chip was rotated with a magnetic stirrer to stir the aqueous sodium chloride solution for 10 minutes with a water aspirator. Degas the aqueous sodium chloride solution for ~15 minutes.

(Procedure 42) The polystyrene sulfonic acid particle stock solution for ζ potential measurement (monitor particle, commercially available product) is diluted 250 times with the degassed sodium chloride aqueous solution prepared in Procedure 11 to prepare 10 mL of a solution.

(Procedure 43) A flat plate quartz cell is installed in a zeta potential measuring system, and a silicone rubber tube sample coated with DDSA-PLL, a silicone rubber tube sample coated with heparin, and DDSA-PLL are placed in the flat plate quartz cell. The uncoated (untreated) silicone rubber tube samples are allowed to stand and the zeta potential on the surface of these samples is measured.

(Procedure 44) After measuring the ζ potential, the ζ potential is analyzed by software (trade name: ELSZ, manufactured by Otsuka Electronics Co., Ltd.).

The results are shown in Fig. 64.
FIG. 64 shows the ζ potentials of the surface of the silicone rubber tube sample coated with DDSA-PLL, the surface of the silicone rubber tube sample coated with heparin, and the surface of the silicone rubber tube sample not coated with DDSA-PLL. It is a figure which shows the measured result.

From FIG. 64, the surface of the silicone rubber tube sample has a positive charge because it contains an amino group derived from PLL when only DDSA-PLL is immobilized, and heparin is immobilized via DDSA-PLL. It was confirmed that the compound had a negative charge because it contained a sulfonic acid group derived from heparin. It was also confirmed that, when DDSA-PLL was immobilized, the surface of the silicone rubber tube sample had a larger positive charge as the introduction rate of the site derived from DDSA increased. Furthermore, when heparin was immobilized via DDSA-PLL in which the introduction rate of the site derived from DDSA was 30% by mass to 40% by mass, it was confirmed that the surface of the silicone rubber tube sample had a negative charge. Was done. Therefore, by immobilizing DDSA-PLL having a high introduction rate of a site derived from DDSA, the surface charge of the silicone rubber tube sample increases, and by immobilizing heparin, the surface charge of the silicone rubber tube sample decreases. It has been suggested.

[Experimental Example 5]
"Preparation of polyvinyl chloride tube samples"
The introduction rate of the site derived from DDSA obtained in Experimental Example 1 was 20% by mass, 30% by mass or 40% by mass of DDSA-PLL was dissolved in dimethyl sulfoxide (DMSO), and 1 w/v% of DDSA-PLL was obtained. A solution was prepared.

A polyvinyl chloride (PVC) tube (trade name: transparent PVC tube, manufactured by As One Co., Ltd.) was immersed in the obtained solution for 1 day, washed with a phosphate buffer (pH=7.0), and dried. Thus, a PVC tube sample whose inner surface was coated with DDSA-PLL was obtained.
Then, after adding an aqueous solution of heparin having a heparin content of 1% by mass into a PVC tube sample coated with DDSA-PLL, the sample was washed with a phosphate buffer solution (pH=7.0), dried, and then dried. A PVC tube sample coated with heparin via DDSA-PLL was obtained.

"Measurement of contact angle"
For the PVC tube sample coated with DDSA-PLL and the PVC tube sample coated with heparin, the contact angle on the inner surface of the PVC tube was measured in the same manner as in Experimental Example 4. Similarly, for comparison, the contact angle on the inner surface of the PVC tube sample not coated with DDSA-PLL (untreated) and the contact angle on the inner surface of the PVC tube sample directly coated with heparin were measured. did. The results are shown in Fig. 65.

FIG. 65 is a diagram showing the analysis results of the contact angles of the surface of the PVC tube sample coated with DDSA-PLL and the surface of the PVC tube sample coated with heparin.

From FIG. 65, it was confirmed that when DDSA-PLL was coated, it showed hydrophobicity with respect to an untreated silicone rubber tube (control). This is probably because DMSO elutes the plasticizer contained in the PVC tube and changes the structure of the PVC tube. Further, it was confirmed that when heparin was coated via DDSA-PLL, the silicone rubber tube coated with DDSA-PLL exhibited hydrophilicity. Since the heparin molecule contains a sulfonic acid group, it is considered that it shows hydrophilicity.

"Fourier transform infrared spectroscopy (FT-IR) surface analysis"
(Surface analysis of PVC tube sample coated with DDSA-PLL)
A surface of a PVC tube sample coated with DDSA-PLL by ATR FT-IR using a Fourier transform infrared spectrophotometer (trade name: FT/IR-400, manufactured by JASCO Corporation) in the same manner as in Experimental Example 4. The infrared absorption spectrum of was measured.

The results are shown in FIGS. 66 to 70.
FIG. 66 is a diagram showing an infrared absorption spectrum of the surface of a PVC tube sample not coated with DDSA-PLL. FIG. 67 is a diagram showing an infrared absorption spectrum of the surface of a PVC tube sample coated with DDSA-PLL in which the introduction rate of the site derived from DDSA was 20 mass %. FIG. 68 is a diagram showing an infrared absorption spectrum of the surface of a PVC tube sample coated with DDSA-PLL in which the introduction rate of the site derived from DDSA was 30% by mass. FIG. 69 is a diagram showing an infrared absorption spectrum of the surface of a SiPVC tube sample coated with DDSA-PLL in which the introduction rate of a site derived from DDSA was 40% by mass. 70 is a diagram in which FIGS. 66 to 69 are overlapped.

From FIG. 66 to FIG. ^70, it was possible to confirm an absorption peak due to N-H stretching vibration in the region of 3000cm ^-1 ~3500cm -1. It was ^also possible to confirm the C = O, the absorption peak due to binding of the C-N, N-H in the region of 1500cm ^-1 ~1600cm -1. Therefore, it was confirmed that DDSA-PLL was immobilized on the surface of the PVC tube sample.

"Evaluation of chemical resistance of polyvinyl chloride against dimethyl sulfoxide"
A polyvinyl chloride (PVC) sheet (product name: PVC sheet, manufactured by Izumi Kogyo Kogyo Co., Ltd.) having a length of 10 mm, a width of 10 mm, and a thickness of 1 mm is immersed in 10 mL of dimethyl sulfoxide (DMSO) at 25° C. for 1 day. did. This PVC sheet is a sheet made of soft PVC.

FIG. 71 is a photograph showing a PVC sheet before being dipped in DMSO. FIG. 72 is a photograph showing a PVC sheet after being immersed in DMSO for 1 day.

From FIG. 72, as a result of immersing the PVC sheet in DMSO for 1 day, the PVC sheet became cloudy and the soft PVC was hardened. Since DMSO is a highly polar solvent and PVC has low chemical resistance, it is considered that PVC was dissolved in DMSO.
As a result of surface analysis by FT-IR, it was confirmed that DDSA-PLL could be immobilized on the PVC tube sample, but when the PVC sheet was immersed in DMSO for 1 day, the PVC sheet became cloudy.

"Fourier transform infrared spectroscopy (FT-IR) surface analysis"
(Surface analysis of PVC tube sample coated with heparin via DDSA-PLL)
In the same manner as in Experimental Example 4, ATR FT-IR using a Fourier transform infrared spectrophotometer (trade name: FT/IR-400, manufactured by JASCO Corporation) was used to remove heparin via heparin and DDSA-PLL. The infrared absorption spectrum of the surface of the coated PVC tube sample was measured.

The results are shown in FIGS. 73 to 76.
FIG. 73 is a diagram showing an infrared absorption spectrum of heparin. FIG. 74 shows the infrared absorption of the surface of a PVC tube sample coated with DDSA-PLL in which the introduction rate of the site derived from DDSA was 20% by mass, and the surface of the PVC tube sample coated with heparin via the DDSA-PLL. It is a figure which shows a spectrum. FIG. 75 shows the infrared absorption of the surface of a PVC tube sample coated with DDSA-PLL in which the introduction rate of the site derived from DDSA was 30% by mass, and the surface of the PVC tube sample coated with heparin via the DDSA-PLL. It is a figure which shows a spectrum. FIG. 76 shows the infrared of the surface of the SiPVC tube sample coated with DDSA-PLL in which the introduction rate of the site derived from DDSA was 40% by mass, and the surface of the PVC tube sample coated with heparin via the DDSA-PLL. It is a figure which shows an absorption spectrum.

In FIG. 73, the absorption peaks at 991 cm ⁻¹ and 1228 cm ⁻¹ represent the sulfonic acid group of the heparin molecule, and the absorption peak at 3370 cm ⁻¹ represents the sulfonic acid amide. From FIG. 74 to FIG. ^76, it was not possible to confirm the absorption peak of the sulfonic acid groups derived from heparin in the region of 900cm ^-1 ~1200cm -1. However, since there is an absorption peak in the vicinity of 3500 cm ^-1 , a peak thought to be sulfonic acid amide derived from heparin was confirmed. Therefore, it is considered possible to immobilize heparin on the surface of a PVC tube sample.

[Experimental example 6]
"Antithrombogenicity evaluation"
(Preparation of silicone rubber tube sample)
The introduction rate of the site derived from DDSA obtained in Experimental Example 1 was 20% by mass, 30% by mass or 40% by mass of DDSA-PLL was dissolved in dimethyl sulfoxide (DMSO), and 1 w/v% of DDSA-PLL was obtained. A solution was prepared.

A 50 cm long silicone rubber tube (trade name: Silicone tube, manufactured by As One Co., Ltd.) was dipped in the obtained solution for 1 day, washed with a phosphate buffer (pH=7.0), and dried. A silicone rubber tube sample whose inner surface was coated with DDSA-PLL was obtained.
Then, after adding a heparin aqueous solution having a heparin content of 1% by mass into a DDSA-PLL-coated silicone rubber tube sample, the sample was washed with a phosphate buffer solution (pH=7.0), and dried, A silicone rubber tube sample having an inner surface coated with heparin via DDSA-PLL was obtained.

The antithrombogenicity was evaluated using the obtained silicone rubber tube sample coated with heparin via DDSA-PLL (hereinafter, also referred to as "silicone rubber tube sample A").

(Antithrombogenicity test)
An aqueous solution of trisodium citrate was added to commercially available bovine blood to prepare citrated bovine blood. The citrated cow blood was centrifuged, and the resulting supernatant was used as blood for antithrombotic test. Hereinafter, this blood is referred to as plasma. The concentration of the trisodium citrate aqueous solution added to bovine blood (the concentration of trisodium citrate dihydrate in the trisodium citrate aqueous solution) was 1.7 mol/L. The amount of the trisodium citrate aqueous solution added to bovine blood was adjusted so that the trisodium citrate aqueous solution was 10 mL per 1 L of plasma. The citric acid concentration in plasma was 0.017 mol/L. The amount of citric acid in the plasma injected into the silicone rubber tube sample A was 6.8×10 ⁻⁶ mol.
The antithrombotic test was performed by the following procedure.

(Procedure 51) One end of the silicone rubber tube sample A is closed with forceps or the like, and 0.4 mL of plasma and 0.1 mL of a 0.125 mol/L calcium chloride aqueous solution are injected into the silicone rubber tube sample A.
(Procedure 52) Thereafter, the other end of the silicone rubber tube sample A is also closed with forceps or the like.
(Procedure 53) After the liquid in the silicone rubber tube sample A is mixed by inversion stirring, the silicone rubber tube sample A is allowed to stand still in water at 37°C.
(Procedure 54) Every 1 minute after standing, the silicone rubber tube sample A is tilted to observe whether plasma is coagulated.
(Procedure 55) When plasma is coagulated, the elapsed time at that time is recorded. If the plasma does not coagulate after 120 minutes or more, the observation is terminated at that time.
The results are shown in Table 4.

In Table 4, control indicates an untreated silicone rubber tube sample.
From Table 4, blood coagulation was confirmed in about 15 minutes in the untreated silicone tube sample. In the silicone rubber tube sample coated with heparin via DDSA-PLL (silicone rubber tube sample A), coagulation of blood was not confirmed even after 120 minutes. Therefore, the silicone rubber tube sample A is considered to have the strong antithrombotic function of heparin.

(Running water test)
Using a micro tube pump (trade name: MP-1000, manufactured by Tokyo Rika Kikai Co., Ltd.), distilled water contained in a beaker is circulated in a silicone rubber tube sample coated with DDSA-PLL at a flow rate of 60 mL/min. Then, the bonding state of the DDSA-PLL and the silicone rubber tube sample was evaluated. The test was terminated when the silicone rubber tube sample failed.

A silicone rubber tube sample coated with DDSA-PLL by ATR FT-IR using a Fourier transform infrared spectrophotometer (trade name: FT/IR-400, manufactured by JASCO Corporation) in the same manner as in Experimental Example 4. The bonding state of the DDSA-PLL and the silicone rubber tube sample was evaluated by measuring the infrared absorption spectrum of the surface.

The results are shown in FIGS. 77 to 82. 77 to 82, control represents an untreated silicone rubber tube sample.
FIG. 77 is a diagram showing an infrared absorption spectrum of the surface of a silicone rubber tube sample coated with DDSA-PLL in which the introduction rate of the site derived from DDSA is 20% by mass. FIG. 78 is a diagram showing an infrared absorption spectrum of the surface of a silicone rubber tube sample coated with DDSA-PLL in which the introduction rate of the site derived from DDSA was 30% by mass. FIG. 79 is a diagram showing an infrared absorption spectrum of the surface of a silicone rubber tube sample coated with DDSA-PLL in which the introduction rate of the site derived from DDSA was 40% by mass. FIG. 80 is a diagram showing an infrared absorption spectrum of the surface of a silicone rubber tube sample coated with heparin via DDSA-PLL in which the introduction rate of the site derived from DDSA after circulating distilled water for 1 day was 20 mass %. Is. FIG. 81 is a diagram showing an infrared absorption spectrum of a surface of a silicone rubber tube sample coated with heparin via DDSA-PLL in which the introduction rate of the site derived from DDSA after circulating distilled water for 1 day was 30 mass %. Is. FIG. 82 is a diagram showing an infrared absorption spectrum of the surface of a silicone rubber tube sample coated with heparin via DDSA-PLL in which the introduction rate of the site derived from DDSA after circulating distilled water for 1 day was 40 mass %. Is.

From FIG. 77 to FIG. ^79, it was possible to confirm an absorption peak due to N-H stretching vibration in the region of 3000cm ^-1 ~3500cm -1. Therefore, it was confirmed that DDSA-PLL was immobilized on the surface of the silicone rubber tube sample.

A silicone rubber tube sample coated with heparin via DDSA-PLL having a DDSA-derived site introduction rate of 20 mass% and heparin coated via DDSA-PLL having a DDSA-derived site introduction rate of 40 mass% Since the silicone rubber tube sample was broken one day after the circulation of distilled water started, the test was terminated at that time. From FIGS. 80 and 82, although the silicone rubber tube was broken, it was confirmed that DDSA-PLL and heparin were immobilized in these samples.

The silicone rubber tube sample coated with heparin through DDSA-PLL with the introduction rate of the site derived from DDSA being 30% by mass was tested at that time because the silicone rubber tube was broken 2 days after the circulation of distilled water was started. finished. From FIG. 81, it can be confirmed that the absorption peak is slightly decreased one day after the circulation of distilled water is started. In addition, it is considered that DDSA-PLL is detached from the silicone rubber tube because it approaches the control peak two days after the start of circulation of distilled water. Therefore, in order to immobilize DDSA-PLL on the silicone rubber tube, it is necessary to change the conditions when dipping the silicone rubber tube in a DMSO solution containing DDSA-PLL. When the DMSO solution containing DDSA-PLL is heated, as shown in FIG. 83, the DDSA-PLL is completely dissolved in DMSO, and the DMSO solution becomes transparent. Moreover, when the temperature of the DMSO solution containing DDSA-PLL is lowered, the DMSO solution containing DDSA-PLL becomes slightly cloudy as shown in FIG. It is considered that this is because the solubility of DDSA-PLL decreases as the temperature decreases. Therefore, when immersing the silicone rubber tube in the DMSO solution containing DDSA-PLL, by allowing the DMSO solution containing DDSA-PLL to stand while heating, more DDSA-PLL can be prepared for the silicone rubber tube. It is thought that it can be immobilized.

As a result of the above antithrombotic test, coagulation of blood was not confirmed even after 120 minutes in the silicone rubber tube sample coated with heparin via DDSA-PLL. Therefore, it is considered that the above-mentioned silicone rubber tube sample has a strong antithrombotic function of heparin. From this result, it was considered that when blood was flown into the silicone rubber tube sample, heparin was released from the silicone rubber tube, the heparin was mixed with the blood, and the blood did not coagulate. Therefore, when the above running water test was performed, the blood did not coagulate because the antithrombotic function of heparin worked because DDSA-PLL did not detach from the silicone rubber tube even one day after the circulation of distilled water started. It is believed that

Further, the silicone rubber tube sample after being coated with heparin via DDSA-PLL and dried was washed with phosphate buffered saline.
In the case of heparin bound in an ionic bond type, it was considered that heparin was dissociated by washing with phosphate buffered saline having high ionic strength. However, from the above experimental results, it is considered that the ionic bond type of PLL and heparin is unlikely to dissociate heparin by washing with phosphate buffered saline. Since the antithrombotic function of heparin is working, it is considered that the contact angle of DDSA-PLL is balanced.

In the running water test, distilled water was circulated very gently at a flow rate of 60 mL/min. Mainly in extracorporeal circulation, blood is circulated at a flow rate of 2.2 L/min to 2.5 L/min in many facilities, and blood is circulated at a flow rate of 4 L/min to 5 L/min particularly in cardiac surgery. .. It has been reported that the blood flow rate in the heart-lung machine also contributes to oxygen supply to the human body. Therefore, it is necessary to examine whether the immobilization of DDSA-PLL can be maintained even if the blood flow rate is increased to the reference value (2.2 L/min to 2.5 L/min) in the extracorporeal circulation.

The heparin-immobilized binder according to the present invention can be used industrially because heparin can be immobilized on the surface of a polymer substrate easily and safely.

10 Medical device 11 Polymeric substrate 12 Heparin-immobilized binder 100 Cardiopulmonary bypass device (blood circulation system)
101 Blood Removal Line 102 Reservoir 103 Blood Line 104 First Blood Delivery Line (Blood Delivery Line)
105 Artificial lung 106 Second blood supply line (blood supply line)
111 Blood flow rate sensor (blood flow rate measuring means)
120 Roller pump (blood pump)
121 Blood removal regulator (flow rate adjustment means)
140 control unit

Claims (11)

A heparin-immobilized binder characterized by having an alkyl group introduced into a polyamine and having both cationic property and hydrophobic property. The heparin-immobilized binder according to claim 1, wherein the polyamine is ε-polylysine, and a part of the amino group of the ε-polylysine is a partial amidation product amidated with dodecylsuccinic anhydride. The heparin-immobilized binder according to claim 2, wherein the introduction rate of the dodecyl succinic anhydride in the partially amidated product is 20% by mass or more and 40% by mass or less. A medical device, wherein at least a part of the blood contact surface is coated with the heparin-immobilized binder according to any one of claims 1 to 3. The medical device according to claim 4, wherein at least one of heparin and a heparin derivative is fixed to at least a part of the blood contact surface via the heparin-immobilized binder. The medical device according to claim 4 or 5, wherein the medical device is a tube. The medical device according to claim 4 or 5, wherein the medical device is a reservoir. A method for producing a heparin-immobilized binder, comprising the step of reacting a polyamine with an acid anhydride to amidate a part of amino groups of the polyamine with the acid anhydride. The method for producing a heparin-immobilized binder according to claim 8, wherein the polyamine is ε-polylysine, and the acid anhydride is dodecyl succinic anhydride. A step of dissolving the heparin-immobilized binder according to any one of claims 1 to 3 in a solvent to prepare a solution containing the heparin-immobilized binder;
A step of applying the solution to at least a part of the blood contact surface of the medical device and coating at least a part of the blood contact surface with the heparin-immobilized binder. Method. The method for producing a medical device according to claim 10, further comprising the step of fixing at least one of heparin and a heparin derivative to at least a part of the blood contact surface via the heparin-immobilized binder.