JP5296416B2 - Antithrombotic material - Google Patents

Description

  The present invention relates to an antithrombotic material, and more particularly to an antithrombotic material used for a medical instrument or the like that comes into direct contact with blood.

  With advances in medical technology, opportunities for contact between medical devices and blood are increasing. For this reason, the importance of compatibility (biocompatibility) with blood and living tissue is increasing. Among these, antithrombogenicity that prevents blood coagulation is very important in medical devices that come into contact with blood. At present, as a method for imparting antithrombogenicity to a medical device, the medical device is mainly covered with a hydrophilic polymer material. There are also known methods of fixing an antithrombotic material such as heparin on the surface of a medical instrument or a polymer material that releases heparin.

  However, when a medical instrument is covered with a polymer material, there is a problem that it is difficult to sufficiently adhere the medical instrument and the polymer material. In many cases, the base material of the medical device is made of metal, and the polymer material is often peeled off from the base material. The same problem occurs when fixing heparin or the like. In particular, when heparin is used, it is known that problems of infectious diseases occur due to animal origin, and problems such as an increase in blood volume due to overdose occur.

  As a method for solving these problems, coating of medical devices with a carbonaceous film typified by a diamond-like thin film (DLC film) has attracted attention. The carbonaceous film can be formed as a strong film on the surface of metals and other materials. For this reason, the effect that it is hard to peel from the base material of a medical device compared with a polymeric material is acquired. Furthermore, since the carbonaceous film is excellent in wear resistance and corrosion resistance, it has a feature that the durability of the medical device can be improved.

Since the carbonaceous film is a smooth and inert material, it itself has a certain degree of biocompatibility. As a method to further improve the antithrombogenicity of the carbonaceous film, a reactive site is formed in the carbonaceous film by irradiating the carbonaceous film covering the medical device with plasma, and the formed reactive site is used. A method of imparting hydrophilicity to a carbonaceous film is known (see, for example, Patent Document 1).
International Publication No. 2005/97673 Pamphlet

  However, the present inventors have found that the improvement of antithrombogenicity by making the conventional carbonaceous film hydrophilic has the following problems.

  Coagulation of blood that causes thrombosis is related to activation of blood coagulation factors by platelet aggregation and activation of blood coagulation factors by foreign substances. For this reason, in order to impart antithrombogenicity to a medical device, it is necessary to prevent adhesion of platelets to the surface of the medical device and to prevent activation of blood coagulation factors by the medical device.

  As a result of the study by the present inventors, a carbonaceous membrane imparted with hydrophilicity by introducing a carboxyl group has a very high effect of reducing the number of platelet adhesion, but has a low effect of preventing activation of blood coagulation factors. It became clear.

  The present invention has been made based on the above findings found by the inventors of the present application, and is intended to realize an antithrombotic material in which both adhesion of platelets and activation of blood coagulation factors are reduced. Objective.

  In order to achieve the above object, the present invention comprises an antithrombotic material by a carbonaceous film having a zeta potential on the surface of −8 mV or more.

  Specifically, the antithrombogenic material according to the present invention is directed to an antithrombogenic material composed of a carbonaceous film formed on the surface of a substrate, and a membrane body formed by bonding carbon to each other, and a membrane body And a zeta potential on the surface of the membrane body is −8 mV or more.

  The zeta potential of the carbonaceous membrane surface greatly affects the antithrombogenicity, and when the zeta potential is low, the amount of TAT generated increases. However, the antithrombotic material according to the present invention has both an amino group and a carboxyl group, and the zeta potential on the surface of the membrane body is −8 mV or more. For this reason, the amount of TAT produced can be suppressed and the number of platelet adhesion can also be reduced.

  In the antithrombogenic material of the present invention, the ratio of the nitrogenous functional group to the carboxyl group is preferably 2.5 or more and 4 or less.

  In the antithrombogenic material of the present invention, the contact angle on the surface of the membrane body is preferably 60 degrees or more and 70 degrees or less.

  The antithrombotic material according to the present invention can realize an antithrombotic material in which both adhesion of platelets and activation of blood coagulation factors are reduced.

  The antithrombogenic material according to the present invention is a carbonaceous film formed on the surface of a substrate. The carbonaceous film is a thin film typified by a so-called diamond-like thin film (DLC film) mainly composed of a carbon skeleton containing carbon bonded with SP2 (graphite) and carbon bonded with SP3 (diamond).

  By irradiating the carbonaceous film with plasma, a carbon-carbon bond or the like is cleaved to form a reactive site. This reactive site and oxygen react to generate a carboxyl group. Thereby, the surface of the carbonaceous film becomes hydrophilic. Conventionally, attempts have been made to improve antithrombogenicity by making the surface of the carbonaceous film hydrophilic.

  By making the surface of the carbonaceous film hydrophilic, adhesion of platelets can be suppressed, and compatibility with platelets is improved. However, as a result of the study by the present inventors, when a carboxyl group is introduced into the carbonaceous membrane, the zeta potential on the surface of the carbonaceous membrane is reduced, and the amount of thrombin-antithrombin III complex (TAT) produced is increased. It became clear that the compatibility with blood coagulation factors was reduced.

  Hereinafter, the antithrombotic material according to the present invention will be described in more detail using examples.

(Example)
-Formation of carbonaceous film-
In this example, a stainless steel (SUS316) plate was used as the base material. A 6 mm square sample was prepared for the platelet adhesion test, and a 46 mm diameter sample was prepared for quantification of the thrombin / antithrombin III complex.

Set the substrate into the chamber of the ionization vapor deposition apparatus, after introducing to argon gas (Ar) pressure becomes ^{10 -1 Pa~10 -3 Pa (10 -3} Torr~10 -5 Torr) in the chamber Then, Ar ions were generated by discharging, and bombard cleaning was performed for about 30 minutes to cause the generated Ar ions to collide with the surface of the substrate.

Subsequently, tetramethylsilane (Si (CH ₃ ) ₄ ) was introduced into the chamber for 3 minutes to form an amorphous intermediate layer having a thickness of 20 nm with silicon (Si) and carbon (C) as main components.

After forming the intermediate layer, C ₆ H ₆ gas was introduced into the chamber, and the gas pressure was adjusted to 10 ⁻¹ Pa. The C ₆ H ₆ ionized by performing continuously introduced while discharging the C ₆ H ₆ at 30ml / min, it was ionized evaporation for about 2 minutes. A carbonaceous film, which is a DLC film having a thickness of 30 nm, was formed on the surface of the substrate.

  When forming the carbonaceous film, the target voltage is 1.5 kV, the target current is 50 mA, the filament voltage is 14 V, the filament current is 30 A, the anode voltage is 50 V, the anode current is 0.6 A, the reflector voltage is 50 V, and the reflector current is The current was 6 mA. Moreover, the temperature of the base material at the time of formation was about 160 ° C.

  The intermediate layer is provided in order to improve the adhesion between the substrate and the carbonaceous film, and may be omitted if sufficient adhesion between the substrate and the DLC film can be secured.

  In this embodiment, metal is used for the base material, but any material may be used. Specifically, although not particularly limited, for example, a metal such as iron, nickel, chromium, copper, titanium, platinum, tungsten, or tantalum can be used as the base material. Further, these alloys are stainless steel such as SUS316L, shape memory alloy such as Ti—Ni alloy or Cu—Al—Mn alloy, Cu—Zn alloy, Ni—Al alloy, titanium alloy, tantalum alloy, platinum alloy or An alloy such as a tungsten alloy can also be used. Moreover, bioactive ceramics, such as oxides, such as aluminum, a silicon | silicone, or a zircon, nitride, or a carbide | carbonized_material, or ceramics which have bioactivity, such as apatite or a biological glass, may be sufficient. Further, it may be a polymer resin such as polymethyl methacrylate (PMMA), high-density polyethylene or polyacetal, a silicon polymer such as polydimethylsiloxane, or a fluorine polymer such as polytetrafluoroethylene.

  Further, the shape is not limited to a plate shape, and may be any shape. The shape may be a state of a medical instrument or the like, or a state of a material before being formed.

  For the formation of the carbonaceous film, instead of sputtering, DC magnetron sputtering, RF magnetron sputtering, chemical vapor deposition (CVD), plasma CVD, plasma ion implantation, superimposed RF plasma ion implantation, An ion plating method, an arc ion plating method, an ion beam evaporation method, a laser ablation method, or the like may be used.

  Although the film thickness of a carbonaceous film is not specifically limited, The range of 0.005 micrometer-3 micrometers is preferable, More preferably, it is the range of 0.01 micrometer-1 micrometer.

  The carbonaceous film may contain silicon (Si). When forming a carbonaceous film, if a silicon source is simultaneously supplied in addition to a carbon source, a carbonaceous film containing Si can be formed.

  The carbonaceous film may contain fluorine (F). When forming a carbonaceous film, if a fluorine source is simultaneously supplied in addition to a carbon source, a carbonaceous film containing F can be formed.

  Various intermediate layers can be used depending on the type of substrate, but silicon (Si) and carbon (C), titanium (Ti) and carbon (C), or chromium (Cr) and carbon (C). A known film such as an amorphous film can be used. Although the thickness is not specifically limited, the range of 0.005 micrometer-0.3 micrometer is preferable, More preferably, it is the range of 0.01 micrometer-0.1 micrometer.

  The intermediate layer may be formed using a CVD method, a plasma CVD method, a thermal spraying method, an ion plating method, an arc ion plating method, or the like instead of the sputtering method.

-Plasma irradiation-
Next, in order to improve the antithrombogenicity of the obtained carbonaceous film, plasma irradiation was performed. Plasma irradiation was performed using a parallel plate type plasma irradiation apparatus as shown in FIG. After setting the base material 11 on which the carbonaceous film was formed in the chamber 10 of the plasma irradiation apparatus, the pressure in the chamber 10 was exhausted to a predetermined pressure. When the pressure in the chamber 10 was changed to a high vacuum state, the turbo molecular pump was used for evacuation. Next, a gas was introduced into the chamber 10 at a predetermined flow rate, and plasma was generated by applying high-frequency power between the parallel plate electrodes 12A and 12B. High frequency power was applied using a high frequency power supply 15 connected via a matching box 14. The gas flow rate was adjusted by the mass flow controller 13.

In this embodiment, argon (Ar), oxygen (O ₂ ), acetylene (C ₂ H ₂ ), ammonia (NH ₃ ), and a mixed gas of C ₂ H ₂ and O ₂ (C ₂ H ₂ / O ₂ The plasma irradiation was performed under the five types of conditions shown in Table 1 using the five types of gases. Note that the plasma irradiation time was 15 seconds per gas.

In Table 1, “high vacuum” indicates that the gas was introduced after the inside of the chamber was once brought into a high vacuum state using a turbo molecular pump. In this example, the ultimate vacuum in a high vacuum state was 5 × 10 ⁻³ Pa, and the ultimate vacuum in normal plasma irradiation was 2 Pa.

  Note that a plasma irradiation apparatus having any structure may be used. Also, any type of discharge may be used. For example, a parallel plate method, an after glow discharge method, an electromagnetic induction type, a magnetic field type, or the like may be used. Plasma irradiation conditions are not particularly limited. For example, as the power source for generating plasma, various power source frequencies such as a commercial frequency (50 Hz or 60 Hz), a high frequency (radio frequency), or a microwave region can be used. Furthermore, the pressure control method and supply structure of the source gas are not particularly limited. However, if plasma irradiation conditions with a very high etching rate are used, the carbonaceous film may be damaged.

-Measurement of contact angle-
The contact angle was measured using an Erma goniometer-type contact angle measuring machine GI type. A 15 μl water droplet was placed on the surface of the medical material, the left contact angle after 50 seconds, and the right contact angle after 70 seconds. The contact angle was measured. The measured value is an average value of 10 points.

-Composition evaluation-
The composition of the obtained plasma-irradiated carbonaceous film was evaluated using an X-ray photoelectron spectroscopy (XPS) method. For the measurement, a photoelectron spectrometer JPS-9010MC manufactured by JEOL Ltd. was used. Al was used as the X-ray source, and X-rays were generated under the conditions of an acceleration voltage of 12.5 kV and an emission current of 17.5 mA. Measurement was performed on an area of 5 mm in diameter arbitrarily selected from the sample. Further, the composition up to a depth of about 5 nm is measured by making X-rays incident on the sample perpendicularly and setting the detection angle to 0 degree.

  The binding energy measurement region was 274 eV to 294 eV, 389 eV to 409 eV, and 522 eV to 542 eV, and peaks of C1s, N1s, and O1s were obtained, respectively. By comparing the area ratios of the obtained peaks, the abundance ratio of oxygen to carbon O / C and the abundance ratio of nitrogen to carbon N / C were determined. In addition, the C1s peak was divided into a C—C component, a C—O component, a C═O component, and an O═C—O component by curve fitting. The abundance ratio O = C—O / C of the carboxyl group component (O═C—O) with respect to the total carbon was determined by determining the area ratio of the O═C—O component to the C1s peak.

-Evaluation of compatibility with platelets-
The suitability for platelets was determined based on the number of platelets adhered to the surface of the sample for a predetermined period of time and the quality of the suitability.

  The number of platelets adhered to the sample was measured as follows. First, 130 μl of platelet suspension plasma is placed on the surface of a sample adjusted to 6 mm × 6 mm. Subsequently, the cells were incubated at 37 ° C. for 60 minutes in a normal state covered with a plastic petri dish. After completion of the incubation, the sample was washed 3 times with a phosphate buffer (PBS) having a pH of 7.4. Subsequently, the sample was immersed in 1% glutaraldehyde / PBS and allowed to stand at 4 ° C. for 60 minutes to fix the adhered platelets. Subsequently, the sample was washed three times using PBS, PBS / distilled water (75/25), PBS / distilled water (50/50), PBS / distilled water (25/75) and distilled water in this order. After lyophilizing the washed sample, platinum vapor deposition was performed on the sample. Using a scanning electron mirror (JEOL, SEM-840), the surface of the platinum-deposited sample was randomly photographed for 5 fields of view at a magnification of 1000 times. Per platelet count was determined.

  Platelet suspension plasma was prepared as follows. Whole blood collected from healthy individuals was citrated (whole blood / 3.8% sodium citrate solution = 95/5 vol / vol), centrifuged at 1500 rpm for 10 minutes at room temperature, and platelets were collected from the supernatant. 1 ml of multi-plasma (PRP) was collected. The remaining blood was further centrifuged at 3000 rpm for 10 minutes, and 1 ml of platelet poor plasma (PPR) was collected from the supernatant. Subsequently, the number of platelets in PRP and PPP was measured with a blood cell counter (Sysmex K-1000). Platelet suspension plasma having a platelet count of 100,000 / μl was prepared by diluting PRP with PPP.

-Evaluation of compatibility with blood coagulation factors-
The suitability for blood coagulation factors was determined by quantifying the amount of thrombin / antithrombin III complex (TAT) produced.

  Thrombin, which is produced by the activation of blood coagulation factor (coagulation system) and is the enzyme mainly responsible for blood coagulation reaction, is quickly deactivated by antithrombin III as thrombin / antithrombin III complex (TAT). . Therefore, it is known that it is difficult to directly quantify the amount of thrombin produced, and it is generally performed to estimate the amount of thrombin produced by determining the blood concentration of TAT. Therefore, the suitability for the coagulation system was judged from the amount of TAT produced when the sample and whole blood were contacted for a predetermined time.

  A polyethylene terephthalate (PET) sheet (φ = 46 mm) coated with poly (2-methoxyethyl acrylate) on one side of an acrylic C-shaped spacer (inner diameter: 40 mm, outer diameter: 46 mm, thickness: 0.8 mm) Adhere with double-sided adhesive tape. A sample with a diameter of 46 mm is attached to the other surface of the C-shaped spacer with a double-sided adhesive tape to produce a sample chamber with a volume of 1000 μl.

  1000 μl of heparinized whole blood (2 IU / ml) collected from a healthy person was injected into the sample chamber and sealed with cellophane tape. In order to prevent blood cell sedimentation, this chamber was attached to a vertical rotating rotor and incubated at 37 ° C. for 120 minutes while rotating (about 4.7 rpm). After the incubation was completed, whole blood was collected from the chamber, added with 110 μl of 3.8% Na citrate aqueous solution, and centrifuged at 10 ° C., 3000 rpm, and 10 minutes.

  The TAT concentration in plasma obtained as the supernatant was quantified using a commercially available enzyme immunoassay kit for TAT (TAT-EIA manufactured by Enzyme Research Laboratories) and a microplate reader (Thermo Max manufactured by Molecular Device). The ELISA operation was performed according to the manual attached to the kit (measurement wavelength: 450 nm). In addition, the case where the PET film which coated poly (2-methoxyethyl acrylate) on both surfaces of the C-type spacer was adhered was used as a control.

-Evaluation of surface potential-
About the obtained plasma irradiation carbonaceous film, the surface potential was measured as follows. For the measurement, a zeta potential / particle size measurement system ELS-Z manufactured by Otsuka Electronics Co., Ltd. was used. The obtained plasma-irradiated carbonaceous film was brought into close contact with a plate sample cell, and monitoring particles were injected into the cell. The monitoring particles used were those manufactured by Otsuka Electronics Co., Ltd. dispersed in a 10 mM sodium chloride (NaCl) solution. The monitor particles were electrophoresed at each level in the cell depth direction, and the apparent velocity distribution inside the cell was measured. Electrophoresis was performed under the conditions of an average electric field of 17.33 V / cm and an average current of 1.02 mA. By analyzing the apparent velocity distribution obtained based on the Mori-Okamoto equation, the surface potential of the plasma-irradiated carbonaceous film surface was determined. The plate sample cell was used after being coated with polyacrylamide in order to suppress the influence of the charge on the cell surface.

-Evaluation results of carbonaceous films-
First, the contact angle with respect to water was measured about the carbonaceous film after plasma irradiation, and it was evaluated how hydrophilic it was. FIG. 2 shows the change with time of the contact angle value of the obtained plasma-irradiated carbonaceous film with respect to water. As shown in FIG. 2, in most of the samples, the contact angle value changed with time, and it became clear that it took about 10 days for the contact angle value to stabilize after the plasma irradiation.

  Thus, the reason why the contact angle changes with time is not clear. However, it is considered that some reaction further proceeds on the surface of the sample exposed to the atmosphere after irradiation with plasma. When a carbonaceous film that has been subjected to plasma irradiation is used in a medical instrument or the like, it is not used immediately after plasma irradiation. Therefore, when evaluating a carbonaceous film that has been subjected to plasma irradiation, it is very important to evaluate a sample after it has reached a stable state. Subsequent evaluation was performed using a sample that had passed 10 days or more after the plasma irradiation in which the value of the contact angle was stable.

FIG. 3 shows the relationship between the zeta potential of the carbonaceous film after plasma irradiation and blood compatibility. As shown in FIG. 3, the platelet adhesion number was low when the zeta potential was low, and gradually increased as the zeta potential increased. However, even when the zeta potential was about +2 mV, the adhesion number of platelets was about 20 × 10 ⁴ μm ² , which was compatible with polyethylene terephthalate (PET) and stainless steel (SUS316). On the other hand, the amount of TAT produced was very high when the zeta potential was low, and rapidly decreased as the zeta potential increased.

  The above results indicate that in order to show compatibility not only with platelets but also with blood coagulation factors, the zeta potential needs to be higher than a certain level and is preferably −8 mV or higher. In addition, as the zeta potential increases, the compatibility with platelets gradually deteriorates, but the compatibility is sufficiently higher than other materials, and there is no problem if it is at least about +2 mV.

Samples having a high zeta potential and a low TAT generation amount are obtained when NH ₃ plasma is irradiated after O ₂ plasma irradiation (O ₂ + NH ₃ : about −8 mV) and when irradiated with NH ₃ plasma (about +2 mV). . This suggests that the increase in zeta potential may be due to the introduction of a functional group that is positively charged when ionized on the surface of the carbonaceous film.

  The state of nitrogen introduced into the carbonaceous film after plasma irradiation is not clear. However, it is considered that functional groups containing nitrogen (nitrogenous functional groups) such as amino groups, amide groups, and amine groups are formed. Although detailed analysis of the nitrogenous functional group is difficult, the N1s peak appeared at 398.9 eV in the XPS measurement. This is deviated from the binding energy (400 ± 1 eV) of N1s of amine and amide, and it is considered that at least a part of the nitrogenous functional groups is an amino group in this sample.

  FIG. 4 shows the relationship between the functional group formed on the surface of the carbonaceous film after plasma irradiation and the zeta potential. In FIG. 4, the horizontal axis represents the ratio of the abundance ratio N / C of nitrogen to carbon and the abundance ratio O = C—O / C of the O═C—O component to carbon. As described above, nitrogen in the carbonaceous film is presumed to be a nitrogenous functional group. Therefore, N / O = C—O represents the ratio of the nitrogenous functional group to the carboxyl group. It is clear that the zeta potential increases as the abundance ratio of nitrogenous functional groups increases.

  Thus, the value of the zeta potential on the surface of the carbonaceous film is determined by the ratio between the nitrogenous functional group and the carboxyl group. Therefore, in order to show compatibility with both platelets and blood coagulation factors, the ratio of the nitrogenous functional group to the carboxyl group is preferably 2.5 or more. A larger amount of the nitrogenous functional group is considered to be preferable, but if it is too high, the zeta potential increases and the compatibility with platelets may deteriorate. Considering the theoretical upper limit of the nitrogenous functional group that can be introduced on the surface of the carbonaceous film, the ratio of the nitrogenous functional group to the carboxyl group is considered to be about 4 as an upper limit.

In the case of irradiation with O ₂ + NH ₃ or NH ₃ having a high zeta potential, the contact angle when 10 days or more passed after plasma irradiation was 60 ° or more. On the other hand, the contact angle of the other samples having a low zeta potential was 50 ° or less. From this, it is considered that the carbonaceous film having a high zeta potential does not have excessive introduction of hydrophilic carboxyl groups.

Thus, it is preferable that a carboxyl group that lowers the zeta potential is not excessively introduced on the surface of the carbonaceous film, and the contact angle is preferably 60 ° or more. On the other hand, the carbonaceous film not irradiated with plasma has a contact angle of about 80 °. The carbonaceous film not irradiated with plasma has a platelet adhesion number of about 55 × 10 ⁴ μm ² and is much less compatible with platelets than the carbonized film irradiated with plasma. Therefore, the contact angle of the carbonaceous film should not be too high, and is preferably 70 ° or less.

  The carbonaceous film having both a carboxyl group and a nitrogenous functional group and having a high zeta potential value may be formed by irradiating ammonia plasma. Further, after irradiating with another plasma, the plasma of ammonia may be irradiated. A higher ultimate vacuum in the chamber during plasma irradiation is preferable because it is not affected by oxygen in the air.

It is also possible to use other basic nitrogen-containing compounds instead of ammonia. Examples of the basic nitrogen-containing compound include organic amines represented by the general formula NR ₁ R ₂ R ₃ (where R ₁ , R ₂ and R ₃ are hydrogen, —CH ₃ , —C ₂ H ₅ , —C ₃ H ₇ or —C ₄ H ₈ , and R ₁ , R ₂ and R ₃ may be the same or different from each other.) Or benzylamine and its secondary and tertiary amines. However, ammonia is preferable because of cost and ease of handling.

  The antithrombotic material according to the present invention can reduce both the adhesion of platelets and the activation of blood coagulation factors, and is particularly useful as an antithrombotic material used for a medical instrument that is in direct contact with blood.

It is the schematic which shows the plasma irradiation apparatus used in one Example of this invention. It is a graph which shows the fluctuation | variation of the contact angle of the carbonaceous film which concerns on one Example of this invention. It is a graph which shows the relationship between the zeta potential of the carbonaceous film which concerns on one Example of this invention, and blood compatibility. It is a graph which shows the relationship between the functional group formed in the carbonaceous film which concerns on one Example of this invention, and zeta potential.

Explanation of symbols

DESCRIPTION OF SYMBOLS 10 Chamber 11 Base material 12A Parallel plate electrode 13 Mass flow controller 14 Matching box 15 High frequency power supply

Claims (3)

An antithrombotic material consisting of a carbonaceous film formed on the surface of a substrate,
A film body formed by bonding carbon to each other;
Comprising an amino group and a carboxyl group introduced into the membrane body,
Antithrombotic material to feature the zeta potential at the surface of the film body or -8 mV, or less + 2 mV. The antithrombogenic material according to claim 1, wherein the ratio of the amino group to the carboxyl group is 2.5 or more and 4 or less.   The antithrombogenic material according to claim 1 or 2, wherein a contact angle on the surface of the membrane body is 60 degrees or more and 70 degrees or less.