JP6041132B2 - Material surface modification method - Google Patents

Description

The present invention relates to a material surface modification method that chemically modifies the surface of a material made of metal, resin, or ceramics with a functional compound that is simple, has wide material selectivity, and has high biological safety.

Attempts have been widely made to provide various functions by modifying the surface of a material made of metal, resin or ceramics. For example, plasma treatment, glow discharge treatment, corona discharge treatment, ozone treatment, surface graft treatment or ultraviolet irradiation treatment are known as methods for hydrophilizing the surface of a material. In addition, a surface treatment method for binding a functional compound such as a functional peptide to the surface of a material is expected to be applied to medical applications such as medical implants and diagnostic devices.

For example, Patent Document 1 includes a base material and a coating layer that is provided on the surface of the base material and includes a natural polymer that includes an anticoagulant component and is partially modified with a crosslinkable functional group. An embedded member is described. The invention described in Patent Document 1 is specific to vascular endothelial progenitor cells, such as natural polymer derivatives such as hyaluronic acid, heparin, chondroitin sulfate, gelatin, and collagen into which a crosslinkable functional group such as a thiol group or vinyl group is introduced. The surface of the substrate is functionalized by complexing proteins that interact with each other, coating it on the surface of the substrate, and crosslinking reaction. The substrate surface modification method described in Patent Document 1 is simple because it is based on a coating method. However, a great deal of labor is required to synthesize a natural polymer derivative having a cross-linking functional group in the preparation stage. In addition, the protein bound to the surface of the substrate is gradually released at an early stage, and it is difficult to exert the effect for a long time. Furthermore, there is a concern about the biocompatibility and safety of the coating layer made of a crosslinked natural polymer.

Patent Document 2 discloses a method for modifying a material surface via dopamine or dihydroxyphenylalanine (DOPA). The invention described in Patent Document 2 is prepared by reacting dopamine or DOPA with a functional compound to prepare a functional compound having a dihydroxyphenyl group, and bringing the functional compound having the dihydroxyphenyl group into contact with a material. The functional compound is fixed on the surface of the material. The material surface modification method described in Patent Document 2 utilizing the high reactivity of a functional compound having a dihydroxyphenyl group can be applied to a wide range of materials such as metals, resins, and ceramics, and has high material selectivity. It is excellent in that it is wide. In addition, the use of dopamine or DOPA, which are biologically derived molecules, provides high biological safety. However, since the functional compound having a dihydroxyphenyl group is extremely reactive, it is difficult to chemically synthesize it or to store the synthesized functional compound having a dihydroxyphenyl group. was there.

JP 2011-92491 A US Patent Application Publication No. 2010/0330025

An object of the present invention is to provide a material surface modification method that chemically modifies the surface of a material made of metal, resin, or ceramics with a functional compound, which is simple, has a wide material selectivity, and has a high biological safety.

The present invention is a material surface modification method for chemically modifying the surface of a material comprising a metal, a resin or a ceramic with a functional compound, wherein the functional compound having a hydroxyphenyl group is converted into a metal in the presence of a catalyst and an oxidizing agent, It is a material surface modification method which has the process made to contact the material which consists of resin or ceramics.
The present invention is described in detail below.

The present inventor has used a functional compound having a hydroxyphenyl group for surface modification of a material in place of a functional compound having a dihydroxyphenyl group, which is extremely reactive and difficult to chemically synthesize and store in advance. We considered using it. A functional compound having a hydroxyphenyl group has a milder reactivity than a functional compound having a dihydroxyphenyl group, and is easy to synthesize and handle. Moreover, since biologically derived molecules such as tyrosine and tyramine can be used as raw materials, safety is also high. However, such a functional compound having a low-reactivity hydroxyphenyl group has been difficult to bond to the surface of a material made of metal, resin or ceramics.
As a result of further intensive studies, the inventor easily brought the functional compound into the material by bringing the functional compound having a hydroxyphenyl group into contact with a material made of metal, resin or ceramics in the presence of a catalyst and an oxidizing agent. The present invention was completed by finding that it can be bonded to the surface.

The material surface modification method of the present invention includes a step of bringing a functional compound having a hydroxyphenyl group into contact with a material comprising a metal, a resin, or a ceramic in the presence of a catalyst and an oxidizing agent.
The functional compound having a hydroxyphenyl group has high stability, is easy to synthesize, and can be stored after synthesis. Therefore, the material surface modification method can be greatly facilitated.

Examples of the functional compound having a hydroxyphenyl group include compounds represented by the following general formula (1).

In said formula (1), R represents the residue of a functional compound.
Examples of the functional compound include natural functional compounds such as functional peptides, nucleic acids, proteins, carbohydrates, and synthetic polymers.

Examples of the functional peptide include fibronectin CS5 domain-derived peptide (REDV, Arg-Glu-Asp-Val), which is a peptide to which vascular endothelial cells specifically adhere, and fibronectin-derived cell adhesive peptide (RGDS, Arg-Gly). -Asp-Ser), laminin-derived cell adhesion peptide (YIGSR, Tyr-Ile-Gly-Ser-Arg), collagen-derived cell adhesion peptide (GFOGER, Gly-Phe-Hyp-Gly-Glu-Arg), yellow grape Examples include a cocci membrane protein-derived complement inhibitory peptide (AHRKAQKAVNLV, Ala-His-Arg-Lys-Ala-Gln-Lys-Ala-Val-Asn-Leu-Val) and the like.
Examples of the nucleic acid include a DNA library for exhaustive analysis.
Examples of the protein include heparin, albumin, avidin, collagen, enzyme, antibody, and recombinant protein.
Examples of the saccharide include hyaluronic acid, chitosan, glucosaminoglycan and the like.
Examples of the synthetic polymer include polyethylene glycol and polyvinyl alcohol.

The functional compound having a hydroxyphenyl group is, for example, a functional compound that is desired to be bound to a biologically derived molecule such as tyrosine represented by the following formula (2) or tyramine represented by the following formula (3). Can be prepared by reacting. The structure of a tyrosine derivative (Ac-YG ₃ REDV) obtained by reacting the REDV with tyrosine is shown in the following formula (4).
Further, when the functional compound itself has a tyrosine residue, it can be used as it is as a functional compound having a hydroxyphenyl group.

Materials for the material surface modification method of the present invention include metals such as stainless steel, cobalt-chromium alloy, gold and titanium, resins such as polyester, polyethylene, polyvinyl chloride, polylactic acid, and polytetrafluoroethylene, and ceramics. A wide range of materials can be used. The material surface modification method of the present invention has a very wide material selectivity, and a wide range of materials can be used.
Examples of the material include DNA and protein array substrates, sensor substrates, fibers, and the like, and materials for medical use such as medical implants and diagnostic devices are particularly suitable.

In the material surface modification method of the present invention, a functional compound having a hydroxyphenyl group is brought into contact with a material comprising a metal, a resin, or a ceramic in the presence of a catalyst and an oxidizing agent. By using a catalyst and an oxidizing agent in combination, even a functional compound having a hydroxyphenyl group having relatively low reactivity can be bonded to the surface of the material with high efficiency.
The immobilization reaction is considered to be due to a coordination bond to a metal element via a quinone as well as a dihydroxyphenyl group or a Michael addition reaction with an amino group, a hydroxyl group and a thiol group. These bonds are highly stable. The chemical formula showing an example of the immobilization reaction is shown in the following formula (5).

Examples of the catalyst include cupric chloride, cuprous chloride, cupric nitrate, cuprous chloride, cupric chloride, ferric sulfate, ferric nitrate, peroxidase, tyrosinase, and the like.

Examples of the oxidizing agent include hydrogen peroxide, potassium permanganate, and nitric acid.

The conditions for bringing the functional compound having a hydroxyphenyl group into contact with the material are not particularly limited, and the reaction is sufficient even under conditions of about 4 to 80 ° C. In addition, since the reaction can be completed in a short time such as several minutes to several hours, even a material that is hydrolyzed, such as polylactic acid, hardly affects the properties of the material itself.
The above reaction is preferably performed in a light-shielded state in a nitrogen atmosphere. Multimerization of the functional compound in the solvent can be prevented by performing the reaction in a nitrogen atmosphere in a light-shielded state.

The material surface modification method of the present invention can be applied to a wide range of materials such as metals, resins, and ceramics, and has a wide material selectivity. In addition, biosafety can be ensured by using biologically derived molecules such as tyrosine and tyramine. Furthermore, since the functional compound having a hydroxyphenyl group is relatively stable, it can be easily synthesized chemically, and the synthesized functional compound having a hydroxyphenyl group can be stored. .

ADVANTAGE OF THE INVENTION According to this invention, the medical polymeric base material which has the adhesiveness and non-adhesiveness of various cells including not only endothelial cells but somatic cells, somatic stem cells, ES cells, and iPS cells can be provided. In addition, anti-inflammatory and antibacterial sutures, implantable medical materials, and wound dressings can be provided. Furthermore, a new scaffold material capable of inducing adhesion to tissues and organs and organ repair can be provided. Further, not only in the medical field, it is also possible to provide industrial materials such as fibers and films having deodorizing properties and antibacterial properties.

According to the present invention, it is possible to provide a material surface modification method that chemically modifies the surface of a material made of metal, resin, or ceramics with a functional compound, which is simple, has wide material selectivity, and has high biological safety.

It is a graph (A) which shows the measurement result of the water contact angle of the surface of the stainless steel test piece obtained in Experimental example 1, and a graph (B) which shows the surface analysis result by XPS. It is a graph (A) which shows the measurement result of the water contact angle of the surface of the stainless steel test piece obtained in Experimental example 2, and a graph (B) which shows the surface analysis result by XPS. Graph showing adhesion test results of human umbilical vein endothelial cells (HUVECs) (A) and human aortic smooth muscle cells (AoSMCs) (B) performed using the stainless steel test piece obtained in Experimental Example 2, and adhesion It is the microscope picture of each done cell. It is a scanning electron microscope (SEM) image of an unmodified stent (A) and an Ac-YG ₃ REDV modified stent (B) taken out after placement in the aorta for one week in Experimental Example 3. 6 is a graph showing the results of surface analysis by XPS of a polyvinyl chloride test piece and a polyethylene test piece obtained in Experimental Example 4. 6 is a graph showing the result of XPS surface analysis of a poly L-lactic acid test piece obtained in Experimental Example 4.

Hereinafter, embodiments of the present invention will be described in more detail with reference to examples. However, the present invention is not limited to these examples.

(Experimental example 1)
The reactivity of tyrosine to a stainless steel (SUS316L) test piece in the presence of a catalyst / oxidant was evaluated.
A stainless steel test piece of 1.0 cm × 1.0 cm was subjected to ultraviolet-ozone treatment for the purpose of cleaning the surface. The washed stainless steel test piece was immersed in a 10 mM aqueous solution of tyrosine, and cupric chloride was used as a catalyst in an amount of 0.02 eq. Hydrogen peroxide as an oxidizing agent is 4.4 eq. After the addition, the reaction was conducted in a light-shielded state in a nitrogen atmosphere at 37 ° C. for 24 hours. After the reaction, the stainless steel specimen was thoroughly washed with ultrapure water.
As a control experiment, a stainless steel specimen was treated in the same manner as above except that the catalyst and the oxidizing agent were not added.

About the surface of the obtained stainless steel test piece, the water contact angle was measured by the sessile drop method using the contact angle meter (CA-X, Kyowa Interface Science company make). Specifically, JIS. R. In accordance with 3257: 1999, 2 μL of water droplets (ultra pure water) were allowed to stand on the surface of a test piece placed horizontally in an environment of room temperature 25 ° C. and humidity 40%, and the contact angle after 1 minute was measured. The results are shown in FIG.
From FIG. 1 (A), the stainless steel test piece reacted with tyrosine in the presence of a catalyst and an oxidizing agent had a significantly smaller water contact angle than that of an untreated sample. In contrast, the stainless steel test piece reacted with tyrosine in the absence of a catalyst and an oxidizing agent had a water contact angle comparable to that of the untreated sample.

About the surface of the obtained stainless steel test piece, the surface analysis by XPS using ESCA-3400 (made by Shimadzu Corp.) was performed. The results are shown in FIG.
From FIG. 1 (B), a peak of nitrogen (N _ls ) derived from tyrosine was detected in the stainless steel test piece reacted with tyrosine in the presence of a catalyst and an oxidizing agent. On the other hand, since the peak of copper (Cu _2p ) used as a catalyst is not detected, it is considered that it was completely removed by washing after the reaction. On the other hand, the peak of nitrogen (N _ls ) derived from tyrosine was not detected in the stainless steel test piece reacted with tyrosine in the absence of a catalyst and an oxidizing agent.

(Experimental example 2)
(1) _Ac-YG 3 Preparation of REDV Fmoc solid phase synthesis, to prepare a tyrosine and fibronectin CS5 domain from a functional compound having from consisting hydroxyphenyl group peptides _{(REDV) Ac-YG 3 REDV} . Specifically, a dehydration condensing agent (4- (4,6-dimethoxy-1,3,5-triazin-2-yl) -4-methyl on a chlorotrityl resin (Code: A00251, manufactured by Watanabe Chemical Co., Ltd.) The Fmoc amino acid derivative was successively extended by a condensation reaction using morpholinium chloride-n-hydrate (manufactured by Kokusan Kagaku Co., Ltd.) and diisopropylethylamine (Code: A00030, manufactured by Watanabe Chemical Co., Ltd.), The peptide was acetylated with acetic anhydride (Wako Pure Chemical Industries, Ltd.), and the crude peptide was released from the resin using trifluoroacetic acid (Code: A00096, Watanabe Chemical Co., Ltd.). The obtained crude peptide was purified by reverse phase HPLC to obtain a purified peptide.
The obtained purified peptide was measured for molecular weight by MALDI-TOF / MS (manufactured by AB SCIEX, 4800 Plus Analyzer) and found to be 894.4. The obtained purified peptide was represented by the above formula (4). it was confirmed -YG is a ₃ REDV.

As a control experiment, negative sequence REVD that did not exhibit physiological activity was also reacted with tyrosine to prepare Ac-YG ₃ REDV (negative), which is a functional compound having a hydroxyphenyl group.

(2) Modification of stainless steel surface A stainless steel test piece having a size of 1.0 cm × 1.0 cm was subjected to ultraviolet-ozone treatment for the purpose of cleaning the surface. The washed stainless steel test piece was immersed in a 10 mM aqueous solution of Ac-YG ₃ REDV, and cupric chloride was used as a catalyst in an amount of 0.02 eq. Hydrogen peroxide as an oxidizing agent is 4.4 eq. After the addition, the reaction was conducted in a light-shielded state in a nitrogen atmosphere at 37 ° C. for 24 hours. After the reaction, the stainless steel specimen was thoroughly washed with ultrapure water.
Ac-YG ₃ REVD (negative) was also bonded to a stainless steel specimen by the same method.

(3) Evaluation About the surface of the obtained stainless steel test piece, the water contact angle was measured by the sessile drop method using the contact angle meter (CA-X, Kyowa Interface Science Co., Ltd.). Specifically, JIS. R. In accordance with 3257: 1999, 2 μL of water droplets (ultra pure water) were allowed to stand on the surface of a test piece placed horizontally in an environment of room temperature 25 ° C. and humidity 40%, and the contact angle after 1 minute was measured. The results are shown in FIG. 2 from _{(A), Ac-YG 3} REDV, also stainless specimens treated with either _Ac-YG 3 REVD (negative), significantly water contact angle was smaller than that of the untreated.
Moreover, about the surface of the obtained stainless steel test piece, the surface analysis by XPS using ESCA-3400 (made by Shimadzu Corp.) was performed. The results are shown in FIG. From FIG. 2 (B), a stainless steel test piece treated with either Ac-YG ₃ REDV or Ac-YG ₃ REVD (negative) showed a peak of nitrogen (N _ls ) derived from the peptide.
These evaluations confirmed that Ac-YG ₃ REDV and Ac-YG ₃ REVD (negative) were bound to the surface of the stainless steel test piece.

Using the obtained stainless steel test pieces, adhesion tests of human umbilical vein endothelial cells (HUVECs) and human aortic smooth muscle cells (AoSMCs) were performed. Specifically, a suspension of cells subcultured in advance was seeded onto a stainless steel test piece so as to be 2.5 × 10 ⁴ cells / sample, and left to stand in a CO ₂ incubator for 3 hours. Thereafter, the non-adherent cells are removed by washing with a phosphate buffer solution, the adherent cells are quantified with WST-1 (manufactured by Takara Bio Inc.), and the morphology of the adherent cells is stained with Cell Tracker Gene (manufactured by Molecular Probes). Later, a confocal laser microscope (manufactured by Olympus) was used. The results are shown in FIGS. 3 (A) and 3 (B).
From FIG. 3 (A), the adhesion number of HUVECs increased remarkably in the stainless steel test piece surface-modified with Ac-YG ₃ REDV. On the other hand, from FIG. 3B, the adhesion number of AoSMCs was similar to other surfaces even in the stainless steel test piece surface-modified with Ac-YG ₃ REDV. From this result, it was revealed that the surface modification using Ac-YG ₃ REDV can specifically improve only the adhesion of HUVECs.

(Experimental example 3)
Ac-YG ₃ REDV was prepared in the same manner as in Experimental Example 2.
A cobalt-chromium alloy stent having an inner diameter of 4.0 mm and a length of 18 mm was subjected to ultraviolet-ozone treatment for the purpose of cleaning the surface. The washed stent was immersed in an Ac-YG ₃ REDV 10 mM aqueous solution, and cupric chloride was used as a catalyst in an amount of 0.02 eq. Hydrogen peroxide as an oxidizing agent is 4.4 eq. After the addition, the reaction was conducted in a light-shielded state in a nitrogen atmosphere at 37 ° C. for 24 hours. After the reaction, the stent was thoroughly washed with ultrapure water.

The obtained stent was inserted into an abdominal aorta of a rabbit using a balloon catheter (ASAHI Douvan, 3.0 / 20 mm, DV30020, manufactured by Asahi Intec Co., Ltd.) and left there. As a control experiment, a non-surface-modified stent was similarly inserted into a rabbit abdominal aorta and left in place.
One week after the placement, the stent was removed and the inside was observed with a scanning electron microscope (SEM). FIG. 4 shows SEM images of the extracted Ac-YG ₃ REDV modified stent and unmodified stent.
From FIG. 4 (A), in the unmodified stent, formation of a thrombus was observed centering on the proximal portion. On the other hand, in the Ac-YG ₃ REDV modified stent of FIG. 4 (B), no thrombus formation was observed, and most of the stent was covered with endothelial cells.

(Experimental example 4)
(1) Preparation of Ac-YG ₃ RGDS Ac is a functional compound having a hydroxyphenyl group, which is composed of tyrosine and a fibronectin-derived cell adhesion sequence (RGDS, Arg-Gly-Asp-Ser) by Fmoc solid phase synthesis. the -YG ₃ RGDS was prepared. Specifically, after the Fmoc amino acid derivative is successively extended by HOBU / HOBt dehydration condensation method on chlorotrityl resin (Code: A00187, manufactured by Watanabe Chemical Industries), acetic anhydride (Wako Pure Chemical Industries, Ltd.) is used. The crude peptide was released from the resin using trifluoroacetic acid (Code: A00096, manufactured by Watanabe Chemical Co., Ltd.). The obtained crude peptide was purified by reverse phase HPLC to obtain a purified peptide.
The molecular weight of the purified peptide thus obtained was measured by MALDI-TOF / MS (manufactured by AB SCIEX, 4800 Plus Analyzer), which was 810.3. The obtained purified peptide was represented by the following formula (6). it was confirmed -YG is a ₃ RGDS.

(2) As a modified resin material on the surface of the resin material, a polyvinyl chloride test piece (Code: 12-547, manufactured by Fisher Scientific), a polyethylene test piece (Code: 162-09311, Wako Pure Chemical Industries, Ltd.) and poly L-lactic acid test pieces (molecular weight 160,000, Mitsui Chemicals) were prepared. Each test piece was subjected to ultrasonic cleaning for 1 minute in an ultrasonic cleaning apparatus filled with ultrapure water (total 3 times) and then subjected to ultraviolet-ozone treatment for the purpose of cleaning the surface. Each test piece after washing was immersed in an Ac-YG ₃ RGDS 10 mM aqueous solution, and cupric chloride was used as a catalyst at 0.05 eq. Hydrogen peroxide as an oxidizing agent is 4.4 eq. After the addition, the reaction was conducted in a light-shielded state in a nitrogen atmosphere at 37 ° C. for 24 hours. In addition, about the poly L-lactic acid test piece, reaction was performed for 2, 6 and 24 hours. After the reaction, each test piece was thoroughly washed with ultrapure water.

(3) Evaluation About the surface of each obtained test piece, the surface analysis by XPS using ESCA-3400 (made by Shimadzu Corporation Corp.) was performed. The result of the polyvinyl chloride test piece and the polyethylene test piece is shown in FIG. 5, and the result of the poly L-lactic acid test piece is shown in FIG.
From FIG. 5, the peak of nitrogen (N _ls ) derived from the peptide was detected in both the polyvinyl chloride test piece and the polyethylene test piece treated with Ac-YG ₃ RGDS.
Moreover, from FIG. 6, the peak of nitrogen (N _ls ) derived from the peptide was detected from the poly L-lactic acid test piece even during the treatment for 2 hours. From this, it was confirmed that the reaction was sufficiently advanced even in a short time of about 2 hours.

According to the present invention, it is possible to provide a material surface modification method that chemically modifies the surface of a material made of metal, resin, or ceramics with a functional compound, which is simple, has wide material selectivity, and has high biological safety.

Claims (2)

A material surface modification method for chemically modifying the surface of a material made of metal, resin or ceramics with a functional compound having a hydroxyphenyl group ,
A functional compound having a hydroxyphenyl group is brought into contact with a material comprising a metal, a resin or a ceramic in the presence of a catalyst and an oxidizing agent, and the hydroxyl group of the functional compound having a hydroxyphenyl group and the metal, resin or ceramic A material surface modification method comprising the step of bonding to a material comprising: 2. The material surface modification method according to claim 1, wherein the functional compound having a hydroxyphenyl group is obtained by reacting tyrosine or tyramine with a functional compound.

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