WO2013038901A1 - 有機ケイ素化合物及びそれを含むシランカップリング剤 - Google Patents
有機ケイ素化合物及びそれを含むシランカップリング剤 Download PDFInfo
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- WO2013038901A1 WO2013038901A1 PCT/JP2012/071697 JP2012071697W WO2013038901A1 WO 2013038901 A1 WO2013038901 A1 WO 2013038901A1 JP 2012071697 W JP2012071697 W JP 2012071697W WO 2013038901 A1 WO2013038901 A1 WO 2013038901A1
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- silane coupling
- cells
- coupling agent
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- C—CHEMISTRY; METALLURGY
- C07—ORGANIC CHEMISTRY
- C07F—ACYCLIC, CARBOCYCLIC OR HETEROCYCLIC COMPOUNDS CONTAINING ELEMENTS OTHER THAN CARBON, HYDROGEN, HALOGEN, OXYGEN, NITROGEN, SULFUR, SELENIUM OR TELLURIUM
- C07F7/00—Compounds containing elements of Groups 4 or 14 of the Periodic Table
- C07F7/02—Silicon compounds
- C07F7/08—Compounds having one or more C—Si linkages
- C07F7/0803—Compounds with Si-C or Si-Si linkages
- C07F7/081—Compounds with Si-C or Si-Si linkages comprising at least one atom selected from the elements N, O, halogen, S, Se or Te
-
- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61L—METHODS OR APPARATUS FOR STERILISING MATERIALS OR OBJECTS IN GENERAL; DISINFECTION, STERILISATION OR DEODORISATION OF AIR; CHEMICAL ASPECTS OF BANDAGES, DRESSINGS, ABSORBENT PADS OR SURGICAL ARTICLES; MATERIALS FOR BANDAGES, DRESSINGS, ABSORBENT PADS OR SURGICAL ARTICLES
- A61L27/00—Materials for grafts or prostheses or for coating grafts or prostheses
- A61L27/28—Materials for coating prostheses
-
- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61L—METHODS OR APPARATUS FOR STERILISING MATERIALS OR OBJECTS IN GENERAL; DISINFECTION, STERILISATION OR DEODORISATION OF AIR; CHEMICAL ASPECTS OF BANDAGES, DRESSINGS, ABSORBENT PADS OR SURGICAL ARTICLES; MATERIALS FOR BANDAGES, DRESSINGS, ABSORBENT PADS OR SURGICAL ARTICLES
- A61L27/00—Materials for grafts or prostheses or for coating grafts or prostheses
- A61L27/50—Materials characterised by their function or physical properties, e.g. injectable or lubricating compositions, shape-memory materials, surface modified materials
- A61L27/54—Biologically active materials, e.g. therapeutic substances
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- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61L—METHODS OR APPARATUS FOR STERILISING MATERIALS OR OBJECTS IN GENERAL; DISINFECTION, STERILISATION OR DEODORISATION OF AIR; CHEMICAL ASPECTS OF BANDAGES, DRESSINGS, ABSORBENT PADS OR SURGICAL ARTICLES; MATERIALS FOR BANDAGES, DRESSINGS, ABSORBENT PADS OR SURGICAL ARTICLES
- A61L29/00—Materials for catheters, medical tubing, cannulae, or endoscopes or for coating catheters
- A61L29/08—Materials for coatings
-
- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61L—METHODS OR APPARATUS FOR STERILISING MATERIALS OR OBJECTS IN GENERAL; DISINFECTION, STERILISATION OR DEODORISATION OF AIR; CHEMICAL ASPECTS OF BANDAGES, DRESSINGS, ABSORBENT PADS OR SURGICAL ARTICLES; MATERIALS FOR BANDAGES, DRESSINGS, ABSORBENT PADS OR SURGICAL ARTICLES
- A61L29/00—Materials for catheters, medical tubing, cannulae, or endoscopes or for coating catheters
- A61L29/14—Materials characterised by their function or physical properties, e.g. lubricating compositions
- A61L29/16—Biologically active materials, e.g. therapeutic substances
-
- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61L—METHODS OR APPARATUS FOR STERILISING MATERIALS OR OBJECTS IN GENERAL; DISINFECTION, STERILISATION OR DEODORISATION OF AIR; CHEMICAL ASPECTS OF BANDAGES, DRESSINGS, ABSORBENT PADS OR SURGICAL ARTICLES; MATERIALS FOR BANDAGES, DRESSINGS, ABSORBENT PADS OR SURGICAL ARTICLES
- A61L31/00—Materials for other surgical articles, e.g. stents, stent-grafts, shunts, surgical drapes, guide wires, materials for adhesion prevention, occluding devices, surgical gloves, tissue fixation devices
- A61L31/08—Materials for coatings
-
- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61L—METHODS OR APPARATUS FOR STERILISING MATERIALS OR OBJECTS IN GENERAL; DISINFECTION, STERILISATION OR DEODORISATION OF AIR; CHEMICAL ASPECTS OF BANDAGES, DRESSINGS, ABSORBENT PADS OR SURGICAL ARTICLES; MATERIALS FOR BANDAGES, DRESSINGS, ABSORBENT PADS OR SURGICAL ARTICLES
- A61L31/00—Materials for other surgical articles, e.g. stents, stent-grafts, shunts, surgical drapes, guide wires, materials for adhesion prevention, occluding devices, surgical gloves, tissue fixation devices
- A61L31/14—Materials characterised by their function or physical properties, e.g. injectable or lubricating compositions, shape-memory materials, surface modified materials
- A61L31/16—Biologically active materials, e.g. therapeutic substances
-
- C—CHEMISTRY; METALLURGY
- C07—ORGANIC CHEMISTRY
- C07F—ACYCLIC, CARBOCYCLIC OR HETEROCYCLIC COMPOUNDS CONTAINING ELEMENTS OTHER THAN CARBON, HYDROGEN, HALOGEN, OXYGEN, NITROGEN, SULFUR, SELENIUM OR TELLURIUM
- C07F7/00—Compounds containing elements of Groups 4 or 14 of the Periodic Table
- C07F7/02—Silicon compounds
- C07F7/08—Compounds having one or more C—Si linkages
- C07F7/18—Compounds having one or more C—Si linkages as well as one or more C—O—Si linkages
- C07F7/1804—Compounds having Si-O-C linkages
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- C—CHEMISTRY; METALLURGY
- C09—DYES; PAINTS; POLISHES; NATURAL RESINS; ADHESIVES; COMPOSITIONS NOT OTHERWISE PROVIDED FOR; APPLICATIONS OF MATERIALS NOT OTHERWISE PROVIDED FOR
- C09D—COATING COMPOSITIONS, e.g. PAINTS, VARNISHES OR LACQUERS; FILLING PASTES; CHEMICAL PAINT OR INK REMOVERS; INKS; CORRECTING FLUIDS; WOODSTAINS; PASTES OR SOLIDS FOR COLOURING OR PRINTING; USE OF MATERIALS THEREFOR
- C09D5/00—Coating compositions, e.g. paints, varnishes or lacquers, characterised by their physical nature or the effects produced; Filling pastes
- C09D5/16—Antifouling paints; Underwater paints
- C09D5/1606—Antifouling paints; Underwater paints characterised by the anti-fouling agent
- C09D5/1612—Non-macromolecular compounds
- C09D5/1625—Non-macromolecular compounds organic
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- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61L—METHODS OR APPARATUS FOR STERILISING MATERIALS OR OBJECTS IN GENERAL; DISINFECTION, STERILISATION OR DEODORISATION OF AIR; CHEMICAL ASPECTS OF BANDAGES, DRESSINGS, ABSORBENT PADS OR SURGICAL ARTICLES; MATERIALS FOR BANDAGES, DRESSINGS, ABSORBENT PADS OR SURGICAL ARTICLES
- A61L2300/00—Biologically active materials used in bandages, wound dressings, absorbent pads or medical devices
- A61L2300/40—Biologically active materials used in bandages, wound dressings, absorbent pads or medical devices characterised by a specific therapeutic activity or mode of action
- A61L2300/424—Anti-adhesion agents
Definitions
- the present invention provides a stable treatment within a molecule that can suppress adhesion of biological substances such as proteins and cells such as platelets by surface treatment of inorganic substances such as silicon and glass, or resins such as polyethylene.
- the present invention relates to a betaine-type or sulfobetaine-type organosilicon compound having both positive and negative charges.
- an anticoagulant such as heparin or a drug such as an immunosuppressant must be used in combination.
- MPC polymer poly (2-methacryloyloxyethylphosphorylcholine) (hereinafter referred to as MPC polymer in this specification) having phosphorylcholine, which is the same amphoteric phospholipid as biological membranes, in the side chain of the polymer chain.
- CMB polymer a medical material using a high molecular weight N-methacryloyloxyethyl-N, N-dimethylammonium- ⁇ -N-methylcarboxybetaine (hereinafter referred to as CMB polymer in this specification) has been proposed (for example, see Patent Literature 3, Patent Literature 4 and Patent Literature 5).
- one aspect of the present invention is the following formula (1): (In the formula, A ⁇ represents the following formula (2) or formula (3): E represents the following formula (4) or formula (5): In the formulas (4) and (5), the carbonyl group is bonded to the carbon atom to which R 3 is bonded, and R 1 and R 2 are each independently an alkyl group having 1 to 5 carbon atoms.
- silane coupling agent surface treatment agent
- organosilicon compound a silane coupling agent containing the organosilicon compound, a polar organic solvent, and water.
- the organosilicon compound represented by the formula (1) can be synthesized, for example, by reacting betaine and mercaptosilane.
- betaine that is a raw material of the organosilicon compound represented by the formula (1) include N-methacryloyloxyethyl-N, N-dimethylammonium- ⁇ -N-methylcarboxybetaine and N-methacryloyloxyethyl-N.
- N-dimethylammonium- ⁇ -N-propylsulfoxybetaine and N-methacryloylaminopropyl-N, N-dimethylammonium- ⁇ -N-propylsulfoxybetaine are preferred.
- Examples of the mercaptosilane that is another raw material of the organosilicon compound represented by the formula (1) include 3-mercaptopropyltrimethoxysilane, 3-mercaptopropyltriethoxysilane, and 3-mercaptopropylmethyldimethoxy. Silane, (mercaptomethyl) dimethoxysilane, (mercaptomethyl) dimethylethoxysilane and mercaptomethyltrimethoxysilane are preferred.
- the organosilicon compound is used for the surface treatment of an inorganic material that is a base material
- the inorganic material is not particularly limited.
- silicon, copper, iron, aluminum, zinc or an alloy thereof, glass, silica, aluminum oxide, aluminum hydroxide, and magnesium oxide can be given.
- the resin is not particularly limited.
- examples include poly (meth) acrylamide, poly (meth) acrylamide derivatives, polysulfone, polycarbonate, cellulose, and cellulose derivatives.
- the organosilicon compound can be used for surface treatment of pharmaceuticals, quasi drugs, medical instruments, and the like.
- medical devices include drug delivery system materials, molding aids, packaging materials, artificial blood vessels, hemodialysis membranes, catheters, guard wires, contact lenses, blood filters, blood storage packs, endoscopes, artificial organs, biotechnology A chip, a cell culture sheet, and a sugar chain synthesizer can be mentioned, but there are no particular limitations on the medical instrument.
- the biological substance in this specification is a basic material constituting a living body, and examples thereof include proteins, nucleic acids, various sugars, amino acids, nucleosides, lipids, and vitamins.
- biological substances whose adhesion is suppressed by using the silane coupling agent (surface treatment agent) containing the organosilicon compound of the present invention include proteins, nucleic acids, various sugars, amino acids, nucleosides, and lipids.
- Vitamins preferably proteins, nucleic acids, and various sugars, and more preferably proteins.
- the cell is the most basic unit constituting a living body, and has a cytoplasm and various organelles inside the cell membrane as its elements.
- the nucleus containing DNA may or may not be contained inside the cell.
- the animal-derived cells in the present invention include germ cells such as sperm and eggs, somatic cells constituting the living body, stem cells, progenitor cells, cancer cells separated from the living body, separated from the living body, and acquired immortalizing ability.
- Cells that are stably maintained outside the body (cell lines), cells that have been isolated from the living body and have been artificially modified, cells that have been isolated from the living body and have been artificially exchanged nuclei, and the like.
- somatic cells constituting a living body include, but are not limited to, fibroblasts, bone marrow cells, B lymphocytes, T lymphocytes, neutrophils, erythrocytes, platelets, macrophages, monocytes, bones Cells, bone marrow cells, pericytes, dendritic cells, keratinocytes, adipocytes, mesenchymal cells, epithelial cells, epidermal cells, endothelial cells, vascular endothelial cells, hepatocytes, chondrocytes, cumulus cells, nervous system cells, Glial cells, neurons, oligodendrocytes, microglia, astrocytes, heart cells, esophageal cells, muscle cells (eg, smooth muscle cells, skeletal muscle cells), pancreatic beta cells, melanocytes, hematopoietic progenitor cells, and single cells Nuclear cells are included.
- the somatic cells are, for example, skin, kidney, spleen, adrenal gland, liver, lung, ovary, pancreas, uterus, stomach, colon, small intestine, large intestine, bladder, prostate, testis, thymus, muscle, connective tissue, bone, cartilage, blood vessel It includes cells taken from any tissue such as tissue, blood, heart, eye, brain, nerve tissue.
- the stem cell is a cell having the ability to replicate itself and the ability to differentiate into cells of other multiple lineages.
- Examples thereof include, but are not limited to, embryonic stem cells (ES cells) ), Embryonic tumor cells, embryonic germ stem cells, induced pluripotent stem cells (iPS cells), neural stem cells, hematopoietic stem cells, mesenchymal stem cells, liver stem cells, pancreatic stem cells, muscle stem cells, germ stem cells, intestinal stem cells, cancer stem cells , Hair follicle stem cells and the like.
- the progenitor cells are cells that are in the process of being differentiated from the stem cells into specific somatic cells or germ cells.
- the cancer cells are cells that have been derived from somatic cells and have acquired infinite proliferation ability.
- examples of cells whose adhesion is suppressed by using the silane coupling agent (surface treatment agent) containing the organosilicon compound of the present invention include germ cells such as sperm and eggs, and somatic cells constituting the living body.
- germ cells such as sperm and eggs
- somatic cells constituting the living body.
- the silane coupling agent (surface treatment agent) containing the organosilicon compound of the present invention can be used as a reagent for researching biological materials and cells because adhesion of biological materials and cells is efficiently suppressed.
- a factor that regulates differentiation or proliferation of a cell or tissue the number and type of cells and a biological substance obtained from the cell when cultured in the presence of the cell and the target factor, a cell surface differentiation marker, Analyze changes in expressed genes.
- the use of the silane coupling agent of the present invention suppresses the adhesion of biological materials and cells, so that the target biological materials and cells can be efficiently recovered.
- the culture conditions, culture apparatus, type of medium, type of silane coupling agent, content, type of additive, content of additive, culture period, culture temperature, etc. for elucidating the target factor are determined by those skilled in the art. It is selected appropriately. Cells that have grown or appeared in culture can be observed using a standard microscope in the technical field according to the present invention. At this time, the cultured cells may be stained with a specific antibody.
- the expressed gene that has changed depending on the target factor can be detected by extracting DNA (deoxyribonucleic acid) or RNA (ribonucleic acid) from cultured cells, and by Southern blotting, Northern blotting, RT-PCR, and the like. Further, the cell surface differentiation marker can be detected by ELISA or flow cytometry using a specific antibody, and the effect on differentiation and proliferation by the target factor can be observed.
- the silane coupling agent (surface treatment agent) containing the organosilicon compound of the present invention is, for example, 0.001% by mass to 20% by adding water and a polar organic solvent such as methanol, ethanol, propylene glycol monomethyl ether to the organosilicon compound. Prepared by diluting to a mass%.
- a polar organic solvent such as methanol, ethanol, propylene glycol monomethyl ether
- an organic acid may be further added. Examples of the organic acid include acetic acid, formic acid, and lactic acid.
- the method for surface-treating the substrate with the silane coupling agent of the present invention is not particularly limited, and can be treated by dipping, coating (spin coating, spray coating, etc.) or vapor deposition, for example.
- the silane coupling agent is applied through a step of applying and baking the silane coupling agent of the present invention on a substrate, a step of washing the substrate with a polar solvent, and a step of drying the substrate. Immobilize.
- the silane coupling agent is immobilized through a step of immersing the substrate in the silane coupling agent, a step of washing the substrate with a polar solvent, and a step of drying the substrate.
- the polar solvent used in the washing step for example, water or a polar organic solvent contained in the silane coupling agent can be used.
- the concentration of CO 2 in the CO 2 incubator (%) was expressed by% by volume of CO 2 in the atmosphere.
- PBS phosphate buffered saline (manufactured by Sigma Aldrich)
- FBS fetal bovine serum (manufactured by Biological Industries).
- Example 2 A glass substrate of the same type as the glass substrate used in Example 1 was immersed in 10 ml of the silane coupling agent containing Compound 4 obtained in Synthesis Example 2, and allowed to stand at 40 ° C. for 24 hours. The glass substrate was taken out of the silane coupling agent, washed with PGME, and air-dried.
- Example 3 0.5 ml of the silane coupling agent containing Compound 2 obtained in Synthesis Example 1 was applied on the same type of glass substrate as that used in Example 1 using a spin coater, and 100 ° C. on a hot plate. And baked for 15 minutes. Thereafter, it was washed with PGME and air-dried.
- Example 1 A glass substrate of the same type as the glass substrate used in Example 1 was washed with PGME and air-dried.
- Example 1 Contact angle measurement
- or Example 4 and the comparative example 1 was performed using water and diiodomethane.
- the contact angle meter manufactured by Kyowa Interface Science Co., Ltd.
- the results are shown in Table 1 below.
- the fluorescence intensity of each glass substrate treated in Example 1 and Example 2 was lower than that of the glass substrate treated in Comparative Example 1 (with fluorescent protein treatment). This was the same as the glass substrate treated in Example 1 (no fluorescent protein treatment). This result is considered to be because the positive and negative zwitterionic sites of Compound 2 and Compound 4 were arranged on the surface layer of the glass substrate, thereby preventing the adsorption of the fluorescent protein.
- the fluorescence intensity of each glass substrate treated in Example 3 and Example 4 was also lower than the fluorescence intensity of the glass substrate treated in Comparative Example 1 (with fluorescent protein treatment), and the adsorption of the fluorescent protein was suppressed.
- Example 5 A glass substrate (TEMPAX Float [registered trademark], ⁇ 12 mm, thickness 1 mm) whose surface was cleaned by O 2 etching was immersed in 100 ml of a 10 mM ethanol solution containing Compound 2 obtained in Synthesis Example 1 for 48 hours at room temperature. Left to stand. The glass substrate was taken out of the solution, washed with ethanol and air dried.
- TEMPAX Float registered trademark
- Example 6 A glass substrate of the same type as the glass substrate used in Example 5 was immersed in 100 ml of a 10 mM ethanol solution containing Compound 4 obtained in Synthesis Example 2, and allowed to stand at room temperature for 48 hours. The glass substrate was taken out of the solution, washed with ethanol and air dried.
- the number of platelet adhesion at five locations in the glass substrate was measured with an electron microscope. By averaging the measured values at each location, the number of platelets adhered to Run-1 or Run-2 was obtained. Further, the platelet adhesion of each glass substrate treated in Example 5, Example 6 and Comparative Example 2 was compared by averaging the number of platelet adhesion of Run-1 and Run-2. The results are shown in Table 2 below.
- Human fetal kidney cell line Hek293 (DS Pharma Biomedical), human liver cancer cell line HepG2 (DS Pharma Biomedical), human cervical cancer cell line HeLa (DS Pharma Biomedical), human fetus Lung cell line MRC5 (DS Pharma Biomedical) and Chinese hamster ovary cell line CHO (DS Pharma Biomedical) were used.
- the culture medium used for culturing these cells was HeLa, Hek293 and MRC5: 10% (v / v) FMEM-containing EMEM medium (manufactured by Wako Pure Chemical Industries, Ltd.), HepG2: 10% (v / v) FBS.
- DMEM / F-12 medium (Sigma Aldrich) containing CHO: 10% (v / v) FBS were used.
- the cells were statically cultured for 2 days or more using a petri dish (10 mL of medium) having a diameter of 10 cm while maintaining a 5% carbon dioxide concentration in a 37 ° C. CO 2 incubator. Subsequently, the cells were washed with 5 ml of PBS, 1 ml of trypsin-EDTA solution (Invitrogen) was added to peel the cells, and the cells were suspended in 10 ml of the above medium. After centrifuging the suspension (the centrifuge used in the preparation of the platelet solution, 1500 rpm / 3 minutes, room temperature), the supernatant was removed, and the above medium was added to prepare a cell suspension.
- trypsin-EDTA solution Invitrogen
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Abstract
Description
すなわち、本発明の態様の一つは、下記式(1):
で表される有機ケイ素化合物である。
また、本発明の他の態様は、前記シランカップリング剤を基材に塗布又は該シランカップリング剤中に基材を浸漬させる工程を含む、シランカップリング剤固定化方法である。
前記式(1)で表される有機ケイ素化合物の原料であるベタインとしては、例えば、N-メタクリロイルオキシエチル-N,N-ジメチルアンモニウム-α-N-メチルカルボキシベタイン、N-メタクリロイルオキシエチル-N,N-ジメチルアンモニウム-α-N-プロピルスルホキシベタイン、N-メタクリロイルアミノプロピル-N,N-ジメチルアンモニウム-α-N-プロピルスルホキシベタインが好ましい。
PGME(プロピレングリコールモノメチルエーテル)/水=9/1体積%濃度の溶媒に酢酸を加えて、pH4.0に調製した。その後、合成例1及び合成例2で得られた有機ケイ素化合物それぞれを、1質量%濃度となるように前記溶媒に溶解させ、シランカップリング剤を調製した。
合成例1で得られた化合物2を含むシランカップリング剤10ml中にガラス基板(TEMPAX Float〔登録商標〕、50×50mm、厚さ1mm)を浸漬させ、40℃で24時間静置した。そのガラス基板を前記シランカップリング剤から取り出した後、PGMEで洗浄し、風乾した。
合成例2で得られた化合物4を含むシランカップリング剤10ml中に、実施例1で使用したガラス基板と同種のガラス基板を浸漬させ、40℃で24時間静置した。そのガラス基板を前記シランカップリング剤から取り出した後、PGMEで洗浄し、風乾した。
合成例1で得られた化合物2を含むシランカップリング剤0.5mlを、実施例1で使用したガラス基板と同種のガラス基板上にスピンコーターを用いて塗布し、ホットプレート上にて100℃で15分間ベークした。その後、PGMEで洗浄し、風乾した。
合成例2で得られた化合物4を含むシランカップリング剤0.5mlを、実施例1で使用したガラス基板と同種のガラス基板上にスピンコーターを用いて塗布し、ホットプレート上にて100℃で15分間ベークした。その後、PGMEで洗浄し、風乾した。
実施例1で使用したガラス基板と同種のガラス基板をPGMEにて洗浄し、風乾した。
実施例1乃至実施例4及び比較例1で処理した各ガラス基板の表面に対する接触角測定を、水及びジヨードメタンを用いて行った。接触角計(協和界面科学(株)製)を用いて液滴法にて測定し、θ/2法にて接触角を算出した。その結果を下記表1に示す。
蛍光標識されたウシ血清アルブミン(FITC-BSA、シグマアルドリッチ社製)をリン酸生理食塩水(PBS、pH7.4)に溶解させ、100μg/mlのFITC-BSA溶液を調製した。このFITC-BSA溶液0.5mlを、実施例1乃至実施例4及び比較例1で処理した各ガラス基板の表面に添加し、37℃で30分間静置した。その後、前記各ガラス基板の表面からFITC-BSA溶液を除き、溶媒として用いたPBSにて洗浄し、風乾した。
合成例1及び合成例2で得られた有機ケイ素化合物それぞれを、10mM溶液となるように99.5体積%エタノール(0.5体積%の水含有)に溶解させ、シランカップリング剤を調製した。
合成例1で得られた化合物2を含む10mMエタノール溶液100ml中に、O2エッチングにて表面洗浄したガラス基板(TEMPAX Float〔登録商標〕、Φ12mm、厚さ1mm)を浸漬させ、室温で48時間静置した。そのガラス基板を前記の溶液から取り出した後、エタノールで洗浄し、風乾した。
合成例2で得られた化合物4を含む10mMエタノール溶液100ml中に、実施例5で使用したガラス基板と同種のガラス基板を浸漬させ、室温で48時間静置した。そのガラス基板を前記の溶液から取り出した後、エタノールで洗浄し、風乾した。
実施例5で使用したガラス基板と同種のガラス基板をプラスチックケース内に1週間保管した。
3.8質量%クエン酸ナトリウム溶液0.5mlに対して、健康なボランティアより採血した血液4.5mLを混和した後、遠心分離にて[冷却遠心機5900((株)久保田製作所製)、1000rpm/10分、室温]上層の多血小板血漿(PRP)を回収した。引き続き、下層について遠心分離を行い(上記遠心機、3500rpm/10分、室温)、上層の乏血小板血漿(PPP)を回収した。多項目自動赤血球分析装置(XT-2000i、シスメックス(株)製)にてPRPの血小板数を計測後、PPPを用いてPRPの血小板濃度が30×104cells/μLになるように調製した。
24穴平底マイクロプレート(コーニング社製)に実施例5、実施例6及び比較例2で処理した各ガラス基板を配置し、上記血小板濃度に調製したPRP溶液300μLを添加した。5%二酸化炭素濃度を保った状態で、37℃で90分間CO2インキュベーター内にて静置した。プレート内のPRPを除いた後、リン酸緩衝生理食塩水(PBS)3mLにて5回洗浄した。その後、2.5体積%グルタルアルデヒドのPBS溶液2mLを添加し、4℃で一昼夜静置後、グルタルアルデヒドのPBS溶液を除き、超純水(Milli-Q水)3mLで5回洗浄した。さらに、70%エタノール水(v/v)1mLで3回洗浄し、風乾した。この実験を2回行った(Run-1、Run-2)。
上記血小板付着実験を行った実施例5、実施例6及び比較例2で処理した各ガラス基板に、イオンスパッター(E-1030、(株)日立ハイテクノロジーズ製)にてPt-Pdを1分間蒸着した。その後、電子顕微鏡(S-4800、(株)日立ハイテクノロジーズ製)にて血小板の付着を1,000倍で観察した。
細胞は、ヒト胎児腎細胞株Hek293(DSファーマバイオメディカル社製)、ヒト肝癌細胞株HepG2(DSファーマバイオメディカル社製)、ヒト子宮頸癌細胞株HeLa(DSファーマバイオメディカル社製)、ヒト胎児肺細胞株MRC5(DSファーマバイオメディカル社製)、及びチャイニーズハムスター卵巣細胞株CHO(DSファーマバイオメディカル社製)を用いた。それらの細胞の培養に用いた培地は、HeLa、Hek293及びMRC5:10%(v/v)FBSを含むEMEM培地(和光純薬工業(株)製)、HepG2:10%(v/v)FBSを含むDMEM培地(和光純薬工業(株)製)、CHO:10%(v/v)FBSを含むDMEM/F-12培地(シグマアルドリッチ社製)を用いた。細胞は、37℃CO2インキュベーター内にて5%二酸化炭素濃度を保った状態で、直径10cmのシャーレ(培地10mL)を用いて2日間以上静置培養した。引き続き、本細胞をPBS5mlで洗浄した後、トリプシン-EDTA溶液 (インビトロジェン社製)1mLを添加して細胞を剥がし、上記の培地10mLにて懸濁した。本懸濁液を遠心分離(上記血小板溶液の調製で用いた遠心機、1500rpm/3分、室温)後、上清を除き、上記の培地を添加して細胞懸濁液を調製した。
24穴平底マイクロプレート(コーニング社製)に実施例5、実施例6及び比較例2で処理した各ガラス基板を配置し、調製した上記細胞懸濁液を2.5×105cells/wellとなるように各1mL加えた。その後、5%二酸化炭素濃度を保った状態で、37℃で24時間CO2インキュベーター内にて静置した。
24時間後、上記細胞付着実験を行った実施例5、実施例6及び比較例2で処理した各ガラス基板を別の24穴平底マイクロプレートに移し、PBS1mLで洗浄した。PBSを除いた後、トリプシン-EDTA溶液 (インビトロジェン社製)500μLを添加した。5分~10分後、10%(v/v)FBSを含むDMEM培地(和光純薬工業(株)製)を500μL添加し、剥がれた細胞を1.5mLマイクロテストチューブ(エッペンドルフ社製)に移した。遠心分離((株)トミー精工製、型番:MX-307、1500rpm/3分、室温)後、上清を除き、10%(v/v)FBSを含むDMEM培地(和光純薬工業(株)製)100μLを添加して細胞懸濁液を調製した。本懸濁液にトリパンブルー染色液を等量添加後、血球計測板(エルマ販売株式会社製)にて生細胞数(細胞付着数)を計測した。
Claims (8)
- 請求項1に記載の有機ケイ素化合物、極性有機溶媒及び水を含むシランカップリング剤。
- さらに有機酸を含む請求項2に記載のシランカップリング剤。
- 請求項2又は請求項3に記載のシランカップリング剤からなる生体物質又は細胞の付着抑制剤。
- 前記細胞が生体を構成する体細胞、生体から分離された癌細胞、又は生体から分離され不死化能を獲得して体外で安定して維持される細胞である、請求項4に記載の付着抑制剤。
- 前記細胞が血小板である請求項4に記載の付着抑制剤。
- 請求項2又は請求項3に記載のシランカップリング剤を基材上に塗布しベークする工程、その後極性溶媒で前記基材を洗浄する工程、及び前記基材を乾燥させる工程を含むシランカップリング剤固定化方法。
- 基材を請求項2又は請求項3に記載のシランカップリング剤中に浸漬する工程、その後極性溶媒で前記基材を洗浄する工程、及び前記基材を乾燥させる工程を含むシランカップリング剤固定化方法。
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