WO2020215307A1 - 材料的表面改性方法及其改性后的材料和应用、医疗产品 - Google Patents

材料的表面改性方法及其改性后的材料和应用、医疗产品 Download PDF

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WO2020215307A1
WO2020215307A1 PCT/CN2019/084522 CN2019084522W WO2020215307A1 WO 2020215307 A1 WO2020215307 A1 WO 2020215307A1 CN 2019084522 W CN2019084522 W CN 2019084522W WO 2020215307 A1 WO2020215307 A1 WO 2020215307A1
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colloidal particles
microspheres
modification
particles
organic polymer
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PCT/CN2019/084522
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French (fr)
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王鹏元
石玥
陶雪莲
金肖特·彼特
泰森·赫尔穆特
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深圳先进技术研究院
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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
    • 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
    • A61L31/08—Materials for coatings
    • A61L31/10—Macromolecular materials
    • 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
    • B—PERFORMING OPERATIONS; TRANSPORTING
    • B05—SPRAYING OR ATOMISING IN GENERAL; APPLYING FLUENT MATERIALS TO SURFACES, IN GENERAL
    • B05D—PROCESSES FOR APPLYING FLUENT MATERIALS TO SURFACES, IN GENERAL
    • B05D1/00—Processes for applying liquids or other fluent materials
    • B05D1/18—Processes for applying liquids or other fluent materials performed by dipping
    • C—CHEMISTRY; METALLURGY
    • C08—ORGANIC MACROMOLECULAR COMPOUNDS; THEIR PREPARATION OR CHEMICAL WORKING-UP; COMPOSITIONS BASED THEREON
    • C08K—Use of inorganic or non-macromolecular organic substances as compounding ingredients
    • C08K3/00—Use of inorganic substances as compounding ingredients
    • C08K3/34—Silicon-containing compounds
    • C08K3/36—Silica
    • 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
    • C09D125/00—Coating compositions based on homopolymers or copolymers of compounds having one or more unsaturated aliphatic radicals, each having only one carbon-to-carbon double bond, and at least one being terminated by an aromatic carbocyclic ring; Coating compositions based on derivatives of such polymers
    • C09D125/02—Homopolymers or copolymers of hydrocarbons
    • C09D125/04—Homopolymers or copolymers of styrene
    • C09D125/06—Polystyrene

Definitions

  • This application relates to the technical field of material surface modification, specifically, to a material surface modification method and modified materials and applications, and medical products.
  • the application range of material surface modification technology is extremely wide, including medical equipment or industrial anti-corrosion fields.
  • the surface modification of materials can be divided into two categories: changes in physical structure and changes in chemical properties.
  • Methods of changing the physical structure of the surface include etching, lithography, or reprinting techniques.
  • the methods for changing surface chemical properties include layer by layer, targeted reactions based on surface chemical functional groups, and substrate independent coating on the surface of materials.
  • etching requires the use of special equipment: plasma, electronics, laser or chemical etching equipment, and the surface chemical properties after etching are uniform.
  • the reprinting technology is relatively easy to operate and can construct a large surface physical structure, the size of the substrate, the reprinting material and the physical structure are limited, and the production of the master still needs to be completed by the etching process.
  • the resulting surface chemistry is also uniform.
  • the lithography technology can build the physical and chemical properties of the surface with high precision, but due to the long time and high cost, it is greatly restricted in the application of large-area surface modification.
  • the existing technology cannot achieve simultaneous control of the physical and chemical properties of the material surface.
  • the common method is to first construct the physical structure and then perform the next chemical modification on the surface with the physical structure, and the existing chemical modification methods are mostly surface Uniform modification, although the multiple chemical regional modification of the surface can be achieved through the method of occlusion-modification-de-occlusion, this method also requires at least three or more steps to achieve.
  • One of the objectives of this application is to provide a method for surface modification of materials, which combines the chemical modification and self-assembly of colloidal particles to make a coating on the surface of the material, which can achieve simultaneous control of the physical and chemical properties of the material surface, and Furthermore, the regional diversity of surface chemistry can be realized by self-assembly of colloidal particles with different chemical modifications.
  • the method is simple and fast, and can construct a large-area surface with multiple levels of physical structure and complex chemistry in one step.
  • the second purpose of the present application is to provide a material that uses the above-mentioned material surface modification method to modify the surface of the material.
  • the third purpose of this application is to provide an application of the above-mentioned material in microorganism or cell culture.
  • the fourth purpose of this application is to provide a medical product including the above-mentioned materials.
  • a method for surface modification of materials which includes the following steps:
  • Coating is formed on the surface of the material by self-assembly of colloidal particles
  • the colloidal particles include inorganic colloidal particles and organic polymer colloidal particles. All or part of the inorganic colloidal particles are chemically modified, and the chemical modification includes chemical functional group modification and/or biologically active molecule modification; the particle size of the inorganic colloidal particles is 1-10 ⁇ m, the particle size of the organic polymer colloidal particles is 0.05-0.75 ⁇ m;
  • the self-assembly uses an evaporation induction method.
  • the inorganic colloidal particles include metal oxide microspheres and/or non-metal oxide microspheres, preferably including SiO 2 microspheres, TiO 2 microspheres and ZnO microspheres.
  • metal oxide microspheres and/or non-metal oxide microspheres preferably including SiO 2 microspheres, TiO 2 microspheres and ZnO microspheres.
  • the chemical modification includes one or several different types of chemical modification
  • the chemical functional group includes one of halogen, amino, methacryloylethyl sulfobetaine, sulfhydryl, epoxy, acryl and azide groups;
  • the biologically active molecule includes one of short peptides with RGD sequence, polypeptides, small chemical molecules, antibiotics, and growth factors.
  • the organic polymer colloidal particles include modified and unmodified polystyrene microspheres, polymethylmethacrylate microspheres, chitosan microspheres, polycaprolactone microspheres, polydimethylsiloxane One or more of alkane microspheres, gelatin microspheres, polylactic acid microspheres and polyacrylic acid microspheres;
  • the organic polymer colloidal particles include chemically modified organic polymer colloidal particles, preferably including carboxylated polystyrene microspheres.
  • the diameter ratio of the inorganic colloidal particles and the organic polymer colloidal particles is 4-200:1, preferably 4-100:1.
  • the surface modification method of the above-mentioned material includes the following steps:
  • the chemical modification method in step (a) includes radical polymerization, anionic polymerization or cationic polymerization, and preferably includes any one of the following polymerization reactions:
  • the further modification in step (e) includes surface initiated polymerization, mercapto-olefin reaction, epoxy-amino group reaction or azide-alkyne reaction.
  • the ratio of the total volume of inorganic colloidal particles to the total volume of organic polymer colloidal particles in the mixed colloidal particle suspension in step (c) is 0.1-77, preferably 0.5-3;
  • step (d) Preferably, natural evaporation is used for drying in step (d);
  • heating is used for fixing in step (d), and the heating temperature is preferably 110-200° C., and the heating time is 1-20 min, preferably 1-5 min.
  • a material is provided, and the surface of the material is modified by the above-mentioned material surface modification method.
  • a medical product including the above-mentioned materials
  • the medical product includes microorganisms or cell culture tools
  • the medical product includes a biomedical antibacterial material.
  • the surface modification method of the material of the present application adopts chemically modified inorganic colloidal particles and organic polymer colloidal particles for self-assembly to form a coating on the surface of the material to realize the surface modification of the material.
  • This application combines the chemical modification and self-assembly of colloidal particles, realizes the construction of surface physical structure through colloidal particle self-assembly, and realizes the construction of surface chemical properties by using chemically modified inorganic colloidal particles before assembly.
  • the physical and chemical properties can be controlled at the same time.
  • multiple chemical modifications can be made on the surface of colloidal particles before self-assembly.
  • the regional diversity of surface chemistry can be realized by selecting different chemically modified colloidal particles for self-assembly, which expands the self-assembly of colloidal particles. Assemble and construct the use range of micro-nano interface.
  • the method of the present application is simple and fast, and can construct a large-area surface with multi-level physical micro-nano structure and complex chemistry in one step.
  • the modified surface can be used for in vitro microorganism and cell culture to regulate the adhesion and growth behavior of microorganisms and cells It can also be used to simulate the behavior of organisms on the surface of materials.
  • FIG. 1 is a schematic diagram of a process of a surface modification method of a material according to an embodiment of the application
  • FIG. 2 is a schematic diagram of a process of self-assembly of colloidal particles according to an embodiment of this application;
  • Fig. 3 is a schematic diagram of a post-modification method of colloidal particles self-assembly according to an embodiment of the application (wherein A in Fig. 3 is a schematic diagram of the post-assembly modification reaction of the self-assembled surface of a double colloidal particle, and B in Fig. 3 is a post-assembly surface of a polycolloid particle Reaction diagram);
  • Figure 4 is a schematic diagram of the water contact angle of the modified surface of Example 3, Example 10 and Example 11 of the application (the left side in Figure 4 is the schematic diagram of the water contact angle of the modified surface of Example 3, and the middle of Figure 4 Is a schematic diagram of the water contact angle of the modified surface of Example 11, and the right side of FIG. 4 is a schematic diagram of the water contact angle of the modified surface of Example 10);
  • Figure 5 is an XPS scan of the modified surface of Example 3 and Example 8 of this application (the left side of Figure 5 is the XPS scan of the modified surface of Example 3, and the right side of Figure 5 is the modified surface of Example 8. XPS scan of the posterior surface);
  • Example 6 is an SEM image of the surface formed after self-assembly in Example 3 to Example 6 of this application (wherein A in FIG. 6 is an SEM image of the surface formed after self-assembly in Example 3, and B in FIG. 6 is a self-assembly surface of Example 5) The SEM image of the surface formed after assembly.
  • C in Figure 6 is the SEM image of the surface formed after self-assembly in Example 4
  • D in Figure 6 is the SEM image of the surface formed after self-assembly in Example 6
  • E in Figure 6 is The SEM image of the surface formed after self-assembly in Example 7
  • F in Figure 6 is the SEM image of the surface formed after self-assembly in Example 8
  • G in Figure 6 is the SEM image of the surface formed after self-assembly in Example 9.
  • H in 6 is the SEM image of the surface formed after self-assembly in Example 11);
  • Figure 7 is a fluorescent staining diagram of the bacterial adhesion test results
  • Figure 8 is a statistical graph showing the area coverage of the bacterial adhesion test results
  • Figure 9 is a graph showing the results of a cell adhesion test
  • Figure 10 is a graph showing the statistical results of cell adhesion
  • Figure 11 is a fluorescence staining diagram of cell growth test results
  • Figure 12 is a statistical graph of cell viability measurement results.
  • a method for surface modification of a material includes the following steps: forming a coating on the surface of the material by self-assembly of colloidal particles; wherein the colloidal particles include inorganic colloidal particles and organic polymers. Colloidal particles, all or part of the inorganic colloidal particles are chemically modified, which includes chemical functional group modification and/or biologically active molecule modification; the particle size of the inorganic colloidal particles is 1-10 ⁇ m, and the particle size of the organic polymer colloidal particles is 0.05 -0.75 ⁇ m.
  • This application proposes a method of using chemically modified inorganic colloidal particles and organic polymer colloidal particles to self-assemble to obtain a coating to achieve simultaneous control of the physical and chemical properties of the material surface.
  • “Material surface” here refers to a material with a two-dimensional surface, that is, to modify the planar surface of the material.
  • the surface of the material is generally a hydrophilic surface.
  • the material of the material is not limited, including but not limited to polymer materials (such as PS (Polystyrene) or PET (Polyethylene terephthalate, polyterephthalate, etc.), inorganic non-metallic materials (such as silicon or quartz, etc.), and metal materials (such as stainless steel or titanium alloy, etc.) surface.
  • Colloidal particles are particles with a particle size of less than 10 ⁇ m.
  • the colloidal dispersion system formed by dispersing in a continuous liquid medium is called colloid.
  • Self-assembly refers to an ordered structure formed by the assembly and regular arrangement of one or more monodisperse colloidal particles.
  • the colloidal particle self-assembly here is the assembly of one or more monodispersed colloidal particles into an ordered two-dimensional planar structure, that is, a coating is formed on the surface (substrate) of the material.
  • a typical but non-limiting method is capillary action (such as evaporation induction: drop a colloidal dispersion on a hydrophilic substrate to form a meniscus. As the solvent evaporates, the capillary action and Under the common influence of convection and migration, the force of the particles will make the particles move to the thin layer area, and finally form a two-dimensional ordered structure) or gravity sedimentation.
  • the self-assembly process will involve capillary phenomena and Brownian motion.
  • the colloidal particles of the present application include inorganic colloidal particles that have been chemically modified in whole or in part.
  • the inorganic colloidal particles have a particle size of 1-10 ⁇ m, and the organic polymer colloidal particles have a particle size of 0.05-0.75 ⁇ m.
  • the typical but non-limiting particle size of the inorganic colloidal particles is, for example, 1 ⁇ m, 2 ⁇ m, 3 ⁇ m, 4 ⁇ m, 5 ⁇ m, 6 ⁇ m, 7 ⁇ m, 8 ⁇ m, 9 ⁇ m, or 10 ⁇ m.
  • Typical but non-limiting examples of the particle size of the organic polymer colloidal particles are 0.05 ⁇ m, 0.1 ⁇ m, 0.15 ⁇ m, 0.2 ⁇ m, 0.25 ⁇ m, 0.3 ⁇ m, 0.35 ⁇ m, 0.4 ⁇ m, 0.45 ⁇ m, 0.5 ⁇ m, 0.55 ⁇ m, 0.6 ⁇ m, 0.65 ⁇ m, 0.7 ⁇ m or 0.75 ⁇ m.
  • inorganic colloidal particles of the present application are chemically modified, that is, as long as there are chemically modified particles in the inorganic colloidal particles, they can be all chemically modified particles or partially chemically modified particles.
  • inorganic colloidal particles is not limited. It can be metal oxide particles (such as TiO 2 microspheres and ZnO microspheres, etc.), or non-metal oxide particles (such as SiO 2 microspheres, etc.), preferably SiO 2 microspheres .
  • the type of chemical modification is also not limited. It can be one type of chemical modification (a type of modification group) or several types of different types of chemical modification (several different modification groups).
  • the number of modification groups contained in a type of chemical modification is also not limited.
  • a type of chemical modification can have one chemical modification group or multiple different chemical modification groups.
  • Chemical modification includes, but is not limited to, chemical functional group modification or biologically active molecule modification.
  • the chemical functional group modified by the chemical functional group is typically but not limited to halogen, amino, methacryloylethyl sulfobetaine, sulfhydryl, epoxy, acryl or azide group; biologically active molecules are typical but not limited For example, short peptides containing arginine-glycine-aspartic acid (RGD) sequence, antibacterial polypeptides (for example, containing tryptophan-arginine-tryptophan-tryptophan-lysine-tryptophan Acid-tryptophan sequence polypeptide), small chemical molecules (such as zwitterionic small molecules [2-(methacryloxy)ethyl] dimethyl-(3-sulfonic acid propyl) ammonium hydroxide, antibiotics Vancomycin-like and signaling molecules like 1-acryloyl-4-(2-fluorophenyl)-5-methylenepyrrolid-2-one, etc.) or growth factors (insulin, insulin-like growth factor, transforming growth factor)
  • the inorganic colloidal particles are of one type (for example, SiO 2 microspheres), then the SiO 2 microspheres may contain the same type of chemically modified SiO 2 microspheres (such as SiO 2 -X), or may contain Many different types of chemically modified SiO 2 microspheres (for example, any two or more combinations of SiO 2 -X, SiO 2 -Y, SiO 2 -Z, etc.), where X, Y and Z represent different modification groups .
  • the SiO 2 microspheres and TiO 2 microspheres may contain the same type of chemically modified SiO 2 microspheres.
  • Balls (e.g. SiO 2 -X) and/or TiO 2 microspheres (e.g. TiO 2 -X) may also contain a variety of different types of chemically modified SiO 2 microspheres and/or TiO 2 microspheres (e.g.
  • SiO 2- Any two or more combinations of X, SiO 2 -Y, SiO 2 -Z, TiO 2 -X 1 , TiO 2 -Y 2 and TiO 2 -Z 3 ), wherein X, Y, Z, X 1 , Y 2 and Z 3 represent different types of modifying groups.
  • organic polymer colloidal particles is not limited. Typical but non-limiting examples are modified and unmodified polystyrene (PS) microspheres, polymethylmethacrylate (PMMA) microspheres, and chitosan microspheres.
  • PS polystyrene
  • PMMA polymethylmethacrylate
  • chitosan microspheres One or more of polycaprolactone microspheres, polydimethylsiloxane microspheres, gelatin microspheres, polylactic acid microspheres and polyacrylic acid microspheres.
  • the organic polymer colloidal particles can be unmodified particles, particles with modified groups, or a combination of unmodified particles and particles with modified groups.
  • the organic polymer colloidal particles are chemically modified, and the organic polymer colloidal particles are preferably carboxylated polystyrene (PSC) microspheres.
  • the surface modification method of the material of the present application adopts chemically modified inorganic colloidal particles and organic polymer colloidal particles for self-assembly to form a coating on the surface of the material to realize the surface modification of the material.
  • This application combines the chemical modification and self-assembly of colloidal particles, realizes the construction of surface physical structure through colloidal particle self-assembly, and realizes the construction of surface chemical properties by using chemically modified inorganic colloidal particles before assembly.
  • the physical and chemical properties can be controlled at the same time. At the same time, multiple chemical modifications can be made on the surface of colloidal particles before self-assembly.
  • the regional diversity of surface chemistry can be realized by selecting different chemically modified colloidal particles for self-assembly, which expands the self-assembly of colloidal particles. Assemble and construct the use range of micro-nano interface
  • the method of the present application is simple and fast, and can construct a large-area surface with multi-level physical micro-nano structure and complex chemistry in one step.
  • the diameter ratio of the inorganic colloidal particles and the organic polymer colloidal particles is 4-200:1, and the diameter ratio refers to a separate comparison between the two particles.
  • the diameter of the inorganic colloidal particles and the diameter of the organic polymer colloidal particles The ratio of diameters is, for example, 4:1, 5:1, 6:1, 7:1, 12.5:1 or 100:1, preferably 4-100:1.
  • an exemplary material surface modification method includes the following steps:
  • the chemical modification in step (S1) is typical but non-limiting, for example, surface grafting of inorganic colloidal particles.
  • the grafting methods are, for example, radical polymerization, anionic polymerization, cationic polymerization, ring-opening polymerization, and controlled radical polymerization. (Including Atom Transfer Radical Living Polymerization (ATRP), Reversible Addition-fragmentation Chain-transfer Polymerization (RAFT) and Single Electron Transfer Polymerization (Single Electron Transfer) Radical Living Polymerization, SET-LRP), etc.).
  • An exemplary way is to achieve surface grafting of inorganic colloidal particles by atom transfer radical polymerization.
  • ATRP is achieved by bonding ATRP initiator to inorganic colloidal particles (such as SiO 2 ) Surface, then surface ATRP polymerization, a living polymerization system composed of initiator (such as alkyl halide RX), catalyst (such as transition metal halide CuBr and CuCl) and complex ligand (such as bipyridine, etc.) , Can graft various homopolymers, block copolymers and hyperbranched polymers on the surface of inorganic particles.
  • initiator such as alkyl halide RX
  • catalyst such as transition metal halide CuBr and CuCl
  • complex ligand such as bipyridine, etc.
  • the organic polymer colloidal particles may also be chemically modified first to obtain chemically modified organic polymer colloidal particles, and then the chemically modified organic polymer colloidal particles are mixed with water.
  • volume of the colloidal particle suspension required to form a single-layer particle coating is calculated as follows:
  • the projected area of the colloid is Ap(cm 2 )
  • the density of the colloidal particle itself is ⁇ (g/cm 3 )
  • the required coating area is A0 (cm 2 )
  • the weight concentration of the colloidal particle suspension is w%
  • the volume V ( ⁇ L) of the colloidal particle suspension required to form a single-layer particle coating is shown in formula (1):
  • the ratio of the total volume of inorganic colloidal particles to the total volume of organic polymer colloidal particles in the mixed colloidal particle suspension in step (S3) is 0.1-77, for example, 0.1:1, 0.2:1, 0.3:1, 0.5: 1, 0.8:1, 1:1, 2:1, 3:1, 4:1, 5:1, 10:1, 20:1, 30:1, 40:1, 50:1, 60:1 70:1 or 77:1, preferably 0.5-3.
  • the volume ratio of 2 ⁇ m large particles to 65nm small particles is about 4.7:1. If two types of polymer particles are used, the volume ratio is about 1.1:1, which is between 0.1-77 It can be spread out, but the effect is not as good as 0.5-3.
  • the surface of the material is pretreated, and different pretreatment methods can be selected according to different materials.
  • a layer of polystyrene (PS) can be spin-coated on the surface of the glass slides to assist in fixing (for example, using 2.5g/mL PS toluene solution dropwise and spin coating on a 1cm diameter glass slide.
  • the spin coating conditions include : The first stage: 800rpm, 3s; the second stage: 1700rpm, 7s).
  • a shielding device that limits the area where the solution is dripped is added to the surface of the material, such as a sealing ring (O-ring), made of PDMS or PS.
  • O-ring sealing ring
  • the shape and material are not limited, as long as it is a device that can restrict the solution to the surface of the required material without spilling.
  • Drop the mixed colloidal particle suspension into the area circled on the surface of the material to make the liquid surface of the mixed solution form a state of negative curvature. After drying to remove the shield, a coating is obtained on the surface of the material ( Figure 2).
  • the drying in step (S5) adopts a natural evaporation method for drying, and the material to which the colloidal particles are dropped is allowed to stand and evaporate and dry naturally (no wind state) to ensure the surface level of the dropped colloidal particles.
  • the regular arrangement of colloidal particles on the surface of the material is triggered by capillary phenomenon and Brownian motion.
  • the fixing of step (S6) can choose different fixing methods according to different materials, preferably heating fixing, for example, when the substrate is a glass sheet, the glass sheet is heated and fixed on the heating plate after the surface coating is completely dried, preferably heating
  • the temperature is 110-130°C, such as 110°C, 120°C or 130°C
  • the heating time is 1-20 min, such as 1 min, 2 min, 5 min, 10 min, 15 min or 20 min, preferably 1-5 min.
  • the appropriate heating temperature and time can be selected according to the different materials of the organic polymer colloidal particles and the final desired effect.
  • the organic polymer colloidal particles are 0.4 ⁇ m PS
  • the heating temperature is 120°C
  • the organic height can be retained after heating for 1 minute.
  • the fixing method can be fixed with an organic solvent. It is preferable to use a mixed solution of toluene and ethanol (toluene:ethanol volume ratio of 1:2-1:4) and drip it on the surface for 1-20 seconds, preferably 2-5 seconds.
  • the further modification in step (S7) is, for example, to further initiate polymerization on the basis of the chemically modified groups on the surface formed by the chemically modified inorganic colloidal particles in step (S1), that is, the chemically modified inorganic colloidal particles
  • the modified group continues to serve as the initiation point of the polymerization reaction to proceed with the polymerization reaction, including but not limited to mercapto-olefin reaction, epoxy-amino group reaction or azide-alkyne reaction.
  • the application range of colloidal particle self-assembly is expanded, and the modification group and modification order (pre-assembly modification or post-assembly modification) can be flexibly selected
  • modification group and modification order pre-assembly modification or post-assembly modification
  • you can choose to modify them after assembly because if the chemical groups with high electrical properties are modified in advance, they will affect the morphology of self-assembly, and the active protein will be modified before assembly. There is a risk of inactivation during the fixation process.
  • This exemplary material surface modification method can simply and quickly construct micro-nano interface coatings with multiple chemistry, and expand the scope of use of colloidal particle self-assembly to construct micro-nano interfaces: that is, multiple chemical functional groups can be self-assembled at the same time.
  • the pre-grafted on the surface of colloidal particles, and these functional groups can also be used for the reaction after assembly.
  • a material is provided, and the surface of the material is modified by the above-mentioned material surface modification method.
  • the surface of the material modified by the method of the present application not only has a multi-level physical micro-nano structure, but also has certain chemical properties, which can realize the regional diversity of surface chemistry and expand the application range of the interface after the material is modified.
  • the surface of the material modified by the method of this application is cultured with microorganisms or cells, which proves that the surface can indeed affect the adhesion behavior of microorganisms or cells. It can be used for in vitro microorganism and cell culture, regulate the growth behavior of microorganisms and cells, and simulate biological interface behavior. .
  • a medical product including the above-mentioned materials.
  • This material can have the characteristics of antibacterial attachment and allowing cell attachment, providing useful information for antibacterial surfaces of biomedical materials.
  • Medical products include, but are not limited to, microorganisms or cell culture tools or medical materials (such as medical antibacterial materials).
  • the medical product containing the modified material of this application has the same advantages as the above-mentioned materials.
  • the surface of the material is modified in a targeted manner. According to the physical and chemical properties of the modified material surface, the material can be used as a medical training tool or material .
  • the reaction was maintained at 37°C for 24 hours; after the reaction, the mixed solution was transferred to Collect the microspheres in the centrifuge tube by centrifugation, wash the microspheres with deionized water three times and then dry at room temperature, the resulting product is SiO 2 -RGD;
  • Step (1) uses SiO 2 spheres with a particle size of 2 ⁇ m, and the remaining steps are the same as in Example 1.
  • a method for surface modification of glass sheet includes the following steps:
  • SiO 2 -Br microspheres are single-layer tiled, colloidal SiO 2 -Br projected area is 19.625 ⁇ m 2 , SiO 2 -Br particle density is 2g/cm 3 , required coating area is 0.79cm 2 , SiO 2- The mass percentage concentration of the Br particle suspension is 10%, and the SiO 2 -Br particle volume is 65 ⁇ m 3. Substituting into the formula (1), the SiO 2 -Br particle suspension required to form a single layer of SiO 2 -Br particle coating is calculated The volume V of the turbid liquid is 4.97 ⁇ L;
  • the projected area of colloidal PSC is 0.126 ⁇ m 2
  • the density of PSC particles is 1.19g/cm 3
  • the required coating area is 0.79cm 2
  • the mass percentage concentration of PSC particle suspension is 10%
  • the volume of PSC particles is 0.033 ⁇ m 3
  • substituting formula (1) into formula (1) calculates that the volume of PSC particle suspension required to form a single-layer particle coating is 0.24 ⁇ L, so the total volume of 0.4 ⁇ m PSC particle suspension required is 1.42 ⁇ L;
  • the glass sheet is heated and fixed on the heating plate, the heating temperature is 120 DEG C, the heating time is 10 minutes, and the surface is coated.
  • Embodiment 3 The difference between this embodiment and Embodiment 3 is that the 5 ⁇ m SiO 2 -Br particles are replaced with the 5 ⁇ m SiO 2 -SBMA particles in Embodiment 1.
  • Embodiment 3 The difference between this embodiment and Embodiment 3 is that the 5 ⁇ m SiO 2 -Br particles are replaced with the 2 ⁇ m SiO 2 -Br particles in Example 2, and the 0.4 ⁇ m PSC particles are replaced with 0.1 ⁇ m PSC particles.
  • this embodiment also includes step (7) to further modify the surface of the SiO 2 -Br colloidal particles in the coating with SBMA (A in Figure 3, that is, the two kinds of particles self-assemble, after assembly only There are large particles with modified polymer brushes on the surface), including:
  • Example 3 The water contact angle and XPS scans of the modified surface of Example 3 and Example 10 are shown in Figures 4 to 5.
  • the difference between this embodiment and the third embodiment is that the 0.4 ⁇ m PSC particles are replaced with a combination of 0.4 ⁇ m PSC particles and 0.4 ⁇ m PMMA particles with a volume ratio of 1:1 to maintain the inorganic colloidal particles in the organic colloidal particle suspension. The total amount remains unchanged.
  • Example 3 differs from Example 10 in that in Example 3, replacing the combination of Example 1 volume ratio of SiO 2 -Br 5 ⁇ m particles and 2:8 embodiment 5 ⁇ m of SiO 2 particles of 5 ⁇ m of SiO 2 particles -Br , Keep the total amount of inorganic colloidal particles in the inorganic colloidal particle suspension unchanged, and the subsequent reaction steps remain unchanged ( Figure 3 B, which is the result of the self-assembly of the three particles, the modified and unmodified particles are pre-mixed before assembly , After assembly, only part of the large particles have modified polymer brushes on the surface).
  • Embodiment 3 The difference between this embodiment and Embodiment 3 is that the 5 ⁇ m SiO 2 -Br particles are replaced with 5 ⁇ m SiO 2 particles.
  • Embodiment 5 The difference between this embodiment and Embodiment 5 is that the 2 ⁇ m SiO 2 -Br particles are replaced with 2 ⁇ m SiO 2 particles.
  • Fig. 6 The SEM observation of the surface formed after self-assembly of Example 3 to Example 6 is shown in Fig. 6. As can be seen from Fig. 6, the colloidal particles of this application have self-assembly capabilities and micro-nano surfaces are formed. structure.
  • Test surface glass sheet, 5SiO 2 : the surface formed after self-assembly in Comparative Example 1; 5SiO 2 -SBMA: the surface formed after self-assembly in Example 4; 5SiO 2 -ATRP: the surface formed after chemical modification in Example 10; 2SiO 2 : The surface formed after self-assembly in Comparative Example 2; 2SiO 2 -SBMA: the surface formed after self-assembly in Example 6; the results are shown in Figs. 7-8.
  • mice pre-osteoblasts (MC 3T3) on the surface formed after self-assembly in Comparative Example 1 and Example 4, a cell climbing slide test was performed.
  • Test method cell inoculation step, the cell culture density reaches about 80-90%, take out the cell culture dish, put it in a sterile operating table, discard the old medium; add about 5mL PBS and shake twice; add an appropriate amount of 0.25% pancreatin Digest the cells and place them in a 37°C incubator for 2 minutes. Observe the cells in a spherical shape and fall off under a microscope. Add 8mL of fresh medium to terminate the digestion; pipette some cells that are not completely removed, transfer the cell solution to a 15mL centrifuge tube, and centrifuge at 1100rpm. Minutes, discard the upper layer of waste liquid, add 4-5mL of fresh medium and mix the bottom cells by pipetting; use a cell counter to calculate the total number of cells.
  • the test samples in Figure 9 are: glass sheet, 5SiO 2 : the surface formed after self-assembly in Comparative Example 1; 5SiO 2 -SBMA: the surface formed after self-assembly in Example 4; 5SiO 2 -ATRP: after chemical modification in Example 10 The formed surface; 2SiO 2 : the surface formed after self-assembly in Comparative Example 2; 2SiO 2 -SBMA: the cytoskeleton staining result of the surface cells formed after self-assembly in Example 6 after 24 hours of culture.
  • test samples in Figure 11 are: glass sheet, 5SiO 2 : the surface formed after self-assembly in Comparative Example 1; 5SiO 2 -SBMA: the surface formed after self-assembly in Example 4; 5SiO 2 -ATRP: after chemical modification in Example 10 The formed surface; 2SiO 2 : the surface formed after self-assembly in Comparative Example 2; 2SiO 2 -SBMA: the surface formed after self-assembly in Example 6 The results of cytoskeleton staining and the cell activity CCK-8 detection results after three days of culture.
  • this application is able to make a surface with multiple physical and chemical properties through the self-assembly of chemically modified inorganic colloidal particles and organic polymer colloidal particles.
  • the reaction of bacteria and cells proves that this surface is indeed realized. It has an effect on the adhesion and proliferation of cells and bacteria. It can provide a good platform for studying the response of bacterial cells to physical and chemical stimuli on the interface.
  • Test Examples 1 and 2 The purpose of Test Examples 1 and 2 is not to select which modification or surface is good, but only to show that this application can prepare a surface with desired physical and chemical properties simply and quickly, which can be applied to the study of bacteria and cell behavior.
  • Example 6 has a reduced number of bacterial adhesion, but it does not affect cell adhesion and proliferation. Based on these two surfaces, it can be Research on the mechanism of bacterial anti-adhesion.
  • the samples of Example 4 and Example 10 have SBMA on their surfaces and can reduce bacterial adhesion, they have different effects on cell proliferation. Based on these two surfaces, the effect of material surface on cell proliferation can be studied.

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Abstract

一种材料的表面改性方法，使用该方法改性后的材料及其应用、包括该材料的医疗产品。材料的表面改性方法包括以下步骤：在材料表面通过胶体颗粒自组装的方法形成涂层；其中，胶体颗粒包括粒径为1-10μm的无机胶体颗粒和粒径为0.05-0.75μm的有机高分子胶体颗粒，全部或部分所述无机胶体颗粒经过化学修饰，化学修饰包括化学官能团修饰和/或生物活性分子修饰。将胶体颗粒的化学修饰和自组装结合，可实现对材料表面物理和化学性质的同时调控，可以通过选取不同化学修饰的胶体颗粒自组装实现表面化学的区域多样性，可一步大面积构建同时具有多级物理结构和复杂化学的表面。

Description

材料的表面改性方法及其改性后的材料和应用、医疗产品 技术领域
本申请涉及材料表面改性技术领域，具体而言，涉及一种材料的表面改性方法及其改性后的材料和应用、医疗产品。
背景技术
材料的表面改性技术应用范围极为广泛，包括医疗器械或工业防腐等领域。对材料表面的改性可以分为两类：物理结构的改变以及化学性质的改变。表面物理结构的改变方法包括刻蚀、平板印刷(lithography)或翻印技术。表面化学性质的改变方法包括逐层镀膜(layerby layer)、根据表面化学官能团进行针对性反应以及材料表面的非特异性镀层(substrate independent coating)。
现有对表面构建可控的物理结构或者化学性质的改变方式过程繁琐，耗时长。例如，刻蚀需要用到特殊的仪器设备：等离子体、电子、激光或化学刻蚀仪器，且刻蚀之后的表面化学性质均一。翻印技术虽操作较为简易且可较大面积构建表面物理结构，但其所选用的基材、翻印材料以及物理结构的尺寸均具有局限性，且母版的制作仍需借助刻蚀工艺完成，翻印所得的表面化学性质也是均一的。平板印刷技术可以高精度地构建表面物理化学性质，但由于所需时间长且成本高，在大面积的表面修饰应用中受到了很大的约束。
此外，现有的技术无法实现对材料表面物理化学性质的同时调控，常见的方法是先构建物理结构然后对于具有物理结构的表面进行下一步的化学修饰，并且现有的化学修饰手段多为表面均一性修饰，虽然可以通过遮挡-修饰-去遮挡的方法实现表面多重化学区域性的修饰，但此方法也至少需要三个甚至更多步骤实现。
因此，所期望的是提供一种表面改性方法，其能够解决上述问题中的至少一个。
有鉴于此，特提出本申请。
发明内容
本申请的目的之一在于提供一种材料的表面改性方法，将胶体颗粒的化学修饰和自组装结合，在材料表面做出涂层，可实现对材料表面物理和化学性质的同时调控，并且进一步可以通过选取不同化学修饰的胶体颗粒自组装实现表面化学的区域多样性，方法简单且快捷，可一步大面积构建同时具有多级物理结构和复杂化学的表面。
本申请的目的之二在于提供一种材料，采用上述材料的表面改性方法对材料的表面进行改性。
本申请的目的之三在于提供一种上述材料在微生物或细胞培养中的应用。
本申请的目的之四在于提供一种医疗产品，包括上述材料。
为了实现本申请的上述目的，特采用以下技术方案：
第一方面，提供了一种材料的表面改性方法，包括以下步骤：
在材料表面通过胶体颗粒自组装的方法形成涂层；
其中，胶体颗粒包括无机胶体颗粒和有机高分子胶体颗粒，全部或部分所述无机胶体颗粒经过化学修饰，化学修饰包括化学官能团修饰和/或生物活性分子修饰；所述无机胶体颗粒的粒径为1-10μm，所述有机高分子胶体颗粒的粒径为0.05-0.75μm；
优选地，自组装采用蒸发诱导法。
优选地，在本申请技术方案的基础上，所述无机胶体颗粒包括金属氧化物微球和/或非金属氧化物微球，优选包括SiO 2微球、TiO 2微球和ZnO微球中的一种或几种，进一步优选为SiO 2微球；
优选地，所述化学修饰包括一类或几类不同的化学修饰；
优选地，在本申请技术方案的基础上，化学官能团包括卤素、氨基、甲基丙烯酰乙基磺基甜菜碱、巯基、环氧基、丙烯酰基和叠氮基团中的一种；
优选地，生物活性分子包括具有RGD序列的短肽、多肽、化学小分子、抗生素和生长因子中的一种。
优选地，所述有机高分子胶体颗粒包括修饰与未修饰的聚苯乙烯微球、聚甲基丙烯酸甲酯微球、壳聚糖微球、聚己内酯微球、聚二甲基硅氧烷微球、明胶微球、聚乳酸微球和聚丙烯酸微球中的一种或几种；
优选地，所述有机高分子胶体颗粒包括化学修饰的有机高分子胶体颗粒，优选包括羧基化的聚苯乙烯微球。
优选地，在本申请技术方案的基础上，无机胶体颗粒和有机高分子胶体颗粒的直径比为4-200∶1，优选为4-100∶1。
优选地，在本申请技术方案的基础上，上述材料的表面改性方法，包括以下步骤：
(a)对无机胶体颗粒进行化学修饰，得到化学修饰的无机胶体颗粒，将化学修饰的无机胶体颗粒与水混合，得到无机胶体颗粒悬浊液；
(b)将有机高分子胶体颗粒与水混合，得到有机高分子胶体颗粒悬浊液；
(c)将无机胶体颗粒悬浊液与有机高分子胶体颗粒悬浊液混合，得到混合胶体颗粒悬浊液；
(d)将混合胶体颗粒悬浊液滴加至材料表面，或将材料表面浸入混合胶体颗粒悬浊液中；干燥和固定后得到涂层；
(e)对涂层中的经过化学修饰的无机胶体颗粒的修饰基团上进行化学修饰，得到表面改性后的材料。
优选地，在本申请技术方案的基础上，步骤(a)中的化学修饰方式包括自由基聚合、阴离子聚合或阳离子聚合，优选包括以下聚合反应的任意一种：
(i)原子转移自由基聚合；
(ii)单电子转移自由基聚合；
(iii)可逆加成-断裂链转移聚合；
优选地，步骤(e)中的进一步修饰方式包括表面引发聚合、巯基-烯烃反应、环氧基-氨基反应或叠氮-炔烃反应。
优选地，步骤(c)中混合胶体颗粒悬浊液中无机胶体颗粒总体积与有机高分子胶体颗粒总体积之比为0.1-77，优选为0.5-3；
优选地，步骤(d)中干燥采用自然蒸发；
优选地，步骤(d)中固定采用加热固定，优选加热温度为110-200℃，加热时间为1-20min，优选为1-5min。
第二方面，提供了一种材料，采用上述材料的表面改性方法对材料的表面进行改性。
第三方面，提供了一种上述材料在微生物或细胞培养中的应用。
第四方面，提供了一种医疗产品，包括上述材料；
优选地，所述医疗产品包括微生物或细胞培养工具；
优选地，所述医疗产品包括生物医药抗菌材料。
与已有技术相比，本申请具有如下有益效果：
本申请材料的表面改性方法通过采用包括化学修饰后的无机胶体颗粒和有机高分子胶体颗粒进行自组装，在材料表面形成涂层，实现对材料表面的改性。本申请将胶体颗粒的化学修饰和自组装相结合，通过胶体颗粒自组装实现表面物理结构的构建，通过组装前采用化学修饰的无机胶体颗粒实现表面化学性质的构建，该方法实现了对材料表面物理和化学性质的同时调控，同时，可以同时将多种化学修饰在自组装前的胶体颗粒表面，可以通过选取不同化学修饰的胶体颗粒自组装实现表面化学的区域多样性，扩大了胶体颗粒自组装构建微纳米界面的使用范围。
本申请的方法简单且快捷，可一步大面积构建同时具有多级物理微纳米结构和复杂化学的表面，改性后的表面可用于体外微生物和细胞培养，调控微生物和细胞的粘附与生长行为，也可用于生物在材料表面界面行为的模拟。
附图说明
图1为本申请一种实施方式的材料的表面改性方法的过程示意图；
图2为本申请一种实施方式的胶体颗粒自组装的过程示意图；
图3为本申请一种实施方式的胶体颗粒自组装后修饰方法示意图(其中图3中A为双胶体颗粒自组装表面组装后修饰反应示意图，图3中B为多胶体颗粒自组装表面组装后反应示意图)；
图4为本申请实施例3、实施例10和实施例11改性后表面的水接触角示意图(其中图4中左侧为实施例3改性后表面的水接触角示意图，图4中中间为实施例11改性后表面的水接触角示意图，图4中右侧为实施例10改性后表面的水接触角示意图)；
图5为本申请实施例3和实施例8改性后表面的XPS扫描图(其中图5中左侧为实施例3改性后表面的XPS扫描图，图5中右侧为实施例8改性后表面的XPS扫描图)；
图6为本申请实施例3-实施例6自组装后形成的表面的SEM图(其中图6中A为实施例3自组装后形成的表面的SEM图，图6中B为实施例5自组装后形成的表面的SEM图，图6中C为实施例4自组装后形成的表面的SEM图，图6中D为实施例6自组装后形成的表面的SEM图，图6中E为实施例7自组装后形成的表面的SEM图，图6中F为 实施例8自组装后形成的表面的SEM图，图6中G为实施例9自组装后形成的表面的SEM图，图6中H为实施例11自组装后形成的表面的SEM图)；
图7为细菌粘附试验结果荧光染色图；
图8为细菌粘附试验结果面积覆盖统计图；
图9为细胞粘附试验结果图；
图10为细胞粘附统计结果图；
图11为细胞生长试验结果荧光染色图；
图12为细胞活性测量结果统计图。
具体实施方式
下面将结合实施例对本申请的实施方案进行详细描述，但是本领域技术人员将会理解，下列实施例仅用于说明本申请，而不应视为限制本申请的范围。实施例中未注明具体条件者，按照常规条件或制造商建议的条件进行。所用试剂或仪器未注明生产厂商者，均为可以通过市售购买获得的常规产品。
根据本申请的第一个方面，提供了一种材料的表面改性方法，包括以下步骤：在材料表面通过胶体颗粒自组装的方法形成涂层；其中，胶体颗粒包括无机胶体颗粒和有机高分子胶体颗粒，全部或部分所述无机胶体颗粒经过化学修饰，化学修饰包括化学官能团修饰和/或生物活性分子修饰；无机胶体颗粒的粒径为1-10μm，有机高分子胶体颗粒的粒径为0.05-0.75μm。
现有技术对于材料表面进行单一的物理或化学修饰的技术已基本纯熟，但针对其表面的物理和化学性质同时进行调控的领域仍缺少一个简易快捷且可对大面积表面进行改性的方法。
本申请提出了一种利用化学修饰的无机胶体颗粒与有机高分子胶体颗粒进行自组装得到涂层的方式实现对材料表面物理和化学性质的同时调控。
这里的“材料表面”指具有二维表面的材料，即在材料的平面表面上进行改性，材料表面一般为亲水性平面，对材料的材质不作限定，包括但不限于高分子材质(如PS(Polystyrene，聚苯乙烯)或PET(Polyethylene terephthalate，聚对苯二甲酸类塑料)等)，无机非金属材质(如硅或石英等)，以及金属材质(如不锈钢或钛合金等)表面。
胶体颗粒(colloidal particle)是粒径在10μm以下的颗粒，分散于连续的液态介质而形成的胶体分散体系称为胶体。
自组装是指由一种或多种单分散胶体粒子组装并规整排列而形成的有序结构。这里的胶体颗粒自组装是由一种或多种单分散胶体粒子组装成有序的二维平面结构，即在材料表面(基体)上形成涂层。
对具体的自组装方式不作限定，典型但非限制性的方式例如为毛细作用(如蒸发诱导：将胶体分散液滴在亲水基片上，形成一弯液，随着溶剂蒸发，在毛细作用和对流迁移作用的共同影响下，粒子所受的力会使粒子向薄层区移动，最终形成二维有序结构)或重力沉降等，自组装过程会涉及毛细现象和布朗运动等作用。
本申请胶体颗粒包括全部或部分经过化学修饰的无机胶体颗粒，无机胶体颗粒的粒径为1-10μm，有机高分子胶体颗粒的粒径为0.05-0.75μm。
无机胶体颗粒的粒径典型但非限制性的例如为1μm、2μm、3μm、4μm、5μm、6μm、7μm、8μm、9μm或10μm。有机高分子胶体颗粒的粒径典型但非限制性的例如为0.05μm、0.1μm、0.15μm、0.2μm、0.25μm、0.3μm、0.35μm、0.4μm、0.45μm、0.5μm、0.55μm、0.6μm、0.65μm、0.7μm或0.75μm。
大颗粒的无机胶体颗粒和小颗粒的有机高分子胶体颗粒通过自组装，形成二维有序结构。
无机胶体颗粒
本申请的无机胶体颗粒中全部或部分颗粒是经过化学修饰的，即只要无机胶体颗粒中有化学修饰的颗粒即可，可以全是化学修饰的颗粒，也可以是部分化学修饰的颗粒。
对无机胶体颗粒的种类不作限定，可以是金属氧化物颗粒(如TiO 2微球和ZnO微球等)，也可以是非金属氧化物颗粒(如SiO 2微球等)，优选为SiO 2微球。
对化学修饰的类别也不作限定，可以是一类化学修饰(一种修饰基团)，也可以是几类不同的化学修饰(几种不同的修饰基团)。对一类化学修饰中所含的修饰基团的个数也不作限定，一类化学修饰中可以具有一个化学修饰基团，也可以具有多个不同的化学修饰基团。化学修饰包括但不限于化学官能团修饰或生物活性分子修饰。化学官能团修饰的化学官能团典型但非限制性的例如为卤素、氨基、甲基丙烯酰乙基磺基甜菜碱、巯基、环氧基、丙烯酰基或叠氮基团；生物活性分子典型但非限制性的例如为含有精氨酸-甘氨酸-天门冬氨酸(RGD)序列的短肽、抗菌多肽(例如含有色氨酸-精氨酸-色氨酸-色氨酸-赖氨酸-色氨酸-色氨酸序列多肽)、化学小分子(例如两性离子小分子类[2-(甲基丙烯酰基氧基)乙基]二甲基-(3-磺酸丙基)氢氧化铵、抗生素类万古霉素以及信号分子类1-丙烯酰基-4-(2-氟苯基)-5-亚甲基吡咯烷-2-酮等)或生长因子(胰岛素、类胰岛素生长因子、转化生长因子、表皮生长因子、血管内皮生长因子、血小板来源生长因子、成纤细胞生长因子、生长激素释放抑制因子、神经生长因子、白细胞介素、红细胞生长素、皮质醇或甲状腺素等)。
例如一种实施方式，无机胶体颗粒为一种(例如为SiO 2微球)，那么SiO 2微球中可以包含同一类的化学修饰的SiO 2微球(例如SiO 2-X)，也可以包含多种不同类的化学修饰的SiO 2微球(例如SiO 2-X、SiO 2-Y和SiO 2-Z等等的任意两种以上组合)，其中X、Y和Z表示不同的修饰基团。
例如另一种实施方式，无机胶体颗粒为几种(例如为SiO 2微球和TiO 2微球的组合)，那么SiO 2微球和TiO 2微球中可以包含同一类化学修饰的SiO 2微球(例如SiO 2-X)和/或TiO 2微球(例如TiO 2-X)，也可以包含多种不同类的化学修饰的SiO 2微球和/或TiO 2微球(例如SiO 2-X、SiO 2-Y、SiO 2-Z、TiO 2-X 1、TiO 2-Y 2和TiO 2-Z 3等等的任意两种以上组合)，其中X、Y、Z、X 1、Y 2和Z 3表示不同类别的修饰基团。
有机高分子胶体颗粒
对有机高分子胶体颗粒的种类不作限定，典型但非限制性的例如为修饰与未修饰的聚 苯乙烯(PS)微球、聚甲基丙烯酸甲酯(PMMA)微球、壳聚糖微球、聚己内酯微球、聚二甲基硅氧烷微球、明胶微球、聚乳酸微球和聚丙烯酸微球中的一种或几种。
有机高分子胶体颗粒可以是未经修饰的颗粒，也可以是带修饰基团的颗粒，还可以是未经修饰的颗粒和带修饰基团的颗粒的组合。有机高分子胶体颗粒是经过化学修饰的，有机高分子胶体颗粒优选为羧基化的聚苯乙烯(PSC)微球。
本申请材料的表面改性方法通过采用包括化学修饰后的无机胶体颗粒和有机高分子胶体颗粒进行自组装，在材料表面形成涂层，实现对材料表面的改性。本申请将胶体颗粒的化学修饰和自组装相结合，通过胶体颗粒自组装实现表面物理结构的构建，通过组装前采用化学修饰的无机胶体颗粒实现表面化学性质的构建，该方法实现了对材料表面物理和化学性质的同时调控，同时，可以同时将多种化学修饰在自组装前的胶体颗粒表面，可以通过选取不同化学修饰的胶体颗粒自组装实现表面化学的区域多样性，扩大了胶体颗粒自组装构建微纳米界面的使用范围。本申请的方法简单且快捷，可一步大面积构建同时具有多级物理微纳米结构和复杂化学的表面。
在一种实施方式中，无机胶体颗粒和有机高分子胶体颗粒的直径比为4-200∶1，直径比是指两种颗粒之间单独对比，无机胶体颗粒的直径和有机高分子胶体颗粒的直径之比，例如为4∶1、5∶1、6∶1、7∶1、12.5∶1或100∶1，优选为4-100∶1。
通过两种微球的尺寸选择合适的体积比有助于获得结构均一有序且厚度适宜的涂层表面。
在一种实施方式中，一种示例性的材料的表面改性方法，如图1所示，包括以下步骤：
(S1)对无机胶体颗粒进行化学修饰，得到化学修饰的无机胶体颗粒，将化学修饰的无机胶体颗粒与水混合，得到无机胶体颗粒悬浊液；
(S2)将有机高分子胶体颗粒与水混合，得到有机高分子胶体颗粒悬浊液；
(S3)将无机胶体颗粒悬浊液与有机高分子胶体颗粒悬浊液混合，得到混合胶体颗粒悬浊液；
(S4)将混合胶体颗粒悬浊液滴加至材料表面，或将材料表面浸入混合胶体颗粒悬浊液中；
(S5)干燥；
(S6)固定，得到涂层；
(S7)对涂层中的经过化学修饰的无机胶体颗粒的修饰基团上进行化学修饰，得到表面改性后的材料。
步骤(S1)中的化学修饰典型但非限制性的例如为对无机胶体颗粒进行表面接枝，接枝的方法例如为自由基聚合、阴离子聚合、阳离子聚合、开环聚合及可控自由基聚合(包括原子转移自由基聚合((Atom Transfer Radical Living Polymerization，ATRP)、可逆加成-断裂链转移聚合(Reversible Addition-fragmentation Chain-transfer Polymerization，RAFT)和单电子转移活性自由基聚合(Single Electron Transfer Radical Living Polymerization，SET-LRP)等)。一种示例性的方式为通过原子转移自由基聚合实现对无机胶体颗粒的表面 接枝。ATRP是通过把ATRP引发剂键接到无机胶体颗粒(例如SiO 2)表面，然后进行表面ATRP聚合，由引发剂(如烷基卤化物RX)、催化剂(如过渡金属卤化物CuBr和CuCl)和络合配位体(如联吡啶等)组成的活性聚合体系，可以在无机颗粒表面接枝各种均聚物、嵌段共聚物和超支化聚合物。
步骤(S2)中也可以先对有机高分子胶体颗粒进行化学修饰，得到化学修饰的有机高分子胶体颗粒，再将化学修饰的有机高分子胶体颗粒与水混合。
一种实施方式中，理论上构成单层颗粒涂层所需胶体颗粒悬浊液的体积的计算方式如下：
设胶体投影面积为Ap(cm 2)，胶体颗粒本身密度为ρ(g/cm 3)，所需涂层面积为A0(cm 2)，胶体颗粒悬浊液重量浓度为w％，胶体颗粒体积为Vp(cm 3)，则构成单层颗粒涂层所需胶体颗粒悬浊液的体积V(μL)如式(1)所示：
Figure PCTCN2019084522-appb-000001
其中，0.95为经验值。
推导过程如下：
令m＝颗粒总重量，N为颗粒总数，则有m＝N×V p×ρ  (2)；
其中：m＝V×w％  (3)；
又令A 0＝涂层面积，A 1＝颗粒投影面积，则有
Figure PCTCN2019084522-appb-000002
其中0.95为经验值。
将式(3)(4)代入式(2)并整理后，可得公式(1)。
优选地，步骤(S3)中混合胶体颗粒悬浊液中无机胶体颗粒总体积与有机高分子胶体颗粒总体积之比为0.1-77，例如0.1∶1、0.2∶1、0.3∶1、0.5∶1、0.8∶1、1∶1、2∶1、3∶1、4∶1、5∶1、10∶1、20∶1、30∶1、40∶1、50∶1、60∶1、70∶1或77∶1，优选为0.5-3。2μm大颗粒与65nm小颗粒体积比约为4.7∶1，如果使用两种高分子小颗粒体积比约为1.1∶1，在0.1-77之间可以铺出来，但效果不如0.5-3之间。
优选地，步骤(S4)对材料表面进行预处理，可根据材料的不同选择不同的预处理方式。例如玻璃片，可以在玻璃片表面旋涂一层聚苯乙烯(PS)起到辅助固定效果(如用2.5g/mL PS的甲苯溶液滴加在1cm直径的玻璃片上旋涂。旋涂条件包括：第一阶段：800rpm、3s；第二阶段：1700rpm、7s)。
优选地，在材料表面加一个限制溶液滴加区域的遮挡装置，比如密封圈(O型圈)，材质为PDMS或PS等。对其形状和材质不作限定，只要是可以将溶液限制在所需材料表面不外溢的装置即可。将混合胶体颗粒悬浊液滴加至材料表面所圈区域内，使混合溶液液面 形成负曲率状态，干燥去遮挡后在材料表面得到涂层(如图2)。
优选地，步骤(S5)的干燥采用自然蒸发法进行干燥，将滴加胶体颗粒的材料静置自然挥发干燥(无风状态)，保证滴加胶体颗粒的表面水平。在干燥过程中，通过毛细现象和布朗运动引发胶体颗粒在材料表面的规则排列。
步骤(S6)的固定可以根据材料的不同选取不同的固定方法，优选采用加热固定，例如当基材为玻璃片时，在表面涂层彻底干燥后将玻璃片在加热板上加热固定，优选加热温度为110-130℃，例如110℃、120℃或130℃，加热时间为1-20min，例如1min、2min、5min、10min、15min或20min，优选为1-5min。可以根据有机高分子胶体颗粒的材质不同和最终所需效果的不同选取合适的加热温度及时间，例如，有机高分子胶体颗粒为0.4μm的PS，加热温度在120℃，加热1min可保留有机高分子颗粒(小颗粒)形貌，加热时间为20min有机高分子颗粒(小颗粒)全部融化仅剩无机颗粒(大颗粒)的形貌。同理，若当基材为高分子材料例如(聚对苯二甲酸类塑料)PET时，固定方法可选用使用有机溶剂固定。优选使用甲苯与乙醇的混合溶液(甲苯∶乙醇体积比为1∶2-1∶4)滴加在表面1-20s，优选为2-5s。
优选地，步骤(S7)中的进一步修饰方式例如为在步骤(S1)化学修饰的无机胶体颗粒形成的表面的已经化学修饰的基团的基础上进一步引发聚合，即以化学修饰的无机胶体颗粒的修饰基团继续作为聚合反应的引发点，进行聚合反应，包括但不限于巯基-烯烃反应、环氧基-氨基反应或叠氮-炔烃反应等。
通过采取组装前无机胶体颗粒的修饰以及组装后对于修饰官能团的针对性修饰的策略，扩大了胶体颗粒自组装的应用范围，可灵活选择修饰基团和修饰顺序(组装前修饰还是组装后修饰)，对于一些电性高的化学基团和一些活性蛋白可以选择组装后修饰，因为电性高的化学基团如果提前修饰将会影响自组装的形貌，而活性蛋白组装前修饰，在之后的固定过程中会有失活的风险。该示例性的材料的表面改性方法能简单快速地构建具有多重化学的微纳米界面涂层，扩大了胶体颗粒自组装构建微纳米界面的使用范围：即可以同时将多种化学官能团在自组装前嫁接于胶体颗粒表面，并且这些官能团也可用于组装后再反应。
根据本申请的第二个方面，提供了一种材料，采用上述材料的表面改性方法对材料的表面进行改性。
经过本申请方法改性后的材料表面不仅具有多级物理微纳米结构，而且具有一定的化学性质，可以实现表面化学的区域多样性，扩大了材料改性后界面的应用范围。
根据本申请的第三个方面，提供了一种上述材料在微生物或细胞培养中的应用。
本申请方法改性后的材料表面进行微生物或细胞培养，证实了该表面确实能够影响微生物或细胞的粘附行为，可用于体外微生物和细胞培养，调控微生物和细胞的生长行为，模拟生物界面行为。
根据本申请的第四个方面，提供了一种医疗产品，包括上述材料。
此材料可具有抗菌贴附与允许细胞贴附的特性，为生医材料表面抗菌提供有用的资讯。
医疗产品包括但不限于微生物或细胞培养工具或医疗材料(例如医药抗菌材料)等。
包含本申请改性后材料的医疗产品具有与上述材料相同的优势，对材料表面进行针对性改性，并根据改性后材料表面的物理化学性质，可将材料作为医疗培养工具或材料去使用。
下面通过具体的实施例和对比例进一步说明本申请，但是，应当理解为，这些实施例仅是用于更详细地说明之用，而不应理解为用于以任何形式限制本申请。本申请涉及的各原料均可通过商购获取。
实施例1 制备粒径5μm的SiO 2-NH 2、SiO 2-Epoxy(表面带有环氧丙基的二氧化硅微球)、SiO 2-RGD、SiO 2-Br和粒径5μm的SiO 2-SBMA
(1)将50mg粒径5μm SiO 2球与5mL无水四氢呋喃(THF)加入圆底烧瓶中，通过反复超声，震荡使微球颗粒分散形成均匀的悬浊液；在悬浊液稳定搅拌下加入200μL 3-氨丙基三甲氧基硅烷，并维持搅拌让其反应24h；反应结束后将混合液转移至离心管中将微球通过离心收集，用THF和酒精分别将微球清洗三次后加热至100℃固定3h，得到SiO 2-NH 2；
(2)将50mg粒径5μm SiO 2球与5mL无水THF加入圆底烧瓶中，通过反复超声，震荡使微球颗粒分散形成均匀的悬浊液；在悬浊液稳定搅拌下加入200μL三乙氧基(3-环氧丙基氧丙基)硅烷，并维持搅拌让其反应24h；反应结束后将混合液转移至离心管中将微球通过离心收集，用无水甲苯和酒精分别将微球清洗三次后加热至100℃固定3h，得到SiO 2-Epoxy；
(3)将9mg RGD(5mM)多肽溶解于1.8mL 2-(N-吗啉代)乙磺酸(MES)缓冲溶液中(pH＝5.5)，将1-(3-二甲氨基丙基)-3-乙基碳二亚胺盐酸盐EDC(160mM)和N-羟基琥珀酰亚胺NHS(80mM)分别加入溶液溶解后置于37℃水浴中搅拌，活化15分钟。之后将(1)中所得SiO 2-NH 2颗粒(10mg)通过反复震荡超声均匀分散在200μLMES缓冲溶液后加入上述混合溶液并维持稳定搅拌，维持37℃反应24h；反应结束后将混合液转移至离心管中将微球通过离心收集，用去离子水将微球清洗3次后常温干燥，所得产物为SiO 2-RGD；
(4)将SiO 2-NH 2通过反复震荡超声在圆底烧瓶中均匀分散至4mL无水二氯甲烷(DCM)内，加入0.3mL三乙胺并将反应体系放至冰上搅拌15min；缓慢滴入0.2mL(用1mL DCM稀释)溴代异丁酰溴；维持反应体系在冰上反应2h后，常温继续反应22h；反应结束后将混合液转移至离心管中将微球通过离心收集，用DCM和丙酮分别将微球清洗3次后常温干燥，所得产物为SiO 2-Br；
(5)将所得产物(SiO 2-Br)通过反复震荡超声在圆底烧瓶中均匀分散至5mL水∶甲醇＝1∶1的混合溶液中，加入2.4g[2-(甲基丙烯酰基氧基)乙基]二甲基-(3-磺酸丙基)氢氧化铵(SBMA)搅拌并通氮气除氧20min；于此同时在另一个圆底烧杯中在始终通氮气的情况下将220mg 2’2-联吡啶52mg溴化亚铜(CuBr)溶解于5mL水∶甲醇＝1∶1的混合溶液中，待完全溶解，溶液呈深红棕色时将此混合液在氮气保护下转移至含有SiO 2-Br微球的溶液 中，通氮气搅拌两分钟后加入0.2mL抗坏血酸溶液(50mg/mL)并将反应容器密封，在油浴锅中加热至60℃反应12h；反应结束后将混合液转移至离心管中将微球通过离心收集，用80℃去离子水，甲醇和常温去离子水分别将微球清洗三次后常温干燥。所得产物为SiO 2-SBMA。
实施例2 制备粒径2μm的SiO 2-Br和粒径2μm的SiO 2-SBMA
步骤(1)采用粒径2μm SiO 2球，其余步骤与实施例1相同。
将实施例1和实施例2合成的胶体颗粒SiO 2-NH 2、SiO 2-Br和SiO 2-SBMA均通过XPS扫描验证，结果如表1所示。
表1 XPS元素扫描结果(atom％)
Figure PCTCN2019084522-appb-000003
表1结果表明，相对于SiO 2，SiO 2-NH 2样品中氮元素的出现可以证明氨基的接枝成功；SiO 2-Epoxy样品中相比于SiO 2样品碳元素与氧元素的比值的增加证明了少量环氧基的接入(测试的SiO 2的碳元素与氧元素的比值为0.16，SiO 2-Epoxy为0.47，而环氧基单体中两元素比值为2.4，证明少量环氧基的接入，并且膜厚度小于10nm，多出的氧元素信号来自于基底)；SiO 2-RGD样品中氮元素的含量相比于SiO 2-NH 2中氮元素的含量明显增加证明了RGD的接枝成功；同理，SiO 2-Br样品(无论5μm还是2μm)中Br元素的出现以及 SiO 2-SBMA中硫元素和氮元素且硫元素与氮元素比值接近1，均证明了各自反应的成功。
实施例3
一种玻璃片的表面改性方法，包括以下步骤：
(1)将实施例1的5μm的SiO 2-Br颗粒与水混合，超声和震荡后形成质量百分浓度10％微球均匀分散的SiO 2-Br颗粒悬浊液；
(2)将0.4μm的PSC(羧基化的聚苯乙烯)颗粒与水混合，超声和震荡后形成质量百分浓度10％微球均匀分散的PSC颗粒悬浊液；
(3)以5μm SiO 2-Br和0.4μm PSC形成直径为1mm的圆形涂层为例，计算悬浊液加入量：
5μm SiO 2-Br微球为单层平铺，胶体SiO 2-Br投影面积为19.625μm 2，SiO 2-Br颗粒密度为2g/cm 3，所需涂层面积为0.79cm 2，SiO 2-Br颗粒悬浊液质量百分浓度为10％，SiO 2-Br颗粒体积为65μm 3，代入公式(1)中计算得出构成单层SiO 2-Br颗粒涂层所需SiO 2-Br颗粒悬浊液的体积V为4.97μL；
0.4μm PSC微球为多层平铺，总高度须达到SiO 2-Br微球颗粒的直径，故，所需层数＝SiO 2-Br微球半径/PSC微球直径，即6层，同理，胶体PSC投影面积为0.126μm 2，PSC颗粒本身密度为1.19g/cm 3，所需涂层面积为0.79cm 2，PSC颗粒悬浊液质量百分浓度为10％，PSC颗粒体积为0.033μm 3，代入公式(1)计算得出构成单层颗粒涂层所需PSC颗粒悬浊液的体积为0.24μL，故所需0.4μm PSC颗粒悬浊液总体积为1.42μL；
将SiO 2-Br颗粒悬浊液与PSC颗粒悬浊液混合，并通过震荡，超声得到混合胶体颗粒悬浊液；
(4)在玻璃片表面旋涂2.5g/mL的PS的甲苯溶液，旋涂条件：第一阶段：800rpm、3s；第二阶段：1700rpm、7s)，表面放密封圈遮挡，将混合胶体颗粒悬浊液滴加至密封圈内，使液面形成负曲率状态；
(5)无风状态下静置自然挥发干燥；
(6)将玻璃片在加热板上加热固定，加热温度为120℃，加热时间为10min，表面形成涂层。
实施例4
本实施例与实施例3的区别在于，将5μm的SiO 2-Br颗粒替换为实施例1中5μm的SiO 2-SBMA颗粒。
实施例5
本实施例与实施例3的区别在于，将5μm的SiO 2-Br颗粒替换为实施例2中2μm的SiO 2-Br颗粒，将0.4μm的PSC颗粒替换为0.1μm的PSC颗粒。
实施例6
本实施例与实施例5的区别在于，将2μm的SiO 2-Br颗粒替换为实施例2中2μm的SiO 2-SBMA颗粒。
实施例7
本实施例与实施例3的区别在于，将5μm的SiO 2-Br颗粒替换为实施例2中5μm的SiO 2-RGD颗粒。
实施例8
本实施例与实施例3的区别在于，将5μm的SiO 2-Br颗粒替换为实施例2中5μm的SiO 2-Epoxy颗粒。
实施例9
本实施例与实施例3的区别在于，将0.4μm的PSC颗粒替换为0.4μm的PMMA颗粒。
实施例10
本实施例在实施例3的基础上，还包括步骤(7)对涂层中含有SiO 2-Br胶体颗粒的表面进一步修饰SBMA(如图3中A，即两种颗粒自组装，组装后仅有大颗粒表面具有修饰高分子刷)，具体包括：
将表面放置于可密封的平底容器中并通氮气将空气置换出来，在平底容器中，加入2.4g SBMA到5mL水∶甲醇＝1∶1的混合溶液中，搅拌并通氮气除氧20min；于此同时在另一个圆底烧杯中在始终通氮气的情况下将220mg 2’2-联吡啶和52mg溴化亚铜(CuBr)溶解于5mL水∶甲醇＝1∶1的混合溶液中，待完全溶解，溶液呈深红棕色时将此混合液在氮气保护下将两溶液混合，通氮气搅拌2min后加入0.2mL后转移至样品放置的容器中，使带有SiO 2-Br的表面完全浸没在反应溶液中(反应溶液根据容器大小等比增加或减少)，加入抗坏血酸溶液(50mg/mL)并将反应容器密封；将容器置于60℃烘箱中反应12h；反应结束后将溶液移除，用80℃去离子水，甲醇和常温去离子水分别将表面清洗3次后常温干燥。
实施例3和实施例10改性后表面的水接触角和XPS扫描图如图4-图5所示。
从图4-图5中可以看出，改性后表面在嫁接SBMA单体反应前后水接触角从113°下降至26°。X射线光电子能谱(XPS)N1s扫描结果也可以看出，反应后SBMA中特征性的季铵基官能团特征峰出现(-N +)，证明了实施例10在实施例3的基础上SBMA单体嫁接成功。
实施例11
本实施例与实施例3的区别在于，将0.4μm的PSC颗粒替换为体积比1∶1的0.4μm的PSC颗粒和0.4μm的PMMA颗粒的组合，保持有机胶体颗粒悬浊液中无机胶体颗粒总量不变。
实施例12
本实施例与实施例10的区别在于将实施例3中，5μm的SiO 2-Br颗粒替换为体积比2∶8的5μm的SiO 2-Br颗粒和实施例1中5μm的SiO 2颗粒的组合，保持无机胶体颗粒悬浊液中无机胶体颗粒总量不变，后续反应步骤不变(如图3中B，即三种颗粒自组装后结果，在组装前将修饰和未修饰的颗粒预混合，在组装后只有部份大颗粒表面具有修饰高分子刷)。
从图4中可以看出，改变组装前颗粒配比后再进行反应后水接触角从113°下降至42°但未达到实施例10中的26°。证明了SBMA的嫁接是区域性的。
对比例1
本实施例与实施例3的区别在于，将5μm的SiO 2-Br颗粒替换为5μm的SiO 2颗粒。
对比例2
本实施例与实施例5的区别在于，将2μm的SiO 2-Br颗粒替换为2μm的SiO 2颗粒。
对实施例3-实施例6自组装后形成的表面进行SEM观察，结果如图6所示，从图6中可以看出，通过SEM证实了本申请胶体颗粒具备自组装能力，表面形成微纳米结构。
试验例1 细菌粘附试验
分别使用大肠杆菌ATCC25922和绿脓杆菌PAO1(带荧光质粒)在实施例4、6以及对比例1、2自组装后形成的表面进行2小时粘附试验。
试验方法：
取10μ在-80℃冻存的菌液于Luria-Bertani培养基(LB培养基)上划线培养。第二天从中取3-5处菌落，加入放有50mL LB液体培养基的150mL锥形瓶中，置于37℃恒温摇床摇动培养(120r/min)18h，获得菌液用LB培养液稀释至1.0×10 8/mL(吸光度值约为A 600＝0.1)，再将此菌液用LB培养液稀释10倍(即最终细菌浓度为1.0×10 7/mL)使用。将样品表面放置于24孔板内(此批样品直径14mm)，修饰后的表面向上，每孔加入1mL菌液后放入37℃恒温孵箱静置培养两小时。两小时候将样品取出，用PBS清洗3遍，去除为黏附的细胞，对于PAO1，由于细菌自带绿色荧光，清洗后直接用显微镜观察，大肠杆菌的样品将被置于新的24孔板内，每孔加入1mL 2.5％的戊二醛水溶液固定15分钟，之后用去离子水清洗一次，每孔加入0.3mL 1.3μg/mL碘化丙锭溶液(PI)染色15分钟。染色完成后用去离子水清洗3次，通过倒置荧光显微镜(奥林巴斯)观察结果。
测试表面：玻璃片，5SiO 2：对比例1自组装后形成的表面；5SiO 2-SBMA：实施例4自组装后形成的表面；5SiO 2-ATRP：实施例10化学修饰后形成的表面；2SiO 2：对比例2自组装后形成的表面；2SiO 2-SBMA：实施例6自组装后形成的表面；结果如图7-图8所示。
从图7-图8中可以看出，所有嫁接SBMA的材料均发现了细菌数量的减少，(对于大肠杆菌和铜绿假单胞杆菌，其修饰后表面细菌粘附量分别为未修饰的34％和50％。)侧面证明了SBMA分子的嫁接无论是在组装前还是在自组装后进行均是可行的并可以影响细菌的粘附行为。
试验例2 细胞粘附及生长试验
使用小鼠前成骨细胞(MC 3T3)在对比例1及实施例4自组装后形成的表面进行细胞爬片试验。
试验方法：细胞接种步骤，细胞培养密度达到约80-90％左右，取出细胞培养皿，至无菌操作台内，弃旧培养基；加入约5mL PBS晃洗2次；添加适量0.25％胰酶消化细胞，37℃培养箱内放置2分钟，显微镜下观察细胞呈圆球状脱落，加入新鲜培养基8mL终止消化；吸管吹打部分没有完全脱的细胞，转移细胞溶液至15mL离心管，1100rpm离心3三分钟，弃上层废液，加入4-5mL新鲜培养基吹打混匀底层细胞；使用细胞计数板，计算细胞总数量。
将样品表面放置于24孔板内(此批样品直径14mm)，修饰后的表面向上，按5×10 3每孔接种细胞于材料表面。培养24小时及72小时后(每24小时更换一次新鲜培养基)，使用4％多聚甲醛固定细胞15分钟。使用F-actin和DAPI分别对细胞骨架和细胞核进行染色20分钟。每孔使用1mL PBS溶液清洗3次后使用荧光显微镜观察结果。
结果如图9所示。图9中测试样品分别为：玻璃片，5SiO 2：对比例1自组装后形成的表面；5SiO 2-SBMA：实施例4自组装后形成的表面；5SiO 2-ATRP：实施例10化学修饰后形成的表面；2SiO 2：对比例2自组装后形成的表面；2SiO 2-SBMA：实施例6自组装后形成的表面细胞培养24小时后细胞骨架染色的结果。
从图10统计的结果可以看出，相比于玻璃片自组装的材料对于MC 3T3细胞一天粘附的面积和细胞形状均有所改变，但对于修饰SBMA和未修饰SBMA的样品差异不大。
图11中测试样品分别为：玻璃片，5SiO 2：对比例1自组装后形成的表面；5SiO 2-SBMA：实施例4自组装后形成的表面；5SiO 2-ATRP：实施例10化学修饰后形成的表面；2SiO 2：对比例2自组装后形成的表面；2SiO 2-SBMA：实施例6自组装后形成的表面细胞培养三天后细胞骨架染色的结果及细胞活性CCK-8检测结果。
从图12中可看出经过三天的培养，5SiO 2-ATRP样品表面细胞数目和活性相较其它表面明显减少，而在组装前修饰SBMA的样品表面细胞数目和细胞活性相比于未修饰样品无明显变化。
可见，本申请通过采用化学修饰的无机胶体颗粒与有机高分子胶体颗粒自组装，能够做出同时具有多种物理和化学性质的表面，细菌和细胞的反应证明了这种表面确实实现了，并对细胞细菌粘附增殖有影响。对于研究细菌细胞对于界面物理化学刺激的反应能提供一个良好的平台。
试验例1与例2目的不是选出哪种修饰或者表面是好的，仅意在说明本申请可简单快捷地制备出具有想要物理化学性质的表面，可应用于细菌与细胞行为的研究。比如结合本试验例1及例2中结果可发现实施例6与对比例2表面相比，虽然细菌粘附数量减少，但并未影响细胞的粘附与增殖，以此两种表面为基础可以进行细菌抗粘附机理的研究。再比如实施例4与实施例10的样品虽然表面均有SBMA且均可减少细菌粘附但对于细胞增殖却有不同的影响。以此两种表面为基础可以进行材料表面对于细胞增殖影响的研究。
尽管已用具体实施例来说明和描述了本申请，然而应意识到，在不背离本申请的精神和范围的情况下可作出许多其它的更改和修改。因此，这意味着在所附权利要求中包括属于本申请范围内的所有这些变化和修改。

Claims (10)

  1. 一种材料的表面改性方法，其特征在于，包括以下步骤：
    在材料表面通过胶体颗粒自组装的方法形成涂层；
    其中，所述胶体颗粒包括无机胶体颗粒和有机高分子胶体颗粒，全部或部分所述无机胶体颗粒经过化学修饰，所述化学修饰包括化学官能团修饰和/或生物活性分子修饰；所述无机胶体颗粒的粒径为1-10μm，所述有机高分子胶体颗粒的粒径为0.05-0.75μm；
    优选地，所述自组装采用蒸发诱导法。
  2. 按照权利要求1所述的材料的表面改性方法，其特征在于，所述无机胶体颗粒包括金属氧化物微球和/或非金属氧化物微球，优选包括SiO 2微球、TiO 2微球和ZnO微球中的一种或几种，进一步优选为SiO 2微球；
    优选地，所述化学修饰包括一类或几类不同的化学修饰；
    优选地，所述化学官能团包括卤素、氨基、甲基丙烯酰乙基磺基甜菜碱、巯基、环氧基、丙烯酰基或叠氮基团中的一种；
    优选地，所述生物活性分子包括具有RGD序列的短肽、多肽、化学小分子、抗生素或生长因子中的一种。
  3. 按照权利要求1所述的材料的表面改性方法，其特征在于，所述有机高分子胶体颗粒包括修饰与未修饰的聚苯乙烯微球、聚甲基丙烯酸甲酯微球、壳聚糖微球、聚己内酯微球、聚二甲基硅氧烷微球、明胶微球、聚乳酸微球和聚丙烯酸微球中的一种或几种；
    优选地，所述有机高分子胶体颗粒是经过化学修饰的，优选所述有机高分子胶体颗粒为羧基化的聚苯乙烯微球。
  4. 按照权利要求1所述的材料的表面改性方法，其特征在于，所述无机胶体颗粒和所述有机高分子胶体颗粒的直径比为4-200∶1，优选为4-100∶1。
  5. 按照权利要求1-4任一项所述的材料的表面改性方法，其特征在于，包括以下步骤：
    (a)对所述无机胶体颗粒进行化学修饰，得到化学修饰的无机胶体颗粒，将化学修饰的无机胶体颗粒与水混合，得到无机胶体颗粒悬浊液；
    (b)将所述有机高分子胶体颗粒与水混合，得到有机高分子胶体颗粒悬浊液；
    (c)将所述无机胶体颗粒悬浊液与所述有机高分子胶体颗粒悬浊液混合，得到混合胶体颗粒悬浊液；
    (d)将所述混合胶体颗粒悬浊液滴加至所述材料表面，或将所述材料表面浸入所述混合胶体颗粒悬浊液中；干燥和固定后得到涂层；
    (e)对所述涂层中的经过化学修饰的无机胶体颗粒的修饰基团上进行化学修饰， 得到表面改性后的材料。
  6. 按照权利要求5所述的材料的表面改性方法，其特征在于，步骤(a)中的化学修饰方式包括自由基聚合、阴离子聚合或阳离子聚合中的一种，优选包括以下聚合反应的任意一种：
    (i)原子转移自由基聚合；
    (ii)单电子转移自由基聚合；
    (iii)可逆加成-断裂链转移聚合；
    优选地，步骤(e)中的进一步修饰方式包括表面引发聚合，包括巯基-烯烃反应、环氧基-氨基反应或叠氮-炔烃反应。
  7. 按照权利要求5所述的材料的表面改性方法，其特征在于，
    优选地，步骤(c)中所述混合胶体颗粒悬浊液中所述无机胶体颗粒总体积与所述有机高分子胶体颗粒总体积之比为0.1-77，优选为0.5-3；
    优选地，步骤(d)中所述干燥采用自然蒸发；
    优选地，步骤(d)中所述固定采用加热固定，优选加热温度为110-200℃，加热时间为1-20min，优选为1-5min。
  8. 一种材料，其特征在于，采用权利要求1-7任一项所述的材料的表面改性方法对所述材料的表面进行改性。
  9. 一种权利要求8所述的材料在微生物或细胞培养中的应用。
  10. 一种医疗产品，其特征在于，包括权利要求8所述的材料；
    优选地，所述医疗产品包括微生物或细胞培养工具；
    优选地，所述医疗产品包括生物医药抗菌材料。
PCT/CN2019/084522 2019-04-26 2019-04-26 材料的表面改性方法及其改性后的材料和应用、医疗产品 WO2020215307A1 (zh)

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