TW583002B - Bio-compatible block copolymer - Google Patents

Abstract

A bio-compatible block copolymer is characterized in comprising different sections each forming a block due to chemical incompatibilities and differences thereof and forming phase separations of the magnitude of 1 to 1000 nm on the surface of the block copolymer caused by different blocks. The phase separations of the magnitude of 1 to 1000 nm on the surface of the block copolymer have properties of bio-compatibility and anti-bacterial, etc. due to their functional size similar to that of the proteins controlling the activities of cells.

Description

V. Description of the invention (丨) Field of the invention The present invention is related to a biocompatible cross-linked copolymer, especially about the chemical incompatibility of different kinds of cross-linked copolymers due to different bond segments. The nature and dissimilarity make them aggregate to form clusters, and form on the surface of the clustered copolymer. 〖～〗 〇 00 Nanometer size The phase separation caused by the different clusters makes it also biological Compatibility and antibacterial. BACKGROUND OF THE INVENTION A general definition of biomedical material refers to "an artificially synthesized material that can be used to replace parts of a living system or perform its functions directly in contact with a living body" or "-a kind of The system is a pharmacologically inactive substance that can be implanted or incorporated into a living system. " According to the above definition, the basic requirements for the application of biomedical materials are also important requirements, that is, they must have good biocompatibility. Generally, this biocompatibility also includes blood compatibility and histocompatibility. The application of biomedical materials in tissue engineering is mainly used as a three-dimensional support to guide cells to grow into appropriate tissues and have normal functions. Therefore, the physical properties of the material (such as size, strength, porosity), or surface chemical properties (such as interaction with cells, degree of decomposition itself), etc., are very important. In addition, the material used as a scaffold must also enable cells to attach to it, interact with body tissues, avoid rejection reactions, and decompose at an appropriate rate, and can finally be replaced by the cells and interstitial of the body to complete Healing eyepiece knife. At present, the most common difficulties encountered in the application of biomedical materials are the problems that arise between the interface of human tissues and the materials, such as foreign body coating.

The size of this paper is ⑽x 297mm) 583002 5. Description of the invention (i) t inflammation reaction. That is, when the environment inside the human body comes into contact with the surface of the foreign material h ′, it will cause the protection of the human system (such as inflammation, agglutination of enterprise fluids, and the immune system). If the human body's physiological response is too violent, the material itself will not only be unable to exert its original X-force effect, it may even cause human body injury. At present, the so-called two biocompatibility, biomedical materials, most of them will cause similar reactions when implanted in the living body. For example, body tissue will form a non-vascular collagen capsule around the implant. The implant is covered, and even after many years of implantation, the presence of macrophages is still found on the surface of the material that can contact the cells. This situation may produce signals that inhibit angiogenesis and allow normal healing and The reconstruction process was blocked. These suppression signals also make it difficult for conventional tissue engineering products to obtain good results. It is known that the interactions that occur at the interface between the cell and the material will determine how well the material is used in the body for biocompatibility. In addition, in some materials that will come into contact with blood, it is necessary to avoid special phenomena such as platelet activation and plasma protein denaturation. Since the 1970s, the improvement of the properties of artificial implanted materials has enabled them to have a longer service life without side effects or to reduce their cost to make them universal 'has become the focus of research and development of related materials. For example, to improve the abrasion resistance and corrosion resistance of printed materials printed by the consumer cooperatives of the Intellectual Property Bureau of the Ministry of Economic Affairs, and improve the artificial crystal spheres.

Intraocular) biocompatibility, improved artificial blood vessels and heart valves, research and development of damaged skin tissue covering (dressing) and artificial skin (scrapping ^^ skin), etc. Because the factors affecting biocompatibility or blood compatibility are quite complicated, 'including the surface properties of ㈣ and cells (or proteins) and the material table: The paper size of k is applicable to the Chinese National Standard (CNS) A4 specification (21G x 297 public directors) 583002

V. Description of the invention (7)) (Please read the precautions on the back before filling out this page). The effect can be summarized into the differences in the local chemical structure of the surface of the material, the surface charge, and the hydrophobicity (such as contact angle and (Potential), surface roughness (roughness) and micro heterogeneity (micr0-heterogeneity) and soft, mechanical properties. Therefore, in order to achieve good biocompatibility, many studies have been performed on the surface of traditional materials by chemical treatment (such as surface grafting of macromolecules or groups), and changes in surface physical properties (such as plasma, ion implantation, or ionization). Bundles) or methods of increasing biological activity to make these materials have acceptable biocompatibility. However, most of the currently known surface modification methods, in addition to the complicated modification process, are not easy to commercialize, nor can they simultaneously take into account all the properties required for use. In addition, the modification of the surface of the material will also increase the manufacturing cost. Most of the conventional synthetic materials printed by the Consumer Cooperatives of the Intellectual Property Bureau of the Ministry of Economic Affairs have mostly homogeneous surfaces, and do not have nano characteristics. A few years ago, some researchers began using materials with micron- or nanometer-sized materials on their surfaces to study their interactions with cells, but have not explored their effects on biocompatibility, and No one uses the technique of surface nanometers as a tool to modify the biocompatibility of materials, and to design and make tissue biomedical materials. As mentioned earlier, materials with nano-sized features on the surface are currently only used to study the signal and behavior of cells (curtjs and Wilkinson; Dalby et al.), And have no biocompatibility, such as blood compatibility. Sexual and inflammatory properties. At present, at home and abroad in the relevant published research on the application of nanomaterials as tissue repair or human biomedical materials, only nanocomposite materials (additives) and the use of non-stoichiometric materials (non_stoichiometric materials) 5 This paper standard applies China National Standard (CNS) A4 (210 X 297 mm) A7 A7

V. Description of the invention (★) Learned =: Nai: The material 'may be applied to the preparation method of the coupler copolymer which is well known in biomedicine. It has the characteristics of nano, so that the characteristics of Bunda are on the surface of the material. Those who control their biocompatibility near the upper production range (several nanometers to hundreds of insect white beetles that have been captured) have not been reported. SUMMARY OF THE INVENTION In view of the above-mentioned disadvantages, the present invention provides a biocompatible pellet such as a copolymer. This cross-linked copolymer contains at least two or more types of groups (Mock) formed by the aggregation of the heterogeneous surface molecule group segments, due to different intermolecular forces. Effect, and the separation forms a phase separation of w_nano size. Because its functional scale (fUnctional scale) is similar to the protein that dominates cell activity, this clustered copolymer has both biocompatibility (cell attachment, anticoagulation and anti-inflammatory) and antibacterial properties. Another object of the present invention is to provide a cross-linked copolymer which can be used as a biomedical material without further surface modification, so as to reduce the complexity of the manufacturing process and the manufacturing cost. The cross-linking copolymer in the present invention is composed of two or more kinds of polymer group-linking segments (such as soft and hard chains) having chemical incompatibility and dissimilarity. Under the influence of inter-species dispersion force or repulsion effect, and the chemical bonding force of polymer segments, the self-assembly of polymer chains is caused, which separates them to form different paper sizes. This paper applies Chinese national standards. (CNS) A4 specification (210 X 297 mm) Read the note on the back and fill out this page. Printed by the Employees ’Cooperatives of the Intellectual Property Bureau of the Ministry of Economic Affairs. 583002 Printed by the Employees’ Cooperatives of the Intellectual Property Bureau of the Ministry of Economics. A7 B7. ) Phase. What differs from the polymer exchange is that covalent polymers still have covalent bonds between them, which limits the size of the phase separation between them. In the state of thermodynamic equilibrium, self-ordering microstructures will be formed between the dissimilar groups, and the size of the molecular segments is limited so that the size of each distinct phase is between 1 and 1000 nanometers. This phenomenon is referred to herein as nanophase separation (see Figure 1). After the nano-phase separation, different types will be produced. For example, the length of the monomer segment will be changed. Due to the different proportions of the two polymer chains, the volume fraction will be different, and according to the composition volume fraction, it will be different. Forms a variety of different ordered microstructures. There are many types of types that have been discovered. As the volume of the polymer clusters changes, they sequentially contain spheres and cylinders (Cyiin (jer)). , Lamellar arrangement (lamella), or special bicontinuous phase (bicontinuus, gyroid), perf0rated layer structure, etc. In addition, the type of monomer of the group-linked copolymer is changed. Structures with different properties can be obtained. Factors affecting protein adsorption on the surface of artificially implanted materials include: surface hydrophilicity, surface softness, and adsorption medium. The soft surface has the advantage of transmitting ring roar, but the disadvantage is that it is easy to detach and make cells It is not easy to attach, but the hard surface will cause the structural reorganization of the protein, making the cells more compact when attached to the surface. Protein adsorption may be reduced by the hydrophilic and soft surface. It increases with water-borne and hard surface. On the surface of polyurethane with nano-phase separation structure, the greater the degree of nano-phase separation, the less protein is adsorbed. The size of the adsorbed protein will decrease the number of inactivated proteins. Conversely, if the size of the hard chain nanophase separation area is far from the size of the adsorbed protein, the inactivated protein (please read the precautions on the back before filling in this page)

Printed by the Shell Consumer Products Co., Ltd. of the Intellectual Property Agency of the Ministry of Economic Affairs 583002 A7 B7 5. The more invention descriptions (U) will be available. In the cross-linked copolymers, the size and distance of nanophase separation can be controlled by controlling the type and length of the monomers, so that its functional scale is close to the protein that controls cell activity. Therefore, the degree and arrangement of protein adsorption can be controlled, and the degree and arrangement of protein adsorption is related to the binding and message transmission of cell surface receptors, which will ultimately manifest in the migration and growth of cells. Therefore, the nanometer-sized phase separation surface has unique biocompatibility, which can simultaneously take into account the characteristics of cell attachment, anticoagulation, anti-inflammatory, and antibacterial infection. The following is a `` polyurethane double-block copolymer, and, polycaprolactone-polyvinyl alcohol double-block copolymer (p〇1caprolactone_p〇lyethylene lactone chblock, PCL-PEG) copolymerization "These two forms of cross-linked polymers are taken as examples to further illustrate the present invention. Among them, the present invention points out that the polyurethane double-stranded copolymers have the main polymerized monomers. The diisocyanate (A) and the polyhydric alcohol (b) also contain a chain extender (c) which can extend the chain. It is used as the main polymerization in the polyurethane double-strand copolymer of the present invention. Examples of the monomer diisocyanate ⑷ 'include diphenylmethane 4,4, _ diisocyanate (MDI), toluene diisocyanate (TDI), naphthyl ^ -diisanoic acid Salt (NDI) or hexamethylene diisocyanate (hdi). As the main polymerized monomer, the alcohol (B) can be polyalcohol, polyvinyl alcohol, polycarbonate diol (for example, 'polyhexyl Polyethylene carbonate (Poly (hexyl,)) ^. Read d ', polyoxyl or polyenol. The chain extender (c) can be a diol, a polyol or a diol. Class' Examples thereof comprising ethylene glycol, butylene di (Read Notes on the back and then fill the page)

583002 A7 Printed by the Consumer Cooperatives of the Intellectual Property Bureau of the Ministry of Economic Affairs 5. Description of the Invention (Λ) 2-butene-l, 4-diol, 1,6-hexanediol, trimethyl alcohol, ethylenediamine, 3 3-dichloro-4,4-diaminodiphenylphosphonium (mOCA). In the present invention, the blend ratio of the diisocyanate (A), the polyol (B) and the chain extender (c) is 100 parts by weight of the diisocyanate (A), and the polyol (b) and The total of the chain extender (C) is 100 parts by weight. Under the conditions of the above blending ratio, the blending ratio of the bond elongating agent (C) is preferably 17 to 100 parts by weight relative to 100. parts by weight of the polyol. The polyurethane double-link copolymer of the present invention can use diisocyanate (A) and polyalcohol (B) as polymerized monomers under the conditions of the above-mentioned blending ratio at 60 to 70 ° C. The reaction is carried out for about 3 to 5 hours to synthesize a prepolymer. Then, the chain elongating agent (C) is mixed with the 'cereal' first and then slowly added. The dropping time is preferably controlled to about 30 minutes. After that, the catalyst and the solvent are mixed and slowly added. At this time, the total solute accounts for about 30% by weight, and the reaction is performed at 60 to 70 ° C for about 4 to 7 hours to complete the polymerization reaction. This reaction-completed polyurethane is diluted with a solvent to prepare the product. "Chendu" can be cast on the substrate by soluti〇11 casting, spin coating, melt coating, or epitaxy. On to prepare a thin film. Examples of the above-mentioned catalyst include di-n-butyltin dilaurate. Examples of the above-mentioned solvent include N, N-dimethyl acetamide (DMAc), tetrahydrofuran (THF), or monomethylformamide (DMF). This solvent must be removed from the water before use. This can be achieved by heating the solvent to a temperature above 10 (rc or by treating with a molecular sieve for more than 2 months.) The crosslinked copolymer will form a separated phase with a size of about 1 to 100 nanometers on the surface. This paper size applies to China National Standard (CNS) A4 (210 x 297 mm) (Please read the precautions on the back before filling This page) I--line · Printed by the Consumer Cooperatives of the Intellectual Property Bureau of the Ministry of Economic Affairs 583002 A7 B7 V. Description of the invention (γ) In addition, the "polycaprolactone-polyvinyl alcohol double-group copolymerization" also specified in the present invention The substance "contains polycaprolactone (D) and polyvinyl alcohol (E) as main polymerized monomers. Polycaprolactone as the main polymerized monomer in the polycaprolactone-polyvinyl alcohol double-group copolymer of the present invention Lactone (D), an example of which includes cyclocaprolactone. Polyvinyl alcohol (E), which is a main polymerizable monomer, includes poly (Ethylene Glycol) Methyl Ether. In the present invention, Blending ratio of polycaprolactone (D) and polyvinyl alcohol (E), polycaprolactone The sum of (D) and polyvinyl alcohol (E) is 100 parts by weight, and polycaprolactone (D) is 20 to 80 parts by weight, preferably 20 to 50 parts by weight. Polycaprolactone of the present invention -Polyvinyl alcohol double-strand copolymer can be added with appropriate amount of catalyst to methylene chloride as a solvent under the above-mentioned mixing ratio condition, and then polyvinyl alcohol is added and stirred at room temperature for about 10 to 14 hours. Toluene was used to replace the previous solvent, and then cyclocaprolactone was added, and the reaction was performed at 60 ° C for about 12 hours. After the reaction was completed, a 0.35 N aqueous acetic acid solution was added to terminate the reaction. Then, n-heptane was added to the above mixture. The polymer is precipitated. The solution is removed and the precipitated polymer is taken out, and a sufficient amount of tetrahydrofuran is added to completely dissolve. After being dissolved, a sufficient amount of n-heptane is added to precipitate the polymer again. Finally, the above-mentioned precipitate is precipitated. The polymer was dissolved in chloroform to prepare a polymer solution of a suitable concentration, and the solution was applied to the substrate at a rotation speed of 1,000 to 3,000 rpm by a spin coating method, and the solvent was evaporated at room temperature. As an example of the above catalyst package [(p-EDBP) AlCH3] 2 10 This paper size is applicable to China National Standard (CNS) A4 (210 X 297 mm) ------------- install ------- -Order --------- Line (Please read the precautions on the back before filling this page) A7

V. Description of the invention (,) 583002 or n-butyl lithium. The above-mentioned "prepared polycaprolactone gg_polyvinyl alcohol double-block copolymer" will form a separation phase with a size of about 100 to 1000 nanometers on the surface. The present invention will be further described by referring to the following examples. These examples of yoke do not limit the content disclosed in the foregoing of the present invention. Those skilled in the art can make some improvements and modifications without departing from the scope of the present invention. Brief description of the figure Figure 1. The self-ordering and self-alignment of the macromolecular cluster-linked co-polymer constitute a micro-phase separation structure with a size between 丨 ~ ... ㈧nm. Fig. 2. An image of the surface of a poly (urethane) double-block copolymer in the modulating force mode of an atomic mechanical microscope. Figure 2. An image of the surface of a polycaprolactone-polyvinyl alcohol double-block copolymer (polycaprolactone: polyvinyl alcohol = 29: 71) was observed using the modulating force mode of an atomic mechanical microscope. Detailed description of the invention Example Example 1 The ester diblock copolymer f is prepared by placing the solid diisocyanate MDI in an oven at 60 ° C. and melting it to form a transparent and clear liquid. In addition, polyhexyl Ethyl carbonate diol is placed in a 60 C oven. After melting to a liquid state, the vacuum is evacuated by a vacuum device. 11 This paper size applies to China National Standard (CNS) A4 (21G x 297------ --I --------- install -------- order --------- line (please read the notes on the back before filling this page) Intellectual Property Bureau of the Ministry of Economic Affairs Printed by employee consumer cooperatives 583002 Printed by A7 of the Intellectual Property Bureau of the Ministry of Economic Affairs Consumer Cooperatives 5. A description of the invention (\ P) And at the same time? Stir and heat '100 ~ U0 .. Maintain for 8 hours to remove the X gas in addition to 3 , Dimethylacetamide is heated to ⑽. [The above is to remove the water vapor contained. The above diisocyanate clarified liquid and the polyhexylethyl carbonate after the removal of water are added to the alcohol at 2: 1. , Λ · ο Γ 1 3 · 2, 4 · 3, 5: 4, 6: 5 and 7: ό were added to the four-necked reaction flask, and the reaction was performed at 65t for 4 hours to form Add the prepolymers described above to a sufficient amount of the above-mentioned dimethyl ethyl amine after water removal to dilute it to completely dissolve. After it is completely dissolved, then dilute the butyl amine pre-dissolved in methyl ethyl amine. Diol (c / ^ s ^ -butene-l, 4-diol) was added slowly within 30 minutes. At this time, the amount of butenediol added was equal to the use of diisocyanate and polyhexyl ethyl carbonate The difference in the amount of diol used, that is, the sum of the amount of butyl butanol and polyhexyl ethyl carbonate diol is equal to the amount of diisocyanate. Dibutyltin dilaurate will be used as a catalyst later After mixing with an appropriate amount of dimethylacetamide, it is slowly added to the above mixed solution so that the final total dissolved mass accounts for about 30% by weight. After reacting at 6 yc for about 5 to 6 hours, the polymerization reaction is completed. The polyurethane double-block copolymer is diluted with dimethyl ethyl amine to an appropriate viscosity, and is dropped into methanol dropwise to form a sink in methanol. The precipitated polyurethane The ester double-strand copolymer is heated and dried under vacuum to constant weight. The obtained dried polyurethane double-strand copolymer The polymer was prepared as a 3% wt solution with diethylethylamine. This 3% wt polyurethane double-chain copolymer solution was dropped onto a 15mm diameter circular glass slide. These 12 paper sizes Applicable to China National Standard (CNS) A4 specification (21〇χ 297 公 --- — — — — — — — — — I i I II! I order ·! ------ (Please read the precautions on the back first Fill out this page again) 583002 A7 ------ B7 V. Description of the invention (\\) The glass slide is heated to 60 ~ 65 ° C to make the solvent volatilize. After that, the residual solvent was completely evaporated under vacuum. The thickness of the resulting film is approximately imm. (Please read the precautions on the back before filling out this page) Observing the surface morphology of the double-stranded copolymer with an atomic mechanical microscope (AFM) can obtain the modulating force mode image. This is a phase diagram of the change in the degree of softness and hardness. It is the amplitude of the Z direction while applying the amplitude in the Z direction while scanning the height change in the Z direction in two-dimensional XY direction. The strength of the feedback signal is used to determine the softness and hardness of the surface The stronger the hardness is, the lower the hardness is. The bright parts on the graph indicate higher hardness, while the dark parts indicate lower hardness. When the surface of this film was observed with an atomic mechanics microscope (AFM), it was found that the polyurethane double-group copolymer prepared after the above steps had a surface characteristic of nanometers having a phase separation of about 50 nanometers. (Figure II). Cell Adhesion The previously prepared polyurethane double-strand copolymer film was sterilized with alcohol (70% v / v) and placed in a 24-well culture plate. Rinse with phytate buffer solution (PBS, pH = 7.4) to remove residual alcohol. Put 5 × 104 endothelial cell lines (human umbilical cord endothelial cells, ECV-304) in each well, and add 1 ml of 10% fetal bovine serum and a compound antibiotic PSA (Penicillin / Streptomycin / Ministry of Economic Affairs, Intellectual Property Office staff) Printed by Consumer Cooperatives

Amphotericin 3-in-1 antibiotic) M199 medium (Medium 199,

HyClone (cell culture grade), cultured at 37 ° C, 5% CO2. After 48 hours of incubation, the culture solution was aspirated from the wells with a pipette. Add 1 ml of scalylate buffer solution to the well, and aspirate the buffered buffer solution from the well after 1 minute. Then add 0.4ml trypsin (0.25%,

Biological Industries), placed in an incubator (37 ° C, 5% C02). 13 This paper size applies to China National Standard (CNS) A4 (210 X 297 mm). 583002 Printed by the Consumer Cooperative of the Intellectual Property Bureau of the Ministry of Economic Affairs A7 ___B7_____ V. Description of the invention (V >>) 10 minutes to peel cells from the polymer surface. The polymer was rinsed with 0.1 ml of a phosphate buffer solution, and the rinse solution was collected. The volume of this rinsing solution was adjusted with a phosphate buffer solution to make it a 0.5 ml cell suspension. Finally, trypan blue (0.05%, trypan blue) was used to stain the cells, and the number of cells attached to the surface of the material was counted using a phase contrast microscope (100x) and a cell counting disk. The results obtained are shown in Table 1. From the data in the table, it can be seen that the polyurethane double-block copolymers prepared by using different proportions of monomers in the above steps are better than the polyurethane biomedical materials produced by Upj0hn (Pellethan, 2363-80A). Or the glass has a higher number of endothelial cells attached. That is, the polyurethane double-group copolymer in the present invention has high cell compatibility when it comes in contact with cells. Hemocompatibility: The film prepared from the prepared polyurethane double-block copolymer was sterilized with alcohol (70% v / v), and then placed in a 24-well culture plate with phosphate. Rinse with a buffer solution (PBS, ρΗ = 7.4) to remove residual alcohol. Add 1 ml of platelet-rich plasma to each well of the culture plate (provided by China Blood Foundation, platelet concentration 600 < 106 to 14000 x 106 / ml), at 37 C, 5% Incubate for 1 hour at CO2. After that, the non-adsorbed platelets were aspirated, and the surface of the material was washed several times with a 1M HBS solution (Hepes Buffer sotin, Gibco BRL), and then the HBS solution was completely aspirated. Then, add 2.5% of glutaraldehyde to each well, 0.05 ml, and fix the platelets adsorbed on the material in the dark, and replace with a new glutaraldehyde solution after 20 minutes. Allow to stand for more than 8 hours at ° C. The above tested materials were immersed in pure water and 30%, 50%, _____ respectively. This paper size is applicable to the Chinese National Standard (CNS) A4 specification (21〇X 297 public love) ---- ^% Φ: — — — — — — — — · 11 (Please read the notes on the back before filling this page) ·.

°. Stage dehydration in 70%, 80%, 90%, 95%, and 100% ethanol. Finally, after the ethanol was removed with a hinged buckle and ^ 4f ^ ° ° boundary point drying method, the scanning electron microscope was used to count again. For the sacrifice, the data obtained after processing the above steps to estimate the number of platelet activation is shown in Table 2. From the results in Table II, it can be known that 'polyurethane double-strand copolymers of different proportions prepared in the above steps in the present invention are all better than the polyamine biomedical materials produced by Upjohn (Peuethane ② 2363 German) ) Or glass has lower blood;;, plate activation number. It is shown that the polyurethane double-block copolymer in the present invention is less likely to cause platelet activation when contacted with blood, that is, it is connected to the blood. 5. Description of the invention) Touching ¥ will not cause blood clotting, so Have better bloodiness. <% φί IIII «--- (Please read the notes on the back before filling out this page) The Consumer Cooperative of the Intellectual Property Bureau of the Ministry of Economic Affairs prints anti-inflammatory properties to cultivate mononuclear spheres in vitro, and transforms them by mononuclear spheres It is the fraction of the macrophages that have been passed through the autumn [the giant rabbit cells / (monocytes + megalocells)], and it is used as an index of the inflammatory response of the test material in vitro. The detailed method is as follows: The film prepared by the aforementioned polyurethane double-strand copolymer was' sterilized with alcohol (70% v / v) and placed in a 24-well culture plate to Rinse with phosphate buffered solution (pBS, ρ7 = 7.4) to remove residual alcohol. Put 1 × 105 human mononuclear spheres (isolated from the blood of healthy volunteers) into each well, and add RPMI-1640 culture solution containing 10% fetal bovine serum and the compound antibiotic PSA, at 37 ° C, 5% C02 for 96 hours. Aspirate the culture medium from the well with a pipette, and then add 1 ml of phosphonate buffer solution (PBS, pH = 7.4). After 1 minute, the phosphate buffer solution in the hole will be 15. The paper size will be in accordance with the Chinese National Standard (CNS) A4 (210 X 297 mm). Printed by the Intellectual Property Bureau of the Ministry of Economic Affairs, and printed by the consumer co-operative of the company. 583002 A7 ____ B7 Note (4) (PBS, PBS = 7.4) Aspirate with a pipette, and then add 0.3 mi trypsin (0.25%, Biological Industries, biochemical reagent grade), at 37 ° C, 5% CO 2 Incubate for 5 minutes in the environment to allow the cells to peel off from the surface of the copolymer film. Finally, the copolymer film was washed with 0.2 ml of RPMI-1640 culture solution, and the washing solution was collected, and its volume was adjusted with RPMI-1640 culture solution to make it a 0.5 ml cell suspension. Then it was stained with 0.05% trypan blue, and the number of monocytes and macrophages attached to the surface of the material were counted with a phase contrast microscope and a cell counting disk, respectively. The data obtained after the above steps are shown in Table III. From the results obtained in Table 3, it can be known that in the present invention, the polyurethane polyamino acid copolymers prepared in the above steps with different proportions of monomers are all better than the polyurethane biomedical materials produced by Upjohn 2363- 80A) or glass has a lower fraction of monocytes to macrophages. It is shown that the polyurethane double-block copolymer of the present invention is less likely to cause a biological rejection reaction, that is, it has higher anti-inflammatory properties. The antibacterial test uses Bacillus subtilis (仏, ATCC6633) as a bacteriostatic test. Bacterium: First, the bacteria are activated, and the stored bacteria are inoculated on a nutrient medium (Nutrient Agar). The culture dish is inverted. Place in an incubator and incubate under the machine for 12 hours. After the colonies are formed, use platinum loops to inoculate the bacteria and add them to sterile water. Dilute the bacteria solution to 10 6 CFU / m, and pray for the prepared polyurethane dual-group copolymer. After the film is sterilized with alcohol (70% v / v), put it into a 24-well culture plate (please read the precautions on the back before filling this page). Order: -I line · 16 583002 A7 ------- -B7 Printed by a member of the Intellectual Property Bureau of the Ministry of Economic Affairs and a Consumer Cooperative. V. Description of the invention (A) Rinse with phosphate buffer solution (PBS, PΗ = 7.4) to remove residual alcohol. It is then placed in a sterilized test tube. Take the aforementioned 1ml of bacterial solution (106 CFU / ml) into the above test tube, and then add liquid culture medium (Tryptic soy broth, TSB) to mix the homogeneous sentence, and place it in a 37.0 incubator with shaking (rotation speed 100rpm) for 12 hours. The test piece was taken out of the above test tube and washed with 2 ml of sterile water, and then placed in another sterile test tube. Add 2mi salt buffer solution and shake with ultrasound (43Hz, 400W) for 1 minute to peel off the bacteria attached to the test material. Dilute the above-mentioned body suspension with sterile water continuously, apply it on a nutrient medium (Tryptic soy agar, TSA), and place the cultured fox in a 37 ° C incubator in an inverted dish. Formation, counting the number of colonies formed. The results obtained after testing Formula 5 are shown in Table 4. From Table 4, it can be seen that although the test samples are in the 4: 3: 1 group (diisocyanate: polyhexyl ethyl carbonate diol: butanol) and the 5 · 4: 1 group with the commercially available peiiethane® The results are similar, but the 0: 5: 1 group and the 7: 6: 1 group, which have relatively low levels of privacy, have obvious results in inhibiting bacterial attachment. It is shown that the polyaminocarbamate S double-group crosslinked polymer having nanophase separation on the surface has better antibacterial adhesion effect than conventional biomedical materials, and the higher the degree of phase separation, the better the effect. Example 2 Preparation of vinegar-polyethylene glycol group copolymers Take a catalyst of o.lg (such as [(μ-ΕΕ) Βρ) Α1 (: Η3] 2) and put 125ml 17 (please read the note on the back first) Please fill in this page for the matters) Installation · · Thread · The paper size is suitable for the national standard (CNS) A4 specification (210 X 297 public love) of the rich country _ 583002 Printed by the consumer property cooperative of the Intellectual Property Bureau of the Ministry of Economic Affairs A7 B7 V. Description of the invention ( v \ 〇) in an Erlenmeyer flask and add 10 ml digas. Mix this mixture with polyethylene glycol fluorenyl ether (Poly (Ethylene) with an average molecular weight of 5000

Glycol) Methyl Ether, Mn = 5000, MPEG), and stirred at room temperature for 12 hours. After removing the solution by suction, 30 ml of toluene was added, the temperature was controlled at 60 ° C, and 0.6 ml of cyclocaprolactone (5 mmol) was added. After the reaction hours, the reaction was terminated with 10 ml of a 0.35N aqueous acetic acid solution. An additional 50 ml of n-heptane was added to precipitate a white polymer. After removing the liquid by filtration, 10 ml of tetrahydrofuran was added to dissolve the polymer, and then 50 ml of n-heptane 'was added to precipitate a white solid. Finally, a white polymer solid product was obtained after the liquid was removed by filtration. 0.25 g of the polymer obtained above was dissolved in 10 ml of chloroform to prepare a polymer solution of 0.25% (w / v), which was spin-coated (200 rpm, 20 seconds). A uniform film is formed on the glass slide. Then, the solvent was volatilized at room temperature to prepare a polycaprolactone-polyvinyl alcohol double-group copolymer having nano-surface characteristics. The polymerization ratio of polycaprolactone-polyvinyl alcohol obtained in the above step was 29:71 (w / w). When the surface of this film is observed with an atomic mechanics microscope (AFM), the polycaprolactone-polyvinyl alcohol double-group copolymer prepared by the above steps can be rolled up, and the surface of the film shows obvious nanophase separation. Structure (Figure 3), in which polycaprolactone is cylindrically distributed on a flat surface of polyvinyl alcohol, and the size of nanophase separation is about 200 ~ 250nm, with an interval of about 500nηΐ-ΐμιη. Cell Adhesion Coexistence of the prepared polycaprolactone · polyvinyl alcohol double group (please read the precautions on the back before filling this page)

18 Printed by the Consumer Cooperative of the Intellectual Property Bureau of the Ministry of Economic Affairs 583002 A7 B7 V. Description of the invention (\ ^ \) The film was sterilized with alcohol (70% v / v) and placed in a 24-well culture dish. Rinse with phosphate buffer solution (PBS, ρΗ = 7.4) to remove residual alcohol. Place 5 × 104 endothelial cell lines (human umbilical cord endothelial cells, ECV-304) in each well, and add 1 ml of M199 culture solution containing 10% fetal bovine serum and the compound antibiotic PSA, at 37 ° C, 5 % C02. After 48 hours of incubation, the culture solution was aspirated from the well with a pipette. Add 1 ml of dish buffer solution to the well and aspirate the phosphate buffer solution from the well after 1 minute. Then add 0.4ml of trypsin (brand Biological Industries, concentration = 0.25%) to the wells and place in an incubator (37 ° C, 5% C02) for 10 minutes to make the cells from the polymer surface Peel off. The polymer was rinsed with 0.1 ml of a phosphate buffer solution, and the rinse was collected. The volume of this washing solution was adjusted with a phosphate buffer solution to make a 0.5 ml cell suspension. Finally, trypan blue (0.05%, trypan blue) was used to stain the cells, and the number of cells attached to the surface of the material was counted using a phase contrast microscope (100χ) and a cell counting disk. The results obtained are shown in Table 5. It is known that polyvinyl alcohol does not have cell attachment. As long as the polymer contains a small amount of polyvinyl alcohol, cells cannot be attached to it, but according to the data in the table, it can be seen that the polycaprolactone pointed out in the present invention -Polyvinyl alcohol double-group copolymer 29/71 (PCL / PEG) group 'experimental results have a good number of endothelial cell attachment, showing that when polyvinyl alcohol is separated into nano-phase, it can have cells. Attachability. That is, the polycaprolactone-polyvinyl alcohol double-group copolymer in the present invention has good cell compatibility when contacted with cells. __ 19 This paper size applies the Zhongguanjia Standard (CNS) A4 specification (210 X 297 male f) ---- (Please read the precautions on the back before filling this page)

583002 Printed by the Consumer Cooperative of the Intellectual Property Bureau of the Ministry of Economic Affairs A7 ______B7_ — V. Description of the Invention u%) Blood Compatibility The previously prepared polycaprolactone-polyvinyl alcohol double-group copolymerization 4 Film not cast After being sterilized with alcohol (70% v / v), it was put into a 24-well culture and rinsed with salt-filled buffer solution (PBS, ρ = 7.4) to remove residual alcohol. Add 1ml of platelet-rich blood to the wells of the culture plate and provide platelets with a platelet concentration of 60 × 106 to 14000 × ΐ () 6 / η ^ at 37 ° C and 5% C02. Incubate for 1 hour. After that, the non-adsorbed platelets were aspirated 'and the surface of the material was washed several times with a 1 M HBS solution, and then the HBS solution was completely aspirated. Then, add 25% glutaraldehyde 0.5mL to each well. Fix the platelets adsorbed on the material in the dark, and replace with a new glutaraldehyde solution after 20 minutes and place in a 4ac environment. Let stand for more than 8 hours. The above tested materials were immersed in pure water and 30%, 50%, 60%, 70%, 80%, 90%, 95%, 100% ethanol respectively for 5 minutes in order to carry out the stage dehydration. Finally, the ethanol was removed by a critical point drying method and then observed with a scanning electron microscope to estimate the number of platelet activations. The data obtained after the above steps are shown in Table 5. From the results obtained in Table 5, it can be known that the polycaprolactone_polyvinyl alcohol double-group copolymer prepared in the above steps in the present invention shows that it is less likely to cause a degenerative reaction in the blood when in contact with blood. That is, it has better blood compatibility. The anti-inflammatory property is a method of culturing monocytes in vitro, through the conversion of monocytes into macrophages, and the meaning of [macrophage monocytes + macrophages]], as another 4 materials in An indicator of an in vitro inflammatory response. The detailed method is as follows: (Please read the precautions on the back before filling out this page): Packing-line · 20

583002 A7 V. Description of the invention (θ) The film made of the prepared polycaprolactone-polyvinyl alcohol double-group copolymer is sterilized with alcohol (70% v / v) and put into 24 The wells were rinsed with phosphate buffer solution (PBS, ρ = 7.4) to remove residual alcohol. Put 1 × 105 human mononuclear spheres (isolated from the blood of healthy volunteers) into each well, and add 1 ml of RPMM640 culture solution containing 10% fetal bovine serum and 1% compound antibiotic PSA. Incubate at 37 ° C and 5% C02 for 96 hours. Aspirate the culture medium in the well with a pipette, and then add 1 ml of PBs solution. After 1 knife and 4 miles, aspirate the PBS solution in the well with a pipette, and then add 0.3 mi trypsin (0.25%, Biological industries, biochemical reagent grade), and incubate under C 5% CO 2 ring 5 Minutes to allow the cells to peel from the surface of the copolymer film. Thread Finally, the copolymer film was washed with 0.2 ml of RPMI-164 ° culture solution, and the washing solution was collected, and its volume was adjusted with RPMI-164 culture solution to make it a 0.5 ml cell suspension. Then it was stained with 0.05% trypan blue, and the number of monocytes and macrophages attached to the surface of the material were counted with a phase contrast microscope and a cell counting disk, respectively. The data obtained after the above steps are shown in Table 5. From the results obtained in Table 5, it can be known that the polycaprolactone-polyvinyl alcohol dual-group copolymer prepared by the above steps in the present invention has a low: the fraction of mononuclear spheres transformed into macrophage cells. It is shown that the polycaprolactone and the vinyl alcohol diblock copolymer of the present invention are less likely to cause a biological rejection reaction, and have higher anti-inflammatory properties. The antibacterial test uses Bacillus subtilis (atq6633) as a bacteriostatic test paper rule—degree tearing home fresh (CNS) A4 g (χ Norwegian public magic-583002 A7) printed by the Consumer Cooperative of the Intellectual Property Bureau of the Ministry of Economic Affairs—— _B7_ _______________ V. Description of the invention (☆) For the tested strains, first activate the g species, inoculate the stored strains into the nutrient medium (Nutrient Agar), and place the culture dish in the incubator in an inverted dish. After 12 hours of colony formation, the colonies were inoculated with platinum rings and inoculated into sterile water after the colonies were formed. The bacterial solution was diluted to ΙΟΟ 6 CFU / m. -The film made of polyvinyl alcohol double-strand copolymer is sterilized with alcohol (70% v / v), put into a well culture dish, and rinsed with linate buffer solution (PBS, pH = 7.4). After removing the residual sperm II, place it in a sterilized test tube. Take the aforementioned 1ml of bacterial solution (106CFU / ml) and add it to the above test tube, and then add 9ml of liquid culture medium (Tryptic soy broth, TSB) Mix well and place in a 37C incubator with shaking (100 rpm) for 12 hours Take out the test piece from the above test tube, wash it with 2ml of sterile water, and place it in another sterile test tube. Add 2ml of phosphonium salt buffer solution and shake it with ultrasound (43Hz, 400W) for 1 minute to make it adhere to The bacterial cells on the test material were peeled off. The above-mentioned bacterial cell suspension was serially diluted with sterile water, and then smeared on a nutrient medium (Tryptic soy agar, TSA). Cultivate until the colony is formed on the medium, and count the number of colony formation. As can be seen from Table 5, there is a polycaprolactone-polyvinyl alcohol double-group copolymer (PCL / PEG = 29) with nanophase separation on the surface. / 71) Polycaprolactone-polyvinyl alcohol double-group copolymer (pCL / pEG == 82/18) or ♦ Caprolactone with less nano-phase separation has better antibacterial attachment number. This The results show that the paper size in the present invention is applicable to the national standard (cns) A4 ^^ (2i〇 ^ mm, ---- (Please read the precautions on the back before filling out this page)-binding---line · 583002 A7 ____ Β7_ " ~ --- 5. Description of the invention (τΛ) Caprolactam-polyacetic alcohol double-group (PCL / PEG = 29/71) has good antibacterial adhesion. That is, the polycaprolactone-polyvinyl alcohol with nano-phase separation on the surface of 矣 I The double-linked copolymer can effectively reduce the adhesion of bacteria. According to the two proposed embodiments of the invention, the "polyurethane double-linked copolymer" prepared through the above steps, and, Inner Turtle_Polyvinyl Double-Band Copolymer "has phase separation of 1 ~ 100nm and 100 ~ 1000nm formed by chemically incompatible and dissimilar groups on its surface, respectively. Although the two embodiments indicated in the present invention have different phase separation sizes, they have been confirmed by experiments to have good biocompatibility and antibacterial properties. That is, a cross-linked copolymer composed of two or more kinds of polymer groups having chemical incompatibility and dissimilarity, which are aggregated together, when the surface has a size of 1 to 1000 nm In phase separation, the functional size is similar to that of the protein that dominates cell activity, which makes the conjugated copolymer both biocompatible (cell attachment, anticoagulation, and anti-inflammatory) and antibacterial. 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583002 Announcement of this table-Cell compatibility of poly (urethane) double-strand copolymers Copolymers with different proportions of polymerized monomers (A: B: C) a Number of endothelial cell attachments (104) 2: 1: 1 8.3 ± 0.2 3: 2: 1 11.8 ± 0.6 4: 3: 1 9.7 ± 1.1 5: 4: 1 9.5 ± 1.1 6: 5: 1 10.1 ± 1.4 7: 6: 1 10.4 ± 0.8 glass 7.9 ± 0.3 Pellethane® 3.3 ± 0.1 Note: a · A: diisocyanate; B: polyhexyl ethyl carbonate diol; C: butene diol. Table Hemocompatibility of diaminoaminobiester copolymers. Copolymer platelet activation number (A: B: C) a (104) 2: 1: 1 2.0 ± 0.1 3: 2: 1 1.4 ± 0.3 4: 3: 1 2.3 ± 0.1 5: 4: 1 4.1 ± 2.2 6: 5: 1 1.5 ± 0.3 7: 6: 1 0.9 ± 0.2 glass 9.3 ± 0.6 Pellethane® 6.3 ± 0.1 583002 Note: a. A: diisocyanate; B: polyhexyl ethyl carbonate diol; C: butene diol. Table Anti-inflammatory properties of the trimeric amino phosphonate double-block copolymers. Copolymer mononuclear spheres with different proportions of polymerized monomers transformed into giant salamander (A: B: C) a cells. 2: 1: 1 0.37 ± 0.03 3: 2: 1 0.21 ± 〇.〇3 4: 3: 1 0.25 ± 0.04 5: 4: 1 0.31 ± 0.01 6: 5: 1 0.19 ± 0.04 7: 6: 1 0.38 ± 0.04 Pellethane ⑧ 0.45 soil 0.08 583002 Note: a · A: diisocyanate; B ·· polyhexylethyl carbonate diol; C: butene diol. Table 4 Antibacterial properties of poly (urethane) double-link copolymers. Copolymers with different proportions of polymerized monomers (A: B: C) a. Number of bacteria attached (CFU) 2: 1: 1 to 3: 2: 1 — 4: 3: 1 1.07χ103 ± 0.16 5: 4: 1 1.6〇103 ± 0.35 6: 5: 1 2.75χ102 ± 0.07 7: 6: 1 UxlOkOJO Pellethane® 1 · 00χ103 ± 0 · 00 Note: a · A : Diisocyanate; B: polyhexylethyl carbonate 583002 ester diol; C: butenediol. 583002 Table 5: Cell adhesion, capacity, and anti-inflammatory properties of the tested materials (polyethylene glycol double-linked copolymers) of endothelial cells (104) Results of platelet activation test Number (106) ----- Fraction of monocytes transformed into giant gift cells! Number of bacteria attached (103CFU) PCL-PEG 29/7 la ----. 11.7 ± U ---- 2.9 0.18 ± 0 · 01 4.55 ± 0.495 PCL-PEG 82 / 18b 10.1 ± 0.5 6.4 0 · 28 ± 0 · 02 9-5〇 ± 0.707 PCLc 10.4 ± 0.7 32.4 0 · 30 ± 0 · 03 42 · 〇 ± 15.56 Note: (Polycaprolactone-polyvinyl alcohol double-group copolymer monomer polymerization ratio (b) M polycaprolactone). ] Is 29 • The ratio of Γ2 hexanol diblock copolymer monomer polymerization is 0 WUK caprolactone: polyvinyl alcohol). Using a commercially available display UniGn to verify the public bribe product name is Tone® p-787 〇

Claims (1)

583002 (Please read the precautions on the back before filling in this page, >!. A biocompatible cross-linked copolymer 'is characterized in that the surface of the cross-linked copolymer has the size of hiOOO nanometers, which is made by different groups The phase separation formed is caused by the different segments contained in the crosslinked copolymer, which are aggregated to form clusters due to chemical incompatibility and dissimilarity with each other. The cross-linking copolymer according to the above item, wherein the chemical incompatibility and dissimilarity are caused by the dispersive force or repulsion effect between different species in the polymer segment, and the chemical bonding force. The cross-linked copolymer according to item 1 in the scope, wherein the biocompatibility means cell attachment, blood compatibility or anti-inflammatory. 4. The cross-linked copolymer as described in item 3 of the scope of patent application The nano-material further has antibacterial properties. 5. The cross-linked copolymer according to item 1 of the scope of patent application, wherein the cross-linked copolymer is a double-group copolymer. 6. According to the scope of patent application The crosslinked copolymer according to item 5, which The double-block copolymer includes polyurethane double-block copolymer or polycaprolactone_polyvinyl alcohol double-block copolymer. Printed by the Consumer Cooperatives of the Intellectual Property Bureau of the Ministry of Economic Affairs. The cross-linked copolymer according to item 1, wherein the cross-linked copolymer is used as a biomedical material. 8. The cross-linked copolymer according to item 7 of the scope of patent application, wherein the biomedical material includes tissue engineering Stents or coating materials used in contact with blood or tissues. 9 · The crosslinked copolymer as described in item 8 of the scope of patent application, wherein the size of the coated paper is applicable to the Chinese National Standard (CNS) M specification (210X297 mm) ) 583002 ^, the patent application material Xiao b is coated on the substrate by means of soiutin casting, spin coating, melt coating or insect crystal method. 10. A kind of biocompatible Biomedical material, which includes the cross-linked copolymer as described in item 1 of the scope of patent application. 11. Biomedical material as described in item 10 of the scope of patent application, which contains a tissue engineering scaffold or is in contact with blood or tissue Used coating materials. 12. The biomedical material according to item 11 of the scope of patent application, wherein the coating material is coated on the solution by> solution casting, spin coating, melt coating, and stupid crystal method. (Please read the notes on the back before filling this page), 1T Printed by the Intellectual Property Bureau Employees Consumer Cooperatives of the Ministry of Economic Affairs 25 This paper size applies to China National Standard (CNS) A4 (210X297 mm)