TW583002B - Bio-compatible block copolymer - Google Patents
Bio-compatible block copolymer Download PDFInfo
- Publication number
- TW583002B TW583002B TW91125194A TW91125194A TW583002B TW 583002 B TW583002 B TW 583002B TW 91125194 A TW91125194 A TW 91125194A TW 91125194 A TW91125194 A TW 91125194A TW 583002 B TW583002 B TW 583002B
- Authority
- TW
- Taiwan
- Prior art keywords
- copolymer
- item
- cross
- patent application
- scope
- Prior art date
Links
Landscapes
- Materials For Medical Uses (AREA)
Abstract
Description
五、發明說明(丨) 發明領域 本么明係有關於一種具生物相容性的團聯共聚物,尤 其是關於-種團聯共聚物因所具有的不同鍵段彼此間的化 學不相容性及相異性使其各自聚集形成團聯,並於此團聯 共聚物的表面形成〗〜〗〇〇〇奈米大小尺寸由不同團聯間所 造成的相分離,而使其兼具有生物相容性及抗菌性。 發明背景 一一般對生醫材料(bi〇matedal)之定義,係指「一種人 工合成材料,可被用來取代活體部份系統,或直接與活體 接觸執行其功⑨」或是「—種有系統合乎藥理的鈍性物質, 其可被植入或合併入活體系統中」。根據上述之定義,生醫 材料於應用上的基本要求也是重要的要求,就是必須具備 良好的生物相容性。一般來說,此生物相容性還包括血液 相容性和組織相容性等。 加生醫材料於組織工程的應用上,主要係作為立體支 架,以引導細胞長成合適的組織,並具備正常功能。因此, 材料的物理特性(如大小、強度 '孔隙),或表面化學性質(如 與細胞的交互作用、本身的分解程度)等,便非常重要、。此 外,用做支架的材料,還必須能使細胞附著於其上,與身 體組織產生交互作用,避免排斥反應,並以適當的速率分 解,且最終能被身體的細胞與間質替換,以完成治療目桿刀。 目前生醫材料在運用上最常遭遇的困難為,當人體組 織與材料接觸時,其界面間所產生的問題,例如異體包覆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.
本紙張尺度適規格⑽x 297公釐) 583002 五、發明說明(i) t發炎反應。亦即,當人體内之環境接觸到外來材料表面 h ’會引起人體系統(如發炎、企液凝集及免疫系統)之防 護。如果人體生理反應過於劇烈、,則材料本身不但無法發 揮原有X力效,甚至還有可能造成人體的傷害。目前所謂的二 生物相容性,,生醫材料,絕大部份植入活體時,或多或少都 會引起類似反應,如身體組織會圍著植入物形成一無血管 的膠原蛋白囊將植入物包覆在内,即使在植入多年後,仍 可f與細胞接觸的材料表面上發現有巨噬細胞的存在,此 -情形可能會產生抑制血管生成的訊號,使正常的癒合與 重建過程受到阻隔。也由於這些抑制訊號的產生,使得習 用的組織工程產品常難以獲得良好的結果。由此得知,在 細胞與材料接觸的界面上所發生之交互作用,將決定材料 應用於人體中生物相容性的好壞。此外,在一些會與血液 接觸的材料上,更要避免如血小板活化、血漿蛋白質變性 專現象的發生。 、 自〇年代起,從事於人工植入材料性能之改良,使 其得以使用壽命更久且無副作用或是降低成本使其普遍 化’已成為相關材料研究開發之重點。例如,改善關節植 經濟部智慧財產局員工消費合作社印製 入材之耐磨性與耐蝕性、改善人工水晶球體(syntheticThe 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)之生物相容性、改良人工血管及心臟瓣膜、研 發文損皮膚組織之覆蓋(dressing)及人工皮膚(抓出^^ skin)等 。 由於影響生物相容性或血液相容性之因素相當複 雜’包含了㈣表面性質及細胞(或蛋白質)與材料表:之 k紙張尺度適用中國國家標準(CNS)A4規格(21G x 297公董 583002Intraocular) 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
五、發明說明(7)) (請先閲讀背面之注意事項再填寫本頁) 交;作用,其影㈣素可概括分成材料表面局部化學結構 的差異、表面電荷、親疏水性(如接觸角及加電位)、表 面粗糙度(roughness)和微異質性(micr〇-heter〇geneity)及柔 ㈣、機械性質料。因此,為達成良好的生物相容性, 目前已有許多研究係針對傳統材料表面進行化學處理(如 表面接枝大分子或基團)、表面物理性質之改變(如等離子 體、離子注入或離子束)或增加生物活性的方法,以使這些 材料具有可接受的生物相容性。然而目前已知的表面改質 法,大部分除了改質過程十分繁複、不易商業化外,亦無 法同時兼顧所有使用上所需要的性質。此外,對材料表面 所進行的改質處理,也會使得製造成本增加許多。 經濟部智慧財產局員工消费合作社印製 大部份習知的人工合成材料,其表面大多是均質態 (homogeneous)的,並不具有奈米特徵。數年前開始有一些 4»者利用表面上具有微米尺寸或奈米尺寸的材料,研究其 與細胞之間的交互作用反應,但並未曾探討其對生物相容 性(biocompatibility)的影響,且並無人使用表面奈米的技巧 做為一工具,對材料進行生物相容性的改質,以及以此設 計製作組織生醫材料。又如前所言,目前表面具有奈米尺 寸特徵的材料僅被用於作為研究細胞的訊號與行為(curtjs and Wilkinson; Dalby et al.),並無對其生物相容性,如血 液相容性及發炎特性,進行研究。 目前國内外於應用奈米材料做為組織修復或人體生 醫材料上的相關已公開的研究中,僅有奈米複合材料(添加 劑)以及使用非化學計量材料(non_stoichiometric materials) 5 本紙張尺度適用中國國家標準(CNS)A4規格(210 X 297公釐) A7 A7V. 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
五、發明說明(★) 學的=:奈:材料’被影射可能可應用於作為生物醫 以習知的圏聯共聚物的製備方法, 出奈米特徵,使並太的特性,於材料表面上製作 範圍附近(數奈米至數百夺 取㊉見的虫白貝 ., 白不未尺寸)而控制其生物相容性 者,亚未被報導。 發明概述 有鑑於上述的缺點,本發明係提供—種具有生物相容 性的團如共聚物。此團聯共聚物至少包含兩種或兩種以上 Ί予不相* ϋ及相異性的面分子團聯鏈段所各自聚集 而成的團聯(M〇ck),因不同分子間作用力的影響,而分離 形成w_奈米尺寸的相分離(phase separati〇n)。又因其 功能尺寸(fUnctional scale)與主宰細胞活性的蛋白質相 近,而使得此團聯共聚物兼具有生物相容性(細胞貼附、抗 凝血及抗發炎)及抗菌性。 本發明的另-目的,則在於提供一種不需再經表面改 質即可應用作為生醫材料使用的團聯共聚物,以降低製造 過程的繁瑣程度及減低製造成本。 本發明中的團聯共聚物係為包含兩種或兩種以上具 有化學不相容性及相異性的高分子團聯鏈段(如軟鏈及硬 鏈),因高分子鏈段中相異物種間的分散力或排斥效應,以 及高分子鏈段化學鍵結作用力的影響下,而造成高分子鏈 自我排列(self assembly)的現象,使其各自分離形成不同的 本紙張尺度適用中國國家標準(CNS)A4規格(210 X 297公釐) 閲 讀 背 面 之 注 項 再 填 寫 本 頁 經濟部智慧財產局員工消費合作社印製 583002 經濟部智慧財產局員工消费合作社印製 A7 B7 五、發明說明(V) 相。與高分子換合物不同的地方,在於此共聚高分子間仍 存在有共價鍵鍵結,因而限制了彼此相分離尺寸的大小。 於熱力學平衡狀態下’相異的團聯間會形成自我有序 (self-ordering)的有序微結構,而侷限分子鏈段的尺寸,使 每個相異相的尺寸介於1〜1000奈米之間,此現象在此稱為 奈米相分離(nanophase separation)(見圖一)。奈米相分離 後會產生不同的型態,例如變換單體的鏈段長度,會因兩 段高分子鏈的比例不同,而導致體積分率的差異,且依照 組成體積分率的不同,會形成各式各樣不同的有序微結 構,已發現的型態有許多種類,隨著高分子團聯鏈段組成 的體積變化,依序包含有圓球(sphere)、圓柱(Cyiin(jer)、層 狀排列(lamella)、或特殊的雙連續相(bic〇ntinu〇us、 gyroid)、穿孔層板(perf0rated layer)結構等。此外,變換團 聯式共聚合物的單體的種類,亦可以得到不同性質的結構。 人工植入材料表面影響蛋白質吸附的因素有:表面親 疏水性、表面柔軟性、吸附介質。柔軟的表面具有傳遞環 土兄吼號的優點,缺點是易產生脫離使細胞不易貼附,而硬 的表面會造成蛋白發生結構性重組,使得細胞附著於表面 時更緊密。蛋白質吸附可能受親水性、柔軟表面而減小, 文疏水性、硬表面而增大。而具有奈米相分離結構的聚胺 酯表面,奈米相分離程度越大,所吸附的蛋白質越少,這 疋由於硬鍵郎奈米相分離區域的尺寸越接近所吸附蛋白質 的尺寸日t,失活的蛋白質越少。反之,若硬鏈節奈米相分 離區域的尺寸遠離所吸附蛋白質的尺寸時,失活的蛋白質 (請先閱讀背面之注意事項再填寫本頁)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)
經 濟 部 智 慧 財 產 局 貝 工 消 費 合 作 社 印 製 583002 A7 B7 五、發明說明(U ) 就會越多。 而團聯共聚合物中可藉由控制單體的種類及鏈段長 度’以控制奈米相分離時的尺度及距離,使其功能性尺寸 (functional scale)與主宰細胞活性的蛋白質相接近,因此得 以控制蛋白質吸附程度及排列情形,而蛋白質吸附程度及 排列情形與細胞膜表面受器的結合及訊息傳遞有關,會最 終表現於細胞的遷移及生長上。因而使此具有奈米尺寸相 分離的表面具有獨特的生物相容性,使之能同時兼顧細胞 貼附、抗凝血、抗發炎、抗細菌感染等特性。 下列以’’聚胺基甲酸酯雙團聯共聚物,,及,,聚己内酯_聚 乙烯醇雙團聯(p〇1ycapr〇lact〇ne_p〇lyethylene 奶㈤ chblock,PCL-PEG)共聚物”,此兩種形式的團聯聚合物為 例’進一步對本發明做說明。 其中,本發明所指出的,,聚胺基甲酸酯雙團聯共聚物,, ^有作為主要聚合單體的二異氰酸鹽(A)及聚醇(b),同時 還含有可使鏈延長的鏈延伸劑(c)。 在本發明的聚胺基甲酸酯雙團聯共聚物中作為主要 聚合單體的二異氰酸鹽⑷’其例子包含二苯基甲烧4,4,_ 二異氰酸鹽(MDI)、甲苯二異氰酸鹽(TDI)、萘基^-二異 亂酸鹽(NDI)或六次甲基二異氛酸鹽(hdi)。作為主要聚合 單體的來醇(B),可以是聚轉醇、聚醋醇、聚碳酸醋二醇(例 如’聚己基聚乙基碳酸酿二醇(p〇ly(hexyl,却)^。讀 d’、聚秒氧烧醇或聚烯醇。作為鏈延伸劑(c)的可以是 二元醇、多元醇或二胺類’其例子包含乙二醇、丁烯二 (請先閲讀背面之注意事項再填寫本頁)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 經濟部智慧財產局員工消費合作社印製 五、發明說明(Λ ) 醇(2-butene-l,4-diol)、1,6-己二醇、三甲基醇、乙二胺、3 3_ 二氯-4,4-二氨基二苯基曱烷(m〇CA)。 在本發明中二異氰酸鹽(A)、聚醇(B)與鏈延伸劑(c) 的掺混比,以100重量份二異氰酸鹽(A)計,聚醇(b)與鏈 延伸劑(C)的總和為1〇〇重量份。於上述摻混比條件下,鍵 延伸劑(C)的摻混比,相對於100.重量份的聚醇,較佳為 17〜100重量份。 本發明的聚胺基甲酸酯雙團聯共聚物可於上述摻混 比的條件下,以二異氰酸鹽(A)及聚醇(B)做為聚合單體於 60〜70°C下反應約3〜5小時以合成預聚物。然後再將鏈延伸 劑(C)與’谷劑先行混合後再緩慢加入,滴入的時間較佳為控 制在30分鐘左右。之後再將催化劑與溶劑混合後緩慢加 入’此時溶質總量約佔30%wt,於60〜70°C下反應約4〜7 小時以完成聚合反應。將此反應完成的聚胺酯以溶劑稀釋 配成所品》辰度’可猎由溶液禱造法(s〇luti〇11 casting)、旋轉 塗佈法、溶融塗佈法或磊晶法鑄造於基材上以製備成薄膜。 作為上述的催化劑的例子包含二丁錫二月桂酸 (di-n-butyltin dilaurate)。作為上述溶劑的例子包含二甲基 二醯胺(N,N-dimethyl acetamide,DMAc)、四氫呋喃(THF) 或一甲基甲醯胺(DMF)。此溶劑於使用前須去除所含水 氣,此可藉由將溶劑加熱至l〇(rc以上或藉由分子篩處理2 個月以上以去除水氣。 經上述製備後的聚胺基甲酸酯雙團聯共聚物,會於表 面形成具有約1〜100奈米尺寸的分離相。 本紙張尺度適用中國國家標準(CNS)A4規格(210 x 297公釐) (請先閱讀背面之注意事項再填寫本頁) 裝 I - -線· 經濟部智慧財產局員工消費合作社印製 583002 A7 B7 五、發明說明(γ) 此外,本發明中另指出的”聚己内酯-聚乙烯醇雙團聯 共聚物”含有作為主要聚合單體的聚己内酯(D)及聚乙烯醇 (E)。 在本發明的聚己内酯-聚乙烯醇雙團聯共聚物中作為 主要聚合單體的聚己内酯(D),其例子包含環己内酯。作為 主要聚合單體的聚乙烯醇(E),其例子包含聚乙二醇甲基醚 (Poly(Ethylene Glycol) Methyl Ether) 〇 在本發明中聚己内酯(D)與聚乙烯醇(E)的摻混比,聚 己内酯(D)與聚乙烯醇(E)兩者之總和以100重量份計,聚 己内酯(D)為20〜80重量份,較佳為20〜50重量份。 本發明的聚己内酯-聚乙烯醇雙團聯共聚物可於上述 摻混比的條件下,先將適量催化劑加入作為溶劑的二氯甲 烷中,再加入聚乙烯醇於室溫下攪拌約10〜14小時。之後 以甲苯取代先前之溶劑,再將環己内酯加入,於60°C下反 應約12小時。待反應完成後,加入0·3 5N醋酸水溶液以終 止反應。 之後於上述混合液中加入正庚烷以使聚合物沉澱析 出。去除溶液取出沉澱析出之聚合物,再加入足量之四氫 呋喃使其完全溶解。待溶解後,再加入足量正庚烷以使聚 合物再度析出。 最後,將上述析出之聚合物溶於氯仿中,配製成適合 濃度的高分子溶液,藉由旋轉塗佈法以1,000〜3,000rpm的 轉速塗佈於基材上,之後於室溫下使溶劑揮發。 可作為上述的催化劑的例子包含[(p-EDBP)AlCH3]2 10 本紙張尺度適用中國國家標準(CNS)A4規格(210 X 297公釐) -------------裝--------訂---------線 (請先閱讀背面之注意事項再填寫本頁) A7583002 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
五、發明說明(、) 583002 或正 丁基經(n-butyl lithium)。 、、’二上述‘備後的聚己内gg _聚乙烯醇雙團聯共聚物, 會於表面形成具有約100〜1000奈米尺寸的分離相。 本發明將藉由參考下列的實施例做進一步的說明,這 些貝轭例並不限制本發明前面所揭示之内容。熟習本發明 之技藝者,可做些許之改良與修飾,但仍不脫離本發明之 範轉。 圖式簡單說明 圖一高分子團聯共聚合物之自我有序及自我排組 成一微觀相分離結構,尺寸介於丨〜…㈧奈米之間。 圖二以原子力學顯微鏡調變力模式觀察聚胺基甲 酸醋雙團聯共聚物的表面之影像。 圖二以原子力學顯微鏡調變力模式觀察聚己内酯_ 聚乙烯醇雙團聯共聚物(聚己内酯:聚乙烯醇=29 : 71)的表 面之影像。 發明的詳細說明 具體實施例 實施例一 酯雙團聯共聚物的f借 將固態二異氰酸鹽MDI置於6(rc的烘箱中,加熱融 化至呈現透明澄清液狀。另外,將聚己基乙基碳酸酯二醇 置於60 C烘箱中,待融化至液狀後,以抽真空裝置抽真空 11 本紙張尺度適用中國國家標準(CNS)A4規格(21G x 297 - ---- ----I---------裝--------訂---------線 (請先閱讀背面之注意事項再填寫本頁) 經濟部智慧財產局員工消費合作社印製 583002 經濟部智慧財產局員工消費合作社印製 A7 五、發明說明(\P ) 並同時?拌加熱’於100〜U0。。下維持8小時,以去除所 3的X氣此外,將二甲基乙醯胺加熱至⑽。〔以上以 去除内含的水氣。 將上述二異氰酸鹽澄清液與除水後的聚己基乙基碳 酸酉日^—醇以2:1、Λ · ο Γ 1 3 · 2、4 · 3、5 : 4、ό : 5 及 7 : ό 之比 例分別加入四頸反應瓶中,於65t下進行反應4小時,以 形成預聚物。 將幻述的預聚物分別加入足量上述經除水後的二甲 基乙蕴胺稀釋使其完全溶解。待完全溶解後,再將預先溶 解於甲基乙酉监胺的丁稀二醇(c/^s^-butene-l,4-diol),於 30分鐘内緩慢加入。此時丁烯二醇加入的量等於二異氰酸 鹽的使用ϊ與聚己基乙基碳酸酯二醇使用量的差值,亦即 丁稀一醇與聚己基乙基碳酸酯二醇使用量的總和與二異氰 酸鹽的使用量相等。之後將作為觸媒的二丁錫二月桂酸與 適置二甲基乙醯胺混合後,緩慢加入上述混合液中,使最 後的總溶質量約佔3〇%wt。於6yc下反應約5〜6小時後即 完成聚合反應。 將上述所得之聚胺基甲酸酯雙團聯共聚物以二甲基 乙酿胺稀釋成適當黏度,逐滴滴入甲醇中,使其於曱醇中 形成沉殿。將此沉澱出的聚胺基甲酸酯雙團聯共聚物於真 空下加熱乾燥至恆重。 將上述所得乾燥的聚胺基甲酸酯雙團聯共聚物以二 曱基乙胺配製成3%wt的溶液。將此3%wt的聚胺基甲酸 醋雙團聯共聚物溶液滴至直徑15mm的圓形玻片上。將此 12 本紙張尺度適用中國國家標準(CNS)A4規格(21〇 χ 297公 ---— — — — — — —--I i I II ! I 訂·!------ (請先閲讀背面之注意事項再填寫本頁) 583002 A7 ------ B7 五、發明說明(\\) 玻片加熱至60〜65°C,以使溶劑揮發。之後再於真空下, 使殘留的溶劑揮發完全。最後所製成的薄膜厚度約為imm。 (請先閱讀背面之注意事項再填寫本頁) 利用原子力學顯微鏡(AFM)觀察雙團聯共聚合物表面 型態可獲得調變力模式影像。此係為相對軟硬程度變化的 相圖,是在二維X-Y方向掃描Z方向高度變化的同時,施 以Z方向的振幅,藉由回饋訊號的強度,判斷表面的軟硬 私度’回饋訊號越強,表示表成硬度越高,反之則越低。 圖上明亮部分表示硬度越高,而陰暗部分表示硬度越低。 當將此薄膜表面以原子力學顯微鏡(AFM)觀察時,可 以發現經上述步驟製備後的聚胺基甲酸酯雙團聯共聚物, 其表面呈現具有約50奈米的相分離之奈米特徵(圖二)。 細胞貼附性 將前述經製備好的聚胺基甲酸酯雙團聯共聚物薄 膜,以酒精(70% v/v)滅菌後,放入24孔培養盤中。再以構 酸鹽緩衝溶液(PBS、ΡΗ=7·4)沖洗,去除殘餘酒精。 於每個孔中放置5χ 104個内皮細胞株(人類臍帶内皮 細胞、ECV-304),並加入lml内含10%的胎牛血清及 的複合抗生素 P-S-A(Penicillin / Streptomycin / 經濟部智慧財產局員工消費合作社印製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 三合一抗生素)的 M199 培養液(Medium 199,Amphotericin 3-in-1 antibiotic) M199 medium (Medium 199,
HyClone⑧細胞培養級),於37°C,5% C02的環境下培養。 培養48小時後,以吸管自孔中將培養液吸出。再於 孔中加入1 ml鱗酸鹽緩衝溶液,1分鐘後再將孔中的填酸 鹽緩衝溶液吸出。然後於孔中加入0.4ml的胰蛋白晦(〇.25%,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),放置於培養箱中(37°C、5% C02)中 13 本紙張尺度適用中國國家標準(CNS)A4規格(210 X 297公釐) 583002 經濟部智慧財產局員工消費合作社印製 A7 ___B7_____ 五、發明說明(V》) 10分鐘,以使細胞自聚合物表面剝離。將此聚合物再以 0.1ml的磷酸鹽緩衝溶液沖洗,收集此沖洗液。以磷酸鹽緩 衝溶液調整此沖洗液體積,使其成為〇.5ml之細胞懸浮液。 最後以錐蟲藍(0.05%,trypan blue)對細胞染色,並以 位相差顯微鏡(100χ)及細胞計數盤,計數貼附在材料表面 的細胞個數。所得之結果示於表一。由表中之數據可以看 出,將不同比例的單體以上述步驟所製備之聚胺基甲酸酯 雙團聯共聚物,皆較Upj0hn所生產的聚胺酯生醫材料 (Pellethan,2363-80A)或玻璃有較高的内皮細胞貼附數。 亦即本發明中的聚胺基甲酸酯雙團聯共聚物與細胞接觸時 有較高之細胞相容性。 血液相容性 將前述經製備後的聚胺基甲酸酯雙團聯共聚物所鑄 成的薄膜,以酒精(70% v/v)滅菌後,放入24孔培養盤中, 以磷酸鹽緩衝溶液(PBS、ρΗ=7·4)沖洗,去除殘餘酒精。 於培養盤的每個孔中分別加入lml富血小板血漿(中 華血液基金會提供,血小板濃度6〇〇><1〇6至14〇〇xl〇6/ml), 於37 C、5% C〇2的環境下培養i小時。之後,將未吸附之 血小板吸出,並以1M的HBS溶液(Hepes Buffer so丨uti〇n, Gibco BRL)沖洗材料表面數次後,將HBS溶液完全吸出。 然後,於每個孔中分別加入2·5%的戊二醛〇 5ml,於避光 下使吸附在材料上的血小板固定,於2〇分鐘後再更換新的 戊一醛溶液,置於4°C的環境下靜置8小時以上。 將上述受試之材料依次分別浸入純水及3〇%、5〇% 、 _____ 14 本紙張尺度適用中國國家標準(CNS)A4規格(21〇 X 297公爱)---- ^ % Φ: — — — — — — — — · 11 (請先閱讀背面之注意事項再填寫本頁) ·. 線·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) ·.
°。70%、80%、90%、95%、1〇〇% 乙醇中各 $ 進行階段脫水。最後,以合 刀釦以 ^ 4f ^ °°界點乾燥方法去除乙醇後,再 以知描式電子顯微鏡 更再 數。仃戒祭,以估计血小板的活化個 〜、、'工上述步驟處理後所得之數據示於表二。由表二中所 ^之結果可得知’本發明中以上述步驟所製備不同比例單 之聚胺基甲酸醋雙團聯共聚物,皆較Upjohn所生產的聚 胺酷生醫材料(Peuethane②2363德)或玻璃有較低的血;;、 板活化個數。顯示本發明中的聚胺基甲酸醋雙團聯共聚物 當^血液接觸時較不會引起血小板的活化,亦即與血液接 五、發明說明) 觸¥較不會產生血液凝結之情形,因此有較佳之血液 性。 < %φί I I I I « --- (請先閱讀背面之注意事項再填寫本頁) 經濟部智慧財產局員工消費合作社印製 抗發炎性 以體外培養單核球的方式,藉由單核球轉變為巨噬細 月己的過秋疋義【巨兔細胞/(單核球+巨嗟細胞)】的分率, 作為叉試材料在體外的發炎反應的指標。詳細的做法如下·· 將前述經製備後的聚胺基甲酸酯雙團聯共聚物所鑄 成的薄膜’以酒精(70% v/v)滅菌後,放入24孔培養盤中, 以磷酸鹽緩衝溶液(pBS、ρΗ=7·4)沖洗,去除殘餘酒精。 於每個孔中放入1χ1〇5個人類單核球(分離自健康自 願者的血液),並加入内含10%的胎牛血清及的複 合抗生素P-S-A的RPMI-1640培養液,於37°C、5% C02 的環境下,培養96小時。 以吸管吸出孔中的培養液,再加入1 ml的鱗酸鹽緩衝 溶液(PBS、PH=7.4)。於1分鐘後將孔中磷酸鹽緩衝溶液 15 本紙張尺度適用中國國家標準(CNS)A4規格(210 X 297公釐) 訂 線 經濟部智慧財產局員工消費合作社印製 583002 A7 ____ B7 五、發明說明(4) (PBS、ΡΗ=7·4)以吸管吸出,再加入〇.3mi的胰蛋白梅 (0.25%,Biological Industries,生化試藥級),於 37°c、5 % C〇2的環境下培養5分鐘,以使細胞自共聚物薄膜的表 面剝離。 最後,以0.2ml RPMI-1640培養液沖洗此共聚物薄 膜,收集此沖洗液,並以RPMI-1640培養液調整其體積, 使其成為0.5ml之細胞懸浮液。再以〇·〇5%錐蟲藍染色,並 以位相差顯微鏡及細胞計數盤,分別計數貼附在材料表面 的單核球與巨噬細胞個數。經上述步驟處理後所得之數據 示於表三。由表三中所得之結果可得知,本發明中以上述 乂驟所‘備不同比例單體之聚胺基甲酸g旨雙團聯共聚物, 皆較Upjohn所生產的聚胺酯生醫材料 2363-80A)或玻璃有較低之單核球轉變為巨噬細胞的分 率。顯示本發明之聚胺基甲酸醋雙團聯共聚物較不易引起 生物產生排斥反應,亦即有較高之抗發炎性。 抗菌性試驗 以枯草桿菌(仏,ATCC6633)作為抑菌測 試,菌種:首先進行菌種的活化,將儲存於代的菌種接種 至營養培養基(Nutrient Agar)上,以倒碟方式將培養皿放置 於培養箱中,於机下培養12小時。待菌落形成後,以接 種用白金環勾取菌體加入無菌水中’將菌液稀釋至1〇6 CFU/m 卜 、將前述經製備後的聚胺基甲酸醋雙團聯共聚物所禱 成的薄膜’以酒精(70%v/v)滅菌後,放人24孔培養盤 (請先閲讀背面之注意事項再填寫本頁) 訂: -I線· 16 583002 A7 --------B7 經濟部智慧財產局員Η消費合作社印製 五、發明說明(A ) 以磷酸鹽緩衝溶液(PBS、ΡΗ=7·4)沖洗,去除殘餘酒精。之 後將其置於已滅菌之試管中。 取前述lml的菌液(106 CFU/ml)加入上述試管中, 再加入液態培養基(Tryptic soy broth, TSB)混合均句, 放置在37。0培養箱震盪培養(轉速lOOrpm) 12小時。 自上述試管中取出試片,並以2ml無菌水清洗後,將 其置於另一無菌試管中。加入2mi填酸鹽緩衝溶液,以超 音波(43Hz、400W)震盪1分鐘,以使附著於受試材料上 的菌體剝離。 將上述勤體懸浮液以無菌水進行連續稀釋後,塗抹於 營養培養基(Tryptic soy agar,TSA)上,並以倒碟方式將培 養狐放置於37°C培養箱中培養,待培養基上有菌落形成, 計數菌落形成的個數。 經測5式後所得之結果示於表四。由表四中可以看出雖 測試樣品4:3:1組(二異氰酸鹽:聚己基乙基碳酸酯二醇: 丁稀一醇)及5 ·4:1組與市售品peiiethane®結果差不多,但 相为_私度較鬲的0:5:1組及7:6:1組此二組則有明顯的抑 制細菌貼附之結果。顯示表面具有奈米相分離之聚胺基甲 酸S旨雙團聯纟聚物比習&之生醫材料有較佳之抗細菌貼附 效果,且相分離程度越高效果越佳。 實施例二 内醋-聚乙越團聯共聚物的製備 取 o.lg 的催化劑(如[(μ-ΕΕ)Βρ)Α1(:Η3]2),置入 125ml 17 (請先閲讀背面之注意事項再填寫本頁) 裝 · · 線· 本紙張尺度適財國國家標準(CNS)A4規格(210 X 297公爱)_ 583002 經濟部智慧財產局員工消費合作社印製 A7 B7 五、發明說明(v\〇 ) 的錐形瓶中’並加入1 〇ml二氣甲烧。將此混合物與平均數 量分子量為5000的聚乙二醇曱基醚(P〇ly(Ethylene°. 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),於室溫下攪拌 12 小時。 以吸官將溶液去除後,加入3 0ml甲苯,並將溫度控 制在60°C,再加入〇.6ml環己内酯(5毫莫耳)。反應小 時後,以10毫升〇·35Ν醋酸水溶液終止反應。再加入5〇 毫升正庚烧’以使白色聚合物沉澱。經過濾去除液體後, 加入10ml四氫呋喃溶解此聚合物後,再加入5〇毫升正庚 烷’以析出白色固體。最後,再以過濾去除液體後可獲得 白色聚合物固體產物。 取上述所得之聚合物〇.25g溶於1〇ml的氯仿中,配製 成〇.25%(w/v)的高分子溶液,以旋轉塗佈(200〇rpm,2〇秒) 法於玻片上形成均勻的薄膜。再於室溫下使溶劑揮發,以 製成表面具有奈米特徵的聚己内酯_聚乙烯醇雙團聯共聚 物。上述步驟所得之聚己内酯-聚乙烯醇聚合比為29 : 71 (w/w)。 將此薄膜表面以原子力學顯微鏡(AFM)觀察時,可以 卷現、、、二上述步驟製備後的聚己内酯_聚乙烯醇雙團聯共聚 物,其表面呈現明顯的奈米相分離的結構(圖三),其中聚 己内醋呈現圓柱形散佈在平坦的聚乙烯醇表面,奈米相分 離尺寸約為200〜250nm,間隔約為5〇〇ηηΐ-ΐμιη。 細胞貼附性 將可述經製備好的聚己内酯·聚乙烯醇雙團聯共 (請先閱讀背面之注意事項再填寫本頁)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 經濟部智慧財產局員工消費合作社印製 583002 A7 B7 五、發明說明(\^\) 薄膜,以酒精(7〇%v/v)滅菌後,放入24孔培養盤中。再以 磷酸鹽缓衝溶液(PBS、ρΗ=7·4)沖洗,去除殘餘酒精。 於每個孔中放置5χ104個内皮細胞株(人類臍帶内皮 細胞、ECV-304),並加入lml内含1〇%的胎牛血清及 的複合抗生素P-S-A的M199培養液,於37°C,5% C02的 環境下培養。 之後於培養48小時後,以吸管自孔中將培養液吸出。 再於孔中加入1 ml碟酸鹽緩衝溶液,1分鐘後再將孔中的 磷酸鹽緩衝溶液吸出。然後於孔中加入0.4ml的胰蛋白晦 (廠牌Biological Industries,濃度=0.25%),放置於培養箱 中(37°C、5% C〇2)中10分鐘,以使細胞自聚合物表面剝離。 將此聚合物再以0.1ml的磷酸鹽緩衝溶液沖洗,收集此沖 洗液。以磷酸鹽緩衝溶液調整此沖洗液體積,使其成為 0.5ml之細胞懸浮液。 最後以錐蟲藍(0.05%,trypan blue)對細胞染色,並以 位相差顯微鏡(ΙΟΟχ)及細胞計數盤,計數貼附在材料表面 的細胞個數。所得之結果示於表五。習知聚乙烯醇不具細 胞貼附性,只要聚合物中含有少量之聚乙烯醇即會使細胞 無法貼附於其上,但由表中之數據可以看出,本發明中所 指出之聚己内酯-聚乙烯醇雙團聯共聚物29/71 (PCL/PEG) 組’經實驗結果具有不錯之内皮細胞貼附個數,顯示當使 聚乙烯醇成奈米相分離時即可使其具有細胞貼附性。亦即 本發明中的聚己内酯-聚乙烯醇雙團聯共聚物與細胞接觸 時有不錯之細胞相容性。 __ 19 本紙張尺度適用中關家標準(CNS)A4規格(210 X 297公f ) ---- (請先閱讀背面之注意事項再填寫本頁)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 經濟部智慧財產局員工消費合作社印製 A7 ______B7_ — 五、發明說明u%) 血液相容性 將前述經製備後的聚己内酯-聚乙烯醇雙團聯共聚4勿 所鑄成的薄膜,以酒精(70% v/v)滅菌後,放入24孔培養般 中,以填酸鹽緩衝溶液(PBS、ρΗ=7·4)沖洗,去除殘餘酒精。 於培養盤的每個孔中分別加入lml富血小板血裝 華血液基金會提供,血小板濃度60〇xl06至14〇〇χΐ()6/η^, 於37°C、5% C〇2的環境下培養1小時。之後,將未吸附之 血小板吸出’並以1Μ的HB S溶液沖洗材料表面數次後, 將HBS溶液完全吸出。然後,於每個孔中分別加入2 5% 的戊二醛0.5mL·於避光下使吸附在材料上的血小板固定, 於20分鐘後再更換新的戊二醛溶液,置於4ac的環境下靜 置8小時以上。 將上述受试之材料依次分別浸入純水及3〇%、5 〇% 、 60%、70%、80% 、90% 、95%、1〇〇% 乙醇中各 5 分鐘以 進行階段脫水。最後,以臨界點乾燥方法去除乙醇後,再 以掃描式電子顯微鏡進行觀察,以估計血小板的活化個 數。經上述步驟處理後所得之數據示於表五。由表五中所 得之結果可得知,本發明中以上述步驟所製備之聚己内酯_ 聚乙烯醇雙團聯共聚物,顯示出其與血液接觸時較不易引 起血液產生减結反應,亦即有較佳之血液相容性。 抗發炎性 以體外培養單核球的方式,藉由單核球轉變為巨噬細 胞的過耘,疋義【巨噬細胞單核球+巨噬細胞)】的分率, 作為又4材料在體外的發炎反應的指標。詳細的做法如下: (請先閱讀背面之注意事項再填寫本頁) :裝 --線· 20583002 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 五、發明說明(θ ) 將前述經製備後的聚己内酯-聚乙烯醇雙團聯共聚物 所鑄成的薄膜,以酒精(7〇% v/v)滅菌後,放入24孔培養盤 中’以磷酸鹽缓衝溶液(PBS、ρΗ=7·4)沖洗,去除殘餘酒精。 於每個孔中放入1χ1〇5個人類單核球(分離自健康自 願者的血液),並加入lml内含1〇%的胎牛血清及1%的複 合抗生素P-S-A的RPMM640培養液,於37°C、5% C02 的環境下,培養96小時。 以吸管吸出孔中的培養液,再加入lml的PBs溶液。 於1刀4里後將孔中PBS溶液以吸管吸出,再加入〇 3mi的 胰蛋白騰(0.25%,Biological industries,生化試藥級),於 C 5% C〇2的環i兄下培養5分鐘,以使細胞自共聚物薄 膜的表面剝離。 線 最後,以0.2ml RPMI-164〇培養液沖洗此共聚物薄 膜,收集此沖洗液,並以RPMI_164〇培養液調整其體積, 使其成為0.5ml之細胞懸浮液。再以〇 〇5%錐蟲藍染色,並 以位相差顯微鏡及細胞計數盤,分別計數貼附在材料表面 的單核球與巨噬細胞個數。經上述步驟處理後所得之數據 示於表五。由表五中所得之結果可得知,本發明中以上述 步驟所製備之聚己内酯_聚乙烯醇雙團聯共聚物,有較低: 單核球轉變為巨嗟細胞的分率。顯示本發明之聚己内能、取 乙烯醇雙團聯共聚物較不易引起生物產生排斥反應,= 有較高之抗發炎性。 抗菌性試驗 以枯草桿菌(細勤仏,atcc6633)作為抑菌測 本紙張尺—度撕家鮮(CNS)A4 g ( χ挪公幻- 583002 A7 經濟部智慧財產局員工消費合作社印製 —— _B7_ _______________五、發明說明(☆) 試之菌種,首先進行g種的活化,將儲存於代的菌種接種 至營養培養基(Nutrient Agar)上,以倒碟方式將培養皿放置 於培養箱中,於抓下培養12小時。待菌落形成後,以接 種用白金環勾取菌體加人無菌水中,將菌液稀釋至ι〇6 CFU/m 卜 將前述經製備後的聚己内醋-聚乙烯醇雙團聯共聚物 所鑄成的薄膜,以酒精(70% v/v)滅菌後,放入以孔培養盤 中,以麟酸鹽緩衝溶液(PBS、pH=7.4)沖洗,去除殘㈣精二 之後將其置於已滅菌之試管中。 取前述lml的菌液(1〇6CFU/ml)加入上述試管中, 再加入9ml液態培養基(Tryptic s〇y br〇th,TSB)混合均勻, 放置在37C培養箱震盪培養(轉速1〇〇rpm) 12小時。 自上述試管中取出試片,並以2ml無菌水清洗後,將 其置於另一無菌試管中。加入2ml鱗酸鹽緩衝溶液,以超 音波(43Hz、400W)震盪i分鐘,以使附著於受試材料上 的菌體剝離。 將上述菌體懸浮液以無菌水進行連續稀釋後,塗抹於 營養培養基(Tryptic soy agar,TSA)上,並以倒碟方式將培 養孤放置於37t培養箱中培養,待培養基上有菌落形成, 計數菌落形成的個數。 從表五中可以看出,表面具有奈米相分離之聚己内酯 -聚乙烯醇雙團聯共聚物(PCL/PEG=29/71)較不具奈米相分 離之聚己内酯-聚乙烯醇雙團聯共聚物(pCL/pEG==82/18)或 ♦己内S曰’有較佳之抗細菌貼附數。此結果顯示本發明中 本紙張尺度適用肀國國家標準(cns)A4^^(2i〇 ^公釐、---- (請先閱讀背面之注意事項再填寫本頁) —裝 訂·- --線· 583002 A7 ____Β7_ " ~—-- 五、發明說明(τΛ) 表面具有奈米相分離之聚己内S旨-聚乙浠醇雙團聯 (PCL/PEG=29/71)有不錯之抗細菌貼附性。亦即矣 I衣面具有奈 米相分離之聚己内酯-聚乙烯醇雙團聯共聚物可有效減小 細菌的貼附。 根據發明上述所提出的兩個實施例,經上述步驟製備 後的”聚胺基甲酸酯雙團聯共聚物,,及,,聚己内龜_聚乙稀醇 雙團聯共聚物”,其表面分別具有化學不相容性及相異性的 團聯所形成之1〜100奈米及100〜1000奈米的相分離。雖本 發明中所指出的兩實施例具有不同之相分離尺寸,但、妙實 驗確認均具有良好之生物相容性及抗菌性。亦即,包含兩 種或兩種以上具有化學不相容性及相異性的高分子團聯鍵 段所各自聚集而成的團聯共聚物,當表面具有1〜1〇〇〇奈米 尺寸的相分離時’因功能尺寸與主宰細胞活性的蛋白質相 近,而使該團聯共聚物得以兼具生物相容性(細胞貼附、抗 凝血及抗發炎)及抗菌性。 (請先閲讀背面之注意事項再填寫本頁) 訂: 線- 經濟部智慧財產局員工消費合作社印製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. (Please read the precautions on the back before filling out this page) Order: Line-Printed by the Consumer Cooperatives of the Intellectual Property Bureau of the Ministry of Economic Affairs
583002 公告本 表一聚胺基曱酸酯雙團聯共聚物的細胞相容性 不同聚合單體比例的共聚物 (A:B:C)a 内皮細胞貼附數 (1〇4 個) 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 玻璃 7.9 ±0.3 Pellethane® 3.3 ±0.1 註:a· A :二異氰酸鹽;B :聚己基乙基碳酸 酯二醇;C : 丁烯二醇。 表二聚胺基曱酸酯雙團聯共聚物的血液相容性 不同聚合單體比例的共聚物 血小板活化個數 (A:B:C)a (1〇4 個) 2:1:1 2.0 ±0.1 3:2:1 1.4 ±0.3 4:3:1 2.3 士0.1 5:4:1 4.1 ±2.2 6:5:1 1.5 ±0.3 7:6:1 0.9 ±0.2 玻璃 9.3 ±0.6 Pellethane® 6.3 ±0.1 583002 註:a· A :二異氰酸鹽;B :聚己基乙基碳酸 酯二醇;C : 丁烯二醇。 表三聚胺基曱酸酯雙團聯共聚物的抗發炎性 不同聚合單體比例的共聚物 單核球轉為巨嗟 (A:B:C)a 細胞的分率 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 士 0.04 7:6:1 0.38 ±0.04 Pellethane ⑧ 0.45 土0.08 583002 註:a· A :二異氰酸鹽;B ··聚己基乙基碳酸 酯二醇;C : 丁烯二醇。 表四聚胺基曱酸酯雙團聯共聚物的抗菌性 不同聚合單體比例的共聚物 (A:B:C)a 細菌貼附個數 (CFU) 2:1:1 一 3:2:1 — 4:3:1 1.07χ103 ±0.16 5:4:1 1.6〇χ103 ±0.35 6:5:1 2.75χ102 ±0.07 7:6:1 UxlOkOJO Pellethane® 1·00χ103±0·00 註:a· A :二異氰酸鹽;B :聚己基乙基碳酸 583002 酯二醇;C : 丁烯二醇。 583002 表五*己内S旨-聚乙稀醇雙團聯共聚物之細胞貼附性、 容性、 受測材料 抗發炎性輿 内皮細胞貼 附數(104個) 性試驗之結果 血小板活化 個數(106個) ----- 單核球轉為 巨禮細胞的 分率 ! 細菌貼附個 數(103CFU) PCL - PEG 29/7 la ----. 11.7±U ---- 2.9 0.18 士 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 士 0·03 42·〇± 15.56 註: 聚己内醋-聚乙烯醇雙團聯共聚物單體聚合之比 b心Μ聚己内聚乙騎)。 ]為29 • Γ2己醇雙團聯共聚物單體聚合之比例為 〇 WUK己内酯:聚乙烯醇)。 使用市售顯UniGn⑸驗公贿品名為Tone® p-787 〇583002 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)
Priority Applications (1)
Application Number | Priority Date | Filing Date | Title |
---|---|---|---|
TW91125194A TW583002B (en) | 2002-10-25 | 2002-10-25 | Bio-compatible block copolymer |
Applications Claiming Priority (1)
Application Number | Priority Date | Filing Date | Title |
---|---|---|---|
TW91125194A TW583002B (en) | 2002-10-25 | 2002-10-25 | Bio-compatible block copolymer |
Publications (1)
Publication Number | Publication Date |
---|---|
TW583002B true TW583002B (en) | 2004-04-11 |
Family
ID=34057952
Family Applications (1)
Application Number | Title | Priority Date | Filing Date |
---|---|---|---|
TW91125194A TW583002B (en) | 2002-10-25 | 2002-10-25 | Bio-compatible block copolymer |
Country Status (1)
Country | Link |
---|---|
TW (1) | TW583002B (en) |
-
2002
- 2002-10-25 TW TW91125194A patent/TW583002B/en not_active IP Right Cessation
Similar Documents
Publication | Publication Date | Title |
---|---|---|
Kannan et al. | The antithrombogenic potential of a polyhedral oligomeric silsesquioxane (POSS) nanocomposite | |
TWI244927B (en) | Biocompatible polymer film | |
Li et al. | Recruitment of multiple cell lines by collagen-synthetic copolymer matrices in corneal regeneration | |
Lai et al. | Characterization of cross-linked porous gelatin carriers and their interaction with corneal endothelium: biopolymer concentration effect | |
Tamada et al. | Fibroblast growth on polymer surfaces and biosynthesis of collagen | |
Schmedlen et al. | Photocrosslinkable polyvinyl alcohol hydrogels that can be modified with cell adhesion peptides for use in tissue engineering | |
Liu et al. | The nanofibrous architecture of poly (L-lactic acid)-based functional copolymers | |
US8343742B2 (en) | Encapsulation of cells in biologic compatible scaffolds by coacervation of charged polymers | |
Bozukova et al. | Improved performances of intraocular lenses by poly (ethylene glycol) chemical coatings | |
Watanabe et al. | Cell adhesion and morphology in porous scaffold based on enantiomeric poly (lactic acid) graft-type phospholipid polymers | |
Costa et al. | Compact saloplastic membranes of natural polysaccharides for soft tissue engineering | |
Tang et al. | Robust MeO2MA/vinyl-4, 6-diamino-1, 3, 5-triazine copolymer hydrogels-mediated reverse gene transfection and thermo-induced cell detachment | |
US10131874B2 (en) | Cell culture support and associated method for cell growth and release | |
US20240279463A1 (en) | Composite material and its method of production | |
WO2013063390A1 (en) | A suturable hybrid superporous hydrogel keratoprosthesis for cornea | |
Davis et al. | Modified PHEMA hydrogels | |
Guo et al. | Anticalcification potential of POSS-PEG hybrid hydrogel as a scaffold material for the development of synthetic heart valve leaflets | |
US6294614B1 (en) | Modified polymers containing poly(2-hydroxyethyl(meth)acrylate segment in the molecule | |
Tovar-Carrillo et al. | Biohydrogels interpenetrated with hydroxyethyl cellulose and wooden pulp for biocompatible materials | |
Woerly et al. | Synthetic polymer derivatives as substrata for neuronal adhesion and growth | |
Fan et al. | Macrophages behavior on different NIPAm‐based thermoresponsive substrates | |
TW583002B (en) | Bio-compatible block copolymer | |
KR20180115531A (en) | Method of preparing three dimensional(3D) structure with cellulose nanofiber for cell culture and the structure prepared by using the method | |
WO2024131924A1 (en) | Broad-spectrum responsive self-assembling peptide and use thereof | |
KR102583866B1 (en) | Antibacterial or antifungal composition |
Legal Events
Date | Code | Title | Description |
---|---|---|---|
MK4A | Expiration of patent term of an invention patent |