201122003 六、發明說明： 【發明所屬之技術領域】 本發明係關於一種抗生物結垢共聚物，特別是關於 抗生物結垢非離子型-兩性離子型無規排列共聚物。 【先前技術】 近年來，兩性離子型高分子因具有抗血漿蛋白質吸附 官能基[例如：填酸醋甜菜驗(phosphobetaine)、硫代甜菜驗 (sufobetaine)、叛基甜菜驗(carboxylbetaine)]，成為新一代 極具潛力之血液相容性材料的選擇。其中，聚硫代甜菜鹼 丙烯酸酯[poly(sulfobetaine methacrylate) ; polySBMA]合成 方式容易’吸引眾多研究人員投入。聚硫代甜菜鹼丙烯酸 醋（polySBMA)係以丙稀酸酯（methacrylate)為主鏈，以及類 似牛續酸甜菜驗（taurine betaine)之懸掛基團（pendant group) « 另外’熱感應型非離子型高分子，在水環境中、特定 溫度下，會進行親水-疏水性性質轉換，此溫度稱為下臨界 溶液溫度[lower critical solution temperature (LCST)]。其 中’聚異丙基丙稀醯胺[poly(N-isopropylacrylamide); polyNIPAAm]係最廣為使用之熱感應型高分子。然而，非 離子型高分子與兩性離子型高分子於水溶液中性質不同。 一般來說’兩性離子型高分子具有上臨界溶液溫度[upper critical solution temperature (UCST)]，例如：聚硫代甜菜驗 丙烯酸酯（polySBMA)，而上臨界溶液溫度會隨著兩性離子 201122003 型高分子濃度增加而提高。 近期研究揭示熱感應型單體與兩性離子型單體所聚合 形成之雙嵌段(diblock)共聚物具有微胞材料特性。並且，上 述之雙嵌段共聚物可同時具有上臨界溶液溫度與下臨界溶 液溫度。但是，這些研究中未討論將其應用於生化領域或 生醫材料之可行性。 、 事實上，上述之雙嵌段共聚物應用於生醫材料上具有 潛在之風險。上述之雙嵌段共聚物包含IJCST嵌段（兩性離 • 子）與LCST嵌段(熱感應）。當雙嵌段共聚物於水溶液中形 成微胞後’改變水溶液溫度，微胞之内部嵌段與外部欲段 即可輕易轉換。然而，LCST嵌段(熱感應）（例如：聚異丙 基丙烯醯胺；polyNIPAAm)具有高蛋白質、細胞、細菌吸 附力。當環境溫度改變導致微胞外部轉為LCST嵌段時， 微胞表面對生物分子的強烈吸附情形快速地發生，造成不 可逆的聚集或阻塞現象。因此，使用雙嵌段共聚物於血液 相容材料上相當危險。 # 由於上述熱感應型單體與兩性離子型單體所聚合形成 之礙段式共聚物於生醫材料應用上可能造成不可逆聚集與 或阻塞現象’因此’仍有必要開發新的適合於生化領域應 用之共聚物。 【發明内容】 鑒於上述之發明背景中，為了符合產業上之要求，本 發明提供一種新的具有熱感應性與抗生物結垢之共聚物以 及水膠。 根據上述目的，本發明提供一種抗生物結垢非離子蜇- 4 201122003 非離子型單體與兩性離 無規排列共聚物係以 子型單體聚合而成。 兩性離子本發贿供—種抗生物結垢_子型- 性離子型單=無規排列共聚物，係以非離子型單體、兩 单體與交聯劑聚合而成。 度與上述之共聚物與水膠具有下臨界溶液溫 為抗生物& 溫度。此外’上述之絲倾水膠可作201122003 VI. Description of the Invention: TECHNICAL FIELD OF THE INVENTION The present invention relates to an anti-biofouling copolymer, and more particularly to an anti-biofouling nonionic-zwitterionic random alignment copolymer. [Prior Art] In recent years, zwitterionic polymers have become anti-plasma protein-adsorbing functional groups [for example, phosphobetaine, sufobetaine, and carboxylbetaine]. A new generation of highly compatible blood compatible materials. Among them, the poly(sulfobetaine methacrylate; polySBMA) synthesis method is easy to attract many researchers. Polythioglycine acrylate (polySBMA) is based on methacrylate and a pendant group similar to taurine betaine «Additional heat-sensitive non-ion The type of polymer, in an aqueous environment, at a specific temperature, undergoes a hydrophilic-hydrophobic property conversion, which is called a lower critical solution temperature (LCST). Among them, 'poly(N-isopropylacrylamide); polyNIPAAm is the most widely used heat-sensitive polymer. However, the nonionic polymer and the zwitterionic polymer have different properties in an aqueous solution. Generally, the 'sexionic polymer has an upper critical solution temperature (UCST), for example, a polythio beet acrylate (polySBMA), and the upper critical solution temperature will be higher with the zwitter ion 201122003. The molecular concentration increases and increases. Recent studies have revealed that diblock copolymers formed by polymerization of heat-sensitive monomers and zwitterionic monomers have microcell material properties. Further, the above diblock copolymer may have both an upper critical solution temperature and a lower critical solution temperature. However, the feasibility of applying it to biochemical fields or biomedical materials has not been discussed in these studies. In fact, the above-mentioned diblock copolymers have potential risks in the application of biomedical materials. The above diblock copolymer comprises an IJCST block (amphoteric ion) and an LCST block (thermal induction). When the diblock copolymer is formed into a microcell in an aqueous solution, the temperature of the aqueous solution is changed, and the inner block of the microcell and the external segment can be easily converted. However, the LCST block (thermal induction) (e.g., polyisopropyl acrylamide; polyNIPAAm) has high protein, cellular, and bacterial adsorption. When the change in ambient temperature causes the external cells to turn into LCST blocks, the strong adsorption of biomolecules on the surface of the cells rapidly occurs, causing irreversible aggregation or blockage. Therefore, the use of diblock copolymers on blood compatible materials is quite dangerous. # Due to the polymerization of the above-mentioned heat-sensitive monomer and zwitterionic monomer, the block copolymer may cause irreversible aggregation and blocking in the application of biomedical materials. Therefore, it is still necessary to develop a new biochemical field. Copolymer used. SUMMARY OF THE INVENTION In view of the above-described background of the invention, in order to comply with industrial requirements, the present invention provides a novel copolymer having heat and biofouling and a water gel. According to the above object, the present invention provides an anti-biofouling nonionic 蜇- 4 201122003 non-ionic monomer and amphoteric-free random copolymer are polymerized by a sub-type monomer. Zwitterionic Bribery - Anti-biofouling_Subtype - Sex Ion Type = Random Arrangement Copolymer, which is formed by polymerizing a nonionic monomer, two monomers and a crosslinking agent. The copolymer with the above and the water gel have a lower critical solution temperature to be antibiotic & temperature. In addition, the above-mentioned silk water-repellent glue can be used as
材料、抑：Γ塗佈材料、可控制細胞吸附/脫附基質之塗層 P市J細胞生長之塗佈層材料或藥物載體材料。 【實施方式】 型共=明在此揭示—種抗生物結垢非離子型_兩性離子 \ 。為了能徹底地瞭解本發明，將在下列的描述中 ，詳盡的步驟及其組成。顯然地，本發明的施行並未限 ^於該領域之技藝者所熟習的特殊細節。另-方面，眾所 周头的組成或步驟並未描述於細節巾，以避免造成本發明 不必要之限制。本發明的較佳實施例會詳細描述如下，然 而除了這些詳細描述之外，本發明還可以廣泛地施行在其 他的實施例中，且本發明的範圍不受限定，其以之後的專 利範圍為準。 本發明之第一實施例揭露一種抗生物結垢非離子型_兩 性離子型無規排列共聚物(random copolymer，學理上亦稱 為statistical copolymer)，可由一非離子型單體與一兩性離 子型單體進行一般習知的自由基聚合反應或原子轉移自由 基聚合反應聚合而成。 其中上述非離子型單體可包含下列族群中之一者或其 201122003 任意組合：N-烧基丙烯醢胺(N-alkylacrylamide)、N，N-二烧 基丙酸胺 （N，N-dialkylacrylamide)、雙丙酮丙酸胺 (diacetone acrylamide)、N-丙稀酿0比洛烧 (N-Materials, sputum coating materials, coatings capable of controlling cell adsorption/desorption matrix, coating layer materials or drug carrier materials for P-cell growth. [Embodiment] The type of total = Ming disclosed here is an anti-biofouling non-ionic type zwitterion \ . In order to fully understand the present invention, in the following description, detailed steps and compositions thereof. It will be apparent that the practice of the invention is not limited to the specific details that are apparent to those skilled in the art. In other instances, the composition or steps of the subject matter are not described in detail to avoid unnecessarily limiting the invention. The preferred embodiments of the present invention are described in detail below, but the present invention may be widely practiced in other embodiments, and the scope of the present invention is not limited by the scope of the following patents. . A first embodiment of the present invention discloses an anti-biofouling nonionic zwitterionic random copolymer, also known as a statistical copolymer, which can be composed of a nonionic monomer and a zwitterionic type. The monomer is polymerized by a conventional radical polymerization or atom transfer radical polymerization. Wherein the above nonionic monomer may comprise one of the following groups or any combination thereof in 201122003: N-alkylacrylamide, N,N-dialkylacrylamide ), diacetone acrylamide, N-propanol 0 洛洛(N-
acryloylpyrrolidine)、醋酸乙烯（vinyl acetate)、苯乙稀 (styrene)、乙稀啼^^(N-vinylimidazoline)、經烧基纖維素 (hydroxy alkyl celluloses)、聚嗯唾烧酮（polyoxazolidones)、 聚乙稀甲基®€ (polyvinyl methyl ethers)、聚環氧乙烧 (polyethylene oxide)、聚甲基丙稀酸(polymethacrylic acid)、 甲基丙稀酸二甲氨基乙醋[dimethylaminoethyl methacrylate(DMAEMA)]以及其衍生物。 其中上述之兩性離子型單體包含兩性離子官能基，而上 述之兩性離子官能基包含下列族群中之一者或其任意組 合：麟酸酯甜菜驗（phosphobetaine)、硫代甜菜驗 (sufobetaine)、叛基甜菜驗(carboxylbetaine)以及其衍生物。 於一範例中，上述之兩性離子型單體係為一混合電荷型 單體（mix-charged monomers)，其包含混合兩種電性相反 的化合物，且該混合電荷型單體整體而言係為電中性。部 分帶正電荷化合物如表一所示： 表一： [S] 6 201122003Acryloylpyrrolidine), vinyl acetate, styrene, N-vinylimidazoline, hydroxy alkyl celluloses, polyoxazolidones, polyethylidene Polyvinyl methyl ethers, polyethylene oxide, polymethacrylic acid, dimethylaminoethyl methacrylate (DMAEMA), and Its derivatives. Wherein the above zwitterionic monomer comprises a zwitterionic functional group, and the zwitterionic functional group comprises one of the following groups or any combination thereof: a phosphobetaine, a sufobetaine, Carboxyl betaine and its derivatives. In one example, the zwitterionic single system is a mixed-charged monomer comprising two compounds having opposite electrical properties, and the mixed charge monomer is Electrically neutral. Some of the positively charged compounds are shown in Table 1: Table 1: [S] 6 201122003
2-(二甲胺基）乙基甲基丙烯酸酯 2-(dimethylamino)ethyl methacrylate 2-(二乙胺基）乙基曱基丙烯酸酯 2-(diethylamino)ethyl methacrylate 2-曱基丙烯醯氧乙基三甲基氣化 錄 [2-(methacryloyloxy)ethyl]trimethylam monium chloride2-(dimethylamino)ethyl methacrylate 2-(diethylamino)ethyl methacrylate 2-(diethylamino)ethyl methacrylate 2-mercaptopropene oxime 2-(methacryloyloxyethyl)trimethylam monium chloride
〇 h2c^J\〇 CH, 〇H2C^^、 CH, CH3—N—CH3 C1_ ch3 部分帶負電荷化合物如表二所示： 表二： 2-羧乙基丙烯酸 2-carboxyethyl acrylate cj ) 〇 3-磺酸丙基曱基丙烯酸鉀 3-sulfopropyl methacrylate potassium salt h2c) c 0 0 r^o八，0K 〇 ：h3 乙二醇甲基丙烯酸鉀 ethylene glycol methacrylate phosphate h2c> c 0 1 :h3 oh 201122003 於另一範例中，上述之非離子型單體與兩性離子型單體 可皆為丙烯類單體。 範例一 製備硫代甜菜鹼丙烯酸酯與異丙基丙烯醢胺共聚物 [poly(SBMA-co-NIPAAm)] 取不同重量比例的異丙基丙烯醯胺(NIPAAm)與硫代甜 菜鹼丙浠酸醋（SBMA) ’總重為8 wt%，溶於1〇.2 ml去離 子水中，並同時通入氮氣以去除殘餘氧氣。加入8 0mg起 始劑（Ammonium peroxodisulfate; APS)與 8.0 mg (0.011 mL)促進劑(TEMED)，使聚合反應開始，並於23〇c氮氣環 境下反應6 hr。於4°C冷卻3 hr後，緩緩加入丙酮。接著， 將產物溶於去離子水中，使聚合物沈漱析出，去除雜質。 最後’將產物至於23°C烘箱中乾燥，獲得白色粉末。 表三、硫代甜菜鹼丙烯酸酯（SBMA)與異丙基丙烯醯胺 (NIPAAm)無規排列共聚物之基本資料表。 單體之反應比例共聚物成分(mol%)b 臨界溶液溫度(〇C)c (wt%)a _ 樣品代瑪 硫代甜 菜鹼丙 稀酸醋 (SBMA) 異丙基丙 烯醯胺 (NIPAAm) 聚硫代甜 菜鹼丙烯 酸 酯 (polySBMA) 聚異丙基丙 稀酿胺 (polyNIPAAm) 上臨界溶 液溫度 (UCST) 下臨界溶 液溫度 (LCST) S100-N0 100 0 100.0 0.0 27 - S70-N30 70 30 45.3 54.7 18 S50-N50 50 50 29.0 71.0 15 41 S30-N70 30 70 15.0 85.0 - 37 S0-N100 0 100 0.0 100.0 32 201122003 硫代甜菜驗丙烯酸酯（SBMA)單體與異丙基丙烯醯胺 (NIPAAm)單體之反應重量比，且總重為〇 8 g。 硫代甜菜鹼丙烯酸酯與異丙基丙烯酿胺共聚物 [poly(SBMA-co-NIPAAm)]之組成比例係由iH NMR所鑑 定’聚硫代甜菜驗丙烯酸酯（polySBMA)之(CH3)2N+氫 原子共振位置δ=3.2 ppm，聚異丙基丙烯醯胺（p〇iyNIpAAm) 異丙基之甲基氫原子共振位置§= 1.14 ppm。 c下臨界溶液溫度與上臨界溶液溫度係由23〇nm紫外可 見光譜定出。 如第二圖所示’聚硫代甜菜鹼丙烯酸酯（p〇lySBMA)僅 具有上臨界溶液溫度[第二團線(a)]，而聚異丙基丙烯醯胺 (polyNIPAAm)僅具有下臨界溶液溫度[第二圖線⑷]。當硫 代甜菜鹼丙烯酸酯（SBMA)與異丙基丙烯醯胺(NIPAAm)反 應比例(wt%)接近1 : 1時’共聚物同時具有上臨界溶液溫 度與下臨界溶液溫度[第二圖線(c)]。隨著非離子型單體比 例增加，共聚物之臨界溶液溫度改變。 比較第二圖中線(a)與線(b)，可知當非離子型單體與兩 性離子型單體反應比例（wt%)大於〇小於1時，隨著非離子 型單體比例增加，上臨界溶液溫度改變。 比較第二圖中線(d)與線(e)，可知當非離子型單體與兩 性離子型單體反應比例(wt°/〇)大於1時，隨著非離子型單體 比例增加，下臨界溶液溫度改變。 上述之共聚物之詳細資料可參見 “Dual-Thermoresponsive Phase Behavior of Blood Compatible Zwitterionic Copolymers Containing Nonionic 201122003〇h2c^J\〇CH, 〇H2C^^, CH, CH3—N—CH3 C1_ ch3 Part of the negatively charged compound is shown in Table 2: Table 2: 2-carboxyethyl acrylate cj ) 〇3 -sulfopropyl methacrylate potassium salt h2c) c 0 0 r^o 八,0K 〇:h3 ethylene glycol methacrylate methacrylate phosphate h2c> c 0 1 :h3 oh 201122003 In another example, the nonionic monomer and the zwitterionic monomer may all be propylene monomers. Example 1 Preparation of thiobetaine acrylate and isopropyl acrylamide copolymer [poly(SBMA-co-NIPAAm)] Isopropyl acrylamide (NIPAAm) and thiobetaine propionate in different weight ratios Vinegar (SBMA) 'Total weight is 8 wt%, dissolved in 1 〇. 2 ml of deionized water, and nitrogen is also introduced to remove residual oxygen. 80 mg of initiator (Ammonium peroxodisulfate; APS) and 8.0 mg (0.011 mL) of accelerator (TEMED) were added to start the polymerization and reacted for 6 hr under a nitrogen atmosphere of 23 ° C. After cooling at 4 ° C for 3 hr, acetone was slowly added. Next, the product is dissolved in deionized water to precipitate the polymer and remove impurities. Finally, the product was dried in an oven at 23 ° C to obtain a white powder. Table 3. Basic data sheets for random alignment copolymers of thiobetaine acrylate (SBMA) and isopropyl acrylamide (NIPAAm). Reaction ratio of monomer Copolymer component (mol%) b Critical solution temperature (〇C)c (wt%)a _ Sample methalin betaine acrylic acid vinegar (SBMA) Isopropyl acrylamide (NIPAAm) Polythiobetaine acrylate (polySBMA) polyisopropylacrylamide (polyNIPAAm) Upper critical solution temperature (UCST) Lower critical solution temperature (LCST) S100-N0 100 0 100.0 0.0 27 - S70-N30 70 30 45.3 54.7 18 S50-N50 50 50 29.0 71.0 15 41 S30-N70 30 70 15.0 85.0 - 37 S0-N100 0 100 0.0 100.0 32 201122003 Sulfur beet acrylate (SBMA) monomer and isopropyl acrylamide (NIPAAm) The reaction weight ratio of the monomers, and the total weight is 〇8 g. The composition ratio of thiobetaine acrylate to isopropyl acrylamide copolymer [poly(SBMA-co-NIPAAm)] was identified by iH NMR 'polysulfide beet acrylate (polySBMA) (CH3) 2N+ Hydrogen atom resonance position δ = 3.2 ppm, polyisopropyl acrylamide (p〇iyNIpAAm) isopropyl group methyl hydrogen atom resonance position § = 1.14 ppm. The critical solution temperature and the upper critical solution temperature are determined from the UV visible spectrum of 23 〇 nm. As shown in the second figure, 'polythiobetaine acrylate (p〇lySBMA) has only the upper critical solution temperature [second group line (a)], while polyisopropyl acrylamide (polyNIPAAm) has only the lower limit. Solution temperature [second line (4)]. When the reaction ratio (wt%) of thiobetaine acrylate (SBMA) to isopropyl acrylamide (NIPAAm) is close to 1:1, the copolymer has both the upper critical solution temperature and the lower critical solution temperature [second line] (c)]. As the proportion of nonionic monomer increases, the critical solution temperature of the copolymer changes. Comparing the line (a) and the line (b) in the second figure, it is understood that when the ratio (wt%) of the nonionic monomer to the zwitterionic monomer is greater than 〇 less than 1, as the proportion of the nonionic monomer increases, The temperature of the upper critical solution changes. Comparing the line (d) and the line (e) in the second figure, it can be seen that when the reaction ratio (wt°/〇) of the nonionic monomer to the zwitterionic monomer is greater than 1, as the proportion of the nonionic monomer increases, The temperature of the lower critical solution changes. For details of the above copolymers, see "Dual-Thermoresponsive Phase Behavior of Blood Compatible Zwitterionic Copolymers Containing Nonionic 201122003.
Poly(N-isopropyl acrylamide)” Biomacromolecules 2009, 1〇, 2092 - 2100, by Yung Chang et al.。 如第1A圖、第IB圖所示，當共聚物濃度5 wt%時， S50-N50於溫度為15°C至41°C之間係為一共聚物水溶 液’但是當溫度小於15°C或大於41°C時，共聚物不溶解 水溶液中[線(c)]。也就是說，當環境溫度大於共聚物的上 臨界溶液溫度，且低於下臨界溶液溫度時，共聚物可溶於 水中；相對地’如果環境溫度超過這個範圍，則共聚物沈 澱析出，其原因係由分子間與分子内的靜電作用力作用， 造成共聚物具有上臨界溶液溫度與下臨界溶液溫度。 上述之共聚物係為血液相容性材料，具有抗蛋白質、抗 細菌、抗細胞吸附特性如第四圖所示，在37°C環境下，表 面塗佈S30-N70、S50-N50、S70-N30或聚硫代甜菜鹼丙 烯酸酯（polySBMA)之材料對人類血纖蛋白原與人類血漿蛋 白具有強烈抗非特定吸附性質。然而表面塗佈疏水性官能 基（CH3-SAMs)或聚異丙基丙稀醢胺（p〇lyNIPAAm)之材 料，具有高蛋白質吸附力。 上述吸附實驗之詳鈿資料可參見 “Dual-Thermoresponsive Phase Behavior of Blood Compatible Zwitterionic Copolymers Containing Nonionic Poly(N-isopropyl acrylamide)” Biomacromolecules 2009, 10, 2092 - 2100, by Yung Chang et al.。另外，某些共聚物特性 與實驗數據的詳細資料亦揭露於上述文獻中。 上述之共聚物可作為抗生物結垢塗佈材料；或是，作為 控制細胞吸附/脫附基質之塗層材料，以快速分離培養細胞 與基質；或是，作為抑制細胞生長、細胞保存之塗佈層材 201122003 料。 另外，具有上臨界溶液溫度之共聚物可作為高分子藥物 共聚(polymer-dnig conjugate)之載體材料。當溫度低於載體 材料的上臨界溶液溫度(UCST)，載體材料係為凝膠態，且 藥物可欲入於載體材料兩性離子鏈段聚集（zwitteH〇nic segment association)。當要使用時，溫度高於上臨界溶液溫 度時’兩性離子鍵段聚集分散，使藥物釋放。 類似地，具有下臨界溶液溫度之共聚物亦可作為作為高 分子藥物共聚(polymer-drug conjugate)之載體材料。當溫 度高於下臨界溶液溫度(LCST)，載體材料係為凝勝態，且 藥物可嵌入於載體材料之非離子鏈段聚集（n〇ni〇nic segment association)。當溫度低於下臨界溶液溫度時，要使 用時，非離子鏈段聚集分散，使藥物釋放。 本發明之第二實施例揭露一種抗生物結垢非離子型-兩 性離子型水膠。上述之抗生物結垢非離子型-兩性離子型水 膠係為無規排列共聚物，可由非離子型單體、兩性離子型 單體與交聯劑’進行一般習知之自由基聚合反應或原子轉 移自由基聚合反應聚合而成。 上述之非離子型單體與兩性離子型單體的選擇如第一 實施例所示。 此外，上述之非離子型單體與兩性離子型單體可皆為丙 烯類單體。上述之交聯劑包含至少兩個烯基團（alkene groups) 0 範例二 製備硫代甜菜驗丙稀酸醋與異丙基丙稀酿胺水膠共聚物 201122003 [poly(SBMA-co-NIPAAm) hydrogels] 參考表4，對硫代甜菜鹼丙烯酸酯（20, 50, 70與100 wt %)、異丙基丙烯醯胺與交聯劑(BIS)進行化學自由基反應， 可得到硫代甜菜鹼丙烯酸酯與異丙基丙烯醯胺水膠共聚物 Poly(NIPAAm-co-SBMA)。其反應步驟如下：首先，將硫代 '* 甜菜鹼丙烯酸酯、異丙基丙烯醯胺與交聯劑（BIS)溶於水 - 中。接著，添加起始劑，起始劑的雙碳鍵被自由基反應打 斷產生自由基，促使聚合反應進行。 表四、水膠之聚合反應條件表 水膠 反應溶液組成（wt%) 重量平均 膨脹率 (w/w) 異丙基丙稀 醯胺 (NIPAAm) 硫代甜 菜鹼丙 烯酸酯 (SBMA) 交聯劑 (BIS) 起始劑 (APS) 促進劑 (TEMED) 去離 子水 s#o 20 0 1.6 0.2 0.2 78 3.8 S#20 16 4 1.6 0.2 0.2 78 3.2 S#50 10 10 1.6 0.2 0.2 78 3.0 S#70 4 16 1.6 0.2 0.2 78 2.7 S#100 0 20 1.6 0.2 0.2 78 2.1 第六圖揭示，於37〇C環境下，水膠對人類蛋白質吸附 力與接觸角關係圖。當水膠包含20 wt °/〇以上硫代甜菜驗丙 稀酸醋（SBMA)時，人類蛋白質（HSA，fibrinogen and gamma globulin)吸附情形明顯下降。 第七圖顯示水膠於不同水溶液鹽度下之重量平均膨脹 度。當鹽度大於或等於0.1 Μ時，上述之水勝之重量平均 膨脹度大於單純以聚異丙基丙稀醢胺(polyNiPAAm)組成水 [S] 12 201122003 膠。此外，當水溶液鹽度大於或等於〇 i M時，非離子型 單體與兩性離子型單體反應重量比例為〇至丨時，隨著鹽 度增加，重量平均膨脹度增加。 第八圖為放大倍率l，〇〇〇X之掃瞄式電子顯微鏡圖。共 聚物水膠與聚異丙基丙烯酿胺(polyNIPAArn)水膠分別於37 °C下與富含血小板血漿溶液（由人類全血製備）接觸12〇分 鐘。與polyNIPAArn比較，共聚物水膠明顯抑制血小板吸 附情形。明顯地，血小板遍佈在p〇lyNIPAAm水膠上，說 明polyNIPAArn對血小板的強烈吸附力；當使用s#2〇共聚 物時，仍有微量血小板吸附於共聚物表面上；當共聚物之 硫代甜菜鹼丙烯酸酯（SBMA)含量大於31 mol %時，具有較 佳的抗血小板吸附力；當使用S#50與S#70共聚物水膠時， 幾乎沒有血小板沾黏現象。 另外，於37°C環境將纖維母細胞(HS 68)於聚苯乙稀組 織培養jiil(TCPS)，共聚物水膠(S#20, S#5〇, and S#70)與聚 硫代甜菜鹼丙烯酸酯（poly SBMA)水膠（S# 100)表面培養3 天，並以具有數位相機(NIKON CCD)之顯微鏡觀察。第九 圖為放大倍率100倍之顯微鏡觀察圖，將水膠表面與聚苯 乙烯表面比較，纖維母細胞大量吸附分散於整個聚苯乙；^ 表面上並形成合流狀結構層（confluent-like layer)。 第十圖顯示於37°C環境使用表4中5種水膠與TCPS plate表面培養纖維母細胞1至8天，其細胞數目與時間關 係圖。當共聚物之硫代甜菜鹼丙烯酸酯（SBMA)含量大於3j mol %時，即使進行細胞培養8天’依然沒有觀察到細胞生 長之情況。另外，當溫度小於25°C，異丙基丙烯醯胺 (polyNIPAArn)鏈段聚集消失，水膠表面轉為親水性，可脫 13 201122003 附細胞（參考第六圖S#20、S#50)。很有趣的現象是，當 溫度由37°C改變為25°C，添加硫代甜菜鹼丙烯酸酯 (SBMA)於異丙基丙烯醯胺(polyNIPAAm)水膠（如S#20)， 增加了細胞生長表面之水合作用，並加快了細胞脫附速 度。於37°C環境下，值得注意的是添加硫代甜菜驗丙烯酸 酯（SBMA)於共聚物中，明顯抑制細胞生長與沾黏^基於上 述之結果’抗生物結垢非離子型-兩性離子型水膠，可作為 控制細胞吸附/脫附基質之塗層材料，以快速分離培養細胞 與基質。 如第十一圖、第十二圖所示，以革蘭氏陽性葡萄球菌 (Gram-positive S. epidermidis)與革蘭氏陰性菌種 (Gram-negative E.coli)兩種菌種，於370C環境下進行長時 間24 h培養實驗，探討表4中5種水膠共聚物表面之細菌 吸附性，並以24格聚苯乙烯組織培養皿(Tcps)作為對照 組，染劑使用Live/DeadBacLight™，再以螢光顯微鏡觀察 分析。聚苯乙烯組織培養皿(TCPS)表面菌種數目最多，可 能原因是革蘭氏陽性葡萄球菌細胞膜外具有蛋白質，而疏 水性的聚苯乙烯(TCPS)具有強烈蛋白質吸附性，推測TCPS 可能是先吸附蛋白質後，接續吸引更多革蘭氏陽性葡萄球 菌。在外觀上’革蘭氏陽性葡萄球菌與革蘭氏陰性菌種之 培養實驗外觀不相同。革蘭氏陽性葡萄球菌呈現薄膜狀， 而革蘭氏陰性菌種呈現網狀結構。實驗結果顯示，硫代甜 菜鹼丙烯酸酯與異丙基丙烯醯胺水膠共聚物 [poly(NIPAAm-CO-SBMA)]與聚硫代甜菜鹼丙烯酸酯 (polySBMA)水膠表面之細菌數目較少，而聚苯乙烯組織培 養皿(TCPS)與聚異丙基丙烯醯胺(p〇lyNIPAAm)表面之細 14 201122003 菌數目較多。 第六圖與第八至十二圖之詳細實驗步驟與數據可參考 wStimuli-Responsive and Tunable-Bioadhesive Hydrogels of Nonionic Poly(N-isopropyl acrylamide) Containing Zwitterionic Polysulfobetaine ” Yung Chang，目前尚在投 稿中。Poly(N-isopropyl acrylamide)" Biomacromolecules 2009, 1〇, 2092-2100, by Yung Chang et al. As shown in Figure 1A and Figure IB, when the copolymer concentration is 5 wt%, S50-N50 is at temperature. Between 15 ° C and 41 ° C is a copolymer aqueous solution 'but when the temperature is less than 15 ° C or greater than 41 ° C, the copolymer does not dissolve in the aqueous solution [line (c)]. That is, when the environment The temperature is greater than the upper critical solution temperature of the copolymer, and below the lower critical solution temperature, the copolymer is soluble in water; relatively 'if the ambient temperature exceeds this range, the copolymer precipitates due to intermolecular and molecular The internal electrostatic force acts to cause the copolymer to have an upper critical solution temperature and a lower critical solution temperature. The above copolymer is a blood compatible material having anti-protein, anti-bacterial and anti-cell adsorption properties as shown in the fourth figure. Surface-coated S30-N70, S50-N50, S70-N30 or polythiobetaine acrylate (polySBMA) materials are strongly resistant to human fibrinogen and human plasma proteins at 37 °C. Adsorption properties. However, the table A material coated with a hydrophobic functional group (CH3-SAMs) or polyisopropyl acrylamide (p〇lyNIPAAm) with high protein adsorption. For details of the above adsorption experiments, see “Dual-Thermoresponsive Phase Behavior”. Of Blood Compatible Zwitterionic Copolymers Containing Nonionic Poly(N-isopropyl acrylamide)" Biomacromolecules 2009, 10, 2092-2100, by Yung Chang et al. In addition, details of certain copolymer properties and experimental data are also disclosed in the above literature. The above copolymer can be used as an anti-biofouling coating material; or as a coating material for controlling a cell adsorption/desorption matrix to rapidly separate cells and a substrate; or, as a cell growth inhibiting, cell preservation Coating material 201122003. In addition, the copolymer with the upper critical solution temperature can be used as a carrier material for polymer-dnig conjugate. When the temperature is lower than the upper critical solution temperature (UCST) of the carrier material, the carrier material Is in a gel state, and the drug may be incorporated into the zwitterionic segment of the carrier material (zwitteH〇nic segment) Association) When used, when the temperature is higher than the temperature of the upper critical solution, the zwitterionic bond segments are aggregated and dispersed to release the drug. Similarly, copolymers having a lower critical solution temperature can also serve as a carrier material for polymer-drug conjugates. When the temperature is higher than the lower critical solution temperature (LCST), the carrier material is in a condensed state, and the drug can be embedded in the non-ionic segmental association of the carrier material. When the temperature is lower than the temperature of the lower critical solution, when used, the nonionic segments are aggregated and dispersed to release the drug. A second embodiment of the present invention discloses an anti-biofouling nonionic-zwitterionic water gel. The above-mentioned anti-biofouling nonionic-zwitterionic hydrocolloid is a randomly arranged copolymer which can be subjected to a conventional radical polymerization or atomization by a nonionic monomer, a zwitterionic monomer and a crosslinking agent. It is formed by polymerization of transfer radical polymerization. The selection of the above nonionic monomer and zwitterionic monomer is as shown in the first embodiment. Further, the above nonionic monomer and zwitterionic monomer may both be propylene monomers. The above cross-linking agent comprises at least two alkene groups. 0 Example 2 Preparation of thio beet acetoacetate and isopropyl propylene-hydrogel copolymer 201222003 [poly(SBMA-co-NIPAAm) Hydrogels] Refer to Table 4 for chemical radical reaction of thiobetaine acrylate (20, 50, 70 and 100 wt%), isopropyl acrylamide and crosslinker (BIS) to obtain thiobetaine. Acrylate and isopropyl acrylamide hydrogel copolymer Poly (NIPAAm-co-SBMA). The reaction steps are as follows: First, thio '* betaine acrylate, isopropyl acrylamide and a crosslinking agent (BIS) are dissolved in water. Next, an initiator is added, and the double carbon bond of the initiator is interrupted by a radical reaction to generate a radical, which causes the polymerization to proceed. Table 4, Polymerization Conditions of Water Glue Table Composition of Water Glue Reaction Solution (wt%) Weight Average Expansion Ratio (w/w) Isopropyl acrylamide (NIPAAm) Thiobetaine Acrylate (SBMA) Crosslinker (BIS) Starter (APS) Accelerator (TEMED) Deionized Water s#o 20 0 1.6 0.2 0.2 78 3.8 S#20 16 4 1.6 0.2 0.2 78 3.2 S#50 10 10 1.6 0.2 0.2 78 3.0 S#70 4 16 1.6 0.2 0.2 78 2.7 S#100 0 20 1.6 0.2 0.2 78 2.1 The sixth figure reveals the relationship between the adsorption force of human water and the contact angle of water gel at 37 °C. The adsorption of human proteins (HSA, fibrinogen and gamma globulin) was significantly reduced when the water gel contained thioacetate (SBMA) at 20 wt ° / 〇 or more. Figure 7 shows the average weight expansion of water gel at different aqueous salinities. When the salinity is greater than or equal to 0.1 Μ, the weight average expansion of the above water is greater than that of polyisopropyl amide (polyNiPAAm) [S] 12 201122003. Further, when the aqueous solution salinity is greater than or equal to 〇 i M , when the weight ratio of the nonionic monomer to the zwitterionic monomer is from 〇 to 丨, the weight average degree of expansion increases as the salinity increases. The eighth figure is a scanning electron microscope image of magnification l, 〇〇〇X. The copolymer water gel and polyisopropyl propylene amine (polyNIPAArn) water gel were contacted with a platelet-rich plasma solution (prepared from human whole blood) at 37 ° C for 12 〇 minutes. Compared to polyNIPAArn, copolymer hydrogel significantly inhibited platelet adsorption. Obviously, platelets are spread on p〇lyNIPAAm water gel, indicating the strong adsorption of polyNIPAArn on platelets; when using s#2〇 copolymer, there are still traces of platelets adsorbed on the surface of the copolymer; When the content of alkali acrylate (SBMA) is more than 31 mol%, it has better anti-platelet adsorption; when S#50 and S#70 copolymer water gel is used, there is almost no platelet adhesion. In addition, fibroblasts (HS 68) were cultured in polystyrene tissue at a temperature of 37 ° C. Jisil (TCPS), copolymer water gel (S#20, S#5〇, and S#70) and polythio Betaine acrylate (poly SBMA) water gel (S# 100) was surface cultured for 3 days and observed under a microscope with a digital camera (NIKON CCD). The ninth picture is a microscopic observation of the magnification of 100 times. The surface of the water gel is compared with the surface of the polystyrene. The fibroblasts are adsorbed and dispersed in a large amount on the surface of the polystyrene; and form a confluent-like layer. ). The tenth graph shows the use of five water gels and the surface of the TCPS plate to culture fibroblasts in Table 4 at 37 ° C for 1 to 8 days, and the number of cells versus time. When the thiobetaine acrylate (SBMA) content of the copolymer was more than 3 j mol%, cell growth was not observed even after cell culture for 8 days. In addition, when the temperature is less than 25 ° C, the aggregation of isopropyl acrylamide (polyNIPAArn) segments disappears, and the surface of the water gel changes to hydrophilic, which can be removed from the cells of 201122003 (refer to the sixth figure S#20, S#50) . It is interesting to note that when the temperature is changed from 37 ° C to 25 ° C, thiobetaine acrylate (SBMA) is added to isopropyl acrylamide (polyNIPAAm) water gel (eg S#20), which increases the cells. The hydration of the growing surface accelerates the rate of cell desorption. At 37 ° C, it is worth noting that the addition of thioacetate acrylate (SBMA) to the copolymer significantly inhibits cell growth and adhesion. Based on the above results, 'antibiotic scale nonionic-zwitterionic type Water gel can be used as a coating material for controlling cell adsorption/desorption matrix to rapidly separate cultured cells from matrix. As shown in the eleventh and twelfth figures, Gram-positive S. epidermidis and Gram-negative E. coli are used at 370C. The culture was carried out for 24 h in a long time to investigate the bacterial adsorption of the surface of the five hydrogel copolymers in Table 4. The 24 polystyrene tissue culture dishes (Tcps) were used as the control group, and the dye was used as Live/DeadBacLightTM. Then observe and analyze with a fluorescent microscope. The number of surface strains on polystyrene tissue culture dishes (TCPS) is the highest, probably because Gram-positive staphylococci have proteins outside the membrane, while hydrophobic polystyrene (TCPS) has strong protein adsorption. It is speculated that TCPS may be the first. After adsorbing the protein, it continues to attract more Gram-positive staphylococci. In appearance, the culture experiment of Gram-positive Staphylococcus and Gram-negative bacteria is different. Gram-positive staphylococci are film-like, while Gram-negative strains exhibit a network structure. The experimental results show that the number of bacteria on the surface of thiobetaine acrylate and isopropyl acrylamide hydrogel copolymer [poly(NIPAAm-CO-SBMA)] and polythiobetaine acrylate (polySBMA) hydrogel is less. However, the surface of polystyrene tissue culture dish (TCPS) and polyisopropyl acrylamide (p〇ly NIPAAm) is relatively large. Detailed experimental procedures and data for the sixth and eighth to twelfth drawings can be found in wStimuli-Responsive and Tunable-Bioadhesive Hydrogels of Nonionic Poly(N-isopropyl acrylamide) Containing Zwitterionic Polysulfobetaine ” Yung Chang, which is currently under submission.
上述之水膠共聚物可作為抗生物結垢塗佈材料；或是， 作為控制細胞吸附/脫附基質之塗層材料，以快速分離培養 細胞與基質；或是，作為抑制細胞生長、細胞保存之塗佈 層材料。 另外，具有上臨界溶液溫度之水膠共聚物可作為高分子 藥物共聚(polymer-drug conjugate)之載體材料。當溫度低於 載體材料的上臨界溶液溫度（UCST)，載體材料係為凝膠 態’且藥物可嵌入於載體材料兩性離子鍵段聚集 當要使用時，溫度高於 (zwitterionic segment association) 〇 上臨界溶液溫度時，兩性離子鏈段聚集分散，使藥物釋放。 此外，具有下臨界溶液溫度之水膠共聚物亦可作為作為 高分子藥物共聚(polymer-drug conjugate)之載體材料。當 溫度高於下臨界溶液溫度(LCST)，載體材料係為凝膠態备 且藥物可彼入於載體材料之非離子鏈段聚，（n〇ni〇nic segment association)。當溫度低於下臨界溶液溫度時要伯 用時，非離子鏈段聚集分散，使藥物釋放。 顯然地，依照上面實施例中的描述，本發明可能有奇 多的修正與差異。因此需要在其附加的權利要求項之範匡 内加以理解’除了上述詳細的描述外，本發明還可以廣 地在其他的實施例中施行。上述僅為本發明之較佳實施你 15 m 201122003 而已，並非用以限定本發明之申請專利範圍；凡其它未脫 離本發明所揭示之精神下所完成的等效改變或修飾，均應 包含在下述申請專利範圍内。 【圖式簡單說明】 第一圖顯示S50-N50共聚物在不同溫度下於水溶液中溶解 與不溶解特性模型示意圖。 第二圖係根據本發明之第一實施例，5 wt%之共聚物溶液 (a)S100-N0、（b)S70-N30、（c)S50-N50、（d)S30-N70 與 (e)S0-N100溫度與吸收度關係圖。 第三圖顯示聚硫代甜菜鹼丙烯酸酯（polySBMA)之上臨界溶 液溫度、S50-N50共聚物之上臨界溶液溫度與下臨界溶液 溫度以及聚異丙基丙稀醯胺(polyNIPAAm)之下臨界溶液溫 度。 第四圖係根據本發明之第一實施例，於37°C緩衝溶液（PBS buffer)環境下，表面接枝疏水性官能基(CH3-SAMs)、聚異 丙基丙稀醯胺(polyNIPAAm brushes)或聚硫代甜菜驗丙婦 酸醋(polySBMA brushes)以及表面塗佈 S30-N70、 S50-N50或S70-N50對1 mg/mL之纖維蛋白原與10%人類 血漿吸附度。 201122003 第五圖係根據本發明第二實施例，水膠之製備反應途徑示 意圖。 第六圖係根據本發明第二實施例，不同成分之水膠於37°C 環境下，溫度與人類蛋白質吸附度關係圖，以及溫度與接 觸角關係圖。 第七圖係根據本發明第二實施例，於不同之鹽度環境下， 水膠之重量平均膨脹度。 第八圖係根據本發明第二實施例，掃描式電子顯微鏡圖顯 示聚異丙基丙烯醯胺水膠(polyNIPAAm gel (S#0)、異丙基 丙烯醯胺與硫代甜菜鹼丙烯酸酯水膠共聚物 [poly(NIPAAm-co-SBMA) gels，S#20, S#50, S#70]以及聚硫 代甜菜鹼丙烯酸酯水膠（polySBMA gel (s# 100))之血小板 吸附程度。 第九圖係根據本發明第二實施例，於聚苯乙烯組織培養皿 (TCPS)，共聚物水膠(S#20，S#50，and S#7〇)與聚硫代甜菜 驗丙稀酸醋（P〇lySBMA)水膠(S#100)表面進行H68細胞培 養吸附實驗結果。 第十圖顯示於聚苯乙烯組織培養孤(TCPS)、聚異丙基丙烯 [S]. 17 201122003 醯胺(polyNIPAAm)水膠(S#0)、共聚物水膠(S#20, S#50, and S#70)與聚硫代甜菜鹼丙烯酸酯（p〇iySBMA)水膠（S# 100)對 H68細胞進行培養吸附實驗1天、3天、8天後，水膠材料 與細胞數關係圖，其中，最初細胞濃度為104cells/mL。 第十一圖係根據本發明第二實施例，於聚苯乙烯組織培養 皿(TCPS)、聚異丙基丙烯醯胺(polyNIPAAm)水膠(S#0)、共 聚物水膠(S#20, S#50, and S#70)與聚硫代甜菜鹼丙烯酸酯 (polySBMA)水膠（S#100)表面培養革籣氏陽性葡萄球菌 (Gram-positive S_ epidermidis)—天後之螢光顯微鏡圖。 第十二圖係根據本發明第二實施例，於聚苯乙烯組織培養 im(TCPS)、聚異丙基丙烯醯胺(polyNIPAAm)水膠(S#0)、共 聚物水膠(S#20, S#50, and S#70)與聚硫代甜菜鹼丙烯酸酯 (polySBMA)水膠(S# 100)表面培養革蘭氏陰性菌種(E. coli) 一天後之螢光顯微鏡圖。The above water-gel copolymer can be used as an anti-biofouling coating material; or, as a coating material for controlling a cell adsorption/desorption matrix, to rapidly separate cells and a substrate; or, as a cell growth inhibiting, cell preservation Coating layer material. In addition, a hydrogel copolymer having an upper critical solution temperature can be used as a carrier material for a polymer-drug conjugate. When the temperature is lower than the upper critical solution temperature (UCST) of the carrier material, the carrier material is in a gel state' and the drug can be embedded in the carrier material. The zwitterionic bond segment aggregates when used, the temperature is higher than (zwitterionic segment association) At the critical solution temperature, the zwitterionic segments are aggregated and dispersed to release the drug. Further, a hydrogel copolymer having a lower critical solution temperature can also be used as a carrier material for polymer-drug conjugate. When the temperature is higher than the lower critical solution temperature (LCST), the carrier material is in a gel state and the drug can be incorporated into the nonionic segment of the carrier material. When the temperature is lower than the temperature of the lower critical solution, the non-ionic segments are aggregated and dispersed to release the drug. Obviously, the present invention may have many modifications and differences in accordance with the description in the above embodiments. Therefore, it is to be understood that the invention may be practiced in other embodiments. The above is only the preferred embodiment of the present invention, which is not intended to limit the scope of the present invention; all other equivalent changes or modifications which are not departing from the spirit of the present invention should be included. Within the scope of the patent application. [Simple description of the diagram] The first figure shows a schematic diagram of the dissolution and insolubility characteristics of S50-N50 copolymer in aqueous solution at different temperatures. The second figure is a 5 wt% copolymer solution (a) S100-N0, (b) S70-N30, (c) S50-N50, (d) S30-N70 and (e) according to the first embodiment of the present invention. ) S0-N100 temperature and absorbance diagram. The third panel shows the critical solution temperature above polythiobetaine acrylate (polySBMA), the critical solution temperature above the S50-N50 copolymer and the lower critical solution temperature, and the criticality of polyisopropyl amide (polyNIPAAm). Solution temperature. The fourth figure is based on the first embodiment of the present invention, surface grafting hydrophobic functional groups (CH3-SAMs), polyisopropylammonium amide (polyNIPAAm brushes) in a 37 ° C buffer solution (PBS buffer) environment. Or polysulfone beet vinegar (polySBMA) and surface coated S30-N70, S50-N50 or S70-N50 to 1 mg/mL fibrinogen and 10% human plasma adsorption. 201122003 The fifth drawing is a schematic diagram of a reaction route for preparing a water gel according to a second embodiment of the present invention. Fig. 6 is a graph showing the relationship between temperature and human protein adsorption degree, and the relationship between temperature and contact angle of water-gel of different compositions in a 37 ° C environment according to a second embodiment of the present invention. The seventh figure is the weight average degree of expansion of the water gel in different salinity environments according to the second embodiment of the present invention. Figure 8 is a scanning electron micrograph showing polyisopropyl acrylamide hydrogel (polyNIPAAm gel (S#0), isopropyl acrylamide and thiobetaine acrylate water according to a second embodiment of the present invention. The degree of platelet adsorption of the gel copolymer [poly(NIPAAm-co-SBMA) gels, S#20, S#50, S#70] and polythiobetaine acrylate water gel (polySBMA gel (s# 100)). The ninth figure is a polypropylene polystyrene culture dish (TCPS), a copolymer water gel (S#20, S#50, and S#7〇) and a polythio beet according to a second embodiment of the present invention. The results of H68 cell culture adsorption experiments on the surface of acid vinegar (P〇lySBMA) water gel (S#100). The tenth figure shows the polystyrene tissue culture orphan (TCPS), polyisopropyl propylene [S]. 17 201122003 醯Amine (polyNIPAAm) water gel (S#0), copolymer water gel (S#20, S#50, and S#70) and polythiobetaine acrylate (p〇iySBMA) water gel (S# 100) H68 cells were cultured for adsorption experiments for 1 day, 3 days, and 8 days, and the relationship between the water gel material and the number of cells, wherein the initial cell concentration was 104 cells/mL. The eleventh figure is according to the second embodiment of the present invention, Styrene tissue culture dish (TCPS), polyisopropyl amide (polyNIPAAm) water gel (S#0), copolymer water gel (S#20, S#50, and S#70) and polythio beet a fluorescent micrograph of the base cultivar (S#100) surface culture leather Gram-positive S_ epidermidis - day after. The twelfth figure is according to the second embodiment of the present invention, In polystyrene tissue culture im (TCPS), polyisopropyl acrylamide (polyNIPAAm) water gel (S#0), copolymer water gel (S#20, S#50, and S#70) and polysulfide Fluorescence microscopy of a day after the growth of Gram-negative bacteria (E. coli) on the surface of a betaine acrylate (polySBMA) water gel (S# 100).
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