- 學位論文
雙離子性硫代甜菜鹼共聚高分子於生物醫學之應用
Zwitterionic Sulfobetaine-based Copolymers and their Biomedical Applications
摘要
研究血液相容性雙離子仿生高分子可分為三種類型：phosphobetaine、sulfobetaine 和 carboxybetaine，這類化學結構與生物體中組成細胞膜外壁的磷脂質 (phosphatidylcholine, PC)類似。其中Poly(sulfobetaine methacrylate) (polySBMA)具有taurine betaine pendent化學官能基，相較於其它雙離子高分子，此結構於合成高分子共聚物時，具有較易合成且較容易控制之特性。此外，於過去的研究和文獻中可得知，當雙離子性polySBMA刷狀高分子接枝於材料表面時，可表現出良好且穩定之抗生物沾黏特性，因此被視為可應用於人體血液接觸以及可植入人體的前瞻性生醫材料。因此，本研究將雙離子性polySBMA合成為新式的共聚高分子，並探討其對於生物醫學之應用，進行生物與血液相容性之探討。本論文分成三部份，擬設計出不同之polySBMA共聚高分子，並依應用分成刺激應答型高分子、智能型基因治療載體和自組裝抗生物沾黏薄膜。 本論文的第一部分為使用原子轉移自由基聚合法 (atom transfer radical polymerization, ATRP)製備雙離子型刺激應答型共聚高分子poly(N-isopropylacrylamide)-block-poly(sulfobetaine methacrylate) (PNIPAAm-b- PSBMA)，並探討其共聚高分子於水溶液中構形的改變與其在不同溫度下血液相容性的影響。在本研究中，由於PNIPAAm高分子鏈段具有低臨界溶解溫度 (lower critical solution temperature, LCST)和PSBMA高分子鏈段具有高臨界溶解溫度 (upper critical solution temperature, UCST)的性質，可透過核磁共振光譜儀 (nuclear magnetic resonance, NMR)與動態光散射儀 (dynamic light scattering, DLS)來探討PNIPAAm-b-PSBMA共聚高分子於水溶液中物理構形的變化。研究發現，PNIPAAm-b-PSBMA共聚高分子會隨著溫度變化，會產生分子鏈與微胞間的構形轉換，當溶液溫度由低溫升至高溫時，構形轉換會由以PSBMA分子鏈段包覆之微胞轉變為PNIPAAm-b- PSBMA線性高分子，再轉換為以PNIPAAm分子鏈段包覆之微胞。將PNIPAAm-b-PSBMA共聚高分子於不同溫度下進行其血液相容性質之探討，使用DLS觀察高分子與蛋白溶液接觸之吸附實驗，並進行高分子於人體血液中之凝血時間與溶血測試實驗。實驗結果顯示，PNIPAAm-b-PSBMA共聚高分子於4 ºC ~ 40 ºC的溫度範圍下均呈現良好的血液相容性，主要其功能展現是由於PNIPAAm-b-PSBMA共聚高分子在低溫或高溫時，其親水性的高分子鏈可以包覆另一端疏水性的高分子鏈，展現良好的抵抗血液分子吸附之特性。這表示具有新穎設計概念之PNIPAAm-b- PSBMA共聚高分子，將可應用於前瞻性與血液接觸之生醫材料設計。 本論文的第二部分為基因治療載體之設計，使用可逆加成鏈轉移聚合法(Reversible addition−fragmentation chain-transfer polymerization, RAFT) 可合成出三種不同鏈段比例之雙離子性PSBMA鏈段與酸鹼感應型PAA鏈段之PAA-b-PSBMA共聚高分子，使其與外層為正電性之PDMAEMA和內部包覆負電性之plasmid DNA之polyplexes結合，並觀察其基因轉殖效能、血液相容性和細胞毒性等特性。結果顯示，基因載體中最外層之雙離子鏈段PSBMA展現出雙離子遮蔽效應，因而無溶血現象發生，代表此基因載體並未造成紅血球破裂，同時並未發現加速凝血的情況發生，表示此基因載體具有良好的血液相容性質。本研究中，同時利用PAA高分子之酸鹼感應的特性，當pH值改變時，因PAA高分子鏈段為電中性而使得PAA-b-PSBMA共聚高分子與polyplexes外層為正電性之PDMAEMA 高分子鏈段間失去靜電作用力，使載體表面之共聚高分子脫附，進而誘發基因轉植。由細胞轉染結果中發現，當pH值改變時，PAA-b-PSBMA與Plasmid DNA/PDMAEMA結合製備而成的基因載體，表現出最佳的細胞轉染效果，並保有與控制組相當的細胞存活率，此兩步驟之基因載體設計，能有效的提高血液相容性與生物相容性，同時保有高效的轉質效率。 本論文的最後一部分，主要是利用多層自組裝的塗佈技術，將聚醚酰亞胺 (poly(ether imide), PEI)薄膜進行孔洞和厚度的有效控制，並使用塗佈技術將負電鏈段之雙離子性共聚高分子PAA-b-PSBMA固定於薄膜表面。改質後之薄膜，在良好控制PAA-b-PSBMA高分子之覆蓋率和接枝密度下，使用表面電位量測高分子薄膜表面之電性、掃描式電子顯微鏡觀察薄膜之結構和蛋白質連續循環蛋白過濾測試其抗生物沾黏之特性。在論文的最後一個章節中，延伸離子性/雙離子特性之 PAA-b-PSBMA 共聚高分子於製備抗沾黏之PEI薄膜，使用多層自組裝的塗佈技術能有效的控制其奈米孔洞結構與水過濾通量，而薄膜表面之表面電性可藉由調控PEI/PAA之比例達到良好的控制效果，由牛血清蛋白 (bovine serum albumin, BSA)與纖維蛋白原 (fibrinogen)過濾測試中，低蛋白質沾黏薄膜於最佳之PAA與PSBMA鏈段比例下表現高效能的抗沾黏特性，且其結果比未經PAA-b-PSBMA 共聚高分子塗佈處理之薄膜佳。當PAA-b-PSBMA 共聚高分子塗佈於帶有正電性之PEI薄膜表面，PAA-b-PSBMA 共聚高分子中PAA之重複鏈段越長時，具有越高的蛋白質吸附，若再次吸附具有短分子鏈段PAA之PAA-b-PSBMA 共聚高分子時，則可達到最優化的抗沾黏效果。在此論文中，發現含有陰離子基團高分子鏈段之雙離子性共聚高分子，可透過靜電作用力塗佈於帶正電性之薄膜時，若表面具有高接枝密度雙離子性基團，可製備出具有高效能抗蛋白質沾黏之薄膜系統。
並列摘要
Biomimetic polymers containing zwitterionic structures similar to phosphatidylcholine, such as phosphobetaine, sulfobetaine, and carboxybetaine, have received growing attention for use in a new generation of blood-contacting materials because of their good plasma-protein-fouling resistance. Poly(sulfobetaine methacrylate) (polySBMA), with a methacrylate main chain and an analogue of the taurine betaine pendent group (CH2CH2N+(CH3)2CH2CH2CH2SO3-), has become the most widely studied zwitterionic polymer due to its ease of synthetic preparation. It is now recognized that grafted dense polymer brushes, composed of zwitterionic polySBMA, formed an effective and stable nonfouling surface, potentially enabling the practical use in human blood-contacting devices and implants. In this dissertation, the new functionalization of zwitterionic sulfobetaine-based copolymers and their potential biomedical applications was developed and investigated. In the first part of the dissertation, “schizophrenic” diblock copolymers containing nonionic and zwitterionic blocks were prepared with well-controlled molecular weights via the atom-transfer radical polymerization (ATRP). In this work, we demonstrate a systematic study of how morphological changes of poly(N-isopropylacrylamide)-block-poly(sulfobetaine methacrylate) (PNIPAAm-b- PSBMA) copolymers affect hemocompatibility in the human blood solution. The “schizophrenic” behavior of PNIPAAm-b-PSBMA was observed by 1H nuclear magnetic resonance (1H NMR), dynamic light scattering (DLS), and turbidity measurement with double morphological transition, exhibiting both lower critical solution temperature (LCST) and upper critical solution temperature (UCST) in aqueous solution. Below the UCST of PSBMA block, micelles were obtained with a core of insoluble PSBMA association and a shell of soluble PNIPAAm whereas the opposite micelle structure was observed above the LCST of PNIPAAm block. In between the UCST and LCST, unimers with both soluble blocks were detected. The hydrodynamic size of prepared polymers and copolymers are determined to illustrate the correlations between intermolecular nonionic/zwitterionic associations and blood compatibility of PNIPAAm, PNIPAAm-b-PSBMA, and PSBMA suspension in human blood. Human fibrinogen adsorption onto the PNIPAAm-b-PSBMA copolymers from single-protein solutions was measured by DLS to determine the nonfouling stability of copolymer suspension. The new nonfouling nature of PNIPAAm-b-PSBMA copolymers was demonstrated to show an extremely high anticoagulant activity and antihemolytic activity in human blood over a wide range of explored temperatures from 4 ºC to 40 ºC. The temperature-independent blood compatibility of the nonionic/zwitterionic block copolymer along with their schizophrenic phase behavior in aqueous solution suggests their potential in blood-contacting applications. In the second part of the dissertation, “intelligent” diblock copolymers containing ionic and zwitterionic blocks were prepared with well-controlled molecular weights via reversible the addition-fragmentation chain transfer (RAFT) polymerization. In this work, we demonstrate a systematic study of how zwitterionic shielding on a new self-assembled cargo of plasmid DNA/poly(dimethylaminoethyl methacrylate) (PDMAEMA) polyplexes conjugated with poly(acrylic acid)-block-poly(sulfobetaine methacrylate) (PAA-b-PSBMA) copolymers affect hemocompatibility in human blood solution and gene transfection onto target cells. The carrier stability, cell toxicity, hemocompatibility, and gene transfection efficiency of DNA/ PDMAEMA/PAA-b-PSBMA polyplexes was investigated as compared with DNA/polyethyleneimine (PEI)/PAA-b-PSBMA. The binding capability of plasmid DNA with PEI, PDMAEMA, and PAA-b-PSBMA were measured by ethidium bromide displacement assays and agarose gel retardation assays at different desired number of polymer nitrogen atoms per nucleotide phosphate (N/P ratio) and solution pH. Hemocompatibility of the prepared polyplexes was evaluated by the anticoagulant activity of the blood coagulant determined by testing the plasma-clotting time and the antihemolytic activity in human blood by measuring red blood-cell hemolysis. The zwitterionic shielding of PAA-b-PSBMA copolymers self-assembled on the shell of DNA/ PDMAEMA polyplexes was demonstrated to show extremely high anticoagulant activity and antihemolytic activity in human blood over a wide range of N/P ratio from 1 to 40. The pH-dependent gene transfection of DNA/ PDMAEMA/PAA-b-PSBMA polyplexes, along with their stimuli-responsive behavior, suggests their potential in blood-contacting gene delivery applications. In the final part of the dissertation, ionic/zwitterionic PAA-b-PSBMA diblock copolymer was extended to prepare anti-fouling poly(ether imide) membranes with well-controlled nano-pore structures and water flux via layer-by-layer self-assembled coating process. The surface charge property of the prepared membrane can be regulated by PEI/PAA ratios. Low protein-fouling membrane surfaces from BSA and fibrinogen solution with respect to uncoated membrane surfaces are achieved with optimized block ratio of PAA and PSBMA. Importantly, the PAA-b-PSBMA copolymer with a longer PAA block weight has high protein adsorption after direct adsorption onto a charged poly(ether imide) surface, but this can be reduced to a very low fouling level after the surface is back-filled with a copolymer with a shorter PAA block. Thus, positively charged membranes, coated with zwitterionic copolymers containing anionic groups via charge-driven, are ideal for highly resisting protein adsorption if the membrane surface density of the zwitterionic groups is controlled to a high-level densities.