雙離子偏差型界面與薄膜之血液相容性控制

Hemocompatible Control of Zwitterionic-bias Interfaces and Membranes

陳聖翰(Sheng-Han Chen)

指導教授 ： 張雍 ； 李魁然

中原大學/工學院/化學工程研究所/博士(2015年)

https://doi.org/10.6840/CYCU.2015.00143

摘要

在血液相容性材料的研究領域中，目前最受到重視的仿生雙離子高分子具有取代傳統血液接觸材料的潛力，藉由雙離子性分子的化學結構類似於細胞膜外壁的磷脂質，利用仿生的概念展現其生物相容性，經由許多研究證實雙離子高分子對於生物分子如蛋白質、細菌、細胞及藻類都具有抗生物分子沾粘之特性。重要的是藉由雙離子結構所形成之水合層可在抵抗血液成份(如血漿蛋白與血球)的貼附與活化上發揮其功能性，充分地展現出其完美的血液惰性，為未來前瞻性血液接觸材料的發展開創出新的方向。一般而言，仿生雙離子性高分子又可區分為以下三大類，phosphobetaine (PB)、sulfobetaine (SB)和carboxybetaine (CB)，本研究論文系統針對SB高分子材料，深入探討與人體血液接觸界面之相容性質，透過分子設計、電性偏差控制與表面改質技術剖析及觀察SB材料界面與人體各種血球之間的作用力，並找出可控制血液相容性的特定結構與組成範圍，期望發展出以雙離子性高分子結構體為基礎的功能性血液過濾材料。本論文主要分為兩大材料系統: (1) 雙離子結構體系統- 以雙離子性PB與SB高分子所具備的電中性化學結構為主，並與疏水基團結合製備成不同類型的共聚高分子，與使用自組裝物理性吸附塗佈達到材料表面的雙離子化，並評估其血液相容性。此外，本研究中亦引入電漿誘導表面起始聚合反應，以化學接枝方式來探討雙離子化改質與控制; (2) 雙離子電荷偏差結構體系統- 調整雙離子性高分子的電性偏差，藉由水凝膠製備與電漿改質技術導入雙離子電荷偏差結構體的概念，並藉由材料表面所帶有不同電荷偏差程度控制特定血球優先貼附之順序，並將電荷偏差特性應用於血球分離效能的分析。
本論文的第一部分是研究雙離子性材料之血液相容性，選擇雙離子單體材料2-methacryloyloxyethyl phosphorylcholine (MPC)、[3-(methacryloylamino)propyl] dimethyl(3-sulfopropyl)ammonium hydroxide inner salt (SBAA)與親水性材料2-hydroxyethyl methacrylate (HEMA)，結合疏水性鏈段n-butyl methacrylate (BMA)，分別使用熱起始自由基聚合法與逆加成斷裂鏈轉移聚合法(RAFT)來合成製備出隨機與雙嵌段共聚高分子，並藉由物理性浸泡塗佈方式將共聚高分子吸附於基材，使疏水性聚丙烯(PP)平板薄膜表面達到雙離子化改質。透過化學合成poly(2-methacryloyloxyethyl phosphorylcholine)-co-poly(n-butyl methacrylate) (PMPC-co-PBMA), poly(sulfobetaine acrylamide)-co-poly(n-butyl methacrylate) (PSBAA-co-PBMA), 與poly(2-hydroxyethyl methacrylate)-co-poly(n-butyl methacrylate) (PHEMA-co-PBMA)共聚高分子的親疏水鏈段比例可控制改質後PP平板薄膜的表面親水性與抵抗蛋白質吸附的能力，更進一步有系統地探討PMPC-co-PBMA, PSBAA-co-PBMA及PHEMA-co-PBMA共聚高分子對人體血液相容性的影響。經由雙離子共聚高分子塗佈改質後的PP表面可有效地降低血漿蛋白的吸附、血小板的貼附、白血球及全血細胞的貼附，本研究證實結合雙離子共聚高分子的分子設計與自組裝塗佈程序可大幅地改善材料的人體血液相容性。此外，利用電漿誘導接枝聚合反應可將聚硫代甜菜鹼高分子(PSBMA)接枝於PP纖維薄膜，成功地達到化學性的雙離子化改質，藉由調整低壓與常壓電漿之操作參數可控制雙離子PSBMA接枝層的電荷平衡、接枝鏈段的構型、表面親水性與水合能力，研究中使用酵素連結免疫吸附分析法(ELISA)檢測接枝PSBMA薄膜的抵抗蛋白質吸附之能力，透過血小板貼附與血漿凝血時間分析可觀察改質薄膜是否會造成血小板的貼附及活化行為，結果指出當PSBMA接枝層可維持正電與負電基團的電中性時，同時可以鍵結更多的水分子進而產生穩定水合層，顯著地抑制血小板貼附與活化行為，且延長血漿凝血的時間，降低血栓反應的發生，即使直接接觸人體全血也可抵抗全血細胞的貼附。
本論文的第二部分則由雙離子硫代甜菜鹼的電中性結構為基礎，藉由帶正電荷之[2-(Methacryloyloxy)ethyl] trimethylammonium chloride (TMA)單體與帶負電荷之3-sulfopropyl methacrylate potassium salt (SA)單體，以聚合反應製備雙離子電荷偏差型水凝膠，使用不同單體的組成比例可控制材料表面的正負電荷相對含量，範圍由100%的負電荷逐漸增加至100%的正電荷，並定義此數值為電荷偏差程度charge-bias level (bias)。研究中探討poly(SBMA-co-SA)和poly(SBMA-co-TMA)水凝膠的電荷偏差程度對蛋白質吸附、血球貼附與澎潤程度的影響，由實驗結果顯示當bias為0%時，電中性的雙離子結構展現優異的抗沾粘、抗凝血與抗血栓活化的性質，由實驗結果指出隨著bias分別偏差至-100%和+100%時，poly(SBMA-co-SA)和poly(SBMA-co-TMA)水凝膠的澎潤程度也隨之增加，而纖維蛋白原的吸附量隨著正電荷偏差程度的增加會有上升的趨勢，但相對於負電荷偏差程度則不影響蛋白質吸附行為。經由血小板、紅血球及白血球的貼附測試發現，bias為負電荷偏差的poly(SBMA-co-SA)表面僅有少量的血球貼附，而當bias為正電荷偏差並介於+8.7與+18.6%之間時，poly(SBMA-co-TMA)表面可優先選擇性吸附白血球，僅微量吸附血小板和紅血球。隨著bias的正電荷偏差上升至+26.3%與+39.2%之間時，poly(SBMA-co-TMA)表面所吸附的白血球數量可達到最高值，同時膜材表面只吸附少量的紅血球，展現出優異的血球優先吸附性特性。接著，延續雙離子電荷偏差結構體的概念，藉由常壓電漿處理不同級數的胺類單體分別為acrylamide (AA, 1o N), N-isopropylacrylamide (NIPAAm, 2o N), 2-(dimethylamino)ethyl methacrylate (DMAEMA, 3o N) 及TMA (4o N)結合雙離子SBMA材料，接枝於疏水性PP纖維薄膜表面，研究中可精準地控制胺類偏差程度amine-bias level (bias)範圍介於30-40%，可符合前一章節電荷偏差水凝膠的設計。由結果顯示，接枝poly(SBMA)的PP薄膜表面顯著地抵抗人類血漿蛋白與血球貼附，添加胺類單體後，隨著接枝層中胺類官能基級數的增加，蛋白質吸附、血小板貼附與白血球貼附量皆逐漸增加，其中以接枝四級胺類poly(TMA)受到正電荷作用力的影響，造成改質膜面具有較多血球吸附量，當雙離子SBMA添加至胺類單體後，控制bias於30-40%的poly(S65T35)表面可達到白血球吸附量的最高值，展現出優先吸附的特性，最後以血小板濃厚液的動態過濾實驗分析接枝薄膜對血球的吸附能力，由過濾數據得知雖然接枝poly(TMA)與poly(S65T35)的薄膜可高達白血球移除率99%的標準，但膜面上貼附過多活化的血小板，卻也導致過濾後損失99%的血小板，為了選擇具有高白血球移除率與高血小板保留率的過濾效能，分析後發現以二級胺類poly(NIPAAm)與SBMA混合二級胺類poly(S80N20)之接枝薄膜可得到較佳的血小板濃厚液過濾效能。
歸納上述結果，對於製備雙離子性與雙離子性偏差結構體的材料表面，本研究除了維持雙離子性高分子所具有的優異血液相容性之外，更凸顯材料電荷偏差程度的重要性，發展出可控制與篩選不同特定血球吸附順序的規律性，並提供發展出功能型血液接觸材料與推展其於先端血球過濾之應用領域。

關鍵字

抗生物分子沾粘 ； 電荷偏差程度 ； 血球優先吸附 ； 血小板 ； 紅血球 ； 白血球 ； 血小板濃厚液 ； 血液過濾 ； 生醫材料 ； 血液相容性 ； 硫代甜菜鹼 ； 高分子薄膜 ； 共聚高分子 ； 雙離子高分子 ； 表面雙離子化

並列摘要

Zwitterionic polymers recently received medical attention to study and design the next generation of hemocompatible materials superior than the typical blood-contacting materials in the advanced healthcare development. Previous works have already demonstrated that the biomimetic inspiration of zwitterionic betaines from the cell membrane nature with original anti-fouling characteristic introducing for the resistance of protein, bacteria, cell, and algae. Importantly, zwitterionic polymers also performed the excellent antithrombogenic properties potentially used as a blood-inert surface. It is generally acknowledgement that the hemocompatibility of betaine polymers might be associated with the formation of bounded water layer on a highly hydrated zwitterionic structure. Zwitterionic polymers containing the pendant groups of phosphobetaine (PB), sulfobetaine (SB), and carboxybetaine (CB) have received growing attention for use in the new generation of blood inert materials because of their excellent inhibition in plasma protein adsorption.
This research thesis systematically lays the focus on the study of bioadhesive control based on zwitterionic and zwitterionic-bias interfaces in human blood. Two case studies: (1) Zwitteironic interfaces - For the physical-immobilized surface zwitterionization, random and block zwitterionic copolymers of PB methacrylate or SB methacrylate combined with hydrophobic moieties were synthesized with a controlled polydispersity. Two types of zwitterionic copolymers were physically self-assembled coating onto the surface of polypropylene (PP) flat films for enhancing blood compatibility. For the chemical-immobilized surface zwitterionization, the hemocompatibility of zwitterionic PP fibrous membranes with varying grafting coverage of poly(sulfobetaine methacrylate) (PSBMA) via plasma-induced surface polymerization was studied. (2) Zwitterionic-bias interfaces - Herein, a zwitterionic structure bearing moieties with extra positive or negative charges is introduced to develop a potential biomaterial for blood cell selections. The charge balance and charge-bias level was effectively regulated through the control of the initial negatively and positively charged monomer ratio. The focus was then laid on the assessment of a variety of essential properties in human blood contacting, including plasma protein adsorption, blood cell attachment and related hemocompatibility.
In the first part of this thesis, two surface zwitterionization processes are presented, aiming at improving the blood compatibility of PP flat substrate and fibrous membranes. For the physical-immobilized surface zwitterionization, zwitterionic diblock copolymers containing hydrophobic n-butyl methacrylate (BMA) and hydrophilic 2-methacryloyloxyethyl phosphorylcholine (MPC),[3-(methacryloylamino)propyl] dimethyl(3-sulfopropyl)ammonium hydroxide inner salt (SBAA) and 2-hydroxyethyl methacrylate (HEMA) blocks were prepared with well-controlled molecular weight via the conventional free radical polymerization and addition-fragmentation chain transfer polymerization (RAFT). Poly(2-methacryloyloxyethyl phosphorylcholine)-co-poly(n-butyl methacrylate) (PMPC-co-PBMA), poly(sulfobetaine acrylamide)-co-poly(n-butyl methacrylate) (PSBAA-co-PBMA), and poly(2-hydroxyethyl methacrylate)-co-poly(n-butyl methacrylate) (PHEMA-co-PBMA) copolymers with varying hydrophilic blocks or segments were self-assembly coated onto the surface of PP flat substrate for comparing surface hydrophilicity and protein resistance. In this work, we report a systematic study of how different hydrophilic moieties of PMPC-co-PBMA, PSBAA-co-PBMA, and PHEMA-co-PBMA copolymers affect hemocompatibility of modified PP membranes in human blood solution. The coating layers of anchoring zwitterionic copolymers highly regulate the PP surfaces to resist the adsorption of plasma proteins, the adhesion of platelets, the attachment of leukocytes and whole blood cells. This work suggests that the hemocompatible nature of self-assembled zwitterionic brushes by controlling anchoring copolymer structures gives them great potential in the molecular design of antithrombogenic substrates for use in human blood. For the chemical-immobilized surface zwitterionization, the hemocompatibility of zwitterionic PP fibrous membranes with varying grafting coverage of PSBMA via plasma-induced surface polymerization were studied. Charge neutrality of PSBMA-grafted layers on PP membrane surfaces was controlled by the low-pressure and atmospheric plasma treatment in this study. The effects of grafting composition, surface hydrophilicity and hydration capability on blood compatibility of the membranes were determined. Protein adsorption onto the different PSBMA-grafted PP membranes from human fibrinogen solutions were measured by enzyme-linked immunosorbent assay (ELISA) with monoclonal antibodies. Blood platelet adhesion and plasma clotting time measurements from a recalcified platelet rich plasma solution were used to determine if platelet activation depends on the charge bias of the grafted PSBMA layer. The charge bias of PSBMA layer deviated from electrical balance of positively- and negatively- charged moieties was well-controlled via atmospheric plasma-induced interfacial zwitterionization and further tested with human whole blood. The optimized PSBMA surface graft layer in overall charge neutrality has a high hydration capability and keeps its original blood-inert property of antifouling, anticoagulant, and antithrombogenic activities when comes into contact with human blood. This work suggests that the hemocompatible nature of grafted PSBMA polymers by controlling grafting quality via atmospheric plasma treatment gives a great potential in the surface zwitterionization of hydrophobic membranes for use in human whole blood.
In the second part of this thesis, the charge neutral structure of zwitterionic polymer was designed as the basis matrix component. Zwitterionic-bias hydrogels were prepared by the chemical cross-linking reaction with positively charged monomer [2-(Methacryloyloxy)ethyl] trimethylammonium chloride (TMA) and negatively charged monomer 3-sulfopropyl methacrylate potassium salt (SA). To further determine the quantitative analysis of charge distribution in the prepared zwitterionic-bias hydrogels, the degree of charge-bias level (bias) from -100 to +100% is defined as the percentage difference in the relative composition amount of each component (PSA or PTMA) compared to zwitterionic component (PSBMA). A systematic study of how charge-bias variations in poly(SBMA-co-SA) and poly(SBMA-co-TMA) hydrogels affect hemocompatibility in human blood plasma and whole blood was reported. It was found that the protein adsorption, blood cell attachment, and swelling capability of prepared hydrogels can be effectively controlled by regulating the charge balance of the SA/SBMA or TMA/SBMA compositions in the copolymeric gel networks. The results suggest that 0% bias of zwitterionic-bias hydrogels with overall charge neutrality has the best antifouling, anticoagulant, and antihemolytic activities while contacting with blood plasma in human body temperature. It was found that the swelling ratio of poly(SBMA-co-SA) and poly(SBMA-co-TMA) hydrogels increased with the increment of negative and positive bias. The relative fibrinogen adsorption on the zwitterionic-bias surfaces increased as the bias shifted from negatively to positively charged level. However, the observed protein adsorption was independent of bias in the negatively charged level. The hemocompatible test results of platelet, erythrocyte and leukocyte attachment showed that only few platelets and erythrocytes adhered on the negatively charged poly(SBMA-co-SA) surfaces. As the bias is controlled in a range between +8.7% and +18.6%, the poly(SBMA-co-TMA) surfaces showed the preferential attachment of leukocytes and the slight adhesion of platelets and erythrocytes. Furthermore, a maximum number of specific leukocytes attached on the poly(SBMA-co-TMA) surfaces was observed as the bias is controlled in a range between +26.3% to +39.2%. Then, the concept of the zwitterionic-bias formulation was further applied to incorporate with various amine monomers from acrylamide (AA, 1o N), N-isopropylacrylamide (NIPAAm, 2o N), 2-(dimethylamino)ethyl methacrylate (DMAEMA, 3o N) to TMA (4o N) combined with the zwitterionic SBMA grafted on the hydrophobic PP fibrous membranes. The degree of amine-bias level (bias) in a range of 30-40% is precisely controlled and defined as the percentage difference in the relative composition amount of each amine component (1o N, 2o N, 3o N or 4o N) compared to zwitterionic component (PSBMA). The results suggested that the poly(SBMA)-grafted fibrous membranes exhibited the ultrahigh resistance of human plasma protein adsorption and blood cell attachment. But the amount of protein adsorption, platelet adhesion and leukocyte attachment was gradually increased from primary amine to quaternary amine groups. Especially for the quaternary amine of poly(TMA), the positively charged surfaces attached high amount of blood cells to adhere than other amine-grafted surfaces including poly(AA), poly(NIPAAm) and poly(DMAEMA). After the zwitterionic SBMA added into the amine monomers, the poly(S65T35)-grafted surfaces adsorbed a most high number of leukocytes in the fixed bias in a range of 3040%, indication the preferential bioadhesion of specific leukocytes from human blood. The performance of platelet concentration (PC) filtration showed that the modified membranes of poly(S65T35)-grafted PP fibrous membranes depleted 99% leukocytes, but lost 99% of platelets due to the serious platelet adhesion and activation. In the consideration between leukocyte deletion and platelet recovery as a balance, the poly(S80N20)-grafted PP fibrous membranes supported the best leukocyte depletion in PC filtration.
This work not only performed the excellent hemocompatibility of zwitterionic polymers, but also demonstrated the controllable charge-bias from zwitterionic neutrality to preferentially capture specific blood cells, which provide a potential possibility in the development of advanced blood filtration systems.

並列關鍵字

platelet ； preferential bioadhesion ； charge-bias level ； biomaterials ； hemocompatibility ； surface zwitterionization ； SBMA ； zwitterionic polymer ； blood filtration ； platelet concentration ； leukocyte ； erythrocyte ； anti-fouling ； polymeric membrane ； copolymer

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