新穎特殊的共聚肽分子之設計、合成、鑑定及其在神經組織工程上的應用

Abstract

神經組織工程為神經損傷和疾病引起的功能障礙或疼痛帶來了前景。許多肽基底生物醫學支架需要額外的生物活性因子和特殊塗層來滿足細胞附著、細胞活性（包括細胞生長和分化）的要求。功能性支架非常適合滿足這些雙重功能的需求：同時具有良好的細胞附著和細胞活性。而且也希望它們可以很容易地由單一種材料製成。我們開發了具有良好生物相容性和無細胞毒性的雙功能性新穎共聚肽高分子。其含有谷氨酸和賴氨酸衍生物的化學結構組成通過開環聚合設計和合成。共聚肽的賴氨酸部分增強細胞貼附，而神經刺激的谷氨酸部分提高細胞活性。因此，單一共聚物可以具有兩種均聚物的優點和特性。且在無需增加額外細胞貼附處理的情況下，也可有效地讓細胞附著、生長、擴散並分化。 而為了找到對於細胞生長之最理想單體比例共聚物，首先合成出不同莫耳比例單體的共聚物並將其製備成薄膜，並鑑定它們的表面特性及評估細胞培養特性。更進一步探討不同化學組成的共聚物所造成不同的薄膜表面特徵與細胞表現之間的影響與關聯性。使用的賴氨酸衍生物包含叔丁氧羰基賴氨酸(BOCL)和羧基芐基賴氨酸(CBZL)。賴氨酸上BOC側鏈的尺寸比CBZ大，這導致薄膜表面粗糙且疏水，細胞貼附降低。由於賴氨酸側鏈上的酰胺鍵，通過增加賴氨酸的組成來增強薄膜親水性及細胞活性。當這些影響因素達到平衡時，可增強細胞生長。因此CBZL和BG在等莫耳比組成的共聚物在嗜鉻細胞瘤(PC-12)神經細胞培養中表現出最佳性能。而共聚物部分水解成聚電解質時，由於存在電荷且增加親水性和細胞識別，進一步可更增強細胞粘附和生長。而人類視網膜色素上皮細胞(ARPE-19) 培養在共聚物薄膜上亦有良好的貼附及活性。ARPE-19 細胞培養在平坦的共聚肽薄膜上的時間拉長到兩個多月後，它可以形成密集排列的單層細胞排列，這證明了它們在視網膜修復組織工程中的潛力。 將最佳組成的共聚肽使用靜電紡絲製程製備成具有對齊纖維排列的纖維支架。PC-12細胞培養在等比例谷氨酸和賴氨酸的共聚物支架上相較於在均聚物支架上展現最好的神經細胞活性和最長的軸突長度，且神經軸突受到基材引導沿著電紡纖維的方向生長並延長。在支架被部分水解而帶正電荷並增加親水性後，細胞活性和神經突生長都進一步增加。這證明了共聚肽不但同時可增加神經細胞貼附力也能讓神經傳導物質谷氨酸有效發揮功能來刺激神經細胞生長並分化。 使用聚谷氨酸纖維支架的體內動物實驗與台大醫院陳偉勵醫師進行合作，初步結果顯示了谷氨酸可刺激角膜神經生長且聚肽高分子支架不會造成兔子眼角膜的發炎反應。聚谷氨酸製成的仿生支架被探索為角膜神經再生和神經營養性角膜炎治療的候選材料。此新穎設計之共聚肽材料有望成為神經組織工程領域的一盞明燈。

Neural tissue engineering brings prospective landscapes for dysfunction or pain caused by nerve damage and disease. Many peptide-based biomedical scaffolds require additional bioactive factors and special coatings to satisfy the requirements of cell attachment, cell viability including the growth and differentiation of cells. Functional scaffolds are highly desirable to meet the demands of these dual function: good cell attachment and cell viability simultaneously. And they are also desirable can be made from a single material easily. We develope the dual-function co-polypeptide which can achieve desired properties simultaneously with good biocompatibility and none cytotoxicity. Novel co-polypeptides containing glutamate and lysine derivative moieties are designed and synthesized by ring-opening polymerization. The lysine moeity of co-polypeptide enhances cell adhesion while the glutamate moiety of neural stimulate improves cell viability. Thus, a single copolymer can have the advantages and characteristics of two homopolymers. And it can effectively allow cells to attach, grow, spread and differentiate without additional treatments to enhanced cell adhesion. In order to find the optimal monomer ratio copolymers for cell growth, the copolymers of different molar ratio of monomers are first synthesized and prepared into thin films, and their surface properties are examined and cell culture properties are evaluated. The influence and correlation between different film surface characteristics and cell performances caused by copolymers with different chemical compositions are explored. The lysine derivatives used in co-polypeptides include tert-butoxycarbonyl lysine (BOCL) and carboxybenzyl lysine (CBZL). The size of the BOC side chain on lysine is larger than that of CBZ, which causes the film surface to be rough and hydrophobic, and cell adhesion is reduced. Due to the amide linkage on the side chain of lysine, the hydrophilicity of the film and cell activity are enhanced by increasing the composition of lysine. When these influencing factors reached equilibrium, cell growth is enhanced. Therefore, the copolymer composed of CBZL and BG in equal molar ratio showed the best performance in pheochromocytoma (PC-12) nerve cell culture. When the copolymer is partially hydrolyzed, it can further enhance cell adhesion and growth due to the presence of electric charge and increase the hydrophilicity and cell recognition. The copolymer film also exhibits good adhesion and activity on adult retinal pigment epithelial (ARPE-19) cells. The ARPE-19 cells cultured on flat co-polypeptide films can form densely packed monolayer cell arrangements for more than two months, demonstrating their potential in tissue engineering for retinal repair. Then, the optimal composition of co-polypeptide is further used to prepare fibrous scaffold with aligned fiber by electrospinning. PC-12 cells cultured on copolymer scaffolds with equal ratios of glutamate and lysine exhibit the best neuronal activity and longest neurite length as compared to their respective homo-polypeptide scaffolds. And the neurites are guided by the substrate to grow and elongate along the direction of the fibers. After the scaffold is partially hydrolyzed to contain positive charge and increase hydrophilicity, both cell viability and neurite growth are further increased. The lysine moiety of co-polypeptide enhances cell attachment while its glutamic acid moiety stimulates neurogenesis effectively. The in vivo animal experiments using poly(γ-benzyl-L-glutamate) (PBG) fibrous scaffold are collaborated with Dr. Wei-Li Chen from National Taiwan University Hospital. The results show that glutamate can stimulate corneal nerve growth and the polypeptide scaffolds did not cause inflammation of the cornea. PBG made biomimic scaffold is explored as candidate material for corneal nerve regeneration and neurotrophic keratopathy treatment. This novel designed co-polypeptide materials are expected to be bright light in the field of neural tissue engineering.