光驅動軟性致動器應用於細胞排列

Abstract

細胞排列對於生物科技及組織工程上的分化及成熟是一個關鍵的要素，而這樣的形態也普遍存在於生物體中。儘管透過機械力、表面型態、電刺激和化學修飾達到細胞排列已經研究了數十年，然而他們仍受限於複雜的結構、困難的製程和培養的方式。在此，由類彈力蛋白、蠶絲蛋白、氧化石墨烯和還原後的氧化石墨烯組成的光驅動軟性致動器搭配近紅外光雷射將首次應用於引導細胞排列。軟性致動器在受到不同的刺激情況下會取決於他們結構的設計和驅動的原理進而能夠以柔韌的結構展現出翻筋斗、爬行和抓取物體等不同的動作。蓬勃發展的軟性致動器在數個應用上取代傳統的硬性致動器而帶來了前景與希望，像是人工肌肉、藥物傳遞和生物感測等。然而，據我們所知，從微米到釐米大小的軟性致動器大部分都著重於控制動作或是設計出仿生肢體的行為。為了開發出操控細胞行為的軟性致動器，這篇論文將研究一個簡易製成、容易操控以及高效能的新穎軟性致動器ESGRG，並應用在引導細胞排列。 ESGRG是由類彈力蛋白、蠶絲蛋白、氧化石墨烯和還原後的氧化石墨烯組成的，其中ELP基於重複的氨基酸序列結構valine-proline-glycine-isoleucine-glycine，具有熱敏感性、韌性以及安插arginine-glycine-aspartate的胜肽提供了生物活性。蠶絲蛋白則可以透過有序的β-sheet結構達到強化ESGRG的機械性質。ESGRG藉由GO和rGO個別形成了雙層結構，GO和rGO在照射近紅外光後會產生光熱轉換效應而驅動ESGRG彎曲。由於GO和rGO會有不同的親疏水性導致ESGRG在成形時在雙層間會有孔洞大小的差異。此外，ESGRG可以透過調控近紅外光的強度和GO與rGO的比例來控制它的行為。其中，提高GO的含量會使得ESGRG受到近紅外光的照射後，由於GO提供更大的光熱轉換效應導致ELP有更好的溫度敏感表現，所以致動彎曲的角度越大。當肌肉細胞C2C12培養於ESGRG表面上時，透過近紅外光照射後，細胞會沿著受力方向進而產生有效的排列。隨著致動彎曲的次數增加，細胞排列的效果也會更好。此外，軟性致動器也可以容易地塑造成圓形和蝴蝶形狀而產生不同的彎曲效果。這篇研究首創了軟性致動器提供近紅外光致動彎曲達到細胞排列的目的，在未來可以應用於軟性機器人，心肌細胞的成熟和生醫工程上。

Cell alignment has been widely observed in organism which is a critical factor to differentiation and maturation in cell biology and tissue engineering. Despite several strategies including mechanical force, surface topography, electrical stimulation, and patterned chemical substrate had been studied over past decades, their complex structure, difficult manufacture, and culture methods are obstacles needed to be overcome. Herein, for the first time, a light-control soft actuator composed of elastin-like polypeptide, silk fibroin, graphene oxide, and reduce graphene oxide, named as ESGRG, is first developed for driving cell orientation on soft actuator via remotely NIR laser exposure. Soft actuators enable to generate diverse motions such as somersault, crawl, and holding an object with flexible structure in response to multiple stimulation depending on their structure design and fundamental principles. The emergence of soft actuators offers prospects and opportunities in various applications, such as artificial muscles, drug delivery and biosensors replacing traditional rigid actuators. However, to our knowledge, most soft actuators ranging from micrometers to centimeter are aimed at controlling the behaviors and motions of actuators or design for prosthesis movement. To develop a soft actuator using for controlling the behaviors in cell level, a novel ESGRG hydrogel actuator system exhibits simple manufacture, easy control and high efficiency has been developed to guide cell orientation in this study. ESGRG was constructed by elastin-like polypeptide, silk fibroin, graphene oxide and reduced graphene oxide which presented thermosensitivity, flexibility and bioactive property provided by ELP owing to the repeated amino acid structure, valine-proline-glycine-isoleucine- glycine and inserted peptide of arginine-glycine-aspartate. Silk fibroin promotes mechanical robust to strengthen ESGRG by the structure of highly organized crystalline β-sheets. ESGRG was designed to form by two layers incorporated with GO and rGO, respectively. Both GO and rGO exhibited photo-thermal conversion which led ESGRG perform bending motion in response to NIR radiation. Anisotropic porosity was observed in the double-layer structure because there are different affinities to water between GO and rGO during ESGRG was fabricated. In addition, the actuation of ESGRG could be manipulated through modulating intensities of NIR and ratio of GO to rGO. When ESGRG consisted higher content of GO, it would perform actuation with larger bending angle due to the higher temperature provided by photo-thermal property of GO cause the better thermoresponsive behavior of ELP. As muscle cells, C2C12, were seeded on ESGRG, they would be guided cell orientation effectively along the direction of bending force by NIR radiation. And the more ordered cell alignment would form by applying more bending motion. Furthermore, hydrogel actuators could be easily molded into shapes of round and butterfly that would generate different motion. This study provides a possibility, for the first time, for cell alignment achieved by bending motion of NIR-driven soft actuator, which can be utilized in applications of soft robotic engineering, maturation of cardiomyocyte, and biomedical engineering.