聚氮-異丙基丙烯醯胺於生醫材料之應用與評估

Evaluation and applications of poly(N-isopropylacrylamide) for biomedical materials

陶鈞德(Chun-Te Tao)

指導教授 ： 楊台鴻

國立臺灣大學/工學院/醫學工程學研究所/博士(2007年)

https://doi.org/10.6342/NTU.2007.02589

摘要

本論文之目的在於研究溫度敏感性聚氮-異丙基丙烯醯胺高分子於生醫材料之應用性評估。研究的範圍主要包括溫度敏感性聚氮-異丙基丙烯醯胺高分子基材的製備及其特性、神經幹細胞於此高分子基材的行為，以及聚氮-異丙基丙烯醯胺高分子對生物細胞的影響。
本研究中，我們首先考慮嘗試用傳統的高分子-溶劑-非溶劑的薄膜製造方法來製備聚氮-異丙基丙烯醯胺的薄膜基材。然而，文獻資料上並無聚氮-異丙基丙烯醯胺在常溫下的非溶劑，但是在文獻上卻有以水-甲醇的聚氮-異丙基丙烯醯胺共非溶劑。因此，我們嘗試利用此共非溶劑系統來製備其薄膜。除了薄膜的製備，我們也完成了此聚氮-異丙基丙烯醯胺-水-甲醇共非溶系統的部分實驗相圖，並且修正Flory-Huggins的理論計算出其完整的理論相圖。共非溶現象的機制，也在此部分加以討論。依據實驗結果，在計算共非溶系統時，三成份作用參數是不可被忽略的。同時，雖然其薄膜強度不佳，我們也證實了高分子薄膜的確可利用共非溶系統來製備。而共非溶的成因，可能起於三種成份同時存在下，其兩兩成份間作用力的改變而造成。
利用上述共非溶系統所製備出來的聚氮-異丙基丙烯醯胺薄膜，其結構是非常的脆弱，並不利於其生醫材料的應用。因此，在應用上，我們是利用氧化還原的起始方式將聚氮-異丙基丙烯醯胺接枝於聚乙烯乙烯醇的薄膜上來做為細胞培養的基材。我們將由大鼠取出的神經幹細胞培養於此溫度敏感性的基材上，研究其在25℃與37℃的環境下於此基材上的行為及溫度敏感性高分子對神經細胞的影響並討論其於組織工程的應用性。結果顯示，神經幹細胞在溫度敏感性基材上的貼附性不佳，而貼附的細胞可在低溫下被誘導脫附此基材。當培養基加入胎牛血清時，幾乎所有的神經幹細胞都會貼附於此基材上形成一層細胞層，且在低溫下，可以成功的誘導此細胞層轉移至塗佈有聚賴氨酸的基材上。
聚氮-異丙基丙烯醯胺線性高分子、水膠、奈(微)米粒子及薄膜等的性質及應用已被廣泛的研究。然而，其作為生醫材料的應用時之對生物組織或細胞的影響卻很少被提及。本研究第三部分便製備聚氮-異丙基丙烯醯胺的奈米粒子並將之與巨噬細胞與骨母細胞培養，研究不同溫度及溫度的波動下此粒子對細胞的毒性、細胞功能以及其所引發的免疫反應。結果顯示，聚氮-異丙基丙烯醯胺的奈米粒子在疏水狀態下其細胞毒性較親水狀態強，且會造成巨噬細胞大量免疫酵素的釋放。而經由幾丁聚醣的改質，會大大的降低其毒性及免疫反應。

關鍵字

聚氮-異丙基丙烯醯胺 ； 生醫材料 ； 溫度敏感性

並列摘要

In this study, we evaluate the applications of the thermosensitive poly(N-isopropylacrylamide) (PNIPAAm) for biomedical materials. The preparation and characterizations of the thermosensitive PNIPAAm membrane substrate, behaviors of neural stem cells (NSCs) on thermosensitive substrate and the influence of the PNIPAAm to the cells were included in this study.
First of all, we tried to prepare PNIPAAm membrane by using the traditional polymer-solvent-nonsolvent system at ambient temperature but it was found that there exist no nonsolvents at room temperature for PNIPAAm. Fortunately, it is shown that there are cononsolvents for PNIPAAm composed of water and methanol. Thus, we were encouraged to prepare PNIPAAm membrane by the cononsolvent system. In addition, the complete phase diagram of PNIPAAm-water-methanol was performed by using modified Flory-Huggins theory and the mechanism of cononsolvency was also discussed. The results show that the ternary interaction parameter can not be neglected to calculate the phase behavior of the cononsolvent system. In addition, although the mechanical strength of the PNIPAAm membrane prepared by the cononsolvent system is poor, it is proved that polymeric membranes can be prepared via the cononsolvent system. From the calculation results, it is suggested that the cononsolvency may be originated from the interaction perturbation of two component in the existence of the third component.
PNIPAAm membrane prepared by the cononsolvent system exerted a very poor mechanical strength due to the macrovoid in the cross-section of the membrane. In this study, PNIPAAm chains were grafted on poly(ethylene-co-vinyl alcohol) (EVAL) membrane by the redox initiation method. Embryoic rat cortical neural stem cells were subjected to culture on this thermosensitive substrate and the behaviors of the cell were explored at both 25 and 37℃. Two dimensional cell sheet manipulations for tissue engineering were also studied in this work. Results show that NSCs hardly attach on PNIPAAm-g-EVAL and the attached NSCs can be induced to detach from the thermosensitive surface via low temperature treatment. When FBS was added, almost all NSCs can attach on PNIPAAm-g-EVAL surface easily and proliferate to form a cell confluence. Consequently, NSCs confluence can be successfully induced to transfer to poly lysine-coated TCPS under low temperature treatment.
Linear PNIPAAm, hydrogels, nano(micron) particles and membranes were extensively studied in the past few years. Although good performance of PNIPAAm was revealed, the influences and disadvantages of the PNIPAAm to tissues or cells were rarely mentioned. In this study, PNIPAAm nanoparticles were prepared and the influence on macrophage and osteoblast at different temperatures, including cytotoxicity, cell function and immune-responses, were explored. Results show that PNIPAAm-based particles exhibit more cytotoxicity at 37 than 25℃, and they stimulate strong immune responses to macrophage. The cytotoxicity and immune-responses can be lowered by introducing chitosan to PNIPAAm particles.

並列關鍵字

poly(N-isopropylacrylamide) ； biomaterial ； thermosensitive

參考文獻

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