將聚乙二醇接枝於聚己內酯基材表面以調控細胞貼附之研究

Surface Grafting of Polyethylene Glycol onto Polycaprolactone to Modulate Cell Attachment

陳敬秉(Jing-Bing Chen)

指導教授 ： 謝學真

國立臺灣大學/工學院/化學工程學研究所/碩士(2015年)

https://doi.org/10.6342/NTU.2015.10449

摘要

生醫材料表面之親疏水性質會影響蛋白質吸附和細胞貼附之情況，而聚乙二醇則常被用於增加材料之親水性質。本研究主要是藉由數種方法將聚乙二醇二甲基丙烯酸酯接枝於聚己內酯緻密膜材表面，增加其親水性，以達到調控細胞貼附及生長為目的。
首先進行聚己內酯緻密膜材的製備，將配製好的聚己內酯混和溶液，倒入玻璃培養皿中，真空乾燥成膜，再進行膜材的機械性質、熱性質、親水性質以及細胞相容性測試。
至於膜材表面改質，第一種接枝方法（EDC/NHS交聯法）是將聚乙二醇二甲基丙烯酸酯與乙二胺以莫耳數1:2的比例於37˚C進行反應，將其末端接上乙二胺以產生胺基。另外以0.5 M之氫氧化鈉對聚己內酯表面進行水解而產生羧基，處理時間約為26小時，再利用EDC/NHS作為交聯劑連接胺基與羧基，達成將聚乙二醇接枝至聚己內酯表面之目的。本研究使用甲苯胺藍染色法測出鹼處理後聚己內酯表面所產生之羧基量為2.83 nmol/cm2。
第二種接枝方法（紫外光接枝法）使用光引發劑二苯基甲酮，將聚己內酯緻密膜材浸泡於由二苯基甲酮(0.7 wt%)及聚乙二醇二甲基丙烯酸酯水溶液(50% v/v)所形成之混合溶液中，利用紫外光將光引發劑激發，使聚己內酯膜表面生成自由基，進而與聚乙二醇二甲基丙烯酸酯進行反應，達成接枝之目的。此外，本研究嘗試將反應之膜材先經鹼處理和電漿處理，以提升膜材之親水性質，再進行紫外光接枝反應以期達到更佳的表面改質功效。
根據水接觸角測定得知利用EDC/NHS交聯法可將膜材的水接觸角由87.8°± 0.5°降至45.5°± 0.5°，由此可以推論聚乙二醇已成功地接至聚己內酯膜上並且使其成為親水表面。而紫外光接枝法可將膜材的水接觸角降至65.6°± 3.2°，若先經過26小時鹼處理則降至54°± 3.5°，若先經電漿處理則可降至50.6°± 5°，亦均為親水表面。
此外透過FTIR、XPS、DSC等分析也證實了聚乙二醇的確存在於聚己內酯基材的表面上。而由膜材機械性質測試顯示除了經過電漿處理後的膜材延展性下降之外，其餘的機械性質如：抗拉強度等並沒有太大的改變。另外，由蛋白質總量、MTT測定以及顯微鏡觀察L929小鼠纖維母細胞生長在膜材上以及貼附的結果顯示，此膜材具有良好的細胞相容性，且可略微調控細胞於膜材上之貼附程度。希望本研究之表面改質技術未來能夠在生醫材料的領域能有更進一步的發展以及應用。

關鍵字

聚己內酯 ； 聚乙二醇 ； 緻密膜 ； EDC/NHS ； 紫外光接枝 ； 細胞貼附

並列摘要

The surface hydrophilicity of biomaterials affects the protein adsorption and cell attachment on the surface. This research aims to carry out biomaterial surface modification by grafting polyethylene glycol dimethacrylate (PEGDMA) onto polycaprolactone (PCL) dense films to increase their surface hydrophilicity and thus regulate the cell attachment and proliferation.
In this study, the PCL solution was poured into glass dishes and vacuum dried overnight to form PCL dense films. After that, the mechanical properties, thermal properties and hydrophilicity of the films were examined.
As for the surface modification, the first method to graft polyethylene glycol (PEG) onto PCL films is an EDC/NHS crosslinking method. PEGDMA reacted with ethylenediamine (EDA) (mole ratio 1:2) at 37°C for 24 hours to form a compound with amino groups at the ends of the polymer chain. In the meantime, the PCL films were immersed in a 0.5 M NaOH solution for 26 hours to make the surface bearing carboxylic groups. The amount of carboxylic groups on the surface of the PCL films, as determined by a Toluidine Blue O stain method, was about 2.83 nmol/cm2. Afterwards the PCL films (bearing carboxylic groups) were activated by the EDC/NHS solution and then reacted with the aforementioned compound (bearing amino groups), thus achieving the grafting of PEG onto the PCL films.
The second method to graft PEG onto PCL films is a UV-induced surface grafting method. Benzophenone (BP) was used as a photoinitiator. A 0.7 wt% BP solution was prepared by adding BP directly into a PEGDMA/water solution (volume ratio 1:1). The reaction was initiated by UV to generate free radicals on the surface of the PCL films which reacted with PEGDMA, thus grafting PEG onto the PCL films. Besides, in some experiments the PCL films were pretreated by plasma or NaOH solution before the UV-induced surface grafting in order to further increase the surface hydrophilicity of the PCL films.
According to water contact angle measurement, the EDC/NHS crosslinking method could decrease the contact angle of PCL films from 87.8°±0.5° to 45.5°±0.5°, indicating that PEG had been grafted onto the PCL films and increased the surface hydrophilicity. The UV-induced surface grafting method could decrease the contact angle of PCL films 65.6°±3.2°. If the PCL films underwent alkaline pretreatment, the contact angle of the films could decrease to 54°±3.5°. Similarly, plasma pretreatment caused the contact angle decreased to 50.6°±5°. These results indicated that UV-induced surface grafting also generated more hydrophilic surfaces.
Besides, the FT-IR, XPS, and DSC analyses confirmed the presence of PEG on the PCL film surfaces. The mechanical properties testing revealed that the plasma treatment decreased the elongation (at maximum load) of the PCL films, but the maximum tensile strength did not change significantly. Furthermore, total protein, MTT assays and microscope observation of mouse fibroblasts indicated that the cytocompatibility of the PCL films was fairly good. The surface modification of PCL films by PEG slightly affected the cell attachment, activity, and proliferation. It is expected that the above-mentioned surface modification methods can be applied to biomaterial-related areas in the future.

並列關鍵字

polycaprolactone ； polyethylene glycol ； dense films ； EDC/NHS ； UV-induced grafting ； cell attachment

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