臺灣博碩士論文加值系統

本研究選用溫度敏感性材料N-isopropylacrylamide (NIPAM)，並與Acrylamide (AM)作自由基加成聚合，其中調整AM的含量，製備成不同比例的poly(N-isopropylacrylamide-co-acrylamide)(P(NIPAM-co-AM))，並將其作為高分子主鍊，之後再添加3-glycidyloxypropyltrimethoxysilane (GLYMO)及Tetraethyl orthosilicate (TEOS)，利用GLYMO與AM作開環反應，再利用GLYMO與TEOS經溶膠-凝膠法(Sol-Gel reaction)，而得到最終產物之溫度敏感型複合奈米粒子P(NIPAM-co-AM)_Silica hybrid nanoparticles，並評估此奈米粒子應用於藥物傳遞系統之可行性。
因本研究所合成的複合奈米粒子具有溫度敏感性，當溫度低於低臨界相分離溫度(lower critical solution temperature, LCST)時，使高分子鍊伸展開來，奈米粒子會呈現親水性，且於環境pH 7.4下，材料帶負電荷，能夠與帶正電的DOX經靜電荷交互作用而相互吸附，反之，若溫度高於LCST時，會呈現疏水性，我們利用此特性，於低溫下進行。不同的AM比例，藥物包覆效率約為45 ~ 55%。
藥物釋放的部分，於不同的酸鹼度下進行，如pH 7.4、pH 6及pH 5，且將環境模擬人體生理溫度37"℃" ，此時複合奈米粒子會收縮，將藥物包覆在核心中，但由於藥物會受到內部擠壓，且酸性的環境會提供質子，此時藥物會藉由質傳釋放出來，並於120小時內的釋放率，不同的AM比例，於pH 7.4、pH 6及pH 5分別達到40%、70%及80%左右。
最後，將複合奈米粒子包覆DOX之樣品與癌細胞(HeLa cells)共同培養，並對此癌細胞進行細胞毒性分析及細胞吞噬分析，綜合以上，探討此複合材料在癌症治療之藥物傳遞系統的應用價值。

In this research, the thermal-sensitive material NIPAM was used, and Acrylamide (AM) was used as a free radical addition polymerization. The content of AM was adjusted to prepare different ratios of poly(N-isopropylacrylamide-co-acrylamide) (P(NIPAM-co-AM)), and as a backbone. Adding 3-glycidyloxypropyltrimethoxysilane (GLYMO) and Tetraethyl orthosilicate (TEOS) Tetraethyl orthosilicate (TEOS), use GLYMO and AM for ring-opening reaction, and use GLYMO and TEOS by Sol-Gel Reaction to get the final product The thermal-sensitive hybrid nanoparticles P(NIPAM-co-AM)_Silica. It was used and the feasibility of applying the nanoparticles to the drug delivery system was evaluated.
Because the hybrid nanoparticle synthesized in the research is temperature sensitive. When the temperature is lower than the low critical solution temperature (LCST), the polymer chain is swelling, and the nanoparticles are hydrophilic. At ambient pH 7.4, the material will have a negative charge and can interact with the positively charged DOX via electrostatic interaction. Conversely, if the temperature is higher than LCST, it will be hydrophobic. We use the property to do load drug. It is carried out at low temperature, the drug coating efficiency is about 45 - 55% for different AM ratios.
The drug release is carried out under different pH value, such as pH 7.4, pH 6 and pH 5, and the environment is simulated at 37 °C. The hybrid nanoparticles will shrink and the drug is coated in the core. However, because the drug will be squeezed internally, and the acidic environment will provide protons, the drug will be released by mass transfer, and the release rate in 120 hours, different AM ratios, at pH 7.4, pH 6 and pH 5 reached 40%, 70% and 80% respectively.
Finally, the drug-coated composite nanoparticles were co-cultured with cancer cells (Hela cells), and the cancer cells were analyzed for cytotoxicity and phagocytosis. In summary, exploring the application value of this hybrid material in drug delivery system for cancer treatment.

摘要I
AbstractII
致謝IV
目錄V
表目錄IX
圖目錄X
第一章 緒論1
1.1 前言1
1.2 研究動機2
第二章 文獻回顧3
2.1水膠3
2.1.1 水膠定義3
2.1.2 水膠分類4
2.2聚氮-異丙基丙烯醯胺(PNIPAM)之性質5
2.3 自由基加成聚合反應6
2.4 複合材料9
2.5 溶膠-凝膠法(Sol-Gel Process)10
2.6 Doxorubicin藥物介紹12
2.7 孔洞材料13
2.7.1 孔洞材料介紹13
2.7.2 孔洞材料於藥物傳遞系統之應用14
2.8 溫度敏感型內核－外殼之奈米粒子15
第三章 實驗藥品、儀器與方法17
3.1 實驗藥品17
3.1.1 合成實驗17
3.1.2 配製緩衝液之藥品19
3.1.3 配製培養基之藥品19
3.1.4 配製藥物釋放緩衝液之藥品21
3.1.5 細胞計數之藥品21
3.1.6 MTT試劑21
3.1.7 DOX (阿黴素)21
3.1.8 細胞螢光染色之藥品22
3.2 實驗儀器23
3.3 實驗流程圖24
3.4 合成實驗25
3.4.1 實驗內容25
3.4.2 實驗步驟25
3.5 鑑定與分析27
3.5.1 動態光散射粒徑分析儀(DLS)27
3.5.2 熱重/熱式差分析儀(TGA)27
3.5.3 高解析度場發射掃描式電子顯微鏡(SEM)28
3.5.4 穿透式電子顯微鏡(TEM)28
3.5.5 表面積及孔徑分析儀(BET)28
3.5.6 雷射界面電位分析儀暨粒徑分析儀28
3.6 體外細胞實驗29
3.6.1 細胞來源29
3.6.2 培養基配製31
3.6.3 磷酸鹽緩衝液(PBS)配製32
3.6.4 Trypsin-EDTA配製33
3.6.5 細胞繼代及培養33
3.6.6 細胞冷凍保存34
3.6.7 細胞解凍及培養34
3.6.8 細胞計數35
3.6.9 MTT溶液配製36
3.6.10 生物相容性測試37
3.6.11 細胞吞噬測試39
3.7 Doxorubicin體外釋放實驗40
3.7.1 DOX檢量線之製備40
3.7.2 DOX包覆實驗與分析40
3.7.3 DOX體外釋放實驗與分析41
第四章 結果與討論42
4.1 鑑定結果42
4.1.1 粒徑與界面電位之特性42
4.1.2 熱分析結果46
4.1.3 孔徑分析48
4.2 藥物包覆及體外藥物釋放分析52
4.2.1 藥物包覆52
4.2.2 體外藥物釋放53
4.3 體外細胞實驗56
4.3.1 細胞吞噬測試56
4.3.2 細胞毒性測試61
第五章 結論66
第六章 參考文獻68

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