- 學位論文
搭載近紅外光螢光劑之聚乙二醇-聚己內酯共聚物奈米微胞於乳癌前哨淋巴結顯影之應用
Application of Poly(ethylene glycol)-b-Poly(𝜺-caprolactone) Polymeric Micelle Loaded with Near-Infrared Fluorescent Dye for the Mapping of Breast Cancer Sentinel Lymph Node
摘要
聚乙二醇廣泛使用在藥品、化妝品、食品中，近年來文獻證實健康人 體內存在聚乙二醇抗體，造成患者使用聚乙二醇修飾藥物後，產生減低其 生物可利用度與療效問題。本研究基於前人建立的近紅外光螢光之聚乙二 醇(分子量 5000 Da)-聚己內酯(分子量 3500 Da)共聚物奈米微胞，以纖維胚 胎細胞 NIH/3T3 及小鼠乳腺癌細胞 4T1 評估微胞之細胞存活率;結果顯示 添加濃度為 14.25 𝜇g/mL 的微胞於這兩種細胞株培養 24 小時後的存活率皆 大於 80%，顯見該濃度對細胞不具細胞毒性。以活體螢光影像系統觀察搭 載吡喃結構代號為 2CN 顯影劑之微胞於小鼠體內器官的分佈，結果顯示注 射 1 mL(2CN 濃度為 28.5 μg/mL;載體濃度為 1000 μg/mL )微胞劑型一小時 後肝臟累積較顯著。以單次靜脈注射於健康 SD 品系大鼠體內評估搭載顯 影劑之微胞劑型最大耐受劑量;結果顯示高劑量微胞(10 mL/kg 顯影劑 285 μg/kg、高分子 10 mg/kg)下均無動物死亡(n = 24);體重及食物消耗量 無明顯增加或減少之趨勢;在給藥七日後，腦、心、肺、肝、腎、脾之臟 器係數比均無明顯上升或下降之變化，心、肺、脾、肝、腎、腦之組織切 片分析也均無發現組織壞死、萎縮及空洞化等病理現象發生。在誘導 Balb/c 小鼠體內產生聚乙二醇抗體(IgG 與 IgM)實驗中，Balb/c 小鼠(n = 15) 注射 0.1 mL 的聚乙二醇-聚己內酯微胞後，於第二周產生聚乙二醇 IgM 抗 體(26.2±9.6 μg/mL)，六週後濃度降至 21.3±6.7 μg/ mL 而 IgG 抗體濃度上升到 25.03±6.2 μg/ mL 。上述實驗後老鼠注射 4T1 細胞一周後觀察到乳癌 腫瘤，犧牲後其前哨淋巴結免疫組織染色(mouse monoclonal anti-cytokeratin) 觀察到 4T1 細胞，因此確認建立乳癌轉移前哨淋巴結的模型。注射 10 μL 攜帶近紅外光染劑(吡喃結構代號為 2CN，激發波長 500 nm/放射波長 780 nm)的微胞後 18 及 24 小時以活體螢光影像系統觀察，控制組小鼠(未誘導 聚乙二醇抗體)在注射後第 18 小時平均螢光強度為 1.52x108，第 24 小時為 1.68x108，實驗組小鼠平均螢光強度分別為 2.13x108(18 小時)及 1.67x108(24 小時)，統計後無顯著差異。因此本研究確立在誘導聚乙二醇抗體下小鼠體 內仍可顯影乳癌前哨淋巴結，未來可朝向臨床應用研發。
並列摘要
Polyethylene glycol (PEG) is widely used in medicines, cosmetics, and foods. In recent years, the paper has confirmed the existence of anti-PEG antibodies in healthy humans, causing patients to use PEG-modified drugs to reduce their bioavailability and treatment effects. This study is based on the near-infrared fluorescent PEG (molecular weight 5000 Da)-Poly(𝜺-caprolactone) (PCL ,molecular weight 3500 Da) copolymer nano micelles established by the predecessors, and the cell survival rate of micelles was evaluated with the fibroblast cell line ( NIH/3T3 ) and mouse breast cancer cell line ( 4T1 ); the results showed that the survival rate of micelles at a concentration of 14.25 𝜇g/mL was over than 80% when these two cell lines were cultured for 24 hours, which shows that the concentration is highly biocompatible to the cells. The biodistribution of the micelles in the organs of Balb/c mice was observed with in vivo image system ( IVIS ) . The results showed that the liver accumulation was more significant one hour after the injection of 1 mL of micelles. The maximum tolerated dose ( MTD ) of this dosage form was evaluated by a single IV injection in healthy rats; the results showed that no animal (n = 24) died at high doses of micelles (10 mL/kg)；there was no significant increase or decrease in body weight and food consumption；seven days after the administration, the organ coefficient ratios of brain, heart, lung, liver, kidney, and spleen did not increase or decrease. Tissue section analysis of liver, kidney, and brain also found no pathological phenomena such as tissue necrosis, atrophy, and cavitation.In the experiment of inducing Balb/c mice (n = 15) to produce anti-PEG antibodies (IgG and IgM), Balb/c mice (n = 15) were injected with 0.1 mL of PEG-PCL micelles； the anti-PEG IgM antibody (26.2±9.6 μg/mL) was produced in the second week. After six weeks, the concentration dropped to 21.3±6.7 μg/mL and the IgG antibody concentration rose to 25.03±6.2 μg/mL . The above-mentioned mice successfully developed breast cancer tumors after being injected with 4T1 cells, and 4T1 cells were also observed in the sentinel lymph node immune tissue staining (mouse monoclonal anti-cytokeratin), thus confirming the establishment of a model of breast cancer metastasis to the sentinel lymph node. 18 and 24 hours after injection of micelles carrying near-infrared dye (2DFL-pyran-2CN, excitation wavelength 500 nm/emission wavelength 780 nm, provided by Professor Lin, Department of Chemistry, NCU) was no statistical difference between the average fluorescence intensity of the control group (18hr: 1.52x108/24hr: 1.68x108) and the average fluorescence intensity of the experimental group (18hr: 2.13x108/1.67x108) for the length of time and the presence or absence of induced antibodies. Therefore, the micelles used in this study have good biocompatibility in vitro and in vivo, and have the application potential of sentinel lymph node imaging in human breast cancer.