臺灣博碩士論文加值系統

腫瘤形成的過程中，為能供給足夠的養分與氧氣，腫瘤內部進行急遽的血管新生，然而多數粗大且不健全，在血管無法供應足夠養分與氧氣的地方將形成缺氧性區域，而此區阻礙化學治療成為腫瘤治療的瓶頸，為此需要新的治療方法將藥物標靶至缺氧性區域。近期研究顯示，單核球及其分化的巨噬細胞會趨向腫瘤並滲透積聚於腫瘤缺氧性區域，因此我們期望利用巨噬細胞之此特性將藥物或基因傳遞至腫瘤內部缺氧缺血區。
本研究之目的是探討利用巨噬細胞滲透至腫瘤之特性，承載包覆藥物相變液滴作為藥物傳遞載體。自製相變液滴以疏水作用力和靜電吸引力包覆一線化療
藥物阿黴素(DOX)於相變液滴脂質殼層之疏水端，作為新式藥物載體(DOX-Droplet)，藉由超音波熱效應或機械效應可將相變液滴內所包覆之液態全氟碳化合物轉變為氣態，此稱為acoustic droplet vaporization(ADV)，並藉由此相變液滴汽化之爆破性膨脹、穴蝕效應、殼層破裂將藥物作釋放。
研究工作首先為製備藥物載體，並針對DOX-Droplet做完整的特性量測。將DOX-Droplet與小鼠巨噬細胞株(RAW 264.7)一同培養，藉由吞噬作用，巨噬細胞將承載包覆藥物相變液滴，對已承載DOX-Droplet之巨噬細胞進行多項評估，包含巨噬細胞對DOX-Droplet之攝取量，其細胞存活率是否受到DOX-Droplet之影響，以及探討承載DOX-Droplet是否阻礙細胞移行之能力，最後利用3.5-MHz高能聚焦式超音波探頭發射極短脈衝(3 cycles, 200 Hz pulse repetition frequency (PRF) , 12 MPa)激發承載DOX-Droplet之巨噬細胞，收集照射後的細胞溶液上清液，加入小鼠攝護腺癌細胞株(Tramp-C1)，待48小時後量測Tramp-C1細胞存活率以評估承載相變液滴之巨噬細胞藉由超音波激發釋藥之效果。
結果顯示以巨噬細胞承載包覆藥物相變液滴作為藥物傳遞載體於未來應用上極具發展潛力。在24小時內巨噬細胞存活率未受承載相變液滴而有所影響，在細胞移行能力上，承載越多的相變液滴將導致細胞移行能力的降低，然而此問題可以採用多次治療的方式或針對細胞進行移行能力訓練作補償。透過高速攝影機拍攝影像，我們可得知高能聚焦式超音波確實能將巨噬細胞體內之相變液滴進行激發，並在汽化的同時將其上藥物作釋放達到毒殺腫瘤細胞之效果。
本研究所提出的以巨噬細胞作為藥物傳遞載體，包含承載藥物、標靶傳遞至腫瘤、超音波激發釋藥之系統，經評估極具可行性，但卻面臨藥物承載量有限導致治療效果不佳之問題，未來工作將改進藥物載體，增進載藥量並著重於藥物滯留能力，並探討相變液滴汽化同時產生之機械行效應，期望本研究於未來可實際應用於腫瘤動物模型治療上。

Tumors normally possess irregular vasculature, and tend to outstrip blood supplement and become hypoxia or ischemia, which results in the resistances of the tumor to chemotherapy treatment. New therapies are required to target hypoxic or ischemic areas in tumors. Recently, many studies have shown that monocytes and derived macrophages can be engineered to actively migrate toward tumors and infiltrate avascular and hypoxic areas. The properties of macrophages prompted us to propose the drug or genes delivery by macrophages to otherwise inaccessible areas within tumors.
The aim of this study is to investigate the feasibility of using macrophages to infiltrate hypoxic or ischemic areas in tumors, as the carriers of drug-loaded phase-change droplets. We develope a drug-loaded droplet formulation (DOX-Droplet) with a high loading capacity of doxorubicin (DOX) drug, which was complexed to the lipid shell by both hydrophobic and electrostatic interactions. Ultrasound induced the transition from liquid droplets to gas bubbles was referred as the effect of acoustic droplet vaporization (ADV), and ADV can trigger encapsulated drug release from the droplet-loaded macrophages.
DOX-Droplets were fabricated via the thin-film hydration method. The DOX drug encapsulation efficiency was 76.55 ± 5.97% estimated by a Fluorescence spectrophotometer. RAW 264.7 cells (mouse leukaemic monocyte macrophage cell line) were used to ingest the drug-loaded phase-change droplets. For evaluating DOX-Droplets uptake efficiency, cell viability and migration mobility of DOX-Droplet loaded macrophages, flow cytometric analysis, alarmarBlueTM assay, and transmembrane cell migration assay were measured, respectively. In vitro ultrasound triggering DOX release, a 3.5-MHz high-intensity focused ultrasound transducer was used. The DOX-Droplets loaded RAW 264.7 cells were insonated by a three-cycle, 200 Hz PRF and 12-MPa HIFU pulses for 10 min. After exposed to ultrasound the supernatant was collected and incubated with Tramp-C1 cells (mouse prostate cancer cell line) for 48 hr. The cytotoxicity of DOX released from DOX-Droplets loaded RAW 264.7 cells was evaluated by Tramp-C1 cell viability.
The results demonstrate the feasibility of using macrophages to deliver DOX-loaded phase-change droplets. Cell viability assays reveals that the loading of droplets did not affect cell viability for 24 hr. But transmembrane cell migration assay reveals that the loading of droplets hampered the migration mobility of RAW 264.7 cells. In addition, Tramp-C1 cell viability analysis performed that ultrasound can trigger DOX release from DOX-droplet loaded RAW 264.7 cells effectively and the release DOX killed the Tramp-C1 cell.
Future works include the drug payload and retention improvement of DOX-Droplet, the assessments of mechanical effect of droplet vaporization on therapy and the liberated drug payload via ultrasound-triggered vaporization in vivo.

第一章 緒論 1
1.1 醫用超音波 1
1.1.1 對比劑-微氣泡 1
1.1.2 超音波與微氣泡之應用 3
1.1.3 新式對比劑-相變液滴 4
1.1.4 相變液滴之應用 5
1.2 惡性腫瘤 6
1.2.1 腫瘤微環境 8
1.2.2 腫瘤治療 10
1.2.3 腫瘤缺氧區與治療瓶頸 10
1.3 細胞調控藥物傳遞(cell-mediated drug delivery) 12
1.3.1單核-巨噬細胞 13
1.3.2 腫瘤巨噬細胞 13
1.3.3 巨噬細胞與腫瘤缺氧區 14
1.3.4 巨噬細胞作為傳遞載體 14
1.4 藥物載體 15
1.4.1 阿黴素(doxorubicin) 15
1.4.2 包覆藥物之相變液滴 17
1.5 研究目的與內容 19

第二章 實驗材料與方法 20
2.1 概論 20
2.2 包覆藥物相變液滴(DOX-Droplet)之製備 20
2.2.1 光學定性分析 22
2.2.2 粒徑分析 22
2.2.3 藥物包覆量定量 22
2.2.4 藥物滯留率量測 23
2.3 細胞培養 24
2.3.1 細胞株 24
2.3.1.1小鼠巨噬細胞株(RAW 264.7) 24
2.3.1.2小鼠攝護腺癌細胞株(Tramp-C1) 24
2.3.2 細胞培養液配製 24
2.3.2.1 RPMI細胞培養液 24
2.3.2.2 DMEM細胞培養液 25
2.3.2.3 Tramp-C1條件培養液(Tramp-C1 condition medium) 25
2.3.3 細胞繼代 25
2.4 巨噬細胞承載包覆藥物相變液滴 26
2.4.1共軛焦顯微鏡影像 26
2.4.2 相變液滴承載量評估 27
2.4.3 巨噬細胞存活率分析 27
2.4.3 巨噬細胞移行能力評估 29
2.5 超音波激發巨噬細胞體內相變液滴汽化並釋藥 30
2.5.1 物理特性 30
2.5.2超音波影像對比 32
2.5.3汽化前後巨噬細胞細胞型態的改變 34
2.5.4汽化前後巨噬細胞細胞存活率分析 34
2.5.5釋藥毒殺攝護腺癌細胞 35
2.6 數據統計 36

第三章 實驗結果與討論 37
3.1 DOX-Droplet製備及其性質量測 37
3.1.1 DOX-Droplet粒徑分布 38
3.1.2 DOX-Droplet物理穩定性 39
3.1.3 DOX-Droplet包藥效率及包藥量 42
3.1.4 DOX-Droplet藥物滯留率 46
3.2 巨噬細胞承載包覆藥物相變液滴 48
3.2.1共軛焦顯微鏡影像 49
3.2.2 巨噬細胞承載量最佳化之結果 50
3.2.3 承載相變液滴後細胞之存活率 52
3.2.4 承載相變液滴後細胞之移行能力 55
3.3 超音波激發巨噬細胞體內相變液滴汽化並釋藥 59
3.3.1 高速攝影機拍攝光學影像 59
3.3.2 聲學影像對比 60
3.3.3 汽化前後巨噬細胞型態的改變 63
3.3.4 汽化前後巨噬細胞之存活率 64
3.3.5 釋藥毒殺攝護腺癌細胞之結果 66

第四章 結論與未來展望 71
4.1 結論 71
4.2 未來展望 72
4.2.1 提升相變液滴載藥量 72
4.2.2 探討相變液滴汽化之機械性效應 73
4.2.3 實際應用於腫瘤動物模型 74

參考資料 75

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