臺灣博碩士論文加值系統

藥物傳遞的主要目標是希望增加藥物在腫瘤組織中的濃度，以達到控制腫瘤成長的效果；且降低藥物在身體其他部位的濃度，減少在正常組織中的副作用，達到所謂的標靶治療。高能量強度聚焦型超音波已用於臨床治療，近年來，許多研究更發現超音波搭配超音波顯影劑所產生的穴蝕效應對區域性的藥物傳遞有益。
本研究使用大腸癌CT-26小鼠皮下腫瘤模式，注射超音波顯影劑(SonoVue○R)，搭配1 MHz、1% duty cycle、10ms脈衝長度、1 Hz的脈衝重複頻率、聲壓1.2 MPa、超音波施打總時間120秒的低強度聚焦型超音波，以誘發穴蝕效應，與使用市售奈米抗癌藥物Doxil○R (Ben Venue, Laboratories, Inc., USA)。實驗結果發現，相較僅注射10 mg/kg Doxil○R，搭配穴蝕效應之實驗組顯著提升了在治療後24小時，doxorubicin於腫瘤組織內的累積量，且藥物搭配穴蝕效應實驗組在實驗後第24小時達其極值；持續給予治療，穴蝕效應對腫瘤組織的影響將會變高，可有效抑制大腫瘤(體積＞100 mm3)的成長(p＜.05)。同時，注射較低劑量5 mg/kg的Doxil○R，在穴蝕效應的加強下，相較無穴蝕效應實驗組，已可有效抑制小腫瘤(體積＜30 mm3)的成長(p＜.05)。實驗同時探討治療方式(額外施加超音波與否)對腫瘤成長的影響。

The main goal of drug delivery is to enhance the drug accumulation in tumor tissue in order to control the tumor growth, and to decrease drug concentration in other sites of the body to avoid side effect on normal tissues. Such treatment is so-called “targeted therapy.” High intensity focused ultrasound (HIFU) has been used for clinical therapy for years. Recently, it was found that the cavitation effect due to ultrasound sonication and ultrasound contrast agent was beneficial to local drug delivery.
In this study, we used s.c. colon carcinoma CT-26 mice tumor model and sonicated the tumors with focused ultrasound (frequency 1 MHz、1% duty cycle、pulse length 10ms、pulse repetition frequency 1Hz、pressure 1.2 MPa、total duration 120 seconds) after the injection of ultrasound contrast agent (SonoVue○R), and then inkected nanoparticles—commercial nanodrug Doxil○R (Ben Venue, Laboratories, Inc., USA). We have found that cavitation effect combined with 10 mg/kg Doxil○R can significantly increase doxorubicin accumulation in tumor tissue 24 hour after treatment as compared to injecting Doxil○R only. Keeping the treatment for a duration time (at least 4 weeks), the effect of cavitaiton on tumor tissue becomes more obvious and able to inhibit big tumor (volume＞100 mm3) growth effectively(p＜.05). In addition, 5 mg/kg Doxil○R injection with cavitation has significant impact (p＜.05) on inhibiting the growth of small tumors (volume＜30 mm3). This study also investigated treatment effects to mouse tumors with applying additional focused ultrasound.

誌 謝 ………………………………………………………………………… i
摘 要 ………………………………………………………………………… ii
目 錄 ……………………………………………………………..………….. iv
圖 目 錄 ……………………………………………………………………….... vii
表 目 錄 ………………………………………………………………………… viii
第 一 章 緒論….………………………………………………………………… 1
1-1 腫瘤…………………………………………………………………… 1
1-2 化學治療……………………………………………………………… 1
1-3 藥物傳輸……………………………………………………………… 2
1-4 腫瘤血管特性………………………………………………………… 2
1-5 奈米粒子……………………………………………………………… 3
1-6 超音波治療………...…………………..……………………………… 5
1-7 機械參數……………………………………………………………… 8
1-8 研究動機與目標……………………………………………………… 9
第 二 章 實驗設備材料與方法………………………………………………… 10
2-1 腫瘤細胞株…………………………………………………………… 10
2-2 實驗動物……………………………………………………………… 10
2-3 腸癌腫瘤模式………………………………………………………… 10
2-4 超音波顯影劑………………………………………………………… 11
2-5 實驗超音波裝置……………………………………………………… 11
2-6 超音波治療儀………………………………………………………… 14
2-7 奈米抗癌藥物………………………………………………………… 15
2-8 治療與組織樣本採集………………………………………………… 15
2-9 腫瘤組織confocal影像………………………………………………. 15
2-10 藥物隨時間在組織內之累積量……………………………………...… 16
2-11 Doxorubicin、Doxil○R與Lipo-Dox之激發螢光波長及強度之關係.....
16
2-12 藥物在腫瘤組織內的分佈量測定…………………………………...… 17
2-13 治療方法、藥物劑量與腫瘤體積之關係…………………………….. 17
2-13-1 腫瘤大小對應同等Doxil○R劑量搭配穴蝕效應治療之反應………….
17
2-13-2 不同Doxil○R劑量搭配穴蝕效應治療………………………………….
18
2-13-3 治療方法(額外聚焦超音波或持續式超音波)對腫瘤的影響……….... 18
2-13-4 各實驗條件所使用之小鼠與樣本數及實驗設計…….……………….. 19
2-14 數值統計與分析方法………………………………………................... 20
第 三 章 結果…………………………………………………………................ 21
3-1 腫瘤組織confocal影像……………………………………………….... 21
3-2 Doxorubicin、Doxil○R之螢光發散波長與螢光強度……...……………..
22
3-3 Doxil○R之標準曲線……………………………………………...………
23
3-4 治療24小時後藥物在腫瘤組織內的分佈量測定…………………….. 24
3-5 藥物短時間在腫瘤組織內的分佈量測定……...……………………… 25
3-6 治療方法與藥物劑量對起始腫瘤體積關係……………...…………… 26
3-6-1 腫瘤大小對應同等Doxil○R劑量搭配穴蝕效應治療之反應………….
26
3-6-2 不同Doxil○R劑量搭配穴蝕效應治療………………………………….
28
3-6-3 治療方法(額外聚焦超音波或持續式超音波)對腫瘤的影響……...…. 30
第 四 章 討論…………………………………………………………................ 32
4-1 腫瘤組織confocal影像……………………………………………...…. 32
4-2 治療24小時後藥物在腫瘤組織內的分佈量測定…………………….. 32
4-3 藥物隨時間在腫瘤組織內的分佈量測定…………………................... 33
4-4 治療方法與藥物劑量對起始腫瘤體積關係………………................... 33
第 五 章 結論與未來工作……………………………………………................ 35
參考資料 ………………………………………………………………………… 37
附錄 …………………………………………………………………………... 41

[1]Byrne J.D., Betancourt T., and Peppas B.L., “Active targeting schemes for nanoparticle systems in cancer therapeutics,” Adv Dru Deli Rev 2008; 60: 1615-1626.
[2]Chen H., Li X., Wan M., and Wang S., “High-speed observation of cavitation bubble cloud structures in the focal region of a 1.2 MHz high-intensity focused ultrasound transducer,” Ultrason Sonochem 2007; 14: 291-297.
[3]C homas J.E., Dayton P., May D., and Ferrara K.W., “Threshold of fragmentation for ultrasonic contrast agents,” J Biomed Opt 2001; 6: 141-150.
[4]黃啟訓(2008)。聚焦式超音波配合超音波顯影劑應用於小鼠正常與腫瘤血管滲透性之探討。碩士論文，國立台灣大學醫學工程研究所。
[5]Frenkel V., Etherington A., Greene M., Quijano J., Xie J., Hunter F., Dromi S., and Li K.C., “Delivery of liposomal doxorubicin (Doxil) in a breast cancer tumor model: investigation of potential enhancement by pulsed-high intensity focused ultrasound exposure,” Acad Radiol. 2006; 13(4): 469-479.
[6]Gabizon A., Shmeeda H., and Barenholz Y., “Pharmacokinetics of pegylated liposomal doxorubicin. Review of animal and human studies,” Clin Pharmacokinet 2003; 42(5): 419-436.
[7]Gao Z.G., Fain H.D., and Rapoport N., “Controlled and targeted tumor chemotherapy by micellar-encapsulated drug and ultrasound,” J Control Release 2005; 102(1): 203-222.
[8]Gao Z., Kennedy A.M., Christensen D.A., and Rapoport N.Y., “Drug-loaded nano/microbubbles for combining ultrasonography and targeted chemotherapy,” Ultrasonics 2008; 48(4): 260-270.
[9]Gregoriadis G. and Ryman B.E., “Liposomes as carriers of enzymes or drugs: a new approach to the treatment of storage diseases,” Biochemical 1971; 124:58P.
[10]Haley B. and Frenkel E., “Nanoparticles for drug delivery in cancer treatment,” Urol.Onc. 2008; 26: 57-64.
[11]Heuser L.S. and Miller F. N., “Differential macromolecular leakage from the vasculature of tumors,” Cancer 1986; 57: 461-464.
[12]Hynynen K., “Review of ultrasound therapy,” Ultrasonics symposium 1997; p. 1305-1313.
[13]Iwanaga K., Tominaga K., Yamamoto K., Habu M., Maeda H., Akifusa S., Tsujisawa T., Okinaga T., Fukuda J., and Nishihara T., “Local delivery system of cytotoxic agents to tumors by focused sonoporation,” Cancer Gene Ther. 2007; 14(4): 354-363.
[14]Kennedy J. E., ter Haar G. R. and Cranston D., “High intensity focused ultrasound: surgery of the future?” British Journal of Radiology 2003; 76: 590–599.
[15]Larkin J.O., Casey G.D., Tangney M., Cashman J., Collins C.G., Soden D.M., and O''Sullivan G.C., “Effective tumor treatment using optimized ultrasound-mediated delivery of bleomycin,” Ultrasound Med Biol. 2008; 34(3): 406-413.
[16]Lasic D. and Martin F., Stealth Liposome, Boca Raton (FL): CRC Press, 1995. p. 103-117.
[17]Lasic D. and Martin F., Stealth Liposome, Boca Raton (FL): CRC Press, 1995. p. 139-147.
[18]Lasic D. and Martin F., Stealth Liposome, Boca Raton (FL): CRC Press, 1995. p. 149-171.
[19]Lin C.Y., Liu T.M., Chen C.Y., Huang Y.L., Huang W.K., Sun C.K., Chang F.H., and Lin W.L., “Quantitative and qualitative investigation into the impact of focused ultrasound with microbubbles on the triggered release of nanoparticles from vasculature in mouse tumors,” J Control Release. 2010; 146(3):291-298.
[20]Maeda H., “SMANCS and polymer-conjugated macromolecular drugs: advantages in cancer chemotherapy,” Adv. Drug Deliv. Rev. 2001; 46: 169-185.
[21]Maeda H., Wu J., Sawa T., Matsumura Y., and Hori K., “Tumor vascular permeability and the EPR effect in macromolecular therapeutics: a review,” J. Control. Release 2000; 65: 271-284.
[22]O''Neill B.E., Vo H., Angstadt M., Li K.P., Quinn T., and Frenkel V., “Pulsed high intensity focused ultrasound mediated nanoparticle delivery: mechanisms and efficacy in murine muscle,” Ultrasound Med Biol. 2009; 35(3): 416-424.
[23]Rapoport N., Gao Z., and Kennedy A., “Multifunctional nanoparticles for combining ultrasonic tumor imaging and targeted chemotherapy,” J Natl Cancer Inst. 2007; 99(14): 1095-106.
[24]Sakakima Y., Hayashi S., Yagi Y., Hayakawa A., Tachibana K., and Nakao A, “Gene therapy for hepatocellular carcinoma using sonoporation enhanced by contrast agents,” Cancer Gene Ther. 2005; 12: 884-889.
[25]Seip R., Chin C.T., Hall C.S., Raju B.I., Ghanem A., and Tiemann K., “Targeted ultrasound-mediated delivery of nanoparticles: on the development of a new HIFU-based therapy and imaging device,” IEEE Trans Biomed Eng. 2010; 57(1): 61-70.
[26]吳聖凱(2008)。單頻與共焦雙頻聚焦式超音波配合超音波顯影劑應用於局部血腦屏障開啟之探討。碩士論文，國立台灣大學醫學工程研究所。
[27]Stieger S.M., Caskey C.F., Adamson R.H., Qin S., Curry F.R., Wisner E.R., and Ferrara K.W., “Enhancement of vascular permeability with low-frequency contrast-enhanced ultrasound in the chorioallantoic membrane model,” Radiology 2007; 243(1): 112-121.
[28]Tang H., Wang C. C. , Blankschtein D., and Langer R., “An investigation of the role of cavitation in low-frequency ultrasound-mediated transdermal drug transport,” Pharm Res 2002; 19: 1160-1169.
[29]王慈吟(2006)。微氣泡輔助之穴蝕效應與超音波治療之應用。碩士論文，國立台灣大學電機工程學研究所。
[30]Yuh E.L., Shulman S.G., Mehta S.A., Xie J., Chen L., Frenkel V., Bednarski M.D., and Li K.C., “Delivery of systemic chemotherapeutic agent to tumors by using focused ultrasound: study in a murine model,” Radiology 2005; 234(2): 431-437.
[31]曾雲龍(1999)。Doxorubicin微脂粒劑型應用於癌症標的治療之探討，博士論文，國立台灣大學生化學研究所。
[32] 李咏馨(2009)。非侵入式聚焦超音波結合超音波顯影劑應用於中樞神經系統藥物傳輸之強化與監控，碩士論文，國立台灣大學醫學工程研究所。
[33] Duck FA., Baker AC., and Starritt HC., Ultrasound in medicine, 1997. P204.