紅血球膜衍伸藥物載體被視為新一代的藥物傳遞平台，因其帶有自我識別分子可避免免疫系統辨識攻擊，以及其他紅血球具有之生理穩定度優勢。而遙控觸發藥物傳遞系統為一深具應用潛力的藥物載體設計，因其能遠端控制藥物釋放的時間、劑量或位置而達到更佳治療效果。本研究結合此兩系統的優勢，成功由紅血球細胞膜製作出多功能、可藉超音波觸發藥物釋放的藥物載體－紅血球細胞膜衍伸相變液滴並探討其醫學診斷與治療之應用。
我們提出假設預期紅血球細胞膜衍伸相變液滴之低沸點液體核心可經由高強度聚焦式超音波照射觸發汽化而促使藥物釋放。並且產生之微氣泡可作為超音波對比劑以增強超音波顯影。
實驗製作出之紅血球細胞膜衍伸相變液滴呈現均一大小，直徑約為1.7 μm，具良好分散性及膜蛋白保留情形，抗癌藥物喜樹鹼 (Camptothecin, CPT) 裝載量約2-3%。在超音波照射觸發下，可促使40%的藥物釋放並造成70%癌細胞死亡。超音波觸發汽化產生的氣泡可顯著提高超音波回聲訊號30分貝，並可藉由爆破力量物理性傷害鄰近癌細胞造成60%的癌細胞死亡。活體測試中同樣能藉由超音波觸發體內紅血球細胞膜衍伸相變液滴之汽化，並增強超音波顯影及造成目標區域出血等物理性損傷。
總結實驗結果顯示我們成功建立一新式多功能可遙控觸發釋放的紅血球膜衍伸藥物載體，並可作為超音波對比劑，對於臨床應用具有相當大的潛力。

Red blood cell (RBC)–derived drug carriers are considered as new generation drug delivery platforms because of its advantages of self-recognition for avoiding immune responses. And remotely triggerable drug delivery systems are advantageous on their ability to control the timing, duration, dosage or location of drug delivery. To combine advantages of these two great delivery systems, a new multi-functional, acoustically-activated drug release droplets made of naturally derived red blood cell membrane (RBCM) were fabricated and tested for the potential theranostic applications in this study. We hypothesized that the RBCM droplets (RBCMDs) with great biocompatibility can be vaporized by insonation using high intensity focused ultrasound (HIFU) and results in sudden droplet-bubble transition for on-demand drug release. Additionally, the generated microbubbles could serve as contrast agent to enhance ultrasound imaging. The as-synthesized RBCMDs exhibited uniform size (1.7 μm in diameter), good dispersity and well preservation of RBC membrane-associated proteins that protect the droplets from macrophage uptake. Camptothecin (CPT), an anti-cancer drug, was successfully loaded in the RBCMD with loading efficiency (L.E.) of 2-3% and encapsulation efficiency (E.E.) of 62-97%. Upon short period (3 min) of HIFU irradiation, up to 40% of the encapsulated CPT can be released from the RBCMDs and caused high cell death rate (up to 69%). Besides, the acoustically vaporized RBCMDs significantly increased ultrasound echo signal to 30 dB, and the HIFU-induced physical droplet explosion was capable of damaging nearby cancer cells and resulted in significant cell death (up to 62%). In vivo examinations also show that RBCMDs can be acoustically vaporized in target region, cause blood vessel damage and enhance ultrasound imaging. To summarize, we have developed a new class of naturally derived RBCMDs with great potential on remotely triggerable drug delivery and ultrasound imaging enhancement.

摘要 1
ABSTRACT 2
目錄 4
圖目錄 8
表目錄 11
1. 緒論 12
1.1 前言 12
1.2 實驗簡述 13
2. 文獻回顧 15
2.1 紅血球 15
2.1.1 紅血球概論 15
2.1.2 紅血球細胞膜 15
2.1.3 紅血球衍伸藥物載體 17
2.2 可遙控釋放藥物載體 23
2.2.1 可遙控釋放藥物載體概論 23
2.2.2 光觸發藥物載體 23
2.2.3 磁觸發藥物載體 24
2.2.4 超音波觸發藥物載體 25
2.3 相變液滴 28
2.3.1 相變液滴概論 28
2.3.2 聲學激發相變液滴汽化 29
2.3.3 相變液滴之應用 30
3. 實驗材料與方法 32
3.1 實驗材料 32
3.2 紅血球細胞膜萃取與純化 32
3.2.1 小鼠紅血球細胞膜萃取與純化 32
3.2.2 人類紅血球細胞膜萃取與純化 33
3.3 製作紅血球細胞膜相變液滴 33
3.4 液滴外觀與粒徑大小分布測量 33
3.4.1 顯微影像觀察 33
3.4.2 粒徑大小分布測量 33
3.5 紅血球膜蛋白保留分析 34
3.5.1 樣本製備 34
3.5.2 十二烷基硫酸鈉聚丙烯酰胺凝膠電泳 (Sodium Dodecyl Sulfate Polyacrylamide Gel Electrophoresis, SDS-PAGE) 34
3.6 藥物攜載效率分析 35
3.6.1 樣本製備 35
3.6.2 標準品檢量線製作 35
3.5.3 樣本測定 35
3.7 液滴穩定度分析 36
3.8 巨噬細胞吞食測試 36
3.8.1 小鼠初代腹腔巨噬細胞分離培養 36
3.8.2 巨噬細胞吞食液滴測試 37
3.9 超音波觸發液滴汽化之高速影像觀察 37
3.10 B-MODE超音波顯影 38
3.10.1 系統架構 38
3.10.2 樣本測定 39
3.11 超音波觸發液滴汽化對癌細胞之物理傷害評估 39
3.11.1 系統架構 39
3.11.2 MTT細胞存活度分析 40
3.12 超音波觸發藥物釋放對癌細胞傷害評估 40
3.12.1 超音波觸發藥物釋放 40
3.12.2 釋放藥物對癌細胞傷害評估 41
3.13 小鼠體內超音波觸發液滴汽化觀察 41
3.13.1 窗型觀測腔顯微影像 41
3.13.2 B-mode 超音波顯影 42
3.14 細胞培養 42
4 結果與討論 43
4.1 小鼠紅血球膜相變液滴 43
4.1.1 光學影像與粒徑大小分析 43
4.1.2 紅血球膜蛋白保留分析 44
4.1.3 藥物攜載效率分析 45
4.1.4 液滴穩定度測試 46
4.1.5 巨噬細胞吞食測試 47
4.1.6 超音波觸發液滴汽化 50
4.1.7 超音波觸發藥物釋放與對癌細胞傷害評估 51
4.1.8 B-mode超音波顯影 54
4.1.9 超音波觸發液滴汽化對癌細胞之物理傷害 55
4.1.10 小鼠體內超音波觸發液滴汽化觀察 57
4.2 人類紅血球膜液滴 61
4.2.1 光學影像與粒徑大小分析 61
4.2.2 細胞膜膜蛋白保留分析 62
4.2.3 藥物攜載效率分析 63
4.2.4 液滴穩定度測試 64
4.2.5 超音波觸發液滴汽化 65
4.2.6 超音波觸發藥物釋放與對癌細胞傷害評估 66
4.2.7 B-mode超音波顯影 69
4.2.8 超音波觸發液滴汽化對癌細胞之物理傷害 70
5 結論 73
6 未來工作 74
7 參考文獻 75

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