臺灣博碩士論文加值系統

微脂粒表面以聚乙烯乙二醇(PEG)等聚合物修飾，可以增進微脂粒藥物在動力學上的優勢，使藥物在血液中的循環時間大幅度增長，以便增高藥物在腫瘤部位的累積量，達到提高治療率的效果。然而，使用穩定的微脂粒組成，即利用distearoylphosphatidylcholine (DSPC) / 膽固醇 (Chol) 構成脂雙層膜，製造較緊密的中性小微脂粒包裹抗癌藥物doxorubicin時，即使沒有PEG修飾，也可以在血液中長時間循環。因此，利用PEG修飾增高藥物在血液中的AUC (area under the time-concentration curve) 的效果，在 3% PEG-DSPE 時，AUC 僅增加一倍，即使增高到 6% 的PEG-DSPE，也不能再有效增高 AUC。可是出現 PEG 在微脂粒表面卻阻礙微脂粒進入腫瘤組織，因而減低藥物在腫瘤的累積量，注射藥物七十二小時後的腫瘤內藥物 AUC，沒有 PEG 的Doxorubicin微脂粒劑型 (NPLD) 是有 6% PEG 的Doxorubicin微脂粒劑型 (PLD) 的1.44 倍。NPLD在腫瘤的累積效率 Te ( Te=AUC Tumor/AUC Plasma)是0.87而 PLD 只有 0.31。此外，進行抑制腫瘤生長、增長存活時間及降低副作用等性質比較時，PLD 和 NPLD 均優於 free doxorubicin； PLD 和 NPLD 相互比較時，雖然 NPLD 略優於 PLD，但沒有統計上的差異。總之，於穩定的微脂粒系統，Pegylation 於小鼠C-26腫瘤模型僅有增加血中AUC的效果，並沒有因為循環時間增長而增加治療指數及降低副作用。
Pegylated 微脂粒有較小組織分配量和較長血中循環時間的特性，利用上述性質與抗體的專一標的能力結合，將有助於癌症的標的性治療。我們利用對淋巴癌細胞 (38C13) 上的 idiotype 抗原有特異辨識能力的 S5A8單株抗體 (MAb)，共價結合到微脂粒表面的長鏈 linker 製造免疫微脂粒。用一種對 pH 靈敏的螢光的染料(HPTS)，可以發現 S5A8 免疫微脂粒不但可以與標的細胞 38C13 特異結合，還可以經由細胞吞噬作用將藥物送入細胞內。此外，以S5A8 免疫微脂粒包裹 doxorubicin (S5A8-Lipo-Dox) 處理細胞24小時後，測量 doxorubicin 的攝取量，發現有抗原的38C13細胞是沒有抗原表達的變異細胞(V1-1)的五倍。因此，在測量免疫微脂粒的藥物細胞毒性時，發現S5A8-Lipo-Dox 具有選擇性的細胞毒殺能力。進行藥物動力學試驗時，S5A8-Lipo-Dox 的血中半衰期僅比 PLD 略短，仍具有長效型微脂粒的功能。利用C3H小鼠的淋巴瘤模式進行治療效果試驗，証明 S5A8-Lipo-Dox 的療效優於 PLD 及 free doxorubicin。我們的結果顯示利用與本研究類似的微脂粒系統進行標的性腫瘤療法，可以提高傳統抗癌藥物的療效，值得更進一步的研究發展。

Steric stabilization by polyethylene glycol (PEG) can reduce opsonization of the liposome by plasma proteins. It has a higher plasma area under the concentration-time curve (AUC), which is believed to correlate with better therapeutic efficacy. However, the presence of large molecules on the liposomal surface may reduce interactions of liposomes with cells and hinder the entry of liposomes into the tumor tissue. Using a stable liposomal system composed of distearoyl phosphatidylcholine (DSPC)/cholesterol, we examined the effect of PEG (mol. wt.: 2000) on the pharmacokinetics and on the efficacy of liposomal doxorubicin with C-26 syngeneic tumor model in the BALB/c mice. The plasma AUC of liposomal doxorubicin with 6 mol % PEG-modified distearoylphosphatidylethanolamine (PEG-DSPE) was about twice that of liposomal doxorubicin without PEG at various dosages, regardless of tumor bearing or not. Paradoxically, the tumor concentration of liposomal doxorubicin without PEG was higher. The 72-hour tumor AUC was 1.44 times that of liposomal doxorubicin with 6% PEG-DSPE. The tumor-accumulation efficiency (Te=AUCTumor/AUCPlasma) of liposomal doxorubicin without PEG was 0.87 and this was more than twice that of the liposomal doxorubicin with 6% PEG-DSPE (0.31). At the dose of 10 mg/kg, although both liposomal groups were better than free drug group in terms of clinic-relevant parameters, including toxicity, tumor shrinkage, and survival, there was no difference between two liposomal drug groups. In this stable liposome system, surface coating with PEG (PEGylation) offered no benefit for liposomal doxorubicin in the C-26 tumor model. To enhance the therapeutic index of liposomal doxorubicin, just increasing plasma AUC by PEGylation may not be satisfactory.
Pegylated liposomes results in smaller distribution volume and longer circulation time in blood and thus may improve drug targeting. The characteristics and therapeutic efficacy of immunoliposomes with similar liposomal formulation have never been studied in lymphoma models. We have developed immunoliposomes conjugated with S5A8 monoclonal antibody (MAb), an anti-idiotype antibody to 38C13 murine B-cell lymphoma, and loaded them with doxorubicin using an ammonium sulfate gradient. Purified antibodies were covalently coupled to the termini of PEG on the surface of small unilamellar liposomes. Cell binding and internalization ability of these immunoliposomes was estimated by a fluorescence assay using a pH-sensitive fluorescent dye (HPTS). In vitro cytotoxicity of doxorubicin encapsulated in immunoliposomes was greater for idiotype-positive 38C13 cells than that for idiotype-negative variant (V1-1) of this cell line. In syngeneic C3H/HeN mice, doxorubicin encapsulated in immunoliposomes exhibited long circulation time and was more effective in prolonging survival of mice bearing 38C13 tumor than non-targeted liposomal doxorubicin or free doxorubicin plus empty immunoliposomes. Our results demonstrate the superiority of targeted therapy with these immunoliposomes, and may have potential in cancer treatment.

目 錄
頁次
中文摘要---------------------1
英文摘要---------------------3
縮 寫---------------------5
一、 緒 論
第一節：微脂粒之簡介---------------------8
第二節：微脂粒在藥物傳輸的應用 --------------15
第三節：長效型微脂粒的發展 --------------19
第四節：研究背景與動機---------------------24
二、實驗材料與方法---------------------27
三、結果------------------------------50
四、討論------------------------------63
五、結論------------------------------69
六、參考文獻------------------------------71
七、圖表------------------------------83
八、附錄------------------------------110

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