- 學位論文
聚乙二醇聚內酯兩性高分子材料應用於喜樹鹼藥物之藥物傳輸應用
Poly(ethylene glycol)-co- Poly(lactone) Amphiphilic Copolymer as A Camptothecin Drug Carrier in Drug Delivery System Application
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摘要
本論文以聚乙二醇（mPEG）作為起始劑將戊內酯（delta- valerolactone）及己內酯（epsilon-caprolactone）單體利用開環聚合的方法合成出聚乙二醇-聚戊內酯共聚物（mPEGPVL）以及聚乙二醇-聚己內酯（mPEGPCL）生物可分解兩性高分子材料，之後利用末端改質將磷酸膽鹼（PC）接在mPEGPCL，改質成聚乙二醇-聚己內酯-磷酸膽鹼（mPEGPCLPC）。利用核磁共振光譜儀、傅立葉紅外光譜儀及凝膠滲透層析儀等各項儀器分析，證實mPEGPVL、mPEGPCL和mPEGPCLPC均成功的合成。經由CDCl3與D2O的H1-NMR圖譜中證明mPEGPVL、mPEGPCL和mPEGPCLPC兩性高分子材料在水中可以自我聚集形成微胞。經由動態光散射儀分析微胞的粒徑，粒徑皆小於200 nm。以穿透式顯微鏡與原子力顯微鏡觀察其外型，微胞呈現圓球狀分佈。隨著疏水端（PVL或PCL）分子量越大，發現臨界微胞濃度（CMC）越小，粒徑越大，可以形成穩定的微胞結構。經由37oC的儲存安定性實驗，結果顯示E5000V5000在儲存42天內微胞都沒有聚集或是分離的現象，顯式此微胞有穩定的儲存安定性。使用溶劑揮發與冷凍乾燥方式進行製備喜樹鹼的高分子微胞劑型（CPT-PM），結果發現冷凍乾燥法比溶劑揮發法包覆更多的喜樹鹼。喜樹鹼的包覆率可高達80%以上，藥物濃度高達0.8 mg/ml。利用動態光散射儀分析CPT-PM的粒徑，粒徑皆小於200 nm。利用透析袋方式進行藥物釋放實驗，CPT-PM可以持續有效的釋放到26天以上。在直接稀釋法進行藥物釋放實驗，CPT-PM可以在16天後仍有30%的喜樹鹼lactone form成分。結果顯示CPT-PM可以緩慢釋放喜樹鹼並且在水溶液中可以減緩喜樹鹼水解的速率，保持較多的喜樹鹼lactone form成分。利用老鼠肌肉細胞株（L929 cell line）進行生物相容性實驗，細胞的存活率高達90％以上，證實mPEGPVL、mPEGPCL和mPEGPCLPC兩性高分子材料不具有細胞毒性。將兔子的紅血球與材料微胞或CPT-PM溶液均勻混合，不會造成紅血球溶血的現象，顯示mPEGPVL、mPEGPCL和mPEGPCLPC微胞具有良好血液相容性。利用人類卵巢癌細胞株（ES-2 cell line）進行CPT-PM的細胞毒性實驗，發現CPT-PM最終會與喜樹鹼產生相同的細胞毒性，顯示利用高分子微胞包覆並不會降低喜樹鹼的藥效。在藥物動力學實驗，發現CPT-PM（E5000V6000、E5000C2000和E5000C2000PC）都提高了喜樹鹼的AUC（area under curve），從原本16 hr*ng/ml增加到62 – 77 hr*ng/ml，最多增加了4.8倍。CPT-PM也延長血液中循環時間的半生期（t1/2），從0.25 hr增加到0.25-2.39 hr，最多增加9.6倍。經由藥物動力學實驗評估發現CPT-PM-E5000C2000PC為最佳的劑型，之後以此劑型進行藥效動物實驗。在藥效動物實驗中發現市售藥品Topotecan (10 mg/kg)、CPT-PM-E5000C2000PC (CPT 9 mg/kg) 及CPT-PM- E5000C2000PC (CPT 18 mg/kg)均具有明顯抗腫瘤活性。在細胞植入後第27天其腫瘤抑制速率（TIR %）分別為88%、77%及97%。發現CPT-PM- E5000C2000PC (CPT 18 mg/kg)劑型具有最顯著的抗癌效果，而且比市售藥物Topotecan在統計上有明顯的差異。綜合以上實驗結果，預期合成出的mPEGPVL、mPEGPCL和mPEGPCLPC高分子共聚物將可有效地作為藥物傳送的載體並被應用於醫藥治療等領域。
並列摘要
Amphiphilic block copolymers, methoxy polyethylene glycol-b-poly valerolactone (mPEGPVL) and methoxy polyethylene glycol-b-polycaprolactone (mPEGPCL), were synthesized via ring-opening polymerization of delta-valerolactone and epsilon-caprolactone in the presence of methoxy polyethylene glycol (mPEG). The mPEGPCL was end-modified with phosphorylcholine (PC) for methoxy polyethylene glycol-b-polycaprolactone (mPEGPCLPC). The mPEGPVL, mPEGPCL, and mPEGPCLPC copolymers were successfully synthesized by detection using Nuclear Magnetic Resonance, Fourier Transform Infrared spectroscopy and Gel permeation Chromatography. These copolymers could form micelle-like nanoparticles and the micellar formation was confirmed by 1H-NMR spectra in CDCl3 and D2O. The sizes of all micelles were smaller than 200 nm as measuring by dynamic light scattering detection. The morphology of micelles was spherical shap by using the transmission electron microscope and atomic force microscope analyses. The longer hydrophobic chains (PVL or PCL segments) could reduce the critical micelle concentration (CMC) of micelles, increase the particle size of micelles, and form the more stable micelle structures. Stability analysis showed that the E5000V5000 micellar solutions maintained their sizes at 37 oC for forty-two days without aggregation or dissociation, this micelle had the stable micellar structure. Using two methods to incorporate camptothecin (CPT) into the micelles, the experiments exhibited that the lyophilization method was better than the evaporation method. The CPT encapsulation efficiency was higher than 80 %, and CPT concentration was more than 0.8 mg/ml. The sizes of CPT loaded polymeric micelle formulations (CPT-PM) were smaller than 200 nm using dynamic light scattering detection. The CPT-PM could continuously release CPT for twenty-six days by using dialysis bag method. Using direct dilution method, the CPT-PM could keep 30% biologically active CPT lactone form for sixteen days. These results suggested that the CPT-PM could slow release and reduce the CPT hydrolysis rate in the water solution and maintain more CPT lactone. Using L929 mouse muscular cell line for cytotoxicity, the mPEGPVL, mPEGPCL and mPEGPCLPC copolymers showed higher than 90% relative cell viability, suggesting these copolymers have low cytotoxicity. Incubation of polymeric micelle or CPT-PM solutions with rabbit RBC showed no hemolysis phenomenon, indicating these copolymers have high hemocompatibility.The CPT-PM had similar cytotoxicity as well as CPT free drug using MTT assay with ES-2 ovarian cancer cell line, showing that the polymeric micelles did not reduce the toxicity of CPT. In the pharmacokinetic study, CPT-PMs raised the value of area under curve (AUC), form 16 hr*ng/ml increased to 62-77 hr*ng/ml, that is more than 4.8 times. CPT-PMs also elongated the blood circulation half time (t1/2), form 0.25 hr increased to 0.25-2.39 hr, that is more than 9.6 times. The CPT-PM-E5000C2000PC was the best formulation in the pharmacokinetic study. This formulation was used to do the animal efficacy study. The result of the animal efficacy study exhibited that CPT-PM-E5000C2000PC at CPT 9 mg/kg dose, Topotecan drug at 10 mg/kg dose, and CPT-PM-E5000C2000PC at CPT 18 mg/kg dose, had evident anti-tumor activity. The tumor inhibition rates (TIR %) were 88%, 77% and 97% after twenty-seven days of the cell embedding in mice. The anti-tumor activity of CPT-PM-E5000C2000PC at CPT 18 mg/kg dose was significantly better than Topotecan drug. Taken together, the mPEGPVL, mPEGPCL and mPEGPCLPC copolymers could be used as the drug delivery systems and for the therapeutic fields.
參考文獻
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