利用Eudragit包覆幾丁聚醣/褐藻酸奈米粒子於大腸直腸癌之偵測

Abstract

大腸直腸癌為國人罹患癌症之前三名，為降低至死率發展有效的偵測方法為 另一重要的方向。內視鏡診斷為臨床上常用之偵測方式，需搭配經驗豐富之醫師加以區分些微變異。目前已發展出利用葉酸接枝之幾丁聚醣並混入褐藻酸做為奈米級藥物載體，用以包覆光感藥物前驅物五胺基酮戊酸。五胺基酮戊酸具有在癌細胞內堆積吡喀紫質的特性，又吡喀紫質為一光感性物質，受激發後可發散出紅色螢光足以做為標定腫瘤之用，而葉酸則做為專一性之鍵結分子增加癌細胞對於奈米粒子的攝取。而以口服方式進行藥物的輸送會面臨複雜的腸胃道環境帶來的破壞，本研究利用具有酸鹼敏感性之EudragitR高分子及冷凍乾燥技術製備出一口服藥丸，並經由餵食人類大腸直腸癌細胞株觀察其吡喀紫質堆積量相對處理前之奈米粒子為90~97%，另由正立螢光顯微鏡可見明顯紅色螢光於經過凍乾處理後之組別；由此結果可知經由冷凍乾燥處理改變奈米粒子形態並不會對其功能造成損失。接著在體外的溶離測試的結果發現經EudragitR高分子的包覆後的藥丸在酸性環境下具有保護內容物之效果；而在中性環境則快速溶解釋放出所含物；以及在藥物釋放實驗中得知在pH 2.5溶液中2小時時釋放出14%五胺基酮戊酸，而在pH 7.4溶液中4小時時則釋放出67%五胺基酮戊酸，此一特性適合做為腸胃道藥物傳輸之用。為了進一步確認在生物體上之應用，需建立原處位的大腸直腸癌小鼠模型，使用手術將BALB/c小鼠的大腸直腸癌細胞株注入盲腸及後段大腸，並於特定時間觀察細胞株所帶之冷光訊號，由H&E染色可發現癌細胞已生長於小鼠腸壁。最後餵食藥丸於已帶有腫瘤之小鼠觀察五胺基酮戊酸標定腫瘤的效果，就目前結果得知在腫瘤位置的紅色螢光訊號並沒有顯著表現，推測原因可以為吸收能力之差異及停留時間，將需要進一步調整以期望達到效果。

Colorectal cancer is the third common cancer in Taiwan, and the most common histologic type is adenomatous polyposis. Removing adenomatous polyps in earlier stage by surgery can reduce the mortality efficaciously. Colonoscopy is a common way to discover the dysplasia of colonic tissues directly. But some small adenomatous polyps or cancers in early-stage could be easily missed during endoscopy examinations due to the little differences between normal tissues and cancerous tissue. The main aim of this study was to prepared an oral formulation for protected nanoparticles from degrade in acidic environment. Because the oral nanoparticles solution has been challenged in the acidic fluids of stomach which might not be able to preserve nanoparticles from destruction. To overcome this problem, freeze-dried nanopartilces and EudragitR L 100-55 coated was used in this study. L100-55 is a well-known pH sensitive polymer and is used as a material for enteric coating for protected the drugs from dissolution or collapse in the acidic environments. And the oral nanoparticles solution had been reported in our previous study, compose by folic acid conjugated chitosan and alginate as the vehicle for 5-ALA and is abbreviated as fCANA. fCANA has shown the accumulations of PpIX in colorectal cancer cells to label out cancer cells. PpIX expression in colorectal cancer cells after freeze-drying process was observed about 90-97 % compared to that before freeze-drying process. In addition, red fluorescence was observed obviously in fluorescent microscopy when fed with nanoparticles after freeze-drying process. These results indicated that changes in the morphology during freeze-drying process would not attribute to damages of fCANA. Next, EudragitR coated pills provided the ability of protection from releasing in acidic environment and rapid release of loading dye in neutral environment via in vitro dissolution tests. Indicate that the prepared pills are suitable for drug delivery in GI tract. For further in vivo applications, an orthotopic colon cancer mice model was established by injected CT-26 cells in the cecum and rectum of BALB/c mice and monitored by bioluminescence image at time intervals. Cancer cells could be found in H&E stain slides and showed that cancer cells grew on the walls of colon. Eventually, pills were fed to tumor bearing mice and the accumulation of PpIX at tumor sites were observed. The results so far showed no significant red fluorescence. The probable reason may be the difference of absorptions and retention time between small intestine and rectum. More improvements are required in the future.