以Luminol作為新型光動力治療光源可行性之研究

Abstract

光動力治療係在患處施以一光敏感藥物(photosensitizer)，再於患處照射特定波長之光線以刺激光敏感藥物，其自激發態回到基態時釋放之能量將透過能量轉換(energy transfer)，使患處的分子反應形成具有細胞毒性的自由基(free radicals)、活性氧族(reactive oxygen species)，或單態氧(singlet oxygen)，進而攻擊患處細胞，造成細胞死亡。為求較深的光穿透深度，目前光動力治療之施行主要係利用紅光雷射作為光源，而施行之標的多為淺層部位，例如頭頸部腫瘤，或皮膚疾病。對於較深層或體積較大的腫瘤，受限於光的穿透深度，光動力治療之施行除必須輔以高度侵入性之內視鏡，施行成效亦較不彰。 為改善光動力治療之療效，並擴大其應用，本研究欲發展新式光源以取代現有之紅光雷射，而利用化學反應所釋出之化學冷光，迄今已在諸多領域獲廣泛應用，遂成為本研究考量替代光源之選項。其中，3-胺鄰苯二甲醯胺(luminol)能在水溶液環境釋出波長420nm之化學冷光，是作為替代光源的良好選項。臨床上常用的光敏感藥物中，五胺基酮戊酸(5-Aminolevulinic Acid; 5-ALA)在420nm有較強吸收值，故以之為本研究使用之光敏感藥物。 為評估細胞實驗之可行性，本研究係利用人類的大腸癌細胞株Caco-2作為實驗模型。首先利用冷光分析儀(chemiluminescence analyzer)分析luminol在細胞培養環境中可釋出冷光之強度。在細胞培養環境中，luminol可經由硫酸亞鐵(FeSO4)之氧化進而發出冷光，強度約為1至3×10-14J/cm2，發光期間長於十分鐘。細胞先加入5-ALA作用3.5小時後，在粒腺體內轉化成具有光毒性(phototoxicity)的原紫環IX (protoporphyrin IX)，此時加入luminol及FeSO4，以釋出之冷光進行能量轉換，進而造成細胞損害。反應後72小時，細胞活性及毒性分析中，實驗組與對照組之間出現統計上之明顯差異，染色後在共軛焦螢光顯微鏡觀察，實驗組除細胞數量明顯減少，細胞亦呈現顯著粗糙、細胞膜破損等受損現象。利用流式細胞儀分析，亦獲得與前述分析吻合之結果。 動物實驗係利用免疫缺陷小鼠及作為模型。在小鼠皮下異種植入(xenografting) Caco-2細胞株，待腫瘤成長後，於患處直接注射5-ALA，反應3.5小時候，再依序注射luminol和FeSO4。異種植入腫瘤後，小鼠在血液及血清生化檢驗均呈現異常，但光動力治療組之小鼠有較佳的肝功能，腫瘤之生長速度相較於控制組亦略受抑制。在組織切片檢驗中，光動力治療組之小鼠之腫瘤細胞分化現象較之控制組明顯，細胞排列亦較規則。總體而言，小鼠之生命跡象均相當活躍，可推論注射之藥物並不造成急性毒性。 目前在細胞實驗已獲致成效，動物實驗則需要更大規模實驗以確認其可行性。

Photodynamic therapy (PDT) is a relatively new cancer treatment method, which involves a two-stage process, including the endocytosis of a light-absorbing photosensitizer (PS) and stimulating the PS by light with adequate wavelength. Presently, the light sources used in photodynamic therapy are high intensity lasers or light emitting diodes (LED), making it unsuitable for large-volume tumors and tumors located deep inside the body. To overcome this limitation, we propose an in situ light source to excite the photosensitizer thus generating cytotoxic species to kill the tumor cells directly. Based on this concept, we selected a reagent that acts as the light emitting source, which might be delivered to the tumor site intravenously after photosensitizer administration. Among the light-emitting chemicals, luminol is widely used in forensic medicine for detecting fingerprints or trace amounts of blood. Luminol emits chemiluminescence with 420nm for more than 10 minutes when oxidized by strong oxidants e.g. trace metals. Therefore, luminol is selected as the in situ light source for 5-aminolevulinic acid (5-ALA) -mediated photodynamic treatment of cancerous cell cultures. The chemiluminescence of luminol was initially analyzed by a chemiluminescence analyzer. Cell viability and cytotoxicity were determined using the water soluble tetrazolium salt assay and lactate dehydrogenase assay respectively. Confocal microscopy and flow cytometry were used to evaluate the cell morphology and cell death pathway. According to the results, cancerous cells exposed to 5-aminolevulinic acid for 3.5 hours and subsequently treated with luminol/FeSO4 underwent apoptosis and necrosis. In an animal study, nude and NOD SCID mice were xenografted with Caco-2 cells subcutaneously and the experiment began when the tumor reached a specific size. 5-ALA was topically injected and allowed to react for 3.5 hours. Luminol and FeSO4 were subsequently topically injected. No acute toxicity was observed during the experiment. As for hematological and blood chemistry tests, abnormalities in many categories were found but the mice remained alive. Histopathologic examination revealed that the PDT treatment enhanced cell differentiation, relative regular cellular patterning and cell polarity. The results from the preliminary study warrant additional studies. This method has the potential to extend photodynamic therapy applications to tumors located deep inside the body.