探討脈衝超音波熱治療結合抗癌奈米藥物對轉移性黑色素腦腫瘤之治療效果

Abstract

研究背景與目的: 人類黑色素瘤是一種不正常增生之惡性腫瘤，晚期黑色素瘤的特性具高度侵略性、轉移性與致死率，其中將近40-50% 患有第四期黑色素瘤的病患確診轉移到腦部，在所有癌症中轉移到腦部排名第三位。一旦黑色素瘤轉移到腦部難以有效控制腫瘤生長，究其無法有效治療的原因是因為血腦障壁/血腫瘤障壁的限制，其調控所有物質進出避免病原體或危害物質入侵大腦，因此黑色素腦腫瘤難以單純使用藥物治療。在本研究中，我們將探討低劑量脈衝超音波結合聚乙二醇化微脂體包覆化療藥物阿黴素 (doxorubicin)，對黑色素瘤轉移到腦部腫瘤的治療效果，低劑量脈衝超音波能非侵入式標的腦中腫瘤組織進行熱升溫效果，在不傷害正常腦組織的情形下，使更多化療藥物累積腫瘤組織，提升整體抗癌效果。 實驗材料與方法: 本研究利用細胞實驗與動物實驗探討合併抗癌效果。細胞實驗使用黑色素癌細胞夾帶綠色螢光蛋白 B16F10-GFP 細胞，利用不同濃度藥物 pegylated liposomal doxorubicin (PLD) 處理不同時間後 (24 與 72 小時)，測試其細胞存活率，之後再結合低劑量脈衝式超音波與固定濃度 PLD 作用下 (共分為控制組、藥物 PLD 組、超音波組以及藥物 PLD+超音波合併治療組)，進一步測試細胞存活率分析。動物實驗方面將 B16F10-GFP 細胞直接種入 C57BL/6 黑鼠腦部，實驗組別分為控制組、藥物 PLD 組、超音波組以及藥物 PLD+超音波合併治療組，動物使用藥物濃度 5 mg/kg 靜脈注射後體循環一分鐘，再施打超音波參數頻率為 0.5 MHz、脈衝重複頻率 1000 Hz，工作週期為 50%，施打時間 10 分鐘，利用非侵入式影像系統觀察動物不同天數腫瘤生長趨勢，並將動物犧牲後取下組織測定其藥物含量以及做切片觀察腫瘤的型態、細胞凋亡情形，最後會測試腫瘤組織內蛋白表現變化。 實驗結果: 從細胞實驗上觀察到單純藥物 PLD 在不同時間作用下對細胞有不同抑制效果，且當固定藥物濃度再結合超音波施打，會對細胞產生更顯著的生長抑制作用，合併治療組會有較好的細胞生長影響。在動物實驗方面確定超音波參數，測試其安全性以及藥物有效傳輸性。腫瘤生長大小方面合併治療組的腫瘤相較其他組別冷光表現最少，有效抑制腫瘤大小；取下組織測定藥物濃度發現，合併治療組的腫瘤區域藥物含量高於其正常腦組織測得藥量，而單純藥物組中不論腫瘤組織或正常腦組織，都低於合併治療組當中腫瘤組織藥物含量；在觀察腫瘤 H&E 染色切片中，控制組與單純藥物組腫瘤會持續分裂，單純超音波組與合併治療組受超音波影響細胞排列，且合併治療組會有較明顯藥物影響情形。切片觀察中，PLD 在組織分布性會集中分布，且根據定量結果顯示合併治療組相對單純藥物組會有更多藥物累積；IHC 標的特殊蛋白 (Ki-67 及 Caspase-3)、TUNEL 檢測以及西方墨點法探討細胞凋亡相關蛋白質的表現變化，都顯示合併治療組有較多的細胞凋亡現象。 結論: 經由細胞與動物實驗證實經由低劑量脈衝式超音波熱作用，奈米藥物能成功穿透血腫瘤障壁，在不影響正常腦組織的情況下藥物累積到腫瘤處，進一步抑制腫瘤的生長。

Background and Purpose: Melanoma is a malignant tumor that arises from uncontrolled proliferation of melanocytes. Advanced melanoma is the most aggressive, metastatic and deadly skin cancer, and 40-50% patients with advanced melanoma are diagnosed with brain metastasis in clinic. The main reason that melanoma brain metastasis remain untreatable is due to the blood-brain-barrier/blood-tumor-barrier (BBB/BTB). In this study, the synergistic effects of low-dose pulsed-wave focused ultrasound hyperthermia (pUH) on the delivery and therapeutic efficacy of pegylated liposomal doxorubicin (PLD) for melanoma brain metastases are investigated. Materials and Methods: In this study, the synergistic effects of pulsed-wave ultrasound hyperthermia and anticancer drug PLD in vitro and in vivo were investigated. B16F10 melanoma cells transfected with GFP were used in the experiments. Different concentration of PLD with/without pUH were used in the cultured cells for 24 and 72 h. Cell viability was evaluated by MTT assay. In animal models, B16F10-GFP cells were injected into the female striatum of C57BL/6 mice, and 6 days later the mice were divided into four groups (control, PLD, pUH, PLD+pUH) and received treatment. The mice were I.V. injected with PLD (5 mg/kg) with/without pUH one-minute after drug injection. The parameters of ultrasound were frequency at 500 kHz, PRF at 1000 Hz, duty cycle at 50%, and sonication duration at 10 min. The tumor growth was captured and assessed every other day by IVIS system. DOX in brain and tumor tissues were quantified after animal sacrification. The tissues were histologically processed with H&E, PLD distribution, IHC, TUNEL assay, and evaluation of apoptosis-related protein expression in the tumor was conducted via Western blot. Results: The inhibitory effects of PLD alone on cancer cells depended on the concentration and treatment time. PLD with ultrasound sonication caused significant inhibition on the cancer cells (p<0.0001). In vivo, the parameters of ultrasound were determined by the safety of hyperthermia on normal brain tissue and the delivery of the drug to the brain tumor. The tumor size and morphology were influenced with PLD and pUH treatment and inhibited the tumor growth development. Drug was cumulated much more in the tumor tissue than in the normal brain for the combined therapy. The therapeutic efficacy of PLD plus ultrasound hyperthermia were demonstrated by IHC (Ki-67 and Caspase-3 staining) and TUNEL assay. PLD+pUH induced more apoptosis on the melanoma brain tumor by Western blot analysis. Conclusion: These results support that low-dose pulsed-wave ultrasound hyperthermia could enhance the PLD delivery into brain tumors. The anticancer drug could be effectively cumulated in the sonicated tumor and further inhibited the tumor growth without damaging the normal brain tissues.