- 學位論文
創新標靶奈米傳輸EGFR靜默RNA技術結合新穎光動力療法於頭頸癌治療
Novel therapy of combining targeting delivery EGFR siRNA nanoparticle and innovative photosensitizer in photodynamic therapy for head and neck cancer treatment
摘要
本研究使用新型脂質磷酸鈣包覆EGFR靜默RNA後標靶到頭頸癌細胞抑制EGFR基因的表達，本微脂體包覆新穎光敏劑藥物焦脫鎂葉氯甲酸(pyropheophorbide、pyro) 並在微脂體表面上，接枝具標靶功用的大茴香醯胺 (anisamide) 使得微脂體具標靶能力可以標靶至口腔癌細胞，並以組合療法抑制EGFR基因的過表達及光動力治療之方式，共同毒殺腫瘤。本研究的LCP-EGFR siRNA微脂體粒徑為34.9 ± 3.0.nm，表面電位為51.8 ± 1.8.mV；LCP-Pyro微脂體粒徑約為20 nm，表面電位為52.0 ± 7.6 mV。體外細胞實驗的目的找出最佳的組合治療順序，以利提升頭頸癌細胞毒殺效果。經研究發現最佳毒殺細胞之順序組合為先施以LCP-EGFR siRNA抑制癌細胞的EGFR表達量，再施以光動力治療，在pyropheophorbide濃度為0.25.µg/ml結合LCP-EGFR siRNA治療的頭頸癌細胞存活率達80%，在西方墨點法亦證實於SCC4及SAS兩株口腔癌，分別抑制 89%及97%的EGFR蛋白質表達量。此細胞研究顯示先施以EGFR siRNA抑制再施以PDT的組合治療確實可提升頭頸癌細胞毒殺功效。 針對體內動物實驗乃是以異種移植人類頭頸癌裸鼠模式驗證組合治療療效，使用Balb/c裸鼠的皮下移植頭頸癌細胞 (SCC4及SAS)，並等待腫瘤成長至200 mm3以五組實驗，分別為對照組 (PBS)、光動力對照組 (LCP-Control siRNA + PDT)及EGFR siRNA對照組 (LCP-EGFR siRNA + LCP-Pyro no light、LCP-EGFR siRNA + PBS with light 2組) 及組合治療組 (LCP-EGFR siRNA + PDT)，以連續13天的實驗期間觀察腫瘤生長情形。結果在於第13天，異種移植SCC4及SAS裸鼠PBS組的腫瘤體積分別為642.1與764.9.mm3；異種移植SCC4及SAS裸鼠LCP-Control siRNA + PDT組腫瘤體積分別為447.8及499.3.mm3，較PBS組腫瘤體積分別減少194.3及265.6.mm3，可推算PDT抑制腫瘤體積比例分別為30.3% (p<0.001).及34.7% (p<0.001)；異種移植SCC4及SAS裸鼠LCP-EGFR siRNA + LCP-Pyro no light組腫瘤體積分別為385.3及440.7.mm3，較PBS組腫瘤體積分別減少256.8及324.2.mm3，抑制腫瘤體積比例分別為40.0% (p<0.001) 和 42.4% (p<0.001)；異種移植SCC4及SAS裸鼠LCP-EGFR siRNA + PBS with light組腫瘤體積分別為406.4及432.2.mm3，較PBS組腫瘤體積分別減少235.7及332.7.mm3，抑制腫瘤體積比例分別為36.7% (p<0.001) 和43.5% (p<0.001)；異種移植SCC4及SAS裸鼠LCP包覆EGFR siRNA + PDT實驗組腫瘤體積分別為238.0及339.7 mm3，較PBS組腫瘤體積分別減少404.1及425.2.mm3，組合治療的腫瘤抑制率分別為為62.9% (p<0.001) 及55.6% (p<0.001)，此組與其他單獨治療 (EGFR siRNA或PDT) 相比，抑制腫瘤體積效果最好。在定量即時聚合酶鏈鎖反應 (QPCR) 分析口腔癌裸鼠腫瘤經組合治療後的基因表達量，亦證實組合治療組可達分別抑制SCC4及SAS口腔癌達 92%及82%的EGFR基因表達量。以免疫染色切片Phosphorylated-EGFR 免疫分析於SCC4及SAS裸鼠腫瘤中，第5組LCP-EGFR siRNA + PDT陽性染色面積百分比分別為0.9% (p<0.001) 及0.8% (p<0.001)，較PBS組EGFR表達量分別減少91%及92%，可得知以LCP-EGFR siRNA + PDT組合治療後可有效地降低磷酸化EGFR蛋白質的陽性染色面積，有效的抑制EGFR蛋白質在腫瘤的過表達現象。針對毒性分析方面，以酵素免疫分析法分析IL-6、IL-12、IFN-gamma、TLR3及其他發炎因子皆無發現有心、肝、腎的異常表達，各組相比皆無顯著差異。以上結果皆表明LCP微脂體包覆靜默RNA治療及包覆光敏劑的光動力療法在治療人類頭頸癌上是種很有潛力的治療方法。
並列摘要
EGFR siRNA was encapsulated into Lipid-calcium-phosphate nanoparticles (LCP NPs) to silence EGFR expression in head-and-neck squamous cell carcinoma (HNSCC). A novel photosensitizer, Pyropheophorbide, was modified with anisamide with LCP NPs. The above 2 treatments were combined for head-and-neck therapy in vitro and in vivo. The LCP-EGFR siRNA and LCP-Pyro particle sizes were 34.9 ± 3.0.nm and 20.nm respectively, zeta potential were 51.8 ± 1.8.mV and 52.0 ± 7.6.mV respectively. In vitro study, the combined therapy of LCP-EGFR siRNA + PDT was showed significantly greater inhibition response as decreased 80% of cell viability with pyro of 0.25 µg/ml in SCC4 and SAS cell lines. The western blot showed combined therapy has greater inhibition rate on SCC4 and SAS in vitro study at 89% and 97% respectively. These results indicated that the EGFR siRNA was effectively knocked down while PDT was successfully inhibited the cancer growth. In vivo study, we used SCC4 and SAS xenografts of Balb/c nude mice to verify the efficacy of the combination therapy. The results showed in 13th day, PBS group both of SCC4 and SAS reached 642.1 and 764.9.mm3 respectively, LCP-Control siRNA + PDT group of SCC4 and SAS reached 447.8 and 499.3.mm3 respectively, while the tumor reduction volume were 194.3 and 265.6 mm3 respectively compared to PBS group. Tumor volume reduction rate were 30.3% (p<0.001) and 34.7% (p<0.001) respectively compared to PBS group. LCP-EGFR siRNA + LCP-Pyro without light group of SCC4 and SAS in term of tumor volume which 385.3 and 440.7.mm3 respectively, reduced tumor volume were 256.8 and 324.2 mm3 respectively compared to PBS group tumor volume, tumor volume reduction rate were 40.0% (p<0.001) and 42.4% (p<0.001) respectively compared to PBS group tumor volume. LCP-EGFR siRNA + PBS with light group of SCC4 and SAS in term of tumor volume which 406.4 and 432.2.mm3 respectively, reduced tumor volume were 235.7 and 332.7.mm3 respectively compared to PBS group tumor, tumor volume reduction rate were 36.7% (p<0.001) and 43.5% (p<0.001) respectively compared to PBS group. LCP-EGFR siRNA + PDT group both of SCC4 and SAS the tumor volume were 238.0 and 332.7.mm3 respectively, reduced tumor volume were 404.1 and 425.2.mm3 respectively compared to PBS group, tumor volume reduction rate were 62.9% (p<0.001) and 55.6% (p<0.001) respectively compared to PBS group. Combined therapy have greater tumor growth inhibition for both SCC4 and SAS in term of tumor volume reduction compared to PBS or control siRNA or PDT treated alone. In vivo QPCR results showed EGFR expression were significantly reduced for groups treated with EGFR siRNA with PDT in SCC4 and SAS were 92.8% and 82.0% respectively compared to control siRNA or PDT treated alone. In vitro and in vivo western blot results indicated EGFR protein expression for group treated with EGFR siRNA decrease compared with the PBS and PDT and Control siRNA. In toxicity study, ELISA and clinical blood biochemistry assay to examine factors relevant factors of potential applications of siRNA and PDT therapy clinically. Serum biomarkers and inflammatory factors were found non-toxic results found in heart, liver and kidney. Overall these results indicating that gene combine PDT therapy encapsulated into LCP NPs could be potential technology of delivery modality to overcome head-and-neck cancer.