- 學位論文
創新光動力治療結合標靶奈米VEGF-A靜默RNA基因微脂體輸送在頭頸癌轉譯模型之組合治療
Novel combination therapy using photodynamic therapy and targeted VEGF-A siRNA nanoparticle delivery in head and neck cancer models
摘要
光動力治療促進腫瘤部位形成缺氧狀態，促使血管內皮新生因子-A(VEGF-A)等血管新生標記物過度表現，因此結合抗血管新生因子治療與光動力治療時，預期可有效提升治療效率。本研究以磷酸鈣奈米微脂體包覆血管內皮新生因子-A的靜默RNA，並於微脂體標記sigma 受體標記物anisamide，治療人類頭頸部鱗狀上皮細胞癌。實驗結果顯示，以磷酸鈣奈米微脂體包覆siVEGF-A，並以Anisamide標靶之磷酸鈣奈米微脂體包覆粒徑為 37.9 ± 2.4nm，zeta電位為46.2 ± 0.6 mV。光動力治療人類頭頸部鱗狀上皮細胞癌細胞株24 小時後，SCC4與SAS細胞內之VEGF-A表現量上升，並過量表現sigma受體，並在氟哌啶醇與標記anisamide之LCP NPs包覆德克薩斯紅DNA片段的競爭試驗中，證實標記anisamide之LCP NPs可經由sigma受體進入細胞中。在25 nM之siVEGF-A細胞存活度試驗中，組合基因治療與光動力治療相較於單光動力治療或單基因治療有顯著差異。在SCC4或SAS細胞株建立之異種移植動物模型試驗中，組合光動力治療與基因治療相較於PBS組，抑制腫瘤體積約30%。VEGF-A表現量檢測顯示，siVEGF-A組相較於PBS組、光動力治療組、亂碼靜默RNA (siScrambled) 組等，有顯著下降。蘇木精-伊紅染色檢驗結果，在組合治療組別中能有效降低有絲分裂發生，並在免疫染色檢測CD31、α-SMA與VEGF-A等蛋白表現時，顯示siVEGF-A治療組有顯著下降。TUNEL檢測細胞凋亡結果顯示，治療組與PBS組比較下，能有效提升細胞凋亡發生並降低Ki-67蛋白表現。動物體內毒性測試結果顯示，有效劑量治療下無明顯毒性。綜合試驗結果，以抗血管新生因子基因治療結合光動力治療之方式，結合標記物質能有效提升治療頭頸癌之效率，為頭頸癌治療中具潛力發展之方式。
並列摘要
Abstract Photodynamic therapy (PDT) promotes hypoxic condition to tumor cells leading to overexpression of angiogenic markers such as vascular endothelial growth factor-A (VEGF-A). Combination with antiangiogenesis therapy is expected to enhance the efficacy of PDT. In this study, PDT was combined with lipid-calcium-phosphate nanoparticles (LCP NPs) to encapsulate VEGF-A siRNA (siVEGF-A) and enhanced with anisamide to target sigma receptor overexpressing human head-and-neck squamous cell carcinoma (HNSCC) The measured LCP NPs size was 37.9 ± 2.4 nm and the zeta potential of 46.2 ± 0.6 mV attributing to the addition of anisamide. VEGF-A was up-regulated 24 hours post-PDT and sigma receptor were found to be expressed in HNSCC cells. In vitro SCC4 and SAS cellular uptake study using haloperidol to compete with the targeted LCP NPs containing Texas-Red Oligo DNA revealed that the nanoparticles were delivered through the sigma receptor. Cell viability was conducted at 25 nM of transfected siVEGF-A for combination therapy. In vitro combined therapy of PDT and siVEGF-A showed higher cell killing effect compared with PDT or gene therapy alone. In vivo xenograft animal model results show tumor growth inhibition for both SCC4 and SAS of decreased tumor volume to ~30% in groups treated with PDT and siVEGF-A compared to PBS group. In vivo VEGF-A expression were significantly reduced for groups treated with siVEGF-A compared with PBS and PDT and siScrambled. H&E staining of tumor showed less mitotic figures for groups treated with PDT and siVEGF-A. Immunostaining of CD31, α-SMA and VEGF-A were significantly decreased for groups treated with siVEGF-A. Cleaved caspase-3 and in situ TUNEL assay show more apoptotic cells and reduced Ki-67 expression for treated groups compared to PBS group. In vivo toxicity assay suggests that there is no animal toxicity of the delivered dose. In summary, results suggest that PDT combined with targeted gene therapy targeting angiogenesis could be a potential therapeutic mean to achieve better therapeutic outcome for head-and-neck cancer.