探討以奈米鑽石於活體內傳送雙股核醣核酸之效率與其生物耐受性及追蹤方式

Delivery, Biosafety and Tracing of Nanodiamond-dsRNA Complexes in vivo

簡大為(Ta-Wei Chien)

指導教授 ： 周涵怡

國立臺灣大學/牙醫專業學院/口腔生物科學研究所/碩士(2013年)

https://doi.org/10.6342/NTU.2013.10515

摘要

由本實驗室先前的研究已證實出奈米鑽石在協助短干擾核醣核酸進入腫瘤細胞抑制目標基因表現的過程中具有相當大的成效，因此，在發展以此基因療法進入活體並治療癌症的過程中，去驗證奈米鑽石是否在活體內也能夠促進短干擾核醣核酸的傳遞並抑制目標基因是必要的。另一方面，奈米鑽石身為一個外來載體，其未來應用在活體內癌症治療上的生物相容性之疑慮仍然存在；因此，我們必須進行奈米鑽石在活體內之生物相容性測試。在本實驗中，我們利用異種移植腫瘤模式，分析奈米鑽石攜帶短干擾核醣核酸之傳遞效率：藉由比較基因抑制程度、抑制速率、抑制間期長短、腫瘤生長趨勢、及鑽石劑量效應，我們證實出在活體內奈米鑽石的確能夠促進目標基因的專一性抑制。就生物相容性測試方面，我們將不同劑量之奈米鑽石由尾靜脈及腹腔注入活體內，再利用組織切片和血液生化測試來觀察指標器官功能指數是否受到影響；同時搭配基礎生理量測監控，我們發現即使在最高劑量之奈米鑽石實驗組別與對照組之間仍沒有顯著差異，顯示奈米鑽石對活體內主要器官功能及形態上是無害的。最後為了發展未來奈米鑽石在活體內追蹤的可行性，我們應用奈米鑽石獨特的高折射率，利用斷層相位顯微鏡記錄相位變化的方式重建出細胞與奈米鑽石的影像，成功辨別出細胞內奈米鑽石的三維分布，對未來於活體內追蹤奈米鑽石的方式建立新的里程碑。綜合以上結果，我們相信奈米鑽石是個具有高潛力之可追蹤藥物載體，期望本研究在未來癌症治療上能帶給人民福祉。

關鍵字

奈米鑽石 ； 短干擾核醣核酸傳遞效率 ； 生物相容性 ； 活體追蹤

並列摘要

Previous studies from our laboratory have demonstrated that nanodiamonds (NDs) can promote delivery of siRNA into cancer cells to enhance target gene suppression. In order to develop ND-mediated delivery into clinically applicable gene therapy for cancer, it is necessary to verify its silencing efficiency in vivo. In this study, we use tumor xenograft model to analyze the delivery efficiency of ND-carried siRNA complexes, by comparing the kinetics of target gene suppression level and its effect on tumor growth. Our results indicate that NDs can greatly promote the delivery of siRNA efficiency in vivo. On the other hand, despites of the high bio-inertness of ND, it still remains to be tested whether NDs display good in vivo biocompatibility under invasive administration scheme. Toward this end, we examined the physiological and pathological responses of mice after administration of ND by intravenous and intraperitoneal injection, and observed no significant abnormality in the ND treated mice. Taken these together, our results suggest that ND can be an excellent siRNA delivery vector for the treatment of cancer, and thus prompt us to develop a practical method for its tracking in vivo. We explored the high refractive index of NDs for imaging by tomographic phase microscopy, and identified intracellular NDs in 3 dimensions. Combining the good delivery efficiency, high biocompatibility and traceable characteristics, we believe that ND promises high potentials as drug vector and might bring great welfare in cancer therapy in the future.

並列關鍵字

nanodiamond ； siRNA delivery ； biocompatibility ； in vivo tracing

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