Multi-functional Nanoparticles for biomedical applications

拉维(Raviraj)

指導教授 ： 黃國柱

國立清華大學/理學院/化學系/博士(2012年)

摘要

Multi-functional nanoparticles such as metal nanoparticles and core/shell metal-filled carbon nanoparticles acquire several important and unique properties such as ultra-high absorption co-efficient, photostability (for metal nanoparticles) and magnetic, fluorescent, biocompatibility and facile surface functionalization capability (for core/shell metal-filled carbon nanoparticles) and making them as novel nanoparticle vectors/cargoes for carrying DNA, siRNA, anti-cancer drugs and photosensitizers for several potential therapeutic technologies.
In brief, the thesis begins with the cellular uptake and gene transfection studies of lipid-Fe@CNPs in various cell lines including HeLa, U87MG and TRAMP-C1. Our results suggest that flexible binding between the DNA and the nanoparticle cargo is the key for high gene transfection efficiency and in contrast, tight binding results in very poor efficiencies. In vivo zebrafish gene transfection also showed that flexible binding has driven a better efficiency in the GFP expression, when compared to the tight binding between nanoparticles functionalized with tri methyl ammonium group (TMAEA) and naked DNA. In the case of mouse embryonic stem cells (MESCs), the lipid-Fe@CNPs has also exhibited a better GFP expression, in the presence of elongation factor (EF-1) promoter rather than cytomegalovirus (CMV) promoter. We further investigated the effects of surface functionality of Fe@CNPs on the cellular cytotoxicity and embryonic development in zebrafish. Both in vitro and in vivo zebrafish studies revealed that the Fe@CNPs with imidazolium, trimethyl ammonium and carboxylic acid functional groups induce most of the cellular events, such as, higher levels of ROS and apoptosis, lowering mitochondrial membrane potential, acidification of cells and blocking the cell cycle at S-phase, which all confers to the cellular death along with several abnormalities in zebrafish. Hence, bearing all these issues in mind, the Fe@CNPs with particular functionality could be used as a suitable candidate for potential therapeutic applications.
Among many nanoparticles, metal nanoparticles (M NPs) possess several unique properties such as tunable surface plasmon resonance (SPR), ultra-high absorption co-efficient and excellent photostability. Herein, we showed that singlet oxygen can be formed via direct photoexcitation of metal nanoparticles without any photosensitizers. Further, we also showed that morphology of metal nanoparticles / nanorods play a critical role in the sensitization of singlet oxygen. Overall, the results indicate that gold nanorods are potentially very promising dual functional nanomaterials with capabilities of simultaneously serving as near infrared (NIR) photodynamic therapy and photothermal therapy reagents for cancer treatments. In the last chapter, we have utilized lipid-coated Au NRs to serve as excellent photodynamic / photothermal reagents in the photo destruction of HeLa cells and also for the in vivo malignant tumors with B16F0 melanoma model. Under low laser fluencies, in the in vitro, we have detected reactive oxygen species (ROS), apoptosis, SOSG, and heat shock protein expression (HSP70) levels under photoexcitation at 550 and 940 nm wavelengths. In the in vivo, when we irradiated melanoma tumors with 780 nm, the tumor temperature has been raised until 46oC and whereas, 915 nm irradiation results in temperature of 43oC. The tumor growth curve shows appreciable growth delay when compared with all the other controls, including, chemotherapeutic drug doxorubicin. The experimental results clearly reveal that ROS mediated cell death is more dominant than the contribution from PTT.
Hence, our research provides a sufficient understanding of demonstrating multi-functional nanoparticles (metal and core/shell carbon nanoparticles) as excellent candidates for various biomedical applications.

關鍵字

多功能纳米和生物医学应用

並列摘要

金屬奈米顆粒以及包裹金屬的奈米碳球均為具多功能性奈米粒子其本身具有幾點重要和獨特的性能，如高吸收係數，光穩定性 (以金屬奈米粒子而言), 磁性，螢光，生物相容性和極簡單表層官能基化 (以包裹金屬的奈米碳球而言）, 同時在一些具有潛力的治療技術上，這些奈米粒子可作為去氧核醣核酸(ＤNA), 短干擾核醣核酸(Small interfering RNA, siRNA), 抗癌藥物以及光治療的新型生物載體
　　此論文則以研究利用脂質（lipid）修飾在具有包裹鐵的奈米碳球上，並以此奈米碳球為載體進行HeLa細胞, U87MG和TRAMP 的细胞吸收和基因轉殖研究為開端。我們的實驗结果闡訴，基因轉殖深深受到去氧核醣核酸(DNA) 與奈米粒子表面的官能基之間的作用力的影響，當其作用較強時，其基因轉殖能力較差；反言之，當作用力較具彈性時，其具有較好的轉植表現。在斑馬魚的實驗中，以綠色螢光蛋白(ＧＦP) 進行基因轉殖的表現的觀測，發現在表面修飾TMAEA的奈米碳球具有較差的螢光表現，而這表示去氧核醣核酸（ＤＮＡ）與TMAEA具有較強的作用力，讓GFP不易在斑馬魚中進行表達。而在小老鼠胚胎幹細胞(mESCs)的實驗中，Lipid-Fe@CNPs 在ＥＦ-1比巨細胞病毒（ＣＭＶ）具有較好的GFP的表徵。我們更進一步探討其奈米碳球表面的官能基對於斑馬魚的細胞毒性與胚胎生長上的影響。　在體內以及體外的斑馬魚研究中顯示，咪唑啉酮（imidazolium）三甲基氨（trimethyl ammonium）以及羧酸 (carboxylic acid group）的官能基團都會導致細胞死亡。因此，總結上述的內容，表面官能基化的包裹鐵金屬粒子的奈米碳球極在醫療上具有無限的潛力。
縱觀許多奈米粒子，金屬奈米粒子具有獨特的性質，如可調式的表面電漿共振(SPR)、高吸收係數以及極為優異的光穩定性…等等。然而，我們實驗證實金屬奈米粒子在沒有修飾任何光敏劑的情況下，照光產生單重態的氧。　我們也同時實驗證實不同形狀的金屬奈米粒子其晶面對於產生單重態的氧扮演著舉足輕重的角色。　總結，實驗的結果指出金奈米棒極有前途可能成為具有雙功能的奈米材料治療癌症，因為金奈米棒同時具有紅外光驅動的光動療以及光熱療的能力，可以成為抗癌試劑。　在最後一個章節，我們利用脂質包裹的金奈米棒當作一個極為優越的光動療/光熱療的試劑，在照光下摧毀HeLa細胞；並也同時進行生物體實驗，在B16F0黑色素惡性腫瘤下，在極低功率的光照射下，我們量測到具高氧化力的物種存在於生物體內及體外實驗中，並且發現加熱停止HSP70的運作，進而導致細胞死亡，而造成細胞死亡的原因為極為可能是光動療以及光熱療。
因此，我們研究提供對這些具功能性的金屬材料(如　金屬　以及　包裹金屬的奈米碳球)的認知，並提供實驗說明這一些材料如何在生化的運用上具有極佳的性質。

並列關鍵字

multi-functional nanoparticles and biomedical applications

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