製備上轉換奈米粒子當作細胞影像追蹤劑之研究

Abstract

傳統的生物螢光標籤大致可分為有機螢光染劑及無機半導體奈米晶體，有機螢光染劑雖具有較小之尺寸及較好之生物相容性，但容易發生光漂白之現象，導致無法長時間觀測螢光，且其光學性質也不利於觀察，而無機半導體奈米晶體雖然具有較佳之光學性質及光化學穩定性，但目前最常使用之選擇為量子點，而量子點內含有鎘離子，因此量子點仍有毒性之疑慮，而造成使用上的限制，而此二類螢光標籤皆是使用較高能量之光源激發出較低能量之螢光，所以此二類屬於下轉換材料，而使用高能量之光源激發生物樣品可能會遇到幾項限制，例如低穿透深度、破壞生物樣品及導致生物自體螢光而不利於觀察等，近年以來，上轉換奈米粒子發展的速度有愈來愈快之趨勢，原因在於上轉換奈米粒子具有以非線性之光學轉換的機制將近紅外光轉變為較高能量之可見光之能力，而使用近紅外光當作激發光源具有許多優勢，例如對生物樣品破壞性較小、高穿透深度及減少生物自體螢光等，除此之外，上轉換奈米粒子也同時具有高化學穩定性及低毒性之優點，雖然以油酸為介面活性劑合成之上轉換奈米粒子為疏水性，但目前已有研究團隊藉由修飾二氧化矽殼層於上轉換奈米粒子表面上改善其疏水性，使得上轉換奈米粒子能夠應用於生醫領域上，雖然目前已有文獻使用上轉換奈米粒子於細胞螢光顯影上，但僅著重於細胞質與細胞膜之螢光顯影，然而對於細胞內之胞器標定的研究仍佔少數，因此，本研究期許藉由分別接枝NLS(nuclear localization signal)及TAT 胜肽序列於二氧化矽包覆上轉換奈米粒子上，能夠成功達成細胞核之螢光顯影與追蹤。

Traditional fluorescent labels can be roughly categorized into two parts. One is fluorescent organic dye, the other is semiconductor nanocrystal. Both of them have advantages and limitations. Organic dye is the most popular fluorescent label due to its small size and good biocompatibility. However, there are some limitations coming out, such as high photobleaching efficiency and worse optical property. Fortunately, semiconductor nanocrystal improves some shortcomings of organic dye, e.g. excellent optical properties, good photo-stability and high quantum yield. However, the most widely used semiconductor nanocrystal is quantum dots (QDs), even though QDs are very toxic due to cadmium ions within their structure. Therefore, QDs are not suitable for long-term development. Besides, organic dye and QDs are down-conversion materials. They all need a high energy light source for excitation, such as ultra-violet. In this case, there are three problems to face including low penetration depth, biomolecules damage and low signal-to-noise ratio. In the last decade, new fluorescent labels started to rising up, called up-conversion nanoparticles (UCNPs). Compared to down-conversion material, UCNPs can transfer lower energy near infrared excitation into higher energy visible light by a nonlinear optical process. Moreover, UCNPs have many advantages such as autofluorescence absence, deeper tissue penetration depth, excellent optical and chemical stability, and low toxicity. However, in most cases, UCNPs are hydrophobic. Besides, research regarding the use of UCNPs as organelle targeting is not abundant. Therefore, in this study, NaYF4:Yb,Er upconversion nanoparticles were synthesized. After silica coating and amino group grafting, NaYF4:Yb,Er up-conversion nanoparticles could be made hydrophilic and later conjugated with biomolecules. For nuclear targeting purpose, nuclear localization signal (NLS) and TAT peptide was used to conjugate with silica coated NaYF4:Yb,Er nanoparticles. Our expectation is that NaYF4:Yb,Er@SiO2-NLS nanoparticles or NaYF4:Yb,Er@SiO2-TAT nanoparticles could be uptaken by cell and act as a nuclear tracker.