| 摘要: | 矽奈米粒為具潛力的藥物載體，本論文分別探討矽奈米粒之藥動學（第二章）及細胞攝入動力學（第三章）。
過去研究指出，一氧化氮可用於疾病的治療。然而，這種反應性高且不穩定的氣態分子很難遞送到目標作用部位。儘管有各種奈米藥物遞送系統已被發展成為一氧化氮釋放劑，但其體內的動力學研究卻不詳盡。第二章的研究目標為：探討一氧化氮矽奈米遞送系統之藥動學和生物相容性。利用兩種不同的方法製備有機矽奈米粒，分別是奈米沉澱法與一鍋法。合成的載體命名為NO-SiNP-1與NO-SiNP-2。含有亞硝基硫醇的有機矽奈米粒具有相似的粒徑大小（~130 nm），但具備不同的形態和表面電位。在體外釋放研究中，NO-SiNP-1的降解速率比NO-SiNP-2更慢（約延長5倍）；因此，NO-SiNP-1被視為一種緩慢的一氧化氮釋放劑，而NO-SiNP-2則是一種快速的一氧化氮釋放劑。但是在藥動學研究中，NO-SiNP-1卻從血液中迅速被排除（20分鐘內）；相比之下，NO-SiNP-2的亞硝基硫醇在血漿循環長達12小時，且其亞硝酸鹽和硝酸鹽的血漿濃度明顯更高。此外，血液、生化分析及組織切片的結果顯示給予二種劑型後並沒有產生顯著變化，表示其具有生物相容性。
奈米遞送系統一直存在著遞送效率普遍偏低的問題。研究指出巨噬細胞吞噬奈米載體具有特定的閾值（threshold），顯示細胞所能吸收的載體有一定的數量。第三章的目標是利用含有螢光團的有機矽奈米粒來評估巨噬細胞之吞噬和滯留奈米粒的程度與速率。研究中製備攜載rhodamine 6G（R6G）的有機矽奈米粒（SiNP-R6G）並探討其基本特徵及細胞攝入動力學（cellular uptake kinetics）。結果顯示，SiNP-R6G為球形粒子，粒徑約為100至200 nm，R6G不但穩定承載於奈米粒中，且螢光強度主要來自包覆的R6G分子，故可藉由螢光強度之測量，追蹤奈米粒子之細胞攝入動態。此外，SiNP-R6G的細胞毒性顯著低於游離態R6G，兩者之細胞耐受濃度相差>150倍。動力學實驗結果顯示巨噬細胞（RAW 264.7）胞吞SiNP-R6G時，呈現飽和動力學效應，即吞噬百分率隨投予的奈米數增加而降低，這樣的結果由直接螢光定量測定與共軛焦雷射掃描顯微圖像得到證實。每顆巨噬細胞的最大吞噬速率（Vmax）為每小時6.9×10^4顆奈米粒，半飽和（half-saturation）奈米粒數量濃度為7.6×10^11/mL（每毫升約1兆顆粒子）。SiNP-R6G在胞吞後的48小時，約有80%仍然滯留在細胞中。本研究發展出細胞內奈米粒子數量之直接定量法，利用低毒性和高螢光強度的螢光奈米粒子，追縱奈米載體進出巨噬細胞的動態變化，以絕對的量化數據，呈現巨噬細胞攝入奈米粒子之能力。
Silica-based nanoparticles (SiNPs) have been extensively studied as promising drug carriers. This dissertation includes two parts: the first part (Chapter 2) describes the pharmacokinetics of SiNPs; the second part (Chapter 3) describes the cellular uptake kinetics of SiNPs.
Previous studies indicate that nitric oxide (NO) has therapeutic potential. However, it is challenging to deliver this reactive and unstable gaseous molecule. A variety of nano drug delivery systems have been proposed as NO donors without comprehensive characterization of their properties in the body. Chapter 2 describes the study on the pharmacokinetics, biodistribution and biocompatibility of two nitric oxide-releasing organosilica nanoparticle formulations. Nanoprecipitation and direct one-pot synthesis were used to prepare two organosilica-based NO nanocarriers, namely NO-SiNP-1 and NO-SiNP-2, respectively. The organosilica particles present similar sizes (~130 nm), but different morphologies and surface charges. The two formulations show distinct in vitro releasing profile of nitric oxide: NO-SiNP-1 is a much slower NO releaser than NO-SiNP-2 (release rate differed by 5-fold). Nevertheless, in the rat pharmacokinetic study, NO-SiNP-1 was rapidly eliminated from the blood (within 20 min); in contrast, NO-SiNP-2 had a prolonged circulation time of 12 h. The rapid circulation elimination of NO-SiNP-1 could be attributed to high distribution of NO-SiNP-1 in the liver and spleen. The two nanoformulations are generally biocompatible, although they may interact with blood cells.
The low delivery efficiency has long been recognized in the field of nanomedicine. Studies have pointed out that phargocytic cells have a certain threshold for internalizing nanocarriers, indicating that cells can take up limited numbers of nanoparticles. Chapter 3 describes the study on the use of a fluorescent SiNP to quantify the cellular uptake kinetics of nanoparticles in macrophages. In this study, a novel method for measuring the absolute number of nanoparticle in cells is proposed. The aim is to measure the rate and extent of cellular uptake and retention in macrophage cells. Thiolated organosilane was used as a precursor for spontaneous encapsulation of a fluorescent probe, rhodamine 6G (R6G). Basic characterizations of R6G-loaded organosilica nanoparticles (SiNP-R6G) were investigated, including hydrodynamic size, surface charge, loading amount, R6G release, and cytotoxicity. The DLS and NTA measurement demonstrated the size is around 100 to 200 nm and the TEM images indicate spherical particles with smooth surface. The R6G molecule is stably encapsulated in the nanoparticles without significant release. In addition, SiNP-R6G is less toxic than free R6G against RAW 264.7 cells (>150-fold difference in maimum tolerated concentration). Unlike free R6G, the endocytosis of SiNP-R6G presents a dose-dependent, saturable kinetic internalization in the macrophages (RAW 264.7 cells), which is confirmed by direct fluorometric measurements and confocal laser scanning microscopic images. The maximum uptake rate (Vmax) for each macrophage cell is 6.9×10^4 per hour. The half-saturation number concentration of SiNP-R6G is around one trillion per milliliter. After internalization, about 80% of SiNP-R6G is still entrapped in the cells for 48 h. In summary, the proposed method enables direct measurement of the absolute number of nanoparticles in cells. The kinetic study demonstrates that macrophages have high capacity in internalizing and retaining organosilica nanoparticles. |
