| 摘要: | 背景：奈米藥物被應用於提高靶向功效和降低使用傳統藥物時的副作用，但其自體內清除的機制仍有待研究。腎臟是身體主要排泄器官之一，負責清除體內的廢物和異物。一般而言，水合直徑大於8奈米的顆粒，會受到腎小球過濾屏障的阻礙而無法被排泄。然而在近期的研究中，我們出乎意料地發現，有一種奈米載體——聚乙二醇 (PEG) 和功能性陽離子矽烷 N-三甲氧基甲矽烷基丙基-N,N,N-三甲基氯化銨 (TA) 修飾的中孔洞二氧化矽奈米粒子 (MSNs) (命名為25 nm RMSN@PEG/TA) ，被全身性注射到小鼠身體後，可經由腎排泄後並保持完整。本研究的目是找出完整的25 nm MSNs 的排泄機制。
方法：首先, 我們合成出帶正電荷的聚乙二醇化MSNs。通過透射電子顯微鏡 (TEM)、動態光散射 (DLS)、熱重分析 (TGA)、氮氣吸附-脫附等溫線 (BET) 和奈米顆粒追蹤分析 (NTA) 鑑定材料的特性。為了研究體內奈米顆粒清除機制, 進一步進行活體實驗。我們透過腹腔注射的方式將一種稱為甲基-β-環糊精（MβCD）內吞作用抑製劑選擇性地給予小鼠並將 RMSN@PEG/TA 靜脈注射到健康和已接種4T1 乳腺腫瘤的 BALB/c 小鼠﹐及後使用非侵入性體內成像系統 (IVIS) 追蹤其生物分佈。最後我們應用了免疫染色、高通量全視野成像系統和共聚焦螢光顯微照片去辨認 25 nm RMSN@PEG/TA 在組織上分佈的現象，以了解其腎臟清除途徑。
結果：我們合成出分散性良好的 25 nm RMSN@PEG/TA 後, 使用NTA 進一步量化25 nm RMSN@PEG/TA 的劑量。在MβCD 預處理的小鼠組中，注射 25 nm RMSN@PEG/TA後，尿液和器官的螢光強度較弱。這意味著腎臟和尿液中的奈米顆粒數量顯著減少。此外，通過免疫螢光顯微鏡可觀察到，25 nm RMSN@PEG/TA 和內皮細胞的共同定位現象, 表示它們沉積在腎絲球基底膜和腎小球繫膜細胞中。而在電子顯微鏡圖不但顯示一致的跡象，更揭示了25 nm RMSN@PEG/TA 在近端小管中積累。
結論：我們的研究結果表明，帶正電荷的25 nm RMSN@PEG/TA (+35 mV) 可以通過小窩介導的白蛋白胞移作用通過近曲腎小管上皮細胞排泄，並在排泄後保持完整。因此，旨在實現「目標或清除」，這項研究為下一代納米醫學領域鋪上一條康莊大道。
Background: Nanomedicines have been applied to improve the targeting efficacy and reduce the side-effects of using traditional drugs; however, their body clearance awaits investigation. Kidneys serve as the major excretion organs play a crucial role in removing wastes and foreign materials in the body. In general, nanoparticles with a hydrodynamic diameter larger than the kidney filtration threshold (~8 nm) are unable to excrete into the urine. Unexpectedly, we noticed that a kind of nanocarriers, 25 nm mesoporous silica nanoparticles (MSNs) coated with polyethylene glycol (PEG) and functional cationic silane N-Trimethoxysilylpropyl-N,N,N-trimethylammonium chloride (TA) (designated as 25 nm RMSN@PEG/TA), could bypass the renal filtration after systemic administration in mice. The objective of this study is to investigate the excretion mechanism of intact 25 nm MSNs in urine.
Methods: First, 25 nm RMSN@PEG/TA were synthesized and characterized by transmission electron microscopy (TEM), dynamic light scattering (DLS), thermogravimetric analysis (TGA), nitrogen adsorption-desorption isotherm (BET) and nanoparticle tracking analysis (NTA). The clearance of nanoparticles in vivo was then studied. Briefly, an endocytosis inhibitor called methyl-β-cyclodextrin (MβCD) was given selectively to mice by intraperitoneal injection. After intravenous injections of RMSN@PEG/TA into 4T1 breast tumor-bearing BALB/c mice, biodistribution was investigated by a non-invasive in vivo imaging system (IVIS). Immunostaining, high-throughput whole-field imaging system and confocal immunofluorescence micrographs were applied to examine the distribution and constitute a plausible renal excretion mechanism of 25 nm RMSN@PEG/TA.
Results: Well-dispersed 25 nm RMSN@PEG/TA were obtained. The particle number of 25 nm RMSN@PEG/TA administered in mice was quantified by NTA. In the group of mice pre-treated with MβCD, weaker fluorescence in organs and urine was obtained after injection of 25 nm RMSN@PEG/TA. It implied that nanoparticles’ accumulation in organs and excretion in urine are relevant to caveolae-mediated endocytosis. Moreover, the co-localization of 25 nm RMSN@PEG/TA and immunofluorescent endothelial cells, either in the glomerular basement membrane or mesangium of glomeruli, was observed by fluorescence microscopy. TEM showed consistent results and further revealed the presence of 25 nm RMSN@PEG/TA in proximal tubules.
Conclusions: We demonstrated that the 25 nm RMSN@PEG/TA (+35 mV, water) could be excreted through proximal tubular epithelial cells by caveolae-mediated albumin transcytosis and retained intact after urinary excretion. This paves the way for next-generation nanomedicine aiming to achieve the goal of “target-or-clear”. |
