文獻：結合共聚焦顯微鏡、dSTORM 和質譜技術揭示與納米結構脂質載體在血腦屏障穿越過程中相關的蛋白質冠層的演變

鏈接：https://pubs.rsc.org/en/content/articlehtml/2022/nr/d2nr00484d

作者：Matteo Battaglini ORCID ，Natalia Feiner bc， Christos Tapeinos a，Daniele De Pasquale a， Carlotta Pucci a，Attilio Marino a，Martina Bartolucci d，Andrea Petretto d，Lorenzo Albertazzi bc和 Gianni Ciofani 

節選：

材料和方法

LMNV制備

脂質磁性納米載體 (LMNV) 的制造協議改編自我們小組以前的研究，結合了熱超聲波和高壓均質化 (HPH) 方法。20簡而言之，我們將不同的脂質混合在一起，包括 2.5 mg 油酸（Sigma-Aldrich）、25 mg 1-硬脂酰-rac -甘油（Sigma-Aldrich）、2.5 mg 油酸（Sigma-Aldrich）、2.5 mg 1,2-二棕櫚酰-rac-甘油-3-磷酸膽堿（Sigma-Aldrich）和 4 mg 1,2-二硬脂酰-sn-甘油-3-磷酸乙醇胺與共軛甲氧基聚乙二醇 (mPEG-DSPE) (5000 Da, Nanocs)，以及 84.5 μl 超順磁性氧化鐵納米粒子 (SPION) 的乙醇溶液 (3 nm 直徑，15 wt%；美國研究納米材料公司) 放入 6 ml 玻璃小瓶中。

將3 ml預熱（70 °C）的Tween? 80 (Sigma-Aldrich) 溶液 (1.0 wt%) 加入脂質/SPION分散體中，并使用超聲波探頭 (Fisherbrand? Q125 Sonicator) 進行超聲處理15分鐘（振幅30%，功率120 W）。超聲處理后，使用均質機以100 [細空格（1/6-em）]000 psi的壓力對混合物進行高壓均質處理（共進行5次高壓均質處理）。納米載體在4 °C下以16 [細空格（1/6-em）]000 g離心90分鐘（共進行3次）進行純化，然后重新分散于水中。為了進行共聚焦成像，LMNV 使用熒光 Vybrant DiO 細胞標記染料（Invitrogen）進行標記。將 5 mg 納米載體與 20 μM DiO 在 37 °C 下孵育 2 小時，然后在 4 °C 下以 16 [細空格（1/6-em）]000 g離心 90 分鐘（三次）進行洗滌。對于直接隨機光學重建顯微鏡 (dSTORM) 分析，在制備過程中將 3 mg mPEG-DSPE（5000 Da，Nanocs）與 1 mg DSPE-PEG-Cy3（5000 Da）混合進行染色。

Materials and methods

LMNV preparation

The protocol for the fabrication of lipid magnetic nanovectors (LMNVs) was adapted from previous works of our group combining hot ultra-sonication and high-pressure homogenization (HPH) methods.20 Briefly, we mixed different lipids including 2.5 mg of oleic acid (Sigma-Aldrich), 25 mg of 1-stearoyl-rac-glycerol (Sigma-Aldrich), 2.5 mg of oleic acid (Sigma-Aldrich), 2.5 mg of 1,2-dipalmitoyl-rac-glycero-3-phosphocholine (Sigma-Aldrich), and 4 mg of 1,2-distearoyl-sn-glycero-3-phosphoethanolamine with conjugated methoxyl poly(ethylene glycol) (mPEG-DSPE) (5000 Da, Nanocs) with 84.5 μl of an ethanol solution of superparamagnetic iron oxide nanoparticles (SPIONs) (3 nm diameter, 15 wt%; US Research Nanomaterials Inc.) into a 6 ml glass vial. 3 ml of pre-warmed (70 °C) Tween? 80 (Sigma-Aldrich) solution (1.0 wt%) were added to the lipid/SPION dispersion and sonicated using an ultrasonic tip (Fisherbrand? Q125 Sonicator) for 15 min (amplitude 30%, 120 W). After the sonication, the mixture underwent high-pressure homogenization with a homogenizer at 100[thin space (1/6-em)]000 psi (5 passages of high-pressure homogenization were performed). The nanovectors were purified by centrifugation at 16[thin space (1/6-em)]000g for 90 min at 4 °C (three passages) and then re-dispersed in water. For confocal imaging, LMNVs were labeled with the fluorescent Vybrant DiO cell-labeling dye (Invitrogen) by incubating 5 mg of nanovectors with 20 μM of DiO for 2 h at 37 °C and then washing them by centrifugation at 16[thin space (1/6-em)]000g for 90 min at 4 °C (three passages). For Direct STochastic Optical Reconstruction Microscopy (dSTORM) analysis, the staining was obtained by mixing 3 mg of mPEG-DSPE (5000 Da, Nanocs) with 1 mg of DSPE-PEG-Cy3 (5000 Da) during the preparation procedure.

西安齊岳生物提供相關產品：

M2pep-PEG-DSPE

EB1-PEG-DSPE

CPP-PEG-DSPE

CCK8-PEG-DSPE

FSHB(QCHCGKCDSDSTDCT)-PEG-DSPE

GRGDS-PEG-DSPE

MMPs-PEG-DSPE

WSW(WSWGPYS)-PEG-DSPE

LyP-1-PEG-DSPE

VIP-PEG-DSPE

CREKA-PEG-DSPE

Asp8-PEG-DSPE

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