文献：结合共聚焦显微镜、dSTORM 和质谱技术揭示与纳米结构脂质载体在血脑屏障穿越过程中相关的蛋白质冠层的演变

链接：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 

节选：

材料和方法

LMNV制备

脂质磁性纳米载体 (LMNV) 的制造协议改编自我们小组以前的研究，结合了热超声波和高压均质化 (HPH) 方法。20简而言之，我们将不同的脂质混合在一起，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 1,2-二棕榈酰-rac-甘油-3-磷酸胆碱（Sigma-Aldrich）和 4 mg 1,2-二硬脂酰-sn-甘油-3-磷酸乙醇胺与共轭甲氧基聚乙二醇 (mPEG-DSPE) (5000 Da, Nanocs)，以及 84.5 μl 超顺磁性氧化铁纳米粒子 (SPION) 的乙醇溶液 (3 nm 直径，15 wt%；美国研究纳米材料公司) 放入 6 ml 玻璃小瓶中。

将3 ml预热（70 °C）的Tween® 80 (Sigma-Aldrich) 溶液 (1.0 wt%) 加入脂质/SPION分散体中，并使用超声波探头 (Fisherbrand™ Q125 Sonicator) 进行超声处理15分钟（振幅30%，功率120 W）。超声处理后，使用均质机以100 [细空格（1/6-em）]000 psi的压力对混合物进行高压均质处理（共进行5次高压均质处理）。纳米载体在4 °C下以16 [细空格（1/6-em）]000 g离心90分钟（共进行3次）进行纯化，然后重新分散于水中。为了进行共聚焦成像，LMNV 使用荧光 Vybrant DiO 细胞标记染料（Invitrogen）进行标记。将 5 mg 纳米载体与 20 μM DiO 在 37 °C 下孵育 2 小时，然后在 4 °C 下以 16 [细空格（1/6-em）]000 g离心 90 分钟（三次）进行洗涤。对于直接随机光学重建显微镜 (dSTORM) 分析，在制备过程中将 3 mg mPEG-DSPE（5000 Da，Nanocs）与 1 mg DSPE-PEG-Cy3（5000 Da）混合进行染色。

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.

西安齐岳生物提供相关产品：

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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