文獻(xiàn)：結(jié)合共聚焦顯微鏡、dSTORM 和質(zhì)譜技術(shù)揭示與納米結(jié)構(gòu)脂質(zhì)載體在血腦屏障穿越過程中相關(guān)的蛋白質(zhì)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(jié)選：

材料和方法

LMNV制備

脂質(zhì)磁性納米載體 (LMNV) 的制造協(xié)議改編自我們小組以前的研究，結(jié)合了熱超聲波和高壓均質(zhì)化 (HPH) 方法。20簡而言之，我們將不同的脂質(zhì)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(yù)熱（70 °C）的Tween? 80 (Sigma-Aldrich) 溶液 (1.0 wt%) 加入脂質(zhì)/SPION分散體中，并使用超聲波探頭 (Fisherbrand? Q125 Sonicator) 進(jìn)行超聲處理15分鐘（振幅30%，功率120 W）。超聲處理后，使用均質(zhì)機(jī)以100 [細(xì)空格（1/6-em）]000 psi的壓力對(duì)混合物進(jìn)行高壓均質(zhì)處理（共進(jìn)行5次高壓均質(zhì)處理）。納米載體在4 °C下以16 [細(xì)空格（1/6-em）]000 g離心90分鐘（共進(jìn)行3次）進(jìn)行純化，然后重新分散于水中。為了進(jìn)行共聚焦成像，LMNV 使用熒光 Vybrant DiO 細(xì)胞標(biāo)記染料（Invitrogen）進(jìn)行標(biāo)記。將 5 mg 納米載體與 20 μM DiO 在 37 °C 下孵育 2 小時(shí)，然后在 4 °C 下以 16 [細(xì)空格（1/6-em）]000 g離心 90 分鐘（三次）進(jìn)行洗滌。對(duì)于直接隨機(jī)光學(xué)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(jìn)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pg電子官方生物提供相關(guān)產(chǎn)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

以上文章內(nèi)容來源各類期刊或文獻(xiàn)，如有侵權(quán)請(qǐng)聯(lián)系我們刪除！