光学汽化全氟化碳纳米液滴的配方和声学调制
Summary
光学活化全氟化碳纳米液滴在血管系统以外的成像应用中显示出前景。本文将演示如何合成这些颗粒，交联聚丙烯酰胺模型，并在声学上调制液滴以增强其信号。
Abstract
微泡是超声中最常用的成像造影剂。然而，由于它们的大小，它们仅限于血管室。这些微气泡可以冷凝或配制成全氟化碳纳米液滴（PFCnD），其小到足以外渗，然后在目标位点以声学方式触发。这些纳米颗粒可以通过包括诸如近红外有机染料或纳米颗粒（例如硫化铜纳米颗粒或金纳米颗粒/纳米棒）的光吸收剂来进一步增强。光学标记的PFCnD可以通过激光照射在称为光滴汽化（ODV）的过程中汽化。这种活化过程允许使用高沸点全氟化碳核心，这些核心在诊断成像的最大机械指数阈值下不能在声学上汽化。较高的沸点核心导致液滴在汽化后重新冷凝，导致“闪烁”PFCnD在蒸发后短暂产生对比度，然后凝结回纳米液滴形式。该过程可以重复以按需产生对比度，从而允许通过光学和声学调制进行无背景成像、多路复用、超分辨率和对比度增强。本文将演示如何利用探针超声合成光学触发的脂壳PFCnD，创建聚丙烯酰胺模型来表征纳米液滴，并在ODV后对PFCnD进行声学调制以提高对比度。
Introduction
微泡是最普遍的超声造影剂，因为与软组织相比，微泡具有生物相容性和出色的回声性。这使它们成为可视化血流、器官描绘和其他应用的宝贵工具1。然而，它们的尺寸（1-10μm）使它们在基于共振频率的成像中非常出色，因此将其应用限制在脉管系统2上。
这种限制导致了PFCnD的发展，PFCnD是由包裹在液体全氟化碳核心周围的表面活性剂组成的纳米乳液。这些纳米颗粒可以以小至200nm的尺寸合成，旨在利用肿瘤脉管系统中发现的“泄漏”脉管系统或孔隙和开窗。虽然这些破坏是肿瘤依赖性的，但这种渗透性允许纳米颗粒从~200nm-1.2μm外渗，具体取决于肿瘤3，4。在它们的初始形式中，这些颗粒几乎不产生超声波对比度。在汽化时 – 通过声学或光学诱导 – 核心相从液体变为气体，导致直径增加两倍半到五倍5，6，7并产生光声和超声波对比。虽然声学汽化是最常见的激活方法，但这种方法会产生声学伪影，从而限制汽化成像。此外，大多数全氟化碳需要机械指数超过安全阈值的聚焦超声才能蒸发8。这导致了低沸点PFCnD的发展，它可以通过将微气泡冷凝成纳米液滴来合成9。然而，这些液滴更易挥发，并且会自发汽化10。
另一方面，光滴汽化（ODV）需要添加光学触发器，例如纳米颗粒11，12，13或染料6，14，15，并且可以使用ANSI安全限值11内的注量蒸发更高沸点的全氟化碳。用更高沸点核心合成的PFCnD更稳定，汽化后会重新凝结，从而实现无背景成像16、多路复用17和超分辨率18。这些技术的主要局限性之一是高沸点PFCnD在汽化后仅在短时间内产生回波，在毫秒19的尺度上，并且相对较暗。虽然这个问题可以通过反复汽化和平均来缓解，但液滴信号的检测和分离仍然是一个挑战。
从脉冲反转中汲取灵感，可以通过修改超声成像脉冲19的相位来增强持续时间和对比度。通过以稀疏相（n脉冲）启动超声成像脉冲，汽化PFCnD的持续时间和对比度都会增加。相反，以压缩期（p脉冲）启动超声成像脉冲会导致对比度降低和持续时间缩短。本文将介绍如何合成光学触发的全氟化碳纳米液滴，即成像中常用的聚丙烯酰胺模型，并通过声学调制演示对比度增强和延长信号寿命。
Protocol
1. 全氟化碳纳米液滴制剂 用氯仿冲洗出 10 mL 圆底烧瓶，然后反复吸出整个注射器体积并排出总共 3 次，用氯仿冲洗 10 μL 和 1 mL 气密玻璃注射器。注意：氯仿是挥发性的，如果吸入可能有毒。所有使用该溶剂的工作都应在通风橱中进行。 使用注射器，将 200 μL DSPE-mPEG2000 （25 mg/mL）、6.3 μL 1，2-二硬脂酰基-sn-甘油-3-磷酸胆碱（DSPC，25 mg/mL）和 1 mL IR 1048（1 mg/mL 氯仿溶液）加入圆?…
Representative Results
PFCnDs的成功配制和离心分离应产生直径约为200-300nm的液滴（图1A）。分离不当的液滴可能在1μm附近显示小峰。这些溶液可以进一步进行浴超声处理，以分解较大的液滴。由于在称为奥斯特瓦尔德成熟21，22的过程中聚结和/或扩散，液滴的大小将随着时间的推移而增加（图1B）。 通过操纵?…
Discussion
探针超声处理是一种相对简单易学的PFCnD制备方法。有几个步骤必须小心。处理氯仿时，必须使用外置活塞式移液器或玻璃注射器，因为它具有挥发性，并且会从标准空气置换式移液器中“泄漏”。此外，如果使用正置换，请确保使用适当的吸头，因为氯仿会溶解大多数塑料吸头，这会将污染物引入溶液中。对于全氟己烷，还建议使用外置活塞式移液器或玻璃注射器，因为它既易挥发又比水密度?…
Divulgations
The authors have nothing to disclose.
Acknowledgements
这项工作得到了乳腺癌研究基金会BCRF-20-043的部分支持。
Materials
| Ammonium Persulfate (APS) | VWR | 97064-592 | |
| 1,2-distearoyl-sn-glycero-3-phosphocholine (DSPC) | Avanti Polar Lipids | 850365C | Lipids, these can be purchased suspended in chloroform or in powder form. For long term storage, powder form is the best but chloroform is more practical. |
| 1,2-distearoyl-sn-glycero-3-phosphoethanolamine-N-[methoxy(polyethylene glycol)-2000] (ammonium salt) (DSPE-PEG) | Avanti Polar Lipids | 880120C | Lipids, these can be purchased suspended in chloroform or in powder form. For long term storage, powder form is the best but chloroform is more practical. |
| Acrylamide : Bisacrylamide solution (19:1) 40% (w/v), OmniPur® | VWR | EM-1300 | acrylamide solution, lower concentration/ powder |
| IR-1048 | Sigma | 405175 | Infrared dye |
| L11-4v | Verasonics | – | ultrasound linear array transducer |
| Microtip 1/8" | Qsonica LLC | 4418 | microtip for probe sonicator |
| N, N, N′, N′ -Tetramethylethylenediamine (TEMED) | VWR | 97064-902 | Used to polymerize polyacrylamide by forming free radicals in the presence of ammonium persulfate |
| Nova II | Ophir-Spiricon | 7Z01550 | laser power meter |
| Perfluorohexane | Fluoromed | APF-60M | perfluorocarbon liquid |
| Phosphate buffered saline (PBS) tablets | VWR | 97062-732 | Tablets used to make PBS |
| Q500 | Qsonica LLC | Q500-110 | Probe sonicator |
| Silica gel | Sigma-Aldrich | 288500 | 2-25 μm particle size |
| Tempest 30 | New wave research | – | Pulsed laser system |
| Vantage 128 | Verasonics | – | research ultrasound imaging system |
| Zetasizer Nano ZS | Malvern Instruments Ltd | – | Makes size measurements based on dynamic light scattering |
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Citer Cet Article
Zhao, A., Lee, J., Emelianov, S. Formulation and Acoustic Modulation of Optically Vaporized Perfluorocarbon Nanodroplets. J. Vis. Exp. (173), e62814, doi:10.3791/62814 (2021).