含有金纳米棒蜂窝车辆的制备及光声分析
1Institute of Applied Physics,Italian National Research Council, 2Department of Experimental and Clinical Biomedical Sciences,University of Florence, Firenze, 3Department of Physics and Astronomy,University of Florence, Sesto Fiorentino, 4Department of Pharmacy and Biotechnology,University of Bologna
Summary
We show the preparation and address the feasibility of cellular vehicles containing gold nanorods for the photoacoustic imaging of cancer.
Abstract
金纳米棒是为一系列的生物医学应用，如光热消融和癌症的光声成像的吸引力，这要归功于在近红外窗口，低细胞毒性和潜在的对置成肿瘤其强烈的光学吸光度。然而，他们交付的肿瘤仍然是一个问题。一种创新的方法包括可装在体外金纳米棒肿瘤相关巨噬细胞的向性的开采。在这里，我们描述的制备方法和含有金纳米棒的细胞车辆的光声检查。 PEG化的金纳米棒被修改与季铵化合物，以实现一种阳离子轮廓。与普通培养皿小鼠巨噬细胞的接触，这些粒子被发现发生巨大摄取进入内吞囊泡。然后将这些细胞被包埋在生物聚合物水凝胶，这是用来验证光声转换的稳定性的颗粒被保持在其纳入蜂窝车辆。我们相信，这些结果可能为新型的发展战略提供等离子粒子肿瘤新的灵感。
Introduction
在过去十年中，各种电浆颗粒如金纳米棒，纳米壳和纳米笼，在生物医学光学1，2，3，4已受到相当关注的应用。在与标准的金纳米球方差，这些较新的粒子共振在于提供用于通过主体最深光学穿透并用内源性组分1最高光学对比度的近红外（NIR）窗口。这个特性已引起对创新应用，例如光声（PA）的成像和癌症的光热消融的兴趣。然而，有几个问题限制这些颗粒的临床渗透。例如，它们的光学活性趋于诱导它们过热和修改他们的功能性形状朝向多个球形轮廓，其驱动一个光稳定性5，6，7，8 </SUP> 9。占主导地位的科学辩论的另一个问题是他们的全身输送到肿瘤。尤其是金纳米棒相结合，是理想渗透了该显示增强通透性和保留和易用性恶性肿瘤标志物的特异性探针结合的肿瘤大小。因此，他们对直接注射到血流中制备被认为是一个可行的方案10，11，12，13。然而，这条路线仍然是个问题，其中大部分粒子变得由单核吞噬细胞系统10，11，12捕获。此外，另一个问题是，通过主体14循环后的粒子的光学和生化稳定性。当颗粒失去胶体稳定性和聚集，其等离子的特点和传热动态可能从电浆子耦合15受苦， </s达> 16，17和交叉过热18。
最近，利用肿瘤相关巨噬细胞的趋性的概念已经成为一个聪明的替代19，20，21。这些细胞持有检测和高特异性的肿瘤普遍存在一种与生俱来的能力。因此，一个角度看可能是这些细胞从病人隔离， 与体外金纳米棒装载它们，然后注入回到患者，以意向使用它们作为负责递送的细胞的车辆。另一个优点是获得对颗粒的光学和生化稳定性更多的控制，因为它们的生物界面将在体外构建。尽管如此，这些细胞汽车作为光造影剂的性能需要一个批判性的分析。
在这项工作中，我们描述了准备和cellul的关键问题含有金纳米棒为癌症的PA的成像芳车辆。 PEG化的金纳米棒被修改与季铵化合物22，以实现该预期以促进与血浆膜23,24及其相互作用的阳离子分布。这些颗粒进行有效和非特异性摄取大部分细胞种类，希望不受外界干扰太多与他们的生物学功能。小鼠巨噬细胞装载有高达每细胞多达200，000阳离子金纳米棒，这成为紧内吞囊泡内密闭。这种配置应该出现的问题，因为这些囊泡内电浆耦合和交叉过热的威胁。因此，巨噬细胞被嵌入模仿生物组织生物聚合物水凝胶，以验证大多数颗粒PA转化的稳定性被保持在从生长培养基转移到内吞囊泡。有效实现了È测量标准，以便测量的PA转化为PA的成像直接兴趣的条件下的稳定性制定。一个重塑阈值设置在光不稳定性的非常发病50的激光脉冲10赫兹的典型重复率一个序列后。
我们相信，这些结果可能为新的发展战略，以实现等离子粒子肿瘤提供动力。
Protocol
注：金纳米棒的全部浓度是名义上金摩尔浓度表示。为与其它工作相比较，注意，1M的金大致相当于20μM的金纳米棒，在我们的情况。 1.阳离子金纳米棒的制备注意：该方法开始于西曲溴铵（CTAB）加帽的金纳米棒通过用抗坏血酸自催化还原氯金酸4的合成，根据根据Ratto 等人 26通过Nikoobakht 等 25介绍，并适于协议?…
Representative Results
这里，含有金纳米棒为癌症的PA的成像蜂窝车辆的可行性与协议的典型的结果一起示出。 图1中的TEM图像显示步骤1之后的颗粒和它们的细胞车辆步骤2.颗粒和细胞的TEM成像的制备别处17描述后的通常外观。阳离子金纳米棒进行巨噬细胞中一个巨大的积累，维持其正常形态。被发现的粒子被严密囊泡内吞局限?…
Discussion
靶向肿瘤相关巨噬细胞的概念正在成为一个强大的概念，以打击癌症34，35，36。在这里，而不是销毁，这些细胞被招募为移动车辆，使金纳米棒成肿瘤，其取向的剥削。这个角度看，需要颗粒的贴心设计，融入细胞及其鉴定。我们已发现，装载有阳离子金纳米棒的鼠巨噬细胞的耐光不会从颗粒的内吞囊泡内的限制，这意味着他们的电浆耦合和交叉过热不是…
Divulgations
The authors have nothing to disclose.
Acknowledgements
这项工作是部分由托斯卡纳大区和欧洲共同体的ERANET +项目LUS泡沫和BI-TRE的框架内支持。
Materials
| Hexadecyltrimethylammonium bromide | Sigma-Aldrich | H6269 | To synthesize gold nanorods |
| Gold(III) chloride trihydrate | Sigma-Aldrich | 520918 | To synthesize gold nanorods |
| Silver nitrate | Sigma-Aldrich | S6506 | To synthesize gold nanorods |
| L-ascorbic acid | Sigma-Aldrich | A5960 | To synthesize gold nanorods |
| Sodium borohydride | Sigma-Aldrich | To synthesize gold nanoseeds | |
| MeO-PEG-SH | Iris Biotech | PEG1171 | To PEGylate gold nanorods. Molecular weight about 5,000 Da |
| Acetic acid | Sigma-Aldrich | 320099 | To PEGylate gold nanorods and solubilize chitosan |
| Sodium acetate | Sigma-Aldrich | S8750 | To PEGylate gold nanorods |
| (11-Mercaptoundecyl)-N,N,N-trimethylammonium bromide | Sigma-Aldrich | 733305 | To modify gold nanorods with quaternary ammonium compounds |
| Dimethyl sulfoxide | Sigma-Aldrich | 276855 | To solubilize (11-mercaptoundecyl)-N,N,N-trimethylammonium bromide |
| Polysorbate 20 | Sigma-Aldrich | P2287 | To centrifuge PEGylated gold nanorods |
| PBS | Lonza | BE17-516F | To suspend gold nanorods before incubation with cells and to treat pellets of cells |
| J774a.1 | ATCC | TIB-67 | Monocyte/macrophage murine cell line |
| DMEM | Lonza | BE12-707F | Cell culture medium |
| FBS | Lonza | DE14-801F | To be added to cell culture medium |
| L-glutamine | Lonza | BE17-605E | To be added to cell culture medium |
| Penicillin/streptomycin | Lonza | DE17-602E | To be added to cell culture medium |
| Petri dish | NEST | 705001 | Cell culture dish |
| Cell scraper | EuroClone | ES7018 | To detach cells |
| Formaldehyde | Fluka | 47630 | To fix cells |
| Chitosan, low molecular weight | Sigma-Aldrich | 448869 | 75-85% deacetylated. Molecular weight about 120,000 Da |
| Sodium hydroxyde | Sigma-Aldrich | 306576 | To insolubilize chitosan and generate the hydrogel |
| Polystyrene cell culture plates | NEST | 702011 | Used as molds to fabricate chitosan hydrogels |
| Optical parametric oscillator pumped by the third harmonic of a Q-switched Nd:YAG laser | Continuum, Santa Clara, USA | Surelite OPO plus | Source of optical excitation for photoacoustic tests |
| Pyroelectric detector | Gentec, Quebec, Canada | QE8SP | To monitor optical fluence for photoacoustic tests |
| Pre amplified needle hydrophone | Precision Acoustic, Dorset, UK | Model with 1 mm sensor diameter and 1-20 MHz frequency range | To measure photoacoustic signals |
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Citer Cet Article
Cavigli, L., Tatini, F., Borri, C., Ratto, F., Centi, S., Cini, A., Lelli, B., Matteini, P., Pini, R. Preparation and Photoacoustic Analysis of Cellular Vehicles Containing Gold Nanorods. J. Vis. Exp. (111), e53328, doi:10.3791/53328 (2016).