Name: surface-enhanced resonance raman scattering nanoprobe ratiometry for detecting microscopic ovarian cancer via folate receptor targeting
Uploaded: 2019-03-25T13:00:00
Description: Watch this Scientific Journal Video about Surface-enhanced Resonance Raman Scattering Nanoprobe Ratiometry for Detecting Microscopic Ovarian Cancer via Folate Receptor Targeting at JoVE.com

确认了以下资金来源 (对 m. f. k.): NIH R01 EB017748、R01 CA222836 和 K08 CA16396;达蒙 Runyon-Rachleff 创新奖 DRR-29-14, 潘兴广场索恩奖, 由潘兴广场索恩癌症研究联盟, MSCCC 分子成像 & 纳米技术中心 (CMINT) 和技术开发赠款。还向 MSCC NIH 核心赠款 (P30-ca008748) 提供的赠款资助支助提供了确认服务。

所有动物研究都得到了纪念斯隆·凯特林癌症中心 (#06-07-011) 机构动物护理和使用委员会的批准。
1. 金纳米星芯合成
请注意:在本实验中, 金纳米星被用作两种类型的 SERS 纳米探针的核心。
<ol><li>在去离子水中制备 800 mM 抗坏血酸 (c6h8o6) 溶液, 在 di 水中制备 20 mm 四氯油酸 (hauccl4) 溶液 8 ml.冷却至4°C。</li>
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<ol>
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本文所述的协议为 SERRS 纳米探针两种 "味道" 的合成及其在小鼠体内的应用提供了指导, 用于应用于卵巢肿瘤过度表达叶酸受体的拉曼成像, 并采用了比率法。与其他光学成像技术 (如荧光) 相比, 拉曼成像的主要优势在于纳米探针信号的高特异性, 而纳米探针信号不能与任何生物来源的信号混淆。在拉曼成像的这一实施例中, 纳米颗粒不是静脉注射的, 而是局部的, 通过腹腔内注射, 然后是一个单一的...

具有 "表面增强拉曼散射" (sers) 纳米粒子的拉曼成像在描述各种环境中的病变以及许多不同类型的肿瘤 1、2、3、4方面显示出巨大的希望.SERS 纳米粒子的主要优点是它们的指纹样光谱特征, 使它们能够毫无疑问地检测到生物背景信号5。此外, 发射信号的强度进一步放大, 使用记者分子 (染料) 与激发激光器一致的吸收极大值, 产生 "表面增强共振拉曼散射" (serrs)具有更高灵敏度的纳米粒子6、7、8、9、10、11、12.
采用 se (r) rs 纳米颗粒13和许多其他纳米颗粒结构 14, 15 临床使用需要解决的一个障碍是它们的给药方式, 因为静脉注射导致系统性剂的暴露, 并需要广泛的测试, 以排除潜在的不利影响。在本文中, 我们提出了一个不同的范式, 基于在体内局部应用纳米粒子, 直接进入腹腔在手术过程中, 然后是一个洗涤步骤, 以消除任何未绑定的纳米粒子1。这种方法符合目前正在研究的新的治疗方法, 这些方法也利用局部注射剂进入腹腔, 称为高温腹腔内化疗 (HIPEC)。因此, 该原则本身应该相对容易融入临床工作流程。我们研究了腹腔内应用后纳米颗粒的生物分布, 但没有观察到任何可检测到的吸收到全身循环1。此外, 局部应用方法规避网状内皮系统对纳米颗粒的固存, 从而显著减少了所需的纳米颗粒的数量。然而, 当局部应用时, 抗体功能化纳米粒子倾向于粘附在内脏表面上, 即使在没有目标的情况下也是如此。为了最大限度地减少由于非特异性纳米粒子粘附而产生的假阳性信号, 我们采用了一种比率测量方法, 即分子靶向纳米探针提供特定信号, 以及具有不同拉曼光谱的非靶向对照纳米探针,帐户的非特定背景16,17。我们最近在弥漫性卵巢癌1的小鼠模型中展示了局部应用表面增强共振拉曼比率光谱法。
该方法的总体目标是开发两个 SERRS 纳米探针, 一个有针对性的, 一个是非特异的, 在小鼠模型中局部应用, 以便利用两个探针的比率测量来成像与癌症相关的生物标志物的显性过度表达通过拉曼成像。在这项工作中, 叶酸受体 (fr) 被选择为目标, 因为这是一个标记在许多卵巢癌18,19。拉曼微光成像与 sers 为基础的纳米粒子也被证明用于癌细胞的鉴定20。合成了拉曼纳米粒子的两种不同的 "味道", 每种化合物都是从不同的有机染料中提取指纹的。纳米粒子由一个星形金芯组成, 周围是一个硅壳, 并在大约710纳米处显示表面等离子体共振。拉曼机 (有机染料) 与硅壳的形成同时沉积。最后, 对于 frs 靶向纳米探针 (Α-Fr-nps), 二氧化硅壳与抗体结合, 而非靶向纳米探针 (nt-NPs) 则与聚乙二醇单层钝化。
该技术成功地用于绘制弥漫性转移性卵巢癌 (SKOV-3) 小鼠异种移植模型中的显微肿瘤图, 证明了其在体内使用的适用性。它还可以扩展用于切除组织, 肿瘤表型, 或脱粒后边缘的确定, 如同源研究21所示。
SERS 纳米探针为创建多个生物标志物的目标标签提供了一个强大的平台, 如图 1所示, 通过简单的化学反应合成。在这里, 我们提出了合成两种类型的 SERRS 纳米探针 (第1-3 节) 的协议, 开发一个合适的卵巢癌小鼠模型 (第4节), 纳米探针的管理和成像 (第5节), 最后的数据分析和可视化 (第6节)。

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			<td style="width:161px;"></td>
			<td style="width:132px;"></td>
			<td style="width:185px;"></td>
		</tr>
		<tr height="25">
			<td height="25" style="height:25px;">Ascorbic acid</td>
			<td>Sigma-Aldrich</td>
			<td>A5960</td>
			<td></td>
		</tr>
		<tr height="25">
			<td height="25" style="height:25px;">3-MPTMS</td>
			<td>Sigma-Aldrich</td>
			<td>175617</td>
			<td></td>
		</tr>
		<tr height="25">
			<td height="25" style="height:25px;">Ammonium hydroxide (28%)</td>
			<td>Sigma-Aldrich</td>
			<td>338818</td>
			<td></td>
		</tr>
		<tr height="25">
			<td height="25" style="height:25px;">Anti-Folate Receptor antibody [LK26]&#160;</td>
			<td>AbCam</td>
			<td>ab3361</td>
			<td></td>
		</tr>
		<tr height="25">
			<td height="25" style="height:25px;">Dimethyl sulfoxide</td>
			<td>Sigma-Aldrich</td>
			<td align="right">276855</td>
			<td></td>
		</tr>
		<tr height="25">
			<td height="25" style="height:25px;">Dimethyl sulfoxide (anhydrous)</td>
			<td>Sigma-Aldrich</td>
			<td>276855</td>
			<td></td>
		</tr>
		<tr height="25">
			<td height="25" style="height:25px;">Ethanol</td>
			<td>Sigma-Aldrich</td>
			<td>792780</td>
			<td></td>
		</tr>
		<tr height="25">
			<td height="25" style="height:25px;">IR140</td>
			<td>Sigma-Aldrich</td>
			<td>260932</td>
			<td></td>
		</tr>
		<tr height="25">
			<td height="25" style="height:25px;">IR780 perchlorate*</td>
			<td>Sigma-Aldrich</td>
			<td>576409</td>
			<td>Discontinued*</td>
		</tr>
		<tr height="25">
			<td height="25" style="height:25px;">Isopropanol</td>
			<td>Sigma-Aldrich</td>
			<td>650447</td>
			<td></td>
		</tr>
		<tr height="25">
			<td height="25" style="height:25px;">N.N.Dimethylformamide</td>
			<td>Sigma-Aldrich</td>
			<td>227056</td>
			<td></td>
		</tr>
		<tr height="25">
			<td height="25" style="height:25px;">PEG crosslinker</td>
			<td>Sigma-Aldrich</td>
			<td>757853</td>
			<td></td>
		</tr>
		<tr height="25">
			<td height="25" style="height:25px;">PEG-maleimide</td>
			<td>Sigma-Aldrich</td>
			<td>900339</td>
			<td></td>
		</tr>
		<tr height="25">
			<td height="25" style="height:25px;">Tetrachloroauric Acid</td>
			<td>Sigma-Aldrich</td>
			<td>244597</td>
			<td></td>
		</tr>
		<tr height="25">
			<td height="25" style="height:25px;">Tetraethyl Orthosilicate</td>
			<td>Sigma-Aldrich</td>
			<td>86578</td>
			<td></td>
		</tr>
		<tr height="25">
			<td height="25" style="height:25px;">*IR792</td>
			<td>Sigma-Aldrich</td>
			<td>425982</td>
			<td>*Alternative</td>
		</tr>
		<tr height="25">
			<td height="25" style="height:25px;">Name of Equipment</td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr height="25">
			<td height="25" style="height:25px;">Dialysis cassette (3,500 MWCO)</td>
			<td>ThermoFIsher</td>
			<td align="right">87724</td>
			<td></td>
		</tr>
		<tr height="25">
			<td height="25" style="height:25px;">Centrifugal filters</td>
			<td>Millipore</td>
			<td>UFC510096</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">inVia confocal Raman microscope</td>
			<td>Renishaw</td>
			<td></td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">MATLAB (v2014b)</td>
			<td>Mathworks</td>
			<td></td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">PLS Toolbox (v8.0)</td>
			<td>Eigenvector research</td>
			<td></td>
			<td></td>
		</tr>
	</tbody>
</table>

surface-enhanced resonance raman scattering nanoprobe ratiometry for detecting microscopic ovarian cancer via folate receptor targeting

卵巢癌是最致命的妇科恶性肿瘤。大多数患者处于晚期 (FIO iii 或 IV 期), 当局部转移扩散已经发生。然而, 卵巢癌有一个独特的转移扩散模式, 因为肿瘤植入物最初包含在腹腔内。这一特点原则上可以使肿瘤植入物的完全切除具有治疗意图。这些转移性病变中的许多都是微观的, 很难识别和治疗。中和这种微转移被认为是消除肿瘤复发和实现长期生存的主要目标。具有表面增强共振的拉曼成像拉曼散射纳米探针由于其明亮和生物正交特征, 可用于高灵敏度的显微肿瘤的描述。在这里, 我们描述了这种纳米探针的两种 "味道" 的合成: 一种是针对叶酸受体的抗体功能化的合成--在许多卵巢癌中过度表达--另一种是非靶向对照纳米探针, 具有明显的光谱。纳米探针在腹腔内联合给转移性人卵巢腺癌的小鼠模型。所有动物研究都得到了纪念斯隆·凯特林癌症中心动物护理和使用委员会的批准。用拉曼显微光谱仪对动物的腹腔进行手术暴露、清洗和扫描。随后, 使用经典最小二乘拟合算法对这两个纳米探针的拉曼特征进行解耦, 并对它们各自的分数进行划分, 以提供叶酸比非目标探针更有针对性的比率信号。通过这种方式, 显微镜转移是可视化的高特异性。这种方法的主要优点是, 局部应用到腹腔-这可以在手术过程中方便地做-标签肿瘤, 而不会使患者受到系统的纳米颗粒暴露。通过采用比率法, 将具有不同拉曼特征的靶向和非靶向纳米探针作为混合物应用, 可以消除纳米探针与内脏表面的非特定结合所产生的假阳性信号。由于缺乏商业广域拉曼成像摄像系统, 这一程序目前仍然受到限制, 一旦可用, 就可以在手术室应用这一技术。

为了质量控制的目的, 纳米粒子可以在合成过程中使用各种方法进行表征, 包括 TEM、DLS、纳米粒子跟踪分析和 UV/Vis 吸收光谱, 如图 2所示。
通过这种方式, 可以验证金纳米岩核的大小 (第1节所述)、硅壳的形成 (第2节) 和随后的表面功能化 (第3节) (图 2)。通常情况下, 金纳米核的大小 (...

Watch this Scientific Journal Video about Surface-enhanced Resonance Raman Scattering Nanoprobe Ratiometry for Detecting Microscopic Ovarian Cancer via Folate Receptor Targeting at JoVE.com

Surface-enhanced Resonance Raman Scattering Nanoprobe Ratiometry for Detecting Microscopic Ovarian Cancer via Folate Receptor Targeting

卵巢癌在腹腔内形成转移。在这里, 我们提出了一个协议, 以制作和使用叶酸受体靶向表面增强共振拉曼散射纳米探针, 揭示这些病变具有高度的特异性,通过比例成像。纳米探针在腹腔内对活着的小鼠进行处理, 所得图像与组织学有很好的相关性。

表面增强共振拉曼散射纳米探针比比法检测微卵巢癌的意义上

Ovarian cancer represents the deadliest gynecologic malignancy. Most patients present at an advanced stage (FIGO stage III or IV), when local metastatic ...

该方法利用纳米粒子对显微肿瘤进行局部应用，特别是在尚未获得血管瘤的恶性肿瘤中，如卵巢癌的转移。该技术是比率，成像靶向和非靶向纳米粒子在一次扫描，以减少假信号，并揭示肿瘤的真实程度。这些纳米粒子可以通过内丙酮或静脉注射进行，用于卵巢癌和许多其他肿瘤类型的高精度成像。对于金纳米星芯合成，在4摄氏度的磁搅拌板上放置含有800毫升新鲜准备的抗坏血酸溶液的圆锥形烧瓶，并产生稳定的涡流。快速添加八毫升新鲜准备的四氯酸溶液到漩涡。在几秒钟内，纳米星将形成，溶液将采用深蓝色。将纳米星悬浮液倒入 50 毫升锥形管中进行离心，并在旋转结束时从每个管中吸出除了最后 200 微升的上经剂。在将所有纳米粒子汇集到单个透析盒中之前，使用微管将纳米粒子重新插入每个管中的纳米粒子。然后将纳米颗粒解洗至少三天，对两升去水每天改变水。对于二氧化硅壳的形成，首先在50毫升锥形管中加入10毫升异丙醇、500微升TEOS、200微升的去维水和60微升染料。在15毫升管中加入3毫升乙醇和200微升氢氧化铵。接下来，对拨号纳米星进行声波化，以分散任何星团，并在管中加入1.2毫升的纳米星。将50毫升管放在涡流混合器上，并诱导稳定的涡流。在室温下以 300 RPM 的速度快速将管转移到摇床 15 分钟之前，只需再混合约 5 秒，即可将纳米星溶液快速添加到 50 毫升管中。两个管必须快速结合，并不断的尼兴下，以确保在纳米星上发生硅反应，并尽量减少自由二氧化硅生产。在孵化结束时，用乙醇填充管子，以淬火反应，通过离心收集纳米星。吸气所有，但关于最后500微升的上超，注意不要打扰颗粒和重新暂停纳米粒子在一毫升新鲜乙醇。然后将溶液转移到1.5毫升离心管中，通过离心洗涤四个一毫升乙醇，每次洗涤之间通过超声波循环重新产生颗粒约一秒钟。将硫醇引入颗粒表面，在上次洗涤后，将颗粒重新用一毫升的85%乙醇、10%3-3-甲基苯丙胺和5%的脱硫水溶液中重新暂停，在室温下孵育一到两小时。在孵化结束时，如所示，通过离心洗硫醇功能化纳米粒子，在洗涤之间通过超声波重新产生颗粒。对于抗体功能化抗叶酸受体纳米程序，在500微升的海信缓冲液中，用10倍PEG交联剂的PEG交叉链接反应200微克抗叶酸结合蛋白抗体。30分钟后，通过将离心过滤器离心浓缩抗体，通过将过滤器倒置到新管中进行额外离心，恢复抗体。然后将硫醇功能化纳米颗粒与浓缩抗体混合，在室温下孵育混合物至少30分钟。为了形成非靶向纳米程序，将溶解在DMSO中的5毫克甲氧氧终止PEG 5000雄性添加到500微升的 HEPES 缓冲液中的硫醇功能化纳米颗粒中，在室温下至少孵育 30 分钟。用于纳米喷头注射，可同时旋转两种纳米蛋白，并在 600 个皮摩尔浓度下将颗粒重新注入新鲜 MES 缓冲液中。混合纳米颗粒，将产生的溶液的一毫升装入一个一毫升注射器中，每只小鼠装有26粒针，内向每只小鼠的腹部注射整个体积。然后轻轻按摩腹部，将纳米粒子分布到腹腔中。至少25分钟后，将安乐死注射动物的四肢固定到手术平台上，并使用锯齿状钳子和解剖器去除皮肤以暴露胸膜。将佩里托内姆化为一体。将腹膜瓣连接到平台，用至少 60 毫升 PBS 清洗腹腔内部。然后将平台传输到具有直立光学配置和电动舞台的拉曼分光光度计，并在适当的成像参数下对腹部进行成像。这一点至关重要，拉曼扫描的焦平面位于大多数内脏的表面。如果器官出平面，获得的信号将没有用处。出于质量控制目的，纳米粒子可以在合成过程中使用各种方法进行特性，包括透射电子显微镜、动态光散射、纳米粒子跟踪分析和超远可见吸收光谱。拉曼测量揭示了纳米粒子在合成过程中存在的独特光谱。典型的反应产量和浓度在很大程度上取决于洗涤步骤中的移液技术。拉曼光谱可以处理以去除荧光，并规范化为单位区域，以补偿信号强度，然后再应用经典最少的正方形。虽然每个纳米诊断参考光谱上的经典最不平方分数不能单独提供肿瘤的特定位置，但点对点比率揭示了传播的微小扩散的存在。在尝试此过程时，重要的是要记住在合成的每个步骤中验证纳米粒子，因为纳米粒子的质量会强烈地影响最终结果。这项技术展示了研究人员使用SERRS纳米蛋白检测动物模型中与肿瘤相关的标记物的潜力，并具有高度的微观精度切除患者组织。

Research

JoVE Journal

Cancer Research

Intra-iliac Artery Injection for Efficient and Selective Modeling of Microscopic Bone Metastasis

内髂动脉注射显微骨转移的效率和选择性建模

Intra-iliac artery (IIA) injection is an efficient approach to introduce metastatic lesions of various cancer cells in animals. Compared to the widely ...

科研

癌症研究

Murine Experimental Model of Original Tumor Development and Peritoneal Metastasis via Orthotopic Inoculation with Ovarian Carcinoma Cells

Murine Experimental Model of Original Tumor Development and Peritoneal Metastasis via Orthotopic Inoculation with Ovarian Carcinoma Cells

原发肿瘤发展小鼠实验模型和腹膜转移<em&gt;通过</em&gt;原位接种卵巢癌细胞

Epithelial ovarian carcinoma (EOC) is associated with a poor prognosis because it shows peritoneal dissemination. To improve the prognosis, it is ...

An Integrated Platform for Genome-wide Mapping of Chromatin States Using High-throughput ChIP-sequencing in Tumor Tissues

肿瘤组织中高通量芯片测序的染色质状态全基因组图谱集成平台

Histone modifications constitute a major component of the epigenome and play important regulatory roles in determining the transcriptional status of ...

A GPC3-targeting Bispecific Antibody, GPC3-S-Fab, with Potent Cytotoxicity

GPC3-targeting 双特异性抗体, GPC3-S-Fab, 具有强大的细胞毒性

This protocol describes the construction and functional studies of a bispecific antibody (bsAb), GPC3-S-Fab. bsAbs can recognize two different epitopes ...

Synthesis and Characterization of Placental Chondroitin Sulfate A (plCSA)-Targeting Lipid-Polymer Nanoparticles

Synthesis and Characterization of Placental Chondroitin Sulfate A plCSA-Targeting Lipid-Polymer Nanoparticles

胎盘硫酸软骨素 A (plCSA) 靶向脂质高分子纳米粒的合成与表征

An effective cancer therapeutic method reduces and eliminates tumors with minimal systemic toxicity. Actively targeting nanoparticles offer a promising ...

Detecting the Ligand-binding Domain Dimerization Activity of Estrogen Receptor Alpha Using the Mammalian Two-Hybrid Assay

利用哺乳动物双杂交法检测雌激素受体α的配联域二维化活性

Estrogen receptor alpha (ER&#945;) is an estrogenic ligand-dependent transcription regulator. The sequence of ER&#945; protein is highly conserved among ...

Manufacturing Chimeric Antigen Receptor (CAR) T Cells for Adoptive Immunotherapy

Manufacturing Chimeric Antigen Receptor CAR T Cells for Adoptive Immunotherapy

制造用于采用免疫疗法的奇美抗原受体（CAR）T细胞

Adoptive immunotherapy holds promise for the treatment of cancer and infectious disease. We describe a simple approach to transduce primary human T cells ...

Preparation of Mitochondria from Ovarian Cancer Tissues and Control Ovarian Tissues for Quantitative Proteomics Analysis

卵巢癌组织线粒体的制备与控制卵巢组织进行定量蛋白质组学分析

Ovarian cancer is a common gynecologic cancer with high mortality but unclear molecular mechanism. Most ovarian cancers are diagnosed in the advanced ...

Quantifying Replication Stress in Ovarian Cancer Cells Using Single-Stranded DNA Immunofluorescence

使用单链DNA免疫荧光量化卵巢癌细胞中的复制应激

Replication stress is a hallmark of several ovarian cancers. Replication stress can emerge from multiple sources, including double-strand breaks, ...

Visualizing DNA Damage Repair Proteins in Patient-Derived Ovarian Cancer Organoids via Immunofluorescence Assays

Visualizing DNA Damage Repair Proteins in Patient-Derived Ovarian Cancer Organoids via Immunofluorescence Assays

通过免疫荧光测定 可视化 患者来源的卵巢癌类器官中的 DNA 损伤修复蛋白

Immunofluorescence is one of the most widely used techniques to visualize target antigens with high sensitivity and specificity, allowing for the accurate ...