The authors would like to thank Drs. Helene Salmon and Miriam Merad from Icahn School of Medicine at Mount Sinai for providing the B16-F10-YFP cells and for their expert advice on melanoma mouse models. The authors further thank the Animal Imaging Core Facility, the Radiochemistry and Molecular Imaging Probes Core Facility, and the Molecular Cytology Core Facility at Memorial Sloan Kettering Cancer Center (MSK) for their support. This work was supported by National Institutes of Health grants NIH 1 R01 HL125703 (W.J.M.M.), R01CA155432 (W.J.M.M.), K25 EB016673 (T.R.) and P30 CA008748 (MSK Center Grant). The authors also thank the Center for Molecular Imaging and Nanotechnology (CMINT) at MSK for their financial support (T.R.).

该过程包括纳米颗粒的双放射性和荧光标记， 体内 PET-CT成像， 离体生物分布的测量，和离体免疫染色和流式细胞仪分析。所有的动物实验是由纪念斯隆 - 凯特琳癌症中心的机构动物护理和使用委员会批准。 1.双标记的脂质体的制备注:同基因的B16黑色素瘤也可以从吸入含-isoflurane-2％的氧气注入300000 B16-F10细胞到C57BL / 6小鼠的背部侧面麻醉下进行诱导。在手术过程中使用无菌的试剂和工具，在无菌的工作区。手术后，仔细观察动物，直到从麻醉其恢复。把动物放回笼子时，才重新获得全意识和流动性。他们的房子在动物移植瘤无菌室等。具有大小肿瘤300毫米3是理想的纳米颗粒的表征，由于其显着增强的渗透性和滞留效应（EPR），这可以增加纳米颗粒累积。下文提供的详细协议。 <ol><li>在一个圆底烧瓶中，溶解20毫克脂质的混合物在2毫升含氯仿1,2- dipalmitoyl- SN -glycero -3- phophocholine（DPPC），胆固醇，1,2- disearoyl- SN -glycero-的3-磷酸乙醇胺-聚（乙二醇）2000（DSPE-PEG2000），DSPE-去铁胺（DSPE-DFO）8，和1,1- diododecyl-3,3,3,3-tetramethylindodicarbocyanine -5,5-二磺酸（ DiIC12 [5] -DS）以分别61.3％，33.4％，5，0.2％和0.1％，摩尔比。 注意:此脂质组合物非常类似于目前在临床2中使用阿霉素的脂质体制剂。从我们的程序所得到的脂质体将密切模仿临床nanomedic 的体内性能国家统计局。 </li><li>使用旋转蒸发器除去有机溶剂，并形成在室温下的薄的脂质膜。加入20毫升的PBS并超声处理30分钟，通过使用了3.8毫米超声尖端和30瓦的幅度，以在冰上充分冷却，以产生脂质体纳米颗粒。 </li><li>离心机在4000 xg离心脂质体溶液10分钟以除去聚集物和洗涤用PBS离心过滤（分子量截止（MWCO纳米颗粒:100 kDa的）以除去游离脂质和残余有机溶剂的浓缩脂质体，这将留在顶室，然后将它们转移到新管中。脂质体可以在随后的步骤之前被存储在PBS中的冰箱长达1周。 </li><li>对于质量控制，通过动态光散射（DLS）表征脂质体。具体地，混合脂质体的50微升用PBS 950微升，然后将该混合物转移到上浆杯中。放置反应杯在分析仪和测量颗粒尺寸和它们的尺寸分布。的粒径和其粒度分布（多分散性指数（PDI））预计将90 - 分别为0.2， - 120纳米和0.1。 </li><li>对于放射性标记，在6.9和7.1之间的pH用1毫居里89 Zr的草酸，在37℃在1.5-mL管中混合纯化的脂质体，相当于2毫克脂质，在PBS中2小时，（总体积:〜 100μL）。通过离心过滤（MWCO = 100 kDa的），类似于步骤1.3除去游离的，未反应的89锆。用无菌PBS洗涤滞留并稀释至所需的体积。放射化学产率应是80 - 95％。 警告！放射性材料的工作是高度管制。机构必须得到认证，并提供必要的设施，人才培养，使用放射性严格监控。研究人员需要接受适当的培训和处理放射性物质时，佩戴个人防护装备和剂量环/徽章。 </li><li&gt;通过尺寸排阻放射性HPLC，其通常应在98％以上10确定放射性标记的脂质体的放射化学纯度。放射化学纯度低于95％都在1.5步洗指示不足。 </li><li>放射性标记后，确定通过动态光散射纳米颗粒的大小。的尺寸和PDI预计是相同的范围内，在步骤1.4 10测量范围内。 注意:如果放射性样品没有在流式细胞仪或荧光显微镜允许的，非放射性的NAT Zr的草酸盐可以用于在步骤1.5以标记的脂质体。这些非放射性脂质体具有其放射性类似物相同的特性。这个过程的描述在图2中提供。 </li></ol> 2. 在双标记的脂质体的体内 PET-CT成像和生物分布<ol><li>注入大约100微升的双标记的脂质体W为第i约300微居里的放射性成抑制黑素瘤小鼠（C57BL / 6，雌性，10 - 26周龄）通过其尾静脉，在约10毫居里89的Zr / kg体重。 </li><li>为了确定半衰期，从尾静脉从麻醉动物抽血下，使用28-G的胰岛素注射器含有2％异氟醚的氧气。收集血液 - 在预称重管（10 20微升）在2分钟，15分钟，1小时，4小时，8小时，24小时，并在注射后48小时。由差权衡血，用γ计数器测定放射性含量，并计算放射性浓度为每克注射剂量（％ID / g）的百分比。 注:在配备有麻醉系统，回收笼，加热，和生物危害罩的房间执行的过程。确保动物完全恢复，并从回收笼转移他们拿着笼子前，手术后获得正常流动。楼荷瘤动物的房间指定用于动物管理放射性材料。 </li><li> 24或48小时的后注射 脂质体，图像小鼠在小动物MicroPET-CT扫描仪10。水平放置在它的肚子麻醉动物，并保持它麻醉。辖2％异氟醚的氧气通过鼻锥。扫描以防止其干燥前申请眼药膏它的眼睛。 </li><li>执行全身PET静态扫描记录不少于5000万元重合的事件，用15分钟的大致时间。在350-700千电子伏和6纳秒分别设置能源和巧合的时间窗口。之后，执行一个5分钟计算机断层扫描（CT）扫描。在X射线管设置为80千伏和500微安的电流的电压;在CT扫描使用总共220度120旋转的步骤，每帧10大约145毫秒。 </li><li>在PET-CT扫描后，立即通过牺牲二氧化碳窒息的老鼠，然后按确认pinchi死亡纳克动物的爪子。一旦确认，进行颈椎脱位。 </li><li>通过首先切开右心房，然后注入20毫升的PBS到左心室除去在小鼠组织中的血液，并随后收集相关组织如肿瘤，肝，脾，肺，肾，肌肉和其他16。 </li><li>称量组织，测量通过γ计数8,9其放射性含量，并计算组织放射性蓄积为每克注射剂量（％ID / g）的百分比。 </li></ol> 3. 离体流式细胞仪和免疫注意:在剂量为0.5毫克每公斤体重染料注入大约100微升荧光脂质体的进黑素瘤小鼠通过其尾静脉。如果放射性样品没有在流式细胞仪，荧光显微镜允许的，必须使用非放射性的脂质体。 <ol><li>牺牲小鼠24小时后注射，如在步骤2.5，并收集肿瘤，它必须存储在冷PBS中。 </li><li>流式细胞仪，请按照下列步骤17: <ol><li>准备在流由纯化的胶原酶I和II（4 U / mL）中，透明质酸酶（60单位/毫升），和DNA酶I（60单位/毫升）的混合物中的流式细胞仪缓冲液（PBS中的0.5％BSA和1的酶鸡尾酒1mM EDTA）中。 </li><li>混合100毫克肿瘤组织与酶鸡尾酒缓冲的200微升中的1.5ml管和切块的组织小片用细剪刀。添加另外1.3毫升酶鸡尾酒的进入管及其内容物转移到6孔板中。 </li><li>摇上放置一个37℃的烘箱内60分钟以60rpm的水平振荡器6孔板中。 </li><li>与流洗组织匀浆仪缓冲器3次，以获得单细胞悬浮液。 </li><li>与抗体的鸡尾酒染色细胞的特异性生物标记物，包括CD45，CD11b的，的Ly6C，CD11c的，CD64，CD3，MHCII，和CD31（1:200稀释的所有抗体;克隆信息是在材料的电子表格提供）。确定相关的细胞类型，并确定DiIC12其平均荧光强度（MFI）（5）在每个细胞群体-DS用流式细胞仪分析软件的适当流动。 </li></ol></li><li>对于免疫染色，请按照下列步骤操作: <ol><li>将肿瘤放入盛有最佳切割介质（OCT）磁带，并冻结它的干冰。 </li><li>从肿瘤组织做6微米的冰冻切片，并将其放置在组织学载玻片;的部分应涵盖肿瘤的最大横截面，其提供了关于组织最有代表性的信息。 </li><li>染色生物标记（ 例如，CD31对内皮细胞）的与相应的抗体（ 例如，抗CD31，克隆MEC13.3）的兴趣。修正了多聚甲醛的部分;血清匹配二级抗体的物种起源阻止他们; incub用12小时的初级抗体吃在4℃，然后用2小时的第二抗体;最后，用PBS洗和盖玻片覆盖。图片的染色切片用Leica共聚焦直立显微镜SP5 13，16。 </li></ol></li></ol>

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The authors have no disclosure to make.

该议定书中的关键步骤: 双标记的脂质体的高品质的关键是产生在一段长期一致的结果。自由荧光染料或89 Zr离子可以产生完全不同的定位方式，而且必须在纯化步骤中被完全去除。另外，如果免疫系统显著影响实验癌症纳米性能，使用免疫小鼠模型的应该是优选的，如在C57BL / 6小鼠的B16-F10黑素瘤模型中，这是在本协议中使用。如果肿瘤微环境或遗传突变的特定组合是?...

纳米医学正在改变癌症治疗的发展1的范例。由以前的癌症纳米医学，如脂质体和基于白蛋白nanotherapies 2,3的巨大临床影响的启发，许多新的配方已在过去十年中产生的。然而，最近这些癌症纳米医学的临床转化成功的分析表明，只有少数已被批准用于临床4,5。以新的癌症纳米医学临床翻译的一个主要障碍是有限的治疗指数的提高与自由治疗化合物6直接管理比较。如在临床前动物模型中在全身性的，组织纳米医学，和细胞水平的体内性能等，准确的评价是IDENTIF必不可少Ÿ那些对未来临床翻译最佳的治疗指数。 纳米材料可以是放射性标记的用于在活的动物与正电子发射断层扫描（PET）成像，其具有所有的临床成像方式7之间极好的灵敏度和重现性定量表征。例如，89 Zr的标记的长循环纳米医学已经表征在小鼠模型中的癌症8，9，10，以及在其它疾病模型11。此外，该纳米医学的血半衰期和生物分布可广泛用在个人组织8利用体外放射性测量评估。因此，単允许纳米医学在全身和组织水平的定量评价。 重要的是，radiolabeleð纳米医学通常不能在单细胞或亚细胞水平进行分析，由于放射性信号的空间分辨率有限。因此，荧光标记证明是纳米颗粒的光学成像技术，如流式细胞仪和荧光显微镜12中的评价的互补方式。为此，标记放射性同位素和荧光标签纳米粒子可以定量在体内通过核成像评估和体外通过放射性计数，而且它们也可广泛表征在光学成像的细胞水平。 以前，我们已经开发了模块式程序，以放射性和荧光标记纳入各种纳米粒子，包括高密度脂蛋白（HDL）11，脂质体9，10，聚合物纳米颗粒，抗体片段，和nanoemulsions 10,13。这些标记纳米粒子已经允许在不同层次的相关动物模型，引导这些纳米材料的优化其具体应用定量表征。在目前的研究中，目的是利用纳米脂质体，最成熟的纳米平台14 -作为一个例子来证明综合程序生成双标记纳米颗粒，并把它在一个典型的同系黑色素瘤B16-F10小鼠模型15彻底表征。从结果来看，我们有信心这种纳米粒子的表征方法可以适用于评估相关的小鼠模型其它癌症纳米医学。

<table><tbody><tr><td>DPPC</td><td>Avantilipids</td><td>850355</td><td></td></tr><tr><td>Cholesterol</td><td>Sigma-Aldrich</td><td>C8667</td><td></td></tr><tr><td>DSPE-PEG2000</td><td>Avantilipids</td><td>880120P</td><td></td></tr><tr><td>DSPE-DFO</td><td>Home made</td><td>110634</td><td>Perez-Medina &lt;em&gt;et al.&lt;/em&gt;, JNM, 2014</td></tr><tr><td>DiIC12[5]-DS</td><td>AAT Bioquest</td><td>22051</td><td></td></tr><tr><td>Centrifugal filter</td><td>Vivaproducts</td><td>VS2061</td><td></td></tr><tr><td>Rotary evaporator</td><td>Buchi</td><td>R-100</td><td></td></tr><tr><td>Radio-HPLC</td><td>Shimadzu HPLC with 2 LC-10AT pumps</td><td>N/A</td><td></td></tr><tr><td>&lt;sup&gt;89&lt;/sup&gt;Zr-oxalate</td><td>MSKCC</td><td>Synthesized in house</td><td>TR19/9 variable beam cyclotron (Ebco Industries Inc.)</td></tr><tr><td>Micro PET-CT</td><td>Siemens</td><td>Inveon Micro-PET/CT</td><td></td></tr><tr><td>Gamma counter</td><td>PerkinElmer</td><td>2470-0150</td><td></td></tr><tr><td>Flow cytometry</td><td>BD Biosciences</td><td>Fortessa</td><td>Any multi-parametric flow cytometry analyzers would suffice</td></tr><tr><td>C57BL/6 mice</td><td>Jackson Laboratories</td><td></td><td></td></tr><tr><td>B16-YFP melanoma cells</td><td>Home made</td><td>N/A</td><td>Salmon &lt;em&gt;et al.&lt;/em&gt;, Immunity, 2016</td></tr><tr><td>Ly6C (clone HK1.4)--APC-Cy7</td><td>128025</td><td>Biolegend</td><td></td></tr><tr><td>MHCII (M5/114/152)--APC</td><td>107613</td><td>Biolegend</td><td></td></tr><tr><td>CD45 (30-F11)--BV510</td><td>103137</td><td>Biolegend</td><td></td></tr><tr><td>CD64 (X54-5/7.1)--PE-Cy7</td><td>139313</td><td>Biolegend</td><td></td></tr><tr><td>CD11b (M1/70)--BV605</td><td>101237</td><td>Biolegend</td><td></td></tr><tr><td>CD3 (17A2)--BV711</td><td>100241</td><td>Biolegend</td><td></td></tr><tr><td>CD31 (13.3)--PE</td><td>561073</td><td>Biolegend</td><td></td></tr><tr><td>CD11c (M418)--PerCP-Cy5.5</td><td>117327</td><td>BD Biosciences</td><td></td></tr><tr><td>CD31 (13.3) no fluorophore</td><td>550274</td><td>BD Biosciences</td><td></td></tr></tbody></table>

a comprehensive procedure to evaluate the in vivo performance of cancer nanomedicines

在诊所以前的癌症纳米医学的成功的启发，研究人员已经在过去的十年中产生了大量新的配方。然而，只有纳米医学少数已被批准用于临床使用，而根据临床开发的大多数纳米医学的已产生令人失望的结果。到的新的癌症纳米医学成功临床翻译的一个主要障碍是缺乏其在体内表现的准确理解。本文设有严格的程序通过正电子发射断层扫描，计算机断层扫描（PET-CT），放射性量化方法的整合来表征在全身性的，组织，单细胞，和亚细胞水平荷瘤小鼠纳米医学的体内行为，流式细胞仪，荧光显微镜。使用这种方法，研究人员可以准确地评价着性相关的小鼠模型的新纳米制剂河这些协议可以具有高平移电位，以确定最有前途的癌症纳米医学或癌症纳米医学的供将来翻译优化以帮助的能力。

图1示出了该过程的概述。 图2表示在步骤1 10中所述的双重标记的脂质体的概略合成方法。 图3显示一个代表的PET-CT图像（ 图3a），从PET成像（ 图3b），血中半衰期（ 图3c），和放射性纳米颗粒的生物分布（ 图3d）放射性量化，如在步骤2中所述在图3a中 ?...

Watch this Scientific Journal Video about A Comprehensive Procedure to Evaluate the In Vivo Performance of Cancer Nanomedicines at JoVE.com

A Comprehensive Procedure to Evaluate the In Vivo Performance of Cancer Nanomedicines

The poor understanding of the in vivo performance of nanomedicines stymies their clinical translation. Procedures to evaluate the in vivo behavior of cancer nanomedicines at systemic, tissue, single-cell, and subcellular levels in tumor-bearing immunocompetent mice are described here. This approach may help researchers to identify promising cancer nanomedicines for clinical translation.

综合程序，以评估<em&gt;在体内</em&gt;癌症纳米医学的性能

Inspired by the success of previous cancer nanomedicines in the clinic, researchers have generated a large number of novel formulations in the past ...

a-comprehensive-procedure-to-evaluate-vivo-performance-cancer

Research

JoVE Journal

Cancer Research

A Comprehensive Procedure to Evaluate the In Vivo Performance of Cancer Nanomedicines

8.8K 次观看.  Memorial Sloan Kettering Cancer Center. The poor understanding of the in vivo performance of nanomedicines stymies their clinical translation. Procedures to evaluate the in vivo behavior of cancer nanomedicines at systemic, tissue, single-cell, and subcellular levels in tumor-bearing immunocompetent mice are described here. This approach may help researchers to identify promising cancer nanomedicines for clinical translation.

视频: A Comprehensive Procedure to Evaluate the In Vivo Performance of Cancer Nanomedicines

Anticancer Efficacy of Photodynamic Therapy with Lung Cancer-Targeted Nanoparticles

Photodynamic therapy (PDT) is a non-invasive and non-surgical method representing an attractive alternative choice for lung cancer treatment. Photosensitizers selectively accumulate in tumor tissue and lead to tumor cell death in the presence of oxygen and the proper wavelength of light.
To increase the therapeutic effect of PDT, we developed both photosensitizer- and anticancer agent-loaded lung cancer-targeted nanoparticles. Both enhanced permeability and retention (EPR) effect-based passive targeting and hyaluronic-acid-CD44 interaction-based active targeting were applied. CD44 is a well-known hyaluronic acid receptor that is often introduced as a biomarker of non-small cell lung cancer. 
In addition, a combination of PDT and chemotherapy is adopted in the present study. This combination concept may increase anticancer therapeutic effects and reduce adverse reactions. 
We chose hypocrellin B (HB) as a novel photosensitizer in this study. It has been reported that HB causes higher anticancer efficacy of PDT compared to hematoporphyrin derivatives1. Paclitaxel was selected as the anticancer drug since it has proven to be a potential treatment for lung cancer2. 
The antitumor efficacies of photosensitizer (HB) solution, photosensitizer encapsulated hyaluronic acid-ceramide nanoparticles (HB-NPs), and both photosensitizer- and anticancer agent (paclitaxel)-encapsulated hyaluronic acid-ceramide nanoparticles (HB-P-NPs) after PDT were compared both in vitro and in vivo. The in vitro phototoxicity in A549 (human lung adenocarcinoma) cells and the in vivo antitumor efficacy in A549 tumor-bearing mice were evaluated.
The HB-P-NP treatment group showed the most effective anticancer effect after PDT. In conclusion, the HB-P-NPs prepared in the present study represent a potential and novel photosensitizer delivery system in treating lung cancer with PDT.

Photodynamic therapy (PDT) is an alternative choice for lung cancer treatment. To increase the therapeutic effect of PDT, lung cancer-targeted nanoparticles combined with chemotherapy were developed. Both in vitro and in vivo anticancer efficacies of PDT with prepared nanoparticles were evaluated.

光动力疗法的抗癌功效肺癌靶向纳米粒子

Photodynamic therapy (PDT) is a non-invasive and non-surgical method representing an attractive alternative choice for lung cancer treatment. ...

科研

癌症研究

Sample Extraction and Simultaneous Chromatographic Quantitation of Doxorubicin and Mitomycin C Following Drug Combination Delivery in Nanoparticles to Tumor-bearing Mice

Combination chemotherapy is frequently used in the clinic for cancer treatment; however, associated adverse effects to normal tissue may limit its therapeutic benefit. Nanoparticle-based drug combination has been shown to mitigate the problems encountered by free drug combination therapy. Our previous studies have shown that the combination of two anticancer drugs, doxorubicin (DOX) and mitomycin C (MMC), produced a synergistic effect against both murine and human breast cancer cells in vitro. DOX and MMC co-loaded polymer-lipid hybrid nanoparticles (DMPLN) bypassed various efflux transporter pumps that confer multidrug resistance and demonstrated enhanced efficacy in breast tumor models. Compared to conventional solution forms, such superior efficacy of DMPLN was attributed to the synchronized pharmacokinetics of DOX and MMC and increased intracellular drug bioavailability within tumor cells enabled by the nanocarrier PLN. 
To evaluate the pharmacokinetics and bio-distribution of co-administered DOX and MMC in both free solution and nanoparticle forms, a simple and efficient multi-drug analysis method using reverse-phase high performance liquid chromatography (HPLC) was developed. In contrast to previously reported methods that analyzed DOX or MMC individually in the plasma, this new HPLC method is able to simultaneously quantitate DOX, MMC and a major cardio-toxic DOX metabolite, doxorubicinol (DOXol), in various biological matrices (e.g., whole blood, breast tumor, and heart). A dual fluorescent and ultraviolet absorbent probe 4-methylumbelliferone (4-MU) was used as an internal standard (I.S.) for one-step detection of multiple drug analysis with different detection wavelengths. This method was successfully applied to determine the concentrations of DOX and MMC delivered by both nanoparticle and solution approaches in whole blood and various tissues in an orthotopic breast tumor murine model. The analytical method presented is a useful tool for pre-clinical analysis of nanoparticle-based delivery of drug combinations.

This protocol describes an efficient and convenient analytical process of sample extraction and simultaneous determination of multiple drugs, doxorubicin (DOX), mitomycin C (MMC) and a cardio-toxic DOX metabolite, doxorubicinol (DOXol), in the biological samples from a preclinical breast tumor model treated with nanoparticle formulations of synergistic drug combination.

纳米颗粒对荷瘤小鼠药相结合的样品提取及同时色谱定量分析

Combination chemotherapy is frequently used in the clinic for cancer treatment; however, associated adverse effects to normal tissue may limit its ...

Evaluation of Nanoparticle Uptake in Tumors in Real Time Using Intravital Imaging

Current technologies for tumor imaging, such as ultrasound, MRI, PET and CT, are unable to yield high-resolution images for the assessment of nanoparticle uptake in tumors at the microscopic level1,2,3, highlighting the utility of a suitable xenograft model in which to perform detailed uptake analyses. Here, we use high-resolution intravital imaging to evaluate nanoparticle uptake in human tumor xenografts in a modified, shell-less chicken embryo model. The chicken embryo model is particularly well-suited for these in vivo analyses because it supports the growth of human tumors, is relatively inexpensive and does not require anesthetization or surgery 4,5. Tumor cells form fully vascularized xenografts within 7 days when implanted into the chorioallantoic membrane (CAM) 6. The resulting tumors are visualized by non-invasive real-time, high-resolution imaging that can be maintained for up to 72 hours with little impact on either the host or tumor systems. Nanoparticles with a wide range of sizes and formulations administered distal to the tumor can be visualized and quantified as they flow through the bloodstream, extravasate from leaky tumor vasculature, and accumulate at the tumor site. We describe here the analysis of nanoparticles derived from Cowpea mosaic virus (CPMV) decorated with near-infrared fluorescent dyes and/or polyethylene glycol polymers (PEG) 7, 8, 9,10,11. Upon intravenous administration, these viral nanoparticles are rapidly internalized by endothelial cells, resulting in global labeling of the vasculature both outside and within the tumor7,12. PEGylation of the viral nanoparticles increases their plasma half-life, extends their time in the circulation, and ultimately enhances their accumulation in tumors via the enhanced permeability and retention (EPR) effect 7, 10,11. The rate and extent of accumulation of nanoparticles in a tumor is measured over time using image analysis software. This technique provides a method to both visualize and quantify nanoparticle dynamics in human tumors.

We present a novel approach to quantify nanoparticle localization in the vasculature of human xenografted tumors using dynamic, real-time intravital imaging in an avian embryo model.

使用实时活体成像在肿瘤纳米粒子吸收的评价

Current technologies for tumor imaging, such as ultrasound, MRI, PET and CT, are unable to yield high-resolution images for the assessment of nanoparticle ...

医学

Viral Nanoparticles for In vivo Tumor Imaging

The use of nanomaterials has the potential to revolutionize materials science and medicine. Currently, a number of different nanoparticles are being investigated for applications in imaging and therapy. Viral nanoparticles (VNPs) derived from plants can be regarded as self-assembled bionanomaterials with defined sizes and shapes. Plant viruses under investigation in the Steinmetz lab include icosahedral particles formed by Cowpea mosaic virus (CPMV) and Brome mosaic virus (BMV), both of which are 30 nm in diameter. We are also developing rod-shaped and filamentous structures derived from the following plant viruses: Tobacco mosaic virus (TMV), which forms rigid rods with dimensions of 300 nm by 18 nm, and Potato virus X (PVX), which form filamentous particles 515 nm in length and 13 nm in width (the reader is referred to refs. 1 and 2 for further information on VNPs).
From a materials scientist's point of view, VNPs are attractive building blocks for several reasons: the particles are monodisperse, can be produced with ease on large scale in planta, are exceptionally stable, and biocompatible. Also, VNPs are "programmable" units, which can be specifically engineered using genetic modification or chemical bioconjugation methods 3. The structure of VNPs is known to atomic resolution, and modifications can be carried out with spatial precision at the atomic level4, a level of control that cannot be achieved using synthetic nanomaterials with current state-of-the-art technologies.
In this paper, we describe the propagation of CPMV, PVX, TMV, and BMV in Vigna ungiuculata and Nicotiana benthamiana plants. Extraction and purification protocols for each VNP are given. Methods for characterization of purified and chemically-labeled VNPs are described. In this study, we focus on chemical labeling of VNPs with fluorophores (e.g. Alexa Fluor 647) and polyethylene glycol (PEG). The dyes facilitate tracking and detection of the VNPs 5-10, and PEG reduces immunogenicity of the proteinaceous nanoparticles while enhancing their pharmacokinetics 8,11. We demonstrate tumor homing of PEGylated VNPs using a mouse xenograft tumor model. A combination of fluorescence imaging of tissues ex vivo using Maestro Imaging System, fluorescence quantification in homogenized tissues, and confocal microscopy is used to study biodistribution. VNPs are cleared via the reticuloendothelial system (RES); tumor homing is achieved passively via the enhanced permeability and retention (EPR) effect12. The VNP nanotechnology is a powerful plug-and-play technology to image and treat sites of disease in vivo. We are further developing VNPs to carry drug cargos and clinically-relevant imaging moieties, as well as tissue-specific ligands to target molecular receptors overexpressed in cancer and cardiovascular disease.

Viral Nanoparticles for In vivo Tumor Imaging

Plant viral nanoparticles (VNPs) are promising platforms for applications in biomedicine. Here, we describe the procedures for plant VNP propagation, purification, characterization, and bioconjugation. Finally, we show the application of VNPs for tumor homing and imaging using a mouse xenograft model and fluorescence imaging.

病毒粒子为<em在体内</em&gt;肿瘤显像

The use of nanomaterials has the potential to revolutionize materials science and medicine. Currently, a number of different nanoparticles are being ...

生物工程

Evaluation of the In vivo Antitumor Activity of Polyanhydride IL-1&#945; Nanoparticles

Cytokine therapy is a promising immunotherapeutic strategy that can produce robust antitumor immune responses in cancer patients. The proinflammatory cytokine interleukin-1 alpha (IL-1&#945;) has been evaluated as an anticancer agent in several preclinical and clinical studies. However, dose-limiting toxicities, including flu-like symptoms and hypotension, have dampened the enthusiasm for this therapeutic strategy. Polyanhydride nanoparticle (NP)-based delivery of IL-1&#945; would represent an effective approach in this context since this may allow for a slow and controlled release of IL-1&#945; systemically while reducing toxic side effects. Here an analysis of the antitumor activity of IL-1&#945;-loaded polyanhydride NPs in a head and neck squamous cell carcinoma (HNSCC) syngeneic mouse model is described. Murine oropharyngeal epithelial cells stably expressing HPV16 E6/E7 together with hRAS and luciferase (mEERL) cells were injected subcutaneously into the right flank of C57BL/6J mice. Once tumors reached 3-4 mm in any direction, a 1.5% IL-1a - loaded 20:80 1,8-bis(p-carboxyphenoxy)-3,6-dioxaoctane:1,6-bis(p-carboxyphenoxy)hexane (CPTEG: CPH) nanoparticle (IL-1&#945;-NP) formulation was administered to mice intraperitoneally. Tumor size and body weight were continuously measured until tumor size or weight loss reached euthanasia criteria. Blood samples were taken to evaluate antitumor immune responses by submandibular venipuncture, and inflammatory cytokines were measured through cytokine multiplex assays. Tumor and inguinal lymph nodes were resected and homogenized into a single-cell suspension to analyze various immune cells through multicolor flow cytometry. These standard methods will allow investigators to study the antitumor immune response and potential mechanism of immunostimulatory NPs and other immunotherapy agents for cancer treatment.

Evaluation of the In vivo Antitumor Activity of Polyanhydride IL-1&#945; Nanoparticles

A standard protocol is described to study the antitumor activity and associated toxicity of IL-1&#945; in a syngeneic mouse model of HNSCC.

聚酐IL-1α纳米颗粒 体内抗肿瘤 活性评价

Cytokine therapy is a promising immunotherapeutic strategy that can produce robust antitumor immune responses in cancer patients. The proinflammatory ...

Evaluating the Effectiveness of Cancer Drug Sensitization In Vitro and In Vivo

Due to the high level of heterogeneity and mutations inherent in human cancers, single agent therapies, or combination regimens which target the same pathway, are likely to fail. Emphasis must be placed upon the inhibition of pathways that are responsible for intrinsic and/or adaptive resistance to therapy. An active field of investigation is the development and testing of DNA repair inhibitors that promote the action of, and prevent resistance to, commonly used chemotherapy and radiotherapy. We used a novel protocol to evaluate the effectiveness of BRCA2 inhibition as a means to sensitize tumor cells to the DNA damaging drug cisplatin. Tumor cell metabolism (acidification and respiration) was monitored in real-time for a period of 72 hr to delineate treatment effectiveness on a minute by minute basis. In combination, we performed an assessment of metastatic frequency using a chicken embryo chorioallantoic membrane (CAM) model of extravasation and invasion. This protocol addresses some of the weaknesses of commonly used in vitro and in vivo methods to evaluate novel cancer therapy regimens. It can be used in addition to common methods such as cell proliferation assays, cell death assays, and in vivo murine xenograft studies, to more closely discriminate amongst candidate targets and agents, and select only the most promising candidates for further development.

Evaluating the Effectiveness of Cancer Drug Sensitization In Vitro and In Vivo

Here, real-time monitoring of tumor cell metabolism, combined with an in vivo chicken embryo chorio-allantoic membrane (CAM) model of metastasis, are used to evaluate novel anti-cancer targets/agents for their ability to sensitize tumor cells to DNA damaging chemotherapeutics.

评估癌症药物过敏的有效性<em&gt;在体外</em&gt;和<em&gt;在体内</em

Due to the high level of heterogeneity and mutations inherent in human cancers, single agent therapies, or combination regimens which target the same ...

Validation of Nanobody and Antibody Based In Vivo Tumor Xenograft NIRF-imaging Experiments in Mice Using Ex Vivo Flow Cytometry and Microscopy

This protocol outlines the steps required to perform ex vivo validation of in vivo near-infrared fluorescence (NIRF) xenograft imaging experiments in mice using fluorophore labelled nanobodies and conventional antibodies.
First we describe how to generate subcutaneous tumors in mice, using antigen-negative cell lines as negative controls and antigen-positive cells as positive controls in the same mice for intraindividual comparison. We outline how to administer intravenously near-infrared fluorophore labelled (AlexaFluor680) antigen-specific nanobodies and conventional antibodies. In vivo imaging was performed with a small-animal NIRF-Imaging system. After the in vivo imaging experiments the mice were sacrificed. We then describe how to prepare the tumors for parallel ex vivo analyses by flow cytometry and fluorescence microscopy to validate in vivo imaging results.
The use of the near-infrared fluorophore labelled nanobodies allows for non-invasive same day imaging in vivo. Our protocols describe the ex vivo quantification of the specific labeling efficiency of tumor cells by flow cytometry and analysis of the distribution of the antibody constructs within the tumors by fluorescence microscopy. Using near-infrared fluorophore labelled probes allows for non-invasive, economical in vivo imaging with the unique ability to exploit the same probe without further secondary labelling for ex vivo validation experiments using flow cytometry and fluorescence microscopy.

Validation of Nanobody and Antibody Based In Vivo Tumor Xenograft NIRF-imaging Experiments in Mice Using Ex Vivo Flow Cytometry and Microscopy

This protocol outlines the steps required to perform ex vivo validation of in vivo near-infrared fluorescence xenograft imaging experiments in mice using fluorophore labelled nanobodies and conventional antibodies.

纳米抗体和基于抗体的验证<em&gt;在体内</em&gt;肿瘤异种移植NIRF成像的小鼠实验使用<em&gt;离体</em&gt;流式细胞仪和显微镜

This protocol outlines the steps required to perform ex vivo validation of in vivo near-infrared fluorescence (NIRF) xenograft imaging experiments in mice ...

<meta charset="utf8"></head><body>靶向致癌 miRNA-10b 治疗转移性乳腺癌的纳米药物

Monitoring of Nanodrug Accumulation in Murine Breast Cancer Metastases

Metastatic breast cancer is a devastating disease with very limited therapeutic options, calling for new therapeutic strategies. Oncogenic miRNAs have been shown to be associated with the metastatic potential of breast cancer and are implicated in tumor cell migration, invasion, and viability. However, it can be difficult to deliver an inhibitory RNA molecule to the tissue of interest. To overcome this challenge and deliver active antisense oligonucleotides to tumors, we utilized magnetic iron oxide nanoparticles as a delivery platform. These nanoparticles target tissues with increased vascular permeability, such as sites of inflammation or cancer. Delivery of these nanoparticles can be monitored in vivo by magnetic resonance imaging (MRI) due to their magnetic properties. Translation of this therapeutic approach into the clinic will be more accessible because of its compatibility with this relevant imaging modality. They can also be labeled with other imaging reporters such as a Cy5.5 near-infrared optical dye for correlative optical imaging and fluorescence microscopy. Here, we demonstrate that nanoparticles labeled with Cy5.5 and conjugated to therapeutic oligomers targeting oncogenic miRNA-10b (termed MN-anti-miR10b, or &#34;nanodrug&#34;) administered intravenously accumulate in metastatic sites, opening a possibility for therapeutic intervention of metastatic breast cancer.

<meta charset="utf8"></head><body>监测小鼠乳腺癌转移灶中纳米药物的积累

Here, we describe the protocol for in vivo delivery of magnetic iron oxide nanoparticles carrying RNA oligomers to metastatic breast cancer in animal models, providing a clinically viable approach for the therapeutic silencing of oncogenic nucleic acids.

监测小鼠乳腺癌转移灶中纳米药物的积累

Metastatic breast cancer is a devastating disease with very limited therapeutic options, calling for new therapeutic strategies. Oncogenic miRNAs have ...

综合程序，以评估

摘要

探索更多视频

此视频中的章节

光动力疗法的抗癌功效肺癌靶向纳米粒子

纳米颗粒对荷瘤小鼠药相结合的样品提取及同时色谱定量分析

使用实时活体成像在肿瘤纳米粒子吸收的评价

病毒粒子为

聚酐IL-1α纳米颗粒体内抗肿瘤活性评价

评估癌症药物过敏的有效性

纳米抗体和基于抗体的验证

监测小鼠乳腺癌转移灶中纳米药物的积累

监测小鼠乳腺癌转移灶中纳米药物的积累

监测小鼠乳腺癌转移灶中纳米药物的积累

综合程序，以评估

摘要

探索更多视频

此视频中的章节

光动力疗法的抗癌功效肺癌靶向纳米粒子

纳米颗粒对荷瘤小鼠药相结合的样品提取及同时色谱定量分析

使用实时活体成像在肿瘤纳米粒子吸收的评价

病毒粒子为

聚酐IL-1α纳米颗粒 体内抗肿瘤 活性评价

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