这项工作是由美国国立卫生研究院/美国国家癌症研究所（R01CA129822 R01CA140995）LKM-K补助。 Medina博士Kauwe感谢C.国王，M. M-Kauwe D. Revetto的的继续支持。

1。全身交付体内的 <ol><li>用无菌生理盐水足够HerDox的混合等同于0.2毫升0.004毫克/千克的剂量注射HerDox每6-8周的老NU / NU鼠标轴承皮下双边侧翼异种移植肿瘤。 </li><li>轻轻绘制HerDox的混合物倒入一个3/10毫升配备29G针头的胰岛素注射器，避免气泡。 </li><li>进行了简短的异氟醚麻醉诱导感应室配备一个气体净化系统（氧气流速:0.5-1升/分钟，异氟醚浓度:3-4％（或更低）。 </li><li>整个混合物注入到麻醉小鼠的尾静脉中（0.2毫升，每次注射）。内敛的，非麻醉鼠标也可以进行静脉注射。 </li><li>六年多的连续天重复注射相同的鼠标，每天一次。 </li></ol> 2。 在体内荧光成像积累的HerDox荧光肿瘤注射的最后一天（7天）使用多模成像仪可以探测到。下面的过程中需要使用自定义的宏照明和探测系统（ 图1）8。 <ol><li>打开模体内光学成像仪 。 </li><li>选择适合阿霉素荧光检测发射带通滤波器（590纳米±30纳米）。 </li><li>打开氩气，氪激光，在激光的光路中放置一个激励的带通滤波器（488纳米±10nm的）。 </li><li>打开麻醉系统上，然后放置到麻醉箱（氧气流速:0.5〜1升/分钟，异氟醚的浓度为3-4％（或更低）的气体清除系统配备鼠标。 </li><li>将鼠标成像室在模体内光学成像仪 ，当鼠标麻醉麻醉室。 </li><li>将鼠标的鼻子，打开一个头锥流管理持续麻醉医师SIA在图像采集（氧气流速:0.5-1升/分钟，异氟醚浓度2-3％（或更低）。 </li><li>获取荧光图像的曝光时间为5-15秒。 </li><li>执行背景校正或对比度调整的图像分析和处理。 </li></ol> 3。 体外荧光成像可以HerDox荧光成像在肿瘤和特定的器官（包括肝，肾，脾，心，骨骼肌）收获安乐死的小鼠在24小时后，最终（7天）注射HerDox。 <ol><li>打开模体内光学成像仪 。 </li><li>选择阿霉素荧光检测发射带通滤波器（580纳米±20 nm的）。 </li><li>打开氩气，氪激光，在激光的光路中放置一个激励的带通滤波器（488纳米±10nm的）。 </li><li>到成像室Ø安排在一个培养皿将肿瘤和特定器官f为模体内光学成像仪。 </li><li>采集的组织中使用的曝光时间为5-15秒的荧光图像。初始荧光图像采集的一个例子示出在图2a中。重复使用空培养皿，这将作为背景（ 图2b）。 </li><li>执行背景校正或对比度调整的图像分析和处理。纠正后的图像的一个例子是如图2c所示，产生从图2a，图2b减法。 </li></ol> 4， 在肿瘤的原位共聚焦成像 在原位共焦成像允许的HerDox肿瘤积累在细胞水平上的检测和分析。 <ol><li>打开一个徕卡SPE共聚焦显微镜。 </li><li>选择488 nm激光激发阿霉素，阿霉素和发射波长（560-620纳米）的光荧光检测。 </li><li>选择一个目标和滴浸油物镜40X或63X。 </li><li>提取新鲜从先前接收HerDox和模拟处理步骤2中描述的安乐死小鼠的肿瘤。 </li><li>在培养皿上冰，以避免将肿瘤组织退化，然后肿瘤转移到三角洲T室共聚焦成像。 </li><li>采集的肿瘤的共聚焦图像，按顺序震源深度（步长:1微米，厚度:20微米）。沿z轴的顺序获得的图像的一个例子示出在图3中，左侧面板中 。 </li><li>进行最大强度z轴投影的图像。 Z-叠图像的最大强度投影显示在图3中，右面板 。 </li><li>在整个视场的图像的平均荧光强度，计算出平均荧光强度的最大强度Z - 投影图像。计算D使用ImageJ的。 </li></ol> 5。比率光谱成像与分析比率光谱成像和分析，可以区分霉素荧光和自发荧光。 <ol><li>激光扫描荧光共聚焦显微镜上的电源。 </li><li>采集15幅图像的HerDox处理和未经处理的肿瘤为510-650纳米的光谱范围内，在指定的深度，步长为10nm时，激发波长为488纳米的光，使用Leica SPE的激光共聚焦显微镜。 </li><li>准备100微米的解决方案的阿霉素。 </li><li>执行的100μM阿霉素溶液的光谱成像获得纯光谱特征的Dox诱导荧光（波长范围510-650纳米，步长大小:10纳米）。从光谱成像和由此产生的荧光光谱图像采集的典型结果绘制的曲线图如图4所示。 </li><li>采集自体荧光光谱特征，从图像CUBE（光谱范围:510-650 nm的步长:10纳米）光谱成像未经处理的肿瘤。从光谱成像和由此产生的荧光光谱图像采集的典型结果绘制的曲线图如图5所示。 </li><li>产生四个的参考光谱特征（纯自发荧光，+0.9 0.1.doxorubin。自发荧光，0.2 +0.8阿霉素自体荧光，+0.7 0.3.doxorubin。自发荧光）使用该程序，我们制定了9。在图6中示出一个典型的曲线示出四个基准光谱签名。 </li><li>执行光谱图像的分类所定义的参考通过使用该程序，我们以前开发的9欧氏距离测度的光谱特征。 </li><li>这些图像进行线性光谱分离，通过使用光谱分离程序（插件在ImageJ），我们开发了7，10，比例光谱成像和分析比较。一封图7中所示的xample分离荧光HerDox的线性光谱分离的自体荧光。 </li></ol>

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作者丹尼尔·法卡斯，分子成像光谱主席。其余的作者有没有利益冲突。

阿霉素荧光可以检测到体内时使用多模成像肿瘤皮下。然而，治疗有效剂量的HerDox（0.004毫克/千克）的检测阈值以下后，单次剂量。相比之下，经过7每天注射（7天1x/day），，肿瘤的积累和保留的粒子是足以使阿霉素荧光可视化。 这是至关重要的工作时，与阿霉素在体内成像，使用清洁技术或任何其他荧光。鼠标的外侧上的滴水应避免，因为成像器将检测到皮肤的外侧上，没有鉴别是否霉素是外部的还是内部的荧光。同样，在手套上的阿霉素污染可以离开鼠标以外的荧光标记的动物处理过程中，从而导致错误的荧光检测。 在多模光纤的系统，一个利用激光束激发HerDox的。的激光束通常具有高斯分布。因此，对于利益的标本一致激励，利用光学扩散。然而，扩散激光有所保留了高斯光束轮廓。因此，对于理想的定量分析中，进行背景校正。修正前和背景图像，典型地具有16位深度的。因此，在执行背景校正，图像深度应被转换为32位，以避免不必要的错误，从得到的图像和输入图像之间的差异的图像的深度。 在收购的背景图像，一个黑色的纸（Artagain）先前将成像板，以防止前照明反射进行荧光成像。然而，由于相机是高度敏感的，低背景信号从黑纸相机检测到的。因此，背景在图2所示的信号来自黑纸，而不是从一个空的培养皿。 高浓度的阿霉素溶液，利用获得纯光谱特征，具有精度高。我们进一步研究了在该系统能够检测足够的荧光从试样产生可靠的光谱特征的光谱分辨率。其结果是，光谱分辨率调整到10nm。在这里，我们发现，高光谱分辨率（&lt;10纳米）的设置，在显微镜下无法检测到足够的荧光得到可靠的光谱特征。与纯的光谱特征，我们可以产生一些比例的光谱特征（ 图6）。特别是，我们可以定量区分HerDox的荧光信号，从自体荧光，由各自的比例7的光谱特征。未混合的光谱获得的图像与纯光谱特征（ 图7）是相似的分类的图像由两个基准光谱签名（蓝色:0.2.Dox 0.8。Autofl绿:Autofl）。总之，比例的共焦/光谱成像原位分辨率高，提供了在肿瘤细胞中对HerDox积累更多的量化信息，从而验证了它的有用性，在传递到HER2 +肿瘤的纳米颗粒的分析。

肿瘤靶向化疗有可能消除癌细胞不相关的药物相比，在​​较低的剂量，因为可以在其预定的目的地，而不是分发非肿瘤组织蓄积的交付治疗。作为后一种情况下，会稀释药的药效，因此需要较高的剂量是有效的，肿瘤靶向治疗和安全性优于标准的非针对性的治疗。 通过封装在自组装纳米颗粒靶向化疗使药物保持化学改性的共价连接到靶向分子的药物相比。因此联动有可能改变的活性的药物，靶向分子非共价键的组件允许保留药物效力。 我们先前的研究显示，这种新型三分量，复杂的自组装，HerDox，针对HER2 +肿瘤<em在体内诱发肿瘤生长消融，同时保留正常组织，包括心脏1。通过受体结合的细胞渗透蛋白，HerPBK10，与化疗剂之间的非共价相互作用，形成HerDox，多柔比星（阿霉素），通过一个小的核酸连接器。 HerPBK10结合人类表皮生长因子受体（HER）和，触发受体介导的内吞作用2-4，而核内体膜的渗透是通过腺病毒衍生的聚氯基地衣壳蛋白4-6成立。甲带正电荷的蛋白质域使核酸结合4，5，通过该DNA插霉素可以用于靶向递送运。亲电，插层，并有可能蛋白低聚相互作用促进自组装成圆形10-20nm的颗粒是稳定的血液和根据扩展存储在不同温度下1。她的优先目标2 +肿瘤细胞是促进增强配体的亲和力，时HER2升高。 我们以前的研究已经表明，在对非肿瘤组织中的肿瘤相比，优惠HerDox产量积累全身给药不相关的Dox诱导1，渗透进入肿瘤细胞在体内 7。我们已经观察到HerDox释放Dox诱导肿瘤细胞的条目后，允许进入细胞核1的阿霉素积累。肿瘤积累与受体水平相对较低HER2表达肿瘤积累减HerDox相比相对较高的HER2水平1。此外，有效的细胞死亡的浓度呈现负相关与HER2显示肿瘤细胞株表达不同的细胞表面HER2水平。 HerDox具有不相关的DOX治疗和安全的优势，作为肿瘤杀伤作用发生在超过10倍，低剂量相比解压geted药物和产生心（通过超声心动图和组织学染色检测）或肝功能（TUNEL检测染色）组织上没有检测的不利影响，相反，没有针对性霉素1。尽管其从病毒衣壳蛋白的推导，HerPBK10展品没有检测免疫原性治疗水平2。鉴于整个腺病毒的预先存在的抗体可以识别HerPBK10，他们是无法防止细胞结合2。 测量肿瘤体积随着时间的推移是一个标准的方法，靶向治疗的疗效评估，并已为评估疗效HerDox的雇用。补充这种方法在体内和体外荧光强度成像已经让我们更好地评估靶向效率7。我们已经专门集成在切除肿瘤的原位共焦成像光谱的DOX荧光分析来验证HerDox只有积累在体内的肿瘤，但渗透到肿瘤细胞和交付阿霉素进入细胞质和细胞核7。此外，频谱分析使我们能够区分霉素荧光自发荧光7。 这里，我们证明我们的方法更详细地用于全身给药后在体内的评估HerDox的，最重要的是，通过多模成像方法和分析评估目标。

<table border="1">
<tbody>
 <tr>
 <td>Name of the reagent</td>
 <td>Company</td>
 <td>Catalogue number</td>
 <td>Comments (optional)</th
 ></tr>
 <tr>
 <td >Fluorescence laser scanning confocal microscope</td>
 <td >Leica</td>
 <td >SPE</td>
 <td></td>
 </tr>
 <tr>
 <td>In Vivo Optical Imager</td>
 <td>Spectral Molecular Imaging</td>
 <td>Multimode In Vivo Optical Imager</td>
 <td></td>
 </tr>
 <tr>
 <td >Doxorubicin-HCl</td>
 <td >Sigma-Aldrich</td>
 <td >D4035</td>
 <td></td>
 </tr>
 <tr>
 <td >Nude (NU/NU) mouse, female, 6-8 week</td>
 <td >Charles River</td>
 <td >Strain code 088</td>
 <td></td>
 </tr>
 <tr>
 <td >MDA-MB-435 human HER2+ tumor cells</td>
 <td >NCI-Frederick Cancer DCTD Tumor/Cell Line Repository</td>
 <td >0507292</td>
 <td></td>
 </tr>
 <tr>
 <td >3/10 cc insulin syringe U-100 with 29G x 1/2" Ultra-FineIV permanently attached needle</td>
 <td >BD</td>
 <td >309301</td>
 <td></td>
 </tr>
 <tr>
 <td >Delta T chamber </td>
 <td >Bioptechs </td>
 <td >04200417B</td>
 <td></td>
 </tr>
 </tbody>
</table>

analysis of targeted viral protein nanoparticles delivered to her2+ tumors

HER2 +肿瘤靶向纳米粒子，HerDox，具有肿瘤优惠的积累和消融治疗HER2 +癌症动物模型中肿瘤生长。 HerDox形成通过非共价的肿瘤靶向与化疗剂，多柔比星的细胞渗透蛋白的自组装体，通过一个小的核酸连接器。电，插层，齐聚相互作用的结合，促进成圆形的10-20纳米粒子自组装。 HerDox显示出在不同温度下的血中稳定性，以及在扩展存储。全身给药HerDox在荷瘤小鼠的肿瘤细胞死亡的结果没有检测到不利影响的非肿瘤组织，包括心脏和肝脏（发生明显损坏由无目标的多柔比星）。 HER2海拔细胞中的表达相比，有利于靶向表达人表皮生长因子受体的细胞，因此，显示升高的HER2水平的肿瘤表现出较大的积累HerDox唱较低的水平，无论是在体外和体内 。 原位共焦和频谱分析相结合的荧光强度成像已经让我们在体内肿瘤靶向肿瘤细胞渗透全身给药后HerDox的验证。在这里，我们详细介绍我们的方法，以评估肿瘤靶向全身给药后通过多模成像。

图1显示了在体内光学成像样机下图像采集多种方式，包括荧光强度，光谱，寿命，双光子，重要的内焦，生物发光成像的目的，这是建。此外，冷却的高灵敏度的摄像头和纳入在本系统中的高功率激光线会产生更高的对比度比商业光学成像系统11的荧光图像，特别是用于检测体内阿霉素荧光。因此，该系统是用于在本研究中，获得多个互补的数据点在体内的评估HerDox的关键。 如图2所示的高肿瘤荧光典型全身给药HerDox后，表示其肿瘤优先定位。骨骼肌中检测到的荧光是非典型的，并可能导致来回米在注射过程中的异常（ 即注入太接近身体）。定性地说，在图2a中所示的图像具有低的背景。然而，为了执行组织相对荧光强度进行定量分析，它是要执行背景校正，用一个空盘，如在图2b中所示。这可能会导致在背景校正" 图2c中所示的图像相比，具有更高的背景信号在图像的边缘的原稿（ 图2a）的背景图像（ 图2b），因为具有较低的边缘强度的图像。因此，虽然背景校正，可以得到在图像的边缘的信号增加，这是一个必要的步骤，用于随后的定量分析。 为了获得的定量评估肿瘤HerDox积累，共聚焦荧光图像获得了在不同焦距深度原位其次是最大强度的z轴投影的图像（ 图3），并取得的投影图像的平均荧光强度。将所得映像包含HerDox的的累积信息的指示在每个像素的z深度，因此，平均荧光强度反映在不同深度的定量整体HerDox在肿瘤中的积累。 在肿瘤中的自体荧光，来区分HerDox的荧光从定量区分两个具有较高的特异性，比例光谱成像和分析进行。纯的多柔比星和获得的自体荧光光谱成像的光谱特征示于图4和5。参考光谱特征（ 图6）也被创建，通过混合不同比例的纯的光谱特征，采用我们开发的程序9。每个参考，NCE光谱特征代表的HerDox累积在肿瘤方面的自体荧光的相对荧光。光谱分类的图像（在本文中未示出数据）显示更好的定量的累积HerDox 7比未混合的图像，使用纯的参考光谱特征（ 图7）得到的光谱， 图4-7表示典型的调查结果，当执行比例光谱成像和分析。 <img alt="图1" fo:content-width="2.5in" fo:src="/files/ftp_upload/50396/50396fig1highres.jpg" src="/files/ftp_upload/50396/50396fig1.jpg" /> 图1。 在体内，光学成像器 。光学成像系统，荧光强度，光谱，荧光寿命和活体的共焦成像能力必须被使用。这里使用的多模光纤成像系统显示，并一直describ以前的8。图片提供施普林格科学与商业媒体BV:黄某，JY， 等摩尔的一种许可。成像生物学报 ，14，431-42（2012）。 <img alt="图2" fo:content-width="6in" fo:src="/files/ftp_upload/50396/50396fig2highres.jpg" src="/files/ftp_upload/50396/50396fig2.jpg" /> 图2。代表荧光强度图像内部从一个鼠标接受HerDox的提取器官和肿瘤 。组织和肿瘤收获从安乐死的小鼠在24小时后，最终（7天）注射前的荧光强度HerDox图像和<a href="https://www.jove.com/files/ftp_upload/50396/50396fig2large.jpg" target="_blank">点击这里查看大</a>背景校正后（二）（一）及（c）分别表示。 <a href="https://www.jove.com/files/ftp_upload/50396/50396fig2large.jpg" target="_blank">弄清楚</a> 。 图3。最大强度z轴投影获得的图像在顺序的z深度 。如左图所示的层叠图像是代表顺序为1μm的深度得到的肿瘤组织的共焦扫描。在右边的图像显示在左侧的重叠影像编译。 <img alt="图4" fo:content-width="6in" fo:src="/files/ftp_upload/50396/50396fig4highres.jpg" src="/files/ftp_upload/50396/50396fig4.jpg" /> 图4。收购一个纯光谱阿霉素签名 。的阿霉素溶液的图像后获得了在顺序510-650纳米的波长范围内，与步骤尺寸为10nm（左），为多柔比星，得到纯的光谱特征<html>使用我们开发的程序。 <a href="https://www.jove.com/files/ftp_upload/50396/50396fig4large.jpg" target="_blank">点击此处查看大图</a> 。 <img alt="图5" fo:content-width="6in" fo:src="/files/ftp_upload/50396/50396fig5highres.jpg" src="/files/ftp_upload/50396/50396fig5.jpg" /> 图5。收购一个纯粹的自体荧光光谱特征 。未经处理的肿瘤图像后获得在连续波长在510-650纳米，一步大小为10纳米（左），纯为自体荧光光谱特征，利用我们开发的程序获得。 <a href="https://www.jove.com/files/ftp_upload/50396/50396fig5large.jpg" target="_blank">点击这里查看大图</a> 。 load/50396/50396fig6.jpg"/&gt; 图6。四个参考比例光谱成像和分析的光谱特征 。参考光谱特征，创建，混合纯光谱特征，利用我们开发的程序。绿色的实线表示纯的自体荧光，红色的实线表示0.9 0.1.doxorubin。自体荧光，蓝色0.2的实线表示。阿霉素0.8。自体荧光，和青色实线0.3.doxorubin 0.7。自体荧光。图转载自作者以前的工作:黄某，JY 等，PLoS一 7（4），（2012）。 <img alt="图7" src="/files/ftp_upload/50396/50396fig7.jpg" /> 图7。 HerDox组织荧光光谱前后未混合 。阿霉素自体荧光，TH后获得的光谱特征ê霉素信号分离采用线性光谱未混合的12,13。联合国前光谱混合和分离后的HerDox图像光谱未混合图显示自发荧光和HerDox的的混合图像。

Watch this Scientific Journal Video about Analysis of Targeted Viral Protein Nanoparticles Delivered to HER2+ Tumors at JoVE.com

Analysis of Targeted Viral Protein Nanoparticles Delivered to HER2+ Tumors

本文详细介绍了肿瘤靶向纳米粒子，HerDox光学成像分析的程序。特别是，使用多模成像装置，用于检测肿瘤靶向和评估肿瘤渗透详细描述在这里。

分析有针对性的病毒蛋白的纳米粒子递送HER2 +肿瘤

The HER2+ tumor-targeted nanoparticle, HerDox, exhibits tumor-preferential accumulation and tumor-growth ablation in an animal model of HER2+ cancer. ...

analysis-targeted-viral-protein-nanoparticles-delivered-to-her2

Universidad San Sebastian

Research

JoVE Journal

Bioengineering

11.0K 次观看.  University of Southern California. 本文详细介绍了肿瘤靶向纳米粒子，HerDox光学成像分析的程序。特别是，使用多模成像装置，用于检测肿瘤靶向和评估肿瘤渗透详细描述在这里。

视频: Analysis of Targeted Viral Protein Nanoparticles Delivered to HER2+ Tumors

Introduction to the Ultrasound Targeted Microbubble Destruction Technique

In UTMD, bioactive molecules, such as negatively charged plasmid DNA vectors encoding a gene of interest, are added to the cationic shells of lipid microbubble contrast agents7-9. In mice these vector-carrying microbubbles can be administered intravenously or directly to the left ventricle of the heart. In larger animals they can also be infused through an intracoronary catheter. The subsequent delivery from the circulation to a target organ occurs by acoustic cavitation at a resonant frequency of the microbubbles. It seems likely that the mechanical energy generated by the microbubble destruction results in transient pore formation in or between the endothelial cells of the microvasculature of the targeted region10. As a result of this sonoporation effect, the transfection efficiency into and across the endothelial cells is enhanced, and transgene-encoding vectors are deposited into the surrounding tissue. Plasmid DNA remaining in the circulation is rapidly degraded by nucleases in the blood, which further reduces the likelihood of delivery to non-sonicated tissues and leads to highly specific target-organ transfection.

Ultrasound Targeted Microbubble Destruction (UTMD) can be used to direct site-specific delivery of bioactive molecules, including therapeutic genes, to target organs accessible to ultrasound, such as the heart and liver1-6.

有针对性的超声微泡销毁技术简介

In UTMD, bioactive molecules, such as negatively charged plasmid DNA vectors encoding a gene of interest, are added to the cationic shells of lipid ...

科研

生物工程

Design of a Cyclic Pressure Bioreactor for the Ex Vivo Study of Aortic Heart Valves

The aortic valve, located between the left ventricle and the aorta, allows for unidirectional blood flow, preventing backflow into the ventricle. Aortic valve leaflets are composed of interstitial cells suspended within an extracellular matrix (ECM) and are lined with an endothelial cell monolayer. The valve withstands a harsh, dynamic environment and is constantly exposed to shear, flexion, tension, and compression. Research has shown calcific lesions in diseased valves occur in areas of high mechanical stress as a result of endothelial disruption or interstitial matrix damage1-3. Hence, it is not surprising that epidemiological studies have shown high blood pressure to be a leading risk factor in the onset of aortic valve disease4. 
The only treatment option currently available for valve disease is surgical replacement of the diseased valve with a bioprosthetic or mechanical valve5. Improved understanding of valve biology in response to physical stresses would help elucidate the mechanisms of valve pathogenesis. In turn, this could help in the development of non-invasive therapies such as pharmaceutical intervention or prevention. Several bioreactors have been previously developed to study the mechanobiology of native or engineered heart valves6-9. Pulsatile bioreactors have also been developed to study a range of tissues including cartilage10, bone11 and bladder12. The aim of this work was to develop a cyclic pressure system that could be used to elucidate the biological response of aortic valve leaflets to increased pressure loads. 
The system consisted of an acrylic chamber in which to place samples and produce cyclic pressure, viton diaphragm solenoid valves to control the timing of the pressure cycle, and a computer to control electrical devices. The pressure was monitored using a pressure transducer, and the signal was conditioned using a load cell conditioner. A LabVIEW program regulated the pressure using an analog device to pump compressed air into the system at the appropriate rate. The system mimicked the dynamic transvalvular pressure levels associated with the aortic valve; a saw tooth wave produced a gradual increase in pressure, typical of the transvalvular pressure gradient that is present across the valve during diastole, followed by a sharp pressure drop depicting valve opening in systole. The LabVIEW program allowed users to control the magnitude and frequency of cyclic pressure. The system was able to subject tissue samples to physiological and pathological pressure conditions. This device can be used to increase our understanding of how heart valves respond to changes in the local mechanical environment.

Design of a Cyclic Pressure Bioreactor for the Ex Vivo Study of Aortic Heart Valves

A cyclic pressure bioreactor capable of subjecting heart valve tissue to physiological and pathological pressure conditions has been designed. A LabVIEW program allows users to control pressure magnitude, amplitude and frequency. This device can be used to study the mechanobiology of heart valve tissue or isolated cells.

循环压力为生物反应器的设计体外主动脉心脏瓣膜的研究

The aortic valve, located between the left ventricle and the aorta, allows for unidirectional blood flow, preventing backflow into the ventricle. Aortic ...

Cellular Lipid Extraction for Targeted Stable Isotope Dilution Liquid Chromatography-Mass Spectrometry Analysis

The metabolism of fatty acids, such as arachidonic acid (AA) and linoleic acid (LA), results in the formation of oxidized bioactive lipids, including numerous stereoisomers1,2. These metabolites can be formed from free or esterified fatty acids. Many of these oxidized metabolites have biological activity and have been implicated in various diseases including cardiovascular and neurodegenerative diseases, asthma, and cancer3-7. Oxidized bioactive lipids can be formed enzymatically or by reactive oxygen species (ROS). Enzymes that metabolize fatty acids include cyclooxygenase (COX), lipoxygenase (LO), and cytochromes P450 (CYPs)1,8. Enzymatic metabolism results in enantioselective formation whereas ROS oxidation results in the racemic formation of products.
While this protocol focuses primarily on the analysis of AA- and some LA-derived bioactive metabolites; it could be easily applied to metabolites of other fatty acids. Bioactive lipids are extracted from cell lysate or media using liquid-liquid (l-l) extraction. At the beginning of the l-l extraction process, stable isotope internal standards are added to account for errors during sample preparation. Stable isotope dilution (SID) also accounts for any differences, such as ion suppression, that metabolites may experience during the mass spectrometry (MS) analysis9. After the extraction, derivatization with an electron capture (EC) reagent, pentafluorylbenzyl bromide (PFB) is employed to increase detection sensitivity10,11. Multiple reaction monitoring (MRM) is used to increase the selectivity of the MS analysis. Before MS analysis, lipids are separated using chiral normal phase high performance liquid chromatography (HPLC). The HPLC conditions are optimized to separate the enantiomers and various stereoisomers of the monitored lipids12. This specific LC-MS method monitors prostaglandins (PGs), isoprostanes (isoPs), hydroxyeicosatetraenoic acids (HETEs), hydroxyoctadecadienoic acids (HODEs), oxoeicosatetraenoic acids (oxoETEs) and oxooctadecadienoic acids (oxoODEs); however, the HPLC and MS parameters can be optimized to include any fatty acid metabolites13.
Most of the currently available bioanalytical methods do not take into account the separate quantification of enantiomers. This is extremely important when trying to deduce whether or not the metabolites were formed enzymatically or by ROS. Additionally, the ratios of the enantiomers may provide evidence for a specific enzymatic pathway of formation. The use of SID allows for accurate quantification of metabolites and accounts for any sample loss during preparation as well as the differences experienced during ionization. Using the PFB electron capture reagent increases the sensitivity of detection by two orders of magnitude over conventional APCI methods. Overall, this method, SID-LC-EC-atmospheric pressure chemical ionization APCI-MRM/MS, is one of the most sensitive, selective, and accurate methods of quantification for bioactive lipids.

This protocol will demonstrate the extraction and analysis of free and esterified bioactive fatty acids from cells. Fatty acids are accurately quantified using stable isotope dilution, chiral liquid chromatography, electron capture atmospheric chemical ionization multiple reaction monitoring mass spectrometry (SID-LC-ECAPCI-MRM/MS).

有针对性的稳定同位素稀释液相色谱 - 质谱法分析细胞脂质提取

The metabolism of fatty acids, such as arachidonic acid (AA) and linoleic acid (LA), results in the formation of oxidized bioactive lipids, including ...

Therapeutic Gene Delivery and Transfection in Human Pancreatic Cancer Cells using Epidermal Growth Factor Receptor-targeted Gelatin Nanoparticles

More than 32,000 patients are diagnosed with pancreatic cancer in the United States per year and the disease is associated with very high mortality 1. Urgent need exists to develop novel clinically-translatable therapeutic strategies that can improve on the dismal survival statistics of pancreatic cancer patients. Although gene therapy in cancer has shown a tremendous promise, the major challenge is in the development of safe and effective delivery system, which can lead to sustained transgene expression. 
Gelatin is one of the most versatile natural biopolymer, widely used in food and pharmaceutical products. Previous studies from our laboratory have shown that type B gelatin could physical encapsulate DNA, which preserved the supercoiled structure of the plasmid and improved transfection efficiency upon intracellular delivery. By thiolation of gelatin, the sulfhydryl groups could be introduced into the polymer and would form disulfide bond within nanoparticles, which stabilizes the whole complex and once disulfide bond is broken due to the presence of glutathione in cytosol, payload would be released 2-5. Poly(ethylene glycol) (PEG)-modified GENS, when administered into the systemic circulation, provides long-circulation times and preferentially targets to the tumor mass due to the hyper-permeability of the neovasculature by the enhanced permeability and retention effect 6. Studies have shown over-expression of the epidermal growth factor receptor (EGFR) on Panc-1 human pancreatic adenocarcinoma cells 7. In order to actively target pancreatic cancer cell line, EGFR specific peptide was conjugated on the particle surface through a PEG spacer.8
Most anti-tumor gene therapies are focused on administration of the tumor suppressor genes, such as wild-type p53 (wt-p53), to restore the pro-apoptotic function in the cells 9. The p53 mechanism functions as a critical signaling pathway in cell growth, which regulates apoptosis, cell cycle arrest, metabolism and other processes 10. In pancreatic cancer, most cells have mutations in p53 protein, causing the loss of apoptotic activity. With the introduction of wt-p53, the apoptosis could be repaired and further triggers cell death in cancer cells 11. 
Based on the above rationale, we have designed EGFR targeting peptide-modified thiolated gelatin nanoparticles for wt-p53 gene delivery and evaluated delivery efficiency and transfection in Panc-1 cells.

Type B gelatin-based engineered nanovectors system (GENS) was developed for systemic gene delivery and transfection in the treatment of pancreatic cancer. By modification with epidermal growth factor receptor (EGFR) specific peptide on the surface of nanparticles, they could target on EGFR receptor and release plasmid under reducing environment, such as high intracellular glutathione concentrations.

交付使用表皮生长因子在人胰腺癌细胞转染基因受体有针对性的明胶基于工程Nanovectors

More than 32,000 patients are diagnosed with pancreatic cancer in the United States per year and the disease is associated with very high mortality 1. ...

High-throughput Synthesis of Carbohydrates and Functionalization of Polyanhydride Nanoparticles

Transdisciplinary approaches involving areas such as material design, nanotechnology, chemistry, and immunology have to be utilized to rationally design efficacious vaccines carriers. Nanoparticle-based platforms can prolong the persistence of vaccine antigens, which could improve vaccine immunogenicity1. Several biodegradable polymers have been studied as vaccine delivery vehicles1; in particular, polyanhydride particles have demonstrated the ability to provide sustained release of stable protein antigens and to activate antigen presenting cells and modulate immune responses2-12.
The molecular design of these vaccine carriers needs to integrate the rational selection of polymer properties as well as the incorporation of appropriate targeting agents. High throughput automated fabrication of targeting ligands and functionalized particles is a powerful tool that will enhance the ability to study a wide range of properties and will lead to the design of reproducible vaccine delivery devices.
The addition of targeting ligands capable of being recognized by specific receptors on immune cells has been shown to modulate and tailor immune responses10,11,13 C-type lectin receptors (CLRs) are pattern recognition receptors (PRRs) that recognize carbohydrates present on the surface of pathogens. The stimulation of immune cells via CLRs allows for enhanced internalization of antigen and subsequent presentation for further T cell activation14,15. Therefore, carbohydrate molecules play an important role in the study of immune responses; however, the use of these biomolecules often suffers from the lack of availability of structurally well-defined and pure carbohydrates. An automation platform based on iterative solution-phase reactions can enable rapid and controlled synthesis of these synthetically challenging molecules using significantly lower building block quantities than traditional solid-phase methods16,17.
Herein we report a protocol for the automated solution-phase synthesis of oligosaccharides such as mannose-based targeting ligands with fluorous solid-phase extraction for intermediate purification. After development of automated methods to make the carbohydrate-based targeting agent, we describe methods for their attachment on the surface of polyanhydride nanoparticles employing an automated robotic set up operated by LabVIEW as previously described10. Surface functionalization with carbohydrates has shown efficacy in targeting CLRs10,11 and increasing the throughput of the fabrication method to unearth the complexities associated with a multi-parametric system will be of great value (Figure 1a).

In this article, a high throughput method is presented for the synthesis of oligosaccharides and their attachment to the surface of polyanhydride nanoparticles for further use in targeting specific receptors on antigen presenting cells.

碳水化合物和聚酸酐纳米粒子功能化的高通量合成

Transdisciplinary approaches involving areas such as material design, nanotechnology, chemistry, and immunology have to be utilized to rationally design ...

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 ...

Evaluation of Polymeric Gene Delivery Nanoparticles by Nanoparticle Tracking Analysis and High-throughput Flow Cytometry

Non-viral gene delivery using polymeric nanoparticles has emerged as an attractive approach for gene therapy to treat genetic diseases1 and as a technology for regenerative medicine2. Unlike viruses, which have significant safety issues, polymeric nanoparticles can be designed to be non-toxic, non-immunogenic, non-mutagenic, easier to synthesize, chemically versatile, capable of carrying larger nucleic acid cargo and biodegradable and/or environmentally responsive. Cationic polymers self-assemble with negatively charged DNA via electrostatic interaction to form complexes on the order of 100 nm that are commonly termed polymeric nanoparticles. Examples of biomaterials used to form nanoscale polycationic gene delivery nanoparticles include polylysine, polyphosphoesters, poly(amidoamines)s and polyethylenimine (PEI), which is a non-degradable off-the-shelf cationic polymer commonly used for nucleic acid delivery1,3 . Poly(beta-amino ester)s (PBAEs) are a newer class of cationic polymers4 that are hydrolytically degradable5,6 and have been shown to be effective at gene delivery to hard-to-transfect cell types such as human retinal endothelial cells (HRECs)7, mouse mammary epithelial cells8, human brain cancer cells9 and macrovascular (human umbilical vein, HUVECs) endothelial cells10.
A new protocol to characterize polymeric nanoparticles utilizing nanoparticle tracking analysis (NTA) is described. In this approach, both the particle size distribution and the distribution of the number of plasmids per particle are obtained11. In addition, a high-throughput 96-well plate transfection assay for rapid screening of the transfection efficacy of polymeric nanoparticles is presented. In this protocol, poly(beta-amino ester)s (PBAEs) are used as model polymers and human retinal endothelial cells (HRECs) are used as model human cells. This protocol can be easily adapted to evaluate any polymeric nanoparticle and any cell type of interest in a multi-well plate format.

A protocol for nanoparticle tracking analysis (NTA) and high-throughput flow cytometry to evaluate polymeric gene delivery nanoparticles is described. NTA is utilized to characterize the nanoparticle particle size distribution and the plasmid per particle distribution. High-throughput flow cytometry enables quantitative transfection efficacy evaluation for a library of gene delivery biomaterials.

高分子基因传递纳米粒子的纳米粒子跟踪分析和高通量流式细胞仪评价

Non-viral gene delivery using polymeric nanoparticles has emerged as an attractive approach for gene therapy to treat genetic diseases1 and as a ...

Harmonic Nanoparticles for Regenerative Research

In this visualized experiment, protocol details are provided for in vitro labeling of human embryonic stem cells (hESC) with second harmonic generation nanoparticles (HNPs). The latter are a new family of probes recently introduced for labeling biological samples for multi-photon imaging. HNPs are capable of doubling the frequency of excitation light by the nonlinear optical process of second harmonic generation with no restriction on the excitation wavelength.
Multi-photon based methodologies for hESC differentiation into cardiac clusters (maintained as long term air-liquid cultures) are presented in detail. In particular, evidence on how to maximize the intense second harmonic (SH) emission of isolated HNPs during 3D monitoring of beating cardiac tissue in 3D is shown. The analysis of the resulting images to retrieve 3D displacement patterns is also detailed.

Protocol details are provided for in vitro labeling human embryonic stem cells with second harmonic generating nanoparticles. Methodologies for hESC investigation by multi-photon microscopy and their differentiation into cardiac clusters are also presented.

纳米谐波再生研究

In this visualized experiment, protocol details are provided for in vitro labeling of human embryonic stem cells (hESC) with second harmonic generation ...

Electronic Tongue Generating Continuous Recognition Patterns for Protein Analysis

In current protocol, a combinatorial approach has been developed to simplify the design and production of sensing materials for the construction of electronic tongues (eT) for protein analysis. By mixing a small number of simple and easily accessible molecules with different physicochemical properties, used as building blocks (BBs), in varying and controlled proportions and allowing the mixtures to self-assemble on the gold surface of a prism, an array of combinatorial surfaces featuring appropriate properties for protein sensing was created. In this way, a great number of cross-reactive receptors can be rapidly and efficiently obtained. By combining such an array of combinatorial cross-reactive receptors (CoCRRs) with an optical detection system such as surface plasmon resonance imaging (SPRi), the obtained eT can monitor the binding events in real-time and generate continuous recognition patterns including 2D continuous evolution profile (CEP) and 3D continuous evolution landscape (CEL) for samples in liquid. Such an eT system is efficient for discrimination of common purified proteins.

A novel approach is described for construction of electronic tongue (eT), which greatly simplifies the design and production of sensing materials, and allows the eT to generate continuous evolution profiles and landscapes for samples in liquid. The obtained eT is efficient for common protein analysis such as discrimination.

电子舌生成连续的识别模式的蛋白质分析

In current protocol, a combinatorial approach has been developed to simplify the design and production of sensing materials for the construction of ...

Production of Metal Nanoparticles by Pulsed Laser-ablation in Liquids: A Tool for Studying the Antibacterial Properties of Nanoparticles

The emergence of multidrug-resistant bacteria is a global clinical concern leading some to speculate about our return to a "pre-antibiotics" era of medicine. In addition to efforts to identify novel small-molecule antimicrobial drugs, there has been great interest in the use of metal nanoparticles as coatings for medical devices, wound dressings, and consumer packaging, due to their antimicrobial properties. The wide variety of methods available for nanoparticle synthesis results in a broad spectrum of chemical and physical properties which can affect antibacterial efficacy. This manuscript describes the pulsed laser-ablation in liquids (PLAL) method to create nanoparticles. This approach allows for the fine tuning of nanoparticle size, composition, and stability using post-irradiation methods as well as the addition of surfactants or volume excluders. By controlling particle size and composition, a large range of physical and chemical properties of metal nanoparticles can be explored which may contribute to their antimicrobial efficacy thereby opening new avenues for antibacterial development.

The antimicrobial properties of metals such as copper and silver have been recognized for centuries. This protocol describes pulsed laser-ablation in liquids, a method of synthesizing metal nanoparticles that provides the ability to fine tune the properties of these nanoparticles to optimize their antimicrobial effects.

液体中脉冲激光烧蚀制备金属纳米粒子:研究纳米粒子抗菌性能的工具

The emergence of multidrug-resistant bacteria is a global clinical concern leading some to speculate about our return to a "pre-antibiotics" era of ...