这项工作是由NIH / NIBIB的补助R00 EB009105（NFS）和P30 EB011317（NFS），NIH / NIBIB的培训津贴T32 EB007509（AMW），一个凯斯西储大学跨学科联盟投资补助（NFS）的支持，案例综合癌症中心授予P30 CA043703（NFS）。我们感谢斯坦梅茨实验室本科学生，研究人员对他们的实际支持：纳迪亚阿亚特Sourav（SID），凯文·陈，戴伊，杨蕙如山姆亚历山大，克雷格·德克鲁兹，斯蒂芬·亨·劳伦兰多夫，布赖恩所以，保罗Chariou 。

1。 VNP（CPMV，BMV，PVX和TMV）的传播<ol><li>将室内植物室设置的控制，以每天15小时（100％光，25°C，湿度为65％）和9小时的夜间（0％光，22°C，湿度60％）。 </li><li>根据表1中的时间线，接种植物。 </li></ol><table border="1"><tbody><tr><td> CPMV </td><td> PVX，TMV，BMV </td></tr><tr><td rowspan="2"> 0三厂豇豆种子/罐。 </td><td> 0天：植物〜30的本生种子/锅。施肥每周一次用1汤匙的肥料/ 5升的水。 </td></tr><tr><td> 第14天：重新锅本生1株/罐。 </td></tr><tr><td> 第10天：感染主要与CPMV（500μg/50μL/叶）叶通过机械接种使用的光尘的碳化硅离开。 </td><td> 第28天：感染到f香港专业教育学院叶TMV，PVX，或BMV（5μg/50μL/叶）通过机械接种使用的光尘的碳化硅。 </td></tr><tr><td> 第20天：收获的叶子和储存在-80°C。 </td><td> 第42天：收获的叶子和储存在-80°C。 </td></tr></tbody></table> 表1。时间轴越来越大，感染，收获的叶子。 注意：仅CPMV传播表明作为一个例子。 2。 VNP（CPMV，BMV，PVX和TMV）净化 注：所有步骤都在冰上或4℃。 <ol><li>均质100g的冷冻用2倍体积的冷缓冲液（ 见表2）在一个标准搅拌机叶片。通过2-3层纱布过滤。 </li><li> PVX的，调整pH值至6.5，用1 M HCl。加入0.2％（w / v的）抗坏血酸和0.2％（重量/体积）硫酸钠sulfi德。 </li><li>在5,500 xg离心20分钟，离心粗制植物匀浆。收集上清。 </li><li>对于BMV，层25毫升上清液中超过5毫升的10％（重量/体积）蔗糖溶液。离心机在9000 XG CsCl溶液中的38.5％（W / V）为3小时，并重新悬浮颗粒。混合振荡5小时，然后继续执行步骤2.12。 </li><li>提取植物材料，通过添加0.7体积的1:1（体积/体积）氯仿:1-丁醇。搅拌混合物30-60分钟。 </li><li>在5,500 xg下20分钟的离心溶液。收集上层水相。 </li><li>添加NaCl至0.2 M和8％（重量/体积）的PEG（分子量8000）。对于烟草花叶病毒，还可以添加1％（体积/体积）的Triton X-100。搅拌至少1小时，然后让参加至少1小时。 </li><li>以15,000 xg离心15分钟的离心溶液。在10毫升的缓冲液重悬沉淀。 PVX的，0.1％β-巯基乙醇和尿素添加到0.5M </li><li> 8000 xg离心30分钟，离心收集上清。 </li><li>超速离心机上清液在160,000 xg离心3小时。重悬浮颗粒在5毫升缓冲Øvernight。 </li><li>用等体积的10％，20％，30％，和40％的蔗糖缓冲液中（最重的第一）准备一个10-40％的蔗糖密度梯度。允许该梯度以在室温下平衡过夜。 </li><li>超速在10万XG蔗糖密度梯度悬浮颗粒超过2小时（24小时BMV）。 </li><li>收集散射光带和透析对缓冲区。 </li><li>特性VNPs（下图），并存储在4°C。对于长期储存，储存在-80°C。 </li></ol><table border="1"><tbody><tr><td> CPMV和TMV </td><td> 0.1M磷酸钾缓冲液（pH7.0） 38.5毫米KH 2 PO 4 61.5毫米K 2 HPO 4 </td></tr><tr><td> PVX </td><td> 0.5M的硼酸盐缓冲液（pH7.8）中 0.5 M硼酸 用NaOH调节pH值</td></tr><tr><td> BMV </td><td> SAMA缓冲液（pH4.5） 250 mM乙酸钠 的10mM的MgCl 2 2毫米β-巯基乙醇（补充新鲜） </td></tr></tbody></table> 表2。每个VNP的缓冲器和他们的食谱。 注意：仅CPMV传播表明作为一个例子。 3。 VNP（CPMV，BMV，PVX和TMV）表征<ol><li>执行UV /可见光谱来确定浓度的VNPs。 <ol><li>测量使用的NanoDrop分光光度计2μl的样品的吸光度。 </li><li>确定使用Beer-Lambert定律（A =εcl，其中A为吸光度的颗粒和染料的浓度，ε是消光系数，c是浓度，和l是路径长度）。的路径长度是的NanoDrop0.1厘米。 的的VNP特定消光系数是： CPMV：8.1厘米-1毫克-1毫升（波长为260 nm） PVX：2.97厘米-1毫克-1毫升（在260 nm） TMV：3.0厘米-1毫克-1毫升（波长为260 nm） BMV：5.15厘米-1毫克-1毫升（在260 nm） </li></ol></li><li>分析颗粒尺寸排阻快速蛋白质液相色谱（FPLC）。 <ol><li>使用的Superose 6尺寸排阻柱和ÄKTA资源管理器中，负载50-100微克VNPs在200μl的0.1M磷酸钾缓冲液（pH7.0）中。 </li><li>设置探测器，260 nm处（核酸），280nm的（蛋白质），和附加任何染料的激发波长。 </li><li> 72分钟，在0.5毫升/分钟的流速运行。 </li><li>流出曲线和A260：A280 nm的表示，，VNP准备是否是纯粹的粒子是否完整和组装。 A260：280比表示一个纯粹的VNP准备： CPMV：1.8±0.1 PVX：1.2±0.1 TMV：1.1±0.1 BMV：1.7±0.1 </li></ol></li><li>执行变性（预铸的NuPAGE）的Bis-Tris聚丙烯酰胺4-12％梯度凝胶电泳分析单独的衣壳蛋白的制备和共轭的纯度。 <ol><li>将3毫升的4倍LDS样品缓冲液添加到10μg9微升磷酸钾缓冲液中的颗粒。添加一个额外的1μl的4倍LDS样品缓冲液的β-巯基乙醇和3μlBMV，以减少大量的二硫键。 </li><li>孵育5分钟，在100℃，在热块</li><li>将样到SDS凝胶。 </li><li>运行1小时，1×MOPS电泳缓冲液样品在200 V。 </li><li>文件的凝胶在紫外光下观察荧光外壳蛋白。 </li><li>对于非荧光蛋白，用考马斯亮蓝（0.25％（重量/体积）考马斯亮蓝R-250，30％（体积/体积）甲醇，10％（体积/体积）乙酸）处理1小时染色。 </li><li>脱色，用30％甲醇，10％乙酸中过夜。陈GE的解决方案，如果需要的话。 </li><li>记录在白色灯光下的凝胶。 </li></ol></li><li>分析通过透射电子显微镜（TEM）的粒子的完整性。 <ol><li> 0.1-1毫克/毫升DI水在20μl稀释样品。 </li><li>将20微升滴封口膜的样品。 </li><li>包括滴用TEM网格，让坐2分钟。威克关闭在网格上的多余的溶液用滤纸。 </li><li>格下降DI水，然后放置在排汗干洗净。 </li><li>染色网格通过放置在20微升的2％（重量/体积）的下拉醋酸双氧铀，持续2分钟。威克掉多余的污点用滤纸。 </li><li>电网再次在水中洗净。 </li><li>透射型电子显微镜下的观察网格。 </li></ol></li></ol> 4。 PEG和荧光，纯化，表征与化学结合的VNPs <ol><li>对于下面的反应中的计算，摩尔质量的VNPs是： CPMV：5.6×10 6 g / mol的 PVX：35×10 6 g / mol的 TMV：41×10 6 g / mol的 BMV：4.6×10 6 g / mol的</li><li>染料和PEG共轭表面赖氨酸CPMV和PVX使用一个单步的N-羟基琥珀酰亚胺基碳酸酯偶联反应：加入2500摩尔当量（所有摩尔过量是指摩尔过量每VNP）的Alexa Fluor 647琥珀酰亚胺基酯和4500当量的NHS- PEG（分子量5000）溶解在DMSO中CPMV在0.1M磷酸钾缓冲液。工作时，PVX，增加了10000摩尔过量的的NHS-染料和NHS-PEG。调整使得CPMV和PVX的最终浓度为2毫克/毫升和DMSO的含量为10％的总反应体积的缓冲液和DMSO卷。孵育反应混合物在室温避光过夜。 CPMV和PVX分别有300和1270寻址赖氨酸，。 （ 进一步阅读的读者可以参考下面的参考化学修饰CPMV和PVX：13日至15日 ）。 </li><li>其次是铜（Ⅰ）催化叠氮炔环加成反应的共轭染料和PEG对TMV的酪氨酸重氮耦合。 <ol><li>准备重氮盐（炔）通过混合400微升0.3M的p-甲苯磺酸一水合物，25微升3.0 M亚硝酸钠，和75微升68万蒸馏的3 -乙炔基苯胺溶解在乙腈中，在4℃下1小时。 </li><li>加入3.3毫升的硼酸盐缓冲液，pH 8.8，含有100mM NaCl的烟草花叶病毒的1.25毫升（20毫克/毫升原液）。 </li><li>起反应用450微升的重氮盐（炔）溶液在冰浴中3小时添加的炔结扎处理由重氮耦合花叶病毒烟草花叶病毒。该解决方案将变成浅棕色的颜色。 TMV 2140提供酪氨酸结合。 </li><li>纯化的最终产物，在步骤4.4中所描述使用蔗糖座垫。 </li><li>附加的Alexa Fluor 647叠氮化物的功能和PEG-叠氮化物（MW 5000）全光照克铜（Ⅰ）催化的叠氮化物 - 炔环加成（CuAAC）。加入2当量的染料和PEG-磷酰基叠氮化物的每外壳蛋白并孵育1 mM的CuSO 4的 ，2mM的AMG，和2mM抗坏血酸钠，在室温下15分钟。调整缓冲体积使得TMV的最终的反应浓度为2毫克/毫升。 （ 读者可以参考以下化学修饰的烟草花叶病毒的进一步阅读的参考：16,17）。 </li></ol></li><li>共轭染料到赖氨酸和PEG BMV半胱氨酸突变体（cBMV）半胱氨酸： <ol><li>添加2000摩尔当量的在0.1M TNKM缓冲液（50mM的Tris碱，50毫摩尔NaCl，10 mM KCl中，5毫摩尔MgCl 2，pH值7.4）溶解在DMSO中cBMV俄勒冈绿488琥珀酰亚胺基酯。调整这样的BMV的最终浓度为1毫克/毫升和DMSO的含量为10％的总反应体积的缓冲液和DMSO卷。孵育反应混合物过夜，在4℃下避光保存。 </li><li>净化颗粒离心遁走曲的过滤器，在步骤4.4所述。 </li><li>加入2000摩尔过量的PEG-马来酰亚胺（分子量2,000）如前使用的相同的反应条件下，孵育2小时，将反应混合物在4℃下cBMV有180反应赖氨酸和半胱氨酸。 （读者可以参考下面引用的BMV化学改性的进一步阅读：18）。 </li></ol></li><li> 纯化：通过该解决方案通过在160,000 xg下2.5小时的40％（重量/体积）蔗糖坐垫。重新溶解在缓冲液中的颗粒。另外，适当的缓冲液透析对10 kDa的截止离心过滤。 </li><li> 表征：分析PEG化和荧光标记的VNPs 然而，使用上述的方法：UV /可见光光谱，SDS凝胶电泳，FPLC，和透射电子显微镜（ 未示出，请参阅图6和图7）。 </li></ol> 5。用小鼠移植瘤模型的肿瘤靶向性和成像<ol><li>文化HT-29人结肠癌细胞的RPMI培养基中补充了5％FBS，1％青霉素-链霉素，1％L-谷氨酰胺，在37℃下在5％CO 2用175厘米2细胞培养瓶中。 </li><li>洗细胞两次，用无菌PBS和收获，用5ml的胰蛋白酶-EDTA在37℃下孵育5分钟。用5毫升的RPMI培养基中，失活胰蛋白酶。经离心分离，在500×g离心5分钟收集细胞。在4℃下，在5×10 6 cells/50微升培养基（确定总细胞计数使用台盼蓝（trypan blue）和血细胞计数）重悬在新鲜RPMI。用等体积的基底膜混合之前注射（查看所有的解决方案和试剂保持无菌）。 </li><li>采购六周龄NCR nu / nu小鼠和维护他们上了苜蓿饮食2周。 [注：所有动物的程序必须IACUC批准。皮下注射诱导肿瘤异种移植5×10 6 cells/100μL/肿瘤的侧面（2肿瘤/鼠标）18 1/2号无菌针。定期监视的动物。使用游标卡尺测量肿瘤的大小，使肿瘤生长的平均量为20毫米3（在接下来的12天之内）。将小鼠两个不同的组：PBS和VNP（N = 3只/组/时间点）。使用1毫升28压力表胰岛素注射器，管理由静脉尾静脉注射100微升无菌PBS或10毫克/公斤，VNP制剂。 </li></ol> 注：组织培养实验和研究活的动物将无法得到证实。示范将被限制在组织处理和数据采集。对于一个参考对HT-29肿瘤异种移植模型上，读者被称为文献19中 三种技术被用来评估肿瘤归巢VNPs： <ol start="4"><li> 荧光成像在不同的时间点（2，24和72小时），使用CO 2 使用Maestro卡成像系统：牺牲小鼠气体。解剖动物和消费的所有主要器官（脑，心，肺，脾，肾和肝），与肿瘤的侧面，将组织石蜡，分析与使用黄色的激发和发射滤光片的荧光成像仪（800毫秒曝光）检测荧光信号的存在，在组织（来自A647标签共轭的VNPs）。保存的图像和分析荧光强度使用ImageJ：1.44o软件（ <a href="http://imagej.nih.gov/ij" target="_blank">http://imagej.nih.gov/ij</a> ）。比较VNPs在随着时间的推移与其他主要组织肿瘤摄取的图案。 </li><li>成像后，削减每个组织中的一半，和，在OCT化合物嵌入一个半冰冻切片和激光共聚焦分析。收藏报错另一半则在预先称重的冷冻小瓶并立即冻结他们使用液N 2。储存于-80℃，直到准备好以供进一步处理。 </li><li> 荧光定量：回复脊髓组织权重。在室温下解冻冷冻的组织，并且将它们放置在单独的50毫升Falcon管中含有1ml的PBS。使用一个手持的组织匀浆，在PBS中的2-3分钟的组织均匀，然后匀浆转移到微量离心管中。离心13000×g离心10分钟，除去非均质的组织匀浆。 </li><li>移液管加入100μl的上清液从从每个组（PBS及的VNP制剂/时间点）到384孔黑色UV板的组织。评估使用读板器的荧光强度（激发/发射波长665分之600）。规格化所获得的荧光值，由组织重量。 </li><li> 免疫组化：准备在-20℃的冷冻切片机切片（10微米），并存储染色细胞核吲哚（DAPI）和血管内皮细胞标记物（FITC-标记的抗小鼠CD31抗体）的组织切片。进行共焦显微镜分析血管瘤内的本地化的荧光标记的VNP映射第</li></ol> 注意：此过程将不会被证明的，有代表性的数据如图8所示。上的参考，免疫组织化学和所描述的染色方法，读者被称为文献19中

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D. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Intravital+imaging+of+human+prostate+cancer+using+viral+nanoparticles+targeted+to+gastrin-releasing+Peptide+receptors.">Intravital imaging of human prostate cancer using viral nanoparticles targeted to gastrin-releasing Peptide receptors.</a> Small. 7, 1664-1672 (2011).</li><li>Wu, C., Barnhill, H., Liang, X., Wang, Q., Jiang, H. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+new+probe+using+hybrid+virus-dye+nanoparticles+for+near-infrared+fluorescence+tomography.">A new probe using hybrid virus-dye nanoparticles for near-infrared fluorescence tomography.</a> Optics Communications. 255, 366-374 (2005).</li><li>Steinmetz, N. F., Cho, C. F., Ablack, A., Lewis, J. D., Manchester, M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Cowpea+mosaic+virus+nanoparticles+target+surface+vimentin+on+cancer+cells.">Cowpea mosaic virus nanoparticles target surface vimentin on cancer cells.</a> Nanomedicine (Lond). 6, 351-364 (2011).</li><li>Maeda, H., Wu, J., Sawa, T., Matsumura, Y., Hori, K. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Tumor+vascular+permeability+and+the+EPR+effect+in+macromolecular+therapeutics:+a+review.">Tumor vascular permeability and the EPR effect in macromolecular therapeutics: a review.</a> Journal of Controlled Release. 65, 271-284 (2000).</li><li>Chatterji, A., Ochoa, W., Paine, M., Ratna, B. R., Johnson, J. E., Lin, T. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=New+addresses+on+an+addressable+virus+nanoblock:+uniquely+reactive+Lys+residues+on+cowpea+mosaic+virus.">New addresses on an addressable virus nanoblock: uniquely reactive Lys residues on cowpea mosaic virus.</a> Chem. Biol. 11, 855-863 (2004).</li><li>Steinmetz, N. F., Mertens, M. E., Taurog, R. E., Johnson, J. E., Commandeur, U., Fischer, R., Manchester, M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Potato+virus+X+as+a+novel+platform+for+potential+biomedical+applications.">Potato virus X as a novel platform for potential biomedical applications.</a> Nano Lett. 10, 305-312 (2010).</li><li>Wang, Q., Lin, T., Tang, L., Johnson, J. E., Finn, M. G. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Icosahedral+virus+particles+as+addressable+nanoscale+building+blocks.">Icosahedral virus particles as addressable nanoscale building blocks.</a> Angew. Chem. Int. Ed. 41, 459-462 (2002).</li><li>Bruckman, M. A., Kaur, G., Lee, L. A., Xie, F., Sepulveda, J., Breitenkamp, R., Zhang, X., Joralemon, M., Russell, T. P., Emrick, T., Wang, Q. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Surface+modification+of+tobacco+mosaic+virus+with+"click"+chemistry.">Surface modification of tobacco mosaic virus with "click" chemistry.</a> Chembiochem. 9, 519-523 (2008).</li><li>Schlick, T. L., Ding, Z., Kovacs, E. W., Francis, M. B. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Dual-surface+modification+of+the+tobacco+mosaic+virus.">Dual-surface modification of the tobacco mosaic virus.</a> J. Am. Chem. Soc. 127, 3718-3723 (2005).</li><li>Yildiz, I., Tsvetkova, I., Wen, A. M., Shukla, S., Masarapu, M. H., Dragnea, B., Steinmetz, N. F. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Engineering+of+Brome+mosaic+virus+for+biomedical+applications.">Engineering of Brome mosaic virus for biomedical applications.</a> RSC Advances. , (2012).</li><li>Brunel, F. M., Lewis, J. D., Destito, G., Steinmetz, N. F., Manchester, M., Stuhlmann, H., Dawson, P. E. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Hydrazone+ligation+strategy+to+assemble+multifunctional+viral+nanoparticles+for+cell+imaging+and+tumor+targeting.">Hydrazone ligation strategy to assemble multifunctional viral nanoparticles for cell imaging and tumor targeting.</a> Nano Lett. 10, 1093-1097 (2010).</li><li>Shukla, S., Ablack, A., Wen, A., Lee, K., Lewis, J., Steinmetz, N. F. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Increased+tumor+homing+and+tissue+penetration+of+the+filamentous+plant+viral+nanoparticle+Potato+virus+X.">Increased tumor homing and tissue penetration of the filamentous plant viral nanoparticle Potato virus X.</a> Molecular Pharmaceutics. , (2012).</li><li>Chatterji, A., Ochoa, W., Shamieh, L., Salakian, S. P., Wong, S. M., Clinton, G., Ghosh, P., Lin, T., Johnson, J. E. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Chemical+conjugation+of+heterologous+proteins+on+the+surface+of+Cowpea+mosaic+virus.">Chemical conjugation of heterologous proteins on the surface of Cowpea mosaic virus.</a> Bioconjug. Chem. 15, 807-813 (2004).</li></ol>

没有利益冲突的声明。

该协议提供了一种方法VNPs和他们的应用程序在体内肿瘤成像的化学改性。动物荧光成像，荧光定量和免疫组化技术可用于研究生物分布和评估肿瘤归巢。这些技术提供了有价值的信息，通过EPR效应的纳米粒子对肿瘤。从不同的分析方法相结合的结果，我们得到了一个强大的方法，评估的定位和生物分布的VNPs。 之前可以进行这些研究，但是，它是必不可少的，以获得纯...

<table border="1">
	<tbody>
		<tr>
			<td>Material Name</td>
			<td>Company</td>
			<td>Catalogue number</td>
			<td>Comments (optional)</td>
		</tr>
		<tr>
			<td>&nbsp;</td>
			<td>&nbsp;</td>
			<td>&nbsp;</td>
			<td>VNP production</td>
		</tr>
		<tr>
			<td>Indoor plant chamber</td>
			<td>Percival Scientific</td>
			<td>E-41L2</td>
			<td>&nbsp;</td>
		</tr>
		<tr>
			<td>V. unguiculata seeds (California black-eye no. 5)</td>
			<td>Burpee</td>
			<td>51771A</td>
			<td>&nbsp;</td>
		</tr>
		<tr>
			<td>N. benthamiana seeds</td>
			<td>&nbsp;</td>
			<td>&nbsp;</td>
			<td>N. benthamiana seeds were a gift from Salk Institute. Seeds are produced through plant propagation.</td>
		</tr>
		<tr>
			<td>Carborundum</td>
			<td>Fisher</td>
			<td>C192-500</td>
			<td>&nbsp;</td>
		</tr>
		<tr>
			<td>Pro-mix BX potting soil</td>
			<td>Premier Horticulture</td>
			<td>713400</td>
			<td>&nbsp;</td>
		</tr>
		<tr>
			<td>Jack's Professional 20-10-20 Peat-Lite Fertilizer</td>
			<td>JR Peters</td>
			<td>77860</td>
			<td>&nbsp;</td>
		</tr>
		<tr>
			<td>&nbsp;</td>
			<td>&nbsp;</td>
			<td>&nbsp;</td>
			<td>Equipment</td>
		</tr>
		<tr>
			<td>50.2 Ti rotor</td>
			<td>Beckman</td>
			<td>337901</td>
			<td>&nbsp;</td>
		</tr>
		<tr>
			<td>SW 32 Ti rotor</td>
			<td>Beckman</td>
			<td>369694</td>
			<td>&nbsp;</td>
		</tr>
		<tr>
			<td>Optima L-90K ultracentrifuge</td>
			<td>Beckman</td>
			<td>365672</td>
			<td>&nbsp;</td>
		</tr>
		<tr>
			<td>SLA-3000 rotor</td>
			<td>Thermo Scientific</td>
			<td>07149</td>
			<td>&nbsp;</td>
		</tr>
		<tr>
			<td>SS-34 rotor</td>
			<td>Thermo Scientific</td>
			<td>28020</td>
			<td>&nbsp;</td>
		</tr>
		<tr>
			<td>Sorvall RC-6 Plus centrifuge</td>
			<td>Thermo Scientific</td>
			<td>46910</td>
			<td>&nbsp;</td>
		</tr>
		<tr>
			<td>Polypropylene bottle</td>
			<td>Beckman</td>
			<td>355607</td>
			<td>For SLA-3000 rotor</td>
		</tr>
		<tr>
			<td>Polycarbonate bottle</td>
			<td>Beckman</td>
			<td>357002</td>
			<td>For SS-34 rotor</td>
		</tr>
		<tr>
			<td>Ultra-Clear tube</td>
			<td>Beckman</td>
			<td>344058</td>
			<td>For sucrose gradient and SW 32 Ti rotor</td>
		</tr>
		<tr>
			<td>Polycarbonate bottle</td>
			<td>Beckman</td>
			<td>355618</td>
			<td>For pelleting and 50.2 Ti rotor</td>
		</tr>
		<tr>
			<td>NanoDrop spectrophotometer</td>
			<td>Thermo Scientific</td>
			<td>NanoDrop2000c</td>
			<td>&nbsp;</td>
		</tr>
		<tr>
			<td>PowerEase 500 pre-cast gel system</td>
			<td>Invitrogen</td>
			<td>EI8675EU</td>
			<td>&nbsp;</td>
		</tr>
		<tr>
			<td>Superose 6 10/300 GL (24 ml) size-exclusion column</td>
			<td>GE Healthcare</td>
			<td>17-5172-01</td>
			<td>&nbsp;</td>
		</tr>
		<tr>
			<td>&Auml;KTA Explorer 100 Chromatograph</td>
			<td>GE Healthcare</td>
			<td>28-4062-66</td>
			<td>&nbsp;</td>
		</tr>
		<tr>
			<td>Allegra X-12R</td>
			<td>Beckman</td>
			<td>392302</td>
			<td>Benchtop centrifuge</td>
		</tr>
		<tr>
			<td>Cryostat</td>
			<td>Leica</td>
			<td>CM1850</td>
			<td>&nbsp;</td>
		</tr>
		<tr>
			<td>Maestro 2</td>
			<td>Caliper Life Sciences</td>
			<td>&nbsp;</td>
			<td>In vivo imaging system</td>
		</tr>
		<tr>
			<td>Tissue-Tearor</td>
			<td>Biospec Products</td>
			<td>985370-395</td>
			<td>&nbsp;</td>
		</tr>
		<tr>
			<td>Microplate reader</td>
			<td>Tecan</td>
			<td>Infinite-200</td>
			<td>&nbsp;</td>
		</tr>
		<tr>
			<td>Transmission electron microscope</td>
			<td>ZEISS</td>
			<td>Libra 200FE</td>
			<td>&nbsp;</td>
		</tr>
		<tr>
			<td>FluoView laser scanning confocal microscope</td>
			<td>Olympus</td>
			<td>FV1000</td>
			<td>&nbsp;</td>
		</tr>
		<tr>
			<td>&nbsp;</td>
			<td>&nbsp;</td>
			<td>&nbsp;</td>
			<td>Chemicals and Reagents</td>
		</tr>
		<tr>
			<td>3-ethynylaniline</td>
			<td>Sigma Aldrich</td>
			<td>498289-5G</td>
			<td>&nbsp;</td>
		</tr>
		<tr>
			<td>384 well black plate</td>
			<td>BD Biosciences</td>
			<td>353285</td>
			<td>&nbsp;</td>
		</tr>
		<tr>
			<td>4-12% Bis-Tris NuPAGE SDS gel</td>
			<td>Invitrogen</td>
			<td>NP0321BOX</td>
			<td>&nbsp;</td>
		</tr>
		<tr>
			<td>4X LDS sample buffer</td>
			<td>Invitrogen</td>
			<td>NP0008</td>
			<td>&nbsp;</td>
		</tr>
		<tr>
			<td>Acetic Acid</td>
			<td>Fisher</td>
			<td>A385-500</td>
			<td>&nbsp;</td>
		</tr>
		<tr>
			<td>Acetonitrile</td>
			<td>Sigma Aldrich</td>
			<td>271004-1L</td>
			<td>&nbsp;</td>
		</tr>
		<tr>
			<td>Alexa Fluor 647 azide</td>
			<td>Invitrogen</td>
			<td>A10277</td>
			<td>&nbsp;</td>
		</tr>
		<tr>
			<td>Alexa Fluor 647 carboxylic acid, succinimidyl ester</td>
			<td>Invitrogen</td>
			<td>A20006</td>
			<td>&nbsp;</td>
		</tr>
		<tr>
			<td>Amicon Ultra-0.5 ml Centrifugal Filters</td>
			<td>Millipore</td>
			<td>UFC501096</td>
			<td>10 kDa cut-off</td>
		</tr>
		<tr>
			<td>Aminoguanidine hydrochloride</td>
			<td>Acros Organics</td>
			<td>36891-0250</td>
			<td>&nbsp;</td>
		</tr>
		<tr>
			<td>Boric acid</td>
			<td>Fisher</td>
			<td>A74-500</td>
			<td>&nbsp;</td>
		</tr>
		<tr>
			<td>Coomassie Brilliant Blue R-250</td>
			<td>Fisher</td>
			<td>BP101-25</td>
			<td>&nbsp;</td>
		</tr>
		<tr>
			<td>CsCl</td>
			<td>Acros Organics</td>
			<td>42285-1000</td>
			<td>&nbsp;</td>
		</tr>
		<tr>
			<td>DAPI</td>
			<td>MP Biomedicals</td>
			<td>157574</td>
			<td>&nbsp;</td>
		</tr>
		<tr>
			<td>Dimethyl sulfoxide</td>
			<td>Fisher</td>
			<td>BP231-100</td>
			<td>&nbsp;</td>
		</tr>
		<tr>
			<td>Filter paper</td>
			<td>Fisher</td>
			<td>09-801K</td>
			<td>P5 grade</td>
		</tr>
		<tr>
			<td>FITC anti-mouse CD31</td>
			<td>BioLegend</td>
			<td>102406</td>
			<td>&nbsp;</td>
		</tr>
		<tr>
			<td>Goat serum</td>
			<td>Invitrogen</td>
			<td>16210-064</td>
			<td>&nbsp;</td>
		</tr>
		<tr>
			<td>KCl</td>
			<td>Fisher</td>
			<td>BP366-500</td>
			<td>&nbsp;</td>
		</tr>
		<tr>
			<td>L-ascorbic acid sodium salt</td>
			<td>Acros Organics</td>
			<td>35268-0050</td>
			<td>&nbsp;</td>
		</tr>
		<tr>
			<td>Methanol</td>
			<td>Fisher</td>
			<td>A412P-4</td>
			<td>&nbsp;</td>
		</tr>
		<tr>
			<td>MgCl2</td>
			<td>Fisher</td>
			<td>BP214-500</td>
			<td>&nbsp;</td>
		</tr>
		<tr>
			<td>Microscope slides</td>
			<td>Fisher</td>
			<td>12-544-3</td>
			<td>&nbsp;</td>
		</tr>
		<tr>
			<td>Microscope cover glass</td>
			<td>VWR</td>
			<td>48366-277</td>
			<td>&nbsp;</td>
		</tr>
		<tr>
			<td>MOPS buffer</td>
			<td>Invitrogen</td>
			<td>NP0001</td>
			<td>&nbsp;</td>
		</tr>
		<tr>
			<td>mPEG-mal</td>
			<td>Nanocs</td>
			<td>PG1-ML-2k</td>
			<td>MW 2000</td>
		</tr>
		<tr>
			<td>mPEG-N3</td>
			<td>Nanocs</td>
			<td>PG1-AZ-5k</td>
			<td>MW 5000</td>
		</tr>
		<tr>
			<td>mPEG-NHS</td>
			<td>Nanocs</td>
			<td>PG1-SC-5k</td>
			<td>MW 5000</td>
		</tr>
		<tr>
			<td>NaCl</td>
			<td>Fisher</td>
			<td>BP358-212</td>
			<td>&nbsp;</td>
		</tr>
		<tr>
			<td>Oregon Green 488 succinimidyl ester *6-isomer*</td>
			<td>Invitrogen</td>
			<td>O-6149</td>
			<td>&nbsp;</td>
		</tr>
		<tr>
			<td>p-toluenesulfonic acid monohydrate</td>
			<td>Acros Organics</td>
			<td>13902-0050</td>
			<td>&nbsp;</td>
		</tr>
		<tr>
			<td>Permount</td>
			<td>Fisher</td>
			<td>SP15-100</td>
			<td>&nbsp;</td>
		</tr>
		<tr>
			<td>Potassium phosphate dibasic</td>
			<td>Fisher</td>
			<td>BP363-1</td>
			<td>&nbsp;</td>
		</tr>
		<tr>
			<td>Potassium phosphate monobasic</td>
			<td>Fisher</td>
			<td>BP362-1</td>
			<td>&nbsp;</td>
		</tr>
		<tr>
			<td>Sodium acetate</td>
			<td>Fisher</td>
			<td>BP333-500</td>
			<td>&nbsp;</td>
		</tr>
		<tr>
			<td>Sodium nitrite</td>
			<td>Acros Organics</td>
			<td>42435-0050</td>
			<td>&nbsp;</td>
		</tr>
		<tr>
			<td>Sodium sulfite</td>
			<td>Amresco</td>
			<td>0628-500G</td>
			<td>&nbsp;</td>
		</tr>
		<tr>
			<td>Sucrose</td>
			<td>Fisher</td>
			<td>S6-500</td>
			<td>&nbsp;</td>
		</tr>
		<tr>
			<td>TEM grid</td>
			<td>Ted Pella</td>
			<td>FCF-400Cu</td>
			<td>&nbsp;</td>
		</tr>
		<tr>
			<td>Tris base</td>
			<td>Fisher</td>
			<td>BP152-500</td>
			<td>&nbsp;</td>
		</tr>
		<tr>
			<td>Triton X-100</td>
			<td>EMD Chemicals</td>
			<td>TX1568-1</td>
			<td>&nbsp;</td>
		</tr>
		<tr>
			<td>&beta;-mercaptoethanol</td>
			<td>Fisher</td>
			<td>O3446I-100</td>
			<td>&nbsp;</td>
		</tr>
		<tr>
			<td>&nbsp;</td>
			<td>&nbsp;</td>
			<td>&nbsp;</td>
			<td>Tissue Culture</td>
		</tr>
		<tr>
			<td>Fetal bovine serum</td>
			<td>Invitrogen</td>
			<td>12483-020</td>
			<td>&nbsp;</td>
		</tr>
		<tr>
			<td>Hemocytometer</td>
			<td>Fisher</td>
			<td>0267110</td>
			<td>&nbsp;</td>
		</tr>
		<tr>
			<td>HT-29 cells</td>
			<td>ATCC</td>
			<td>HTB-38</td>
			<td>&nbsp;</td>
		</tr>
		<tr>
			<td>L-glutamine</td>
			<td>Invitrogen</td>
			<td>25030-080</td>
			<td>&nbsp;</td>
		</tr>
		<tr>
			<td>PBS</td>
			<td>Cellgro</td>
			<td>21-040-CV</td>
			<td>&nbsp;</td>
		</tr>
		<tr>
			<td>Penicillin-streptomycin</td>
			<td>Invitrogen</td>
			<td>10378-016</td>
			<td>&nbsp;</td>
		</tr>
		<tr>
			<td>RPMI-1640</td>
			<td>Invitrogen</td>
			<td>31800-089</td>
			<td>&nbsp;</td>
		</tr>
		<tr>
			<td>Tissue culture flasks</td>
			<td>Corning</td>
			<td>431080</td>
			<td>175 cm2</td>
		</tr>
		<tr>
			<td>Trypan Blue</td>
			<td>Thermo Scientific</td>
			<td>SV30084.01</td>
			<td>&nbsp;</td>
		</tr>
		<tr>
			<td>Trypsin, 0.05% (1X) with EDTA 4Na, liquid</td>
			<td>Invitrogen</td>
			<td>25300-054</td>
			<td>&nbsp;</td>
		</tr>
		<tr>
			<td>&nbsp;</td>
			<td>&nbsp;</td>
			<td>&nbsp;</td>
			<td>Animal Studies</td>
		</tr>
		<tr>
			<td>18% Protein Rodent Diet</td>
			<td>Harlan Teklad</td>
			<td>Teklad Global 2018S</td>
			<td>Alfalfa free diet</td>
		</tr>
		<tr>
			<td>Insulin syringe</td>
			<td>BD Biosciences</td>
			<td>329410</td>
			<td>28 gauge</td>
		</tr>
		<tr>
			<td>Isoflurane</td>
			<td>Baxter</td>
			<td>AHN3637</td>
			<td>&nbsp;</td>
		</tr>
		<tr>
			<td>Matrigel Matrix basement membrane</td>
			<td>BD Biosciences</td>
			<td>356234</td>
			<td>&nbsp;</td>
		</tr>
		<tr>
			<td>NCR nu/nu mice</td>
			<td>&nbsp;</td>
			<td>&nbsp;</td>
			<td>CWRU School 
			of Medicine Athymic Animal and Xenograft Core Facility</td>
		</tr>
		<tr>
			<td>Sterile syringe</td>
			<td>BD Biosciences</td>
			<td>305196</td>
			<td>18 1/2 gauge</td>
		</tr>
		<tr>
			<td>Tissue-Tek CRYO-OCT Compound</td>
			<td>Andwin Scientific</td>
			<td>4583</td>
			<td>&nbsp;</td>
		</tr>
	</tbody>
</table>

viral nanoparticles for in vivo tumor imaging

纳米材料的使用有可能彻底改变材料科学和医学。目前，正在调查一些不同的纳米粒子成像和治疗中的应用。来自植物的病毒粒子（VNPs）可以被视为与定义的尺寸和形状的自组装bionanomaterials。植物病毒正在调查在斯坦梅茨实验室包括二十面体由豇豆花叶病毒 （CPMV）， 雀麦花叶病毒 （BMV），这两者都是在直径为30nm的颗粒。我们也正在开发棒状，丝状的结构衍生从下列植物病毒： 烟草花叶病毒 （TMV），其形成为300nm的尺寸为18nm，和马铃薯X病毒 （PVX），形成丝状颗粒515与刚性杆在长度和宽度为13 nm的纳米（读取器被称为文献。VNPs进一步信息的1和2）。 在植物易于大规模生产，异常稳定的，和生物相容性。此外，VNPs“可编程”的单位，可专门设计使用遗传修饰或化学生物耦合方法3。原子分辨率的结构VNPs是众所周知的，并且可以进行修改与空间精度在原子水平上4，一定程度的控制不能使用合成纳米材料与当前国家的最先进的技术来实现的。 在本文中，我们描述了CPMV，PVX，TMV，和BMV， 豇豆ungiuculata和烟草植物的繁殖。 VNP对每个提取和纯化协议。纯化和化学标记VNPs表征方法描述。在这项研究中，我们专注于通道emical标签VNPs与荧光团（ 如的Alexa Fluor 647）和聚乙二醇（PEG）。该染料促进VNPs 5-10的跟踪和检测，和PEG降低免疫原性的蛋白质纳米粒子，同时增强其药动学8,11。我们展示的聚乙二醇化VNPs使用小鼠移植瘤模型的肿瘤归巢。组织体外荧光成像技术的使用Maestro成像系统，荧光定量同质化组织，和共聚焦显微镜的组合被用于研究生物分布。 VNPs通过网状内皮系统（RES）被清零;肿瘤归巢实现被动地通过增强的渗透性和保持率（EPR）效果12。 “的VNP纳米技术是强大的插件和播放技术，图像和治疗的疾病在体内网站。我们进一步发展VNPs携带药物的货物和临床相关的摄像部分，以及组织特异性的配体目标分子的受体过度表达，在癌症和心血管疾病。

<img alt="Figure 1" src="/files/ftp_upload/4352/4352fig1.jpg" /> 
 Figure 1. Plant virus-infected plants. Vigna unguiculata plants infected with CPMV (A). Nicotiana benthamiana plants infected with PVX (B), TMV (C), and BMV (D). The pictures were taken about 10 days post infection by mechanical inoculation.
<img alt="Figure 2" fo:content-width="3in" fo:src="/files/ftp_upload/4352...

Watch this Scientific Journal Video about Viral Nanoparticles for In vivo Tumor Imaging at JoVE.com

Viral Nanoparticles for In vivo Tumor Imaging

植物病毒粒子（VNPs），是有前途的平台，在生物医学中的应用。在这里，我们将介绍程序植物VNP传播，净化，表征和生物标记。最后，我们的应用VNPs肿瘤归巢和成像用小鼠移植瘤模型和荧光成像。

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 ...

viral-nanoparticles-for-in-vivo-tumor-imaging

Research

JoVE Journal

Bioengineering

Viral Nanoparticles for In vivo Tumor Imaging

17.1K 次观看.  Case Western Reserve University. 植物病毒粒子（VNPs），是有前途的平台，在生物医学中的应用。在这里，我们将介绍程序植物VNP传播，净化，表征和生物标记。最后，我们的应用VNPs肿瘤归巢和成像用小鼠移植瘤模型和荧光成像。

视频: Viral Nanoparticles for In vivo Tumor Imaging

Simple Microfluidic Devices for in vivo Imaging of C. elegans, Drosophila and Zebrafish

Micro fabricated fluidic devices provide an accessible micro-environment for in vivo studies on small organisms. Simple fabrication processes are available for microfluidic devices using soft lithography techniques 1-3. Microfluidic devices have been used for sub-cellular imaging 4,5, in vivo laser microsurgery 2,6 and cellular imaging 4,7. In vivo imaging requires immobilization of organisms. This has been achieved using suction 5,8, tapered channels 6,7,9, deformable membranes 2-4,10, suction with additional cooling 5, anesthetic gas 11, temperature sensitive gels 12, cyanoacrylate glue 13 and anesthetics such as levamisole 14,15. Commonly used anesthetics influence synaptic transmission 16,17 and are known to have detrimental effects on sub-cellular neuronal transport 4. In this study we demonstrate a membrane based poly-dimethyl-siloxane (PDMS) device that allows anesthetic free immobilization of intact genetic model organisms such as Caenorhabditis elegans (C. elegans), Drosophila larvae and zebrafish larvae. These model organisms are suitable for in vivo studies in microfluidic devices because of their small diameters and optically transparent or translucent bodies. Body diameters range from ~10 &mu;m to ~800 &mu;m for early larval stages of C. elegans and zebrafish larvae and require microfluidic devices of different sizes to achieve complete immobilization for high resolution time-lapse imaging. These organisms are immobilized using pressure applied by compressed nitrogen gas through a liquid column and imaged using an inverted microscope. Animals released from the trap return to normal locomotion within 10 min.
We demonstrate four applications of time-lapse imaging in C. elegans namely, imaging mitochondrial transport in neurons, pre-synaptic vesicle transport in a transport-defective mutant, glutamate receptor transport and Q neuroblast cell division. Data obtained from such movies show that microfluidic immobilization is a useful and accurate means of acquiring in vivo data of cellular and sub-cellular events when compared to anesthetized animals (Figure 1J and 3C-F 4).
Device dimensions were altered to allow time-lapse imaging of different stages of C. elegans, first instar Drosophila larvae and zebrafish larvae. Transport of vesicles marked with synaptotagmin tagged with GFP (syt.eGFP) in sensory neurons shows directed motion of synaptic vesicle markers expressed in cholinergic sensory neurons in intact first instar Drosophila larvae. A similar device has been used to carry out time-lapse imaging of heartbeat in ~30 hr post fertilization (hpf) zebrafish larvae. These data show that the simple devices we have developed can be applied to a variety of model systems to study several cell biological and developmental phenomena in vivo.

Simple Microfluidic Devices for in vivo Imaging of C. elegans, Drosophila and Zebrafish

A simple microfluidic device has been developed to perform anesthetic free in vivo imaging of C. elegans, intact Drosophila larvae and zebrafish larvae. The device utilizes a deformable PDMS membrane to immobilize these model organisms in order to perform time lapse imaging of numerous processes such as heart beat, cell division and sub-cellular neuronal transport. We demonstrate the use of this device and show examples of different types of data collected from different model systems.

简单的微流控芯片的<em在体内</em&gt;成像<em&gt; C.线虫</em&gt;<em&gt;果蝇</em&gt;和斑马鱼

Micro fabricated fluidic devices provide an accessible micro-environment for in vivo studies on small organisms. Simple fabrication processes are ...

科研

生物工程

Analysis of Targeted Viral Protein Nanoparticles Delivered to HER2+ Tumors

The HER2+ tumor-targeted nanoparticle, HerDox, exhibits tumor-preferential accumulation and tumor-growth ablation in an animal model of HER2+ cancer. HerDox is formed by non-covalent self-assembly of a tumor targeted cell penetration protein with the chemotherapy agent, doxorubicin, via a small nucleic acid linker. A combination of electrophilic, intercalation, and oligomerization interactions facilitate self-assembly into round 10-20 nm particles. HerDox exhibits stability in blood as well as in extended storage at different temperatures. Systemic delivery of HerDox in tumor-bearing mice results in tumor-cell death with no detectable adverse effects to non-tumor tissue, including the heart and liver (which undergo marked damage by untargeted doxorubicin). HER2 elevation facilitates targeting to cells expressing the human epidermal growth factor receptor, hence tumors displaying elevated HER2 levels exhibit greater accumulation of HerDox compared to cells expressing lower levels, both in vitro and in vivo. Fluorescence intensity imaging combined with in situ confocal and spectral analysis has allowed us to verify in vivo tumor targeting and tumor cell penetration of HerDox after systemic delivery. Here we detail our methods for assessing tumor targeting via multimode imaging after systemic delivery.

This article details the procedures for optical imaging analysis of the tumor-targeted nanoparticle, HerDox. In particular, detailed use of the multimode imaging device for detecting tumor targeting and assessing tumor penetration is described here.

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. ...

PLGA Nanoparticles Formed by Single- or Double-emulsion with Vitamin E-TPGS

Poly(lactic-co-glycolic acid) (PLGA) is a biocompatible member of the aliphatic polyester family of biodegradable polymers. PLGA has long been a popular choice for drug delivery applications, particularly since it is already FDA-approved for use in humans in the form of resorbable sutures. Hydrophobic and hydrophilic drugs are encapsulated in PLGA particles via single- or double-emulsion. Briefly, the drug is dissolved with polymer or emulsified with polymer in an organic phase that is then emulsified with the aqueous phase. After the solvent has evaporated, particles are washed and collected via centrifugation for lyophilization and long term storage. PLGA degrades slowly via hydrolysis in aqueous environments, and encapsulated agents are released over a period of weeks to months. Although PLGA is a material that possesses many advantages for drug delivery, reproducible formation of nanoparticles can be challenging; considerable variability is introduced by the use of different equipment, reagents batch, and precise method of emulsification. Here, we describe in great detail the formation and characterization of microparticles and nanoparticles formed by single- or double-emulsion using the emulsifying agent vitamin E-TPGS. Particle morphology and size are determined with scanning electron microscopy (SEM). We provide representative SEM images for nanoparticles produced with varying emulsifier concentration, as well as examples of imaging artifacts and failed emulsifications. This protocol can be readily adapted to use alternative emulsifiers (e.g. poly(vinyl alcohol), PVA) or solvents (e.g.&#160;dichloromethane, DCM).

We describe the production and characterization of nanoparticles and microparticles composed of poly(lactic-co-glycolic acid) using vitamin E-TPGS as an emulsifier. By varying formulation parameters such as the concentration of emulsifier, it is possible to produce nanoparticles with mean diameters ranging from 220 nm to&#160;1.98 &#181;m.

Poly(lactic-co-glycolic acid) (PLGA) is a biocompatible member of the aliphatic polyester family of biodegradable polymers. PLGA has long been a popular ...

Imaging Denatured Collagen Strands In vivo and Ex vivo via Photo-triggered Hybridization of Caged Collagen Mimetic Peptides

Collagen is a major structural component of the extracellular matrix that supports tissue formation and maintenance. Although collagen remodeling is an integral part of normal tissue renewal, excessive amount of remodeling activity is involved in tumors, arthritis, and many other pathological conditions. During collagen remodeling, the triple helical structure of collagen molecules is disrupted by proteases in the extracellular environment. In addition, collagens present in many histological tissue samples are partially denatured by the fixation and preservation processes. Therefore, these denatured collagen strands can serve as effective targets for biological imaging. We previously developed a caged collagen mimetic peptide (CMP) that can be photo-triggered to hybridize with denatured collagen strands by forming triple helical structure, which is unique to collagens. The overall goals of this procedure are i)&#160;to image denatured collagen strands resulting from normal remodeling activities in vivo, and ii) to visualize collagens in ex vivo tissue sections using the photo-triggered caged CMPs. To achieve effective hybridization and successful in vivo and ex vivo imaging, fluorescently labeled caged CMPs are either photo-activated immediately before intravenous injection, or are directly activated on tissue sections. Normal skeletal collagen remolding in nude mice and collagens in prefixed mouse cornea tissue sections are imaged in this procedure. The imaging method based on the CMP-collagen hybridization technology presented here could lead to deeper understanding of the tissue remodeling process, as well as allow development of new diagnostics for diseases associated with high collagen remodeling activity.

Imaging Denatured Collagen Strands In vivo and Ex vivo via Photo-triggered Hybridization of Caged Collagen Mimetic Peptides

This procedure demonstrates in vivo near IR fluorescence imaging of collagen remodeling activities in mice as well as ex vivo staining of collagens in tissue sections using caged collagen mimetic peptides that can be photo-triggered to hybridize with denatured collagen strands.

成像变性的胶原股<em&gt;在体内</em&gt;和<em&gt;体外</em&gt;通过光引发杂交笼胶原蛋白仿生肽

Collagen is a major structural component of the extracellular matrix that supports tissue formation and maintenance. Although collagen remodeling is an ...

Biofunctionalized Prussian Blue Nanoparticles for Multimodal Molecular Imaging Applications

Multimodal, molecular imaging allows the visualization of biological processes at cellular, subcellular, and molecular-level resolutions using multiple, complementary imaging techniques. These imaging agents facilitate the real-time assessment of pathways and mechanisms in vivo, which enhance both diagnostic and therapeutic efficacy. This article presents the protocol for the synthesis of biofunctionalized Prussian blue nanoparticles (PB NPs) - a novel class of agents for use in multimodal, molecular imaging applications. The imaging modalities incorporated in the nanoparticles, fluorescence imaging and magnetic resonance imaging (MRI), have complementary features. The PB NPs possess a core-shell design where gadolinium and manganese ions incorporated within the interstitial spaces of the PB lattice generate MRI contrast, both in T1 and T2-weighted sequences. The PB NPs are coated with fluorescent avidin using electrostatic self-assembly, which enables fluorescence imaging. The avidin-coated nanoparticles are modified with biotinylated ligands that confer molecular targeting capabilities to the nanoparticles. The stability and toxicity of the nanoparticles are measured, as well as their MRI relaxivities. The multimodal, molecular imaging capabilities of these biofunctionalized PB NPs are then demonstrated by using them for fluorescence imaging and molecular MRI in vitro.

This protocol describes the synthesis of biofunctionalized Prussian blue nanoparticles and their use as multimodal, molecular imaging agents. The nanoparticles have a core-shell design where gadolinium or manganese ions within the nanoparticle core generate MRI contrast. The biofunctional shell contains fluorophores for fluorescence imaging and targeting ligands for molecular targeting.

Biofunctionalized普鲁士蓝纳米粒子多式联运分子成像应用

Multimodal, molecular imaging allows the visualization of biological processes at cellular, subcellular, and molecular-level resolutions using multiple, ...

Microwave-driven Synthesis of Iron Oxide Nanoparticles for Fast Detection of Atherosclerosis

A fast and reproducible microwave-driven protocol has been developed for the synthesis of neridronate-functionalized nanoparticles. Starting from the synthesis of hydrophobic nanoparticles, our method is based on an adaptation from thermal decomposition method to microwave driven synthesis. The new methodology produces a decrease in the reaction times in comparison with traditional procedures. Moreover, the use of the microwave technology increases the reproducibility of the reactions, something important from the point of view of clinical applications. The novelty of this iron oxide nanoparticle is the attachment of Neridronate. The use of this molecule leads a bisphosphonate moiety towards the outside of the nanoparticle that provides Ca2+ binding properties in vitro and selective accumulation in vivo in the atheroma plaque. The protocol allows the synthesis and plaque detection in about 3 hr since the initial synthesis from organic precursors. Their accumulation in the atherosclerotic area in less than 1 hr provides a contrast agent particularly suitable for clinical applications.

Microwave technology enables extremely fast synthesis of iron oxide nanoparticles for atherosclerosis plaque characterization. The use of an aminobisphosphonate in the external side of the nanoparticle provides a fast accumulation in the atherosclerotic area.

动脉粥样硬化的快速检测的氧化铁纳米颗粒微波驱动综合

A fast and reproducible microwave-driven protocol has been developed for the synthesis of neridronate-functionalized nanoparticles. Starting from the ...

Visualizing Angiogenesis by Multiphoton Microscopy In Vivo in Genetically Modified 3D-PLGA/nHAp Scaffold for Calvarial Critical Bone Defect Repair

The reconstruction of critically sized bone defects remains a serious clinical problem because of poor angiogenesis within tissue-engineered scaffolds during repair, which gives rise to a lack of sufficient blood supply and causes necrosis of the new tissues. Rapid vascularization is a vital prerequisite for new tissue survival and integration with existing host tissue. The de novo generation of vasculature in scaffolds is one of the most important steps in making bone regeneration more efficient, allowing repairing tissue to grow into a scaffold. To tackle this problem, the genetic modification of a biomaterial scaffold is used to accelerate angiogenesis and osteogenesis. However, visualizing and tracking in vivo blood vessel formation in real-time and in three-dimensional (3D) scaffolds or new bone tissue is still an obstacle for bone tissue engineering. Multiphoton microscopy (MPM) is a novel bio-imaging modality that can acquire volumetric data from biological structures in a high-resolution and minimally-invasive manner. The objective of this study was to visualize angiogenesis with multiphoton microscopy in vivo in a genetically modified 3D-PLGA/nHAp scaffold for calvarial critical bone defect repair. PLGA/nHAp scaffolds were functionalized for the sustained delivery of a growth factor pdgf-b gene carrying lentiviral vectors (LV-pdgfb) in order to facilitate angiogenesis and to enhance bone regeneration. In a scaffold-implanted calvarial critical bone defect mouse model, the blood vessel areas (BVAs) in PHp scaffolds were significantly higher than in PH scaffolds. Additionally, the expression of pdgf-b and angiogenesis-related genes, vWF and VEGFR2, increased correspondingly. MicroCT analysis indicated that the new bone formation in the PHp group dramatically improved compared to the other groups. To our knowledge, this is the first time multiphoton microscopy was used in bone tissue-engineering to investigate angiogenesis in a 3D bio-degradable scaffold in vivo and in real-time.

Visualizing Angiogenesis by Multiphoton Microscopy In Vivo in Genetically Modified 3D-PLGA/nHAp Scaffold for Calvarial Critical Bone Defect Repair

Here, we present a protocol to visualize blood vessel formation in vivo and in real-time in 3D scaffolds by multiphoton microscopy. Angiogenesis in genetically modified scaffolds was studied in a murine calvarial critical bone defect model. More new blood vessels were detected in the treatment group than in controls.

多显微镜下的血管生成术在基因修饰的 3 d-PLGA/nHAp 支架上进行颅骨临界骨缺损修复

The reconstruction of critically sized bone defects remains a serious clinical problem because of poor angiogenesis within tissue-engineered scaffolds ...

Initial Evaluation of Antibody-conjugates Modified with Viral-derived Peptides for Increasing Cellular Accumulation and Improving Tumor Targeting

Antibody-conjugates (ACs) modified with virus-derived peptides are a potentially powerful class of tumor cell delivery agents for molecular payloads used in cancer treatment and imaging due to increased cellular accumulation over current ACs. During early AC in vitro development, fluorescence techniques and radioimmunoassays are sufficient for determining intracellular localization, accumulation efficiency, and target cell specificity. Currently, there is no consensus on standardized methods for preparing cells for evaluating AC intracellular accumulation and localization. The initial testing of ACs modified with virus-derived peptides is critical especially if several candidates have been constructed. Determining intracellular accumulation by fluorescence can be affected by background signal from ACs at the cell surface and complicate the interpretation of accumulation. For radioimmunoassays, typically treated cells are fractionated and the radioactivity in different cell compartments measured. However, cell lysis varies from cell to cell and often nuclear and cytoplasmic compartments are not adequately isolated. This can produce misleading data on payload delivery properties. The intravenous injection of radiolabeled virus-derived peptide-modified ACs in tumor bearing mice followed by radionuclide imaging is a powerful method for determining tumor targeting and payload delivery properties at the in vivo phase of development. However, this is a relatively recent advancement and few groups have evaluated virus-derived peptide-modified ACs in this manner. We describe the processing of treated cells to more accurately evaluate virus-derived peptide-modified AC accumulation when using confocal microscopy and radioimmunoassays. Specifically, a method for trypsinizing cells to remove cell surface bound ACs. We also provide a method for improving cellular fractionation. Lastly, this protocol provides an in vivo method using positron emission tomography (PET) for evaluating initial tumor targeting properties in tumor-bearing mice. We use the radioisotope 64Cu (t1/2 = 12.7 h) as an example payload in this protocol.

Viral-derived peptides coupled to antibody-conjugates (ACs) is an approach gaining momentum due to the potential of delivering molecular payloads with increased tumor cell accumulation. Utilizing common methods to evaluate peptide conjugation, AC and payload intracellular accumulation, and tumor targeting, this protocol helps researchers during the key initial development phases.

病毒衍生肽修饰抗体结合物对增强细胞积累和改善肿瘤靶向性的初步评价

Antibody-conjugates (ACs) modified with virus-derived peptides are a potentially powerful class of tumor cell delivery agents for molecular payloads used ...

Correlative Light Electron Microscopy (CLEM) for Tracking and Imaging Viral Protein Associated Structures in Cryo-immobilized Cells

Due to its high resolution, electron microscopy (EM) is an indispensable tool for virologists. However, one of the main difficulties when analyzing virus-infected or transfected cells via EM are the low efficiencies of infection or transfection, hindering the examination of these cells. In order to overcome this difficulty, light microscopy (LM) can be performed first to allocate the subpopulation of infected or transfected cells. Thus, taking advantage of the use of fluorescent proteins (FPs) fused to viral proteins, LM is used here to record the positions of the "positive-transfected" cells, expressing a FP and growing on a support with an alphanumeric pattern. Subsequently, cells are further processed for EM via high pressure freezing (HPF), freeze substitution (FS) and resin embedding. The ultra-rapid freezing step ensures excellent membrane preservation of the selected cells that can then be analyzed at the ultrastructural level by transmission electron microscopy (TEM). Here, a step-by-step correlative light electron microscopy (CLEM) workflow is provided, describing sample preparation, imaging and correlation in detail. The experimental design can be also applied to address many cell biology questions.

Correlative Light Electron Microscopy CLEM for Tracking and Imaging Viral Protein Associated Structures in Cryo-immobilized Cells

A correlative light electron microscopy (CLEM) method is applied to image virus-induced intracellular structures via electron microscopy (EM) in cells that are previously selected by light microscopy (LM). LM and EM are combined as a hybrid imaging approach to achieve an integrated view of virus-host interactions.

低温固定化细胞跟踪和成像病毒蛋白相关结构的相关光电子显微镜 (克莱)

Due to its high resolution, electron microscopy (EM) is an indispensable tool for virologists. However, one of the main difficulties when analyzing ...

Rigid Embedding of Fixed and Stained, Whole, Millimeter-Scale Specimens for Section-free 3D Histology by Micro-Computed Tomography

For over a hundred years, the histological study of tissues has been the gold standard for medical diagnosis because histology allows all cell types in every tissue to be identified and characterized. Our laboratory is actively working to make technological advances in X-ray micro-computed tomography (micro-CT) that will bring the diagnostic power of histology to the study of full tissue volumes at cellular resolution (i.e., an X-ray Histo-tomography modality). Toward this end, we have made targeted improvements to the sample preparation pipeline. One key optimization, and the focus of the present work, is a straightforward method for rigid embedding of fixed and stained millimeter-scale samples. Many of the published methods for sample immobilization and correlative micro-CT imaging rely on placing the samples in paraffin wax, agarose, or liquids such as alcohol. Our approach extends this work with custom procedures and the design of a 3-dimensional printable apparatus to embed the samples in an acrylic resin directly into polyimide tubing, which is relatively transparent to X-rays. Herein, sample preparation procedures are described for the samples from 0.5 to 10 mm in diameter, which would be suitable for whole zebrafish larvae and juveniles, or other animals and tissue samples of similar dimensions. As proof of concept, we have embedded the specimens from Danio, Drosophila, Daphnia, and a mouse embryo; representative images from 3-dimensional scans for three of these samples are shown. Importantly, our methodology leads to multiple benefits including rigid immobilization, long-term preservation of laboriously-created resources, and the ability to re-interrogate samples.

We developed protocols and designed a custom apparatus to enable embedding of millimeter-scale specimens. We present sample preparation procedures with an emphasis on embedding in acrylic resin and polyimide tubing to achieve rigid immobilization and long-term storage of specimens for the interrogation of tissue architecture and cell morphology by micro-CT.

用微计算机断层扫描法对无节段3D 组织学进行固定染色、全尺寸、毫米度试样的刚性嵌入

For over a hundred years, the histological study of tissues has been the gold standard for medical diagnosis because histology allows all cell types in ...