病毒粒子为<em在体内</em>肿瘤显像
Summary
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。
Abstract
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）。
<p class="jove_content">出发的材料科学家的角度来看，VNPs是有吸引力的积木有以下几个原因：颗粒是单分散的，可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。
Protocol
1。 VNP（CPMV，BMV，PVX和TMV）的传播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％）。 根据表1中的时间线，接种植物。 CPMV PVX，TMV，BMV 0三厂豇豆种子/罐。 0天：植物〜30的本生种子/…
Representative Results
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…
Discussion
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…
Divulgazioni
The authors have nothing to disclose.
Acknowledgements
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 。
Materials
| Material Name | Company | Catalogue number | Comments (optional) |
| VNP production | |||
| Indoor plant chamber | Percival Scientific | E-41L2 | |
| V. unguiculata seeds (California black-eye no. 5) | Burpee | 51771A | |
| N. benthamiana seeds | N. benthamiana seeds were a gift from Salk Institute. Seeds are produced through plant propagation. | ||
| Carborundum | Fisher | C192-500 | |
| Pro-mix BX potting soil | Premier Horticulture | 713400 | |
| Jack’s Professional 20-10-20 Peat-Lite Fertilizer | JR Peters | 77860 | |
| Equipment | |||
| 50.2 Ti rotor | Beckman | 337901 | |
| SW 32 Ti rotor | Beckman | 369694 | |
| Optima L-90K ultracentrifuge | Beckman | 365672 | |
| SLA-3000 rotor | Thermo Scientific | 07149 | |
| SS-34 rotor | Thermo Scientific | 28020 | |
| Sorvall RC-6 Plus centrifuge | Thermo Scientific | 46910 | |
| Polypropylene bottle | Beckman | 355607 | For SLA-3000 rotor |
| Polycarbonate bottle | Beckman | 357002 | For SS-34 rotor |
| Ultra-Clear tube | Beckman | 344058 | For sucrose gradient and SW 32 Ti rotor |
| Polycarbonate bottle | Beckman | 355618 | For pelleting and 50.2 Ti rotor |
| NanoDrop spectrophotometer | Thermo Scientific | NanoDrop2000c | |
| PowerEase 500 pre-cast gel system | Invitrogen | EI8675EU | |
| Superose 6 10/300 GL (24 ml) size-exclusion column | GE Healthcare | 17-5172-01 | |
| ÄKTA Explorer 100 Chromatograph | GE Healthcare | 28-4062-66 | |
| Allegra X-12R | Beckman | 392302 | Benchtop centrifuge |
| Cryostat | Leica | CM1850 | |
| Maestro 2 | Caliper Life Sciences | In vivo imaging system | |
| Tissue-Tearor | Biospec Products | 985370-395 | |
| Microplate reader | Tecan | Infinite-200 | |
| Transmission electron microscope | ZEISS | Libra 200FE | |
| FluoView laser scanning confocal microscope | Olympus | FV1000 | |
| Chemicals and Reagents | |||
| 3-ethynylaniline | Sigma Aldrich | 498289-5G | |
| 384 well black plate | BD Biosciences | 353285 | |
| 4-12% Bis-Tris NuPAGE SDS gel | Invitrogen | NP0321BOX | |
| 4X LDS sample buffer | Invitrogen | NP0008 | |
| Acetic Acid | Fisher | A385-500 | |
| Acetonitrile | Sigma Aldrich | 271004-1L | |
| Alexa Fluor 647 azide | Invitrogen | A10277 | |
| Alexa Fluor 647 carboxylic acid, succinimidyl ester | Invitrogen | A20006 | |
| Amicon Ultra-0.5 ml Centrifugal Filters | Millipore | UFC501096 | 10 kDa cut-off |
| Aminoguanidine hydrochloride | Acros Organics | 36891-0250 | |
| Boric acid | Fisher | A74-500 | |
| Coomassie Brilliant Blue R-250 | Fisher | BP101-25 | |
| CsCl | Acros Organics | 42285-1000 | |
| DAPI | MP Biomedicals | 157574 | |
| Dimethyl sulfoxide | Fisher | BP231-100 | |
| Filter paper | Fisher | 09-801K | P5 grade |
| FITC anti-mouse CD31 | BioLegend | 102406 | |
| Goat serum | Invitrogen | 16210-064 | |
| KCl | Fisher | BP366-500 | |
| L-ascorbic acid sodium salt | Acros Organics | 35268-0050 | |
| Methanol | Fisher | A412P-4 | |
| MgCl2 | Fisher | BP214-500 | |
| Microscope slides | Fisher | 12-544-3 | |
| Microscope cover glass | VWR | 48366-277 | |
| MOPS buffer | Invitrogen | NP0001 | |
| mPEG-mal | Nanocs | PG1-ML-2k | MW 2000 |
| mPEG-N3 | Nanocs | PG1-AZ-5k | MW 5000 |
| mPEG-NHS | Nanocs | PG1-SC-5k | MW 5000 |
| NaCl | Fisher | BP358-212 | |
| Oregon Green 488 succinimidyl ester *6-isomer* | Invitrogen | O-6149 | |
| p-toluenesulfonic acid monohydrate | Acros Organics | 13902-0050 | |
| Permount | Fisher | SP15-100 | |
| Potassium phosphate dibasic | Fisher | BP363-1 | |
| Potassium phosphate monobasic | Fisher | BP362-1 | |
| Sodium acetate | Fisher | BP333-500 | |
| Sodium nitrite | Acros Organics | 42435-0050 | |
| Sodium sulfite | Amresco | 0628-500G | |
| Sucrose | Fisher | S6-500 | |
| TEM grid | Ted Pella | FCF-400Cu | |
| Tris base | Fisher | BP152-500 | |
| Triton X-100 | EMD Chemicals | TX1568-1 | |
| β-mercaptoethanol | Fisher | O3446I-100 | |
| Tissue Culture | |||
| Fetal bovine serum | Invitrogen | 12483-020 | |
| Hemocytometer | Fisher | 0267110 | |
| HT-29 cells | ATCC | HTB-38 | |
| L-glutamine | Invitrogen | 25030-080 | |
| PBS | Cellgro | 21-040-CV | |
| Penicillin-streptomycin | Invitrogen | 10378-016 | |
| RPMI-1640 | Invitrogen | 31800-089 | |
| Tissue culture flasks | Corning | 431080 | 175 cm2 |
| Trypan Blue | Thermo Scientific | SV30084.01 | |
| Trypsin, 0.05% (1X) with EDTA 4Na, liquid | Invitrogen | 25300-054 | |
| Animal Studies | |||
| 18% Protein Rodent Diet | Harlan Teklad | Teklad Global 2018S | Alfalfa free diet |
| Insulin syringe | BD Biosciences | 329410 | 28 gauge |
| Isoflurane | Baxter | AHN3637 | |
| Matrigel Matrix basement membrane | BD Biosciences | 356234 | |
| NCR nu/nu mice | CWRU School of Medicine Athymic Animal and Xenograft Core Facility |
||
| Sterile syringe | BD Biosciences | 305196 | 18 1/2 gauge |
| Tissue-Tek CRYO-OCT Compound | Andwin Scientific | 4583 |
Riferimenti
- Carrillo-Tripp, M., Shepherd, C. M., Borelli, I. A., Venkataraman, S., Lander, G., Natarajan, P., Johnson, J. E., Brooks, C. L., Reddy, V. S. VIPERdb2: an enhanced and web API enabled relational database for structural virology. Nucl. Acids Res. 37, 436-442 (2009).
- Pokorski, J. K., Steinmetz, N. F. The art of engineering viral nanoparticles. Mol. Pharm. 8, 29-43 (2011).
- Steinmetz, N. F., Lin, T., Lomonossoff, G. P., Johnson, J. E. Structure-based engineering of an icosahedral virus for nanomedicine and nanotechnology. Curr. Top Microbiol. Immunol. 327, 23-58 (2009).
- Jung, B., Rao, A. L., Anvari, B. Optical Nano-Constructs Composed of Genome-Depleted Brome Mosaic Virus Doped with a Near Infrared Chromophore for Potential Biomedical Applications. ACS Nano. , (2011).
- Leong, H. S., Steinmetz, N. F., Ablack, A., Destito, G., Zijlstra, A., Stuhlmann, H., Manchester, M., Lewis, J. D. Intravital imaging of embryonic and tumor neovasculature using viral nanoparticles. Nat. Protoc. 5, 1406-1417 (2010).
- Leopold, P. L., Ferris, B., Grinberg, I., Worgall, S., Hackett, N. R., Crystal, R. G. Fluorescent virions: dynamic tracking of the pathway of adenoviral gene transfer vectors in living cells. Hum. Gene Ther. 9, 367-378 (1998).
- Lewis, J. D., Destito, G., Zijlstra, A., Gonzalez, M. J., Quigley, J. P., Manchester, M., Stuhlmann, H. Viral nanoparticles as tools for intravital vascular imaging. Nat. Med. 12, 354-360 (2006).
- Steinmetz, N. F., Ablack, A. L., Hickey, J. L., Ablack, J., Manocha, B., Mymryk, J. S., Luyt, L. G., Lewis, J. D. Intravital imaging of human prostate cancer using viral nanoparticles targeted to gastrin-releasing Peptide receptors. Small. 7, 1664-1672 (2011).
- Wu, C., Barnhill, H., Liang, X., Wang, Q., Jiang, H. A new probe using hybrid virus-dye nanoparticles for near-infrared fluorescence tomography. Optics Communications. 255, 366-374 (2005).
- Steinmetz, N. F., Cho, C. F., Ablack, A., Lewis, J. D., Manchester, M. Cowpea mosaic virus nanoparticles target surface vimentin on cancer cells. Nanomedicine (Lond). 6, 351-364 (2011).
- Maeda, H., Wu, J., Sawa, T., Matsumura, Y., Hori, K. Tumor vascular permeability and the EPR effect in macromolecular therapeutics: a review. Journal of Controlled Release. 65, 271-284 (2000).
- Chatterji, A., Ochoa, W., Paine, M., Ratna, B. R., Johnson, J. E., Lin, T. New addresses on an addressable virus nanoblock: uniquely reactive Lys residues on cowpea mosaic virus. Chem. Biol. 11, 855-863 (2004).
- Steinmetz, N. F., Mertens, M. E., Taurog, R. E., Johnson, J. E., Commandeur, U., Fischer, R., Manchester, M. Potato virus X as a novel platform for potential biomedical applications. Nano Lett. 10, 305-312 (2010).
- Wang, Q., Lin, T., Tang, L., Johnson, J. E., Finn, M. G. Icosahedral virus particles as addressable nanoscale building blocks. Angew. Chem. Int. Ed. 41, 459-462 (2002).
- 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. Surface modification of tobacco mosaic virus with “click” chemistry. Chembiochem. 9, 519-523 (2008).
- Schlick, T. L., Ding, Z., Kovacs, E. W., Francis, M. B. Dual-surface modification of the tobacco mosaic virus. J. Am. Chem. Soc. 127, 3718-3723 (2005).
- Yildiz, I., Tsvetkova, I., Wen, A. M., Shukla, S., Masarapu, M. H., Dragnea, B., Steinmetz, N. F. Engineering of Brome mosaic virus for biomedical applications. RSC Advances. , (2012).
- Brunel, F. M., Lewis, J. D., Destito, G., Steinmetz, N. F., Manchester, M., Stuhlmann, H., Dawson, P. E. Hydrazone ligation strategy to assemble multifunctional viral nanoparticles for cell imaging and tumor targeting. Nano Lett. 10, 1093-1097 (2010).
- Shukla, S., Ablack, A., Wen, A., Lee, K., Lewis, J., Steinmetz, N. F. Increased tumor homing and tissue penetration of the filamentous plant viral nanoparticle Potato virus X. Molecular Pharmaceutics. , (2012).
- Chatterji, A., Ochoa, W., Shamieh, L., Salakian, S. P., Wong, S. M., Clinton, G., Ghosh, P., Lin, T., Johnson, J. E. Chemical conjugation of heterologous proteins on the surface of Cowpea mosaic virus. Bioconjug. Chem. 15, 807-813 (2004).
Tags
NanoparticlesIn Vivo Tumor ImagingNanomaterialsMaterials ScienceMedicinaViral Nanoparticles (VNPs)Plant VirusesCowpea Mosaic Virus (CPMV)Brome Mosaic Virus (BMV)Tobacco Mosaic Virus (TMV)Potato Virus X (PVX)Monodisperse ParticlesLarge-scale ProductionStabilityBiocompatibilityGenetic ModificationChemical Bioconjugation MethodsAtomic Resolution
Citazione di questo articolo
Wen, A. M., Lee, K. L., Yildiz, I., Bruckman, M. A., Shukla, S., Steinmetz, N. F. Viral Nanoparticles for In vivo Tumor Imaging. J. Vis. Exp. (69), e4352, doi:10.3791/4352 (2012).