Ce travail a été soutenu par le NIH / NIBIB subventions R00 EB009105 (pour NFS) et P30 EB011317 (pour NFS), un NIH / NIBIB bourse de formation T32 EB007509 (à AMW), un Case Western Reserve University interdisciplinaire Alliance Investment Grant (pour NFS), et un cas Comprehensive Cancer Center de subvention P30 CA043703 (pour NFS). Nous remercions les chercheurs de laboratoire Steinmetz étudiants de premier cycle pour leur soutien pratique: Nadia Ayat, Kevin Chen, Sourav (Sid) Dey, Alice Yang, Sam Alexander, Craig D&#39;Cruz, Stephen Hern, Lauren Randolph, Brian Alors, et Paul Chariou .

1. VNP (CPMV, BMV, PVX et TMV) Propagation <ol><li> Réglez la chambre de contrôle plante d&#39;intérieur à 15 h du jour (100% de la lumière, 25 ° C, 65% d&#39;humidité) et 9 h de nuit (lumière 0%, 22 ° C, 60% d&#39;humidité). </li><li> Inoculer plantes en fonction de l&#39;échéancier dans le tableau 1. </li></ol><table border="1"><tbody><tr><td> CPMV </td><td> PVX, TMV, et BMV </td></tr><tr><td rowspan="2"> Jour 0: Usine 3 niébé graines / pot. </td><td> Jour 0: Usine ~ 30 N. benthamiana graines / pot. Fertiliser une fois par semaine avec 1 cuillère à soupe d&#39;engrais / 5 L d&#39;eau. </td></tr><tr><td> Jour 14: Re-pot N. benthamiana à 1 plant / pot. </td></tr><tr><td> Jour 10: infecter les feuilles feuilles primaires avec CPMV (5 μg/50 pl / feuille) par inoculation mécanique à l&#39;aide d&#39;une fine couche de carborundum. </td><td> Jour 28: Infect trois à five laisse avec PVX, TMV, ou BMV (5 μg/50 pl / feuille) par inoculation mécanique à l&#39;aide d&#39;une fine couche de carborundum. </td></tr><tr><td> Feuilles de récolte et de stocker à -80 ° C: Jour 20 </td><td> Feuilles de récolte et de stocker à -80 ° C: Jour 42 </td></tr></tbody></table> Tableau 1. Calendrier de plus en plus, l&#39;infection et la récolte des feuilles. Remarque: seule la propagation CPMV est démontrée à titre d&#39;exemple. 2. VNP (CPMV, BMV, PVX et TMV) Purification Remarque: Toutes les étapes sont effectuées sur de la glace ou à 4 ° C. <ol><li> Homogénéiser 100 g de feuilles congelées dans un mélangeur standard à l&#39;aide de 2 volumes de tampon froid (voir le tableau 2). Filtrer à travers 2-3 couches de gaze. </li><li> Pour PVX, ajuster le pH à 6,5 avec 1 M HCl. Ajouter 0,2% (p / v) d&#39;acide ascorbique et 0,2% de sodium (p / v) sulfite. </li><li> Centrifuger homogénat de plante brute à 5500 xg pendant 20 min. Recueillir le surnageant. </li><li> Pour BMV, couche 25 ml de surnageant au-dessus de 5 ml de 10% (p / v) une solution de saccharose. Centrifugeuse à 9.000 g pendant pastilles h et remettre en suspension dans une solution de CsCl 3 38,5% (p / v). Mélanger en agitant pendant 5 heures, puis passez à l&#39;étape 2.12. </li><li> Extraire la matière végétale par ajout de 0,7 volumes de 1:1 (v / v) chloroforme :1-butanol. Incorporer le mélange pendant 30-60 min. </li><li> Centrifuger à 5500 xg solution pendant 20 min. Recueillir la phase aqueuse supérieure. </li><li> Ajouter du NaCl à 0,2 M et 8% (p / v) de PEG (PM 8.000). Pour TMV, ajouter 1% (v / v) de Triton X-100. Agiter pendant au moins 1 heure, puis laisser reposer au moins 1 heure. </li><li> Centrifuger la solution à 15.000 xg pendant 15 min. Resuspendre le culot dans 10 ml de tampon. Pour PVX, ajouter 0,1% β-mercaptoéthanol et de l&#39;urée à 0,5 M. </li><li> Centrifuger à 8000 xg pendant 30 minutes et recueillir le surnageant. </li><li> Ultracentrifugeuse surnageant à 160.000 xg pendant 3 heures. Resuspendre le culot dans 5 ml de tampon overnight. </li><li> Préparer un gradient de saccharose 10-40% en utilisant des volumes égaux de 10%, 20%, 30% et 40% de saccharose dans le tampon (plus lourd en premier). Laissez le gradient de s&#39;équilibrer pendant la nuit à température ambiante. </li><li> Ultracentrifugeuse culot remis en suspension sur gradient de saccharose à 100.000 xg pendant 2 h (24 h pour le BMV). </li><li> Recueillir bande diffusion de la lumière et de dialyser contre du tampon. </li><li> Caractériser les VNPs (ci-dessous) et conserver à 4 ° C. Pour stockage à long terme, stocker à -80 ° C. </li></ol><table border="1"><tbody><tr><td> CPMV et TMV </td><td> 0,1 M de tampon phosphate de potassium (pH 7,0) 38,5 mM KH 2 PO 4 61,5 mM de K 2 HPO 4 </td></tr><tr><td> PVX </td><td> 0,5 M tampon borate (pH 7,8) 0,5 M d&#39;acide borique Ajuster le pH avec NaOH </td></tr><tr><td> BMV </td><td> SAMA tampon (pH 4,5) 250 mM d&#39;acétate de sodium MgCl2 10 mM 2 mM de β-mercaptoéthanol (ajouter frais) </td></tr></tbody></table> Tableau 2. Tampons et leurs recettes pour chaque VNP. Remarque: seule la propagation CPMV est démontrée à titre d&#39;exemple. 3. VNP (CPMV, BMV, PVX et TMV) Caractérisation <ol><li> Effectuer la spectroscopie UV / visible de déterminer la concentration de VNPs. <ol><li> Mesurer l&#39;absorbance de 2 ul d&#39;échantillon à l&#39;aide d&#39;un spectrophotomètre NanoDrop. </li><li> Déterminer la concentration de particules et de colorants à l&#39;aide de la loi de Beer-Lambert (A = εcl, où A est l&#39;absorbance, ε est le coefficient d&#39;extinction, c est la concentration, et l est la longueur du trajet). La longueur du trajet est de 0,1 cm pour le NanoDrop. Les coefficients d&#39;extinction VNP-spécifiques sont les suivants: CPMV: 8,1 cm -1 mg -1 ml (à 260 nm) PVX: 2,97 cm -1 mg -1 ml (à 260 nm) TMV: 3,0 cm -1 mg -1 ml (à 260 nm) BMV: 5,15 cm -1 mg -1 ml (à 260 nm) </li></ol></li><li> Analyser des particules par chromatographie d&#39;exclusion stérique des protéines liquide rapide (FPLC). <ol><li> L&#39;utilisation d&#39;un Superose 6 exclusion de taille de colonne et l&#39;explorateur ÄKTA, charge de 50 à 100 mg de VNPs dans 200 ul de 0,1 M de tampon phosphate de potassium (pH 7,0). </li><li> Définir détecteurs à 260 nm (acide nucléique), 280 nm (protéines) et la longueur d&#39;onde d&#39;excitation de tous les colorants fixés. </li><li> Fonctionner à un débit de 0,5 ml / min pendant 72 min. </li><li> Le profil d&#39;élution et A260: A280 nm indique si la préparation VNP est pur et si les particules sont intacts et assemblé. L&#39;A260 suivante: 280 ratios indiquent une préparation VNP pur: CPMV: 1,8 ± 0,1 PVX: 1,2 ± 0,1 TMV:1,1 ± 0,1 BMV: 1,7 ± ​​0,1 </li></ol></li><li> Effectuer dénaturant (préfabriqué NuPAGE) Bis-Tris Polyacrylamide électrophorèse sur gel en gradient 4-12% d&#39;analyser la pureté de la préparation et de la conjugaison de protéines d&#39;enveloppe individuels. <ol><li> Ajouter 3 ml de tampon d&#39;échantillon LDS 4x à 10 ug de particules dans 9 pi de tampon phosphate de potassium. Ajouter un supplément de 1 ul de tampon d&#39;échantillon LDS 4x et 3 pl de β-mercaptoéthanol à BMV pour réduire le nombre élevé de liaisons disulfures. </li><li> Incuber dans un bloc chauffant pendant 5 min à 100 ° C. </li><li> Charger les échantillons sur un gel SDS. </li><li> Exécuter des échantillons à 200 V pendant 1 heure dans du tampon MOPS 1x. </li><li> Documenter le gel sous lumière UV pour visualiser les protéines fluorescentes manteau. </li><li> En cas de non-protéine fluorescente, coloration au bleu de Coomassie (0,25% (p / v) de bleu de Coomassie R-250, l&#39;acide 30% (v / v) de méthanol, 10% (v / v) acétique) pendant 1 heure. </li><li> Décolorer avec du méthanol 30%, 10% d&#39;acide acétique pendant une nuit. Change de la solution si nécessaire. </li><li> Documenter le gel sous lumière blanche. </li></ol></li><li> Analyser l&#39;intégrité des particules par microscopie électronique à transmission (MET). <ol><li> Diluer les échantillons à 0,1-1 mg / ml dans 20 pl d&#39;eau DI. </li><li> Placez gouttes de 20 ul des échantillons sur Parafilm. </li><li> Couvrir gouttes avec une grille de MET et laisser reposer pendant 2 min. Évacuent l&#39;excès de solution sur la grille d&#39;un papier filtre. </li><li> Laver la grille en plaçant sur une goutte d&#39;eau DI, puis mèche sèche. </li><li> Tache grille en plaçant une goutte de 20 pl de 2% (p / v) de l&#39;acétate d&#39;uranyle pendant 2 min. Évacuent l&#39;excès tache avec un papier filtre. </li><li> Laver la grille une fois de plus dans l&#39;eau. </li><li> Observez la grille sous un microscope électronique à transmission. </li></ol></li></ol> 4. Conjugaison chimique de VNPs avec le PEG et fluorophores, de purification et de caractérisation <ol><li> Pour les calculs pour les réactions ci-dessous, la masse molaire des VNPs sont: CPMV: 5.6x 10 6 g / mol PVX: 35 x 10 6 g / mol TMV: 41 x 10 6 g / mol BMV: 4,6 x 10 6 g / mol </li><li> Conjuguer les colorants et les lysines PEG à la surface de CPMV et PVX en utilisant une seule étape de N-hydroxy succinimide réaction de couplage: Ajouter 2.500 équivalents molaires (tous les excès molaires se référer à un excès molaire par VNP) de Alexa Fluor 647 ester de succinimidyle et 4.500 équivalents de NHS- PEG (MW 5.000) dissous dans du DMSO à CPMV dans 0,1 M de tampon phosphate de potassium. Lorsque vous travaillez avec le PVX, ajouter un excès molaire de 10.000 NHS-colorant et NHS-PEG. Régler les volumes de tampon et le DMSO tels que la concentration finale de CPMV et PVX est de 2 mg / ml et la teneur en DMSO est 10% du volume réactionnel total. Incuber le mélange réactionnel pendant une nuit à température ambiante à l&#39;abri de la lumière. CPMV et PVX ont 300 et 1270 lysines adressables, respectivement. (Le lecteur est renvoyé aux références suivantes pour en savoir plus surmodification chimique de CPMV et PVX: 13-15). </li><li> Des colorants et des conjugués de PEG à tyrosines du TMV par couplage de diazonium suivi par le cuivre (I)-catalysée azide-alcyne cycloaddition. <ol><li> Préparer sel de diazonium (alcyne) en mélangeant 400 ul de 0,3 M monohydrate d&#39;acide p-toluène, 25 ul de nitrite de sodium 3,0 M et 75 ul de 0,68 M distillée 3-éthynylaniline dissous dans l&#39;acétonitrile à 4 ° C pendant 1 heure. </li><li> Ajouter 3,3 ml de tampon borate, pH 8,8, contenant 100 mM de NaCl à 1,25 ml de TMV (20 mg / ml de solution mère). </li><li> Réagir à l&#39;TMV 450 ul du sel (alcyne) solution de diazonium dans un bain de glace pendant 3 heures pour ajouter une ligature alcyne poignée de TMV par couplage de diazonium. La solution va se transformer en une couleur brun clair. TMV a 2140 tyrosines disponibles pour la conjugaison. </li><li> Purifier le produit final en utilisant un coussin de sucrose comme décrit à l&#39;étape 4.4. </li><li> Fixez azide fonctionnelle Alexa Fluor 647 et PEG-azide (MW 5000) using de cuivre (I)-catalysée azide-alcyne cycloaddition (CuAAC). Ajouter 2 équivalents d&#39;un colorant et de PEG-protéine d&#39;enveloppe par l&#39;azide et l&#39;incuber avec 1 mM de CuSO 4, 2 mM AMG, et 2 mM de l&#39;ascorbate de sodium à température ambiante pendant 15 min. Régler le volume tampon de telle sorte que la concentration finale de réaction est de TMV 2 mg / ml. (Le lecteur est renvoyé aux références suivantes pour en savoir plus sur la modification chimique des TMV: 16,17). </li></ol></li><li> Conjuguer colorants à lysines et de PEG à cystéines de la cystéine BMV mutant (cBMV): <ol><li> Ajouter 2.000 équivalents molaires de l&#39;Oregon Green 488 succinimidylester dissous dans du DMSO à cBMV dans 0,1 M de tampon TNKM (50 mM Tris base, 50 mM de NaCl, KCl 10 mM, MgCl2 5 mM, pH 7,4). Régler les volumes de tampon et le DMSO tels que la concentration finale de BMV est de 1 mg / ml et la teneur en DMSO est 10% du volume réactionnel total. Incuber le mélange réactionnel pendant une nuit à 4 ° C à l&#39;abri de la lumière. </li><li> Purifier les particules à l&#39;aide centrifiltres fugue comme décrit à l&#39;étape 4.4. </li><li> Ajouter un excès molaire de 2.000 PEG-maléimide (MW 2.000) en utilisant les mêmes conditions de réaction que précédemment et incuber le mélange réactionnel pendant 2 heures à 4 ° C. cBMV dispose de 180 lysines réactifs et cystéines. (Le lecteur est renvoyé aux références suivantes pour en savoir plus sur la modification chimique de BMV: 18). </li></ol></li><li> Purification: Faire passer la solution à travers un coussin de saccharose à 40% (p / v) à 160.000 xg pendant 2,5 heures. Re-dissoudre le culot dans un tampon. Alternativement, on dialyse contre un tampon approprié en utilisant 10 filtres kDa de spin cut-off. </li><li> Caractérisation: VNPs PEGylées et marquée par fluorescence sont analysées en utilisant les méthodes décrites ci-dessus: UV / visible spectroscopie, électrophorèse sur gel SDS, FPLC, et TEM (non montré, cependant, se référer aux figures 6 et 7). </li></ol> 5. Le ciblage de la tumeur et d&#39;imagerie en utilisant un modèle de xénogreffe de souris<ol><li> Culture HT-29 des cellules humaines de cancer du côlon dans le milieu RPMI supplémenté avec 5% de FBS, 1% de pénicilline-streptomycine et 1% de L-glutamine à 37 ° C dans 5% de CO 2 en utilisant 175 cm 2 flacons de culture cellulaire. </li><li> Laver les cellules deux fois avec du PBS stérile et la récolte par incubation avec 5 ml de trypsine-EDTA à 37 ° C pendant 5 min. Inactiver la trypsine avec 5 ml de milieu RPMI. Recueillir les cellules par centrifugation à 500 xg pendant 5 min. à 4 ° C et remettre en suspension dans RPMI frais à 5x10 6 cellules/50 ul de milieu (déterminer le nombre total de cellules en utilisant du bleu trypan et un hémocytomètre). Mélanger avec un volume égal de matrigel avant l&#39;injection (garder toutes les solutions et les réactifs stériles). </li><li> Achat des vieilles de six semaines NCR souris nu / nu et les maintenir à un régime de luzerne gratuit pendant 2 semaines. [Note:. Toutes les procédures sur les animaux doit être approuvé IACUC] Provoquer des xénogreffes de tumeurs par injection sous-cutanée de 5x10 6 cellules/100 ul / tumeur dans les flancs (2 tumeurs / souris) à l&#39;aide d&#39;une jauge de 18 1/2 stérileaiguille. Surveiller régulièrement les animaux. Mesurer la taille de la tumeur à l&#39;aide des étriers et de permettre aux tumeurs de se développer à un volume moyen de 20 mm 3 (dans les 12 prochains jours). Attribuer des souris à deux groupes différents au hasard: PBS et VNP (n = 3 animaux / groupe / l&#39;heure du point). En utilisant un 1 ml 28 seringue à insuline de calibre, administrer par injection intraveineuse dans la veine caudale 100 pi de PBS stérile ou 10 mg / kg de formulation VNP. </li></ol> Remarque: les expériences de culture de tissus et d&#39;études avec des animaux vivants ne sera pas démontrée. Démonstration pratique sera limitée au traitement des tissus et d&#39;acquisition de données. Pour une référence sur le HT-29 modèle de xénogreffe de tumeur, le lecteur est renvoyé à la réf 19. Trois techniques sont utilisées pour évaluer la tumeur ralliement de VNPs: <ol start="4"><li> L&#39;imagerie par fluorescence à l&#39;aide du système d&#39;imagerie Maestro: Sacrifice souris à différents moments (2, 24, et 72 h) en utilisant le CO 2gaz. Disséquer les animaux et accises tous les principaux organes (cerveau, cœur, poumons, la rate, les reins et le foie) ainsi que les tumeurs sur les flancs, placer les tissus sur du parafilm, et analyser avec l&#39;instrument d&#39;imagerie de fluorescence par excitation jaune et filtres d&#39;émission (800 ms exposition) pour détecter la présence de signaux de fluorescence dans des tissus provenant de A647 (marqueur conjugué à la VNPs). Enregistrez les images et d&#39;analyser les intensités de fluorescence à l&#39;aide du logiciel ImageJ 1.44o ( <a href="http://imagej.nih.gov/ij" target="_blank">http://imagej.nih.gov/ij</a> ). Comparez le motif de l&#39;absorption des VNPs dans les tumeurs avec d&#39;autres tissus importants avec le temps. </li><li> Après l&#39;imagerie, couper chaque tissu en deux et incorporez la moitié dans du composé OCT pour l&#39;analyse cryo-coupes et confocale. Recueillir l&#39;autre moitié en pré-pesés cryo-flacons et geler immédiatement les utiliser N 2 liquide. Conserver à -80 ° C jusqu&#39;au moment de transformation. </li><li> Quantification de fluorescence: Recordon tissus poids. Décongeler les tissus congelés à la température ambiante et les placer dans différents tubes Falcon de 50 ml contenant 1 ml de PBS. En utilisant un homogénéisateur de tissus de poche, d&#39;homogénéiser les tissus pour 2-3 min dans du PBS puis transférer le broyat de microtubes. Centrifuger les homogénats pendant 10 min à 13000 xg pour éliminer les tissus non homogénéisé. </li><li> Pipeter 100 ul du surnageant à partir des tissus de chaque groupe (formulations PBS et VNP / points dans le temps) dans une plaque de 384 puits noir UV. Évaluer l&#39;intensité de fluorescence (Ex / Em longueurs d&#39;onde 600/665) en utilisant un lecteur de plaque. Normaliser les valeurs obtenues par les poids fluorescentes tissus. </li><li> Immunohistochimie: cryo-microtome Préparer sections (10 mm) et conserver à -20 ° C. Colorer les coupes de tissus pour les noyaux cellulaires (DAPI) et le marqueur des cellules endothéliales (marqué au FITC anti-souris CD31 anticorps). Effectuer des analyses de microscopie confocale pour cartographier la localisation vasculaire et intra-tumorale marquée par fluorescence de VNPs. </li></ol> Remarque: Cette procédure ne sera pas démontrée, les données représentatives sont présentés dans la figure 8. Pour une référence sur l&#39;immunohistochimie et les méthodes de coloration décrites, le lecteur est renvoyé à la réf 19.

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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>

Aucun conflit d&#39;intérêt déclaré.

Ce protocole fournit une approche pour la modification chimique de VNPs et leurs applications pour l&#39;imagerie in vivo de tumeurs. Les techniques d&#39;imagerie de fluorescence des animaux, la quantification de fluorescence, et l&#39;immunohistochimie présentés ici sont utiles pour étudier la biodistribution et l&#39;évaluation de la tumeur de ralliement. Ces techniques fournissent des informations précieuses concernant l&#39;accès des nanoparticules dans la tumeur via l&#39;effet EPR. En comb...

<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

L&#39;utilisation des nanomatériaux a le potentiel de révolutionner la science des matériaux et de la médecine. À l&#39;heure actuelle, un certain nombre de différentes nanoparticules sont étudiés pour des applications en imagerie et la thérapie. Nanoparticules virales (VNPs) dérivés de plantes peut être considérée comme auto-assemblées bionanomatériaux avec les tailles et formes définies. Les virus des plantes à l&#39;étude dans le laboratoire Steinmetz comprendre des particules icosaédriques formés par virus de la mosaïque du niébé (CPMV) et virus de la mosaïque de Brome (BMV), qui sont tous deux à 30 nm de diamètre. Nous développons également des structures en forme de tige et filamenteux provenant des virus végétaux suivants: virus de la mosaïque du tabac (TMV), qui forme des tiges rigides avec des dimensions de 300 nm 18 nm par le virus de la pomme de terre, et X (PVX), qui forment des particules filamenteuses 515 nm de longueur et de 13 nm de largeur (le lecteur est renvoyé aux refs. 1 et 2 pour de plus amples informations sur VNPs). <p class="jove_content"&gt; Du point un scientifique des matériaux de vue, VNPs sont des éléments attractifs pour plusieurs raisons: les particules sont monodisperses, peut être produit avec facilité sur une grande échelle dans la plante, sont exceptionnellement stable et biocompatible. En outre, VNPs sont «programmables» des unités, qui peuvent être spécialement conçus en utilisant des méthodes de modification génétique ou bioconjugaison chimiques 3. La structure de VNPs est connu pour une résolution atomique, et des modifications peuvent être effectuées avec une précision spatiale à l&#39;échelle atomique 4, un niveau de contrôle qui ne peut pas être réalisé en utilisant des nanomatériaux synthétiques actuels avec state-of-the-art technologies. Dans cet article, nous décrivons la propagation de CPMV, PVX, TMV, et BMV en Vigna ungiuculata et Nicotiana benthamiana. Protocoles d&#39;extraction et de purification pour chaque VNP sont donnés. Méthodes de caractérisation des purifiées et chimiquement VNPs marqués sont décrits. Dans cette étude, nous nous concentrons sur chétiquetage des emical VNPs avec fluorophores (par exemple Alexa Fluor 647) et de polyéthylène glycol (PEG). Les colorants de faciliter le suivi et la détection des VNPs 5-10, et PEG réduit immunogénicité des nanoparticules protéiques tout en améliorant leurs propriétés pharmacocinétiques 8,11. Nous démontrons tumeur ralliement de VNPs PEGylées aide d&#39;un modèle de xénogreffe de tumeur de souris. Une combinaison de l&#39;imagerie de fluorescence des tissus ex vivo avec une carte Maestro du système d&#39;imagerie, la quantification de fluorescence dans les tissus homogénéisés et la microscopie confocale est utilisée pour étudier la biodistribution. VNPs sont effacées par le système réticulo-endothélial (RES); tumeur homing est atteint passive par une perméabilité accrue et de rétention (EPR) Effet 12. La nanotechnologie VNP est un puissant plug-and-play à l&#39;image de la technologie et de traiter les sites de la maladie in vivo. Nous développons davantage VNPs d&#39;effectuer des cargaisons de drogue et des fragments d&#39;imagerie clinique pertinents, ainsi que des ligands spécifiques d&#39;un tissu àcibler des récepteurs moléculaires surexprimés dans le cancer et les maladies cardiovasculaires.

<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

Nanoparticules virales végétales (VNPs) sont prometteurs plates-formes pour des applications dans le domaine biomédical. Ici, nous décrivons les procédures pour les installations VNP propagation, la purification, la caractérisation et la bioconjugaison. Enfin, nous montrons l&#39;application de VNPs prise d&#39;origine tumorale et d&#39;imagerie en utilisant un modèle de xénogreffe de souris et l&#39;imagerie de fluorescence.

Les nanoparticules virales pour<em&gt; In vivo</em&gt; Imagerie des tumeurs

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 vues.  Case Western Reserve University. Nanoparticules virales végétales (VNPs) sont prometteurs plates-formes pour des applications dans le domaine biomédical. Ici, nous décrivons les procédures pour les installations VNP propagation, la purification, la caractérisation et la bioconjugaison. Enfin, nous montrons l'application de VNPs prise d'origine tumorale et d'imagerie en utilisant un modèle de xénogreffe de souris et l'imagerie de fluorescence.

Vidéo: 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.

Simple Dispositifs microfluidiques pour<em&gt; In vivo</em&gt; Imagerie des<em&gt; C. elegans</em&gt;,<em&gt; Drosophila</em&gt; Et le poisson-zèbre

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

Recherche

Bio-ingénierie

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.

Analyse de la protéine virale ciblée nanoparticules administrées à HER2 + Tumeurs

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.

Imagerie collagène dénaturé Strands<em&gt; In vivo</em&gt; Et<em&gt; Ex vivo</em&gt; Par photo-déclenchée hybridation de Caged collagène Mimetic peptides

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 bleu de Prusse nanoparticules pour l&#39;imagerie moléculaire multimodale Applications

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.

Synthèse entraînée par micro-ondes de l&#39;oxyde de fer Nanoparticules pour la détection rapide de l&#39;athérosclérose

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.

Visualisation de l’angiogenèse par microscopie Multiphoton In Vivo en échafaudage de 3D-PLGA/PNVS génétiquement modifiés pour la réparation osseuse défaut critique substance

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.

Évaluation initiale des anticorps-conjugués modifié avec Peptides viraux dérivés pour accroître l’Accumulation cellulaire et améliorer le ciblage tumoral

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.

Corrélatives microscopie électronique léger (CLEM) de suivi et d’imagerie protéine virale associés Structures des cellules immobilisées Cryo

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.

Rigide incrustation du fixe et tachée, ensemble, échelle millimétrique spécimens pour histologie exempt de Section 3D de 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 ...