Diese Arbeit wurde unterstützt durch Zuschüsse zu LKM-K von den National Institutes of Health / National Cancer Institute (R01CA129822 und R01CA140995) finanziert. Dr. Medina-Kauwe dankt C. Rey, M. M-Kauwe und D. Revetto für anhaltende Unterstützung.

1. Systemische Lieferung In vivo <ol><li> Mischen genug mit steriler Kochsalzlösung HerDox bis 0,2 ml einer 0,004 mg / kg Dosis HerDox pro Injektion eine 6-8 Wochen alten nu / nu-Maus, die subkutan bilateral Flanke Xenograft gleichzusetzen. </li><li> Vorsichtig ziehen die HerDox Mischung in eine 3/10 cc Insulinspritze mit einer 29G Nadel ausgestattet Blasenbildung vermeiden. </li><li> 0,5-1 L / min, Isoflurankonzentration: 3-4% (oder weniger) Anästhesie wird durch kurze Exposition Isofluran in einer Induktion Kammer mit einem Gas Spülsystem (Oxygen Flussraten ausgestattet induziert. </li><li> Injizieren Sie die gesamte Mischung in die Schwanzvene einer narkotisierten Maus (0,2 ml pro Injektion). IV Injektion kann auch in einer zurückhaltenden, narkotisierten Maus durchgeführt werden. </li><li> Wiederholen Injektionen am selben Maus für sechs aufeinander folgenden Tagen, einmal pro Tag. </li></ol> 2. Fluorescence Imaging in vivo Die Anhäufung von HerDox Fluoreszenz in Tumorenwerden bis zum letzten Tag der Injektion (Tag 7) mit einem Multimode-Imager nachweisbar. Die folgenden Verfahren beinhalten die Verwendung eines kundenspezifischen Makro-Beleuchtung und Detektion (Abbildung 1) 8. <ol><li> Schalten Sie den Multimode Optical In vivo Imager. </li><li> Wählen Sie eine Emission Bandpassfilter (590 nm ± 30 nm) für Doxorubicin Fluoreszenz-Detektion geeignet. </li><li> Schalten Sie Argon-Krypton-Laser und platzieren Sie eine Anregung Bandpassfilter (488 nm ± 10 nm) bei der Laser-Strahlengang. </li><li> Schalten Sie den Anästhesie-System und legen Sie dann eine Maus in die betäubende Kammer (Oxygen Flussraten: 0,5-1 L / min, Isoflurankonzentration: 3-4% (oder weniger) mit einem Gas-Spülung ausgestattet. </li><li> Übertragen Sie die Maus aus der betäubende Kammer zur Bildgebung Kammer des Multimode Optical In-vivo-Imager, wenn die Maus narkotisiert ist. </li><li> Legen Sie eine nosecone über die Nase der Maus und öffnen Sie den Durchfluss auf kontinuierliche Narkosemitteln verwaltensia während der Bildaufnahme (Oxygen Flussraten: 0,5-1 L / min, Isoflurankonzentration: 2-3% (oder weniger). </li><li> Erwerben Fluoreszenz-Aufnahmen mit einer Belichtungszeit von 5-15 sec. </li><li> Führen Bildanalyse und-verarbeitung einschließlich Hintergrund Korrektur oder Kontrast. </li></ol> 3. Ex vivo Fluoreszenz-Imaging HerDox Fluoreszenz in Tumoren und bestimmte Organe abgebildet werden (einschließlich der Leber, Niere, Milz, Herz-und Skelettmuskel) von eingeschläfert Mäuse nach 24 Stunden geerntet nach der letzten (Tag 7) Injektion von HerDox. <ol><li> Schalten Sie den Multimode Optical In vivo Imager. </li><li> Wählen einer Emission Bandpassfilter (580 nm ± 20 nm) für Doxorubicin Fluoreszenzdetektion. </li><li> Schalten Sie Argon-Krypton-Laser und platzieren Sie eine Anregung Bandpassfilter (488 nm ± 10 nm) bei der Laser-Strahlengang. </li><li> Platz Tumoren und bestimmten Organen auf einer Petri-Schale in die Kammer o imaging angeordnetf die Multimode Optical In vivo Imager. </li><li> Erwerben Fluoreszenz-Aufnahmen von Geweben mit einer Belichtungszeit von 5-15 sec. Ein Beispiel für anfängliche Fluoreszenz Bildaufnahme ist in Abbildung 2a dargestellt. Wiederholen Sie das gleiche mit einem leeren Petrischale, die als Hintergrund (Abbildung 2b) dienen wird. </li><li> Führen Bildanalyse und-verarbeitung einschließlich Hintergrund Korrektur oder Kontrast. Ein Beispiel für eine korrigierte Bild wird in 2c dargestellt, aus der Subtraktion von 2b aus Figur 2a. </li></ol> 4. In situ konfokale Bildgebung von Tumoren In situ konfokale Abbildung ermöglicht die Erfassung und Analyse von HerDox Tumorakkumulation auf zellulärer Ebene. <ol><li> Schalten Sie eine Leica SPE konfokalen Mikroskop. </li><li> Wählen Sie 488 nm Laserlicht zur Anregung von Doxorubicin und Emissions-Wellenlängen (560-620 nm) für DoxorubicinFluoreszenzdetektion. </li><li> Wählen Sie einen 40X oder 63X objektiv und Tropfen Immersionsöl auf dem Objektiv. </li><li> Auszug frisch Tumoren von Mäusen, die eingeschläfert zuvor empfangenen HerDox und Mock Behandlungen wie in Verfahren 2 beschrieben. </li><li> Platz Tumoren auf einer Petrischale auf Eis zu vermeiden Gewebeabbau und übertragen dann die Tumoren zu einem Delta T Kammer für die konfokale Bildgebung. </li><li> Erwerben konfokale Bilder der Tumoren bei sequentiellen Fokustiefen (Schrittweite: 1 um, Dicke: 20 um). Ein Beispiel nacheinander aufgenommenen Bildern entlang der z-Achse ist in Fig. 3 linken Seite gezeigt. </li><li> Führen maximale Intensität z-Projektion der Bilder. Ein Maximum Intensity Projection der z-gestapelten Bilder ist in Fig. 3, rechte Tafel gezeigt. </li><li> Berechnung der mittleren Fluoreszenzintensitäten der maximalen Intensität Z -. Projektion Bilder Mittlere Fluoreszenzintensitäten der Bilder über das gesamte Sichtfeld wurden berechnetd mit ImageJ. </li></ol> 5. Ratiometric Spectral Imaging and Analysis Ratiometric spektralen Bildgebung und Analyse ermöglicht Diskriminierung zwischen Dox Fluoreszenz und Autofluoreszenz. <ol><li> Leistung auf einem Laser-Scanning-Fluoreszenz-konfokalen Mikroskop. </li><li> Erwerben 15 Bilder der HerDox-behandelten und unbehandelten Tumoren in einer bestimmten Tiefe im Spektralbereich von 510-650 nm, mit einer Schrittweite von 10 nm und Anregung bei 488 nm Licht unter Verwendung eines Leica SPE konfokalen Mikroskop. </li><li> Bereiten Sie eine 100 uM Lösung von Doxorubicin. </li><li> Führen spektralen Bildgebung des 100 pM Doxorubicin-Lösung, um das reine spektrale Signatur von Dox Fluoreszenz (Spektralbereich: 510-650 nm, ein Schritt Größe: 10 nm) zu erhalten. Typische Ergebnisse der Bildaufnahme von Spectral Imaging und die daraus resultierende Fluoreszenzspektrum aufgetragen als ein Graph in Abbildung 4 dargestellt. </li><li> Erwerben Sie die Autofluoreszenz spektrale Signatur von einem Bild cube (Spektralbereich: 510-650 nm, ein Schritt: 10 nm) von Spectral Imaging von unbehandelten Tumoren erhalten. Typische Ergebnisse der Bildaufnahme von Spectral Imaging und die daraus resultierende Fluoreszenzspektrum aufgetragen als ein Graph in Abbildung 5 dargestellt. </li><li> Generieren vier Referenz spektralen Signaturen (reine Autofluoreszenz 0.1.doxorubin +0.9. Autofluoreszenz, 0.2. Doxorubicin +0.8. Autofluoreszenz 0.3.doxorubin +0.7. Autofluoreszenz) mit dem Programm, das wir entwickelt 9. Eine typische Kurve vier Referenz spektralen Signaturen ist in Abbildung 6 dargestellt. </li><li> Führen spektrale Klassifizierung der Bilder durch den Verweis spektralen Signaturen durch Euklidische Distanzmaß mit dem Programm, das wir zuvor entwickelten 9 definiert. </li><li> Die lineare Entmischung dieser Bilder durch die Verwendung eines Entmischung Programm (Plug-in in ImageJ) entwickelten wir 7, 10, zum Vergleich mit der ratiometrischen Spectral Imaging und Analyse. Ein example der Trennung HerDox Fluoreszenz von Autofluoreszenz durch lineare Entmischung ist in Abbildung 7 dargestellt. </li></ol>

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Biochem. Eng. Biotechnol. , 95245-95265 (2005).</li></ol>

Der Autor, Daniel Farkas, ist Vorsitzender des Spectral Molecular Imaging. Die übrigen Autoren haben keine Interessenkonflikte.

Dox Fluoreszenz in vivo nachweisbar sein mit der Multimode-Imager, wenn Tumoren subkutane sind. Allerdings ist die therapeutisch wirksame Dosis von HerDox (0,004 mg / kg) unterhalb der Nachweisgrenze nach einer Einzeldosis. Im Gegensatz dazu, nach 7 tägliche Injektionen (1x/day für 7 Tage), ist der Tumor Akkumulation und Retention der Partikel ausreicht, um die Visualisierung von Dox Fluoreszenz ermöglichen. Es ist wichtig bei der Arbeit mit Dox oder einem anderen Fluorophor für...

Tumor-Targeting der Chemotherapie hat das Potenzial, Krebszellen zu niedrigeren Dosis im Vergleich zu beseitigen ungezielte Drogen, weil mehrere gelieferte Therapie am Bestimmungsort anstatt zu verteilen, um Nicht-Tumorgewebe ansammeln können. Da dieser Situation verdünnen sich die Wirksamkeit des Arzneimittels und somit höhere Dosen wirksam zu sein, hat Tumor-Targeting sowohl therapeutische als auch sicherheitstechnische Vorteile gegenüber herkömmlichen nicht-zielgerichtete Behandlung. Targeting Chemotherapie durch Verkapselung in selbstorganisierte Nanopartikel ermöglicht das Medikament zu bleiben chemisch im Gegensatz zu Medikamenten, die kovalent an Targeting-Moleküle gebunden sind unverändert. Als solche Verbindung hat das Potential, die Aktivität von sowohl dem Wirkstoff und dem Zielmolekül zu ändern; nichtkovalente Anordnung ermöglicht Arzneistoffwirksamkeit beibehalten werden. Wir haben bereits gezeigt, dass das neue Drei-Komponenten, self-assembled komplex, HerDox, HER2 + Tumoren zielt <em&gt; In vivo und entlockt Tumor-Wachstum Ablation unter Schonung gesunden Gewebes, einschließlich des Herzens 1. HerDox durch nicht-kovalente Wechselwirkungen zwischen den Rezeptor-Bindungs-Zelle Penetrationsprotein, HerPBK10 und des chemotherapeutischen Mittels gebildet, Doxorubicin (DOX), über eine kleine Nukleinsäure Linker. HerPBK10 bindet den humanen epidermalen Wachstumsfaktor-Rezeptor (HER) und löst Rezeptor-vermittelte Endozytose 2-4, während endosomalen Membran Penetration durch Einbau des Adenovirus-abgeleiteten Pentonbasis Kapsidprotein 4-6 erreicht wird. Eine positiv geladene Domäne des Proteins ermöglicht Nukleinsäurebindung 4, 5, durch die DNA-interkalierten Dox zum gezielten Transport transportiert werden können. Elektrophile, Einlagerung und möglicherweise Protein-Interaktionen erleichtern Oligomerisierung Selbstorganisation zu rund 10-20 nm großen Partikeln, die stabil sind in Blut und unter längerer Lagerung bei unterschiedlichen Temperaturen 1. Bevorzugte Targeting HER2 + Tumorzellen durch die erhöhte Ligandenaffinität erleichtert, wenn HER2 erhöht ist. Unsere bisherigen Untersuchungen haben gezeigt, dass die systemische Verabreichung von HerDox Ausbeuten bevorzugte Anreicherung in Tumoren über Nicht-Tumorgewebe und im Vergleich zu Dox 1 und Eindringen in Tumorzellen ungezielte in vivo 7. Wir haben beobachtet, dass HerDox Veröffentlichungen Dox nach Tumorzelle Eintrag, so dass Dox Akkumulation in den Zellkern ein. Tumor-Akkumulation erscheint mit Rezeptor korrelieren, als relativ gering HER2-positive Tumoren anreichern weniger HerDox Vergleich zu denen mit vergleichsweise höheren HER2 Stufen 1. Darüber hinaus zeigt die effektive Konzentration Zelltod eine inverse Korrelation mit HER2 Anzeige auf Tumor-Zelllinien unterschiedlicher Zelloberfläche HER2-Stufen 1. HerDox zeigt eine therapeutische Sicherheit und Vorteil gegenüber ungezielte Dox, wie Tumorabtötung tritt bei über 10-mal niedriger Dosis im Vergleich zum entpackengeted Wirkstoff und zeigt keine nachweisbare negative Wirkung auf Herz (nachgewiesen durch Echokardiographie und histologische Färbung) oder Leber (nachgewiesen durch TUNEL-Färbung) Gewebe, im Gegensatz zu ungezielte Dox 1. Trotz seiner Herkunft aus einer Virushülle Protein weist HerPBK10 keine nachweisbare Immunogenität im therapeutischen Bereich 2. Während bereits vorhandenen Antikörper zu ganzen Adenovirus HerPBK10 erkennen können, sind sie nicht in der Lage Zellbindung 2 zu verhindern. Tumorvolumen über die Zeit gemessen wird, eine Standard-Methode zur Bewertung der therapeutischen Wirksamkeit einer Therapie erfolgen und ist für die Beurteilung der therapeutischen Wirksamkeit von HerDox eingesetzt. Ergänzend diesen Ansatz mit in vivo und ex vivo Fluoreszenz Bildgebung hat uns erlaubt, besser zu beurteilen Targeting Effizienz 7. Wir haben insbesondere in situ konfokale Abbildung entfernten Tumoren mit Spektralanalyse Dox Fluoreszenz integriert, dass keine Verifizierung HerDoxt nur bei Tumoren in vivo angesammelt aber drang in Tumorzellen und geliefert Dox in das Zytoplasma und Zellkern 7. Spektralanalyse ermöglichte es uns, weiterhin Dox Fluoreszenz von Autofluoreszenz 7 unterscheiden. Hier zeigen wir näher unser Ansatz für die Beurteilung HerDox in vivo nach systemischer Lieferung, und vor allem für die Beurteilung durch Targeting Multimode bildgebende Verfahren und Analysen.

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

analysis of targeted viral protein nanoparticles delivered to her2+ tumors

Der HER2 + Tumor-Targeting-Nanopartikel, HerDox weist Tumor-und Tumor-Akkumulation bevorzugte Wachstum Ablation in einem Tiermodell der HER2 + Krebs. HerDox durch nichtkovalente Selbstorganisation eines Tumors Zielzelle Penetrationsprotein mit dem Chemotherapeutikum Doxorubicin ausgebildet ist, über eine kleine Nukleinsäure Linker. Eine Kombination von elektrophilen, Einlagerung und Oligomerisierung Interaktionen erleichtern self-assembly in runde 10-20 nm großen Partikeln. HerDox aufweist Stabilität im Blut sowie einer längeren Lagerung bei verschiedenen Temperaturen. Systemische Abgabe HerDox in tumortragenden Mäusen zur Tumor-Zelltod ohne feststellbare nachteilige Auswirkungen auf Nicht-Tumor-Gewebe, einschließlich des Herzens und der Leber (der unterziehen gekennzeichnet Schäden durch ungezielte Doxorubicin). HER2 Höhe erleichtert Targeting von Zellen, die den humanen epidermalen Wachstumsfaktor-Rezeptor, damit Tumoren Anzeige erhöhten HER2 Ebenen größere Ansammlung von HerDox weisen im Vergleich zu Zellen, Expressionsingen unteren Ebenen, sowohl in vitro als auch in vivo. Fluoreszenzintensität Bildgebung mit in situ konfokalen und Spektralanalyse kombiniert hat uns erlaubt, in vivo Tumor-Targeting und Tumorzelle Eindringen von HerDox nach systemischer Lieferung überprüfen. Hier haben wir ausführlich unsere Methoden zur Beurteilung der Tumor-Targeting über Multimode-Bildgebung nach systemischer Lieferung.

Abbildung 1 zeigt die in vivo optische Scanner-Prototyp, der für die Zwecke der Bildaufnahme unter mehreren Modalitäten, einschließlich Fluoreszenz-Intensität, spektrale, Lebensdauer, 2-Photonen-, intra-vital konfokalen und Biolumineszenz-Bildgebung gebaut wurde. Darüber hinaus liefert die gekühlte hochempfindliche Kamera und Hochleistungslaser Linien in diesem System eingebaut höheren Kontrast Fluoreszenzbilder Vergleich zu kommerziellen optischen Abbildungssystemen 11, insbe...

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

Analysis of Targeted Viral Protein Nanoparticles Delivered to HER2+ Tumors

Dieser Artikel beschreibt die Verfahren für die optische Bildgebung Analyse der Tumor-Targeting-Nanopartikel, HerDox. Insbesondere wird eine detaillierte Verwendung der Multimode-Bildaufnahmevorrichtung zum Erfassen Tumor-Targeting und Bewertung Tumorpenetration beschrieben.

Gezielte Analyse von viralen Proteins Nanopartikel HER2 + Tumoren Lieferung

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

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

Research

JoVE Journal

Bioengineering

10.9K Aufrufe.  University of Southern California. Dieser Artikel beschreibt die Verfahren für die optische Bildgebung Analyse der Tumor-Targeting-Nanopartikel, HerDox. Insbesondere wird eine detaillierte Verwendung der Multimode-Bildaufnahmevorrichtung zum Erfassen Tumor-Targeting und Bewertung Tumorpenetration beschrieben.

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

Introduction to the Ultrasound Targeted Microbubble Destruction Technique

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

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

Einführung in die Ultraschall-gezielte Mikroblasen Zerstörung Technik

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

Forschung

Biotechnik

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

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

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

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

Entwurf eines zyklischen Druck Bioreaktor für die Ex Vivo Studium der Aorten-Herzklappen

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

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

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

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

Cellular Lipidextraktion für Targeted Stable Isotope Dilution Flüssigchromatographie-Massenspektrometrie-Analyse

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

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

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

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

Gentransfer und Transfektion in humanen Pankreaskarzinom-Zellen mit Epidermal Growth Factor Gelatin-Based Engineered Nanovektoren Rezeptor-gezielte

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

Viral Nanoparticles for In vivo Tumor Imaging

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

Viral Nanoparticles for In vivo Tumor Imaging

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

Viral Nanopartikel für<em&gt; In vivo</em&gt; Tumor Imaging

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

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

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

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

Evaluation von polymeren Gene Delivery-Nanopartikeln durch Nanopartikel Tracking-Analyse und High-throughput Durchflusszytometrie

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

Harmonic Nanoparticles for Regenerative Research

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

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

Harmonic Nanopartikel für Regenerative Forschung

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

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

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

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

Herstellung von Metallnanopartikeln durch gepulste Laserablation in Flüssigkeiten: Ein Werkzeug zum Studium der antibakteriellen Eigenschaften von Nanopartikeln

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

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.

Korrelative Licht Elektronenmikroskopie (CLEM) für die Verfolgung und Imaging virales Protein verbundenen Strukturen in den Cryo-immobilisierten Zellen

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

Protein Kinase C-delta Inhibitor Peptide Formulation using Gold Nanoparticles

Protein kinase C-delta inhibitor (PKC&#948;i) is a promising drug to prevent ischemia-reperfusion-induced organ injury. It is usually conjugated to a cell-penetrating peptide, TAT, for intracellular delivery. However, TAT has shown non-specific biological activities. Gold nanoparticles (GNPs) can be used as drug delivery carriers without recognized toxicity. Therefore, we have used a GNP/peptide hybrid to deliver PKC&#948;i. Two short peptides (P2: CAAAAE and P4: CAAAAW), at a 95:5 ratio, were used to modify the surface properties of GNP. GNPs conjugated with PKC&#948;i (GNP/PKCi) are stable in distilled water, 0.9% NaCl, and phosphate-buffered saline (PBS) containing bovine serum albumin or fetal bovine serum. Intravenous injection of GNP-PKCi was previously shown to prevent ischemia-reperfusion injury of the lung. This article outlines a protocol to formulate GNP/PKCi and assess the physiochemical properties of GNP/PKCi. We have used similar methods to formulate other peptide-based drugs with GNP. This article will hopefully draw more attention to this novel intracellular drug delivery technology and its applications in vivo.

We have previously used a gold nanoparticle peptide hybrid to intravenously deliver a synthetic peptide, protein kinase C-delta inhibitor, which reduced ischemia-reperfusion-induced acute lung injury. Here we show the detailed protocol of the drug formulation. Other intracellular peptides can be formulated similarly.

Protein Kinase C-Delta-Inhibitor Peptid Formulierung mit Gold-Nanopartikeln

Protein kinase C-delta inhibitor (PKC&#948;i) is a promising drug to prevent ischemia-reperfusion-induced organ injury. It is usually conjugated to a ...