Diese Arbeit wurde von der Prinzessin Margaret Hospital Foundation, der National Science and Engineering Research Council of Canada, der kanadischen Institut für Gesundheitsforschung, der kanadischen Stiftung für Innovation, und Joey und Toby Tanenbaum / brasilianischen Ball Chair in Prostate Cancer Research, unterstützt ein Zuschuss von der SUNY Research Foundation und Startup-Unterstützung von der University at Buffalo.

1. Synthese von Pyropheophorbid-Lipid <ol><li> Kombinieren 200 mmol Pyrophäophorbid (von Spirulina Pacifica Algen hergestellt, wie zuvor beschrieben;... Zheng et al, Bioconj Chem 2002, 13 -392) 4, 98,7 mg 16.00 lysophophatidylcholine (1-Palmitoyl-2-hydroxy-sn-glycero -3-phosphocholin, Avanti Polar Lipids # 855675), 76,3 mg EDC, 48,7 mg DMAP (4 - (Dimethylamino) pyridin) in 5 ml Amylen stabilisiertem Chloroform für ein Verhältnis von 1:1:2:2 Pyro: Lipid: EDC DMPA. </li><li> Das Reaktionsgemisch wird bei Raumtemperatur unter Argon 24 Stunden. Verdampfe das Chloroform unter vermindertem Druck mit einem Rotationsverdampfer unter Erwärmen auf 40 ° C. Die Mischung aus pyro-Lipid-Isomeren (pyro an der sn-1 oder sn-2-Position gebunden ist) erzeugt wird. </li><li> Resuspendieren pyro-Lipid-Isomerengemisch in 1 mM CaCl 2, 50 mM Tris (pH 8), 0,5% Triton X-100 und 10% Methanol in einer Konzentration von 5 mg / ml. In Phospholipase A2 (PLA2) aus Honig Bienengift bei0,1 mg / ml Konzentration und Inkubation der Lösung bei 37 ° C für 24 h bis sn-1-pyro-Lipid verdauen und um die reine sn-2-pyro-Lipid-Isomer zu produzieren. </li><li> Fügen Sie zusätzliche 2 Volumen Chloroform und 1,25 Vol. Methanol-Extrakt, und dann das Lösemittel am Rotationsverdampfer bei vermindertem Druck bei 40 ° C Resuspendieren Sie das Spaltprodukt in 1% MeOH in DCM und Reinigung über eine kleine Diol Silica-Säule für die isomerenreine pyro-Lipid. </li></ol> 2. Vorbereitung der Porphysomes <ol><li> Auflösen Pyropheophorbid-Lipid Distearoyl-sn-glycero-3-phosphoethanolamin-N-methoxy (polyethylenglykol) (PEG-2000-PE) und Cholesterin in Chloroform getrennt, und zeichnen die Konzentrationen sind. </li><li> Bereiten porphysome Lipidfilm in einem 12x75 mm Borosilikatglas Reagenzglas mit einem Gesamtlipidgewicht von 5 mg / Rohr durch die Kombination von 65 Mol% Porphyrin-Lipid-, 5 mol% PEG-PE-2000 und 30 Mol-% Cholesterin. </li><li> Trocknen der Lipidfilme unter einem Strom von NitroStoffgas und lassen Sie sie weiter unter Vakuum für 1 Stunde getrocknet. Speichern die Lipidfilme bei -20 ° C unter Argon bis zur Zukunft Hydratation und Extrusion. </li><li> 1 ml phosphatgepufferter Salzlösung (PBS, 150 mM NaCl, 10 mM Phosphat, pH 7,4) zu jeder Lipidfilm Rohr. Einfrieren der Teströhrchen in flüssigen Stickstoff, bis sie vollständig gefroren, aufgetaut und dann das Rohr in 65 ° C Wasserbad, bis die Probe alle geschmolzen. Wirbel das Rohr für insgesamt 30 Sekunden (3x an jeder 10 sec, mit 5 sec Wiedererwärmung in 65 ° C-Wasserbad zwischen jedem Vortexen). </li><li> Wiederholen Sie den Frost-Tau-5 Zyklen oder mehr bei Bedarf, bis keine großen Aggregate im hydrierten Lösung beobachtet werden. Verlassen Sie die porphysome Suspension auf Eis, wenn Sie fertig. </li><li> Montieren Sie den Hochdruck-Extruder (10 ml) mit zwei gestapelte 100 nm Polycarbonatfilter zu einem thermostatisierten Zirkulator für 65 ° C zirkulierenden Wasserbad installiert in. Schließen Sie die thermobarrel Extruder, und schließen Sie das Extrudersystem zu einem Stickstofftank für Hochdrucksicher Versorgung. </li><li> Übertragen Sie die porphysome Suspension auf die Extruderkammer mit einem 9-Zoll-Glas-Transferpipette. Stellen Sie den Druck, indem Sie den Regler an der Stickstofftank Regler, bis ein Lese zwischen 200 und 300 psig wird als Anfangsextrusionsdruck beobachtet. Öffnen des Ventils des Druckreglers schrittweise bis ausreichend porphysome Strömungsrate erzielt wird oder ein Maximum von 800 psig (5.440 kPa) Druck erreicht ist. </li><li> Nachdem die erste Extrusions Pass abgeschlossen ist, schließen Sie die Druckregelventil und den Druck des Extruders durch Öffnen der Druckentlastungsventil am Extruder Spitze. Nachfüllen des Extruders mit der frisch extrudierten porphysome Lösung und wiederholen die Extrusion für mindestens 10 Mal, um sicherzustellen, dass die endgültige extrudierte Lösung homogen ist. </li><li> Bestimmung der Konzentration des endgültigen porphysome Lösung durch Messung der Absorption einer verdünnten Probe in Methanol durch UV-Vis-Spektrometer. Berechnen Sie die Konzentration mit Hilfe der folgenden Gleichung Witzh der Extinktionskoeffizient 97.000 M -1 cm -1 bei 410 nm für Pyropheophorbid-Lipid. </li></ol><img alt="Gleichung 1" src="/files/ftp_upload/50536/50536eq1.jpg" /><ol start="10"><li> Messen Sie die endgültige Größe Verteilung der porphysomes mit einem Malvern Zetasizer ZS90 durch dynamische Lichtstreuung. Verdünnen porphysome Lösung in PBS und führen drei Messungen mit jeweils 15 läuft für die gemittelten Ergebnisse. </li><li> Bestimmung der Fluoreszenzlöschung porphysome durch Vergleich der Fluoreszenzemission des abgeschreckten porphysome (1 mM in PBS) und nicht abgeschreckte porphysome (1 mM in 1% Triton X-100) unter Verwendung Spektrofluorometer. Nehmen Sie die Spektren vor und nach der Reinigungsmittel (Tirton X-100) hinzugefügt wird, und normalisieren das Signal zu lesen, um maximalen Fluoreszenz. Stellen Sie die Erregung bis 410 nm und Emissionswellenlängenbereich von 600-800 nm zu sein. </li><li> Halten porphysomes Lösung unter Argon bei 4 ° C bis zukünftige Verwendung, und schützen Sie es vor Licht durch Aluminium fÖl. </li></ol> 3. Herstellung von Tier Xenograft Modell <ol><li> Kultur die KB-Zellen in RPMI-1640 (mit 10% FBS) und die Sustain-Zellen in 5% CO 2 und befeuchteter Atmosphäre bei 37 ° C. </li><li> Ernte-KB-Zellen unter Verwendung von Standard-Zellkulturtechnik. Halten der Zellsuspension (20000 Zellen / ml) in phosphatgepufferter Salzlösung (PBS) auf Eis für kurzfristige Lagerung bis zum Zeitpunkt der Injektion. </li><li> Bereiten Sie eine KB Xenografts auf Nacktmäusen. Bestrahlte LM-485:;: Nackte Mäuse von Charles River Laboratories, wenn sie 20 g (Kategorie # CRL NU (NCR)-Foxn1 nu, 6-7 Wochen alt) Diät, da Standard-Diät und Bettzeug (Harlan Laboratories # 7912; Schlafmöglichkeiten: Maiskolben-Bettwäsche, ¼ &quot;, # 7097). Sterilisieren Sie alle Tier-Handling-Werkzeugen (Spritze, Pinzetten, Tier-und Pad) vor Experimenten. Betäuben Mäuse mit 2% Isofluran v / v in 100% Sauerstoff-Trägergas. Alle Tier Protokolle werden von der Institution (University Health Ne genehmigttwork in diesem Fall) Tierpflege und Verwendung Ausschusses. </li><li> Injizieren Tumorzellen subkutan in PBS an die rechte Flanke der Mäuse suspendiert. Mäuse sind bereit, nach 10 Tagen verwendet werden, wenn Tumordurchmesser von 4-5 mm und eine Dicke von 2-3 mm erreicht. </li></ol> 4. In-vivo-Photothermische Therapie <ol><li> Inject porphysomes (mit 750 nmol pyro bei 200 ul Volumen) intravenös über die Schwanzvene. Führen Laserbestrahlung 24 Stunden nach der Injektion von porphysomes. </li><li> In diesem Abschnitt führen alle Verfahren in einem geschlossenen Laserschutzraum. Laserschutzbrillen tragen während der Laserbestrahlung. </li><li> Stellen Sie den Laserbestrahlungsgeräte. Halten Sie die Laserfaser und einen Diffusor mit einer Klammer auf einem Ständer. Stellen Sie die Laserleistung auf niedrigem Niveau, und schalten Sie den Laser. Stellen Sie die Höhe der Laserfaser, eine Bestrahlungsfläche von 9 mm im Durchmesser erreichen (DPSS-Laser, LaserGlow Technologies, Toronto, Kanada), kalibrieren die Laserleistung von 750 mW (1,18 W / cm 2) mit apower Meter an und schalten Sie den Laser, um die Tiere vor. </li><li> Anesthetize die Tiere mit 2% Isofluran v / v in 100% Sauerstoff Trägergas durch Nasenkegel offensichtlich kein Abfangen von Isofluran eine Gefährdung der menschlichen Gesundheit Gefahr). Bestätigen Sie, dass das Tier tief genug durch Ausführen einer Prise Zeh mit einer Pinzette oder den Fingern Hinterpfoten Tier betäubt. Die Tiere sind bereit für Laserbestrahlung, wenn sie nicht mehr auf dieses Verfahrens sind. </li><li> Überwachen Sie die Temperatur Tumor durch eine Infrarot-Wärmebildkamera (Mikroshot, LumaSense Technologies). Legen Sie die Kamera in der Nähe des Tumors, konzentrieren sie gut und nehmen eine vorge PTT Bild sowie eine regelmäßige Weißlichtbild vor PTT Bestrahlung beginnt. </li><li> Schalten Sie den Laser wieder zu 750 mW und erhöhen die Lichtleistung. Positionieren Sie den Tumor in der Mitte des Laserstrahls auf der Tumor vollständig abgedeckt zu bekommen, und die Startzeitbestrahlung. Nehmen Sie die Tumortemperatur Bild alle 5 Sekunden eine Minute Laserbehandlung. Schalten Sie den Laser bei 1 min. Stop die Gaszufuhr von 2% Isofluran, bis das Tier aus der Narkose erholt warten und ihn wieder in den Käfig. </li><li> Halten Mäusen bei Normothermie Tierraum unter 0.12 h dunkel und Licht-Zyklus nach von der University Health Network genehmigten Tier Protokolle. Inject Buprenorphin (0,05 mg / kg, subkutan zweimal am Tag für eine Woche nach der Laserbestrahlung, um die Schmerzen von der Narbe zu reduzieren. Alle zwei Tagen, sind die Tumordurchmesser mit einer Schieblehre und ein Foto. Berechnen Sie das Tumorvolumen mit Hilfe der folgende Gleichung: V = π / 6 · a · b 2, wobei a der Längsdurchmesser, und b der kurzen. </li><li> Opfere die Tiere, indem sie in CO 2-Kammer, wenn der lange Durchmesser des Tumors erreicht 10 mm, wie es der definierten Endpunkt in unserem Labor. Führen Genickbruch nach dem CO2-Behandlung. Zeichnen Sie die Überlebenskurve. </li></ol>

<ol>
	<li>Lovell, J. F., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Porphysome+nanovesicles+generated+by+porphyrin+bilayers+for+use+as+multimodal+biophotonic+contrast+agents.">Porphysome nanovesicles generated by porphyrin bilayers for use as multimodal biophotonic contrast agents.</a> Nat. Mater. 10, 324-332 (2011).</li><li>Lovell, J. F., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Enzymatic+regioselection+for+the+synthesis+and+biodegradation+of+porphysome+nanovesicles.">Enzymatic regioselection for the synthesis and biodegradation of porphysome nanovesicles.</a> Angew Chem Int Ed Engl. 51, 2429-2433 (2012).</li><li>Liu, T. W., MacDonald, T. D., Shi, J., Wilson, B. C., Zheng, G. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Intrinsically+Copper-64-Labeled+Organic+Nanoparticles+as+Radiotracers.">Intrinsically Copper-64-Labeled Organic Nanoparticles as Radiotracers.</a> Angewandte Chemie. , (2012).</li><li>Zheng, G., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Low-density+lipoprotein+reconstituted+by+pyropheophorbide+cholesteryl+oleate+as+target-specific+photosensitizer.">Low-density lipoprotein reconstituted by pyropheophorbide cholesteryl oleate as target-specific photosensitizer.</a> Bioconjug Chem. 13, 392-396 (2002).</li><li>Chen, B., Pogue, B. W., Hasan, T. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Liposomal+delivery+of+photosensitising+agents.">Liposomal delivery of photosensitising agents.</a> Expert Opin Drug Deliv. 2, 477-487 (2005).</li><li>Jin, C. S., Zheng, G. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Liposomal+nanostructures+for+photosensitizer+delivery.">Liposomal nanostructures for photosensitizer delivery.</a> Lasers Surg Med. 43, 734-748 (2011).</li><li>Olson, F., Hunt, C. A., Szoka, F. C., Vail, W. J., Papahadjopoulos, D. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Preparation+of+liposomes+of+defined+size+distribution+by+extrusion+through+polycarbonate+membranes.">Preparation of liposomes of defined size distribution by extrusion through polycarbonate membranes.</a> Biochim Biophys Acta. 557, 9-23 (1979).</li><li>Berger, N., Sachse, A., Bender, J., Schubert, R., Brandl, M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Filter+extrusion+of+liposomes+using+different+devices:+comparison+of+liposome+size,+encapsulation+efficiency,+and+process+characteristics.">Filter extrusion of liposomes using different devices: comparison of liposome size, encapsulation efficiency, and process characteristics.</a> Int J Pharm. 223, 55-68 (2001).</li><li>Jesorka, A., Orwar, O. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Liposomes:+technologies+and+analytical+applications.">Liposomes: technologies and analytical applications.</a> Annu Rev Anal Chem (Palo Alto Calif). 1, 801-832 (2008).</li><li>Lapinski, M. M., Castro-Forero, A., Greiner, A. J., Ofoli, R. Y., Blanchard, G. J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Comparison+of+liposomes+formed+by+sonication+and+extrusion:+rotational+and+translational+diffusion+of+an+embedded+chromophore.">Comparison of liposomes formed by sonication and extrusion: rotational and translational diffusion of an embedded chromophore.</a> Langmuir. 23, 11677-11683 (2007).</li><li>Liu, D., Huang, L. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Size+Homogeneity+of+a+Liposome+Preparation+is+Crucial+for+Liposome+Biodistribution+In+Vivo.">Size Homogeneity of a Liposome Preparation is Crucial for Liposome Biodistribution In Vivo.</a> Journal of Liposome Research. 2, 57-66 (1992).</li><li>Zhang, Y., Lovell, J. F. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Porphyrins+as+Theranostic+Agents+from+Prehistoric+to+Modern+Times.">Porphyrins as Theranostic Agents from Prehistoric to Modern Times.</a> Theranostics. 2, 905-915 (2012).</li></ol>

Wir haben nichts zu offenbaren.

Bei der Entwicklung von Drug-Delivery-Technologien, sind multifunktionelle Nanopartikel derzeit in weiten Untersuchungen für eine genaue Überwachung der Drug-Delivery-Fahrzeug unter Beibehaltung einer hohen Wirkstoff Nutzlast. Porphyrin beladene Liposomen wurden zur besseren pharmakokinetischen Eigenschaften und effiziente Lieferung als direkte Verabreichung von Porphyrin entwickelt worden, aber Hindernisse vorhanden sind, einschließlich der begrenzten Beladungsmenge von Porphyrin und schnelle Verteilung von Liposome...

Porphysomes sind neuartige multifunktionale nanovesicles, die wir vor kurzem entwickelt, die in der Lage, multimodale Bildgebung und Therapie 1 sind. Sie werden aus selbstorganisierten Porphyrin-Doppelschichten gebildet und enthalten eine extrem hohe Dichte von Porphyrin (über 83 000/porphysome Partikel), die große Extinktionskoeffizienten und die Ergebnisse in einzigartigen Struktur-abhängige Fluoreszenz Selbstabschreckung erzeugt. Porphysomes haben gute in-vivo-Pharmakokinetik und Bioverteilungseigenschaften: sie zeigen eine Bluthalbwertszeit von 12 Stunden folgende systematische Verwaltung und passiv sammeln sich in Xenotransplantattumoren mit 7,5% ID / g bei 24 Stunden nach der Injektion 2. Ihre einzigartige Struktur und die physikalisch-chemischen Eigenschaften machen porphysome ein guter Kandidat für die multimodale Bildgebung und bildgeführte Therapie. Zuallererst Porphyrin enthalten, sind porphysomes eigen für Fluoreszenz-Bildgebung von Tumoren auf die Tumorakkumulation 1.Darüber hinaus hat jedes Porphyrin ein stabiles Chelat-Website für Radioisotope können daher leicht porphysomes mit Radioisotopen wie etwa 64Cu für PET-Bildgebung 3 gekennzeichnet werden. Darüber hinaus wird die absorbierte Lichtenergie thermisch unter Laserbestrahlung Exposition abgeführt, wenn porphysome Struktur ist intakt, so porphysomes einzigartigen photoakustische Bildgebung und PTT-Funktionen zeigen auch. Es hat sich gezeigt, dass 24 h nach der intravenösen Injektion von porphysomes, Laserbestrahlung des porphysome-Tumor akkumuliert wurde, eine schnelle Temperaturerhöhung und starke photothermische Tumor Ablation. Dies zeigte, dass porphysomes sind effiziente photothermische Verstärker mit Extinktionskoeffizienten so hoch wie Gold-Nanopartikel (AuNPs) ein. Auf der anderen Seite, im Vergleich mit anderen anorganischen photothermische Mittel, einschließlich AuNPs, porphysomes zeigen eine herausragende Vorteil in der biologischen Sicherheit aufgrund ihrer organischen Natur. Porphysomes enzymatisch biologisch abbaubar und induzieren minimale akute toxicity in Mäusen mit intravenösen Dosen bis zu 1000 mg / kg 1. Außerdem ähnlich Liposomen, die große wässrigen Kern porphysomes könnte passiv oder aktiv mit therapeutischen oder Kontrastmittel geladen. Die optischen Eigenschaften und Biokompatibilität von porphysomes zeigen die multimodale Potenzial organischer Nanopartikel für Biophotonik Bildgebung und Therapie. In diesem Papier präsentieren wir die Synthese Verfahren Pyropheophorbid-Lipid-Konjugate, die Herstellung und die Charakterisierung Verfahren porphysomes mit Hochdruckextrusion. PTT In vivo an Mäusen ist ebenfalls durchgeführt, um die Effizienz der porphysome PTT-fähigen im Tumor zeigen, Behandlung mit einer subkutanen Xenograft-Tumormodell.

<table border="1">
	<tbody>
		<tr>
			<td>Name of Equipment</td>
			<td>Company</td>
			<td>Catalog Number</td>
			<td>Comments (optional)</td>
		</tr>
		<tr>
			<td>Rotary evaporator</td>
			<td>Thermo-Savant</td>
			<td>SPD131DDA</td>
			<td>&nbsp;</td>
		</tr>
		<tr>
			<td>Votex</td>
			<td>Scientific Industries</td>
			<td>SI-0236</td>
			<td>Model number: G560</td>
		</tr>
		<tr>
			<td>High pressure extruder</td>
			<td>LIPEX, Northern Lipids Inc.</td>
			<td>T.001</td>
			<td>10 ml Thermobarrel Extruder</td>
		</tr>
		<tr>
			<td>Heated bath circulators (Thermostatted circulator)</td>
			<td>Thermo SCIENTIFIC</td>
			<td>SC100-S5P</td>
			<td>&nbsp;</td>
		</tr>
		<tr>
			<td>Polycarbonate filters</td>
			<td>Avanti Polar Lipids</td>
			<td>610005</td>
			<td>Pore size 100 nm, and membrane diameter 19 mm</td>
		</tr>
		<tr>
			<td>Zetasizer</td>
			<td>Malvern Instruments</td>
			<td>ZS90</td>
			<td>&nbsp;</td>
		</tr>
		<tr>
			<td>UV/Visible Spectrophototmeter</td>
			<td>Varian Australia</td>
			<td>Cary 50 Bio UV/ Visible Spectrophotometer</td>
			<td>&nbsp;</td>
		</tr>
		<tr>
			<td>Spectrofluorometer</td>
			<td>HORIBA Scientific</td>
			<td>FluoroMax-4</td>
			<td>&nbsp;</td>
		</tr>
		<tr>
			<td>Transmission Electron Microscopy (TEM)</td>
			<td>Hitachi</td>
			<td>H-7000</td>
			<td>&nbsp;</td>
		</tr>
		<tr>
			<td>Cell culture incubator</td>
			<td>SANYO</td>
			<td>MCO-18AIC</td>
			<td>&nbsp;</td>
		</tr>
		<tr>
			<td>Powermeter</td>
			<td>Thorlabs</td>
			<td>PM100D</td>
			<td>with sensor S142C</td>
		</tr>
		<tr>
			<td>671nm Laser</td>
			<td>LaserGlow Technologies</td>
			<td>LRS-0671_PFN-02000-05</td>
			<td>S/N:10097270</td>
		</tr>
		<tr>
			<td>Infrared Thermometer</td>
			<td>Mikroshot, LUMASENSE Technologies</td>
			<td>9102409</td>
			<td>&nbsp;</td>
		</tr>
		<tr>
			<td>Laser protective eye goggles</td>
			<td>LaserGlow Technologies</td>
			<td>AGF6602XX</td>
			<td>Optical Density: 1.5+ at 630-700 nm</td>
		</tr>
	</tbody>
</table>

one minute, sub-one-watt photothermal tumor ablation using porphysomes, intrinsic multifunctional nanovesicles

Wir haben vor kurzem porphysomes als eigen multifunktionale nanovesicles entwickelt. Ein Photosensibilisator, Pyropheophorbid α, wurde zu einer Phospholipid-konjugiert und Liposomen-wie kugelförmige Vesikel dann selbst zusammengebaut. Durch die extrem hohe Dichte von Porphyrin im Porphyrin-Lipid-Doppelschicht erzeugt porphysomes großen Extinktionskoeffizienten, Struktur-abhängige Fluoreszenz Selbstlöschung und ausgezeichnete photothermische Wirksamkeit. In unserer Formulierung wurden porphysomes mit Hochdruckextrusion hergestellt und zeigte eine mittlere Teilchengröße ca. 120 nm auf. Vierundzwanzig Stunden nach der intravenösen Injektion von porphysomes, die lokale Temperatur des Tumors schnell auf 1 Minute Belichtung von 750 mW (1,18 W / cm 2), 671 nm-Laser-Bestrahlung erhöhte sich von 30 ° C bis 62 ° C. Nach der vollständigen thermischen Ablation des Tumors, Schorf gebildet und heilte innerhalb von 2 Wochen, während in den Kontrollgruppen die Tumoren weiter zu wachsen und alle erreichten das definierte end Punkt innerhalb 3 Wochen. Diese Daten zeigen, wie porphysomes können als starke photothermische Therapie (PTT)-Mittel verwendet werden.

Pyrophäophorbid ist mit dem Phospholipid als stabile Lipid-Monomer (1a) konjugiert und die Konjugate durch Selbstorganisation zu porphysomes durch Membranextrusionsverfahren zu bilden, das eine Hochdruck-Extruder. Es ist normalerweise schwierig, porphysome-Lipid-Suspension in den ersten 3 Zyklen der Extrusion mit relativ langsamen Fließgeschwindigkeit zu extrudieren. Als weitere Extrusionen werden wiederholt, die Strömungsgeschwindigkeit der extrudierten Lösung allmählich erhöht, und der Druck kan...

Watch this Scientific Journal Video about One Minute, Sub-One-Watt Photothermal Tumor Ablation Using Porphysomes, Intrinsic Multifunctional Nanovesicles at JoVE.com

One Minute, Sub-One-Watt Photothermal Tumor Ablation Using Porphysomes, Intrinsic Multifunctional Nanovesicles

Wir entwickelten neuartigen multifunktionalen Eigen nanovesicles porphysomes genannt, die strukturabhängigen Fluoreszenz Selbstlöschung und einzigartigen photothermischen Eigenschaften haben, so als potente photothermische Therapie Mittel. Wir formuliert porphysomes mit Hochdruckextrusion und untersucht ihre photothermische Therapie Wirksamkeit in einem Xenotransplantat-Tumor-Modell.

Eine Minute, Sub-One-Watt-Photothermische Tumor Ablation mit Porphysomes, Eigen Multifunktionale Nanovesicles

We recently developed porphysomes as intrinsically multifunctional nanovesicles. A photosensitizer, pyropheophorbide &alpha;, was conjugated to a ...

one-minute-sub-one-watt-photothermal-tumor-ablation-using-porphysomes

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JoVE Journal

Bioengineering

10.7K Aufrufe.  University of Toronto.  Wir entwickelten neuartigen multifunktionalen Eigen nanovesicles porphysomes genannt, die strukturabhängigen Fluoreszenz Selbstlöschung und einzigartigen photothermischen Eigenschaften haben, so als potente photothermische Therapie Mittel. Wir formuliert porphysomes mit Hochdruckextrusion und untersucht ihre photothermische Therapie Wirksamkeit in einem Xenotransplantat-Tumor-Modell. 

Serie: One Minute, Sub-One-Watt Photothermal Tumor Ablation Using Porphysomes, Intrinsic Multifunctional Nanovesicles

Optical Frequency Domain Imaging of Ex vivo Pulmonary Resection Specimens: Obtaining One to One Image to Histopathology Correlation

Lung cancer is the leading cause of cancer-related deaths1. Squamous cell and small cell cancers typically arise in association with the conducting airways, whereas adenocarcinomas are typically more peripheral in location. Lung malignancy detection early in the disease process may be difficult due to several limitations: radiological resolution, bronchoscopic limitations in evaluating tissue underlying the airway mucosa and identifying early pathologic changes, and small sample size and/or incomplete sampling in histology biopsies. High resolution imaging modalities, such as optical frequency domain imaging (OFDI), provide non-destructive, large area 3-dimensional views of tissue microstructure to depths approaching 2 mm in real time (Figure 1)2-6. OFDI has been utilized in a variety of applications, including evaluation of coronary artery atherosclerosis6,7 and esophageal intestinal metaplasia and dysplasia6,8-10.
Bronchoscopic OCT/OFDI has been demonstrated as a safe in vivo imaging tool for evaluating the pulmonary airways11-23 (Animation). OCT has been assessed in pulmonary airways16,23 and parenchyma17,22 of animal models and in vivo human airway14,15. OCT imaging of normal airway has demonstrated visualization of airway layering and alveolar attachments, and evaluation of dysplastic lesions has been found useful in distinguishing grades of dysplasia in the bronchial mucosa11,12,20,21. OFDI imaging of bronchial mucosa has been demonstrated in a short bronchial segment (0.8 cm)18. Additionally, volumetric OFDI spanning multiple airway generations in swine and human pulmonary airways in vivo has been described19. Endobronchial OCT/OFDI is typically performed using thin, flexible catheters, which are compatible with standard bronchoscopic access ports. Additionally, OCT and OFDI needle-based probes have recently been developed, which may be used to image regions of the lung beyond the airway wall or pleural surface17.
While OCT/OFDI has been utilized and demonstrated as feasible for in vivo pulmonary imaging, no studies with precisely matched one-to-one OFDI:histology have been performed. Therefore, specific imaging criteria for various pulmonary pathologies have yet to be developed. Histopathological counterparts obtained in vivo consist of only small biopsy fragments, which are difficult to correlate with large OFDI datasets. Additionally, they do not provide the comprehensive histology needed for registration with large volume OFDI. As a result, specific imaging features of pulmonary pathology cannot be developed in the in vivo setting. Precisely matched, one-to-one OFDI and histology correlation is vital to accurately evaluate features seen in OFDI against histology as a gold standard in order to derive specific image interpretation criteria for pulmonary neoplasms and other pulmonary pathologies. Once specific imaging criteria have been developed and validated ex vivo with matched one-to-one histology, the criteria may then be applied to in vivo imaging studies. Here, we present a method for precise, one to one correlation between high resolution optical imaging and histology in ex vivo lung resection specimens. Throughout this manuscript, we describe the techniques used to match OFDI images to histology. However, this method is not specific to OFDI and can be used to obtain histology-registered images for any optical imaging technique. We performed airway centered OFDI with a specialized custom built bronchoscopic 2.4 French (0.8 mm diameter) catheter. Tissue samples were marked with tissue dye, visible in both OFDI and histology. Careful orientation procedures were used to precisely correlate imaging and histological sampling locations. The techniques outlined in this manuscript were used to conduct the first demonstration of volumetric OFDI with precise correlation to tissue-based diagnosis for evaluating pulmonary pathology24. This straightforward, effective technique may be extended to other tissue types to provide precise imaging to histology correlation needed to determine fine imaging features of both normal and diseased tissues.

Optical Frequency Domain Imaging of Ex vivo Pulmonary Resection Specimens: Obtaining One to One Image to Histopathology Correlation

A method to image ex vivo pulmonary resection specimens with optical frequency domain imaging (OFDI) and obtain precise correlation to histology is described, which is essential to developing specific OFDI interpretation criteria for pulmonary pathology. This method is applicable to other tissue types and imaging techniques to obtain precise imaging to histology correlation for accurate image interpretation and assessment. Imaging criteria established with this technique would then be applicable to image assessment in future in vivo studies.

Optical Frequency Domain Imaging der<em&gt; Ex vivo</em&gt; Pulmonary Resektionsproben: Beziehen One to One Image to Histopathologie Correlation

Lung cancer is the leading cause of cancer-related deaths1. Squamous cell and small cell cancers typically arise in association with the conducting ...

Forschung

Biotechnik

Rapid Isolation of Viable Circulating Tumor Cells from Patient Blood Samples

Circulating tumor cells (CTC) are cells that disseminate from a primary tumor throughout the circulatory system and that can ultimately form secondary tumors at distant sites. CTC count can be used to follow disease progression based on the correlation between CTC concentration in blood and disease severity1. As a treatment tool, CTC could be studied in the laboratory to develop personalized therapies. To this end, CTC isolation must cause no cellular damage, and contamination by other cell types, particularly leukocytes, must be avoided as much as possible2. Many of the current techniques, including the sole FDA-approved device for CTC enumeration, destroy CTC as part of the isolation process (for more information see Ref. 2). A microfluidic device to capture viable CTC is described, consisting of a surface functionalized with E-selectin glycoprotein in addition to antibodies against epithelial markers3. To enhance device performance a nanoparticle coating was applied consisting of halloysite nanotubes, an aluminosilicate nanoparticle harvested from clay4. The E-selectin molecules provide a means to capture fast moving CTC that are pumped through the device, lending an advantage over alternative microfluidic devices wherein longer processing times are necessary to provide target cells with sufficient time to interact with a surface. The antibodies to epithelial targets provide CTC-specificity to the device, as well as provide a readily adjustable parameter to tune isolation. Finally, the halloysite nanotube coating allows significantly enhanced isolation compared to other techniques by helping to capture fast moving cells, providing increased surface area for protein adsorption, and repelling contaminating leukocytes3,4. This device is produced by a straightforward technique using off-the-shelf materials, and has been successfully used to capture cancer cells from the blood of metastatic cancer patients. Captured cells are maintained for up to 15 days in culture following isolation, and these samples typically consist of &gt;50% viable primary cancer cells from each patient. This device has been used to capture viable CTC from both diluted whole blood and buffy coat samples. Ultimately, we present a technique with functionality in a clinical setting to develop personalized cancer therapies.

Circulating tumor cells are isolated from the blood of cancer patients without inflicting cellular damage. Isolation of tumor cells is accomplished using a bimolecular surface of E-selectin in addition to antibodies against epithelial markers. A nanotube coating specifically promotes cancer cell adhesion resulting in high capture purities.

Schnelle Isolierung von lebensfähigen zirkulierende Tumorzellen von Patientenblutproben

Circulating tumor cells (CTC) are cells that disseminate from a primary tumor throughout the circulatory system and that can ultimately form secondary ...

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 ...

Construction of a Preclinical Multimodality Phantom Using Tissue-mimicking Materials for Quality Assurance in Tumor Size Measurement

World Health Organization (WHO) and the Response Evaluation Criteria in Solid Tumors (RECIST) working groups advocated standardized criteria for radiologic assessment of solid tumors in response to anti-tumor drug therapy in the 1980s and 1990s, respectively. WHO criteria measure solid tumors in two-dimensions, whereas RECIST measurements use only one-dimension which is considered to be more reproducible 1, 2, 3,4,5. These criteria have been widely used as the only imaging biomarker approved by the United States Food and Drug Administration (FDA) 6. In order to measure tumor response to anti-tumor drugs on images with accuracy, therefore, a robust quality assurance (QA) procedures and corresponding QA phantom are needed.
To address this need, the authors constructed a preclinical multimodality (for ultrasound (US), computed tomography (CT) and magnetic resonance imaging (MRI)) phantom using tissue-mimicking (TM) materials based on the limited number of target lesions required by RECIST by revising a Gammex US commercial phantom 7. The Appendix in Lee et al. demonstrates the procedures of phantom fabrication 7. In this article, all protocols are introduced in a step-by-step fashion beginning with procedures for preparing the silicone molds for casting tumor-simulating test objects in the phantom, followed by preparation of TM materials for multimodality imaging, and finally construction of the preclinical multimodality QA phantom. The primary purpose of this paper is to provide the protocols to allow anyone interested in independently constructing a phantom for their own projects. QA procedures for tumor size measurement, and RECIST, WHO and volume measurement results of test objects made at multiple institutions using this QA phantom are shown in detail in Lee et al. 8.

This paper describes in-house procedures of constructing a preclinical multimodality phantom made of tissue-mimicking (TM) materials for quality assurance (QA) of tumor size measurement in animal imaging modalities such as ultrasound (US), computed tomography (CT) and magnetic resonance imaging (MRI).

Bau eines Präklinische Multimodalität Phantom Mit Tissue-ähnlichen Materialien für die Qualitätssicherung in der Tumor Größe Maß

World Health Organization (WHO) and the Response Evaluation Criteria in Solid Tumors (RECIST) working groups advocated standardized criteria for ...

Production of Large Numbers of Size-controlled Tumor Spheroids Using Microwell Plates

Tumor spheroids are increasingly recognized as an important in vitro model for the behavior of tumor cells in three dimensions. More physiologically relevant than conventional adherent-sheet cultures, they more accurately recapitulate the complexity and interactions present in real tumors. In order to harness this model to better assess tumor biology, or the efficacy of novel therapeutic agents, it is necessary to be able to generate spheroids reproducibly, in a controlled manner and in significant numbers.
The AggreWell system consists of a high-density array of pyramid-shaped microwells, into which a suspension of single cells is centrifuged. The numbers of cells clustering at the bottom of each microwell, and the number and ratio of distinct cell types involved depend only on the properties of the suspension introduced by the experimenter. Thus, we are able to generate tumor spheroids of arbitrary size and composition without needing to modify the underlying platform technology. The hundreds of microwells per square centimeter of culture surface area in turn ensure that extremely high production levels may be attained via a straightforward, nonlabor-intensive process. We therefore expect that this protocol will be broadly useful to researchers in the tumor spheroid field.

We introduce a protocol for the generation of large numbers (thousands to hundreds of thousands) of uniform size- and composition-controlled tumor spheroids, using commercially available microwell plates.

Die Produktion der großen Zahlen von Größe gesteuerte Tumorsphäroide Mit Microwell Platten

Tumor spheroids are increasingly recognized as an important in vitro model for the behavior of tumor cells in three dimensions. More physiologically ...

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.

PLGA-Nanopartikel durch Einzel-oder Doppel-Emulsion mit Vitamin E-TPGS gegründet

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

Longitudinal Measurement of Extracellular Matrix Rigidity in 3D Tumor Models Using Particle-tracking Microrheology

The mechanical microenvironment has been shown to act as a crucial regulator of tumor growth behavior and signaling, which is itself remodeled and modified as part of a set of complex, two-way mechanosensitive interactions. While the development of biologically-relevant 3D tumor models have facilitated mechanistic studies on the impact of matrix rheology on tumor growth, the inverse problem of mapping changes in the mechanical environment induced by tumors remains challenging. Here, we describe the implementation of particle-tracking microrheology (PTM) in conjunction with 3D models of pancreatic cancer as part of a robust and viable approach for longitudinally monitoring physical changes in the tumor microenvironment, in situ. The methodology described here integrates a system of preparing in vitro 3D models embedded in a model extracellular matrix (ECM) scaffold of Type I collagen with fluorescently labeled probes uniformly distributed for position- and time-dependent microrheology measurements throughout the specimen. In vitro tumors are plated and probed in parallel conditions using multiwell imaging plates. Drawing on established methods, videos of tracer probe movements are transformed via the Generalized Stokes Einstein Relation (GSER) to report the complex frequency-dependent viscoelastic shear modulus, G*(&#969;). Because this approach is imaging-based, mechanical characterization is also mapped onto large transmitted-light spatial fields to simultaneously report qualitative changes in 3D tumor size and phenotype. Representative results showing contrasting mechanical response in sub-regions associated with localized invasion-induced matrix degradation as well as system calibration, validation data are presented. Undesirable outcomes from common experimental errors and troubleshooting of these issues are also presented. The 96-well 3D culture plating format implemented in this protocol is conducive to correlation of microrheology measurements with therapeutic screening assays or molecular imaging to gain new insights into impact of treatments or biochemical stimuli on the mechanical microenvironment.

Particle-tracking microrheology can be used to non-destructively quantify and spatially map changes in extracellular matrix mechanical properties in 3D tumor models.

Längs Messung der extrazellulären Matrix Steifigkeit in 3D Tumormodelle mit Particle-Tracking Mikrorheologie

The mechanical microenvironment has been shown to act as a crucial regulator of tumor growth behavior and signaling, which is itself remodeled and ...

Real-time Monitoring of High Intensity Focused Ultrasound (HIFU) Ablation of In Vitro Canine Livers Using Harmonic Motion Imaging for Focused Ultrasound (HMIFU)

Harmonic Motion Imaging for Focused Ultrasound (HMIFU) is a technique that can perform and monitor high-intensity focused ultrasound (HIFU) ablation. An oscillatory motion is generated at the focus of a 93-element and 4.5 MHz center frequency HIFU transducer by applying a 25 Hz amplitude-modulated signal using a function generator. A 64-element and 2.5 MHz imaging transducer with 68kPa peak pressure is confocally placed at the center of the HIFU transducer to acquire the radio-frequency (RF) channel data. In this protocol, real-time monitoring of thermal ablation using HIFU with an acoustic power of 7 W on canine livers in vitro is described. HIFU treatment is applied on the tissue during 2 min and the ablated region is imaged in real-time using diverging or plane wave imaging up to 1,000 frames/second. The matrix of RF channel data is multiplied by a sparse matrix for image reconstruction. The reconstructed field of view is of 90&#176; for diverging wave and 20 mm for plane wave imaging and the data are sampled at 80 MHz. The reconstruction is performed on a Graphical Processing Unit (GPU) in order to image in real-time at a 4.5 display frame rate. 1-D normalized cross-correlation of the reconstructed RF data is used to estimate axial displacements in the focal region. The magnitude of the peak-to-peak displacement at the focal depth decreases during the thermal ablation which denotes stiffening of the tissue due to the formation of a lesion. The displacement signal-to-noise ratio (SNRd) at the focal area for plane wave was 1.4 times higher than for diverging wave showing that plane wave imaging appears to produce better displacement maps quality for HMIFU than diverging wave imaging.

Real-time Monitoring of High Intensity Focused Ultrasound HIFU Ablation of In Vitro Canine Livers Using Harmonic Motion Imaging for Focused Ultrasound HMIFU

This article describes real-time monitoring of HIFU ablation in canine liver with high frame rate ultrasound imaging using diverging and plane wave imaging. Harmonic Motion Imaging for Focused Ultrasound is used to image the decrease of acoustic radiation force induced displacement in the ablated region.

Echtzeit-Überwachung von hochintensiven fokussierten Ultraschall (HIFU) Ablation<em&gt; In-vitro-</em&gt; Canine Livers Verwendung harmonische Bewegung Imaging für fokussierten Ultraschall (HMIFU)

Harmonic Motion Imaging for Focused Ultrasound (HMIFU) is a technique that can perform and monitor high-intensity focused ultrasound (HIFU) ablation. An ...

Tissue Engineering by Intrinsic Vascularization in an In Vivo Tissue Engineering Chamber

In reconstructive surgery, there is a clinical need for an alternative to the current methods of autologous reconstruction which are complex, costly and trade one defect for another. Tissue engineering holds the promise to address this increasing demand. However, most tissue engineering strategies fail to generate stable and functional tissue substitutes because of poor vascularization. This paper focuses on an in vivo tissue engineering chamber model of intrinsic vascularization where a perfused artery and a vein either as an arteriovenous loop or a flow-through pedicle configuration is directed inside a protected hollow chamber. In this chamber-based system angiogenic sprouting occurs from the arteriovenous vessels and this system attracts ischemic and inflammatory driven endogenous cell migration which gradually fills the chamber space with fibro-vascular tissue. Exogenous cell/matrix implantation at the time of chamber construction enhances cell survival and determines specificity of the engineered tissues which develop. Our studies have shown that this chamber model can successfully generate different tissues such as fat, cardiac muscle, liver and others. However, modifications and refinements are required to ensure target tissue formation is consistent and reproducible. This article describes a standardized protocol for the fabrication of two different vascularized tissue engineering chamber models in vivo.

Tissue Engineering by Intrinsic Vascularization in an In Vivo Tissue Engineering Chamber

This is a guideline for constructing in vivo vascularized tissue using a microsurgical arteriovenous loop or a flow-through pedicle configuration inside a tissue engineering chamber. The vascularized tissues generated can be employed for organ regeneration and replacement of tissue defects, as well as for drug testing and disease modeling.

Tissue Engineering von Intrinsic Vaskularisation in ein<em&gt; In Vivo</em&gt; Tissue Engineering Chamber

In reconstructive surgery, there is a clinical need for an alternative to the current methods of autologous reconstruction which are complex, costly and ...

A Rapid and Chemical-free Hemoglobin Assay with Photothermal Angular Light Scattering

Photo-thermal angular light scattering (PT-AS) is a novel optical method for measuring the hemoglobin concentration ([Hb]) of blood samples. On the basis of the intrinsic photothermal response of hemoglobin molecules, the sensor enables high-sensitivity, chemical-free measurement of [Hb]. [Hb] detection capability with a limit of 0.12 g/dl over the range of 0.35 - 17.9 g/dl has been demonstrated previously. The method can be readily implemented using inexpensive consumer electronic devices such as a laser pointer and a webcam. The use of a micro-capillary tube as a blood container also enables the hemoglobin assay with a nanoliter-scale blood volume and a low operating cost. Here, detailed instructions for the PT-AS optical setup and signal processing procedures are presented. Experimental protocols and representative results for blood samples in anemic conditions ([Hb] = 5.3, 7.5, and 9.9 g/dl) are also provided, and the measurements are compared with those from a hematology analyzer. Its simplicity in implementation and operation should enable its wide adoption in clinical laboratories and resource-limited settings.

A photo-thermal angular light scattering (PT-AS) sensor enables the rapid and chemical-free hemoglobin assay of nanoliter-scale blood samples. Here, details of the PT-AS setup and a measurement protocol for the hemoglobin concentration in blood are provided. Representative results for anemic blood samples are also presented.

Eine schnelle und Chemiefreie Hämoglobin-Assay mit Photothermische Winkellichtstreuung

Photo-thermal angular light scattering (PT-AS) is a novel optical method for measuring the hemoglobin concentration ([Hb]) of blood samples. On the basis ...