Zuschüsse hat NHLBI HL080532, NHLBI HL073449, NCRR RR16453 und ein AHA Nationale Grant-in Aid Award (RVS) enthalten. Ein besonderer Dank geht an die Entfernung Course Design und Consulting (DCDC)-Gruppe, dcdcgroup.org, für ihre Unterstützung mit Video-Produktion und dem US Department of Education Grant No P336C050047, dass die DCDC gegründet verlängert.

1. Mikroblasen Stoffaufbereitung <ol><li> In 10 ml PBS-Mix 200 mg 1,2-Dipalmitoyl-sn-glycero-3-Phosphatidylcholin und 50 mg 1,2-Dipalmitoyl-sn-glycero-3-Phosphatidylethanolamin mit 1 g Glucose. </li><li> Erhitzen Sie die Mischung in kochendes Wasserbad 20-30 Minuten, Pipette Mischen alle 5 Minuten. </li><li> Die Lösung kann bei 4 ° C gelagert werden für bis zu 6 Monaten. </li></ol> 2. Mikroblasen Vorbereitung <ol><li> Werfen Sie einen 250 ul der vorbereiteten Mikroblasen Stammlösung und Inkubieren bei 40 ° C für 15 Minuten. </li><li> Die vorgewärmte Mikroblasen Lösung wird dann auf einem 1,5 mL Mikroröhrchen mit 50 ul von Glycerin übertragen. </li><li> 1-2 mg der gereinigten Plasmid-DNA, die einen Ausdruck für das Gen von Interesse (gereinigt in diesem Beispiel von Qiagen Endotoxin frei MegaPrep Kit, Qiagen, Germantown, MD, mit einer optimalen Konzentration von 4mg/ml) zu konstruieren. 2,4) Phosphat-gepufferte Salzlösung ist, um ein Endvolumen von 500 ul zugegeben. Endotoxin frei Qiagen maxipreps verwendet werden, sowie sterile PBS, um die Sterilität zu gewährleisten. </li><li> Die Luft in der Mikroröhrchen wird dann mit Octafluorpropan Gas ersetzt. </li><li> Die Mikroröhrchen wird dann kräftig in eine zahnärztliche Amalgamator für 20 Sekunden geschüttelt. </li><li> Der Unterstand mit Rest-DNA und Puffer, die nicht der Mikrobläschen ist gebunden wird dann vorsichtig entfernt und die Mikrobläschen-Schicht wird dreimal mit sterilem PBS gewaschen, um ungebundene DNA zu entfernen, und auf Eis zwischen jedem Waschgang. Wir in der Regel erreichen eine verbindliche Effizienz von 30-40%. Alle Reagenzien sind steril und Sorgfalt gemacht, um eine Kontamination zu minimieren. </li><li> Die Plasmid-DNA-gebundenen Mikrobläschen werden dann auf Eis gelegt bis zu zwei Stunden bis zur Verwendung. </li><li> Der Unterstand aus dem Mikroröhrchen nach dem Mischen und dem PBS Waschungen entfernt werden, können verwendet werden, um die Konzentration der ungebundenen DNA, und ebenso die Menge gebunden, auf die bekannten ursprünglichen Konzentration zu bestimmen, durch Messung der optischen Dichte der Lösung bei einer Wellenlänge von 260nm mit einem Spektralphotometer. </li></ol> 3. Gerätekalibrierung <ol><li> Vor dem ersten Gebrauch muss ein 1 MHz Kavitation Wandler zu kalibrieren, um eine ordnungsgemäße mechanische Index und Impulsfolgefrequenz zu gewährleisten. Eine Tauchpumpe 1 MHz, 13mm, unscharf Wandler ist ein 20 MHz Funktion / Arbitrary Waveform Generator über einen Leistungsverstärker verbunden. </li><li> Der Wandler ist in einem Kunststoff-Behälter voller Wasser platziert, direkt an einem Hydrophon, was zu einem 500-MHz-Oszilloskop wurde über einen Ladungsverstärker verbunden abzielen. </li><li> Waveform können Frequenz, Amplitude, Burst-Zyklus, und Leistungsverstärkung alle geändert, um die ordnungsgemäße duty cycle und mechanischen Index optimal auf die Mikrobläschen kavitieren erhalten. Für dieses spezielle Experiment haben wir das System auf einem mechanischen Index entspricht ~ 1,3 bei 1 MHz kalibriert. </li></ol> 4. Mikroblasen Lieferung &amp; UTMD <ol><li> Vor Mikroblasen Lieferung und UTMD wurden C57BL / 6 Mäuse mit 100mg/kg Ketamin und Xylazin 5mg/kg durch IP Injektion. </li><li> Mikroblasen Lieferung wurde intravenös über eine direkte Injektion in den linken Ventrikel des Herzens oder durch Schwanzvene Katheterisierung verabreicht. Für die direkte kardiale Injektion wird ein Volumen von bis zu 100 ul der Plasmid-DNA-beladenen Mikrobläschen Lösung durch eine 30-Gauge-Nadel an der vorderen 4 th Interkostalraum unter Ultraschall-Visualisierung eingefügt in die linke Herzkammer injiziert. </li><li> Die Mikrobläschen Lösung Bolus aus dem linken Ventrikel Injektion wird visualisiert mit Visual Sonics &quot;38 MHz Hochfrequenz-Schallkopf in einer stationären Position auf dem Thorax der Maus in eine lange Achse Ansicht mit VisualSonics gelegt&quot; Vevo 2100 Imaging System. Alle Spritzen und Nadeln sind steril, die Versicherung der sterilen Umgebung möglich, dass diese Injektionen. </li><li> Unmittelbar nach der Injektion wird Mikroblasen Zerstörung out für ca. 5 Minuten mit einem zweiten, kleineren niedrige Frequenz von 1,0 MHz Schallkopf statt direkt über das gewünschte Organ, Targeting Zerstörung dieser Region durchgeführt. In diesem Beispiel wurde der Ultraschall in die Leber bei einer Pulsfrequenz von 1,0 MHz verabreicht wird, mit einem mechanischen Index entspricht ca. 1,3-1,5 und Impulsfolgefrequenz Zeitraum von 100 ms für alle 20 Zyklen. Alternativ kann der Puls gated die EKG der Maus (in diesem Experiment nicht gezeigt), um einen Ausbruch von 3 Bilder von Ultraschall, alle 4-6 Herzzyklen. Wir erhalten eine höhere Effizienz der Transfektion mit Protokollen, die das Kapillarbett können zum Nachfüllen mit Blasen zwischen den Bursts von Ultraschall. </li></ol> 5. Alternative Lieferung Methode Wir entschieden uns für die interventrikulären Injektion aufgrund der Komplexität des Verfahrens Highlight, aber in vielen Fällen, wie zB längere Infusion von Mikrobläschen, eine Schwanzvene injection ist die bevorzugte Methode. Für die Schwanz-Vene Methode der Mikrobläschen Lieferung, ist die Maus betäubt die gleiche Weise. Eine Spritze mit der Plasmid-DNA-gebundenen Mikrobläschen ist es, eine 27-Gauge-Nadel / Schwanzvene Katheter verbunden. Die Schwanzvene Katheter in die distalen Drittel der entweder den rechten oder linken seitlichen Adern, die entlang der Schwanz der Maus eingefügt. Die Spritze mit der Mikrobläschen in einer Infusionspumpe, die automatisch verwaltet eine einheitliche voreingestellten Volumen der Lösung über eine vorgegebene Zeit gelegt. Wir ziehen in der Regel 200-300μl einer Rate von 3ml/hour. Tierversuche Alle Tiere wurden in Übereinstimmung mit guter Tier Praxis von den zuständigen nationalen und / oder lokale Tierschutz Körper definiert behandelt, und alle tierischen Arbeit wurde durch den zuständigen Ausschuss genehmigt (University of Hawaii Institutional Animal Care und Verwenden Committee, Zulassungsnummer 07-100 -3). Entsprechende Narkose (Ketamin / zylazine) verwendet wurde und Analgetika (Bupivacain und Buprenorphin) zur Verfügung standen, aber nicht erforderlich. 6. Repräsentative Ergebnisse: Die Wirksamkeit des UTMD-vermittelte Plasmid-DNA Lieferung kann durch eine Vielzahl von Methoden abhängig von der Gene in das Konstrukt, wie codierte ausgewertet werden, aber nicht beschränkt; Luciferase in-vivo-Bildgebung, B-gal ex vivo-Färbung, und / oder Immunhistologie. Insbesondere in vivo Biolumineszenz-Bildgebung ermöglicht es, die Anwesenheit und die Dauer der Genexpression seriell in Mäusen mit einem Plasmid kodierend für eine Biolumineszenz-Reportergen (Luciferase) transfiziert überwachen. Die Xenogen In Vivo Imaging System (IVIS) (Caliper Life Sciences, Hopkinton, MA) ist für die Biolumineszenz Bildgebung verwendet. Die Bilder werden in der Regel von allen Mäusen ersten Tag nach UTMD vermittelte Transfektion genommen und wiederholt sich alle drei vor vier Tagen bis Biolumineszenz Genexpression ist nicht mehr visuell erkennbar durch das System (Abbildung 1). Zur Vorbereitung Mäuse für Biolumineszenz Bildgebung, Mäuse zuerst eine IP Injektion des Luciferase-Reporter-Sonde D-Luciferin (Caliper Life Sciences) und dann narkotisiert ~ 3 Minuten später. Bioverteilung der D-Luciferin-Substrat darf für ~ 10 Minuten ablaufen, bevor das Tier in der IVIS Bildgebung Kammer platziert und ein Ganzkörper-Bild zu scannen genommen wird. Während der Akquisition werden die Photonen aus der Firefly-Luciferase / D-Luciferin photochemische Reaktion emittiert gemessen. Abbildung 1 zeigt auch, ähnlich IVIS Biolumineszenz Bildgebung der Leber nach UTMD und Abbildung 2 ist ein Epifluoreszenz (100X) Bild der transfizierten Leber mit Hilfe eines anti-Luciferase primärer Antikörper (Sigma-Aldrich) und AlexFluor-568 konjugierten sekundären Antikörper (Invitrogen) . Es ist klar zu sehen, dass die UTMD vermittelte Transfektion Leber hat nicht nur die Endothelzellen betroffen, aber die hepatocyes auch. <img src="/files/ftp_upload/2963/2963fig1.jpg" alt="Abbildung 1" /> Abbildung 1 Xenogen / IVIS Bildgebung des Herzens UTMD behandelten Mäusen (A) Negative Control Mouse:.. Plasmid + PBS, gefolgt von Herz gerichtet UTMD, (B) Behandlung Mouse: Plasmide + Mikrobläschen durch Herz gefolgt gerichtet UTMD und (C) Behandlung Maus : Plasmid + Mikrobläschen von Leber, gefolgt gerichtet UTMD. <img src="/files/ftp_upload/2963/2963fig2.jpg" alt="Abbildung 2" /> Abbildung 2. Immunhistochemie der Leber UTMD (anti-Luciferase in rot). (A) Plasmid ohne UTMD und (B) Plasmid mit UTMD. Konfokale Bild (100X); Kerne sind DAPI blau angefärbt.

<ol>
	<li>Bekeredjian, R., Chen, S., Frenkel, P. A., Grayburn, P. A., Shohet, R. V. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Ultrasound-targeted+microbubble+destruction+can+repeatedly+direct+highly+specific+plasmid+expression+to+the+heart.">Ultrasound-targeted microbubble destruction can repeatedly direct highly specific plasmid expression to the heart.</a> Circulation. 108, 1022-1026 (2003).</li><li>Bekeredjian, R., Katus, H. A., Kuecherer, H. F. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Therapeutic+use+of+ultrasound+targeted+microbubble+destruction:+a+review+of+non-cardiac+applications.">Therapeutic use of ultrasound targeted microbubble destruction: a review of non-cardiac applications.</a> Ultraschall Med. 27, 134-140 (2006).</li><li>Chen, S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Regeneration+of+pancreatic+islets+in+vivo+by+ultrasound-targeted+gene+therapy.">Regeneration of pancreatic islets in vivo by ultrasound-targeted gene therapy.</a> Gene Ther. 17, 1411-1420 (2010).</li><li>Miao, C. H. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Ultrasound+enhances+gene+delivery+of+human+factor+IX+plasmid.">Ultrasound enhances gene delivery of human factor IX plasmid.</a> Hum Gene Ther. 16, 893-905 (2005).</li><li>Shimoda, M., Chen, S., Noguchi, H., Matsumoto, S., Grayburn, P. A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=In+vivo+non-viral+gene+delivery+of+human+vascular+endothelial+growth+factor+improves+revascularisation+and+restoration+of+euglycaemia+after+human+islet+transplantation+into+mouse+liver.">In vivo non-viral gene delivery of human vascular endothelial growth factor improves revascularisation and restoration of euglycaemia after human islet transplantation into mouse liver.</a> Diabetologia. 53, 1669-1679 (2010).</li><li>Shohet, R. V. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Echocardiographic+destruction+of+albumin+microbubbles+directs+gene+delivery+to+the+myocardium.">Echocardiographic destruction of albumin microbubbles directs gene delivery to the myocardium.</a> Circulation. 101, 2554-2556 (2000).</li><li>Sirsi, S., Borden, M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Microbubble+Compositions,+Properties+and+Biomedical+Applications.">Microbubble Compositions, Properties and Biomedical Applications.</a> Bubble Sci Eng Technol. 1, 3-17 (2009).</li><li>Li, H. L. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Ultrasound-targeted+microbubble+destruction+enhances+AAV-mediated+gene+transfection+in+human+RPE+cells+in+vitro+and+rat+retina+in+vivo.">Ultrasound-targeted microbubble destruction enhances AAV-mediated gene transfection in human RPE cells in vitro and rat retina in vivo.</a> Gene Ther. 16, 1146-1153 (2009).</li><li>Lindner, J. R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Microbubbles+in+medical+imaging:+current+applications+and+future+directions.">Microbubbles in medical imaging: current applications and future directions.</a> Nat Rev Drug Discov. 3, 527-532 (2004).</li><li>Newman, C. M., Bettinger, T. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Gene+therapy+progress+and+prospects:+ultrasound+for+gene+transfer.">Gene therapy progress and prospects: ultrasound for gene transfer.</a> Gene Ther. 14, 465-475 (2007).</li><li>Vancraeynest, D. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Myocardial+injury+induced+by+ultrasound-targeted+microbubble+destruction:+evidence+for+the+contribution+of+myocardial+ischemia.">Myocardial injury induced by ultrasound-targeted microbubble destruction: evidence for the contribution of myocardial ischemia.</a> Ultrasound Med Biol. 35, 672-679 (2009).</li></ol>

UTMD stellt einen neuartigen Ansatz zur Gentransfer. Als Plattform-Technologie kann es mit jedem der vielen möglichen Gentherapie-Strategien kombiniert werden, um eine Vielzahl von bioaktiven Molekülen, wenn ein hohes Maß an Gewebsspezifität gewünscht zu liefern. Die wichtigsten biologischen Einschränkung der Technik ist die geringe Effizienz der Transfektion. Ein weiterer wichtiger Aspekt ist die Zugänglichkeit der Zielorgan zu Ultraschall, die deutlich durch Eingriffe Knochen oder in der Luft verringert werden ...

<table border="1"><tbody><tr><th> Name des Reagenzes / Ausrüstung </th><th> Firma </th><th> Katalog-Nummer </th><th> Kommentare </th></tr><tr><td> 1,2-Dipalmitoyl-sn-glycero-3-Phosphatidylcholin </td><td> Sigma-Aldrich </td><td> P-5911 </td><td> Bestandteil der Mikrobläschen Lipidhülle </td></tr><tr><td> 1,2-Dipalmitoyl-sn-glycero-3-Phosphatidylethanolamin </td><td> Sigma-Aldrich </td><td> P-3275 </td><td> Bestandteil der Mikrobläschen Lipidhülle </td></tr><tr><td> Glucose </td><td> Sigma-Aldrich </td><td> G5400 </td><td> Gedanken über die Stabilisierung von Mikrobläschen </td></tr><tr><td> Phosphat-gepufferter Kochsalzlösung </td><td> Sigma-Aldrich </td><td> P5368 </td><td></td></tr><tr><td> Glycerin </td><td> Sigma-Aldrich </td><td> G5516 </td><td> vermutlich Mikrobläschen aus Koaleszenz zu verhindern </td></tr><tr><td> Octafluorpropan Gas </td><td> Airgas </td><td> N / A </td><td> Inertgas in klinischen Anwendungen eingesetzt </td></tr><tr><td> Vialmix zahnärztliche Amalgamator </td><td> Bristol-Myers Squibb </td><td> N / A </td><td></td></tr><tr><td> 1 MHz, 13mm, unscharf Wandler </td><td> Olymp </td><td> A303S-SU </td><td></td></tr><tr><td> 20 MHz Funktion / Arbitrary Waveform Generator </td><td> Agilent </td><td> 33220A </td><td></td></tr><tr><td> Power Amplifier </td><td> Krohn-Hite Co. </td><td> Modell 7500 </td><td></td></tr><tr><td> Hydrophone </td><td> Bruel und Kjaer </td><td> Type 1803 </td><td></td></tr><tr><td> Ladungsverstärker </td><td> Bruel und Kjaer </td><td> Type 2634 </td><td></td></tr><tr><td> 500 MHz Oszilloskop </td><td> LeCroy </td><td> 9354L </td><td></td></tr><tr><td> VisualSonics Vevo 2100 Imaging System mit 34-MHz-Schallkopf </td><td> VisualSonics </td><td> 2100 </td><td></td></tr><tr><td> 27G ein Zoll Schwanzvene Kathetern </td><td> VisualSonics </td><td> N / A </td><td></td></tr><tr><td> Genie Plus-Infusionspumpe </td><td> Kent Scientific </td><td> GENIE </td><td></td></tr></tbody></table>

introduction to the ultrasound targeted microbubble destruction technique

In UTMD, bioaktive Moleküle, wie negativ geladenen Plasmid-DNA-Vektoren kodieren ein Gen von Interesse sind, um die kationische Schalen von Lipid-Mikrobläschen-Kontrastmittel 7-9 aufgenommen. In Mäusen diesen Vektor-tragenden Mikrobläschen kann intravenös oder direkt an den linken Ventrikel des Herzens verabreicht werden. In größeren Tieren können sie auch durch eine intrakoronare Katheter infundiert werden. Die Nachlieferung aus dem Kreislauf zu einem Zielorgan erfolgt durch akustische Kavitation bei einer Resonanzfrequenz der Mikrobläschen. Es scheint wahrscheinlich, dass die mechanische Energie, die durch die Zerstörung von Mikrobläschen Ergebnisse in transient Porenbildung in oder zwischen den Endothelzellen der Mikrovaskulatur der Zielregion 10 erzeugt. Als Ergebnis dieser Sonoporation Effekt ist die Transfektionseffizienz in und über den Endothelzellen verbessert und Transgen-kodierende Vektoren sind in das umgebende Gewebe abgelagert. Plasmid-DNA im Blutkreislauf schnell abgebaut wird durch Nukleasen in das Blut, das reduziert die Wahrscheinlichkeit von Lieferungen in Nicht-beschallten Gewebe und führt zu sehr spezifischen Ziel-Orgel Transfektion.

Watch this Scientific Journal Video about Introduction to the Ultrasound Targeted Microbubble Destruction Technique at JoVE.com

Introduction to the Ultrasound Targeted Microbubble Destruction Technique

Ultraschall gezielte Mikroblasen Destruction (UTMD) können verwendet werden, um ortsspezifische Abgabe von bioaktiven Molekülen, einschließlich therapeutischer Gene zu richten, um Organe zugänglich Ultraschall, wie Herz und Leber Ziel sein<sup&gt; 1-6</sup&gt;.

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 ...

introduction-to-ultrasound-targeted-microbubble-destruction

Research

JoVE Journal

Bioengineering

15.5K Aufrufe.  University of Hawaii. Ultraschall gezielte Mikroblasen Destruction (UTMD) können verwendet werden, um ortsspezifische Abgabe von bioaktiven Molekülen, einschließlich therapeutischer Gene zu richten, um Organe zugänglich Ultraschall, wie Herz und Leber Ziel sein 1-6.

Serie: Introduction to the Ultrasound Targeted Microbubble Destruction Technique

Contrast Ultrasound Targeted Treatment of Gliomas in Mice via Drug-Bearing Nanoparticle Delivery and Microvascular Ablation

We are developing minimally-invasive contrast agent microbubble based therapeutic approaches in which the permeabilization and/or ablation of the microvasculature are controlled by varying ultrasound pulsing parameters.  Specifically, we are testing whether such approaches may be used to treat malignant brain tumors through drug delivery and microvascular ablation.  Preliminary studies have been performed to determine whether targeted drug-bearing nanoparticle delivery can be facilitated by the ultrasound mediated destruction of "composite" delivery agents comprised of 100nm poly(lactide-co-glycolide) (PLAGA) nanoparticles that are adhered to albumin shelled microbubbles. We denote these agents as microbubble-nanoparticle composite agents (MNCAs). When targeted to subcutaneous C6 gliomas with ultrasound, we observed an immediate 4.6-fold increase in nanoparticle delivery in MNCA treated tumors over tumors treated with microbubbles co-administered with nanoparticles and a 8.5 fold increase over non-treated tumors. Furthermore, in many cancer applications, we believe it may be desirable to perform targeted drug delivery in conjunction with ablation of the tumor microcirculation, which will lead to tumor hypoxia and apoptosis. To this end, we have tested the efficacy of non-theramal cavitation-induced microvascular ablation, showing that this approach elicits tumor perfusion reduction, apoptosis, significant growth inhibition, and necrosis. Taken together, these results indicate that our ultrasound-targeted approach has the potential to increase therapeutic efficiency by creating tumor necrosis through microvascular ablation and/or simultaneously enhancing the drug payload in gliomas.

Insonation of microbubbles is a promising strategy for tumor ablation at reduced time-averaged acoustic powers, as well as for the targeted delivery of therapeutics. The purpose of the present study is to develop low duty cycle ultrasound pulsing strategies and nanocarriers to maximize non-thermal microvascular ablation and payload delivery to subcutaneous C6 gliomas.

Contrast Ultrasound gezielte Behandlung von Gliomen in Mäusen über Drug-Bearing Nanopartikel Liefer-und mikrovaskuläre Ablation

We are developing minimally-invasive contrast agent microbubble based therapeutic approaches in which the permeabilization and/or ablation of the ...

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 ...

Analysis of Targeted Viral Protein Nanoparticles Delivered to HER2+ Tumors

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

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

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. ...

Wideband Optical Detector of Ultrasound for Medical Imaging Applications

Optical sensors of ultrasound are a promising alternative to piezoelectric techniques, as has been recently demonstrated in the field of optoacoustic imaging. In medical applications, one of the major limitations of optical sensing technology is its susceptibility to environmental conditions, e.g. changes in pressure and temperature, which may saturate the detection. Additionally, the clinical environment often imposes stringent limits on the size and robustness of the sensor. In this work, the combination of pulse interferometry and fiber-based optical sensing is demonstrated for ultrasound detection. Pulse interferometry enables robust performance of the readout system in the presence of rapid variations in the environmental conditions, whereas the use of all-fiber technology leads to a mechanically flexible sensing element compatible with highly demanding medical applications such as intravascular imaging. In order to achieve a short sensor length, a pi-phase-shifted fiber Bragg grating is used, which acts as a resonator trapping light over an effective length of 350 &#181;m. To enable high bandwidth, the sensor is used for sideway detection of ultrasound, which is highly beneficial in circumferential imaging geometries such as intravascular imaging. An optoacoustic imaging setup is used to determine the response of the sensor for acoustic point sources at different positions.

Optical detection of ultrasound is impractical in many imaging scenarios because it often requires stable environmental conditions. We demonstrate an optical technique for ultrasound sensing in volatile environments with miniaturization and sensitivity levels appropriate for optoacoustic imaging in restrictive scenarios, e.g. intravascular applications.

Wideband Optical Detector von Ultraschall für medizinische Imaging-Anwendungen

Optical sensors of ultrasound are a promising alternative to piezoelectric techniques, as has been recently demonstrated in the field of optoacoustic ...

Manufacture of Concentrated, Lipid-based Oxygen Microbubble Emulsions by High Shear Homogenization and Serial Concentration

Gas-filled microbubbles have been developed as ultrasound contrast and drug delivery agents. Microbubbles can be produced by processing surfactants using sonication, mechanical agitation, microfluidic devices, or homogenization. Recently, lipid-based oxygen microbubbles (LOMs) have been designed to deliver oxygen intravenously during medical emergencies, reversing life-threatening hypoxemia, and preventing subsequent organ injury, cardiac arrest, and death. We present methods for scaled-up production of highly oxygenated microbubbles using a closed-loop high-shear homogenizer. The process can produce 2 L of concentrated LOMs (90% by volume) in 90 min. Resulting bubbles have a mean diameter of ~2 &#956;m, and a rheologic profile consistent with that of blood when diluted to 60 volume %. This technique produces LOMs in high capacity and with high oxygen purity, suggesting that this technique may be useful for translational research labs.

We describe methods for the manufacture of large volumes of lipid-based oxygen microbubbles (LOMs) designed for intravenous oxygen delivery using high-shear homogenization and serial concentration.

Herstellung von konzentrierten, Lipid-basierte Sauerstoffmikroblasen-Emulsionen von High-Shear-Homogenisierung und Serien Konzentration

Gas-filled microbubbles have been developed as ultrasound contrast and drug delivery agents. Microbubbles can be produced by processing surfactants using ...

A Microfluidic Technique to Probe Cell Deformability

Here we detail the design, fabrication, and use of a microfluidic device to evaluate the deformability of a large number of individual cells in an efficient manner. Typically, data for ~102 cells can be acquired within a 1 hr experiment. An automated image analysis program enables efficient post-experiment analysis of image data, enabling processing to be complete within a few hours. Our device geometry is unique in that cells must deform through a series of micron-scale constrictions, thereby enabling the initial deformation and time-dependent relaxation of individual cells to be assayed. The applicability of this method to human promyelocytic leukemia (HL-60) cells is demonstrated. Driving cells to deform through micron-scale constrictions using pressure-driven flow, we observe that human promyelocytic (HL-60) cells momentarily occlude the first constriction for a median time of 9.3 msec before passaging more quickly through the subsequent constrictions with a median transit time of 4.0 msec per constriction. By contrast, all-trans retinoic acid-treated (neutrophil-type) HL-60 cells occlude the first constriction for only 4.3 msec before passaging through the subsequent constrictions with a median transit time of 3.3 msec. This method can provide insight into the viscoelastic nature of cells, and ultimately reveal the molecular origins of this behavior.

We demonstrate a microfluidics-based assay to measure the timescale for cells to transit through a sequence of micron-scale constrictions.

Ein Mikrofluidiktechnik to Cell Verformbarkeit Sonde

Here we detail the design, fabrication, and use of a microfluidic device to evaluate the deformability of a large number of individual cells in an ...

A Microfluidic Chip for ICPMS Sample Introduction

This protocol discusses the fabrication and usage of a disposable low cost microfluidic chip as sample introduction system for inductively coupled plasma mass spectrometry (ICPMS). The chip produces monodisperse aqueous sample droplets in perfluorohexane (PFH). Size and frequency of the aqueous droplets can be varied in the range of 40 to 60 &#181;m and from 90 to 7,000&#160;Hz, respectively. The droplets are ejected from the chip with a second flow of PFH and remain intact during the ejection. A custom-built desolvation system removes the PFH and transports the droplets into the ICPMS. Here, very stable signals with a narrow intensity distribution can be measured, showing the monodispersity of the droplets. We show that the introduction system can be used to quantitatively determine iron in single bovine red blood cells. In the future, the capabilities of the introduction device can easily be extended by the integration of additional microfluidic modules.

We present a discrete droplet sample introduction system for inductively coupled plasma mass spectrometry (ICPMS). It is based on a cheap and disposable microfluidic chip that generates highly monodisperse droplets in a size range of 40&#8722;60 &#181;m at frequencies from 90 to 7,000 Hz.

Eines mikrofluidischen Chips für ICPMS Probenaufgabe

This protocol discusses the fabrication and usage of a disposable low cost microfluidic chip as sample introduction system for inductively coupled plasma ...

An Experimental Protocol for Assessing the Performance of New Ultrasound Probes Based on CMUT Technology in Application to Brain Imaging

The possibility to perform an early and repeatable assessment of imaging performance is fundamental in the design and development process of new ultrasound (US) probes. Particularly, a more realistic analysis with application-specific imaging targets can be extremely valuable to assess the expected performance of US probes in their potential clinical field of application.
The experimental protocol presented in this work was purposely designed to provide an application-specific assessment procedure for newly-developed US probe prototypes based on Capacitive Micromachined Ultrasonic Transducer (CMUT) technology in relation to brain imaging.
The protocol combines the use of a bovine brain fixed in formalin as the imaging target, which ensures both realism and repeatability of the described procedures, and of neuronavigation techniques borrowed from neurosurgery. The US probe is in fact connected to a motion tracking system which acquires position data and enables the superposition of US images to reference Magnetic Resonance (MR) images of the brain. This provides a means for human experts to perform a visual qualitative assessment of the US probe imaging performance and to compare acquisitions made with different probes. Moreover, the protocol relies on the use of a complete and open research and development system for US image acquisition, i.e. the Ultrasound Advanced Open Platform (ULA-OP) scanner.
The manuscript describes in detail the instruments and procedures involved in the protocol, in particular for&#160;the calibration, image acquisition and registration of US and MR images. The obtained results prove the effectiveness of the overall protocol presented, which is entirely open (within the limits of the instrumentation involved), repeatable, and covers the entire set of acquisition and processing activities for US images.

The development of new ultrasound (US) probes based on Capacitive Micromachined Ultrasonic Transducer (CMUT) technology requires an early realistic assessment of imaging capabilities. We describe a repeatable experimental protocol for US image acquisition and comparison with magnetic resonance images, using an ex vivo bovine brain as an imaging target.

Ein experimentelles Protokoll zur Bewertung der Leistung des neuen Ultraschallsonden basierend auf CMUT Technologie in Anwendung auf die Bildgebung des Gehirns

The possibility to perform an early and repeatable assessment of imaging performance is fundamental in the design and development process of new ...

Comprehensive Evaluation of the Effectiveness and Safety of Placenta-Targeted Drug Delivery Using Three Complementary Methods

No effective treatments currently exist for placenta-associated pregnancy complications, and developing strategies for the targeted delivery of drugs to the placenta while minimizing fetal and maternal side effects remains challenging. Targeted nanoparticle carriers provide new opportunities to treat placental disorders. We recently demonstrated that a synthetic placental chondroitin sulfate A binding peptide (plCSA-BP) could be used to guide nanoparticles to deliver drugs to the placenta. In this protocol, we describe in detail a system for assessing the efficiency of drug delivery to the placenta by plCSA-BP that employs three separate methods used in combination: in vivo imaging, high-frequency ultrasound (HFUS), and high-performance liquid chromatography (HPLC). Using in vivo imaging, plCSA-BP-guided nanoparticles were visualized in the placentas of live animals, while HFUS and HPLC demonstrated that plCSA-BP-conjugated nanoparticles efficiently and specifically delivered methotrexate to the placenta. Thus, a combination of these methods can be used as an effective tool for the targeted delivery of drugs to the placenta and development of new treatment strategies for several pregnancy complications.

We describe a system that utilizes three methods to evaluate the safety and effectiveness of placenta-targeted drug delivery: in vivo imaging to monitor nanoparticle accumulation, high-frequency ultrasound to monitor placental and fetal development, and HPLC to quantify drug delivery to tissue.

Umfassende Bewertung der Wirksamkeit und Sicherheit von Plazenta-gezielte Medikamentenverabreichung mit drei komplementäre Methoden

No effective treatments currently exist for placenta-associated pregnancy complications, and developing strategies for the targeted delivery of drugs to ...

Preparing Protein Producing Synthetic Cells using Cell Free Bacterial Extracts, Liposomes and Emulsion Transfer

The bottom-up assembly approach for construction of synthetic cells is an effective tool for isolating and investigating cellular processes in a cell mimicking environment. Furthermore, the development of cell-free expression systems has demonstrated the ability to reconstitute the protein production, transcription and translation processes (DNA&#8594;RNA&#8594;protein) in a controlled manner, harnessing synthetic biology. Here we describe a protocol for preparing a cell-free expression system, including the production of a potent bacterial lysate and encapsulating this lysate inside cholesterol-rich lipid-based giant unilamellar vesicles (GUVs) (i.e., stable liposomes), to form synthetic cells. The protocol describes the methods for preparing the components of the synthetic cells including the production of active bacterial lysates, followed by a detailed step-by-step preparation of the synthetic cells based on a water-in-oil emulsion transfer method. These facilitate the production of millions of synthetic cells in a simple and affordable manner with a high versatility for producing different types of proteins. The obtained synthetic cells can be used to investigate protein/RNA production and activity in an isolated environment, in directed evolution, and also as a controlled drug delivery platform for on-demand production of therapeutic proteins inside the body.

This protocol describes the method, materials, equipment and steps for bottom-up preparation of RNA and protein producing synthetic cells. The inner aqueous compartment of the synthetic cells contained the S30 bacterial lysate encapsulated within a lipid bilayer (i.e., stable liposomes), using a water-in-oil emulsion transfer method.

Herstellung von Proteinproduktion synthetischer Zellen mit zellfreien bakteriellen Extrakten, Liposomen und Emulsionstransfer

The bottom-up assembly approach for construction of synthetic cells is an effective tool for isolating and investigating cellular processes in a cell ...