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

Universidad de Cartagena UDEC

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

Contrast Imaging in Mouse Embryos Using High-frequency Ultrasound

Ultrasound contrast-enhanced imaging can convey essential quantitative information regarding tissue vascularity and perfusion and, in targeted applications, facilitate the detection and measure of vascular biomarkers at the molecular level. Within the mouse embryo, this noninvasive technique may be used to uncover basic mechanisms underlying vascular development in the early mouse circulatory system and in genetic models of cardiovascular disease. The mouse embryo also presents as an excellent model for studying the adhesion of microbubbles to angiogenic targets (including vascular endothelial growth factor receptor 2 (VEGFR2) or &#945;v&#946;3) and for assessing the quantitative nature of molecular ultrasound. We therefore developed a method to introduce ultrasound contrast agents into the vasculature of living, isolated embryos. This allows freedom in terms of injection control and positioning, reproducibility of the imaging plane without obstruction and motion, and simplified image analysis and quantification. Late gestational stage (embryonic day (E)16.6 and E17.5) murine embryos were isolated from the uterus, gently exteriorized from the yolk sac and microbubble contrast agents were injected into veins accessible on the chorionic surface of the placental disc. Nonlinear contrast ultrasound imaging was then employed to collect a number of basic perfusion parameters (peak enhancement, wash-in rate and time to peak) and quantify targeted microbubble binding in an endoglin mouse model. We show the successful circulation of microbubbles within living embryos and the utility of this approach in characterizing embryonic vasculature and microbubble behavior.

Here, we present a protocol to inject ultrasound microbubble contrast agents into living, isolated late-gestation stage murine embryos. This method enables the study of perfusion parameters and of vascular molecular markers within the embryo using contrast-enhanced high-frequency ultrasound imaging.

Contrast Imaging in Maus Embryonen Verwendung von Hochfrequenz-Ultraschall

Ultrasound contrast-enhanced imaging can convey essential quantitative information regarding tissue vascularity and perfusion and, in targeted ...

Entwicklungsbiologie

MRI-guided Disruption of the Blood-brain Barrier using Transcranial Focused Ultrasound in a Rat Model

Focused ultrasound (FUS) disruption of the blood-brain barrier (BBB) is an increasingly investigated technique for circumventing the BBB1-5. The BBB is a significant obstacle to pharmaceutical treatments of brain disorders as it limits the passage of molecules from the vasculature into the brain tissue to molecules less than approximately 500 Da in size6. FUS induced BBB disruption (BBBD) is temporary and reversible4 and has an advantage over chemical means of inducing BBBD by being highly localized. FUS induced BBBD provides a means for investigating the effects of a wide range of therapeutic agents on the brain, which would not otherwise be deliverable to the tissue in sufficient concentration. While a wide range of ultrasound parameters have proven successful at disrupting the BBB2,5,7, there are several critical steps in the experimental procedure to ensure successful disruption with accurate targeting. This protocol outlines how to achieve MRI-guided FUS induced BBBD in a rat model, with a focus on the critical animal preparation and microbubble handling steps of the experiment.

Microbubble-mediated focused ultrasound disruption of the blood-brain barrier (BBB) is a promising technique for non-invasive targeted drug delivery in the brain1-3. This protocol outlines the experimental procedure for MRI-guided transcranial BBB disruption in a rat model.

MRI-geführten Störung der Blut-Hirn-Schranke mit Hilfe der transkraniellen fokussiertem Ultraschall in einem Rattenmodell

Focused ultrasound (FUS) disruption of the blood-brain barrier (BBB) is an increasingly investigated technique for circumventing the BBB1-5. The BBB is a ...

Medizin

Induction of Microstreaming by Nonspherical Bubble Oscillations in an Acoustic Levitation System

When located near biological barriers, oscillating microbubbles may increase cell membrane permeability, allowing for drug and gene internalization. Experimental observations suggest that the temporary permeabilization of these barriers may be due to shear stress that is exerted on cell tissues by cavitation microstreaming. Cavitation microstreaming is the generation of vortex flows which arise around oscillating ultrasound microbubbles. To produce such liquid flows, bubble oscillations must deviate from purely spherical oscillations and include either a translational instability or shape modes. Experimental studies of bubble-induced flows and shear stress on nearby surfaces are often restricted in their scope due to the difficulty of capturing shape deformations of microbubbles in a stable and controllable manner. We describe the design of an acoustic levitation chamber for the study of symmetry-controlled nonspherical oscillations. Such control is performed by using a coalescence technique between two approaching bubbles in a sufficiently intense ultrasound field. The control of nonspherical oscillations opens the way to a controlled cavitation microstreaming of a free surface-oscillating microbubble. High-frame rate cameras allow investigating quasi-simultaneously the nonspherical bubble dynamics at the acoustic timescale and the liquid flow at a lower timescale. It is shown that a large variety of fluid patterns may be obtained and that they are correlated to the modal content of the bubble interface. We demonstrate that even the high-order shape modes can create large-distance fluid patterns if the interface dynamics contain several modes, highlighting the potential of nonspherical oscillations for targeted and localized drug delivery.

A fast and reliable technique is proposed to control the shape oscillations of a single, trapped acoustic bubble that is based on coalescence technique between two bubbles. The steady-state, symmetry-controlled bubble shape oscillations allow analysis of the fluid flow generated in the vicinity of the bubble interface.

Induktion von Mikroströmungen durch nichtsphärische Blasenschwingungen in einem akustischen Levitationssystem

When located near biological barriers, oscillating microbubbles may increase cell membrane permeability, allowing for drug and gene internalization. ...

Ingenieurwesen

Fabricating and Labeling Microbubbles with Fluorescent and Radioactive Tracers

Microbubbles are lipid-shelled, gas-filled particles that have evolved from vascular ultrasound contrast agents into revolutionary cancer therapy platforms. When combined with therapeutic focused ultrasound (FUS), they can safely and locally overcome physiological barriers (e.g., blood-brain barrier), deliver drugs to otherwise inaccessible cancers (e.g., glioblastoma and pancreatic cancer), and treat neurodegenerative diseases. The therapeutic arsenal of microbubble-FUS is advancing in new directions, including synergistic combination radiotherapy, multimodal imaging, and all-in-one drug loading and delivery from microbubble shells.
Labeling microbubbles with radiotracers is key to establishing these expanded theranostic capabilities. However, existing microbubble radiolabeling strategies rely on purification methodologies known to perturb microbubble physicochemical properties, use short-lived radioisotopes, and do not always yield stable chelation. Collectively, this creates ambiguity surrounding the accuracy of microbubble radioimaging and the efficiency of tumor radioisotope delivery.
This protocol describes a new one-pot, purification-free microbubble labeling methodology that preserves microbubble physicochemical properties while achieving &#62;95% radioisotope chelation efficiency. It is versatile and can be applied successfully across custom and commercial microbubble formulations with differing acyl lipid chain length, charge, and chelator/probe (porphyrin, DTPA, DiI) composition. It can be adaptively applied during ground-up microbubble fabrication and to pre-made microbubble formulations with modular customizability of fluorescence and multimodal fluorescence/radioactive properties. Accordingly, this flexible method enables the production of tailored, traceable (radio, fluorescent, or radio/fluorescent active) multimodal microbubbles that are useful for advancing mechanistic, imaging, and therapeutic microbubble-FUS applications.

This protocol outlines the fabrication of lipid microbubbles and a compatible one-pot microbubble radiolabeling method with purification-free &gt;95% labeling efficiency that conserves microbubble physicochemical properties. This method is effective across diverse lipid microbubble formulations and can be tailored to generate radioactive and/or fluorescent microbubbles.

Herstellung und Markierung von Mikrobläschen mit fluoreszierenden und radioaktiven Tracern

Microbubbles are lipid-shelled, gas-filled particles that have evolved from vascular ultrasound contrast agents into revolutionary cancer therapy ...

Krebsforschung

Studying Cavitation Enhanced Therapy

Interest in the therapeutic applications of ultrasound is significant and growing, with potential clinical targets ranging from cancer to Alzheimer&#8217;s disease. Cavitation - the formation and subsequent motion of bubbles within an ultrasound field - represents a key phenomenon underpinning many of these treatments. There remains, however, considerable uncertainty regarding the detailed mechanisms of action by which cavitation promotes therapeutic effects and there is a need to develop reliable monitoring techniques that can be implemented clinically. In particular, there is significant variation between studies in the exposure parameters reported as successfully delivering therapeutic effects and the corresponding acoustic emissions. The aim of this paper is to provide design guidelines and an experimental protocol using widely available components for performing studies of cavitation-mediated bioeffects, and&#160;include real-time acoustic monitoring. It is hoped that the protocol will enable more widespread incorporation of acoustic monitoring into therapeutic ultrasound experiments and facilitate easier comparison across studies of exposure conditions and their correlation to relevant bio-effects.

The presented experimental protocol can be used to perform real time measurements of cavitation activity in a cell culture device with the aim of enabling investigation of the conditions required for successful drug delivery and/or other bioeffects.

Untersuchung der Kavitationstherapie

Interest in the therapeutic applications of ultrasound is significant and growing, with potential clinical targets ranging from cancer to ...

Real-Time Intravital Multiphoton Microscopy to Visualize Focused Ultrasound and Microbubble Treatments to Increase Blood-Brain Barrier Permeability

The blood-brain barrier (BBB) is a key challenge for the successful delivery of drugs to the brain. Ultrasound exposure in the presence of microbubbles has emerged as an effective method to transiently and locally increase the permeability of the BBB, facilitating para- and transcellular transport of drugs across the BBB. Imaging the vasculature during ultrasound-microbubble treatment will provide valuable and novel insights on the mechanisms and dynamics of ultrasound-microbubble treatments in the brain.
Here, we present an experimental procedure for intravital multiphoton microscopy using a cranial window aligned with a ring transducer and a 20x objective lens. This set-up enables high spatial and temporal resolution imaging of the brain during ultrasound-microbubble treatments. Optical access to the brain is obtained via an open-skull cranial window. Briefly, a 3-4 mm diameter piece of the skull is removed, and the exposed area of the brain is sealed with a glass coverslip. A 0.82 MHz ring transducer, which is attached to a second glass coverslip, is mounted on top. Agarose (1% w/v) is used between the coverslip of the transducer and the coverslip covering the cranial window to prevent air bubbles, which impede ultrasound propagation. When sterile surgery procedures and anti-inflammatory measures are taken, ultrasound-microbubble treatments and imaging sessions can be performed repeatedly over several weeks. Fluorescent dextran conjugates are injected intravenously to visualize the vasculature and quantify ultrasound-microbubble induced effects (e.g., leakage kinetics, vascular changes). This paper describes the cranial window placement, ring transducer placement, imaging procedure, common troubleshooting steps, as well as advantages and limitations of the method.

This protocol describes the surgical and technical procedures that enable real-time in vivo multiphoton fluorescence imaging of the rodent brain during focused ultrasound and microbubble treatments to increase blood-brain barrier permeability.

Intravitale Multiphotonenmikroskopie in Echtzeit zur Visualisierung fokussierter Ultraschall- und Mikroblasenbehandlungen zur Erhöhung der Durchlässigkeit der Blut-Hirn-Schranke

The blood-brain barrier (BBB) is a key challenge for the successful delivery of drugs to the brain. Ultrasound exposure in the presence of microbubbles ...

Multi-timescale Microscopy Methods for the Characterization of Fluorescently-labeled Microbubbles for Ultrasound-Triggered Drug Release

Microbubble contrast agents hold great promise for drug delivery applications with ultrasound. Encapsulating drugs in nanoparticles reduces systemic toxicity and increases circulation time of the drugs. In a novel approach to microbubble-assisted drug delivery, nanoparticles are incorporated in or on microbubble shells, enabling local and triggered release of the nanoparticle payload with ultrasound. A thorough understanding of the release mechanisms within the vast ultrasound parameter space is crucial for efficient and controlled release. This set of presented protocols is applicable to microbubbles with a shell containing a fluorescent label. Here, the focus is on microbubbles loaded with poly(2-ethyl-butyl cyanoacrylate) polymeric nanoparticles, doped with a modified Nile Red dye. The particles are fixed within a denatured casein shell. The microbubbles are produced by vigorous stirring, forming a dispersion of perfluoropropane gas in the liquid phase containing casein and nanoparticles, after which the microbubble shell self-assembles. A variety of microscopy techniques are needed to characterize the nanoparticle-stabilized microbubbles at all relevant timescales of the nanoparticle release process. Fluorescence of the nanoparticles enables confocal imaging of single microbubbles, revealing the particle distribution within the shell. In vitro ultra-high-speed imaging using bright-field microscopy at 10 million frames per second provides insight into the bubble dynamics in response to ultrasound insonation. Finally, nanoparticle release from the bubble shell is best visualized by means of fluorescence microscopy, performed at 500,000 frames per second. To characterize drug delivery in vivo, the triggered release of nanoparticles within the vasculature and their extravasation beyond the endothelial layer is studied using intravital microscopy in tumors implanted in dorsal skinfold window chambers, over a timescale of several minutes. The combination of these complementary characterization techniques provides unique insight into the behavior of microbubbles and their payload release at a range of time and length scales, both in vitro and in vivo.

The presented protocols can be used to characterize the response of fluorescently-labeled microbubbles designed for ultrasound-triggered drug delivery applications, including their activation mechanisms as well as their bioeffects. This paper covers a range of in vitro and in vivo microscopy techniques performed to capture the relevant length and timescales.

Multi-Timescale-Mikroskopie-Methoden zur Charakterisierung fluoreszenzmarkierter Mikroblasen für die ultraschallgesteuerte Wirkstofffreisetzung

Microbubble contrast agents hold great promise for drug delivery applications with ultrasound. Encapsulating drugs in nanoparticles reduces systemic ...

Preparation and Characterization of Targeted Microbubbles

Targeting of microbubbles (ultrasound contrast agents for molecular imaging) has been researched for more than two decades. However, methods of microbubble preparation and targeting ligand attachment are cumbersome, complicated, and lengthy. Therefore, there is a need to simplify the targeted microbubble preparation procedure to bring it closer to clinical translation. The purpose of this publication is to provide a detailed description and explanation of the steps necessary for targeted microbubble preparation, functional characterization and testing. A sequence of the optimized and simplified procedures is presented for two systems: a biotin-streptavidin targeting pair model, and a cyclic RGD peptide targeting the recombinant &#945;v&#946;3 protein, which is overexpressed on the endothelial lining of the tumor neovasculature.
Here, we show the following: covalent coupling of the targeting ligand to a lipid anchor, assessment of the reagent quality, and tests that confirm the successful completion of the reaction; preparation of the aqueous precursor medium containing microbubble shell components, followed by microbubble preparation via amalgamation; assessment of the efficacy of lipid transfer onto the microbubble stabilizer shell; adjustment of microbubble size distribution by flotation at normal gravity to remove larger microbubbles that might be detrimental for in vivo use; assessment of microbubble size distribution by electrozone sensing; evaluation of targeted binding of the microbubbles to receptor-coated surface in a static binding assay test (in an inverted dish); and evaluation of targeted binding of the microbubbles to receptor-coated surface in a parallel plate flow chamber test.

The goal of this protocol is to prepare, purify and characterize gas-filled microbubbles (targeted contrast agents for ultrasound molecular imaging). Two targeting systems are described: biotinylated bubbles adherent to streptavidin, and cyclic RGD peptide microbubbles that bind to &#945;v&#946;3, a known tumor neovasculature biomarker.

Targeting of microbubbles (ultrasound contrast agents for molecular imaging) has been researched for more than two decades. However, methods of ...

A High-Throughput Image-Guided Stereotactic Neuronavigation and Focused Ultrasound System for Blood-Brain Barrier Opening in Rodents

The blood-brain barrier (BBB) has been a major hurdle for the treatment of various brain diseases. Endothelial cells, connected by tight junctions, form a physiological barrier preventing large molecules (&#62;500 Da) from entering the brain tissue. Microbubble-mediated focused ultrasound (FUS) can be used to induce a transient local BBB opening, allowing larger drugs to enter the brain parenchyma.
In addition to large-scale clinical devices for clinical translation, preclinical research for therapy response assessment of drug candidates requires dedicated small animal ultrasound setups for targeted BBB opening. Preferably, these systems allow high-throughput workflows with both high-spatial precision as well as integrated cavitation monitoring, while still being cost effective in both initial investment and running costs.
Here, we present a bioluminescence and X-ray guided stereotactic small animal FUS system that is based on commercially available components and fulfills the aforementioned requirements. A particular emphasis has been placed on a high degree of automation facilitating the challenges typically encountered in high-volume preclinical drug evaluation studies. Examples of these challenges are the need for standardization in order to ensure data reproducibility, reduce intra-group variability, reduce sample size and thus comply with ethical requirements and decrease unnecessary workload. The proposed BBB system has been validated in the scope of BBB opening facilitated drug delivery trials on patient-derived xenograft models of glioblastoma multiforme and diffuse midline glioma.

The blood-brain barrier (BBB) can be temporarily disrupted with microbubble-mediated focused ultrasound (FUS). Here, we describe a step-by-step protocol for high-throughput BBB opening in vivo using a modular FUS system accessible for non-ultrasound experts.

Ein hochdurchsatzgesteuertes, stereotaktisches Neuronavigations- und fokussiertes Ultraschallsystem für die Öffnung der Blut-Hirn-Barriere bei Nagetieren

The blood-brain barrier (BBB) has been a major hurdle for the treatment of various brain diseases. Endothelial cells, connected by tight junctions, form a ...