Subvention de soutien a inclus NHLBI HL080532, NHLBI HL073449, NCRR RR16453, et une nationale AHA Grant-in de l&#39;aide (aux RVS). Un merci tout spécial est étendu à la conception de cours à distance et de conseil (DCDC) du groupe, dcdcgroup.org, pour leur aide à la production vidéo et à l&#39;US Department of Education Grant No. P336C050047 qui a fondé la DCDC.

1. Préparation de la pâte de microbulles <ol><li> Dans 10 ml de PBS mélange de 200 mg de 1,2-dipalmitoyl-sn-glycéro-3-phosphatidylcholine et 50 mg 1,2-dipalmitoyl-sn-glycéro-3-phosphatidyléthanolamine avec 1 g de glucose. </li><li> Chauffez le mélange au bain d&#39;eau bouillante 20-30 minutes, une pipette mélangeant toutes les 5 minutes. </li><li> La solution peut être conservé à 4 ° C pour un maximum de 6 mois. </li></ol> 2. Préparation des microbulles <ol><li> Prenez un ul 250 de la solution stock de microbulles préparée et incuber à 40 ° C pendant 15 minutes. </li><li> La solution pré-chauffé microbulles est ensuite transféré dans un microtube de 1,5 ml contenant 50 ul de glycérol. </li><li> 1-2 mg d&#39;ADN plasmidique purifié codant pour une construction d&#39;expression pour le gène d&#39;intérêt (dans cet exemple purifiée par Qiagen endotoxines libres Bluetooth Megaprep, Qiagen, Germantown, MD, avec une concentration optimale de 4mg/ml). 2.4) du tampon phosphate salin est ajouté à un volume final de 500 pl. Endotoxines gratuitement maxipreps Qiagen sont utilisés, ainsi que du PBS stérile afin d&#39;en garantir la stérilité. </li><li> L&#39;air dans le microtube est alors remplacé par du gaz Octafluoropropane. </li><li> Le microtube est alors secoué vigoureusement dans un amalgamateur dentaire pour 20 secondes. </li><li> Le subnatant ADN contenant résiduelle et le tampon qui n&#39;a pas forcément les microbulles est ensuite soigneusement enlevé et la couche de microbulles est lavé trois fois avec du PBS stérile pour éliminer l&#39;ADN seules, et placé sur la glace entre chaque cycle de lavage. Nous généralement atteindre une efficacité de liaison de 30-40%. Tous les réactifs sont stériles et les soins sont faits pour minimiser la contamination. </li><li> Le plasmide d&#39;ADN liés microbulles sont ensuite placés sur la glace pendant près de deux heures jusqu&#39;à l&#39;utilisation. </li><li> Le subnatant retiré de la microtube après mélange et le PBS lavages, peut être utilisé pour déterminer la concentration de l&#39;ADN non lié, et également le montant lié basé sur la concentration initiale connue, en mesurant la densité optique de cette solution à une longueur d&#39;onde de 260 nm à l&#39;aide un spectrophotomètre. </li></ol> 3. Étalonnage de l&#39;équipement <ol><li> Avant la première utilisation, un transducteur de 1 MHz cavitation doit être calibré pour assurer un bon indice de mécanique et de répétition des impulsions. Un submersible de 1 MHz, 13mm, capteur flou est connecté à une fonction 20 MHz / Générateur de signaux arbitraires à travers un amplificateur de puissance. </li><li> Le transducteur est placé dans un récipient en plastique rempli d&#39;eau, visant directement à un hydrophone, qui a été relié à un oscilloscope 500 MHz via un amplificateur de charge. </li><li> Waveform, fréquence, amplitude, le cycle de l&#39;éclatement, et l&#39;amplification de puissance peuvent tous être modifiés pour obtenir le cycle de travail approprié et index mécanique optimale à la cavitation des microbulles. Pour cette expérience particulière, nous avons calibré le système à un index mécanique équivalente à ~ 1,3 à 1 MHz. </li></ol> 4. Livraison &amp; microbulles UTMD <ol><li> Avant la livraison de microbulles et UTMD, des souris C57BL / 6 ont été anesthésiés avec 100mg/kg kétamine et xylazine 5mg/kg par injection IP. </li><li> La livraison des microbulles a été administré par voie intraveineuse par une injection directe dans le ventricule gauche du cœur ou par cathétérisme de la veine de la queue. Pour l&#39;injection directe cardiaque, un volume de 100 ul du plasmide ADN chargés solution de microbulles est injecté par une aiguille de calibre 30 insérées à la partie antérieure 4 ème espace intercostal, sous visualisation par ultrasons dans le ventricule gauche du cœur. </li><li> Le bolus solution de microbulles résultant de l&#39;injection interventriculaire gauche est visualisée à l&#39;aide de Visual Sonics 38 MHz de fréquence à ultrasons transducteur haute placée dans une position stationnaire sur le thorax de la souris dans une vue à long axe en utilisant VisualSonics système Vevo Imaging 2100. Toutes les seringues et les aiguilles sont stériles, assurant l&#39;environnement le plus stérile possible pour ces injections. </li><li> Immédiatement après l&#39;injection, la destruction des microbulles est effectuée pour ~ 5 minutes à l&#39;aide d&#39;une seconde, plus petite taille basse fréquence 1,0 MHz transducteur lieu directement sur l&#39;organe désiré, en ciblant la destruction de cette région. Dans cet exemple, l&#39;échographie a été administré pour le foie à une fréquence de répétition des impulsions de 1,0 MHz, avec un équivalent de l&#39;indice à environ 1.3 à 1.5 mécaniques, et la période de répétition des impulsions de 100 ms pour 20 cycles. Alternativement, le pouls peut être fermée à l&#39;ECG de la souris (non montré dans cette expérience) pour une rafale de 3 images de l&#39;échographie, tous les 4-6 cycles cardiaques. Nous obtenons une plus grande efficacité de la transfection avec des protocoles qui permettent au lit capillaire à recharger avec des bulles entre les salves d&#39;ultrasons. </li></ol> 5. Livraison Méthode alternative Nous avons choisi de mettre en évidence l&#39;injection interventriculaire en raison de la complexité de la procédure, mais dans de nombreux cas, comme une perfusion prolongée de microbulles, une veine de la queue deprojection est la méthode préférée. Pour la méthode de la queue-veineuse de la livraison de microbulles, la souris est anesthésiée de la même façon. Une seringue contenant le plasmide d&#39;ADN liés microbulles est relié à un cathéter de calibre 27 veine d&#39;aiguille / queue. Le cathéter veine de la queue est insérée dans le tiers distal de la veine soit droite ou gauche latéral qui le long de la queue de la souris. La seringue contenant les microbulles est placée dans une pompe à perfusion qui administre automatiquement un volume uniforme prédéfini de solution sur une période de temps prédéterminée. En général, nous infuser 200-300μl à un taux de 3ml/hour. L&#39;utilisation d&#39;animaux Tous les animaux ont été traités conformément à la pratique des animaux de bonnes telles que définies par l&#39;autorité nationale et / ou organismes locaux de protection des animaux, et tous les travaux d&#39;animaux a été approuvé par le comité approprié (Université de Hawaï Soins des animaux et du Comité institutionnel utilisation, 07-100 numéro d&#39;agrément -3). L&#39;anesthésie appropriée (la kétamine / zylazine) a été utilisé et des analgésiques (bupivicaïne et buprénorphine) étaient disponibles, mais pas nécessaire. 6. Les résultats représentatifs: L&#39;efficacité de la livraison UTMD médiée par ADN plasmidique peut être évalué à travers une variété de méthodes selon les gènes codés dans la construction, tels que, mais non limité à, la luciférase imagerie in vivo, B-gal ex vivo coloration, et / ou immunohistologie. En particulier, dans l&#39;imagerie par bioluminescence in vivo permet de surveiller la présence et la durée de l&#39;expression des gènes chez les souris série transfectées avec un plasmide codant pour un gène rapporteur bioluminescent (luciférase). Le Xenogen l&#39;imagerie in vivo du système (IVIS) (Caliper Life Sciences, Hopkinton, MA) est utilisé pour l&#39;imagerie de bioluminescence. Les images sont généralement prises de toutes les souris du premier jour après la transfection médiée UTMD et est répété tous les trois à quatre jours jusqu&#39;à l&#39;expression des gènes de bioluminescence n&#39;est plus détectable visuellement par le système (figure 1). Pour préparer les souris pour l&#39;imagerie de bioluminescence, des souris d&#39;abord recevoir une injection IP de la reporter de la luciférase de sonde D-luciférine (Caliper Life Sciences) et sont ensuite anesthésiés ~ 3 minutes plus tard. Biodistribution du substrat D-luciférine est autorisé à procéder pour ~ 10 minutes avant que l&#39;animal est placé dans la chambre IVIS imagerie et un scan complet du corps image est prise. Lors de l&#39;acquisition, les photons émis par la luciférase de luciole / D-luciférine réaction photochimique sont mesurés. La Figure 1 illustre également similaire IVIS imagerie bioluminescente du foie suivantes UTMD, et la figure 2 est une épifluorescence (100X) l&#39;image du foie transfectées en utilisant un anticorps anti-luciférase primaire (Sigma-Aldrich) et AlexFluor-568 anticorps secondaire conjugué (Invitrogen) . Il est évident de voir que la transfection médiée UTMD foie a affecté non seulement les cellules endothéliales, mais le hepatocyes ainsi. <img src="/files/ftp_upload/2963/2963fig1.jpg" alt="Figure 1" /> Figure 1 Xenogen / IVIS imagerie cardiaque UTMD souris traitées (A) Souris Contrôle négatif:.. Plasmidique + PBS suivie cardiaques dirigé UTMD, Souris traitement (B): Plasmide + microbulles suivie cardiaques dirigé UTMD, et la souris de traitement (C) : + microbulles plasmidique suivie par le foie dirigé UTMD. <img src="/files/ftp_upload/2963/2963fig2.jpg" alt="Figure 2" /> Figure 2. Immunohistochimie de foie UTMD (anti-luciférase en rouge) (A). Plasmidique sans UTMD, et (B) plasmide avec UTMD. Image confocale (100X); noyaux sont DAPI bleue tachée.

<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 représente une nouvelle approche pour la livraison de gènes. Comme une plateforme technologique qu&#39;elle peut être combinée avec l&#39;une des nombreuses stratégies possibles de la thérapie génique, pour offrir une multitude de molécules bioactives où un degré élevé de spécificité tissulaire est souhaitée. La principale limitation biologique de la technique est la faible efficacité de la transfection. Un autre facteur important est l&#39;accessibilité de l&#39;organe cible à l&#39;échographie,...

<table border="1"><tbody><tr><th> Nom du réactif / équipements </th><th> Société </th><th> Numéro de catalogue </th><th> Commentaires </th></tr><tr><td> 1,2-dipalmitoyl-sn-glycéro-3-phosphatidylcholine </td><td> Sigma-Aldrich </td><td> P-5911 </td><td> composante de l&#39;enveloppe lipidique de microbulles </td></tr><tr><td> 1,2-dipalmitoyl-sn-glycéro-3-phosphatidyléthanolamine </td><td> Sigma-Aldrich </td><td> P-3275 </td><td> composante de l&#39;enveloppe lipidique de microbulles </td></tr><tr><td> le glucose </td><td> Sigma-Aldrich </td><td> G5400 </td><td> pensé pour stabiliser les microbulles </td></tr><tr><td> tampon phosphate salin </td><td> Sigma-Aldrich </td><td> P5368 </td><td></td></tr><tr><td> glycérol </td><td> Sigma-Aldrich </td><td> G5516 </td><td> cru pour empêcher des microbulles d&#39;coalescence </td></tr><tr><td> De gaz Octafluoropropane </td><td> Airgas </td><td> N / A </td><td> gaz inerte utilisé dans les applications cliniques </td></tr><tr><td> Vialmix dentaires amalgamateur </td><td> Bristol-Myers Squibb </td><td> N / A </td><td></td></tr><tr><td> 1 MHz, 13mm, capteur flou </td><td> Olympus </td><td> A303S-SU </td><td></td></tr><tr><td> 20 Fonction MHz / Générateur de signaux arbitraires </td><td> Agilent </td><td> 33220A </td><td></td></tr><tr><td> Amplificateur de puissance </td><td> Krohn-Hite Co. </td><td> Modèle 7500 </td><td></td></tr><tr><td> Hydrophone </td><td> Bruel et Kjaer </td><td> Tapez 1803 </td><td></td></tr><tr><td> Amplificateur de charge </td><td> Bruel et Kjaer </td><td> Tapez 2634 </td><td></td></tr><tr><td> 500 MHz Oscilloscope </td><td> LeCroy </td><td> 9354L </td><td></td></tr><tr><td> VisualSonics système Vevo Imaging 2100 avec 34 capteur MHz </td><td> VisualSonics </td><td> 2100 </td><td></td></tr><tr><td> 27G d&#39;un pouce cathéters veine de la queue </td><td> VisualSonics </td><td> N / A </td><td></td></tr><tr><td> Genie plus une pompe à perfusion </td><td> Kent scientifique </td><td> GENIE </td><td></td></tr></tbody></table>

introduction to the ultrasound targeted microbubble destruction technique

En UTMD, molécules bioactives, comme chargée négativement vecteurs ADN plasmidique codant pour un gène d&#39;intérêt, sont ajoutés à la coquille de lipides cationiques agents de contraste microbulles 7-9. Chez les souris ces microbulles vecteur de transport peut être administré par voie intraveineuse ou directement dans le ventricule gauche du cœur. Dans les grands animaux qu&#39;ils peuvent aussi être perfusé par un cathéter intracoronaire. La livraison ultérieure de la circulation à un organe cible se fait par cavitation acoustique à une fréquence de résonance des microbulles. Il semble probable que l&#39;énergie mécanique générée par les résultats de destruction des microbulles dans la formation de pores transitoires dans ou entre les cellules endothéliales de la microvascularisation de la région ciblée 10. En conséquence de cet effet sonoporation, l&#39;efficacité de la transfection dans et à travers les cellules endothéliales est augmentée, et transgène vecteurs codant sont déposés dans les tissus environnants. L&#39;ADN plasmidique restant dans la circulation est rapidement dégradé par les nucléases dans le sang, ce qui réduit encore la probabilité de la livraison à la non-soniqué tissus et conduit à très spécifique des organes cibles de la transfection.

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

Échographie destruction ciblée des microbulles (UTMD) peut être utilisé pour diriger spécifique au site de livraison de molécules bioactives, notamment des gènes thérapeutiques, pour cibler les organes accessibles à l&#39;échographie, comme le cœur et le foie<sup&gt; 1-6</sup&gt;.

Introduction à l&#39;échographie destruction des microbulles ciblées Technique

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

introduction-to-ultrasound-targeted-microbubble-destruction

Research

JoVE Journal

Bioengineering

15.4K vues.  University of Hawaii. Échographie destruction ciblée des microbulles (UTMD) peut être utilisé pour diriger spécifique au site de livraison de molécules bioactives, notamment des gènes thérapeutiques, pour cibler les organes accessibles à l'échographie, comme le cœur et le foie 1-6.

Vidéo: 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.

L&#39;échographie de contraste ciblées traitement des gliomes chez la souris via la livraison de drogue nanoparticules-Bearing et l&#39;ablation microvasculaire

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

Recherche

Bio-ingénierie

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.

Conception d&#39;un bioréacteur pour la pression cyclique Ex Vivo Étude des valves cardiaques aortiques

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.

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

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

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.

Fabrication de concentré, de l&#39;oxygène à base de lipides microbulles émulsions par cisaillement élevé homogénéisation et la concentration de série

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.

Une technique microfluidique à la sonde cellulaire Déformabilité

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.

Une puce microfluidique pour ICPMS Présentation échantillon

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

Synthesis of Gold Nanoparticle Integrated Photo-responsive Liposomes and Measurement of Their Microbubble Cavitation upon Pulse Laser Excitation

Photo-responsive nanoparticles (NPs) have received considerable attention because of their potential in providing spatial, temporal, and dosage control over the drug release. However, most of the relevant technologies are still in the development process and are unprocurable by clinics. Here, we describe a facile fabrication of these photo-responsive NPs with commercially available gold NPs and thermo-responsive liposomes. Calcein is used as a model drug to evaluate the encapsulation efficiency and the release kinetic profile upon heat/light stimulation. Finally, we show that this photo-triggered release is due to the membrane disruption caused by microbubble cavitation, which can be measured with hydrophone.

This protocol describes a simple preparation method for gold nanoparticle integrated photo-responsive liposomes with the commercially available materials. It also shows how to measure the microbubble cavitation process of the synthesized liposomes upon the treatment of pulsed laser.

Synthèse de l&#39;or nanoparticules liposomes intégré photosensible et mesurer leur microbulles cavitation sur Pulse Laser excitation

Photo-responsive nanoparticles (NPs) have received considerable attention because of their potential in providing spatial, temporal, and dosage control ...

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.

Un protocole expérimental pour évaluer la Performance des nouvelles sondes ultrasons basés sur la technologie CMUT en Application à l’imagerie cérébrale

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.

Évaluation globale de l’efficacité et l’innocuité de la délivrance de médicaments ciblés Placenta en utilisant trois méthodes complémentaires

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.

Préparation des cellules synthétiques produisant des protéines à l’aide d’extraits bactériens exempts de cellules, de liposomes et de transfert d’émulsion

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