Sovvenzioni ha incluso NHLBI HL080532, NHLBI HL073449, NCRR RR16453, e una nazionale AHA Grant-in dell&#39;aiuto (a RVS). Un particolare ringraziamento è esteso al Corso di Progettazione e consulenza a distanza (DCDC) gruppo, dcdcgroup.org, per la loro assistenza con la produzione video e al US Department of Education Concessione n. P336C050047 che fondò la DCDC.

1. Microbolle magazzino preparazione <ol><li> In 10 ml di PBS mix di 200 mg di 1,2-dipalmitoil-sn-glicero-3-fosfatidilcolina e 50 mg di 1,2-dipalmitoil-sn-glicero-3-fosfatidiletanolamina con 1 g di glucosio. </li><li> Riscaldare il miscuglio a bagno d&#39;acqua bollente 20-30 minuti, mescolando pipetta ogni 5 minuti. </li><li> La soluzione può essere conservato a 4 ° C per un massimo di 6 mesi. </li></ol> 2. Microbolle di preparazione <ol><li> Prendere un 250 microlitri della soluzione preparata magazzino microbolle e incubare a 40 ° C per 15 minuti. </li><li> La pre-riscaldato soluzione microbolle viene poi trasferito in un microtubo 1,5 ml contenente 50 ml di glicerolo. </li><li> 1-2 mg di DNA plasmidico purificato codifica per costruire l&#39;espressione del gene di interesse (purificato in questo esempio da endotossine Qiagen libero MegaPrep kit, Qiagen, Germantown, MD, con una concentrazione ottimale di 4mg/ml). 2.4) PBS viene aggiunto a un volume finale di 500 microlitri. Endotossina libero maxipreps Qiagen vengono utilizzati, così come PBS sterile in modo da garantire la sterilità. </li><li> L&#39;aria nella microtubo viene poi sostituito con il gas Octafluoropropane. </li><li> Il microtubo è poi agitato energicamente in un amalgamatore dentale per 20 secondi. </li><li> Il subnatant DNA contenente residui e buffer che non è legato al microbolle viene poi accuratamente rimosso e lo strato di microbolle viene lavato tre volte con PBS sterile per rimuovere il DNA distaccati, e immessi sul ghiaccio tra ogni ciclo di lavaggio. Noi di solito raggiungere una efficienza di legame del 30-40%. Tutti i reagenti sono sterili e la cura è fatta per minimizzare la contaminazione. </li><li> Il plasmide DNA legato microbolle vengono poi immessi sul ghiaccio per un massimo di due ore fino al momento dell&#39;uso. </li><li> Il subnatant rimosso dal microtubo dopo la miscelazione e la PBS lavaggi, può essere usato per determinare la concentrazione di DNA non legato, così come la quantità limite in base alla concentrazione nota iniziale, misurando la densità ottica di questa soluzione alla lunghezza d&#39;onda di 260 nm utilizzando uno spettrofotometro. </li></ol> 3. Gli apparecchi di taratura <ol><li> Prima del primo utilizzo, un 1 trasduttore cavitazione MHz deve essere calibrato per assicurare un corretto indice meccanico e la ripetizione di impulso. Un sommergibile 1 MHz, 13mm, sfocata trasduttore è collegato ad una funzione 20 MHz / generatore di forme d&#39;onda arbitrarie attraverso un amplificatore di potenza. </li><li> Il trasduttore è posto in un contenitore di plastica pieno d&#39;acqua, rivolte direttamente a un idrofono, che è stato collegato ad un oscilloscopio a 500 MHz tramite un amplificatore di carica. </li><li> Forma d&#39;onda, frequenza, ampiezza, ciclo burst, e di amplificazione di potenza possono essere modificati per ottenere il ciclo proprio dovere e indice meccanico ottimale per la cavitazione microbolle. Per questo particolare esperimento, abbiamo calibrato il sistema ad un indice meccanico pari a ~ 1,3 a 1 MHz. </li></ol> 4. Consegna microbolle &amp; UTMD <ol><li> Prima della consegna microbolle e UTMD, topi C57BL / 6 sono stati anestetizzati con 100mg/kg xylazina ketamina e 5mg/kg mediante iniezione IP. </li><li> Consegna microbolle è stato somministrato per via endovenosa attraverso una iniezione diretta nel ventricolo sinistro del cuore o attraverso il cateterismo coda vena. Per l&#39;iniezione diretta cardiaco, un volume di 100 microlitri della plasmide DNA caricato soluzione microbolle viene iniettato attraverso un ago 30 gauge inserito anteriore 4 ° spazio intercostale, sotto visualizzazione ultrasonica nel ventricolo sinistro del cuore. </li><li> Il bolo soluzione microbolle derivanti dalla iniezione interventricolare sinistro è visualizzata utilizzando Visual Sonics &#39;38 MHz trasduttore ad ultrasuoni ad alta frequenza collocato in una posizione stazionaria sul torace del mouse in un asse lungo con VisualSonics&#39; Vevo 2100 Imaging System. Tutte le siringhe e aghi sterili, assicurando l&#39;ambiente più sterile possibile per queste iniezioni. </li><li> Subito dopo l&#39;iniezione, la distruzione delle microbolle viene effettuata per ~ 5 minuti con un secondo, più piccolo di dimensioni trasduttore a bassa frequenza di 1,0 MHz detenute direttamente sopra l&#39;organo desiderato, il targeting distruzione di questa regione. In questo esempio, l&#39;ecografia è stato somministrato al fegato ad una frequenza di ripetizione degli impulsi di 1,0 MHz, con un indice pari a circa 1,3-1,5 meccanico, e la ripetizione periodo di impulso di 100 ms per ogni 20 cicli. In alternativa, l&#39;impulso può essere controllato al l&#39;ECG del mouse (non mostrato in questo esperimento) ad una raffica di 3 fotogrammi degli ultrasuoni, ogni 4-6 cicli cardiaci. Si ottiene una maggiore efficienza di trasfezione con i protocolli che permettono il letto capillare di ricarica con le bolle tra raffiche di ultrasuoni. </li></ol> 5. Consegna Metodo Alternativo Abbiamo scelto per evidenziare l&#39;iniezione interventricolare a causa della complessità della procedura, ma in molti casi, come ad esempio l&#39;infusione prolungata di microbolle, una vena codaproiezione è il metodo preferito. Per la coda vena metodo di consegna microbolle, il mouse è anestetizzato allo stesso modo. Una siringa contenente il plasmide DNA legato microbolle è collegato ad un calibro 27 ago / catetere venoso coda. Il catetere venoso coda è inserita nel terzo distale di entrambi le vene laterale destra o sinistra che lungo la coda del mouse. La siringa contenente le microbolle si trova in una pompa di infusione che gestisce automaticamente un volume uniforme preset di soluzione in un periodo di tempo predefinito. Noi di solito infondere 200-300μl ad un tasso di 3ml/hour. Animal uso Tutti gli animali sono stati trattati in conformità alla prassi buon animale come definito dalle autorità nazionali e / o enti locali il benessere degli animali, e tutto il lavoro degli animali è stato approvato dalla commissione competente (Università di cura degli animali alle Hawaii e del Comitato Istituzionale Usa, numero di omologazione 07-100 -3). Anestesia appropriata (ketamina / zylazine) è stato utilizzato e analgesici (Bupivicaine e buprenorfina) erano disponibili, anche se non richiesto. 6. Rappresentante dei risultati: L&#39;efficacia del UTMD-mediata consegna DNA plasmidico può essere valutato attraverso una varietà di metodi a seconda dei geni codificati nel costruire, come ad esempio, ma non limitato a, luciferasi imaging in vivo, B-gal ex colorazione in vivo, e / o immunohistology. In particolare, nella diagnostica per immagini bioluminescenza in vivo permette di monitorare la presenza e la durata dell&#39;espressione genica nei topi serie transfettate con un plasmide codifica un gene reporter bioluminescenti (luciferasi). Il Xenogen In Vivo Imaging System (IVIS) (Caliper Life Sciences, Hopkinton, MA) è utilizzato per l&#39;imaging bioluminescenti. Le immagini sono in genere conto di tutti i topi del primo giorno dopo UTMD trasfezione mediata e viene ripetuto ogni tre o quattro giorni fino a quando l&#39;espressione genica bioluminescenti non è più percepibile a vista attraverso il sistema (Figura 1). Per preparare i topi bioluminescenti per l&#39;imaging, i topi prima ricevere un&#39;iniezione IP del reporter luciferasi sonda D-luciferina (Caliper Life Sciences) e sono poi anestetizzati ~ 3 minuti più tardi. Biodistribuzione del D-luciferina substrato è autorizzato a procedere per circa 10 minuti prima che l&#39;animale si trova nella camera di imaging IVIS e una scansione completa immagine del corpo è presa. Durante l&#39;acquisizione, i fotoni emessi dalla luciferasi lucciola / D-luciferina reazione fotochimica sono misurati. La figura 1 illustra simili immagini bioluminescenti IVIS del fegato seguenti UTMD, e nella Figura 2 è un epifluorescenza (100X), l&#39;immagine del fegato transfettate con un anti-luciferasi anticorpo primario (Sigma-Aldrich) e AlexFluor-568 coniugato secondario (Invitrogen) . È chiaro a vedere che il fegato UTMD transfezione mediata ha colpito non solo le cellule endoteliali, ma il hepatocyes pure. <img src="/files/ftp_upload/2963/2963fig1.jpg" alt="Figura 1" /> Figura 1 Xenogen / IVIS imaging cardiaco UTMD topi trattati (A) Mouse Controllo negativo:.. Plasmidi + PBS seguita da cardiaco diretto UTMD, (B) Mouse Trattamento: plasmidi + microbolle seguita da cardiaco diretto UTMD, e (C) Mouse trattamento : microbolle + plasmidi seguiti da fegato diretto UTMD. <img src="/files/ftp_upload/2963/2963fig2.jpg" alt="Figura 2" /> Figura 2. Immunoistochimica del fegato UTMD (anti-luciferasi in rosso). (A) plasmidi senza UTMD, e (B) plasmidi con UTMD. Immagine confocale (100X); nuclei sono blu DAPI colorati.

<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>

Nessun conflitto di interessi dichiarati.

UTMD rappresenta un nuovo approccio per la consegna del gene. Come una piattaforma tecnologica che può essere combinato con qualsiasi delle strategie di terapia genica molte potenzialità, per offrire una miriade di molecole bioattive, quando un alto grado di specificità del tessuto è voluta. Il principale limite biologico della tecnica è la bassa efficienza di trasfezione. Un&#39;altra considerazione importante è l&#39;accessibilità dei organo bersaglio a ultrasuoni, che possono essere notevolmente diminuita, int...

<table border="1"><tbody><tr><th> Nome del reagente / attrezzature </th><th> Azienda </th><th> Numero di catalogo </th><th> Commenti </th></tr><tr><td> 1,2-dipalmitoil-sn-glicero-3-fosfatidilcolina </td><td> Sigma-Aldrich </td><td> P-5911 </td><td> componente del guscio dei lipidi microbolle </td></tr><tr><td> 1,2-dipalmitoil-sn-glicero-3-fosfatidiletanolamina </td><td> Sigma-Aldrich </td><td> P-3275 </td><td> componente del guscio dei lipidi microbolle </td></tr><tr><td> glucosio </td><td> Sigma-Aldrich </td><td> G5400 </td><td> pensato per stabilizzare la microbolle </td></tr><tr><td> PBS </td><td> Sigma-Aldrich </td><td> P5368 </td><td></td></tr><tr><td> glicerina </td><td> Sigma-Aldrich </td><td> G5516 </td><td> creduto per evitare microbolle di coalescenza </td></tr><tr><td> Octafluoropropane gas </td><td> Airgas </td><td> N / A </td><td> gas inerte utilizzato in applicazioni cliniche </td></tr><tr><td> Vialmix dentale amalgamatore </td><td> Bristol-Myers Squibb </td><td> N / A </td><td></td></tr><tr><td> 1 MHz, 13mm, trasduttore unfocused </td><td> Olimpo </td><td> A303S-SU </td><td></td></tr><tr><td> 20 Funzione MHz / Generatore di forme d&#39;onda arbitrarie </td><td> Agilent </td><td> 33220A </td><td></td></tr><tr><td> Amplificatore di potenza </td><td> Krohn-Hite Co. </td><td> Modello 7500 </td><td></td></tr><tr><td> Idrofoni </td><td> Bruel e Kjaer </td><td> Tipo 1803 </td><td></td></tr><tr><td> Amplificatore di carica </td><td> Bruel e Kjaer </td><td> Tipo 2634 </td><td></td></tr><tr><td> 500 MHz Oscilloscopio </td><td> LeCroy </td><td> 9354L </td><td></td></tr><tr><td> VisualSonics &#39;Vevo 2100 Imaging System con 34 MHz trasduttore </td><td> VisualSonics </td><td> 2100 </td><td></td></tr><tr><td> 27G un pollice coda vena cateteri </td><td> VisualSonics </td><td> N / A </td><td></td></tr><tr><td> Genie In più pompa di infusione </td><td> Kent scientifico </td><td> GENIE </td><td></td></tr></tbody></table>

introduction to the ultrasound targeted microbubble destruction technique

In UTMD, molecole bioattive, come carica negativa vettori di DNA plasmide che codifica per un gene di interesse, vengono aggiunti i gusci di lipidi cationici mezzi di contrasto microbolle 7-9. Nei topi questi vettori che trasportano microbolle può essere somministrato per via endovenosa o direttamente al ventricolo sinistro del cuore. In animali più grandi possono anche essere infusa attraverso un catetere intracoronarico. La successiva consegna effettuata dalla circolazione per un organo bersaglio avviene per cavitazione acustica ad una frequenza di risonanza delle microbolle. Sembra probabile che l&#39;energia meccanica generata dalla distruzione delle microbolle risultati transitori della formazione di pori o tra le cellule endoteliali del microcircolo della regione di destinazione 10. Come risultato di questo effetto sonoporazione, l&#39;efficienza di trasfezione all&#39;interno e attraverso le cellule endoteliali è rafforzata, e transgene codifica vettori sono depositati nel tessuto circostante. DNA plasmidico rimanendo in circolo è rapidamente degradato dalla nucleasi nel sangue, che riduce ulteriormente la probabilità di consegna non sonicato tessuti e conduce ad altamente specifici degli organi bersaglio trasfezione.

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

Ecografia distruzione mirata microbolle (UTMD) può essere utilizzato per dirigere site-specific di consegna di molecole bioattive, tra cui i geni terapeutici, di indirizzare gli organi accessibile a ultrasuoni, come il cuore e il fegato<sup&gt; 1-6</sup&gt;.

Introduzione alla tecnica di distruzione mirata ad ultrasuoni microbolle

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 Views.  University of Hawaii. Ecografia distruzione mirata microbolle (UTMD) può essere utilizzato per dirigere site-specific di consegna di molecole bioattive, tra cui i geni terapeutici, di indirizzare gli organi accessibile a ultrasuoni, come il cuore e il fegato 1-6.

Video: 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.

Mezzo di contrasto ecografico mirato trattamento dei gliomi nei topi tramite Drug-Bearing consegna delle nanoparticelle e ablazione microvascolari

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

Ricerca

Bioingegneria

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.

Progettazione di un bioreattore per la pressione ciclica Ex Vivo Studio delle valvole cardiache aortica

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.

Analisi delle Targeted proteina virale nanoparticelle distribuito HER2 + Tumori

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.

Fabbricazione di concentrato, ossigeno a base di lipidi microbolle emulsioni da High Shear omogeneizzazione e concentrazione seriale

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.

Una tecnica Microfluidic per sondare deformabilità cellulare

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.

Un chip microfluidico per ICPMS introduzione del campione

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.

Sintesi di nanoparticelle d&#39;oro liposomi Integrated Photo-reattiva e la misurazione del loro microbolle cavitazione su impulso laser di eccitazione

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 protocollo sperimentale per valutare le prestazioni delle nuove sonde ad ultrasuoni basato sulla tecnologia CMUT nell'applicazione di formazione immagine del cervello

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.

Valutazione globale dell'efficacia e della sicurezza di Placenta-Targeted Drug Delivery utilizzando tre metodi complementari

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.

Preparazione di proteine che producono cellule sintetiche utilizzando estratti batterici cell free, liposomi e trasferimento di emulsione

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