Apoio financeiro incluiu NHLBI HL080532, NHLBI HL073449, NCRR RR16453, e um Nacional AHA Grant-in do auxílio (a RVS). Um agradecimento especial é estendido para o Projeto Curso a Distância e grupo Consulting (DCDC), dcdcgroup.org, por sua assistência com a produção de vídeo e ao Departamento de Educação dos EUA Grant No. P336C050047 que fundou a DCDC.

1. Preparação de microbolhas <ol><li> Em 10 ml de PBS misturar 200 mg 1,2-dipalmitoil-sn-glicero-3-fosfatidilcolina e 50 mg 1,2-dipalmitoil-sn-glicero-3-fosfatidiletanolamina com 1 g de glicose. </li><li> Aqueça a mistura em ebulição água do banho de 20-30 minutos, pipetar mistura a cada 5 minutos. </li><li> A solução pode ser armazenada a 4 ° C por até 6 meses. </li></ol> 2. Preparação de microbolhas <ol><li> Dê uma mL 250 da solução estoque preparada microbolhas e incubar a 40 ° C por 15 minutos. </li><li> A solução pré-aquecido microbolhas é então transferido para um microtubo 1,5 mL contendo 50 mL de glicerol. </li><li> 1-2 mg de DNA plasmidial purificado codificação de uma expressão construir para o gene de interesse (neste exemplo purificada por endotoxina Qiagen livre MegaPrep kit, Qiagen, Germantown, MD, com uma concentração ideal de 4mg/ml). 2.4) salina tamponada com fosfato é adicionado a um volume final de 500 mL. Maxipreps endotoxina livre Qiagen são usados, bem como PBS estéril, a fim de assegurar a esterilidade. </li><li> O ar no microtubo é então substituída por gás Octafluoropropane. </li><li> O microtubo é então agitada vigorosamente em um amalgamador dental por 20 segundos. </li><li> O DNA contendo subnatant residual e buffer que não tenha vinculado à microbolhas é então cuidadosamente removido ea camada de microbolhas é lavado três vezes com PBS estéril para remover DNA solto, e colocado no gelo entre cada ciclo de lavagem. Normalmente, alcancem uma eficiência de ligação de 30-40%. Todos os reagentes são estéreis e cuidados é feito para minimizar a contaminação. </li><li> O plasmídeo DNA-bound microbolhas são então colocados em gelo por até duas horas até o uso. </li><li> O subnatant removido do microtubo após a mistura ea PBS lavagens, pode ser usado para determinar a concentração de DNA não ligado, e também a quantidade limite baseado na concentração conhecida inicial, através da medição da densidade óptica da solução no comprimento de onda de 260nm utilizando um espectrofotômetro. </li></ol> 3. Calibração de equipamentos <ol><li> Antes do primeiro uso, a um transdutor de cavitação MHz precisa ser calibrado para garantir índice mecânico adequado e de repetição de pulso. A submersível 1 MHz, transdutor, 13 milímetros unfocused está ligado a uma função de 20 MHz / Gerador de Formas de Onda Arbitrárias através de um amplificador de potência. </li><li> O transdutor é colocado em um recipiente de plástico cheio de água, voltado diretamente para um hidrofone, que tem estado ligado a um osciloscópio de 500 MHz através de um amplificador de carga. </li><li> Forma de onda, freqüência, amplitude, ciclo de burst, e amplificação de potência podem ser modificados para obter o ciclo de trabalho adequado e índice mecânico ideal para cavitar as microbolhas. Para este experimento em particular, temos calibrado o sistema para um índice mecânico equivalente a ~ 1,3 MHz a 1. </li></ol> 4. Entrega de microbolhas &amp; UTMD <ol><li> Antes da entrega de microbolhas e UTMD, camundongos C57BL / 6 foram anestesiados com quetamina e xilazina 100mg/kg 5mg/kg através de injeção IP. </li><li> Entrega de microbolhas foi administrado por via intravenosa através de uma injecção directa para o ventrículo esquerdo do coração ou por meio de cateterização da veia da cauda. Para a injeção direta cardíaca, um volume de até 100 l da solução de DNA-plasmídeo carregado de microbolhas é injetado através de uma agulha de calibre 30 inserido no espaço de 4 ª intercostal anterior sob visualização ultra-som para o ventrículo esquerdo do coração. </li><li> O bolus solução de microbolhas resultantes da injeção interventricular esquerdo é visualizado usando o Visual Sonics &#39;38 MHz transdutor de ultra-som de alta freqüência colocado em uma posição estacionária sobre o tórax do mouse em uma visão de longo eixo usando VisualSonics &quot;Vevo Imaging System 2100. Todas as seringas e agulhas são esterilizadas, garantindo o ambiente mais estéril possível para essas injeções. </li><li> Imediatamente após a injeção, a destruição das microbolhas é realizado para ~ 5 minutos, utilizando um segundo, menor tamanho de baixa freqüência 1,0 MHz transdutor realizada diretamente sobre o órgão desejado, visando a destruição desta região. Neste exemplo, o ultra-som foi administrado para o fígado em uma freqüência de repetição do pulso de 1,0 MHz, com um índice mecânico equivalente a cerca de 1,3-1,5 e período de repetição de pulso de 100 ms para cada 20 ciclos. Alternativamente, o pulso pode ser fechado para o ECG do mouse (não é mostrado neste experimento) para uma explosão de três quadros de ultra-som, a cada 4-6 ciclos cardíacos. Obtemos uma maior eficiência de transfecção com os protocolos que permitem que o leito capilar para reabastecer com bolhas entre rajadas de ultra-som. </li></ol> 5. Método de entrega alternativa Optamos por destacar a injeção interventricular, devido à complexidade do procedimento, mas em muitos casos, como infusão prolongada de microbolhas, uma veia da cauda emprojecção é o método preferido. Para o método de cauda veia de entrega de microbolhas, o mouse é anestesiado do mesmo modo. A seringa contendo o plasmídeo DNA-bound microbolhas é conectado a um cateter da veia 27 gauge agulha / cauda. O cateter da veia da cauda é inserida no terço distal das veias ou direito ou lateral esquerdo, que ao longo da cauda do rato. A seringa contendo as microbolhas é colocado em uma bomba de infusão que, automaticamente, administra um volume uniforme predefinida de solução ao longo de um período de tempo predefinido. Normalmente, infundir 200-300 ul a uma taxa de 3ml/hour. Uso de animais Todos os animais foram tratados de acordo com a prática bom animal, tal como definido pelas autoridades nacionais e / ou órgãos de bem-estar animal local, e todo o trabalho animal foi aprovado pela comissão competente (University of Hawaii Animal Care Institucional e Comitê de uso, número de aprovação 07-100 -3). Anestesia adequada (ketamina / zylazine) foi usado e analgésicos (Bupivicaine e buprenorfina) estavam disponíveis, embora não seja necessário. 6. Resultados representativos: A eficácia da entrega de DNA mediada por plasmídeo UTMD pode ser avaliado através de uma variedade de métodos, dependendo dos genes codificados na construção, tais como, mas não limitado a, luciferase in vivo de imagens, B-gal ex coloração vivo, e / ou imuno-histologia. Em particular, em imagem de bioluminescência vivo permite monitorar a presença e duração da expressão do gene em camundongos transfectados em série com um plasmídeo um gene repórter bioluminescentes (luciferase). O Xenogen In Vivo Imaging System (IVIS) (Caliper Life Sciences, Hopkinton, MA) é usado para geração de imagens bioluminescente. As imagens são normalmente tomadas de todos os camundongos do primeiro dia após transfecção mediada UTMD e é repetida a cada 3-4 dias até a expressão do gene bioluminescentes não é mais visualmente detectável pelo sistema (Figura 1). Para preparar os ratos para imagens bioluminescentes, camundongos primeiro receber uma injeção IP do repórter luciferase sonda D-luciferina (Caliper Life Sciences) e são, então, anestesiados ~ 3 minutos depois. Biodistribuição do substrato D-luciferina é permitido prosseguir para ~ 10 minutos antes de o animal é colocado na imagem IVIS câmara e um exame de imagem de corpo inteiro é tomado. Durante a aquisição, os fótons emitidos a partir do luciferase do firefly / D-luciferina reação fotoquímica são medidos. A Figura 1 também ilustra imagem IVIS semelhante bioluminescentes do fígado após UTMD, e Figura 2 é uma imagem (100X) epifluorescência do fígado transfectadas com um anticorpo anti-luciferase primária (Sigma-Aldrich) e AlexFluor-568 anticorpo conjugado secundário (Invitrogen) . Está claro para ver que a transfecção mediada fígado UTMD tem afetado não só as células endoteliais, mas o hepatocyes também. <img src="/files/ftp_upload/2963/2963fig1.jpg" alt="Figura 1" /> Figura 1 Xenogen / IVIS imagem de cardíaco UTMD camundongos tratados (A) Mouse Controle Negativo:.. Plasmid + PBS seguido por cardíaca dirigido UTMD, (B) Mouse Tratamento: Plasmid + Microbolhas seguido por cardíaca dirigido UTMD, e (C) Mouse Tratamento : Microbolhas + Plasmid seguido pelo fígado dirigida UTMD. <img src="/files/ftp_upload/2963/2963fig2.jpg" alt="Figura 2" /> Figura 2. Imunohistoquímica do fígado UTMD (anti-luciferase em vermelho). (A) Plasmid sem UTMD, e (B) Plasmid com UTMD. Imagem confocal (100X); núcleos são DAPI azul manchado.

<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>

Não há conflitos de interesse declarados.

UTMD representa uma nova abordagem para a entrega do gene. Como uma tecnologia de plataforma que pode ser combinado com qualquer uma das muitas estratégias de terapia potencial gene, para entregar uma miríade de moléculas bioativas quando um alto grau de especificidade do tecido é desejado. A principal limitação biológica da técnica é a baixa eficiência de transfecção. Outra consideração importante é a acessibilidade do órgão alvo ao ultra-som, que pode ser acentuada diminuição, intervindo osso ou ar....

<table border="1"><tbody><tr><th> Nome do reagente / equipamentos </th><th> Companhia </th><th> Número de catálogo </th><th> Comentários </th></tr><tr><td> 1,2-dipalmitoil-sn-glicero-3-fosfatidilcolina </td><td> Sigma-Aldrich </td><td> P-5911 </td><td> componente do shell de lipídios microbolhas </td></tr><tr><td> 1,2-dipalmitoil-sn-glicero-3-fosfatidiletanolamina </td><td> Sigma-Aldrich </td><td> P-3275 </td><td> componente do shell de lipídios microbolhas </td></tr><tr><td> glicose </td><td> Sigma-Aldrich </td><td> G5400 </td><td> pensamento para estabilizar as microbolhas </td></tr><tr><td> tampão fosfato </td><td> Sigma-Aldrich </td><td> P5368 </td><td></td></tr><tr><td> glicerina </td><td> Sigma-Aldrich </td><td> G5516 </td><td> Acredita-se evitar microbolhas de coalescência </td></tr><tr><td> Gás Octafluoropropane </td><td> Airgas </td><td> N / A </td><td> gás inerte utilizado em aplicações clínicas </td></tr><tr><td> Vialmix dental amalgamador </td><td> Bristol-Myers Squibb </td><td> N / A </td><td></td></tr><tr><td> 1 MHz, transdutor, 13 milímetros unfocused </td><td> Olimpo </td><td> A303S-SU </td><td></td></tr><tr><td> Função 20 MHz / Gerador de Formas de Onda Arbitrárias </td><td> Agilent </td><td> 33220A </td><td></td></tr><tr><td> Amplificador de potência </td><td> Krohn-Hite Co. </td><td> Modelo 7500 </td><td></td></tr><tr><td> Hidrofone </td><td> Bruel e Kjaer </td><td> Tipo de 1803 </td><td></td></tr><tr><td> Amplificador de carga </td><td> Bruel e Kjaer </td><td> Tipo de 2634 </td><td></td></tr><tr><td> 500 MHz Osciloscópio </td><td> LeCroy </td><td> 9354L </td><td></td></tr><tr><td> Vevo VisualSonics &quot;Imaging System 2100 com 34 MHz. </td><td> VisualSonics </td><td> 2100 </td><td></td></tr><tr><td> 27G cateteres uma veia da cauda polegadas </td><td> VisualSonics </td><td> N / A </td><td></td></tr><tr><td> Genie Além disso bomba de infusão </td><td> Kent Scientific </td><td> GENIE </td><td></td></tr></tbody></table>

introduction to the ultrasound targeted microbubble destruction technique

Em UTMD, moléculas bioativas, tais como vetores de DNA carregados negativamente plasmídeo codificando um gene de interesse, são adicionados às conchas de lipídios catiônicos agentes de contraste de microbolhas 7-9. Em camundongos essas microbolhas vetor carregando pode ser administrado por via intravenosa ou diretamente para o ventrículo esquerdo do coração. Em animais maiores também podem ser infundido através de um cateter intracoronário. A entrega posterior da circulação de um órgão-alvo ocorre por cavitação acústica em uma freqüência de ressonância de microbolhas. Parece provável que a energia mecânica gerada pelos resultados destruição das microbolhas na formação dos poros transitórios ou entre as células endoteliais da microvasculatura da região de destino 10. Como resultado deste efeito sonoporação, a eficiência de transfecção em e através de células endoteliais é reforçada, e transgene codificação de vetores são depositados no tecido circundante. DNA plasmídeo restantes na circulação é rapidamente degradado por nucleases no sangue, o que reduz ainda mais a probabilidade de entrega para não-tecidos e sonicado leva a altamente específico transfecção de órgãos-alvo.

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

Destruição de ultra-som Targeted microbolhas (UTMD) pode ser usado para direcionar site-specific entrega de moléculas bioativas, incluindo genes terapêuticos, para órgãos-alvo acessível ao ultra-som, como o coração eo fígado<sup&gt; 06/01</sup&gt;.

Introdução à Técnica Destruição Ultrasound Targeted microbolhas

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 visualizações.  University of Hawaii. Destruição de ultra-som Targeted microbolhas (UTMD) pode ser usado para direcionar site-specific entrega de moléculas bioativas, incluindo genes terapêuticos, para órgãos-alvo acessível ao ultra-som, como o coração eo fígado 06/01.

Vídeo: 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.

O ultra-som contraste Targeted Tratamento de Gliomas em camundongos através de Drogas Levando-entrega de nanopartículas e Ablação Microvascular

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

Bioengenharia

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.

Projeto de um biorreator de pressão cíclica para o Ex Vivo Estudo de válvulas cardíacas Aórtica

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.

Análise de Proteína Viral alvejado Nanopartículas entregue para tumores HER2 +

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.

Fabricação de concentrado, oxigênio à base de microbolhas Lipid Emulsões pelo Alto cisalhamento homogeneização e Concentração 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.

A técnica de microfluídica para sondar Deformabilidade celular

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.

Um chip microfluídico para ICPMS Amostra Introdução

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.

Síntese de nanopartículas de ouro lipossomas Integrated Photo-reactivos e medição das suas microbolhas cavitação em cima do pulso laser de excitação

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.

Um protocolo Experimental para avaliar o desempenho de novas sondas de ultrassom baseado na tecnologia CMUT na aplicação de imagens do cérebro

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.

Avaliação global da eficácia e segurança de entrega de droga Placenta-alvo usando três métodos complementares

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.

Preparando proteínas que produzem células sintéticas usando extratos bacterianos livres de células, liposomos e transferência de emulsão

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